Quantitative insights into grapevine anthracnose (Elsinoë ampelina) epidemiology: impact of temperature and leaf age on incubation, lesion development, and sporulation.

Grapevine anthracnose, caused by Elsinoe 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) were 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 RDL 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 relative lesion development and relative sporulation 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.

Survey of Phytophthora Diversity Reveals P. abietivora as a Potential Phytophthora Root Rot Pathogen in Québec Christmas Tree Plantations.

Christmas trees are an economically and culturally important ornamental plant in North America. Many microorganisms are pathogens of firs cultivated as Christmas trees. Among those, Phytophthora causes millions of dollars in damage to plantations annually. In Canada, it is unknown which species are responsible for Phytophthora root rot (PRR) of cultivated Abies species. Between 2019 and 2021, soil and root samples were collected from 40 Christmas tree plantations in Québec province. We used soil baiting and direct isolation from unidentified root fragments to assess the diversity of culturable Phytophthora spp. The obtained isolates were identified using a multilocus sequencing and phylogenetic approach. A total of 44 isolates were identified, including eight P. chlamydospora, eight P. abietivora, seven P. gonapodyides, three P. gregata, six P. megasperma, and two P. kelmanii isolates, plus 10 isolates belonging to a previously unknown taxon that is phylogenetically close to P. chlamydospora and P. gonapodyides. Among the known species, P. abietivora was the most prevalent isolated species associated with trees showing aboveground PRR-like symptoms. Pathogenicity trials confirmed the pathogenicity potential of P. abietivora on both Fraser fir and balsam fir seedlings. Our study provides a first snapshot of the Phytophthora diversity in Québec's Christmas tree productions and describes multiple potential first associations between Phytophthora species and Abies balsamea and A. fraseri.[Formula: see text] Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Natural Resources Canada. This is an open access article distributed under the CC BY-NC-ND 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.

A HRM Assay for Rapid Identification of Members of the Seedcorn Maggot Complex (Delia florilega and D. platura) (Diptera: Anthomyiidae) and Evidence of Variation in Temporal Patterns of Larval Occurrence.

The seedcorn maggot Delia platura (Meigen), and the bean seed maggot Delia florilega (Zetterstedt) can cause considerable feeding damage to a wide range of cultivated crops. The recent discovery of two distinct genetic lines of D. platura, each with a unique distribution pattern overlapping only in eastern Canada, suggests the presence of a new cryptic species for the group. The reliable identification of the three species/lines in the seedcorn maggot complex is crucial to our understanding of their distribution, phenology, and respective contribution to crop damage as well as to the development of specific integrated pest management approaches. As these taxa are morphologically indistinguishable in the immature stages, we developed a high-resolution melting PCR (HRM) assay using primers amplifying a variable 96-bp PCR product in the CO1 mitochondrial gene for rapid and economical identification of specimens. The three species/lines exhibited distinguishable melting profiles based on their different Tm values (between 0.4 and 0.9°C) and identification results based on HRM and DNA sequencing were congruent for all specimens in the validation data set (n = 100). We then used the new, highly sensitive HRM assay to identify survey specimens from the seedcorn maggot complex collected in Quebec, Canada, between 2017 and 2019. Progress curves developed to document the temporal occurrence patterns of each species/lines indicate differences between taxa, with the N-line (BOLD:AAA3453) of D. platura appearing approximately 17 d before D. florilega (BOLD:ACR4394) and the H-line (BOLD:AAG2511) of D. platura.

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.

Soilborne Inoculum Density and Environmental Parameters Influence the Development of Pythium Stunt Caused by Pythium tracheiphilum in Head Lettuce Crops.

In Quebec muck soils, Pythium stunt (Pythium tracheiphilum Matta) is responsible for important yield losses in head lettuce crops each year, which can reach up to 50% in certain cases. Despite the significance of the disease, factors influencing its development remain poorly documented, and no disease risk indicators are available, which makes the development of management strategies difficult. Hence, growers systematically use chemical fungicides throughout the growing season to reduce crop losses. However, it is known that soilborne disease incidence or severity may be influenced by soil inoculum density and environmental parameters. Therefore, the objectives of this study were to investigate the influence of inoculum density on lettuce growth under controlled conditions and evaluate the influence of soil inoculum density, air temperature, relative humidity, and rainfall on disease incidence under field conditions. In particular, this study aims to develop accurate predictors for Pythium stunt incidence. Results showed that, under controlled environment, thresholds of inoculum density of 97 and 46 propagules per gram of dry soil were needed to reduce lettuce dry weight by one-half for cultivars Estival and Prestige, respectively. These results were confirmed under field conditions, where a soil inoculum density >132 propagules per gram of dry soil combined with air temperatures <18°C for the first 2 weeks and rain accumulation >64 mm for the first 3 weeks after transplanting accurately predicted disease incidence 79% of the time. These relationships improve understanding of seasonal Pythium stunt development and will provide useful tools to develop sustainable management strategies.

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.

Host Range and Genetic Characterization of Ditylenchus dipsaci Populations from Eastern Canada.

The stem and bulb nematode, Ditylenchus dipsaci, is a plant-parasitic nematode affecting over 500 plant species worldwide. Since 2012, garlic producers from Ontario and Quebec have been particularly affected with economic losses caused by this pest. Reproduction of D. dipsaci on a particular host depends on its biological race, and races are unknown for these populations from eastern Canada. As a polyphagous pest, D. dipsaci can possibly be a threat and have negative impact on many crops grown in Quebec, such as field and vegetable crops (e.g., onion). In this study, the host range of four populations of D. dipsaci from Quebec and Ontario was determined in a greenhouse experiment using 11 crops. Garlic, onion, and green onion showed high susceptibility to the nematode, whereas reproduction on potato was poor. No reproduction was observed on corn, soybean, barley, alfalfa, mustard, carrot, and lettuce. These crops could therefore be used as rotational crops in a control program. Thirty-two populations of D. dipsaci were also genetically characterized using genotyping-by-sequencing. The comparison of allele frequencies at 481 loci showed that most of the populations had a genotype similar to a reference population from northern Ontario. However, a sample from eastern Quebec exhibited a distinct genotype and will require further phenotyping in a greenhouse to preclude the possibility of a different race.

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.

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.

Infection Efficiency of Four Phytophthora infestans Clonal Lineages and DNA-Based Quantification of Sporangia.

The presence and abundance of pathogen inoculum is with host resistance and environmental conditions a key factor in epidemic development. Therefore, several spore-sampling devices have been proposed to monitor pathogen inoculum above fields. However, to make spore sampling more reliable as a management tool and to facilitate its adoption, information on infection efficiency and molecular tools for estimating airborne sporangia concentration are needed. Experiments were thus undertaken in a growth chamber to study the infection efficiency of four clonal lineages of P. infestans (US-8, US-11, US-23, and US-24) by measuring the airborne sporangia concentration and resulting disease intensity. The relationship between the airborne sporangia concentration and the number of lesions per leaf was exponential. For the same concentration, the sporangia of US-23 caused significantly more lesions than the sporangia of the other clonal lineages did. Under optimal conditions, an airborne sporangia concentration of 10 sporangia m-3 for US-23 was sufficient to cause one lesion per leaf, whereas for the other clonal lineages, it took 15 to 25 sporangia m-3 to reach the same disease intensity. However, in terms of diseased leaf area, there was no difference between clonal lineages US-8, US-23 and US-24. Also, a sensitive quantitative real-time polymerase chain reaction (qPCR) tool was developed to quantify P. infestans airborne sporangia with detection sensitivity of one sporangium. The specificity of the qPCR assay was rigorously tested for airborne inoculum and was either similar to, or an improvement on, other published PCR assays. This assay allows rapid and reliable detection and quantification of P. infestans airborne sporangia and thereby, facilitates the implementation of spores-sampling network.