NEW SEQUENCE-BASED TECHNOLOGIES FOR THE DETECTION OF PLANT PATHOGENS: FOCUS ON PSEUDOMONAS SYRINGAE PV. ACTINIDIAE
Rapid and accurate identification of unwanted microorganisms is needed for disease management and border biosecurity. Traditional isolation and identification techniques are time consuming and rely on prior knowledge of the disease-causing agent. Next generation sequencing technologies offer an alternative for the rapid detection of pathogens in a wide range of samples. To trial this method we have used amplicon pyrosequencing to identify Pseudomonas spp. in kiwifruit (Actinidia deliciosa Hayward and A. chinensis Hort16A) leaf lesions. An important question within this study was whether we could distinguish P. syringae pv. actinidiae (Psa) in such samples. Kiwifruit leaf samples were pre-screened with microbiological and quantitative PCR (qPCR) techniques to determine the presence of the low virulent Psa-LV (biovar 4), or the outbreak virulent Psa-V (biovar 3). Ribosomal 16S and 16S-23S internal transcribed spacer (ITS) regions were PCR amplified from selected samples and sequenced using Roche 454 technology. The resulting reads were filtered based on sequence quality, error corrected, and clustered into operational taxonomic units (OTUs) before taxonomy assignment. Samples of DNA from a mixture of Pseudomonas species, with varying amounts of Psa-V DNA, were examined as controls. The ITS region provided better segregation of the Pseudomonas species into separate OTUs compared with the 16S region. Psa-V was identified in all control samples containing Psa-V DNA, regardless of the concentration. Using this technique, Psa was identified in orchard samples that were shown by traditional methods to be infected. We could also differentiate between orchards that were infected with Psa-V or with Psa-LV. A range of P. syringae pathovars were found to be associated with Actinidia leaves, as well as a range of Pantoea, Burkholderia, and Methylobacterium species. The use of regions with greater variation, such as the ITS, enabled closely related species to be differentiated.
Frampton, R.A., Beard, S.S. and Bulman, S.R. (2015). NEW SEQUENCE-BASED TECHNOLOGIES FOR THE DETECTION OF PLANT PATHOGENS: FOCUS ON PSEUDOMONAS SYRINGAE PV. ACTINIDIAE. Acta Hortic. 1095, 81-88
amplicon sequencing, 16S, ITS