mohammed ibrahim elbashir ali · investigation of entomopathogenic fungi (beauveria bassiana and...
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Investigation of Entomopathogenic Fungi (Beauveria bassiana
and Metarhizium anisopliae) for Control of Bactrocera dorsalis
(Hendel) (Diptera: Tephritidae), and Development of
Formulation
MOHAMMED IBRAHIM ELBASHIR ALI
DIVISION OF ENTOMOLOGY
INDIAN AGRICULTURAL RESEARCH INSTITUTE
NEW DELHI – 110 012
2014
Investigation of Entomopathogenic Fungi (Beauveria bassiana
and Metarhizium anisopliae) for Control of Bactrocera dorsalis
(Hendel) (Diptera: Tephritidae), and Development of
Formulation
A Thesis By
MOHAMMED IBRAHIM ELBASHIR ALI
Submitted to the Faculty of Post-Graduate School,
Indian Agricultural Research Institute, New Delhi
In partial fulfilment of the requirements
For the award of the degree of
DOCTOR OF PHILOSOPHY
IN
ENTOMOLOGY
2014
Approved by the Advisory committee:
Chairman
Dr. Bishwajeet Paul
________________________________
Members Dr. Pratibha Sharma
________________________________
Dr. K. Shankarganesh
________________________________
Dr. S.D. Singh
________________________________
CERTIFICATE
This is to certify that the thesis entitled “Investigation of Entomopathogenic Fungi
(Beauveria bassiana and Metarhizium anisopliae) for Control of Bactrocera dorsalis
(Hendel) (Diptera: Tephritidae), and Development of Formulation” submitted to the
Faculty of the Post-Graduate School, Indian Agricultural Research Institute, New Delhi, in
partial fulfilment of the requirements for the award of the degree of DOCTOR OF
PHILOSOPHY IN ENTOMOLOGY is a record of bona fide research work carried out by
Mr. MOHAMMED IBRAHIM ELBASHIR ALI, Roll No. 9764 under my guidance and
supervision, and that no part of this thesis has been submitted for any other degree or
diploma. It is further certified that all the assistance and help availed during the course of
investigation as well as all sources of information have been duly acknowledged by him.
Date: /2/2014 (Dr. Bishwajeet Paul)
New Delhi-110012 Chairman of Advisory Committee
Dedication
To the pure soul of my beloved mother whom her wishes, was
to see me a great human being, beautified with noble manners
ACKNOWLEDGMENTS
First of all I thank Almighty Allah for giving me health and support to complete this work. I
wish to thank my previous supervisor Dr. R.D. Gautam Division of Entomology IARI New
Delhi, for accepting me in this subject of my interest and for his kind support and
encouragement. I acknowledge my current supervisor Dr. Bishwajeet Paul, Division of
Entomology IARI New Delhi, for his help, support and effort he has made to complete this
work. His critical views are of utmost importance.
Advisory committee especially Dr. Shankarganesh , Division of Entomology IARI New
Delhi -the ever smiling person- who has given me moral support also his tactfulness and
wisdom is greatly appreciated. Biological Control Laboratory, Division of Plant Pathology,
where I initiated this work and learnt mass production technique is highly acknowledged.
Words cannot express my thanks to Professor Dr V.V. Ramamurthy, Division of Entomology
IARI New Delhi whom without his help, close monitoring, systematic arrangement and
sincere sponsoring and fatherly encouragement this work would not see the light. Besides,
many administrative lessons have been learnt from his wisdom and tactfulness.
I would like to express my profound greeting for Indian Council for Cultural Relations
(ICCR) for providing me Scholarship to study In India.
National Council for Training- Sudan has played invaluable role during the course of this
study by providing me with monthly allowance without which it would be almost impossible
to bear the expenses of living in India. so I record my hearty respect for this esteemed
organization.
I thank Dr G.K. Mahapatro whom I call unofficial member of my advisory committee for his
constructive criticism and sharp discussion since starting of this work up to final submission.
I thank Dr Suresh M. Nebapure, Scientist at Division of Entomology IARI New Delhi who
has been very kind to me as a colleague and helpful as a class mate and as a Scientist.
Dr (Ms) Vinay Kalia Senior Scientist, who has been always helpful, welcoming, cooperative
and advising whenever necessary. I do thank her also for making the equipment and facilities
of her lab, such as autoclave and microscopes particularly inverted microscope. Her lab
members are also acknowledged particularly madam Monika, whose her experience has been
useful to me.
I do appreciate the effort and sincere advices provided by Dr S. Subramanian Principal
Scientist, Division of Entomology, IARI New Delhi. I also thank him for availing his lab
facilities particularly lyophiliser, deep freezer -20 and other lab equipment. Section of
Taxonomy Division of Entomology, IARI New Delhi is heartily acknowledged. The facilities
of this section have been very valuable to me particularly ever tidy microscopes and the staff
who has been welcoming and cooperative as well.
All my colleagues, seniors and juniors are very valuable to me with whom I interacted during
course work particularly Dr. Sujay Anand Dr. Suresh Dr. Gundappa Baradevanal Mr. Vivek
Shah, Mr. Guru Prasanna pandey Miss Soumia and Madam Sooji. Their valuable discussion
during the course of study particularly in seminar series has been very valuable to me. Special
thanks are due to my colleague Dr. Reddy who has been working on fruit flies, for his
valuable discussions.
Dr. Subhash Chandra- who has been for some time in advisory committee- thanks to him for
his keenness to know my progress and his follow-up is highly appreciated.
Of course all the teachers in Division of Entomology and supporting staff have been helpful
to me without their support I would not have been able to proceed.
I do thank Dr (Mrs) Chitra Srivastava for supplying me with equipment such as pH meter.
Her advice and recommendations has been of great importance.
I thank Head Division of Entomology Dr G.T. Gujar for his follow-up keenness to see me
taking degree.
I recognize the role of previous dean Dr. H.S. Guar who has been always solving problems.
His encouragements and administrative skills have been of great help. Moreover he used to
pray full attention whenever approached.
I acknowledge the great role of Director of IARI Dr. H.S. Gupta who has been always acute
to ensure that, his institute is efficiently functioning. I do thank him also for the best
environment he had provided by his wise management which was reflected in systematic and
highly organized institute.
Laboratory of Dr. Dr Kirti Sharma is acknowledged where with help of technician there, I got
trained on rearing of fruit flies and handling them.
Special thanks are due to Mr. Tolosi a worker at the office of professor Division of
Entomology, IARI New Delhi who has been always smiling, polite and cooperative while
doing office work.
My friends Dr. K.M. Anes Dr. Mohammed Azhar and Dr.Mohammed Jameel have been very
kind to me, they have provided valuble advices and sincere help whenever required.
Also I thank my Neighbours at Saraswati apartment from different countries
Specially Dr, Abdulrahman Mustafa and Dr, Mohammed Ibrahim Farag from Egypt, Dr.
Nouri kushlaf and Dr. Sulyman Fadl from Libya,Dr. Ibrahim Sabeki from Iran and Madam
Dr. Parasanna and Dr. Shaker from India and all neighbours for making the residence area as
beatifull as their fascinating characters.
All IARI staff and facilities are acknowledged particulary dispensary Dr. Anita and her kind
staff.
I am very thankful to all my class mates at IARI from various disciplines with whom I had
interacted and learned a lot.
Dr. Sami Ibrahim Mohammed Nour Sudanese friend, faculty of university of Gezira who had
joined IARI as Ph.D. his help and support for frequent technical problems in computer and
moral support during the course of this study. Moreover he and his family provided Sudanese
atmosphere.
I express gratitude to Professor Mazoub Omer Bashir who had supervised me during M.Sc.,
from him I learned this subject. Also show appreciation to Dr. Abasher Aawad Abasher, Dr.
Edur Zahran and Ustaz Gasim University of Gezira from them I had learned the basics of
plant pathology. Not only this but moreover they had grown the spirit of thirst to knowledge.
My kind father has been always supporting me, without his sincere supplication to almighty
Allah this work could not have been completed. My dear wife and beloved kids have
sacrificed their evenings and weekends while I am carrying out this research so they deserve
my profound thanks and acknowledgements.
CONTENTS
CHAPTER No. TITLE PAGE No.
1 INTRODUCTION 1-4
2 BACKGROUND 5-13
3 MATERIALS AND METHODS 14-19
4 RESEARCH PAPER I 20-25
5 RESEARCH PAPER II 26-33
6 RESEARCH PAPER III 34-44
7 GENERAL DISCUSSION 45-52
8 SUMMARY AND CONCLUSIONS 53-56
9
ABSTRACTS
ENGLISH 57-58
HINDI 59-60
10 BIBLIOGRAPHY 61-70
LIST OF TABLES
TABLE No. TITLE PAGE AFTER
3.1 Origin of fungal isolates tested against the insects. Source:
Catalogue of Fungal & Bacterial Cultures 1936-2012 (VIII
Edition)
14
3.2 Three commercial mycoinsecticides (Bio-Power®, Bio-
Magic® and Bio-Catch
®) were supplied by T. Stanes and
Company Tamil Nadu, India
14
3.3 General information on adjuvants and their sources 14
3.4 General information on carriers and their sources 14
3.5 The list of 45 recipes based on five carriers 15
4.1 Origin of fungal isolates tested against the insect. Source
Catalogue of Fungal & Bacterial Cultures 1936-2012 (VIII
Edition)
22
4.2 Pathogenicity and percentage mortality of entomopathogenic
fungi against the tested insects
23
4.3 Percentage mortality of adult Bactrocera dorsalis due to
entomopathogenic fungi
23
4.4 Average mortality of Corcyra cephalonica and Coccinella
septumpunctata
23
4.5 LT50 values of entomopathogenic fungi isolates against
Corcyra cephalonica.
23*
5.1 Origin of fungal isolates tested against the three stages of
Bactrocera dorsalis,Source: Catalogue of Fungal & Bacterial
Cultures 1936-2012 (VIII Edition)
27
5.2 Products tested against the oriental fruit fly, Bactrocera
dorsalis
27
5.3 Average mortality of Bactrocera dorsalis (last larval stage,
pupae and adults) treated with three commercial
mycoinsecticides.
29
5.4 LC50 values of three virulent isolates of entomopathogenic
fungi against adults of Bactrocera dorsalis
29
5.5. Percentage of emergent adults out of last larval stage and
pupae of Bactrocera dorsalis treated with different
concentrations of three strains of Beauveria bassiana.
29
5.6 LC50 values of entomopathogenic fungi isolate ITCC No.
6645 against last larval stage of Bactrocera dorsalis
29
5.7 Cumulative mortality of adult fruit fly Bactrocera dorsalis
treated with different strains of Beauveria bassiana
29
6.1 General information on carriers 36
6.2 The list of 45 recipes based on five carriers 36
6.3 General information on adjuvants 36
6.4 Results of tests carried out on physico-chemical properties of
the carriers
41
6.5 Germination percentage of ten products selected for viability
test
41
6.6 Result of bioassay of three products on three stages of fruit fly
Bactrocera dorsalis
41
LIST OF FIGURES
FIGURE No. TITLE PAGE AFTER
4.1. Pathogenicity of different isolates of entomopathogenic
fungi against different insect pests and natural enemies
23
4.2. Percentage mortality of adults of Bactrocera dorsalis due
to treatments of different isolates of entomopathogenic
fungi
23
4.3. Average mortality of Corcyra cephalonica treated with
different isolates entomopathogenic fungi.
23
4.4. Average mortality of Coccinella septumpunctata treated
with different isolates of entomopathogenic fungi
23
4. 5 LT50 (Days) Values of different isolates of
entomopathogenic fungi against larvae of Corcyra
cephalonica
23
5.1. Pathogenicity of entompathogenic fungi isolate ITCC No.
6628 to adult of Bactrocera dorsalis
29
5.2. Pathogenicity of entompathogenic fungi isolates ITCC No.
6645 to adult of Bactrocera dorsalis
29
5.3 Pathogenicity of entomopathogenic fungi isolate
B.NCIPM to adult of Bactrocera dorsalis
29
5.4. Per cent mortality of Bactrocera dorsalis treated with three
commercial mycoinsecticides (Bio-Power®,
Bio-Magic®,
Bio-Catch®)
29
5.5 Pathogenicity of entompathogenic fungi isolates ITCC No.
6645 to last larval stage of Bactrocera dorsalis
29
LIST OF PLATES
PLATE No. TITLE PAGE AFTER
3.1 Eight fungal isolates screened by this study 14
3.2 Entomopathogenic fungus, Beauveria bassiana ITCC No.
6628
14
3.3 Mass culture of Beauveria bassiana on sorghum grains 15
3.4 spores washed off with 0.05% Criton X-100 in conical
flasks
15
3.5 Sigma laboratory centrifuge 3K18 15
3.6 The collected spores of Beauveria bassiana by
centrifugation
15
3.7 The fungus kept in -200
C before lyophilisation 15
3.8 Lyophilisation of the centrifuged fungus for 24 hours 15
3.9 Calcite, Dolomite, Pyrophylite, Soapstone and Talcum
powder
15
3.10 Perfume sprayers 19
3.11 Soil and fungi vigorously mixed with a mixer for 30
seconds
19
3.12 Condition of experiments kept in, B.O.D,27±10
C, 65± 5%
R.H and 12:12 h
19
3.13 Newly developed cages, Plastic jars (15cm diameter ×
21cm height)
19
3.14 Heated ironic round piece of metal for cutting holes
through plastic jars
19
3.15 Heated rod kept on electrical heater, for puncturing holes
around the cut opening
19
4.1 Three EPF isolates of Beauveria bassiana that pathogenic
against Bactrocera dorsalis
27
4.2 Adults of B. dorsalis fully covered with white mycelium 29
4.3 Pathogenicity of ITCC No. 6628 to C. cephalonica 29
4.4 Pathogenicity of Beauveria bassiana isolate ITCC No.
6645 to Spodoptera litura
26
4.5 Pieris brassicae infected by Beauveria bassiana, ITCC
No. 6628
29
4.6 Growth of white mycelium on the intersegmental parts of
dead larvae of C. cephalonica treated by Beauveria
bassiana, ITCC No. 6628
29
4.7 Pink to reddish colour observed on cadavers of Corcyra
cephalonica treated by Beauveria bassiana
29
5.1 Commercial mycoinsecticides , Bio-Power®
, Bio-Magic®
and Bio-Catch ®
30
5.2 Ten-fold serial dilution which was prepared form different
fungal isolates of Beauveria bassiana
29
5.3 Mycosis of isolate B. NCIPM on B.dorsalis 29
5.4 Mycosis of isolate ITCC No. 6628 on B.dorsalis 29
5.5 Mycosis of isolate ITCC No. 6645 on B.dorsalis 29
5.6 Pathogenicity of entompathogenic fungi isolate ITCC 6645
to 3rd
instar larvae of Bactrocera dorsalis
29
6.1 Germination percentage of the product, PA above 90% 44
6.2. Gemination percentage of the product, PD above 90% 44
6.3 Gemination percentage of the product, PG above 90% 44
Fig. 4.3. Average mortality of Corcyra cephalonica treated with different isolates
entomopathogenic fungi.
Fig. 4.1. Pathogenicity of different isolates of entomopathogenic fungi against different insect
pests and natural enemies
1= Not tested
2= Tested but not pathogenic
3= Pathogenic
Fig. 4. 5. LT50 (Days) Values of different isolates of entomopathogenic fungi against larvae
of Corcyra cephalonica
Fig. 4.4. Average mortality of Coccinella septumpunctata treated with different isolates of
entomopathogenic fungi
Fig. 4.2. Percentage mortality of adults of Bactrocera dorsalis due to treatments of
different isolates of entomopathogenic fungi
Fig. 5.4. Per cent mortality of Bactrocera dorsalis treated with three commercial mycoinsecticides
(Bio-power®,
Bio-magic®
, Biocatch®
)
Fig.5.1. Pathogenicity of entompathogenic fungi isolate ITCC No. 6628 to adults of
Bactrocera dorsalis
Fig. 5.2. Pathogenicity of entompathogenic fungi isolates ITCC No. 6645 to adults of
Bactrocera dorsalis
Fig. 5.3. Pathogenicity of entompathogenic fungi isolate B.NCIPM to adult of Bactrocera
dorsalis
Fig. 5.5. Pathogenicity of entompathogenic fungi isolates ITCC No. 6645 to last larval stage
of Bactrocera dorsalis
Plate. 3.1. Eight fungal isolates screened by this study
Plate. 3.2. Entomopathogenic fungus, Beauveria bassiana ITCC No. 6628,the most
virulent one.
Plate. 3.3. Mass culture of Beauveria bassiana on sorghum grains
Plate. 3.4. Spores were washed off with 0.05% Criton X-100 in conical flasks
Plate. 3.5. Sigma laboratory centrifuge 3K18 Plate. 3.8. Lyophilisation of the centrifuged,
fungus for 24h
Plate. 3.6. The collected spores of Beauveria bassiana by centrifugation
Plate. 3.7.The fungus was kept in -200
C before lyophilisation
Plate. 3.9. Calcite, dolomite, pyrophylite, soapstone and talcum powder
Plate 3.11. Soil and fungi vigorously mixed with a mixer for 30s
Plate. 3.10. Perfume sprayers
Plate.3.12. Condition of experiments kept in, B.O.D, 27±10
C, 65± 5% R.H and 12:12 h
Plate.3.13. Newly developed cages, Plastic jars (15cm diameter × 21cm height)
Plate 3.14. Heated ironic round piece of metal for cutting holes through plastic jars
Plate 3.15. Heated rod kept on electrical heater, for puncturing holes around the cut opening
Plate. 4.1. Three EPF isolates of Beauveria bassiana which were found to be pathogenic
against Bactrocera dorsalis
Plate. 4.3. Pathogenicity of ITCC No. 6628 on C. cephalonica
Plate. 4.4. Pathogenicity of Beauveria bassiana isolate No ITCC No 6645 to S. litura
Plate. 4.2. Adults of B. dorsalis fully covered with white mycelium
Plate. 4.5. Pieris brassicae infected by Beauveria bassiana, ITCC No. 6628
Plate 4.6. Growth of white mycelium on the intersegmental parts of dead larvae of C.
cephalonica treated by Beauveria bassiana, ITCC No. 6628
Plate. 4.7. Pink to reddish colour observed on cadavers of Corcyra cephalonica treated by
Beauveria bassiana, this colouration is thought to be due to metabolite oosporein
Plate. 5.1. Commercial mycoinsecticides , Bio-power®
, Bio-magic®
and Bio-catch ®
Plate. 5.2. Ten-fold serial dilution was prepared form different fungal isolates of Beauveria
bassiana
Plate. 5.3. Mycosis of isolate B. NCIPM on B. dorsalis
Plate. 5.6. Pathogenicity of entompathogenic fungi isolate ITCC 6645 to 3rd
instar larvae of
Bactrocera dorsalis
Plate. 5.4. Mycosis of isolate ITCC No. 6628 on B. dorsalis
Plate. 5.5. Mycosis of isolate ITCC No. 6645 on B. dorsalis
Plate 6.1.Germination percentage of the product, PA above 90%
Plate. 6.2. Gemination percentage of the product, PD above 90%
Plate. 6.3. Gemination percentage of the product, PG, above 90%
Table 4.1.Origin of fungal isolates tested against the insect. Source: Catalogue of Fungal & Bacterial Cultures 1936-2012 (VIII Edition)
Isolates
No
Isolate name Accession number Deposition
date
Host Isolator and address
1 Beauveria bassiana (Balsamo)
Vuillemen
ITCC No.6552 29.3.2010 Tetranychus
urticae
TNAU, Head
2 Beauveria bassiana (Balsamo)
Vuillemen
ITCC No.6628 3.8.2010 Phyllocotruta
oloeivora
Banglore, P.P. Sreerama Kumar
3 Beauveria bassiana (Balsamo)
Vuillemen
ITCC No.6645 18.8.2010 White grub
Jorhat, B. Bhattacharya
4 Beauveria bassiana (Balsamo)
Vuillemen
ITCC No.4512 1994 Sugarcane borer
Karnal, H.R. Sardana
5 Metarrhizium anisopliae (Metschin)
Sorokin
ITCC No.4514 1994 Sugarcane root
borer
Karnal, H.R. Sardana
6 Metarrhizium anisopliae (Metschin)
Sorokin
ITCC No.6377 27.10.2008 Insects,
Junagadh, Plant Pathology,
Prof. & Head
7 Beauveria bassiana (Balsamo)
Vuillemen
Beauveria bassiana
N. CIPM
----- --------------- NCIPM, New Delhi
8 Metarrhizium anisopliae (Metschin)
Sorokin
Metarrhizium
anisopliae N. CIPM
----- --------------- NCIPM, New Delhi
Table 4. 2. Pathogenicity and percentage mortality of entomopathogenic fungi against the tested insects
Fungal isolates
Insect species
Fungal isolates
ITCC No.
6552
ITCC No.
6628
ITCC No.
6645
ITCC No.
4512
ITCC No.
4514
ITCC No.
6377
M. NCIPM B. NCIPM mortality %
Bactrocera dorsalis
Corcyra cephalonica
Coccinella septempunctata
Spilarctia obliqua
Spodoptera litura
Pieris brassicae
Drosicha mangiferae
(-ve)
(-ve)
-----
-----
(-ve)
-----
-----
(+ve
(+ve)
(+ve)
(+ve)
(+ve)
(+ve)
(-ve)
(+ve
(+ve)
(+ve)
(+ve)
(+ve)
(+ve)
(-ve)
(-ve)
(-ve)
-----
-----
(-ve)
-----
-----
(-ve)
(-ve)
-----
-----
(-ve)
-----
-----
(-ve)
(-ve)
-----
-----
(-ve)
-----
-----
(-ve)
(+ve)
----
----
----
----
----
(+ve)
-----
-----
------
(+ve)
-----
(-ve)
100
31-98
38-100
30-50
60
60
0
*M. NCIPM= Metarhizium anisopliae B. NCIPM= Beauveria bassiana ----- = not tested
Table 5.5. Percentage of emergent adults out of last larval stage and pupae of Bactrocera dorsalis treated with different concentrations
of three strains of Beauveria bassiana.
Stage Larvae Pupae
Concentrations B. NCIPM ITCC No. 6628 ITCC No. 6645 B. NCIPM ITCC No. 6628 ITCC No. 6645
Control 90 90 90 90 90 90
106 100 87 93 70 87 90
107 93 97 87 87 83 90
108 90 87 93 83 90 90
109 93 87 90 90 90 87
1010
93 87 87 83 87 93
1011
90 100 93 80 87 90
Note: concentration for ITCC No. 6628 were 105 to 10
10
Table 3.1.Origin of fungal isolates tested against the insect. Source: Catalogue of Fungal & Bacterial Cultures 1936-2012 (VIII Edition)
Isolates
No
Isolate name Accession number Deposition
date
Host Isolator and address
1 Beauveria bassiana (Balsamo)
Vuillemen
ITCC No.6552 29.3.2010 Tetranychus
urticae
TNAU, Head
2 Beauveria bassiana (Balsamo)
Vuillemen
ITCC No.6628 3.8.2010 Phyllocotruta
oloeivora
Banglore, P.P. Sreerama Kumar
3 Beauveria bassiana (Balsamo)
Vuillemen
ITCC No.6645 18.8.2010 White grub
Jorhat, B. Bhattacharya
4 Beauveria bassiana (Balsamo)
Vuillemen
ITCC No.4512 1994 Sugarcane borer
Karnal, H.R. Sardana
5 Metarrhizium anisopliae (Metschin)
Sorokin
ITCC No.4514 1994 Sugarcane root
borer
Karnal, H.R. Sardana
6 Metarrhizium anisopliae (Metschin)
Sorokin
ITCC No.6377 27.10.2008 Insects,
Junagadh, Plant Pathology, Prof. &
Head
7 Beauveria bassiana (Balsamo)
Vuillemen
Beauveria
bassiana N.CIPM
----- --------------- NCIPM, New Delhi
8 Metarrhiziumanisopliae(Metschin)
Sorokin
Metarrhizium
anisopliae
NCIPM
----- --------------- NCIPM, New Delhi
Table 3.2 Three commercial mycoinsecticides (Bio-power®, Bio-magic
® and Bio-catch
®)
were supplied by T. Stanes and Company Tamil Nadu, India.
Product Entomopathogenic
fungus
Concentration Manufacturer
Bio-Power Beauveria
bassiana
1 × 1010
conidia/ml T. Stanes& Company Ltd., India
Bio-Magic Metarhizium
anisopilae
1 × 1010
conidia/ml T. Stanes& Company Ltd. India
Bio-Catch Lecanicillium
lecanii
1 × 1010
conidia/ml T. Stanes& Company Ltd. India
Table 3.3 General information on adjuvants and their sources
1) Binders material Nature Source
Acacia Gum White powder
odorless
Titan Biotech Ltd,
Rajasthan, India
Corboxy methyl
Celullose
Completely
water solubale
Loba-ChemieIndoaustranial
Co. Bombay, India
Xanthan Gum White to cream
free flowing
powder
Titan Biotech Ltd,
Rajasthan, India
2) Wetting agent Sodium
lignosulphate
Brown powder Titan Biotech Ltd,
Rajasthan, India
3) Moisturizer Glycerol A clear viscous
liquid , more
than 10 hazan
units in color
General Drug House Ltd
New Delhi, India
4) Spreading agents Tween 20 Yellowish
color liquid
Titan Biotech Ltd,
Rajasthan, India
Teepol Viscous liquid Titan Biotech Ltd,
Rajasthan, India
Criton X100 A clear
colorless to
pale yellow
liquid
General Drug House Ltd.
New Delhi, India
Table 3.4 .General information on carriers and their sources
S.No Carrier Chemical Nature General Formula Source Remarks
1 Calcite Carbonate mineral CaCO3 Stake cera, ahouse of
complete ceramic
solutions New
Delhi- India
White
color
2 Dolomite Calcium and
Magnisium carbonate
Ca(Co3)Mg(Co3) -do- White
color
3 Pyrophylite Dioctahedral
aluminium silicate
Al2 SI2O5(OH)4 -do- White
color
4 Soapstone Hyderated
Magnesium silicate
Mg3Si4O16(OH)2 -do- White
color
5 Talcum
Powder
Hyderated magnisium
silicate (2:1)
Mg3(OH)2Si4O10 Titan Biotech
Company
White
color
Table 3.5.The list of 45 reciepes based on five carriers.
TA Talcum powder Sodium lignosulphate Criton-x100 Acacia Gum Glycerol
TB Talcum powder Sodium lignosulphate Criton-x100 CMC Glycerol
TC Talcum powder Sodium lignosulphate Criton-x100 Xanthan Gum Glycerol
TD Talcum powder Sodium lignosulphate Tween-20 Acacia Gum Glycerol
TE Talcum powder Sodium lignosulphate Tween-20 CMC Glycerol
TF Talcum powder Sodium lignosulphate Tween-20 Xanthan Gum Glycerol
TG Talcum powder Sodium lignosulphate Teepol Acacia Gum Glycerol
TH Talcum powder Sodium lignosulphate Teepol CMC Glycerol
TI Talcum powder Sodium lignosulphate Teepol Xanthan Gum Glycerol
PA Pyrophylite Sodium lignosulphate Criton-x100 Acacia Gum Glycerol
PB Pyrophylite Sodium lignosulphate Criton-x100 CMC Glycerol
PC Pyrophylite Sodium lignosulphate Criton-x100 Xanthan Gum Glycerol
PD Pyrophylite Sodium lignosulphate Tween-20 Acacia Gum Glycerol
PE Pyrophylite Sodium lignosulphate Tween-20 CMC Glycerol
PF Pyrophylite Sodium lignosulphate Tween-20 Xanthan Gum Glycerol
PG Pyrophylite Sodium lignosulphate Teepol Acacia Gum Glycerol
PH Pyrophylite Sodium lignosulphate Teepol CMC Glycerol
P1 Pyrophylite Sodium lignosulphate Teepol Xanthan Gum Glycerol
SA Soapstone Sodium lignosulphate Criton-x100 Acacia Gum Glycerol
SB Soapstone Sodium lignosulphate Criton x100 CMC Glycerol
SC Soapstone Sodium lignosulphate Criton x100 Xanthan Gum Glycerol
SD Soapstone Sodium lignosulphate Tween-20 Acacia Gum Glycerol
SE Soapstone Sodium lignosulphate Tween-20 CMC Glycerol
SF Soapstone Sodium lignosulphate Tween-20 Xanthan Gum Glycerol
SG Soapstone Sodium lignosulphate Teepol Acacia Gum Glycerol
SH Soapstone Sodium lignosulphate Teepol CMC Glycerol
SI Soapstone Sodium lignosulphate Teepol Xanthan Gum Glycerol
DA Dolomite Sodium lignosulphate Criton-x100 Acacia Gum Glycerol
DB Dolomite Sodium lignosulphate Criton x100 CMC Glycerol
DC Dolomite Sodium lignosulphate Criton x100 Xanthan Gum Glycerol
DD Dolomite Sodium lignosulphate Tween-20 Acacia Gum Glycerol
DE Dolomite Sodium lignosulphate Tween-20 CMC Glycerol
DF Dolomite Sodium lignosulphate Tween-20 Xanthan Gum Glycerol
DG Dolomite Sodium lignosulphate Teepol Acacia Gum Glycerol
DH Dolomite Sodium lignosulphate Teepol CMC Glycerol
DI Dolomite Sodium lignosulphate Teepol Xanthan Gum Glycerol
CA Calcite Sodium lignosulphate Criton-x100 Acacia Gum Glycerol
CB Calcite Sodium lignosulphate Criton x100 CMC Glycerol
CC Calcite Sodium lignosulphate Criton x100 Xanthan Gum Glycerol
CD Calcite Sodium lignosulphate Tween-20 Acacia Gum Glycerol
CE Calcite Sodium lignosulphate Tween-20 CMC Glycerol
CF Calcite Sodium lignosulphate Tween-20 Xanthan Gum Glycerol
CG Calcite Sodium lignosulphate Teepol Acacia Gum Glycerol
CH Calcite Sodium lignosulphate Teepol CMC Glycerol
CI Calcite Sodium lignosulphate Teepol Xanthan Gum Glycerol
Table 4. 3. Percentage mortality of adult Bactrocera dorsalis due to entomopathogenic fungi
(within 5-6 days).
Isolate accession number Per cent mortality
ITCC No. 6552 0
ITCC No. 6628 100
ITCC No. 6645 100
ITCC No.4512 0
ITCC No.4514 0
ITCC No.6377 0
B. NCIPM 100
M. NCIPM 0
Control 0
Table 4.4 Average mortality of Corcyra cephalonica and Coccinella septumpunctata
Treatments Corcyra cephalonica Coccinella septumpunctata
Average mortality
ITCC No. 6628 17.0 (4.18) 20.0(4.4)
ITCC No. 6645 19.6 (4.48) 19.0 (4.3)
M. NCIPM 3.4 (1.94) 7.6 (2.7)
CONTROL 1.8 (1.41) 2.2 (1.4)
C.D. (0.486) (0.463)
SE(m)+ (0.161) (0.153)
SE(d) (0.227) (0.217)
C.V. (11.542) (9.999)
*Fig in parenthesis is square root transformed values
Table 5. 1 Origin of fungal isolates tested against the three stages of Bactrocera dorsalis,
Source: Catalogue of Fungal & Bacterial Cultures 1936-2012 (VIII Edition)
Table. 5. 2. Products tested against the oriental fruit fly, Bactroceradorsalis
Product Entomopathogenic
fungus
Concentration Manufacturer
Bio-Power Beauveria bassiana 1 × 1010
conidia/ml T. Stanes& Company Ltd.
Bio-Magic Metarhizum anisopilae 1 × 1010
conidia/ml T. Stanes& Company Ltd.
Bio-Catch Lecanicillium lecanii 1 × 1010
conidia/ml T. Stanes& Company Ltd.
Table 5.3 Average mortality of Bactrocera dorsalis (last larval stage, pupae and adults)
treated with three commercial mycoinsecticides.
Treatments Average mortality
Isolate name Accession number Deposition
date
Host Isolator and
address
Beauveria bassiana
(Balsamo) Vuillemen
ITCC No. 6628 3.8.2010 Phyllocotruta
oloeivora
Banglore, P.P.
Sreerama
Kumar
Beauveria bassiana
(Balsamo) Vuillemen
ITCC No. 6645 18.8.2010 White grub
Jorhat, B.
Bhattacharya
Beauveria bassiana
(Balsamo) Vuillemen
Beauveria bassiana
NCIPM
----- --------------- National
Center for
Integrated Pest
Management
New Delhi
Adult Last larval stage Pupae
Biopower®
2.020 (26.6%) 3.160 3.209
Biomagic®
2.205 (40%) 2.879 3.265
Biocatch®
2.333 (46.6%) 3.202 3.263
Control 1.000 (0%) 3.214 3.314
C.D. 0.918 N/A N/A
SE(m)+ 0.277 0.137 0.099
SE(d) 0.392 0.193 0.139
C.V. 25.413 7.598 5.232
Table. 5.4. LC50 values of three virulent isolates of entomopathogenic fungi against adults of
Bactrocera dorsalis
Strains Heterogeneity Regression equation LC50 Fiducial limits
ITCC No.6628 1.922 Y= 1.358195 + 0.674319x 2.5x105 5.3x10
4 6.8x10
5
ITCC No. 6645 3.955 Y= -3.063075 + 0.885878x 1.2x109 4.5x10
8 2.8x10
9
B. NCIPM 6.634 Y= 0.857657 + 0.602183x 7.5x106 1.9x10
6 2x10
7
Table 5.7. Cumulative mortality of adult fruit fly Bactrocera dorsalis treated with different
strains of Beauveria bassiana within seven days
Conc./Treat ITCC No.
6645
%
Mortality B. NCIPM
%
Mortality
ITCC No.
6628
%
Mortality
Mortality
% Mycosis% Mortality% Mycosis% Mortality% Mycosis%
1x 1011
100 90 100 50 100 66
1x 1010
73 60 93 70 100 70
1x 109 50 30 90 23 97 56.
1x 108 6 0 23 0 24 50
1x107 4 10 17 0 17 50
1x106 1 0 3 0 7 50
control 0 0 0 0 0 0
Note: concentration for ITCC No. 6628 were 105 to 10
10
Table 6.3 General information on adjuvants
1) Binders material Nature Source
Acacia Gum White powder
odorless
Titan Biotech Ltd,
Rajasthan, India
Corboxy methyl
Celullose
Completely
water solubale
Loba-ChemieIndoaustranial
Co. Bombay, India
Xanthan Gum White to cream
free flowing
powder
Titan Biotech Ltd,
Rajasthan, India
2) Wetting agent Sodium
lignosulphate
Brown powder Titan Biotech Ltd,
Rajasthan, India
3) Moisturizer Glycerol A clear viscous
liquid , more
than 10 hazan
units in color
General Drug House Ltd
New Delhi, India
4) Spreading agents Tween 20 Yellowish
color liquid
Titan Biotech Ltd,
Rajasthan, India
Teepol Viscous liquid Titan Biotech Ltd,
Rajasthan, India
Criton X100 A clear
colorless to
pale yellow
liquid
General Drug House Ltd.
New Delhi, India
Table 6. 1 .General information on carriers
S.No Carrier Chemical Nature General Formula Source Remarks
1 Calcite Carbonate mineral CaCO3 Stake cera, ahouse of
complete ceramic
solutions New
Delhi- India
White
color
2 Dolomite Calcium and magnisium
carbonate
Ca(Co3)Mg(Co3) -do- White
color
3 Pyrophylite Dioctahedral aluminium
silicate
Al2 SI2O5(OH)4 -do- White
color
4 Soapstone Hydrated magnesium
silicate
Mg3Si4O16(OH)2 -do- White
color
5 Talcum
Powder
Hydrated magnisium
silicate (2:1)
Mg3(OH)2Si4O10 Titan Biotech
Company
White
color
Table. 6.2 recipes of 45 ingredients of blank formulation based five carriers
TA Talcum powder Sodium lignosulphate Criton-x100 Acacia Gum Glycerol
TB Talcum powder Sodium lignosulphate Criton-x100 CMC Glycerol
TC Talcum powder Sodium lignosulphate Criton-x100 Xanthan Gum Glycerol
TD Talcum powder Sodium lignosulphate Tween-20 Acacia Gum Glycerol
TE Talcum powder Sodium lignosulphate Tween-20 CMC Glycerol
TF Talcum powder Sodium lignosulphate Tween-20 Xanthan Gum Glycerol
TG Talcum powder Sodium lignosulphate Teepol Acacia Gum Glycerol
TH Talcum powder Sodium lignosulphate Teepol CMC Glycerol
TI Talcum powder Sodium lignosulphate Teepol Xanthan Gum Glycerol
PA Pyrophylite Sodium lignosulphate Criton-x100 Acacia Gum Glycerol
PB Pyrophylite Sodium lignosulphate Criton-x100 CMC Glycerol
PC Pyrophylite Sodium lignosulphate Criton-x100 Xanthan Gum Glycerol
PD Pyrophylite Sodium lignosulphate Tween-20 Acacia Gum Glycerol
PE Pyrophylite Sodium lignosulphate Tween-20 CMC Glycerol
PF Pyrophylite Sodium lignosulphate Tween-20 Xanthan Gum Glycerol
PG Pyrophylite Sodium lignosulphate Teepol Acacia Gum Glycerol
PH Pyrophylite Sodium lignosulphate Teepol CMC Glycerol
P1 Pyrophylite Sodium lignosulphate Teepol Xanthan Gum Glycerol
SA Soapstone Sodium lignosulphate Criton-x100 Acacia Gum Glycerol
SB Soapstone Sodium lignosulphate Criton x100 CMC Glycerol
SC Soapstone Sodium lignosulphate Criton x100 Xanthan Gum Glycerol
SD Soapstone Sodium lignosulphate Tween-20 Acacia Gum Glycerol
SE Soapstone Sodium lignosulphate Tween-20 CMC Glycerol
SF Soapstone Sodium lignosulphate Tween-20 Xanthan Gum Glycerol
SG Soapstone Sodium lignosulphate Teepol Acacia Gum Glycerol
SH Soapstone Sodium lignosulphate Teepol CMC Glycerol
SI Soapstone Sodium lignosulphate Teepol Xanthan Gum Glycerol
DA Dolomite Sodium lignosulphate Criton-x100 Acacia Gum Glycerol
DB Dolomite Sodium lignosulphate Criton x100 CMC Glycerol
DC Dolomite Sodium lignosulphate Criton x100 Xanthan Gum Glycerol
DD Dolomite Sodium lignosulphate Tween-20 Acacia Gum Glycerol
DE Dolomite Sodium lignosulphate Tween-20 CMC Glycerol
DF Dolomite Sodium lignosulphate Tween-20 Xanthan Gum Glycerol
DG Dolomite Sodium lignosulphate Teepol Acacia Gum Glycerol
DH Dolomite Sodium lignosulphate Teepol CMC Glycerol
DI Dolomite Sodium lignosulphate Teepol Xanthan Gum Glycerol
CA Calcite Sodium lignosulphate Criton-x100 Acacia Gum Glycerol
CB Calcite Sodium lignosulphate Criton x100 CMC Glycerol
CC Calcite Sodium lignosulphate Criton x100 Xanthan Gum Glycerol
CD Calcite Sodium lignosulphate Tween-20 Acacia Gum Glycerol
CE Calcite Sodium lignosulphate Tween-20 CMC Glycerol
CF Calcite Sodium lignosulphate Tween-20 Xanthan Gum Glycerol
CG Calcite Sodium lignosulphate Teepol Acacia Gum Glycerol
CH Calcite Sodium lignosulphate Teepol CMC Glycerol
CI Calcite Sodium lignosulphate Teepol Xanthan Gum Glycerol
Table 6.4 Results of tests carried out on physicochemical properties of the carriers
S.No Carrier Bulk density (g/100cc) Moisture
content %
Particle
mesh size
Sorptivity pH
Before After
1 Calcite 89 140 0 150 9.3 8.6
2 Dolomite 113.6 175 0 150 9.3 8.8
3 Pyrophylite 54.7 97.2 0 150 18.3 6.7
4 Soapstone 58.55 108 0 150 18.6 8.5
5 Talcum
powder
45.18 79.8 0 150 19.06 7.7
Table. 6.5. Germination percentage of ten products selected for viability test
Product % Germination
PA 95
PD 95
PE 85
PG 95
P1 80
TA 30
TD 10
TE 10
TG 40
TI 35
Table 6.6. Result of bioassay of three products on three stage of fruit fly Bactrocera dorsalis
Treatment Adults Pupae 3rd
larvae
Mean/S.E Mean/ S.E Mean/S.E
Control 0.000, 0.000 3.054, 0.054 2.997, 0.096
PA 2.667, 0.667 2.943, 0.057 3.093, 0.224
PD 1.667, 0.882 3.214, 0.051 3.108, 0.054
PG 2.667, 1.333 2.930, 0.200 2.930, 0.200
C.D N/A N/A N/A
SE(m) 0.866 0.111 0.160
SE(d) 1.225 0.156 0.160
C.V 85.714 6.312 9.136
Table 4.5. LT50 values of entomopathogenic fungi isolates against Corcyra cephalonica.
Strains Heterogenity Regression equation LT50 Fiducial limits
ITCC No.6628 2.162 Y=-7.083380 + 4.944646x 11.57 days 10.785 12.329
ITCC No. 6645 4.674 Y=-5.292781+ 4.562572x 7.51 days 6.785 8.117
M. NCIPM 6.193 Y=-17.107702 + 8.064309x 22.97 days 20.503 37.967
Table 5. 6 LC50 values of entomopathogenic fungi isolate ITCC No. 6645 against last larval
stage of Bactroceradorsalis
Strain Heterogeneity Regression equation LC50 Fiducial limits
ITCC
No. 6645
3.159 y=1.468583+0.353618x 9x109 1.x10
9 4.2x10
11
1
INTRODUCTION
The genus Bactrocera consists of at least 440 tephritid species are distributed primarily in
tropical Asia, Australia and the South Pacific (White and Elson-Harris 1994). Oriental
fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is considered to be among
the five most damaging and aggressive pest fruit flies in the world (Leblanc and Putoa
2000). In India, the loss in fruit yield ranges from 1 to 31% with a mean of 16%
(Verghese et al., 2002). B. dorsalis is a serious pest of a wide range of fruit crops in the
Indian sub continent. On mango (Mangifera indica L.), it causes enormous losses up to
80% (Jayanthi and Verghese, 2011). Fruit flies affect the production of fruits by
infestation which causes both direct and indirect loss of product. Direct loss occurs
through fruit flies feeding on the plant which causes fruit drop and renders the fruit
inedible (Du Toit, 1998). Indirect loss arises from phytosanitary restrictions imposed by
importing countries (Ekesi et al., 2007). The pest is controlled by a variety of methods
such as cultural, behavioral, genetic (Ekesi et al., 2007) and biological control (Du Toit,
1998). Use of synthetic insecticides such as diazinon (organophosphate) to control fruit
fly larvae/puparia is associated with various ecological problems such as environmental
contamination, adverse effects on non-target organisms and the development of resistance
(Croft, 1990). Moreover, persistence of diazinon in the soil is known to decrease within 2
weeks, therefore repetition of application is required (Roessler, 1989). Therefore
biological control by various means remains the safest and environmentally friendly
method to control insect pest among which entomopathogenic fungi is one of the
important components.
Over 500 species of fungi are known to have insect pathogenic properties. Interestingly,
Beauveria and Metarhizium (Deuteromycotina: Hyphomycetes) represent the most
frequently used genera (Burges and Hussey, 1971) and are known to infect a broader
range of insect pests of crops belonging to Lepidoptera, Homoptera, Hymenoptera,
Coleoptera and Diptera. Most research on entomopathogenic fungi has been directed to
Beauveria and Metarhizium (Greathead and Prior, 1990; Whitten and Oakeshott, 1991),
which are cosmopolitan and do not leave undesirable residues hence can be used even
close to harvest. Besides, they are compatible with other methods of pest management
tactics. These fungi also have potential for better commercialization and offer excellent
alternative to chemical pesticides in the world market (Gautam, 2008). Additionally, their
production is easy and cheap and do not require high input technology (Prior, 1988).
2
Recent studies on Metarhizium anisopliae (Metschn.) (Sorokin) revealed that this fungal
species had rhizosphere competence (St Leger, 2008). Whereas, Beauveria bassiana
(Balsamo) (Vuillemin) has been included in the spectrum of fungi with endophytic
activity; and its natural occurrence has been found within corn, cocoa, poppy, coffee and
tomato (Meyling and Eilenberg, 2007). This facultative entomopathogenic fungus play
additional roles in nature including antagonists of plant pathogens (Goettel et al., 2008)
and plant growth promoters (Vega et al., 2009). Commercially, B. bassiana and B.
brongniartii (Sacc.) are produced by more than 14 companies, and Metarhizium (M.
anisopliae and M. anisopliae var. acridum) by more than 10 (including some companies
in Africa), aimed at controlling various insect pests including termites, cockroaches, black
vine weevil, whiteflies, aphids, corn borers, cockchafers, and other insects (Wraight et al.,
2001).
Soil-inhabiting entomopathogenic fungi are an important and widespread component of
most terrestrial ecosystems and play a key role in regulating some soil-dwelling insect
populations (Meyling and Eilenberg, 2007) consequently; numerous studies have
demonstrated the success of soil treatment with fungal pathogens for the control of
different agricultural pests (Booth and Shanks, 1998). Since most entomopathogenic
fungi are soil-borne microorganisms, their incorporation into the soil targeting
pupariating larvae and puparia can form an important component of an integrated pest
management strategy for fruit flies. For example puparia of rose fruit fly Rhagoletis
alternate (Meigen) are attacked by the fungus Scopulariopsis brevicaulis (Sacc.) Bainier,
(Lipa et al., 1976). Moreover, entomopathogenic fungi can control adult of fruit flies
where it was found that various isolates of M. anisopliae and Paecilomyces fumosoroseus
(Wize.) were found to be pathogenic to adult Ceratitis capitata (Wied.) and infection was
reported to reduce fecundity and fertility (Castillo et al., 1999). Subsequent steps in the
development of entomopathogenic fungi as mycoinsecticides are the inoculum production
and formulation (Butt et al., 2001).
Soil application of entomopathogenic fungi has been undertaken in various parts of the
world as a cost-effective management technique for many insect pests. Wojciechowska et
al., (1977) showed increased mortality of pupating Colorado potato beetle populations up
to 2 years after a single application of B. bassiana. Watt and Le Brun (1984)
demonstrated in USA that soil application of B. bassiana successfully controlled first and
second generation Colorado potato beetle with 74 and 77% respectively. Recently
numerous studies have demonstrated the potential success of soil treatments with
3
entomopathogenic fungi for the management strategies of fruit flies (Ekesi et al., 2002;
Ekesi et al., 2005; Ekesi et al., 2007). High level of sporulation of fungal cultures, on host
cadavers, is also considered a desirable trait because each pupa dying in the soil
constitutes an infection site (Ekesi et al., 2002).
According to (Ekesi, 2002) soil drenches with entomopathogenic fungi may be an
effective integrated pest management component for the control of C. capitata, C. rosa
fasciventris and C. cosyra (Walker) in mango orchards. Muñoz (2000) evaluated 16
strains of B. bassiana against C. capitata adults and found mortality levels between 20
and 98.7%. Dimbi et al., (2003) reported 7 to 100% mortality in adults of C. capitata and
C. rosa var. fasciventris when treated with B. bassiana. Besides horizontal auto
dissemination which occurs when fungal infected individual flies mate with healthy flies.
To prove so, uninfected C. capitata females were paired with Metarhizium inoculated
males for 24 hrs, these females showed a higher density of conidia in several parts of
insect body. Horizontal transmission is more effective when infected male flies mate with
healthy females because female C. capitata fruit flies are monogamous or oligogamous
(Quesada-Moraga et al., 2008). In addition to the possible transmission of
entomopathogenic fungi between male and female fruit flies, male flies also show the
tendency to homosexual mating (Ekesi et al., 2007).
Keeping in view the applicability of entomopathogenic fungi to control adults of fruit
flies, attraction of insect pests using visual, chemical and food lures to the focal point of
entomopathogenic fungi can be benefited from, in which the insects, accidentally pick up
the fungal inoculum and disseminate the infective conidia via horizontal transmission to
other healthy individuals of the pest population (Globe, 2009). The combined application
of M. anisopliae and food bait resulted in a huge reduction (92%) of B. invadens (Drew,
Tsura and White) population on mango in Kenya (Ekesi et al., 2010).
In India (Jiji et al., 2006) reported that Paecilomyces lilacinus (Thom) (ITCC No. 6064)
@ 1.0x109 spores/ml caused 96.67%, and 100 % cumulative mortality in fruit flies on
second and third day respectively. While treated pupae were black in colour and failed to
emerge. B. bassiana (ITCC No. 6063) developed disease symptoms on adult Bactrocera
cucurbitae (Coquillett) and B. dorsalis and killed them within 4-5 days. Whereas, adult
fruit flies sprayed with Aspergillus candidus (Link. ex Fr) (ITCCNo. 5428) were found to
die within three to four days.
A granular formulation of M. anisopliae, BioGreenTM
, has recently been developed and
registered in Australia against the soil inhabiting pest Adoryphorus couloni Burmeister
4
(Jackson, 1999). It is reported that a single soil application of this product can suppress
the pest for 5- 10 years, considerably reducing the cost of control (Rath et al., 1995). In
field caged experiment (Ekesi et al., 2007) tested three formulation based on
entomopathogenic fungi besides diazinon against three species of fruit flies. They found
that the entomopathogenic fungi are better than diazinon particularly granular
formulation, which was found effective up to 668 days after soil inoculation and yielding
reduction of 54% in some fruit fly species. This initiated the author’s interest to carry out
laboratory work with the following objectives:
Objectives
1. To screen different isolates of Beauveria bassiana and Metarhizium anisopliae
against fruit flies.
2. To carryout bioassay studies on virulent isolates of Beauveria bassiana and
Metarhizium anisopliae against different stages of fruit flies.
3. To develop biopesticide formulation of virulent fungal entomopathogens for the
management of fruit flies.
5
BACKGROUND
More than 700 species of fungi have been reported to be entomopathogenic (Hajek and
St. Leger, 1994). They are known to infect a broader range of insect pests of crops
belonging to Lepidoptera, Homoptera, Hymenoptera, Coleoptera and Diptera (Burges and
Hussey, 1971).Though, insect pathology dates back to 2700 B.C, when the Chinese
recorded disease of silk worm. However, the first experimental demonstration that an
insect pathogen, a fungus, which now known as Beauveria bassiana (Balsamo)
Vuillemin, caused an infectious disease in insect was made by an Italian scientist, Bassi in
1835, who also suggested the use of pathogens for insect pest control. Since then workers
began to demonstrate the infectiousness of the white muscardine fungus to other insects
including important insect pests of agriculture (Tanada and Kaya, 1993). For instance, the
first, field attempt to control a pest with a fungal agent was carried out in Russia in 1888,
when the fungus now known as Metarhizium anisopliae (Metschn.) Sorokin
(Deuteromycotina: Hyphomycetes) was mass produced on beer mash and sprayed in the
field for control of the beet weevil Cleonus punctiventris (Germar) (Lord, 2005). It is well
known that B. bassiana has a wide host range, occurring on several hundred arthropod
species; however, host specificity is really a strain-specific trait. This is evident by the
fact that most isolates of this fungus have a restricted host range. Therefore, it is
necessary to screen the virulence of different isolates against a target insect species in
order to select the most virulent one (Zimmermann, 2007). Virulence is the most
important indicator to measure the potential of fungi against pests and on the basis of
laboratory bioassays highly virulent fungi is choosen (Li et al., 2012). As an alternative to
chemical control or as part of IPM programs, there is a resurgence of interest in the use of
microbial insecticides for biological control of insect pests. In fly populations, under
laboratory and field conditions, the Deuteromycete fungi have long been known to cause
epizootics (Reithinger et al, 1997). One of the most important advantages of
entomopathogenic fungi, compared to other entomopathogenic microbial organisms is
that, they infect their host via contact and do not need to be ingested by the insect to cause
infection (Goettel et al., 2005). Tephritid fruit flies are among the major pests of fruits
throughout the world and represent the most economically important group of
phytophagous Diptera (White and Elson-Harris 1994). Bactrocera dorsalis (Hendel)
(Tephritidae: Diptera) is among these tephritids which was identified as one of the three
most important agricultural pests in South East Asia (Waterhouse, 1993). Recently,
6
several studies have demonstrated the effectiveness of autochthonous isolates of B.
bassiana and M. anisopliae for the control of Ceratitis capitata (Weidemann) adults in
Africa (Ekesi et al., 2002, 2007; Dimbi et al., 2003), Greece (Konstantopoulos and
Mazomenos, 2005) and Spain (Quesada-Moraga et al., 2006). Faria et al., (2007) reported
that during the last four decades, over 80 companies worldwide have developed 171
mycoinsecticides and mycoacaricides products. Among these B. bassiana based products
represent (33.9%), M. anisopliae (33.9%) whereas, 26.3% out of these are wettable
powders. The research areas for using entomopathogenic fungi and development of
formulation against fruit fly are reviewed here under.
2.1. Screening of different isolates of Beauveria bassiana and Metarhizium anisopliae
against fruit flies, and bioassay studies of virulent strains against different
stages of fruit flies.
2.1.1. Screening and bioassay of entomopathogenic fungi against adult stage
Castillo et al., (1999) reported that various isolates of M. anisopliae and Paecilomyces
fumosoroseus (Wize) Brown and Smith were found to be pathogenic to adult C. capitata
and infection was reported to reduce fecundity and fertility. Castillo et al., (2000)
evaluated the effectiveness of seven strains of entomopathogenic fungi, against C.
capitata adults in the laboratory by topical application. They observed that the adults
were susceptible to five aqueous suspensions of conidia. M. anisopliae and strain CG-260
of P. fumosoroseus were the most pathogenic fungi, with 10-day LD50 values of 5.1x103
and 6.13 x103 conidia /fly, respectively. Muñoz (2000) screened 16 strains of B. bassiana
against three species of fruit flies adults; on C. capitata he found mortality levels between
20.0 to 98.7%. Mortality of Bactrocera zonata (Saunders) varied between 12.0 to 98.0%
and 2.0 to 94.0% in Bactrocera cucurbitae (Coquillett) at five days post-treatment. De La
Rosa et al., (2002) sprayed adult of Anastrepha ludens (Loew) (Tephritidae: Diptera) with
conidial suspension of 1x108
conidia/ml for 30 seconds and achieved 100, 98 and 98%
mortality with B. bassiana isolates Bb 16, Bb24 and Bb26, respectively at 10 days post-
inoculation. The LC50 values for the three strains used, respectively were 5.13x105,
3.12x106, and 9.07x10
6 conidia/ml. Dimbi et al., (2003) found adult mortality in C.
capitata and C. rosa var. fasciventris treated with different isolates of B. bassiana and M.
anisopliae were 7 to 100% and 11.4 to 100% respectively, at four days post inoculation.
Konstantopoulou and Mazomenos (2005) assessed the virulence of two isolates of B.
7
bassiana and B. brongniartii (Sacc.) in addition to Mucor hiemalis (Wehmer),
Penicillium aurantiogriseum (Dierckx), P. chrysogenum (Thom) against adults of the
Bactrocera oleae (Gmelin) and C. capitata. Contact and oral bioassays revealed that
moderate to high mortality rates for the olive fruit fly occurred when the adults were
exposed to conidia of M. hiemalis, P. aurantiogriseum, P. chrysogenum and B. bassiana
isolates. M. hiemalis was the most toxic resulting in 85.2% mortality to the olive fruit fly
adults. B. brongniartii and B. bassiana were the most pathogenic to the C. capitata adults
causing 97.4 and 85.6% mortality. Quesada-Moraga et al., (2006) tested 10 isolates of B.
bassiana and 5 of M. anisopliae by inoculating the ventral surface of the abdomen with
1x108cfu/ml and recorded 30-100% mortality in adults at 20 days post-inoculation. In this
study mycosis was observed from B. bassiana and M. anisopliae. In India (Jiji et al.,
2006) reported that Paecilomyces lilacinus (Thom) (ITCC No. 6064) at 1.0x109 spores/ml
caused 96.67%, and 100% cumulative mortality in fruit flies on second and third day
respectively. B. bassiana (ITCC No. 6063) developed disease symptoms on adult B.
cucurbitae and B. dorsalis and killed them within 4-5 days. Sookar et al., (2008) screened
the pathogenicity of seven isolates of M. anisopliae, five isolates of B. bassiana and two
isolates of P. fumosoroseus towards the adults of B. zonata and B. cucurbitae by topical
application of conidial suspension of 1x106
conidia /ml. All the isolates tested were
pathogenic to the two fruit fly species. Mortality of B. zonata varied between 12.0 and
98.0% and between 2.0 and 94.0% in B. cucurbitae at 5 days post-treatment. Dimbi et al.,
(2009) carried out bioassays in the laboratory to investigate the effect of inoculation by
M. anisopliae on mating behaviour of three species of fruit flies, Ceratitis cosyra
(Walker), C. fasciventris and C. capitata. In all three species, inoculation by the fungus
resulted in significant delay in the beginning of calling and mating of treated males as
they spent substantial amount of time in grooming activity. Díaz-Ordaz et al., (2010)
screened three isolates, two strains of B. bassiana (Bb26 and BbJLSV) and a strain of M.
anisopliae (MaCENGICAÑA) against adults of A. obliqua in laboratory. The Bb26 strain
caused the highest infection rate (99.8%), followed by the BbJLSV strain (93.5%), and
the MaCENGICAÑA strain which caused the lowest infection (89.8%). Both males and
females were susceptible to the strains tested, with a similar mortality in both sexes. The
median lethal time (LT50) was 3.9, 5.3 and 6.4 days for MaCENGICAÑA, Bb26 and
BbJLSV. Yousef et al., (2013) reported that the susceptibility of adult, B. oleae, to a
strain of the M. brunneum (Petch) when it was sprayed with 1.0x108 conidia per ml.
Strain EAMb09/01-Su caused 60% mortality to B. oleae adults, with average survival
8
time of 8.8 days. He also reported that metabolites of crude extract of EAMb 09/01-Su
strain caused 80.0% adult mortality when administered per os, with average survival time
of 27.7 h. Castillo et al., (2000) tested the culture broth dichloromethane extracts from
entomopathogenic fungi, for insecticide activity against C. capitata, including effects on
fecundity and fertility. The extract from M. anisopliae was the most toxic, resulting in
about 90% mortality at a concentration of 25 mg/g of diet; under these conditions,
fecundity and fertility of treated females were reduced by 94 and 53%, respectively,
compared with untreated controls. Sublethal effects on fecundity and fertility of the
fungal-exposed females were also studied. The most effective fungus in reducing
fecundity was P. fumosoroseus CECT 2705, with reductions on the order of 65% at
1.3x106
conidia /fly. M. anisopliae and Aspergillus ochraceus (Wilhelm) also showed
significant reductions of fecundity (40–50% for most of the assayed concentrations).
Fertility was moderately affected by the fungi. M. anisopliae at 1.3 x106 conidia /fly was
the most effective fungus, showing egg eclosion reduction of over 50% compared with
the control. The effects of six fungus isolates, M. anisopliae 714, M. anisopliae 786, I.
fumosorosea (Wize) 531, I. fumosorosea Apopka 97, B. bassiana ATCC 74040 and I.
farinosa (Dicks) 954, on the mortality of adults Rhagoletis cerasi (Meigen). They were
found to be highly susceptible to all fungus isolates, with 90 to 100% mortality, induced
by B. bassiana and I. fumosorosea (Daniel, 2008). Hadi et al., (2013) examined the
combination of B. bassiana with concentration of 108spores/ml and insect growth
regulator lufenuron 1.5 ml/l on adults of fruit fly B. carambolae (Drew dan Hancock).
The result of this treatment significantly reduced the fecundity, egg fertility and
reproduction of the pest up to 95.69%. Imoulan et al, (2010) tested various isolates of
Moroccan B. bassiana isolates against emerged adults of C. capitata. He inoculated the
adults with 2 x106
conidia /ml then coupled them with clean opposite sex. Horizontal
transmission was proved among medfly adults at the laboratory and varied significantly in
relation to the B. bassiana isolates. In oral bioassay, Konstantopoulou and Mazomenos
(2005) evaluated suspension of B. bassiana and B brongniartii and four wild-type fungal
species (two isolates of M. hiemalis, P. aurantiogriseum and P. chrysogenum) at 1x108
conidia/ml in solid diet against B. oleae and C. capitata. Mycosis in adult flies after
feeding on the fungus ranged from 79 to 100% in B. oleae and 85 to 99% in C. capitata
21 days post-inoculation. When both insects were fed on metabolic extract of the four
wild type of fungi for 24 h, M. hiemalis (SMU-21) and P. chrysogenum caused respective
mortalities of 100 and 61% in B. oleae and 100 and 47% in C. capitata. This study
9
suggested that extracts of fungal inoculums hold promise for utilization in bait sprays in
the management of fruit flies. The combined application of M. anisopliae and food bait
resulted in a huge reduction (92%) of B. invadens population on mango in Kenya (Ekesi
et al. 2010).
2.1.2. Screening and bioassay of entomopathogenic fungi against preimaginal stages
Many of the chemical insecticides currently used to control fruit flies are listed among the
most persistent organic pollutants (POPs) by the United Nations Environmental
Programme (UNEP). There is a need to develop effective replacements for these toxic
chemicals. Biological control, including entomopathogens, is being considered as one of
the alternatives. Soil is a rich environment for microbes and an important reservoir for
insect pathogenic fungi. It is also generally considered as an excellent environment for
use of fungi as biological control agents because it offers protection from ultraviolet
irradiation and extreme temperature fluctuations as soil humidity is high and stable
(Ekesi, 2007). (Meyling and Eilenberg (2007) declared that soil-inhabiting
entomopathogenic fungi are an important and widespread component of most terrestrial
ecosystems and play a key role in regulating some soil-dwelling insect populations.
Meyling and Eilenberg (2007) mentioned that soil application of entomopathogenic fungi
has been undertaken in various parts of the world as a cost-effective management
technique for many insect pests. Wojciechowska et al., (1977) showed increased
mortality of pupating Colorado potato beetle populations for up to 2 years after a single
application of B. bassiana. Watt and Le Brun (1984), in the USA, demonstrated that soil
application of B. bassiana successfully controlled first and second generation Colorado
potato beetle with 74 and 77% reduction in populations, respectively. Since most
entomopathogenic fungi are soil-borne microorganisms, their incorporation into the soil
targeted at pupariating larvae and puparia can form an important component of an
integrated pest management strategy for fruit flies. For example puparia of rose fruit fly
Rhagoletis alternata (Meigen) are attacked by the fungus Scopulariopsis brevicaulis
(Sacc.), Bainier (Lipa et al., 1976). Fruit flies spend part of their life cycle in contact with
soil as pupariating larvae and puparia, suggesting that application of fungi in this
environment has potential for management of fruit flies (Ekesi 2007). Recently numerous
studies have demonstrated the potential success of soil treatments with EP fungi for
control management strategies against fruit flies. Ekesi et al., (2002) screened
pathogenicity of 13 isolates of M. anisopliae and two isolates of B. bassiana to C.
10
capitata and C. var. rosa fasciventris exposed as late third instar larvae in sand in the
laboratory. All isolates caused a significant reduction in adult emergence and a
corresponding large mortality on puparia of both species on C. capitata, seven isolates
(M. anisopliae ICIPE 18, 20, 32, 60 and 69 and B. bassiana ICIPE 44 and 82) caused
significantly higher mortality on puparia than other isolates. M. anisopliae ICIPE 18 and
20 were equally pathogenic to all pupal ages tested against C. capitata and C. cosyra but
ICIPE 18 was more pathogenic to older puparia of C. rosa fasciventris than ICIPE 20.
High level of sporulation of fungal cultures, on host cadavers, is also considered a
desirable trait because each pupa dying in the soil constitutes an infection site so the
author suggested that soil drenches with entomopathogenic fungi may be an effective
integrated pest management component for the control of C. capitata, C. rosa fasciventris
and C. cosyra in mango orchards. Yousef et al., (2013) reported that in soil treatments M.
brunneum Strain EAMb09/01-Su against pupariating third-instar larvae and preimaginal
B. oleae, mortality reached 82.3%, whereas mortality targeting puparia was 33.3%.
Almeida et al., (2007) screened six B. bassiana and M. anisopliae isolates against C.
capitata prepupae with application concentration of 5×108 conidia/ml, in soil pots. It was
verified that B. bassiana and M. anisopliae were pathogenic to C. capitata prepupae, with
the isolates IBCB 66 and IBCB 425, were the most virulent, respectively. In the
greenhouse, the B. bassiana fungus reached a prepupal control efficiency of 66.6%. Ali et
al., (2009) evaluated the potential of the entomopathogenic fungus B. bassiana strain 412
against pupae of C. capitata in semi field conditions, by spraying concentration of 4x108
spores/ml to soil surface (1.3x107 spores/cm
2). The emergent fly out of treated pupae was
reduced by 46%. Ali et al., (2010) evaluated entomopathogenic fungi of L. muscarium
and B. bassiana against the old larvae of C. capitata. They applied (1×108
spores/ml) on
soil surface then they released old larvae, though all larvae have developed to pupae, but
the death occurred by pupae stage. The mortality of pupae ranged between 51.6 % by L.
muscarium and 46.7 % by B. bassiana in comparison to the control with 18.4 %. It was
also observed that the development of infected flies inside the pupae was stopped 2 to 3
days after the pupation. Amala et al., (2010) studied the efficacy of entomopathogenic
fungus P. lilacinus on one day old pupae of melon flies under in vitro conditions. Glass
troughs (30 cm diameter) were filled with 1.3x108 and 1.3x10
9spores/ml then left for
three days after that pupae were introduced to them. The mortality results obtained were
highly significant i.e., 92.45% at 1.3x109 followed by 72.28% at 1.3x10
8. Mar and
Lumyong (2012) reported the activities of six entomopathogenic fungal isolates, against
11
pupa of fruit fly Bactrocera spp. All tested isolates were pathogenic with mortality of
pupa varied from 25.89% to 100% in M. flavoviride, 22 to 100% in P. lilacinus and
29.67% to 100% in B. bassiana. M. flavoviride CMUCDCT01, P. lilacinus
CMUCDMT02 and B. bassiana CMUCDMF03 had the highest pathogenicity at the
conidial concentration of (1x108 spore/ml). Daniel (2008) screened six fungus isolates,
M. anisopliae 714, M. anisopliae 786, I. fumosorosea 531, I. fumosorosea Apopka 97, B.
bassiana ATCC 74040) and I. farinose 954, on third larvae of R. cerasi. None of the
fungus isolates induced mortality in more than 25% of the larvae. Cossentine et al.,
(2010) reported that last-instar larvae of the western cherry fruit fly, R. indifferens
(Curran), subjected to B. bassiana GHA incorporated into sterile sand and non-sterile
orchard soil. They reported that, mycosis in the pupal stage was observed in >20% of
buried R. indifferens pupae and >80% of larvae entering sand treated with B. bassiana
GHA. Imoulan and Elmeziane (2013) used third late instar larvae of C. capitata to
investigate the effectiveness of 15 B. bassiana strains. Results showed that all isolates
were able to infect the larval stage. Large mortality rate in puparia was produced, ranging
from 65 to 95%, caused significant reduction in adult emergence. The fungal treatment
revealed that the mycosis occurred also in adult escaping infection as puparating larvae.
The percentage of mycosed puparia was highest in strain TAM6.2 (95%) followed by
ERS4.16 (90%).
2.2. Development of biopesticide formulation of virulent fungal entomopathogens for
the management of fruit flies
Increased public concern about the potential adverse environmental effects associated
with the heavy use of chemicals insecticides has promoted the examination of alternative
methods for insect pest control; one such alternative is the use of biopesticides. According
to US Environmental Protection Agency (EPA); biopesticides are pesticides from natural
materials such as animals, plants, bacteria and minerals. They include microbial
pesticides, entomopathogenic nematodes, baculoviruses, plant-derived pesticides and
insect pheromone. While microbial pesticides, have been defined as: products derived
from various microscopic organisms. They may consist of the organisms themselves
and/or the metabolites they produce (Rathore and Nollet, 2012). Among several hundred
species of entomopathogenic fungi, Beauveria and Metarhizium represent the most
frequently used genera (Burges and Hussey, 1971). They are known to infect a broader
range of insect pests of crops including Diptera. Therefore, most research on fungi has
12
been directed to Beauveria and Metarhizium (Whitten and Oakeshott, 1991). Historically,
countries in Asia, Latin America, and Eastern Europe have accounted for the greatest use
of fungal pathogens (Faria et al 2007). The first attempt to control a pest with a fungal
agent was carried out in Russia in 1888, when the fungus now known as M. anisopliae
was mass produced on beer mash and sprayed in the field for control of the beet weevil
Cleonus punctiventris (Germar) (Lord, 2005). On adult fruit flies Daniel and Grunder
(2012) reported that the effectiveness of the mycoinsecticide Naturalis based on the B.
bassiana strain ATCC 74040 against the C. capitata. Under laboratory conditions, the
bioinsecticide protected fruits from the insect ovipositions. Moreover field trials proved
that it was as effective as a pyrethroid. Daniel and Wyss (2009) recorded that B.
bassiana and I. fumosorosea with concentration of 107conidia/ml against R. cerasia
caused 90 to 100% mortality. Lohmeyer and Miller, (2006) evaluated powder
formulations of three species of entomopathogenic fungi for their pathogenic effect upon
adult horn flies, Hematobia irritans (L.) (Muscidae: Diptera). The flies were treated with
conidia and blastospores of the entomopathogenic fungi B. bassiana (strain GHA), M.
anisopliae (strain ESCI), and P. fumosoroseus (strain ARSEF 3581) in the laboratory. At
seven day post exposure, flies treated with B. bassiana had an average of 100% mortality
compared with 73.0% from treatment with M. anisopliae and 33.3% from treatment with
P. fumosoroseus. Mean lethal time (LT50) was 2.70, 4.98, and 7.97 days for B. bassiana,
M. anisopliae, and P. fumosoroseus, respectively. Ortu et al., (2009) reported that
bioinsecticide Naturalis-L, based on the B. bassiana strain ATCC 74040, was effective
against C. capitata. When the fruits were covered uniformly (5.4 ml/fruit) with the
mycoinsecticide in laboratory, they were protected from medfly ovipositions. In the field
the product was as effective as pyrethroid, in reducing adult medfly populations and
protecting orange fruits. Under field conditions, foliar applications of Naturalis-L at seven
day intervals significantly reduced the number of infested fruit by 60 to 70%. The authors
concluded that, the application of Naturalis-L is a suitable and economically feasible
strategy for controlling R. cerasi. Moreover they have reported that the mycoinsecticide
Naturalis-L is currently registered for cherry fruit fly control in Italy and Switzerland
(Daniel, 2008). Mahmoud (2009) tested the pathogenicity of three commercial
biopesticides based on entomopathogenic fungi. B. bassiana, Bio-Power(R)
, Metarhizum
anisopilae, Bio-Magic(R)
and Lecanicillium lecanii Bio-Catch(R)
, against adults of the
olive fruit fly B. oleae by contact bioassays under laboratory conditions. Their virulence
reflected by LT50, was 12.59 for, L. lecanii, 16.6 days for B. bassiana and 26.07 days for
13
M. anisopilae. The efficacy of soil treatments using different formulations of
entomopathogenic fungi was evaluated in semi-field trials. Soil treatments with barley
grain-formulated entomopathogenic fungi had no effect on fly emergence rate. However,
adult mortality was significantly increased. The oviposition rate was thus reduced by up
to 90% (Ekesi et al., 2007). Ekesi et al., (2007) emphasized that entomopathogenic fungi
must be formulated to control different target pests with distinct biological aspects and
this must take place with two basic objectives in mind: ease of field application to target
insects within their habitats and, enhancement of shelf-life and environmental persistence
after application. In general, ingredients selected for formulation are crucial and they
should not interfere with infection process and at best should enhance fungal viability,
virulence, disease transmission and field persistence. Ekesi et al., (2005) evaluated the
persistence and infectivity of three formulations (aqueous, oil/aqueous [50:50] and
granular) of M. anisopliae against, pupariating larvae of three species of fruit flies (C.
capitata, C. fasciventris and C. cosyra) in field cage experiments. Compared with
untreated control, all formulations of the fungus, the chemical insecticide diazinon,
significantly reduced emergence of fruit flies from the soil. Exposure of pupariating
larvae to treated soil samples collected from the field at 183 and 366 days after the
treatment showed that the three formulations were more effective than diazinon in
reducing adult emergence. By 668 days after soil inoculation, the granular formulations of
conidia achieving 37, 42 and 54% reduction in emergence in C. capitata, C. fasciventris
and C. cosyra, respectively. The entomopathogenic fungi formulations proved their safety
on the associated parasitoid, Psyttalia concolor (Szepligeti) and P. cosyrae (Wilkinson)
whereas no emergence of the parasitoid was recorded from the plots treated by diazinon.
14
MATERIALS AND METHODS
3.1 Culture of Oriental fruit fly, Bactrocera dorsalis
Larvae of the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) were
obtained from Biological Control Laboratory, Division of Entomology, Indian Agricultural
Research Institute, New Delhi. The larvae were reared on ripe bananas whereas; adult flies
were maintained on sugar and yeast autolysate (CDH Bioscience Pvt. Ltd. New Delhi). They
were kept in ventilated acrylic cages (20x20x20 cm) at 27±10C, 65±5% R.H and 12:12
photoperiod. Water was supplied in vials with cotton wicks.
3.2 Culture of Lepidopteran, Coleopteran and Hemipteran insect pests and natural
enemies
Rearing of Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae), Spodoptera litura (Fab.)
(Lepidoptera: Noctuidae), and Spilarctia obliqua (Walker) (Lepidoptera; Arctiidae) was
carried out as per the protocols given by Gautam, (2008). Whereas, Coccinella septempunctata
(L.) (Coleoptera: Coccinellidae), Pieris brassicae (L.) (Lepidoptera: Pieridae) and Drosicha
mangiferae (Green); Hemiptera: Monophlebidae) were collected from the IARI fields, mustard
crop, cabbage crop and mango tree respectively. C. septempunctata were fed with Brevicoryne
brassicae (L.) (Homoptera: Aphididae). While P. brassicae and D. mangifera were reared on
cabbage and mango respectively. Thirty insects of D. mangiferae were tested prelimilary, using
the three virulent strains. Wherein ten insects were contaminated by one of three isolates ITCC
No. 6628; ITCC No. 6645 and B. NCIPM. Adults of C. septumpunctata and full grown larvae
(30 days old) of C. cephalonica (100 insects) were used in five replications in complete
randomized design layout format. Whereas, fifty insects of 3rd
and 4th
instars larvae were used
for each S. litura and P. brassicae. Temperature was maintained at 27+10C and R.H. at 60-65%
respectively.
3.3. Entomopathogenic fungi isolates and their original sources
Eight fungal isolates were used in this experiment (Table 3.1 Plate 3.1). Six of them were
obtained from Indian type culture collection (ITCC) Division of Plant Pathology, IARI New
Delhi. Two were obtained from National Centre for Integrated Pest Management IARI
Campus, Pusa New Delhi-110012. The fungi were cultured on Potato Carrot Agar (PCA) in
slant and kept in fridge at 40C as a stock culture then grown on Potato Dextrose Agar (PDA) in
Petri dishes and maintained at ambient temperature (27±10C) till usage. Fifteen to twenty one
days old-cultures were used in bioassay or as inoculums for mass culture in sorghum grains.
15
3.4 Mass culture, lyophilisation and harvest protocol of the fungus
The entomopathogenic fungus, Beauveria bassiana, ITCC No. 6628 (Plate 3.2) was mass
cultured on sorghum grains (Plate 3.3) which had been washed and half-cooked by
boiling. Two hundred grams of wet sorghum was maintained in each autoclavable,
transparent polyethylene bags, a pinch of chloramphenicol, was added to the bags. Then
the sorghum was autoclaved according to standard protocol, the materials left for 24
hours before inoculation. Loop-full fungus from 15 days-old culture was inoculated in
each bag. Then the bags were plugged with cotton, kept for one month at temperature
27±10C, 65±5% relative humidity and complete darkness. Afterwards the conidia were
washed off with, 0.05% Criton X-100 in conical flasks, under laminar flow (Plate 3.4.).
Then kept in 30 ml centrifuge tubes and centrifuged (Sigma laboratory centrifuge 3K18),
(Plate 3.5 for five minutes at 50C temperature at 10.000 RPM. The collected conidia
(Plate 3.6) kept in -200C, (Plate 3.7) after that they were lyophilised for 24 hours (Plate
3.8) got powdered then it was finely sieved with piece of cloth.
3.5 Preparation of blank formulation
An array of combination of different material was used viz., carriers (Calcite, Dolomite,
Pyrophylite, Soapsone and Talcum powder (Plate 3.9) The binders used were Acacia
gum, Carboxy Methyl Cellulose and Xanthan gum. Wetting agent was Sodium
lignosulphate and moisturizer was Glycerol. Spreading agent used were Tween 20,
Teepol and Criton X100. For general information on adjuvants and their sources please
refer (Table 3.3). The list of 45 reciepes based on five carriers is shown in (Table 3.4).
Component of each recipe were mixed together with a mixer and autoclaved before
adding the active ingredient.
3.6 Preparation of final product
Conidia (0.15 g) were added to five grams of each of the prepared autoclaved blank
formulations that based on Pyrophylite and Talcum powder and mixed manually to form
final products. Then one g from each ready product was added to 3.5 ml sterilized
distilled water to prepare the stock solution which was serially diluted to be easily
quantified by Neubauer Improved haemocytometer. Finally the products were titrated up
1010
ml, using the haemocytometer, then evaluated against different stages of fruit flies.
Small, well-cleaned perfume sprayers (Plate 3.10) were used in case of treatment of
adults while the preimaginal stages were immersed, for 30 seconds in 0.5 ml and 1ml for
third larval stage and pupae respectively.
16
3.7 Preparation of conidial suspension
Conidia were harvested from 15 days old surface cultures by scraping. Conidia were
suspended in sterile distilled water containing 0.05% Criton X-100 in bottles. The bottles
were shaken vigorously until homogeneous conidial suspension was obtained. Ten-fold
serial dilution was prepared and quantified with Neubauer Improved Haemocytometer.
For commercial mycoinsecticides the dose 1010
was adjusted, using the Neubauer
Improved Haemocytometer.
3.8 Germination test of entomopathogenic fungi and newly developed products
Viability of each product or unformulated fungi was determined by spread-plating. 0.1 ml
of product’s suspension, titrated to 1x106 conidia /ml, was spread on PDA plates, with
three replications. Then incubated in complete darkness at temperature 27±10C relative
humidity 65±5% for 24 hours then, using cork borer round pieces of media were cut, kept
in cavity slides and covered with cover slips then observed and percentage germination
was examined, from 100 conidia under 40x.
3.9 The experimental conditions and inoculation of insects preliminarily
Inoculation of insects in first preliminary screening was conducted on adults of B.
dorsalis. Two weeks old of fungus culture kept in Petri dish without lid (15x90mm)
containing one isolate was kept in plastic jar measured (13x10cm) containing saturated
sand water at 27±1°C and 85±5% R.H. Twenty adults (three replications) of B. dorsalis
were released and provided with banana fruits and water in cotton wicks. The experiment
lasted for 9 days and repeated twice. For the rest of the test insects, they were kept in
each Petri dish (15x90 mm) shaken for 3 minutes to get full coverage by conidia powder,
then transferred into small jars and given food as described in rearing method for C.
cephalonica and C. septumpunctata. For the insect apart from fruit fly, results were
restricted to (+ve) and (-ve) which means susceptible/not susceptible respectively,
however percentage mortality was estimated.
3.10 Inoculation of adult of B. dorsalis
Four days old adults were used for all bioassays. The insect were immobilised by keeping
them in fridge for 5-7 minute. Then they were kept in Petri dishes (90x15mm) having
filter paper in which they were sprayed with 1 ml from each concentration of each strain.
Immediately treated insects were transferred to 21x15cm jars. Three replications with 10
insects were maintained for each treatment as well as control. All food source and
condition were same as in rearing conditions.
17
3.11 Confirmation of mycosis on the cadavers
Insects died during experimentation were taken surface sterilized using 2% sodium
hypochlorite then washed with three rinse of sterilized distilled water and kept in
sterilized Petri dish in which sterilized moisturized filter paper had been kept, then the
dishes were kept at temperature 27±10C and relative humidity 65±5% in complete
darkness for appearance of mycosis.
3.12 Treatment of preimaginal stages using immersion method
Thirty individuals of either third-instars larvae or 24 hours-old pupae were treated with
different concentrations of different strains in three replications. A group of 30 insects,
were immersed in 2ml of specified treatment for 30 seconds then transferred, 10 insects
per replication, to sterilized Petri dish (15x90mm) having autoclaved moisturized sand
(17g sand + 3ml water). All Petri dishes were kept at temperature 27±10C and relative
humidity 65±5%. Emergent adults were counted and recorded.
3.13 Prophylactic treatment of third instars larvae of B. dorsalis
Jar containing 50g, of autoclaved sand, inoculated with 5ml of specified concentrations,
and vigorously mixed with a mixer for 30 seconds (Plate 3.11). For each treatment, forty
last instar larvae of B. dorsalis (four replications) which were going to pupate within 24
hours were used. The jars were kept at 27±10C temperature, 65±5% relative humidity and
photoperiod 12:12 (Plate 3.12) till emergence of adult insects (8-9) days.
3.14 Statistical Analysis
Opstat statistical programme was used for analysis.The data were subjected to square root
transformation. For calculating LC50 and LT50 values, EPA Probit Analysis Program
(Version 1.5) was used.
3.15. Studies on different physico-chemical properties of carriers
A group of carriers were subjected to numerous tests. Table 3.4 gives general information
on the chemical nature and sources of carriers. The test carried out as follows;
3.15.1. Bulk density
To determine the bulk density of the carriers before and after compaction a known
volume (cylinder 4.8 cm diameters x 1.95cm height) was filled with the carrier before
compaction, then the carries occupied that volume, was weighed. Kept again in same
volume and pressed till got compacted, then it was weighed again. The volume values and
weighed value were calculated as per the following formula.
18
Bulk density (g/100cc) (before compaction):
Weight of the carrier x 100
Volume occupied by the same carrier before compaction
Bulk density (g/100cc) (after compaction):
Weight of the carrier x 100
Volume occupied by the same carrier after compaction
Particle size
The carriers were dry sieved through a 250 mesh sieve (aperture 105 μ) using manual
shaking and with help of a brush and tissue paper.
3.15.2. Reaction pH
One gram of each carrier was added to 10 ml of sterilized distilled water. Then the digital
pH meter (pHepR original) was used to check the pH of each carrier by keeping the probe
of pH meter in suspension formed. The instrument was frequently washed in sterilize
distilled water before using it again.
3.15.2 Sorptivity
Sorptivity (%) was determined by ASTM rubout method. To a known weight of carrier
commercially available linseed oil was added drop by drop through micropipette and
worked consistently by camel hair brush until the powder slipped freely from the tip of
brush. The volume of the linseed oil absorbed by the carrier was noted down and
sorptivity was calculated as:
Sorptivity (%) =
Millilitre of linseed oil required to slip the material from spatula x 0.93 x 100
Weight of the carrier taken
3.15.3 Moisture content
19
Moisture content was determined by gravimetric method. A known weight of the carrier
was spread uniformly in Petri dish and kept in microwave for 15 minutes. Then the
carriers were weighed again. Loss in weight was noted and per cent moisture content in
the carrier was calculated on microwave dry weight basis.
3.15.4 Wettability and suspensibility
One gram of the WP formulation was taken in aluminum foil. The powder was poured
rapidly and gently on the top of the surface of 100 ml water taken in to more than 1200
ml capacity cup with internal diameter 4.3cm and height 10.3 cm. The time was
measured with a stop watch from the moment the powder was placed on the surface of
the water until more than 95% of the powder had become wet and submerged below the
surface of the water. The time (in seconds) was the wetting time of the formulation.
3.1.6. Physico-chemical properties of newly developed WP formulation
3.1.6.1 Particle size
The particle size of the newly developed WP formulation was reduced by milling and
remilling.
3.1.6.2. Flowability
The flowability of newly developed WP formulation was normally determined with the
help of dusting appliances. However, in the absence of such an appliance it was
determined by visual observation.
Development of low cost efficient cages for fruit flies
Generally acrylic cages (20×20×20cm) are used in rearing of fruit flies. Instead of that
plastic cages in the form of plastic jars were prepared with size 15cm diameter × 21cm
height (Plate 3.13). In this jar 11±.05 cm hole was cut using heated ironic ring (Plate
3.14) then around that rounded hole, punctures were made using heated rod (Plate 3.15)
with wooden handle. Then with the help of needle and thread a sleeve of markin cloth
was stitched around that hole through which handling of insect are made. The lid of the
jar was also cut in the middle using same equipments with above mentioned
measurements and methodology however the hole prepared was covered with nylon
mesh, through which one can see. A source of light was kept above the jar to attract
insects while handling (taking cadavers out or changing the feed or water).
New sprayers used
Sprayer (Plate 3.10) used by this study, were perfume sprayers with capacity of 10 ml.
This sprayer was able to deliver fine droplets compared to the big droplets produced by
hand glass automizers.
20
Research Paper I
Pathogenicity of Indian Isolates of Entomopathogenic Fungi against Important
Insect Pests and Natural Enemies
Abstract
Entomopathogenic fungi (EPF) are one of the best alternatives to chemical pesticides and
important component of IPM. Eight isolates of Beauveria bassiana (Balsamo) Vuillemin
and Metarhizium anisopliae (Metsch.) Sorokin, were obtained from Indian Type Culture
Collection (ITCC) and National Centre of Integrated Pest Management, New Delhi India.
They were screened against adults of, Bactrocera dorsalis, larvae of Corcyra cephalonica
(Stainton) and larvae of Spodoptera litura (Fab). By exposing the insects to 2-3 weeks old
culture of EPF, The pathogenicity of four isolates was proved, using contact method.
Three isolates ITCC No. 6628; ITCC No. 6645 and B. NCIPM were found pathogenic to
fruit flies; whereas, in case of C. cephalonica, pathogenicity of first two isolates in
addition to (M. NCIPM) was proved. Mortality of adult fruit flies was 100% within 5-6
days of exposure, however, in case C. cephalonica (31-98%) within three weeks.
Subsequently the pathogenic isolates were tested against Coccinella septumpunctata (L.)
and C. cephalonica.Significant differences were observed among isolates, and the isolate
B. NCIPM was found relatively safer to C. Septumpunctata.
Key words: Biological control, entomopathogenic fungi, Beauveria bassiana, natural
enemies, Bactrocera dorsalis, Coccinella septumpunctata, Corcyra cephalonica.
21
4.1. Introduction
As an alternative to chemical control or as part of IPM programs, there is a resurgence of
interest in the use of microbial insecticides for biological control of insect pests. Fungal
agents are among the most promising group of biological control agents against insect pests
(Reithinger et al., 1997). Over 500 species of fungi are known to have insect pathogenic
properties. Interestingly, Beauveria and Metarhizium (Deuteromycotina, Hyphomycetes)
represent the most frequently used genera (Burges and Hussey, 1971) and are known to infect
a broader range of insect pests of crops belonging to Lepidoptera, Homoptera, Hymenoptera,
Coleoptera and Diptera. Most research on fungi has been directed to Beauveria and
Metarhizium (Greathead and Prior, 1990; Whitten and Oakeshott, 1991). Entomopathogenic
fungi (EPF), compared to other entomopathogenic microbial organisms can infect their host
via contact i.e., invade via epicuticle of integument, and not required to be ingested by the
insect to cause infection (Goettel et al., 2005; Ali et al., 2010).These fungi are cosmopolitan
and do not leave undesirable residues hence can be used, even close to harvest. Besides that,
they are compatible with other pest management tactics. Additionally, their production is
easy and economical and do not require high input technology (Prior, 1988). Commercially,
Beauveria bassiana (Balsamo) Vuillemin and Beauveria brongniartii (Saccardo) are
produced by more than 14 companies, and Metarhizium (M. anisopliae and M. anisopliae
var. acridum) by more than 10 companies world-wide. Virulence is the most important
indicator to measure the potential of fungi against pests and the basis of choosing highly
virulent fungi in laboratory bioassays (Li et al., 2012). Therefore it was planned to evaluate
the pathogenicity of different strains of B. bassiana and Metarhizum anisopilae
(Metchnikoff) Sorokin, on some important insect pests of crops and natural enemies.
4.2. Materials and methods
4.2.1. Rearing of fruit flies
Larvae of the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) were
obtained from Biological Control Laboratory, Division of Entomology, Indian Agricultural
Research Institute, New Delhi. The larvae were reared on ripe bananas whereas; adult flies
were maintained on sugar and yeast autolysate (CDH Bioscience (P) Ltd New Delhi)). They
were kept in ventilated acrylic cages (20x20x20 cm) at 27±10C, 65±5% R.H. and 12:12
photoperiod. Water was supplied in vials with cotton wicks.
22
4.2.2. Rearing of other insects
Rearing of Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae), Spodoptera litura (Fab.)
(Lepidoptera: Noctuidae) and Spilarctia obliqua (Walker) (Lepidoptera; Arctiidae) was
carried out as per the protocols given by Gautam, (2008). Whereas, Coccinella
septempunctata (L.) (Coleoptera: Coccinellidae), Pieris brassicae (L.), (Lepidoptera:
Pieridae) and Drosicha mangiferae (Green); Hemiptera: Monophlebidae) were collected
from the IARI fields, mustard crop, cabbage crop and mango tree
respectively. C.septempunctata was fed with Brevicoryne brassicae (L.) (Homoptera:
Aphididae). While P. brassicae and D. mangifera were reared on cabbage and mango
respectively. Ten insects were used for D. mangiferae for each isolate. Adults of C.
septumpunctata and full grown larvae, 30 days old, of C. cephalonica (100 insects) were
used. Five replications were used, in complete randomized design layout format. Whereas, 50
individuals of 3rd
and 4th
instar larvae were used for each S. litura and P. brassicae.
Temperature was maintained at 27+10C and R.H. at 60+5% respectively. Experiments were
carried out in 2011 and 2012.
4.2.3. Entopathogenic fungi source and culture
Eight fungal isolates were used in this experiment (Table 4.1). Six of them were obtained
from Indian type culture collection (ITCC) Division of Plant Pathology, IARI, New Delhi.
Two were obtained from National Centre for Integrated Pest Management IARI Campus,
Pusa New Delhi. The fungi were grown on potato dextrose agar (PDA) in Petri dishes and
maintained at 27±10Ctemperatures in B.O.D. incubator for 15 to 21 days.
4.2.4. Inoculation of the insect
The experimental conditions, inoculation of insects and first preliminary screening were
conducted on adults of B. dorsalis. Two week old, Petri dish without lid (15x90mm)
containing one isolate was kept in a plastic jar measured (13x10cm) containing saturated sand
with water at 27±1°C and 85±5% R.H. Twenty adults (three replications) of B. dorsalis were
released and provided with banana fruits and water in cotton wicks. The experiment lasted for
9 days and repeated twice. For other insects they were kept in each Petri dish (15x90mm)
shaken for 3 minutes to get full coverage by conidia powder, then transferred into small jars
and given food as described in rearing method.
4.2.5. Statistical analysis
Opstat statistical programme was used for analysis, in which the data were subjected to
square root transformation. For the rest of the insects results were restricted to (+ve) and (-ve)
23
which means susceptible/not susceptible respectively, however percentage mortality was
estimated. For calculating LT50 values, EPA Probit Analysis Program (Version 1.5) was used.
4.3. Results
The eight isolates of entomopathogenic fungi mentioned in (Table 4.1, Plate 4.1), were
screened against different insect pests and their pathogenicity was reported (Table 4.2) (Fig4.
1). When the eight entomopathogenic fungi screened against adults of B. dorsalis (4-5 days
old) three out of eight isolates were found to be pathogenic. (Fig 4.2 Plate 4. 1), whereas the
remaining five were not pathogenic, (Table 4.3). It is worth mentioning that 100% insects
died within 4-6 days and the cadavers of adults were found fully covered with white
mycelium within nine days (Plate 4.2). All isolates presented in (Table 4.1) except B. NCIPM
was tested against full grown larvae of C. cephalonica. All the isolates were non-pathogenic
to C. cephalonica however; ITCC No. 6628, ITCC No. 6645 and M. NCIPM were virulent
(Plate 4.3) and registered average mortality of 17, 19.6 and 3.4% respectively (Table 4.4, Fig
4. 1). There was a significant difference between the treatment and control and the isolates
significantly differed among themselves. Results of the efficacy of three isolates of B.
bassiana on C. septumpunctata (Fig 4.2) and C. cephalonica (Fig 4.3 and 4.4) are presented
in (Table 4.4). The highest average mortality was 20, 19 and 7.6 % for the isolates ITCC No.
6628, ITCC No. 6645 and B. NCIPM, respectively. LT50 values of entomopathogenic fungi
isolates against C. cephalonica was calculated and reported (Table. 4.5). The isolate, B.
NCIPM, showed minimum mortality rate to C. septumpunctata which reflects it is relative
safety. Efficacy of virulent isolates also was shown on S. litura (Plate 4.5) and Pieris
brassicae (Plate4. 6), whereas no mortality was observed on D. mangiferae.
Discusion
Since cadavers of adults of fruit fly were found fully covered with white mycelium within
nine days, this indicated that the inoculum picked up during walking was sufficient to kill the
insect within the mentioned time. The results were in accordance with that of Dimbi et al.,
(2003) who found adult mortality in Ceratitis capitata (Weidemann) and Ceratitis rosa var.
fasciventris (Karsch) treated with different isolates of B. bassiana and M. anisopliae were 7
to 100% and11.4 to 100% respectively, at four days post inoculation. The mycelium appeared
on the abdominal side of the insect as well as mouth parts and joint of the leg (Plate 4.3).
These results were in conformity with that of Muñoz (2000), wherein he evaluated 16 strains
of B. bassiana against C. capitata adults and found mortality levels between 20.0to 98.7%.
Also Quesada-Moraga et al., (2006) reported 30-100% mortality after 20 days, while testing
10 isolates of B. bassiana and five isolates of M. anisopliae against adult fruit fly. Sookar et
24
al., (2008) reported the pathogenicity of seven isolates of M. anisopliae, five isolates of B.
bassiana and two isolates of Paecilomyces fumosoroseus (Wise) in adults of Bactrocera
zonata (Saunders) and Bactrocera cucurbitae (Coquillett). Mortality of B. zonata varied
between 12.0 to 98.0% and 2.0 to 94.0% in B. cucurbitae at five days post-treatment.
The symptoms observed on treated larvae of C. cephalonica were growth of white mycelium
on the intersegmental parts (Plate 4.7), pink to reddish colour cadavers (Plate 4.8). C.
cephalonica larvae colour indicated secretion of a metabolite called oosporein registered by
infections caused by ITCC No. 6645 and ITCC No. 6628. These findings are in agreement
with results reported by Zimmermann (2007), wherein he reported presence of a major
secondary metabolite dihydroxybenzoquinone, an extracellular secretion, of many isolates of
B. bassiana. Samodra and Ibrahim (2006) noted up to 90% C. cephalonica larval mortality
within 15 days when treated with isolates of formulated B. bassiana. Rice (1999) reported an
isolate of B. bassiana to be pathogenic to adults of Sitophilus oryzae (L.), Rhyzopertha
dominica (F.) and Tribolium castaneum (Herbst). Khashaveh et al., (2011) reported the
potential of a commercial product based on B. bassiana against adults of S.granarius (L.)
and Oryzaephilus surinamensis ( L.) and T. castaneum and they found that mortality record
within 15 days was 88, 78 and 64% respectively.
Safety of some B.bassiana isolates was proved by Zimmermann, (2007) where he stated that
host specificity is a strain-specific trait. For example, B. bassiana isolates from the lady
beetle, Olla v-nigrum (Mulsant), were pathogenic to adult O. v-nigrum but not to adults of the
Asian lady beetle, Harmonia axyridis (Pallas). Also the GHA strain of B. bassiana was not
significantly pathogenic to either O. v-nigrum or H. axyridis. Moreover non-target insects
which are infected under laboratory conditions may not necessarily be infected in nature, in
other words, difference between physiological host and ecological host range plays an
important role in pathogenicity. None of the isolates used against D. mangiferae were found
pathogenic to this insects despite their virulence against other insect pests which confirmed
the specificity of the isolates used.
On the other hand it was observed (Table 4.1) that the recently deposited (6-18 months)
cultures were more virulent compared to those deposited long back (more than three years).
The reason for losing the virulence needs to be worked out and appropriate measures must be
taken to conserve it. Isolates which have been proved to be pathogenic to fruit fly and other
insect pests if used later may not be virulent, due to longer storage period. That leads to loss
of biological wealth, efforts and resources. According to peer reviewed literature this is the
25
first report of evaluation of pathogenicity of entomopathogenic fungi against fruit fly and C.
cephalonica in India.
The virulence of the three isolates (ITCC No. 6628, ITCC No. 6645 and B. NCIPM) was
proved against above mentioned insect pests. Therefore, further detailed studies needs to be
conducted using these isolates on different insect pests. Entomopathogenic fungi can be used
to control storage pests where worldwide trials are being conducted. Incorporation of this
very important component in IPM programmes for fruit flies in India in specific and Asia at
large where this component seems to be lacking.
26
Research Paper II
Performance of three Indian isolates of Beauveria bassiana (Balsamo) vuillemin and
three commercial mycoinsecticides against all stages of Bactrocera dorsalis (Hendel)
(Diptera: Tephritidae)
Abstract
Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) has been identified as
one of the three most important agricultural pests in South East Asia. In India, on mango
alone, it causes losses up to 80%. In this study three isolates of B. bassiana and three
commercial mycoinsecticides were screened against larva, pupa and adults of B. dorsalis.
The isolates ITCC No. 6628 and B. NCIPM were found pathogenic to adult stage with LC50
2.5x105 and 7.5x 10
6 conidia /ml,
respectively. While, ITCC No. 6645 were found effective
against adult stage at 1.2x109 and larvae with LC50 9x10
9 conidia/ml. The three commercial
mycoinsecticides evidenced their efficacy only on adult stages with 26.6%, 40% and 46.6%
mortality for, Bio-power®
, Bio-magic®, Bio-catch
®, respectively. There was a significant
difference between treatment and control. Mycosis was recorded on the cadavers.
Key words: B. dorsalis, Beauveria bassiana, fruit flies, biological control
5.1. Introduction
Oriental fruit fly, B. dorsalis (Hendel) (Diptera: Tephritidae) is considered to be among the
five most damaging and aggressive pest fruit flies in the world (Leblanc and Putoa 2000).
Waterhouse (1993) identified it as one of the three most important agricultural pests in South
East Asia. It infests hundred twenty-four hosts in tropical Asia (Allwood et al., 1999). In
India the mean of yield fruit loss is 16% (Verghese et al., 2002). On mango (Mangifera
indica L.), it causes up to 80% economic loss (Verghese and Jayanthi, 2001). Fruit flies
causing damage by fruit drop and renders the fruit inedible (Du Toit 1998). Indirect loss
arises from phytosanitary restrictions imposed by importing countries. The intensive use of
synthetic pesticides for crop protection causes a number of undesirable effects on human
health (Perry et al., 1998). Moreover, the development of resistance in insect populations
results in decreasing the effectiveness of insecticides (Vontas et al., 2011). Besides, current
social and environmental problems are associated with insecticide use for fruit fly control,
(Penrose 1993). The chemical insecticides currently used to control fruit flies are listed
among the most Persistent Organic Pollutants (POPs) by the United Nations Environmental
Programme (UNEP) (Roessler, et al., 1989). Therefore, biological control methods, including
the use of entomopathogenic microbial organisms have been developed, as an alternative to
synthetic pesticides (Lacey and Shapiro-Ilan 2008). Entomopathogenic fungi (EPF),
27
compared to other entomopathogenic microbial organisms, have the advantage that they can
infect their host via contact, and do not need to be ingested by the insect to cause infection
(Goettel et al., 2005; Ali et al., 2010). Several studies have demonstrated the effectiveness of
autochthonous isolates of B. bassiana and Metarhizium anisopliae (Metschn.) for the control
of various fruit fly species. The conidial phase of a large number of strains of both species,
coming from different geographic regions, have been assessed, under laboratory conditions,
for the control of different fruit fly species and on different life stages (Garcia et al., 1984;
Espin et al., 1989; Campos, 2000; Castillo et al., 2000; Lezama-Gutierrez et al., 2000; De la
Rosa et al., 2002 and Ekesi et al., 2002). The most common method used has been the
immersion of any insect stage (larva, pupa or adult) in a conidia solution although topical,
oral or contact applications have also been tested (De la Rosa et al., 2002; Toledo et
al.,2007). Preliminary study proved that the current isolate evaluated by this study had been
effective against adult of B. dorsalis (Elbashir et al., in press).
Therefore, we evaluated the virulence of three isolates of B. bassiana against different stages
of B. dorsalis and assessed the performance of three commercial mycoinsecticides namely,
Bio-power®, Bio-magic
® and Bio-catch
® against different stages of B. dorsalis.
5.2. Materials and Methods
5.2.1. Rearing of fruit fly
Larvae of the Oriental fruit fly, B. dorsalis were obtained from Biological Control
Laboratory, Division of Entomology, Indian Agricultural Research Institute, New Delhi. The
larvae were reared on ripe bananas whereas; adult flies were maintained on sugar and yeast
autolysate (CDH Bioscience Pvt. Ltd., New Delhi). They were kept in ventilated acrylic
cages (20 x 20 x 20 cm) at 27±10C, 65±5% R.H. and 12:12 photoperiod. Water was supplied
in vials with cotton wicks.
5.2.2.1 Fungi
The two fungal isolates ITCC No. 6628 and ITCC No. 6645 used in our experiments (Table.5
1) were obtained from the Indian Type Culture Collection (ITCC), Division of Plant
Pathology, Indian Agricultural Research Institute New Delhi, India and one isolate B.
NCIPM was obtained from National Centre for Integrated Pest Management (NCIPM), New
Delhi. The fungal isolates were cultured on Potato Carrot Agar (PCA) in slant and kept at 40C
as a stock culture. Then grown on Potato Dextrose Agar (PDA) in Petri dishes and
maintained at ambient temperature (27±10C) till usage. Three commercial mycoinsecticides
viz., Bio-power®, Bio-magic
® and Bio-catch
® (Plate 5.1) were obtained from T. Stanes and
Company Tamil Nadu, India, (Table. 2).
28
5.2.2.2. Preparation of conidial suspension
Conidia of fungal isolates were harvested from 15 days old surface cultures, by scraping.
Conidia were suspended in sterile distilled water containing 0.05% octylphenol ethoxylate
(Criton X-100) in small bottles. The bottles were shaken vigorously until homogeneous
conidial suspension was created. Ten-fold serial dilution was prepared (Plate 5.2) and
quantified with haemocytometer. Viability of conidia was determined by spread-plating, 0.1
ml of conidial suspension on PDA Petri dish, with three replications. Then the dishes were
incubated in B.O.D. at 27±10C and 65±5% R.H. for 24 hours. The viability percentage was
estimated by counting 100 conidia from each plate at 40× magnification. In case of
commercial mycoinsecticides the doses 1010
were adjusted by Neubauer Improved
haemocytometer.
5.2.3. Inoculation of insects
5.2.3.1 Adult bioassay
Four day old adults were used for all bioassays, the insect were immobilised by exposing
them in 40C for 5-7 minute. Then they were transferred to filter paper kept in Petri dishes
(90x15mm) in which they were sprayed with 1 ml from each concentration for each strain.
Treated insects were immediately transferred to 21x15cm jars. Three replications with 10
insects were maintained for each treatment as well as control. All food source and condition
were same as in rearing conditions. The cadavers of insects died during the course of
experiment were collected after and surface sterilized by using 2% sodium hypochlorite
followed by washing with sterilized distilled water. Thereafter, cadavers were transferred to
sterilized Petri dish containing sterilized moisturized filter paper at temperature 27±10C and
65±5% R.H. in complete darkness for appearance of mycosis.
Thirty individuals of third instar larvae or 24 hours old pupae were treated with different
concentrations of each isolates. A group of 30 insects were immersed in 2ml of specified
concentration for 30 seconds. Then immediately transferred to sterilized Petri dish having
autoclaved (17g) of sand moisten with (3ml) sterilized distilled water in three replicate
with10 insects per replication. All Petri dishes were kept at temperature 27±10C and 65±5%
relative humidity. Emergence of adults was counted nine days later, onwards.
5.2.3.2 Inoculation of preimaginal stages
Each jar containing 50g autoclaved sand was inoculated with 5ml of specified concentrations
(Plate 5.3), and vigorously mixed with a mixer for 30 seconds. For each treatment forty-last
instar larvae/prepupa of B. dorsalis was selected in four replications. Jars were kept at
29
temperature 27±10C and 65±5% R.H. and photoperiod of 12:12 hour till emergence of adult
insects.
Statistical analysis
Opstat statistical programme was used for analysis. The data were subjected to square root
transformation. For calculating LC50 EPA Probit Analysis (Version 1.5) was used.
5.3. Results
5.3.1. Germination test
In the viability tests, germination for the three strains and three commercial products was
above 90%.
5.3.2. Thre effect of isolates against adults of fruit fly
The three isolates of B. bassiana were found to be effective against the adult stage. The LC50
obtained for these isolates were 2.5x105, (Fig 5.1)
1.2x10
9 (Fig 5.2) and 7.5x10
6 (Fig 5.3) for
ITCC No. 6628, ITCC No. 6645 and B. NCIPM respectively, (Table 5 .4). In case of strain
ITCC No. 6645 with range of concentrations 105 to 10
11 conidia/ml the mortality varied
between 1% and 100%. While 3% to 100% for the strain B. NCIPM, whereas 7% to 100%
mortality was achieved, using strain ITCC No. 6628 with concentration of 105 to 10
10 (Table
5. 7).
5.3.3. Mycosis on the cadavers
Mycosis followed a general trend and it reached up to 90% of the cadavers of the adult fruit
fly, B. dorsalis treated by the three virulent strains ( Plate 5.3, 5.4, and 5.5) The mycoses first
appeared on the cavity of the mouthparts and the ovipositor area. Fungal mycelium emerged
from the soft parts of the body, such as wing bases, mouthparts, and base of the legs,
intersegmental regions, and membranous regions of the abdomen, coxa, and neck.
5.3.4. Efficacy of commercial mycoinsecticides against adults of fruit fly
Percentage mortality of commercial mycoinsecticides viz., Bio-power®, Bio-magic
® and Bio-
catch® against adults of B. dorsalis were 26.6%, 40%, 46.6% respectively (Table 5.3, Fig 5
.4). The results obtained by the three products were significantly different compared to
control.
5.3.5. Last larval stage of fruit fly with strain ITCC No. 6645
All last larval stages pupated normally, however dose- dependent-mortality was observed in
pupal stages. LC50 of the last larval stage, when treated with ITCC No. 6645, was 9 x 109
conidia /ml (Table 5.6, Fig 5.5). Two to three days later visible white mycosis was observed
on pupae (Plate.5.5).
30
5.3.6. Efficacy of commercial mycoinsecticides and the three virulent fungi on
preimaginal stages
Regarding last larvae and pupae which were dipped either in three fungi tested (Table 5. 5) or
the mycoinsecticides used (Table 5.3), there was no effect despite usage of higher
concentrations. All larvae pupated normally and emergence of adult was comparable to
control.
5.4. Discussion
The LC50 obtained for three tested entomopathogenic fungi shows that the most virulent one
was ITCC No. 6628 having the least LC50 value followed by B. NCIPM. The cumulative
mortality recorded, during 7 days; for the treated adults with the different doses, revealed the
dose response relationship (Table 5.7). Death of adult fruit fly within one week is of practical
implications which reflect reduction of the adult population before the first egg laying, a
critical moment of insect attack. Sensitivity and susceptibility of mycosed areas to fungal
infection particularly the mouth parts would guide for practical application using
mycoinsecticide. Similar results were obtained by several workers worldwide for instance
LC50 of three strains of B. bassiana against adult of Ceratitis capitata (Wied.) had the range
of 3.8 to 10.5 log conidia/ml (Qazzaz et al., 2012). Also Jiji et al., (2006) reported that LC50
values of B. bassiana on B. dorsalis were 7.0x108, 2.0x10
7, and 5.0 x10
6 conidia /ml on 3
rd,
4th
and 5th
day, respectively. De La Rosa et al., 2002, documented LC50 values as 5.13x105,
3.12x106, and 9.07x10
6 conidia /ml when he tested three strains of B. bassiana against adults
of Mexican fruit fly Anastrepha ludens (Loew) under laboratory conditions. The difference in
virulence among B. bassiana isolates in the experiment may be due to differences in the
production of enzymes such as chitinase, chyemolestase, chymotrypsin, and esterase which
degrade the insect cuticle. These enzymes are considered an essential pre requisite for
successful fungal infection (De La Rosa et al., 2002). Among the isolates tested, ITCC No.
6628 registered the least LC50 which indicate high virulence compared to other isolates. It is
worth mentioning that coloration of pink to reddish colour was reported on cadavers of larvae
of Corcyra cephalonica (Stainton) when it was treated with this isolate (Elbashir et al., in
press). This coloration was thought to be due to excretion of metabolite called oosporein.
This red-coloured pigment is dihydroxy benzoquinone which is the major secondary
metabolite produced by B. brongniartii and is also produced by many isolates of B. bassiana
(Zimmermann, 2007). This coloration was more visible in case of treatment with ITCC No.
6628 compared to other isolate tested. This may show more excretion of oosporein, and
indicate correlation between coloration and high virulence of this fungus.
31
The results obtained for three commercial mycoinsecticides were in conformity with the
results obtained by several authors from different parts of the world. Mahmoud (2009)
evaluated same three mycoinsecticides against adults of Bactrocera oleae (Gmelin) recorded
27.2, 18.4 and 34.4% mortality for B. bassiana, M. anisopilae and Verticillium lecanii
(Zimmermann) Zareand W. Gams, respectively. Other similar studies reported in laboratory
and field which may match with our results are exemplified by; Dimbi et al., (2003) recorded
mortality of between 70–93%. When they evaluated M. anisopliae ICIPE 20 against C.
capitata and, C. rosa var. fasciventris, using several autoinoculative device in laboratory.
Dimbi et al., (2013), studied horizontal transmission of entomopathogenic fungi, where they
exposed adult of C. capitata C. fasciventris and C. cosyra to velvet material, resulted in
100% mortality within 5-6 days post-exposure besides fecundity was reduced drastically. In
organic agriculture, repeated applications of Naturalis-L against R. cerasi (L.) shown to
reduce the infestation level of fruits by 60%-70%. Besides it is a suitable and economically
reasonable strategy (Daniel and Grunder, 2012). Ortu et al., (2009) revealed that Naturalis is
based on the B. bassiana strain ATCC 74040, when used against C. capitata, under
laboratory conditions, protected fruits from the insect ovipositions. Moreover field trials
proved that it was as effective as a pyrethroid. Daniel and Wyss (2009) recorded that B.
bassiana and Isaria fumosorosea (Wize) with concentration 107 conidia/ml against R. cerasia
caused 90–100% mortality. Flores et al., (2013) reported that sterile C. capitata males which
were evaluated as vectors to spread B. bassiana conidia to wild C. capitata populations under
field conditions; in an area 7000 ha. They succeeded in transmitting fungal conidia to 44% of
the wild C. capitata flies. In this study mycosis was observed from B. bassiana and M.
anisopliae products but not from V. lecanii. Concentration used by this study (1010
)
conidia/ml may be the reason for reporting higher mortality compared to mortality reported
by Mahmoud (2009) or due to susceptibility of B. dorsalis compared to B. oleae.
The concentration required to kill 50% of the last larval stage when treated with isolate ITCC
No. 6645 was very high indicated low virulence of the isolate used and raised a question
about economic feasibility of using this particular strain in treating preimaginal stages of this
insect. However prophylactic method using inoculation of the soil before releasing the last
larvae was effective compared to dipping method used for the other strains. Moreover this
method is more valid in terms of its applicability; particularly it is mimicking the natural
behaviour of the larvae such that they drop from fruit into the soil where it encounters the
fungus-contaminated soil. Lack of virulence of of tested entomopathogenic fungi and
commercial mycoinsecticides against preimaginal stages of fruit fly may be attributed to no
32
virulence of the mycoinsecticides and tested strains against these immature stages. Similar
results were obtained by De La Rosa et al., (2002), using dipping method for 30 seconds,
evaluated the effect of eight strains of the entomopathogenic fungus B. bassiana upon larvae
and pupae, of the Mexican fruit fly, Anastrepha ludens (Loew). Mortality was 2-8% in larvae
and 0% in pupae. M. anisopliae and B. bassiana produced no mortality in larvae and pupae of
tse tse flies, (Kaaya and Okech, 1990). Beris et al., (2012) carried out laboratory experiment
against pupae and adults of C. capitata via different routes of exposure. Average mortality of
pupae after immersing them into conidia suspensions was low and ranged from 18.7 to 23.9
%. The results obtained contradicted results obtained by various authors worldwide. Espin et
al., (1989) applied M. anisopiliae directly to pupae of C. capitata and observed that 65.8% of
the pupae were infected with the fungus. Ekesi et al., (2002) investigated, in the laboratory,
the pathogenicity of 13 isolates of M. anisopliae and two isolates of B. bassiana against C.
capitata and C. var. rosa fasciventris exposed as late third instar larvae in sand. All isolates
caused a significant reduction in adult emergence and a corresponding large mortality on
puparia of both species. Mar and Lumyong (2012) evaluated six entomopathogenic fungal
isolates against fruit fly Bactrocera spp. All tested isolates were pathogenic to fruit fly pupa.
Mortality of pupa varied from 25.89% to 100% in Metarhizium flavoviride (Gams and
Rozsypal), 22.22 to 100% in Paecilomyces lilacinus (Thom) and 29.67% to 100% in B.
bassiana. On one hand Hajek and St. Leger, (1994) demonstrated that the low pathogenicity
of these fungi against many insect species was due to the nature of the cuticle, in terms of its
density and thickness and the degree of sclerotization, among other factors. Moreover an
infection can be aborted on the epicuticle if a factor essential for a phase of adhesion,
microbial development or pathogenesis is absent. Also the failure of fungi to invade insect
cuticle has been attributed to the presence of inhibitory compounds (phenols, quinones and
lipids) on the cuticle surface. Ferron (1981) reported that the degree of sclerotization of the
insect cuticle, the method of application of the fungus, the life stage involved, are all factors
that could affect the efficacy of fungal pathogens to control insect pests. On the other hand,
though, no efficacy of the tested isolates against neither last larval stage nor pupae,
nevertheless efficacy might be initiated in adult stages. Despite that the experiments was
terminated up to the emergence of adult without further follow up. But this can be predicted
based on several previous studies which was carried against preimaginal stages and proved
efficacy on adults as well. Ekesi et al., (2002) found that adult flies emerged from treated
pupae may be contaminated either by contact with conidia on the pupal integument during
emergence or by fungal penetration of the adult before emergence. Other researchers
33
observed, after exposing pupae of C. capitata, as well as other insect species, to
entomopathogenic fungal conidia, high levels of post emergence mortality and mycosis in
adults which had avoided the infection as pupae (Poprawski et al., 1985; Ekesi et al., 2002).
Ekesi et al., (2002) found high levels of mortality and mycosis on emerged adults after
exposing pupae of C. capitata, C. var rosa fasciventris and C. cosyra (Walker) to conidia of
two isolates of M. anisopliae.
5.5. Conclusions
In this study, three entomopathogenic fungal isolates along with three commercial
mycoinsecticides products were evaluated against different life stages of B. dorsalis. Results
showed that third instar larvae and pupae were not susceptible to all commercial products.
The three isolates used were also not effective against preimaginal stages when immersion
method was used. However the strain ITCC No 6645 was effective against prepuating larvae
when it was applied to the soil prophylactically. Therefore control of B. dorsalis should be
focused on adult flies if these strains are to be used against the pest. Adult flies were found to
be the only life stage susceptible to fungi infection. B. bassiana from all strains and products
tested showed a high virulence against adult of fruit flies causing the pest death during the
pre-oviposition period. These findings are of practical implications which reflect reduction of
the adult population before the first egg laying, a critical moment of insect attack. So based
on our finding it is clear that mycoinsecticides can be used against adult stages of fruit flies.
This can be used in further studies in combination with available novel application methods.
Isolate, ITCC No. 6645 was the only strain could be used prophylactically, proved its
efficacy against last instar larvae. However the high Lc50 (Lc50 9x109
conidia/ml) indicates its
efficacy at higher concentrations.
34
Paper III
Development of Wettable Powder Mycoinsecticide and its Efficacy against All Stages of
Fruit fly Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)
Abstract
Five clay minerals were screened for their suitability as carriers for aerial conidia of
Beauveria bassiana (Balsamo) (Vuillemin) isolate ITCC. No 6628. A series of physico-
chemical tests were carried out to study the suitability of the best carrier. According to
mosaic of tests, Pyrophylite and talcum powder were found best for use in formulation,
whereas calcite dolomite and soapstone were abandoned due to their failure either to fulfill
the physio-chemical properties or the fungus requirements. An array of combination was
added to the three qualified carriers, viz, wetting agents, spreaders, binders, and moisturizer.
Out of ten products only three maintained the viability of conidia with germination above
90%. Those products were further tested against three stages of fruit fly Bactrocera dorsalis
(Hendel). The result of bio-efficacy of three products on all stages of the pests was not
significant, compared to control. This failure was attributed to lack of factors responsible of
attachment of spores to the insect integument or the production of enzymes responsible for
causing the mortality. So the formulations were responsible in a way or another, for this
failure keeping into consideration the success of unformulated fungi.
6.1. Introduction
Rathore and Nollet (2012) revealed that annual sales of microbial pesticides are reported to
be US$750 million globally, amounting only to 2.5% of the chemical market. The global
market for biopesticides is forecast to reach US$2.8 billion by the year 2015. This segment is
expected to grow at a 15%.6% compound annual growth rate from US$ 1.6 billion in 2009 to
$3.3 billion in 2014. Key factors driving market growth include increasing environmental
concerns and consumer preference towards chemical free crops. Besides, acceptance of
substitutes to conventional pesticides and declining market for harmful organophosphate
insecticides. Worldwide, United States represents the largest region for biopesticides with
279 registered biopesticides while in Europe 77 registered products in 2008. While in Asia-
pacific constitutes the other leading market with biopesticides sales projected to reach
US$362 million in 2012. For all crops types, bacterial biopesticides claim about 74% of the
market; fungal biopesticides about 10%, viral biopesticides 5%, predator biopesticides 8%
35
and other biopesticides 3%. At present there are approximately 73 microbial active
ingredients that have been registered by the US EPA. The registered biopesticides include 35
bacterial products, 15 fungi, 6 nonviable (genetically engineered) microbial pesticides, 8
plant incorporated protectants, 1 protozoan, 1 yeast and 6 viruses. Gupta and Dikshit (2010)
reported that India has a vast potential for biopesticides, representing only 2.89% of the
overall pesticide market, in 2005. However it is expected to exhibit an annual growth rate of
about 2.3% in the coming years. Biopesticide consumption, in India has shown its increased
use over the time, in 2005-06, stands at 1920 MT. So far only 12 types of biopesticides,
including mycoinsecticide based on B. bassiana, were registered under the Insecticide Act,
1968, in India. The use of some of these bio pesticides, including B. bassiana have been
crowned with success in Indian agriculture. This usage is driven by large area under organic
cultivation (crops), estimated to be around 1, 00,000 hectares. Besides lakhs of hectares of
forest area being certified as organic. However Sharma et al., (2013) mentioned that, in India,
the information for microbial insecticides including entomopathogenic fungi in term of their
inefficacy, mass production, formulation and field application technologies is quite scanty
and scattered. Therefore there is a need for centralized national agency for proper evaluation
of strains/races of microbial biocontrol agents.
Increased public concern about the potential adverse environmental effects associated with
the heavy use of chemicals insecticides has promoted the examination of alternative methods
for insect pest control; one such alternative is the use of biopesticides. A major advantage of
biopesticides is their lack of toxicity to pollinators and compatibility with other natural
enemies, such as hymenopteran parasitoids. Changes in political and social attitudes towards
safer, more environmentally compatible pest control alternatives have increased opportunities
for biopesticides. Among several hundred species of entomopathogenic fungi, Beauveria and
Metarhizium (Deuteromycotina, Hyphomycetes) represent the most frequently used genera
(Burges and Hussey, 1971), and are known to infect a broader range of insect pests of crops
including Diptera. Therefore, most research on fungi has been directed to Beauveria and
Metarhizium (Whitten and Oakeshott, 1991). Historically, countries in Asia, Latin America,
and Eastern Europe have accounted for the greatest use of fungal pathogens (Faria et al.,
2007). The first attempt to control a pest with a fungal agent was carried out in Russia in
1888, when the fungus now known as Metarhizium anisopliae (Metschn.) Sorokin was mass
produced on beer mash and sprayed in the field for control of the beet weevil Cleonus
punctiventris (Germar) (Lord, 2005). In the 1980s approximately 0.8-1.3 million hectares of
36
forests in China were treated annually with B. bassiana for control of numerous pests (Feng,
2003). In Africa Metarhizium anisopliae var. acridum 330189 (Deuteromycetes) which is
originally isolated from Africa used against desert locust and registered in many African
countries including Sudan (Elbashir and Bashir, 2008). Among these products 33.9%, were
based on B. bassiana, and (26.3%) among them were wettable powders (Faria et al., 2007).
Oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae), is a serious pest of a
wide range of fruit crops in the Indian subcontinent. On mango (Mangifera indica L.), it
causes enormous losses up to 80% (Jayanthi and Verghese, 2011).Waterhouse (1993)
reported that this pest is one of the three most important agricultural pests in South East Asia.
Numerous control methods are applied to suppress infestation of fruit fly in the field (Ekesi et
al., 2007), including use of mycoinsecticides products against various stages of the pest. Ortu
et al., (2009) reported that the effectiveness of the mycoinsecticide Naturalis-L based on the
B. bassiana strain ATCC 74040 against the C. capitata. Daniel and Wyss (2010) observed
65% reduction in Rhagoletis cerasi infested fruits by foliar applications of B. bassiana ATCC
74040 (Naturalis-L), under field conditions. Conidia of entomopathogenic fungi are strongly
hydrophobic and difficult to suspend in water. This feature prevents the suspension formation
in water. The isolate ITCC No. 6628 has been the best among three virulent isolates, having
LC50, 2.5x105. It has proved efficacy against adults of B. dorsalis (Elbashir et al, in press).
Therefore, the objective of this study was to develop a wettable powder formulation based on
this virulent isolate and carry out bioassays using newly developed biopesticide against three
stages of fruit fly B. dorsalis.
6.2. Materials and methods
6.2.1. Fungus culture
The fungal isolate ITCC No. 6628 used in these experiments was obtained from the Indian
Type Culture Collection, Division of Plant Pathology, Indian Agricultural Research Institute
New Delhi India. The fungus was cultured on potato carrot agar (PCA) in slant and kept in
refrigerator at 40C as a stock culture, then grown on potato dextrose agar (PDA) in Petri
dishes and maintained at ambient temperature (27±10C).
6.2.2. Mass culture of the fungus
The fungus was mass cultured on sorghum grains which had been washed and half-cooked by
boiling 200 g of wet sorghum in each autoclavable, transparent polyethylene bags, added to
that a pinch chloramphenicol as antibiotic. Then the sorghum was autoclaved according to
standard protocols, the material left for 24 hours before inoculation. Loop full of the fungus
37
from 15 days-old culture of the fungus was inoculated in each bag. The bags were plugged
with cotton and kept for one month at temperature 27±10C, 65±5 % R.H. and complete
darkness. Afterwards the conidia developed were washed off with 0.05% Criton X-100 in
conical flasks under laminar air flow. Then kept in 30 ml centrifuge tubes and centrifuged
(Sigma laboratory centrifuge 3K18), for five minutes at 50C temperature at 10000 RPM.
Then kept in -200C, after that they were lyophilised for 24hours till a fine powder was
obtained, then it was sieved through a very fine piece of cloth. The powder product thus
obtained was then added to the prepared autoclaved blank formulations, all the component of
which had been mixed properly, using mixer, for 1-2 minutes. To each five grams of blank
formulation (Pyrophylite and Talcum based products), 0.15 g of conidia was added. Then one
gram from each ready product was added to 3.5 ml sterilized distilled water to prepare the
stock solution, which was serially diluted to be easily quantified by haemocytometer. Finally
the products were calibrated to 1010
conidia/ml, using haemocytometer, then evaluated
against different stages of fruit flies. Small, well-cleaned perfume sprayer was used in case of
treatment of adults while the preimaginal stages were immersed, for 30 seconds in 0.5 ml and
1ml for third larval stage and pupae respectively.
6.2.3. Studies on different physico-chemical properties of carriers
A group of carriers were subjected to numerous tests (Table 6.1) gives general information on
these carriers) carried out as follows;
6.2.3.1. Bulk density
To determine the bulk density of the carriers before and after compaction a known volume
(cylinder 4.8 diameters cm x 1.95 cm height) was filled with the carrier before compaction,
then the carries occupied that volume was weighed. Then the carrier was kept again in same
volume and pressed till it get compacted, then it was weighed again. The volume values and
weighed value were calculated as per the following formula.
Bulk density (g/100cc) (before compaction):
Weight of the carrier x 100
Volume occupied by the same carrier before compaction
Bulk density (g/100cc) (after compaction):
Weight of the carrier x 100
38
Volume occupied by the same carrier after compaction
6.2.3.2. Particle size
The carriers were dry sieved through a 250 mesh sieve (aperture 105 μ) using manual shaking
and with help of a brush and tissue paper.
6.2.3.3. Reaction pH
One gram of each carrier was added to 10 ml of sterilized distilled water. Then the digital pH
meter (pHepR original) was used to check the pH of each carrier by keeping the probe of pH
meter in water then taking the reading. Then the instrument was washed in sterilize distilled
water and used again.
6.2.3.4. Sorptivity
Sorptivity (%) was determined by ASTM rubout method. To a known weight of carrier
commercially available linseed oil was added drop by drop through micropipette and worked
consistently by camel brush until the powder slipped freely from the tip of brush. The volume
of the linseed oil absorbed by the carrier was noted down and sorptivity was calculated as:
Sorptivity (%) =
Millilitre of linseed oil required to slip the material from spatula x 0.93 x 100
Weight of the carrier taken
6.2.3.5. Moisture content
Moisture content was determined by gravemetric method. A known weight of the carrier was
spread uniformly in Borosil® Petri dish and kept in microwave for 15 minutes. Then the
carriers were weighed again. Loss in weight was noted and pe rcent moisture content in the
carrier was calculated on microwave dry basis.
6.2.4. Preparation of blank formulation
An array of combination of different material were used viz., carriers (calcite, dolomite,
pyrophylite, soapsone and talcum powder). The binders used were acacia gum, carboxy
methyl cellulose and xanthan gum. Wetting agent was sodium lignosulphate and moisturizers
glycerol. Spreading agent were Tween 20, Teepol and Criton X-100. For general information
on adjuvants used refer (Table 6.2). The list of different recipes based on talcum powder is
presented in (Table 6. 3).
6.2.5. Physico-chemical properties of newly developed WP formulation
6.2.5.1. Wettability and suspensibility
39
One gram of the WP formulation was taken in aluminum foil. The powder was poured
rapidly and gently on the top of the surface of 100 ml water taken in to more than 1200 ml
capacity cup with internal diameter 4.3cm and height 10.3 cm. The time was measured with a
stop watch from the moment the powder was placed on the surface of the water until more
than 95% of the powder had become wet and submerged below the surface of the water. The
time (in seconds) was the wetting time of the formulation. The shorter the time required for
wetting the better was wettability of the formulation in addition the powder must remain
uniformly suspended in water
6.2.5.2. Particle size
The particle size of the newly developed WP formulation was reduced by milling and re-
milling.
6.2.5.3. Flowability
The flowability of newly developed WP formulation was normally determined with the help
of dusting appliances. However, in the absence of such an appliance it was determined by
visual observation.
6.2.5.4. Germination test
Viability of each product was determined by spread-plating 0.1 ml of product’s suspension
calibrated to 1x106 conidia /ml, on PDA plate with three replications. Then incubated in
complete darkness at temperature 27±10C relative humidity 65±5% for 24 hours then, using
cork borer round pieces of media were cut, kept in cavity slides and covered with cover slips
then observed and percentage germination was examined, from 100-conida under 40x.
6.2.6. Insect culture
Larvae of the Oriental fruit fly, B. dorsalis were obtained from Biological Control
Laboratory, Division of Entomology, Indian Agricultural Research Institute, New Delhi
(IARI). The larvae were reared on bananas whereas adult flies were maintained on sugar and
yeast autolysate (CDH Bioscience (P) Ltd. New Delhi -110002). They were kept in ventilated
acrylic cages (20x20x20 cm) at temperature 27±10C, relative humidity 65±5% and
photoperiod 12:12. Water was supplied in vials with cotton wicks.
40
6.2.7. Inoculation of insects
6.2.7.1. Adults
Adults aged four days were used for all bio assays, the insect were anesthetized in fridge for
5-7 minute. Then they were kept in Petri dishes (90x15mm) having filter paper in which they
were sprayed with 0.8-1 ml. Immediately treated insects were transferred to 21x15cm jars.
Three replications with 10 insects were maintained for each treatment as well as control. All
food source and condition are same as in rearing conditions. The insects which died during
the course of experiment were taken, surface sterilized using 2% sodium hypochlorite then
washed with three rinse of sterilized distilled water and kept in sterilized Petri dish in which
sterilized moisturized filter paper had been kept. The dishes were kept at temperature 27±10C
and relative humidity 65±5% in complete darkness for appearance of mycosis.
6.2.7.2. Inoculation of pre-imaginal stages
Thirty 24 hours old pupae or last larval stage were treated with concentration 1010
conidia/ml
of ITCC No. 6628 from each product. Thirty insects replicated thrice (10 insects), were
immersed in 2 ml of specified treatment for 30 seconds. Then transferred to sterilized Petri
dish containing moistened, autoclaved sand (17g of sand moistened with 3 ml sterilized
distilled water). The dishes were kept in B.O.D. at temperature 27±10C, relative humidity
65±5% and photoperiod 12:12.
6.2.8. Statistical analysis
Opstat statistical programme was used for analysis. In which the data were subjected to
square root transformation.
6.3. Results
Results of tests carried out on physic-chemical properties of the carriers are presented in
(Table 6.2)
6.3.1. Carrier reaction
The pH of the five carriers tested was determined. Result showed that pH values ranged from
6.7 to 8.8 (table.4) followed by dolomite (8.8) > calcite (8.6) > soapstone (8.5) > talcum
powder (7.7) > pyrophylite (6.7). Among the test carriers, dolomite was highly alkaline while
pyrophylite was acidic however closer to neutral which is preferred for biological agents such
as B. bassiana. So it could be used safely for preparation of formulation based on
entomopathogenic fungi.
41
6.3.2. Moisture content of newly developed formulations
Result of per cent moisture content of the five carriers tested in the present investigation
showed that those carriers have moisture content in the following descending order dolomite
(12.0) > pyrophylite (7.0) > soapstone ( 3.09) > talcum powder (2.6) > calcite (0.1). On the
basis of this result it was inferred that the calcite had less moisture content while dolomite is
had the highest moisture content. The low moisture contents indicated that the carrier is free
from anti-caking properties. However the moisture content alone was not the single factor
which determined the suitability of the carrier rather it was a combination of properties that
determined the suitability of a carrier. The caking phenomenon was obvious in case of
dolomite which was having highest moisture content. When it was mixed with other
formulation materials it had formed cake to muddy formulation. So it was excluded from
further evaluation.
6.3.3. Flowability
On the basis of visual observation it was found that flowability of all carriers was good.
Except dolomite and to some extent calcite, the rest of the carriers were of free-flowing
nature. Since this study was restricted to laboratory level, therefore real flowability test which
was supposed to be done with appliances was not carried out.
6.3.4. Bulk density
Bulk density values (g/100cc) of carriers before and after compaction were estimated (table 6.
4). The values were recorded in the following descending order dolomite (113.6, 175) >
calcite (89, 140) > soapstone (58.55, 108.45) > pyrophylite (54.7, 97.2) > talcum powder
(45.18, 79.8).
The bulk density before compaction for all tested carriers ranged from 45.18 to 113.6.
Whereas, their bulk density, after compaction ranged from 45 to 175. Dolomite was found to
be the most bulky carrier as the value was (113.6, 175). The lightest carrier registered was
Talcum powder as the value was (45.18, 79.8) followed by Pyrophylite (113.6, 175). So
based on this result it was concluded that Talcum powder was the best carrier as light carrier
provides more dispersibility.
6.3.5. Sorptivity
Sorptivity (%) of five carriers (w/w) was estimated based on the per cent sorption of linseed
oil. The values ranged from 9.3 to 19.06. Results of the sorptivity of the carriers tested are
reported in the following descending order, talcum powder (19.06) > soapstone (18.6) >
42
pyrophylite (18.6) > dolomite (9.3) > calcite (9.3). Results showed that percentage sorptivity
for talcum powder was highest indicating that it was better carrier followed by pyrophylite
(Table 6.4).
6.3.6. Moisture content of carriers
Result of percent moisture content of the five carriers (Table 6.4) tested indicated the five
carriers having 0% moisture content according to the microwave method used. In general the
low moisture content indicates that the carriers are free from anti-caking properties.
6.3.7. Particle size: The particle size of the five carriers was less than 150 mesh size (Table.
4)
6.3.8. Results of wettability and suspensibility for newly developed product
Results showed that the wettability of all products tested was very good. That was reflected
by the shorter time required for the product to be submerged in water where all the products
submerged in less than 30 seconds with slight variation among them. Moreover the
suspensibility was recorded for them based on the five carriers as descending order talcum
powder, pyrophylite, calcite, soapstone and dolomite.
6.3.3. Bio assay on all insect stage using three products (PA, PD and PG):
There were no significant differences between treatments of the product and control when the
insects were treated with concentration 1010
conidia/ml of all products (Table 6. 6)
6.4. Discussion
Dolomite failed to be selected for further due to cake formation which was actually observed
when it was mixed with other component where it formed not only cakey formulation but
muddy. Moreover it has high bulk density which was not preferred for dispersibility.
Moreover Dolomite had the highest pH amongst all the carriers tested, recording (8.8) which
was alkaline and not suitable for biological control agent such as B. bassiana. Besides,
sorptivity of this mineral was very less reporting only 9.3%. So this formulation with these
physicochemical properties was not eligible for further study. Therefore it was abandoned.
Second is calcite, though this carrier had 0% moisture content, but it ranked second, after
dolomite, in terms of bulk density which was (89, 140) before and after compaction
respectively. Moreover its pH was high (8.6) and harm biological control agents such as B.
bassiana. Regarding sorptivity this mineral recorded only 9.3%. Moreover the flowability of
dolomite and calcite were poor compared to rest of the carriers tested.; flowability of soap
stone was good compared to talcum and pyrophylite, having 0% moisture, failed in two
43
subsequent tests which were bulk density where its record was (58.55, 108.45) before and
after compaction respectively. Sorpitivity of Soapstone was 18.6%. However its pH was high
(8.5), which alkaline and it may affect B. bassiana negatively. So based on these tests it was
excluded from being tested further. The flowability of talcum powder was good, its moisture
content was 0% and did not form cake while mixing, the pH was 7.7 alkaline but more closer
to neutral which was preferred for biological control agents such as B. bassiana. Sorptivity of
this carrier was 18.6 %, recording least bulk density among all tested carriers which was
(45.18, 79.8) before and after compaction respectively. It is well known that the less the bulk
density the more preferred to be used as carrier because this feature was required while
application is made in the field. So based on these features and tests this carrier proved its
eligibility to be used in the formulation. Flowability of pyrophylite was good; its pH was the
least (6.7) among all carriers tested. Its bulk density was 54.7 and 97.2, before and after
compaction respectively. Moisture content was zero so the overall combination of features
enlisted this carrier for further tests. This carrier recorded the best sorpitivity among all
carrier tested which was 19.06. It is well known that the higher the sorptivity would make a
better carrier. Since the carriers viz calcite, dolomite and soapstone were not qualified enough
to be formulated they had been abandoned. Further tests were carried out using 18
formulations based on two carriers, viz., pyrophylite and talcum powder. To those 18
formulations active ingredient was added. However 10 out of those were tested for viability
of the fungus only three formulations based on pyrophylite registered germination above 90%
per cent (Table 6.5) so bioassay was carried out using those three products against three
stages of B. dorsalis. Results showed that the wettability of all products tested was very good.
All newly developed formulations had very good wettability; this reflected the best
performance of the wetting agent used that in its compatibility with the other components of
the formulation materials. The time required for the all formulations sampled five carriers
was less than 30 seconds. It is worth mentioning that dolomite based formulations were pellet
shape due to cake formation therefore it had settles at the bottom of the container and
released other materials. The suspensibility for all five formulation presented in descending
order was talcum powder, pyrophylite, calcite, soapstone and dolomite. It is worth mention
that again pyrophylite and talcum-based formulation were the best among all.
Bio assay of three products on adult stages of Bactrocera dorsalis
There were no significance differences between treatments of the product and control when
the insect were treated with concentration 1010
conidia/ml of all products (Table 6.6). It is
44
very important to keep in record that the viability of conidia in most three viable products
(PA, PD and PG) (Plate 6.1, 6.2 and 6.3 ) was higher than 90% which was confirmed by
viability test nevertheless there was no mortality on adult insect despite the same fungus was
virulent against same insect with same dose recording LC50 2.5x105. So this may raise a big
question about the effect of the products component on the virulence of the fungi, but not on
the viability which was confirmed. Therefore further research is required to investigate the
effect of formulation on the virulence of this fungus.
Preimaginal stages of Bactrocera dorsalis
The results of the three products PA, PD and PG on pre-imaginal were not significant
compared to control. When same isolate was tested in its unformulated form, using
immersion method, against preimaginal stage it was not ineffective totally. Since the insect
has been proven susceptible, therefore all the possible scenarios of failure are from the fungus
formulation side, all are attributed to formulation this categorized in several scenarios as
follows first a factor essential for the adhesion of the fungus to surface of the cuticle of insect
or the cuticle-degrading enzymes that actively destroy or modify structural integrity of the
host have been affected by the formulation ingredients. Because failure of either adhesion or
production of those enzymes would lead to failure by abortion of infection (Hajek and Leger,
1994) so this failure may be attributed to lack of factors responsible of attachment of conidia
to the insect integument or the production of enzymes responsible for causing the mortality.
This was because the germination percentage was more than 90% in the three products. And
the crude (unformulated) fungi were working and have registered highest mortality. Then the
hindrance of virulence must be caused by the formulation in a way or another.
45
GENERAL DISCUSION
Entomopathogenic fungi (EPF) in general and the genera Metarhizium and Beauveria in
particular are used worldwide for control of different insect pests including Dipterans.
Literature has proved that Beauveria bassiana (Balsamo) (Vuillemin) is cosmopolitan (EPF)
which have been exploited as microbial control agent since its discovery in 1835. Oriental
fruit fly Bactrocera dorsalis (Hendel) is a noxious insect pest of quarantine importance that
affects international trade. This pest is controlled with chemical insecticides some of them are
banned, while continuous deregistering process for the hazardous ones is going on. The
environmental concerns, public health problems, consumer awareness, economic feasibility
forced to look for other eco-friendly alternatives. Entomopathogenic fungi represent one of
those alternatives that can substitute hazardous insecticides. Recently control of different
species of fruit flies using entomopathogenic fungi mainly B. bassiana and Metarhizium
anisopliae (Metschn.) (Sorokin) is reported from different parts of the world. The
International Centre for Insect Physiology and Ecology represents the main hub of this kind
of work. Screening of several isolates took place there, which has lead to development of
formulations that proved its efficacy in the field. Moreover safety of virulent isolates was
proven against some important natural enemies besides proving performance far better than
chemical insecticides such as diazinon. In Asia it has been observed that, according to
published literature, absent to meagre work to exploitation of these entomopathogenic in the
IPM for management of fruit fly this may be due to high success of methyl eugenol and other
methods of combating this pest. Based on this literature and success worldwide using
entomopathogenic fungi for management of insect pests particularly fruit fly, we have been
inspired to screen some entomopathogenic fungi of Indian origin. With hope to get virulent
ones that can be used for development of mycoinsecticdes for the management of insect pests
with special reference to fruit fly. So eight isolates have been brought and tested against
several insect orders with special emphasis on fruit fly B. dorsalis. Preliminary screening was
conducted on Lepidopteran, Hemipteran insect pests whereas Coccienella septumpunctata
(L.) represented coleopteran as important natural enemy. However detailed work using
selected virulent entomopathogenic fungi was exclusively on different stage of fruit fly B.
dorsalis. Keeping into consideration that development of mycoinsecticide based on the
virulent isolates found by this study that be registered and commercialized, may require time.
Therefore commercial mycoinsecticides were tested, and if found effective, may be further
tested in the field for incorporation in IPM.
46
So in the present study, to begin with; preliminary screening was carried out, using eight
isolate of entomopathogenic fungi against adult of B. dorsalis.Three out of eight isolates were
found to be pathogenic to fruit flies, whereas the remaining five are not. It is worth
mentioning that 100% insects died within 4-6 days and the cadavers of adults were found
fully covered with white mycelium within nine days. This indicated that the inoculums
picked up, while the adults were walking on the open Petri dish was sufficient to kill the
insect within the mentioned time. The results are in accordance with that of Dimbi et al.,
(2003) who found adult mortality in Ceratitis capitata (Weidemann) and Ceratitis rosa var.
fasciventris (Karsch) treated with different isolates of B. bassiana were 7 to 100% at four
days post inoculation. Also Muñoz (2000) evaluated 16 strains of B. bassiana against C.
capitata adults and found mortality levels between 20.0 to 98.7%. Sookar et al., (2008)
evaluated five isolates of B. bassiana against adults of Bactrocera zonata (Saunders) and
Bactrocera cucurbitae (Coquillett). Mortality of B. zonata varied between 12.0 to 98.0% and
2.0 to 94.0% in B. cucurbitae at five days post-treatment. The eight isolates screened against
adult fruit fly except B. NCIPM were tested against larvae of Corcyra cephalonica
(Stainton). ITCC No. 6628, ITCC No. 6645 and M. NCIPM were virulent and registered
average mortality of 17, 19.6 and 3.4 respectively with significant difference among them.
This in conformity with Samodra and Ibrahim (2006) where they noted up to 90% larval
mortality of C. cephalonica within 15 days when treated with isolates of formulated B.
bassiana. The first two isolates were also pathogenic to adult fruit fly whereas the third, B.
NCIPM, which was effective against fruit fly, was not tested against C. cephalonica.
However M. NCIPM which was not effective against adult of fruit fly proved low
pathogenicity against C. cephalonica. Spilarctia obliqua (Walker) proved susceptible to two
isolates tested that is ITCC No. 6628 and ITCC No. 6645. Spodoptera litura (Fab.) was
susceptible to only three entomopathogenic fungi out of seven tested, while Drosicha
mangiferae (Green) was not susceptible to even three virulent ones. This may indicate the
specificity of entomopathogenic fungi which was reported by (Zimmermannn, 2007). It is
worth mentioning that the symptoms observed on cadavers of larvae of C. cephalonica,
treated with isolates ITCC No. 6645 and ITCC No. 6628, were growth of white mycelium on
the intersegmental regions, accompanied by pink to reddish colour with more coloration from
the isolate ITCC No. 6628. This coloration indicates secretion of a metabolite called
oosporein. These findings are in agreement with results reported by Zimmermannn (2007),
wherein he reported presence of a major secondary metabolite dihydroxy benzoquinone, an
extracellular secretion of many isolates of B. bassiana. The isolate B. NCIPM proved its
47
relative safety for C. septumpunctata registering only 7.6% average mortalities, compared to
ITCC No. 6628 and, ITCC No. 6645 which they recorded 20, 19% respectively. This in
accordance with Zimmermann, (2007) who reported B. bassiana isolates from the lady
beetle, Olla v-nigrum (Mulsant) , were pathogenic to adult O. v-nigrum but not Asian lady
beetle, Harmonia axyridis (Pallas), also The GHA strain of B. bassiana was not significantly
pathogenic to either O. v-nigrum or H. axyridis. Moreover physiological and ecological host
range plays an important role in pathogenicity. Regarding entomopathogenic fungi obtained
from culture collection, based on deposition date, it was observed that the recently deposited,
6-18 months, cultures were more virulent compared to those deposited three years or more.
Longer storage period or frequent sub-culturing followed, may have been the reason so
appropriate measures must be taken. This also may give an alarm that isolates which has been
proved to be pathogenic to fruit fly and other insect pests, if used later may not be virulent,
due to same above mentioned reason. That obviously leads to loss of biological wealth,
efforts and resources.
The three virulent isolates of EPF in addition to three commercial mycoinsecticides were
taken for further study against three stages of fruit fly B. dorsalis. First viability test was
carried out to check their viability where they had proved viability with more than 90%. The
three isolates of B. bassiana were found to be effective against the adult stage. The LC50
obtained for these isolates were 2.5x105, 1.2x10
9 and 7.5x10
6 for ITCC No. 6628, ITCC No.
6645 and B. NCIPM respectively. The cumulative mortality recorded, during 7 days; for the
treated adults with the different doses, revealed the dose response relationship. The short
period required for the fungi to kill the pest may of practical implications which reflect
reduction of the adult population before the first egg laying, a critical moment of insect
attack. These results are in agreement of results obtained by De La Rosa et al, (2002) where
he documented LC50 values as 5.13x105, 3.12x10
6, and 9.07 x10
6 conidia/ml strains of B.
bassiana against adults of Anastrepha ludens (Loew) under laboratory conditions. The
difference in virulence among the three isolates of B. bassiana tested may be due to
differences in the production of enzymes such as chitinase, chyemolestase, chymotrypsin, and
esterase which degrade the insect cuticle. These enzymes are considered an essential
prerequisite for successful fungal infection (De La Rosa et al., 2002). ITCC No. 6628
registered the least LC50 and evident its high virulence compared to other isolates. It is worth
mentioning that, same isolate, showed coloration of pink to reddish colour on cadavers of
larvae of C. cephalonica (Elbashir et al., in press). This coloration was thought to be due to
48
excretion of metabolite called oosporein. Red-coloured pigment is reported in literature as
dihydroxy benzoquinone which is the major secondary metabolite produced by Beauveria
brongniartii (Sacc ) and is also produced by many isolates of B. bassiana (Zimmermann,
2007). Production of this metabolite may indicate correlation between coloration and
virulence of this fungus. On cadavers of fruit fly, mycosis first appeared on the cavity of the
mouthparts and the ovipositor area. Fungal mycelium emerged from the soft parts of the
body, such as wing bases, mouthparts, and base of the legs, intersegmental regions, and
membranous regions of the abdomen, leg joints, and neck. Sensitivity and susceptibility of
these areas to fungal infection particularly the mouth parts would guide for practical
application to using mycoinsecticides. Regarding efficacy of commercial mycoinsecticides
against adults of fruit fly Bio-power®, Bio-magic
® and Bio-catch
® against adults of B.
dorsalis were 26.6%, 40%, 46.6% respectively. These results are in conformity with the
results obtained by several authors from different parts of the world. Mahmoud (2009)
reported 27.2, 18.4 and 34.4% mortality using same mycoinsecticides against adults of
Bactrocera oleae (Gmelin) (Dimbi et al., 2003) recorded mortality of between 70–93% when
evaluated M. anisopliae ICIPE 20 against C. capitata and, C. rosa var. fasciventris. Only the
isolate ITCC No. 6645 was tested against third instar larvae using prophylactic method,
treating soil and releasing larvae. In this case, though all larvae pupated normally, however
dose dependent mortality was proved with LC50 9x109..This high LC50 required, indicates low
virulence of the isolate used and raises a question about economic feasibility of using this
particular strain in treating preimaginal stages of this insect. However prophylactic method
using inoculation of the soil before releasing the last larvae was effective compared to
dipping method. Moreover this method is more valid in terms of its applicability; particularly
it simulates nature, such that, larvae drop from fruit into the soil where it encounters the
fungus-contaminated soil. Regarding preimaginal stages which were dipped either in three
fungi tested or the mycoinsecticides used showed no response to the treatment. This is due to
no virulence of the mycoinsecticides tested strains against these immature stages. This is in
conformity of De La Rosa et al., (2002), when he dipped preimaginal stages of A. ludens in
suspension of B. bassiana, mortality was 2-8% in larvae and 0% in pupae. Kaaya and Okech,
(1990) reported that no mortality in larvae and pupae of tse tse flies due to treatments by M.
anisopliae and B. bassiana. Beris et al. (2012) reported that average mortality of pupae of C.
capitata which had been immersed in suspension of B. bassiana ranged from 18.7 to 23.9 %.
On the other hand the results obtained contradicted results obtained by various authors
worldwide. Espin et al., (1989) applied M. anisopiliae directly to pupae of C. capitata and
49
observed that 65.8% of the pupae were infected with the fungus. Ekesi et al., (2002)
investigated the pathogenicity of 13 isolates of M. anisopliae and two isolates of B. bassiana
against C. capitata and C. var. rosa fasciventris exposed as late third instar larvae in sand,
under laboratory conditions. All isolates caused a significant reduction in adult emergence
and a corresponding large mortality on puparia of both species. Hajek and St. Leger (1994)
demonstrated that the low pathogenicity of many entomopathogenic fungi against many
insect species was due to the nature of the cuticle, in terms of its density and thickness and
the degree of sclerotization, among other factors. Moreover an infection can be aborted on the
epicuticle if a factor essential for a phase of adhesion, microbial development or pathogenesis
is absent. Also the failure of fungi to invade insect cuticle has been attributed to the presence
of inhibitory compounds (phenols, quinones and lipids) on the cuticle surface. Ferron (1981)
reported that the degree of sclerotization of the insect cuticle, the method of application of the
fungus, the life stage involved, are all factors that could affect the efficacy of fungal
pathogens to control insect pests.
Having selected the most virulent fungi, ITCC No. 6628, the next logical step would be
development of formulation based on this isolate. To do that it was decided the most
appropriate formulation type would be a wettable powder which can be applied for both
stages of fruit fly. To do that various carriers were selected on which an array of
physicochemical properties were conducted with the aim to select the one that would be
having mosaic of features that suits our biological control agent, B. bassiana and at the same
time to fulfil other necessary aspects for good formulation. First Dolomite failed to be
selected for further due cake formation which actually observed when it was mixed with
other component where it formed not only caky formulation but muddy. Moreover it has high
bulk density which is not preferred for dispersibility. Dolomite had the highest pH among all
the carriers tested, recording (8.8) which is alkaline that is not suitable for biological control
agent such as B. bassiana. Besides, sorptivity of this mineral is very less reporting only 9.3%
so this formulation with these physicochemical properties is not eligible for further study.
Therefore, it was abandoned. Second is Calcite, though this carrier is having 0% moisture
content, but it ranked second, after Dolomite, in term of bulk density which is (89, 140)
before and after compaction respectively moreover its pH is high which was (8.6) and may
harm biological control agents such as B. bassiana. Regarding sorptivity this mineral
recorded only 9.3%. Moreover the flowability of Dolomite and Calcite were poor compared
to rest of the carriers used. Thirdly, soap stone; flowability is good compare to Talcum and
50
Pyrophylite, having 0% moisture, however it failed in two subsequent tests which were bulk
density where its records was (58.55, 108.45) before and after compaction respectively.
Sorpitivity of Soapstone is 18.6%. However its pH was high recording 8.5 which alkaline and
it may affect B. bassiana negatively. So based on this mosaic of tests it was excluded from
being tested further. Fourthly, Talcum powder the flowability of it was good, its moisture
content was 0% which is not going to form cake while mixing, the pH was 7.7 alkaline but
more closer to neutral the later is preferred for biological control agents such as B. bassiana.
Sorptivity of this carrier was 18.6 %, moreover recording least bulk density among all tested
carriers which was (45.18, 79.8) before and after compaction respectively. It is well known
that the less the bulk density the more preferred to be used as carrier because this feature is
required while application in the field. So based on this mosaic of features and tests this
carrier had its eligibility to be used in the formulation. Finally the Pyrophylite carrier, the
flowability of this carrier was good its pH was the least recording 6.7 which acidic and the
closest, among all tested carriers to the neutral having bulk density (54.7, 97.2) before and
after compaction respectively. Moisture content for this carrier was zero so the overall
combination of features enlisted these carriers for further test. This carrier recorded the best
sorpitivity among all carrier tested which was 19.06. It is well known that the higher the
sorptivity the better the carrier. Since the carriers viz Calcite, Dolomite and Soapstone were
not qualified enough to be formulated they had been abandoned. Further tests were carried
out using 18 formulations based on two carriers, viz., Pyrophylite and Talcum. To those 18
formulations active ingredient was added. However 10 out of those were tested for viability
of the fungus. Only three formulations based on Pyrophylite given germination above 90%
percentage so bioassay was carried out using those three products against three stages of B.
dorsalis. Results showed that the wettability of all products tested was very good. All newly
developed formulation had very good wettability, this reflect the best performance of the
wetting agent used that in its compatibility with the other component of the formulation
materials. The time required for wetting the all formulations sampled by five carriers was less
than 30 seconds. It is worth mentioning that Dolomite based formulation were pellet shape
due to cake formation therefore it had gone to the bottom of the container and starts release
other materials. Among the five Pyrophylite and Talcum based formulation were the best in
wettability test. Despite viability of three newly developed formulation was above 90% but
they failed in their bioefficacy against all stages of fruit fly. There was no mortality on adult
insect despite the same fungus was virulent against same insect with same dose (1010
)
recording LC50 2.5x105. So this may raise a big question about the effect of the products
51
component on the virulence of the fungi, but not on the viability which was confirmed as it
was shown above. Therefore further research is required to investigate the effect of
formulation on the virulence of this fungus. The possible scenarios of failure are from the
fungus formulation side. First a factor essential for the adhesion of the fungus to surface of
the cuticle of insect or the cuticle-degrading enzymes that actively destroy or modify
structural integrity of the host have been affected by the formulation substances. Because
failure of either adhesion or production of those enzymes would lead to abortion of infection
(Hajek and Leger, 1994) so this failure may be attributed to lack of factors responsible of
attachment of spores to the insect integument or the production of enzymes responsible for
causing the mortality. According to peer reviewed literature this is the first report of
evaluation of pathogenicity of entomopathogenic fungi against fruit fly and C. cephalonica in
India. Since pathogenicity of these isolates screened by this study was proven preliminarily,
therefore further qualitative study should be carried out against the insects, apart from B.
dorsalis. Further study should be on fruit fly with the aim to incorporate this very important
component in IPM programmes for fruit flies in India in specific and Asia at large where this
component seems to be lacking.
Newly developed cages for fruit fly (Plastic jars)
The newly developed cages were low cost, the price of commercial cage is Rupees 600
whereas in case of these jars about Rupees 100- 120 that is only 20% of the price of
commercial available cages. Moreover this can be developed any time required whereas
commercial cages required time to be prepared by the company agents or person involved
which take ages and very killing bureaucracy and hinder the work and experiments keeping
in mind dealing with biological agents which needs synchronizations in lay out of
experiments arranging all requirments of equipment which is either unknown by the company
agents who look only for their profits margin ignoring the value of the work so they keep
delaying giving ever excuses. Keeping in mind the material used in preparation of these jars
is plastic so they are easily repairable compared to acrylic or other glass that is prone to be
broken. Expiry date or depreciation of the commercial would be faster than newly developed
cages. These jars can be developed further to have two rooms, in the bottom room would be
saturated sand with some small hole through which water can be supplied to maintain
moisture in the sand. The purpose of second room is to maintain humidity and temperature;
those can be measured with hygrometer and thermometer respectively. This in the case of
lack of power and advance equipment such as controlled champers. In case of the availability
52
of these controlled chamber the efficiency of use of this chambers would be doubled if we
consider that the B.O.D can accommodate double the numbers of commercial cages.
Moreover these cages can be redesigned to be smaller particularely in height; in that case it
would be more efficient and more economic. These jars may be used also for other flies
species whenever suitable and may be other insect species. They can be developed by lay
man; there is no need for sophisticated equipment or high tech.
Sprayers used
These sprayers can deliver 0.5 ml to 1 ml with high percisions. In the absence of Potter
Tower These sprayers can beused. They were cleaned with hexane followed by repeated
washes of detergent so as to remove the remains of chemical used that may affect the active
ingriedent, conidia of Beauveria bassiana.
53
SUMMARY AND CONCLUSION
According to literature the genera Metarhizium and Beauveria are cosmopolitan (EPF)
which have been exploited as microbial control agents against different species of fruit
flies worldwide.
Eight isolates of Beauveria bassiana and Metarhizium anisopliae have been provided by
Indian Agricultural Research Institute and National Centre for Integrated Pest
Management and tested against several insect orders with special emphasis on fruit fly
Bactrocera dorsalis.
Preliminary screening was conducted on lepidopteran, Hemipteran insect pests and
Coleopteran insect represented by Coccienella septumpunctata
The eight isolates of entomopathogenic fungi first screened preliminarily against adults of
B. dorsalis. ITCC No. 6628, ITCC No. 6645 and M. NCIPM out of eight isolates were
found to be pathogenic to fruit flies, whereas the remaining five were not.
All adult fruit flies died within 4-6 days and the cadavers were found fully covered with
white mycelium within nine days.
Contact method proved its efficacy, by exposing the adults by walking on the open Petri
dish.
Eight isolates screened against adult fruit fly revealed that ITCC No. 6628, ITCC No.
6645 and M. NCIPM were virulent and registered average mortality of 17, 19.6 and 3.4
respectively.
M. NCIPM which was not effective against adult fruit fly exhibited pathogenicity against
Corcyra cephalonica.
Spilarctia obliqua was found to be susceptible to two isolates (ITCCNo. 6628, and ITCC
No. 6645) tested.
Spodoptera litura was susceptible to only three entomopathogenic fungi out of seven
tested. Drosicha mangiferae was not succsptible to even three virulent ones. This may
indicate the specificity of entomopathogenic fungi which was reported in literature.
54
The symptoms observed on cadavers of larvae of C. cephalonica, treated with isolates
ITCC No. 6645 and ITCC No. 6628, were growth of white mycelium on the
intersegmental regions, accompanied by pink to reddish colour with more coloration from
the isolate ITCC No. 6628. This coloration was thought to be a metabolite called
oosporein, a red-coloured pigment chemically known as dihydroxy benzoquinone.
The isolate B. NCIPM proved its relative safety for C. septumpunctata registering only
7.6 average mortalities, compared to ITCC No. 6628 and ITCC No. 6645 which they
recorded 20 and 19% respectively.
It was observed that the recently deposited entomopathogenic fungus i.e., 6-18 months,
in culture collection were more virulent compared to those deposited three years or more.
Longer storage period or frequent sub-culturing followed, may have been the reason. This
also may give an alarm that isolates which has been proved to be pathogenic to fruit fly
and other insect pests, if used later may not be virulent, due to same above mentioned
reason.
The LC50 obtained for B. bassiana isolates viz., ITCC No. 6628, ITCC No. 6645 and B.
NCIPM were 2.5x105, 1.2x10
9 and 7.5x10
6 respectively.
The cumulative mortality recorded, during 7 days; for the treated adults shows that a short
period is required for the fungi to kill the pest.
The difference in virulence among the three isolates of B. bassiana tested may be due to
differences in the production of enzymes such as chitinase, chyemolestase, chymotrypsin,
and esterase which degrade the insect cuticle. ITCC No. 6628 registered the least LC50
and was the most virulent isolate tested. This may be correlated with red pigment
observed on larvae of Corcyra cephalonica.
On cadavers of fruit fly, mycosis first appeared on the cavity of the mouthparts and the
ovipositor area. Fungal mycelium emerged from the soft parts of the body, such as wing
bases, mouthparts, and base of the legs, intersegmental regions, and membranous regions
of the abdomen, leg joints, and neck. Sensitivity and susceptibility of these areas to fungal
infection particularly the mouth parts would guide for practical application to using
mycoinsecticides.
55
Efficacy of commercial mycoinsecticides against adults of fruit fly Bio-power®, Bio-
magic® and Bio-catch
® against adults of B. dorsalis were 26.6%, 40%, 46.6%
respectively.
The isolate ITCC No. 6645 was tested against third instar larvae using prophylactic
method, treating soil and releasing larvae. In this case, though all larvae pupated
normally, however dose dependent mortality was proved with LC50 9 x 109.
Prophylactic method using inoculation of the soil before releasing the last larvae was
effective compared to dipping method.
Preimaginal stages, dipped separately in three fungi tested or the mycoinsecticides used
showed no response to the treatment. This was due to no virulence of the
mycoinsecticides tested strains against these immature stages.
ITCC No. 6628 the most effective isolate was selected as an active ingredient for
development of wettable powder formulation was mass produced on sorghum grains.
Various carriers were selected on which an array of physicochemical properties were
conducted with the aim to select the one that would be having mosaic of features that
suits our biological control agent, B. bassiana .
First Dolomite failed to be selected for further due to cake formation which actually
observed when it was mixed with other component where it became not only caky
formulation but muddy also.
Dolomite has high bulk density which is not preferred for dispersibility. Moreover had the
highest pH among the all carriers tested, recording (8.8) which is alkaline that is not
suitable for biological control agent such as B. bassiana.
Sorptivity of this mineral is very less reporting only 9.3% so this formulation with these
physicochemical properties is not eligible for further study.
Second was Calcite; though this carrier had 0% moisture content, but it ranked second
after Dolomite, in term of bulk density which it was (89, 140) before and after
compaction respectively moreover its pH was high (8.6) and would harm biological
control agents such as B. bassiana. Sorptivity of this mineral recorded only 9.3%.
56
Flowability of both carriers Dolomite and Calcite was poor compared to rest of the
carriers used.
Thirdly, soap stone; flowability was good compared to Talcum and Pyrophylite, having
0% moisture, however it failed in two subsequent tests viz., bulk density where it
recorded 58.55 and 108.45 before and after compaction respectively. Sorpitivity of
Soapstone was 18.6%. However its pH was high (8.5) which would affect B. bassiana
negatively. So based on this mosaic of tests it was excluded from being tested further.
Fourthly, Talcum powder the flowability of it was good, its moisture content was 0%
and did not form cake while mixing, the pH was 7.7, closer to neutral the later is preferred
for biological control agents such as B. bassiana. Sorptivity of this carrier was 18.6%,
moreover it registered least bulk density among all tested carriers i.e., 45.18 and 79.8
before and after compaction respectively.
Finally the Pyrophylite carrier, the flowability of this carrier was good its pH was the
least (6.7) which acidic and the closest, among all tested carriers to the neutral had bulk
density (54.7, 97.2) before and after compaction respectively. Moisture content for this
carrier was zero so the overall combination of features enlisted this carrier for further
tests. This carrier recorded the best sorpitivity among all carrier tested which was 19.06.
Further tests were carried out using 18 formulations based on two carriers, viz.,
Pyrophylite and Talcum powder. To those 18 formulations active ingredient was added.
However 10 out of those were tested for viability of the fungus. Only three formulations
based on Pyrophylite registered germination above 90% percentage so bioassay was
carried out using those three products against three stages of B. dorsalis.
Results showed that the wettability of all products tested was very good. The time
required for wetting of the all formulations sampled by five carriers was less than 30
seconds. It is worth mentioning that Dolomite based formulation were pellet in shape due
to cake formation therefore it had gone to the bottom of the container and released other
materials. Among the five Pyrophylite and Talcum powder -based formulations were the
best in wettability test. The viability of B. bassiana in three newly developed
formulations was above 90% but they failed in their bioefficacy against all stages of fruit
fly.
57
ABSTRACT
Eight isolates of Beauveria bassiana and Metarhizium anisopliae had been obtained from
Indian Agricultural Research Institute- New Delhi and National Centre for Integrated Pest
Management New Delhi. They were tested against several insect orders with special
emphasis on fruit fly Bactrocera dorsalis. Preliminary screening was conducted using
contact method, by exposing the insects to inoculums in a lid removed Petri dish. The
tested insect represented different insect orders viz, Lepidopteran, Hemipteran, Dipteran
insect pests and Coleopteran insect was represented by Coccienella septumpunctata.
ITCC No. 6628, ITCC No. 6645 and B. NCIPM were found to be pathogenic to adult
fruit flies; B. dorsalis registered 100% mortality within 4-6 days. While ITCC No. 6628,
ITCC No. 6645 and M. NCIPM were virulent against larvae of Corcyra cephalonica and
registered 17, 19.6 and 3.4% average mortality respectively. Spilarctia obliqua proved
susceptible to two isolates tested that ITCC No. 6628, and ITCC No. 6645. Spodoptera
litura was susceptible to only three entomopathogenic fungi out of seven tested. Drosicha
mangiferae was not succsptible to three virulent ones. Cadavers of adults fruit flies were
found fully covered with white mycelium within nine days. The symptoms observed on
larvae cadavers of C. cephalonica, treated with isolates ITCC No. 6645 and ITCC No.
6628 were, growth of white mycelium on the intersegmental regions, accompanied by
pink to reddish colour with more coloration from the isolate ITCC No. 6628. The isolate
B. NCIPM proved its relative safety for C. septumpunctata registerd only 7.6% average
mortality. In subsequent detailed tests the LC50 obtained for these isolates were 2.5x105,
1.2x109
and 7.5x106
for ITCC No. 6628, ITCC No. 6645 and B. NCIPM respectively
against the adults of B. dorsalis. The difference in virulence among the three isolates of B.
bassiana tested was thought to be due to differences in the production of enzymes such as
chitinase, chyemolestase, chymotrypsin, and esterase which degrade the insect cuticle.
ITCC No. 6628 registered the least LC50 and indicated its high virulence among the
virulent isolate tested. This may be correlated with red pigment observed on larvae of C.
cephalonica. Efficacy of commercial mycoinsecticides Bio-power®, Bio-magic
® and Bio-
catch® against adults of B. dorsalis were 26.6%, 40%, 46.6% respectively. The isolate
ITCC No. 6645 was tested against third instars larvae using prophylactic method, treating
soil and releasing larvae. In this case, though all larvae pupated normally, however dose
dependent mortality was proved with LC50 9x109. Preimaginal stages, dipped either in
three fungi tested or the mycoinsecticides used showed no response to the treatment. This
58
is due to no virulence of the mycoinsecticides tested strains against these immature
stages. Since isolate ITCC No. 6628 proved to be the most effective isolate with least
LC50, it has been selected as an active ingredient for development of wettable powder
formulation. So it has been mass produced on sorghum grains. Physicochemical
properties of five carriers were tested for their suitability for B. bassiana. Dolomite had
formed cakey formulation, has had highest bulk density, highest pH while sorptivity was
low, 9.3%. Calcite had 0% moisture content high bulk density, alkaline and sorptivity of
this mineral recorded only 9.3%. Flowability of carriers, Dolomite and Calcite was poor
compared to rest of the carriers used. Flowability of soap stone was good, its moisture
content was 0%, bulk density was relatively high. Sorpitivity of Soapstone is good 18.6%.
However its pH was high recorded 8.5 which is alkaline. So based on this mosaic of tests
it was excluded from being tested further. Flowability of Talcum powder was
comparatively good, its moisture content was 0%, and the pH was 7.7. Sorptivity of this
carrier was high having least bulk density among all tested carriers. The flowability of the
last carrier, Pyrophylite, was good; its pH was the least recording 6.7 which is acidic and
the closest, among all tested carriers to the neutral having less bulk density. Moisture
content for this carrier was zero and also recorded the best sorptivity among all carriers
tested which was 19.06%. Wettability of all products tested was very good it took less
than 30 seconds. Eighteen formulations, based on Talcum powder and Pyrophylite were
tested for viability of the fungus. Only three formulations based on Pyrophylite given
germination above 90%, so bioassay was carried out using those three products against
three stages of B. dorsalis. Despite high viability of spores of Beauveria bassiana, in
these three formulations, but they failed in their bioefficacy against all stages of fruit fly.
59
Hkkjrh; d`f"k vuqla/kku laLFkku ubZ fnYyh ,oa lesfdr ihM+d izca/ku gsrq jk"Vªh; dsUnz] ubZ fnYyh ls csosfj;k
cSfl;kuk ,oa esVkjkbft+;e ,uvkblksIyh ds vkB foyx izkIr fd, x,A dhVksa ds dbZ x.kksa] fo'ks"k :Ik ls
Qy&ef{kdk cSDVªkslsjk Mkslsfyl ds fo:) mudk ijh{k.k fd;k x;kA vkjafHkd fofoDrdj fujh{k.k esa lEidZ fof/k
dk mi;ksx fd;k x;k ftlesa fcuk <Ddu okyh iSVªks IysV esa dhVksa dks fuos'knzO;ksa ds lEidZ esa vkus fn;k x;kA
ijh{k.k fd, x, dhV] fofHkUu dhV&x.kksa dk izfrfuf/kRo djrs Fks ;Fkk] ysfi;ksIVsju] gsehIVsju] dhV ihM+d rFkk
dksfy;ksIVsju dhVksa dk izfrfuf/kRo djus okyk dkWDlhusyk lsIVeiaDVsVk FkkA o;Ld Qy&ef{kdk] cS-MkslsZfyl gsrq
vkbZVhlhlh ua- 6628] vkbZVhlhlh ua- 6645 ,oa ach,ulhvkbZih,e jksxtud ik, x, vkSj 4&6 fnu ds Hkhrj budh
100% eR;Zrk ns[kh xbZ tcfd vkbZVhlhlh ua- 6628] vkbZVhlhlh ua- 6625 ,oa ,e,ulhvkbZih,e dkslkZ;jk flQsyksfudk
ds ykoksaZ ds fo:) mxz ik, x, vkSj buds }kjk dze'k% 17] 19-6 ,oa 3-4 vkSlr eR;Zrk jsdkMZ dh xbZA
LikbZykdZf'k;k vkWCyhDok] ijh{k.k fd, x, nks foyxksa] vkbZVhlh ua- 6628 ,oa vkbZVhlhlh ua- 6645 ds izfr lqxzkgh
ik;k x;kA LiksMksIVsjk fyV~;wjk] ijh{k.k fd, x, lkr dhVksa esa jksxtud dodksa esa ls dsoy rhu ds izfr lqxzkgh FkkA
rhu mxz jksxtudksa ds izfr Mªksflpk esathQsjh lqxzkgh ugha FkkA ukS fnu ds Hkhrj o;Ld Qy&ef{kdkvksa ds e`r 'ko
lQsn dodtky ls iw.kZr;k <d ik, x,A vkbZVhlhlh ua- 6646 ,oa vkbZVhlhlh ua- 6628 foyxksa ls mipkfjr dks-
flQsyksfudk ds ykoksZa ds e`r'koksa ij bl izdkj ds y{k.k ns[ks x, & varj[kaM {ks=ksa ij lQsn dod tky dh of̀)
ftlesa lkFk gh xqykch ls ysdj ykfyek fy, jax Hkh Fkk vkSj ;g jax] foyx vkbZVhlhlh ua- 6628 esa vf/kd FkkA dkW-
lsIVeiaDVsVk gsrq foyx ch,ulhvkbZih,e vis{kkd`r de ykfudkjd Fkk vkSj blds lkFk vkSlr eR;Zrk dsoy 7-6 FkhA
rRi'pkr fd, x, foLr`r ijh{k.kksa esa bu foyxksa] vkbZVhlhlh ua- 6682] vkbZVhlhlh ua- 6645 ,o ach,ulhvkbZih,e
gsrq cS-MkslsZfyl ds o;Ldksa ds fo:) izkIr ,ylh50 eku dze'k% 2-5x105] 1-2x10
9 ,oa 7-5x10
6 FksA ijh{k.k fd, x,
cs- cSfl;kuk ds rhuksa foyxksa dh mxzrk esa vUrj dk dkj.k] dhV dh D;wfVd irZ dk fuEuhdj.k djus okys ,Utk;eksa
;Fkk] dkbfVust] dkbZeksysLVst] dk;eksfVªfIlu ,oa ,LVjst ds mRiknu esa fHkUurk ds dkj.k le>k x;kA vkbZVhlhlh
ua- 6628 ls U;wure ,ylh50 eku jsdkMZ fd;k x;k tks bl ckr dks n'kkZrk gS fd ijh{k.k fd, x, mxz foyxksa esa
budh mxzrk vf/kd FkhA budk laca/k dks-flQsyksfudk ds ykoksZa ds Åij ns[ks x, yky o.kZd ls gks ldrk gSA 'osr
ef{kdk] cS-MkslsZfyl ds o;Ldksa ds fo:) okf.kfT;d doddhVukf'k;ksa] ck;ks&ikojvkj
] ck;ks&eSftdvkj
,oa
ck;ks&dSpvkj
dh izHkkfork dze'k% 26-6%] 40% ,oa 46-6% FkhA r`rh; bUlVkj ykoksZa ds fo:)] foyx vkbZVhlhlh ua-
6645 dk ijh{k.k] fujks/kksipkj fof/k dk mi;ksx dj fd;k x;k ftlesa e`nk dks mipkfjr dj ykoksZa dks NksM+k tkrk
gSA bl iz;ksx esa ;|fi lHkh ykosZ lkekU; :Ik ls I;wiksa esa :ikarfjr gq, fdUrq ,ylh50 9x109 ds lkFk
[kqjkd&vk/kkfjr eR;Zrk ns[kh xbZA ijh{k.k fd, x, rhuksa dodksa esa vo;Ld voLFkkvksa dks Mqcksus vFkok iz;qDr
doddhVukf'k;ksa us mipkj ds izfr dksbZ vuqfdz;k ughas n'kkZ;hA ,slk] ijh{k.k fd, x, doddhVuk'kh foHksnksa dh bu
60
vo;Ld voLFkkvksa ds fo:) mxzrk u gksus ds dkj.k FkkA pwafd foyx vkbZVhlhlh ua- 6628] U;wure ,ylh50 lfgr
lokZf/kd izHkkoh foyx ik;k x;k blfy, oSVscy ikmMj QkewZys'ku ds fodkl gsrq lfdz; ?kVd ds :Ik esa budk
oj.k fd;k x;kA bldk vf/kd ek=k esa mRiknu] lksj?ke ds nkuksa ij fd;k x;kA cs-cSfl;kuk gsrq mudh mi;qDrrk
ds ijh{k.kkFkZ ikap dSfj;lZ ds HkkSfrd&jklk;fud xq.kksa dk ijh{k.k fd;k x;kA MksyksekbV ls dsd ds :Ik esa
QkeZwys'ku fufeZr gqbZ] budk LFkwy ?kuRo vf/kdre Fkk] ih,p eku Hkh vf/kdre Fkk tcfd 'kks"k.k ;ksX;rk de ¼9-3%½
FkhA dSYlkbV dk ueh va'k 0% Fkk] LFkwy ?kuRo vf/kd ;k {kkjh; Fkk vkSj bl [kfut dh 'kks"k.k;ksX;rk ek= 9-3%
jsdkMZ dh xbZA MksyksekbV ,oa dSYlkbV dh izokg ;ksX;rk] iz;ksx fd, x, 'ks"k dSfj;lZ dh rqyuk esa de FkhA
lksiLVksu dh izokg ;ksX;rk vPNh Fkh] budk ueh va'k 0% Fkk] LFkwy ?kuRo vis{kkd`r vf/kd Fkk rFkk 'kks"k.k;ksX;rk
vPNh ¼18-6%½ FkhA oSls bldk ih,p eku vf/kd jsdkMZ fd;k x;k tks 8-5 gksus ds dkj.k {kkjh; FkkA bl dkj.k ls
vkxs bldk ijh{k.k ugha fd;k x;kA VsYde ikmMj dh izokg;ksX;rk vis{kkd`r mRre Fkh] bldk ueh va'k 0% Fkk ,oa
ih,p eku 7-7 FkkA bl dSfj;j dh 'kks"k.k;ksX;rk vf/kd Fkh rFkk ijh{k.k fd, x, lHkh dSfj;lZ dh rqyuk esa bldk
LFkwy ?kuRo U;wure FkkA ,d vU; dSfj;j] ik;jksQkbykbV dh izokg ;ksX;rk mRre Fkh] bldk ih,p eku 6-7 jsdkMZ
fd;k x;k tks vEyh; gksrs gq, Hkh ijh{k.k fd, x, lHkh dSfj;lZ dh rqyuk esa mnklhu ds fudVre Fkk rFkk bldk
LFkwy ?kuRo de FkkA bl dSfj;j dk ueh&va'k 'kwU; Fkk rFkk bldh 'kks"k.k ;ksX;rk 19-06 jsdkMZ dh xbZ tks
ijh{k.k fd, x, lHkh dSfj;lZ dh rqyuk esa loZJs"B FkhA ijh{k.k fd, x, lHkh mRiknksa dh oSVsfcfyVh cgqr vPNh Fkh
vkSj blesa 30 lSdsM ls Hkh de le; yxkA VSYde ikmMj ,oa ik;jksQk;ykbV ij vk/kkfjr vBkjg QkeZwys'kuksa dk
dod dh thou{kerk gqrq ijh{k.k fd;k x;kA dsoy ik;jksQkbykbV ij vk/kkfjr rhu QkewZys'kuksa ls 90% ls vf/kd
vadqj.k ik;k x;k blfy, bu rhuksa mRiknksa dk mi;ksx dj cS-MkslsZfyl dh rhuksa voLFkkuksa ds fo:) tSovkekiu
fd;k x;kA ;|fi cs-cSfl;kuk dod ds chtk.kqvksa dk vadqj.k 90% ls vf/kd gqvk fdUrq 'osr ef{kdk cS-MkslsZfyl dk
rhuksa voLFkkvksa ds fo:) mudh tSo&izHkkfork ugha ik;h xbZA
61
Bibliography
Ali, A., Sermann, H., Lerche, S. and Büttner, C. (2009). Soil application of Beauveria
bassiana to control Ceratitis capitata in semi field conditions. Commun. Agric. Appl.
Biol. Sci. 74(2):357-361.
Ali, S., Huang, Z. and Ren, S.X. (2010). Production of cuticle degrading enzymes by Isaria
fumosorosea and their evaluation as a biological agent against diamondback moth. J.
Pest. Sci., 83(4): 361-370.
Allwood, A.J., Chinajariyawong, R.A.I., Drew, E.L., Hamacek, D.L., Hancock, C.,
Hengsawad, J.C., Jipanin, M., Jirasurat, C., Kong Krong, S., Kritsaeneepaiboon,
C.T.S., Leong, and S. Vijaysegaran. (1999). Host plant records for fruit flies
(Diptera: Tephritidae) in South East Asia. Raffles Bull. Zool. Suppl. 7: 1–92. Cross
reference .
Alves, S.B. (1998). Fungos entomopatogeˆnicos. In: Alves, S.B. (Ed.), Controle Microbiano
de Insetos. FEALQ, Piracicaba, pp. 289–381. Cross reference.
Almeida, J.E.M., Filho, A.B., Fernanda, C., Oliveira, E. and Raga, A. (2007). Pathogenicity
of the entomopathogenic fungi and nematode on Medfly Ceratitis capitata (Wied.)
(Diptera: Tephritidae). Bio Assay: 2-7.
Amala, U., Jiji, T., Naseema, T., Jacob, Arthur and Sheela, M.S. (2010). Effect of Soil
Application of Paecilomyces lilacinus (Thom) Samson in controlling Melon Fly
Bactrocera cucurbitae Coq. 8th Intl. Symp on Fruit Flies of Economic Importance.
Valencia.
Beris, E. I., Papachristos, D.P., Fytrou, A., Antonatos, S.A. and Kontodimas, D.C. (2012).
Pathogenicity of three entomopathogenic fungi on pupae and adults of the
Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae). J. Pest. Sci. 86(2):
275- 284.
Booth, S.R. and Shanks, J.R. (1998). Potential of a dried rice/mycelium formulation of
entomopathogenic fungi to suppress subterranean pests in small fruits. Biocontrol
Sci. Technol. 8(2): 197- 206.
Burges, H.D. and Hussey, H.W. (1971). Microbial control of insects and mites. In Control of
insect pests and weeds, with special reference to developing countries. FAO Plant
Protect. Bull. 39: 155-181.
Burges, H.D. (1998). Formulation of Microbial Pesticides, Beneficial Microorganisms,
Nematodes and Seed Treatments. Kluwer Academic, Dordrecht, 412pp.
62
Butt, T.M., Jackson, C.W. and Magan, N. (2001). Introduction - fungi as biological control
agents: progress, problems and potential. In: Butt, T.M., Jackson, C.W. and Magan,
N. (Eds.) Fungi as Bio-control Agents: progress, problems and potential. 1-8. CABI
International, Wallingford, United 390pp.
Campos, C.S.E. (2000). Seleccion de cepas de Metarhizium anisopliae (Metsch) Sorokin
virulent as a la moscamexicana de la fruta, Anastrepha ludens (Loew) econdiciones
de laboratorio. Tesis de Licenciatura. Facultad de Ciencias Agricolas.Univ. Aut.de
Chiapas.Huehuetan, Chis., Mexico.60 p. Cross reference.
Castillo, M.A., Moya, P. and Primo-Yufera, E. (1999). Laboratory evaluation of
entomopathogenic fungi for the control of Ceratitis capitata Weidemann
(Dipt.:Tephritidae). Effects on fecundity and fertility, in Abstracts, Int. Symp,
Biological Control Agents in Crop and Animal Production, 24th- 28th August, 1999,
University of Wales, Swansea, p. 13.
Castillo, M.A., Moya, P., Hernandez, E. and Primo-Yufera, E. (2000). Susceptibility of
Ceratitis capitata Wiedemann (Diptera: Tephritidae) to entomopathogenic Fungi and
Their Extracts. Biol. Control. 19(3): 274-282.
Cossentine, J., Thistlewood, H., Goettel, M. and Jaronski, S. (2010). Susceptibility of
preimaginal western cherry fruit fly, Rhagoletis indifference (Diptera: Tephritidae) to
Beauveria bassiana (Balsamo) Vuillemin Clavicipitaceae (Hypocreales). J.
Invertebr. Pathol. 104(2): 105-109.
Croft, B.A. (1990). Arthropod Biological Control Agents and Pesticides, (eds). Wiley-
Interscience, New York, 723pp.
Daniel, C. (2008). Entomopathogenic fungi as a new strategy to control the European cherry
fruit fly Rhagoletis cerasi Loew (Diptera: Tephritidae). Ph.D. Thesis, Technische
Universität München Fachgebiet Obstbau, 171pp.
Daniel, C. and Grunder, J. (2012). Integrated management of European cherry fruit fly
Rhagoletis cerasi (L.): Situation in Switzerland and Europe. Insects. 3(4): 956-988.
Daniel, C. and Wyss, E. (2010). Field applications of Beauveria bassiana to control the
European Cherry Fruit Fly Rhagoletis cerasi. J. Appl. Entomol. 134(9-10): 675-681.
Daniel, C. and Wyss, E. (2009). Susceptibility of different life stages of the European cherry
fruit fly, Rhagoletis cerasi, to entomopathogenic fungi. J. Appl. Entomol. 133(6):
473-483.
63
De La Rosa, W., Lopez, F.L. and Liedo, P. (2002). Beauveria bassiana as a pathogen of the
Mexican Fruit Fly (Diptera: Tephritidae), under laboratory conditions. J. Econ.
Entomol., 95(1): 36-43.
Díaz-Ordaz, N.H., Pérez, N. and Toledo, J. (2010). Patogenicidad De Tres Cepas De Hongos
EntomopatógenosA Aadultos De Anastrepha oblique (Macquart) (Diptera:
Tephritidae) En Condiciones De Laboratorio. Acta Zoológ. Mexic. (n.s.) 26(3): 481-
494.
Dimbi, S., Maniana, N. and Ekesi, S. (2013). Horizontal Transmission of Metarhizium
anisopliae in fruit flies and effect of fungal Infection on Egg Laying and Fertility.
Insects, 4(2): 206-216.
Dimbi, S., Maniania, N.K. and Ekesi .S. (2009). Effect of Metarhizium anisopliae inoculation
on the mating behavior of three species of African Tephritid fruit flies, Ceratitis
capitata, Ceratitis cosyra and Ceratitis fasciventris. Biol. Control, 50(2): 111-116.
Dimbi, S., Maniania, N.K., Lux, A.S., Ekesi, S. and Mueke, K.J. (2003). Pathogenicity of
Metarhizium anisopliae (Metsch.) Sorokin and Beauveria bassiana (Balsamo)
Vuillemin, to three adult fruit fly species: Ceratitis capitata (Wiedemann), C. rosa
var.fasciventris Karsch and C. cosyra (Walker) (Diptera: Tephritidae).
Mycopathologia. 156(4): 375-382.
Du toit, W.J. (1998). Fruit Flies. In: E.C.G. Bedford, M.A. Van den Berg and E.A. de Villiers
(Eds.) Citrus Pests in the Republic of South Africa. 229-233. Dynamic Ad: Nelspruit,
South Africa. Cross reference.
Ekesi, S., Dimbi, S. and Maniania, N.K. (2007). The role of entomopathogenic fungi in the
integrated management of fruit flies (Diptera: Tephritidae) with emphasis on species
occurring in Africa. In: S. Ekesi and N.K. Maniania (Eds.) Use of entomopathogenic
fungi in biological pest management. Research Signpost, 37/661 (2): 239-274.
Ekesi, S., Maniania, N.K. and Lux, S.A. (2002). Mortality in three African tephritid fruit fly
puparia and adults caused by the entomopathogenic fungi, Metarhizium anisopliae
and Beauveria bassiana. Bio-control Sci. Technol. 12(1): 7-17.
Ekesi, S., Maniania, N.K., Mohamed, S.A. and Lux. S.A. (2005). Effect of soil application of
different formulations of Metarhizium anisopliae on African tephritid fruit flies and
their associated endoparasitoids. Biol. Control 35(1): 83-91.
Ekesi, S., Mohamed, S. and Hanna, R. (2010). Rid fruits and vegetables in Africa of
notorious fruit flies, Techn Innova Bri. No. 4 June 2010.
64
Elbashir, M.I. and Bashir, M.O. (2008). Effect of Desert Locust adult aggregation pheromone
alone, and integrated with fractional doses of Metarhizium anisopliae var acridium
on some non-target arthropods in Red Sea Coast. M. Sc. Thesis, Sudan Academy of
Sciences, 181pp.
Elbashir, M.I., Paul B., Shankarganesh, K., Gautam, R.D. and Sharma, P. (2013).
Pathogenicity of Indian Isolates of Entomopathogenic fungi against important insect
pests and natural enemies. Indian. J. Entomol. in press.
Espin, G.A.T., Laghi de Suza H.M., Messias, C.L. and Piedrabuena A.E. (1989):
Patogenicidad de Metarhizum anisopilae nas diferentesfases de desenvolvimento de
Ceratitis capitata (Wied.) (Diptera: Tephritidae). Rev. Bras. Entomologia, 33: 17-23.
Cross reference.
Faria, M. R. de. and Wraight, .S.P. (2007). Mycoinsecticides and Mycoacaricides: A
comprehensive list with worldwide coverage and international classification of
formulation types. Biol. Control. 43(3): 237-256.
Feng, M.G., (2003). Microbial control of insect pests with entomopathogenic fungi in China:
a decade’s progress in research and utilization. In: Upadhyay, R.K. (Ed.), Advances
in Microbial Control of Insect Pests. Kluwer Academic, New York, NY, 213-234,pp.
Feng, M.G., Poprawski, T.J. and Khachatourians, G.C. (1994). Production, formulation and
application of the entomopathogenic fungus Beauveria bassiana for insect control:
current status. Biocontrol Sci. Technol. 4: 3-34.
Ferron, P. (1981). Pest control by the fungi Beauveria and, In Burges H. D. [ed.], Microbial
control of pest and plant diseases 1970-1980. Academic Press, New York. 465-482,
pp.
Flores, S., Campos, S., Villaseñor, A., Villaseñor, A., Valle, A., Enkerlin, W., Toledo, J.,
Liedo, P. and Montoya, P. (2013). Sterile males of Ceratitis capitata (Diptera:
Tephritidae) as disseminators of Beauveria bassiana conidia for IPM strategies.
Biocontrol Sci. Technol. 23(10): 1186-1198.
Garcia, A.S., Messias, C.L., de Souza, H.M.L. and Piedrabuena, A.E. (1984). Patogenicidade
de Metarhizium anisopliae var. anisopliae a Ceratitis capitata (Wied.) (Diptera,
Tephritidae). Rev. Bras. Entomol. 28(4): 421-424.
Gautam, R.D. (2008). Biological Pest Suppression. (Second Edition-enlarged). Westville
Publishing House, New Delhi, 304p.
65
Glare,T., Caradus. J., Gelernter, W., Jackson, T., Keyhani, N., Kohl, J., Marrone, P., Morin,
L. and Stewart. A. (2012). Have biopesticides come of age, Trends Biotechnol. 30(5):
250-258.
Goble, T. A. (2009) Investigation of entomopathogenic fungi for control of false codling
moth, Thaumatotibia leucotreta, Mediterranean fruit fly, Ceratitis capitata and Natal
fruit fly, C. rosa in South African Citrus. M.Sc Thesis. Rhodes University. 147pp.
Goettel, M.S., Eilenberg, J. and Glare, T.R. (2005). Entomopathogenic fungi and their role in
regulation of insect populations. In: Gilbert, L.I., Iatrou, K., Gill, S. (eds) Compre.
Mol. Insect Sci., 6: Elsevier, Oxford, pp 361–406.
Goettel, M.S., Koike, M., Kim, J.J., Aiuchi, D., Shinya, R. and Brodeur , J. (2008). Potential
of Lecanicillium spp. for management of insects, nematodes and plant diseases. J.
Invertebr. Pathol. 98(3): 256-261.
Greathead, D.J. and Prior, C. (1990). the regulation of pathogens for biological control with
special reference to locust control. In Somme, L., and Bie, S. (eds.). Proc. of the
Workshop on health and environmental impact of alternative control agents for
desert locust control. NORAGRIC Occasional Papers Series C: Dev. Env. 5: 65-80.
Gupta, S. and Dikshit. A.K. (2010). Biopesticides: An ecofriendly approach for pest control.
J. Biopest. (Special Issue) 3(1): 186 -188.
Hadi, M.S., Himawan, T. and Aini, L.Q. (2013). The application of Beauveria bassiana and
Lufenuron Could Reduce the Reproduction of Fruit Fly Bactrocera carambolae
(Drew dan Hancock) (Diptera: Tephritidae). J. Trop. Plant Prot. 1(1): 22-29.
Hajek, A.E. and ST. Leger R.J. (1994). Interactions between fungal pathogens and insect
hosts. Ann. Review. Entomol. 39: 293-322.
Imoulan, Abdessamad, Elmeziane. and Abdellatif. (2010). Horizontal transmission of fungal
infection between Ceratitis capitata adults under laboratory conditions. 8th Int.
Symp. on Fruit Flies of Economic Importance. Valencia.
Jackson, T.A. (1999). Factors in the success and failures of microbial control agents for soil
dwelling pests. Integ. Pest Manag. Rev., 4(4): 281- 285.
Jayanthi P.D.K. and Verghese, A. (2011) Host-plant phenology and weather based
forecasting models for population prediction of the oriental fruit fly, Bactrocera
dorsalis Hendel. Crop Protection 30:1557-1562.
66
Jiji, T., Praveena, R., Babu,K., Naseema, A. and Anitha, N. (2006). Fruit Flies of Economic
Importance: From Basic to Applied Knowledge. In Proc. of the 7th Int. Symp. on
Fruit Flies of Economic Importance 10-15 September, Salvador, Brazil pp. 175-177.
Kaaya, G.P. and Okech, M.A. (1990). Horizontal transmission of mycotic infection in adult
tsetse, Glossina morsitans morsitans. Entomoph. 35(4): 589-600.
Khashaveh, A., Ghosta, Y. and Safaralizadeh, H. (2011). The Use of Entomopathogenic
Fungus, Beauveria bassiana (Bals.) Vuill. in Assays with Storage Grain Beetles J.
Agr. Sci. Tech. Vol. 13: 35-43
Konstantopoulou, M.A. and Mazomenos, B.E. (2005). Evaluation of Beauveria bassiana and
B. brongniartii strains and four wild-type fungal species against adults of Bactrocera
oleae and Ceratitis capitata. Biol. Control. 50(2): 293-305.
Lacey, L.A. and Shapiro-Ilan, D.I. (2008). Microbial control of insect pests in temperate
orchard systems: potential for incorporation into IPM. Ann. Rev. Entomol. 53: 121-
144.
Leblanc, L. and Putoa, R. (2000). Fruit flies in French Polynesia and Pitcairn Islands.
Secretariat of the Pacific Community Pest Advisory Leafl., Suva, Fiji.
Lezama-Gutierrez, R., Trujillo-DeLaLuz, A., Molina-Ochoa, J., Rebolledo-Dominguez, O.,
Pescador.A.R., Lopez-Edwards. M. and Aluja, M. (2000). Virulence of Metarhizium
anisopliae (Deuteromycotina: Hyphomycetes) on Anastrepha ludens (Diptera:
Tephritidae): Laboratory and field trials. J. Econ. Entomol. 93(4): 1080-1084.
Li, M., Lin, H., Li, S., Chen, P., Jin, L. and Yang, J. (2012). Virulence of entomopathogenic
fungi to adults and eggs of Nilaparvata lugens(Stal) (Homopera: Delphacidae). Afr.
J. Agri. Res. 7(14): 2183-2190.
Lipa, J.J., Borusiewicz, K. and Balazy, S. (1976). Noxiousness of the rose fruit fly Rhagoletis
alternate (Meigen) and infection of its puparia by a fungus Scopulariopsis
brevicualis (Sacc.) Bainer. Bulletin de l’ Academie Polonaise de Sciences, Sci. Biolo.
24: 451- 456.
Lipa, J.J. (1985). Progress in biological control of the Colorado beetle (Leptinotarsa
decemlineata) in Eastern Europe. Bulletin OEPP 15: 207-211.
Lohmeyer, K.H. and Miller, J.A. (2006). Pathogenicity of Three Formulations of
Entomopathogenic Fungi for Control of Adult Haematobia irritans (Diptera:
Muscidae). J. Econ. Entomol. 99(6): 1943-1947.
Lord, J.C. (2005). From Metchnikoff to Monsanto and beyond: the path of microbial control.
J. Invertebr. Pathol. 89(1): 19-29.
67
Mahmoud, M.F. (2009). Pathogenicity of three commercial products of entomopathogenic
fungi, Beauveria bassiana, Metarhizum anisopilae and Lecanicillium lecanii against
adults of olive fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae) in the
laboratory. Plant Protect. Sci. 45(3): 98-102.
Mar, T. T. and Lumyong, S. (2012). Evaluation of effective entomopathogenic fungi to fruit
fly Pupa, Bactrocera spp. and their antimicrobial activity. Chiang Mai J. Sci., 39(3):
464-477.
Meyling, N.V. and Eilenberg, J. (2007). Ecology of the entomopathogenic fungi Beauveria
bassiana and, Metarhizium anisopliae in temperate agro-ecosystems: potential for
conservation biological control. Biol. Control. 43(2): 145-155.
Muñoz, R. J.A. (2000). Patogenicidad de Beauveria bassiana (Bals.) Bullí.sobre la
moscadelmediterráneo, Ceratitis capitata (Wied.) en condiciones de laboratorio.
Tesis de Licenciatura. Facultad de Ciencias Agrícolas. Univ. Aut. de Chiapas.
Huehuetán, Chis., México. 55 p.
Ortu, S., Cocco, A. and Dau, R. (2009). Evaluation of the entomopathogenic fungus
Beauveria bassian strain ATCC 74040 for the management of Ceratitis capitata.
Bull. Insectol. 62(2): 245-252.
Penrose, D. (1993). The 1989/1990 Mediterranean fruit fly eradication program in California,
pp. 441-406. In M. Aluja and P. Liedo [eds.]. Fruit flies: Biology and Management.
Springer-Verlag, New York. Cross reference.
Perry, A.S., Yamamoto, I., Ishaaya, I. and Perry R.Y. (1998). Insecticides in Agriculture and
environment: Retrospects and prospects, Springer Berlin, p261.
Poprawski, T.J., Robert, P.H., Majchrowicz, I. and Bovin, G. (1985). Susceptibility of Delia
antiqua (Diptera:Anthomyiidae) to eleven isolates of entomopathogenic
hyphomycetes. Envl. Entomol. 14(5): 557- 561.
Prior, C. (1988). Biological pesticides for low external-input agriculture. Biocontrol News
and Information 10(1): 17- 22.
Qazzaz, F.O. and Barakat, R. (2012). Isolation of Beauveria bassiana from Soil and
evaluation of its entomopathogenic and biocontrol efficacy against the Mediterranean
fruit fly (Ceratitis capitata). M.Sc. Thesis, College of Graduate Studies and
Academic Research Hebron University, Hebron- Palestine.
Quesada-Moraga, E., Ruiz-García, A. and Santiago-Álvarez, C. (2006). Laboratory
evaluation of entomopathogenic fungi Beauveria bassiana and Metarhizium
68
anisopliae against puparia and adults of C. capitata (Diptera: Tephritidae). J. Econ.
Entomol. 99(6): 1955–1966.
Quesada-Moraga, E, Martin-Carballo, I., Garrido-Jurado, I. and Santiago-Álvarez, C. (2008).
Horizontal transmission of Metarhizium anisopliae among laboratory populations of
Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Biol. Control. 47(1): 115-
124.
Rath, A.C., Koen, T.B., Anderson, G.C. and Worledge, D. (1995). Long term efficacy of the
entomogenous fungus, Metarhizium anisopliae against the subterranean scarab,
Adoryphorus couloni. Bicontrol Sci. Technol. 5(4): 439- 452.
Rathore, H.S. and Nollet, M.L. (2012). Pesticides evaluation for environmental pollution.
CRC Press Page No. 499.
Reithinger, R., Davies, C.R., Cadena, H. and Alexander, B. (1997). Evaluation of the fungus
Beauveria bassianaas a potential biological control agent against phlebotomine sand
flies in Colombian coffee plantations. J. Invertebr. Pathol. 70(2): 131-135.
Rice W.C., Cogburn R.R. (1999). Activity of entomopathogenic fungus Beauveria bassiana
(Deuteromycota: Hyphomycetes) against three coleopteran pests of stored grain. J.
Econ. Entomol. 92(1): 691- 694.
Roessler, Y. (1989) Insecticidal bait and cover sprays. In A. S. Robinson and G. Hooper
(eds), Fruit flies, their Biology, Natural Enemies and Control. World Crop Pests, 3B,
Elsevier, Amsterdam, pp. 329-336. Cross reference.
Samroda, H. and Ibrahim, Y. (2006). Effectiveness of selected entomopathogenic fungi in
packed rice grain at room temperature against Corcyra cephalonica stainton. Asian J.
Sci. Technol. Developt. 23(3): 183-192.
Sharma, H.C., Dhillon, M.K. and Sahrawat, K.L. (2013). Environmental safety of Biotech
and Conventional IPM Technologies. Stadium press LLC, Houston Texas-77072
USA, Page 88-89.
Sookar, P., Bhagwant, S. and Awuor Ouna, E. (2008). Isolation of entomopathogenic fungi
from the soil and their pathogenicity to two fruit fly species (Diptera: Tephritidae). J.
Appl. Entomol. 132(9-10): 778-788.
ST leger, R. J. (2008). Studies on adaptations of Metarhizium anisopliae to life in the soil, J.
Invertebr. Pathol. 98(3): 271-276.
Tanada, Y. and Kaya, H.K. (1993). Insect Pathology, Academic Press, San Diego. 666 pp.
Toledo, J., Campos, S.E. Flores, S., Liedo, P., Barrera, JF.Villasenior., A and Montoya, P.
(2007). Horizontal transmission of Beauveria bassiana in Anastrepha ludens
69
(Diptera: Tephritidae), under laboratory and field-cage conditions.J. Eco. Entomol.
100(2): 291-297.
Vega, F.E., Goettel, M.S., Blackwell, M., Chandler, D., Jackson, M.A., Keller, S., Koike, M.,
Maniania, N.K., Monzon, A., Ownley, B.H., Pell, J.K., Rangel, D. and Roy, H.E.
(2009). Fungal entomopathogens: new insights on their ecology. Fung. Ecol. 2(4):
149-159.
Vega, O.F.L., 2005. Development, production and use of biopesticides in Cuba. In: Roettger,
U., Muschler, R. (Eds.), Int. Symp on Biopesticides for Developing Countries 2003.
CATIE/GTZ, Turrialba, Costa Rica, pp. 85–91. Cross reference.
Verghese, A. and Jayanthi, P.D.K., (2001). Integrated pest management in fruits. In: Parvatha
Reddy, P., Verghese, A., Krishna Kumar, N.K. (Eds.), Pest Management in
Horticultural Ecosystems. Capital Publishing Company, New Delhi, pp. 1-23.
Verghese, A., Madhura, H.S., Jayanthi, P.D. K. and Stonehouse J. M. (2002). Fruit flies of
economic Significance in India, with special reference to Bactrocera dorsalis
(Hendel). Proc. of 6th International Fruit fly Symposium, 6–10 May 2002,
Stellenbosch, South Africa. pp. 317 – 324.
Vontas, J., Herna´ndez-Crespo, P., Margaritopoulos, J.T., Ortego, F., Feng, H.T.,
Mathiopoulos, K.D., and Hsu, J.C. (2011). Insecticide resistance in Tephritid flies.
Pestic. Biochem. Phys. 100(3): 199-205. Cross reference.
Waterhouse, D.F. (1993). The major arthropod pests and weed of agriculture in Southeast
Asia. Aciar. Monograph No. 21.
Watt, B.A. and Lebrun, R.A. (1984). Soil effects of Beauveria bassiana on pupal
populations of the Colorado potato beetle (Coleoptera: Chrysomelidae). Env.
Entomol. 13(1): 15-18.
White, I.M and Elson-Harris. M. (1994). Fruit flies of economic significance: their
identification and bionomics. CAB International, Wallingford, Oxon, UK. P. xii +
601
Whitten, M.J. and Oakeshott, J.G. (1991). Opportunities for modern biotechnology with
special reference to locust control. In Proc. of the workshop on health and
environmental impact of alternative control agents for desert locust control (eds.
Somme, L. and Bie, S.). Noragric Noragric Occasional Papers Series C: Dev. Env. 5:
65-80.
70
Wojciechowska, M., Kmitowa, K., Fedorko, A. and Bajan, C. (1977). Duration of activity of
entomopathogenic microorganisms introduced into the soil. Pol. Ecol. Stu. 3(2): 141-
156.
Wraight, S.P., Jackson, M.A. and De Kock S.L. (2001). Production, stabilization and
formulation of biocontrol agents. In: Butt, T.M., Jackson, C. and Magan, N., (eds).
Fungi as Biocontrol Agents, Progress, Problems and Potential. CABI Publishing,
New York. 253-288.
Yousef, M., Lozano-Tovar, M.D., Garrido-Jurado, I. and Quesada, E. (2013). Biocontrol of
Bactrocera oleae (Diptera: Tephritidae) With Metarhizium brunneum and its
extracts. J. Econ. Entomol. 106(3): 1118-1125.
Zimmermann, G. (2007). Review on safety of the entomopathogenic fungi Beauveria
bassiana and Beauveria brongniartii. Bio-control Sci. Technol. 17(6): 553-596.