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

Abstract

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.

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

“…Isolation and identification of endohyphal bacteria from Ffuji was carried out as previously described by Obasa et al () with slight modifications. Briefly, 5 mm‐diameter agar discs containing fungal mycelia were grown in quarter‐strength PDB at 28 °C with shaking at 180 rpm for 72 h. Subsequently, the mycelia were surface‐sterilized once in 70% alcohol for 2 min and rinsed in five changes of filter‐sterilized water autoclaved for 20 min at 121 °C.…”

“…The sequences of the four genes from two Enterobacter strains isolated from Ffuji (henceforth referred to as En1‐Ff and En2‐Ff, respectively) were concatenated in the same order and aligned to concatenated representatives, including sequences from an Enterobacter sp. (En‐Cren; NCBI GenBank accession ) isolated from a Rhizoctonia solani AG 2‐2IIIB fungus reported by Obasa et al (; Table ) selected from the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn%26PAGE_TYPE=BlastSearch%26LINK_LOC=blasthome) to generate an MLSA‐based phylogenetic tree using the Tamura–Nei genetic distance model and the default settings of the geneious tree builder application in geneious R11 v. 11.1.4 (http://geneious.com) (Kearse et al , ).…”

“…To visualize the intrahyphal localization of the bacteria, the two bacterial strains were separately transformed to express the green fluorescent protein (GFP) and restored separately into the cured fungus as previously described by Obasa et al (). Briefly, the two Enterobacter sp.…”

“…GFP‐expressing bacteria within the restored fungal hyphae were visualized using the ×40 objective of an Olympus IX81 inverted epifluorescence microscope at 488 nm. Scanning electron microscopy of the restored hyphae was done as previously described by Obasa et al (). Briefly, intact hyphae of the restored strains were fixed in 4% paraformaldehyde, post‐fixed with 2% osmium tetroxide for 10 min, dehydrated in a graded alcohol series and critical‐point dried.…”

“…In another study, inoculation of maize cobs with a fungal antagonist, Clonostachys rosea , was found to significantly reduce fumonisin B 1 contamination by up to 70% regardless of the ripening stage of the treated maize ear (Samsudin et al , ). Natural endohyphal bacteria have also been reported to play important roles in the functional traits of their fungal hosts (Partida‐Martinez & Hertweck, ; Obasa et al , ). The present study therefore investigated two endohyphal Gram‐negative bacteria that were observed to be associated with mycelia of F. fujikuroi strain W343, isolated from wheat in Nebraska (Parikh et al , ), for roles in any of the functional traits of F. fujikuroi W343.…”