Biodiversity of Bacteria Associated with Eight Pleurotus ostreatus (Fr.) P. Kumm. Strains from Poland, Japan and the USA

Various types of relationship between two or more organisms are well known among plants and in the animal kingdom. However, little is known about the relationship and interactions between fungi and bacteria. There are three most common types of these relationships. Bacteria can live in the same environment as fungi where both organisms live close to each other but not in direct contact. This kind of relationship is common in various environments like forests, fermented food or animal tissues affected by diseases (Frey-Klett et al. 2011). More complicated type of bacterial-fungal association is mixed biofilms, where bacteria may live on fungal hyphae and are held by the extracellular matrix formed from molecules secreted by both organisms (Donlan and Costerton 2002). This kind of coexistence can be found in medical equipment (Pierce 2005) as well as in some infections (Hogan et al. 2007) or mycorrhizal systems (Sarand et al. 1998). The third type of interactions between these two groups of organisms is endosymbiosis when bacteria live inside fungal cells and do not produce any specific structures. It is the most complex type of relationship and often, in contrast to free-living fungi-associated bacteria it is impossible to cultivate such bacteria outside a fungal host. The presence of unculturable endosymbiotic bacteria was described mostly in cells of arbuscular mycorrhizal fungi belonging to Gigasporaceae family (Bianciotto et al. 1996, 2000, 2003; Bonfante 2003; Cruz et al. 2008). The endosymbiotic bacteria were also observed in the cells of pathogenic fungus Rhizopus microsporus although in this case, authors were able to grow isolated bacteria on microbiological media (Partida-Martinez et al. 2007a; 2007b). Among Basidiomycota such relationship was found in Laccaria bicolor. Similarly to Gigaporaceae endosymbionts, these bacteria were also unculturable (Bertaux et al. 2003). Only one report described bacteria of Burkholderia cepacia complex related with oyster mushroom P. ostreatus. Authors, however, did not establish if these bacteria are endosymbionts or just fungi-related organisms (Yara et al. 2006).

The aim of this work was to identify bacteria associated with eight P. ostreatus strains, and also the description of their biodiversity and assessment of their ability for N₂ fixation, cellulose, xylanase, and laccase-like multicopper oxidase activity.

A number of small bacteria-like organisms were observed inside and outside of cells of all analyzed oyster mushroom strains. They were visible as small green or yellow-green rods. The microscopic image is shown in Fig. 1. The bacteria-like organisms were marked with white arrows. Several of the cells observed were motile inside of fungal hyphae what is presented in materials published online (PxS – https://youtu.be/G93Rm0tHIgg; P234 – https://youtu.be/pxApJvCshQ0; PB7’96 – https://youtu.be/JZdGXSyDdG4; PB63S – https://youtu.be/A4U4MtUeIiU). After 10 days of cultivation, a lot of small objects were observed growing deeply in the medium around and outside fungal mycelia of all inoculates (Fig. 2). Such growth was not observed on TSA medium without addition of Tween 20. Tween 20 is used usually as a surfactant to disperse cells in solutions. Some of the bacteria also use Tween 20 as a source of fatty acids. No objects were observed in control samples that were not inoculated with fungi. Similar results were described previously by Yara et al. (2006). Blocks of media containing objects were used for cells isolation. After 14 days of stationary cultivation in the liquid TSBt medium, a white precipitate was observed on flasks bottom for all oyster mushrooms strains analyzed. After centrifugation, this precipitate was used for DNA amplification protocol.

Fig. 1.

Fig. 2.

As a result of PCR performed with DNA obtained from precipitant and 799f and 1492r primers, a 700 bp product was obtained (Fig. 3). Product that size is characteristic only for bacterial DNA as it is a part of the bacterial 16S rRNA gene. For comparison, the reaction with maize DNA provided two bands: one of 1100 bp which is characteristic for plant mitochondrial DNA and second of size about 700 bp, characteristic for bacteria. The reason for this phenomenon is that maize cultivar used in experiment carry endophytic bacteria in its tissues (Pisarska and Pietr 2015).

Fig. 3.

The total number of 307 sequences was obtained as a result of the sequencing of plasmid clones. The detailed number of identified clones with the most similar strains from NCBI database is presented in Table II. The species or genus was determined for 233 of them and for the remaining 74 sequences we were not able to identify higher taxonomic level than domena of Bacteria. All clones were identified only for PUSAS strain originated from the USA. Among other seven fungal strains, the unidentified clones occurred in the range from 7 to 15. Among tested strains of fungi, the lower biodiversity of bacteria was found for the strain PUSAS, which contained 64 clones closely related to E. coli and only two clones related to Cupriavidus genus. The only one genus, which occurred in all fungal strains analyzed but PUSAS, was Curvibacter and it was represented by 38 clones. Propionibacterium genus was found in six of eight fungal strains analyzed. It was not present only in PUSAS and PB8 strains. The total number of identified Propionibacterium strains was 14. Another genus found in six of P. ostreatus strains was Cupriavidus. It occurred in a total number of 13 clones in all fungal strains except PxS and PB63S. Bacteria belonging to genus Pseudomonas were also identified in a number of 12 clones from six fungal strains (except PUSAS and PBo6S). Genus Pelomonas was identified as nine clones from five fungal strains: P234, PB7’96, PB8, PxS, and P112. Bacteria from genus Paracoccus in a number of six were found in almost all of P. ostreatus strains from Poland (except P112). The remaining clones identified were as follows: Bacillus – 13 clones (in P234, PBo6S, PB8, P112), Staphylococcus – nine clones (in P234, PB8, PxS, P112), Acidovorax – seven clones (in PB7’96, PBo6S, PB63S, P112), Weisella – six clones (in PB7’96, PBo6S, PxS, P112), Micrococcus – six clones (in P234, PB8, PxS, P112), Nocardioides – five clones (in PB7’96, PBo6s, PxS), Ralstonia – three clones (in PBo6S, PB8), Corynebacterium – three clones (in PBo6S, PxS), Lactobacillus – three clones (in PBo6S, PxS), Acidobacteria – two clones (in P234, PB7’96), Paenibacillus – two clones (in P112), Acinetobacter – two clones (in P234), Streptomyces – two clones (in PBo6S), Legionella – one clone (PxS), Finegoldia – one clone (PxS), Sporosarcina – one clone (PxS), Streptococcus – one clone (PBo6S), Kocuria – one clone (PB8), Lactococcus – one clone (PB8), Delftia – one clone (P234), Pectobacterium – one clone (P234), Oryzihumus – one clone (P234), Tumebacillus – one clone (P234), Achromobacter – one clone (P112), Herbaspirillum – one clone (PB7’96), Citrococcus – one clone (PxS), Sphingomonas – one clone (PB7’96). The detailed number of identified clones with the most similar strains from NCBI database is presented in Table II. Phylogenetic tree of representatives of all identified genotypes with most similar sequences from NCBI database is presented in Fig. 4. As an out-group sequence, the Thermosipho sp. MV1063 (AJ419874) was used. The detailed number of identified clones with the most similar strains from NCBI database is presented in Table II.

Fig. 4.

The PCR reactions designed for detection of NifH, β-glucosidase and β-xylanase genes gave no product for all analyzed fungal strains. However, LMCO genes were detected (Fig. 5). LMCO is a complex of enzymes, which include laccases (EC 1.10.3.2) among others.

Fig. 5.

Yara et al. (1999, 2006) reported that bacteria or bacteria-like organisms could interact with only one strain of P. ostreatus. However, authors did not confirm the endocellular character of these bacteria, they described them only as bacteria associated with P. ostreatus G2 hyphae. Using fluorescent microscopy technique, which was described by Bianciotto et al. (2000), we found bacteria associated with all eight tested strains of P. ostreatus originating from different geographic zones (Japan, Poland, and the USA). Every specimen was prepared from living cells and their movement was possible to observe under a microscope. These observations and lack of growth of isolated organisms without the presence of host on microbiological media strongly suggested the endosymbiotic character of the observed bacteria. This hypothesis was also strongly supported by the fact that cultivation of the fungal strains on media with wide range of antibacterial antibiotics (ampicillin, neomycin, gentamicin, penicillin G, streptomycin, polymyxin B, kanamycin, ciprofloxacin, detreromycin, tobramycin (200 ppm), meropenem, and ceftadizine (30 ppm), data unpublished) did not eradicate bacteria from fungal mycelia. Similar techniques used for the isolation of bacteria from five strains of Lentinula edodes not revealed the presence of associated bacteria (data unpublished). Due to the fact that it was not possible to fully distinguish between endosymbiotic and associated bacteria among the isolates tested we decided to use in this study the term “bacteria associated with”. Literature of subject shows the presence of one or two species of bacteria associated with P. ostreatus. Yara et al. (2006) described bacteria associated with one strain of P. ostreatus belonging to B. cepacia complex, which is complex of at least 20 different species from Burkholderia genus (LiPuma 2005). In this work, we report the presence of bacteria related to at least 34 different genera living in association with eight P. ostreatus strains originating from different geographical regions. It was noticeable that bacteria from genus Curvibacter were isolated from almost all analyzed fungal strains. The only one strain that did not contain these bacteria was a PUSAS strain. This strain also was the only one associated with E. coli bacteria what is difficult to explain due to lack of knowledge of history of this strain, The bacteria from the Curvibacter genus were reported endosymbionts of Oryza sativa roots (Singh et al. 2006), similarly as an uncultured bacterium from Chlorella cultures (Otsuka et al. 2008) and tomato rhizosphere (Lioussanne et al. 2010) and the bacteria associated with Hydra, which serve as a protective factor against pathogen infections (Fraune et al. 2015). Bacteria of genera Bacillus and Pseudomonas are well-known plant growth-promoting bacteria due to competitive and antagonistic activity versus several pathogens (Compant et al. 2010). The occurrence of Curvibacter, Bacillus and Pseudomonas bacteria associated with P. ostreatus could be related to the known lower susceptibility of this edible mushroom when compare to Agaricus bisporus (J.E. Lange) Imbach. Gigasporacea sp. endosymbionts were described as N₂ fixing bacteria (Bianciotto et al. 2003). However, our study did not show the presence of nitrogenase reductase genes using PolF and PolR primers; however, the occurrence of clones similar to Cupriavidus suggest that these bacteria could be N₂-fixers associated with oyster mushroom. Strains of Cupriavidus taiwanensis, previously were isolated from nodules of Mimosa, can nodulate also legumes and fix N₂. However, the nifH gene is only distantly related to other alphaproteobacterial rhizobial strains (Gyaneshwar et al. 2011), what was probably a reason that used primers were not specific in this case. The bacteria associated with P. ostreatus could also influence their hosts’ capability for lignocellulolytic substrates utilization. PCR reactions with specific primers for β-glucosidase, β-xylanase genes did not reveal their presence, which suggests that these bacteria did not play important role in degradation of polysaccharides. However, the presence of LMCO genes was identified in this study. Laccases are a group of enzymes able to use a variety of substrates, including lignin (Rekuc et al. 2006). This suggests that although oyster mushroom-associated bacteria were able to produce neither β-glycosidase nor β-xylanase, they still could be able to support the lignocellulolytic activity of the strains tested due to laccase activity.

The origin of identified P. ostreatus-associated bacteria, their phylogenesis, and relationship with hosts were difficult to determine. In the natural environment, the oyster mushroom is a saprophyte, which develops on dead trunks and deciduous trees. Occasionally, it can develop in places of cuts on living trees. Such a variety of hosts may be the reason for the biodiversity of bacterial associations. Different non-pathogenic species probably inhabited mycelium of P. ostreatus during colonization of various hosts.