Studies on Groundnut rosette disease in Ghana and genomic analysis of a novel Phasey bean virus in Australia

Viruses present a major challenge to the production of major food crops worldwide, including legumes. The diseases they cause have profound effects on both plant growth and the quality of produce, resulting in significant losses. The current study investigated the proximate and elemental composition of four groundnut (peanut, Arachis hypogaea L.) cultivars infected with groundnut rosette disease (GRD), screened local cultivars of groundnut for resistance to GRD, detected Groundnut ringspot virus for the first time in Ghana and assessed the genetic diversity within Ghanaian isolates of Groundnut rosette assistor virus (GRAV), Groundnut rosette virus (GRV) and satellite RNA of GRV and compare those with known isolates from other African countires. In a related study, the complete genome of an isolate of novel virus infecting Phasey bean (Macroptilium lathyroides L.) in Australia; Phasey bean mild yellows virus (PhBMYV) was sequenced with evidence of genomic recombination found and its transmission to other legumes demonstrated. Proximate analysis of seeds from GRD-infected groundnuts showed a decrease in moisture and ash content, while fat and energy content increased. Protein and carbohydrate content varied inconsistently between seeds of diseased and healthy plants of the different cultivars. Instrumental neutron activation analysis (INAA) of ten elements within leaves, stems and seeds revealed elevated levels of K, Al and Cl in leaves, stems and seeds in at least three of the four GRD-infected cultivars while Na was decreased in stems but increased in seeds. Despite significant differences, Mg, Mn, Ca and Zn did not show any consistent change with respect to plant part or genotype, between diseased and healthy plants. V and Fe were not detected in seeds but were found at low levels in leaves and stems. This work has been published in Annals of Applied Biology (2015) and represents the first report on the effect of GRD on the nutritive quality of groundnuts. Twelve cultivars of groundnut were screened in field trials for resistance to GRD in the coastal savannah agro-ecological zone of Ghana. Cultivar ‘Oboshie’ was rated as highly resistant; ‘Bremaowuo’, ‘Nkatefufuo’, and ‘Behenase’ as resistant; and ‘Nkosuor’, ‘Kumawu’, and ‘Otuhia’ as moderately resistant. GRAV infection rates of 11.8 to 61.8% and 13.9 to 100% were found within the field trial for dry and wet seasons respectively. These included symptomless plants suggesting that some lacked co-infection with GRV and its satellite RNA which are responsible for symptom induction. Some plants exhibited chlorotic and line pattern symptoms suggestive of Groundnut ringspot virus (GRSV) infection, which was confirmed by enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction, and amplicon sequencing. This represents the first report of GRSV in Ghana. GRSV infection rates within the field trial were 0.0 to 69.5% (dry season) and 26.1 to 69.5% (wet season) and was commonly found in mixed infections with GRAV in all cultivars except Nkosuor and Bremaowuo in the dry season. Graft-inoculated groundnut cultivars showed significantly reduced height, leaf area, chlorophyll content, dry haulm weight, and seed yield compared to healthy plants. The sources of resistance to GRV and possibly GRAV and GRSV identified in this study could be exploited in groundnut breeding programs. This work has been published in Plant Disease (2015). GRAV incidence in farmers’ fields was assessed through crop surveys in the three northern groundnut-producing regions of Ghana. High (69.5 to 75.0%) but insignificantly different incidences were found between the regions. Isolates of GRAV, GRV and sat RNA collected during the survey were sequenced. There were no obvious isolate diversity patterns among the Ghanaian isolates of all three agents of GRD based on the regions from where they were collected. Nucleotide sequences of the coat protein gene of GRAV showed 99-100% identity among the Ghanaian isolates and 97-100% similarity to GRAV sequences from Nigeria and Malawi for both nucleotide and predicted amino acids. Ghanaian GRV isolates were closer in nucleotide sequence identity to Nigerian isolates (95 - 98%) than Malawian isolates (88 - 90%). Similarly, Ghanaian satRNA isolates shared close nucleotide identities (94-100%), but were distinct from Nigerian (82 - 87%) and Malawian (82 - 86%) isolates. This work has been accepted for publication in Tropical Plant Pathology (2017) and presents the first report on the distribution and genetic diversity of GRD agents in Ghana. The complete genome of a QLD isolate of the novel PhBMYV was determined. The genome consisted of six open reading frames (ORFs) typical of Poleroviruses, with their respective putative proteins closely related to two previously reported PhBMYV isolates from New South Wales (NSW) and Western Australia (WA), except within the RNA-dependent-RNA-polymerase (RdRp) and Coat protein-Read through (CP-RT). The RdRp only shared ~63% amino acid identity with the NSW and WA isolates and the CP-RT was distinct (33 – 34% amino acid identity with other PhBMYV isolates) and shared 53% identity with Chickpea chlorotic stunt virus (CpCSV). Recombination analysis using RDP4 suggested the QLD isolate was an evolutionary product of recombination between the NSW (minor parent) and WA (major parent) isolates. The virus was successfully transmitted from Phasey bean to pea (Pisum sativum), chickpea (Cicer arietinum) and to Phasey bean plants using both vector and graft transmission methods. Based on the results of this study, PhBMYV QLDCL 16 is suggested as a genetic variant of PhBMYV and perhaps represents a distinct species.