Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars.

Recombinase polymerase amplification (RPA) is a rapid, isothermal amplification method with high specificity and sensitivity. In this study, an assay was developed and evaluated for the detection of banana bunchy top virus (BBTV) in infected banana plants. Three oligonucleotide primer pairs were designed from the replicase initiator protein gene sequences of BBTV to function both in RPA as well as in polymerase chain reaction (PCR). A total of 133 symptomatic as well as asymptomatic banana leaf samples from various cultivars were collected from the different regions of India and evaluated for BBTV infection using the RPA assay. BBTV was efficiently detected using crude leaf sap in RPA and the results obtained were consistent with PCR-based detection using purified DNA as template. To our knowledge, this is the first report of reliable diagnosis of BBTV infection by RPA using crude leaf sap as a template.

First record of Pepper cryptic virus 2 in chilli (Capsicum annuum) in India.

The occurrence of Pepper cryptic virus 2 was confirmed by RT-PCR and partial sequencing of coat protein gene in chilli cultivars Jwalamukhi and Jwalasakhi grown in the experimental farm at IARI, New Delhi. To the best of our knowledge this is the first report of occurrence of cryptovirus in chilli in India.

Molecular Characterization of Tomato leaf curl Palampur virus and Pepper leaf curl betasatellite Naturally Infecting Pumpkin (Cucurbita moschata) in India.

Pumpkin cultivation in India is affected by severe incidence of a yellow vein mosaic disease. Tomato leaf curl New Delhi virus and Squash leaf curl China virus are known to be associated with this disease in India. We were able to identify a third begomovirus-Tomato leaf curl Palampur virus (ToLCPMV), from pumpkin showing typical symptoms of the disease at Varanasi based on the sequence of complete DNA-A genome of the virus. The complete DNA-A sequence of the virus shared more than 99% sequence identity with other ToLCPMV isolates available in the GenBank and clustered with them in the phylogenetic analysis. This betasatellite amplified from the same infected sample has been identified as Pepper leaf curl betasatellite (PepLCB) which also infects chilli in India. There was 92% sequence identity between the two isolates. This is the first report of natural infection of ToLCPMV on pumpkin and association of PepLCB with yellow vein mosaic disease of pumpkin in India.

Nucleotide sequence of the S and M RNA segments of a Groundnut bud necrosis virus isolate from Vigna radiata in India.

Nucleotide sequence of the S and M RNA segments of a Groundnut bud necrosis virus isolate from Vigna radiata (mungbean isolate, GBNV-MB) was determined and compared with another isolate from Arachis hypogaea (groundnut isolate, GBNV-type). Comparative sequence analysis revealed that the genome organization of the S and M RNA segments of both GBNV-MB and GBNV-type isolates was similar. However, considerable differences were observed in their intergenic regions (IGRs) and the glycoprotein precursors (Gn/Gc) of the M RNA segments. M RNA IGRs of GBNV-MB and GBNV-type isolates differed in size by 14 nucleotides. This difference was of 75 nucleotides in another GBNV isolate from Lycopersicon esculentum (tomato). Sequence comparison of the M RNA IGRs of GBNV isolates from mungbean, groundnut and tomato from India revealed 56-89% sequence identity. The topology of Gn/Gc revealed the presence of both N-glycosylation (5) and O-glycosylation (1) sites in GBNV-MB isolate, whereas only N-glycosylation sites (4) were present in GBNV-type isolate.