novel toxicity of bacillus thuringiensis strains against

Original

Biocontrol Science, 2015, Vol. 20, No. 2, 115－123

＊Corresponding author. Tel: +88-01717083673, E-mail : mmzhoq（a）gmail.com

Novel Toxicity of Bacillus thuringiensis Strains against the Melon Fruit Fly, Bactrocera cucurbitae（Diptera: Tephritidae）

MD. ASADUZZAMAN SHISHIR1, ASMA AKTER1, MD. BODIUZZAMAN1,M. AFTAB HOSSAIN2, MD. MUSFIQUL ALAM1, SHAKIL AHMED KHAN2,

SHAKILA NARGIS KHAN1 AND M. MOZAMMEL HOQ1＊

1Department of Microbiology, University of Dhaka, Dhaka- 1000, Bangladesh2Insect Biotechnology Division, Institute of Food and Radiation Biology,

Atomic Energy Research Establishment, Savar- 1349, Bangladesh.

Received 23 September, 2014/Accepted 25 December, 2014

　Bactrocera cucurbitae （melon fruit fly） is one of the most detrimental vegetable-damaging pests in Bangladesh. The toxicity of Bacillus thuringiensis （Bt） has been reported against a few genera of Bactrocera in addition to numerous other insect species. Bt strains, harbouring cry1A-type genes were, therefore, assayed in vivo against the 3rd instar larvae of B. cucurbitae in this study. The biotype-based prevalence of cry1 and cry1A genes was calculated to be 30.8% and 11.16%, respectively, of the test strains （n=224） while their prevalence was greatest in biotype kurstaki. Though three indigenous Bt strains from biotype kurstaki with close genetic relationship exhibited higher toxicity, maximum mortalities were recorded for Btk HD-73 （96%） and the indigenous Bt JSc1 （93%）. LC50 and LC99 values were determined to be 6.81 and 8.32 for Bt JSc1, 7.30 and 7.92 for Bt SSc2, and 6.99 and 7.67 for Btk HD-73, respectively. The cause of toxicity and its variation among the strains was found to be correlated with the synergistic toxic effects of cry1, cry2, cry3 and cry9 gene products, i.e. relevant Cry proteins. The novel toxicity of the B. thuringiensis strains against B. cucurbitae revealed in the present study thus will help in developing efficient and eco-friendly control measures such as Bt biopesticides and transgenic Bt cucurbits.

Key words ： Pest management / Bacillus thuringiensis （Bt）, / Cry genes / Synergistic toxicity / Bactrocera cucurbitae.

INTRODUCTION

　B. thuringiensis （Bt） biopesticides, for their highly specific mode of actions, are the key components of Integrated Pest Management （IPM） strategies aimed at preserving natural enemies of pests and managing insect resistance （Kumar et al., 2008）. Bt biopesticides are eco-friendly as they are free of recalcitrant residues which, upon bioaccumulation and biomagnification, might become carcinogenic, mutagenic, teratogenic or allergenic （Zahm et al., 1997）. Hence, Bt biopesticides have served as valuable alternatives to synthetic chem-

ical pesticides in agriculture, forestry and mosquito control for last many decades （Mohan and Gujar, 2000）.　B. thuringiensis, a gram-positive spore forming bacterium, synthesizes proteinaceous insecticidal crys-tals or δ- endotoxins during sporulation which can specifically kill the insects belonging to the Lepidoptera, Coleoptera, Diptera, Hymenoptera, Hemiptera, and Mallophaga as well as some invertebrates at their larval stage （Ben-Dov et al., 1997; Schnepf et al., 1998; Feitelson et al., 1999）. 　Bangladesh, a country of subtropical climate, produces various vegetable crops covering an area of ca. 498,073 acres. However, the yield per unit area is low as 25% annual yield losses occur due to the pests

H. M. MOZAMMEL ET AL.116

Bacterial strains and growth conditions　Indigenous Bt strains obtained from different locations of Bangladesh were studied in terms of their abundance and diversity （Shishir et al., 2014）. These Bt strains are preserved in the Bt Collection Centre, Bangladesh, in the Department of Microbiology, University of Dhaka. Two hundred and twenty four Bt strains from this collec-tion were used in this study. The biotype kurstaki （Btk） strain HD-73 and the biotype sotto （Bts） strain T84A1 were kindly provided from Bt stock collection of Okayama University, Japan and used as reference strains. LB agar （per litre: tryptone 10 g, yeast extract 5 g, NaCl 10 g, agar 15 g） was used for culture mainte-nance and T3- broth （per litre: Tryptone 3.0 g, tryptose 2.0 g, yeast extract 1.5 g, MnCl2 0.005 g, phosphate buffer 50 mM）（Travers et al., 1987） was used to promote sporulation. Phase contrast microscopy was used to observe sporulation as well as endotoxin synthesis （Fig. 1）. Incubation temperature was main-tained at 30 ℃ for all types of culture conditions.

Total DNA preparation　Total DNA was prepared from the indigenous Bt isolates streaked on LB agar medium （Bravo et al., 1998）. After 12 hours of incubation at 30℃, a single colony was transferred into 100 µl sterile de-ionized water in a microfuge tube, vortexed and kept at -70℃for 20 min. It was then incubated in boiling water for 10 min to lyse the cells and briefly centrifuged for 20 s at 12,000×g. The upper aqueous phase transferred into sterile microfuge tubes was used as a template and preserved at –20℃ for further use. 50-100 ng of DNA from this suspension was used as a template in PCR.

PCR detection of cry1 and cry1A genes　Bt strains were characterized on the basis of cry1 and cry1A genes by PCR detection with respective p r i m e r s i . e . c r y 1 , f w d （ 5 ’- CATGATTCATGCGGCAGATAAAC-3’）, rev （5’- TTGTGACACTTCTGCTTCCCATT-3’）; cry1A, fwd （5’- C C G G T G C T G G AT T T G T G T TA - 3’）, r e v （5’- AATCCCGTATTGTACCAGCG-3’）（Carozzi et al., 1991; Ben-Dov et al., 1997）. PCR detection of the genes were performed in 25 µ l reaction mixture [containing 10 mM Tris, 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTPs, 0.5 µM of each primer, 50-100 ng of the template and 0.5 U of Taq DNA polymerase （Promega, USA）] in a thermal cycler （MJ mini, Bio-Rad, USA） for 35 cycles （95℃ for 50 s, 53℃ for 45 s, 72℃ for 60 s） with an initial denaturation step at 95℃ for 4 min and a final extension step at 72℃ for 10 min （Ben-Dov et al., 1997）. PCR products were analysed by electrophoresis in 1.5% （w/v） agarose gel submerged in 1× TBE buffer （89 mM Tris （pH 7.6）, 89

alone （Rahman, 2000）. More than 200 major species of insects and mites from different field crops, fruit trees, and stored products have been recorded in Bangladesh （Rahman, 2000）. The melon fruit fly, B. cucurbitae （Diptera: Tephritidae）, is one of the widely distributed and detrimental pests that damages about 81 host plants （Hollingsworth and Allwood, 2000） mainly from cucurbitaceous crops （Dhillon et al., 2005）, and it causes significant losses in different cucurbits （including cucumber, melon, watermelon, squash, pumpkin, gourds, etc.） in Bangladesh also. The female melon fly is capable of destroying large numbers of fruits in its lifespan of 10 months to a year as one may deposit up to 1,000 eggs in soft tender fruit tissues by piercing them with the ovipositor （Mohan and Gujar, 2000）. Maggots feed inside the fruits, flowers and stems, and young larvae cause necrotic regions, often introducing various pathogens and hastening fruit decomposition （Dhillon et al., 2005）.　A lot of control measures have been reported against the melon fruit fly such as the bagging of fruit, field sani-tation, monitoring and control with parapheromone lures/cue-lure traps, increasing the host plant resis-tance, chemical control, wide area management, male-sterile technique, transgene based embryo-specific lethality system, quarantine and also biological control （Dhillon et al., 2005）. In Bangladesh, with no exception, chemical insecticides are the major and most widely administered control measure against the melon fruit fly. Continuous and indiscriminate use of these insecticides due to their long residual action and toxicity against a wide spectrum of insects has polluted the environment largely by bioaccumulation and biomagnifications, and simultaneously led to the development of resistance in agricultural pests and the spread of human diseases （Marrone and MacIntosh, 1993）. As a result, eco-friendly pest management or organic farming is an undeniable necessity in agriculture （Frankenhuyzen 1993; Margalit et al., 1995）; Salehi et al., 2005）. The Bt biopesticide, therefore, should be the most appropriate choice for insect control. 　Although a few species from genus Bactrocera have been reported to be killed by the δ- endotoxins of Bt, no such development has been found against B. cucur-bitae （Ansari et al., 2012）. Worldwide continuous screening programs for new strains with different combinations of crystal proteins as well as the discovery of new toxins have broadened the activity spectrum of Bt toxins. The present study was therefore designed by combining in vitro and in vivo molecular techniques with a view to finding potential Bt strains that are toxic to B. cucurbitae （melon fruit fly）.

MATERIALS AND METHODS

NOVEL TOXICITY OF BT AGAINST B. CUCURBITAE 117

mM boric acid, 2 mM EDTA） and visualized against UV after Ethidium Bromide （EtBr） staining in a Gel Doc system （Alpha Imager Mini, USA）.

Insect rearing　Larvae were maintained on a locally developed semi liquid artificial diet. Adult melon flies were stocked in a stainless steel framed cage （120×120×90 cm） covered with a stainless steel net. The insects were usually supplied with a laboratory diet （yeast extract: casein: sugar- 1: 1: 2） and water soaked cotton. In general, 2000-2500 adult fruit flies were maintained in a stock cage. Temperature and relative humidity （RH） of the rearing room were maintained at 28±2℃ and 70-80%, respectively. To collect huge numbers of eggs the matured flies in the cage were provided with a piece of sweet gourd for oviposition. The piece of sweet gourd was removed after 2 hours from the adult cage and placed in a plastic bowl with sawdust for further larval development.

Bioassay　The toxicity of the Bt isolates was analysed in vivo by bioassay against the 3rd instar larvae of melon fruit fly, B. cucurbitae. Spore-Cry protein mixtures were prepared with cry1A gene positive isolates （Obeidat et al., 2004） and mixed with 10g of boiled and smashed sweet gourd paste on which 20 larvae were kept and fed at 25±2℃ and 70±10% RH, with a photoperiod of 16:8 （L: D）. The mortality was then scored （Fig 4A and 4B） for the Bt strains along with a parallel control prepared with sterile distilled water, and it was used to correct test mortality using Abbot’s formula （Daffonchio et al., 1998）. The concentration of spores in the suspension, determined by spore count technique, was the basis for estimating LC50 and LC99 values for the strains causing more than 50% mortality. Bioassays were performed in triplicate in all cases and LC50 and LC99 values were determined by Probit analysis using Statplus 2009 soft-ware for Windows.

16S rRNA gene sequence analysis　16S rRNA genes from indigenous Bt strains were amplified by PCR with universal primers for Bacillus spp.: fwd （20F）; 5'-GAGTTTGATCCTGGCTCAG-3' （ p o s i t i o n 9 - 2 7）, a n d r e v （ 1 5 0 0 R）; 5'-GTTACCTTGTTACGACTT-3' （position 1492-1509）（Soufiane and Cote, 2009）. The PCR was conducted in a thermal cycler by performing 35 cycles （96℃ for 50 s, 50℃ for 45 s, 72℃ for 2 min） with an initial denatur-ation step at 96℃ for 10 min and a final extension step at 72℃ for 10 min in 25 µl reaction mixture （fwd and rev primers 0.5 µM each, 50-100 ng of template, 0.5 U of Taq DNA polymerase, 200 µM dNTPs, 10 mM Tris,

50 mM KCl and 1.5 mM MgCl2）. 　5 µl of the PCR product was electrophoresed on 1.5% （w/v） agarose gel （Promega, USA） submerged in 1× TBE buffer at 60 V for 1 hour and stained with 0.5 µg/mL EtBr （Sigma, USA） for visualization against a UV light in a gel documentation system （Alpha imager mini, USA）. Purified PCR products （Wizard® SV Gel and PCR Clean-Up System, Promega, USA） were sequenced at CARS （ABI PRISM® 310 Genetic Analyzer）, University of Dhaka, Bangladesh, and the sequences were submitted to the NCBI database （GenBank KF741358- KF741360 and GenBank KF812552- KF812557）.Based on the sequences, multiple alignments and phylogenetic analysis were carried out to compare the Bt strains among each other and to determine the evolutionary relatedness as well as the genetic distance.

Identification of putative cry genes responsible for toxicity　The amplicons for the cry1 gene from the potential stra ins were pur i f ied and sequenced, and the sequences were aligned using MEGA 5.2 software to see if any differences persisted. Moreover, we evaluated whether or not the variation in toxicity of the strains against B. cucurbitae was due to the expression of some other cry genes. This was done by analyzing their cry genes profile. PCR was performed to detect cry2, cry3, cry4, cry8, cry9, cry10 and cry11 genes （Shishir et al., 2014）, and the PCR products were then analyzed by agarose （1.5%） gel electrophoresis following staining and destaining in EtBr. The image was then captu red aga ins t UV by the ge l doc sys tem （Alphaimager mini, USA）. The molecular weight of the amplicons was determined from the images with the aid of Alphaview SA software （version 3.4.0.0）. Amplicons of certain sizes were considered to be the desired PCR products, and the strains were assumed to harbour those particular genes.

Cry protein profile analysis　The Cry proteins were partially purified from the Bt strains （Öztürk et al., 2009） harbouring cry1A genes and were analysed by SDS-PAGE in a 10% separating gel （Sambrook et al., 1989）. Colonies formed on T3-agar medium upon incubation at 30℃ for 7 days were scraped off and resuspended in cold sterile de-ionized water. Washing was performed twice with cold sterile de-ionized water to remove exotoxins, once with 1.0 M NaCl containing 5.0 mM EDTA and finally with 5.0 mM EDTA alone. The pellet, after being resus-pended in 1×Laemmli buffer lacking Bromophenol blue （50 mM Tris-HCl （pH 6.8）, 10% （v/v） glycerol, 2% （w/v） SDS and 100 mM dithiothreitol）, was incubated at


presence of cry1 and cry1A genes, 30.8% and 11.16% （n=224） were detected, respectively （Fig. 2A & 2B）. Bt strains producing 277 bp and 490 bp amplicons were considered positive for the cry1 and cry1A gene, respectively. 　Biotype-based prevalence study of these genes revealed that 78% and 42% of the strains （n=42） of Bt biotype kurstaki harboured the cry1 and cry1A gene, respectively （Fig. 3）. Significant prevalence was also observed with the biotype thuringiensis （28% and 10% for cry1 and cry1A genes, respectively）.

Bioassay　Six Bt strains out of twenty five harbouring cry1A gene were found to exhibit significant toxicity against the 3 rd instar larvae of B. cucurbitae （Fig. 4C）. Mortalities were recorded （Fig. 4A and 4B） as JSc1-93%, SSc2- 80%, SSe2- 63% and JaS8- 56% among the indigenous Bt strains while reference strain, Btk HD-73 exhibited 96% mortality and Bts T84A1 only 16% （Fig. 4C）. Hence, the bioassay was repeated with the Bt strains causing more than 50% mortality for further determination of LC50 and LC99 values. 　The LC50 and LC99 values varied from 6.81 to 7.61 and from 7.67 to 9.13, respectively （Table 1）. The lowest LC50 value was observed for the indigenous Bt strain JSc1 （LC50- 6.81） whereas for reference strain Btk HD-73, it was slightly higher （LC50- 6.99）. On the other hand, Bt strain SSc2 （LC99- 7.92） demonstrated a comparable LC99 value with Btk HD-73 （LC99- 7.67）.

boiling temperature for 5 min and the supernatant was collected by centrifugation for 5min at 10,000 rpm （Eppendorf centrifuge, 5415D）. Protein concentration in the supernatant was estimated by the Bradford method （Bradford, 1976） prior to SDS-PAGE analysis to ensure that an equal amount of proteins was loaded in each lane. Upon the completion of electrophoresis, the gel was stained in a staining solution （0.02% Coomassie Brilliant Blue- G250 in 2% （w/v） phosphoric acid, 5% aluminum sulfate and 10% ethanol）（Kang et al. 2002） for 2 hours. The molecular weight of the proteins was determined with the help of Alphaview SA software.

RESULTS

Identification of cry genes in B. thuringiensis　Glowing spores and juxtaposed dark crystal shaped proteins were observed with phase contrast microscopy upon sporulation of the Bt strains （Fig. 1）. As for the

FIG. 1．Spores and crystal proteins of indigenous B. thuringiensis strains are distinguished under phase contrast microscopy. Glowing parts are spores and juxtaposed dark parts are crystal proteins. （Bar= 2µm）

FIG. 2．A） Detection of the cry1 gene in the Bt strains as amplicons of about 270 bp were visualized by agarose gel electrophoresis. （Marker= 100 bp DNA ladder, Bioneer, Korea）. B） Detection of the cry1A gene with 490 bp amplicons （M= 100 bp DNA ladder, TaKaRa, Japan）. Names of the strains have been written over each lane.

FIG. 3．Prevalence and distribution of cry1 and cry1A genes in different biotypes of indigenous Bt from Bangladesh. （thu= thuringiensis, kur= kurstaki, ind= indiana, gal= galleriae, sot= so t to , den= dend ro l imus , mo r= mor r i son i , da r= darmstadiensis, ost= ostriniae, isr= israelensis, 9, 10, 11, 13, 15, 16= undefined biotypes）（n= number of isolates available in each biotype, tested for the presence of cry1 and cry1A genes）.


16S rRNA gene sequence analysis　Sequences of amplicons obtained for the 16S rRNA gene from the indigenous Bt strains were aligned to analyse the phylogenetic relationship between them. Following the bootstrap neighbor joining method, a phylogenetic tree was constructed with 19 indigenous Bt strains and reference Btk HD-73 （Fig. 5）. The tree was observed to contain 2 major distinct phylogenetic groups consisting of clusters A and B. Cluster A, the largest one, contained 16 native Bt strains and 1 refer-ence strain whereas cluster B contained the other 3. Nine of the indigenous Bt strains from biotype kurstaki and reference Btk HD-73 remained in the same sub-cluster A1 among which were strains JSc1, SSc2 and SSe2 that exhibited toxicity against B. cucurbitae.

Causes of toxicity and its variation based on the gene and protein profile　It was observed from the alignment of sequences of the cry1 conserved region, obtained from the indige-nous Bt strains JSc1, SSc2, SSe2 and two reference strains Btk HD-73 and Bts T84A1, that the indigenous strains were more similar to Bts T84A1. Mismatches were observed at 24 and 231 base positions with Btk HD-73 （Fig. 6A）. 　Upon agarose gel electrophoresis of the PCR prod-ucts, the presence of cry1, cry2, cry3 and cry9 genes in the indigenous Bt strains was revealed whereas refer-ence Btk HD-73 （cry1Ac） and Bts T84A1 （cry1Aa） were positive for only the cry1 gene （Table 2）. Bt JSc1 was found to harbour cry1, cry2 and cry9 genes （Fig. 6B） whereas Bt SSc2 and SSe2 harboured cry1 and cry3 genes （Table 2）. The correlation between the cry gene profiles and LC50 values of these Bt strains was observed in this study （Table 2）. The presence of cry2 and cry9 genes had a positive effect on the toxicity as the LC50 value for Bt JSc1 was found to be the lowest among all the cry1 gene-harbouring Bt strains. The effect of the cry3 gene was also observed since the toxicity was found to be more intense in strains SSc2 and SSe2 than JaS8 that lacked the genes. The toxicity of Bt strains against B. cucurbitae can therefore be concluded as the synergistic effects of cry1, cry2, cry3 and cry9 gene products.

FIG. 4．Bioassay performed with 3rd instar larvae of B. cucurbitae. Fate of the larvae that were fed with the diet supplemented with A） Sterile distilled water, or B） Spore- Cry protein mixture of Bt. C） Efficiency of the indigenous and reference Bt strains in causing death to the test larvae.

TABLE 1．LC50 and LC99 values estimated for indigenous and reference Bt strains

Strains LC50 LC99 X2 Df P

Bt SSc2Bt SSe2Bt JSc1Bt JaS8Btk HD-73

7.307.426.817.616.99

7.928.818.329.137.67

0.8523.0050.2781.4530.364

11111

0.3550.2220.5970.2280.545

LC50 and LC99: log （spore concentration ml-1）. X2: Chi-square

FIG. 5．Neighbor-joining tree showing the phylogenetic relationship between indigenous Bacillus thuringiensis strains and reference strain Btk HD-73 based on the 16S rRNA gene sequence analysis. This is an un-rooted tree reconstructed with 1000 bootstrap repl icates based on maximum composite likelihood using tree construction software MEGA version 5.22.


Cry protein profile of the potential Bt strains　SDS-PAGE analysis （Fig. 7） of the partially purified Cry proteins revealed that diverse Cry proteins are synthesized by the indigenous Bt strains at different expression levels. Based on the molecular weight of the proteins （Table 3）, Cry1, Cry2, Cry3 and Cry9 proteins could be presumed to be expressed. From the analysis, common 23, 45, 50 kDa protein bands were observed in all Bt strains which were not considered as Cry proteins. Another 67 kDa common band was observed in all Bt strains except strain JaS8 which might be the

FIG. 6．A） The sequences obtained from the conserved regions of cry1 genes of indigenous and reference Bt strains were aligned by ClustalW. Mismatches at 24 and 231 base positions of Btk HD-73 are visible with the sequences of other strains. B） Investigation of causative cry genes in Bt JSc1 rendering toxicity against the melon fruit fly. Amplicons of approximately 490, 639 and 492 bp were observed in detecting cry1A, cry2 and cry9 genes as indicated with arrows whereas spurious amplicons of about 564 and 365 for cry4A/4B, 667 for cry9, 480, 700 bp for cry10 and 200, 270 for cry11 genes were observed.

TABLE 2．Comparison of the cry gene profiles of Bt strains tested in bioassay

Strains cry1 cry2 cry3 cry4A/4B cry8 cry9 cry10 cry11 LC50

Bt SSc2 ✓ × ✓ × × × × × 7.30

Bt SSe2 ✓ × ✓ × × × × × 7.42

Bt JSc1 ✓ ✓ × × × ✓ × × 6.81

Bt JaS8 ✓ × × × × × × × 7.61

Btk HD-73 ✓ × × × × × × × 6.99

Bts T84A1 ✓ × × × × × × × －

FIG. 7．SDS-PAGE analysis of partially purified Cry proteins of Bt strains. Lanes have been labelled with the names of the strains. （HD73: Reference strain Btk HD-73）（M: Precision plus protein standards; All blue; Bio-Rad, USA）.

TABLE 3．Determination of MW of the partially purified Cry proteins

StrainMW of the visible protein bands （kDa）

Presumptive Cry proteins

Bt SSc2

Bt SSe2

Bt JSc1

Bt JaS8Btk HD-73

23, 45, 50, 67, 73, 90, 109, 20423, 45, 50, 67, 73, 90, 109, 20423, 45, 50, 67, 73, 90, 109, 134, 20423, 45, 56, 10923, 45, 50, 66, 134

Cry1, Cry3

Cry1, Cry3

Cry1, Cry2, Cry9

Cry1Cry1Ac


strains were further searched for the presence of cry1A genes. The targeted amplicons of about 490 bp were observed in 25 out of 69 cry1 positive Bt strains. It was found from the previous studies that the cry1 gene was present mainly in the biotypes of kurstaki, thuringiensis, sotto, dendrolimus, morrisoni, galleriae and darmstadi-ensis （Carozzi et al., 1991）. As exceptions, in our study we detected the cry1 gene in the biotypes indiana and israelensis and in 11 undescribed biotypes at percent-ages of 31%, 18% and 21%, respectively, and the absence of the cry1 gene in the biotype galleriae.　Bt toxin was reported to cause mortality （at more than 65-80%） to the olive fruit fly, Bactrocera oleae （Ansari et al., 2012）. However, there has been no report of Bt toxicity against the melon fly, B. cucurbitae, nor is it listed in the toxin specificity data summary. Though Dipteran insect orders have been found to be susceptible to Bt subsp. israelensis and mostly to Cry4, Cry10 and Cry11 proteins, Cry1Ab and Cry1Ac proteins were also found to exert toxicity against them （The Canadian Forest Service: http://cfs.nrcan.gc.ca/projects/119/6）. In this connection, the toxicity of Bt strains harbouring cry1A-type gene was tested against the 3rd instar larvae of B. cucurbitae in this study. 　The larvae were fed on sweet gourd paste in which a Bt spore-Cry protein suspension was mixed. The larvae were observed up to 7 days as the unaffected larvae grew up into pupae and finally matured into fly. On the other hand, the effect of Cry toxins over the larvae was evidenced as their movement and feeding gradually stopped, and finally they turned black and died （Fig. 4A and 4B）. The result of the bioassay performed in our experiment revealed that the indigenous Bt JSc1 and reference Btk HD-73 were highly toxic to the melon fly larvae （Fig. 4C）.　The experiment was repeated thrice, and each time it was done in triplicate to evaluate the results statistically. Bt strains causing more than 50% mortality were studied to determine the lethal concentrations. Thus, four indigenous Bt strains and one reference strain, Btk HD-73, were used. The logarithmic value of the concen-tration of spores was the basis for LC50 and LC99 deter-mination instead of proteins because actual amount of the active proteins should be confirmed for the strains expressing more than one protein. From this analysis, the LC50 value of Bt JSc1 and the LC99 value of Bt SSc2 were found to be highly comparable to those of Btk HD-73 （Table 1）. 　16S rRNA gene sequence analysis has been used as a molecular identification tool for Bt and the claims of its ability to discriminate Bt in different H-serotypes also was reported （Joung and Cote, 2002; Soufiane and Cote, 2009; Poornima et al., 2010）. In the present study, 16S rRNA gene analysis was performed with 19

degraded product of Cry1 protein （Armengol et al., 2006）. An unusual but prominent band at 109 kDa was common in all indigenous strains. A faint band in the range of 130-140 kDa was observed in Bt JSc1 which could be either the Cry1 and Cry9 protein whose expression level seems to be low. The molecular weight of Cry1Ac protein of reference Btk HD-73 was deter-mined at 133 kDa.

DISCUSSION

　B. thuringiensis, the entomo-pathogenic bacterium, has gained considerable attention since 1960 and is widely preferred over chemical insecticides for eco-friendly pest management as it is environmentally benign （Sezen et al. 2010）. Discovery of novel potential Bt strains is necessary to solve the problem of resis-tance that has been reported in many pests against many Bt biopesticide formulations and transgenic Bt crops （Shishir et al., 2014）. As a result, the screening for potential Bt strains that are highly toxic against different insect orders including resistant species is a rudimentary and continuous process all over the world, and many such novel Bt strains have been recovered from numerous sources such as soil, grain dust, diseased insect larvae, animal feed mills and aquatic environments （Sezen, et al. 2010）. 　Bt is widely distributed and approximately 70% of soil samples from all continents have been found to produce this bacteria, and are especially plentiful in Asian samples. Because of this, the particular interest of this research work was to isolate and identify poten-tial Bt strains with novel toxicity against the melon fruit fly （B. cucurbitae）, an important vegetable-damaging pest in Bangladesh. 　Bt strains used in this study were observed with the phase contrast microscope upon sporulation （Fig. 1）. Crystal proteins and the spores were distinguished from each other as the lights of different phases were passed through the specimens that revealed glowing spores and dark crystal proteins. 　Bt has also been reported to produce parasporin, another type of parasporal crystal protein which has anti-cancer cell activity. It was shown that parasporal proteins from non-hemolytic Bt strains are mainly non-insecticidal but may have anti-cancer cell activity （Mizuki et al. 1999）. That is why, hemolytic strains were mainly used in cry1 and cry1A gene detection. 　As all of the biotypes describing different subspecies were found to consist of both hemolytic and non-hemo-lytic Bt strains （Shishir et al., 2014）, all of the 16 biotypes were included in the process of detecting cry genes. Initially, the cry1 gene was detected in the strains producing amplicons of ca. 277 bp, and the positive


cidal potential of a Bt strain can be more appropriately ascertained by detection of cry genes present followed by analysis of crystal proteins produced by that strain （Sarvjeet, 2006）. This method can supplement the PCR strategy to determine which genes are actually expressed. Thus, it was observed from the protein profile of the indigenous Bt strains that multiple protein bands ranging from 23 kDa to more than 200 kDa were present. Proteins bands with molecular weights similar to those of the reported Cry proteins were considered as Cry proteins, and thus the presence of Cry proteins in both protoxin and toxin forms were observed. The Cry protein profile of the indigenous Bt strains revealed the presence of Cry1, Cry2, Cry3 and Cry9 proteins （Table 3）. Thus the Cry protein profile of Bt JSc1 with Cry1, Cry2 and Cry9, strain JaS8 with Cry1 and strain SSc2 as well as SSe2 with Cry1 and Cry3 comply with the cry gene profiles. 　It can, therefore, be concluded that the synergistic effects of Cry proteins encoded by cry1, cry2, cry3 and cry9 are the causes of novel toxicity of the indigenous Bt strains against B. cucurbitae and that the Cry1Ac protein is the toxic agent in Btk HD-73.

CONCLUSION

　The report of the novel toxicity of the Bt strains against the larvae of melon fly, B. cucurbitae, will help to develop efficient and eco-friendly control measures which in turn will prevent bioaccumulation and biomag-nification of toxic substances through the food chain and enhance the food safety and security.

ACKNOWLEDGEMENT

　This work was supported by a Grant-in-Aid from the USDA as a project entitled ‘‘Production of Bacillus thuringiensis biopesticides by biotechnological approach for the control of vegetable pests in Bangladesh.’’ We thank Okayama University, Japan for providing the reference strains. We also thank CARS, University of Dhaka, Dhaka-1000, Bangladesh, for providing the sequencing facility.

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Bt strains （16 harbouring the cry1 gene, 10 harbouring the cry1A gene, 3 without the cry1 gene） and one reference strain Btk HD-73. Two main clusters （A and B） were generated from the dendrogram analysis （Fig. 5） with two sub-clusters in cluster A. Sub-cluster A1 was the largest with 12 strains in which Bt DSf3, harbouring no cry1 gene, was present and 7 Bt strains harbouring the cry1A gene were found. In cluster A2, two Bt strains were found without any cry1 gene and the rest of them did not harbour the cry1A gene as well. In cluster B, all three Bt strains were found to harbour both cry1 and cry1A genes. Interestingly, all the Bt strains that were found to be highly toxic against the larvae of melon fruit fly were observed in the same sub-cluster A1（Fig. 5）.　The alignment of sequences of the cry1 gene from the potential strains obtained from bioassay revealed that the sequences are more similar to that of cry1Aa of Bts T84A1 rather than Btk HD-73. Hence, the variation in toxicity is very likely. However, the toxicity of Bts T84A1 was very low compared to the indigenous strains and reference Btk HD-73 （Fig. 4C）. That is why, some other gene products, i.e. Cry proteins, might be involved in the toxicity against B. cucurbitae. To get an insight into this, potential strains were investigated for the genes cry2, cry3, cry4, cry8, cry9, cry10 and cry11 as most of the lepidopteran and dipteran vegetable-damaging pests were reported to be susceptible to the proteins encoded by these genes. Interestingly, the indigenous strains were found to possesssome of the genes searched for in this regard. Besides the cry1 gene, Bt JSc1 was positive for cry2 and cry9 genes and Bt SSc2 as well as SSe2 were positive for cry3 genes. The PCR products of expected sizes were the only basis for the presumption of the presence of a gene, and non-specific products were disregarded （Shishir et al., 2014）. From this analysis, the toxicity was found to be correlated with the cry gene profiles of the Bt strains （Table 2）. The lowest LC50 value was obtained for Bt JSc1 which was detected with cry1, cry2 and cry9 genes whereas it was slightly higher for reference Btk HD-73 that harboured only the cry1Ac gene. The LC50

values of Bt SSc2 and SSe2 were higher than that of Bt JSc1 and Btk HD-73 as they lacked cry2 and cry9 genes, and the cry1 gene in them was not similar to cry1Ac. The presence of the cry3 gene enhanced their toxicity more than Bt JaS8 harbouring only the cry1 gene. 　The Cry protein profiles also support the idea as the correlation between Cry proteins and cry genes was observed, and the presence of a PCR band alone was not sufficient to guarantee its expression. Cryptic cry genes can arise by frequent recombination events among the genes present in the Bt strains. The insecti-


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