Disease Control Attributes of Oxathiapiprolin Fungicides for Management of Cucurbit Downy Mildew.

Oxathiapiprolin, a novel oomycete fungicide recently registered by DuPont, was reported to have high intrinsic activity against cucurbit downy mildew (Pseudoperonospora cubensis). The goal of this study was to characterize disease control attributes of oxathiapiprolin-based fungicides critical to effective management of cucurbit downy mildew. In growth chamber and greenhouse studies, oxathiapiprolin-based fungicides were compared with mandipropamid, mefenoxam + mancozeb, fluopicolide + propamocarb, cymoxanil + mancozeb, and ametoctradin + dimethomorph products for pre- and postinfection activity, local systemic movement, and protection of new growth produced after fungicide application. In preventive application, oxathiapiprolin-based fungicides significantly (P < 0.0001) inhibited downy mildew development, with the highest level of disease observed being 0.4% compared with 86.7% observed for mandipropamid. When applied postinfection, oxathiapiprolin-based fungicides significantly (P < 0.0001) suppressed disease development, but disease control was reduced relative to that observed for preventive application. There was a significant effect of formulation on the postinfection activity of oxathiapiprolin, whereby the oil dispersion (OD) formulation was more inhibitory than the water-dispersible granule formulation (0.001 ≤ P ≤ 0.049). Disease severity on the outer half leaf portion protected from spray deposition during fungicide application was lower for oxathiapiprolin-based fungicides (1.6 to 6.6%) than observed for fluopicolide + propamocarb (10.9 to 23.7%), mefenoxam + mancozeb (40.3 to 51.4%), and the nontreated controls (83.3 to 84.9%), which indicates significant acropetal movement within the leaf. Postinfection applications of oxathiapiprolin-based formulations had the greatest effect on lesion growth and sporangia production compared with the other fungicides in the experiment. When applied preventively to rapidly growing plants in a greenhouse, oxathiapiprolin-based fungicides consistently protected new growth that was not present at the time of application, with the OD formulation reducing disease severity by >75% relative to nontreated plants. The practical implications of these observations are discussed.

Rootstock-regulated gene expression patterns associated with fire blight resistance in apple.

BACKGROUND: Desirable apple varieties are clonally propagated by grafting vegetative scions onto rootstocks. Rootstocks influence many phenotypic traits of the scion, including resistance to pathogens such as Erwinia amylovora, which causes fire blight, the most serious bacterial disease of apple. The purpose of the present study was to quantify rootstock-mediated differences in scion fire blight susceptibility and to identify transcripts in the scion whose expression levels correlated with this response. RESULTS: Rootstock influence on scion fire blight resistance was quantified by inoculating three-year old, orchard-grown apple trees, consisting of 'Gala' scions grafted to a range of rootstocks, with E. amylovora. Disease severity was measured by the extent of shoot necrosis over time. 'Gala' scions grafted to G.30 or MM.111 rootstocks showed the lowest rates of necrosis, while 'Gala' on M.27 and B.9 showed the highest rates of necrosis. 'Gala' scions on M.7, S.4 or M.9F56 had intermediate necrosis rates. Using an apple DNA microarray representing 55,230 unique transcripts, gene expression patterns were compared in healthy, un-inoculated, greenhouse-grown 'Gala' scions on the same series of rootstocks. We identified 690 transcripts whose steady-state expression levels correlated with the degree of fire blight susceptibility of the scion/rootstock combinations. Transcripts known to be differentially expressed during E. amylovora infection were disproportionately represented among these transcripts. A second-generation apple microarray representing 26,000 transcripts was developed and was used to test these correlations in an orchard-grown population of trees segregating for fire blight resistance. Of the 690 transcripts originally identified using the first-generation array, 39 had expression levels that correlated with fire blight resistance in the breeding population. CONCLUSIONS: Rootstocks had significant effects on the fire blight susceptibility of 'Gala' scions, and rootstock-regulated gene expression patterns could be correlated with differences in susceptibility. The results suggest a relationship between rootstock-regulated fire blight susceptibility and sorbitol dehydrogenase, phenylpropanoid metabolism, protein processing in the endoplasmic reticulum, and endocytosis, among others. This study illustrates the utility of our rootstock-regulated gene expression data sets for candidate trait-associated gene data mining.

Orchard factors associated with resistance and cross resistance to sterol demethylation inhibitor fungicides in populations of Venturia inaequalis from Pennsylvania.

Orchard management practices, such as destroying of overwintered inoculum and limiting the number of fungicide applications, are often recommended as tactics for slowing the development of resistance to sterol demethylation-inhibitor (DMI) fungicides in populations of Venturia inaequalis. However, there is little quantitative evidence relating the use of such practices to levels of resistance in orchards. The aim of this study was to evaluate the sensitivity of V. inaequalis isolates from Pennsylvania to DMI fungicides, and to identify orchard management factors related to the incidence of resistant isolates. In total, 644 single-spore V. inaequalis cultures obtained from 20 apple orchards in 2008 or 2009 were tested for sensitivity to myclobutanil, fenbuconazole, or difenoconazole. Growers provided management history of the sampled plots. Widespread shifts toward resistance to the three fungicides were noted, with mean effective concentration for 50% inhibition (EC(50)) values of 2.136, 0.786, and 0.187 µg/ml for myclobutanil, fenbuconazole, and difenoconazole, respectively. Cross resistance to the three fungicides was documented in high correlation (Spearman's r > 0.6) between mean EC(50) values for 14 orchards. Based on a 0.5-µg/ml threshold, 66 and 26% of isolates were resistant to myclobutanil and fenbuconazole, respectively, and 22% were cross resistant to the two fungicides. A significant between-year shift toward increased resistance was noted in two of three orchards surveyed in both years. Failure to use dormant copper sprays, older trees, larger orchards, orchards with ≤10 cultivars, and application of >4 DMI sprays were positively correlated (0.0001 < P < 0.05) with the incidence of resistant isolates. Isolates from orchards with >4 DMI sprays were four times as likely to be resistant to fenbuconazole (odds ratio = 4.57; P = 0.015). Isolates from orchards without dormant copper sprays were twice as likely to be cross-shifted toward resistance to all three fungicides (odds ratio = 1.76; P = 0.048). Results identify management practices that can reduce the risk of V. inaequalis developing resistance to DMI fungicides.

Assessment of Application Timing of Bacillus spp. to Suppress Pre- and Postharvest Diseases of Apple.

Four isolates of Bacillus spp. were tested in a 2-year field study for biological control of pre- and postharvest diseases of apple. For the preharvest test, bacteria were applied to 'Golden Delicious' and 'Rome Beauty' trees in May or May and June. Foliar apple scab severity was assessed weekly. After harvest, fruit were wounded and then either left untreated or given a postharvest application of the bacteria. Wounded fruit were then inoculated with the bitter rot pathogen and lesion size was measured over 8 days. Bacillus megaterium isolate A3-6, B. mycoides isolate A1-1, and B. cereus isolate FLS-5 applied in May or May and June significantly reduced fruit and foliar apple scab severity in both years. A postharvest application of the bacteria (alone or in combination with a preharvest application) resulted in the greatest suppression of bitter rot on both cultivars (P < 0.04). The May + June + postharvest application of isolate A3-6 resulted in the greatest suppression of bitter rot, with an average of 45 and 95% reduction in lesion size compared with nontreated controls on 'Golden Delicious' and 'Rome Beauty' fruit, respectively. Results from this study indicate that preharvest applications of the bacteria were able to reduce foliar and fruit scab and an additional postharvest application was effective in reducing bitter rot severity.

FIBRILLIN4 is required for plastoglobule development and stress resistance in apple and Arabidopsis.

The fibrillins are a large family of chloroplast proteins that have been linked with stress tolerance and disease resistance. FIBRILLIN4 (FIB4) is found associated with the photosystem II light-harvesting complex, thylakoids, and plastoglobules, which are chloroplast compartments rich in lipophilic antioxidants. For this study, FIB4 expression was knocked down in apple (Malus 3 domestica) using RNA interference. Plastoglobule osmiophilicity was decreased in fib4 knockdown (fib4 KD) tree chloroplasts compared with the wild type, while total plastoglobule number was unchanged. Compared with the wild type, net photosynthetic CO(2) fixation in fib4 KD trees was decreased at high light intensity but was increased at low light intensity. Furthermore, fib4 KD trees produced more anthocyanins than the wild type when transferred from low to high light intensity, indicating greater sensitivity to high light stress. Relative to the wild type, fib4 KD apples were more sensitive to methyl viologen and had higher superoxide levels during methyl viologen treatment. Arabidopsis (Arabidopsis thaliana) fib4 mutants and fib4 KD apples were more susceptible than their wild-type counterparts to the bacterial pathogens Pseudomonas syringae pathovar tomato and Erwinia amylovora, respectively, and were more sensitive to ozone-induced tissue damage. Following ozone stress, plastoglobule osmiophilicity decreased in wild-type apple and remained low in fib4 KD trees; total plastoglobule number increased in fib4 KD apples but not in the wild type. These results indicate that FIB4 is required for plastoglobule development and resistance to multiple stresses. This study suggests that FIB4 is involved in regulating plastoglobule content and that defective regulation of plastoglobule content leads to broad stress sensitivity and altered photosynthetic activity.

Meta-analysis to determine the effects of plant disease management measures: review and case studies on soybean and apple.

The continuing exponential increase in scientific knowledge, the growing availability of large databases containing raw or partially annotated information, and the increased need to document impacts of large-scale research and funding programs provide a great incentive for integrating and adding value to previously published (or unpublished) research through quantitative synthesis. Meta-analysis has become the standard for quantitative evidence synthesis in many disciplines, offering a broadly accepted and statistically powerful framework for estimating the magnitude, consistency, and homogeneity of the effect of interest across studies. Here, we review previous and current uses of meta-analysis in plant pathology with a focus on applications in epidemiology and disease management. About a dozen formal meta-analyses have been published in the plant pathological literature in the past decade, and several more are currently in progress. Three broad research questions have been addressed, the most common being the comparative efficacy of chemical treatments for managing disease and reducing yield loss across environments. The second most common application has been the quantification of relationships between disease intensity and yield, or between different measures of disease, across studies. Lastly, meta-analysis has been applied to assess factors affecting pathogen-biocontrol agent interactions or the effectiveness of biological control of plant disease or weeds. In recent years, fixed-effects meta-analysis has been largely replaced by random- (or mixed-) effects analysis owing to the statistical benefits associated with the latter and the wider availability of computer software to conduct these analyses. Another recent trend has been the more common use of multivariate meta-analysis or meta-regression to analyze the impacts of study-level independent variables (moderator variables) on the response of interest. The application of meta-analysis to practical problems in epidemiology and disease management is illustrated with case studies from our work on Phakopsora pachyrhizi on soybean and Erwinia amylovora on apple. We show that although meta-analyses are often used to corroborate and validate general conclusions drawn from more traditional, qualitative reviews, they can also reveal new patterns and interpretations not obvious from individual studies.

Use of Gibberellic Acid for Management of Bunch Rot on Chardonnay and Vignoles Grape.

Harvest bunch rot of wine grape, caused primarily by Botrytis cinerea, is a perennial problem limiting the productivity of eastern vineyards, especially on cultivars with compact clusters. The aim of the present study was to evaluate the effectiveness of gibberellic acid (GA) sprays at reducing the compactness of Chardonnay and Vignoles clusters and minimizing bunch rot. Applications of GA reduced the number of berries per centimeter and the incidence and severity of bunch rots in Vignoles and, to a lesser extent, in Chardonnay over three consecutive years; however, the magnitude of GA effects often depended on the timing and rate of application. Bloom GA applications were more effective (P < 0.001) at reducing compactness and bunch rots than prebloom applications. Significantly, negative effects of GA applications on yield were negligible based on data from 4 years of trials on single vines and 2 years of data on 24-vine plots of Vignoles, provided the rates did not exceed 25 ppm. Regression analysis showed that berries per centimeter accounted for between 89 and 94% of variation in the incidence of Botrytis rot on Vignoles. On Chardonnay, compactness accounted for 53% of the variation in incidence, and the estimated compactness level at which no bunch rot would occur was 4.40 ± 1.05 (mean ± standard error) berries per centimeter. The relationship between cluster compactness and spray coverage of berries was also investigated in two separate experiments. Spray coverage of individual berries decreased linearly as cluster compactness increased within the range tested (3 to 18 berries per centimeter). Cluster compactness accounted for two-thirds of the variation in individual berry coverage, and coverage was reduced by 40 to 50% for clusters with about 18 berries per centimeter. These results strongly support the use of GA in integrated management of bunch rot on Vignoles and Chardonnay in eastern U.S. vineyards.

Virulence characteristics accounting for fire blight disease severity in apple trees and seedlings.

The gram-negative bacterium Erwinia amylovora is the causal agent of fire blight, the most destructive bacterial disease of rosaceous plants, including apple and pear. Here, we compared the virulence levels of six E. amylovora strains (Ea273, CFBP1367, Ea581a, E2002a, E4001a, and HKN06P1) on apple trees and seedlings. The strains produced a range of disease severity, with HKN06P1 producing the greatest disease severity in every assay. We then compared virulence characteristic expression among the six strains, including growth rates in immature apple fruit, amylovoran production, levansucrase activity, biofilm formation, carbohydrate utilization, hypersensitive cell death elicitation in tobacco leaves, and protein secretion profiles. Multiple regression analysis indicated that three of the virulence characteristics (amylovoran production, biofilm formation, and growth in immature apple fruit) accounted for >70% of the variation in disease severity on apple seedlings. Furthermore, in greenhouse-grown 'Gala' trees, >75% of the variation in disease severity was accounted for by five of the virulence characteristics: amylovoran production, biofilm formation, growth in immature apple fruit, hypersensitive cell death elicitation, and sorbitol utilization. This study demonstrates that virulence factor expression levels account for differences in disease severity caused by wild isolates of E. amylovora on apple trees.

Bee Vectoring: Development of the Japanese Orchard Bee as a Targeted Delivery System of Biological Control Agents for Fire Blight Management.

Fire blight, which is caused by the bacteria Erwinia amylovora, remains one of the most important diseases limiting the productivity of apple and pear orchards in the United States. In commercial orchards, in-season fire blight management relies exclusively on the use of antibiotic treatments (such as streptomycin and oxytetracycline) and on bacterial biocontrol agents whose efficacy is limited. We hypothesize that the efficacy of the biocontrol agents can be greatly enhanced through targeted delivery to flowers, which serve as initial infection courts, using the Japanese orchard bee, Osmia cornifrons. Many factors, such as the synchrony of life cycle with plant phenology and specificity to pomaceous plants, suggest that O. cornifrons could be an excellent vector of the biocontrol products during bloom in pome tree fruits. However, deployment of this pollinator species to deliver biocontrol agents for fire blight control has not been attempted previously due to the lack of an efficient system to pack the bodies of the bees exiting nesting tubes with the biocontrol products. In this study, we design and test a dispenser system to facilitate the use of O. conifrons as a vector for commercially available biocontrol products for fire blight control. The effectiveness of O. conifrons to deliver biocontrol agents to flowers, and to effect secondary dissemination from treated to untreated flowers is also evaluated in greenhouse experiments. We found that the O. conifrons bees were able to use the nest dispenser designed for the delivery of biological control products, and are effective in vectoring and delivering the Bacillus subtilis-based biological control product (Serenade®) to apple blossoms. We also found that the O. cornifrons were effective in secondary inoculation of this biological control product to newly-opened flowers. These findings suggest the potential use of commercially available O. conifrons and other orchard bees in targeted delivery of biological control products for fire blight, and possibly other diseases, in different fruit crops.

Relationship Between Cluster Compactness and Bunch Rot in Vignoles Grapes.

The impact of cluster compactness and debris retention on harvest bunch rot of Vitis interspecific hybrid 'Vignoles' was investigated between 2001 and 2005 near Lake Erie, in Pennsylvania. Cluster compactness was characterized subjectively based on the OIV code 204 standard and objectively by determining the number of berries per centimeter of rachis. In 2001, 2002, and 2004, the median number of berries per centimeter for loose clusters was 6.3, 7.0, and 6.4 compared with 10.2, 12.7, and 12.4 for the compact clusters, respectively. Kolmogorov-Smirnoff and the Mann-Whitney U two-samples tests confirmed that the distribution of the berries per centimeter was significantly (90 ≤ χ2 ≤ 184.3; P < 0.0001) different between the two subjective compactness categories. Cluster compactness was strongly correlated with bunch rot incidence (χ2 = 73.1 and 62.2 for 2001 and 2002, respectively; P < 0.0001), whereby disease incidence was higher in compact than in loose clusters. Logistic regression analysis indicated that every additional berry per centimeter unit of compactness almost doubled the likelihood of a cluster becoming infected with bunch rot (odds ratio = 1.828, 95% confidence interval [CI] = 1.392 to 2.399 in 2001 and odds ratio = 1.705, 95% CI = 1.394 to 2.085 in 2002). In 2004, bunch rot severity in compact clusters was nearly four times that of loose clusters. Linear regression analysis revealed that berries per centimeter accounted for >89% of the variation in bunch rot severity (R2 = 0.893, P < 0.0001, n = 30) and >74% in cluster weight (R2 = 0.745, P < 0.0001, n = 30). Accumulations of dehiscent floral debris contributed to greater bunch rot severity, and the effect was more pronounced in compact clusters than in loose clusters. Removal of basal leaves at trace bloom reduced berries per centimeter by 13% in 2004 and >25% in 2005, with corresponding reductions in bunch rot severity of 60% in 2004 and 62.5 to 82% in 2005. These results indicate that berries per centimeter is a good indicator of cluster compactness in Vignoles, and that practices that reduce cluster tightness would be effective in an integrated program for control of bunch rot on this cultivar.

Biology of flower-infecting fungi.

The ability to infect host flowers offers important ecological benefits to plant-parasitic fungi; not surprisingly, therefore, numerous fungal species from a wide range of taxonomic groups have adopted a life style that involves flower infection. Although flower-infecting fungi are very diverse, they can be classified readily into three major groups: opportunistic, unspecialized pathogens causing necrotic symptoms such as blossom blights (group 1), and specialist flower pathogens which infect inflorescences either through the gynoecium (group 2) or systemically through the apical meristem (group 3). This three-tier system is supported by life history attributes such as host range, mode of spore transmission, degree of host sterilization as a result of infection, and whether or not the fungus undergoes an obligate sexual cycle, produces resting spores in affected inflorescences, and is r- or K-selected. Across the three groups, the flower as an infection court poses important challenges for disease management. Ecologically and evolutionarily, terms and concepts borrowed from the study of venereal (sexually transmitted) diseases of animals do not adequately capture the range of strategies employed by fungi that infect flowers.

Mimicry in plant-parasitic fungi.

Mimicry is the close resemblance of one living organism (the mimic) to another (the model), leading to misidentification by a third organism (the operator). Similar to other organism groups, certain species of plant-parasitic fungi are known to engage in mimetic relationships, thereby increasing their fitness. In some cases, fungal infection can lead to the formation of flower mimics (pseudo flowers) that attract insect pollinators via visual and/or olfactory cues; these insects then either transmit fungal gametes to accomplish outcrossing (e.g. in some heterothallic rust fungi belonging to the genera Puccinia and Uromyces) or vector infectious spores to healthy plants, thereby spreading disease (e.g. in the anther smut fungus Microbotryum violaceum and the mummy berry pathogen Monilinia vaccinii-corymbosi). In what is termed aggressive mimicry, some specialized plant-parasitic fungi are able to mimic host structures or host molecules to gain access to resources. An example is M. vaccinii-corymbosi, whose conidia and germ tubes, respectively, mimic host pollen grains and pollen tubes anatomically and physiologically, allowing the pathogen to gain entry into the host's ovary via stigma and style. We review these and other examples of mimicry by plant-parasitic fungi and some of the mechanisms, signals, and evolutionary implications.