Ectomycorrhizal fungi slow soil carbon cycling.

Respiration of soil organic carbon is one of the largest fluxes of CO2 on earth. Understanding the processes that regulate soil respiration is critical for predicting future climate. Recent work has suggested that soil carbon respiration may be reduced by competition for nitrogen between symbiotic ectomycorrhizal fungi that associate with plant roots and free-living microbial decomposers, which is consistent with increased soil carbon storage in ectomycorrhizal ecosystems globally. However, experimental tests of the mycorrhizal competition hypothesis are lacking. Here we show that ectomycorrhizal roots and hyphae decrease soil carbon respiration rates by up to 67% under field conditions in two separate field exclusion experiments, and this likely occurs via competition for soil nitrogen, an effect larger than 2 °C soil warming. These findings support mycorrhizal competition for nitrogen as an independent driver of soil carbon balance and demonstrate the need to understand microbial community interactions to predict ecosystem feedbacks to global climate.

An endophytic Coniochaeta velutina producing broad spectrum antimycotics.

An endophyte (PC27-5) was isolated from stem tissue of Western hemlock (Tsuga heterophylla) in a Pacific Northwest temperate rainforest. Phylogenetic analyses, based on ITS-5.8S rDNA and 18S rDNA sequence data, combined with cultural and morphological analysis showed that endophyte PC27-5 exhibited all characteristics of a fungus identical to Coniochaeta velutina. Furthermore, wide spectrum antimycotics were produced by this endophyte that were active against such plant pathogens as Sclerotinia sclerotiorum, Pythium ultimum, and Verticillium dahliae and lethal to Phythophthora cinnamomi, Pythium ultimum, and Phytophthora palmivora in plate tests. The bioactive components were purified through organic solvent extraction, followed by silica column chromatography, and finally preparative HPLC. The minimum inhibitory concentration of the active fraction to Pythium ultimum, which was gained from preparative HPLC, was 11 µg/ml. UPLC-HRMS analysis showed there were two similar components in the antimycotic fraction. Their molecular formulae were established as C30H22O11 (compound I) and C30H22O10 (compound II) respectively, and preliminary spectral results indicate that they are anthroquinone glycosides. Other non-biologically active compounds were identified in culture fluids of this fungus by spectral means as emodin and chrysophanol--anthroquinone derivatives. This is the first report that Coniochaeta velutina as an endophyte produces bioactive antifungal components.

Characterization of an Endophytic Gloeosporium sp. and Its Novel Bioactivity with "Synergistans".

Gloeosporium sp. (OR-10) was isolated as an endophyte of Tsuga heterophylla (Western hemlock). Both ITS and 18S sequence analyses indicated that the organism best fits either Hypocrea spp. or Trichoderma spp., but neither of these organisms possess conidiophores associated with acervuli, in which case the endophytic isolate OR-10 does. Therefore, the preferred taxonomic assignment was primarily based on the morphological features of the organism as one belonging to the genus Gloeosporium sp. These taxonomic observations clearly point out that limited ITS and 18S sequence information can be misleading when solely used in making taxonomic assignments. The volatile phase of this endophyte was active against a number of plant pathogenic fungi including Phytophthora palmivora, Rhizoctonia solani, Ceratocystis ulmi, Botrytis cinerea, and Verticillium dahliae. Among several terpenes and furans, the most abundantly produced compound in the volatile phase was 6-pentyl-2H-pyran-2-one, a compound possessing antimicrobial activities. When used in conjunction with microliter amounts of any in a series of esters or isobutyric acid, an enhanced inhibitory response occurred with each test fungus that was greater than that exhibited by Gloeosporium sp. or the compounds tested individually. Compounds behaving in this manner are hereby designated "synergistans." An expression of the "median synergistic effect," under prescribed conditions, has been termed the mSE50. This value describes the amount of a potential synergistan that is required to yield an additional median 50% inhibition of a target organism. In this report, the mSE50s are reported for a series of esters and isobutyric acid. The results indicated that isoamyl acetate, allyl acetate, and isobutyric acid generally possessed the lowest mSE50 values. The value and potential importance of these microbial synergistic effects to the microbial environment are also discussed.

Sharing rotting wood in the shade: ectomycorrhizal communities of co-occurring birch and hemlock seedlings.

Coarse woody debris (CWD) is an important nursery environment for many tree species. Understanding the communities of ectomycorrhizal fungi (ECMF)and the effect of ECMF species on tree seedling condition in CWD will elucidate the potential for ECMF-mediated effects on seedling dynamics. In hemlock-dominated stands, we characterized ECMF communities associated with eastern hemlock (Tsuga canadensis (L.) Carr) and yellow birch (Betula alleghaniensis Britt) seedling pairs growing on CWD. Seedling foliage and CWD were analyzed chemically, and seedling growth, canopy cover, and canopy species determined. Thirteen fungal taxa, 12 associated with birch, and 6 with hemlock, were identified based on morphology and ITS sequencing. Five species were shared by co-occurring birch and hemlock, representing 75% of ectomycorrhizal root tips. Rarified ECMF taxon richness per seedling was higher on birch than hemlock. Nonmetric multidimensional scaling revealed significant correlations between ordination axes, the mutually exclusive ECMF Tomentella and Lactarius spp., foliar N and K, CWD pH, and exchangeable Ca and Mg. Seedlings colonized by Lactarius and T. sublilacina differed significantly in foliar K and N, and CWD differed in exchangeable Ca and Mg. CWD pH and nutrient concentrations were low but foliar macro-nutrient concentrations were not. We hypothesize that the dominant ECMF are adapted to low root carbohydrate availability typical in shaded environments but differ in their relative supply of different nutrients.

Fine-scale distribution of ectomycorrhizal fungi colonizing Tsuga diversifolia seedlings growing on rocks in a subalpine Abies veitchii forest.

Numerous species of ectomycorrhizal (ECM) fungi coexist under the forest floor. To explore the mechanisms of coexistence, we investigated the fine-scale distribution of ECM fungal species colonizing root tips in the root system of Tsuga diversifolia seedlings in a subalpine forest. ECM root tips of three seedlings growing on the flat top surface of rocks were sampled after recording their positions in the root system. After the root tips were grouped by terminal-restriction fragment length polymorphism (T-RFLP) analysis of ITS rDNA, the fungal species representing each T-RFLP group were identified using DNA sequencing. Based on the fungal species identification, the distribution of root tips colonized by each ECM fungus was mapped. Significant clustering of root tips was estimated for each fungal species by comparing actual and randomly simulated distributions. In total, the three seedlings were colonized by 40 ECM fungal species. The composition of colonizing fungal species was quite different among the seedlings. Twelve of the 15 major ECM fungal species clustered significantly within a few centimeters. Some clusters overlapped or intermingled, while others were unique. Areas with high fungal species diversity were also identified in the root system. In this report, the mechanisms underlying generation of these ECM root tip clusters in the root system are discussed.

Phylogenetic structure of ectomycorrhizal fungal communities of western hemlock changes with forest age and stand type.

On Vancouver Island, British Columbia, fertilization with nitrogen (N) and phosphorus (P) following clearcutting increases growth of western hemlock. To explore whether fertilization also resulted in ectomycorrhizal fungal communities that were more or less similar to neighboring unlogged stands, we sampled roots from western hemlock from three replicate plots from each of five different, well-characterized, forest stand types that differed in site type, and in logging and fertilization history. We harvested four samples of 100 ectomycorrhizal root tips from each plot, a total of 60 samples per stand type. From each sample, we analyzed fungal ribosomal internal transcribed spacers and 28S DNA, sequencing 15-29 clones per sample and 60-116 clones per plot. We detected 147 fungal operational taxonomic units among a total of 1435 sequences. Craterellus tubaeformis was frequently present and resulted in a pattern of phylogenetic overdispersion in the fungal communities. Fungal species composition was strongly correlated with foliar nitrogen concentration. However, other site quality factors were also important because the fertilized regenerating hemlock and mature hemlock-amabilis fir forests had similar foliar nitrogen content but little overlap in fungal species. Compared with unfertilized regenerating forests, fungal communities in N + P-fertilized regenerating forests had significantly more species overlap with old growth forests. However, the fungal communities of all regenerating forest were similar to one another and all differed significantly from older forests. By correlating fungal clades with habitats, this research improves understanding of how forest management can contribute to maintaining diverse ectomycorrhizal fungal communities across a landscape.

Characterization of a strong CCA-treated wood degrader, unknown Crustoderma species.

In this study, basidiomycete isolates that possessed a strong ability to degrade chromated copper arsenate (CCA)-treated wood were characterized. These fungal isolates, which were collected from CCA-treated pine log wastes, showed no recognizable morphological properties on culture media. Nucleotide sequence analysis of the large subunit rDNA of the isolates revealed that they were one species. Based on the high sequence similarity (>95%) and close phylogenetic relationship with several known species of Crustoderma, the fungal isolates characterized in this study were classified as a Crustoderma sp. In a wood degradation test, Crustoderma isolate KUC8611 produced a remarkably higher weight loss in CCA-treated Pinus radiata (68.7%), Pseudotsuga menziesii (39.7%), and Tsuga heterophylla (38.5%) wood than other evaluated basidiomycete species, including Crustoderma flavescens and Crustoderma corneum. In addition, extracellular enzymes for cellulose and protein degradation were detected when the isolates were cultured in chromogenic media, which supports the finding that isolate KUC8611 is a wood degrader. Furthermore, an in vitro test for metal tolerance revealed that isolate KUC8611 showed strong arsenic tolerance, but that it could not tolerate copper. Finally, isolate KUC8611 produced lower amounts of oxalic acid than copper-tolerant fungi such as Fomitopsis palustris and Antrodia vaillantii. To the best of our knowledge, this is the first study to report the degradation of CCA-treated wood by a Crustoderma species.

The effect of fertilization on the below-ground diversity and community composition of ectomycorrhizal fungi associated with western hemlock (Tsuga heterophylla).

Fertilization typically reduces ectomycorrhizal diversity shortly after its application but less is known about its longer-term influence on fungal species. Long-term effects are important in forests where fertilizer is rarely applied. We compared fungal species composition in western hemlock control plots with plots last fertilized 7 years ago with nitrogen (N) or nitrogen plus phosphorus (N + P). The N + P fertilization had a significant lingering effect, increasing the tree size and foliar P content of the western hemlocks. From ectomycorrhizal roots of 24-year-old trees from northern Vancouver Island, Canada, we identified fungi from 12 samples per treatment, by amplifying, cloning, and sequencing fungal ribosomal DNA fragments, placing sequences with 97% or more identity in the same operational taxonomic unit (OTU). Diversity was high across treatments; we detected 77 fungal OTUs, 52 from ectomycorrhizal genera, among 922 clone sequences. The five most frequent OTUs were similar in abundance across treatments. Only 19 OTUs matched any of the 197 previously reported ectomycorrhizal species of western hemlock. Species composition but not diversity in nitrogen plus phosphorus plots differed significantly from control or nitrogen plots. Two Cortinarius OTUs were indicator species for nitrogen plus phosphorus plots and presence of Cortinarius cinnamomeus was correlated with control or nitrogen plots. After 7 years, fertilization history had made no detectable difference in ectomycorrhizal fungal diversity, but long-lasting changes in environment resulting from fertilization had a lingering effect on fungal ectomycorrhizal species composition.

Forest stand age and the occurrence of chanterelle (Cantharellus) species in Oregon's central Cascade Mountains.

We describe watershed-scale habitat associations of three Cantharellus species with respect to stand age. During the 1998 autumn fruiting season we collected chanterelle sporocarps from 18 forest stands in and adjacent to the H.J. Andrews experimental forest in the central Cascade Mountains of Oregon. Sampled stands represented two age categories: old growth ( approximately 350+ y) and 40-60-y-old second growth naturally regenerated from clear-cut harvest. Old growth and second growth stands were spatially paired to reduce the chance of spurious habitat relationships caused by unmeasured correlated variables. We found stand age to be a good predictor of the distribution of C. subalbidus and C. formosus, but only marginally useful for predicting the occurrence of C. cascadensis. The odds that a randomly located chanterelle sporocarp will be C. subalbidus, compared to other chanterelles, are 3-23.5 times higher in old growth than in second growth. Alternatively, there is only a 4-38% chance that a randomly located sporocarp will be C. formosus in old growth. C. cascadensis was found to be uncommon throughout the study area and showed no significant habitat associations. The abundance of C. cascadensis increased substantially with decreasing elevation indicating that landscape features other than stand age may be more useful in predicting its occurrence.

Species diversity of polyporoid and corticioid fungi in northern hardwood forests with differing management histories.

Effects of forest management on fungal diversity were investigated by sampling fruit bodies of polyporoid and corticioid fungi in forest stands that have different management histories. Fruit bodies were sampled in 15 northern hardwood stands in northern Wisconsin and the upper peninsula of Michigan. Sampling was conducted in five old-growth stands, five uneven-age stands, three even-age unthinned stands and two even-age thinned stands. Plots 100 m x 60 m were established and 3000 m2 within each plot was sampled during the summers of 1996 and 1997. A total of 255 polyporoid and corticioid morphological species were identified, 46 (18%) of which could not be assigned to a described species. Species accumulation curves for sites and management classes differed from straight lines, although variability from year to year suggests that more than 2 y of sampling are needed to characterize annual variation. Mean species richness and diversity index values did not vary significantly by management class, although mean richness on large diameter wood (> or = 15 cm diam) varied with moderate significance. Richness values on small diameter debris varied significantly by year, indicating that a large part of year-to-year variability in total species richness is due to small diameter debris. Ten species had abundance levels that varied by management class. Two of these species. Changes in the diversity and species composition of the wood-inhabiting fungal community could have significant implications for the diversity, health and productivity of forest ecosystems.

Douglas-fir ectomycorrhizae in 40- and 400-year-old stands: mycobiont availability to late successional western hemlock.

We investigated ectomycorrhizal (EM) fungi in forest stands containing both early successional Douglas-fir and late successional western hemlock at two points in the typical stand development by identifying EM fungi from roots of Douglas-fir and western hemlock in mixed stands. In an early seral stage forest, EM roots of western hemlock seedlings and intermingling 40-year-old Douglas-fir were sampled. In a late seral stage forest, EM roots of trees of both species were sampled in a 400-year-old stand. We use molecular approaches to identify the symbionts from field samples in this descriptive study. In the early seral stage study, >95% of the western hemlock root tips by biomass were colonized by fungi also colonizing Douglas-fir roots. This result supports the prediction that western hemlock can associate with fungi in Douglas-fir EM networks. In the same study, fungi specific to Douglas-fir colonized 14% of its EM root tips. In the late seral stage study, 14% of the western hemlock root tips were colonized by fungi also observed in association with Douglas-fir, a result strongly influenced by sampling issues and likely represents a conservative estimate of multiple host fungi in this old growth setting. Fungi specific to Douglas-fir colonized 25% of its root tip biomass in the old growth study, in tight coralloid clusters within five of the 24 soil samples. The trends revealed in this study corroborate earlier studies suggesting a predominance of multiple host fungi in mixed communities of EM plants. The role of host-specific fungi in these stands remains unclear.

Thuja plicata exclusion in ectomycorrhiza-dominated forests: testing the role of inoculum potential of arbuscular mycorrhizal fungi.

The ability of trees dependent on arbuscular mycorrhizal (AM) fungi to establish in ectomycorrhizal forests is unknown. On northern Vancouver Island, Canada, there are sharp boundaries between mixed red cedar (Thuja plicata)-hemlock (Tsuga heterophylla) (CH) stands, and stands of hemlock and amabilis fir (Abies amabilis) (HA). We tested differences in AM colonization of red cedar between ectomycorrhiza-dominated (HA) stands and stands containing red cedar (CH), across a range of light levels. We used a soil bioassay approach to determine whether there was sufficient AM fungal inoculum in the HA tree stands to colonize red cedar seedlings. Seeds of hemlock and red cedar were sown in forest floor samples collected from the two types of forests, and shade treatments ranging from < 1 to 53% of full sunlight were imposed. After 6 months, seedling survival and root and shoot biomass were quantified, and red cedar seedlings were sampled for AM fungal colonization. Hemlock survival and growth did not differ between soil types, suggesting there was no substrate-associated limitation to its establishment in either forest type. Red cedar colonization by AM fungi was significantly correlated with light levels in CH soils but arbuscular mycorrhizas were absent in roots of red cedar seedlings grown in HA soil. Red cedar survival and relative growth rate were significantly greater in the CH than in HA soil; higher growth was due primarily to greater shoot growth in CH soils at high light levels. The low soil inoculum potential for red cedar in ectomycorrhiza-dominated stands may account for the virtual exclusion of red cedar seedlings from these forests.