A novel Bacillus thuringiensis isolate toxic to cotton pink bollworm (Pectinophora gossypiella Saunders).

In this study, we report a novel indigenous Bacillus thuringiensis (Bt) isolate, T26, which showed spores and crystals under scanning electron microscope and pathogenicity against the pink bollworm (Pectinophora gossypiella Saunders) in artificial diet based bioassay. SDS-PAGE analysis of the spore-crystal mixture of the Bt isolate, T26 revealed presence of three major protein bands of approximate molecular weights of 80, 55 and 40 kDa. The draft genome assembly consists of 56 scaffolds with an entire draft genome size of 5,054,095 bp. NCBI blast analysis revealed that assembled draft genome is spread over in a chromosome (4,818,543 bp) and one plasmid (235,552 bp). NCBI Prokaryotic Genome Annotation Pipeline (PGAP) showed presence of 5033 coding gene sequences and 159 RNAs genes. None of the known lepidopteran active genes (cry1, cry2 and cry9) could be detected with PCR or with whole genome sequence analysis using Bt toxin scanner tool or CryProcessor tool. Thus, presence of protein crystals and toxicity towards cotton pink bollworm and absence of any known cry/vip/cyt type of genes in draft genome indicates it is a novel type of Bt isolate. Further investigation of this genome sequence along with protein sequencing will lead to understand the novel factors responsible for its virulence and could be a useful tool for the insect resistance management in pink bollworm.

Genome sequencing of Bacillus thuringiensis isolate T414 toxic to pink bollworm (Pectinophora gossypiella Saunders) and its insecticidal genes.

Bacillus thuringiensis is a spore-forming bacterium that is pathogenic towards a range of insect and nematode species and had been widely used as a biopesticide. In this study, we present the morphological, molecular and genetic characteristics of an indigenous Bt isolate T414 which displayed an effective toxicity against Pectinophora gossypiella. Scanning electron microscopy revealed the presence of bipyramidal, spherical and cubic shaped protein crystals in its spore-crystal suspension. SDS-PAGE analysis of its spore-crystal mixture showed the presence of two major protein bands viz.130 and 65 kDa. Whole genome sequencing with MiSeq divulged that it contains a chromosome and many plasmids. The assembled genome finally contained 6493494bp. Automated annotation of this genome draft predicted 6877 coding sequences and 152 RNAs (rRNAs, tRNAs and ncRNAs). NCBI blast analysis showed that assembled genome was distributed in a chromosome and 15 different types of plasmids. Further analysis of draft sequence revealed it harbors parasporal crystal protein genes (cry1Aa, cry1Ab, cry1Ac, cry1IAa, cry2Aa, cry2Ab and cyt1), vegetative insecticidal protein gene (vip3Aa), all plasmid borne and various additional virulence factors such as chitinases, proteases, bacteriocins and hemolysins. From the analysis it is evident that all the Cry, Cyt or Vip toxins are plasmid borne and are present on two types of plasmids named as p414A and p414E in the present study. A cry2A type gene was cloned and sequenced. It was named as cry2Aa21 by Bt nomenclature committee.

A new observation on feeding behaviour of pink bollworm and its application in screening Bt-resistant population.

The pink bollworm (PBW), Pectinophora gossypiella (Saund.) does not feed on leaves in natural circumstances. We made an attempt with the first instar larva (5 days old) and found it eats the leaf in the absence of other foods. Utilizing this new feeding behaviour, a simple methodology was developed for screening of resistance in PBW against Bt cotton plants. The PBW collected from BGII Bt cotton fields (BGII-resistant population) and NBAIR culture (susceptible population) were reared under laboratory conditions for two generations. Laboratory reared 5-day old larvae of PBW were released on cotton leaf discs individually for screening. The BGII-resistant and susceptible larvae fed an average leaf area of 75.52 ± 16.68 and 5.95 ± 0.93 mm2 with survival rate of 90 and 4% respectively, in BGII Bt cotton leaves. Whereas in case of non-Bt cotton (MCU13), the BGII-resistant and susceptible larvae consumed average leaf area of 114.84 ± 23.70 and 116.80 ± 24.14 mm2 with survival rate of 93.34 and 95.33%, respectively. In addition to the screening process, the larval survivors were transferred to an artificial diet after 7 days of experiment and observed up to their emergence as adults. To confirm the development of resistance in PBW, sequencing of larval DNA amplicons was carried out and it revealed mutation in the cadherin gene of the BGII-resistant PBW population. Hence, the detached leaf bit feeding assay described here could be used in a simple manner for screening of resistance developed by PBW against Bt cotton and also for evaluating Bt cotton plants for their inherent resistance to PBW. This method could also be used for studying toxicity of Bt isolates by coating spore-crystal mixture on non-transgenic cotton leaf discs.

Transgenic rice expressing the cry2AX1 gene confers resistance to multiple lepidopteran pests.

A chimeric Bacillus thuringiensis toxin (Bt) gene, cry2AX1was cloned in a bi-selectable marker free binary vector construct. The cry2AX1 gene, driven by the Chrysanthemum rbcS1 promoter, was introduced into JK1044R, the restorer line (Oryza sativa L. ssp. Indica) of a notified commercially grown rice hybrid in India, by Agrobacterium-mediated transformation. Its effect against two major lepidopteran insect pests viz., yellow stem borer (YSB) Scirpophaga incertulas, rice leaf folder (RLF) Cnaphalocrocis medinalis and one minor insect pest, oriental army worm (OAW) Mythimna separata was demonstrated through bioassays of transgenic rice plants under laboratory and greenhouse conditions. The rbcS1 promoter with chloroplast signal peptide was used to avoid Cry2AX1 protein expression in rice seed endosperm tissue. A total of 37 independent transformants were generated, of which after preliminary molecular characterization and YSB bioassay screening, five events were selected for their protein expression and bioefficacy against all three rice insect. One elite transgenic rice line, BtE15, was identified with Cry2AX1 expression ranging from 0.68 to 1.34 µg g(-1) leaf fresh weight and with 80-92 % levels of resistance against rice pests at the vegetative and reproductive stages. Increase in Cry2AX1 protein concentration was also observed with crop maturity. The Cry2AX1protein concentration in the de-husked seeds was negligible (as low as 2.7-3.6 ng g(-1)). These results indicate the potential application of cry2AX1 gene in rice for protection against YSB, RLF and OAW.

Development of leaffolder resistant transgenic rice expressing cry2AX1 gene driven by green tissue-specific rbcS promoter.

The insecticidal cry genes of Bacillus thuringiensis (Bt) have been successfully used for development of insect resistant transgenic rice plants. In this study, a novel cry2AX1 gene consisting a sequence of cry2Aa and cry2Ac gene driven by rice rbcS promoter was introduced into a rice cultivar, ASD16. Among 27 putative rice transformants, 20 plants were found to be positive for cry2AX1 gene. The expression of Cry2AX1 protein in transgenic rice plants ranged from 5.95 to 122.40 ng/g of fresh leaf tissue. Stable integration of the transgene was confirmed in putative transformants of rice by Southern blot hybridization analysis. Insect bioassay on T0 transgenic rice plants against rice leaffolder (Cnaphalocrosis medinalis) recorded larval mortality up to 83.33%. Stable inheritance and expression of cry2AX1 gene in T1 progenies was demonstrated using Southern and ELISA. The detached leaf bit bioassay with selected T1 plants showed 83.33-90.00% mortality against C. medinalis. The whole plant bioassay for T1 plants with rice leaffolder showed significant level of resistance even at a lower level of Cry2AX1 expression varying from 131 to 158 ng/g fresh leaf tissue during tillering stage.

Transgenic rice plants expressing synthetic cry2AX1 gene exhibits resistance to rice leaffolder (Cnaphalocrosis medinalis).

Bacillus thuringiensis is a major source of insecticidal genes imparting insect resistance in transgenic plants. Level of expression of transgenes in transgenic plants is important to achieve desirable level of resistance against target insects. In order to achieve desirable level of expression, rice chloroplast transit peptide sequence was fused with synthetic cry2AX1 gene to target its protein in chloroplasts. Sixteen PCR positive lines of rice were generated by Agrobacterium mediated transformation using immature embryos. Southern blot hybridization analysis of T0 transgenic plants confirmed the integration of cry2AX1 gene in two to five locations of rice genome and ELISA demonstrated its expression. Concentration of Cry2AX1 in transgenic rice events ranged 5.0-120 ng/g of fresh leaf tissue. Insect bioassay of T0 transgenic rice plants against neonate larvae of rice leaffolder showed larval mortality ranging between 20 and 80 % in comparison to control plant. Stable inheritance and expression of cry2AX1 gene was demonstrated in T1 progenies through Southern and ELISA. In T1 progenies, the highest concentration of Cry2AX1 and mortality of rice leaffolder larvae were recorded as 150 ng/g of fresh leaf tissue and 80 %, respectively. The Cry2AX1 expression even at a very low concentration (120-150 ng/g) in transgenic rice plants was found effective against rice leaffolder larvae.

[Characterization and cloning of the cry2A gene from indigenous isolates of Bacillus thuringiensis].

Discovery of novel cry genes of Bacillus thuringiensis (Bt) with higher toxicity is important for the development of transgenic Bt crops resistant to target pests. Two new indigenous isolates of Bt were characterized for their colony type, crystal inclusion and toxicity with Helicoverpa armigera Hubner and Spodoptera litura Linn. Screening of cry2A genes from the two new isolates of Bt, T30 and T48, by PCR with cry2A family primers showed the presence of cry2A genes only in the isolate T30. Further screening of these isolates of Bt with three different cry2A gene specific primers showed the presence of cry2Aa, cry2Ab and cry2Ac genes in both of the new Bt isolates. The cry2A open reading frame (ORF) of Bt T48 (~1.9 kb) was amplified and cloned in a T/A vector (~2.8 kb). All the E. coli transformants showed only the cry2Aa gene. Comparison of the nucleotide sequence data generated from the cry2Aa (~1.9 kb) gene showed a 99 percent homology and one amino acid variation when compared with the holotype sequence of Cry2Aa1.

Characterization of parasporin gene harboring Indian isolates of Bacillus thuringiensis.

Bacillus thuringiensis (Bt) is popularly known as insecticidal bacterium. However, non-insecticidal Bt strains are more extensively available in natural environment than the insecticidal ones. Parasporin (PS) is a collection of genealogically heterogeneous Cry proteins synthesized in non-insecticidal isolates of Bt. An important character generally related with PS proteins is their strong cytocidal activity preferentially on human cancer cells of various origins. Identification and characterization of novel parasporin protein which are non-hemolytic and non-insecticidal but having selective anticancer activity raise the possibility of a novel application of Bt in medical field. In the present study, seven new indigenous isolates (T6, T37, T68, T98, T165, T186, and T461) of Bt showed variation in colony morphology, crystal characters and protein profiles with each other. Out of the seven new isolates screened for parasporin (ps) and cry genes, two of the new indigenous isolates (T98 and T186) of Bt showed the presence of ps4 gene. Partial ps4 gene was cloned from the two new isolates and the sequence of partial ps4 gene showed high homology with its holotype ps4Aa1. These two isolates were characterized based on the proteolytic processing of the inclusion proteins and the proteolytic products were found to be comparable to the PS4 reference strain A1470. The two isolates of Bt did not show toxicity toward Spodoptera litura and Helicoverpa armigera. Based on the results of this study, it can be concluded that the isolates T98 and T186 are parasporin producers.

Cloning and characterization of truncated cry1Ab gene from a new indigenous isolate of Bacillus thuringiensis.

The insecticidal crystal protein(s) encoded by cry gene(s) of Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. A new 3'-truncated cry1Ab gene was cloned from an indigenous isolate of Bt, A19-31. Nucleotide sequencing and homology search revealed that the deduced amino acid sequence of Cry1Ab toxin of Bt strain A19-31 had a variation of two amino acid residues with the holotype sequence, Cry1Ab1. Expression of the 3'-truncated cry1Ab gene was studied in an acrystalliferous strain of Bt (4Q7). SDS-PAGE and immunostrip analysis of spore-crystal mixture revealed a low level expression of the 3'-truncated cry1Ab gene. Insecticidal activity assay showed that the recombinant 3'-truncated cry1Ab gene product was toxic to larvae of both Helicoverpa armigera and Spodoptera litura.

Diversity of Bacillus thuringiensis isolated from Western Ghats of Tamil Nadu State, India.

The Western Ghats of India is the one of the world's 10 "Hottest biodiversity hotspots" that runs along the western part of India through four states including Tamil Nadu. The only biodiversity reserve in the Western Ghats is the Nilgiri biosphere located in the Tamil Nadu state. In the present study, 525 soil samples were collected from all the 14 different divisions of the Western Ghats in Tamil Nadu state, India. A total of 316 new isolates of Bacillus thuringiensis (Bt) that produce parasporal crystalline inclusions were isolated from 525 soil samples. Seven different types of crystalline inclusions were observed in the 316 new isolates of Bt. Cuboidal inclusion was predominantly present in 26.9% of the Bt isolates when compared to other shapes. Further characterization of 70 of the 316 Bt isolates for crystal protein profile through SDS-PAGE revealed six different types of crystal protein profile viz., 135 and 65, 135, 95, 65, 43, and 30 kDa crystal proteins. Variation in the mass of crystal protein(s) purified from the isolates of Bt revealed molecular diversity of this bacterium prevalent in the Western Ghats of Tamil Nadu, India.

Cloning, characterization, and expression of a new cry2Ab gene from Bacillus thuringiensis strain 14-1.

Bacillus thuringiensis is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of B. thuringiensis are promising candidates for management of resistance development in insects owing to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. The cry2Ab gene was found to lack a functional promoter and, hence, is cryptic in nature. The cry2Ab7 gene was cloned from a new indigenous B. thuringiensis strain, 14-1. Nucleotide sequencing of the cry2Ab gene cloned from B. thuringiensis strain 14-1 revealed an open reading frame of 1902 bp. The deduced amino acid sequence of Cry2Ab of B. thuringiensis strain 14-1 showed a variation in three amino acid residues in comparison to the holotype sequence, Cry2Ab1. Expression of the newly cloned cry2Ab gene was studied in an acrystalliferous strain of B. thuringiensis (4Q7) by fusing the cry2Ab gene downstream of cry2Aa promoter and orf1 + orf2 sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a spore-crystal mixture obtained from transformants of B. thuringiensis strain 4Q7 showed production of Cry2Ab protein of about 65 kDa. Alkali solubilized Cry2Ab7 protein showed toxicity against Helicoverpa armigera neonates.

Transfer of an insecticidal protein gene ofBacillus thuringiensis into plant-colonizingAzospirillum.

A fusion plasmid, pRKC, was constructed, using pACYC184, RSF1010 and a kanamycin-resistance cartridge from pUC4K, to convey thecryIA(a) gene intoAzospirillum spp. With the pRKC plasmid, the number of putative transconjugants obtained inA. lipoferum was about 300-fold higher than inA. brasilense. Conjugation frequency and plasmid stability inA. lipoferum were less for pBTF8, which carries thecryIA(a) gene in the correct orientation for a constitutive promoter, than for pBTF9, which carries the gene in the opposite orientation. Expression of thecryIA(a) gene was not apparent in SDS-PAGE analysis ofA. lipoferum transconjugants harbouring pBTF8. However,Escherichia coli transformants with the pBTF8 rescued fromA. lipoferum transconjugants produced an approximately 135 kDa Cry protein, indicating that thecry gene is intact in the transconjugants.

Cloning of a new cryIA(a) gene from Bacillus thuringiensis strain FU-2-7 and analysis of chimaeric CryIA(a) proteins for toxicity.

We cloned the cryIA(a) gene from Bacillus thuringiensis strain FU-2-7, one of the toxin genes encoding lepidopteran-specific protoxins. Sequence analysis of the gene showed two amino acid differences (Pro77 to Leu and Phe965 to Ser) from the CryIA(a) of B. thuringiensis strain HD-1. We constructed chimaeric cryIA(a) genes using FU-2-7 and HD-1 cryIA(a) genes and isolated the chimaeric protoxins, as well as the parental ones, from Escherichia coli cells harboring the recombinant plasmids to examine the effects of the two amino acid variations on the toxicity. FU-2-7 CryIA(a) protein was about half as toxic against the smaller tea tortrix, Adoxophyes sp., and one-third as toxic against the silkworm, Bombyx mori, as that of HD-1. On the other hand, a chimaeric CryIA(a) protein with a single replacement of Phe965 to Ser had nearly the same toxicity as the HD-1 CryIA(a) against the smaller tea tortrix and one-third the toxicity against silkworm as that of HD-1. This improved property of the chimaeric CryIA(a) protoxin may be useful for widening its application to crop protection in sericultural countries.