Variation Among Phytophthora cinnamomi Isolates from Oak Forest Soils in the Eastern United States.

Phytophthora cinnamomi isolates from geographically diverse oak forest soils in the Mid-Atlantic regions were studied to determine the extent of genotypic, phenotypic, and pathogenic variation. Four microsatellite loci were targeted for genetic analysis. Phenotypic characteristics measured included sexual and asexual spore dimensions and colony growth rate and morphology. Red oak (Quercus rubra) logs were inoculated with selected isolates to determine relative pathogenicity. Microsatellite analysis showed that the genetic variability of P. cinnamomi isolates was low, with two predominant microsatellite fingerprint groups (MFG). Isolates in MFG1 (48% of the total isolates examined) were characterized by DNA fragment lengths of 120 and 122 bp at locus d39, 169 and 170 bp at locus e16, and 254 and 255 bp at locus g13. MFG2 isolates were characterized by marker sizes of 122 and 124 bp at locus d39, 161 and 163 bp at locus e16, and 247 and 248 bp at locus g13. Asexual and sexual spore dimensions varied greatly among isolates but were similar to previously published descriptions. Phenotypic differences were most pronounced when data were grouped by MFG; the most significant were colony morphology and growth rate. Neither characteristic was a reliable predictor of isolate genotype. Differences in growth rates of MFGs were observed, with MFG1 being less tolerant at higher incubation temperatures. No variation in pathogenicity was observed on red oak logs. The low level of phenotypic and genotypic variation of P. cinnamomi suggest that other factors such as climate might play a more important role in its northern distribution and the diseases it causes.

Phytophthora spp. Associated with Forest Soils in Eastern and North-Central U.S. Oak Ecosystems.

A survey of soils associated with oak species was conducted in 2003 and 2004 in Indiana, Illinois, Maryland, Michigan, Minnesota, Pennsylvania, Ohio, West Virginia, and Wisconsin to investigate the occurrence of Phytophthora spp. Soils taken from around the base of healthy and declining oak trees were flooded with H2O and Quercus robur leaflets were used as bait for Phytophthora spp. From 829 soil samples collected near trees, 21% were positive for Phytophthora spp., with 55% of the 125 sites surveyed yielding a Phytophthora sp. Phytophthora cinnamomi was the most frequently isolated species, representing 69.4% of the Phytophthora-infested sites surveyed. Other species, in decreasing order of isolation frequency were Phytophthora sp. 2, P. citricola, P. europaea, P. cambivora, P. quercina-like isolates, and Phytophthora sp. 1. No significant association was found between the presence of Phytophthora organisms and site characteristics such as latitude, elevation, soil pH, or the crown condition of the trees. However, in P. cinnamomi-infested sites, a significant association was found with the deteriorating crown status of Q. alba and the presence of P. cinnamomi. The absence of P. cinnamomi above the 40°N latitude range also was noteworthy.

First Report of Phytophthora europaea in Oak Forests in the Eastern and North-Central United States.

In 2003 and 2004, soils in oak forest ecosystems in nine central and eastern states of the United States were surveyed for Phytophthora spp. Soil samples were collected around healthy and symptomatic trees. Symptoms included dieback of branches, gaps in lateral branch systems, yellowing of foliage, wilting and clustering of leaves, and the presence of epicormic shoots. Soil subsamples were collected in each of the four cardinal directions and at a distance of 1 to 2 m from the base of a tree. The four subsamples were bulked to produce a sample of approximately 2,000 ml. In the laboratory, each sample was mixed thoroughly and a single 250-g subsample was flooded with 500 ml of distilled water and baited with Quercus robur leaflets for 3 to 5 days at 17 to 20°C. Discolored leaflets were examined microscopically (×200) and those with sporangia typical of Phytophthora spp. were plated on PARPNH selective medium (1). Phytophthora europaea was recovered from soil samples collected from Q. alba in West Virginia, Q. rubra in Minnesota, West Virginia, and Wisconsin, Q. phellos in Ohio, and Q. velutina in Pennsylvania. Cultures were identified as P. europaea by their morphological, physiological, and molecular characteristics (4). Average dimensions of nine isolates were determined. Oogonia were 40 ± 3.9 µm in diameter and often had few bullet protuberances and tapered bases; oospores mostly filled the oogonia and averaged 36 ± 3.7 µm; sporangia dimensions averaged 42 ± 6.1 × 30 ± 4.1 µm with a length/width ratio of 1:4. Isolates produced larger oogonia and oospores but had similar sporangia length/width ratios comparable to the species description (4). Growth optimum (5.8 to 6.9 mm day-1) on V8 juice agar (V8A) occurred at 25°C. On potato dextrose agar, colonies produced dense, felt-like mycelia, often with a central mound of aerial hyphae. DNA also was extracted from eight representative isolates and the internal transcribed spacer (ITS) region of rDNA from each isolate was amplified and sequenced. ITS sequences were identical to those of P. europaea in the NCBI GenBank database (Accession No. DQ313222). Pathogenicity of six isolates (one from each site) was confirmed by wounding stems of 2-year-old Q. alba, Q. rubra, and Q. velutina seedlings and inoculating wounds with V8A plugs (6 mm) containing mycelia; V8A plugs without mycelia were used for controls. Two months after inoculation, P. europaea was reisolated on PARPNH medium from advancing lesions on all inoculated seedlings but was not isolated from control plants. Mean lesion lengths on seedlings inoculated with P. europaea were significantly greater (P < 0.05) than those on control plants; lesions averaged 0.46 cm on Q. alba, 1.38 cm on Q. rubra, and 1.01 cm on Q. velutina. Previously, P. europaea only was reported from oak trees and soil in forests of Austria, France, and Germany (1-4). These findings extend the current distribution of P. europaea and raise questions about its origin and role in the health of oak forests in eastern and north-central United States. Q. alba, Q. phellos, Q. rubra, and Q. velutina are new host associations for P. europaea. References: (1) Y. Balci and E. Halmschlager. For. Pathol. 33:157, 2003. (2) E. Hansen and C. Delatour. Ann. Sci. For. 56:539, 1999. (3) G. Hartmann and R. Blank. Forst Holz. 57:539, 2002. (4) T. Jung et al. Mycol. Res. 106:397, 2002.

Heterokaryon formation and parasexual recombination between vegetatively incompatible lineages in a population of the chestnut blight fungus, Cryphonectria parasitica.

Heterokaryosis was recently reported in the chestnut blight fungus, Cryphonectria parasitica, in which individuals contain nuclei that are isogenic except at the mating-type locus (MAT). MAT heterokaryons were found in several natural populations, including a putatively clonal population in West Salem, Wisconsin, providing an opportunity to address the question of how heterokaryons arise. We represented relationships among RFLP fingerprint haplotypes as networks in which loop formation is considered evidence of recombination. From 1990 to 1995, this population was clonal, as indicated by a simple haplotype network without loops, and the correlation of vegetative compatibility (vc) types and mating types with haplotype lineages. By 1999, we observed loops in the haplotype network involving isolates of two vc types (WS-2 and WS-3). Isolates with haplotypes in the loops were either MAT heterokaryons, carried the opposite mating type from other isolates of the same vc type, and/or had two alleles at two or more codominant SCAR (sequence-characterized amplified region) loci. Segregation of markers and recombination were evident among single-spore isolates from one heterokaryon; these single-spore isolates had novel fingerprint haplotypes, also within the loops. In contrast, vc type WS-1, which comprises 85% of the population, was represented by a simple network with no loops, indicating a clonal lineage varying only by mutation. Almost all isolates of WS-1 had the same mating type; the exceptions were five isolates that were MAT heterokaryons. These results are consistent with the hypothesis that heterokaryons formed between vegetatively incompatible individuals, and recombination occurred by a parasexual process.

Situational factors and attitudes toward voluntary euthanasia.

This study examines how attitudes toward voluntary euthanasia vary across categories of four situational factors: (1) type of assistance; (2) type of assistant; (3) type of illness; (4) age of the patient. The data, based on a random sample of 514 adult residents of Ohio, indicate that more active assistance is favored over less active assistance, and that voluntary euthanasia for cancer patients receives more support than does voluntary euthanasia for victims of Alzheimer's disease. The findings also suggest that, for persons who do not strongly adhere to the belief that life belongs to God, physician-assistants are preferred over nonphysician-assistants, and that voluntary euthanasia for children receives less support than does voluntary euthanasia for adults. For those who strongly adhere to the belief that life belongs to God. however, these two situational factors have less of an influence. At the microlevel, the findings appear to reflect a concern about safeguarding patients' autonomy in the decision-making process, and a concern about authoritative control of the procedure of voluntary euthanasia. At the macrolevel, the findings suggest that the influence of cultural ideology on a social movement's direction is not independent, but is instead moderated by the internal dynamics and struggles of movement organizations. This is indicated by the finding that movement leaders' attitudes toward active assistance on the part of the physician are more consistent with the attitudes of physicians than with the attitudes of the public.