Quantitative Insights into Grapevine Anthracnose (Elsinoë ampelina) Epidemiology: Impact of Temperature and Leaf Age on Incubation, Lesion Development, and Sporulation.

Grapevine anthracnose, caused by Elsinoë ampelina, is one of the most devastating diseases for wine and table grapes, particularly in hot, humid regions. This study explores how temperature and leaf age affect incubation and how temperature affects lesion development and sporulation. The influence of temperature and leaf age on incubation period (days) was tested under controlled conditions. Leaves from 1 to 8 days old were inoculated and maintained at temperatures of 5, 10, 15, 20, 25, and 30°C. The time elapsed between inoculation and the emergence of initial lesions was recorded. The effect of temperature on lesion development and sporulation was investigated under vineyard conditions. This was achieved through artificial inoculations, with 17, 11, and 11 inoculations conducted in 2016, 2017, and 2018, respectively. The average incubation period, considering all leaf ages, was 27.50 days at 5°C, 15.10 days at 10°C, 9.70 days at 15°C, 5.90 days at 20°C, 3.70 days at 25°C, and 2.26 days at 30°C. Regardless of temperature, the average incubation period was 3.6, 5.9, 8.3, 9.8, 11.9, 13.4, 15.6, and 17.1 days for leaves 1, 2, 3, 4, 5, 6, 7, and 8 days old, respectively. The exponential decay model accurately describes the incubation period as a function of both temperature and leaf age. On average, the relative lesion development (RLD) was 0.00, 0.00, 0.23, 0.47, 0.72, 0.93, 0.92, 0.90, 0.94, and 1.0 at 0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 days after inoculation, respectively. The average relative sporulation (RSPO) was 0.03, 0.36, 0.82, 0.96, and 1.0 at 5, 10, 15, 20, and 25 days after inoculation, respectively. Both RLD and RSPO as a function of degree-days (Tbase = 0°C) since inoculation were well described by the logistic function. The rates of change in RLD and RSPO were 0.055 and 0.032, respectively. The results of this study provide new quantitative insights into three important stages (monocyclic processes) in the development of grapevine anthracnose caused by E. ampelina.

Cluster Zone Leaf Removal Reduces the Rate of Anthracnose (Elsinöe ampelina) Progress and Facilitates Its Control.

Anthracnose caused by Elsinöe ampelina is an economically important disease that affects certain hardy and semihardy grapevine cultivars. The control of this disease requires repeated application of fungicides, which has financial and environmental consequences. In this study, leaf removal in the cluster area was studied with a view to facilitating integrated anthracnose management. First, the effect of leaf removal timing (BBCH stage 53 or 71) and intensity (one or both sides of rows) on the progression of anthracnose and on the microclimate was studied in plots planted with Vidal blanc (Vitis vinifera) at two sites in both 2020 and 2021. Overall, at both sites and in both years, anthracnose on leaves was more severe in plots without cluster zone leaf removal. Regardless of the timing of leaf removal, anthracnose severity on leaves and incidence of infected berries at harvest were significantly lower in plots where leaves had been removed on both sides of the rows compared with plots where leaves were removed on one side only. Second, anthracnose management programs with leaf removal, with or without disease risk estimation, were evaluated. All anthracnose management programs including leaf removal in the cluster zone reduced anthracnose development compared with the standard program without leaf removal. Overall mean leaf anthracnose severity, severity at harvest, and anthracnose incidence on clusters at harvest were lower in plots with leaf removal than in the standard program, but the differences between the two treatments were not significant (P > 0.05). More fungicide applications were made in plots managed using the standard programs, specifically 13 applications, compared with plots managed based on assessing the weather-related risk of anthracnose, with 9 and 10 applications made at sites 1 and 2 for the risk-based program, respectively, and 5 and 7 applications made at sites 1 and 2, respectively, when microclimate within the cluster zone was considered. The results of this study clearly show the important role that leaf removal can play in managing grape anthracnose.

Why Do We Need Alternative Methods for Fungal Disease Management in Plants?

Fungal pathogens pose a major threat to food production worldwide. Traditionally, chemical fungicides have been the primary means of controlling these pathogens, but many of these fungicides have recently come under increased scrutiny due to their negative effects on the health of humans, animals, and the environment. Furthermore, the use of chemical fungicides can result in the development of resistance in populations of phytopathogenic fungi. Therefore, new environmentally friendly alternatives that provide adequate levels of disease control are needed to replace chemical fungicides-if not completely, then at least partially. A number of alternatives to conventional chemical fungicides have been developed, including plant defence elicitors (PDEs); biological control agents (fungi, bacteria, and mycoviruses), either alone or as consortia; biochemical fungicides; natural products; RNA interference (RNAi) methods; and resistance breeding. This article reviews the conventional and alternative methods available to manage fungal pathogens, discusses their strengths and weaknesses, and identifies potential areas for future research.

Combining Desirable Traits for a Good Biocontrol Strategy against Sclerotinia sclerotiorum.

The fungal pathogen Sclerotinia sclerotiorum (Helotiales: Sclerotiniaceae) causes white mold, a disease that leads to substantial losses on a wide variety of hosts throughout the world. This economically important fungus affects yield and seed quality, and its control mostly relies on the use of environmentally damaging fungicides. This review aimed to present the latest discoveries on microorganisms and the biocontrol mechanisms used against white mold. A special focus is put on the identification of biocontrol desirable traits required for efficient disease control. A better understanding of the mechanisms involved and the conditions required for their action is also essential to ensure a successful implementation of biocontrol under commercial field conditions. In this review, a brief introduction on the pathogen, its disease cycle, and its main pathogenicity factors is presented, followed by a thorough description of the microorganisms that have so far demonstrated biocontrol potential against white mold and the mechanisms they use to achieve control. Antibiosis, induced systemic resistance, mycoparasitism, and hypovirulence are discussed. Finally, based on our actual knowledge, the best control strategies against S. sclerotiorum that are likely to succeed commercially are discussed, including combining biocontrol desirable traits of particular interest.

Competition Between Plasmopara viticola Clade riparia and Clade aestivalis: A Race to Lead Grape Downy Mildew Epidemics.

There is evidence of five clades of Plasmopara viticola in the world. Only two clades, riparia and aestivalis, have been identified as responsible for downy mildew epidemics in Quebec, Canada. It was reported in 2021 that epidemics caused by clade riparia start 2 or 3 weeks before those caused by clade aestivalis and that clade aestivalis was more aggressive than clade riparia. The objective of this work was to study the competition between P. viticola clade riparia (A) and clade aestivalis (B) and to compare the aggressiveness of both clades in mono- and coinfection situations. Suspensions of sporangia from both clades with six percentage combinations (AB 100-0; AB 89-11; AB 74-26; AB 46-54; AB 23-77; and AB 0-100) were inoculated on leaf discs (cultivar Vidal), and three other combinations (AB 88-12; AB 68-32; and AB 47-53) were inoculated on living leaves of grape plants (cultivar Vidal). Then, sporangium production, expressed as the percentage of sporangia produced by each clade, was estimated on leaf discs after eight cycles of infection-sporulation and then validated on living grape leaves after five cycles. The aggressiveness of clades in monoinfection situations on leaf discs was compared with that in coinfection situations. The results show that the percentage of sporangia produced by clade aestivalis increases with the infection-sporulation cycle while that produced by clade riparia decreases. The area under the sporangium production progress curve (AUSPPC) of clade aestivalis was significantly higher than that of clade riparia. The aggressiveness of P. viticola clades riparia and aestivalis in coinfection situations was different from that in monoinfection situations and was strongly influenced by the percentage of each clade in competition. These results suggest that, on the grapevine cultivar Vidal, P. viticola clade aestivalis is more competitive than clade riparia and that the percentage of each clade present in the vineyard should be considered for management of downy mildew.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Genotyping by sequencing suggests overwintering of Peronospora destructor in southwestern Québec, Canada.

Several Peronospora species are carried by wind over short and long distances, from warmer climates where they survive on living plants to cooler climates. In eastern Canada, this annual flow of sporangia was thought to be the main source of Peronospora destructor responsible for onion downy mildew. However, the results of a recent study showed that the increasing frequency of onion downy mildew epidemics in eastern Canada is associated with warmer autumns, milder winters, and previous year disease severity, suggesting overwintering of the inoculum in an area where the pathogen is not known to be endogenous. In this study, genotyping by sequencing was used to investigate the population structure of P. destructor at the landscape scale. The study focused on a particular region of southwestern Québec-Les Jardins de Napierville-to determine if the populations were clonal and regionally differentiated. The data were characterized by a high level of linkage disequilibrium, characteristic of clonal organisms. Consequently, the null hypothesis of random mating was rejected when tested on predefined or nonpredefined populations, indicating that linkage disequilibrium was not a function of population structure and suggesting a mixed reproduction mode. Discriminant analysis of principal components performed with predefined population assignment allowed grouping P. destructor isolates by geographical regions, while analysis of molecular variance confirmed that this genetic differentiation was significant at the regional level. Without using a priori population assignment, isolates were clustered into four genetic clusters. These results represent a baseline estimate of the genetic diversity and population structure of P. destructor.

Anthracnose Risk Establishment Based on Age-Related Susceptibility of Grape Leaves, Flowers, and Berries to Infection by Elsinoë ampelina.

Anthracnose is an important disease of grapevines caused by the fungus Elsinoë ampelina. In recent years, there have been regular outbreaks in humid grape-growing regions around the world. Young leaves and berries are reported to be highly susceptible to E. ampelina, but detailed and seasonal development of age-related susceptibility remains unclear. Experiments were conducted under greenhouse and vineyard conditions by inoculating 1- to 19-day-old leaves, flowers, and berries at different phenological stages of three grapevine cultivars (Vandal-Cliche, Marquette, and Vidal). Leaf susceptibility was highest when inoculated at 1 to 2 days old, and inoculated leaves were moderately susceptible at 3 to 6 days old and almost resistant when older than 6 days. The influence of leaf age on anthracnose relative severity was adequately described by an exponential decay model. The susceptibility of the inflorescences was high when inoculated from their initiation to the full flowering (50% fall of the caps), and the inflorescences/flowers were moderately susceptible until veraison, after which the berries were practically resistant. The flower/berry susceptibility as a function of degree-days accumulated since 1 April was modeled using a sigmoid model. Based on this model, 50% disease incidence is reached when 656, 543, and 550 degree days are accumulated for the cultivars Vandal-Cliche, Marquette, and Vidal, respectively. These results suggest that the risk of anthracnose development is high from bud-break to fruit set, and on newly emerged leaves either early in the season or following pruning. More knowledge on anthracnose epidemiology is needed, but these results could be used to improve timing of fungicide applications and pruning activities.

Development and Evaluation of a Model that Predicts Grapevine Anthracnose Caused by Elsinoë ampelina.

Grapevine anthracnose caused by Elsinoë ampelina is a serious threat in many vineyards, and its control requires repeated application of fungicides, usually on a calendar basis. A better understanding of the pathogen life cycle would help growers manage anthracnose more safely and effectively. After conducting a systematic literature search of grape anthracnose, we used the retrieved information and data to develop a mechanistic model based on systems analysis. The model simulates production and maturation of primary inoculum, infection caused by both primary and secondary conidia, and lesion formation and production of secondary inoculum. The model was validated for its ability to predict first seasonal onset of anthracnose lesions by using 8 years of data collected at Auckland, New Zealand, and disease progress during the season by using 3 years of data collected at Frelighsburg, Canada. Overall, the model provided accurate predictions of infection occurrence, with 0.96 accuracy, 0.91 sensitivity, and 0.97 specificity. The model also showed good accuracy for predicting disease progress, with a concordance correlation coefficient between observed and predicted disease severities of 0.92, a root mean square error of 0.14, and a coefficient of residual mass of 0.06. Although the model failed to predict 10 of 110 real infection periods, these missed infections led to only mild disease symptoms. We therefore conclude that the model is reliable and can be used to reduce the costs of anthracnose management by improving the timing of fungicide applications.

Monitoring of Peronospora destructor Primary and Secondary Inoculum by Real-Time qPCR.

Onion downy mildew (ODM), caused by Peronospora destructor, is a serious threat for onion growers worldwide. In southwestern Québec, Canada, a steady increase in occurrence of ODM has been observed since the mid-2000s. On onion, P. destructor can develop local and systemic infections producing numerous sporangia which act as initial inoculum locally and also for neighboring areas. It also produces oospores capable of surviving in soils and tissues for a prolonged period of time. A recent study showed that ODM epidemics are strongly associated with weather conditions related to production and survival of overwintering inoculum, stressing the need to understand the role of primary (initial) and secondary inoculum. However, P. destructor is an obligate biotrophic pathogen, which complicates the study of inoculum sources. This study aimed at developing a molecular assay specific to P. destructor, allowing its quantification in environmental samples. In this study, a reliable and sensitive hydrolysis probe-based assay multiplexed with an internal control was developed on the internal transcribed spacer (ITS) region to quantify soil- and airborne inoculum of P. destructor. The assay specificity was tested against 17 isolates of P. destructor obtained from different locations worldwide, other members of the order Peronosporales, and various onion pathogens. Validation with artificially inoculated soil and air samples suggested a sensitivity of less than 10 sporangia g-1 of dry soil and 1 sporangium m-3 of air. Validation with environmental air samples shows a linear relationship between microscopic and real-time quantitative PCR counts. In naturally infested soils, inoculum ranged from 0 to 162 sporangia equivalent g-1 of dry soil, which supported the hypothesis of overwintering under northern climates. This assay will be useful for primary and secondary inoculum monitoring to help characterize ODM epidemiology and could be used for daily tactical and short-term strategic decision-making.

A Diverse Virome of Leafroll-Infected Grapevine Unveiled by dsRNA Sequencing.

Quebec is the third-largest wine grape producing province in Canada, and the industry is constantly expanding. Traditionally, 90% of the grapevine cultivars grown in Quebec were winter hardy and largely dominated by interspecific hybrid Vitis sp. cultivars. Over the years, the winter protection techniques adopted by growers and climate changes have offered an opportunity to establish V. vinifera L. cultivars (e.g., Pinot noir). We characterized the virome of leafroll-infected interspecific hybrid cultivar and compared it to the virome of V. vinifera cultivar to support and facilitate the transition of the industry. A dsRNA sequencing method was used to sequence symptomatic and asymptomatic grapevine leaves of different cultivars. The results suggested a complex virome in terms of composition, abundance, richness, and phylogenetic diversity. Three viruses, grapevine Rupestris stem pitting-associated virus, grapevine leafroll-associated virus (GLRaV) 3 and 2 and hop stunt viroid (HSVd) largely dominated the virome. However, their presence and abundance varied among grapevine cultivars. The symptomless grapevine cultivar Vidal was frequently infected by multiple virus and viroid species and different strains of the same virus, including GLRaV-3 and 2. Our data show that viruses and viroids associated with the highest number of grapevines expressing symptoms included HSVd, GLRaV-3 and GLRaV-2, in gradient order. However, co-occurrence analysis revealed that the presence of GLRaV species was randomly associated with the development of virus-like symptoms. These findings and their implications for grapevine leafroll disease management are discussed.

Influence of Leaf Wetness Duration and Temperature on Infection of Grape Leaves by Elsinoë ampelina under Controlled and Vineyard Conditions.

On susceptible varieties, indirect damage to vines infected by Elsinoë ampelina range from reduced vigor to complete defoliation while, on berries, damage ranges from reduced quality to complete yield loss. Limited knowledge about the relationship between weather conditions and infection makes anthracnose management difficult and favors routine application of fungicides. The influence of leaf wetness duration and temperature on infection of grape leaves by E. ampelina was studied under both controlled and vineyard conditions. For the controlled conditions experiments, the five youngest leaves of potted vines (Vidal) were inoculated with a conidia suspension and exposed to combinations of six leaf wetness durations (from 0 to 24 h) and six constant temperatures (from 5 to 30°C). A week after each preset infection period, the percent leaf area diseased (PLAD) was assessed. At 5°C, regardless of the leaf wetness duration, no disease developed. At 10 and at 15 to 30°C, the minimum leaf wetness durations were 4 and 6 h, respectively. Above the minimum wetness duration, at temperatures from 10 to 30°C, PLAD increased linearly, with increasing leaf wetness up to 12 h, and then at a lower rate from 12 to 24 h. The optimal temperature for infection was 25°C. Relative infection was modeled as a function of both temperature and wetness duration using a Richards model (R2 = 0.93). The predictive capacity of the model was evaluated with data collected in experimental vineyard plots exposed to natural wetness durations or artificial wetness durations created using sprinklers. In total, 264 vineyard infection events were used to validate the controlled experiments model. There was a linear relationship between the risk of infection estimated with the model and the observed severity of anthracnose (R2 = 90); however, the model underestimated disease severity. A risk chart was constructed using the model corrected for vineyard observations and three levels of risk, with light, moderate, and severe risks corresponding to ≤5, >5% to ≤25, and >25% leaf area diseased, respectively. Overall, 93.9% of 132 independent observations were correctly classified, with 100, 29.4, and 9.4% of the light, moderate, and severe risks, respectively.

Detection and Quantification of Pythium tracheiphilum in Soil by Multiplex Real-Time qPCR.

In Canada, head lettuce (Lactuca sativa capitata) is extensively produced in the muck soils of southwestern Québec. However, yields are increasingly affected by various soilborne pathogens, including Pythium spp., which cause wilt and damping off. In a survey conducted in Québec muck soils in 2010 and 2011, Pythium tracheiphilum Matta was identified as the predominant Pythium sp. in the root of head lettuce showing Pythium stunt symptoms. Therefore, to improve risk assessment and help further understanding of disease epidemiology, a specific and sensitive real-time quantitative polymerase chain reaction (qPCR) assay based on TaqMan-minor groove binder (MGB) technology was developed for P. tracheiphilum. The PCR primers along with a TaqMan-MGB probe were designed from the ribosomal internal transcribed spacer 2 region. A 100-bp product was amplified by PCR from all P. tracheiphilum isolates tested while no PCR product was obtained from 38 other Pythium spp. or from a selection of additional lettuce pathogens tested. In addition to P. tracheiphilum, the assay was multiplexed with an internal control allowing for the individual validation of each PCR. In artificially infested soils, the sensitivity of the qPCR assay was established as 10 oospores/g of dry soil. P. tracheiphilum was not detected in soils in which lettuce has never been grown; however, inoculum ranged from 0 to more than 200,000 oospores/g of dry soil in commercial lettuce fields. Also, disease incidence was positively correlated with inoculum concentration (r = 0.764). The results suggest that inoculum concentration should be considered when making Pythium stunt management decisions. The developed qPCR assay will facilitate reliable detection and quantification of P. tracheiphilum from field soil.

Development of Action Threshold to Manage Common Leaf Spot and Black Seed Disease of Strawberry Caused by Mycosphaerella fragariae.

The fungus Mycosphaerella fragariae is responsible for two strawberry diseases: common leaf spot (CLS) and black seed disease (BSD). In June-bearing strawberry plantings, CLS influences vigor, yield, and winter survival. During production years, BSD causes black lesions around strawberry seeds, reducing the market value of the berries. The objective of this study was to characterize the relationships between CLS and BSD and to develop action thresholds for the management of BSD. Data on the number of lesions per leaf, number of black seeds per berry, and percentage of diseased berries were collected at two experimental and six commercial sites from 2000 to 2011, corresponding to 50 farm-years. First, logistic regression was used to model the relationship between BSD occurrence in its binary data form and the number of lesions per leaf assessed at 7, 14, 21, and 28 days before 10% bloom. Second, linear regression was used to model the relationship between BSD severity, BSD incidence, and number of lesions per leaf assessed at 7, 14, 21, and 28 days before 10% bloom. Resulting action thresholds of 15, 25, or 33 lesions per leaf at 21, 14, or 7 days before 10% bloom, respectively, were compared with the recommended practice at three commercial sites in 2014, 2015, and 2016. The percentage of diseased berries was significantly (P = 0.0016; least significant difference = 7.140) higher in the sections of the fields that were not managed for BSD, with an average of 15.22% diseased berries, in comparison with 3.22 and 2.44% diseased berries in sections managed according to the recommendations and the thresholds, respectively. Overall, 40% less fungicide was used when the thresholds were applied. Hence, these thresholds can be used as an additional decision tool to optimize fungicide applications during the prebloom period.

Assessing Performance of Spore Samplers in Monitoring Aeromycobiota and Fungal Plant Pathogen Diversity in Canada.

Spore samplers are widely used in pathogen surveillance but not so much for monitoring the composition of aeromycobiota. In Canada, a nationwide spore-sampling network (AeroNet) was established as a pilot project to assess fungal community composition in air and rain samples collected using three different spore samplers in the summers of 2010 and 2011. Metabarcodes of the internal transcribed spacer (ITS) were exhaustively characterized for three of the network sites, in British Columbia (BC), Québec (QC), and Prince Edward Island (PEI), to compare performance of the samplers. Sampler type accounted for ca. 20% of the total explainable variance in aeromycobiota compositional heterogeneity, with air samplers recovering more Ascomycota and rain samplers recovering more Basidiomycota. Spore samplers showed different abilities to collect 27 fungal genera that are plant pathogens. For instance, Cladosporium spp., Drechslera spp., and Entyloma spp. were collected mainly by air samplers, while Fusarium spp., Microdochium spp., and Ustilago spp. were recovered more frequently with rain samplers. The diversity and abundance of some fungi were significantly affected by sampling location and time (e.g., Alternaria and Bipolaris) and weather conditions (e.g., Mycocentrospora and Leptosphaeria), and depended on using ITS1 or ITS2 as the barcoding region (e.g., Epicoccum and Botrytis). The observation that Canada's aeromycobiota diversity correlates with cooler, wetter conditions and northward wind requires support from more long-term data sets. Our vision of the AeroNet network, combined with high-throughput sequencing (HTS) and well-designed sampling strategies, may contribute significantly to a national biovigilance network for protecting plants of agricultural and economic importance in Canada.IMPORTANCE The current study compared the performance of spore samplers for collecting broad-spectrum air- and rain-borne fungal pathogens using a metabarcoding approach. The results provided a thorough characterization of the aeromycobiota in the coastal regions of Canada in relation to the influence of climatic factors. This study lays the methodological basis to eventually develop knowledge-based guidance on pest surveillance by assisting in the selection of appropriate spore samplers.

Association Between Weather Variables, Airborne Inoculum Concentration, and Raspberry Fruit Rot Caused by Botrytis cinerea.

Botrytis fruit rot (BFR), one of the most important diseases of raspberry (Rubus spp.), is controlled primarily with fungicides. Despite the use of fungicides, crop losses due to BFR are high in most years. The aim of this study was to investigate the association between airborne inoculum, weather variables, and BFR in order to improve the management of the disease as well as harvest and storage decisions. Crop losses, measured as the percentage of diseased berries during the harvest period, were monitored in unsprayed field plots at four sites in three successive years, together with meteorological data and the number of conidia in the air. Based on windowpane analysis, there was no evidence of correlation between crop losses and temperature, vapor pressure deficit, wind, solar radiation, or probability of infection. There were significant correlations between crop losses and airborne inoculum and between crop losses and humidity-related variables, and the best window length was identified as 7 days. Using 7-day average airborne inoculum concentration combined with 7-day average relative humidity for periods ending 6 to 8 days before bloom, it was possible to accurately predict crop losses (R2 of 0.86 to 0.89). These models could be used to assist with managing BFR, timing harvests, and optimizing storage duration in raspberry crops.

Identification of Weather Conditions Associated with the Occurrence, Severity, and Incidence of Black Seed Disease of Strawberry Caused by Mycosphaerella fragariae.

Black seed disease (BSD) of strawberry is a sporadic disease caused by Mycosphaerella fragariae. Because little is known about potential crop losses or the weather conditions conducive to disease development, fungicides are generally not applied or are applied based on a preset schedule. Data collected from 2000 to 2011 representing 50 farm-years (total of 186 strawberry fields) were used to determine potential crop losses and to study the influence of weather on disease occurrence and development. First, logistic regression was used to model the relationship between occurrence of BSD and weather variables. Second, linear and nonlinear regressions were used to model the number of black seed per berry (severity) and the percentage of diseased berries (incidence). Of the 186 fields monitored, 78 showed black seed symptoms, and the number of black seed per berry ranged from 1 to 10, whereas the percentage of diseased berries ranged from 3 to 32%. The most influential weather variable was total rainfall (in millimeters) in May, with a threshold of 103 mm of rain (absence of BSD < 103 mm < presence of BSD). Similarly, nonlinear models with the total rainfall in May accurately predicted both disease severity and incidence (r = 0.94 and 0.97, respectively). Considering that management actions such as fungicide application are not needed every year in every field, these models could be used to identify fields that are at risk of BSD.

Development of Real-Time Isothermal Amplification Assays for On-Site Detection of Phytophthora infestans in Potato Leaves.

Real-time loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) assays were developed targeting the internal transcribed spacer 2 region of the ribosomal DNA of Phytophthora infestans, the potato late blight causal agent. A rapid crude plant extract (CPE) preparation method from infected potato leaves was developed for on-site testing. The assay's specificity was tested using several species of Phytophthora and other potato fungal and oomycete pathogens. Both LAMP and RPA assays showed specificity to P. infestans but also to the closely related species P. andina, P. mirabilis, P. phaseoli, and P. ipomoeae, although the latter are not reported as potato pathogen species. No cross-reaction occurred with P. capsici or with the potato pathogens tested, including P. nicotianae and P. erythroseptica. The sensitivity was determined using P. infestans pure genomic DNA added into healthy CPE samples. Both LAMP and RPA assays detected DNA at 50 fg/µl and were insensitive to CPE inhibition. The isothermal assays were tested with artificially inoculated and naturally infected potato plants using a Smart-DART platform. The LAMP assay effectively detected P. infestans in symptomless potato leaves as soon as 24 h postinoculation. A rapid and accurate on-site detection of P. infestans in plant material using the LAMP assay will contribute to improved late blight diagnosis and early detection of infections and facilitate prompt management decisions.

A Quantitative Dynamic Simulation of Bremia lactucae Airborne Conidia Concentration above a Lettuce Canopy.

Lettuce downy mildew, caused by the oomycete Bremia lactucae Regel, is a major threat to lettuce production worldwide. Lettuce downy mildew is a polycyclic disease driven by airborne spores. A weather-based dynamic simulation model for B. lactucae airborne spores was developed to simulate the aerobiological characteristics of the pathogen. The model was built using the STELLA platform by following the system dynamics methodology. The model was developed using published equations describing disease subprocesses (e.g., sporulation) and assembled knowledge of the interactions among pathogen, host, and weather. The model was evaluated with four years of independent data by comparing model simulations with observations of hourly and daily airborne spore concentrations. The results show an accurate simulation of the trend and shape of B. lactucae temporal dynamics of airborne spore concentration. The model simulated hourly and daily peaks in airborne spore concentrations. More than 95% of the simulation runs, the daily-simulated airborne conidia concentration was 0 when airborne conidia were not observed. Also, the relationship between the simulated and the observed airborne spores was linear. In more than 94% of the simulation runs, the proportion of the linear variation in the hourly-observed values explained by the variation in the hourly-simulated values was greater than 0.7 in all years except one. Most of the errors came from the deviation from the 1:1 line, and the proportion of errors due to the model bias was low. This model is the only dynamic model developed to mimic the dynamics of airborne inoculum and represents an initial step towards improved lettuce downy mildew understanding, forecasting and management.

Identification of the Dominant Genotypes of Phytophthora infestans in Canada Using Real-Time PCR with ASO-PCR Assays.

Phytophthora infestans, a pathogenic oomycete that is the causal agent of potato and tomato late blight, has devastating effects worldwide. The genetic composition of P. infestans populations in Canada has changed considerably over the last few years, with the appearance of several new genotypes showing different mating types and sensitivity to the fungicide metalaxyl. Genetic markers allowing for a rapid assessment of genotypes from small amounts of biological material would be beneficial for the early detection and control of this pathogen throughout Canada. Mining of the P. infestans genome revealed several regions containing single-nucleotide polymorphisms (SNP) within both nuclear genes and flanking sequences of microsatellite loci. Allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) assays were developed from 14 of the 50 SNP found by sequencing. Nine optimized ASO-PCR assays were validated using a blind test comprising P. infestans and other Phytophthora spp. The assays revealed diagnostic profiles unique to each of the five dominant genotypes present in Canada. The markers developed in this study can be used with environmental samples such as infected leaves, and will contribute to the genomic toolbox available to assess the genetic diversity of P. infestans at the intraspecific level. For late blight management, early warning about P. infestans genotypes present in potato and tomato fields will help growers select the most appropriate fungicides and application strategies.

A pyrosequencing-based method to quantify genetic substitutions associated with resistance to succinate dehydrogenase inhibitor fungicides in Botrytis spp. populations.

BACKGROUND: The genetic underlying resistance mechanisms in the population of the phytopathogenic fungus Botrytis cinerea are well documented. Specifically, several genetic substitutions associated with succinate dehydrogenase inhibitor (SDHI)-based fungicide resistance have been identified in the succinate dehydrogenase gene. The objective of the present work was to develop a molecular tool for accurate quantification of these genetic substitutions within Botrytis populations. A test using the PyroMark Q24 instrument was designed to detect and quantify five genetic substitutions associated with SDHI resistance. RESULTS: The technique is based on sequencing by synthesis, and it generated quantitative and accurate data with a limit of quantification of a minimum of 500 spores. There was a linear relationship between the known and estimated percentages of spores with the targeted genetic substitutions and wild-type strains at ratios of 0-100%, with a 20% increment. CONCLUSION: With the pyrosequencing assay developed in this study, a large number of Botrytis spp. individuals can be characterised in a timely fashion with greater accuracy than by commonly used methods. Hence, pyrosequencing-based methods will be useful for improving our understanding of fungicide resistance, detecting the arrival of new genetic substitutions, monitoring shifts in fungal populations and assessing the effectiveness of antiresistance strategies, and for routine monitoring of fungicide resistance.