Empirical vs. mechanistic models for primary infections of Plasmopara viticola*

Plasmopara viticola, the causal agent of grapevine downy mildew, requires fungicide treatments to avoid severe yield losses, so epidemiological models have been elaborated to better manage fungicide scheduling. Primary oosporic inoculum plays a key role in epidemic development, and some of these models have been elaborated following an empirical approach to define quantitative relationships between occurrences of primary infections and weather factors influencing them. Recently, a mechanistic dynamic model was elaborated which accounts for the biological effects of weather on the different stages of the primary infection chain, from the progressive breaking of dormancy in the overwintering oospore population to infection establishment. In this work, three widely used models (3–10 rule, EPI and DMCast) were compared with this model (named UCSC model) on the basis of their theoretical approaches and their accuracy was tested against real data collected over a 9‐year period in Sardinia (Italy). The two empirical models, 3–10 and EPI, seem to be too over‐simplified to simulate correctly a complex biological phenomenon such as the sexual stage of P. viticola they lack consideration of some key stages of the infection chain and simultaneously simulate various biological events, each of which are influenced by specific weather conditions. EPI, in particular, needs specific calibrations and ongoing processes of adaptation that make its use in disease warning difficult. DMCast overcomes these problems because of its mechanistic approach; nevertheless, the empirical method used to model oospore maturation requires validations and, as in the present case, modifications before practical use. The UCSC model, due to its fully mechanistic approach, does not present this problem and provides accurate dynamic simulations of the sexual stage of P. viticola, with a high degree of detail and requiring neither calibrations nor corrections.     

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.     

Estimating the germination dynamics of Plasmopara viticola oospores using hydro-thermal time

The effects of environmental conditions on the variability in germination dynamics of Plasmopara viticola oospores were studied from 1999 to 2003. The germination course was determined indirectly as the relative infection incidence (RII) occurring on grape leaf discs kept in contact with oospores sampled from a vineyard between March and July. The time elapsed between 1 January and the infection occurrence was expressed as physiological time, using four methods: (i) sums of daily temperatures > 8°C; (ii) hourly temperatures > 10°C; (iii) sums of hourly rates from a temperature-dependent function; or (iv) sums of these rates in hours with a rain or vapour pressure deficit ≤ 4·5 hPa (hydro-thermal time, HT). An equation of Gompertz in the form RII = exp[− a  · exp(− b  · HT)] produced an accurate fit for both separate years ( R ² = 0·97 to 0·99) and pooled data ( R ² = 0·89), as well as a good accuracy in cross-estimating new data ( r between observed and cross-estimated data were between 0·93 and 0·99, P  < 0·0001). It also accounted for a great part of the variability in oospore germination between years and both between and within sampling periods. Therefore, the equation of Gompertz (with a  = 15·9 ± 2·63 and b  = 0·653 ± 0·034) calculated over hydro-thermal time, a physiological time accounting for the effects of both temperature and moisture, produced a consistent modelling of the general relationships between the germination dynamics of a population of P. viticola oospores and weather conditions. It represents the relative density of the seasonal oospores that should have produced sporangia when they have experienced favourable conditions for germination.     

葡萄霜霉病菌PCR检测方法的建立与应用

通过对已测序和已报道的葡萄霜霉病菌［Plasmopara viticola (Berk et Curtis) Ber.et de Toni］细胞色素c氧化酶亚型2（cytochrome c oxidase Ⅱ,cox2）的基因序列进行同源性比对分析,设计合成了一对用于P.viticola的检测引物F Pv/R Pv。利用该引物对包括P.viticola在内的30种常见植物病原真菌（包括15种Plasmopara属真菌、8种葡萄常见致病菌）的基因组DNA进行了PCR扩增,验证引物F Pv/R Pv的特异性,结果表明：该引物特异性强,仅P.viticola基因组DNA作为模板的PCR扩增产物呈现一条600 bp左右的特异性条带,其他参照菌株及阴性对照均无任何条带;灵敏度验证结果表明,该检测法可以检测出3.3 pg/μL 水平的P.viticola基因组DNA;应用该方法对来自全国不同葡萄产区的不同品种的78份葡萄霜霉病菌进行了检测,结果表明,该检测法适用范围广,且检测准确率达100%。     

Assessment of QoI resistance in Plasmopara viticola oospores

QoI fungicides, inhibitors of mitochondrial respiration at the Qo site of cytochrome b in the mitochondrial bc(1) enzyme complex, are commonly applied in vineyards against Plasmopara viticola (Berk. & MA Curtis) Berl. & De Toni. Numerous treatments per year with QoI fungicides can lead to the selection of resistant strains in the pathogen population owing to the very specific and efficient mode of action. In order to evaluate the resistance risk and its development, two different methods, biological and molecular, were applied to measure the sensitivity of oospores differentiated in vineyards, both treated and untreated with azoxystrobin, from 2000 to 2004. Assays using oospores have the advantage of analysing the sensitivity of bulked samples randomly collected in vineyards, describing accurately the status of resistance at the end of the grapevine growing season. Both methods correlated well in describing the resistance situation in vineyards. QoI resistance was not observed in one vineyard never treated with QoI fungicides. In the vineyard where azoxystrobin had been used in mixture with folpet, the selection of QoI-resistant strains was lower, compared with using solely QoI. In vineyards where QoI treatments have been stopped, a decrease in resistance was generally observed.     

Sensitivity of European Plasmopara viticola populations to cymoxanil

Between 1995 and 1997, 278 grape downy mildew (Plasmopara viticola) populations originating from European vineyards were characterised for their sensitivity to cymoxanil in a leaf-disc assay. The sensitivity profile revealed a wide distribution, with minimum inhibitory concentrations (MIC) ranging from 10 to more than 800 mg litre⁻¹. EC₅₀ values ranged from 1 to more than 800 mg litre⁻¹ with an average of 125 mg litre⁻¹. The sensitivity distribution was stable between 1995 and 1997. Surprisingly, populations from Portugal appeared significantly more sensitive than those from France or Italy, which could not be linked to differential cymoxanil usage in these countries. P viticola populations collected outside Europe and never exposed to cymoxanil appeared significantly more sensitive than exposed European populations, with an average EC₅₀ value of 10 mg litre⁻¹. The level of sensitivity of European P viticola populations was relatively unaffected by the number of cymoxanil applications made during a season or by the number of years of cymoxanil use. No link was found between the level of sensitivity in the leaf-disc assay and the level of performance of the cymoxanil mixtures used in the fields where the populations originated. Specific field trials conducted in Italy and Portugal have shown that the performance of cymoxanil-based mixtures remained good even on populations of the grape downy mildew fungus characterised as less sensitive in the leaf-disc assay. While there are no baseline sensitivity data for pre-commercialisation P viticola populations, the results of our study suggest that a shift in sensitivity (12.5-fold) may have occurred in some areas since introduction of cymoxanil on grapes nearly 20 years ago. Because cymoxanil is never used alone, it is difficult to determine whether or not practical resistance is occurring in European vineyards. © 1999 Society of Chemical Industry     

Effect of water on germination of Plasmopara viticola oospores

The effect of moisture in grape leaf litter holding overwintering Plasmopara viticola oospores was investigated. Oospores were incubated under different regimes of water activity ( a_W 0·991 to 0·123) for 2 to 15 days and their ability to germinate and cause infection was determined using a sensitive leaf disk assay. Reduction of a_W caused a significant shift in the infection dynamics, with maximum effect when a_W  ≤ 0·56. Dynamics of a_W in the leaf litter under natural conditions were estimated from moisture data using a Chen-Clayton equation. Daily patterns of leaf litter moisture (M in % weight) were determined in non rainy periods between mid February and mid June, while the Chen-Clayton equation was calculated using data of a_W and M measured in both sorption and desorption conditions, at different temperatures. Water activity was highest at 08·00 hours, decreased progressively until 14·00 hours, and then increased. Water activity was favourable for oospores to develop in about 25% of the measurements, all made between 18·00 hours and 08·00 hours. A close relationship was found between vapour pressure deficit (VPD in hPa) and a_W of the leaf litter, so that when VPD is lower than 2·13 hPa there is sufficient water for oospores to develop. Results showed that leaf litter moisture due to water from the atmosphere makes oospore development possible during non rainy periods.     

Development of overwintering oospores of Plasmopara viticola and severity of primary foci in relation to climate*

The French Plant Protection Service uses different techniques to provide advice on treatments against grapevine downy mildew: observation of primary foci, development of overwintering oospores and climate‐based forecasting models. This system is generally satisfactory but is time‐consuming, and the models used sometimes give a false evaluation of the severity of primary foci. We have accumulated 30 years of observations on the latter, together with the maturation of overwintering oospores. The severity of primary foci is linked to spring and autumn rainfall. Oospore maturation is affected by low autumn and warm spring temperatures. Long dry periods can block maturation. While there is little prospect that oospore maturation can be modelled in the near future, success can be expected for primary foci.     

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.     

葡萄霜霉病菌拮抗放线菌PY-1发酵条件优化

为探讨暗黑链霉菌Streptomyces atratus PY-1液体摇瓶发酵条件,提高其活性次级代谢产物的产量,以菌体生物量和发酵液抑制葡萄霜霉病菌Plasmopara viticola活性为指标,采用单因子试验和正交试验对菌株PY-1的最适发酵培养基成分及发酵条件进行优化。结果表明,菌株PY-1最适发酵培养基为玉米粉50 g/L、葡萄糖5 g/L、蛋白胨5 g/L、氯化铵5 g/L、氯化钠 0.5 g/L;最佳发酵培养条件为培养温度28 ℃、培养时间5 d、初始pH 7.0、250 mL三角瓶装液量90 mL、接种量体积分数5%、摇床转速180 r/min。在最佳发酵培养基和培养条件下,菌株PY-1发酵液抑菌率达到99.26%,抑菌能力提高8.35%。     

Genetic diversity and population structure of Plasmopara viticola in China

Grape downy mildew is a destructive oomycete disease worldwide for viticulture. A total of 440 infected leaf lesions were collected from nineteen vineyards distributing in the major viticulture regions in China. Six specific SSR markers were used to genetically characterize the Plasmopara viticola populations. A total of 60 alleles were generated and 324 distinct genotypes were identified based on these multi-locus markers. Most populations had high levels of genetic and genotypic diversity. AMOVA analysis showed that majority of the total genetic diversity was distributed within the vineyards while moderate to high genetic differentiation was also observed among some P. viticola populations. Three populations (BJ, GX, and GS2) were clearly separated from the rest of the populations by using PCA and Nei’s genetic distance analysis. Mantel test showed no significant correlation between unbiased Nei’s genetic distance and geographical distance.     

Selection for fungicide resistance throughout a growing season in populations of Plasmopara viticola

A method for evaluating the potential threat of selection for resistance to organically-based fungicides in populations of P. viticola is needed to screen a large panel of products alternative to copper in organic viticulture. Populations from an unexposed plot were compared throughout one season with a population sprayed with azoxystrobin (Quadris), reported as engendering selection pressure and resistance, and a population sprayed with an organically-based fungicide (Mycosan). The evolution of the three populations was followed with neutral specific SSR markers and with the specific marker for strobilurin resistance, as control of selection for resistant mutants. A reduction in genetic diversity of the P. viticola population was observed in the population sprayed with azoxystrobin, consistent with directional selection toward higher resistance, confirmed by an enhanced frequency of resistant mutants with respect to the unexposed population. In contrast, a higher diversity and a reduced frequency of resistant mutants were observed in the population sprayed with the organically-based fungicide. Assessing a reduction of genotypic diversity allows the detection of selection for resistance and constitutes a valid instrument for screening a large panel of products with non-specific, different and possibly indirect modes of action.     

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.     

Sensitivity to cymoxanil in populations of Plasmopara viticola in northern Italy

In 1993, control failures were reported on grapevine in northern Italy under severe downy mildew pressure after postinfection application of cymoxanil in mixtures with copper or mancozeb. A monitoring survey was started immediately in Piedmont (north-western Italy) in order to determine the sensitivity of populations of Plasmopara viticola to cymoxanil from those vineyards where the fungicide was not controlling the disease satisfactorily. In 1994 and 1995, monitoring surveys were extended to north-eastern Italy, where cymoxanil mixtures were not performing as well as in the past. Sampled populations were tested on detached leaf discs and on whole potted plants under controlled conditions. In 1993, 12 populations, sampled in Piedmont, showed MIC values (minimum inhibitory concentration) varying from 10 to more than 100 mg L⁻¹ cymoxanil. With a baseline reference population having a MIC value of 3 mg L⁻¹, resistance factors ranged from 3 to more than 30. In 1994, 17 populations out of 27 sampled in Trentino (north-eastern Italy) showed MIC values of 100 mg L⁻¹ or higher and in 1995, 32 populations out of 38 showed the same behaviour. In similar experiments, the MIC values of populations from nontreated plots were between 3 and 10 mg L⁻¹. In whole potted plant tests, populations with MIC values higher than 200 mg L⁻¹ in a leaf disc test were not controlled by 500 mg L⁻¹ of cymoxanil. The results of our study suggest that resistance to cymoxanil in P. viticola may contribute to a lack of disease control in Italian vineyards.     

Particular Structure of Plasmopara viticola Populations Evolved Under Greek Island Conditions

ABSTRACT Plasmopara viticola populations collected from three islands in the Ionian Sea-an arm of the Mediterranean Sea to the west of Greece-were analyzed with microsatellite molecular markers in order to investigate the pathogen population structure. Downy mildew populations from mainland regions previously studied were found to have high genotypic diversity and limited clonality; however, populations under Mediterranean island conditions mostly showed limited variation and the epidemics basically were driven by the multiple clonal infections of one or a few genotypes. Populations from different islands were differentiated from each other, whereas genetic divergence also was found among subpopulations of the same plot. Polyploid individuals and individuals that overwintered in asexual form were observed in some cases. The findings obtained by this population genetics study improve our understanding of the biology of the pathogen and lead to potential alternative control measures for the disease.     

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.     

云南省葡萄霜霉病菌群体遗传结构的SSR分析

为明确云南特色葡萄产区葡萄霜霉病菌的群体遗传结构,本研究采用7对SSR分子标记对采自云南3个地区的155株葡萄霜霉病菌进行了群体遗传多样性研究,并分析了病原菌群体遗传结构与地域之间的关系。7对引物共检测出41个等位基因,114种基因型,病原菌群体的Shannon’s信息指数和Nei’s无偏基因多样性指数分别为0.942和0.600。分子方差分析和群体连锁不平衡分析显示,不同群体间存在相当大的遗传变异。遗传分化系数、基因流以及Structure分析表明,元谋群体与宾川、寻甸两群体之间均具有较高的基因交流,宾川和寻甸两群体间则存在一定的遗传分化。上述结果表明,云南葡萄霜霉病菌群体存在较高水平的遗传多样性,且群体遗传结构与地理距离之间存在一定相关性。