PCR detection of Xanthomonas fragariae and studies on its survival
1) A PCR technique (nested PCR) and specific primers have been developed in Florida (US) for the detection of Xanthomonas fragariae (EPPO A2 quarantine pest). With this technique, bacteria can be detected in either symptomatic or asymptomatic strawberry tissues. The authors felt that their technique is sufficiently sensitive to provide a useful tool for the detection of X. fragariae in asymptomatic plants in nurseries for certification purposes or in consignments moving in trade. This method was also used to study the survival of the bacterium in nurseries during summer in Florida. For this purpose, strawberry plants were inoculated with a rifampicin-resistant strain of X. fragariae and planted in the field. Bacteria were detected by PCR and by recovery onto growing media, at 2 weeks interval during 92 days after planting. Recovery of the rifampicin-marked strain from some samples indicated that the bacteria could remain viable throughout the summer. During summer, it was observed that the number of positive samples declined and then increased when more favourable conditions occurred (cooler temperatures). Authors stressed the importance of starting a new crop with plants free of X. fragariae. In addition, bacteria were also detected in daughter plants. Dissemination could be due either to systemic movement through vascular system of runners or to mechanical means.
2) Another new PCR method (rep-PCR) has been developed in California (US) to detect Xanthomonas fragariae (EPPO A2 quarantine pest). The aim was to provide a rapid and accurate identification of the pathogen, as pathogenicity testing is slow and ELISA may give some false positive results. The authors have found that the isolation of the bacterium could be improved by using a culture medium (PDM) similar to that used for Xylella fastidiosa. PCR primers which anneal to dispersed repetitive bacterial sequences (rep-PCR), genomic fingerprints of reference strains of X. fragariae were generated. These fingerprints where then used to identify X. fragariae in strawberry plants collected in Californian nurseries over the last 5 years. This method was found to be rapid (1 week) and accurate, as all field isolates which tested positive were also pathogenic to strawberry. The authors concluded that rep-PCR is a useful tool, especially for the production of pathogen-free strawberry planting material in nurseries.
Sources
Roberts, P.D.; Jones, J.B.; Chandler, C.K.; Stall, R.E.; Berger, R.D. (1996) Survival of Xanthomonas fragariae on strawberry in summer nurseries in Florida detected by specific primers and nested polymerase chain reaction.
Plant Disease, 80(11), 1283-1288.
Opgenorth, D.C.; Smart, C.D.; Louws, F.J.; de Bruijn, F.J.; Kirkpatrick, B.C. (1996) Identification of Xanthomonas fragariae field isolates by rep-PCR genomic fingerprinting.
Plant Disease, 80(8), 868-873.