Contribution of molecular studies to botanical epidemiology and disease modelling: grapevine downy mildew as a case-study

After being accidentally introduced from the USA at the end of the 19^th century, downy mildew caused by Plasmopara viticola (Berk. et Curt.) Berlese et De Toni became one of the most damaging diseases affecting Vitis vinifera in Europe. Downy mildew causes both direct and indirect losses and can lead to severe reduction of yield. Our understanding of the life cycle and epidemiology of P. viticola has been recently altered by molecular studies that revealed that the overwintering inoculum (i.e., the oospores) does more than initiate disease, as was previously thought. A mechanistic model was developed for predicting the entire chain of processes leading to primary infections, and this primary infection model was linked to other models of secondary infection cycles. The model for primary infections defines the length of the primary inoculum season and a seasonal oospore dose consisting of several cohorts of oospores that progressively mature. The model was evaluated by means of Bayesian analysis in both Italy and eastern Canada, and showed high sensitivity, specificity, and accuracy both for potted plants and vineyards. Fungicide applications are necessary to control downy mildew because preventive agronomic practices are not very effective, including host resistance. The use of warning systems based on weather-driven models leads to a reduction in the use and cost of chemicals and a reduction in their environmental impact.     

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     

Management of wheat blast with synthetic fungicides,partial resistance and silicate and phosphite minerals

Field trials conducted on a yellow-red latossol (pH 6.0), replicated in 2010 and 2011, sought to examine the effect of silicon, phosphite minerals, synthetic fungicides and genetic resistance for wheat blast management (Magnaporthe grisea) in Central Brazil. Disease intensity was measured on cvs. BRS 264 and BR18 subjected to the following Si treatments: pre-plant furrow application of Ca & Mg silicate (300 kg ha^-1); post-plant scattered application of Ca & Mg silicate on top of the soil (1 ton ha^-1); multiple foliar SiO₂ applications (30 g l ^-1); and non-treated control. Blast incidence and severity were scored. Further experiments were conducted on cv. BR-264, for examination of the effect of potassium phosphite and synthetic fungicides on wheat blast intensity, with the following treatments: K₂HPO₃ (1ml l ^-1); epoxinazole + pyraclostrobin (700 ml ha^-1); tebuconazole (600 ml ha^-1); tebuconazole + trifloxystrobin (750 ml ha^-1); and non-treated control. In 2010, disease intensity was lower than in 2011. In the silicate experiments, disease was significantly lower when plants were treated with foliar or furrow silicate. Si applications significantly reduced disease in BRS-264. While BR-18 consistently demonstrated lower disease levels, cv. BRS-264 generally responded more markedly to silicon applications. In the phosphite/fungicide experiment of 2010, all treatments reduced disease when compared with the control, and in 2011 phosphite efficiency was not significantly different from some fungicide treatments. Synthetic fungicides demonstrated an average blast control of 55% by severity values. Yields were increased in the phosphite-treated plots (by 9–80%), in the Si treatments (by 26–92%), and more so, and more consistently, with synthetic fungicides (by 90–121%). Combined results of all field studies, carried out under environmental conditions highly conducive to disease, indicated that control of wheat blast necessitates the joint integration of several alternatives for efficient disease management.     

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.     

Co-inoculated Plasmopara viticola genotypes compete for the infection of the host independently from the aggressiveness components

During Plasmopara viticola epidemics only few genotypes produce most of the secondary lesions and dominate in the population. Selection of dominant genotypes is hypothesized to be linked to environmental conditions and can occur rapidly, particularly if there is also difference between genotypes in terms of fitness and aggressiveness. Measurements of aggressiveness components can largely determine the rate of epidemic development, although the components of aggressiveness do not take into account potential direct competition between genotypes. Differences in aggressiveness have been also reported to be greater under non-optimal conditions suggesting for genotype adaptation to different conditions. To evaluate differences in latency at non-optimal conditions, we characterized genotypes deriving from different climatic regions at three different temperatures (15, 25 and 35 °C) and we found no differences. To investigate whether other factors may impact on competition between P. viticola genotypes, we evaluated polycyclic infections of P. viticola by co-inoculating three genotypes with similar aggressiveness components in two different co-inoculation experiments and an increasing prevalence of one of the two genotypes was observed. Competition was not related to the origin of the genotype and we hypothesize that competitive selection is modulated by differences in the secretion of effector molecules which can contribute to the establishment of dominant genotypes over an epidemic season.     

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.     

Application of fuzzy logic for the simulation of Plasmopara viticola using agrometeorological variables*

A mechanistic model called PLASMO was developed earlier to simulate grapevine downy mildew (Plasmopara viticola) and has been applied in several viticultural areas of Italy since 1988 by the collaboration of several research institutions of Firenze. In this study, a new simulation model based on fuzzy logic has been developed for the same structure (biological cycle of P. viticola). This approach allows classical quantitative information to be used together with qualitative information. Vague concepts can also be handled. Agrometeorological data is used, with an hourly time step, starting from budbreak to the end of the growing season. Air temperature, relative humidity, rainfall and leaf wetness are required. The simulated processes are the growth of grapevine leaf area and the main phases of the biological cycle of the pathogen: incubation, sporulation, germination, spore survival and inoculation. The main epidemiological outputs are timing of infection events and disease intensity. The performance of the model is evaluated and the mechanistic and fuzzy logic approaches are compared.     

Genetic Dissection of Plasmopara viticola Population from a Greek Vineyard in Two Consecutive Years

Oilspots of the Plasmopara viticola population in a Greek vineyard over 2 years were collected and the pathogen genotyped with the use of four microsatellite loci. In 2001, five samplings occurred and 327 lesions were collected, which were classified to 23 genotypes. In 2002, the 426 lesions collected in four samplings belonged to 54 genotypes. A reproducible pattern of the evolution of the epidemic was exhibited that can be described as 'predominance-of-one-clone'. In 2001, the predominant clone covered 72–92% of each sample, while in 2002 the incidence of the predominant clone was 38–90%. The remaining genotypes showed low clonal reproduction and dispersal. Oosporic infections occurred throughout the season. The severity and the diversity in 2002 were, however, higher than in 2001 and this was not in accordance with the climatic conditions, suggesting that other factors, like the epidemic of the previous year, also influence the epidemic of the disease. These results produce new concepts about the epidemiology of the pathogen.     

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.     

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.     

Haustorial development test to characterize metalaxyl resistance and genetic variability in Plasmopara viticola1

A new method is described for assessing haustorium formation in Plasmopara viticola on metalaxyl-treated grape leaves. Using this method, three types of sensitivity of P. viticola to metalaxyl could be distinguished and the precise ratio of sensitive, ‘reduced sensitive’ and resistant zoospores of selected isolates could be determined.     

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.     

The control of Plasmopara viticola with the 1:1 complex of zineb with 2-(2-aminoethylamino)ethanol and the potential effect of the use of this complex on ethylenethiourea residues

Zineb 1:1 complex with 2-(2-aminoethylamino)ethanol was shown to be more active than zineb in glasshouse and field trials against Plasmopara viticola. The effects of this increase in efficacy on the potential reduction of ethylenethiourea residues are discussed.     

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.     

„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”.     

葡萄生单轴霉重寄生菌F3的鉴定及防治效果测定

为明确从生长异常的葡萄霜霉层上分离获得的菌株F3对葡萄生单轴霉Plasmopara viticola的重寄生作用及对葡萄霜霉病的防治效果,通过光学和扫描电子显微镜观察菌株F3对葡萄生单轴霉的重寄生作用,并对菌株F3进行形态特征观察及28S rDNA序列分析,采用孢子囊萌发抑制和离体叶片点样法测定该菌株对葡萄霜霉病的防治效果。结果表明,菌株F3对葡萄生单轴霉表现出覆盖、缠绕的重寄生现象。结合形态特征与序列分析结果将该菌株最终鉴定为层生镰刀菌Fusarium proliferatum。菌株F3分生孢子悬浮液及其无菌发酵液对葡萄霜霉菌孢子囊萌发具有显著的抑制作用,抑制率分别为86.8%和83.1%;经菌株F3分生孢子悬浮液预防处理后的离体葡萄叶片发病率为10.9%,显著低于对照的发病率98.8%,防治效果达88.9%;菌株F3分生孢子悬浮液与葡萄霜霉病菌孢子囊混合后点样接种处理的叶片发病率为50.0%,也显著低于对照,防治效果为49.1%;而治疗处理的叶片发病率与对照间无显著差异。表明层生镰刀菌对葡萄生单轴霉具有重寄生作用,且对葡萄霜霉病具有较强的预防控制作用。     

Temporal disease dynamics and relative importance of sexual and asexual reproduction of grape downy mildew (Plasmopara viticola) in an isolated vineyard in the North Georgia Mountains,USA

The North Georgia Mountains are the southernmost region along the United States East Coast where European wine grapes (Vitis vinifera) are grown commercially. Epidemics of downy mildew, caused by Plasmopara viticola, are frequent and severe, but little is known about the epidemiology and population biology of the pathogen in this region. Disease monitoring in an experimental vineyard from 2015 to 2017 indicated that times of disease onset and progress rates were highly variable across years and cultivars. Oospores were observed microscopically, and simulation with a process-based model indicated presence of conditions favourable for oospore germination in the spring and early summer each year. A total of 409 P. viticola isolates collected over three  years were genotyped with seven microsatellite markers, revealing very high genotypic diversity, which when combined with the observation of oospores is indicative of a sexually reproducing population. Among the 409 isolates, 225 multilocus genotypes (MLGs) were identified, of which 164 were detected only once and 61 were repeated (clonal). Eight MLGs (represented by 28 isolates) were detected across years, suggesting the possibility of asexual overwintering of P. viticola in this region. Across sampling dates, the percentage of isolates belonging to nonrepeated (unique) MLGs ranged from 27.3% to 63.2%. Even towards the end of the annual epidemic, the percentage of isolates in nonrepeated MLGs was still relatively high, around 30%. These MLGs may have originated from oospores germinating late during the growing season, although incomplete sampling at earlier dates and contribution by immigration cannot be fully excluded.     

Rapid method for detecting resistance to a QoI fungicide in Plasmopara viticola populations

BACKGROUND: The increasing occurrence of Qo inhibitor (QoI)‐fungicide‐resistant Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew worldwide. RESULTS: The authors have developed a rapid method for detecting resistance to a QoI fungicide, azoxystrobin, in P. viticola populations using the nested PCR‐RFLP method. With this method, a glycine‐to‐alanine substitution was discovered at codon 143 in the cytochrome b gene of P. viticola populations found in Japan. CONCLUSION: It is proposed that the nested PCR‐RFLP method is a high‐speed, sensitive and reliable tool for detecting azoxystrobin‐resistant P. viticola populations. Copyright © 2009 Society of Chemical Industry     

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.     

Cryptic species and population genetic structure of Plasmopara viticola in São Paulo State,Brazil

Downy mildew (Plasmopara viticola) is one of the most important diseases in grape-growing areas worldwide, including Brazil. To examine pathogen population biology and structure, P. viticola was sampled during the 2015/16 growing season from 516 lesions on nine grape cultivars in 11 locations in subtropical areas of São Paulo State, Brazil. For identification of cryptic species, a subsample of 130 isolates was subjected to cleaved amplified polymorphic sequence (CAPS) analysis, and for 91 of these isolates the ITS1 region was sequenced. These analyses suggest that the population of P. viticola in São Paulo State consists of a single cryptic species, P. viticola clade aestivalis. Seven microsatellite markers were used to determine the genetic structure of all 516 P. viticola isolates, identifying 23 alleles and 55 multilocus genotypes (MLGs). Among these MLGs, 34.5% were clonal and represented 93% of the isolates sampled. Four dominant genotypes were present in at least five different locations, corresponding to 65.7% of the isolates sampled. Genotypic diversity (Ĝ = 0.21–0.89) and clonal fraction (0.58–0.96) varied among locations (populations). Most populations showed significant deviation from Hardy–Weinberg expectations; in addition, excess of heterozygosity was verified for many loci. However, principal coordinate analysis revealed no clusters among locations and no significant isolation by distance was found, suggesting high levels of migration. The results indicate that downy mildew epidemics result from multiple clonal infections caused by a few genotypes of P. viticola, and reproduction of P. viticola in São Paulo State is predominantly asexual.