# Endofungal bacteria: Emerging paradigms and future directions

Adrian Wallner, Utkarsh Talukdar, Aurélie Deveau, Julia Buchner, Deepak Kumar, Pooja Shukla, Stéphane Compant, Satish K Verma

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## To cite this version:

Adrian Wallner, Utkarsh Talukdar, Aurélie Deveau, Julia Buchner, Deepak Kumar, et al. Endofungal bacteria: Emerging paradigms and future directions. Microbiological Research, 2025, 10.1016/j.micres.2025.128361. hal-05316608

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Submitted on 15 Oct 2025

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# Endofungal bacteria: Emerging paradigms and future directions

Adrian Wallner<sup>a,*</sup>, Utkarsh Talukdar<sup>b</sup>, Aurélie Deveau<sup>c</sup>, Julia Buchner<sup>d</sup>, Deepak Kumar<sup>b</sup>, Pooja Shukla<sup>b</sup>, Stéphane Compant<sup>a</sup>, Satish K. Verma<sup>a,b</sup>

## ARTICLE INFO

**Keywords:**

Endosymbiosis

Endohyphal

Fungal holobiont

Tripartite interactions

Symbiont-host coevolution

Genome reduction

## ABSTRACT

The colonization of fungal hyphae and spores by bacteria represents a widespread phenomenon with significant ecological and biotechnological implications across all surveyed fungal phyla. First thought to be restricted to *Mollicutes* and *Burkholderiaceae*-Related Endobacteria, these endofungal associations exhibit remarkable diversity, from simple uniform populations to complex communities, contradicting earlier assumptions of uniform populations. Acquisition dynamics demonstrate both ancient co-evolutionary relationships and recent horizontal transfer events, with environmental factors driving strain-level variation in symbiont presence even within the same fungal species. Fungi can harbor either uniform or diverse bacterial communities, sometimes within specialized structures, and exhibit varying degrees of dependence on their symbionts. These interactions can be mutualistic, commensal, or parasitic, influencing fungal physiology, metabolism, and ecological function. Yet the underlying mechanisms have been thoroughly characterized in only a few model systems in which endofungal bacteria have been shown to affect nutrient acquisition, stress tolerance, secondary metabolite production, and even fungal pathogenicity. In return, fungi offer a protective niche and promote dispersion. These concepts collectively illustrate the evolutionary flexibility and ecological importance of fungi-bacteria partnerships across terrestrial ecosystems.

This review synthesizes emerging paradigms in endofungal bacteria research, integrating recent discoveries that challenge traditional assumptions about these symbioses. We examine host specificity patterns, acquisition mechanisms, and functional impacts while identifying critical knowledge gaps requiring investigation. A deeper understanding of these associations is essential to establish standardized frameworks for their applications in agriculture, medicine, and environmental sustainability.

## 1. Introduction

With a predicted diversity of around 10 million species (Wu et al., 2019; Baldrian et al., 2022), the fungal kingdom is among the most diverse currently present on the planet. While the large majority of fungal species around the globe resides in soils (Anthony et al., 2023), they are also found in aquatic environments, and by some estimates, on average, one different fungal species can be found inside each animal species (Larsen et al., 2017). The capacity of some of them to associate with autotrophic organisms in lichen structures has allowed them to colonize additional highly inhospitable environments (Spribille et al., 2022).

The initial observation of bacterial bodies enclosed in fungal tissues

is widely attributed to Barbara Mosse, who described bacteria-like organisms inside the spores of the glomeromycotan *Acaulospora laevis* (Mosse, 1970). However, Fritz von Wettstein had already reported the endohyphal presence of cyanobacteria within *Geosiphon pyriformis* in 1915 (von Wettstein, 1915). Due to its peculiar appearance, he misidentified the rare glomeromycotan host as an alga but noted the surprising absence of cellulose in cell walls, replaced by chitin, and the absence of chloroplasts, which he justified through the presence of the photosynthetic symbionts. Von Wettstein correctly identified these symbionts as *Nostoc*, which at the time were also classified as algae. Although it was not recognized as such then, we can assume today that von Wettstein made the first observation of bacteria residing inside a fungus. The remaining timeline of endofungal bacteria-related

<footnote><sup>a</sup> Center for Health & Bioresources, Bioresources Unit, AIT Austrian Institute of Technology, Konrad Lorenz Str. 24, Tulln 3430, Austria</footnote>

<footnote><sup>b</sup> Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi, UP 221005, India</footnote>

<footnote><sup>c</sup> Université de Lorraine, INRAE, IAM, Nancy 54000, France</footnote>

<footnote><sup>d</sup> Plant Development, Ludwig-Maximilians-University Munich, Planegg-Martinsried, 82152, Germany</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: adrian.wallner@ait.ac.at (A. Wallner).</footnote>
discoveries is fully described by Alabi, Uehling and collaborators (Alabid et al., 2019; Uehling et al., 2023).

Bacteria have been shown to form endosymbiotic associations with every clade of eukaryotes, including multicellular plants, animals, and unicellular protists. In comparison, their association with fungi has been overlooked for surprisingly long but promises to be a common occurrence. Bacteria form associations with fungi in mixed biofilms, exchanging nutrients or adhering to their surfaces and benefiting from these long-distance transport routes (Pawlowska, 2024). In addition, the endocellular presence of bacteria inside fungi was increasingly scrutinized in the past years. Endofungal bacteria (EFB) can form stable or transient associations but occur in every fungal phylum surveyed to date.

The pervasiveness of EFB has been suggested by recurrent bacterial proliferation in fungal fermentor cultures (Bertaux et al., 2003) or bacterial reads in fungal sequencing data (Robinson et al., 2021) but was often dismissed as contaminations. Since 2003, a handful of laboratories have accumulated much of the current knowledge on endofungal bacteria, which has been summarized in several excellent reviews (Kobayashi and Crouch, 2009; Desirò et al. 2018; Alabid et al., 2019; Arora and Riyaz-Ul-Hassan, 2019; Steffan et al., 2020; Uehling et al., 2023). Their pioneering work has paid off as the field is rapidly gaining interest, and an increasing number of scientists are immersing themselves in the still enigmatic world of endofungal ecology. Here, we integrate the findings of the last years and undertake an extensive appraisal of the current knowledge on obligate and transient bacterial endosymbionts of fungi. We describe the expanding diversity of symbiotic associations, the underlying mechanisms uncovered to date, and the diverse impacts of the symbiotic bacteria on their host.

## 2. *Mucoromyceta* associated EFB communities are more complex than previously thought

The classification of fungi has been subjected to continuous change, regularly updating the number and status of included taxa. Here, we will treat *Glomeromycota*, *Mortierellomycota*, and *Mucoromycota* as separate phyla, part of the subkingdom *Mucoromycota* (Tedersoo et al., 2018; Wijayawardene et al., 2020; Zhao et al., 2023). *Mucoromycota* species have been disproportionately studied for EFB compared to the remaining fungal clades (Table 1). There are several reasons for this increased

scrutiny. They were the first fungi in which EFB were evidenced (von Wettstein, 1915; Mosse, 1970), and they are favored models in fungi-plant interaction studies, both mutualistic and parasitic.

Endobacteria of *Mucoromycota* have generally been associated with taxonomic groups closely related to either *Burkholderiaceae* or *Mollicutes*. *Mollicutes*-Related Endobacteria (MRE) differ from other *Mollicutes* in the presence of an electron-dense cell wall, they are coccoid-shaped and directly embedded in the host cytoplasm (Naito et al., 2017). No MRE was successfully cultured on artificial media yet, a hurdle likely ascribed to their extremely reduced genome, reaching sizes < 1 Mbp (Fig. 1; Supplementary Table S1). While MRE are detected in all three *Mucoromycota* phyla, their lack of cophylogeny with fungal hosts supports the late invasion hypothesis, where MRE were acquired through independent lineage-specific invasions and/or horizontally transferred (Longley et al., 2023). *Burkholderiaceae*-Related Endobacteria (BRE) are usually larger in size and can be entrapped in a fungal membrane, as is the case of *Candidatus* Glomeribacter gigasporarum (CaGg). Although they often display signs of genome reduction (Fig. 1), several BRE were successfully

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Genome size and GC content of endofungal bacteria. This is an extensive overview of the genomic size and GC content of all EHB (n = 82) for which whole genome sequences or metagenome-assembled genomes were deposited in public databases. The dashed line delineates reduced genomes as inferred through size comparisons with free-living outgroups. Ellipses were inserted manually to group bacterial genera for which at least two genomes are available. A detailed list of represented strains and additional genome and isolation information can be found in Supplementary Table S1.</figcaption></figure>

**Table 1**

Total fungal diversity compared to species with confirmed endofungal bacteria. The number of described species was obtained from the Catalogue of Life 2023 (Bánki et al., 2023). Total number of assemblies was accessed on NCBI's GenBank on the 18 of March 2024. The NCBI classification does not recognize *Mortierellomycota* and *Glomeromycota* as fungal phyla but as sub-phyla of *Mucoromycota*. Assemblies for *Mortierellomycotina* and *Glomeromycotina* are reported instead, and their numbers deducted from the total assemblies reported for *Mucoromycota*. As several assemblies can be available for a same species, the numbers cannot be considered as a sequenced proportion of the known diversity. The number of species hosting EFB was determined by manually reviewing bibliographic sources. A fungal species was counted as EFB host only if supported by fluorescence or electron microscopy observations (Supplementary Table S2) or being an obligate endosymbiont i.e. MRE and CaGg.

<table><thead><tr><th>Taxon</th><th>Genome assemblies (GenBank)</th><th>Described species</th><th>Species hosting EFB (confirmed)</th></tr></thead><tbody><tr><td><strong>Fungi</strong></td><td><strong>18,546</strong></td><td><strong>154,537</strong></td><td><strong>86</strong></td></tr><tr><td><em>Ascomycota</em></td><td>15,215</td><td>98,335</td><td>23</td></tr><tr><td><em>Basidiomycota</em></td><td>2,377</td><td>52,981</td><td>5</td></tr><tr><td><em>Mucoromycota</em></td><td>259</td><td>369</td><td>10</td></tr><tr><td><em>Glomeromycota</em></td><td>82</td><td>362</td><td>27</td></tr><tr><td><em>Mortierellomycota</em></td><td>95</td><td>142</td><td>20</td></tr><tr><td><em>Zoopagomycota</em></td><td>218</td><td>223</td><td>1</td></tr><tr><td><em>Blastocladiomycota</em></td><td>11</td><td>216</td><td>0</td></tr><tr><td><em>Chytridiomycota</em></td><td>118</td><td>1,032</td><td>0</td></tr></tbody></table>
isolated from their host (Partida-Martinez et al., 2007a; Ohshima et al., 2016).

Mucoromycota were often reported to harbor one prominent endosymbiotic bacterial species either belonging to MRE or BRE (Table S2) and in some cases, bacteria from both taxa were found to coexist within a same fungus (Lastovetsky et al., 2018). It is only recently that the first community analyses of EFB were performed. A study on 84 environmental *Glomeromycota* spores revealed the presence of up to 277 bacterial OTUs in a single spore, in addition to MRE and *CaGg* (Lastovetsky et al., 2024). Altogether, 10 bacterial phyla were identified, with some spores dominated by other bacteria than MRE or *CaGg*. The next most common bacterium was *Escherichia coli*, present in 90% of analyzed spores. In several *Glomeromycota* but also *Mortierellomycota* and *Mucoromycota*, Okrasińska and colleagues (2021) identified a wider diversity of bacterial 16S rRNA gene signatures than previously reported, notably *Paraburkholderia*. The role of this large spore-associated diversity is unknown, but its presence might be opportunistic, taking advantage of shelter and dispersal purposes provided by the spore (Venkatesh et al., 2022). It is likely that the increased depth offered by amplicon sequencing will reveal a wider diversity of EFB in fungal models initially thought to be hosting a homogeneous bacterial population.

Although the endofungal community might be dominated by a single taxon, such as MRE or *CaGg*, rare bacteria could also play a role for their host. With the diversification of fungal taxa screened for endosymbiotic bacteria, new associations beyond the canonical MRE and BRE are revealed such as *Serratia marcescens* in *Mucor irregularis* (Hazarika et al., 2021), SakBRE in *Saksenaea boninensis* (Takashima et al., 2023) and *Pandoraea sputorum* in *Rhizopus arrhizus* (Liu et al., 2024).

### 3. Fungal taxa below the EFB radar

Comparatively to *Mucoromycota*, little is known about the presence and role of EFB in the remaining fungal phyla although studies on *Ascomycota* are becoming another focal point (Table 1).

In *Basidiomycota*, EFB screenings have dominantly targeted mycorrhizal species belong to *Boletales*, *Laccaria*, *Russula*, *Sebacina* or *Serenidipita* but also the phytopathogen *Ustilago maydis* (Bertaux et al., 2005; Izumi et al., 2006; Sharma et al., 2008; Guo et al., 2017; Paul et al., 2020; Bai et al., 2021; Liang et al., 2023; Zhang et al., 2024). Both ascomycetous and basidiomycetous yeasts are largely unexplored except for a study on the human opportunists *Candida albicans* and *Coniochaeta polymorpha* (Tavakolian et al., 2018; Heydari et al., 2021).

Only few studies were dedicated to fungi of the *Zoopagomycota* phylum. EFB were detected in the entomopathogenic *Massospora cicutdena*, *Pandora neoaphidis* and *Pandora nouryi* (Chen et al., 2016; Ettinger et al., 2022) and the amoebae feeding *Acaulopage tetraceros* (Davis et al., 2019). Using single-cell DNA sequencing, reads with close identity to *Mycoavidus* were obtained from spores of the predatory fungus *Stylopage hadra* (Davis et al., 2019; Amses et al., 2023). As *Mycoavidus* is well studied in *Mortierellomycota* models and was identified by metabarcoding approaches in *Ascomycota*, its identification in *Zoopagomycota* makes it a highly adaptable colonizer of fungi.

To the best of our knowledge, there have been no studies on EFB in *Blastocladiomycota* or *Chytridiomycota*. However, these lineages have been included in a meta-analysis screening bacterial sequences in fungal genomic data (Robinson et al., 2021). Although validations through microscopy observations are still required, the results suggest that EFB are also common in these phyla and, to some extent, comparable to what is found in better-investigated lineages with the presence of *Pseudomonas*, *Paenibacillus*, *Escherichia*, and *Curtobacterium*.

The increasing reports of EFB in various, phylogenetically distant fungal models may generate the impression that they are ubiquitous. While genetic approaches are powerful to identify and characterize fungi-associated bacteria, they are not sufficient to establish their endosymbiotic nature. Indeed, episymbiotic bacteria can go unnoticed

in supposedly pure fungal cultures, and microscopy approaches are required to unequivocally establish the existence of endofungal symbionts.

### 4. Symbiotic associations are guided by environmental factors

Interestingly, numerous reports have identified discrepancies in the presence of EFB within strains of a same species. This raises evolutionary questions on the selective processes that drive the associations of fungi with EFB. Despite having a strong impact on their host's physiology and an evolutionary origin dating back as far as 400 million years (Mondo et al., 2012), some selective forces lead to differential repartition of EFB at the host strain level.

EFB studies initially gained momentum through the demonstration that the bacterial symbiont *Mycetohabitans rhizoxinica* was the original producer of rhizoxin, a toxin used by its fungal host *Rhizopus microsporus* to infect rice seedlings (Partida-Martínez et al., 2005). However, only a minority of *R. microsporus* are hosts to *Mycetohabitans* (Dolatabadi et al., 2016). Furthermore, out of the eight *R. microsporus* - *M. rhizoxinica* symbiotic pairs isolated so far, only one is linked to rice seedling blight disease (Lackner et al., 2009). Plant pathogenicity evolved only as a secondary role and the primary purpose of rhizoxin, appears to lie in the defense against fungivorous predators (Richter et al., 2022). The existence of non-toxinogenic *R. microsporus* strains could be explained by a lack of predatory pressure in their natural environment, reducing the need for the symbiosis. In the eight toxinogenic *R. microsporus* strains, *Mycetohabitans* is in full control of the asexual reproduction (Partida-Martinez et al., 2007b) and strongly influences sexual spore formation (Mondo et al., 2017). By contrast, non-host *R. microsporus* strains resist colonization by *M. rhizoxinica* and are able to sporulate freely in the absence of endosymbionts (Lastovetsky et al., 2016). Non-host *R. microsporus* are likely resistant to the active penetration step of *M. rhizoxinica* as the bacterium was able to propagate through the hyphae and into spores once forcefully introduced into the mycelium (Richter et al., 2024). The adaptation of non-host fungi to the acquired EFB required mutations in as little as four genes. Again, it was shown that only in the presence of a high selective force, would the fitness-cost of the newly acquired symbiont be outbalanced by its benefits. The steps leading to bacterial dependence for sporulation remain to be explored.

*Candidatus* Glomeribacter gigasporarum is a rare symbiont of *Glomeromycota* fungi which is surprising when the bacterium offers clear growth benefits for its host and was suggested to be a positive driver for the competitive selection of spores holding the highest density of symbionts (Lumini et al., 2007). In a large survey on foredune arbuscular mycorrhizal fungi (AMF), only 2.6% of *Gigaspora* spores hosted *CaGg* (Lastovetsky et al., 2018). In an independent study, the presence of *CaGg* in *Cetraspora pellucida* and *Gigaspora rosea* was dependent on the geographical origin of the fungal spores (Toomer et al., 2015). Interestingly, in those strains, the presence of *CaGg* was strictly correlated with that of MRE, suggesting that some strains might have an overall increased tolerance to the invasion by bacteria. Alternatively, the colonization by a primary EFB could facilitate the entry of a secondary symbiont. Overall, the positive selection of spores holding EFB is strongly context dependent.

In a large scale study on *Mortierella* isolates, only 12 out of 394 fungi, from Australian, Kenyan, Dutch and USA soils were positive for MRE symbionts (Desirò et al. 2018). Independent screenings on Japanese and Austrian samples found no MRE at all in a total of 627 *Mortierellomycota* isolates (Takashima et al., 2018; Telagathoti et al., 2021). Similarly, *Paraburkholderia* were detected in only 11 out of 76 *Mortierella* samples, almost all originating from the Polish Tatra mountains (Okrasińska et al., 2021), suggesting that environmental and geographical conditions could be decisive for the type of association between *Mucoromycota* and EFB.

The fungal models described in this section are amongst the few that have been sampled in sufficient depth to draw conclusions as to their
association rate with EFB. The contrasted patterns observed between strains of the same species strongly suggest that hosting EFB comes at a cost for the fungus, which is compensated through various benefits offered by the symbiont in specific conditions, both of which remain to be fully explored. Increased scrutiny for EFB in *Dikarya* models will rapidly enable to make similar observations for this still underexplored group of fungi.

Beyond biogeography and life-cycle stage, local physicochemical conditions are likely to tip the balance between exclusion, transient residency, and stable endosymbiosis. Nutrient scarcity promotes entry and persistence, while starvation increases reassociation success, favors chlamydospore formation, and enriches EFB within protective structures (Arendt et al., 2016; Venkatesh et al., 2022). Acidity and redox status modulate cell wall chemistry and secretory activity, while temperature and osmotic stress alter membrane fluidity and lipid composition. While not yet demonstrated, such modulators may plausibly impact the dynamics of association between fungi and bacteria on a microenvironmental scale.

## 5. The understudied role of EFB in animal tripartite interactions

The most studied endofungal models were isolated from endophytic fungi such as *Mycetohabitans rhizoxinica* from rice pathogenic strains of *Rhizopus microsporus*, *Candidatus* Glomeribacter gigasporarum from arbuscular mycorrhizal fungi or *Luteibacter mycovicinius* sp. nov. from the leaf endophytic *Pestalotiopsis* aff. *neglecta*. A review recently discussed the implication of EFB in fungal phytopathogens (Moses and Carter, 2025). In contrast, animal colonizing fungi and human pathogens have only marginally been explored. Their pathogenicity is also influenced by EFB, as in the case of the aphid parasite *P. neoaphidis*, where an increased diversity of the EFB population resulted in a significantly higher mortality rate when the fungus was inoculated into the insects (Chen et al., 2016).

Invasive fungal diseases in humans are on the rise, but diagnosis and treatment lag behind those for bacterial diseases (Fisher and Denning, 2023). Only recently did a study investigate how endofungal populations are related to the high phenotypic variability observed in *Aspergillus fumigatus* strains, a WHO priority pathogen (*Piontkivska et al.*, 2024). Sequencing of the 16S rRNA gene revealed complex EFB communities, conserved across 37 *A. fumigatus* strains (*Piontkivska et al.*, 2024). As for *Glomeromycota* (*Lastovetsky et al.*, 2024), the endofungal community is dominated by only few taxa, and nine were identified as core genera using both short-read and long-read approaches. These include taxa that were reported in other fungi i.e. *Ralstonia*, *Burkholderia-Caballeronia-Paraburkholderia*, and *Bradyrhizobium*, but also bacterial genera which are less frequently found as endofungal symbionts i.e. *Acidibacter*, *Gemmata*, *Puia*, and *Afipia*. The capacity to harbor diverse bacteria in its vacuole was further suggested for the opportunistic yeast *Candida albicans* (Tavakolian et al., 2018). Sequencing of taxonomic marker genes identified the bacteria as *Staphylococcus hominis*, *Staphylococcus haemolyticus*, and *Helicobacter pylori*. However, microscopy approaches suggest the presence of an even wider diversity of EFB, which are identified as alive and moving inside yeast vacuoles. In *Coniochaeta polymorpha*, another yeast found in the human gastrointestinal tract, a *Nocardia* species was identified (Heydari et al., 2021). Again, microscopy observations suggested a wider diversity of EFB, as generic staining revealed more bacteria than the *Nocardia*-specific probe. Whether the BRE found in animal-colonizing fungi have metabolic capacities that improve their host's fitness remains to be fully explored. *R. microsporus* has an increasing incidence as an opportunistic pathogen in clinical settings (Pandey et al., 2018). While the mechanisms underlying the evasion of the mammalian host immune system by *R. microsporus* had remained elusive, a recent study underlines the EFB-enabled anti-phagocyte activity, which is also used for amoebae evasion (Itabangi et al., 2022). In this clinical setting, the endosymbiont of *R. microsporus* was *Ralstonia pickettii*, suggesting that

the fungus adapts its endosymbiotic interactions in a context-dependent manner. Curing the fungus had large-scale physiological impacts, reducing the sporangiospore production, altering the cell wall structure, and putatively the ergosterol content in the cell membrane, increasing fungal sensibility to the antibiotic amphotericin B (Itabangi et al., 2022).

An emerging role of bacteria for their fungal host lies in the predator evasion capacities they enable. Independently of their ecological niche, one of the benefits *R. microsporus* strains seem to reap from their association with EFB is efficient protection from predation. The phytotoxin rhizoxin was additionally evidenced to be involved in defending *R. microsporus* from predation by fungi-feeding amoebae and nematodes (Richter et al., 2022). This feature is also found in the EFB *Candidatus* Mycoavidus necrohimicus that lives inside the fungal host *Podilla verticillata* and produces necroxime, a powerful cytotoxin with bioactivity against *Caenorhabditis elegans* and the fungivorous nematode *Aphelenchus avenae* (Büttner et al., 2021, 2023). The anthelminthic activity of necroxime is boosted by the simultaneous production of symbiosin, a surfactant suggested to improve the permeability of necroxime into the nematodes (Büttner et al., 2023). While no role in host defense has been found, the production of spiromarmycin by the marine fungus *Spiromastix* sp. is triggered by the presence of *Alcaligenes faecalis* (Shao et al., 2020). This metabolite could be useful for host protection as it has strong antibacterial and antifungal activities.

## 6. A diversity of co-adaptive strategies between symbiotic partners

There are several reasons why a bacterium might benefit from associating with a fungus. Whether it is for protection inside chlamydospores (Venkatesh et al., 2022) or dispersion using hyphal growth (Zhang et al., 2024), several bacteria were found to take an opportunistic advantage of fungi. Also, several free-living bacteria were observed to invade fungal tissues *in vitro* (del Barrio-Duque et al., 2020; Venkatesh et al., 2022). Still, the literature abounds with signs of co-evolution and mutual adaptations between both partners.

Genome reduction is a frequent phenomenon occurring in vertically transmitted symbionts and has been reported for diverse bacteria in numerous animal and plant models (Rocha, 2002; Carlier and Eberl, 2012; McCutcheon and Moran, 2012; Gupta and Nair, 2020). In fungi, it was so far only observed in EFB of *Mucoromyceta* (Fig. 1). Different nonexclusive mechanisms are proposed to explain genome shrinking. Some, such as the bias towards higher numbers of gene deletions than insertions, are pervasive throughout the bacterial realm (Mira et al., 2001), while others are adaptive. Host tissues offer a stable environment with reduced selective pressure compared to a free-living state, allowing the loss of specialized functions and the associated genes (D'Souza et al., 2014). Although stable, the host environment can also be limiting in nutrients, favoring the evolution of 'streamlined' genomes that reduce metabolic expenses (Giovannoni et al., 2014). Finally, the population bottlenecks arising during vertical transmission of obligate symbionts tend to fix deleterious mutations, a phenomenon also known as Muller's ratchet (Moran, 1996). A bacterial strategy to evade extinction through the progressive accumulation of slightly deleterious mutations has been proposed for MRE, obligate fungal symbionts that are predominantly transmitted vertically and experience a strong bottleneck of about 1,000 bacterial cells per spore (Desirò et al. 2014). While the exact mechanisms are still unclear, MRE appear to avoid the erosion of their recombination machinery, maintaining their sexual competence and genome plasticity (Naito and Pawlowska, 2016).

Even when compared to other Mollicutes, a class characterized by their small genomes, MRE display a genome size range at the lower end of the spectrum (Uehling et al., 2023). Reduced genomes compared to free-living outgroup species were further described in the BRE *Mycetohabitans* (Yu et al., 2011; Liu et al., 2024) and *Mycoavidus* (Fujimura et al., 2014; Guo et al., 2020). Genome reduction in EFB is not only characterized by a loss of genes but also by a reduction of protein length.
Compared to non-EFB congeners, protein length reduction can reach between 3.1 % in BRE and 20 % in MRE (Longley et al., 2023). Adenine-thymine content and coding density typically increase in long-term bacterial endosymbionts. However, these markers could not be conclusively applied to solve the ancestry of MRE invasion in *Mucoromyceta* taxa. *Candidatus* Glomeribacter gigasporarum lacks closely related congeners for comparison, but it displays the smallest genome known for betaproteobacteria (Jargeat et al., 2004). Thus, the facultative state of an endofungal association can be approximated through the analysis of the symbiont's genome. Indeed, one study on eleven facultative EFB demonstrated the absence of genome reduction (Baltrus et al., 2017). The authors also underlined the lack of conserved encoded mechanisms allowing the interaction with fungi. This raises the question as to how, and why, bacteria transiently colonize fungi. Clear benefits could be identified for the host in some cases underlined in the previous sections. However, especially in *Dikarya*, the hyphal tissues seem to be hosting a staggering diversity of different bacteria, much of which remains functionally unexplored. Fungal hyphae are suggested to be an easy environment to colonize for bacteria, as exemplified by their widespread occurrences in fungi from various habitats. The spontaneous loss of EFB over the course of subcultivation in *Ascomycota* suggests a transient interaction that needs to be replenished by a natural source of surrounding bacteria (Hoffman and Arnold, 2010). In the basidiomy- cetous *L. bicolor*, loss of EFB by subcultivation could be explained by the overall low incidence of bacteria, increasing the chances of selecting an axenic stretch of hyphae for fungal propagation (Bertaux et al., 2003, 2005). The stimulation of chlamydospore formation and their subse- quent colonization was observed for some bacteria (Li et al., 2012; Spraker et al., 2016). This demonstrates how the colonization of fungi can improve bacterial fitness under harsh conditions such as nutrient starvation and cold stress (Venkatesh et al., 2022).

Specificity in endofungal associations emerges from the intersection of genomic reduction, transmission mode, and host filtering. Obligate endobacteria have streamlined genomes and reduced biosynthetic capacity (Jargeat et al., 2004; Naito et al., 2017). They also exhibit frequent vertical inheritance with tight intracellular bottlenecks,

canalizing host range and co-adaptation, despite evidence for late, lineage-specific invasion (Desirò et al. 2014; Longley et al., 2023). By contrast, facultative endobacteria retain environmental competencies and show no consistent genome reduction, aligning with broader host spectra and recurrent horizontal acquisitions into receptive fungi (Baltrus et al., 2017). One working model is that endobacteria–fungus specificity reflects a continuum with genome-reduced, vertically trans- mitted symbionts at one end and community-level, facultative assem- blages shaped by ecological exposure and host permeability at the other.

The obligate EFB *CaGg* acquired six toxin-antitoxin (TA) systems through horizontal gene transfer (Di Fossalunga et al., 2017). TAs include a stable toxin and an unstable antitoxin that counteracts the effect of the toxin. In *CaGg*, two TA systems were demonstrated to have a stronger expression during the non-symbiotic life stage of *CaGg*'s host, *Gigaspora margarita*, and are suggested to promote persistence of the EFB during these stages as well as restricting their proliferation in this nutrient limiting steps before colonizing a plant host (Di Fossalunga et al., 2017). The presence of several TA modules was also noted in other persistent EFB such as *M. cysteinexigens* B1-EB and Mc-AG77 (Sharmin et al., 2018) as well as in *M. rhizoxinica* (Lackner et al., 2011b). Comparing the prevalence of TA modules in EFB of *Mucoromyceta* and *Dikarya* could yield further hints about their involvement in the sym- biont's strategy to persist in their host.

In some cases, specific physiological adaptations are observed in the context of endofungal associations. The most striking is the formation of “bladder-like” vesicles by *Geosiphon pyriformis* to accommodate *Nostoc punctiforme* (Fig. 2A-B; Schüßler and Kluge, 2001). An interesting phe- nomenon was observed using scanning electron micrographs of *G. decipiens* inoculated with *B. pseudomallei*. The presence of bacteria triggered the formation of fibrillar material emanating from the fungus and connecting it with the bacteria (Fig. 2C; Levy et al., 2003). Similar structures were detected connecting *Pelomonas* EFB to their *Metarhizium* host (Fig. 2D; Ying et al., 2022). The exact function of these fiber-like structures has not been elucidated yet but highlights the importance of visually investigating the physical interactions between host and endosymbiont in addition to their molecular exchanges.

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** *Geosiphon pyriformis* bladder like structures containing cyanobacteria (A) as visualized by von Wettstein (von Wettstein, 1915) and (B) photographed by Kluge and colleagues (Kluge et al. 2002). Fibrillar material connecting (C) *Burkholderia pseudomallei* to *Gigaspora decipiens* (Levy et al. 2003); B, bacterium; S, spore; and (D) *Pelomonas* to *Metarhizium bibionidarum* indicated by arrowheads (Ying et al. 2022). Endofungal bacteria inside a basidiomycetous host (E) visualized using Syto9 and (F) transmission electron microscopy (pictures were taken by the authors). EFB can be seen enclosed in a membrane (arrow) and in direct contact of the fungal cytoplasm (arrowheads). (G) Multiple sub-cellular bodies in *Mortierella humilis* identified as MRE (arrowhead), lipid droplets (ld) and mitochondria (m) (Desirò et al. 2018).</figcaption></figure>
For the initiation of host colonization, *M. rhizoxinica* uses chitin degrading enzymes secreted through the T2SS (Moebius et al., 2014). Once inside *R. microsporus*, *M. rhizoxinica* relies on its T3SS to induce sporulation and ensure its endohyphal persistence (Lackner et al., 2011a). Among the fifty-nine T3SS-associated candidate effectors identified in *M. rhizoxinica* (Lastovetsky et al., 2020), three are homologues of transcription activator like (TAL) effectors (Lange et al., 2014). These TALs are not essential for fungal colonization but contribute to evade fungal defenses and avoid entrapment by *R. microsporus*, as they drastically reduce septum formation (Richter et al., 2023b, 2023a). When confronted with *Mycetohabitans* isolates, host and non-host *R. microsporus* strains displayed differential transcriptomic adaptations (Lastovetsky et al., 2016). Notably, host fungi increased the pools of triacylglycerol and phosphatidylethanolamine, and failing to do so shifted the interaction towards antagonism. On the bacterial side, *M. rhizoxinica* mimics the components of its host cell by adopting a poly-D-galactofuranose O-antigen structure (Leone et al., 2010). The loss of O-antigen leads to a reduced reinfection and sporulation rate of *R. microsporus*. The endobacteria's capacity to keep its cell count below a certain threshold appears to also be of importance. When forcefully introduced into *R. microsporus*, *E. coli* cells were trapped through septum formation and denied access to spores when growing densely, but not at lower densities (Giger et al., 2024).

In the *S. indica* – *R. radiobacter* F4 model, the symbiont adapts its morphology when inside the hyphae compared to its free-living state (Sharma et al., 2008). While F4 has a rod shape of up to 2 µm in length in isolated culture, it adopts a coccoid shape of less than 1 µm in diameter as an endosymbiont (Guo et al., 2017). As *R. radiobacter* F4 cannot be completely removed from the hyphae by antibiotic treatment, it was suggested that some cells enter an inactive state, which could explain the morphological change and also make them insensitive to antibiotics (Glaeser et al., 2017). Hosts are known to actively influence their symbiont's physiology, such as legumes forcing rhizobia into bacteroid differentiation through specific peptides (Van de Velde et al., 2010). Whether similar mechanisms exist in endofungal symbioses remains to be explored.

## 7. Symbiosis without mutualism

Given de Bary's inclusive definition of symbiosis, which does not impose behavioral constraints (Martin and Schwab, 2012), the classification of endofungal symbionts should likewise encompass endocellular biotrophs, including mycophagous and parasitic bacteria (Leveau and Preston, 2008). The frontiers between mutualism and pathogenicity are seldom clearly defined, as the same mechanisms can be used to reach different ends. Endocellular biotrophs can use cell wall-degrading enzymes and secretion systems in a similar way as beneficial symbionts (Levy et al., 2003). Using confocal fluorescence microscopy on non-axenic samples of *L. bicolor*, Bertaux and colleagues (2005) observed several instances of unidentified bacteria breaching the fungal cell wall. This might be a widespread phenomenon in environmental settings, and not always playing in the fungus' favor. Also, fungi growth-promoting activities could be purported at providing more tissue to colonize.

Symbiotic relationships can rapidly turn depending on environmental factors. In the mutualistic interaction between *G. pyriformis* and *Nostoc*, an increase in phosphate availability will cause the cyano-bacterial symbiont to proliferate and ultimately overpower its fungal host (Kluge et al., 2002). *Burkholderia* species are known to colonize a wide range of habitats and hosts, and fungi are no exception. *Burkholderia pseudomallei*, *Burkholderia cepacia*, and *Burkholderia vietnamiensis* are usually found in animal and plant hosts, but can also penetrate inside the glomeromycotan *Gigaspora decipiens* (Levy et al., 2003). The germination rate was the only parameter tested to evaluate the impact of *Burkholderia* on their fungal host. The presence of *B. pseudomallei*, a well-known pathogen in both animal and plant

models, was found to reduce this rate. The *Burkholderia*-related *Myce-tohabitans* symbionts of *R. microsporus* will turn antagonistic if the fungus does not adapt its lipid profile at the onset of symbiosis (Lastovetsky et al., 2016). Thus, the correct metabolic adaptations on the fungal side are essential to control EFB behavior.

Hosting *M. cysteinexigens* comes at great cost for *M. elongata*, which increases its respiration and fatty acid catabolism while reducing its growth rate in the presence of the symbiont (Uehling et al., 2017b). But these *in vitro* results might not reflect the competitive advantage in natural settings offered by the secondary metabolite gene clusters found in the symbiont. Similarly, inoculating *Pestalotiopsis* with *Luteibacter* sp. strain 9143 reduces fungal growth. Transcriptomics approaches revealed that the bacterium induces a defense response from the fungus in the form of drug resistance proteins (Shaffer et al., 2022). The study further demonstrates the bacterial dependence on organic sulfur compounds produced by the fungus. However, *Luteibacter* sp. strain 9143 displays multiple contextual benefits for its host as discussed before (Arendt, 2015; Araldi-Brondolo et al., 2017).

Multiple examples relate the mycophagous behaviors of bacteria but almost exclusively focus on extracellular predation (Leveau and Preston, 2008; Leveau et al., 2010; Ballhausen et al., 2015; Swain et al., 2017). For instance, *Serratia marcescens* is able to migrate along the hyphae of *Mucoromycota*, killing the mycelium in the process (Hover et al., 2016). *S. marcescens* strain D1 is found as a natural symbiont of *Mucor irregularis* SS7 but retains a predatory capacity against non-host fungi such as *Fusarium oxysporum* (Hazarika et al., 2020). As D1 still reduces sporulation in its natural host *M. irregularis*, it is likely a fungal predator to which this particular fungus has built a level of tolerance. In comparison, the endocellular parasitism of fungi by bacteria has rarely been explored and is poorly understood. Using theoretical models based on intra-host symbiont-relatedness measures, horizontal dispersion capacity, and codivergence with fungal partners, MRE endosymbionts were suggested to be antagonistic parasites of AMF (Toomer et al., 2015). A later study found that more carbon is directed from fungal host to MRE than CaGg in *G. margarita*, further supporting the assumption that harboring MRE comes with an increased cost for the host compared to other EFB (Kuga et al., 2021). Despite their largely reduced genomes, MRE display gene acquisitions from their fungal host, such as methyl-transferases, kinases, and LRR domains (Naito et al., 2015; Torres-Cortés et al., 2015; Longley et al., 2023). These genes might be used by MRE to manipulate their host, explaining their persistence. In *L. bicolor*, strong EFB densities were observed in live but also dead hyphae (Bertaux et al., 2005). These bacteria could be involved in the degradation of mycelium and become active only at the death of the fungal tissues. This could also explain the asymptomatic presence of diverse bacteria in different *Dikarya*.

Active invasion of fungi by bacteria has been demonstrated and observed on several occasions as described before. As for other eukaryotic hosts of bacterial life, a central question revolves around the fungi's capacity to distinguish between mutualistic and parasitic symbionts. The fungal immune system is still poorly understood, and it is all the more challenging to predict if fungi can differentiate between the types of bacteria that colonize them (Daskalov, 2023). *Mucoromycota* are amongst the few fungal lineages lacking cytosolic surveillance modules comparable to nucleotide oligomerization domain (NOD)-like receptors (NLR) found in animals and plants (Dyrka et al., 2014). While the involvement of fungal NLRs in innate immunity is not clear (Uehling et al., 2017a), their absence in *Mucoromycota* could have facilitated the establishment of obligatory endobacteria in this lineage. Interestingly, among the fungal genomes screened, the most NLRs were found in *S. indica* (Dyrka et al., 2014). This could explain why no other endosymbiont than *R. rhizobacter* F4 has been detected in *S. indica* and further supports the hypothesis that the fungus actively recognizes its symbiont and influences its physiology.

The bacteriophagous behavior of fungi has rarely been explored (Barron, 1988; Pion et al., 2013), and was never demonstrated to occur
through the absorption of bacterial cells and subsequent digestion in the vacuole. Rather, fungi colonize or even farm bacterial colonies and lyze them extracellularly before absorbing the nutrients. Still, further investigations might reveal that some EFB originate from active endocytosis and withstood digestion which would explain the presence of membranes of fungal origin surrounding certain EFB like *Candidatus* Glomeribacter gigasporarum (Desirò et al. 2014).

## 8. Fungi curing and EFB cultivation approaches

Curing the host from its symbiont can offer decisive perspectives into the functioning of the association. In some cases, this can be readily achieved by applying bacteria-targeting antibiotics. The application of diverse methods for EFB isolation and fungal curing was recently reviewed for *Mucoromycota* but can in many cases be applied to a wider range of fungal lineages (Desirò et al. 2023). For the removal of a single bacterial genotype, one type of antibiotic can be sufficient, as in the curing of *Pestalotiopsis* from *Luteibacter* using ciprofloxacin (Hoffman

et al., 2013). In hosts displaying genetic diversity among endosymbionts, multiple antibiotics can be used to generate axenic fungi (Arendt et al., 2016; Desirò et al. 2018; Büttner et al., 2021). It can be mentioned here that ciprofloxacin has been frequently included in antibiotic cocktails to successfully cure diverse fungi from their EFB (Table 2). In cases where antibiotics were ineffective, alternative strategies have been employed, such as successive isolations of single spores or mycelial fragments to dilute the transmitted bacterial population until a cured fungal line could be obtained (Lumini et al., 2007; Mondo et al., 2017).

The failure to completely remove a symbiont could hint towards a strong dependency of the host on its bacterial partner, as is suggested for *S. indica* and its symbiont *R. radiobacter* F4 or *Spiromastix* sp. and its symbiont *A. faecalis* (Sharma et al., 2008; Shao et al., 2020). Alternatively, bacteria could reside in an inactive state within the hyphae, making them insensitive to the antibiotic (Glaeser et al., 2017). This is a common strategy observed in the rhizosphere where antimicrobial pressure is important (Navarro Llorens et al., 2010). Fungal cell walls could furthermore restrict the diffusion of antibiotics and reduce their

<caption>**Table 2**</caption>

All antibiotic concentration are expressed in µg.mL⁻¹. Unless stated otherwise (*), all antibacterial substances were applied simultaneously. Not all specificities of the antibiotic treatments applied in the different studies can be reflected in this table. Please refer to the respective publications for a detailed description of the employed methodology. Antibiotic abbreviations: Ciprofloxacin (Cip), Ampicillin (Amp), Kanamycin (Kan), Tetracycline (Tet), Chloramphenicol (Cam), Streptomycin (Stp). NA: Information not available.

<table><thead><tr><th>Fungus</th><th>Bacteria</th><th>Cip</th><th>Amp</th><th>Kan</th><th>Tet</th><th>Cam</th><th>Stp</th><th>Others</th><th>Duration</th><th>Culture</th><th>Result</th><th>Reference</th></tr></thead><tbody><tr><td>Mortierellomycota spp.</td><td>MRE</td><td>120</td><td>80</td><td>50</td><td></td><td></td><td>50</td><td></td><td>NA</td><td>liquid</td><td>successful</td><td>(Desirò et al. 2018)</td></tr><tr><td>Rhizopus microsporus</td><td>Mycetohabitans rhizoxinica</td><td>20–40</td><td></td><td></td><td></td><td></td><td></td><td></td><td>continuous</td><td>NA</td><td>successful</td><td>(Partida-Martinez and Hertweck, 2005)</td></tr><tr><td>Rhizopus microsporus Mortierella elongata</td><td>Mycetohabitans</td><td>20–40</td><td></td><td></td><td></td><td></td><td></td><td></td><td>continuous</td><td>NA</td><td>successful</td><td>(Mondo et al. 2017)</td></tr><tr><td>Rhizopus microsporus Pestalotiopsis / Microdiplodia</td><td>Mycetohabitans endofungorum Luteibacter</td><td>20–60</td><td></td><td></td><td></td><td></td><td></td><td></td><td>1 week cycles</td><td>liquid / solid</td><td>successful</td><td>(Uehling et al. 2017b)</td></tr><tr><td>Pestalotiopsis aff. neglecta</td><td>Luteibacter</td><td>40</td><td></td><td></td><td></td><td></td><td></td><td></td><td>NA</td><td>liquid</td><td>successful</td><td>(Partida-Martinez et al. 2007a)</td></tr><tr><td>Rhizopus arrhizus</td><td>Pandoraea sputorum / Mycetohabitans endofungorum Chitinophaga</td><td>40</td><td>100</td><td>50</td><td>10</td><td></td><td></td><td></td><td>10 days</td><td>solid</td><td>successful</td><td>(Arendt et al. 2016)</td></tr><tr><td>Fusarium keratoplasticum</td><td>Chitinophaga</td><td>40</td><td>100</td><td>50</td><td>10</td><td></td><td></td><td></td><td>33 days</td><td>solid</td><td>successful</td><td>(Hoffman et al. 2013)</td></tr><tr><td>Piriformospora indica</td><td>Rhizobium radiobacter</td><td>200</td><td></td><td></td><td></td><td></td><td></td><td></td><td>NA</td><td>solid</td><td>successful</td><td>(Shaffer et al. 2017)</td></tr><tr><td>Piriformospora indica protoplasts</td><td>Rhizobium radiobacter</td><td>300</td><td></td><td></td><td></td><td></td><td></td><td></td><td>Spectinomycin (300)</td><td>2 months</td><td>liquid</td><td>unsuccessful</td></tr><tr><td>Ustilago maydis</td><td>Bacillus spp.</td><td>20</td><td>100</td><td></td><td>20</td><td>30</td><td></td><td></td><td>Spectinomycin (300)</td><td>3 weeks</td><td>liquid</td><td>unsuccessful</td></tr><tr><td>Mortierella verticillata</td><td>Mycoavidus</td><td>40*</td><td></td><td></td><td></td><td>50*</td><td></td><td></td><td>Trimethoprim (1.5)</td><td>48 h</td><td>liquid</td><td>unsuccessful</td></tr><tr><td>Fusarium fujikuroi</td><td>Enterobacter</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>several months</td><td>solid</td><td>successful</td><td>(Büttner et al. 2021)</td></tr><tr><td>Spiromastix sp. CBS13827</td><td>Alcaligenes faecali</td><td>100</td><td>100</td><td>100</td><td></td><td></td><td></td><td></td><td>12 days</td><td>liquid</td><td>successful</td><td>(Obasa et al. 2020)</td></tr><tr><td>Veronaeopsis simplex</td><td>Rhizobium</td><td>50</td><td>100</td><td>50</td><td></td><td>100</td><td></td><td></td><td>7 days</td><td>liquid</td><td>unsuccessful</td><td>(Shao et al. 2020)</td></tr><tr><td>Aspergillus fumigatus</td><td>community</td><td>100</td><td></td><td></td><td></td><td></td><td></td><td></td><td>24 h</td><td>liquid</td><td>successful</td><td>(Guo et al. 2018)</td></tr><tr><td>Echinochaete brachypora</td><td>Ralstonia</td><td></td><td></td><td></td><td></td><td></td><td>50</td><td>Penicillin G (50)</td><td>48 h</td><td>liquid</td><td>unsuccessful</td><td>(Piontkivska et al. 2024)</td></tr><tr><td>Ralstonia solani</td><td>Enterobacter</td><td></td><td></td><td></td><td></td><td>30</td><td></td><td></td><td>NA</td><td>solid</td><td>likely successful</td><td>(Hassan et al. 2022)</td></tr><tr><td>Benniella erionia</td><td>MRE</td><td>NA</td><td></td><td></td><td></td><td></td><td></td><td></td><td>5 days</td><td>liquid</td><td>successful</td><td>(Obasa et al. 2017)</td></tr><tr><td>Staphyliodium bicolor</td><td>Burkholderia contaminans</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>NA</td><td>liquid</td><td>successful</td><td>(Longley et al. 2023)</td></tr><tr><td>Rhodotorula mucilaginosa</td><td>Pseudomonas stutzeri / Bradyrhizobium spp.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>&lt; 6 months</td><td>solid</td><td>unsuccessful</td><td>(Almeida et al. 2018)</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>NA</td><td>solid</td><td>successful</td><td>(Paul et al. 2020)</td></tr></tbody></table>
concentration inside the hyphae, resulting in ineffective doses for bacterial removal. The association between *A. faecalis* and its host was maintained in the presence of antibiotics, but the bacterium displayed high sensitivity to them in pure culture (Shao et al., 2020). Similarly, *S. indica* could not be cured from *R. radiobacter* using ciprofloxacin and spectinomycin, although the pure bacterial culture is sensitive to these antibiotics (Sharma et al., 2008). To increase the cell wall's permeability, calcium chloride and DMSO can be added in combination with antibiotics to successfully cure a fungus (Cheng et al., 2022). The maintained detection of bacterial signal in antibiotic-treated hyphae has further been used to validate the endofungal nature of the symbiont (Li et al., 2010; Ali et al., 2022). Also, during reassociation procedures, antibiotics are added to selectively remove extrahyphal bacteria, although it is unsure if and how this affects EFB (Baltrus et al., 2018; Desirò et al. 2023).

Different approaches have been successfully employed to isolate EFB. Mechanic and enzymatic disruption of the hyphae followed by plating on bacterial growth media complemented with fungi-directed antibiotics such as cycloheximide (Venkatesh et al., 2022) or filtration to remove fungal particles (Itabangi et al., 2022). In some cases, spontaneous bacterial emergence can be triggered by exposing the hyphae to a heat stress (Arendt et al., 2016). Some symbionts resist isolation, likely due to the advancement of their genome reduction (Fig. 1). Despite various attempts, *CaGg* remains uncultured, although spore suspension could be kept viable for several weeks (Jargeat et al., 2004). MRE are likely to remain uncultured due to their extremely eroded genomes generally lacking amino acid and nucleotide biosynthesis genes as well as enzymes for essential metabolic processes such as the citric acid cycle (Naito et al., 2017). By contrast, *Mycetohabitans* species are cultivable despite showing signs of genome reduction compared to free-living congeners (Lackner et al., 2011b). Several *Mycoavidus* EFB are yet to be cultured, but their genomes were successfully assembled through metagenomics approaches (Büttner et al., 2021, 2023). Still, *Mycoavidus cysteinexigens* and *Mycoavidus* sp. B2-EB were successfully isolated on conventional media (Ohshima et al., 2016; Guo et al., 2020). Interestingly, all *Mycoavidus* EFB are phylogenetically close, but the genomic determinants enabling or restricting their cultivation have not been uncovered (Amses et al., 2023).

Reassociation of the host and potential bacterial isolates can further be attempted to confirm a phenotype observed in axenic culture. While endosymbionts were successfully reintroduced into their original host in several models (Partida-Martinez et al., 2007b; Arendt et al., 2016; Obasa et al., 2017, 2020; Baltrus et al., 2018; Hazarika et al., 2020), reassociation by co-culturing is not always successful. As the penetration or establishment steps of EFB inside their host are likely influenced by environmental conditions, they can be restricted in artificial settings. GFP-tagged *R. radiobacter* cells were not able to colonize the hyphae or spores of their original host, *S. indica*, in co-cultivation assays (Guo et al., 2017). Although the bacteria can be vertically transmitted, the absence of genome reduction suggests an additional horizontal transmission to avoid population bottlenecks. The underlying mechanisms remain to be uncovered but might be restricted in artificial environments. Higher reassociation ratios under dilute nutrient conditions further suggest that particular conditions, such as starvation, promote the endosymbiotic establishment of bacteria in fungal hosts (Arendt et al., 2016). For bacteria that do not spontaneously reinfect their original host, or to establish non-specific endosymbiosis, a fluidic force microscopy approach was recently devised and used to successfully inject both *M. rhizoxinica* and *E. coli* into *R. microsporus* (Giger et al., 2024). Alternatively, forceful incorporation of bacteria into fungi was also performed using electroporation (Cheng et al., 2022). These procedures are highly promising for the study of symbiont-host adaptation and the evolution of endofungal symbioses.

## 9. Final considerations and future perspectives

Endosymbiotic bacteria are present in all major fungal taxa explored so far, but their distribution varies among fungal phyla and species. While some fungal species host genetically homogeneous endosymbiotic populations, others contain diverse EFB. The community resolution levels offered by amplicon sequencing and shotgun metagenomics might, however, uncover additional and rare EFB that have so far been overlooked. A substantial proportion of fungal taxa remain to be explored and the environmental conditions controlling their association with EFB are still enigmatic. Several association-promoting traits were uncovered both in EFB and their hosts. Still, the fungal mechanisms enabling the bacterial invasion and allowing perennial colonization are not well understood. Since associations between EFB and fungi have emerged independently at several time points, the underlying mechanisms likely vary depending on the fungal taxon involved.

There is increasing evidence for diversified endofungal populations, especially in *Dikarya*, which are also frequent hosts of transient colonizers. The endofungal space is not restricted to long-term symbionts but is an additional level of the dynamic fungal holobiont (Partida-Martínez, 2017). Whether they serve a purpose for their host or opportunistically colonize the hyphae for temporary shelter is still unresolved in most cases. In plants, for instance, multiple facultative endosymbionts have a strong, sometimes context-dependent impact on their host. Future investigations might uncover similar roles for endosymbionts of fungi. Dual-transcriptomics studies in this field are still rare but could shed light on the role of bacteria as well as on the still elusive immune system of fungi.

An increasing number of biotechnological applications are found for fungi in the agricultural, medical, and industrial sectors (Niego et al., 2023). While they are contributing to the development of food and drugs, fungi are also a growing and understudied cause of human disease. Endobacteria represent a non-negligible layer of fungal biology and must be considered in fungal applications. Some fungal features were already disavowed as being of bacterial origin, and multiple examples testify to severe behavioral changes for fungi in the absence of their natural bacterial symbionts. To thoroughly understand fungi and hope to harness their capacities efficiently, one cannot disregard their metaorganism status.

Leveraging EFB that modulate nutrient flows, stress tolerance, or antipredator defenses could enhance plant-fungus symbioses and disease suppression in agricultural settings. Candidates include symbionts that induce nitrogen assimilation in plant-associated fungi, trigger the production of defensive secondary metabolites, or deter fungivores in soil systems. These candidates are not restricted to isolates from fungi, as exogenous bacteria have also demonstrated the ability to colonize fungal hosts de novo (del Barrio-Duque et al., 2020). However, resident EFB are frequently overlooked because bacterial reads from fungal sequencing are routinely treated as contaminants. Yet these bacteria may carry antimicrobial resistance genes or toxins, which must be assessed and monitored when fungi are developed or approved for agricultural bioformulations. Routine monitoring and stabilization or curing of EFB strains used in the food and fermentation industry may further reduce phenotypic drift and batch variability. Conversely, the deliberate addition of beneficial EFB could be employed to adjust metabolite profiles in targeted fermentations. In clinical mycoses, disrupting pathogenicity-enhancing EFB may improve prognoses, as endobacteria can mediate immune evasion and antifungal susceptibility. Diagnostic pipelines should anticipate the presence of mixed endobacterial communities in priority pathogens and consider their impact on virulence and therapy.

A frequent phenomenon occurring in fungi subjected to long courses of sub-cultivation on artificial media is phenotypic degeneration. It is described in AMF and various pathogens where it can affect sporulation and virulence. Genetic, epigenetic and stress have been suggested as causes for degeneration but the potential role of EFB was so far
overlooked (Danner et al., 2023). Indeed, we have seen that *in vitro*
replication could lead to EFB loss, altering fungal phenotypes.

Identifying and describing EFB requires a range of different ap-
proaches and competencies. In this age of accessible genomics, micro-
scopy observations are not to be neglected as they are essential for the
validation of the symbiont's endofungal localization (Fig. 2E-F). Fungal
organelles can have shapes and a staining profile highly similar to
endobacteria (Fig. 2G), thus requiring *in situ* hybridization to validate
the status of EFB. Visual observations might further reveal morpholog-
ical adaptations and behaviors of bacteria inside fungi that would be
overlooked otherwise.

Plant-interacting fungi were disproportionately sampled in the
exploration of EFB. In the future, it will be interesting to learn more
about the reliance on EFB of animal-colonizing fungi and especially
human pathogens. Considering the overall diversity of fungi, there is
still a substantial amount of additional knowledge to be gained for
*Mucoromyceta* and especially *Dikarya*. Many studies rely on fungal iso-
lates that have been stored and replicated for years to decades. To avoid
a reduction in endofungal diversity through these unintended purifica-
tion steps, it is important to consider fresh isolates or, if the model al-
lows, to perform *in situ* studies. A growing body of literature shows that
fungi can host complex bacterial communities, but also physiologically
important viruses (Espino-Vázquez et al., 2020). The endofungal
ecosystem is therefore likely to be the site of diverse interactions be-
tween residents, which fully remain to be explored. The knowledge
gained on the mechanisms of fungal-bacterial symbioses will be useful
for the still outstanding investigation of similar associations formed by
oomycetes, and their importance in the economically important plant
diseases they cause.

CRediT authorship contribution statement

**Pooja Shukla:** Writing – original draft, Conceptualization. **Julia Buchner:** Writing – review & editing, Resources, Investigation. **Deepak Kumar:** Writing – original draft, Conceptualization. **Adrian Wallner:** Writing – review & editing, Writing – original draft, Resources, Conceptualization. **Utkarsh Talukdar:** Writing – original draft, Conceptualization. **Aurélie Deveau:** Writing – review & editing, Conceptualization. **Stéphane Complant:** Writing – review & editing, Resources, Conceptualization. **Verma Satish Kumar:** Writing – original draft, Supervision, Conceptualization.

Funding

The authors acknowledge the support of the Department of
Biotechnology (DBT), for providing the JRF to UT to carry out this work.
SKV also acknowledges the support from DBT, India as project P07/
1265. The authors thank IoE-BHU for financial help as incentive grant.
SKV acknowledges the support from SERB-SIRE fellowship. This
research was funded in whole or in part by the Austrian Science Fund
(FWF) [https://doi.org/10.55776/ESP254]. For open access purposes,
the author has applied a CC BY public copyright license to any author
accepted manuscript version arising from this submission.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Acknowledgments

Authors are thankful to the Head and Coordinator CAS, FIST of Botany, B.H.U., Varanasi, India for providing research facilities and gratefully acknowledge the Plant Developmental Biology Lab at the Ludwig-Maximilians Universität München under the direction of Prof. Dr. Andreas Klingl for their precious contribution for imaging endofungal bacteria using transmission electron microscopy.

**Appendix A. Supporting information**

Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.micres.2025.128361.

Data availability

No data was used for the research described in the article.

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