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Calonectria pseudonaviculata causes lesions on boxwood leaves and twigs. Controlled-environment experiments were conducted to determine the effects of temperature and leaf wetness period on C. pseudonaviculata sporulation on diseased (cv. Suffruticosa) leaves and of dryness periods and high temperature on conidial survival. Infected leaves were incubated in moist chambers and subjected to six temperatures (9, 13, 17, 21, 25, and 29°C) and six leaf wetness periods (0, 12, 24, 40, 48, and 72 h). Spore production was influenced significantly by wetness period, temperature, and their interaction. Increasing duration of leaf wetness and increasing temperature generally increased sporulation, with no sporulation occurring at 29°C or 9 and 13°C, except at 72 h of wetness exposure, while it was optimal at 21°C. Detached leaves with profuse conidia were subjected to a range of drying (relative humidity at 65%) times (0, 2, 4, 6, and 8 h) at two temperatures of 21 and 29°C. Conidia were then harvested and plated on water agar. Germinating conidia were counted to measure the spore viability. Spore mortality increased with increasing dryness duration at both temperatures but occurred more quickly and severely at 29 than 21°C. Overall, this study extended biological knowledge of conditions required for crucial stages of the C. pseudonaviculata disease cycle and the obtained results will be vital for developing boxwood blight forecasting and management tools. Accepted version

Burkholderia sp. strain SSG is a boxwood endophyte with potent antagonistic activities against a variety of plant pathogens. Here we present its complete genome sequence that is 8.6 Mb long with a GC content of 66.9%, 10,209 predicted protein-coding sequences, and 866 secondary metabolism gene clusters. Many of these genes and clusters involve antibiosis and other antagonistic activities against plant pathogens and insect pests as well as plant growth promoting traits but none for the Burkholderia cepacia epidemic strain marker. This genome sequence supports SSG as a potent biocontrol agent and source of other biotechnological applications. Published version

Calonectria pseudonaviculata (Cps) poses an increasing threat to boxwood, a major nursery crop and iconic landscape plant worldwide. Here, we report on a potent biocontrol agent that produces small sage green (SSG) colonies on potato dextrose agar. SSG is a bacterial strain recovered from Justin Brouwers boxwood leaves with unusual response to Cps inoculation. Water-soaked symptoms developed on leaves 2 days after inoculation then disappeared a few days later. This endophyte affected several major steps of the boxwood blight disease cycle. SSG at 107 cfu/mL lysed all conidia in mixed broth culture. SSG at 108 cfu/mL reduced blight incidence by >98% when applied one day before or 3 h after boxwood were inoculated with Cps. Its control efficacy decreased with decreasing bacterial concentration to 103 cfu/mL and increasing lead time up to 20 days. When applied on diseased leaf litter under boxwood plants, SSG reduced Cps sporulation and consequently mitigated blight incidence by 90%. SSG was identified as a new member of the Burkholderia cepacia complex with distinct characters from known clinical strains. With these protective, curative, and sanitizing properties, this Burkholderia endophyte offers great promise for sustainable blight management at production and in the landscape. Published version

Zoospore exudates play important roles in promoting zoospore communication, homing and germination during plant infection by Phytophthora. However, it is not clear whether exudates affect plant immunity. Zoospore-free fluid (ZFF) and zoospores of P. nicotianae were investigated comparatively for effects on resistance of Arabidopsis thaliana Col-0 and mutants that affect signaling mediated by salicylic acid (SA) and jasmonic acid (JA): eds16 (enhanced disease susceptibility16), pad4 (phytoalexin deficient4), and npr1 (nonexpressor of pathogenesis-related genes1). Col-0 attracted more zoospores and had severe tissue damage when flooded with a zoospore suspension in ZFF. Mutants treated with ZFF alone developed disease symptoms similar to those inoculated with zoospores and requirements of EDS16 and PAD4 for plant responses to zoospores and the exudates was apparent. Zoospore and ZFFs also induced expression of the PR1 and PDF1.2 marker genes for defense regulated by SA and JA, respectively. However, ZFF affected more JA defense signaling, down regulating PR1 when SA signaling or synthesis is deficient, which may be responsible for Arabidopsis mutant plants more susceptible to infection by high concentration of P. nicotianae zoospores. These results suggest that zoospore exudates can function as virulence factors and inducers of plant immune responses during plant infection by Phytophthora. Published version

Provides a set of guidelines for production nurseries with boxwood blight to contain and manage the disease in the most effective manner and avoid the spread of the disease to new locations.

Provides a set of guidelines for retail nurseries with boxwood blight to contain and manage the disease in the most effective manner and avoid the spread of the disease to new locations.

Provides information about boxwood blight so that growers can be aware of the symptoms, signs, and impacts of the pathogen and monitor nursery and landscape boxwoods for symptoms.

Powdery mildew and gray mold diseases have potential to cause significant economic losses to cut flower growers. Information is needed on the effects of these diseases on growers’ returns and the effectiveness of practices to control the diseases. A survey was conducted to examine powdery mildew and gray mold disease management practices on cut flower farms in North Carolina and Virginia. Twenty cut flower farmers completed an online survey and eight of those respondents also participated in an interview. The survey gathered information about the farms, such as its acreage and number of years growing cut flowers, flower genera affected by the diseases, fungicide use and efficiency, and cultural control use and efficiency. The survey and subsequent interviews provided several major findings. First, the floral genera most affected by powdery mildew in North Carolina and Virginia were Zinnia, followed by Dahlia. The genera most affected by gray mold were Ranunculus, followed by Anemone and Dahlia. Second, crop loss due to each disease was mostly at the 0 to 10% level, with one farm losing 91 to 100% of Phlox crops to powdery mildew and another losing 81 to 90% of Tulipa crops to gray mold. Third, 40% of respondents have not used any fungicides for disease management; and for those who have, neem oil was the product they used most frequently. Fourth, all respondents considered sustainability very important when making disease management decisions; this was supported by their extensive use of cultural practices for disease control, in addition to their preference of nonchemical approaches and biorational products over synthetic pesticides. The commonly used cultural controls included, crop rotation, proper watering, disease resistant cultivars, sanitation, proper spacing, and weeding. Findings from this survey will help cut flowers growers in the Mid-Atlantic region to better manage these two important diseases. MALS

The genus Phytophthora includes many agriculturally and ecologically important plant pathogens. Characterization of new Phytophthora species is the first and a most critical step to understanding their biology, ecology and economic importance. Six novel Phytophthora species recovered from irrigation systems at ornamental plant nurseries in Mississippi and Virginia were described based on morphological, physiological and molecular characters: 1. Phytophthora mississippiae sp. nov. produces a mix of non-papillate and semi-papillate sporangia, and catenulate hyphal swellings. It is a heterothallic species. All examined isolates of P. mississippiae are A1. When paired with A2 mating type testers, P. mississippiae produces ornamented oogonia and amphigynous antheridia. It is phylogenetically grouped in Phytophthora subclade 6b based on sequences of the rRNA internal transcribed spacer (ITS) region and the mitochondrially encoded cytochrome c oxidase 1 (cox 1) gene. 2. Phytophthora hydrogena sp. nov. is heterothallic. It produces non-caducous and non-papillate sporangia. It is characterized by frequently producing widening at the pedicel tip of sporangiophores or tapered sporangial based toward the point of attachment. This species is phylogenetically placed in a high-temperature tolerant cluster in Phytophthora clade 9. All members in this cluster grow well at 35 C. 3. Phytophthora virginiana sp. nov. is a self-sterile species. All examined isolates are silent A1. It produces non-caducous and non-papillate sporangia and is also placed in the high-temperature tolerant cluster in clade 9. Morphologically, it is characterized by producing abundant thin-walled, lateral chlamydospores in carrot agar and clarified V8 juice agar. 4. Phytophthora macilentosa sp. nov. is a heterothallic species. Only A1 isolates have been found. It produces characteristic elongated, non-papillate sporangia. It is also a member of the high-temperature cluster in clade 9. 5. Phytophthora stricta sp. nov. is a heterothallic species. It produces unique non-papillate and slightly caducous sporangia with one to three constrictions on its sporangiophore. Phylogenetically, P. stricta represents a new ITS clade within the genus. 6. Phytophthora Xstagnum nothosp. nov. is a novel hybrid species with P. taxon PgChlamydo as its paternal parent and a P. mississippiae-like species as its maternal parent. This new hybrid produces intercalary chlamydospores and catenulate hyphal swellings, which are morphological characters of P. taxon. PgChlamydo and P. mississippiae, respectively. It also produces both smooth-walled and ornamented oogonia, which may be indicative of oogonial characters of its paternal and maternal parents, respectively. By incorporating new Phytophthora species, clusters and clades, phylogenies including approximately 128 Phytophthora taxa were constructed based on sequences of five genetic markers. Among the selected genetic markers, the beta-tubulin (B-tub) gene provided the highest phylogenetic resolution. General phylogenetic structure of the B-tub phylogeny was similar to that in previous multi-locus phylogenies, except that P. cinnamomi, P. parvispora, P. quercina, P. stricta, and a provisional species, P. sp. e1, were not clustered in any of the 10 known Phytophthora clades and represented new clades. The B-tub phylogeny was also used to study the correlations between phylogeny and morphological characteristics including sporangial papillation, caducity, homothallism, and antheridial configuration, as well as maximum growth temperature. The results indicated that the character of sporangial papillation was mostly consistent among species within individual subclades. Maximum growth temperature was also generally correlated with phylogenetic positions. Consistency in caducity, homothallism or antheridial configuration was not found. A new multi-locus phylogeny based on sequences of 11 genetic markers of more than 146 Phytophthora species was proposed to validate new clades and clusters, as well as investigate detailed phylogenetic relations among species in this quickly expanding, taxonomically complex group of plant pathogens. Ph. D.

The potential of increasing disease problems through the use of recycled irrigation water in horticultural operations is a serious concern, yet basic research on waterborne plant pathogens in Virginia is lacking. In this work seasonal fluctuations and locations of Pythiaceae in a recycled water irrigation system at a container nursery were determined. Pythium spp. were recovered more frequently and in greater numbers than Phytophthora spp. Species of Phytophthora recovered in filtering assays were identified as P. capsici, P. citricola, P. citrophthora, P. cryptogea, P. drechsleri, and P. nicotianae. P. cryptogea and P. drechsleri were the only Phytophthora spp. recovered from baits placed on the surface of the irrigation reservoir, whereas a greater diversity of species was recovered from baits placed at depths. Hymexazol-amended medium was found to have limitations in recovery of Phytophthora spp. In pathogenicity tests, P. cactorum, P. capsici, P. citrophthora, and P. nicotianae caused significant mortality of Salvia officinalis and P. cactorum showed limited pathogenicity on Gerbera jamesonii. Asymptomatic (aboveground) plants were found to harbor inoculum long after Phytophthora-inoculation. Fresh weight analyses of roots and shoots of asymptomatic plants demonstrated that Phytophthora inoculation may either reduce or stimulate plant shoot growth, but little effect is apparent on roots. Irrigation with naturally infested irrigation water reduced plant growth. This research provides data for prioritizing development of detection technology and management practices for plant pathogens in irrigation water. The results may also lead to improvements in conventional water assay protocols for plant pathogens. Master of Science