Introduction
In nature, some microorganisms have the potential to produce useful biological agents capable of infecting other living organisms including insects. Many of these infectious agents have a narrow host range and are non toxic to beneficial insects or vertebrates (Glazer and Nikaido, 1995). Therefore, the use of these microorganisms has been developed as the biological way of pest control. Viruses (baculoviruses), some fungi, protozoa and bacteria have been used as biological pest control agents for insects. Amongst all, Bt is the most important microorganism having entomopathogenic activity against certain insect orders.

In addition to the crystal associated toxic polypeptides, some insecticidal proteins produced during vegetative growth of the bacteria have also been identified called as vegetative insecticidal proteins (VIPs). These proteins were reported from about 15 per cent of the Bt strains analyzed (Estruch et al., 1996). According to the encoded amino acid sequence similarity the Bt nomenclature committee classified these 103 kinds of vegetative insecticidal protein genes into four groups viz. vip1, vip2, vip3 and vip4, eight subgroups, 29 classes and 103 subclasses (Crickmore, 2012). The vip1 and vip2 proteins are the components of the binary toxin that exhibits toxicity to the coleopterans (Warren, 1997). Vip1Aa1 and vip2Aa1 are very active against corn rootworms, particularly Diabrotica virgife. longicornis (Hann et al., 1999); whereas, vip3 toxins have Lepidoperan specificity (Estruch et al., 1996, Warren, 1997). The transgenic cotton, VipCot, developed by Syngenta in 1994 provides cotton growers a way to control bollworms which offers the added benefit of protecting cotton plants from armyworm and loopers.

Worldwide, there are several reports of field evolved resistance in H. armigera against Cry toxins (Van Rensburg, 2007, Dhurua and Gujar, 2011). Resistance has been reported to develop in Australia (Mahon et al., 2007); USA (http://www.epa.gov/ oppbppd1 /biopesticides/ pips/Bt_corn_refuge_2007.html.); Arizona state of USA (Van Rensburg, 2007, Tabashnik et al., 2008); China (Liu et al., 2008). Also, in India resistance scenario is becoming a matter of great concern, pink bollworm has already developed resistance against Cry1Ac (Dhurua and Gujar, 2011, Tabashnik and Carriere, 2010). Similarly, H. armigera from Bathinda and Muktsar (Punjab) have reported to develop resistance against Cry1Ac (Kaur and Dilawari, 2011). Apart from using various field adopted insect resistance management (IRM) strategies, molecular IRM strategies have potent genes/protein to overcome the resistant population. It is a suitable alternative to sustain the success of Bt transgenic technology in India.

The discovery of novel genes from native Bt can overcome the unavoidable resistance development in insects. The present study has explored the native isolates of Bt to screen the presence of vip genes and its diversity. This study also promises to yield more potent strain of Bt than standard Bt. By exploring the possibilities of getting new and more potent Bt vegetative insecticidal protein genes, we can overcome or prolong by increasing insect resistance against Bt toxins.

1 Results
1.1 Molecular screening of local Bt isolates
Four isolates viz. PDKV-08, PDKV-27, PDKV-28 and NCIM-5112, showed amplification for vip1/vip2 gene with expected size of 742 bp (Figure 1). vip1/vip2 proteins have prime future prospects due to potent insecticidal activity against coleopteran insect pest. Warren in 1997 reported 11.9% a bundance of vip1/vip2 gene amongst 463 Bt isolates. Shi et al. (2006) also confirmed that these two genes are located on single operon and co-expressed.
 

Figure 1 Screening of Bt isolates for the presence of vip1/vip2 binary toxin gene

Twenty eight local isolates, eleven different Bt strains and reference strain HD1 were surveyed for the presence of vip3A genes. Amongst 40 strains 5 strains viz., PDKV-08, PDKV-21, NCIM-5110, NCIM-5132 and HD-1 showed the presence of vip3A gene by using vip3A (partial) primer (Figure 2A). 
 

Figure 2 Screening of Bt isolates for the presence of vip3A genes

However, vip3A (full length) primer showed the presence of vip3A gene in four isolates viz. PDKV-21, NCIM-5132, NCIM-5130 and HD-1. Percent abundance data showed 15% abundance of vip3A gene in local Bt isolates (Figure 2B). Out of 40 isolates five isolates viz. PDKV-3, PDKV-8, NCIM-5110, NCIM-5132 and HD-1 showed successful amplification of vip3Aa1 gene with expected size of 2400 bp.

In total, three types of vip genes (vip1, vip2, and vip3) were identified from the available Bt strains with the primer pairs of vip1/vip2, vip3A partial, vip3A full length, and vip3Aa1. Some types of vip gene were not identified in certain strains. 

1.2 SDS PAGE profiling of different vip proteins
SDS-PAGE was performed for the total protein extracted from all 40 supernatants although several common bands (Figure 3) were detected among some strains. Overall results clearly demonstrated a strain-specific pattern of polypeptide secretion in the culture medium, which reflect final insecticidal potential of the respective isolate. The PDKV-8 isolate displayed significant high larvae mortality against lepidopteran pest (Table 1). PDKV-08 have particularly relevant band in the position of the putative VIP3A-like polypeptide (88 kD,) appeared much more intense than in all others lanes (Figure 3). Also, strains PDKV-21, NCIM-5132, NCIM-5110 and HD-1 showed the presence of 88 kD band which already depicts positive results in molecular screening for vip3A gene specific primer. 
 

Figure 3 SDS PAGE electrophoretogram of different vip proteins M- unstained protein molecular weight marker

Table 1 Toxicity of partially purified toxin from different Bt isolates for H. armigera

1.3 Insect bioassay to study the insecticidal potential
Four isolates of Bt were subjected for insect bioassay viz. PDKV-8, PDKV-21, NCIM-5110 and NCIM-5132 with HD-1 as standard reference strain. Four isolates were selected as a representative samples as PDKV-08, NCIM-5110 and NCIM-5132 showed vip3A and vip3Aa1 apart from this, PDKV08 also found to harbour vip1/vip2. PDKV-21 showed the presence of very prominent vip3A gene (Figure 4). 
 

Figure 4 Insect bioassay of vip toxins against H. armigera

The molecular analysis and protein electrophoretogram of vip proteins strongly supported the presence of vip3 proteins in selected isolates to study their insecticidal activity. Though there was no high magnitude difference in the LC₅₀ values. The all test isolates showed lower LC₅₀ values than reference strain as showed in table 1. Insect bioassay is considered as very important and confirmation studies with respect to the insecticidal protein. 

2 Discussins
The present investigation was carried out to characterize Bt isolates obtained from native ecological niche for the presence of different vip genes and also to explore the possibility of getting any new vip gene (s). The recent insect resistance scenario prompted us to attempt this study. It is first attempt to study the distribution of different vip genes in local Bt isolates from Vidarbha region of Maharashtra (India).

It has been reported that vip proteins account for about 15% of the Bt strains (30). With the three pairs of primers, we found that the average rates of vip3A (12.5%) was the most and the average rates of vip1/vip2 (10%) were similar with the results reported by Herna´ndez-Rodrı´guez et al. (2009). Whereas in present finding, abundance of vip like genes are not more than 12.5%. Other researchers have reported such lower frequencies of vip genes: 23.2% (Rice, 1999), 2% (Selvapandiyan et al., 2001) and 33.3% (Bhalla et al., 2005). However, Loguericio et al. (Loguercio et al., 2002) and Fang et al. (Fang et al., 2007) reported the presence of vip like genes in all the isolates, Beard et al. (Beard et al., 2008) also showed the similar trends. Yu et al. (2010), found 67.4% abundance of vip3 type of genes in from Sichuan basin in China. Similarly, Liu et al. (2008), found 63.03%. High abundance of vip genes in these reports are contradictory to our findings. This might be due to difference in habitat, geographical location and other environmental conditions.

The main purpose of our research was finding insecticidal proteins for Lepidoptera. Therefore, this study concentrated on finding novel vip3-type genes. We have found five novel vip genes, which could enrich the available categories of insecticidal genes. However, our isolates had very few novel vip genes. The reason why there were only five novel vip3 genes in screened Bt strains might be that the vip genes (three groups of vip genes have been researched) were more conserved than cry genes (67 groups of cry genes have been researched), and the referenced method may not be the best way to probe for vip genes (Fang et al., 2007). Therefore, we should find better ways to identify novel genes.

To find toxicity of vip3 for H. armigera, the four novel vip proteins of the screened Bt strains were assayed with H. armigera. The partially purified proteins of Bt were bioassayed against H. armigera with positive and negative controls. The bioassay results indicated that the 4 screened Bt samples really include novel vip3 genes. The presence of different insecticidal genes detected, may account for variation in the toxicity of these isolates. According to Monnerat et al. (Monnerat et al., 2007) the greater potency of these isolates than Bt-kurastaki HD 1 against the target insects may be due to (i) variation in the number of genes harbour by the isolates, (ii) higher level of accumulation of toxins, (iii) the presence of other Bt toxin genes that are not detected by the primers used in the screening (iv) combination of all these factors.

3 Materials and Methods
3.1 Local Bt strains used in present investigation
40 local Bt strains were subjected for molecular screening. Out of these, 28 isolates (PDKV-01 to 28) were isolated by Kedar, S.B. 2011 from the campus of Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola and Nagpur campus of Dr. PDKV. Reference strain HD1 was kindly supplied by Dr. Donald Dean (Bacillus Genetic Stock Centre, Columbus, Ohio). Similarly eleven different subspecies of Bt were obtained from NCIM (National Center for Industrially useful Microbes (NCIM)), NCL, Pune.

3.2 Total genomic DNA isolation and PCR screening of Bt isolates
Genomic DNA was isolated from the Bt isolates as per the method given by Kate Wilson (Wilson, 1997). All PCR amplifications were performed by using the gradient thermal cycler (EP gradient, Eppendorf). Isolates were tested for the presence of vip genes with primers indicated in Table 2.
 

Table 2 The primers for identification of vip genes and finding novel vip3-type genes

After the positive screening of Bt isolates for the presence of vip genes attempts are made to harbour the full length vip genes. Three primers are designed on the basis of full length vip gene sequences to amplify the coding DNA sequences (cds) of vip genes. PCR results were analysed by using 1.2% agarose gel.

3.3 Isolation of vegetative insecticidal proteins
According to the method given by Sattar et al. (2008) vegetative insecticidal proteins were obtained for insect bioassay. Proteins present in the supernatant were precipitated with ammonium sulphate [(NH₄)₂SO₄] (80% saturation) and collected by centrifugation at 12 000 g for 10 min at 4℃. The pellet was re-suspended in minimum volume of 20 mM of Tris-HCl buffer (pH 7.4) and dialyzed overnight at 4℃ against 20 mM Tris HCl (pH 7.4).

The purified proteins were estimated by the Bradford method (Bradford, 1976) and used for insect toxicity assay.

3.4 SDS-PAGE profiling of vip proteins of Bt
Vegetative insecticidal proteins were performed by using 10% Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) according to method given in protocol Sambrook and Russell (Sambrook and Russell, 2007).

3.5 Insect Bioassay for determining insecticidal potential of vip proteins
Biological activity of vip3A toxins were confirmed by using standard bioassay procedures. Lepidoptera bioassays against H. amigera neonates were conducted with diet incorporation method (Yu et al., 1997). Bt subsp. kurstaki was used as positive control. In each experimental set, ten such neonates were released and observed after each 12 hrs.

4 Conclusions
The study led to the identification of a novel vip3A gene, which is known to effective, against coleopterans. It is necessary to study the effect against insect pests, clone these genes into a plant expression vector, transform crop plants and study their effectiveness against different insect pests.

Authors’ contributions
PS conducted all the research for this paper and prepared the manuscript. DD participated in bioassays and molecular characterization. AD coordinated the protein isolation, purification and profiling experiments. Molecular screening and characterization was performed in supervision of PJ and MS. The insect bioassays were carried out under supervision of NS. MM reviewed the manuscript and coordinated the project.

Acknowledgements
Authors are thankful to Head, Department of Agril Botany and Officer In-charge, Biotechnology Centre, Dr. Panjabrao Deshmukh Agricultural University, Akola, for their support.

References
Beard C.E., Court, L., Boets A., Mourant R., Rie V.J., and Akhurst R.J., 2008, Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bt collection, Curr. Microbiol., 57(3):195-199
http://dx.doi.org/10.1007/s00284-008-9173-1  PMid: 18592309

Bhalla R., Dalal M., Panguluri S.K., Jagadish B., Mandackar A.D., Singh A.K., and Kumar P.A., 2005, Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bt, FEMS Microbiol. Lett., 243(2): 467-472
http://dx.doi.org/10.1016/j.femsle.2005.01.011  PMid:15686851

Bradford M.M., 1976, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analytical Biochemistry, 17: 248-254
http://dx.doi.org/10.1016/0003-2697(76)90527-3

Bravo A., and Soberon M., 2008. How to cope with insect resistance to Bt toxins? Trends Biotechnol., 26(10): 573-579
http://dx.doi.org/10.1016/j.tibtech.2008.06.005  PMid:18706722

Dhurua S., and Gujar G.T., 2011, Field-evolved resistance to Bt toxin Cry1Ac in the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), from India, Pest Management Science. 67(8): 898-903
http://dx.doi.org/10.1002/ps.2127  PMid:21438121

Estruch J.J., Warren G.W., Mullins M.A., Nye G.J., Craig J.A., and Koziel M.G., 1996, Vip3A a novel Bt vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects, Proc. Natl. Acad. Sci. USA. 93(11): 5389-5394
http://dx.doi.org/10.1073/pnas.93.11.5389  PMid:8643585 PMCid:PMC39256

Jun Fang, Xiaoli Xu, Ping Wang, Jian-Zhou Zhao, Anthony M. Shelton, Jiaan Cheng, Ming-Guang Feng, and Zhi-cheng Shen, 2007. Characterization of chimeric Bt Vip3 Toxins. Appl. Environ. Microbiol., 73(3): 956-961
http://dx.doi.org/10.1128/AEM.02079-06  PMid:17122403 PMCid:PMC1800787

Glazer A.N., and Nikaido H., 1995, Microbial insecticides, Microbial Biotechnology Fundamentals of Applied Microbiology, W.H. Freeman and Company, New York, pp.209-229

Han S., Craig J.A., Putnam C.D., Carozzi N.B., and Tainer J.A., 1999, Evolution and mechanism from structures of an ADP-ribosylating toxin and NAD complex, Nature Struct. Biol., 6(10): 932-936
http://dx.doi.org/10.1038/13300 PMid:10504727

Herna´ndez-Rodrı´guez C.S., Boets A., Van-Rie J., and Ferre J., 2009, Screening and identification of vip genes in Bt strains, J. Appl. Microbiol., 107(1): 219-225
http://dx.doi.org/10.1111/j.1365-2672.2009.04199.x  PMid:19302326

Kaur P., and Dilawari V.K., 2011, Inheritance of resistance to Bt Cry1Ac toxin in Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) from India, Pest Man Science. 67(10): 1294-1302
http://dx.doi.org/10.1002/ps.2185 PMid:21567889

Liu F., Xu Z., Chang J., Chen J., Meng F., Zhu Y., and Shen J., 2008, Resistance Allele Frequency to Bt Cotton in Field Populations of Helicoverpa armigera (Lepidoptera: Noctuidae) in China, J. Econ. Entomol., 101(3): 933-943
http://dx.doi.org/10.1603/0022-0493(2008)101[933:RAFTBC]2.0.CO;2

Loguercio L.L., Barreto M.L., Rocha T.L., Santos C.G., Teixeira F.F., and Paiva E., 2002, Combined analysis of supernatant-based feeding bioassay and PCR as a first-tier screening strategy for Vipderived activities in Bt strains effective against tropical fall armyworm, J. Appl. Microbiol., 93(2): 269-277
http://dx.doi.org/10.1046/j.1365-2672.2002.01694.x  PMid:12147075

Mahon R.J., Olsen K.M., Downes S., and Addison S., 2007, Frequency of Alleles Conferring Resistance to the Bt Toxins Cry1Ac and Cry2Ab in Australian Populations of Helicoverpa armigera Lepidoptera: Noctuidae), Journal of Economic Entomology, 100(6): 1844-1853
http://dx.doi.org/10.1603/0022-0493(2007)100[1844:FOACRT]2.0.CO;2

Monnerat R.G., Batista A.C., Medeiros P.T., Martins E.S., Viviane M.M., Praca L.B., Dumas V.F., Moringa C., Demo C., Gomes C.M., Siqueira C.B., Silva-Werneck J.O., and Berry C., 2007, Screening of Bt isolates active against Spodoptera frugiperda, Plutella xylostella and Anticarsia gemmatalis, Biological control, 41: 291-295
http://dx.doi.org/10.1016/j.biocontrol.2006.11.008

Rang C., Gil P., Neisner N., Rie V.J., and Ftuos R., 2005, Novel Vip3-related protein from Bt, Appl. Environ. Microbiol., 71(10): 6276-6281
http://dx.doi.org/10.1128/AEM.71.10.6276-6281.2005 PMid:16204549 PMCid:PMC1265940

Neil crickmore, 2012, Recent classification of the Bacillus thuringinesis, http://www.biols.susx.ac.uk/Home/Neil Crickmore/BtBt/vip html.2012

Rice W.C., 1999, Specific primers for the detection of Vip3A insecticidal gene in a Bt collection, Letters in Applied Microbiology, 28: 378-382
http://dx.doi.org/10.1046/j.1365-2672.1999.00536.x

Sambrook and Russell, 2007, Molecular cloning: a laboratory manual, 3^rd ed., Cold Spring Harbor Laboratory Press, New York. pp.1.31-1.162

Sattar S., Biswas P. K., Hossain M.A., Maiti M.K., Sen S. K., and Basu A., 2008, Search for Vegetative Insecticidal Proteins (VIPs) from local isolates of Bt effective against lepidopteran and homopteran insect pests, Journal of Biopesticides, 1(2): 216-222

Selvapandiyan A., Arora N., Rajagopal R., Jalali R., Venkatesan T., Singh S., and Bhatnagar R., 2001, Toxicity Analysis of N- and C-Terminus-Deleted Vegetative Insecticidal Protein from Bt. Appl. Environ. Microbio., 67(12): 5855–5858
http://dx.doi.org/10.1128/AEM.67.12.5855-5858.2001 PMid:11722946 PMCid:PMC93383

Shi Y., Wenli M., Meijin Y., Fan S., and Pang Y., 2006, Cloning of vip1/vip2 genes and expression of Vip1Ca/Vip2Ac proteins in Bt, World Journal of Microbiology and Biotechnology, 23(4): 501-507
http://dx.doi.org/10.1007/s11274-006-9252-z

Tabashnik B.E., and Carriere Y., 2010. Field evolved resistance to Bt Cotton: bollworm in the U.S. and pink bollworm in India, Southwestern Entomologist. 35(3): 417-424
http://dx.doi.org/10.3958/059.035.0326

Tabashnik B.E., and Gassmann A.J., Crowder, D.W., and Carriere, Y., 2008, Insect resistance to Bt crops: evidence versus theory, Nature Biotechnology, 26(2):199-202
http://dx.doi.org/10.1038/nbt1382PMid:18259177

Van Rensburg J. B. J., 2007, First reports of field resistance by the stem borer, Busseola fusca (Fuller) to Bt-transgenic maize, S. Afr. J. Plant Soil, 24: 147-151
http://dx.doi.org/10.1080/02571862.2007.10634798

Warren G.W., 1997, Vegetative insecticidal proteins: novel proteins for control of corn pests, In: Carozzi N.B., Koziel M., (eds.), Advances in insect control, the role of transgenic plants, Taylors & Francis Ltd., London, pp.109-121
http://dx.doi.org/10.1201/9780203211731.ch7

Wilson K., 2001, Preparation of Genomic DNA from Bacteria, Current Protocols in Molecular Biology, 2.4.1-2.4.5

Wu J.Y., Zhao F.Q., Bai J., Deng G., Qin S., and Bao Q.Y., 2007, Evidence for positive Darwinian selection of vip gene in Bt, J. Gene Genom., 34(7):649-660
http://dx.doi.org/10.1016/S1673-8527(07)60074-5

Wu Z.L., Guo W.Y., Qiu J.Z., Huang T.P., Li X.B., and Guan X., 2004. Cloning and localization of vip3A gene of Bt, Biotechnological Letters, 26(18): 1425-1428
http://dx.doi.org/10.1023/B:BILE.0000045645.45536.3f PMid:15604775

Yu C.G., Mullins M.A., Warren G.W., Koziel M.G., and Estruch J.J., 1997, The Bt vegetative insecticidal protein Vip3 lyses midgut epithelium cells of susceptible insects, Appl. Environ. Microbiol.,63(2): 532-536
PMid:9023933 PMCid:PMC168345

Yu X., Zheng A., Zhu J., Wang S., Wang L., Deng Q., Li S., Liu H., and Li P., 2010, Characterization of vegetative insecticidal protein vip Genes of Bt from Sichuan Basin in China, Curr. Microbial., 62(3):752-757
http://dx.doi.org/10.1007/s00284-010-9782-3 PMid:20963416