Comprehensive survey of common bean viruses in Tanzania using next generation and Sanger sequencing techniques

<p>Common bean (<em>Phaseolus vulgaris</em> L.) is an important legume crop in Tanzania and elsewhere in the tropics and subtropics. We employed a next-generation sequencing technique to detect viruses in common bean plant samples collected from five agricultural research zones in the country. The aim was to target and sequence virus-derived small RNAs. To achieve this, total RNA was isolated from dry leaf samples using the CTAB method. The CTAB buffer contained 2% CTAB, 100 mM Tris–HCl, 20 mM EDTA, 2.5 M NaCl, freshly prepared 1% sodium sulfite, 2% PVP and 2.5% 2-mercaptoethanol in nuclease-free water. Total RNA was sent to Fasteris in Switzerland where the small RNA was purified by electrophoresis in an acrylamide gel. The small RNA library was prepared using the Illumina TrueSeq small RNA  sample preparation kit (Illumina Inc., San Diego, CA, USA). Viruses were detected using VirusDetect software (v.1.6 and v.1.7) (available at http://bioinfo.bti.cornell.edu/cgi-bin/virusdetect/index.cgi) and supercomputer at CSC.fi. Viruses detected belonged to at least 11 genera.</p

Symptoms observed in common bean plants in La Compañia, Nicaragua.

<p>(a), Stunting of the plant, malformation and blistering of leaves. (b), Mild epinasty and vein reversion. (c), Green-yellow chlorosis. (d), Green-yellow mosaic.</p

Identification of PvEV-2 in the sample pool HXH8 from the Southern Highland zone of Tanzania based on small-RNA deep sequencing.

<p>(a), Viral contigs (red bars) mapped to the sequence of PvEV-2-Okada (AB719398) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178242#pone.0178242.ref025" target="_blank">25</a>] using VirusDetect [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178242#pone.0178242.ref051" target="_blank">51</a>]. Each nucleotide in the contigs was covered by siRNA reads at least 5 times. (b) The 21- to 24-nt reads mapped to the sequence of PvEV-2. The <i>x</i> axis and the scale below the figure depict the viral genome and nucleotide positions, respectively. The <i>y</i> axis indicates the number of siRNA reads derived from the coding strand (blue bars above the <i>x</i> axis) and complementary strand (red bars below the <i>x</i> axis).</p

Conserved domains in the polyprotein encoded by PvEV-1 and PvEV-2 from Nicaragua.

<p>Numbers indicate the residues defining the conserved domains. Hel-1, helicase; CPS, putative capsular polysaccharide synthase; UGT, UDP-glycosyltransferase; RdRp, RNA-dependant RNA polymerase; and MTR, methyltransferase.</p

Viruses detected in the pools of common bean samples from Nicaragua (NI) and Tanzania (TZ) by VirusDetect.

<p>Viruses detected in the pools of common bean samples from Nicaragua (NI) and Tanzania (TZ) by VirusDetect.</p

Detection of PvEV-1 by RT-PCR in common beans in Tanzania.

<p>In the list below, landraces are marked with asterisk (*). Other samples represent improved varieties (origin of samples shown in parenthesis). Lane labelled ‘M’ represents a O'GeneRuler 1 kb Plus DNA ladder. The expected size of PCR products was 374 bp. Lanes 1, ‘Njugu’* (Southern Highlands zone); 2, ‘pooled RNA’ (Southern Highlands zone); 3, ‘pooled RNA’ (Eastern zone); 4, ‘pooled RNA’ (Northern zone); 5, ‘Rosekoko’/’Lyamungu 85’ (Eastern zone); 6, ‘Salundi’ (Southern Highlands zone); 7, ‘E 36’ (Southern Highlands zone); 8, ‘Msafiri’* (Southern Highlands zone); 9, ‘Msafiri’* (Eastern zone); and 10, ‘Mshindi’ (Eastern zone).</p