Everett Hansen

Multiple new phenotypic taxa from trees and riparian ecosystems in Phytophthora gonapodyides-P. megasperma ITS Clade 6, which tend to be high-temperature tolerant and either inbreeding or sterile

Phytophthora isolates associated with Phytophthora major ITS Clade 6 were grouped into 11 phenotypic taxa. These comprised the described morphospecies P. gonapodyides, P. megasperma s. str. and P. humicola; four previously identified but so far undescribed taxa, informally designated here P. sp. O-group, P. sp. Apple-cherry, P. taxon Pgchlamydo, and P. taxon Walnut; and four previously unknown taxa, designated P. taxon Oaksoil, P. taxon Raspberry, P. taxon Forestsoil, and P. taxon Riversoil. With the exception of P. gonapodyides, each phenotypic taxon represented an unique ITS lineage. Two isolates morphologically identical to P. gonapodyides comprised a separate lineage and probably represent another taxon, designated here P. taxon Salixsoil, P. humicola, P. sp. O-group, P. sp. Apple-cherry and P. taxon Walnut grouped together as subclade I. Within subclade II, P. taxon Oaksoil, P. taxon Raspberry, P. taxon Forestsoil, P. taxon Riversoil and P. taxon Pgchlamydo formed a cluster of closely related but phenotypically distinct lineages basal to P. gonapodyides and P. megasperma, P. taxon Salixsoil being the most basal member. The taxonomy, adaptation and breeding systems of Clade 6 taxa are discussed. They show a strong association with forests and riparian ecosystems, only a limited association with agriculture and an ability to tolerate high temperatures. Also, in contrast to most other Phytophthora clades, Clade 6 taxa are predominantly sterile or inbreeding in culture. Only one taxon, P. sp. O-group, appears classically A1/A2 heterothallic.

Susceptibility of Oregon Forest Trees and Shrubs to Phytophthora ramorum: A Comparison of Artificial Inoculation and Natural Infection

Phytophthora ramorum is an invasive pathogen in some mixed-hardwood forests in California and southwestern Oregon, where it causes sudden oak death (SOD) on some members of Fagaceae, ramorum shoot dieback on some members of Ericaceae and conifers, and ramorum leaf blight on diverse hosts. We compared symptoms of P. ramorum infection resulting from four different artificial inoculation techniques with the symptoms of natural infection on 49 western forest trees and shrubs; 80% proved susceptible to one degree or another. No single inoculation method predicted the full range of symptoms observed in the field, but whole plant dip came closest. Detached-leaf-dip inoculation provided a rapid assay and permitted a reasonable assessment of susceptibility to leaf blight. Both leaf age and inoculum dose affected detached-leaf assays. SOD and dieback hosts often developed limited leaf symptoms, although the pattern of midrib and petiole necrosis was distinctive. Stem-wound inoculation of seedlings correlated with field symptoms for several hosts. The results suggested that additional conifer species may be damaged in the field. Log inoculation provided a realistic test of susceptibility to SOD, but was cumbersome and subject to seasonal variability. Pacific rhododendron, salmonberry, cascara, and poison oak were confirmed as hosts by completing Kochs postulates. Douglas-fir was most susceptible to shoot dieback shortly after budburst, with infection occurring at the bud.

Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction.

ABSTRACT Phaeocryptopus gaeumannii is a widespread foliar parasite of Douglas-fir. Although normally innocuous, the fungus also causes the defoliating disease Swiss needle cast in heavily infected needles. The extent of P. gaeumannii colonization in Douglas-fir foliage was estimated with real-time quantitative polymerase chain reaction (PCR) using TaqMan chemistry. In order to derive a normalized expression of colonization, both pathogen and host DNA were simultaneously amplified but individually detected by species-specific primers and TaqMan probes labeled with different fluorescent dyes. Detection of host DNA additionally provided an endogenous reference that served as both an internal positive control and adjusted for variation introduced by sample-to-sample differences in DNA extraction and PCR efficiencies. The genes employed for designing the TaqMan probes and primers were beta-tubulin for the pathogen and a LEAFY/FLORICAULA-like gene involved in floral development for the tree host. Both probe/primer sets exhibited high precision and reproducibility over a linear range of 4 orders of magnitude. This eliminated the need to analyze samples in multiple dilutions when comparing lightly with heavily infected needles. Quantification of the fungus within needles was successful as early as 1 month after initial infection. Real-time PCR is the only method currently available to quantify P. gaeumannii colonization early in the first year of the colonization process.

Three new species of Phytophthora from European oak forests

In several studies of oak decline in Europe, one semi-papillate ( Phytophthora psychrophila sp. nov.) and two non-papillate homothallic Phytophthora species ( P. europaea and P. uliginosa spp. nov.) were isolated, together with other Phytophthora species, from rhizosphere soil samples which could not be assigned to existing taxa. P. psychrophila differs from other semi-papillate species of Waterhouses morphological Group IV, like P. ilicis and P. hibernalis , by its uniform, dome-shaped and cottonwool-like colony growth pattern on V8 juice agar and malt extract agar, the occurrence of sympodially branched primary hyphae, the high variation in size and shape of the sporangia, shorter pedicels, lower optimum temperature for growth, and ITS sequences. P. europaea is distinguished from related non-papillate Group V and VI species, namely P. fragariae, P. cambivora , and the ‘alder phytophthora’, by producing oogonia with tapered bases, irregular walls and exclusively paragynous antheridia, its cardinal temperatures for growth, and ITS sequences. P. uliginosa differs from related Group V and VI species by its large oogonia with exclusively paragynous antheridia, the predominant occurrence of ellipsoid sporangia with markedly wide exit pores, its slow growth, low cardinal temperatures, its colony growth patterns, and ITS sequences. P. uliginosa is separated from P. europaea by its larger oogonia without tapering bases, lower cardinal temperatures and growth rates, different colony growth patterns, and greater aggressiveness on Q. robur.

Incidence and Impact of Swiss Needle Cast in Forest Plantations of Douglas-fir in Coastal Oregon

An epidemic of Swiss needle cast, caused by the ascomycete Phaeocryptopus gaeumannii, is causing defoliation and growth reductions in Douglas-fir forest plantations along the Oregon Coast. The area of symptomatic plantations has been monitored annually since 1996 by aerial survey; in spring 1999, 119,500 ha were affected. Pathogen and symptom development have also been monitored on nine permanent plots in stands of differing disease severity. Infection levels and symptom severity are greatest in low elevation plantations close to the coast. In areas of severe disease, trees retain only current year needles. Defoliation is proportional to the number of stomata occluded by pseudothecia of the fungus, with needles being shed when about 50% of stomata are occupied, regardless of needle age. Fungus sporulation and premature needle abscission are greatest on the upper branches of trees. Annual application of fungicides increases needle retention significantly. Tree height and diameter growth and total tree volume are reduced by disease, and tree volume is significantly correlated with needle retention on our plot trees. The epidemic continues to be most severe in Douglas-fir plantations established on sites where Sitka spruce and western hemlock or red alder predominated in earlier times.

Population dynamics of the sudden oak death pathogen Phytophthora ramorum in Oregon from 2001 to 2004

Phytophthora ramorum (Oomycetes) is an emerging plant pathogen in forests in southwestern Oregon (Curry County). Moreover, since 2003 it has been repeatedly isolated from plants in Oregon nurseries. In this study, we analysed the genetic diversity of the P. ramorum population in Oregon from 2001 to 2004 by using microsatellites. A total of 323 isolates (272 from the infested forest; 51 from nurseries) were screened at 10 loci. The overall P. ramorum population in Oregon is characterized by low genetic diversity and has all the hallmarks of an introduced organism. All isolates within the A2 mating type belonged to the same clonal lineage and no recombinant genotypes were found. The forest population (24 genotypes) was dominated by a single multilocus genotype which persisted over years, indicating that eradication efforts in the forest have not completely eliminated inoculum sources. In contrast, genotypic evidence suggests that eradication was effective in nurseries. In 2003 and 2004, a total of 11 genotypes were found in the nurseries (one belonged to the European lineage of P. ramorum) but no genotype was recovered in both sampling years. Significant differentiation and low gene flow were detected between nursery and forest populations. Only two nursery genotypes were also found in the forest, and then at low frequency. Thus, the nursery infestation is not caused by the genotypes observed in Curry County, but likely resulted through introduction of novel genotypes from nurseries out‐of‐state. This highlights the continued importance of sanitation and quarantine in nurseries to prevent further introduction and spread of P. ramorum.

The taxonomic structure of Phytophthora megasperma: evidence for emerging biological species groups.

Nomenclatural uncertainty surrounds P. megasperma as various authors, working with limited groups of isolates, offer their interpretations of this species based on pathology, morphology, or cytology. We compared 93 isolates, including many described by others, for classical morphological features, growth behaviour and appearance, electrophoretic pattern of total proteins, chromosome number and nuclear DNA content. Nine distinct sub-groups were distinguished. While most groups could be distinguished by each of the criteria, protein electrophoresis was the most sensitive. The groups included: ALF, pathogenic to alfalfa, n = 12–15; SOY, pathogenic to soybean, n = 12–15; CLO, pathogenic to clover, n = 11–15; DF, pathogenic to Douglas fir, n = 17–24; AC, isolated from rosaceous fruit trees; and BHR, a major group obtained from a broad range of hosts. The last two groups, distinguished primarily by protein pattern, comprised at least four karyotypes: KI, n = 12–17; KII, n = 15–23; KIII, n = 22–28; and KIV, n = 26–34. All four karyotypes occur within the BHR protein group, suggesting a polyploid series within a closely related genotype. Two broad lines of evolution are hypothesized, a legume line comprising ALF, SOY, CLO, and perhaps DF isolates, and a Broad Host Range line of AC and BHR isolates. Sub-groups within each line may represent emerging biological species, isolated by host specificity or karyotype. Taxonomic designation for the various groups must await confirmation of the hypothesis by demonstration of the extent of barriers to gene flow between the groups.

Phytophthora beyond agriculture.

Little is known about indigenous Phytophthora species in natural ecosystems. Increasing evidence, however, suggests that a diverse, trophically complex Phytophthora community is important in many forests. The number of described species has steadily increased, with a dramatic spike in recent years as new species have been split from old and new species have been discovered through exploration of new habitats. Forest soil, streams, and the upper canopies of trees are now being explored for Phytophthora diversity, and a new appreciation for the ecological amplitude of the genus is emerging. Ten to twenty species are regularly identified in temperate forest surveys. Half or more of this Phytophthora diversity comes from species described since 2000. Taxa in internal transcribed spacer (ITS) Clade 6 are especially numerous in forest streams and may be saprophytic in this habitat. Three ecological assemblages of forest Phytophthora species are hypothesized: aquatic opportunists, foliar pathogens, and soilborne fine-root and canker pathogens. Aggressive invasive species are associated with all three groups.

Phytophthora species in forest streams in Oregon and Alaska

Eighteen Phytophthora species and one species of Halophytophthora were identified in 113 forest streams in Alaska, western Oregon and southwestern Oregon that were sampled by baiting or filtration of stream water with isolation on selective media. Species were identified by morphology and DNA characterization with single strand conformational polymorphism, COX spacer sequence and ITS sequence. ITS Clade 6 species were most abundant overall, but only four species, P. gonapodyides (37% of all isolates), P. taxon Salixsoil, P. taxon Oaksoil and P. pseudosyringae, were found in all three regions. The species assemblages were similar in the two Oregon regions, but P. taxon Pgchlamydo was absent in Alaska and one new species present in Alaska was absent in Oregon streams. The number of Phytophthora propagules in Oregon streams varied by season and in SW Oregon, where sampling continued year round, P. taxon Salixsoil, P. nemorosa and P. siskiyouensis were recovered only in some seasons.

Sudden oak death caused by Phytophthora ramorum in Oregon.

Sudden oak death, caused by Phytophthora ramorum (1,2), has been found for the first time in Oregon, killing tanoak, Lithocarpus densiflorus, trees. To our knowledge, this is the first report of the disease outside of the San Francisco to Monterey area in California, (300 km to the south). Nine areas of infestation, all within a 24-km2 area, were discovered on forest lands near Brookings, in southwest Oregon. Mortality centers ranged in size from 0.2 to 4.5 ha and included 5 to approximately 40 diseased trees. P. ramorum was isolated from stem cankers using Phytophthora-selective medium. Isolates had distinctive morphological features characteristic of P. ramorum, including abundant production of chlamydospores and caducous, semipapillate sporangia on solid media. Internal transcribed spacer (ITS) sequences of isolates of P. ramorum from Oregon were identical to ITS sequences of isolates from California (1). The pathogen also was isolated from necrotic lesions on leaves and stems of native Rhododendron macrophyllum and Vaccinium ovatum growing beneath diseased tanoaks. In July 2001, the disease was located by an aerial survey conducted cooperatively by the USDA Forest Service and Oregon Department of Forestry. All lands within 1.6 km (1 mile) of the mortality centers are subject to Oregon quarantine, which bars the transport of any host plant materials. An eradication effort is currently underway. Symptomatic plants and all known host plants within 15 to 30 m of symptomatic plants are being cut and burned in the first phase of this operation. The total treated area is approximately 16 ha. References: (1) D. M. Rizzo et al. Plant Dis. In press. (2) S. Werres et al. Mycol. Res. 105:1155, 2001.