Detection, Diversity, and Population Dynamics of Waterborne Phytophthora ramorum Populations.

Sudden oak death, the tree disease caused by Phytophthora ramorum, has significant environmental and economic impacts on natural forests on the U.S. west coast, plantations in the United Kingdom, and in the worldwide nursery trade. Stream baiting is vital for monitoring and early detection of the pathogen in high-risk areas and is performed routinely; however, little is known about the nature of water-borne P. ramorum populations. Two drainages in an infested California forest were monitored intensively using stream-baiting for 2 years between 2009 and 2011. Pathogen presence was determined both by isolation and polymerase chain reaction (PCR) from symptomatic bait leaves. Isolates were analyzed using simple sequence repeats to study population dynamics and genetic structure through time. Isolation was successful primarily only during spring conditions, while PCR extended the period of pathogen detection to most of the year. Water populations were extremely diverse, and changed between seasons and years. A few abundant genotypes dominated the water during conditions considered optimal for aerial populations, and matched those dominant in aerial populations. Temporal patterns of genotypic diversification and evenness were identical among aerial, soil, and water populations, indicating that all three substrates are part of the same epidemiological cycle, strongly influenced by rainfall and sporulation on leaves. However, there was structuring between substrates, likely arising due to reduced selection pressure in the water. Additionally, water populations showed wholesale mixing of genotypes without the evident spatial autocorrelation present in leaf and soil populations.

First Report of the NA2 Lineage of Phytophthora ramorum from an Ornamental Rhododendron in the Interior of California.

In July 2012, we collected a rhododendron var. Trilby with twig dieback symptoms in the lower canopy, consistent with the disease "ramorum blight" caused by Phytophthora ramorum. The symptomatic plant had been planted a year earlier to replace a dead rhododendron in a landscape setting in Placer County, California (Lat: 39.036216°; Long: -120.999274°), Sierra Nevada foothills at ~600 m elevation. Isolations yielded a culture with a fast growth rate and overall morphology resembling the P. ramorum NA2 lineage described by Ivors et al. (4). DNA was extracted from the culture as described previously (4) and six SSR loci: MS18, MS39, MS43, MS45, MS64, MS145, were amplified (2,4). Allelic patterns were compared with those of three testers from each of the three lineages NA1, NA2, and EU1 known to be present in ornamental plants in North America, and they unambiguously confirmed the isolate belongs to the NA2 lineage of the pathogen. Although the symptomatic plant was confined to a landscape setting, it had been planted in that location for a year, providing a possible source of inoculum for the surrounding area. This is the first report of P. ramorum from the Sierra Nevada eco-region in the interior of California. It is also the first report of a NA2 isolate from a plant outside of commercial nurseries in California. The mating type of the isolate was not determined, but NA2 isolates are normally A2, the same mating type of NA1 isolates. The only other report of a NA2 isolate found outside of a nursery is from Washington State (1). Although there is no evidence the pathogen may have infected other plants, the infected rhododendron was found at a location situated over 100 km east of the closest known infestation (www.sodmap.org). Additionally, this is the first report of the pathogen outside the coast mountain range of California. Because the three lineages are genetically and phenotypically distinct (3), the escape of NA2 or EU1 isolates, both still absent from plants in natural settings, could have significant implications for California ecosystems. This finding highlights that introductions of P. ramorum via ornamental plants are still possible, in spite of current regulations. References: (1) G. Chastagner et al. Phytopathology 101:S32, 2011. (2) P. P. Croucher et al. Biol. Invasions 15:2281, 2013. (3) N. J. Grünwald et al. Trends Microbiol. 20:131, 2012. (4) K. Ivors et al. Mol. Ecol. 15:1493, 2006.

A First Generation Heterobasidion Hybrid Discovered in Larix lyalli in Montana.

On September 25, 2010, a wood sample was collected from an entirely decayed root ball of an alpine larch (Larix lyallii Parl.), 10 cm in diameter at breast height, recently downed, but still green. No attempts were made to determine whether the decay progressed into the stem. The discovery occurred in a stand in the Bitterroot Mountains, south of Darby, Montana (elev. 2,530 m; 45.893528° N, 114.278322° W). Several adjacent alpine larches were either dead or displayed thin crowns, and an old Heterobasidion basidiocarp was found on the decayed root ball of a neighboring dead tree, suggesting the presence of a root disease pocket. The stand is mature and composed of alpine larch, whitebark pine (Pinus albicaulis Engelm.), and a few subalpine firs (Abies lasiocarpa (Hooker) Nuttall), but only larches were symptomatic. No stumps were visible, and the site is in a designated wilderness area characterized by minimal forest management. Wood chips displaying a white rot with bleached speckles were plated on 2% malt agar, and cultures displaying the typical Heterobasidion anamorph (Spiniger meineckellus) were visible after 7 days. DNA was extracted from two distinct cultures, and the sequences of three nuclear loci, namely the internal transcribed spacer, the elongation factor 1-alpha, and the glyceraldehyde 3-phosphate dehydrogenase, were analyzed. The sequence of the mitochondrial ATPase was also sequenced. All loci were amplified using the primers indicated in Linzer et al. (2). Sequences of all three nuclear loci (GenBank Accession Nos. KF811480 to 82) unequivocally indicated both isolates to be first generation hybrids between H. irregulare (Underw.) Garbel. & Otrosina and H. occidentale Otrosina & Garbel. Cumulatively, sequences were heterozygous at over 40 positions in all three loci, and for the presence of two indels (one in ITS, one in EF 1-alpha). Polymorphisms and indels indicated alleles from both species were present in these heterokaryotic (ploidy n+n) isolates. The mitochondrial ATPase (KF811483 to 84) indicated instead the cytoplasm belonged to H. occidentale, suggesting that species was the first to be established in the infected tree and was either dikaryotized by a basidiopsore of the other species, or subject to nuclear re-assortment through di-mon mating with a genotype of H. irregulare. This is the first report of a Heterobasidion sp. in L. lyalli, and it is the second report of a natural Heterobasidion hybrid in North America (1). This finding indicates Alpine larch may be a host for both Heterobasidion species, as described for pine stumps in California (1). Thus, this conifer may have provided a substrate for the hybridization and interspecific gene introgression documented to have occurred before stumps were generated in high frequency by modern forestry practices (2). References: (1) M. Garbelotto et al. Phytopathology 86:543, 1996. (2) R. Linzer et al. Mol. Phylogenet. Evol. 46:844, 2008.

Lineage, Temperature, and Host Species have Interacting Effects on Lesion Development in Phytophthora ramorum.

There are four recognized clonal lineages of the pathogen Phytophthora ramorum. The two major lineages present in North America are NA1 and NA2. With a few exceptions, NA1 is found in natural forest ecosystems and nurseries, and NA2 is generally restricted to nurseries. Isolates from the NA1 and NA2 lineages were used to infect rhododendron, camellia, and California bay laurel in detached leaf assays to study the effects of lineage, temperature, and host on pathogenicity and host susceptibility. Isolates within both lineages were highly variable in their ability to form lesions on each host. There was also a tendency toward reduced lesion size in successive trials, suggesting degeneration of isolates over time. Temperature had a significant effect on lesion size, with a response that varied depending on the host and isolate. Phenotypic differences between lineages appear to be heavily influenced by the representation of isolates used, host, and temperature. The importance of temperature, host, and lineage are discussed with respect to disease management, as well as future range expansions and migrations of the pathogen.

Population genetic analyses provide insights on the introduction pathway and spread patterns of the North American forest pathogen Heterobasidion irregulare in Italy.

A population genetics approach is used to identify the most likely introduction site and introduction pathway for the North American forest pathogen Heterobasidion irregulare using 101 isolates from six sites in Italy and 34 isolates from five sites in North America. Diversity indices based on sequences from ten loci indicate the highest diversity in Italy is found in Castelfusano/Castelporziano and that diversity progressively decreases with increasing distance from that site. AMOVA, Bayesian clustering and principal coordinates analyses based on 12 SSR loci indicate high levels of gene flow among sites, high frequency of admixing, and fail to identify groups of genotypes exclusive to single locations. Cumulatively, these analyses suggest the current infestation is the result of multiple genotypes expanding their range from a single site. Based on two sequenced loci, a single source site in North America could provide enough variability to explain the variability observed in Italy. These results support the notion that H. irregulare was introduced originally in Castelporziano: because Castelporziano has been sealed off from the rest of the world for centuries except for a camp set up by the US military in 1944, we conclude the fungus may have been transported in infected wood used by the military. Finally, spatial autocorrelation analyses using SSR data indicate a significant under-dispersion of alleles up to 0.5-10 km, while a significant overdispersion of alleles was detected at distances over 80 km: these ranges can be used to make predictions on the likely dispersal potential of the invasive pathogen.

Population dynamics of aerial and terrestrial populations of Phytophthora ramorum in a California forest under different climatic conditions.

Limited information is available on how soil and leaf populations of the sudden oak death pathogen, Phytophthora ramorum, may differ in their response to changing weather conditions, and their corresponding role in initiating the next disease cycle after unfavorable weather conditions. We sampled and cultured from 425 trees in six sites, three times at the end of a 3-year-long drought and twice during a wet year that followed. Soil was also sampled twice with similar frequency and design used for sampling leaves. Ten microsatellites were used for genetic analyses on cultures from successful isolations. Results demonstrated that incidence of leaf infection tripled at the onset of the first wet period in 3 years in spring 2010, while that of soil populations remained unchanged. Migration of genotypes among sites was low and spatially limited under dry periods but intensity and range of migration of genotypes significantly increased for leaf populations during wet periods. Only leaf genotypes persisted significantly between years, and genotypes present in different substrates distributed differently in soil and leaves. We conclude that epidemics start rapidly at the onset of favorable climatic conditions through highly transmissible leaf genotypes, and that soil populations are transient and may be less epidemiologically relevant than previously thought.

Amplified fragment length polymorphism and sequence analyses reveal massive gene introgression from the European fungal pathogen Heterobasidion annosum into its introduced congener H. irregulare.

The paucity of fungal species known to be currently hybridizing has significantly hindered our understanding of the mechanisms driving gene introgression in these eukaryotic microbes. Here, we describe an area of hybridization and gene introgression between the invasive plant pathogen Heterobasidion irregulare (introduced from North America) and the native H. annosum in Italy. A STRUCTURE analysis of amplified fragment length polymorphism data for 267 individuals identified gene introgression in 8-42% of genotypes in the invasion area, depending on site. Data indicate that introgression is mostly occurring unilaterally from the native to the invasive species and is responsible for 5-45% of genomes in admixed individuals. Sequence analysis of 11 randomly selected and unlinked loci for 30 individuals identified introgression at every locus, thus confirming interspecific gene flow involves a large number of loci. In 37 cases, we documented movement of entire alleles between the two species, but in 7 cases, we also documented the creation of new alleles through intralocus recombination. Sequence analysis did not identify enrichment of either transcriptionally different nonsynonymous alleles or of transcriptionally identical synonymous alleles. These findings may suggest introgression is occurring randomly for extant alleles without an obvious enrichment process driven by selection. However, further studies are needed to ensure selection is not at work elsewhere in the genome.

Sequence and simple-sequence repeat analyses of the fungal pathogen Seiridium cardinale indicate California is the most likely source of the cypress canker epidemic for the Mediterranean region.

Seiridium cardinale is the pathogenic fungus of unknown origin responsible for a world pandemic known as cypress canker affecting several species of Cupressaceae in both the Northern and Southern Hemisphere. In this study, a comparative genetic analysis of worldwide populations was performed using sequence analysis of a portion of the ?-tubulin locus and seven polymorphic simple-sequence repeat (SSR) loci on 96 isolates. Sequence analysis identified two distinct ?-tubulin alleles, both present in California. Only one of the two alleles was detected in the Mediterranean basin, while two isolates from the Southern Hemisphere were characterized by the presence of the allele absent from the Mediterranean. SSRs identified a total of 46 multilocus genotypes (MGs): genotypic diversity was always higher in the California population, and calculations of the index of association (I(A)) determined the presence of linkage disequilibrium associated with the absence of sexual reproduction only in the Mediterranean population but not in California. In 50 instances, the same MG was found at great geographic distances, implying a role played by humans in spreading the disease. Network analysis performed on SSR data identified three clusters of MGs: California, Morocco, and the rest of the Mediterranean. Both the Morocco and the Mediterranean clusters were linked to the California cluster. Coalescent analysis identified insignificant migration between California and Italy, as expected in the presence of a single introduction event, and very high migration from Italy into Greece, as expected of an outbreak still in exponential growth phase and starting from an Italian source.

First Report of Seiridium unicorne Causing Bark Cankers on a Monterey Cypress in California.

In June 2009, dieback of distal branches and resin exudation associated with bark lesions were observed in an adult Cupressus macrocarpa tree in Sonoma County, California (Glenn Ellen; 38°21'N, 122°31'W, elevation 233 m). The fungal pathogen, Seiridium unicorne (Cooke and Ellis) Sutton, was obtained by plating fragments of necrotic bark from the margins of branch cankers on potato dextrose agar (PDA). Identification was based on cultural, morphological, and molecular traits (2,3). Colonies on PDA were dense, cottony, off-white at first and then turning pale gray-green, and 2.3 and 4.3 cm in diameter after 1 and 2 weeks of growth at 20°C, respectively. Colonies of the fungus showed a faster radial growth at 20°C than at 25°C. Acervuli were abundantly produced on water agar amended with autoclaved cypress seeds after 2 to 3 weeks at 18°C under a mixture of fluorescent and near UV light. Conidia were six celled (five euseptate), fusiform, 20.9 to 35.2 × 7.11 to 10.57 µm, straight or slightly curved, with four, brown median cells, and with end cells bearing unbranched appendages 2 to 5 µm long. The DNA sequence of a portion of the ß-tubulin locus (GenBank Accession No. HQ678171) revealed a 100% homology with sequences of S. unicorne isolates from Portugal and South Africa, while being clearly distinct from sequences of S. cupressi and S. cardinale isolates (2). Greenhouse stem inoculations were performed by underbark placement of a 3-mm plug taken from the margins of a colony of the fungus grown on PDA. Inoculations were repeated twice in the spring and fall of 2010 on 10 C. macrocarpa saplings grown in pots for 3 years. Three months postinoculation, the pathogen could be successfully reisolated from the edges of 15 to 30 mm long elliptical lesions, present on each one of the inoculated saplings. The observed S. unicorne isolate is atypical because of its shorter appendages compared with the form reported in the literature (2,3). Because of its shorter conidial appendages and in vitro temperature optimum of 18 to 20°C, the fungus described here is similar to an unnamed Coryneum sp. observed by Wagener on C. macrocarpa (4). S. unicorne is a pathogen of many Cupressaceae in Africa, New Zealand, Japan, and some U.S. states (Georgia, South Carolina, Kansas, and Texas) (3), and although it was mentioned in a USDA Plant Quarantine Division report from 1963 as found on cypress in San Francisco (1), it has never been officially reported from California. Since similar disease symptoms were observed on many Cupressaceae in the course of an extensive survey performed in 2009 in California, it may be important to evaluate the relative incidence of S. unicorne compared with that of S. cardinale, a pathogen more commonly reported in association with the disease (4). References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm , 1/19/2011. (2) P. Krokene et al. Mycologia 96:1352, 2004. (3) N. A. Tisserat. Plant Dis. 75:138, 1991. (4) W. W. Wagener. J. Agric. Res. 58:1, 1939.

Optimization of sampling procedures for DNA-based diagnosis of wood decay fungi in standing trees.

AIMS: To develop fast and reliable sampling procedures for DNA-based diagnosis of wood decay fungi in standing trees. METHODS AND RESULTS: A total of 250 trees were tested for the presence of a suite of wood decay fungi by collecting wood frass obtained by drilling each tree once with a 4-mm-diameter, 43-cm-long bit. We identified at least one of 11 target wood decay fungi in 56 trees through multiplex PCR assays. The presence of target wood decay taxa was further investigated in these 56 trees, by analysing independently wood from each of six drillings. Results were then compared with those obtained using sampling schemes differing in terms of number and position of drillings. Samples of 1-4 drillings were either analysed separately, and the results were combined, or pooled together before analysis was performed. In comparison with taxa identified by the analysis of six drillings, diagnostic efficiency ranged from 56.6% for the scheme based on a single drill to 96.8% for the scheme based on four drillings analysed separately. Both schemes significantly differ (P < 0.05) from those based on two and three drillings, whose efficiency was 72.6% and 83.9%, respectively. Diagnostic efficiency of pooled samples was comparable to that of samples analysed separately. CONCLUSIONS: Highest diagnostic efficiency was obtained by analysing wood from four drillings. It is advisable to pool samples deriving from different drillings to reduce laboratory costs. SIGNIFICANCE AND IMPACT OF THE STUDY: Fast and reliable sampling procedures make DNA-based diagnosis more suitable for tree inspection procedures.

Genetic epidemiology of the sudden oak death pathogen Phytophthora ramorum in California.

A total of 669 isolates of Phytophthora ramorum, the pathogen responsible for Sudden Oak Death, were collected from 34 Californian forests and from the ornamental plant-trade. Seven microsatellite markers revealed 82 multilocus genotypes (MGs) of which only three were abundant (>10%). Iteratively collapsing based upon minimum Phi(ST), yielded five meta-samples and five singleton populations. Populations in the same meta-sample were geographically contiguous, with one exception, possibly explained by the trade of infected plants from the same source into different locations. Multidimensional scaling corroborated this clustering and identified nursery populations as genetically most distant from the most recent outbreaks. A minimum-spanning network illustrated the evolutionary relationships among MGs, with common genotypes at the centre and singletons at the extremities; consistent with colonization by a few common genotypes followed by local evolution. Coalescent migration analyses used the original data set and a data set in which local genotypes were collapsed into common ancestral genotypes. Both analyses suggested that meta-samples 1 (Santa Cruz County) and 3 (Sonoma and Marin Counties), act as sources for most of the other forests. The untransformed data set best explains the first phases of the invasion, when the role of novel genotypes may have been minimal, whereas the second analysis best explains migration patterns in later phases of the invasion, when prevalence of novel genotypes was likely to have become more significant. Using this combined approach, we discuss possible migration routes based on our analyses, and compare them to historical and field observations from several case studies.

Evaluation of molecular markers for Phytophthora ramorum detection and identification: testing for specificity using a standardized library of isolates.

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as beta-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.

A revision of the genus Psathyrella, with a focus on subsection Spadiceogriseae.

Specimens belonging to taxa traditionally assigned to the subsection Spadiceogriseae of the genus Psathyrella were analyzed both morphologically and molecularly. Samples included mainly European collections, selected GenBank accessions, and specimens of various North American taxa described by Smith (1972) and deposited at the Herbarium of the University of Michigan (MICH). Three additional taxa from Africa and Central America were also included. Bayesian and Maximum Likelihood analyses of two loci (ITS and Tef-1α) independently and together supported the monophyletic nature of the subsection Spadiceogriseae, and identified nine statistically supported clades within the subsection. North American and European species often fell within the same clade, suggesting a relatively recent origin of the subsection or human induced intercontinental movement. While this study determines for the first time that the presence of a white veil is diagnostic for the entire subsection, very few morphological traits were associated with individual clades, but clades were often distinctively different in terms of habitat association, suggesting that trophic interactions may have driven the evolution of this group of fungi. Combined, morphological and DNA analyses revealed both expected and unexpected synonymies. The new combinations P. vesiculosa, P. ochrofulva and P. sanjuanensis are proposed, and the new species P. rogersiae is described. New information is provided on the taxonomic status and distribution of several species including P. agrariella, P. albescens, P. atrifolia, P. bivelata, P. fatua, P. kauffmanii, P. aff. kauffmanii, P. incondita, P. infida, P. nitens, P. niveobadia, P. phegophila, P. pseudocorrugis sensu Kits van Waveren, P. subnuda. In total, 13 synonymies were proposed. Based on DNA data, five species of uncertain validity were confirmed as valid, while six species may be ambiguous and may require an in-depth re-analysis. The information gathered in this study was used to generate a key to the species of the subsection Spadiceogriseae.

Reconstruction of the Sudden Oak Death epidemic in California through microsatellite analysis of the pathogen Phytophthora ramorum.

The genetic structure of the clonally reproducing Sudden Oak Death (SOD) pathogen in California was investigated using seven variable microsatellites. A total of 35 multilocus genotypes were identified among 292 samples representative of populations from 14 forest sites and of the nursery trade. amova indicated significant genetic variability both within (44.34%) and among populations (55.66%). Spatial autocorrelation analyses indicated that Moran's index of similarity reached a minimum of 0.1 at 350 m, increased to 0.4 at 1500 m and then decreased to zero at 10 km. These results suggest a bimodal pattern of spread, with medium range dispersal (1500-10,000 m) putatively attributed to the presence of strong winds. Lack of genetic structure was identified for three groups of populations. One group notably included the nurseries' population and two forest populations, both linked to early reports of the pathogen. A neighbour-joining analysis based on pairwise Phi(ST) values indicated that the clade inclusive of the nurseries' populations is basal to all California populations. A network analysis identified three common genotypes as the likely founders of the California infestation and proposes a stepwise model for local evolution of novel genotypes. This was supported by the identification in the same locations of novel genotypes and of their 1- or 2-step parents. We hypothesize that the few undifferentiated population groups indicate historical human spread of the pathogen, while the general presence of genetically structured populations indicates that new infestations are currently generated by rare medium or long-range natural movement of the pathogen, followed by local generation of new genotypes.

Variation in rates of spore deposition of Fusarium circinatum, the causal agent of pine pitch canker, over a 12-month-period at two locations in Northern California.

Patterns of spore deposition by Fusarium circinatum, the causal agent of pine pitch canker (PPC) of Monterey pine (Pinus radiata) and other conifers, were studied between May 2003 and April 2004 at two sites in Northern California using a novel spore trapping method combined with a real-time polymerase chain reaction (PCR) approach. At each study site, two plots were sampled by placing spore traps at 100 m intervals along transects 600 m in length. The air was sampled continuously by exchanging the spore traps every 2 weeks. The spore deposition rate (DR), ranged from 0 to 1.3 x 10(5) spores m(2). Spores were detected throughout the year, with higher trapping frequencies (TF) during the rainy season (November to April), than during the dry season (May to October). The detection of spores on traps at distances larger than 200 m from any Monterey pine, suggests at least midrange aerial dispersal. Finally, different inoculum loads were associated with trees displaying different levels of disease symptoms, suggesting infectiousness of the pathogen varies as the disease progresses. This study represents one of the first documenting continuous inoculum pressure values over an entire year for a forest pathogen, and provides important epidemiological information that will be invaluable in the development of disease progression models.

First Report on an Infestation of Phytophthora cinnamomi in Natural Oak Woodlands of California and its Differential Impact on Two Native Oak Species.

During an intense survey of natural woodlands around Lake Hodges (33°N, 117°W) in June 2001, symptoms typical of root and collar rot caused by Phytophthora spp. were observed on 27% of 474 coast live oaks (Quercus agrifolia Nee.) and on none of 86 Engelmann oaks (Q. engelmannii Greene), in spite of complete spatial intermixing of the two species. Symptoms on coast live oaks included viscous exudates emerging through intact bark matched by underbark dark lesions with irregular margins. Lesions were delineated by a dark line and present on the root collar or the buttress of symptomatic trees. Crowns of trees with lesions ranged from completely healthy to declining or dead. All symptomatic trees were in proximity of the lake or streams. Phytophthora cinnamomi Rands was isolated from four trees in three distinct sites by plating tissues from lesion margins on PARP selective medium and from four soil samples by using standard pear baiting and plating lesions from pear tissue onto PARP. Identification of the isolates was obtained from microscopic observations and direct sequencing of the internal transcribed spacer region of the rDNA (Genbank Accession Nos. AY302148, MC2 and AY302149, MC3). P. citricola Sawada was also isolated once. Pathogenicity tests were completed to compare the susceptibility of the two species of oaks growing in the Lake Hodges region with P. cinnamomi. Two P. cinnamomi isolates from Lake Hodges (MC2, ATCC MYA-3711; MC3) and one isolate from an avocado orchard in San Diego County (MC6) were used to inoculate separately 10 5-year-old trees each of Q. agrifolia and Q. engelmannii grown in 5-gallon containers. Inoculations were performed in two lath-house experiments during February and September 2002 by placing an 8-mm diameter V8-agar plug from the margin of a P. cinnamomi colony underbark and sealing the wound with Parafilm and grafting wax. Lesion lengths were measured 2 months after inoculation, and the presence of the pathogen confirmed by reisolation on PARP. Mean average, maximum, and minimum temperatures were 14, 19, and 9°C and 21, 24, and 18°C for the February and September inoculations, respectively. The February inoculation resulted in small lesions only on Q. agrifolia (26 ± 15 mm, SD). The September inoculation resulted in 135 ± 68 mm (SD) lesions on Q. agrifolia and 49 ± 35 mm (SD) lesions on Q. engelmannii. Controls did not show any lesions. The length of lesions was significantly different between the two hosts (P < 0.0001) and significant differences were observed among the three isolates (P = 0.0018). Although Q. agrifolia is a known host for P. cinnamomi in California (2,3), to our knowledge, this is the first report of widespread infestation of P. cinnamomi in natural oak woodlands in the western United States. Survey and inoculation results indicated Q. engelmannii to be less susceptible to infection. Inoculation results confirm previous research that cold temperatures are unfavorable to this pathogen and isolates differed in pathogenicity toward Q. agrifolia. Decline of oaks infected by P. cinnamomi was observed only in conjunction with other factors, in particular with the presence of the oak twig girdler, Agrilus angelicus Horn., an insect favored by stress conditions such as drought. Similar effects have been reported for Mediterranean oaks infected by the same pathogen (1). References: (1) C. M. Brasier. Nature 360:539, 1992. (2) P. A. Miller. Western Shade Tree Conf. Proc. 8:39, 1941. (3) S. M. Mircetich et al. Plant Dis. Rep. 61:66, 1977.

A New Report of Phytophthora ramorum on Rhamnus purshiana in Northern California.

Rhamnus purshiana, or cascara, is a deciduous tall shrub or small tree as much as 9 m high with thin, smooth, silver-gray bark. It is often present in shady sites in redwood and mixed evergreen forests of the North Ameri-can west coast, from British Columbia to northern California. In July 2005, symptomatic leaves with irregular, black spots, 2 to 5 mm in diameter and concentrated toward the tips, were collected from four cascara plants in the Samuel P. Taylor State Park, Marin County, California. There was no evidence of defoliation. Pieces of necrotic tissue were plated on selective medium (PARP) and maintained at 19°C for 2 weeks. A Phytophthora sp. was consistently isolated and it was identified as P. ramorum on the basis of morphological and molecular traits published previously (3,4). The P. ramorum isolate Pr-418 has been deposited in the American Type Culture Collection (ATCC MYA-3676) and a portion of the internal transcribed sequence (ITS) of rDNA has been deposited in the NCBI database (GenBank Accession No. DQ168874). Koch's postulates were completed using the leaf-dip method (2) on detached leaves collected from three cascara plants growing at the University of California Botanical Garden at Berkeley. Zoospore inoculum was prepared by flooding a 2-week-old culture growing on V8 agar with sterile water for 4 days. The liquid was filtered after cold shocking at 4°C for 30 min and incubated at room temperature for 1 h. Fifteen leaves were dipped in the resulting zoospore suspension (1.6 × 104 zoospores per ml) for either 1 min (experiment 1) or overnight (experiment 2). Leaves used as negative controls were dipped in sterile water. After removal from the inoculum, excess liquid was allowed to drain. Leaves were maintained in a moist chamber at 19°C with 13 h of natural light for 1 week. After 3 days of incubation, necrotic spots similar to those observed in the field had developed on leaves in experiment 2, while no symptoms were observed in experiment 1. Necrotic lesions were observed on 12 and 15 of 15 leaves in experiments 1 and 2, respectively, after 7 days of incubation. For each leaf, the necrotic area and percent necrosis was determined by placing the leaves in a flatbed scanner and processing the images with Assess (Version 1.01; The American Phytopathological Society, St. Paul, MN). Lesions extended from the tip of the leaves and covered 3 ± 1% of the total leaf area in experiment 1 and 33 ± 3% in experiment 2. Reisolation of P. ramorum on PARP was successful for all inoculated leaves. P. ramorum was never isolated from negative controls and no symptoms of infection were observed. The leaf-dip inoculation method is a rapid and reliable indicator of host susceptibility to P. ramorum, with many species developing necrosis when exposed to high concentrations of zoospores (3). Our results show that exposure time to the pathogen can play an important role in the development of symptoms. R. purshiana has been previously reported as a host in Oregon (1,2), but to our knowledge, this is the first report of cascara as a natural host of P. ramorum in the state of California. Our results confirm those from Oregon (2). The impact of infection by P. ramorum on cascara is unknown. References: (1) J. M. Davidson et al. Plant Health Prog. DOI:10.1094/PHP-2003-0707-01-DG, 2003. (2) E. Hansen et al. Plant Dis. 89:63, 2005. (3) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (4) S. Werres et al. Mycol Res. 105:1155, 2001.

First Report of Infection of Maiden-Hair Fern (Adiantum jordanii and A. aleuticum) by Phytophthora ramorum in California.

During July 2005, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from nine native Adiantum jordanii plants growing at two forest sites (Samuel P. Taylor State Park, Marin County and Peachland Road, Mendocino County) and from seven A. aleuticum plants at one forest site (Peachland Road) in California. At both locations, symptomatic plants were distributed close to rivers and roads and in association with infected bay laurel trees (Umbellularia californica), toyon (Heteromeles arbutifolia), and tanoaks (Lithocarpus densiflorus). Symptomatic leaflets showed brown spots that sometimes coalesced, killing entire leaves, but the disease did not appear to be fatal to the ferns. Necrotic tissues were plated on PARP and maintained in the dark at 18°C for 1 to 2 weeks. Isolates were identified as P. ramorum on the basis of colony morphology, the presence of chlamydospores and caducous, semipapillate sporangia, and the internal transcribed spacer (ITS) rDNA sequences (1,2). The P. ramorum isolates, Pr-419 from A. jordanii and Pr-422 from A. aleuticum, have been deposited in the American Type Culture Collection (ATCC MYA-3677 and MYA-3679, respectively) and a region of the ITS rDNA sequence deposited in the NCBI database (GenBank Accession No. DQ173082 and DQ219821, respectively). To test the pathogenicity, the tips of freshly detached leaves of A. jordanii and A. aleuticum were dipped into a solution of 1 × 103 zoospores per ml of Pr-419 and Pr-422 for 1 min. The wounded end of the leaves was not exposed to the inoculum. The zoospores were produced by flooding agar disks (1 cm in diameter) from the margin of 8- to 14-day-old colonies growing on V8 juice agar with sterile deionized water. After 3 days of incubation at 20°C in the dark, zoospore release was induced by placing dishes at 4°C for 20 min and then at room temperature for 60 min. For each Adiantum species and P. ramorum isolate, 15 leaves collected from five potted nursery plants were tested. Control leaves were dipped in sterile deionized water. Leaves were maintained in a moist chamber at 19°C with 13 h of natural light for 9 days. Brown lesions similar to those seen in the forest developed on approximately 60 and 33% of the A. jordanii and A. aleuticum leaves, respectively, inoculated with Pr-419 and on approximately 73 and 40% of the leaves inoculated with Pr-422. Under these experimental conditions, A. aleuticum appeared to be slightly more susceptible than the A. jordanii, with a necrotic leaf area of approximately 38% compared with 20%. The pathogen was reisolated on PARP after surface sterilization from all symptomatic leaves. Control leaves did not develop symptoms and P. ramorum was not recovered. A. jordanii and A. aleuticum have already been listed as associated hosts for P. ramorum on the APHIS (USDA Animal and Plant Health Inspection Service) website ( http://www.aphis.usda.gov/ ). To our knowledge, this is the first report of ferns as natural hosts of P. ramorum. References: (1) D. M. Rizzo et al. Plant Dis. 86:205, 2002. (2) S. Werres et al. Mycol Res. 105:1155, 2001.

First Report of Foliar Infection of Maianthemum racemosum by Phytophthora ramorum.

In May 2003, Phytophthora ramorum S. Werres & A.W.A.M. de Cock was isolated from the leaf tips of a single plant of false Solomon's seal (Maianthemum racemosum (L.) Link, formely known as Smilacina racemosa (L.) Desf.), a native, herbaceous perennial of the Liliaceae family, at the Jack London State Park in Sonoma County, California. Affected leaves had cream-to-brown lesions on the tips that were delimited by a yellow chlorotic zone. Lesions on the stems were not observed. The isolate (American Type Culture Collection [ATCC], Manassas, VA, MYA-3280; Centraal Bureau voor Schimmelcultures, Baarn, the Netherlands, CBS 114391) was typical of P. ramorum with large chlamydospores and caduceus, semipapillate sporangia, and the sequence (GenBank Accession No. AY526570) of the internal transcribed spacer region of the rDNA matched those published previously (4). The site, from which wood rose (Rosa gymnocarpa) was recently identified as a host, is a mixed forest containing confirmed P. ramorum-infected coast redwood (Sequoia sempervirens), California bay laurel (Umbellularia californica), and tanoak (Lithocarpus densiflora) trees (2,3). Two leaves per asymptomatic, pesticide free, potted plant of false Solomon's seal were inoculated with zoospores of the P. ramorum isolate obtained from infected false Solomon's seal (1). Five plants were inoculated in trial 1, and the following day, three plants were inoculated in trial 2. A control leaf of each plant was dipped in sterile deionized water. Plants were enclosed in plastic bags, misted regularly with sterile distilled water, and maintained at 16 to 21°C in the greenhouse. In both trials, plants did not have lesions on the leaves after 16 days and were reinoculated on separate days for each trial with higher concentrations of zoospores (1 × 105 [trial 1] and 2 × 105 [trial 2] zoospores/ml). Cream-colored lesions, similar to those observed in the field, were evident 1 week after the second inoculation and stopped progressing in both trials by 17 days. Lesions starting from the leaf tips averaged 13 mm (range 8 to 24 mm) long, and P. ramorum was reisolated on Phytophthora-selective agar medium modified with 25 mg of pentachloronitrobenzene from 44% (trial 1) and 83% (trial 2) of all lesions (4). Control leaves had no lesions, and P. ramorum was not reisolated. Sporangia were not observed on any leaves when examined with the dissecting microscope. The fact that lesions developed only after a second inoculation with higher concentrations of zoospores, and these lesions stopped progressing after 17 days, suggests that false Solomon's seal is much less susceptible than other hosts such as western starflower (Trientalis latifolia) (1) and wood rose (2). To our knowledge, this is the first report of a plant from the Liliaceae as a natural host for P. ramorum, although Smilax aspersa was identified as being susceptible in artificial inoculations of detached leaves (E. Moralejo and L. Hernández, personal communication). False Solomon's seal is popular in the horticultural industry. References: (1) D. Hüberli et al. Plant Dis. 87:599, 2003. (2) D. Hüberli et al. Plant Dis. 88:430, 2004. (3) P. E. Maloney et al. Plant Dis. 86:1274, 2002. (4) D. M. Rizzo et al. Plant Dis. 86:205, 2002.

Genetic Structure of Heterobasidion annosum in White Fir Mortality Centers in California.

ABSTRACT The structure of Heterobasidion annosum populations was studied in 15 mixed-conifer sites in central and northern California. Study sites displayed mortality of white fir trees in enlarging discrete patches (mortality centers). At each site, fungal genotypes were defined by somatic compatibility tests. In two sites, further genetic and molecular analyses were performed on field genotypes and on homokaryons obtained by dedikaryotization of field heterokaryons. Isolates were found to be colonizing mostly the roots and the bole sapwood of white fir trees, and no significant infections of other tree species were observed. Each mortality center was characterized by the presence of several fungal genotypes, all belonging to the S intersterility group. Both homokaryotic and heterokaryotic strains were present in all sites. Multiple genotypes were retrieved in individual trees or stumps. Out of 228 fungal genotypes, 86% were found only within a single tree or stump, while 14% had spread to adjacent trees. The two largest genotypes had diameters of 9 and 10 m, and had colonized five and nine trees, stumps, or both, respectively. The maximum distance between two adjacent trees colonized by the same genotype was 6 m, and a highly significant correlation was found between tree diameter and distance of fungal "vegetative" spread. The largest clones were found in areas characterized by high tree and stump densities, and secondary spread of the fungus was more significant in denser stands. In most cases, original infection courts of existing genotypes could be traced to standing trees and not to stumps. The genetic analysis performed in two mortality centers revealed that most local genotypes had different mating alleles, and thus originated from unrelated basidiospores. In a few cases, the same mating allele was shared by two heterokaryons (n+n genome) or by a homokaryon (n genome) and a heterokaryon. Molecular analysis showed that nuclei bearing the same mating allele were identical, providing evidence that the two nuclei forming heterokaryons can act independently in the field and can be shared among isolates, presumably via di-mon mating or by separate matings of different portions of widespread homokaryons.