Ectomycorrhizal fungal community assembly on regenerating Douglas-fir after wildfire and clearcut harvesting.

Wildfire severity in forests is projected to increase with warming and drying conditions associated with climate change. Our objective was to determine the impact of wildfire and clearcutting severity on the ectomycorrhizal fungal (EMF) community of Douglas-fir seedlings in the dry forests of interior British Columbia, Canada. We located our study within and surrounding the area of the McLure fire (August 2003). We hypothesized that disturbance would affect EMF community assembly due to reductions in fungal inoculum. Five treatments representing a range of disturbance severities were compared: high severity burn, low severity burn, screefed clearcut (manual removal of forest floor), clearcut, and undisturbed forest. EMF communities in the undisturbed forest were more complex than those in all disturbance treatments. However, aspects of community assembly varied with disturbance type, where the burn treatments had the simplest communities. After 4 months, regenerating seedlings in the burn treatments had the lowest colonization, but seedlings in all treatments were fully colonized within 1 year. EMF communities were similar among the four disturbance types, largely due to dominance of Wilcoxina throughout the study period. However, forest floor retention influenced community assembly as the EMF in the clearcut treatment, where forest floor was retained, had levels of diversity and richness comparable to the undisturbed forest. Overall, the results suggest that increasing forest floor disturbance can alter EMF community assembly in the first year of regeneration. A correlation between poorly colonized seedlings and seedling productivity also suggests a role for productivity in influencing community assembly.

Rediscovery of the vesicles that characterized Rhizopogon vesiculosus.

Molecular distinction between Rhizopogon vinicolor and R. vesiculosus has been made recently, but the diagnostic "yellow-brown (fresh) inflated cells" of R. vesiculosus, originally described by AH Smith, were not observed. These distinctive hyphal cells (vesicles) have not been reported since the type description. In that description they were said to collapse upon drying and described as being difficult to find. Here we report the rediscovery of these vesicles and describe their specific location on sporocarps of R. vesiculosus. We also report an original discovery that coiled, dark-walled hyphae on the sporocarps and in the mycorrhizae of R. vinicolor are of taxonomic value. The coiled hyphae, combined with the presence or absence of the elusive vesicles, allow R. vesiculosus and R. vinicolor to be morphologically distinguished with increased accuracy.

Diversity and species distribution of ectomycorrhizal fungi along productivity gradients of a southern boreal forest.

Coniferous forests with diverse ectomycorrhizal fungus (EMF) communities are associated with nutrient-poor, acidic soils but there is some debate whether EMF can be equally adapted to more productive, nitrogen-rich sites. We compared EMF species distribution and diversity along a replicated productivity gradient in a southern boreal forest of British Columbia (Canada). Roots from subalpine fir (Abies lasiocarpa) saplings of the understory were sampled and EMF species were identified by morphotypes supplemented with ITS rDNA analysis. There were significant changes in the distribution and abundance of 74 EMF species along the productivity gradient, with as little as 24% community similarity among contrasting sites. Species richness per plot increased asymptotically with foliar nitrogen concentrations of subalpine fir, demonstrating that many EMF species were well suited to soils with high rates of nitrogen mineralization. EMF species abundance in relation to site productivity included parabolic, negative linear, and positive exponential curves. Both multi-site and more narrowly distributed EMF were documented, and a diverse mix of mantle exploration types was present across the entire productivity gradient. The results demonstrate strong associations of EMF fungal species with edaphic characteristics, especially nitrogen availability, and a specialization in EMF communities that may contribute to the successful exploitation of such contrasting extremes in soil fertility by a single tree host.

Decomposition of 14 C-labelled substrates by ectomycorrhizal fungi in association with Douglas fir.

Ectomycorrhizas of Pseudotsuga menziesii (Mirb.) Franco were synthesized in pure culture with Cenococcum geophilum Fr., Laccaria bicolor (Maire) Orton, Rhizopogon vinicolor Smith, or Suillus lakei (Murr.) Smith and Thiers. The mycorrhizal plants were grown in sterile vessels with peat/vermiculite plus 14 C labelled hemicellulose, cellulose, Douglas fir needles, or humic polymers as an organic substrate. The sum of the 14 C respired and that found in the seedling after 240 d of growth was used to quantify decomposition of the labelled substrates. All mycobionts released at least 20-30% of the 14 C in hemicellulose and all were able to release 14 C from cellulose and needles, escept L. buolttr, which failed to release 14 C from needles. S. lakei released the largest amount of 14 C from cellulose. C. geophilum, S. lakei and R. filticolor released similar amounts of 14 C from needles. Generally, substrates could be ranked in terms of ease of breakdown by the ectomycorrhizal fungi as follows: humic polymers < needles < cellulose < hemicellulose.

Morphological changes and photosynthate allocation in ageing Hebeloma crustuliniforme (Bull.) Quel. and Laccaria bicolor (Maire) Orton mycorrhizas of Pinus ponderosa Dougl. ex. Laws.

Seedlings of Pinus Ponderosa Dougl, ex. Laws were grown in root observation containers. They were inoculated with either Hebeloma crustuliniforme (Bull.) Quel. or Laccaria bicolor (Maire) Orton or left uninoculated (control). Oil a monthly basis starting at the eighth month of a 12-month growing period, roots (mycorrhizal root tips from inoculated plants, non-mycorrhizal from control plants) were traced onto acetate sheets. Each root tip was classified as light, 'intermediate' or 'dark' in colour. Roots initiated between months eight and nine were monitored for the next 90 d. All root tips progressed from light-brown to dark-brown to black for all three treatments. Hebeloma crustuliniforme, and to a lesser extent L. bicolor, retarded this progression relative to the control. At the end of 12 months, seedlings were labelled with 14 CO2 to determine the effects of ectomycorrhizal inoculation on carbon supply to roots of the different morphological categories. The amount of 14 C in 'light'L. bicolor and H. crustuliniforme mycorrhizas was 2.3 and 1.8 times greater, respectively, than that in 'light' control root tips. The amount of 14 C in mycorrhizas of the inoculated treatments and, to a lesser extent, roots of the control seedlings decreased as they progressed from 'light' to 'dark'. It is concluded that pondersa pine seedlings continue to allocate photosynthate to morphologically older roots, perhaps to meet maintenance requirements or to supply carbon for growth and metabolism of extra-matrical hyphae. Such allocation may enhance root longevity, which would have an important influence on tree, forest and soil carbon budgets.

Allocation of 14 C-carbon in ectomycorrhizal willow.

The flow of carbon from plant to fungus in ectomycorrhizal associations has not been well quantified. The objective of this study was to use 14 C to quantify the increase in fixed carbon translocated below ground in ectomycorrhizal relative to non-mycorrhizal willow (Salix viminalis L, Bowles hybrid). Rooted cuttings were inoculated with Thelephora terrestris (Ehrh). Fr. or left non-mycorrhizal. Non-mycorrhizal plants were grown at the same (4 mg kg-l bicarbonate-extractable P) (NM-P) or at a higher (21 mg kg-1 ) soil P concentration (NM +P), one at which the non-mycorrhizal plants were similar in size to the mycorrhizal (M-P) plants. At 41, 51, 76 and 89 days after planting, the shoots were exposed to a pulse of 14 CO2 . Plants were harvested after a 202 h chase period. The 14 Cactivity was quantified in live fractions: shoot tissue, shoot respiration, 'root' tissue (= roots plus fungi), 'root' respiration (= CO2 released below ground) and soil. Of the total 14 C detected in these five fractions, M-P plants allocated from 3.9% (harvest 1) to 11.5% (harvest 4) more to the below-ground fractions ('root' tissue, soil and 'root' respiration), than did the NM-P plants. Differences between NM+P and M-P plants were only half of those above (3.1 % and 4.4% at harvests 2 and 3, respectively, compared to 6.4 % and 7.4%, respectively for the difference between NM-P and M-P). Correction for differences in root/shoot ratio between M-P and NM-P plants eliminated the observed differences in carbon distribution only at the first three harvests. There was no evidence for increased 'root' respiration fates or rhizodeposition being responsible for the increased carbon diverted below ground by M-P plants.

Carbon use efficiency in mycorrhizas theory and sample calculations.

The benefit to a fungus by a mycorrhizal association is that it gains carbon from its host. A benefit to a host is usually a nutritional one, but any resulting increase in dry weight may be counteracted by the carbon consumed by the fungus. The carbon costs of mycorrhizal fungi have been calculated using 14 C pulse-chase experiments in the laboratory or by estimating turnover rates in the field. Both of these techniques have their limitations, but estimates have been remarkably consistent amongst most laboratory studies. Carbon demands of the fungus may not reduce theoretical growth enhancement in plants which are sink-limited but would be expected to do so iii source-limited plants. A model of carbon use efficiency is developed based on the economic concepts of cost and benefit. Efficiency is defined in terms of carbon gained via the growth response to infection, and the carbon expended supporting the fungus. Practical considerations of measuring carbon allocation, and calculating carbon use efficiency are discussed. In an experiment on Salix viminalis L., colonized by Thelephora terrestris (Ehrh.) Fr., the carbon use efficiency calculated by this method was on overage 85% based on shoot tissue production, and 95% based on whole plant production.

Fluxes of carbon and phosphorus between symbionts in willow ectomycorrhizas and their changes with time.

One way of viewing a mycorrhizal symbiosis is as a balance between the nutritional 'benefits' and carbon 'costs' to the phytobiont. Phosphorus acquisition efficiency (the amount of phosphorus taken up per unit of carbon allocated belowground) can be used as an indicator of this balance. In this study, phosphorus uptake and belowground carbon allocation were measured using ectomycorrhizal (M) (Thelephora terrestris (Ehrh.) Fr.) and non-mycorrhizal (NM) Salix viminalis L. cv. Bowles Hybrid. Following 50, 60, 85 or 98 d of growth in a gamma-irradiated soil/sand mixture containing 4 mg bicarbunate-extractable P kg-1 , seven randomly-selected cuttings of each treatment were harvested and their P contents determined. Nine d prior to each harvest, the three median plants from the group of seven were pulse labelled with 14 C to determine the relative allocation of C aboveground and belowground. Mycorrhizal colonization of willow caused a two-fold increase in growth owing to substantially higher P uptake. Phosphorus inflow rates were almost three times as high for M root systems as for NM root systems over the interval up to the first harvest (3.2 × 10 -12 and 1.2 × 1012 mol m-1 s-1, respectively). Over the interval from 50 to 98 d, inflows into M plants were 50% higher than into NM plants (1.4 × 1012 and 0.9 × 10-13 mol m-1 s-1 respectively). The M plants allocated about 25 times as much carbon belowground as the NM plants for both periods. The P acquisition efficiency was higher in M than in NM plants during the first interval (16% and 40% higher using two different calculation methods), whereas during the second interval it was higher in NM than in M plants (33% and 44% higher using the two different methods). Thus, ectomycorrhizas can be very effective in supplying P to their hosts even at an early stage of infection. Furthermore, it is suggested that a temporal separation exists in the maximal fluxes of P and C between the fungus and the host of the mycorrhizal association. The results are discussed in the context of the nutrient requirements and carbon economies of field-grown woody plants.

Phosphorus relationships and production of extrametrical hyphae by two types of willow ectomycorrhizas at different soil phosphorus levels.

There is much circumstantial evidence for a role of increased P uptake in the growth response of plants to ectomycorrhizas. Full response curves with and without mycorrhizal infection along a P gradient in soil are, however, required to test this hypothesis fully. In this experiment, rooted cuttings of Salix viminalis L. cv. Bowles Hybrid were grown in a 1:2 mixture by volume of gamma-irradiated soil and sterile sand, with bicarbonate-extractable P concentrations of 4, 6, 10, 21, 60 or 90 mg P kg-1 . The cuttings were inoculated by mixing peat/vermiculite spawn of Laccaria proxima (Boud.) Pat., or Thelephora terrestris (Ehrh.) Fr., or autoclaved spawn 1: 5 by volume with the soil: sand mixture. The plants showed a positive growth response to mycorrhizal infection by either fungus at the two lower P levels, and to L. proxima only at 10 mg P kg-1 . At 21 mg kg-1 and above, infection was reduced and neither mycorrhizal inoculation nor further P additions caused significant growth increases. These results imply that the growth responses to ectomycorrhizas in this experiment were solely due to increases in P uptake. Cuttings infected with L. proxima tended to be larger than those infected by T. terrestris. Estimates of percent mycorrhizal infection did not differ between the fungi at the lower P levels. However, overall production of extramatrical hyphae per gram of soil was highest in soil inoculated with L. proxima. At 10 mg P kg-1 the length of L. proxima hyphae per unit length of mycorrhizal root, P uptake per unit root weight, and total P content in plants infected with L. proxima were significantly higher than for T. terrestris. This study confirms that increased P uptake can be an important component of growth stimulation by ectomycorrhizas. It also presents the first quantification of extramatrical ectomycorrhizal hyphae in soil and suggests a role for them in the growth response.

Temporal allocation of (14)C to extramatrical hyphae of ectomycorrhizal ponderosa pine seedlings.

Ponderosa pine seedlings were inoculated with Hebeloma crustuliniforme either in growth pouches before they were transplanted to root-mycocosms (P seedlings), or at the time of transfer to root-mycocosms (V seedlings). Uninoculated seedlings served as controls (U seedlings). The use of root-mycocosms allowed examination of portions of hyphae separate from roots and rooting substrate but still in symbiosis with the host. The results thus provided a quantitative basis for estimating hyphal mass and carbon allocation to extramatrical hyphae. The amount of (14)CO(2) fixed after a 2-h exposure was greatest for P seedlings and least for uninoculated seedlings. Four and nine days after exposure, (14)C content was greatest in uninoculated seedlings and least in inoculated seedlings. In isotope distribution and dry mass accumulation, V seedlings were more similar to U than to P seedlings. Calculated on a dry weight basis, the allocation of isotope to mycelium suggested that extramatrical hyphae of P seedlings were a stronger sink for carbon than extramatrical hyphae of V seedlings. Differences in inoculation methods resulted in differences in carbon allocation and physiology of extramatrical hyphae that could affect seedling establishment and survival. Seedlings inoculated by one method cannot serve as surrogates for mycorrhizal seedlings produced by other inoculation techniques.

Effects of ozone and sulfur dioxide on phyllosphere fungi from three tree species.

Short-term effects of ozone (O(3)) on phyllosphere fungi were studied by examining fungal populations from leaves of giant sequoia (Sequoiadendron giganteum (Lindl.) Buchholz) and California black oak (Quercus kelloggii Newb.). Chronic effects of both O(3) and sulfur dioxide (SO(2)) were studied by isolating fungi from leaves of mature Valencia orange (Citrus sinensis L.) trees. In this chronic-exposure experiment, mature orange trees were fumigated in open-top chambers at the University of California, Riverside, for 4 years with filtered air, ambient air plus filtered air (1:1), ambient air, or filtered air plus SO(2) at 9.3 parts per hundred million. Populations of Alternaria alternata (Fr.) Keissler and Cladosporium cladosporioides (Fres.) de Vries, two of the four most common fungi isolated from orange leaves, were significantly reduced by chronic exposure to ambient air. In the short-term experiments, seedlings of giant sequoia or California black oak were fumigated in open-top chambers in Sequoia National Park for 9 to 11 weeks with filtered air, ambient air, or ambient air plus O(3). These short-term fumigations did not significantly affect the numbers of phyllosphere fungi. Exposure of Valencia orange trees to SO(2) at 9.3 parts per hundred million for 4 years reduced the number of phyllosphere fungi isolated by 75% compared with the number from the filtered-air treatment and reduced the Simpson diversity index value from 3.3 to 2.5. A significant chamber effect was evident since leaves of giant sequoia and California black oak located outside of chambers had more phyllosphere fungi than did seedlings within chambers. Results suggest that chronic exposure to ambient ozone or SO(2) in polluted areas can affect phyllosphere fungal communities, while short-term exposures may not significantly disturb phyllosphere fungi.