Lessons learned from the virus indexing of Musa germplasm: insights from a multiyear collaboration

Abstract

The Bioversity International Transit Center (ITC) for banana hosts more than 1500 accessions largely covering the genetic diversity of the genus Musa. Its objective is to conserve this genetic diversity and to supply plant materials to users worldwide. All the Musa accessions must be tested for virus presence and, if infected, virus elimination must be attempted, to enable the supply of virus-free plant material. An international collaborative effort launched under the auspices of Bioversity International (2007)(2008)(2009)(2010)(2011)(2012)(2013) finally led to the implementation of a two-step process to test the accessions. The first step, called pre-indexing, involved only molecular tests and was designed as a pre-screen of new germplasm lines or existing accessions to reduce the need for post-entry virus therapy and repeated virus indexing. The second step, called full indexing, was performed on either older existing accessions or newer accessions which tested negative during pre-indexing, and involved molecular tests, transmission electron microscopy (TEM) and symptom observation. In total, 270 germplasm lines (434 samples) were pre-indexed; while full indexing was carried out on 243 accessions (68 of which had been pre-indexed). A significant proportion of the samples tested during pre-indexing was infected with at least one virus (68%), showing the utility of this early pre-screening step. Banana streak OL virus and Banana mild mosaic virus were the most commonly detected viruses during both pre-and full indexing. For 22 accessions, viral particles were observed by TEM in full indexing while the molecular tests were negative, underlining the importance of combining various detection techniques. After full indexing, viruses were not detected in 166 accessions, which were then released for international distribution from the ITC. This publication exemplifies how the practical application of diagnostic protocols can raise fundamental questions related to their appropriate use in routine practice and the need for their continuous monitoring and improvement after their first publication.

Safeguarding and using global banana diversity: a holistic approach

“…The virus testing is particularly important as several banana growing countries are currently free of damaging viral diseases. The banana bunchy top virus is not present in South America while the banana bract mosaic virus is only present in some Asian countries, and the virus prevalence in collected germplasms reaches 68% (De Clerck et al 2017). Therefore, guaranteeing the movement of pathogen-tested germplasm is an Van den houwe et al CABI Agric Biosci (2020) 1:15 important task to minimize the risk of pest introduction to new countries.…”

The impact of high throughput sequencing on plant health diagnostics

“…Subsequent investigations have shown that MLBVV is the more probable cause of big-vein disease (Lot et al 2002), but that LBVaV may still be associated with other symptoms such as localised necrosis (Verbeek et al 2013). This eight-decade arc of investigation moved on with advances in diagnostic methods, but ultimately progress was hampered by the limitations of these techniques: Electron microscopy can only observe the morphology of viral particles present, but cannot be used to give a conclusive diagnosis at species level; Biological indexing can give an indication of the pathogens present in a sample, but is prone to failure where a virus is labile or is not amenable to mechanical transmission; Targeted methods can only detect the targets the assays have been designed against, at best a broad number of species in a given genus (Adams et al 2013;De Clerck et al 2017;Rott et al 2017).…”

Identification of Divergent Isolates of Banana Mild Mosaic Virus and Development of a New Diagnostic Primer to Improve Detection

“…Banana accession ITC0763 from the International Musa Germplasm Transit Centre (ITC) (Bioversity International) presented discrepancies between diagnostic techniques used for indexing when assessed under post entry quarantine in Australia [ 23 ]. No viral symptoms were observed for the accession.…”

“…Routine germplasm screening molecular diagnostic tests were carried out on the crude extracts following the procedure described by [ 23 ]. Then, an IC-RT assay was conducted to produce the complementary DNAs, on which the PCR test was carried out.…”

“…Partially purified and concentrated viral minipreps were prepared as described previously [ 23 ]. ‘Necoloidine’ solution (Stanvis)-coated copper grids were coated with a mixture of BSV, BanMMV and BBrMV antibodies before being floated over a droplet of viral miniprep.…”