Assessment of fungicide resistance and pathogen diversity in Erysiphe necator using quantitative real-time PCR assays

BACKGROUND: Management of grapevine powdery mildew Erysiphe necator Schw. requires fungicide treatments such as sterol demethylation inhibitors (DMIs) or mitochondrial inhibitors (QoIs). Recently, reduction in the efficacy of DMIs or QoIs was reported in Europe and the United States. The aim of the present study was to develop real‐time qPCR tools to detect and quantify several CYP51 gene variants of E. necator: (i) A versus B groups (G37A) and (ii) sensitive versus resistant to sterol demethylase inhibitor fungicides (Y136F). RESULTS: The efficacy of the qPCR tools developed was better than the CAPS method, with a limit of 2 pg for E necator DNA, 0.06 ng for genetic group A and 1.4 ng for the DMI‐resistant allele. The detection limits of qPCR protocols (LOD) ranged from 0.72 to 0.85%, and the quantification limits (LOQ) ranged from 2.4 to 2.85% for the two alleles G47A and Y136F respectively. The application of qPCR to field isolates from French vineyards showed the presence of DMI‐resistant and/or QoI‐resistant alleles in French pathogen populations, linked to genetic group B. CONCLUSION: The real‐time PCR assay developed in this study provides a potentially useful tool for efficient quantification of different alleles of interest for fungicide monitoring and for population structure of E. necator. Copyright © 2010 Society of Chemical Industry     

Crop damage caused by powdery mildew on hop and its relationship to late season management

Powdery mildew of hop (Podosphaera macularis) may cause economic loss due to reductions in cone yield and quality. Quantitative estimates of crop damage from powdery mildew remain poorly characterized, especially the effect of late season disease management on crop yield and quality. Field studies in Washington State evaluated cone yield, bittering acid content and quality factors when fungicide applications were ceased at different stages of cone development. The incidence of cones with powdery mildew was linearly correlated with yield of cones, bittering acids and accelerated cone maturation. In cultivar Galena, the cumulative effect of every 1% increase in cones powdery mildew incidence was to reduce alpha‐acid yield by 0·33%, which was due to direct effects on cone yield but also indirect effects mediated by dry matter. In the more susceptible cultivar Zeus, alpha‐acid yield was increased 20% by controlling powdery mildew through the transition of bloom to early cone development compared to ceasing fungicide applications at bloom: additional applications provided only modest improvements in alpha‐acid yield. In both cultivars, the impact of powdery mildew on aroma characteristics and bittering acid content were less substantial than cone yield. The damage caused by powdery mildew to cone colour and alpha‐acid yield, as well as the effectiveness of fungicide applications made to manage the disease, appears inseparably linked to dry matter content of cones at harvest. Realising achievable yield potential in these cultivars requires control of the disease through early stages of cone development and harvest before maturity exceeds c. 25% dry matter.     

PCR detection of Pseudoperonospora humuli in air samples from hop yards

Downy mildew of hop, caused by Pseudoperonospora humuli , is an important disease in most regions of hop production and is managed largely with regular fungicide applications. A PCR assay specific to P . humuli and the related organism P .  cubensis was developed and used to monitor airborne inoculum in hop yards to initiate fungicide applications. The PCR amplified as little as 1 fg of genomic DNA of P . humuli , and yielded an amplicon in 70% of reactions when DNA was extracted from single sporangia. In the presence of 25 mg of soil, an amplicon was amplified in 90% of reactions when DNA was extracted from 10 or more sporangia. During nine location-years of validation, PCR detection of the pathogen in air samples occurred no later than 8 days after the appearance of trace levels of disease signs and/or detection of airborne spores in a volumetric spore sampler. Inoculum was detected on average 4·5 days before (range −8 to 14 days) the first appearance of basal spikes in six commercial yards, or 1·3 days after (range −5 to 1 days) sporangia were detected in a volumetric spore sampler in experimental plots. In commercial yards, use of PCR to initiate the first fungicide application led to enhanced disease control or a reduction in fungicide use in four of six yards compared to growers' standard practices. These results indicate that the efficiency and efficacy of hop downy mildew management can be improved when control measures are timed according to first detection of inoculum.     

New tools for studying epidemiology and resistance of grape powdery mildew to DMI fungicides

Using a Random Amplified Polymorphic DNA (RAPD) assay, we investigated the genetic polymorphism existing among 62 European isolates of the grape powdery mildew fungus (Uncinula necator [Schw.] Burr.). Isolates overwintering as mycelium in buds were genetically distinct from isolates overwintering as ascospores, suggesting the existence of two genetically isolated powdery mildew populations, and consequently of two independent sources of inoculum in the vineyard. Isolates resistant to fungicides inhibiting sterol 14α-demethylation (DMIs) were found in both populations, suggesting that resistance to DMIs may arise independently in the two powdery mildew populations. A PCR assay targeting the gene encoding U. necator 14α-demethylase has been developed which will permit an early, specific detection of U. necator infections, and may be useful for spraying programmes. ©1997 SCI     

Relating disease progress to cumulative numbers of trapped spores: apple powdery mildew and scab epidemics in sprayed and unsprayed orchard plots

A technique for relating the progress of plant diseases caused by airborne fungal pathogens to cumulative numbers of trapped spores is proposed. The relationship involves two epidemiological parameters—a disease asymptote and the infection efficiency (disease units/spore) of inoculum. The technique was evaluated using data on apple powdery mildew and scab epidemics in sprayed and unsprayed apple orchard plots. For powdery mildew, the observed relationships were close to those proposed in the unsprayed plot, but changed after or during the period of fungicide application in sprayed plots. Parameter estimates gave useful comparative information on the epidemics. The technique was not useful for scab because of the discontinuous patterns of infection.     

Is the onset of septoria tritici blotch epidemics related to the local pool of ascospores?

To elucidate the early epidemic stages of septoria tritici blotch, especially the relationship between the onset of epidemics, the local availability of primary inoculum, and the presence of wheat debris, the early disease dynamics and airborne concentration in Zymoseptoria tritici ascospores were concomitantly assessed at a small spatiotemporal scale and over two years, using spore traps coupled with a qPCR assay. One plot, with the crop debris left, provided a local source of primary inoculum, while the other plot, without debris, lacked any. According to the assay's limits of detection, daily spore trap samples were classified as not detectable or not quantifiable, detectable, and quantifiable. The proportions of samples assigned to the different classes and numbers of spores in samples classified as quantifiable were significantly different between years, time periods (from November to March) and spore trap location (field with or without debris). The effect of year on the airborne ascospore concentration was high: 22 daily peaks with more than 230 ascospores m^?3 day^?1 were identified in the autumn of 2012/13, but none in the autumn of 2011/12. The local presence of wheat debris had no obvious effect on the amount of airborne ascospores or on the earliness of the two epidemics, especially in the year with high inoculum pressure (2012/13). These results suggest that the amount of primary airborne inoculum available in a wheat crop is not a limiting factor for the onset of an epidemic.     

采用孢子萌发法测定化合物对黄瓜白粉病菌的生物活性

接种黄瓜白粉菌后16 h的黄瓜叶片用透明胶带在子叶表面粘孢子取样的平均发芽率与用台盼蓝染色后在光学显微镜下调查的发芽率分别为83.7%和85.0%,t测验无显著差异,表明透明胶带取样的发芽率可以代表黄瓜叶片上白粉病菌孢子的发芽率。采用孢子萌发法和叶碟法分别测定了醚菌酯、植物源活性组分大黄素甲醚(P3D)对8个黄瓜白粉菌菌株的EC₅₀。统计结果表明:采用两种方法测定醚菌酯P、3D对黄瓜白粉菌的EC₅₀的相关系数的平方值分别为0.880、.99,表明两种方法的测定结果有很强的相关性;采用该孢子萌发法可评价化合物对植物专性寄生病菌如白粉菌孢子萌发的生物活性。     

Efficiency of inorganic fungicides against the formation of Erysiphe necator chasmothecia in vineyards

BACKGROUND

A reduction in chasmothecia, an important inoculum of grape powdery mildew (Erysiphe necator Schwein.), is essential for disease control in vineyards; the use of fungicides during the formation of chasmothecia on vine leaves, late in the growing season, may accomplish this. Inorganic fungicides, such as sulphur, copper, and potassium bicarbonate, are very useful for this purpose because of their multisite mode of action. The aim of this study was to evaluate chasmothecia reduction using different fungicide applications late in the growing season in commercially managed vineyards and in an exact application trial.

RESULTS

Chasmothecia on vine leaves were reduced in commercial vineyards by four copper (P = 0.01) and five potassium bicarbonate (P = 0.026) applications. The positive effect of potassium bicarbonate was also confirmed in the application trial, where two applications showed lower chasmothecia numbers than the control (P = 0.002).

CONCLUSION

The application of inorganic fungicides reduced the amount of chasmothecia as the primary inoculum source. Potassium bicarbonate and copper are of further interest for disease control as these fungicides can be used by organic and conventional wine growers. The application of these fungicides should be carried out as late as possible before harvest to reduce chasmothecia formation and, consequently, the potential for powdery mildew infestation in the subsequent season. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

A model for the development of Erysiphe necator chasmothecia in vineyards

A temperature‐driven, mechanistic model predicting the development of Erysiphe necator chasmothecia in vineyards was developed and validated in 38 vineyards in the Po Valley (northern Italy), Baden‐Württemberg (Germany), and South Australia between 2005 and 2011. The model, which begins operating when the first ascocarp initials are formed, predicts on a daily basis the proportions of chasmothecia at the yellow, brown and black maturity stage. The initialization date was estimated with an iterative procedure that minimized the residuals of predicted versus observed values. In all vineyards, a drop to more favourable temperatures for ascocarp production over 2–4 days in the week or in the 2 weeks before the model initialization date probably triggered chasmothecia production. Model predictions provided a good fit of observed data (coefficients of determination, model accuracy, efficacy and efficiency were all ≥0·90), with some overestimation. When predicted production of black chasmothecia (on leaves) was compared with observed dispersal of chasmothecia from vines, lack of splashing rain was probably the main cause of overestimation. When observed numbers of yellow, brown or black chasmothecia on leaves were compared with model predictions, removal of the developing chasmothecia by rainfall was probably the main cause of overestimation. Inclusion of the effect of rainfall on the removal of immature and mature chasmothecia from the powdery mildew colonies could improve the model. The model could be used to time the application of fungicides or biocontrol agents for reducing ascocarp formation and reducing primary inoculum in the following season.     

The control of sclerotinia stem rot on oilseed rape (Brassica napus): current practices and future opportunities

Sclerotinia stem rot (SSR) caused by the phytopathogenic fungus Sclerotinia sclerotiorum is a major disease of oilseed rape (Brassica napus). During infection, large, white/grey lesions form on the stems of the host plant, perturbing seed development and decreasing yield. Due to its ability to produce long‐term storage structures called sclerotia, S. sclerotiorum inoculum can persist for long periods in the soil. Current SSR control relies heavily on cultural practices and fungicide treatments. Cultural control practices aim to reduce the number of sclerotia in the soil or create conditions that are unfavourable for disease development. These methods of control are under increased pressure in some regions, as rotations tighten and inoculum levels increase. Despite their ability to efficiently kill S. sclerotiorum, preventative fungicides remain an expensive gamble for SSR control, as their effectiveness is highly dependent on the ability to predict the establishment of microscopic infections in the crop. Failure to correctly time fungicide applications can result in a substantial cost to the grower. This review describes the scientific literature pertaining to current SSR control practices. Furthermore, it details recent advances in alternative SSR control methods including the generation of resistant varieties through genetic modification and traditional breeding, and biocontrol. The review concludes with a future directive for SSR control on oilseed rape.     

Morphological,molecular phylogenetic and pathogenic analyses of Erysiphe spp. causing powdery mildew on cashew plants in Brazil

Cashew powdery mildew is presently the most important disease of cashew trees in all Brazilian growing regions. Although it was described over a century ago, it had never threatened the Brazilian cashew industry until the first decade of the 21st century. Morphological and pathogenic evidence indicated the possibility of different pathogen species being involved in early and late types of cashew powdery mildew. This study was designed to elucidate this issue by comparing two different powdery mildew fungi occurring on cashew plants in Brazil according to the morphological characteristics, phylogenetic relationships with closely related powdery mildew fungi and pathogenic relationships. Based on morphology, molecular phylogenetics and pathogenicity on cashew, it was shown that two species of powdery mildew specimens are without question associated with cashew trees. One species, which infects young immature tissues such as shiny leaves, flowers and young fruits, is Erysiphe quercicola, while Erysiphe necator is associated exclusively with mature leaves. This is the first report of both E. quercicola and E. necator causing cashew powdery mildew, and the first detection of E. necator on cashew.     

Comparison of techniques for estimation of resting spores of Plasmodiophora brassicae in soil

Clubroot (Plasmodiophora brassicae) is an important disease of canola (Brassica napus) and other brassica crops. Accurate estimation of inoculum load in soil is important for evaluating producer risk in planting a susceptible crop, but also for evaluation of management practices such as crop rotation. This study compared five molecular techniques for estimating P. brassicae resting spores in soil: quantitative polymerase chain reaction (qPCR), competitive positive internal control PCR (CPIC-PCR), propidium monoazide PCR (PMA-PCR), droplet digital PCR (ddPCR) and loop-mediated isothermal DNA amplification (LAMP). For ddPCR and LAMP, calibrations were developed using spiked soil samples. The comparison was carried out using soil samples collected from a long-term rotation study at Normandin, Québec, with replicated plots representing 0-, 1-, 2-, 3-, 5- and 6-year breaks following susceptible canola infested with clubroot. CPIC-PCR and ddPCR provided repeatable estimates of resting spore numbers in soil compared with estimates from qPCR or LAMP alone. CPIC-PCR provided the most robust measurement of spore concentration, especially in the 2 years following a crop of susceptible canola, because it corrected for effects of PCR inhibitors. PMA-PCR demonstrated that a large proportion of the DNA of P. brassicae detected in soil after the susceptible canola crop was derived from spores that were immature or otherwise not viable. Each assay provided a similar pattern of spore concentration in soil, which supported the conclusion of a previous study at this site that resting spore numbers declined rapidly in the first 2 years after a susceptible crop, but much more slowly subsequently.     

Coincidence of maximum severity of powdery mildew on grape leaves and the carbohydrate sink‐to‐source transition

Grapevine leaves infected with powdery mildew are a source of inoculum for fruit infection. Leaves emerging on a single primary shoot of Vitis vinifera cv. Cabernet Sauvignon were exposed to average glasshouse temperatures of 18°C (0·23 leaves emerging/day) or 25°C (0·54 leaves emerging/day). All leaves on 8–10 shoots with approximately 20 leaves each were inoculated with Erysiphe necator conidia to assess disease severity after 14 days in the 25°C glasshouse. Two photosynthetic ‘source’ leaves per shoot on the remaining 8–10 shoots were treated with ¹⁴CO₂ to identify, by autoradiography, the leaf position completing the carbohydrate sink‐to‐source transition. There was a clear association between the mean modal leaf position for maximum severity of powdery mildew (position 3·7 for 18°C; position 4·4 for 25°C) and the mean position of the leaf completing the sink‐to‐source transition (position 3·8 for 18°C; position 4·7 for 25°C). The mean modal leaf position for the maximum percentage of conidia germinating to form secondary hyphae was 4·2 for additional plants grown in the 25°C glasshouse. A higher rate of leaf emergence resulted in a greater proportion of diseased leaves per shoot. A Bayesian model, consisting of component models for disease severity and leaf ontogenic resistance, had parameters representing the rate and magnitude of pathogen colonization that differed for shoots developing in different preinoculation environments. The results support the hypothesis that the population of leaves in a vineyard capable of supporting substantial pathogen colonization will vary according to conditions for shoot development.     

Relationship between Spongospora subterranea f. sp. subterranea soil inoculum level,host resistance and powdery scab on potato tubers in the field

The relationship between initial soil inoculum level of Spongospora subterranea f. sp. subterranea (Sss) and the incidence and severity of powdery scab on potato tubers at harvest was investigated. In all experiments soil inoculum level of Sss (sporeballs/g soil) was measured using a quantitative real‐time PCR assay. Of 113 commercial potato fields across the UK, soil inoculum was detected in 75%, ranging from 0 to 148 Sss sporeballs/g soil. When arbitrary soil inoculum threshold values of 0, <10 and >10 sporeballs/g soil were set, it was observed that the number of progeny crops developing powdery scab increased with the level of inoculum quantified in the field soil preplanting. In four field trials carried out to investigate the link between the amount of inoculum added to the soil and disease development, disease incidence and severity on progeny tubers was found to be significantly (P < 0·01) greater in plots with increasing levels of inoculum incorporated. There was a cultivar effect in all years, with disease incidence and severity scores being significantly greater in cvs Agria and Estima than in Nicola (P < 0·01).     

Baseline sensitivity to proquinazid in Blumeria graminis f. sp. tritici and Erysiphe necator and cross‐resistance with other fungicides

BACKGROUND: Proquinazid is a new quinazolinone fungicide from DuPont registered in most European countries for powdery mildew control in cereals and vines. The aim of this paper is to present baseline sensitivity data in populations of Blumeria graminis f. sp. tritici EM Marchal and Erysiphe necator (Schw) Burr as well as results from cross‐resistance studies with other fungicides. RESULTS: Proquinazid exhibited a high intrinsic activity on B. graminis f. sp. tritici isolates at rates ranging from 0.000078 to 0.02 mg L^?1. Erysiphe necator isolates were comparatively less sensitive to proquinazid, with EC₅₀ values ranging from 0.001 to 0.3 mg L^?1. Proquinazid controlled equally well B. graminis f. sp. tritici isolates sensitive and resistant or less sensitive to tebuconazole, fenpropimorph, fenpropidin, cyprodinil and kresoxim‐methyl. A positive correlation (r = 0.617) between quinoxyfen and proquinazid sensitivities was found among 51 B. graminis f. sp. tritici isolates. Quinoxyfen‐resistant B. graminis f. sp. tritici isolates were slightly less sensitive to proquinazid than the quinoxyfen‐sensitive isolates; however, proquinazid remained much more active than quinoxyfen on these isolates. A stronger sensitivity relationship (r = 0.874) between proquinazid and quinoxyfen was found among 65 E. necator isolates tested in a leaf disc assay. The sensitivity values for proquinazid were significantly lower than those for quinoxyfen, confirming the higher intrinsic activity of proquinazid on both pathogens. CONCLUSION: Given the history of resistance development in powdery mildew and the observed sensitivity relationship with quinoxyfen, specifically in E. necator, we conclude that the risk of resistance developing to proquinazid might be influenced by the use of quinoxyfen. Based on these results, the authors recommend that proquinazid and quinoxyfen be managed together for resistance management. Copyright © 2009 Society of Chemical Industry     

Combining sanitation and disease modelling for control of grapevine powdery mildew

Chasmothecia of Erysiphe necator form in one season, survive winter and discharge ascospores that cause primary infections and trigger powdery mildew epidemics in the next season. A strategy for powdery mildew control was developed based on (i) the reduction in overwintering chasmothecia and on (ii) spring fungicide applications to control ascosporic infections timed based on estimate risk (two to five sprays per season). Several fungicides, the hyperparasite Ampelomyces quisqualis, and a mineral oil product were first tested as separate applications in a greenhouse and in vineyards. In the greenhouse, A. quisqualis suppressed chasmothecia formation by 41 %; fungicides and mineral oil suppressed chasmothecia formation by 63 % and ascospore viability by 71 %. In vineyards, application of boscalid + kresoxim-methyl or meptyldinocap once after harvest, as well as application of A. quisqualis pre- and post-harvest, delayed disease onset and epidemic development in the following season by 1 to 3 weeks and lowered disease severity (up to the pea-sized berry stage) by 56 to 63 %. Risk-based applications of sulphur and of synthetic fungicides provided the same control as the grower spray program but required fewer applications (average reduction of 47 %). Sanitation strategies were then tested by combining products and application times (late-season, and/or pre-bud break, and/or spring). Adequate disease control with a reduced number of sprays was achieved with the following combination: two applications of A. quisqualis (pre- and post-harvest), one application of mineral oil before bud break, and model-based applications of sulphur fungicides between bud break and fruit set.     

The effect of fungal density on fungicide dose–response curves in barley powdery mildew (Erysiphe graminis f.sp. hordei)

The effect of inoculation density on fungicide dose–response curves and the estimated ED₅₀ value (the fungicide concentration needed to halve the infected leaf area relative to the infected area of leaves that had not been treated with a fungicide) were investigated theoretically, and predictions were tested experimentally using powdery mildew of barley ( Erysiphe graminis [syn. Blumeria graminis ] f.sp. hordei ). A host leaf was assumed to consist of a number of independent compartments, in each of which only a single fungal spore could germinate successfully. The number of fungal spores landing in a compartment was assumed to be Poisson-distributed. The spores were assumed to vary in their sensitivity toward a fungicide, and the sensitivity of the spore population was assumed to be normally distributed around a mean sensitivity. Under these assumptions, the ED₅₀ value was shown to be a positively biased estimate of the mean sensitivity in the fungal population, and the bias increased with inoculum density. Consequently, the estimated ED₅₀ value is expected to vary between experiments and laboratories if the inoculation density varies over a considerable range. The correlation between inoculum density and estimated ED₅₀ value is expected to be strongest when the assayed fungal population consists of several genotypes differing in sensitivity. This expectation was tested by estimating the ED₅₀ values for one barley powdery mildew isolate at different inoculation densities for three different fungicides. These ED₅₀ values were positively correlated strongly with the inoculum density for fenpropimorph and more weakly for triadimenol, whereas no correlation was observed for propiconazole.     

宁夏温室瓜菜白粉病菌鉴定及病害流行预测模型构建

为明确宁夏回族自治区温室瓜菜白粉病菌的分类地位,对采自该地区温室的南瓜、黄瓜和甜瓜上的白粉病菌基于ITS序列分析进行分子鉴定;利用孢子捕捉器对温室中甜瓜白粉病菌的孢子量进行监测,分析环境因子、孢子量和病情指数之间的关系,并采用逐步回归分析法构建温室甜瓜白粉病的流行预测模型。结果表明,基于ITS序列的分子鉴定结果,3种瓜菜白粉病的病原菌均为单囊壳白粉菌Podosphaera xanthii。发病期间,每日温室中甜瓜白粉病菌的孢子量在12:00—16:00时段最多,占24 h内总孢子量的34%~81%,20:00—08:00时段最少;白粉病菌孢子的释放与光照强度有关,相关系数为0.602。第t天的病情指数与标准累积温度、标准累积湿度、t-4 d前08:00—12:00时段的累积孢子量、第t-4天16:00—20:00时段的孢子量均具有显著的相关性,相关系数分别为0.935、0.938、0.956和0.921。以标准累积湿度和第t-4天16:00—20:00时段的孢子量为预测变量构建了温室甜瓜白粉病流行预测模型,决定系数为0.962,表明该模型具有较好的实际应用价值。     

Spatiotemporal variation in airborne sporangia of Phytophthora infestans: characterization and initiatives towards improving potato late blight risk estimation

This study investigated the value of using real‐time monitoring of Phytophthora infestans airborne inoculum as a complement to decision support systems (DSS). The experiment was conducted during the 2010, 2011 and 2012 potato production seasons in two locations in New Brunswick, Canada. Airborne sporangia concentrations (ASC) of P. infestans were monitored using 16 rotating‐arm spore samplers placed 3 m above the ground. The first cases of late blight (2010 and 2011) were detected 6–7 days after the first ASC peak, and all samplers captured their first sporangia within the same week (at 3‐ and 9‐day periods). The cumulative ASC curve and the risk curves from two DSS (PLANT‐Plus and Pameseb Late Blight) had the same shape but different magnitudes. In both locations, the negative binomial distribution fitted the data better than the Poisson distribution, which is indicative of heterogeneity, and based on Taylor's power law, the heterogeneity increased with increasing ASC. Therefore, the present results suggest that spore‐sampling network devices may be a suitable approach for early detection of incoming inoculum and, when combined with DSS, represent a potential aid for targeting the optimal time to apply a disease‐control product. In this context, cumulative ASC can be a counterweight to the DSS risk estimate: a high risk combined with significant ASC will trigger fungicide spraying. Moreover, spore sampling can be used to assess the efficiency of management strategies by means of examining the area under the inoculum progress curve.