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<p>The management of gray mold, caused by the fungus <em>Botrytis cinerea</em>, on ornamental plants relies heavily on fungicide applications. To improve fungicide efficacy, the manipulation of nozzle type, spray volume, and pressure influence droplet size (µm) and density (droplets/cm2) on the leaf’s surface. However, leaf wettability dictates how well the application droplets adhere and spread across the surface. When leaf surfaces are waxy (hydrophobic) or hairy (tomentose), droplets fail to adhere, impacting fungicide sorption.</p> <p>The goal of this research was to evaluate how the interaction of droplet density and leaf wettability impact the efficacy of chemical and biological fungicides against <em>Botrytis cinerea</em>. Leaf surfaces vary between species, within species, leaf age, and leaf sides (abaxial or adaxial). Hydrophobic leaf surfaces influence fungicide efficacy by reducing fungicide droplet spread compared to the wettable and hydrophilic leaf surfaces. The presence of trichomes on the leaf surface can inhibit droplets from reaching the surface.</p> <p>To quantify droplet density, a fine and coarse spray of fungicide treatments was applied with a yellow fluorescent dye. After application, <em>Begonia</em> x <em>hybrida</em> ‘Dragon wing’ leaves were placed on black, blackout curtains below a blacklight. Images were analyzed by ImageJ, using an image processing method. The number of lesions, disease incidence, were counted to observe fungicide efficacy. Results show there was no interaction between the actual droplet density within treatments applied with fine and coarse sprays. However, the interaction between spray type (fine and coarse) and treatments can have a significant effect on disease incidence. Disease incidence was significantly different between the systemic and contact fungicides for fine and coarse sprays. However, the systemic fungicide treatment had the highest disease incidence compared to the contact fungicide.</p> <p>To assess leaf wettability impact on fungicide efficacy, five <em>Begonia </em>species (<em>B. scharffii, B. erythrophylla, B. </em>x<em> hybrida ‘</em>Dragon Wing’<em>, B. epipsila, and B. goldingiana</em>) were used based on their observed leaf surface type. A contact angle goniometer was used to take pictures of a droplet on <em>Begonia</em> leaf surfaces. The quantification of the leaf surface took place by using the ImageJ program ‘Drop-Snake’ within the plugin ‘Drop Analysis’. The number of lesions, an indicator of disease incidence, were counted to observe fungicide efficacy. Results showed the contact angles were different between the <em>Begonia</em> species. There was a significant interaction between the <em>Begonia</em> species and treatments, where <em>Begonia</em> ‘hairy’ and ‘waxy’ leaf surfaces can influence fungicide efficacy. However, there was no significance for the interaction between <em>Begonia</em> species’ contact angles and treatments.</p> <p>These studies advance our understanding of how droplet density and leaf surfaces influence fungicide efficacy, thus improving our ability to manage <em>Botrytis</em> for diverse ornamental plants. </p>

Mefenoxam has been a premier compound for Phytophthora disease control in the nursery industry for 30 years. The primary objectives of this research were to examine whether Phytophthora species have developed resistance to this compound and to investigate fungicide resistance management strategies. Phytophthora nicotianae, a destructive pathogen of numerous herbaceous and some woody ornamental plants, was used as a model system. P. cinnamomi, a major pathogen of a wide range of tree species and shrub plants, was also included for comparison. Twenty-six isolates of P. nicotianae were highly resistant to mefenoxam with a mean EC50 value of 326.5 µg/ml while the remaining 70 were sensitive with an EC50 of <0.01 µg/ml (Label rate: 0.08µg/ml). All resistant isolates were recovered from herbaceous annuals and irrigation water in 3 Virginia nurseries. Resistant isolates were compared with sensitive ones using seedlings of Lupinus "Russell Hybrids" in the absence of mefenoxam for relative competitive ability. Resistant isolates out-competed sensitive ones within 3 to 6 sporulation cycles. Resistant isolates exhibited greater infection rate and higher sporulation ability than sensitive ones. No mefenoxam resistant isolates were identified in P. cinnamomi. All 65 isolates of P. cinnamomi were sensitive to mefenoxam with an EC50 of < 0.04 ï ­g/ml. Attempts to generate mutants with high resistance to mefenoxam through UV mutagenesis and mycelial adaptation were not successful. However, there were significant reductions in sensitivity to mefenoxam; those slightly resistant mutants carried fitness penalties, which may explain why P. cinnamomi remains sensitive to mefenoxam. The effect of propamocarb hydrochloride on different growth stages of Phytophthora nicotianae was evaluated in search for an alternative fungicide. Propamocarb greatly inhibited sporangium production, zoospore motility, germination and infection. However, it has little inhibition of mycelial growth and infections. Propamocarb can be used as an alternative fungicide to mefenoxam where mefenoxam resistance has become problematic. However, it must be used preventively; i.e. before infections occur. The genetic inheritance of mefenoxam resistance in P. nicotianae was studied using F1 progenies of a cross between resistant and sensitive isolates. The F1 progenies segregated for mefenoxam resistance in ratio of 1R:1S, indicating the mefenoxam resistance is controlled by a single dominant gene. One RAPD marker putatively linked to resistant locus in repulsion phase was obtained by bulked segregant analysis and was converted to the SCAR marker. This marker is capable of differentiating mefenoxam resistant populations from sensitive populations included in this study. / Ph. D.

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Low temperature injury is among the greatest challenges facing golf courses with ultradwarf bermudagrass (UDB) (Cynodon dactylon (L.) Pers. x C. transvaalensis Burtt-Davy) putting greens in Virginia. This research focused on the impact of turf covers, fungicide programming, core aeration, and trinexapac-ethyl (TE) on UDB cold tolerance, winter quality, and cold de-acclimation (CD). Our results indicate that the use of turf covers significantly increased UDB canopy and soil temperatures when air temperatures were below -3.9°C. Air gaps under covers and the use of double turf covers increased soil and canopy temperatures compared to single covers alone in some instances, but results were inconsistent. Late fall and early winter fungicide applications of chlorothalonil and azoxystrobin improved UDB quality throughout winter dormancy and spring green up. The addition of a pigmented phosphonate significantly improved winter and spring UDB quality. The addition of acibenzolar-S-methyl to fungicide programs did not improve winter UDB quality or spring green up. Summer core aeration programs were evaluated for their impact on spring green up, turfgrass quality, surface firmness, and moisture retention. Spring UDB green up was improved incrementally as surface disruption increased. Treatments with 20%, 15%, and 10% surface disruption produced higher color vs treatments with lower surface disruption. Surface firmness and volumetric water content of UDB were impacted by construction method but were not significantly impacted by core aeration programs. Field research revealed that 'fall only' and 'fall and winter' TE applications improved UDB quality but only 'fall and winter' delayed UDB premature CD in early spring when UDB can be susceptible to low temperature injury. Growth chamber studies evaluated the impact of TE on UDB cold tolerance to -9.4°C x time duration. Regression analysis predicted a 50% mortality exposure point for UDB under TE treatments of 9.84 hours at -9.4°C (r2=0.836) compared to 11.38 hours at -9.4°C (r2=0.671) for non-treated UDB during cold acclimation. Winter and spring scenarios resulted in delayed CD under TE but no differences in cold tolerance when exposed to -9.4°C. Together, these results increase our understanding of the impact of management practices on UDB winter quality, CD, and low temperature injury. / Doctor of Philosophy / Ultradwarf bermudagrass putting greens are commonly found on golf courses in warm climates. These grasses thrive in heat and humidity but are susceptible to injury or death when exposed to cold temperatures. This research is focused on evaluating management practices that may impact bermudagrass' susceptibility to injury from cold temperature exposure. The cultural practices evaluated include turf covers, fungicide programming, core aeration, and the use of plant growth regulators to manipulate the turfgrasses own self defense mechanisms. Our results show that the use of turf covers significantly increased putting green canopy and soil temperatures when air temperatures were below -3.9°C. Air gaps under covers and the use of double turf covers increased soil and canopy temperatures compared to single covers alone in some instances, but results were inconsistent. Late fall and early winter fungicide applications of commonly-used fungicides improved putting green quality throughout winter dormancy and spring green up. The addition of a green-pigmented phosphonate fungicide significantly improved winter and spring putting green quality. The addition of a plant defense activator, acibenzolar-S-methyl to fungicide programs did not improve winter quality or spring green up. Summer core aeration programs were evaluated for their impact on spring green up, turfgrass quality, surface firmness, and moisture retention. Spring green up was improved incrementally as surface disruption increased. Treatments with 20%, 15%, and 10% surface disruption produced higher color vs treatments with lower surface disruption. Surface firmness and soil moisture content of the putting greens were impacted by construction method but were not significantly impacted by core aeration programs. Field research revealed that 'fall only' and 'fall and winter' plant growth regulator applications improved ultradwarf bermudagrass quality but only 'fall and winter' delayed premature green-up in early spring when the turfgrass can be susceptible to low temperature injury. Growth chamber studies revealed that plants treated with the growth regulator, trinexapac-ethyl were more sensitive to low-temperature exposure than non-treated plants. Together, these results increase our understanding of the impact of management practices on UDB winter quality, CD, and low temperature injury.

This study was undertaken to determine the sensitivity of grape downy mildew (DM, Plasmopara viticola) and powdery mildew (PM, Erysiphe necator) to commonly used single-site fungicides in Virginia and nearby states. DM and PM isolates were collected from 2005 to 2007. In grape leaf disc bioassays, 92% of the DM isolates were QoI (azoxystrobin)-resistant, but none were resistant to mefenoxam. Eighty-two percent of the PM isolates were QoI-resistant, but none were resistant to boscalid and quinoxyfen. The frequency of the G143A point mutation, which confers high levels of QoI resistance, was quantified in DM and PM isolates by real-time PCR. Most of the QoI-resistant DM and PM isolates contained >95% of the 143A allele. QoI-sensitive DM isolates contained less than 1% of 143A. One out of 145 and 14 out of 154 QoI-resistant DM and PM isolates (able to grow on azoxystrobin concentration ï ³ 1 µg/ml), respectively, contained less than 1% 143A. Most PM isolates exhibited reduced sensitivity to five DMI fungicides when compared to a sensitive subgroup (n=9) and compared to published reports for unexposed populations; the resistance factor (median EC50 of the entire isolate collection divided by median EC50 of sensitive subgroup) was highest for tebuconazole (360) and myclobutanil (350), followed by triflumizole (79), triadimefon (61), and fenarimol (53). Sensitivities to all five DMI fungicides, but also azoxystrobin, were moderately to strongly correlated (pairwise r-values ranging from 0.60 to 0.88). / Master of Science in Life Sciences

Development of fungicide resistance in fungal and oomycete pathogens is a serious problem in grape production. Quinoxyfen is a fungicide widely used against grape powdery mildew (Erysiphe necator). In 2013, E. necator isolates with reduced quinoxyfen sensitivity (designated as quinoxyfen lab resistance or QLR) were detected in Virginia. Field trials were conducted in 2014, 2015, and 2016 at the affected vineyard to determine to what extent quinoxyfen might still contribute to disease control. Powdery mildew control by quinoxyfen was good, similar to, or only slightly less, than that provided by myclobutanil and boscalid in all three years. The frequency of QLR in vines not treated with quinoxyfen declined only slowly over the three years, from 65% to 46%. Information about the mode of action of quinoxyfen is limited; previous research suggests that quinoxyfen interferes with the signal transduction process. We profiled the transcriptomes of QLR and sensitive isolates in response to quinoxyfen treatment, providing support for this hypothesis. Additional transcriptional targets of quinoxyfen were revealed to be involved in the positive regulation of the MAPK signaling cascade, pathogenesis, and sporulation activity. Grape downy mildew (Plasmopara viticola), another important grape pathogen, is commonly controlled by phosphite fungicides. A field trial and laboratory bioassays were conducted to determine whether P. viticola isolates from vineyards with suspected control failures showed reduced sensitivity against phosphite fungicides. Prophyt applied at 14-day intervals under high disease pressure provided poor downy mildew control in the field. Next-generation sequencing technologies were utilized to identify 391,930 single nucleotide polymorphisms (SNPs) and generated a draft P. viticola genome assembly at ~130 megabase (Mb). Finally, field isolates of P. viticola collected from a Virginia vineyard with suspected mandipropamid control failure were bioassayed. The EC50 values of the isolates were >240 μg.ml-1 for mandipropamid, well above the field rate. The PvCesA3 gene of two resistant isolates was sequenced revealing that these isolates had a GGC-to-AGC substitution at codon 1105, the same mutation that has been found associated with CAA resistance elsewhere. / PHD / Powdery and downy mildew are two diseases of grapes that can cause large yield losses, and are usually controlled by regular fungicide applications. Development of fungicide resistance has been a growing challenge. Quinoxyfen is a protectant fungicide commonly used against powdery mildews. Unusual grape powdery mildew isolates that grew well on quinoxyfen-treated plants in the laboratory (designated as quinoxyfen lab resistance or QLR) were detected in a Virginia vineyard. In 2014, the first year of this study, 65% of powdery mildew isolates from parts of this vineyard that received no further quinoxyfen treatments had the QLR type of resistance, and this declined only slowly to 46% by the third year. Field trials were conducted in 2014, 2015, and 2016 to determine the efficacy of quinoxyfen in the presence of QLR. Powdery mildew control by quinoxyfen on both grape clusters and leaves was similar to, or only slightly less, than that provided by the standard anti-powdery mildew fungicides myclobutanil and boscalid in all three years. In order to gain a better understanding of the mode(s) of action and resistance mechanism(s) of quinoxyfen, gene expression of QLR and sensitive isolates, both in the presence and absence of quinoxyfen, was analyzed by nucleic acid sequencing. This study confirms previous research suggesting that quinoxyfen interferes with the important biological process signal transduction, and revealed additional gene targets of quinoxyfen. The phosphites are a group of fungicides commonly used to control grape downy mildew. Control failures after phosphite application have occasionally been suspected, and downy mildew isolates from vineyards with and without suspected control failures were tested in laboratory bioassays to determine if any level of resistance could be demonstrated. There was a limited range of sensitivity, and none of the isolates showed a notable loss of sensitivity. A field trial was conducted to determine the efficacy of one phosphite fungicide, Prophyt, applied at 14-day intervals under conditions favorable for disease development. Prophyt provided poor downy mildew control, suggesting that it has to be applied more frequently. Next-generation sequencing technologies were utilized to identify genetic markers for clade identification and generated a draft genome assembly of grape downy mildew, which improves the understanding of grape downy mildew genome. Grape downy mildew isolates collected from a vineyard in Virginia where mandipropamid provided poor control of downy mildew were bioassayed. The isolates tolerated mandipropamid rates well above the field rate, showing that they were indeed resistant. The mutation that confers mandipropamid resistance on other continents was found in the PvCesA3 gene of two resistant isolates.

La lutte chimique est la principale méthode utilisée pour contrôler les maladies causées par les champignons phytopathogènes. Dans certains cas, desphénomènes de résistance envers les fongicides se développent au sein despopulations, altérant parfois l’efficacité des molécules. La compréhension du moded’action des fongicides et des mécanismes de résistance sous-jacents participe à élaboreret à adapter des stratégies de management anti résistance ; et ainsi permettre depérenniser la durée de vie des molécules. Le fenhexamid est un fongicide récent (BayerCropScience, 2000), avec un mode d’action unique. Il est le seul fongicide commercialisébloquant l’étape de C4-déméthylation de la biosynthèse de l’ergostérol. Plusieurs typesde résistance (naturelle et acquises) ont été détectées dans les vignobles européens chez lecomplexe d’espèces Botrytis spp. responsable de la pourriture grise de la vigne. Lestravaux développés durant la thèse s’inscrivent dans l’objectif de la caractérisation dumode d’action et de l’élucidation des mécanismes de résistance. Le premier axe s’estattaché à la caractérisation fonctionnelle de deux gènes impliqués dans la C-4déméthylation de la biosynthèse de l’ergostérol : le gène erg27 codant la 3-céto réductase,cible du fenhexamid, et le gène erg28 codant une protéine qui interagirait en partie avecla 3-céto réductase. Concernant la résistance au fenhexamid, il a été démontré que, pargénétique inverse, les mutations détectées dans le gène erg27 de différents types d'isolatsrésistants issus du vignoble (phénotypes de résistance HydR3- et HydR3+) conféraient larésistance. Par ailleurs, une analyse de fitness du phénotype le plus préoccupant(phénotype HydR3+) a été réalisée en conditions contrôlées sur des souches isogéniquesartificielles afin d’apporter une réponse sur la persistance possible de ces souches auvignoble. Une méthode fine de quantification moléculaire de ces mêmes isolats aégalement été mise au point pour faciliter le suivi de leur évolution et de la persistancedes populations naturelles à l’échelle des vignobles. Cette nouvelle méthode, nomméeASPPAA PCR, exploite le polymorphisme nucléotidique du gène erg27, à l’origine de larésistance. Enfin, la résistance naturelle au fenhexamid de l’espèce apparentée à Botrytiscinerea, appelée Botrytis pseudocinerea a été élucidée. La résistance au fongicide de cetteespèce a été expliquée par la combinaison de modifications de cible (mécanismeminoritaire) et d’une dégradation du fongicide par un cytochrome P450 nomméCyp68.4 (mécanisme majeur). Il s’agit de la première identification et caractérisationgénétique d’un mécanisme de résistance à un fongicide conférée par un processus dedétoxification chez un champignon phytopathogène. / Chemical control is the main method used to control diseases caused byphytopathogenic fungi. In some cases, the resistance phenomena towardfungicides occur within fungal populations, which might alter practicalefficiency of molecules. Understanding modes of action of fungicides andunderlying resistance mechanisms participate to the development and adaptationof management strategies against resistance, and thus help to sustain the life ofmolecules. Fenhexamid is a recent fungicide (Bayer CropScience, 2000), with aparticular mode of action. It is the only fungicide marketed blocking the C4-demethylation step of ergosterol biosynthesis. Several types of resistance (naturaland acquired) were detected in European vineyards in the Botrytis spp speciescomplex, causing grey mold disease. This work focused on the characterization ofthe mode of action and the elucidation of resistance mechanisms. The first aspectinvestigated the functional characterization of two genes involved in the C4-demethylation of ergosterol biosynthesis. The erg27 gene potentially encoding the3-keto reductase which is the fenhexamid target and the erg28 gene encoding aprotein that interact in part with the 3-ketoreductase. Concerning fenhexamidresistance, we shown by reverse genetics that mutations detected in the erg27 genefrom different resistant isolates from the vineyards (phenotypes HydR3- andHydR3+) confer resistance. Furthermore, a fitness analysis under controlledconditions on the most worrying resistant phenotype (HydR3+) was performed onisogenic artificial strains in order to predict the possible persistence of these strainsin vineyards. A fine molecular method to quantify these isolates was developed tofacilitate the follow-up of evolution and persistence of resistant populations in thevineyard. This new method, named ASPPAA PCR is based on the nucleotidepolymorphism of the erg27 gene, responsible for fenhexamid resistance. Finally,the natural resistance to fenhexamid of the related species to Botrytis cinerea, B.pseudocinerea, was elucidated. Fungicide resistance of this species is explained bythe combination of target site modifications (minor mechanism) and fungicidedegradation mediated by a cytochrome P450 named Cyp68.4 (major mechanism).This is the first characterization of a genetic resistance mechanism to a fungicideconferred by detoxification in a phytopathogenic fungus.

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