Raju 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.

New sources of resistance to Fusarium wilt and sterility mosaic disease in a mini-core collection of pigeonpea germplasm

Fusarium wilt (FW) and Sterility mosaic disease (SMD) are important biotic constraints to pigeonpea production worldwide. Host plant resistance is the most durable and economical way to manage these diseases. A pigeonpea mini-core collection consisting of 146 germplasm accessions developed from a core collection of 1290 accessions from 53 countries was evaluated to identify sources of resistance to FW and SMD under artificial field epiphytotic conditions during 2007–08 and 2008–09 crop seasons. Resistant sources identified in the field were confirmed in the greenhouse using a root dip screening technique for FW and a leaf stapling technique for SMD. Six accessions (originated from India and Italy were found resistant to FW (<10% mean disease incidence). High level of resistance to SMD was found in 24 accessions (mean incidence <10%). These SMD resistant accessions originated from India, Italy, Kenya, Nepal, Nigeria, Philippines and United Kingdom. Combined resistance to FW and SMD was found in five accessions (ICPs 6739, 8860, 11015, 13304 and 14819). These diverse accessions that are resistant to FW or SMD will be useful to the pigeonpea resistance breeding program.

Comparative genomics and prediction of conditionally dispensable sequences in legume–infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors

BackgroundSoil-borne fungi of the Fusarium oxysporum species complex cause devastating wilt disease on many crops including legumes that supply human dietary protein needs across many parts of the globe. We present and compare draft genome assemblies for three legume-infecting formae speciales (ff. spp.): F. oxysporum f. sp. ciceris (Foc-38-1) and f. sp. pisi (Fop-37622), significant pathogens of chickpea and pea respectively, the world’s second and third most important grain legumes, and lastly f. sp. medicaginis (Fom-5190a) for which we developed a model legume pathosystem utilising Medicago truncatula.ResultsFocusing on the identification of pathogenicity gene content, we leveraged the reference genomes of Fusarium pathogens F. oxysporum f. sp. lycopersici (tomato-infecting) and F. solani (pea-infecting) and their well-characterised core and dispensable chromosomes to predict genomic organisation in the newly sequenced legume-infecting isolates. Dispensable chromosomes are not essential for growth and in Fusarium species are known to be enriched in host-specificity and pathogenicity-associated genes. Comparative genomics of the publicly available Fusarium species revealed differential patterns of sequence conservation across F. oxysporum formae speciales, with legume-pathogenic formae speciales not exhibiting greater sequence conservation between them relative to non-legume-infecting formae speciales, possibly indicating the lack of a common ancestral source for legume pathogenicity. Combining predicted dispensable gene content with in planta expression in the model legume-infecting isolate, we identified small conserved regions and candidate effectors, four of which shared greatest similarity to proteins from another legume-infecting ff. spp.ConclusionsWe demonstrate that distinction of core and potential dispensable genomic regions of novel F. oxysporum genomes is an effective tool to facilitate effector discovery and the identification of gene content possibly linked to host specificity. While the legume-infecting isolates didn’t share large genomic regions of pathogenicity-related content, smaller regions and candidate effector proteins were highly conserved, suggesting that they may play specific roles in inducing disease on legume hosts.

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.

Dry root rot (Rhizoctonia bataticola (Taub.) Butler): an emerging disease of chickpea – where do we stand?

Dry root rot caused by Rhizoctonia bataticola (Macrophomina phaseolina) of chickpea (Cicer arietinum L.) is gaining importance in the changed scenario of climate when growing crop is predisposed to high temperature and moisture stress. Being mainly a soil-inhabiting pathogen, many environmental and soil factors are responsible for the development of disease. No systematic research related to the biology, ecology and epidemiology of dry root rot in chickpea has been conducted so far. Research is needed to improve the identification and characterisation of variability within its epidemiological and pathological niches. Limited literature available on host plant resistance for dry root rot indicated lack of resistant sources for this disease. The present article discusses current status of the disease in the context of climate change and possible management options to alleviate the problem.

Development of DArT markers and assessment of diversity in Fusarium oxysporum f. sp. ciceris, wilt pathogen of chickpea (Cicer arietinum L.)

BackgroundFusarium oxysporum f. sp. ciceris (Foc), the causal agent of Fusarium wilt of chickpea is highly variable and frequent recurrence of virulent forms have affected chickpea production and exhausted valuable genetic resources. The severity and yield losses of Fusarium wilt differ from place to place owing to existence of physiological races among isolates. Diversity study of fungal population associated with a disease plays a major role in understanding and devising better disease control strategies. The advantages of using molecular markers to understand the distribution of genetic diversity in Foc populations is well understood. The recent development of Diversity Arrays Technology (DArT) offers new possibilities to study the diversity in pathogen population. In this study, we developed DArT markers for Foc population, analysed the genetic diversity existing within and among Foc isolates, compared the genotypic and phenotypic diversity and infer the race scenario of Foc in India.ResultsWe report the successful development of DArT markers for Foc and their utility in genotyping of Foc collections representing five chickpea growing agro-ecological zones of India. The DArT arrays revealed a total 1,813 polymorphic markers with an average genotyping call rate of 91.16% and a scoring reproducibility of 100%. Cluster analysis, principal coordinate analysis and population structure indicated that the different isolates of Foc were partially classified based on geographical source. Diversity in Foc population was compared with the phenotypic variability and it was found that DArT markers were able to group the isolates consistent with its virulence group. A number of race-specific unique and rare alleles were also detected.ConclusionThe present study generated significant information in terms of pathogenic and genetic diversity of Foc which could be used further for development and deployment of region-specific resistant cultivars of chickpea. The DArT markers were proved to be a powerful diagnostic tool to study the genotypic diversity in Foc. The high number of DArT markers allowed a greater resolution of genetic differences among isolates and enabled us to examine the extent of diversity in the Foc population present in India, as well as provided support to know the changing race scenario in Foc population.

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.

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.