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Characterization of lepidopteran-specific cry1 and cry2 gene harbouring native Bacillus thuringiensis isolates toxic against Helicoverpa armigera

Highlights • The manuscript deals with the characterization of lepidopteran-specific cry1 and cry2 gene harbouring Bt isolates.• We were able to find certain Bt isolates containing both cry1 and cry2 genes.• Both cry1 and cry2 genes were found in isolates containing vip3A genes, hence can result in complementation of toxicity.• 13.63% of the Bt isolates were found to be toxic against Helicoverpa armigera.


Introduction
As the world's population is increasing geometrically, achieving global food security-making safe and nutritious food accessible to everyone, and achieving so sustainably is a challenging task. Feeding estimated 9.2 billion people in 2050 would require raising overall food production by about 70% [1]. A major bottleneck in achieving this challenge is the competition from the insect pests. Insect pests are responsible for destroying one fifth of the world's total crop production annually, leading to heavy economic losses. The major damaging insect pests of crops belong to the order Lepidoptera [2] and Helicoverpa armigera is one of the most significant lepidopteran pests with potential to attack more than 180 species of plants [3]. It is widely distributed in Asia, Europe, Africa and Australasia causing damages worth 2 billion US dollars annually, excluding the socio-economic and environmental costs associated with the use of chemical insecticides and the introduction of GM crops [4][5][6]. H. armigera has over the years developed resistance to various chemical insecticides [7,8] and of late, its resistance to genetically modified crops expressing insecticidal protein from B. thuringiensis has also been reported [9,10].
The most common method to control insect pest populations is the use of chemical insecticides. Two of their properties, long residual action and toxicity to a wide spectrum of organisms made chemical insecticides very useful against insect pests. However, extended use of certain chemical insecticides have caused many environmental problems like persistence, toxicity to non-target organisms including humans and development of insect resistance [11,12]; reviewed in [13]. One of the most promising alternatives to the man-made chemical pesticides is the use of natural insect pathogen, Bacillus thuringiensis (Bt). The entomopathogenic potential of Bt is primarily due to its ability to produce insecticidal crystalline proteins (Cry and Cyt) [14] and in certain cases due to the production of vegetative insecticidal proteins (Vips) [15]. The crystalline and vegetative insecticidal proteins are respectively produced during the sporulation and vegetative stages of Btgrowth. Upto November 2016, seventy four classes of Cry proteins (Cry1-Cry74), three classes of Cyt proteins (Cyt1-Cyt3) and four classes of Vip proteins (Vip1-Vip4) have been designated based on their amino acid sequence homology [16]. These toxins are highly specific in action, harmless to humans and other vertebrates and are biodegradable. Presently, there are more than 50,000 known strains of B. thuringiensis isolated from diverse environments around the world [17,18]. These strains exhibit varying degree of toxicity against different pests. Despite the availability of such large collection of B. thuringiensis strains and their insecticidal genes, three events have rendered the search for novel insecticidal strains/genes more urgent. First, a significant number of pests are not controlled with the available Cry proteins. Second, at times the level of expressed toxins is not high enough to kill the host and third, after many years of successful use in the field, the first cases of resistance to B. thuringiensis have appeared [19].
Jammu and Kashmir (32°00′-36°10′ North and 73°22′-77°40′ East) falls in the great North-Western complex of the Himalayan ranges having complex geomorphology. The variations in topographical features along longitude, latitude and altitude of the region create climatic variations resulting in unique and rich biodiversity [20], thereby making this North-Western Himalayan region a critical biodiversity hotspot of the world. These distinctive features and diversity of insects in the region provide an opportunity for prospecting novel B. thuringiensis strains with novel combinations of crystalline protein coding genes having wide insecticidal spectrum. The ecological distribution of this bacterium in Jammu and Kashmir region remains largely unexplored. The aim of this study was to isolate B. thuringiensis strains from Jammu and Kashmir region and to assess their geographical diversity with respect to the presence of lepidopteran-specific (cry1 and cry2) genes content to assess their toxicity against H. armigera.

Sample collection and isolation of Bacillus thuringiensis
A total of 86 isolates were analysed in this study which were obtained from various soil samples, collected from forests, lake sediments and agricultural fields of Kashmir valley in our previous study [21]. Sites with no history of Bt application were selected ( Table 1). Isolation of Bt was done by enrichment using acetate selection as described by Travers et al. [22]. Briefly, 1 g of soil samples were incubated in 10 ml of LB broth buffered with sodium acetate solution (0.3 M, pH 6.8) at 30°C for 4 h. In order to eliminate non-sporulated microbes that germinated during incubation, 2 ml aliquot of each sample was heated at 80°C for 10 min. The surviving spores were diluted (10-1000 folds) in sodium acetate buffer (pH 6.8) from which 300 μl of each was spread on T3 agar plates and incubated at 30°C to grow for 72 h. For each plated sample, well isolated colonies representing Bacillus like morphology were picked and purified on T3 agar plates containing penicillin at a concentration of 10 μgml −1 .

Scanning electron microscopy
Following acetate selection, isolates that tested positive for growth on T3 agar medium amended with penicillin at a concentration of 10 μgml −1 were examined for the presence of parasporal crystals [23,24]. The different stages of bacterial growth as well as the crystal production was analysed by Scanning Electron Microscopy (SEM) as described previously [21]. Bt index was calculated as described by Baig et al. [25].

16S rRNA gene sequencing
Total cell DNA was extracted from the native and reference strains using GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, USA) as per the manufacturer's instructions. PCR amplification of 16S rRNA gene from the selected Bacillus isolates was performed using the universal primers: forward (27f) 5′-AGAGTTTGATCCTGGCTCAG-3′, reverse (5210r) 5′-AAGGAGCTGATCCAGCCGCA-3′. The purified amplicons of 16S rRNA gene were sequenced using primers 27f and 5210r with fluorescent terminators (Big Dye, Applied Bio systems). The identity of the sequences obtained were determined by NCBI BLAST [26].

PCR amplification of cry1 and cry2 genes
Using total DNA as template, the cry genes were amplified using primers (+)5′-MDATYTCTAKRTCTTGACTA-3' and (−)5′-TRACRHTDDBDGTATTAGAT-3′ [27] for the detection of cry1 and (+) 5′-GTTATTCTTAATGCAGATG-AATGGG-3′ and (−)5′-CGGATAAAAT-AATCTGGGAAATAGT-3′ [28] for the detection of cry2 class of genes. PCR was performed in an Eppendorf Mastercycler and amplification conditions were as follows: an initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 60 s, annealing at 47°C and 59°C for cry1 and cry2 primers, respectively for 60 s, and extension at 72°C for 60 to 90 s (depending on the expected size of amplicon), and a final extension step at 72°C for 10 min. An aliquot of the samples were mixed with 6X DNA loading buffer so that the final concentration of the mixture is 1X, electrophoresis was carried out at 70-80 mA current in 1X TAE, for 45-90 min. An appropriate DNA marker was run alongside in one of the lanes. To visualize the DNA bands gels were viewed under UV light (λ = 254 nm) and images captured using AlphaImager HP (ProteinSimple, USA) gel documentation system.

Insect bioassays
The insecticidal activity of cry gene harbouring Bt isolates was evaluated against 2nd instar larvae of H. armigera by "quick screen" method [29]. The spore crystal mixtures were obtained as described for microscopy analysis. Higher doses of spore crystal mixtures (1 mgml −1 ) were smeared on young chick pea leaves in triplicates. The larvae were initially left for 10 h with the leaves smeared with spore crystal mixtures and then allowed to feed on fresh leaves for 12 h. Based on their ability to subsequently feed on the fresh diet, the treated larvae were scored as: not eaten was assigned a score of 3; partially eaten a score of 2 and completely eaten a score of 1. Based on the scores assigned, the toxicity potential of the isolates was classified as, non-toxic for isolates displaying score of 1.0, uncertain toxicity for isolates with score > 1.0 but < 2.0 and toxic for isolates exhibiting score of ≥2.0. B. thuringiensis subsp. kurstaki HD-1 was used as positive control and solubilisation buffer as negative control. The conditions for bioassay were as; 25°C, 50 ± 10% RH and a 14:10 (light/dark [h]) photoperiod.

Identification of B. thuringiensis
Eighty six Bacillus isolates were recovered by applying acetate-penicillin selection methodology to 30 soil samples collected from three types of soils (Table 1). Forty four isolates were found to synthesize crystal protein during sporulation by phase contrast microscopy. The cell shape of B. thuringiensis was recorded at different stages of growth (Fig. 1). B. thuringiensis was successfully isolated from all types of samples, recovery was highest from lake sediments (0.75) followed by forest soils (0.60) and the least from agricultural soils (0.33) ( Table 1). The average Bt index of the study was 0.51 (Table 1). A 1500 bp amplicon corresponding to 16S rRNA was successfully amplified in all the Table 1 Features of sampling sites, success of Bt isolation and the distribution of cry1 and cry2 genes in the native isolates.   isolates using total cell DNA as template (Fig. 2). The amplicons were sequenced and the partial sequences were aligned with the 16S rRNA gene sequences of Bt available from GenBank. It was observed that the obtained sequences showed 95-100% identity with the reported sequences on BLAST analysis ( Table 2). The partial 16S rRNA gene sequences were submitted to GenBank and accession numbers were obtained [21].

Amplification of cry genes
PCR screening using two pairs of universal primers was performed to detect cry1 and cry2 gene families in our collection. Primers for the detection of both cry1 and cr2 showed successful amplification as indicated by their specific product sizes of 1500 and 700 bp, respectively on agarose gel electrophoresis (Fig. 3). Twenty six isolates of the collection were found to contain either or both the cry genes tested while as 18 isolates did not show the presence of either of the genes. Compared to cry2, cry1 genes were found to be more abundant and in most of the isolates (12) they were found together.

"Quick screen" and toxicity assay against Helicoverpa
All the 44 native B. thuringiensis isolates and the reference strain HD1 were subject to toxicity screening using concentrated spore crystal suspensions (1 mgml −1 ). Twenty two out of 44 isolates were assigned a score of 1.0-2.0 as the leaves were partially eaten, 16 isolates were assigned a score of 1.0 because the leaves were completely eaten in this case while as only six isolates and the reference strain were assigned a score of 2 as the leaves were not eaten at all by the larvae. The scores indicate the mild toxicity, absence of toxicity and high toxicity of the isolates, respectively (Fig. 4).

Discussion
Although, distribution of cry genes of B. thuringiensis in some geographical regions of India has been investigated earlier [30,31], no researcher has systematically analysed the distribution of cry genes in soils of Jammu and Kashmir region. So, it is of interest to determine the distribution and diversity of cry genes and identify the type of cry genes. Since lepidopteran pests are the most predominant among all other insect pests and cause heavy economic loss, this study was carried to characterize and study the distribution of Bt isolates containing lepidopteran-specific cry1 and cry2 genes. Further, the toxicity of the isolates was evaluated against an economically important lepidopteran pest H. armigera.
The presence of a parasporal inclusion body is a diagnostic feature to discriminate B. thuringiensis species form its close relatives in the Bacillus cereus group [32]. Based on the presence of parasporal crystals,  [25,21,33,34]. The likely reason for difference in Bt index is hard to access, however, the difference in geomorphology and the interaction of the bacterium with their insect hosts could influence their numbers in different environments. 16S rRNA gene sequencing is a routine method used for the taxonomic identification of bacteria [35]. However, the method has not been successful in clearly distinguishing the members of the B. cereus group of which B. thuringiensis is one of the member [35]. So, the partial 16S rRNA gene sequences were considered B. thuringiensis for the statistical counting if they showed maximum similarity to either B. thuringiensis or B. cereus.
PCR-based approach has been extensively utilized for the identification of known and novel cry genes in B. thuringiensis since its introduction by Carozzi et al. [36]. The rapidity and reproducibility of PCR-based detection of cry genes makes it a very useful tool even today. Both cry1 and cry2 genes were detected in the isolates, cry1 was found to be more prevalent than cry2 and most of the cry1 harbouring isolates also contained cry2 gene. The predominance of cry1 gene compared to all other cry genes in the native B. thuringiensis has been reported extensively [37,38]. However, predominance of cry genes other than cry1 has also been reported [25,28,39,40]. Twenty six out of 44 B. thuringiensis isolates showed the presence of either cry1 or cry2 genes among which 12 isolates showed the presence of cry1 and cry2 in combination ( Table 2). The possible reason for both cry1 and cry2 genes being found together is that both the genes may be closely located on the genome and have evolved together [41]. Isolates containing both the cry genes are ideal for development of biocontrol agent as both the proteins encoded by these genes are specific against lepidopteran pests. Eighteen of the 44 isolates did not show presence of either cry1 or cry2, suggesting that they may contain other types of cry genes not tested in this study. A large number of isolates harbouring no cry genes has been reported in various B. thuringiensis collections previously [42,43].
All the putative B. thuringiensis isolates were subjected to toxicity screening against 2nd instar larvae of H. armigera using higher doses of spore crystal mixtures. Only 13.63% of the isolates exhibited toxicity as evidenced by the treated larvae not being able to feed on fresh leaves, 50% of the isolates were mildly toxic whereas 36.36% were found to be non-toxic (Fig. 4). The difference in toxicity is multifactorial including the absence of toxin proteins and due the poor expression of the Cry proteins.
In the present study, B. thuringiensis isolates from various soil samples were found to harbour either or both of the cry1 and cry2 genes and some of them were found to be toxic against H. armigera. More precise bioassays using different doses of spore crystal mixtures against more number of larvae shall lead to the identification of potentially toxic isolates for the control of H. armigera.

Conflict of interest
The authors declare no conflict of interest.