Warren E. Copes

Genetic Characterization of Binucleate Rhizoctonia Species Causing Web Blight on Azalea in Mississippi and Alabama

Web blight on containerized azalea is an annual problem for commercial nurseries during summer months in the southern United States. Losses to web blight are associated with the cost of fungicide applications, delayed marketing of diseased plants, and plant death. Two hundred and eleven isolates of binucleate Rhizoctonia were recovered from azalea leaves with web blight symptoms from two nurseries in Mississippi and Alabama over 3 years. The internal transcribed spacer region (ITS) of the ribosomal DNA (rDNA) was sequenced from all isolates to determine genetic identity. A single anastomosis group (AG) of binucleate Rhizoctonia represented 92% of the samples collected from infected leaves. Genetic data and hyphal fusion experiments confirmed that these isolates belong to AG-U, which was recently identified from root and stem infections on miniature rose in Japan. Isolates of binucleate Rhizoctonia belonging to anastomosis groups AG-R, CAG-7 (=AG-S), and AG-G were also identified in the sample in low frequency. This is the first report of the occurrence of binucleate Rhizoctonia AG-U in the United States.

Phytophthora mississippiae sp. nov., a New Species Recovered from Irrigation Reservoirs at a Plant Nursery in Mississippi

A previously unknown Phytophthora species was recovered from irrigation water in Mississippi. This novel species produced both nonpapillate and semipapillate sporangia, and catenulate hyphal swellings. All examined isolates were compatibility type A1. Ornamented oogonia with amphigynous antheridia and plerotic oospores were produced when this novel species was paired with A2 mating type testers of P. cryptogea and P. nicotianae in polycarbonate membrane tests. Sequence analyses of the rDNA internal transcribed spacer (ITS) region and the mitochondrially encoded cytochrome c oxidase 1 (cox 1) gene placed this species in clade 6 of the genus Phytophthora. Based on the morphological, physiological and molecular features, this new species is named as Phytophthora mississippiae sp. nov. The implications of these results are discussed.

Two novel species representing a new clade and cluster of Phytophthora

Phytophthora stricta sp. nov. and Phytophthora macilentosa sp. nov. are described based on morphological, physiological and molecular characters in this study. Phytophthora stricta represents a previously unknown clade in the rRNA internal transcribed spacer (ITS)-based phylogeny. Phytophthora macilentosa, along with nine other species, consistently forms a high temperature-tolerant cluster within ITS clade 9. These observations are supported by the sequence analysis of the mitochondrial cytochrome c oxidase 1 gene. Both species are heterothallic and all examined isolates are A1 mating type. Phytophthora stricta produces nonpapillate and slightly caducous sporangia. This species is named after its characteristic constrictions on sporangiophores. Phytophthora macilentosa produces nonpapillate and noncaducous sporangia, which are mostly elongated obpyriform with a high length to breadth ratio. Both species were recovered from irrigation water of an ornamental plant nursery in Mississippi, USA and P. stricta was also recovered from stream water in Virginia, USA.

Spore Germination, Infection Structure Formation, and Colony Development of Erysiphe pulchra on Dogwood Leaves and Glass Slides

Spore germination, infection structure formation, and colony development of Erysiphe pulchra on glass slides and leaf disks of a susceptible flowering dogwood line were examined using light and scanning electron microscopes. On both glass slides and leaf disks, germination of conidia started within 2 h after inoculation (hai). One to four germ tubes grew from two poles of a conidium, one or two of the germ tubes formed initial appressoria, and only one of the germ tubes with initial appressoria formed secondary appressoria. However, formation of initial and secondary appressoria was delayed on glass slides (48 and 72 hai, respectively) compared with that on dogwood leaf disks (3 and 24 hai, respectively). Branching hyphae did not grow from germinated conidia on glass slides. However, on dogwood leaf disks, branched hyphae were observed 48 hai. In epidermal cells, the fungus formed compact and globose haustoria. Conidia formation on conidiophores started on leaf disks 7 days after inoculation.

Effects of hydrostatic pressure, agitation and CO2 stress on Phytophthora nicotianae zoospore survival.

BACKGROUND Phytophthora nicotianae Breda de Haan is a common pathogen of ornamental plants in recycled irrigation systems. In a previous study, annual vinca (Catharanthus roseus Don) inoculated with zoospore suspensions using a CO(2)-pressurized sprayer had less foliage blight than plants inoculated using a hand sprayer. Here, the impact of hydrostatic pressure, agitation and aeration with CO(2) on the survival of P. nicotianae zoospores was examined. RESULTS Exposure of zoospores to 840 kPa hydrostatic pressure for 8 min or agitation at a mixing intensity (G) of 6483 s(-1) for 4 min at 22-23 degrees C did not kill zoospores, but resulted in viable cysts. Motile and forcefully encysted zoospores of P. nicotianae were equally infectious on vinca or lupine (Lupinus polyphylus Lindl.). Bubbling CO(2) into zoospore-infested water at 110.4 mL (0.2 g) min(-1) for 5 min caused 81% reduction in the number of germinated zoospores. Pressure at 630 kPa (16.3 g CO(2)) or 70 kPa (3.85 g CO(2)) facilitated CO(2) injection and shortened the zoospore inactivation time to 30 s. When air was bubbled through the suspension, germination was similar to the control. CONCLUSIONS Exposure to CO(2) killed P. nicotianae zoospores in water. Neither pressure nor agitation had an effect on zoospore viability or infectivity. Based on results of this study, the authors designed a recycling CO(2) water treatment system that is currently under evaluation.

Microscopic and macroscopic studies of the development of Puccinia hemerocallidis in resistant and susceptible daylily cultivars

Infection and colonization of eight daylily cultivars, which varied in resistance to daylily rust, by Puccinia hemerocallidis was studied macroscopically and microscopically. After germination of urediniospores, appressoria formed at the tip of germ tubes and the fungus penetrated the host through stomatal openings 2 days after inoculation (DAI). Under the infection sites, intercellular hyphae aggregated and formed uredia, which released urediniospores 8 DAI. Resistant cultivars, characterized by the development of rapid death of host cells, were separated into three qualitative categories based on absence and presence of necrotic lesions without or with sporulation. In highly resistant cvs. Prairie Blue Eyes and Bertie Ferris, no macroscopic disease symptoms were observed on leaf surfaces although a few collapsed cells were detected microscopically. Both resistant and moderately resistant reactions were characterized by necrotic lesions with many collapsed cells under infection sites. The difference between these two reactions was that uredia and urediniospores were observed in the moderately resistant cv. Chicago Apache, but not in resistant cvs. Buttered Popcorn and Stella De Oro. Susceptible cultivars, characterized by the absence of a hypersensitive response, were separated into two qualitative categories based on restriction of intercellular hyphal growth that delayed development of uredia and formation of urediniospores. Compared to the susceptible cv. Pardon Me, moderately susceptible cvs. Mary Todd and Chorus Line had a delayed latent period and reduced amount of sporulation. The results indicate that hypersensitive cell death is one of the resistance responses to daylily rust. Necrotic lesions on leaf surfaces are associated with the number of collapsed host cells. Delayed latent period and reduced sporulation that resulted from restriction of intercellular hyphal growth could represent another type of resistance response in the daylily-rust pathosystem.

Development of Erysiphe pulchra, the causal agent of powdery mildew, on leaf disks of susceptible and resistant flowering dogwood (Cornus florida)

Understanding the relative contribution of the different resistance components is necessary to develop selection schemes and accelerate resistant-cultivar development. This study was conducted to investigate spore germination, infection-structure formation, and fungal development of Erysiphe pulchra, the causal agent of powdery mildew, on leaf disks of six cultivars or lines of flowering dogwood (Cornus florida) with different levels of resistance. The cultivars and lines tested were grouped into the following three resistance categories: highly susceptible (‘Cherokee Daybreak’ and MW 94-60), moderately susceptible (‘Cherokee Princess’ and MW 95-25), and resistant (‘Cherokee Brave’ and ‘Karen’s Appalachian Blush’). Percentages of spore germination and secondary-appressoria formation were not significantly different among the cultivars and lines. Significantly less percent germinated conidia with branched hyphae were observed on resistant cultivars than on the moderately susceptible cultivar or line, which was less than on the highly susceptible cultivar or line. Infection efficiencies were significantly different among cultivars and lines in the three resistance categories, except that there were no differences between ‘Cherokee Princess’ and the resistant cultivars. Resistant cultivars supported shorter latent periods than moderately and highly susceptible cultivars or lines, but no differences in latent period were detected between the later two resistance categories. The recently released ‘Karen’s Appalachian Blush’ expressed higher levels of resistance to powdery mildew than did ‘Cherokee Brave’, as indicated by the longer latent period and reduced relative sporulation of the pathogen.

A Pictorial Disease Severity Key and the Relationship Between Severity and Incidence for Black Root Rot of Pansy Caused by Thielaviopsis basicola

A pictorial key was developed and the relationship between disease severity (S) and incidence (I) was examined to aid in the assessment of black root rot of pansy caused by Thielaviopsis basicola. The key consisted of photographs of root segments that represented nine disease severity levels ranging from 1 to 91%. Pansies that had received different fertility treatments, as part of seven separate experiments, were inoculated with T. basicola. Four weeks after inoculation, roots were washed, and incidence and severity of black root rot were visually assessed using a grid-line-intersect method. Disease incidence ranged from 1.3 to 100%, and severity ranged from 0.1 to 21.4% per plant. Four different mathematical models were compared to quantitatively describe the I-S relationship for the combined data from all seven experiments. Although all models provided an adequate fit, the model that is analogous to the Kono-Sugino equation provided the most reliable estimate of severity over the entire range of disease incidence values. The predictive ability and accuracy of this model across data sets was verified by jackknife and cross-validation techniques. We concluded that incidence of black root rot in pansy can be assessed more objectively and with greater precision than disease severity and can be used to provide reliable estimates of disease severity based on derived regression equations that quantify the I-S relationship for black root rot.

Spread Potential of Binucleate Rhizoctonia from Nursery Propagation Floors to Trays Containing Azalea Stem Cuttings and Sanitary Control Options

Binucleate Rhizoctonia sp. (BNR), the cause of web blight, can be spread on azalea stem cuttings into propagation houses, but also can be eliminated from stems by submerging cuttings in 50°C water for 21 min. The overall objective was to evaluate risk of stem cuttings in trays becoming contaminated from inoculum on polypropylene fabric and gravel floors of propagation houses by evaluating four spread aspects in separate experiments. In experiment one, BNR was recovered from 1 to 50% samples from floor areas. In experiment two, BNR survival was higher under 70% shade than full sun, with the presence of organic media than its absence, and on gravel than on fabric. In experiment three in both years, BNR was never recovered from peat in trays beside or on colonized floor materials. In experiment four, disinfestants greatly reduced BNR on ground fabric and significantly on gravel. Results show BNR persists in propagation houses, yet spread into propagation trays is very low. Results imply the importance of cleaning floor surfaces of organic matter. Disinfestants further reduce propagule survival, but may not completely eliminate BNR, especially on gravel.

Nutrient, pH, Alkalinity, and Ionic Property Levels in Runoff Containment Basins in Alabama, Louisiana, Maryland, Mississippi, and Virginia Ornamental Plant Nurseries

USDA National Institute of Food and Agriculture Specialty Crop Research Initiative [2010-51181-21140]; U.S. Department of Agriculture, Agricultural Research Service, Thad Cochran Southern Horticultural Laboratory, Southern Horticultural Research Unit [6404-21430-001-00D]; Virginia Agricultural Experiment Station