Staining methods for the investigation of Plasmopara viticola and its infection structures in semi-thin sections

The investigation of Plasmopara viticola, that spends most of its life cycle inside of leaves, requires specific staining techniques. It is desirable to visualize both, host cells and parasitic structures, given their silimar chemical constitutions, especially in reference to the cell wall. On the basis of appropriate staining procedures it was possible to demonstrate parasitic structures during the infection process. Several dyes and optical brighteners were successfully used to identify hyphae, vesicles, haustoria, gametangia and oospores. The chemical determination of the stained structures is discussed

Epicoccum nigrum Link: A biological control agent of Plasmopara viticola (Berk. et Curt.) Berl. et De Toni?

Research Not

Effect of sterol biosynthesis inhibitors and azole-type inducers on growth and development of Plasmopara viticola on grapevine

The effect of various azole fungicides (demethylation inhibitors, DMIs), mainly triazoles, that interfere with sterol metabolism in higher fungi, triazole-type inducers and an inhibitor of plant brassinosteroid biosynthesis on growth and spore production of the downy mildew pathogen of grapevine (Plasmopara viticola) was tested in a leaf disc-based system. Application of DMIs and azole-type inducers two days before inoculation resulted in a delayed mycelial growth during the first three days pot inoculation, which was not observed at day 5 or 7 post inoculation, and in a weakly reduced sporulation of the pathogen. These effects were not observed when compounds were applied during or post inoculation. Thus, azole treatments do not interfere with infections and epidemics caused by the downy mildew pathogen as observed in the field. However, alteration of brassinosteroid metabolism of the plant induced by the inhibition of a 5α-reductase with finasteride resulted in a markedly reduced sporulation. This indicates that plant derived sterols or those processes modulated by sterols interfere with the reproduction of the pathogen

Comparison of PR1 expression in grapevine cultures after inoculation with a host- and a non-host pathogen

PR1 gene induction was monitored by RT-PCR in three different grapevine cultures that were inoculated with Plasmopara viticola as a host pathogen and Pseudoperonospora cubensis as a non-host pathogen. The results of the expression analysis showed that the PR1 mRNA level in Vitis vinifera cv. Riesling and Vitis riparia cv. Gloire de Montpellier is mainly affected by the culture system. PR1 is constitutively expressed in callus cultures. In vitro plants showed a low basal expression of PR1 that is enhanced after challenge with both downy mildews in the two species tested. In greenhouse plants, PR1 is only expressed 24 h post inoculation (hpi) but not 12 hpi. Heavily infected leaves (‘oilspots‘) of the susceptible cv. Riesling, exhibiting well developed infection structures in the intercellular space as indicated by microscopical methods, also showed a high PR1 expression rate three weeks post inoculation. Thus, the role of PR1 expression in impeding the downy mildew pathogen remains equivocal. It seems that expression of PR1 is a general stress response in some grapevine culture systems and that their use as a reference for gene expression analysis is limited

The role of callose deposits during infection of two downy mildew-tolerant and two-susceptible Vitis cultivars

Semithin sections of leaves from four cultivars were analysed for callose deposition after infection with Plasmopara viticola. Two of these cultivars are tolerant to this pathogen in the field (Orion, Phoenix), while the other two are susceptible (Kerner, Riesling). Callose was not detectable during the first 5 days after experimental inoculation. During late stages of the infection cycle however, callose deposits could be found in tolerant as well as in susceptible grapes. During the fungal infection of tolerant plants hyphal development stops 3 to 4 days post infection indicating that mechanisms other than callose deposition are the main factors of tolerance

The role of hairs on the wettability of grapevine (Vitis spp.) leaves

Hairs represent an important physico-chemical factor on abaxial leaf surfaces of grapevine and can influence the wettability of leaves. This was demonstrated by scanning electron micrographs and monitored with a new method using a torsion balance. Forces necessary to remove water droplets of distinct size from leaves were lower in case of pubescent (0,2-0,4 mN) compared to non-pubescent vine leaves (0,4-1,2 mN). Depending on the number of hairs and their surface structure the deposition and distribution of water droplets originating from dew and rain can be prevented due to weak adhesion. Thus, surface features enabling water droplets to pearl off may reduce the probability of infection by fungal spores

Searching new targets for the control of Black Rot: following the role of host factors modulating the infection process of Phyllosticta ampelicida

Black Rot is a grapevine disease caused by the ascomycete Phyllosticta ampelicida. Neglected so far, this is developing into a pertinent problem in organic viticulture as resistant varieties are still lacking. Here, we follow cellular details of the infection process in the susceptible vinifera variety Müller-Thurgau and screen the ancestral European wild grapevine (V. vinifera sylvestris) for resistance to Black Rot. Using a standardized infection assay, we follow fungal development using LTSEM and quantify key stages on different hosts using fluorescence microscopy. There is considerable variation in susceptibility, which is associated with more rapid leaf maturation. Hyphal growth on different carbon sources shows a preference for pectins over starch, cellulose or xylans. In the resistant sylvestris genotypes Ketsch 16 and Ketsch 18 we find that neither spore attachment nor appressorium formation, but hyphal elongation is significantly inhibited as compared to Müller-Thurgau. Moreover, defence-related oxidative burst and accumulation of phenolic compounds is stimulated in the resistant genotypes. We arrive at a model, where more rapid maturation of the cell wall in these sylvestris genotypes sequesters pectins as major food source and thus block hyphal elongation. This paves the way for introgression of genetic factors responsible for cell wall maturation into V. vinifera to develop Black Rot-resistant varieties of grapevine

Powdery mildew responsive genes of resistant grapevine cultivar 'Regent'

The ascomycete Erysiphe necator causes powdery mildew disease of grapevine, a disastrous infection which is commonly defeated with multiple fungicide applications in viticulture. Breeding for natural resistance of quality grapes (Vitis vinifera) is thus a major aim of current efforts. The cultivar 'Regent' is resistant to powdery mildew due to an introgression from an American Vitis sp. resistance donor. To identify key regulatory elements in defense responses of 'Regent' we performed transcript analyses after challenging with E. necator inoculation in comparison with a susceptible grapevine. A set of genes selected from preliminary microarray hybridization results were investigated by RT-qPCR. The data indicate an important role of transcription factors MYB15, WRKY75, WRKY33, WRKY7, ethylene responsive transcription factors ERF2 and ERF5 as well as a CZF1/ZFAR transcripton factor in regulating the early defense when the fungus starts the interaction with its host by the formation of haustoria

Strategien zur Regulation der Schwarzfäule (Guignardia bidwellii) im ökologischen Weinbau

Die Schwarzfäule tritt in einigen deutschen Weinbaugebieten flächendeckend auf und kann gravierende Ertragsausfälle verursachen. Um die Produktionssicherheit im ökologischen Weinbau zu gewährleisten, wurde ein Kooperationsprojekt mit der Zielsetzung initiiert, Informationen über die Biologie des Schadpilzes zu erarbeiten und Strategien zur Prävention und Bekämpfung der Krankheit unter den spezifischen Bedingungen des ökologischen Weinbaus zu entwickeln. Die Biologie des Schaderregers und seiner Interaktionen mit der Rebe wurde in Hinblick auf die Fruchtkörperentwicklung, die Sporenbildung und den Infektionsprozess eingehend untersucht und in Beziehung zu Witterungsbedingungen und Bewirtschaftungsparametern gesetzt. Die Ergebnisse bilden eine Grundlage für die Einschätzung des Infektionsrisikos und die Entwicklung von Entscheidungshilfen für den Rebschutz. Traditionelle und „pilzwiderstandsfähige“ Rebsorten wurden hinsichtlich ihrer Anfälligkeit für die Schwarzfäule charakterisiert, wodurch den Winzern Informationen für die Auswahl weniger Anfälliger Sorten zum Anbau in Befallsgebieten zur Verfügung stehen. Befallenes Rebholz und befallene Ranken wurden als bedeutende Quellen des Primärinokulums der Schwarzfäule identifiziert. Das Entfernen befallener Blätter bei Laubarbeiten erwies sich als Möglichkeit, das Infektionsrisiko für die Trauben zu reduzieren. Aus einer Vielzahl von Mikroorganismen, Pflanzenextrakten, Pflanzenschutz- und –stärkungsmitteln wurden wirksame Agenzien selektiert und im Freiland unter Praxisbedingungen geprüft. Die Kombination von Pflanzenschutzmitteln auf der Basis von Schwefel und Kupfer war besonders wirksam. Wurde, abhängig vom Entwicklungsstadium und dem Infektionsrisiko, das Kupfer mit Gesteinsmehl ersetzt, ließ sich der Kupferaufwand erheblich reduzieren. Im Gegensatz zu Gewächshausversuchen war die Wirkung saponinhaltiger Pflanzenextrakte im Freiland unzureichend. Bei entsprechender Formulierung zur Verbesserung der Regenfestigkeit könnte das große Potential dieser Pflanzenextrakte jedoch genutzt werden. Aufgrund der Ergebnisse des Forschungsprojekts stehen dem ökologischen Weinbau Informationen als Grundlage eines umfassenden Managementkonzepts für die Schwarzfäule zur Verfügung