Comparisons of ectomycorrhizae on pinyon pines (Pinus edulis; Pinaceae) across extremes of soil type and herbivory

Using field and greenhouse studies, we examined the relationships among pinyon pines (Pinus edulis), their ectomycorrhizal mutualists, and their moth herbivores as a function of soil fertility. We studied two soil types—the ash and cinder soils of the San Francisco volcanic field and nearby sandy loam soils. In the field, pinyons growing in cinders suffered from reduced moisture, negative nitrogen mineralization rates, low phosphate levels, reduced growth, and high moth herbivory relative to pinyons growing in sandy loam. Pinyons growing in cinders also had twofold higher levels of ectomycorrhizal colonization than their noncinder counterparts. Similarly, in the greenhouse, seedlings grown in cinders had higher levels of ectomycorrhizal colonization and greater numbers of ectomycorrhizae than seedlings grown in sandy loam. Seedling shoot growth was significantly enhanced by ectomycorrhizae in both soils. These patterns support three conclusions. First, field and greenhouse studies demonstrated that trees growing in nutrient-poor soils had higher levels of ectomycorrhizal colonization than trees growing in better soils. Second, across soil types, variation in ectomycorrhizal colonization was better predicted by soil fertility than by herbivory. However, herbivory negatively affected ectomycorrhizae in the stressful cinder environment. Third, although mycorrhizae can be parasitic under some conditions, ectomycorrhizae had mutualistic impacts on pinyon seedlings across the environmental extremes we studied.     

Habitat matters: The role of spore bank fungi in early seedling establishment of Florida slash pines

This study investigated broad patterns in communities of ectomycorrhizal fungi from three Florida habitats (sandhills, scrub, and pine rocklands) and the ability of spore bank fungi to associate with Pinus elliottii (slash pine) and Pinus densa (south Florida slash pine). Efforts to replant pines in the endangered pine rocklands are vital to the persistence of this habitat, yet little is known about the ectomycorrhizal fungi communities or how they may differ from those in other pine-dominated habitats in Florida. We used high-throughput amplicon sequencing (HTS) to assess baseline fungal communities and greenhouse bioassays to bait ectomycorrhizal fungi using seedlings. HTS soil data recovered 188 ectomycorrhizal species but only a few subsequently colonized the bioassay seedlings. We recovered 21 ectomycorrhizal species on pine seedlings including common spore bank fungi such as Cenococcum, Suillus, and Tuber, but Rhizopogon species were dominant across all sites and habitats. Habitat type and site were significant variables influencing the community composition of the total soil fungal community, soil ectomycorrhizal community, and the fungi found on seedling root tips. However, we found no significant differences between the ectomycorrhizal communities on seedling roots from the two Pinus species.     

The co-occurrence of ectomycorrhizal,arbuscular mycorrhizal,and dark septate fungi in seedlings of four members of the Pinaceae

Although roots of species in the Pinaceae are usually colonized by ectomycorrhizal (EM) fungi, there are increasing reports of the presence of arbuscular mycorrhizal (AM) and dark septate endophytic (DSE) fungi in these species. The objective of this study was to determine the colonization patterns in seedlings of three Pinus (pine) species (Pinus banksiana, Pinus strobus, Pinus contorta) and Picea glauca x Picea engelmannii (hybrid spruce) grown in soil collected from a disturbed forest site. Seedlings of all three pine species and hybrid spruce became colonized by EM, AM, and DSE fungi. The dominant EM morphotype belonged to the E-strain category; limited colonization by a Tuber sp. was found on roots of Pinus strobus and an unknown morphotype (cf. Suillus–Rhizopogon group) with thick, cottony white mycelium was present on short roots of all species. The three fungal categories tended to occupy different niches in a single root system. No correlation was found between the percent root colonized by EM and percent colonization by either AM or DSE, although there was a positive correlation between percent root length colonized by AM and DSE. Hyphae and vesicles were the only AM intracellular structures found in roots of all species; arbuscules were not observed in any roots.     

Cultivation of mushrooms of edible ectomycorrhizal fungi associated with Pinus densiflora by in vitro mycorrhizal synthesis

Edible mushroom fungi in the genera Lyophyllum, Tricholoma, Leucopaxillus, Suillus, Rhizopogon, Lactarius, and Morchella were tested for mycorrhization with Pinus densiflora in vitro. Most of the tested fungi in the genera Lyophyllum, Tricholoma, Suillus, Rhizopogon, and Lactarius formed ectomycorrhizas 2–4 months after fungal inoculation. Mycorrhizal seedlings were then acclimatized in open-pot soil under growth-chamber conditions. Almost all mycorrhizal seedlings sustained their symbiont and developed new mycorrhizas for 8–9 months after transplantation. Under these conditions, more than half of the tested species formed primordia and Tricholoma flavovirens, Rhizopogon rubescens, and Lactarius akahatsu developed basidiocarps with young host plants. Accepted: 28 November 2000     

A new Rhizopogon species associated with Pinus amamiana in Japan

Pinus amamiana is an endangered Pinus species found only on Yakushima Island and Tanegashima Island, Japan. We surveyed remaining P. amamiana forests and found some sporocarps of Rhizopogon (Boletales), many species of which exhibit strict host specificity to a narrow range of Pinaceae trees and play critical roles in host establishment. Based on morphological characteristics and molecular phylogeny, here we describe Rhizopogon yakushimensis sp. nov. This new species belongs to a new clade, phylogenetically related to the subgenera Versicolores and Rhizopogon. We also confirmed its ectomycorrhizal association with P. amamiana by comparing rDNA ITS sequences between the sporocarps and ectomycorrhizal root tips.     

Ectomycorrhizal fungi of Pinus pinea L. in northeastern Spain

 Although Pinus pinea L. is an important forest species in the Mediterranean region, few reports exist on its ectomycorrhizal associates. Sixty isolates, obtained from fungal sporocarps collected in mixed forests of P. pinea in Catalonia (northeastern Spain), were tested for ectomycorrhiza formation on containerized P. pinea seedlings when applied as mycelial inoculum produced in peat-vermiculite. A total of 17 isolates, in 8 genera (Amanita, Hebeloma, Laccaria, Lactarius, Pisolithus, Rhizopogon, Scleroderma and Suillus), formed ectomycorrhizas and the percentages of mycorrhizal short roots varied among isolates and species from 13% to 89%. Some of these fungi are cited for the first time in association with P. pinea. The results indicate further fungal candidates for controlled inoculation of P. pinea seedlings in the nursery. Accepted: 29 October 1998     

Fungal selectivity of two mycorrhiza helper bacteria on five mycorrhizal fungi associated with <Emphasis Type="Italic">Pinus thunbergii</Emphasis>

The fungal selectivity of helper effect was revealed using four ectomycorrhizal fungi (Rhizopogon sp., Pisolithus tinctorius, Cenococcum geophilum and Suillus granulatus), and one ectoendomycorrhizal fungus (Wilcoxina mikolae). Previously, we isolated a rhizobacteria, Ralstonia basilensis and Bacillus subtilis, which have the ability to enhance the mycorrhizal symbiosis between S. granulatus and Pinus thunbergii. However, the characteristics of each bacterium on mycorrhizal fungi are still unclear. Therefore, we tried to examine the fungal selectivity of helper effect. A confrontation assay revealed that R. basilensis significantly promoted the in vitro hyphal growth of W. mikolae and S. granulatus, but it had no effect on Rhizopogon sp., P. tinctorius and C. geophilum. These results were consistent with the effects shown by R. basilensis on the mycorrhizal formation of these fungi. On the other hand, B. subtilis promoted the hyphal growth of W. mikolae and C. geophilum but suppressed growth of Rhizopogon sp. B. subtilis significantly stimulated the mycorrhizal formation of S. granulatus. Thus the effects of B. subtilis on hyphal growth and on mycorrhizal formation were inconsistent. These results suggest that R. basilensis and B. subtilis have fungal selective and different mechanisms in their roles as mycorrhiza helper bacteria.     

Cultivation of mushrooms of edible ectomycorrhizal fungi associated with Pinus densiflora by in vitro mycorrhizal synthesis

The morphology and anatomy of ectomycorrhizas of edible mushroom fungi in association with Pinus densiflora seedlings are described. These include species of Lyophyllum, Tricholoma, Suillus, Rhizopogon, and Lactarius. Almost all mycorrhizas synthesized in vitro could be acclimatized in open-pot soil conditions after 8–9 months. Although mycorrhizal anatomy was almost identical under in vitro and open-pot culture conditions, external morphology, such as the development of rhizomorphs and hydrophobic aerial hyphae, differed between the two conditions in some fungal species. Fully developed, mature mycorrhizas of different fungal species could be distinguished as ectomycorrhizal morphotypes, which could also be distinguished by PCR-RFLP analysis of their rDNA.     

Diversity of fungi in hair roots of Ericaceae varies along a vegetation gradient

Ericaceous dwarf shrubs including Calluna vulgaris and Vaccinium spp. occur both in open heathland communities and in forest ecosystems as understory vegetation. Ericaceous shrubs were once thought to form ericoid mycorrhizal associations with a relatively narrow range of ascomycetous fungi closely related to, and including, Rhizoscyphus ericae. However, perceptions have recently changed since the realization that a broader range of ascomycete fungi, and in some cases basidiomycete fungi, can also form associations with the roots of ericaceous plants. We used a combination of molecular approaches, including denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism, cloning and sequencing, to investigate the diversity of fungi associated with C. vulgaris roots collected across a heathland/native Scots pine forest vegetation gradient. We also determined differences in fungal community composition between roots of co-occurring C. vulgaris and Vaccinium myrtillus in the forest understory. Collectively, the data show that a large diversity of potentially ericoid mycorrhizal fungal taxa associate with roots of C. vulgaris and V. myrtillus, and that ascomycetes were about 2.5 times more frequent than basidiomycetes. The assemblages of fungi associated with C. vulgaris and V. myrtillus were different. In addition, the community of fungi associated with C. vulgaris hair roots was different for samples collected from the forest, open heathland and a transition zone between the two. This separation was partly, but not entirely, due to the occurrence of typical ectomycorrhizal basidiomycetes associated with the hair roots of C. vulgaris in the forest understory. These data demonstrate that forest understory ericaceous shrubs associate with a diverse range of ascomycete and basidiomycete taxa, including typical ectomycorrhizal basidiomycetes.     

Ontogenetic differentiation between Mediterranean and Eurasian pines (sect. Pinus) at the seedling stage

Heteroblastic development in pine seedlings includes extreme morphological changes with still unclear adaptive and evolutionary significance. In particular, Mediterranean and Eurasian pines (section Pinus) living in the Mediterranean basin seem to follow quite distinct developmental trajectories at the seedling stage. Aiming to confirm this ontogenetic differentiation we performed a nursery experiment with seedlings of five Mediterranean pines (Pinus pinaster, P. brutia, P. halepensis, P. pinea and P. canariensis) and three Eurasian pines (P. sylvestris, P. uncinata, and P. nigra subsp. salzmannii), also including P. radiata as an outgroup. After destructive analyses at two harvest times (9 and 32 weeks), we found sharp differentiation between Mediterranean and Eurasian pines in a combination of traits linked to shoot heteroblasty. In particular, Mediterranean pines showed a marked delay in the proportion of adult needles to total needles in the shoot compared to Eurasian species, especially at the second harvest. However, two Mediterranean pines, P. pinaster and P. brutia showed a slightly higher proportion of secondary needles, and a higher rate of budset at a more advanced stage (68 weeks) compared to the other three Mediterranean species. Meaningfully, the outgroup P. radiata was the only species combining a high proportion of adult foliage since the first harvest with a delayed formation of the first terminal bud. We discussed the adaptive significance of these findings at the light of species’ climatic niches and life histories.     

Persistence of ecto- and ectendomycorrhizal fungi associated with <Emphasis Type="Italic">Pinus montezumae</Emphasis> in experimental microcosms

Ectomycorrhizal (ECM) and ectendomycorrhizal fungal species associated with Pinus montezumae were recorded in 8 year-old trees established in microcosms and compared with those associated with 2 year-old trees, in order to determine their persistence over the long-term. Mycorrhizal root tips were morphologically and anatomically characterized and sequenced. The extension of extramatrical mycelium of ECM fungi with long exploration strategies was evaluated. In total, 11 mycorrhizal species were registered. Seven mycorrhizal species were detected on both 2 and 8 year-old pines: Atheliaceae sp., Rhizopogon aff. fallax, R. aff. occidentalis, Suillus pseudobrevipes, Tuber separans, Wilcoxina mikolae and Wilcoxina rehmii. One species, Thelephora terrestris, was exclusively associated with two year–old seedlings, while Cenococcum geophilum, Pezizaceae sp. and Pyrenomataceae sp. were exclusively found on 8 year-old trees. Atheliaceae sp. was the ECM fungal species that presented the most abundant mycelium. Finally, we report one new fungal species of Pezizaceae occurring as a symbiont of P. montezumae.     

The charcoal effect in Boreal forests: mechanisms and ecological consequences

Wildfire is the principal disturbance regime in northern Boreal forests, where it has important rejuvenating effects on soil properties and encourages tree seedling regeneration and growth. One possible agent of this rejuvenation is fire-produced charcoal, which adsorbs secondary metabolites such as humus phenolics produced by ericaceous vegetation in the absence of fire, which retard nutrient cycling and tree seedling growth. We investigated short-term ecological effects of charcoal on the Boreal forest plant-soil system in a glasshouse experiment by planting seedlings of Betula pendula and Pinus sylvestris in each of three humus substrates with and without charcoal, and with and without phenol-rich Vaccinium myrtillus litter. These three substrates were from: (1) a high-productivity site with herbaceous ground vegetation; (2) a site of intermediate productivity dominated by ericaceous ground vegetation; and (3) an unproductive site dominated by Cladina spp. Growth of B. pendula was stimulated by charcoal addition and retarded by litter addition in the ericaceous substrate (but not in the other two), presumably because of the high levels of phenolics present in that substrate. Growth of P. sylvestris, which was less sensitive to substrate origin than was B. pendula, was unresponsive to charcoal. Charcoal addition enhanced seedling shoot to root ratios of both tree species, but again only for the ericaceous substrate. This response is indicative of greater N uptake and greater efficiency of nutrient uptake (and presumably less binding of nutrients by phenolics) in the presence of charcoal. These effects were especially pronounced for B. pendula, which took up 6.22 times more nitrogen when charcoal was added. Charcoal had no effect on the competitive balance between B. pendula and P. sylvestris, probably due to the low intensity of competition present. Juvenile mosses and ferns growing in the pots were extremely responsive to charcoal for all sites; fern prothalli were entirely absent in the ericaceous substrate unless charcoal was also present. Charcoal stimulated active soil microbial biomass in some instances, and also exerted significant although idiosyncratic effects on decomposition of the added litter. Our results provide clear evidence that immediately after wildfire fresh charcoal can have important effects in Boreal forest ecosystems dominated by ericaceous dwarf shrubs, and this is likely to provide a major contribution to the rejuvenating effects of wildfire on forest ecosystems. Received: 23 May 1997 / Accepted: 24 February 1998     

Genetic analysis of Pinus strobus and Pinus monticola populations from Canada using ISSR and RAPD markers: development of genome-specific SCAR markers

Pinus is the largest genus of conifers, containing over 100 species and is also the most widespread genus in the Northern Hemisphere. Pinus monticola and P. strobus are two closely related and economically important species in Canada. Morphological and allometric characteristics have been used to assess genetic variation within these two species but these markers are not reliable due to ecological variations. The purpose of the present study was to determine the level of genetic diversity within and among Canadian populations from the two species using molecular markers and to identify and characterize genome-specific inter-simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers. The level of genetic variation among populations was much lower for P. monticola than P. strobus. For both species, the among population variation values were smaller than within population variation. The populations from P. monticola were more closely genetically related than populations from P. strobus based on ISSR and RAPD analyses. Six ISSR and four RAPD markers specific to either P. monticola or P. strobus were cloned and sequenced. Primer pairs flanking these specific sequences were designed and genome specific SCAR markers for P. monticola and P. strobus were developed and characterized.     

Mycorrhizal networks mediate overstorey-understorey competition in a temperate forest

In forests, common mycorrhizal networks (CMNs) often connect the roots of neighbouring plants. Observations of material flows between hosts connected by CMNs have given rise to the hypothesis that CMNs limit the negative effects of competition by overstorey trees on seedlings recruiting underneath them. I conducted an experiment in a temperate forest dominated by ectomycorrhizal conifers and hardwoods to isolate the effects of CMNs on the growth and survival of four tree species that co‐occur in the understorey. Ectomycorrhizal networks had strong negative effects on the survival of an arbuscular mycorrhizal species, Acer rubrum, and neutral effects on the survival of three ectomycorrhizal species, Betula allegheniensis, Pinus strobus, and Tsuga canadensis. CMNs had positive effects on the growth of at least one ectomycorrhizal species, P. strobus. Interspecific differences in juvenile responses to CMNs may influence forest community development, promoting coexistence of some tree species while limiting the success of others.     

Diversity of ectomycorrhizal fungi naturally established on containerised Pinus seedlings in nursery conditions

The study examined the diversity of ectomycorrhizal fungi, naturally established on roots of containerised Pinus seedlings in a nursery, using PCR-RFLP and sequencing of the nuclear ribosomal internal transcribed spacer. Seventy-two samples, including ectomycorrhizae and fruit bodies, were examined. Molecular typing assigned the fungal symbionts to four ectomycorrhizal Boletales: Rhizopogon rubescens, Suillus bovinus, S. variegatus, and R. luteolus. R. rubescens was abundant (37.5%), while Suillus and R. luteolus species were moderately established (25-26%) and rare (2.8%), respectively. In addition, Rhizopogon species colonised P. nigra ssp. salzmannii seedlings, whereas Suillus species were identified on Pinus nigra ssp. nigra seedlings. The diversity and the ability of these naturally established symbionts under artificial nursery conditions were discussed. The molecular survey investigated here should contribute to successful monitoring of mycorrhizal application under both nursery and plantation conditions.     

A comparative cytogenetic analysis of five pine species from North America, Pinus banksiana, P. contorta, P. monticola, P. resinosa, and P. strobus

The genus Pinus comprises more than 100 species, which are widely distributed in the Northern hemisphere. Cytogenetic information on North American pines is very limited despite their economic importance. In the present study, a detailed comparative cytogenetic analysis is presented for five pine species from North America, P. resinosa, P. monticola, P. contorta, P. banksiana, and P. strobus. Morphometric analysis and physical mapping of rDNA probes were performed. The karyotype of P. monticola was considered ancestral with small difference in relative chromosome lengths. P. banksiana, P. contorta, and P. strobus karyotypes were considered semi-asymmetrical and less ancestral type. P. banksiana showed five secondary constrictions, P. strobus six, and P. contorta eight. P. resinosa karyotype was semi-asymmetrical and derived with 14 secondary constrictions identified on eight different chromosomes. Karyological data were consistent with molecular cytogenetic information. A significant association was observed between the number and locations of secondary constrictions and the 18S-5.8S-26S rDNA sites detected using fluorescence in situ hybridization. The two methods were used to establish a reliable comparative karyotype of the selected pines. In general, karyotype and chromosome evolution were not related to genetic relationships among pine species studied.     

Ostryopsis davidiana seedlings inoculated with ectomycorrhizal fungi facilitate formation of mycorrhizae on Pinus tabulaeformis seedlings

Reforestation in China is important for reversing anthropogenic activities that degrade the environment. Pinus tabulaeformis is desired for these activities, but survival and growth of seedlings can be hampered by lack of ectomycorrhizae. When outplanted in association with Ostryopsis davidiana plants on reforestation sites, P. tabulaeformis seedlings become mycorrhizal and survival and growth are enhanced; without O. davidiana, pines often remain without mycorrhizae and performance is poorer. To better understand this relationship, we initiated an experiment using rhizoboxes that restricted root and tested the hypothesis that O. davidiana seedlings facilitated ectomycorrhizae formation on P. tabulaeformis seedlings through hyphal contact. We found that without O. davidiana seedlings, inocula of five indigenous ectomycorrhizal fungi were unable to grow and associate with P. tabulaeformis seedlings. Inocula placed alongside O. davidiana seedlings, however, resulted in enhanced growth and nutritional status of O. davidiana and P. tabulaeformis seedlings, and also altered rhizosphere pH and phosphatase activity. We speculate that these species form a common mycorrhizal network and this association enhances outplanting performance of P. tabulaeformis seedlings used for forest restoration.     

Mycobiont overlap between two mycoheterotrophic genera of Monotropoideae (Pterospora andromedea and Sarcodes sanguinea) found in the Greater Yellowstone Ecosystem

Pterospora andromedea, a mycoheterotroph, has been shown to form obligate symbioses with only three species of Rhizopogon in section Amylopogon: R. salebrosus, R. arctostaphyli and an undescribed molecular taxon. Sarcodes sanguinea, another my coheterotroph in Ericaceae, and sister taxon to Pterospora andromedea, has been found to form symbioses with two species of Rhizopogon in section Amylopogon: R. ellenae and R. subpurpurascens. To date no overlap has been recorded between these two achlorophyllous plants and their associated mycobionts. Tissue from Pterospora andromedea rootballs and Rhizopogon spp. basidiocarps were collected from the Greater Yellowstone Ecosystem. The mycobionts were identified using sequence analysis of the ITS locus and compared with sequences of Rhizopogon spp. section Amylopogon from GenBank. Sequences of two additional loci, ATP6 and RPB2 were also generated and analyzed. In addition to Rhizopogon salebrosus, Pterospora andromedea was found for the first time in association with a fourth mycobiont, Rhizopogon ellenae, a known associate of Sarcodes sanguinea. The discovery of a new symbiont may provide evidence for an undiscovered lineage of Pterospora andromedea inhabiting the Greater Yellowstone Ecosystem. In addition, overlap in obligate mycobionts between closely related mycoheterotrophs provides interesting new information on the phylogenetic history and coevolution of the mycoheterotrophs in the Monotropoideae (Ericaceae).     

Kinetics of phosphate absorption by mycorrhizal and non-mycorrhizal Scots pine seedlings

Kinetics of net phosphate (P_i) uptake was measured on intact ectomycorrhizal and non‐mycorrhizal Pinus sylvestris seedlings using a semihydroponic cultivation method. The depletion of P_i in a nutrient solution was assessed over a 160–0.2 μM P_i gradient. Growth of the pine seedlings was P limited and measurements were performed 7 and 9 weeks after inoculation. Three ectomycorrhizal fungi were studied: Paxillus involutus, Suillus bovinus and Thelephoraterrestris. P_i uptake was extremely fast in plants colonised by P. involutus. The P_i concentration dropped below 0.2 μM within 4–5 h. In plants colonised with S. bovinus this occurred in 5–6 h and in plants associated with T. terrestris 8 h were needed to run through the whole concentration range. Non‐mycorrhizal plants of similar size and nutrient status decreased P_i to a concentration between 1 and 2 μM in 18 h. Data were curve fitted to a two‐phase Michaelis‐Menten equation. The apparent kinetic constants, K_m and V_max, for the high affinity P_i uptake system of the pine roots could be estimated accurately. V_max of this system was up to 7 times higher in pines associated with P. involutus than in non‐mycorrhizal seedlings. The intact extraradical mycelium greatly increased the absorption surface area of the roots (V_max). Non‐mycorrhizal plants had a K_m between 7.8 and 16.4 μM P_i. Plants mycorrhizal with P. involutus had K_m values between 2.4 and 7.2, plants colonised with S. bovinus had a K_m between 5.1 and 12.3, and seedlings associated with T. terrestris had a K_m from 4.6 to 10.1 μM P_i. All 3 ectomycorrhizal fungi had a strong impact on the P_i absorption capacity of the pine seedlings. The results also demonstrated that there is substantial heterogeneity in kinetic parameters among the different mycorrhizal root systems.