W. L. MacDonald

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.

The Origin of Ceratocystis fagacearum, the Oak Wilt Fungus*

The oak wilt pathogen, Ceratocystis fagacearum, may be another example of a damaging, exotic species in forest ecosystems in the United States. Though C. fagacearum has received much research attention, the origin of the fungus is unknown. The pathogen may have been endemic at a low incidence until increased disturbances, changes in land use, and forest management created conditions favorable for disease epidemics. The host genus Quercus contains some relatively resistant species native to the United States, further supporting the hypothesis that the pathogen is native in origin. However, there are also many common, highly susceptible Quercus species--a characteristic typical of introduced pathogens. Most convincingly, studies have shown that the known populations of C. fagacearum have experienced a severe genetic bottleneck that can only be explained by a single introduction. The weight of evidence indicates that C. fagacearum is an introduced pathogen, with possible origins in Central or South America, or Mexico.

Phytophthora quercetorum sp. nov., a novel species isolated from eastern and north-central USA oak forest soils.

Isolates belonging to an undescribed Phytophthora species were frequently recovered during an oak forest soil survey of Phytophthora species in eastern and north-central USA in 2004. The species was isolated using an oak leaf baiting method from rhizosphere soil samples collected from Quercus rubra, Q. macrocarpa, and Q. phellos. This species is formally described as P. quercetorum. It is homothallic and has aplerotic oogonia and paragynous antheridia. It produces papillate sporangia (occasionally bipapillate) of ovoid-elongated shapes. Its temperature optimum for growth is ca 22.5 degrees C with the upper limit of ca 32.5 degrees C. P. quercetorum differs from the morphologically related P. quercina in producing distinct submerged colony-patterns, different growth-temperature requirements, and oogonial shapes and sizes. Phylogenetic analyses using seven nuclear loci supported P. quercetorum as a novel species within clade 4, closely related to P. arecae, P. palmivora, P. megakarya, and P. quercina.

Challenges and Successes in Managing Oak Wilt in the United States

Oak wilt, caused by the fungus Ceratocystis fagacearum (Bretz) J. Hunt, is an important disease of oaks (Quercus spp.) in the eastern United States. It has been particularly destructive in the North Central states and Texas. Oak wilt is one of several significant oak diseases that threaten oak health worldwide. The significant gains made in our knowledge of the biology and epidemiology of this vascular wilt disease during the past six decades has led to development of various management strategies.

Multilocus PCR Assays Elucidate Vegetative Incompatibility Gene Profiles of Cryphonectria parasitica in the United States

ABSTRACT Chestnut blight is a devastating disease of Castanea spp. Mycoviruses that reduce virulence (hypovirulence) of the causative agent, Cryphonectria parasitica, can be used to manage chestnut blight. However, vegetative incompatibility (vic) barriers that restrict anastomosis-mediated virus transmission hamper hypovirulence efficacy. In order to effectively determine the vegetative incompatibility genetic structure of C. parasitica field populations, we have designed PCR primer sets that selectively amplify and distinguish alleles for each of the six known diallelic C. parasitica vic genetic loci. PCR assay results were validated using a panel of 64 European tester strains with genetically determined vic genotypes. Analysis of 116 C. parasitica isolates collected from five locations in the eastern United States revealed 39 unique vic genotypes and generally good agreement between PCR and tester strain coculturing assays in terms of vic diversity and genotyping. However, incongruences were observed for isolates from multiple locations and suggested that the coculturing assay can overestimate diversity at the six known vic loci. The availability of molecular tools for rapid and precise vic genotyping significantly improves the ability to predict and evaluate the efficacy of hypovirulence and related management strategies.

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.

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.

Natural regeneration in oak wilt centers

Abstract Openings, 0.2 ha and less, and 3–12 years old, caused by the oak wilt fungus Ceratocystis fagacearum had abundant oak reproduction, averaging 4700 stems per ha. Species of other woody reproduction made up over 27 000 stems per ha. Seedling sprouts comprised 54%, seedlings 30%, and stumps sprouts the remainder of the reproduction. No infections were found except a few on stump sprouts of diseased oak stumps. Oakhickory reproduction was abundant on all site qualities, but especially so on the poorer sites with over 8000 stems per ha. Sugar maple reproduction was much more abundant on high quality sites, also with over 8000 stems per ha. The reproduction in the oak wilt openings was judged to be similar to that of selection cuttings in disease-free but otherwise similar stands, and should respond in like manner.