Macroptilium erythroloma (Fabaceae): A Natural Weed Host of Bean Golden Mosaic Virus in Brazil

Abstract

Bean golden mosaic virus (BGMV) is a widely distributed Begomovirus species (family Geminiviridae), being responsible for severe yield losses in the bean (Phaseolus vulgaris L.) crop in Brazil. Isolates of BGMV infecting cultivated as well as weed hosts have been also reported (Bracero et al. 2003; Sobrinho et al. 2014). Macroptilium erythroloma [(Mart. ex Benth.) Urban] leaves displaying golden mosaic–like symptoms were collected from Brasilia–DF area in 2016. Total DNA extraction was performed from three symptomatic plants using a CTAB protocol (Boiteux et al., 1999). Sanger dideoxy sequencing of the maturase (matK) gene (99.11% identity) and morphological analyses confirmed the symptomatic plants as being M. erythroloma. Purified DNA was also submitted to Rolling Circle Amplification (Inoue-Nagata et al. 2004) and sequenced on an Illumina Hiseq 2500 platform (Macrogen, South Korea). Sequence analysis of the isolate ‘RDF_327 JB_09’ was performed in the CLC Genomics Workbench program after manually refining and assembling the reads in higher quality contigs. Contigs were imported into the Geneious software and compared via BLASTn algorithm to a GenBank viral database. The Illumina–derived viral contigs for full–length DNA–A from symptomatic M. erythroloma (MN822294) displayed high identity levels (≈ 99%) with DNA–A components from an array of BGMV isolates. This DNA–A sequence was also obtained by direct Sanger dideoxy sequencing of overlapping PCR amplicons employing a primer walking strategy. The final sequence (MT319763) was identical to the original Illumina–derived contigs and displayed nucleotide identities above 99% with a collection of BGMV isolates from common beans (e.g. KJ939798) and M. lathyroides (e.g. KJ939766), indicating the absence of artefactual chimeric sequences. The 2617 nts of the DNA–A component displayed all six ORFs: AV1 (CP) in the viral sense, and AC1 (Rep), AC2 (TrAP), AC3 (Ren), AC4, and AC5 in the complementary sense. The contigs for full–length DNA–B (MN822293) displayed 98.2% identity to one bean BGMV isolate from Minas Gerais State, Brazil (MG334553). The 2594 nts of the DNA–B component displayed two ORFs: BV1 (NSP) in the viral sense and BC1 (MP) in the complementary-sense. Plants of bean (P. vulgaris cv. Carioca), soybean (Glycine max cv. Monsoy 9144), peanut (Arachis hypogaea cv. BRS Havana), cowpea (Vigna unguiculata cv. Seridó), and tomato (Solanum lycopersicum cv. Santa Clara) were employed in infectivity assays. One week before, a BGMV–infected M. erythroloma plant (one of the plants previously employed in the molecular BGMV characterization) was caged with aviruliferous adults of Bemisia tabaci MEAM 1. These whiteflies were subsequently transferred to cages with five plants of each plant species. Non–inoculated plants of each species were employed as negative controls (i.e. BGMV–free plants of bean, soybean, cowpea, and tomato that were exposed to aviruliferous adult whiteflies). One apical leaf sample per test plant was collected at 21 days after inoculation and the presence of BGMV was studied employing PCR assays with specific primers. PCR results indicated that BGMV isolate from M. erythroloma was transmitted to all five plant species. In the genus Macroptilium, BGMV has been reported thus far infecting only the species M. lathyroides (L.) Urban (Bracero et al. 2003; Sobrinho et al. 2014). Macroptilium erythroloma was assigned in the early 1980s as a putative BGMV host based solely upon symptoms and biological tests (Chagas et al. 1981). Therefore, we provide here the first formal confirmation of M. erythroloma as a natural BGMV host. The BGMV isolate from M. erythroloma was able to infect accessions of bean, soybean, peanut, cowpea, and tomato under our experimental conditions. Therefore, the presence of Macroptilium weeds in association with commercial bean fields might be a major epidemiological concern, since they may serve as year–round sources of BGMV inoculum for this crop.