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Venturia inaequalis is the causal pathogen of apple scab, one of the most economically important fungal diseases of apples. Understanding how this devastating pathogen evolves over time and space can help plant pathologists predict new pathogen populations and their effective management. In this thesis, I describe my study of the Malus-Venturia pathosystem and the genetics of pathogen evolution and host resistance to apple scab. I present an overview of the pathosystem, including modern apple production, V. inaequalis disease cycle, and previous genetic diversity studies within the species. I describe my analysis of the population genetic study of Venturia spp. and its evolutionary patterns as it coevolved with its Malus spp. hosts. Sequence data was compiled from publicly available sources and locally collected Venturia spp. isolates, and SNPs were identified and filtered within these sequences. These polymorphisms identified population differentiation and genetic diversity differences between Venturia isolates collected on various Malus spp. hosts. I also present the experiments for the identification of genetic recombinants amongst the F1 progeny of two mapping populations for the eventual fine mapping of scab resistance in the apple cultivar ‘Honeycrisp’. The common resistant parent in both populations, MN1980, contains the major scab resistance genes Rvi6 and Vhc1 on linkage group 1 in repulsion. The identification of recombinant progeny with these two genes in the coupling phase will allow development of markers tightly linked with Vhc1 for marker assisted selection to pyramid these loci for apple breeding.

Apples are an important specialty crop in New York State, and NYS is the second largest producers of apple nationwide. Of economic importance is the impact of managing the fungal pathogen causing apple scab, Venturia inaequalis. If left unmanaged, this disease will render the fruit unmarketable due to the malformation and commercially unacceptable olive-green to brown lesions that develop on fruit. The disease can also have negative impacts on overall tree health by reducing photosynthesis when the severity of leaf infection is high. In the absence of durable host resistance, commercial management relies heavily on the use of fungicides to prevent and control apple scab infections. Misuse of fungicides targeting a single site within fungi has resulted in sequential development of resistance to many classes of fungicide, resulting in ineffective fungal disease management. The first goal of this research was to characterize the efficacy and target site of succinate dehydrogenase inhibitors (SDHIs), a class of fungicides in which new chemistries have been released. This work demonstrated high in vitro efficacy of SDHI fungicides and provided the framework for future phenotypic and genotypic screening for fungicide resistance. The second goal of this research was to improve our understanding of how to best use these fungicides to prevent resistance development. We investigated practices involving application at both low and high doses as well as uses in different mixtures. We found that application at the higher doses often led to isolates with sensitivity shifted towards resistance. Additionally, this work reiterated the importance of mixing fungicide modes of action to prevent selection for resistance. Finally, the third goal of this research was to create a management plan that uses SDHIs with more sustainable biopesticide chemistries. We found potential for a program where SDHIs rotated with biopesticides could provide season-long management of apple scab. The work completed in this dissertation will help contribute to sustainable use of SDHI fungicides for the management of apple scab.

Reducing apple (Malus x domestica Borkh.) crop load during bloom is a reliable option for increasing fruit quality and return bloom. In this thesis, multiple approaches to improving bloom thinning practices are discussed. The first project analyzed the pollen tube growth of several crabapple cultivars. Previous research had improved the use of bloom thinning chemicals, by coordinating the application timing with the pollen tube growth between pollination and fertilization. However, pollen tube growth rates have only been measured in a few genotypes. In Chapter 2, the pollen tube growth rates of five crabapple cultivars were measured in the styles of 'Fuji', 'Golden Delicious', and 'Pink Lady' flowers, at four temperatures 12, 18, 24 and 30 C. Complex relationships were found among paternal pollen tube growth, maternal cultivar, and temperature. Chapters 3 and 4 describe projects where organically-approved chemicals, including the biofungicide, Regalia, were evaluated for their ability to simultaneously reduce crop load and decrease early season disease infection. These chemicals were applied in conventionally managed orchards (Chapter 3), and in an organically-managed 'Honeycrisp' orchard (Chapter 4). The number of chemicals approved for bloom thinning is limited, especially in the Eastern U.S. where lime sulfur and oil applications are not permitted during bloom. These studies indicate Regalia, applied during bloom, can reduce crop load and provide early season disease control. The research presented in this thesis provides new knowledge that can be incorporated into crop load management practices in both conventional and organic apple orchards.

Apple scab, caused by the fungal pathogen Venturia inaequalis is among the most prevalent and economically important diseases in commercial apple orchards in regions with temperate climates worldwide. The absence of durable apple scab resistance in the majority of commercially desired cultivars often mandates numerous fungicide applications each season to successfully manage the disease. Highly effective single-site fungicides have enhanced the ability to manage a number of phytopathogenic fungi, including V. inaequalis. Unfortunately the highly specific nature and repetitive use of these fungicides has led to their diminished efficacy and widespread resistance in V. inaequalis. The identification of the prevalence of single-site resistance in populations of V. inaequalis and an understanding of molecular mechanisms of resistance to these fungicides can aid in the rapid detection of fungicide resistance, the deployment of fungicide anti-resistance management strategies, and the development of efficient and effective chemical management programs for apple scab control. In this dissertation, a combination of applied and basic research was implemented to gain further understanding of the molecular mechanisms associated with practical resistance to DMI and QoI fungicides in isolates and/or populations of V. inaequalis. Microscopy-aided mycelial growth assays were used to determine sensitivity phenotypes to the DMI fungicides myclobutanil and difenoconazole and to the QoI fungicide trifloxystrobin. The majority of populations of V. inaequalis collected throughout the Northeastern, Mid-Atlantic, and Mid-Western United States were found to have reduced sensitivity or practical resistance to myclobutanil, but not difenoconazole. Illumina sequencing and reverse-transcriptase quantitative PCR were used to identify the contribution of target gene overexpression and the presence of upstream repeated elements in myclobutanil and difenoconazole resistance. Another molecular technique, allelespecific quantitative PCR was used to evaluate the role of target gene heteroplasmy in the quantitative resistance response and practical resistance to trifloxystrobin in isolates and populations of V. inaequalis. Additionally, baseline sensitivity of V. inaequalis to three SDHI fungicides was determined, and the sdhB gene, a target for SDHI fungicides, was identified and characterized. In summary, the results of this dissertation contribute to the identification and understanding of mechanisms associated with single-site fungicide resistance in V. inaequalis.

Two novel apple scab resistance loci have been identified in the apple cultivar Honeycrisp, an emerging cultivar in North America that is utilized in apple breeding programs worldwide. Greenhouse inoculation experiments with the apple scab fungal pathogen, Venturia inaequalis, identified a resistance defense response in `Honeycrisp' and its ancestors `Keepsake', `Frostbite', and `Northern Spy' seven days after inoculation. The defense response ranged from necrotic and chlorotic lesions to stellate necrosis and class 3b lesions (with sporulation). A hallmark of the resistance defense response is autofluorescence at the infection site in cleared leaf tissue. Several `Honeycrisp' progeny populations were screened with monoconidial isolates of V. inaequalis and segregated 3:1 for resistance, suggesting two resistance genes inherited from `Honeycrisp'. A consensus `Honeycrisp' linkage map with 1091 SNP markers was constructed for use in mapping. Two resistance loci were mapped using linkage mapping and quantitative trait loci mapping approaches. Marker haplotypes were constructed to trace the inheritance of resistance loci. Rvi19 mapped on linkage group 1 at ~50 cM. In the Rvi19 haplotype, the 138 bp allele for the Ch-Vf1 marker cosegregates with resistance, and is identical by state (IBS) with the Rvi17 resistance in `Antonovka'. Rvi19 is transmitted from `Frostbite' to `Keepsake' to `Honeycrisp', and into the resistant progeny of `Honeycrisp'. The other locus, Rvi20, mapped onto linkage group 15, and is IBS with a marker haplotype found in the susceptible cultivar Golden Delicious. Rvi20 is transmitted to `Honeycrisp' from an unknown parent. Molecular marker haplotypes were used to identify advanced selections in the University of Minnesota breeding program with pyramided scab resistance. Candidate genes were identified at each haplotype that can serve as starting points for identifying the functional genes conferring resistance. A collection of V. inaequalis isolates was assembled and curated from six locations in Minnesota for screening scab resistance. These 80+ isolates were examined for genetic diversity and population structure and provide a snapshot of the diversity of the pathogen present at this time.

Apple scab (causal organism: Venturia inaequalis) is an economically devastating disease of apples that is predominantly controlled with fungicides. Of the chemical classes currently available, the sterol-inhibiting (SI) and strobilurin (QoI) fungicides are the most commonly used. Recent observations indicate that V. inaequalis populations in Virginia have developed resistance to myclobutanil and other SIs. However, little is known about the frequency and distribution of SI and QoI resistance in Virginia's scab populations. The first objective of this research was to evaluate V. inaequalis populations in Virginia for SI and QoI resistance. Fungal isolates were collected from experimental orchards at the Alson H. Smith Jr., Agricultural Research and Extension Center (AHS AREC) and from commercial orchards in Virginia and Maryland. Sensitivities were determined by assessing colony growth at 19°C on potato dextrose agar (PDA) amended with 0 or 1.0 µg ml-1 of myclobutanil (SI) (N=87) or trifloxystrobin (QoI) (N=25) at 28 days. A range of fungicide sensitivity was observed for both chemical classes. The second objective of this research was to monitor the temporal dynamics of SI resistance over five sequential field seasons. To monitor shoot growth, neon rubber bands were placed over actively growing shoot tips following myclobutanil application or sample collection. Fungal isolates were collected from the same trees from 2007 through 2010 (N=176) and compared with isolates collected from wild apple seedlings (N=3). A continuum of SI resistance was observed for each year, and the V. inaequalis population exhibited a baseline shifted toward reduced sensitivity. The third objective of this research was to examine the spatial distribution of SI fungicide resistance within the tree canopy in a lower-density orchard (less than 150 trees A-1). Leaves collected from larger trees (>8m) in a lower-density orchard at the AHS AREC were analyzed for manganese deposition, pre- and post-mancozeb application. Fungal isolates (N=105) were collected from several locations within the canopy in replicated trees in the same orchard. Weather sensors also monitored the microclimates within those tree canopies. Spray deposition, microclimate and SI resistance were influenced by canopy location. The fourth objective of this research was to investigate potential SI resistance mechanisms. Previously classified isolates were screened for point mutations within the CYP51A1 gene (Appendix C), differences in polymorphic bands (alleles) (Appendix D), and differences in metabolism of myclobutanil (Appendix E). The consensus sequences for the CYP51A1 gene were identical for all isolates tested (N=9), and results from amplified fragment length polymorphism experiment (N=82) were inconclusive. There were, however, significant differences among incubation time and myclobutanil concentration in the bioassay (N=11). Our results indicate that myclobutanil is still an effective compound for control of apple scab in many areas of Virginia.

Alternaria blotch of apples emerged in the late 1980s in North Carolina threatening the nations seventh largest apple producing state. Alternaria blotch, causal agent Alternaria mali, has since spread throughout the entire Southeast apple growing region. Alternaria blotch proved to be difficult to control with the management programs in place at the time. Filajdic et al developed a weather-based disease-forecasting model to aid in the timing of fungicide applications to manage the disease. However, this model was based on the use of iprodione (Rovral), which never became fully registered for use on apples. The purpose of this project is to improve the prediction of the onset and progression of Alternaria blotch to aid in the timing of fungicide applications. The ability of four Alternaria models for predicting the occurrence of Alternaria blotch was examined over three growing seasons. Five sites across the apple production area of Henderson County, North Carolina were established to track disease progress and collect weather information. The data collected on-site were compared to the output of the disease models generated from on-site weather data as well as geospatial satellite based weather data. The initial results of the project show that none of the examined models exhibit a strong correlation with either the onset or the progression of the disease. However, examining the individual disease model attributes revealed key information about the disease progression. The detailed model analysis suggests that the initial inoculum level, in addition to the increasing susceptibility of the host, most readily influences the progression of the disease over the course of a growing season. The traditional approach of plant disease model validations is usually limited in scope to counties, states, or regions. With the possible threat of bio-terrorist attacks on our nation's agricultural system, the ability to create and validate models on a national scale is of paramount importance in protecting America's immense agro-economic infrastructure. The nearly non-existent national disease incidence and severity data sets for foliar fungal pathogens are a serious limitation for the accurate validation of risk prediction models such as The North Carolina State University / Animal and Plant Health Inspection Service Plant Pathogen Forecasting System (NAPPFAST). The future of rapid disease model validations is dependent on the existence of research tools to give researchers the ability to accurately predict the potential establishment of exotic diseases. NAPPFAST is a template based modeling tool that uses daily 10-km2 geospatial weather input to create empirical infection models. An adequate validation of NAPPFAST models requires a data set of disease observations with national coverage for multiple years, pathogens and crops. The object of this project was to investigate the potential suitability of meta-analysis techniques to validate the NAPPFAST infection model using disease observation data from fungicide trials available in Fungicide and Nematicide Tests reports. Data on the incidence of apple scab, caused by Venturia inaequalis, were obtained for 10 years for six locations and 25-years for one location. There was a poor correlation between the output from the NAPPFAST model and the observed incidence of apple scab for the locations and years examined. The model appears to be missing variables such as inoculum density or phenological data that may play a key role in apple scab forecasting.