Original articleExpression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen
Introduction
Grapevine (Vitis vinifera) is the world's largest fruit crop with an annual production of more than 60 million tons [22]. Unfortunately, all cultivars currently used for grape or wine production are susceptible to several pathogens, such as the downy mildew pathogen. Resistant sources are available, but breeding efforts take a long time to produce new resistant cultivars that also give good quality in tasting.
Downy mildew caused by Plasmopara viticola (Berk. and Curtis) Berl. et de Toni is a common disease of grapevine anywhere in the world where vines are grown. This pathogen causes significant yield losses because attack occurs on leaves (oil spots) and young berries. Primary infections result from zoospores originating from oospores which represent the resting spores of P. viticola. The encysted zoospores produce germ tubes, which invade the host through stomata and colonise intercellularly the leaf parenchyma producing intracellular haustoria. Later on, masses of hyaline sporangia are produced on sporangiophores at the lower leaf surface and are released through wind currents or raindrops. These sporangia can start secondary infections as soon as weather conditions are favourable for fungal growth and development, and if protective chemical protection is omitted. During the growing season, up to ten fungicide applications have to be brought out to prevent fungal diseases on leaves and berries. Unfortunately, the application of copper-containing fungicides to control downy mildew may result in residual copper in musts, leading to lagging fermentation and a detrimental effect on wine quality [42]. Furthermore, a high frequency of copper applications causes accumulation of this heavy metal in soil and groundwater resulting in toxic effects on micro- and macroorganisms. Although copper is an essential trace element for photosynthetic organisms, it causes severe toxic effects at high concentrations. One mechanism is the inhibition of photosynthesis caused by the substitution of Mg2+ in the chlorophyll molecule [28]. This heavy metal-substituted chlorophyll is unsuitable for photosynthesis. The replacement of copper-based fungicides by synthetic fungicides with specific mode of actions promoted the development of resistant isolates of P. viticola [14], [18], [20]. Therefore, alternative concepts are badly needed. Despite numerous reports of plants extracts exhibiting antifungal properties against downy mildews, no alternative natural compound or mixture of compounds has been developed exhibiting sufficient effectiveness under practical conditions, which is also the case for plant strengtheners and inducers of acquired resistance. Therefore, an investigation of the preformed and induced anti-microbiological barriers of the host may lead scientists to alternative and suitable disease management strategies. In grapevine, hairy and water repellent leaf surfaces seem to be successful by reducing the probability of infection, especially in wild species [24], [25]. Unfortunately, those mechanisms are not established in most cultivars used for crop production. Previous data with downy mildew-resistant in vitro plants had shown that infection with P. viticola was associated with an expression of distinct reactions in a chronological order, such as increased production of reactive oxygen species, hypersensitive response, increased peroxidase activity, and accumulation of phenolic compounds [26]. The pathway leading to the diverse group of phenolic compounds begins with the PAL-controlled conversion of phenylalanine into 4-coumaroyl-CoA which is then used for the production of lignins, coumarins and stilbenes. Mainly the deposition of lignin [9] and other phenolic compounds, accompanied by high peroxidase activity, and the production of stilbene phytoalexins represent important components of a defence response in grapevine [11], [35], [36]. Preformed isoflavonoids (flavonoid derivatives) and induced flavonoids may also play a role in protecting the young berries or leaves from various phytopathogens, such as P. viticola [8], [9], or as feeding deterrents to insects [6]. In addition, plants combat fungal infections by the synthesis of a number of pathogenesis-related (PR) proteins [43]. The constitutive levels of PR proteins are low in healthy plants but become highly elevated after pathogen attack. Some PR proteins may act directly against the pathogen or its infection structures, whereas others lead to the production of elicitors which in turn trigger additional defence mechanisms in the plant. These mechanisms also seem to be present in susceptible varieties, but, in general, they are not activated or are delayed during the infection process. In that case, they are often established when a successful restriction of the pathogen is difficult or impossible.
As the grapevine plant is susceptible to a few specialised parasites but resistant to a wide range of phytopathogens, the present study was conducted:
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to investigate the expression of PR-protein encoding genes and genes involved in the phenylpropanoid metabolism after an inoculation with the compatible host-pathogen P. viticola and the incompatible non-host pathogen Pseudoperonospora cubensis, the downy mildew pathogen of cucumber;
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to verify the occurrence of the corresponding compounds produced after induction of genes involved in the phenylpropanoid pathway with specific staining methods;
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to test the disease development after induction with the non-host pathogen Ps. cubensis followed by an inoculation with P. viticola.
The argument behind these experiments was that if the grapevine plant susceptible to grape downy mildew exhibits the same or similar defence mechanisms as the downy mildew-resistant plant, these mechanisms may become activated in the susceptible plant after inoculation with the non-host pathogen leading to an enhanced resistance against the host pathogen.
Section snippets
Expression analysis
Expression of grapevine defence-related genes was analysed on leaves of greenhouse plants 0, 12, and 24 h after inoculation with Plasmopara viticola and Pseudoperonospora cubensis. Inoculations were done with zoospore-producing sporangia of both pathogens. This allowed the analysis of gene expression in a synchronous time course experiment. Grapevine β-tubulin gene was selected as an internal control to analyse quality of RNA and to normalise the different samples for differences in the amount
Discussion
Structural and biochemical mechanisms are both involved in passive and active defence in grapevine. Passive defence mechanisms include structural characteristics of leaves [19], [24] and berries [13] and preformed antifungal compounds such as anthocyanins and other phenolic compounds. Fungal growth may therefore be impeded or at least retarded allowing for a subsequent establishment of active defence mechanisms such as accumulation of phytoalexins and PR-proteins or deposition of lignin in the
Plant material and pathogens
Two grapevine species, Vitis vinifera Riesling (susceptible to Plasmopara viticola) and Vitis riparia ‘Gloire de Montpellier’ (resistant to P. viticola), were used. Plants were grown in 12 l pots filled with sand and loamy soil (3:1, v/v) in the greenhouse at 19 °C and a 16 h photoperiod, and fertilized weekly with 100 ml of a 1% solution of Hakaphos blau (N/P/K/Mg, 15:10:15:2; Compo, Münster, Germany) supplemented with microelements (Fetrilon Combi, Compo). Sporangiospores of P. viticola
Acknowledgements
I would like to thank Heike Popovitsch for excellent technical assistance, Margit Schmidtke for taking care of the plants in the greenhouse, Regina Belz for her help in statistical analysis, and Annette Rabsilber for her help in collecting sporangiospores of P. viticola. Thanks are also extended to Heinrich Buchenauer for supporting this work and Birgitta Fischer for critically reading the manuscript and language-related corrections.
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