Concepts and consequences of the hyphosphere core microbiome for arbuscular mycorrhizal fungal fitness and function
Summary
Arbuscular mycorrhizal (AM) fungi-associated hyphosphere microbiomes can be considered as the second genome of the mycorrhizal phosphorus uptake pathway. Their composition can be thought of as a stably recurring component of a holobiont, defined by the hyphosphere core microbiome, which is thought to benefit AM fungal fitness. Here, we review evidence indicating the existence of the hyphosphere core microbiome, highlight its functions linked to those functions lacking in AM fungi, and further explore the mechanisms by which different core members ensure their stable coexistence. We conclude that deciphering and utilizing the hyphosphere core microbiome provides an entry point for understanding the complex interactions among plants, AM fungi, and bacteria.
| Contents | ||
|---|---|---|
| Summary | 1529 | |
| I. | Introduction | 1529 |
| II. | The hyphosphere: An existing core microbiome | 1530 |
| III. | Linking the core microbiome to potential ecological functions | 1530 |
| IV. | Potential mechanisms for maintaining stable coexistence of different core members | 1532 |
| V. | Concluding remarks | 1532 |
| Acknowledgements | 1532 | |
| References | 1533 | |
I. Introduction
Arbuscular mycorrhizal (AM) fungi are widely found in terrestrial ecosystems and have been considered as a vital component of plant-associated microbiomes for decades (Smith & Read, 2008). They offer host plants multiple benefits, including defense against pathogens, enhanced tolerance to salinity, low pH, and heavy metals, as well as improved nutrient uptake. Their most-recognized benefit is facilitating plant uptake of relatively immobile nutrients, particularly phosphorus (P). Accumulating evidence suggests that the root and the fungus provide two independent P uptake pathways for mycorrhizal plants, that is the direct pathway (DP) and the mycorrhizal pathway (MP; Chu et al., 2020; Shi et al., 2023). The MP compensates for, or entirely supplants, the P acquisition function of the DP, leading to a downregulation of gene expression associated with root P uptake (Chu et al., 2020).
In the MP, the extensive extraradical hyphae significantly expands the nutrient absorption range of the roots. Notably, the interaction between AM fungi and the soil microbes also profoundly affects nutrient turnover. Like plant roots, AM fungi obtain photo-assimilates from plant shoots, part of which is rapidly released into the soil surrounding the hyphae, the hyphosphere, in the form of exudates. Diversified bacterial taxa, feeding on carbon (C)-rich hyphal exudates, grow and reproduce in the hyphosphere, form unique microbial communities and structures that are distinct from the bulk soil and rhizosphere microbiome, that is the hyphosphere microbiome (Wang et al., 2022a; Faghihinia et al., 2023). Hyphosphere bacteria, beyond being mere C consumers, crucially aid AM fungi in soil nutrient uptake, acting as a secondary genome of the MP (Zhang et al., 2022a). Although the extensive literature exists on the rhizosphere microbiome, the hyphosphere microbiome has not yet received the attention it deserves. Deciphering the ecological characteristics and potential functions of the hyphosphere microbiome for AM fungi and their associated host plants is important for a comprehensive understanding of AM symbiosis.
In this review, our objectives are to (1) review evidence that there is a core microbiome in the hyphosphere of AM fungi, (2) highlight the functional significance of the core members related to the fitness and function of AM fungi, and (3) discuss potential mechanisms for core members to ensure their stable co-occurrence.
II. The hyphosphere: An existing core microbiome
The quest to identify a core microbiome has gained momentum in microbial ecology. The core microbiome refers to the set of microbial species that are consistently present in a particular environment or associated with a specific host, regardless of external factors or conditions (Shade & Handelsman, 2012; Zhang et al., 2022b). These species are hypothesized to represent the most functionally crucial microbial associates of that environment or host (Neu et al., 2021). The core microbiome is still a young concept for the hyphosphere of AM fungi. Based on what we currently know, previous studies have mainly focused on the portion of the hyphosphere microbiome that changes in response to AM fungal species and some environmental factors, while neglecting the stable members, the hyphosphere core microbiome (Faghihinia et al., 2023).
Recently, two pieces of pioneering research have enabled understanding of the hyphosphere core microbiome (Emmett et al., 2021; Wang et al., 2022b). Emmett et al. (2021) examined the effect of AM fungal species, soil types, and timing on the composition and structure of the hyphosphere microbiome based on 16S rRNA gene sequencing. The results showed that, some orders as core members were consistently enriched in the hyphosphere across soil types and AM fungal species. In addition, a more concise meta-analysis by Zhang et al. (2022a) found that hyphosphere microbiomes associated with different AM fungal species were mainly composed of Proteobacteria and Actinobacteria. Faghihinia et al. (2023) further pointed out that certain bacterial taxa at phylum level consistently increased significantly in the hyphosphere across different studies. Wang et al. (2022b) developed a research pipeline to effectively collect AM fungal hyphae in situ and established on-site field trials to decipher AM fungal–hyphosphere bacterial co-occurrence patterns. Despite being from a range of crop-soil systems and climate zones, specific taxa were co-enriched in the hyphosphere. These results further suggested that AM fungal communities co-cultivated a taxonomically conserved hyphosphere core microbiome across environments. Together plant and AM fungi as a holobiont have been described as an evolutionary individual, and the above discoveries suggest that this holobiont must also include the bacterial community tightly related to AM fungal hyphae (Johnson & Marin, 2023).
When comparing the core members identified by Wang et al. (2022b) and Emmett et al. (2021) in distinct tested environments, the noteworthy overlap highlights the dominance of Myxococcales, Betaproteobacteriales, Fibrobacterales, Cytophagales, and Chloroflexales. In real-world environments, a single plant root is simultaneously infected by multiple AM fungal species, while the hyphosphere provide an enormous yet invisible scaffolding for bacterial growth, reproduction, and even evolution, which together form an intricate hairball-like interaction network. In this scenario, the interaction between AM fungi and hyphosphere bacteria is ‘many-to-many’ rather than simply ‘one-to-one’. Existing studies focusing on only a few AM fungal species under controlled conditions are far from adequate. The next step is to identify the core members of the hyphosphere interaction network, which encompasses the environment, plants, AM fungi, and bacteria. This needs to be achieved by upscaling AM fungal–bacterial interactions from the laboratory to the field (Wang et al., 2022b; Johnson & Marin, 2023).
III. Linking the core microbiome to potential ecological functions
Functional redundancy occurs much more frequently than was previously thought in microbial communities across a range of environments (Allison & Martiny, 2008). This suggests that a very tiny proportion of bacteria, termed the hyphosphere core microbiome, are required to fulfil a variety of ecological services to AM fungi. Given our current knowledge of the genome and biology of AM fungi, we emphasize that the hyphosphere core microbiome may possess the following four potential functions that are of major relevance to the fitness of AM fungi: (1) improving organic P (Po) mineralization; (2) improving organic N mobilization; (3) preying on bacteria; and (4) providing C sources (Fig. 1).
Based on knowledge gleaned from the genomes of several AM fungi, they have a limited capacity to mineralize Po (Tisserant et al., 2013). Correspondingly, in sterile microcosm and in vitro culture conditions, supplying the AM fungi with phytate, it was evident that AM fungi did not secrete phosphatase and were unable to utilize this Po resource (Zhang et al., 2014, 2016). However, the ability of AM fungi to access soil P resources is critical for maintaining AM symbiosis, as P is the ‘capital’ they exchange for C from plant hosts, subsequently used for growth and development. This implies that AM fungi inevitably need to resort to other functional soil microorganisms to compensate for this missing key function so that they can stabilize the partnership with plants, especially in Po-dominated soil environments.
A series of studies documented that a single bacterial species or complex bacterial community stimulated by hyphal exudates contributed to increased rates of soil Po mineralization (Zhang et al., 2014, 2018; Wang et al., 2016, 2023). It can be concluded that AM fungi and hyphosphere bacteria establish a stable C–P reciprocal relationship in the face of Po sources: AM fungi supply the C source for bacteria, and in return, bacteria provide P to AM fungi (Zhang et al., 2022a). Since the hyphosphere core members are repeatedly occurring and prevalent across multiple AM fungal species and broad environments, it can be speculated that they are tightly linked to the Po mineralization function. To test this hypothesis, Wang et al. (2022b) examined the relationship between individual core members or core modules singled out from the fungal–bacterial network and phosphatase activity. Significant positive correlations suggested that the hyphosphere core microbiome's capacity for Po exploitation, which compensated for the lack of AM fungal function and thus maintained the stability of AM symbiosis.
Unlike the vital role of P in the maintenance of reciprocal plant–fungal partnerships, the amount of N supplied to the plant through AM symbiosis is insufficient to satisfy plant requirement, making it a less significant plant N capture mechanism (Reynolds et al., 2005). The reason is that AM fungi require large amounts of N for their own metabolism and growth. Hyphae are shown to be N-rich: having a concentration of N that was 4–7 times greater than that of the plant shoots (Hodge & Fitter, 2010). Previous studies have found that organic material is a principal source of N for AM fungi (Hodge & Fitter, 2010). Due to a limited exo-enzymatic repertoire, AM fungi also cannot effectively utilize complex organic N (Tisserant et al., 2013). Moreover, there is also evidence that suggests hyphosphere bacteria, such as Paenibacillus sp., may aid AM fungi in acquiring N from the organic source chitin (Rozmoš et al., 2022). Based on these, we highlight that the hyphosphere core microbiome should also have the ability to mineralize organic N to meet AM fungal own nutritional need.
As mentioned above, efforts to define the hyphosphere core microbiome based on the repeated enrichment criteria underscored the significance of Myxococcales (Emmett et al., 2021; Wang et al., 2022b). Although the interaction between Myxococcales and AM fungi has yet to be explored, their strong increase in abundance in the hyphosphere is thought-provoking. As bacterivores in soil food webs, Myxococcales are known for their predation on bacteria through ‘wolf-pack’ hunting combined with the secretion of lytic enzymes (Petters et al., 2021). Their predation on bacteria might aid in the mobilization of nutrients from microbial biomass, potentially supplying nutrients to AM fungi. Moreover, the well-developed hyphal network of AM fungi is inevitably attacked by pathogenic bacteria in soil. If the predatory behavior of Myxococcales is finely regulated by AM fungi, they may act as the ‘immune system’ of the hyphal network.
Plants deliver C to AM fungal partners in the form of fatty acids (Jiang et al., 2017). Consistently, the genes lacking for their own biosynthesis of long-chain fatty acids mean that AM fungi depend on an external supply of lipids for metabolism and growth (Tisserant et al., 2013). Sugiura et al. (2020) found that, when provided with myristates, AM fungi could complete their life cycle without the host. Mixtures of myristate and palmitate could further accelerate fungal growth by contrast with a single application of myristate. The importance of these pioneering results for understanding the biology of AM fungi is clear (Rillig et al., 2020). At the same time, this has brought some new insights into the interaction between AM fungi and hyphosphere bacteria. Although myristate and palmitate are the types of saturated fatty acid found mainly in plants and animals, some bacteria may also synthesize them (Quan et al., 2020). Whether the hyphosphere core microbiome has the ability to synthesize these acids to compensate for loss of supply in host C and help AM fungi survive extreme environments is a priority to explore in the future.
In summary, an intriguing picture emerges that plant–AM fungal–hyphosphere bacterial systems foster interkingdom dependency and that some conserved core bacterial taxa induced by C source or signals in hyphal exudates tend to be reoccurring in the hyphosphere across a broad range of environments. Core guilds may react to P availability for the stability of plant–AM fungal symbiosis, the N nutrient requirements for AM fungi's own growth, and the C requirements for the loss of supply in host C under some extreme conditions.
IV. Potential mechanisms for maintaining stable coexistence of different core members
The hyphosphere microbiome can be regarded as the stable backbone for the overall hyphosphere community. Different core taxa occur together in the hyphosphere and therefore inevitably interact with each other. This raises the question of what mechanisms contribute to the stable co-occurrence of these core microbes in the hyphosphere, especially under changing environment–plant–AM fungal gradients. Although this issue is still to be studied, the mechanistic understanding of interactions and coexistence among diverse bacteria observed in other systems can provide us with some insights (Fig. 1).
First, resource partitioning, which involves each community member metabolizing a different set of nutrients to avoid direct competition for available substrates, is a typical mechanism of niche differentiation (Adler et al., 2013). Secretion of C-rich hyphal exudates by AM fungi constitutes a strong driver for the assembly of the hyphosphere core microbiome. The composition of hyphal exudates contain various kinds of carbohydrates and carboxylates (Zhang et al., 2022a). Assuming that core species differ in their C source metabolic preferences, that is one species predominantly occupies a specific ecological niche to minimize competition with another, a stable coexistence of several core species can be fostered. Second, syntrophyis described as a one- or two-way metabolic interaction between consortia where one member uses intermediate products that are produced by the other (Shahab et al., 2020). Because they can significantly change the habitat's nutritional quality, syntrophic interactions are also expected to play a significant role in microbial community composition (Levy & Borenstein, 2013; Morris et al., 2013). Cross-feeding of metabolic by-products such as ethanol and acetate, for instance, is essential to the variety of cellulose-degrading communities (Kato et al., 2008). A systematic survey of the extent of this metabolic cross-feeding found that metabolically interdependent groups are widespread in natural microbial communities across diverse habitats, and highlighted that metabolic dependencies could be major driver of species co-occurrence (Zelezniak et al., 2015). Thus, syntrophic interactions can help core taxa in the hyphosphere efficiently utilize limited hyphal exudates through metabolite exchange, providing a survival advantage and allowing them to coexist across complex environments. Finally, the formation of bacterial biofilms on the hyphal surfaces of some AM fungal species has already been reported, and it is the key factor for the stable colonization of hyphal surfaces by bacteria (Scheublin et al., 2010). Core members that live as biofilms in the hyphosphere have the dual benefits of protecting bacteria from deleterious conditions and enabling bacterial guilds into functional sub-communities (Guennoc et al., 2018).
V. Concluding remarks
Arbuscular mycorrhizal fungal extraradical hyphae provide an extensive and invisible scaffold for fungal–bacterial interactions. We argue that, due to the functional deficiencies of AM fungi, the hyphosphere functional core microbiome is a consequence of the AM fungi's own nutrient requirements and need to maintain a stable plant-AM fungal symbiosis. We suggest several crucial areas that merit further exploration. First, an effective method for defining and prioritizing the core microbiome is required. Second, employing techniques such as metagenomics, metatranscriptome, and metabolomics is pivotal to uncover the potential functions of the hyphosphere microbiome. Third, we need more bacterial cultures from the hyphosphere. Constructing reproducible synthetic communities will help establish the causal relationship between the hyphosphere microbiome, AM fungi, and plant phenotypes. Deciphering the hyphosphere core microbiome in the environment–plant–AM fungi–bacteria nexus will provide important fundamental understanding of the mycorrhizal symbiosis and further maximize the beneficial functions of AM fungi.
Acknowledgements
This study is financially supported by the National Natural Science Foundation of China (32272807). The contribution of TSG was supported by the Rural & Environment Science & Analytical Services Division of the Scottish Government.
Competing interests
None declared.
Author contributions
GF and LW planned and designed the framework of this review. The first draft of the manuscript was written by LW and GF. GF, LW and TSG authors wrote and commented on successive versions of the manuscript. All authors read and approved the final manuscript.
