A nitrogen fertilization field study of carbon-13 and nitrogen-15 transfers in ectomycorrhizas of Pinus sabiniana.

Ectomycorrhizal (EM) fungi form relationships with higher plants; plants transfer C to fungi, and fungi transfer nutrients to their host. While evidence indicates that this interaction is largely mutualistic, less is known about how nutrient supply and EM associates may alter C and nutrient exchanges, especially in intact plant-soil-microbe systems in the field. In a dual-labeling experiment with N fertilization, we used C and N stable isotopes to examine in situ transfers in EM pine trees in a Pinus sabiniana woodland in northern California. We added (15)NH4SO2 and (13)CO2 to track (13)C transfer from pine needles to EM roots and (15)N transfer from soil to EM roots and pine needles. Transfers of (13)C and (15)N differed with EM morphotype and with N fertilization. The brown morphotype received the least C per unit of N transferred (5:1); in contrast red and gold morphotypes gained more C and transferred less N (17:1 and 25:1, respectively). N fertilization increased N retention by ectomycorrhizas (EMs) but did not increase N transfer from EMs to pine needles. Therefore N fertilization positively affected both nutrient and C gains by EMs, increasing net C flows and N retention in EMs. Our work on intact and native trees/EM associations thereby extends earlier conclusions based on pot studies with young plants and culturable EM fungi; our results support the concept that EM-host relationships depend on species-level differences as well as responses to soil resources such as N.

A molecular survey of ectomycorrhizal hyphae in a California Quercus-Pinus woodland.

Ectomycorrhizal (ECM) hyphal communities have not been well characterized. Furthermore, there have been few studies where the ECM hyphal community is compared to fungi detected as sporocarps or ECM-colonized root tips. We investigated fungi present as hyphae in a well-studied California Quercus-Pinus woodland. Hyphal species present were compared to those found as sporocarps and ECM root tips at the same site. Hyphae were extracted from root-restrictive nylon mesh in-growth bags buried in the soil near mature Quercus douglasii, Quercus wislizeni, and Pinus sabiniana. Taxa were identified using PCR, cloning, and DNA sequencing of internal transcribed spacer and 28s rDNA. Among the 33 species detected, rhizomorph-forming ECM fungi dominated the hyphal community, especially species of Thelephoraceae and Boletales. Most fungi in soils near Quercus spp. and P. sabiniana were ECM basidiomycetes, but we detected two ECM ascomycetes and three non-mycorrhizal fungi. Many ECM species present as hyphae were also previously detected at this site as sporocarps (18%) or on ECM root tips (58%). However, the hyphal community was mostly dominated by different taxa than either the sporocarp or ECM root communities.

Contrasting ectomycorrhizal fungal communities on the roots of co-occurring oaks (Quercus spp.) in a California woodland.

Plant host species is considered an important factor influencing ectomycorrhizal (EM) communities. To gain insights into the role of host species in structuring EM communities, EM communities on sympatric oak (Quercus) species were compared in the Sierra Nevada foothills of California. Using molecular methods (polymerase chain reaction, cloning, restriction fragment length polymorphism and DNA sequencing), EM fungi on roots of deciduous Quercus douglasii and evergreen Quercus wislizeni trees were identified from 64 soil cores. The total EM species richness was 140, of which 40 taxa were detected on both oak hosts. Greater diversity and frequency of EM fungi with epigeous fruiting habit were found on Q. wislizeni, while taxa in the Ascomycota were more frequent and diverse on Q. douglasii. Using ordination, it was determined that both soil extractable phosphorus and oak host species explained a significant proportion of the variation in EM species distribution. These results indicate that plant host species can be an important factor influencing EM fungal community composition, even within congeneric trees.

Root hydraulic conductivity and xylem sap levels of zeatin riboside and abscisic acid in ectomycorrhizal Douglas fir seedlings.

Mechanistic hypotheses to explain mycorrhizal enhancement of root hydraulic conductivity (Lp ) suggest that phosphorus (P) nutrition, plant growth substances and/or altered morphology may be responsible. Such ideas are based on work with VA (vesicular-arbuscular) mycorrhizas. Since VA mycorrhizas and ectomycorrhizas differ in many respects, they may alter host plant water uptake via different mechanisms. This paper examines LT in various ectomycorrhizal associations while considering factors which are important to the VA mycorrhizal effect on Lp . Douglas fir Pseudotsuga menziesii (Mirb.) Franco] seedlings inoculated with the ectomycorrhizal fungi Laccaria bicolor (Maire) Orton and Hebeloma crustuliniforme (Bull, ex St. Amans) Quel. and non-inoculated seedlings infected naturally with Thelephora were grown under three low levels of P fertilization (1, 10 and 100 fim P). Seedling morphology, tissue P levels, Lp and plant growth substance levels in xylem sap were measured after nine months growth. Increased tissue P and decreased root/shoot ratio correlated with increased Lp in each of the mycorrhizal treatments. When adjusted for the effect of these two factors, Lp of Laccaria and Hebeloma seedlings was still lower than the Thelephora seedlings. In a subsequent experiment, the Lp of seedlings with Hebeloma and Rhizopogon vinicolor Smith mycorrhizas was compared to the Lp of non-mycorrhizal seedlings (grown at 100 mM P) and no differences were found among treatments. The lack of an ectomycorrhizal effect on Lp is quite different from the enhancement of host Lp by VA mycorrhizas. Zeatin riboside concentrations of Thelephora- and Hebeloma-iniected seedlings were similar, yet higher than with Laccaria. There was no relationship between plant growth substances and Lp in ectomycorrhizal Douglas fir, despite lower zeatin riboside concentrations for Laccaria-inoculated plants.

Influence of host species on ectomycorrhizal communities associated with two co-occurring oaks (Quercus spp.) in a tropical cloud forest.

Interactions between host tree species and ectomycorrhizal fungi are important in structuring ectomycorrhizal communities, but there are only a few studies on host influence of congeneric trees. We investigated ectomycorrhizal community assemblages on roots of deciduous Quercus crassifolia and evergreen Quercus laurina in a tropical montane cloud forest, one of the most endangered tropical forest ecosystems. Ectomycorrhizal fungi were identified by sequencing internal transcribed spacer and partial 28S rRNA gene. We sampled 80 soil cores and documented high ectomycorrhizal diversity with a total of 154 taxa. Canonical correspondence analysis indicated that oak host was significant in explaining some of the variation in ectomycorrhizal communities, despite the fact that the two Quercus species belong to the same red oak lineage (section Lobatae). A Tuber species, found in 23% of the soil cores, was the most frequent taxon. Similar to oak-dominated ectomycorrhizal communities in temperate forests, Thelephoraceae, Russulaceae and Sebacinales were diverse and dominant.

Multiple species of ectomycorrhizal fungi are frequently detected on individual oak root tips in a tropical cloud forest.

The ecological importance of ectomycorrhizal (EM) fungi in tropical ecosystems is increasingly recognized, but few studies have used molecular methods to examine EM fungal communities in tropical forests. The diversity and composition of the EM community on Quercus crassifolia in a tropical montane cloud forest in southern Mexico were characterized using DNA sequencing of single root tips. Individual root tips commonly harbored multiple fungal species that resulted in mixed polymerase chain reaction (PCR) products. By cloning and performing gel extractions on mixed PCR samples, we identified two or more EM fungi on 26% of the root tips. When non-EM fungi were considered, this figure increased to 31% of root tips. A total of 44 EM taxa and nine non-EM taxa were detected on roots from 21 soil cores (104 root tips). Taxa in the families Russulaceae, Cortinariaceae, Inocybaceae, and Thelephoraceae were frequent. This is the first study to characterize the belowground EM community in a tropical montane cloud forest.

Rapid nitrogen transfer from ectomycorrhizal pines to adjacent ectomycorrhizal and arbuscular mycorrhizal plants in a California oak woodland.

Nitrogen transfer among plants in a California oak woodland was examined in a pulse-labeling study using 15N. The study was designed to examine N movement among plants that were mycorrhizal with ectomycorrhizas (EM), arbuscular mycorrhizas (AM), or both. Isotopically enriched N (K15NO3-) was applied to gray pine (Pinus sabiniana) foliage (donor) and traced to neighboring gray pine, blue oak (Quercus douglasii), buckbrush (Ceanothus cuneatus) and herbaceous annuals (Cynosurus echinatus, Torilis arvensis and Trifolium hirtum). After 2 wk, needles of 15N-treated pines and foliage from nearby annuals were similarly enriched, but little 15N had appeared in nontreated (receiver) pine needles, oak leaves or buckbrush foliage. After 4 wk foliar and root samples from pine, oak, buckbrush and annuals were significantly 15N-enriched, regardless of the type of mycorrhizal association. The rate of transfer during the first and second 2-wk periods was similar, and suggests that 15N could continue to be mobilized over longer times.

Contrasting root associated fungi of three common oak-woodland plant species based on molecular identification: host specificity or non-specific amplification?

An increasingly popular approach used to identify arbuscular mycorrhizal (AM) fungi in planta is to amplify a portion of AM fungal small subunit ribosomal DNA (SSU-rDNA) from whole root DNA extractions using the primer pair AM1-NS31, followed by cloning and sequencing. We used this approach to study the AM fungal community composition of three common oak-woodland plant species: a grass (Cynosurus echinatus), blue oak (Quercus douglasii), and a forb (Torilis arvensis). Significant diversity of AM fungi were found in the roots of C. echinatus, which is consistent with previous studies demonstrating a high degree of AM fungal diversity from the roots of various hosts. In contrast, clones from Q. douglasii and T. arvensis were primarily from non-AM fungi of diverse origins within the Ascomycota and Basidiomycota. This work demonstrates that caution must be taken when using this molecular approach to determine in planta AM fungal diversity if non-sequence based methods such as terminal restriction fragment length polymorphisms, denaturing gradient gel electrophoresis, or temperature gradient gel electrophoresis are used.

Contribution of relative growth rate to root foraging by annual and perennial grasses from California oak woodlands.

Plants forage for nutrients by increasing their root length density (RLD) in nutrient-rich soil microsites through root morphological changes resulting in increased root biomass density (RBD), specific root length (SRL), or branching frequency (BF). It is commonly accepted that fast-growing species will forage more than slow-growing species. However, foraging responses may be due solely to differences in relative growth rates (RGR). There is little evidence, after the effects of RGR are removed, that the fast versus slow foraging theory is correct. In a pot study, we evaluated foraging of four grass species that differed in RGR: one fast-growing annual species, Bromus diandrus, two intermediate-growing species, annual Bromus hordeaceus and perennial Elymus glaucus, and one slow-growing perennial species, Nassella pulchra. We harvested plants either at a common time (plants varied in size) or at a common leaf number (plants similar size, surrogate for common biomass). By evaluating species at a common time, RGR influenced foraging. Conversely, by evaluating species at a common leaf number, foraging could be evaluated independent of RGR. When RGR was allowed to contribute to foraging (common time harvest), foraging and RGR were positively correlated. B. diandrus (fast RGR) foraged to a greater extent than did E. glaucus (intermediate RGR) and N. pulchra (slow RGR). E. glaucus (intermediate RGR) foraged to a greater extent than N. pulchra (slow RGR). Root growth within nutrient-rich microsites was due to significant increases in RBD, not to modifications of SRL or BF. However, when RGR was not allowed to influence foraging (common leaf number harvest), none of the four species significantly enhanced RLD in nutrient-rich compared to control microsites. This suggests that RGR strongly influenced the ability of these grass species to forage and also supports the need to evaluate plastic root traits independent of RGR.

Epiphytic and terrestrial mycorrhizas in a lower montane Costa Rican cloud forest.

The epiphyte community is the most diverse plant community in neotropical cloud forests and its collective biomass can exceed that of the terrestrial shrubs and herbs. However, little is known about the role of mycorrhizas in this community. We assessed the mycorrhizal status of epiphytic (Araceae, Clusiaceae, Ericaceae, and Piperaceae) and terrestrial (Clusiaceae, Ericaceae) plants in a lower montane cloud forest in Costa Rica. Arbuscular mycorrhizas were observed in taxa from Araceae and Clusiaceae; ericoid mycorrhizas were observed in ericaceous plants. This is the first report of intracellular hyphal coils characteristic of ericoid mycorrhizas in roots of Cavendishia melastomoides, Disterigma humboldtii, and Gaultheria erecta. Ericaceous roots were also covered by an intermittent hyphal mantle that penetrated between epidermal cells. Mantles, observed uniquely on ericaceous roots, were more abundant on terrestrial than on epiphytic roots. Mantle abundance was negatively correlated with gravimetric soil water content for epiphytic samples. Dark septate endophytic (DSE) fungi colonized roots of all four families. For the common epiphyte D. humboldtii, DSE structures were most abundant on samples collected from exposed microsites in the canopy. The presence of mycorrhizas in all epiphytes except Peperomia sp. suggests that inoculum levels and environmental conditions in the canopy of tropical cloud forests are generally conducive to the formation of mycorrhizas. These may impact nutrient and water dynamics in arboreal ecosystems.