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Over the last decades the use of pesticides in vine protection, e.g. copper is under severe discussion and is becoming a major concern in viticulture. Since the effectiveness of oxygenates against various microorganisms had been proven in the medical field a strategy for oxygenate-based plant protection was developed and evaluated over three vintages. The production of the oxygenate is following the Criegee-mechanism using O3 and unsaturated natural plant derived fatty acids forming so called ozonides. Therefore the effect of the treatment has been evaluated in a holistic approach, covering the efficiency against fungal diseases, protection of desired beneficial insects, the micro flora, various secondary metabolites of the grapevine and the resulting sensory profile of the wines. The biological effectiveness has been measured by using different in-vivo and in-vitro studies. The influence on desired berry compounds, e.g. anthocyanins, have been determined by classical GC-MS and HPLC methods. Positive effects against downy and powdery mildew could be demonstrated. No negative effects against insects, naturally occurring microorganisms, and desired berry compounds were observed. Even spontaneous fermentation was not inhibited. Quantitative descriptive sensory analysis as well as CATA/RATA showed no negative effect of the treatment.

Esca is a grapevine trunk disease (GTD) that is caused by filamentous fungi. It is responsible for considerable economic losses in viniculture on a global scale. Despite many unknown factors contributing to the development of symptoms in affected plants, Phaeoacremonium minimum (PMI), Phaeomoniella chlamydospora (PCH) and Fomitiporia mediterranea (FMED) are generally considered as the main causative fungal species. Early detection and specific identification of these pathogens therefore play an important role in disease control and evaluation of suitable countermeasures. In this study, loop‐mediated isothermal amplification (LAMP) assays were developed for each of the three pathogens. A genome‐based approach was applied for detection and selection of unique target DNA sequences. The designed primer sets showed overall good specificities, with some observed cross‐reactions towards closely related Phaeoacremonium species for the PMI primer set. The developed assays had detection limits of 100 pg (FMED, PMI) and 1 pg (PCH) per reaction (corresponding to 1460 [FMED]; 1950 [PMI]; 342 [PCH] genome copies per reaction). The application of the assays to field samples was demonstrated by testing individual infected grapevine trunks from two European viticultural regions using crude DNA obtained in a rapid sample preparation step. LAMP assay results matched those of PCR following a conventional DNA extraction protocol. The study showed that LAMP‐based rapid molecular detection of major Esca agents can serve as a useful tool for further research and surveillance of a highly devastating grapevine disease. The application of computer‐based whole genome comparison between target and non‐target species for the identification of unique target sequences as the basis for LAMP (or PCR) primer design was demonstrated to be a useful approach in species for which scarce sequence information is available. Moreover, the developed method for rapid DNA preparation from grapevine trunks may potentially be adapted to the DNA‐based detection also of other fungal species that cause grapevine trunk diseases. [ABSTRACT FROM AUTHOR]

In this study, an amplicon metagenomic approach was used to determine the effect of repeated treatments with ozonized oleic acid on the microbial community of grapevine carpoplane. Differences in community composition of treated vineyards were compared to non-treated and conventionally treated samples regarding the prokaryotic and eukaryotic microbiome at two developmental stages (BBCH 83, BBCH 87). The results showed effects both on occurrence and on abundance of microorganisms and the community assembly. Wine-relevant genera such as Acetobacter and members of the former genus Lactobacillus could be identified as part of the natural microbiota. The impact of the new viticultural treatment on these organisms was assessed in liquid culture-based microtiter assays. Therefore, we investigated an array of two acetic acid bacteria (AAB), four lactic acid bacteria (LAB) and nine saccharomyces and non-saccharomyces yeasts. Brettanomyces bruxellensis, Saccharomyces cerevisiae, Pediococcus sp. and Acetobacter aceti revealed the highest sensitivities against ozonized oleic acid (LIQUENSO® Oxygenat). Culture growth of these organisms was significantly reduced at an ozonide concentration of 0.25% (v/v), which corresponded to a quarter of the concentration used in the vineyard. The metabarcoding approach in combination with complementary in vitro assays allow new insights into treatment effects on the community and species scale. [ABSTRACT FROM AUTHOR]

Abstract Background The oomycete Plasmopara viticola (Berk. and Curt.) Berl. and de Toni causes downy mildew in grapevine (Vitis vinifera L.). This pathogen is strictly biotrophic, thus completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. We have carried out a large-scale cDNA-AFLP analysis to identify grapevine and P. viticola genes associated with the infection process. Results We carried out cDNA-AFLP analysis on artificially infected leaves of the susceptible cultivar Riesling at the oil spot stage, on water-treated leaves and on a sample of pure sporangia as controls. Selective amplifications with 128 primer combinations allowed the visualization of about 7000 transcript-derived fragments (TDFs) in infected leaves, 1196 of which (17%) were differentially expressed. We sequenced 984 fragments, 804 of which were identified as grapevine transcripts after homology searching, while 96 were homologous to sequences in Phytophthora spp. databases and were attributed to P. viticola. There were 82 orphan TDFs. Many grapevine genes spanning almost all functional categories were downregulated during infection, especially genes involved in photosynthesis. Grapevine genes homologous to known resistance genes also tended to be repressed, as were several resistance gene analogs and carbonic anhydrase (recently implicated in pathogen resistance). In contrast, genes encoding cytoskeletal components, enzymes of the phenylpropanoid and beta-oxidation pathways, and pathogenesis related proteins were primarily upregulated during infection. The majority of P. viticola transcripts expressed in planta showed homology to genes of unknown function or to genomic Phytophthora sequences, but genes related to metabolism, energy production, transport and signal transduction were also identified. Conclusion This study provides the first global catalogue of grapevine and P. viticola genes expressed during infection, together with their functional annotations. This will help to elucidate the molecular basis of the infection process and identify genes and chemicals that could help to inhibit the pathogen.

Stilbenes, as important secondary metabolites of grapevine, represent central phytoalexins and therefore constitute an important element of basal immunity. In this study, potential genetic variation in Vitis vinifera ssp. sylvestris, the ancestor of cultivated grapevine, was sought with respect to their output of stilbenes and potential use for resistance breeding. Considerable variation in stilbene inducibility was identified in V. vinifera ssp. sylvestris. Genotypic differences in abundance and profiles of stilbenes that are induced in response to a UV-C pulse are shown. Two clusters of stilbene 'chemovars' emerged: one cluster showed quick and strong accumulation of stilbenes, almost exclusively in the form of non-glycosylated resveratrol and viniferin, while the second cluster accumulated fewer stilbenes and relatively high proportions of piceatannol and the glycosylated piceid. For all 86 genotypes, a time dependence of the stilbene pattern was observed: piceid, resveratrol, and piceatannol accumulated earlier, whereas the viniferins were found later. It was further observed that the genotypic differences in stilbene accumulation were preceded by differential accumulation of the transcripts for chalcone synthase (CHS) and stilbene-related genes: phenylalanine ammonium lyase (PAL), stilbene synthase (StSy), and resveratrol synthase (RS). A screen of the population with respect to susceptibility to downy mildew of grapevine (Plasmopara viticola) revealed considerable variability. The subpopulation of genotypes with high stilbene inducibility was significantly less susceptible as compared with lowstilbene genotypes, and for representative genotypes it could be shown that the inducibility of stilbene synthase by UV correlated with the inducibility by the pathogen. [ABSTRACT FROM AUTHOR]

Grapevine (Vitis vinifera ssp. vinifera) is one of the most important fruit species; however, it is highly susceptible to various pathogens, which can cause severe crop losses in viticulture. It has been shown that several WRKY class transcription factors (TFs) are part of the signal transduction cascade, which leads to the activation of plant defense reactions against various pathogens. In the present investigation, a full‐length cDNA was isolated from V. vinifera leaf tissue encoding a predicted protein, designated VvWRKY33, which shows the characteristics of group I WRKY protein family. VvWRKY33 induction correlates with the expression of VvPR10.1 (pathogenesis‐related 10.1) gene in the leaves of the resistant cultivar ‘Regent’ after infection with Plasmopara viticola, whereas in the susceptible cultivar ‘Lemberger’ VvWRKY33 and VvPR10.1 are not induced. Corresponding expression of the TF and VvPR10.1 was even obtained in uninfected ripening berries. In planta, analysis of VvWRKY33 has been performed by ectopic expression of VvWRKY33 in grapevine leaves of greenhouse plants mediated via Agrobacterium tumefaciens transformation. In consequence, VvWRKY33 strongly increases resistance to P. viticola in the susceptible cultivar ‘Shiraz’ and reduces pathogen sporulation of about 50–70%, indicating a functional role for resistance in grapevine. Complementation of the resistance‐deficient Arabidopsis thaliana Columbia‐0 (Col‐0) mutant line wrky33‐1 by constitutive expression of VvWRKY33 restores resistance against Botrytis cinerea to wild‐type level and in some complemented mutant lines even exceeds the resistance level of the parental line Col‐0. Our results support the involvement of VvWRKY33 in the defense reaction of grapevine against different pathogens. [ABSTRACT FROM AUTHOR]

Hot water treatment (HWT) applied on dormant woody plant material to control Grapevine Trunk Diseases (GTD) showed variable results in previous studies. In this project, a field experiment was carried out to investigate the effect of HWT on GTD pathogens (Phaeomoniella chlamydospore (Pch), Phaeoacremonium minimum (Pmi), Botryosphaeriaceae (BOT) species). Inoculated wood cuttings were treated in a hot water tank under practical conditions, grafted onto healthy rootstocks and planted in nurseries. Pathogen development was analysed in wood samples at distinct time points after treatment. Pch and BOT could be successfully eliminated through HWT. However, only a reduced infection by Pmi was observed in the treated samples. To determine the influence of HWT on the growth of Trichoderma and its long-term antagonistic effect following HWT on GTD pathogens, a commercially available biocontrol agent product, Vintec®, [Trichoderma atroviride strain SC1 (TASC1), Belchim Crop Protection Deutschland GmbH] was tested. No negative impact of HWT could be detected on the growth of TASC1, and thus its antagonistic ability against the GTD pathogens was also not affected. Heat tolerance of the distinct developmental stages of each GTD pathogens was assessed at different temperature-time combinations under laboratory conditions. In general, Pch and BOT were more sensitive to HWT than Pmi at both tested developmental stages. Pmi showed reduced sensitivity to HWT at the ungerminated spore stage. Conidial germination was not inhibited following an incubation of the spore suspension for 45 minutes at 50°C, whereas no survival of treated mycelia of Pmi could be detected. These results suggest that HWT can be a useful tool to reduce infections with GTD pathogens in propagation material without affecting the antagonistic effects of TASC1. [ABSTRACT FROM AUTHOR]

European grapevine cultivars (Vitis vinifera spp.) are highly susceptible to the downy mildew pathogen Plasmopara viticola. To reduce the dependence of viticulture on chemical inputs, and thereby reduce the ecological and economic burden of wine production, a number of breeding programs have introgressed resistance loci from wild North American and Asian Vitis species into V. vinifera resulting in new fungus-resistant grapevine cultivars (FRCs). These FRCs are a promising strategy to reduce the impact of disease management. Due to variety-specific resistance differences, little is known about the required amount and the potential reduction of fungicide applications. The aim of the project was to investigate the degree of resistance of new FRCs and thus the capability of fungicide reduction in the vineyard over a 6 year time period. For this purpose, the infection and sporulation ability as well as the development of P. viticola in different FRCs and susceptible cultivars were investigated and compared. Additionally, FRCs with reduced plant protection treatments were compared to traditional cultivars. On the basis of the results obtained, adjusted plant protection recommendations for FRCs were developed. The on-farm experiments showed that the use of FRCs in combination with reduced plant protection management strategies offers the possibilities to significantly reduce the number of fungicide treatments required for grape production. Results obtained from this study demonstrated that the deployment of FRCs can save 50-85 % of fungicide applications in viticulture depending on the degree of the vairiety's resistance level. The omission of all plant protection applications can ultimately lead to negative effects on yield, quality and even resistance durability. The latter was demonstrated by the identification of new P. viticola isolates capable of overcoming Rpv3- and Rpv12-mediated resistance. Therefore, this study demonstrates the importance of sustainable breeding and crop protection strategies. [ABSTRACT FROM AUTHOR]

The high susceptibility of European grapevine varieties (Vitis vinifera) to downy mildew (Plasmopara viticola) leads to intensive use of fungicides in viticulture. To reduce the use of fungicides, resistance loci from wild Vitis species have been crossed into Vitis vinifera varieties. The resulting new fungusresistant grapevine cultivars (FRCs) represent an important tool for reducing pesticide applications in viticulture. Due to variety-specific resistance differences, little is known about the type and timing of the plant defense responses mediated by different resistance loci. Since the long-term goal of resistance breeding is to pyramidize several resistance loci, these should be based on different resistance mechanisms to increase the durability of the resistance. Therefore, detailed knowledge of the different defense mechanisms and resistance genes conferred by the respective Rpv-loci is essential to ensure sustainable resistance management and durable as well as stable resistance breeding. Therefore, the resistance mechanisms mediated by the Rpv10-, Rpv3-and/or Rpv12-loci on downy mildew development, sporulation ability, onset of programmed cell death (PCD), production of hydrogen peroxide and stilbene levels were evaluated and compared. Furthermore, resistancebreaking isolates were used as a tool to additionally evaluate whether the resistance loci are based on different mechanisms. In order to understand the mechanistic basis of the defense responses mediated by the Rpv12-locus, cultivars containing this locus were examined in more detail. The experiments revealed an early and locally precise defense response in Rpv10-, Rpv12- and Rpv12/Rpv3-genotypes, whereas a delayed defense response in Rpv3-genotypes was observed. These temporal differences correlated with an increase in the trans-resveratrol level and the formation of hydrogen peroxide shortly before onset of PCD. The differences in timing of onset of Rpv-loci specific defense reactions following downy mildew infection could be responsible for the observed differences in hyphal growth, sporulation and cultivar-specific susceptibility to this pathogen in the vineyard. Regarding the Rpv12-locus described first by Venuti et al. 2013, we were able to further narrow down the region, which confers resistance by new resistance markers. In this reduced region 10 putative disease resistance R-genes were found and are analyzed bioinformatically. [ABSTRACT FROM AUTHOR]