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.

Multiple Ceratocystis smalleyi infections associated with reduced stem water transport in bitternut hickory.

Hundreds of cankers caused by Ceratocystis smalleyi are associated with hickory bark beetle-attacked bitternut hickory exhibiting rapid crown decline in the north-central and northeastern United States. Discolored sapwood colonized by the fungus commonly underlies the cankers. Field studies were conducted to test the hypothesis that C. smalleyi infections cause vascular system dysfunction in infected trees. Fifty C. smalleyi inoculations made at 1.8 to 3.8 m in height on stems of healthy bitternut hickory trees (13 to 28 cm in diameter at 1.4 m in height) resulted in extensive canker formation and sapwood discoloration 12 to 14 months after treatment compared with water-inoculated and noninoculated controls. Sap flow velocity (midday) was significantly lower in the infected trees compared with that in the controls. Sap flow velocity also was inversely correlated with the proportion of bark area with cankered tissues and with tylose abundance in the youngest two growth rings. Tylose formation in current-year vessels associated with C. smalleyi infections is likely responsible for much of the water transport disruption. It is hypothesized that multiple stem infections of C. smalleyi and the resulting xylem dysfunction contribute to crown wilt development in bitternut hickory exhibiting rapid crown decline.

Fusarium Canker of Bitternut Hickory Caused by Fusarium solani in the North-Central and Northeastern United States.

Multiple annual cankers were observed on the upper main stems of bitternut hickory (Carya cordiformis) exhibiting top dieback in Indiana, Iowa, Minnesota, New York, Ohio, and Wisconsin during a 2006 to 2008 survey of declining hickory. The top-killed trees had normal-sized, green leaves below and the cankers were oval, sunken, and bounded by heavy callus that seemed to arrest further canker expansion. Fusarium solani was consistently isolated from the margins of inner bark lesions or discolored sapwood of the cankers. When cultured on potato dextrose agar, the isolates grew rapidly with abundant aerial mycelium. On carnation leaf agar, thick-walled macroconidia with 4 to 5 septa were produced in cream, blue-green, or blue sporodochia. Macroconidia were generally cylindrical with a blunt or rounded apical cell and a rounded or foot-shaped basal cell. Microconidia were oval to kidney shaped with 0 to 1 septa and were produced in false heads on elongate monophialides. Chlamydospores were formed singly or in pairs. These morphological characteristics are consistent with descriptions of F. solani (2). The identities of 42 representative isolates were confirmed by sequencing the translation elongation factor (tef) 1-α gene. BLAST analysis of the sequences from each isolate against the GenBank and FUSARIUM-ID database found 98 to 100% similarities to F. solani isolates (GenBank Accession Nos. DQ246841, DQ247025, DQ247282, and DQ247436 and FUSARIUM-ID isolate FD01041). Two haplotypes (BB and BC) were distinguished based on the tef 1-α gene sequences that differed by 10 bp. Pathogenicity tests were conducted with two isolates of each haplotype on asymptomatic C. cordiformis (12 to 21 cm in diameter) in forest stands. In May 2009 in Wabasha County, MN, 0.1-ml spore suspensions (1 × 104 macroconidia/ml) or sterile water was placed in one of three holes (0.6 cm in diameter) drilled to the cambium of 12 trees. The holes were sealed with moist cotton and moldable putty. A duplicate trial, but with BB and BC isolates from Wisconsin, was initiated in Chippewa County, WI in June 2009. The extent of inner bark necrosis was assessed 13 months after inoculation in both sites. Inoculations with F. solani in Minnesota resulted in inner bark lesions with average lengths of 20 and 30 mm for the BB and BC haplotypes, respectively. In Wisconsin, BB and BC haplotypes caused inner bark lesions with average lengths of 34 and 38 mm, respectively. While sunken or open cankers were found for all the BC isolate inoculations, relatively small and callus-bounded cankers were found for BB isolate inoculations. All control wounds were callus-closed with average wound lengths of 12 and 23 mm in Minnesota and Wisconsin, respectively. The same haplotype of F. solani used for inoculation was recovered from each canker as confirmed by analysis of tef 1-α gene sequences. F. solani was not obtained from control wounds. To our knowledge, this is the first report of a canker caused by F. solani on bitternut hickory (1). The same fungus has been previously reported to cause cankers on stems of other hardwood tree genera in the eastern United States and Canada. We hypothesize that numerous main-stem cankers caused by F. solani lead to top dieback of bitternut hickory. References: (1) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

Decline as a disease category: is it helpful?

Many, but not all, forest pathologists use "decline" to describe forest tree diseases of complex etiology. We contend that this distinction from abiotic or biotic diseases is completely arbitrary, has caused undue confusion, and provides no practical insights for forest managers. All diseases are complex and can be characterized within the conceptual framework of the disease triangle. Why do we use a simple label ("decline") to describe disease situations of complex abiotic and biotic origin when we need to know which damaging agents are present, whether the environment is conducive for disease progression, and host susceptibility over time to understand the origins and management of disease? We propose that forest pathologists discontinue the use of "decline" as a distinct category of disease. Furthermore, we suggest that new diseases should be named based on the affected host, characteristic symptom, and, once known, major determinant. We believe that clearer communication in describing complex diseases is a prerequisite to finding effective management options.

Ceratocystis Canker of Bitternut Hickory Caused by Ceratocystis smalleyi in the North-Central and Northeastern United States.

Between 2006 and 2008, diffuse cankers without surrounding callus and callus-edged sunken cankers were observed on main stems of poletimber (13 to 28 cm in diameter at 1.4 m high) and sawtimber (>28 cm in diameter) bitternut hickory (Carya cordiformis) in Iowa, Indiana, Minnesota, New York, Ohio, and Wisconsin. Reddish, inner bark necrosis and reddish brown sapwood discoloration were associated with the cankers. Entry or exit holes of the hickory bark beetle (Scolytus quadrispinosus) were commonly associated with the cankers. Ceratocystis smalleyi was consistently isolated from the margins of cankers or discolored sapwood of assayed samples. When cultured on 2% malt yeast extract agar (MYEA), the isolates produced perithecia, ascospores, endoconidiophores, and conidia diagnostic of C. smalleyi (1). To confirm identification, the translation elongation factor (tef) 1-α gene and the internally transcribed spacer (ITS) regions were sequenced. DNA for both regions was extracted from mycelia growing on MYEA. The tef sequences (GenBank Accession Nos. GU201529-201539) and the ITS sequences (GenBank Accession Nos. GU190734-190745) were 98 to 100% similar to sequences of C. smalleyi isolates (GenBank Accession Nos. EF070408 and AY9907030-907032). Pathogenicity tests were conducted with local isolates (two per site) on healthy Carya cordiformis (13 to 28 cm in diameter) in forest stands in three states. In May 2007, one fungus-colonized MYEA disk or sterile MYEA agar was placed in one of two holes (0.6 cm in diameter) drilled to the cambium on stems of 10 trees in Allamakee County, IA. Sterile moist cotton and laboratory film held the disks in place. After 12 months, diffuse cankers were found for all but one fungus inoculation site; no cankers occurred with control inoculations. Reddish brown, inner bark necrosis (mean area 22.4 cm2) and sapwood discoloration (mean volume 38.1 cm3) were associated with the cankers. C. smalleyi was recovered from five of nine cankers, but not from the control wounds. In June 2008, 0.1 ml of spore suspension (1 × 104 ascospores/ml) of C. smalleyi or sterile dH20 was pipetted into four drilled holes (to the outer sapwood) of four trees in Chippewa County, WI. Holes were sealed with moist cotton and masking tape. Two months later, diffuse cankers with reddish inner bark (mean 49.7 cm2) surrounded 16 inoculation points; no cankers or inner bark necrosis was observed for the control points. In a similar trial, a spore suspension or sterile water was placed into four drilled holes covered with moist cotton and moldable putty on six trees in Olmstead County, MN. Fourteen months later, either diffuse or sunken cankers with reddish, inner bark necrosis (mean 22.3 cm2) were observed surrounding all inoculated points while all control points were callus closed. For the latter two trials, long, narrow discoloration (reddish brown) was found in the sapwood associated with each canker; no sapwood discoloration was observed for the control points. In addition, C. smalleyi was reisolated from all cankered stems in Wisconsin and from 21 of 24 cankers in Minnesota, but not from the controls. This report verifies the ability of C. smalleyi to cause a newly described canker disease on poletimber-sized hickories. We hypothesize that this disease is contributing to the death of hickory bark beetle-attacked Carya cordiformis in the eastern United States. Reference: (1) J. Johnson et al. Mycologia 97:1067, 2005.

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.

Phytophthora Species in Soils Associated with Declining and Nondeclining Oaks in Missouri Forests.

Periodic episodes of oak decline have occurred in the Missouri Ozark forests since the early 1900s and the disease is currently severe (2). Several Phytophthora spp. contribute to oak decline in Europe (1), but the role of Phytophthora spp. in oak decline in the eastern United States is not known. Mineral soils collected around the bases of declining and nondeclining oaks in paired sites in central Missouri forests were assayed for the presence of these taxa by baiting flooded soil with Quercus robur leaves. Q. rubra and Q. velutina were the oak species on three sites and Q. alba was on the fourth. Isolates from symptomatic baits plated on PARPNH selective medium were identified tentatively on classical taxonomic characteristics. Five isolates of P. cambivora, two of P. quercina, and eight of P. cinnamomi were obtained from soils around one, one, and three trees, respectively, on decline sites. The internal transcribed spacer sequences for each isolate were compared to those in GenBank; BLAST searches for all isolates had nucleotide identities of 99% and E values of 0, which confirmed the identifications. Greenhouse pot trials were conducted to assess pathogenicity of isolates on stems of 2-year-old Q. alba and Q. rubra. A mycelial agar plug was inserted into a 1-cm long slit cut into the cambium 5 cm above the root collar and covered with sterile, moist cotton and wrapped with laboratory film. The treatments included two isolates of P. cambivora, one of P. quercina, three of P. cinnamomi, and a sterile agar plug. Each host × isolate combination was replicated four times, and the experiment was conducted twice in the greenhouse (natural lighting; temperature ≤32°C in summer and ≥7°C in winter). Stem lesions were produced commonly by P. cambivora (28 of 32 seedlings) and P. cinnamomi (46 of 48 seedlings) within 3 months; none was found on control seedlings or those inoculated with P. quercina. Mean lengths (cm) of lesions caused by P. cinnamomi were greater for Q. rubra (5.6) than for Q. alba (4.3) and lesion lengths for P. cambivora were greater for Q. alba (5.2) than for Q. rubra (4.4). Seven Q. alba seedlings inoculated with P. cambivora and one Q. alba inoculated with P. cinnamomi died before 3 months. All Phytophthora species were recovered from inoculated stems at 3 months except that P. quercina was not recovered in one trial. To our knowledge, this is the first report of Phytophthora species in soils of Missouri oak forests, of P. quercina in the United States, and of the ability of P. cambivora to cause stem lesions on Q. alba. P. cinnamomi and P. cambivora should be investigated in situ as possible contributing factors of oak decline in Missouri. References: (1) T. Jung et al. Plant Pathol. 49:706, 2000. (2) R. Lawrence et al. MO. Conserv. 63:11, 2002.

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.