2007 SOUTHEASTERN NATURALIST 6(1):67–82 Microfungi from Bark of Healthy and Damaged American Beech, Fraser Fir, and Eastern Hemlock Trees During an All Taxa Biodiversity Inventory in Forests of the Great Smoky Mountains National Park Richard E. Baird1,*, Clarence E. Watson2, and Sandra Woolfolk1 Abstract - The assemblage of microfungi associated with bark samples of healthy and damaged Fagus grandifolia (American beech), Abies fraseri (Fraser fir), and Tsuga canadensis (eastern hemlock) trees was evaluated during an All Taxa Biodiversity Inventory of the Great Smoky Mountains National Park in 2003 and 2004. Bark samples were collected from sampling points 0.3, 0.6, 0.9, and 1.2 m above the ground surface on the bole of each replicate tree. Patterns of species composition and diversity (species richness) were evaluated from bark samples over three sampling dates (May, July, and September) each year. A total of 4814 isolates were obtained, with greater than 95% belonging to the Deuteromycota. Over 94 species of fungi were identified from bark of the three tree species, which were either healthy or were damaged or under pressure from exotic pests. The most common genus was Trichoderma, for which a total of 13 species were identified during the two-year study. Frequencies of microfungi between healthy and damaged trees were similar across years, but when data was compared by year, frequencies were significantly greater in 2004 than 2003. Species richness was almost always significantly greater in September than in May and July. Frequencies of microfungi isolated from bark samples collected 1.2 m above the ground were significantly greater than in samples collected at 0.9, 0.6 and 0.3 m. Increased species richness at the higher bole positions was likely related to changes in microenvironment, as proposed by previous researchers. All other comparisons of species richness were similar. Introduction Microfungi species, which produce microscopic spore-bearing structures, belong primarily to the Ascomycota and Deuteromycota, but may also include species of the Basidiomycota and Zygomycota (Cannon and Sutton 2004). Large numbers of species and genera of microfungi have been reported from bark and litter of forest trees (Bills et al. 2004, Fernandez and Boyer 1989). However, studies to determine species richness of microfungi in different forest ecosystems have been limited due to the small size of microfungi and lack of occurrence on economically important hosts. To overcome this disparity in information for fungi and other organisms, the All Taxa Biological Inventory (ATBI) was developed to inventory all organisms in unique or diverse habitats around the world (Sharkey 2001). Fungi were selected as one 1Entomology and Plant Pathology Department, Box 9655, Mississippi State University, Mississippi State, MS 39762. 2MAFES Administration, Box 9740, Mississippi State University, Mississippi State, MS 39762. *Corresponding author - rbaird@plantpath.msstate.edu. 68 Southeastern Naturalist Vol. 6, No. 1 of the groups of organisms to be included as a pilot program for an ATBI in the Area de Conservación Guanacaste, located in the northwestern corner of Costa Rica (Janzen 1996, Rossman et al. 1998). Following that initiative, an ATBI was started in the Great Smoky Mountains National Park (GSMNP) in 1997 (Sharkey 2001). What limited data on fungi that existed was included in a list for the GSMNP that was compiled from various sources over many years (Petersen 1979) and for myxomycetes and select phyla known from specific habitats (H.A. Raja and C.A. Shearer, unpubl. data; Stephenson et al. 2001). Prior to these studies, little data on microfungi had been obtained in the park due to cost of the work and lack of mycological expertise. Data for microfungi from forest ecosystems in the GSMNP are needed especially from sites where loss of habitat or tree species are occurring from exotic pests, since loss of these host trees may result in the elimination of fungi specific to the hosts. The surface of bark from living trees contains a wide variety of organisms including bacteria, bryophytes, and lichens. Fungi also comprise a large percentage of the microflora (Bier 1963a, b), and research on the occurrence of bark fungi has been conducted for many different tree species (Garner 1967, Sivak and Person 1973). While Butin and Kowalski (1986) found fungi in the Ascomycota and Deuteromycota were the most common taxa present on five hardwood species, Kliejunas and Kuntz (1974) identified many fungal genera from healthy bark with the most common genera being Alternaria, Epicoccum, Fusarium, Penicillium, Phoma, and Trichoderma. Other researchers have catalogued caulosphere microorganisms to determine if bark microfungi inhibit or reduce invasion by pathogenic fungi (Baird 1991, Cox and Hall 1978, Weir et al. 1996). Unfortunately, very few studies evaluated changes in microflora following invasion of exotic fungal pathogens or insect pests. In the Southern Appalachian Mountains, pests cause significant losses to three major forest tree species. Nectria coccinea var. faginata Lohm. Wats. & Ay., the causal organism of beech bark disease, currently attacks Fagus grandifolia Ehrh. (American beech). Adelges tsuga Annand. (hemlock woolly adelgid) is attacking Tsuga canadensis (L.) Carr. (eastern hemlock), and Adelges piceae Ratzeburg (balsam woolly adelgid) is killing Abies fraseri (Pursh) Poir. (Fraser fir). American beech occurs in eastern North American forests and is an important component of the GSMNP. This tree species occurs in areas predominantly composed of beech, termed “beech gaps.” Since many of these clusters of beech occur at high elevations, the gaps are considered a rare forest community type (Whittaker 1956). Unfortunately, the exotic fungus N. coccinea var. faginata, which entered North America near Nova Scotia around 1890 (Ehrlich 1934), has spread southward and entered the GSMNP in 1993 (Houston 1994). Since the introduction of this pathogen, the population of American beech in the GSMNP has been drastically reduced. Permanent plots were established in 1994 to monitor tree health and mortality (Wiggins et al. 2004). As of 1997, 26% tree mortality occurred within the plots, but in one 2007 R.E. Baird, C.E. Watson, and S. Woolfolk 69 area, all 77 overstory trees died (G. Taylor, USDA/National Park Service[NPS]-GSMNP, pers. comm.). Eastern hemlock is a dominant tree species that is widely distributed in the southern Appalachian Mountains and covers 3820 acres, or 1% of the GSMNP (Johnson et al. 1999). The exotic insect, hemlock woolly adelgid, was first reported in the Pacific Northwest in 1924 and spread to the northeast in the 1950s. The insect moved southward through the mountains of Virginia and Tennessee around 2001. In the following year, the insect entered the GSMNP and now infests large areas of the park where Eastern hemlock occurs (R. Miller 2002, USDA/NPS-GSMNP, unpubl. data). Fraser fir is the only fir species endemic to the southern Appalachian Mountains and has a disjunct distribution that restricts it to higher elevations in southwest Virginia, eastern Tennessee, and western North Carolina. The GSMNP has approximately 74% of the total spruce-fir forest in the southern Appalachians. The balsam woolly adelgid, which entered the eastern portion of the GSMNP at Mount Sterling, has killed over 90% of the mature Fraser fir over the last 35 years in these areas, but regeneration from remnant trees appears to hold hope for survival of this species (Dull et al. 1988). However, many of the surviving larger trees continue to be newly infested with this pest, which results in their eventual death (M. Kloster, USDA/NPSGSMNP, pers. comm.). As the health of American beech, Fraser fir, and eastern hemlock declines, changes in the caulosphere fungi can be expected. Unfortunately, no studies to compare bark fungi are available for healthy and damaged trees. Development of baseline data to establish identities and frequencies of fungi present on healthy and damaged trees may be useful for evaluation of tree health and development of biological control strategies for the pests. The objectives of this study were: 1) to develop baseline data and catalog the fungal microflora present on bark tissues of healthy and damaged American beech, Eastern hemlock, and Fraser fir for the ATBI in the GSMNP; and 2) to determine if sampling positions on boles and dates of sampling influence species richness. Materials and Methods Field collections of bark were obtained from American beech, Fraser fir, and eastern hemlock stands located within the boundaries of the GSMNP. Although, it was difficult to establish a similar range of tree sizes for sampling during the investigation, all trees sampled were 􀂕 20 cm in diameter at 1.3 m above the ground. Additional criteria for sampling are discussed below. Table 1 gives exact dates and locations sampled in 2003 and 2004. On each sampling date, bark samples were obtained from five healthy and five damaged trees of each species. Healthy trees exhibited no symptoms of damage as a result of exotic fungal or insect pest. Damaged American beech trees selected for sampling included those with greater that 50% canopy defoliation and die-back (Durr et al. 1988) and a high rating of Nectria spp. 70 Southeastern Naturalist Vol. 6, No. 1 Table 1. Sampling dates and locations within GSMNP of bark samples collected from three tree species over a two-year period. Coordinates are listed on UTM NAD27 CONUS and are within UTM zone 17S. Healthy Damaged Sampling date American beech Fraser fir Eastern hemlock American beech Fraser fir Eastern hemlock 2003 (May 6–7, 2003) Fork Ridge Trail Mt. Sterling A Sugarlands Center Fork Ridge Trail Mt. Sterling A Laurel Falls 3939907N 3952818N 3951831N 3939907N 3952586N 3950395N 0278128E 0307907E 0270532E 0278128E 0307796E 0266283E (July 1–3, 2003) Mt. Sterling C Mt. Sterling A Sugarlands Center Fork Ridge Trail Mt. Sterling A Laurel Falls 3952349N 3952818N 3951831N 3939907N 3952586N 3950395N 0307646E 0307907E 0270532E 0278128E 0307796E 0266283E (Sept. 9–10, 2003) Beech Gap A Clingman’s Dome A Rainbow Falls A Beech Gap B Clingman’s Dome B Rainbow Falls B 3946910N 3938104N 3950552N 3946822N 3938080N 3950550N 0300490E 0272998E 0274930E 0300636E 0273302E 0274993E 2004 (May 15, 2004) Double Gap Appal. Trail Twin Creeks Double Gap Appal. Trail Twin Creeks 393 8507N 3938128N 3951948N 393 8507N 3938128N 3951948N 026 9497E 0271952E 0273776E 0269497E 0271952E 0273776E (July 8–9, 2004) Beech Forest on Mt. Buckley Cove Mt. Trail, Beech Forest on Mt. Buckley Cosby Campground New Found Gap Near Sugarland New Found Gap on Cosby Nature Trail Hdq. Trail Trail 3943462N 3938089N 3952482N 3943462N 3938089N 3958992N 0278482E 272790E 0270634E 0278482E 0272790E 0300416E (Aug. 11–12, 2004) Cataloochee, Clingman’s Dome Cataloochee, Cataloochee, Clingman’s Dome Cataloochee, GSMNP-Horse Area GSMNP on GSMNP-Big GSMNP-Horse Area GSMNP on GSMNP-Rough Camp area just Appalachian Trail Fork Ridge Trail Camp area just Appalachian Trail Fork Trail before Little before Little Cataloochee Trail Cataloochee Trail 3944395N 3938112N 3939633N 3944395N 3938112N 3942239N 0308693E 0273087E 0307911E 0273087E 0273087E 0307212E 2007 R.E. Baird, C.E. Watson, and S. Woolfolk 71 levels (Vance 1995). Ratings for Nectria spp. (presence of perithecia) were made on the north and south side of each tree using a rating scale within a 33- x 33-cm area, centered 122 cm above the ground surface. Damaged Fraser fir selected for sampling included those with greater than 50% canopy defoliation and die-back, and high numbers of A. piceae present on 100 cm2 of each bole as determined by applying a 2-cm2 template at 25 points around the circumference of each tree at 1.5 m above the ground surface (G. Taylor and K. Johnson, GSMNP, unpubl. USDA/FS BWA Survey Protocol and pers. comm.). Damaged eastern hemlock trees include those that were rated as heavily infested with A. tsuga. This included trees that had up to 80% of the branches infested by the insects (J. Pickering, University of Georgia, pers. comm.). Following tree selection, four bark samples, each 5 x 5 cm and 5 mm deep, were taken at 0.3, 0.6, 0.9, and 1.2 m above the ground on the north side of each bole. Each bark piece was placed into an envelope, kept cool until returned to the laboratory, and stored at 4􀃝 C until processed. For isolation and identification of fungi, each of the bark samples were subdivided into four 1-cm2 pieces and surface-sterilized in sodium hypoclorite (0.524% w/v) for 3 min. Two pieces were then plated onto potato-dextrose agar (PDA) and water agar (WA) as previously described by Baird et al. (2004). The plates were incubated for 7 days at room temperature to allow for growth of fungi. All fungi growing in the plates were subcultured onto PDA and stored for later identification using standard mycological methods. More than one fungus was often isolated from a single piece of bark. Isolation frequencies based on numbers of bark samples infected were determined for genera and species isolated. Single spores of cultures initially identified as Fusarium spp. were transferred to carnation leaf agar using original ingredients as previously defined (Nelson et al. 1983). Keys for general identification of fungi were those developed by Barnett and Hunter (1998), Ellis (1971, 1976), and Sutton (1980). In addition, an unpublished guide to Trichoderma spp. by G. Samuels, USDA/ Agricultural Research Service-Beltsville, MD, was used. Statistical analysis The experimental design was a completely randomized design (CRD) with five replications (trees) within each tree species, sampling date, and condition (healthy or diseased). Data were analyzed by analysis of variance as a series of combined CRDs over years using the GLM procedure of SAS (SAS Institute, Cary, NC). Mean separation was carried out using Fisher’s protected least significant difference (LSD). Results Ninety-four species of fungi were isolated from the bark of healthy and damaged American beech, Fraser fir, and eastern hemlock during the study (Appendix 1). A total of 2195 fungi was isolated in 2003 and 2619 in 2004. Greater than 95% of total fungi were from the Deuteromycota, 2.3% Ascomycota, 1.2% Zygomycota, and 1% others or unknowns. 72 Southeastern Naturalist Vol. 6, No. 1 The most common species identified during the study were Curvularia lunata (Wakk.) Boedijn, Pestalotia clavipora (Atk.) Steyaert, Pestalotia funerea (Desmaz) Steyaert, Trichoderma aggressivum Samuels and W. Gams., Trichoderma aureoviride Rifai, Trichoderma hamatum (Bonord.) Bainier, Trichoderma harzianum Rifai, Trichoderma koningii Oudem., Trichoderma virens (J. Miller et al.) Arx, and Trichoderma viride Pers.:Fr. Yeast species were obtained from American beech, but none were found on the bark of the other two tree species. When the percentages of isolation were compared between bark samples from healthy and damaged trees across years, no differences were observed. However, the percent isolation data pooled across three tree species, two tree conditions, and four sampling points were significantly greater in 2004 than 2003. When the 20 most common fungi isolated were evaluated across years, bark positions, and tree conditions, no differences were observed. Furthermore, when isolation frequencies were analyzed separately by tree condition, no differences were observed in 2003 or 2004 (data not shown). Isolation frequencies were compared between the four bark sampling points for each tree species and year (Table 2). No differences were observed between frequencies from bark sample locations collected in 2003, but differences in species richness occurred in 2004. Isolation frequencies from the samples collected at 1.2 m above the ground were significantly greater than bark samples collected from 0.3 and 0.6 m. Percent frequencies from bark samples from 0.9 m were almost always significantly greater than at 0.3 and 0.6 m, with the exception that the frequencies from eastern hemlock were similar between 0.6 and 0.9 m bark sample points from both healthy and damaged trees. When individual fungal species were compared by sampling position, only Cephalosporium spp. species had significantly different isolation frequencies on American Beech. No other position differences by fungal species for the three trees occurred during the study. Table 2. Percent occurrence of mycobiota isolated from bark at four locations on three tree species over two years. Position means within a year, condition, and tree species not followed by a common letter (a–c) are significantly (P 􀂔 0.05) different according to Fisher’s protected LSD. Bark sample American beech Fraser fir Eastern hemlock position DA HB D H D H 2003 0.3m 2.05 1.62 1.76 1.80 1.95 1.62 0.6m 1.86 1.95 1.90 1.80 1.71 2.67 0.9m 1.86 1.80 1.33 2.05 2.05 1.76 1.2m 2.19 2.04 1.67 2.05 2.28 1.95 2004 0.3m 1.50c 1.15c 1.41c 1.19c 1.67c 1.59c 0.6m 1.81c 1.72c 2.03c 1.67c 2.34bc 1.81bc 0.9m 2.91b 2.87b 2.69b 2.60b 3.00b 2.47b 1.2m 4.37a 4.55a 4.64a 4.31a 4.55a 3.31a AD = Trees damaged from exotic pests (fungi or insect). BH = healthy trees without occurrence of a pest. 2007 R.E. Baird, C.E. Watson, and S. Woolfolk 73 Percent isolation frequencies across all three tree species and their conditions were compared by sampling date (Table 3). The largest number of isolates almost always occurred in September and the percentages were either numerically or significantly greater than from the May or July samplings. Two exceptions were that the September frequencies were numerically, but not significantly lower than for May and July for healthy American beech and damaged Fraser fir in 2004. Percent isolation frequencies were similar compared across tree species, tree conditions, and bark positions. When percent isolations were compared by tree species across both tree conditions and all bark positions, all data were similar by year. Discussion In December 1997, the ATBI was initiated to collect all species of organisms in GSMNP (Sharkey 2001), but research on microfungi was not conducted until 2002 (Raja et al. 2003). Because of this initial effort, microfungi were sampled over two years from bark of select, potentially endangered trees that occur in GSMNP. Ninety-four species of fungi were identified from the bark of the three tree species. Isolation frequencies were almost always higher during the September sampling than in May and July. The greater percentages might be expected due to the increase of inoculum and colonization by fungi towards the end of the growing season. All bark collections were georeferenced, and the species data with sampling information were added to the database list of organisms identified for GSMNP. Previously, Bills and Polishook (1994) collected 300 to 400 species of fungi within each replicate 1-ml leaf-litter sample collected from a Costa Rican rainforest. Since the current study of bark samples were much lower, isolation methods (e.g., media) or substrate differences, such as litter compared to bark, may have limited species richness. Table 3. Percent occurrence of mycobiota from three tree species by sampling dates over two years. Sampling date means with a year, condition, and tree species not followed by a common letter (a–b) are significantly (P 􀂔 0.05) different according to Fisher’s protected LSD. American beech Fraser fir Eastern hemlock Sampling date DA H D H D H 2003 May 1.86ab 1.71b 1.25b 1.46b 1.43b 1.57b July 1.67b 1.17b 1.75ab 1.93ab 1.43b 1.57b September 2.43a 2.68a 2.00a 2.39a 3.14a 2.86a 2004 May 2.09b 2.65a 2.95a 2.21a 3.01ab 2.05a July 2.78ab 2.81a 3.15a 2.478a 2.38b 2.25a September 3.08a 2.25a 1.98b 2.88a 3.28a 2.58a AD = Trees damaged from exotic pests (fungi or insect). BH = healthy trees without occurrence of a pest. 74 Southeastern Naturalist Vol. 6, No. 1 The majority of fungi isolated in the current study were members of the Deuteromycota, which coincides with results from past investigations (Baird 1991, Cotter and Blanchard 1982, Fernandez and Boyer 1989). More than 13 species of Trichoderma, a common hyphomycete, were identified, and collectively had the greatest isolation frequencies of any genus during this study. Other common fungi on these three tree species included C. lunata, two Pestalotia spp. and five Penicillium spp. Previously, Cotter and Blanchard (1982) showed that Trichoderma spp. could be isolated from American beech bark, but Alternaria, Aureobasidium, and Epicoccum spp. were the most common fungi identified. Since this previous study was conducted in the northeastern United States, variations in species richness from the present study could be expected. In Canada, the most common fungi isolated from bark of American beech were Alternaria, Aposphaeria, Aureobasidium, and Cladosporium spp. (Fernandez and Boyer 1989). In the current investigation, isolation frequencies compared between healthy and damaged trees were similar. These results also differed from the two previous studies where species richness was greater from American beech trees damaged by insects and Nectria canker than healthy ones (Cotter and Blanchard 1982, Fernandez and Boyer 1989). In those studies, however, similar fungal microflora occurred on damaged trees as was found in the current investigation. Trichoderma was the most common genus identified during this study. Trichoderma spp. are ubiquitous, occurring in soil, on roots or on aboveground parts of plants (G. Samuels-USDA/ARS, unpubl. guide to Trichoderma). Species within the genus are commonly found sporulating on moist wood and other environments in a forest ecosystem and are believed to be anamorphs of Hypocrea spp. (Chaverri and Samuels 2003). A study with Theobroma gileri L. (cocao trees) reported that Trichoderma spp. commonly occur as endophytes in trunks (Evans et al. 2003). In that study, several of the endophytic Trichoderma spp. evaluated in vitro were found to be antagonistic to pathogens of cocao. Previous studies reported that Trichoderma spp. have antifungal or plant-growth-stimulating potential and are being tested or used as biological control agents (Chaverri and Samuels 2003). Trichoderma species are also known to be cellulose-degrading fungi, that rapidly colonize dying and dead plant tissues (bark) until their depletion (Harman 2000). This depletion would be expected to limit growth of other microorganisms. Even though Trichoderma frequencies were higher on damaged American beech trees, control of N. coccinea var. faginata was not evident. Trichoderma spp. may be operating as opportunists degrading dying and dead tissue following invasion by the pathogen. A rarely isolated fungus, Thysanophora canadensis Stolk and Hennebert, occurred only on Eastern hemlock. Ellis (1971) reported that T. canadensis was only found on Tsuga in Canada. The potential loss of the eastern hemlock throughout Canada and the eastern United States, may also result in the loss or extinction of T. canadensis. 2007 R.E. Baird, C.E. Watson, and S. Woolfolk 75 Microenvironmental variation that occurs within and among different microhabitats affects species richness and diversity (Stephenson 1989). The position of bark samples from each tree affected the isolation frequency of microfungi during this investigation. Bark samples taken at 0.9 and 1.2 m had significantly greater percentages of fungi than at 0.3 and 0.6 m. A previous study surveying myxomycetes showed that aerial leafy litter (1.5– 2.5 m above the ground) had greater species diversity and frequencies than the substratum of the forest floor (Schnittler and Stephensen 2000). It was suggested that at lower bole positions, increased moisture levels inhibit spore development, dispersal, and germination. Furthermore, it was believed that increased moisture may enhance colonization of a substrate by parasitic fungi, reducing the survival of other species at lower positions on a tree (Alexopoulus 1970, Stephenson et al. 2004). In this study, the bark from the lower sampling points may have received less inoculum dispersal due to higher moisture levels, resulting in lower fungal populations. In addition, if moisture levels were greater at the lower bark sampling points, Trichoderma spp. might be actively growing on the bark surface, thus inhibiting growth and sporulation of other fungi and reducing species richness. It was uncertain why the isolation frequencies from the four sampling positions on the trees varied significantly for the three tree species. It is likely related to microenvironment factors such as moisture and other parameters such as pH, but further studies evaluating different microenvironments must be conducted to clarify the results from the present study. The similarity of the bark mycobiota between damaged and healthy trees showed that there is a common group present on American beech, Fraser fir, and eastern hemlock, even in the presence of exotic pests. Presence of many Trichoderma spp. on American beech bark did not appear to provide natural biological control of N. coccinea var. faginata. Potentially, when N. coccinea var. faginata becomes established on American beech, the pathogen grows into the inner bark and phloem where other fungi do not actively grow. When this occurs, control of the pathogen by Trichoderma spp. would be minimal and insufficient to prevent girdling of the trees. Isolation frequencies of Penicillium spp., which are ubiquitous in soil, decaying vegetation, seeds or wood (Pitt 2000), varied across tree species, damaged or healthy, with no apparent trends being observed during the study. The results of this and previous studies suggest that a common mycobiota of Deuteromycota occur on bark of temperate trees (Cotter and Blanchard 1982), and the majority of species identified occurred across the three tree species. Even though frequencies of fungi differed at four sampling points on trees, genera and species of fungi were similar. Furthermore, trees damaged by one of the exotic pests did not contain a different level or diversity of microfungi than healthy trees during the investigation. It is well known that cultural methods for isolating fungi limit the accuracy of species richness due to selectivity. Therefore, molecular methods appear to have the greatest potential for maximizing species richness determination for future ATBI investigations. 76 Southeastern Naturalist Vol. 6, No. 1 Acknowledgments I would like to acknowledge Discover Life in America for providing research support both years of the study under Project Numbers GSM # 2003-01 and GSM # 2004-01. Also, thanks go to Chelsey Wilson and Becky Reed for laboratory support during the investigation. Literature Cited Alexopoulos, C.J. 1970. Rain forest myxomycetes. Pp. 21–23, In H.T. Odum (Ed.). Tropical Rain Forest. United States Atomic Energy Commission, Washington, DC. 87 pp. Baird, R.E. 1991. Mycobiota of bark fungi associated with seven strains of Cryphonectria parasitica on two hardwood tree species. Mycotaxon 15:23–33. Baird, R.E., D.E. Carling, C.E. Watson, M.L. Scruggs, and P. Hightower. 2004. Effect of nematicides on cotton root mycobiota. Mycopathologia 157:191–199. Barnett, H.L., and B.B. Hunter. 1998. Illustrated Genera of Imperfect Fungi, 4th Edition. American Phytopathological Society Press, St. Paul, MN. 218 pp. Bier, J.E. 1963a. Tissue saprophytes and possibility of biological control of some tree species. Forest Chronicle 39:82–84 Bier, J.E. 1963b. Further effects of bark saprophytes of Hypoxylon canker. Forest Science 9:263–269. Bills, G.F., and J.D. Polishook. 1994. Abundance and diversity of microfungi in leaf litter of a lowland rain forest in Costa Rica. Mycologia 86:187–198. Bills, G.F., M. Christensen, M. Powell, and G. Thorn. 2004. Saprobic soil fungi. Pp. 271–302, In G.M. Mueller, G.F. Bills, and M.S. Foster (Ed.). Biodiversity of Fungi: Inventory and Monitoring Methods, Elsevier Academic Press, San Diego, CA. 777 pp. Butin, H., and T. Kowalski. 1986. The natural pruning of branches and their natural preconditioning III. The fungus flora of common maple, grey alder, silver birch, hornbeam, and common ash. Environment Journal Forest Pathology 16:129–138. Cannon, P.F., and B.C. Sutton. 2004. Microfungi of wood and plant debris. Pp. 217– 240, In G.M. Mueller, G.F. Bills, and M.S. Foster (Ed.). Biodiversity of Fungi: Inventory and Monitoring Methods. Elsevier Academic Press, San Diego, CA. 777 pp. Chaverri, P., and G.J. Samuels. 2003. Hypocrea/Trichoderma (Ascomycota, Hypocreales, Hypocreaceae): Species with green ascospores. Studies in Mycology 48:1–119. Cotter, V.T., and R.O. Blanchard. 1982. The fungal flora of bark of Fagus grandifolia. Mycologia 74:836–843. Cox, L.A., and A.M. Hall. 1978. Phylloplane fungi of Quercus robur. Annals Applied Biology 89:119–122. Dull, C.W., J.D. Ward, H.D. Brown, G.W. Ryan, W.H. Clerke, and R.J. Uhler. 1988. Evaluation of spruce and fir mortality in the southern Appalachian Mountains. USDA/Forest Service Protection Report R8-PR13, Atlanta, GA. 92 pp. Durr, P.C., L. Richmond, and C. Edgar. 1988. Site classification and field measurements methods manual. Internal document. Resource Management Division, Great Smoky Mountain National Park, Gatlinburg, TN. 83 pp. Ehrlich, J. 1934. The beech bark disease: A Nectria disease of Fagus, following Cryptococcus fagi (Baer.). Canadian Journal of Forest Research 10:593–701. Ellis, M.B. 1971. Dematiaceous Hyphomycetes. Commonweath Mycological Institute, Kew, Surrey, UK. 608 pp. 2007 R.E. Baird, C.E. Watson, and S. Woolfolk 77 Ellis, M.B. 1976. More Dematiaceous Hyphomycetes. Commonweath Mycological Institute, Kew, Surrey, UK. 507 pp. Evans, H.C., K.A. Holmes, and S.E. Thomas. 2003. Endophytes and mycoparasites associated with indigenous forest tree Theobroma gileri L. in Ecuador and a preliminary assessment of their potential as a biocontrol agent of cocao diseases. Mycological Progress 2:149–160. Fernandez, M.R., and M.G. Boyer. 1989. Beech bark mycoflora and its distribution in relation to the presence of the scale insect Cryptococcus-fagisuga. Canadian Plant Disease Survey 69:101–104. Garner, J.B. 1967. Some notes on the study of bark fungi. Canadian Journal Botany 45:540–541. Harman, G.E. 2000. The myths and dogmas of biocontrol: Changes in perceptions derived from research on Trichoderma harzianum strain T-22. Plant Disease 84:377–393. Houston, D.R. 1994. Major new tree disease epidemics: Beech bark disease. Annual Revue Phytopathology 32:75–87. Janzen, D.H. 1996. Prioritization of major groups of taxa for the All Taxa Biodiversity Inventory (ATBI) of the Guanocaste Conservation Area in Northwestern Costa Rica, a biodiversity development project. Association of Systematics Collections Newsletter 24:49–56. Johnson, K.D., E.P. Hain, K.S. Johnson, and F. Hartings. 1999. Proceeding: Symposium on sustainable management of hemlock ecosystems in eastern North America (June 22–23), Durham, NH. USDA/Forest Service General Technical Report NE-267:111–112. Kliejunas, J.T., and J.E. Kuntz. 1974. Microorganisms associated with Eutypella parasitica in Acer saccharium and A. rubrum. Canadian Journal Forest Research 4:207–212. Nelson, P.E., T.A. Toussoun, and M. Maraas. 1983. Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University Press, University Park, PA. 193 pp. Petersen, R.H. 1979. Checklist of the fungi of the Great Smoky Mountains National Park. US Department of the Interior, National Park Service Southeast Region, Gatlinburg, TN. Management Report No. 29. 133 pp. Pitt, J.I. 2000. A Laboratory Guide to Common Penicillium Species. Food Science Australia, CSIRO/AFISC. 197 pp. Raja, H.A., J. Campbell, and C.A. Shearer. 2003. Freshwater ascomycetes: Cyanoannulus petersenii a new genus and species on submerged wood. Mycotaxon 88:1–17. Rossman, A.Y., R.E. Tulloss, T.E. O’Dell, and R.G. Thorn. 1998. Protocols For an All Taxa Biodiversity Inventory of Fungi in a Costa Rican Conservation Area. Parkway Publishing Inc., Boone, NC. 195 pp. Schnittler, M., and S.L. Stephenson. 2000. Myxomycete biodiversity in four different forest types in Costa Rica. Mycologia 92:626–637. Sharkey, M.J. 2001. The All Taxa Biological Inventory of the Great Smoky Mountains National Park. Florida Entomologist 84:556–564. Sivak, B., and C.O. Person. 1973. The bacterial and fungal flora of the bark, wood, and pith of alder, black cottonwood, maple, and willow. Canadian Journal Botany 51:1985–1988. 78 Southeastern Naturalist Vol. 6, No. 1 Stephenson, S.L. 1989. Distribution and ecology of myxomycetes in temperate forests. II. Patterns of occurrence on bark surface of living trees, leaf litter, and dung. Mycologia 81:608–621. Stephenson, S.L., M. Schnittler, D.W. Michell, and Y.K. Novozhilov. 2001. Myxomycetes of the Great Smoky Mountains National Park. Mycotaxon 78:1–15. Stephenson, S.L., M. Schnittler, and C. Lado. 2004. Ecological characterization of tropical myxomycete assemblage-Maquipucuna Cloud Forest Reserve, Ecuador. Mycologia 96:488–497. Sutton, S.C. 1980. The Coelomycetes. Commonweath Mycological Institute, Kew, Surrey, UK. 696 pp. Vance, R.A. 1995. Incidence and life history of beech scale, initiator of beech bark disease in the Great Smoky Mountains National Park. M.Sc. Thesis. The University of Tennessee, Knoxville, TN. 72 pp. Weir, A.W., M.A. Schnitzler, T.A. Tattar, E.J. Klekowski, Jr., and A.I. Stern. 1996. Wound periderm development in red mangrove, Rhizophora periderm L. International Journal Plant Science 157:63–70. Whittaker, R. 1956. Vegetation of the Great Smoky Mountains. Ecological Monographs 26:1–80. Wiggins, G.J., J.F. Grant, M.T. Windham, R.A. Vance, B. Rutherford, R. Klein, K. Johnson, and G. Taylor. 2004. Associations between causal agents of the beech bark disease complex (Cryptococcus fagisuga [Homptera: Cryptococcidae] and Nectria spp.) in the Great Smoky Mountains National Park. Environmental Entomology 33:1274–1281. 2007 R.E. Baird, C.E. Watson, and S. Woolfolk 79 Appendix 1. Mean percent occurrence of fungi from bark of American beech, Fraser fir and eastern hemlock from the Great Smoky Mountains National Park. 2003 2004 A. Beech F. Fir E. Hemlock A. Beech F. Fir E. Hemlock Taxa DA H DHDHDHDHDH Fungi imperfectiB Acremonium crotocinigenum (Schol-Schwartz) W. Gams 0.0C 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 Alternaria tenuissima (Kunze:Fr.) Wiltshire 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Aspergillus niger Tiegh. 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 0.0 Aureobasidium pullulans (deBary) G. Arnaud 0.0 5.0 0.0 0.0 3.3 0.0 1.7 5.0 5.0 0.0 0.0 0.0 Botrytis cinereaB Pers.:Fr. 3.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Botryoderma sp. Papendorf & Upadhyay 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Botryosphaeria sp. Ces. & De Not. 3.3 3.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Botryodipodia sp. (Sacc.) Sacc. 0.0 1.7 0.0 0.0 1.7 1.7 0.0 3.3 0.0 0.0 0.0 0.0 Calcarisporium arbuscula Preuss 0.0 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Candida guilliermondii (Castellani) Langeron & Guerra 0.0 0.0 0.0 0.0 0.0 0.0 3.3 1.7 5.0 0.0 0.0 0.0 Coryneum stromatoideum (Dearn.) Sutton 0.0 0.0 0.0 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Cephalosporium sp. Corda. 5.0 1.7 0.0 0.0 0.0 0.0 6.7 8.3 8.3 3.3 3.3 0.0 Chaetomella oblonga Fuckel 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.3 3.3 0.0 Cladosporium herbarumB (Pers.:Fr.) Link 1.7 0.0 0.0 1.7 0.0 0.0 3.3 8.3 6.7 1.7 0.0 0.0 Curvularia lunata (Wakk.) Boedijn. 15.0 18.2 9.9 1.7 1.7 0.0 6.7 5.0 15.0 11.7 6.7 3.3 C. oryzae Bugnicourt 3.3 0.0 3.3 0.0 1.7 3.3 3.3 5.0 10.0 6.7 5.0 3.3 C. fallax Boedijn. 0.0 1.7 1.7 0.0 0.0 0.0 3.3 8.9 16.7 6.7 5.0 3.3 Curvularia sp. Boedijn. 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 3.3 1.7 1.7 3.3 Dinemasporium strigosum (Pers.:Fr.) Sacc. 0.0 0.0 3.3 1.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 0.0 Epicoccum nigrum Link 1.7 0.0 1.7 5.0 0.0 0.0 6.7 26.7 3.3 6.7 0.0 0.0 Fusarium equisiti (Corda) Sacc. 28.3 1.7 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 F. graminearum Schwabe 8.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 F. lateritium Nees.:Fr. 0.0 0.0 0.0 0.0 0.0 0.0 16.7 25.0 3.3 6.7 0.0 0.0 F. oxysporum Schlechtend.:Fr. 3.3 3.3 5.0 0.0 0.0 3.3 0.0 3.3 0.0 1.7 0.0 0.0 80 Southeastern Naturalist Vol. 6, No. 1 2003 2004 A. Beech F. Fir E. Hemlock A. Beech F. Fir E. Hemlock Taxa DA H DHDHDHDHDH Fusarium nivale (Fr.) Ces. 3.3 0.0 3.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 F. solani (Mart.) Sacc. 10.0 1.7 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 F. sambusinum Fuckel 6.7 0.0 1.7 3.3 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 F. semitectum Berk. & Ravenel 0.0 5.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 Fusarium spp. Link:Fr. 11.7 6.7 3.3 6.7 0.0 0.0 20.0 0.0 1.7 3.0 0.0 0.0 Gloeosporium sp.B Desmaz. & Mont. 1.7 0.0 0.0 0.0 1.7 1.7 0.0 0.0 0.0 0.0 0.0 0.0 Hansfordia ovalispora S. J. Hughes 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 Haplographium delicatum Berk. & Br. 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 1.7 0.0 3.3 0.0 Hapalosphaeria sp. Syd. in Died. & Syd. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 Hyalodendron lignicola Diddens. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 Humicola spp. Tragen. 1.7 0.0 0.0 0.0 0.0 3.0 3.3 0.0 0.0 0.0 0.0 0.0 Libertella faginea Desmaz. 5.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 Melanconium atrum Link 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Memnoniella echinata (Rivolta) G. Sm. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.3 1.7 0.0 Monochaetia concentrica (Berk. & Br.) Sacc. 5.0 5.5 6.0 3.8 2.5 9.5 0.0 0.0 0.0 0.0 0.0 0.0 Nigrospora sphaerica (Sacc.) E. Mason 8.3 10.0 11.7 10.8 3.3 1.7 6.7 15.0 3.3 3.3 0.0 1.7 Nodulisporium spp. G. Preuss 0.0 3.3 1.7 0.0 15.0 6.7 0.0 0.0 0.0 0.0 0.0 0.0 Paecilomyces sp. Bainier 0.0 0.0 0.0 0.0 0.0 5.0 0.0 0.0 0.0 0.0 1.7 0.0 Papulospora sepedonioidesB G. Preuss 3.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Penicillium spp. Link:Fr. 5.0 13.3 23.0 30.0 33.0 30.0 0.0 1.7 8.3 0.0 8.3 0.0 Penicillium oxalicum Currie & Thom 1.7 1.7 5.0 1.7 28.3 16.6 1.7 1.7 0.0 0.0 10 6.7 P. lividum Westling 0.0 3.3 1.7 0.0 15.0 6.7 1.7 3.3 8.3 5.0 5.0 3.3 P. arenicola Chalabuda 0.0 0.0 0.0 0.0 5.0 0.0 0.0 1.7 8.3 0.0 8.3 0.0 P. islandicum Sopp. 0.0 0.0 0.0 0.0 0.0 1.7 0.0 1.7 0.0 0.0 5.0 3.3 Penicillium sp. A 0.0 1.7 0.0 0.0 10.0 0.0 0.0 0.0 5.0 0.0 1.7 3.4 Periconiella verrucosa E. Stewart & M. corden 0.0 0.0 0.0 0.0 0.0 0.0 3.3 3.3 0.0 0.0 0.0 0.0 Pestalotia clavipora (Atk.) Steyaert 15.0 18.0 0.0 0.0 5.0 15.0 15.0 10.0 0.0 0.0 10.0 20.0 2007 R.E. Baird, C.E. Watson, and S. Woolfolk 81 2003 2004 A. Beech F. Fir E. Hemlock A. Beech F. Fir E. Hemlock Taxa DA H DHDHDHDHDH Pestalotia funerea (Desmaz.) Steyaert 0.0 0.0 12.3 7.0 0.0 0.0 0.0 0.0 3.3 1.7 0.0 0.0 Phialophora verrucosa Medlar 0.0 0.0 0.0 0.0 0.0 0.0 1.7 5.0 1.7 1.7 6.7 3.3 Phoma dura Sacc. 5.0 1.7 0.0 0.0 3.3 0.0 0.0 6.2 0.0 0.0 0.0 Phomopsis occultra (Sacc.) Traverso 0.0 0.0 0.0 0.0 1.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 P. diachenii Sacc. 1.7 5.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 Pithomyces atro-olivaceous (Cooke & Harkn.) M. B. Ellis 0.0 0.0 0.0 1.7 0.0 3.3 0.0 0.0 1.7 0.0 0.0 0.0 Pleospora sp. Rabenh. ex Ces. & De Not. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 13.3 0.0 0.0 Rhinocladiella atrovirens Nannf. in Melin & Nannf. 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 1.7 0.0 0.0 0.0 Rhizoctonia (CAG) D.C. 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Scolicosporium macrosporium (Berk.) Sutton 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 0.0 Septonema faciculare (Corda) S. J. Hughes 0.0 0.0 6.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Sphaeropsis sapineab (Fr.:Fr.) Dyko & Sutton in Sutton 0.0 1.7 0.0 13.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Sporothrix schenckii Hektoen & Perkins 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 Stigmella sp. Lév. In Demidoff 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 Stillbospora sp. Pers. ex Mérat. 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Thysanophora canadensis Stolk & Hennebert 0.0 0.0 0.0 0.0 0.0 3.3 0.0 0.0 0.0 0.0 10.0 0.0 Trichoderma spp. Pers.:Fr. 25.0 48.3 38.3 43.3 48.3 66.7 0.0 0.0 0.0 1.7 3.3 8.3 T. aggressivum Samuels & W. Gams 0.0 0.0 0.0 0.0 0.0 0.0 25.0 17.5 13.3 25 26.7 21.7 Trichoderma atroviride P. Karst. 0.0 0.0 0.0 0.0 0.0 0.0 6.7 7.5 8.3 10.0 11.7 13.3 T. aureoviride Rafai 10.0 13.0 0.0 11.7 23.3 16.7 33.3 8.3 31.7 15.0 26.7 35.0 T. cremeum Chaverri & Samuels 0.0 0.0 0.0 0.0 0.0 0.0 8.3 22.5 6.7 16.7 15.0 15.0 T. ghanense Y. Doi, Y. Abe & J. Sugiyama 0.0 0.0 0.0 0.0 0.0 0.0 6.7 15.0 15.0 16.7 20.0 13.3 T. koningii Oudem. 3.3 13.3 1.7 10.0 15.0 18.3 30.0 31.7 30.0 11.7 31.7 21.7 T. hamatum (Bon.) Bain. 6.7 13.3 6.7 20.0 13.3 5.0 10.0 15.0 21.7 21.7 38.3 25.0 T. harzianum Rifai 15.0 20.0 25.0 21.7 15.0 0.0 38.3 20.0 38.3 45.0 48.3 58.3 T. polysporum (Link:Fr.) Rifai 0.0 0.0 0.0 0.0 0.0 0.0 7.5 7.5 7.5 7.5 8.3 5.0 T. strigosum Bissett 0.0 0.0 0.0 0.0 0.0 0.0 15.0 5.0 2.5 5.0 5.0 7.5 82 Southeastern Naturalist Vol. 6, No. 1 2003 2004 A. Beech F. Fir E. Hemlock A. Beech F. Fir E. Hemlock Taxa DA H DHDHDHDHDH Trichoderma virens (J. Miller et al.) Arx 0.0 0.0 0.0 0.0 0.0 0.0 18.3 23.3 25.0 11.7 33.3 6.7 T. viride Pers.:Fr. 0.0 1.7 0.0 0.0 0.0 0.0 21.7 18.3 26.7 33.3 48.3 51.7 Trichoderma sp. A 0.0 1.7 0.0 0.0 0.0 0.0 15.0 0.0 0.0 0.0 0.0 0.0 Truncatella sp. Stey. 5.0 6.5 5.0 4.2 4.2 6.5 0.0 0.0 0.0 0.0 0.0 0.0 Verticillium sp. Nees. 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Virgaria nigra (Link) Nees. 0.0 0.0 0.0 1.7 0.0 0.0 25.0 28.3 13.3 3.3 3.3 1.7 Ascomycetes Chaetomium spirale Zorf 3.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.7 Gelasinospora tetrasperma Dowding 1.7 3.3 5.0 1.7 0.0 0.0 3.3 0.0 0.0 10 0.0 0.0 Nectria coccinea var. faginata (Lohman, A.M. Watson & Ayers)0.0 1.7 0.0 0.0 0.0 0.0 3.3 1.7 0.0 0.0 0.0 0.0 Other spp. 0.0 0.0 0.0 1.7 1.7 3.3 0.0 0.0 0.0 0.0 1.7 0.0 Zygomycetes Cunninghamella sp. Matr. 0.0 0.0 0.0 8.3 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 Mucor microsporus Naumov. 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 1.7 0.0 0.0 0.0 Mortierella sp. Coem. 0.0 0.0 1.7 0.0 0.0 0.0 0.0 1.7 1.7 6.7 0.0 0.0 Rhizopus arrhizus A. Fisher 0.0 0.0 0.0 1.7 0.0 1.7 3.3 3.3 0.0 1.7 0.0 0.0 R. monosporus Tiegh. 0.0 0.0 0.0 1.7 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 R. solonifera (Ehrenb.:Fr.) Vuill. 0.0 0.0 1.7 2.4 3.3 1.7 3.3 6.7 0.0 3.3 0.0 0.0 Rhizopus sp. Ehrenb. 0.0 0.0 0.0 0.0 0.0 0.0 1.7 0.0 0.0 0.0 1.7 0.0 Basidiomycetes 0.0 5.0 1.7 5.0 5.0 3.3 0.0 3.3 3.3 11.7 1.7 0.0 Unknowns 2.0 1.0 0.0 0.0 3.3 0.0 10.0 18.3 10.0 15.0 6.7 0.0 AD = damaged bark from infected/infested trees, H = bark from healthy trees. BPetersen, R. 1979. Checklist of fungi GSMNP, Management Report # 29. CPercent occurrence is based on isolation of a fungus from 60.0 bark samples per year covering 3 sampling dates, 5 replicate trees (infested or damaged), and 4 pieces per replicate tree.