Colonization patterns in a mycorrhiza-defective mutant tomato vary with different arbuscular-mycorrhizal fungi


  • L-L. Gao,

    Corresponding author
    1. Department of Soil and Water and the Centre for Plant Root Symbioses, Adelaide University, Waite Campus, PMB 1, Glen Osmond, South Australia, 5064, Australia;
      Author for correspondence: L-L. Gao Tel: +61 8830 36529 Fax: +61 8830 36511
    Search for more papers by this author
  • G. Delp,

    1. Present address: Sveavägen 164 G21, 11346 Stockholm, Sweden
    Search for more papers by this author
  • S. E. Smith

    1. Department of Soil and Water and the Centre for Plant Root Symbioses, Adelaide University, Waite Campus, PMB 1, Glen Osmond, South Australia, 5064, Australia;
    Search for more papers by this author

Author for correspondence: L-L. Gao Tel: +61 8830 36529 Fax: +61 8830 36511


  •  Interactions between a mycorrhiza-defective tomato (Lycopersicon esculentum) mutant, rmc, and different species of arbuscular mycorrhizal (AM) fungi were investigated and compared with those with the wild-type cv. 76R.
  •  Both cv. 76R and rmc were challenged with Glomus intraradices, G. mosseae, G. coronatum, G. versiforme, G. etunicatum, G. fasciculatum, Gigaspora margarita and Scutellospora calospora using a nurse pot inoculation system.
  •  Cv. 76R demonstrated normal colonization patterns for all fungal species. By contrast, the development of different fungal species with rmc was impaired at different steps. Development of G. intraradices, G. etunicatum and G. fasciculatum was arrested on the root surface. However, G. mosseae, G. coronatum, G. margarita and S. calospora frequently penetrated the root epidermis, but colonization of the cortex was rare. G. versiforme achieved relatively normal colonization in rmc compared with the other species.
  •  This is the first report on the variation of colonization patterns in a mycorrhiza-defective mutant by different species of AM fungi, and highlights the need for previously described mutants in legumes to be challenged by more than one fungus.


Arbuscular mycorrhizas (AM) are probably the most widespread plant symbioses. They are formed by c. 80% of land plant species and fungal species belonging to the order Glomales (Zygomycotina) (Smith & Read, 1997). The symbiosis is of ancient origin, as shown by fossils in the Devonian Rhynie Chert (450 million yr BP) (Nicolson, 1975; Remy et al., 1994), the Ordovician of Wisconsin (460 million yr BP) (Redecker et al., 2000) and by molecular phylogeny of the fungi (Simon et al., 1993). Compared with the interactions between plants and other microorganisms, AM symbioses have extremely low specificity and high compatibility between plant and fungal symbionts, which may reflect both their mutualistic biotrophic nature and the very long period of coevolution. Studies on the mechanisms controlling the development of AM associations by both plant and fungal symbionts are significant for understanding the interactions between plants and microbes in general.

The development of AM colonization involves several well-defined stages. The phenotypic characteristics of each stage have been summarized by Smith (1995). The interactions between plant and fungus start before they come into physical contact. Hyphal growth and branching are induced by the roots of host plants, followed by the formation of appressoria which are thought to be the key event in recognition (Staples & Macko, 1980), leading to the hyphal penetration of the roots (Giovannetti et al., 1994; Smith & Read, 1997). By contrast, in nonhost plants, the signals inducing hyphal growth and branching, and recognition at the root surface, are apparently switched off (Giovannetti & Sbrana, 1998). Following the penetration of host root epidermal cells, hyphae grow inter- or intracellularly in the root cortex and intracellular hyphae form arbuscules. Exchange of P and carbohydrate between plant cells and fungal hyphae occurs across these plant–fungus interfaces (Smith & Read, 1997).

Plant mutants in which the formation of AM structures is impaired have been identified in both legume and nonlegume plants and indicate the existence of plant genes controlling AM development. Among these mutants, three main phenotypes have been characterized in legumes. Those referred to as myc−1 are most frequently found among nodfix mutants. In this phenotype, fungal growth is blocked on the root surface following formation of appressoria (Duc et al., 1989; Bradbury et al., 1991; Gianinazzi-Pearson et al., 1991; Bradbury et al., 1993; Balaji et al., 1994; Sagan et al., 1995; Shirtliffe & Vessey, 1996; Senoo et al., 2000). Mutants with fungal development arrested in the epidermal cells have been identified in Lotus japonicus (Wegel et al., 1998; Senoo et al., 2000). Another phenotype, designated as myc−2, has been identified among Nod+fix mutants in Pisum sativum (Gianinazzi-Pearson et al., 1991), Medicago truncatula (Sagan et al., 1995) and M. sativa (Bradbury et al., 1993). In this phenotype, fungi can penetrate the roots and colonize cortical cells, but arbuscular development is reduced to a few stumpy branches.

These different phenotypes of mycorrhiza mutants were thought to be entirely controlled by the altered genes in the plants (Gianinazzi-Pearson et al., 1991; Wegel et al., 1998). The influence of the identity of AM fungi on the phenotypes has not been reported. Gianinazzi-Pearson et al. (1996) suggested that no isolate of microsymbiont (either mycorrhizal fungus or Rhizobium) had been found to infect myc−1 pea mutants. However, in most cases only one or two fungal species have been used to assess the phenotypes of each mutant. One exception is the M. sativa which were challenged by G. versiforme, G. intraradices, G. monosporum, G. fasciculatum and Gi. margarita. The phenotypes differed only in the number and the morphology of appressoria, and not in the extent to which root tissues could be penetrated by the fungi (Bradbury et al., 1991; Bradbury et al., 1993).

A mycorrhiza-defective mutant (rmc) has been identified in a nonlegume plant species, Lycopersicon esculentum (Barker et al., 1998). The phenotype was initially tested against G. mosseae (also used to test the Pisum mutants) and found to be similar to myc−1. Some differences in the development of G. mosseae, G. intraradices and Gi. margarita were observed (Barker et al., 1998), which suggested that the interactions with rmc might vary with fungal genotype. In order to confirm the phenotype of this mutant and to extend the study to include a wider range of AM fungal genotypes, we challenged the mutant rmc with eight species of AM fungi. These were G. intraradices, G. mosseae, G. coronatum, G. versiforme, G. etunicatum, G. fasciculatum, Gi. margarita and S. calospora. Except for G. coronatum and S. calospora, the species were chosen from among those used to characterize phenotypic features of other mycorrhiza-defective mutant plants. The results showed differential colonization patterns of species of AMF on rmc, highlighting diversity in the interactions, influenced by the genotypes of both plant and fungal partners.

Materials and Methods

Plants and fungi

A mycorrhiza-defective tomato (Lycopersicon esculentum Mill.) mutant, rmc (Barker et al., 1998), was studied for its interactions with different species of AM fungi (see below). Wild-type tomato cv. 76R (the near isogenic line of rmc; Peto Seed Company, CA, USA) was included as a control genotype.

The species of AM fungi included in the experiments were Glomus intraradices Schenck and Smith (DAOM 181602) subcultured from an axenic culture on transformed roots obtained from Professor J. A. Fortin, University of Montreal, Canada; G. mosseae (Nicholson & Gerdemann) Gerdemann and Trappe (NBR4-1), obtained from Dr P. McGee, University of Sydney, NSW, Australia; G. coronatum Giovannetti (WUM16, formerly known as G. ‘City Beach’), obtained from Associate Prof. L. K. Abbott, University of Western Australia, WA, Australia; G. versiforme (Karsten) Berch, obtained from Professor Paola Bonfante, Centro Di Studio Sulla Micologia Del Terrena, Torino, Italy, prior to the establishment of the BEG collection; G. etunicatum Becker and Gerdemann (UT 316 A-2), obtained from Dr Joe Morton, INVAM, University of West Virginia, USA; G. fasciculatum (Thaxter) Gerd. & Trappe emend. Walker & Koske (LPA7), obtained from the Turin Botanic Garden, Italy; Gigaspora margarita Becker and Hall, obtained from Dr V. Gianinazzi-Pearson, INRA, Dijon, France, before the establishment of the BEG collection; and Scutellospora calospora (Nicolson & Gerdemann) Walker & Sanders (WUM 12(2)), obtained from Mr Chris Gazey, University of Western Australia, WA, Australia. All the inocula were grown as pot cultures on Trifolium subterraneum L. cv. Mt Barker and used to prepare nurse pots for the experiments (see below).

Experimental design

A preliminary experiment was carried out using all eight species of AM fungi. Each combination of plant genotype (rmc or 76R) and fungal species was set up with one pot containing three plants. This design therefore had three pseudo-replicated plants per treatment. The experiment was repeated with six fungi (G. etunicatum and G. fasciculatum were omitted) and full replication, as follows. For each fungal species, there were three pots each containing four plants of rmc or 76R. To avoid cross contamination in the growth room or glasshouse, the pots were grouped into blocks according to fungal species and each species was placed on a separated bench (approx. 50 cm between benches). Pots were watered carefully to prevent spores or fungal hypha from ‘jumping’ between pots. Blocks and pots in each block were randomized twice a week.

Inoculation and plant growth

The ‘Nurse pot’ inoculation system (Rosewarne et al., 1997) with minor modifications was used in the experiment in order to produce synchronous and rapid mycorrhizal colonization and to avoid potential problems caused by the differences in inoculum potential between fungal species. Specifically, leek (Allium porrum L. cv. Vertina) plants were grown for 8 wk in pots with inoculum of each species of AM fungus to establish a fungal network in the growth medium for inoculation of tomato plants.

Nurse pots, with free drainage, contained 700 g of a mixture of sterilized sand (3 parts coarse sand and 1 part fine sand) and soil (9 : 1 w/w). For the species of Glomus, the soil came from Mallala, South Australia (pH 7.1) and for Gi. margarita and S. calospora from Kuitpo, South Australia (pH 5.0) (Dickson et al., 1999). Pot culture inoculum (10% by weight) of each fungus including soil, hyphae, spores and colonized root pieces was incorporated into the sterile mix. Calcium hydrogen orthophosphate (CaHPO4) was also incorporated at the rate of 0.025 g per kg to provide 2.5 ppm bicarbonate extractable P (Colwell, 1963). This very low P supply improves the growth of plants in the low nutrient sand and soil mix and does not reduce percentage colonization of 76R by any of the fungi used, or of rmc by G. versiforme (L-L Gao, unpublished).

Ten leek seeds were planted into each pot and thinned to five plants after 2 wk. After 8 wk growth and just before the transplantation of the tomato seedlings, the shoots of four remaining leek plants were cut off to minimize the competition for nutrients from the growth medium between leek and tomato plants.

Tomato plants for transplantation into the nurse pots were produced as follows: seeds of rmc or 76R were sterilized with 4% sodium hypochlorite for 15 min and rinsed in reverse osmosis (RO) water before germination by incubation at 25°C for 3 d on moist filter paper. Tomato seedlings were transplanted into compartments of a seedling tray, each containing c. 50 g of sterilized sand (3 parts of coarse and 1 part of fine sand) mixed with 0.025 g CaHPO4 per kg. After 16 d growth these plants were transplanted into the 8-wk-old nurse pots.

Plants (both nurse pots and seedling trays) were watered with 50–120 ml per pot or seedling tray compartment (depending on the size of the plants) of half strength modified Long Ashton solution (Cavagnaro et al., 2001a) minus P every day for the seedling trays and three times per week for the nurse pots. The preliminary experiment was carried out in a glasshouse with ambient light and average temperature c. 24°C. The main experiment was undertaken in a growth chamber with a 14-h photoperiod (500 µmol m−2 s−1 photon flux density) and temperatures of 18 and 25°C in the dark/light phases, respectively.

Evaluation of fungal structures and colonization

The tomato plants were harvested 6 wk after transplanting to the nurse pots. After thorough washing with water, root samples (c. 300 mg per pot) were cleared in 10% KOH for 7 d at room temperature and stained with trypan blue for 15 min at 80°C (a modification of the method of Phillips & Hayman (1970), omitting phenol from the solutions). For assessment of mycorrhizal colonization, the method of McGonigle et al. (1990) was used. Two slides were made from the root samples of each plant (approximately 20, 1 cm long, root pieces). Approximately 100 intersections between roots and an eyepiece crosshair arranged perpendicular to the root axis were observed. At each intersection between the root and the crosshair, the incidences of external hyphae, appressoria, hyphae aborted in epidermal cells without further mycorrhizal structures in the intersections, hyphae in the cortex alone, arbuscules, vesicles, colonization of cortex and colonization of any root cells were recorded and percentage incidence of each structure over total intersections (colonized or not) was calculated. Total percent colonization was based on the presence of any colonized cells, whether the morphology was typical of the pattern in wild-type cells or not. A bright field microscope (Olympus IX70, Olympus Optical Co., Ltd, Tokyo, Japan) was used to quantify the colonization at × 200 magnification and photograph fungal morphology in root squashes. Data were analysed statistically using two-way ANOVA, Genstat 5, release 4.1, 4th Edition (1998), Lawes Agricultural Trust (IACR Rothamsted).

Laser scanning confocal microscopy (LSCM)

Laser scanning confocal microscopy (LSCM) was used in order to confirm the colonization patterns observed by the bright field microscope and the cell layer involved in fungal interactions. The details of LSCM have been described by Barker et al. (1998). Briefly, after washing the roots, samples were taken and treated in one of the following ways: root pieces were stained with 1% acid fuchsin for 30 min or segments of root were embedded in 15% gelatin blocks containing 2% glycerol, frozen on a freezing stage (Zeiss, Carlzeiss, Oberkochen, Germany) and sectioned (120 µm) in the longitudinal plane using a Leitz freezing microtome (Ernst Leitz, Wetzlar GmbH, Wetzlar, Germany). Sections were stained with 1% acid fuchsin overnight. After staining, root pieces or sections were mounted in lactoglycerol and examined under a dissecting microscope. Those that showed mycorrhizal colonization were mounted on slides and the coverslips sealed with nail polish. Images were visualized using a BioRad MRC 1000 Laser Scanning Confocal Microscope (BioRad Microscopy Division, Hemel Hampstead, UK) system combined with a Nikon Diaphot 300 inverted microscope Nikon, (Tokyo, Japan) with fluorescence optics using 488/10 nm excitation and 522/32 emission wavelengths and × 40 water immersion lens NA 1.15. Images were captured as computer files and analysed with Comos Image analysis software (BioRad) and Confocal Assistant Version 4.02 (Todd Clarke Brelje, free software from website: The data were either constructed as 3D images or presented as montage of simple confocal pictures composed of a varying number of optical section in the z axis.


Results of the preliminary glasshouse experiment were very similar to the main, replicated growth-room experiment. Colonization patterns in both 76R and rmc were identical in both experiments and percent colonization was very similar, with the exception of G. coronatum, which showed much higher colonization in the glasshouse (c. 50%, compared with c. 20%). In the preliminary experiment colonization patterns of G. etunicatum and G. fasciculatum were the same as G. intraradices and these fungi were omitted from the main experiment. The numerical results of the main experiment (G. intraradices only) are presented, but some additional observations on these two fungi from the preliminary experiment are included.

Mycorrhizal colonization in wild-type tomato 76R

All species of AM fungi formed normal colonization in the wild-type L. esculentum 76R with significant differences between the fungi in total colonization, which ranged from 20.1 to 96% and arbuscular colonization from 19.7 to 82.7% (Table 1). Vesicles were observed in all roots colonized by Glomus species, but were so sparse in those colonized by G. coronatum in the main experiment that they were not detectable by the scoring method used (results not shown). Typical colonization by G. intraradices in 76R is shown in Fig. 1a, including external hyphae, appressoria, arbuscules and vesicles.

Table 1.  Characteristics of mycorrhizal colonization in Lycopersicon esculentum wild-type 76R and mutant rmc by six species of AM fungi after 6 wk inoculation
Fungal speciesInternal colonization (%)External colonization (%)
TotalEpidermal onlyCorticalArbuscular1HyphalAppressorial
  • 1

    Arbuscular colonization includes both arbuscular coils for colonization by Glomus coronatum, Gigaspora margarita and Scutellospora calospora and arbuscules for Glomus intraradices, Glomus mosseae and Glomus versiforme.

  • 2

    Means (n = 3) followed by the same letter(s) are not significantly different (P < 0.05) between each other using LSD test: A, B, C, D for comparison of means of 76R and a, b, c, d for rmc with different species of AM fungi; X, Y for comparison between 76R and rmc with each species of fungus. -

  • 3

    3 , Indicates absence of the structure.

  • 4

    ns, not significant at P < 0.05.

G. intraradices93.0DX2 8.7aY0.0AX 0.0aX93.0CDX 8.7aY82.7CX 4.3aY 2.3AX 6.3abY0.0AX0.7abX
G. mosseae66.3BX 4.3aY0.0AX 1.7aX66.3BX 2.7aY58.3BX 0.7aY 4.7AX11.0bY0.7AX2.7bX
G. coronatum20.7AX 3.7aY0.0AX 0.3aX20.7AX 3.3aY19.7AX 2.7aY 1.0AX 2.7aX0.0AX0.3aX
Gi. margarita68.0BCX28.3bY0.0AX27.0bY68.0BX 2.0aY68.0 BCX 1.0aY 5.0AX18.3cY3
S. calospora82.0CDX30.0bY0.3AX23.0bY81.7CX 7.0aY78.0CX 0.0aY14.7BX30.0dY
G. versiforme96.0DX76.0cY0.0AX 0.7aX96.0DX75.3bY71.3 BCX38.7bY 6.0AX19.3cY1.7AX7.0cY
Plant genotype< 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 
Fungal species< 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.05 
Interaction< 0.001 < 0.001 < 0.001 < 0.001 ns4 < 0.05 
Figure 1.

A typical mycorrhizal colonization in Lycopersicon esculentum wild-type 76R (a) and deficient colonizations in mutant rmc (b–f) with various colonization patterns by three Glomus species. (a) 76R colonized by Glomus intraradices, showing external hyphae (eh), appressoria (ap), intercellular hyphae (ih), arbuscules (ar) and vesicles (v). (b) rmc showing colonization by G. intraradices aborted at the point of penetration (pa). (c, d) rmc showing that external hyphae (eh) of G. mosseae penetrated (p) the root epidermis and aborted in the outer cortical cells (cc) accompanied by hyphal swelling (*) and branching (hb). (e, f) rmc showing that G. coronatum formed swellings on the root surface (*) and penetrated the root epidermal cells. Fungal hyphae aborted (ah) or confined within a single epidermal cell. Scale bar, 50 µm.

The fungal hyphae of Glomus species often formed swellings (appressoria) on the root surface. This step was followed by penetration of the root epidermis. Gi. margarita and S. calospora did not form obvious appressorial structures, but appeared to penetrate the roots directly. Following penetration and colonization of the epidermal cells of 76R, fungal hyphae of all species rapidly colonized the root cortex. The cortical colonization therefore accounted for most of the total root colonization (Table 1). The morphology of the cortical colonization in 76R fell into two patterns, depending on the fungal species used. G. intraradices, G. mosseae, G. versiforme, G. fasciculatum and G. etunicatum formed intercellular hyphae which subtended intracellular arbuscules. By contrast, G. coronatum, Gi. margarita and S. calospora grew directly from cell to cell and formed well-defined intracellular hyphal coils. Details of these differences are presented in Cavagnaro et al. (2001b).

Reduced mycorrhizal colonization in mutant rmc

Mycorrhizal colonization, assessed as colonization of any root cell layer, was significantly reduced (P < 0.05) in roots of the mutant rmc with all species of fungi, however the percentage root length associated with external hyphae increased (Table 1). As shown in Table 1, this effect was particularly marked for G. intraradices (and G. fasciculatum and G. etunicatum in the preliminary experiment), G. mosseae and G. coronatum. Colonization by Gi. margarita and S. calospora in rmc was somewhat higher, with up to 30% of the root length containing some colonized cells. However, although colonization by G. versiforme was significantly reduced in rmc compared with 76R, it was still much higher than the other species of fungi. These overall trends in the extent of colonization were accompanied by clear differences in the details of colonization pattern and fungal morphological characteristics. The fungi fell into three groups on the basis of the interactions with root cells, and details are described below (see also Tables 1 and 2 and Figs 1, 2, 3 and 4).

Table 2.  Summary of mycorrhizal phenotypes formed by eight species of AM fungi with mutant rmc tomato, Lycopersicon esculentum Mill.
FungusAppressoriaEpidermal colonizationArbusculesVesiclesPhenotype
  1. a Relative ‘intensity’ of development of structures: ++, always present in significant numbers; + always present; (+) very rare, only present after cortical colonization has occurred; –, absent. b Pen: normal external colonization and aborted penetration. cCort: fungal hyphae penetrated root epidermis and failed to colonize the cortex. dMyc+: relatively normal mycorrhizal colonization, but less colonized than wild-type 76R.

Glomus intraradicesa+(+)(+) 
Glomus fasciculatum+(+)(+)bPen
Glomus etunicatum+(+)(+) 
Glomus mosseae+Intracellular, swollen hyphae(+)(+) 
Glomus coronatum+Intracellular, swollen hyphae(+)(+) 
 atypicalaborted  cCort
Gigaspora margaritaIntercellular, aborted(+)_ 
Scutellospora calosporaIntercellular and intracellular abortions(+)_ 
Glomus versiforme+ (complex)Intracellular, swollen++++dMyc+
  some abortions   
Figure 2.

Bright field microscopy (a) and laser scanning confocal microscopy (LSCM) (b–f) of longitudinal root squashes showing penetration and abortion of Gigaspora margarita in rmc. (a) Bright field microscopy and (b) extended focus image of 10 optical sections 2 µm apart in the z axis, showing that hyphae formed many cross walls (cw) and aborted in epidermal cells (ec) accompanied by hyphal swelling (*). (c–f) Montage of four optical sections 6 µm apart in the z axis starting at the surface of the root. Scale bar, 50 µm. External hyphae (eh); epidermal cells (ec); fungal hyphae aborted (ah); hyphal branching (hb); penetrated (p).

Figure 3.

Bright field microscopy (a, c) and laser scanning confocal microscopy (LSCM) (b, d) of longitudinal squashes of roots of rmc. (a, b) Hyphae of Scutellodpora calospora penetrated root epidermal cells, accompanied by swelling (*) and branching. The hyphae formed many cross walls (cw) and aborted in epidermal or outer cortical cells. (c, d) Colonization pattern of Glomus versiforme at the surface and outer cell layers of the roots. Some hyphae branched on the root surface and penetrated root epidermal and cortical cells, where they swelled (*), branched and frequently aborted. Scale bar, 50 µm. External hyphae (eh); fungal hyphae aborted (ah); hyphal branching (hb); penetrated (p).

Figure 4.

Comparison of colonization patterns in Lycopersicon esculentum wild-type 76R (a, c, e) and mutant rmc (b, d, f) by Glomus versiforme. Bright field microscopy of longitudinal squashes of roots (a, b, c, d) and laser scanning confocal microscopy (LSCM) (e, f) of root sections (120 µm). (a) 76R and (b) rmc, overview of the colonization patterns formed by G. versiforme (c) 76R, direct entry of G. versiforme into the roots (d) rmc, abnormal colonization in the outer cortical layers, showing hyphal swelling (*) or abortion at the site of penetration, and the presence of swollen, branched hyphae in the epidermal or outer cortical cells (e) 76R, and (f) rmc, arbuscules formed by G. versiforme in the cortical cells. Scale bar, 50 µm. External hyphae (eh); epidermal cells (ec); fungal hyphae aborted (ah); hyphal branching (hb); penetrated (p); appressoria (ap); intercellular hyphae (ih); arbuscules (ar); point of penetration (pa); and vesicles (v).

Colonization of rmc by G. intraradices, G. fasciculatum and G. etunicatum

Results of the preliminary experiment showed that the development of these three species of AM fungi in rmc was arrested on the root surface, following relatively normal growth of external hyphae (results not shown). G. intraradices grew along the root surface with few attempts at entry (Fig. 1b). The size and shape of the appressoria and hyphal structures formed at the attempted entry points did not differ from those formed with the wild-type 76R (compare Fig. 1a,b). Normal arbuscules and vesicles were observed, but were very rare (Tables 1 and 2).

Colonization of rmc by G. mosseae, G. coronatum, Gi. margarita and S. calospora

This species group of fungi was able to penetrate the root epidermis. Following the penetration, fungal hyphae formed swollen structures with irregular branches that were often confined within one or a few adjacent epidermal or outer cortical cells (Figs 1c–f; 2; 3a,b). Fungal hyphae rarely succeeded in colonizing the root cortex and when this occurred arbuscules and vesicles were formed (Tables 1 and 2). However, the frequency of penetration and hyphal morphology differed significantly between species. G. mosseae and G. coronatum penetrated the epidermal cells of the root, but at a low frequency (Table 1). Fungal swelling at the penetration of rmc was often observed with these two species (Fig. 1c–f).

Both Gi. margarita and S. calospora frequently penetrated the root epidermis (Table 1, Figs 2a,b; 3a,b). This was confirmed by LSCM of root pieces stained with acid fuchsin (Figs 2b–f; 3b). The colonization was often preceded by aborted attempts at entry of epidermal cells and/or outer cortical cells, accompanied by formation of cross walls (Figs 2a; 3a). If hyphae of Gi. margarita successfully penetrated the cells they branched and soon aborted (Fig. 2). Hyphae of S. calospora penetrated epidermal cells more frequently, some of which aborted soon after penetration but others extended into one or a few cells accompanied by numerous fine hyphal branches (Fig. 3a,b). This colonization of the epidermis accounted for most of the total colonization of rmc by these two fungi (c. 30%, see Table 1).

Colonization of rmc by G. versiforme

Once the surface layers of the root had been penetrated, G. versiforme was able to form a much more extensive and apparently normal colonization pattern with rmc than the other fungal species (Table 1; Fig. 4b,d,f). As shown in Table 1, the total colonization of rmc by G. versiforme was even higher than that achieved by G. mosseae, G. coronatum or Gi. margarita in 76R. The most obvious difference was in the surface and epidermal interactions with the root. Once G. versiforme came into contact with 76R, it penetrated epidermal cells rapidly forming normal appressoria and further colonized the cortical cells (Fig. 4a,c,e). In rmc, in contrast, this fungus formed extensive branches and swellings of external hyphae that often penetrated epidermal cells (Fig. 3c,d; Fig. 4b,d). Some of these hyphae aborted in root epidermal cells or outer cortical cells, but others frequently succeeded in colonizing the cortex and thereafter a normal colonization pattern was established, including the formation of intercellular hyphae, arbuscules and vesicles (Fig. 4b,f). These internal structures in rmc appeared normal, and very similar to those in 76R (compare Fig. 4a,b,e,f). Although the percentage of the total root length containing arbuscules was lower in rmc than in 76R (Table 1), arbuscule and vesicle densities per unit length of colonized root in rmc were 51.6% and 45.2%, respectively, and not significantly different from the values of 74.1% and 34.6% observed in 76R (P < 0.05).


Colonization of wild-type 76R by different species of AM fungi

All fungi used in this investigation formed normal mycorrhizas in the roots of wild-type tomato 76R, with the formation of arbuscules and (for Glomus spp.) vesicles. Percent root length colonized was generally high (c. 70% or above), with the exception of G. coronatum, which colonized poorly in the main experiment. This was probably due to poor establishment of the mycorrhizal network, as the leek ‘nurse plants’ were also poorly colonized. The low colonization does not devalue the data on interactions between rmc and G. coronatum and all conclusions from the growth room experiment are supported by data from the preliminary experiment in which colonization was much higher (c. 50%). CaHPO4 was supplied in both preliminary and main experiments so elevated P is not therefore the explanation for reduced colonization in the main experiment.

Again depending on the identity of the fungus, colonization of 76R fell clearly into either Arum- or Paris-types (Gallaud, 1904). This surprising finding will not be discussed further here, but is the subject of the second paper (Cavagnaro et al. 2001b).

Resistance of rmc to AM fungi

The extent of colonization of the roots of rmc by different species of AM fungi varied, confirming the preliminary results of Barker et al. (1998). Such variation has not been observed in investigations of other mycorrhiza-deficient mutants, possibly because few have challenged the mutants with more than one or a few fungi. The colonization of rmc by most of the fungal species tested was aborted either on the root surface, within the epidermal cells or outer cortical cell (see Table 2), again confirming the previous characterization of the mutant when challenged with G. mosseae and G. intraradices (Barker et al., 1998). Even G. versiforme, which achieved relatively high colonization in rmc, showed evidence of altered growth patterns in these outer root cell layers. This general response is very similar to the myc−1 type mutations identified in several species of legumes (Gianinazzi-Pearson et al., 1994; Gianinazzi-Pearson, 1996; Wegel et al., 1998; Ruiz-Lozano et al., 1999; Bonfante et al., 2000; Senoo et al., 2000) suggests that a gene involved in the ‘acceptance’ or ‘susceptibility’ of the root to colonization by AM fungi has been mutated in rmc and the other genotypes, resulting in ‘exclusion’ of or ‘resistance’ to a broad range of species of AM fungi. The site of action of this gene is presumably the epidermis and/or outer cortical cells of the root and this is similar to the Lotus japonicus mutants (Bonfante et al., 2000; Senoo et al., 2000). The similarity of the phenotypes in rmc and myc−1 mutants does not necessarily mean that a common gene has been mutated, although it does appear that the genes may involve steps in the same process of acceptance in the outer cell layers.

In our experiments, if any fungus was successful in penetrating beyond the outer cell layers, normal colonization of the root cortex occurred, including formation of arbuscules and sometimes vesicles (see Table 2). Structurally normal colonization of the root cortex, although restricted in extent, is a common feature of several legume mutants, particularly the Lotus japonicus mutants (Wegel et al., 1998; Bonfante et al., 2000; Senoo et al., 2000), although Ljsym4–2 completely excludes both G. intraradices and Gi. margarita (Bonfante et al., 2000). In rmc successful cortical colonization was particularly extensive for G. versiforme, which appears to have a high ability to overcome the ‘resistance’ to colonization in rmc, and also suggests the existence of a degree of specificity in interactions between plants and AM fungi.

The mechanism of the ‘resistance’ is not clear. All mutants so far isolated are recessive, which does not immediately appear consistent with increased transcript accumulation of defence-related genes (Ruiz-Lozano et al., 1999) or with the increased deposition of phenolics and β(1–3) glucans observed in the interaction between G. mosseae and P. sativum myc−1 (Gollotte et al., 1993). A similar response, evidenced by autofluorescence in epidermal cells of rmc in contact with G. mosseae, G. coronatum and S. calospora, has also been observed (L-L Gao, unpublished). Gollotte et al. (1993) suggested that the mutant might lack a receptor that normally exerts control on defence pathways and enables tissue penetration by the mycorrhizal fungi. In any event, the responses are specific for AM fungi (see below). Furthermore, neither rmc (L-L Gao, unpublished) nor the mutation in P. sativum (Gianinazzi-Pearson et al., 1994) affects the interaction with root-infecting plant pathogens, such as Rhizoctonia spp. or Meloidogyne spp., Agrobacterium tumefaciens and Aphanomyces euteiches in nonmycorrhizal mutants of P. sativum as well (Gianinazzi-Pearson et al., 1994; Ruiz-Lozano et al., 1999).

The details of structures formed by the species of AM fungi with rmc were different, resulting in different colonization phenotypes, defined in terms of the framework suggested by Smith (1995) and shown in Table 2. The variations highlight the diversity of the fungal interactions with a single plant genotype and suggest that the colonization of different cell types by AM fungi may not necessarily be under control only of specific plant genes, as proposed by Wegel et al. (1998). This is an important discovery. Fungal responses to rmc did not involve formation of multiple or deformed appressoria on the surface of the root, but rather branched or swollen structures at various steps of colonization. As other plant mutants have not been systematically or quantitatively assessed for their effects on the development of different fungal species, it is difficult to compare them with rmc. There are general similarities with mutants of the myc−1 type and others in various plant/fungus combinations. For example, the response of G. mosseae and G. coronatum to rmc in forming deformed intracellular swellings is very similar to the response of G. intraradices and Gi. margarita to the Lotus japonicus mutants Ljsym2, 3 and 4 (Wegel et al., 1998; Bonfante et al., 2000). In general terms the formation of deformed structures during colonization seems to be a response to failure of tissue colonization, as suggested by Bradbury et al. (1993) and can be akin to the altered morphogenesis of G. mosseae when colonization of normal host plants is physically prevented (Giovannetti et al., 1993). Despite these similarities, in the few cases where the same fungi have been used to challenge different mutants, they do not necessarily respond in the same way. For example, G. intraradices is completely excluded by rmc, but penetrates the epidermal cells of the L. japonicus mutants (Wegel et al., 1998) and G. versiforme colonizes rmc extensively but cannot colonize M. sativa mutants (Bradbury et al., 1991). These findings imply that despite having the same site of action and other potential similarities, the operation of the genes may be different, giving rise to the apparent differences in specificity.

Bonfante et al. (2000) suggested that the difference between Ljsym4–2 and Ljsym4–1, with respect to the ability of G. mosseae and Gi. margarita to colonize the cortex might be related to retention of some gene function in Lysym4–1, which allows occasional successful penetration through the epidermal cells. It is possible that in the case of rmc, partial gene function is also retained, and the apparent specificity of the fungi in colonizing the mutant is because they have different sensitivity to the reduced amount of RMC protein synthesized.

Further work is required to determine if the mycorrhizas formed by any of the fungi, and particularly G. versiforme, on rmc are functional in terms of nutrient exchange between the partners. It could be suggested that successful growth and colonization of G. versiforme on rmc must be supported by organic carbon from the leek nurse plants. However, G. versiforme also colonizes rmc following conventional inoculation with spores and colonized root pieces (L-L Gao, unpublished), providing strong evidence of carbon transfer from rmc. Similar indications are available for the myc−2 of P. sativum, which colonizes the cortex intercellularly, but does not form arbuscules (Smith & Read, 1997). At this stage we have no evidence relating to involvement of G. versiforme in mineral uptake by rmc.

We conclude that rmc is altered in a gene controlling the early stages of colonization by AM fungi, as the development of AM fungi is normally blocked in the stage of penetration or colonization of cortex. Individual fungi respond to the mycorrhiza-resistant mutant by colonizing certain cell layers. This implies that mutant plants exert resistance through multiple cell types and/or that the fungi vary in their abilities to overcome the ‘resistance’ operating in each type. Such a cell–specific interaction has also been suggested by Bonfante et al. (2000). Future work comparing the way in which different fungi colonize rmc with respect to cell wall modifications and other defence responses in the cells in which colonization is blocked should shed light on how these interactions operate.

From a practical standpoint, the variations in colonization pattern between the different fungi, and in particular the extensive colonization by G. versiforme, on rmc highlight the difficulties of defining the interactions of different fungi with individual host genotypes. It now appears that definition of a mutant phenotype must include several species of AM fungi and cannot rely on the interaction between a single pair of symbionts, as suggested by Wegel et al. (1998). The same variation between fungi may have implications for the outcome of interactions between wild-type plant and fungi at molecular, physiological and ecological levels.


This work was financially supported by the Australian Research Council. L-L Gao thanks Adelaide University, Australia for an International Postgraduate Research Scholarship and Shanxi Agricultural University, P. R. China for study leave. The authors also thank F. Andrew Smith for critical reading of the manuscript, Debbie Miller for technical help, Peter Kolesik and Sandy Dickson for help with confocal microscopy and Tatsu Ezawa, Yongguan Zhu and Tim Cavagnaro for useful discussions. We are all grateful to referees of this paper for valuable suggestions. These data was presented in a preliminary form at the Third International Conference on Symbiosis, Marburg, Germany, August 2000 and we thank participants for their helpful comments.