Detection and isolation of a new member of Burkholderiaceae-related endofungal bacteria from Saksenaea boninensis sp. nov., a new thermotolerant fungus in Mucorales

Abstract

Thermotolerance in Mucorales (Mucoromycotina) is one of the factors to be opportunistic pathogens, causing mucormycosis. Among thermotolerant mucoralean fungi, Burkholderiaceae-related endobacteria (BRE) are rarely found and the known range of hosts is limited to Rhizopus spp. The phylogenetic divergence of BRE has recently expanded in other fungal groups such as Mortierellaceae spp. (Mortierellomycotina); however, it remains unexplored in Mucorales. Here, we found a thermotolerant mucoralean fungus obtained from a litter sample collected from Haha-jima Island in the Ogasawara (Bonin) Islands, Japan. The fungus was morphologically, phylogenetically, and physiologically characterized and proposed as a new species, Saksenaea boninensis sp. nov. Besides the fungal taxonomy, we also found the presence of BRE in isolates of this species by diagnostic PCR amplification of the 16S rRNA gene from mycelia, fluorescence microscopic observations, and isolation of the bacterium in pure culture. Phylogenetic analysis of the 16S rRNA gene of BRE revealed that it is distinct from all known BRE. The discovery of a culturable BRE lineage in the genus Saksenaea will add new insight into the evolutional origin of mucoralean fungus-BRE associations and emphasize the need to pay more attention to endofungal bacteria potentially associated with isolates of thermotolerant mucoralean fungi causing mucormycosis.

Keywords: Endohyphal bacteria; Mucormycosis; Mucoromycotina; One new taxon; Opportunistic pathogens; Saksenaeaceae; Thermotolerant fungi.

Fig. 1

The landscape of collection site and microscopic characters of Saksenaea boninensis Sak4. The landscape of Haha-jima Island where the litter was collected A The morphologies of the isolate were observed after incubation for 1 month on CZA at 23 °C, unless otherwise specified. B–I. B: A sporangium developed from a stolon. C A sporangium. D–F: Stages of sporangia showing formation of the apical portion of the neck with a mucilaginous plug after incubation for 7 d D, the gradual dissolution of apical mucilage E, and liberation of sporangiospores F. G Sporangiospores. H: A dichotomously branched rhizoid partitioned by a septum from a stolon. I: An early stage of a rhizoid formed when the tip of the stolon touched the surface of the medium. Scale bars: B, I 100 μm; C 50 μm; D–G 10 μm; H 30 μm

Fig. 2

Colony growth of Saksenaea boninensis Sak4. A: Colony appearance of Saksenaea boninensis Sak4 incubated for 4 d at 30 °C on different media [CMA a, CZA b, _LCA c, MEA d, and PDA e]. Left and right photos of each medium were front and reverse sides of colonies, respectively. B: Diametrical colony growth of Saksenaea boninensis Sak4 incubated on CZA for 3 d at different temperatures. Optimum growth was observed around 28–30 °C. No growth was observed above 40 °C. Three replicates (N = 3) were measured for each temperature except for 23, 28, and 30 °C (N = 9). Pinkish diamonds indicate the mean value

Fig. 3

Maximum likelihood (ML) phylogenetic trees of Saksenaea spp. based on the concatenated sequences. A: dataset 1 (1,276 positions in total) consisted of the ITS2 region (168 positions), the partial LSU region (637 positions), and the partial tef1 gene (471 positions). The value of the log likelihood was − 3633.090024. B: dataset 2 (1,662 positions in total) consisted of the ITS1-5.8S-ITS2 region (554 positions), the partial LSU region (637 positions), and the partial tef1 gene (471 positions). The value of the log likelihood was − 5163.803276. Bootstrap values ≥ 70% are shown at nodes. Apophysomyces elegans was used as the outgroup. Saksenaea spp. were clustered into three clades as defined by Alvarez et al. (2010). “T” beside each strain name indicates the strains as ex-type strains

Fig. 4

FISH image of SakBRE associated with Saksenaea boninensis Sak4. FISH was performed using the Cy3-labeled (Red) EUB338 probe (top). DAPI (center) and bright field (bottom) images are also shown. A representative host nucleus and bacterial cell within a hypha are indicated by an arrow and arrowhead, respectively. Scale bars: 10 μm

Fig. 5

LIVE/DEAD stained fluorescence images of SakBRE associated with Saksenaea boninensis Sak4. Bright field images are shown beside each fluorescence image. Bacterial cells are indicated by arrowheads. Rod-shaped endofungal bacterium-like cells were localized within asexual stages such as aerial hypha A, rhizoid B, stalk C, sporangium D, sporangiospore E, and germinated sporangiospore incubated for 12 h on _LCA F. Scale bars: A–D 20 μm; E, F 10 μm

Fig. 6

Maximum likelihood (ML) phylogenetic tree of Burkholderiaceae based on 16S rRNA gene sequences (1329 positions). Bootstrap values ≥ 70% are shown at nodes. The value of the log likelihood was − 12,214.778642. Wolbachia pipientis was used as the outgroup. “T” beside each strain name indicates the strains as ex-type strains. The SakBRE clade consisted of the obtained sequences in this study, indicated by a square with dashed red lines. In the square,16S rRNA gene sequences determined from the DNA templates prepared from BRE-harboring mycelia and pure cultures of the BRE are shown in black and red letters, respectively

Fig. 7

Pure culture of SakBRE grown on BCYEα medium after 14-d incubation at 30 °C