The diagnosis of novel unidentified viral plant diseases can be problematic, as the conventional methods such as real‐time PCR or ELISA may be too specific to a particular species or even strain of a virus, whilst alternatives such as electron microscopy (EM) or sap inoculation of indicator species do not usually give species level diagnosis. Next‐generation sequencing (NGS) offers an alternative solution where sequence is generated in a non‐specific fashion and identification is based on similarity searching against GenBank. The conventional and NGS techniques were applied to a damaging and apparently new disease of maize, which was first identified in Kenya in 2011. ELISA and TEM provided negative results, whilst inoculation of other cereal species identified the presence of an unidentified sap transmissible virus. RNA was purified from material showing symptoms and sequenced using a Roche 454 GS‐FLX+. Database searching of the resulting sequence identified the presence of Maize chlorotic mottle virus and Sugarcane mosaic virus, a combination previously reported to cause maize lethal necrosis disease. Over 90% of both viral genome sequences were obtained, allowing strain characterization and the development of specific real‐time PCR assays which were used to confirm the presence of the virus in material with symptoms from six different fields in two different regions of Kenya. The availability of these assays should aid the assessment of the disease and may be used for routine diagnosis. The work shows that next‐generation sequencing is a valuable investigational technique for rapidly identifying potential disease‐causing agents such as viruses.
Ullucus tuberosus (ulluco) is a tuber‐forming species that has become a novel crop in highland and temperate maritime climates. Eight viruses have been previously reported infecting Ullucus, including Andean potato latent virus (APLV), a quarantine virus within the European Union. No reference sequences have been published for the viruses previously described from U. tuberosus. Plants grown in the UK for the internet trade were tested for the presence of quarantine viruses using ELISA and real‐time RT‐PCR. ELISA positive results were obtained for APLV and multiple other viruses. A similar suite of viruses was detected at a second outbreak site linked to horticultural trade. Virus identification was by high‐throughput sequencing (HTS) using a ribosomal RNA (rRNA)‐depleted total RNA approach. Analysis of viral contigs indicated the presence of several novel viruses closely related to, but not consistent with, the viruses indicated by ELISA. Further confirmatory testing by real‐time RT‐PCR indicated that two tymoviruses, tentatively named Ullucus tymovirus 1 and Ullucus tymovirus 2, were more closely related to each other (85% identity), than to APLV or Andean potato mild mosaic virus (63–66% identity). APLV could not be confirmed from either site by either HTS or PCR. A novel tobamovirus (Ullucus tobamovirus 1) was only detected at the initial outbreak site. A novel polerovirus (Ullucus polerovirus 1) and a distinct genotype of Papaya mosaic virus were detected from both outbreak sites. Deploying HTS during a plant health outbreak demonstrates the potential of this approach to give rapid, accurate diagnosis.
A new potyvirus has been found in canna. A 1700-nucleotide region at the 3' end of the genomic RNA has been sequenced from two isolates. The sequence reveals the virus to be a distinct member of the genus Potyvirus but most closely related to Johnsongrass mosaic virus. A specific primer pair was designed that enabled canna material to be screened specifically for this virus. The virus was consistently found in cannas showing severe virus symptoms. This virus has been found in different canna varieties from the UK, Belgium, Netherlands, France and Israel. The name Canna yellow streak virus (CaYSV) has been proposed for this new virus.
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