Chamber bioaerosol study: human emissions of size-resolved fluorescent biological aerosol particles.

Humans are a prominent source of airborne biological particles in occupied indoor spaces, but few studies have quantified human bioaerosol emissions. The chamber investigation reported here employs a fluorescence-based technique to evaluate bioaerosols with high temporal and particle size resolution. In a 75-m(3) chamber, occupant emission rates of coarse (2.5-10 µm) fluorescent biological aerosol particles (FBAPs) under seated, simulated office-work conditions averaged 0.9 ± 0.3 million particles per person-h. Walking was associated with a 5-6× increase in the emission rate. During both walking and sitting, 60-70% or more of emissions originated from the floor. The increase in emissions during walking (vs. while sitting) was mainly attributable to release of particles from the floor; the associated increased vigor of upper body movements also contributed. Clothing, or its frictional interaction with human skin, was demonstrated to be a source of coarse particles, and especially of the highly fluorescent fraction. Emission rates of FBAPs previously reported for lecture classes were well bounded by the experimental results obtained in this chamber study. In both settings, the size distribution of occupant FBAP emissions had a dominant mode in the 3-5 µm diameter range.

Metatranscriptomic analysis of ectomycorrhizal roots reveals genes associated with Piloderma-Pinus symbiosis: improved methodologies for assessing gene expression in situ.

Ectomycorrhizal (EM) fungi form symbiotic associations with plant roots that regulate nutrient exchange between forest plants and soil. Environmental metagenomics approaches that employ next-generation sequencing show great promise for studying EM symbioses; however, metatranscriptomic studies have been constrained by the inherent difficulties associated with isolation and sequencing of RNA from mycorrhizae. Here we apply an optimized method for combined DNA/RNA extraction using field-collected EM fungal-pine root clusters, together with protocols for taxonomic identification of expressed ribosomal RNA, and inference of EM function based on plant and fungal metatranscriptomics. We used transcribed portions of ribosomal RNA genes to identify several transcriptionally dominant fungal taxa associated with loblolly pine including Amphinema, Russula and Piloderma spp. One taxon, Piloderma croceum, has a publically available genome that allowed us to identify patterns of gene content and transcript abundance. Over 1500 abundantly expressed Piloderma genes were detected from mycorrhizal roots, including genes for protein metabolism, cell signalling, electron transport, terpene synthesis and other extracellular activities. In contrast, Piloderma gene encoding an ammonia transporter showed highest transcript abundance in soil samples. Our methodology highlights the potential of metatranscriptomics to identify genes associated with symbiosis and ecosystem function using field-collected samples.

Serpentine and non-serpentine ecotypes of Collinsia sparsiflora associate with distinct arbuscular mycorrhizal fungal assemblages.

Although plant adaptation to serpentine soils has been studied for several decades, the mechanisms of plant adaptation to edaphic extremes are still poorly understood. Arbuscular mycorrhizal fungi (AMF) are common root symbionts that can increase the plant hosts' establishment and growth in stressful environments. However, little is known about the role plant-AMF interactions play in plant adaptation to serpentine. As a first step towards understanding this role, we examined the AMF assemblages associated with field populations of serpentine and non-serpentine ecotypes of California native plant Collinsia sparsiflora. We sampled roots of C. sparsiflora from three serpentine and three non-serpentine sites in close proximity (110 m to 1.94 km between sites) and analysed the small subunit ribosomal DNA gene amplified from root DNA extracts using AMF-specific primers. A total of 1952 clones from 24 root samples (four from each site) were sequenced. We used sequence similarity and phylogenetic analysis to determine operational taxonomic units (OTU) resulting in 19 OTUs representing taxa from six AMF genera, including one serpentine-specific OTU. We used Bray-Curtis similarity, multidimensional scaling and analysis of similarity to compare root sample AMF assemblages. These analyses clearly showed that plant ecotypes associated with distinct AMF assemblages; an Acaulospora OTU-dominated serpentine, and a Glomus OTU-dominated non-serpentine assemblages. Species diversity and evenness were significantly higher in serpentine assemblages. Finally, relate analysis showed a relationship between ecotype AMF assemblages and soil nutrients. This study reveals a strong relationship between AMF associates and plant adaptation to edaphic extremes.

Genetic Structure of Heterobasidion annosum in White Fir Mortality Centers in California.

ABSTRACT The structure of Heterobasidion annosum populations was studied in 15 mixed-conifer sites in central and northern California. Study sites displayed mortality of white fir trees in enlarging discrete patches (mortality centers). At each site, fungal genotypes were defined by somatic compatibility tests. In two sites, further genetic and molecular analyses were performed on field genotypes and on homokaryons obtained by dedikaryotization of field heterokaryons. Isolates were found to be colonizing mostly the roots and the bole sapwood of white fir trees, and no significant infections of other tree species were observed. Each mortality center was characterized by the presence of several fungal genotypes, all belonging to the S intersterility group. Both homokaryotic and heterokaryotic strains were present in all sites. Multiple genotypes were retrieved in individual trees or stumps. Out of 228 fungal genotypes, 86% were found only within a single tree or stump, while 14% had spread to adjacent trees. The two largest genotypes had diameters of 9 and 10 m, and had colonized five and nine trees, stumps, or both, respectively. The maximum distance between two adjacent trees colonized by the same genotype was 6 m, and a highly significant correlation was found between tree diameter and distance of fungal "vegetative" spread. The largest clones were found in areas characterized by high tree and stump densities, and secondary spread of the fungus was more significant in denser stands. In most cases, original infection courts of existing genotypes could be traced to standing trees and not to stumps. The genetic analysis performed in two mortality centers revealed that most local genotypes had different mating alleles, and thus originated from unrelated basidiospores. In a few cases, the same mating allele was shared by two heterokaryons (n+n genome) or by a homokaryon (n genome) and a heterokaryon. Molecular analysis showed that nuclei bearing the same mating allele were identical, providing evidence that the two nuclei forming heterokaryons can act independently in the field and can be shared among isolates, presumably via di-mon mating or by separate matings of different portions of widespread homokaryons.

The effect of different pine hosts on the sampling of Rhizopogon spore banks in five Eastern Sierra Nevada forests.

Our primary goal was to determine whether detection of Rhizopogon internal transcribed spacer (ITS) groups is affected by the pine species used in seedling bioassays. Our secondary goal was to investigate composition of Rhizopogon spore banks in the Eastern Sierra Nevada of California, a previously unsampled region. We used seedlings of Pinus contorta, Pinus jeffreyi, Pinus lambertiana, and Pinus muricata as bioassay plants and identified the Rhizopogon retrieved by internal transcribed spacer (ITS) sequence analysis. We found that each of the pine species retrieved all of the abundant Rhizopogon ITS groups, but there were significant differences among pines in the richness of Rhizopogon ITS groups recovered. Pinus muricata recovered all ITS groups found in this study and was significantly better than P. lambertiana. Rhizopogon communities from the five sampled sites contained six to eight ITS groups per site, with two unique sequence groups and a higher abundance of the Rhizopogon ellenae and Rhizopogon arctostaphyli groups than at previously sampled sites. These results show high cross-receptivity between Rhizopogon and pine species, and regional patterns in spore bank composition.

On the origins of extreme mycorrhizal specificity in the Monotropoideae (Ericaceae): performance trade-offs during seed germination and seedling development.

Fungal-induced seed germination is a phenomenon characteristic of mycorrhizal plants that produce dust-like seeds with only minimal nutritional reserves. In such systems, fungi trigger germination and/or subsidize development. We studied mycorrhizal germination in relation to mycorrhizal specificity in the Monotropoideae, a lineage of dust-seeded non-photosynthetic plants that are dependent upon ectomycorrhizal fungi of forest trees. A total of 1695 seed packets, each containing two to five compartments with seeds from different sources, were buried for up to 2 years near known ectomycorrhizal fungi in six different native forest locations. Upon harvest, seedlings were analysed by cultivation-independent molecular methods to identify their mycorrhizal fungi. We report that (i) germination is only induced by the same fungus that associates with mature plants or by closely related congeners; (ii) seedlings associated with the latter fungi develop less than those associated with maternal fungal species in most settings; and (iii) exceptions to this pattern occur in allopatric settings, where novel plant-fungal associations can result in the greatest seedling development. We interpret these results as evidence of performance trade-offs between breadth of host range and rate of development. We propose that in conjunction with host-derived germination cues, performance trade-offs can explain the extreme mycorrhizal specificity observed at maturity. The allopatric exceptions support the idea that performance trade-offs may be based on a coevolutionary arms race and that host range can be broadened most readily when naive fungal hosts are encountered in novel settings.

Rate and mode differences between nuclear and mitochondrial small-subunit rRNA genes in mushrooms.

Sequences from homologous regions of the nuclear and mitochondrial small-subunit rRNA genes from 10 members of the mushroom order Boletales were used to construct evolutionary trees and to compare the rates and modes of evolution. Trees constructed independently for each gene by parsimony and tested by bootstrap analysis have identical topologies in all statistically significant branches. Examination of base substitutions revealed that the nuclear gene is biased toward C-T transitions and that the distribution of transversions in the mitochondrial gene is strongly effected by an A-T bias. When only homologous regions of the two genes were compared, base substitutions per nucleotide were roughly 16-fold greater in the mitochondrial gene. The difference in the frequency of length mutations was at least as great but was impossible to estimate accurately because of their absence in the nuclear gene. Maximum likelihood was used to show that base-substitution rates vary dramatically among the branches. A significant part of the rate inconstancy was caused by an accelerated nuclear rate in one branch and a retarded mitochondrial rate in a different branch. A second part of the rate variability involved a consistent inconstancy: short branches exhibit ratios of mitochondrial to nuclear divergences of less than 1, while longer branches had ratios of approximately 4:1-8:1. This pattern suggests a systematic error in the branch length calculation. The error may be related to the simplicity of the divergence estimates, which assumes that all base positions have an equal probability of change.

Independent, specialized invasions of ectomycorrhizal mutualism by two nonphotosynthetic orchids.

We have investigated the mycorrhizal associations of two nonphotosynthetic orchids from distant tribes within the Orchidaceae. The two orchids were found to associate exclusively with two distinct clades of ectomycorrhizal basidiomycetous fungi over wide geographic ranges. Yet both orchids retained the internal mycorrhizal structure typical of photosynthetic orchids that do not associate with ectomycorrhizal fungi. Restriction fragment length polymorphism and sequence analysis of two ribosomal regions along with fungal isolation provided congruent, independent evidence for the identities of the fungal symbionts. All 14 fungal entities that were associated with the orchid Cephalanthera austinae belonged to a clade within the Thelephoraceae, and all 18 fungal entities that were associated with the orchid Corallorhiza maculata fell within the Russulaceae. Restriction fragment length polymorphism and single-strand conformational polymorphism analysis of ectomycorrhizal tree roots collected adjacent to Cephalanthera showed that (i) the fungi associated internally with Cephalanthera also form typical external ectomycorrhizae and that (ii) ectomycorrhizae formed by other Basidiomycetes were abundant where the orchid grows but these fungi did not associate with the orchid. This is the first proof of ectomycorrhizal epiparasitism in nature by an orchid. We argue that these orchids are cheaters because they do not provide fixed carbon to associated fungi. This view suggests that mycorrhizae, like other ancient mutualisms, are susceptible to cheating. The extreme specificity in these orchids relative to other ectomycorrhizal plants agrees with trends seen in more conventional parasites.

Fine-level mycorrhizal specificity in the Monotropoideae (Ericaceae): specificity for fungal species groups.

The Monotropoideae (Ericaceae) are non-photosynthetic angiosperms that obtain fixed carbon from basidiomycete ectomycorrhizal fungi. In previous work, we showed that each plant species is associated with a single genus or a set of closely related genera of ectomycorrhizal fungi. Here we show that the level of specificity is much higher. We used a molecular phylogenetic approach to contrast specificity patterns among eight plant lineages and three fungal genera. We relied on fungal nuclear internal transcribed spacer (nrITS) sequence data obtained from 161 basidiocarps and 85 monotropoid roots representing 286 sampled plants screened using restriction length polymorphisms. From the phylogenetic placement of fungal symbionts in fungal phylograms, we found that three basal (Sarcodes, Pterospora, Pleuricospora) and one derived lineage (Allotropa) of plants target narrow clades of closely related species groups of fungi, and four derived lineages (Monotropa hypopithys species group, Pityopus) target more distant species groups. Within most plant lineages, geography and photobiont association constrain specificity. Specificity extended further in Pterospora andromedea, in which sequence haplotypes at the plastid trn L-F region of 73 plants were significantly associated with different fungal species groups even in sympatry. These results indicate that both the macro- and microevolution of the Monotropoideae are tightly coupled to their mycorrhizal symbionts.

Evolution of mushroom mitochondrial DNA: Suillus and related genera.

Mapping studies were performed with 18 cloned probes on mitochondrial DNA (mtDNA) from 15 species of Suillus and four species from three related genera of fleshy pore mushrooms (Boletaceae). Within Suillus, mtDNAs vary in size from 36 to 121 kb, differ in gene order by only one major rearrangement, and have diverged in nucleotide sequence within the large subunit ribosomal RNA gene region by up to 2.9%. Three additional gene orders exist in related genera. Two of the three can be transformed into the predominant Suillus order by either one or two rearrangements. The fourth requires two to three rearrangements to be converted to any of the others. The minimum estimates of nucleotide divergence within the large subunit ribosomal RNA gene region vary from 8.3% to 11% in comparisons between Suillus and these related species. Trees based on restriction-site and size differences within the mitochondrial ribosomal RNA genes were consistent with the hypothesized sequence of genome rearrangements and provide suggestive evidence for a major expansion of the mitochondrial genome within Suillus. Structural and sequence changes in mtDNA provided information about phylogenetic relationships within the Boletaceae.

The molecular revolution in ectomycorrhizal ecology: peeking into the black-box.

Molecular tools have now been applied for the past 5 years to dissect ectomycorrhizal (EM) community structure, and they have propelled a resurgence in interest in the field. Results from these studies have revealed that: (i) EM communities are impressively diverse and are patchily distributed at a fine scale below ground; (ii) there is a poor correspondence between fungi that appear dominant as sporocarps vs. those that appear dominant on roots; (iii) members of Russulaceae, Thelephoraceae, and/or non-thelephoroid resupinates are among the most abundant EM taxa in ecosystems sampled to date; (iv) dissimilar plants are associated with many of the same EM species when their roots intermingle--this occurs on a small enough spatial scale that fungal individuals are likely to be shared by dissimilar plants; and (v) mycoheterotrophic plants have highly specific fungal associations. Although, these results have been impressive, they have been tempered by sampling difficulties and limited by the taxonomic resolution of restriction fragment length polymorphism methods. Minor modifications of the sampling schemes, and more use of direct sequencing, has the potential to solve these problems. Use of additional methods, such as in situ hybridization to ribosomal RNA or hybridization coupled to microarrays, are necessary to open up the analysis of the mycelial component of community structure.

Extreme specificity in epiparasitic Monotropoideae (Ericaceae): widespread phylogenetic and geographical structure.

The Monotropoideae (Ericaceae) are nonphotosynthetic plants that obtain fixed carbon from their fungal mycorrhizal associates. To infer the evolutionary history of this symbiosis we identified both the plant and fungal lineages involved using a molecular phylogenetic approach to screen 331 plants, representing 10 of the 12 described species. For five species no prior molecular data were available; for three species we confirmed prior studies which used limited samples; for five species all previous reports are in conflict with our results, which are supported by sequence analysis of multiple samples and are consistent with the phylogenetic patterns of host plants. The phylogenetic patterns observed indicate that: (i) each of the 13 plant phylogenetic lineages identified is specialized to a different genus or species group within five families of ectomycorrhizal Basidiomycetes; (ii) mycorrhizal specificity is correlated with phylogeny; (iii) in sympatry, there is no overlap in mature plant fungal symbionts even if the fungi and the plants are closely related; and (iv) there are geographical patterns to specificity.

ITS primers with enhanced specificity for basidiomycetes--application to the identification of mycorrhizae and rusts.

We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a 'universal' primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the 'universal' ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.

Molecular tools for the identification of ectomycorrhizal fungi--taxon-specific oligonucleotide probes for suilloid fungi.

Five taxon-specific oligonucleotide probes are described that can be used to help identify the fungal components of ectomycorrhizae. Comparisons among partial sequence from the mitochondrial large subunit rRNA gene (mt-LrRNA) were used to select the probes, which were intended to be specific to several taxa within the suilloid group of the Boletales (Basidiomycota). Probes S1, R1, and G1 were targeted at the genera Suillus, Rhizopogon and Gomphidius; probe G2 was designed to recognize the family, Gomphidiaceae, and probe US1 was designed to recognize all of these taxa and any other members of the suilloid group. The specificity of each probe was determined empirically by testing their ability to hybridize to PCR amplified fragments derived from 84 species of basidiomycetes. Although none of the probes exhibited their intended specificity, all specifically hybridized to useful subsets of taxa, and collectively they can be used to identify many suilloid taxa to the generic level or below. The probes were also tested for their ability to identify field collected mycorrhizae and were found to perform well.

Community structure of ectomycorrhizal fungi in a Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communities.

We have investigated colonization strategies by comparing the abundance and frequency of ectomycorrhizal fungal species on roots in a mature Pinus muricata forest with those present as resistant propagules colonizing potted seedlings grown in the same soil samples. Thirty-seven fungal species were distinguished by internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs); most were identified to species level by sporocarp RFLP matches or to genus/family level by using sequence databases for the mitochondrial and nuclear large-subunit rRNA genes. The below-ground fungal community found in the mature forest contrasted markedly with the resistant propagule community, as only four species were found in both communities. The dominant species in the mature forest were members of the Russulaceae, Thelephorales and Amanitaceae. In contrast, the resistant propagule community was dominated by Rhizopogon species and by species of the Ascomycota. Only one species, Tomentella sublilacina (Thelephorales), was common in both communities. The spatial distribution of mycorrhizae on mature roots and propagules in the soil differed among the dominant species. For example, T. sublilacina mycorrhizae exhibited a unique bias toward the organic horizons, Russula brevipes mycorrhizae were denser and more clumped than those of other species and Cenococcum propagules were localized, whereas R. subcaerulescens propagules were evenly distributed. We suggest that species differences in resource preferences and colonization strategies, such as those documented here, contribute to the maintenance of species richness in the ectomycorrhizal community.

Use of atp6 in fungal phylogenetics: an example from the boletales.

Complete nucleotide sequences have been determined for atp6 from Suillus luteus and cox3 from Suillus sinuspaulianus (Boletales, Hymenomycetes, Basidiomycota), which code for ATPase subunit 6 and cytochrome oxidase subunit 3, respectively. These sequences were used to design PCR primers for the amplification of partial atp6 and cox3 sequences from other members of the Boletales and outgroup taxa. In atp6 and cox3 from Russula rosacea, one of the outgroup taxa, we observed a number of in-frame TGA(trp) codons, which imply a Neurospora crassa-type mitochondrial code in R. rosacea and possibly in basidiomycetes in general. Interestingly, however, most basidiomycetes other than R. rosacea appear to strongly prefer the TGG(trp) codon, which is unusual, given the strong A + T bias in fungal mitochondrial genomes. Pairwise comparisons were performed between atp6 sequences from increasingly divergent fungal lineages, and results show that all three codon positions become saturated in substitutions after an estimated divergence time of approx 300 Ma. This means that atp6 is likely to provide phylogenetic resolution within fungal classes but not at higher taxonomic levels. Also, because of the strong A + T bias in fungal mitochondrial genomes, A/T transversions were found to be more common than any other type of substitution, resulting in transversions being about two to three times more common in most pairwise sequence comparisons. Finally, atp6 sequences were used to infer phylogenetic relationships between 27 taxa from the Boletales and 4 outgroup taxa. Analyses were performed (i) on nucleotide sequence data using parsimony (successive approximation) as well as maximum likelihood methods and (ii) on deduced amino acid sequences using distance methods based on empirical substitution probabilities. Results from the various analyses are largely concordant with each other as well as with prior analyses of partial mitochondrial large-subunit rDNA (mtLSU rDNA). Analysis of the combined atp6 and mtLSU rDNA sequences results in increased bootstrap support for several key branches. Relationships that have been resolved for the first time in the current analysis are discussed.

Ancestral lineages of arbuscular mycorrhizal fungi (Glomales).

Using new and existing 18S rRNA sequence data, we show that at least five species of glomalean fungi lie outside the previously defined families and diverged very early in the evolution of that group. These five fungi would have been missed by many previous ecological studies because their sequences are not well matched to available taxon-specific primers and they do not stain well with the standard reagents used for morphological analysis. Based upon spore morphology, these species are currently assigned to Glomus and Acaulospora, and two of the species are dimorphic, exhibiting spore stages of both genera. This suggests that dimorphic spores are the ancestral state for the order and that one or the other morphology was lost in various lineages. Our analyses also show that Geosiphon pyriforme, a symbiont with cyanobacteria, is not necessarily a sister group of the Glomales; instead, it may be derived from mycorrhizal ancestors.

Specific amplification of 18S fungal ribosomal genes from vesicular-arbuscular endomycorrhizal fungi colonizing roots.

The first DNA sequences obtained from arbuscular endomycorrhizal fungi are reported. They were obtained by directly sequencing overlapping amplified fragments of the nuclear genes coding for the small subunit rRNA. These sequences were used to develop a polymerase chain reaction primer (VANS1) that enables the specific amplification of a portion of the vesicular-arbuscular endomycorrhizal fungus small subunit rRNA directly from a mixture of plant and fungal tissues. The specificity of this primer for arbuscular endomycorrhizal fungi was demonstrated by testing it on a number of organisms and by sequencing the fragment amplified from colonized leek (Allium porum) roots. This approach, coupled with other molecular techniques, will facilitate rapid detection, identification, and possibly quantitation of arbuscular endomycorrhizal fungi.

High root concentration and uneven ectomycorrhizal diversity near Sarcodes sanguinea (Ericaceae): a cheater that stimulates its victims?

Sarcodes sanguinea is a nonphotosynthetic mycoheterotrophic plant that obtains all of its fixed carbon from neighboring trees through a shared ectomycorrhizal fungus. We studied the spatial structuring of this tripartite symbiosis in a forest where Sarcodes is abundant, and its only fungal and photosynthetic plant associates are Rhizopogon ellenae and Abies magnifica, respectively. We found disproportionately high concentrations of Abies roots adjacent to Sarcodes roots compared to the surrounding soil. Rhizopogon ellenae colonizes the vast majority of those Abies roots (86-98%), and its abundance tends to decrease with increasing distance from Sarcodes plants. At 500 cm from Sarcodes plants we did not detect R. ellenae, and the ectomycorrhizal community instead was dominated by members of the Russulaceae and Thelephoraceae, which are commonly dominant in other California pinaceous forests. The highly clumped distribution of Abies-R. ellenae ectomycorrhizas indicates that Sarcodes plants either establish within pre-existing clumps, or they stimulate clump formation. Several lines of evidence favor the latter interpretation, suggesting an unexpected mutualistic aspect to the symbiosis. However, the mechanism involved remains unknown.