Characterization of single-nucleotide-polymorphism markers for Plasmopara viticola, the causal agent of grapevine downy mildew

We report 34 new nuclear single-nucleotide-polymorphism (SNP) markers that have been developed from an expressed sequence tag library of Plasmopara viticola, the causal agent of grapevine downy mildew. This newly developed battery of markers will provide useful additional genetic tools for population genetic studies of this important agronomic species.     

Population genetic structure of Plasmopara viticola after 125 years of colonization in European vineyards

To examine the within- and among-population genetic structure of Plamopara viticola oosporic populations in Europe, 8991 lesions from 32 vineyard plots were collected and analysed. Four multi-allelic microsatellite markers were used to genotype the pathogen. All populations had high levels of gene and genotypic diversity. Most populations were in Hardy–Weinberg equilibrium and thus randomly mating. Among P. viticola populations, significant low to moderate genetic differentiation was observed, even between geographically close populations. This genetic differentiation was also evident in the neighbour-joining phylogenetic genetic distance tree, showing clear substructure and distinguishing mainly five clusters based on geographical origin. Significant isolation by distance was found in central European P. viticola populations, suggesting a step-wise migration model. No significant isolation by distance was found within Greek populations, most probably owing to natural geographical barriers such as the sea and mountains, as well as the frequent population bottlenecks occurring in these populations, preventing natural migration among populations. The high variability of P. viticola provides explanation for its successful infestation of the heterogeneous European vineyards in the last 125 years after its introduction.     

Identification of effector genes from the phytopathogenic Oomycete Plasmopara viticola through the analysis of gene expression in germinated zoospores

Grapevine downy mildew caused by the Oomycete Plasmopara viticola is one of the most important diseases affecting Vitis spp. The current strategy of control relies on chemical fungicides. An alternative to the use of fungicides is using downy mildew resistant varieties, which is cost-effective and environmentally friendly. Knowledge about the genetic basis of the resistance to P. viticola has progressed in the recent years, but little data are available about P. viticola genetics, in particular concerning the nature of its avirulence genes. Identifying pathogen effectors as putative avirulence genes is a necessary step in order to understand the biology of the interaction. It is also important in order to select the most efficient combination of resistance genes in a strategy of pyramiding. On the basis of knowledge from other Oomycetes, P. viticola effectors can be identified by using a candidate gene strategy based on data mining of genomic resources. In this paper we describe the development of Expressed Sequence Tags (ESTs) from P. viticola by creating a cDNA library from in vitro germinated zoospores and the sequencing of 1543 clones. We present 563 putative nuclear P. viticola unigenes. Sequence analysis reveals 54 ESTs from putative secreted hydrolytic enzymes and effectors, showing the suitability of this material for the analysis of the P. viticola secretome and identification of effector genes. Next generation sequencing of cDNA from in vitro germinated zoospores should result in the identification of numerous candidate avirulence genes in the grapevine/downy mildew interaction.     

与葡萄抗霜霉病基因紧密连锁的分子遗传标记

以种间杂交组合88-110［83-4-96（毛葡萄,抗霜霉病）×粉红玫瑰（欧洲葡萄,感霜霉病）］的F     

Development of a transient expression system in grapevine via agro-infiltration

Transient expression of genes using Agrobacterium is a powerful tool for the analysis of gene function in plants. We have developed this method for the analysis of genes involved in disease resistance in grapevine leaves. Our research showed that the quality of the plant material, the plant genotype used for agro-infiltration and the presence of additional virulence factors (carried on plasmid pCH32) in the Agrobacterium strain are all important factors for success of the procedure. After optimising these factors, we consistently achieve sufficient acceptable levels of expression of the markers beta-glucuronidase (GUS) and green fluorescent protein (GFP) using vacuum infiltration of grapevine leaves from plants grown in vitro. We used this procedure to investigate the proposed role of stilbenes in defense against grapevine downy mildew (Plasmopara viticola) by transiently overexpressing stilbene synthase in grapevine leaves, before infection with P. viticola. We found that agro-infiltration itself induces the synthesis of stilbenes in grapevine leaves, thus preventing us to test the effect of the overexpression of stilbene synthase in defense. However, our results revealed that agro-infiltration before P. viticola inoculation had an effect on the development of the infection. Further research is required to show whether stilbenes or some other factor are the causal agent restricting pathogen development. The method described here provides and excellent tool to exploit at the many grapevine genomic resources now available, and will contribute to a better understanding of many areas of grapevine biology.     

A Basic Peroxidase Isoenzyme, Marker of Resistance Against Plasmopara viticola in Grapevines, is Induced by an Elicitor from Trichoderma viride in Susceptible Grapevines

The constitutive expression of basic peroxidase isoenzymes in the Plasmopara viticola -resistant ( Vitis vinifera × Vitis rupestris) × Vitis riparia crossing and in the P. viticola -susceptible V. vinifera parent species was studied. The results illustrate that both leaves and stems of the ( V. vinifera × V. rupestris) × V. riparia crossing showed the differential expression of a basic peroxidase isoenzyme B₃ (pl = 8.9), this being almost completely absent from the P. viticola -susceptible V. vinifera parent species. To test whether the basic peroxidase isoenzyme B₃ may be considered as a molecular marker of disease resistance in Vitis spp., suspension cell cultures derived from the P. viticola -susceptible V. vinifera parent species were treated with an elicitor (cellulase Onoztika R-10) from the soil fungus Trichoderma viride , a specific and well-known elicitor of disease resistance reactions in grapevines. The results showed that treatment with the elicitor induces, simultaneously with the activation of the disease resistance mechanism, the appearance of B₃ in the cell cultures. These results suggest that the basic peroxidase isoenzyme B₃ may be considered as a marker of disease resistance in Vitis species since it is present in the P. viticola -resistant ( V. vinifera × V. rupestris) × V. riparia hybrid and is induced by the elicitor Onozuka R-10 in cell cultures of the P. viticola -susceptible Vitis vinifera parent species. This conclusion is supported by the presence of this isoenzyme in other resistant and its absence in other susceptible Vitis spp.     

Association of RGA-SSCP markers with resistance to downy mildew and anthracnose in grapevines

Downy mildew (Plasmopara viticola) and anthracnose (Sphaceloma ampelinum) are two major diseases that severely affect most grapevine (Vitis vinifera) cultivars grown commercially in Thailand. Progress of conventional breeding programs of grapevine for improved resistance to these diseases can be speeded up by selection of molecular markers associated with resistance traits. We evaluated the association between 13 resistance gene analog (RGA)-single-strand conformation polymorphism (SSCP) markers with resistance to downy mildew and anthracnose in 71 segregating progenies of seven cross combinations between susceptible cultivars and resistant lines. F(1) hybrids from each cross were assessed for resistance to downy mildew and anthracnose (isolates Nk4-1 and Rc2-1) under laboratory conditions. Association of resistance traits with RGA-SSCP markers was evaluated using simple linear regression analysis. Three RGA-SSCP markers were found to be significantly correlated with anthracnose resistance, whereas significant correlation with downy mildew resistance was observed for only one RGA-SSCP marker. These results demonstrate the usefulness of RGA-SSCP markers. Four candidate markers with significant associations to resistance to these two major diseases of grapevine were identified. However, these putative associations between markers and resistance need to be verified with larger segregating populations before they can be used for marker-assisted selection.     

Characterization of Plasmopara-resistance in grapevine using in vitro plants

Although the exact mechanisms by which grapevine cells operate to reduce disease incidence caused by the downy mildew fungus Plasmopara viticola are not fully elucidated, our cytological results obtained from infected in vitro-plants confirm that enhanced disease resistance is associated with an expression of distinct reactions in a chronological order. An increased production of reactive oxygen species (superoxide radicals, 4-6 hours post infection, hpi) was followed by a hypersensitive response (6-8 hpi), an increased activity of peroxidase in cells flanking the infection area and in the vascular tissue (10-12 hpi) and an increased production, accumulation or conversion of phenolic compounds (12-15 hpi). These mechanisms seem also to be present in susceptible varieties as shown after an inoculation with non-host oomycetic pathogens on the basis of peroxidase activity, but they do not become activated after P. viticola infection. The investigation of the peroxidase activity in leaves at several time points after an infection with P. viticola indicated that there is a strong correlation between the POX activity in leaves of in vitro-plants and the resistance of grapevine plants to P. viticola in the field.     

Synthesis and anti-oomycete activity of novel quinazolin- and benzothiazol-6-yloxyacetamides: Potent aza-analogs and five-ring analogs of quinoline fungicides

Novel quinazolin- and benzothiazol-6-yloxyacetamides show excellent in vivo activity against the three economically most important Oomycete pathogens Phytophthora infestans, Plasmopara viticola and Pythium ultimum. They are polar analogs of known quinolin-6-yloxyacetamides, which are not active against the soil-borne damping-off disease caused by Pythium ultimum. The Bogert quinazoline synthesis, an almost forgotten heterocyclization technique, proved to be highly useful for the concise construction of required quinazolin-6-ol building blocks.     

Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola

Grapevine (Vitis vinifera L.) is susceptible to many pathogens, such as Botrytis cinerea, Plasmopara viticola, Uncinula necator, and Eutypa lata. Phytochemicals are used intensively in vineyards to limit pathogen infections, but the appearance of pesticide-resistant pathogen strains and a desire to protect the environment require that alternative strategies be found. In the present study, the beta-1,3-glucan laminarin derived from the brown algae Laminaria digitata was shown both to be an efficient elicitor of defense responses in grapevine cells and plants and to effectively reduce B. cinerea and P. viticola development on infected grapevine plants. Defense reactions elicited by laminarin in grapevine cells include calcium influx, alkalinization of the extracellular medium, an oxidative burst, activation of two mitogen-activated protein kinases, expression of 10 defense-related genes with different kinetics and intensities, increases in chitinase and beta-1,3-glucanase activities, and the production of two phytoalexins (resveratrol and epsilon-viniferin). Several of these effects were checked and confirmed in whole plants. Laminarin did not induce cell death. When applied to grapevine plants, laminarin reduced infection by B. cinerea and P. viticola by approximately 55 and 75%, respectively. Our data describing a large set of defense reactions in grapevine indicate that the activation of defense responses using elicitors could be a valuable strategy to protect plants against pathogens.     

分离自海藻的青霉属三个中国新记录种

报道了分离自青岛沿海潮间带海藻的青霉属3个中国新记录种:产红青霉Penicillium rubens、苏门答腊青霉P.sumatrense和葡萄生青霉P.viticola,并对其进行了形态学描述和系统发育分析。研究菌株保存于中国海洋大学海洋生物标本室(OUCMB)。     

The host guides morphogenesis and stomatal targeting in the grapevine pathogen <Emphasis Type="Italic">Plasmopara viticola</Emphasis>

The oomycete grape downy mildew (Plasmopara viticola Berk. & Curt. Ex de Bary) is a serious pathogen of grapevine and spreads by extremely efficient cycles of asexual propagation. The high efficiency must involve efficient sensing of the host. We therefore analyzed the time course and morphology of the early development of this pathogen in a host system, by infection of leaf discs of grapevine (Vitis vinifera L. cv. Müller-Thurgau), and in a host-free system. Host factors were demonstrated to influence pathogen development in the following ways: (i) the release of zoospores from mature sporangia was accelerated, (ii) the morphogenesis of the germ tube was coordinated, and (iii) the zoospores were targeted to the stomata by factors that depended on stomata closure. The findings show that the early development of P. viticola is regulated, specifically and coordinately, by factors originating from the host plant.     

More years of field trials against Plasmopara viticola in organic viticolture

The organic viticulture assumes a decisive role in the national agricultural sector. More impelling problems in the management of organic vineyards are represented from the plants pathology defence and particularly of Plasmopara viticola containment. Copper represents one of the few usable fungicides in the organic farming and the only effective against downy mildew. With Regulation EC n. 473/2002, fixed maximum quantity usable of copper compounds, owing to the environment problems due to the copper accumulation in the soil. To reduce quantity of metal copper or replace it with natural products, are conducted field trials with copper compounds at a low rate or alternative to copper products. Besides, we are estimating possibility to reduce the operations against P. viticola optimizing fungicidal treatments. Field trials in the organic farms located near Rome, have been carried out. Guidelines EPPO/OEPP PP 1/31 (3) have been carried out. The results of the trials have showed that, using cupric products with low metallic content, to reduce copper quantities used, always allowed to respect the limits established by Regulation EC. The alternative products that were investigated have not guaranteed, instead, an adequate protection in high pressure of grapevine downy mildew. It was possible to reduce treatments against P. viticola through control of different environmental parameters. The trials confirm that the copper is indispensable for plant protection in organic farming as it is not possible to replace it with natural extracts substances. We can reduce, instead, the copper quantities used trough the use of new products with low quantity metal copper or through the evaluation of climatic and pedologic data that allow to rationalize the fungicidal treatments.     

A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.     

Botryosphaeriaceae as potential pathogens of prunus species in South Africa, with descriptions of Diplodia africana and Lasiodiplodia plurivora sp. nov

Botryosphaeriaceae are common dieback and canker pathogens of woody host plants, including stone fruit trees. In the present study the diversity of members of the Botryosphaeriaceae isolated from symptomatic wood of Prunus species (plum, peach, nectarine and apricot) was determined in stone fruit-growing areas in South Africa. Morphological and cultural characteristics as well as DNA sequence data (5.8S rDNA, ITS-1, ITS-2 and EF-1a) were used to identify known members and describe novel members of Botryosphaeriaceae. From the total number of wood samples collected (258) 67 isolates of Botryosphaeriaceae were obtained, from which eight species were identified. All species were associated with wood necrosis. Diplodia seriata (= "Botryosphaeria" obtusa) was dominant, and present on all four Prunus species sampled, followed by Neofusicoccum vitifusiforme and N. australe. First reports from Prunus spp. include N. vitifusiforme, Dothiorella viticola and Diplodia pinea. This is also the first report of D. mutila from South Africa. Two species are newly described, namely Lasiodiplodia plurivora sp. nov. from P. salicina and Diplodia africana sp. nov. from P. persica. All species, except Dothiorella viticola, caused lesions on green nectarine and/or plum shoots in a detached shoot pathogenicity assay.     

Phenotypic and histochemical traits of the interaction between Plasmopara viticola and resistant or susceptible grapevine varieties

ABSTRACT: BACKGROUND: Grapevine downy mildew, caused by Plasmopara viticola, is a very serious disease affectingmainly Vitis vinifera cultivated varieties around the world. Breeding for resistance throughthe crossing with less susceptible species is one of the possible means to reduce the diseaseincidence and the application of fungicides. The hybrid Bianca and some of its siblings areconsidered very promising but their resistance level can vary depending on the pathogenstrain. Moreover, virulent strains characterized by high fitness can represent a potential threatto the hybrid cultivation. RESULTS: The host response and the pathogen virulence were quantitatively assessed by artificiallyinoculating cv Chardonnay, cv Bianca and their siblings with P. viticola isolates derived fromsingle germinating oospores collected in various Italian viticultural areas. The hostphenotypes were classified as susceptible, intermediate and resistant, according to the AreaUnder the Disease Progress Curve caused by the inoculated strain. Host responses in cvBianca and its siblings significantly varied depending on the P. viticola isolates, which in turndiffered in their virulence levels. The fitness of the most virulent strain did not significantlyvary on the different hybrids including Bianca in comparison with the susceptible cvChardonnay, suggesting that no costs are associated with virulence. Among the individualfitness components, only sporangia production was significantly reduced in cv Bianca and insome hybrids. Comparative histological analysis revealed differences between susceptibleand resistant plants in the pathogen diffusion and cytology from 48 h after inoculation onwards. Defence mechanisms included callose depositions in the infected stomata, increasein peroxidase activity, synthesis of phenolic compounds and flavonoids and the necrosis ofstomata and cells immediately surrounding the point of invasion and determined alterations inthe size of the infected areas and in the number of sporangia differentiated. CONCLUSIONS: Some hybrids were able to maintain an intermediate-resistant behaviour even wheninoculated with the most virulent strain. Such hybrids should be considered for further fieldtrials.     

In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves

Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5-6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues.     

On the presence of calmodulin-like protein in mycobacteria

Abstract Endogenous elemental sulfur (S⁰) has been studied in dormant spores and in spores in the early stages of germination, of Phomopsis viticola . S⁰ was measured by high-pressure liquid chromatography (HPLC). The rapid and almost total disappearance of endogenous S⁰ during the early stages of spore germination was directly related to a sharp increase of the respiratory activity and the ATP concentration. This was followed by the synthesis of DNA, RNA, proteins and lipids. Respiratory activity, S⁰ reduction and germination were inhibited in high concentrations of spores. Endogenous S⁰ disappearance, due to its reduction at the level of the mitochondrial respiratory chain with hydrogen sulfide production, may play a key role in the breaking of dormancy and the induction of germination in spores of P. viticola .