A multiplex nested-PCR assay for sensitive and simultaneous detection and direct identification of phytoplasma in the Elm yellows group and Stolbur group and its use in survey of grapevine yellows in France

摘要

Flavescence doree and Bois noir (or Vergilbungs-krankheit), are two main yellows diseases of grapevines in Europe. The two diseases cannot be distinguished on the basis of symptoms but they are associated with two different phytoplasmas which belong to the Elm yellows (16SrV) group and Stolbur (16SrⅫ) group, respectively. Their spreading areas are overlapping in France, Italy and Spain but they have different vector insects. Flavescence doree is an epidemic disease and a quarantine organism. National surveys conducted annually in France require straightforward and sensitive assays to detect phytoplasma that sometimes occur in grapevine with a low titre and to characterize them readily. A bi-specific multiplex nested-PCR procedure was developed, to amplify simultaneously two non-ri-bosomal DNA fragments, 1150 bp and 720 bp in length, specific for Elm yellows-group and Stolbur-group phytoplasmas, respectively. They were identified using agarose gel electrophoresis of amplification products. The procedure is quick, sensitive and reliable. It was used on 2,525 grapevine samples from the field, in the frame of the French survey in 2002. Mixed samples containing both phytoplasmas displayed a mixed profile in the gel. It was confirmed that the nested-PCR amplimer obtained in the FD9 DNA region with Elm yellows-group phytoplasmas, though shorter than the initial FD9 fragment, nevertheless contained the restriction sites that permit the RFLP identification of geographic phytoplasma isolates already characterized in former studies.