Characterization of bean common mosaic virus isolates infecting three leguminous bean crops from South and Southeast Asia.

Bean common mosaic virus (BCMV) is causing economically important diseases in leguminous crops worldwide. In this study, BCMV isolates from country bean (CB, Lablab purpureus), yard-long bean (YLB, Vigna unguiculata) and rajma bean (RB, Phaseolus vulgaris) collected from Bangladesh, Nepal and Cambodia were characterized. Samples tested positive for BCMV in serological assays were subjected to high-throughput sequencing to generate near full-length genome sequences. In pair-wise comparisons of the polyprotein open reading frame, thirteen BCMV isolates from Bangladesh, Cambodia, and Nepal showed sequence identity of 92.1 to 98.8% at the nucleotide and 94.2 to 99% at the amino acid level among themselves and with corresponding sequences of BCMV reported previously. In phylogenetic analyses using the global BCMV sequences, they segregated into five distinct lineages, with RB isolates from Nepal clustering with US1/NL1-clade of common bean isolates from different countries, YLB isolates aligning with blackeye cowpea strain sequences reported from China, and CB isolates from Nepal and Bangladesh clustering with soybean isolates from China. One YLB isolate from Nepal was identified as a putative recombinant. None of the BCMV sequences aligned with isolates representing the RU1 or PStV clades. In grow-out tests, seed samples from local markets showed 14.3 to 38.1% transmission efficiency rate of BCMV with CB seed lots and from 9.5% to 33.3% with YLB seed lots.

Characterization of a New, Country Bean (Lablab purpureus) Lineage of Bean Common Mosaic Necrosis Virus.

Country bean (Lablab purpureus, family Fabaceae) is grown in subsistence agriculture in Bangladesh as a multipurpose crop for food, animal feed, and green manure. This study was undertaken to investigate the genetic diversity of bean common mosaic necrosis virus (BCMNV, genus Potyvirus, family Potyviridae) in country beans. Leaf samples from country beans showing yellowing, vein banding, and mosaic symptoms were collected during field surveys between 2015 and 2019 cropping seasons from farmers' fields in different geographic regions. These samples were tested by serological and molecular diagnostic assays for the presence of BCMNV. Virus-positive samples were subjected to high-throughput Illumina sequencing to generate near-complete genomes of BCMNV isolates. In pairwise comparisons, the polyprotein sequences of BCMNV isolates from Bangladesh showed greater than 98% identities among themselves and shared less than 84% sequence identity at the nucleotide level with virus isolates reported from other countries. In the phylogenetic analysis, BCMNV isolates from Bangladeshi country beans formed a separate clade from virus isolates reported from common beans in other countries in the Americas, Africa, Europe, and from East Timor. Grow-out studies showed seed-to-seedling transmission of BCMNV, implying a possible seedborne nature of the virus in country beans.

First Report of Cucumber mosaic virus from Eggplant (Solanum melongena) in Bangladesh.

In Bangladesh, eggplant (Solanum melongena L.) is largely cultivated by subsistence farmers for domestic consumption and generating family income. During a survey of family-owned farms in April of 2014 in Barisal region of Bangladesh, we observed a farmer's field (0.25 acres) of 3-month-old eggplants with nearly 90% of plants showing mild mosaic and mottling of leaves. Symptomatic plants showed reduced growth, with nearly 50% fewer fruits than from healthy plants. Symptomatic leaves tested positive for Cucumber mosaic virus (CMV; genus Cucumovirus, family: Bromoviridae) by immunostrip diagnostic kit (Agdia, Elkhart, IN). For confirmation of the virus identity, leaf samples were pressed on FTA Plant Cards (Whatman International, Maidstone, UK) and air-dried at room temperature. For eluting total nucleic acids, four to eight disks were punched from the spotted circles of each FTA card using a Harris micropunch (2-mm diameter, Sigma-Aldrich, USA) and soaked for 1 h in 300 µl of extraction buffer (15 mM Na2CO3, 35 mM NaHCO3, 2% [w/v] PVP40, 0.2% [w/v] BSA, 0.05% [v/v] Tween 20, pH 9.6). After vortexing followed by a brief centrifugation, 10 µl of the supernatant was mixed with denaturing buffer (0.1M glycine-NaOH, pH 9.0, 50 mM NaCl, 1 mM EDTA, pH 8.0, 0.5% [v/v] Triton X-100) containing 1% ß-mercaptoethanol, incubated at 95°C for 10 min, and kept in ice until use. Denatured sample (2 µl) was subsequently used in reverse-transcription (RT)-PCR using primers CMV-RNA3F (5'-GTAGACATCTGTGACGCGA-3') and CMV-RNA3R (5'-GCGCGAAACAAGCTTCTTATC-3') previously reported (2) to amplify a 529-nucleotide (nt) fragment representing the 210-nt intergenic region and the 319-nt partial coat protein (CP) gene of the RNA 3 segment. The amplicons were cloned into pCR2.1 (Invitrogen Corp., Carlsbad, CA), and DNA isolated from four independent clones per amplicon was sequenced in both orientations. The derived sequences (GenBank Accession Nos. KM516898 to KM516901) showed close to 100% identity among themselves and 97% identity with the corresponding sequence of CMV isolate BK16 from cucumber in Thailand (FN552546). These results supported immunostrip diagnostic assays in confirming the presence of CMV in symptomatic samples of eggplants from Barisal. For additional confirmation, a second primer pair (CMV-CP-F: 5'-ATGGACAAATCTGAATCAACCAG-3' and CMV-CP-R: 5'-TCAAACTGGGAGCACCCCAGAC-3') was designed using CMV sequences from JN054635 and GU906293 to amplify the full-length CP gene from the same nucleic acid preparations used above. The approximately 657-nt amplicons, representing the full-length CP gene, were cloned, and plasmid DNA from four independent colonies per amplicon wa s sequenced as described above. The derived CP sequences (KM516902 to KM516905) shared 96 and 95% nucleotide and 98.6 and 99.5% amino acid sequence identities with corresponding sequences of CMV isolates from banana (EF178298) and eggplant (GU906293), respectively, from India. Phylogenetic analysis of CP sequences derived from this study with corresponding sequences available in GenBank indicated that CMV from eggplant in Bangladesh aligned closely with CMV subgroup 1B. CMV was previously reported in chili pepper, and tomato from Bangladesh (1) and in eggplant from Israel (4) and India (3). To our knowledge this is the first confirmed report of the occurrence of CMV subgroup 1B in eggplant in Bangladesh. Since no aphids were observed on eggplants, it is likely that CMV was introduced into the farmer's field through seedlings raised from seed carrying the virus. References: (1) A. M. Akanda et al. J. Fac. Agric., Kyushu Univ. 35:151, 1991. (2) C. De Blas et al. J. Phytopathol. 141:323, 1994. (3) S. Kumar et al. Virus Dis. 25:129, 2014. (4) E. Tanne and S. Zimmerman-Gries. Plant Dis. 64:371, 1980.

A multiplex polymerase chain reaction method for reliable, sensitive and simultaneous detection of multiple viruses in citrus trees.

A multiplex polymerase chain reaction (mPCR) assay was developed to detect six RNA and one DNA citrus virus: Citrus leaf rugose virus (CLRV), Citrus psorosis virus (CPsV), Citrus tatter leaf virus (CTLV), Citrus tristeza virus (CTV), Citrus variegation virus (CVV), Citrus yellow mosaic virus (CYMV), and Indian citrus ringspot virus (ICRSV) from citrus plants. These seven viruses are classified in six different virus genera. Degenerate primers were designed based on the respective virus isolate sequence data available from the GenBank and were used for reliable detection of the different viruses by simplex- and mPCR. The sensitive and simultaneous detection of RNA and DNA viruses using the mPCR decreases the risk of contamination, saves time and reduces the cost as compared to other conventional methods for citrus virus detection. Seven different fragments (245-942 bp) specific to the viruses were simultaneously amplified using mPCR and were identified on the basis of their molecular sizes. The consistent results of the mPCR were compared with simplex PCR for detection of each virus pathogen. The mPCR results were confirmed with sequencing analysis. The mPCR provides a useful rapid method for detecting multiple viruses in citrus plants that will aid in the production of virus-free citrus plants for certification programs.