Subrata Kumar Ghosh

An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants.

A relatively quick and inexpensive modified cetyl trimethylammonium bromide method for extraction of DNA from leaf materials containing large quantities of mucilage is described. The modification including use of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 M NaCl, reduced markedly the viscosity of the mucilage and thus in the final purification step yielded a larger quantity of mucilage-free DNA suitable for subsequent PCR-based detection of begomoviruses. The method was standardized with jute samples with yellow mosaic disease and validated with different other mucilaginous-hosts with low titre of begomoviruses. DNA isolated using this method showed consistency in yield and compatibility with PCR for detection of begomoviruses from different mucilaginous plant species. The method was compared for efficacy with other reported methods and it was found to be superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus the method described could be used on a wider scale for reliable and consistent detection of begomoviruses from mucilaginous hosts for characterization and variability study.

Sequence variability and phylogenetic relationship of betasatellite isolates associated with yellow vein mosaic disease of mesta in India.

Six betasatellite isolates associated with the yellow vein mosaic disease in mesta crops grown under three different geographical locations of India have been characterized. These six isolates and the one previously reported from eastern India could be divided into two distinct Types. The first Type, consisted of four betasatellite isolates characterized from northern and southern regions of India, was observed to be the newer isolates of Ludwigia leaf distortion betasatellite. The second Type, comprised three betasatellite isolates obtained from the eastern part of India, showed highest sequence identity with Cotton leaf curl Multan betasatellite and appeared to be the newer isolates of it. These isolates present within each of these two betasatellite species showed limited variability with respect to their individual group. The results thus indicated the association of two different betasatellite species with yellow vein mosaic disease of mesta in India and highlighted the possible adaptation of mesta crops as a newer hosts by these two betasatellite species.

Distribution, epidemiology and molecular variability of the begomovirus complexes associated with yellow vein mosaic disease of mesta in India.

Yellow vein mosaic disease of mesta (Hibiscus spp.) poses a serious threat to the cultivation of this crop in India. The disease was found to be associated with two different whitefly-transmitted monopartite begomoviruses, Mesta yellow vein mosaic virus and Mesta yellow vein mosaic Bahraich virus, together with two betasatellite species, Cotton leaf curl Multan betasatellite and Ludwigia leaf distortion betasatellite. These begomovirus complexes were detected in different combinations throughout the mesta growing regions of India. All the eight cultivars tested were highly susceptible to the disease. The effect of the disease in terms of loss in fibre yield was greatest (around 70%) in plants that were inoculated at an early stage of growth. A regression approach was adopted to consider the relationship of whitefly vector populations with weather conditions and disease spread which explained that different conducive weather factors facilitated the build up of whitefly populations and contributed to the spread of the disease.

Alterations in biochemical components in mesta plants infected with yellow vein mosaic disease

Yellow vein mosaic disease of mesta (kenaf, Hibiscus cannabinus L.; and roselle, H. sabdariffa L.) is a new entrant to the disease scenario and it is associated with a novel monopartite Begomovirus. Changes in different biochemical parameters in diseased mesta plants were observed as compared to healthy ones. Isozyme pattern and assays of different enzymes, namely catalase, acid phosphatase, peroxidase, esterase, polyphenol oxidase and superoxide dismutase, revealed lower activities of catalase, acid phosphatase and peroxidase enzymes and enhanced activities of esterase, polyphenol oxidase and superoxide dismutase in diseased plants as compared to healthy ones. Due to the infection, chlorophyll content, phenolics and total soluble protein decreased whereas free amino acid, proline and disease-related proteins increased in the host plants. Differential responses of polyacetylene and isoflavone content as well as SDS-PAGE band profiling of total soluble proteins were also observed in plants due to the infection.

Complete nucleotide sequence of a monopartite begomovirus associated with yellow vein mosaic disease of mesta from north India

AbstractYellow vein mosaic disease of mesta in northern India was found to be associated with a distinct begomovirus species. Except the AC1 gene, this begomovirus isolate shares low sequence identity with the Mesta yellow vein mosaic virus reported to be associated with a similar disease of mesta from eastern India.

Detection of Corchorus golden mosaic virus Associated with Yellow Mosaic Disease of Jute (Corchorus capsularis)

Yellow mosaic disease, caused by a whitefly transmitted New World Begomovirus, named Corchorus golden mosaic virus (CoGMV), is emerging as a serious biotic constraint for jute fibre production in Asia. For rapid and sensitive diagnosis of the Begomovirus associated with this disease, a non-radiolabelled diagnostic probe, developed against the DNA A component of the east Indian isolate of CoGMV, detected the presence of the virus in infected plants and viruliferous whiteflies following Southern hybridization and nucleic acid spot hybridization tests. Presence of the virus was also confirmed when polymerase chain reaction amplification was performed using virus-specific primers on DNA templates isolated from infected plants and viruliferous whiteflies.

SSR and RAPID Profile Based Grouping of Selected Jute Germplasm with Respect to Fibre Fineness Trait

With an aim to develop mapping population on fibre fineness trait, grouping of 16 selected jute accessions, eight each from Corchorus olitorius and Corchorus capsularis which showed promising agronomic characteristics, was carried out using fibre fineness data and DNA fingerprinting using SSR and RAPID primers. Based on fibre fineness trait two subgroups depicting the fine and coarse fibre yielding accessions were obtained in each species. A total of 26 RAPID primers and 22 pairs of SSR primers yielded 277 and 41 scorable bands, respectively. High level of polymorphism was detected between fine and coarse fibre yielding jute accessions. Dendrogram showed that all the accessions formed two main clusters between C. olitorius and C. capsularis and each main cluster subdivided in two clusters containing fine and coarse fibre jute accessions. RAPID and SSR marker data-sets showed high levels of positive correlation (Mantel test, r = 0.97). Grouping of jute accessions based on morphological and molecular data was highly correlated. This study will be useful in future jute breeding programs.

Production of nitric oxide in host-virus interaction: A case study with a compatible begomovirus-kenaf host-pathosystem

Nitric oxide (NO) plays a key role in plant diseases resistance. Here we have first time demonstrated that begomovirus infection in susceptible H. cannabinus plants, results in elevated NO and reactive nitrogen species production during early infection stage not only in infected leaf but also in root and shoot. Production of NO was further confirmed by oxyhemoglobin assay. Furthermore, we used Phenyl alanine ammonia lyase as marker of pathogenesis related enzyme. In addition evidence for protein tyrosine nitration during the early stage of viral infection clearly showed the involvement of nitrosative stress.

Development of linkage map in F2 population of selected parents with respect to Macrophomina phaseolina resistance trait using screened polymorphic RAPD and developed SCAR markers of jute

Two molecular markers (RAPD and simple sequence repeat (SSR)) were applied on 12 Corchorus capsularis jute samples. Two of them were Macrophomina phaseolina-resistant and the remaining eight were M. phaseolina-susceptible accessions. Eleven SSR primer combinations out of 18 gave the polymorphic results between M. phaseolina-resistant and -susceptible accessions. Five pairs of sequence characterised amplified region (SCAR) primers designated as SCP-4, SCS-3, SCS-13, SCG-10 and SCU-10 were designed based on the polymorphic loci obtained between JRC-412 and CIM-036. Only SCU-10 and SCS-13 produced polymorphic markers corresponding to OPU-10 and OPS-13 amplified from ‘CIM-036’ and JRC-412, respectively. RAPD and SCAR markers were employed for construction of a linkage map using a set of 67 F2 population of a cross between JRC-412 and CIM-036 as a mapping population. Nine markers were assigned to two linkage groups (LGs) covering a total length of 628.4 cM with an average distance of 28 cM between markers.

Analysis of coat protein gene sequences of begomoviruses associated with different weed species in India

Sida cordifolia L., Croton bonplandianum L., Malachra capitata L., Eclipta prostrata L., Clerodendron inerme L., Acalypha indica L., and Urena lobata L. are common weeds found all over India. They are often infected by different begomovirus complexes and may act as reservoirs of crop-infecting begomoviruses. Cloning and sequencing were done to partially characterize the begomovirus complexes associated with these weed species from the eastern part of India and their genetic pattern was compared with respective geographical isolates throughout India. Coat protein (CP) genes were found to be amplified from all the infected samples tested in this study, whereas betasatellite molecules amplified only from four infected samples (Sida, Croton, Malachra and Urena). Sequence analysis using CP genes and betasatellites of the present begomovirus complexes showed significant variation among their geographical isolates and also exhibited closeness to different crop-infecting begomovirus complexes. The majority of the weed-infecting begomovirus complexes characterized in this paper are reported for the first time from India.