Summary auto-generated
This study developed and applied molecular methods to assess genetic variability of bean golden mosaic geminivirus (BGMV) in the Dominican Republic. Researchers used polymerase chain reaction (PCR) and asymmetric PCR (A-PCR) to amplify a hypervariable region of the DNA-B component from five field-collected bean samples infected with BGMV, then sequenced these products. The field isolates showed 95-98% nucleotide sequence identity to the laboratory clone pDRB1, indicating very low genetic variability among Dominican Republic isolates. In contrast, BGMV isolates from Guatemala and Puerto Rico showed 75-86% identity, while more distant isolates from Brazil and bean dwarf mosaic virus showed only 42-46% identity to the Dominican Republic strain. These results demonstrate that the infectious clones of BGMV-DR are representative of the virus circulating in the Dominican Republic and establish a rapid, efficient PCR-based method for viral detection and differentiation applicable to other plant viruses.
Key findings
- BGMV isolates from five Dominican Republic field locations showed 95-98% sequence identity in the hypervariable region, confirming they represent a single virus species (BGMV-DR)
- Laboratory infectious clones of BGMV-DR are representative of field isolates in the Dominican Republic, validating their use for resistance screening and epidemiological studies
- PCR and asymmetric PCR combined with DNA sequencing provides a rapid, efficient method for detecting and differentiating geminivirus isolates from dried leaf tissue
- The hypervariable region of BGMV-DR showed substantial divergence from geographically distinct BGMV isolates (75-86% identity with Guatemala and Puerto Rico isolates)
- The method successfully amplified and sequenced viral DNA from samples stored at room temperature for four months, demonstrating flexibility for field sample collection and processing
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Abstract
A combination of the polymerase chain reaction (PCR), asymmetric PCR (A- PCR) and DNA sequencing was used to determine the nucleotide sequence of a hypervariable region of the bipartite genome of bean golden mosaic geminivirus (BGMV). This region, which was part of the intergenic region of the DNA-B component, was amplified using primers designed from the nucleotide sequence of a DNA-B component clone (pDRB1) of an isolate of BGMV from the Dominican Republic (BGMV-DR). pDRB1 is infectious on beans when coinoculated with the DNA-A component of BGMV- DR (pDRA1), and typical bean golden mosaic symptoms are observed on infected plants. Bean leaf tissue infected with BGMV was collected at five separate field locations in the Dominican Republic and the hypervariable region was amplified by PCR, ssDNA was produced using A- PCR, and partial nucleotide sequences were determined. The sequences of the hypervariable region from the field-collected samples ranged from 95% (one sample) to 98% (four samples) identical to the sequence of pDRB1. This contrasts with sequence identities of 86, 75 and 46% between the pDRB1 hypervariable region and the hypervariable regions of BGMV isolates from Guatemala, Puerto Rico and Brazil respectively, and 42% with bean dwarf mosaic geminivirus. These results indicate that Dominican Republic isolates of BGMV are very similar and should be considered isolates of the same virus (BGMV-DR), and that the infectious clones of BGMV-DR are representative of BGMV isolates in the Dominican Republic. The procedures described for DNA extraction from leaf tissue and for production of high quality ssDNA using PCR and A- PCR are rapid and efficient and could be applied to studies of variability and epidemiology of other viruses.