We evaluated sporangium and zoospore production by three isolates of Phytophthora ramorum on Rhododendron 'Cunningham's White' leaves under light and dark conditions at both variable and constant (14 C) temperature. P. ramorum-infected leaves were detached and placed in funnels inside of a 62-L plastic storage container located in a growth chamber. Cool mist was introduced to the container to create a high-humidity environment. Sporangia and zoospores were collected over a 4-day period by misting leaves with 5 mL of distilled water, which was collected in conical test tubes that also contained runoff from the misting. Spores were collected daily just before a 13-h light period and again just before an 11-h dark period. Sporangia and zoospores in the collection tubes were counted using a dissecting microscope following staining with lactoglycerin/aniline blue. Large differences in sporangium and zoospore numbers observed for the dark versus light periods were observed on days 2, 3, and 4. A diurnal effect has been observed for production of propagules of other oomycetes, but such effects have not been previously reported for P. ramorum. This information will help provide a better understanding of patterns of inoculum production by P. ramorum and resulting fluctuations in inoculum density that will influence sudden oak death epidemics in forest ecosystems in the United States and other countries where it occurs.

We examined the impact of relative humidity (RH) on Phytophthora ramorum sporangia production on Rhododendron 'Cunningham's White'. When diseased plants were maintained under continuous moisture in a mist tent, sporangia were collected from some plants for 22 weeks. More than 3,000 sporangia/leaf/week were collected over the first 3 weeks but levels declined to <100 sporangia/leaf/week after 7 weeks. We also examined the impact of drying on P. ramorum sporangia production. Diseased, detached leaves were maintained in humidity chambers (100, 96.2, 84.5, 74.9, and 56.2% RH) for up to 9 weeks and removed weekly to assess sporulation. For comparison, diseased leaves were harvested from plants maintained with dry foliage or subjected to 10 h of simulated dew nightly. All leaves supported sporulation following 5 weeks at 100% RH, 3 weeks at 96.2% RH, and 1 week at 84.5% RH. All leaves collected from plants subjected to nightly dew supported sporulation for 3 weeks; however, only 66.7% of leaves collected from plants with dry foliage supported sporulation after 1 week. Knowledge of the effects of RH levels on P. ramorum sporulation capacity will prove useful in terms of disease management recommendations and for development of predictive models and pest risk assessments.

Sporangia of three isolates of Phytophthora ramorum representing three different clonal lineages were subjected to relative humidity (RH) levels between 80 and 100% for exposure periods ranging from 1 to 24h at 20 degrees C in darkness. Plastic containers (21.5x14.5x5cm) were used as humidity chambers with 130ml of glycerine solution added to each container. Glycerine concentrations corresponded to 100, 95, 90, 85 and 80% RH based on refractive index measurements. Sporangia suspensions were pipeted onto nitrile mesh squares (1.5x1.5cm, 15 micron pore size) which were placed in the humidity chambers and incubated at 20 degrees C in darkness. Following exposure periods of 1, 2, 4, 8, 12 and 24h, mesh squares were inverted onto Petri dishes of selective medium and sporangia germination assessed after 24 and 48h. At 100% RH, we observed a mean value of 88% germination after 1h exposure declining to 18% germination following 24h incubation. At 95% RH, a steeper decline in germination was noted, with means ranging from 79% at 1h to less than 1% at 24h exposure. At 90% RH, no germination was noted after 8 or more h exposure, and values were 57%, 22% and 3% germination for the 1, 2 and 4h exposures, respectively. Germination was only observed at 1h exposure for both the 85% RH treatment (52% germination) and the 80% RH treatment (38% germination). The three isolates responded similarlyover the range of RH values tested. The germination response of P.ramorum sporangia to RH values between 80% and 100% was comparable to that reported for other Phytophthora species. Knowledge of conditions that affect P.ramorum sporangia germination can shed light on pathogenesis and epidemic potential and lead to improved control recommendations.

Colonization of the fleshy fruit of Cornus florida, C. kousa, Laurus nobilis, Malus hupehensis, and Pyracantha 'Mohave' was observed following inoculation with sporangia of Phytophthora ramorum. However, abundant production of chlamydospores was only observed in the fruit of Pyracantha 'Mohave'. Pyracantha 'Mohave' fruit that had been inoculated with a P. ramorum sporangia suspension were placed in pots containing rooted cuttings of Viburnum tinus in a misting tent or in water-filled trays in a climate-controlled greenhouse. Runoff was collected for 24 to 30 days, and roots were plated after the final collection. Mean percent recovery from infected roots was not significantly different (P = 0.05, Tukey's test) between bottom-watered treatments in trays and misted treatments, averaging 58% for bottom-watered and 54% for mist treatments. The number of CFU collected in runoff from bottom-watered plants was consistently lower than that obtained from plants held under mist, likely due to desiccation of the fruit. The results show that root infection of V. tinus can occur by P. ramorum via infected fruit of Pyracantha 'Mohave'. This phenomenon represents a pathway of infection for P. ramorum not previously reported, which may play a role in disease epidemiology.

The effect of temperature and moist period on the onset of sporangia production by Phytophthora ramorum on Rhododendron Cunningham's White' was examined with misted detached leaves held in humid chambers. Following wound inoculation with sporangia, leaves were pre-incubated at 20 degrees C for either 24 or 72h prior to placement at six different temperatures (4, 10, 15, 20, 25 and 30 degrees C). The overall mean moist period required for first occurrence of sporulation over all six temperatures was 3.24days with the 24-h pre-incubation time, compared with 1.49days for the 72-h pre-incubation time. Following 24h pre-incubation at 20 degrees C and at an incubation temperature of 15 degrees C, sporangia were first collected from leaves following a 24h incubation. At 10 and 20 degrees C, sporangia were first collected after 48h, whereas at 4, 25 and 30 degrees C, sporangia were first collected after 3days. Following 72h pre-incubation at 20 degrees C, sporulation generally occurred within 1day, even at temperatures such at 4 and 30 degrees C that are suboptimal for sporulation. The highest levels of P.ramorum sporulation were observed at 20 degrees C. P.ramorum formed sporangia on host tissue under moist conditions within the same time frame reported for P.phaseoli, P.palmivora and P.nicotianae, but substantially more slowly than certain other species such as P.infestans. Quantifying moisture and temperature conditions for initiation of sporangia production provides knowledge which leads to a greater understanding of the epidemic potential of P.ramorum.

Mycelium-free chlamydospores of 12 isolates of P. ramorum representing three clonal lineages were produced with a method involving incubation in nonsterile sand at 20 C in darkness for 30 d. Chlamydospores were incubated on selective agar medium at 5, 10, 15, 20, 25 and 30 C and germination assessed after 1, 2, 4, 6 and 8 d incubation. The optimal temperature for germination based on 8 d incubation was 20 C for all three clonal lineages tested (NA1 NA2, EU1). Mean germination rates were 2, 21, 44, 67, 32 and 0 percent at 5, 10, 15, 20, 25 and 30 C respectively for all isolates combined. The highest mean germination rate was scored by isolates of the EU1 clonal lineage at 20 C (85%) after 8 d incubation However, substantial variation was observed among isolates within each clonal lineage. Overall temperatures and days of incubation on which germination was assessed isolates of the NA1 clonal lineage had the lowest mean germination, even though one isolate had the highest germination of any isolate in any lineage. The results indicate that 20 C is the optimal germination temperature for P. ramorum chlamydospores and that a great disparity in germination percentage can exist within isolates, even within a single clonal lineage.

Seedlings of three Eastern US forest species Quercus rubra (northern red oak), Quercus prinus (chestnut oak) and Acer rubrum (red maple) were inoculated by applying Phytophthora ramorum sporangia to stems at different inoculum densities with and without wounding. Disease occurred in all treatments involving wounds, and no disease was observed in unwounded treatments. Younger seedlings (2-3years old) did not differ significantly from older seedlings (5-6years old) in disease incidence, but older seedlings sustained smaller lesions compared with younger seedlings. For both old and young seedlings, disease on wounded stems was observed down to the lowest sporangia concentration utilized (500 sporangia/ml for old seedlings and 100 sporangia/ml for young seedlings). The results show that in the presence of wounding, even very low sporangia concentrations can result in disease, and further suggest that wounding caused by insects and other factors may play an important role in P.ramorum epidemiology in forest environments.

The objective of this work was to establish inoculum density relationships between Phytophthora ramorum and selected hosts based on whole plant inoculations. Knowledge of levels of initial inoculum needed to generate epidemics is needed for disease prediction and development of pest risk assessments. Sporangia of six P. ramorum isolates representing the NA1 and EU1 clonal lineages were produced by incubating 20 percent V8- juice agar plugs containing mycelium, in 1 percent soil extract for 48 hours and adjusting the suspensions to 0, 50, 100, 500, 1,000, 2,000, and 3,000 sporangia/ml. Whole plants (2- to 3-year-old) of chestnut oak (Quercus prinus L.), northern red oak (Q. rubra L.), red maple (Acer rubrum L.), mountain laurel (almia latifolia L.), and Rhododendron 'Cunningham's White' were dip-inoculated and incubated in a 20 °C dew chamber in darkness for 5 days. The total number of diseased and healthy leaves was recorded and leaves were scanned. A linear model, as well as, a two-parameter asymptotic regression analysis through the origin were fit to the data. For all five species, the percentage of infected leaves increased from 0 to 2,000 sporangia/ml and then leveled off. Calibration threshold estimates for obtaining 50 percent infected leaves based on the linear analysis ranged from 36 to 750 sporangia/ml for the five hosts. Half-life (LD50) estimates from the asymptotic regression analysis ranged from 94 to 319 sporangia/ml. Multiple regression analysis revealed statistically significant differences (p = 0.0076) among hosts in increases in infection in response to increased inoculum density. Our results provide estimates of initial inoculum levels necessary to cause disease on these five P. ramorum hosts and will be useful in disease prediction and for development of pest risk assessments. Spore concentrations occurring in nature have rarely been determined experimentally. Thus, it is not known whether the level of spores determined experimentally to result in a given level of disease occurs commonly in native ecosystems.

Little is known of the parameters associated with the onset of sporangia production by P. ramorum on its hosts following infection and on leaves with established dis- ease. We conducted experiments to examine the relationship between lesion size, moisture period, and temperature with the first appearance of dehisced sporangia. Leaves exhibiting small lesions induced via artificial inoculation using a variety of techniques were positioned on 15 µm-mesh nylon screens in a mist chamber inside a greenhouse cubicle (15-18°C). Lesions were measured daily, and sporangia col- lected and counted. Lesion size was not a reliable predictor for onset of sporangia production. We observed a wide range of lesion sizes (1-54 mm2) corresponding to the initial collection of dehisced sporangia from diseased leaves. In contrast, the longer the moist period duration, the higher the percentage of diseased leaves supporting sporangia production by P. ramorum; 17-22% within 24 h, and an ad- ditional 39-64% within 48 h. On leaves with established disease, dehisced sporan- gia were collected from ≥ 50% of leaves within a 6-12 h moist period. In tempera- ture studies, sporangia were collected from misted detached leaves within 1 day at 15°C, 2 days at 10° and 20°C, and 3 days at 4°, 25°, and 30°C. When infected leaves were first preincubated at 20°C for 72 h allowing lesions to expand uniform- ly, sporangia were collected from some leaves following a 1 day incubation at all temperatures.

We investigated the temperature and moisture conditions that allow Phytophthora ramorum to infect Rhododendron 'Cunningham's White'. Most experiments were performed with a single P. ramorum isolate from the NA1 clonal lineage. For whole plants incubated in dew chambers at 10 to 31 degrees C, the greatest proportion of diseased leaves, 77.5%, occurred at the optimum temperature of 20.5 degrees C. Disease occurred over the entire range of temperatures tested, although amounts of disease were minor at the temperature extremes. For whole plants exposed to varying dew periods at 20 degrees C and then incubated at 20 degrees C for 7 days, a dew period as short as 1 h resulted in a small amount of disease; however, at least 4 h of dew were required for >10% of the leaves to become diseased. Moisture periods of 24 and 48 h resulted in the greatest number of diseased leaves. In detached-leaf, temperature-gradient-plate experiments, incubation at 22 degrees C resulted in the greatest disease severity, followed by 18 degrees C and then 14 degrees C. In detached-leaf, moisture-tent experiments, a 1-h moisture period was sufficient to cause disease on 67 to 73% of leaves incubated for 7 days at 20 degrees C. A statistical model for disease development that combined the effects of temperature and moisture period was generated using nonlinear regression. Our results define temperature and moisture conditions which allow infection by P. ramorum on Cunningham's White rhododendron, and show that P. ramorum is able to infect this host over a wide range of temperatures and moisture levels. The results indicate that P. ramorum has the potential to become established in parts of the United States that are outside its current range.

Glufosinate-resistant transgenic creeping and velvet bentgrass plants expressing a bar gene under the control of the maize ubiquitin promoter were inoculated separately with the fungal pathogens, Rhizoctonia solani and Sclerotinia homoeocarpa, before or after treatment with 560 ing L-1 of glufosinate at a rate of 0.56 kg ha(-1). Application of the herbicide 3 h before or 1 d after fungal inoculation significantly reduced infection of these transgenic grasses by R. solani and S. homoeocarpa. Assessment of the in vitro antifungal activity of the herbicide showed that 336 and 448 mg L-1 glufosinate completely inhibited the mycelial, growth of S. homoeocarpa and R. solani, respectively. The results suggest that the nonselective herbicide glufosinate may also be used to suppress the activity of some fungal pathogens in turf composed of these transgenic glufosinate-resistant creeping and velvet bentgrasses.

Twenty-five plant species (21 genera, 14 families), which comprise a portion of the understory in forests of the Eastern United States, were evaluated for susceptibility to infection by Phytophthora ramorum. The degree to which P. ramorum is able to form sporangia and chlamydospores was also assessed on these hosts. Seedlings were spray-inoculated with a mixture (4,000 sporangia/ml) of four P. ramorum isolates followed by incubation in a dew chamber at 20°C in darkness for 5 days. Percent infection on individual leaves/leaflets was assessed visually. Mean percent leaf area infected ranged from 0.7% for Smilax rotundifolia to 93.8% for Kalmia latifolia. Eight plant species tested developed significantly larger lesion areas than those found on susceptible control Rhododendron 'Cunningham's White'. Fourteen species in addition to the susceptible control exhibited infection of over 90% of their leaves. Sporangia production by P. ramorum varied considerably among plant species, ranging from 36 per cm2 lesion area on Myrica pennsylvannica to 2,001 per cm2 lesion area on Robinia pseudoacacia. Numbers of chlamydospores produced per 6-mm-diameter leaf disk incubated in a P. ramorum sporangia suspension ranged from 25 on Ilex verticillata to 493 on Rhus typhina. The results indicate that many common understory species in Eastern U.S. forests are susceptible to P. ramorum and capable of providing ample sources of inoculum (sporangia and chlamydospores) for forest epidemics should the pathogen be introduced and should temperature and moisture conditions exist that are conducive to disease development.

We examined the effect of short-term exposure to high and low temperatures and a range of relative humidity (RH) on survival of Phytophthora ramorum hyphae. Spore-free hyphal colonies were grown on dialysis squares atop V8 medium. Colonies were transferred to water agar plates positioned at 27.5-50 C on a thermal gradient plate and incubated 2.5-480 min. For low temperature trials colonies were transferred to vials of distilled water and incubated in a water bath at -5 to -25 C for 1-24 h. In the relative humidity trials hyphal colonies were transferred to sealed humidity chambers containing various concentrations of glycerin for 1-8 h. Relative humidity was 41-93% at 20 C and 43-86% at 28 C. Survival in all trials was characterized by growth from dialysis squares into V8 medium. Temperatures of 37.5-40 C were lethal to P. ramorum hyphae within several hours, and temperatures of 42.5-50 C were lethal within minutes. Exposure to 32.5 and 35 C resulted in reduced survival over 8 h, while 30 C had no effect on three of four isolates. Hyphal colonies demonstrated considerable tolerance to cold, with all isolates surviving a 24 h exposure to -5 C. Survival diminished over time at lower temperatures, however a few colonies survived 24 h exposure to -25 C. Temperature also affected the ability of hyphal colonies to withstand reduced humidity. A RH of 41-43% was lethal in 2 h at 28 C compared to 8 h at 20 C. Three of four isolates were unaffected by an 8 h exposure to 81 and 95% RH at 20 C, and 73 and 86% RH at 28 C. Isolate differences were apparent in tolerance to freezing temperatures and reduced humidity. From these results it is apparent that the cold temperatures found in the northeastern USA are not likely to prevent the establishment of P. ramorum. There is also the potential for hyphae, and presumably spores, to survive periods of high humidity on the leaf surface in the absence of free water.

We examined the impact of exposure to high and low temperature extremes on recovery of Phytophthora ramorum both as free chlamydospores and within infected rhododendron tissue over a 7-day period. Chlamydospores held in moistened sand were incubated at 30, 35, 40, 0, -10, and -20°C for up to 7 days. Infected Rhododendron 'Cunningham's White' leaf disks held in sandy loam, loam, or sand at two different soil moisture levels also were subjected to these temperatures for up to 7 days, and to a variable temperature regimen for 12 weeks. Recovery was characterized by growth of P. ramorum on selective agar medium following exposures to temperature treatments. Chlamydospores held in moistened sand showed a high rate of recovery at 30°C, steadily declining recovery at 35°C, and no recovery at 40°C over the 7-day period. Chlamydospores were recovered from 0°C after 7 days, with little or no recovery observed at -10 or -20°C. In infected rhododendron tissue, P. ramorum was recovered at 20 and 30°C after 7 days but, at 35°C, the pathogen showed a decline within 2 days and no recovery by 4 days. A 40°C treatment allowed no recovery of P. ramorum from infected tissue after 2 days. For cold treatments, P. ramorum was recovered from infected leaf disks at 0 and -10°C after 7 days. At -20°C, recovery declined rapidly after 1 to 3 days and no recovery was obtained after 4 days. P. ramorum showed nearly 100% recovery from leaf disks subjected to a 12-week variable temperature treatment based on ambient summer temperatures in Lewisburg, TN. The results suggest that P. ramorum is capable of surviving some highly adverse temperature conditions for at least 7 days both as free chlamydospores in sand and within infected host tissue. Thus, P. ramorum present as free chlamydospores or within tissue of infected plants shipped to the eastern United States has the potential to survive some of the adverse conditions encountered in summer and winter in many eastern states.

Chlamydospores were produced as described by Colburn and Shishkoff (Phytopathology 96:S25). Samples (5cc) of chlamydospores in sand inoculum were placed in 15 ml conical plastic test tubes and incubated at selected temperatures for 1, 2, 3, 4, and 7 days. Following incubation, tube contents were resuspended in 0.2 percent water agar and 1 ml was plated onto PARPH selective medium amended with 4 percent clarified V8 juice. Numbers of colonies resulting from germinated chlamydospores were assessed microscopically. High temperature treatments included 30, 35, and 40°C while low temperature treatments included 0, -10, and -20°C. All experiments also included chlamydospores placed at 20°C as positive controls. Near 100 percent survival was observed at temperatures of 0°C and for the 20°C controls for up to 7 days in the low temperature treatments, while almost no survival occurred at -10 or -20°C over the 7 day period. For the high temperature treatments, high levels of chlamydospore germination were observed over the 7 day period at 30°C and for the 20°C controls, while no growth was observed at 40°C. At 35°C, high levels of chlamydospore germination were observed at day 1, but growth declined steadily and was zero by 7 days. These results help define the temperature conditions under which chlamydospores of P. ramorum survive, and provide information to help define treatments aimed at inactivating chlamydospores in soil substrates.

Phytophthora ramorum, recently found in the US, is causing concern for hardwood forests and the nursery industry. In an effort to identify some of the environmental limitations to growth and sporulation we undertook a laboratory study of four US and three European (EU) isolates. On V8 media, isolates grew when incubated at 2-28 C and produced chlamydospores at 8-28 C. Sporangia were produced at all temperatures tested: 10-30 C for US isolates and 6-26 C for EU isolates. Optimal temperatures were 16-26 C for growth, 14-26 C for chlamydospore production and 16-22 C for sporangia production. US isolates grew less and produced fewer spores when exposed to increasing doses of near-UV radiation (50-300 microW/cm(2)) and visible radiation (250-1500 microW/cm(2)). EU isolates were exposed to 300 microW/cm(2) near-UV only, which significantly reduced growth of one of three isolates and had no significant effect on spore production. In our studies P. ramorum tolerated a broad range of temperature and light conditions, which suggests that it is capable of establishment in a wide geographic area.

Bacterial wilt of Poa annua has been seen increasingly in the Northeast and mid-Atlantic United States in the past few years. The disease causes severe injury to putting greens and can kill large stands of turfgrass. For some time, however, both the bacterial origin of the disease and the causal agent were in doubt. In order to investigate the identity of the causal agent, isolation of the pathogen was undertaken and pathogenicity was confirmed using Koch's postulates on P. annua. Additional pathogenicity trials then were undertaken to determine the host range of the causal bacterium. Ability of the bacterium to cause disease was restricted to P. annua var. annua and P. attenuata. However, the bacterium was able to survive asymptomatically in vascular systems of P. annua var. reptans and P. trivialis. Experiments to determine the optimal growth temperature of the organism demonstrated that the bacterial growth peaked between 30 and 35°C. Fatty acid analysis suggested that the bacterium might be a species of Xanthomonas but was inconclusive. Ribosomal RNA analysis demonstrated significant similarity to the American Type Culture Collection isolate of Xanthomonas translucens pv. poae at 99.8%. Comparison of the host range to previously reported data agrees with our molecular findings and indicates that the likely casual organism of bacterial wilt of annual bluegrass is X. translucens pv. poae.

Leptosphaerulina leaf blight has been sporadically documented affecting turfgrasses. Recently, a Leptosphaerulina species has been seen on diseased turfgrass samples from New England and New York State. In all cases where Leptosphaerulina has been observed in New England, it has been accompanied by a number of other turf pathogens on highly stressed hosts, so its pathogenic potential has been unclear. The purpose of this study was to identify isolates to species, determine their pathogenicity in the greenhouse, and characterize their fungicide sensitivity in vitro in the event that significant pathogenicity was demonstrated. Microscopic observation of spores and culture characteristics indicated that all examined isolates were Leptosphaerulina australis. The optimum temperature for growth was assessed and the relationship between temperature and sporulation was examined. After repeated experiments utilizing several grass species under a variety of environmental conditions, all four isolates examined were determined to be nonpathogenic. Isolates produced pseudothecia on senescent tissue but infection of live tissue was never observed. The fungicide sensitivity assay demonstrated significant variation among isolates. Based on the results of this study and other observational evidence, it is likely that this species is saprobic on senescent turfgrass leaves but not pathogenic.

Glufosinate-resistant transgenic creeping and velvet bentgrass plants expressing a bar gene under the control of the maize ubiquitin promoter were inoculated separately with the fungal pathogens, Rhizoctonia solani and Sclerotinia homoeocarpa, before or after treatment with 560 mg L−1 of glufosinate at a rate of 0.56 kg ha−1. Application of the herbicide 3 h before or 1 d after fungal inoculation significantly reduced infection of these transgenic grasses by R. solani and S. homoeocarpa. Assessment of the in vitro antifungal activity of the herbicide showed that 336 and 448 mg L−1 glufosinate completely inhibited the mycelial growth of S. homoeocarpa and R. solani, respectively. The results suggest that the nonselective herbicide glufosinate may also be used to suppress the activity of some fungal pathogens in turf composed of these transgenic glufosinate-resistant creeping and velvet bentgrasses.