First report of Yambean mosaic virus in Brazil
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Potyvirus can cause losses in several important crops worldwide. This study describes the identification of a potyvirus infecting Jack bean plants and causing severe mosaic in Piauí state, Brazil. The virus reproduced original symptoms in mechanically inoculated Jack bean plants. Further molecular and transmission electron microscopy assays enabled the identification of the causal virus as Yambean mosaic virus (YBMV). To the best of our knowledge, this report is the first to describe YBMV in Brazil.
KeywordsCanavalia ensiformis Cylindrical inclusions Jack bean Potyvirus
The leaf samples of symptomatic Jack beans were collected at the experimental unit (S 05° 2′ 33.55″ and W 42° 47′ 3.08″) of the Department of Plant Science at the UFPI, Teresina, Piauí, Brazil. Symptomatic leaves were collected for further analysis by transmission electron microscopy (TEM) and for molecular diagnosis by amplification and sequencing of genome regions. Samples were identified and preserved at −80 °C. TEM was employed to examine negatively stained leaf extracts (Kitajima 1965) and to observe the cytopathic effects on leaf tissues exhibited by symptomatic Jack bean plants. For direct observation of leaf extract, symptomatic leaves were macerated in the presence of phosphate buffer (0.01 M, pH 7.0), transferred onto carbon-Formvar coated grids and negatively contrasted with uranyl acetate. For ultrastructural analysis by TEM, small fragments of symptomatic leaves of Jack bean were fixed in a glutaraldehyde (2.5%)/paraformaldehyde (2%) mixture in cacodylate buffer (0.05 M, pH 7.2) for several hours, post-fixed with 1% OsO4, dehydrated in a graded ethanol series, and subjected to infiltration and embedding in the Spurr epoxy resin. The blocks were sectioned in a Leica Ultracut UCT ultramicrotome equipped with a Diatome diamond knife, and 70–100-nm-thick sections were collected on 300 mesh copper grids. Sections were contrasted with uranyl acetate and lead citrate and examined in a TEM JEOL 1011. The images were digitally recorded (Kitajima and Nome 1999). Total tissue leaf RNA from symptomatic plants was extracted with the Trizol® reagent (Thermo Fisher Scientific, USA) following the manufacturer’s recommendations. The pellets were resuspended in 50 μL of ultrapure distilled water free of nucleases. The RNA samples were analysed by 1% agarose gel electrophoresis in 0.5x TBE (Tris-Borate-EDTA) buffer, stained with ethidium bromide and visualized under UV light in the UVB transilluminator (Loccus, São Paulo, Brazil). The RNAs were stored at −20 °C. The reactions to amplify the CI and NIb genes were performed in two steps. First-strand cDNAs were synthesized using GoScript® reverse transcriptase enzyme (Promega, Madison, WI, USA) according to the manufacturer’s recommendations and using CIRev and NIb3R as the initial primers. Polymerase chain reaction (PCR) assays were performed with the universal oligonucleotides CIFord and CIRev (Ha et al. 2008) and the NIb2F and NIb3R (Zheng et al. 2010) of potyviruses. For synthesis of the PCR, the GoTaq® Hot Start Polymerase (Promega, Madison, WI, USA) was used according to the manufacturer’s recommendations. Samples were also tested by reverse transcription PCR (RT-PCR) using specific or universal primers for different viruses that incite similar symptoms on legume plants: Cucumber mosaic virus (Kim et al. 2014), Cowpea severe mosaic virus (Brioso et al. 1996), and Cowpea mild mottle virus (Lamas et al. 2017). The reactions were performed in AmpliTherm® thermal cyclers (Axygen). The amplified fragments were purified and sequenced in both directions (forward and reverse) by Macrogen Inc. (Seoul, South Korea). The edited sequences were compared to sequences deposited in GenBank by the BLASTn tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments were generated using the MUSCLE algorithm implemented by the MEGA v.8.0 program (Kumar et al. 2018). The alignments were corrected manually and analysed with the MEGA v.8.0 program using the neighbour-joining method.
Leaf samples from Jack beans plants that were previously confirmed to be infected with only one virus species were mechanically inoculated via buffered plant extract (0.01 M potassium phosphate, pH 7.5) and abrasive (carborundum) in five healthy Jack bean plants. The inoculation occurred 20 days after emergence of the seedlings. The plants were cultivated in 2.8 L pots containing sterile substrate (70% soil, 15% burned rice straw and 15% cattle manure) and kept in insect-free cages throughout the entire experiment period. Two plants were used as controls.
IAO is recipient of a CAPES scholarship and JEABJ and EWK are recipients of PQ fellowship from CNPq. Financial support from FAPESP Biota Program 2017/18910-4.
Compliance with ethical standards
Disclosure of potential conflicts of interest
We declare that the authors have no conflicts of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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