Detection and identification of bacteria intimately associated with fungi of the orderSebacinales

Abstract

Because of their beneficial impact on plants, the highly diverse mycorrhizal fungi grouped in the order Sebacinales lay claim to high ecological and agricultural significance. Here, we describe for the first time associations of Sebacinoid members with bacteria. Using quantitative PCR, denaturating gradient gel electrophoresis and fluorescence in situ hybridization, we detected an intimate association between Piriformospora indica and Rhizobium radiobacter, an alpha-Proteobacterium. The stability of the association, vertical transmission of the bacteria during asexual fungal reproduction and fungal plant colonization was monitored using R. radiobacter-specific primers. Treatment of mycelium or fungal protoplasts with antibiotics highly efficient against the free bacteria failed to cure the fungus. Barley seedlings dip-inoculated with R. radiobacter showed growth promotion and systemic resistance to the powdery mildew fungus Blumeria graminis comparable to P. indica inoculation. By screening additional isolates of the Sebacina vermifera complex, three species-specific associations with bacteria from the genera Paenibacillus, Acinetobacter and Rhodococcus were found. These findings suggest that Sebacinales species regularly undergo complex interactions involving host plants and bacteria reminiscent of other ectomycorrhizal and endomycorrhizal associations.

Non-pathogenic Rhizobium radiobacter F4 deploys plant beneficial activity independent of its host Piriformospora indica

“…Agrobacterium tumefaciens) strain F4 (RrF4). Fluorescence in situ hybridization using a Rhizobium-specific probe confirmed the stable endocellular association of small numbers of RrF4 cells within P. indica chlamydospores and hyphae, and a ratio of 0.035 ng of bacterial DNA per 100 ng of P. indica DNA was determined by quantitative PCR (qPCR) analysis (Sharma et al, 2008). This result coincides well with the low number of different bacteria (1-20 per fungal cell) that were detected in the ectomycorrhiza fungus Laccaria bicolor (Bertaux et al, 2003;Bertaux et al, 2005).…”

“…Treatment of barley roots with free-living RrF4 increased plant biomass and powdery mildew resistance (Sharma et al, 2008). To further expand this finding, we assessed the potential of RrF4 to mediate growth promotion in Arabidopsis.…”

“…Agrobacterium radiobacter; syn. Agrobacterium tumefaciens) F4(RrF4), a subculture of strain PABac-DSM isolated from P. indica DSM 11827 (Sharma et al, 2008). Generation of RrF4 strains expressing ß-glucuronidase (GUS) and green fluorescent protein (GFP) is described in the Supplementary Materials and Methods.…”

“…The relative abundance of RrF4 in root tissues was quantified by qPCR targeting the internal transcribed spacer (ITS) between the 16S rRNA and 23S rRNA genes of the ribosomal RNA operon of RrF4 (Sharma et al, 2008;see Supplementary Table S1 and Supplementary Materials and Methods).…”

“…In 2008, Sharma and coworkers (Sharma et al, 2008) reported for the first time that members of the Serendipitaceae regularly undergo complex symbioses involving plants and endofungal bacteria of different genera. Endofungal bacteria were previously detected in Glomeromycotan arbuscular mycorrhiza symbioses (Bonfante and Anca, 2009;Naumann et al, 2010), in the ectomycorrhizal fungus Laccaria bicolor (Bertaux et al, 2003(Bertaux et al, , 2005, in the rice pathogenic fungus Rhizopus microsporus (Partida-Martinez and Hertweck, 2005), in hyphae of phylogenetically diverse foliar fungal endophytes (Hoffman and Arnold, 2010), in the soil isolate Mortierella alpina (Kai et al, 2012) and the plant symbiotic Endogone Mucoromycotina fungi (Desirò et al, 2015).…”