Distributionand Characterization Of Groundnut Rosette Associated Virusesin Western Kenya

ABSTRACT

Groundnut (Arachis hypogaea L.) 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 constraint of groundnuts

in Sub-Saharan Africa (SSA) causing up to 100% yield losses. The disease is caused

by two synergistic viruses; groundnut rosette assistor virus (GRAV, genus Luteovirus)

and groundnut rosette virus (GRV, genus Umbravirus) associated with a satelliteribonucleic

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 the causal agents is possible by

multiplex RT-PCR but this depends on the availability of specific primers to known

agents that occur in a specific area. Information on occurrence and distribution of

GRD in western Kenya was not documented and little was known about the

characteristics of associated viruses. This study determined the distribution and

characterized GRD associated viruses in western Kenya. Two surveys were conducted

(2016/2017) in six counties; Bungoma, Busia, Homabay, Kakamega, Siaya and

Vihiga. Symptomatic and asymptomatic groundnut and some bean leafy samples were

collected for laboratory analysis. Total RNA was extracted from the leaf samples

using RNeasy Mini Kit (Qiagen) according to the manufacturers’ instructions and

used for double stranded cDNA synthesis using the SuperScript II kit. 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.

Trimmed reads were 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 against the GenBank database. Phylogenetic

analyses and comparisons were performed using MEGA X. Primers were designed

using Primer3Plus from consensus sequences. Biological characterization of GRD

was done through sap inoculation on leguminous hosts. Average incidence was 53%

and 41% in the short and long rain seasons, respectively. Chlorotic rosette was the

dominant symptom followed by Green rosette and Mosaic. Most farmers (65%)

sourced groundnut seeds from open air market. Complete nucleotide sequences of

Sat-RNA revealed identities of 88-100% with those from Malawi, Nigeria and Ghana.

Isolate EG16-5 clustered together 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 Luteoviruses in phylogenetic analysis. Leguminous hosts showed varied

symptoms and tested positive for Sat-RNA and GRAV using the designed primers.

The variations of GRD symptoms observed on groundnuts were due to the existence

of different variants of Sat-RNA. Sat-RNA and GRV are more diverse than GRAV.

The GRD viruses have hosts among the commonly grown legumes and this enhance

the perpetuation of the disease. The study recommends an urgent need to curb GRD,

possibly through the exploitation of pathogen derived resistance (PDR).