Detection of sugar beet-infecting beet mild yellowing luteovirus isolates with a specific RNA probe

A complementary RNA (cRNA) probe, BM1, was prepared by transcription of a 1,061 nucleotide cDNA fragment complementary to nucleotides 1 to 1,061 (open reading frame [ORF] 1) within the sequence of a French sugar beet—infecting beet mild yellowing luteovirus (BMYV) -2ITB isolate. This probe detected specifically the homologous isolate as well as 14 other BMYV isolates collected from sugar beet grown in various areas, mainly in Europe. It did not hybridize with nonbeet-infecting isolates of the closely related beet western yellows luteovirus (BWYV) or cucurbit aphid-borne yellows luteovirus (CABYV) -N isolate, but reacted weakly with two English BMYV isolates that do not infect Capsella bursa-pastoris or Montia perfoliata. The probe BM1 detected BMYV in single Myzus persicae, giving no reaction with nonviruliferous individuals. As a comparison, a second BMYV probe (BM2) was produced to the coat protein gene (ORF 4) of the French BMYV-21TB isolate. This probe detected all BMYV, BWYV and CABYV isolates, highlighting the closer sequence homology within this region among Subgroup 2 luteoviruses. The dilution end-point for the detection of virus from infected material by radioactively labeled probes was 1:500, and about 250 fg of viral RNA could be detected from purified virions preparations. Non-radioactively labeled (digoxigenin [DIG]) probes were found to be 30-fold less sensitive than radioactive cRNA probes. Probe BM1 has potential for large-scale screening with applications in epidemiology and sugar beet-breeding programs. This report shows that heterogeneity at the 5’ proximal regions of the genomes of BMYV and BWYV offers the potential for discriminating between the two viruses and identifying the sugar beet—infecting BMYV isolates

Virus preparations from the mixed-infected P70 Pinot Noir accession exhibit GLRaV-1/GVA ‘end-to-end’ particles

P70 is a Pinot Noir grapevine accession that displays strong leafroll disease symptoms. A high-throughput sequencing (HTS)-based analysis established that P70 was mixed-infected by two variants of grapevine leafroll-associated virus 1 (GLRaV-1, genus Ampelovirus) and one of grapevine virus A (GVA, genus Vitivirus) as well as by two viroids (hop stunt viroid [HSVd] and grapevine yellow speckle viroid 1 [GYSVd1]) and four variants of grapevine rupestris stem pitting-associated virus (GRSPaV). Immunogold labelling using gold particles of two different diameters revealed the existence of ‘hybrid’ particles labelled at one end as GLRaV-1, with the rest labelled as GVA. In this work, we suggest that immunogold labelling can provide information about the biology of the viruses, going deeper than just genomic information provided by HTS, from which no recombinant or ‘chimeric’ GLRaV-1/GVA sequences had been identified in the dataset. Our observations suggest an unknown interaction between members of two different viral species that are often encountered together in a single grapevine, highlighting potential consequences in the vector biology and epidemiology of leafroll and rugose-wood diseases