Endohyphal Bacteria Research Articles

Isolation of Endohyphal Bacteria from Foliar Ascomycota and In Vitro Establishment of Their Symbiotic Associations.

Endohyphal bacteria (EHB) can influence fungal phenotypes and shape the outcomes of plant-fungal interactions. Previous work has suggested that EHB form facultative associations with many foliar fungi in the Ascomycota. These bacteria can be isolated in culture, and fungi can be cured of EHB using antibiotics. Here, we present methods for successfully introducing EHB into axenic mycelia of strains representing two classes of Ascomycota. We first establish in vitro conditions favoring reintroduction of two strains of EHB (Luteibacter sp.) into axenic cultures of their original fungal hosts, focusing on fungi isolated from healthy plant tissue as endophytes: Microdiplodia sp. (Dothideomycetes) and Pestalotiopsis sp. (Sordariomycetes). We then demonstrate that these EHB can be introduced into a novel fungal host under the same conditions, successfully transferring EHB between fungi representing different classes. Finally, we manipulate conditions to optimize reintroduction in a focal EHB-fungal association. We show that EHB infections were initiated and maintained more often under low-nutrient culture conditions and when EHB and fungal hyphae were washed with MgCl2 prior to reassociation. Our study provides new methods for experimental assessment of the effects of EHB on fungal phenotypes and shows how the identity of the fungal host and growth conditions can define the establishment of these widespread and important symbioses.

Mycoavidus cysteinexigens gen. nov., sp. nov., an endohyphal bacterium isolated from a soil isolate of the fungus Mortierella elongata.

An endohyphal bacterium (strain B1-EBT) living in association with the fungus Mortierella elongata FMR23-6 I-B1 was isolated from a fungal cell homogenate and studied for its taxonomic allocation. Cells were Gram-stain-negative, rod-shaped, non-spore-forming, non-motile, and negative for oxidase and catalase. Strain B1-EBT required cysteine for growth and grew at temperatures between 4 and 35 °C. A comparative analysis of 16S rRNA gene sequences revealed that strain B1-EBT forms a distinct clade in the family Burkholderiaceae, encompassing a group of endosymbionts associated with several soil isolates of M. elongata. The most closely related genus is 'Candidatus Glomeribacter gigasporarum', an endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita. The major cellular fatty acids of strain B1-EBT were C16 : 0, summed feature 3 (C16 : 1ω7c and C16 : 1ω6c) and summed feature 8 (C18 : 1ω7c or C18 : 1ω6c). Ubiquinone Q-8 was the only quinone detected. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unknown aminophospholipid and two unknown aminolipids. The DNA G+C content was 49.8 mol%. On the basis of phenotypic, chemotaxonomic, and phylogenetic characteristics, strain B1-EBT represents a novel genus and novel species in the family Burkholderiaceae, for which the name Mycoavidus cysteinexigens gen. nov., sp. nov. is proposed. The type strain is B1-EBT ( = JCM 30646T = LMG 28693T = NBRC 110909T).

Endohyphal Bacterium Enhances Production of Indole-3-Acetic Acid by a Foliar Fungal Endophyte

Numerous plant pathogens, rhizosphere symbionts, and endophytic bacteria and yeasts produce the important phytohormone indole-3-acetic acid (IAA), often with profound effects on host plants. However, to date IAA production has not been documented among foliar endophytes -- the diverse guild of primarily filamentous Ascomycota that live within healthy, above-ground tissues of all plant species studied thus far. Recently bacteria that live within hyphae of endophytes (endohyphal bacteria) have been detected, but their effects have not been studied previously. Here we show not only that IAA is produced in vitro by a foliar endophyte (here identified as Pestalotiopsis aff. neglecta, Xylariales), but that IAA production is enhanced significantly when the endophyte hosts an endohyphal bacterium (here identified as Luteibacter sp., Xanthomonadales). Both the endophyte and the endophyte/bacterium complex appear to rely on an L-tryptophan dependent pathway for IAA synthesis. The bacterium can be isolated from the fungus when the symbiotic complex is cultivated at 36°C. In pure culture the bacterium does not produce IAA. Culture filtrate from the endophyte-bacterium complex significantly enhances growth of tomato in vitro relative to controls and to filtrate from the endophyte alone. Together these results speak to a facultative symbiosis between an endophyte and endohyphal bacterium that strongly influences IAA production, providing a new framework in which to explore endophyte-plant interactions.

Diverse bacteria inhabit living hyphae of phylogenetically diverse fungal endophytes.

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.