Devendra Jain

Rapid Identification of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) Using Ribosomal RNA Internal Transcribed Spacer 1

Rapid identification of invasive species is crucial for deploying management strategies to prevent establishment. Recent Helicoverpa armigera (Hübner) invasions and subsequent establishment in South America has increased the risk of this species invading North America. Morphological similarities make differentiation of H. armigera from the native Helicoverpa zea (Boddie) difficult. Characteristics of adult male genitalia and nucleotide sequence differences in mitochondrial DNA are two of the currently available methods to differentiate these two species. However, current methods are likely too slow to be employed as rapid detection methods. In this study, conserved differences in the internal transcribed spacer 1 (ITS1) of the ribosomal RNA genes were used to develop species-specific oligonucleotide primers that amplified ITS1 fragments of 147 and 334 bp from H. armigera and H. zea, respectively. An amplicon (83 bp) from a conserved region of 18S ribosomal RNA subunit served as a positive control. Melting temperature differences in ITS1 amplicons yielded species-specific dissociation curves that could be used in high resolution melt analysis to differentiate the two Helicoverpa species. In addition, a rapid and inexpensive procedure for obtaining amplifiable genomic DNA from a small amount of tissue was identified. Under optimal conditions, the process was able to detect DNA from one H. armigera leg in a pool of 25 legs. The high resolution melt analysis combined with rapid DNA extraction could be used as an inexpensive method to genetically differentiate large numbers of H. armigera and H. zea using readily available reagents.

Viral, Fungal and Bacterial Disease Resistance in Transgenic Plants

Continuing attention is being devoted to the development of substitute strategies in plant-disease management and reducing dependency on synthetic chemicals. Viral, fungal and bacterial diseases are unquestionably the most versatile for environmental adaption and in the destruction of plant growth. Among the strategies, resistance breeding has generated proven data and been exploited in depth. However, conventional methods alone are not sufficient to control the novel races of viral, fungal and bacterial pathogens in crops due to a scarcity in required crop variations. The current situation encourages the search for variation against biotic stress through identification of genes across species. Over the last two decades, significant efforts have been initiated in plant-disease management via genetic engineering. In addition, several molecular techniques have emerged to disentangle multifaceted plant-pathogen systems and associated disease-resistance candidate genes. Besides describing many promising candidate genes from viruses, fungi and bacteria, numerous plant disease-resistance genes have been identified and evaluated in crop improvement programs by transformation. Advancement in plant transformation techniques enables transferring useful genes for the rational creation of disease-resistant plants. Success has been achieved in transgenic crops against various diseases of important crop plants. This chapter describes genetically engineered plants and their resistant to viral, fungal and bacterial pathogens.

Biosynthesised silver nanoparticles using aqueous leaf extract of Tagetes patula L. and evaluation of their antifungal activity against phytopathogenic fungi

In the recent decades, nanotechnology is gaining tremendous impetus due to its capability of modulating metals into their nanosize, which drastically changes the chemical, physical, biological and optical properties of metals. In this study, silver nanoparticles (AgNPs) synthesis using aqueous leaf extracts of Tagetes patula L. which act as reducing agent as well as capping agent is reported. Synthesis of AgNPs was observed at different parameters like temperature, concentration of silver nitrate, leaf extract concentration and time of reduction. The AgNPs were characterized using UV-vis spectroscopy, scanning electron microscope with energy dispersive spectroscopy, transmission electron microscopy with selected area electron diffraction, X-ray diffraction, Fourier transform infrared and dynamic light scattering analysis. These analyses revealed the size of nanoparticles ranging from 15 to 30 nm as well revealed their spherical shape and cubic and hexagonal lattice structure. The lower zeta potential (-14.2mV) and the FTIR spectra indicate that the synthesized AgNPs are remarkably stable for a long period due to the capped biomolecules on the surface of nanoparticles. Furthermore, these AgNPs were found to be highly toxic against phytopathogenic fungi Colletotrichum chlorophyti by both in vitro and in vivo and might be a safer alternative to chemical fungicides.

Molecular characterization and PCR-based screening of cry genes from Bacillus thuringiensis strains

Novel cry genes are potential candidates for resistance management strategies, due to their different structures and modes of action. Therefore, it is desirable to clone and express novel cry genes from several new isolates of Bacillus thuringiensis (Bt). In the present study, 28 Bt strains were characterized at morphological and molecular level. All these strains are Gram positive, endospore forming and had shown different crystal morphologies when viewed under the microscope. The ARDRA (16S rDNA PCR-RFLP technique) with AluI, HaeIII, HinfI and TaqI produced unique and distinguishable restriction patterns used for the molecular characterization of these isolates. Based on UPGMA clustering analysis, Bt strains showed significant molecular diversity and the dendrogram obtained differentiated 28 Bt strains into 1 major cluster at a similarity coefficient 0.56. PCR analysis demonstrated that the Bt strains showed diverse cry gene profiles with several genes per strain. The Bt strain G3C1 showed the presence of maximum cry-type genes by PCR. The toxicological characterization of these cry genes will have huge importance in transgenic technology and will be useful in transgenesis of crop plants for better resistance management.

Current Status of Bacillus thuringiensis : Insecticidal Crystal Proteins and Transgenic Crops

Bacillus thuringiensis (Bt) is used to control agriculturally-important pests. It is a Gram positive spore-forming bacterium which produces parasporal proteinaceous inclusions during the sporulation phase. These crystalline parasporal inclusions are toxic to a wide spectrum of insects including the orders Lepidoptera, Coleopteran, Diptera, etc. The Bt insecticide proteins are toxic only after ingestion by the susceptible insects. The main steps involved when the Cry protein is ingested by the insect is comprised of solubilization of the protoxin, its enzymatic activation by terminal cleavage, receptor binding in brush border membrane of the midgut, pore formation, consequent disruption of ionic potential and destruction of the epithelial membrane leading to cell death. The first discovery of Bt was in 1901 when Ishiwata discovered a bacterium in Japan and in 1915, Berliner in Germany renamed it as Bacillus thuringiensis. Following a brief introduction, this chapter addresses the classification, the general structure of Cry toxin, its mode of action, strategies to improve the insecticidal activity of Cry proteins, transgenic plants developed using Bt genes, resistance to Bt toxins and resistance management, and an overall brief account of Bt and its insecticidal proteins, from 1901 to the present.

Assessment of genetic diversity in black gram [Vigna mungo (L.) Hepper] genotypes based on ISSR

A study was carried out among 22 black gram genotypes to study the genetic diversity using 20 ISSR primers. Screening of the primers revealed that only 15 out of the 20 primers produced amplification. A total of 84 amplified bands were obtained, out of which 72 were polymorphic 85.71 percent polymorphism. The total number of amplified bands varied between 1 (UBC-813 and UBC-878) and 9 (UBC-826) with an average of 5 bands per primer. The overall size of PCR amplified products ranged between 250 bp to 2000 bp. PIC values ranged from 0.00 to 0.51 with an average of 0.285 across all genotypes. Five unique bands were detected in four genotypes, out of which the genotype U-9 gave maximum number of distinct bands. The size of these unique bands ranged from 450 bp to 2000 bp. Based on the UPGMA derived dendrogram and PCA, the 22 genotypes could be divided into four main clusters. While Cluster I included 16 genotypes, the Clusters II, III and IV included two genotypes each. Genotypes IC-16511 and UTTARA, UH-177 and IPU2K-21, STY-2834 and UH-177 were found to be genetically distant from each other with a minimum similarity value of 0.42. The results are encouraging with the suggestion that the ISSR marker could prove to be a versatile tool in further screening of the Vigna germplasm pool for study of genetic divergence and the establishment of phylogenetic relationship amongst accessions.

SSR-Marker assisted evaluation of Genetic Diversity in Mungbean ( Vigna radiata (L.) Wilcezk) genotypes

Mungbean (Vigna radiata (L.) Wilczek) also known as green gram is an important source of protein in the category of food legumes. In the present study, SSR marker is used to analyze the genetic diversity amongst 23 genotypes of mungbean. Out of a total of 10 primers used for SSR analysis revealed generation of 15 alleles. The number of alleles per locus ranged from one (CEDG006, CEDG010, CEDG050, CEDG088, CEDG092 and CEDG232) to three (CEDG 214), with an average of 1.5 allele per primer. The index for expected heterozygosity was 0.29 ranging from 0.15 to 0.49 revealed a deficit in heterozygosity. The size of amplification products varied in case of each primer and the range was found to be 100 bp to 190 bp. 13 out of 15 alleles were found polymorphic. The average PIC value of SSR marker was found to be 0.205. The value of Jaccard’s similarity coefficient had ranged from 0.28-1.00 with an average value of 0.64. The dendrogram constructed on SSR molecular marker data through UPGMA method and PCA using average linkage, had enabled grouping of the genotypes into three main clusters. Clustering pattern based on SSR marker data clearly indicated the narrow genetic base of mungbean genotypes that emphasizes the need to explore and exploit more number of germplasm from additional source to study genetic variation in mungbean for genetic improvement. The results indicated the marked usefulness of SSR in the assessment of genetic diversity in mungbean crop.

Assessment of Genetic Diversity in Mungbean Genotypes using ISSR Markers

Assessment of Genetic Diversity in Mungbean Genotypes using ISSR Markers Molecular assessment of genetic diversity was done for 23 mungbean (Vigna radiata (L.) Wilczek) genotypes using ISSR marker system. Twenty ISSR primers were used in the study out of which thirteen primers enabled DNA amplification in all genotypes. A total of 75 amplified and reproducible amplicons were obtained from 13 primers, out of which 60 were found polymorphic. The total number of amplified bands varied between 2 (UBC-813) and 11 (UBC-810) with an average of 6.5 per primer. The overall size of PCR amplified products ranged between 200 to 2500 bp. PIC values ranged from 0.07 to 0.35 with an average value of 0.208 across all genotypes. Four unique bands were detected in four genotypes with 3 (UBC-818, UBC-820 and UBC-826) out of 13 ISSR primers. The genotype IC-393407 gave maximum number of distinct bands. The similarity indices between the 23 genotypes ranged from 0.38 to 0.94. The extent of diversity among genotypes was also estimated in relation to their source and a set of genotypes with narrow genetic bases developed from various region were identified. Based on a dendrogram generated through UPGMA method and PCA, most of the genotypes could be divided into two main clusters. Cluster I included twenty genotypes, while cluster II included three genotypes. The genotype GM-9925 lay apart from all the two clusters. A minimum similarity co-efficient of 0.38 was observed between genotypes GM-9925 and EC-398885 thereby indicating maximum genetic divergence. The Mantel statistical analysis (r = 0.92) also supported cluster analysis..