Kaolin Particle Film Protects Grapevine cv. Cabernet Sauvignon Against Downy Mildew by Forming Particle Film at the Leaf Surface, Directly Acting on Sporangia and Inducing the Defense of the Plant

Downy mildew is a major threat to viticulture, leading to severe yield loss. The use of traditional copper-based fungicides is effective, but has adverse effects on the environment and human health, making it urgent to develop an environmentally friendly disease management program. Multi-functional kaolin particle film (KPF) is promising as an effective and safer treatment strategy, since this material lacks chemically active ingredients. In this study, ability of Kaolin particle film (KPF) pretreatment to protect grapevine leaves from Plasmopara viticola was tested and the mode of action of KPF was analyzed. KPF application reduced the disease severity and the development of intercellular hyphae. Additionally, there was reduced accumulation of H2O2 and malondialdehyde (MDA) with pretreatment. The observation of ultrastructure on the leaf surface showed KPF deposition and stomatal obstruction, indicating that KPF protected plants against disease by preventing the adhesion of pathogens to the leaf surface and blocking invasion through the stomata. KPF pretreatment also activated host defense responses, as evidenced by increased activities of anti-oxidative enzymes [superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)] and defense-related enzymes [phenylalanine ammonia-lyase (PAL), chitinases, and β-1,3-glucanases], increased phytohormone signals [abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA)] and the up-regulation of defense genes related to plant defense. Overall, these results demonstrate that KPF treatment counters grapevine downy mildew by protecting leaves and enhancing plant defense responses.

High-Throughput Phenotyping of Leaf Discs Infected with Grapevine Downy Mildew Using Shallow Convolutional Neural Networks

Objective and standardized recording of disease severity in mapping crosses and breeding lines is a crucial step in characterizing resistance traits utilized in breeding programs and to conduct QTL or GWAS studies. Here we report a system for automated high-throughput scoring of disease severity on inoculated leaf discs. As proof of concept, we used leaf discs inoculated with Plasmopara viticola ((Berk. and Curt.) Berl. and de Toni) causing grapevine downy mildew (DM). This oomycete is one of the major grapevine pathogens and has the potential to reduce grape yield dramatically if environmental conditions are favorable. Breeding of DM resistant grapevine cultivars is an approach for a novel and more sustainable viticulture. This involves the evaluation of several thousand inoculated leaf discs from mapping crosses and breeding lines every year. Therefore, we trained a shallow convolutional neural-network (SCNN) for efficient detection of leaf disc segments showing P. viticola sporangiophores. We could illustrate a high and significant correlation with manually scored disease severity used as ground truth data for evaluation of the SCNN performance. Combined with an automated imaging system, this leaf disc-scoring pipeline has the potential to considerably reduce the amount of time during leaf disc phenotyping. The pipeline with all necessary documentation for adaptation to other pathogens is freely available.

High-throughput phenotyping of leaf discs infected with grapevine downy mildew using shallow convolutional neural networks

Objective and standardized recording of disease severity in mapping crosses and breeding lines is a crucial step in characterizing resistance traits utilized in breeding programs and to conduct QTL or GWAS studies. Here we report a system for automated high-throughput scoring of disease severity on inoculated leaf discs. As proof of concept, we used leaf discs inoculated with Plasmopara viticola causing grapevine downy mildew (DM). This oomycete is one of the major grapevine pathogens and has the potential to reduce grape yield dramatically if environmental conditions are favorable. Breeding of DM resistant grapevine cultivars is an approach for a novel and more sustainable viticulture. This involves the evaluation of several thousand inoculated leaf discs from mapping crosses and breeding lines every year. Therefore, we trained a shallow convolutional neural-network (SCNN) for efficient detection of leaf disc segments showing P. viticola sporangiophores. We could illustrate a high and significant correlation with manually scored disease severity used as ground truth data for evaluation of the SCNN performance. Combined with an automated imaging system, this leaf disc-scoring pipeline has the potential to reduce the amount of time during leaf disc phenotyping considerably. The pipeline with all necessary documentation for adaptation to other pathogens is freely available.

Sequence and Gene Expression Analysis of Recently Identified NLP from Plasmopara viticola

Grapevine downy mildew, evoked by the obligate biotrophic oomycete Plasmopara viticola, is one of the most challenging diseases in viticulture. P. viticola establishes an infection by circumvention of plant immunity, which is achieved by the secretion of effector molecules. One family of potential effectors are the necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP). NLP are most abundant in plant pathogenic microorganisms and exist in cytotoxic and non-cyctotoxic forms. Cytotoxic NLP often act as virulence factors and are synthesized in necrotrophic or hemibiotrophic pathogens during the transition from biotrophic to necrotrophic growth. In addition to these cytotoxic NLP, many non-cytotoxic NLP have been identified; their function in biotrophic pathogens is still unknown. In 2020, eight different NLP coding genes were identified in P. viticola and named PvNLP1 to PvNLP8 (Plasmopara viticola NLP 1–8). In the present study, PvNLP4 to PvNLP8 were characterized by using qPCR analysis and transient expression in the model plant Nicotiana benthamiana. Gene expression analysis showed high PvNLP expression during the early stages of infection. Necrosis-inducing activity of PvNLP was not observed in the nonhost N. benthamiana.

Transcriptional profiling reveals multiple defense responses in downy mildew-resistant transgenic grapevine expressing a TIR-NBS-LRR gene located at the MrRUN1/MrRPV1 locus

Grapevine downy mildew (DM) is a destructive oomycete disease of viticulture worldwide. MrRPV1 is a typical TIR-NBS-LRR type DM disease resistance gene cloned from the wild North American grapevine species Muscadinia rotundifolia. However, the molecular basis of resistance mediated by MrRPV1 remains poorly understood. Downy mildew-susceptible Vitis vinifera cv. Shiraz was transformed with a genomic fragment containing MrRPV1 to produce DM-resistant transgenic Shiraz lines. Comparative transcriptome analysis was used to compare the transcriptome profiles of the resistant and susceptible genotypes after DM infection. Transcriptome modulation during the response to P. viticola infection was more rapid, and more genes were induced in MrRPV1-transgenic Shiraz than in wild-type plants. In DM-infected MrRPV1-transgenic plants, activation of genes associated with Ca2+ release and ROS production was the earliest transcriptional response. Functional analysis of differentially expressed genes revealed that key genes related to multiple phytohormone signaling pathways and secondary metabolism were highly induced during infection. Coexpression network and motif enrichment analysis showed that WRKY and MYB transcription factors strongly coexpress with stilbene synthase (VvSTS) genes during defense against P. viticola in MrRPV1-transgenic plants. Taken together, these findings indicate that multiple pathways play important roles in MrRPV1-mediated resistance to downy mildew.

The Durability of Quantitative Host Resistance and Variability in Pathogen Virulence in the Interaction Between European Grapevine Cultivars and Plasmopara viticola

European grapevine, Vitis vinifera, carries no major resistances against Plasmopara viticola, the causal agent of grapevine downy mildew. The introgression of quantitative trait loci conferring resistance to P. viticola (Rpv) from American and Asian donor species has resulted in a range of resistant cultivars. In light of the perennial nature of grapevine and the high evolutionary potential of P. viticola, the durability of this quantitative resistance is an important challenge. Durability of host resistance and variability in pathogen virulence may be evaluated by describing interactions between pathogen isolates and grapevine cultivars in terms of Rpv loci. A set of 16 cultivars carrying different combinations of Rpv loci, was challenged with five P. viticola isolates, obtained from susceptible or Rpv3.1+V. vinifera cultivars. Based on the severity of sporulation, different host and pathogen phenotypes might be distinguished, which could be related to the presence of different Rpv loci. The hormonal responses before and during some interactions were compared to assess the resistance mechanisms underlying Rpv3.1, Rpv10, and Rpv12 and the infection mechanisms of the different isolates. This paper reports on the strength of some of the commonly used Rpv loci, single or stacked. The isolates derived from Rpv3.1+ hosts, GREPv1 and GPHPv1, were able to sporulate intensely on cultivars carrying Rpv3.1, without triggering necrosis. Moreover, Rpv10 was not able to efficiently halt the development of the Rpv3.1-breaking isolate GPHPv1. Cultivars carrying Rpv12, however, were resistant to all five P. viticola isolates. Phytohormones might be implicated in the basal resistance against this pathogen, but during the early defense response, no significant hormonal responses to the isolates were observed. The isolate-specificity of the Rpv3- and Rpv10-mediated resistance suggests that these loci do not result in the most sustainable resistance. Furthermore, the isolate-specific behavior of the pathogen emphasizes the need for a characterization system for P. viticola. A standardized phenotyping assay may be used to determine P. viticola pathogen phenotypes or measure the durability, strength, and isolate-specificity of the host quantitative resistances. The characterization of both components of the pathosystem may lead to an increased understanding of the resistance mechanisms, beneficial for a durable deployment of resistance genes.