Species Diversity of Uncultured and Cultured Populations of Soil and Marine Ammonia Oxidizing Bacteria

Although molecular techniques are considered to provide a more comprehensive view of species diversity of natural microbial populations, few studies have compared diversity assessed by molecular and cultivation-based approaches using the same samples. To achieve this, the diversity of natural populations of ammonia oxidising bacteria in arable soil and marine sediments was determined by analysis of 16S rDNA sequences from enrichment cultures, prepared using standard methods for this group, and from 16S rDNA cloned from DNA extracted directly from the same environmental samples. Soil and marine samples yielded 31 and 18 enrichment cultures, respectively, which were compared with 50 and 40 environmental clones. There was no evidence for selection for particular ammonia oxidizer clusters by different procedures employed for enrichment from soil samples, although no culture was obtained in medium at acid pH. In soil enrichment cultures, Nitrosospira cluster 3 sequences were most abundant, whereas clones were distributed more evenly between Nitrosospira clusters 2, 3, and 4. In marine samples, the majority of enrichment cultures contained Nitrosomonas, whereas Nitrosospira sequences were most abundant among environmental clones. Soil enrichments contained a higher proportion of identical sequences than clones, suggesting laboratory selection for particular strains, but the converse was found in marine samples. In addition, 16% of soil enrichment culture sequences were identical to those in environmental clones, but only 1 of 40 marine enrichments was found among clones, indicating poorer culturability of marine strains represented in the clone library, under the conditions employed. The study demonstrates significant differences in species composition assessed by molecular and culture-based approaches but indicates also that, employing only a limited range of cultivation conditions, 7% of the observed sequence diversity in clones of ammonia oxidizers from these environments could be obtained in laboratory enrichment culture. Further studies and experimental approaches are required to determine which approach provides better representation of the natural community.     

Ion Torrent PGM as Tool for Fungal Community Analysis: A Case Study of Endophytes in Eucalyptus grandis Reveals High Taxonomic Diversity

The Kingdom Fungi adds substantially to the diversity of life, but due to their cryptic morphology and lifestyle, tremendous diversity, paucity of formally described specimens, and the difficulty in isolating environmental strains into culture, fungal communities are difficult to characterize. This is especially true for endophytic communities of fungi living in healthy plant tissue. The developments in next generation sequencing technologies are, however, starting to reveal the true extent of fungal diversity. One of the promising new technologies, namely semiconductor sequencing, has thus far not been used in fungal diversity assessments. In this study we sequenced the internal transcribed spacer 1 (ITS1) nuclear encoded ribosomal RNA of the endophytic community of the economically important tree, Eucalyptus grandis, from South Africa using the Ion Torrent Personal Genome Machine (PGM). We determined the impact of various analysis parameters on the interpretation of the results, namely different sequence quality parameter settings, different sequence similarity cutoffs for clustering and filtering of databases for removal of sequences with incomplete taxonomy. Sequence similarity cutoff values only had a marginal effect on the identified family numbers, whereas different sequence quality filters had a large effect (89 vs. 48 families between least and most stringent filters). Database filtering had a small, but statistically significant, effect on the assignment of sequences to reference sequences. The community was dominated by Ascomycota, and particularly by families in the Dothidiomycetes that harbor well-known plant pathogens. The study demonstrates that semiconductor sequencing is an ideal strategy for environmental sequencing of fungal communities. It also highlights some potential pitfalls in subsequent data analyses when using a technology with relatively short read lengths.     

Tracking Fungal Community Responses to Maize Plants by DNA- and RNA-Based Pyrosequencing

We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age) in pots associated with four maize cultivars, including two genetically modified (GM) cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA). The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most “active” fungi (as recovered via RNA). Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production). Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.     

PCR primers that amplify fungal rRNA genes from environmental samples

Two PCR primer pairs were designed to amplify rRNA genes (rDNA) from all four major phyla of fungi: Ascomycota, Basidiomycota, Chytridomycota, and Zygomycota. PCRs performed with these primers showed that both pairs amplify DNA from organisms representing the major taxonomic groups of fungi but not from nonfungal sources. To test the ability of the primers to amplify fungal rDNA from environment samples, clone libraries from two avocado grove soils were constructed and analyzed. These soils possess different abilities to inhibit avocado root rot caused by Phythophthora cinnamomi. Analysis of the two rDNA clone libraries revealed differences in the two fungal communities. It also revealed a markedly different depiction of the soil fungal community than that generated by a culture-based analysis, confirming the value of rDNA-based approaches for identifying organisms that may not readily grow on agar media. Additional evidence of the usefulness of the primers was obtained by identifying fungi associated with avocado leaves. In both the soil and leaf analyses, no nonfungal rDNA sequences were identified, illustrating the selectivity of these PCR primers. This work demonstrates the ability of two newly developed PCR primer sets to amplify fungal rDNA from soil and plant tissue, thereby providing unique tools to examine this vast and mostly undescribed community of organisms.     

墓室酥碱砖壁画及其环境的真菌多样性分析

【背景】酥碱是威胁古代墓室砖壁画保存的主要病害之一,水分和盐分共同作用下壁画产生酥碱病害,而其中真菌类群、来源及耐盐性鲜有报道。【目的】比较敦煌汉墓、晋墓和嘉峪关五号墓内砖壁画酥碱样品及其赋存环境空气中可培养真菌的群落组成、多样性及耐盐性,为砖壁画的防护提供理论依据。【方法】运用扫描电镜观察酥碱样品微观形貌,并结合能谱、X射线衍射进行成分分析;对样品培养、分离、纯化获得真菌纯菌株,运用形态和分子生物学技术鉴定种属;利用梯度含盐培养基研究菌株的耐盐性。【结果】墓室酥碱样品及环境空气中青霉属(Pencillium)、曲霉属(Aspergillus)和枝孢属(Cladosporium)丰度较高,为优势属;其他菌属包括镰孢菌属(Fusarium)和毛壳菌属(Chaetomium)为稀有属。三座墓室砖壁画酥碱中均存在烟曲霉(A.fumigatus)、花斑曲霉(A.versicolor)以及黄灰青霉(P.aurantiogriseum);酥碱样品与墓室内外空气中分离获得的真菌类群间有一定相似性。多数菌株可在含15%NaCl的培养基上生长,一株产黄青霉(P. chrysogenum)甚至可在30%盐浓度条件下生长。【结论】青霉属和曲霉属为墓室共有优势菌,多数菌株有较强的耐盐性。     

Fungal Communities Respond to Long-Term CO2 Elevation by Community Reassembly

Fungal communities play a major role as decomposers in the Earth''s ecosystems. Their community-level responses to elevated CO₂ (eCO₂), one of the major global change factors impacting ecosystems, are not well understood. Using 28S rRNA gene amplicon sequencing and co-occurrence ecological network approaches, we analyzed the response of soil fungal communities in the BioCON (biodiversity, CO₂, and N deposition) experimental site in Minnesota, USA, in which a grassland ecosystem has been exposed to eCO₂ for 12 years. Long-term eCO₂ did not significantly change the overall fungal community structure and species richness, but significantly increased community evenness and diversity. The relative abundances of 119 operational taxonomic units (OTU; ∼27% of the total captured sequences) were changed significantly. Significantly changed OTU under eCO₂ were associated with decreased overall relative abundance of Ascomycota, but increased relative abundance of Basidiomycota. Co-occurrence ecological network analysis indicated that eCO₂ increased fungal community network complexity, as evidenced by higher intermodular and intramodular connectivity and shorter geodesic distance. In contrast, decreased connections for dominant fungal species were observed in the eCO₂ network. Community reassembly of unrelated fungal species into highly connected dense modules was observed. Such changes in the co-occurrence network topology were significantly associated with altered soil and plant properties under eCO₂, especially with increased plant biomass and NH₄⁺ availability. This study provided novel insights into how eCO₂ shapes soil fungal communities in grassland ecosystems.     

Fungal Diversity in Permafrost and Tallgrass Prairie Soils under Experimental Warming Conditions

Soil fungi play a major role in terrestrial ecosystem functioning through interactions with soil structure, plants, micro- and mesofauna, and nutrient cycling through predation, pathogenesis, mutualistic, and saprotrophic roles. The diversity of soil fungi was assessed by sequencing their 28S rRNA gene in Alaskan permafrost and Oklahoma tallgrass prairie soils at experimental sites where the effect of climate warming is under investigation. A total of 226,695 reads were classified into 1,063 genera, covering 62% of the reference data set. Using the Bayesian Classifier offered by the Ribosomal Database Project (RDP) with 50% bootstrapping classification confidence, approximately 70% of sequences were returned as “unclassified” at the genus level, although the majority (∼65%) were classified at the class level, which provided insight into these lesser-known fungal lineages. Those unclassified at the genus level were subjected to BLAST analysis against the ARB-SILVA database, where ∼50% most closely matched nonfungal taxa. Compared to the more abundant sequences, a higher proportion of rare operational taxonomic units (OTU) were successfully classified to genera at 50% bootstrap confidence, indicating that the fungal rare biosphere in these sites is not composed of sequencing artifacts. There was no significant effect after 1 year of warming on the fungal community structure at both sites, except perhaps for a few minor members, but there was a significant effect of sample depth in the permafrost soils. Despite overall significant community structure differences driven by variations in OTU dominance, the prairie and permafrost soils shared 90% and 63% of all fungal sequences, respectively, indicating a fungal “seed bank” common between both sites.     

Amplification of soil fungal community DNA using the ITS86F and ITS4 primers

Internal transcribed spacer (ITS) 86F and ITS4 and the ITS1-F and ITS86R primer pairs were tested to specifically amplify fungal community DNA extracted from soil. Libraries were constructed from PCR-amplified fragments, sequenced and compared against sequences deposited in GenBank. The results confirmed that the ITS86F and ITS4 primer pair was selectively specific for the Ascomycetes, Basidiomycetes and Zygomycetes fungal clades. Amplified products generated by the ITS1F and ITS86R primer pair also aligned with sequences from a range of species within the Ascomycete and Basidiomycete clades but not from the Zygomycete. Both primer sets demonstrated fungal specificity and appear to be well suited for rapid PCR-based (fingerprinting) analysis of environmental fungal community DNA. This is the first reported use and assessment of the ITS86F and ITS4 and the ITS1-F and ITS86R primer pairs in amplifying fungal community DNA from soil.     

The rpb2 gene represents a viable alternative molecular marker for the analysis of environmental fungal communities

Although the commonly used internal transcribed spacer region of rDNA (ITS) is well suited for taxonomic identification of fungi, the information on the relative abundance of taxa and diversity is negatively affected by the multicopy nature of rDNA and the existence of ITS paralogues. Moreover, due to high variability, ITS sequences cannot be used for phylogenetic analyses of unrelated taxa. The part of single‐copy gene encoding the second largest subunit of RNA polymerase II (rpb2) was thus compared with first spacer of ITS as an alternative marker for the analysis of fungal communities in spruce forest topsoil, and their applicability was tested on a comprehensive mock community. In soil, rpb2 exhibited broad taxonomic coverage of the entire fungal tree of life including basal fungal lineages. The gene exhibited sufficient variation for the use in phylogenetic analyses and taxonomic assignments, although it amplifies also paralogues. The fungal taxon spectra obtained with rbp2 region and ITS1 corresponded, but sequence abundance differed widely, especially in the basal lineages. The proportions of OTU counts and read counts of major fungal groups were close to the reality when rpb2 was used as a molecular marker while they were strongly biased towards the Basidiomycota when using the ITS primers ITS1/ITS4. Although the taxonomic placement of rbp2 sequences is currently more difficult than that of the ITS sequences, its discriminative power, quantitative representation of community composition and suitability for phylogenetic analyses represent significant advantages.     

Organic acids production by white rot Basidiomycetes in the presence of metallic oxides

The purpose of the present work was to determine if selected fungal strains belonging to wood-rotting Basidiomycetes are able to grow on and to solubilize different insoluble oxides in solid media. Twenty-eight strains of white rot fungi were checked for their growth on oxide-amended media (ZnO, CaO, Cu2O). All strains displayed growth on Zn-amended plates but to a different extent, and Cu2O-amended plates turned out to be the most toxic oxide. Most of the tested strains solubilized oxalates and produced noticeable clear zones under the mycelium. These clear zones were tested for the presence of organic acids, the level of which was clearly elevated upon exposure of fungal strains to insoluble oxides. We determined the presence of oxalic, malic, and formic acids, with oxalic acid the predominant one.     

ANCHOR: a 16S rRNA gene amplicon pipeline for microbial analysis of multiple environmental samples

Analysis of 16S ribosomal RNA (rRNA) gene amplification data for microbial barcoding can be inaccurate across complex environmental samples. A method, ANCHOR, is presented and designed for improved species-level microbial identification using paired-end sequences directly, multiple high-complexity samples and multiple reference databases. A standard operating procedure (SOP) is reported alongside benchmarking against artificial, single sample and replicated mock data sets. The method is then directly tested using a real-world data set from surface swabs of the International Space Station (ISS). Simple mock community analysis identified 100% of the expected species and 99% of expected gene copy variants (100% identical). A replicated mock community revealed similar or better numbers of expected species than MetaAmp, DADA2, Mothur and QIIME1. Analysis of the ISS microbiome identified 714 putative unique species/strains and differential abundance analysis distinguished significant differences between the Destiny module (U.S. laboratory) and Harmony module (sleeping quarters). Harmony was remarkably dominated by human gastrointestinal tract bacteria, similar to enclosed environments on earth; however, Destiny module bacteria also derived from nonhuman microbiome carriers present on the ISS, the laboratory's research animals. ANCHOR can help substantially improve sequence resolution of 16S rRNA gene amplification data within biologically replicated environmental experiments and integrated multidatabase annotation enhances interpretation of complex, nonreference microbiomes.     

Community structure of bacteria and fungi in aerosols of a pig confinement building

Modern intensive husbandry practices can create poor indoor air quality, with high levels of airborne dust, endotoxins, ammonia, and microorganisms. Air in a sow breeding barn was investigated to determine the biomass composition of bioaerosols using molecular methods supplemented with microscopic and cultivation-dependent approaches. A total of 2.7?±?0.7?×?10(7) bacterial cells?m(-3) air and 1.2?±?0.3?×?10(6) fungi spores?m(-3) were detected, corresponding to the fungal biovolume constituted 98% of the total microbial biovolume (fungal and bacterial). Fifty-two percent of all 4',6-diamidino-2-phenyl indole-stained cells were detectable with fluorescence in situ hybridization (FISH) with a general bacterial probe mixture. Quantitative FISH of the bacterial consortium revealed Firmicutes as the dominant group with Streptococcus as the major genus, while Actinobacteria constituted 10% of the detectable bacteria. Additionally, the study revealed an abundant and diverse fungal community including species not previously found in similar environments. The most abundant fungal 18S rRNA gene clone sequences identified affiliated with the Aspergillus-Eurotium cluster, but among others, species of Wallemia, Mucorales, and Russulales were detected. For both fungi and anaerobic bacteria, a hitherto undescribed diversity was found in bioaerosols from a modern sow breeding barn, which potentially could create poor indoor air quality, although their effect on the health of farmworkers and stock still is not resolved.     

Cyanoseq: A database of cyanobacterial 16S rRNA gene sequences with curated taxonomy

Cyanobacteria are photosynthetic bacteria that occupy various habitats across the globe, playing critical roles in many of Earth's biogeochemical cycles both in both aquatic and terrestrial systems. Despite their well-known significance, their taxonomy remains problematic and is the subject of much research. Taxonomic issues of Cyanobacteria have consequently led to inaccurate curation within known reference databases, ultimately leading to problematic taxonomic assignment during diversity studies. Recent advances in sequencing technologies have increased our ability to characterize and understand microbial communities, leading to the generation of thousands of sequences that require taxonomic assignment. We herein propose CyanoSeq ( https://zenodo.org/record/7569105 ), a database of cyanobacterial 16S rRNA gene sequences with curated taxonomy. The taxonomy of CyanoSeq is based on the current state of cyanobacterial taxonomy, with ranks from the domain to genus level. Files are provided for use with common naive Bayes taxonomic classifiers, such as those included in DADA2 or the QIIME2 platform. Additionally, FASTA files are provided for creation of de novo phylogenetic trees with (near) full-length 16S rRNA gene sequences to determine the phylogenetic relationship of cyanobacterial strains and/or ASV/OTUs. The database currently consists of 5410 cyanobacterial 16S rRNA gene sequences along with 123 Chloroplast, Bacterial, and Vampirovibrionia (formally Melainabacteria) sequences.     

Diversity of fungi in organic soils under a moorland--Scots pine (Pinus sylvestris L.) gradient

The conservation and regeneration of native Scots pine (Pinus sylvestris L.) woodlands is being actively encouraged by conservation agencies in the UK because of their high biodiversity value. In the present study, the consequences of regeneration on terrestrial fungal communities was determined in three parallel transects running from open moorland, through an intermediate zone showing seedling colonization, into a mature Scots pine forest at Abernethy Forest, Cairngorm, Scotland. Soil cores were taken at 18 m intervals along each 180 m transect, and the diversity of the soil fungal community was investigated by DGGE and sequence analysis of ITS fragments PCR-amplified from DNA extracted from soil. Analysis of DGGE profiles generated for two of the three transects indicates a clear shift in the community from the moorland region of the transects to the forest region. Whereas a few bands were present at all sampling points across the transects, the majority of bands were unique to either the moorland or forest samples. FASTA database searches of ITS sequence data generated from excised DGGE bands revealed the closest species match for each band. In some cases, the similarity of ITS sequences to database sequences was poor, but the remaining sequences were most closely related to ITS sequences of both mycorrhizal and non-mycorrhizal fungi. The data are discussed in relation to the effect of native pine woodland expansion on the soil fungal community.     

Objective criteria to assess representativity of soil fungal community profiles

Soil fungal community structures are often highly heterogeneous even among samples taken from small field plots. Sample pooling is widely used in order to overcome this heterogeneity, however, no objective criteria have yet been defined on how to determine the number of samples to be pooled for representatively profiling a field plot. In the present study PCR/RFLP and T-RFLP analysis of fungal 18S rDNA in ten soil samples obtained from a grassland plot of 400 m(2) also revealed this known heterogeneity in fungal community structures. Based on these data a three-step approach to assess representativity of fungal community profiles was established. First, soil DNA quantities needed for robust community profiling were determined. Second, profiles of single or multiple samples were theoretically averaged to test for statistically significant clustering in order to determine the minimal number of samples to be pooled to achieve representativity. Third, DNA extracts of single or multiple samples were pooled prior to profiling in order to test for statistically significant clustering. Analyses revealed robust profiles for 50 ng soil DNA but not for 5 ng. Averaged T-RFLP profiles from five or more soil samples and experimental T-RFLP profiles from pools of seven or more samples formed one significant branch. Theoretical averaging and experimental pooling revealed that five to seven samples have to be pooled for robustly representing the field plot. Our results demonstrate that representativity of soil fungal community profiles can objectively be determined for a field plot with only little deviation between theoretical and experimental approaches. This three-step approach will be of assistance for designing sampling and pooling strategies for comparative analyses of soil fungal communities in ecological studies.     

High‐throughput sequencing provides insight into manipulated soil fungal community structure and diversity during temperate forest restoration

The process of community assembly in fungal communities is poorly understood and may have important implications for restoration. However, there is a shortage of data describing fungal community composition at various stages of restoration. This study describes how microbial inoculation with field‐collected soils or a commercial inoculum influenced fungal communities during temperate tree restoration. We utilized Illumina Mi‐Seq sequencing technology to examine fungal community structure in the rhizosphere soils of trees at the conclusion of one growing season. Inoculation treatment was found to be a significant determinant of fungal community structure in one of our three experimental tree species (Liriodendron tulipifera). We also found a marginally significant influence of inoculation method on fungal community structure in the rhizosphere soils of Quercus rubra, an ectomycorrhizal tree species. Importantly, within these taxa, the use of commercial inocula, while failing to lead to detectable abundances of the inoculated taxa, strongly influenced the resulting fungal community structure after 4 months in the field, relative to control trees that received no such inoculation. We observed lower abundances of Hebeloma, a potentially important ectomycorrhizal genera, in Quercus trees receiving the commercial inoculum compared with control trees; thus, the commercial inoculum might have unexpected consequences for fungal community assembly. Such unintended legacy effects of soil inoculation should be considered in ecological restoration. Furthermore, by taking a time series approach to sampling, high‐throughput sequencing approaches could be used to test the principles of ecological assembly theory, including legacy effects of taxa no longer detectable in the community.     

白蚁巢菌圃真菌多样性研究

培菌性白蚁能在存在于蚁巢或分散在其周围土壤中的菌圃上培养真菌。菌圃在无白蚁存在下培养会生长出炭角菌的子实体。对分别采集自我国西南四川、云南两省的4个土白蚁属菌圃采用原位培养法分离并纯化得到40株炭角菌,划分为13个形态型,ITS1-5.8S-ITS2序列分析确定为两种炭角菌。采用建立ITS基因文库的方法分析了白蚁菌圃真菌群落多样性,结果表明有白蚁存在的菌圃,蚁巢伞为单一优势菌;废弃的蚁巢中的菌圃,木霉、炭角菌等其他真菌成为优势菌。     

Molecular characterizations of microbial communities fouling painted and unpainted concrete structures

This study reports the use of culture-independent and culture-dependent approaches to identify naturally occurring communities of Bacteria and Fungi fouling the surfaces of concrete structures with and without an acrylic paint coating in Georgia, USA. Genomic DNA was extracted from four different sites and PCR amplification of bacterial ribosomal RNA (16S rRNA) genes and the internal transcribed spacer (ITS) region of fungal rRNA genes was conducted. Bacterial and fungal community composition was determined by restriction analysis of amplified DNA of eight clone libraries and sequencing. Five bacterial phyla were observed, and representatives of the phylum Cyanobacteria and the classes Betaproteobacteria and Gammaproteobacteria dominated the bacterial clone libraries. The ITS region of rRNA gene sequences revealed the dominant phylotypes in the fungal clone libraries to be most closely related to Alternaria, Cladosporium, Epicoccum and Udeniomyces. The majority of these fungal genera could be cultured from the sites and successfully used to foul concrete in laboratory-based experiments. While the fungal sequences were most closely related to cultured isolates, the vast majority of bacterial sequences in the libraries were related to uncultured environmental clones. Results show phylogenetically distinct microbial populations occurring at the four sites.     

Molecular analysis of the Cryptococcus neoformans ADE2 gene, a selectable marker for transformation and gene disruption.

Cryptococcus neoformans is an important fungal pathogen of man. The incidence of cryptococcal disease has increased dramatically in patients immunocompromised because of HIV infection, organ transplantation, or treatment with cytotoxic chemotherapy or corticosteroids. This organism is an excellent model for molecular dissection of fungal pathogenesis and virulence factors. Here we report the nucleotide sequence of the C. neoformans serotype D genomic ADE2 gene, which encodes a phosphoribosylaminoimidazole carboxylase required for purine biosynthesis. Importantly, this version of the ADE2 gene has been used as the selectable marker for virtually all gene disruptions by transformation and homologous recombination in C. neoformans. We compare the nucleotide and amino acid sequences of the ADE2 gene and product to other highly related adenine biosynthetic genes and enzymes from other yeasts and fungi. We also describe a series of convenient ADE2 cassettes for gene disruption construct preparation. Finally, we have identified the ade2 mutations in strains M001 and M049, adenine auxotrophic mutants derived from the serotype A strain H99. These mutant strains have served as recipients for targeted gene disruptions using the ADE2 gene. These studies should facilitate transformation and gene disruption approaches using the ADE2 selectable marker in this important human fungal pathogen.     

Fungal diversity associated with hawaiian Drosophila host plants

Hawaiian Drosophila depend primarily, sometimes exclusively, on specific host plants for oviposition and larval development, and most specialize further on a particular decomposing part of that plant. Differences in fungal community between host plants and substrate types may establish the basis for host specificity in Hawaiian Drosophila. Fungi mediate decomposition, releasing plant micronutrients and volatiles that can indicate high quality substrates and serve as cues to stimulate oviposition. This study addresses major gaps in our knowledge by providing the first culture-free, DNA-based survey of fungal diversity associated with four ecologically important tree genera in the Hawaiian Islands. Three genera, Cheirodendron, Clermontia, and Pisonia, are important host plants for Drosophila. The fourth, Acacia, is not an important drosophilid host but is a dominant forest tree. We sampled fresh and rotting leaves from all four taxa, plus rotting stems from Clermontia and Pisonia. Based on sequences from the D1/D2 domain of the 26S rDNA gene, we identified by BLAST search representatives from 113 genera in 13 fungal classes. A total of 160 operational taxonomic units, defined on the basis of ≥97% genetic similarity, were identified in these samples, but sampling curves show this is an underestimate of the total fungal diversity present on these substrates. Shannon diversity indices ranged from 2.0 to 3.5 among the Hawaiian samples, a slight reduction compared to continental surveys. We detected very little sharing of fungal taxa among the substrates, and tests of community composition confirmed that the structure of the fungal community differed significantly among the substrates and host plants. Based on these results, we hypothesize that fungal community structure plays a central role in the establishment of host preference in the Hawaiian Drosophila radiation.