MSE FINDR: A Shiny R Application to Estimate Mean Square Error Using Treatment Means and Post Hoc Test Results.

Research synthesis methods such as meta-analysis rely primarily on appropriate summary statistics (i.e., means and variance) of a response of interest for implementation to draw general conclusions from a body of research. A commonly encountered problem arises when a measure of variability of a response across a study is not explicitly provided in the summary statistics of primary studies. Typically, these otherwise credible studies are omitted in research synthesis, leading to potential small-study effects and loss of statistical power. We present MSE FINDR, a user-friendly Shiny R application for estimating the mean square error (i.e., within-study residual variance, [Formula: see text]) for continuous outcomes from analysis of variance (ANOVA)-type studies, with specific experimental designs and treatment structures (Latin square, completely randomized, randomized complete block, two-way factorial, and split-plot designs). MSE FINDR accomplishes this by using commonly reported information on treatment means, significance level (α), number of replicates, and post hoc mean separation tests (Fisher's least significant difference [LSD], Tukey's honest significant difference [HSD], Bonferroni, Sidák, and Scheffé). Users upload a CSV file containing the relevant information reported in the study and specify the experimental design and post hoc test that was applied in the analysis of the underlying data. MSE FINDR then proceeds to recover [Formula: see text] based on user-provided study information. The recovered within-study variance can be downloaded and exported as a CSV file. Simulations of trials with a variable number of treatments and treatment effects showed that the MSE FINDR-recovered [Formula: see text] was an accurate predictor of the actual ANOVA [Formula: see text] for one-way experimental designs when summary statistics (i.e., means, variance, and post hoc results) were available for the single factor. Similarly, [Formula: see text] recovered by the application accurately predicted the actual [Formula: see text] for two-way experimental designs when summary statistics were available for both factors and the sub-plot factor in split-plot designs, irrespective of the post hoc mean separation test. The MSE FINDR Shiny application, documentation, and an accompanying tutorial are hosted at https://garnica.shinyapps.io/MSE_FindR/ and https://github.com/vcgarnica/MSE_FindR/. With this tool, researchers can now easily estimate the within-study variance absent in published reports that nonetheless provide appropriate summary statistics, thus enabling the inclusion of such studies that would have otherwise been excluded in meta-analyses involving estimates of effect sizes based on a continuous response.

Effects of Host and Weather Factors on the Growth Rate of Septoria nodorum Blotch Lesions on Winter Wheat.

Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum, is a major disease of winter wheat that occurs frequently in the central and southeastern United States. Quantitative resistance to SNB in wheat is determined by various disease resistance components and their interaction with environmental factors. A study was conducted in North Carolina from 2018 to 2020 to characterize SNB lesion size and growth rate and to quantify the effects of temperature and relative humidity on lesion expansion in winter wheat cultivars with different levels of resistance. Disease was initiated in the field by spreading P. nodorum-infected wheat straw in experimental plots. Cohorts (groups of foliar lesions arbitrarily selected and tagged as an observational unit) were sequentially selected and monitored throughout each season. Lesion area was measured at regular intervals, and weather data were collected using in-field data loggers and the nearest weather stations. Final mean lesion area was approximately seven times greater on susceptible than on moderately resistant cultivars, and lesion growth rate was approximately four times higher on susceptible than on moderately resistant cultivars. Across trials and cultivars, temperature had a strong effect of increasing lesion growth rates (P < 0.001), while relative humidity had no significant effect (P = 0.34). Lesion growth rate declined slightly and steadily over the duration of cohort assessment. Our results demonstrate that restricting lesion growth is an important component of SNB resistance in the field and suggest that the ability to limit lesion size may be a useful breeding goal.

A Systematic Review and Quantitative Synthesis of the Efficacy of Quaternary Ammonium Disinfestants Against Fungal Plant Pathogens.

Quaternary ammonium compounds (QACs) have been used as disinfestants in plant production systems since the late 20th century. In studies on the control of fungal pathogens in agricultural and horticultural crop production systems, the efficacy of QAC disinfestants is variable, ranging from very high to ineffective. A systematic review and meta-analysis were performed to establish and understand how pathogen- and application-related factors influenced product efficacy. The meta-analysis was based on 124 studies involving 14 fungal plant pathogen genera, eight target materials, and four generations of QAC products that contained different mixtures of active ingredients. A significant (P < 0.0001) reduction in either disease intensity or propagule viability resulted following disinfestation using QAC products. Hedges' g standardized mean difference (g̅+) across the studies was 2.16, indicating that QACs, on average, were highly effective against fungal pathogens. Heterogeneity was significant (P < 0.0001), indicating that effect sizes (g) were not representative of a common mean effect size and supported selection of a random effects model. In all, 78.5% of the observed variance consisted of variance in true effects with a high estimate of between-study variability (τ2 = 2.15). For fungal genus, subgroup g̅+ for genera Pseudonectria and Calonectria was significantly (P < 0.0038) higher than for all other genus subgroups, except Fusarium. For target materials, subgroup g̅+ for solution, cloth, plant, and metal were significantly (P > 0.0071) higher than for inorganic material or wood. For product generation, subgroup g̅+ for fifth-generation products was significantly (P > 0.0071) higher than for fourth-, third-, and second-generation products. Dose and time accounted for only 8 and 4%, respectively, of the true variance in effect sizes in the regression model dose, time, and dose-time (P = 0.0004). Genus accounted for 40 and 51% of the true variance in effect sizes in the regression models dose and genus (P = 0.0008) and time and genus (P = 0.0007), respectively. Target material accounted for 18 and 19% of the true variance in effect sizes in the regression models dose and target (P = 0.0001) and time and target (P = 0.0001), respectively. QAC product generation accounted for 24 and 21% of the true variance in effect sizes in the regression models dose and QAC generation (P = 0.0034) and time and QAC generation (P = 0.0189), respectively. These results show that the current recommended rates for dose and contact time are generally expected to result in effective disinfestation for commercial QAC products. However, the efficacy against fungal plant pathogens is likely to be influenced by the fungal genus and target being treated and the generation of the QAC product that is used for disinfestation.

A Systematic Review and Quantitative Synthesis of the Efficacy of Quaternary Ammonium Compounds in Disinfesting Nonfungal Plant Pathogens.

This quantitative review and systematic analysis of the effectiveness of quaternary ammonium compounds (QACs) in disinfesting nonfungal plant pathogens in agricultural and horticultural cropping systems is a complementary follow-up to a previous study that evaluated the efficacy of QACs against fungal plant pathogens. In the present study, a meta-analysis involving 67 studies was conducted to assess the overall efficacy of QACs against plant pathogenic bacteria, oomycetes, and viruses and to identify factors associated with observed differences in product efficacy. Across all studies, QACs resulted in a significant (P < 0.0001) reduction in either disease intensity or propagule viability with a mean Hedges' g ([Formula: see text]) of 1.75, indicating that overall QAC treatments were moderately effective against nonfungal pathogens. Significant differences in product efficacy were observed between organism types (P = 0.0001), with QAC interventions resulting in higher efficacy (P = 0.0002) against oomycetes ([Formula: see text] = 4.20) than against viruses ([Formula: see text] = 1.42) and bacteria ([Formula: see text] = 1.07), which were not different (P = 0.2689) from each other. As a result, bacterium and virus types were combined into a composite set (BacVir). QAC intervention against BacVir resulted in significant differences in efficacy within categorical moderator subgroups for genus (P = 0.0133), target material (P = 0.0001), and QAC product generation (P = 0.0281). QAC intervention against oomycetes resulted in significant differences in efficacy only for genus (P < 0.0001). For the BacVir composite, five random effect (RE) meta-regression models were significant (P = 0.05), where models with dose and time, dose and genus, time and genus, dose and target, and time and target accounted for 62, 61, 52, 83, and 88%, respectively, of the variance in true effect sizes (R2) associated with [Formula: see text]. For oomycetes, three RE meta-regression models were significant (P = 0.05), where models with dose and time, dose and genus, and time and genus accounted for 64, 86, and 90%, respectively, of R2 associated with [Formula: see text]. These results show that while QACs are moderately effective against nonfungal plant pathogens, the observed variability in their efficacy due to dose of active ingredient and contact time of these products can be influenced by organism type, genus within organism type, the target being treated, and the generation of QAC products.

Within-Season Shift in Fungicide Sensitivity Profiles of Pseudoperonospora cubensis Populations in Response to Chemical Control.

Cucurbit downy mildew, caused by Pseudoperonospora cubensis, is an important disease affecting cucurbits worldwide. Chemical control is an effective method for disease control but P. cubensis has a high risk for developing resistance to fungicides. Alternating fungicides with different modes of action is recommended to avoid an increase of resistant subpopulations. Thus, this study was conducted to establish shifts in the sensitivity profiles of P. cubensis isolates during the growing season, wherein chlorothalonil was applied in alternation with either cymoxanil, fluopicolide, or propamocarb in field experiments conducted from 2018 to 2020 at Rocky Mount, NC and in 2018 and 2020 at Charleston, SC. The sensitivity of baseline isolates sampled early in the season or exposed isolates sampled late in the season to these single-site fungicides was determined using a detached-leaf assay, where tested isolates were classified as sensitive or resistant based on the relative disease severity. Based on the Kruskal-Wallis test, the distribution profile of relative disease severity among baseline and exposed isolates was significantly different where chlorothalonil was alternated with fluopicolide (χ2 = 10.82; P = 0.001) but not with cymoxanil (χ2 = 1.39; P = 0.238) or propamocarb (χ2 = 2.37; P = 0.412). Although there was a directional selection toward resistance for isolates sampled from plots that were treated with fluopicolide or propamocarb alternated with chlorothalonil during a growing season, a significant shift in fungicide sensitivity distribution based on combined data were observed for fluopicolide (χ2 = 8.25; P = 0.004) but not propamocarb (χ2 = 1.05; P = 0.461). Baseline and exposed isolates sampled from the cymoxanil-treated plots were all resistant to this fungicide and there was no significant shift in their fungicide sensitivity profile during a growing season (χ2 = 0.06; P = 1.000). These results indicate that a shift toward reduced sensitivity in P. cubensis can occur during a growing season and the efficacy of fluopicolide is likely to decrease as the frequency of the less sensitive subpopulations increases during a production season. The resultant effect on disease severity and selection of an insensitive subpopulation may accelerate the development of resistance to propamocarb in the southeastern United States.

Evaluation of a Model for Predicting Onset of Septoria nodorum Blotch in Winter Wheat.

Prediction models that aid growers in making decisions on timing of fungicide application are important components of integrated management programs for several foliar diseases of wheat. The risk of Septoria nodorum blotch (caused by Parastagonospora nodorum) onset in winter wheat has been reported to be influenced by location, amount of wheat residue in the field, and cumulative daily infection values 2 weeks prior to day of year (DOY) 102. A model previously developed based on these predictor variables was evaluated for its ability to predict disease onset under field conditions. An experiment was conducted at three locations in North Carolina in 2018, 2019, and 2020, where plots were either treated with >20% wheat residue or received no residue treatment. Plots were monitored for disease symptoms, and disease onset was defined to have occurred when mean disease incidence in a plot was 50%. Of the 298 disease cases recorded, disease onset occurred early (i.e., prior to DOY 102) in 257 cases, while onset was late (i.e., on or after DOY 102) in 41 cases. Model accuracy based on correct classification ranged from 0.67 to 0.95, with a mean of 0.87 across the study period. Similarly, sensitivity rates of the model ranged from 0.88 to 1.0 with a mean of 0.98 across all years. However, the model had low specificity, with a mean rate of 0.15 across the study period. Overall, there was no significant difference in the frequency of observed and predicted cases in the study (χ2 = 0.50, P = 0.7788, df = 2). Time to disease onset was significantly correlated with grain yield and explained 26% of variation in yield (P < 0.0001). Results indicated that the disease onset model performs well in predicting early disease onset but requires further evaluation and improvement, particularly in the Piedmont, where it over-predicted early onset in 2 successive years.

Field Occurrence and Overwintering of Oospores of Pseudoperonospora cubensis in the Southeastern United States.

In the United States, the cucurbit downy mildew pathogen, Pseudoperonospora cubensis, has been shown to form oospores under laboratory conditions, but there are no reports on the formation of oospores in naturally infected cucurbit plants in the field. This study investigated the occurrence of oospores in naturally infected leaves from cucurbit fields in North Carolina and South Carolina from 2018 to 2020. Oospore viability and survival was also determined outdoors during the winter in North Carolina during this study period using soil containing leaves infested with oospores. About 5% of 1,658 naturally infected cucumber and cantaloupe leaves sampled during the study had oospores, with a mean density of 585 oospores per cm2 of infected leaf tissue. Absolute oospore viability, as assessed using the plasmolysis method, declined linearly (slope = -0.27; P < 0.0001) over the 6-month exposure period from 67.8% in November to 19.3% in May. Other variables being equal, the decrease in oospore viability was significantly affected by soil temperature (b = -0.03 to -0.05; P < 0.0001) and number of rainy days (b = 21.6 to 40.46; P < 0.05), while the effects of soil moisture on oospore viability were less clear. About 20% of the oospores exposed to outdoor conditions at the end the study period were putatively viable and deemed potentially infective. However, these putatively viable oospores failed to germinate or initiate disease when inoculated onto cucumber or cantaloupe leaves. These results indicate that oospores might require some unrecognized stimuli or physiological factors to initiate germination and infection. Nonetheless, viability of oospores at the end of the winter season suggests that once exposed to the right conditions that stimulate germination, these oospores could potentially serve as a primary inoculum source in the southeastern United States where winter temperatures are cold enough to kill cucurbits plants.

Temporal Dynamics and Severity of Cucurbit Downy Mildew Epidemics as Affected by Chemical Control and Cucurbit Host Type.

Cucurbit downy mildew caused by the oomycete Pseudoperonospora cubensis is an important disease that affects members of Cucurbitaceae family globally. However, temporal dynamics of the disease have not been characterized at the field scale to understand how control strategies influence disease epidemics. Disease severity was assessed visually on cucumber and summer squash treated with weekly alternation of chlorothalonil with cymoxanil, fluopicolide, or propamocarb during the 2018 spring season and 2019 and 2020 fall seasons in North Carolina and the 2018 and 2020 fall seasons in South Carolina. Disease onset was observed around mid-June during the spring season and early September during the fall season, followed by a rapid increase in severity until mid-July in the spring season and late September or mid-October in the fall season, typical of polycyclic epidemics. The Gompertz, logistic, and monomolecular growth models were fitted to disease severity using linear regression and parameter estimates to compare the effects of fungicide treatment and cucurbit host type on disease progress. The Gompertz and logistic models were more appropriate than the monomolecular model in describing temporal dynamics of cucurbit downy mildew, with the Gompertz model providing the best description for 34 of the 44 epidemics examined. Fungicide treatment and host type significantly (P < 0.0001) affected the standardized area under disease progress curve (sAUDPC), final disease severity (Final DS), and weighted mean absolute rates of disease progress (ρ), with these variables, in most cases, being significantly (P < 0.05) lower in fungicide-treated plots than in untreated control plots. Except in a few cases, sAUDPC, Final DS, and ρ were lower in cases where chlorothalonil was alternated with fluopicolide or propamocarb than in cases where chlorothalonil was alternated with cymoxanil or when chlorothalonil was applied alone. These results characterized the temporal progress of cucurbit downy mildew and provided an improved understanding of the dynamics of the disease at the field level. Parameters of disease progress obtained from this study could serve as inputs in simulation studies to assess the efficacy of fungicide alternation in managing fungicide resistance in this pathosystem.

Need for speed: bacterial effector XopJ2 is associated with increased dispersal velocity of Xanthomonas perforans.

Bacterial spot caused by Xanthomonas perforans (Xp) is an economically important disease in tomato. Previous studies have shown that the recently isolated Xp strains have acquired and retained the effector gene, xopJ2, which has been reported to increase fitness of the pathogen in the field. To elucidate the fitness benefit of xopJ2, we quantified the effect of xopJ2 on the dispersal and evolution of Xp populations on tomato. We compared movement of two wild-type Xp strains expressing xopJ2 to their respective xopJ2 mutants when co-inoculated in the field. We developed a binary logistic model to predict the presence of Xp over spatial and temporal dimensions with or without xopJ2. Based on the model, wild-type bacteria were dispersed approximately three times faster than the xopJ2 mutants. In a simulation experiment, the selective advantage due to increased dispersal velocity led to an increase in the frequency of xopJ2 gene in the Xp population and its apparent fixation within 10 to 12 cropping seasons of the tomato crop. Our results show that the presence of a single gene can affect the dispersal of a bacterial pathogen and significantly alter its population dynamics.

Efficacy of Hypochlorite as a Disinfestant Against Fungal Pathogens in Agricultural and Horticultural Plant Production: A Systematic Review and Meta-Analysis.

Hypochlorite is often used as a disinfestant of fungal pathogens in a range of agricultural and horticultural settings. However, reports of its effectiveness are variable across studies and it is unclear what factors could potentially influence the reported estimates of its efficacy. A systematic review and meta-analysis was conducted to assess the efficacy of hypochlorite against fungal pathogens and explore factors that may explain the observed heterogeneity in estimates of efficacy. Standardized mean effect size, Hedges' g, was calculated for each of the 109 selected studies, published from 1972 to 2019, that met the criteria defined for the systematic review. A random-effects model was used to estimate the overall mean effect size ([Formula: see text]) and determine the heterogeneity in g among studies. Hypochlorite resulted in a significant (P < 0.001) reduction in either disease intensity or propagule viability with [Formula: see text]= 2.25, suggesting a large overall effect. However, 95% prediction intervals ranged from -0.18 to 4.68, indicating that hypochlorite could be ineffective against some fungi or when targeting some substrate materials. An estimate of the within-study variability, τ2, was 1.48 and the proportion of heterogeneity in g among studies due to true effects was 71.5%. Inclusion of categorical moderator variables in the random effects model showed that hypochlorite treatments were significantly (P < 0.0062) more effective when used to disinfest spores in an aqueous solution ([Formula: see text]= 4.58) than when used on plastic ([Formula: see text]= 2.13), plant ([Formula: see text]= 2.13), and wood ([Formula: see text]= 0.79). Similarly, hypochlorite treatments were significantly (P < 0.0083) more effective in disinfesting fungal propagules of Thielaviopsis spp. ([Formula: see text]= 2.51) than those of Verticillium spp. ([Formula: see text]= 1.21). A meta-regression indicated that the effect of dose (ß = -3.54; P = 0.0398) and contact time (ß = -0.05; P = 0.0001) on [Formula: see text] were highly significant. Further, [Formula: see text]was significantly affected by the dose × time interaction (ß = -0.017; P = 0.0269). In the meta-regression models, dose and time explained 0 and 16% of the variance in true effects, respectively. In meta-regression models with a continuous variable of dose or time, a categorical variable of target or genus and their interaction term, genus and target explained an additional 7 to 19% of the variance in true effects. These results show that although the current recommended dose and contact time for commercial bleach products are expected to result in effective disinfestation, the target material and genera of the fungal pathogen of interest will likely influence their efficacy.

Efficacy of Hypochlorite in Disinfesting Nonfungal Plant Pathogens in Agricultural and Horticultural Plant Production: A Meta-Analysis.

Bleach products containing hypochlorite are commonly used as disinfectants to eliminate nonfungal plant pathogens from production surfaces, tools, plant surfaces, irrigation water, and produce dump tanks. Although bleach products are useful, their effectiveness has been reported to vary under specific settings. A meta-analysis was conducted of 86 studies to assess the overall efficacy of hypochlorite against plant pathogenic bacteria, oomycetes, and viruses and to identify factors that explain differences in product efficacy. Hypochlorite resulted in a significant (P < 0.0001) reduction in disease intensity or propagule viability, with a mean Hedges' g standardized difference ([Formula: see text]) of 3.01, indicating that overall, hypochlorite treatments are highly effective. However, heterogeneity in g was significant (P < 0.0001) between studies, wherein 69.8% of the variance observed in g was attributed to true effects. Furthermore, an estimate of between-study variability was moderate (τ2 = 1.46). Random effects (REs) metaregression showed limited effects of moderator variables dosage, contact time, targeted material of treatment, and organism type on product efficacy when all organism types were considered together. Because subgroup [Formula: see text] was significantly higher (P = 0.0070) for oomycetes ([Formula: see text] = 3.30) than for bacteria ([Formula: see text] = 2.19), subsequent metaregressions were performed by organism type. For oomycetes, five RE metaregression models, each containing two moderators and their interaction, resulted in significant (P = 0.05) effects, where models with dosage and time, dosage and genus, time and genus, dosage and target, and time and target accounted for ≤50, 71, 57, 48, and 47%, respectively, of the variance in true effect sizes (R2) associated with [Formula: see text]. For viruses, only the RE metaregression model containing time and target and their interaction resulted in significant (P = 0.0435) effects accounting for 38% of the variance in true effect sizes associated with [Formula: see text]. None of the RE metaregression models for bacteria were significant, although they still accounted for ≤28% of the variance in true effect sizes associated with [Formula: see text]. These results show that although the current recommended rates for dosage and contact time for commercial bleach products are generally expected to result in effective disinfestation, the efficacy against nonfungal plant pathogens is expected to be influenced by the organism type and target being treated with hypochlorite.

Field Characterization of Partial Resistance to Gray Leaf Spot in Elite Maize Germplasm.

Forty-eight inbred lines of maize with varying levels of resistance to gray leaf spot (GLS) were artificially inoculated with Cercospora zeina and evaluated to characterize partial disease resistance in maize under field conditions from 2012 to 2014 across 12 environments in western Kenya. Eight measures of disease epidemic-that is, final percent diseased leaf area (FPDLA), standardized area under the disease progress curve (SAUDPC), weighted mean absolute rate of disease increase (ρ), disease severity scale (CDSG), percent diseased leaf area at the inflection point (PDLAIP), SAUDPC at the inflection point (SAUDPCIP), time from inoculation to transition of disease progress from the increasing to the decreasing phase of epidemic increase (TIP), and latent period (LP)-were examined. Inbred lines significantly (P < 0.05) affected all measures of disease epidemic except ρ. However, the proportion of the variation attributed to the analysis of variance model was most strongly associated with SAUDPC (R2 = 89.4%). Inbred lines were also most consistently ranked for disease resistance based on SAUDPC. Although SAUDPC was deemed the most useful variable for quantifying partial resistance in the test genotypes, the proportion of the variation in SAUDPC in each plot was most strongly (R2 = 93.9%) explained by disease ratings taken between the VT and R4 stages of plant development. Individual disease ratings at the R4 stage of plant development were nearly as effective as SAUDPC in discerning the differential reaction of test genotypes. Thus, GLS rankings of inbred lines based on disease ratings at these plant developmental stages should be useful in prebreeding nurseries and preliminary evaluation trials involving large germplasm populations.

Cultural and Genetic Approaches to Manage Aflatoxin Contamination: Recent Insights Provide Opportunities for Improved Control.

Aspergillus flavus is a morphologically complex species that can produce the group of polyketide derived carcinogenic and mutagenic secondary metabolites, aflatoxins, as well as other secondary metabolites such as cyclopiazonic acid and aflatrem. Aflatoxin causes aflatoxicosis when aflatoxins are ingested through contaminated food and feed. In addition, aflatoxin contamination is a major problem, from both an economic and health aspect, in developing countries, especially Asia and Africa, where cereals and peanuts are important food crops. Earlier measures for control of A. flavus infection and consequent aflatoxin contamination centered on creating unfavorable environments for the pathogen and destroying contaminated products. While development of atoxigenic (nonaflatoxin producing) strains of A. flavus as viable commercial biocontrol agents has marked a unique advance for control of aflatoxin contamination, particularly in Africa, new insights into the biology and sexuality of A. flavus are now providing opportunities to design improved atoxigenic strains for sustainable biological control of aflatoxin. Further, progress in the use of molecular technologies such as incorporation of antifungal genes in the host and host-induced gene silencing, is providing knowledge that could be harnessed to develop germplasm that is resistant to infection by A. flavus and aflatoxin contamination. This review summarizes the substantial progress that has been made to understand the biology of A. flavus and mitigate aflatoxin contamination with emphasis on maize. Concepts developed to date can provide a basis for future research efforts on the sustainable management of aflatoxin contamination.

Evaluation of a Model for Predicting the Infection Risk of Squash and Cantaloupe by Pseudoperonospora cubensis.

Infection risk models of downy mildew of cucumber caused by Pseudoperonospora cubensis were evaluated for their performance in predicting the infection risk of squash and cantaloupe plants under field conditions. Experiments were conducted from 2012 to 2014 in Clayton, NC and Charleston, SC, where disease-free potted plants were exposed to weather conditions during a 24- and 48-h period (hereafter 24- and 48-h models, respectively) within a plot with naturally occurring inoculum. Exposed plants were subsequently placed in a growth chamber where they were monitored for disease symptoms, which was indicative of a successful infection. Disease severity was assessed after 7 days as the proportion of leaf area with disease symptoms. Two predictor variables, day temperature and hours of relative humidity >80% during each exposure were used as inputs to generate model predictions that were compared with observed data. The threshold probability on the receiver operating characteristic (ROC) curve that minimized the overall error rate for the 24-h model was 0.85 for both squash and cantaloupe. The 24-h model was consistently more accurate than the 48-h model in predicting the infection risk for the two hosts. The accuracy of the 24-h model as estimated using area under ROC curve ranged from 0.75 to 0.81, with a correct classification rate ranging from 0.69 to 0.74 across the two hosts. Specificity rates for the model ranged from 0.81 to 0.84, while the sensitivity rates ranged from 0.58 to 0.67. Optimal decisions thresholds (POT) developed based on estimates of economic damage and costs of management showed that POT was dependent on the probability of disease occurrence, with the benefit of using the 24-h model for making management decisions being greatest at low levels of probability of disease occurrence. This 24-h model, previously developed using cucumber as the host, resulted in accurate estimates of the daily infection risk of squash and cantaloupe and could potentially be useful when incorporated into a decision support tool to guide fungicide applications to manage downy mildew in these other cucurbit host types.

Occurrence and Distribution of Mating Types of Pseudoperonospora cubensis in the United States.

During the past two decades, a resurgence of cucurbit downy mildew has occurred around the world, resulting in severe disease epidemics. In the United States, resurgence of the disease occurred in 2004 and several hypotheses, including introduction of a new genetic recombinant or pathotype of the pathogen, have been suggested as potential causes for this resurgence. Occurrence and distribution of mating types of Pseudoperonospora cubensis in the United States were investigated using 40 isolates collected from cucurbits across 11 states from 2005 to 2013. Pairing of unknown isolates with known mating-type tester strains on detached leaves of cantaloupe or cucumber resulted in oospore formation 8 to 10 days after inoculation. Isolates differed in their ability to form oospores across all coinoculation pairings, with oospore numbers ranging from 280 to 1,000 oospores/cm2 of leaf tissue. Oospores were hyaline to golden-yellow, spherical, and approximately 36 µm in diameter. Of the 40 isolates tested, 24 were found to be of the A1 mating type, while 16 were of the A2 mating type. Mating type was significantly (P < 0.0001) associated with host type, whereby all isolates collected from cucumber were of the A1 mating type, while isolates from squash and watermelon were of the A2 mating type. Similarly, mating type was significantly (P = 0.0287) associated with geographical region, where isolates from northern-tier states of Michigan, New Jersey, New York, and Ohio were all A1, while isolates belonging to either A1 or A2 mating type were present in equal proportions in southern-tier states of Alabama, Florida, Georgia, North Carolina, South Carolina, and Texas. Viability assays showed that oospores were viable and, on average, approximately 40% of the oospores produced were viable as determined by the plasmolysis method. This study showed that A1 and A2 mating types of P. cubensis are present and the pathogen could potentially reproduce sexually in cucurbits within the United States. In addition, the production of viable oospores reported in this study suggests that oospores could have an important role in the biology of P. cubensis and could potentially influence the epidemiology of cucurbit downy mildew in the United States.

Virulence Structure Within Populations of Pseudoperonospora cubensis in the United States.

Cucurbit downy mildew (CDM), caused by the obligate oomycete Pseudoperonospora cubensis, has resurged around the world during the past three decades. A new pathotype or genetic recombinant of P. cubensis have been suggested as possible reasons for the resurgence of CDM in the United States in 2004. In total, 22 isolates collected between 2004 and 2014, mainly in the eastern United States, were tested for their compatibility with a set of 15 cucurbit host types. The virulence structure within these isolates was evaluated on a set of 12 differential genotypes from eight genera. All isolates were highly compatible with the susceptible cultivar of Cucumis sativus, whereas the least compatibility was observed with Luffa cylindrica and Momordica charantia. Based on the compatibility with the differential host set, five pathotypes (1, 3, 4, 5, and 6) were identified among the 22 isolates examined. Pathotypes 1 and 3 had not been previously described in the United States and isolates of these two new pathotypes were also compatible with 'Poinsett 76', a cultivar of C. sativus known to be resistant to CDM prior to 2004. Virulence within the pathogen population was expressed based on virulence factors, virulence phenotypes, and virulence complexity. The number of virulence factors ranged from two to eight, indicating a complex virulence structure, with 77% of the isolates having five to eight virulence factors. Thirteen virulence phenotypes were identified; the mean number of virulence factors per isolate and mean number of virulence factors per virulence phenotype was 5.05 and 5.23, respectively, indicating that complex isolates and phenotypes contributed equally to the complex virulence structure of P. cubensis. Gleason and Shannon indices of diversity were 3.88 and 2.32, respectively, indicating a diverse virulence structure of P. cubensis within the United States population. The diverse virulence and high virulence complexity within the pathogen population indicate that host resistance alone in available cucurbit cultivars will not be effective to control CDM. An integrated approach involving a combination of fungicide application and introduction of cultivars with new resistance genes will be required for effective management of CDM.

Resurgence of Pseudoperonospora cubensis: The Causal Agent of Cucurbit Downy Mildew.

The downy mildew pathogen, Pseudoperonospora cubensis, which infects plant species in the family Cucurbitaceae, has undergone major changes during the last decade. Disease severity and epidemics are far more destructive than previously reported, and new genotypes, races, pathotypes, and mating types of the pathogen have been discovered in populations from around the globe as a result of the resurgence of the disease. Consequently, disease control through host plant resistance and fungicide applications has become more complex. This resurgence of P. cubensis offers challenges to scientists in many research areas including pathogen biology, epidemiology and dispersal, population structure and population genetics, host preference, host-pathogen interactions and gene expression, genetic host plant resistance, inheritance of host and fungicide resistance, and chemical disease control. This review serves to summarize the current status of this major pathogen and to guide future management and research efforts within this pathosystem.

Resurgence of Cucurbit Downy Mildew in the United States: A Watershed Event for Research and Extension.

In 2004, an outbreak of cucurbit downy mildew (CDM) caused by the oomycete Pseudoperonospora cubensis (Berk. & M. A. Curtis) Rostovzev resulted in an epidemic that stunned the cucumber (Cucumis sativus L.) industry in the eastern United States. The disease affects all major cucurbit crops, including cucumber, muskmelon, squashes, and watermelon. Although the 2004 epidemic began in North Carolina, the cucumber crop from Florida to the northern growing regions in the United States was devastated, resulting in complete crop loss in several areas. Many cucumber fields were abandoned prior to harvest. The rapid spread of the disease coupled with the failure of fungicide control programs surprised growers, crop consultants, and extension specialists. The epidemic raised several fundamental questions about the potential causes for the resurgence of the disease. Some of these questions revolved around whether the epidemic would recur in subsequent years and the possible roles that changes in the host, pathogen, and environment may have played in the epidemic.

Identifying highly connected sites for risk-based surveillance and control of cucurbit downy mildew in the eastern United States.

Objective: Surveillance is critical for the rapid implementation of control measures for diseases caused by aerially dispersed plant pathogens, but such programs can be resource-intensive, especially for epidemics caused by long-distance dispersed pathogens. The current cucurbit downy mildew platform for monitoring, predicting and communicating the risk of disease spread in the United States is expensive to maintain. In this study, we focused on identifying sites critical for surveillance and treatment in an attempt to reduce disease monitoring costs and determine where control may be applied to mitigate the risk of disease spread. Methods: Static networks were constructed based on the distance between fields, while dynamic networks were constructed based on the distance between fields and wind speed and direction, using disease data collected from 2008 to 2016. Three strategies were used to identify highly connected field sites. First, the probability of pathogen transmission between nodes and the probability of node infection were modeled over a discrete weekly time step within an epidemic year. Second, nodes identified as important were selectively removed from networks and the probability of node infection was recalculated in each epidemic year. Third, the recurring patterns of node infection were analyzed across epidemic years. Results: Static networks exhibited scale-free properties where the node degree followed a power-law distribution. Betweenness centrality was the most useful metric for identifying important nodes within the networks that were associated with disease transmission and prediction. Based on betweenness centrality, field sites in Maryland, North Carolina, Ohio, South Carolina and Virginia were the most central in the disease network across epidemic years. Removing field sites identified as important limited the predicted risk of disease spread based on the dynamic network model. Conclusions: Combining the dynamic network model and centrality metrics facilitated the identification of highly connected fields in the southeastern United States and the mid-Atlantic region. These highly connected sites may be used to inform surveillance and strategies for controlling cucurbit downy mildew in the eastern United States.

Impact of frequency of application on the long-term efficacy of the biocontrol product Aflasafe in reducing aflatoxin contamination in maize.

Aflatoxins, produced by several Aspergillus section Flavi species in various crops, are a significant public health risk and a barrier to trade and development. In sub-Saharan Africa, maize and groundnut are particularly vulnerable to aflatoxin contamination. Aflasafe, a registered aflatoxin biocontrol product, utilizes atoxigenic A. flavus genotypes native to Nigeria to displace aflatoxin producers and mitigate aflatoxin contamination. Aflasafe was evaluated in farmers' fields for 3 years, under various regimens, to quantify carry-over of the biocontrol active ingredient genotypes. Nine maize fields were each treated either continuously for 3 years, the first two successive years, in year 1 and year 3, or once during the first year. For each treated field, a nearby untreated field was monitored. Aflatoxins were quantified in grain at harvest and after simulated poor storage. Biocontrol efficacy and frequencies of the active ingredient genotypes decreased in the absence of annual treatment. Maize treated consecutively for 2 or 3 years had significantly (p < 0.05) less aflatoxin (92% less) in grain at harvest than untreated maize. Maize grain from treated fields subjected to simulated poor storage had significantly less (p < 0.05) aflatoxin than grain from untreated fields, regardless of application regimen. Active ingredients occurred at higher frequencies in soil and grain from treated fields than from untreated fields. The incidence of active ingredients recovered in soil was significantly correlated (r = 0.898; p < 0.001) with the incidence of active ingredients in grain, which in turn was also significantly correlated (r = -0.621, p = 0.02) with aflatoxin concentration. Although there were carry-over effects, caution should be taken when drawing recommendations about discontinuing biocontrol use. Cost-benefit analyses of single season and carry-over influences are needed to optimize use by communities of smallholder farmers in sub-Saharan Africa.