Introduction

Arbuscular mycorrhiza are associations formed between the majority (80 %) of terrestrial plant species and arbuscular mycorrhizal (AM) fungi in the soil (Smith and Read 2008). The formation of mycorrhiza can benefit plants through enhanced acquisition of nutrients such as phosphorus (P), nitrogen, (N) and zinc (Zn) (Cavagnaro 2008; Clark and Zeto 2000; Gyaneshwar et al. 2002; Marschner and Dell 1994; Rillig 2004a; Smith and Read 2008). In addition to their beneficial effects on plant nutrition, mycorrhiza provide other ecosystem services: for example, improvement of soil structure (Miller and Jastrow 1990; Rillig 2004b; Rillig and Mummey 2006; Tisdall 1991; Tisdall and Oades 1980), reduction of soil nutrient losses through leaching (Asghari and Cavagnaro 2011, 2012; Asghari et al. 2005; Bender et al. 2015; Bender and van der Heijden 2015; van der Heijden 2010) and the suppression of weeds (Rinaudo et al. 2010; Veiga et al. 2011), improvement of plant acquisition of nutrients from compost (Cavagnaro 2014, 2015), as well as other benefits in the context of a changing climate and increased abiotic stress (Smith et al. 2010). Consequently, mycorrhiza have an important role in influencing plant communities, ecosystem productivity and potentially agricultural productivity (Hartnett and Wilson 1999; O’Connor et al. 2002; van der Heijden et al. 1998a, b; Wagg et al. 2011).

In mycorrhizal legume species, where plants can be colonised by mycorrhizal fungi and nodulating bacteria simultaneously, common signalling pathways for the formation of mycorrhizal and rhizobial associations have been well studied (Hirsch and Kapulnik 1998; Horváth et al. 2011; Parniske 2008). This work has resulted in the identification of numerous genotypes defective for AM colonisation (referred to as a ‘mycorrhiza-defective mutants’ hereafter) in model legume species. While the present review focuses on non-legume mycorrhiza-defective mutant genotypes, it is important to mention that much of the research on the genetic basis of the AM symbiosis has been conducted using legume mutants (Ané et al. 2004; Endre et al. 2002; Imaizumi-Anraku et al. 2005; Lévy et al. 2004; Stracke et al. 2002), and thus they have been invaluable to the study of the AM symbiosis. For example, a symbiotic ‘toolkit’ has been collated using model legume species, containing 25 molecular components that work in concert to control AM colonisation (Delaux et al. 2013; Table 1). This symbiotic toolkit provides useful information for developing mutant genotypes in non-legume plant species by looking for orthologues of genes in non-legumes that have a known function in AM symbiosis.

Table 1 List of non-legume mycorrhiza-defective mutant genotypes and their properties. ‘Stage affected’ category follows the steps in AM symbiosis proposed by Delaux et al. (2013)
Full size table

The advantage of using non-legume mycorrhiza-defective mutant genotypes is that they do not form associations with nodulating bacteria, thereby avoiding complications of multi-trophic interactions (Barker et al. 1998; Cavagnaro et al. 2004a). As well as being important tools for investigating the molecular basis of AM colonisation (Barker and Larkan 2002), the mutant and wild-type pairs are also useful for studying the functioning of mycorrhiza because it is possible to compare mycorrhizal and non-mycorrhizal plants in native soil without any other experimental manipulation or intervention.

The intention of this review is to explore the potential for, and advantages of, using pairs of mycorrhiza-defective mutants (as non-mycorrhizal controls) and corresponding wild-type genotypes to study the role of mycorrhiza in various aspects of plant and soil ecology, with the aim to stimulate more work using such genotype pairs. In this context, various alternative methods for establishing non-mycorrhizal controls are summarised, before describing different non-legume plant species that have mycorrhiza-defective mutant genotypes characterised and the nature of the research they are used for. Emphasis is placed on non-legume mycorrhiza-defective mutants as legume mycorrhiza-defective mutants have been reviewed in detail previously. The review concludes with a brief discussion of research activities that could benefit from the use of mycorrhiza-defective mutant and wild-type pairs of non-legumes.

Non-mycorrhizal treatments in physiological and ecological studies

Most information on the functioning of mycorrhiza has come from studies in which plants colonised by AM fungi are compared to those that are not colonised by AM fungi (Rillig et al. 2008; Smith and Smith 1981b). However, there is no universally accepted method for establishing treatments in which AM fungi are absent but the remainder of the soil biota are present. This is especially challenging under field conditions, where the elimination of a single group of soil biota is extremely difficult (Carey et al. 1992; West et al. 1993). The various techniques used in an attempt to overcome this challenge include soil disinfection or sterilisation, comparison of constitutively mycorrhizal and non-mycorrhizal plant species, comparison of plants grown in soils with different inoculum potential and the comparison of mycorrhiza-defective mutant genotypes with their mycorrhizal wild-type progenitors. Each option has its inherent advantages and limitations, but in many cases, they are the only option available and are therefore most appropriate.

The most widely used method for establishing non-mycorrhizal control treatments is that of modifying the soil via soil disinfection or sterilisation to inactivate the AM fungal propagules (Endlweber and Scheu 2006; Koide and Li 1989; Smith and Smith 1981a, b). While these methods effectively suppress viable AM fungi, they also adversely affect or eliminate other members of the soil biota. Consequently, such soil manipulation approaches introduce non-target effects into experiments, which may be potentially confounding (Koide and Li 1989; Rillig 2004a) and should be taken into account when interpreting results.

Using a mutant approach to control AM development, by comparing a mycorrhiza-defective mutant plant genotype to its mycorrhizal wild-type counterpart, avoids the need to sterilise or disinfect soil, or compare different plant species (Rillig et al. 2008). A number of mycorrhiza-defective mutant and wild-type genotype pairs have been described, both in legume and non-legume plant species (see Table 1). The present review focuses on non-legume mycorrhiza-defective mutants since the use of legume mutants to compare interactions in mycorrhizal and rhizobial symbioses has been amply reviewed previously (see Barker et al. 2002; Marsh and Schultze 2001; Paszkowski 2006; Shtark et al. 2010; Stacey et al. 2006).

Generation and screening of mycorrhiza-defective mutants

Mycorrhiza-defective mutants can be generated in a number of ways, including via fast neutron bombardment (Li et al. 2001) and ethyl methanesulphonate (EMS) generally used to generate mutant plant genotypes (Engvild 1987; Froese-Gertzen et al. 1963; Koornneeff et al. 1982). Whatever the method used, M2 generation mutants are screened in the mutagenised populations for non-mycorrhizal phenotypes by growing the entire population of plants in soil containing AM fungal inoculum, together with the wild-type genotype, in order to compare their AM colonisation phenotype. Potential mycorrhiza-defective mutant genotypes are assessed at the M3 generation and later (up to M9 in David-Schwartz et al. (2001)) to ensure that a stable non-mycorrhizal phenotype persists. Paszkowski et al. (2006) screened for mycorrhiza-defective mutant genotypes in maize (Zea mays) in a novel manner. Maize roots that are colonised by AM fungi accumulate yellow pigment, which can be detected macroscopically (Klingner et al. 1995). Potential mycorrhiza-defective mutant genotypes from a mutator-mutagenised population of maize were grown in soil inoculated with Glomus mosseae. Plants with roots that displayed altered intensity or distribution of yellow pigmentation relative to the wild-type genotype underwent further microscopic visual screening, ultimately revealing several non-mycorrhizal mutant maize plants.

The fast neutron bombardment method is a classical reverse genetics technique (Li et al. 2001). In consequence, the gene sequence(s) controlling mycorrhizal colonisation is not known until further research is undertaken. Both map-based sequencing and transcriptomic analyses have been used to identify gene sequences that had been disrupted using this approach in mycorrhiza-defective plant mutants (see below). Creation of fast neutron mutagenised seed libraries, and their subsequent screening for desired phenotypes, is a labour-intensive, albeit effective, method for generating and identifying mycorrhiza-defective mutant genotypes.

There are a number of desirable phenotypes that need to be considered when identifying potential mycorrhiza-defective mutant genotypes, and suitable criteria have been summarised by Rillig et al. (2008) as follows: (1) a non-mycorrhizal genotype should not, while the mycorrhizal genotype should, be colonised by AM fungi in the presence of a full suite of soil biota, and (2) the mutant and wild-type genotype pair should have matched growth properties, and similar soil microbial communities, when grown in a soil where AM fungi are absent. With these criteria in mind, currently reported legume mutant genotypes are first briefly listed (see Marsh and Schultze (2001) for details), and non-legume mutant genotypes are reviewed in more detail, including their method of mutagenesis, colonisation phenotype (where relevant) and use in research.

Currently described mycorrhiza-defective mutant genotypes

A number of mycorrhiza-defective mutant and wild-type genotype pairs, in both legume and non-legume species, are available, although many more have been characterised in legumes. In legumes, mycorrhiza-defective mutants obtained using different mutagenic approaches have been identified in several plant species including pea (Pisum sativum) and fababean (Vicia faba) (Duc et al. 1989), lucerne (Medicago sativa) (Bradbury et al. 1991), barrel medic (Medicago truncatula) (Sagan et al. 1995), bean (Phaseolus vulgaris) (Shirtliffe and Vessey 1996) and Lotus japonicus (Senoo et al. 2000), but these are not the focus of this review. In terms of non-legume species, there are currently reports of mycorrhiza-defective mutant and wild-type genotype pairs in tomato (Solanum lycopersicum) (Barker et al. 1998; David-Schwartz et al. 2001; David-Schwartz et al. 2003, Kapulnik and Bonfante, unpublished), maize (Paszkowski et al. 2006), rice (Oryza sativa) and petunia (Petunia hybrid) (Chen et al. 2007, 2008; Gutjahr et al. 2008; Reddy et al. 2007) (see Table 1).

Loss-of-function mycorrhiza-defective mutant genotypes

In addition to the identification and characterisation of mutant genotypes that cannot be colonised by AM fungi, mutants that are defective in an aspect of mycorrhizal functioning have also been characterised. A mutant in M. truncatula that is defective in the gene encoding for the mycorrhiza-induced phosphate transporter, MtPT4, and affected in colonisation by AM fungi (Javot et al. 2007), has been used in a number of studies (Grønlund et al. 2013; Javot et al. 2011, Watts-Williams et al. 2015a). In rice (O. sativa) and Chinese milk vetch (Astragalus sinicus), similar mutants have been characterised for the genes OsPT11 and AsPT4, respectively, orthologues of MtPT4 (Xie et al. 2013; Yang et al. 2012). Isotope tracer studies, used in conjunction with the MtPT4 and OsPT11 mutants, confirmed that the mycorrhizal pathway of P uptake had been successfully shut down (Yang et al. 2012, Watts-Williams et al. 2015a). Future work using these mutants and work on developing other loss-of-function mutants in mycorrhiza-induced nutrient transporter genes (including nitrate and ammonium transporters) will contribute considerably to the understanding of plant-AM fungus nutrient relations.

Mycorrhiza-defective tomato mutants

76R and rmc

The mycorrhiza-defective tomato mutant rmc (reduced mycorrhizal colonisation) was first identified and described by Barker et al. (1998), and it has since been used widely by researchers, alongside its wild-type progenitor 76R, in a number of field and glasshouse studies covering many aspects of soil and plant ecology. Field studies have been undertaken on sites in Australia and the USA, and glasshouse studies have used a range of AM fungal species and soils containing native AM fungal communities (from Europe, Australia and the USA).

The degree to which the 76R and rmc genotypes are colonised, and the colonisation phenotypes they express, is highly influenced by fungal identity (Gao et al. 2001). Consequently, a number of different colonisation phenotypes have been described (see Gao et al. 2001 for photos of colonisation phenotypes). Before discussing these phenotypes, it is important to note that there has recently been a major revision of the nomenclature of AM fungi (Krüger et al. 2012; Redecker et al. 2013). In this review, for the sake of clarity, the names of the AM fungi are used as in the original publications; however, the revised species names are also provided, for ease of comparison with future work.

Several species of AM fungi that colonise the wild-type 76R genotype normally are unable to colonise the rmc genotype, giving the Pen− phenotype: Rhizophagus irregularis (formerly known as Glomus intraradices Schenck and Smith [DAOM 181602]), Glomus fasciculatum [Thaxter] Gerd. & Trappe emend. Walker & Koske [LPA7] and Glomus etunicatum Becker and Gerdemann [UT316 A-2]) (Gao et al. 2001; Manjarrez et al. 2008). The rmc genotype displays the Coi− phenotype with other species of AM fungi which can penetrate the root epidermal cells but cannot colonise cortical cells (Scutellospora calospora [Nicolson & Gerdemann] Walker & Sanders [WUM 12(2)], Gigaspora margarita Becker and Hall, Glomus coronatum Giovannetti [WUM16], formerly known as G. ‘City Beach’, and G. mosseae [Nicholson & Gerdemann] Gerdemann and Trappe [NBR4-1]) (Gao et al. 2001; Manjarrez et al. 2008). For S. calospora, the AM fungal symbiosis can be functional (in terms of C transfer from plant to fungi) but colonisation is of an intermediate morphology and is restricted to root epidermal cells (Gao et al. 2001; Manjarrez et al. 2008, 2010; Poulsen et al. 2005). Interestingly, for G. intraradices Schenck and Smith WFVAM23 (referred to as G. versiforme [Karsten] Berch in (Gao et al. 2001), see (Gao et al. 2006)), full, functional mycorrhizal development (Myc+) has been shown to occur in rmc roots, although the rate of colonisation is much slower than in 76R roots (Gao et al. 2001; Manjarrez et al. 2008; Poulsen et al. 2005).

Recently, a meta-analysis was conducted on 22 published studies that have compared the 76R and rmc genotype pair in terms of growth and tissue nutrient concentrations (Watts-Williams and Cavagnaro 2014). Tissue P concentrations were generally higher (often significantly so) in the 76R genotype than the rmc genotype, in both root and shoot tissue, in soils with low and high P concentrations. A similar trend was recorded for tissue copper and sulphur concentrations, with concentrations in the 76R plants higher than those in the rmc plants. Furthermore, the meta-analysis confirmed that the colonisation phenotype displayed by the AM fungi had a significant influence on the extent to which roots were colonised. The results of the meta-analysis also highlighted that there was no substantial mycorrhizal growth response in the 76R genotype relative to the rmc genotype. It is important to note that, with respect to the criteria for assessing suitable mycorrhiza-defective mutant and wild-type pairs by Rillig et al. (2008), the 76R/rmc pair are matched in terms of growth in the absence of AM fungi in all studies (Cavagnaro et al. 2004a; Facelli et al. 2010; Poulsen et al. 2005) except one (Marschner and Timonen 2005).

The precise genome location of the Rmc locus has been identified and found to include a close match to the CYCLOPS/IPD3 gene (Larkan et al. 2013). This gene is essential for intracellular regulation of both rhizobial and mycorrhizal symbioses in legumes (Larkan et al. 2013). So far, nearly all cloned legume genes required for nodulation and AM colonisation have their putative orthologues in non-legume plants (Zhu et al. 2006). This is because the two symbioses share some signalling pathways (Zhu et al. 2006), suggesting that the more recent symbiosis between nodulating bacteria and plants may have evolved from the ancient symbiosis between AM fungi and plants (Doyle 1998; Parniske 2008; Wang et al. 2010). Further identification of the gene sequences associated with the Rmc locus will be useful information for past and future work using the rmc mutant (Larkan et al. 2013).

The 76R and rmc genotypes continue to be valuable for numerous studies focusing on different aspects of plant nutrition (Cavagnaro et al. 2007b, 2010; Poulsen et al. 2005; Watts-Williams and Cavagnaro 2012; Watts-Williams et al. 2013, 2014, 2015b), plant competition (Cavagnaro et al. 2004a; Facelli et al. 2010; Neumann and George 2005), mycorrhizal formation and colonisation phenotypes (Cavagnaro et al. 2004b; Gao et al. 2001; Manjarrez et al. 2008, 2009, 2010), soil ecology (Cavagnaro et al. 2006, 2007a, 2012; Hallett et al. 2009; Marschner and Timonen 2005), soil greenhouse gas emissions (Cavagnaro et al. 2008, 2012; Lazcano et al. 2014) and plant genetics (Barker et al. 2005; Gao et al. 2006; Larkan et al. 2007; Ruzicka et al. 2012, 2013).

Micro-Tom mutants

Micro-Tom, a model tomato genotype that has been used extensively in genetic studies because of its small size and rapid life cycle (Carvalho et al. 2011; Meissner et al. 1997), has also been used to create three mycorrhiza-defective mutant genotypes (David-Schwartz et al. 2001, 2003). The mutants M20 and M161 were obtained by fast neutron bombardment mutagenesis, while the BC1 mutant is an F2 segregant of the cross between wild-type and M161 genotypes. All mycorrhiza-defective mutant genotypes of the Micro-Tom variety are pre-mycorrhizal infection (pmi) mutants. Specifically, the M161 mutant displayed the Myc− phenotype and was unable to form mycorrhiza when grown in soil inoculated with G. intraradices spores (David-Schwartz et al. 2001). However, a low level of AM colonisation (vesicular and arbuscular) occurred when ‘whole’ inoculum (spores, root segments, external hyphae) was applied to the soil or when M161 was grown in a field soil (Rillig et al. 2008). When the M161 mutant was grown in the presence of its AM-colonised wild-type progenitor, arbuscules, vesicles and internal hyphae developed in roots at a rate similar to that of the wild-type. Similarly, the M20 mutant displayed the Myc− colonisation phenotype and was able to resist AM fungal colonisation in the presence of spores or (dead) pieces of mycorrhizal root, but was not resistant to colonisation in the presence of a live mycorrhizal wild-type progenitor plant (David-Schwartz et al. 2003). A third Micro-Tom mutant (BC1) has been identified, which is highly resistant to AM fungal colonisation when grown in field soil (1.2 % root length colonised) (Rillig et al. 2008). However, this genotype has not yet been tested for resistance to AM fungal colonisation when grown in the presence of the mycorrhizal wild-type plant.

That mycorrhiza-defective Micro-Tom mutants can be colonised when grown in the presence of the wild-type plant needs to be taken into consideration when using these mutant genotypes. In contrast to Micro-Tom mutant genotypes, the rmc mutant genotype cannot be colonised in the presence of its wild-type progenitor or other nurse plants (Cavagnaro et al. 2004a). Rillig et al. (2008) tested the Micro-Tom mutant genotypes for the selection criteria (see above) for mycorrhiza-defective mutant plants. They found that only the BC1 mutant met all the prescribed criteria while the other two mutants did not, for the following reasons: the M161 plants had a larger root biomass than wild-type when both were grown in the absence of AM fungi, and M20 gave rise to more soil microbial biomass than the wild-type. The Micro-Tom mutant M161 has so far been used in two published studies comparing mycorrhizal and non-mycorrhizal plants for root exudates involved in signal exchange between host plants and AM fungi (Gadkar et al. 2003; Sun et al. 2012). These mycorrhiza-defective mutant and wild-type Micro-Tom pairs could be of considerable utility in the study of mycorrhizal functioning.

Other non-legume mutants

In maize, mutator-mutagenised F2 families of the normal line W64A were screened by Paszkowski et al. (2006) for alteration in yellow root pigmentation compared to wild-type roots (see above for detail on screening). From this screen, the authors described seven mycorrhiza-defective mutants in maize and categorised them into three colonisation phenotype classes: nope1 (no perception 1) mutants, which showed a marked reduction in intra-radical colonisation by G. mosseae, but displayed occasional root sectors containing normal mycorrhizal structures (appressoria and arbuscules); taci1 (taciturn 1) mutants, which had lower colonisation levels than the wild-type genotype (45 % compared to 86 % root length colonised) and slightly modified fungal structures; and Pram1 (Precocious arbuscular mycorrhiza 1) mutants, which are in fact colonised more rapidly and intensely than the wild-type, becoming saturated with intra-radical fungal structures (arbuscules and vesicles) much earlier. These maize mutants represent the first mycorrhiza-defective mutant plants to be characterised in an agronomically important cereal crop. Future research using these mutants will be very useful for research into nutrient uptake in cereal crops, especially in field trials. However, to our knowledge, no such studies have yet been reported in the literature.

Reddy et al. (2007) used a transposon-mutagenised population of petunia (W138, Gerats et al. 1990) in order to identify and characterise a mycorrhiza-defective genotype in this line. The resulting mycorrhiza-defective petunia genotype, pam1 (penetration and arbuscule morphogenesis1), displayed a strong decrease in AM fungal colonisation compared to its wild-type progenitor. G. intraradices formed complex appressoria on pam1 roots but could not easily penetrate the epidermal cells. Where the fungus did penetrate epidermal cells, the resulting hyphae were distorted compared to those in the wild-type roots and did not progress further except in the rare instance where hyphae produced lateral branches between cells and small lateral appendages. In addition, two stabilised pam1 mutant lines (pam1S1 and pam1S2) were isolated and characterised. The two stabilised mutant lines displayed reduced extra- and intra-radical AM fungal colonisation compared to pam1, and thus the pam1S1 line was used for subsequent experiments (Reddy et al. 2007). As with the Micro-Tom mutants, the pam1S1 mutant could be colonised by AM fungi when grown in the presence of a nurse plant. However, intra-radical colonisation lacked arbuscule formation, and there was no contribution to the plant’s shoot P or Cu nutrition as a result of root colonisation.

In rice, a large number of mycorrhiza-defective mutant genotypes were characterised by Gutjahr et al. (2008), who were interested in identifying rice lines that were defective in one of a number of signalling steps in the common SYM pathway both upstream and downstream of Ca2+ spiking (see Parniske (2008) for recent review). The authors searched for relevant insertion lines in both T-DNA and Tos17 databases and found one insertion in CASTOR, three in POLLUX, two in CCMAK and three in CYCLOPS (IPD3, see Table 1). The nine sym mutants were then grown in soil inoculated with spores of G. intraradices and assessed for colonisation phenotype. Root colonisation in all the mutants was restricted to hyphal colonisation in the epidermal cells, with no cortical colonisation and thus no arbuscules or vesicles forming in any of the mutants.

A gene required for mycorrhizal colonisation in rice, OsDMI3 (does not make infections 3), has been identified. Chen et al. (2007) searched a rice Tos17 mutant database for OsDMI3 insertion lines, identifying two, but ultimately using just one (NF8513) for subsequent experiments. When grown in soil inoculated with G. intraradices, the OsDMI3 mutant roots showed occasional penetration of the cortical cells, without any arbuscule formation. However, most observed fungal growth (appressoria and external hyphae) was restricted to the root surface.

Similarly, Chen et al. (2008) characterised three knockout mutants defective in AM fungal colonisation (NC0263, NC2713, NC2794), by searching for putative Tos17 insertion lines available for OsIPD3, another gene required for mycorrhiza formation in rice. When inoculated with G. intraradices, the root epidermal cells of the three OsIPD3 mutants could not be penetrated, and there was no intra-radical colonisation of the roots by the AM fungi (i.e. no hyphae, arbuscules or vesicles) except in one root segment of a NC2713 mutant that displayed aborted intracellular fungal hyphae. There is no explanation given for this observation in NC2713, but it is assumed that the observed aborted hyphae did not confer functionality of the symbiosis. Chen et al. (2008) noted that the colonisation phenotype displayed by the OsIPD3 mutants was comparable to that of the previously identified OsDMI3 mutant genotype (Chen et al. 2007).

Future directions

Mycorrhiza-defective plant mutant genotypes have the potential to be used in a broad range of studies. Future uses of both legume and non-legume mutant genotypes may be extended to areas of study where mycorrhiza have previously been shown to improve plant or soil health, but hypotheses have not yet been tested using a mycorrhiza-defective mutant, such as soil nutrient cycling (Jeffries and Barea 1994; Read and Perez-Moreno 2003) and interactions with foliar-feeding insects (Gange and West 1994; Gehring and Whitham 1994; Wamberg et al. 2003) and foliar pathogens (Campos-Soriano et al. 2012; Nair et al. 2015; West 1997). Research that directly compares plant nutrient uptake via the direct (i.e. via root epidermal cells) and mycorrhizal pathways could utilise appropriate mycorrhiza-defective mutant and wild-type genotype pairs (Poulsen et al. 2005), in conjunction with the use of stable or radioactive isotopes (Merrild et al. 2013; Watts-Williams et al. 2015b). It would also be useful for future studies using mycorrhiza-defective mutant and wild-type pairs to continue to integrate molecular biology methods (e.g. quantification of gene expression) with more commonly reported physiological variables (e.g. plant nutrient concentration).

The intention of this review was to explore the potential to use mycorrhiza-defective mutant genotypes to study the formation and functioning of mycorrhiza in non-legumes. This approach has both strengths and limitations. Nevertheless, such mutant and wild-type genotype pairs are proving to be useful tools in the study of arbuscular mycorrhiza, and it is hoped that this review will stimulate and inform further research using this approach.