Friend or foe? Evolutionary history of glycoside hydrolase family 32 genes encoding for sucrolytic activity in fungi and its implications for plant-fungal symbioses.

BACKGROUND: Many fungi are obligate biotrophs of plants, growing in live plant tissues, gaining direct access to recently photosynthesized carbon. Photosynthate within plants is transported from source to sink tissues as sucrose, which is hydrolyzed by plant glycosyl hydrolase family 32 enzymes (GH32) into its constituent monosaccharides to meet plant cellular demands. A number of plant pathogenic fungi also use GH32 enzymes to access plant-derived sucrose, but less is known about the sucrose utilization ability of mutualistic and commensal plant biotrophic fungi, such as mycorrhizal and endophytic fungi. The aim of this study was to explore the distribution and abundance of GH32 genes in fungi to understand how sucrose utilization is structured within and among major ecological guilds and evolutionary lineages. Using bioinformatic and PCR-based analyses, we tested for GH32 gene presence in all available fungal genomes and an additional 149 species representing a broad phylogenetic and ecological range of biotrophic fungi. RESULTS: We detected 9 lineages of GH32 genes in fungi, 4 of which we describe for the first time. GH32 gene number in fungal genomes ranged from 0-12. Ancestral state reconstruction of GH32 gene abundance showed a strong correlation with nutritional mode, and gene family expansion was observed in several clades of pathogenic filamentous Ascomycota species. GH32 gene number was negatively correlated with animal pathogenicity and positively correlated with plant biotrophy, with the notable exception of mycorrhizal taxa. Few mycorrhizal species were found to have GH32 genes as compared to other guilds of plant-associated fungi, such as pathogens, endophytes and lichen-forming fungi. GH32 genes were also more prevalent in the Ascomycota than in the Basidiomycota. CONCLUSION: We found a strong signature of both ecological strategy and phylogeny on GH32 gene number in fungi. These data suggest that plant biotrophic fungi exhibit a wide range of ability to access plant-synthesized sucrose. Endophytic fungi are more similar to plant pathogens in their possession of GH32 genes, whereas most genomes of mycorrhizal taxa lack GH32 genes. Reliance on plant GH32 enzyme activity for C acquisition in these symbionts supports earlier predictions of possible plant control over C allocation in the mycorrhizal symbiosis.

Expression of genes involved in symbiotic carbon and nitrogen transport in Pinus taeda mycorrhizal roots exposed to CO2 enrichment and nitrogen fertilization.

As atmospheric carbon dioxide (CO(2)) concentrations rise, one important mechanism by which plants can gain greater access to necessary soil nutrients is through greater investment in their mycorrhizal symbionts. In this study, we tested the hypotheses that (1) plants increase C allocation to ectomycorrhizal fungi (EMF) under elevated CO(2) conditions, (2) N fertilization decreases C allocation to EMF, and (3) EMF activity at the site of symbiotic C and nutrient exchange is enhanced with CO(2) enrichment. To test these hypotheses, we examined expression levels of Pinus taeda genes encoding monosaccharide transport (MST) and ammonium transport (AMT) proteins thought to be involved in symbiotic C and N movement, respectively, from mycorrhizal root tips exposed to CO(2) and N fertilization. We also examined EMF ribosomal RNA expression (18S rRNA) to determine EMF activity. There was a trend toward lower relative MST expression with increased CO(2). AMT expression levels showed no significant differences between control and treatment plots. EMF 18S rRNA expression was increased in CO(2)-enriched plots and there was a marginally significant positive interactive effect of CO(2) and N fertilization on expression (p = 0.09 and 0.10, respectively). These results are consistent with greater C allocation to EMF and greater EMF metabolic activity under elevated CO(2) conditions, although selective allocation of C to particular EMF species and greater fungal biomass on roots are plausible alternative hypotheses.

Biomass and compositional responses of ectomycorrhizal fungal hyphae to elevated CO2 and nitrogen fertilization.

The extramatrical mycelia (EMM) of ectomycorrhizal fungi make up a large proportion of the microbial diversity and biomass in temperate forest soils. Thus, their response to elevated CO(2) can have large effects on plant nutrient acquisition and carbon movement through forests. Here, the effects of CO(2) and nitrogen (N) fertilization on EMM biomass and community structure in Pinus taeda forest plots were examined using sand-filled mesh bags buried in the field, the contents of which were analyzed by phospholipid fatty acid (PLFA) and DNA sequencing. A total of 2138 sequences comprising 295 taxa were recovered; most (83.5%) were from ectomycorrhizal fungal taxa. No biomass increase was detected in elevated CO(2) plots relative to control plots, but individual taxa responded to both CO(2) and N fertilization, four of the six most abundant taxa were less frequent in N-fertilized plots. Thelephoroid and athelioid taxa were both frequent and abundant as EMM, and thelephoroid richness was extremely high. Russula and Cortinariaceae taxa were less abundant and boletoid taxa were more abundant as EMM relative to ectomycorrhizas. The EMM community, sampled across seasons and years, was dynamic with a high degree of interspecific variation in response to CO(2) enrichment and N fertilization.

CO2-enrichment and nutrient availability alter ectomycorrhizal fungal communities.

Ectomycorrhizal fungi (EMF), a phylogenetically and physiologically diverse guild, form symbiotic associations with many trees and greatly enhance their uptake of nutrients and water. Elevated CO2, which increases plant carbon supply and demand for mineral nutrients, may change the composition of the EMF community, possibly altering nutrient uptake and ultimately forest productivity. To assess CO2 effects on EMF communities, we sampled mycorrhizae from the FACTS-I (Forest-Atmosphere Carbon Transfer and Storage) research site in Duke Forest, Orange County, North Carolina, USA, where Pinus taeda forest plots are maintained at either ambient or elevated CO2 (200 ppm above ambient) concentrations. Mycorrhizae were identified by DNA sequence similarity of the internal transcribed spacer ribosomal RNA gene region. EMF richness was very high; 72 distinct phylotypes were detected from 411 mycorrhizal samples. Overall EMF richness and diversity were not affected by elevated CO2, but increased CO2 concentrations altered the relative abundances of particular EMF taxa colonizing fine roots, increased prevalence of unique EMF species, and led to greater EMF community dissimilarity among individual study plots. Natural variation among plots in mean potential net nitrogen (N) mineralization rates was a key determinant of EMF community structure; increasing net N mineralization rate was negatively correlated with EMF richness and had differential effects on the abundance of particular EMF taxa. Our results predict that, at CO2 concentrations comparable to that predicted for the year 2050, EMF community composition and structure will change, but diversity will be maintained. In contrast, high soil N concentrations can negatively affect EMF diversity; this underscores the importance of considering CO2 effects on forest ecosystems in the context of background soil chemical parameters and other environmental perturbations such as acid deposition or fertilizer runoff.

Fungal community analysis by large-scale sequencing of environmental samples.

Fungi are an important and diverse component of soil communities, but these communities have proven difficult to study in conventional biotic surveys. We evaluated soil fungal diversity at two sites in a temperate forest using direct isolation of small-subunit and internal transcribed spacer (ITS) rRNA genes by PCR and high-throughput sequencing of cloned fragments. We identified 412 sequence types from 863 fungal ITS sequences, as well as 112 ITS sequences from other eukaryotic microorganisms. Equal proportions of Basidiomycota and Ascomycota sequences were present in both the ITS and small-subunit libraries, while members of other fungal phyla were recovered at much lower frequencies. Many sequences closely matched sequences from mycorrhizal, plant-pathogenic, and saprophytic fungi. Compositional differences were observed among samples from different soil depths, with mycorrhizal species predominating deeper in the soil profile and saprophytic species predominating in the litter layer. Richness was consistently lowest in the deepest soil horizon samples. Comparable levels of fungal richness have been observed following traditional specimen-based collecting and culturing surveys, but only after much more extensive sampling. The high rate at which new sequence types were recovered even after sampling 863 fungal ITS sequences and the dominance of fungi in our libraries relative to other eukaryotes suggest that the abundance and diversity of fungi in forest soils may be much higher than previously hypothesized. All sequences were deposited in GenBank, with accession numbers AY 969316 to AY 970290 for the ITS sequences and AY 969135 to AY 969315 for the SSU sequences.

Population genetic structure of the polypore Datronia caperata in fragmented mangrove forests.

Datronia caperata, a basidiomycete fungus, is one of the dominant polypore species found in neotropical mangrove forest fragments, where it is locally specialized on Laguncularia racemosa. We examined the genetic structure of D. caperata populations from four Panamanian mangrove forests using AFLP markers. Using five primer pair combinations, 145 loci were detected, 98.6% of which were polymorphic. Each of the populations showed a high degree of genetic diversity (Nei's h ranging from 0.146 to 0.223). Results from minimum spanning trees and Mantel tests showed little evidence for small-scale spatial structure within sites. A significant amount of total genetic variation was partitioned among populations (phi(ST) = 0.21) separated by 10s to 100s of km, a considerably greater amount than has been detected in other mushroom and wood-decaying fungi sampled at equal or greater geographic distances. These results suggest that despite production of copious basidiospores capable of long distance dispersal, some homobasidiomycete fungi may be susceptible to genetic isolation due to habitat fragmentation.