Anirban Roy

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.

Grouping and comparison of Indian citrus tristeza virus isolates based on coat protein gene sequences and restriction analysis patterns

Summary. Citrus tristeza virus (CTV) is an aphid-transmitted closterovirus, which causes one of the most important citrus diseases worldwide. Isolates of CTV differ widely in their biological properties. CTV-infected samples were collected from four locations in India: Bangalore (CTV-B), Delhi (CTV-D), Nagpur (CTV-N), and Pune (CTV-P), and were maintained by grafting into Kagzi lime (Citrus aurantifolia (Christm. Swing.). All isolates produced typical vein clearing and flecking symptoms 6–8 weeks after grafting. In addition, CTV-B and CTV-P isolates produced stem-pitting symptoms after 8–10 months. The CTV coat protein gene (CPG) was amplified by RT-PCR using CPG specific primers, yielding an amplicon of 672 bp for all the isolates. Sequence analysis of the CPG amplicon of all the four Indian isolates showed 93–94% nucleotide sequence homology to the Californian CTV severe stem pitting isolate SY568 and 92–93% homology to the Japanese seedling yellows isolate NUagA and Israeli VT p346 isolates. In phylogenetic tree analysis, Indian CTV isolates appeared far different from other isolates as they formed a separate branch. Comparison among the Indian isolates was carried out by restriction analysis and restriction fragment length polymorphism (RFLP). Specific primers to various genome segments of well-characterized CTV isolates were used to further classify the Indian CTV isolates.

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.

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.

Molecular Cloning, Sequencing and Phylogenetic Analysis of Coat Protein Gene of a Biologically Distinct Citrus Tristeza Virus Isolate Occurring in Central India

Citrus tristeza virus (CTV) culture, collected from a fifteen-year-old wilted and declined mosambi sweet orange [Citrus sinensis (L) Osb] plant, was maintained under green house into acid lime [C. aurantifolia Swing] and mosambi seedlings. It gave positive reaction in ELISA, both with CTV specific polyclonal antibodies (G604) and monoclonal antibody MCA13, which specifically detects severe CTV strains. Coat protein gene (CPG) of the virus was amplified by RT PCR using CPG specific primers yielding an amplicon of 672 bp. Sequence analysis of the CPG amplicon showed 97% nucleotide sequence homology with Chinese isolate CTV-0002 (Acc. No. AJ518841) and isolate S4 (Acc. No. EF063109). In phylogenetic analysis, the present CTV isolate was displayed in different clade than other reported Indian CTV isolates, but it shared a separate clade with isolates from China, Israel, Jordan and New Zealand.

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.