Evidence for Differences in the Temporal Progress of Plasmopara viticola Clades riparia and aestivalis Airborne Inoculum Monitored in Vineyards in Eastern Canada Using a Specific Multiplex Quantitative PCR Assay.

Four clades of Plasmopara viticola isolated from wild and cultivated Vitis species were described in 2013. Only P. viticola clades riparia and aestivalis have been detected in eastern Canada. To increase our understanding of the epidemiology of these clades of P. viticola, airborne sporangia were monitored with spore samplers at two experimental vineyards from 2015 to 2018 and at 11, 14, and 15 commercial vineyards in 2016, 2017, and 2018, respectively. At each vineyard and in each year, airborne sporangia were assessed three times weekly from grapevine budbreak to harvest. To accurately monitor airborne inoculum, a specific and sensitive quantitative PCR assay for simultaneous quantification of P. viticola clades riparia and aestivalis was developed. At the experimental site, in the vineyard planted with the hybrid grape variety Chancellor, mostly P. viticola clade riparia was detected. In vineyards planted with multiple grape varieties, airborne sporangia of P. viticola clade riparia were prevalent at the beginning of the season, whereas P. viticola clade aestivalis was mostly detected from midseason to harvest. At the commercial sites, airborne sporangia of P. viticola clade riparia were more prevalent in 2016, whereas P. viticola clade aestivalis was more prevalent in 2017 and 2018. The only significant difference between the inoculum progress curves was the time at which 50% of the seasonal inoculum was reached, with an average for the 3 years of 100.8 and 117.9 days since 1 May for P. viticola clade riparia and clade aestivalis, respectively. When airborne sporangium concentrations were expressed as the proportion of the two clades, in general, the proportion of clade aestivalis to that of clade riparia was low at the beginning of the season and increased to reach approximately 0.9 to 1.0 at the end of the season. These results suggest that both clades of P. viticola coexist, but that clade aestivalis is predominant and that downy mildew epidemics caused by P. viticola clade riparia occur 2 to 3 weeks before those caused by clade aestivalis.

Bremia lactucae Infection Efficiency in Lettuce is Modulated by Temperature and Leaf Wetness Duration Under Quebec Field Conditions.

More than 80% of Canadian lettuce production is located in the province of Quebec. Yet most of our knowledge on the epidemiology of lettuce downy mildew (Bremia lactucae) is derived from controlled-condition experiments or field experiments conducted in subtropical climates and, thus, cannot readily be applied to Quebec lettuce production. The influence of temperature and leaf wetness duration on the infection efficiency (IE) of B. lactucae was studied for 4 years (2003, 2004, 2012, and 2013) under field and growth-chamber conditions. IE was defined as the ratio of the number of lesions/leaf to the airborne conidia concentration (ACC). B. lactucae ACC was measured with rotating-arm samplers three times/week. In addition, 72 lettuce trap plants/sampling day were exposed to the potential airborne B. lactucae inoculum and disease intensity was assessed after 7 days of incubation in greenhouse. Under growth-chamber conditions, an ACC of 1 conidium/m3 was sufficient to cause 1 lesion/leaf, and IE ranged from 0.25 to 1.00. Under field conditions, an ACC of 10 to 14 conidia/m3 was required to cause 1 lesion/leaf, and IE ranged from 0.02 to 0.10, except in 2004, when IE ranged from 0.03 to 1.00. IE increased with increasing leaf wetness duration but decreased with increasing temperature. Also, considering an observed average temperature range from 10 to 20°C in the area of Quebec, 2 h of leaf wetness was sufficient for infection by B. lactucae. Therefore, under Quebec lettuce production conditions, a leaf wetness period of 2 h and an ACC of 10 to 14 conidia/m3 can be used as risk indicators to facilitate disease management decisions. Also, under typical Quebec weather conditions, measuring both morning and evening leaf wetness events could be used to improve the reliability of leaf wetness duration as a downy mildew risk indicator. Further research is needed to validate these risk indicators for integration into management strategies.

Relationship of Airborne Botrytis cinerea Conidium Concentration to Tomato Flower and Stem Infections: A Threshold for De-leafing Operations.

Gray mold, caused by Botrytis cinerea, is an important threat for tomato greenhouse producers. The influence of airborne conidia concentration (ACC) on both flower and stem-wound infections was studied in a greenhouse maintained at a temperature of 15, 20, or 25°C using diseased tomato leaves as the unique source of dry inoculum. Spore samplers were used to monitor ACC, and a previously developed real-time qPCR assay was used to quantify airborne B. cinerea conidia. The proportion of infected flowers remained low at ACC < 10 conidia/m3; above this concentration, flower infection increased with increasing ACC. The influence of ACC on proportion of infected flowers was well described by a sigmoid model (R2 = 0.90 to 0.92). The mean proportion of infected stem wounds over the three trials was 0.021; no infected wounds were observed at ACC < 100 conidia/m3. Based on logistic regression, the probability that a stem becomes infected increased rapidly with mean probabilities of 0.24 and 0.87 at ACCs of 315 and 3,161 conidia/m3, respectively. The results suggest that the amount of airborne B. cinerea inoculum in the greenhouse is often above the action threshold for flower infection and that monitoring airborne B. cinerea inoculum could help in timing de-leafing operations.

Spatial distribution of single-nucleotide polymorphisms related to fungicide resistance and implications for sampling.

Spatial distribution of single-nucleotide polymorphisms (SNPs) related to fungicide resistance was studied for Botrytis cinerea populations in vineyards and for B. squamosa populations in onion fields. Heterogeneity in this distribution was characterized by performing geostatistical analyses based on semivariograms and through the fitting of discrete probability distributions. Two SNPs known to be responsible for boscalid resistance (H272R and H272Y), both located on the B subunit of the succinate dehydrogenase gene, and one SNP known to be responsible for dicarboximide resistance (I365S) were chosen for B. cinerea in grape. For B. squamosa in onion, one SNP responsible for dicarboximide resistance (I365S homologous) was chosen. One onion field was sampled in 2009 and another one was sampled in 2010 for B. squamosa, and two vineyards were sampled in 2011 for B. cinerea, for a total of four sampled sites. Cluster sampling was carried on a 10-by-10 grid, each of the 100 nodes being the center of a 10-by-10-m quadrat. In each quadrat, 10 samples were collected and analyzed by restriction fragment length polymorphism polymerase chain reaction (PCR) or allele specific PCR. Mean SNP incidence varied from 16 to 68%, with an overall mean incidence of 43%. In the geostatistical analyses, omnidirectional variograms showed spatial autocorrelation characterized by ranges of 21 to 1 m. Various levels of anisotropy were detected, however, with variograms computed in four directions (at 0°, 45°, 90°, and 135° from the within-row direction used as reference), indicating that spatial autocorrelation was prevalent or characterized by a longer range in one direction. For all eight data sets, the ß-binomial distribution was found to fit the data better than the binomial distribution. This indicates local aggregation of fungicide resistance among sampling units, as supported by estimates of the parameter θ of the ß-binomial distribution of 0.09 to 0.23 (overall median value = 0.20). On the basis of the observed spatial distribution patterns of SNP incidence, sampling curves were computed for different levels of reliability, emphasizing the importance of sample size for the detection of mutation incidence below the risk threshold for control failure.

Spatial Pattern of Strawberry Powdery Mildew (Podosphaera aphanis) and Airborne Inoculum.

The relationship between strawberry powdery mildew and airborne conidium concentration (ACC) of Podosphaera aphanis was studied using data collected from 2006 to 2009 in 15 fields, and spatial pattern was described using 2 years of airborne inoculum and disease incidence data collected in fields planted with the June-bearing strawberry (Fragaria × ananassa) cultivar Jewel. Disease incidence, expressed as the proportion of diseased leaflets, and ACC were monitored in fields divided into 3 × 8 grids containing 24 100 m2 quadrats. Variance-to-mean ratio, index of dispersion, negative binomial distribution, Poisson distribution, and binomial and beta-binomial distributions were used to characterize the level of spatial heterogeneity. The relationship between percent leaf area diseased and daily ACC was linear, while the relationship between ACC and disease incidence followed an exponential growth curve. The V/M ratios were significantly greater than 1 for 100 and 96% of the sampling dates for ACC sampled at 0.35 m from the ground (ACC0.35m) and for ACC sampled at 1.0 m from the ground (ACC1.0m), respectively. For disease incidence, the index of dispersion D was significantly greater than 1 for 79% of the sampling dates. The negative binomial distribution fitted 86% of the data sets for both ACC1.0m and ACC0.35m. For disease incidence data, the beta-binomial distribution provided a good fit of 75% of the data sets. Taylor's power law indicated that, for ACC at both sampling heights, heterogeneity increased with increasing mean ACC, whereas the binary form of the power law suggested that heterogeneity was not dependent on the mean for disease incidence. When the spatial location of each sampling location was taken into account, Spatial Analysis by Distance Indices showed low aggregation indices for both ACCs and disease incidence, and weak association between ACC and disease incidence. Based on these analyses, it was found that the distribution of strawberry powdery mildew was weakly aggregated. Although a higher level of heterogeneity was observed for airborne inoculum, the heterogeneity was low with no distinct foci, suggesting that epidemics are induced by well-distributed inoculum. This low level of heterogeneity allows mean airborne inoculum concentration to be estimated using only one sampler per field with an overall accuracy of at least 0.841. The results obtained in this study could be used to develop a sampling scheme that will improve strawberry powdery mildew risk estimation.

Lagged association between powdery mildew leaf severity, airborne inoculum, weather, and crop losses in strawberry.

Knowledge about epidemiology and the impact of disease on yield is fundamental for establishing effective management strategies. The purpose of this study was to investigate the relationship between foliar strawberry mildew severity, Podosphaera aphanis airborne inoculum concentration, weather, and subsequent crop losses for day-neutral strawberry. The experiment was conducted at three, five, and four sites in 2006, 2007, and 2008, respectively, for a total of 12 epidemics. At each site, data were collected on 25 plants at 2-day intervals from the end of May to early October for a total of 60 to 62 samplings annually. First, seasonal crop losses were statistically described; then, a lagged regression model was developed to describe crop losses from the parameters that were significantly associated with losses. There was a strong positive linear relationship between seasonal crop losses and the area under the leaf disease progress curve (R(2) = 0.90) and daily mean airborne conidia concentration (R(2) = 0.86), and a negative linear relationship between crop losses and time to 5% loss (R(2) = 0.76) and time to 5% leaf area diseased (R(2) = 0.61). Among the 53 monitoring- and weather-based variables analyzed, percent leaf area diseased, log10-transformed airborne inoculum concentration, and weather variables related to temperature were significantly associated with crop losses. However, polynomial distributed lag regression models built with weather variables were not accurate in predicting losses, with the exception of a model based on a combined temperature and humidity variable, which provided accurate prediction of the data used to construct the model but not of independent data. Overall, the model based on log10-transformed airborne inoculum concentration did not provide accurate crop loss predictions. The model built using percent leaf area diseased with a time lag of 8 days (n = 4) and a polynomial degree of 2 provided a good description of the crop-loss data used to construct the model (r = 0.99 and 0.90) and of independent data (r = 0.92). For the 12 epidemics studied, 5% crop loss was reached when an average of 17% leaf area diseased was observed since the beginning of symptom development. These results indicate that information on foliar powdery mildew must be considered when making strawberry powdery mildew management decisions.

Analysis of Incidence-Severity Relationships for Strawberry Powdery Mildew as Influenced by Cultivar, Cultivar Type, and Production Systems.

The relationships between strawberry powdery mildew incidence (I) and severity (S) were investigated for various cultivars, for June-bearing and day-neutral cultivars, and for production systems (open-field and plastic-tunnel) with the objective of deriving a simple relationship for predicting severity (proportion of leaf area diseased [PLAD]) from incidence (proportion of diseased leaves). Data were collected from 2006 to 2011 at 11 commercial and experimental sites, for a total of 2,326 observations (n). For the cultivars grown in open fields, higher severity was observed on 'Seascape', with mean PLAD of 0.299 (n = 427); followed by 'Chambly', with 0.133 (n = 334); 'Cavendish', with 0.115 (n = 250); 'Darselect', with 0.111 (n = 321); and 'Jewel', with 0.105 (n = 276). In general, mean severity was higher when the strawberry plants were grown in plastic tunnels, with PLAD of 0.204, 0.199, and 0.181 for Chambly (n = 204), Darselect (n = 261), and Jewel (n = 253), respectively. A linear model based on complementary log-log transformation of I and S provided a good fit of the data (coefficient of determination [R2] adjusted for degrees of freedom from 0.82 to 0.96). A covariance analysis indicated that the sampling year and site of sampling did not significantly influence the estimated slope of the I-S relationship, nor did the specific cultivar among the June-bearing ones, whereas the production system (open-field versus plastic-tunnel) and the cultivar type (June-bearing versus day-neutral) significantly influenced the estimated slope. From this analysis, we were able to develop three specific models for open-field-grown June-bearing cultivars (R2 = 0.90), for the open-field-grown day-neutral cultivar (Seascape, R2= 0.91), and for June-bearing cultivars grown in plastic tunnels (R2 = 0.92). From these results, it was concluded that strawberry powdery mildew leaf severity can be accurately estimated from incidence of diseased leaves. The I-S relationships developed in the present study may be used in making practical disease management decisions, especially for management programs that use information on disease level in the field to initiate fungicide spraying programs or to time the interval between sprays.

Risk factors for crop health under global change and agricultural shifts: a framework of analyses using rice in tropical and subtropical Asia as a model.

Plant disease epidemiology requires expansion of its current methodological and theoretical underpinnings in order to produce full contributions to global food security and global changes. Here, we outline a framework which we applied to farmers' field survey data set on rice diseases in the tropical and subtropical lowlands of Asia. Crop health risks arise from individual diseases, as well as their combinations in syndromes. Four key drivers of agricultural change were examined: labor, water, fertilizer, and land availability that translate into crop establishment method, water shortage, fertilizer input, and fallow period duration, respectively, as well as their combinations in production situations. Various statistical approaches, within a hierarchical structure, proceeding from higher levels of hierarchy (production situations and disease syndromes) to lower ones (individual components of production situations and individual diseases) were used. These analyses showed that (i) production situations, as wholes, represent very large risk factors (positive or negative) for occurrence of disease syndromes; (ii) production situations are strong risk factors for individual diseases; (iii) drivers of agricultural change represent strong risk factors of disease syndromes; and (iv) drivers of change, taken individually, represent small but significant risk factors for individual diseases. The latter analysis indicates that different diseases are positively or negatively associated with shifts in these drivers. We also report scenario analyses, in which drivers of agricultural change are varied in response to possible climate and global changes, generating predictions of shifts in rice health risks. The overall set of analyses emphasizes the need for large-scale ground data to define research priorities for plant protection in rapidly evolving contexts. They illustrate how a structured theoretical framework can be used to analyze emergent features of agronomic and socioecological systems. We suggest that the concept of "disease syndrome" can be borrowed in botanical epidemiology from public health to emphasize a holistic view of disease in shifting production situations in combination with the conventional, individual disease-centered perspective.

Development of a TaqMan real-time PCR assay for quantification of airborne conidia of Botrytis squamosa and management of botrytis leaf blight of onion.

The use of a DNA-based method for quantifying airborne inoculum of Botrytis squamosa, a damaging pathogen of onion, was investigated. A method for purifying DNA from conidia collected using rotating-arm samplers and quantifying it using a TaqMan real-time quantitative polymerase chain reaction (qPCR) assay is described. The sensitivity of the qPCR assay was high, with a detection limit of 2 conidia/rod. A linear relationship between numbers of conidia counted with a compound microscope and those determined with the qPCR assay was obtained. Receiver operating characteristic curve analysis was used to evaluate the reliability of the two methods of conidia quantification (microscope examination and qPCR assay) to predict the risk of disease being below or above a damage threshold (D(th)). In total, 142 field samples from commercial onion fields were analyzed. At damage thresholds of 5 or 10 lesions/leaf, conidia quantification with the qPCR assay was more reliable at predicting disease risk than conidia quantification based on microscope counts. The proportion of decisions where the disease was present and predicted was higher for the qPCR assay than for the microscope counts, with values of 0.95 and 0.89 compared with 0.79 and 0.81 for D(th) of 5 and 10 lesions/leaf, respectively. The proportion of decisions where the disease was present but not predicted was lower for the qPCR assay than for microscope counts, with values of 0.05 and 0.11 compared with 0.20 and 0.19 for D(th) of 5 and 10 lesions/leaf, respectively. The results demonstrated that this new qPCR assay was reliable for quantifying B. squamosa airborne inoculum in commercial onion fields and that molecular conidia quantification could be used as a component of a risk management system for Botrytis leaf blight.

Spatiotemporal relationships between disease development and airborne inoculum in unmanaged and managed Botrytis leaf blight epidemics.

Comparatively little quantitative information is available on both the spatial and temporal relationships that develop between airborne inoculum and disease intensity during the course of aerially spread epidemics. Botrytis leaf blight and Botrytis squamosa airborne inoculum were analyzed over space and time during 2 years (2002 and 2004) in a nonprotected experimental field, using a 6 x 8 lattice of quadrats of 10 x 10 m each. A similar experiment was conducted in 2004 and 2006 in a commercial field managed for Botrytis leaf blight using a 5 x 5 lattice of quadrats of 25 x 25 m each. Each quadrat was monitored weekly for lesion density (LD) and aerial conidium concentration (ACC). The adjustment of the Taylor's power law showed that heterogeneity in both LD and ACC generally increased with increasing mean. Unmanaged epidemics were characterized in either year, with aggregation indices derived from SADIE (Spatial Analysis by Distance Indices). For LD, the aggregation indices suggested a random pattern of disease early in the season, followed by an aggregated pattern in the second part of the epidemic. The index of aggregation for ACC in 2002 was significantly greater than 1 at only one date, while it was significantly greater than 1 at most sampling dates in 2004. In both years and for both variables, positive trends in partial autocorrelation were observed mainly for a spatial lag of 1. In 2002, the overall pattern of partial autocorrelations over sampling dates was similar for LD and ACC with no significant partial autocorrelation during the first part of the epidemic, followed by a period with significant positive autocorrelation, and again no autocorrelation on the last three sampling dates. In 2004, there was no significant positive autocorrelation for LD at most sampling dates while for ACC, there was a fluctuation between significant and non-significant positive correlation over sampling dates. There was a significant spatial correlation between ACC at given date (t(i)) and LD 1 week later (t(i + 1)) on most sampling dates in both 2002 and 2004 for the unmanaged and managed sites. It was concluded that LD and ACC were not aggregated in the early stage of epidemics, when both disease intensity and airborne conidia concentration were low. This was supported by the analysis of LD and ACC from a commercial field, where managed levels of disease were low, and where no aggregation of both variables was detected. It was further concluded that a reliable monitoring of airborne inoculum for management of Botrytis leaf blight is achievable in managed fields using few spore samplers per field.

Incidence of Myclobutanil- and Kresoxim-Methyl-Insensitive Isolates of Venturia inaequalis in Quebec Orchards.

Sensitivity of baseline and exposed populations of Venturia inaequalis to myclobutanil and to kresoxim-methyl were evaluated in vitro. For myclobutanil, the population was constructed with 238 monoconidial isolates of V. inaequalis collected from 48 orchards. For kresoxim-methyl, the population was constructed with 251 monoconidial isolates collected from 49 orchards. Baseline populations were constructed with 34 and 29 monoconidial isolates collected from apple trees that had never been treated for myclobutanil and kresoxim-methyl, respectively. Sensitivity to fungicides was evaluated based on 50% effective dose (ED50) values. The V. inaequalis population that was not exposed to myclobutanil had a baseline sensitivity (mean ED50) of 0.064 µg/ml and showed a lognormal distribution. The V. inaequalis population constructed with isolates from commercial orchards had a mean ED50 of 2.600 µg/ml, which was significantly higher than the baseline sensitivity. The distribution of ED50 values did not follow a lognormal distribution. In response to declining levels of scab control with myclobutanil and other sterol demethylation inhibitor fungicides (DMIs), three orchards were more deeply investigated. The mean ED50 values were 1.618 (n = 23), 3.079 (n = 29), and 1.500 µg/ml (n = 20) in orchards one, two, and three, respectively. Resistant isolates, according to criteria set by other studies, accounted for 39, 76, and 85% of the isolates tested. The V. inaequalis population that had never been exposed to kresoxim-methyl had a baseline sensitivity (mean ED50) of 0.092 µg/ml and showed a lognormal distribution. The V. inaequalis population constructed with isolates from commercial orchards had a mean ED50 of 6.093 µg/ml, which was significantly higher than the baseline sensitivity. The distribution of ED50 values followed a lognormal distribution. However, when a subsample of isolates was retested for their sensitivity to kresoxim-methyl with the addition of salicylhydroxamic acid (an inhibitor of alternative oxidase) at 100 µg/ml to the growth medium, more than 98% inhibition was observed for all isolates. The results from in vitro tests showed a high level of resistance to myclobutanil and a low level of resistance to kresoxim-methyl, suggesting that the use of myclobutanil and DMIs should be discontinued or significantly reduced before practical resistance is reached.

Incidence and Significance of Iprodione-Insensitive Isolates of Botrytis squamosa.

Botrytis leaf blight, caused by Botrytis squamosa, is an economically important disease of onion. The principal means of controlling the disease is by applying fungicides. Typical fungicide programs include applications of dithiocarbamates, chloronitriles, carboxamides, and dicarboximides such as iprodione (Rovral). Onion fields were surveyed in 2002, 2003, and 2004 for insensitivity to iprodione. Tests for insensitivity to iprodione were conducted on 62, 58, and 60 monoconidial field isolates using the automated quantitative (AQ) method with a discriminatory dose of 1.78 ppm of iprodione active ingredient (a.i.) in 2002, 2003, and 2004, respectively. Overall, insensitive isolates were detected in 51% of the fields, and the proportions of insensitive isolates were 8.1, 20.7, and 18.3% in 2002, 2003, and 2004, respectively. The aggressiveness of 10 insensitive and 18 sensitive isolates and the efficacy of iprodione was tested in planta. Onion leaves were inoculated with 750 µl of a conidial suspension of 75,000 conidia per ml and incubated in a growth chamber at 15°C. Aggressiveness was measured as lesion density (average number of lesions per cm2 of onion leaf). Lesion density varied from 2.82 to 8.04 lesions per cm2 of leaf. There was a significant effect (P < 0.0001) of isolates on lesion density. However, there was no significant correlation between lesion density and sensitivity to iprodione (r = 0.08). When onion leaves were sprayed with 1,875, 3,750, and 7,500 ppm of iprodione, percent inhibition of lesion density was higher for sensitive isolates with means of 43.04, 61.42, and 74.59, respectively. Accordingly, percent inhibition was lower for insensitive isolates with means of 13.81, 28.26, and 44.37 for iprodione concentrations of 1,875, 3,750, and 7,500, respectively. It was concluded that the incidence of insensitive isolates was relatively low, but insensitive isolates were capable of infecting onion leaves. There was a good relationship between insensitivity to iprodione in B. squamosa populations measured in vitro with the AQ method, and the reduced efficacy of iprodione in controlling Botrytis leaf blight.

Quantification of Airborne Inoculum as an Aid in the Management of Leaf Blight of Onion Caused by Botrytis squamosa.

Botrytis leaf blight, caused by Botrytis squamosa, is a common and frequently damaging disease of onion crops, but the severity of epidemics varies widely from year to year. The disease is initiated and spread by airborne conidia. The relationship between airborne conidium concentration (ACC) and lesion development was studied in the field. A linear relationship was found between ACC and number of lesions per leaf: ACC values of 10 to 15 and 25 to 35 conidia m-3 were associated with 1 and 2.5 lesions per leaf, respectively. In 2000 and 2001, at three sites, four different criteria were used to start a fungicide spray program and their effect on epidemic development was compared with that of a grower's conventional schedule. The criteria were: at the fourth-true-leaf growth stage; according to an inoculum production index; when the ACC reached 10 to 15 conidia m-3; and when the ACC reached 25 to 35 conidia m-3. A nonsprayed control plot was included in the trial. Fungicide programs started when the ACC reached 10 to 15 conidia m-3 were as effective as the conventional program, but used fewer sprays. A fungicide spray program based on measurements of ACC and disease severity was evaluated in 2002 and 2003 in five and three commercial onion fields, respectively. At each site, half of the field was sprayed according to the grower's schedule and, in the other half, fungicide sprays were initiated when a threshold of 10 to 15 conidia m-3 or five lesions on the lower leaf (whichever came first) was reached. Overall, the number of fungicide applications was reduced by 75 and 56% in 2002 and 2003, respectively, without causing significant yield reduction. In both years, the reduction in number of fungicide applications was due mainly to the delay in initiation of the fungicide program.

Effect of Timing of Application of the Biological Control Agent Microsphaeropsis ochracea on the Production and Ejection Pattern of Ascospores by Venturia inaequalis.

ABSTRACT Field and in vitro trials were conducted to establish the influence of the biological control agent Microsphaeropsis ochracea on the ejection pattern of ascospores by Venturia inaequalis and on apple scab development, and to establish the best timing of application. The ejection pattern of ascospores was similar on leaves sprayed with M. ochracea and on untreated leaves. Fall application of M. ochracea combined with a delayed-fungicide program was evaluated in orchards with intermediate and high scab risk. For both orchards, it was possible to delay the first three and two infection periods in 1998 and 1999, respectively, without causing significant increase or unacceptable leaf and fruit scab incidence. To evaluate the best timing of application, sterile leaf disks were inoculated with V. inaequalis and then with M. ochracea 0, 2, 4, 6, 8, 10, 12, 14, and 16 weeks later. After incubation under optimal conditions for pseudothecia development, the number of ascospores was counted. Similarly, M. ochracea was sprayed on scabbed leaves on seven occasions from August to November 1999 and 2000. Leaves were overwintered on the orchard floor and ascospore production was evaluated the following spring. Ascospore production was reduced by 97 to 100% on leaf disks inoculated with M. ochracea less than 6 weeks after inoculation with V. inaequalis, but ascospore production increased with increasing period of time when M. ochracea was applied 8 to 16 weeks after the inoculation with V. inaequalis. In the orchard, the greatest reduction in production of ascospores (94 to 96% in 2000 and 99% in 2001) occurred on leaves sprayed with M. ochracea in August. The production of ascospores was reduced by 61 to 84% in 2000 and 93% in 2001 on leaves sprayed with M. ochracea in September, reduced by 64 to 86% in 2000 and 74 to 89% in 2001 on leaves sprayed in October, and reduced by 54 and 67% in 2000 and 2001, respectively, on leaves sprayed in November. It was concluded that M. ochracea should be applied in August or September and that ascospore maturation models and delayed-fungicide program could be used in orchards treated with this biological control agent.

Sensitivity of Botrytis squamosa to Different Classes of Fungicides.

An automated quantitative (AQ) assay was compared with radial growth on solid media and with dry weight in liquid culture for assaying fungicide sensitivity in Botrytis squamosa, the causal agent of onion leaf blight. Five isolates of B. squamosa were assayed for sensitivity to mancozeb (Dithane DG) and iprodione (Rovral) at five concentrations (0.5, 1.0, 5.0, 10.0, and 50 ppm). For mancozeb, the correlations between 50% effective concentration (EC50) values obtained with the three assays were not significant; however for iprodione, correlations between EC50 values for AQ and radial growth and for AQ and dry weight were significant (r = 0.98 and 0.99, respectively). The AQ method was less time consuming and more reliable than the two standard assays. The AQ method was used to evaluate the sensitivity of 35 field isolates of B. squamosa to mancozeb (Dithane DG), iprodione (Rovral), vinclozolin (Ronilan DF), and chlorothalonil (Bravo 500). All isolates were sensitive to mancozeb (EC50 ranged from 3.36 to 12.97) and chlorothalonil (EC50 < 1.5 µg/ml), but four isolates were insensitive to both iprodione (EC50 ≥ 3.98 µg/ml) and vinclozolin (EC50 ≥ 17.49 µg/ml). The ratio of the EC50 values of the least-sensitive and the most-sensitive isolates of B. squamosa was 1.08, 3.86, 6.98, and 37.59 for chlorothalonil, mancozeb, iprodione, and vinclozolin, respectively. Cross-resistance was observed for the two dicarboximide fungicides, iprodione and vinclozolin, with a significant correlation (r = 0.94) in the sensitivity of the 35 isolates to these two fungicides.

Spatial Distribution of Venturia inaequalis Airborne Ascospores in Orchards.

ABSTRACT Apple scab (Venturia inaequalis) causes important economic losses in many apple production areas of the world. The disease is controlled by numerous fungicide applications regardless of the presence of ascospores in the orchard. Airborne ascospore concentration (AAC) can be measured in real time to time fungicide applications. However, the level of heterogeneity of the AAC in commercial orchards was unknown. Consequently, the spatial distribution of V. inaequalis ascospores was studied in a commercial apple orchard of 0.43 ha. The potential ascospore dose (PAD) and AAC were measured in 40 quadrats each of 108 m(2). In each quadrat, the AAC was monitored during the major rain events in spring 1999 and 2000 using spore samplers. The variance-to-mean ratio for the PAD and for most of the AAC sampling dates was >1, indicating an aggregated pattern of distribution. None of the frequency distributions of the most important ascospore ejection events followed the Poisson probability distribution, indicating that the pattern of distribution was not random. For all events, AAC had an aggregated pattern of distribution as suggested by the negative binomial distribution. The PAD followed neither the Poisson nor the negative binomial distribution. Geostatistical analyses confirmed the aggregated pattern of distribution. The cultivars had an effect on the PAD and AAC distribution pattern, but both PAD and AAC were not uniformly distributed within a block of the same cultivar. Therefore, the number, location, and height of samplers required to estimate AAC in orchards need to be investigated before using information on AAC for decision making.

Effect of Microsphaeropsis sp. Strain P130A on Germination and Production of Sclerotia of Rhizoctonia solani and Interaction Between the Antagonist and the Pathogen.

Microsphaeropsis sp. strain P130A was evaluated for the control of tuber-borne inoculum of Rhizoctonia solani based on the viability of sclerotia produced in vitro and on both the viability and production of tuber-borne sclerotia. The interactions between the antagonist and the pathogen, as well as the effect of the toxins produced by the antagonist on mycelial growth of R. solani were studied using transmission electron microscopy. On sclerotia produced in vitro, for all incubation periods (1 to 42 days), Microsphaeropsis sp. significantly reduced germination. Percent germination of sclerotia treated with Microsphaeropsis sp. decreased with increasing incubation period from an average of 82.0% after 1 day to stabilize at an average of 5.8% after 35 days. Similarly, percent germination of tuber-borne sclerotia was significantly lower when tubers were treated with Microsphaeropsis sp. Both 2% formaldehyde and Microsphaeropsis sp. treatments significantly reduced sclerotia germination to approximately 10% after 42 days of incubation at 4 degrees C. Furthermore, on tubers treated with the antagonist, the number of sclerotia per square centimeter decreased from 1.6 to 0.5 during the 8 months of storage at 4 degrees C, whereas an increase from 1.2 to 7.8 sclerotia per square centimeter was observed on untreated tubers. Microsphaeropsis sp. (strain P130A) colonized hyphae of R. solani within 4 days after contact on culture media. Transmission electron microscopic observations showed that the antagonist induced a rupture of the pathogen plasma membrane and that a chitin-enriched matrix was deposited at sites of potential antagonist penetration. Host penetration was not associated with pathogen cell wall alterations, which occurred at the time of progress of the antagonist in the pathogen cytoplasm. In the presence of a crude extract of Microsphaeropsis sp., cells of R. solani showed cytoplasm disorganization and breakdown of plasma membranes. Antibiosis and mycoparasitism were involved in the antagonism of R. solani by Microsphaeropsis sp., but the sequence by which these events occur, as well as the significance of wall appositions produced by R. solani, is yet to be established.

Effect of Microsphaeropsis sp. on the Production of Perithecia and Ascospores of Gibberella zeae.

The potential of Microsphaeropsis sp. (isolate P130A) as an antagonist of Gibberella zeae was tested under in vitro and field conditions. Firstly, an in vitro method of ascospore production was developed on wheat and corn residues. The plant type (corn or wheat), residue type (straw/stalk or grain), and incubation conditions (closed or open) had a significant effect on ascospore production. Perithecia were more abundant on wheat and corn grain incubated under open conditions. On these two substrates, the application of Microsphaeropsis sp. significantly reduced ascospore production. On wheat, the antagonist had a significant effect when applied 2 weeks before (-2), at the same time (0), and 4 weeks after (+4) inoculation with G. zeae, with 1.73, 0.31, 1.11, and 1.36 log ascospores per cm2 for the control, -2, 0, and +4 weeks treatments, respectively. On corn, Microsphaeropsis sp. had a significant effect when applied 2 weeks before, at the same time, 4 weeks after, and 6 weeks after inoculation with G. zeae with 3.02, 0.23, 1.29, 2.35, and 2.22 log ascospores per cm2 for the control, -2, 0, +4, and +6 weeks treatments, respectively. When applied to crop residues in the field as postharvest or preplanting applications, Microsphaeropsis sp. had no effect on the pattern of perithecia maturation, but significantly reduced the number of perithecia produced on two sampling dates, May 1998 and July 1999. There is a potential to biologically reduce the initial inoculum of G. zeae; however, more work is needed to optimize the efficacy of the biocontrol agent.

A New Semi-Selective Medium for Xanthomonas campestris pv. vitians, the Causal Agent of Bacterial Leaf Spot of Lettuce.

A semi-selective medium containing maltose, methyl green, and antibiotics (MMG) was developed for the isolation of Xanthomonas campestris pv. vitians. The semi-selective medium was evaluated based on plating efficiency of X. campestris pv. vitians in cell suspensions of pure cultures from leaves and soil. MMG medium allowed recovery of 5.7 to 30.6% of the X. campestris pv. vitians colonies recovered on nutrient agar and 0.1 to 8.4% of those recovered on 1/10-strength tryptic soy agar. MMG inhibited growth of most background bacteria and allowed reliable identification of X. campestris pv. vitians. The semi-selective medium contained (per liter) maltose (10 g), tryptone (5 g), K2HPO4 (3.5 g), KH2PO4 (2.75 g), trace elements (0.02 to 1.0 mg), methyl green (2 ml of a 1% aqueous solution), amoxicillin (32 mg), cephalothin (32 mg), cycloheximide (50 mg), and agar (15 g).

Evaluation of the Effect of Seed Treatments, Bactericides, and Cultivars on Bacterial Leaf Spot of Lettuce Caused by Xanthomonas campestris pv. vitians.

As a part of a broader program for management of bacterial leaf spot, the effects of lettuce-seed treatments, greenhouse application of bactericides, and cultivars were evaluated. Seed artificially inoculated with Xanthomonas campestris pv. vitians was treated with bactericides or heat treated and evaluated for the incidence of contaminated seed and seed germination. Seed soaked in 1% sodium hypochlorite for 5 and 20 min had an incidence of contaminated seed of less than 10%. Dry-heat (1 h), hot-water (50°C, 2 h), and organic-acid treatments significantly reduced seed germination. Considering both the effects on incidence of contaminated seed and seed germination, the best treatments were soaking the seeds in 1% sodium hypochlorite for 5 or 20 min. Copper sulfate, alone or mixed with Zineb or Dithane, failed to control the disease and caused phytotoxicity. All of the other bactericides significantly reduced the severity of bacterial leaf spot. However, the differences among bactericide efficacy were too small to allow comparison between the different forms of copper used, as well as the effect of adding manganese and zinc (Dithane) or zinc alone (Zineb) to the copper product. Nevertheless, copper hydroxide alone, mixed with Zineb or mixed with Dithane, and basic copper sulfate reduced disease severity by 86.89, 78.67, 80.42, and 81.82%, respectively, without causing phytotoxicity. For the two years of cultivar evaluation, no significant difference in mean disease severity was observed among the cultivars. Based on disease incidence, the most susceptible cultivar was Bellagreen. Cvs. Ideal cos, Grand Teton, Great Lakes, Paris Island, Ithaca, and Optima showed intermediate susceptibility, and the least-susceptible cultivars were Waldmann's and Grand Rapids, both green-leaf type. There was no significant difference between the three romaine (cos) cultivars and between the two crisphead cultivars, but a significant difference was observed between the two butterhead types, Bellagreen and Optima, which had 80.04 and 48.01%, respectively, of their leaves diseased at the time of harvest.