department of entomology, faculty of agriculture,...
TRANSCRIPT
ENTOMOPATHOGENS AS POTENTIAL BIOCONTROL AGENTS AGAINST
RED PALM WEEVIL, RHYNCHOPHORUS FERRUGINEUS (OLIVIER)
By MUHAMMAD YASIN
M.Sc. (Hons.) Entomology
A thesis submitted in partial fulfillment of the
requirements for the degree of
DOCTOR OF PHILOSPHY (Ph.D.)
In
ENTOMOLOGY
DEPARTMENT OF ENTOMOLOGY, FACULTY OF AGRICULTURE, UNIVERSITY OF AGRICULTURE,
FAISALABAD, PAKISTAN 2016
DECLARATION
I hereby declare that that the contents of the thesis “Entomopathogens as Potential Biocontrol
Agents Against Red Palm Weevil, Rhynchophorus ferrugineus (Olivier)” are product of my own
research and no part has been copied from published sources (except references, standard
mathematical or genetic models/equations/formulas/protocols etc.). I further declare that this
work has not been submitted for the award of any other degree/diploma. The University may
take action if the information provided found inaccurate at any stage (In case of any default, the
scholar will be proceeded against as per HEC plagiarism policy).
Muhammad Yasin
The Controller of Examinations,
University of Agriculture,
Faisalabad.
We, the Supervisory Committee , certify that the contents and
form of thesis submitted by Muhammad Yasin, Registration No. 2005-
ag-1886 have been found satisfactory and recommend that it be
processed for evaluation, by the External Examiner (s) for the award of
degree.
Supervisory Committee
Chairman ____________________________________
Dr. Waqas Wakil
Member ____________________________________
Prof. Dr. Muhammad Jalal Arifl
Member ____________________________________
Prof. Dr. Shahbaz Talib Sahil
This thesis is dedicated with Love and Respect to My Parents
ACKNOWLEDGEMENTS
First of all I would like to bow my head before “ALMIGHTY ALLAH” the Omnipotent, the Omnipresent, the Merciful, the Beneficial who presented me in a Muslim community and also bestowed and blessed me with such a lucid intelligence as I could endeavor my services toward this manuscript. Countless salutations are upon the HOLY PROPHET MUHAMMAD (May Peace Be Upon Him), the fountains of knowledge, who has guided his “Ummah” to seek knowledge for cradle to grave.
The work presented in this manuscript was accomplished under the sympathetic attitude, animate directions, observant pursuit, scholarly criticism, cheering perspective and enlightened supervision of Dr. Waqas Wakil, Assistant Professor, Department of Entomology, University of Agriculture, Faisalabad. His thoughtful guidance helped me in all the time of research, writing of dissertation/publications etc. and his rigorous critique improved my overall understanding of the subject. I am grateful to his ever inspiring guidance, keen interest, scholarly comments and constructive suggestions throughout my PhD studies.
I wish to acknowledge my deep sense of profound gratitude to the worthy member of my research
supervisory panel Dr. Muhammad Jalal Arif and Dr. Shahbaz Talib Sahi, for their constructive criticism, illuminating and inspiring guidance and continuous encouragement throughout course of my study. I really have no words to express my sincere thankful feelings and emotions for all my Teachers, seniors and friends especially Dr. M. Usman Ghazanfar, Dr. M. Khalid Bashir, Dr. M. Ejaz Ashraf, Dr. Mirza Abdul Qayyum, Dr. Kashif Ali, Dr. M. Tahir and Dr. M. Irfan Akram for their cooperation, well wishes and moral support from time to time during the course of study. I am also thankful to my junior fellows specially Muhammmad Farooq, Muhammad Asif, Muhammad Ismail, Muhammad Usman, Muhammad Shoaib Qazi, Muhammad Ahmad Rana, Shumaila Rasoool, Sehrish Gulzar, Zeshna Khaliq, Muhammad Tufail, Mehwish Ayaz, Kanza Syed and Ayesha Faraz for their support towards my PhD studies. Words are lacking to express my humble obligation to my affectionate grandparents, Father, Mother, Brothers, Sisters, uncles, aunts, cousins and specially my lovely wife and my children Rehmin Fatima and Muhammad Abdullah for patience in absence of their Dad, and all family members for their love, good wishes, inspirations and unceasing prayers for me, without which the present destination would have been mere a dream.
I would like to express my deepest gratitude to the Higher Education Commission, Islamabad
(Pakistan) for the scholarship under PhD Indigenous Fellowship Program is greatly acknowledged. I would like to say special thanks to Dr. Richard Stouthamer and Paul Rugman-Jones UCR, USA for accepting me towards IRSIP as an international student. Last but least I will pay my special thanks to Ms. Aima Bashir who always prayed for my success, may God bless her.
Muhammad Yasin
i
CONTENTS
Chapter 1. Introduction……………………………………………………………………. 01
Chapter 2. Literature Reviewed
2.1 Invasive Red Palm Weevil (RPW)……………………………… 07
2.2 Taxonomic position ……………………………………………... 07
2.3 Classification…………………………………………………….. 07
2.4 Spatial distribution………………………………………………. 07
2.5 Control measures………………………………………………… 08
2.5.1 Microbial control………………………………………………… 08
2.5.2 History of microbial control……………………………………... 08
2.5.3 Entomopathogenic Fungi (EPFs)………………………………... 10
2.5.3.1 History…………………………………………………………… 10
2.5.3.2 Geographical distribution and occurrence……………………….. 11
2.5.3.3 Classification…………………………………………………….. 11
2.5.3.4 Host range……………………………………………………….. 11
2.5.3.5 Mode of infection………………………………………………... 12
2.5.3.6 Enzymes and toxins of EPFs…………………………………….. 12
2.5.3.7 Chitinases………………………………………………………... 12
2.5.3.8 Proteases and peptidases………………………………………… 13
2.5.3.9 Lipases…………………………………………………………… 13
2.5.3.10 Toxins……………………………………………………………. 13
2.5.3.11 Destruxins………………………………………………………... 13
2.5.3.12 Oosporein………………………………………………………... 14
2.5.3.13 Beauvericin and beauveriolide…………………………………... 14
2.5.3.14 Bassianolide……………………………………………………... 14
2.5.3.15 Beauveriolide……………………………………………………. 14
2.5.3.16 Host range……………………………………………………….. 14
2.5.3.17 Effect of abiotic factors………………………………………….. 15
2.5.3.17.1 Temperature……………………………………………………... 15
2.5.3.17.2 Relative humidity………………………………………………... 15
2.5.3.18 Effect of EPFs on non-target organisms………………………… 15
2.5.3.19 Integration of EPFs with other control measures……………….. 16
2.5.3.20 EPF against RPW………………………………………………... 17
2.5.3.21 Natural incidence of EPFs on RPW……………………………... 17
2.5.3.22 Susceptibility of RPW to EPFs infections under lab condition…. 18
2.5.3.23 Field and Semi-field assessment of fungi for RPW management.. 19
2.5.4 Endophytic fungi………………………………………………… 20
2.5.5 Future prospects of entomopathogenic fungi.................................. 20
2.5.6 Entomopathogenic Nematodes (EPNs)........................................... 21
2.5.6.1 Natural incidence………………………………………………… 21
2.5.6.2 Susceptibility of RPW to EPNs infections under lab conditions... 22
2.5.6.3 Field and semi-field assessment of EPNs for RPW management.. 22
2.5.6.4 Interactions between EPNs and pesticides...................................... 23
2.5.7 Entomopathogenic Bacteria............................................................. 24
ii
2.5.7.1 History............................................................................................... 24
2.5.7.2 Classification…………………………………………………….. 24
2.5.7.3 Life cycle………………………………………………………… 25
2.5.7.4 Ecology…………………………………………………………... 25
2.5.7.5 Mechanism of action…………………………………………….. 26
2.5.7.6 Commercial formulations………………………………………... 26
2.5.7.7 Methods of applications of Bt products…………………………. 27
2.5.7.8 Superiority of Bt products over synthetic insecticides…………... 27
2.5.7.9 Concerns to use of Bt……………………………………………. 27
2.5.7.10 Interaction of Bt products and other toxins……………………… 28
2.5.7.11 Effect of Bacillus thuringiensis on non-target invertebrates…….. 28
2.5.7.12 Mode of infection………………………………………………... 29
2.5.7.13 Important entomopathogenic bacteria…………………………… 29
2.5.7.13.1 Bacillus thuringiensis……………………………………………. 29
2.5.7.13.1 Paenibacillus popilliae…………………………………………... 30
2.5.7.13.2 Brevibacillus laterosporus………………………………………. 30
2.5.7.13.3 Bacillus subtilis………………………………………………….. 30
2.5.7.13.4 Bacillus sphaericus……………………………………………… 30
2.5.7.13.5 Wolbachia………………………………………………………... 31
2.5.7.14 Host range of B. thuringiensis…………………………………… 31
2.5.7.15 Natural incidence………………………………………………… 32
2.5.7.16 Susceptibility of RPW to EPB under lab conditions….………… 32
2.5.7.17 Field and Semi-field assessment of EPB for RPW management.. 32
2.5.8 Microbial control agents as a component of RPW IPM…………. 33
2.5.9 Ecological engineering and agricultural practices to conserve
microbial control agents………………………………………….
33
2.5.10 Biotechnological approaches to enhance virulence of microbial
control agents…………………………………………………….
34
2.6 References……………………………………………………….. 35
Chapter 3
Genetic variation among populations of Red Palm Weevil
Rhynchophorus ferrugineus (Olivier) (Coleoptera:
Curculionidae) from the Punjab and Khyber Pakhtunkhwa
provinces of Pakistan
Abstract………………………………………………………….. 61
3.1 Introduction ……………………………………………………... 62
3.2 Materials and methods…………………………………………... 63
3.2.1 Specimen collections…………………………………………….. 63
3.2.2 DNA extraction and amplification………………………………. 63
3.2.3 Cleaning and sequencing………………………………………… 64
3.2.4 Genetic analysis………………………………………………….. 64
3.3 Results…………………………………………………………… 64
3.4 Discussion……………………………………………………….. 65
3.5 References……………………………………………………….. 68
iii
Chapter 4 Resistance to commonly used insecticides and phosphine
(PH3) against Rhynchophorus ferrugineus (Olivier)
(Coleoptera: Curculionidae) in Punjab and Khyber
Pakhtunkhwa, Pakistan
Abstract………………………………………………………….. 74
4.1 Introduction……………………………………………………… 75
4.2 Materials and methods…………………………………………... 76
4.2.1 RPW collection and rearing……………………………………... 76
4.2.2 Test chemicals…………………………………………………… 76
4.2.3 Generation of phosphine gas…………………………………...... 76
4.2.4 Bioassay…………………………………………………………. 77
4.3 Statistical analysis……………………………………………….. 77
4.4 Results…………………………………………………………… 78
4.4.1 Imidacloprid……………………………………………………... 78
4.4.2 Spinosad…………………………………………………………. 78
4.4.3 Lambda cyhalothrin……………………………………………… 78
4.4.4 Chlorpyrifos…………………………………………………....... 78
4.4.5 Profenophos……………………………………………………… 78
4.4.6 Deltamethrin……………………………………………………... 78
4.4.7 Cypermethrin…………………………………………………….. 79
4.4.8 Phosphine………………………………………………………... 79
4.5 Discussion……………………………………………………….. 79
4.6 References……………………………………………………….. 81
Chapter 5
Insecticidal potential of Beauveria bassiana and Metarhizium
anisopliae isolates against Rhynchophorus ferrugineus
(Olivier) (Coleoptera: Curculionidae)
Abstract………………………………………………………….. 89
5.1 Introduction……………………………………………………… 90
5.2 Materials and methods…………………………………………... 91
5.2.1 RPW collection and rearing……………………………………... 91
5.2.2 Culture collection………………………………………………... 91
5.2.3 Isolation from RPW cadavers…………………………………… 92
5.2.4 Screening assay …………………………………………………. 92
5.2.5 Virulence assay………………………………………………….. 92
5.2.6 Statistical analysis……………………………………………….. 93
5.3 Results…………………………………………………………… 93
5.3.1 Screening assay …..……………………………………………… 93
5.3.2 Virulence assay………………………………………………….. 93
5.4 Discussion……………………………………………………….. 94
5.5 References……………………………………………………….. 97
iv
Chapter 6 Combined effectiveness of endophytically colonized
Beauveria bassiana and Bacillus thuringiensis against
Rhynchophorus ferrugineus (Olivier) (Coleoptera:
Curculionidae)
Abstract………………………………………………………….. 111
6.1 Introduction ……………………………………………………... 112
6.2 Materials and methods…………………………………………... 113
6.2.1 RPW collection and rearing…………………………………….. 113
6.2.2 Preparation of fungi……………………………………………… 113
6.2.3 Preparation of Bacillus thuringiensis spore-crystal mixtures…… 114
6.2.4 Screening of fugal isolates……………………………………… 114
6.2.5 Bioassay procedure……………………………………………… 114
6.2.6 Bioassay on development of R. ferrugineus……………………... 115
6.2.7 Bioassay on larval development ………………………………… 115
6.2.8 Statistical analysis……………………………………………….. 115
6.3 Results …………………………………………………………... 115
6.3.1 Fungal colonization of date palm petioles……………………….. 115
6.3.2 Toxicity of microbial agents…………………………………….. 116
6.3.3. Development of R. ferrugineus………………………………….. 116
6.3.4 Effect on larval development……………………………………. 117
6.4 Discussion ……………………………………………………….. 117
6.5 References……………………………………………………….. 120
Chapter 7 Integrated effect of entomopathogenic fungi and
entomopathogenic nematodes against Rhynchophorus
ferrugineus (Olivier) (Coleoptera: Curculionidae)
Abstract………………………………………………………….. 134
7.1 Introduction……………………………………………………… 135
7.2 Materials and methods…………………………………………... 136
7.2.1 RPW collection and rearing……………………………………... 136
7.2.2 Nematode………………………………………………………... 136
7.2.3 Preparation of fungi……………………………………………… 136
7.2.4 Treatment with entomopathogenic fungi………………………... 136
7.2.5 Treatment with H. bacteriophora ………………………………. 137
7.2.6 Treatment with entomopathogenic fungi and nematode ………... 137
7.2.7 Effects of entomopathogens on R. ferrugineus development…… 137
7.2.8 Effects of entomopathogens on larval development…………….. 138
7.2.9 Statistical analysis……………………………………………….. 138
7.3 Results………………………………………………………….... 138
7.3.1 Entomopathogenic fungi and nematode interaction……………... 138
7.3.2 Development of R. ferrugineus …………………………………. 139
7.3.3 Effect on larval development ………………………………….... 139
7.4 Discussion……………………………………………………….. 139
7.5 References……………………………………………………….. 142
v
Chapter 8 Combined toxicity of Beauveria bassiana, Bacillus
thuringiensis and entomopathogenic nematodes against red
palm weevil Rhynchophorus ferrugineus (Olivier)
(Coleoptera: Curculionidae)
Abstract………………………………………………………….. 155
8.1 Introduction……………………………………………………… 156
8.2 Materials and methods…………………………………………... 157
8.2.1 RPW collection………………………………………………….. 157
8.2.2 Preparation of B. thuringiensis spore-crystal mixtures………….. 157
8.2.3 Entomopathogenic nematode……………………………………. 157
8.2.4 Preparation of fungi……………………………………………… 158
8.2.5 Treatment with B. bassiana……………………………………… 158
8.2.6 Treatment with B. thuringiensis…………………………………. 158
8.2.7 Treatment with H. bacteriophora……………………………….. 158
8.2.8 Treatment with B. bassiana, Bt-k and H. bacteriophora………... 159
8.2.9 Sporulation and Nematode production………………………….. 159
8.2.10 Statistical analysis……………………………………………….. 159
8.3 Results…………………………………………………………… 159
8.3.1 Mortality of larvae and adult…………………………………….. 159
8.3.2 Mycosis and sporulation…………………………………………. 160
8.3.3 Insects affected by EPNs and EPNs production…………………. 160
8.4 Discussion……………………………………………………….. 160
8.5 References……………………………………………………….. 162
Summary………………………………………………………… 172
vi
List of Tables
No Title Page
No.
Table 3.1 Sampling information for RPW populations collected from date palm
Phoenix dactylifera in Punjab and KPK provinces of Pakistan……………...
70
Table 3.2 Genetic characterization of five RPW populations from the Punjab and
KPK provinces of Pakistan based on a 528 bp section of the mitochondrial
COI gene. For population abbreviations, see Table 1………………………..
70
Table 3.3 Variation in a 528 bp segment of the cytochrome oxidase subunit I (COI)
region of mitochondrial DNA (mtDNA) of Rhynchophorus ferrugineus.
Average number of pairwise nucleotide differences (k) within (diagonal
element) and between (below diagonal) populations in the Punjab and KPK
provinces of Pakistan. For population abbreviations………………………...
71
Table 4.1 Geographical characteristics of the localities from where R. ferrugineus
populations were collected in Punjab and Khyber Pakhtunkhwa, Pakistan….
84
Table 4.2 Resistance to commonly used insecticides and phosphine against
susceptible strains and field-collected populations of R. ferrugineus………..
85
Table 5.1 Characterization of B. bassiana and M. anisopliae isolates obtained from
soils and insect cadavers……………………………………………………..
103
Table 5.2 Factorial analysis of screening and mycosis of R. ferrugineus exposed to B.
bassiana and M. anisopliae isolates………………………………………….
104
Table 5.3 Percentage pathogenicity (%±SE) of 19 isolates of B. bassiana and M.
anisopliae isolates against R. ferrugineus larvae after 12 days of incubation.
104
Table 5.4 Percentage pathogenicity (%±SE) of 19 isolates of B. bassiana and M.
anisopliae isolates against R. ferrugineus adult after 12 days of incubation...
105
Table 5.5 Factorial analysis for virulence of B. bassiana and M. anisopliae isolates
against larvae and adult of R. ferrugineus……………………………………
106
Table 5.6 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 7 days of
exposure treated with B. bassiana and M. anisopliae isolates…………….…
106
Table 5.7 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 14 days
of exposure treated with B. bassiana and M. anisopliae isolates…………….
107
Table 5.8 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 21 days
of exposure treated with B. bassiana and M. anisopliae…………………….
107
Table 5.9 LC50 and LC90 values of B. bassiana and M. anisopliae isolates tested
against larvae and adult of R. ferrugineus …………………………………..
108
Table 5.10 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested
against larvae of R. ferrugineus………………………………………………
109
Table 5.11 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested
against adult of R. ferrugineus……………………………………………….
110
Table 6.1 Percentage of petiole fragments colonized by entomopathogenic (E) and
other (O) fungi in live palm petioles experiments……………………………
124
Table 6.2 Factorial analysis of mortality, pupation, adult emergence and egg eclosion
of R. ferrugineus exposed to endophytically colonized B. bassiana and B.
thuringiensis………………………………………………………………….
125
Table 6.3 Mean mortality (%±SE) of 2nd, 4th and 6th instar larvae of R. ferrugineus
vii
treated with endophytic B. bassiana (Bb: 2 cm away from inoculation site)
and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination
(means followed by the same letter within each treatment are not
significantly different; HSD test P≤0.05) …………………………………...
126
Table 6.4 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar
larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 2 cm
away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg
ml-1) alone and in combination (means followed by the same letter within
each treatment are not significantly different; HSD test P≤0.05)……………
127
Table 6.5 Growth parameters e.g. larval duration (days), larval weight (grams) pre-
pupal duration (days), pre-pupal weight (grams), pupal duration (days),
pupal weight (grams), adult longevity (days) and adult weight (grams)
(%±SE) of 2nd instar larvae of R. ferrugineus treated with endophytic B.
bassiana (Bb: 4 cm away from inoculation site) and Bt-k (Bt1: 10 µg; Bt2:
15 µg; Bt3: 20 µg ml-1) alone and in combination (means followed by the
same letter within each treatment are not significantly different; HSD test
P≤0.05) ……………………………………………………………………..
128
Table 6.6 Analysis of Co-variance for 10th instar larvae of R. ferrugineus regarding
weight gain, frass production and diet consumption when treated with
endophytic B. bassiana (Bb: 4 cm away from inoculation site) and Bt-k (Bt:
10 µg ml-1). Initial weight of larvae and diet consumption were taken as
covariate……………………………………………………………………...
129
Table 7.1 Mean mortality (%±SE) of 2nd instar larvae of R. ferrugineus treated with B.
bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M.
anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was
applied @ 100 IJs ml-1………………………………………………………..
146
Table 7. 2 Mean mortality (%±SE) of 4th instar larvae of R. ferrugineus treated with B.
bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M.
anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was
applied @ 100 IJs ml-1………………………………………………………..
147
Table 7.3 Mean mortality (%±SE) of 6th instar larvae of R. ferrugineus treated with B.
bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M.
anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was
applied @ 100 IJs ml-1………………………………………………………..
148
Table 7.4 Factorial analysis of pupation, adult emergence and egg eclosion of R.
ferrugineus exposed to B. bassiana, M. anisopliae and H. Bacteriophora…..
148
Table 7.5 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar
R. ferrugineus larvae treated with B. bassiana, M. anisopliae and H.
Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106
spore ml-1 and H. Bacteriophora was applied @ 100 IJs ml-1. Mean sharing
the same letters are not significantly different. Means sharing the same
letters within columns are not significantly different……………………….
149
Table 7.6 Effect of B. bassiana, M. anisopliae and H. Bacteriophora on the
development of R. ferrugineus. B. bassiana and M. anisopliae were used
each @ 1×104 spore ml-1 and H. Bacteriophora was applied @ 50 IJs ml-1.
Mean sharing the same letters are not significantly different………………..
150
viii
Table 7.7 Analysis of co-variance for 2nd, 4th and 6th instar larvae of R. ferrugineus
regarding weight gain and frass production at a given level of diet
consumption when treated with B. bassiana and H. Bacteriophora alone
and in combination. Initial weight of larvae and diet consumption were
taken as covariate…………………………………………………………….
151
Table 8.1 ANOVA parameters for the main effects and associated interactions for
mortality levels of R. ferrugineus larvae and adults………………………….
166
Table 8.2 Mean mortality (%±SE) of R. ferrugineus populations collected from
Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg
g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs)
applied alone or in combination after 7 days of exposure (means followed
by the same letter within each treatment and insect populations not
significantly different; HSD test P≤0.05) …………………………………...
166
Table 8.3 Mean mortality (%±SE) of R. ferrugineus populations collected from
Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg
g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs)
applied alone or in combination after 14 days of exposure (means followed
by the same letter within each treatment and insect populations not
significantly different; HSD test P≤0.05) …………………………………...
167
Table 8.4 Mean mortality (%±SE) of R. ferrugineus populations collected from
Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg
g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs)
applied alone or in combination after 21 days of exposure (means followed
by the same letter within each treatment and insect populations not
significantly different; HSD test P≤0.05)……………………………………
167
ix
List of Figures
No. Title Page
No.
Figure 3.1 Map of collection sites in Punjab and KPK provinces of Pakistan………….. 71
Figure 3.2 Distribution of mitochondrial haplotypes across five populations of RPW
from the Punjab and KPK provinces of Pakistan……………………………
72
Figure 3.3 Relationships between four Pakistani COI haplotypes and 48 others are
occurring around the world. Haplotype network constructed from 539 COI
sequences (each 528 bp long) generated by the present study and three
earlier studies (see text). Each haplotype is represented by an oval or for
that with the highest outgroup probability, a rectangle. Size of each
haplotype is indicative of the number of specimens sharing that haplotype;
also given inside each haplotype. H1-43 is numbered according to El
Mergawy et al. (2011) and Rugman-Jones et al. (2013); H44-50
corresponds to additional haplotypes from Wang et al. (2015); and H51-52
are new to this study…………………………………………………………
73
Figure 4.1 Map of collection sites in Punjab and Khyber Pakhtunkhwa provinces of
Pakistan (1. Bahawalpur 2. Rahim Yar Khan 3. Vehari 4. Dera Ghazi Kahn
5. Muzaffargarh 6. Layyah 7: Dera Ismail Khan)………………………….
87
Figure 4.2 Resistance ratio (RR) of chemical insecticides and phosphine against
susceptible strains and field-collected populations of R. ferrugineus
populations of R. ferrugineus from various localities in Punjab and Khyber
Pakhtunkhwa, Pakistan………………………………………………………
88
Figure 6.1 Mean mycosis (%±SE) in cadavers of R. ferrugineus treated with
endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k
(Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means
followed by the same letter within each treatment are not significantly
different; HSD test P≤0.05)………………………………………………….
130
Figure 6.2 Sporulation (conidia ml-1) on R. ferrugineus cadavers treated with
endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k
(Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means
followed by the same letter within each treatment are not significantly
different; HSD test P≤0.05)………………………………………………….
130
Figure 6.3 Diet consumption (grams) in 10th instar larvae of R. ferrugineus treated
with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-
k (Bt: 10 µg ml-1) ……………………………………………………………
131
Figure 6.4 Frass production (grams) in 10th instar larvae of R. ferrugineus treated with
endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-k (Bt:
10 µg ml-1) …………………………………………………………………..
132
Figure 6.5 Weight gain (grams) in 10th instar larvae of R. ferrugineus treated with
endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-k (Bt:
10 µg ml-1) ………………………………………………………………….
133
Figure 7.1 Diet consumption in last instar larvae of R. ferrugineus when treated with
B. bassiana and H. bacteriophora…………………………………………...
152
Figure 7.2 Frass production in last instar larvae of R. ferrugineus when treated with B.
x
bassiana and H. bacteriophora……………………………………………… 153
Figure 7.3 Weight gain in last instar larvae of R. ferrugineus when treated with B.
bassiana and H. bacteriophora………………………………………………
154
Figure 8.1a Mean mycosis (%±SE) in larvae of R. ferrugineus populations collected
from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k
(70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs)
applied alone or in combination (means followed by the same letter within
each treatment are not significantly different; HSD test
P≤0.05)………………………………………………………………………
168
Figure 8.1b Mean mycosis (%±SE) in adults of R. ferrugineus populations collected
from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k
(70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs)
applied alone or in combination (means followed by the same letter within
each treatment are not significantly different; HSD test
P≤0.05)…………………………..…………………………………………
168
Figure 8.2a Sporulation (conidia ml-1) in larvae of R. ferrugineus populations collected
from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k
(70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs)
applied alone or in combination (means followed by the same letter within
each treatment are not significantly different; HSD test
P≤0.05)………………………………………………………………………
169
Figure 8.2b Sporulation (conidia ml-1) in adult of R. ferrugineus populations collected
from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k
(70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs)
applied alone or in combination (means followed by the same letter within
each treatment are not significantly different; HSD test
P≤0.05)……………………………………………………………………….
169
Figure 8.3a R. ferrugineus larvae affected by H. bacteriophora (%±SE) from different
populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y.
Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H.
bacteriophora (300 IJs) applied alone or in combination (means followed
by the same letter within each treatment are not significantly different; HSD
test P≤0.05) ………………………………………………………………….
170
Figure 8.3b R. ferrugineus adult affected by H. bacteriophora (%±SE) from different
populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y.
Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H.
bacteriophora (300 IJs) applied alone or in combination (means followed
by the same letter within each treatment are not significantly different; HSD
test P≤0.05) …………………............................................................... ..........
170
Figure 8.4a Nematode production (IJs ml-1) in larvae of R. ferrugineus affected by H.
bacteriophora from different populations collected from Layyah, D.G.
Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.
bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone
or in combination (means followed by the same letter within each treatment
are not significantly different; HSD test P≤0.05)……………………………
171
Figure 8.4b Nematode production (IJs ml-1) in adult of R. ferrugineus affected by H.
xi
bacteriophora from different populations collected from Layyah, D.G.
Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.
bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone
or in combination (means followed by the same letter within each treatment
are not significantly different; HSD test P≤0.05)……………………………
171
Abstract
The red palm weevil red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera:
Curculionidae) is one of the voracious pest among invasive insect pests. Pakistani populations of
R. ferrugineus distributed among different areas have been found genetically diverse. Four
different haplotypes were recorded across Punjab and Khyber Pakhtunkhwa (KPK) provinces.
Haplotype H1, H5, H51 were found from all collection sites while H52 was rare haplotype that
was only present in populations of Layyah and Dera Ghazi Khan only. Study indicated the native
range of R. ferrugineus instead of invaded from other parts of the world, since the weevil was
recorded from Pakistan in 1913 and may have been present before that. The populations of R.
ferrugineus have gained resistance to commonly used chemical insecticides and phosphine due
to the excessive and unwise use of these chemical insecticides. Resistance against seven different
populations of R. ferrugineus was determined from very low to low and moderate to high level
of resistance against commonly used insecticides. Phosphine, cypermethrin and deltamethrin
exhibited high resistance against almost all the populations. To overcome the insecticide
resistance entomopathogens were evaluated against R. ferrugineus. Nineteen different isolates of
Beauveria bassiana s.l. and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales) were
screened which exhibited varying level of susceptibility towards larvae and adult. Three isolates
of B. bassiana (WG-41, WG-42 and WG-43) and two isolates of M. anisopliae (WG-44 and
WG-45) exhibited highest larval and adult mortality after 12 days of application and the same
isolates were tested for their virulence at different exposure intervals against larvae and adult
which caused almost 100% mortality for certain isolates. B. bassiana are capable of colonizing
endophytically in live date palm petioles even after 30 days of inoculation and can significantly
reduce the weevil population when exposed to the endophytically colonized date palm petiole
pieces. Moreover Bacillus thuringiensis var. kurstaki (Bt-k) is also an effective agent that can
cause detrimental effects on R. ferrugineus survival alone and in combination with
endophytically colonized date palm. Both agents exerted influence on developmental parameters
such as larval duration, larval weight, pre-pula duration, pre-pupal weight, pupal duration, pupal
weight, adult longevity (male and female) and adult weight (male and female) etc. Moreover,
diet consumption, frass production and weight gain was affected by the treatments applied.
Entomopathogenic fungi in integration with Heterorhabditis bacteriophora Poinar applied either
simultaneously or in sequential manners exert detrimental effects on growth and development of
R. ferrugineus larvae. Combined application of three agents i.e. B. bassiana, Bt-k and H.
bacteriophora also suppress the larvae and adult population collected from 4 different areas of
Punjab and KPK, Pakistan under laboratory conditions. Hence we can use microbial
entomopathogens against this voracious pest which are safer to human beings and compatible to
environment.
1
CHAPTER 1
Introduction
Date palm is a tropical fruit crop which belongs to the family Arecaceae. It is a main
source of dietary fiber and provides livelihood to a number of peoples in the old and new world.
The tree is named as “tree of life” in the Bible due to its long life (100 year), productivity and
elevated nutritional value (UN, 2003). It is one of the oldest cultivated plants on the earth (Lee,
1963; Riad, 2006). Date fruits have key importance in the Muslim culture and among the few
fruits repeatedly mentioned in the Holy Quran, the tree has been mentioned as a humble tree as it
does not affect the growth and development of any other plant (Goes straight and does not pour
the shade on other plants or inhibit their growth). The date palm cultivation may have been
practiced 7000 year ago (Popenoe, 1924), but the domestication of date palm is assumed to be
started in Mesopotamia by 3000 B.C. (Nixon, 1951). Excavation of godowns from Mohenjo
Daro indicated the presence of date seeds which depict the date palm cultivation in Sindh
province of Pakistan since 5000 year back (Marshal, 1931).
Some school of thought believes that Alexander the Great brought the date palm to the
Indian subcontinent (Nixon, 1951; Pasha et al., 1972). But some scientists believe that the date
palm prevailed in subcontinent before the time of Alexander as Greek army use to eat harvested
date from the gardens of Kech valley (Balochistan) when they travel through the Makran coasts
during 4th century BC (Qasim and Naqvi, 2012). Latter on the mass spread of date seeds came to
existence by the arrival of Mohammed Bin Qasim in Sindh as early as 712 AD, when he came
for the preach of Islam. During camping the Arab soldiers (threw) discarded date seeds at that
site which caused spread of date palm cultivation in Sindh valley (Ahmad and Tahir, 2005;
Dhillon et al., 2005; Jatoi et al., 2010). In Indian subcontinent off-shoots of highly potent verities
of date palm (Dayri, Halawy, Khadrawy, Zahidi and Sayer) were imported from Basra (Iraq)
during the colonial period (1910-1912) by the British Indian Government and were planted in
Muzaffargarh and Multan (Punjab, Pakistan) (Milne, 1918). Pakistan is ranked 6 th in date
production in the world which produced 0.6 million tones dates in 2013 cultivated over 93,088
ha (Al-Khayri et al., 2015), largest presence of the dates in the world. The major importer
countries include India, UK, USA, Canada, Malaysia, Germany, Indonesia and Denmark
(Faostat, 2013). In Pakistan the major cultivars are Begum Jangi (Baluchistan), Aseel (Sindh)
and Dhakki of (Dera Ismail Khan). The major date growing areas in Pakistan are Kech (the
administrative center is Turbat), Panjgur, Sukkur and Khairpur, Jhang, Dera Ismail Khan, Dera
Ghazi Khan, Multan, Muzaffargarh and Bahawalpur.
Date palm is attacked by a numerous insect pests and diseases (Al-Doghairi, 2004).
Among these insects Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier)
(Coleoptera: Curculionidae) caused 10-20% loss in production to different varieties of dates in
Pakistan (Baloach et al., 1992). The pest is cryptic in nature and has been found devastating 29
different palm species belonging to 18 genera and 3 families (Malumphy and Moran, 2009;
Hussain et al., 2013). It is a voracious feeder and most prolific. A single female may give birth to
about five million weevils in four generations, within 14 months (Nirula, 1956) which is a very
high reproduction rate (Rahalkar et al., 1972; Avand-Faghih, 1996; Esteban-Duran et al., 1998;
Cabello, 2006). Male RPW produces aggregation pheromones which attract weevils to damaged
plants (Gunawardena and Bandarage, 1995). Larvae are damaging stages that remain confined
within tree trunk, exploiting the stem vascular system and bore into the trunk (heart of the host)
causing death of the palm (Ju et al., 2011; Hussain et al., 2013). Usually 3-4 generations
comprised of different stages of the insect may be seen inside an infested palm (Rahalkar et al.,
2
1972) but in Egypt 21 generations have been reported in a single year by Salama et al. (2002).
This high rate of multiplication may be attributed to continuous egg laying throughout the year,
with some periods being more intense than others. During her life span a single RPW female can
lay 58-760 eggs (Avand-Faghih 1996; Abraham et al. 2002; Kaakeh, 2005; Faleiro, 2006,
Prabhu and Patil, 2009) which incubate for 1-6 days, before hatching into whitish yellow larvae
(grubs), which live for 25-105 days depending on the weather conditions (Wattanapongsiri,
1966; Avand-Faghih, 1996; Abraham et al., 2002). The neonate larvae chew plant fibers and
penetrate the interior leaving behind the chewed-up frass that has a typical fermented odor. The
completely developed grubs pupate in a cocoon fabricated from chewed fibers and the pupal
period lasts for 11-45 days. The life cycle of the pest may vary from just 45-139 days reported
from Philippines and Spain respectively (Esteban-Duran et al., 1998; Murphy and Briscoe,
1999). Adult weevils can interbreed and live within the same host until they are required to
colonize a new palm. If the plant remains untreated the palm can die within 6-8 months (Kurian
and Mathen, 1971; Avand-Faghih, 1996; Rajamanickam et al., 1995).
By now the beetle is distributed over more than 50% of the date palm cultivated areas
worldwide causing wide-spread damage. The ancient records of the beetle date back to the 1750s
when Rhumph first found this species on sago palm Sagu Campas, in Ceylon in 1750-1755. He
also first described the larvae, cocoon and dorsal and ventral views of the adult as Cossus
sagurios. This name was not valid according to the ICZN article 3, 11 (a), and 86. In 1776,
Sulzer identified and described the weevil from India as Curculios hemipterus Linnaeus, 1758.
The weevil was later described as Curculios ferrugineus by Olivier in 1790 and this name is
currently used. When Herbst erected the genus Rhynchophorus, he pointed out that R.
ferrugineus (Olivier) was commonly mistaken for C. hemipterus Linn. He also pointed out the
difference between two species. In 1797 Thunberg described a male specimen from India as
Cordyle sexmaculatus. It was placed in synonymy with R. ferrugineus by Csiki in 1936.
Chevrolate 1882 described the male and female specimens from Assam as “R. indostanus” and a
male from Ceylon as “R. signaticillis” based on the shapes and number of spots on the pronotum
(Wattanapongsiri, 1966).
As described earlier the beetle is native to the Indian sub-continent that was identified by
Olivier in 1790. It was observed first time from India in 1891, but devastation to coconut palms
was not reported until 1906 (Lefroy, 1906). Until 1917, RPW was considered the only pest of
coconut palm but latter on it was found attacking date palms in Punjab, India (Mohan, 1917;
Buxton, 1920). During the same period (1918) RPW also inflicted harmful effects to date palm
in Mesopotamia, but damage authentication was not confirmed by collecting any insect
specimen. Furthermore, RPW is generally considered to be invasive in Pakistan, although it was
first formerly reported in what are now the Multan, Muzaffargarh and Dera Ghazi Khan Districts
of the Pakistani province of Punjab, and the neighboring Indian state of Punjab, almost a century
ago (Mohan, 1917; Milne, 1918). The native range of RPW is thought to be restricted to
Southeast Asia and Melanesia, stretching: through the countries bordering the Bay of Bengal
from Sri-Lanka to the Malayan peninsula and Singapore; through Thailand, Cambodia and
Vietnam; across the South China Sea to Taiwan and the Philippines; and down through the
Sunda Islands (Java, Sumatra and Borneo) (Wattanapongsiri, 1966). Latter on worldwide
distribution of the pest was recorded from Japan in 1975 (Matsuura, 1993).
Since the mid-1980s, weevils advanced westward rapidly from Southern Asia and
Melanesia (Gomez and Ferry, 1999) and the Kingdom of United Arab Emirates (UAE),
Kingdom of Saudi Arabia (KSA) and Oman in 1985 (El-Ezaby, 1997), south of Spain in 1994
3
(Barranco et al., 1996), Savaran region of Iran in 1996 (Avand-Faghih, 1996), Palestine, Israel
and Jordan 1999 (Kehat, 1999) Sharquiya region of Egypt in 1992 (Cox, 1993) in China the
weevil was detected in 2007 (Li et al., 2009) and recently been reported in Cyprus, Morocco,
Italy, France, Turkey, Greece, Portugal, Aruba and Syria (Zhang et al., 2008). The weevil has
invaded every country of Southern, South Eastern and Western Asia (EPPO 2005, 2008) and
lastly in Australia (Li et al., 2009) and California USA (NAPPO, 2010). It was added to the
EPPO Alert list in 1999, since 2006 has been included on the A2 list of pests recommended for
regulation (no. 339) (Melifronidou-Pantelidou, 2009; Nardi et al., 2011).
This pest has spread to different climatic regions including Mediterranean, Monsoon,
coastal, Arid and Semi-Arid (Avand-Faghih, 1996; Faleiro, 2006; El-Mergawy et al., 2011;
Hussain et al., 2014). According to Rahalkar et al. (1972), the environment does not have a
marked influence on the growth and development of the weevil. However, Ramachandran (1991,
1998) revealed variations in morphology and habit of RPW samples collected from different
parts of India and suggested that fecundity and sex ratio may influence F1 and F2 progeny. DNA
finger prints of three morphologically different forms of RPW collected from Egyptian date
plantations indicated major genetic variations in the three forms (Salama and Saker, 2002).
Agro-climatic conditions of the region, morphology of the date palm and modern farming
systems have provided an environment conducive to the rapid establishment of RPW in the
Middle East (Abraham et al., 1998).
Different control tactics have been employed against RPW within an IPM strategy. The
main component used against RPW is phyto-sanitation, mechanical control and deployment of
chemical insecticides (Nirula, 1956; Abraham, 1971; Butani, 1975; Faleiro, 2006; Ajlan et al.,
2000), use of plant extracts (Nassar and Abdullah, 2001), and pheromone trapping (Hagley,
1965; Hallet et al., 1993; Oehlschlager et al., 1993). Chemical insecticides are efficient in RPW
control but they are short-lived and need to be applied periodically with possible negative
consequences for human health and the induction of resistance in the insect (Abraham et al.,
1998; Ferry and Gomez, 2002; Faleiro, 2006; Llácer et al., 2012a). Moreover, the unwise use of
chemical insecticides has led the resistance against this pest (Abraham et al., 1998);
To combat this problem some control measures should be initiated that are
environmentally friendly and compatible with human health. Bio-control agents are an alternate
method to potentially replace many chemical pesticides now used against RPW (Gauglar and
Kaya, 1990). Entomopathogenic fungi (EPFs), entomopathogenic bacteria and
entomopathogenic nematodes (EPNs) have been found very effective against a vast array of
insect orders. Microorganisms have been successfully used to control a number of insect pests of
economic importance (Francesca et al., 2015). Among them EPFs are an important microbial
control agent and their effectiveness has been studied by a number of scientists (Murphy and
Briscoe, 1999; Faleiro, 2006), particularly Beauveria bassiana (Balsamo) Vuillemin
(Ascomycota: Clavicipitaceae) and, to a lesser extent, Metarhizium anisopliae (Metschnikoff)
Sokorin (Ascomycota: Clavicipitaceae) (Deadman et al., 2001; Ghazavi and Avand-Faghih,
2002). Microbiological treatments with B. bassiana and M. anisopliae offer an alternative and
bio-rational pest management strategy (Inglis et al., 2001) and this is the alternate method where
potential replacement of many chemical pesticides may occur against RPW (Gauglar and Kaya,
1990; Merghem, 2011; Deadman et al., 2001;Gindin et al., 2006; El-Sufty et al., 2007; 2009;
2011;Sewify et al., 2009; Torta et al., 2009; Vitale et al., 2009; Dembilio et al., 2010; Francardi
et al., 2012).
4
EPFs cause natural epizootics in insect pests through contact to the host body following
penetrating, germination and proliferation into the host body and ultimately killing the host
(Zimmermann, 2007). Infection is caused by direct application or by horizontal transmission
from one developmental stage to the other (subsequent developmental stages) (Lacey et al.,
1999; Quesada-Moraga et al., 2004). Similarly, mechanical transmission within populations has
also been recorded for M. anisopliae, B. bassiana and Isaria fumosorosea (Lacey et al., 1999;
Quesada-Moraga et al., 2004, 2008). These peculiar characteristics enable EPFs to combat
concealed insect pests. Same is the case with RPW, whose most stages live into the tree trunk,
enabling the pest to direct contact with the treatments applied except the adult stages which can
be infected on emergence.
Being the main pathogens of Lepidopteran pests EPFs can actively participate in the
control of coleopteran pests as well due to its active mode of infestation on the outer surface of
host cuticle (Hajek and St Leger, 1994; Lacey et al., 1999). Some researchers believe that EPFs
can be effective as bio-control agents against RPW (Lacey et al., 1999; Dembilio et al., 2010;
Francardi et al., 2013; Ricaño et al., 2013). The recent identification of strains of M. anisopliae
and B. bassiana with high virulence against the RPW has increased the possibility of a more
efficient microbiological control of the Curculionid. Entomopathogenic bacteria also play a
significant role in managing insect pest populations which include the members of genus
Bacillus (Salama et al., 2004). A number of species from this genus are successfully deployed
against a variety of insect pests including the member of order Coleoptera; the species are stage
specific. Bacillus thuringiensis (Bt), B. lentimorbus, B. sphaericus and B. popilliae synthesize
insecticidal proteins (Bulla et al., 1975).
B. thuringiensis is a spore-forming gram positive bacterium is the selectively toxic
products of Bt; products that are harmless to mammals and acceptable to environment (Entwistle
et al., 1993). B. thuringiensis produces protein crystal when larvae ingested this crystal protein
through his food, crystal protein dissolved in the alkaline environment of larval midgut. Actual
toxic fragment (protein) is produced when the dissolved crystal protein is proteolytically
processed. This proteoltically processed protein adheres to the intima membrane of midgut
columnar cells. The spores are produced on the epithelial cell membranes by membrane bound
proteins. Finally, as a result of spore formation the cells of the larvae die (Bauer, 1995; Aronson
et al., 1986; Gill et al., 1992). Manachini et al. (2009) evaluated a Bt based commercial product
registered against coleopteran pests for the control of RPW and found the pathogenicity with
small increase in concentrations than the recommended dose for Coleoptera. Experiments
showed that the total number of circulating hemocytes (mainly the plasmatocytes) gradually
decreased after 19 hours when RPW larvae fed with Bt spores. In this experiment for the first
time a very high number of Bt vegetative forms were recorded in the hemolymph of RPW larvae
after exposure to the Bt commercial product.
As far as the mode of action is concerned, the Cry toxins produced after the ingestion of
Bt spores in alkaline media that lyse larval midgut epithelial cells (Bravo et al., 2007). Cell
contents together with other components promote spore germination which leads to the severe
septicemia and ultimately cause the insect death. It has been proposed that during vegetative
growth, Bt release some kind of new insecticidal proteins (Soberón, 2005; Salamitou et al., 2000;
De Maagd et al., 2001; Bravo et al., 2007). The findings that bacteria, as a vegetative form, are
in RPW hemolymph suggests that Bt is able to bypass the various above described steps to reach
the hemolymph and affect the defense system. THC were dramatically reduced, especially the
plasmatocytes. Many parasites must avoid hemocyte-mediated immune responses to growth in
5
host larvae, and many species achieve this by suppressing one or more components of the host
immune defense system, e.g. alteration of THC, inhibition of hemocyte spreading, apoptosis in
circulating hemocytes (Adamo, 2005; Eleftherianos et al., 2008; Ericsson et al., 2009). The
interaction between entomopathogenic bacteria and hemocytes is little studied in insects and the
available literature is mainly on Lepidoptera. However similar results to our findings have been
found in insect response to other entomopathogenic bacteria, for example Bt-k in Trichoplusia ni
(Ericsson et al., 2009) and Photorhabdus in fifth-stage larvae of Manduca sexta (Eleftherianos et
al., 2008). Differences in antibacterial responses have been attributed to bacterial species and
virulence levels (Dettloff et al., 2001; Giannoulis et al., 2007), however some quite important
gaps in understanding general mode of action of Bt still exist (Then, 2009). Indeed, there are
several contradictions among the different models (Then, 2009). Thus, question of how bacteria
act in RPW larvae is still open.
EPNs are obligate parasites in the families Steinernematidae and Heterorhabditidae. They
kill insects with the aid of mutualistic bacteria, which are carried in their intestine (Xenorhabdus
spp. and Photorhabdus spp. are associated with Steinernema spp. and Heterorhabditis spp.,
respectively) (Poinar, 1990). The nematodes complete 2-3 generations within the host, after
which free-living Infective Juveniles (IJs) emerge to seek new hosts (Poinar, 1990). The
pathogenicity of EPNs to Helicoverpa sp. has been demonstrated previously (Bong, 1986).
Furthermore, they have been found effective against a variety of insect pests including foliage
feeders and they have been effective mainly against soil-inhabiting pests (Kaya, 1990). Several
formulations have been developed to improve the activity of nematodes on plant and in stored
products. In coleopteran pests larvae of several weevil species (Coleoptera: Curculionidae) such
as the black vine weevil, Otiorhynchus sulcatus (F.), and the Diaprepes root weevil, Diaprepes
abbreviatus (L.) (Shapiro-Ilan et al., 2002) are susceptible to EPNs. One approach to controlling
H. armigera with EPNs would be to target the larvae when they drop to the ground or after
burrowing into the soil for pupate.
Currently efforts are focused on developing integrated control strategies against RPW by
combining more than one control agent e.g. integrated use of EPFs, EPNs and entomopathogenic
bacteria. Several researchers have demonstrated successful control of multivoltine coleopterans
by combining microbial agents. In view of the extent of damage to the date palms attributed to
RPW, it was considered worthwhile to exploit bio-rational approaches. The present study was
carried out for evaluating the efficacy of different microbial control agents with the aim to meet
the following objectives;
To check the genetic variation among populations of Red Palm Weevil Rhynchophorus
ferrugineus (Olivier) (Coleoptera: Curculionidae) from the Punjab and Khyber
Pakhtunkhwa provinces of Pakistan
To check the resistance to commonly used insecticides and phosphine (PH3) against
Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) in Punjab and Khyber
Pakhtunkhwa, Pakistan
To check the Insecticidal potential of Beauveria bassiana and Metarhizium anisopliae
isolates against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)
6
To check the combined effectiveness of endophytically colonized Beauveria bassiana
and Bacillus thuringiensis against Rhynchophorus ferrugineus (Olivier) (Coleoptera:
Curculionidae)
To check the integrated effect of entomopathogenic fungi and entomopathogenic
nematode against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)
To evaluate the combined toxicity of Beauveria bassiana, Bacillus thuringiensis and
Heterorhabditis bacteriophora against red palm weevil Rhynchophorus ferrugineus
(Olivier) (Coleoptera: Curculionidae)
7
CHAPTER 2
2.1 Invasive Red Palm Weevil (RPW)
Some invasive insect pests are of great importance due to their habit of severely
damaging the agricultural products, and imposing severe threats to ecology and causing serious
economic losses (Kenis et al., 2009; Simberloff et al., 2013). Resultantly, these annoying pests
cause direct losses of worth thousand million dollars annually and money involved in
management efforts to reduce populations below economic thresh hold level (Pimentel et al.,
2005; Kovacs et al., 2010; Van Driesche et al., 2010; Simberloff et al., 2013). RPW is an
important invasive pest that has invaded more than 50% of the date palm growing areas of the
world. This is attributed to a higher fecundity than most species (Faleiro, 2006), capability to live
and interbreed in the same tree even for several years (Avand-Faghih, 1996; Rajamanickam et
al., 1995), adult flight capacity (Wattanapongsiri, 1966) and pest tolerance to a wide range of
climatic conditions due to its protected habit within palm trees.
2.2 Taxonomic position
A key to revision of this species and related genera was previously provided by
Wattanapongsiri (1966). RPW was classified under order Coleoptera, the family Curculionidae
and the subfamily Rhynchophorinae (Wattanapongsiri, 1966; EPPO, 2007). Synonymously it is
also called Asian Palm Weevil, Indian Palm Weevil or Pakistani Weevil. This genus has 10 other
species, three of them identified from New World, two African and five tropical Asian countries.
Among these R. bilineatus, R. quadrangulus, R. palmarum, R. bilineatus, R. lobatus, R.
distinctus R. ritcheri, R. vulneratus are severe pest of palms (Booth et al., 1990; Hallet et al.,
2004).
2.3 Classification
Kingdom: Animalia (Animals)
Phylum: Arthropoda (Arthropods)
Subphylum: Hexapoda (Hexapods)
Class: Insecta (Insects)
Order: Coleoptera (Beetles)
Suborder: Polyphaga (Water, Rove, Scarab, Leaf and Snout Beetles)
Superfamily: Curculionoidea (Snout and Bark Beetles)
Family: Curculionoidea (Snout and Bark Beetles)
Subfamily: Dryophthorinae
Tribe: Rhynchophorini
Genus: Rhynchophorus
Species: ferrugineus (Red Palm Weevil)
2.4 Spatial Distribution
The aboriginal home of RPW is considered to be the Southern Asia and Melanesia, it is
cryptic in nature and has been reported to attack more than 29 different palm species belonging
to 18 genera and 3 families (Malumphy and Moran, 2009; Hussain et al., 2013), including date
palms and coconut palms, as well as the Mediterranean fan palms, the native Cretan date palms
(Kontodimas et al., 2006; Dembilio et al., 2011) and Canary Islands date palms (Dembilio et al.,
2009). Currently, this pest has spread to many areas of the world; its range now includes much of
8
Asia, regions of Oceania, Southern Europe, Middle-East, North Africa, the Caribbean, and in
October 2010, five specimens belonging to the genus Rhynchophorus sp., were found in southern
California (EPPO, 2008; 2009; 2011).
2.5 Control Measures
Different control practices have been deployed to combat RPW among date palm
growing areas of the world. Treatments revolve around the use of conventional chemical
insecticides, sterile insect techniques and use of semio-chemicals (Paoli et al., 2014) and bio-
control agents (Wattanapongsiri, 1966; Murphy and Briscoe, 1999; Faleiro, 2006a, b).
Integration of RPW associated microbial control agents with other control practices such as bio-
control agents with chemical insecticides and attract-and-kill techniques.
2.5.1 Microbial control
Microbial pest control relies on use of microbes such as EPFs, EPNs, entomopathogenic
bacteria and viruses. Very few researchers have systematically studied the effect of
entomopathogens on RPW (Murphy and Briscoe, 1999; Faleiro, 2006). Conversely,
entomopathogenic microorganisms, in particular mitosporic ascomycetes, have been reported to
naturally regulate RPW populations (Dembilio et al., 2010b). The deployment of microbial
control agents in pest control is an important step towards mitigating reliance on conventional
chemical insecticides. Microorganism exhibit high degrees of host specificity that accounts for
their distinguish ability to search their host. The use of entomopathogenic microbes has a number
of advantages such as safer to environment and non-target organisms, cheaper and self-
perpetuation. Environment friendly control practices against RPW are getting serious attention in
many parts of the world. Concentrations are focused on use of entomopathogens such as EPFs,
EPNs, entomopathogenic bacteria and their integration with the chemical insecticides and plant
extracts.
2.5.2 History of microbial control
Insects and microorganisms have ancient relationships well described by insects
conserved in amber 15 to 20 million years ago, as the collection of several insect cadavers
dressed with entomopathogens like neucleopolyhedrovirus (NPV), EPNs, trypanosomes is
reported (Poinar and Poinar, 2005). It has been an old profession; however, its roots can be
traced back to the time of Aristotle (2700 BC), who observed the diseased silk worm with
whitish growth on the dead larvae of silk worm during 335 BC. It was not until the work of
Agostino Bassi (1773-1856) an Italian lawyer and scientist reported the fungus, B. bassiana on
the larvae as a whitish sooty growth. This led to the germ theory of disease and named
“Calcinaccio” disease because the dead larvae exhibit whitish calcium powder like coverings
(Steinhaus, 1956, 1975).
Agostino Bassi observed that the causal agent of the disease as “vegetable parasite” a
fungus now called B. bassiana that may be transferred through inoculation, contact or by the
ingestion of the leaves by the caterpillar. This was the first research of Bassi which confirmed
that microorganisms could cause disease and also it was the important contribution towards
disproving the idea of spontaneous generation. Calcinaccio disease was found plaguing the silk
industry first in Italy (1805) and then in France (1841). Bassi conducted scientific studies on
Calcinaccio disease in 1807. After long and comprehensive observations, in 1835 Bassi
9
confirmed that this disease causing entity is a living organism which produced whitish growth on
the dead larvae.
He was honored for rescuing the precious and economically important silk industry by
suggesting separation of the rows of caterpillars feeding on the mulberry leaves, disinfection
process, destroying dead cadavers and keeping the rearing room clean and infection free. His
findings were translated and distributed throughout the Europe and greatly helped Louis Pasteur
(1822-1895) to study the cause and potential cure of the disease in Europe (Porter, 1973). In the
same year, a famous Italian naturalist Giuseppe Gabriel Balsamo-Crivelli studied and named the
fungus, Botrytis bassiana in the honor of Bassi (Steinhaus, 1949; Müller-Kögler, 1965; Rehner,
2005). The species B. bassiana came into existence when in 1911 Beauverie studied the fungus
again and Vuillemin created the new genus Beauveria in honor of Beauverie in 1912, since then
the species B. bassiana became the type.
In 1865, French silk industry was badly devastated and Louis Pasteur was asked to
identify the disease. He was not reluctant to accept the offer, although he was not fully aware of
silk worms, he was persuaded by his teacher and friend Senator Jean-Baptist Dumas to move and
consult the famous entomologist Jean Henri Fabre (1823-1915) in Alés Village in the south of
France. After several years he came to the conclusion that two silk worm diseases "pébrine" and
"flacherie" (thought to be caused by bacterium) are responsible for the decline of silk industry.
He proposed that pébrine is characterized by tiny black spots on the surface of dead larvae of silk
worm caused by the microorganism Nosema bombycis, previously described by Nägeli (1857).
For the potential elimination of the disease, he proposed that careful handling, segregating of
healthy and diseased larvae, and well maintained sanitation conditions may be helpful in disease
prevention (Debré, 1998).
During the study it is found that disease can be transmitted by contaminated food, contact
with the infected caterpillar and even from mother to the offspring. This is the first study
demonstrating the vertical transmission of the disease (Pasteur, 1874). He published his findings
in two series to make people aware of silk worm disease and its prevention (Pasteur, 1874). This
work laid the foundation for advances in sericulture in Japan dealing with the molecular and
biochemical biology of the silk worm. The scientists like, Agostino Bassi, Louis Pasteur and Elie
Metchnikoff, the19th century pioneers also proposed that these micro-organisms can be a good
solution for controlling economically important insect pests (Steinhaus, 1956, 1975). The first
half and end of the 19th century was the period of most development in invertebrate pathology, it
was not until the discovery of B. thuringiensis (Bt) Berliner, that practical and massive use of
entomopathogens started (Lacey and Goettel, 1995).
In 1879, Metchnikoff discovered the diseased larvae of wheat cockchafer and later on
Cleonus punctiventris near Odessa (Ukraine). He named this fungus the green muscardine
fungus. The genus Metarhizium was first established by Sorokin (1883). For this fungus, he first
proposed the name Entomophthora anisopliae, and later renamed as Isaria destructor. The
history of the description, discovery the scientific research and on the use of fungus in biological
control is described in detail by Steinhaus (1949) and Müller-Kögler (1965). In the start of 20th
century B. thuringiensis was recovered first time from infected silk worm larvae by a Japanese
bacteriologist (Ishiwata, 1901) and subsequently in 1911 German biologist Berliner re-
discovered the disease. He isolated the bacterium from infected larvae of Mediterranean flour
moth (Berliner, 1915) so named it B. thuringiensis. Because of high and knock down mortality
effects with small amount of B. thuringiensis preparations, the agronomist get aware about the
insecticidal properties of this bacterium. The first B. thuringiensis based commercial formulation
10
“Sporéine” was developed in France in 1938, but the 1st well documented record of commercial
procedure for producing Bt-based product dates from 1959 by the “Bactospéine” under the 1st
French patent as a bio-pesticide formulation. Since after, a vast array of microorganisms like
fungi, bacteria, viruses and protozoans has been identified as potential biocontrol agent against
insect pests (Riba and Silvy, 1989). So far, even though more than 100 species of
entomopathogenic bacteria have been identified, only a few Bacillus species have met with
commercial success, B. thuringiensis in particular (Starnes et al., 1993).
Today a vast array of entomopathogens are deployed against insect pests of agriculture
importance such as fruits, cereals, ornamentals, stored commodities, insect pests of households
and insect vector of medical and veterinary importance (Tanada and Kaya,1993; Lacey and
Kaya, 2007). Microbial control agents used against insect pests includes EPFs, EPNs, viruses,
protozoa and bacteria. Keeping in mind the harmful effects of chemical insecticides and their
impact on the environment and human health, insecticides based on entomopathogens exert only
a small fraction of hazards on environment and human health as compared to the conventional
insecticides. The share of bio-pesticide in crop protection market is about 600 million US$ which
accounts for only 2% of the total pesticides, with about 90% of all bio-pesticide sales involving
products based on B. thuringiensis.
The comparison of microbial pesticides with chemical pesticides is usually exclusively
cost effectiveness. These microbial insecticides particularly offer unique advantages when there
are environmental and human safety concerns along with the increasing need of enriched
biodiversity in an ecosystem and increased activity of natural enemies (Shahid et al., 2012).
Furthermore, ease of application, production on artificial medium and long term storage are
further distinct features of these bio-insecticides over other insect control tactics.
2.5.3 Entomopathogenic Fungi (EPFs)
2.5.3.1 History
The history and research on mycopathogens invading insect pests is ancient. Before the
invention of microscopes fungi could be seen with naked eye and this observation helped to
establish invertebrate pathology as a modern study. Fungi are categorized in a number of taxa
that exhibit greater diversity in properties, requirements and found in all arthropod habitats. As a
result, great attention was diverted to possible use of fungi as microbial control of insect pests.
The fungi are heterotrophic, eukaryotic, absorptive individuals which may develop in different
patterns like diffuse, branched, or tubular body that can reproduce sexually as well as asexually
(Kendrick, 2000). The primitive studies regarding entomopathogenic fungi were conducted
during start of 18th century with an aim to develop control strategies for managing muscardine
diseases of silk worm (Steinhaus, 1975). Bassi (1835 as cited by Steinhaus, 1975) proposed the
germ theory using silkworm and invading fungus, later this was named Beauveria bassiana in
the honor of Bassi. His studies on silkworm disease assisted him to introduce the fungal
biocontrol agents for the control of insect vector that elicit disease in human beings.
The silkworm diseases provided gross root foundations for the control of insect pests by
employing entomopathogens. Nevertheless, the major efforts were attempted in deploying EPFs
for the control of insect pests carried out during 1950’s when chemical insecticides were
invented. There are many fungal based products commercially available worldwide now-a-days
(Shah and Goettel, 1999; Copping, 2001).
EPFs have a long primordial historic recognition; their illustrated descriptions can be
seen centuries back, infection of B. bassiana and Cordyceps sp. to silk worm described in ancient
11
Japanese paintings infections of insects date from the 19th century (Samson et al., 1988). As a
vocation, invertebrate pathology is an organized discipline. Historic stories can be drawn from
the solution of silk worm and honey bee diseases prevention from entomopathogens (Steinhaus,
1956, 1975). Very first reports of managing insect pests in insect pathology with
entomopathogenic fungi were proposed by the legend pioneers like Louis Pasteur, Elie
Metchnikoff and Agostino Bassi (Steinhaus, 1975). Currently numerous entomopathogens are
deploying for managing insect pests in lawn and turf, orchards, glasshouse, ornamentals, row
crops, forestry, range lands, stored products, pest and insect vectors of medical and veterinary
importance (Tanada and Kaya, 1993; Lacey and Kaya, 2007).
2.5.3.2 Geographical distribution and occurrence
Soil is vital source for a number of EPFs especially species belonging to Ascomycota
thus they serve to regulate the insect populations in soil (Keller and Zimmermann, 1989; Hajek,
1997) as most arthropods spent some of their life stages into the soil. The knowledge about the
indigenous isolates of EPFs, their diversity, distribution and composition is key factor to
conserve these indigenous fungal species for the natural control of the insect pest populations
within the agro-ecosystem. The detailed studies have been conducted on the 14 occurrences? of
soil dwelling entomopathogenic fungi in different countries, the data from around the world
suggest them to be ubiquitous inhabitants of the soil (Chandler et al., 1997). The effect of
different factors like climatic conditions, geographical distribution, habitat type, and soil
properties, soil pH (Foth, 1984; Ali-Shtayeh et al., 2002; Padmavathi et al., 2003), soil organic
matter (Milner, 1989; Mietkiewski et al., 1997), soil type (Storey and Gardner, 1988; Rath et al.,
1992; Inglis et al., 2001; Derakhshan, 2008), soil moisture contents (Ali-Shtayeh et al., 2002) on
fungal incidence and dispersal has been studied several times (Vänninen et al., 1989; Chandler et
al., 1997; Meyling and Eilenberg, 2006; Zimmerman, 2007).
The worldwide distribution of EPFs in insects from different habitats is also reported by
many authors (MacLeod, 1954; Evans, 1982; Wraight et al., 1993; Aung et al., 2008; Thakur and
Sandhu, 2010). The naturally occurring EPFs can be obtained by collecting insects from the field
then incubating them under laboratory conditions and checking for outgrowth of fungi (Meyling
and Eilenberg, 2007). EPFs from eight genera (Entomphthora, Batkoa, Conidiobolus, Pandora,
Erynia, Neozygites, Zoospora and Tarichium) have been isolated from aphids. The most
common entomopathogenic fungi, B. bassiana, P. fumosoroseus, and P. farinosus were recently
isolated from some new insect hosts such as beetles of Agrilus species and hairy caterpillar of
Lymantria species from Central India (Thakur and Sandhu, 2010).
2.5.3.3 Classification
Among different fungal divisions, EPFs belongs to Ascomycota, Zygomycota and
Deuteromycota (Samson et al., 1988), Oomycota and Chytridiomycota (classifies within fungi
previously). Many of the genera of EPFs currently under research belong either to the class
Hyphomycetes in the Deuteromycota or to the class Entomophthorales in the Zygomycota.
2.5.3.4 Host range
Fungal infections to the most insect orders with all life stages have been observed, while
infection to the immatures of holometabolous insects have been reported more commonly
(Tanada and Kaya, 1993). The host range may differ significantly among different species of
EPFs and even among different strains of the same single species. For obligate pathogens,
12
specifically restricted to a narrow host range and complicated life cycles associated to their insect
host like Strongwellsea castrans (Phycomycetes: Entomophthoraceae), restricted to flies like
anthomyiid (Eilenberg and Michelsen, 1999) and Entomophthorales, Massospora sp. are
restricted to a single genus belonging to cicadas (Soper, 1974). In contrast, Deuteromycetes,
particularly B. bassiana, have wide host range including numerous genera of insects (McCoy et
al., 1988). It must be kept under consideration that description of host range to some extent
mainly relies on laboratory studies which do not reflect the true picture in nature. Some factors
like insect host, fungal biology and ecology may be responsible for reducing infection in insect
host. It is important to mention that fungi are capable of infecting several other arthropods,
insects and the species which are not pests of cultivated crops (Gibellula spp. predator of spiders
and, Erynia and Cordyceps sp. infects ants).
2.5.3.5 Mode of infection
The fungal infection of insect hosts is a complex process, involving chemical and
physical procedures starting from spore attachment to host death. Following steps are undertaken
during infection process: (1) spore attachment to the host cuticle, (2) germination of fungal
spore, (3) diffusion into the host cuticle, (4) overcoming the immune defense mechanism, (5)
formation and proliferation of hyphal bodies into the hemocoel, (6) saprophytic outgrowth from
the dead host, production and dissemination of new conidia. For the successful attachment,
mainly hydrophobicity of the spore and cuticular surface play significant role. Furthermore, the
germination and infection is influenced by a number of factors e.g., humidity, optimal
temperature, susceptible host stage and cuticular lipids, such as aldehydes, ketones, wax, short-
chain fatty acids, alcohols and esters which may exhibit antimicrobial activity. Generally, fungal
spores breach through the non-sclerotised parts of the cuticle such as joints, between segments or
the mouthparts. The conidial germination starts after 10 h of attachment and may complete by 20
h at 20-25 oC. Before infection process, germ tube produce appressorium or penetration pegs
which is accompanied by mechanical and chemical processes by the production of several
enzymes (Ortiz-Urquiza and Keyhani, 2013).
2.5.3.6 Enzymes and toxins of EPFs
Along with different degrading enzymes (such as lipase, protease, chitinase) which
account for the virulence of different entomopathogenic fungi (Joshi et al., 1995; Fan et al.,
2007), certain secondary metabolites of these fungi also possess insecticidal activities and
contribute to the pathogenesis of the fungal strains (Mollier et al., 1994). Some metabolites may
also act as the defensive tool by protecting the fungi from certain hostile factors such as
competitive micro-organism (Dowd, 1992; Bandani et al., 2000). The type of toxins produced
may also be helpful in defining the mode of action of the entomopathogenic fungi (Vey et al.,
1993). The toxins produced by different entomopathogenic fungi, their role in fungal efficacy
and safety concerns about the utilization of these compounds have been discussed by several
researchers (Roberts, 1981; Strasser et al., 2000; Vey et al., 2001).
2.5.3.7 Chitinases
The chitin is a major constituent of the insect cuticle, therefore, endo and exo chitanses
are important enzymes for the breakdown of N-acetylglucosamine polymer of insect cuticle into
monomers and a key factor determining the fungal virulence (Khachatourians, 1991).
13
Endochitinases, N-acetyl-β-D-glucosaminidases and chitinolytic enzymes from M. anisopliae
and M. flavovirid and B. bassiana were presented in broth culture nourished with insect cuticles.
2.5.3.8 Proteases and peptidases
Chitin and protein are the main constituents of insect cuticle; hence proteases and
peptidases of EPFs is considered key component in degradation of insect cuticle, saprophytic
growth, initiation of prophenol oxidase in insect hemolymph, furthermore they are also
responsible for virulence in EPFs. Chymotrypsin (CHY1) of 374 amino acids, with pI of 5.07
and MW38279 were investigated from M. anisopliae by Screen and St Leger (2000). Some
genes of overlapping response with a unique expiration pattern were observed when encountered
with the cuticle of Blaberus giganteus, Popilla japonica and Lymantria dispar and using cDNA
counted gene expression responses to the cuticles of number of host insects and constructed
microarrays from expressed sequence tags, clone of 837 genes (Freimoser et al., 2005).
2.5.3.9 Lipases
The epicuticle of the insects is chiefly composed of non-polar lipids which play an
important role in chemical signaling between insect host and EPFs (Blomquist and Vogt, 2003),
and keeps cuticular outer surface dry which aids to avoid the penetration of chemicals and
insecticides (Blomquist et al., 1987; Juárez, 1994). They are chemically stable with high
molecular mass, mainly due to the presence of specific physicochemical characteristics, like
number of carbons, length of the chain and the kind and position of double bond and the
functional groups. The long chain HC, free fatty acids, fatty alcohols and wax esters are ample
components of the insect epicuticle. It also contains fats, waxy layers and lipoproteins which act
as a barrier to the action of lipoxygenases and lipases of eontomopathogenic fungus. Among
these compounds some have anti-fungal activities (Khachatourians, 1996) while some other
possess saturated fatty acids which can inhibit the fungal growth.
2.5.3.10 Toxins
The biochemical properties and structure of some major fungal metabolites have been
investigated in detail (Vey et al., 2001), but very few studies have been conducted regarding the
metabolite production under field conditions (Bandani et al., 2000; Strasser et al., 2000). One
major problem to fungal toxins is that one type of fungi can produce variety of bioactive
metabolites and risk assessment to these entire compounds would be enormous. Furthermore,
fate of their toxins is little known in the environment, which would be the key question for their
registration.
2.5.3.11 Destruxins
Destruxins are moderately dissimilar compounds which exist in the form of isomers.
Basically destruxins contain 5 amino acids and α-hydroxy acid which may be found in many
different forms. Till now 28 different but structurally similar destruxins have been isolated from
different EPFs and most of these are discovered from M. anisopliae isolates (Vey et al., 2001).
Insects exhibit varying susceptibility levels to destruxins and Lepidopterans have been reported
as the most susceptible amongst the all studied insect orders (Samuels et al., 1988; Kershaw et
al., 1999). The toxicosis symptoms also vary among insect pests the most peculiar symptom are
an immediate tetanus; which at low concentrations develops for up to three minutes period,
while, brief or no paralysis is depicted at high dose rates (Abalis, 1981; Samuels et al., 1988).
14
2.5.3.12 Oosporein
Oosporein is produced mainly from the soil inhibiting fungi like Beauveria spp. which
contain red colored di-benzoquinone (Eyal et al., 1994). It reacts with amino acids and proteins
through redox reaction by altering the SH-groups and results malfunctioning in enzymes
(Wilson, 1971). Like bassianin and tenellin, oosporein also inhibit the activity of erythrocyte
membrane ATPase which is directly proportional to the dose rate of oosporein. Up to 50%
activity can be ceased at 200g/ml. All these pigments greatly influenced Ca2+-ATPases compared
to the activity of Na+/K+-ATPase. Antibiotic effect of oosporein against gram-positive bacteria
has also been observed with no or little effect on gram negative bacteria (Taniguchi et al., 1984;
Wainwright et al., 1986).
2.5.3.13 Beauvericin and beauveriolide
Beauvericin is also an important toxin isolated from Beauveria, Paecilomyces sp., the
plant pathogenic fungi Polyporus fumosoroseus and Fusarium sp. (Gupta et al., 1991; Plattner
and Nelson, 1994). Gupta et al. (1995) described two different forms of these toxicants
Beauvericin A and B forms K+ and Na+ complexes, which increase the membranes permeability
(Ovchinnikov et al., 1971). It also exhibits antibiotic activity against a number of bacteria like,
Mycobacterium phlei, Escherichia coli, Sarcinea lutea, Bacillus subtilis, Staphylococcus aureus
and Streptococcus faecalis (Ovchinnikov et al., 1971).
2.5.3.14 Bassianolide
Another toxin cyclo-octadepsipeptide also called bassianolide is secreted by B. bassiana
(Suzuki et al., 1977). Bassianolide is also an ionophore which exhibits different reactions with
different hosts (Kanaoka et al., 1978). Very little knowledge about the toxic nature of
bassianolide against plants and animals, the synergistic interaction with the structurally
associated myco-toxin moniliformin may be possible.
2.5.3.15 Beauveriolide
Beauveriolide are isolated from Beauveria spp. which is structurally similar to
bassianolide and beauvericin (Namatame et al., 1999). The toxic effect of beauveriolid towards
plants and animals is still unknown except beauveriolide I (Mochizuki et al., 1993). Overall
these cyclodepsipeptides may still have an unreported health hazard effects is common. Except
the above mentioned metabolites these B. bassiana also produce bassianin, tenellin and two non-
peptide toxins isolated from Beauveria spp. which aid in inhibiting the erythrocyte membrane
ATPases (Jeffs and Khachatourians, 1997).
2.5.3.16 Host range
EPFs are the diverse group of insect pathogens that contains a large number of genera
and species, the exact figure is unidentified but according to some estimation there are
approximately 700 species in 100 genera of entomopathogenic fungi, however, Onofre et al.
(2001) described it as 90 genera. These insect pathogens have broader host range and have been
found naturally occurring in various populations of insect pests. The more extensive work
regarding the natural host identification in order to recognize the biological activity of the
entomopathogenic fungi is done on B. bassiana and M. anisopliae. The host range for B.
bassiana is stated as 700 species including beneficial insects too (Goettel et al., 1990).
15
2.5.3.17 Effect of abiotic factors
2.5.3.17.1 Temperature
Among different abiotic factors affecting the fungal propagation and survival (Roberts
and Campbell, 1977; Fuxa, 1995); temperature is very important in determining the germination,
growth rate and viability of the fungal conidia not only in the host but in the environment as
well. Furthermore; the efficacy of fungal biopesticides has been highly influenced by the
environmental temperatures (Inglis et al., 1996; Klass et al., 2007). The awareness about the
fungal growth in association with the prevailing temperature is the preliminary step in selection
of any fungal strain (Fargues et al., 1992). Ferron (1978) described that optimum values for
different entomopathogenic fungi are between 20-30 ºC, which has been verified also by certain
other researchers as well such as Ekesi et al. (1999), Dimbi et al. (2004) and Kiewnick (2006).
The ability to tolerate different temperature profiles not only varies between the strains but the
thermal tolerance between the isolates is also significant (Parker et al., 2003; Dimbi et al., 2004).
Bugeme et al. (2008) observed the variable responses of B. bassiana and M. anisopliae isolates
and preeminent germination was seen at 25 and 30 ºC whereas 30 ºC was best for the radial
growth of the colony. The strains of M. anisopliae had better adoptability to tolerate high
temperatures for germination (Inglis et al., 1997; Milner, 1997).
The temperature is also considered as a factor which influences the virulence of different
fungal isolates (Tefera and Pringle, 2003). As far as the stored grain insect pest management is
concerned, mostly the virulence of different entomopathogenic fungi is evaluated under the
mutual effect of temperature and relative humidity (Sheeba et al., 2001; Batta, 2004;
Athanassiou et al., 2008). The geographical location of the isolates also account for their thermal
tolerance as studied by Vidal et al. (1997) who found that isolates of I. fumosorosea (P.
fumosoroseus) collected from Europe depicted growth at temperatures between 8-30 ºC
(optimum growth rates 20-25 ºC), the isolates from southern United States and West Asia
tolerated 8-35 ºC (optimum growth rates 25-28 ºC) whereas Indian isolates showed optimum
growth at relatively high temperature range i.e. 32-35 ºC.
2.5.3.17.2 Relative humidity
Humidity is also a key abiotic factor which greatly influences the efficacy and viability of
entomopathogenic fungi. The role of relative humidity (r.h.) is more significant in spore
germination and post mortal sporulation of the entomopathogens (Inglis et al., 2001). The
viability of the conidia over a period of time is the important parameter which is mainly
influenced by the interaction of relative humidity with different temperature levels
(Zimmermann, 2007). The prolonged stability of fungal conidia is reported mostly under cool
and dry conditions (Hedgecock et al., 1995; Hong et al., 1997). High relative humidity is the
prerequisite for the mycosis on dead cadavers (Fernandes et al., 1989) as optimum sporulation on
dead locusts (Schistocera gregaria) at >96% r.h. was observed by Arthurs and Thomas (2001).
After the treatment of different stored grain beetle’s eggs with B. bassiana among different
relative humidity levels, 92% r.h. reduced eggs hatchability up to 83 and 87% in R. dominica and
T. castaneum, respectively (Lord, 2009). Some studies have also revealed that entomopathogenic
fungi can germinate and infect the host even at low relative humidity levels (Inglis et al., 2001).
2.5.3.18 Effect of EPFs on non-target organisms
EPFs are developed as commercial formulations (biopesticides) to combat the insect
pests of agriculture and veterinary importance (Goettel et al., 1990; Kooyman et al., 1997;
16
Thungrabeab and Tongma, 2007; Reddy et al., 2008; Mahmoud, 2009). Different laboratory
trials had also been conducted to test the biological activity of formulated conidia against various
insect pests both under laboratory and field conditions (Ibrahim et al., 1999; Inglis et al., 2002;
Batta, 2003; Ugine et al., 2005). The effect on non-targeted organisms is one of the basic
principles for the evaluation of biopesticides. The broad spectrum activity of fungal
entomopathogens (Zimmermann, 2007) is the key point for their successful adaptation as
biological control agents but at the same time it might have some effects on non-target or
beneficial insects. Peveling and Demba (1997) recommended the mycopesticide as economically
sound and ecologically safe control measure of desert locust in date palm as M. flavoviride was
safe to the natural enemy (Pharoscymnus anchorago F.) of scale insects of date palm. Cottrell
and Shapri-Ilan (2003) also found that exotic Asian lady beetle (Harmonia axyridis) was less
susceptible to GHA strain of B. bassiana. On the other hand the negative effect of EPFs has also
been reported by several authors. The pathogenicity of eight EPFs isolated from natural
populations of Coccinellids was revealed against Coccinella septempunctata (Kubilay et al.,
2008). Some previous studies indicated the pathogenicity of EPFs to C. septempunctata (Manjula
and Padmavathamma, 1996; Haseeb and Murad, 1997; Cagan and Uhlik, 1999) and other
coccinellids as well (James and Lighthart, 1994; Pell and Vandenberg, 2002; Ashouri et al.,
2003).
2.5.3.19 Integration of EPF with other control measures
The publication “Silent Spring” by Rachel Carson in 1962, not only realized the side
effects of chemical insecticides to human health (Purwar and Sachan, 2006) and environment but
also lead a way to search for eco-friendly control strategies for the insect pest management. The
continuous search for biologically safe and ecologically sound control measures identified some
natural agents as potential candidates for insect control. The microbial control of insect pests
using viruses, bacteria, EPNs and EPFs (Bhattacharya et al., 2003; Sabbour and Sahab, 2005)
has become an important element of integrated pest management (Inglis et al., 2001). Among
these microbial agents, EPFs are known as promising alternative to conventional insecticides
which can effectively be used against a large number of pest species (McCoy et al., 1988;
Zimmermann, 1993; Kaur and Padmaja, 2008). The potential role of EPFs as microbial control
agents has been reviewed by various researchers (Evan, 1989; Ferron et al., 1991; Tanada and
Kaya, 1993; Boucias and Pendland, 1998; Inglis et al., 2001). One of the best strategies is the
combining EPFs with low lethal doses of chemical insecticides (Anderson et al., 1989). As
pesticides may have a variety of effects on EPFs (Alves and Leucona, 1998), therefore, care
should be taken while selecting the chemical substance that it should enhance the efficacy
without any adverse impact on the fungal strain (Inglis et al., 2001).
The first report of carbofuran being effectively combined with B. bassiana against
Ostrinia nubilalis (European corn borer) came from Lewis et al. (1996). In another study from
two carbamate insecticides (carbosulfan and carbofuran), carbosulfan inhibited the growth of B.
bassiana and B. brongniartii but overall effect revealed the possibility of using these insecticides
in IPM of Melolontha melolontha L. (Bednarek et al., 2004). Among organophosphates,
chlopyriphos 20 EC was less toxic while triazophos 40 EC and profenophos 50 EC were
moderately toxic to B. bassiana (Amutha et al., 2010). In addition to the chemical stressors
various EPFs have also been evaluated in combination with botanicals (phytoproducts); the most
prominent of them is neem and neem based insecticides. Akbar et al. (2005) evaluated the
effectiveness of B. bassiana for T. castaneum in integrated manner with plant essential oils and
17
organosilicone carriers. Azadirachtin was combined with P. fumosoroseus against Bemisia
argentifolii (James, 2003). The interaction and compatibility of different insect growth regulators
(IGRs) and herbicides with EPFs is in vague (Inglis et al., 2001). The fungus Lecanicillium
muscarium when simultaneously applied with buprofezin a higher mortality of B. tabaci 2nd
instar larvae was seen (Cuthbertson et al., 2010).
Triflumuron, a benzoylphenyl urea (BPU) which is a chitin synthesis inhibiter acted as
“general stressor” and made the Lepidopteran larvae more susceptible to fungal infection by M.
anisopline (Hassan and Charnley, 1989). Along with other agrochemicals; the herbicides are also
considered as potential inhibitor to entomopathogenic fungi (Inglis et al., 2001). While studying
the effects of four commonly used herbicides on vegetative growth and sporulation of six EPFs
under laboratory conditions. Poprawski and Majchrowicz (1995) found totally impaired fungal
growth at all tested temperatures. The EPFs have also been integrated with some other microbial
pathogens as an alternative use of chemical substances (Zimmermann, 1993). The interaction of
B. bassiana and B. thuringiensis var. israelensis for the control of Musca domestica in poultry
houses was studied in field trials (Mwamburi et al., 2009). In the same scenario the B.
thuringiensis has been incorporated with EPFs against a number of insect pests under different
agro ecosystems (Kryukov et al., 2009; Lawo et al., 2008; Wraight and Ramos, 2005; Lacey et
al., 2001; Brousseau et al., 1998; Molina et al., 2007).
2.5.3.20 EPF against RPW
EPFs are commonly found in the nature and cause epizootics in insect populations, thus
play a significant role in regulating insect population. Mostly, Entomophthorales and
Hyphomycetes attack on terrestrial insects. EPFs from various strains of B. bassiana and M.
anisopliae have been found in association with RPW. EPFs are among the most relevant
biological agents suggested to control RPW (Faleiro, 2006). Some of these EPF strains were
tested against RPW, M. anisopliae being more effective than the latter one (Gindin et al., 2006).
However, no strain was originally isolated from RPW in this study. A number of studies were
carried out to investigate the effectiveness of EPFs against RPW, the investigations led to the
detection of more active isolates against RPW individuals in laboratory and field trials (Gazavi
and Avand-Faghih, 2002; Shawir and Al-Jabr, 2010; Shaju et al., 2003; El-Sufty et al., 2007;
Tarasco et al., 2007; El-Sufty et al., 2009; Sewify et al., 2009; Dembilio et al., 2010).
Usually, EPFs infect their host through contact action which makes them superior than
the other entomopathogens (Butt and Goettel, 2000). In RPW infection mostly occur via direct
contact to the inoculum, transmission from diseased to the healthy ones (horizontal transmission)
and transmission from the subsequent developmental stages (vertical transmission) via new
generation of spores (Lacey et al., 1999; Quesada-Moraga et al., 2004). Thus, EPFs must be put
forward as potential bio-control agents in IPM to control RPW current outbreaks (Faleiro, 2006;
Murphy and Briscoe, 1999).
2.5.3.21 Natural incidence of EPFs on RPW
In the beginning, intentions were focused on isolation of fungal strains from RPW;
different strains of B. bassiana and M. anisopliae were recovered from pupae and adults of RPW
(Ghazavi and Avand-Faghih, 2002). Very first natural infection of M. anisopliae was recorded
from R. bilineatus as a result of accidental infection when treatments were applied against
Scapanes australis (Bedford, 1974) with M. anisopliae spore based commercial formulation
(Prior and Arur, 1985). Latter on different researchers found naturally infected RPW specimens
18
with B. bassiana and M. anisopliae (Salama et al., 2001; Shaiju-Simon and Gokulapalan, 2003;
Salama et al., 2004; Gindin et al., 2006; Güerri-Agulló et al., 2007, unpublished; El-Sufty et al.,
2007; Güerri-Agulló et al., 2008; Merghem, 2011). In 2007, RPW fungal infected pupae were
reported from date palm garden in Spain (Dembilio et al., 2010b). Colonies of B. bassiana,
Aspergillus sp., Metarhizium sp., Fusarium sp., Trichothecium sp. and Penicillium sp. were also
recovered from different developmental stages of RPW in Italy (Torta et al., 2009; Tarasco et al.,
2008). A B. bassiana isolate (B-SA3) isolated from Al-Qatif province (Saudi Arabia) from dead
RPW by Hegazy et al. (2007) which latter on was used against RPW in laboratory study.
Lo-Verde et al. (2014) isolated B. bassiana from RPW adults collected from Villagrazia
and Cinisi (Palermo Province, Sicily). Recently, M. pingshaense recovered from RPW in
Vietnam which kill the adults in a very short time (Cito et al., 2014). Thanks to an efficient
enzymes and toxin production.
2.5.3.22 Susceptibility of RPW to EPFs infections under laboratory conditions
Laboratory studies found that M. anisopliae strains caused 80-100% mortality in RPW
larvae and adult. Adults withstand for 4-5 week against spore suspension, while dried
formulation took 2-3 weeks to kill 100% RPW adults. Moreover, M. anisopliae caused 100%
larval mortality within 6 and 7 days, but this took longer time to B. bassiana for getting the same
level of mortality (Gindin et al., 2006). Similar findings were recorded in Egypt (Merghem,
2011) and Italy (Francardi et al., 2012, 2013) against RPW larvae and adults. Research studies
suggested that effectiveness of EPFs against RPW was about 85% under controlled and semi-
natural conditions (El-Sufty et al., 2009; Dembilio et al., 2010). Significantly higher mortality
was recorded for the bio-control of RPW by Vitale et al. (2009) when treated with the
commercial formulations of using a commercial product of B. bassiana and M. anisopliae alone
and an integrated manners, whereas sole application of B. bassiana recovered from dead RPW
cadavers did not gave promising results against adults. This might be attributed to the fact that
polar extracts of adult may inhibit adhesion and germination of pores.
In contrast efficient results were recorded by deploying B. bassiana as a bio-control agent
recovered from naturally infected RPW (Sewifi et al., 2009; Dembilio et al., 2010; Güerri-
Agulló et al., 2010). Thus, we cannot deny its importance in bio-controls of RPW. Dembilio et
al. (2010b) performed laboratory experiments to check the vulnerability of RPWs to B. bassiana.
The strain was infective to eggs, larvae and adult stages. They further reported adult lifespan was
reduced from 1/2 to 1/10 and adults of either sex transmitted 55 and 60% disease to the healthy
adults during courtship. B. bassiana not only induced mortality but also significantly affect the
fecundity (approximately 62.6%) and egg hatchability (32.8%). Likewise, larvae obtained from
infected female eggs exhibited 30-35% more mortality than the healthy ones, overall 78% less
progeny were recorded as compared to the check treatment (Dembilio et al., 2010a). These
finding are in accordance with the findings of Torta et al. (2009) and El-Sufty et al. (2009) who
reported significantly higher mortality of RPW small larvae and adults to indigenous strains of B.
bassiana.
They further revealed that susceptibility was more evident in young larvae than old ones
that might be due to the scarcity of antimicrobial cuticular compounds in younger larvae (Mazza
et al., 2011a). Most recently Hussain et al. (2014) reported varied susceptibility level of different
larval instars to four different isolates of B. bassiana. Other scientists also revealed the same
results with EPFs against different developmental stages of RWP (Ghazavi and Avand-Faghih
2002; Shaiju-Simon and Gokulapalan, 2003; Gindin et al., 2006; Dembilio et al., 2010; Cito et
19
al., 2014). Lo-Verde et al. (2014) evaluated B. bassiana against egg, larvae and adult, the strain
was isolated from infected adults and were found quite effective against all the tested stages. For
the very first time 3 isolates of another different EPF Isaria fumosorosea showed promising
results against RPW (Sabbour and Abdel-Raheem, 2014).
2.5.3.23 Field and Semi-field assessment of fungi for RPW management
The efficacy of EPFs depends to a great extent on formulation. Sewify et al. (2009)
reported successful reduction in RPW incidence in Egypt with indigenous strain of B. bassiana
isolated from a RPW cadaver under field conditions. In field trials El-Sufty et al. (2009) reported
13-47% mortality in adult RPW population using a strain of B. bassiana isolated in UAE. Later
on this strain was effectively deployed in auto-dissemination traps in date palm groves (El-Sufty
et al., 2011). Field studies were conducted using two formulations of local strains of B. bassiana
at UAE. The oil-based formulation of EPF exhibited 13.7-19.2% adult mortality, while dust
formulations imparted only 8.9% mortality (El-Sufty et al., 2007). However, Abdel-Samad et al.
(2011) observed little effect of oil based commercial formulation of B. bassiana on RPW, hence
not recommended for formulation and field application, since it was quite expensive as compared
to the other formulations. Moreover, polar extracts from adults were found to inhibit the spore
germination of B. bassiana commercial formulation (Mazza et al., 2011a).
Besse et al. (2011) reported high pathogenic potential of an indigenous strain of B.
bassiana against RPW, hence recommended as promising agent for bio-control. A preventive
and curative treatment with solid formulation of highly virulent strain of B. bassiana with high
persistence was applied against RPW under semi filed conditions on 5 year old P. canariensis
palms. The efficacies were >85.7%, confirming the pathogenic potential of this strain as a bio-
control agent against RPW (Güerri-Agulló et al., 2011). However, the few field studies carried
out so far lacked adequate experimental designs, used fewer replication, had high infection rates,
etc. (Güerri-Agulló et al., 2011). Lecanicillium (Verticillium) lecanii significantly affected the
mortality of various larval instars and adults and the egg hatching percentages of adult females.
Moreover, yield losses in date production decreased from 56 and 60% to 22 and 22% in El-Esraa
(Nobarya) and El-Kassaseen (Ismailia) respectively (Sabbour and Solieman, 2014). Solid state
formulations of two B. bassiana isolates deployed against RPW under field condition exhibited
100% mortality even after 30 days post application and the efficacy persisted for 3 months
(Ricaño et al., 2013).
Sabbour and Abdel-Raheem (2014) applied Iseria fumosorosea in date palm plantations
and reported significant reductions in date palm weight loss. Results revealed that palm weight
significantly increased in El-Kassaseen compared to El-Esraa to 5341±40.30 kg Feddan-1 as
compared to 1981±80.54 kg Feddan-1 in the control during season 2012. During 2012 season
yield losses were 59 and 62% in El-Esraa and El-Kassaseen which decreased to 28 and 27% in
these respective regions; the same results obtained during 2013 season. These results called for
expanded open field trials with I. fumosorosea strains to explore their bio-control potential.
Most recently, Jalinas et al. (2015), using acoustic recording methods, found greatly reduced
feeding activity after infesting palm trees with B. bassiana treated larvae. Retarded larval
movement and feeding noises suggested that B. bassiana infection weakened RPW larvae, which
reduced detectable feeding activity. The main difficulty in the implementation of acoustical
detection methods is accessibility of these bio-control agents to the pest insects. Given the
concealed nature of RPW, a systemic distribution of the agent, while highly desirable, is difficult
to achieve practically. Consequently, adults are the targeted stage for fungal delivery because
20
this is the only free living stage and future research should be focused at attracting-and-infecting
RPW adults could be most effective in managing this pest (Dembilio et al., 2010b).
2.5.4 Endophytic fungi
Fungal endophytes may be beneficial in preventing disease by induction of host defense
mechanisms (Sivasithamparam, 1998) or by directly affecting plant pests (Arnold et al., 2003).
Reports on endophytic colonization of EPFs in date palms have been published which may be
useful in the future (Gómez-Vidal et al., 2006). They inoculated B. bassiana, Lecanicillium
dimorphum and L. c.f. psalliotae within young and adult date palms petioles and exhibited the
fungal survival even after 30 days of inoculation; fungi were detected inside the parenchyma and
sparsely within vascular tissue using microscopy techniques without any detrimental effect on
date palm. Arab and El-Deeb (2012) applied endophytic fungi on date palm seedlings after 6
months the date palm pulp was offered to the larvae in laboratory which inflicted 80.3%
mortality after 14 days. Ben Chobba et al. (2013) provided the report on 13 different fungal
isolates from date palms from roots and leaves in Tunisia, although these might be affiliated to
some fungal diseases in date palms but provided the way to the researchers towards endophytic
colonization of EPFs. Nevertheless, the deployment of EPF strains with endophytic behavior is
not well understood for systemic protection of palms against RPW. Field trials with B. bassiana
strains to explore their bio-control potential are urgently needed.
2.5.5 Future prospects of entomopathogenic fungi
The successful utilization of EPFs is well recognized in biocontrol programs but the
inconsistent performance of these control agents is attributed by a variety of factors and the
major one is their great dependency on the environmental conditions. Different fungal strains
have limiting temperature ranges for germination, infection and post mortal sporulation. Same is
the case for humidity conditions as many strains require high humidity for spore germination and
sporulation (therefore oil formulations have been developed). The other restricting factors for the
broad spectrum application of the mycoinsecticides include; the limited production of toxins
(from the view point of registration authorities, the production of toxins is a hurdle for
registration and practical use) by the fungi, the slow rate of activity of the fungal conidia, the
higher doses of the conidia required for the effective control which ultimately yields inconsistent
results compared to the chemical insecticides (Gressel, 2001), and the pathogenicity of various
fungal strains to non-target organisms. Therefore, different approaches have been proposed to
tackle these limitations especially with regard of decreasing application rates and increasing
virulence of the fungal pathogens. The most significant among them is the integrated use of EPFs
with other biological control agents.
The second and the most promising approach is the implementation of biotechnology
which has great potential to play a vital role in the EPFs development process from the
identification of virulent strains to final formulation (Glare, 2003). Moreover, the high
production rates of commercialized fungal formulations can be compensated if the equivalent
control is attained at lower concentrations. The transgenic and genomic recombinant approaches
yielding the reduced median lethal concentration (LC50) of the pathogens and shorter survival
time of the target species (St. Leger et al., 1996) not only tend to improve the infection rate but
also reduce the cost of the applied formulation. The herbicide and fungicide resistant genes have
also been induced to various fungal strains (Bernier et al., 1989; Fang et al., 2004) which
21
ultimately make use of the fungal pathogens in combination with certain herbicides and
fungicides.
2.5.6 Entomopathogenic Nematodes (EPNs)
Interest in the use of EPNs as bio-control agents against a variety of pests has increased
in last two decades (Dolinski and Lacey, 2007; Lacey and Shapiro-Ilan, 2008). Researchers are
expanding the pathogenic potential of EPNs against a variety of plant nematodes, harmful
insects, soil-borne plant pathogens and mollusks (Grewal et al., 2005). So far more than 30
families of nematodes, associated with insects have been reported, but because of the bio-control
potential concentrations are focused on seven families of nematode including Sphaerularidae,
Rhabditidae, Allantonematidae, Mermithidae, Heterorhabditidae, Steinernematidae and
Neotylenchidae. For the biological control of RPW, Steinernematidae and Heterorhabditidae,
received the most attention. These nematodes carry species-specific pathogenic bacteria,
Photorhabdus by Heterorhabditidae and Xenorhabdus by Steinernematidae, which are released
into the insect hemocoel when infective juveniles (IJ), penetrate into the insect host body. The
third infective juvenile (IJ) stage of these EPNs actively searches for a suitable host, invades it,
and releases symbiotic bacteria into the insect hemocoel. This process kills the invaded insect via
bacterial septicemia and/or toxemia (Kaya and Gaugler, 1993).
2.5.6.1 Natural incidence
Few species of EPNs have been recorded as naturally infecting RPW. The efforts to
infect RPW with EPNs started with the recovery of parasitic species, Praecocilenchus
rhaphidophorus Poinar, from Rhynchophorus bilineatus in Papua New Guinea and New Britain
(Poinar, 1969) and Praecocilenchus ferruginophorus, isolated from infected RPW in India (Rao
and Reddy, 1980). P. ferruginophorus was recovered from hemocoel and hemocoel of adults,
and fat tissue, trachea and intestine of RPW larvae. Usually, the nematodes are released out of
the body from infected insect during oviposition or may also be released with the feces via
intestine. As a consequence of being released from the body the ovaries of infected weevils are
harmed due to the production of eggs (Triggiani and Cravedi, 2011). The practical importance of
nematode fauna associated with the Rhynchophorus palmarum lead researchers to study the
Bursaphelenchus cocophilus (Cobb) Baujard a causative agent of red ring disease in palms in
neo tropics (Giblin-Davis, 1993). Morover, Bursaphelenchus gerberae, Caenorhabditis angaria
and Mononchoides sp. have also been reported from R. palmarum (Gerber and Giblin-Davis,
1990; Giblin-Davis et al., 2006; Kanzaki et al., 2008; Sudhaus et al., 2011). But none beyond
Rhynchophorus spp. has been effectively surveyed.
Other non-pathogenic nematodes inflicted no harmful effects on RPW are known from
three Rhynchophorus spp.: Acrostichus rhynchophori (named Diplogasteritus in older
publications; Kanzaki et al., 2009) and Teratorhabditis palmarum were isolated from R.
palmarum and R. cruentatus respectively (Gerber and Giblin-Davis, 1990), while
Teratorhabditis synpapillata Sudhaus was recovered from RPW in India and Japan (Kanzaki et
al., 2008). Salama and Abd-Elgawad (2001) isolated Heterorhabditis spp. from five sites in
Egypt. However, only two out of the five isolated nematode strains survived for 24 hours
exposure in RPW infested palm tissue, nematode had a low viability of only 14-19%. The
retarded growth of nematodes is thought to be due to the generation of acetic acid, ethyl acetate
and ethyl alcohol from the infested palm tissue that limit the use of EPNs especially
Heterorhabditis indica (Monzer and El-Rahman, 2003) in addition to the concealed nature of
22
RPW (Abraham et al., 2002). On the other hand, El-Bishry et al. (2000) reported that the host
finding ability of juveniles decreased when the palm tissues were washed and sterilized.
Anti-desiccants such as Leaf Shield and Liqua-Gel were reported to improve the efficacy
of EPNs isolated from date plantations in Saudi Arabia (Hanounik et al., 2000). Steinernema sp.
was isolated from naturally infected field collected adult of RPW inform the eastern province in
Saudi Arabia (Saleh et al., 2011). Recently, Mononchoides sp., Teratorhabditis sp. and
Koerneria sp. were found infecting pupae and adults of RPW in southern Italy, but their species
identification, clarification on their biological parameters and type of association between RPW
and these nematode species are still in progress (Oreste et al., 2013).
2.5.6.2 Susceptibility of RPW to EPNs infections under laboratory conditions
Laboratory studies revealed that both larvae and adults of RPW were infected by
Steinernema riobrave, S. carpocapsae and Heterorhabditis sp. (Abbas and Hononik, 1999).
Similar results were reported by Salama and Abd-Elgawad (2001) when tested five strains of
Heterorhabditis, were more virulent to RPW than other tested entomophilic nematode species
(Salama and Abd-Elgawad, 2001). Laboratory studies showed that RPW larvae were suitable
host for H. indicus (Banu et al., 1998). Similarly, Elawad et al. (2007) reported high mortality of
H. indicus against RPW in UAE under laboratory conditions. Laboratory studies performed in
Italy H. bacteriophora Poinar was reported to be the most effective against RPW larvae and
adults (Triggiani and Tarasco, 2011). Additionally, exposure of RPW larvae to genetically
modified strains of Heterorhabditis and Steinernema exhibited 95-100% and 50% mortality
under and laboratory and field conditions respectively (Hanounik, 1998).
Similar findings were reported from Turkey in which H. bacteriophora inflicted 69% and
80% larval and pupal mortality respectively in RPW (Atakan et al., 2009). This might be
attributed to the fact that S. carpocaspsae was not encapsulated by RPW hemocytes, thus it is
necessary to discover the phenomenon which contributed to the lack of reproduction in larvae
and adult of RPW (Manachini et al., 2013). Shahina et al. (2009) evaluated seven Pakistani
strains of EPNs against eggs, first, third, sixth and final larval instar, and adults of RPW under
laboratory conditions. Significant differences were observed in the mortality of various life
stages of the weevil, while the highest egg mortality was found from S. siamkayai and H.
bacteriophora (95±2.1 and 97±2.2% at 150 infective juveniles (IJs ml-1). Recently, Atwa and
Hegazi, 2014 evaluated 12 EPNs, that all were infective against first instars of RPW larvae.
Some species were selective for a specific host stage while others were effective against all
stages.
2.5.6.3 Field and semi-field assessment of EPNs for RPW management
For EPNs applications in the field a preliminary agarose assay is preferred. This is a
simple and rapid laboratory test for measuring chemo-attraction of nematodes to host diffusates
and host recognition as a predictive screening tool for field testing of new Heterorhabditis
isolates (Monzer, 2004). Earlier field studies with EPNs such as Steinernematid and
Heterorhabditid in date palms did not exhibit efficacious results due to the environmental
constraints and RPW ecology (Hanounik, 1998; Abbas et al., 2001). Similarly, Koppenhöfer and
Fuzy (2004) reported gradually decreased susceptibility of RPW larvae to S. carpocapsae IJs.
However, later studies in the date palms from Middle East reported adult and larval mortality
from the same nematode spp. isolated from respective hosts. The soil treatment of these
nematodes around palms with 8×106 IJs palm-1 led to 33-87% adult mortality, while spraying the
23
palm trunk with the same nematode suspension resulted in only 8-13% adult mortality (Abbas et
al., 2000).
Similar results were recorded by Santhi et al. (2015) who evaluated S. carpocapsae and
H. bacteriophora against the RPW under simulated natural conditions and reported that pupae in
cocoons and adults exhibited a high susceptibility to S. carpocapsae. These findings are
important when considering optimal use of EPNs for the control of RPW under natural
conditions. Studies in curative and preventive assays of S. carpocapsae with chitosan by
spraying the product at a dose of 3.6×106 IJs + 36 ml chitosan palm-1 in about 2 l of water on
trunk and the bases of the fronds of each palm until the run off with the help of a manually
operated backpack compact sprayer. The treatment showed efficacies of around 80% in curative
assays and 98% in preventive assays in various palms.
Accordingly, Llácer et al. (2009) and Dembilio et al. (2010a) performed experiments on
P. canariensis and P. theophrasti respectively by using S. carpocapsae with chitosan, efficacies
ranged from 83.8-99.7% and palm survival significantly increased as compared to the check
treatments. From these experiments it was proved that chitosan as adjuvant can be effectively
used with EPNs, particularly S. carpocapsae, which extend their period and protect them from
environmental conditions (Llácer et al., 2009; Dembilio et al., 2010a). Apart from weather
factors, other organisms associated with RPW can interfere with the efficiency of EPNs. The
RPW associated organisms can interfere with the effectiveness of entomopathogens such as
RPW predatory mites Centrouropoda almerodai Wisniewski and Hirschmann can reduce the
efficacy of the nematode S. carpocapsae (Morton and Garcia-del-Pino, 2011; Mazza et al.,
2011b).
In Egypt, encouraging results were also recorded with another Steinernema sp. recovered
from pupae and adults of RPW; this strain along with two other indigenous strains of the same
genus inflicted considerable mortality against larvae and adults of RPW both under laboratory
and field conditions (Shamseldean and Atwa, 2004). Shamseldean (2002) performed field studies
on date palm trees with Egyptian isolates H. indicus (strain EGBB) H. bacteriophora (strain
EKB20) and Steinernema sp. (strain EBNUE). He reported no symptoms of old or new
infestation in treated palm trees at all treatments. The Saudi Arabian strain of H. indica induced
60 and 46% larval and adult mortalities, respectively, when the nematode was applied at the base
of tree into the soil (Saleh and Alheji, 2003). Similar finding were observed from the field trials
in France but raised an important point regarding defining optimal application standards (Chapin
and André, 2010; Pérez et al., 2010). Because the efficacies in the field study of Dembilio et al.
(2010a) were not significantly different when S. carpocapsae was applied singly or integrated
manner with imidacloprid.
Two successive application of Steinernema sp. by trunk injection resulted in significant
reduction in RPW population after 3 weeks. Efficacies ranged from 48-88% in the curative assay
and significant increase in palm survival was recorded as compared to the control treatment
(Atwa and Hegazi, 2014). Recently, 42 billion worms were imported from Germany to Israel to
combat voraciously feeding RPW spp. in Israel (Anonymous, 2015). The use of EPNs should be
considered when designing integrated management strategies against RPW.
2.5.6.4 Interactions between EPNs and pesticides
Sole or the integrated application S. carpocapsae and imidacloprid under natural
conditions were not significantly different from each other with RPW mortalities ranging from
73-95% and significant increase in plant growth (Dembilio et al., 2010a). Similar results were
24
also observed by Tapia et al. (2011) and suggested application of S. carpocapsae and
imidacloprid after every 60 days as preventive measure during field studies in Southern Spain. In
the light of above findings the results suggested that the combined effect of S. carpocapsae in
chitosan formulation and imidacloprid greatly enhanced the efficacy against RPW under field
conditions and significantly reduced the reproductive potential of the RPW (Tapia et al., 2011).
2.5.7 Entomopathogenic Bacteria
2.5.7.1 History
Existence of bacteria is as old as the history of life on earth. Evidence of bacterial fossils
dates back to the Devonian period (416-359.2 million years ago) and considerable signs depict
their presence from Precambrian time, about 3.5 billion years ago. The fossils found in north-
west Australia's Pilbara region are thought to be nearly 3.5 billion years old and considered the
oldest ones on earth planet. In Proterozoic Eon (about 1.5 billion years ago), when the activity of
cyanobacteria resulted in oxygen production, bacteria became widespread (Anonymous, 2013).
The gradual evolution of the bacteria made them able to survive under a wide range of
environmental conditions with several descendent forms. As a result of this, today an
uncountable and immeasurable diversity in morphology, physiology and taxonomy of bacteria
prevails. Bacteria have been found living very close to every living organism including human
beings. Both beneficial and harmful forms of bacteria have been thriving in various climates like
soil, water, air and hot water springs etc.
Confirmatory evidence of using entomopathogens for the control of insect pests are not
known in ancient times, however, human interest in exploiting microbes particularly bacteria
rose to its extreme after the discovery and the commercial availability of microscope in late 19 th
and early 20th century. Scientific efforts for the survival of the famous Japanese silk industry
against sudden death of caterpillars proved fruitful resulting in the discovery of a spore forming
bacterium, Bacillus sotto, by Sigetane Ishiwata (1868-1941) (Aizawa, 2001). This discovery lead
to the world’s first ever demonstration of toxins when many other scientists including Aoki and
Chigasaki (1915) and Mitani and Watari (1916) found enhanced lethal actions of bacterial
cultures on silk worms when they were applied in alkaline solution (Aizawa, 2001). Doors of
discoveries were opened for man and a German scientist Ernst Berliner in 1909 isolated a
bacterium named by him as B. thuringiensis that killed the flour moth, Ephestia kuhniella
Guenée (Lepidoptera: Pyralidae).
Within the Prokaryotes, bacteria are the microorganisms that lack a nuclear membrane
which separates genetic material from cytoplasmic contents and other membrane bounded
organelles. Bacteria surround us all around and thus, can be isolated from any environment and
hence their enriched flora can be given the name of metabolic strategy which they use to earn
energy such as phototrophs (gain energy from sunlight), lytotrophs (obtain energy form
inorganic material) and organotrophs (receive energy from organic material). The variation in
their size is from one to few microns, and depending upon the morphologies, they can be
grouped as cocci (spherical), bacilli (rod shaped) and spirochetes (spiral shaped). Propagation in
bacteria is carried out through binary fission, a mode of asexual reproductions in which daughter
cells are produced from mother cell as clonal copies (Jurat-Fuentes and Jackson, 2012).
2.5.7.2 Classification
Entomopathogenic bacteria mostly belong to the families Bacillaceae,
Enterobacteriaceae, Pseudomonadaceae, Micrococaceae and Streptococcaceae (Tanada and
25
Kaya, 1993). Although many bacteria are beneficial and essential but members from families
Eubacteriales, Bacillus and Serratia have been registered against insect pests (Tanada and Kaya,
1993). For the successful control of RPW, bacterium has been exclusively isolated from different
developmental stages of RPW and deployed under laboratory conditions and field conditions.
The recognized factor for classifying bacteria involves the sequence of 16S ribosomal RNA.
Two important groups of bacteria are Eubacteria (true bacteria) and Archaea containing bacteria
having similar features of DNA replication, transcription and translation as exhibited by
eukaryotes. Three major divisions within Eubacteria are primarily based on the presence or
structure of cell wall: Gracilicutes (gram negative typed cell wall bacteria), Firmicutes (gram
positive typed cell wall bacteria) and Tenericutes (Eubacteria which are devoid of cell wall).
Most recent classification within Eubacteria mostly relies on the use of polyphasic taxonomy that
includes analysis of nucleotide sequence of RNA (16S rDNA), DNA-DNA hybridization,
genotypic, phenotypic and phylogenetic aspects (Brenner et al., 2005).
Entomopathogenic bacteria; greatly concerned with entomological studies are grouped in
Eubacteria. The cell wall of bacteria greatly serves the purpose to classify, support the molecules
and organelles. In gram-positive bacteria, the cell wall is formed of cross-linked peptidoglycan
while on the other hand, cell wall in gram-negative bacteria is formed of rather complex thin
layer of peptidoglycan and lipoproteins and an outer polysaccharide membrane. Gram-negative
bacteria are distinguished from gram-positive bacteria by lacking the ability to retain crystal
violet dyes. Gram-positive are endospore forming, rod and cocci shaped bacteria often
undergoing sporulation. Gram-negative bacteria on the other hand appear to be in rod or
cocciform. They are much more diverse in their distribution and hence isolation can be
successful from diseased and dead insect specimens (Jurat-Fuentes and Jackson, 2012).
2.5.7.3 Life cycle
Life cycle of B. thuringiensis can be divided into different phases for convenience in
understanding; Phase-I (vegetative growth); Phase-II (transition to sporulation); Phase-III
(sporulation); and Phase-IV (spore maturation and cell lysis) (Berbert-Molina et al., 2008).
Specific insecticidal (Cry) proteins lying deposited in crystals within mother cells starts to form
with the onset of sporulation (Pérez-García et al., 2010). There are some evidences of the
production of the insecticidal proteins within culture medium during vegetative growth (Singh et
al., 2010; Abdelkefi-Mesrati et al., 2011). Distinctive characteristic insecticidal properties to Bt
are conferred by another additional virulence factor phospholipase C, proteases and hemolysins
(George and Crickmore, 2012) which are under control of pleiotropic regulator plc R. Removal
of plc R gene results in drastic reduction in the virulence of Bt in orally infected insects
(Salamitou et al., 2000). Sporulation leads to the production of two types of insecticidal proteins
(cry toxins and cyt-toxins) within crystalline bodies. A single Bt strain is naturally provided with
one or more toxins packaged into a single or multiple crystals (de Maagd et al., 2001). The Cry
toxin is named for its production within crystals whereas Cyt-toxin got the mnemonic Cyt due to
their in vitro cytolytic activity (Crickmore et al., 1998). The Cry toxins acquired the mnemonic
Cry from the fact that they are found in the crystal while the Cyt-toxins acquired the mnemonic
Cyt because of their in vitro cytolytic activity (Crickmore et al., 1998).
2.5.7.4 Ecology
Earlier, B. thuringiensis was thought to have confined to soil only, but advanced isolation
techniques in ‘Insect Pathology’ discovers its various sources of origin (Chaufaux et al., 1997).
26
Now this school of thought has expired and Bt has been isolated from dust, stored grain and silos
materials (Iriarte et al., 1998). The probability of isolation of Bt from a site varies with its
climate, geographical and environmental conditions. In much of the findings till today reveal
most common shapes of crystals as bipyramidal and spherical. More often, Bt is regarded as soil
inhabiting, but it lacks the capacity to multiply in soil or water that offers healthy environment
for other bacteria to compete (Furlaneto et al., 2000). Reproduction in regular practice is carried
within host insects. Most of the commercial Bt formulations are isolation from infected insects,
so it would be obvious to say that soil acts as a reservoir for bacterium instead of multiplication
site.
2.5.7.5 Mechanism of action
The infection cycle start with the ingestion of Bt spores by insects making its way to
alkaline environment of midgut (pH >9.5). The exposure to higher pH of the gut solublizes the
inactive proteins that on the other hand remain insoluble. This activation results in the release of
crystal proteins that produces δ-endotoxins. Insecticidal activities of δ-endotoxins get magnifies
as a result of proteolytic activation and activated toxin readily get bound to specific receptors
present at apical brush border of the midgut microvillae in target insects (Hofmann et al., 1988).
The toxic action of proteins is because of N-terminal half consisting of seven anti-parallels α-
helices. Loss of integrity of insect’s gut is the outcome of Bt activity that ultimately leads to
death of insect due to starvation and septicemia (Kumar et al., 2013).
2.5.7.6 Commercial formulations
A rapid development of interest in bio-pesticides has led to the commercial preparation of
several Bt products. Now a day, wide variety of commercial products is accessible to farmers
infecting a wide range of host insects. While developing commercial products, Bt strains used
against lepidopterous insects belong to subspecies; thuringiensis, kurstaki, morrisoni and
aizawai. For Dipterous insects, Bt strains include subspecies israelensis while coleopterans pests
infecting products include subspecies tenebrionis. The total bio-pesticides used in agriculture
globally, Bt products contribute the share of about 80% (Whalon and Wingerd, 2003). A series of
complex changes are involved in Preparation of Bt products require standard fermentation batch
process including vegetative phase, a sporulation phase release of spores and sporangia in final
phase. Fermented solids at this phase are concentrated and mixed with inert material for packing
as finished product (JuratFuentes and Jackson, 2012). Recently, over 400 of Bt originated
commercial products are marketed over the world in different names registered against pests in
different formulations (solid and liquid). These products contain in them various insecticidal
proteins and viable spores, yet some products are also available with inactivated spores
(Ahmedani et al., 2008). Several valuable products have been prepared from B. thuringiensis var.
israelensis (Gnatrol, Aquabee, Bactimos, LarvX, Teknar AND Mosquito Attack etc.), B.
thuringiensis var. kurstaki (Bioworm, Bactur, Dipel, Topside, Caterpillar Killer, Javelin, Futura,
Thuricide, Worthy Attack and Tribactur), B. thuringiensis var. tenebrionis (M-One, Foil,
Novardo, M-Track and Trident), B. sphaericus (Vectolex WDG and Vectolex CG etc.), B.
thuringiensis var. aizawai (Certan) B. lentimorbus and B. popilliae (Japidemic, Milky Spore
Disease, Doom and Grub Attack etc.).
27
2.5.7.7 Methods of applications of Bt products
B. thuringiensis is undoubtedly regarded as biologically active pesticide effective against
important insect pests well suited to IPM strategies. Based on the ecological aspects of target
pest and infection cycle of active insecticidal proteins, Bt products in solid form as well as liquid
form are applied. Bt products in solid form are either dust or granules (spread over the area of
infection) while in liquid form (foliar sprays), Bt products are sprayed directly (Ali et al., 2010)
to point of infection. A more persistent and biotechnologically advanced way of supplying toxins
to target insect is to genetically express toxin-encoding genes within transgenic plants (Walter et
al., 2010; Chen et al., 2011). This practice leaves no chances of escape of insect from active Bt
components as toxin remained concealed along with the preferred diet of insects. Moreover, the
presence of toxin-encoding genes leaves no harm to non-target fauna, no obvious changes in the
physiology of host plant and no chemical change in the products and byproducts from host plant.
2.5.7.8 Superiority of Bt products over synthetic insecticides
Environmental concern of pesticides has ever remained the hot issue. The hazards posed
with the use of insecticides predominantly broad spectrum nature, quick onset of resistance in
insects (Ahmad et al., 2008), environmental persistence, effect to non-target individuals, and
phenomenon of bio-magnification have declared pesticides an evil for mankind. Another peculiar
reason for the considering bio-control agents as key weapon against notorious insect pests is the
selective (Stevens et al., 2011) and environmental friendly nature (Chen et al., 2011). Several
laboratory and field studies have declared Bt toxins as a necessary component of insect control
strategies and a widely preferred tool over the synthetic chemistries. Bt toxins have specific
insecticidal impacts on insect pests of order coleopteran (Sharma et al., 2010), Diptera (Roh et
al., 2010), Hymenoptera (Sharma et al., 2008), Lepidoptera (Baig et al., 2010) and non-insect
hosts like nematodes (Hu et al., 2010). Although no such reports of harm to non-target individual
has been reported, yet some studies provide insight into reduction in reproduction capacity in
bumblebee (Bombus terrestris) workers after using commercial Bt aizawai strain (Mommaerts et
al., 2010). Beside of this, Bt products still rule over bio-pesticide market and remains hub of bio-
control policies launched against insect pests.
2.5.7.9 Concerns to use of Bt
Bt has several advantages over chemical insecticides: it is host specific and highly toxic
to the target insects highly toxic to insects and yet highly specific. Bt toxins are safer to
environment, animals, human beings and vast array of non-target pests. Therefore, Bt can be
considered an ideal component for IPM programs (Nester et al., 2002). Besides these
advantages, Bt formulations have some reservations to be considered (McGaughey and Whalon,
1992). Most of the microbial based products need repeated application for effective control of
insect pests and sometimes officious only against immature stages immature stages feeding
externally. This seems to be a concern limiting its worth for internal plant feeding insects. This
can be overcome by incorporation of Bt gene (s) in transgenic plants (Krattiger, 1997). Some
recent studies are reporting the development of resistance and cross resistance developed after
the continuous use of Bt toxins is one of the emerging concern about the future of biological
control. Even the laboratory investigations are confirming the incidence of resistance
development in some insects (Pereira et al., 2010) and field populations (Sayyed et al. 2004).
The term ‘cross resistance’ is used by some researchers (Gong et al., 2010; Xu et al.,
2010) for the previously resistant insects (for a specific toxin) showing resistance to other
28
toxin/toxins they have not been exposed yet. Different routes and mechanism of resistance have
been discovered, the most important explanation includes the reduced binding of toxins to
receptors in midgut lining of insects, reduction in solubilisation of protoxin, precipitation by
proteases, toxin degradation and or alteration in proteolytic processing of protoxins (Bruce et al.,
2007). Several mode of resistance has been verified about the development of resistance, the
‘Mode 1’ being the most accepted that hypothesizes that reduction is the outcome of reduction in
binding of Cry1A toxin to specific receptors (Heckel et al., 2007). The alteration of protease
profile in the midgut of Cry1Ac resistant American boll worm is due to proteolytic processing of
Cry1Ac ultimately producing 95 and 68 kDa toxins normally producing active 65 kDa toxins by
midgut protease in vulnerable insects (Rajagopal et al., 2009). Apart from resistance, another
constraint in use of Bt is the narrow host range and limited efficacy. Bt toxins are very limited in
regard of host infection (Shu et al., 2009) and in most of Bt strains, infections remain limited to a
single specie only a few Bt strains exhibit activity span against two or more insect orders (Zhong
et al. 2000).
2.5.7.10 Interaction of Bt products and other toxins
Narrow spectrum of activity in case of Bt is one of the constraint making biopesticides a
second choice after synthetic insecticides. Most of Bt isolates show rather poor control over
insects but their pathogenicity can be magnified by using in synchrony with some other suitable
toxin. For instance, Cyt1Aa is a weak toxin to mosquitoes but synergistic action is found when
combined with toxins like Cry4Ba and Cry11Aa (Fernandez-Luna et al., 2010). Combining Bt
insecticidal toxins in combination with other proteins not only boosts their pathogenic effect but
also helps to lower the resistance developed by insect pests. Proteins like cadherin fragments
have been found to be successfully synergizing the efficacy of several insecticidal toxins (Peng
et al., 2010). The simultaneous use of Cry1Ac and Cry2Ab results in powerful synergistic
interaction against H. armigera (Ibargutxi et al., 2008). Mixing of spores and crystal proteins
from same strain also yields synergistic insecticidal action (Johnson et al., 1998). A dose
dependent interaction of Bt was also recorded against H. armigera in Pakistan from soil isolated
M. anisopliae which integrated synergistically as well as antagonistically at lower and higher
doses (Wakil et al., 2013). Kalantari et al. (2013) interpreted synergistic effect of Bt and
HaSNPV by combining a suitable dose among several tested doses against H. armigera. Future
research will open the horizon of success in integrating Bt with several agents to boost up its
efficacy against a wide host range.
2.5.7.11 Effect of Bacillus thuringiensis on non-target invertebrates
From the last few decades, Bt pesticides are being studied to control crop, forest and
aquatic insect pests. Most of Cry toxins are specific to insects belonging to one of the insect
orders either Lepidoptera, coleopteran and Diptera. Cry2 is an exception to this fact as it exhibits
insecticidal activity against several families of Diptera and Lepidoptera (Schnepf et al., 1998).
Many of the Bt formulations containing purified Cry toxins registered against Lepidopteran
orders show no harm to non-lepidopterous insects (MacIntosh et al., 1990; Sims, 1997).
Conversely, there is an exception as non-target Lepidoptera are not necessarily secure from Bt
treated plants especially in forests (Sample et al., 1996; Herms et al., 1997). The drifting of
aerially applied Bt subsp. kurstaki (Bt-k) to control gypsy moth was also found to be lethal to
non-target Lepidoptera 3000 m away from treated site as demonstrated by Whaley et al. (1998).
However no or negligible effect was found for aquatic habitats in Bt treated sites when
29
Kreutzweiser et al. (1992) demonstrated high concentrations of Bt-k on drift and mortality of
Ephemeroptera, Plecoptera, and Prichoptera. Predators that preyed upon Bt treated hosts were
not found susceptible except the Chrysoperla carnea. So in this regard, it would rather be
justified statement to declare Bt toxin rather safe, specific in action and compatible to non-target
individuals.
2.5.7.12 Mode of infection
The ingestion of B. thuringiensis compounds by insects follows the route of midgut to
expose it to alkaline environment of gut (pH >9.5). Here the higher pH of the gut solublizes the
inactive, otherwise insoluble proteins resulting in the release of crystal proteins that produces δ-
endotoxins. This proteolytic activation of δ-endotoxins offers an extraordinary insecticidal
activity to insects and this activated toxin readily gets bound to specific receptors present at
apical brush border of the midgut microvillae in target insects (Hofmann et al., 1988). The toxic
action of proteins is attributed to N-terminal half consisting of seven anti-parallels α-helices.
These α-helices offers potential gradient by penetrating the membrane and forming an ion
channel in apical brush border membrane allowing rapid flux of ions. Loss of integrity of insect’s
gut is the outcome of B. thuringiensis activity causes starvation and septicemia which leads to
the death of insects (Kumar et al., 2013). The penetration of α-helices in the apical brush border
membrane forms an ion channel (Knowles and Dow, 1993). As a result, rapid flux of ions takes
place because of toxin-induced pores formation (Wolfersberger, 1989). Consequently the gut
integrity gets lost that resultant starvation and/or septicemia leads to insect death.
A wide array of B. thuringiensis products formulated for commercial uses have an
extended spectrum of action effective to secure food crops, forest trees, stored grains and
ornamentals (Meadows, 1993). Contrary to hazards associated with chemical pesticides, B.
thuringiensis formulation offers a wide range of benefits. Although it is highly virulent to target
insects, yet it is harmless to non-target insects due to its specificity. In spite of decades of use in
field, B. thuringiensis toxins are still reported as non-hazardous to animals, human beings and
other non-target pests. All these characteristics render it highly suited to include IPM programs
(Nester et al., 2002). Besides these benefits, B. thuringiensis formulations have some associated
limitations (McGaughey and Whalon, 1992). One of the limitations is its effectiveness against
specific stage of insect especially immature stage. For this reason, an effective control of targeted
insect requires repeated application. B. thuringiensis products perform better against insect
exposed pests than insects concealed within plant parts or some other structures. But the
expression of B. thuringiensis gene (s) using transgenic cultivars (Krattiger, 1997) may be able to
address such concerns.
2.5.7.13 Important entomopathogenic bacteria
2.5.7.13.1 Bacillus thuringiensis
Bacillus thuringiensis (Bt) holds a prominent position among commercial chemical
compounds important for agricultural insect pests. It is a naturally occurring spore forming,
gram-positive bacterium. It has been found as a source and reservoir of several important
insecticidal proteins like δ-endotoxins, vegetative insecticidal proteins (vip) and cytolytic
proteins etc. Among these proteins, δ-endotoxins have a vital role in protecting number of
important crops from various insect pests. B. thuringiensis based insecticides have proved their
worth as a bio-pesticide to protect food crops, cash crops, ornamentals, forest trees and stored
commodities (Meadows, 1993). For convenience, life cycle of B. thuringiensis can be divided
30
into different phases; Phase-I: vegetative growth; Phase-II: transition to sporulation; Phase-III:
sporulation; and Phase-IV: spore maturation and cell lysis (Berbert-Molina et al., 2008). More
than 150 genes of exhibiting insecticidal nature have been identified from Bt δ-endotoxins family
of proteins (Crickmore et al., 1998). These crystalline (cry) proteins remain inactive until the
exposure to alkaline contents (pH >9.5) of insect mid gut, solubilize them (Milne and Kaplan,
1993) and ultimately liberating δ-endotoxins proteins.
2.5.7.13.1 Paenibacillus popilliae
Paenibacillus popilliae previously known as B. popilliae is a gram-positive spore-
forming bacterium which was initially isolated from infected Japanese beetle (Popillia japonica)
(Coleoptera: Scarabaeidae) larvae in the late 1930s and then named after the name of its first
host. The spore forming capability of bacterium protects it from heat, cold, drying and other
harsh environmental regimes. P. popilliae plays a major role in biologically managing scarabs,
particularly Japanese beetle (Petterson et al., 1999). B. popilliae has been reported from at least
29 scarabs, mostly from Melolonthinae and Rutelinae. P. popilliae causes milky spore disease in
P. japonica and it is the first pathogen registered as insect biological control in USA.
2.5.7.13.2 Brevibacillus laterosporus
Brevibacillus laterosporus is a gram-positive, rod-shaped, endospore-forming bacterium
and is considered an important entomopathogenic and antimicrobial agent. It is morphologically
distinguished by producing characteristic canoe-shaped parasporal body (CSPB) firmly attached
at one end of the spore imparting it lateral position in the sporangium. Ubiquitous existence of
this bacterium has enabled its isolation from various reservoirs particularly soils, insect bodies,
fresh and sea water, leaf surfaces, compost, milk, honey, factory effluents, animal hide, wool and
many other materials (Ruiu, 2013). It was discovered by White (1912) during 20 th century
associated with honey bees determined during an investigation on European foulbrood.
2.5.7.13.3 Bacillus subtilis
German botanist Ferdinand Cohn in 1877, while working on hay Bacillus, discovered two
new forms of Bacillus strain named Bacillus subtilis; one of them was heat sensitive (without
endospore) while other was heat tolerant (endospore). A significant genomic diversity in the
bacterium has been publicized using genomics analysis based on microarray-based techniques. It
is competent for growth in many environmental conditions and is often considered as soil
dweller. Most common sources of its isolation are air, soil, water and decomposing plants.
However in most of the cases, it is not found naturally in biologically active but occurs in spore
forms. Bacillus subtilis is scientifically fabulous for its ability to produce a number of antibiotics
especially bacitracin and iturin. It regulates the development of adult mosquitoes by inhibiting
their growth (Ramathilaga et al., 2012).
2.5.7.13.4 Bacillus sphaericus
Bacillus sphaericus is a naturally occurring spore-forming gram positive bacterium that
exhibits strong insecticidal properties. It possesses efficient larvicidal properties against
mosquito by producing delta-endotoxins via sporulation that binds strongly to receptors in
midgut epithelial lining of mosquito larvae. The bacterium has narrow spectrum and quite
specific activity that sometimes decreases its demand for use in field. Enhanced time of lethal
action against some mosquito species and recycling of toxin within dead mosquito sometimes
31
works as limiting factors for its use. One of the advantages exhibits over B. thuringiensis var.
israelensis is its longer persistence that provides long lasting control (Filha et al., 2008).
2.5.7.13.5 Wolbachia
Wolbachia are α-protobacteria, the members of the order Rickettsiales; a varied group of
intracellular bacteria that comprises species exhibiting parasitic, mutualistic and commensal
associations with their hosts. With its pathogenic nature extended to arthropods and filarial
nematodes, it is regarded as the most common endosymbiotic bacterial species on the globe. The
only member contained with genus Wolbachia in family Anaplasmataceae and order
Rickettsiales is Wolbachia pipientis; the rest of the species; W. melophagi and W. persica have
been recently declared as unrelated (Dumler et al., 2001). An insight into the intracellular life
study of the bacterium ensures its obligate nature of infection to hosts and it has been found
successfully infesting about 66% of the insect species (Hilgenboecker et al., 2008). Wolbachia
being intracellular bacterium are vertically transmitted through the egg. Wolbachia sometimes
manipulate the reproduction of host insects by cytoplasmic incompatibility. One of the vital
reasons behind the successful propagation of Wolbachia in arthropods is its inherent ability to
take control of the host’s reproductive cycle by providing nutrients and protecting host from
other pathogens (Hosokawa et al., 2010).
The genera closely related to Wolbachia; Anaplasma, Ehrlichia and Neorickettsia during
their life stages include an invertebrate ‘vector’ and mammalian ‘host’ and in some cases
invertebrate associations in some species have also been found. But contrary to unlike members,
Wolbachia does not necessarily affect vertebrates. One of the important reasons behind increased
interest for Wolbachia is their immense diversity, interesting phenomena shown while infecting
their hosts such as reproductive manipulation, and their possible exploitations for pest and
disease vector control (Bourtzis, 2008).
2.5.7.14 Host range of B. thuringiensis
Different commercial products of B. thuringiensis for use in crops, forests and aquatic
system do not necessarily contain β-exotoxin, but most of the B. thuringiensis products
registered against insect pests contain Cry toxins (also known as δ-endotoxins). Normally, a
single Cry protein works perfectly against a single order and sometimes against several families
within an order. The Cry2 is an exception to this fact as it exhibits insecticidal nature against
several families of Diptera and Lepidoptera (Schnepf et al., 1998). Most of the commercial B.
thuringiensis products or purified Cry toxins formulated for lepidopterous insects are non-
hazardous to a vast variety of non-target organisms (Sims, 1997). However, non-target
Lepidopterans are mostly at risk in B. thuringiensis treated plants particularly in forests (Herms
et al., 1997). For instance, the aerial spraying of B. thuringiensis subsp. kurstaki (Bt-k) to control
gypsy moth was found to be lethal to non-target Lepidoptera 3000 m away from treated site
(Whaley et al., 1998). However, no or a negligible effect was found for aquatic habitats in Bt
treated sites when Kreutzweiser et al. (1992) demonstrated high concentrations of Bt-k on drift
and mortality of Ephemeroptera, Plecoptera, and Trichoptera. Predators that preyed upon B.
thuringiensis treated hosts were not found susceptible except the Chrysoperla carnea. So in this
regards, it would rather be justified statement to declare B. thuringiensis toxin rather safe,
specific in action and compatible to non-target individuals.
32
2.5.7.15 Natural incidence
Effect of different bacterial spp. on RPW has been reported by many scientists. Dangar
and Banerjee (1993) discovered some bacteria species belonging to Serratia sp., Bacillus sp. and
the coryneform group from adult and larval stages of RPW in India, while Bacillus sphaericus
Meyer and Neide and B. thuringiensis Berliner were isolated from larvae and adult of RPW in
Egypt (Alfazairy et al., 2003; Alfazariy, 2004). Later on (Banerjee and Dangar, 1995) isolated
Pseudomonas aeruginosa from larvae infected with this agent from Kerala, India. In Egypt,
Salama et al. (2004) recovered three potential spore-forming bacilli from RPW larvae. The three
bacteria belonged to the genus Bacillus and were identified as variants of B. laterosporus
Laubach (strain 27), B. sphaericus (strain 73) and B. megaterium de Bary (strain 15).
Under in vitro conditions, B. sphaericus caused 40% and 60% mortality in 2nd instar
larvae of RPW when use different isolates of Bt. B. sphaericus considered being the most active
culture which produces crystalline endotoxins and spherical endospores which are responsible
for disease production in RPW. In Italy (Sicily), B. sphaericus B. thuringiensis and B.
megaterium were recovered from RPW cadavers, but these isolates exhibited weaker pathogenic
effect against eggs of RPW (Francesca et al., 2008), although lacking antimicrobial compounds
(Mazza et al., 2011a). Most recently Francesca et al. (2015) isolated distinct strains of seven
species from RPW beetle cadavers (B. cereus, B. amyloliquefaciens, B. pumilus, B. licheniformis,
B. subtilis, B. megaterium and Lysinibacillus sphaericus) from Sicily.
2.5.7.16 Susceptibility of RPW to entomopathogenic bacteria under laboratory conditions
Regarding the pathogenicity of Bt strains against different developmental stages of RPW
very few studies has been conducted so for. In laboratory study application of P. aeruginosa
suspension, either by inoculation by forced feeding, injecting and wading RPW larvae in the
suspension. Complete mortality occurred eight days post inoculation in case of forced feeding
and wading, while injection took 6 days, moreover, younger larvae were more vulnerable than
the older ones (Banerjee and Dangar, 1995). This might be attributed to the fact that younger
larvae probably lacking antimicrobial cuticular compounds (Mazza et al., 2011a). Alfazariy
(2004) revealed successful control of RPW in laboratory conditions by infecting with B.
thuringiensis var kurstaki (Bt-k). Albite this, other scientists revealed different susceptibility of
RPW to the same bacterium (Bauce et al., 2002; Sivasupramaniam et al., 2007; Birda and
Akhursta, 2007; Manachini et al., 2008a, b; Manachini et al., 2009).
Evidences suggested that feeding cessation and midgut damage were observed amongst
surviving larvae. Manachini et al. (2009) integrated commercially available B. thuringiensis into
RPW larvae diet and revealed moderate pathogenicity against RPW larvae. Similar results were
also recorded by Dembilio and Jacas (2013). Under laboratory conditions this bacterium can be
effective against RPW larvae when ingested but their commercial application does not give
satisfactory control (Manachini et al., 2009). However, retarded larval growth and its effect on
hemocytes was primarily described, exhibiting that the bacterium is capable of growing in the
hemolymph when uptake by the larvae (Manachini et al., 2011).
2.5.7.17 Field and Semi-field assessment of bacteria for RPW management
Sequentially, several investigators tested certain commercial products based on Bt against
RPW and reported the difficulty of using such products as a good control agent against the insect
due to different reasons (Dembilio and Jacas, 2013). So for no solid evidence regarding
susceptibility of RPW to entomopathogenic bacteria and confirmation of successful control
33
under field or semi-field conditions has been reported. The reduced susceptibility might be due
to the host defense mechanism. Manachini et al. (2011) conducted preliminary study by
deploying B. thuringiensis and Saccharomyces cerevisiae against the cellular immune response
of RPW and exhibited that Bt is a stress factor for RPW. Future experiments to control RPW by
deploying microorganisms must be designed to explore the molecular mechanism of disease
resistance among RPWs and interaction between them and RPW immune system. Most recently
Francesca et al. (2015) tested nine distinct stains of bacterium which significantly reduced the
egg hatching, while B. licheniformis exhibited significant insecticidal activity against RPW
larvae.
Dangar (1997) evaluated the bio-control potential of free living unidentified yeast
recovered from RPW haemolymph. The calculated lethal dose was 8,000,000 yeasts insect-1,
while lethal time was recorded 4 days. Latter on Salama et al. (2004) isolated yeast from infected
pupae of RPW in Egypt which caused 20-35% mortality in 2nd instar larvae of RPW after 7 days
of application.
2.5.8 Microbial control agents as a component of RPW IPM
As components of RPW IPM, entomopathogens can provide significant and selective
control. An integrated approach is needed which can provide maximum effectiveness when
combined with other control practices (Edwards, 1989). In the near future efforts are being made
to study the synergistic interaction between entomopathogens and other pest control tactics
(integration with soft chemical pesticides, semiochemicals, resistant plants, other natural
enemies, remote sensing and chemigation etc.). These efforts will enhance the efficacy and
sustainability of entomopathogens.
Different formulations of EPNs have been employed in order to enhance the bio-control
potential of EPN against RPW. A commercial formulation (Biorend-R® Palmeras) S.
carpocapsae has been found effective both for preventive and curative measure under semi-filed
conditions (Llácer et al., 2009). EPNs exhibit varying degrees of dispersal to find and invade the
insect host in their habitat, and this affects the fitness traits of parasitism and infectivity
(Koppenhöfer and Fuzy, 2008).
Nematodes disperse by following long-distance cues to search for hosts. The IJs use
distinctive foraging patterns to discover potential prey; either vigorously hunting for insect hosts
(cruisers), or standing on their tails in an upright position over the surface and waiting for the
host insect to pass by (ambushers) (Lewis et al., 2006). These foraging and dispersal strategies
have a significant impact on effective traits of EPNs and on determining their relationship with
the host, an important aspect in predicting EPN efficacy. Moreover, EPFs particularly B.
bassiana and M. anisopliae have been reported to be effective against RPW but promising
results under field conditions are not recorded except with solid formulation of B. bassiana
which exhibited high RPW pathogenicity and persistence for preventive and curative treatments.
The integration of microbial control agents with suitable control measures can be effectively
used to combat RPW both under laboratory and field conditions.
2.5.9 Ecological engineering and agricultural practices to conserve microbial control agents
Ecological Engineering (EE) in IPM of pest control is a comprehensive strategy
integrating modern technologies with traditional cultural techniques to promote
entomopathogens as a cornerstone for sustainable agricultural productivity (Gurr et al., 2004). It
is argued that these ecological based approaches for managing insect populations of agricultural
34
importance can be safer and more sustainable than sole dependence on conventional chemical
insecticides, hence the need to examine the concept and practice of ecological engineering as a
component of modern agricultural activity. Ecological based pest control strategies are more
appealing to the researchers, conservation bio-control in particular that increase the number and
activity of natural enemies in the natural conditions by manipulation of habitat (Gurr et al.,
2004). The conservation of antagonistic organisms in date palm gardens mostly targets
indigenous strains with knowledge of local natural microbe communities instead of inundative
applications of microbial antagonists.
The idea behind conservation is to increase the number of entomopathogens and protect
refuges in the orchards which resultantly will encourage entomopathogens to reduce pest
infestation. Unluckily, in date palm pest control intention are mostly focused to inundative
applications rather than conservation. The enhanced efficacy of EPNs can be achieved by
providing supplementary food sources such as organic amendments in the soil enhance the
efficacy and persistence of EPNs. Contrarily, some amendments with manures and plants
containing allelopathic compounds can exert also hazardous effects to EPNs. In case of
endophytic colonization of entomopathogens plant genotype can interfere with rhizosphere
colonization and antagonist’s metabolites production, as well as the expression of induced
resistance by plants. Ultimately, indicators will need to be identified, such as the presence of
particular antagonists, which can guide decisions on where it is practical to use conservation
biological control. Combination of entomopathogens with conservation practices can be helpful
in improving the effectiveness and persistence of entomopathogens. Future research must be
focused on the greater use of bioassays that accounts for the RPW suppression as effectiveness
of a particular entomopathogen against RPW is not significantly affected by their abundance.
2.5.10 Biotechnological approaches to enhance virulence of microbial control agents
Biotechnology provides magnificent opportunities to enhance the virulence of microbial
control agents through incorporation of gene of interest exhibiting excellent control against
insect pests e.g. the transformation of M. anisopliae by Aspergillus nidulans. To understand the
virulence mechanism efforts are being made, emphasizing the cuticuler area where mostly
penetrations occur and possessing a key enzyme, an endoprotease (St. Leger et al., 1986). In the
future, insect killing speed of EPFs may be enhanced by inserting delta-endotoxin genes from B.
thuringiensis into fungi which certainly will achieve improved strains. Except from the Bt delta-
endotoxin there are several other proteins of insecticidal properties such as alpha-endotoxin,
Vegetative Insecticidal Proteins (VIP) and numerous secondary metabolites that are prone
genetic modification a (Attathom, 2002).
The introduction of gene coding for proteinaceous insect toxins (scorpion toxin, mite
toxin, trypsin inhibitor), hormones (eclosion hormone, diuretic hormone) or metabolic enzymes
(juvenile hormone esterase) into nucleopolyhedroviruses genome are some approaches to
increase speed of kill, enhanced virulence and extend host specificity of the virus (Attathom,
2002). Future, attention should be focused on formulation development and targeting RPW
populations. Advantages of this approach include reduced risk for development of resistance and
greater safety to the environment, and lack of effect on non-target and beneficial organisms. We
believe that biotechnology and genetic engineering will come up with the effective use of insect
antagonists as an integral part of integrated pest management program worldwide.
35
2.6 References
Abbas, M.S.T. and S.P. Hanonik, 1999. Pathogenicity of entomopathogenic nematodes to red
palm weevil, Rynchophorus ferrugineus. Inter. J. Nematol., 9: 84-86.
Abbas, M.S.T., S.B. Hanounik, S.A. Mousa and M.I. Mansour, 2001. Pathogenicity of
Steinernema abbasi and Heterorhabditis indicus isolated from adult Rhynchophorus
ferrugineus (Coleoptera). Inter. J. Nematol., 11: 69-72.
Abbas, M.S.T., S.B. Hanounik, S.A. Mousa and S.H. Albagham, 2000. Soil application of
entomopathogenic nematodes as a new approach for controlling Rhynchophorus
ferrugineus on date palm. Inter. J. Nematol., 10: 215-218.
Abdelkefi Mesrati, L., H. Boukedi, M. Dammak-Karray, T. Sellami-Boudawara, S. Jaoua and S.
Tounsi, 2011. Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and
determination of its putative binding proteins in the midgut of Spodoptera littoralis. J.
Inver. Pathol., 106(2): 250-254.
Abdel-Samad, S.S.M., B.A. Mahmoud and M.S.T. Abbas, 2011. Evaluation of the fungus,
Beauveria bassiana (Bals.) Vuill as a bio-control agent against the red palm weevil,
Rhynchophorus ferrugineus (Oliv.) (Coleoptera: Curculionidae). Egyp. J. Biol. Pest
Control, 21: 125-129.
Abraham, V.A., 1971. Prevention of red palm weevil entry into coconut palms through wounds.
Mysore J. Agric. Sci., 5:121-122.
Abraham, V.A., J.R. Faleiro, C.P.R. Nair and S.S. Nair, 2002. Present management technologies
for red palm weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) in
palms and future thrusts. Pest Manag. Hort. Ecosy., 8: 69-82.
Abraham, V.A., M.A. Al-Shuaibi, J.R. Faleiro, R.A. Abozuhairah and P.S.P.V. Vidyasagar,
1998. An integrated approach for the management of red palm weevil Rhynchophorus
ferrugineus Oliv. A key pest of date palm in the Middle East. Sultan Qaboos Uni. J. Sci.
Res., 3: 77-83.
Adamo, A.S., 2005. Parasitic suppression of feeding in the tobacco hornworm, Manduca sexta:
parallels with feeding depression after an immune challenge. Arch. Insect Biochem.
Physiol., 60: 185-197.
Ahmad, M., A.H. Sayyed, M.A. Saleem and M. Ahmad, 2008. Evidence for field evolved
resistance to newer insecticides in Spodoptera litura (Lepidoptera: Noctuidae) from
Pakistan. Crop Prot., 27: 1367-1372.
Ahmad, S. and A. Tahir, 2005. Dates culture. In: Ahmad, S. (Ed.). Date palm. Horticulture
Foundation of Pakistan, Islamabad, and Pakistan Science Foundation, Islamabad. Pp.
4145.
Ahmedani, M.S., M.I. Haque, S.N. Afzal, U. Iqbal and S. Naz, 2008. Scope of commercial
formulations of Bacillus thuringiensis Berliner as an alternative to Methyl bromide
against Tribolium castaneum adults. Pak. J. Bot., 40: 2149-2156.
Aizawa, S.I., 2001. Bacterial flagella and type III secretion systems. FEMS Microbiol. Lett., 202:
157-164.
Ajlan, A.M., M. Shawir, M.A. Abo-El-Saad Rezk and K.S. Abdulsalam, 2000. Laboratory
evaluation of certain organophosphorus insecticides against the red palm weevil
(Olivier). Sci. J. King Faisal Uni., 1: 15-26.
Akbar, W., J.C. Lord, J.R. Nechlos and T.M. Loughin, 2005. Efficacy of Beauveria bassiana for
red flour beetle when applied with plant essential oils or mineral oil and organosilicone
carriers. J. Econ. Entomol., 98: 683-688.
36
Al-Doghairi, M.A., 2004. Effect of eight acaricides against the date dust mite, Oligonychus
afrasiaticus (McGregor) (Acari: 13 Tetranychidae). Pak. J. Biol. Sci., 7(7): 1168-1171.
Alfazairy, A.A., 2004. Notes on the survival capacity of two naturally occurring
entomopathogens of the red palm weevil Rhynchophorus ferrugineus (Olivier)
(Coleoptera: Curculionidae). Egyp. J. Biol. Pest Control, 14: 423.
Alfazairy, A.A., R. Hendi, A.M. El-Minshawy and H.H. Karam, 2003. Entomopathogenic agents
isolated from Coleopteran insect pests in Egypt. Egyp. J. Biol. Pest Control, 13: 125.
Ali, S., Z. Huang and S.X. Ren, 2010. Production of cuticle degrading enzymes by Isaria
fumosorosea and their evaluation as a biocontrol agent against diamondback moth. J. Pest
Sci., 83: 361-370.
Ali-Shtayeh, M.S., B.M. Abdel-Basit, B.M. Marai and R.M. Jamous, 2002. Distribution,
occurrence and characterization of entomopathogenic fungi in agricultural soil in the
Palestinian area. Mycopathol., 156: 235-244.
Alves, S.B. and R.E. Leucona, 1998. Epizootiologia aplicada ao controle microbiano de insetos.
In: Alves, S.B. (Ed.). Controle microbiano de insetos. Piracicaba: FEALQ. Pp. 97-170.
Amutha, M., J.G. Banu, T. Surulivelu and N. Gopalakrishnan, 2010. Effect of commonly used
insecticides on the growth of white Muscardine fungus, Beauveria bassiana under
laboratory conditions. J. Biopesti., 3(1): 143-146.
Anderson, T.E., A.E. Hajek, D.W. Roberts, K. Preisler and J.L. Robertson, 1989. Colorado
potato beetle (Coleoptera: Chrysomelidae): effects of combinations of Beauveria
bassiana with insecticides. J. Econ. Entomol., 82: 83-89.
Anonymous, 2013. Problems of agriculture in Pakistan. Available at: www.zaraimedia.com.
Accessed on May 06, 2013.
Anonymous, 2015. 42 billion worms airlifted from Germany to combat palm weevil crisis.
Avaialable at: http://www.jpost.com/Israel-News/42-billion-worms-airlifted-from-
Germany-to-combat-palm-weevil-crisis-391817. Assessed on 10 Feb 2016.
Aoki, K. and Y. Chigasaki, 1915. Uber des toxin von sog. Sotto-bacillen Mitt. Med. Fak. Kais.
Univ. Tokyo, 14: 59-80.
Arab, Y.A., and H.M. El-Deeb, 2012. The Use of endophyte Beauveria Bassiana for bio-
protection of date palm seedlings against red palm weevil and Rhizoctonia Root-Rot
Disease. Sci. J. King Faisal Uni., 13(2): 91-101.
Arnold, A.E., L.C. Mejía, D. Kyllo, E.I. Rojas, Z. Maynard, N. Robbins and E.A. Herre, 2003.
Fungal endophytes limit pathogen damage in a tropical tree. Proc. Nat. Acad. Sci. USA.,
100: 15649-15654.
Aronson, A.I., W. Beckman and P. Dunn, 1986. Bacillus thuringiensis and related insect
pathogens. Microbiol. Rev., 50: 1-24.
Arthurs, S. and M.B. Thomas, 2001. Behavioral changes in Schistocerca gregaria following
infection with a fungal pathogen: implications for susceptibility to predation. Ecol .
Entomol., 26: 227-234.
Ashouri, A., N. Arzanian and H. Askary, 2003. Interactions of Verticillium lecanii (Zimm.)
Viegas and Adonia variegata (Col.: Coccinellidae), pathogen and predator of aphids.
Colloque international tomate sous abri, protection integree agriculture biologique,
Avignon, France. Pp. 158-162.
Atakan, E., H. Elekçioğlu, U. Gözel and O. Yüksel, 2009. First report of Heterorhabditis
bacteriophora (Poinar, 1975) (Nematoda: Heterorhabditidae) isaloted from
37
Rhynchophorus ferrugineus (Oliver, 1790) (Coleoptera: Curculionidae) in Turkey. Bull.
OEPP/EPPO., 39: 155-160.
Athanassiou, C.G., N.G. Kavallieratos, B.J. Vayias, J.B. Tsakiri, N.H. Mikeli, C.M. Meletsis and
Z. Tomanovic, 2008. Persistence and efficacy of Metarhizium anisopliae (Metschnikoff)
Sorokin (Deuteromycotina: Hyphomycetes) and diatomaceous earth against Sitophilus
oryzae (L.) (Coleoptera: Curculinoidae) and Rhyzopertha dominica (F.) (Coleoptera:
Bostrichidae) on wheat and maize. Crop Prot., 27(10): 1303-1311.
Attathom, T., 2002. Biotechnology for insect pest control. In: Proc. Sat. Forum, "Sustainable
Agricultural System in Asia," Nagoya. Pp. 73-84.
Atwa, A.A. and E.M. Hegazi, 2014. Comparative susceptibilities of different life stages of the
red palm weevil (Coleoptera: Curculionidae) treated by entomopathogenic nematodes. J.
Econ. Entomol., 107(4): 1339-1347.
Aung, O.M., K. Soytong and K.D. Hyde, 2008. Diversity of entomopathogenic fungi in
rainforests of Chiang Mai Province, Thailand. Fungal Diver., 30: 15-22.
Avand-Faghih, A., 1996. The biology of red palm weevil, Rhynchophorus ferrugineus Oliv.
(Coleoptera: Curculionidae) in Saravan region (Sistan and Balouchistan Province, Iran).
Appl. Entomol. Phytopathol., 63: 16-18.
Azam, K.M. and S.A. Razvi, 2001. Control of red palm weevil, Rhynchophorus ferrugineus
Oliver using prophylactic spraying of date palms and trunk injection. In: Proceedings of
the 2nd international conference on date palms, Al-Ain, UAE, March 2001. Pp. 216-222.
Baig, D.N., D.A. Bukhari and A.R. Shakoori, 2010. Cry genes profiling and the toxicity of
isolates of Bacillus thuringiensis from soil samples against American bollworm,
Helicoverpa armigera. J. Appl. Microbiol., 109(6): 1967-1978.
Baloach, H.B., M.A. Rustamani, R.D. Khuro, M.A. Talpur and T. Hussain, 1992. Incidence and
abundance of date palm weevil in different cultivars of date palm. Proc. of 12 th Cong.
Zool. Pak., 12: 445-447.
Bandani, A.R., B.P.S. Khamba, J. Faul, R. Newton, M. Deadman and T.M. Butt, 2000.
Production of efrapeptins by Tolypocladium species and evaluation of their insecticidal
and antimicrobial properties. Mycol. Res., 104: 537-544.
Banerjee A. and T.K. Dangar, 1995. Pseudomonas aeruginosa, a facultative pathogen of red
palm weevil, Rhynchophorus ferrugineus. World J. Microb. Biot., 11: 618-620.
Banu, J.G., V.K. Sosamma and P.K. Koshy, 1998. Natural occurrence of an entomopathogenic
nematode, Heterorhabditis indicus from Kerala, India. Nematology: Challenges and
Opportunities in 21st
Century. Proceedings of the 3rd International Symposium of Afro-
Asian Society of Nematologists (TISAASN), Sugarcane Breeding Institute (ICAR),
Coimbatore, India. Pp. 274-280.
Barranco, P., J. de la Peña and T. Cabello, 1996. El picudo rojo de las palmeras, Rhynchophorus
ferrugineus (Olivier), nueva plaga en Europa. Phytoma España, 67:36-40.
Batta, Y.A., 2003. Production and testing of novel formulations of the entomopathogenic fungus
Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina: Hyphomycetes).
Crop Prot., 22: 415-422.
Batta, Y.A., 2004. Control of rice weevil (Sitophilus oryzae L. Coleoptera: Curculinoidae) with
various formulations of Metarhizium anisopliae. Crop Prot., 23: 103-108.
Bauce E., Y. Bidon and R. Berthiaume, 2002. Effects of food nutritive quality and Bacillus
thuringiensis on feeding behaviour, food utilization and larval growth of spruce budworm
38
Choristoneura fumiferana (Clem.) when exposed as fourth-and sixth-instar larvae. Agri.
Forest Entomol., 4: 57-70.
Bauer, L.S., 1995. Resistance: a threat to the insecticidal crystal proteins of Bacillus
thuringiensis. Florida Entomol., 78(3): 414-442.
Bedford, G.O., 1974. Parasitism of the palm weevil Rhynchophorus bilineatus (Montrouzier)
(Coleoptera: Curculionidae) by Praecocilenchus rhaphidophorus (Poinar) (Nematoda:
Aphelenchoidea) in New Britain. J. Austr. Entomol. Soc., 13: 155-156.
Bednarek, A., E. Popowska-nowak, E. Pezowicz and M. Kamionek, 2004. Integrated methods in
pest control: effect of insecticides on entomopathogenic fungi (Beauveria bassiana
(Bals.) Vuill., B. Brongniartii (Sacc.) and nematodes (Heterorhabditis megidis Poinar,
Jackson, Klein, Steinernema feltiae Filipjev, S. glaseri Steiner). Polish J. Ecol., 52(2):
223-228.
Ben-Chobba, I., A. Elleuch, I. Ayadi, L. Khannous, A. Namsi, F. Cerqueira, N. Drira, N.
Gharsallah and T. Vallaeys, 2013. Fungal diversity in adult date palm (Phoenix
dactylifera L.) revealed by culture-dependent and culture-independent approaches. J.
Zhejiang Uni. Sci., 14(12): 1084-1099.
Berbert-Molina, M.A., A.M.R. Prata, L.G. Pessanha and M.M. Silveira, 2008. Kinetics of
Bacillus thuringiensis var. israelensis growth on high glucose concentrations. J. Indust.
Microbiol. Biotech., 35(11): 1397-1404.
Berliner, E., 1915. Ueber die Schlaffsucht der Mehlmottenraupe (Ephestia kuhniella Zell) und
ihren Erreger Bacillus thuringiensis n. sp. Zeitschrift für Angewandte Entomologie, 2:
29-56.
Bernier, L., R.M. Cooper, A.K. Charnley and J.M. Clarkson, 1989. Transformation of the fungus
Metarhizium anisopliae to benomyl resistance. FESM Microbiol. Lett., 60(3): 261-265.
Besse, S., L. Crabos and K. Panchaud, 2011. Efficacite de 2 souches de Beauveria bassiana sur
le charançon rounge du palmier, Rhynchophorus ferrugineus. In: AFPPNeuvième
Conférence Internationale sur les ravageurs en agriculture, Montpellier, France. Pp. 404-
409.
Bhattacharya, A.K., P. Mondal, V.V. Ramamurthy and R.P. Srivastava, 2003. Beuveria
bassiana. In: Srivastava, R.P. (Ed.). A potential bioagent for innovative integrated pest
management programme. Biopesticides and Bioagents in Integrated Pest Management of
Agricultural Crops. International Books Distributing Co., Lucknow. Pp. 381-491.
Birda, L.J. and J.R. Akhursta, 2007. Variation in susceptibility of Helicoverpa armigera
(Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) in
Australia to two Bacillus thuringiensis toxins. J. Inver. Pathol., 94: 84-94.
Blomquist, G. and R. Vogt, 2003. Insect pheromone biochemistry and molecular biology: the
biosynthesis and detection of pheromones and plant volatiles. Boston (MA): Academic
Press.
Bong, C.F., 1986. Field control of Heliolhis zeo (Boddie) (Lepidoptera: Noctuidae) using a
parasitic Nematode. Ins. Sci. Appl., 7:23-25.
Booth, R.G., M.L. Cox and R.B. Madge, 1990. IIE Guides to insects of importance to Man. 3
New Guinea records of economically important beetles (Coleoptera). CABI Publishing,
Walllingford, UK. Pp. 384.
Boucias, D.G., J.C. Pendland and J.P. Latge, 1988. Non-specific factors involved in attachment
of entomopathogenic deuteromycetes to host insect cuticle. Appl. Environ. Microbiol.,
54: 1795-1805.
39
Bourtzis, K., 2008. Wolbachia based technologies for insect pest population control. Adv. Exp.
Med. Biol., 627: 104-113.
Bravo, A., S.S. Gill and M. Soberon, 2007. Mode of action of Bacillus thuringiensis Cry and Cyt
toxins and their potential for insect control. Toxicon, 49: 423-435.
Brenner, C., R. Deplus, C. Didelot, A. Loriot, E. Vire, C. De-Smet, A. Gutierrez, D. Danovi, D.
Bernard, T. Boon, P. Giuseppe Pelicci, B. Amati, T. Kouzarides, Y. de Launoit, L. Di
Croce and F. Fuks, 2005. Myc represses transcription through recruitment of DNA
methyltransferase corepressor. EMBO. J., 24: 336-346.
Brousseau, C., G. Charpentier and S. Belloncik, 1998. Effects of Bacillus thuringiensis and
Destruxins (Metarhizium anisopliae Mycotoxins) combinations on Spruce Budworm
(Lepidoptera: Tortricidae). J. Inver. Pathol., 72(3): 262-268.
Bruce, M.J., R. Gatsi, N. Crickmore and A.H. Sayyed, 2007. Mechanisms of resistance to
Bacillus thuringiensis in the Diamondback Moth. Biopesti. Inter., 3(1): 1-12.
Bugeme, D.M., N.K. Maniania, M. Knapp and H.I. Boga, 2008. Effect of temperature on
virulence of Beauveria bassiana and Metarhizium anisopliae isolates to Tetranychus
evansi. Experi. Appl. Acarol., 46: 275-285.
Bulla, L.A. Jr, R.A. Rhodes and G.S. Julian, 1975. Bacteria as insect pathogens. Annu. Rev.
Microbiol., 29: 163-190.
Bunerjee, A. and T.K. Dangar, 1995. Pseudomonas aeruginosa, a facultative pathogen of red
palm weevil, Rhynchophorus ferrugineus. World J. Microbiol. Biotechnol. 11: 618-620.
Butani, D.K., 1975. Insect pests of fruit crops and their control, sapota-11. Pesticide Res. J., 9:
40-42.
Butt, T.M. and M.S. Goettel, 2000. Bioassays of entomogenous fungi In: Navon, A. and K.R.S.
Ascher (Eds.). Bioassays of Entomopathogenic Microbes and Nematodes, CABI. Pp.
141-195.
Buxton, P.A., 1920. Insect pests of dates and the date palm in Mesopotamia and elsewhere. Bull.
Entomol. Res., 11: 287-303.
Cabello, T.P., 2006. Biology and population dynamics of red palm weevil in Spain. In
Proceedings of the 1st International Workshop on Red Palm Weevil, 28-29, November
2005, IVIA, Valencia, Spain (in press).
Cagan, L. and V. Uhlik, 1999. Pathogenicity of Beauveria bassiana strains isolated from
Ostrinia nubilalis Hbn. (Lepidoptera: Pyralidae) to original host larvae and to ladybirds
(Coleoptera: Coccinellidae). Plant Prot. Sci., 35: 108-112.
Chandler, D., D. Hay and A.P. Reid, 1997. Sampling and occurrence of entomopathogenic fungi
and nematodes in UK soils. Appl. Soil Ecol., 5: 133-141.
Chapin, E. and N. André, 2010. Nouveau moyen de lutte biologique contre le papillon
palmivore. Phytoma La Défense des Végétaux, 635: 27-30.
Chaufaux, J., M. Marchal, N. Gilois, I. Jehanno and C. Buisson, 1997. Recherche de souches
naturelles du Bacillus thuringiensis dans differents biotopes, átravers le monde. Canad. J.
Microbiol., 43: 337-343.
Chen, M., A. Shelton and G.Y. Ye, 2011. Insect-resistant genetically modified rice in China:
from research to commercialization. Ann. Rev. Entomol., 56: 81-101.
Cito, A., G. Mazza, A. Strangi, C. Benvenuti, G.P. Barzanti, E. Dreassi, T. Turchetti, V.
Francardi and P.F. Roversi, 2014. Characterization and comparison of Metarhizium
strains isolated from Rhynchophorus ferrugineus. FEMS Microbiol. Lett., 355: 108-115.
40
Cottrell, T.E. and D.I. Shapiro-Ilan, 2003. Susceptibility of a native and an exotic lady beetle
(Coleoptera: Coccinellidae) to Beauveria bassiana. J. Inver. Pathol., 84: 137-144.
Cox, M.L., 1993. Red palm weevil, Rhynchophorus ferrugineus in Egypt. FAO Plant Prot. Bull.,
41: 30-31.
Crickmore, N., D.R. Zeigler, J. Feitelson, E. Schnepf, J.V. Rie, D. Lereclus, J. Baum and D.H.
Dean, 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal
proteins. Microbiol. Molecul. Biol. Rev., 62(3): 807-813.
Dangar T. K. and A. Banerjee, 1993. Infection of red palm weevil by microbial pathogens. In:
Nair, M.K., H.H. Khan, P. Gopalasundaram and E.V.V. Bhaskara Rao (Eds.). Advances
in coconut research and development. Oxford IBM Publishing Co., New Delhi. Pp. 531-
533.
Dangar, T.K., 1997. Infection of red palm weevil, Rhynchophorus ferrugineus, by a yeast. J.
Plant. Crops, 25: 193-196.
Deadman, M.L., K.M. Azam, S.A. Ravzi and W. Kaakeh, 2001. Preliminary investigation into
the biological control of the red palm weevil using Beauveria bassiana. Proceedings of
the Second International Conference on Date Palm, Al-Ain, UAE. March 25-27. Pp. 225-
232.
Debré, P., 1998. Louis Pasteur. E. Forster, translator. Johns Hopkins University Press, Baltimore,
Maryland, USA.
De-Maagd, R.A., A. Bravo and N. Crickmore, 2001. How Bacillus thuringiensis has evolved
specific toxins to colonize the insect world. Trends Genet., 17: 193-199.
Dembilio, Ó., E. Llácer, D.E. Martinez, M.M. Altube and J.A. Jacas, 2010a. Field efficacy of
imidacloprid and Steinernema carpocapsae in a chitosan formulation against the red
palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) in Phoenix
canariensis. Pest Manag. Sci., 66: 365-370.
Dembilio, Ó. and J.A. Jacas, 2013. Biological control of Rhynchophorus ferrugineus. AFPP
palm pest mediterranean conference nice-16, 17 and 18 January, 2013.
Dembilio, Ó., E. Quesada-Moraga, C. Santiago-Alvarez and J.A. Jacas, 2010b. Potential of an
indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological
control agent against the red palm weevil, Rhynchophorus ferrugineus. J. Inver. Pathol.,
104(3): 214-221.
Dembilio, Ó., J.A. Jacas and E. Llacer, 2009. Are the new palms Washintonia filifera and
Chamaerops humilis suitable hosts for the red palm weevil, Rhynchophorus ferrugineus
(Col. Curculionidae). J. Appl. Entomol., 133(7): 565-567.
Derakhshan, A., 2008. Natural occurrence and distribution of soil borne entomopathogenic fungi
in Shahrood Region, Northeast of Iran. International Meeting on Soil Fertility Land
Management and Agroclimatology. Turkey. Pp. 873-877.
Dettloff, M., B. Kaiser and A. Wiesner, 2001. Localization of injected apolipophorin III in-vivo
new insights into the immune activation process directed by this protein. J. Insect
Physiol., 47: 789-797.
Dhillon, B.S., R.K. Tyaagi and S. Saxena, 2005. Plant genetic resources: horticultural crops.
Narosa Publishing House, New Delhi. Pp. 174-176.
Dimbi, S., N.K. Maniania, S.A. Lux and J.M. Mueke, 2004. Effect of constant temperatures on
germination, radial growth and virulence of Metarhizium anisopliae to three species of
African tephritid fruit flies. Biocontrol, 49: 83-94.
41
Dolinski, C. and L.A. Lacey, 2007. Microbial control of arthropod pests of tropical tree
fruit. Neotropi. Entomol., 36: 161-179.
Dowd, P.F., 1992. Insect interactions with mycotoxin-producing fungi and their hosts. In:
Bhatnagar, D. E.B. Lillehoj and D.K. Arora (Eds.). Handbook of Applied Mycology.
Volume 5. Mycotoxins in Ecological Systems. Marcel Dekker, New York. Pp. 137-155.
Dumler, J.S., A.F. Barbet, C.P. Bekker, G.A. Dasch, G.H. Palmer, S.C. Ray, Y. Rikihisia and
F.R. Rurangirwa, 2001. Reorganization of genera in the families Rickettsiaceae and
Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with
Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six
new species combinations and designation of Ehrlichia equi and ’HGE agent’ as
subjective synonyms of Ehrlichia phagocytophila. Inter. J. Syst. Evol. Microbiol., 51:
2145-2165.
Edwards, S., 1989. Real Exchange Rates, Devaluation, and Adjustment, Cambridge,
Massachusetts: MIT Press.
Eilenberg, J. and V. Michelsen, 1999. Natural host range and prevalence of the genus
Strongwellsea (Zygomycota: Entomophthorales) in Denmark. J. Inver. Pathol., 73: 189-
198.
Ekesi, S., N.K. Maniania and K. Ampong-Nyarko, 1999. Effect of temperature on germination,
radial growth and virulence of Metarhizium anisopliae and Beauveria bassiana on
Megalurothrips sjostedti. Biocontrol Sci. Technol., 9: 177-185.
Elawad, S.A., S.A. Mousa, A.S. Shahbad, S.A. Alawaash and A.M.A. Alamari, 2007. Efficacy of
entomopathogenic nematodes against red palm weevil in UAE. Acta Horti., 736: 415-
420.
El-Bishry, M.H., Y. El-Sebay and M.H. Al-Elimi, 2000. Impact of the environment in date palm
infested with Rhynchophorus ferrugineus on five entomopathogenic nematodes
(Rhabditida). Inter. J. Nematol., 10(1): 75-80.
Eleftherianos, I., H. Baldwin, R.H. French-Constant, S.E. Reynolds, 2008. Developmental
modulation of immunity: changes within the feeding period of the fifth larval stage in the
defence reactions of Manduca sexta to infection by Photorhabdus. J. Insect Physiol., 54:
309-318.
El-Ezaby, F.A, 1997. A biological in-vitro study on the red Indian date palm weevil. Arab J.
Plant Prot., 15(2): 84-87.
El-Mergawy, R.A.A.M. and A.M. Al-Ajlan, 2011. Red palm weevil, Rhynchophorus ferrugineus
(Olivier): economic importance, biology, biogeography, and integrated pest management.
J. Agri. Sci. Technol., 1: 1-23.
El-Sufty, R., S. Al-Bgham, S. Al-Awash, A. Shahdad and A. Al-Bathra, 2011. A trap for auto
dissemination of the entomopathogenic fungus Beauveria bassiana by red palm weevil
adults in date palm plantations. Egyp. J. Biol. Pest Control, 21: 271-276.
El-Sufty, R., S.A. Al-Awash, A.M. Al-Amiri, A.S. Shahdad, A.H. Al-Bathra and S.A. Musa,
2007. Biological control of red palm weevil, Rhynchophorus ferrugineus (Col.:
Curculionidae) by the entomopathogenic fungus Beauveria bassiana in United Arab
Emirates. Proceeding of the 3rd International Conference on Date Palm. Acta Horti., 736:
399-404.
El-Sufty, R., S.A. Al-Awash, S. Al-Bgham, A.S. Shahdad and A.H. Al-Bathra, 2009.
Pathogenicity of the fungus Beauveria bassiana (Bals.) Vuill to the Red Palm Weevil,
42
Rhynchophorus ferrugineus (Oliv.) (Col.: Curculionidae) under laboratory and field
conditions. Egyp. J. Biol. Pest Control, 19: 81-85.
Entwistle, P.F., J.S. Cory, M. Bailey and S. Higgs, 1993. Bacillus thuringiensis, an
environmental bio-pesticide: theory and practice. New York, NY: Wiley.
EPPO (European and Mediterranean Plant Protection Organization), 2008. Data sheets on
quarantine pests. Rhynchophorus ferrugineus. EPPO Bull., 38: 55-59.
EPPO (European and Mediterranean Plant Protection Organization), 2009. EPPO Reporting
Service. 2009/002 - First record of Rhynchophorus ferrugineus in Curacao, Netherlands
Antilles, January 26, 2009.
EPPO (European and Mediterranean Plant Protection Organization), 2010. EPPO Reporting
Service. 2010/176 - First record of Rhynchophorus ferrugineus in the USA, November 1,
2010.
EPPO (European and Mediterranean Plant Protection Organization), 2008. Data sheets on
quarantine pests. Rhynchophorus ferrugineus. EPPO Bull., 38: 55-59.
Ericsson, J.D., A.F. Janmaat, C. Lowenberger and J.H. Myers, 2009. Is decreased generalized
immunity a cost of Bt resistance in cabbage loopers Trichoplusia ni? J. Inver. Pathol.,
100: 61-67.
Esteban-Duran J., J.L. Yela, F. Beitia Crespo and A. Jimenez Alvarez, 1998. Biology of red palm
weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae:
Rhynchophorinae), in the laboratory and field, life cycle, biological characteristics in its
zone of introduction in Spain, biological method of detection and possible control.
Boletin de Sanidad Vegetal Plagas, 24: 737-748.
Esteban-Duran, J., J.L. Yela, F. Beitia-Crespo and A. Jimenez-Alvarez, 1998. Biologia del
Curculionido ferruginoso de las palmeras Rhynchophorus ferrugineus (Olivier) en
laboratorio y campo: Ciclo en cautividad, peculiaridades biologicas en su zona de
introduccion en Espana y metodos biologicos de deteccion y posible control (Coleoptera:
Curculionidae: Rhynchophorinae). Boletín de Sanidad Vegetal - Plagas, 24: 737-748.
Evans, H.C., 1989. Mycopathogens of insects of epigeal and aerial habitats. In: Wilding, N.N.M.,
P.M. Collins and J.F.W. Hammond (Eds.). Insect-Fungus interactions. Pp. 205-238.
Eyal, M.D., A. Mabud, K.L. Fishbein, J.F. Walter, L.S. Osbourne and Z. Landa, 1994.
Assessment of Beauveria bassiana Nov. EO-1 Strain, Which Produces a Red Pigment for
Microbial Control. Appl. Biochem. Biochem., 44: 65-80.
Faghih, A.A., 1996. The biology of red palm weevil, Rhynchophorus ferrugineus Oliver
(Coleopter, Curculionidae) in Savaran region (Sistan province, Iran). Appl. Entomol.
Phytopathol., 63: 16-18.
Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil
Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm
during the last one hundred years. Inter. J. Tropi. Insect Sci., 26: 135-154.
Fan, Y., W.S. Fang, S. Guo, X. Pei, Y. Zhang, Y. Xiao, D. Li, K. Jin, M.J. Bidochka and Y. Pei,
2007. Increased insect virulence in Beauveria bassiana strains over expressing an
engineered chitinase. Appl. Environ. Microbiol., 73: 295-302.
Fang, W., Y. Zhang, X. Yang, X. Zheng, H. Duan, Y. Li, and Y. Peia, 2004. Agrobacterium
tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance
gene as a selection marker. J. Inver. Pathol., 85: 18-24.
FAO Statistics Division, 2013. Production, harvested area, import and export of dates in Pakistan
in 2011. Available at: http://faostat.fao.org.
43
Fargues, J., N.K. Maniania, J.C. Delmas and N. Smits, 1992. Influence de la température sur la
croissance in vitro d’hyphomycètes entomopathogènes. Agronomie, 12: 557-564.
Fernandes, P.M., B.P. Magalhaes, S.B. Alves and D.W. Roberts, 1989. Effect of physical and
biological factors on the conidiogenisis and survival of Beauveria bassiana (Bals.) Vuill.
inside cadavers of Cerotoma arcuata (Olivier, 1791). (Coleoptera: Chrysomelidae).
Anais da Sociedade Entomologia do Brasil, 18: 147-156.
Fernandez-Luna, M.T., H. Lanz-Mendoza, S.S. Gill, A. Bravo, M. Soberon and J. MirandaRios,
2010. An alpha-amylase is a novel receptor for Bacillus thuringiensis ssp. israelensis
Cry4Ba and Cry11Aa toxins in the malaria vector mosquito Anopheles albimanus
(Diptera: Culicidae). Environ. Microbiol., 12: 746-757.
Ferron, P., 1978. Biological control of insect pests by entomopathogenic fungi. Ann. Rev.
Entomol., 23: 409-423.
Ferron, P., J. Fargues and G. Riba, 1991. Fungi as microbial insecticides against pests. In: Arora,
D.K., L. Ajello and K.G. Mukerji (Eds.). Handbook of Applied Mycology, Vol. 2:
Humans, Animals and Insects. Marcel Dekker, Inc, New York. Pp. 665-706.
Ferry, M. and S. Gomez, 2002. The red palm weevil in the Mediterranean area. Palms, 46(4):
172-178.
Foth, H.D., 1984. Fundamentals of Soil Science. John Wiley & Sons, London.
Francardi, V., C. Benventi, G.P. Barzanti and P.F. Rovers, 2013. Auto contamination trap with
entomopathogenic fungi: A possible strategy in the control of Rhynchophorus ferrugineus
(olivier) (Coleoptera Curculionidae). REDIA, XCVI: 57-67.
Francardi, V., C. Benvenuti, P.F. Roversi, P. Rumine and G. Barzanti, 2012.
Entomopathogenicity of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae
(Metsch.) Sorokin isolated from different sources in the control of Rhynchophorus
ferrugineus (Olivier) (Coleoptera Curculionidae). Redia, 95: 49-55.
Francesca, N., A. Alfonzo, G.L. Verde, L. Settanni, M. Sinacori, P. Lucido and G. Moschetti,
2015. Biological activity of Bacillus spp. evaluated on eggs and larvae of red palm weevil
Rhynchophorus ferrugineus. Ann. Microbiol., 65: 477-485.
Francesca, N., C.G. Caldarella and G. Moschetti, 2008. Indagini preliminari su bacilli sporigeni
associati ad adulti di Punteruolo rosso e loro possibili impieghi in lotta biologica, In: La
ricerca scientifica sul punteruolo rosso e gli altri fitofagi delle palme in Sicilia, Regione
Siciliana-Assessorato Agricoltura e Foreste Dipartimento Interventi Infrastrutturali,
Servizi allo Sviluppo. Pp. 69-72.
Freimoser, F.M., G. Hu and R.J. St Leger, 2005. Variation in gene expression patterns as the
insect pathogen Metarhizium anisopliae adapts to different host cuticles or nutrient
deprivation in vitro. Microbiol., 151: 361-371.
Furlaneto, L., H.O. Saridakis and O.M.N. Arantes, 2000. Survival and conjugal transfer between
Bacillus thuringiensis strains in aquatic environment. Braz. J. Microbiol., 31: 233-238.
Fuxa, J.R., 1995. Ecological factors critical to the exploitation of entomopathogens in pest
control. In: Franklin R. Hall and J.W. Barry (Eds.), Biorational Pest Control Agents
Formulation and Delivery, Vol. 595. American Chemical Society.
Gauglar, R. and H.K. Kaya, 1990. Entomopathogenic nematodes in biological control. Boca
Raton, Florida, USA: CRC press. Pp. 365.
George, Z. and N. Crickmore, 2012. Bacillus thuringiensis Applications in Agriculture. In:
Estibaliz S.E. (Ed.). Bacillus thuringiensis Biotechnology. Springer Science + Business
Media B.V. Springer Netherlands.
44
Gerber, K. and R.M. Giblin-Davis, 1990. Association of the red ring nematode and other
nematode species with the palm weevil, Rhynchophorus palmarum. J. Nematol., 22: 143-
149.
Ghazavi, M., A. Avand-Faghih, 2002. Isolation of two entomopathogenic fungi on red palm
weevil, Rhynchophorus ferrugineus (Olivier) (Col., Curculionidae) in Iran. Appl.
Entomol. Phytopathol., 9: 44-45.
Giannoulis, P., C.L. Brooks, G.B. Dunphy, C.A. Mandato, D.F. Niven and R.J. Zakarian, 2007.
Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus
subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta:
Lepidoptera: Lasiocampidae). J. Inver. Pathol., 94: 20-30.
Giblin-Davis, R.M., 1993. Interactions of nematodes with insects, In: W. Khan (Ed.). Nematode
Interactions. Chapman & Hall, London. Pp. 302-344.
Giblin-Davis, R.M., N. Kanzaki, W. Ye, B.J. Center and W.K. Thomas, 2006. Morphology and
systematics of Bursaphelenchus gerberae n. sp. (Nematoda: Parasitaphelenchidae), a rare
associate of the palm weevil, Rhynchophorus palmarum in Trinidad. Zootaxa, 1189: 39-
53.
Gill, S.S., E.A. Cowles and P.V. Pietrantonio, 1992. The mode of action of Bacillus thuringiensis
δ-endotoxins. Ann. Rev. Entomol., 37: 615-636.
Gindin, G., S. Levski, I. Glazer and V. Soroker, 2006. Evaluation of the entomopathogenic fungi
Metarhizium anisopliae and Beauveria bassiana against the red palm weevil
Rhynchophorus ferrugineus. Phytoparasitica, 34: 370-379.
Glare, T.R., 2003. Biotechnology potential of entomopathogenic fungi. In: Arora, D.K., D.
Bridge and D. Bhatnagar (Eds.). Fungal biotechnology in agricultural, food and
environmental applications. CRC Press.
Goettel, M.S., T.J. Poprawski, J.D. Vandenberg, Z. Li and D.W. Roberts, 1990. Safety to non-
target invertebrate of fungal bicontrol agents. In: Laird, M., L.A. Lacey and E.W.
Davidson (Eds.). Safty of Microbial insecticides. Boca Raton, CA: CRS Press. Pp. 209-
232.
Gómez-Vidal, S. and M. Ferry, 1999. Attempts at biological control of date palm pests recently
found in Spain. In: M. Canard and V. Beyssatarnaouty (Eds.). Proceedings of the First
Regional Symposium for Applied Biological Control in Mediterranean Countries. Cairo,
25-29 October 1998. Imprimerie Sacco, Toulouse, France. Pp. 121-125.
Gómez-Vidal, S., L.V. Lopez-Llorca, H.B. Jansson and J. Salinas, 2006. Endophytic
colonization of date palm (Phoenix dactylifera L.) leaves by entomopathogenic fungi.
Micron, 37: 624-632.
Gong, Y.J., C.L. Wang, Y.H. Yang, S.W. Wu and Y.D. Wu, 2010. Characterization of resistance
to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella from China. J. Inver. Pathol.,
104(2): 90-96.
Gressel, J., 2001. Potential failsafe mechanisms against the spread and introgression of
transgenic hyper virulent biocontrol fungi. Trend. Biotech., 19: 149-154.
Grewal, P.S., R.U. Ehlers and D.I. Shapiro-Ilan, 2005. Nematodes as Biocontrol Agents, CABI,
New York, Wallingford. Pp. 513.
Güerri-Agulló, B., L. Asensio., P. Barranco, S. Gómez-Vidal and L.V. Lopez-Llorca, 2008. Use
of Beauveria bassiana as a tool for biological control of Rhynchophorus ferrugineus, In:
41st Annual Meeting of Society for Invertebrate pathology and 9th international
Conference on Bacillus thuringiensis, Warwick, UK, 3-7 August, 2008. Pp. 125.
45
Güerri-Agulló, B., R. López-Follana, L. Asensio, P. Barranco and L.V. Lopez-Llorca, 2011. Use
of a solid formulation of Beauveria bassiana for biocontrol of the red palm weevil
(Rhynchophorus ferrugineus) (Coleoptera: Dryophthoridae) under field conditions in SE
Spain. Florida Entomol., 94: 737-747.
Güerri-Agulló, B., S. Gómez-Vidal, L. Asensio, P. Barranco and L.V. Lopez-Llorca, 2010.
Infection of the red palm weevil (Rhynchophorus ferrugineus) by the entomopathogenic
fungus Beauveria bassiana: a SEM study. Micros. Res. Tech., 73: 714-725.
Gunawardena, N.E. and U.K. Bandarage, 1995. 4-methyl-5-nonanol (ferrugineol) as an
aggregation pheromone of the coconut pest, Rhynchophorus ferrugineus F. (Coleoptera:
Curculionidae): Synthesis and use in a preliminary field assay. J. Natural Sci. Council of
Sri Lanka, 23: 71-79.
Gupta, J.J., B.P.S. Yadav and H.K. Gupta, 1995. Nutritional value of jack bean for broiler.
Indian J. Poult. Sci., 30(2): 112-116.
Gupta, S.C., T.D. Leathers, G.N. El-Sayed and C.M. Ignoffo, 1991. Production of degradative
enzymes by Metarhizium anisopliae during growth on defined media and insect cuticle.
Exp. Mycol., 15: 310-315.
Gurr, G.M., S.D. Wratten and M.A. Altieri, 2004. Ecological engineering for pest management:
habitat manipulation for arthropods. CSIRO Publishing, Collingwood, Australia.
Hagley, E., 1965. Test of attractant for the palm weevil. J. Econ. Entomol., 58: 1002-1003.
Hajek, A.E. and R.J. St-Leger, 1994. Interactions between fungal pathogens and insect host.
Ann. Rev. Entomol., 39: 293-322.
Hajek, A.E., 1997. Ecology of terrestrial fungal entomopathogens. Adv. Microbiol. Ecol., 15:
193-249.
Hallet, R.H., G. Gries, J.H. Borden, E. Czyzewska, A.C. Oehlschlager, H.D. Pierce Jr., N.P.D.
Angerilli and A. Rauf, 1993. Aggregation pheromones of two Asian palm weevils,
Rhynchophorus ferrugineus and R. vulneratus. Naturwissen, 80: 328-331.
Hallett, R.H., B.J. Crespi and J.H. Borden, 2004. Synonymy of Rhynchophorus
ferrugineus (Olivier), 1790 and R. vulneratus (Panzer), 1798 (Coleoptera, Curculionidae,
Rhynchophorinae). J. Natural History, 38(22): 2863-2882.
Hanounik, S.B., 1998. Steinemematids and Heterorhabditids as biological control agents for the
red palm weevil, Rhynchophorus ferrugineus. Sultan Qaboos Uni. J. Sci. Res. Agri. Sci.,
3: 95-102.
Hanounik, S.B., M.M.E. Saleh, R.A. Abuzuairah, M. Alheji, H. Aldhahir and Z. Aljarash, 2000.
Efficacy of entomopathogenic nematodes with antidesiccants in controlling the red palm
weevil Rhynchophorus ferrugineus on date palm trees. Inter. J. Nematol., 10: 131-134.
Haseeb, M. and H. Murad, 1997. Susceptibility of the predator, Coccinella septempunctata to the
entomogenous fungus, Beauveria bassiana. Ann. Plant Prot. Sci., 5: 188-219.
Hassan, A.E.M. and A.K. Charnley, 1989. Ultrastructural study of the penetration by
Metarhizium anisopliae through Dimilin affected cuticle of Manduca sexta. J. Inver.
Pathol., 54: 117-124.
Heckel, D.G., L.J. Gahan, S.W. Baxter, J.Z. Zhao, A.M. Shelton, F. Gould and B.E. Tabashnik,
2007. The diversity of Bt resistance genes in species of Lepidoptera. J. Inver. Pathol.,
95(3): 192-197.
Hedgecock, S., D. Moore, P.M. Higgins and C. Prior, 1995. Influence of moisture content on
temperature tolerance and storage of Metarhizium flavoviride conidia in an oil
formulation. Biocontrol Sci. Technol., 5: 371-377.
46
Hegazy, G., O. Al-Muhanna , S.B. Hanounik , T.S. Al-Gumaiah and A.A. Aldossary, 2007.
Efficacy of new isolates of the entomopathogenic fungus Beauveria bassiana against
RPW Rhynchophorus ferrugineus in Saudi Arabia. Egyp. J. Agri. Res., 85(1): 61-71.
Herms, C.P., D.G. McCullough, L.S. Bauer, R.A. Haack, D.L. Miller and N.R. Dubois, 1997.
Susceptibility of the endangered Karner blue butterfly (Lepidoptera: Lycaenidae) to
Bacillus thuringiensis var. kurstaki used for gypsy moth suppression on Michigan. Great
Lakes Entomol., 30: 125-141.
Hilgenboecker, K., P. Hammerstein, P. Schlattmann, A. Telschow and J.H. Werren, 2008. How
many species are infected with Wolbachia? a statistical analysis of current data. Fems.
Microbiol. Lett., 281: 215-220.
Hofmann, C., H. Vanderbruggen, H. Hofte, J.V. Rie, S. Jansens and H.V. Mellaert, 1988.
Specificity of Bacillus thuringiensis δ-endotoxins is correlated with the presence of high-
affinity binding sites in the brush border membrane of target insect midguts. Proc. Nati.
Acad. Sci. USA., 85: 7844-7848.
Hong, T.D., R.H. Ellis and D. Moore, 1997. Development of a model to predict the effect of
temperature and moisture on fungal spore longevity. Annal. Botany, 79: 121-128.
Hosokawa, T., R. Koga, Y. Kikuchi, X.Y. Meng and T. Fukatsu, 2010. Wolbachia as a
bacteriocyte-associated nutritional mutualist. Proc. Nat. Acad. Sci., 107: 769-774.
Hu, Y., S.B. Georghiou, A.J. Kelleher and R.V. Aroian, 2010. Bacillus thuringiensis Cry5B
protein is highly efficacious as a single-dose therapy against an intestinal roundworm
infection in mice. PLOS Negl. Trop. Dis., 4(3): e614.
Hussain, A., M. Rizwan-Ul-Haq, H. Al-Ayedh, S. Ahmed and A.M. Al-Jabr, 2015. Effect of
Beauveria bassiana infection on the feeding performance and antioxidant defence of red
palm weevil, Rhynchophorus ferrugineus. BioControl, 60: 849-859.
Hussain, A., M.R.U. Haq, A.M. Al-Jabr and H.Y. Al-Ayied, 2013. Managing Invasive
Populations of Red Palm Weevil: A Worldwide Perspective. J. Food Agri. Envir., 11:
456-463.
Ibargutxi, M.A., D. Muñoz, I.R.D. Escudero and P. Caballero, 2008. Interactions between
Cry1Ac, Cry2Ab, and Cry1Fa Bacillus thuringiensis toxins in the cotton pests
Helicoverpa armigera (Hübner) and Earias insulana (Boisduval). Biol. Control, 47(1):
89-96.
Ibrahim, L., T.M. Butt, A. Beckett and S.J. Clark, 1999. The germination of oil-formulated
conidia of the insect pathogen, Metarhizium anisopliae. Mycol. Res., 103(7): 901-907.
Inglis, D.G., D.L. Johnson and M.S. Goettel, 1996. Effect of bait substrate and formulation on
infection of grasshopper nymphs by Beauveria bassiana. Biocontrol Sci., Tech., 6: 35-50.
Inglis, G.D., G.M. Duke, P. Kanagaratnam, D.L. Johnson and M.S. Goettel, 1997. Persistence of
Beauveria bassiana in soil following application of conidia through crop canopies. Mem.
Entomol. Soci. Canada, 171: 253-263.
Inglis, G.D., M.S. Goettel, M.T. Butt and A. Strasser, 2001. Use of hyphomycetous fungi for
managing insect pests. In: Butt, T.M., C. Jackson and N. Magan (Eds.). Fungi as
Biocontrol Agents. CAB International, Wallingford, UK. Pp. 24-69.
Inglis, G.D., S.T. Jaronski and S.P. Wraight, 2002. Use of spray oils with entomopathogens. In:
Beattie, G.A.C., D.M. Watson, M.L. Stevens, D.J. Rae and R.N. SpoonerHart (Eds.).
Spray Oils Beyond 2000-Sustainable Pest and Disease Management. University of
Western Sydney Press. Pp. 302-312.
47
Iriarte, J., Y. Bel, M.D. Ferrandis, R. Andrew, J. Murillo, J. Ferré and P. Caballero, 1998.
Environmental distribution and diversity of Bacillus thuringiensis in Spain. System.
Appl. Microbiol., 21: 97-106.
Ishiwata, S., 1901. On a kind of severe flacherue (sotto disease). Dainihan Sanshi, Kaiho, 114: 1-
5.
Jalinas, J., B. Güerri-Agulló, R.W. Mankin, R. López-Follana and L.V. Lopez-Llorca, 2015.
Acoustic assessment of Beauveria bassiana (Hypocreales: Clavicipitaceae) effects on
Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) larval activity and mortality. J.
Econ. Entomol., 108(2): 444-453.
James, R.R. and B. Lighthart, 1994. Susceptibility of the convergent lady beetle (Coleoptera:
Coccinellidae) to four entomogenous fungi. Environ. Entomol., 23: 190-192.
James, R.R., J.S. Buckner and T.P. Freeman, 2003. Cuticular lipids and silverleaf whitefly stage
affect conidial germination of Beauveria bassiana and Paecilomyces fumosoroseus. J.
Inver. Pathol., 84(2): 67-74.
Jatoi, M.A., N. Solangi and Z. Markhand, 2010. Dates in Sindh: facts and figures. In: Markhand
G.S. and A.A. Abul-Soad (Eds.). Proceedings international dates seminar, 28 July 2009,
Khairpur. Pp. 59-71.
Jeffs, L.B. and G.G. Khachatourians, 1997. Toxic properties of Beauveria pigments on
erythrocyte membranes. Toxicon, 35: 1351-1356.
Joshi, L., R.J. St. Leger and M.J. Bidochka, 1995. Cloning of a cuticle-degrading protease from
the entomopathogenic fungus, Beauveria bassiana. FEMS Microbiol. Lett., 125: 211-
217.
Ju, R.T., F. Wang, F.H. Wan and B. Li, 2011. Effect of host plants on development and
reproduction of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). J. Pest
Sci., 84: 33-39.
Juárez, M.P., 1994. Inhibition of cuticular lipid synthesis and its effect on insect survival. Arch.
Insect Biochem. Physiol., 25: 177-191.
Jurat-Fuentes, J. and T. Jackson, 2012. Bacterial entomopathogens. In: Vega, F. and H. Kaya
(Eds.). Insect Pathology, 2nd edition. Edited by Elsevier. Pp. 266-349.
Kaakeh, W., 2005. Longevity, fecundity, and fertility of the red palm weevil, Rhynchophorus
ferrugineus Olivier (Coleoptera: Curculionidae) on natural and artificial diets. Emirates J.
Agri. Sci., 17: 23-33.
Kaakeh, W., A. Khamis and M.M. Aboul-Nour, 2001. The Red Palm Weevil. The most
dangerous agricultural pest. UAE University Press. Pp. 163.
Kalantari, M., R. Marzban, S. Imani and H. Askari, 2014. Effects of Bacillus thuringiensis
isolates and single nuclear polyhedrosis virus in combination and alone on Helicoverpa
armigera. Archi. Phytopathol. Plant Prot., 47(1): 42-50.
Kanaoka, M., A. Isogai, S. Murakoshi, M. Ichione, A. Suzuki and S. Tamura, 1978.
Bassianolide, a new insecticidal cylcodepsipeptide from Beauveria bassiana and
Verticillium lecanii. Agric. Biol. Chem., 42: 629-635.
Kanzaki, N., F. Abe, R.M. Giblin-Davis, K. Kiontke, D.H.A. Fitch, K. Hata and K. Soné, 2008.
Teratorhabditis synpapillata Sudhaus, 1985 (Rhabditida: Rhabditidae) is an associate of
the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae). Nematol.,
10: 207-218.
Kanzaki, N., R.M. Giblin-Davis, Y. Zeng, W. Ye and B.J. Center, 2009. Acrostichus
rhynchophori n. sp. (Rhabditida: Diplogastridae): a phoretic associate of Rhynchophorus
48
cruentatus Fabricius and R. palmarum L. (Coleoptera: Curculionidae) in the Americas.
Nematol., 11: 669 688.
Kaur, G. and V. Padmaja, 2008. Evaluation of Beauveria bassiana isolates for virulence against
Spodoptera litura (Fab.) (Lepidoptera; Noctuidae) and their characterization by RAPD-
PCR. Afri. J. Microbiol. Res., 2: 299-307.
Kaya, H.K. and R. Gaugler, 1993. Entomopathogenic nematodes. Ann. Rev. Entomol., 38: 181-
206.
Kaya, H.K., 1990. Soil ecology. In: Gaugler, R., H.K. Kaya (Eds.). Entomopathogenic
Nematodes in Biological Control. CRC Press, Boca Raton, FL. Pp. 93-115.
Kehat, M., 1999. Threat to date palms in Israel, Jordan and the Palestinian Authority by the red
palm weevil, Rhynchophorus ferrugineus. Phytoparasitica, 27: 107-108.
Keller, S. and G. Zimmermann, 1989. Mycopathogens of soil insects. In: Wilding, N., N.M.
Collins, P.M. Hammond and J.F. Webber (Eds.). Insect-Fungus Interactions. Academic
Press, London, UK. Pp. 239-270.
Kendrick, M., 2000. The Fifth Kingdom, 3rd Edition. Mycologue Publications, Sydney,
Australia.
Kenis, M., M.A. Auger-Rozenberg, A. Roques, L. Timms, C. Péré, M.J.W. Cock, J. Settele, S.
Augustin and C. Lopez-Vaamonde, 2009. Ecological effects of invasive alien insects.
Biol. Invasi., 11: 21-45.
Kershaw, M.J., E.R. Moorhouse, R. Bateman, S.E. Reynolds and A.K. Charnley, 1999. The role
of destruxins in the pathogenicity of Metarhizium anisopliae for three species of insects.
J. Inver. Pathol., 74: 213-223.
Khachatourians, G.G., 1986. Production and use of biological pest control agents. Trends
Biotech., 4: 120-124.
Khachatourians, G.G., 1991. Physiology and genetics of entomopathogenic fungi. In: Arora,
D.K., L. Ajello and K.G. Mukerji (Eds.). Handbook of Applied mycology, vol 2: humans,
animals, and insects. Marcel Dekker Inc, New York. Pp. 613-661.
Kiewnick, S., 2006. Effect of temperature on growth, germination, germ-tube extension and
survival of Paecilomyces lilacinus strain 251. Biocontrol Sci. Technol., 16: 535-546.
Klass, J.I., S. Blanford and M.B. Thomas, 2007. Development of a model for evaluating the
effects of environmental temperature and thermal behavior on biological control of
locusts and grasshoppers using pathogens. Agri. Forest Entomol., 9: 189-199.
Knowles, B.H. and J.A.T. Dow, 1993. The crystal δ-endotoxins of Bacillus thuringiensis: models
for their mechanism of action on the insect gut. BioEssays, 15: 469-476.
Kontodimas, D.C., P. Milonas, V. Vassiliou, N. Thymakis and D. Economou, 2006. The
occurrence of Rhynchophorus ferrugineus in Greece and Cyprus and the risk against the
native Greek palm tree Phoenix theophrasti. Entomol. Hellenica, 16: 11-15.
Kooyman, C, P.R.P. Bateman, J.R. Langewald, C.J. Lomers, Z. Ouambama and M.B. Thomas,
1997. Operational-scale application of entomopathogenic fungi for control of Sahelian
grasshoppers Proceedings of Royal Society of London, The Royal Soc. Printed in Great
Britain, 264: 541-546.
Koppenhöfer, A.M. and E.M. Fuzy, 2004. Effect of white grub developmental stage on
susceptibility to entomopathogenic nematodes. J. Econ. Entomol., 97: 1842-1849.
Koppenhöfer, A.M. and E.M. Fuzy, 2008. Early timing and new combinations to increase the
efficacy of neonicotinoid-entomopathogenic nematode (Rhabditida: Heterorhabditidae)
49
combinations against white grubs (Coleoptera: Scarabaeidae). Pest Manag. Sci. 64: 725-
735.
Kovács, Á.M., E. Téglás and A.D. Endress, 2010. The social sense: susceptibly to others’ beliefs
in human infants and adults. Science, 330: 1830-1834.
Krattiger, A.F., 1997. Insect resistance in crops: a case study of Bacillus thuringiensis (Bt) and
its transfer to developing countries. ISAAA Briefs No. 2. ISAAA: Ithaca, NY. Pp. 42.
Kreutzweiser, D.P., S.B. Holmes, S.S. Capell and D.C. Eichenberg, 1992. Lethal and sublethal
effects of Bacillus thuringiensis var. kurstaki on aquatic insects in laboratory bioassays
and outdoor stream channels. Bull. Environ. Contam. Toxicol., 49(2): 252-258.
Kryukov, V.Y., V.P. Khodyrev, O.N. Yaroslavtseva, A.S. Kamenova, B.A. Duisembekov and
V.V. Glupov, 2009. Synergistic action of entomopathogenic hyphomycetes and the
bacteria Bacillus thuringiensis sp. morrisoni in the infection of colorado potato beetle
Leptinotarsa decemlineata. Appl. Bioch. Microbiol., 45: 511-516.
Kubilay, M.E.R., H. Tunaz, A.A. Isikber, S. Satar, C. Mart and N. Uygun, 2008. Pathogenicity
of entomopathogenic fungi to Coccinella septempunctata L. (Col.: Coccinellidae) and a
survey of fungal diseases of coccinellids. KSU J. Sci. Eng., 11(1): 118-122.
Kumar, K., K.S. Yashaswini and N. Earanna, 2013. Molecular characterization of Lepidopteran
specific Bacillus thuringiensis strains isolated from hilly zone soils of Karnataka, India.
Afri. J. Biotech., 2(20): 2924-2931.
Kurian, C. and K. Mathen, 1971. Red palm weevil Hidden enemy of coconut palm. Ind. Farmi.,
21: 29-31.
Lacey, L.A. and D.I. Shapiro-Ilan, 2008. Microbial control of insect pests in temperate orchard
systems: Potential for incorporation into IPM. Ann. Rev. Entomol., 53: 121-144.
Lacey, L.A. and M.S. Goettel, 1995. Current developments in microbial control of insect pests
and prospects for the early 21st century. Entomophaga, 40: 3-27.
Lacey, L.A., A.A. Kirk, L. Millar, G. Mereadier and C. Vidal, 1999. Ovicidal and larvicidal
activity of conidia and blastospores of Paecilomyces fumosoroseus (Deuteromycotina:
Hyphomycetes) against Bemisia argentifolii (Homoptera: Aleyrodidae) with a description
of a bioassay system allowing prolonged survival of control insects. Biocontrol Sci.
Technol., 9: 9-18.
Lacey, L.A., R. Frutos, H.K. Kaya and P. Vail, 2001. Insect pathogens as biological control
agents: Do they have a future? Biol. Control, 21: 230-248.
Lacey, L.A. and H.K. Kaya, 2007. Field Manual of Techniques in Invertebrate Pathology:
Application and Evaluation of Pathogens for Control of Insects and Other Invertebrate
Pests. (Springer, The Netherlands).
Lawo, N.C., R.J. Mahon, R.J. Milner, B.K. Sarmah, T.J.V. Higgins and J. Romeis, 2008.
Effectiveness of Bacillus thuringiensis-transgenic chickpeas and the entomopathogenic
fungus Metarhizium anisopliae in controlling Helicoverpa armigera (Lepidoptera:
Noctuidae). Appl. Environ. Microbiol., 74(14): 4381-4389.
Lee, D.R., 1963. Date cultivation in the Coachella Valley California. The Ohio J. Sci., 63(2): 82-
87.
Lefroy, H.M., 1906. More Important Insects Injurious to Indian Agriculture, Govt. Press,
Calcutta.
Lewis, L.C., E.C. Berry, J.J. Obrycki and L.A. Bing, 1996. Aptness of insecticides (Bacillus
thuringiensis and carbofuran) with endophytic Beauveria bassiana, in suppressing the
larval populations of the European corn borer. Agri. Ecosy. Environ., 57: 27-34.
50
Lewis, R.S., N.Y. Weekes and T.H.Y. Wang, 2006. The relationship among a naturalistic
stressor, frontal asymmetry, stress, and health, Manuscript submitted for publication.
Llácer, E., M. Negre and J.A. Jacas, 2012. Evaluation of an oil dispersion formulation of
Imidacloprid against Rhynchophorus ferrugineus (Coleoptera, Curculionidae) in young
palm trees. Pest Manag. Sci., 68(6): 878-882.
Llácer, E., M.M. Martínez de Altube and J.A. Jacas, 2009. Evaluation of the efficacy of
Steinernema carpocapsae in a chitosan formulation against the red palm weevil,
Rhynchophorus ferrugineus, in Phoenix canariensis. BioControl, 54(4): 559-565.
Lord, J.C., 2009. Beauveria basssiana infection of eggs of stored-product beetles. Entomol. Res.,
39: 155-157.
MacIntosh, S.C., T.B. Stone, S.R. Sims, P.L. Hunst, J.T. Greenplate, P.G. Marrone, F.J. Perlak,
D.A. Fischhoff and R.L. Fuchs, 1990. Specificity and efficacy of purified Bacillus
thuringiensis proteins against agronomically important insects. J. Inver. Pathol., 56: 258-
266.
MacLeod, D.M., 1954. Investigations on the genera Beauveria Vuill. and Tritirachium Limber.
Canad. J. Bot., 32: 818-890.
Mahmoud, M.F., 2009. Pathogenicity of three commercial products of entomopathogenic fungi,
Beauveria bassiana, Metarhizum anisopliae and Lecanicillium lecanii against adults of
olive fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae) in the laboratory. Plant Prot.
Sci., 45(3): 98-102.
Malumphy, C. and H. Moran, 2009. Red palm Weevil, Rhynchophorus ferrugineus. Plant Pest
Factsheet. Available online at: www.fera.defra.gov.uk/plants/publications/
documents/factsheets/redPalmWeevil.pdf. Accessed on 7 June 2011.
Manachini, B., D. Schillaci and V. Arizza, 2013. Biological responses of Rhynchophorus
ferrugineus (Coleoptera: Curculionidae) to Steinernema carpocapsae (Nematoda:
Steinernematidae). J. Econ. Entomol., 106: 582-1589.
Manachini, B., P. Lo-Bue, E. Peri and S. Colazza, 2009. Potential effects of Bacillus
thuringiensis against adults and older larvae of Rhynchophorus ferrugineus. IOBC/WPRS
Bull., 45: 239-242.
Manachini, B., V. Arizza and N. Parrinello, 2008a. Sistema immunitario del Punteruolo Rosso
(Rhynchophorous ferrugineus). In: La ricerca scientifica sul punteruolo rosso e gli altri
fitofagi delle palme in Sicilia, Regione Siciliana-Assessorato Agricoltura e Foreste
Dipartimento Interventi Infrastrutturali, Servizi allo Sviluppo. Pp. 133-136.
Manachini, B., V. Arizza, D. Parrinello and N. Parrinello, 2011. Hemocytes of Rhynchophorus
ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces
cerevisiae and Bacillus thuringiensis. J. Inver. Pathol., 106(3): 360-365.
Manachini, B., V. Mansueto, V. Arizza and N. Parrinello, 2008b. Preliminary results on the
interaction between Bacillus thuringiensis and red palm weevil. In: 41st Annual Meeting
of Society for Invertebrate Pathology and 9th International Conference on Bacillus
thuringiensis, Warwick, UK. Pp. 45.
Manjula, K. and K. Padmavathamma, 1996. Effect of microbial insecticides on the control of
Maruca testulalis and on the predators of redgram pest complex. Entomon., 21: 269-271.
Marshall, J., 1931. Mohenjo-Daro and the Indus Civilization. Arthur Probsthain, London.
Matsuura, H., 1993. Weevils associated with palms. Kobe Plant Prot., 901: 46-47.
51
Mazza, G., A. Cini, R. Cervo and S. Longo, 2011b. Just phoresy? reduced lifespan in red palm
weevils Rhynchophorus ferrugineus (Coleoptera: Curculionidae) infested by the mite
Centrouropoda almerodai (Uroactiniinae: Uropodina). Ital. J. Zool., 78: 101-105.
Mazza, G., V. Arizza, D. Baracchi, G.P. Barzanti, C. Benvenuti, V. Francardi, A. Frandi, F.
Gherardi, S. Longo, B. Manachini, B. Perito, P. Rumine, D. Schillaci, S. Turillazzi and R.
Cervo, 2011a. Antimicrobial activity of the red palm weevil Rhynchophorus ferrugineus.
Bull. Insectol., 64: 33-41.
McCoy, C.W., R.A. Samson and D.G. Boucias, 1988. Entomopathogenic fungi. In: Ignoffo,
C.M. (Ed.). CRC Handbook of Natural pesticides; Vol. 5-Microbial Insecticides: Part A-
Entomopathogenic Protozoa and Fungi. CRC, Press, Boca Raton, FL. Pp.151-243.
McGaughey, W.M. and M.E. Whalon, 1992. Managing insect resistance to Bacillus thuringiensis
toxins. Science, 258: 1451-1455.
Meadows, M. P. 1993. Bacillus thuringiensis in the environment: ecology and risk assessment.
In: Entwistle, P.F., J.S. Cory, M.J. Bailey and S. Higgs (Eds.). Bacillusthuringiensis, an
Environmental Biopesticide: Theory and Practice. John Wiley and Sons, New York. Pp.
193-220.
Melifronidou-Pantelidou, A., 2009. Eradication campaign for Rhynchophorus ferrugineus in
Cyprus. Bull. OEPP., 39(2): 155-160.
Merghem, A., 2011. Susceptibility of the red palm weevil, Rhynchophorus ferrugineus (Olivier)
to the green muscardine fungus, Metarhizium anisopliae (Metsch.) in the laboratory and
in palm tree orchards. Egyp. J. Biol. Pest Control, 21: 179-183.
Merghem, A., 2011. Susceptibility of the red palm weevil, Rhynchophorus ferrugineus (Olivier)
to the green muscardine fungus, Metarhizium anisopliae (Metsch.) in the laboratory and
in palm tree orchards. Egyp. J. Biol. Pest Control, 21: 179-183.
Meyling, N.V. and J. Eilenberg, 2006. Occurrence and distribution of soil borne
entomopathogenic fungi within a single organic agroecosystem. Agri. Ecosys. Environ.,
113: 336-341.
Mietkiewski, R.T., J.K. Pell and S.J. Clark, 1997. Influence of pesticide use on the natural
occurrence of entomopathogenic fungi in arable soils in the UK: field and laboratory
comparisons. Biocontrol Sci., Tech., 7: 565-575.
Milne, D., 1918. The Date Palm and Its Cultivation in the Punjab. The Punjab Government. Pp.
153.
Milne, R. and H. Kaplan, 1993. Purification and characterization of a trypsin like digestive
enzyme from spruce budworm (Christoneura fumiferana) responsible for the activation
of δ-endotoxin from Bacillus thuringiensis. Insect Biochem. Molecul. Biol., 23: 663-673.
Milner, R.J., 1989. Ecological considerations in the use of Metarhizium for control of
soildwelling pests. In: Robertson L.N. and P.G. Allsopp (Eds.). Proceedings of a
Soilinvertebrate Workshop, Queensland Department of Primary Industries Conference
and Workshop series QC 89004, Indooroopilly, Queensland. Pp. 10-13.
Milner, R.J., 1997. Insect pathogens-how effective are they against soil insect pests? In: Allsopp,
P.G., D.J. Rogers and L.N. Robertson (Eds.). Soil Invertebrates in 1997. Proceedings of
the 3rd Brisbane Workshop on Soil Invertebrates. Bureau of Sugar Experiment Station,
Brisbane Paddington, Australia. Pp. 63-67.
Mitani, K. and J. Watari, 1916. A new method to isolate the toxin of Bacillus sotto Ishiwata by
passing through a bacerial filter and a preliminary report on the toxic action of this toxin
to the silkworm larvae. Archi. Gensanshu Serzojo Hokoku, 3: 33-42.
52
Mochizuki, M., A. Kawanishi, H. Sakamoto, S. Tashiro, R. Fujimoto and M. Ohwaki, 1993. A
calicivirus isolated from a dog with fatal diarrhoea. Veter. Record, 132: 221-222.
Mohan, L.M., 1917. Rept. Asst. Prof. Entomol; Rept. D Sagr. Punjab, for the year ended 30th
June, 1917.
Molina, A.J.P., R.I. Samuels, I.R. Machado and C. Dolinski, 2007. Interactions between isolates
of the entomopathogenic fungus Metarhizium anisopliae and the entomopathogenic
nematode Heterorhabditis bacteriophora JPM4 during infection of the sugar cane borer
Diatraea saccharalis (Lepidoptera: Pyralidae). J. Inver. Pathol., 96(2):187-92.
Mollier, P., J. Lagnel, B. Fournet, A. Aïoun and G. Riba, 1994. A Glycoprotein highly toxic for
Galleria melonela larvae secreted by the entomopathogenic fungus Beauveria
sulfurecens. J. Inver. Pathol., 64: 200-207.
Mommaerts, V., K. Jans and G. Smagghe, 2010. Impact of Bacillus thuringiensis strains on
survival, reproduction and foraging behaviour in bumblebees (Bombus terrestris). Pest
Manag. Sci., 66(5): 520-525.
Monzer, A.E. and R. Abd El-Rahman, 2003. Effect on Heterorhabditis indica of substance
occurring in decomposing palm tissues infested by Rynchophorus ferrugineus. Nematol.,
5(5): 647-657.
Monzer, M.A., 2004. Response of Heterorhabditis indica infective juveniles to host diffusates in
a modified laboratory bioassay. Egyp. J. Biol. Pest Control, 14: 309-313.
Morton, A. and F. Garcia-Del-Pino, 2011. Possible interaction of the phoretic mite
Centrouropoda almerodai on the control of Rhynchophorus ferrugineus by
entomopathogenic nematodes. IOBC-WPRS Bull., 66: 363-366.
Müller-Kögler, E., 1965. Pilzkrankheiten bei Insekten. Paul Parey, Berlin. Pp. 186.
Murphy, S.T. and B.R. Briscoe, 1999. The red palm weevil as an alien invasive: Biology and the
prospects for biological control as a component of IPM. Biocontrol New. Info., 20(1): 35-
45.
Mwamburi, L.A., M.D. Laing and R. Miller, 2009. Interaction between Beauveria bassiana and
Bacillus thuringiensis var. israelensis for the control of house fly larvae and adults in
poultry houses. Poult. Sci., 88: 2307-2314.
Nägeli, K.W., 1857. Uber die neue Krankheit der Seidenraupe und verwandte Organismen. Bot.
Z., 15: 760-761.
Namatame, I., H. Tomoda, N. Tabata, S.Y. Si, S. Omura, 1999. Structure elucidation of fungal
Beauveriolide-III, a novel inhibitor of lipid droplet formation in mouse macrophages. J.
Antibiot., 52: 7-12.
Nardi, S., E. Ricci, R. Lozzi, F. Marozzi, E. Ladurner, F. Chiabrando, L. Granchelli, E.
Verdolini, N. Isidoro and P. Riolo, 2011. Control of Rhynchophorus ferrugineus (Olivier,
1790) according to EU Decision 2007/365/EC in the Marche region (Central-Eastern
Italy) Bull. OEPP., 41(2): 103-115.
Nassar, M., and M.A. Abdllahi, 2001. Evaluation of Azadiractin fo the control of red palm
weevil, Rhynchophorus ferrugineus (Oliever) (Curculionidae: Coleoptera) J. Egypt., 6:
163-173.
Nester, E.W., L.S. Thomashow, M. Metz and M. Girdon, 2002. 100 years of Bacillus
thuringiensis: a critical scientific assessment. American Society for Microbiology,
Washington DC, USA. Pp. 1-22.
Nirula, K.K., 1956. Investigations on the pests of coconut palm. Part. Rhynchophorus
ferrugineus. Ind. Coc. J., 9: 229-247.
53
Nixon, R.W., 1951. The date palm: ‘‘Tree of Life’’ in the subtropical deserts. Econ. Bot., 5: 274-
301.
Oehlschlager, A.C., C.M. Chinchilla and L.M. Gonzalez, 1993. Optimization of a pheromone-
based trap for the American palm weevil, Rhynchophorus palmarum. In: Proceedings,
International Oil Palm Congress, 20-25 September 1993. Palm Oil Research Institute of
Malaysia, Kuala Lumpur, Malaysia.
OEPP/EPPO, 2005. Data sheets on quarantine pests Rhynchophorus palmarum. EPPO Bull., 35:
468-471.
Onofre, S.B., C.M. Miniuk, N.M. Barros and J.L. Azevedo, 2001. Pathogenicity of four strains
of entomopathogenic fungi against the bovine tick Boophilus microplus. Amer. J. Vet.
Res., 63: 1478-1480.
Oreste, M., F. De Luca, E. Fanelli, A. Troccoli and E. Tarasco, 2013. New nematodes associated
to Rhynchophorus ferrugineus (Coleoptera: Curculionidae): preliminary description.
IOBC-WPRS Bull., 90: 271.
Ortiz-Urquiza, A. and N.O. Keyhani, 2013. Action on the surface: Entomopathogenic fungi
versus the insect cuticle. Insects, 4: 357-374.
Ovchinnikov, Yu. A., A.A. Kiryushkin and I.V. Kozhevnikova, 1971. Gen. Chem. USSR., 41:
2105-2116.
Padmavathi, J., K.U. Devi and C.U.M. Rao, 2003. The optimum and tolerance PH range is
correlated to colonial morphology in isolates of the entomopathogenic fungus Beauveria
bassiana, a potential biopesticide. World J. Microbiol. Biotech., 19: 469-477.
Paoli, F., R. Dallai, M. Cristofaro, S. Arnone, V. Francardi and P.F. Roversi, 2014. Morphology
of the male reproductive system, sperm ultrastructure and cirradiation of the red palm
weevil Rhynchophorus ferrugineus Oliv. (Coleoptera: Dryophthoridae). Tissue and Cell,
46 (4): 274-285.
Pasha, S.A., A. Hussain and I.B. Gajani, 1972. Date Palm of Sindh. Punjab Fruit. J., 33(4): 9-14.
Pasteur, L., 1874. Observations (au sujet des conclusions de M. Dumas) relatives au phylloxera.
Comptes rendus hebdomadaires des séances de l’Académie des Sci., 79: 1233-1234.
Pell, J.K. and J.D. Vandenberg, 2002. Interactions among the aphid Diuraphis noxia, the
entomopathogenic fungus Paecilomyces fomosoroseus and the coccinellid Hippodamia
convergens. Biocontrol Sci. Technol., 12: 217-224.
Peng, D.H., X.H. Xu, L.F. Ruan, Z.N. Yu and M. Sun, 2010. Enhancing Cry1Ac toxicity by
expression of the Helicoverpa armigera cadherin fragment in Bacillus thuringiensis.
Research in Microbiol., 161(5): 383-389.
Pereira, E.J.G., H.A.A. Siqueira, M. Zhuang, N.P. Storer and B.D. Siegfried, 2010.
Measurements of Cry1F binding and activity of luminal gut proteases in susceptible and
Cry1F resistant Ostrinia nubilalis larvae (Lepidoptera: Crambidae). J. Inver. Pathol.,
103(1): 1-7.
Pérez, L., N. André, C. Gutleben, J. Vendeville, A.I. Lacordaire, A. Maury and E. Chapin, 2010.
Palmier, efficacité curative du nématode Steinernema carpocapsae contre le papillon
palmivore Paysandisia archon: résultats d'essais conduits dans des jardins et espaces
verts. Phytoma La Défense des Végétaux, 637: 14-17.
Pérez-García, G., R. Basurto-Ríos and J.E. Ibarra, 2010. Potential effect of a putative σHdriven
promoter on the over expression of the Cry1Ac toxin of Bacillus thuringiensis. J. Inver.
Pathol., 104(2): 140-146.
54
Peterson, A.T., J. Soberón and V. Sánchez-Cordero, 1999. Conservatism of ecological niches in
evolutionary time. Science, 285: 1265-1267.
Peveling, R. and S.A. Demba, 1997. Virulence of the entomopathogenic fungus Metarhizium
flavoviride Gams and Rozsypal and toxicity of diflubenzuron, fenitrothionesfenvalerate
and profenofos-cypermethrin to nontarget arthropods in mauritania. Archi. Environ.
Contami. Toxicol., 32: 69-79.
Plattner, R.D. and P.E. Nelson, 1994. Production of beauvencin by a stram of Fusarium
proliferatum Isolated from corn fodder for Swine. Applied and Environmental
MicrobIology, 60(10): 3894-3896.
Poinar Jr., G.O., 1990. Taxonomy and biology of Steinernematidae and Heterorhabditidae. In:
Gaugler, R. and H.K. Kaya (Eds.). Entomopathogenic Nematodes in Biological Control.
CRC Press, Boca Raton, FL. Pp. 23-61.
Poinar, Jr. G.O., 1969. Praecocilenchus rhaphidophorus n. gen., n. sp. (Nematoda:
Aphelenchoidea) parasitizing Rhynchophorus bilineatus (Montrouzier) (Coleoptera:
Curculionidae) in New Britain. J. Inver. Pathol., 1: 227-231.
Popenoe, P., 1924. ‘The date palm in antiquity’. Sci. Month., 19: 313-325.
Poprawski, T.J. and l. Majchrowicz, 1995. Effects of herbicides on in vitro vegetative growth
and sporulation of entomopathogenic fungi. Crop Prot., 14(1): 81-87.
Porter, J.R., 1973. Agostino Bassi bicentennial (1773-1973). Bacteriol. Rev., 37: 284-288.
Prabhu, S.T. and R.S. Patil, 2009. Studies on the biological aspects of red palm weevil,
Rhynchophorus ferrugineus (Oliv.), Karnataka J. Agri. Sci., 22: 732-733.
Prior, C. and M. Arura, 1985. The infectivity of Metarhizium anisopliae to two insect pests of
coconuts. J. Inver. Pathol., 45: 187-194.
Purwar, J.P. and G.C. Sachan, 2006. Synergistic effect of entomogenous fungi on some
insecticides against Bihar hairy caterpillar, Spilarctia obliqua (Lepidoptera: Arctiidae).
Microbiol. Res., 161: 38-42.
Qasim, M. and S.A. Naqvi, 2012. A fruit from heaven. In: Manickavasagan, A., M.M. Essa and
E. Sukumar (Eds.). Dates: production, processing, food, and medicinal values, CRC
Press, Taylor & Francis Group, 6000 Broken Sound Parkway NW, Suite 300, Boca
Raton. Pp. 341-349.
Quesada-Moraga, E., I. Martín-Carballo, I. Garrido-Jurado and C. Santiago-Álvarez, 2008.
Horizontal transmission of Metarhizium anisopliae among laboratory populations of
Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Biol. Control, 47: 115-124.
Quesada-Moraga, E., R. Santos-Quiros, P. Valverde-Garcia and C. Santiago-Álvarez, 2004.
Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium
anisopliae (anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). J.
Inver. Pathol., 87: 51-58.
Rahalkar, G.W., M.R. Harwalkar and H.D. Rananvare, 1972. Development of red palm weevil,
Rhynchophorus ferrugineus Oliv. on sugarcane. Ind. J. Ent., 34: 213-215.
Rajagopal, R., N. Arora, S. Sivakumar, N.G.V. Rao, S.A. Nimbalkar and R.K. Bhatnagar, 2009.
Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus thuringiensis is due
to improper processing of the protoxin. Biochem. J., 419: 309-316.
Rajamanickam K., J.S. Kennedy and A. Christopher, 1995.Certain components of integrated
management for red palm weevil, Rhynchophorus ferrugineus F. (Curculionidae:
Coleoptera) on coconut. Mededelingen Faculteit Landbouwkundige en Toegepaste
Biologische Wetenschappen Universiteit Gent., 60: 803-805.
55
Ramachandran, C.P., 1998. Biotypic variability among four populations of red palm weevil,
Rhynchophorus ferrugineus Oliv. from different parts of India. Coc. Res. Develop.
(CORD), 14: 26-41.
Ramachandran, C.P.,1991. Effect of gamma radiation on various stages of red palm weevil,
Rhynchophorus ferrugineus F. J. Nuclear Agri. and Biol., 20: 218-221.
Ramathilaga, A., A.G. Murugesan and C. Sathesh Prabu, 2012. Biolarvicidal activity of
Peanibacillus macerans and Bacillus subtilis isolated from the dead larvae against Aedes
aegypti. Vector for Chikungunya. Proc. Inter. Acad. Ecol. Environ. Sci., 2(2): 90-95.
Rao, P.N. and Y.N. Reddy, 1980. Description of a new nematode Praecocilienchus
ferruginophorus n. sp. from weevil pests (Coleoptera) of coconut palms in South India.
Rivista di Parassitologia, 44: 93-98.
Rath, A.C., 1992. Metarhizium anisopliae for control of the Tasmanian pasture scarab
Adoryphorus couloni. In: T.A. Jackson and T.R. Glare (Eds.). Use of Pathogens in Scarab
Pest Management. Intercept, Andover. Pp. 217-227.
Reddy, N.P., A.P.A Khan, U.D. Koduru, S.V. John and H.C. Sharma, 2008. Assessment of the
suitability of Tinopal as an enhancing adjuvant in formulations of the insect pathogenic
fungus Behauveria bassiana (Bals.) Vuillemin. J. Pest Manag. Sci., 3: 15-19.
Reid, T.W. and I.B. Wilson, 1971. Enzymes. 3rd Ed. 4: 373-415.
Riad, M., 2006. ‘The date palm sector in Egypt’. CIHEAM- Options Mediterraneennes. Pp. 45-
53.
Riba, G. and C. Silvy, 1989. Combattre les ravageurs des cultures enjeux et perspectives, INRA,
Paris.
Ricaño, J., B. Güerri-Agulló, M.J. Serna-Sarriás, G. Rubio-Llorca, L. Asensio, P. Barranco and
L.V. Lopez-Llorca, 2013. Evaluation of the pathogenicity of multiple isolates of
Beauveria bassiana (Hypocreales: Clavicipitaceae) on Rhynchophorus ferrugineus
(Coleoptera: Dryophthoridae) for the assessment of a solid formulation under simulated
field conditions. Florida Entomol., 96(4): 1311-1324.
Roberts, D.W. and A.S. Campbell, 1977. Stability of entomopathogenic fungi. Miscellaneous
Publications of the Entomol. Soc. Ameri., 10: 19-76.
Roberts, D.W., 1989. World picture of biological control of insects by fungi. Memoirs Institute
Oswaldo Cruz Rio de Janeiro, 84: 89-100.
Roh, J.Y., Y.S. Kim, Y. Wang, Q. Liu, X. Tao, H.G. Xu, H.J. Shim, J.Y. Choi, K.S. Lee, B.R.
Jin and Y.H. Je, 2010. Expression of Bacillus thuringiensis mosquitocidal toxin in an
antimicrobial Bacillus brevis strain. J. Asia-Pacific Entomol., 13(1): 61-64.
Ruiu, L., 2013. Brevibacillus laterosporus, a Pathogen of Invertebrates and a Broad-Spectrum
Antimicrobial Species. Insects, 4: 476-492.
Sabbour, M. and M. Abdel-Raheem, 2014. Evaluations of Isaria fumosorosea isolates against the
Red Palm Weevil Rhynchophorus ferrugineus under laboratory and field conditions.
Curr. Sci. Inter., 3(3): 179-185.
Sabbour, M.M. and A.F. Sahab, 2005. Efficacy of some microbial control agents against cabbage
pest in Egypt. Pak. J. Biol. Sci., 8(10): 1351-1356.
Sabbour, M.M. and N.Y. Solieman, 2014. Preliminary investigations into the biological control
of Red Palm Weevil Rhynchophorus ferrugineus by using three isolates of the fungus
Lecanicillium (Verticillium) lecanii in Egypt. Inter. J. Sci. Res., 3(8): 2016-2066.
56
Salama, H., M. Hamdy and M. Magd El-Din, 2002. The thermal constant for timing the
emergence of the red palm weevil, Rhynchophorus ferrugineus (Oliv.), (Coleoptera,
Curculionidae). J. Pest Sci., 75: 26-29.
Salama, H.S. and M.M. Abd-Elgawad, 2001. Isolation of heterorhabditid nematodes from palm
tree planted areas and their implications in the red palm weevil control. Anzeiger für
Schädlingskunde, 74: 43-45.
Salama, H.S. and M.M. Saker, 2002. DNA fingerprints of three different forms of red palm
weevil collected from Egyptian date palm orchards. Archi. Phytopathol. Plant Prot., 35:
299-306.
Salama, H.S., M.S. Foda, M.A. El-Bendary and A. Abdel-Razek, 2004. Infection of red palm
weevil Rhynchophorus ferrugineus, by spore-forming bacilli indigenous to its natural
habitat in Egypt. J. Pest Sci., 77: 27-31.
Salamitou, S., F. Ramisse, M. Brehelin, D. Bourguet, N. Gilois, M. Gominet, E. Hernandez and
D. Lereclus, 2000. The plcR regulon is involved in the opportunistic properties of
Bacillus thuringiensis and Bacillus cereus in mice and insects. Microbiol., 146(11): 2825-
2832.
Saleh, M.M.E. and M. Alheji, 2003. Biological control of red palm weevil with
entomopathogenic nematodes in the eastern province of Saudi Arabia. Egyp. J. Biol. Pest
Control, 13: 55-59.
Saleh, M.M.E., M.A. Alheji, M.H. Alkhazal, H. Alferdan and A. Darwish, 2011. Evaluation of
Steinernema sp. SA a native isolate from Saudi Arabia for controlling adults of the red
palm weevil. Rhynchophorus ferrugineus (Oliver). Egyp. J. Biol. Pest Control, 21: 277-
282.
Sample, B.E., L. Butler, C. Zivkovich, R.C. Whitmore and R. Reardon, 1996. Effects of
Bacillus thuringiensis Berliner var. kurstaki and defoliation by the gypsy moth
[Lymantria dispar L. (Lepidoptera: Lymantriidae)] on native arthropods in West
Virginia. Canad. Entomol., 128: 573-592.
Samson, R.A., H.C. Evans and J.P. Latg, 1988. Atlas of entomopathogenic fungi. Springer,
Berlin Heidelberg New York.
Samuels, R.I., A.K. Charnley and S.E. Reynolds, 1988. The role of destruxins in the
pathogenicity of 3 strains of Metarhizium anisopliae for the tobacco hornworm Manduca
sexta. Mycopathol., 104: 51-58.
Santhi, V.S., L. Salame, Y. Nakache, H. Koltai, V. Soroker and I. Glazer, 2015. Attraction of
entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis
bacteriophora to the red palm weevil (Rhynchophorus ferrugineus). Biol. Control, 83:
75-81.
Sayyed, A.H., B. Raymond, M.S. Ibiza-Palacios, B. Escriche and D.J. Wright, 2004. Genetic and
biochemical characterization of field-evolved resistance to Bacillus thuringiensis toxin
Cry1Ac in the diamondback moth, Plutella xylostella. Appl. Environ. Microbiol., 70(12):
7010-7017.
Schnepf, E., N. Crickmore, J.V. Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler and D.H.
Dean, 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Molecul.
Biol. Rev., 62: 775-806.
Screen, S.E. and R.J. St. Leger, 2000. Cloning, expression, and substrate specificity of a fungal
chymotrypsin. Evidence for lateral gene transfer from an actinomycete bacterium. J. Biol.
Chem., 275: 6689-6694.
57
Sewify, G.H., M.H. Belal and S.A. Al-Awash, 2009. Use of the entomopathogenic fungus,
Beauveria bassiana for the biological control of the red palm weevil, Rhynchophorus
ferrugineus Olivier. Egyp. J. Biol. Pest Control, 19(2):157-163.
Shah, P.A. and M. S. Goettel, 1999. Directory of Microbial Control Products and Services, 2 nd
edn. Division on Microbial Control. Society for Invertebrate Pathology, Division on
Microbial Control, Gainesville, USA. Pp. 81.
Shahid, M., S. Hameed, A. Imran, S. Ali and J.D. Van Elsas, 2012. Root colonization and growth
promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter
sp. Fs-11. World J. Microbiol. Biotech., 28: 2749-2758.
Shahina, F., J. Salma, G. Mehreen, M.I. Bhatti and K.A. Tabassum, 2009. Rearing of
Rhynchophorus ferrugineus in laboratory and field conditions for carrying out various
efficacy studies using EPNs. Pak. J. Nematol., 27: 219-228.
Shaiju-Simon, R.K.K. and C. Gokulapalan, 2003. Occurrence of Beauveria sp. on red palm
weevil, Rhynchophorus ferrugineus (Oliv.) of coconut. Insect Environ., 9: 66-67.
Shamseldean, M.M. and A.A. Atwa, 2004. Virulence of Egyptian steinernematid nematodes used
against the red palm weevil, Rhynchophorus ferrugineus (Oliv.). Egyp. J. Biol. Pest
Control, 14: 135-140.
Shamseldean, M.M., 2002. Laboratory trails and field applications of Egyptian and foreign
entomopathogenic nematodes used against the red palm weevil Rhynchophorus
ferrugineus Oliv., In: Proceedings of the First International Workshop on
Entomopathogenic Nematodes, Sharm ElSheikh, Egypt. Pp. 57-68.
Shapiro-Ilan, D.I., D.H. Gouge and A.M. Koppenhöfer, 2002. Factors affecting commercial
success: case studies in cotton, turf and citrus. In: Gaugler, R. (Ed.). Entomopathogenic
nematology. Wallingford: CABI Publishing. Pp. 333-355.
Sharma, H.C., M.K. Dhillon and R. Arora, 2008. Effects of Bacillus thuringiensis
deltaendotoxin-fed Helicoverpa armigera on the survival and development of the
parasitoid Campoletis chlorideae. Entomol. Experi. et Appli., 126(1): 1-8.
Sharma, P., V. Nain, S. Lakhanpaul and P.A. Kumar, 2010. Synergistic activity between Bacillus
thuringiensis Cry1Ab and Cry1Ac toxins against maize stem borer (Chilo partellus
Swinhoe). Lett. Appl. Microbiol., 51(1): 42-47.
Shawir, M.S. and A.M. Al-Jabr, 2010. The infectivity of entomopathogenic fungi Beauveria
bassiana and Metarhizium anisopliae to Rhynchophorus ferrugineus (Olivier) stages
under laboratory conditions. Proceeding of the 4th International Date Palm Conference.
Acta Horti., 882: 431-436.
Shu, C., G. Yan, R. Wang, J. Zhang, S. Feng, D. Huang and F. Song, 2009. Characterization of a
novel cry8 gene specific to Melolonthidae pests: Holotrichia oblita and Holotrichia
parallela. Appl. Microbiol. Biotech., 84(4): 701-707.
Silva-Filha, M.H., L. Regis, C. Nielsen-LeRoux and J.F. Charles, 1995. Lowlevel resistance to
Bacillus sphaericus in a field-treated population of Culex quinquefasciatus (Diptera:
Culicidae). J. Econ. Entomol., 88: 525-530.
Simberloff, D., J.L. Martin, P. Genovesi, et al., 2013. Impacts of biological invasions: what’s
what and the way forward. Trends Ecol. Evol., 28: 58-66.
Sims, S.R., 1997. Host activity spectrum of the cryIIa Bacillus thuringiensis subsp. kurstaki
protein: effects on Lepidoptera, Diptera, and non-target arthropods. Southwestern
Entomol., 22: 395-404.
58
Singh, G., B. Sachdev, N. Sharma, R. Seth and R.K. Bhatnagar, 2010. Interaction of Bacillus
thuringiensis vegetative insecticidal protein with ribosomal S2 protein triggers larvicidal
activity in Spodoptera frugiperda. Appl. Environ. Microbiol., 76(21): 7202-7209.
Sivasithamparam, K., 1998. Root cortex the final frontier for the biocontrol of root-rot with
fungal antagonists: a case study on a sterile red fungus. Ann. Rev. Phytopathol., 36: 439-
452.
Sivasupramaniam, S., G.P. Head, L. English, Y.J. Li and T.T. Vaughn, 2007. A global approach
to resistance monitoring. J. Inver. Pathol., 95: 224-226.
Soberón, M., 2005. Bacillus thuringiensis mechanisms and use. In: Comprehensive Molecular
Insect Science. Elsevier BV, Amsterdam. Pp. 175-206.
Soper, R.S., 1974. The genus massospore, entomopathogenic for cicadas, Part I, taxonomy of the
genus mycotaxon, 1: 13-40.
St. Leger, R.J., L. Joshi, M.J. Bidochka, N.W. Rizzo and D.W. Roberts, 1996. Biochemical
characterization and ultrastructural localization of two extracellular trypsins produced by
Metarhizium anisopliae in infected insect cuticles. Appl. Environ. Microbiol., 62: 1257-
1264.
St. Leger, R.J., R.M. Cooper, and A.K. Charnley, 1986. Cuticle degrading enzymes of
entomopathogenic fungi: regulation of production of chitinolytic enzymes. J. Gen.
Microbiol., 132: 1509-1517.
Starnes, R.L., C.L., Liu and P.G., Marrone, 1993. History and future of microbial insecticides.
Amer. Entomol., 38-40: 83-91.
Steinhaus, E.A., 1949. Principals of Insect Pathology. Mc Graw Hill Book co. Inc., New York.
Pp. 757.
Steinhaus, E.A., 1956. Microbial control: The emergence of an idea. Hilgardia, 26: 107-160.
Steinhaus, E.A., 1975. Disease in a Minor Cord. Ohio State Univ. Press, Columbus, OH.
Stevens, C.B., D.A. Cameron and D.L. Stenkamp, 2011. Plasticity of photoreceptor-generating
retinal progenitors revealed by prolonged retinoic acid exposure. BMC. Dev. Biol., 11:
51.
Storey, G.K. and W.A. Gardner, 1987. Vertical movement of commercially formulated
Beauveria bassiana conidia through four Georgia soil types. Environ. Entomol., 16: 178-
181.
Strand, M.R., 2008. The insect cellular immune response. Insect Sci., 15: 1-14.
Strasser, R.J., A. Srivastava and M. Tsimilli-Michael, 2000. The Fluorescence Transient As a
Tool to Characterize and Screen Photosynthetic Samples. In: Yunus, M., U. Pathre and P.
Mohanty (Eds.). Probing Photosynthesis: Mechanisms, Regulation and Adaptation.
Taylor & Francis: London. Pp. 445-483.
Sudhaus, W., K. Kiontke, K. and R.M. Giblin-Davis, 2011. Description of Caenorhabditis
angaria n. sp. (Nematoda: Rhabditidae), an associate of sugarcane and palm weevils
(Coleoptera: Curculionidae). Nematol., 13: 61-78.
Suzuki, T., S. Tominaga, S. Standgaad and T. Nakamura, 1975. Fluorescein cineangiography of
the pial microcirculation in the rat in acute angiotensin-induced hypertension. Blood
Flow and Metabolism in the Brain. In: Harper, A.M. et al. (Eds.). Churchill Livingstone,
Edinburgh, London and New York. Pp. 8-5.
Tanada, Y. and H.K. Kaya, 1993. Insect pathology. Academic Press, London. Pp. 319-385.
59
Tapia, G., M.A. Ruiz and M.M. Téllez, 2011. Recommendations for a preventive strategy to
control red palm weevil (Rhynchophorus ferrugineus, Olivier) based on the use of
insecticides and entomopathogenic nematodes. OEPP/EPPO. Bull., 41: 136-141.
Tarasco, E., F. Porcelli, M. Poliseno, E. Quesada-Moraga, C. Santiago Álvarez and O. Triggiani,
2008. Natural occurrence of entomopathogenic fungi infecting the red palm weevil
Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera, Curculionidae) in Southern
Italy. IOBC/WPRS Bull., 31: 195-197.
Tefera, T. and K. Pringle, 2003. Germination, radial growth, and sporulation of Beauveria
bassiana to Chilo partellus (Lepidoptera: Pyralidae) at different temperatures. Biocontrol
Sci. Technol., 13: 699-704.
Thakur, R. and S.S. Sandhu, 2010. Distribution, occurrence and natural invertebrate hosts of
indigenous entomopathogenic fungi of Central India. Ind. J. Microbiol., 50(1): 89-96.
Then, C., 2009. Risk assessment of toxins derived from Bacillus thuringiensis synergism,
efficacy, and selectivity. Environ. Sci. Pollut. Res., 17(3): 791-797.
Thungrabeab, M. and S. Tongma, 2007. Effect of entomopathogenic fungi, Beauveria bassiana
(Balsam) and Metarhizuim anisopliae (Metsch). KMITL Sci. Technol., 7(1):
Torta, L., V. Leone, G. Caldarella, G. Lo-Verde and S. Burruano, 2009. Microrganismi fungini
associati a Rhynchophorus ferrugineus (Olivier) in Sicilia e valutazione dell’efficacia
entomopatogena di un isolato di Beauveria bassiana (Bals.) Vuill, Ossevazioni
preliminari. Micologia Italiana, 2: 49-56.
Triggiani, O. and E. Tarasco, 2011. Evaluation of the autochthonous and commercial isolates of
Steinernematidae and Heterorhabditidae on Rhynchophorus ferrugineus. Bull. Insectol.,
64: 175-180.
Triggiani, O. and P. Cravedi, 2011. Entomopathogenic nematodes. Redia, XCIV: 119-122.
Ugine, T.A., S.P. Wraight and J.P. Sanderson, 2005. Acquisition of lethal doses of Beauveria
bassiana conidia by western flower thrips, Frankliniella occidentalis, exposed to foliar
spray residues of formulated and unformulated conidia. J. Inver. Pathol., 90: 10-23.
Van-Driesche, R.G., R.I. Carruthers, T. Center, M.S. Hoddle, J. Hough-Goldstein, L. Morin, L.
Smith, D.L. Wagner, et al. 2010.Classical biological control for the protection of natural
ecosystems. Biol. Control, 54: 2-33.
Vey, A., J.M. Quiot, I. Mazet and C.W. McCoy, 1993. Toxicity and pathology of crude broth
filtrate produced by Hirsutella thompsonii var. thompsonii in shake culture. J. Inver.
Pathol., 61:131-137.
Vey, A., R. Hoagland and T.M. Butt, 2001. Toxic metabolites of fungal biocontrol agents. In:
Butt, T.M., C. Jackson and N. Magan (Eds.). Fungal Biocontrol Agents: Progress
Problem and Potential. CABI, Wallingford. Pp. 311-346.
Vidal, C., J. Fargues and L.A. Lacey, 1997. Intraspecific variability of Paecilomyces
fumosoroseus: effect of temperature on vegetative growth. J. Inver. Pathol., 70: 18-26.
Vitale, A., V. Leone, L. Torta, S. Burruano and G. Polizzi, 2009. Prove preliminari di lotta
biologica con Beauveria bassiana e Metarhizium anisopliae nei confronti del punteruolo
rosso. In: La ricerca scientifica sul punteruolo rosso e gli altri fitofagi delle palme in
Sicilia, Vol.1. Regione Siciliana, Assessorato Agricoltura e Foreste Dipartimento
Interventi Infrastrutturali, Italy. Pp. 169-172.
Wakil, W., M.U. Ghazanfar, T. Riasat, M.A. Qayyum, S. Ahmed and M. Yasin, 2013. Effects of
interactions among Metarhizium anisopliae, Bacillus thuringiensis and
60
chlorantraniliprole on the mortality and pupation of six geographically distinct
Helicoverpa armigera field populations. Phytoparasitica, 41(2): 221-234.
Walter, C., M. Fladung and W. Boerjan, 2010. The 20-year environmental safety record of GM
trees. Nature Biotech., 28(7): 656-658.
Wattanapongsiri, A.L., 1966. Revision of the genera Rhynchophorus and Dynamis (Coleoptera:
Curculionidae),. Bulletin 1, Department of Agriculture Science, Bangkok, Thailand. Pp.
328.
Whaley, W.H., J. Anhold and G.B. Schaalje, 1998. Canyon drift and dispersion of Bacillus
thuringiensis and its effects on select nontarget Lepidopterans in Utah. Environ.
Entomol., 27: 539-548.
Whalon, M.E. and B.A. Wingerd, 2003. Bt: mode of action and use. Arch. Insect Biochem.
Physiol., 54: 200-211.
Wolfersberger, M.C., 1989. Neither barium nor calcium prevents the inhibition by Bacillus
thuringiensis S-endotoxin of sodiumor potassium-gradient-dependent amino acid
accumulation by tobacco hornworm midgut brush border membrane vesicles, Arch.
Insect Biochem. Physiol., 12: 267-277.
Wraight, S.P. and M.E. Ramos, 2005. Synergistic interaction between Beauveria bassianaand
Bacillus thuringiensis tenebrionis based biopesticides applied against field populations of
Colorado potato beetle larvae. J. Inver. Pathol., 90: 139-150.
Xu, L., Z. Wang, J. Zhang, K. He, N. Ferry and A.M.R. Gatehouse, 2010. Cross-resistance of
Cry1Ab-selected Asian corn borer to other Cry toxins. J. Appl. Entomol., 134(5): 429-
438.
Zhang, G.L., W.D. Fu and K. Liu, 2008. Agricultural invasive pest in China. Science Press,
Beijing. Pp. 172.
Zhong, C.H., D.J. Ellar, A. Bishop, C. Johnson, S.S. Lin and E.R. Hart, 2000. Characterization of
a Bacillus thuringiensis delta-endotoxin which is toxic to insects in three orders. J. Inver.
Pathol., 76(2): 131-139.
Zimmermann, G., 1993. The entomopathogenic fungus Metarhizium anisopliae and its potential
as a biocontrol agent. Pesti. Sci., 37: 375-379.
Zimmermann, G., 2007. Review on safety of the entomopathogenic fungi Beauveria bassiana
and Beauveria brongniartii. Biocontrol Sci. Tech., 17: 553-596.
61
CHAPTER 3
Genetic variation among populations of Red Palm Weevil Rhynchophorus ferrugineus
(Olivier) (Coleoptera: Curculionidae) from the Punjab and Khyber Pakhtunkhwa
provinces of Pakistan
Abstract
The red palm weevil (RPW) Rhynchophorus ferrugineus is a voracious pest of various palm
species. In recent decades its geographic range has expanded greatly, particularly impacting the
date palm industries in the countries of the Middle East. This has led to conjecture regarding the
origins of invasive RPW populations. For example, in parts of the Middle East, RPW is
commonly referred to as the “Pakistani weevil” in the belief that it originated there. We sought
evidence to support or refute this belief. The first reports of the weevil in Pakistan were from the
Punjab region in 1918, but it is unknown whether RPW is native or invasive there. We estimated
genetic variation across 5 populations of RPW from the Punjab and Khyber Pakhtunkhwa
provinces of Pakistan, using sequences of the mitochondrial cytochrome oxidase subunit I gene.
Four haplotypes were detected, of which, two (H1, H5) were abundant (accounting for >88% of
specimens) across all five sampled populations. There was no geographic overlap in the
distribution of the remaining “rare” haplotypes (H51 and H52) which were restricted to three
(Bahawalpur, Muzaffargarh and Dera Ismail Khan) and two (Dera Ghazi Khan and Layyah)
populations respectively. Levels of mitochondrial haplotype diversity reported herein were much
lower than those previously recorded in accepted parts of the native range of RPW, suggesting
that the weevil may indeed be invasive in Pakistan. In a “global” analysis, the close affinity of
Pakistani haplotypes to those reported from India (and of course the geographical proximity of
the two countries), make the latter a likely “native” source. With regards the validity of the name
“Pakistani weevil”, we found little genetic evidence to justify the name.
Key words: Rhynchophorus ferrugineus, Middle East, Pakistani weevil, Punjab, Khyber
Pakhtunkhwa
62
3.1 Introduction
The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) (Coleoptera:
Curculionidae: Rhynchophorinae) has been recognized as a major economically important pest
of palm species for more than a century. It has been found devastating > 40 different commercial
and ornamental palm tree species, belonging to 23 different genera and 3 families (Faleiro et al.
2012; Giblin-Davis et al., 2013). These include date palm Phoenix dactylifera L. (Mukhtar et al.,
2011), oil palm Elaeis guineensis (Murphy and Briscoe, 1999), coconut Cocos nucifera (Faleiro,
2006) and Canary Island date palm P. canariensis, (El-Mergawy and Al-Ajlan, 2011). The larval
stages of RPW typically reside within the trunk of an infested palm tree, destroying the vascular
system and boring into the heart of the host. Voracious feeding by these larvae may subsequently
lead to tree collapse (Ju et al., 2011). In favorable climates, the reproductive biology of RPW is
such that an infestation of just a single female has the potential to turn into about five million
weevils in just four generations (about one year) (Nirula, 1956; Rahalkar et al., 1972; Avand-
Faghih, 1996; Esteban-Durán et al., 1998; Cabello, 2006). In India, yield loses of 10-25% have
been reported in coconut plantations (Murphy and Brisco, 1999). In the Arabian Peninsula (an
area that accounts for 30% of global date production), RPW is estimated to damage up to 5% of
the date palm plantations, resulting in losses of 5-20 million US dollars (El-Sabea et al., 2009).
In Pakistan, production losses of 10-20% have been reported in different varieties of dates
(Baloch et al., 1992).
Around the world, RPW is also variously referred to as the Asiatic palm weevil, coconut
weevil, sago palm weevil, and red stripe weevil (although the actual specific identity of many
reported populations, particularly those in SE Asia, is likely to be wrong; Rugman-Jones et al.,
2013). Furthermore, because of the cryptic, internal nature of the beetle’s attack, and the
resulting slow death of the palm tree, it has also been referred to as “the hidden enemy” and even
date palm AIDS (Khamiss and Abdel-Badeea, 2013). In the Middle East, RPW is often referred
to as the “Pakistani weevil” in the belief that it invaded the former from Pakistan. However, this
is somewhat controversial nomenclature, since there is little empirical evidence supporting a
causative link (Rugman-Jones et al., 2013). Furthermore, RPW is generally considered to be
invasive in Pakistan, although it was first formerly reported in what are now the Multan,
Muzaffargarh and Dera Ghazi Khan Districts of the Pakistani province of Punjab, and the
neighboring Indian state of Punjab, almost a century ago (Lal, 1917; Milne, 1918).
Wattanapongsiri (1966) has since defined the native range of RPW as an area stretching east
from India throughout SE Asia (although its occurrence in Indonesia has recently been thrown
into doubt; Rugman-Jones et al., 2013), and in his detailed revision of the genus Rhynchophorus,
based on several extensive museum collections, he recorded only a single un-dated specimen
from modern day Pakistan. However, it remains a possibility that RPW was “always” present in
Pakistan.
The objective of this study was to characterize genetic diversity within and between
different RPW populations in Pakistan with the hope of identifying: 1) whether Pakistan forms
part of the native range of RPW; or if not, 2) the most likely origin of Pakistani RPW
populations; and finally, 3) whether there is any conclusive evidence that Middle Eastern
populations of RPW originated from Pakistan. We used sequences of the mitochondrial
cytochrome oxidase I (COI) gene to investigate genetic variation in RPW from 5 different
geographically isolated populations in the Punjab and Khyber Pakhtunkhwa (KPK) provinces of
Pakistan, and also compared these with publically available sequences from RPW populations
from around the world. If Pakistan is part of the native range, we expect to find relatively high
63
levels of diversity (i.e. a large number of haplotypes; see Rugman-Jones et al., 2013). In contrast,
relatively low levels of diversity are typical of invasive populations and by comparison with
other populations, can be used to make inferences about their potential origins.
3.2 Materials and methods
3.2.1 Specimen collections
During February and March, 2015, RPW were collected from five districts spread across
the Punjab and KPK provinces of Pakistan (Table 3.1; Figure 3.1). Live insects were collected
from infested or fallen date palm plants, with the permission of the orchard’s owners or farmers,
and permission from the Director of the Regional Agricultural Research Institute (RARI)
(Bahawalpur). A total of 80 RPW adults were collected (Table 3.1), stored collectively in plastic
jars containing 95% ethanol (one per location), and maintained at -20 °C in the Microbial
Control Laboratory, University of Agriculture, Faisalabad. At the end of March 2015, the
ethanol-preserved insects were transported to the laboratory of Dr. Richard Stouthamer
(University of California Riverside, USA [UCR]). During transport, the insects were not kept
under controlled temperatures. At UCR each weevil was transferred to an individual plastic vial,
labelled accordingly, and kept in the freezer at -20 °C until processing.
3.2.2 DNA extraction and amplification
Each specimen was extracted using the protocol detailed in Rugman-Jones et al. (2013).
Specifically, a small piece (2-5 mm3) of muscle tissue was dissected from a single tibia using
flame-sterilized scissors and forceps, and allowed to air dry for 1 min on sterile filter paper. The
tissue was then transferred to a sterile 0.6 ml microcentrifuge tube and ground up in 6 µl
proteinase-K (>600mAU/mL; Qiagen, Valencia, CA) using a glass pestle. To this was added 120
µl of a 5% (w/v) suspension of Chelex® 100 resin (Bio-Rad Laboratories, Hercules, CA) and the
reaction was incubated at 55 °C for 1 h followed by 10 min at 99 °C. After this the tubes were
centrifuged for 4 minutes at 14000 rpm to pellet the Chelex. Subsequently, 80 µl of supernatant
was carefully transferred to a new eppendorf tube.
The polymerase chain reaction (PCR) was used to amplify a section of the mitochondrial
gene (mtDNA) cytochrome oxidase subunit 1 (COI) from each specimen. PCR was performed in
25 µl reactions containing 2 µl of DNA template (concentration not determined), ddH2O,1X
ThermoPol PCR Buffer (New England BioLabs, Ipswich, MA), an additional 1 mM MgCl2, 400
µM dUTP, 200 µM each dATP, dCTP and dGTP, 10 µg BSA (NEB), 1 U Taq polymerase
(NEB), and 0.2 µM of each PCR primer. Initial reactions utilized the primers C1-J-1718 and C1-
N-2329 (Simon et al., 1994). Reactions were performed in a Mastercycler® ep gradient S
thermocycler (Eppendorf North America Inc., New York, NY) programmed for an initial
denaturing step of 2 min at 94 °C; followed by five cycles of 30 s at 94 °C, 1 min 30 s at 45 °C,
and 1 min at 72 °C; followed by a further 35 cycles of 30 s at 94 °C, 1 min 30 s at 51 °C, and 1
min at 72°C; and, a final extension of 5 min at 72 °C. Amplification was verified by standard
agarose gel electrophoresis, and samples that failed to amplify were subject to two further
attempted amplifications, this time using the primer sets SIMON and BRON (El-Mergawy et al.,
2011) or BRON and C1-N-2329 (see Rugman-Jones et al., 2013). The integrity of the DNA
extracted from any specimen that still failed to yield a COI amplicon was tested in a 4 th PCR, this
time targeting the 28S rRNA region with the primers (28sF3633 and 28sR4076) and protocol
detailed in Rugman-Jones et al. (2013).
64
3.2.3 Cleaning and sequencing
PCR products were purified using ExoSAP-IT® (Affymetrix, Santa Clara, CA), and
sequenced in both directions at the Institute for Integrative Genome Biology core instrumentation
facility (UCR). Sequences were aligned manually and trimmed to 528 bp (removing the primers
and ambiguous tails) using BioEdit version 7.0.9.0 (Hall, 1999), and then translated using the
EMBOSS-Transeq website (http://www.ebi.ac.uk/Tools/emboss/transeq/index.html) to confirm
the absence of nuclear pseudogenes (Song et al., 2008). All sequences were deposited in
GenBank (Benson et al., 2008) (accession numbers KU696489-KU696537).
3.2.4 Genetic analysis
Sequences of the COI gene generated in this study were collapsed into haplotypes, and
the number and nature of polymorphic sites was characterized, using DnaSP v5.10.01 (Librado
and Rozas, 2009). Genetic variation within each Pakistani population (Table 3.2), was
characterized by calculating the number of COI haplotypes, haplotype diversity (Hd; the
probability that two randomly sampled haplotypes are different), and the average number of
nucleotide differences in pairwise comparisons among COI sequences (k) using DnaSp. Since
most estimators of population differentiation can be highly unreliable when using a single locus
and relatively small sample sizes, genetic variation between topographical populations was also
investigated simply by obtaining population-pairwise estimates of k, again in DnaSP. In order to
put variation within Pakistan into a global context, we then combined our sequences with those
from three earlier studies (El-Mergawy et al., 2011; Rugman-Jones et al., 2013; Wang et al.,
2015; GenBank accessions GU581319-GU581628, KF311358-KF311740, KF413063-
KF413073, respectively). All sequences were trimmed to a uniform length, resulting in a matrix
of 539 sequences, each 528bp long. Sequences were again collapsed into haplotypes using
DnaSP v5.10.01 and a “global” haplotype network was constructed using the statistical
parsimony method of Templeton et al. (1992) in the software program TCS, version 1.21
(Clement et al., 2000).
3.3 Results
Using various combinations of four PCR primers, sequences of the COI gene were
successfully obtained from 50 of our 80 RPW specimens collected from the Punjab and KPK
provinces of Pakistan. For the remaining 30 “failed” specimens, attempts to amplify the highly
conserved 28S rRNA also failed, suggesting that our extractions from those specimens had
yielded no amplifiable DNA. Among the 50 specimens successfully sequenced, we found four
haplotypes. The four haplotypes were very closely related, with only three polymorphic
nucleotides (positions 63, 156, and 174) (see GenBank accessions). All substitutions were
synonymous. Just two haplotypes accounted for the majority (88%) of the specimens and
corresponded to haplotypes H1 (n=20) and H5 (n=24) previously encountered by El-Mergawy et
al. (2011) and Rugman-Jones et al. (2013). These haplotypes were common in all five Pakistani
populations (Table 3.2; Figure 3.2). The remaining two haplotypes had restricted, and non-
overlapping distributions, being found in three and two populations, and our “global” haplotype
analysis revealed that neither had been encountered in the earlier studies of El-Mergawy et al.
(2011), Rugman-Jones et al. (2013), or Wang et al. (2015). Hereafter they are referred to as H51
(n=4) and H52 (n=2), respectively.
Given the very similar nature of the four Pakistani haplotypes, and the prevalence of just
two of those haplotypes across the five Pakistani RPW populations, estimates of genetic
65
variation within and between populations were very low (k was <1 in all comparisons; Table
3.3). In global terms, the haplotypes detected in our Pakistani samples were most similar to
native haplotypes from other parts of the Indian sub-continent; haplotypes H9-16 from Rugman-
Jones et al. (2013) (Figure 3.3). Outside of the native range, the most common Pakistani
haplotypes (H1 and H5) were also invasive in the United Arab Emirates, Oman, and Syria
(Figure 3).
3.4 Discussion
In the Middle East, RPW is often referred to as the “Pakistani weevil” in the belief that it
invaded from Pakistan. However, strong empirical evidence to justify this belief has not been
forthcoming (e.g., Rugman-Jones et al., 2013). Furthermore, there is some doubt as to the actual
status (invasive or native) of RPW in Pakistan, although it has typically been considered an
invasive pest there. In this study we found four haplotypes across 50 specimens, from five
sampled populations located in the Pakistani provinces of Punjab and KPK. Of these haplotypes,
two (H1 and H5) were common in all the populations sampled. The other two were relatively
rare, and non-overlapping, with one (H51; Fig 3.3) represented by only four specimens (two
from Bahawalpur and one form Muzaffargarh and DI Khan), and the other (H52; Fig 3.3) by two
specimens (one from Layyah and another from DG Khan). In global terms, the four Pakistani
haplotypes were very similar to native haplotypes in the remainder of the Indian sub-continent
(Fig 3.3), and H1 and H5 were also common in invasive populations in parts of the Middle East
(UAE, Oman, and Syria).
Is the red palm weevil native to Pakistan?
Low levels of genetic diversity are atypical of RPW populations across its described
native range, but very characteristic of invasive populations of this species around the globe (see
Rugman-Jones et al., 2013). Levels of genetic diversity detected in our study lay somewhere
between the two extremes. If we consider RPW to be an invasive, this suggests that the RPW
populations in the Punjab and KPK provinces of Pakistan have resulted from: a) the influx of a
large number of weevils during a single invasion event; and/or b) multiple invasions from one or
more sources. The genetic similarity between our Pakistani haplotypes and those previously
reported from the Indian state of Goa suggests that India would have been the most likely source
of any invasion. This is also the most likely scenario in a biogeographical sense. Commercial
cultivation of dates in India is focused largely in the western states of Gujarat, Rajasthan and
Punjab, adjacent to the border with Pakistan, and (to a much lesser extent) the southernmost
states of Tamil Nadu and Kerala.
RPW is a strong flyer and it is easy to imagine that Pakistan may have been invaded by
weevils from one or more of these Indian states. However, convincing support for such a
hypothesis will require a much bigger sample from India, which sadly remains a genetic “black
hole” since the country does not allow researchers to collect and export specimens, due to Indian
claims of intellectual property rights over genetic resources. In contrast to the invasive
“argument”, our results could also be interpreted as evidence that the Punjab and KPK provinces
of Pakistan actually fall within the native range of the weevil. At least three known hosts of RPW
are native to Pakistan [Nannorhops ritchiana, Phoenix loureirii and P. sylvestris (Champion et
al., 1960; Mughal, 1992; Malumphy and Moran, 2007; Malik, 2015), and the date palm Phoenix
dactylifera has been cultivated in the Sindh province of Pakistan for more than a thousand years
(http://edu.par.com.pk/wiki/dates/).
66
Furthermore, the presence of RPW in the Punjab province of Pakistan, and what is now
the neighboring Indian state of Punjab, was first documented almost a century ago (Lal, 1917;
Milne, 1918). Therefore, we must consider the possibility that small populations of RPW have
always been present in Pakistani Punjab, but have simply gone ignored, or unnoticed, because of
the relative isolation of the region, and/or because their economic impact was (at that time) not
significant. In light of this information, it is perhaps surprising that Wattanapongsiri (1966), in
his revision of the genus Rhynchophorus, considered only a single R. ferrugineus specimen from
anywhere in Pakistan (a specimen from the Kalat District of the modern Balochistan province,
held in the Bavarian State Collection of Zoology, Munich). Prior to the Partition of India in
1947, the two “Punjabs” were considered a single province under the governance of the British
Raj, and British collectors described vast numbers of insects from the entire Indian sub-continent
(including Pakistan), depositing the bulk of their specimens at the British Museum of Natural
History, London. Had RPW been abundant at that time, it seems unlikely that such a conspicuous
insect would have escaped collection. However, despite having access to the BMNH collections
(among many others), Wattanapongsiri (1966) included only the single “Balochistan” specimen,
in his work. Unfortunately, that specimen was without a collection date, and so sheds little
further light on the history of RPW in Pakistan.
Whether native or invasive, RPW has certainly been present in Pakistan for some time.
The recent “rise” of RPW in the Punjab and KPK provinces has likely been exacerbated by
anthropogenic movement of date palm germplasm from the neighboring provinces of Sindh and
Baluchistan where date palms have been cultivated for centuries, and/or the rise of date
cultivation in neighboring Indian states. Again, this is difficult to substantiate without sampling
of those areas, and that should be a priority for further genetic work.
The Pakistani weevil?
It has been claimed by some Middle Eastern countries that RPW originally crossed into
Arabia in ornamental plants imported from Pakistan in 1985 (Dawn News 2003). While our data
cannot completely refute this hypothesis it cannot fully support it either. Although both of the
abundant Pakistani haplotypes detected in our study have been recorded in the Middle Eastern
countries of UAE and Oman (and H1 only also in Syria), a third haplotype H8, has not been
detected in Pakistan, but was found to be widespread in Saudi Arabia (El Mergawy et al. 2011;
Rugman-Jones et al., 2013). Indeed, El Mergawy et al. (2011) detected three further haplotypes
from Oman and UAE. If the Middle East was invaded solely by RPW from Pakistan it is hard to
explain why there are additional haplotypes in the Middle East that have not been detected in
Pakistan.
One answer, originally put forward by Rugman-Jones et al. (2013), is that whether or not
the Middle East has been invaded from Pakistan, it has also been invaded from somewhere else
in the native range of RPW (most likely Thailand). It should also be noted that RPW-like
damage was recorded in Iraq around the same time RPW was first recorded in the Punjab,
although no specimens were collected to confirm this (Buxton, 1920). There is currently no
genetic data available for Iraqi populations of RPW, but given its relative proximity to the
Middle East, it is possible that the latter (and indeed Pakistan) were invaded by RPW from Iraq.
Intensive sampling of Iraq should be a priority for future genetic work.
67
Conclusions The present study showed that the red palm weevil is native to Pakistan and had been
present in Pakistan for a long time. It may have been invaded from India and Sri Lanka. The
population present in KSA is called the "Pakistani Weevil’’ but in my study I have indicated that
this weevil might have been invaded from Thialand or Vietnam instead of Pakistan. In Pakistan
four different groups of haplotypes are present which are commonly found in al the collection
areas of the country. Only the haplotype H52 was the rare haplotype that was only present in the
populations collected from Layyah and Dera Ghazi Khan districts of Punjab.
Acknowledgements
This research work was supported by the scholarship from Higher Education
Commission (HEC), Islamabad, Pakistan (112-30536-2AV1-263) under Indigenous Ph.D.
Fellowship Program.
68
3.5 References
Baloch, H.B., M.A. Rustamani, R.D. Khuhro, M.A. Talpur and T. Hussain, 1992. Incidence and
abundance of date palm weevil in different cultivars of date palm. Proc. Pak. Cong. Zool.,
12: 445-447.
Benson, D.A., I. Karsch-Mizrachi, D.J. Lipman, J. Ostell and D.L. Wheeler, 2008. GenBank.
Nucleic Acids Res., 36: 25-30.
Buxton, P.A., 1920. Insect pests of dates and the date palm in Mesopotamia and elsewhere. Bull.
Entomol. Res., 11: 287-304.
Champion, H.G., S.K. KSeth and G.M. Khattak, 1960. Manual of silviculture for Pakistan.
Clement, M., D. Posada and K.A. Crandall, 2000. TCS: a computer program to estimate gene
genealogies. Mol. Ecol., 9: 1657-1660.
DAWN News, 2003. Insect pests ravage red date palm trees. Available at:
http://www.dawn.com/news/89129/insect-pests-ravage-red-date-palm-trees.
El-Mergawy, R.A.A.M. and A.M. Al-Ajlan, 2011. Red palm weevil, Rhynchophorus ferrugineus
(Olivier): economic importance, biology, biogeography and integrated pest management.
J. Agric. Sci. Tech., 1: 1-23.
El-Mergawy, R.A.A.M., M.I. Nasr, N. Abdallah and J.F. Silvain, 2011. Mitochondrial genetic
variation and invasion history of red palm weevil, Rhynchophorus ferrugineus
(Coleoptera: Curculionidae), in Middle-East and Mediterranean Basin. Inter. J. Agric.
Biol., 13: 631-637.
El-Sabea, A.M.R., J.R. Faleiro and M.M. Abo-El-Saad, 2009. The threat of red palm weevil
Rhynchophorus ferrugineus to date plantations of the Gulf region in the Middle-East: an
economic perspective. Outlooks on Pest Manag., 20: 131-134.
Esteban-Durán, J., J.L. Yela, F. Beitia-Crespo and A. Jiménez-Alvarez, 1998. Biology of red
palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae:
Rhynchophorinae), in the laboratory and field, life cycle, biological characteristics in its
zone of introduction in Spain, biological method of detection and possible control.
Boletin de Sanidad Vegetal Plagas, 24: 737-748.
Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil
Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm
during the last one hundred years. Inter. J. Trop. Insect Sci., 26: 135-154.
Faleiro, J.R., A. Ben Abdullah, M. El-Bellaj, A.M. Al-Ajlan and A. Oihabi, 2012. Threat of red
palm weevil, Rhynchophorus ferrugineus (Olivier) to date palm plantations in North
Africa. Arab J. Plant Prot., 30: 274-280.
Giblin-Davis, R.M., J.R. Faleiro, J.A. Jacas, J.E. Peña and P.S.P.V. Vidyasagar, 2013.
Coleoptera: Biology and management of the red palm weevil, Rhynchophorus
ferrugineus pp. 1-34 In: J.E. Peña (Ed.). Potential Invasive Pests of Agricultural Crop
Species. CABI. Wallingford, UK.
Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis
program for Windows 95/98/NT. Nucleic Acids Sym. Ser., 41: 95-98.
Ju, R.T., F. Wang, F.H. Wan and B. Li, 2011. Effect of host plants on development and
reproduction of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). J. Pest
Sci., 84: 33-39.
Khamiss, O. and A. Abdel Badeea, 2013. Initiation, characterization and karyotyping of a new
cell line from red palm weevil Rhynchophorus ferrugineus adapted at 27 °C in AFPP -
palm pest Mediterranean conference 16, 17 and 18 January 2013, NICE.
69
Lal, M.M., 1917. Report of assistant professor of entomology, department of agriculture Punjab
for year ended 30th June 1917.
Librado, P. and J. Rozas, 2009. DnaSP v5: A software for comprehensive analysis of DNA
polymorphism data. Bioinformatics, 25: 1451-1452.
Malik, K.A., 2015. Flora of Pakistan. Available at:
http://www.efloras.org/florataxon.aspx?flora_id=5&taxon_id=20427.
Malumphy, C. and H. Moran, 2007. Red palm weevil Rhynchophorus ferrugineus. Plant Pest
Notice, Central Sci. Lab., 50: 1-3.
Milne, D., 1918. The date palm and its cultivation in the Punjab. The Punjab government,
Lyallpur. Pp. 153.
Mughal, M.S., 1992. Spotlight on species: Nannorrhops ritchieana. Pak. J. For., 42: 162-166.
Mukhtar, M., K.G. Rasool, M.P. Parrella, Q.I. Sheikh, A. Pain, L.V. Lopez-Llorca, Y.N.
Aldryhim, R.W. Mankin and A.S. Aldawood, 2011. New initiatives for management of
red palm weevil threats to historical Arabian data palms. Florida Entomol., 94: 733-736.
Murphy, S.T. and B.R. Briscoe, 1999. The red palm weevil as an alien invasive: biology and the
prospects for biological control as a component of IPM. Biocontrol News and Info., 20:
35-46.
Nirula, K.K., 1956. Investigations on the pests of coconut palm. Part-IV. Rhynchophorus
ferrugineus. Ind. Coc. J., 9: 229-247.
Rahalkar, G.W., M.R. Harwalkar and H.D. Rananavare, 1972. Development of red palm weevil
Rhynchophorus ferrugineus (Oliv.) on sugarcane. Ind. J. Entomol., 34: 213-215.
Rugman-Jones, P.F., C.D. Hoddle, M.S. Hoddle and R. Stouthamer, 2013. The lesser of two
weevils: molecular-genetics of pest palm weevil populations confirm Rhynchophorus
vulneraturs (Panzer 1798) as a valid species distinct from R. ferrugineus (Olivier 1790),
and reveal the global extent of both. PLoS ONE, 8: 1-15.
Simon, C., F. Frait, A. Bechenback, B. Crespi, H. Liu and P.K. Flook, 1994. Evolution,
weighting and phylogentic utility of mitochondrial gene sequence and a compilation of
conserved polymerase chain reaction primers. Ann. Entomol. Soc. Amer., 87: 651-701.
Song, H., J.E. Buhay, M.F. Whiting and K.A. Crandall, 2008. Many species in one: DNA
barcoding overestimates the number of species when nuclear mitochondrial pseudogenes
are coamplified. Proc. Nati. Acad. Sci. USA., 105: 13486-13491.
Tamura, K., G. Stecher, D. Peterson, A. Filipski, and S. Kumar, 2013. MEGA6: Molecular
Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol., 30: 2725-2729.
Templeton, A.R., K.A. Crandall and C.F. Sing, 1992. A cladistic analysis of phenotypic
associations with haplotypes inferred from restriction endonuclease mapping and DNA
sequence data III. Cladogram estimation. Genetics, 132: 619-633.
Wang, G., X. Zhang, Y. Hou and B. Tang, 2015. Analysis of the population genetic structure of
Rhynchophorus ferrugineus in Fujian, China, revealed by microsatellite loci and
mitochondrial COI sequences. Entomol. Exp. et Appli., 155: 28-38.
Wattanapongsiri, A., 1966. A revision of the genera Rhynchophorus and Dynamis (Coleoptera:
Curculionidae). Dept. Agric. Sci. Bull., 1: 1-328.
70
Table 3.1 Sampling information for RPW populations collected from date palm Phoenix
dactylifera in Punjab and KPK provinces of Pakistan
Population Location Collection date
No. of specimens
Province Geographical Characteristic
Alt. (m) Lat. Long.
LY Layyah 27-Feb-2015 8 Punjab 143 30°58'N 70°56'E
BWP Bahawalpur 14-Mar-2015 17 Punjab 252 29°59′N 73°15′E
DGK D.G. Khan 10-Feb-2015 21 Punjab 150 29°57'N 70° 29'E
MG Muzaffargarh 8-Mar-2015 18 Punjab 114 30°50'N 71°54'E
DIK D.I. Khan 1-Feb-2015 16 KPK 166 31°49'N 70°52'E
Table 3.2 Genetic characterization of five RPW populations from the Punjab and KPK
provinces of Pakistan based on a 528 bp section of the mitochondrial COI gene.
For population abbreviations, see Table 3.1.
Population N No. of haplotypes Haplotypes Haplotype diversity (Hd)
LY 9 3 H1, H5, H52 0.556
BWP 11 3 H1, H5, H51 0.691
DGK 8 3 H1, H5, H52 0.464
MG 15 3 H1, H5, H51 0.590
DIK 7 3 H1, H5, H51 0.668
Table 3.3 Variation in a 528 bp segment of the cytochrome c oxidase subunit I (COI) region
of mitochondrial DNA (mtDNA) of Rhynchophorus ferrugineus. Average number
of pairwise nucleotide differences (k) within (diagonal element) and between
(below diagonal) populations in the Punjab and KPK provinces of Pakistan. For
population abbreviations, see Table 3.1.
LY BWP DGK MG DIK
LY 0.722 - - - -
BWP 0.747 0.836 - - -
DGK 0.833 0.909 0.500 - -
MG 0.689 0.733 0.667 0.667 -
DIK 0.778 0.792 0.714 0.686 0.857
71
Figure 3.1 Map of collection sites in Punjab and KPK provinces of Pakistan.
72
Figure 3.2 Distribution of mitochondrial haplotypes across five populations of RPW from the
Punjab and KPK provinces of Pakistan.
73
Figure 3.3 Relationships between four Pakistani COI haplotypes and 48 others occurring
around the world. Haplotype network constructed from 539 COI sequences (each
528 bp long) generated by the present study and three earlier studies (see text).
Each haplotype is represented by an oval or for that with the highest outgroup
probability, a rectangle. Size of each haplotype is indicative of the number of
specimens sharing that haplotype; also given inside each haplotype. H1-43 are
numbered according to El Mergawy et al. (2011) and Rugman-Jones et al. (2013);
H44-50 correspond to additional haplotypes from Wang et al. (2015); and H51-52
are new to this study.
74
CHAPTER 4
Resistance to commonly used insecticides and phosphine (PH3) against Rhynchophorus
ferrugineus (Olivier) (Coleoptera: Curculionidae) in Punjab and Khyber Pakhtunkhwa,
Pakistan
Abstract In the first ever survey of insecticide resistance in field populations of Red Palm Weevil
Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) in Pakistan were collected
from seven date palm growing areas across Punjab and Khyber Pakhtunkhwa (KPK), Pakistan,
and assessed by the diet incorporation method against the formulated commonly used chemical
insecticides profenophos, imidacloprid, chlorpyrifos, cypermethrin, deltamethrin, spinosad,
lambda-cyhalothrin and a fumigant phosphine (or hydrogen phosphide) (PH3). Currently, there is
no IRAC approved bioassay method for R. ferrugineus, so this study aimed to develop a suitable
susceptibility test. Elevated levels of resistance were recorded for cypermethrin, deltamethrin
and PH3 in R. ferrugineus after a long history of use in Pakistan. Resistance Ratios (RRs)
documented for PH3 were 63- to 79-fold for cypermethrin 16- to 74-fold for deltamethrin 13- to
58-fold for profenophos 2.6- to 44-fold for chlorpyrifos 3- to 24-fold for lambda-cyhalothrin 2-
to 12-fold and for Spinosad 1- to 10-fold as compared to the control. Resistant populations of R.
ferrugineus mainly belonged to southern Punjab and to some extent from the KPK populations.
The populations from Bahawalpur, Vehari, Layyah and Dera Ghazi Khan were found most
resistant to chemical insecticides, while all populations exhibited high levels of resistant to
phosphine. Of the eight agents tested, lower LC50 and LC90 values were recorded for spinosad
and lambda-cyhalothrin. Resistance levels were very low to low against imidacloprid, very low
to moderate against profenophos and chlorpyrifos, low to high against cypermethrin and
deltamethrin and high against phosphine. These results suggest that spinosad and lambda-
cyhalothrin exhibit unique modes of action and given their better environmental profile could be
used in insecticide rotation or assist in discarding the use of older insecticides.
Keywords: Rhynchophorus ferrugineus, insecticide resistance, profenophos, imidacloprid,
chlorpyrifos, cypermethrin, deltamethrin, spinosad, lambda-cyhalothrin,
phosphine
75
4.1 Introduction
The Red Palm Weevil’s (RPW), Rhynchophorus ferrugineus (Olivier) (Col.,
Curculionidae) invasive potential is a consequence of the elevated female fecundity (Faleiro,
2006), the ability to complete several generations in a year even in the same tree (Rajamanickam
et al., 1995; Avand Faghih, 1996). It is one of the most destructive pests of ornamental and
economically important palms, which is currently present in 50% of date growing countries and
15% coconut producing countries of the world. The weevil is concealed in nature and all their
life stages remain inside the tree, usually found up to 1m in the tree trunk (Azam et al., 2001).
The beetles quite often interbreed and reproduce within the same plant and continue devastating
their host to death.
The aboriginal home of this pest is South and Southeast Asia and Melanesia where it has
been found destructing coconut palms (Lefroy, 1906; Brand, 1917; Viado and Bigornia, 1949;
Nirula, 1956). Lately in 1918 the beetle was found inflicting date palm in India, during the same
year weevil was found from some southern districts of Punjab, Pakistan (Multan, Muzaffargarh
and Dera Ghazi Khan) (Milne, 1918). Two year latter Buxton (1920) reported this pest on date
palm plantation from Mesopotamia (Iraq). It was only during the mid-1980s that RPW attained a
major pest status on date palms, in the Middle Eastern region (Abraham et al., 1998).
Subsequently, the weevil moved from North Africa into Europe, where it was reported for the
first time in the South of Spain (Cox, 1993; Barranco et al., 1995). So far pest has been
distributed to many areas worldwide; its range now includes much of Asia, regions of Oceania,
the Middle-East and North Africa, southern Europe, the Caribbean, and most recently it has been
found in southern California in 2010 from the canary Island.
To combat this voracious pest synthetic pesticide remained the mainstay since decades
but this offers a challenge due to the cryptic nature of the pest, moreover insecticidal treatments
with fumigants, soil treatments with insecticides, frond axil filling, trunk injections, wound
dressing and crown drenching remains the main strategy for R. ferrugineus control (Hussain et
al., 2013). Different scientists evaluated insecticidal potential of various chemical insecticides
against this pest successfully through different application methods and their combinations
(Cabello et al., 1997; Azam et al., 2000; Ajlann et al., 2000; Khalifa et al., 2001; AboEl-Saad et
al., 2001; Abdul-salam et al., 2001; Al-Rajhy et al., 2005; Kaakeh, 2006; Llácer and Jacas,
2010).
In Pakistan, the application of insecticides on date palms has an ancient history to combat
R. ferrugineus infestation because more than hundred year history of R. ferrugineus presence, but
their published records was not available. Shar et al. (2012) evaluated ten different insecticides
against R. ferrugineus under field conditions and found fipronil, spirotetramat, chlorpyrifos and
methidathion the most effective among all the tested insecticides. Al-Jabr et al. (2013) tested ten
different chemical insecticides against R. ferrugineus midgut cell line, emamectin benzoate was a
highly potent that significantly reduce the growth inhibition and increased the mortality. In
Pakistan effectiveness of commercially used insecticides has complained by the date palm
grower, reduced effectiveness may be because of the development of resistance. Although,
resistance mechanism in R. ferrugineus against commonly used insecticides is poorly
understood. The limited exploration of resistance mechanism in R. ferrugineus is focused in the
design of the current investigation. We selected seven commonly used insecticides and
phosphine (PH3), and checked their effectiveness against different distinct populations of R.
ferrugineus collected from seven different areas of the Pakistan.
76
4.2 Materials and methods
4.2.1 RPW collection and rearing
Different developmental stages of R. ferrugineus were collected from fallen and infested
date palm trees from various areas of Punjab and KPK during 2014-2015 (Table 1). The areas
were selected on the basis of ancient history of date palm cultivation and long term use of
insecticides and phosphine. During collection adults, larvae and pupae were kept in separate
plastic jars for each location until brought to the laboratory. In the laboratory larvae were
provided with sugarcane (Saccharum officinarum L.; Poales: Poaceae) stems for feeding and
pupation, while adults were offered shredded sugarcane pieces for both feeding and as an
oviposition substrate. Pupae were kept in separate boxes for adult emergence in incubators
(Sanyo Corporation Japan) at 27±2 oC, 60±5 RH and photoperiod of 12: 12 (D: L) hours. On
emergence adults were transferred to jars for feeding and mating. The colonies were maintained
in plastic boxes (15×30×30 cm) having a lid whose center (8 cm diameter covered with mesh
wire gauze (60 mesh) for aeration. Rearing was carried out in IPM laboratory, Department of
Entomology, University of Agriculture, Faisalabad, Pakistan. The adult food was changed after
every three days and replaced sugarcane pieces were kept in separate jars for egg hatch. After
egg hatching neonate larvae were transferred to sugarcane pieces for feeding until freshly molted
4th instar larvae were recovered. The laboratory strain was used as reference strain which was
reared in the IPM laboratory since year 2009. The strain was maintained for more than 25
generations in the laboratory without any insecticidal exposure before these tests commenced.
Preliminary laboratory bioassays showed high susceptibly of tested insecticides, being as
susceptible as used by Ahmad et al. (2003).
4.2.2 Test chemicals
For bioassay, commercial formulations of Curacron® (profenophos, 500 g/liter, 500 EC;
Syngenta Pakistan Ltd., Karachi, Pakistan); Confidor® (jmidacloprid, 700g/kg, 70 WG; Bayer
Crop Sciences, Pakistan Pvt. Ltd., Karachi, Pakistan); Lorsban® (chlorpyrifos, 400 g/liter, 40 EC;
Arysta Life Science Pakistan Pvt. Ltd., Karachi, Pakistan); Arrivo® (cypermethrin, 100 g/liter,
10% EC; FMC United Pvt. Ltd., Lahore, Pakistan), Deltamethrin® (deltamethrin, 25 g/ liter,
2.5% EC; Target Agro Chemicals, Lahore, Pakistan); Tracer® (spinosad, 240 g/liter, 240 SC;
Arysta LifeScience, Pakistan Pvt. Ltd.), Karate® (lambda-cyhalothrin 50g/liter, 5 EC; Syngenta
Pakistan Ltd., Karachi, Pakistan). Phosphine (PH3) was generated by using aluminum phosphide
(Celphos 56%; Jaffer Brothers (PVT) Ltd., Lahore, Pakistan) tablets.
4.2.3 Generation of phosphine gas
The PH3 gas was generated using the FAO method (Anonymous, 1975). The apparatus
for generation of PH3 gas consisted of a 5 liter beaker, a collection tube (cylinder), an inverted
funnel, Aluminum phosphide tablets and muslin cloth. The tube for collection of gas was sealed
from one side with an air-tight rubber stopper and then was filled with 5% sulphuric acid
(H2SO4) solution. Half of the beaker was also filled with 5% H2SO4 solution. The gas collecting
tube was placed carefully into the beaker over the inverted funnel in such a way that there is no
loss of H2SO4 solution from the collection tube, while dipping into the beaker. Before generating
PH3 gas all air in collection tube was removed within collection tube. Then aluminum phosphide
tablets (wrapped in muslin cloth) were placed under inverted funnel. PH3 gas was then collected
in the gas collecting tube inverted over the funnel. As the funnel filled with generated gas, the
77
level of solution went down. When the collecting tube was filled, 5 ml gas were sucked out with
the help of an air tight syringe and was injected into sealed desiccators of known volume, then 50
ml of gas was taken out from the desiccators and injected into a Phosphine meter to measure gas
concentration allowing the required concentrations of PH3 gas to be obtained.
4.2.4 Bioassay
From each population freshly molted F1 fourth-instar (L4) larvae of R. ferrugineus were
challenged with the test insecticides. The F1 generation was obtained by mass mating of the field
collected beetles and the beetles emerged from field-collected larvae and pupae. Toxicity
bioassays were performed using artificial diet (Martín and Cabello, 2006). For each bioassay
artificial diets were prepared by diluting the respective concentrations of commercial products in
distilled water (mg a.i./liter of water) previously determined for each bioassay. In the control
treatment diet was prepared using distilled water. A piece of artificial diet from each diet was
offered to ten freshly molted L4 larvae of R. ferrugineus individually in plastic cups measuring
(6×6 cm) with 40 mesh/inch screen lids for aeration and the avoid insects escape. All the
treatments were replicated three time and incubated at 27±2 °C with 65±5% RH and a
photoperiod of 12: 12 (L: D) hours.
FAO Method No. 16 (FAO 1975) was used on 4th instar larvae of R. ferrugineus with
slight modifications. For each population, 10 larvae were placed in each of 10 glass cups and
were placed in 4 liter air tight glass boxes (serving as fumigation chambers) before PH3
fumigation. A small quantity of artificial diet (10 g) was added to each cup. The boxes were
centrally equipped with a port on the metal screw on lid which was fitted with a rubber injection
point which served as an entry point for PH3. Before the lid was screwed onto the box, a rubber
gasket was placed in it, and a thin layer of vacuum grease applied for a tight seal between the
metal lid and the top edge of the box to increase gas tightness. Five phosphine doses (mg l -1)
were measured in preliminary toxicity assays against 4th instar larvae of R. ferrugineus. Another
4 liter box without any treatment served as control. The gas was introduced into each box
containing R. ferrugineus larvae through the rubber septum by using a gas tight syringe after first
removing an equivalent volume of air from the jar using a syringe. Two drops of water were
added to each box using a syringe in order to maintain 70% RH inside the boxes. Boxes were
then placed in an incubator maintained at 27±2 °C. The boxes were opened 24 hours after
application, and all larvae from each treatment were placed on damp filter paper and maintained
at 27±2 °C and 65±5% RH for an extra 5 days to allow recovery.
4.3 Statistical Analysis
For insecticidal treatments three days post-release of insects to treated diets, mortality
counts were made in each treatment and at 24h +(5 recovery days) after PH3 application. Results
for mortality were converted into percentages, corrected using mortality in the untreated check
using Abbott’s (1925) formula and analyzed statistically in Probit analysis by Polo-Plus (LeOra,
2003). LC50, LC90 and 95% fiducial limits were estimated for each insecticide at each location.
Resistance ratios (RRs) were determined at LC50 and LC90 by dividing the lethal concentration
(LC) values of each insecticide by the respective LC values determined for the laboratory
susceptible strain. The distinction devised by Ahmad and Arif (2009) was used for categorizing
the RRs, which were summarized as “no resistance” if (RR≤ 1), “very low” (RR = 2–10), “low”
(RR = 11-20), “moderate” (RR = 21-50), “high” (RR = 51-100), and “very high” (RR> 100).
78
4.4 Results
4.4.1 Imidacloprid
Resistance levels varied among the seven distinct field populations of R. ferrugineus. The
degree of resistance varied from very low to low levels i.e RR 2.10- to 17.68-fold for all the
tested populations (Figs. 4.1 and 4.2; Table 42). A very low resistance level was recorded in
Bahawalpur (2.10-fold), Dera Ismail Kahn (2.86-fold), Vehari (4.20-fold) and Muzaffargarh
(5.60-fold) populations at LC50 level. Low level of resistance were observed in populations of R.
ferrugineus from Rahim Yar Khan (11.70-fold), Dera Ghazi Khan (15.12-fold) and Layyah
(17.68-fold) at LC50 values.
4.4.2 Spinosad
Very low level (1.02- to 9.77-fold) resistance ratios were recorded to spinosad among
different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). The resistance levels were
detected in Dera Ghazi Khan (1.02-fold), Bahawalpur (2.03-fold), Layyah (2.37-fold), Rahim
Yar Khan (3.41-fold), Muzaffargarh (4.40-fold), Vehari (7.15-fold) and Dera Ismail Khan (9.77-
fold) populations of R. ferrugineus at LC50 values.
4.4.3 Lambda cyhalothorin
Very low to low levels (1.96- to11.88-fold) RR to Lambda-cyhalothrin were recorded
(Fig. 4.2; Table 4.2). A very low level of resistance was found in Bahawalpur (1.96-fold), Dera
Ghazi Khan (2.01-fold), Layyah (2.77-fold), Rahim Yar Khan (3.76-fold), Muzaffargarh (4.72-
fold) and Vehari (9.21-fold) populations, while low level of resistance was recorded in Dera
Ismail Khan (11.88-fold) populations of R. ferrugineus at LC50 values.
4.4.4 Chlorpyrifos
Very low and low to moderate levels (3.03- to 24.47-fold) RR to chlorpyrifos were
recorded among tested populations of R. ferrugineus (Fig. 4.2; Table 4.2). A very low level of
resistance was found in Dera Ghazi Khan (3.03- fold) and Bahawalpur (5.45-fold) populations,
while low level of resistance was recorded in Layyah (10.65-fold), Muzaffargarh (17.91-fold)
and Rahim Yar Khan (15.98-fold), and moderate level of resistance was recorded in Dera Ismail
Khan (21.84-fold) and Vehari (24.47-fold) populations of R. ferrugineus at LC50 values.
4.4.5 Porfenophos
Very low and low to moderate levels (RR 2.65- to 44.10-fold) were recorded for
profenophos among different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). A very
low level of resistance was found in Bahawalpur (2.65-fold), Dera Ismail Khan (4.57-fold) and
Vehari (6.90-fold) populations, while low level of resistance was recorded in Muzaffargarh
(10.50-fold), and moderate level of resistance was recorded in Rahim Yar Khan (30.31-fold),
Dera Ghazi Khan (35.22-fold) and Layyah (44.10-fold) populations of R. ferrugineus at LC50
values.
4.4.6 Deltamethrin
Low and moderate to high levels (RR 12.90- to 57.97-fold) of resistance to deltamethrin
were recorded among different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). A low
level of resistance was found in Dera Ghazi Khan (12.90-fold) and Dera Ismail Khan (16.42-
79
fold) populations, while moderate level of resistance was recorded in Layyah (26.76-fold),
Rahim Yar Khan (34.52-fold), and high level of resistance was recorded in Muzaffargarh (51.37-
fold), Bahawalpur (53.81-fold) and Vehari (57.97-fold) populations of R. ferrugineus at LC50
values.
4.4.7 Cypermethrin
Low and moderate to high levels (RR 15.89- to 73.82-fold) of resistance to cypermethrin
were recorded among different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). A low
level of resistance was found in Dera Ghazi Khan (15.89-fold) population, while moderate level
of resistance was recorded in Dera Ismail Khan (22.59-fold), Layyah (31.11-fold), Rahim Yar
Khan (39.41-fold), and high level of resistance was recorded in Muzaffargarh (64.29-fold),
Bahawalpur (69.49-fold) and Vehari (73.82-fold) populations of R. ferrugineus at LC50 values.
4.4.8 Phosphine
High levels of resistance were recorded against PH3 in all the tested populations of R.
ferrugineus, ranging from 63.09- to 79.46-fold (Fig. 4.2; Table 4.2) with very little difference
between populations. The highest levels of resistance were recorded in Rahim Yar Khan (63.09-
fold), Muzaffargarh (63.78-fold), Bahawalpur (68.76-fold), Dera Ghazi Khan (72.29-fold), Dera
Ismail Khan (73.64-fold), Vehari (76.30-fold) and (79.46-fold) in Layyah populations of R.
ferrugineus at LC50 values. The slope of regression line was >2 in all the insect populations,
indicating significant differences in RR from the reference strain.
4.5 Discussion
Knowledge of the resistance status of pests of economic importance is imperative for
researchers to guide the farming community in combating pest problems. It could be helpful for
the farming community in partially reduce or completely suspend the use of particular chemical
in their farming system. In our study we considered seven populations of R. ferrugineus collected
from different areas of Punjab and Khyber Pakhtunkhwa provinces of Pakistan and tested
resistance to seven commonly used chemical insecticides and PH3 against laboratory reared F1 of
these populations. These areas are reported to be the major date producing areas of the country
and contribute the major share of the in country’s date production. Among these areas most have
the history of R. ferrugineus infestation stretching back almost 100 years (Milne, 1918). To
combat this voracious pest, farmers have mainly used conventional insecticides and fumigants,
particularly PH3, throughout the country over decades, resulting in R. ferrugineus.
This is the very first report of a resistance investigation in R. ferrugineus against
conventional insecticides and PH3 in Pakistan. Seven laboratory populations of R. ferrugineus
were established from field collections and all tested strains exhibited significantly different
susceptibility levels to all tested insecticides through dose-mortality bioassays. In our study
laboratory population was considered reference strain which exhibited no resistance to the tested
chemicals. Among the tested chemicals spinosad and lambda-cyhalothrin were the most effective
and revealed very low level of resistance in any tested population. The LC50 value of spinosad
and lambda-cyhalothrin was significantly lower for all the tested populations as compared to rest
of the insecticides. The chemical insecticides fed with artificial diet to R. ferrugineus larvae
caused mortality in a dose-dependent manner. The most consistent resistance across seven
populations was recorded for deltamethrin and cypermethrin. High level of resistance against
PH3 were observed for all the seven populations. The current study provides a base line of
80
resistance to cypermethrin, deltamethrin and PH3 against R. ferrugineus. In the strains examined
low and moderate to high level of resistance was due to the excessive application of these
chemical insecticides to manage R. ferrugineus in Pakistan.
The cryptic habit of R. ferrugineus facilitates almost year-round activity in date palm
plantations which has forced the farmers to expose field populations to different chemical
insecticides and fumigants in order to successfully control pest infestations. The repeated
application of these chemical insecticides could then lead to the resistance in R. ferrugineus
populations. Resistance to cypermethrin and deltamethrin is common amongst the arthropod
pests worldwide (Mueller-Beilschmidt, 1990). In Pakistan resistance against these commonly
used insecticides in various crop pests such as in Spodoptera exigua, Brevicoryne brassicae,
Spodoptera litura, Helicoverpa armigera and Bemisia tabaci has been reported by many
scientists (Ahmad 2008, 2009; Ahmad and Akhtar, 2013; Ahmad and Mehmood, 2015; Ahmad
et al., 2001; Sayyed et al., 2008; Ishtiaq et al., 2012; Qayyum et al., 2015). So far only one
published record of cypermethrin resistance against R. ferrugineus is reported from Saudi Arabia
by Al-Ayedh et al. (2015). Resistance to PH3 is not surprising because deployment of PH3 in the
form of aluminum phosphide tablets is common practice among date palm farming community
in the country. Resistance to PH3 is common in stored grain insets such as Tribolium castaneum,
Rhyzopertha dominica and psocid species worldwide e.g. Opit et al. (2012) reported 119 fold
and 1500 fold resistance in T. castaneum and R. dominica strains respectively, collected from
Oklahoma. Many other researchers have reported PH3 resistance in stored product pests from all
over the world.
It is advised not to use cypermethrin and deltamethrin against this pest and alternative
chemicals such as spinosad and lambda-cyhalothrin should be utilized in control strategies. So
far, there is no understanding of the molecular mechanisms in this species not any understanding
of cross-resistance pattern. This gap should be filled in order to design strategies to mitigate the
resistance problem. Spinosad and lambda-cyhalothrin possesses high efficacy against the
devastating stage of R. ferrugineus in laboratory assays (Abo-El-Saad et al., 2001). Treatment
strategies with chemical insecticides which include spinosad in control programs would be worth
considering. Moreover, integrated use of microbial control agents with newer chemistry
insecticides with novel modes of action could successfully replace the resisted chemistries in
field control programs.
Conclusions The present study showed that the population of red palm weevil in Pakistan has
resistance against commonly used chemical insecticides and phosphine. The unwise use of these
insecticides can lead to this problem. Almost all the insecticides exhibited resistance which was
varied form very low to, low and moderate to high for all the population tested. Delatmethrin,
cypermethrin and phosphine exhibited moderate to high resistance to almost all the populations.
Populations from Layyah Dera Ismail Khan and Vehari showed most resistance as compared to
the other populations tested.
Acknowledgements
This research work was supported by the scholarship from Higher Education
Commission (HEC), Islamabad, Pakistan (112-30536-2AV1-263) under Indigenous Ph.D.
Fellowship Program.
81
4.6 References
Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ.
Entomol., 18: 265-267.
Abdulsalam, K.S., M.S. Shawir, M.M. Abo-El-Saad, M.A. Rezk and A.M. Ajlan, 2001. Regent
(fipronil) as a candidate insecticide to control red palm weevil (Rhynchophorus
ferrugineus Olivier). Ann. Agric., 46: 841-849.
Abo-El-Saad, M.M., A.M. Ajlan, M.S. Shawir, K.S. Abdul-salam and M.A. Rezk, 2001.
Comparative toxicity of four pyrethroid insecticides against red palm weevil,
Rhynchophorus ferrugineus (Olivier) under laboratory conditions. J. Pest Cont. Environ.
Sci., 9: 63-76.
Abo-El-Saad, M.M., H.A. Elshafie, J.R. Faleiro and I.A. Bou-Khowh, 2011. Toxicity evaluation
of certain insecticides against the red palm weevil, Rhynchophorus ferrugineus (Olivier),
under laboratory conditions. ESA Annual Meeting, 2011.
Abraham, A., M.A. Al-Shuhaibi, J.R. Faleiro, R.A. Abozuhairah and P.S.P. Vidyasagar, 1998.
An integrated management approach for red palm weevil Rhynchophorus ferrugineus
Oliv. as key pest of date Palm in the Middle East .Agri. Sci., 3: 77- 83.
Ahmad, M., 2008. Potentiation between pyrethroid and organophosphate insecticides in resistant
field populations of cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) in
Pakistan. Pesti. Biochem. Physiol., 91: 24-31.
Ahmad, M., 2009. Observed potentiation between pyrethroid and organophosphorus insecticides
for the management of Spodoptera litura (Lepidoptera: Noctuidae). Crop Prot., 28: 264-
268.
Ahmad, M. and M.I. Arif, 2009. Resistance of Pakistani field populations of spotted bollworm
Earias vittella (Lepidoptera: Noctuidae) to pyrethroid, organophosphorus and new
chemical insecticides. Pest Manag. Sci., 65: 433-439.
Ahmad, M. and R. Mehmood, 2015. Monitoring of resistance to new chemistry insecticides in
Spodoptera litura (Lepidoptera: Noctuidae) in Pakistan. J. Econ. Entomol., 108(3): 1279-
1288.
Ahmad, M. and S. Akhtar, 2013. Development of insecticide resistance in field populations of
Brevicoryne brassicae (Hemiptera: Aphididae) in Pakistan. J. Econ. Entomol., 106(2):
954-958.
Ahmad, M., M.I. Arif, Z. Ahmad and I. Denholm, 2001. Cotton whitefly (Bemisia tabaci)
resistance to organophosphate and pyrethroid insecticides in Pakistan. Pest Mang. Sci.,
58: 203-208.
Ahmad, M., M. I. Arif, and Z. Ahmad, 2003. Susceptibility of Helicoverpa armigera
(Lepidoptera: Noctuidae) to new chemistries in Pakistan. Crop Prot., 22: 539-544
Ajlann, A.M., M.S. Shawir, M.M. Abo-El-Saad, M.A. Rezk and K.S. Abdulslam, 2000.
Laboratory evaluation of certain organophosphorus insecticides against the red palm
weevil, Rhynchophorus ferrugineus (Oliver). Sci. J., 1: 15-16.
Al-Ayedh, H., A. Hussain, M. Rizwan-ul-Haq and A.M. Al-Jabr, 2015. Status of insecticide
resistance in field-collected populations of Rhynchophorus ferrugineus (Olivier)
(Coleoptera: Curculionidae). Int. J. Agric. Biol., 17(6): 1-9.
Al-Jabr, A.M., M. Rizwan-ul-Haq, A. Hussain, A.I. Al-Mubarak and H.Y. Al-Ayed, 2013.
Establishing midgut stem cell culture from Rhynchophorus ferrugineus (Olivier) and
toxicity assessment against 10 different insecticides. In Vitro Cell. Dev. Biol. Anim., 50:
296-303.
82
Al-Rajhy, D.H., H.I. Hussein and A.M.A. Al-Shawaf, 2005. Insecticidal activity of carbaryl and
its mixture with piperonylbutoxide against red palm weevil Rhynchophorus ferrugineus
(Olivier) (Curculionidae: Coleoptera) and their effects on acetylcholinesterase activity.
Pak. J. Biol. Sci., 8: 679-682.
Anonymous, 1975. Recommended methods for detection and measurement of resistance in
agricultural pests to pesticides. Tentative methods for adults of some major pest species
to stored cereals with methyl bromide and phosphine. FAO Plant Prot. Bull., 23:12-35.
Avand-Faghih, A., 1996. The biology of red palm weevil, Rhynchophorus ferrugineus Oliv.
(Coleoptera, Curculionidae) in Saravan region (Sistan &Balouchistan Province, Iran).
Appl. Entomol. Phytopathol., 63: 16-18.
Azam, K.M., S.A. Razvi and I. Al-Mahmuli, 2000. Management of red date palm weevil,
Rhynchophorus ferrugineus Oliver on date palm by Prophylactic measures. In
proceedings of first workshop on control of date palm red weevil. Ministry of Higher
Education, King Faisal University, Date Palm Research Centre, Kingdom of Saudi
Arabia. Pp. 26-34.
Azam, K.M., S.A. Razvi and I. Al-Mahmuli, 2001. Survey of red palm weevil, Rhynchophorus
ferrugineus Oliver. Infestation in date palm in Oman. 2nd International Conf. on Date
Palms. (Al-Ain, UAE). Pp. 25-27.
Barranco, P., J.P. DeLa and T. Cabello, 1995. Un Nuevo Curculiónido tropical para la fauna
Europa, Rhynchophorus ferrugineus (Olivier 1790), (Curculionidae: Coleoptera). Boletin
de la Asociaction Espanola de Entomol., 20: 257-258.
Brand, E., 1917. Coconut red weevil. Some facts and fallacies. Tropic. Agric. Mag. Ceylon
Agricul. Soc., 49: 22-24.
Buxton, P.A., 1920. Insect pests of dates and the date palm in Mesopotamia and elsewhere. Bull.
Entomol. Res., 11: 287-304.
Cabello, T.P., J. de la Peña, P. Barranco and J. Belda, 1997. Laboratory evaluation of
imidacloprid and oxamyl against Rhynchophorus ferrugineus. Tests of Agrochem. Culti.,
18: 6-7.
Cox, M.L. 1993. Red palm weevil, Rhynchophorus ferrugineus, in Egypt. FAO Plant Prot. Bull.,
41: 30-31.
European and Mediterranean Plant Protection Organisation (EPPO), 2008. Rhynchophorus
ferrugineus, EPPO Bulletin Volume 38, Issue 1, pages 55-59, April 2008.
Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil
Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm
during the last one hundred years. Inter. J. Trop. Insect Sci., 26: 135-154.
Food and Agriculture Organization (FAO), 1975. Tentative method for adults of some major pest
species of stored cereals with methyl bromide and phosphine, FAO method no. 16. FAO
Plant Prot. Bull., 23: 12-25.
Hussain, A., M.R.U. Haq, A.M. Al-Jabr and H.Y. Al-Ayied, 2013. Managing Invasive
Populations of Red Palm Weevil: A Worldwide Perspective. J. Food Agric. Environ., 11:
456-463.
Ishtiaq, M., M.A. Saleem and M. Razaq, 2012. Monitoring of resistance in Spodoptera exigua
(Lepidoptera: Noctuidae) from four districts of the southern Punjab, Pakistan to four
conventional and six new chemistry insecticides. Crop Prot., 33: 13-20.
83
Kaakeh, W., 2006. Toxicity of imidacloprid to developmental stages of Rhynchophorus
ferrugineus (Curculionidae: Coleoptera): Laboratory and field tests. Crop Prot., 25: 432-
439.
Khalifa, O., A.H. El-Assal, F.A.A. Ezaby, M.A. Murse, S.M.A. Nuaimi and N.S. Al-Zehli, 2001.
Database for infestation of date palm by red palm weevil (Rhynchophorus ferrugineus
(Oliver). In U.A.E. and Oman. 2nd International Conf. on Date Palms. (Al-Ain, UAE). Pp.
25-27.
Lefroy, H.M., 1906. The more important insects injurious to Indian agriculture. Govt. Press,
Calcutta. Pp. 151.
LeOra, 2003. Polo plus, A User’s Guide to Probit and Logit Analysis. LeOra Software, Barkeley,
CA.
Llácer, E. and J.A. Jacas, 2010. Efficacy of phosphine as a fumigant against Rhynchophorus
ferrugineus (Coleoptera: Curculionidae) in palms. Spanish J. Agri. Res., 8: 775-779.
Martín, M.M. and T. Cabello, 2006. Manejo de la cría del picudo rojo de la palmera,
Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera, Dryophthoridae), en dieta
artificial y efectos en su biometría y biología. Boletín de Sanidad Vegetal de Plagas, 32:
631-641.
Milne, D., 1918. The Date Palm and Its Cultivation in the Punjab. The Punjab Government. Pp.
153.
Mueller-Beilschmidt, D., 1990, "Resistance of insect pests and disease vectors to synthetic
pyrethroids", J. Pesti. Ref., 10(4): 34-38.
Nirula, K.K., 1956. Investigations on the pests of coconut palm. Part. Rhynchophorus
ferrugineus. Ind. Coc. J., 9: 229-247.
Opit, G.P., T.W. Phillips, M.J. Aikins, and M.M. Hasan, 2012. Phosphine resistance in Tribolium
castaneum and Rhyzopertha dominica from stored wheat in Oklahoma. J. Econ.
Entomol., 105(4): 1107-1114.
Rajamanickam, K., J.S. Kennedy and A. Christopher, 1995. Certain components of integrated
management for red palm weevil, Rhynchohphorus ferrugineus F. (Curculionidae:
Coleoptera) on coconut. Mededelingen Faculteit Landbouwkundige en Toegepaste
Biologische Wetenschappen, 60: 803-805.
Sayyed, A.H., M. Ahmad and M.A. Saleem, 2008. Cross-resistance and genetics of resistance to
indoxacarb in Spodoptera litura (Lepidoptera: Noctuidae). J. Econ. Entomol., 101(2):
472-479.
Shar, M.U., M.A. Rustamani, S.M. Nizamani and L.A. Bhutto, 2012. Red palm weevil
(Rhynchophorus ferrugineus Olivier) infestation and its chemical control in Sindh
province of Pakistan. Afri. J. Agri. Res., 11: 1666-1673.
Viado, G.B.S. and A.E. Bigornia, 1949. A biological study of the Asiatic palm weevil,
Rhynchophorus ferrugineus Oliv. (Curculionidae: Coleoptera). Philip. Agri., 33: 1-27.
84
Table 4.1 Geographical characteristics of the localities where R. ferrugineus populations
were collected in the Punjab and Khyber Pakhtunkhwa, Pakistan
Location Province Host plant Geographical Characteristic
Alt. (m) Lat. Long. Layyah Punjab Date palm 143 30°58'N 70°56'E Bahawalpur Punjab Date palm 252 29°59′N 73°15′E Dera Ghazi Khan Punjab Date palm 150 29°57'N 70°29'E Muzaffargarh Punjab Date palm 114 30°50′N 71°54′E Dera Ismail Khan KPK Date palm 166 31°49'N 70°52'E Vehari Punjab Date palm 135 29°58'N 71°58'E Rahim Yar Khan Punjab Date palm 83 27°40'N 60°45'E
85
Table 4.2 Resistance to commonly used insecticides and phosphine against susceptible
strains and field-collected populations of R. ferrugineus
Insecticide Localities LC50 (mg liter-1)
(95% fiducial limits)
LC90 (mg liter-1)
(95% fiducial limits)
Slope Resistance
Ratio (RR)
Imidacloprid
Bahawalpur 16.59 (12.09-23.41) 250.87 (197.42-314.98) 1.12±0.18 2.10
Muzaffargarh 44.22 (36.31-51.39) 669.08.16 (581.35.793.45) 1.42±0.19 5.60
Layyah 139.52 (108.54-195.02) 2112.86 (1984.81-2358.44) 2.50±0.22 17.68 Dera Ismail Khan 22.64 (18.76-29.78) 341.52 (285.16-459.49) 1.34±0.20 2.86
Dera Ghazi Khan 119.37 (94.62-137.87) 1806.54 (1685.43-2132.38) 2.21±0.23 15.12
Vehari 33.19 (26.14-42.81) 501.51 (435.32-651.32) 1.23±0.19 4.20
Rahim Yar Khan 92.35 (79.37-117.69) 1397.43 (1258.15-1606.42) 1.36±0.21 11.70
Laboratory 7.89 (5.81-12.25) 119.48(106.03-148.11) 0.74±0.14 -
Spinosad
Bahawalpur 7.98 (3.03-10.08) 128.22 (114.34-165.10) 0.92±0.16 2.03
Muzaffargarh 17.26 (14.96-22.82) 276.34 (204.28-385.40) 1.14±0.18 4.40
Layyah 9.32 (4.67-13.92) 150.22 (101.34-236.10) 1.06±0.15 2.37
Dera Ismail Khan 38.30 (30.57-46.74) 620.75 (502.22-844.95) 1.42±0.19 9.77
Dera Ghazi Khan 3.98 (2.80-5.55) 64.41 (42.16-97.84) 0.61±0.13 1.02
Vehari 28.06 (24.87-33.91) 454.23 (357.81-648.35) 1.12±0.17 7.15
Rahim Yar Khan 13.37 (10.75-17.21) 216.72 (141.38-321.18) 1.17±0.15 3.41
Laboratory 3.92 (2.89-5.63) 63.56 (49.34-88.32) 0.69±0.14 -
Lambda cyhalothrin
Bahawalpur 13.85 (10.51-18.83) 221.59 (165.41-352.82) 0.72±0.17 1.96
Muzaffargarh 32.29 (26.54-42.12) 535.51 (448.32-679.32) 1.21±0.19 4.72
Layyah 19.64 (15.31-24.44) 313.82 (217.46-416.10) 1.07±0.17 2.77
Dera Ismail Khan 83.44 (73.39-95.72) 1347.16 (1081.84-1564.82) 1.61±0.21 11.88 Dera Ghazi Khan 14.15 (10.97-19.21) 228.76 (151.39-318.30) 0.72±0.15 2.01
Vehari 64.76 (56.34-71.72) 1045.11 (803.22-1281.35) 1.41±0.21 9.21
Rahim Yar Khan 26.44 (20.31-75.63) 425.93 (319.32-620.47) 1.33±0.20 3.76
Laboratory 7.02 (5.21-11.27) 113.48(102.03-130.10) 0.90±0.15 -
Chlorpyrifos
Bahawalpur 44.21 (37.57-53.40) 611.92 (499.40-881.55) 1.22±0.19 5.45
Muzaffargarh 145.36 (117.57-174.38) 2011.35 (1861.65-2351.88) 2.42±0.21 17.91
Layyah 86.42 (73.70-105.08) 1195.43 (996.15-1440.42) 1.37±0.20 10.65
Dera Ismail Khan 177.25 (145.86-197.65) 2452.54 (2275.47-2865.36) 2.31±0.21 21.84
Dera Ghazi Khan 24.61 (20.48-30.22) 340.55 (249.42-481.64) 1.72±0.19 3.03
Vehari 198.52 (146.54-237.53) 2747.67 (2505.43-3018.38) 2.55±0.20 24.47
Rahim Yar Khan 129.64 (97.54-182.38) 1794.75 (1598.89-2121.52) 2.31±0.18 15.98
Laboratory 8.11 (4.01-11.92) 112.29 (78.34-208.10) 0.97±0.16 -
Profenophos
Bahawalpur 23.31 (19.18-27.71) 307.01 (261.52-377.90) 1.25±0.18 2.65
Muzaffargarh 92.19 (78.12-115.08) 1220.43 (1161.15-1383.42) 1.30±0.21 10.50
Layyah 387.26 (330.65-424.59) 5125.41 (4871.21-5561.27) 3.22±0.23 44.1
Dera Ismail Khan 40.20 (32.98-46.87) 531.87 (414.23-680.45) 1.41±0.18 4.57
Dera Ghazi Khan 309.37 (286.17-353.53) 4093.08 (3814.56-4405.82) 2.53±0.18 35.22 Vehari 60.64 (52.34-67.72) 801.21 (719.22-1029.35) 1.41±0.17 6.90
Rahim Yar Khan 266.28 (224.23-305.29) 3522.54 (3368.83-3917.76) 2.56±0.20 30.31
Laboratory 8.78 (4.44-12.35) 116.22 (90.45-174.59) 0.99±0.16 -
Deltamethrin
Bahawalpur 348.28 (296.65-374.59) 5729.41 (5204.21-6480.27) 3.61±0.23 53.81
Muzaffargarh 332.43 (283.65-365.59) 5469.41 (5108.21-5911.27) 3.36±0.21 51.37
Layyah 173.29 (144.54-199.65) 2849.54 (2617.47-3227.36) 2.31±0.20 26.76
Dera Ismail Khan 106.34 (96.12-121.05) 1748.22 (1553.08-2092.29) 1.73±0.19 16.42
Dera Ghazi Khan 83.54 (73.24-92.11) 1373.16 (1202.84-1591.82) 1.54±0.17 12.90
Vehari 375.95 (329.65-411.59) 6172.41 (5728.21-6798.27) 3.52±0.23 57.97
Rahim Yar Khan 223.41 (194.36-284.66) 3675.76 (3339.78-4105.55) 2.72±0.21 34.52
Laboratory 6.47 (4.97-9.12) 106.48(93.03-122.13) 0.71±0.14 -
Bahawalpur 503.12 (454.22-583.48) 8127.65 (7875.63-8587.39) 3.72±0.23 69.49
Muzaffargarh 565.54 (391.43-605.66) 7519.76 (7256.32-7991.18) 3.83±0.22 64.29
86
Cypermethrin
Layyah 225.39 (201.57-267.29) 3638.02 (3425.25-4058.32) 2.59±0.20 31.11
Dera Ismail Khan 163.65 (139.23-204.32) 2641.58 (2470.43-2911.43) 2.48±0.19 22.59
Dera Ghazi Khan 115.18 (94.12-128.11) 1859.64 (1715.76-2119.64) 2.16±0.21 15.89
Vehari 534.47 (489.22-622.48) 8633.65 (7247.63-9211.39) 3.65±0.23 73.82
Rahim Yar Khan 255.42 (233.23-311.23) 4609.54 (4266.83-5165.76) 2.81±0.21 39.41
Laboratory 7.24 (5.39-11.23) 116.96(103.77-134.12) 0.71±0.19 -
Phosphine
Bahawalpur 4008.70 (3280.23-
4955.42)
51177.95 (43719.73-
59088.76) 5.46±0.27 68.76
Muzaffargarh 3718.37 (3011.23-4604.56)
47679.37 (42165.65-55248.54) 5.82±0.24 63.78
Layyah 4632.51 (3846.43-
5517.72)
59402.62 (51484.42-
64804.32) 6.37±0.34 79.46
Dera Ismail Khan 4293.21 (3618.43-
5180.41)
55048.84 (50342.23-
62268.54) 6.20±0.30 73.64
Dera Ghazi Khan 4214.50 (3529.43-
5065.54)
54039.67 (5045534-
621180.23) 5.82±0.28 72.29
Vehari 4448.33 (3750.43-
5309.52)
57038.53 (50118.76-
63308.37) 6.02±0.31 76.3
Rahim Yar Khan 3678.14 (2972.23-
4545.56)
47163.47 (42540.65-
54540.84) 4.83±0.23 63.09
Laboratory 58.3 (52.45-64.54) 747.56 (692.54-894.89) 1.48±0.21 -
87
Figure 4.1 Map of collection sites in Punjab and Khyber Pakhtunkhwa provinces of Pakistan
(1. Bahawalpur 2. Rahim Yar Khan 3. Vehari 4. Dera Ghazi Khan 5.
Muzaffargarh 6. Layyah 7: Dera Ismail Khan)
1. Bahawalpur
2. Rahim Yar Khan
3. Vehari
4. Dera Ghazi Kahn
5. Muzaffargarh
6. Layyah
7. Dera Ismail Khan
88
Figure 4.2 Resistance ratios (RRs) of chemical insecticides and phosphine against
susceptible strains and field-collected populations of R. ferrugineus populations of
R. ferrugineus from various localities in Punjab and Khyber Pakhtunkhwa,
Pakistan
89
CHAPTER 5
Insecticidal potential of Beauveria bassiana and Metarhizium anisopliae isolates against
Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)
Abstract
Entomopathogenic fungi are amongst the most common microbial control agents in nature
against insect pests. Their efficacy against a variety of arthropod pests had been witnessed since
for many years. The aim of this study was to screen 19 different isolates of Beauveria bassiana
s.l. and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales), recovered from different soil
samples (field crops, fruit orchards, vegetable fields and forests) and insect cadavers at two
different spore concentrations (1×107 and 1×108 conidia ml-1). Three isolates of B. bassiana
(WG-41, WG-42 and WG-43) and two isolates of M. anisopliae (WG-44 and WG-45) exhibited
˃88% larval and ˃75% adult mortality of Rhynchophorus ferrugineus (Olivier) (Coleoptera:
Curculionidae) on their highest dose rate. On the other hand more sporulating cadavers were
observed at high dose rate compared to low dose on both life stages of R. ferrugineus. The
current study confirmed the lethal action of B. bassiana, and M. anisopliae isolates with
differential mortality levels, usually directly proportional to the conidial concentration. This
study further confirmed that the isolates recovered from R. ferrugineus dead cadavers exhibited
more mortality compared to the other sources. In virulence assay WG-41 and WG-42 caused
highest percentage of both larval and adult mortality at all the exposure intervals which suggest
that these two isolates may be the most promising for their use in sustainable management
programs aimed at microbial control in date palm plantation.
Key words: Entomopathogens, Beauveria bassiana, Metarhizium anisopliae, Red Palm
Weevil sporulating
90
5.1 Introduction
The red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier 1790) (Coleoptera:
Curculionidae) is a devastating palm pest that has caused large economic losses in palm farming
worldwide (Murphy and Briscoe, 1999; Faleiro, 2006). This beetle can affect a wide range of
palms (Barranco et al., 2000) including economically important species such as the date palm
(Phoenix dactylifera L.), Canary Islands date palm (P. canariensis Hort), coconut (Cocos
nucifera L.), African oil palm (Elaeis guineensis Jacq.) and chusan palm (Trachycarpus fortunei)
(Sabbour and Solieman, 2014). The weevil has been found devastating palm plantations almost
50% of the date palm growing countries of the world (Faleiro, 2006) resulting yield losses from
0.7-10 tons hac-1 (Singh and Rethinam, 2005), while in Pakistan it caused 10-20% production
losses to different varieties of dates (Baloach et al., 1992). Presently its distribution is reported in
Oceania, Asia, Africa and Europe and was found in Curaçao and Marruecos, in 2008, and USA,
in 2010 (EPPO 2006, 2007, 2009a, 2009b, 2010).
The females lay eggs at the base of the fronds in separate holes made with their rostrum.
Neonate larvae bore into the palm core and upon completion of development move back to the
base of the fronds to pupate. A new generation emerges and adults may remain within the same
host and reproduce until the palm eventually dies and after that adults emerge out of the plant for
infection to the new plants (Dembilio et al., 2010). Furthermore, R. ferrugineus is a strong flyer
that increases the weevil’s ability to disperse, colonize and breed at new sites (Murphy and
Briscoe, 1999). However, longevity, activity and behavior of adult weevils are greatly affected
by humidity.
The most commonly used control treatments for this voracious pest are chemical
insecticides such as Diazinon, Imidacloprid, Phosmet and phosphine (Llácer and Jacas, 2010;
Llácer et al., 2010). These pesticides are applied with numerous application methods, including
wound dressing, frond axil filing, fumigation, injection, and spraying, are being tried for the
control of RPW infestations (Cabello et al., 1997; Abraham et al., 1998; Al-Rajhy et al., 2005).
Undoubtedly, the use of synthetic pesticides will continue to reduce RPW infestations. However,
several factors, including the evolution of resistance, residue persistence, applicator safety,
environmental hazards and harms non-target organisms have urged researchers to explore
alternatives to control RPW which can be compatible to human health and environment friendly
(Gindin et al., 2006; Hussain et al., 2013; Jalinas et al., 2015).
Alternatively, a number of biological control agents like predators, parasites, parasitoids,
and microbial control agents (bacteria, fungi and nematodes) are deployed to combat this pest. In
laboratory studies of the infection of RPW with different microorganisms, including
entomopathogenic nematodes (EPNs) (Gerber and Giblin-Davis, 1990; Llácer et al., 2009;
Dembilio et al., 2010a), entomopathogenic bacteria (Banerjee and Dangar, 1995; Salama et al.,
2004; Manachini et al., 2009) and entomopathogenic fungi (EPFs) (Ghazavi and Avand-Faghih,
2002; Shaiju-Simon and Gokulapalan, 2003; Gindin et al., 2006; Dembilio et al., 2010b; Cito et
al., 2014) have shown variable results in terms of larval and adult mortality.
Among these microorganisms, EPFs are considered promising microbial control agents
against RPW due to their epizootic potential, transmitted horizontally, natural dispersion and safe
to non-target organisms, environment friendly and ability to maintain lasting control once
established in the environment (Van Driesche et al., 2007; Hussain et al., 2015). Microbiological
treatments with Beauveria bassiana s.l. (Ascomycota: Hypocreales) and Metarhizium anisopliae
s.l. (Ascomycota: Hypocreales) offer an alternative and ecologically compatible pest
management strategy (Inglis et al., 2001). Several research programs have also been initiated for
91
studying the biological control of RPW. Specifically, by deploying these two agents, have been
detected on the RPW and tested under laboratory and field conditions (Deadman et al., 2001;
Gindin et al., 2006; El-Sufty et al., 2007; 2009; 2011; Sewify et al., 2009; Torta et al., 2009;
Vitale et al., 2009; Dembilio et al., 2010; Güerri-Aguilló et al., 2010; 2011; Merghem, 2011;
Francardi et al., 2012; Ricaño et al., 2013; Cito et al., 2014). The main advantage of using EPFs
is their unique mode of action. Unlike other insect pathogens, fungi infect the host by contact,
penetrating the insect cuticle. The host can be infected by direct treatment or by transmission of
inoculum from treated insects/cadavers to untreated insects or to subsequent developmental
stages via the new generation of spores (Quesada-Moraga et al., 2004).
A number of studies were carried out to isolate the EPFs from different developmental
stages of RPW. Entomopathogenic M. anisopliae was isolated from Rhynchophorus bilineatus
(Montrouzier) (Coleoptera: Curculionidae) in New Guinea, after treatment of young palms
against the Scapanes australis Boisduval (Coleoptera: Scarabaeidae: Dynastinae) with a
formulation based on M. anisopliae spores (Murphy and Briscoe, 1999). Other published report
(Ghazavi and Avand-Faghih, 2002) from Iran; showed the recovery of M. anisopliae and B.
bassiana from RPW adults and pupae. The pupae of R. ferrugineus presumed to be infected with
entomopathogenic fungi were collected in a date palm grove in Spain during 2007 which later on
proved to be infected with the entomopathogenic fungus B. bassiana (Dembilio et al., 2010b).
The aim of this study was to screen and identify pathogenicity of 19 isolates of M. anisopliae and
B. bassiana recovered from different soils, stored grain insects and cadavers of infected R.
ferrugineus. This study also aimed to determine the exposure time dose mortality relationships of
virulent fungal isolates, and to confirm infection against RPW under laboratory conditions.
5.2 Materials and Methods
5.2.1 RPW collection and rearing
Different life stages of RPW were collected from fallen and infested date palm trees with
the permission of farmers (owners) from date palm growing areas of west Punjab and Khyber
Pakhtunkhwa (KPK), Pakistan. During collection adult, larvae and pupae were kept in 2 liter
plastic jars until brought to the laboratory. After arriving to the laboratory larvae were provided
with sugarcane (Saccharum officinarum L.; Poales: Poaceae) stems for feeding and pupation,
while adult were offered with the shredded sugarcane pieces as both for feeding and substrate for
oviposition. Pupae were kept in separate boxes (15×30×30 cm) for adult emergence in incubator.
As the adults were emerged, they were transferred to the adult’s jars for feeding and mating.
Colony was developed in plastic boxes (15×30×30 cm) having a lid covered with mesh wire
gauze (60 mesh size) in the middle (7 cm diameter) for aeration. Rearing was carried out in
Microbial Control Laboratory, Department of Entomology, University of Agriculture,
Faisalabad, Pakistan. The rearing conditions were maintained at 25±2oC and 65±5% RH and
12:12 (D: L) h photoperiod in an incubator. Adults diet was changed after every three days and
replaced sugarcane pieces were kept in separate jars for egg hatching. After egg hatching neonate
larvae were transferred to the new sugarcane pieces for feeding and pupation.
5.2.2 Culture collection
The virulence of 19 isolates of entomopathogenic fungi B. bassiana and M. anisopliae,
14 of them (WG-2, WG-3, WG-4, WG-5, WG-6, WG-7, WG-10, WG-11, WG-12, WG-13, WG-
15, WG-16, WG-17, WG-18) belonging to the culture collection of Microbial Control
92
Laboratory, Department of Entomology, University of Agriculture, Faisalabad, Pakistan. All
these isolates were recovered from soils of different origin (field crops, fruit orchards, vegetable
fields and forest) and stored grain insect pests (Wakil et al., 2013; 2014). Other five isolates,
three of B. bassiana (WG-41, WG-42 and WG-43) and two of M. anisopliae (WG-44 and WG-
45) were isolated from infected RPW cadavers collected from Layyah, Bahawalpur, Dera Ismail
Khan and Muzaffargarh districts (Table 4.1).
5.2.3 Isolation from RPW cadavers
The B. bassiana and M. anisopliae isolates were obtained from naturally infected RPW
adults collected from different areas of Punjab, Pakistan. All the dead cadavers were washed
with 75% ethanol for 30 sec, rinse with distilled water and transferred to 3% NaClO for 1 min,
followed by rinse in distilled water for 2 times. Cadavers were then dried on filter paper and
placed in petri dishes containing either Sabouraud Dextrose Agar (Merck, Germany) or Potato
Dextrose Agar (BD, France) supplemented with 0.1 g liter-1 of streptomycin sulfate. Plates were
sealed with parafilm and incubated at 25°C for 7 days. Insects were examined under a
microscope every 24 h for the appearance of any fungal outgrowth on the medium (Wakil et al.
2014). Where more than one fungal colony was present on the medium, the culture was purified
following single spore method (Choi et al. 1999). The identification of the isolated fungi was
done with the taxonomic keys (Barnett & Hunter 1999; Domsch et al. 2007). The culture was
sub-cultured and stored in petri dishes at 4°C in refrigerator. Spore concentration was determined
with an improved Neubauer haemocytometer and the conidial viability was confirmed <90%
before each assay.
5.2.4 Screening assay
Nineteen fungal isolates (Table 4.1) were assayed against larvae and adults of R.
ferrugineus. Two conidial concentrations (1×107 and 1×108 conidia ml-1) were employed against
4th instar larvae and adult by dipping method. The larvae were immersed in conidial suspension
for 60s and adults for 90s (Dembilio et al., 2010b). After treatment insects were transferred to
the moistened filter paper for 24 h and shifted individually to 150 ml plastic cups (6×6 cm). The
cups were covered with screening lids and treated larvae were offered with artificial diet (Martín
and Cabello 2006) and adults with shredded sugarcane pieces (3×3 cm). The control individuals
were treated with 0.01% Tween-80 solution. Each treatment consisted of 10 individuals and
three replicates in each treatment, a total of 30 insects per treatment were used. Mortality was
checked daily for 12 days and dead individuals were transferred to SDA medium for 10 days to
observe mycosed individuals. The whole experiment was repeated twice.
5.2.5 Virulence assay
Potential strains which showed high pathogenicity in preliminary screening assays (WG-
41, WG-42, WG-43, WG-44 and WG-45) were further tested against both larvae and adult of R.
ferrugineus at 1×106, 1×107, 1×108 and 1×109 conidia ml-1. Ten 6th instar larvae were dipped in a
conidial suspension for 60s and adults for 90s and air dried in sterile petri dish lined with damp
filter paper for 24 h and transferred to the plastic cups individually (Dembilio et al., 2010b). The
conditions in an incubator were adjusted to a photoperiod 12:12 h (L: D) and 65±5% RH at
25±2°C. Then the larvae were allowed to feed on artificial diet (Martín and Cabello, 2006) in
150 ml plastic cups (6×6 cm) individually and adults with shredded sugarcane pieces (3×3 cm).
The larval mortality was recorded after 7, 14 and 21 days. For control treatment the individuals
93
were immersed in distilled water containing 0.01% Tween-80. Three replicates were used for
each treatment and entire experiment was repeated thrice independently. Median lethal
concentration LC50, LC90 and lethal time LT50, LT90 were calculated for different exposure
intervals.
5.2.6 Statistical analysis
Mortality for each treatment was corrected for control mortality using Abbott's (1925)
formula and subjected to one way analysis of variance (ANOVA) in Minitab (Minitab, 2002)
using Tukey’s Kramer test (HSD) (Sokal and Rohlf, 1995) at 5% significance level. Probit
analysis was used to estimate the LC50, LC90, LT50 and LT90 of isolates using Confidence Limit
95% (CL).
5.3 Results
5.3.1 Screening assay
The results revealed that all the tested isolates of B. bassiana and M. anisopliae were
pathogenic to larvae and adult of R. ferrugineus under laboratory conditions. Overall both fungal
isolates inflicted greater mortality of R. ferrugineus larvae (Table 5.3) compared to the adults
(Table 5.4). For example, five isolates caused highest mortality ranging 65.55-88.33% at 1×108
conidia ml-1 after 12 days post incubation, whereas adult mortality was ranged between 46.03-
75.95% at the same concentration. All the fungal isolates differ significantly in their virulence
against larvae (F18, 113 = 31.2, P≤0.05) at 1×107 conidia ml-1 and (F18, 113 = 56.8, P≤0.05) at 1×108
conidia ml-1 and for beetles (F18, 113 = 41.4, P≤0.05) at 1×107 conidia ml-1 and (F18, 113 = 46.8, P≤0.05)
at 1×108 conidia ml-1. More numbers of mycosed individuals were recorded from the treatments
where lower spore concentration (1×107 conidia ml-1) was applied than higher concentration
(1×108 conidia m l-1). Mycosis in larvae was also significantly different (F18, 113 = 757, P≤0.05) at
1×107 conidia ml-1 and (F18, 113 = 433, P≤0.05) at 1×108 conidia ml-1 and for beetles (F18, 113 = 763,
P≤0.05) at 1×107 conidia ml-1 and (F18, 113 = 423, P≤0.05) at 1×108 conidia ml-1. Main effects and
their associated interactions for mortality and mycosis were significantly different (Table 5.2).
Among five most virulent isolates three belonged to B. bassiana (WG-41, WG-42 and WG-43)
and two to M. anisopliae (WG-44 and WG45). These five isolates were selected for further
virulence assays.
5.3.2 Virulence assay
In virulence assay the mortality of larvae and adult was recorded after 7, 14 and 21 days
after application. Until 7th day of application no isolate caused 100% mortality in either larvae or
adult, highest activity was recorded for WG-41 76.95% and 63.96% in larvae and adult
respectively at highest spore concentration (Table 5.6). After last count all isolates caused 100%
mortality both in larvae and adult at highest dose rate used except WG-43 and WG-45 which
caused 98.41and 91.83 % adult mortality respectively. Overall WG-41 was the most virulent
isolate followed by WG-42, WG-44, WG-43 and WG-45. The mortality of larvae were
significantly affected by the dose rates of fungal isolates at 5days exposure (WG-41: F3, 35 = 71.1,
P≤0.05; WG-42: F3, 35 = 101, P≤0.05; WG-43: F3, 35 = 63.3, P≤0.05; WG-44: F3, 35 = 105, P≤0.05
and WG-45: F3, 35 = 60.8, P≤0.05). Similarly significant difference was also recorded in case of
adults at this exposure (WG-41: F3, 35 = 133, P≤0.05; WG-42: F3, 35 = 108, P≤0.05; WG-43: F3, 35 =
106, P≤0.05; WG-44: F3, 35 = 56.6, P≤0.05; WG-45: F3, 35 = 58.3, P≤0.05). After 14 days post
94
application, only WG-41 exhibited 100% larval mortality at highest dose rate used (Table 5.7),
while highest adult mortality (93.22%) was also recorded for the same isolate at highest fungal
dose rate (Table 5.8). Significant differences have been observed between the mortalities of the
larvae at all the dose rates (WG-41: F3, 35 = 220, P≤0.05; WG-42: F3, 35 = 193, P≤0.05; WG-43: F3,
35 = 120, P≤0.05; WG-44: F3, 35 = 170, P≤0.05; WG-45: F3, 35 = 203, P≤0.05). Significant
differences were also recorded in case of adults at this exposure (WG-41: F3, 35 = 166, P≤0.05;
WG-42: F3, 35 = 162, P≤0.05; WG-43: F3, 35 = 112, P≤0.05; WG-44: F3, 35 = 172.0, P≤0.05; WG-45:
F3, 35 = 135, P≤0.05). After last count, all tested isolates caused 100% mortality both in larvae and
adult except WG-43 and WG-45, and significant differences were observed between all the
tested isolates for larvae (WG-41: F3, 35 = 138, P≤0.05; WG-42: F3, 35 = 245, P≤0.05; WG-43: F3, 35
= 179, P≤0.05; WG-44: F3, 35 = 168, P≤0.05; WG-45: F3, 35 = 223, P≤0.05) and adults (WG-41: F3,
35 = 164, P≤0.05; WG-42: F3, 35 = 312, P≤0.05; WG-43: F3, 35 = 261, P≤0.05; WG-44: F3, 35 = 165,
P≤0.05; WG-45: F3, 35 = 271.0, P≤0.05). Factorial analysis shows that the main effects were
significant, while their associated interactions Isolate × Treatment and Isolate × Interval for
larvae were non-significant and Isolate × Interval were also non-significant for adults (Table
5.5).
The results of our study revealed that all isolates of B. bassiana and M. anisopliae are
virulent to larvae and adults of R. ferrugineus at the dose rate of 106, 107, 108, and 109 conidia
ml-1. The lethal concentrations LC50 and LC90 at the 15th day after treatment were assessed
(Table 5.9). The isolates (WG-41 and WG-42) exhibited half of the larval mortality with a
concentration of 105 conidia ml-1 and isolates (WG-43, WG-44 and WG-45) revealed LC50
values at 106 conidia ml-1. While half of the adult mortality for all the tested isolates were
recorded for a concentration of 106 conidia ml-1. The isolates WG-41 and WG-42 caused a high
percentage of adult mortality, thus requiring a lower concentration of fungal conidia to cause an
average percentage of adult or larval mortality; hence, these isolates were considered the most
virulent among all the isolates investigated in this study.
The increased virulence, shown by the reduction in LT50, increased mortality and
proportion of mycosed cadavers were closely related to the conidial concentration. LT50
decreased when conidial concentration increased from 106-109 conidia ml-1. Estimated LT50 of
all fungal isolates against R. ferrugineus larvae varied from 17.75 to 27.92 days at 106 conidia
ml-1, while less time 5.27 to 7.82 days were recorded in case of highest dose rate used (Table
5.10). In case of estimated LT50 of all fungal isolates against R. ferrugineus adults varied from
20.41 to 30.06 days at 106 conidia ml-1, while less time 4.93 to 9.57 days were recorded in case
of 109 conidia ml-1 (Table 5.11). Overall WG-41 was considered most virulent isolate against
both larvae and adult which inflicted highest adult and larval mortality at almost all the dose rate
used within short period of time as compared to the other isolates used.
5.4 Discussion
Fungal entomopathogens are important biological control agents against insect pests
worldwide and have been the subject of intense research for more than 100 years (Vega et al.,
2012). Some of the advantages offered by the use of entomopathogenic fungi in microbial
control programs are their specificity, contact transmission, natural dispersion, safety for non-
target organisms and the ability to maintain lasting control once established in the environment
(Van Driesche et al., 2007). Laboratory screening of fungal isolates bring down to manageable
number is a vital step in identifying virulent strains prior to field use (Cherry et al., 2005). The
two stage approach to screening adopted in this study proved a robust mechanism that has been
95
used effectively by many other workers (Moino et al., 1998; Kassa et al., 2002). In our studies, a
panel of 19 isolates that were never screened were selected and selected 5 isolates based on their
pathogenicity potential for critical assays in vivo which exhibited ˃ 85% and ˃ 75% larval and
adult mortality respectively. Via this hierarchical approach we ultimately identified three B.
bassiana (WG-41, W-42 and WG-43) and two M. anisopliae (WG-44, WG-45) isolates,
exhibited shortest LC50 of 105 conidia ml-1 with potential as a novel bio-pesticide for use against
R. ferrugineus.
The conidial viability is very important in the host infection process because it permits
success at the beginning of the early stages of fungal infection process of the host cuticle,
followed by conidial germination and the formation of a germ tube (Schrank and Vainstein,
2010). In the present study, the conidia viability of the 19 isolates was 100%, which ensured the
quality of conidia present in the fungal suspensions used to treat R. ferrugineus larvae. The re-
isolation of fungal isolates after treatment confirmed the infection capacity of the studied
isolates. Our results indicated the B. bassiana isolates (WG-41, WG-42) exhibited high larval
and adult mortality, suggesting an enormous potential for this fungal species to be used for pest
control. Moreover, five isolates presented the better results, with average almost 100% larval and
adult mortality by the 21st day post treatment at highest dose rate used and they should be
considered as ideal isolates to be used in formulations for field studies, exhibited LC50 of high
virulence range within 6.47×105-3.66×106 conidia ml-1 for larvae and 1.04×106-9.30×106 conidia
ml-1. These LC50 values are practical for the development of a myco-insecticide aimed to control
R. ferrugineus in integrated management programs.
The individuation of virulent strains of entomopathogenic fungi towards the R.
ferrugineus in the countries of introduction represent a precious opportunity to increase studies
on the microbiological control efficacy in view of a possible field applications. Within fungal
taxa, individual isolates can exhibit substantially restricted host range (Inglis et al., 2001) and
isolates recovered from a target host and closely related species are generally more virulent than
isolates from non-related species or from soil. Ricaño et al. (2013) reported high efficacy of B.
bassiana isolates recovered from RPW as compared to the other sources (Insects and soil
samples). Moreover, B. bassiana strains recovered from 30 day old RPW were most pathogenic
strains among all tested isolates. These isolates were also the most virulent on RPW adults. Lo
Verde et al. (2014) also reported the significantly more pathogenic action of RPW isolated B.
bassiana strains. El-Sufty et al. (2009) obtained a mortality of 12.8-47.1% in adult R.
ferrugineus population in field assays using a strain of B. bassiana isolated in the United Arab
Emirates. These published reports are supportive to our findings as our results showed that B.
bassiana isolates (WG-41, WG-42) recovered from RPW cadavers inflicted highest mortality.
Further corroborating our results, an indigenous strain of B. bassiana obtained from mycosed
RPW collected in field showed good results in laboratory and field tests (El-Sufty et al., 2007;
Sewify et al., 2009). Our results are in accordance with Cherry et al. (2005) who reported,
indigenous isolates that had been recovered from C. maculatus were more virulent in laboratory
bioassays against C. maculatus than exotic isolates from other insects. Similarly Goettel et al.
(1990) reported, a fungal isolate may be more pathogenic against the host from which it was
obtained than to other novel hosts.
Contrarily, Monteiro et al. (1998a, b) reported that an ant derived isolate was more
effective against ticks than a tick derived isolate. Ángel-Sahagún et al. (2010) indicate that
isolates with high pathogenicity against ticks could be found from samples derived from soil on
the last instars of the great wax moth, Galleria mellonella. Fernandes et al. (2011), observed that
96
several B. bassiana isolates obtained from naturally infected ticks were not significantly more
virulent against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) than isolates obtained
from other arthropod orders. With regard to entomopathogenic fungi important factors in
reaching these goals are: intra alia, the availability of an isolate highly virulent towards the
target insect, suitability for mass spore production on an appropriate contamination substratum,
an efficient delivery system and inoculum stability and germinability in field conditions (Ibrahim
et al., 1999; Zhang et al., 2011).
On contrary, Gindin et al. (2006) observed high mortality of R. ferrugineus adults treated
with dry spores of M. anisopliae than the B. bassiana treatments after two weeks. Tests carried
out with the experimental traps showed that M. anisopliae was the more virulent pathogen,
causing 75% cumulative mortality in R. ferrugineus adults, while B. bassiana gave a 45%
cumulative mortality (Francardi et al., 2103). Similar findings were reported by the Francardi et
al. (2012) who evaluated field collected strain of B. bassiana and M. anisopliae isolated in Italy
from naturally infected R. ferrugineus adults, and tested under laboratory conditions. M.
anisopliae obtained from R. ferrugineus showed the highest efficacy against RPW larvae and
adults which showed values of cumulative larval mortality of 100% and adult mortality of 90%.
Our study indicated that the indigenous strain of B. bassiana recovered from R.
ferrugineus were more infective to larvae and adult of R. ferrugineus. This study was further
supported by the findings of Dembilio et al. (2010b) who reported potential effect of indigenous
strain of B. bassiana against different developmental stages of R. ferrugineus. The use of
entomopathogenic fungi, in particular indigenous strains of B. bassiana and M. anisopliae,
obtained from naturally infected weevils, should be seriously considered for biological control
because both have provided encouraging results for the control of certain economic pests
(Jaronski, 2010). This suggests that the identification of indigenous entomopathogenic fungi
already active on the weevil may offer better prospects for its biological control.
Conclusion
The present study showed that B. bassiana and M. anisopliae isolates recovered from the
RPW used in laboratory bioassays caused high mortality in larvae and adults compared to the
other tested fungal isolates. More number of mycosed individuals was also observed from the
same isolates. For these reasons, the use of entomopathogenic fungi can be considered to be
useful tool as an integral part of successful IPM program. Moreover, the inoculum of
entomopathogenic fungi may be transmitted by R. ferrugineus due to their low mortality which
may further be helpful in reducing RPW population in date palm systems, further research is
needed to support this thought.
Acknowledgements
This research work was supported by Higher Education Commission, Islamabad
(Pakistan) (2AV1-263) under Indigenous Ph.D. Fellowship Program.
97
5.5 References
Abraham V.A., A.S., Mahmood, J.R. Faleiro, R.A. Abozuhairah and P.S.P.V. Vidyasagar, 1998.
An integrated approach for the management of red palm weevil Rhynchophorus
ferrugineus Oliv. A key pest of date palm in the Middle East. Sultan Qaboos University,
J. Sci. Res. Agric. Sci., 3: 77-83.
Al-Rajhy, D.H., H.I. Hussein and A.M.A. Al-Shawaf, 2005. Insecticidal activity of carbaryl and
its mixture with piperonylbutoxide against red palm weevil Rhynchophorus ferrugineus
(Olivier) (Curculionidae: Coleoptera) and their effects on acetylcholinesterase activity.
Pak. J. Biol. Sci., 8: 679-682.
Ángel-Sahagún, C.A., R. Lezama-Gutiérrez, J. Molina-Ochoa, A. Pescador-Rubio, S.R. Skoda,
C. Cruz-Vázquez, A.G. Lorenzoni, E. Galindo-Velasco, H. Fragoso-Sánchez and J.E.
Foster, 2010. Virulence of Mexican isolates of entomopathogenic fungi (Hypocreales:
Clavicipitaceae) upon Rhipicephalus = Boophilus microplus (Acari: Ixodidae) larvae and
the efficacy of conidia formulations to reduce larval tick density under field conditions.
Vet. Parasitol., 170: 278-286.
Baloach, H.B., M.A. Rustamani, R.D. Khuro, M.A. Talpur and T. Hussain, 1992. Incidence and
abundance of date palm weevil in different cultivars of date palm. Proc. 12 th Cong. Zool.
Soc. Pak., 12: 445-447.
Banerjee, A. and T.K. Dangar, 1995. Pseudomonas aeruginosa, a facultative pathogen of red
palm weevil, Rhynchophorus ferrugineus. World J. Microbiol. Biotech., 11: 618-620.
Barnett, H.L. and B.B. Hunter, 1999. Illustrated Genera of Imperfect Fungi, 4th Edn. APS Press,
The American Phytopathological Society, St. Paul, MN. p 218.
Barranco, P., J.A. De La Peña, M.M. Martin and T. Cabello, 2000. Rango de hospedantes de
Rhynchophorus ferrugineus (Olivier, 1790) y diametro de la palmera hospedante.
(Coleoptera, Curculionidae). Boletín de Sanidad Vegetalde Plagas, 26: 73-78.
Cabello, T.P., J.A. De La Peña, P. Barranco and J. Belda, 1997. Laboratory evaluation of
imidacloprid and oxamyl against Rhynchophorus ferrugineus. Test. Agrochem. Culti., 18:
6-7.
Carruters, R.I. and R.S. Soper, 1987. Fungal Diseases. In Fuxa, J.R. and Y.T. Tanada, (eds.)
Epizootology of Insect Diseases. Wiley and Sons, New York. pp. 357-416.
Cherry, A.J., P. Abalo and K. Hell, 2005. A laboratory assessment of the potential of different
strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and
Metarhizium anisopliae (Metschnikoff) to control Callosobruchus maculatus (F.)
(Coleoptera: Bruchidae) in stored cowpea. J. Stored Prod. Res., 41: 295-309.
Choi, I., R.E. Nisbett and A. Norenzayan, 1999. Causal attribution across cultures: Variation and
universality. Psychol. Bull., 125: 47-63.
Cito, A., G. Mazza, A. Strangi, C. Benvenuti, G.P. Barzanti, E. Dreassi, T. Turchetti, V.
Francardi and P.F. Roversi, 2014. Characterization and comparison of Metarhizium
strains isolated from Rhynchophorus ferrugineus. FEMS Microbiol. Lett., 355: 108-115.
Deadman, M.L., K.M. Azam, S.A. Ravzi and W. Kaakeh, 2001. Preliminary investigation into
the biological control of the red palm weevil using Beauveria bassiana. Proc. 2nd Inter.
Conf. Date Palm, Al-Ain, UAE. March 25-27. pp. 225-232.
Dembilio Ó., E. Llácer, M.M. Martínez de Altube and J.A. Jacas, 2010a. Field efficacy of
imidacloprid and Steinernema carpocapsae in a chitosan formulation against the red
palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) in Phoenix
canariensis. Pest Manag. Sci., 66: 365-370.
98
Dembilio Ó., E. Quesada-Moraga, C. Santiago-Álvarez and J.A. Jacas, 2010b. Biocontrol
potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana
(Ascomycota; Hypocreales) against the red palm weevil, Rhynchophorus ferrugineus
(Coleoptera: Curculionidae). J. Inver. Pathol., 104: 214-221.
Domsch, K.H., W. Gams and T.H. Anderson, 2007. Compendium of soil fungi. 2. ed. London:
Lubrecht & Cramer Ltd.
El-Sufty, R., S. Al Bgham, S.A. Al-Awash, A.S. Shahdad and A.H. Al Bathra, 2011. A trap for
auto-dissemination of the entomopathogenic fungus Beauveria bassiana by the red palm
weevil adults in date palms plantations. Egyp. J. Biol. Pest Control, 21(2): 271-276.
El-Sufty, R., S.A. Al-Awash, A.M. Al-Amiri, A.S. Shahdad, A.H. Al-Bathra and S.A. Musa,
2007. Biological control of red palm weevil, Rhynchophorus ferrugineus (Col.:
Curculionidae) by the entomopathogenic fungus Beauveria bassiana in United Arab
Emirates. Proc. of the 3rd Inter. Conf. Date Palm. Acta Horti., 736: 399-404.
El-Sufty, R., S.A. Al-Awash, S. Al Bgham, A.S. Shahdad, A.H. Al-Bathra, 2009. Pathogenicity
of the fungus Beauveria bassiana (Bals.) Vuill to the red palm weevil, Rhynchophorus
ferrugineus (Oliv.) (Col.: Curculionidae) under laboratory and field conditions. Egyp. J.
Biol. Pest Control, 19: 81-85.
EPPO. (European and Mediterranean Plant Protection Organization), 2009a. EPPO Reporting
Service. 2009/002 - First record of Rhynchophorus ferrugineus in Curaçao, Netherlands
Antilles January 26, 2009.
EPPO. (European and Mediterranean Plant Protection Organization) 2009b. Rhynchophorus
ferrugineus found on Howea forsteriana in Sicilia, Italy. No. 3 2009/051. European and
Mediterranean Plant protection Organization, Paris, France. Available at:
https://archives.eppo.int/EPPOReporting/2009/Rse-0912.pdf. Accessed on: 12 June 2015.
EPPO. (European and Mediterranean Plant Protection Organization) 2010. EPPO Reporting
Service. 2010/176 - First record of Rhynchophorus ferrugineus in the USA, November 1,
2010.
EPPO. (European and Mediterranean Plant Protection Organization) 2006. Reporting Service,
No.112006-11-01/2006/225, First record of Rhynchophorus ferrugineus in France and
2006/226, First report of Rhynchophorus ferrugineus in Greece and 2006/226,
Rhynchophorus ferrugineus found in Lazio region, Italy.
Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil
Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm
during the last one hundred years. Inter. J. Tropi. Insect Sci., 26: 135-154.
Fernandes, E.K.K., I.C. Angelo, D.E.N. Rangel, T.C. Bahiense, A.M.L. Moraes, D.W. Roberts
and V.R.E.P. Bittencourt, 2011. An intensive search for promising fungal biological
control agents of ticks, particularly Rhipicephalus microplus. Vet. Parasitol., 182: 307-
318.
Francardi, V., C. Benvenuti, P.F. Roversi, P. Rumine and G. Barzanti, 2012.
Entomopathogenicity of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae
(Metsch.) Sorokin isolated from different sources in the control of Rhynchophorus
ferrugineus (Olivier) (Coleoptera Curculionidae). Redia, 95: 49-55.
Francardi, V., C. Benventi, G.P. Barzanti and P.F. Rovers, 2013. Auto contamination trap with
entomopathogenic fungi: A possible strategy in the control of Rhynchophorus ferrugineus
(olivier) (Coleoptera Curculionidae). REDIA, XCVI: 57-67.
99
Gerber, K. and R.M. Giblin-Davis, 1990. Association of the red ring nematode and other
nematode species with the palm weevil, Rhynchophorus palmarum. J. Nematol., 22: 143-
149.
Ghazavi, M. and A. Avand-Faghih, 2002. Isolation of two entomopathogenic fungi on red palm
weevil, Rhynchophorus ferrugineus (Olivier) (Col., Curculionidae) in Iran. Appl.
Entomol. Phytopathol., 9: 44-45.
Gindin, G., S. Levski, I. Glazer and V. Soroker, 2006. Evaluation of the entomopathogenic fungi
Metarhizium anisopliae and Beauveria bassiana against the red palm weevil
Rhynchophorus ferrugineus. Phytoparasitica, 34(4): 370-379.
Goettel, M.S., T.J. Poprawski, J.D. Vandenberg, Z. Li and D.W. Roberts, 1990. Safety to non-
target invertebrate of fungal bicontrol agents. p 209-232. In: Laird, M., L.A. Lacey and
E.W. Davidson (eds.). Safty of Microbial insecticides. Boca Raton, CA: CRS Press.
Güerri-Agulló, B., S. Gómez-Vidal, L. Asensio, P. Barranco and L.V. Lopez-Llorca, 2010.
Infection of the red palm weevil (Rhynchophorus ferrugineus) by the entomopathogenic
fungus Beauveria bassiana: a SEM study. Microscopy Res. Tech., 73: 714-725.
Hussain, A., M. Rizwan-Ul-Haq, H. Al-Ayedh, S. Ahmed and A.M. Al-Jabr, 2015. Effect of
Beauveria bassiana infection on the feeding performance and antioxidant defence of red
palm weevil, Rhynchophorus ferrugineus. BioControl, 60: 849-859.
Hussain, A., M.R.U. Haq, A.M. Al-Jabr and H.Y. Al-Ayied, 2013. Managing Invasive
Populations of Red Palm Weevil: A Worldwide Perspective. J. Food Agri. Environ., 11:
456-463.
Hussain, A., M.Y. Tian, Y.R. He and S. Ahmed, 2009. Entomopathogenic fungi disturbed the
larval growth and feeding performance of Ocinara varians Walker (Lepidoptera:
Bombycidae) larvae. Insect Sci., 16: 511-517.
Hussain, A., M.Y. Tian, Y.R. He, J.M. Bland and W.X. Gu, 2010 Behavioral and
electrophysiological responses of C. formosanus towards entomopathogenic fungal
volatiles. Biol. Control, 55: 166-173.
Hussain, A., S. Ahmed and M. Shahid, 2011. Laboratory and field evaluation of Metarhizium
anisopliae var. anisopliae for controlling subterranean termites. Neotrop. Entomol.,
40(2): 244-250.
Inglis, D.G., D.L. Johnson and M.S. Goettel, 1996. Effect of bait substrate and formulation on
infection of grasshopper nymphs by Beauveria bassiana. Biocontrol Sci. Tech., 6: 35-50.
Inglis, G.D., M.S. Goettel, T.M. Butt and H. Strasser, 2001. Use of hyphomycetous fungi for
managing insect pests. p 23-69. In: Butt, T.M., C.W. Jackson and N. Magan (eds), Fungi
as Biocontrol Agents: Progress, Problems and Potential. CABI International/ AAFC,
Wallingford, United Kingdom.
Jalinas, J., B. Güerri-Agulló, R.W. Mankin, R. López-follana and L.V. Lopez-Llorca, 2015.
Acoustic assessment of Beauveria bassiana (Hypocreales: Clavicipitaceae) effects on
Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) larval activity and mortality. J.
Eco. Entomol., 108(2): 444-453.
James, R.R., J.S. Buckner and T.P. Freeman, 2003. Cuticular lipids and silverleaf whitefly stage
affect conidial germination of Beauveria bassiana and Paecilomyces fumosoroseus. J.
Inver. Pathol., 84(2): 67-74.
Jaronski, S.T., 2010. Ecological factors in the inundative use of fungal entomopathogens.
Biocontrol, 55: 159-185.
100
Kassa, A., G. Zimmermann, D. Stephan and S. Vidal, 2002. Susceptibility of Sitophilus zeamais
(Motsch.) (Coleoptera: Curculionidae) and Prostephanus truncatus (Horn) (Coleoptera:
Bostrichidae) to entomopathogenic fungi from Ethiopia. Biocontrol Sci. Tech., 12: 727-
736.
Llácer, E. and J.A. Jacas, 2010. Short communication. Efficacy of phosphine as a fumigant
against Rhynchophorus ferrugineus (Coleoptera: Curculionidae) in palms. Spanish J.
Agri. Res., 8(3): 775-779.
Llácer, E., Ó. Dembilio and J.A. Jacas, 2010. Evaluation of the efficacy of an insecticidal paint
based on chlorpyrifos and pyriproxyfen in a microencapsulated formulation against
Rhynchophorus ferrugineus (Coleoptera: Curculionidae). J. Econ. Entomol., 103: 402-
408.
Lo Verde, G., L. Torta, V. Mondello, C.G. Caldarella, S. Burruano and V. Caleca, 2014.
Pathogenicity bioassays of isolates of Beauveria bassiana on Rhynchophorus
ferrugineus. Pest Manag. Sci., 71: 323-328.
Manachini, B., P. Lo Bue, E. Peri and S. Colazza, 2009. Potential effects of Bacillus
thuringiensis against adults and older larvae of Rhynchophorus ferrugineus. IOBC/ wprs
Bull., 45: 239-242.
Martín, M.M. and T. Cabello, 2006. Manejo de la cría del picudo rojo de la palmera,
Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera, Dryophthoridae), en dieta
artificial y efectos en su biometría y biología. Boletín de Sanidad Vegetal de Plagas, 32:
631-641.
Merghem, A., 2011. Susceptibility of the red palm weevil, Rhynchophorus ferrugineus (Olivier)
to the green muscardine fungus, Metarhizium anisopliae (Metsch.) in the laboratory and
in palm tree orchards. Egyp. J. Biol. Pest Control, 21: 179-183.
Mohan, C.M., K.A. Lakshmi and K.U. Devi, 1999. Laboratory evaluation of the pathogenicity of
three isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin on
the American cockroach (Periplaneta americana). Biocontrol Sci. Tech., 9(1): 29-33.
Moino Jr, A., S.B. Alves and R.M. Pereira, 1998. Efficacy of Beauveria bassiana (Balsamo)
Vuillemin isolates for control of stored grain pests. J. Appl. Entomol., 122: 301-305.
Monteiro, A.C., A.C. Fiorin and A.C.B. Correia, 1998a. Pathogenicity of isolates of Metarhizium
anisopliae (Metsch.) Sorokin towards the cattle tick Boophilus microplus (Can.) (Acari:
Ixodidae) under laboratory conditions. Rev. Microbiol., 29: 109-112.
Monteiro, S.G., W.R.E.P. Bittencourt, E. Daemon and J.L.H. Faccini, 1998b. Pathogenicity
under laboratory conditions of the fungi Beauveria bassiana and Metarhizium anisopliae
on larvae of the tick Rhipicephalus sanguineus (Acari: Ixodidae). Revista brasileira de
parasitologia veterinaria, 7: 113-116.
Murphy, S.T. and B.R. Briscoe, 1999. The red palm weevil as an alien invasive : Biology and the
prospect for biological control as component of IPM. Bio-control News Info., 20: 35-46.
Quesada-Moraga E, R. Santos-Quiros, P. Valverde-Garcia and C. Santiago-Álvarez, 2004.
Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium
anisopliae (anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). J.
Inver. Pathol., 87: 51-58.
Ricaño J., B. Güerri-Agulló, M.J. Serna-Sarriás, G. Rubio-Llorca, L. Asensio, P. Barranco, L.V.
Lopez-Llorca, 2013. Evaluation of the pathogenicity of multiple isolates of Beauveria
bassiana (Hypocreales: Clavicipitaceae) on Rhynchophorus ferrugineus (Coleoptera:
101
Dryophthoridae) for the assessment of a solid formulation under simulated field
conditions. Florida Entomol., 96: 1311-1324.
Sabbour, M.M. and N.Y. Solieman, 2014. Preliminary Investigations into the Biological Control
of Red Palm Weevil Rhynchophorus ferrugineus by using three isolates of the fungus
Lecanicillium (Verticillium) lecanii in Egypt. Inter. J. Sci. Res., 3(8): 2016-2066.
Salama, H.S., M.S. Foda, M.A. El-Bendary and A. Abdel-Razek, 2004. Infection of red palm
weevil Rhynchophorus ferrugineus, by spore-forming bacilli indigenous to its natural
habitat in Egypt. J. Pest Sci., 77: 27-31.
Schrank, A. and M.H. Vainstein, 2010. Metarhizium anisopliae enzymes and toxins. Toxicon,
56: 1267-1274.
Sevim, A., B.G. Donzelli, D. Wu, Z. Demirbaq, D.M. Gibson and B.G. Turqueon, 2012.
Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are
differentially expressed and corresponding mutants are decreased in virulence. Current
Genetics, 58: 79-92.
Sewify, G.H., M.H. Belal and S.A. Al-Awash, 2009. Use of the entomopathogenic fungus,
Beauveria bassiana for the biological control of the Red Palm Weevil, Rhynchophorus
ferrugineus Olivier. Egyp. J. Biol. Pest Control, 19(2):157-163.
Shah, F.A., M.A. Ansari, M. Prasad and T.M. Butt, 2007. Evaluation of black vine weevil
(Otiorhynchus sulcatus) control strategies using Metarhizium anisopliae with sublethal
doses of insecticides in disparate horticultural growing media. Biol. Control. 40: 246-252.
Shaiju-Simon, K.R.K. and C. Gokulapalan, 2003. Occurrence of Beauveria sp. on red palm
weevil, Rhynchophorus ferrugineus (Oliv.) of coconut. Insect Environ., 9: 66-67.
Shawir, M.S. and A.M. Al-Jabr, 2010. The infectivity of entomopathogenic fungi Beauveria
bassiana and Metarhizium anisopliae to Rhynchophorus ferrugineus (Olivier) stages
under laboratory conditions. Proceeding of the 4th International Date Palm Conference,
Acta Horti., 882: 431-436.
Singh, S.P., and P. Rethinam, 2005. Trapping-a major tactic of BEPM strategy of palm weevils.
Cord., 21(1): 57-79.
Torta, L., V. Leone, C.G. Caldarella, G. Lo Verde and S. Burruano, 2009. Microrganismi fungini
associati a Rhynchophorus ferrugineus (Olivier) in Sicilia e valutazione dell’efficacia
entomopatogena di 484 Ann Microbiol., 65: 477-485.
Van Driesche, R.G., M.S. Hoddle and T.D. Center, 2007. Use of Insect Pathogens as Pesticides
in Control of Pests and Weeds by Natural Enemies. p 443-462. In: Van Driesche, R.G.
and M.S. Hoddle (eds.). Center TD Forest Health Technology Enterprise Team, New
York.
Vega, F.E., N.V., Meyling, J.J. Luangsa-Ard, and M. Blackwell, 2012. Fungal
Entomopathogens. P. 171-220. In: Vega F.E. and H.K. Kaya (eds.). Insect Pathology
London: Elsevier.
Vitale, A, V. Leone, L. Torta, S, Burruano and G. Polizzi, 2009. Prove preliminari di lotta
biologica con Beauveria bassiana e Metarhizium anisopliae nei confronti del punteruolo
rosso. In Regione Siciliana - Assessorato Agricoltura e Foreste. La ricerca scientifica sul
Punteruolo rosso e gli altri fitofagi delle palme in Sicilia, Palermo, Italy, 1:169-172.
Wakil, W., M.U. Ghazanfar and M. Yasin, 2014. Naturally occurring entomopathogenic fungi
infecting stored grain insect species in Punjab, Pakistan. J. Insect Sci., 14(182): 1-7.
102
Zhang, H., W. Yin, J. Zhao, L. Jin, Y. Yang, S. Wu, B.E. Tabashnik and Y. Wu, 2011. Early
warning of cotton bollworm resistance associated with intensive planting of Bt cotton in
China. PLoS ONE, 6(8): e22874.
103
Table 5.1 Characterization of B. bassiana and M. anisopliae isolates obtained from soils and insect cadavers
*Geographical attributes based on web source indicating the nearest point
Species Isolate
No.
Host/substrate Location Geographical attributes
Altitude (m)* Latitude* Longitude*
Metarhizium anisopliae WG-02 Soil (Vegetables) Changa Manga 191 31°08′N 73°96′E
Metarhizium anisopliae WG-03 Tribolium castaneum Murree 2300 33°56'N 73°28'E
Metarhizium anisopliae WG-04 Soil (Vegetables) Chichawatni 159 30°53′N 72°70′E
Metarhizium anisopliae WG-05 Rhyzopertha dominica Khanewal 128 30°71'N 71°55'E
Metarhizium anisopliae WG-06 Soil (Forests) Lal Sohanra 114 29°28′N 71°58′E
Metarhizium anisopliae WG-07 Soil (Forests) Bahawalpur 109 29°24'N 71°40'E
Metarhizium anisopliae WG-10 Soil (Crop fields) Rawalpindi 497 33°58′N 73°08′E
Beauveria bassiana WG-11 Soil (Crop fields) Lal Sohanra 114 29°28′N 71°58′E
Beauveria bassiana WG-12 Soil (Fruits) Chichawatni 159 30°53′N 72°70′E
Beauveria bassiana WG-13 Sitophilus oryzae Changa Manga 191 31°08'N 73°96'E
Beauveria bassiana WG-15 Soil (Forests) Faisalabad 184 31°30′N 73°05′E
Beauveria bassiana WG-16 Tribolium castaneum Sargodha 193 32°10'N 72°40'E
Beauveria bassiana WG-17 Callosobruchus maculates Gujranwala 223 32°10'N 72°12'E
Beauveria bassiana WG-18 Soil (Forests) Rawalpindi 497 33°58′N 73°08′E
Beauveria bassiana WG-41 Rhynchophorus ferrugineus Layyah 143 30°58'N 70°56'E
Beauveria bassiana WG-42 Rhynchophorus ferrugineus Dera Ismail Khan 166 31°49'N 70°52'E
Beauveria bassiana WG-43 Rhynchophorus ferrugineus Bahawalpur 109 29°24'N 71°40'E
Metarhizium anisopliae WG-44 Rhynchophorus ferrugineus Layyah 143 30°58'N 70°56'E
Metarhizium anisopliae WG-45 Rhynchophorus ferrugineus Muzaffargarh 114 30°50'N 71°54'E
104
Table 5.2 Factorial analysis of screening and mycosis of R. ferrugineus exposed to B. d M.
anisopliae isolates
S.O.V.
Df
Mortality Mycosis
Larvae Adult Larvae Adult
F P F P F P F P
Treatment 1 158.20 ≤0.05 733.72 ≤0.05 178.89 ≤0.05 733.72 ≤0.05
Isolate 18 146.69 ≤0.05 110.84 ≤0.05 18.28 ≤0.05 110.84 ≤0.05
Treatment ×
Isolate
18 29.90 ≤0.05 8.49 ≤0.05 1.87 0.02 8.49 ≤0.05
Error 185 - - - - - - - -
Total 227 - - - - - - - -
Table 5.3 Percentage pathogenicity (%±SE) and mycosis (%±SE) of 19 isolates of B.
bassiana and M. anisopliae isolates against R. ferrugineus larvae after 12 days
post incubation
Isolate 1×107 Conidia ml-1 1×108 Conidia ml-1
% Mortality % Mycosis % Mortality % Mycosis
WG-02 3.49±0.21j 0.00±0.00j 8.01±0.66k 17.8±0.87h
WG-03 9.90±0.89ghij 3.50±0.42ij 25.23±1.12hi 47.16±1.68g
WG-04 18.35±1.31fghi 12.50±0.56gh 48.25±1.48ef 87.33±1.42bc
WG-05 21.69±1.17 33.33±0.71d 55.23±2.16de 93.66±1.35ab
WG-06 9.90±0.85ghij 15.83±0.94g 27.61±1.55ghi 63.83±1.90e
WG-07 13.75±1.09fghij 21.33±1.22f 35.63±1.62fgh 54.50±1.54f
WG-10 15.88±0.98fghij 25.83±0.94e 41.50±1.19efg 73.66±2.04d
WG-11 5.46±0.34ij 4.50±0.42i 24.12±1.38hij 42.50±1.17g
WG-12 8.96±1.09ghij 10.66±0.84h 29.92±1.52ghi 56.66±1.42f
WG-13 16.99±1.08fghij 31.33±1.22d 49.28±1.43ef 88.50±1.70bc
WG-15 4.27±0.24j 3.50±0.42ij 9.04±0.39jk 15.33±1.04h
WG-16 26.21±1.40def 56.66±1.38c 56.42±2.16cde 95.50±1.78a
WG-17 19.46±1.21efgh 32.16±1.25d 46.98±1.70ef 93.33±1.71ab
WG-18 7.68±0.53hij 9.50±0.76h 19.44±1.17ijk 41.66±1.28g
WG-41 53.71±1.28a 72.50±2.78a 88.33±2.47a 96.50±1.99a
WG-42 46.80±1.07ab 65.33±1.45b 79.04±1.54ab 95.66±1.62a
WG-43 35.52±1.55bcd 56.66±1.88c 71.27±1.69bc 87.33±1.38bc
WG-44 41.33±1.21abc 61.83±1.67b 74.68±2.56ab 84.66±1.67c
WG-45 32.11±1.60cde 53.50±1.99c 65.55±1.47bcd 90.66±1.73abc
105
Table 5.4 Percentage pathogenicity (%±SE) and mycosis (%±SE) of 19 isolates of B. M.
anisopliae isolates against R. ferrugineus adults after 12 days post incubation
Isolate 1x107 Conidia ml-1 1x108 Conidia ml-1
% Mortality % Mycosis % Mortality % Mycosis
WG-02 0.00±0.00i 0.00±00m 5.63±0.65k 4.83±0.30l
WG-03 4.44±0.22ghi 2.33±0.33lm 20.65±1.12hij 47.50±1.23i
WG-04 16.94±1.09def 8.50±0.42jk 32.06±1.37efgh 72.50±1.88ef
WG-05 21.58±1.05de 29.66±1.26g 38.88±1.88ef 74.16±2.10rf
WG-06 5.55±0.64ghi 6.66±0.66kl 21.82±1.00ghij 43.33±1.40i
WG-07 8.96±1.06fghi 11.50±0.69j 23.96±1.63ghi 56.50±1.52h
WG-10 10.15±1.10fghi 16.33±1.08i 27.54±1.42fghi 65.83±1.90g
WG-11 0.00±0.00i 0.00±00m 17.06±1.09ijk 30.33±1.08j
WG-12 3.33±0.37ghi 1.66±0.22m 21.74±1.29ghij 48.50±1.76i
WG-13 11.34±0.81efgh 17.50±1.08hi 35.47±1.18efgh 71.33±1.54fg
WG-15 0.00±0.00i 0.00±00m 8.968±0.88jk 16.16±1.10k
WG-16 21.50±1.21de 36.66±1.14f 43.49±2.01de 78.66±2.52de
WG-17 13.65±1.03efg 21.50±0.98h 36.58±1.85efg 77.50±1.85def
WG-18 2.22±0.24hi 0.00±00m 13.65±1.07ijk 31.16±1.24j
WG-41 41.90±1.88a 78.16±1.19a 75.95±2.78a 95.33±1.62a
WG-42 36.19±1.36ab 62.83±1.42b 69.04±2.73ab 87.16±2.12bc
WG-43 27.22±1.16bcd 55.83±1.37c 55.00±1.37bcd 91.50±2.14ab
WG-44 32.93±1.65abc 49.66±1.54d 59.68±2.93bc 83.33±1.70cd
WG-45 24.92±1.04cd 41.50±1.20e 46.03±2.07cde 76.83±1.44ef
106
Table 5.5 Factorial analysis for virulence of B. bassiana and M. anisopliae isolates against
larvae and adult of R. ferrugineus
S.O.V. df Larvae Adult
F P F P
Isolate 4 153.27 ≤0.05 163.71 ≤0.05
Treatment 3 2192.69 ≤0.05 2202.71 ≤0.05
Interval 2 2700.31 ≤0.05 2852.28 ≤0.05
Isolate × Treatment 12 1.24 0.25 1.94 0.02
Isolate × Interval 8 0.66 0.72 1.92 ≤0.05
Treatment × Interval 6 90.92 ≤0.05 78.87 ≤0.05
Isolate × Treatment ×
Interval
24 6.26 ≤0.05 5.60 ≤0.05
Error 472 - - - -
Total 539 - - - -
Table 5.6 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 7 days of
exposure treated with B. bassiana and M. anisopliae isolates
Stage Isolates Dose (Conidia ml-1)
106 107 108 109
Larvae
WG-41 19.52±1.11Ca 26.57±1.36Ca 47.17±2.12Ba 76.95±2.41Aa
WG-42 16.56±1.27Cab 21.23±1.41Cab 40.29±2.19Bab 65.73±2.28Ab
WG-43 11.21±1.03Cab 15.82±1.25Cb 28.79±1.53Bcd 52.48±1.78Ac
WG-44 13.43±1.32Cab 19.75±1.37Cab 36.60±1.26Bbc 58.62±1.90Abc
WG-45 8.14±1.15Cb 13.49±1.20Cb 22.71±1.04Bd 43.54±1.50Ad
Adult
WG-41 12.69±1.32Ca 19.47±1.23Ca 38.30±1.11Ba 63.96±1.78Aa
WG-42 9.68±1.10Cab 15.02±1.28Cab 32.22±1.69Bab 54.76±1.66Ab
WG-43 5.92±1.03Cab 10.47±1.08Cbc 21.79±1.45Bcd 40.63±1.62 Acd
WG-44 8.14±1.20Cab 12.69±1.19Cabc 26.98±1.17Bbc 47.35±2.09Abc
WG-45 2.91±0.45Cb 7.46±0.76Cc 18.73±1.14Bd 35.23±1.56Ac
107
Table 5.7 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 14 days of
exposure treated with B. bassiana and M. anisopliae isolates
Stage Isolates Dose (Conidia ml-1)
106 107 108 109
Larvae
WG-41 37.35±1.19Ca 58.83±2.22Ba 93.28±1.92Aa 100.0±0.00Aa
WG-42 31.95±1.37Dab 49.68±2.06Cab 84.70±2.34Bab 98.46±2.01Aab
WG-43 23.49±1.26Dbc 38.78±1.37Cc 73.12±2.58Bc 92.38±1.78Ab
WG-44 27.46±1.18Dbc 43.49±1.73Cbc 78.46±2.84Bbc 95.45±1.59Aab
WG-45 19.78±1.07Dc 35.02±1.26Cc 71.69±2.19Bc 84.60±1.34Ac
Adult
WG-41 28.04±1.14Ca 47.40±1.60Ba 84.76±2.90Aa 93.22±1.97Aa
WG-42 21.27±1.46Dab 41.16±1.34Cab 76.98±2.51Bab 86.87±2.26Aa
WG-43 14.44±1.08Cbc 35.82±1.34Bbc 67.19±1.98Abc 75.29±2.49Abc
WG-44 17.46±1.19Db 38.14±1.48Cbc 70.05±1.54Bb 83.12±2.10Aab
WG-45 8.30±0.78Dc 32.06±1.96Cc 57.24±1.46Bc 71.53±1.51Ac
Table 5.8 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 21 days of
exposure treated with B. bassiana and M. anisopliae
Stage Isolates Dose (Conidia ml-1)
106 107 108 109
Larvae
WG-41 57.67±2.00Ca 88.50±2.15Ba 100.0±0.00Aa 100.0±0.00Aa
WG-42 52.16±1.56Cab 83.16±2.00Bab 100.0±0.00Aa 100.0±0.00Aa
WG-43 37.54±1.34Cc 65.93±2.16Bc 94.70±1.86Aa 100.0±0.00Aa
WG-44 46.88±2.09Cb 77.75±2.86Bb 98.41±1.64Aa 100.0±0.00Aa
WG-45 31.40±1.31Dc 59.10±2.00Cc 85.49±2.47Bb 100.0±0.00Aa
Adult
WG-41 49.78±2.05Ca 80.79±2.73Ba 100.0±0.00Aa 100.0±0.00Aa
WG-42 41.48±1.34Cab 72.32±2.62Bab 96.98±2.13Aab 100.0±0.00Aa
WG-43 30.74±1.94Dcd 59.15±1.93Cc 85.55±2.27Bc 98.41±1.94Aa
WG-44 38.30±1.55Dbc 68.62±2.32Cb 91.53±2.55Bbc 100.0±0.00Aa
WG-45 22.22±1.29Dd 43.86±1.61Cd 76.08±2.15Bd 93.81±2.40Ab
108
Table 5.9 LC50 and LC90 values of B. bassiana and M. anisopliae isolates tested against larvae and adult R. ferrugineus
Stage Isolate LC50 (Conidia ml-1) (CI) LC90
(Conidia ml-1) (CI) Slope Intercept ᵪ2 (df = 2) P
Larvae
WG-41 6.42×105 (3.61×105-9.49×105) 6.52×106 (4.51×106-1.03×107) 0.54±0.07 -6.84 0.53 <0.01
WG-42 9.46×105 (5.67×105-1.39×106) 1.34×107 (8.95×106-2.36×107) 0.47±0.05 -7.95 3.56 <0.01
WG-43 2.22×106 (1.50×106-3.11×106) 3.90×107 (2.52×107-6.81×107) 0.44±0.06 -10.11 6.98 <0.01
WG-44 1.24×106 (7.92×105-1.76×106) 1.81×107 (1.20×107-3.15×107) 0.46±0.05 -8.65 6.05 <0.01
WG-45 3.63×106 (2.41×106-5.22×106) 1.01×108 (6.21×107-1.80×108) 0.37±0.04 -10.81 4.07 <0.01
Adult
WG-41 1.04×106 (6.35×105-1.51×106) 1.53×107 (1.02×105-6.81×104) 0.47±0.05 -8.21 4.68 <0.01
WG-42 1.61×106 (1.02×106-2.35×106) 3.34×107 (2.16×107-5.95×107) 0.42±0.06 -9.44 3.27 <0.01
WG-43 3.92×106 (2.61×106-5.64×106) 1.23×108 (7.55×107-2.30×108) 0.37±0.04 -10.9 4.62 <0.01
WG-44 2.13×106 (1.33×106-3.15×106) 6.25×107 (3.90×107-1.15×108) 0.37±0.03 -9.88 2.16 <0.01
WG-45 9.28×106 (6.51×106-1.25×107) 2.63×108 (1.60×108-5.02×108) 0.38±0.03 -12.24 10.55 <0.01
109
Table 5.10 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested against larvae of R. ferrugineus
Isolate Dose LT50 (Conidia ml-1)
(CI)
LT90 (Conidia ml-1)
(CI)
Slope Intercept x2 (df =2) P
WG-41
106 17.75 (15.90-20.33) 35.70 (30.34-45.82) 0.07± 0.01 -7.07 0.00 ˂0.01
107 11.62 (10.41-12.69) 21.79 (20.04-24.26) 0.12± 0.01 -8.02 0.30 ˂0.01
108 7.14 (6.07-7.98) 12.89 (11.88-14.33) 0.22± 0.02 -6.15 0.16 ˂0.01
109 6.01* 7.66* 0.77±108.49 -0.01 0.00 0.99
WG-42
106 19.64 (17.55-22.96) 38.08 (32.03-49.87) 0.06±0.01 -7.43 0.03 ˂0.01
107 13.24 (12.09-14.35) 23.84 (21.86-26.69) 0.12±0.01 -8.70 0.57 ˂0.01
108 8.16 (7.10-9.03) 14.77 (13.66-16.28) 0.19±0.02 -7.01 1.39 ˂0.01
109 5.27 (3.61-6.26) 10.40 (9.45-11.95) 0.24±0.04 -3.74 0.00 ˂0.01
WG-43
106 25.22 (21.64-32.94) 47.08 (37.54-69.54) 0.05±0.01 -7.59 0.01 ˂0.01
107 16.35 (15.00-17.94) 29.66 (26.45- 34.79 0.09±0.01 -8.48 0.00 ˂0.01
108 10.07 (8.96-11.03) 18.41 (17.08-20.19) 0.15±0.01 -8.06 0.24 ˂0.01
109 6.53 (5.19-7.52) 12.95 (11.88-14.48) 0.19±0.02 -5.22 0.34 ˂0.01
WG-44
106 21.53 (19.11-25.73) 40.25 (33.55-53.72) 0.06±0.01 -7.77 0.04 ˂0.01
107 14.34 (13.15-15.57) 25.88 (23.53-29.39) 0.11±0.01 -8.67 0.94 ˂0.01
108 8.85 (7.72-9.79) 16.44 (15.23-18.08) 0.16±0.01 -7.33 0.44 ˂0.01
109 5.81 (4.30-6.84) 11.89 (10.85-13.42 0.21±0.02 -4.45 0.20 ˂0.01
WG-45
106 27.92 (23.52-38.35) 50.31 (39.43-77.72) 0.05±0.01 -7.80 0.11 ˂0.01
107 17.86 (16.35-19.85) 32.19 (28.33-38.66) 0.08±0.01 -8.46 0.02 ˂0.01 108 11.21 (9.98-12.29) 21.19 (19.54-23.50) 0.12±0.01 -7.87 7.41 ˂0.01
109 7.82 (6.66-8.75) 14.74 (13.61-16.30) 0.18±0.02 -6.51 1.66 ˂0.01
*Unable to estimate confidence limits from the data
110
Table 5.11 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested against adult of R. ferrugineus
Isolate Dose LT50 (Conidia ml-1)
(CI)
LT90 (Conidia ml-1)
(CI)
Slope Intercept x2 (df = 2) P
WG-41
106 20.41 (18.46-23.42) 36.38 (31.26-45.68) 0.08± 0.01 -8.43 0.00 ˂0.01
107 13.89 (12.77-15.00 24.38 (22.37-27.26) 0.12±0.01 -9.09 0.24 ˂0.01
108 8.47 (7.49-9.29) 14.79 (13.71-16.25) 0.20±0.02 -7.49 1.15 ˂0.01
109 4.93 (2.82-6.29) 12.35 (11.19-14.02) 0.17±0.02 -3.41 0.91 ˂0.01
WG-42
106 23.17 (20.65-27.56) 39.98 (33.70-52.11) 0.07±0.01 -8.61 0.031 ˂0.01
107 15.55 (14.40-16.81) 26.73 (24.35-30.27) 0.11±0.01 -9.34 0.00 ˂0.01
108 9.53 (8.46-10.45) 17.20 (15.97-18.83) 0.16±0.01 -7.97 0.04 ˂0.01
109 6.36 (4.71-7.53) 14.12 (12.91-15.82) 0.16±0.02 -4.72 1.88 ˂0.01
WG-43
106 26.96 (23.45-34.03) 44.57 (36.59-61.63) 0.07±0.01 -8.67 0.00 ˂0.01
107 17.90 (16.55-19.61) 30.46 (27.25-35.54) 0.10±0.01 -9.23 1.03 ˂0.01
108 11.81 (10.66-12.84) 21.50 (19.87-23.76) 0.13±0.01 -8.43 4.45 ˂0.01
109 8.68 (7.43-9.69) 16.89 (15.59-18.66) 0.15± 0.01 -6.82 1.93 ˂0.01
WG-44
106 24.25 (21.55-29.12) 40.79 (34.31-53.47) 0.07±0.01 -8.80 0.22 ˂0.01
107 16.38 (15.20-17.72) 27.69 (25.15-31.48) 0.11±0.01 -9.52 0.04 ˂0.01
108 10.72 (9.59-11.71) 19.63 (18.19-21.58 0.14±0.01 -8.20 0.98 ˂0.01
109 7.52 (6.21-8.54) 14.99 (13.80-16.64) 0.17±0.01 -5.97 2.61 ˂0.01
WG-45
106 30.06 (25.65-39.89) 47.23 (38.10-68.46) 0.07± 0.01 -8.60 0.03 ˂0.01
107 21.17 (19.18-24.31) 36.51 (31.45-45.68) 0.08±0.01 -8.67 4.74 ˂0.01 108 13.54 (12.32-14.72) 24.90 (22.70-28.12) 0.11±0.01 -8.34 3.70 ˂0.01
109 9.57 (8.268-10.66) 18.97 (17.48-21.02) 0.13±0.01 -7.02 0.00 ˂0.01
111
CHAPTER 6
Combined effectiveness of endophytically colonized Beauveria bassiana and Bacillus
thuringiensis against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)
Abstract Research study was carried out to investigate the insecticidal properties of endophytically
colonized Beauveria bassiana and Bacillus thuringiensis var. kurstaki (Bt-k) against 2nd, 4th and
6th instar larva of red palm weevil (RPW) Rhynchophorus ferrugineus. Initially five isolates of B.
bassiana (WG-11, WG-40, WG-41, WG-42 and WG-43) were screened by inoculating
endophytically in date palm leaf petioles. Only one B. bassiana isolate (WG-41) recovered from
up to the 10 cm after 30 days during both years was considered to be effective. Both agents were
applied alone and in combination to tested instars and pupation, adult emergence and egg
eclosion was recorded from survivers. Moreover development, diet consumption, frass
production and weight gain were also observed. Mortality was low in sole treatments, while in
combined treatments increase in mortality, decrease in pupation, adult emergence and egg
eclosion found inversely correlated to toxic levels of both microbial agents. Second instar larvae
exhibited more susceptibility followed by 4th and 6th instar larvae. Synergistic effect (CTF≥20)
on the mortality was observed when larvae were exposed to simultaneous application of WG-41
with 40 µg ml-1 of Bt-k in case of all three larval instars tested. All the tested instars exhibited
varying level of growth and development when exposed to the sub-lethal doses of palm petiole
piece (6 cm away from inoculated point) inoculated with WG-41 and dipped in Bt-k
concentrations; moreover significant variations were recorded for larval duration, larval weight,
pupal duration, pupal weight, pre-pupal duration, pre-pupal weight, adult longevity (male and
female) and adult weight (male and female). The toxic nature of microbial agents also influenced
the frass production and diet consumption. Larvae treated with Bt-k gained more weight than the
WG-41 and their combined application. Initial weight of larvae exerted its impact on the weight
gain and diet consumption and the trend was found linked to pathogenicity of applied agents. It
can be surmised from the findings that microbial agents exhibit a reliable level of mortality
against R. ferrugineus. Hence it would be fruitful to replace the conventional reliance on
chemical approaches.
Keywords: Rhynchophorus ferrugineus, Beauveria bassiana, Bacillus thuringiensis,
development, diet consumption, frass production
112
6.1 Introduction
The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) (Coleoptera:
Curculionidae) is considered one of the most destructive pests of variety of palm species,
including date palms (Giblin-Davis, 2001). The weevil lives and breed into the tree trunk and
devastate its vascular system which lead to the tree collapse and death of host plant. Until 1917,
it was considered that RPW is the only pest of coconut palm but latter on it was found
devastating date palms in Pakistan, India and Iraq (Mohan, 1917; Milne, 1918; Buxton, 1920). In
Pakistan infestation of the RPW was observed during 1913 when researcher found the beetle
attack on the date palm trees imported from Middle East and later on Milne an Economist of
Punjab Agriculture College, Lyallpur (Presently University of Agriculture, Faisalabad, Pakistan)
observed RPW infestation in date palms and collected insect specimens from Multan,
Muzaffargarh and Dera Ghazi Khan Districts of Punjab (Milne, 1918). Since last 30 years RPW
has caused huge economic losses and no effective control measure has been invented so for
(Murphy and Briscoe, 1999; Faleiro, 2006).
For the successful control of RPW trapping, deployment of chemical insecticides and
fumigants has been considered a core component since long time. The unwise use of these
chemical insecticides and fumigants lead the resistance against this voracious pest which lead the
researchers to search for the alternative control strategies which are safer for human beings and
compatible to environment (Abraham et al., 1998). Moreover, RPW live in the self-made tunnels
which make them less vulnerable to Chemical and mechanical control. Thus, self-pathogens have
been proposed for the successful control of RPW (Dangar, 1997). Entomopathogens are the
safer, environment friendly and economic alternative to the chemical insecticides and getting
serious attention against RPW control. Fungal entomopathogens play a key role in managing
plant pathogens and herbivorous insects by improving the plant host defense mechanism or by
directly affecting plant pathogens (Sivasithamparam, 1998; Arnold et al., 2003).
Entomopathogenic fungi are preferred to the other entomopathogens due to its unique mode of
action by infecting their host through contact action and penetrating into insect hemocoel by
breaching the host cuticle. Fungal infection can be transformed by direct contact of infected
individuals to the healthy ones or subsequent development via new generation of spores (Lacey
et al., 1999; Quesada-Moraga et al., 2004).
On the other hand, an entomopathogenic bacterium from the genus Bacillus is also an
economical and potential alternative to the chemical insecticides. They are key antagonists of
insect pests of economic importance, their products and by products (Salama et al., 2004). It is
often an integral part of products used in biological control strategies worldwide, about 95%
microbial pesticides being used globally are bacterial in origin with annual sale of about $100
million (Federici et al., 2006). A number of species of this genus particularly Bacillus
thuringiensis (Berliner) are frequently used against vast array of insect pests from the order
Coleoptera, Lepidoptera and Diptera etc, which exhibit specificity towards the host and specific
stage of the host (Salama et al., 2004). It is a gram-positive soil bacterium, spore forming
mesophile having ability to produce proteinaceous parasporal inclusions during sporulation. It
produces δ-endotoxins in the form parasporal crystals which may vary from one to several types
and formed from different δ-endotoxins that are related to each other (Aronson et al., 1986).
So for more than 350 genes from B. thuringiensis has been discovered which are encoded for
specific toxic proteins which are specific to the larvae of various orders (Schnepf et al., 1998).
Both pathogens are widely distributed in the environment and found from the soil of
different origin and insect cadavers (Martin and Travers, 1989). The intervention of more than
113
one biocontrol agent can enhance the effectiveness of the other partner, many studies have been
conducted in this regard. The combined effect of B. bassiana and B. thuringiensis working
synergistically delivers more harm to insect pests (Wraight and Ramos, 2005) and hence can be a
hint for those willing to manage it. Combine effect of B. thuringiensis and Entomopathogenic
fungi has synergistic effect performed by several researchers (Navon, 2000). Wraight and Ramos
(2005) observed that combinations of B. thuringiensis and B. bassiana have been successful in
increasing mortality in some insects. Sandner and Cichy (1967) applied a mixture of B.
thuringiensis var. kurstaki (Bt-k) and B. bassiana against larvae of the Mediterranean flour moth.
Results revealed that the two agents acted independently (mean mortalities from B. bassiana, B.
thuringiensis and the mixture were 57, 44, and 71% respectively).
The present study aiming at the endophytically colonizing B. bassiana isolates in date
palm and their evaluation against R. ferrugineus alone and in integrated manners with B.
thuringiensis
6.2 Materials and Methods
6.2.1 RPW collection and rearing
Different developmental stages (larvae, pupae and adult) of RPW were collected from
fallen and infested date palm trees with the permission of farmers (owners) from date palm
growing areas of west Punjab, Pakistan. During collections, adult, larvae and pupae were kept in
separate plastic jars until brought to the laboratory Microbial Control Laboratory, Department of
Entomology, University of Agriculture, Faisalabad, Pakistan. After arriving to the laboratory
larvae were provided with sugarcane (Saccharum officinarum L.; Poales: Poaceae) stems for
feeding and pupation, while adults were offered shredded sugarcane pieces for feeding and
oviposition substrates. On pupation, pupal cocoons were kept in separate boxes for adult
emergence in incubator at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod. As adults
emerged, they were transferred to the adult’s jars for feeding and mating with shredded sugracne
pieces. Colony was developed in plastic boxes (15×30×30 cm) having a lid covered with mesh
wire gauze (60 mesh size) in the middle (7 cm diameter) for aeration. Rearing was carried out in
Microbial Control Laboratory. The rearing conditions were maintained as mentioned above.
Adult’s diet was changed after every three days and replaced sugarcane pieces were kept in
separate jars (8×8×12 cm) for egg hatching. On egg hatching, neonate larvae were allowed to
feed for 3 days in the same sugarcane set and then were transferred to the sugarcane sets for
feeding and pupation. Larvae were shifted to the new sugarcane sets biweekly until pupation.
6.2.2 Fungal isolates
Five isolates of B. bassiana used in the study belonging to the culture collection of
Microbial Control Laboratory, originally isolated from soil samples from crop fields (WG-11),
recovered endophytically from tomato plants (WG-40) and R. ferrugineus cadavers (WG-41,
WG-42 and WG-43). The cultures were maintained continuously on slants and sub-cultured for
14 days at 25±2 °C under a 12:12 (D: L) hours, tubes were tightly sealed and the culture was
stored at 4 °C. Mass culturing was done by inoculating Petri plates containing Sabourad
Dextrose Agar (SDA, from BD, Becton, Dickinson and Company Spark, MD 21152 USA)
media. Spore concentration of 1×106 spore ml-1 was determined with a Neubauer
haemocytometer.
114
6.2.3 Preparation of Bacillus thuringiensis spore-crystal mixtures
The commercial formulation of B. thuringiensis var. kurstaki (Bt-k) was obtained from
Microbial Control Laboratory, originally obtained from National Center for Genetic Engineering
and Biotechnology (BIOTEC) in Thailand. This strain was then subjected to sporulation by
culturing in 50 ml nutrient broth media. Harvesting of culture was carried out by centrifugation
at 6000 rpm for 15 min (Crecchio and Stotzky, 2001; Hernández et al., 2005). The pellets formed
resultantly were washed twice in cold 1M NaCl and thrice in sterile distilled water (SDW), re-
suspended in distilled water (5 ml). From the suspension formed, 1 ml was centrifuged for 5 min
at 10,000 rpm, dried for 4 hours at 37 °C and weighed (Wakil et al., 2013).
6.2.4 Screening of fugal isolates
Date palm trees less than one year old were selected in the date palm plantations located
at Faisalabad during 2014 and 2015. Inoculation of B. bassiana isolates were carried out
following the method of Gómez-Vidal et al., (2006). From each palm, three petioles were rubbed
with 70% ethanol and 30 µl of conidial suspension was injected using insulin syringe. To avoid
sun drying and external contamination the inoculated area were wrapped with Parafilm. For re-
isolation, after 15 and 30 days post inoculation, petioles were sampled from inoculation site, 2, 4,
6, 8 and 10 cm above or below the inoculation site with the help of sterilize cork borer (0.6 cm
diameter). The samples were surface-sterilized with 1% sodium hypochlorite for 1 min, followed
by rinsing three times with sterile distilled water (Gómez-Vidal et al., 2006). Samples were then
placed on PDA for 8 days at 25±2 ͦ C for spore germination. Different fungi were recovered from
petiole pieces that were further purified by sub-culturing on SDA medium, and fungal isolates
were identified following the keys developed by Barnett and Hunter (1999).
6.2.5 Bioassay procedure
To evaluate the effect of endophytically colonized B. bassiana WG-41, a piece of
inoculated palm petiole (2×2 cm2), 2 cm away from the inoculated site was offered to the 2nd, 4th
and 6th instar larvae of R. ferrugineus separately in 150 ml plastic cups measuring (6×6 cm)
individually. While in control treatment untreated palm petiole was offered to the respective
larval stages. The larvae were allowed to feed on palm piece for 48 hours and then shifted to
artificial diet (Martín and Cabello, 2006) for rest of the period and provided with fresh diet every
day. For Bt-k treatments, three concentrations (30, 40 and 50 µg ml-1) were prepared. An
untreated piece of date palm petiole (2×2 cm2) immersed in respective doses of Bt-k suspension
for 90s and offered to the respective larval stages individually. The larvae were allowed to feed
on treated palm piece for 48 hours and shifted to the artificial diet for rest of the period
individually. In combined treatments WG-41 inoculated date palm petiole piece was immersed in
respective doses of Bt-k suspensions for 90s and then offered to the larvae. After last instar dry
coir was provided for pupation to the last instar larvae. Larval mortality was counted daily until
larvae pupated or died. Larvae that failed to respond on slight prodding by blunt needle were
considered as dead. From the surviving individual percent pupation, adult emergence and egg
eclosion were also recorded. Three replicates of ten larvae were used for each treatment and
same count of larvae fed on normal petiole served as untreated check. All the treatments were
incubated at 27±2 °C and 65±5 % RH and a 12: 12 (D: L) hours photoperiod in an incubator
(Sanyo, Japan). Entire experiment was repeated thrice.
115
6.2.6 Bioassay on development of R. ferrugineus
To determine the effect of sub-lethal doses of WG-41 and Bt-k on developmental
parameters viz. larval duration, larval weight, pre-pupal duration, pre-pupal weight, adult
longevity (male and female) and adult weight (male and female) was recorded on 4th instar larvae
of R. ferrugineus. The piece of endophytically colonized WG-41 (6 cm away from inoculation
site) and Bt-k (10, 15 and 20 µg ml-1) were applied alone and in combination against 4 th instar
larvae and maintained at above motioned conditions. The larvae were allowed to feed on treated
petiole piece for 48 hours and then shifted to the artificial diet for rest of the period. After last
instar larvae was provided with dry coir (coconut coir) for pupal development. All the above
mentioned parameters were observed hereafter.
6.2.7 Bioassay on larval development
A new batch of 10th instar larvae of R. ferrugineus (L10) were encountered with sub-lethal
dose of WG-41 and Bt-k (10 µg ml-1). A piece of endophytically colonized palm petiole (6 cm
away from inoculated area) alone and in combination with Bt-k was offered to the larvae. The
larvae were allowed to feed for the whole 10th instar on the treated diet. Before exposure to the
palm petiole each larval instar were weighed and transferred to rearing vial with palm piece.
Every day until the larvae pupated or died; larvae were changed to new cups individually and
provided with fresh diet every day. Frass produced during this period was separated from vials
using tip of fine camel hair brush and weighed. Diet left unused in each vial was recovered, dried
in drying oven at 80 °C. Prior to assay, diet in 30 cups was dried to obtain an estimate of the dry
weight. Diet consumption of each larva was determined by subtracting the after feeding mass of
diet from before feeding mass. Moreover, frass production and weight gains during this period
were also determined.
6.2.8 Statistical analysis
Mortality for each treatment was corrected for control mortality using Abbott's (1925)
formula and subjected to one way analysis of variance (ANOVA) in Minitab (Minitab, 2002) and
means were separated using Tukey’s Kramer test (HSD) (Sokal and Rohlf, 1995) at 5%
significance level. Type of interaction among combined treatments of Bt-k and WG-41 was
determined by equation; CTF = (Oc-Oe)/ Oe×100, where CTF is the co-toxicity factor, Oc is the
observed mortality (%) in combined application, and Oe the expected mortality (%), that is the
sum of individual mortality (%) encountered in each of the treatments used in the combination
(Mansour et al., 1966). The interactions were categorized as additive, synergistic or antagonistic:
CTF≥20 meaning synergism, CTF>20 - –20 meaning additive, and CTF<-20 meaning
antagonism (Mansour et al., 1966; Wakil et al., 2012). To inspect the impact of microbial agents
on the diet consumption, weight gain and frass production were analyzed by ANCOVA using
initial larval weight and diet consumption as covariates (Janmaat et al., 2014).
6.3 Results
6.3.1 Fungal colonization of date palm petioles
All the fungal isolates successfully colonized endophytically in date palm petioles more
or less from the inoculation point during both years. At the injection site hydrophobic and
necrotic lesions appeared above and below the injection site. Different entomopathogenic fungi
were recovered from the site of injection up to 10 cm above and below. At the injection site all
116
the inoculated fungi were recovered (90-100%) from all the plants after 15 days of inoculation
during 2014 and (80-100%) following year (Table 6.1). B. bassiana isolate WG-41 was
recovered 10 cm above and below the injection site, while no entomopathogenic fungi were
recovered from non-inoculating petioles. Non-entomopathogenic fungi were less near the
inoculation site and gradually increased towards the ends in both years.
6.3.2 Toxicity of microbial agents
Toxicity assay was conducted on 2nd, 4th and 6th instar larvae of R. ferrugineus by
deploying B. bassiana inoculated date palm piece and Bt-k alone and in integrated manners.
Significant differences (Table 6.2) were recorded for mortality among different treatments and
larval instars (treatment: F7, 172 =201.87, P≤0.05; instar: F2, 161=63.34, P≤0.05) but non-
significant interaction was recorded for (instar × treatment: F14, 161=1.56, P=0.093). Synergistic
effect (CTF≥20) on the mortality was observed when larvae were exposed to simultaneous
application of endophytic B. bassiana and 30 µg of Bt-k in case of all the three instars tested.
Second instar larvae of R. ferrugineus were more susceptible to both pathogenic agents followed
by 4th and 6th instar larvae in all the treatments tested. Highest level of larval mortality
(83.17±2.28%) was observed in second instar larvae with the simultaneous application of B.
bassiana and Bt-k (50 µg ml-1) followed by 71.01±2.39% when treated with B. bassiana and Bt-k
(40 µg ml-1) and 54.63±2.26% when treated with B. bassiana and Bt-k (30 µg ml-1) (Table 6.3).
Additive effect (CTF≤20) was recorded when tested instar were treated with low and high dose
of Bt-k in integration with endophytic B. bassiana. A similar trend in mortality was recorded for
4th and 6th instar larvae of R. ferrugineus while mortality was found increasing with increase of
concentration of Bt-k. Combined application of endophytic B. bassiana and Bt-k proved more
fatal to all the instar larvae as compared to their sole application. Both microbial agents were
found working additively and synergistically. Percent pupation, adult emergence and egg
eclosion from surviving individuals was found inversely correlated to toxic level of microbial
agents in all instar larvae. Increase in mortality, while decrease in pupation, adult emergence and
egg eclosion was found in concentration dependent manner.
6.3.3. Development of R. ferrugineus
Development of 4th instar larvae was adversely affected by the toxic effect of microbial
agents. When larvae were exposed to different concentrations of Bt-mixed diets and B. bassiana,
significant variations were recorded for larval duration, larval weight, pre-pupal duration, pre-
pupal weight, pupal duration, pupal weight, adult longevity and adult and adult weight (larval
duration: F7, 71 =72.73, P≤0.05; larval weight F7, 71 =47.54, P≤0.05; pre-pupal duration: F7, 71
=53.12, P≤0.05; pre-pupal weight: F7, 71 =33.80, P≤0.05; pupal duration F7, 71 =43.95, P≤0.05;
pupal weight F7, 71 =26.61, P≤0.05; adult longevity (female F7, 71 =58.20, P≤0.05 and male F7, 71
=65.03, P≤0.05); adult weight (male F7, 71 =12.50, P≤0.05 and female F7, 71 =7.76, P≤0.05).
Increase in larval and pupal duration while decrease in pupal weight and adult duration was
recorded depending upon the lethal action of the applied agent. When compared to control,
significantly increased larval and pupal duration was recorded for combined application of Bb
and highest concentration of Bt-k followed by Bb and middle concentration of Bt-k. Similarly
extended pre-pupal and pupal period and reduced adult life span was recorded in concentration
dependent manner. While decrease in pre-pupal and adult weight was also recorded in
concentration dependent manners (Table 6.5).
117
6.3.4 Effect on larval development
Diet consumption by 10th instar larvae was significantly influenced by the treatments
applied diet consumption was low in combined treatments as compared to sole applications.
Least diet consumption was recorded for combined treatments followed by B. bassiana and Bt-k.
While, highest diet consumption was recorded for the control treatment (Fig 6.1a). Frass
production was influenced by treatments applied with lowest frass production for combined
treatments of Bt-k and B. bassiana from (0.61±0.05 to 0.00±0.00g) during experimental period.
More frass production was recorded during the initial days of treatments which gradually
decreased to zero before pupation. On the other hand, highest level of frass production was found
in untreated larvae for all the period of last instar larvae till pupation (Fig 6.1b). Larvae treated
with sub-lethal concentrations of B. bassiana and Bt-k gained more weight as compared to their
combined application (Single agent versus combined treatment). Initial weight of larvae (10th
instar: 4.35±0.12g) exerted its impact on the weight gain and among treatments, there was a
trend of weight gain linked to pathogenicity. Combined application of B. bassiana and Bt-k had
an adverse impact on the weight gain and lowest gain (-0.54±.04g) was recorded for the
combined treatment while highest gain (-0.11±.02g) was recorded in untreated larvae (Fig 6.1c).
6.4 Discussion
The endophytic colonization by B. bassiana is commonly practiced to combat voracious
pests of different field crops. Endophytic fungi are also capable of colonizing date palm tissues
after petiole wounding. The B. bassiana isolate WG-41 was recovered from up to 10 cm away
from the inoculation site during both years. The results confirmed that entomopathogenic fungi
survived endophytically and colonized petiole tissue of date palms. Gómez-Vidal et al., (2006)
also confirmed the endophytic colonization of entomopathogenic fungi in live and detached palm
petioles of date palm. They confirmed the movement in the parenchyma and sparsely within
vascular tissue using microscopy techniques.
An effective remedy to combat voracious insect pests is influenced by a number of
factors which includes the toxic nature, compatibility to other control agents, speed of kill,
feeding deterrence to insect, effect on development of insect, acceptance to insect and
environmental persistence. Integration of two or more entomopathogens to fight against insect
pests may brighten the chances of targeting multiple hosts (Pingel and Lewis, 1999). Marzban et
al. (2009) reported that the integration of two or more myco-pathogens interact positively than
their individual effect. In most combinations the virulence of an agent is rectified by the action of
the other agent which resultantly increases the speed of kill, retard growth, feeding sesation,
improve virulence and broaden host range.
The findings of our study revealed higher mortality levels of RPW in combined
treatments of B. bassiana inoculated date palm piece and Bt-k as compared to their sole
application. Moreover, these deterrent effects increased with the increase of number of Bt spores.
Similar findings were revealed by Khalique and Ahmad (2002), who reported the extended larval
duration with the increase in Bt-k concentration. While, larvae in check treatments consumed
more food which are in accordance with the findings of Marzban et al. (2009). The studies of Ma
et al. (2008) and Marzban et al. (2009) also support our findings who reported the growth
retardation of Ostrinia furnacalis and H. armigera when challenged with Cry1Ac from Bt-
treated diets and combined action of Bt (Cry1Ac) and HaCPV respectively.
The mode of action of entomopathogens is the key to success factor which make them fit
for the use in IPM program to combat any insect pest. For infection to occur Bt toxins attach to
118
the specific bindings sites of the insect’s midgut which then leads to cell lysis. This lysis may
result in the insect to stop feeding, become lethargic and ultimately die (Marzban et al., 2009).
While B. bassiana exhibit novel mode of action by adhering to the insect cuticle; later on
breaching the insect cuticle with the aid of enzyme complexes (beauvericin, bassianolide and
oosporein) followed by infection to the hemocoel where fungal conidia germinate and proliferate
in the insect body resultanting in insect death (Vey and Fargues, 1977). In combined treatments
of Bt and B. bassiana both agents work synergistically weakening the insect and affecting the
insect immune response to allow entomopathogens to infect the host more efficiently. Moreover,
conidial concentrations of both agents are the key factor in the degree of disease severity.
B. thuringiensis is rather quick in action which gets amplified when a larger number of
fungal spores are available to target the insect host. When B. bassiana gains access to the insect
gut, it boosts the infection of Bt toxins. In this way both agents help each other in retardation of
normal physiological functions of an insect host. These findings are further supported by Allee et
al. (1990) who found B. bassiana germinating and invading the insect favors the Bt toxins to
increase severity in grubs of Colorado potato beetle. Synergistic interaction was reported by
Wraight and Ramos (2005) when B. bassiana (GHA) and Bt-k were sprayed on potatoes to
protect against potato beetle (Leptinotarsa decemlineata). Similar findings were also reported by
Furlong and Groden (2003) who reported synergistic interaction between B. bassiana and B.
thuringiensis. Other researchers have also observed the similar results when combined B.
bassiana and B. thuringiensis withsynthetic insecticides (Fargues, 1975; Lewis and Bing, 1991;
Sander and Chichy, 1967). Contrarily, no synergistic interaction was observed between B.
bassiana and B. thuringiensis against 4th stage larvae of L. decemlineata (Costa et al., 2001).
Here the method of fungal spore application may retard the synergistic effect and respond
differently in terms of time and level of mortality. Different response of Chilo partellus was
reported when fungal spores were applied on leaf disk (Tefera and Pringle, 2003a).
In our present study synergistic interaction may be due to the treatment of RPW larvae
with fungal spores by larval dip method in which dorso-ventral infection of fungal spores may
weaken the larvae and render it more susceptible to Bt-k spores. Tefera and Pringle (2003b)
observed that loss of feeding H. armigera after exposure to Bt mixed diet make it easier for B.
bassiana to grab the physiology and hence proliferate rather easily. Similar loss of feeding was
observed by Tefera and Pringle (2003b) when second and third instar C. partellus were exposed
to simultaneous action of Bt (Cry1Ac) and B. bassiana as behavioral response elucidated reduced
diet uptake. Jadhav et al. (2012) found similar retardation in growth and survival of H. armigera
and Spodoptera litura after treatment with three flavonoids namely chlorogenic acid, quercetin
and rutin. The feeding experiments on RPW exhibited that B. thuringiensis invades the
hemolymph and the total circulating hemocytes decreased, mainly the plasmatocytes, after 19 h,
and for the first time, many Bt vegetative forms were recorded in the hemolymph of RPW after
Bt commercial product ingestion (Manachini et al., 2011).
Integrated action of Bt and fungi may also hypothesized by delayed larval molts with Bt
treatments which enhance the inter-molt period, thereby providing fungus extended period of
time for infection before next molt. The reduced food consumption with Bt treatments (Nathan et
al., 2005; Ramalho et al., 2011), food utilization (Prutz and Dettner, 2005; Ramalho et al., 2011)
and reduced larval weight has been observed. On the other hand in healthy larvae loss of conidia
may happen during molting, hence conferring interruption in fungal colonization (Wraight and
Ramos, 2005). Contrarily, Slansky (1993) reported surprising results by observing the reduction
in larval weight, food consumption and frass production in fungal treated larvae as compared to
119
the untreated control. Our findings suggest that assimilation activities were found related to toxic
effect of pathogen as indicated by decreased quantity of frass produced. Our results are in
accordance with the study of Janmaat et al. (2014) who reported reduced frass production in
Trichoplusia ni with the increase of concentrations. This may be attributed to the fact that
enhanced Bt concentrations may alter the protein to carbohydrate ratio of the diet as required
optimally which resultantly disturb the growth response (Simpson and Raubenheimer, 1995).
Moreover, the toxicant larvae try to repair its midgut lining and the lysis effect is directly
proportional to the concentration of pathogen (Tanaka et al., 2012). Muñoz et al. (2014) reported
reduced food intake, growth and weight gain in H. armigera exposed to the sublethal doses of Bt
which in response did not allowed most of them to get the critical weight and pupate in time.
This type of feeding behavior may be the result of metabolic interference of the
entomopathogens with larva’s growth.
A greater knowledge of RPW biology and, in particular, of the interaction between
potential pathogens and immunocytes would be useful to improve RPW-IPM programs, which
should focus on the identification of more virulent natural pathogen strains and on improving the
virulence capacity of Bt (Manachini et al., 2011). While choosing microbial control agents to
fight insect pests, several uncontrolled factors must be considered as the comprehensive nature of
entomopathogens requires multidisciplinary targeted efforts. This study may help to apply
laboratory study to field conditions covering almost all the questions required to be answered.
However field study regarding the persistence of entomopathogens is required to have a detailed
knowledge about some of the factors that cannot be imagined under laboratory or green house
condition. This study could be a foundation and direction for the future researches.
Conclusions The present study showed that B. bassiana can colonize endophytically in date palm
petiole even after 30 days post inoculation. WG-41 isolate was recovered up to 10 cm from the
site of inoculation even after 30 days. Endophytic B. bassiana in integration with Bt-k can be
effectively used against 2nd, 4th and 6th instar larvae of this pest under laboratory conditions.
Moreover they also exert detrimental effect on their growth and development parameters such as
diet consumption, frass production and weight gain.
Acknowledgements
This research work was supported by the scholarship from Higher Education
Commission (HEC), Islamabad, Pakistan (112-30536-2AV1-263) under Indigenous Ph.D.
Fellowship Program.
120
References
Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ.
Entomol., 18: 265-267.
Abraham, V.A., M.A. Al-Shuaibi, M.A. Faleiro, J.R. Abozuhairah, R.A. and P.S.P. Vidyasagar,
1998. An integrated management approach for red palm weevil Rhynchophorus
ferrugineus Oliv. a key pest of date palm in the Middle East. Proc. Inter. Conf. on
Integrated Pest Management (Muscat, Sultanate of Oman). Sultan Qaboos Uni. J. Sci.
Res., 3: 77-83.
Allee, L.L., R.S. Soper and D.W. Roberts, 1990. Germination and infection processes of the
entomophthoralean fungus Erynia radicans on the potato leafhopper Empoasca fabae. J.
Inver. Pathol., 56: 157-174.
Arnold, A.E., L.C. Mejía, D. Kyllo, E.I. Rojas, Z. Maynard, N. Robbins and E.A., Herre, 2003.
Fungal endophytes limit pathogen damage in a tropical tree. Proc. Nati. Acad. Sci. USA.,
100: 15649-15654.
Aronson, A.I., W. Beckman and P. Dunn, 1986. Bacillus thuringiensis and related insect
pathogens. Microbiol. Rev., 50: 1-24.
Barnett, H.L. and B.B. Hunter, 1999. Illustrated Genera of Imperfect Fungi, 4th Edn. APS Press,
The American Phytopathological Society, St. Paul, MN. Pp. 218.
Buxton, P.A., 1920. Insect pests of dates and the date palm in Mesopotamia and elsewhere. Bull.
Entomol. Res., 11: 287-304.
Costa, S.D., M.E. Barbercheck and G.G. Kennedy, 2001. Mortality of Colorado potato beetle
(Leptinotarsa decemlineata) after sub-lethal stress with the CRYIIIA delta-endotoxin of
Bacillus thuringiensis and subsequent exposure to Beauveria bassiana. J. Inver. Pathol.,
77: 173-179.
Crecchio, C. and G. Stotzky, 2001. Biodegradation and insecticidal activity of the toxin from
Bacillus thuringiensis subsp. kurstaki bound on complexes of montmorillonite-humic
acids-Al hydroxypolymers. Soil Biol. Biochem., 33: 573-581.
Dangar, T.K., 1997. Infection of red palm weevil, Rhynchophorus ferrugineus, by a yeast. J.
Plant. Crops, 25: 193-196.
Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil
Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm
during the last one hundred years. Int. J. Trop. Insect Sci., 26: 135-154.
Fargues, T., 1975, Etude experimental dans la nature de Beauveria and Metarhizium a dose
reduite contre Lephinotarsa decemlineata. Annal. Zool. Ecol. Ani., 7: 247-264.
Federici, B.A., H.W. Park and Y. Sakano, 2006. Insecticidal protein crystals of Bacillus
thuringiensis. In: Inclusions in Prokaryotes. Shively, J.M. (Ed.). Springer-Verlag, Berlin-
Heidelberg. Pp. 195-235.
Furlong, M.J. and E. Groden, 2003. Starvation induced stress and the susceptibility of the
Colorado potato beetle, Leptinotarsa decemlineata, to infection by Beauveria bassiana. J.
Inver. Pathol., 83: 127-138.
Giblin-Davis, R.M., 2001. Borers of palms. In: Howard F.W., D. Moore, R.M. Giblin-Davis,
R.G. Abad (Eds.). Insects on Palms. CABI Publishing, Wallingford, UK. Pp. 267-305.
Gómez-Vidal, S., L.V. Lopez-Llorca, H.B. Jansson and J. Salinas, 2006. Endophytic colonisation
of date palm (Phoenix dactylifera L.) leaves by entomopathogenic fungi. Micron, 37:
624-632.
121
Hernández, C.S., R. Andrew, Y. Bel and J. Ferre, 2005. Isolation and toxicity of Bacillus
thuringiensis from potato-growing areas in Bolivia. J. Inver. Pathol., 88: 8-16.
Jadhav, D.R., N. Mallikarjuna, A. Rathore and D. Pokle, 2012. Effect of some flavonoids on
survival and development of Helicoverpa armigera (Hübner) and Spodoptera litura (Fab)
(Lepidoptera: Noctuidae). Asi. J. Agric. Sci., 4(4): 298- 307.
Janmaat, A.F., L. Bergmann and J. Ericsson, 2014. Effect of low levels of Bacillus thuringiensis
exposure on the growth, food consumption and digestion efficiencies of Trichoplusia ni
resistant and susceptible to Bt. J. Inver. Pathol., 119: 32-39.
Khalique, F. and K. Ahmad, 2002. Retarding effect of spore-δ-endotoxin complex of Bacillus
thuringiensis (Berliner) strains on the development of Helicoverpa armigera (Hubner).
Pak. J. Biol. Sci., 5(8): 853-857.
Lacey, L.A., A.A. Kirk, L. Millar, G. Mereadier and C. Vidal, 1999. Ovicidal and larvicidal
activity of conidia and blastospores of Paecilomyces fumosoroseus (Deuteromycotina:
Hyphomycetes) against Bemisia argentifolii (Homoptera: Aleyrodidae) with a description
of a bioassay system allowing prolonged survival of control insects. Biocontrol Sci.
Tech., 9: 9-18.
Lewis, L.C. and L.A. Bing, 1991. Bacillus thuringiensis Berliner and Beauveria bassiana
(Balsamo) Vuillemin for European Corn Borer Control: Potential for Immediate and
Season-long Suppression. Canad. Entomol., 123: 387-393.
Lezama-Guttierez, R., J.J. Hamm, J. Molina-Ochoa, M. Lopez-Edwards, A. Pescador-Rubio, M.
Gonzalez-Ramirez and E.L. Styer, 2001. Occurrence of entomopathogens of Spodoptera
frugiperda (Lepidoptera: Noctuidae) in the Mexican states of Michoacan, Colima,
Jalisco, Tamaulipas. Florida Entomol., 84: 23-30.
Ma, X.M., X.X. Liu, X. Ning, B. Zhang, F. Han, X-M. Guan, Y.F. Tan and Q.W. Zhang, 2008.
Effects of Bacillus thuringiensis toxin Cry1Ac and Beauveria bassiana on Asiatic corn
borer (Lepidoptera: Crambidae). J. Inver. Pathol., 99: 123-128.
Manachini, B., V. Arizza, D. Parrinello and N. Parrinello, 2011. Hemocytes of Rhynchophorus
ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces
cerevisiae and Bacillus thuringiensis. J. Inver. Pathol., 106:360-365.
Mansour, N.A., M.E. Eldefrawi, A. Toppozada and M. Zeid, 1966. Toxicological studies on the
Egyptian cotton leafworm, Prodenia litura Vl potentiation and antagonism of carbamate
insecticide. J. Econ. Entomol., 59: 307-311.
Martin, P. and R. Travers, 1989. Worldwide abundance and distribution of Bacillus thuringiensis
isolates Appl. Environ. Microbiol., 55(10): 2437-2442.
Marzban, R., Q. He, X. Liu and Q. Zhang, 2009. Effects of Bacillus thuringiensis toxin Cry1Ac
and cytoplasmic polyhedrosis virus of Helicoverpa armigera (Hübner) (HaCPV) on
cotton bollworm (Lepidoptera: Noctuidae). J. Inver. Pathol., 101: 71-76.
Milne, D., 1918. The Date Palm and Its Cultivation in the Punjab. The Punjab Government. Pp.
153.
Minitab, 2003. MINITAB Release 14 for Windows. Minitab Inc., State College, Pennsylvania,
USA.
Mohan, L.M., 1917. Rept. Asst. Prof. Entomol; Rept. D Sagr. Punjab, for the year ended 30th
June, 1917.
Murphy, S.T. and B.R. Briscoe, 1999. The red palm weevil as an alien invasive: biology and
prospects for biological control as a component of IPM. BioControl, 20: 35-45.
122
Nathan, S.S., P.G. Chung and K. Murugan, 2005. Effect of biopesticides applied separately or
together on nutritional indices of the rice leaffolder, Cnaphalocrocis medinalis.
Phytoparasitica, 33: 187-195.
Navon, A., 2000. Bacillus thuringiensis insecticides in crop protection reality and prospects.
Crop Prot., 19: 669-676.
Pingel, R.L. and L.C. Lewis, 1999. Effect of Bacillus thuringiensis, Anagrapha falcifera multiple
nucleopolyhedrovirus, and their mixture on three Lepidoptera corn ear pests. J. Econ.
Entomol., 92: 91-96.
Prutz, G. and K. Dettner, 2005. Effects of various concentrations of Bacillus thuringiensis Corn
leaf material on food utilization by Chilo partellus larvae of different ages.
Phytoparasitica, 33: 467-479.
Quesada-Moraga, E., R. Santos-Quiros, P. Valverde-Garcia and C. Santiago-Álvarez, 2004.
Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium
anisopliae (anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). J.
Inver. Pathol., 87: 51-58.
Ramalho F.D., T.L. Azeredo, A.R.B. de Nascimento, F.S. Fernandes, J.L. Jr. Nascimento, J.B.
Malaquias, C.A.D. da Sliva and J.C. Zanuncio, 2011. Feeding of fall armyworm,
Spodoptera frugiperda, on Bt transgenic cotton and its isoline. Entomol. Exper. Appl.,
139: 207-214.
Salama, H.S., M.S. Foda, M.A. El-Bendary and A. Abdel-Razek, 2004. Infection of red palm
weevil Rhynchophorus ferrugineus, by spore-forming bacilli indigenous to its natural
habitat in Egyp. J. Pest Sci., 77: 27-31.
Sandner, H. and D. Cichy, 1967. Research on the effectiveness of fungal and bacterial
insecticides. Ekol. Pol. Ser., 15: 325-333.
Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler and D.H.
Dean, 1998. Bacillus thuringiensis and its pesticidal crystal proteins, Microbiol. Mol.
Biol. Rev., 62: 775-806.
Simpson, S.J. and D. Raubenheimer, 1995. The geometric analysis of feeding and nutrition: a
user's guide. J. Insect Physiol., 41: 545-553.
Sivasithamparam, K., 1998. Root cortex - the final frontier for the biocontrol of root-rot with
fungal antagonists: a case study on a sterile red fungus. Ann. Rev. Phytopathol., 36: 439-
452.
Slansky, F. Jr., 1993. Nutritional ecology: the fundamental quest for nutrients. In: Stamp, N.E.
and T.M. Casey (Eds.). Caterpillars: ecological and evolutionary constraints on foraging.
Chapman & Hall, London. Pp. 29-91.
Sokal, R.R. and F.J. Rohlf, 1995. Biometry. Freeman, New York.
Tanaka, S., Y. Yoshizawa and R. Sato, 2012. Response of midgut epithelial cells to Cry1Aa is
toxin-dependent and depends on the interplay between toxic action and the host apoptotic
response. FEBS J., 279: 1071-1079.
Tefera, T. and K.L. Pringle, 2003a. Effect of exposure method to Beauveria bassiana and
conidia concentration on mortality, mycosis, and sporulation in cadavers of Chilo
partellus (Lepidoptera: Pyralidae). J. Inver. Pathol., 84: 90-95.
Tefera, T. and K.L. Pringle, 2003b. Food consumption by Chilo partellus (Lepidoptera:
Pyralidae) larvae infected with Beauveria bassiana and Metarhizium anisopliae and
effects of feeding natural versus artificial diets on mortality and mycosis. J. Inver.
Pathol., 84: 220-225.
123
Vey, A. and J. Fargues, 1977. Histological and ultrastructural studies of Beauveria bassiana
infection in Leptinotarsa decemlineata larvae during ecdysis. J. Inver. Pathol., 30: 207-
221.
Wakil, W., M.U. Ghazanfar, T. Riasat, M.A. Qayyum, S. Ahmed and M. Yasin, 2013. Effects of
interactions among Metarhizium anisopliae, Bacillus thuringiensis and
chlorantraniliprole on the mortality and pupation of six geographically distinct field
populations. Phytoparasitica, 41: 221-234.
Wraight, S.P. and M.E. Ramos, 2005. Synergistic interaction between Beauveria bassianaand
Bacillus thuringiensis tenebrionis-based biopesticides applied against field populations of
Colorado potato beetle larvae. J. Inver. Pathol., 90: 139-150.
124
Table 6.1 Percentage of petiole fragments colonized by entomopathogenic (E) and other (O)
fungi in live palm petioles experiments
Isolate
Site
2014 2015 15 days 30 days 15 days 30 days
E O E O E O E O
WG-11
A5 - 100 - 100 - 100 - 100 A4 - 100 - 100 - 100 - 100 A3 20 80 20 80 15 85 25 75 A2 60 40 40 60 45 55 30 70 A1 100 - 60 40 90 10 50 50 INS 90 10 60 40 80 20 45 55 B1 80 20 60 40 70 30 40 60 B2 40 60 20 80 33 66 15 85 B3 - 100 - 100 - 100 - 100 B4 - 100 - - - 100 - - B5 - 100 - - - 100 - -
WG-40
A5 - 100 - 100 - 100 - 100 A4 35 65 20 80 25 75 15 85 A3 66 33 40 60 50 50 30 70 A2 80 20 40 60 66 33 55 45 A1 100 - 60 40 90 10 60 40 INS 100 - 100 - 100 - 100 75 B1 100 - 80 20 95 05 70 30 B2 75 25 80 20 66 33 66 33 B3 50 50 40 60 40 60 30 70 B4 25 75 15 85 20 80 - 100 B5 - 100 - 100 - 100 - 100
WG-41
A5 50 50 25 75 40 60 20 80 A4 70 30 50 50 66 33 40 60 A3 75 25 50 50 70 30 40 60 A2 90 10 75 25 75 25 65 35 A1 100 - 90 10 90 10 80 20 INS 100 - 100 - 100 - 100 - B1 100 - 80 20 90 10 70 30 B2 95 5 75 25 80 20 60 40 B3 80 20 66 33 70 30 50 50 B4 65 35 40 60 50 50 30 70 B5 40 60 33 66 40 60 20 80
WG-42
A5 - 100 - 100 - 100 - 100 A4 33 66 - 100 25 75 - 100 A3 60 40 33 66 40 60 25 75 A2 85 15 55 45 70 30 40 60 A1 100 0 65 35 90 10 50 50 INS 100 0 85 15 100 0 85 15 B1 100 0 60 40 85 15 50 50 B2 75 25 50 50 66 33 40 60 B3 66 33 20 80 50 50 15 85 B4 35 65 10 90 25 75 - 100 B5 - 100 - 100 - 100 - 100
125
WG-43
A5 - 100 - 100 - 100 - 100 A4 33 66 - 100 25 75 - 100 A3 55 45 40 60 40 60 30 70 A2 67 33 55 45 55 45 40 60 A1 90 10 75 25 75 25 60 40 INS 100 - 90 10 100 - 90 10 B1 85 15 60 40 70 30 50 50 B2 67 33 45 55 50 50 35 65 B3 50 50 33 66 40 60 20 80 B4 15 85 - 100 - 100 - 100 B5 - - - 100 - - - 100
Control
A5 - 100 20 80 - 100 - 100 A4 - 100 - 100 - 100 - 100 A3 10 90 - 100 - 100 20 80 A2 - 100 - 100 - 100 - 100 A1 - 100 - 100 20 80 10 90 INS - 100 - 100 - 100 - 100 B1 - 100 15 85 - 100 - 100 B2 - 100 - 100 - 100 - 100 B3 - 100 - 100 - 100 - 100 B4 - 100 - 100 - 100 - 100
B5 - 100 - 100 - 100 - 100 INS: site of injection; A1-A5 and B1-B5: site 2-10 cm above or below site of injection.
Table 6.2 Factorial analysis of mortality, pupation, adult emergence and egg eclosion of
R. ferrugineus exposed to endophytically colonized B. bassiana and B.
thuringiensis
S.O.V.
df
Mortality Pupation Adult Emergence
Egg Eclosion
F P F P F P F P
Instar 2 63.34 ≤0.05 59.23 ≤0.05 51.45 ≤0.05 34.56 ≤0.05 Treatment 7 201.87 ≤0.05 229.10 ≤0.05 233.45 ≤0.05 249 ≤0.05 Instar × Treatment
14 1.56 0.093 0.68 0.625 0.61 0.635 0.78 0.515
Error 149 - - - - - - - - Total 172 - - - - - - - -
126
Table 6.3 Mean mortality (%±SE) of 2nd, 4th and 6th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 2 cm
away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means
followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)
Treatments Second Instar Fourth Instar Sixth Instar
Actual Mortality (%)
Expected Mortality
CTF Actual Mortality (%)
Expected Mortality
CTF Actual Mortality (%)
Expected Mortality
CTF Type of Interaction
Bb 29.90±1.27de - - 23.36±1.18cde - - 20.67±1.17cde - - - Bt1 17.65±1.16e - - 12.53±1.09e - - 8.27±0.89e - - - Bt2 25.89±1.61de - - 18.58±1.12de - - 14.19±1.22de - - - Bt3 41.20±1.01cd - - 33.01±1.45cd - - 25.02±1.38cd - - - Bb + Bt1 54.63±2.26bc 47.55 14.88 42.13±1.32bc 35.89 17.38 34.07±1.50bc 28.94 17.72 Add. Bb + Bt2 71.01±2.39ab 55.79 27.28 54.07±1.65ab 41.94 28.92 47.87±1.42ab 34.86 37.32 Syn. Bb + Bt3 83.17±2.28a 71.1 16.97 65.86±1.50a 55.42 18.83 52.68±1.57a 44.96 17.17 Add. Control 2.4 - - 1.5 - - 1.00 - - - df 6 - - 6 - - 6 - - - F 33.3 - - 18.7 - - 26.1 - - - P ≤0.05 - - ≤0.05 - - ≤0.05 - - -
127
Table 6.4 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar larvae of R. ferrugineus treated with
endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone
and in combination (means followed by the same letter within each treatment are not significantly different; HSD test
P≤0.05)
Treatments Second Instar Fourth Instar Sixth Instar
Pupation (%) Adult
Emergence (%)
Egg Eclosion
(%)
Pupation
(%)
Adult
Emergence (%)
Egg Eclosion
(%)
Pupation
(%)
Adult
Emergence (%)
Egg
Eclosion (%)
Bb 63.31±2.35c 57.72±2.77c 51.13±2.20c 71.16±2.60cd 64.46±2.93c 57.73±2.23b 76.64±3.33bc 70.06±2.88c 64.46±2.93b
Bt1 78.82±3.15b 70.00±2.92b 63.37±2.48b 84.44±3.36ab 76.62±2.35b 65.97±3.01b 87.73±2.94ab 84.42±2.93ab 75.51±2.42b
Bt2 66.67±2.87c 58.83±2.60bc 52.28±2.73bc 75.56±2.75bc 67.73±3.13bc 58.75±2.23b 80.10±2.88bc 73.36±3.12bc 66.65±2.88b
Bt3 49.02±2.21d 37.96±1.53d 30.02±1.88d 58.81±2.60de 50.07±2.88d 43.31±2.35c 68.85±3.21c 52.24±2.77d 51.16±2.60c Bb + Bt1 32.24±2.12e 25.53±1.42e 18.84±1.15de 46.64±1.72e 38.83±2.09d 32.24±1.85c 53.36±2.40d 45.54±2.42d 37.72±2.77d
Bb + Bt2 18.79±1.12f 11.10±1.02f 7.72±0.86ef 31.15±1.51f 17.96±1.11e 15.56±1.05d 44.42±2.93de 31.16±1.88e 24.46±2.42e
Bb + Bt3 13.31±1.26f 6.66±0.63f 3.38±0.45f 24.41±1.34f 13.30±1.09e 8.83±0.69d 35.54±1.75e 22.27±1.23e 15.53±1.15e
Control 94.48±1.75a 91.14±2.60a 85.52±2.93a 96.64±1.66a 92.18±2.24a 90.08±2.35a 95.49±1.91a 93.32±2.35a 87.72±2.22a df 7 7 7 7 7 7 7 7 7
F 119.0 117 122 83.1 103 90.1 46.0 73.6 100
P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05
128
Table 6.5 Growth parameters e.g. larval duration (days), larval weight (grams), pre-pupal duration (days), pre-pupal weight
(grams), pupal duration (days), pupal weight (grams), adult longevity (days) and adult weight (grams) (%±SE) of 2nd
instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-k
(Bt1: 10 µg; Bt2: 15 µg; Bt3: 20 µg ml-1) alone and in combination (means followed by the same letter within each
treatment are not significantly different; HSD test P≤0.05)
Treatments
Larval Duration (days)
Larval Weight (g)
Pre-Pupal Duration
(days)
Pre-Pupal Weight
(g)
Pupal Duration
(days)
Pupal Weight (g)
Adult Longevity (days) Adult Weight (g)
Male Female Male Female
Bb 109.16±3.56c 3.34±0.08bc 17.60±1.13de 3.20±0.06cd 24.16±1.12de 3.27±0.07cd 38.27±1.31a 42.16±1.15a 1.39±0.09abc 1.19±0.07bc Bt1 99.72±2.25d 4.07±0.08a 15.83±0.76fg 3.82±0.11ab 21.72±0.92fg 3.93±0.10ab 36.94±1.17ab 40.27±1.07ab 1.58±0.10ab 1.35±0.06ab Bt2 105.27±3.07cd 3.66±0.11b 16.48±0.57ef 3.52±0.12bc 22.94±1.18ef 3.65±0.10bc 35.50±1.05abc 39.16±1.25ab 1.50±0.11ab 1.28±0.06abc Bt3 111.71±3.10c 3.21±0.12cd 18.76±0.65cd 3.03±0.10de 25.83±1.04cd 3.12±0.11de 33.16±1.21bcd 37.94±1.03abc 1.37±0.08abcd 1.17±0.07bcd Bb + Bt1 118.62±2.80b 3.08± 0.10cde 20.16±1.38bc 2.77±0.06def 26.94±1.35bc 2.93±0.08def 30.94±0.91cde 35.61±1.14bcd 1.21±0.07bcd 1.02±0.06cde
Bb + Bt2 121.27±2.56ab 2.96±0.09de 21.41±0.70ab 2.61±0.09ef 28.27±1.26ab 2.78±0.08ef 28.83±1.06de 33.00±0.98cd 1.07±0.05cd 0.91±0.04de Bb + Bt3 125.41±3.23a 2.74±0.07e 22.72±1.33a 2.38±0.04f 29.61±1.41a 2.54±0.09f 25.72±1.11e 30.38±1.03d 0.98±0.04d 0.88±0.03e Control 85.72±2.19e 4.42±0.15a 14.16±0.86g 4.02±0.11a 20.27±0.94g 4.14±0.11a 39.50±1.40a 43.72±1.40a 1.70±0.08a 1.51±0.06a Df 7 7 7 7 7 7 7 7 7 7 F 72.73 47.54 53.12 33.8 43.95 26.61 65.03 58.20 7.76 12.5 P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05
129
Table 6.6 Analysis of Co-variance for 10th instar larvae of R. ferrugineus regarding weight
gain, frass production and diet consumption when treated with endophytic B.
bassiana (Bb: 6 cm away from inoculation site) and Bt-k (Bt: 10 µg ml-1). Initial
weight of larvae and diet consumption were taken as covariate
S.O.V. df F P Covariate: Diet Consumption 1 0.193 0.660 Covariate: Weight Gain 1 3.062 0.081 Frass Production × Diet Consumption 24 2.318 ≤0.05 Diet Consumption × Weight Gain 1 0.138 0.710 Frass Production × Weight Gain 27 2.733 ≤0.05 Frass Production × Diet Consumption × Weight Gain
24 2.704 ≤0.05
Error 687 - - Total 828 - -
130
Figure 6.1 Mean mycosis (%±SE) in cadavers of R. ferrugineus treated with endophytic B.
bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg;
Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter
within each treatment are not significantly different; HSD test P≤0.05)
.
Figure 6.2 Sporulation (conidia ml-1) on R. ferrugineus cadavers treated with endophytic B.
bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg;
Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter
within each treatment are not significantly different; HSD test P≤0.05)
131
Figure 6.3 Diet consumption (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away
from inoculation site) and Bt-k (Bt: 10 µg ml-1)
132
Figure 6.4 Frass production (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away
from inoculation site) and Bt-k (Bt: 10 µg ml-1)
133
Figure 6.5 Weight gain (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away from
inoculation site) and Bt-k (Bt: 10 µg ml-1)
134
CHAPTER 7
Integrated Effect of Entomopathogenic fungi and Entomopathogenic Nematode against
Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)
Abstract
Research study was carried out to investigate the insecticidal properties of entomopathogenic
fungi Beauveria bassiana s.l. (Ascomycota: Hypocreales) strain WG-11, Metarhizium anisopliae
s.l. (Ascomycota: Hypocreales) strain WG-02 and the entomopathogenic nematode,
Heterorhabditis bacteriophora Poinar (Heterorhabditidae) for their virulence against 2nd, 4th and
6th instar larva of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Both agents
were either applied alone or in combination, with H. bacteriophora 1 and 2 weeks after fungi
application. Moreover, the complete spectrum of toxicity, development, diet consumption, frass
production and weight gain were observed at sub lethal doses of both agents. In combined
treatments, additive and synergistic interactions were observed for all the three instars and effects
were not significantly different among the treatments either applied simultaneously or in
sequential combinations with each other. Enhanced morality was recorded for the combined
treatments when delayed application of H. bacteriophora was made 1 or 2 weeks after fungus
treatment as compared to their sole application. Decrease in pupation, adult emergence and egg
hatching were also found related to the toxic effect of treatments. Duration of different
developmental stages was significantly affected by the treatments applied. Decreased larval
weight, increased larval duration, increased pre-pupal and pupal period and decreased weight,
decreased adult weight and life span were recorded and compared to the control. Larvae fed on
sub lethal amounts of both agents revealed reduced food ingestion, reduced growth and weight
gain, preventing most of them from achieving the critical weight. Initial weight of larvae exerted
its impact on weight gain and diet consumption, and the trend was found linked to pathogenicity
of applied agents. A result of the present study suggests that R. ferrugineus can be successfully
managed by applying entomopathogenic fungi and H. bacteriophora. Additionally, their
simultaneous and sequential application may offer enhanced mortality as compare to the
application of either of them alone.
Keywords: Rhynchophorus ferrugineus, Beauveria bassiana, Metarhizium anisopliae,
Heterorhabditis bacteriophora, diet consumption, frass production
135
7.1 Introduction
The coleopteran insects are ranked among the most voracious pests of economically
important crops. Among these the Red Palm weevil (RPW) Rhynchophorus ferrugineus (Olivier)
(Coleoptera: Curculionidae) is most destructive to 29 different palm species particularly date
palms of economic importance in the Middle East, Africa and South East Asia (Malumphy and
Moran, 2009). Synonymously it is known as Asiatic palm weevil, coconut weevil, red stripe
weevil, hidden enemy and also called AIDS palm because of the damage and the slow death of
the palm tree (Khamiss and Abdel-Badeea, 2013). The pest has cryptic nature and mostly
damages the palm trees younger than 20 years (Nirula, 1956; Abraham et al., 1998) in which
crown, trunk and bole are the natural sites of damage, while the crown is the site of infestation in
older plantations. The larval stages destroy the vascular system while boring into the heart of the
host leading to tree collapse (Ju et al., 2011).
Insecticides and fumigants remained the mainstay of date palm growers for decades but
the cryptic nature of RPW presented an access challenge to treatments (Hussain et al., 2013).
Moreover, insecticides exert negative effects on the environment and human health and more
importantly pests have developed resistance against these chemicals (Abraham et al., 1998).
Alternatively entomopathogens can be used for suppression of this notorious pest in a wide array
of management approaches in versatile manners. Among microbial control agents
entomopathogenic fungi (EPFs) particularly, Beauveria bassiana s.l. (Ascomycota: Hypocreales)
and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales) are considered promising
alternatives to conventional chemical insecticides. They pose negligible detrimental effects on
the environment and human health (Khan et al., 2012), and have been reported to be effective
against a number of arthropod pests (Charnley and Collins, 2007; de Faria and Wraight, 2007).
Several researchers have isolated and successfully deployed these two strains against
different developmental stages of RPW as bio-control agents both under laboratory and field
conditions (Deadman et al., 2001; Gindin et al., 2006; El-Sufty et al., 2007, 2009, 2011; Sewify
et al., 2009; Torta et al., 2009; Vitale et al., 2009; Güerri-Aguilló et al., 2010; Merghem, 2011;
Francardi et al., 2012; Ricaño et al., 2013; Cito et al., 2014). EPFs are preferred over the other
microorganisms due to their novel mode of action by direct contact to the host cuticle instead of
ingestion or engulfing and their ability to transfer inoculum from treated insects to untreated
insects or to subsequent developmental stages via the new generation of spores (Quesada-
Moraga et al. 2004). Similarly, entomopathogenic nematodes (EPNs) are also promising
microbial control agents and declared efficient control agents against vast array of insect pests
(Abbas et al., 2001; Llácer et al., 2009; Dembilio et al., 2010a). They are obligate parasites in
the families Steinernematidae and Heterorhabditidae which kill insects with the aid of
mutualistic bacterium, which is carried in their intestine (Xenorhabdus spp. and Photorhabdus
spp. are associated with Steinernema spp. and Heterorhabditis spp., respectively) (Poinar, 1990).
Both agents are considered safer to non-target organisms (vertebrates and invertebrates) and
compatible to environment, and they are successfully integrated with each other exhibiting
strong additive and synergistic interactions (Thurston et al., 1993, 1994; Koppenhöfer and Kaya,
1997; Koppenhöfer et al., 1999).
This study aimed at the integration of B. bassiana, M. anisopliae and H. bacteriophora to
examine the mortality; development and growth of R. ferrugineus under laboratory conditions to
select the suitable application times of both agents for future field trials to successfully manage
R. ferrugineus populations in Pakistan.
136
7.2 Materials and Methods
7.2.1 RPW collection and rearing
Survey was conducted for collection of R. ferrugineus in date palm growing areas of west
Punjab, Pakistan. Different developmental stages (larvae, pupae and adults) were collected from
fallen and infested date palm trees with the permission of farmers (owners). All the stages
collected were kept separately in plastic jars until brought to the Microbial Control Laboratory,
Department of Entomology, University of Agriculture, Faisalabad (UAF), Pakistan. Larvae were
fed with sugarcane (Saccharum officinarum L.; Poales: Poaceae) sets and the same were used for
pupation after last instar, while shredded sugarcane pieces were offered to adults for feeding and
substrate for oviposition. After pupation pupal cocoon were kept in separate plastic jars for adult
emergence in incubator set at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod. After
adult emergence beetles were shifted to the adult’s jar for feeding, mating and oviposition.
Colony was developed in plastic boxes (30×60×60 cm) having a lid covered with mesh wire
gauze (60 mesh size) in the middle (10 cm diameter) for aeration. Adult’s diet was changed after
every three days and replaced sugarcane pieces were kept in separate jars (8×8×12 cm) for egg
hatching. After egg hatching neonate larvae were allowed to feed for some time in the same set
after 3 days larvae were transferred to the same sugarcane sets for feeding and pupation. Larvae
were shifted to the new sugarcane sets after every week until pupation. The rearing conditions
were maintained at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod.
7.2.2 Entomopathogenic Nematode
Infective juveniles (IJs) of H. bacteriophora culture was obtained from Microbial Control
Laboratory which was used for the bioassay against 2nd, 4th and 6th instar larvae of R.
ferrugineus. H. bacteriophora was maintained in 3rd instar Galleria mellonella L. (Lepidoptera:
Pyralidae) following the procedure of Kaya and Stock (1997).
7.2.3 Entomopathogenic fungi
Two isolates of entomopathogenic fungi B. bassiana (WG-11) and M. anisopliae (WG-
02) used in the study were taken from the culture collection of Microbial Control Laboratory,
originally isolated from soils of vegetables and crop fields respectively. Mass culturing was done
by inoculating Petri plates containing Potato Dextrose Agar (PDA) media (BD, France). Spore
concentration of 1×106 spore ml-1 was determined with a Neubauer haemocytometer.
7.2.4 Treatment with entomopathogenic fungi
A spore concentration of 1×106 spore ml-1 was prepared from conidial powder of B.
bassiana and M. anisopliae using haemocytometer. Second, 4th and 6th instar larvae were directly
immersed in 100 ml conidial suspension for 60s individually and control was treated in aqueous
solution of 0.01% Tween-80 (Merck, KGaA, Darmstadt, Germany) (Dembilio et al., 2010b). The
fungal isolate treated and control larvae were individually shifted to 150 ml cylindrical plastic
cups, each measuring 6 cm in height with 6 cm diameter. The top of the cups were covered with
a fine mesh in order to avoid the insects to escape. A piece of 2×2 cm2 artificial diet (Agar,
brewer’s yeast, wheat germ, corn flour, ascorbic acid, benzoic acid, amino acid-vitamin mix,
chloramphenicol and nipagin) (Martín and Cabello, 2006) were kept in the center of cups and
incubated at 27±2 °C, 65±5 % RH and 12:12 (D: L) hours photoperiod in an incubator (Sanyo,
Japan). Three replicates of 10 larvae were treated to the fungal suspension. Each cup was opened
137
daily and checked for mortality, and the old diet was replaced with fresh artificial diet until dying
or pupation. After last instar dry coir (coconut coir) was provided to the surviving larvae for
pupation. The bioassay was repeated thrice to avoid the pseudo-replication phenomenon.
7.2.5 Treatment with H. bacteriophora
Nematode suspension was prepared with a concentration of 100 IJs ml-1 in glass jars and
1 ml of suspension was poured into the cylindrical plastic cups lined with Whatman filter paper.
After pouring 30 minutes were given for evenly distribution of nematodes on filter paper. A
small piece of artificial diet 2×2 cm2 was placed in middle of the cups as a food source. Ten
larvae for each treatment were used separate in each cup and each treatment was replicated three
times, while control treatment received 1 ml of distilled water. The cups were maintained at
above mentioned conditions. Each cup was opened daily and checked for mortality, and the old
diet was replaced with fresh artificial diet until dying or pupation. After last instar dry coir was
provided to the surviving larvae for pupation. Whole bioassay was repeated thrice to avoid the
pseudo replication phenomenon.
7.2.6 Treatment with entomopathogenic fungi and nematode
In combined treatments both agents were applied simultaneously or at different time
intervals as follows:
B. bassiana, M. anisopliae and H. bacteriophora were applied simultaneously: larvae
were immersed in fungal suspensions and transferred to the cylindrical plastic cups lined
with moisten filter paper treated with H. bacteriophora IJs and maintained at 27±2 °C
and 65±5% RH at 12: 12 (D: L) hours photoperiod.
Insects were first inoculated with B. bassiana and M. anisopliae, maintained at 27±2 °C
and 65±5% RH for one week, transferred to cylindrical plastic cups lined with moisten
filter paper treated with H. bacteriophora IJs and maintained at above mentioned
conditions.
Insects were first inoculated with B. bassiana and M. anisopliae, maintained at 27±2 °C
and 65±5% RH for two weeks, transferred to cylindrical plastic cups lined with moisten
filter paper treated with H. bacteriophora IJs and maintained at above mentioned
conditions.
Control insects were immersed in aqueous solution with 0.01% Tween-80 and maintained
in cylindrical plastic cups lined with moistened filter paper using conditions stated above.
Larval mortality was recorded after one, two and three weeks post application. For all treatments,
artificial diet was offered to the larvae as food source. Larvae that failed to respond on slight
prodding by blunt needle were considered dead. After the last instar dry coir was provided for
pupation. Percent pupation, adult emergence and egg eclosion were also recorded.
7.2.7 Effects of entomopathogens on R. ferrugineus development
To check the effect of entomopathogens on development of RPW, 4th instar larvae were
exposed to the sub-lethal dose of fungal entomopathogens (1×104 spore ml-1) and H.
bacteriophora (50 IJs ml-1). The larvae were fed on artificial diet and transferred to the treatment
cups with 1 ml of. Dry coir was provided to the each larva before pupation for cocoon formation.
While adult on emergence were offered with shredded sugarcane pieces. Developmental
138
parameters of each stage (Larval duration, larval weight, pre-pupal duration, pre-pupal weight,
pupal duration, pupal weight, adult longevity (male and female) and adult weight (male and
female) was recorded.
7.2.8 Effects of entomopathogens on larval development
For the larval development last instar larvae of RPW was exposed to sub-lethal doses of
B. bassiana (1×104 spore ml-1) and H. bacteriophora (50 IJs ml-1). Before exposure in all the
treatments larvae were weighed first and transferred to the rearing cups with artificial diet.
Larvae continued to feed until pupated under experimental conditions maintained at 25±2 °C,
65±5% RH and L: D (12: 12) hours photoperiod. Every day until the larvae pupated, larvae were
changed to a new clean cup and new piece of artificial diet was offered. Frass produced during
this period was separated from vials using tip of fine camel hair brush and weighed. Diet left
unused in each vial was recovered, dried in drying oven at 80 °C. Prior to assay, diet in fifteen
vials was dried to obtain an estimate of the dry weight. Diet consumption of each larva was
determined by subtracting after feeding mass of diet from before feeding mass. Three replicates
of ten insects were used for each treatment and same count of larvae fed on normal diet served as
untreated check while entire experiment was repeated thrice.
7.2.9 Statistical analysis
The fungus nematode interactions (synergistic, additive or antagonistic) were calculated
using formula devised by Nishimatsu and Jackson (1998). The type of interaction was
determined through a comparison of expected and observed percentage mortality of RPW.
Expected mortality was calculated using formula PE = P0 + (1- P0) (P1) + (1- P0) (1- P1) (P2),
where PE is the expected mortality of the combination, P0 is the control mortality, P1 is the
mortality from one pathogen treatment applied alone, and P2 is the mortality from the
other pathogen applied alone. A X2 test was applied to the observed and expected results: X2 =
(L0 - LE)2 / LE + (D0 - DE)2 / DE, where L0 is the number of living larvae observed, LE the number
of living larvae expected, D0 the number of dead larvae observed, and DE the number
of dead larvae expected. Interactions were additive if X2 < 3.84, antagonistic if X2 > 3.84 and PC
< PE, and synergistic if X2 > 3.84 and PC > PE, where PC is the observed mortality from the
combination and PE is the expected mortality from the combination. Data for pupation, adult
emergence, egg eclosion and developmental parameters were subjected to one way analysis of
variance (ANOVA) in Minitab (Minitab, 2003) means were separated using Tukey’s Kramer test
(HSD) (Sokal and Rohlf, 1995) at 5% significance level. To inspect the impact of microbial
agents on the diet consumption, weight gain and frass production were analyzed by ANCOVA
using initial larval weight and diet consumption as covariates (Janmaat et al., 2014).
7.3 Results
7.3.1 Entomopathogenic fungi and nematode interaction
In integrated applications of H. bacteriophora with B. bassiana and M. anisopliae
additive to synergistic interaction were observed when both agents were applied simultaneously
or delayed nematode application for all the three instars tested (Table 7.1, 7.2 and 7.3). During
simultaneous application, B. bassiana and H. bacteriophora produced additive lethality to 2nd
instar larvae for first two weeks, while synergistic interactions were observed the third week
after application. The degree of synergism increased with the delayed application of H.
139
bacteriophora one or two weeks after initial B. bassiana treatments. For M. anisopliae additive
effects were recorded for simultaneous application, while interactions were shifted towards
synergism when delayed nematode application was made after one and two weeks of fungal
spore application (Table 7.1). Similar trends were recorded for 4th and 6th instar larvae but 2nd
instars were less susceptible to treatments. Percent pupation, adult emergence and egg eclosion
from surviving individuals was found inversely related to toxic level of microbial agents and
delayed application of H. bacteriophora for all instars tested (Table 7.5). In factorial analysis
main effects for pupation adult emergence and egg eclosion were significant while their
interaction effects were non-significant except pupation (Table 7.4)
7.3.2 Development of R. ferrugineus
Growth and development of 4th instar RPW larvae was adversely affected by the toxic
effect of the microbial agents. When larvae were exposed to the sub-lethal doses of H.
bacteriophora, B. bassiana and M. anisopliae, significant variations were recorded for larval
duration, larval weight, pre-pupal duration, pre-pupal weight, pupal duration, pupal weight, adult
longevity and adult weight (larval duration: F5, 53 =9.92, P≤0.05; larval weight F5, 53 =27.3,
P≤0.05; pre-pupal duration: F5, 53 =6.59, P≤0.05; pre-pupal weight: F5, 53 =6.94, P≤0.05; pupal
duration F5, 53 =5.15, P≤0.05; pupal weight F5, 53 =11.10, P≤0.05; adult longevity (female F5, 53
=3.93, P≤0.05 and male F5, 53 =5.58, P≤0.05 ); adult weight (female F5, 53 =4.26, P≤0.05 and
male F5, 53 =12.7, P≤0.05). Increase in larval, pre-pupal and pupal duration while decrease in
weight was recorded for all the treatments tested. On the other hand decrease in adult life span
and weight (male and female) was also recorded. Highest detrimental effect on growth was
recorded for combined application of B. bassiana and H. bacteriophora followed by M.
anisopliae and H. bacteriophora, H. bacteriophora alone, B. bassiana and M. anisopliae (Table
7.6).
7.3.3 Effect on larval development
Diet consumption by 10th instar larvae was significantly influenced by the treatments
applied; diet consumption was low in combined treatments of H. bacteriophora and B. bassiana
as compared to their individual applications (Fig 6.1). Similarly frass production was influenced
by treatments applied with lowest frass production for combined treatments of H. bacteriophora
and B. bassiana (0.57±0.04 to 0.00±0.00g) during the experimental period. After treatment frass
production gradually decreased to zero before pupation. On the other hand, the highest frass
production was found in untreated larvae during the last instar larvae until pupation (Fig 6.2).
Larvae treated with sub-lethal concentrations of either B. bassiana or H. bacteriophora alone
gained more weight compared to larval treated with a combined application. Initial weight of
larvae (10th instar: 4.31±0.14g) exerted its impact on the weight gain and among treatments,
there was a trend of weight gain was linked to pathogenicity. Combined application of B.
bassiana and H. bacteriophora had an adverse impact on the weight gain. The lowest weight
gain (-0.54±0.04g) was recorded for the combined treatment while the highest gain (-
0.12±0.02g) was recorded in untreated larvae (Fig 6.3).
7.4 Discussion
This is very first study to investigate the combined effect of fungal isolates and H.
bacteriophora against larvae of RPW. The results revealed that both agents can effectively
control the larval stages, either applied simultaneously or delayed application of H.
140
bacteriophora 1 or 2 weeks of fungal treatment. Additive to synergistic interactions were
recorded for combined applications of both agents. Greater additive or synergistic interactions
were observed when fungi were applied 1 or 2 weeks before H. bacteriophora treatment. Our
study corroborates the findings of Ansari et al. (2004, 2006) who reported similar results with
combined application of H. megidis or S. glaseri with M. anisopliae CLO 53 against 3rd instar H.
philanthus under laboratory and greenhouse conditions, and between H. bacteriophora and M.
anisopliae CLO 53 under field conditions respectively. Similarly additive and synergistic effects
were observed in combined treatments of H. bacteriophora and M. anisopliae isolate MM
against barley chafer grub, C. curtipennis (Anbesse et al., 2008). They have suggested exposing
grubs 3 or 4 weeks before addition of nematodes to get stronger synergistic interaction. However
in our study enhanced efficacy and stronger interactions were recorded 1 or 2 weeks delayed
application of H. bacteriophora.
It is tempting to speculate that longer grubs were exposed to the fungus, the more
debilitated they become and subsequently were more susceptible to the EPN. The debilitated
insects respired more, attracting the EPNs, which followed a CO2 gradient to their hosts (Ansari
et al., 2008). Steinhaus, (1958) also suggested that the stressed insects were more vulnerable to
pathogen infection, hence enhancing insect mortality or facilitating the speed of kill and
enhancing additive or synergistic effects in combined treatments. For example, Paenibacillus
popilliae (Dutky) against scarab larvae acted as a stressor to nematode infection that caused
elevated larval mortality (Thurston et al., 1993; Thurston et al., 1994). Other authors also have
reported similar results during their studies (Kermarrec and Mauleon, 1989; Barbercheck and
Kaya, 1990; Thurston et al., 1993, 1994; Koppenhöfer and Kaya, 1997; Koppenhöfer et al.,
1999). Contrarily Shapiro-Ilan et al. (2004) found antagonism between EPNs and P.
fumosoroseus. This antagonism may be due to pathogen interactions prior to or during infection.
In case of Sternima marcescens and P. fumosoroseus, it is possible that these organisms are
directly pathogenic to EPNs, therefore nematodes may have been killed or their fitness reduced
prior to infection. The negative interactions may also be due to antagonistic toxins produced by
the entomopathogens after infection was initiated. The synergy shown between fungi and H.
bacteriophora provided an opportunity to reduce the cost of RPW control while increasing the
overall efficacy of the control strategy.
For performing normal daily functions, an insect needs to have proper growth and
development and any delay may render the insect susceptible to biotic and abiotic factors such as
natural enemies, and environmental regimes that ultimately influence the growth, development,
diet consumption and frass production. In this regard larval stages are considered vulnerable
towards these agents (Marzban et al., 2009). The extended larval and pupal period reduces time
span left for adult stage that directly affects the insect’s fecundity and in other words threatens
the survival in next generation. Outcomes of our present study indicate that entomopathogenic
fungi and H. bacteriophora can be applied successfully against RPW but their efficacy varies
depending upon the interval of nematode application. Larval development is greatly influenced
by the lethal action of both entomopathogenic fungi and H. bacteriophora affecting the degree of
diet consumption that establishes the foundation for insect control. The alone and combined
concentrations of both agent offers a range of toxicity that exerts corresponding effect on the
development and survival of target host.
In present study detrimental effects imposed by EPFs and H. bacteriophora decreased
larval, pre-pupal, pupation rate, pupal weight and prolonged the developmental period. It is
generally accepted that major part of the insect energy consumed fighting pathogens lead to the
141
weakness and growth retardation in insects (Sikorowski and Thampson, 1979; Wiygul and
Sikorowski, 1991). Similar observations have been made during the current research and the
integrated application of H. bacteriophora and EPFs increased the larval mortality compared
with their respective individual treatments. Therefore, in an IPM program they can be
recommended for pest control where this entomopathogens are important natural enemies.
Conclusions The present study showed that B. bassiana and M. anisopliae isolates in integration with
H. bacteriophora under laboratory conditions caused high mortality against larvae of red palm
weevil. The pathogens exerted detrimental effects on growth and development of different
developmental stages of R. ferrugineus. Hence, integrated application of H. bacteriophora in
sequential manners with B. bassiana and M. anisopliae can be effectively used for the successful
control of red palm weevil.
Acknowledgements
This research work was supported by the scholarship from Higher Education
Commission (HEC), Islamabad, Pakistan (112-30536-2AV1-263) under Indigenous Ph.D.
Fellowship Program.
142
7.5 References
Abraham, V.A., M.A. Al-Shuaibi, M.A. Faleiro, J.R. Abozuhairah, R.A. and P.S.P. Vidyasagar,
1998. An integrated management approach for red palm weevil Rhynchophorus
ferrugineus Oliv a key pest of date palm in the Middle East. Proc. Inter. Conf. on
Integrated Pest Management (Muscat, Sultanate of Oman). Sultan Qaboos Uni. J. Sci.
Res., 3: 77-83.
Anbesse, S.A., B.J. Adge, W.M. and Gebru, 2008. Laboratory screening for virulent
entomopathogenic nematodes (Heterorhabditis bacteriophora and Steinernema
yirgalemense) and fungi (Metarhizium anisopliae and Beauveria bassiana) and
assessment of possible synergistic effects of combined use against grubs of the barley
chafer Coptognathus curtipennis. Nematol., 10: 701-709
Ansari, M.A., B.N. Adhikari, F. Ali and M. Moens, 2008. Susceptibility of Hoplia philanthus
(Coleptera: Scarabaeidae) larvae and pupae to entomopathogenic nematodes (Rhabditida:
Steinernematidae, Heterorhabditidae). Biol. Control, 47: 315-321.
Ansari, M.A., F.A. Shah, L. Tirry and M. Moens, 2006. Field trials against Hoplia philanthus
(Coleoptera: Scarabaeidae) with a combination of an entomopathogenic nematode and
the fungus Metarhizium anisopliae CLO 53. Biol. Control, 39: 453-459.
Ansari, M.A., S. Vestergaard, L. Tirry and M. Moens, 2004. Selection of a highly virulent fungal
isolate, Metarhizium anisopliae CLO 53, for controlling Hoplia philanthus. J. Inver.
Pathol., 85: 89-96.
Barbercheck, M.E. and H.K. Kaya, 1990. Interactions between Beauveria bassiana and the
entomogenous nematodes Steinernema feltiae and Heterorhabditis heliothidis. J. Inver.
Pathol., 55: 225-234.
Charnley, A. and S.A. Collins, 2007. Entomopathogenic fungi and their role in pest control. In:
Howard, D.H. and J.D. Miller (Eds.), The Mycota IV: Environmental and Microbial
Relationships, Springer-Verlag, Berlin, Heidelberg, pp.159-187.
Cito, A., G. Mazza, A. Strangi, C. Benvenuti, G.P. Barzanti, E. Dreassi, T. Turchetti, V.
Francardi and P.F. Roversi. 2014. Characterization and comparison of Metarhizium
strains isolated from Rhynchophorus ferrugineus. FEMS Microbiol. Lett., 355: 108-115.
de Faria, M.R. and S.P. Wraight, 2007. Mycoinsecticides and Mycoacaricides: a comprehensive
list with worldwide coverage and international classification of formulation types. Biol.
Control, 43: 237-256.
Deadman, M.L., K.M. Azam, S.A. Ravzi and W. Kaakeh. 2001. Preliminary investigation into
the biological control of the red palm weevil using Beauveria bassiana. Proceedings of
the Second International Conference on Date Palm, Al-Ain, UAE. March 25-27: 225-
232.
Dembilio, Ó., E. Quesada-Moraga, C. Santiago-Alvarez and J.A. Jacas, 2010b. Potential of an
indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological
control agent against the red palm weevil, Rhynchophorus ferrugineus. J. Inver. Pathol.,
104(3): 214-221.
El-Sufty, R., S. Al Bgham, S.A. Al-Awash, A.S. Shahdad and A.H. Al Bathra, 2011. A trap for
auto-dissemination of the entomopathogenic fungus Beauveria bassiana by the red palm
weevil adults in date palms plantations. Egyp. J. Biol. Pest Control, 21(2): 271-276.
El-Sufty, R., S.A. Al-Awash, A.M. Al-Amiri, A.S. Shahdad, A.H. Al-Bathra and S.A. Musa,
2007. Biological control of red palm weevil, Rhynchophorus ferrugineus (Col.:
Curculionidae) by the entomopathogenic fungus Beauveria bassiana in United Arab
143
Emirates. Proceeding of the 3rd International Conference on Date Palm. Acta
Horticulturae. 736: 399-404.
El-Sufty, R., S.A. Al-Awash, S. Al Bgham, A.S. Shahdad, A.H. Al-Bathra, 2009. Pathogenicity
of the fungus Beauveria bassiana (Bals.) Vuill to the red palm weevil, Rhynchophorus
ferrugineus (Oliv.) (Col.: Curculionidae) under laboratory and field conditions. Egyp. J.
Biol. Pest Control, 19: 81-85.
Francardi, V., C. Benvenuti, P.F. Roversi, P. Rumine and G. Barzanti, 2012.
Entomopathogenicity of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae
(Metsch.) Sorokin isolated from different sources in the control of Rhynchophorus
ferrugineus (Olivier) (Coleoptera Curculionidae). Redia, 95: 49-55.
Gindin, G., S. Levski, I. Glazer and V. Soroker, 2006. Evaluation of the entomopathogenic fungi
Metarhizium anisopliae and Beauveria bassiana against the red palm weevil
Rhynchophorus ferrugineus. Phytoparasitica, 34(4): 370-379.
Güerri-Agulló, B., S. Gómez-Vidal, L. Asensio, P. Barranco and L.V. Lopez-Llorca, 2010.
Infection of the red palm weevil (Rhynchophorus ferrugineus) by the entomopathogenic
fungus Beauveria bassiana: a SEM study. Microsco. Res. Tech., 73: 714-725.
Hussain, A., M.R.U. Haq, A.M. Al-Jabr and H.Y. Al-Ayied, 2013. Managing Invasive
Populations of Red Palm Weevil: A WorldwidePerspective. J. Food Agri. Environ., 11:
456-463.
Janmaat, A.F., L. Bergmann and J. Ericsson, 2014. Effect of low levels of Bacillus thuringiensis
exposure on the growth, food consumption and digestion efficiencies of Trichoplusia ni
resistant and susceptible to Bt. J. Inver. Pathol., 119: 32-39.
Ju, R.T., F. Wang, F.H. Wan and B. Li, 2011. Effect of host plants on development and
reproduction of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). J. Pest
Sci., 84(1): 33-39.
Kaya, H.K. and S.P. Stock, 1997. Techniques in insect nematology. Manual of Techniques in
Insect Pathology (ed. by LA Lacey). Academic Press, London, UK. pp. 281-324.
Kermarrec, A. and H. Mauleon, 1989. Synergy between chlordecone and Neoaplectana
carpocapsae Weiser (Nematoda: Steinernematidae) in the control of Cosmopolites
sordidus (Coleoptera: Curculionidae). Rev. Nematol., 12: 324-325.
Khamiss, O. and A. Abdel Badeea, 2013. Initiation, characterization and karyotyping of a new
cell line from red palm weevil rhynchophorus ferrugineus adapted at 27°c. AFPP - palm
pest mediterranean conference nice - 16, 17 and 18 january 2013.
Khan, S., L. Guo, Y. Maimaiti, M. Mijit and D. Qiu, 2012. Entom opathogenic fungi as
microbial biocontrol agent. Mol. Plant Breed, 3(7): 63-79.
Koppenhöfer, A.M. ans H.K. Kaya, 1997. Additive and Synergistic interactions between
entomopathogenic nematodes and Bacillus thuringiensis for scarab grub control. Biol.
Control, 8: 131-137.
Koppenhofer, A.M., H.Y. Choo, H.K. Kaya, D.W. Lee and W.D. Gelernter, 1999. Increased
field and greenhouse efficacy against scarab grubs with a combination of an
entomopathogenic nematode and Bacillus thuringiensis. Biol. Control, 14: 37-44.
Malumphy, C. and H. Moran, 2009. Red palm Weevil, Rhynchophorus ferrugineus. Plant Pest
Factsheet. Available online at
www.fera.defra.gov.uk/plants/publications/documents/factsheets/redPalmWeevil.pdf
(accessed 19 September 2012).
144
Martín, M.M. and T. Cabello, 2006. Manejo de la cría del picudo rojo de la palmera,
Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera, Dryophthoridae), en dieta
artificial y efectos en su biometría y biología. Boletín de Sanidad Vegetal de Plagas, 32:
631-641.
Marzban, R., Q. He, X. Liu and Q. Zhang, 2009. Effects of Bacillus thuringiensis toxin Cry1Ac
and cytoplasmic polyhedrosis virus of Helicoverpa armigera (Hübner) (HaCPV) on
cotton bollworm (Lepidoptera: Noctuidae). J. Inver. Pathol., 101: 71-76.
Merghem, A., 2011. Susceptibility of the red palm weevil, Rhynchophorus ferrugineus (Olivier)
to the green muscardine fungus, Metarhizium anisopliae (Metsch.) in the laboratory and
in palm tree orchards. Egyp. J. Biol. Pest Control, 21: 179-183.
Minitab, 2003. MINITAB Release 14 for Windows. Minitab Inc., State College, Pennsylvania,
USA. Mirza, I. 2007. Tomato paste plant to be set up at Killa Saifullah. Available at url;
http://www.pakissan.com/english/news/newsDetail.php?newsid=15041. Accessed on
August 31, 2007.
Nirula, K.K., 1956. Investigations on the pests of coconut palm. Part IV. Rhynchophorus
ferrugineus. Ind. Coc. J., 9: 229-247.
Nishimatsu, T., J.J. Jackson, 1998. Interaction of insecticides, entomopathogenic nematodes, and
larvae of the western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol., 91:
410-418.
Poinar Jr, G.O., 1990. Taxonomy and biology of Steinernematidae and Heterorhabditidae. In:
Gaugler, R., H.K. Kaya (Eds.), Entomopathogenic Nematodes in Biological Control.
CRC Press, Boca Raton, FL, pp. 23-61.
Ricaño, J., B. Güerri-Agulló, M.J. Serna-Sarriás, G. Rubio-Llorca, L. Asensio, P. Barranco, L.V.
Lopez-Llorca. 2013. Evaluation of the pathogenicity of multiple isolates of Beauveria
bassiana (Hypocreales: Clavicipitaceae) on Rhynchophorus ferrugineus (Coleoptera:
Dryophthoridae) for the assessment of a solid formulation under simulated field
conditions. Florida Entomol., 96: 1311-1324.
Sewify, G.H., M.H. Belal and S.A. Al-Awash, 2009. Use of the entomopathogenic fungus,
Beauveria bassiana for the biological control of the red palm weevil, Rhynchophorus
ferrugineus Olivier. Egyptian Journal of Biological Pest Control. 19(2):157-163.
Shapiro-Ilan, D.I., M. Jackson, C.C. Reilly and M.W. Hotchkiss, 2004. Effects of combining an
entomopathogenic fungi or bacterium with entomopathogenic nematodes on mortality of
Curculio caryae (Coleoptera: Curculionidae). Biol. Control, 30: 119-126.
Sikorowski, P.P. and A.C. Thampson, 1979. Effects of cytoplasmic polyhedrosis virus on
diapausing Heliothis virescens. J. Inver. Pathol., 33: 66-70.
Sokal, R.R. and F.J. Rohlf, 1995. Biometry. Freeman, New York.
Steinhaus, E.A., 1958. Stress as a factor in insect disease. Proceedings of the Xth Inter. Cong.
Entomol., 4: 725-730.
Thurston, G.S., H.K. Kaya and R. Gaugler, 1994. Characterization of enhanced susceptibility of
milky disease infected scarabaeid grubs to entomopathogenic nematodes. Biol. Control,
4: 67-73.
Thurston, G.S., H.K. Kaya, T.M. Burlando and R.E. Harrison, 1993. Milky disease bacteria as a
stressor to increase susceptibility of scarabaeid larvae to an entomopathogenic nematode.
J. Inver. Pathol., 61: 167-172.
145
Torta, L., V. Leone, C.G. Caldarella, G. Lo Verde and S. Burruano, 2009. Microrganismi fungini
associati a Rhynchophorus ferrugineus (Olivier) in Sicilia e valutazione dell’efficacia
entomopatogena di 484 Ann Microbiol., 65: 477-485.
Vitale, A, V. Leone, L. Torta, S, Burruano and G. Polizzi, 2009. Prove preliminari di lotta
biologica con Beauveria bassiana e Metarhizium anisopliae nei confronti del punteruolo
rosso. In Regione Siciliana - Assessorato Agricoltura e Foreste. La ricerca scientifica sul
Punteruolo rosso e gli altri fitofagi delle palme in Sicilia, Palermo, Italy. pp. 1:169-172.
Wiygul, G. and P.P. Sikorowski, 1991. Oxygen uptake in larval bollworm (Heliothis zea)
infected with iridescent virus. J. Inver. Pathol., 58: 252-256.
146
Table 7.1 Mean mortality (%±SE) of 2nd instar larvae of R. ferrugineus treated with B.
bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae
were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs
ml-1.
Treatments Intervalsa Weekb Observed mortality
Expected mortality
Chi Sq. Type of interaction
Bb
- 1 11.22 - - - - 2 14.28 - - - - 3 20.40 - - -
Ma
- 1 8.16 - - - - 2 12.24 - - - - 3 17.34 - - -
EPN
- 1 14.28 - - - - 2 21.42 - - - - 3 29.59 - - -
Bb+EPN
0 1 27.55 23.90 0.48 Additive 0 2 43.87 32.65 2.87 Additive 0 3 61.22 43.96 4.86 Synergistic
Ma+EPN
0 1 23.71 21.28 0.24 Additive 0 2 32.98 38.21 0.82 Additive 0 3 48.45 36.94 2.73 Additive
Bb+EPN
7 1 32.99 26.53 1.26 Additive 7 2 51.54 37.46 3.84 Synergistic 7 3 73.19 51.86 6.21 Synergistic
Ma+EPN
7 1 28.86 24.78 0.57 Additive 7 2 45.36 35.05 2.33 Additive 7 3 64.94 48.27 4.28 Synergistic
Bb+EPN
14 1 51.54 37.46 3.84 Synergistic 14 2 69.07 51.86 4.28 Synergistic 14 3 88.65 62.51 7.70 Synergistic
Ma+EPN
14 1 44.32 35.05 1.93 Additive 14 2 64.94 48.99 3.92 Synergistic 14 3 75.25 53.95 6.02 Synergistic
a Intervals between the application of EPFs and EPNs. b Week after fungal application.
147
Table 7.2 Mean mortality (%±SE) of 4th instar larvae of R. ferrugineus treated with B.
bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae
were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs
ml-1.
Treatments Intervalsa Weekb Observed mortality
Expected mortality
Chi Sq. Type of interaction
Bb
- 1 9.18 - - - - 2 11.22 - - - - 3 16.32 - - -
Ma
- 1 6.12 - - - - 2 9.18 - - - - 3 14.28 - - -
EPN
- 1 12.24 - - - - 2 17.34 - - - - 3 23.46 - - -
Bb+EPN
0 1 24.29 20.30 0.71 Additive 0 2 34.69 26.62 1.87 Additive 0 3 51.02 35.96 4.44 Synergistic
Ma+EPN
0 1 19.58 17.61 0.19 Additive 0 2 28.86 24.93 0.53 Additive 0 3 44.32 34.40 2.22 Additive
Bb+EPN
7 1 27.83 22.09 1.18 Additive 7 2 43.29 30.84 3.58 Additive 7 3 61.85 42.99 5.75 Synergistic
Ma+EPN
7 1 23.71 20.30 0.48 Additive 7 2 38.14 29.15 2.11 Additive 7 3 55.67 40.64 4.05 Synergistic
Bb+EPN
14 1 42.26 30.84 3.08 Additive 14 2 58.76 42.99 4.23 Synergistic 14 3 80.41 57.39 6.58 Synergistic
Ma+EPN
14 1 36.08 29.15 1.33 Additive 14 2 54.63 40.64 3.58 Additive 14 3 72.16 53.95 4.50 Synergistic
a Intervals between the application of EPFs and EPNs. b Week after fungal application.
148
Table 7.3 Mean mortality (%±SE) of 6th instar larvae of R. ferrugineus treated with B.
bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae
were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs
ml-1.
Treatments
Intervalsa Weekb Observed mortality (%)
Expected mortality
Chi Sq. Type of interaction
Bb
- 1 7.14 - - - - 2 9.18 - - - - 3 13.26 - - -
Ma
- 1 4.081 - - - - 2 7.14 - - - - 3 11.22 - - -
EPN
- 1 9.18 - - - - 2 14.28 - - - - 3 18.36 - - -
Bb+EPN
0 1 17.34 15.67 0.16 Additive 0 2 28.57 22.15 1.43 Additive 0 3 42.85 29.19 4.35 Synergistic
Ma+EPN
0 1 13.40 12.89 0.01 Additive 0 2 22.68 20.40 0.22 Additive 0 3 35.05 27.53 1.61 Additive
Bb+EPN
7 1 21.64 17.52 0.78 Additive 7 2 35.05 25.65 2.51 Additive 7 3 50.51 35.02 4.74 Synergistic
Ma+EPN
7 1 17.52 15.67 0.19 Additive 7 2 30.92 23.90 1.59 Additive 7 3 44.32 31.69 3.60 Additive
Bb+EPN
14 1 34.02 25.65 2.05 Additive 14 2 48.45 35.02 3.72 Additive 14 3 71.13 51.86 5.22 Additive
Ma+EPN
14 1 28.86 23.90 0.85 Additive 14 2 42.26 31.69 2.64 Additive 14 3 61.85 46.03 4.04 Additive
a Intervals between the application of EPFs and EPNs. b Week after fungal application.
Table 7.4 Factorial analysis for pupation, adult emergence and egg eclosion of R.
ferrugineus exposed to B. bassiana, M. anisopliae and H. Bacteriophora
S.O.V. df Pupation Adult emergence Egg eclosion
F P F P F P
Instar 2 18.78 ≤0.05 39.94 ≤0.05 35.0 ≤0.05 Treatment 9 114.28 ≤0.05 84.44 ≤0.05 90.89 ≤0.05 Instar × Treatment 18 10.49 ≤0.05 0.48 0.96 0.46 0.97 Error 232 - - - - - - Total 269 - - - - - -
149
Table 7.5 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar R. ferrugineus larvae treated with B.
bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106 spore ml-1 and
H. Bacteriophora was applied @ 100 IJs ml-1. Mean sharing the same letters are not significantly different. Means
sharing the same letters within columns are not significantly different
Treatments
Interval
Second instar Fourth instar Sixth instar
Pupation
(%)
Adult
emergence (%)
Egg eclosion
(%)
Pupation
(%)
Adult
emergence (%)
Egg eclosion
(%)
Pupation
(%)
Adult
emergence (%)
Egg eclosion
(%)
Bb - 62.22±2.23bc 57.77±2.77bc 53.33±2.33bc 71.11± 3.54b 66.66±3.08b 59.55±2.92b 80.00±2.88bc 74.44±3.37b 66.33±3.10b
Ma - 67.77±2.64b 61.11±3.21b 58.88±2.23b 73.33±3.40b 68.88±3.51b 62.22±2.77b 83.33±2.35ab 78.88±3.88ab 72.22±3.22b
EPN - 56.66±2.33bcd 50.55±2.69bcd 45.55±1.67bcd 62.22±2.77bc 57.77±2.33bc 51.11±2.51bc 69.44±3.42cd 65.55±2.75bc 59.33±2.44bc
Bb+EPN 0 45.55±1.93def 40.33±2.35def 36.66±1.88def 48.88±2.09cd 43.33±3.08cd 38.88±1.60cd 54.44±2.76ef 49.44±2.57de 43.33±2.35de Ma+EPN 0 51.11±1.51cde 44.77±2.89cde 39.44±1.36cde 54.44±2.12cd 49.44±2.16cd 44.44±2.23cd 60.00±2.88de 56.66±2.72cd 51.11±2.51cd
Bb+EPN 7 39.44±1.69ef 36.66±1.33def 31.11±1.51def 45.55±2.42d 40.55±1.93d 35.55±1.93d 51.11±2.51ef 45.55±2.93de 40.55±1.42de
Ma+EPN 7 43.55±1.73def 38.88±1.51def 34.44±1.37def 51.11±2.88cd 46.11±2.32cd 41.11±1.51cd 57.77±2.77def 52.22±2.23cde 46.66±1.33cde
Bb+EPN 14 32.22±1.27f 27.77±1.77f 22.22±1.46f 39.44±2.42d 36.66±1.33d 31.11±1.60d 44.44±2.93f 40.22±2.79e 34.44±1.93e Ma+EPN 14 35.55±1.43f 30.55±3.37ef 26.66±1.68ef 47.77±2.23cd 42.22±2.12cd 37.77±1.22cd 54.44±2.76ef 47.77±2.64de 42.22±1.22de
Control 90.55±2.11a 86.66±2.88a 81.11± 2.60a 93.33±2.66a 90.55±2.11a 83.33±3.33a 95.55±1.75a 92.22±2.22a 85.55±2.93a
df 9 9 9 9 9 9 9 9 9
F 35.7 31.3 31.0 23.4 23.6 28.8 30.6 28.1 32.4 P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05
150
Table 7.6 Effect of B. bassiana, M. anisopliae and H. Bacteriophora on the development of R. ferrugineus. B. bassiana and
M. anisopliae were used each @ 1×104 spore ml-1 and H. Bacteriophora was applied @ 50 IJs ml-1. Mean sharing the
same letters are not significantly different
Treatmen
ts
Larval duration
(days)
Larval
weight
(g)
Pre-pupal
duration
(days)
Pre-pupal
weight
(g)
Pupal duration
(days)
Pupal weight
(g)
Adult longevity (days) Adult weight (g)
Male
Female
Male
Female
Bb 98.16±3.21b 4.01±0.12bc 15.16±0.58bc 4.02±0.20ab 22.94±1.20bc 3.92±0.18abc 39.05±1.54ab 42.83±1.78ab 1.411±0.12ab 1.14±0.11abc
Ma 96.50±3.88bc 4.41±0.15ab 15.94±0.79bc 4.07±0.19ab 23.72±1.36abc 4.11±0.15ab 41.83±1.40a 43.61±1.67a 1.33±0.14ab 1.28±0.15ab
EPN 101.16±3.63ab 3.72±0.12cd 16.72±0.95abc 3.85±0.14ab 24.16±1.41abc 3.67±0.10bcd 37.16±1.23ab 40.38±1.45ab 1.18±0.1bc 1.05±0.12abc Bb+EPN 109.27±4.16a 3.08±0.12e 20.16±1.14a 3.07±0.11c 27.61±1.24a 3.21±0.11d 34.94±1.29b 36.50±1.78b 0.84±0.10d 0.78±0.11c
Ma+EPN 104.38±3.32ab 3.27±0.11de 18.50±0.98ab 3.48±0.12bc 25.50±1.41ab 3.43±0.13cd 36.16±1.17b 38.71±1.68ab 1.01±0.17cd 0.92±0.16bc
Control 87.05±1.08c 4.87±0.14a 14.38±0.74c 4.17±0.25a 21.27±1.36c 4.24±0.10a 42.83±1.61a 45.27±1.43a 1.56±0.13a 1.37±0.11a
df 5 5 5 5 5 5 5 5 5 5 F 9.92 27.3 6.59 6.94 5.15 11.1 5.58 3.93 12.7 4.26
P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05
151
Table 7.7 Analysis of co-variance for 2nd, 4th and 6th instar larvae of R. ferrugineus
regarding weight gain and frass production at a given level of diet consumption
when treated with B. bassiana and H. Bacteriophora alone and in combination.
Initial weight of larvae and diet consumption were taken as covariate
S.O.V. df F P Covariate: Diet Consumption 1 0.191 0.62 Covariate: Weight Gain 1 3.060 0.078 Frass Production × Diet Consumption 24 2.312 ≤0.05 Diet Consumption × Weight Gain 1 0.132 0.73 Frass Production × Weight Gain 27 2.730 ≤0.05 Frass Production × Diet Consumption × Weight Gain 24 2.701 ≤0.05 Error 687 - - Total 828 - -
152
.
Figure 7.1 Diet consumption in last instar larvae of R. ferrugineus when treated with B. bassiana and H. bacteriophora
153
.
Figure 7.2 Frass production in last instar larvae of R. ferrugineus when treated with B. bassiana and H. bacteriophora
154
.
Figure 7.3 Weight gain in last instar larvae of R. ferrugineus when treated with B. bassiana and H. bacteriophora
CHAPTER 8
Combined toxicity of Beauveria bassiana, Bacillus thuringiensis and Heterorhabditis
bacteriophora against red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera:
Curculionidae)
Abstract
Laboratory studies were carried out to evaluate the insecticidal effect of Beauveria bassiana
(Bb), Bacillus thuringiensis var. kurstaki (Bt-k) and an entomopathogenic nematode (EPN)
Heterorhabditis bacteriophora against distinct populations of Red Palm Weevil (RPW)
Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Four populations of RPW
were collected from different districts of Punjab, Pakistan including Layyah, Dera Ghazi Kahn,
Muzaffargarh and Rahim Yar Khan. All the three agents were used alone and in all possible
combinations (Bt-k+Bb, Bt-k+EPN, Bb+EPN and Bt-k+Bb+EPN) against 6th instar larvae and
adults of RPW. The experiments were carried out at 25±2 °C and 70±5% RH and 12:12 (D: L)
hours, mortality counts were taken after 7, 14 and 21 days post incubation. H. bacteriophora was
more effective followed by B. bassiana and Bt-k in alone treatments, while in combined
treatments increased mortality was recorded. Combined treatments of Bb+Bt-k exhibited lowest
mortality followed by Bt-k+ EPN, Bb+EPN and BB+Bt-k+EPN. The maximum rate of mycosis
and sporulation in the cadavers of RPW was observed where B. bassiana was applied alone and
similar trend was recorded for nematode production. The results of the present study indicate that
all three control measures may provide effective control against RPW. But need of the hour is to
evaluate these agents under field conditions.
Keywords: Beauveria bassiana, Heterorhabditis bacteriophora, Bacillus thuringiensis
Rhynchophorus ferrugineus
8.1 Introduction
The Red Palm Weevil (RPW) is an important invasive pest which has almost been
invaded and fully established in more than 50% of the date palm growing areas of the world
which attributes to the high fecundity than the normal species (Faleiro, 2006), capability to live
and interbreed in the same tree even for several generations (Rajamanickam et al., 1995; Avand-
Faghih, 1996), adult flight capacity to a longer distance (Wattanapongsiri, 1966) and pest
tolerance to a wide range of climatic conditions due to its hidden habit in palm tree. To combat
RPW different control practices have been deployed among date palm growing areas of the
world. Treatments revolve around the deployment of conventional chemical insecticides, sterile
insect techniques, use of semio-chemicals (Paoli et al., 2014) and bio-control agents
(Wattanapongsiri, 1966; Murphy and Briscoe, 1999; Faleiro, 2006). Most commonly used
control treatments are insecticides such as Diazinon, Imidacloprid, and Phosmet (Abbas, 2010).
However, heavy use of chemical treatments causes environmental damage and harms non-target
organisms, and also leads to the development of insecticide resistance against RPW (Jalinas et
al., 2015).
Very few studies have been conducted on the natural entomophagous enemies of R.
ferrugineus or other Rhynchophorus species (Murphy and Briscoe, 1999; Faleiro, 2006).
Entomopathogenic fungi (EPFs) are commonly found in the nature and cause epizootics in insect
populations, thus play significant role in regulating insect population. Mostly, the member of
Entomophthorales and Hyphomycetes attack on terrestrial insects. EPFs from various strains of
B. bassiana and M. anisopliae have been found in association with RPW and found among the
most relevant biological agents suggested to control RPW (Faleiro, 2006). Unlike the other
entomopathogens, entomopathogenic fungi infect the host by contact, then germinate and
penetrate the insect cuticle. The host can be infected both by direct treatment and by horizontal
transmission from infected insects or cadavers to healthy insects. Subsequently, infection can
occur via the new generation of spores (Lacey et al., 1999; Quesada-Moraga et al., 2004). These
unique characters make EPF especially important for the control of concealed insects such as
RPW.
Bacillus thuringiensis (Bt) is another important microbial control agent which holds a
prominent position among commercial chemical compounds important for agricultural insect
pests. Different researchers have evaluated the pathogenic potential of Bt against RPW and
revealed successful control (Banerjee and Dangar, 1995; Alfazariy, 2004; Bauce et al., 2002;
Sivasupramaniam et al., 2007; Birda and Akhursta, 2007; Manachini et al., 2008; Manachini et
al., 2009). Studies reveled that feeding seasation and midgut damage were observed amongst the
larvae survived after treatments. Entomopathogenic nematodes (EPNs) have been declared an
efficient entomopathogen against variety of insect in integrated pest management program
against RPW (Abbas et al., 2001; Llácer et al., 2009; Dembilio et al., 2010a). They are obligate
parasites in the families Steinernematidae and Heterorhabditidae which kill their host with the
aid of mutualistic bacterium present in their intestine (Poinar, 1990). As for as the life cycle is
concerned the nematodes complete 2-3 generations within the host, after which free-living
infective juveniles emerge to seek new hosts (Poinar, 1990). Several formulations have been
developed to improve the activity of nematodes against insect pests (Georgis, 1990; Georgis and
Kaya, 1998). In coleopteran pests larvae of several weevil species (Coleoptera: Curculionidae)
such as the black vine weevil, Otiorhynchus sulcatus (F.), and the Diaprepes root weevil,
Diaprepes abbreviatus (L.) was successfully controlled with EPNs (Shapiro-Ilan et al., 2002).
The intervention of more than one biocontrol agent can enhance the effectiveness of the
other partner; many studies have been conducted in this regard. The combined effect of B.
bassiana and B. thuringiensis working synergistically delivers more harm to insect pests
(Wraight and Ramos, 2005). Similarly combined application of EPNs and EPFs have been
evaluated against different insect pests (Thurston et al., 1993, 1994; Koppenhöfer and Kaya,
1997; Koppenhöfer et al., 1999; Yadav et al., 2004; Ansari et al., 2004, 2006, 2008). Hence
integrated practices can be a hint for those willing to manage RPW.
8.2 Materials and Methods
8.2.1 RPW collection and rearing
Four different populations of R. ferrugineus were collected from Layyah, Dera Ismail
Khan (D.I. Khan), Muzaffargarh and Rahim Yar Khan (R.Y. Khan) districts of Punjab
(Pakistan). Different developmental stages were collected from infested and fallen trees with the
permission of farmer (owner). From each area insects were collected and kept in different plastic
boxes assigned for a specific stage and brought to the laboratory until enough collection was
done. Further multiplication for one generation was carried out in Microbial Control Laboratory,
Department of Entomology, University of Agriculture, Faisalabad, Pakistan. Larvae were offered
with pieces of sugarcane (Saccharum officinarum L.; Poales: Poaceae) stem for feeding and
pupation, while shredded sugarcane pieces were offered to adults for feeding and substrate for
oviposition. After pupation, pupal cocoon were kept in separate plastic jars for adult emergence
in an incubator (Sanyo, Japan). After emergence beetles were shifted to the adult’s jar for
feeding, mating and oviposition. Colony was developed in plastic boxes (30×60×60 cm) having a
lid covered with mesh wire gauze (60 mesh size) in the middle (10 cm diameter) for aeration.
Adult’s diet was changed after every three days and replaced sugarcane pieces were kept in
separate jars for egg hatching. After egg hatching neonate larvae were allowed to feed for 3 days
in the same set and then shifted to the sugarcane sets for feeding and pupation. Larvae were
shifted to the new sugarcane sets after every week until pupation. The rearing conditions were
maintained at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod.
8.2.2 Preparation of B. thuringiensis spore-crystal mixtures
The commercial formulation of B. thuringiensis var. kurstaki (Bt-k) was obtained from
Microbial Control Laboratory, originally obtained from National Center for Genetic Engineering
and Biotechnology (BIOTEC) in Thailand. This strain was then subjected to sporulation by
culturing in 50 ml nutrient broth media. Harvesting of culture was carried out by centrifugation
at 6000 rpm for 15 min (Crecchio and Stotzky, 2001; Hernández et al., 2005). The pellets formed
resultantly were washed twice in cold 1M NaCl and thrice in sterile distilled water (SDW), re-
suspended in distilled water (5 ml). From the suspension formed, 1 ml was centrifuged for 5 min
at 10,000 rpm, dried for 4 hours at 37 °C and weighed (Wakil et al., 2013).
8.2.3 Entomopathogenic nematode
The Infective Juveniles (IJs) of H. bacteriophora were obtained from the culture
collection of Microbial Control Laboratory. Second instar larvae and adult of R. ferrugineus
were encountered with 300 IJs under laboratory conditions. H. bacteriophora was maintained in
3rd instar Galleria mellonella L. (Lepidoptera: Pyralidae) following the procedure of Kaya and
Stock (1997).
8.2.4 Preparation of fungi
The fungal isolate of B. bassiana (WG-43) used in the study was taken from the culture
collection of Microbial Control Laboratory, originally isolated from dead cadaver of RPW. Fungi
were sub-cultured on Sabouraud Dextrose Agar (BD, Becton, Dickisonand Company sparks, MD
21152 USA). Conidial suspension was prepared with 0.01% Tween-80 (Merck, KGaA,
Darmstadt, Germany) in sterile distilled water and conidial concentration of 1×107 conidia ml-1
determined using a Neubauer haemocytometer.
8.2.5 Treatment with B. bassiana
Sixth instar larvae and adults of uniform age from each population were directly
immersed into the conidial suspensions for 60 and 90s respectively and control was treated in
aqueous solution with 0.01% Tween-80. Isolate-treated and control insects were individually
shifted to 150 ml cylindrical plastic cups, each measuring 6 cm in height with 6 cm diameter.
The top of cups were covered with fine mesh in order to avoid the insects to escape. A piece of
2×2 cm2 artificial diet (Agar, brewer’s yeast, wheat germ, corn flour, ascorbic acid, benzoic acid,
amino acid-vitamin mix, chloramphenicol and nipagin) (Martín and Cabello, 2006) was kept in
the center of the cups for larvae and a shredded sugarcane piece was offered to the adults. All the
treatments were incubated at 25±2 °C, 70±5 % RH and a 12:12 (D: L) hours photoperiod and
mortality counts were made after 7, 14 and 21 days post-incubation. The causal agent of dead
larvae or adults were confirmed by shifting the cadavers into a Petri dish lined with wet filter
paper and incubating them at 25±2 °C and 70±5 % RH for up to 15 days.
8.2.6 Treatment with B. thuringiensis var. kurstaki (Bt-k)
Sixth instar larvae form each population was individually offered with artificial diets
(Martín and Cabello, 2006), mixed with the diluted spore-crystal (70 µg g-1). To each larvae, Bt-k
treated diet piece of (2×2 cm2) was provided to feed. For adults shredded sugarcane pieces were
dipped in known concentration of Bt-k for 90s and offered to respective populations.
8.2.7 Treatment with H. bacteriophora
H. bacteriophora suspension was prepared with a concentration of 300 IJs in glass jars
and 1 ml of suspension was poured into the cylindrical plastic cups lined with damp Whatman
filter paper. The top of the cups were covered with a fine mesh in order to avoid the insects to
escape. After pouring nematodes 30 minutes time was given for their even distribution on filter
paper (Atwa et al., 2014). A small piece of artificial diet 2×2 cm2 was placed in middle of the
cups as a food source for larvae and provided with new food every day. In each cup one 6 th instar
larvae from each population was placed on top of the filter paper. Same procedure was repeated
for adult population and shredded sugarcane pieces were offered as food source. Each treatment
was replicated three times, while control treatment received 1 ml of distributed water. The cups
were placed in an incubator at 25±2 °C and 70±5% RH at 12: 12 (D: L) hours photoperiod.
Mortality data was recorded after 7, 14 and 21 days after treatment and whole bioassay was
repeated thrice to avoid the pseudo-replication phenomenon. Dead individuals were transferred
to the modified White traps (White, 1927) and left for 10 more days for IJs emergence. The
insects exhibiting typical odor and color (signs for nematode infestation) were considered to be
killed by nematodes (Woodring and Kaya, 1988).
8.2.8 Treatment with B. bassiana, Bt-k and H. bacteriophora
In combined treatments of B. bassiana, Bt-k and H. bacteriophora 6th instar larvae and
adults were directly immersed in 100 ml of conidial suspensions for 60 and 90s respectively and
control was treated in aqueous solution with 0.01% Tween-80 solution. After fungal treatments
larvae and adults were offered with Bt-k treated artificial diet and sugarcane pieces respectively
on H. bacteriophora treated plastic cups lined with damp Whatman filter paper. Experimental
conditions were maintained at 25±2 °C, 70±5% RH and 12:12 (L: D) hours photoperiod and
mortality data was recorded after 7, 14 and 21 days. Three replicates of ten insects were used for
each treatment and same count of larvae fed on normal diet served as untreated check while
entire experiment was repeated thrice. Larvae that exhibiting fungal infection symptoms
(hardening of the cadaver or emergence of conidiophores) were maintained as described above
and production of spores on the cadavers were evaluated following 14 day of incubation at 25±2
°C. Larvae demonstrating symptoms of EPN infection (changes in pigmentation) were
maintained in White traps for 10 days for production of IJs.
8.2.9 Sporulation and Nematode production
Mycosed larvae after 14 days of incubation were vortexed for 30 minutes in distilled
water containing 0.01% Tween-80 and number of spores was estimated in 1 ml from the
suspension using a haemocytometer. Concentration of IJs was measured by 1 ml sample from the
final solution and counting IJs with the help of a Peters’ slide and microscope.
8.2.10 Statistical analysis
Mortality for each treatment was corrected for control mortality using Abbott's (1925)
formula and subjected to one way analysis of variance (ANOVA) in Minitab (Minitab, 2003)
means were separated using Tukey’s Kramer test (HSD) (Sokal and Rohlf, 1995) at 5%
significance level.
8.3 Results
8.3.1 Mortality of larvae and adult
The results of present study revealed that the larval and adult mortality was significantly
affected by the main effects and their associated interactions for all the population tested (Table
8.1). The mortality of both larvae and adult was non-significant (P≤0.05) among all the
population tested after each exposure interval, except the Bt-k+EPN for larvae after 7 days of
exposure and EPN, Bt-k+EPN and Bb+EPN for larvae and Bb+EPN for adults after 14 days of
exposure, after 21 days of exposure the treatments Bb and EPN for larvae and EPN, Bt-k+EPN
and Bb+EPN for adult were significantly different (P≤0.05). Overall the mortality was higher on
combined treatments as compared to individual applications of either B. bassiana, Bt-k or H.
bacteriophora for both larvae and adult, while the larval mortality was higher as compared to the
adult beetles in all the treatments applied at all the exposure intervals (Table 8.2, 8.3 and 8.4).
The laboratory population was more susceptible followed by R.Y. Khan, D.G. Khan and
Muzaffargarh at all the exposure intervals. After the last count Bb+EPN treatment exhibited
100% larval and adult mortality for all the populations tested, while Bt-k+EPN exhibited 100%
mortality for the laboratory population after 21 days post incubation.
8.3.2 Mycosis and sporulation
The maximum mycosed larvae (85.74%) and adults (69.07%), and sporulation in larvae
and (189.22 conidia ml-1) adults (164.56 conidia ml-1) was observed in treatments where B.
bassiana was applied alone against larvae and adults respectively in laboratory population
(Figure 8.1a, b and 8.2a, b), however low rate of mycosis and sporulation was observed in the
treatments where H. bacteriophora and B. bassiana were applied in combined manners. Similar
trend was recorded for the R.Y. Khan, D.G. Khan and Muzaffargarh populations.
8.3.3 Insects affected by EPF and EPN and their production
The maximum lethality in larvae affected by nematode was 92.40% and in adults
81.29%. The maximum number of nematode production on white trap was (178 IJs ml-1) adult
(153 IJs ml-1) was observed in treatments where H. bacteriophora was applied alone against
larvae and adult respectively in laboratory population (Figure 8.4a and 8.4b), however low rate
of nematode affected and production was recorded in the treatments where H. bacteriophora and
B. bassiana were applied in combined manners. Similar trend was recorded for R.Y. Khan, D.G.
Khan and Muzaffargarh population.
8.4 Discussion
Evidence suggests that entomopathogens play a key role in the host biology like
production of essential nutrients (amino acids and vitamins) and indispensable compounds which
influence some essential parameters such as growth, development, longevity, fertility, vector
capability, immunological competences and deliver protection against natural enemies (Valzano
et al., 2012). Numerous studies have documented sole and integrated applications of
entomopathogens against a number of insect pests. Generally the combined treatment of
entomopathogens exhibit enhanced mortality as compared to their individual application.
Therefore, simultaneous use of these agents did not cause any harmful effects on the efficiency
of the other agent. All agents have different modes of actions which enhance the disease severity
in a short period of time, hence reduce the time span to inflict damage to the host crop.
The entomopathogens that curtail RPW infestations are more effective in managing
weevil a population as compared to the plant protection (Salama et al., 2004; Dembilio et al.,
2010; El-Sufty et al., 2011). A number of entomopathogens are available worldwide that are
very effective against RPW including entomopathogenic fungi, bacteria and nematodes. Among
bacteria members of genus Bacillus like B. sphaericus, B. lentimorbus, B. popilliae are important
antagonists of RPW. These bacteria produce insecticidal proteins that target specific
developmental stages of RPW (Bulla et al., 1975; Salama et al., 2004). The entomopathogenic
fungi M. anisopliae and B. bassiana inflict mortality in different developmental stages of RPW
(Gindin et al., 2006; Dembilio et al., 2010). Moreover, the combined treatments of B. bassiana
and Bt exhibit enhanced larval mortality as compared to their individual applications. Similar
findings were observed by a number of researchers (Sander and Chichy, 1967; Kaliuga, 1968;
Fargues, 1973, 1975; Kalvish and Krivstova, 1978; Lewis and Bing, 1991).
Synergistic effects resulting from a combination of entomopathogenic nematodes with
other entomopathogens have been reported in a number of studies (Thurston et al., 1993, 1994;
Koppenhöfer and Kaya, 1997; Koppenhöfer et al., 1999). Contrarily Shapiro-Ilan et al. (2004)
found antagonism between entomopathogenic nematodes and P. fumosoroseus. Such
antagonisim may be due to pathogen interactions prior to or during infection. In case of Sternima
marcescens and P. fumosoroseus, it is possible that these organisms are directly pathogenic to
entomopathogenic nematodes, and therefore the nematodes may have been killed or their fitness
reduced prior to infection. These negative interactions may also be due to antagonistic toxins
produced by the entomopathogens after infection was initiated.
In our present study high mortality was recorded after different exposure intervals in
combined treatments as compared to their sole application. Higher mortalities were recorded for
(Bb+EPN) followed by (Bt-k+EPN) and (Bt-k+Bb) at all the exposure intervals. While in sole
treatments H. bacteriophora was found more effective followed by B. bassiana and Bt-k. Our
results are in accordance with the findings of Koppenhöfer and Kaya (1997) who reported
additive or synergistic interactions among entomopathogens when applied simultaneously.
Koppenhöfer and his collogues also recorded positive interaction between H. bacteriophora and
B. thuringiensis against Cyclocephala pasadenae, C. hirt and Anomala orientalis after different
exposure intervals (Koppenhöfer et al., 1999). Similar findings were reported by many other
scientists (Barbercheck and Kaya, 1990; Kermarrec and Mauleon, 1989).
Ansari et al. (2008) found synergistic interaction against black vine weevil larvae when
applied M. anisopliae and EPNs simultaneously. They also found similar results with H.
philanthus Füessly white grubs and revealed that type of interaction between EPN and fungal
entomopathogens depends on the time of application and specie of EPNs (Ansari et al., 2004,
2006). Similarly additive or slight synergistic interaction was recorded between M. anisopliae
and EPNs against Holotrichia consanguinea larvae (Yadav et al., 2004) and the larvae of pecan
weevil (Shapiro-Ilan et al., 2004).
A good knowledge of biological parameters of RPW and most importantly the interaction
among entomopathogens could play a key role to expand RPW-IPM programs. This, calls for the
isolation and identification of more virulent strains of entomopathogens (Manachini et al., 2011).
Moreover, the field evaluation of these substances in combined manners can provide substantial
information and help in developing new strategies by deploying IPM production systems (Neves
et al., 2001). In summary, the results of the present study indicate that the integration of
entomopathogens may be preferable to the use of a single agent. The integration takes advantage
of the positive characteristics of each agent. For example, the Bt treatments lead the gut to
septicemia causing the insect to stop feeding, and weakening the host immune system. This will
favor the B. bassiana to work efficiently with very low resistant of the host immune system
thereby increasing mortality.
Conclusions The present study showed that B. bassiana and Bt-k and H. bacteriophora can kill the
larvae and adult of R. ferrugineus from different populations collected across Punjab and Khyber
Pkhtunkhwa, Pakistan. They also exert the detrimental effect on their growth and development
which can be use effectively against this pest.
Acknowledgements
This research work was supported by the scholarship from Higher Education
Commission (HEC), Islamabad, Pakistan (112-30536-2AV1-263) under Indigenous Ph.D.
Fellowship Program.
8.5 References
Abbas, M.S.T., S.B. Hanounik, S.A. Mousa and S.H. Al-Bagham, 2000. Soil application of
entomopathogenic nematodes as a new approach for controlling Rhynchophorus
ferrugineus on date palm. Inter. J. Nematol., 10: 215-218.
Abbas, M.S.T., 2010. IPM of the red palm weevil, Rhynchophorus ferrugineus. In: Ciancio, A.
and K.G. Mukerji (Eds.). Integrated management of arthropod pests and insect borne
diseases, integrated management of plant pests and diseases 5, Springer, Berlin. Pp. 209-
233.
Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ.
Entomol., 18: 265-267.
Alfazariy, A.A., 2004. Notes on the survival capacity of two naturally occurring
entomopathogens on the red palm weevil Rhynchophorus ferrugineus (Olivier)
(Coleoptera: Curculionidae). Egyp. J. Biol. Pest Control, 14: 423.
Ansari, M.A., F.A. Shah and T.M. Butt, 2008. Combined use of entomopathogenic nematodes
and Metarhizium anisopliaeas a new approach for black vine weevil, Otiorhynchus
sulcatus, control. Entomol. Experi. et Appli., 129: 340-347.
Ansari, M.A., S. Vestergaard, L. Tirry and M. Moens, 2004. Selection of a highly virulent fungal
isolate, Metarhizium anisopliae CLO 53, for controlling Hoplia philanthus. J. Inver.
Pathol., 85: 89-96.
Ansari, M.A., F.A. Shah, L. Tirry and M. Moens, 2006. Field trials against Hoplia philanthus
(Coleoptera: Scarabaeidae) with a combination of an entomopathogenic nematode and
the fungus Metarhizium anisopliae CLO 53. Biol. Control, 39: 453-459.
Avand-Faghih, A., 1996. The biology of red palm weevil, Rhynchophorus ferrugineus Oliv.
(Coleoptera, Curculionidae) in Saravan region (Sistan & Balouchistan Province, Iran).-
Appl. Entomol. Phytopathol., 63: 16-18.
Banerjee, A. and T.K. Dangar, 1995. Pseudomonas aeruginosa a facultative pathogen of red
palm weevil Rhynchophorus ferrugineus. World J. Microbiol. Biotech., 11: 618-620.
Barbercheck, M.E. and H.K. Kaya, 1990. Interactions between Beauveria bassiana and the
entomogenous nematodes Steinernema feltiae and Heterorhabditis heliothidis. J. Inver.
Pathol., 55: 225-234.
Bauce, E., Y. Bidon and R. Berthiaume, 2002. Effects of food nutritive quality and Bacillus
thuringiensis on feeding behaviour, food utilization and larval growth of spruce budworm
Choristoneura fumiferana (Clem.) when exposed as fourth- and sixth instar larvae. Agri.
Forest Entomol., 4: 57-70.
Birda, L.J. and J.R. Akhursta, 2007. Variation in susceptibility of Helicoverpa armigera
(Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) in
Australia to two Bacillus thuringiensis toxins. J. Inver. Pathol., 94: 84-94.
Bulla, Jr L.A., R.A. Rhodes and G.S. Julian, 1975. Bacteria as insect pathogens. Ann. Rev.
Microbiol., 29: 163-190.
Crecchio, C. and G. Stotzky. 2001. Biodegradation and insecticidal activity of the toxin from
Bacillus thuringiensis subsp. kurstaki bound on complexes of montmorillonite-humic
acids-Al hydroxypolymers. Soil Biol. Biochem., 33: 573-581.
Dembilio, Ó., E. Quesada-Moraga, C. Santiago-Álvarez and J.A. Jacas, 2010. Potential of an
indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological
control agent against the red palm weevil, Rhynchophorus ferrugineus. J. Inver. Pathol.,
104: 214-221.
El-Sufty, R., S. Al-Bgham, S. Al-Awash, A. Shahdad and A. Al-Bathra, 2011. A trap for auto
dissemination of the entomopathogenic fungus Beauveria bassiana by red palm weevil
adults in date palm plantations. Egyp. J. Biol. Pest Control, 21: 271-276.
Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil
Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm
during the last one hundred years. Int. J. Trop. Insect Sci., 26: 135-154.
Fargues, J., 1973. Susceptibility of Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)
towards Beauveria bassiana (Bals.) Vuill. (Fungi Imperfecti, Moniaiales) in presence of
reduced doses of insecticide. Ann. Zool. Ecol. Anim. 5, 231-246.
Fargues, J., 1975. Field experiments on the combination of Beauveria bassiana with insecticides
for the control of Leptinotarsa decemlineata. Ann. Zool. Ecol. Anim., 7: 247-264.
Georgis, R., 1990. Formulation and application technology. In: Gaugler, R. and K.H. Kaya
(Eds.). Entomopathogenic Nematodes in Biological Control. Boca Raton, Fl: CRC Press.
Georgis, R. and H.K. Kaya, 1998. Advances in entomopathogenic nematode formulation. In:
Burges, H.D. (Ed.). Formulation of microbial biopesticides, beneficial microorganisms
nematodes and seed treatments. Kluwer Academic Publishers, Dordrecht, the
Netherlands. Pp. 289-308.
Gindin, G., S. Levski, I. Glazer and V. Soroker, 2006. Evaluation of the entomopathogenic fungi
Metarhizium anisopliae and Beauveria bassiana against the red palm weevil
Rhynchophorus ferrugineus. Phytoparasitica, 34(4): 370-379.
Hernández, C.S., R. Andrew, Y. Bel and J. Ferre, 2005. Isolation and toxicity of Bacillus
thuringiensis from potato-growing areas in Bolivia. J. Inver. Pathol., 88: 8-16.
Jalinas, J., B. Güerri-Agulló, R.W. Mankin, R. López-Follana and L.V. Lopez-Llorca, 2015.
Acoustic assessment of Beauveria bassiana (Hypocreales: Clavicipitaceae) effects on
Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) larval activity and mortality. J.
Econ. Entomol., 108(2): 444-453.
Kaliuga, M.V., 1968. Combined use of microbiological preparations in the control of pests. Ent.
Obozr., 47: 450-453.
Kalvish, T.K., and N.V. Krivtsova, 1978. Interaction of Muscardine fungi and Bacillus
thuringiensis var. Galleriae in vitro and in vivo. Izv. Sib. Otd. Akad. Nauk. SSSR Ser.
Biol. Nauk, 5: 40-46.
Kaya, H.K. and S.P. Stock, 1997. Techniques in insect nematology. Manual of Techniques in
Insect Pathology (ed. by LA Lacey). Academic Press, London, UK. pp. 281-324.
Kermarrec, A. and H. Maul eon, 1989. Synergy between chlordecone and Neoaplectana
carpocapsae Weiser (Nematoda: Steinernematidae) in the control of Cosmopolites
sordidus (Coleoptera: Curculionidae). Rev. Nematol., 12: 324-325.
Koppenhöfer, A.M. and H.K. Kaya, 1997. Additive and synergistic interaction between
entomopathogenic nematodes and Bacillus thuringiensis for scarab grub control. Biol.
Control, 8: 131-137.
Koppenhöfer, A.M., H.Y. Choo, H.K. Kaya, D.W. Lee and W.D. Gelernter, 1999. Increased
field and greenhouse efficacy with combination of an entomopathogenic nematode and
Bacillus thuringiensis against scarab grubs. Biol. Control, 14: 37-44.
Lacey, L.A., A.A. Kirk, L. Millar, G. Mereadier and C. Vidal, 1999. Ovicidal and larvicidal
activity of conidia and blastospores of Paecilomyces fumosoroseus (Deuteromycotina:
Hyphomycetes) against Bemisia argentifolii (Homoptera: Aleyrodidae) with a description
of a bioassay system allowing prolonged survival of control insects. Biocontrol Sci.
Tech., 9: 9-18.
Lewis, L.C. and L.A. Bing, 1991. Bacillus thuringiensis and Beauveria bassiana (Balsamo)
Vuillemin for European corn borer control: Program for immediate and season-long
suppression. Can. Ent., 123: 387-393.
Llácer, E., D.E. Martínez, M.M. Altube and J.A. Jacas, 2009. Evaluation of the efficacy of
Steinernema carpocapsae in a chitosan formulation against the red palm weevil,
Rhynchophorus ferrugineus, in Phoenix canariensis. BioControl, 54(4): 559-565.
Manachini, B., V. Arizza, D. Parrinello and N. Parrinello, 2011. Hemocytes of Rhynchophorus
ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces
cerevisiae and Bacillus thuringiensis. J. Inver. Pathol., 106: 360-365.
Manachini, B., V. Mansueto, V. Arizza and N. Parrinellom, 2008. Preliminary results on the
interaction between Bacillus thuringiensis and red palm weevil, In: 41st Annual Meeting
of Society for Invertebrate pathology and 9th international Conference on Bacillus
thuringiensis, Warwick, UK. Pp. 45
Manachini, B., P. Lo Bue, E. Peri and S. Colazza, 2009. Potential effects of Bacillus
thuringiensis against adults and older larvae of Rhynchophorus ferrugineus. IOBC/WPRS
Bull., 45: 239-242.
Mansour, N.A., M.E. Eldefrawi, A. Toppozada and M. Zeid, 1966. Toxicological studies on the
Egyptian cotton leafworm, Prodenia litura Vl potentiation and antagonism of carbamate
insecticide. J. Econ. Entomol., 59: 307-311.
Martín, M.M. and T. Cabello, 2006. Manejo de la cría del picudo rojo de la palmera,
Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera, Dryophthoridae), en dieta
artificial y efectos en su biometría y biología. Boletín de Sanidad Vegetal de Plagas, 32:
631-641.
Minitab, 2003. MINITAB Release 14 for Windows. Minitab Inc., State College, Pennsylvania,
USA.
Murphy, S.T. and B.R. Briscoe, 1999. The red palm weevil as an alien invasive: Biology and the
prospects for biological control as a component of IPM. Biocontrol New. Info., 20(1): 35-
45.
Neves, P.O.J., E. Hirose, P.T. Tchujo and A. Moino Jr, 2001. Compatibility of
entomopathogenic fungi with neonicotinoid insecticides. Neotro. Entomol., 30: 263-268.
Paoli, F., R. Dallai, M. Cristofaro, S. Arnone, V. Francardi and P.F. Roversi, 2014. Morphology
of the male reproductive system, sperm ultrastructure and cirradiation of the red palm
weevil Rhynchophorus ferrugineus Oliv. (Coleoptera: Dryophthoridae). Tissue and Cell,
46 (4): 274-285.
Poinar Jr., G.O., 1990. Taxonomy and biology of Steinernematidae and Heterorhabditidae. In:
Gaugler, R. and H.K. Kaya (Eds.). Entomopathogenic Nematodes in Biological Control.
CRC Press, Boca Raton, FL. Pp. 23-61.
Quesada-Moraga, E., R. Santos-Quiros, P. Valverde-Garcia and C. Santiago-Álvarez, 2004.
Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium
anisopliae (anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). J.
Inver. Pathol., 87: 51-58.
Rajamanickam, K., J.S. Kennedy and A. Christopher, 1995.Certain components of integrated
management for red palm weevil, Rhynchophorus ferrugineus F. (Curculionidae:
Coleoptera) on coconut. Mededelingen Faculteit Landbouwkundige en Toegepaste
Biologische Wetenschappen Universiteit Gent, 60: 803-805.
Salama, H.S., M.S. Foda, M.A. El-Bendary and A. Abdel-Razek, 2004. Infection of red palm
weevil, Rhynchophorus ferrugineus, by spore-forming bacilli indigenous to its natural
habitat in Egypt. J. Pest Sci. 77: 27-31.
Sander, H., and D. Cichy, 1967. Research on the effectiveness of fungal and bacterial
insecticides. Ekolog. Polska Ser., 15: 325-333.
Shapiro-Ilan, D.I., D.H. Gouge and A.M. Koppenhofer, 2002. Factors affecting commercial
success, case studies in cotton, turf and citrus. In: Gaugler, R. (Ed.). Entomopathogenic
Nematology, New York: CABI. Pp. 333-355.
Shapiro-Ilan, D.I., M. Jackson, C.C. Reilly and M.W. Hotchkiss, 2004. Effects of combining an
entomopathogenic fungi or bacterium with entomopathogenic nematodes on mortality of
Curculio caryae (Coleoptera: Curculionidae). Biol. Control, 30: 119-126.
Sivasupramaniam, S., G.P. Head, L. English, Y.J. Li and T.T. Vaughn, 2007. A global approach
to resistance monitoring. J. Inver. Pathol., 95: 224-226.
Sokal, R.R. and F.J. Rohlf. 1995. Biometry, 3rd edn. Freedman and Company, New York.
Thurston, G.S., H.K. Kaya, T.M. Burlando and R.E. Harrison, 1993. Milky disease bacteria as a
stressor to increase susceptibility of scarabaeid larvae to an entomopathogenic nematode.
J. Inver. Pathol., 61: 167-172.
Thurston, G.S., H.K. Kaya and R. Gaugler, 1994. Characterization of enhanced susceptibility of
milky disease infected scarabaeid grubs to entomopathogenic nematodes. Biol. Control,
4: 67-73.
Valzano, M.G., F. Achille, I. Burzacca, C. Ricci, P. Damiani, G. Scuppa and G. Favia, 2012.
Deciphering microbiota associated to Rhynchophorus ferrugineus in Italian samples: a
preliminary study. J. Entomol. Acarol. Res., 44: e16
Wakil, W., M.U. Ghazanfar, F. Nasir, M.A. Qayyum and M. Tahir, 2012. Insecticidal efficacy of
Azadirachta indica, nucleopolyhedrovirus and chlorantraniliprole singly or combined
against field populations of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae).
Chil. J. Agricul. Res., 72(1): 53-61.
Wakil, W., M.U. Ghazanfar, T. Riasat, M.A. Qayyum, S. Ahmed and M. Yasin, 2013. Effects of
interactions among Metarhizium anisopliae, Bacillus thuringiensis and
chlorantraniliprole on the mortality and pupation of six geographically distinct field
populations. Phytoparasitica, 41: 221-234.
Wattanapongsiri, A., 1966. A revision of the genera Rhynchophorus and Dynamis (Coleoptera:
Curculionidae). Department of Agriculture Science Bulletin (Bangkok). Pp. 328.
White, G.F., 1927. A method for obtaining infective nematode larvae from cultures. Science, 66:
302-303.
Woodring, L. and H. K. Kaya, 1988. Steinernematid and heterorhabditid nematodes: a handbook
of techniques. Series Bull. vol. 331. Arkansas Agricultural Experiment Station,
Fayetteville, AR.
Yadav, B.R., V. Singh and C.P.S. Yadava, 2004. Application of entomogenous nematode,
Heterorhabditis bacteriophora and fungi, Metarhizium anisopliae and Beauveria
bassiana for the control of Holotrichia consanguinea by soil inoculation method. Annal.
Agri. Bio. Res., 9: 67-69.
Table 8.1 ANOVA parameters for the main effects and associated interactions for mortality
levels of R. ferrugineus larvae and adults
S.O.V. Larvae Adult
df F P F P
Treatment 5 454.81 ≤0.05 437.56 ≤0.05
Interval 2 1546.71 ≤0.05 1099.79 ≤0.05
Location 4 25.54 ≤0.05 17.88 ≤0.05
Treatment × Interval 10 19.98 ≤0.05 26.11 ≤0.05
Treatment × Location 20 0.58 0.92 0.64 0.88
Interval × Location 8 0.93 0.49 1.24 0.27
Treatment × Interval ×
Location
40 0.73 0.89 0.41 0.99
Error 550 - - - -
Total 647 - - - -
Table 8.2 Mean mortality (%±SE) of R. ferrugineus populations collected from Layyah,
D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.
bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in
combination after 7 days of exposure (means followed by the same letter within
each treatment and insect populations not significantly different; HSD test
P≤0.05)
Stage Treatments Insect Populations
Layyah D.G. Khan Muzaffargarh R.Y. Khan F P
Larvae
Bt-k 9.47±0.78e 6.47±0.55d 6.08±0.78d 7.93±0.71e 0.38 0.77 Bb 13.48±1.18de 10.47±1.12cd 8.39±1.01cd 11.68±1.16de 0.30 0.82
EPN 25.57±1.39cd 21.57±1.38bc 19.58±1.17bc 23.45±1.13cd 0.62 0.60
Bt-k + Bb 32.28±1.61bc 28.36±1.46ab 25.06±1.30b 30.17±1.65bc 1.28 0.29
Bt-k + EPN 45.37±2.15ab 33.73±1.50ab 28.08±1.57ab 42.11±2.36ab 6.46 ≤0.05
Bb + EPN 51.68±2.34a 42.19±2.51a 39.40±2.14a 46.82±2.54a 2.05 0.12
F 24.8 30.5 18.8 14.4 - -
P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -
Adult
Bt-k 6.54±0.70d 4.69±0.49d 4.02±0.53c 5.46±0.74d 0.24 0.87
Bb 10.46±1.12d 8.41±0.87cd 7.56±1.04bc 9.29±1.09cd 0.16 0.92
EPN 19.18±1.24cd 14.90±1.01bcd 13.22±1.35bc 16.51±1.21cd 0.72 0.54
Bt-k + Bb 24.70±1.65bc 21.24±1.96abc 19.69±1.56ab 22.06±1.56bc 0.31 0.81
Bt-k + EPN 35.41±2.35ab 28.84±2.11ab 27.09±1.75b 31.52±2.13ab 0.90 0.45
Bb + EPN 39.73±2.09a 32.37±2.03a 30.89±2.05a 36.08±2.45a 1.38 0.26
F 19.2 8.90 12.4 14.4 - -
P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -
Table 8.3 Mean mortality (%±SE) of R. ferrugineus populations collected from Layyah,
D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.
bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in
combination after 14 days of exposure (means followed by the same letter within
each treatment and insect populations not significantly different; HSD test
P≤0.05)
Stage Treatments Insect Populations
Layyah D.G. Khan Muzaffargarh R.Y. Khan F P
Larvae
Btk 22.50±1.58c 20.67±1.39c 17.70±1.08c 19.50±1.23c 0.43 0.73
Bb 31.02±1.81c 27.76±1.85c 23.39±1.92c 25.64±1.08c 0.59 0.62
EPN 60.38±2.71b 55.66±2.44b 49.22±2.65b 51.57±2.18b 1.38 0.26
Bt-k + Bb 65.24±3.10b 59.10±2.18b 52.55±2.77b 55.09±3.07b 3.39 ≤0.05
Bt-k + EPN 89.66±3.26a 83.10±3.72a 74.57±3.14a 78.42±3.87a 4.07 ≤0.05
Bb + EPN 97.37±2.73a 91.55±3.27a 82.68±3.86a 86.17±3.03a 3.67 ≤0.05
F 86.2 73.1 46.3 60.3 - -
P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -
Adult
Btk 17.40±1.46c 15.78±1.04c 12.01±0.88c 13.76±1.07c 0.73 0.54
Bb 24.07±1.50c 21.85±1.28c 17.85±1.33cd 20.52±1.32c 0.37 0.77
EPN 45.06±2.48b 40.40±1.65b 34.54±2.03bc 37.62±1.99b 0.93 0.43
Bt-k + Bb 52.54±2.49b 48.01±2.14b 39.98±2.19b 43.27±2.75b 2.56 0.07
Bt-k + EPN 72.37±3.21a 67.77±3.10a 61.64±2.93a 65.80±2.57a 1.48 0.23
Bb + EPN 81.29±3.37a 75.74±3.34a 64.43±3.06a 70.18±2.28a 3.87 ≤0.05
F 50.5 42.1 27.9 41.8 - -
P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -
Table 8.4 Mean mortality (%±SE) of R. ferrugineus populations collected from Layyah,
D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.
bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in
combination after 21 days of exposure (means followed by the same letter within
each treatment and insect populations not significantly different; HSD test
P≤0.05)
Stage Treatments Insect Populations
Layyah R.Y. Khan Muzaffargarh D.G. Khan F P
Larvae
Bt-k 58.36±4.55d 54.66±4.46c 46.86±4.53d 49.78±2.45c 1.55 0.22
Bb 72.63±3.88c 66.79±3.45c 57.27±2.75cd 55.55±2.92c 6.04 ≤0.05
EPN 84.54±2.71bc 81.06±3.14b 70.45±3.19bc 73.92±2.74b 4.75 ≤0.05
Bt-k + Bb 87.01±3.04b 82.17±2.90b 75.77±4.75b 78.24±2.39b 2.09 0.12
Bt-k + EPN 100.00±0.00a 98.41±1.58a 93.35±3.06a 95.57±2.85a 1.75 0.17
Bb + EPN 100.0±0.00a 100.0±0.00a 100.0±0.00a 100.0±0.00a - -
F 29.8 34.9 35.5 69.0 - -
P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -
Adult
Bt-k 39.04±2.41d 32.56±3.80d 26.79±3.68f 30.74±2.96f 2.45 0.08
Bb 47.55±2.60d 43.07±4.15d 38.13±4.09e 36.12±3.31e 2.03 0.12
EPN 61.13±3.81c 58.15±3.25c 52.37±2.20d 54.35±1.82d 1.83 0.16
Bt-k + Bb 80.53±2.66b 76.69±3.11b 67.96±3.92c 71.04±3.18c 2.99 ≤0.05
Bt-k + EPN 94.24±2.28a 90.03±2.82a 81.27±2.28b 86.48±1.46b 5.88 ≤0.05
Bb + EPN 100.0±0.00a 100.0±0.00a 100.0±0.00a 100.0±0.00a - -
F 95.3 70.9 80.5 130 - -
P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -
Figure 8.1a Mean mycosis (%±SE) in larvae of R. ferrugineus populations collected from
Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.
bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in
combination (means followed by the same letter within each treatment are not
significantly different; HSD test P≤0.05)
Figure 8.1b Mean mycosis (%±SE) in adults of R. ferrugineus populations collected from
Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.
bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in
combination (means followed by the same letter within each treatment are not
significantly different; HSD test P≤0.05)
Figure 8.2a Sporulation (conidia ml-1) in larvae of R. ferrugineus populations collected from
Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.
bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in
combination (means followed by the same letter within each treatment are not
significantly different; HSD test P≤0.05)
Figure 8.2b Sporulation (conidia ml-1) in adult of R. ferrugineus populations collected from
Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B.
bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in
combination (means followed by the same letter within each treatment are not
significantly different; HSD test P≤0.05)
Figure 8.3a R. ferrugineus larvae affected by H. bacteriophora (%±SE) from different
populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan
treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H.
bacteriophora (300 IJs) applied alone or in combination (means followed by the
same letter within each treatment are not significantly different; HSD test P≤0.05)
Figure 8.3b R. ferrugineus adult affected by H. bacteriophora (%±SE) from different
populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan
treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H.
bacteriophora (300 IJs) applied alone or in combination (means followed by the
same letter within each treatment are not significantly different; HSD test P≤0.05)
Figure 8.4a Nematode production (IJs ml-1) in larvae of R. ferrugineus affected by H.
bacteriophora from different populations collected from Layyah, D.G. Khan,
Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107
conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination
(means followed by the same letter within each treatment are not significantly
different; HSD test P≤0.05)
Figure 8.4b Nematode production (IJs ml-1) in adult of R. ferrugineus affected by H.
bacteriophora from different populations collected from Layyah, D.G. Khan,
Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107
conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination
(means followed by the same letter within each treatment are not significantly
different; HSD test P≤0.05)
Summary
The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) (Coleoptera:
Curculionidae) is one of the major and destructive insect pests of 29 different palm species all
around the world. It is an important invasive pest that has invaded and established in more than
50% of the date palm growing areas of the world attributed to the high fecundity of this species
(Faleiro, 2006), ability to live and interbreed in the same tree for several generations
(Rajamanickam et al., 1995; Avand-Faghih, 1996), adult flight capacity (Wattanapongsiri, 1966)
and pest tolerance to a wide range of climatic conditions due to its hidden habit in palm tree. To
combat RPW different control practices has been deployed among date palm growing areas of
the world. Treatments revolve around the deployment of conventional chemical insecticides,
sterile insect techniques, use of semio-chemicals (Paoli et al., 2014) and bio-control agents
(Wattanapongsiri, 1966; Murphy and Briscoe, 1999; Faleiro, 2006). Integration of RPW
associated microbial control agents with other control practices such as entomopathogens,
chemical insecticides and attract-and-kill techniques.
Various management strategies have been adopted for controlling this pest mostly relying
upon the use of broad-spectrum insecticides, but the injudicious use of such chemicals raises
various environmental and human health related issues that necessitates review of prevailing
control measures and evaluation of the new and alternative control methods. The utilization of
entomopathogenic microorganisms such as entomopathogenic fungi, entomopathogenic bacteria
and entomopathogenic nematodes are considered to be promising alternatives to conventional
insecticides in managing this voracious pest.
Prior to the application of any control strategy, sampling or monitoring of the pest
population and their genetic analysis can give a better idea of the exact status of the insect
populations and can facilitate the adaptation of appropriate curative measures. In order to have
base line data about the genetic diversity of R. ferrugineus from local populations and their
comparison with the rest of the world populations can give the idea of their native and invaded
range and their distribution pattern. Moreover, local populations of R. ferrugineus have gained
resistance to commonly used chemical insecticides and phosphine due to the excessive and
unwise use of these chemical insecticides. Resistance against seven different populations of R.
ferrugineus was determined from very low to low and moderate to high level against agents
commonly used insecticides. Phosphine, cypermethrin and deltamethrin exhibited highest
resistance against almost all populations of this insect pest.
Entomopathogenic fungi are potent alternatives to these chemical insecticides. Screening
of 19 different fungal isolates of B. bassiana and M. anisopliae exhibited variable ranges of
mortality against larvae and adults. Five best isolates that caused highest mortality against larvae
and adult after 5, 10 and 15 days of incubation were screened by virulence assays. WG-41 and
WG-42 were the best isolates that caused highest mortality and significantly reduced the
developmental parameters. B. bassiana are capable of colonizing endophytically in live date
plam petioles even after 30 days of inoculation and can significantly reduce the weevil
population when exposed to the endophytically colonized date palm pieces. Moreover Bt-k is
also an effective agent that can also cause detrimental effects of larval and adult survival alone
and in combination with endophytically colonized date palm pieces. Both agents also had great
influence on the developmental parameters such as larval duration, larval weight, prepupal
duration, prepupal weight, pupal duration, pupal weight, adult longevity and adult weight etc. the
agents also affect the developmental parameters like, diet consumption, frass production and
weight gain.
The alone and integrated use of entomopathogenic fungi, Bt-k and nematodes can also
cause to suppress the weevil population collected from 4 different areas of Punjab and Khyber
Pkhtunkhwa, Pakistan under laboratory conditions. Hence we can use microbial
entomopathogens against this voracious pest which are safer to environment and compatible to
environment.