David M. Rizzo

Phytophthora ramorum as the cause of extensive mortality of Quercus spp. and Lithocarpus densiflorus in California.

A new canker disease, commonly known as sudden oak death, of Lithocarpus densiflorus, Quercus agrifolia, Q. kelloggii, and Q. parvula var. shrevei in California is shown to be caused by Phytophthora ramorum. The pathogen is a recently described species that previously was known only from Germany and the Netherlands on Rhododendron spp. and a Viburnum sp. This disease has reached epidemic proportions in forests along approximately 300 km of the central coast of California. The most consistent and diagnostic symptoms on trees are cankers that develop before foliage symptoms become evident. Cankers have brown or black discolored outer bark and seep dark red sap. Cankers occur on the trunk at the root crown up to 20 m above the ground, but do not enlarge below the soil line into the roots. Individual cankers are delimited by thin black lines in the inner bark and can be over 2 m in length. In L. densiflorus saplings, P. ramorum was isolated from branches as small as 5 mm in diameter. L. densiflorus and Q. agrifolia were inoculated with P. ramorum in the field and greenhouse, and symptoms similar to those of naturally infected trees developed. The pathogen was reisolated from the inoculated plants, which confirmed pathogenicity.

Sudden oak death: endangering California and Oregon forest ecosystems

Sudden oak death is a new disease affecting tanoak (Lithocarpus densiflora) and oaks (Quercus spp) in California and Oregon, caused by the recently described pathogen Phytophthora ramorum. It has reached epidemic proportions in several counties in central California, leading to the death of tens of thousands of trees. In addition to oaks and tanoak, P ramorum has been found in nearly all woody plant species in mixed evergreen and redwood forests from central California to southern Oregon. Plant species that are not killed appear to serve as a reservoir for the pathogen. The high susceptibility of tanoak to infection and death suggests that P ramorum is an exotic pathogen, but its origins, and most details of its biology and ecology, remain unknown. Our limited knowledge only compounds our concern over the long-term implications of this epidemic for the ecology of coastal forests.

Transmission of Phytophthora ramorum in Mixed-Evergreen Forest in California

ABSTRACT During 2001 to 2003, the transmission biology of Phytophthora ramorum, the causal agent of sudden oak death, was studied in mixedevergreen forest, a common forest type in northern, coastal California. Investigation of the sources of spore production focused on coast live oak (Quercus agrifolia) and bay laurel (Umbellularia californica), dominant hosts that comprised 39.7 and 46.2% of the individuals at the study site, respectively. All tests for inoculum production from the surface of infected coast live oak bark or exudates from cankers were negative. In contrast, sporangia and chlamydospores were produced on the surface of infected bay laurel leaves. Mean number of zoospores produced from infected bay laurel leaves under natural field conditions during rainstorms was 1,173.0 +/- SE 301.48, and ranged as high as 5,200 spores/leaf. P. ramorum was recovered from rainwater, soil, litter, and streamwater during the mid- to late rainy season in all 3 years of the study. P. ramorum was not recovered from sporadic summer rains or soil and litter during the hot, dry summer months. Concentrations of inoculum in rainwater varied significantly from year to year and increased as the rainy season progressed for the two complete seasons that were studied. Potential dispersal distances were investigated for rainwater, soil, and streamwater. In rainwater, inoculum moved 5 and 10 m from the inoculum source. For soil, transmission of inoculum was demonstrated from infested soil to bay laurel green leaf litter, and from bay laurel green leaf litter to aerial leaves of bay laurel seedlings. One-third to one-half of the hikers tested at the study site during the rainy season also were carrying infested soil on their shoes. In streamwater, P. ramorum was recovered from an unforested site in pasture 1 km downstream of forest with inoculum sources. In total, these studies provide details on the production and spread of P. ramorum inoculum in mixed-evergreen forest to aid forecasting and managing disease transmission of this environmentally destructive pathogen.

AFLP and phylogenetic analyses of North American and European populations of Phytophthora ramorum

The genetic structure within and between USA and European populations of the emerging phytopathogen Phytophthora ramorum was examined. Four primer combinations were used for amplified fragment length polymorphism (AFLP) fingerprinting of 67 USA isolates from California and Oregon, and 18 European isolates from Belgium, Germany, The Netherlands, Spain and the UK. In addition, three DNA regions (ITS, cox II, and nad 5) of additional Phytophthora species were amplified by polymerase chain reaction, sequenced, and analysed to provide better phylogenetic understanding of P. ramorum within the genus Phytophthora. AFLP banding patterns indicate that the 85 isolates form two distinct lineages within a monophyletic group, distinct from the closely related outgroup species P. lateralis. With the exception of two isolates from an Oregon nursery, European and USA isolates clustered separately within individual clades. The AFLP profiles also indicate that a single clonal lineage dominates the North American population, while the European population consists of an array of mainly unique, closely related AFLP types. Sequences from the three DNA regions were identical among all P. ramorum isolates, and phylogenetic analysis indicates that P. ramorum is closely related to P. lateralis and P. hibernalis.

Contrasting ectomycorrhizal fungal communities on the roots of co-occurring oaks (Quercus spp.) in a California woodland.

Plant host species is considered an important factor influencing ectomycorrhizal (EM) communities. To gain insights into the role of host species in structuring EM communities, EM communities on sympatric oak (Quercus) species were compared in the Sierra Nevada foothills of California. Using molecular methods (polymerase chain reaction, cloning, restriction fragment length polymorphism and DNA sequencing), EM fungi on roots of deciduous Quercus douglasii and evergreen Quercus wislizeni trees were identified from 64 soil cores. The total EM species richness was 140, of which 40 taxa were detected on both oak hosts. Greater diversity and frequency of EM fungi with epigeous fruiting habit were found on Q. wislizeni, while taxa in the Ascomycota were more frequent and diverse on Q. douglasii. Using ordination, it was determined that both soil extractable phosphorus and oak host species explained a significant proportion of the variation in EM species distribution. These results indicate that plant host species can be an important factor influencing EM fungal community composition, even within congeneric trees.

Detection and Quantification of Phytophthora ramorum from California Forests Using a Real-Time Polymerase Chain Reaction Assay

ABSTRACT The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.

Epidemiological modeling of invasion in heterogeneous landscapes: spread of sudden oak death in California (1990–2030)

The spread of emerging infectious diseases (EIDs) in natural environments poses substantial risks to biodiversity and ecosystem function. As EIDs and their impacts grow, landscape- to regional-scale models of disease dynamics are increasingly needed for quantitative prediction of epidemic outcomes and design of practicable strategies for control. Here we use spatio-temporal, stochastic epidemiological modeling in combination with realistic geographical modeling to predict the spread of the sudden oak death pathogen (Phytophthora ramorum) through heterogeneous host populations in wildland forests, subject to fluctuating weather conditions. The model considers three stochastic processes: (1) the production of inoculum at a given site; (2) the chance that inoculum is dispersed within and among sites; and (3) the probability of infection following transmission to susceptible host vegetation. We parameterized the model using Markov chain Monte Carlo (MCMC) estimation from snapshots of local- and regional-scale data on disease spread, taking account of landscape heterogeneity and the principal scales of spread. Our application of the model to Californian landscapes over a 40-year period (1990–2030), since the approximate time of pathogen introduction, revealed key parameters driving the spatial spread of disease and the magnitude of stochastic variability in epidemic outcomes. Results show that most disease spread occurs via local dispersal (<250 m) but infrequent long-distance dispersal events can substantially accelerate epidemic spread in regions with high host availability and suitable weather conditions. In the absence of extensive control, we predict a ten-fold increase in disease spread between 2010 and 2030 with most infection concentrated along the north coast between San Francisco and Oregon. Long-range dispersal of inoculum to susceptible host communities in the Sierra Nevada foothills and coastal southern California leads to little secondary infection due to lower host availability and less suitable weather conditions. However, a shift to wetter and milder conditions in future years would double the amount of disease spread in California through 2030. This research illustrates how stochastic epidemiological models can be applied to realistic geographies and used to increase predictive understanding of disease dynamics in large, heterogeneous regions.

Intra-specific and intra-sporocarp ITS variation of ectomycorrhizal fungi as assessed by rDNA sequencing of sporocarps and pooled ectomycorrhizal roots from a Quercus woodland.

The Internal Transcribed Spacer (ITS) regions of ribosomal DNA are widely used as markers for phylogenetic analyses and environmental sampling from a variety of organisms including fungi, plants, and animals. In theory, concerted evolution homogenizes multicopy genes so that little or no variation exists within populations or individuals. However, contrary to theory, ITS variation has been confirmed in populations and individuals from a diverse range of eukaryotes. The presence of intraspecific and intra-individual variation in multicopy genes has important implications for ecological and phylogenetic studies, yet relatively little is known about natural variation of these genes, particularly at the community level. In this study, we examined intraspecific and intra-sporocarp ITS variation by DNA sequencing from sporocarps and pooled roots from 68 species of ectomycorrhizal fungi collected at a single site in a Quercus woodland. We detected ITS variation in 27 species, roughly 40% of the taxa examined. Although intraspecific ITS variation was generally low (0.16–2.85%, mean = 0.74%), it was widespread within this fungal community. We detected ITS variation in both sporocarps and ectomycorrhizal roots, and variation was present within species of Ascomycota and Basidiomycota, two distantly related lineages within the Fungi. We discuss the implications of such widespread ITS variability with special reference to DNA-based environmental sampling from diverse fungal communities.

Early detection of emerging forest disease using dispersal estimation and ecological niche modeling

Distinguishing the manner in which dispersal limitation and niche requirements control the spread of invasive pathogens is important for prediction and early detection of disease outbreaks. Here, we use niche modeling augmented by dispersal estimation to examine the degree to which local habitat conditions vs. force of infection predict invasion of Phytophthora ramorum, the causal agent of the emerging infectious tree disease sudden oak death. We sampled 890 field plots for the presence of P. ramorum over a three-year period (2003-2005) across a range of host and abiotic conditions with variable proximities to known infections in California, USA. We developed and validated generalized linear models of invasion probability to analyze the relative predictive power of 12 niche variables and a negative exponential dispersal kernel estimated by likelihood profiling. Models were developed incrementally each year (2003, 2003-2004, 2003-2005) to examine annual variability in model parameters and to create realistic scenarios for using models to predict future infections and to guide early-detection sampling. Overall, 78 new infections were observed up to 33.5 km from the nearest known site of infection, with slightly increasing rates of prevalence across time windows (2003, 6.5%; 2003-2004, 7.1%; 2003-2005, 9.6%). The pathogen was not detected in many field plots that contained susceptible host vegetation. The generalized linear modeling indicated that the probability of invasion is limited by both dispersal and niche constraints. Probability of invasion was positively related to precipitation and temperature in the wet season and the presence of the inoculum-producing foliar host Umbellularia californica and decreased exponentially with distance to inoculum sources. Models that incorporated niche and dispersal parameters best predicted the locations of new infections, with accuracies ranging from 0.86 to 0.90, suggesting that the modeling approach can be used to forecast locations of disease spread. Application of the combined niche plus dispersal models in a geographic information system predicted the presence of P. ramorum across approximately 8228 km2 of Californias 84785 km2 (9.7%) of land area with susceptible host species. This research illustrates how probabilistic modeling can be used to analyze the relative roles of niche and dispersal limitation in controlling the distribution of invasive pathogens.

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.