Development of rapid direct PCR assays to identify downy and powdery mildew and grey mould in <em>Vitis vinifera</em> tissues

Abstract

Aims: The development of a rapid and reliable direct PCR method to detect fungal propagules in grapevine tissues without prior DNA purification steps, and illustration of its potential use with different examples. Methods and results: Different grapevine samples crushed in the presence of polyvinylpolypyrrolidone (PVPP) were used as templates for direct PCR amplification with primers specifie for Erysiphe necator, Plasmopara viticola, Botrytis cinerea and Vitis vinifera. Sequencing of the PCR products confirmed the specificity of the amplifications. The sensitivity tested using conidia/sporangia dilution series was high, ranging from five sporangia for P. viticola to one conidium for E. necator. The potential of this technique is illustrated through the study of four epidemiological questions. Fungal propagules were observed in dormant buds using microscopy, but the responsible species could not be identified. Direct PCR revealed the presence of E. necator and B. cinerea in 29 % and 65 % of the buds, respectively. Downy mildew could be detected in asymptomatic leaves sampled in fields after potentially infectious events. In bunch, microscopic analysis of rachis sections showed the presence of hyphae growing in the green tissue. Direct PCR identified the presence of P. viticola. Conclusion: A direct PCR method without DNA purification was demonstrated to be a simple and reliable method for the detection and identification of fungal pathogens in grapevine tissues. This method, together with microscopy, is a very interesting tool that can be used to study various epidemiological problems in the grapevine, including important unanswered questions such as the route of infection that leads to brown rot caused by downy mildew. Significance and impact of the study: Direct PCR was shown to be a simple and versatile technique for the study of epidemiological questions in the grapevine. This technique could be extended to other pathosystems with minor adaptations.

Keywords

• Plasmopara viticola
• Erysiphe necator
• Botrytis cinerea
• electron microscopy
• direct PCR