DISTRIBUTIONAND CHARACTERIZATION OF GROUNDNUT ROSETTE ASSOCIATED VIRUSESIN WESTERN KENYA

Abstract

Groundnut (Arachis hypogaeaL.) is an economically important edible oilseed legume in Sub-Saharan Africa (SSA). Smallholder farmers, who account for 75% of producers, depend on it for food and income. However the yields are far below the world averages. Groundnut rosette disease (GRD)is a major constraintofgroundnutsin Sub-Saharan Africa (SSA)causingup to 100%yieldlosses. The disease is caused by two synergistic viruses; groundnut rosette assistor virus(GRAV, genus Luteovirus)andgroundnut rosette virus(GRV, genusUmbravirus)associated with a satellite-ribonucleic acid (Sat-RNA).Some of the setbacks in the epidemiological studies of GRD associated viruses include the complex etiology of the disease and lack of specific diagnostic tools.Simultaneous detection of thecausal agents is possible by multiplex RT-PCR but this depends on the availability of specific primers to known agents that occur ina specific area. Information on occurrence and distribution of GRDin western Kenyawas not documentedand little was knownabout thecharacteristics ofassociated viruses.This study determinedthedistributionand characterizedGRD associated virusesinwestern Kenya.Twosurveyswereconducted(2016/2017)in sixcounties; Bungoma, Busia, Homabay, Kakamega, Siaya and Vihiga. Symptomatic and asymptomatic groundnut and some bean leafy samples werecollected for laboratory analysis.Total RNA was extracted from the leaf samples using RNeasy Mini Kit(Qiagen)accordingto the manufacturers’ instructionsand used for double stranded cDNA synthesis using the SuperScript IIkit.The cDNA was column-purified with the DNA Clean & ConcentratorTM-5 –DNA kit. The samples were then processed with the transposon-based chemistry library preparation kit (Nextera XT, Illumina) following manufacturer’s instructions. The fragment sizes structure of the DNA libraries was assessed using the Agilent 2100 Bioanalyzer. The indexed denatured DNA libraries were sequenced (200-bp paired-end sequencing) on the Illumina MiSeq platform (Illumina).Reads quality check was done using FastQC. Trimmedreadswere used for denovo assembly and contigs aligned to the viral genomes database using CLC Genomics Workbench 10.1.2. The assembled contigs were subjected to a BLASTn search againstthe GenBank database. Phylogenetic analyses and comparisons were performed usingMEGA X. Primers were designed using Primer3Plus from consensus sequences. Biological characterization of GRD wasdone through sapinoculation on leguminous hosts.Average incidence was 53% and41% in the short and long rainseasons,respectively. Chlorotic rosette was the dominantsymptom followed by Green rosette and Mosaic.Most farmers (65%) sourced groundnut seeds from open air market.Complete nucleotide sequencesof Sat-RNA revealed identities of 88-100%withthosefrom Malawi, Nigeria and Ghana.Isolate EG16-5clusteredtogether with chlorotic M24S, all chlorotic isolates and yellow blotch.The GRV isolates shared 84-98% sequence identity with those available GeneBank. The GRAV coat protein (GRAV-CP)gene sequences revealed 97-100%identity with GeneBank isolates.Complete GRAV sequences clustered closest with Luteovirusesinphylogenetic analysis.Leguminoushosts showed varied symptoms and tested positive for Sat-RNA and GRAV using the designed primers.The variations of GRD symptoms observed on groundnutsweredue to the existence of different variants of Sat-RNA.Sat-RNA and GRV are more diverse than GRAV.The GRD viruseshave hosts among the commonly grown legumes and this enhance the perpetuationof the disease. The study recommends an urgent need to curb GRD, possibly through the exploitation of pathogen derived resistance (PDR).