Glycosylation and oxidative dimerization of resveratrol are respectively associated to sensitivity and resistance of grapevine cultivars to downy mildew

Levels of resistance to Plasmopara viticola, from susceptible to highly resistant, of different grapevine cultivars were observed in vineyards and confirmed by the symptoms developed after inoculations. On the abaxial surface of infected leaves, P. viticola developed abundant sporangiophores on susceptible cultivars (Chasselas, Gamay, Gamaret and Pinot Noir), whereas on less susceptible cultivars (Seyval Blanc and Johanniter) the parasite produced few sporangiophores and some necrotic spots at the site of infection. On resistant cultivars (Bronner, Solaris, IRAC 2091), P. viticola induced a hypersensitive response and only necrotic spots were visible and the disease ceased to develop. Stilbenes were analyzed 4, 7, 24, 48 h post-infection (hpi) on small leaf samples cut from the site of infection. Large differences were observed between the cultivars at 24 and 48 hpi. Susceptible grapevines produced resveratrol and its glycoside, piceide. In contrast, resistant plants produced high concentration of ε- and δ-viniferin. Resveratrol and piceide have little or no toxicity activity against P. viticola, whereas δ-viniferin is highly toxic and can be considered an important marker for resistance of grapevine to downy mildew. The importance of oxidative dimerization of resveratrol in comparison to the extent of its glycosylation in defense reaction of grapevines against P. viticola is discussed.     

暗黑链霉菌PY-1活性产物分析及其对葡萄霜霉病田间防效评价

为探究暗黑链霉菌Streptomyces atratus PY-1对葡萄霜霉病菌Plasomopara viticola的抑制作用,通过扫描电镜观察PY-1发酵滤液对葡萄霜霉病菌孢子囊的影响,检测PY-1的活性物质种类,并测定其对葡萄霜霉病的田间防效。结果表明,PY-1发酵滤液能够导致葡萄霜霉病菌孢子囊和孢子囊梗出现褶皱、破裂和畸形,进而使其丧失侵染功能。PY-1菌株代谢产物中包含几丁质酶、蛋白酶、嗜铁素、1-氨基环丙烷-1-羧酸脱氨酶、氰化氢、吲哚乙酸,不含纤维素酶。PY-1菌株对葡萄霜霉病具有较好的田间防效,发酵原液对葡萄霜霉病的田间中期防效可达到89.17%以上,末期防效达86.28%以上,比52.5%噁唑菌酮·霜脲氰2 000倍液的防效略低,但显著高于58%甲霜·锰锌1 000倍液的防效;PY-1菌株发酵液稀释700倍后对葡萄霜霉病的末期防效与甲霜锰锌1 000倍液防效相当。表明PY-1菌株具有研制防治葡萄霜霉病生防制剂的潜力。     

Role of stilbenes in the resistance of grapevine to powdery mildew

Stilbene phytoalexins are identified as defence response in pathogen–grapevine interactions, but little information is available on the role of stilbenes on Erysiphe necator, causal agent of grapevine powdery mildew. Analysis of stilbenes in artificially infected leaf discs from susceptible to highly resistant cultivars was performed and compared to the development of the pathogen. Results indicate that stilbene synthesis is confined in infected cells, penetrated by an appressorium–peg. Stilbene amounts expressed by infection site allow discriminating susceptible and resistant cultivars. Highest viniferins concentrations on resistant cultivars are in correspondence with the observed inhibition of the pathogen growth. The analysis of stilbenes at the infection site and viniferins accumulation in grapevine defence reaction is discussed.     

新疆不同地区葡萄霜霉病菌致病性分化及遗传多样性的分析

为了解新疆不同地区葡萄霜霉病菌Plasmopara viticola的遗传特征,分别采用叶盘接菌法及SSR分子标记技术对采自新疆吐鲁番、阿克苏、石河子等11个不同产区的葡萄霜霉病菌菌株的致病性进行测定,并分析各菌株的遗传多样性。结果显示,来自新疆不同地区的菌株种内存在着致病性分化现象,依据其在鉴别寄主上致病性的差异,将供试菌株划分为强、中、弱3类,其中强致病性菌株为优势菌株,且种间致病性分化与菌株的地理来源无关;SSR标记结果表明,供试菌株之间存在遗传变异现象,并且菌株之间亲缘关系都较近,在相似系数为0.93时,48株菌株聚为4大类,且遗传分化与地理分布具有一定的相关性。研究表明新疆不同地区的葡萄霜霉病菌菌株之间存在致病性分化与遗传变异现象。     

宁夏葡萄霜霉病菌致病型鉴定及葡萄品种抗性评价

为合理利用葡萄抗性品种以及田间病害综合治理,采用离体叶盘接种法分别对2018年和2019年自宁夏回族自治区银川市、石嘴山市和吴忠市采集的43株葡萄霜霉病菌Plasmopara viticola菌株进行致病型鉴定及聚类分析,并对17个主栽葡萄品种进行抗性评价及聚类分析。结果表明：2018、2019年供试菌株致病力和供试葡萄品种抗性之间均存在显著差异,菌株平均病情指数与发病率呈显著正相关,皮尔逊相关系数r和回归系数R²分别介于0.45~0.96和0.20~0.92之间;2018、2019年供试菌株的强致病力菌株和弱致病力菌株分别为CS-YL和VI-BYDD、RG-JSJG和CS-YM;2018年和2019年供试菌株致病型被划分为强、中、弱3种类群,2年所占比例分别为10.00%、15.00%、75.00%和34.78%、39.13%、26.09%;供试17个葡萄品种的抗性类型可被划分为感病、抗病、高抗3种类群,2年所占比例均为17.65%、29.41%和52.94%。不同葡萄品种对不同菌株的抗性类型多样化。表明宁夏回族自治区葡萄霜霉病菌菌株致病力分化明显,不同菌株的致病型与其地域来源无关,不同葡萄品种的抗感程度不同。     

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.     

芽胞杆菌与嘧菌酯协同应用对葡萄叶部微生物群落及霜霉菌线粒体功能的影响

为明确生防菌剂枯草芽胞杆菌Bacillus subtilis HMB20428与化学杀菌剂嘧菌酯协同应用对葡萄霜霉菌Plasmopara viticola的抑制机制,结合生物学测定与显微表型观察方法分析其对葡萄霜霉病菌及活孢子囊的抑制作用,采用高通量测序技术分析其对葡萄叶部微生物种群多样性的影响,通过测定生理生化指标研究其对葡萄霜霉菌线粒体功能的作用。结果显示,枯草芽胞杆菌HMB20428与嘧菌酯协同应用对葡萄霜霉病菌的抑制作用显著,对活孢子囊的抑制率为99.05%;两者协同应用对微生物细菌种群有显著影响,枯草芽胞杆菌的优势占位作用明显;但对微生物真菌种群无显著影响;两者协同应用造成葡萄霜霉菌线粒体膜通透性转运孔开放度增加至90.59%,对跨膜电位的抑制率为94.29%,呼吸链酶复合物Ⅰ～Ⅴ的活性分别显著下降56.39%、22.47%、47.90%、60.52%和90.91%,说明葡萄霜霉菌线粒体膜在受到明显损伤和出现代谢功能障碍的同时,呼吸链酶活性明显被抑制,从而导致葡萄霜霉菌的能量代谢和生长受阻。表明枯草芽胞杆菌HMB20428与嘧菌酯协同应用时主要通过促进枯草芽胞杆菌竞争占位和增...     

沈阳地区葡萄霜霉病流行速率、空中孢子囊密度与环境因素的相关性

为明确葡萄霜霉病流行速率、空中孢子囊密度与环境因素之间的相关性,以感病品种红地球为试材,采用田间小区试验系统调查了沈阳地区葡萄霜霉病的流行动态,并对空中孢子囊密度进行定时捕捉,结合田间环境监测分析不同变量之间的相关性。结果显示,葡萄霜霉病的流行规律为:指数增长期为5月1日至7月23日,逻辑斯蒂增长期为7月23日至8月19日,衰退期为8月19日至葡萄生育末期。整个生长季葡萄霜霉病流行速率呈正态分布,其变化动态与该病害的流行时期特点基本一致。单季病害流行期,流行速率与温度、前5 d平均相对湿度均呈显著正相关,相关系数分别为0.442和0.409;在始发期和盛发期,空中孢子囊密度与流行速率、前5 d平均相对湿度、前5 d平均叶面湿润时数、前5 d积累降雨量基本均呈显著正相关,相关系数分别为0.753和0.768、0.836和0.651、0.651和0.834、0.954和0.747;与当日降雨量呈负相关,相关系数分别为-0.473和-0.542。表明上述与葡萄霜霉病流行相关性较高的因素可作为构建葡萄霜霉病预测模型的关键因子。     

樟树枝叶提取物防治葡萄霜霉病的效果

为明确樟树枝叶提取物对葡萄霜霉病的防治效果,采用混合液滴显微镜观察法研究了樟树枝叶提取物及其活性成分对病原菌孢子囊萌发的抑制作用,利用高效液相色谱法鉴定了枝叶乙酸乙酯萃取物的化学成分,测定了其活性成分对葡萄霜霉病的室内和田间防治效果。结果表明,浓度为0.1 g/mL以上的樟树枝叶提取物对孢子囊萌发均有很好的抑制效果,抑制率达100.0%;樟树枝叶提取物经不同极性有机溶剂萃取后,分别获得正己烷、乙酸乙酯、正丁醇和水相分离物,浓度为1.0%时各分离物对孢子囊萌发的抑制率分别为58.4%、92.4%、32.5%和58.7%,乙酸乙酯相富集了主要抑菌活性成分,经液相色谱分析并与色谱库数据比对,确定其活性成分中含有芦丁和槲皮素;浓度为1.0%的芦丁和槲皮素对孢子囊具有抑制作用;田间试验显示,浓度为0.3 g/mL以上的枝叶提取物对葡萄霜霉病预防效果显著,治疗效果在60.0%以上。研究表明,樟树枝叶提取物可以有效控制葡萄霜霉病,提取物的活性成分芦丁和槲皮素可以作为植物源药剂。     

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.     

Evidence for sporangial dispersal leading to a single infection event and a sudden high incidence of grapevine downy mildew

In a German vineyard (PRO) between 10 and 13 June 2004, the incidence of grapevine downy mildew ( Plasmopara viticola ) increased abruptly from 0 to 99%. Infected vines bore on average between 45 and 60 lesions each, corresponding to about 220 000 lesions ha⁻¹ in a non-aggregated distribution. A second vineyard (FUT), approximately 50 m distant from PRO, had been inoculated 3 weeks before the abrupt increase in incidence of disease in PRO. Using microsatellites to ascertain the sources of inoculum and likelihood and extent of interplot spread from FUT to PRO, 555 samples were collected and 20 unique genotypes were identified, of which one caused 80% of the sampled lesions in both vineyards. Three genotypes responsible for 95% of the lesions in FUT and PRO were identified as the genotypes originally established through earlier inoculations in FUT. This is the first report of definitive and quantitative evidence of sporangial migration up to 130 m in a single infection event. The utility of molecular tools to address practical epidemiological issues in this pathosystem is illustrated. The results of this study provide an example of how P. viticola was able to rapidly colonize European vineyards after the pathogen was introduced from North America in 1878.     

葡萄叶提取物对葡萄霜霉病的防治作用及有效成分分析

为探究葡萄叶提取物对葡萄霜霉病的防治作用,测定了葡萄叶提取物及其有效物质对葡萄生单轴霉Plasmopara viticola孢子囊和游动孢子的抑制作用以及对葡萄霜霉病的防治效果,观察了其对葡萄生单轴霉在叶片中扩展、产生孢子囊能力的影响,并对葡萄叶提取物组成成分进行了分析。结果表明,葡萄叶提取物对葡萄生单轴霉孢子囊的形成和萌发均有明显的抑制作用。当葡萄叶提取物浓度为10 mg/mL时,其对孢子囊形成的抑制率为82.4%,葡萄生单轴霉孢子囊和游动孢子的萌发率仅为7.6%和1.1%,均显著低于无菌水处理。荧光显微观察发现,葡萄叶提取物可以抑制葡萄生单轴霉在叶片内的扩展。当葡萄叶提取物浓度为10 mg/mL和50 mg/mL时,对葡萄霜霉病的室内防治效果分别为80.1%和100.0%;且浓度为10 mg/mL的葡萄叶提取物对葡萄霜霉病的田间预防效果为67.8%,与烯酰吗啉处理无显著差异。液质联用技术检测显示,葡萄叶提取物中含有白藜芦醇和紫檀芪,其对葡萄生单轴霉均有显著的抑制作用,且紫檀芪的抑制效果强于白藜芦醇。     

葡萄霜霉病抗病性鉴定方法及品种抗病性测定

本文通过对葡萄霜霉病抗病性鉴定方法进行优化,建立了一种更为快捷、方便、可靠的鉴定方法,并对32个葡萄品种对霜霉病抗病性进行了鉴定,为葡萄抗病品种的选育和应用提供依据。结果显示:以田间混合的霜霉病菌为接种体采用叶盘法鉴定葡萄品种的抗病性更加快捷、方便。供试的32个葡萄品种对葡萄霜霉病的抗性存在显著差异。其中免疫品种有‘康拜尔早生';高抗的有‘阳光玫瑰'、‘美乐'、‘Ms27-31'等5个品种;中抗的有‘贝达'、‘小芒森'、‘2E-16-2'等6个品种;低抗的有‘瑞都红玉'、‘早黑宝'、‘摩尔多瓦'等10个品种;感病的有‘里扎马特'、‘玫瑰香'、‘香妃'等10个品种。     

First report of oospore formation in Plasmopara viticola,the causal agent of grapevine downy mildew,in highland regions of southern Brazil

This work evaluates the formation of oospores of Plasmopara viticola, the causal agent of grape downy mildew (DM), in highland regions in southern Brazil. Leaves of susceptible and resistant grape genotypes naturally infected with the pathogen were collected in the autumn of 2017, 2018, and 2019 from vineyards located in the highlands of Santa Catarina state. Leaf tissues were evaluated by light microscopy and scanning electron microscopy. Oospores of P. viticola were identified in both susceptible and resistant host genotypes. They were concentrated in the central regions of the DM lesions, close to the leaf veins, and exhibited a rounded shape, yellowish colour, thick wall, and a diameter ranging from 16.28 to 49.15 µm. The formation of oospores is strong evidence that sexual reproduction is occurring in P. viticola in the climatic conditions of the highlands of southern Brazil. Sexual reproduction contributes to the maximization of genetic diversity via meiosis. Populations with high genetic variability are more likely to break resistance mechanisms conferred by resistance genes and to develop resistance to fungicides applied for disease control. To our knowledge, this is the first scientific study to prove the formation of P. viticola oospores in Brazil. The results presented provide a solid basis for further studies on sexual recombination in P. viticola. Genetic improvement programmes for grapevines, disease management methods, and disease prediction models need to consider the sexual reproduction of this pathogen, otherwise their effectiveness may be compromised.     

Fungicide modes of action and resistance in downy mildews

Among oomycetes, Plasmopara viticola on grape and Phytophthora infestans on potato are agronomically the most important pathogens requiring control measures to avoid crop losses. Several chemical classes of fungicides are available with different properties in systemicity, specificity, duration of activity and risk of resistance. The major site-specific fungicides are the Quinone outside inhibitors (QoIs; e.g. azoxystrobin), phenylamides (e.g. mefenoxam), carboxylic acid amides (CAAs; e.g. dimethomorph, mandipropamid) and cyano-acetamide oximes (cymoxanil). In addition, multi-site fungicides such as mancozeb, folpet, chlorothalonil and copper formulations are important for disease control especially in mixtures or in alternation with site-specific fungicides. QoIs inhibit mitochondrial respiration, phenylamides the polymerization of r-RNA, whereas the mode of action of the other two site-specific classes is unknown but not multi-site. The use of site-specific fungicides has in many cases selected for resistant pathogen populations. QoIs are known to follow maternal, largely monogenic inheritance of resistance; they bear a high resistance risk for many but not all oomycetes. For phenylamides, inheritance of resistance is based on nuclear, probably monogenic mechanisms involving one or two semi-dominant genes; resistance risk is high for all oomycetes. The molecular mechanism of resistance to QoIs is mostly based on the G143A mutation in the cytochrome b gene; for phenylamides it is largely unknown. Resistance risk for CAA fungicides is considered as low to moderate depending on the pathogen species. Resistance to CAAs is controlled by two nuclear, recessive genes; the molecular mechanism is unknown. For QoIs and CAAs, resistance in field populations of P. viticola may gradually decline when applications are stopped.     

Induction of resistance against downy mildew pathogen in pearl millet by a synthetic jasmonate analogon

The synthetic 1-oxo-indanoyl-l-isoleucine methyl ester (Ind-Ile-Me) represents a highly active mimic of octadecanoic phytohormones, which are involved in plant defenses against pathogens and pests. Seed treatments and foliar spray with Ind-Ile-Me were tested for induced resistance against downy mildew disease caused by the phytopathogenic oomycete Sclerospora graminicola in pearl millet (Pennisetum glaucum) under greenhouse and field conditions. Under greenhouse conditions, a 50% protection level was achieved after seed treatment. Seed treatment in combination with foliar spray resulted in 60% protection. The induction of resistance was correlated with the enhanced activities of defense-related proteins such as phenylalanine-ammonia-lyase, peroxidase, and enhanced level of hydroxyproline-rich glycoproteins. Under field conditions, a maximum protection of 62% was recorded upon seed treatment along with foliar spray. Hence, it infers that Ind-Ile-Me can be used as a valuable protection compound at least in downy mildew disease management.     

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。     

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.     

Microscopical studies of the effect of metalaxyl on the interaction between sunflower,Helianthus annuus L. and downy mildew,Plasmopara halstedii

The mode of action of the fungicide metalaxyl againstPlasmopara halstedii, the causal agent of sunflower downy mildew, was studied following its application before, during and after artificial contamination of seedlings. The development of the fungus within the treated seedlings was examined microscopically and compared to that occuring in untreated genetically susceptible or resistant genotypes. Hypersensitive-like reactions and necrotic zones leading to the inhibition of fungus growth within the hypocotyl were observed for the three modes of application. This suggests that sunflower defence mechanisms activated by genetical resistance are also involved in the control of downy mildew by metalaxyl.