A New, Virulent, Broad Host Range Bean-Infecting Begomovirus from Puerto Rico: Rhynchosia Mild Mosaic Virus

A NEW, VIRULENT, BROAD HOST RANGE BEAN-INFECTING BEGOMOVIRUS FROM PUERTO RICO: RHYNCHOSIA MILD MOSAIC VIRUS

Judith K. Brown and Ali M. Idris

Department of Plant Sciences, The University of Arizona, Tucson, AZ 85721 USA

INTRODUCTION

Members of the genus Begomovirus {Geminiviridae) are circular, single-stranded (ss) DNA viruses that are naturally infect numerous ruderal, indigenous and introduced dicotyledonous plants in tropical/subtropical habitats. Begomoviral genome arrangement can be bipartite (DNA-A and DNA- B; -2.6 kb each) or monopartite (~2.8 kb). All New World indigenous begomoviruses have been shown to contain a bipartite genome. Begomoviruses are transmitted by the whitefly vector Bemisia tabaci (Genn.) complex, which comprises divergent haplotypes and biotypes that vary in aggressiveness and other behaviors that can influence begomovirus spread (Brown 2007). Some begomoviruses have host-shifted (species-, genus-, family-levels), enabling infection of domesticated plants in which they can cause significant crop loss.

Previously, two begomoviruses were identified in Puerto Rico that infected bean {Phaseolus vulgaris), Bean golden yellow mosaic virus (BGYMV) (Bird and Sanchez, 1971) and Macroptilium mosaic Puerto Rico virus (MaMPRV) (Idris et al., 2003). Also MaMPRV has been identified there in native fabaceous species and in bean. Until recently, BGYMV and its close relatives were prevalent in bean throughout the Caribbean region and in Florida (Blair et al., 1995; Bird Maramorosch, 1978; Faria et al., 1994). However, following the introduction of the Old World B biotype whitefly Bemisia tabaci (Genn.) in Puerto Rico during -1989, BGYMV diminished in prevalence, presumably because this non-native vector does not effectively transmit BGYMV between P. vulgaris in which the virus is presumed to have been bottlenecked. Several other begomoviruses infect P. vulgaris in the tropical Americas and SW-US, including Bean golden mosaic virus (BGMV), Bean calico mosaic virus (BCaMV), Chino del tomato virus (CdTV), Cotton leaf crumple virus (CLCrV), Melon chlorotic leaf curl virus (MCLCV), and Squash leaf curl virus (SLCV). Also, in Puerto Rico several begomoviruses naturally and/or experimentally infect bean and one more indigenous species, including MaMPRV [Macroptilium lathyroides], Rhynchosia mosaic virus [Rhynchosia minima] and Jatropha mosaic virus [Jatropha gossypifolia; P.foetida],

MATERIALS AND METHODS

R. minima plants exhibiting mild mosaic symptoms reminiscent of begomovirus infection were observed in PR during the summer, 1997. Total nucleic acids were extracted and circular ssDNA was subjected to rolling circle ampHfication (RCA). RCA products were subjected to restriction endonuclease digestion and Sad was found to linearize both the DNA-A and DNA-B component (-2.6 kb, each). Clone viral inserts (n = 8) subjected to capillary DNA sequencing.

RESULTS AND DISCUSSION

Eight clones bearing a fi-agment of-2.6 KB were sequenced using primer walking. Comparative sequence analysis revealed that five and three clones were of the DNA-A and DNA-B components, respectively. The genome organization was similar to that of other bipartite begomoviruses in that 30 six and two open reading frames (ORF) of characteristic size, position and orientation were identified in the DNA-A and DNA-B components, respectively. The DNA-A (n = 5) and DNA-B components (n=3), respectively, shared 98-99% nucleotide (nt) identity, indicating the presence of a single species. The common region (ca. 200 bases) was 99% identical, indicating that they are cognate components. This newly described begomoviral species is herein referred to as Rhynchosia mild mosaic virus (RhMMV). Phylogenetic analysis of the DNA-A component with well-studied begomoviruses revealed that the closest relative of RhMMV are Macroptilium mosaic Puerto Rico virus (MaMPRV) and Rhynchosia golden mosaic virus (RhGMV), at 80%. The RhMMV DNA-B component shared 64% and 62%) nt identity with RhGMV and Cabbage leaf curl virus (CaLCV), respectively.

Biolistic inoculation of R. minima and P. vulgaris seedlings with the dimeric RhMMV clones indicated the clones were infectious based on symptom development 7-10 days post-inoculation. RhMMV caused severe mottling of 'Red Kidney' bean leaves (Fig. 1), compared to the mild foliar symptoms characteristic of BGYMV. R. minima leaves exhibited mild mosaic symptoms reminiscent of those observed in the field, thereby fulfilling Koch's Postulates. This newly described begomovirus has potential to become problematic in cultivated bean in Puerto Rico. In addition, the infectious clones may be useful for germplasm screening, given the extinction of BGYMV where bean varietal screening has been traditionally Figure 1. Symptoms ofRhMMV in'Red Kidney' carried out in Puerto Rico. bean biolistically inoculated with infectious clones.

REFERENCES

Bird, J., and Sanchez, J. 1971. Whitefly-transmitted viruses in Puerto Rico. Tech Paper, Agrie Exp Stn, Univ. of Puerto Rico 55:461-467.

Blair, M. W., A. M., E. Hiebert, J. E. Polston, R. T. Graves, and M. Lamberts. 1995. Occurrence of bean golden mosaic virus in Florida. Plant Dis. 79:529-533.

Brown, J.K. 2007. The Bemisia tabaci complex: genetic and phenotypic variability drives begomovirus spread and virus diversification. Plant Dis. http://www.apsnet.org/online/feature/btabaci/.

Faria, J. C., R. L. Gilbertson, S. F. Hanson, F. J. Morales, P. Ahlquist, A. O. Loniello and D. P. Maxwell, 1994. Bean golden mosaic geminivirus type II isolates from Dominican Republic and Guatemala: nucleotide sequence, infectious pseudorecombinants, and phylogenetic relationships. Phytopathology 84:321-329.

Idris, A. M., E. Hiebert, J. Bird and J. K. Brown, 2003. Two newly described begomoviruses of Macroptilium lathyroides and common bean. Phytopathology. 93:744-783.