Identification and Analysis of Toxins in Novel Bacillus thuringiensis Strain Bt S3076-1 against Spodoptera frugiperda and Helicoverpa armigera

doi:10.21203/rs.3.rs-2475119/v1

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Identification and Analysis of Toxins in Novel Bacillus thuringiensis Strain Bt S3076-1 against Spodoptera frugiperda and Helicoverpa armigera

https://doi.org/10.21203/rs.3.rs-2475119/v1

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published 05 Apr, 2023

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Despite the successful application of toxins from Bacillus thuringiensis as biological control agents against pests, new toxins having higher toxicity and broad-spectrum activity to insects are identified more and more. To find new toxins, in this study, based on the whole genome sequence of novel Bacillus thuringiensis strain Bt S3076-1, ten predicted toxic genes were identified, including six cry genes, two tpp genes, one cyt gene and one vip gene, among which six were novel toxins. Subsequently, SDS-PAGE analysis showed that the major proteins at spore maturation stage were about 120 kDa, 70 kDa, 67 kDa, 60 kDa and 40 kDa, while approximately 70 kDa and 40 kDa active proteins after trypsin digestion with the LC50 of 149.64 µg/g and 441.47 µg/g against Spodoptera frugiperda and Helicoverpa armigera larvae, respectively. Furthermore, pathological observation results showed the peritrophic membrane of Spodoptera frugiperda and Helicoverpa armigera larvae was degraded. These findings will provide experimental reference for further research on insecticidal activity, toxicity spectrum as well as the synergism of these toxins in Bt S3076-1.

Bacillus thuringiensis

Bt S3076-1

Toxin

Spodoptera frugiperda

Helicoverpa armigera

Insecticidal activity

Spodoptera frugiperda and Helicoverpa armigera are important agriculture pests, and the long-term use of microbial pesticides can easily be prone to increase the pest resistance to Spodoptera frugiperda and Helicoverpa armigera. Finding novel Bacillus thuringiensis insecticidal strains and toxic proteins can be more effectively controled. Bt S3076-1, which was isolated from Diaoluoshan National Nature Reserve in Hainan Province, exhibited insecticidal activity against Spodoptera frugiperda and Helicoverpa armigera. Based on the whole genome sequence of Bt S3076-1, 10 predicted toxin genes were analyzed and identified via bioinformatics method. It will provide new resources for accurate prevention and control the agricultural lepidopteran pests.

Spodoptera frugiperda (Lepidoptera: Noctuidae, Spodoptera) is an important migratory pest, originating in tropical and subtropical America(Sparks. 1979)(Mehrkhou et al. 2012), which has the characteristics of strong reproduction, fast migration, and wide host range(Fu et al. 2017). It is a worldwide widespread pest, and its host plants include 350 species belonging to 76 families, including Gramineae, Leguminosae, and Compositae(Montezano et al. 2018). In the larval stage, they feed on corn leaves and ears, and one adult female of Spodoptera frugiperda can lay up to 2,000 eggs, which can directly damage one acre of corn. In the last two decades, severe outbreaks of this pest have been reported in many parts of Asia, Africa, Europe, and America(Zhang et al. 2008)(Ehler 2004)(Mehrkhou et al. 2012)][. Helicoverpa armigera, another kind of worldwide omnivorous pest, is also a major pest in China, destroying cotton, tomato, pepper, tobacco, sunflower, corn and sorghum and other crops, causing significant economic losses(Chen and Liu 2021). Currently, chemical pesticides are mainly used to control lepidopterous pests like Spodoptera frugiperda and Helicoverpa armigera. However, the long-term use of chemical pesticides can easily be prone to increase the pest resistance and environmental pollution, and even cause food safety problems(Casida and Durkin 2017). Therefore, green, safe and environmentally friendly biopesticides are more and more popular.

At present, Bacillus thuringiensis (Bt) is the most widely used microbial insecticide, and many toxic proteins have been applied to agricultural pest control(Kumar et al. 2008). Since the first cry1Ab1 transgenic maize was used to control the European corn borer in 1996, lepidopteran pests have been controlled for nearly two decades(Meissle et al. 2011). Previous studies have found that the Bt insecticidal genes against Spodoptera frugiperda are mainly cry and vip like toxic proteins(Ingber et al. 2018)(Grossi-De-Sa et al. 2007), among which, Cry1Ab, Cry1Ac, Cry1Ea, Cry2Aa, Cry2Ab, Cry3Aa, Cry3Ca, Vip3Aa and Vip3Ca all have high insecticidal activity against Spodoptera frugiperda. Although the amino acid sequences of each toxin differed very little, their insecticidal activity against Spodoptera frugiperda is quite different (Chakroun et al. 2016). In addition, long-term single use led to the gradual emergence of resistance of Spodoptera frugiperda(Jakka et al. 2014). For example, in Brazil, transgenic soybeans with cry1Ac and cry1F genes have been unable to effectively control Spodoptera frugiperda(Machado et al. 2020). Transgenic corn expressing cry2Ab2 also needs to be co-expressed with other toxic proteins to keep its insecticidal activity(Yang et al. 2017). In addition, there is no report about the resistance of Vip3Aa transgenic corn to Spodoptera frugiperda(Fatoretto et al. 2017), therefore, the synergistic effects of Cry-type and Vip-type toxic proteins have been used to against Spodoptera frugiperda (Bergamasco et al. 2013). Such as, corn with multi-gene of Cry1Ab+Vip3Aa has a good effect on controlling Spodoptera frugiperda(Siebert et al. 2012). Although transgenic Bt cotton can effectively resist agricultural pests including Helicoverpa armigera, long-term, single and large-scale planting of transgenic Bt cotton will increase the selection pressure of Helicoverpa armigera, which will lead to the increase of resistance of Helicoverpa armigera to transgenic Bt cotton(Wei et al. 2021). Consequently, it is very important to explore new Bacillus thuringiensis strains and novel toxic proteins for the effective control of agricultural pests.

With the rapid development of sequencing technology, whole genome sequencing has provided a convenient way to mine the toxic protein genes of Bacillus thuringiensis. Up to now, 195 strains of Bacillus thuringiensis have been reported and completed whole genome sequencing, and 1110 insecticidal toxic genes have been published online (https://www.bpprc-db.org/), including 753 Cry genes, 42 Cyt genes and 133 Vip genes, and 182 other types of genes such as App, Tpp and Mpp(Soaresdasilva et al. 2015)(Hayakawa et al.2016)(Beltrao and Silvafilha 2007)][. To identify the types and effects of the major toxic genes in the target strains, we can modify the dominant toxic gene to improve its insecticidal activity. Such as, genetic modification of toxin proteins, containing protein domain transformation, site-directed mutation and promoter replacement, may improve the effect or insecticidal spectrum of Bacillus thuringiensis (Berry et al. 2002).

Strain Bt S3076-1, isolated from Diaoluoshan National Nature Reserve in Hainan Province, exhibited insecticidal activity against Spodoptera frugiperda and Helicoverpa armigera. In order to explore the insecticidal gene resources in strain Bt S3076-1, illumina HiSeq 2000 sequencing platform was used to carry out the whole genome sequence of this strain and assembled the whole genome data in previous stage(Wu et al. 2016). Here, on the one hand, the parasporal crystal shapes were observed by scanning electron microscope, and the expression of total protein as well as the intestinal tissue identification of Spodoptera frugiperda and Helicoverpa armigera after feeding on spores and crystal mixtures were analyzed, respectively. On the other hand, based on the genome sequence of strain Bt S3076-1, predicted toxin genes were analyzed and identified via bioinformatics method, which will subsequently provide new resources for accurate prevention and control the agricultural lepidopteran pest.

2.1 Gene prediction and identification of Bt 3076-1whole genome sequence

Firstly, With Glimmer（http://ccb.jhu.edu/software/glimmer/index.shtml; version 3.02） tools, An Open Reading Frame (ORF) prediction was performed based on Bt 3076-1 whole genome sequence. Gene prediction was carried out using MetaGeneMark (http://exon.gatech.edu/ GeneMark/metagenome/index.cgi; version1.0) tool. KEGG automatic annotation server (KAAS, http://www.genome.jp/tools/kaas/; Ver.1.67x, BBH method) was applied for function annotation and enrichment analysis of predicted protein coding genes. And the automatic functional annotation was analyzed through RAST (http://rast.nmpdr.org/). Subsequently, 1110 amino acid sequences and 192 nucleotide sequences of toxic proteins were obtained from international toxic protein database (https://www.bpprc-db.org). Meanwhile, local alignment database was constructed for Blast. The predicted toxic genes were screened out through the comparison program of Blastp and Blastn with the threshold of significance level as E-value < 0.0001.

4.2 Identification and structure analysis of toxin

Functional domain of predicted toxic protein was analyzed by CDD of NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), and the homology alignment analysis of all the predicted toxins of Bt S3076-1 strain were performed on the Bacterial Pesticidal Protein Resource Center (https://www.bpprc-db.org/search_ database_home/). The high homology amino acid sequences of toxic proteins were selected to construct the phylogenetic tree using MEGA 11.0(Kumar et al. 2016).

2.3 SDS-PAGE identification of Bt 3076-1

According to Qiu's method(Qiu et al. 2017), the total protein of Bt S 3076-1 strain cultured for 72 h were extracted by using the ultrasonic apparatus (Ningbo Xinzhi JY 92-II) under the working conditions of 3 s working, 7 s rest, 150 cycles and 200 W of power. After digested by trypsin for 30 minutes, 60 minutes and 120 minutes, respectively, the digestion products were analyzed by 10% SDS-PAGE gel electrophoresis at 80 V for 30 min and 120 V for 1 h. And then dyed with Coomassie brilliant blue for 30 min and decolorized in the decolored solution with glacial acetic acid: methanol: water at 1:1:8 for 6 h. Finally, BIO-RAD protein imaging system was used for observation and analysis.

2.4 Bioassay

Bt S3076-1 strain was cultured on NB medium at 30℃ with 200 r/min for 72 h till forming spores and quantities of parasporal crystal proteins were produced. Then harvest the bacteria by centrifugation at 8000 r/min for 5 min at 4℃ and discard the supernatant to add 10 mL 50 mM Tris-Hcl (50 mM Tris-HCl, 500 mM NaCl, 10% glycerol, pH=7.8) resuspension for bioassay. Subsequently, the 2nd instar larvae of Spodoptera frugiperda and Helicoverpa armigera in good physiological conditions were selected as target insects for biological activity determination. The serial-treated protein concentrations were set for each kind of larvae, and 20 2nd instar larvae were treated in each concentration, setting distilled water as negative control. Each treatment was repeated for 3 times independently. They were kept at 28°C and 75% relative humidity at 12 h light/dark for 4 days. Mortality was observed and recorded after incubation for 96 h. Finally, the 50% lethal concentration (LC50) and 95% confidence interval were determined by Probit analysis using SPSS 26.0 software.

2.5 Pathological observations on midgut tissue of target larvae

The just dead larvae after treating with Bt S3076-1 total protein were immediately fixed in 2.5% glutaraldehyde solution (pH7.4) at room temperature for 2 to 4 hours. And then the fixed larvae were directly put into a dehydration box, which was placed in a dehydrator (Donatello) for dehydration, followed by gradient alcohol dehydration and then waxing. Subsequently, the wax-impregnated larvae were embedded in an embedding machine (JB-P5). After the wax was solidified, the wax block was taken out from the embedding frame and trimmed, and then placed on microtomes (RM 2016) for slicing, keeping the thickness of the slice of 4 to 10 μm. the slices were treated with Xylene for 20 min twice and absolute ethanol for 5 min twice, as well as 75% ethanol for 5 min, followed by washing with tap water. Subsequently, the slices were placed in hematoxylin dyeing solution for 3~5 min, followed washing with tap water, differentiating with differentiation solution, washing with tap water, blued with return solution, and washing with running water. The slices were then successively dehydrated using 85% and 95% ethanol each for 5 min, and stained in Yihong dyeing solution for 5 min. Transparency was next ensured by sequential treatment with a gradient of alcohol concentrations and a transparent agent. Finally, the slices were observed and recorded by microscopy (Leica DM3000).

3.1 Observation the parasporal crystal of Bt S3076-1

The previous results demonstrated that Bt S3076-1 had high insecticidal activity against Spodoptera frugiperda and Helicoverpa armigera. Phase contrast microscope and scanning electron microscope (SEM) observation showed that Bt S3076-1 strain produced numerous parasporal crystals with various shapes including square, irregular, spherical during sporulation (Figure. 1). Therefore, it was speculated that the insecticidal activity of this strain might be attributed to these many shapes of parasporal crystals.

3.2 ORF and coding gene prediction of Bt S3076-1 whole genome

Based on the complete genome data of Bt S3076-1, 7700 ORF sequences were identified by using Glimmer, including 6662 in the chromosome genome, 365 and 673 in two plasmids, respectively. Subsequently, 6,421 coding genes of Bt S3076-1 whole genome were predicted by MetaGeneMark, corresponding 6,421 coding proteins, of which 5,711 were distributed in chromosome genome S3076-1chromosome, 188 in S3076-1plasmid1 and 522 S3076-1plasmid2 (Table 1).

Table 1

ORF and coding genes of Bt S3076-1 whole genome
Genome	ORF	ORF average length (bp)	CDSs	CDSs average length (bp)
S3076-1Chromosome	6662	811	5711	839
S3076-1plasmid1	365	443	188	618
S3076-1plasmid2	673	709	522	732

3.3 Coding gene KAAS functional annotation analysis of Bt S3076-1 genome

In addition, 6,421 predicted genes encoded proteins were annotated by KAAS. The results showed that 2,609 proteins were annotated to the known functions, and the others were hypothetical proteins. Subsequently, functional enrichment analysis was performed on the 2,609 functional proteins. It was found that functional proteins were mainly enriched in biological processes such as biosynthesis of amino acid, ABC transporter, carbon metabolism, purine metabolism, ribosome, pyrimidine metabolism, and so on. Some proteins also involved in the control of cell wall, protein and lipid metabolism, as well as spore formation. Furthermore, some proteins participated in pathways including plasmids, bacteriophage, signal transduction regulation and stress response (Figure. 2).

3.4 Identification and analysis of toxin sequence

Based on the prediction of the Bt S3076-1 strain whole genome gene sequence, as well as the KAAS functional annotation analysis, the amino acid sequence of predicted toxins was blasted in the Bacterial Insecticidal Protein Resource Center (http://bpprc.Org/). The result showed that 10 predicted toxic genes were in Bt S3076-1 strain, including six cry genes, two tpp genes, one cyt gene and one vip gene (Table 2). Among them, the similarity of toxin 1044 and 2041 to homology protein was 80.4% and 92.3%, respectively, which belonged to the third classification level (Rank 3 with the similarity between 78% and 95%) of the Bt toxin protein classification system, indicating that they are the homologous genes to the known toxic genes. Similarly, the similarity of toxin 1035 and 1139 shared 77.3% and 66.7%, respectively, which is belonged to the second classification level (Rank 2 with the similarity from 45–78%), demonstrating that these two toxins are important homologous proteins to the known toxins. as for the remaining six predicted proteins, the similarity of them were less than 45%, belonging to the first classification level (Rank 1), which was presumed to be novel toxin protein genes (Table 2).

According to the gene information, it was found that, except 5295 and 5303 toxic genes located on chromosome, the remaining eight toxic genes were all located on S3076-1 plasmid1 and S3076-1 plasmid 2 (Figure. 3). Subsequently, the phylogenetic tree analysis of the predicted toxins with their homologous proteins showed that (Figure. 4) all other Cry like toxins belong to one large branch but toxin 2041 and 2521, which probably due to the lack of Endotoxin_M domain. The Cyt, Tpp and Vip like toxins were located on an individual branch, respectively. Of which the similarity of toxin 2041 with Cry70Ba1 was 92.3%, indicating the closest genetic relationship. Although the highest similarity of toxin 2219 was 33.6% to Vip1Aa1, phylogenetic tree displayed that 2219 was clustered into one branch with Vpb4Aa1 and Vpb4Ab2, not Vip1Aa, which represented a potential Vip like toxic protein of Bt S3076-1. The other evolutionary relationship between toxins and the corresponding known homologous proteins was relatively conservative.

Moreover, protein conserved domain analysis present that (Figure. 5) among the six Cry toxins, 1139, 1035, 1044 and 2044 toxins had typical 3D domains Endotoxin_N, Endotoxin_M and Endotoxin_C, while 2041 and 2521 toxic proteins only contained Endotoxin_N and Endotoxin_C, which shared the same phenomenon in toxins Cry39Aa1 and Cry70Aa1; meanwhile, only one Toxin 10 domain are both in toxin 595 and 5303; yet a tandem RicinB_lectin_2 domain was present in Cyt-like toxin 1073, and it was found that this structural domain had the inhibited toxicity and signal transduction(Wu et al. 2007). Besides, 2219 toxin contained three Binary toxB domains and one PA14 domain.

Table 2

Homology alignment information of 10 predicted toxic genes of Bt S3076-1 strain
Gene ID	Length (bp)	Homologous protein	Identify
5295	1092	Tpp49Aa1	31.7%
5303	1080	Tpp49Aa1	31.9%
1035	1677	Cry56Aa4	77.3%
1044	1596	Cry19Aa1	80.4%
1073	1695	Cyt1Ca1	19.4%
1139	2097	Cry69Aa2	66.7%
2041	1638	Cry70Ba1	92.3%
2044	2427	Cry79Aa1	35.3%
2219	2931	Vip1Aa1	33.6%
2521	3735	Cry39Aa1	27.8%

3.5 Total protein of spore maturation and trypsin digestion analysis

Spore-crystal mixtures were collected cultured for 72 h on NB medium, and then were prepared for the total protein of Bt S3076-1 by ultrasonic apparatus and analyzed the expression of the protein by SDS-PAGE. Simultaneously, the total protein was digested by trypsin (1:300, from Porcine Pancreas, Sangon Biotech, Shanghai) to validate the active peptide. SDS-PAGE analysis showed that the major protein bands produced at spore maturation stage were 120 kDa, 70 kDa, 67 kDa, 60 kDa and 40 kDa. After trypsin digestion for 2 h, the protein was hydrolyzed to approximately 70 kDa and 40 kDa active proteins (Figure. 6), which was consistent with the reference(Navon and Ascher 2000).

3.6 Bioassay of Bt S3076-1

Total protein of against 2nd instar larvae of Spodoptera frugiperda and Helicoverpa armigera was performed, setting ddH₂O as the negative control. We found that Bt S3076-1 total protein exhibited insecticidal activity against the larvae of Spodoptera frugiperda and Helicoverpa armigera, with the LC₅₀ of 149.64 µg/g (95% confidence limits: 100.565-166.836 µg/g) and 441.47 µg/g (95% confidence limits: 275.572-676.228 µg/g), respectively (Table 3), indicating that the total protein of Bt S3076-1 showed relatively high insecticidal activity against Spodoptera frugiperda larvae, but relatively weak against Helicoverpa armigera larvae.

Table 3

Bioassays of Bt S3076-1 isolates against the neonates of *Helicoverpa armigera* and *Spodoptera frugiperda*
Target insect	Regression equation	LC50 (ug/g)	95% Confidence interval	Correlation coefficient
Helicoverpa armigera	Y = 2.134X-5.664	441.47	275.572-676.228	0.726
Spodoptera frugiperda	Y = 2.001X-4.353	149.64	100.565-166.836	0.759

3.7 Effect on midgut structure of target larvae

The effect on midgut structure of Spodoptera frugiperda and Helicoverpa armigera larvae treated and untreated Bt S3076-1 total protein, respectively. The just dead larvae and the negative control were fixed immediately and paraffin-embedded so that the morphology could be observed under a microscopy. The pathological observation results showed that the midgut structure of the negative control was intact, presenting the dense, neat and distributed evenly intestinal wall cells. While the peritrophic membrane of Spodoptera frugiperda and Helicoverpa armigera larvae that had died following Bt S3076-1 total protein treatment was degraded. It is proposed that the midgut structure became incomplete, causing an imbalance in cell osmotic pressure, resulting in larval died eventually (Figure. 7).

Bacillus thuringiensis can produce parasporal crystals in many shaped during sporulation phase, including bipyramidal, square, irregular and spherical, which are closely related to its target insect species(Maeda et al. 2000). Moreover, determination of the crystal morphology can identify certain toxin types such as the typical bipyramidal crystals proteins are toxic to Lepidopteran insects(Huang et al. 2006). The scanning electron microscope observation showed that Bt S3076-1 produced mainly spherical and irregular shaped parasporal crystals. Similarly, Bt MC28, which is toxic to the Lepidopteran and Diptera, produces a large number of spherical crystals during sporulation(Guan et al. 2014). The insecticidal proteins exist as protoxins and are converted to active insect toxins by proteases in the alkaline environment of the target insect. When ingested by susceptible insect larvae, the C-terminal inactive region will be proteolytically digested to yield the active toxic form of about 60 kDa to 70 kDa or 25 kDa to 40 kDa(Bravo et al. 2013). The active toxic form of 70 kDa and 40 kDa are commonly found in Cry toxins, such as, Cry1Cb2 can be digested into 65 kDa active protein(Torres-Quintero et al. 2022); the active protein form of Cry52Ca1 is 77.2 kDa(Panwar et al. 2018); while Cry78Ba1 can be enzymatically cleaved to produce a active protein of 40 kDa(Cao et al. 2022). Similarity to the reference, active toxic form of 70 kDa and 40 kDa of Bt S3076-1 strain was produced after trypsin digestion. It was speculated that insecticidal activity of Bt S3076-1 against Spodoptera frugiperda and Helicoverpa armigera might be related to these active proteins.

The toxin types and location on genome in different Bacillus thuringiensis strains is likely different. Currently, among the whole genome data of public Bt strains, it is rare that Bt strain harboring various Cry, Cyt or Vip genes simultaneously. As reported, HBF-18 contained multiple toxin proteins including Cry8-like, Vip1Ad1, and Vip2Ag1(Bi et al. 2015). Here, 10 predicted toxic genes of Bt S3076-1 strain included cry, tpp, cyt and vip genes. Cry toxin is generally comprised of three conserved domains (3D) with Endotoxin_N, Endotoxin_M and delta_endotoxin_C, but there are many phenomena without typical three conserved domains. For instance, Cry11 toxin is only composed of Endotoxin_N(Fatima et al. 2022), while Cry70Aa1 has Endotoxin_N and delta_endotoxin_C. Likewise, Vip toxins also have their unique conserved domain(Fu et al. 2022). In Bt S3076-1 strain, toxins 2041 and 2521 only exist Endotoxin_N and Endotoxin_C conserved domains, but missing Endotoxin_M domain. However, it is found that Endotoxin_M is an important structure for specific recognition of Cry toxins(Boonserm et al. 2006), when missing may affect the larvicidal activity of toxin. Nevertheless, it also was found that the number of toxic protein domains is not directly related to the level of toxicity. For example, Cry11Aa only existed the Endotoxin_N domain, while Cry4Aa contained Endotoxin_N, Endotoxin_M, and Endotoxin_C domains, nonetheless the insecticidal spectrum and toxicity of Cry4Aa were relatively low(Beltrao and Silvafilha 2007)(Revina et al. 2004). Furthermore, here, among the four Cry toxins with 3D domain, toxin 1139 also had a Cry1Ac-D5 domain, which was considered to bind to the ALP and APN sites in the midgut of the target insect, thereby increasing the toxicity(Peña-Cardeña et al. 2018). Interestingly, we also found that the gene encoding Cry1Ac-D5 domain was located on 30-40bp downstream of the ORF of toxins 1035 and 1044, forming ORF1 + Gap + ORF2(Hernández-Soto et al. 2009), which was found in Bacillus thuringiensis H3 and also really expressed in the strain by mass spectrometry identification(Fayad et al. 2020). In this research, we will successively identify these toxins by MS, and determine the insecticidal activity against the target pests.

Different from Cry toxins, Cyt toxins exhibited low toxicity to all mosquito species, whereas they can synergism with other Cry proteins to produce high larvicidal activity(Thiery et al. 1997)(Elleuch et al. 2015). Toxin 1073 contained a tandem domain of RicinB_lectin_2, which was a carbohydrate-binding domain with certain enzyme activity, toxicity inhibition, and signal transduction(Wu et al. 2007). Moreover, it is also possible to lose toxicity by changing the conformation of Cyt toxin(Ben-dov 2014). However, this speculation has not yet been validated. Both Tpp toxins 5295 and 5303 contain a Toxin_10 domain, and many Toxin_10 family members have been reported with toxic activity, which is a pore-forming domain and located on N-terminal domain that may be responsible for binding different receptors(Cao et al. 2020).

In addition, one Vip1A-like toxin was found on Bt S3076-1plasmid2. Nevertheless, Vip1A and Vip2A are components of a binary toxin that is specifically active against coleopteran insects. Generally, vip2A is located upstream of vip1A with four bases space(Warren. 1997). However, in this study, vip2A gene was not found upstream of vip1A gene. Previous investigations revealed that the phenomenon of only vip1A or vip2A gene exist in some Bt strains, whereas no more detailed information was not provided in the reference(Hernández-Rodríguez et al. 2009). Here, whether it is the same situation, it requires more detail investigation on vip1A in the future.in Conclusion, Bt S3076-1 had high insecticidal activity against agricultural pests of Spodoptera frugiperda and Helicoverpa armigera, and showed various toxins based on the whole genome analysis, which would provide experimental reference for further research on insecticidal activity, toxicity spectrum as well as the synergism of these toxins. Thus, Bt S3076-1 strain can be well applied to the control of agricultural and forestry pests.

Bacillus thuringiensis

°C

Degree centigrade

Relative humidity

Potential of hydrogen

rpm

Revolution per minute

Hour/hours

min

Minutes

Gram/grams

µg

Microgram

Millilitres

µl

Microlitres

Molar

Millimolar

Ultraviolet

Kilo base

Base pair

kDa

Kilo Dalton.

Author Contributions:

Tianbao Yang completed the strain culture, and proteome identification and their analysis, conducted the prokaryotic expression of the predicted proteins, performed the larvae bioassays experiments, and wrote and revised the whole manuscript. Zhongqi Wu and Liuping Li complete genome sequenced and made genome-wide analysis of strain Bt S3076-1. Mingguo Jiang and Xuanjun Fang were involved in project design and manuscript modification. Wenshan Huang participated in the bioassay analysis. Yan Zhou is responsible for writing, revising, and finalizing the manuscript. All authors have read and approved the final manuscript.

Competing interests: Authors declare no competing interests.

Acknowledgments:

This work was supported by grants by Guangxi Minzu University Introduction of Talent Research Start-up Fund (2020KJQD23), Guangxi Natural Science Foundationunder (Grant No.2022GXNSFBA035446), The Basic Ability Enhancement Program for Young and Middle-aged Teachers of Guangxi (2022KY0159). All the Intellectual property rights are owned and reserved by Hainan Institute of Tropical Agricultural Resources.

Availability of data and materials :

Not applicable.

Ethics approval and consent to participate:

Not applicable.

Consent for publication:

Not applicable.

Competing interests:

The authors declare that they have no competing interests.

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No competing interests reported.

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Identification and Analysis of Toxins in Novel Bacillus thuringiensis Strain Bt S3076-1 against Spodoptera frugiperda and Helicoverpa armigera

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Abstract

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Key Contribution

1. Background

2. Materials And Methods

3. Results And Analysis

3.1 Observation the parasporal crystal of Bt S3076-1

3.2 ORF and coding gene prediction of Bt S3076-1 whole genome

3.3 Coding gene KAAS functional annotation analysis of Bt S3076-1 genome

3.4 Identification and analysis of toxin sequence

3.5 Total protein of spore maturation and trypsin digestion analysis

3.6 Bioassay of Bt S3076-1

3.7 Effect on midgut structure of target larvae

4. Discussion

Abbreviations

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References

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Journal Publication

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