Loop-mediated isothermal amplification assay for the detection of Plasmopara viticola infecting grapes

Grapes downy mildew caused by obligate oomycete plant pathogen Plasmopara viticola is a devastating disease worldwide, resulting in significant yield and quality losses. A field survey was conducted in two major grapes cultivated areas of Tamil Nadu for the incidence of grapevine downy mildew. The disease incidence was 43.42%–76.69%, and the highest disease incidence of 76.69% was observed in the Theni district. Totally eight P. viticola isolates were collected from different places in Coimbatore and Theni districts. These isolates were confirmed through microscopic observation and sequencing of COX 2 gene, and the phylogenetic tree was developed to study their phylogenetic relationship among the isolates which shows 97–100% sequence similarity with other P. viticola isolates and less sequence similarity with Plasmopara species. The loop-mediated isothermal amplification (LAMP) assay was developed based on the CesA4 gene sequence of P. viticola. The assay developed was more sensitive as it detected P. viticola genomic DNA up to 20 fmg. LAMP assay specificity was proved by carrying out the assay with genomic DNA extracted from other Oomycetes and fungal plant pathogens. Finally, LAMP assay was validated by testing seventy-eight grapevine leaf samples collected from seven different locations. LAMP assay showed a positive reaction in sixty-two samples tested out of seventy-eight samples tested. Therefore, the LAMP assay described should helpful for early and specific detection of downy mildew pathogen and help in mitigating disease incidence.     

Breakdown of resistance to grapevine downy mildew upon limited deployment of a resistant variety

Background  

Natural disease resistance is a cost-effective and environmentally friendly way of controlling plant disease. Breeding programmes need to make sure that the resistance deployed is effective and durable. Grapevine downy mildew, caused by the Oomycete Plasmopara viticola, affects viticulture and it is controlled with pesticides. Downy mildew resistant grapevine varieties are a promising strategy to control the disease, but their use is currently restricted to very limited acreages. The arising of resistance-breaking isolates under such restricted deployment of resistant varieties would provide valuable information to design breeding strategies for the deployment of resistance genes over large acreages whilst reducing the risks of the resistance being defeated. The observation of heavy downy mildew symptoms on a plant of the resistant variety Bianca, whose resistance is conferred by a major gene, provided us with a putative example of emergence of a resistance-breaking isolate in the interaction between grapevine and P. viticola.     

Isolation and characterization of Bacillus subtilis KS1 for the biocontrol of grapevine fungal diseases

Bacillus subtilis KS1 was isolated from grape berry skin as a biological control agent against grapevine fungal diseases. KS1 was identified as a new strain of B. subtilis according to morphological, biochemical, and genetic analyses. In vitro bioassay demonstrated that KS1 suppressed the growth of Botrytis cinerea (the casual agent of grape grey mold) and Colletotrichum gloeosporioides (the casual agent of grape ripe rot). The biocontrol activity of KS1 against grapevine fungal diseases in vineyards was evaluated over a 3-year span (from 2007 to 2009). Downy mildew, caused by Plasmopara viticola, was reduced on berry skins and leaves by treatment with KS1. The KS1 genome possesses ituD and lpa-14 genes, both of which play a role in iturin A production followed by iturin A production in the culture. In contrast, mutants lacking both genes lost the antagonistic activity against B. cinerea and C. gloeosporioides and the activity in iturin A production, suggesting that the antagonistic activity of KS1 against grapevine fungal pathogens may depend on iturin A production. As KS1 showed tolerance to various chemical pesticides, chemical pesticides could be applied before and/or after KS1 treatment in vineyards. Due to its potential as a biological control agent against grape downy mildew, KS1 is expected to contribute to the further improvement of integrated pest management systems and to potentially reduce the amount of chemical fungicides applied in vineyards.     

Grapevine VpPR10.1 functions in resistance to Plasmopara viticola through triggering a cell death‐like defence response by interacting with VpVDAC3

As one of the most serious diseases in grape, downy mildew caused by Plasmopara viticola is a worldwide grape disease. Much effort has been focused on improving susceptible grapevine resistance, and wild resistant grapevine species are important for germplasm improvement of commercial cultivars. Using yeast two‐hybrid screen followed by a series of immunoprecipitation experiments, we identified voltage‐dependent anion channel 3 (VDAC3) protein from Vitis piasezkii ‘Liuba‐8’ as an interacting partner of VpPR10.1 cloned from Vitis pseudoreticulata ‘Baihe‐35‐1’, which is an important germplasm for its resistance to a range of pathogens. Co‐expression of VpPR10.1/VpVDAC3 induced cell death in Nicotiana benthamiana, which accompanied by ROS accumulation. VpPR10.1 transgenic grapevine line showed resistance to P. viticola. We conclude that the VpPR10.1/VpVDAC3 complex is responsible for cell death‐mediated defence response to P. viticola in grapevine.     

Identification of a Vitis vinifera endo‐β‐1,3‐glucanase with antimicrobial activity against Plasmopara viticola

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis‐related (PR) proteins. Endo‐β‐1,3‐glucanases (EGases) belong to the PR‐2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti‐P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR‐2 family from Vitis.     

Surfactin and fengycin contribute to the protection of a Bacillus subtilis strain against grape downy mildew by both direct effect and defence stimulation

Bacillus subtilis GLB191 (hereafter GLB191) is an efficient biological control agent against the biotrophic oomycete Plasmopara viticola, the causal agent of grapevine downy mildew. In this study, we show that GLB191 supernatant is also highly active against downy mildew and that the activity results from both direct effect against the pathogen and stimulation of the plant defences (induction of defence gene expression and callose production). High-performance thin-layer chromatography analysis revealed the presence of the cyclic lipopeptides fengycin and surfactin in the supernatant. Mutants affected in the production of fengycin and/or surfactin were thus obtained and allowed us to show that both surfactin and fengycin contribute to the double activity of GLB191 supernatant against downy mildew. Altogether, this study suggests that GLB191 supernatant could be used as a new biocontrol product against grapevine downy mildew.     

Exploiting medicinal plants and phylloplane microflora for the management of grapevine downy mildew

Abstract

Grapevine downy mildew is the most devastating disease throughout the world causing huge monetary losses. Twenty medicinal plant extracts and six phylloplane microfloras were evaluated for their efficacy against sporangial germination of grapevine downy mildew pathogen Plasmopara viticola in vitro. The results revealed that the Neem Seed Kernel Extract (NSKE) at 5% significantly inhibited the sporangial germination (75.36%) of P. viticola. Among the phylloplane microflora Pseudomonas fluorescens was highly effective in reducing the sporangial germination (64.26%). Post inoculation spraying of NSKE (5%) and P. fluorescens (0.2%) effectively inhibited the disease development in the greenhouse. Three sprays with NSKE (5%) and phylloplane P. fluorescens (0.2%): first spray after initial appearance of disease and the second and third at 10 day intervals were found to be promising in reducing disease incidence in the field.     

Control of Downy Mildew, Plasmopara viticola (de Bary) Berlese et de Toni, on Grapevine Leaves through Water Extracts from Composted Organic Wastes

Watery extracts of composted manure-straw-soil mixtures induced increased resistance of grapevine leaves against downy mildew, Plasmopara viticola, if applied by dipping or spraying. The extracts had no direct fungicidal or fungitoxic effects.     

葡萄生单轴霉菌围可培养微生物多样性及潜在生防菌研究

【目的】揭示葡萄生单轴霉(Plasmoparaviticola)菌围可培养细菌和真菌的多样性特征,筛选对葡萄霜霉病有较强稳定防治效果的生防菌。【方法】连续两年从我国南北方具有代表性的7个葡萄产区采集葡萄霜霉病叶,镊子夹取经保湿培养获得的新鲜霉层并配制孢子囊悬浮液,采用传统分离培养法,结合形态分类、BOX-PCR指纹图谱分析以及分子鉴定结果,对葡萄生单轴霉菌围的可培养细菌和真菌进行聚类分析;采用菌株及其发酵液与病原菌孢子囊悬浮液等体积混合培养测定其对孢子囊的抑制作用,离体叶片接种法检测该菌株及其发酵液对霜霉病的防治效果。【结果】分离获得了90株细菌和110株真菌,分别归属于8个细菌属和14个真菌属,且相同地区不同葡萄品种葡萄生单轴霉菌围的细菌和真菌在同年处于同一分支。假单胞菌属(Pseudomonas)和枝孢属(Cladosporium)稳定存在于各地区不同品种葡萄霜霉病叶上葡萄生单轴霉菌围;在两年间稳定存在的菌株占比多数在80.0%以上且均具有较高的生防作用;其中,广泛分布的6株枝顶孢属(Acremonium)真菌对葡萄霜霉病的防治效果均较好,最高可达100.0%;防治效果较高的11个菌株的无菌发酵液中,黑曲霉(Aspergillusniger) NX2F、苋楔孢黑粉菌(Thecaphora amaranthi) BJ1G和匍枝根霉(Rhizopus stolonifer) BM1L的无菌发酵液防治效果均为100.0%。【结论】葡萄生单轴霉菌围的可培养细菌和真菌群落主要受地区因素影响,有较高的稳定性和生防作用,揭示了枝顶孢属真菌在我国葡萄主要产区葡萄生单轴霉菌围附生的普遍性,为葡萄霜霉病的防治提供了丰富和宝贵的资源。     

Construction of a reference linkage map of Vitis amurensis and genetic mapping of Rpv8, a locus conferring resistance to grapevine downy mildew

Downy mildew, caused by the oomycete Plasmopara viticola, is one of the major threats to grapevine. All traditional cultivars of grapevine (Vitis vinifera) are susceptible to downy mildew, the control of which requires regular application of fungicides. In contrast, many sources of resistance to P. viticola have been described in the Vitis wild species, among which is V. amurensis Rupr. (Vitaceae), a species originating from East Asia. A genetic linkage map of V. amurensis, based on 122 simple sequence repeat and 6 resistance gene analogue markers, was established using S1 progeny. This map covers 975?cM on 19 linkage groups, which represent 82% of the physical coverage of the V. vinifera reference genetic map. To measure the general level of resistance, the sporulation of P. viticola and the necrosis produced in response to infection, five quantitative and semi-quantitative parameters were scored 6?days post-inoculation on the S1 progeny. A quantitative trait locus (QTL) analysis allowed us to identify on linkage group 14 a major QTL controlling the resistance to downy mildew found in V. amurensis, which explained up to 86.3% of the total phenotypic variance. This QTL was named ??Resistance to Plasmopara viticola 8?? (Rpv8).     

Grapevine leaf tissue colonization by the fungal entomopathogen <Emphasis Type="Italic">Beauveria bassiana</Emphasis><Emphasis Type="Italic">s.l.</Emphasis> and its effect against downy mildew

A study was conducted to examine whether Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) can colonize grapevine leaf tissues and subsequently confer protection against downy mildew caused by Plasmopara viticola (Berk. and Curt.) Berl. and de Toni. Following the foliar inoculation of plants with conidial suspensions of selected B. bassiana strains, colonization of leaves by the fungus was determined using culture-based and PCR techniques at different time intervals. Seven days following B. bassiana inoculation, grapevine plants were challenged with P. viticola and symptoms were assessed by calculating the disease incidence and severity. Although all tested strains were able to colonize grapevine plants, percent colonization differed significantly among strains. Disease incidence and severity were, on the other hand, significantly reduced in B. bassiana-inoculated plants compared to control plants irrespective of strain. This study is one of very few studies investigating the promising role B. bassiana could play as a plant disease antagonist.     

Genetic dissection of a TIR‐NB‐LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR‐NB‐LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated r esistance to P lasmopara v iticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south‐eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1‐mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR‐NB‐LRR genes at this locus share a common ancestor.     

The efficacy of spraying programmes and treatment methods against downy mildew in two grapevine cultivars in Kosovo

The present study aimed to assess the efficacy of applied programmes and treatment methods against downy mildew (Plasmopara viticola). Two vine cultivars (Frankovka and Game) were tested in an experimental field (in Rahovec, Kosovo) for a two-year period (2010–2011). The fungicides used were Quadris (azoxystrobin), Antracol EP-70 (propineb), Dithan M-45 (mancozeb), Ridomil (metalaxyl), Curzate (cymoxanil), Bordeaux mixture (calcium hydroxide and copper sulphate) and Mikal (fosetyl-aluminium) applied three treatment methods. The evaluation of the disease severity was performed using the McKinney index. In both tested cultivars and in both treatment years (2010 and 2011), all of the programmes tested significantly reduced the severity of grape downy mildew compared to the control. The highest efficacy against grape downy mildew was achieved with the combined use of Ridomil and Dithan (programme 3); this treatment was more than 80% effective in both cultivars and in both years.     

Histological and Ultrastructural Studies on the Curative Effects of Mandipropamid on Plasmopara viticola

The mandelic acid amide, mandipropamid, which belongs to the carboxylic acid amide (CAA) fungicides, is active against Plasmopara viticola, the causal agent of grapevine downy mildew. The fungicide primarily inhibits the germination of encysted zoospores, thus preventing the pathogen’s penetration into the host tissues, but it also shows curative effects. In this study, the infection structures of P. viticola in both leaves and berries were investigated to detect the histological and ultrastructural alterations induced by mandipropamid when applied after inoculation. Compared to the untreated samples characterized by a diffuse colonization of the tissues and by a normal ultrastructure of the pathogen, the application of mandipropamid 24 h after inoculation with P. viticola reduced pathogen colonization in leaves and berries. In addition, detachment of the plasmalemma from the hyphal and haustorial walls was observed 72 h after inoculation. In the berries, an abnormal proliferation of the pathogen plasma membrane was observed. Collapsed hyphae and haustoria in treated leaves were surrounded by callose or encapsulated in an amorphous material inside the host cell 72 h after inoculation, while a similar effect was observed in later stages (7 days) in berries. The results confirm that mandipropamid, which acts at the interface between the pathogen plasmalemma and cell wall, has curative activity against P. viticola, appearing more rapidly in leaves than in berries.     

Putative new plant viruses associated with Plasmopara viticola-infected grapevine samples

In this study, we analysed a total of 16 libraries from over 150 grapevine leaf and grape samples infected with Plasmopara viticola (downy mildew of grapevine) to characterise the virome associated to this oomycete. Samples were collected in five distinct regions in Italy and in four different regions in Spain, representative of different pedoclimatic conditions and different grapevine cultivars during 2018 growing season. Due to the metagenomics nature of the samples (containing at least both downy mildew hyphae and spores, and grapevine cells residues), we were able to assemble several plant viruses and a few possible novel plant virus genomes with our in silico analysis. We detected several plant virus variants already reported in grapevine, and a putative new ilarvirus previously unreported in grapevine. Furthermore, we characterised three new phenui-like viruses (in the order Bunyavirales), one of which shares some commonalities with plant coguviruses. Finally, we report a new strict association of three viral segments (one flavi-like and two virga-like) that we propose to be a new virus taxon named jivivirus.     

Depsidones and other constituents from Phomopsis sp. CAFT69 and its host plant Endodesmia calophylloides with potent inhibitory effect on motility of zoospores of grapevine pathogen Plasmopara viticola

In our search for secondary metabolites regulating the motility behavior of zoospores of the grapevine downy mildew pathogen Plasmopara viticola, we found that extracts from an endophytic fungus Phomopsis sp. CAFT69 and its host plant Endodesmia calophylloides remarkably impaired motility of zoospores followed by lysis. The active principles in the extracts were isolated and identified as two new compounds, namely excelsional (1a) and 9-hydroxyphomopsidin (2a), together with excelsione (1b), phomopsidin (2b), alternariol (3a), alternariol-5-O-methyl ether (3b), the hitherto undescribed 5′-hydroxyalternariol (3c), altenusin (4) from the fungus, xanthochymol (5) and 1,5-dihydroxy-3-methoxyxanthone (mesuaxanthone, 6) from the plant. Bioassays revealed that compounds 1a/b, 2a/b, and 3a–6 displayed motility inhibition and lytic activities against zoospores of the grapevine downy mildew pathogen P. viticola in a dose- and time-dependent manner from 1 to 10 μg/mL. Their structures were elucidated by extensive spectroscopic analyses including 2D NMR techniques. This is the first report of an endophyte and its natural products from E. calophylloides and the first isolation of compounds 5 and 6 from this plant.     

Cryptic diversity of <Emphasis Type="Italic">Plasmopara viticola</Emphasis> (Oomycota,Peronosporaceae) in North America

Plasmopara viticola is the causal agent of grapevine downy mildew and is among the most important diseases in viticulture. It originates from North America, where it coevolved with wild Vitis species. Beginning in the 1870s it turned into a global epidemic that has been causing severe yield losses. It is generally believed that a single species is causing downy mildew on a large variety of economically important cultivars. Here we report, based on one nuclear and two mitochondrial markers, that isolates from vineyards in the United States fall into three highly distinct phylogenetic lineages. One of these contains European strains and affects Vitis vinifera cultivars, while the other two lineages affect also other species of Vitis. The divergence between these lineages is high, and, judging from the genetic variation in other Plasmopara lineages, might reflect distinct species. Due to the potentially significant implications for quarantine regulations and resistance breeding, detailed studies will be necessary to clarify whether these genetically distinct lineages occur outside of North America or are still confined there.     

Comparative evaluation of Pseudomonas fluorescens and their lipopolysaccharides as implicated in induction of resistance against pearl millet downy mildew

Two plant growth promoting Pseudomonas fluorescens isolates namely UOM SAR 14 and UOM SAR 80 most effectively induced resistance against downy mildew disease of pearl millet both under greenhouse and field conditions. Relative assessment of live cultures of P. fluorescens UOM SAR 14 and UOM SAR 80 and their lipopolysaccharides (LPS) extracted from their cell walls were evaluated for their ability to induce resistance against pearl millet downy mildew. Treatment with P. fluorescens and their LPS enhanced the seed germination and seedling vigour considerably. Although both live cultures and their LPS treatment induced resistance in pearl millet against downy mildew disease both under greenhouse and field conditions as evidenced by the significant reduction of the disease, live cultures were more effective than the LPS in level of resistance induced. Live cultures of UOM SAR 14 and UOM SAR 80 induced 66% and 57% protection while their respective LPS extracts offered 59 and 53% protection against downy mildew disease under greenhouse conditions. Similarly, under field conditions with very heavy inoculum pressure live cultures offered 75% and 70%, and their LPS offered 71% and 67% protection, respectively. In either case, the time gap required for the building up of resistance was found to be 3 days and nature of the resistance induced was systemic and durable with both live cultures and their lipopolysaccharides. It was also noticed that the live bacteria significantly varied in the degree of protection offered and so also their respective LPS.