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Cited by 4 publications
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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 is observed since the mid 2000s. On onion, P. destructor can develop local and systemic infections producing numerous sporangia which are acting as initial inoculum locally, but 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 to 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 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 linear relationship between microscopic and real-time qPCR 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.
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 is observed since the mid 2000s. On onion, P. destructor can develop local and systemic infections producing numerous sporangia which are acting as initial inoculum locally, but 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 to 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 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 linear relationship between microscopic and real-time qPCR 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.
Advances in Diagnostics of Downy Mildews: Lessons Learned from Other Oomycetes and Future Challenges
Downy mildews are plant pathogens that damage crop quality and yield worldwide. Among the most severe and notorious crop epidemics of downy mildew occurred on grapes in the mid-1880s, which almost destroyed the wine industry in France. Since then, there have been multiple outbreaks on sorghum and millet in Africa, tobacco in Europe, and recent widespread epidemics on lettuce, basil, cucurbits, and spinach throughout North America. In the mid-1970s, loss of corn to downy mildew in the Philippines was estimated at US$23 million. Today, crops that are susceptible to downy mildews are worth at least $7.5 billion of the United States’ economy. Although downy mildews cause devastating economic losses in the United States and globally, this pathogen group remains understudied because they are difficult to culture and accurately identify. Early detection of downy mildews in the environment is critical to establish pathogen presence and identity, determine fungicide resistance, and understand how pathogen populations disperse. Knowing when and where pathogens emerge is also important for identifying critical control points to restrict movement and to contain populations. Reducing the spread of pathogens also decreases the likelihood of sexual recombination events and discourages the emergence of novel virulent strains. A major challenge in detecting downy mildews is that they are obligate pathogens and thus cannot be cultured in artificial media to identify and maintain specimens. However, advances in molecular detection techniques hold promise for rapid and in some cases, relatively inexpensive diagnosis. In this article, we discuss recent advances in diagnostic tools that can be used to detect downy mildews. First, we briefly describe downy mildew taxonomy and genetic loci used for detection. Next, we review issues encountered when identifying loci and compare various traditional and novel platforms for diagnostics. We discuss diagnosis of downy mildew traits and issues to consider when detecting this group of organisms in different environments. We conclude with challenges and future directions for successful downy mildew detection.
Bremia lactucae is an obligate, oomycete pathogen of lettuce that causes leaf chlorosis and necrosis and adversely affects marketability. The disease has been managed with a combination of host resistance and fungicide applications with success over the years. Fungicide applications are routinely made under the assumption that inoculum is always present during favorable environmental conditions. This approach often leads to fungicide resistance in B. lactucae populations. Detection and quantification of airborne B. lactucae near lettuce crops provides an estimation of the inoculum load, enabling more judicious timing of fungicide applications. We developed a quantitative polymerase chain reaction (qPCR)-based assay using a target sequence in mitochondrial DNA for specific detection of B. lactucae. Validation using amplicon sequencing of DNA from 83 geographically diverse isolates, representing 14 Bremia spp., confirmed that the primers developed for the TaqMan assays are species specific and only amplify templates from B. lactucae. DNA from a single sporangium could be detected at a quantification cycle (C) value of 32, and C values >35 were considered to be nonspecific. The coefficient of determination (R) for regression between sporangial density derived from flow cytometry and C values derived from the qPCR was 0.86. The assay was deployed using spore traps in the Salinas Valley, where nearly half of U.S. lettuce is produced. The deployment of this sensitive B. lactucae-specific assay resulted in the detection of the pathogen during the 2-week lettuce-free period as well as during the cropping season. These results demonstrate that this assay will be useful for quantifying inoculum load in and around the lettuce fields for the purpose of timing fungicide applications based on inoculum load.
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