Critical research challenges facing Mucoromycotina ‘fine root endophytes’

Abstract

Mucoromycotina ‘fine root endophytes’ (MFRE), referred to previously as Glomus tenue (Greenall) or more recently Planticonsortium tenue (Walker et al., 2018), are a globally distributed group of soil fungi (Orchard et al., 2017a) that form endosymbioses with plants from across most of the land plant phylogeny (Hoysted et al., 2018, 2019; Rimington et al., 2019). Despite much progress having been made in characterizing plant–MFRE symbioses in the last decade, significant challenges remain. Here, we mark out these challenges and discuss future directions for promoting research in this rapidly developing field. MFRE, within Endogonales (Mucoromycotina, Mucoromycota), are recognized as phylogenetically (Bidartondo et al., 2011; Spatafora et al., 2016; Orchard et al., 2017b) and functionally (Field et al., 2015, 2019; Hoysted et al., 2019) distinct from the more commonly studied arbuscular mycorrhizal fungi (AMF), which belong to the Glomeromycotina (or Glomeromycota) (Spatafora et al., 2016). Research using isotope tracers has shown that MFRE exchange both phosphorus and nitrogen for plant-fixed carbon when in association with liverworts (Field et al., 2015, 2016, 2019) and with the vascular plant Lycopodiella inundata (Hoysted et al., 2019, 2021b), while a cryo-scanning electron microscopy (SEM) and X-ray microanalysis study suggests MFRE may play a role in phosphorus assimilation in Trifolium subterraneum (Albornoz et al., 2020). Where it has been measured, MFRE have been shown to transfer a significant amount of nitrogen to their host plant (Field et al., 2016, 2019; Hoysted et al., 2019, 2021a), suggesting that there may be a complementary role for these fungal symbionts alongside AMF. In contrast to their well-established role in plant phosphorus nutrition, the extent to which AMF contribute directly to host plant nitrogen nutrition has been subject to some debate (Smith & Smith, 2011; Hodge & Storer, 2015; Thirkell et al., 2016) which is now pertinent given the widespread misidentification of fungal endosymbionts, including MFRE, as AMF (Orchard et al., 2017a; Field et al., 2019). A meta-analysis of the literature on MFRE revealed that many past studies have neglected to focus on MFRE due to difficulties in distinguishing between MFRE and AMF morphologies (Orchard et al., 2017a), the challenge of isolating MFRE, and the absence of MFRE from plant specimens as a result of degradation brought about by sample storage conditions and duration (Orchard et al., 2017c). As the importance of MFRE in plant nutrition is increasingly recognized, further research into their form and function has become critical for understanding of the flows of carbon and nutrients through plant and soil communities. Such findings may have potentially important implications for applications of mycorrhizal fungi in sustainable agriculture (Thirkell et al., 2017). The choice of plant host for MFRE in experiments represents a critical consideration for researchers, particularly given that relatively little is known about compatibility and variability in function of MFRE symbionts across plant clades. To date, the majority of experiments have been conducted using a relatively limited range of plant hosts, focusing on species where MFRE but not AMF have been detected molecularly across multiple wild populations (e.g. Lycopodiella inundata and some Haplomitriopsida liverwort species), or those which are readily colonized by MFRE in soil-based inocula (e.g. Trifolium spp.). The breadth of host range for MFRE symbionts, inclusive of compatibility, structure and function of plant–MFRE associations, warrants further investigation (Sinanaj et al., 2020). Experiments involving the use of plants, particularly those where genomes are available, that might be considered as models for symbiosis research (e.g. Medicago, Lotus) would be especially valuable in unpicking the molecular and physiological mechanisms underpinning the symbiosis. Using light microscopy, MFRE are generally recognizable by their fine hyphae (< 1.5 µm diameter) with small intercalary and terminal swellings and ‘fan-like’ branching structures (Thippayarugs et al., 1999). These contrast with the relatively coarse hyphae (> 3 µm diameter) of AMF (or ‘coarse root endophytes’) (Field & Pressel, 2018). Arbuscules (highly branched intracellular fungal structures) are characteristic of plant–AMF symbioses; however, their occurrence and appearance in MFRE symbioses across host plants and even plant lifecycles (Hoysted et al., 2021a), is variable (Orchard et al., 2017b; Hoysted et al., 2019). Morphological plasticity has also been noted in transmission and scanning electron micrographs of the ultrastructure of symbioses in plants where only MFRE were detected (Field et al., 2015; Hoysted et al., 2019), making it challenging to distinguish them in planta in co-colonizations with AMF (Field et al., 2016). In contrast with the generally very well-characterized AMF spores, those of MFRE are poorly documented. Brief descriptions of their appearance and size occur but are unaccompanied by images (Hall, 1977; McGee, 1987); in fact, only a single unvalidated image of an Endogonales MFRE spore has been published to date (Orchard et al., 2017a). The prevailing symbiotic scenario among mycorrhiza-forming vascular plants is colonization by multiple fungal symbionts (Hoysted et al., 2019; Teste et al., 2020). Over the years, techniques for the detection and characterization of mycorrhizal fungi have been refined, including molecular detection methods using fungal-specific primers that target marker genes (White et al., 1990), the MaarjAM curated database dedicated to AMF sequences (Öpik et al., 2010), and inoculation methods using either axenic fungal cultures (Mugnier & Mosse, 1987) or fungal spores extracted from soil (Gerdemann & Nicolson, 1963) to generate plants colonized exclusively by specific species of mycorrhizal fungi. This approach is particularly challenging for MFRE, as their spores are poorly characterized and difficult to isolate, and available fungal isolates are few (Field et al., 2015). This represents perhaps the most pressing obstacle to MFRE research progress, highlighting the need for MFRE–plant experimental systems that allow researchers greater control over biotic and abiotic factors that may influence form and function of MFRE symbioses. Here, we discuss the three state-of-the-art approaches currently available to investigate these associations, including use of soil sieving, wild plants and axenic fungal isolates, together with the caveats that should be considered where each method is employed. Inoculum production through soil sieving (Gerdemann & Nicolson, 1963; An et al., 1990; Orchard et al., 2017b) is currently the only published technique for colonizing experimental vascular plants with MFRE, while excluding the other arbuscule-forming symbionts, AMF (Albornoz et al., 2020). This method, based on the observation that AMF spores appear to be much larger than those produced by MFRE, involves wet-sieving soil collected from a site known to contain MFRE to obtain the material that accumulates between sieves of pore sizes 200 µm and 50 µm. This is then dried and used as a soil inoculum enriched in MFRE. The inoculum is mixed with autoclaved sand or soil at a ratio of 1 : 81 (Orchard et al., 2017b) or 1 : 162 (Albornoz et al., 2020) to produce a substrate for plant growth (Fig. 1). There are several factors with this method that require consideration (Table 1). The diameters of AMF spores typically range from 91 µm to > 300 µm (Gerdemann & Nicolson, 1963) but can be smaller (see supplementary material of Aguilar-Trigueros et al., 2019). As such, AMF spores and other propagules such as hyphal fragments (Bingle & Paul, 1986) cannot be consistently excluded from inocula produced using the sieve sizes specified earlier, which generate inoculum containing spores and/or hyphae ≥50 µm and up to 200 µm. MFRE spore diameters are reported to range from 10 to 12 µm (Hall, 1977) or 25 to 35 µm (McGee, 1987) and thus could pass through a 50 µm sieve. As a result of this uncertainty and the ambiguity in descriptions of MFRE spore morphologies, it is difficult to determine the quality of a soil inoculum immediately after it is produced. Checks on inoculum quality and/or viability for monoxenic AMF cultures or AMF spores extracted from soil involve the microscopic quantification of spore density and morphological confirmation of spore identities (Daniels & Skipper, 1982). This is currently not possible for MFRE inocula and is a limitation across methodologies for obtaining MFRE-colonized plants (Table 1). The quality of an MFRE-enriched soil inoculum only becomes apparent when it is used in a substrate to grow plants. Dilution of soil-based inocula aims to ensure a plant will ‘more likely encounter a single unit of inoculum and contain a single fungus’ (Orchard et al., 2017b). While this may be the case, the unit of inoculum that colonizes the plant is determined largely by chance and can equally be MFRE or AMF if the growth substrate retains AMF propagules c. 50–200 µm. As such, this inoculation strategy may not be effective at exposing plants to similar amounts of fungal propagules and generating roots with consistent MFRE colonization, which can be a limitation to ensuring replicable experimental conditions. An improvement to this method, as used in Albornoz et al. (2020), is to first use the soil inoculum to produce an MFRE pot culture. Plants are grown in pots containing diluted soil inoculum, after which the substrate within pots containing the highest amount of MFRE and lowest amount of AMF is sieved again to produce soil inoculum to grow more plants. This process encourages the proliferation of MFRE hyphal networks in the substrate and can be repeated until plants grown in the sieved substrate are consistently colonized by MFRE. This process can be labour intensive and time consuming, especially when propagating a pot culture to produce enough material for an experiment with a large number of replicate plants. Appropriate quality control measures are necessary when using any soil-based inoculation method, regardless of whether the resultant soil inoculum comes directly from wild soil or from a sieved soil pot culture. AMF contamination, which is the biggest issue of this method, should be monitored through molecular and morphological identification of fungi in plants and their substrate. As with other methodologies for obtaining MFRE-colonized plants, the more quality control checks that are carried out, the more accurate the data on the fungal symbionts present. Entire root systems, which inevitably have patchy colonization regardless of inoculation method, cannot be simultaneously analysed molecularly if other informative but destructive techniques, e.g. root clearing and staining for microscopy, are used. This means symbionts may go undetected molecularly due to the limited amount of root material used for sequencing. However, co-colonizations can go undiagnosed due to the morphological plasticity of AMF and MFRE (Field et al., 2016). As such, it is critical that enough plant material is available to adequately sample (multiple plants, multiple pots, multiple time points) for these checks when using soil inoculation methods. We tested the effectiveness of soil sieving protocols for growing vascular plants predominantly or exclusively colonized by MFRE in four experiments using long-term pasture soil (Supporting Information Fig. S1; see Methods S1). In Experiment 1, we followed the methods of Albornoz et al. (2020) and grew Trifolium repens in pots containing sieved soil inoculum combined with either autoclaved soil or autoclaved sand. In Experiment 2, we grew Medicago truncatula using the methods of Orchard et al. (2017b). Given the necessary considerations outlined earlier, in both of these experiments we used smaller sieve sizes than those published. To refine the protocols further and increase the chance of MFRE colonization, we explored the effect of growing M. truncatula in substrate containing colonized root fragments taken from Experiment 2, with or without sieved soil inoculum (Experiment 3). In Experiment 4, we grew Trifolium repens in substrate containing root fragments and sieved soil inoculum. We hypothesized that supplementing the growth substrate with root fragments (derived from plants grown in MFRE-enriched substrate), in addition to sieved soil inoculum, would increase colonization of plant roots by MFRE through exposure to more MFRE propagules and hyphal networks. At the harvest stage of each experiment, plant roots were stained with acidified ink to quantify fungal colonization (Vierheilig et al., 1998) and hyphal extractions with Trypan blue staining were carried out on substrate from each pot (Brundrett et al., 1994). In Experiment 1, we found no evidence of fungal colonization in plant roots, while soil hyphal extractions revealed that six pots out of a total of 35 contained few AMF-like hyphal fragments (Fig. S2). As no MFRE were detected in the substrate of any pots, we were not able to generate any inoculum to grow a second generation of plants to recreate the second stage of the methods of Albornoz et al. (2020). In Experiment 2, root staining revealed colonization with MFRE and AMF in one pot; colonization with only MFRE in two pots; and no fungal colonization in one pot (Table S1). Values for the percentage total root length (%TRL) colonized by MFRE varied, ranging between 1.4% and 92.9% (Fig. 2a). This wide range for MFRE was similar to results reported in Orchard et al. (2017b) where %TRL colonization ranged between c. 18% and c. 77% between pots (n = 3). Using molecular methods (see Methods S1 for details), we detected MFRE in root samples from all four pots using the Endogonales-specific primers EndAD1f and EndAD2r (Desirò et al., 2013). The inclusion of root fragments in the substrates of Experiments 3 and 4 resulted in plant roots with variable levels of MFRE colonization, and increased AMF colonization within roots and in the substrate (Figs 2a–d, S3). Our data confirm methods using soil inoculum obtained from wild soils are prone to result in inconsistent colonization of plant roots by MFRE fungi. Plants colonized by MFRE have been sourced from the wild for use in several experiments examining the nutritional significance of MFRE symbioses (Fig. 1) (Field et al., 2015, 2016, 2019; Hoysted et al., 2019). To do this, a field site is identified where MFRE are prevalent by collecting environmental samples, including plant roots, and identifying the fungal symbionts using staining and molecular methods. Following this, whole plants are carefully removed from the site and transferred into pots containing autoclaved substrate. Although the use of wild plants allows glasshouse experiments to be more representative of natural habitats, there are critical biological considerations that must not be overlooked in such experiments (Table 1). The soil adhering to the roots of wild plants contains microbial communities that are transferred to the pots, adding a further level of complexity to the experimental system. Rhizosphere bacteria are known to associate with mycorrhizal fungi (Garbaye, 1994; Bonfante et al., 2019); however, the extent of bacterial impacts on fungal fitness and function under different environmental conditions is largely unknown, particularly for MFRE. For researchers wishing to disentangle the effects that MFRE have on plants from those of rhizobacteria, the use of wild-collected plants presents difficulties that can only be mitigated with complex and sometimes expensive experimental features. Custom-made plastic cores with mesh-covered windows accessible to fungal hyphae but not plant roots (Johnson et al., 2001), can be buried within the substrate of pots to account for microbial nutrient cycling (Fig. 1) (Field et al., 2015). Comparisons between the same plant species obtained from different field sites should be drawn with caution, as variations in microbial community, including MFRE diversity, between sites could lead to ambiguity in results. This is also applicable when comparing data from independent experiments on wild plants colonized by different fungal symbionts, e.g. MFRE and AMF may host distinct microbiomes. The types of research questions that wild-collected plants would be suitable for addressing include those exploring the function, recruitment and competition of MFRE in an ecological context where various other biota may be present. Although staining and molecular identification of fungal symbionts in roots must be carried out as a quality control when selecting sites to source wild plants from and at the end of experiments when wild plants are harvested, this does not guarantee a complete picture of fungal colonization or the absence of AMF. Some stained fungal structures may be only remnant cell walls, lacking cytoplasm and functional capabilities. Patchy fungal colonization is a limitation when DNA sequencing fungi from small segments of root, as primers can fail to detect fungi due to their spatial distribution. The specificity of primers to certain clades of fungi is also an issue (Bidartondo et al., 2011), which can be overcome by using a selection of primer sets to capture fungal diversity. Despite these measures, there is always a chance that fungal symbionts may go undetected, which also brings into question whether plant species, populations or individuals can truly form exclusive MFRE, or AMF, associations in nature. Currently, candidate MFRE-specialist plants include several Haplomitriopsida liverwort species (Field et al., 2015; Rimington et al., 2020) and the lycophyte Lycopodiella inundata (Rimington et al., 2015; Hoysted et al., 2019) where, so far, AMF have never been detected across repeated sampling from multiple, geographically separated populations at various time points across a number of years. A single report of rare AMF occurrence in Haplomitrium mnioides mainly colonized by MFRE (Yamamoto et al., 2019) was based on limited molecular evidence and without and thus further investigation (Rimington et al., 2020). The for the production of fungal inocula is and axenic culture of symbiotic which result in propagules of the without with other fungi or is now critical that a of MFRE isolates, potentially within AMF is inclusive of fungi from the breadth of MFRE plant hosts, across environmental and to truly capture the of symbiotic MFRE fungi. techniques for of MFRE currently and require dedicated in developing protocols and for their effective and The first axenic culture of MFRE was and reported in Field et al. where segments of colonized from the liverwort were fungal to allow MFRE hyphae to grow out and in the The was then to and the of liverwort in These MFRE grew in without a plant host to (Field et al., suggesting may have in nature. This could researchers the so for AMF, to in experiments to questions the function and of MFRE in for the range of nutrient that MFRE are able to and such as fungal and that may be specific to this MFRE cultures could be used as inoculum in experiments (Fig. but this method As with other should be taken when data from experiments using fungal isolates (Table 1). cultures of MFRE do not contain the microbial communities that occur alongside fungi in While this allows of the of MFRE on plant hosts, it may not their role in natural A understanding of the of MFRE is to ensure that the are representative and with the experimental plants. The of cultures is also an important that should be taken into as production and the viability of cultures can over For it has been that mycorrhiza-forming fungi may and as a result of the growth conditions that are to while in which may influence their to plant roots et al., 2020). The three experimental methods have each important into the form and function of plant–MFRE associations and will to do particularly if used in to determine the of MFRE fungi to plants alongside those from sieving methods have the to the selection of plant species we are able to study MFRE with variable colonization plants more consistent fungal colonization and are a valuable into the significance of in their natural but do not with AMF in most vascular plants. and axenic culture techniques will allow the function of plant–MFRE associations to be determined directly in a of Although this may ecological it is likely to be the only by which we may study the of MFRE–plant symbioses (e.g. and the including a of MFRE isolates from a wide of plants, is for the future of the field. The from the Research to and and the Research and are by a The are to the and to the for their on the and and the and the experiments and analysed the results. the all results and on the to as the for and ensure Fig. S1 Methods used to out soil sieving Experiments Fig. AMF-like coarse hyphal in the soil-based substrate of Experiment Fig. micrographs of roots of Trifolium from Experiment Methods S1 and methods for Experiments S1 total root length (%TRL) colonized by fungi in Medicago truncatula from Experiment are not for the or of any Information by the than should be to the The is not for the or of any by the than should be to the for the

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