Timothy L. Widmer

Standardizing the Nomenclature for Clonal Lineages of the Sudden Oak Death Pathogen, Phytophthora ramorum

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.

Enhanced Growth and Seed Properties in Introduced Vs. Native Populations of Yellow Starthistle (Centaurea Solstitialis)

Abstract There is much discussion as to why a plant becomes invasive in a new location but is not problematic in its native range. One example is yellow starthistle, which originates in Eurasia and is considered a noxious weed in the United States. We grew yellow starthistle originating from native and introduced regions in a common environment to test whether differences in growth would be observed. In growth chamber studies, seedlings originating from the invasive range were larger than seedlings from the native range after 2 wk. Seed starch content is an important component of initial seedling growth. The starch content of seeds from introduced populations was higher than that of seeds from native populations. Regression analysis showed a relationship between the amount of starch in the seeds and the weight of yellow starthistle seedlings after 2 wk growth. There was no difference in chromosome number, except in accessions originating from Sicily and Sardinia. Field studies conducted in France and Russia revealed that rosettes and mature plants grown under natural conditions were larger when grown from seeds originating from the invasive range than from seeds originating from the native range. The number of capitula per plant and stem diameters were not significant among all populations, but differences were noted. The F1 progeny of plants originating from U.S. seed, but grown and pollinated in France, showed no differences in seedling growth, mature plant characteristics, and seed starch content from the plants grown from field-collected U.S. seed. The changes in seed starch resource allocation and its relation to plant growth is useful in understanding factors that contribute to yellow starthistles invasibility. Nomenclature: Yellow starthistle; Centaurea solstitialis L. CENSO

Phytophthora kernoviae oospore maturity, germination, and infection.

Limited information is known on the basic biology of the recently described Phytophthora kernoviae that produces homothallic oospores. In this study, different P. kernoviae isolates were used to investigate oospore maturity, germination, and infection. All isolates produced oospores in V8 broth at 20°C in the dark by 6d. Oospores also formed at 10 and 15°C, but did not form at 25 and 28°C. Continuous light inhibited oospore production of some isolates but had no negative effect on others. Maturation time of the oospores, as noted by germination and staining with tetrazolium bromide, was not much different among the isolates between 2 and 14 weeks. Oospore germination was optimal at 18 and 20°C, and did not occur at 5, 25, and 30°C. Oospore germination under continuous light was higher than in the dark, but individual isolates showed variable results. Rhododendron leaf disks inoculated with oospores and maintained in the dark at 20°C were necrotic after 1 week, while those kept under continuous light did not develop necrosis. The percentage of leaf disks infected with P. kernoviae was lower in the leaves exposed to continuous light (40%) compared to those kept in the dark (100%).

Synchytrium solstitiale sp. nov. causing a false rust on Centaurea solstitialis in France

A new species of Synchytrium, S. solstitiale, infecting leaves of Centaurea solstitialis in France, is described and illustrated. Synchytrium solstitiale causes development of orange to red galls on the leaves and petioles of living plants. It differs microscopically from all previously described species of the genus mainly in having larger sporangia and zoospores and resting spores that are formed in succession without an evanescent prosoral stage.

Synchytrium solstitiale: Reclassification based on the function and role of resting spores.

Studies were made about resting spores of Synchytrium solstitiale, a chytrid that causes false rust disease of yellow starthistle (YST). During evaluation of this fungus for biological control of YST, a protocol for resting spore germination was developed. Details of resting spore germination and study of long-term survival of the fungus were documented. Resting spores from dried leaves germinated after incubating them on water agar at least 7 d at 10–15 C. Resting spores were viable after storage in air-dried leaves more than 2 y at room temperature, suggesting they have a role in off-season and long-term survival of the fungus. Each resting spore produced a single sorus that contained a single sporangium, which on germination released zoospores through a pore. YST inoculated with germinated resting spores developed symptoms typical of false rust disease. All spore forms of S. solstitiale have been found to be functional, and the life cycle of S. solstitiale has been completed under controlled laboratory and greenhouse conditions. Resting spore galls differ from sporangial galls both morphologically and biologically, and in comparison, each sporangial gall cleaves into several sori and each sorus produces 5–25 sporangia that rupture during release of zoospores. For this reason S. solstitiale should be reclassified as diheterogallic sensu Karling (Am J Bot 42:540–545). Because resting spores function as prosori and produce an external sorus, S. solstitiale is best placed in into the subgenus Exosynchytrium.

Infectivity and Inoculum Production of Phytophthora ramorum on Roots of Eastern United States Oak Species

Little is known about colonization of roots of trees by Phytophthora ramorum. We examined zoospore concentration and exposure time needed to infect six Quercus (oak) species and the inoculum produced from their roots. Sprouted acorns, exposed to zoospores (3,000/ml) for different times and transplanted to potting soil, were susceptible to infection within 1 h of exposure but root weights were not impacted after 4 weeks (P = 0.952). Roots of Quercus prinus seedlings, inoculated with sporangia, had 0.6 to 3.2% colonization of the total root mass after 5 months. Neither root lesions nor obvious root sloughing were observed. Inoculum threshold levels were tested by exposing radicles to varying zoospore concentrations for 24 h. Results showed that radicle infection occurred even at 1 zoospore/ml. To test inoculum production, roots were inoculated with sporangia and transplanted into pots. Periodically, samples of runoff were collected and plated on selective medium. Afterward, root segments were plated to calculate percent colonization. After 16 and 35 days, root colonization and inoculum production from oak was lower than that of Viburnum tinus, a positive control. This study shows that P. ramorum is able to infect sprouted oak acorns and produce secondary inoculum, which may be important epidemiologically.

Temperature effects on Phytophthora kernoviae germination of sporangia, infection, and protein secretion

Phytophthora kernoviae is reported to be a pathogen on a wide range of plants, but there is little knowledge of the optimal infection conditions. Rhododendron ponticum leaves were inoculated with six different isolates of P. kernoviae sporangia and set at different temperatures from 10-28°C. After 1 week, lesion development and pathogen recovery were only observed from all isolates at 15 and 20°C and a few isolates at 10°C. In an experiment with a more narrow temperature range of 20-25°C, lesion development and pathogen recovery on R. ponticum, Magnolia stellata, and Viburnum tinus occurred consistently at 20 and 21°C, was limited at 22°C, and did not occur at all at 23°C and above. There was no difference in sporangia and zoospore germination at 20-25°C. In a temperature fluctuation experiment, the necrotic area of inoculated R. ponticum leaves was larger the longer leaves were stored at 20°C and smaller the longer leaves were stored at 24°C. To determine if temperature affects secreted proteins that are involved in pathogenicity, crude purifications of secreted proteins from P. kernoviae cultures grown at 20 and 24°C were compared. When spot tested on R. ponticum leaves, the crude protein suspension isolated from cultures grown at 20°C induced necrosis while the proteins secreted from a culture grown at 24°C did not. Proteomic analysis confirmed a 10 kDa protein secreted at both 20 and 24°C shared sequence homology to the conserved domains of known elicitins of other Phytophthora spp. The protein secreted at 20°C that was responsible for the necrosis has not been identified. This article is protected by copyright. All rights reserved.

Recovery of Phytophthora ramorum in plant tissue with mixed infections

This study was performed to investigate the frequency with which P. ramorum would be isolated from host tissue co-infected with P. ramorum as well as an indigenous Phytophthora species or P. kernoviae. Three separate experiments were tested in a similar manner using different combinations of pathogens and hosts. Overall for any of the individual experiments, very few segments did not have any growth of Phytophthora spp. For mixed infections of P. ramorum and P. kernoviae, a difference was observed between isolating both of the species and P. ramorum only on rhododendron. The data showed that P. ramorum or P. kernoviae will not be detected 29 or 12% of the time, respectively, in infected Rhododendron sp. Phytophthora kernoviae was not detected alone in mixed infections with P. ramorum on Pieris japonica. When two different P. ramorum isolates were co-inoculated individually with one P. citricola isolate, there was a significant difference between isolating P. ramorum and isolating both species. These results confirm that choice of host species used for baiting can strongly influence detection results. For example, if P. japonica were used for baiting in mixed infections, there is a 55% chance that P. kernoviae would not be detected. This study highlights the difficulty in being confident in isolating and identifying an individual Phytophthora sp. from host material when mixed infections are present, and emphasizes the importance of a large sample size in order to increase the chances to recover all possible different species in a mixed infection.

Phytophthora stricta isolated from Rhododendron maximum in Pennsylvania

During a survey in October 2013 in the Michaux State Forest in Pennsylvania (Pennsylvania Department of Conservation and Natural Resources Permit number SFRA-1351), mature plants of Rhododendron maximum were noticed alongside a stream with nondescript necrotic lesions at the tips of some mature leaves that had been in contact with the stream water. Several leaves were collected and taken back to the laboratory for processing. Segments from 10 leaves were removed at the interface of the lesion area, surface-sterilized for 20 s in 70% ethanol and rinsed three times in sterile water. The segments were plated on PARPH+V8 Phytophthora selective medium (Ferguson and Jeffers, 1999). The plates were kept in the dark at 20oC. After approximately 3 d, colonies resembling a Phytophthora sp. were observed growing from the segments of two leaves. Mycelial tips were transferred to 20% V8 agar (isolates MSF-C and MSF-F). The isolates were identified as P. stricta based on sequences from the internal transcribed spacer (...

Bioassay Conditions for Infection of Pinus radiata Seedlings with Phytophthora pinifolia Zoospores

Phytophthora pinifolia is known to cause a devastating disease on Monterey pine in Chile. Although this pathogen is not yet present in the United States, there is reason for concern. The main source of Monterey pine genetic material is found in California and there is potential for other important tree species to be hosts. The study presented here was conducted to develop a method to produce zoospores and determine optimal conditions for infection to be used in future host range studies. Abundant zoospores were produced when agar plugs containing P. pinifolia mycelia were ground into suspension prior to transfer in a solution of carrot broth. These zoospores then were used to inoculate Monterey pine seedlings under various conditions. Infected plants displayed necrotic crowns and stems, often resulting in wilting of the seedling. Consistent infection occurred when seedlings were wounded by trimming needles prior to inoculation and exposure of inoculated seedlings to constant dew for 5 days. Dew chamber temperature (15, 20, or 25°C) did not affect the infection rate. Information obtained from this study will be useful in screening other hosts for susceptibility to P. pinifolia infection.