- Top of page
- Materials and Methods
The association between plants and fungi of the Glomeromycota is one of the most widespread mutualistic symbioses between plants and soil microorganisms. Arbuscular mycorrhiza (AM) fungal colonization assists the plant in mineral element uptake (George, 2000) because of the spread of nutrient-absorbing mycelium in the soil (Li et al., 1991; Neumann & George, 2004).
The colonization of plant roots by AM fungi starts with the formation of appressoria on the epidermal cell wall, from which AM fungal hyphae penetrate through the epidermis to spread inter- and intracellularly within the root cortex. The intimacy of the association between plant and AM fungi implies selective recognition processes enabling the plant to distinguish the symbiotic fungus from harmful microorganisms (Gianinazzi-Pearson, 1996). Numerous studies indicate that plant defence mechanisms are induced in response to the invasion of AM fungi (Blee & Anderson, 1996; Salzer et al., 1999; Bonanomi et al., 2001). However, these responses appear to occur mainly during the initial stages of root colonization, and appear to be locally restricted and transient compared with defence responses to plant pathogens (García-Garrido & Ocampo, 2002). It is therefore assumed that plant recognition of AM fungi leads to the activation of an ‘AM fungus accommodation programme’, which includes the suppression of defence mechanisms against the invading symbiont (Gianinazzi-Pearson & Dénarié, 1997).
Several symbiosis-defective plant mutants have been identified, in which AM fungal root colonization is restricted to early events such as appressoria formation and penetration of epidermal cells (Myc−1 phenotype), or which allow only restricted cortex colonization or abnormal arbuscule formation (Myc−2 phenotype; Marsh & Schultze, 2001). These mutants (which lack the ability to host AM fungi at different stages of AM development) have been used as a tool to investigate mechanisms involved in plant accommodation to AM fungi in many molecular and cytochemical studies (Marsh & Schultze, 2001).
The Lycopersicon esculentum Mill. mutant rmc (Barker et al., 1998), which was used in the present study, was found to restrict colonization by Glomus intraradices, Glomus mosseae and various other AM fungi to the formation of appressoria, abortive penetrations of epidermal cells and extraradical hyphae (surface colonization, Myc−1), but allows extensive cortical root colonization by Glomus sp. WFVAM23 (formerly called Glomus versiforme; Gao et al., 2001).
The Myc−1 phenotype is considerably different from naturally occurring ‘nonhosts’, of which the majority do not promote appressoria formation or development of AM fungal hyphae associated with the root surface (Giovannetti & Sbrana, 1998). As arbuscules and intraradical hyphae are known to be necessary for the carbohydrate supply to AM fungi (Bago & Bécard, 2002), it can be assumed that surface colonization in the mutants is established from ‘external’ carbohydrate sources, which could be either fungal storage lipids or carbohydrates provided by the connection to a host plant which allows normal AM fungal root colonization. Appressoria formation is the first stage of physical contact between AM fungi and the root in the formation of AM. It appears likely that the infective potential of an AM fungal inoculum, and the amount of appressoria which can be formed, are dependent on the fungal carbohydrate resources. We therefore hypothesized that AM fungal access to additional carbohydrate sources from a viable host plant will lead to increased AM fungal inoculum potential on the rmc roots.
In this study we investigated the influence of the presence of roots of the tomato cultivar Golden Queen (GQ), allowing normal development of cortical AM fungal structures, on AM fungal surface colonization of rmc roots. Furthermore, it was investigated whether AM fungal inoculation of rmc plants, in the presence or absence of GQ roots, leads to differences in growth or nutrient acquisition compared with noninoculated plants. Also, the AM fungal contribution to plant growth and nutrient uptake in GQ plants, in the presence or absence of rmc roots, was assessed. To reduce competitive effects between tomato plants sharing the same soil volume, two plants were grown together in a horizontal three-compartment split-root system, sharing the middle compartment with one part of their root system while the other part of the root system was grown in a separate pot.
Materials and Methods
- Top of page
- Materials and Methods
In a preliminary experiment, where GQ and rmc plants were grown on a commercial culture medium (Floragard TKS-2, Floragard Gmbh, Oldenburg, Germany), both genotypes appeared to have a similar phenotype during vegetative growth. However, GQ is not the wild-type progenitor (L. esculentum cv. 76R), from which the rmc mutant was generated, and which was used in previous experiments on the rmc mutant (Gao et al., 2001; Cavagnaro et al., 2004).
Seeds of GQ (Bruno Nebelung Pflanzenzüchtung Gmbh & Co, Everswinkel, Germany) and of the mycorrhiza-defective plant mutant rmc (Barker et al., 1998) were surface sterilized in 4% H2O2 for 10 min and pregerminated on filter paper soaked with saturated CaSO4 solution before they were transferred to plastic pots (100 cm2, one plant per pot) filled with autoclaved perlite. The perlite was watered with nutrient solution (concentration of element/applied form: N, 5 mm/Ca(NO3)24H2O; P, 0.5 mm/KH2PO4; K, 3.7 mm/KH2PO4 and K2SO4; Ca, 3.5 mm/Ca(NO3)2 and CaSO42H2O; Mg, 1.6 mm MgCl26H2O; S, 1.72 mm/CaSO4 and K2SO4; Fe, 0.3 mm/Fe-EDTA; B, 1 m/H3BO3; Mn, 0.5 m/MnSO4; Zn, 0.5 m/ZnSO4; Cu, 0.2 m/CuSO4; Mo, 0.07 m/(NH4)6Mo7O24) once per day in sufficient amounts to allow free drainage from the bottom of the pots. At 10 d after planting, tomato plants were removed from the pots and perlite was gently washed from the root system. All plants had one main root of ≈10 cm length. The lower 1 cm of the main root was cut off to break apical dominance. Thereafter, the main root of each plant was transferred to the middle (combi) compartment and four to five lateral roots (length ≈5–8 cm each) emerging at the base of the main root were transferred to one of the outer (solo) compartments.
Either two GQ plants (GQ × GQ); two rmc plants (rmc ×rmc); or one GQ and one rmc plant (GQ × rmc) were planted together in one three-compartment split-root pot. The soil in the three-compartment split-root pots was either prepared with fertile (+M) or autoclaved (–M) AM fungal inoculum. Four replicates were prepared for each treatment, but in the (–M/GQ × rmc) treatment one plant died 2 wk after transplanting, thus only three replicates remained until harvest.
The three-compartment split-root pot consisted of a row of three black 700 cm3 plastic pots (Teku Tainer 0.7, Pöppelmann Teku, Germany) fastened together with adhesive tape. Each pot was filled with 810 g dry soil at a bulk density of 1.3 g cm−3. The soil had been dry-heated twice for 24 h at 85°C, with an interval of 48 h at room temperature, to eliminate AM fungal propagules. Before heating, the sieved (2 mm) soil contained (mg kg−1): 5.2 and 3.4 CaCl2 (0.0125 m)-extractable NH4+ and NO3−, respectively; 4.4 acetate lactate-extractable (CAL, Schüller, 1969) P; 58 CAL-extractable K; and 1.93 (Fe), 1.75 (Mn), 0.10 (Zn) and 0.16 (Cu) DTPA-extractable micronutrients. The soil had a pH (0.01 m CaCl2) of 7.3 and 0.2% organic matter. It was classified as loamy sand (45.2% sand, 42.0% silt, 12.8% clay). The soil in all treatments was fertilized with 200 mg K (K2SO4), 200 mg N (NH4NO3), 100 mg Mg (MgSO4), 40 mg P (Ca(H2PO4)2H2O), 10 mg Zn (ZnSO4H2O), 10 mg Cu (CuSO4), and 4 mg Fe (FeNH4-citrate) kg−1 dry soil.
The AM fungal inoculum (CAU collection code ‘Henan Fengaiu’) consisted of a mixture of the two AM fungal species G. mosseae and G. intraradices, both isolated from an alkaline (pH 7–8) soil in Beijing (PR China) and provided by the Department of Plant Nutrition of the China Agricultural University in Beijing. The inoculum was propagated on maize plants in open-pot culture in the glasshouse for ≈8 wk, using the same soil as described above.
A mixture of maize root pieces with an AM fungal colonized root length of ≈70% and adhering air-dried soil containing external mycelium and spores was used for inoculation of tomato plants. Inoculum, representing 10% w/w of the growth substrate, was homogeneously mixed with the soil before it was filled into the three-compartment split-root pots. The inoculum for the (–M) treatments was filtered with deionized water (70 ml per 50 g dry inoculum through Blue Ribbon filter paper, Schleicher & Schüll, Germany) before being autoclaved. The filtrate was added to the soil of (–M) treatments to encourage a microflora similar to that in the (+M)-treatments.
The inoculum propagation and the experiment were conducted in a glasshouse at Hohenheim University in Stuttgart, Germany (48°25′ N, 9°11′ E), from June to August (inoculum propagation) and from August to October (experiment). The experiment received 16 h supplemental lighting of 400 mol photons m−2 s−1 at bench height during the day, provided by Osram HQL-R 400 W lamps. Daily water loss from the three-compartment split-root pots was estimated gravimetrically, and was replaced with deionized water to maintain an average soil-water content of ≈20% w/w. Differences in water uptake from the three compartments could not be quantified, thus the water added was distributed over the three compartments according to visual appraisal. 4 wk after the plants were transferred to the split-root pots, the soil in all compartments was fertilized with an additional 100 mg K (K2SO4), 200 mg N (NH4NO3), 5 mg Zn (ZnSO4H2O), 5 mg Cu (CuSO4), and 4 mg Fe (FeNH4-citrate) kg−1 dry soil.
Plants were grown in the three-compartment split-root pots for 7 wk. At the time of harvest, roots were washed from the soil. Roots of the two plants sharing the (combi) compartment were placed in a water basin after the soil had been removed, and the two root parts were carefully separated using forceps and a preparation needle. Detached roots that could not be assigned to a given plant [pooled over all treatments, 8.3% of total root dry weight obtained from the (combi) compartments] were not included in further analysis.
Representative samples (0.5 g) of each of the two plant root parts were taken immediately after harvest, and stained with trypan blue in lactic acid (Koske & Gemma, 1989) to evaluate the AM fungal colonized root length by a modified intersection method (Tennant, 1975; Kormanik & McGraw, 1982). Between 300 and 350 intersections were counted per sample. In the (+M) rmc treatments, scores were taken separately for AM fungal surface colonization and cortical colonization. As the rates for AM fungal cortical root colonization in (+M) rmc plants were <0.5% of the colonized root length, both values were combined for statistical analysis, and are referred to as ‘surface colonization’.
All other plant material was freeze-dried at −30°C sample temperature for 1 wk. After obtaining dry weights of shoots and roots, samples of 200 mg ground shoot material were dry-ashed at 500°C, oxidized with 5 ml 1 : 3 diluted HNO3, and taken up into 25 ml 1 : 30 diluted HCl. Phosphorus concentrations in the samples were analysed colorimetrically with a spectrophotometer at 436 nm wavelength, after staining with ammonium–molybdate–vanadate solution (Gericke & Kurmies, 1952). Potassium and Ca were quantified using a flame photometer (Eppendorf ELEX 6361/Eppendorf Vertrieb, Deutschland GmbH, Germany).
Concentrations of Mg, Cu, Zn, Fe and Mn were measured by atomic absorption spectrometry (AAS; ATI Unicam 939/Solaar, Thermo Electron, USA). A Cs–La buffer was added to the samples before they were analysed for Mn and Fe.
Data obtained for pairs of plants of the same genotype were averaged. A two-way anova was performed on balanced data sets (n = 4). The cross-classified data were unbalanced when the (–M/GQ × rmc) treatment (n = 3) was included. In this case a two-way anova with adjusted sums of squares was performed (Searle, 1987). Statistics were calculated using the sigmastat 2.03 program.
- Top of page
- Materials and Methods
The AM fungal colonization of rmc plants was restricted to the root surface, with very rare exceptions where fungal hyphae penetrated into the root cortex and arbuscules and vesicles were formed. Apart from the observation that Glomus sp. WFVAM23 formed apparently normal arbuscules and vesicles in the cortex of rmc plants, Gao et al. (2001) also demonstrated that the phenotype of rmc root surface colonization (Myc−1) was different depending on the AM fungal species used for inoculation. While rmc colonization by G. intraradices, Glomus fasciculatum and Glomus etunicatum was restricted to appressoria formation, hyphae of G. mosseae, Glomus coronatum, Gigaspora margarita and Scutellospora calospora penetrated the epidermal cells but aborted before entering the cortex.
Inter- or intracellular hyphal growth within epidermal cells was not observed in rmc roots in the present experiment, although the inoculum contained propagules of G. mosseae. However, hyphal tip penetration of epidermal cells below the appressoria without further growth would not have been detected by the methods used for AM observation in this study.
Root exudates such as flavonoids, phenolic acids or polyamines have been shown to promote preinfective growth of AM fungi (Chabot et al., 1992; Nagahashi & Douds, 1999), while appressoria have been shown to be induced upon contact recognition, depending on the topology of the epidermal surface (Nagahashi & Douds, 1997). Naturally occurring nonhosts neither release AM fungi-stimulating compounds (Giovannetti et al., 1994; David-Schwartz et al., 2003) nor promote appressorium formation (Nagahashi & Douds, 1997). The observation of appressoria on (+M) rmc roots and, in particular, the high percentage of surface colonization in the (+M/GQ × rmc/combi) rmc root part, showing appressoria and hyphae growing in close contact to the root surface, supports the hypothesis that, in contrast to nonhosts, Myc−1 roots do not fail to attract AM fungal attempts to root colonization (Gollotte et al., 1993; Gianinazzi-Pearson et al., 1996).
The presence of GQ roots strongly increased rmc root surface colonization compared with rmc roots grown in the absence of GQ roots. At the same time, growth and total plant P upake of GQ (+M/GQ × rmc) plants was decreased compared with GQ (+M/GQ × GQ) plants. Bago et al. (2004) found strong evidence that the AM fungal extraradical mycelium can specialize on different functions and alter its morphology accordingly. It is possible that when rmc roots were present in the (+M/GQ × rmc/combi) compartment, the formation of AM fungal hyphae functioning in P uptake was decreased in favour of mycelium functioning in the spread of infection. The need of AM fungal hyphae to forage for uncolonized host-plant roots as new carbohydrate sources is discussed by Olsson et al. (2003) in an ecological context.
Although it is possible that carbohydrates were transferred from rmc plants to AM fungi in places where cortical colonization was established, it can be assumed that surface colonization of rmc roots in the (+M/GQ × rmc/combi) compartment was established mainly at the expense of carbohydrates derived from associated GQ plants. This might be the reason why growth of (+M) GQ plants was decreased compared with the (+M/GQ × GQ) treatment when rmc roots were present in the (combi) compartment. However, this assumption needs to be confirmed by further studies using, for example, 13C/12C or 14C discrimination methodology.
The reasons for a lower rate of AM fungal surface colonization of rmc roots in the (+M/GQ × rmc/solo) compared with the (+M/rmc × rmc/solo) compartment also need further investigation. It is possible that the induction of some systemic defence responses was increased in rmc plants when surface colonization of roots in the (combi) compartment was high.
Compared with standard values cited by Bergmann (1992), macro- and micronutrient concentrations in the shoot were in a sufficient range for plant growth, except for P, where values of all (–M) plants and (+M) rmc plants were indicative of P deficiency. Shoot P concentrations in (+M) GQ were higher compared with (–M) GQ plants, but still not in an optimal range. It can thus be assumed that, in all plants, P was a major growth-limiting factor.
The total plant P uptake of (+M/rmc × rmc) plants was 67% lower than for the (–M/rmc × rmc) plants. This suggests that P uptake was specifically decreased in response to AM fungal inoculation in rmc plants. Increased concentrations of Ca, Cu, Fe and Mn in the tissue of (+M) rmc compared with (–M) rmc plants can be explained by the decreased growth of (+M) rmc plants, leading to a smaller dilution of nutrients in the plant tissue. Smith et al. (2004) obtained experimental evidence for the assumption that the direct P-uptake pathway, via root hairs and epidermis, can be largely inactivated upon AM fungal root colonization. To date, not much is known about the precise mechanisms behind this effect. It could be speculated that in the (+M/rmc × rmc) plants, direct root P-uptake mechanisms were inactivated in response to AM fungal surface colonization, or in response to the rare events of cortical colonization observed in rmc roots.
It is also possible that the inability of the rmc mutant to suppress defence mechanisms induced upon AM fungal attempts to root colonization not only restricted AM fungal rmc root colonization to the root surface, but also decreased the P-uptake capacity of rmc roots. For example, a rapid general defence response involving the synthesis of reactive oxygen species and subsequent hypersensitive cell death would probably lead to a dysfunction of affected cells and, in consequence, to a decreased ability of the root to take up sufficient amounts of nutrients (Levine et al., 1994). Similarly, the penetration of hyphal tips into epidermal cells, as observed by Gao et al. (2001) for colonization attempts of G. mosseae on rmc roots, might also decrease root P uptake, particularly when the soil P availability is low. In our study, AM fungal attempts to colonize rmc roots often appeared to increase in the area behind the root tip, which is a major site of cell elongation and nutrient uptake. However, the reasons for a decreased P uptake of rmc plants in response to AM fungal inoculation clearly need further investigation.
In the (+M/GQ × rmc) treatment, GQ plants might have gained an advantage over rmc through their ability to form a functional AM symbiosis, enabling them to acquire soil P from both soil compartments more efficiently compared with rmc plants (Cavagnaro et al., 2004). The lower total plant P uptake of rmc (+M/GQ × rmc) plants compared with respective (–M) plants might, therefore, also be partly explained by the better ability of GQ (+M/GQ × rmc) plants to extract the soil in the (combi) compartment for P. Indeed, the total amount of P taken up by both plants in the (–M/GQ × rmc) treatment was not significantly different (t-test, P < 0.05) from the total P uptake of both plants in the (+M/GQ × rmc) treatment (data and statistics not shown).
In conclusion, our results indicate that rmc surface colonization can be increased in the presence of wild-type roots, suggesting that available carbohydrates may be crucial for fungal attempts at root colonization. The Myc−1 mutants may be suitable plants to test inoculum quality if further experiments confirm that rmc growth depression and AM fungal surface colonization are correlated with AM fungal inoculum strength.
Inoculation with AM fungi reduced total plant P uptake and growth of rmc plants. The reasons for this effect need further investigation. However, the results of the present study indicate that, under some conditions, Myc−1 mutants may be unsuitable as ‘nonmycorrhizal controls’ in nutrient-uptake experiments on nonsterilized soil.