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Keywords:

  • Root exudate;
  • Mycorrhizal mutant;
  • Non-legume;
  • Glomus intraradices;
  • Gigaspora gigantea;
  • Gigaspora rosea

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgements
  8. References

Soluble factors released from roots of the pre-mycorrhizal infection (pmi) myc tomato mutant M161 were analyzed and compared with normal wild-type released factors. Aseptic whole exudates from the M161 mutant retarded the proliferation of Glomus intraradices in vitro. When the whole exudate was further fractionated on a C18 SEPAK cartridge, the 50/70% methanol fraction showed an activity against hyphal tip growth of Gigaspora gigantea and Gl. intraradices. Preliminary characterization of the exudate suggests that the inhibitory moieties are heat labile, bind to PVPP (polyvinyl polypyrrolidone), and are not volatile. This is the first reported instance of the inhibition by a myc plant being ascribed to inhibitory component(s) released in root exudate.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgements
  8. References

The arbuscular mycorrhizal (AM) symbiosis is a mutualistic symbiosis between higher plants and Glomeromycota [1]. The fungal partners are characterized by their lack of host specificity and their obligate biotrophy. The initial stages of colonization of roots by the mycorrhizal fungus are dependent on fungal growth and on rhizospheric signals [2]. Morphogenetic changes in both plant and fungus during colonization indicate that there must be an exchange of signals between the partners, with regulation occurring at several control points [3]. Mycorrhizal host plant mutants (myc) provide excellent starting material for categorizing the complex steps that occur during the establishment of the symbiosis [4]. The current repertoires of myc mutants, isolated mainly from legumes, are invariably defective in post-penetration stages. The recent identification of the pre-mycorrhizal infection (pmi) tomato mutant [5], impaired in its ability to manifest the early stages of infection, has provided us with the opportunity to examine the early stages of the infection process. This pmi mutant reduces AM fungus spore germination and hyphal proliferation, thereby implicating a role for root exudate components, including volatile compounds.

Root exudates affect hyphal growth of AM fungi [6]. Several studies have established that the growth of germinating AM fungus spores was stimulated in the presence of host root exudates [7–9], whereas exudates from non-mycotrophic species had no effect [10–12] or were inhibitory [6,13]. The precise mechanism responsible for the non-mycorrhizal phenotype has not been identified to date, but some of the acceptable reasons for this phenotype include: release of inhibitory compound(s), lack of an active growth stimulant, presence of an elevated defense response, and inhibitory topographic determinants [6,12,14].

We present for the first time evidence that the exudate of the pmi M161 mutant contains a methanol-soluble fraction that inhibits hyphal tip growth of Gigaspora gigantea and Glomus intraradices. Furthermore, the inhibition phenomenon was studied by developing an in vitro root organ culture (ROC) [15] with non-transformed and Ri T-DNA (root inducing transfer DNA)-derived hairy roots of wild-type (WT) and M161 plants. This enabled us to confirm the susceptible and resistive phenotypes of the WT and M161 plants, respectively, and thus to gain a better insight into AM fungus colonization in vitro.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgements
  8. References

2.1Establishment of ROCs

ROCs were established from WT and M161 roots by following the protocols described by Bécard and Piché[15], with M medium solidified with 0.4% Phytagel (Sigma). Phosphorus was routinely omitted from the original recipe because a significant amount, about 0.1% by weight, was contributed by the gelling agent [16]. Hairy roots of WT and M161 plants were generated by using Agrobacterium rhizogenes ATCC 15834. Clonal selection and growth conditioning were done by serial subculturing in the low-nutrient M medium containing the antibiotic Claforan (500 μg l−1). The transformation was confirmed by amplifying the rolA/B and ags fragments located on the TL and TR regions of the Ri plasmid by polymerase chain reaction (PCR) (data not shown).

Inoculum of Gl. intraradices Schenck and Smith (DAOM 181602) consisted of cubes of medium, containing 50–60 spores and hyphae, cut from the distal side of a previously established split plate monoxenic culture [17]. Gigaspora rosea Nicolson and Schenck (DAOM 194757) and G. gigantea (Nicol. and Gerd.) Gerdemann and Trappe (isolated from the Rodale Institute Experimental Farm, Kutztown, PA, USA) spores were produced in pot cultures with Paspalum notatum Flugge as host. Spores were harvested, selected and surface sterilized [15]. At least five Petri dishes were established for ROCs of each genus of AM fungi.

2.2Effect of root volatiles upon hyphal growth

Spores of Gl. intraradices were utilized to study the effects of volatile compounds produced by the two tomato root clones upon hyphal growth and branching. Spores were isolated from the distal compartments of split plate cultures [17] of Gl. intraradices and Ri T-DNA transformed roots of carrot. Gelled medium containing spores was blended at high speed with 10 mM sodium citrate (pH 6.0) [18]. Spores with short, broken subtending hyphae were selected and inserted into M medium in one side of split plate Petri dishes. There were three treatments: control (no roots present in the second compartment), and plates with non-transformed roots of either WT or M161 tomato in the second compartment. Two experiments were conducted: one at 24°C and ambient CO2 (n=24 spores per treatment) and the second at 29°C and 2% CO2 (n=16 spores per treatment). The second experiment was designed to indicate the effects of volatiles other than CO2.

2.3Exudate interaction bioassay

2.3.1Whole exudate assays

Axenic exudates from WT and M161 were generated from excised roots grown in liquid culture as described previously [6]. The liquid M media containing the root-released components were added in a ratio of 1:1 (v/v) to slightly warm, autoclaved, double strength M medium, gelled with 0.6% Phytagel and poured into standard 9-cm Petri plates. The final concentration of the gellan after pouring was 0.3%. A single gel plug from an actively growing carrot dual culture, approximately 9 cm3 in volume, containing 120–150 spores with mycelia and roots, was inserted into the amended medium in each dish. The dual cultures then were incubated at 26°C in darkness. The mycelial strands emerging from the inoculum plug were recorded every other day in both groups (WT and M161) of exudate-amended Petri dishes. Additionally, the axenic exudates from WT and M161 were treated separately in two different ways: (a) with insoluble polyvinyl polypyrrolidone (PVPP) (1 g l−1; Sigma) and (b) autoclaved at 121°C at 1.2 atm for 20 min, before preparing media as above. Five separate plates were prepared for each genotype×treatment combination, each with one plug of inoculum, and data were recorded every other day for 2 weeks.

2.3.2Effect of exudate fractions upon hyphal growth

Non-transformed tomato roots were grown in liquid M medium for 10 days, and then transferred to liquid M medium minus P for 10 days to facilitate production of exudate signals [6]. The fresh weight and dry weight of the root biomass were recorded. Exudates then were fractionated and concentrated on a SEPAK C18 column with methanol. Methanol fractions (25, 50, and 70%) then were evaporated under N2 and resuspended in 70% ethanol [6]. The final volume of ethanol was adjusted according to the respective root dry weight data, specifically, 200 μl/136.8 mg for WT, and 240 μl/163.8 mg for M161. Therefore, results of bioassays using 5-μl volumes of these concentrated fractions were indicative of differential production of branching stimulators/inhibitors by WT and M161 roots on a per root weight basis. Three experiments were conducted with these preparations.

In the first, surface sterilized spores of G. gigantea were inserted into square Petri dishes (9 cm) and allowed to germinate at 32°C in a 2% CO2 atmosphere. Three to four days after germination, 5 μl of the concentrated 50/70% methanol fraction was injected into a small well near the growing tips of the primary germ tube (n=4) and the main secondary branch hyphae (n=8). The effects of the exudates upon hyphal growth were measured 24 h later. In the second experiment, the 50/70% methanol exudate fraction of M161 was diluted 10-fold and 100-fold and injected near primary germ tube tips as above (n=6). Growth was measured after 24 h. In the third experiment, the 25, 50 and 70% methanol fractions of WT and M161 tomato root exudates were injected into small wells (5 μl) near the dominant hypha of germinating spores of Gl. intraradices, isolated from split plate cultures as above. The spores were incubated at 32°C in a 2% CO2 atmosphere for 12 days (six spores per dish, two dishes per treatment) after which the length of the dominant germ tube hypha of each spore was measured with the ocular micrometer (20×) and total branch hyphae on the main hypha were quantified.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgements
  8. References

The validity of the continued use of ROCs for studying some of the most challenging questions regarding biochemical, genetic and physiological relationships between AM fungi and their hosts is supported by the fact that these roots show the same mycorrhizal characteristics as the plants from which they were developed [19]. The WT tomato hairy roots were readily susceptible to infection by anti-muscle factor (AMF) of two different genera, Gl. intraradices (Fig. 1b) and G. rosea (Fig. 1c), which completed their life cycles by forming viable progeny spores. The extraradical mycelium developed profusely, and quickly covered the Petri dishes with concomitant spore formation. The production of resting spores is an indication, from the fungal viewpoint, of successful mycorrhizal establishment [20]. Spore formation in association with WT roots shows that key signals, hallmarks of a typical host, were available to the fungi.

image

Figure 1. Monoxenic culture of AM fungi with WT and M161 roots in vitro. a: Formation of Gl. intraradices spores in culture with WT non-transformed roots (bar=210 μm) and b: Ri T-DNA transformed roots (bar=120 μm). c: Progeny spores of G. rosea with WT transformed roots (bar=255 μm) and d: abortive spores of Gl. intraradices in association with non-transformed M161 roots (bar=140 μm).

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In contrast, M161 showed a resistant phenotype when roots from non-transformed or hairy roots were used for ROC. No colonization was detected in the M161 mutant roots, where few active mycelia were seen emerging from the inoculum plugs of Gl. intraradices. This could have been due to the lack of appropriate hyphal growth stimulators and/or release of an active inhibitor from the M161 roots. Interestingly, spores that managed to germinate in the presence of either M161 non-transformed or hairy roots bore hollow spores on hyphal branches (Fig. 1d). These hollow spores (of which there were 8 to 15 per plate on average), reminiscent of abortive spores, were formed with no specific preference in terms of location and number. They were characterized by their lack of lipid contents and did not germinate when transferred to fresh M medium under a 2% CO2 atmosphere at 32°C (data not shown). Prolonged incubation, typically 6 months, of the M161 ROC plates did not alter either the hollowness or the dysfunctional character of these spores.

There was a large difference in susceptibility between the non-transformed and the Ri T-DNA transformed (‘hairy’) WT roots. The non-transformed WT roots were not colonized readily by Gl. intraradices and required a longer incubation period for fungal development than hairy roots (Fig. 1a). This was evident in the difference between their sporulation rates: 999±263 per dish (n=5) in 13 weeks of incubation vs. 11 625±836 per dish (n=5) in 12 weeks, for non-transformed and transformed roots, respectively. The difference in susceptibility could be attributed to the better adaptation of the hairy roots to the low-nutrient regime, to a high rate of nutrient turnover, or to altered phytohormone levels. The low success rate of non-transformed ROCs – about 53% as compared with 100% for hairy roots – was comparable to the earlier results of Bago et al. [21], who obtained a success rate of 20–60% when non-transformed tomato roots were used for developing dual cultures. No successful sporulation of G. rosea was obtained with ROCs of non-transformed WT roots in the present study, for unknown reasons. The fungi invariably failed to proliferate actively in vitro after the initial contact phase of the mycelia, and did not exhibit the subsequent development steps. Manipulation of the basic M medium recipe, changes in the carbon level, and incubation of the dual cultures under a 2% CO2 atmosphere were some of the measures employed without success. With transformed roots, however, sporulation was achieved within 6 weeks of establishment (Fig. 1c), and ontogenic events as described previously [22] for this fungus were observed.

Exudates derived from host and non-host plants can modify hyphal growth characteristics. The WT and M161 exudates influenced the hyphal proliferation of Gl. intraradices mycelia emerging from the carrot dual culture medium plug: hyphal proliferation was greater in medium amended with WT exudate than in that treated with M161 exudate (Table 1). The emergence of the mycelia was observed within 24 h of initiation of the dual culture in both the treatments, albeit at different rates of emergence. Significantly more hyphae emerged from inoculum plugs in WT- than in M161-treated dishes (α=0.05, Duncan's multiple range test) (Table 1). When the WT and M161 exudates were treated with PVPP, which has been shown to bind phenolics [23], there was no significant difference in hyphal emergence at any date of hyphal enumeration (Table 1). This suggests that the active components in WT and M161 exudates, which stimulate or inhibit, respectively, are removed by PVPP treatment and thus could be phenolics. Exposure of the exudate to high temperature presumably destroyed the active component as no difference was observed between treatments.

Table 1.  Number of emerging hyphae from plugs of Gl. intraradices embedded in M media amended with WT and M161 exudate
  1. Treatment of exudates included control, boiling and PVPP as described in the text. The number of emerging mycelial strands were enumerated every 2–3 days post initiation (dpi) of the culture. Data followed by different letters within a row are significantly different (P<0.05) using Duncan's multiple range test. Values are means of five replicates and LSD refers to the least significant difference at 5% significance level.

Time (dpi)ControlHeatPVPPLSD (0.05)
 WTM161WTM161WTM161 
22.6 a0.0 c0.8 bc0.8 bc1.4 b0.8 bc0.95
44.6 a0.2 c1.8 b2.0 b2.6 b2.2 b1.33
67.4 a0.2 c3.0 b3.4 b4.2 b3.8 b1.78
914.4 a0.8 d4.8 c5.4 bc7.2 b6.0 bc2.18
1119.0 a1.6 d8.4 bc10.0 b10.2 b7.6 c2.17
1433.2 a8.2 c10. bc12.0 bc14.0 b10.0 bc4.3

Spores of Gl. intraradices, germinating under ambient CO2 in the distal side of split plates containing non-transformed roots of WT or M161, exhibited more hyphal growth and branching than controls incubated without roots (Table 2, top). This could have been due to build-up of CO2 from root respiration prior to diffusion out of the parafilm wrappings on the Petri dishes [8]. A second experiment was conducted at 2% CO2, designed to overwhelm any effect of CO2 contributed by root respiration; the results again showed increased hyphal growth and branching in the presence of root volatiles (Table 2, bottom). This indicates the presence of a volatile signal other than CO2: a signal that does not play any significant role in the pmi inhibition phenotype for the M161 mutant. Previous studies have demonstrated the possibility of volatile chemical signals in the AM symbiosis [24].

Table 2.  Effect of volatiles from non-transformed tomato roots on the growth of Gl. intraradices
  1. Means of 24 or 16 observations for Experiments I and II respectively, ±S.E.M. GT=germ tube.

TreatmentLength (mm) of primary GTNumber of branches off dominant GTsubtending hyphae
Experiment I: 24°C and ambient CO2
Control10.5±1.13.5±0.72.7±0.2
WT13.5±1.07.2±1.23.1±0.2
M16115.1±1.09.4±1.73.4±0.3
Experiment II: 29°C and 2% CO2
Control11.3±0.82.5±0.63.1±0.3
WT15.7±0.711.6±1.73.6±0.2
M16112.7±1.314.2±2.73.2±0.3

The branching of germ tubes and secondary branch hyphae of AM fungi in the presence of host root exudate has been shown previously [6,10]. The root exudate components from both transformed and non-transformed roots of host plants, in either the semi-purified, concentrated form, or the fractionated form, stimulate hyphal branching in G. gigantea and Gl. intraradices. For example, exudates of non-transformed roots of maize and tomato contained branching signals primarily in the 50/70% and 0/50% methanol fractions, respectively, whereas exudates of transformed carrot roots contained signals primarily in the 50/70% methanol fraction [6].

Interestingly, exudates from roots of a non-host plant, Beta vulgaris, inhibited fungal hyphal tip growth [6]. This indicated that exudates from the non-host plants contain component(s), which are detrimental to AM fungal proliferation. In the present study, non-transformed WT and M161 tomato root exudates were fractionated and injected near growing hyphae of germinating spores of Gl. intraradices (Table 3). Fractions of the WT exudate slightly stimulated hyphal growth relative to controls, but appeared to inhibit hyphal branching. Concentrated exudate fractions of the mutant also inhibited branching. Furthermore, the 50/70% methanol fraction inhibited hyphal growth. This inhibition of germ tube growth was verified by using germinating spores of G. gigantea: the 50/70% methanol fraction of M161 root exudates inhibited growth of both the primary germ tube (Fig. 2) and the main secondary branch hyphae (data not shown), compared with the corresponding fraction of the WT exudate. There was little hyphal growth in the presence of concentrated M161 exudate, and that growth there was occurred after the germ tube tip had been inhibited and a ‘recovery branch’ formed. Ten- and 100-fold dilutions of this fraction still inhibited hyphal growth, but allowed the development of recovery branches, which resumed growth. A similar finding has been reported for the fractionated exudate isolated from the non-host, B. vulgaris[6].

Table 3.  Effect of exudates from non-transformed roots of tomato on the growth of the main germ tube of Gl. intraradices
  1. Injection of the exudate fractions was performed as in Table 2 and observations were recorded after 12 days of growth, *=no growth after injection. Means of 10 or 11 observations, ±S.E.M.

TreatmentLength (mm)Total branches
Control9.5±1.07.4±1.5
WT 25%11.0±1.32.8±0.7
WT 50%12.0±1.03.2±0.7
WT 70%14.8±1.22.5±0.5
M161 25%8.4±0.83.8±1.0
M161 50%14.6±1.24.0±1.2
M161 70%5.9±1.0*1.7±0.7
image

Figure 2. Growth of primary germ tube hyphae (mm) of G. gigantea 24 h after exposure to: 5 μl of 70% ethanol (control, n=6); concentrated 50/70% methanol-soluble exudate fraction (1:0) of non-transformed WT tomato (evaporated and dissolved in 70% ethanol, n=4); or concentrated (1:0), or 10-fold (1:10) or 100-fold (1:100) diluted 50/70% methanol-soluble exudate fraction of M161 tomato roots (n=6). Values are expressed as means±S.E.M.

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4Conclusions

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgements
  8. References

One of the fundamental problems in rhizosphere biology is to understand the mechanism governing the signaling between AM fungi and host plants, which leads to successful colonization. Myc mutants, which block colonization at various stages, provide us with the opportunity to dissect the mechanism(s) involved in the communication between the symbionts [25]. Recently we identified another mutant of tomato, M20, sharing similar phenotype with M161, which we designated as pmi2 [26]. This suggests that the pmi phenotype can be dissected into many substages, all leading to a different degree of pmi phenotype intensity. Natural non-hosts such as B. vulgaris can completely inhibit ontogenic stages of AM fungi [13], thus severely limiting their utility as a tool with which to address this question. We used the pmi tomato mutant M161 [5] since it affects the initial stages of AMF ontogenic development, and little is known about this stage. The primary mechanism responsible for the pmi mutant phenotype was hypothesized to be factor(s) present in the root exudate, the presence of which was investigated in greater detail. The whole root exudate of the M161 mutant was examined and was found to significantly decrease the proliferation of Gl. intraradices in vitro (Table 1). When this exudate was treated with PVPP, which has been shown to bind phenolics [23], the inhibition was significantly reduced. After C18 fractionation of the mutant exudate, the ‘active’ component in the 50/70% methanol fraction inhibited fungal tip growth of G. gigantea and Gl. intraradices (Fig. 2 and Table 3). The inhibitory activity on the hyphal tip growth was neutralized when this fraction was diluted by a factor of 100. Taken together, the above results give rise to speculation about the chemical nature of the inhibitory compound: it appears to be somewhat hydrophobic, heat labile, and non-volatile.

Finally, both transformed and non-transformed WT and M161 roots were used to establish monoxenic cultures with AM fungi of two different genera, Gl. intraradices and G. rosea. The formation of progeny spores in WT and abortive spores in M161 cultures was further evidence that the signals necessary for the fungus to complete its life cycle were not emitted by the pmi mutants. To enhance our understanding of the greater colonization of transformed WT roots compared to non-transformed WT roots, we plan to analyze soluble root factors of the transformed WT roots.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgements
  8. References

We thank J.A. Fortin for kindly providing the original monoxenic cultures of Gl. intraradices and transformed carrot roots.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. 4Conclusions
  7. Acknowledgements
  8. References
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