Islands in the shade: scattered ectomycorrhizal trees influence soil inoculum and heterospecific seedling response in a northeastern secondary forest.

The eastern deciduous forest is a mix of arbuscular (AM) and ectomycorrhizal (EM) trees, but land use legacies have increased the abundance of AM trees like Acer spp. (maple). Although these legacies have not changed the abundance of some EM trees like Betula spp. (birch), EM conifers like Tsuga canadensis (hemlock), and Pinus strobus (pine) have declined. We used a soil bioassay to investigate if the microbial community near EM birch (birch soil) contains a greater abundance and diversity of EM fungal propagules compatible with T. canadensis and P. strobus compared to the community associated with the surrounding AM-dominated secondary forest matrix (maple soil). We also tested the effectiveness of inoculation with soil from a nearby EM-dominated old-growth forest as a restoration tool to reintroduce EM fungi into secondary forest soils. Finally, we examined how seedling growth responded to EM fungi associated with each treatment. Seedlings grown with birch soil were colonized by EM fungi mostly absent from the surrounding maple forest. Hemlock seedlings grown with birch soil grew larger than hemlock seedlings grown with maple soil, but pine seedling growth did not differ with soil treatment. The addition of old-growth soil inoculum increased hemlock and pine growth in both soils. Our results found that EM trees are associated with beneficial EM fungi that are mostly absent from the surrounding AM-dominated secondary forest, but inoculation with old-growth soil is effective in promoting the growth of seedlings by reintroducing native EM fungi to the AM-dominated forests.

Transgenic American Chestnuts Do Not Inhibit Germination of Native Seeds or Colonization of Mycorrhizal Fungi.

The American chestnut (Castanea dentata) was once an integral part of eastern United States deciduous forests, with many environmental, economic, and social values. This ended with the introduction of an invasive fungal pathogen that wiped out over three billion trees. Transgenic American chestnuts expressing a gene for oxalate oxidase successfully tolerate infections by this blight fungus, but potential non-target environmental effects should be evaluated before new restoration material is released. Two greenhouse bioassays evaluated belowground interactions between transgenic American chestnuts and neighboring organisms found in their native ecosystems. Potential allelopathy was tested by germinating several types of seeds, all native to American chestnut habitats, in the presence of chestnut leaf litter. Germination was not significantly different in terms of number of seeds germinated or total biomass of germinated seedlings in transgenic and non-transgenic leaf litter. Separately, ectomycorrhizal associations were observed on transgenic and non-transgenic American chestnut roots using field soil inoculum. Root tip colonization was consistently high (>90% colonization) on all plants and not significantly different between any tree types. These observations on mycorrhizal fungi complement previous studies performed on older transgenic lines which expressed oxalate oxidase at lower levels. Along with other environmental impact comparisons, these conclusions provide further evidence that transgenic American chestnuts are not functionally different with regard to ecosystem interactions than non-transgenic American chestnuts.

A single ectomycorrhizal fungal species can enable a Pinus invasion.

Like all obligately ectomycorrhizal plants, pines require ectomycorrhizal fungal symbionts to complete their life cycle. Pines introduced into regions far from their native range are typically incompatible with local ectomycorrhizal fungi, and, when they invade, coinvade with fungi from their native range. While the identities and distributions of coinvasive fungal symbionts of pine invasions are poorly known, communities that have been studied are notably depauperate. However, it is not yet clear whether any number of fungal coinvaders is able to support a Pinaceae invasion, or whether very depauperate communities are unable to invade. Here, we ask whether there is evidence for a minimum species richness of fungal symbionts necessary to support a pine/ectomycorrhizal fungus coinvasion. We sampled a Pinus contorta invasion front near Coyhaique, Chile, using molecular barcoding to identify ectomycorrhizal fungi. We report that the site has a total richness of four species, and that many invasive trees appear to be supported by only a single ectomycorrhizal fungus, Suillus luteus. We conclude that a single ectomycorrhizal (ECM) fungus can suffice to enable a pine invasion.

Ectomycorrhizal fungal communities coinvading with Pinaceae host plants in Argentina: Gringos bajo el bosque.

Coinvasive ectomycorrhizal (ECM) fungi allow Pinaceae species to invade regions otherwise lacking compatible symbionts, but ECM fungal communities permitting Pinaceae invasions are poorly understood. In the context of Pinaceae invasions on Isla Victoria, Nahuel Huapi National Park, Argentina, we asked: what ECM fungi are coinvading with Pinaceae hosts on Isla Victoria; are some ECM fungal species or genera more prone to invade than others; and are all ECM fungal species that associate with Northern Hemisphere hosts also nonnative, or are some native fungi compatible with nonnative plants? We sampled ECMs from 226 Pinaceae host plant individuals, both planted individuals and recruits, growing inside and invading from plantations. We used molecular techniques to examine ECM fungal communities associating with these trees. A distinctive subset of the ECM fungal community predominated far from plantations, indicating differences between highly invasive and less invasive ECM fungi. Some fungal invaders reported here have been detected in other locations around the world, suggesting strong invasion potential. Fungi that were frequently detected far from plantations are often found in early-successional sites in the native range, while fungi identified as late-successional species in the native range are rarely found far from plantations, suggesting a means for predicting potential fungal coinvaders.

Comparisons of ectomycorrhizal colonization of transgenic american chestnut with those of the wild type, a conventionally bred hybrid, and related fagaceae species.

American chestnut (Castanea dentata [Marsh.] Borkh.) dominated the eastern forests of North America, serving as a keystone species both ecologically and economically until the introduction of the chestnut blight, Cryphonectria parasitica, functionally eradicated the species. Restoration efforts include genetic transformation utilizing genes such as oxalate oxidase to produce potentially blight-resistant chestnut trees that could be released back into the native range. However, before such a release can be undertaken, it is necessary to assess nontarget impacts. Since oxalate oxidase is meant to combat a fungal pathogen, we are particularly interested in potential impacts of this transgene on beneficial fungi. This study compares ectomycorrhizal fungal colonization on a transgenic American chestnut clone expressing enhanced blight resistance to a wild-type American chestnut, a conventionally bred American-Chinese hybrid chestnut, and other Fagaceae species. A greenhouse bioassay used soil from two field sites with different soil types and land use histories. The number of colonized root tips was counted, and fungal species were identified using morphology, restriction fragment length polymorphism (RFLP), and DNA sequencing. Results showed that total ectomycorrhizal colonization varied more by soil type than by tree species. Individual fungal species varied in their colonization rates, but there were no significant differences between colonization on transgenic and wild-type chestnuts. This study shows that the oxalate oxidase gene can increase resistance against Cryphonectria parasitica without changing the colonization rate for ectomycorrhizal species. These findings will be crucial for a potential deregulation of blight-resistant American chestnuts containing the oxalate oxidase gene.

Phylogenetic trait conservation in the partner choice of a group of ectomycorrhizal trees.

Ecological interactions are frequently conserved across evolutionary time. In the case of mutualisms, these conserved interactions may play a large role in structuring mutualist communities. We hypothesized that phylogenetic trait conservation could play a key role in determining patterns of association in the ectomycorrhizal symbiosis, a globally important trophic mutualism. We used the association between members of the pantropical plant tribe Pisonieae and its fungal mutualist partners as a model system to test the prediction that Pisonieae-associating ectomycorrhizal fungi will be more closely related than expected by chance, reflecting a conserved trait. We tested this prediction using previously published and newly generated sequences in a Bayesian framework incorporating phylogenetic uncertainty. We report that phylogenetic trait conservation does exist in this association. We generated a five-marker phylogeny of members of the Pisonieae and used this phylogeny in a Bayesian relaxed molecular clock analysis. We established that the most recent common ancestors of Pisonieae species and Pisonieae-associating fungi sharing phylogenetic conservation of their patterns of ectomycorrhizal association occurred no more recently than 14.2 Ma. We therefore suggest that phylogenetic trait conservation in the Pisonieae ectomycorrhizal mutualism association represents an inherited syndrome which has existed for at least 14 Myr.

A revision of the Alpova diplophloeus complex in North America.

Alpova diplophloeus (Boletales, Paxillaceae) is the only currently recognized Alpova in North America with a brownish peridium, large gleba chambers and which forms ectomycorrhizas with Alnus. However, A. diplophloeus as currently circumscribed is a polyphyletic species, with at least three distinct genetic entities. Using a combination of molecular and morphological characters, we examined the type collections of A. diplophloeus, as well as species synonymized with it, including A. cinnamomeus and Rhizopogon parvisporus. We also examined several other collections of A. diplophloeus complex basidiomata. We describe A. diplophloeus sensu stricto; we also resurrect A. cinnamomeus, synonymized with R. parvisporus and describe a new species, A. concolor, from the complex.

Ectomycorrhizal inoculum potential of northeastern US forest soils for American chestnut restoration: results from field and laboratory bioassays.

American chestnut (Castanea dentata) was once a dominant overstory tree in eastern USA but was decimated by chestnut blight (Cryphonectria parasitica). Blight-resistant chestnut is being developed as part of a concerted restoration effort to bring this heritage tree back. Here, we evaluate the potential of field soils in the northern portion of the chestnut's former range to provide ectomycorrhizal (EM) fungus inoculum for American chestnut. In our first study, chestnut seedlings were grown in a growth chamber using soil collected from three sites dominated by red oak (Quercus rubra) as inoculum and harvested after 5 months. Of the 14 EM fungi recovered on these seedlings, four species dominated in soils from all three sites: Laccaria laccata, a Tuber sp., Cenococcum geophilum, and a thelephoroid type. Seedlings grown in the nonsterilized soils were smaller than those growing in sterilized soils. In the second study, chestnut seedlings were grown from seed planted directly into soils at the same three sites. Seedlings with intermingling roots of established trees of various species were harvested after 5 months. Seventy-one EM fungi were found on the root tips of the hosts, with 38 occurring on chestnut seedlings. Multiple versus single host EM fungi were significantly more abundant and frequently encountered. The fungi observed dominating on seedlings in the laboratory bioassay were not frequently encountered in the field bioassay, suggesting that they may not have been active in mycelial networks in the field setting but were in the soils as resistant propagules that became active in the bioassay. These results show that soil from red oak stands can be used to inoculate American chestnut with locally adapted ectomycorrhizal fungi prior to outplanting, a relatively cost effective approach for restoration efforts.

Exotic mammals disperse exotic fungi that promote invasion by exotic trees.

Biological invasions are often complex phenomena because many factors influence their outcome. One key aspect is how non-natives interact with the local biota. Interaction with local species may be especially important for exotic species that require an obligatory mutualist, such as Pinaceae species that need ectomycorrhizal (EM) fungi. EM fungi and seeds of Pinaceae disperse independently, so they may use different vectors. We studied the role of exotic mammals as dispersal agents of EM fungi on Isla Victoria, Argentina, where many Pinaceae species have been introduced. Only a few of these tree species have become invasive, and they are found in high densities only near plantations, partly because these Pinaceae trees lack proper EM fungi when their seeds land far from plantations. Native mammals (a dwarf deer and rodents) are rare around plantations and do not appear to play a role in these invasions. With greenhouse experiments using animal feces as inoculum, plus observational and molecular studies, we found that wild boar and deer, both non-native, are dispersing EM fungi. Approximately 30% of the Pinaceae seedlings growing with feces of wild boar and 15% of the seedlings growing with deer feces were colonized by non-native EM fungi. Seedlings growing in control pots were not colonized by EM fungi. We found a low diversity of fungi colonizing the seedlings, with the hypogeous Rhizopogon as the most abundant genus. Wild boar, a recent introduction to the island, appear to be the main animal dispersing the fungi and may be playing a key role in facilitating the invasion of pine trees and even triggering their spread. These results show that interactions among non-natives help explain pine invasions in our study area.

Ectomycorrhizal fungal succession coincides with shifts in organic nitrogen availability and canopy closure in post-wildfire jack pine forests.

Successional changes in belowground ectomycorrhizal fungal (EMF) communities have been observed with increasing forest stand age; however, mechanisms behind this change remain unclear. It has been hypothesized that declines of inorganic nitrogen (N) and increases of organic N influence changes in EMF taxa over forest development. In a post-wildfire chronosequence of six jack pine (Pinus banksiana) stands ranging in age from 5 to 56 years, we investigated EMF community composition and compared shifts in taxa with detailed soluble inorganic and organic N data. Taxa were identified by internal transcribed spacer rDNA sequencing, and changes in community composition evaluated with non-metric multi-dimensional scaling (NMDS). Dissimilarities in the community data were tested for correlations with N variables. We observed a successional shift along NMDS axis 1 from such taxa as Suillus brevipes and Thelephora terrestris in sites age 5 and 11 to species of Cortinarius and Russula, among others, in the four older sites. This change was positively correlated with soluble organic N (SON) (r(2) = 0.902, P = 0.033) and free amino-acid N (r(2) = 0.945, P = 0.021), but not inorganic N. Overall, our results show a successional shift of EMF communities occurring between stand initiation and canopy closure without a change in species of the dominant plant-host, and associated with SON and free amino-acid N in soil. It is uncertain whether EMF taxa are responding to these organic N forms directly, affecting their availability, or are ultimately responding to changes in other site variables, such as belowground productivity.

Edaphic factors do not govern the ectomycorrhizal specificity of Pisonia grandis (Nyctaginaceae).

Pisonia grandis (Nyctaginaceae), a widespread tree of Pacific coral atolls and islands, displays one of the more restrictive ranges of ectomycorrhizal (ECM) fungus associates among autotrophic plants. Only five ECM fungi are currently known associates; our study adds one. In many habitats, P. grandis is restricted to large seabird colonies where nitrogen and phosphorus inputs in the form of guano are substantial. It has been suggested that the ECM specificity displayed by P. grandis is the result of the unusual nutrient-rich habitat in which P. grandis grows. On Rota, Commonwealth of the Northern Mariana Islands, P. grandis grows in habitats heavily influenced by guano additions and also in upland forests where seabirds do not roost or nest. To test the hypothesis that the ECM specificity displayed by P. grandis is the result of nutrient-related or toxicity-related factors associated with guano inputs, we sampled P. grandis growing in both guano-rich and guano-poor habitats on Rota, Commonwealth of the Northern Mariana Islands. We identified ECM symbionts of P. grandis from both habitats as well as two symbionts of Intsia bijuga (Fabaceae) from nutrient-rich habitats. We identified three ECM symbionts of P. grandis from Rota; all three were found in both guano-rich and guano-poor habitats. No differences in community diversity were detected between guano-rich and guano-poor habitats. We also detected two ECM fungal species associating with I. bijuga but not associating with P. grandis inside guano-rich habitats. From these results, we infer that edaphic factors are not responsible for limiting the ECM community associating with P. grandis to its observed level of specificity.

Is rarity of pinedrops (Pterospora andromedea) in eastern North America linked to rarity of its unique fungal symbiont?

Like other myco-heterotrophic plants, Pterospora andromedea (pinedrops) is dependent upon its specific fungal symbionts for survival. The rarity of pinedrops fungal symbiont was investigated in the eastern United States where pinedrops are rare. Wild populations of eastern pinedrops were sampled, and the plant haplotypes and fungal symbionts were characterized with molecular techniques; these data were compared to those from the West with phylogenetic analyses. The frequency of the fungal symbiont in eastern white pine forests was assessed using a laboratory soil bioassay and in situ pinedrops seed baiting. Only one plant haplotype and fungal symbiont was detected. The plant haplotype was not unique to the East. The fungal symbiont appears to be a new species within the genus Rhizopogon, closely related to the western symbionts. This fungal species was not frequent in soils with or without pinedrops, but was less frequent in the latter and in comparison to the fungal symbionts in western forests. Seed baiting resulted in few germinants, suggesting that mycelial networks produced by the eastern fungal symbiont were rare. Results suggest that eastern pinedrops rarity is influenced by the distribution and rarity of its fungal symbiont.

95% of basidiospores fall within 1 m of the cap: a field-and modeling-based study.

Plant establishment patterns suggest that ectomycorrhizal fungal (EMF) inoculant is not found ubiquitously. The role of animal vectors dispersing viable EMF spores is well documented. Here we investigate the role of wind in basidiospore dispersal for six EMF species, Inocybe lacera, Laccaria laccata, Lactarius rufus, Suillus brevipes, Suillus tomentosus and Thelephora americana. Basidiospores adhered to microscope slides placed on three 60 cm transects radiating from sporocarps. Morphological characteristics of species as well as average basidiospore volume were recorded. Number of basidiospores was quantified at specific distances to produce actual dispersal gradients. We found a negative exponential decay model using characteristics for each species fit the field data well. The 95% modeled downwind dispersal distance of basidiospores was calculated for each species. The 95% modeled downwind dispersal distance increased with increasing cap height and decreasing basidiospore volume for the species sampled, with 95% of basidiospores predicted to fall within 58 cm of the cap. Differences in anatomical characteristics of EMF species influence how far basidiospores are dispersed by wind. We discuss the role of wind dispersal leading to patterns of EMF establishment during primary succession.

Quercus rubra-associated ectomycorrhizal fungal communities of disturbed urban sites and mature forests.

The presence and quality of the belowground mycorrhizal fungal community could greatly influence plant community structure and host species response. This study tests whether mycorrhizal fungal communities in areas highly impacted by anthropogenic disturbance and urbanization are less species rich or exhibit lower host root colonization rates when compared to those of less disturbed systems. Using a soil bioassay, we sampled the ectomycorrhizal fungal (EMF) communities associating with Quercus rubra (northern red oak) seedlings in soil collected from seven sites: two mature forest reference sites and five urban sites of varying levels of disturbance. Morphological and polymerase chain reaction-restriction fragment length polymorphism analyses of fungi colonizing root tips revealed that colonization rates and fungal species richness were significantly lower on root systems of seedlings grown in disturbed site soils. Analysis of similarity showed that EMF community composition was not significantly different among several urban site soils but did differ significantly between mature forest sites and all but one urban site. We identified a suite of fungal species that occurred across several urban sites. Lack of a diverse community of belowground mutualists could be a constraint on urban plant community development, especially of late-successional woodlands. Analysis of urban EMF communities can add to our understanding of urban plant community structure and should be addressed during ecological assessment before pragmatic decisions to restore habitats are framed.

Lack of belowground mutualisms hinders Pinaceae invasions.

Why particular invasions succeed and others fail is not well understood. The role of soil biota has been proposed as important. However, the role of mutualists has received much less attention than that of pathogens. Here we report that lack of adequate ectomycorrhizal fungi hinders invasion by exotic Pinaceae on Isla Victoria, Argentina, by reducing both the probability of establishment and growth of invading individuals. More than one hundred exotic tree species were introduced to this island ca. 80 years ago, but invasive trees are found in high densities only in areas adjacent to plantations. With a series of greenhouse and field experiments we found lower mycorrhizal colonization levels and few fungal species far from original plantings, and key fungal mutualists are confined to areas near plantations, probably owing to dispersal limitations. Low inoculum levels far from the plantations are retarding the invasion. Our experiments indicate that positive interactions belowground can play a key but underappreciated role in invasion dynamics.

The number of nuclei in basidiospores of 63 species of ectomycorrhizal Homobasidiomycetes.

The production of even a limited number of heterokaryotic spores would be advantageous for establishing new individuals after long distance dispersal. While Suillus and Laccaria species are known to produce binucleate, heterokaryotic spores, this condition is poorly studied for most ectomycorrhizal fungi. To begin addressing this matter the number of nuclei in basidiospores was recorded from 142 sporocarps in 63 species and 20 genera of ectomycorrhizal (EM) fungi. The mean proportion of binucleate basidiospores produced by sporocarps within a species ranged from 0.00 to 1.00, with most genera within a family showing similar patterns. Basidiospores from fungi in Amanita, Cortinariaceae and Laccaria were primarily binucleate but were likely still homokaryotic. Basidiospores from fungi in Boletaceae, Cantharellus, Rhizopogonaceae, Russulaceae, Thelephorales and Tricholoma were primarily uninucleate, but binucleate basidiospores were observed in many genera and in high levels in Boletus. Further research is needed to relate basidiospore nuclear number to reproductive potential in ectomycorrhizal species.