New Endohyphal Relationships between Mucoromycota and Burkholderiaceae Representatives

Interactions between fungi and bacteria are common and widespread in the environment, as these organisms often occupy similar niches, and together, they are responsible for most decomposition processes in soil (1). However, not only can bacteria live in close proximity with the fungus, sometimes they are also present inside the fungal hyphae. Bacteria occupying this specific niche are referred to as endohyphal bacteria (EHB). EHB were first observed in spores of Endogone by Mosse in 1970 (2), although they were then described as “bacteria-like structures.”

Despite observing these bacteria-like structures in Endogone, the first formally described endobacterium associated with the Mucoromycota representative was “Candidatus Glomeribacter gigasporarum,” which was found to be living inside the hyphae of Gigasporales (3), and its prevalence seems to be limited to this order. “Candidatus Glomeribacter gigasporarum” belongs to Burkholderiaceae family and is considered one of the Burkholderia-related endobacteria (BRE). These bacteria are known to be vertically transmitted and were detected in both mycelium and spores (3). They help the fungus form a relationship with the plant by increasing ATP production and inducing detoxification of reactive oxygen forms (4). Another similar partnership involves Rhizopus microsporus (Mucoromycotina) and at least two species of BRE (Mycetohabitans rhizoxinica and Mycetohabitans endofungorum [Paraburkholderia rhizoxinica {synonym}] and Paraburkholderia endofungorum) (5, 6). In this relationship, bacteria were demonstrated to completely control asexual reproduction as well as partially control sexual reproduction of their host (7). More recently, a partnership between Mortierella elongata (Mortierellomycotina) and Mycoavidus cysteinexigens was discovered and described (8, 9). This partnership has been studied extensively, with both partners having their genomes assembled and annotated and series of physiological experiments carried out. It has been demonstrated that the bacterium relies on the fungal cysteine and that the host growth rate is higher in cured strains (9). There are also reports of endohyphal bacteria in Dikarya (10). In this group, single fungal hosts were shown to harbor few different bacterial endosymbionts from evolutionarily distant lineages (e.g., reference 11), which is not the case of Mucoromycota. Fungi from this phylum usually harbor one or two different lineages of bacteria, and these are quite uniform across the whole phylum, suggesting their evolutionary ancient relationship.

All of the above-mentioned interactions involving Mucoromycota representatives also involve bacteria from the Burkholderiaceae family (Burkholderiales, Betaproteobacteria, Proteobacteria), which are Gram-negative rod-shaped bacteria. Representatives of this clade are omnipresent in the environment, colonizing a wide range of ecological niches, from soil ecosystems to the human body; this family consists of obligately aerobic, facultatively anaerobic chemoorganotrophs as well as chemolithotrophs, both obligate and facultative (12). Recent phylogenomic studies of Burkholderia sensu lato led to the division of this genus into several new genera that seem to reflect the most prevalent lifestyle within the genus as well as phylogenetic grouping (13). The genus Paraburkholderia comprises environmental strains known to be sometimes beneficial to plants and Burkholderia sensu stricto comprises human and animal pathogens, while the newly established genus Mycetohabitans comprises two BRE isolated from Rhizopus microsporus (Mycetohabitans rhizoxinica and Mycetohabitans endofungorum) (14).

Another lineage of bacteria detected in the representatives of Glomeromycotina as well as in the Mortierella genus and Endogonales order is Mycoplasma-related (Mycoplasmataceae, Mollicutes, Tenericutes) endobacteria (MRE). Nauman et al. (15) identified MRE in Glomeromycotina, which was later confirmed by Naito et al. (16). Then, Desirò et al. (17) found MRE in Mortierella strains and concluded, after curing fungi of their endosymbionts, that these bacteria seem to be mild parasites. MRE were also detected in three out of four recently studied genomes of Endogonales (Mucoromycotina) representatives (18). Apart from being endosymbionts, bacteria from the family Mycoplasmataceae can lead a saprotrophic or parasitic lifestyle as well.

Together, these findings indicate that interactions between Mucoromycota fungi and bacteria are common and that they have been neglected for years (or there were no methods of studying them). Pawlowska et al. (19) suggested that it is inevitable to find new Mucoromycota-endosymbiont partnerships, and thus, looking for endosymbiotic bacteria as a part of primary research for each new species is recommended. In the same year, Takashima et al. (20) published a study in which they screened 238 strains of environmental Mortierella isolates originating from Japan using PCR and fluorescence in situ hybridization (FISH). They report that about 20% of the strains harbored BRE, which can be separated into three new subclades (called A, B, and C), but the authors were not able to draw conclusions about the factors driving these interactions. Moreover, they performed a FISH procedure confirming location of BRE inside the hyphae for five isolates. MRE seem to be less common within Mortierellomycotina, as Desirò et al. (17) report that only 12 out of 394 strains (ca. 3%) possess the bacteria in question.

Clearly, studying Mucoromycota-bacterial relationships can also help our understanding of the evolutionary history of interactions between fungi and bacteria. Mucoromycota are commonly described as early diverging, as they are one of the most ancient groups of land fungi (21). The Mucoromycota phylum comprises three subphyla: Glomeromycotina, Mortierellomycotina, and Mucoromycotina (22). The first one is rather uniform in the trophic mode of its representatives—almost all Glomeromycotina fungi are obligate endomycorrhizal partners of plants (with the exception of Geosiphon pyriformis, which forms a relationship with endosymbiotic cyanobacteria) (23). Mortierellomycotina are common and ubiquitous soil saprotrophs with a worldwide distribution (24), and are thought to form nonobligatory relationships with plant roots (25–27). Mucoromycotina is the most diverse subphylum in the phylum and encompasses the following three clearly distinct orders: Endogonales, Mucorales, and Umbelopsidales. Endogonales are mainly obligatory plant symbionts (28), while representatives of the other two orders are mostly common soil saprotrophs. However, many representatives of Mucorales are also isolated from spoiled fruits, vegetables, mushrooms, or bread (e.g., Rhizopus spp., Mucor spp., Choanephora cucurbitarum). There are also rare examples of Mucorales acting as opportunistic pathogens in immunocompromised patients (causing mucormycosis) (29).

The patterns of presence and absence of BRE and MRE in Mucoromycota representatives enabled Bonfante and Desirò (30) to propose the following two different hypotheses on the evolution of bacterial-fungal interactions: early and late bacterial invasion. While these hypotheses apply to both types of EHB, in this paper, we focus on BRE. The early bacterial invasion hypothesis states that the common ancestor of all present BRE interacted with the common ancestor of extant Mucoromycota representatives. The diversity of BRE that we can observe today is thus a result of a codiversification of hosts and endosymbionts. The late bacterial invasion hypothesis states that there is at least some level of horizontal acquisition of BRE by representatives of different Mucoromycota lineages, which can also explain present day diversity of BRE. However, both scenarios are based on scarce data, especially considering the lack of information about EHB in the representatives of Mucoromycotina other than Endogonales and Rhizopus.

Therefore, the main goal of our study was to screen chosen fungal representatives of the Mucoromycota phylum for the presence of endohyphal bacteria and identify potential coevolution patterns in order to support or modify the current hypotheses on the evolution of bacterial-fungal partnerships within Mucoromycota. We included representatives of genera and orders underrepresented in endobacterial studies, such as Umbelopsis.

Among 196 strains belonging to 16 genera within the Mucoromycota phylum, 42 were demonstrated to be positive for bacteria from the Burkholderiaceae family, which constitutes 21% of all screened isolates (Table 1). As expected and previously reported (20), nearly 20% of Mortierella strains harbored bacteria from Burkholderiaceae. We also observed interactions between Umbelopsis and Burkholderiaceae. More than half (23 of 40) of screened strains of this genus tested positive for Burkholderiaceae. There was one strain (out of 15) of Mucor which seemed to have a relationship with Burkholderiaceae bacteria as well. As was expected, none of the screened arbuscular mycorrhizal fungi tested positive for the presence of bacteria from this group. However, we also identified Mycoplasma-related bacteria in Mortierella formicae, Diversispora sp., and two species of Glomus (data not shown). Some of the strains have had PCR bacterial product, but the identified bacteria belong to neither of the described groups. All of the data can be found in Table 1.

After selection of bacteria belonging to the Burkholderiaceae family, we reconstructed the phylogeny using 16S rRNA gene sequences of previously found Burkholderia-related endobacteria as well as free-living Burkholderiaceae. Our analysis showed that the identified Burkholderiaceae do not form a uniform group (Fig. 1). All obtained sequences split into the following two groups within Burkholderiaceae: one comprised strictly of endohyphal strains from Rhizopus microsporus, Mortierella spp., and arbuscular mycorrhizal fungi (Mycoavidus cysteinexigens [clades A, B, and C], M. rhizoxinica/M. endofungorum, and “Candidatus Glomeribacter gigasporarum” in Fig. 1; hereinafter referred to as BRE); and the other consisting mainly of free-living, environmental Paraburkholderia strains and endohyphal clones from this study (highlighted in green in Fig. 1). In the BRE clade, three main lineages can be distinguished, corresponding to Mycetohabitans spp., “Candidatus Glomeribacter gigasporarum,” and Mycoavidus cysteinexigens. Takashima et al. (20) further divided the Mycoavidus clade into three subclades, A, B, and C, and this pattern is also visible in our analysis. Two out of 15 endohyphal clones from our Mortierella spp. grouped within subclade A, and the same number grouped within subclade B (Fig. 1). None of our Burkholderiaceae sequences grouped within subclade C. Sequences obtained from Rhizopus microsporus all grouped within the Mycetohabitans spp. clade. The remaining 11 sequences of Burkholderiaceae from Mortierella strains, as well as all sequences from Umbelopsis spp. and one from Mucor moelleri, grouped with environmental sequences of Paraburkholderia sensu stricto (highlighted in green in Fig. 1).

ParaFit analyses of bacteria and fungus phylogenies indicated an overall pattern of cospeciation in the BRE clade (ParaFitGlobal, P = 0.004), whereas it was not observed in the Paraburkholderia group (ParaFitGlobal, P = 1). Individual links tested with ParaFit can be found in Fig. 2.

FISH confirmed the presence of endohyphal bacteria inside the living mycelia of Rhizopus microsporus (picture not shown), Umbelopsis sp. (Fig. 3B), and Mortierella elongata (picture not shown). For Mortierella elongata WA50687, three-dimensional visualization of hyphae with endohyphal bacteria was prepared (31). Although the identity of detected EHB was not confirmed by species-specific probe, it is highly likely that visualized bacteria are the ones detected by PCR, as usually only one strain of bacteria was identified in one fungal strain. Even in EHB-positive strains, bacterial cells were not present in all hyphae; for each strain, multiple empty hyphae were also observed (Fig. 3A).

In this study, we aimed to expand knowledge about endohyphal bacteria living within hyphae of Mucoromycota representatives. To achieve this goal, we decided to use molecular methods for screening fungal isolates for the presence of bacteria and fluorescence microscopy for the confirmation of the endohyphal nature of the detected bacteria.

Currently, representatives of Mucoromycota are known to harbor endosymbionts from two distinct bacterial lineages—Burkholderia- and Mycoplasma-related ones (30)—in their hyphae, with the former probably being more widespread than the latter. In our search for EHB from previously undersampled lineages of this phylum, we detected bacteria that were previously undescribed. However, we also detected numerous endobacteria from already established clades of BRE and report similar percentage (20%) of BRE-harboring Mortierella as in the study conducted by Takashima et al. (20). All previously known lineages of BRE, including “Candidatus Glomeribacter gigasporarum” and Mycoavidus cysteinexigens, as well as a portion of BRE detected in Mortierella during this study, form a sister clade to Mycetohabitans. Notably, all endosymbionts of Umbelopsis spp. detected in our study grouped with environmental Paraburkholderia strains, and the endohyphal nature of this relationship is postulated.

At first glance, the prevalence of EHB in different lineages of Mucoromycota does not seem to be correlated with the phylogenetic position of the fungal host. Even though on a subphylum level, the colonization frequency is similar in Mortierellomycotina and Mucoromycotina (ca. 20% and ca. 24%, respectively); on an order level, the highest prevalence of EHB was observed in the representatives of Umbelopsidales (ca. 58%). The highest percentage of BRE-positive strains was observed among the strains isolated from soil (ca. 31%), and the lowest (apart from substrates represented by less than 5 strains) from clinical strains (ca. 4%). We want to elucidate, however, that the number of strains in our study does not allow us to draw conclusions about the impact of ecological niche on the prevalence of EHB and that the influence of fungal isolation substrate on prevalence and identity of EHB within Mucoromycota should be further investigated.

Paraburkholderia spp. seem to be plant associated (usually being isolated from rhizosphere), and some strains can potentially have a positive impact on plant health (13, 14). As more than one-half of the screened strains of Umbelopsis spp. tested positive for Paraburkholderia sp. and one-third of them were isolated from the plant material, we hypothesize that the relationship between fungus and bacteria could be beneficial for plants, especially since it is postulated that the fungal role in relationships with endohyphal bacteria is providing a safe environment for them. However, data are still scarce, and physiological experiments using cleared and infected isogenic isolates, similar to those performed by Uehling et al. (9), as well as comparative transcriptomics experiments of such strains (32) and sampling more strains from different locations around the world, are needed to assess the actual impact of Umbelopsis on plants as well as to examine how this impact changes with the presence of endobacteria.

The ancient origin (350 to 400 million years ago [MYA]) of BRE (Mycoavidus-Glomeribacter-Mycetohabitans lineage) in Mucoromycota was postulated by Mondo et al. (7) and Uehling et al. (9). Their results would thus support the early BRE invasion hypothesis in Mucoromycota as proposed by Bonfante and Desirò (30). In our study, the coevolution of EHB and fungal hosts may be observed on the order level in the BRE clade (i.e., Mortierellales, Mycoavidus sensu lato; Gigasporales, “Candidatus Glomeribacter sp.”; Mucorales, Mycetohabitans spp.). We also prove that there is a significant coevolutionary pattern between Mucoromycota and BRE sensu stricto, which suggests that the common ancestor of this clade interacted with an ancestor of Mucoromycota and they coevolved from this moment onwards. Conversely, our results support the late invasion hypothesis for bacteria identified as Paraburkholderia spp. It seems that symbionts from this genus may be recruited from the environment when it is advantageous for partners and form more transient relationships with Mucoromycota than BRE. Our hypothesis is in agreement with the final conclusion of Bonfante and Desirò (30). They hypothesize that soil, with its living components, has acted as a facilitator in transferring free-living bacteria inside fungi. We also speculate that the event of interaction between ancestors of Mucoromycota and BRE may have enabled fungi to interact with different types of bacteria. That would explain why Paraburkholderia representatives, closely related to BRE, were found in closely related Mortierella and Umbelopsis. As the current state of knowledge is largely incomplete, further studies are required to fully understand the nature of initiating and maintaining relationships between fungi and bacteria as well as their evolutionary origin.

In conclusion, screening of 196 fungal strains of Mucoromycota revealed EHB from the Burkholderiaceae family in ca. 20% of them. Some of the detected bacteria could be assigned to previously described endosymbiotic clades (Mycoavidus sensu lato, Mycetohabitans spp.), but others clustered with free-living Paraburkholderia. Most importantly, this study allowed for identification of potentially endohyphal bacteria in Umbelopsis spp. belonging to Paraburkholderia spp. The hypotheses regarding the time of invasion of EHB in Mucoromycota could not be resolved with certainty. However, we lean toward the early invasion hypothesis for BRE and the late invasion hypothesis for Paraburkholderia spp.

We want to thank Michał Gorczak, Łukasz Istel, Grzegorz Ostrowski, Lidia Serewa, Igor Siedlecki, Grit Walther, and Szymon Zubek for providing Mucoromycota strains. We also acknowledge Bartosz Kiersztyn for his assistance with FISH, and University of Chemistry and Technology, Prague for access to the fluorescent microscopy facility. We want to thank Gregory Bonito and Anna Muszewska for valuable comments on this manuscript. We also want to acknowledge Kacper Maciszewski and Kaley Scott for the text revisions. Finally, we thank the anonymous reviewers whose comments contributed to the improvement of the manuscript.

The research was supported by the National Science Centre, Poland, under grants 2017/25/B/NZ8/00473 (PI: Julia Pawłowska) and 2016/23/B/NZ8/00897 (PI: Marta Wrzosek). Aleksandra Miłobędzka, the researcher working on this project, was supported by the European Structural and Investment 671 Funds, OP RDE-funded project “ChemJets” (number CZ.02.2.69/0.0/0.0/16_027/0008351).

A.O. and J.P. conceived the idea of the study. K.D., A.B., A.G., and B.S. performed molecular laboratory work. A.M. performed FISH experiments. A.O. performed phylogenetic and statistical analysis. A.O. and J.P. wrote the manuscript and prepared figures. A.M. and M.W. edited the manuscript.