A Simplified Multiplex PCR Assay for Simultaneous Detection of Six Viruses Infecting Diverse Chilli Species in India and Its Application in Field Diagnosis.

Chilli is infected by at least 65 viruses globally, with a mixed infection of multiple viruses leading to severe losses being a common occurrence. A simple diagnostic procedure that can identify multiple viruses at once is required to track their spread, initiate management measures and manage them using virus-free planting supplies. The present study, for the first time, reports a simplified and robust multiplex PCR (mPCR) assay for the simultaneous detection of five RNA viruses, capsicum chlorosis orthotospovirus (CaCV), chilli veinal mottle virus (ChiVMV), large cardamom chirke virus (LCCV), cucumber mosaic virus (CMV), and pepper mild mottle virus (PMMoV), and a DNA virus, chilli leaf curl virus (ChiLCV) infecting chilli. The developed mPCR employed six pairs of primer from the conserved coat protein (CP) region of the respective viruses. Different parameters viz., primer concentration (150-450 nM) and annealing temperature (50 °C), were optimized in order to achieve specific and sensitive amplification of the target viruses in a single reaction tube. The detection limit of the mPCR assay was 5.00 pg/µL to simultaneously detect all the target viruses in a single reaction, indicating a sufficient sensitivity of the developed assay. The developed assay showed high specificity and showed no cross-amplification. The multiplex PCR assay was validated using field samples collected across Northeast India. Interestingly, out of 61 samples collected across the northeastern states, only 22 samples (36%) were positive for single virus infection while 33 samples (54%) were positive for three or more viruses tested in mPCR, showing the widespread occurrence of mixed infection under field conditions. To the best of our knowledge, this is the first report on the development and field validation of the mPCR assay for six chilli viruses and will have application in routine virus indexing and virus management.

Genetic diversity of Chilli veinal mottle virus infecting different chilli landraces in North East India indicates the possibility of transboundary movement of virus.

Diverse chilli genetic resources of North East India occupy a unique niche in the Nation's chilli gene pool. Widely cultivated chilli landraces (King chilli: Capsicum chinense, bird eye chilli: Capsicum frutescens and Capsicum annuum) of North East India in general and Manipur in particular suffer from decline complex due to high incidence of viral diseases. With this background and the known prominent distribution of Chilli veinal mottle virus (ChiVMV) in Asian region, we studied its prevalence and association with diverse symptoms of chilli landraces. Molecular indexing of samples from 40 chilli plantation groves of Manipur using reverse transcription (RT)-PCR targeting the coat protein (CP) region of ChiVMV genome revealed a high incidence and wide prevalence. Out of a total of 127 chilli samples collected from different groves of Manipur, 81 chilli samples (63.78%) were positive for ChiVMV. Interestingly, ChiVMV infection rate was comparatively higher in the hilly groves (69.23% samples positive) compared to the valley groves (60% samples positive). Present study through the extensive surveys and molecular indexing work, conclusively reported the association of ChiVMV with diverse symptoms like cupping of leaf lamina with mottling, vein banding and puckering in different chilli landraces. Further, five representative ChiVMV isolates sampled from different groves of Manipur upon mechanical inoculation showed significant variation in symptom expression, indicating wide pathogenic diversity among them. Partial coat protein (CP) sequence analysis of five ChiVMV isolates from Manipur although indicated genetic homogeneity among them, but distinctiveness from ChiVMV isolates reported from the other parts of India. Phylogenetic clustering of ChiVMV isolates from Manipur near Chinese isolates rather than other Indian isolates suggested the possibility of transboundary movement.

CIBMAN: Database exploring Citrus biodiversity of Manipur.

UNLABELLED: The rich wealth of Citrus genetic resources makes India to enjoy a remarkable position in the "Citrus belt of the world". We have developed CIBMAN, a unique database on Citrus biodiversity of Manipur which comprises 33 accessions collected through extensive survey for more than three years. CIBMAN provides integrated access to Citrus species through sophisticated web interface which has following capabilities a) morphological details, b) socio-economic details, c) taxonomic details and d) geographical distribution. Morphological variability among Citrus accessions is due to variance in their genome which contributes to diverse agronomical traits and diverse bioactive compounds of high value. This diverse gene pool can be potential source for genetic improvement of existing cultivars and rootstocks. Systematic collection, characterization and conservation of the underutilized or lesser exploited varieties is required for incorporating in breeding program and conserve the germplasm from ever going on genetic erosion. This database will be useful for scientific validations and updating of traditional wisdom in bioprospecting aspects especially industrialization of Citrus found in the state. Further, the features will be suited for detailed investigation on potential medicinal and edible Citrus that make CIBMAN a powerful tool for sustainable management. AVAILABILITY: http://ibsd.gov.in/cibman.