Occurrence, Distribution And Molecular Diversity Of Groundnut Rosette Assistor Virus Causing Groundnut Rosette Disease In Western Kenya

ABSTRACT

Groundnut (Arachis hypogaea Linn) is an important legume in western Kenya, but yields are

low and declining. Pests and diseases are ranked high among the yield reducing factors.

Groundnut rosette disease (GRD) is the main disease and can cause up to 100% yield loss.

Rosette is transmitted mainly by the groundnut aphid, Aphis craccivora Koch and to a lesser

extent by Aphis gosypii Glover and Myzus persicae Sulzer. Rosette is caused by two

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

groundnut rosette virus (GRV, genus Umbravirus) associated with a satellite-ribonucleic acid

(sat-RNA). Inadequate current information on the occurrence, distribution and diversity of

GRAV causing GRD in western Kenya, is a limiting factor on proper diagnosis and

management of GRD which gave the impetus for this study. This study determined the

occurrence, distribution and diversity of GRAV on groundnuts in western Kenya. A survey of

GRD was conducted in Bungoma and Kakamega Counties during the short rains (October –

December 2016) and long rains (May – June 2017). Symptomatic leafy samples were

collected in falcon tubes containing RNALater solution, and preserved for laboratory

analysis. The data collected on incidence and severity was subjected to analysis of variance

and pairwise comparison of means done using Least Significance Difference at P ≤ 0.05.

Screening for resistance to GRAV was done on five popular legume varieties and one

solanaceous Physalis peruviana Linn. The plants at three leaf-stage were mechanically

inoculated with GRD inoculum prepared from leaves of RT-PCR positive samples. The

plants were monitored for symptom development in the screenhouse for 8 weeks. Total RNA

was extracted from the leaf samples using RNeasy Mini Kit (Qiagen) according to the

manufacturers’ protocol. The extracted total RNA was used for double stranded cDNA

synthesis using the SuperScript II kit. DNA libraries were prepared and sequenced on the

MiSeq platform (Illumina). Quality check on reads was done using FastQC. Trimmed reads

were used for de novo 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

the MEGA X software. Primers used in RT-PCR were designed using Primer3Plus software

from consensus sequences. A total of 144 farms were surveyed. Rosette was observed in all

the surveyed areas with chlorotic symptoms being dominant followed by green rosette and

mosaic. Mean rosette incidence was higher in Bungoma (66.51%) than Kakamega (60.52%).

Short rains had higher mean incidence than the long rains season. Nucleotide sequences of

GRAV coat protein (CP) gene revealed 97-99% identity among the western Kenya isolates

and those from Ghana, Malawi and Nigeria. All tested plants developed viral symptoms and

tested positive for GRAV by RT-PCR. The fact that GRD occurs wherever groundnuts are

grown in western Kenya, is of great concern and may be the reason for the observed low

yields. Incorporation of GRD resistant genes in the local cultivars/varieties may be the only

practical solution.