Endohyphal Bacteria Research Articles

Genome analysis and hyphal movement characterization of the hitchhiker endohyphal Enterobacter sp. from Rhizoctonia solani.

Bacterial-fungal interactions are pervasive in the rhizosphere. While an increasing number of endohyphal bacteria have been identified, little is known about their ecology and impact on the associated fungal hosts and the surrounding environment. In this study, we characterized the genome of an Enterobacter sp. Crenshaw (En-Cren), which was isolated from the generalist fungal pathogen Rhizoctonia solani, and examined the genetic potential of the bacterium with regard to the phenotypic traits associated with the fungus. Overall, the En-Cren genome size was typical for members of the genus and was capable of free-living growth. The genome was 4.6 MB in size, and no plasmids were detected. Several prophage regions and genomic islands were identified that harbor unique genes in comparison with phylogenetically closely related Enterobacter spp. Type VI secretion system and cyanate assimilation genes were identified from the bacterium, while some common heavy metal resistance genes were absent. En-Cren contains the key genes for indole-3-acetic acid (IAA) and phenylacetic acid (PAA) biosynthesis, and produces IAA and PAA in vitro, which may impact the ecology or pathogenicity of the fungal pathogen in vivo. En-Cren was observed to move along hyphae of R. solani and on other basidiomycetes and ascomycetes in culture. The bacterial flagellum is essential for hyphal movement, while other pathways and genes may also be involved.IMPORTANCEThe genome characterization and comparative genomics analysis of Enterobacter sp. Crenshaw provided the foundation and resources for a better understanding of the ecology and evolution of this endohyphal bacteria in the rhizosphere. The ability to produce indole-3-acetic acid and phenylacetic acid may provide new angles to study the impact of phytohormones during the plant-pathogen interactions. The hitchhiking behavior of the bacterium on a diverse group of fungi, while inhibiting the growth of some others, revealed new areas of bacterial-fungal signaling and interaction, which have yet to be explored.

Klebsiella michiganensis: a nitrogen-fixing endohyphal bacterium from Ustilago maydis

Ustilago maydis is a pathogenic fungus in Basidiomycota causing corn smut disease. A strain of U. maydis YZZF202006 was isolated from the tumor of corn smut collected from Jingzhou city in China. The intracellular bacteria were confirmed inner hyphal of the strain YZZF202006 by PCR amplification and fluorescence in situ hybridization (FISH) and SYTO-9. An endohyphal bacterium YZUMF202001 was isolated from the protoplasts of the strain YZZF202006. It was gram-negative, short rod-shaped with smooth light yellow colony. The endohyphal bacterium was genomic evidenced as Klebsiella michiganensis on the basis of average nucleotide identity (ANI) analysis and the phylogram. Then K. michiganensis was GFP-Labeled and reintroduced into U. maydis, which confirmed the bacterium can live in hyphae of U.maydis. The bacterium can grow on N-free culture media. Its nitrogenase activity was reached av. 646.25 ± 38.61 nmol·mL− 1·h− 1 C2H4 by acetylene reduction assay. A cluster of nitrogen fixation genes (nifJHDKTXENXUSVWZMFLABQ) was found from its genome. The endohyphal K. michiganensis may play an important role to help nitrogen fixation for fungi in the future.

Prebiotics: A Solution for Improving Plant Growth, Soil Health, and Carbon Sequestration?

Soil fertility and productivity are severely impacted by exploitation and degradation processes. These threats, coupled with population growth and climatic changes, compel us to search for innovative agroecological solutions. Prebiotics, a type of soil biostimulant, are used to enhance soil conditions and plant growth and may play a role in carbon (C) sequestration. Two commercial prebiotics, K1® and NUTRIGEO L® (referred to as SPK and SPN, respectively), were assessed for their effects on agricultural soil cultivated with Zea mays L., compared to untreated soil or control (SP). Analyses were performed at two harvesting dates: three weeks (D1) and ten weeks (D2) after the application of prebiotics. Plant growth parameters and soil characteristics were measured, focusing on soil organic matter, soil bacterial and fungal communities, and plant root mycorrhization. Regarding physicochemical parameters, both prebiotic treatments increased soil electrical conductivity, cation exchange capacity, and soluble phosphorus (P) while decreasing nitrates. Meanwhile, the SPN treatment was distinct in elevating specific cationic minerals, such as calcium (Ca) and boron (B), at D2. At the microbial level, each prebiotic induced a unique shift in the indigenous bacterial and fungal communities’ abundance and diversity, evident at D2. Simultaneously, specific microbial taxa were recruited by each prebiotic treatment, such as Caulobacter, Sphingobium, and Massilia from bacteria and Mortierella globalpina and Schizothecium carpinicola from fungi in SPK as well as Chitinophaga, Neobacillus, and Rhizomicrobium from bacteria and Sordariomycetes and Mortierella minutissima from fungi in SPN. These biomarkers were identified as (a) saprotrophs, (b) plant growth-promoting bacteria and fungi, (c) endohyphal bacteria, and (d) endophytic and symbiotic microbiota. This result was reflected in the increase in glomalin content and mycorrhization rate in the treated soils, especially by SPN. We observed that these effects led to an increase in plant biomass (shoots by 19% and 22.8% and roots by 47.8% and 35.7% dry weights for SPK and SPN, respectively) and contributed to an increase in soil C content (organic C by 8.4% and total C by 8.9%), particularly with SPN treatment. In light of these findings, the use of prebiotics ten weeks after application not only increased plant growth by improving soil characteristics and shaping its native microbial community but also demonstrated the potential to enhance C sequestration.

Characterization of a Plant Growth-Promoting Endohyphal Bacillus subtilis in Fusarium acuminatum from Spiranthes sinensis

Successful seed germination and seedling growth in orchids require an association with mycorrhizal fungi. An endophytic Fusarium fungal strain YZU 172038 exhibiting plant growth-promoting (PGP) ability was isolated from the roots of Spiranthes sinensis (Orchidaceae). The harboring endohyphal bacteria were detected in the hypha by SYTO-9 fluorescent nucleic acid staining, fluorescence in situ hybridization (FISH), and PCR amplification of the 16S rDNA gene's region. Consequently, one endohyphal bacterium (EHB) - a strain YZSR384 was isolated and identified as Bacillus subtilis based on morphology, phylogenetic analysis, and genomic information. The results indicated that the strain YZSR384 could significantly promote the growth of rice roots and shoots similar to its host fungus. Its indole acetic acid (IAA) production reached a maximum of 23.361 μg/ml on the sixth day after inoculation. The genome annotation revealed several genes involved in PGP traits, including the clusters of genes encoding the IAA (trpABCDEFS), the siderophores (entABCE), and the dissolving phosphate (pstABCS and phoABDHPR). As an EHB, B. subtilis was first isolated from endophytic Fusarium acuminatum from S. sinensis.

The Wheat Head Blight Pathogen Fusarium graminearum Can Recruit Collaborating Bacteria from Soil.

In nature, fungal endophytes often have facultative endohyphal bacteria (FEB). Can a model plant pathogenic fungus have them, and does it affect their phenotype? We constructed a growth system/microcosm to allow an F. graminearum isolate to grow through natural soil and then re-isolated it on a gentamicin-containing medium, allowing endohyphal growth of bacteria while killing other bacteria. F. graminearum PH-1 labelled with a His1mCherry gene staining the fungal nuclei fluorescent red was used to confirm the re-isolation of the fungus. Most new re-isolates contained about 10 16SrRNA genes per fungal mCherry gene determined by qPCR. The F. graminearum + FEB holobiont isolates containing the bacteria were sub-cultured several times, and their bacterial contents were stable. Sequencing the bacterial 16SrRNA gene from several Fg-FEB holobiont isolates revealed endophytic bacteria known to be capable of nitrogen fixation. We tested the pathogenicity of one common Fg-FEB holobiont association, F. graminearum + Stenatrophomonas maltophilia, and found increased pathogenicity. The 16SrRNA gene load per fungal His1mCherry gene inside the wheat stayed the same as previously found in vitro. Finally, strong evidence was found for Fg-S. maltophilia symbiotic nitrogen fixation benefitting the fungus.

Plant Growth-Promoting Ability of Mycorrhizal Fusarium Strain KB-3 Enhanced by Its IAA Producing Endohyphal Bacterium, Klebsiella aerogenes.

Fusarium oxysporum KB-3 had been reported as a mycorrhizal fungus of Bletilla striata, which can promote the seed germination and vegetative growth. Endohyphal bacteria were demonstrated in the hyphae of the KB-3 by 16S rDNA PCR amplification and SYTO-9 fluorescent nucleic acid staining. A strain Klebsiella aerogenes KE-1 was isolated and identified based on the multilocus sequence analysis. The endohyphal bacterium was successfully removed from the wild strain KB-3 (KB-3−), and GFP-labeled KE-1 was also transferred to the cured strain KB-3− (KB-3+). The production of indole-3-acetic acid (IAA) in the culturing broths of strains of KE-1, KB-3, KB-3−, and KB-3+ was examined by HPLC. Their IAA productions were estimated using Salkowski colorimetric technique. The highest concentrations of IAA were 76.9 (at 48 h after inoculation), 31.4, 9.6, and 19.4 μg/ml (at 60 h after inoculation), respectively. Similarly, the three fungal cultural broths exhibited plant promoting abilities on the tomato root and stem growth. The results indicated that the ability of mycorrhizal Fusarium strain KB-3 to promote plant growth was enhanced because its endohyphal bacterium, Klebsiella aerogenes KE-1, produced a certain amount of IAA.

Linnemannia elongata (Mortierellaceae) stimulates Arabidopsis thaliana aerial growth and responses to auxin, ethylene, and reactive oxygen species.

Harnessing the plant microbiome has the potential to improve agricultural yields and protect plants against pathogens and/or abiotic stresses, while also relieving economic and environmental costs of crop production. While previous studies have gained valuable insights into the underlying genetics facilitating plant-fungal interactions, these have largely been skewed towards certain fungal clades (e.g. arbuscular mycorrhizal fungi). Several different phyla of fungi have been shown to positively impact plant growth rates, including Mortierellaceae fungi. However, the extent of the plant growth promotion (PGP) phenotype(s), their underlying mechanism(s), and the impact of bacterial endosymbionts on fungal-plant interactions remain poorly understood for Mortierellaceae. In this study, we focused on the symbiosis between soil fungus Linnemannia elongata (Mortierellaceae) and Arabidopsis thaliana (Brassicaceae), as both organisms have high-quality reference genomes and transcriptomes available, and their lifestyles and growth requirements are conducive to research conditions. Further, L. elongata can host bacterial endosymbionts related to Mollicutes and Burkholderia. The role of these endobacteria on facilitating fungal-plant associations, including potentially further promoting plant growth, remains completely unexplored. We measured Arabidopsis aerial growth at early and late life stages, seed production, and used mRNA sequencing to characterize differentially expressed plant genes in response to fungal inoculation with and without bacterial endosymbionts. We found that L. elongata improved aerial plant growth, seed mass and altered the plant transcriptome, including the upregulation of genes involved in plant hormones and "response to oxidative stress", "defense response to bacterium", and "defense response to fungus". Furthermore, the expression of genes in certain phytohormone biosynthetic pathways were found to be modified in plants treated with L. elongata. Notably, the presence of Mollicutes- or Burkholderia-related endosymbionts in Linnemannia did not impact the expression of genes in Arabidopsis or overall growth rates. Together, these results indicate that beneficial plant growth promotion and seed mass impacts of L. elongata on Arabidopsis are likely driven by plant hormone and defense transcription responses after plant-fungal contact, and that plant phenotypic and transcriptional responses are independent of whether the fungal symbiont is colonized by Mollicutes or Burkholderia-related endohyphal bacteria.

Transcriptional Profiles of a Foliar Fungal Endophyte (Pestalotiopsis, Ascomycota) and Its Bacterial Symbiont (Luteibacter, Gammaproteobacteria) Reveal Sulfur Exchange and Growth Regulation during Early Phases of Symbiotic Interaction.

ABSTRACTSymbiosis with bacteria is widespread among eukaryotes, including fungi. Bacteria that live within fungal mycelia (endohyphal bacteria) occur in many plant-associated fungi, including diverse Mucoromycota and Dikarya. Pestalotiopsis sp. strain 9143 is a filamentous ascomycete isolated originally as a foliar endophyte of Platycladus orientalis (Cupressaceae). It is infected naturally with the endohyphal bacterium Luteibacter sp. strain 9143, which influences auxin and enzyme production by its fungal host. Previous studies have used transcriptomics to examine similar symbioses between endohyphal bacteria and root-associated fungi such as arbuscular mycorrhizal fungi and plant pathogens. However, currently there are no gene expression studies of endohyphal bacteria of Ascomycota, the most species-rich fungal phylum. To begin to understand such symbioses, we developed methods for assessing gene expression by Pestalotiopsis sp. and Luteibacter sp. when grown in coculture and when each was grown axenically. Our assays showed that the density of Luteibacter sp. in coculture was greater than in axenic culture, but the opposite was true for Pestalotiopsis sp. Dual-transcriptome sequencing (RNA-seq) data demonstrate that growing in coculture modulates developmental and metabolic processes in both the fungus and bacterium, potentially through changes in the balance of organic sulfur via methionine acquisition. Our analyses also suggest an unexpected, potential role of the bacterial type VI secretion system in symbiosis establishment, expanding current understanding of the scope and dynamics of fungal-bacterial symbioses.IMPORTANCE Interactions between microbes and their hosts have important outcomes for host and environmental health. Foliar fungal endophytes that infect healthy plants can harbor facultative endosymbionts called endohyphal bacteria, which can influence the outcome of plant-fungus interactions. These bacterial-fungal interactions can be influential but are poorly understood, particularly from a transcriptome perspective. Here, we report on a comparative, dual-RNA-seq study examining the gene expression patterns of a foliar fungal endophyte and a facultative endohyphal bacterium when cultured together versus separately. Our findings support a role for the fungus in providing organic sulfur to the bacterium, potentially through methionine acquisition, and the potential involvement of a bacterial type VI secretion system in symbiosis establishment. This work adds to the growing body of literature characterizing endohyphal bacterial-fungal interactions, with a focus on a model facultative bacterial-fungal symbiosis in two species-rich lineages, the Ascomycota and Proteobacteria.

Whole-Genome Sequence of Entomortierella parvispora E1425, a Mucoromycotan Fungus Associated with Burkholderiaceae-Related Endosymbiotic Bacteria.

ABSTRACTSome mucoromycotan fungi establish symbiotic associations with endohyphal bacteria. Here, the genome of Entomortierella parvispora E1425 (synonymously known as Mortierella parvispora E1425), which harbors a cultured Burkholderiaceae-related endobacterium (BRE) designated Mycoavidus sp. strain B2-EB, was sequenced. We provide genomic information to elucidate fungal-BRE symbiotic features.

Symbiotic Associations: Key Factors That Determine Physiology and Lipid Accumulation in Oleaginous Microorganisms.

Symbiosis naturally provides an opportunity for microorganisms to live together by mutual or one-way benefit. In symbiotic relationships, the microorganisms usually overcome the limitations of being free-living. Understanding the symbiotic relationships of oleaginous microorganisms provides potential route for the sustainable production of microbial-based alternative fuels. So far, several studies have been conducted in oleaginous microorganisms for the production of alternative fuels. However, some oleaginous microorganisms require high quantity of nutrients for their growth, and high level of energy and chemicals for harvest and separation of lipid bodies. Symbiotic associations can successfully be applied to address these issues. Of symbiotic associations, lichens and selective species of oleaginous endosymbiotic mucoromycotina have received substantial interest as better models to study the evolutionary relationships as well as single-cell oil production. Construction of artificial lichen system composed of cyanobacteria and oleaginous yeast has been achieved for sustainable production of lipids with minimum energy demand. Recently, endosymbiotic mucoromycotina species have been recognized as potential sources for biofuels. Studies found that endohyphal bacterium influences lipid profiling in endosymbiotic mucoromycotina species. Studies on the genetic factors related to oleaginous characteristics of endosymbiotic mucoromycotina species are scarce. In this regard, this review summarizes the different forms of symbiotic associations of oleaginous microorganisms and how symbiotic relationships are impacting the lipid formation in microorganisms. Further, the review also highlights the importance of evolutionary relationships and benefits of co-culturing (artificial symbiosis) approaches for sustainable production of biofuels.

Extending Plant Defense Theory to Seeds

Plant defense theory explores how plants invest in defenses against natural enemies but has focused primarily on the traits expressed by juvenile and mature plants. Here we describe the diverse ways in which seeds are chemically and physically defended. We suggest that through associations with other traits, seeds are likely to exhibit defense syndromes that reflect constraints or trade-offs imposed by selection to attract dispersers, enable effective dispersal, ensure appropriate timing of seed germination, and enhance seedling performance. We draw attention to seed and reproductive traits that are analogous to defense traits in mature plants and describe how the effectiveness of defenses is likely to differ at pre- and postdispersal stages. We also highlight recent insights into the mutualistic and antagonistic interactions between seeds and microbial communities, including fungi and endohyphal bacteria, that can influence seed survival in the soil and subsequent seedling vigor.

Endohyphal bacteria associated with virulence, increased expression of fumonisin biosynthetic genes, and production of fumonisin and macroconidia in Fusarium fujikuroi W343

Until recently, Fusarium fujikuroi was widely described as a non‐producer or a producer of little B‐series fumonisin toxins despite having a complement of fumonisin biosynthetic genes similar to those in F. verticillioides and F. proliferatum. Although high and low fumonisin‐producing F. fujikuroi strains have been shown to induce typical disease symptoms, the relationship between fumonisin production and virulence in F. fujikuroi has not been fully established. Following recent reports on the roles of bacterial endosymbionts in influencing the biology of their fungal hosts, including virulence, reproduction and the production of secondary metabolites, this study investigated the association of two strains of bacteria of the genus Enterobacter with a F. fujikuroi strain isolated from wheat in Nebraska. Results demonstrated the intracellular localization of bacteria within the hyphae of the fungus. The association with the bacteria was also correlated with macroconidia production and higher levels of virulence in the fungus. Additionally, fumonisin production was consistently and significantly (α = 0.05) higher in the bacteria‐containing F. fujikuroi strain compared with the bacteria‐free strain. Furthermore, higher levels of fumonisin production by bacteria‐containing F. fujikuroi correlated with higher expression levels of the fumonisin biosynthetic genes fum1, fum8 and fum21 under fumonisin‐inducing and non‐inducing conditions. Expression of fum15, which encodes a P450 monooxygenase, was similarly observed to be significantly (α = 0.05) higher in the bacteria‐containing fungus. Analysis of transcripts also revealed significantly (α = 0.05) higher expression of the sexual and asexual development activator protein, VeA, in the bacteria‐containing F. fujikuroi under fumonisin‐inducing conditions.

Context-dependent and variable effects of endohyphal bacteria on interactions between fungi and seeds

Abstract Plant-associated fungi often harbor endohyphal bacteria (EHB) that modulate fungal phenotypes. We quantified the effects of EHB on interactions between fungi and seeds of neotropical pioneer trees, which fungi colonize naturally in forest soil. Seeds were exposed to six fungal isolates that harbored EHB, and to clones of those fungi from which EHB were removed by antibiotic treatment. Seed colonization by fungi was evaluated for five tree species, and germination and viability were evaluated for three tree species. EHB influenced seed colonization by fungi in 5 of 30 fungus-tree species combinations, but the magnitude of their effects was small and the direction of effects depended upon fungal isolate-tree species pairs. EHB had rare and context-dependent effects on seed germination and viability, but their effects were strong when observed. Rare but powerful effects of EHB on fungal interactions with seeds highlight important and context-dependent aspects of plant and fungal ecology.

Quantifying Re-association of a Facultative Endohyphal Bacterium with a Filamentous Fungus.

We present here a method to quantify reassociation between facultative endohyphal bacteria and filamentous fungal hosts. Our method takes advantage of the capabilities of fungal cell walls to selectively protect internal bacteria from gentamicin treatment, an assay adapted from studies of internalized bacterial pathogens in cell culture. We report the efficacy of gentamicin to kill planktonic bacteria treated during fungal coculture, and also describe and characterize a sampling scheme to recover and quantify culturable bacteria from the growing edge of fungal mycelium in vitro. This assay enables qualitative and quantitative tests of reassociation capabilities for facultative endohyphal bacteria with host fungi and provides a means to investigate the genetic basis for these associations in a repeatable way.

Bacterial Endosymbionts: Master Modulators of Fungal Phenotypes.

The ecological modes of fungi are shaped not only by their intrinsic features and the environment in which they occur, but also by their interactions with diverse microbes. Here we explore the ecological and genomic features of diverse bacterial endosymbionts-endohyphal bacteria-that together are emerging as major determinants of fungal phenotypes and plant-fungi interactions. We first provide a historical perspective on the study of endohyphal bacteria. We then propose a functional classification of three main groups, providing an overview of their genomic, phylogenetic, and ecological traits. Last, we explore frontiers in the study of endohyphal bacteria, with special attention to those facultative and horizontally transmitted bacteria that associate with some of the most diverse lineages of fungi. Overall, our aim is to synthesize the rich literature from nearly 50 years of studies on endohyphal bacteria as a means to highlight potential applications and new research directions.

An Endohyphal Bacterium (Chitinophaga, Bacteroidetes) Alters Carbon Source Use by Fusarium keratoplasticum (F. solani Species Complex, Nectriaceae).

Bacterial endosymbionts occur in diverse fungi, including members of many lineages of Ascomycota that inhabit living plants. These endosymbiotic bacteria (endohyphal bacteria, EHB) often can be removed from living fungi by antibiotic treatment, providing an opportunity to assess their effects on functional traits of their fungal hosts. We examined the effects of an endohyphal bacterium (Chitinophaga sp., Bacteroidetes) on substrate use by its host, a seed-associated strain of the fungus Fusarium keratoplasticum, by comparing growth between naturally infected and cured fungal strains across 95 carbon sources with a Biolog® phenotypic microarray. Across the majority of substrates (62%), the strain harboring the bacterium significantly outperformed the cured strain as measured by respiration and hyphal density. These substrates included many that are important for plant- and seed-fungus interactions, such as D-trehalose, myo-inositol, and sucrose, highlighting the potential influence of EHB on the breadth and efficiency of substrate use by an important Fusarium species. Cases in which the cured strain outperformed the strain harboring the bacterium were observed in only 5% of substrates. We propose that additive or synergistic substrate use by the fungus-bacterium pair enhances fungal growth in this association. More generally, alteration of the breadth or efficiency of substrate use by dispensable EHB may change fungal niches in short timeframes, potentially shaping fungal ecology and the outcomes of fungal-host interactions.

Absence of genome reduction in diverse, facultative endohyphal bacteria.

Fungi interact closely with bacteria, both on the surfaces of the hyphae and within their living tissues (i.e. endohyphal bacteria, EHB). These EHB can be obligate or facultative symbionts and can mediate diverse phenotypic traits in their hosts. Although EHB have been observed in many lineages of fungi, it remains unclear how widespread and general these associations are, and whether there are unifying ecological and genomic features can be found across EHB strains as a whole. We cultured 11 bacterial strains after they emerged from the hyphae of diverse Ascomycota that were isolated as foliar endophytes of cupressaceous trees, and generated nearly complete genome sequences for all. Unlike the genomes of largely obligate EHB, the genomes of these facultative EHB resembled those of closely related strains isolated from environmental sources. Although all analysed genomes encoded structures that could be used to interact with eukaryotic hosts, pathways previously implicated in maintenance and establishment of EHB symbiosis were not universally present across all strains. Independent isolation of two nearly identical pairs of strains from different classes of fungi, coupled with recent experimental evidence, suggests horizontal transfer of EHB across endophytic hosts. Given the potential for EHB to influence fungal phenotypes, these genomes could shed light on the mechanisms of plant growth promotion or stress mitigation by fungal endophytes during the symbiotic phase, as well as degradation of plant material during the saprotrophic phase. As such, these findings contribute to the illumination of a new dimension of functional biodiversity in fungi.

A Dimorphic and Virulence-Enhancing Endosymbiont Bacterium Discovered in Rhizoctonia solani

The ubiquitous soilborne and plant-pathogenic basidiomycete Rhizoctonia solani, although classified as a single species, is a complex of anastomosis groups (AGs) that cause disease in a broad range of higher plants. Here, we investigated the persistent co-isolation of bacteria with R. solani from brown patch-infected, cool-season turfgrasses, and report the presence of endo-hyphal bacteria, related to members in the genus Enterobacter, in an isolate of R. solani AG 2-2IIIB. The intracellular localization of the bacteria was corroborated by fluorescence, confocal and electron microscopy, and DNA analysis. Furthermore, the Enterobacter sp., which is rod-shaped in the free-living form, exists as an L-form (spheroid) within the fungus, a phenomenon not previously reported in endosymbionts. Our findings also indicate that the bacterium is required for full virulence of R. solani on creeping bentgrass and production of wild type levels of the toxin phenylacetic acid in fungal cultures. The possible presence of bacterial endosymbionts in R. solani AG 2-2IIIB may portend the presence of bacteria in additional AGs as well as other Rhizoctonia species, and may help resolve some of the complexities of R. solani pathogenicity. A closely associated bacterium could influence aspects of plant host pathology.

Diversity, Specificity, and Phylogenetic Relationships of Endohyphal Bacteria in Fungi That Inhabit Tropical Seeds and Leaves

Interactions between fungi and tropical trees help shape some of the most biodiverse communities on earth. These interactions occur in the presence of additional microbes that can modify fungal phenotypes, such as endohyphal bacteria (EHB). Here we examine the occurrence, diversity, and taxonomic composition of EHB in fungi that colonize seeds and leaves of plants in tropical forests. We use PCR and fluorescence microscopy to detect EHB in fungi, and a phylogenetic approach to explore evolutionary relationships among seed- and leaf-inhabiting fungi and their bacterial partners. Analyses focusing on two prevalent orders of fungi (Hypocreales and Xylariales) revealed that seed- and leaf-inhabiting fungi have a shared evolutionary history, yet differ in the prevalence, richness, and composition of their endohyphal symbionts. Phylogenetic analyses detected that the endohyphal habit is widespread, here encompassing members of seven phyla of bacteria (including three classes of Proteobacteria). Occurring in seed- vs. leaf-associated fungi has not resulted in detectable structure in the evolution of EHB, and no congruence was observed in the phylogenetic relationships of these apparently facultative, horizontally transmitted symbionts and their fungal hosts. Our results are consistent with multiple origins of fungus-bacterium associations and argue for evaluating focal pairs to determine how particular EHB affect the establishment or maintenance of fungal symbioses in seeds and leaves.

Endohyphal bacteria from fungal endophytes of the Mediterranean cypress (Cupressus sempervirens) exhibit in vitro bioactivity

SummaryEndohyphal organisms of endophytic fungi can induce, or promote, beneficial effects of their respective host fungi to the host plant that harbours them. The Cupressaceae plant family (Coniferales) hosts highly bioactive endophytic fungi. Here, we show that a fraction of such endophytic fungi harbours bioactive endohyphal bacteria with a non‐obligatory symbiotic lifestyle. Indeed, 5 of 16 (31.25%) endophytic fungi of Cupressus sempervirens harboured endofungal bacterial strains of Bacillaceae (G+, Bacilli) and Sphingomonadaceae (G−, α‐Proteobacteria), that is Bacillus pumilus (from the fungi Leptosphaeria CSE211 and Pyrenochaeta CSE134), Bacillus subtilis (from the fungi Leptosphaeria CSE212 and Ascorhizoctonia CSE148) and Sphingomonas paucimobilis (from the fungus Ascorhizoctonia CSE195). Notably, each endophytic fungal species contained only one endofungal bacterial species that was stably maintained in symbiosis over several rounds of subculturing. Moreover, we investigated whether cypress endofungal bacteria (CEB) could benefit their host fungus competing with other fungi and bacteria or the host plant against the invading microorganisms. In vitro assays indicated that CEB possessed antagonistic activity against cypress endophytic and pathogenic microbiome. Also, CEB metabolites and volatile compounds (VOCs) exhibited antifungal and antibacterial activity against the target microbiome. Bioactivity of CEB was less than that of the endophytic microbiome of Cupressaceae, on which we reported earlier. In conclusion, our work is the first to document endohyphal bacteria of fungal endophytes of C. sempervirens and the bioactivity of such endohyphal symbionts. These findings implicate a complicated interrelationship among host plant, endophytic microbiome and endofungal bacteria, which might be of high importance for evolutionary, as well as environmental and agricultural studies. Eventually, endohyphal bacteria may be introduced as a novel source for lead molecule discovery.