Genome profiling of an indigenous Bacillus thuringiensis isolate, T405 toxic against the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae).

In this study, we present the molecular and insecticidal characteristics of an indigenous Bt isolate T405 toxic against the maize fall armyworm (FAW), Spodoptera frugiperda. The presence of cry1, cry2 (cry2Aa & cry2Ab) and vip3A1 genes in T405 was confirmed. The SDS-PAGE gel analysis confirmed the occurrence of Cry and Vip proteins with molecular masses of 130, ∼88 and 65 kDa in T405. LC50 estimates of T405 and HD1 were 161.37 and 910.73 µg ml-1 for neonates whereas, 412.29 and 1014.95 µg ml-1 correspondingly for 2nd instar FAW larvae. Scanning Electron Microscopy depicted the existence of bipyramidal, spherical and cubic crystals in T405 spore suspension. The whole genome sequencing and assembly of T405 produced a total of 563 scaffolds with a genome size of 6,673,691 bp. The BLAST similarity search showed that 12 plasmids were distributed in this genome. Genome annotation revealed the presence of 6174 protein coding genes, 13 rRNA and 98 tRNA, in which 6126 genes were completely annotated for their functions through sequence similarity search, domains/motifs identification and gene ontology studies. Further analysis of these genes identified the presence of many insecticidal toxin protein coding genes viz., cry1Ac32, cry1Ab9, cry1Aa6, cry1Ac5, cry1Aa18, cry1Ab8, cry1Ab11, cry2Aa9, cry1Ia40, cry2Aa9, cry1Ia40, cry2Ab35, cyt, vip3Aa7 and tpp80Aa and several additional virulence assisted factors viz., immune inhibitor A, phospholipase C, sphingomyelinase, cell wall hydrolases, chitinase, hemolysin XhlA and seven urease subunit coding genes (ureA, ureB, ureC, ureD, ureE, ureF, ureG) in the annotated genome.

Comparative Analysis of Chitin SynthaseA dsRNA Mediated RNA Interference for Management of Crop Pests of Different Families of Lepidoptera.

RNA interference (RNAi) is a sequence-specific down-regulation in the expression of a particular gene, induced by double-stranded RNA (dsRNA). Feeding of dsRNA either directly or through transgenic plants expressing dsRNA of insect genes has been proven successful against lepidopteran and coleopteran pests, establishing an additional alternative to control insect pests. Lepidopteran crop pests including Spodoptera litura (Fabricius) (Noctuidae), Chilo partellus (Swinhoe) (Crambidae), Plutella xylostella (Linnaeus) (Plutellidae), and Maruca vitrata (Fabricius) (Pyralidae) are the devastating pests of a variety of crops. To tap the potential of RNAi against insect pests, a gene coding for the key enzyme in chitin biosynthesis in arthropods, the chitin synthaseA (CHSA), has been targeted through an exogenous delivery of dsRNA and plant-mediated RNAi. The introduction of dsCHSA caused "Half ecdysis" and "Black body" type lethal phenotypes and a significant reduction in larval body weight. Subsequent RT-qPCR analysis demonstrated the down-regulation of CHSA gene transcripts from 1.38- to 8.33-fold in the four target species. Meanwhile, when S. litura larvae fed with leaves of transgenic tobacco plants expressing dsSlCHSA, the mRNA abundance of CHSA gene was significantly decreased resulting in lethal phenotypes like "Double head formation," "Half ecdysis," and "Black body." In addition, abnormalities in pupal-adult and adult stage were also documented, strongly suggesting the RNAi effect of CHSA gene at late developmental stages. Overall, the results demonstrated that CHSA gene expression in Lepidopteran crop pests could be suppressed by application of dsRNA either as feeding or through transgenic crop plants.

Analysis of variation in virulence of Beauveria bassiana against insect pests of pigeonpea using qPCR.

Beauveria bassiana is a broad spectrum microbial bioagent used for the control of agriculturally important insect pests. However, in our experiments, two virulent isolates of B. bassiana (B2 and B10) showed specific preference toward Maruca vitrata and Helicoverpa armigera of pigeonpea. To better understand this feature, we developed a qPCR assay to quantify the chitinase (virulence factor) of B. bassiana during the infection process. Isolates of B. bassiana were grown on insect cuticle amended medium and minimal medium (without insect cuticle) to assess the induction of chitinase. Our results revealed a positive correlation between expression of chitinase by B. bassiana and the substrates (with or without cuticles of M. vitrata and H. armigera) used. This study showcases the methodology to quantify the chitinase and analysis of variation in virulence of B. bassiana (B2 and B10) against M. vitrata and H. armigera.

Application of comparative genomics in developing molecular markers tightly linked to the virus resistance gene Rsv4 in soybean.

The Rsv4 gene confers resistance to all the known strain groups of soybean mosaic virus in soybean (Glycine max (L.) Merr.). To construct a fine genetic map near Rsv4 in soybean, we employed a comparative genomics approach that used genome sequence information of the model legume Lotus japonicus. Sequences of the soybean expressed sequence tags (ESTs) AI856415 and BF070293 mapping to one side of the Rsv4 gene showed high similarity with gene sequences of the transformation-competent artificial chromosome (TAC) clone LjT32P24 of Lotus. LjT32P24 is tightly linked to another sequenced TAC clone, LjT26I01, in Lotus. A new marker, AW307114A, developed from soybean EST AW307114, which is homologous to a Lotus gene within LjT26I01, was mapped to the other side of the Rsv4 gene. The identification of the microsyntenic relationship facilitated the development of additional 2 EST markers between BF070293-S and AW307114A bracketing the Rsv4 gene. Several other markers developed in this study were mapped to putative homoeologous or duplicated chromosomal regions in soybean. Alignment between the soybean maps indicated that Rsv4 is located near a local chromosomal rearrangement. This targeted comparative mapping serves to provide a foundation for marker-assisted selection and cloning of the Rsv4 gene.