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

摘要

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. IntroductionThe study of complex relationships between organisms, such as microorganisms, and their substrates, environment or one another can currently be accomplished using methods that are completely independent of classical culture techniques. Methods such as polymerase chain reaction (PCR) (van Belkum et al., 1998; Sachse, 2004), restriction fragment length polymorphism (RFLP) (Vaneechoutte, 1996), pyrosequencing (Petrosino et al., 2009) and, more recently, metagenomics (Miller et al., 2013; Ross-Davis et al., 2013), metatranscriptomics (McGrath et al., 2010) and other meta’omics methods (Lepage et al., 2013) represent new ways to study complex ecosystems and their interactions (Segata et al., 2013). The development of such methods has enabled the detection of specific organisms within a complex matrix.PCR has become a widely used technique with applications in all biological and medical fields. However, in most cases, PCR involves prior purification of nucleic acids. A direct PCR method, without any DNA purification steps, was previously developed to detect latent Botrytis cinerea in young grapevine berries (Gindro et al., 2005). This method, combined with microscopic analyses (Keller et al., 2003), enabled the study of the infection at the bloom stage and during the following latency period. These data provided new insights into the epidemiology of grey mould and confirmed the importance of protecting the grapevine from B. cinerea infections during the bloom stage.Among the approximately ten species of fungi that are considered to be major grapevine pathogens, three are responsible for most damage in vineyards: grey mould (B. cinerea), downy mildew (Plasmopara viticola) and powdery mildew (Erysiphe necator). A specific problem lies in the obligate biotrophic nature of P. viticola and E. necator, which prohibits their cultivation on artificial media, making their study much more complicated and time consuming. PCR amplification methods have been described for the identification or quantification of B. cinerea in air samples (Carisse et al., 2009), E. necator in wine grapes and must (Stummer et al., 2006) and air samples (Thiessen et al., 2013), and P. viticola in grapevine leaves (Valsesia et al., 2005). Several studies have successfully developed PCR reactions without prior DNA purification, relying upon lysis at the high temperature of amplification. For example, these techniques have been applied to whole bacterial cells (Gussow and Clackson, 1989), tobacco leaves and root pieces (Berthomieu and Meyer, 1991), fungal spores (Aufauvre-Brown et al., 1993) and in direct diagnostics of bacteria and viruses in animal tissues (Olive, 1989). However, it is known that the amplification reaction can be inhibited by different parameters such as excessively high cell numbers, cell wall fragments, DNA-binding proteins, polysaccharides, phenols, detergents or insufficient cell lysis (Moreira, 1998; Dewey and Yohalem, 2004). Plant tissues are very rich in polysaccha