Plant pathogens and crops from tropical areas provide a disproportionally large number of diagnostic challenges, as they have been relatively less-studied. Bananas provide such an example, with novel pathogens regularly being discovered. Recently, during indexing of germplasm, Closteroviridae-like particles were identified by electron microscopy (EM) in five south-east Asian accessions.
Isolates were tested with degenerate PCR primers for three Closteroviridae genera. No amplification was achieved in the closterovirus and crinivirus assays. Ampelovirus primers targeting the conserved HSP70 region amplified 580 nt fragments from isolates Q6191 from Vietnam and Q6196 from Indonesia. Fragments were cloned and sequenced. Translated amplicon sequences within each isolate were 97.8-100% identical at the amino acid (aa) level across 193 residues. The two isolates were 96.6-98.3% identical. Sequences from both isolates were 52% identical to Plum bark necrosis stem pitting-associated virus (PBNSPaV; GenBank ID AIH06908), the best database match using BLASTp, and 86.0% identical to novel sequence we obtained for the tentative ampelovirus Sugarcane mild mosaic virus (SMMV).
In order to obtain further genomic sequence, preliminary high throughput sequencing for one isolate, Q6191, was conducted. Primers designed to sequences obtained within ORF1a allowed amplification of overlapping 1064 nt from Q6191 and 812 nt from isolate Q6230. These translated sequences were 93.7% identical, and had the best BLASTp matches of 39% and 40% identity over 353 and 270 aa residues, respectively, to PBNSPaV (AMH87475).
In EM decoration tests using SMMV antiserum, SMMV particles were heavily decorated, while individual particles from the banana accessions had moderate, little or no decoration, suggesting a diversity of ampeloviruses or strains in the banana accessions.
Additional high throughput sequencing has been undertaken for isolates from all five accessions. Identification of further ampelovirus sequence will allow the development of a molecular diagnostic assay for use in the safe importation of new banana germplasm into Australia.