Use of Nested and Real-Time PCR for the Detection of Ceratocystis fagacearum in the Sapwood of Diseased Oak Species in Minnesota

Oak wilt caused by Ceratocystis fagacearum is a significant disease of Quercus spp. in the eastern United States. Early and accurate detection of the pathogen is particularly important when disease control is planned. Nested and real-time polymerase chain reaction (PCR) methods utilizing fungal DNA extracted from sapwood drill shavings of red, bur, and white oak at different stages of disease development were compared with culture-based detection from sapwood. The pathogen was detected in all (n = 3) actively wilting branches of each of nine red oak trees using all three methods. The lowest detection rate (33% of assayed branches; 6 of 8 trees) for actively wilting branches was found for white oak using isolation while nested PCR had a branch detection rate of 100% (8 of 8 trees) and real-time PCR of 87% (8 of 8 trees) for the same samples. For both bur and white oak, the pathogen was not detected by isolation in branches over 1 year after their death but was detected using both PCR methods. Only the PCR assays detected the fungus in sapwood samples underlying remnants of sporulation mats (n = 21; 90%, nested and 62%, real-time) on red oak. These PCR methods offer several significant improvements for laboratory-based detection methods of C. fagacearum.

Propiconazole Distribution and Effects on Ceratocystis fagacearum Survival in Roots of Treated Red Oaks

We investigated the interaction between the oak wilt pathogen (Ceratocystis fagacearum) and propiconazole in lower stems and roots of Quercus rubra to better understand published reports of fungicide failure after 2 years. Propiconazole was infused into mature oaks in July 2004 and roots were inoculated with pathogen endoconidia 1.0 m from injection sites at ±2 weeks of fungicide treatment. Pathogen presence in wood samples was determined by isolation and fungicide concentrations measured using gas chromatography-mass spectrometry. Propiconazole was detected in the roots (≤1.0 m from injection sites) of all treated trees at 2, 12, and 24 months. Propiconazole was detected in all samples (n = 68) at 2 and 12 months and in 93% of samples (n = 72) at 24 months with concentrations ranging from 815 ppm (12 months in lower stem) to 0.7 ppm (24 months in most distal root segment). Although pathogen isolation incidence was lower in treated than disease control trees at 2 and 12 months, at no time did an infused oak fail to yield the fungus upon isolation. The results document basipetal movement and degradation of propiconazole, as well as the survival of the pathogen, over time in roots and lower stems of infused red oaks.

Spatial and Temporal Distribution of Ceratocystis fagacearum in Roots and Root Grafts of Oak Wilt Affected Red Oaks

The spatial and temporal distributions of the oak wilt fungus, Ceratocystis fagacearum, in the roots of symptomatic and adjacent asymptomatic northern red oaks (Quercus rubra) were investigated. Root systems of 12 pairs of such trees were exposed to 1 m (3.3 ft) depth using an air excavation tool. In only one case was the fungus found in more than two cardinal directions of symptomatic trees based on isolation attempts from 192 root segments excised from 1.2 m (3.5 ft) lengths of primary roots extending in four cardinal directions from the root collar. Many (162) self grafts occurred in all oaks. Few (13) inter-tree grafts were found in less than one-third of the tree pairs. The fungus was isolated from the roots involved in two of thirteen inter-tree grafts and 14 of 62 self grafts assayed. The sporadic and unpredictable distribution of the fungus in roots of wilting red oaks supports current control approaches that assume the fungus is present in roots extending out in all directions once crown symptoms are evident. Additionally, self grafts may serve as a conduit for circumferential movement of the fungus around the main stem, but outside the root collar, through the interconnected roots.

First Report of the Oak Wilt Fungus, Ceratocystis fagacearum, in New York State

Ceratocystis fagacearum (T.W. Bretz) J. Hunt is considered to be the most destructive vascular pathogen of oaks (Fagaceae: Quercus spp.) in the eastern, north-central, and south-central United States. (1,2,4). All red oak species (subgenus Quercus: section Lobatae) are highly susceptible to C. fagacearum, and infected trees typically die within 3 months of first symptom expression. However, members of the white oak group (subgenus Quercus: section Quercus) are moderately to highly resistant to C. fagacearum and rarely die from oak wilt (1,3). In early August of 2008, we received branch samples taken from wilting red oaks (Q. rubra L.) growing in a residential neighborhood in Scotia, NY (Schenectady County). The endoconidial state of the oak wilt fungus, Thielaviopsis quercina (B.W. Henry) A.E. Paulin, T.C. Harr. & McNew, was consistently isolated from the xylem in the branches. The cultures were identified based on hyphal and conidial morphology on acidified potato dextrose agar (aPDA) as well as sequences of the internal transcribed spacer (ITS) (GenBank Accession No. FJ347031) and large subunit (LSU) (GenBank Accession No. FJ347030) regions of nuclear ribosomal DNA (rDNA). The nucleotide identities for the ITS and LSU sequences were a precise match, 99 and 100%, to rDNA sequences (GenBank Accession Nos. AF043598 and AF222483, respectively) of other isolates of C. fagacearum. According to the homeowners at the site, 12 red oaks have died during the last 3 years, and each tree died within one growing season after oak wilt-like symptoms were noted. In a brief survey of nearby properties in late August of 2008, we found 12 additional trees that were either expressing crown symptoms of oak wilt (e.g., premature leaf casting, bronzing of leaf margins, and water-soaked leaves) or were standing dead and within close proximity (5 to 10 m) to symptomatic trees. Branch samples from four of the symptomatic trees revealed limited (spotted) or no vascular discoloration; however, C. fagacearum was isolated from each suspect tree on aPDA. Remnants of gray mycelial mats and associated pressure cushions were observed beneath the bark of one standing dead oak. The sweet fruit-like odor characteristic of the oak wilt fungus was immediately evident once the bark overlying the mats was removed. Prior to this discovery, the Susquehanna River in north-central Pennsylvania was considered to be the northeastern limit for oak wilt occurrence in the United States (2,4). To our knowledge, this is the first report of the fungus from New York and expands the known range of C. fagacearum to the northeast by at least 300 km, supporting the hypothesis that the range of this fungus continues to expand via animal vectors and/or human activities (2). An isolate of C. fagacearum from New York has been deposited at the Centraalbureau voor Schimmelcultures (CBS 123913). References: (1) D. N. Appel. Ann. Rev. Phytopathol. 33:103, 1995. (2) J. Juzwik et al. Ann. Rev. Phytopathol. 46:13, 2008. (3) W. L. MacDonald et al. European oaks-susceptible to oak wilt? Page 131 in: Shade Tree Wilt Diseases. C. L. Ash, ed. The American Phytopathological Society, St. Paul, MN, 2001. (4) USDA Forest Service. Oak Wilt Distribution. Northeast Area, State and Private Forestry, St. Paul, MN. Online publication, 2005.

Three Colopterus Beetle Species Carry the Oak Wilt Fungus to Fresh Wounds on Red Oak in Missouri

Beetles in the family Nitidulidae can transmit the oak wilt fungus, Ceratocystis fagacearum, to fresh wounds on healthy oak trees, leading to infection and disease development. Historically, nitidulid beetles have not been considered important vectors of the pathogen in Missouri. Studies were conducted in the spring of 2005 and 2006 to determine frequencies of nitidulid beetle species contaminated with C. fagacearum visiting fresh wounds on red oak trees in central Missouri. Colopterus truncatus, C. niger, and C. semitectus were the most abundant species collected from fresh wounds and the only species found to be contaminated with Ceratocystis fagacearum. Of 230 beetles assayed for C. fagacearum, 23 yielded the fungus. Contamination frequencies were higher for beetles collected in April than May; no beetles collected in June were contaminated. We hypothesize that Colopterus truncatus, C. niger, and C. semitectus are principal nitidulid beetle vector species in Missouri during spring. The risk for pathogen transmission by these beetles appears to be greatest in April and least in June.