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ABSTRACTWe evaluated sporangium and zoospore production by three isolates of Phytophthora ramorumon 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. ramorumand 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.

Platelets bind to exposed vascular matrix at a wound site through a highly specialized surface receptor, glycoprotein (GP) Ib-IX-V complex, which recognizes von Willebrand factor (VWF) in the matrix. GPIb-IX-V is a catch bond for it becomes more stable as force is applied to it. After attaching to the wound site, platelets generate cytoskeletal forces to compact and reinforce the hemostatic plug. Here, we evaluated the role of the GPIb-IX-V complex in the transmission of cytoskeletal forces. We used arrays of flexible, silicone nanoposts to measure the contractility of individual platelets on VWF. We found that a significant proportion of cytoskeletal forces were transmitted to VWF through GPIb-IX-V, an unexpected finding given the widely held notion that platelet forces are transmitted exclusively through its integrins. In particular, we found that the interaction between GPIbαand the A1 domain of VWF mediates this force transmission. We also demonstrate that the binding interaction between GPIbαand filamin A is involved in force transmission. Furthermore, our studies suggest that cytoskeletal forces acting through GPIbαare involved in maintaining platelet adhesion when external forces are absent. Thus, the GPIb-IX-V/VWF bond is able to transmit force, and uses this force to strengthen the bond through a catch-bond mechanism. This finding expands our understanding of how platelets attach to sites of vascular injury, describing a new, to the best of our knowledge, mechanism in which the catch bonds of GPIb-IX-V/VWF can be supported by internal forces produced by cytoskeletal tension.

Mycelium-free chlamydospores of 12 isolates of P. ramorumrepresenting 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. ramorumchlamydospores and that a great disparity in germination percentage can exist within isolates, even within a single clonal lineage.

We examined the effect of short-term exposure to high and low temperatures and a range of relative humidity (RH) on survival of Phytophthora ramorumhyphae. 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. ramorumhyphae 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.

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 μW/cm2) and visible radiation (250–1500 μW/cm2). EU isolates were exposed to 300 μW/cm2near-UV only, which significantly reduced growth of one of three isolates and had no significant effect on spore production. In our studies P. ramorumtolerated a broad range of temperature and light conditions, which suggests that it is capable of establishment in a wide geographic area.

The phylogenetic relationships of 51 isolates representing 27 species of Phytophthorawere assessed by sequence alignment of 568 bp of the mitochondrially encoded cytochrome oxidase II gene. A total of 1299 bp of the cytochrome oxidase I gene also were examined for a subset of 13 species. The coxII gene trees constructed by a heuristic search, based on maximum parsimony for a bootstrap 50% majority-rule consensus tree, revealed 18 species grouping into seven clades and nine species unaffiliated with a specific clade. The phylogenetic relationships among species observed on coxII gene trees did not exhibit consistent similarities in groupings for morphology, pathogenicity, host range or temperature optima. The topology of coxI gene trees, constructed by a heuristic search based on maximum parsimony for a bootstrap 50% majority-rule consensus tree for 13 species of Phytophthora, revealed 10 species grouping into three clades and three species unaffiliated with a specific clade. The groupings in general agreed with what was observed in the coxII tree. Species relationships observed for the coxII gene tree were in agreement with those based on ITS regions, with several notable exceptions. Some of these differences were noted in species in which the same isolates were used for both ITS and coxII analysis, suggesting either a differential rate of evolutionary divergence for these two regions or incorrect assumptions about alignment of ITS sequences. Analysis of combined data sets of ITS and coxII sequences generated a tree that did not differ substantially from analysis of ITS data alone, however, the results of a partition homogeneity test suggest that combining data sets may not be valid.

AbstractThe intron 3 region of the β-tubulin gene, and intron 4 of the translation elongation factor gene were PCR-amplified, cloned, and sequenced to determine relationships among Clavicepsspecies and characterize isolates of Clavicepscausing ergot of sorghum in the USA and other countries. The β-tubulin gene intron 3 region and intron 4 of the EF-1α gene allowed clear differentiation of five species (C. africana, C. sorghicola, C. purpurea, C. fusiformis, and C. paspali), two of which (C. africanaand C. sorghicola) are pathogens of sorghum, with almost no intraspecific variation observed among isolates. Clavicepsisolates obtained from sorghum in the USA contained β-tubulin gene intron 3 region sequences identical to those of C. africanaisolates from India, Australia, and South Africa. PCR primers were designed from unique sequences within the β-tubulin intron 3 region that can differentiate the five Clavicepsspecies used in this study. We describe primers that allow direct PCR detection of C. africanafrom honeydew produced on infected sorghum plants, providing a useful tool for analysis of field samples. The β-tubulin gene intron 3 region and EF-1α intron 4 should prove useful in phylogenetic and epidemiological studies of additional Clavicepsspecies.