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Appl Environ Microbiol. Jun 2010; 76(12): 4063–4075.
Published online Apr 30, 2010. doi:  10.1128/AEM.02928-09
PMCID: PMC2893488

Diverse Bacteria Inhabit Living Hyphae of Phylogenetically Diverse Fungal Endophytes[down-pointing small open triangle]

Abstract

Both the establishment and outcomes of plant-fungus symbioses can be influenced by abiotic factors, the interplay of fungal and plant genotypes, and additional microbes associated with fungal mycelia. Recently bacterial endosymbionts were documented in soilborne Glomeromycota and Mucoromycotina and in at least one species each of mycorrhizal Basidiomycota and Ascomycota. Here we show for the first time that phylogenetically diverse endohyphal bacteria occur in living hyphae of diverse foliar endophytes, including representatives of four classes of Ascomycota. We examined 414 isolates of endophytic fungi, isolated from photosynthetic tissues of six species of cupressaceous trees in five biogeographic provinces, for endohyphal bacteria using microscopy and molecular techniques. Viable bacteria were observed within living hyphae of endophytic Pezizomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes from all tree species and biotic regions surveyed. A focus on 29 fungus/bacterium associations revealed that bacterial and fungal phylogenies were incongruent with each other and with taxonomic relationships of host plants. Overall, eight families and 15 distinct genotypes of endohyphal bacteria were recovered; most were members of the Proteobacteria, but a small number of Bacillaceae also were found, including one that appears to occur as an endophyte of plants. Frequent loss of bacteria following subculturing suggests a facultative association. Our study recovered distinct lineages of endohyphal bacteria relative to previous studies, is the first to document their occurrence in foliar endophytes representing four of the most species-rich classes of fungi, and highlights for the first time their diversity and phylogenetic relationships with regard both to the endophytes they inhabit and the plants in which these endophyte-bacterium symbiota occur.

Traits related to the establishment and outcome of plant-fungus symbioses can reflect not only abiotic conditions and the unique interactions of particular fungal and plant genotypes (49, 50, 56, 59, 62, 67) but also additional microbes that interact intimately with fungal mycelia (4, 12, 42). For example, mycorrhizosphere-associated actinomycetes release volatile compounds that influence spore germination in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita (Glomeromycota) (14). Levy et al. (34) describe Burkholderia spp. that colonize spores and hyphae of the AM fungus Gigaspora decipiens and are associated with decreased spore germination. Diverse “helper” bacteria have been implicated in promoting hyphal growth and the establishment of ectomycorrhizal symbioses (23, 26, 57, 70). Minerdi et al. (43) found that a consortium of ectosymbiotic bacteria limited the ability of the pathogen Fusarium oxysporum to infect and cause vascular wilts in lettuce, with virulence restored to the pathogen when ectosymbionts were removed.

In addition to interacting with environmental and ectosymbiotic bacteria, some plant-associated fungi harbor bacteria within their hyphae (first noted as “bacteria-like organisms” of unknown function) (38). These bacteria, best known from living hyphae of several species of the Glomeromycota and Mucoromycotina, can alter fungal interactions with host plants in diverse ways (see references 12, 31, and 51). For example, the vertically transmitted bacterium “Candidatus Glomeribacter gigasporarum” colonizes spores and hyphae of the AM fungus Gigaspora gigasporarum (9, 10). Removal of the bacterial partner from the fungal spores suppresses fungal growth and development, altering the morphology of the fungal cell wall, vacuoles, and lipid bodies (37). In turn, the discovery of phosphate-solubilizing bacteria within Glomus mossae spores (44), coupled with the recovery of a P-transporter operon in Burkholderia sp. from Gigaspora margarita (54), suggests a competitive role in phosphate acquisition and transport by these bacteria within the AM symbiosis. Within the Mucoromycotina, Partida-Martinez and Hertweck (51) reported that a soilborne plant pathogen, Rhizopus microsporus, harbors endosymbiotic Burkholderia that produces a phytotoxin (rhizoxin) responsible for the pathogenicity of the fungus.

These examples, coupled with the discovery of bacteria within hyphae of the ectomycorrhizal Dikarya (Tuber borchii; Ascomycota; Laccaria bicolor and Piriformospora indica; Basidiomycota) (5-8, 58), suggest that the capacity to harbor endohyphal bacteria is widespread among fungi. To date, however, endocellular bacteria have been recovered only from fungi that occur in the soil and rhizosphere (12, 31). Here we report for the first time that phylogenetically diverse bacteria occur within living hyphae of foliar endophytic fungi, including members of four classes of filamentous Ascomycota. We use a combination of light and fluorescence microscopy to visualize bacterial infections within living hyphae of representative strains. Then, drawing from surveys of endophytes from asymptomatic foliage of cupressaceous trees in five biogeographic provinces, we provide a first characterization of the phylogenetic relationships, host associations, and geographic distributions of endohyphal bacteria associated with focal fungal endophytes.

MATERIALS AND METHODS

Conifers (Pinophyta or Coniferae), comprising ca. 630 species, form the dominant tree component in major biomes such as boreal forests, temperate tree savannas, and temperate rainforests (20, 21, 22). The most widely distributed family of conifers, the Cupressaceae (cypresses and relatives), includes 30 genera and ca. 142 species in seven proposed subfamilies (25). Eleven genera are placed in the subfamily Cupressoideae, including three examined in this study (Cupressus, Juniperus, and Platycladus).

We collected foliar endophytes from six species of Cupressoideae, as available, in five localities (Table (Table1):1): semideciduous forest in the piedmont of North Carolina and four distinct regions of Arizona, selected on the basis of distinctive biogeographic histories and the occurrence of desired plant species. These sites included the Chuska Mountains (CHU) of northeastern Arizona, with petran montane coniferous forest featuring significant elements of the Rocky Mountain flora; the Mogollon Rim (MOG) and central mountains, including Mingus Mountain and the Bradshaw Mountains, which lie at the interface of the Great Basin coniferous woodlands, interior chaparral, and petran montane forest; the Sky Island archipelago (SKY) of southeastern Arizona, including the Santa Catalina and Chiricahua Mountains, characterized by madrean evergreen forest; and the Campus Arboretum at the University of Arizona (UA), a semiurban, cultivated setting within the Arizona upland province of the Sonoran Desert.

TABLE 1.
Locations and characteristics of sampling sites and plant species surveyed for endophytes in each location

Endophyte collection.

Fresh, asymptomatic photosynthetic tissue was collected from at least three healthy, mature individuals of each focal species in each locality during the growing seasons of 2004 to 2007. From each tree, healthy, mature photosynthetic tissue was collected from three haphazardly selected branches at the outer canopy ca. 1 to 2 m above ground. Material was transferred to the laboratory for processing within 6 to 12 h of collection.

Tissue samples were washed in running tap water and then cut into 2-mm segments, corresponding to infection domains for individual fungi (3). Segments were surface sterilized by rinsing in 95% ethanol for 30 s, 10% Clorox (0.6% sodium hypochlorite) for 2 min, and 70% ethanol for 2 min (1), allowed to surface dry under sterile conditions, and plated on 2% malt extract agar (MEA), which encourages growth by a diversity of endophytes (24). In sum, 384 tissue segments (96 per individual) were plated per species at each location, with the exceptions of Juniperus deppeana in the Chiricahua Mountains (144 segments), and Platycladus orientalis and Juniperus virginiana from North Carolina (96 segments/species).

Plates were incubated at room temperature and inspected for hyphal growth daily for 18 weeks. Emerging fungi were isolated on 2% MEA, archived as living vouchers in sterile water, and deposited at the Robert L. Gilbertson Mycological Herbarium at the University of Arizona (ARIZ; accession numbers are available from M. T. Hoffman). Seven isolates of bacteria obtained directly from surface-sterilized plant tissue (i.e., bacterial endophytes) were accessioned as sterile cultures and maintained in sterile 80% glycerol at −80°C.

PCR amplification and sequencing.

Total genomic DNA was extracted directly from axenic fungal cultures, following the method of Arnold et al. (2). In addition, DNA was extracted a second time from mycelium of five isolates using the Qiagen DNeasy Plant Mini Kit to ensure that none of our records of endohyphal bacteria represented contamination of reagents in our standard DNA extraction protocol. Our two extraction methods were consistent in terms of DNA extraction quality, sequence quality, and resulting diagnoses of bacterial infection.

For each fungal isolate, we used PCR to amplify the nuclear ribosomal internal transcribed spacers (ITS) and the 5.8S gene (ITS ribosomal DNA [rDNA]) and when possible the first 600 bp of the large subunit (LSU rDNA) as a single fragment (ca. 1,000 to 1,200 bp in length) using the primers ITS1F and ITS4 or LR3 (27, 55, 66, 69). Each 25-μl reaction mixture included 12.5 μl of Sigma Readymix REDTaq, 1 μl of each primer (10 μM), 9.5 μl of PCR-quality water, and 1 μl of DNA template. Cycling reactions were run with MJ Research PTC200 thermocyclers and consisted of 94°C for 3 min, 36 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 1 min, and 72°C for 10 min.

The presence or absence of bacteria within the surrounding matrix was determined initially using light microscopy. Fungal isolates were examined after 1 week of growth in pure culture on 2% MEA using a Leica DM4000B microscope with bright-field imaging (400×; numerical aperture [NA] = 0.75). Once visual examination ruled out nonendohyphal bacteria (i.e., contaminants in the medium or microbes on hyphal surfaces), total genomic DNA extracted from fresh mycelia was examined using PCR primers specific to bacterial 16S rRNA genes: 10F and 1507R (1,497 bp) or 27F and 1429R (1,402 bp) (33, 45). PCR mixes and cycling parameters were as described above, except that annealing temperatures were 58°C (10F and 1507R) or 55°C (27F and 1429R).

SYBR green I stain (Molecular Probes, Invitrogen) was used to detect DNA bands on 1.5% agarose gels. Positive PCR products were cleaned, quantified, and normalized at the University of Arizona Genomic Analysis and Technology Core facility (GATC), followed by bidirectional sequencing with PCR primers (5 μM) using an Applied Biosystems 3730XL DNA analyzer. PCR products of insufficient concentration for sequencing were cloned using Agilent Technologies StrataClone cloning kits by following the manufacturer's instructions. Water was used in place of template for negative controls. Positive clones were amplified and sequenced using the primers M13F and M13R.

The software applications Phred and Phrap (18, 19) were used to call bases and assemble bidirectional reads into contiguous consensus sequences, with automation provided by ChromaSeq (39), implemented in Mesquite, v. 2.6 (40). Base calls were verified by inspection of chromatograms in the Sequencher (v. 4.5) software program (Gene Codes Corp., Ann Arbor, MI). ITS rDNA and partial LSU rDNA sequence data and bacterial 16S rRNA gene sequences have been submitted to GenBank (see below) or were published previously by the authors (28).

Diversity and taxonomic placement of endophytes.

Previous studies have used 90 to 97% ITS rDNA sequence similarity to estimate species boundaries for environmental samples of fungi (97% [O'Brien et al. {48}], 90% [Hoffman and Arnold {28}], and 95% [Arnold et al. {1}]). Empirical estimates of percent sequence divergence between sister species for four endophyte-containing ascomycetous genera (Botryosphaeria, Colletotrichum, Mycosphaerella, and Xylaria) indicated that groups based on 95% ITS rDNA sequence similarity conservatively estimate species (65). We used Sequencher v. 4.5 (Gene Codes Corp., Ann Arbor, MI; default settings except for the requirement of a 20-character minimum overlap; see Arnold et al. [3]) to estimate fungal operational taxonomic units (OTU) at 95% ITS rDNA sequence similarity and bacterial OTU at 97% 16S sequence similarity (61).

Taxonomic placement for fungi at the level of order and above was estimated by BLAST comparisons with the curated ITS rDNA database for fungi maintained by the Alaska Fungal Metagenomics Project (FMP) (http://www.borealfungi.uaf.edu/) and phylogenetic analysis (see below). Bacterial taxonomy was estimated by BLAST comparisons with GenBank and the Ribosomal Database Project (RDP) (release 9) classifier program (Naive Bayesian rRNA Classifier, version 2.0, July 2007) (15, 68), combined with phylogenetic analyses (see below). All statistical analyses were performed in JMP 7.0 software program (SAS Institute Inc. Cary, NC).

Live/Dead stain.

Live/Dead stain was used to confirm the presence and viability of endohyphal bacteria for fungal isolates identified as containing bacteria on the basis of light microscopy and PCR (above). We treated fresh mycelia in sterile water with 2 μl of Molecular Probes Live/Dead fluorescent stain for 15 min and prepared slides with Vectashield HardSet 1400 medium (Vector Laboratories, Inc.) to prevent photobleaching. A Leica DM4000B microscope with a Luminera camera and 100-W mercury arc lamp was used for fluorescent imaging with a Chroma Technology filter set, 35002 (480-nm excitation/520-nm emission) and 1,000× APO oil objective (NA = 1.40 to 0.70; 0.13-mm working distance) at room temperature. Visible fluorescence of bacterial nucleic acids within living hyphae that was not consistent with fungal mitochondrial or nuclear DNA, coupled with positive PCR results and a lack of extrahyphal or ectosymbiotic bacteria, provided evidence of viable endohyphal bacteria (46, 63).

FISH.

To rule out misinterpretation of fluorescence results from Live-Dead analyses, we used fluorescent in situ hybridization (FISH) with a probe specific for eubacteria to confirm endocellular infection in two focal isolates (9084b and 9143). In each case, we examined hyphae from cultures grown on 2% MEA and on 2% MEA amended with antibiotics, which removed evident bacterial infections (27a).

Fresh mycelium was harvested and suspended in 1,000 μl 1× phosphate-buffered saline (PBS). After centrifugation at 8,000 rpm for 5 min, PBS buffer was replaced with fresh 4% formaldehyde solution and incubated for 1.5 h at 4°C. Samples then were centrifuged at 8,000 rpm for 5 min, and the pellet was resuspended in PBS buffer twice prior to storage in absolute ethanol at −20°C. Fixed mycelium was collected in 0.5-ml tubes and dehydrated in a series of ethanol-PBS solutions beginning with 50%, 70%, and finally 95% ethanol. Dehydrated mycelium was incubated for 90 min at 46°C in 10 μl of hybridization buffer using 40% formamide hybridization stringency (800 μl formamide, 800 μl diethyl pyrocarbonate [DEPC] water, 500 μl EDTA) with 2 μl of EUB338 probe (10 μM), a universal 16S rRNA gene oligonucleotide probe (labeled with 5′ 6-carboxytetramethylrhodamine [TAMRA] fluorochrome tag; 5′ GCTGCCTCCCGTAGGAGT 3′; excitation, 559 nm/emission, 583 nm; Integrated DNA Technologies, Inc.). Each sample was rinsed in 100 μl wash buffer (460 μl 5 M NaCl, 1 ml 1 M Tris, 50 μl SDS, DEPC water, filled to 50 ml) and warmed to 46°C twice. This method was repeated using the following: (i) antibiotic-cured isolates of each strain, which showed no evidence of endohyphal bacteria, and (ii) hybridization buffer and PBS only, but no EUB338 probe, to control for autofluorescence. Mycelium was mounted on gelatin-coated glass slides using 4′,6-diamidino-2-phenylindole (DAPI) as a counterstain (41) and examined with either an Olympus BX61 microscope with a mercury arc lamp or a Leica DMI 6000 confocal system equipped with a 543-nm laser (63× oil; 1,024 × 1,024 format; xyz acquisition; line average = 1; frame average = 4). Leica software, LAS-AF v. 1.8.2, was used to capture images.

Phylogenetic analyses.

Based on positive 16S rRNA gene PCR results and the lack of any extrahyphal bacteria in our axenic cultures, endohyphal bacteria were observed in 75 of 414 fungal isolates. Twenty-one infected isolates, representing a broad diversity of the Pezizomycotina, and 49 isolates in which infections were never observed and which represented a broad array of ITS rDNA genotype groups were sequenced for 1,200 bp of LSU rDNA, following the method of Arnold et al. (3) (primers LROR or LR3R and LR7; protocols as described above).

These 70 sequences were integrated into core alignments of 157 representative Ascomycota, partitioned by class into Sordariomycetes, Dothideomycetes, and Eurotiomycetes (28) using the Mesquite software program, v. 2.6 (40). Details of taxon sampling are given in Table Table22 and also in Table S1 in the supplemental material. Because our sample of Pezizomycetes contained only one isolate with strong evidence of endohyphal bacteria, we did not address phylogenetic relationships of pezizomycetous endophytes here.

TABLE 2.
Taxonomic classification for 29 fungal endophytes and associated endohyphal bacteriaa

Alignment for each class, based on LSU rDNA secondary structure defined by Saccharomyces cerevisiae (13), was performed using ClustalW with manual adjustment in Mesquite. All ambiguously aligned regions were excluded, with matrix details given in Table S2 in the supplemental material. Alignments are available online at http://arnoldlab.net/alignments.html.

Phylogenetic relationships for members of each class were inferred using parsimony and Bayesian Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analyses. For the former, five sets of 200 heuristic searches with random stepwise addition and tree bisection-reconnection (TBR) were implemented using the PAUPRat software program, which incorporates the “parsimony ratchet” method (28, 47, 60) in PAUP* 4b10 (64). The “filter trees” command in PAUP* 4b10 was used to select all shortest trees, which were used to assemble strict consensuses. Bootstrap support was determined using 1,000 replicates and the default settings for nonparametric neighbor-joining (NJ) analysis as implemented in PAUP* 4b10. Clades with ≥70% bootstrap support were considered strongly supported. Details of parsimony searches are given in Table S2 in the supplemental material.

For Bayesian analyses, the Modeltest 3.7 software program (53) was used to select the appropriate model of evolution; in each case, GTR+I+G was selected using the Akaike information criterion. Analyses were executed in MrBayes 3.1.2 (30) for four runs of up to 6 million generations each, initiated with random trees, four chains, and sampling every 1,000th generation. Likelihoods converged to a stable range for each data set, and all trees prior to convergence were discarded as burn-in. Clades with ≥95% posterior probability values were considered to have significant support.

Twenty-nine bacterial 16S DNA sequences, derived from total genomic DNA extractions of fungal endophyte mycelia with no evidence of extrahyphal bacteria (Table (Table2),2), and seven 16S sequences from bacterial endophytes obtained directly from plant material (Table (Table3)3) were incorporated into a core alignment of 30 sequences of named bacterial taxa obtained from the Ribosomal Database Project 9 website (Cole et al. [15]; http://rdp.cme.msu.edu) using Seqmatch (type strains, isolates, ≥1,200 bp, good quality; ca. 1,400 bp), together with eight additional sequences from GenBank that represented endosymbionts of fungi isolated in previous studies (see Table S3 in the supplemental material) (9-11, 52). Data were aligned using the NAST server and screened for chimeric sequences (minimum sequence length = 300 bp) (http://greengenes.lbl.gov/) (17). Ambiguous regions were excluded, resulting in a matrix of 6,300 characters (http://arnoldlab.net/alignments.html).

TABLE 3.
Bacterial endophytes isolated from foliage of focal species of Cupressaceaea

For the bacterial data set, a heuristic search using maximum parsimony with random stepwise additions and TBR branch swapping was implemented in PAUP* 4b10, resulting in 167 optimal trees of 3,668 steps. Support was assessed using a nonparametric neighbor-joining bootstrap (1,000 replicates). Bayesian MCMCMC analysis was implemented in MrBayes v. 3.1.2 for 2.5 million generations, initiated with random trees, four chains, and sampling every 1,000th tree, using GTR+I+G based on evaluation in Modeltest 3.7 (53). After elimination of the first 1,400 trees as burn-in, the remaining 1,100 trees were used to infer a majority-rule consensus. These results were complemented by phylogenetic inference in the ARB software program, v. 7.12.07 (36), using the AxML maximum-likelihood (ML) method (FastDNAML [F84; G. J. Olsen and J. Felsenstein, developers]; no filter: all positions included in phylogenetic analysis; −ln likelihood = 22,430.51). Branch support was assessed using parametric ML bootstrapping (100 replicates) and Bayesian posterior probabilities.

RESULTS

Cultivable endophytes were isolated from healthy foliage of all plant species and from every study site (Table (Table1),1), yielding 414 isolates from 4,560 tissue segments. ITS rDNA data for all isolates were assembled at 95% sequence similarity to yield 113 OTU (putative species; Fisher's alpha = 51.2). Representatives of 5 classes and approximately 10 orders, 13 families, and 28 genera were recovered. The most common orders differed among host species and sites, but the entire data set comprised a high frequency of Pezizales (Pezizomycetes), Capnodiales, Pleosporales, Botryosphaeriales, Dothideales (Dothideomycetes), Helotiales (Leotiomycetes), and Xylariales (Sordariomycetes) (see Table S4 in the supplemental material). Host specificity, diversity, and geographic distributions of these fungi will be addressed in a forthcoming paper (M. T. Hoffman and A. E. Arnold, unpublished data).

Endohyphal bacteria.

Endohyphal bacteria initially were observed in 75 of 414 endophyte isolates (18%; determined by positive PCR results using 16S primers and no evidence of extrahyphal bacteria), including isolates from all four classes of Ascomycota recovered in our surveys (Pezizomycetes, Dothideomycetes, Eurotiomycetes, and Sordariomycetes), isolates from all three host genera (Cupressus, Juniperus, and Platycladus), and isolates from plants in all biogeographic regions (Table (Table2).2). After 47 of these isolates were subcultured, however, they later were screened as negative for bacteria. In general, length of time in culture (after ca. 2 weeks) appeared to adversely affect the detectability or persistence of bacterial infections.

Based on examination with light microscopy and Live/Dead stain, no isolate scored as positive for endohyphal bacteria had evident extrahyphal or ectosymbiotic bacteria. Live/Dead staining indicated that bacteria within hyphae were viable and confirmed viability of the hyphae that housed them (data not shown). Fluorescence in situ hybridization (FISH) for isolates 9084b and 9143 further confirmed the presence of endohyphal bacteria within living mycelia. Isolate 9084b is shown with the EUB338 fluorescent probe mounted in antifading mount medium only (Fig. (Fig.11 A). Isolate 9143 is shown with the EUB338 probe and DAPI counterstain (Fig. (Fig.1B),1B), illustrating the presence of small numbers of bacterial cells in comparison to significant amounts of total genomic DNA contained in the hyphal structure.

FIG. 1.
Fluorescent in situ hybridization (FISH) microscopy showing hyphae of two isolates of endophytic fungi harboring endohyphal bacteria. Panel A (isolate 9084b; Dothideomycetes) shows the TAMRA fluorophore at the site of internal structure in hyphal cells. ...

Phylogenetic inferences and taxonomic placement.

Phylogenetic analyses confirmed BLAST-level taxonomy at the class level for fungal endophytes in the Dothideomycetes, Eurotiomycetes, and Sordariomycetes (see Fig. Fig.22 to to4).4). No pattern regarding the structure of endophyte lineages as a function of host plant taxonomy was evident (i.e., the sister relationship of Cupressus and Juniperus and their sister relationship to Platycladus are not reflected in the phylogenetic relationships of their fungal associates). Isolates containing bacterial associates were spread broadly across endophyte-containing clades in each class, indicating a phylogenetically widespread capacity to harbor bacteria.

FIG. 2.
Phylogenetic relationships of fungal endophytes with representative Dothideomycetes based on a majority rule consensus tree from Bayesian analysis. Branch support values indicate parsimony bootstrap proportions (≥70%; before slash) and ...
FIG. 4.
Phylogenetic relationships of endophytes with representative Sordariomycetes based on a majority rule consensus tree from Bayesian analysis of 95 LSU rDNA sequences. Branch support values indicate parsimony bootstrap proportions (≥70%; ...

Taxonomic placement of endohyphal bacteria initially was assessed using BLAST comparisons in GenBank, typically yielding matches to unidentified or nameless environmental samples. The RDP classifier (68) placed these bacteria in two phyla, the Proteobacteria (including the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria) and Firmicutes. Overall, we recovered five orders and 10 families, putatively identified as Sphingomonadaceae (Alphaproteobacteria), Burkholderiaceae, Comamonadaceae, and Oxalobacteraceae (Betaproteobacteria), Moraxellaceae, Xanthomonadaceae, Pasteurellaceae, and Enterobacteriaceae (Gammaproteobacteria) and Bacillaceae and Paenibacillaceae (Firmicutes) (Table (Table22).

Based on 97% 16S rRNA gene identity, these bacteria represented 15 OTU. Of these, nine were found only once. Three of the remaining six OTU were found in fungi from multiple genera of Cupressaceae, four were found in fungi from more than one biogeographic region, and all were found in fungi representing multiple genera. Four genotypes each were found in fungi representing different classes of Pezizomycotina (genotypes A, B, C, and E) (Table (Table22).

Proteobacteria were observed in association with the Dothideomycetes, Eurotiomycetes, and Sordariomycetes and in endophytes from all three plant genera representing hosts in Arizona and North Carolina. Firmicutes were found in the Eurotiomycetes and one member of the Pezizomycetes. Nominal logistic analysis provided no evidence for a significant effect of host genus (Cupressus, Juniperus, or Platycladus), region (Arizona versus North Carolina), or fungal class on the incidence of major bacterial groups (Alpha-, Beta-, and Gammaproteobacteria; Firmicutes) (P > 0.05 for all effects).

Phylogenetic relationships of endohyphal bacteria from fungal endophytes were not congruent with those of the endophytes they inhabited (Fig. (Fig.22 to to5),5), nor with relationships among host plants (data not shown). The bacterial lineages recovered here were distinct from those recognized previously as endosymbionts of fungi (Fig. (Fig.55).

FIG. 5.
Phylogenetic relationships of 29 endohyphal bacteria, 7 bacterial endophytes (paired circles), and 38 named taxa based on Bayesian analysis of 16S ribosomal RNA gene sequences. Branch support values indicate parsimony bootstrap proportions (≥70%; ...

Comparison of bacterial endophytes and endohyphal bacteria.

Only one bacterial genotype was represented both among bacterial endophytes (isolated from surface-sterilized plant tissue only) and putative endohyphal bacteria (genotype A, Bacillales; Tables Tables22 and and3).3). This genotype was isolated directly from foliage of Juniperus osteosperma (Chuska Mountains) and Cupressus arizonica and Juniperus deppeana (Sky Islands) and was amplified from genomic DNA from a fungal culture recovered from J. deppeana (Sky Islands). Two additional endophytes recovered here also were members of the Bacillaceae, representing genotypes that never were found as endohyphal bacteria (genotypes H and J) (Tables (Tables22 and and3).3). One bacterial endophyte represented the Enterobacteriaceae (putative Erwinia). It was not found as a bacterial endosymbiont within fungal hyphae.

DISCUSSION

Recent studies have highlighted the potential of endosymbiotic microbes, including bacteria and viruses, to shape the ecological roles of fungi (see, e.g., references 16, 42, and 51). Endohyphal bacteria have been found previously in living hyphae of plant-associated Glomeromycota, Mucoromycotina, and up to three taxa of mycorrhizal Dikarya. Our study is the first to compare their phylogenetic relationships with diverse fungal hosts and the first to document their occurrence in ascomycetous endophytes of foliage representing four of the most species-rich classes of Ascomycota (Pezizomycetes, Sordariomycetes, Dothideomycetes, and Eurotiomycetes), which together include numerous plant pathogens, saprotrophs, and pathogens and parasites of animals (30). Coupled with previous studies demonstrating the presence of bacteria in living mycelia of mycorrhizal or soilborne fungi (6, 7, 9, 51), our data provide strong evidence that the ability of fungi to harbor endohyphal bacteria is phylogenetically widespread.

Our survey data show that endohyphal bacterium-endophyte associations occur in regions with divergent biogeographic histories and markedly different environmental conditions (e.g., diverse, arid-land montane systems versus piedmont forest in southeastern North America) and in fungi associated with three genera of plants. Screening of a small number of isolates obtained in previous studies (Fig. (Fig.22 to to4)4) confirmed that endohyphal bacteria occur in endophytes from additional biogeographic provinces and host plant lineages: infections were observed in endophytes isolated from the Pinaceae in boreal forest (endophyte 4466 from Picea mariana in Canada) and two families of angiosperms from a tropical forest (endophyte 6722 from Faramea occidentalis, Rubiaceae, and endophyte 6731 from Swartzia simplex, Fabaceae, recovered at Barro Colorado Island, Panama). Because infections either were lost or became more difficult to detect following subculturing and because of potential limitations of our microscopy, screening methods, and primer selection, our results likely underestimate the incidence of endohyphal bacteria in these plant-associated fungi.

Examination with Live/Dead stain provided evidence that bacteria within fungal hyphae were viable and showed that such bacteria occur within living fungal tissues. FISH confirmed these results by diagnosing the presence of bacteria using 16S rRNA gene-specific tags (Fig. (Fig.1).1). In turn, our phylogenetic inferences show that endohyphal bacteria include a greater phylogenetic diversity—both within and beyond the Proteobacteria—than was previously known (23, 32, 52) (Fig. (Fig.55).

In contrast to previous studies, which have focused primarily on bacteria associated with a particular fungal species or closely related group of species, we addressed the diversity of bacteria associated with a phylogenetically broad sample of ecologically similar fungi. Our analyses provide no evidence of cocladogenesis: fungal and bacterial trees were obviously incongruent, and neither matched the phylogenetic relationships among the plant species surveyed here. Our phylogenetic results are consistent with the hypothesis of horizontal transmission both for fungal endophytes, as expected (1, 28), and for bacterial symbionts (Fig. (Fig.22 to to55).

Using cultivation media infused with antibiotics, we have found that many of these fungi can be cured of their endosymbionts (Hoffman and Arnold, unpublished data), and we currently are investigating methods to reinfect cured mycelia. These attempts will shed light on our hypotheses regarding the facultative nature of these associations, their transmission modes, and their costs and benefits with regard to the inhabited fungi. Notably, some endophytes were unable to grow when transferred to media containing antibiotics (Hoffman and Arnold, unpublished data), suggesting that the relationship may be more intimate or tend toward greater obligacy in some pairs of fungi/bacteria than others. For one isolate (9143), we have successfully isolated the bacterial partner from the fungus and confirmed its identity using 16S rRNA gene sequencing (27a). Attempts to reinfect the endophyte with this bacterium have been ineffective to date.

Discovery of the diversity and ecological roles of fungal endophytes encompasses a trove of future research that will be important for understanding plant ecology and evolution. So too does elucidation of the incidence, diversity, and ecological importance of their endohyphal bacteria, which appear to be common but previously overlooked inhabitants of plant-symbiotic fungi. Evidence from a variety of studies (reviewed in references 12 and 32) indicates that endohyphal bacteria have the capacity to alter the outcome of plant-fungus interactions in a phylogenetically diverse array of symbioses. Experimental assessment of such effects will provide key evidence for understanding the degree to which bacterial associates influence the nature of endophytic symbioses. In turn, ancestral-state reconstructions and further sampling of both endohyphal bacteria (of fungi) and bacterial endophytes (of plants) will elucidate whether bacteria have transitioned from endophytic to endohyphal lifestyles (or the reverse) or whether they have followed a different and yet-to-be-identified evolutionary trajectory.

FIG. 3.
Phylogenetic relationships of endophytes with representative Eurotiomycetes based on a majority-rule consensus tree from Bayesian analysis of 25 LSU rDNA sequences. Branch support values indicate parsimony bootstrap proportions (≥70%; ...

Supplementary Material

[Supplemental material]

Acknowledgments

We thank the Division of Plant Pathology and Microbiology, School of Plant Sciences, and College of Agriculture and Life Sciences at the University of Arizona and the Arizona-Nevada Academy of Sciences for supporting this work. Additional support from the National Science Foundation (NSF-0626520 and NSF-0702825 to A.E.A. and NSF-IGERT to M.T.H.) is gratefully acknowledged.

We thank D. R. Maddison for sharing prerelease versions of Mesquite and Chromaseq; M. Gunatilaka, M. Shimabukuro, D. Grippi, M. J. Epps, and C. Weeks-Galindo for lab assistance and helpful discussion; J. U'Ren, F. Lutzoni, E. Gaya, J. Miadlikowska, and F. Santos-Rodriguez for isolation of samples from the Chiricahua Mountains; and B. Klein and undergraduate students at Diné College for isolation of samples from the Chuska Mountains on the Navajo Nation. We are especially grateful to H. VanEtten and R. Palanivelu for access to microscopy facilities, A. Estes for EUB338 probes and helpful discussion about FISH, and J. L. Bronstein for comments on the manuscript.

Footnotes

[down-pointing small open triangle]Published ahead of print on 30 April 2010.

Supplemental material for this article may be found at http://aem.asm.org/.

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