First clues on a jasmonic acid role in grapevine resistance against the biotrophic fungus Plasmopara viticola

European Journal of Plant Pathology - Plants are sessile organisms being constantly under a wide array of environmental pressures. Their resistance against biotic stress is regulated by...     

Riboflavin (Vitamin B2) induces defence responses and resistance to Plasmopara viticola in grapevine

Grapevine (Vitis vinifera) is susceptible to a variety of pathogenic fungi that affect yield and wine quality. Their control is generally achieved by widespread application of fungicides in the vineyards. The economic costs and negative environmental impact associated with these applications has led to a quest for alternative strategies, focusing on manipulation of host defence mechanisms. Here, we evaluated the ability of riboflavin (i.e. Vitamin B₂) to induce resistance against downy mildew in grapevine. Our results showed that 2 mM riboflavin applied 1–3 days before pathogen inoculation provided a disease reduction efficiency of 86 %. A microscopic analysis of the time course of P. viticola colonization in riboflavin-treated leaf discs suggests early inhibition of hyphae spreading in the intercellular space. This resistance does not result from a direct fungitoxic effect of riboflavin. However, this vitamin activates host-defence responses including H₂O₂ generation, upregulation of an array of defence-related genes and synthesis of callose in stomata cells, while stilbene synthesis is not affected. Using a pharmacological approach, we deduced that both jasmonic acid and callose biosynthesis pathways are significantly involved in riboflavin-induced resistance against downy mildew in grapevine.     

Involvement of phenolic compounds in the resistance of grapevine callus to downy mildew (Plasmopara viticola)

Callus tissue of different grapevines (Vitis spp.) was inoculated withPlasmopara viticola. Short, highly-branched hyphae with necrosis, and long hyphae with heavy sporulation were observed on resistant and susceptible callus respectively. Thin-layer chromatography and spectrophotometric analysis showed that resistant callus contained greater quantities of gallocatechin derivatives than susceptible callus. Regression analysis between the field disease rating of each variety and its gallocatechin derivatives content indicated 92.2% correlation. Histochemical studies showed that, after infection withP. viticola, flavonoids appeared in the superficial cell walls of the callus, to a lesser degree on susceptible callus than on resistant callus. At a late stage of infection, the superficial cells of resistant callus were suberized, which did not occur in susceptible callus. This study showed that the preformed gallocatechin derivatives, the induced flavonoids and suberized superficial cells might play a role in the resistance of grapevine callus tissue to this fungus.Abbreviations CallusI Callus ofV. riparia var. Gloire de Montpellier - CallusV Callus ofV. vinifera var. Grenache - TLC Thin Layer Chromatography - var. variety - GAD Gallic acid derivatives - GD gallocatechin derivatives - RC resistant callus - SC susceptible callus     

Controlling downy mildew (Plasmopara viticola) in field-grown grapevine with β-aminobutyric acid (BABA)

Two foliar sprays of BABA (DL-3-amino-n-butanoic acid, DL-β-aminobutyric acid), or a mixture of BABA and different fungicides at reduced rates, effectively controlled (>90%) downy mildew, caused byPlasmopara viticola, in the foliage of field-grown grapevines. In five field trials BABA sprays resulted in a significant reduction of infectious leaf area and fungal sporulation, and of necrosis of oilspots. The level of disease control for BABA in four trials was similar to that achieved by metalaxyl-Cu or ‘Acrobat Plus’ (dimethomorph + mancozeb). Two-way tank-mixtures of BABA + fosetyl-AI, BABA + folpet, or BABA + Bion (benzothiadiazole), each at half the recommended rate, provided an additive effect againstP. viticola, performing as well as the full rate of each fungicide alone. BABA was not phytotoxic and did not affect pH, total titratable acids, or °Brix of the juice, as determined by commercial fungicidal standards. The results indicate that foliar applications of BABA can efficiently be integrated into a downy mildew control program in vineyards.     

Importance of secondary inoculum of Plasmopara viticola to epidemics of grapevine downy mildew

To quantify the magnitude and the spatial spread of grapevine downy mildew secondary sporangia, 4685 Plasmopara viticola single lesion samples were collected from 18 plots spread across central Europe. Disease symptoms were collected on two to 22 sampling dates per plot between 2000 and 2002. Four multiallelic microsatellite markers were used for genotypic identification of pathogen samples. Genetic analysis showed more than 2300 site-specific P. viticola genotypes, indicating that populations are genetically rich demographic units. Approximately 70% of the genotypes were sampled once and 14% were sampled twice throughout the various epidemics. In the 18 populations only seven genotypes (0.3%) were identified more than 50 times. Three genotypes particularly successful in causing disease through secondary cycles showed mainly a clustered distribution. The distance of sporangial migration per secondary cycle was less than 20 m and their plot colonization rate was calculated at around 1–2 m² day⁻¹. Downy mildew epidemics of grapevine are therefore the result of the interaction of a multitude of genotypes, each causing limited (or a few) lesions, and of a dominant genotype able to spread stepwise at plot-scale. These findings contrast with current theories about grapevine downy mildew epidemiology, which postulate that there is massive vineyard colonization by one genotype and long-distance migration of sporangia.     

Efficacy of copper alternatives applied as stop-sprays against Plasmopara viticola in grapevine

Journal of Plant Diseases and Protection - The six products, Armicarb, Vitisan, lime sulphur, Caso, Calce Fiocco and Ulmasud, were tested for their ability to control downy mildew (Plasmopara...     

Characterization of European Plasmopara viticola isolates with reduced sensitivity to cymoxanil

Cymoxanil has been used for over 30 years to control grape downy mildew (Plasmopara viticola) in European vineyards, prevalently in mixture with other fungicides active on this disease. In the 1990’s cases of P. viticola resistant to cymoxanil were detected using a leaf disc assay. In this study, we establish that the presence of only 1 % of resistant isolates in a P. viticola population will allow the detection of cymoxanil resistance in the leaf disc assay. A poor correlation (R?=?0.194) was observed between the leaf disc assay and a whole- plant test for 38 P. viticola field populations collected in 2004. Over 60 % of these populations were characterized as fully sensitive in a whole-plant assay compared to 10 % in the leaf disc assay. Five P. viticola field isolates resistant to cymoxanil reverted to full sensitivity after six to nine transfers to untreated vines, indicating that cymoxanil resistance in P. viticola is unstable. Two European P. viticola populations sensitive to cymoxanil became resistant when transferred 12–14 times on vines treated with cymoxanil. In contrast, two populations originating from the USA and three monozoospore isolates from France retained full sensitivity to the fungicide after 13 cycles on cymoxanil-treated plants. Whole-plant experiments were conducted in the laboratory to compare the efficiency of spray programs to delay the development of cymoxanil resistance. Whereas the continuous use of cymoxanil alone quickly selected for resistance, the mixture of cymoxanil and folpet applied either continuously or in strict or block alternation effectively prevented the development of resistance over 10 generations of the fungus. These results demonstrate that resistance to cymoxanil in P. viticola can be managed with appropriate spray programs.     

不同葡萄品种对霜霉病的田间苗期抗性评价

对36个葡萄品种(品系)苗期对霜霉病的田间抗病性进行了评价, 结果表明, 不同葡萄品种(品系) 对葡萄霜霉病的抗性存在明显差异, 绝大多数欧美杂交种的抗性要强于欧亚种葡萄, 供试的10个欧美杂种中, ‘瑞峰无核’对葡萄霜霉病表现为高抗; ‘DEMIR’、‘龙宝’、‘红富士’、‘黑奥林’、‘峰后’5个品种表现为中抗。     

Endophytic Beauveria bassiana activates expression of defence genes in grapevine and prevents infections by grapevine downy mildew Plasmopara viticola

Fungal entomopathogens like Beauveria bassiana (Ascomycota: Hypocreales) are known as antagonists of insects with multiple functional and ecological roles, and have attracted increased attention as biocontrol agents in integrated pest management programmes. For some crop plants, it has been proven that endophytic B. bassiana, besides its entomopathogenic habit, can provide protection against plant pathogens or limit their damaging effects. However, for grapevine, limited knowledge is available on the influence of endophytic B. bassiana on fungal pathogens and about the mechanisms underlying putative protection effects. This study assessed the protective potential of endophytic B. bassiana against grapevine downy mildew Plasmopara viticola in greenhouse experiments. Three and seven days after a B. bassiana treatment, potted grapevine plants were inoculated with P. viticola and the evolving disease severity was assessed. Disease severity was significantly reduced in B. bassiana-treated plants compared to control plants, depending on the age of leaves. Furthermore, a microarray and an RT-qPCR analysis were performed to work out fundamental aspects of genes involved in the interaction between grapevine and B. bassiana. The results indicate an up-regulation of diverse defence-related genes in grapevine as a response to endophytic establishment of B. bassiana. Thus, endophytic establishment of an entomopathogenic fungus such as B. bassiana in grapevine plants would represent an alternative and sustainable plant protection strategy, with the potential for reducing pesticide applications in viticulture.     

NO和H2O2提高葡萄抗霜霉病的生理作用机制

 以对霜霉病具有不同抗性的3个葡萄品种Fredonia、西拉和赤霞珠为材料,研究一氧化氮（NO）和过氧化氢（H₂O₂）在调控葡萄抵御霜霉病菌感染过程中的生理机制。结果表明：接种葡萄霜霉病菌后3个葡萄品种叶片中NO和H₂O₂含量均有猝发现象,H₂O₂猝发早于NO,抗性强的品种Fredonia的变化快而显著;外施一定浓度的NO供体硝普钠（SNP）和H₂O₂均可减缓霜霉病菌侵染过程,降低感病率和平均病情指数,并且能够不同程度地提高抗性弱的葡萄品种赤霞珠叶片的过氧化物酶（POD）、苯丙氨酸解氨酶（PAL）、β-1,3-葡聚糖酶（Glu）和几丁质酶（Cht）活性,增强葡萄过敏性坏死反应;而NO和H₂O₂的清除剂2-4,4,5,5-苯-四甲基咪唑-1-氧-3-氧化物（cPTIO）和抗坏血酸（AsA）在一定程度上能够提高感病率和平均病情指数。推测NO和H₂O₂可以通过提高POD、PAL、Glu和Cht病程相关蛋白活性,进而增强葡萄对霜霉病的抗性。     

A method for measuring the level of sensitivity of Plasmopara viticola populations to cymoxanil

A leaf-disk bioassay was developed for monitoring the level of sensitivity of Plasmopara viticola populations to the fungicide cymoxanil. Grapevine leaf disks were treated with various concentrations of fungicide and inoculated one day later with a suspension of P. viticola sporangia. Fungal inhibition was strongest when the leaf disks had their upper side in contact with the fungicide solution and were sprayed on the other side with the same solution. The dose response was influenced by the level of disease on the untreated leaf disks and the date of evaluation which required standardization. Test-to-test variation was small but differences existed between laboratories where the assay was performed. There was good agreement between the results obtained in the leaf-disk assay and those of a whole-plant assay.     

我国云南、湖北和山东葡萄产区霜霉病菌对甲霜灵的抗性检测

为明确我国云南省宾川县、湖北省公安县和山东省烟台市葡萄产区霜霉病菌对甲霜灵的抗性发生态势,采用叶盘漂浮法测定了这3个产区共127株葡萄霜霉病菌对甲霜灵的抗性频率及抗性水平。结果显示,不同区域间病菌的抗性频率和抗性水平均存在差异。其中,湖北省公安县霜霉病菌的抗性频率和抗性水平均较高,抗性频率达92.0%,高抗菌株占76.0%,低抗菌株占16.0%,敏感菌株占8.0%;山东省烟台市霜霉病菌的抗性频率为74.0%,低抗菌株占64.0%,高抗菌株占10.0%,敏感菌株占26.0%;云南省宾川县霜霉病菌的抗性频率和抗性水平均较低,抗性频率为29.6%,敏感菌株占70.4%,低抗菌株占29.6%,无高抗菌株。     

H2S位于H2O2下游介导葡萄抗霜霉病过程

 以霜霉病抗性葡萄品种左优红和感病品种霞多丽为材料,研究硫化氢(H₂S)在葡萄应答霜霉病过程中的作用及其与过氧化氢(H₂O₂)的相互关系。结果表明,接种霜霉病菌后,抗性品种左优红和感病品种霞多丽叶片均出现H₂S含量的猝发、H₂S合成酶基因表达量增加、抗病相关蛋白多酚氧化酶(PPO)和β-1,3葡聚糖酶(Glu)活性显著升高现象;且H₂S清除剂次牛磺酸(HT)可以显著抑制霜霉病菌诱导的PPO和Glu活性升高,并导致葡萄的感病率和病情指数显著升高;同时外源H₂S可降低两个葡萄品种的感病率和病情指数。说明,H₂S是参与葡萄抗霜霉病过程的新的信号物质。受霜霉病菌侵染后,葡萄叶片H₂O₂含量猝发早于H₂S,并且H₂O₂清除剂抗坏血酸(AsA)可显著抑制霜霉病菌所诱导的H₂S含量的上升,而H₂S清除剂HT对霜霉病菌诱导的H₂O₂含量变化影响不显著,推测,H₂S作用于H₂O₂的下游参与葡萄抵御霜霉病过程。     

Inheritance of resistance to carboxylic acid amide (CAA) fungicides in Plasmopara viticola

Mandipropamid is a new mandelic acid amide fungicide expressing high activity against foliar infecting oomycetes, including the grapevine downy mildew, Plasmopara viticola . Because cross-resistance with the valinamide fungicides iprovalicarb and benthiavalicarb and the cinnamic acid amide fungicides dimethomorph and flumorph was postulated, all five compounds are classified as carboxylic acid amide (CAA) fungicides. To support this classification, cross-resistance among these compounds with field isolates and the segregation of resistance in F₁ and F₂ progeny of P. viticola were evaluated. A bimodal distribution of sensitivity in field isolates and cross-resistance among all CAAs for the vast majority of isolates were detected. Crosses between sensitive (s) and CAA-resistant (r) isolates of opposite mating types, P1 and P2, yielded abundant oospores. All F₁-progeny isolates were sensitive to CAAs (s:r segregation 1:0), whereas in F₂ progeny segregation of about 9:1 (s:r) was observed suggesting that resistance to CAA fungicides is controlled by two recessive nuclear genes. Mating type segregated in a ratio P1:P2 of c . 2:1 in F₁ and 1:1 in F₂ progeny. In the same crosses, resistance to the phenylamide fungicide mefenoxam segregated in a ratio of c . 1:3:2 (sensitive:intermediate:resistant), reflecting the monogenic, semidominant nature of resistance. The risk of resistance in P. viticola was classified as high for phenylamide and moderate for CAA fungicides. This is the first report on the inheritance of phenotypic traits in P. viticola .     

A single nucleotide polymorphism in the β-tubulin gene distinguishing two genotypes of Erysiphe necator expressing different symptoms on grapevine

The biotrophic fungus Erysiphe necator (formerly Uncinula necator ), for which two genetic groups have been described in European vineyards, is the causal agent of grapevine powdery mildew. By analysing the pathogen population with respect to polymorphism in the sequence of the β-tubulin gene, which distinguishes two groups of isolates, a new tool was developed for epidemiological and population studies and tested in the vineyard. As in many ascomycetes, the β-tubulin gene of E. necator ( Entub ) includes six introns and seven exons and encodes a 447-amino-acid protein. A single nucleotide polymorphism (SNP) in the intron-3 region of the Entub gene distinguished two genetic groups (A and B). This method was used to examine differences in the ratio of the two groups from a total of 289 grape powdery mildew samples collected at the beginning of the growing season from either flag shoots or leaves with sparse-spot symptoms in four different vineyards. The SNP in the intron-3 region of the β-tubulin gene, similar to SNPs in the CYP51 gene, was associated with genotypes A and B of E. necator and confirmed the existence of two sympatric populations of the pathogen in the French vineyards. Differences in the relative proportions of each group varied with the presence or absence of flag-shoot symptoms and with the region in which isolates had been collected.     

A mechanistic model simulating ascosporic infections by Erysiphe necator,the powdery mildew fungus of grapevine

A new dynamic model for Erysiphe necator ascosporic infections on grapevine was developed. Between budbreak of vines and the time when the pool of ascospores is depleted, the model uses weather data for calculating, at daily intervals: curve of ascospore maturation; ascospore discharge events and relative proportion of the discharged ascospores; infection periods and their relative infection severity; and progress of latency period and time when secondary infections should begin. The model was validated over a 4‐year period (2005–2008) in 26 vineyards in Italy by comparing model predictions with actual observations of the first seasonal symptoms of powdery mildew. The model showed high sensitivity, specificity and accuracy. Proportions of true and false positive predictions were TPP = 0·94 and FPP = 0·26, respectively. Because a proportion of predicted infection periods did not result in actual disease onset, confidence was higher for prediction of non‐infections than for prediction of infections. Most of the false positive predictions occurred in the earlier growth stages of the host, when the surface area of susceptible tissue may be very small so that the probability that ejected ascospores land on susceptible tissue is low. An equation was then developed to describe the probability that a predicted infection period results in disease onset as a function of the growth stage of vines at the time of prediction. The new model should improve early season powdery mildew management by helping vineyard managers schedule fungicide sprays or schedule the scouting of the vineyard for detection of first disease signs.     

Efficiency of resistance elicitors in the management of grapevine downy mildew Plasmopara viticola: epidemiological, biochemical and economic aspects

This paper aimed to evaluate the efficiency of resistance elicitors in the management of grapevine downy mildew (Plasmopara viticola), identify the action of the elicitors on host metabolism, and determine their economic viability. The experiments were performed in a commercial vineyard variety ‘Isabel’ (Vitis labrusca) at Vale do Sirijí [Natuba, Paraiba State in Brazil, in the period of September 2009 to January 2010 (first season) and February to June 2010 (second season)]. The statistical design of randomized blocks consisted of eight treatments (untreated control, fungicide (pyraclostrobin+metiram), potassium phosphite, Agro-Mos?, Fungicide+potassium phosphite, Fungicide+Agro-Mos?, potassium phosphite+AgroMós? and Fungicide+potassium phosphite+Agro-Mos?) with four replications, with the experimental unit consisting of 45 leaves. Applications were made every 7?days, starting 20?days after pruning (DAP) with a total of 12 sprays. The evaluations were carried out biweekly, analyzing the following variables: incubation period, disease incidence and severity, area under the disease progress curve, and efficiency of control. The enzymatic determination was performed using pulp extracts from three fruits harvested at 45, 60, 90 and 120 DAP for each treatment. The resistance elicitors were able to reduce the disease incidence under different climatic conditions, indicating their viability as an alternative for the management of Plasmopara viticola.