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

Abstract

bEndohyphal 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 MgCl 2 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.A s appreciation for their diversity and importance grows, plant microbiomes are increasingly of interest for diverse medical, industrial, and agricultural applications (1, 2). However, many plant-associated microbes remain undescribed (3, 4) and/or are found in association with, or as symbionts of, other microorganisms (5-14, 49-52). One example of these microbial symbioses is that between plant-associated fungi and their bacterial endosymbionts (endobacteria, endofungal bacteria, or endohyphal bacteria [EHB]). Recent studies have indicated that EHB are widespread in rhizosphere fungi from diverse fungal phyla (e.g., mycorrhizal and pathogenic fungi from the Basidiomycota, Glomeromycota, and Mucoromycotina [6][7][8][9][10][11][12][13]) and in the highly diverse Ascomycota that infect roots, stems, and leaves as endophytes (14) (class 3 endophytes, sensu [15]). However, functional relationships have been studied for only a few associations, limiting inferences regarding the scope and potential importance of EHB-fungal associations in ecological interactions and human applications (but see references 16 and 17).Most studies aimed at understanding functional relationships between EHB and fungi have focused on root-associated fungi, especially arbuscular mycorrhizal fungi in the Glomeromycota and plant-pathogenic Rhizopus in the Mucoromycotina (6-11, 18-23). EHB in rhizosphere fungi are often vertically transmitted and host specific, and they frequently maintain obligate relationships with their hosts (18)(19)(20). Although EHB can alter the phenotypes of root-associated fungi and profoundly i...

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

“…Comparisons between the phylogenies of bacteria and those of their fungal and plant hosts revealed a lack of concordance, consistent with facultative associations and horizontal transmission proposed previously for EHB of endophytic Ascomycota (Hoffman and Arnold, 2010;Arendt et al, 2016). Many species of Fusarium, Xylaria, and related fungi have a saprotrophic life phase, permitting colonization by EHB in soil or leaf litter.…”

“…The potential for horizontal transmission by EHB in Ascomycota has been verified experimentally in vitro in members of two classes (Sordariomycetes and Dothideomycetes) (Arendt et al, 2016). This is in contrast to endohyphal Burkholderiaceae observed in Rhizopus spp.…”

“…Visible fluorescence of nucleic acids distinct from fungal mitochondrial or nuclear DNA, combined with the absence of extrahyphal bacteria and successful amplification of the bacterial 16S rRNA gene from fungal genomic DNA, provided evidence for the presence of viable EHB (Hoffman and Arnold, 2010;Arendt et al, 2016). We next evaluated the richness, phylogenetic placement, and composition of these EHB.…”

A novel experimental system using the liverwort Marchantia polymorpha and its fungal endophytes reveals diverse and context‐dependent effects

“…A PCR was run using the 16S ribosomal primers 27F (Lane, 1991) and 1492R (Turner et al, 1999) (Methods S1). We also grew both cultures at 37°C for 3 d to check for bacterial emergence (Arendt et al, 2016).…”

Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens

“…Endobacterial symbioses have been reported from several major fungal phyla including Ascomycota and Basidiomycota (Bertaux et al, 2003;Hoffman and Arnold, 2010;Ruiz-Herrera et al, 2015;Arendt et al, 2016) and are especially frequent among early diverging lineages in the Mucoromycota (Bianciotto, 2003;Partida-Martinez and Hertweck, 2005;Sato et al, 2010;Desiro et al, 2013;Naito et al, 2015;Torres-Cortes et al, 2015;Spatafora et al, 2016). Five groups of bacterial endosymbionts of fungi, defined as bacteria living within viable or active fungal cells, have genome sequences available: (1) Burkholderia rhizoxinica from Rhizopus microsporus (2) Candidatus Glomeribacter gigasporarum from Gigaspora margarita (Ghignone et al, 2012); (3) Mollicutes/Mycoplasma-related endosymbionts associated with different genera of Glomeromycotina (Naito et al, 2015;Torres-Cortes et al, 2015); (4) Mycoavidus cysteinexigens from Mortierella elongata (Fujimura et al, 2014); and (5) Rhizobium radiobacter from Serendipita indica (Sharma, 2008;Glaeser et al, 2015).…”