Population genetic structure of Phytophthora cinnamomi associated with avocado in California and the discovery of a potentially recent introduction of a new clonal lineage.

Phytophthora root rot (PRR) of avocado (Persea americana), caused by Phytophthora cinnamomi, is the most serious disease of avocado worldwide. Previous studies have determined that this pathogen exhibits a primarily clonal reproductive mode but no population level studies have been conducted in the avocado-growing regions of California. Therefore, we used amplified fragment length polymorphism based on 22 polymorphic loci and mating type to investigate pathogen diversity from 138 isolates collected in 2009 to 2010 from 15 groves from the Northern and Southern avocado-growing regions. Additional isolates collected from avocado from 1966 to 2007 as well as isolates from other countries and hosts were also used for comparative purposes. Two distinct clades of A2 mating-type isolates from avocado were found based on neighbor joining analysis; one clade contained both newer and older collections from Northern and Southern California, whereas the other clade only contained isolates collected in 2009 and 2010 from Southern California. A third clade was also found that only contained A1 isolates from various hosts. Within the California population, a total of 16 genotypes were found with only one to four genotypes identified from any one location. The results indicate significant population structure in the California avocado P. cinnamomi population, low genotypic diversity consistent with asexual reproduction, potential evidence for the movement of clonal genotypes between the two growing regions, and a potential introduction of a new clonal lineage into Southern California.

First Report of Basal Leaf Blight of Kikuyugrass Caused by Waitea circinata var. prodigus in Southern California.

Kikuyugrass (Pennisetum clandestinum) is a C4 grass and invasive weed adopted for use as a primary turf species in some golf course fairways and roughs in southern California. In September 2008, a new Rhizoctonia-like fungus was isolated from a diseased kikuyugrass sample received from a golf course fairway in Oceanside, CA. The kikuyugrass was from a mature stand (>20 years old) that was maintained at a height of approximately 1.25 cm. Symptoms on kikyuygrass developed initially as irregular, blighted, chlorotic patches, several centimeters to one meter, which occurred during a period of warm, humid weather (27 to 29°C maximum daytime temperature, 75 to 85% average relative humidity) on a small part of one fairway. Affected areas became brown and necrotic as the disease progressed. Leaf chlorosis and stem rot were observed on affected plants. The organism was isolated by placing symptomatic leaves on acidified one-quarter-strength potato dextrose agar (PDA) (600 µl of 85% lactic acid per liter of medium) in a petri dish (1). A colony of a Rhizoctonia-like fungus with yellow aerial hyphae, multinucleate hyphal cells, and irregularly shaped, golden brown sclerotia (4 to 7 mm) developed within 30 days at 28°C. The rDNA internal transcribed spacer (ITS) sequence was obtained (GenBank Accession No. HQ850254) using PCR amplification with primers ITS1F and ITS4 (1,2), and a BLAST search showed 100% similarity with Waitea circinata var. prodigus (GenBank Accession No. HM597145), which had recently been described as the cause of basal leaf blight of seashore paspalum (Paspalum vaginatum), another C4 grass (3), in Florida. Colony morphology and other physical characteristics were similar but not completely identical to those from Florida, reflecting the reported morphological variation inherent in the pathogen (3). Koch's postulates were performed by growing this isolate on PDA in a petri dish for 7 days, homogenizing the culture with 100 ml of sterilized water, filtering the suspension through two layers of cheesecloth, and pipetting 10 ml of the mycelial suspension onto the foliage and stems of 4-week-old AZ-1 kikuyugrass plants grown in UC-soilless-mix in 7.5-cm-diameter pots (4). Control plants were treated with a homogenized and filtered dish of PDA only. There were three replicate pots for inoculated and noninoculated treatments and the experiment was repeated independently three times. All of the pots were incubated in a moist chamber with a 12-h light period at 28°C. Yellow lesions were observed on leaves and stems of inoculated plants 4 days postinoculation and necrosis developed 8 days later in all experiments. The same organism was isolated from symptomatic plants. The control plants did not exhibit any symptoms. To our knowledge, this is the first report of basal leaf blight caused by W. circinata var. prodigus on kikuyugrass in California and the first report of this pathogen affecting turfgrass in the western United States. References: (1) C. M. Chen et al. Plant Dis. 93:906, 2009 (2) K. de la Cerda et al. Plant Dis. 91:791, 2007. (3) S. J. Kammerer et al. Plant Dis. 95:515, 2011. (4) T. Toda et al. J. Gen. Plant Pathol. 73:379, 2007.

Multigene analysis suggests ecological speciation in the fungal pathogen Claviceps purpurea.

Claviceps purpurea is an important pathogen of grasses and source of novel chemical compounds. Three groups within this species (G1, G2 and G3) have been recognized based on habitat association, sclerotia and conidia morphology, as well as alkaloid production. These groups have further been supported by Random Amplification of Polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers, suggesting this species may be more accurately described as a species complex. However, all divergent ecotypes can coexist in sympatric populations with no obvious physical barriers to prevent gene flow. In this study, we used both phylogenetic and population genetic analyses to test for speciation within C. purpurea using DNA sequences from ITS, a RAS-like locus, and a portion of beta-tubulin. The G1 types are significantly divergent from the G2/G3 types based on each of the three loci and the combined dataset, whereas the G2/G3 types are more integrated with one another. Although the G2 and G3 lineages have not diverged as much as the G1 lineage based on DNA sequence data, the use of three DNA loci does reliably separate the G2 and G3 lineages. However, the population genetic analyses strongly suggest little to no gene flow occurring between the different ecotypes, and we argue that this process is driven by adaptations to ecological habitats; G1 isolates are associated with terrestrial grasses, G2 isolates are found in wet and shady environments, and G3 isolates are found in salt marsh habitats.

First Report of Waitea circinata var. circinata Causing Brown Ring Patch on Poa trivialis in California.

Rough bluegrass (Poa trivialis L.) is a C3 (cool-season) turfgrass used on golf course putting greens. It is often used to overseed C4 (warm-season) turf for fall through early-summer use. In March 2007, at maximium daytime air temperatures of approximately 30 to 35°C, irregular, thin, yellow rings approximately 10 to 20 cm in diameter were reported on P. trivialis in putting greens from two golf courses in the Coachella Valley of southern California. Affected plants had a blight of the leaves and stems and a rot of the crown, with initial symptoms being a yellowing of the tissue followed by the development of a dark, water-soaked appearance of the whole plant. Plants turned reddish brown as the water-soaked tissue dessicated. A Rhizoctonia-like fungus was found to be colonizing the leaves, stems, and upper roots and thatch. Three isolates were obtained from the diseased turf samples. All were identified as Waitea circinata var. circinata based on colony morphology (2) and rDNA internal transcribed spacer (ITS) region sequences (1). The sequences of the three isolates were more than 99% similar to those of W. circinata var. circinata deposited in the NCBI database (1,2). To confirm pathogenicity, each isolate was inoculated onto 4-week-old P. trivialis (2 mg of seed per cm2) grown in 10-cm-diameter pots containing steam-sterilized UC-mix at 28°C. Five 8-mm-diameter plugs of each isolate were taken from 7-day-old cultures grown on potato dextrose agar (PDA) and placed in direct contact with the bases of the stems. The pots were then incubated in a moist chamber at 28°C. Five noncolonized PDA plugs were used as a negative control. Three pots were used for each of the three isolates and the check treatment. For all isolates, chlorosis of leaf and stems were observed 5 days after inoculation and the tissue appeared water soaked after 7 days with the production of abundant aerial mycelia being observed. All plants, except the negative controls, died after 10 days. W. circinata var. circinata was reisolated from symptomatic tissue from the inoculated plants. W. circinata var. circinata was previously reported as the causal agent of brown ring patch on creeping bentgrass (Agrostis palustris) in Japan (2) and as a pathogen of annual bluegrass (P. annua) in the United States (1). To our knowledge, this is the first report of W. circinata var. circinata infecting P. trivialis in California. References: (1) K. de la Cerda et al. Plant Dis. 91:791, 2007. (2) T. Toda et al. Plant Dis. 89:536, 2005.

Revising the standard wisdom of C. elegans natural history: ecology of longevity.

Here, we consider that most of the research concerning Caenorhabditis elegans has been laboratory focused and that only limited research has directly considered the worm's biology relative to its natural history in the wild. We describe that, although the worm has traditionally been considered a soil nematode, we could not find it in soil but frequently recovered it from snails. Finally, we discuss how a better understanding of the natural history of C. elegans may enhance its usefulness as a model organism for studying aging and other phenomena.

Phylogenetic divergence in a local population of the ectomycorrhizal fungus Cenococcum geophilum.

Cenococcum geophilum is a widely distributed mycorrhizal species associated with diverse gymnosperm and angiosperm hosts. In previous studies, a significant amount of genetic and genotypic diversity has been detected in this species, despite the fact that C. geophilum is not thought to reproduce by meiotic or mitotic spores. We conducted a phylogenetic analysis of 103 C. geophilum isolates from a California oak woodland and seven non-California isolates using a glyceraldehyde 3-phosphate dehydrogenase gene. In addition, a subset of isolates was analyzed using sequences from ITS-rDNA, a Group I intron located in the 3' end of the SSU-rDNA and a portion of the mitochondrial SSU-rDNA. Phylogenetically distinct lineages, or cryptic species, of C. geophilum were detected at the scale of a single soil sample within our field site. As much genetic diversity was found within a soil sample as was found for isolates collected across the USA. Our results help explain the large amount of physiological, phenotypic, and genetic differences reported among isolates of C. geophilum from similar as well as diverse geographic regions. The ecological role that these sympatric cryptic species play remains to be determined.

Population Genetic Structure of Tapesia acuformis in Washington State.

ABSTRACT Eyespot of wheat is caused by Tapesia yallundae and T. acuformis. Historically, T. yallundae has been considered the more important causal agent of the disease in Washington state and consists of a large homogeneous population with a genetic structure consistent with both sexual and asexual reproduction. T. acuformis has increased significantly in Washington in the past 10 years and apothecia were found recently under natural field conditions, indicating that T. acuformis may have a more important role in eyespot of wheat than previously was thought. To determine the genetic structure of T. acuformis in Washington, 141 single conidial isolates were sampled from four subpopulations in the eastern wheat-growing region of the state. Isolates were scored for mating type and six amplified fragment length polymorphism markers. All markers segregated in a 1:1 ratio and were determined to be unlinked based on genetic analysis of 24 progeny from an in vitro cross. No significant differences in allele frequencies (0.127 < P < 0.809) were found among individual loci across the four subpopulations and over all loci based on contingency table analysis of the log-likelihood ratio statistic G(2). Likewise, no overall differences between subpopulations were detected using the population differentiation statistic theta (theta = -0.004, P = 0.537). Random mating could not be rejected within each subpopulation or for the combined data using clone-corrected data sets based on (i) 1:1 ratio of mating-type, (ii) multilocus gametic disequilibrium analyses (index of association), (iii) phylogenetic analyses (parsimony tree length permutation test), and (iv) genotypic diversity analyses. T. acuformis has a genetic structure similar to that of sympatric populations of T. yallundae in Washington, with both sexual and asexual reproduction contributing to the structuring of this species.

Multilocus population structure of Tapesia yallundae in Washington State.

Population genetic structure of the fungal wheat pathogen Tapesia yallundae in Washington State was determined using genetically characterized amplified fragment length polymorphic (AFLP) markers and mating-type (MAT1-1 or MAT1-2). Segregation and linkage relationships among 164 AFLP markers and MAT were analysed using 59 progeny derived from an in vitro cross. Alleles at 158 AFLP loci and the mating-type locus segregated in a 1:1 ratio. Ten unlinked markers were chosen to determine genetic and genotypic diversity and to test the hypothesis of random mating and population differentiation among five subpopulations of T. yallundae representative of the geographical distribution of wheat production in eastern Washington. Among 228 isolates collected, overall gene diversity was high (h = 0.425) and a total of 91 unique multilocus genotypes (MLG) were identified, with 32 MLG occurring at least twice. The overall population genetic structure was consistent with random mating based on the segregation of mating-type, index of association (IA), parsimony tree length permutation test (PTLPT) and genotypic diversity analyses. However, clonal genotypes were found within each subpopulation and were also distributed among the five subpopulations. No significant differences in allele frequencies were found among the five subpopulations for all 10 loci based on contingency table analysis (G2) and Wier & Cockerham's population differentiation statistic theta (theta = -0.008, P = 0.722). T. yallundae appears to consist of a large homogeneous population throughout eastern Washington with both sexual and asexual reproduction contributing to the observed population genetic structure despite no report of sexual fruiting bodies of T. yallundae occurring under natural field conditions.

Species and Mating-Type Distribution of Tapesia yallundae and T. acuformis and Occurrence of Apothecia in the U.S. Pacific Northwest.

ABSTRACT Eyespot of wheat is caused by the discomycete fungi Tapesia yallundae and T. acuformis. T. yallundae is considered the most important causal agent of the disease in this region but no apothecia of either species have been found in the U.S. Pacific Northwest (PNW). Two compatible isolates of T. yallundae from the PNW were used to inoculate a field plot in the fall of 1998 and apothecia developed in the spring and fall of 2000 on standing wheat stubble. In the spring of 2000, wheat stubble from eight naturally infected fields was examined for the presence of apothecia of T. yallundae and T. acuformis. Apothecia of T. acuformis were found in two fields but no apothecia of T. yallundae were found. This is the first report of apothecia of the eyespot pathogens occurring in the PNW. Species and mating-type distribution of T. yallundae and T. acuformis in the PNW were determined from 817 isolates collected from diseased wheat over 3 years at spatial scales ranging from within fields to across states. In all, 460 isolates were identified as T. yallundae and 357 isolates were identified as T. acuformis with MAT1-1/MAT1-2 ratios not significantly different from 1:1 based on chi(2) tests at most scales tested. The apparent increase in frequency of T. acuformis from previous surveys may indicate a shift in the predominant species causing eyespot. The occurrence of apothecia under field conditions, along with the widespread distribution of mating types of both species, suggests that sexual reproduction may be occurring in both species.

Infection of Winter Wheat by a beta-Glucuronidase-Transformed Isolate of Cephalosporium gramineum.

ABSTRACT Field-grown winter wheat was inoculated with a beta-glucuronidase-transformed isolate of Cephalosporium gramineum in two field seasons to elucidate the mode of infection in resistant and susceptible cultivars. Colonization of viable root epidermis and cortical cells occurred as soon as 15 days postinoculation and the pathogen was found in the vascular tissues by 20 days postinoculation, well before freezing soil temperatures occurred. Penetration occurred directly through the root epidermis and through wounds adjacent to emerging secondary roots. The pathogen also penetrated through root cap cells and colonized meristematic tissues near root tips to gain access to the vascular system. Lower stem base colonization was observed where the pathogen penetrated directly through the epidermis, wounds, or senescent tissues. Appressorium-like structures, which appeared to aid penetration of cell walls, were often found within cells of both roots and stems after initial colonization. The mechanisms of resistance were not apparent, but less colonization occurred in resistant than in susceptible cultivars.