Molecular characterization and toxicity evaluation of indigenous Bacillus thuringiensis isolates against key lepidopteran insect pests

Background: The indiscriminate use of synthetic insecticides is not affordable and poses collateral damage to humans, non-target organisms, and environmental health. Bacillus thuringiensis ( Bt) is a cosmopolitan, antagonistic soil bacterium employed as an alternative to chemical insecticides in pest management. In this study, genetic diversity and insecticidal activity of 50 indigenous Bt isolates were investigated in order to find the effective Bt isolates with a broad spectrum of insecticidal activity against lepidopteran insects. Results: Most of the Bt isolates investigated appeared as creamy white in colour and fried egg or mucoid-type colonies having a flat or raised elevation with entire or undulated margins. Out of 50 isolates investigated, crystal shapes varied from cuboidal (40.00%), spherical (36.00%), bipyramidal (30.00%), rectangular (10.00%), and minute crystal attached to the spores (28.00%). SDS-PAGE analysis revealed that the molecular weight of the crystal proteins of Bt iso-lates ranged from ~ 30 to ~ 200 kDa in size. Among the Bt isolates screened, ~ 135 kDa size representing Cry1 protein was observed in 12 isolates (24.00%) and 65 kDa representing Cry2 was observed in 14 isolates (28.00%). PCR analysis was performed for cry1, cry2, cry3, cry4, cry9, vip1, vip2, and vip3 genes, which revealed the presence of cry1 gene alone in 5 isolates, and cry2 alone in 7 isolates, whereas 7 isolates were positive for cry1, cry2, and vip3 genes together. None of the Bt isolates showed the presence of cry3, cry4, cry9, vip1, and vip2 genes. Toxicity of Bt isolates was tested against four species of lepidopteran larvae, viz. Plutella xylostella Linnaeus , Helicoverpa armigera Hubner , Spodoptera litura Fabricius , and S. frugiperda Smith . Among them, 6 isolates (T29, T30, T31, T357, T381, and T388) produced 100% larval mortality against all four species of insects. Conclusion: The present study showed the diversity of Bt isolates and confirmed the significance of relentless exploration of Bt isolates for novel genes. Further investigations need to be carried out to disclose the hidden potential of these toxic isolates.


Background
Bacillus thuringiensis (Bt) is a gram-positive, spore-forming entomopathogenic bacterium that produces different proteins toxic to many insects, nematodes, mites, and also protozoans (Santos et al. 2022).It is diversified all over the world usually found in soil, leaf surfaces, grain dust, dead insects, and aquatic environment (Gupta et al. 2021).The insecticidal proteins were produced during the stationary phase as crystal inclusions (crystal (Cry) and cytolytic (Cyt) proteins) or secreted during the vegetative phase (vegetative insecticidal proteins (Vip)) of bacterial growth (Adang et al. 2014).Among these, Cry and Cyt proteins are most explored as biopesticides for their toxicity against various agricultural pests and vectors of human diseases.Crystalline toxins or δ-endotoxins are considered as the main factor conferring insecticidal properties to Bt (Bouslama et al. 2020).Commercialized Bt delta toxins have been used as pesticides over decades as they have constitutive blocks of amino acids and different specificities against different orders of insects (Van Frankenhuyzen 2009).δ-endotoxins of Bt attack the insects' membrane pores and form channels (Melo et al. 2016).The three-domain proteins have various complementary aspects of insect toxification.The first domain is responsible for the formation of pores; the second domain is specific in binding to the receptors in the epithelial cells of the insects' midgut; the third domain is functioning in stabilizing the bond between toxin and receptor that results in osmatic discrepancy and finally death of insect (Melo et al. 2016).Numerous Bt strains that showed toxicity towards a wide range of insect pests, viz.lepidopterans, dipterans, coleopterans, homopterans, hymenoptera, mallophaga, and nematodes, have been isolated from different sources and used for developing biopesticides (Abo-Bakr et al. 2020).The crystal proteins, viz.Cry1, Cry2, Cry9, and Vip3A, were found to be toxic and specific to various lepidopteran insect pests.At present, about 400 Bt-based biopesticide formulations made up of insecticidal proteins and spores have been registered and commercially used in the market.These toxins are employed in pest management in the form of biopesticides or by expressing the toxin-encoded genes in commercial crops against target insect pests (George and Crickmore 2012).
Based on the amino acid sequence homology, the insecticidal proteins were classified into different groups and so far 80 holotypes of crystal proteins (Cry1-Cry 80), four holotypes of vegetative insecticidal proteins (Vip1-Vip4), and seven holotypes of cytolytic proteins (Cyt1-Cyt7) of Bt have been identified and reported (Crickmore et al. 2018).Cry toxins are highly selective and specific to their target insect, inoffensive to non-target pests, vertebrates, and humans, and expeditiously degrade in the environment.Therefore, Bt is an effective alternative to synthetic insecticides for the management of agriculturally important insect pests.However, long-term use of Bt toxins leads to the development of resistance to the toxins by the target insect pests (Hassan et al. 2021).As a result, exploring new strains of Bt with novel toxins will provide another way to mitigate these problems.With this background, the present investigation was carried out to characterize 50 indigenous Bt isolates to explore their diversity and toxicity against key lepidopteran insect pests.

Indigenous Bt isolates and growth conditions
A total of 50 indigenous Bt isolates were obtained from Bt laboratory stock, Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, India (Additional file 1: Table S1).The reference strains for a different group of Cry toxins originally received from Bacillus Genetic Stock Center (BGSC, Columbus, Ohio) were used as a positive check.The Bt cultures were revived from glycerol stocks on T3 agar medium (per litre: 3.0 g Tryptone, 2.0 g Tryptose, 1.5 g Yeast extract powder, 6.9 g NaH 2 PO 4 ; 8.9 g Na 2 HPO 4 and 0.005 g MnCl; 10.0 g agar (pH 6.8-7.0) and incubated at 30 °C for 14 h.In order to get single colonies, the overnight grew Bt cultures were subcultured on T3 agar medium by quadrant streak method and incubated at 30 °C for overnight.Purified single colonies were inoculated separately in test tubes containing 5 ml of T3 broth and incubated at 30 °C for 24 h with 200 rpm (Orbitek, Scigenics Biotech India Pvt Ltd., Chennai, India).

Bacterial colony and crystal morphology
The colony morphology of individual Bt isolates was examined visually.A loop full of Bt culture was inoculated in 5 ml of T3 broth and incubated at 30 °C for 48 h at 200 rpm.To check the spore-crystal inclusions in the Bt isolates, a loop full of culture smears was prepared in glass slides, heat-fixed, and stained with the 0.133% Coomassie Brilliant Blue stain (G250).Then, the stained-glass slides were washed gently with water, blot dried with tissue paper, and observed under a brightfield microscope for the existence of crystalline inclusion (Leica DM 1000LED, DFC295, Germany).

Preparation of spore-crystal mixtures from Bt isolates
A single colony of Bt cultures was inoculated into 5 ml of T3 broth and incubated at 30 °C overnight at 200 rpm.After the overnight incubation, 1% grown cultures were inoculated into a 250-ml conical flask containing 25 ml of T3 broth and incubated at 30 °C with shaking of 200 rpm for 48-60 h.The bacterial sporulation was observed under a bright-field microscope (Leica DM 1000LED, DFC295, Germany).When more than 90% of cells were lysed, the sporulated cultures were transferred into 50-ml falcon tubes and incubated at 4 °C for half-an-hour before harvest.The sporulated bacterial cultures were centrifuged at 8000 rpm for 10 min at 4 °C.The supernatant was discarded, and the pellet was resuspended in 25 ml of ice-cold Tris-EDTA buffer (10 mM Tris; 1 mM EDTA and pH 8.0) containing 1 mM PMSF (phenyl methyl sulphonyl fluoride) and washed thrice with Tris-EDTA buffer and once with 25 ml of ice-cold 0.5 M NaCl solution containing 0.5 mM PMSF by centrifugation at 8000 rpm for 10 min at 4 °C (Eppendorf centrifuge 5810R, Germany).The final pellet was dissolved in nuclease-free water containing 1 mM PMSF and stored at − 20 °C (Ramalakshmi and Udayasuriyan, 2010).

Protein profiling of Bt isolates
Protein profiling was done using SDS-PAGE (Sodium dodecyl sulphate poly acrylamide gel electrophoresis) by the method of Laemmli (1970) using 10% separating and 4% stacking gel.The spore crystal mixture samples were prepared by mixing with 4× loading dye (0.25 M Tris HCl pH 6.8; 8% SDS, 40% glycerol, 0.5% bromophenol blue) in a ratio of 4:1.Then, the samples were incubated in boiling water for 2 min before loading.The molecular weight of the protein was estimated by using three colour pre-stained protein marker (PG PMT 2922, Puregene, Genetix Biotech Asia Pvt Ltd.,) covering a broad range of molecular weights from 10 to 250 kDa.

Bacterial genomic DNA isolation and polymerase chain reaction
Purified single colonies of Bt isolates were used for genomic DNA isolation by following Sambrook and Russell's (2001) method.Extracted genomic DNA was used as a template DNA (30-50 ng) for the amplification of cry1, cry2, cry3, cry4, cry9, vip1, vip2, and vip3 genes with their specific primers (Additional file 1: Table S2).A total reaction volume of 20 µl consists of 1 µl of template DNA, 1 µM of each primer, 10 µl of 2X PCR master mix (SmartPrime) consisting of dNTPs, Taq polymerase, and 7 µl of nuclease-free water.PCR analysis was performed in Mastercycler Nexus GX2 (Eppendorf, Germany), and the PCR products were resolved in agarose gel electrophoresis with ethidium bromide staining.The products were visualized under a UV trans-illuminator for the expected size of amplification of different cry and vip genes (Additional file 1: Table S2).

Insect cultures
The insect cultures of Plutella xylostella (pupae), Spodoptera litura (eggs), S. frugiperda (eggs), and Helicoverpa armigera (eggs) were obtained from NBAIR (National Bureau of Agricultural Insect Resources, Bangalore, India) and established in insect bioassay laboratory at Department of Plant Biotechnology, CPMB&B, TNAU, Coimbatore, India.The newly emerged adult P. xylostella was supplemented with a 10% sugar solution along with Vitamin E, and two leaf-stage mustard seedlings were provided as an oviposition substrate.Newly hatched neonate larvae were transferred into Cauliflower leaf (maintained under controlled conditions) and were maintained at the laboratory with fresh cauliflower leaves as and when required.The cultures of S. litura, S. frugiperda and H. armigera larvae were reared on an artificial diet.Pupae were collected and allowed for adults' emergence in an insect-rearing cage (2.5 × 2.5 Ft).Newly emerged adult moths were supplemented with 10% sugar solution along with Vitamin E. Young maize seedlings for S. frugiperda and H. armigera, and Nerium oleander shoots for S. litura were placed inside the cage as an oviposition substrate.Eggs were collected and maintained in Petri plates for hatching.Newly hatched neonates were transferred into an artificial diet by using a camel hairbrush and maintained.

Toxicity of Bt isolates against lepidopteran insects
The concentration of spore-crystal mixtures was estimated by Broadford's reagent method (Bradford 1976).Estimated spore-crystal mixtures were equalized to a concentration of 20 µg/ml and used for in vitro insect bioassay.Detached leaf bit bioassay was carried out with insect-specific host plants.Cauliflower leaves were used for P. xylostella and S. litura, while maize leaves were used for S. frugiperda and H. armigera.Circular leaf bits of cauliflower (2 cm dia) were prepared and coated with the spore-crystal mixture on both sides (10 µl/side) of the leaf disc and air-dried.Similarly, 2 cm leaf bits of maize leaves were treated with spore-crystal mixtures (10 µl/ side).Treated leaf bits were placed into a 3 cm dia plastic container containing moist filter paper and pre-starved neonate larvae were released.Ten larvae were used per treatment and replicated thrice.The larval mortality was observed up to 72 h after treatment and expressed in percentage.The entire in vitro insect bioassay experiments were conducted under a controlled environment with 25 ± 1 °C and 65 ± 2% RH.

Statistical analysis
The laboratory experiments were performed in a completely randomized design (CRD).The larval mortality data were computed using Abbott's formula (Abbott 1925), and subjected to one-way variance (ANOVA) in AGRES statistical software version 7.01, and significant differences between means were determined by Duncan's multiple range test (DMRT) at p = 0.05.

Bacterial colony and crystal morphology
All the bacterial colonies screened were creamy white in colour.Colony type was observed as fried egg type with raised elevation in 12 Bt isolates and flat elevation in 31 isolates, whereas mucoid-type colonies were found in six isolates with flat elevation and one isolate with raised elevation only in margins.The colony shape appeared circular in 34 Bt isolates and irregular in 16 isolates.The colony margin of 20 isolates was undulated and the remaining were entire (Table 1).Sporecrystal inclusions were observed under a bright-field microscope (100×) (Leica DM 1000LED, DFC295, Germany).Different types of crystal protein morphology were observed in 50 isolates varying from cuboidal (40.00%), spherical (36.00%), bipyramidal (30.00%), rectangular (10.00%), and minute crystal attached to the spores (28.00%) (Table 2; Fig. 1).

Protein profiling of Bt isolates
SDS-PAGE analysis revealed that the molecular weight of the parasporal crystal proteins of Bt isolates ranged from 30 to > 200 kDa in size.Among the Bt isolates screened, 135 kDa size representing Cry1 protein was observed in 12 isolates (24.00%) and 65 kDa representing Cry2 was observed in 14 isolates (28.00%) (Table 3; Fig. 2).Protein banding patterns were distributed as one prominent band in 16 isolates, two in 16 isolates, and more than three bands were recorded in 11 isolates, whereas seven isolates did not show any discrete band (Additional file 1: Table S3).

Discussion
The diversified Bacillus thuringiensis (Bt) are found worldwide in different ecological habitats, viz.soil, sediment, stored products, insect cadaver, phylloplane, and aquatic environments (Baig et al. 2010).Bt producing insecticidal proteins are highly specific to the target insect pests (De Maagd et al. 2001).Hence, the characterization of Bt isolates from different geographical areas may lead to getting novel Bt proteins with a high level of insecticidal activity or broaden the insect spectrum.
The Bt isolates investigated appeared as creamy white in colour and fried egg or mucoid type colonies having flat or raised elevation with entire or undulated margins.Navya et al. (2021) observed creamy white to off-white colour colonies with fried egg appearance, irregular shape, flat and undulate margin in the Bt isolates screened.
The Bt bacterium was identified mainly based on the presence of parasporal crystalline inclusions.In the present study, out of the 50 isolates investigated, crystal shapes varied from cuboidal, spherical, bipyramidal, rectangular, and crystal attached to the spores.These findings are in accordance with reports by Ramalakshmi and Udayasuriyan (2010) wherein the Bt isolates with cuboidal (26.90%) and bipyramidal (21.00%) shapes were predominant when compared to other shapes, whereas these findings varied from the previous reports by Navya et al. (2021) from India, andNair et al. (2018) from Qatar wherein more frequency of spherical crystals were observed.Bt isolates producing bipyramidal and cuboidal shapes of crystal were found to be highly toxic to lepidopteran insect pests (Boonmee et al. 2019).Variations observed in the morphology of the crystal shapes in the Bt isolates suggest the presence of diversity in Bt isolates in Tamil Nadu.
Analysis of crystal protein(s) profile could be useful to predict the presence of insecticidal genes.Among the Bt isolates screened, 135 kDa size representing Cry1 protein was observed in 12 isolates (24.00%) and 65 kDa representing Cry2 was observed in 14 isolates (28.00%).There are protein bands of different molecular weights between 30 and > 200 kDa in size observed which need to be characterized.Diversified electrophoretic patterns in Cry protein with molecular weights ranging from 20 to 160 kDa were reported earlier by Navya et al. (2021).In the present study, 16 isolates were found to produce one protein band (32.00%) and 16 isolates produce two protein bands (32.00%).Most of the isolates (28.00%) produced protein bands at ~ 65 kDa size representing Cry2 protein, followed by Cry1 protein (135 kDa) (Additional file 1: Table S3).Ramalakshmi and Udayasuriyan (2010) found that out of 70 isolates analysed, 17 isolates (24.20%) had two major protein bands with molecular weights in the range of ~ 135 and ~ 65 kDa.The diversity in the protein profile of Bt isolates indicates the possibility of diverse cry genes and corresponding insecticidal activities.
PCR analysis of 50 isolates indicates that five isolates were positive for cry1 gene alone and seven for cry2 gene alone, whereas seven isolates were positive for cry1, cry2, and vip3 genes together.These findings were in

Conclusion
The present investigation revealed the diversity among the indigenous Bt isolates in terms of colony and crystal morphology, protein content, gene content, and toxicity to lepidopteran pests.Six out of 50 isolates produced 100% larval mortality against all the tested species, viz.P. xylostella, H. armigera, S. litura, and S. frugiperda.These results suggest that some of these isolates after proper evaluation of toxicity, the spectrum of activity, and speed of kill against a different group of lepidopteran insects can be developed into broad-spectrum biopesticides.
Although some of the isolates had more than one cry/vip gene did not produce any toxic protein; some of the Bt isolates did not produce any amplicon with the primer     sets used but produced distinct protein bands; some of the isolates showed distinct protein bands but did not show any toxicity; all these necessitates further investigations for the presence of novel cry genes and novel protein.

Fig. 3
Fig. 3 PCR amplification of cry and vip genes

Figures
Figures in parentheses are arcsine transformed values of percentages.Values followed by the same letters in a column are not significantly different (p = 0.05) by DMRT

Fig. 4
Fig. 4 Variation in mortality of lepidopteran larva with 20 µg/ml of spore crystal mixtures of indigenous Bt isolates

Fig. 5
Fig. 5 Distribution of indigenous Bt isolates producing 100 per cent mortality in four species of lepidopteran insects

Table 1
Colony morphology of indigenous Bt isolates

Table 2
Crystal shapes of Bt isolates

Table 3
Protein profile of indigenous Bt isolates from SDS-PAGE analysis

Table 4
Insecticidal gene profile of indigenous Bt isolates from PCR screening

Table 5
Toxicity of indigenous Bt isolates against Lepidopteran pests Sahin et al. (2018)21)2)imilarresults were reported earlier, and they found spore crystal mixtures of Bt isolates able to cause 100% mortality in P. xylostella(Navya et al. 2021), H. armigera(Lone et al. 2017), S. litura(Nayanganesh et al. 2018) and S. frugiperda(Karuppaiyan et al. 2022).In this present study, diversified larval mortality was observed among the Bt isolates screened against the test insects.These results revealed that the Bt isolates are not always effective against all the lepidopteran insects, especially the species of Spodoptera, which could be due to natural resistance developed against specific proteins.S. frugiperda populations showed to be naturally resistant to the Bt proteins Cry1Ab and Cry1Ac(Huang et al. 2014).The fall armyworm population from Puerto Rico exhibited stable Cry1F and Cry1Ac resistance but was still vulnerable to Cry1A.105 and Cry2Ab2 proteins (Gutierrez-Moreno et al. 2020).Besides, the Bt isolates with cry1, cry2, and vip3 gene combinations produced 100% toxicity to all the tested insects, as reported earlier byMaheesha et al. (2021)in S. frugiperda.Federici (1999)reported that Bt products are not always effective against certain noctuid pests, especially S. litura, S. frugiperda, and S. littoralis, whereasSahin et al. (2018)reported that Cry1 and Cry2 type proteins are toxic to many insects viz., S. exigua, S. littoralis, S. frugiperda, H. armigera, and Grapholita molesta.Hence, the observed toxicity of tested Bt isolates against lepidopteran insects is presumed to be mainly due to the expression of Cry/ Vip proteins.