Identification, Pathogenicity, and Spore Trapping of Colletotrichum karstii Associated with Twig and Shoot Dieback in California.

Colletotrichum Corda, 1831 species are well-documented pathogens of citrus that are associated with leaf and fruit anthracnose diseases. However, their role in twig and shoot dieback diseases of citrus has recently become more prominent. Recent surveys of orchards in the Central Valley of California have revealed C. gloeosporioides and a previously undocumented species, C. karstii, to be associated with twig and shoot dieback. Pathogenicity tests using clementine (cv. 4B) indicated that both C. karstii and C. gloeosporioides are capable of producing lesions following inoculation of citrus stems. Pathogenicity tests also revealed C. karstii to be the most aggressive fungal species producing the longest lesions after 15 months. The majority of spores trapped during this study were trapped during or closely following a precipitation event with the majority of spores being trapped from January through May. These findings confirm C. karstii as a new pathogen of citrus in California.

Genetic Structure of the Rice Blast Pathogen (Magnaporthe oryzae) over a Decade in North Central California Rice Fields.

Rice blast, caused by the ascomycete Magnaporthe oryzae, is one of the most destructive rice diseases worldwide. Even though the disease has been present in California since 1996, there is no data for the pathogen population biology in the state. Using amplified fragment length polymorphisms and mating-type markers, the M. oryzae population diversity was investigated using isolates collected when the disease was first established in California and isolates collected a decade later. While in the 1990 samples, a single multilocus genotype (MLG) was identified (MLG1), over a decade later, we found 14 additional MLGs in the 2000 isolates. Some of these MLGs were found to infect the only rice blast-resistant cultivar (M-208) available for commercial production in California. The same samples also had a significant decrease of MLG1. MLG1 was found infecting the resistant rice cultivar M-208 on one occasion whereas MLG7 was the most common genotype infecting the M-208. MLG7 was identified in the 2000 samples, and it was not present in the M. oryzae population a decade earlier. Our results demonstrate a significant increase in genotypic diversity over time with no evidence of sexual reproduction and suggest a recent introduction of new virulent race(s) of the pathogen. In addition, our data could provide information regarding the durability of the Pi-z resistance gene of the M-208. This information will be critical to plant breeders in developing strategies for deployment of other rice blast resistance genes/cultivars in the future.

A pulse of summer precipitation after the dry season triggers changes in ectomycorrhizal formation, diversity, and community composition in a Mediterranean forest in California, USA.

Rapid responses of microbial biomass and community composition following a precipitation event have been reported for soil bacteria and fungi, but measurements characterizing ectomycorrhizal fungi remain limited. The response of ectomycorrhizal fungi after a precipitation event is crucial to understanding biogeochemical cycles and plant nutrition. Here, we examined changes in ectomycorrhizal formation, diversity, and community composition at the end of a summer drought and following precipitation events in a conifer-oak mixed forest under a semiarid, Mediterranean-type climate in CA, USA. To study the effects of different amounts of precipitation, a water addition treatment was also undertaken. Ectomycorrhizal fungal diversity and community composition changed within 6 days following precipitation, with increased simultaneous mortality and re-growth. Ectomycorrhizal diversity increased and community composition changed both in the natural rainfall (less than 10 mm) and water addition (50 mm) treatments, but larger decreases in ectomycorrhizal diversity were observed from 9 to 16 days after precipitation in the water addition treatment. The changes were primarily a shift in richness and abundance of Basidiomycota species, indicating higher drought sensitivity of Basidiomycota species compared with Ascomycota species. Our results indicate that ectomycorrhizal formation, diversity, and community composition rapidly respond to both precipitation events and to the amount of precipitation. These changes affect ecosystem functions, such as nutrient cycling, decomposition, and plant nutrient uptake, in semiarid regions.

Revisiting phylogenetic diversity and cryptic species of Cenococcum geophilum sensu lato.

The fungus Cenococcum geophilum Fr. (Dothideomycetes, Ascomycota) is one of the most common ectomycorrhizal fungi in boreal to temperate regions. A series of molecular studies has demonstrated that C. geophilum is monophyletic but a heterogeneous species or a species complex. Here, we revisit the phylogenetic diversity of C. geophilum sensu lato from a regional to intercontinental scale by using new data from Florida (USA) along with existing data in GenBank from Japan, Europe, and North America. The combination of internal transcribed spacer (ITS) ribosomal DNA and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene resolved six well-supported lineages (87-100 % bootstrap values) that are closely related to each other and a seventh lineage that is phylogenetically distinct. A multi-locus analysis (small subunit (SSU), large subunit (LSU), translational elongation factor (TEF), and the largest and second-largest subunits of RNA polymerase II (RPB1 and RPB2)) revealed that the divergent lineage is the sister group to all other known Cenococcum isolates. Isolates of the divergent lineage grow fast on nutrient media and do not form ectomycorrhizas on seedlings of several pine and oak species. Our results indicate that C. geophilum sensu lato includes more phylogenetically distinct cryptic species than have previously been reported. Furthermore, the divergent lineage appears to be a non-mycorrhizal sister group. We discuss the phylogenetic diversity of C. geophilum sensu lato and argue in favor of species recognition based on phylogenetic and ecological information in addition to morphological characteristics. A new genus and species (Pseudocenococcum floridanum gen. et sp. nov.) is proposed to accommodate a divergent and putatively non-mycorrhizal lineage.

Huanglongbing: An overview of a complex pathosystem ravaging the world's citrus.

Citrus huanglongbing (HLB) has become a major disease and limiting factor of production in citrus areas that have become infected. The destruction to the affected citrus industries has resulted in a tremendous increase to support research that in return has resulted in significant information on both applied and basic knowledge concerning this important disease to the global citrus industry. Recent research indicates the relationship between citrus and the causal agent of HLB is shaped by multiple elements, in which host defense responses may also play an important role. This review is intended to provide an overview of the importance of HLB to a wider audience of plant biologists. Recent advances on host-pathogen interactions, population genetics and vectoring of the causal agent are discussed.

Mycosphaerella musicola Identified as the Only Pathogen of the Sigatoka Disease Complex Present in Minas Gerais State, Brazil.

A thorough assessment of the distribution of Mycosphaerella spp. associated with banana in Minas Gerais State, Brazil, was conducted after Mycosphaerella fijiensis was first reported to occur in this region in 2005. From 2009 to 2011, 80 fields located in 20 municipalities including the same fields where the disease was first reported were sampled. A total of 800 samples of leaf tissue with symptoms similar to those of yellow or black Sigatoka diseases were examined, and 239 isolates were obtained. The identification of the fungi was based on morphological characters combined with DNA sequences obtained after amplification with species-specific primers and phylogeny inferred from the internal transcribed spacer region of Mycosphaerella strains from banana. All 239 isolates were identified as Mycosphaerella musicola. The absence of M. fijiensis in the samples may have been due to misidentification of M. fijiensis or the displacement of M. fijiensis by M. musicola. It is now apparent that yellow Sigatoka caused by M. musicola is the prevailing leaf spot disease of bananas in Minas Gerais State and that regulatory/legislative control measures need to be revised based on our findings.

Inferring dispersal patterns of the generalist root fungus Armillaria mellea.

Investigating the dispersal of the root-pathogenic fungus Armillaria mellea is necessary to understand its population biology. Such an investigation is complicated by both its subterranean habit and the persistence of genotypes over successive host generations. As such, host colonization by resident mycelia is thought to outcompete spore infections. We evaluated the contributions of mycelium and spores to host colonization by examining a site in which hosts pre-date A. mellea. Golden Gate Park (San Francisco, CA, USA) was established in 1872 primarily on sand dunes that supported no resident mycelia. Genotypes were identified by microsatellite markers and somatic incompatibility pairings. Spatial autocorrelation analyses of kinship coefficients were used to infer spore dispersal distance. The largest genotypes measured 322 and 343 m in length, and 61 of the 90 total genotypes were recovered from only one tree. The absence of multilocus linkage disequilibrium and the high proportion of unique genotypes suggest that spore dispersal is an important part of the ecology and establishment of A. mellea in this ornamental landscape. Spatial autocorrelations indicated a significant spatial population structure consistent with limited spore dispersal. This isolation-by-distance pattern suggests that most spores disperse over a few meters, which is consistent with recent, direct estimates based on spore trapping data.

Geographic structure of a bolete-infecting cryptic species within the Hypomyces microspermus species complex in California.

Fungi in the genus Hypomyces are mycoparasites of various fungal groups. Studies within this genus have dealt primarily with taxonomic descriptions, and no studies have been published on the population biology of these fungi in natural ecosystems. In this study, genetic structure within and among six California (CA) bolete-infecting H. microspermus populations were analyzed with amplified fragment length polymorphism (AFLP). Four of the populations were collected south of the transverse mountain ranges of southern CA, whereas the other populations were from the central coast of CA (Pismo Beach) and from northern CA (Humboldt County). Thirty-nine genotypes were found among 68 isolates based on 30 AFLP markers. This was a higher degree of genotypic diversity than expected because no sexual fruiting bodies were observed in the field, suggesting that sexual reproduction occurs infrequently. These observations were also supported by multilocus disequilibrium analyses. Overall the populations were significantly differentiated (θ = 0.321, P < 0.001), and most population pairwise F(st) values were also significantly different. The populations sampled also fit an isolation by distance model based on a Mantel test (P < 0.001). The overall results suggest that this pathogen has a primarily clonal population genetic structure and that spore dispersal was restricted across the sampled populations.

Phylogenetic placement of the ectomycorrhizal genus Cenococcum in Gloniaceae (Dothideomycetes).

Cenococcum is a genus of ectomycorrhizal Ascomycota that has a broad host range and geographic distribution. It is not known to produce either meiotic or mitotic spores and is known to exist only in the form of hyphae, sclerotia and host-colonized ectomycorrhizal root tips. Due to its lack of sexual and asexual spores and reproductive structures, it has proven difficult to incorporate into traditional classification within Ascomycota. Molecular phylogenetic studies of ribosomal RNA placed Cenococcum in Dothideomycetes, but the definitive identification of closely related taxa remained elusive. Here we report a phylogenetic analysis of five nuclear loci (SSU, LSU, TEF1, RPB1, RPB2) of Dothideomycetes that placed Cenococcum as a close relative of the genus Glonium of Gloniaceae (Pleosporomycetidae incertae sedis) with strong statistical support. Glonium is a genus of saprobic Dothideomycetes that produces darkly pigmented, carbonaceous, hysteriate apothecia and is not known to be biotrophic. Evolution of ectomycorhizae, Cenococcum and Dothideomycetes is discussed.

How well do ITS rDNA sequences differentiate species of true morels (Morchella)?

Arguably more mycophiles hunt true morels (Morchella) during their brief fruiting season each spring in the northern hemisphere than any other wild edible fungus. Concerns about overharvesting by individual collectors and commercial enterprises make it essential that science-based management practices and conservation policies are developed to ensure the sustainability of commercial harvests and to protect and preserve morel species diversity. Therefore, the primary objectives of the present study were to: (i) investigate the utility of the ITS rDNA locus for identifying Morchella species, using phylogenetic species previously inferred from multilocus DNA sequence data as a reference; and (ii) clarify insufficiently identified sequences and determine whether the named sequences in GenBank were identified correctly. To this end, we generated 553 Morchella ITS rDNA sequences and downloaded 312 additional ones generated by other researchers from GenBank using emerencia and analyzed them phylogenetically. Three major findings emerged: (i) ITS rDNA sequences were useful in identifying 48/62 (77.4%) of the known phylospecies; however, they failed to identify 12 of the 22 species within the species-rich Elata Subclade and two closely related species in the Esculenta Clade; (ii) at least 66% of the named Morchella sequences in GenBank are misidentified; and (iii) ITS rDNA sequences of up to six putatively novel Morchella species were represented in GenBank. Recognizing the need for a dedicated Web-accessible reference database to facilitate the rapid identification of known and novel species, we constructed Morchella MLST (http://www.cbs.knaw.nl/morchella/), which can be queried with ITS rDNA sequences and those of the four other genes used in our prior multilocus molecular systematic studies of this charismatic genus.

Contrasting genetic structure between Magnaporthe grisea populations associated with the golf course turfgrasses Lolium perenne (perennial ryegrass) and Pennisetum clandestinum (kikuyugrass).

Gray leaf spot (GLS) disease of perennial ryegrass (Lolium perenne) and kikuyugrass (Pennisetum clandestinum) in golf courses in California was first noted in 2001 and 2003, respectively, and within 5 years had become well established. The causal agent of the disease is the fungus Magnaporthe grisea, which is known to consist primarily of clonal lineages that are highly host specific. Therefore, our objective was to investigate host specificity and population dynamics among isolates associated primarily from perennial ryegrass and kikuyugrass since the disease emerged at similar times in California. We also obtained isolates from additional hosts (tall fescue, St. Augustinegrass, weeping lovegrass, and rice) and from the eastern United States for comparative purposes. A total of 38 polymorphic amplified fragment length polymorphism makers were scored from 450 isolates which clustered by host with high bootstrap support (71 to 100%). Genetic structure between kikuyugrass and perennial ryegrass isolates differed significantly. Isolates from kikuyugrass were genotypically diverse (n = 34), possessed both mating types, and some tests for random mating could not be rejected, whereas isolates from perennial ryegrass were less genotypically diverse (n = 10) and only consisted of a single mating type. Low genotypic diversity was also found among the other host specific isolates which also only consisted of a single mating type. This is the first study to document evidence for the potential of sexual reproduction to occur in M. grisea isolates not associated with rice (Oryza sativa). Moreover, given the significant host specificity and contrasting genetic structures between turfgrass-associated isolates, the recent emergence of GLS on various grass hosts in California suggests that potential cultural practices or environmental changes have become conducive for the disease and that the primary inoculum may have already been present in the state, despite the fact that two genotypes associated with perennial ryegrass and St. Augustinegrass in California were the same as isolates collected from the eastern United States.

Development of molecular markers and preliminary investigation of the population structure and mating system in one lineage of black morel (Morchella elata) in the Pacific Northwestern USA.

Phylogenetic analysis of LSU/ITS sequence data revealed two distinct lineages among 44 morphologically similar fruiting bodies of natural black morels (Morchella elata group) sampled at three non-burn locations in the St Joe and Kanisku National Forests in northern Idaho. Most of the sampled isolates (n = 34) represented a dominant LSU/ITS haplotype present at all three sites and identical to the Mel-12 phylogenetic lineage (GU551425) identified in a previous study. Variation at 1-3 nucleotide sites was detected among a small number of isolates (n = 6) within this well supported clade (94%). Four isolates sampled from a single location were in a well supported clade (97%) distinct from the dominant haplotypes and may represent a previously un-sampled, cryptic phylogenetic species. Species-specific SNP and SCAR markers were developed for Mel-12 lineage isolates by cloning and sequencing AFLP amplicons, and segregation of AFLP markers were studied from single ascospore isolates from individual fruiting bodies. Based on the segregation of AFLP markers within single fruiting bodies, split decomposition analyses of two SCAR markers, and population genetic analyses of SNP, SCAR, and AFLP markers, it appears that members of the Morchella sp. Mel-12 phylogenetic lineage are heterothallic and outcross in nature similar to yellow morels. This is the first set of locus-specific molecular markers that has been developed for any Morchella species, to our knowledge. These markers will prove to be valuable tools to study mating system, gene flow and genetic structure of black morels at various spatial scales with field-collected fruiting bodies and eliminate the need to culture samples in vitro.

First report of the ectomycorrhizal status of boletes on the Northern Yucatan Peninsula, Mexico determined using isotopic methods.

Despite their prominent role for tree growth, few studies have examined the occurrence of ectomycorrhizal fungi in lowland, seasonally dry tropical forests (SDTF). Although fruiting bodies of boletes have been observed in a dry tropical forest on the Northern Yucatan Peninsula, Mexico, their occurrence is rare and their mycorrhizal status is uncertain. To determine the trophic status (mycorrhizal vs. saprotrophic) of these boletes, fruiting bodies were collected and isotopically compared to known saprotrophic fungi, foliage, and soil from the same site. Mean δ(15)N and δ(13)C values differed significantly between boletes and saprotrophic fungi, with boletes 8.0‰ enriched and 2.5‰ depleted in (15)N and (13)C, respectively relative to saprotrophic fungi. Foliage was depleted in (13)C relative to both boletes and saprotrophic fungi. Foliar δ(15)N values, on the other hand, were similar to saprotrophic fungi, yet were considerably lower relative to bolete fruiting bodies. Results from this study provide the first isotopic evidence of ectomycorrhizal fungi in lowland SDTF and emphasize the need for further research to better understand the diversity and ecological importance of ectomycorrhizal fungi in these forested ecosystems.

Genetic diversity of Labyrinthula terrestris, a newly emergent plant pathogen, and the discovery of new Labyrinthulid organisms.

Labyrinthuloid organisms are thought almost exclusively to be only associated with marine environments. However in 1995, a disease of turfgrass suddenly appeared that was eventually determined to be caused by a new Labyrinthula species (Labyrinthula terrestris). The disease is primarily thought to be caused by the use of elevated salinity irrigation water, making it a unique example of an emergent plant disease potentially induced by human activity. Our objective was to examine diversity of L. terrestris from broadly distributed isolates using AFLP, sequence analysis of two rDNA loci (SSU & LSU-ITS), and pathogenicity tests since previous research on a limited number of isolates found no variability based in ITS and SSU. In contrast to previous work, 18 unique genotypes were found out of a total of 29 analyzed based on AFLP. Sequence variability was only found in a single pathogenic isolate (Laby 31) that was isolated from the United Kingdom. The divergence based on AFLP and sequence analysis suggests that this isolate is a distinct species but closely related to the other L. terrestris isolates examined. Two putatively new nonpathogenic Labyrinthulid species were also found (Laby 13 & 32). Our results suggest that these organisms may be widely distributed in terrestrial environments based on the diversity found in this study and may have long been associated with terrestrial plants. Our results also suggest that more Labyrinthulid organisms may potentially emerge as new plant pathogens in the future if salinification of agricultural systems continues to increases worldwide.

Geographic Distribution and rDNA-ITS Region Sequence Diversity of Waitea circinata var. circinata Isolated from Annual Bluegrass in the United States.

Waitea circinata var. circinata is the causal agent of brown ring patch, an emergent disease of turfgrass in the United States. Forty-two isolates from annual bluegrass were obtained from California, Connecticut, Idaho, Illinois, Massachusetts, New York, Ohio, Oregon, and Rhode Island. Almost all isolates produced white to orange sclerotia (bulbils), 2 to 5 mm in size, that turned dark brown after 21 days on »-strength potato dextrose agar. The ribosomal DNA internal transcribed spacer regions and 5.8S region (ITS) were analyzed by restriction fragment length polymorphism (RFLP) analysis using MspI and sequencing to attempt identification of the isolates. Some isolates were heterozygous at the MspI restriction site, results not found in previous reports using the RFLP technique for identification. Four additional nucleotide positions were found to be variable within ITS based on sequence analysis, including two indels and two additional heterozygous positions. A total of 17 ITS haplotypes were found, and there was no obvious relationship between ITS haplotype and the geographic distribution of the isolates. Results of this work indicate that W. circinata var. circinata is present in multiple states and provide an initial understanding of the diversity of the pathogen in the United States.

Microsatellite characterization and marker development for the fungus Penicillium digitatum, causal agent of green mold of citrus.

Penicillium digitatum is one of the most important postharvest pathogens of citrus on a global scale causing significant annual losses due to fruit rot. However, little is known about the diversity of P. digitatum populations. The genome of P. digitatum has been sequenced, providing an opportunity to determine the microsatellite distribution within P. digitatum to develop markers that could be valuable tools for studying the population biology of this pathogen. In the analyses, a total of 3,134 microsatellite loci were detected; 66.73%, 23.23%, 8.23%, 1.24%, 0.16%, and 0.77% were detected as mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats, respectively. As consistent with other ascomycete fungi, the genome size of P. digitatum does not seem to correlate with the density of microsatellite loci. However, significantly longer motifs of mono-, di-, and tetranucleotide repeats were identified in P. digitatum compared to 10 other published ascomycete species with repeats of over 800, 300, and 900 motifs found, respectively. One isolate from southern California and five additional isolates from other countries ("global isolates") were used to initially screen microsatellite markers developed in this study. Twelve additional isolates, referred to as the "local isolates," were also collected from citrus at the University of California Riverside agricultural experiment station and were subsequently used to screen the primers that sequenced well and were polymorphic based on the global isolates. Thirty-six primers were screened, and nine trinucleotide loci and one hexanucleotide locus were chosen as robust markers. These loci yielded two to seven alleles and will be useful to study population genetic structure of P. digitatum populations.

Isolation source matters: sclerotia and ectomycorrhizal roots provide different views of genetic diversity in Cenococcum geophilum.

Cenococcum geophilum forms sclerotia and ectomycorrhizas with host plants in forest soils. We demonstrated the differences in genetic diversity of C. geophilum between cultured isolates from sclerotia and those from ectomycorrhizal roots in the same 73 soil samples based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene sequences and newly developed microsatellite markers. Based on GAPDH sequences, 759 cultured isolates (553 from sclerotia and 206 from ectomycorrhizas) were classified into 107 "genotypes" with sequence variation of up to 8.6%. The total number of GAPDH genotypes per soil sample ranged from 1 to 9, but genotypes that were shared between sclerotia and ectomycorrhizas were uncommon (0-3 per soil sample). More than 50% of GAPDH genotypes were unique to one source in most soil samples. Unique GAPDH genotypes were detected from either scleotia or ectomycorrhizal roots in most of the soil samples. Multilocus analysis using nine microsatellite markers provided additional resolution to differentiate fungal individuals and supported the results of GAPDH genotyping. The results indicated that sampling both sclerotia and ectomycorrhizal roots maximizes the detection of diversity at the soil core scale. On the other hand, when all isolates were viewed together, 82 GAPDH genotypes were unique to sclerotia whereas only 6 GAPDH genotypes were unique to ectomycorrhizas. Rarefaction analysis indicated that GAPDH genotypic diversity is significantly higher in sclerotia than ectomycorrhizal roots and the diversity within sclerotia is nearly the same as that of both sclerotia and ectomycorrhizas together. These findings suggest that sampling sclerotia alone is likely to detect the majority of GAPDH genotypes in Cenococcum at the regional scale. When deciding whether to sample sclerotia, ectomycorrhizas, or both types of tissues from Cenococcum, it is critical to consider the spatial scale and also the main questions and hypotheses of the study.

Significant diversity and potential problems associated with inferring population structure within the Cenococcum geophilum species complex.

Cenococcum geophilum is perhaps the most widely distributed and most recognized ectomycorrhizal fungus with a host range of more than 200 tree species from 40 genera of both angiosperms and gymnosperms. We conducted a phylogenetic analysis on a large collection of isolates (n=74) from North America and Europe based on glyceraldehyde 3-phosphate dehydrogenase (gpd). A subset of isolates (n=22) also was analyzed with the more conservative LSU-rDNA locus. Significant nucleotide diversity was detected (approximately 20%) in the gpd region and the LSU-rDNA analysis supported that the C. geophilum isolates studied were monophyletic but distinct from two isolates, Am5-1 and N2-10, which previously were used in population genetic studies of this species. These results suggest that Am5-1 and N2-10 are likely two undescribed species or even genera. Our results suggest that C. geophilum sensu lato is a species complex and support previous molecular, physiological and morphological studies that have shown significant diversity in C. geophilum. This study also revealed that caution is advised when conducting population genetic studies in C. geophilum due to the possibility of pooling unrelated isolates. This potential problem also has implications for other fungal taxa because cryptic species routinely have been found in recent years based on molecular data.

Discovery and Characterization of Waitea circinata var. circinata Affecting Annual Bluegrass from the Western United States.

Waitea circinata var. circinata was identified as the causal agent of a new disease of annual bluegrass (Poa annua) in the United States. This pathogen is also known to cause brown ring patch on creeping bentgrass (Agrostis stolonifera) in Japan, but it had not been reported on any turf species outside of Japan. Symptoms on annual bluegrass caused by this fungus included regular to irregular yellow rings several centimeters to 1 m in diameter, typically at maximum daytime temperatures of 15 to 35°C. A total of 26 isolates were collected from diseased annual bluegrass. Twenty-two of these isolates were multinucleate, grew optimally at 25 to 30°C, and in culture formed irregular sclerotia approximately 2 to 5 mm in size that were white to orange and remained orange or turned brown to dark brown over a 28-day period. The remaining four isolates were characterized as being W. circinata var. zeae (Rhizoctonia zeae), which is a known pathogen of annual bluegrass in the United States. Sequence analysis of the internal transcribed spacer (ITS) regions of rDNA on a subset of isolates confirmed the identifications of W. circinata var. circinata (n = 8) and W. circinata var. zeae (n = 1) based on deposited sequences in GenBank. The identity of the remaining 14 isolates suspected to be W. circinata var. circinata was confirmed by HapII digestion of the amplified rDNA ITS region. Pathogenicity of four W. circinata var. circinata isolates was confirmed on both annual bluegrass and creeping bentgrass. This study is the first morphological and molecular confirmation of the presence of W. circinata var. circinata as a pathogen of turfgrass in the United States.