Variability in the sensitivity of biotrophic grapevine pathogens (Erysiphe necator and Plasmopara viticola) to acibenzolar-S methyl and two phosphonates

The antifungal properties of two phosphonates (fosetyl-Al and a fertilizer) and acibenzolar-S-methyl (ASM) were evaluated to assess their potential for protecting grapevine leaves against grapevine mildews (Plasmopara viticola and Erysiphe necator), and to determine their effects on the development of various mildew isolates, taking into account the inter- and intra-species variability of the pathogens. The phosphonates directly and significantly inhibited the growth of these pathogens. By contrast, ASM had no direct effect on spore production and growth of P. viticola and of E. necator at 1.9 mM. Applied before inoculation, the mean EC₅₀ of ASM was 0.50?±?0.04 mM and 1.00?±?0.07 mM for downy and powdery mildew isolates, respectively. The EC₅₀ of the fosetyl-aluminium (FOS) was 0.50?±?0.02 mM for downy mildew and the EC₅₀ for powdery mildew varied depending on the genetic group under consideration (0.89?±?0.32 mM for group B 3.30?±?0.46 mM for group A, respectively). The EC₅₀ of the potassium phosphonate fertilizer (PK₂) was 0.96?±?0.45 mM for downy and 6.9?±?0.76 mM for powdery mildew isolates. These compounds showed differences in their efficacy depending on the variability of mildews and could be an alternative or additional method to traditional pest management in the grapevine.     

Mechanisms of induced resistance in lettuce against <Emphasis Type="Italic">Bremia lactucae</Emphasis> by DL<Emphasis Type="Bold">-</Emphasis>β<Emphasis Type="Bold">-</Emphasis>amino<Emphasis Type="Bold">-</Emphasis>butyric acid (BABA)

BABA induced local and systemic resistance in lettuce (Lactuca sativa) against the Oomycete Bremia lactucae. Structure-activity analysis showed no induced resistance by related amino-butanoic acids or β-alanine. The R-enantiomer of BABA induced resistance whereas the S-enantiomer did not, suggesting binding to a specific receptor. Other compounds known to be involved in SAR signaling, including abscisic acid, methyl-jasmonate, ethylene, sodium-salicylate and Bion® (BTH) did not induce resistance. Systemic translocation of ¹⁴C-BABA and systemic protection against downy mildew were tightly correlated. BABA did not affect spore germination, appressorium formation, or penetration of B. lactucae into the host. Epifluorescence and confocal microscopy revealed that BABA induced rapid encasement with callose of the primary infection structures of the pathogen, thus preventing it from further developing intercellular hyphae and haustoria. Invaded host cells treated with BABA did not accumulate phenolics, callose or lignin, or express HR. In contrast, cells of genetically-resistant cultivars accumulated phenolics, callose and lignin and exhibited HR within one day after inoculation. The callose synthesis inhibitor DDG did not inhibit callose encasement nor compromised the resistance induced by BABA. PR-proteins accumulated too late to be responsible for the induced resistance. DAB staining indicated that BABA induced a rapid accumulation of H₂O₂ in the penetrated epidermal host cells. Whether H₂O₂ stops the pathogen directly or via another metabolic route is not known.     

The reliability of leaf bioassays for predicting disease resistance on fruit: a case study on grapevine resistance to downy and powdery mildew

This study was designed to assess the reliability of grapevine leaf bioassays for predicting disease resistance on fruit in the field. The efficacy of various grapevine quantitative trait loci (QTLs) for conferring resistance to downy and powdery mildew was evaluated in bioassays and in a 2‐year field experiment for downy mildew. The resistance genes studied were inherited from Muscadinia rotundifolia (Rpv1 and Run1) and from American Vitis species through cv. Regent (QTLRgP and QTLRgD). In bioassays, genotypes carrying Run1 blocked powdery mildew development at early stages. Genotypes combining Run1 with QTLRgP displayed no greater level of resistance. For downy mildew, genotypes carrying Rpv1 and/or QTLRgD were more resistant than the susceptible cv. Merlot, and showed a high level of leaf resistance in the field (<10% severity). Disease levels on bunches were much higher than those on leaves, with a high variability between Rpv1 genotypes (1–48%). A Bayesian decision theory framework predicted that an OIV‐452 threshold of 5 in leaf bioassays allowed accurate selection of grapevine genotypes (P = 0·83) with satisfactory disease severity on bunches. Therefore, this study validates that the use of early bioassays on leaves, as currently performed by grapevine breeders, ensures a satisfactory level of resistance to downy mildew of bunches in the field.     

葡萄应答霜霉病过程中多磷酸肌醇激酶基因与过氧化氢的作用机制

以对霜霉病不同抗性的葡萄品种左优红和霞多丽为材料,利用分子生物学和植物生理学试验手段,结合药理学试验,探讨葡萄在应答霜霉病过程中葡萄多磷酸肌醇激酶基因（VvIPK2）和H2O2的作用机制。接种霜霉病菌后15 h葡萄叶片VvIPK2表达量是正常水平的12倍,接种后3 hH2O2含量达最大值,同时苯丙氨酸解氨酶和几丁质酶活性升高;多磷酸肌醇激酶（IPK2）抑制剂、外源H2O2及H2O2清除剂均能改变霜霉病菌所引起的抗性葡萄品种左优红叶片PAL和几丁质酶活性的变化,同时可以影响不同抗性品种叶片的感病情况;IPK2抑制剂对葡萄霜霉病菌引起的H2O2水平变化没有影响;清除H2O2可减弱葡萄霜霉病菌对VvIPK2表达量的诱导效应。研究表明H2O2位于IPK2的上游,通过调控PAL和几丁质酶活性参与葡萄应答霜霉病过程。     

Downy mildew resistance evaluation in 28 grapevine hybrids promising for breeding programs in Trentino region (Italy)

Downy mildew is a major grapevine disease caused by the biotrophic oomycete, Plasmopara viticola. Numerous disease resistance studies of diverse Vitis germplasm have been previously carried out to identify downy mildew resistance sources; however, ratings were mainly reported using leaf disc in vitro testing and foliage field assessment, or upon leaf and cluster field evaluations. In the current study, 28 grapevine hybrid cultivars were screened using leaf disc bioassay, for disease resistance characterization of both existing and wild-collected materials. 16 hybrids were identified as highly resistant or resistant, and will serve as relevant resistance donors in future pre-breeding and breeding programs. All grapevine hybrids were evaluated for foliar and cluster downy mildew resistance in an untreated field trial over three successive years. This study showed that the leaf disc bioassay provided some information on the resistance level of the genotypes under scrutiny, but it was a weak predictor of their resistance level under field conditions on leaves and even more on bunches. These findings are relevant to future applications in both traditional and marker-assisted breeding programs which promote sustainable viticulture.     

Characterization of resistance mechanisms activated by Trichoderma harzianum T39 and benzothiadiazole to downy mildew in different grapevine cultivars

Downy mildew, caused by Plasmopara viticola, is one of the most destructive diseases of grapevine and is controlled with intense application of chemical fungicides. Treatment with Trichoderma harzianum T39 (T39) or benzothiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) has been previously shown to activate grapevine resistance to downy mildew and reduce disease symptoms in the Pinot noir cultivar. However, enhancement of plant resistance can be affected by several factors, including plant genotype. In order to further extend the use of resistance inducers against downy mildew, the physiological and molecular properties of T39‐ and BTH‐activated resistance in different cultivars of table and wine grapes were characterized under greenhouse conditions. T39 treatment reduced downy mildew symptoms, but the degree of efficacy differed significantly among grapevine cultivars. However, efficacy of BTH‐activated resistance was consistently high in the different cultivars. Expression profiles of defence‐related genes differed among cultivars in response to resistance inducers and to pathogen inoculation. T39 treatment enhanced the expression of defence‐related genes in the responsive cultivars, before and after P. viticola inoculation. A positive correlation between the efficacy of T39 and the expression level of defence‐related genes was found in Primitivo and Pinot noir plants, while different genes or more complex processes were probably activated in Sugraone and Negroamaro. The data reported here suggest that the use of a responsive cultivar is particularly important to maximize the efficacy of resistance inducers and new natural inducers should be explored for the less responsive cultivars.     

Post infection application of DL-3-amino-butyric acid (BABA) induces multiple forms of resistance against <Emphasis Type="Italic">Bremia lactucae</Emphasis> in lettuce

DL-3-amino-butyric acid (BABA) induces local and systemic resistance against disease in numerous plant species. In a recent study we showed that preventive application of BABA to lettuce (Lactuca sativa) plants induced resistance against downy mildew caused by the oomycete Bremia lactucae by callose encasement of the primary infection structures of the pathogen. Now we show that post-infection application of BABA to the foliage or the roots, even at progressive stages of disease development, is highly protective against B. lactucae. Resistance induced by BABA is manifested in multiple microscopic forms, depending on the time of its application. When applied at 1 day post inoculation (dpi) BABA induced HR in penetrated epidermal cells; at 2 dpi it caused massive encasement with callose of the primary haustoria; and, at 3 or 4 dpi it enhanced the accumulation of H₂O₂ in the developing mycelia runners and altered their colour to red. The pronounced change in the colour of the mycelium was visually apparent to the naked eye. In all cases the pathogen failed to sporulate on the treated plants. This is the first indication that an immunizing compound may be protective at advanced stages of disease development.     

Activity of the New Fungicide Benthiavalicarb Against Plasmopara viticola and its Efficacy in Controlling Downy Mildew in Grapevines

Benthiavalicarb is a new fungicide active against Oomycetes fungal plant pathogens. The present study shows that benthiavalicarb is effective for controlling the Oomycete fungal pathogen Plasmopara viticola, which causes downy mildew in grapevines. The fungicide did not affect zoospore discharge from sporangia of P. viticola, but strongly inhibited zoospore encystment, cystospore germination in vitro and mycelial growth, together with sporangial production in vivo. Benthiavalicarb showed strong prophylactic and local activity in intact plants or detached leaves and low translaminar activity. The compound was not translocated from leaf to leaf in either a acropetal or basipetal direction. Benthiavalicarb applied at 1, 3 and 6 days post-inoculation protected grapevine plants against downy mildew and inhibited sporulation of the pathogen. Similar results were obtained on leaf disks if benthiavalicarb was applied up to 96 h post-inoculation. Benthiavalicarb diminished the sporulation of P. viticola when applied to established disease in the tissue. Benthiavalicarb remained active on leaves for a period up to 28 days. Two foliar applications of benthiavalicarb, 2 weeks apart, to field-grown grapevines inhibited downy mildew development and were as effective as the standard metalaxyl-Cu treatment in controlling the disease. A formulated mixture of benthiavalicarb + Folpet was similar or superior in performance to metalaxyl-Cu and the new strobilurin trifloxystrobin in controlling downy mildew. The effectiveness of benthiavalicarb makes it well suited for integration into a control programme against downy mildew disease in vineyards, and as a component to delay resistance buildup.     

Avirulent strain of Colletotrichum induces a systemic resistance in strawberry

Strawberry plants exposed to an avirulent isolate of Colletotrichum fragariae acquired strong resistance against a virulent strain of C. acutatum. Biochemical, morphological and molecular markers indicated that the strong defence response was associated with an oxidative burst and a transient accumulation of salicylic acid (SA). A maximum accumulation of H₂O₂ and O₂ ^? was observed 8 h after inoculation (hai), callose was detected 48 hai, and a peak of SA was observed 48 hai. Biochemical and phytopathogenic analyses carried out in non-treated tissues revealed that the defence response was systemic and remained fully active 60 days after the first inoculation. Experiments also showed that the resistance acquired by mother plants after the inoculation with the avirulent isolate could be passed to daughter plants through runners. Further characterization of the induced systemic resistance showed that the resistance was not only effective against a virulent strain of C. acutatum but also against Botrytis cinerea.     

„VitiMeteo Plasmopara“ – Prognosemodell zur Bekämpfung von Plasmopara viticola (Rebenperonospora) im Weinbau

Downy mildew, caused by Plasmopara viticola, is the most important disease of European grapevine (Vitis vinifera) in humid climates. On the basis of weather data the forcasting model “VitiMeteoPlasmopara” calculates the most important steps of the life cycle of downy mildew. A growth model for the simulation of leaf development is integrated in the pure forcasting model for downy mildew. “VitiMeteoPlasmopara” was elaborated by the Swiss Research Station ACW, Changins-Wädenswil and the State Institute of Viticulture and Enology Freiburg in Germany. Since 2003 the system is scientifically tested in Switzerland and Baden-Württemberg and applied in practice. Once or twice a day, the actual results of the forecasting system are visualised on the Internet. “Vitimeteo Plasmopara” is used to forecast downy mildew in Switzerland and in the south of Germany (Baden Württemberg), comprising approx. 42,000?ha of grapevine (15,000?ha CH, 27,000?ha D), and about 100 weather stations located in the area. The forecasting system is widely used by both advisory service and growers. It has proven a very useful tool to estimate the epidemiological situation and it allows the definition of spray intervals in relation with the epidemics. The open structure of the database allows the use and the integration of weather data into any other expert software moduls to forecast the development of other fungal diseases, pests or completely other models, for example irrigation models. “VitiMeteo Growth ”, a vine growth model, “VitiMeteo Insects”, a model for pests and “VitiMeteo DataGraph”, a software to analyze and present weather data, are already developed. The expert software moduls are summarized under designation “VitiMeteo Suite”.     

Nitric oxide is involved in chitosan‐induced systemic resistance in pearl millet against downy mildew disease

BACKGROUND: The nature and durability of resistance offered by chitosan and the involvement of nitric oxide (NO) in chitosan‐induced defence reactions in pearl millet against downy mildew disease were investigated. RESULTS: It had previously been reported that chitosan seed priming protected pearl millet plants against downy mildew disease. Further elucidation of the mechanism of resistance showed that chitosan seed priming protects the plants systemically. A minimum 4 day time gap is required between the chitosan treatment and pathogen inoculation for maximum resistance development, and it was found to be durable. Chitosan seed priming elevated NO accumulation in pearl millet seedlings, beginning from 2 h post‐inoculation, and it was found to be involved in the activation of early defence reactions such as hypersensitive reaction, callose deposition and PR‐1 protein expression. Pretreatment with NO scavenger C‐PTIO and nitric oxide synthase (NOS) inhibitor L‐NAME before pathogen inoculation reduced the disease‐protecting ability of chitosan, and defence reactions were also downregulated, which indicated a possible role for NO in chitosan‐induced resistance. CONCLUSION: Protection offered by chitosan against pearl millet downy mildew disease is systemic in nature and durable. Chitosan‐induced resistance is activated via NO signalling, as defence reactions induced by chitosan were downregulated under NO deficient conditions. Copyright © 2009 Society of Chemical Industry     

Auswirkungen des Herbizids Basta® auf die Weinrebe und den Falschen Mehltau

A field experiment was conducted in order to analyse the effects of the herbicide Basta^® with glufosinate-ammonium as the active ingredient (that inhibits specifically the enzyme glutamine-synthetase) on the grapevine plant and the infestation with Plasmopara viticola, the downy mildew pathogen. The herbicide was applied at sublethal concentrations and the toxic effects on leaves and berries based on different quality parameters were estimated. Low concentrations of glufosinate-ammonium (<?0,625?mM) did not affect the chlorophyll and amino acid concentrations in leaves. This was also the case for the content of salicylic acid and phenolic compounds. Furthermore, must parameters such as sugars (glucose and fructose), acids (tartaric acid, malic acid, total acids and volatile acids), and alcohol (ethanol and glycerine) also remained unchanged compared to the fungicide-treated or untreated control, whereas the concentrations of nitrogenous components metabolically available to yeasts increased with increasing concentrations of glufosinate-ammonium. The amount of berries harvested at the end of the experiment depended on the glufosinate-ammonium concentration applied. The harvest was highest when glufosinate-ammonium was applied as a 0.1?mM solution and differed not significantly from the fungicide-treated control, but decreased with increasing herbicide doses. However, the infestation of leaves with P. viticola was significantly reduced by the application of the herbicide, even though not each infection was eradicated due to the non-systemic mode of action of the herbicide. The particular influence of the herbicide onto this host-parasite interaction still remains unclear. Since applications of glufosinate-ammonium at low concentrations seem not to affect grapevine plants negatively, but were able to reduce the infestation with downy mildew, the specific mode of action may reveal new alternatives for the control of fungal pathogens.     

Isolation, screening and identification of antagonistic downy mildew endophytic bacteria from cucumber

Cucumber is a common vegetable all over the world; however, its production is seriously affected by cucumber downy mildew caused by Pseudoperonospora cubensis. Endophytes have great potential to control such fungal diseases. The aim of the study was to isolate, screen and identify endophytic bacteria, which show antagonistic activity to downy mildew, in cucumbers. Here, surface sterilized leaves were used for endophytic bacteria isolation. All the isolates were further screened for the antagonistic activities against P. cubensis by leaf discs assay, separate leaves assay and sporangia releasing inhibition assay. The control efficacies of the endophytic bacteria (screened above) were finally evaluated through field tests. In this study, a total of 81 endophytic bacteria were isolated from cucumber leaves sampled at blossoming and fruiting stages, respectively, among which, six (strains CE1, CE8, CE9, Y1, Y2 and Y22) were tested for their strong antagonistic activity in vitro. In the field test, strain CE8, identified as belonging to Bacillus sp. based on 16S rDNA phylogenetic analysis, showed a strong control efficacy of 42.1 %, a value almost two times as high as that using 69 % metalaxyl-mancozeb WG, a widely used antifungal chemical in China. Based on the results above, CE8 was finally screened out for its great potential in cucumber downy mildew control, implicating a powerful value of the strain in the production practice.     

弱光胁迫下黄瓜霜霉病抗性评价与分析

为明确弱光胁迫下黄瓜霜霉病田间发病规律及遗传特性,筛选抗霜霉病黄瓜品种,以15份黄瓜自交系为试材,在弱光条件下通过子叶接菌诱导寄主植株发病,同时结合田间发病率及病情指数调查进行复合抗性鉴定,进而筛选抗病组合,并对其分离世代进行抗性鉴定与遗传特征分析。结果表明,供试15份材料中共筛选出4个典型品系HB1、HB2、HB3和HB4,其病情指数分别是14.17、28.71、63.33和78.33,分别属于高抗、抗病、感病和高感病类型。这4个亲本的F1代及分离世代群体中霜霉病抗性与亲本抗性均存在正相关关系,抗性亲本的后代表现出较强的抗性;F2代群体病株分离现象比较明显,呈偏态分布,有明显的主基因+多基因存在特征,表明弱光胁迫下黄瓜霜霉病受主基因+多基因控制,主基因效应值较大。经田间抗病性鉴定分析,初步筛选出组合HB12、HB13和HB21为抗霜霉病组合,可用于后续抗病品种的选育。     

Nitric oxide donor seed priming enhances defense responses and induces resistance against pearl millet downy mildew disease

Nitric oxide (NO) donors Nitroso-R-Salt, 2-Nitroso-1-Naphthol and Sodium Nitro Prusside (SNP) were evaluated for their effectiveness in protecting pearl millet [(Pennisetum glaucum L.) R. Br.] plants against downy mildew disease caused by Sclerospora graminicola [(Sacc). Schroet]. Optimization experiments with NO donors showed no adverse effect either on the host or pathogen. Aqueous SNP seed treatment with or without polyethylene glycol (PEG) priming was the most effective in inducing the host resistance against downy mildew both under greenhouse and field conditions. Potassium Ferrocyanide, a structural analog of NO donor lacking NO moiety failed to protect the pearl millet plants from downy mildew indicating a role for NO in induced host resistance. Spatio-temporal studies corroborated that the protection offered by NO donor treatment was systemic in nature and a minimum of 3-day time gap between the inducer treatment and subsequent pathogen inoculation was necessary for maximum resistance development. Disease protection ability of NO donors was also validated as durable in nature. Conversely, prior-treatment with NO scavenger 2-4-carboxyphenyl-4,4,5,5 tetrazoline-1-oxyl-3-oxide potassium salt (C-PTIO) rendered the pearl millet plants relatively susceptible for pathogen infection. Expression of primary defense responses like hypersensitive response, lignin deposition and defense related enzyme phenylalanine ammonialyase −EC 4.3.1.5 (PAL) were enhanced by NO donor treatments.     

Detection and analysis of genetic variations in GOB locus of Plasmopara viticola by DNA sequence analysis

The population genetic structure of Plasmopara viticola in Japan was analyzed using grapevine downy mildew specimens collected from two islands, Honshu and Hokkaido. By simple sequence repeat analysis with the GOB microsatellite marker and DNA sequencing, an accurate copy number of the (CT) _n (CTAT) _n repeat was determined. Consequently, we found a large number of genetic variations in (CT) _n and (CTAT) _n repeats. Also, a single nucleotide polymorphism in the (CTAT) _n repeat of the GOB locus was detected. These genetic variations may serve as a valuable tool to understand the population structure of P. viticola.     

Chitosan enhances disease resistance in pearl millet against downy mildew caused by Sclerospora graminicola and defence-related enzyme activation

BACKGROUND: The present study investigated the effect of chitosan seed priming on the induction of disease resistance in pearl millet against downy mildew disease caused by Sclerospora graminicola (Sacc.) Schroet. RESULTS: Pearl millet seeds were primed with chitosan at different concentrations: 0.5, 1.5, 2.5 and 3 g kg^?1 seed. Of the different concentrations, 2.5 g kg^?1 was found to be optimum, with enhanced seed germination of 99% and seedling vigour of 1782, whereas the untreated control recorded values of 87% and 1465 respectively. At optimum concentration, chitosan did not inhibit sporulation and release of zoospores from sporangia. Furthermore, pearl millet seedlings raised after seed treatment with chitosan showed an increased level of the defence‐related enzymes chitosanase and peroxidase as compared with the untreated pearl millet seedlings on downy mildew pathogen inoculation. The effect of chitosan in reducing downy mildew incidence was evaluated in both greenhouse and field conditions, in which respectively 79.08 and 75.8% disease protection was obtained. CONCLUSION: Chitosan was effective in protecting pearl millet plants against downy mildew under both greenhouse and field conditions by inducing resistance against the pathogen. Thus, chitosan formulation can be recommended for seed treatment in the management of downy mildew disease. Copyright © 2008 Society of Chemical Industry     

Monitoring of QoI fungicide resistance in Plasmopara viticola populations in Japan

BACKGROUND: The increasing occurrence of QoI fungicide resistance in Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew. In Japan, the existence of QoI‐fungicide‐resistant P. viticola was reported in 2009. RESULTS: The QoI fungicide resistance in P. viticola samples collected from vineyards in Japan in 2008 and 2009 was monitored. Resistant P. viticola were detected in the regions where QoI fungicides have been introduced in accordance with the pest management programme, whereas in Hokkaido vineyards, where QoI fungicides have not yet been introduced, QoI‐fungicide‐resistant P. viticola were not found. CONCLUSION: Japan comprises thousands of islands and is physically isolated from other countries by the sea. Monitoring the emergence, incidence and distribution of QoI fungicide resistance in P. viticola populations in Japan is necessary to improve pest management strategies for downy mildew disease in Japanese vineyards. Copyright © 2010 Society of Chemical Industry     

PLASMO: a simulation model for control of Plasmopara viticola on grapevine1

The problem of protecting grapevine against diseases is an old one, but in the last few years new techniques have been developed to reduce cost to the farmer and damage to the ecosystem. These are based on mathematical models describing the state of the plant-parasite environment system. A model for forecasting development of grapevine downy mildew (Plasmopara viticola) is presented. The input variables are temperature, rainfall and leaf wetness (determining infection by sporangia), and RH and temperature (for incubation period). The model also takes into account the limited survival of spores. The output is expressed as %, disease progress. Field validation tests, performed in 1990, 1991 and 1992 in several vineyards in Toscana (central Italy) showed a good correlation between observed and simulated infections. The model allowed the number of treatments to be reduced without any increase in downy mildew damage. It could in future be integrated with grapevine growth and development simulation models in an expert system to determine infected tissue area and thus the economic damage threshold.