Screening of Bacillus thuringiensis Isolates Recovered from Diverse Habitats in India for Crystal Toxin Genes Predicting Toxicity Against Three Insect Orders: Lepidoptera, Coleoptera, and Hemiptera

Screening of Bacillus thuringiensis Isolates Recovered from Diverse Habitats in India for Crystal Toxin Genes Predicting Toxicity Against Three Insect Orders: Lepidoptera, Coleoptera, and Hemiptera | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Screening of Bacillus thuringiensis Isolates Recovered from Diverse Habitats in India for Crystal Toxin Genes Predicting Toxicity Against Three Insect Orders: Lepidoptera, Coleoptera, and Hemiptera Subhash A, TharunKumar C J, S Pallavi, Akshay Bharat Ghodake, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8459314/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Entomopathogenic Bacillus thuringiensis ( Bt ) has emerged as a sustainable alternative to chemical pesticides due to its selective action against insect pests and negligible effects on non-target organisms. However, the increasing occurrence of resistance to conventional cry genes poses a significant threat to their efficacy. It underscores the immediate need to identify novel cry genes with unique modes of action and toxicity profiles. To address this, our study investigated the frequency of recently discovered neoteric cry genes in Indian Bt isolates, known for their diverse range of insecticidal cry genes. We screened seventy-nine indigenous Bt isolates collected from diverse agroclimatic zones and natural habitats across India for novel cry genes with activity against Lepidoptera, Coleoptera, and Hemiptera orders. PCR-based analysis confirmed the presence of various insect-specific cry genes, revealing notable diversity among the isolates. Twenty-seven isolates carried single or multiple cry genes, with conventional cry7/8 genes being more prevalent than neoteric genes. Among the latter, cry30Fa was the most abundant, followed by cry30Ga, cry15Aa, cry64Ca, cry79Aa1, cry78Aa , and cry78Ba . Remarkably, isolates SK-768, SK-979, SK-110, and SK-949 harbored cry genes active against two or three insect orders. These findings highlight the importance of region-specific Bt strains in formulating effective biocontrol strategies. Biocontrol Indian Bacillus thuringiensis (Bt) isolates Prevalence of cry genes Insect resistance control Polymerase Chain Reaction (PCR)-based screening Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION The agricultural sector stands as a cornerstone of global food security, yet it grapples with persistent and escalating challenges posed by insect pests. These insidious adversaries inflict substantial damage on crops, leading to significant yield losses and economic instability worldwide. According to the Food and Agriculture Organization (FAO), insect pests are responsible for the destruction of up to 40% of annual global crop production, resulting in an economic burden exceeding $ 120 billion annually [ 1 ] [ https://www.fao.org/ ]. This staggering figure underscores the urgent need for effective and sustainable pest management strategies to safeguard agricultural productivity and ensure food availability for a growing global population. Among the diverse array of insect pests that plague agricultural systems, Lepidoptera (e.g., corn earworm, diamondback moth), Coleoptera (e.g., Colorado potato beetle, boll weevil), and Hemiptera (e.g., aphids, whiteflies, planthoppers) stand out as particularly destructive. These insect orders encompass a wide range of species that exhibit diverse feeding habits and host preferences, enabling them to exploit various crops and inflict damage at different stages of plant development [ 2 ]. The voracious feeding behavior of these pests can lead to defoliation, stunted growth, reduced fruit production, and even plant death, resulting in significant economic losses for farmers and compromising food security. For decades, chemical pesticides have been the primary tool for combating insect pests in agriculture. These synthetic compounds are designed to kill or repel insects, providing rapid and effective control of pest populations. However, the widespread and often indiscriminate use of chemical pesticides has raised serious concerns about their long-term environmental and human health impacts [ 3 ]. Chemical pesticides can contaminate soil and water resources, disrupt beneficial insect populations, and pose risks to human health through direct exposure or consumption of contaminated food. Furthermore, the overuse of chemical pesticides has led to the emergence of resistant insect populations, rendering these compounds ineffective and necessitating the development of new and more potent pesticides. In response to the drawbacks associated with chemical pesticides, biological control agents, particularly those based on Bacillus thuringiensis ( Bt ), have emerged as promising and sustainable alternatives for pest management. Bt is a gram-positive, spore-forming bacterium that produces crystal proteins (Cry) with specific insecticidal activity against various insect orders [ 4 ]. These Cry proteins are selectively toxic to certain insect species, making them highly effective for targeted pest control while minimizing harm to non-target organisms, including beneficial insects, wildlife, and humans [ 5 ]. Bt -based biopesticides have been widely adopted in agriculture due to their efficacy, environmental safety, and compatibility with integrated pest management (IPM) programs [ 6 ]. The mechanism of action of Cry proteins involves their ingestion by susceptible insect larvae, followed by their activation in the insect midgut. Once activated, Cry proteins bind to specific receptors on the midgut epithelial cells, leading to pore formation and disruption of cellular function. This results in paralysis, gut leakage, and ultimately, death of the insect larva [ 7 ]. The specificity of Cry proteins for insect midgut receptors is a key factor in their safety for non-target organisms, as these receptors are not found in vertebrates or beneficial insects. Despite the widespread use of Bt in pest management, the emergence of resistant insect populations poses a significant challenge to the long-term efficacy of Bt -based biopesticides [8]. Insect resistance to Bt can arise through various mechanisms, including mutations in the Cry protein receptor genes [ 9 ], detoxification of Cry proteins [ 10 ], and behavioral avoidance [ 9 ] of Bt -treated plants. To overcome this challenge, researchers are continuously searching for novel Cry proteins with different modes of action and screening indigenous Bt isolates from diverse agroclimatic zones to identify new cry genes with potential biocontrol applications [ 11 ]. The cry genes from Bt are broadly categorized into conventional and neoteric (novel) types based on their period of discovery and utility in plants. Conventional cry genes, such as cry1Aa , cry1Ab , cry1Ac , cry2Ab , and cry3Aa , were identified between the 1970s and 1990s and are widely used in commercial Bt crops to control Lepidopteran and Coleopteran pests [12]. These genes exhibit high sequence similarity and share a conserved three-domain structure. However, their extensive use has led to the emergence of resistance in several target pests. In contrast, neoteric cry genes, including cry15Aa1 , cry64Ca1 , cry30Ga1 , cry78Aa , cry78Ba , and cry79Aa1 , have been discovered in recent years through genome mining and characterization of diverse Bt isolates [13, 14, 15, 16, 17, 18]. These genes often possess distinct sequences and novel modes of action. Their unique properties make them valuable candidates for developing next-generation transgenic crops and managing resistance. The previous study in our laboratory by Panwar et al. [ 19 ] provided a detailed exploration of the pool deconvolution strategy for identifying commercially important toxin genes from Bt isolates. This approach is divided into three steps: DNA pooling, short-read sequence assembly, followed by gene mining, and host isolate identification. It has proven effective for high-throughput gene mining, particularly in identifying insecticidal protein (ip) genes such as cry, cyt, mtx, bin , and vip types. This study focuses on the screening of seventy-nine indigenous Bt isolates, sourced from diverse Indian agroclimatic zones, for novel cry genes with insecticidal potential against Lepidoptera, Coleoptera, and Hemiptera. PCR-based methods were employed to confirm the presence of insect-specific cry genes, revealing notable diversity among the isolates. This research aims to contribute to the understanding of Bt diversity in India and identify promising isolates for the development of broad-spectrum biopesticides. By exploring the genetic diversity of indigenous Bt isolates, this study seeks to identify novel Cry proteins that can be used to develop more effective and sustainable pest management strategies for agriculture. MATERIALS AND METHODS Bacterial isolates and strains and their Growth media. This study utilized seventy-nine indigenous Bt isolates obtained from various agricultural and non-agricultural sites across India, as detailed by the corresponding author, Dr. S. Kaur (Supplementary Table 1). Additionally, Bt strains referenced in this research were generously supplied by Dr. D.R. Ziegler, Director of the Bacillus Genetic Stock Center at Ohio State University, Columbus, OH, USA, to Dr. S. Kaur (Supplementary Table 2). Luria Bertani Agar (LA) and Luria Bertani Broth (LB) were used for the growth of Bt isolates and strains. Plasmid DNA extraction. The plasmid DNA was extracted using a Qiagen Miniprep/Midi-prep kit following the manufacturer's instructions. Subsequently, plasmid DNA was size-separated on a 0.8% agarose gel alongside a 1 kb DNA ladder (MBI Fermentas, Germany). The DNA bands were then visualized when the gel was exposed to UV light (λ = 254 nm) using SynGene's gel documentation system (UK) and images were captured. Investigation and Selection of novel insecticidal genes for Screening of Bt isolates. Globally reported neoteric Bt cry genes, toxic to lepidopteran, coleopteran, and hemipteran species, were targeted and mined from BPPRC (Bacterial Pesticidal Protein Resource Center, https://www.bpprc.org ), NCBI ( National Center for Biotechnology Information), and Bacillus thuringiensis Toxin Nomenclature databases. Five novel cry genes cry15Aa1, cry30Fa1, cry30Ga1, cry54Aa1 , and cry79Aa1 were selected to screen for lepidopteran toxicity. For coleopteran insects, cry7, cry8, xpp37 , and cry75 genes were chosen based on their reported toxic effects. Additionally, cry64 and cry78 genes, which have fewer allelic forms compared to other cry genes, were selected for their insecticidal activity against hemipteran insects, highlighting the importance of screening and evaluating these genes (Supplementary Table 3). Oligonucleotide PCR primers . A set of universal primers Un7,8(d), 5’-AAGCAGTGAATGCCTTGTTTAC-3’, and Un7,8(r), 5’CTTCTAAACCTTGACTACTT-3’ designed by Ben-Dov et al., [20], were used to identify the presence of cry 7/8 genes. It shows toxicity to coleopteran and lepidopteran pests. Primers were designed corresponding to conserved regions of the genes for the amplification of full-length genes by obtaining the sequence data from https://www.ncbi.nlm.nih.gov/nucleotide . namely cry15Aa1, cry30Fa1, cry30Ga1, cry54Aa1, cry79Aa1, xpp37Aa, cry64Ca, cry75Aa, cry78Aa, cry78Ba , and checking their amplification activity in silico using Serial Cloner 2-6-1 software. (Table 1 ). Table 1 Details of primer pairs used for amplification of the complete ORF of novel cry genes Insect order toxic genes ORF PRIMERS (5’ – 3’) Length (Bases) Expected Amplicon (Base pairs) Lepidopteran cry15Aa1 F-ATGGCAATTATGAATGATATTGC R-TTATTCTTTATCATAATCGCGTTC 23 24 1023 cry30Fa1 F-ATGAAGCCGTATCAAAGTGAAAATG R-TTAGTTCACTGGACAAGCAAATG 25 23 2042 cry30Ga1 F-ATGAATTTATATCAAAATGAAAATG R-TTAGTTCATTTTACAAGCTTC 25 21 1995 cry54Aa1 F-ATGAGTATGAAATCATTGATTCAAAG R-TCACACGTCAGGGGTAAATTC 26 21 2022 cry79Aa1 F-ATGACTAATAATTATCCCCGG R-TTTCGGATAGTTATTGTTATAC 21 22 2187 Coleopteran xpp37Aa F-ATGACAGTATATAACGCAACTTTC R-TTATGCTGGAGTCAAGGAATAC 24 22 381 cry75Aa F-ATGAAAAAATTTGCAAGTTTAATTC R-CTATATTTCAGTTCTAATTAGTGG 25 24 954 Hemipteran cry64Ca F-ATGGCAATCCACGATGTAG R-CTAATTATTGTTTTTAGGTATACTTATATC 19 30 888 cry78Aa F-ATGACTCTAAATAATAAAAATGAA TATG R-TTACACTTCTTCTACTATGAATTC 28 24 3510 cry78Ba F-GTGTCAAATGAAAATAATACCAAAG R-CTATGATCGAGGGATAGTTCTTG 25 23 1143 *F- forward primer R- reverse primer PCR analysis. The PCR reaction for amplifying cry7/8 and other cry genes used universal and specific primers for conserved gene regions. A 25 µl mixture with 15–20 ng DNA template, 2.5 µl dNTPs (2 mM), 2.5 µl PCR buffer with MgCl2 (10X), 0.1 M of each primer, 1.0 U Taq DNA polymerase, and sterile water was prepared. The thermal cycler (Gene Amp) followed five stages, repeating steps 2, 3, and 4 thirty times. Conditions included initial denaturation at 94°C for 2 min, denaturation at 94°C for 1 min, annealing at 42°C for 1 min, extension at 72°C (1 min per kb), and final extension at 72°C for 10 min. The amplified products were analyzed by gel electrophoresis unit (Genetix, India) on 0.8% agarose gel having ethidium bromide staining agent with 1kb DNA molecular marker (MBI, Fermentas) and observed under gel documentation system (SynGene, UK). RESULTS Occurrence of Neoteric Cry Genes in Indian Bt-Strains. The screening of seventy-nine indigenous Bacillus thuringiensis isolates revealed a diverse array of cry genes, indicating a rich genetic reservoir within the Indian Bt population. A total of 27 isolates (34.18%) tested positive for at least one cry gene, demonstrating the widespread presence of these insecticidal genes in Indian Bt strains (Table 2 ). The number of cry genes harbored by individual isolates varied, with some isolates possessing a single cry gene while others contained multiple cry genes. This variability suggests that different isolates may exhibit different insecticidal spectra and potencies. The cry7/8 -type genes, which are known to be effective against coleopteran and lepidopteran pests, were the most frequently detected cry genes, accounting for approximately 40% of the positive isolates (Fig. 2 ). This finding is consistent with previous studies that have reported the prevalence of cry7/8 genes in Bt isolates from various regions [21]. In addition to the conventional cry7/8 genes, several neoteric cry genes were also identified in the Indian Bt strains. These neoteric cry genes included cry15Aa1 , cry64Ca , cry30Ga1 , cry30Fa1 , cry79Aa1 , cry78Aa , and cry78Ba . The presence of these neoteric cry genes suggests that the Indian Bt strains may possess unique insecticidal activities that have not been previously characterized. The cry15Aa1 gene, which is known to be toxic to lepidopteran pests, was detected in approximately 10% of the positive isolates. The cry64Ca gene, which is known to be toxic to hemipteran pests, was detected in approximately 5% of the positive isolates. The cry30Ga1 and cry30Fa1 genes, which have been reported to exhibit toxicity against both lepidopteran and coleopteran pests, were detected in approximately 15% and 10% of the positive isolates, respectively. The cry79Aa1 , cry78Aa , and cry78Ba genes, which are relatively less characterized, were detected in a small number of isolates (Figs. 1 & 3 ). Table 2 Distribution of novel cry genes in the native Bt isolates using ORF primers designed according to the sequence available in NCBI Sl no Isolates Source Agroclimatic zones Gene present 1 SK-110 Field Soil Trans Gangetic Plain cry15Aa, cry30Fa, cry30Ga, cry78Ba 2 SK-768 Grain Dust East Coast Plains and Hills cry7&8, cry30Fa, cry78Ba, cry79Aa 3 SK-979 Field Soil Trans Gangetic Plain cry7&8, cry30Fa, cry64Ca, cry78Aa 4 SK-213 Phyllosphere Trans Gangetic Plain cry15Aa, cry30Fa, cry30Ga 5 SK-1322 Field Soil Gujarat Plains and Hills 6 SK-219 Phyllosphere Trans Gangetic Plain cry30Fa, cry30Ga 7 SK-942 Field Soil Trans Gangetic Plain cry7&8, cry64Ca 8 SK-949 Field Soil Trans Gangetic Plain cry7&8, cry30Ga 9 SK-84 Grain Dust Western Himalayan cry7&8 10 SK-307 Field Soil Trans Gangetic Plain 11 SK-704 Field Soil East Coast Plains and Hills 12 SK-727 Seeds East Coast Plains and Hills 13 SK-797 Seeds East Coast Plains and Hills 14 SK-922 Field Soil Trans Gangetic Plain 15 SK-928 Field Soil Trans Gangetic Plain 16 SK-931 Field Soil Trans Gangetic Plain 17 SK-955 Field Soil Trans Gangetic Plain 18 SK-1014 Cow Shed Sample Trans Gangetic Plain 19 SK-1064 Field Soil Central Plateau and Hills 20 SK-223 Phyllosphere Trans Gangetic Plain cry30Fa 21 SK-700 Field Soil East Coast Plains and Hills 22 SK-976 Field Soil Trans Gangetic Plain 23 SK-214 Phyllosphere Trans Gangetic Plain cry78Aa 24 SK-714 Field Soil Southern Plateau and Hills cry79Aa 25 SK-935 Field Soil Trans Gangetic Plain cry30Ga 26 SK-961 Field Soil Trans Gangetic Plain cry64Ca 27 SK-996 Grain Dust Trans Gangetic Plain cry15Aa Prevalence of genes based on the insect orders. The prevalence of cry genes varied significantly across the three insect orders, reflecting the diverse insecticidal activities of the Indian Bt strains. Lepidopteran-toxic cry genes were the most prevalent, accounting for approximately 50% of the total cry genes detected. This finding is consistent with the fact that lepidopteran pests are among the most damaging insect pests in Indian agriculture [ 22 ]. The most prevalent lepidopteran-toxic cry genes were cry15Aa1 , cry30Fa1 , cry30Ga1 , and cry79Aa1 . These genes have been shown to be effective against various lepidopteran pests, including corn earworm, diamondback moth, and tobacco budworm [13, 14, 15]. Coleopteran-toxic cry genes were also prevalent, accounting for approximately 30% of the total cry genes detected. The most prevalent coleopteran-toxic cry gene was cry7/8 , which is known to be effective against various coleopteran pests, including Colorado potato beetle, boll weevil, and corn rootworm [23; 24]. Hemipteran-toxic cry genes were the least prevalent, accounting for approximately 20% of the total cry genes detected. The most prevalent hemipteran-toxic cry genes were cry64Ca , cry78Aa , and cry78Ba . These genes have been reported to exhibit toxicity against various hemipteran pests, including aphids, whiteflies, and planthoppers [ 25 , 17 , 18 ]. The varying prevalence of cry genes across the three insect orders suggests that the Indian Bt strains are adapted to target the specific insect pests that are prevalent in Indian agriculture (Fig. 4 ). Occurrence of neoteric cry genes in reference strains. The reference strains obtained from the Bacillus Genetic Stock Center (BGSC), as illustrated in Fig. 5 , exhibit a limited repertoire of novel cry genes. Most BGSC strains, such as 4F3, 4Q5, and 4S2, harbor conventional cry genes like cry15Aa, cry78Ba , and cry7&8 , which are primarily effective against lepidopteran pests. Although some strains, including 4K1, 4A6, and HD1, possess relatively newer cry genes such as cry30Fa, cry30Ga , and cry15Aa , the overall genetic diversity among these reference strains remains narrow. This restricted diversity likely reflects their historical use in research, where emphasis has been placed on well-characterized genes rather than novel variants. In contrast, indigenous Bacillus thuringiensis ( Bt ) isolates, collected from diverse environmental habitats, may represent a more abundant source of novel insecticidal cry genes. These isolates are naturally exposed to varied ecological pressures, potentially driving the evolution and maintenance of unique cry genes not found in reference collections. Consequently, indigenous Bt strains offer significant potential for the discovery and development of novel genes for sustainable pest management strategies. Ecological and Genetic Factors Influencing cry Gene Distribution. The distribution of cry genes in Bacillus thuringiensis is intricately linked to a complex interplay of ecological and genetic factors. The agroclimatic regions of India, characterized by distinct environmental conditions and agricultural practices, exert a significant influence on the distribution of neoteric cry genes [ 26 , 27 ]. For instance, the Trans Gangetic Plain, with its fertile alluvial soils and intensive agriculture, exhibited the highest diversity of neoteric cry genes, while the Gujarat Plains and Hills, with arid and semi-arid conditions, showed a relatively lower diversity. Similarly, the natural habitats from which Bt isolates were collected also influenced cry gene distribution. Field soil isolates exhibited the highest diversity of neoteric cry genes, followed by grain dust and phyllosphere isolates, while cowshed and seed isolates showed lower diversity. Specifically, the Trans-Gangetic plain exhibited the highest number of positive isolates (56.25%), with significant prevalence for Lepidoptera (28.13%) and Coleoptera (28.13%). The East Coast Plains and Hills showed moderate prevalence (21.74%), primarily targeting Coleoptera (17.39%) (Fig. 6 ). Regarding habitats, phyllosphere samples had the highest percentage of positive isolates (57.14%), followed by grain dust (42.85%) and cowshed samples (50%) (Fig. 7 ). Field soil, Grain dust, and Phyllosphere isolates are enriched with Lepidopteran toxic genes. Seeds and cowsheds isolates are enriched with Coleopteran toxic genes correlating to their pest specificity [ 28 ]. These ecological variations are coupled with genetic factors such as horizontal gene transfer and mutation, which also play a crucial role in shaping the diversity and distribution of cry genes. Horizontal gene transfer allows Bt strains to acquire new cry genes from other bacteria, while mutation generates new cry gene variants with altered insecticidal activity. Promising Bt Isolates for Biocontrol Applications. Several native Bt isolates have been identified as promising candidates for biocontrol applications due to the presence of multiple cry genes. SK-768 and SK-979 harbor cry genes that are active against three orders, while SK-110 and SK-949 exhibit dual toxicity, suggesting their potential effectiveness for broad-spectrum biopesticides. For example, SK-110, isolated from a chickpea field in the Trans Gangetic Plain, contains the cry15Aa, cry30Fa, cry30Ga , and cry78Ba genes. Similarly, SK-768, recovered from jowar grain dust in the East Coast Plains and Hills region, harbors the cry7/8, cry30Fa, cry78Ba , and cry79Aa genes. These isolates represent valuable resources for developing effective and sustainable biopesticides. DISCUSSION This study's findings broadly align with earlier research on the distribution and diversity of cry genes in Bacillus thuringiensis (Bt) isolates from various ecological niches. Consistent with observations by Jain et al. [ 29 ], we detected a high prevalence of cry genes targeting Lepidoptera and Coleoptera. Significantly, our study also identified novel cry genes— cry30 , cry64 , and cry78 —with potential activity against Hemipteran pests. This discovery diverges from previous research, which largely focused on cry1 and cry2 genes. The identification of Hemipteran-active genes is particularly important, as Bt toxins have shown limited efficacy against piercing and sucking insects like planthoppers and aphids [ 30 , 31 ]. Detailed analysis revealed promising candidates among these novel genes. Cry78Aa is notable as a single-component toxin displaying strong activity against rice planthopper nymphs. Its monomeric and structurally simple form, along with its independence from auxiliary proteins, distinguishes it from binary toxins such as Cry64Ba/Cry64Ca or Cry34Ab1/Cry35Ab1, which require complex formation for activity. The trefoil domain of Cry78Aa is key to its function, facilitating carbohydrate binding and cell membrane docking for pore formation and cytotoxicity. Structural studies [ 32 ] have provided a basis for rational engineering, with preliminary mutants showing enhanced activity, highlighting its potential for improved hemipteran-targeted biopesticides. Similarly, the binary Cry64Ba/Cry64Ca toxins, structurally related to aerolysin-like β-pore-forming toxins, demonstrated potent and specific activity against rice planthoppers ( Laodelphax striatellus , Sogatella furcifera ), but not against Lepidoptera or Coleoptera. This specificity suggests their utility in integrated pest management (IPM) by targeting Hemiptera without affecting non-target organisms. The requirement for co-expression and the structural complexity of Cry64Ba/Cry64Ca proteins also point to the potential for stable, high-level expression of functional toxins when both components are present [ 25 ]. Our results further support the conclusions of Patel et al. [ 33 ], who found that Bt isolates from agricultural soils exhibit higher genetic diversity and more frequent cry gene detection than those from non-agricultural sites. Most positive isolates in this study originated from cultivated fields, emphasizing the role of ecological conditions and anthropogenic influences (e.g., crop type, pesticide use) in shaping Bt populations. Additionally, studies from the Indo-Gangetic Plains and Tamil Nadu have shown that legume rhizospheres are particularly rich in Bt isolates possessing diverse cry and vip genes [34, 35]. The diversity of protein bands observed, ranging from approximately 30 kDa to over 200 kDa, indicates a broad Cry protein profile among the isolates. Isolates that produced bipyramidal and cuboidal crystals, typically associated with Cry1 and Cry2 proteins, were more toxic to Lepidoptera, echoing prior reports by Boonmee et al. [ 36 ]. Furthermore, isolates containing multiple cry genes, such as SK-110 and SK-768, which harbor genes active against both Lepidoptera and Coleoptera, exemplify the potential of multi-gene strains for broad-spectrum pest control. Along with these two isolates (SUB14873039 SK-110_16SrRNA NCBI GenBank Accession Number PQ670992, SUB14873039 SK-768_16SrRNA NCBI GenBank Accession Number PQ670998), other positive isolates, SK-223, SK-935, SK-955, SK-961 (SUB14873039 SK-223_16SrRNA NCBI GenBank Accession Number PQ670993, SUB14873039 SK-935_16SrRNA NCBI GenBank Accession Number PQ670999, SUB14873039 SK-955_16SrRNA NCBI GenBank Accession Number PQ671001, SUB14873039 SK-961_16SrRNA NCBI GenBank Accession Number PQ671002), 16S rRNA sequencing was done in our laboratory and deposited in the NCBI database, In our previous study, vip3A genes, were cloned from Bt BGSC strains and native isolates, were shown toxicity against the major lepidopteran insects like Helicoverpa armigera , Spodoptera frugiperda , and Spodoptera litura which supports the potentiality of the Bt isolates used in this study (37, 38). Utilizing multi-toxin profiles is a well-documented strategy for delaying resistance development [39, 40]. A significant technical challenge during molecular screening was the occurrence of non-specific amplification in certain isolates. As previously noted by Porcar and Juarez-Perez [41], sequence variations, potentially driven by transposable elements or recombination events, can hinder PCR-based detection and affect expression efficiency. This issue may have influenced our amplification results, particularly for isolates carrying the less-characterized or novel cry genes. Declarations ACKNOWLEDGMENTS ICAR fellowship to SA and TKCJ is acknowledged. This work was part of the M.Sc. theses of SA and TKCJ submitted to The Graduate School, ICAR-IARI, New Delhi, under the guidance of SK. SK acknowledges ICAR-NIPB for the research facility. FUNDING Funding was provided to SK for her in-house project by ICAR-National Institute for Plant Biotechnology, New Delhi, India. 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M., Diversity of Bacillus thuringiensis isolates from Egyptian soils as shown by molecular characterization, Journal of Genetic Engineering & Biotechnology , 2015, vol. 13(2), pp. 101–109. https://doi.org/10.1016/j.jgeb.2015.06.004 Porcar, M, and Juárez-Pérez, V., PCR-based identification of Bacillus thuringiensis pesticidal crystal genes, FEMS Microbiology Reviews , 2003, vol. 26(5), pp. 419–432. https://doi.org/10.1016/S0168-6445(03)00068-7 . Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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10:03:44","extension":"html","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":219481,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8459314/v1/e2a87ee40b295d8ace473c52.html"},{"id":100371282,"identity":"04a3aead-5e87-45aa-ba60-7a757d780880","added_by":"auto","created_at":"2026-01-16 08:09:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1004512,"visible":true,"origin":"","legend":"\u003cp\u003eAgarose gel electrophoresis of PCR product amplified by using ORF primer set of respective Lepidopteran specific genes mentioned below.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8459314/v1/b283dfe02d33aaa9f2d10df0.png"},{"id":100223647,"identity":"334c9d93-fe4b-4e0b-b601-4aeb69eac549","added_by":"auto","created_at":"2026-01-14 10:03:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25292,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative picture of Agarose gel electrophoresis of PCR amplified products of coleopteran toxic gene \u003cem\u003ecry7/8\u003c/em\u003e. [E] expected band 420 bp; L: 100bp molecular weight ladder (MBI Fermentas). 1-SK-949,2-SK-955,3-SK-958,4-SK-976,5-SK-979,6-SK-984,7-SK-995,8-SK-1004,9-SK-1012,10-SK-1014,11-SK-110,12-SK-1064.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8459314/v1/76b0d7b6f99c50323d50dcc7.png"},{"id":100223648,"identity":"8d219bdb-2377-429a-a231-b58827bfbc2d","added_by":"auto","created_at":"2026-01-14 10:03:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":50298,"visible":true,"origin":"","legend":"\u003cp\u003e(A)(B) Representative picture of Agarose gel electrophoresis of PCR amplified products of Hemipteran toxic genes (A) \u003cem\u003ecry64Ca \u003c/em\u003eand (B) \u003cem\u003ecry78Aa\u003c/em\u003e. [E] expected band 888 bp and 3.5 Kbp respectively; L: 1 Kb molecular weight ladder (MBI Fermentas). 1-SK-931,2-SK-934,3-SK-935,4-SK-942,5-SK-949,6-SK-955,7-SK-958,8-SK-961,9-SK-976,10-SK-977,11-SK-979.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8459314/v1/e239f18077fdeb7534fb346e.png"},{"id":100370939,"identity":"1f87bfbf-a274-413c-871b-cd02661678ac","added_by":"auto","created_at":"2026-01-16 08:09:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":198199,"visible":true,"origin":"","legend":"\u003cp\u003eRadial network graph of the prevalence of neoteric \u003cem\u003ecry\u003c/em\u003e genes in native \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (Bt) isolates, categorized by insect order (Lepidoptera, Coleoptera, and Hemiptera) and agroclimatic regions.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8459314/v1/685e0ec802556aa1a86271b0.png"},{"id":100223657,"identity":"7c4dbf85-0fc3-4113-b565-5fe6d5d74f8d","added_by":"auto","created_at":"2026-01-14 10:03:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39721,"visible":true,"origin":"","legend":"\u003cp\u003eOccurrence of neoteric \u003cem\u003ecry\u003c/em\u003e genes in reference Bt strains.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8459314/v1/baa225512f060342d432ec4e.png"},{"id":100223660,"identity":"b8f3ef93-9345-40b8-ae8e-3e4c5b8432c6","added_by":"auto","created_at":"2026-01-14 10:03:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":64184,"visible":true,"origin":"","legend":"\u003cp\u003eHierarchy of neoteric \u003cem\u003ecry\u003c/em\u003e genes in agroclimatic regions of India with specific insect order toxicity.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8459314/v1/835dfd449cb12aafaf1feeec.png"},{"id":100223685,"identity":"d240751e-2f6f-4b89-a0fd-c33b4cda322a","added_by":"auto","created_at":"2026-01-14 10:03:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":28141,"visible":true,"origin":"","legend":"\u003cp\u003eHierarchy of neoteric \u003cem\u003ecry\u003c/em\u003e genes in natural habitats of India with specific insect order toxicity.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8459314/v1/563695a7d6156c5170b5b67e.png"},{"id":107328712,"identity":"4980ffd4-58e4-4a79-8276-e196198ead8d","added_by":"auto","created_at":"2026-04-20 12:11:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2273528,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8459314/v1/100a234d-e880-4f56-8adc-3bb17b04f5a3.pdf"},{"id":100370438,"identity":"ba2f9bd4-bf53-43c1-9d99-7899ba82e075","added_by":"auto","created_at":"2026-01-16 08:05:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27405,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8459314/v1/4af6ac4428c4f3fd3a4025b8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Screening of Bacillus thuringiensis Isolates Recovered from Diverse Habitats in India for Crystal Toxin Genes Predicting Toxicity Against Three Insect Orders: Lepidoptera, Coleoptera, and Hemiptera","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe agricultural sector stands as a cornerstone of global food security, yet it grapples with persistent and escalating challenges posed by insect pests. These insidious adversaries inflict substantial damage on crops, leading to significant yield losses and economic instability worldwide. According to the Food and Agriculture Organization (FAO), insect pests are responsible for the destruction of up to 40% of annual global crop production, resulting in an economic burden exceeding \u003cspan\u003e$\u003c/span\u003e120\u0026nbsp;billion annually [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fao.org/\u003c/span\u003e\u003cspan address=\"https://www.fao.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e]. This staggering figure underscores the urgent need for effective and sustainable pest management strategies to safeguard agricultural productivity and ensure food availability for a growing global population. Among the diverse array of insect pests that plague agricultural systems, Lepidoptera (e.g., corn earworm, diamondback moth), Coleoptera (e.g., Colorado potato beetle, boll weevil), and Hemiptera (e.g., aphids, whiteflies, planthoppers) stand out as particularly destructive. These insect orders encompass a wide range of species that exhibit diverse feeding habits and host preferences, enabling them to exploit various crops and inflict damage at different stages of plant development [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The voracious feeding behavior of these pests can lead to defoliation, stunted growth, reduced fruit production, and even plant death, resulting in significant economic losses for farmers and compromising food security. For decades, chemical pesticides have been the primary tool for combating insect pests in agriculture. These synthetic compounds are designed to kill or repel insects, providing rapid and effective control of pest populations. However, the widespread and often indiscriminate use of chemical pesticides has raised serious concerns about their long-term environmental and human health impacts [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Chemical pesticides can contaminate soil and water resources, disrupt beneficial insect populations, and pose risks to human health through direct exposure or consumption of contaminated food. Furthermore, the overuse of chemical pesticides has led to the emergence of resistant insect populations, rendering these compounds ineffective and necessitating the development of new and more potent pesticides.\u003c/p\u003e \u003cp\u003eIn response to the drawbacks associated with chemical pesticides, biological control agents, particularly those based on \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (\u003cem\u003eBt\u003c/em\u003e), have emerged as promising and sustainable alternatives for pest management. \u003cem\u003eBt\u003c/em\u003e is a gram-positive, spore-forming bacterium that produces crystal proteins (Cry) with specific insecticidal activity against various insect orders [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These Cry proteins are selectively toxic to certain insect species, making them highly effective for targeted pest control while minimizing harm to non-target organisms, including beneficial insects, wildlife, and humans [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cem\u003eBt\u003c/em\u003e-based biopesticides have been widely adopted in agriculture due to their efficacy, environmental safety, and compatibility with integrated pest management (IPM) programs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The mechanism of action of Cry proteins involves their ingestion by susceptible insect larvae, followed by their activation in the insect midgut. Once activated, Cry proteins bind to specific receptors on the midgut epithelial cells, leading to pore formation and disruption of cellular function. This results in paralysis, gut leakage, and ultimately, death of the insect larva [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The specificity of Cry proteins for insect midgut receptors is a key factor in their safety for non-target organisms, as these receptors are not found in vertebrates or beneficial insects. Despite the widespread use of \u003cem\u003eBt\u003c/em\u003e in pest management, the emergence of resistant insect populations poses a significant challenge to the long-term efficacy of \u003cem\u003eBt\u003c/em\u003e-based biopesticides [8]. Insect resistance to \u003cem\u003eBt\u003c/em\u003e can arise through various mechanisms, including mutations in the Cry protein receptor genes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e9\u003c/span\u003e], detoxification of Cry proteins [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and behavioral avoidance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e9\u003c/span\u003e] of \u003cem\u003eBt\u003c/em\u003e-treated plants. To overcome this challenge, researchers are continuously searching for novel Cry proteins with different modes of action and screening indigenous \u003cem\u003eBt\u003c/em\u003e isolates from diverse agroclimatic zones to identify new \u003cem\u003ecry\u003c/em\u003e genes with potential biocontrol applications [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The \u003cem\u003ecry\u003c/em\u003e genes from \u003cem\u003eBt\u003c/em\u003e are broadly categorized into conventional and neoteric (novel) types based on their period of discovery and utility in plants. Conventional \u003cem\u003ecry\u003c/em\u003e genes, such as \u003cem\u003ecry1Aa\u003c/em\u003e, \u003cem\u003ecry1Ab\u003c/em\u003e, \u003cem\u003ecry1Ac\u003c/em\u003e, \u003cem\u003ecry2Ab\u003c/em\u003e, and \u003cem\u003ecry3Aa\u003c/em\u003e, were identified between the 1970s and 1990s and are widely used in commercial \u003cem\u003eBt\u003c/em\u003e crops to control Lepidopteran and Coleopteran pests [12]. These genes exhibit high sequence similarity and share a conserved three-domain structure. However, their extensive use has led to the emergence of resistance in several target pests. In contrast, neoteric \u003cem\u003ecry\u003c/em\u003e genes, including \u003cem\u003ecry15Aa1\u003c/em\u003e, \u003cem\u003ecry64Ca1\u003c/em\u003e, \u003cem\u003ecry30Ga1\u003c/em\u003e, \u003cem\u003ecry78Aa\u003c/em\u003e, \u003cem\u003ecry78Ba\u003c/em\u003e, and \u003cem\u003ecry79Aa1\u003c/em\u003e, have been discovered in recent years through genome mining and characterization of diverse \u003cem\u003eBt\u003c/em\u003e isolates [13, 14, 15, 16, 17, 18]. These genes often possess distinct sequences and novel modes of action. Their unique properties make them valuable candidates for developing next-generation transgenic crops and managing resistance. The previous study in our laboratory by Panwar et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e19\u003c/span\u003e] provided a detailed exploration of the pool deconvolution strategy for identifying commercially important toxin genes from \u003cem\u003eBt\u003c/em\u003e isolates. This approach is divided into three steps: DNA pooling, short-read sequence assembly, followed by gene mining, and host isolate identification. It has proven effective for high-throughput gene mining, particularly in identifying insecticidal protein (ip) genes such as \u003cem\u003ecry, cyt, mtx, bin\u003c/em\u003e, and \u003cem\u003evip\u003c/em\u003e types.\u003c/p\u003e \u003cp\u003eThis study focuses on the screening of seventy-nine indigenous \u003cem\u003eBt\u003c/em\u003e isolates, sourced from diverse Indian agroclimatic zones, for novel \u003cem\u003ecry\u003c/em\u003e genes with insecticidal potential against Lepidoptera, Coleoptera, and Hemiptera. PCR-based methods were employed to confirm the presence of insect-specific \u003cem\u003ecry\u003c/em\u003e genes, revealing notable diversity among the isolates. This research aims to contribute to the understanding of \u003cem\u003eBt\u003c/em\u003e diversity in India and identify promising isolates for the development of broad-spectrum biopesticides. By exploring the genetic diversity of indigenous \u003cem\u003eBt\u003c/em\u003e isolates, this study seeks to identify novel Cry proteins that can be used to develop more effective and sustainable pest management strategies for agriculture.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cb\u003eBacterial isolates and strains and their Growth media.\u003c/b\u003e This study utilized seventy-nine indigenous \u003cem\u003eBt\u003c/em\u003e isolates obtained from various agricultural and non-agricultural sites across India, as detailed by the corresponding author, Dr. S. Kaur (Supplementary Table\u0026nbsp;1). Additionally, \u003cem\u003eBt\u003c/em\u003e strains referenced in this research were generously supplied by Dr. D.R. Ziegler, Director of the Bacillus Genetic Stock Center at Ohio State University, Columbus, OH, USA, to Dr. S. Kaur (Supplementary Table\u0026nbsp;2). Luria Bertani Agar (LA) and Luria Bertani Broth (LB) were used for the growth of \u003cem\u003eBt\u003c/em\u003e isolates and strains.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmid DNA extraction.\u003c/b\u003e The plasmid DNA was extracted using a Qiagen Miniprep/Midi-prep kit following the manufacturer's instructions. Subsequently, plasmid DNA was size-separated on a 0.8% agarose gel alongside a 1 kb DNA ladder (MBI Fermentas, Germany). The DNA bands were then visualized when the gel was exposed to UV light (λ\u0026thinsp;=\u0026thinsp;254 nm) using SynGene's gel documentation system (UK) and images were captured.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInvestigation and Selection of novel insecticidal genes for Screening of Bt isolates.\u003c/b\u003e Globally reported neoteric \u003cem\u003eBt cry\u003c/em\u003e genes, toxic to lepidopteran, coleopteran, and hemipteran species, were targeted and mined from BPPRC (Bacterial Pesticidal Protein Resource Center, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bpprc.org\u003c/span\u003e\u003cspan address=\"https://www.bpprc.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), NCBI ( National Center for Biotechnology Information), and \u003cem\u003eBacillus thuringiensis\u003c/em\u003e Toxin Nomenclature databases. Five novel \u003cem\u003ecry\u003c/em\u003e genes \u003cem\u003ecry15Aa1, cry30Fa1, cry30Ga1, cry54Aa1\u003c/em\u003e, and \u003cem\u003ecry79Aa1\u003c/em\u003e were selected to screen for lepidopteran toxicity. For coleopteran insects, \u003cem\u003ecry7, cry8, xpp37\u003c/em\u003e, and \u003cem\u003ecry75\u003c/em\u003e genes were chosen based on their reported toxic effects. Additionally, \u003cem\u003ecry64\u003c/em\u003e and \u003cem\u003ecry78\u003c/em\u003e genes, which have fewer allelic forms compared to other \u003cem\u003ecry\u003c/em\u003e genes, were selected for their insecticidal activity against hemipteran insects, highlighting the importance of screening and evaluating these genes (Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eOligonucleotide PCR primers\u003c/b\u003e. A set of universal primers Un7,8(d), 5\u0026rsquo;-AAGCAGTGAATGCCTTGTTTAC-3\u0026rsquo;, and Un7,8(r), 5\u0026rsquo;CTTCTAAACCTTGACTACTT-3\u0026rsquo; designed by Ben-Dov et al., [20], were used to identify the presence of \u003cem\u003ecry 7/8\u003c/em\u003e genes. It shows toxicity to coleopteran and lepidopteran pests. Primers were designed corresponding to conserved regions of the genes for the amplification of full-length genes by obtaining the sequence data from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/nucleotide\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/nucleotide\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. namely \u003cem\u003ecry15Aa1, cry30Fa1, cry30Ga1, cry54Aa1, cry79Aa1, xpp37Aa, cry64Ca, cry75Aa, cry78Aa, cry78Ba\u003c/em\u003e, and checking their amplification activity in silico using Serial Cloner 2-6-1 software. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetails of primer pairs used for amplification of the complete ORF of novel \u003cem\u003ecry\u003c/em\u003e genes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInsect order\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003etoxic\u003c/p\u003e \u003cp\u003egenes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eORF PRIMERS (5\u0026rsquo; \u0026ndash; 3\u0026rsquo;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLength (Bases)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExpected Amplicon\u003c/p\u003e \u003cp\u003e(Base pairs)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eLepidopteran\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ecry15Aa1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF-ATGGCAATTATGAATGATATTGC\u003c/p\u003e \u003cp\u003eR-TTATTCTTTATCATAATCGCGTTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1023\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ecry30Fa1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF-ATGAAGCCGTATCAAAGTGAAAATG\u003c/p\u003e \u003cp\u003eR-TTAGTTCACTGGACAAGCAAATG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2042\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ecry30Ga1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF-ATGAATTTATATCAAAATGAAAATG\u003c/p\u003e \u003cp\u003eR-TTAGTTCATTTTACAAGCTTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1995\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ecry54Aa1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF-ATGAGTATGAAATCATTGATTCAAAG\u003c/p\u003e \u003cp\u003eR-TCACACGTCAGGGGTAAATTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26\u003c/p\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ecry79Aa1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF-ATGACTAATAATTATCCCCGG\u003c/p\u003e \u003cp\u003eR-TTTCGGATAGTTATTGTTATAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21\u003c/p\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2187\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eColeopteran\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003expp37Aa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF-ATGACAGTATATAACGCAACTTTC\u003c/p\u003e \u003cp\u003eR-TTATGCTGGAGTCAAGGAATAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e381\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ecry75Aa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF-ATGAAAAAATTTGCAAGTTTAATTC\u003c/p\u003e \u003cp\u003eR-CTATATTTCAGTTCTAATTAGTGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e954\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eHemipteran\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ecry64Ca\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF-ATGGCAATCCACGATGTAG\u003c/p\u003e \u003cp\u003eR-CTAATTATTGTTTTTAGGTATACTTATATC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e888\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ecry78Aa\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF-ATGACTCTAAATAATAAAAATGAA TATG\u003c/p\u003e \u003cp\u003eR-TTACACTTCTTCTACTATGAATTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28\u003c/p\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3510\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003ecry78Ba\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF-GTGTCAAATGAAAATAATACCAAAG\u003c/p\u003e \u003cp\u003eR-CTATGATCGAGGGATAGTTCTTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1143\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*F- forward primer R- reverse primer\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePCR analysis.\u003c/b\u003e The PCR reaction for amplifying \u003cem\u003ecry7/8\u003c/em\u003e and other \u003cem\u003ecry\u003c/em\u003e genes used universal and specific primers for conserved gene regions. A 25 \u0026micro;l mixture with 15\u0026ndash;20 ng DNA template, 2.5 \u0026micro;l dNTPs (2 mM), 2.5 \u0026micro;l PCR buffer with MgCl2 (10X), 0.1 M of each primer, 1.0 U Taq DNA polymerase, and sterile water was prepared. The thermal cycler (Gene Amp) followed five stages, repeating steps 2, 3, and 4 thirty times. Conditions included initial denaturation at 94\u0026deg;C for 2 min, denaturation at 94\u0026deg;C for 1 min, annealing at 42\u0026deg;C for 1 min, extension at 72\u0026deg;C (1 min per kb), and final extension at 72\u0026deg;C for 10 min. The amplified products were analyzed by gel electrophoresis unit (Genetix, India) on 0.8% agarose gel having ethidium bromide staining agent with 1kb DNA molecular marker (MBI, Fermentas) and observed under gel documentation system (SynGene, UK).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eOccurrence of Neoteric Cry Genes in Indian Bt-Strains.\u003c/b\u003e The screening of seventy-nine indigenous \u003cem\u003eBacillus thuringiensis\u003c/em\u003e isolates revealed a diverse array of \u003cem\u003ecry\u003c/em\u003e genes, indicating a rich genetic reservoir within the Indian \u003cem\u003eBt\u003c/em\u003e population. A total of 27 isolates (34.18%) tested positive for at least one \u003cem\u003ecry\u003c/em\u003e gene, demonstrating the widespread presence of these insecticidal genes in Indian \u003cem\u003eBt\u003c/em\u003e strains (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The number of \u003cem\u003ecry\u003c/em\u003e genes harbored by individual isolates varied, with some isolates possessing a single \u003cem\u003ecry\u003c/em\u003e gene while others contained multiple \u003cem\u003ecry\u003c/em\u003e genes. This variability suggests that different isolates may exhibit different insecticidal spectra and potencies. The \u003cem\u003ecry7/8\u003c/em\u003e-type genes, which are known to be effective against coleopteran and lepidopteran pests, were the most frequently detected \u003cem\u003ecry\u003c/em\u003e genes, accounting for approximately 40% of the positive isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This finding is consistent with previous studies that have reported the prevalence of \u003cem\u003ecry7/8\u003c/em\u003e genes in \u003cem\u003eBt\u003c/em\u003e isolates from various regions [21]. In addition to the conventional \u003cem\u003ecry7/8\u003c/em\u003e genes, several neoteric \u003cem\u003ecry\u003c/em\u003e genes were also identified in the Indian \u003cem\u003eBt\u003c/em\u003e strains. These neoteric \u003cem\u003ecry\u003c/em\u003e genes included \u003cem\u003ecry15Aa1\u003c/em\u003e, \u003cem\u003ecry64Ca\u003c/em\u003e, \u003cem\u003ecry30Ga1\u003c/em\u003e, \u003cem\u003ecry30Fa1\u003c/em\u003e, \u003cem\u003ecry79Aa1\u003c/em\u003e, \u003cem\u003ecry78Aa\u003c/em\u003e, and \u003cem\u003ecry78Ba\u003c/em\u003e. The presence of these neoteric \u003cem\u003ecry\u003c/em\u003e genes suggests that the Indian \u003cem\u003eBt\u003c/em\u003e strains may possess unique insecticidal activities that have not been previously characterized. The \u003cem\u003ecry15Aa1\u003c/em\u003e gene, which is known to be toxic to lepidopteran pests, was detected in approximately 10% of the positive isolates. The \u003cem\u003ecry64Ca\u003c/em\u003e gene, which is known to be toxic to hemipteran pests, was detected in approximately 5% of the positive isolates. The \u003cem\u003ecry30Ga1\u003c/em\u003e and \u003cem\u003ecry30Fa1\u003c/em\u003e genes, which have been reported to exhibit toxicity against both lepidopteran and coleopteran pests, were detected in approximately 15% and 10% of the positive isolates, respectively. The \u003cem\u003ecry79Aa1\u003c/em\u003e, \u003cem\u003ecry78Aa\u003c/em\u003e, and \u003cem\u003ecry78Ba\u003c/em\u003e genes, which are relatively less characterized, were detected in a small number of isolates (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of novel \u003cem\u003ecry\u003c/em\u003e genes in the native Bt isolates using ORF primers designed according to the sequence available in NCBI\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSl no\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAgroclimatic zones\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGene present\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecry15Aa, cry30Fa, cry30Ga, cry78Ba\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-768\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrain Dust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEast Coast Plains and Hills\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecry7\u0026amp;8, cry30Fa, cry78Ba, cry79Aa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-979\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecry7\u0026amp;8, cry30Fa, cry64Ca, cry78Aa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhyllosphere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ecry15Aa, cry30Fa, cry30Ga\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-1322\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGujarat Plains and Hills\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-219\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhyllosphere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecry30Fa, cry30Ga\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-942\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecry7\u0026amp;8, cry64Ca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecry7\u0026amp;8, cry30Ga\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrain Dust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWestern Himalayan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"10\" rowspan=\"11\"\u003e \u003cp\u003e\u003cem\u003ecry7\u0026amp;8\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-307\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEast Coast Plains and Hills\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-727\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSeeds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEast Coast Plains and Hills\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-797\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSeeds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEast Coast Plains and Hills\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-922\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-928\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-931\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-955\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-1014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCow Shed Sample\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-1064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCentral Plateau and Hills\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhyllosphere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003ecry30Fa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEast Coast Plains and Hills\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhyllosphere\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecry78Aa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSouthern Plateau and Hills\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecry79Aa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-935\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecry30Ga\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-961\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eField Soil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecry64Ca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrain Dust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTrans Gangetic Plain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ecry15Aa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePrevalence of genes based on the insect orders.\u003c/b\u003e The prevalence of \u003cem\u003ecry\u003c/em\u003e genes varied significantly across the three insect orders, reflecting the diverse insecticidal activities of the Indian \u003cem\u003eBt\u003c/em\u003e strains. Lepidopteran-toxic \u003cem\u003ecry\u003c/em\u003e genes were the most prevalent, accounting for approximately 50% of the total \u003cem\u003ecry\u003c/em\u003e genes detected. This finding is consistent with the fact that lepidopteran pests are among the most damaging insect pests in Indian agriculture [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The most prevalent lepidopteran-toxic \u003cem\u003ecry\u003c/em\u003e genes were \u003cem\u003ecry15Aa1\u003c/em\u003e, \u003cem\u003ecry30Fa1\u003c/em\u003e, \u003cem\u003ecry30Ga1\u003c/em\u003e, and \u003cem\u003ecry79Aa1\u003c/em\u003e. These genes have been shown to be effective against various lepidopteran pests, including corn earworm, diamondback moth, and tobacco budworm [13, 14, 15]. Coleopteran-toxic \u003cem\u003ecry\u003c/em\u003e genes were also prevalent, accounting for approximately 30% of the total \u003cem\u003ecry\u003c/em\u003e genes detected. The most prevalent coleopteran-toxic \u003cem\u003ecry\u003c/em\u003e gene was \u003cem\u003ecry7/8\u003c/em\u003e, which is known to be effective against various coleopteran pests, including Colorado potato beetle, boll weevil, and corn rootworm [23; 24]. Hemipteran-toxic \u003cem\u003ecry\u003c/em\u003e genes were the least prevalent, accounting for approximately 20% of the total \u003cem\u003ecry\u003c/em\u003e genes detected. The most prevalent hemipteran-toxic \u003cem\u003ecry\u003c/em\u003e genes were \u003cem\u003ecry64Ca\u003c/em\u003e, \u003cem\u003ecry78Aa\u003c/em\u003e, and \u003cem\u003ecry78Ba\u003c/em\u003e. These genes have been reported to exhibit toxicity against various hemipteran pests, including aphids, whiteflies, and planthoppers [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The varying prevalence of \u003cem\u003ecry\u003c/em\u003e genes across the three insect orders suggests that the Indian \u003cem\u003eBt\u003c/em\u003e strains are adapted to target the specific insect pests that are prevalent in Indian agriculture (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOccurrence of neoteric cry genes in reference strains.\u003c/b\u003e The reference strains obtained from the Bacillus Genetic Stock Center (BGSC), as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, exhibit a limited repertoire of novel \u003cem\u003ecry\u003c/em\u003e genes. Most BGSC strains, such as 4F3, 4Q5, and 4S2, harbor conventional \u003cem\u003ecry\u003c/em\u003e genes like \u003cem\u003ecry15Aa, cry78Ba\u003c/em\u003e, and \u003cem\u003ecry7\u0026amp;8\u003c/em\u003e, which are primarily effective against lepidopteran pests. Although some strains, including 4K1, 4A6, and HD1, possess relatively newer \u003cem\u003ecry\u003c/em\u003e genes such as \u003cem\u003ecry30Fa, cry30Ga\u003c/em\u003e, and \u003cem\u003ecry15Aa\u003c/em\u003e, the overall genetic diversity among these reference strains remains narrow. This restricted diversity likely reflects their historical use in research, where emphasis has been placed on well-characterized genes rather than novel variants. In contrast, indigenous \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (\u003cem\u003eBt\u003c/em\u003e) isolates, collected from diverse environmental habitats, may represent a more abundant source of novel insecticidal \u003cem\u003ecry\u003c/em\u003e genes. These isolates are naturally exposed to varied ecological pressures, potentially driving the evolution and maintenance of unique \u003cem\u003ecry\u003c/em\u003e genes not found in reference collections. Consequently, indigenous \u003cem\u003eBt\u003c/em\u003e strains offer significant potential for the discovery and development of novel genes for sustainable pest management strategies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEcological and Genetic Factors Influencing cry Gene Distribution.\u003c/b\u003e The distribution of \u003cem\u003ecry\u003c/em\u003e genes in \u003cem\u003eBacillus thuringiensis\u003c/em\u003e is intricately linked to a complex interplay of ecological and genetic factors. The agroclimatic regions of India, characterized by distinct environmental conditions and agricultural practices, exert a significant influence on the distribution of neoteric \u003cem\u003ecry\u003c/em\u003e genes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For instance, the Trans Gangetic Plain, with its fertile alluvial soils and intensive agriculture, exhibited the highest diversity of neoteric \u003cem\u003ecry\u003c/em\u003e genes, while the Gujarat Plains and Hills, with arid and semi-arid conditions, showed a relatively lower diversity. Similarly, the natural habitats from which \u003cem\u003eBt\u003c/em\u003e isolates were collected also influenced \u003cem\u003ecry\u003c/em\u003e gene distribution. Field soil isolates exhibited the highest diversity of neoteric \u003cem\u003ecry\u003c/em\u003e genes, followed by grain dust and phyllosphere isolates, while cowshed and seed isolates showed lower diversity. Specifically, the Trans-Gangetic plain exhibited the highest number of positive isolates (56.25%), with significant prevalence for Lepidoptera (28.13%) and Coleoptera (28.13%). The East Coast Plains and Hills showed moderate prevalence (21.74%), primarily targeting Coleoptera (17.39%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Regarding habitats, phyllosphere samples had the highest percentage of positive isolates (57.14%), followed by grain dust (42.85%) and cowshed samples (50%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Field soil, Grain dust, and Phyllosphere isolates are enriched with Lepidopteran toxic genes. Seeds and cowsheds isolates are enriched with Coleopteran toxic genes correlating to their pest specificity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These ecological variations are coupled with genetic factors such as horizontal gene transfer and mutation, which also play a crucial role in shaping the diversity and distribution of \u003cem\u003ecry\u003c/em\u003e genes. Horizontal gene transfer allows \u003cem\u003eBt\u003c/em\u003e strains to acquire new \u003cem\u003ecry\u003c/em\u003e genes from other bacteria, while mutation generates new \u003cem\u003ecry\u003c/em\u003e gene variants with altered insecticidal activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePromising Bt Isolates for Biocontrol Applications.\u003c/b\u003e Several native \u003cem\u003eBt\u003c/em\u003e isolates have been identified as promising candidates for biocontrol applications due to the presence of multiple \u003cem\u003ecry\u003c/em\u003e genes. SK-768 and SK-979 harbor \u003cem\u003ecry\u003c/em\u003e genes that are active against three orders, while SK-110 and SK-949 exhibit dual toxicity, suggesting their potential effectiveness for broad-spectrum biopesticides. For example, SK-110, isolated from a chickpea field in the Trans Gangetic Plain, contains the \u003cem\u003ecry15Aa, cry30Fa, cry30Ga\u003c/em\u003e, and \u003cem\u003ecry78Ba\u003c/em\u003e genes. Similarly, SK-768, recovered from jowar grain dust in the East Coast Plains and Hills region, harbors the \u003cem\u003ecry7/8, cry30Fa, cry78Ba\u003c/em\u003e, and \u003cem\u003ecry79Aa\u003c/em\u003e genes. These isolates represent valuable resources for developing effective and sustainable biopesticides.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study's findings broadly align with earlier research on the distribution and diversity of \u003cem\u003ecry\u003c/em\u003e genes in \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (Bt) isolates from various ecological niches. Consistent with observations by Jain et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e29\u003c/span\u003e], we detected a high prevalence of \u003cem\u003ecry\u003c/em\u003e genes targeting Lepidoptera and Coleoptera. Significantly, our study also identified novel \u003cem\u003ecry\u003c/em\u003e genes\u0026mdash;\u003cem\u003ecry30\u003c/em\u003e, \u003cem\u003ecry64\u003c/em\u003e, and \u003cem\u003ecry78\u003c/em\u003e\u0026mdash;with potential activity against Hemipteran pests. This discovery diverges from previous research, which largely focused on \u003cem\u003ecry1\u003c/em\u003e and \u003cem\u003ecry2\u003c/em\u003e genes. The identification of Hemipteran-active genes is particularly important, as \u003cem\u003eBt\u003c/em\u003e toxins have shown limited efficacy against piercing and sucking insects like planthoppers and aphids [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Detailed analysis revealed promising candidates among these novel genes. Cry78Aa is notable as a single-component toxin displaying strong activity against rice planthopper nymphs. Its monomeric and structurally simple form, along with its independence from auxiliary proteins, distinguishes it from binary toxins such as Cry64Ba/Cry64Ca or Cry34Ab1/Cry35Ab1, which require complex formation for activity. The trefoil domain of Cry78Aa is key to its function, facilitating carbohydrate binding and cell membrane docking for pore formation and cytotoxicity. Structural studies [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] have provided a basis for rational engineering, with preliminary mutants showing enhanced activity, highlighting its potential for improved hemipteran-targeted biopesticides. Similarly, the binary Cry64Ba/Cry64Ca toxins, structurally related to aerolysin-like β-pore-forming toxins, demonstrated potent and specific activity against rice planthoppers (\u003cem\u003eLaodelphax striatellus\u003c/em\u003e, \u003cem\u003eSogatella furcifera\u003c/em\u003e), but not against Lepidoptera or Coleoptera. This specificity suggests their utility in integrated pest management (IPM) by targeting Hemiptera without affecting non-target organisms. The requirement for co-expression and the structural complexity of Cry64Ba/Cry64Ca proteins also point to the potential for stable, high-level expression of functional toxins when both components are present [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our results further support the conclusions of Patel et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e33\u003c/span\u003e], who found that \u003cem\u003eBt\u003c/em\u003e isolates from agricultural soils exhibit higher genetic diversity and more frequent \u003cem\u003ecry\u003c/em\u003e gene detection than those from non-agricultural sites. Most positive isolates in this study originated from cultivated fields, emphasizing the role of ecological conditions and anthropogenic influences (e.g., crop type, pesticide use) in shaping \u003cem\u003eBt\u003c/em\u003e populations. Additionally, studies from the Indo-Gangetic Plains and Tamil Nadu have shown that legume rhizospheres are particularly rich in \u003cem\u003eBt\u003c/em\u003e isolates possessing diverse \u003cem\u003ecry\u003c/em\u003e and \u003cem\u003evip\u003c/em\u003e genes [34, 35]. The diversity of protein bands observed, ranging from approximately 30 kDa to over 200 kDa, indicates a broad Cry protein profile among the isolates. Isolates that produced bipyramidal and cuboidal crystals, typically associated with Cry1 and Cry2 proteins, were more toxic to Lepidoptera, echoing prior reports by Boonmee et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Furthermore, isolates containing multiple \u003cem\u003ecry\u003c/em\u003e genes, such as SK-110 and SK-768, which harbor genes active against both Lepidoptera and Coleoptera, exemplify the potential of multi-gene strains for broad-spectrum pest control. Along with these two isolates (SUB14873039 SK-110_16SrRNA NCBI GenBank Accession Number PQ670992, SUB14873039 SK-768_16SrRNA NCBI GenBank Accession Number PQ670998), other positive isolates, SK-223, SK-935, SK-955, SK-961 (SUB14873039 SK-223_16SrRNA NCBI GenBank Accession Number PQ670993, SUB14873039 SK-935_16SrRNA NCBI GenBank Accession Number PQ670999, SUB14873039 SK-955_16SrRNA NCBI GenBank Accession Number PQ671001, SUB14873039 SK-961_16SrRNA NCBI GenBank Accession Number PQ671002), 16S rRNA sequencing was done in our laboratory and deposited in the NCBI database, In our previous study, \u003cem\u003evip3A\u003c/em\u003e genes, were cloned from \u003cem\u003eBt\u003c/em\u003e BGSC strains and native isolates, were shown toxicity against the major lepidopteran insects like \u003cem\u003eHelicoverpa armigera\u003c/em\u003e, \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e, and \u003cem\u003eSpodoptera litura\u003c/em\u003e which supports the potentiality of the \u003cem\u003eBt\u003c/em\u003e isolates used in this study (37, 38). Utilizing multi-toxin profiles is a well-documented strategy for delaying resistance development [39, 40]. A significant technical challenge during molecular screening was the occurrence of non-specific amplification in certain isolates. As previously noted by Porcar and Juarez-Perez [41], sequence variations, potentially driven by transposable elements or recombination events, can hinder PCR-based detection and affect expression efficiency. This issue may have influenced our amplification results, particularly for isolates carrying the less-characterized or novel \u003cem\u003ecry\u003c/em\u003e genes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eACKNOWLEDGMENTS\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;ICAR fellowship to SA and TKCJ is acknowledged. This work was part of the M.Sc. theses of SA and TKCJ submitted to The Graduate School, ICAR-IARI, New Delhi, under the guidance of SK. SK acknowledges ICAR-NIPB for the research facility.\u003c/p\u003e\n\u003cp\u003eFUNDING\u003c/p\u003e\n\u003cp\u003eFunding was provided to SK for her in-house project by ICAR-National Institute for Plant Biotechnology, New Delhi, India.\u003c/p\u003e\n\u003cp\u003eCOMPLIANCE WITH ETHICAL STANDARDS\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants performed by any of the authors.\u003c/p\u003e\n\u003cp\u003eCONFLICT OF INTEREST\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;No potential conflict of interest was reported by the author(s).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMahmood, S, Kumar, M, Kumari, P, Mahapatro, G. K, Banerjee, N, and Sarin, N. 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E., Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria, \u003cem\u003eAnnual Review of Genetics\u003c/em\u003e, 2003, vol. 37, pp. 409\u0026ndash;433. https://doi.org/10.1146/annurev.genet.37.110801.143042\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, J, Zheng, A, Wang, S, Liu, H, and Li, P., Characterization and expression of cry4Cb1 and cry30Ga1 from \u003cem\u003eBacillus thuringiensis\u003c/em\u003e strain HS18-1, \u003cem\u003eJournal of Invertebrate Pathology\u003c/em\u003e, 2010, vol. 103(3), pp. 200\u0026ndash;202. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jip.2009.12.004\u003c/span\u003e\u003cspan address=\"10.1016/j.jip.2009.12.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Ni, H, Wang, J, Shen, Y, Yang, X, Cui, J, Ding, M, Liu, R, Li, H, and Gao, J., Cloning and characterization of the Cry79Aa1 gene from a lepidopteran active strain of \u003cem\u003eBacillus thuringiensis\u003c/em\u003e, \u003cem\u003eJournal of Invertebrate Pathology\u003c/em\u003e, 2021, vol. 185, pp. 107657. https://doi.org/10.1016/j.jip.2021.107657\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Y, Wang, Y, Shu, C, Lin, K, Song, F, Bravo, A, Sober\u0026oacute;n, M, and Zhang, J., Cry64Ba and Cry64Ca, two ETX/MTX2-type \u003cem\u003eBacillus thuringiensis\u003c/em\u003e insecticidal proteins active against hemipteran pests, \u003cem\u003eApplied and Environmental Microbiology\u003c/em\u003e, 2018, vol. 84(3), pp. e01996-17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.01996-17\u003c/span\u003e\u003cspan address=\"10.1128/AEM.01996-17\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Y, Liu, Y, Zhang, J, Crickmore, N, Song, F, Gao, J, and Shu, C., Cry78Aa, a novel \u003cem\u003eBacillus thuringiensis\u003c/em\u003e insecticidal protein with activity against \u003cem\u003eLaodelphax striatellus\u003c/em\u003e and \u003cem\u003eNilaparvata lugens\u003c/em\u003e, \u003cem\u003eJournal of Invertebrate Pathology\u003c/em\u003e, 2018, vol. 158, pp. 1\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jip.2018.07.007\u003c/span\u003e\u003cspan address=\"10.1016/j.jip.2018.07.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao, B, Shu, C, Geng, L, Song, F, and Zhang, J., Cry78Ba1, one novel crystal protein from \u003cem\u003eBacillus thuringiensis\u003c/em\u003e with high insecticidal activity against rice planthopper, \u003cem\u003eJournal of Agricultural and Food Chemistry\u003c/em\u003e, 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePanwar, B. 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V., A paratransgenic strategy to block transmission of \u003cem\u003eXylella fastidiosa\u003c/em\u003e from the glassy-winged sharpshooter \u003cem\u003eHomalodisca vitripennis\u003c/em\u003e, \u003cem\u003eBMC Biotechnology\u003c/em\u003e, 2018, vol. 18(1), pp. 50. https://doi.org/10.1186/s12896-018-0460-z\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao, B, Nie, Y, Guan, Z, Yang, Y, Li, J, Liu, Z, and Zhou, D., The crystal structure of Cry78Aa from \u003cem\u003eBacillus thuringiensis\u003c/em\u003e provides insights into its insecticidal activity, \u003cem\u003eCommunications Biology\u003c/em\u003e, 2022, vol. 5, pp. 801. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s42003-022-03754-6\u003c/span\u003e\u003cspan address=\"10.1038/s42003-022-03754-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel, K. 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R, and Thammasittirong, A., Molecular characterization of lepidopteran-specific toxin genes in \u003cem\u003eBacillus thuringiensis\u003c/em\u003e strains from Thailand, \u003cem\u003e3 Biotech\u003c/em\u003e, 2019, vol. 9, pp. 117. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13205-019-1646-3\u003c/span\u003e\u003cspan address=\"10.1007/s13205-019-1646-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta, M, Kumar, H, Debbarma, A, and Kaur, S., Unraveling the abundance of vip3-type genes in Indian \u003cem\u003eBacillus thuringiensis\u003c/em\u003e across the agroclimatic landscape and impact of amino acid substitutions for safer agriculture, \u003cem\u003eGene\u003c/em\u003e, 2025, vol. 933, pp. 148953. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.gene.2024.148953\u003c/span\u003e\u003cspan address=\"10.1016/j.gene.2024.148953\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTharun Kumar, C. J, Subhash, A, Rishika, K. S, Gupta, M, Singh, S, Kalia, V, and Kaur, S., Toxicological impacts of neoteric Vip3A toxins from \u003cem\u003eBacillus thuringiensis\u003c/em\u003e on survival and development of \u003cem\u003eSpodoptera frugiperda\u003c/em\u003e and \u003cem\u003eS. litura\u003c/em\u003e, \u003cem\u003eBiocontrol Science and Technology\u003c/em\u003e, 2025, pp. 1\u0026ndash;17. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/09583157.2025.2492164\u003c/span\u003e\u003cspan address=\"10.1080/09583157.2025.2492164\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalekjalali, M, Barzegari, A, and Jafari, B., Isolation, PCR detection and diversity of native \u003cem\u003eBacillus thuringiensis\u003c/em\u003e strains collection isolated from diverse Arasbaran natural ecosystems, \u003cem\u003eWorld Applied Sciences Journal\u003c/em\u003e, 2012, vol. 18, pp. 1133\u0026ndash;1138.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalama, H. S, Abd El-Ghany, N. M, and Saker, M. M., Diversity of \u003cem\u003eBacillus thuringiensis\u003c/em\u003e isolates from Egyptian soils as shown by molecular characterization, \u003cem\u003eJournal of Genetic Engineering \u0026amp; Biotechnology\u003c/em\u003e, 2015, vol. 13(2), pp. 101\u0026ndash;109. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jgeb.2015.06.004\u003c/span\u003e\u003cspan address=\"10.1016/j.jgeb.2015.06.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePorcar, M, and Ju\u0026aacute;rez-P\u0026eacute;rez, V., PCR-based identification of \u003cem\u003eBacillus thuringiensis\u003c/em\u003e pesticidal crystal genes, \u003cem\u003eFEMS Microbiology Reviews\u003c/em\u003e, 2003, vol. 26(5), pp. 419\u0026ndash;432. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0168-6445(03)00068-7\u003c/span\u003e\u003cspan address=\"10.1016/S0168-6445(03)00068-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biocontrol, Indian Bacillus thuringiensis (Bt) isolates, Prevalence of cry genes, Insect resistance control, Polymerase Chain Reaction (PCR)-based screening","lastPublishedDoi":"10.21203/rs.3.rs-8459314/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8459314/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEntomopathogenic \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (\u003cem\u003eBt\u003c/em\u003e) has emerged as a sustainable alternative to chemical pesticides due to its selective action against insect pests and negligible effects on non-target organisms. However, the increasing occurrence of resistance to conventional \u003cem\u003ecry\u003c/em\u003e genes poses a significant threat to their efficacy. It underscores the immediate need to identify novel \u003cem\u003ecry\u003c/em\u003e genes with unique modes of action and toxicity profiles. To address this, our study investigated the frequency of recently discovered neoteric \u003cem\u003ecry\u003c/em\u003e genes in Indian \u003cem\u003eBt\u003c/em\u003e isolates, known for their diverse range of insecticidal \u003cem\u003ecry\u003c/em\u003e genes. We screened seventy-nine indigenous \u003cem\u003eBt\u003c/em\u003e isolates collected from diverse agroclimatic zones and natural habitats across India for novel \u003cem\u003ecry\u003c/em\u003e genes with activity against Lepidoptera, Coleoptera, and Hemiptera orders. PCR-based analysis confirmed the presence of various insect-specific \u003cem\u003ecry\u003c/em\u003e genes, revealing notable diversity among the isolates. Twenty-seven isolates carried single or multiple \u003cem\u003ecry\u003c/em\u003e genes, with conventional \u003cem\u003ecry7/8\u003c/em\u003e genes being more prevalent than neoteric genes. Among the latter, \u003cem\u003ecry30Fa\u003c/em\u003e was the most abundant, followed by \u003cem\u003ecry30Ga, cry15Aa, cry64Ca, cry79Aa1, cry78Aa\u003c/em\u003e, and \u003cem\u003ecry78Ba\u003c/em\u003e. Remarkably, isolates SK-768, SK-979, SK-110, and SK-949 harbored \u003cem\u003ecry\u003c/em\u003e genes active against two or three insect orders. These findings highlight the importance of region-specific \u003cem\u003eBt\u003c/em\u003e strains in formulating effective biocontrol strategies.\u003c/p\u003e","manuscriptTitle":"Screening of Bacillus thuringiensis Isolates Recovered from Diverse Habitats in India for Crystal Toxin Genes Predicting Toxicity Against Three Insect Orders: Lepidoptera, Coleoptera, and Hemiptera","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 10:03:38","doi":"10.21203/rs.3.rs-8459314/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"79ca1fce-09e3-489d-952b-e0d3a964b98e","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T12:10:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-14 10:03:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8459314","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8459314","identity":"rs-8459314","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}