Molecular characterisation of ampeloviruses associated with mealybug wilt of pineapple disease in Ghana

Abstract Background: Mealybug wilt of pineapple (MWP) is the most destructive viral disease of pineapple worldwide. The disease is caused by pineapple mealybug wilt-associated virus (PMWaV), a member of the family Closteroviridae and the genus Ampelovirus, and transmitted by mealybugs. Methods: In order to understand the association between closteroviruses and MWP in Ghana, 24 pineapple plant samples showing typical symptoms of MWP were collected during a survey of the Central Region in 2019. Three quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays with PMWaV species specific primers were performed to amplify the heatshock protein 70 gene (HSP70) of PMWaV-1,-2 and -3 in the pineapple samples. Purified qRT-PCR products of thirteen isolates which consist of PMWaV-1 (7 isolates), PMWaV-2 (4 isolates) and PMWaV-3 (2 isolates) were sequenced in both directions. Sequence and phylogenetic analyses were then conducted.Results: Three different species of Ampelovirus namely PMWaV-1, PMWaV-2, and PMWaV-3, were detected from the plant samples, with abundance of mixed infections. Sixteen out of the 24 samples (66.7%) were infected with at least one of the three species of Ampelovirus identified. PMWaV-2 had the highest infection rate of 62.5% across the districts; this was followed by PMWaV-1 and PMWaV-3 with infection rates of 33.3% and 8.3% respectively. Purified qRT-PCR products of thirteen isolates which consist of PMWaV-1 (7 isolates), PMWaV-2 (4 isolates) and PMWaV-3 (2 isolates) were sequenced in both directions. Sequence comparison using BlastN showed that all the seven sequences of the Ghanaian PMWaV-1 isolates (GenBank Accession Nos. MN427634 - MN427639 and MN399973) shared 95.2% to 99.7% nucleotide identity with each other and 95.2-100% with sequences of isolates previously published in GenBank. The four sequences of the Ghanaian PMWaV-2 isolates (GenBank Accession Nos. MN427642 - MN427645) shared nucleotide identity of 98.9–100% with each other and 98.2-100% nucleotide identity with sequences of isolates previously published in GenBank. Also, the two sequences of the Ghanaian PMWaV-3 isolates (GenBank Accession Nos. MN427640 and MN427641) shared 98.3% nucleotide identity to each other and 97.5- 99.3% nucleotide identity with sequences of isolates previously published in GenBank. Phylogenetic analyses of the nucleotide sequences of HSP70 gene of the 13 Ghanaian isolates and 24 sequences previously published in GenBank, clustered the PMWaV-1, PMWaV-2 and PMWaV-3 isolates into three distinct genetic groups with > 95% bootstrap support. Conclusion: The present study shows for the first time the occurrence of PMWaV-1, PMWaV-2 and PMWaV-3 in Ghana pineapple fields as well as in Africa.