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Departamento de Microbiología del Suelo y Sistemas Simbióticos, Estación Experimental del Zaidín (CSIC), calle Profesor Albareda 1, 18008 Granada, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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Intra- and extraradical colonization competition and hyphal interactions among arbuscular mycorrhizal fungi (AMF) Glomus intraradices, Glomus proliferum and Gigaspora margarita were investigated in two in vitro experimental systems. AMF were polyxenically cultured with a Ri T-DNA transformed carrot root organ culture (ROC) in either big Petri plates containing three culture compartments and a common hyphal compartment (i.e. an independent host root for each AMF) or two by two in the culture compartment of regular bicompartmented Petri dishes (i.e. a common host root and a common hyphal compartment). Maps of the extraradical mycelial development of the three AMF were obtained. Two distinct substrate colonization strategies (Glomus-type and Gigaspora-type) were identified, reflecting intrinsic differences among AMF genera/families. Our data reveal a general lack of antagonism between the isolates when extraradical hyphae explore and exploit the substrate outside the root influence zone; however certain growth restrictions were imposed by Gi. margarita extraradical mycelium when developing near the host root and by G. proliferum intraradical hyphae. This work highlights once more the appropriateness of AM in vitro culture systems to perform in vivo studies on the biology of this symbiosis and opens new avenues to the formulation of in vitro AMF inoculants.
Key words: AMF colony development, AM polyxenic cultures, in vitro culture, competition, coloniztion strategies
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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and Mugnier and Mosse (1987), for successful in vitro culturing of arbuscular mycorrhizas (i.e. AM monoxenic culturing) so that these became easy to maintain under such highly controlled conditions. Since then a change of mood—from profound skepticism to general acceptance, from a residual, to a widespread use (Bago and Cano 2005)—has occurred among mycorrhizologists in respect to AM monoxenic cultures. The development of either two-(St-Arnaud et al 1996), three- (Bago et al 1996) or multicompartmented (Bago et al 2004) monoxenic cultures has opened new prospects in the study of the AM symbiosis. Research areas such as fungal colony architecture, physiology, biochemistry, cytology and molecular biology, traditionally affected by the intrinsic problems presented by culturing AM in soil, especially have benefited from this in vitro revolution (see Fortin et al 2002 and Declerck et al 2005 for recent reviews). Although up to now research with AM monoxenic cultures has been focused mainly on extraradical mycelium biology, recent reports also indicate the idoneity of this experimental system for studying intraradical colonization and fungal-host interactions (Declerck et al 2000). Taking this one step further, based on the advantageous direct, nondestructive symbiosis follow-up allowed by AM monoxenic cultures, we were interested in investigating the poorly known field of AM fungal interspecies interaction and competition for space (and therefore for resources), either extraradically (i.e. spreading and media colonization strategies to scavenge mineral/organic nutrients) or intraradically (i.e. root cell colonization to acquire host-derived carbon supplies).
To the best of our knowledge only two attempts have been made to study AM species interactions with AM monoxenics. The first one was by Douds and Bécard (1993) with a Gi. margarita/Gi. gigantea coculture; a negative competition between both species was reported. More recently Tiwari and Adholeya (2002) cocultured Glomus intraradices and Gi. margarita in experimental box systems to find no apparent antagonism between these two AM fungi. In both of the above-mentioned apparently contradictory reports, results were based in qualitative observations; no quantification and/or mapping of fungal development were done. Moreover no indication was provided about species interaction at the sites where AM fungi should be expected to compete for space and resources, which are the intraradical host cells, where carbon supplies are acquired for life.
The aim of the research presented here was to investigate interactions among three different AM fungal species when competing for space (and therefore for resources) both intra- and extraradically. To do this two experimental systems were developed, which allowed the mapping of the three codeveloping extraradical fungal colonies. Here we report the effects of coculture in germination, presymbiotic hyphal development and intra- and extraradical symbiotic hyphal spread, revealing intrinsic fungal strategies for space colonization. Attention is paid to any possible antagonistic/synergistic interaction between fungal isolates that could be of interest in future in vitro AM inocula formulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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, left column) and filled with 20 mL of sterilized (121 C, 20 min) minimal medium (M medium, Chabot et al 1992). These will be referred to as the "culture compartments" (CC) and were used to establish mycorrhizal monoxenic cultures among a transformed carrot root organ culture (ROC, DC-2 clone) and three AM fungi: (i) Glomus intraradices Schenck and Smith (DAOM 197198, Bio-systematic Research Centre, Ottawa, Canada); (ii) Glomus proliferum Dalpé and Declerck (MUCL 41827); and (iii) Gigaspora margarita Becker and Hall (BEG 34) (FIG. 2, left column). We selected these three AM fungi due to their different spore morphology, which should aid colony follow-up and final spore count for each species (FIG. 1a, b).
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). Monoxenic cultures were initiated by placing a 5 cm long piece of root on each CC. For G. intraradices and G. proliferum cultures a cube of medium from a previous monoxenic culture, containing ca 250 spores, as well as fragments of mycorrhizal roots and external hyphae, was placed near the root apex; for Gi. margarita three spores subcultured from a previous monoxenic culture were placed on top of a subapical zone of the ROC.
Experiment 2: Dixenic culture of three AMF (two-by-two combinations) in plates having common culture and hyphal compartments..
90 mm bicompartmented Petri plates were used (FIG. 4, top left). A transformed carrot ROC was placed in the center of one compartment (the culture compartment CC), and two monoxenic inoculum cubes containing two different AM fungi placed at both sides of it. The AM fungi were two-by-two combinations of those used in Experiment 1, resulting in three types of dixenic cultures: (i) carrot ROC/G. proliferum/G. intraradices; (ii) carrot ROC/G. proliferum/Gi. margarita; and (iii) carrot ROC/G. intraradices/Gi. margarita. After symbiosis establishment and mycorrhizal development, AM fungal hyphae, but not roots, were allowed to pass over the plastic barrier and develop on the second compartment of the Petri plate (the hyphal compartment, HC). Therefore this system allowed interactions of the extraradical mycelium (ERM) of each pair of AMF in the presence (CC) or the absence (HC) of host root exudates.
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Observation and measurements of extraradical fungal growth.— – In both experiments germination rates (in terms of hyphal development out of the inoculum cubes for G. intraradices and G. proliferum and germ-tube emergence for Gi. margarita) were assessed. Once the symbiosis established extraradical hyphae development was controlled weekly, with attention to any developmental feature that might indicate competition for external substrate exploitation among the strains. At the end of the experiment total number of spores (and of auxiliary cells in the case of Gi. margarita) produced by each AM fungus was determined for each Petri plate. All measurements were done under a Nikon AFX stereomicroscope. Microphotographs (Kodak 100 ISO slide film) of hyphal morphology and extraradical structures in the different culture media were taken with a Leica DMRB microscope fitted with a Leica MPS-60.
As mentioned above the three AMF in this study were selected due to their different spore morphology, which a priori let us easily distinguish each of them under the stereomicroscope. Indeed there was no problem when comparing Gi. margarita with the two Glomus isolates because Gigaspora spores and auxiliary cells are clearly different from Glomus spores (FIG. 1a). However more problems were encountered when trying to differentiate between G. proliferum and G. intraradices spores: Although when mature G. intraradices spores were darker and bigger and appeared in much less compact and profuse bunches (FIG. 1b), when immature they could be mistaken if using just the transmitted light of the stereomicroscope (FIG. 1c). This confusion was overcome by using combined top and bottom illumination, which let us distinguish clearly between spores of both species (FIG. 1d).
Substrate colonization maps.— –
One of the main objectives of this study was to compare the substrate colonization strategy and interactions between the AM fungi selected. To do this a 1 cm2 gridline was drawn (with a permanent marker) at the bottom of each Petri plate (both for experiments 1 and 2; FIG. 2, bottom left, and FIG. 4, top left). Rows of cells in each Petri plate were named as letters, and for each column a number was assigned (see plate on FIG. 4); therefore each cell of the gridline was identified perfectly (i.e. a1, b6, d5, etc.; FIG. 2, bottom left, and FIG. 4, top left). Spore count was carried out for each cell of each Petri plate and notated separately.
Diagrams representing each Petri plate were drawn (FIGS. 2, 4). A color was assigned to each fungus (G. proliferum = red; G. intraradices = blue; Gi. margarita = green) and a color intensity scale in terms of spore number was prepared: the higher the spore amount, the darker the color on the scale (FIG. 4). The scales (FIG. 4) are valid both for Experiment 1 (FIG. 2) and Experiment 2 (FIG. 4). Due to the low spore number produced by Gi. margarita (compared to the two Glomus isolates) green in diagrams corresponds to auxiliary cell counting rather than spores occurrence for this fungus; spores are represented by small yellow dots.
Tracking intraradical colonization of the different species.— –
After spore count intraradical AM colonization was assessed in two selected zones, close to the AMF inoculum source of the G. proliferum/Gi. margarita Petri plates (FIG. 4, Plate 7, sites A and B) and of the G. intraradices/Gi. margarita Petri plates (FIG. 4, Plate 1, sites C and D). To do this ROC of the indicated sites were removed from the culture medium and trypan blue-stained to view intraradical colonization. Evaluation of internal colonization of G. proliferum/G. intraradices was carried out, but no attempt was made to differentiate among intraradical structures of these closely related fungi. However distinguishing Gigaspora and Glomus intraradical structures was feasible (FIG. 5), so that a separate quantification of fungal colonization was done for each of those fungi. Roots were removed from the culture medium, cut into 1 cm lengths, stained with trypan blue (Phillips and Hayman 1970 with modifications) and mounted in lactic acid on microscope slides. At least 30 root pieces per Petri dish were used to estimate the percentage of root length containing intraradical AM structures, using the colonization intensity method of Trouvelot et al (1986) with some modifications. Microphotographs of species interaction and of the most relevant colonization features were taken with a Leica DMRB microscope fitted with a Leica MPS-60.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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, plates 1 and 5, and FIG. 4, plates 4, 7 and 1), which is to the best of our knowledge the first report on this AM fungus having such an ability on in vitro compartmentalized systems. Development of the ERM of all the three AM fungi within the M-C media was followed weekly, and pictures of the most relevant events were taken (FIG. 3). Although Gi. margarita had a certain tendency of developing aerially (according to its well known negative geotropism) its ERM most often grew within the culture medium, thus frequently meeting hyphae from the two Glomus species. Negative interactions among extraradical hyphae of the three AMF developing within the M-C culture medium were never observed, neither in terms of hyphal morphology modification nor in mycelial growth restriction. Instead hyphae from the different isolates seemed to sense each other, usually avoiding each other. When colonizing the same medium zone, hyphae usually manage to grow in different layers of the culture medium (i.e. one closer to the medium surface, the other close to the Petri plate bottom) and this irrespective to the AMF combination chosen. Nevertheless in some occasions hyphae of different species grew close and seemed to collide (FIG. 3d e, f; dashed arrow) and even some contact between them could be appreciated (FIG. 3b, inset, dashed arrow). Sporulation of cultures started about 2 mo into the experiment for the two Glomus species and 4 mo into the experiment for Gi. margarita. There was no indication that the presence of one fungus hindered the sporulation of another one, the spores (or auxiliary cells) of the three species appearing quite often mixed in few micrometers3 of the M-C culture medium (FIG. 3d, g, h).
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) in sporulation maps. Only three out of 10 replicate Petri plates are shown, these being representative of the others. As specified in M&M G. intraradices is represented in blue, G. proliferum in red and Gi. margarita in green. The darker the color, the higher spore (or auxiliary cell) number, following the scale (FIG. 4).
According to the maps, substrate colonization strategy of the two Glomus species differs in important ways from that of Gi. margarita. Whereas the former extend their extraradical mycelium mainly out of the CC and sporulate out of the influence of the root, Gi. margarita extraradical hyphae remain mainly within the CC near the root, mostly sporulating there. Few Gi. margarita extraradical hyphae grew out the CC, despite the ability of this fungus for overpass the CCs plastic barrier. In no case did Gi. margarita enter any Glomus CC; extraradical hyphae of both Glomus species seemed also to avoid entering a foreign CC, although this occasionally occurred (FIG. 3). As stated above some Gi. margarita hyphae were "aerial" (i.e. they grew to the top of the Petri plate and even coiled on the plate lid between condensation drops of the culture medium). Such behavior never was shown by either of the two Glomus species, which represents another difference in ERM developmental strategy between both AM fungal genera.
Quantification of ERM growth of the different AM fungi was carried out in terms of spore/auxiliary cell formation (FIG. 2), which accurately reflected hyphal spreading within the culture medium because spores are formed in a homogeneous manner on extraradical hyphae in vitro (Bago et al 1998). Such quantification revealed that G. intraradices colonized a mean of 62.6(48.7–73.4)% of the total Petri plate surface, of which 78.7% corresponded to HC only; G. proliferum extended over 53.7(42.2–59.7)%of the total Petri plate surface, of which 67.1% corresponded to HC; and Gi. margarita colonized a mean of 25.5(21.4–32.5)%of the total Petri plate surface, of which only a 22.5% corresponded to the HC (77.5% CC).
Experiment 2: Dixenic cultures in common culture and hyphal compartments.— –
Ten days into the experiment most G. proliferum and G. intraradices inoculum cubes presented hyphae that had grown out of them, irrespective of the dixenic culture they were growing in. Some spores of Gi. margarita also had germinated by that time and more germinated in the next 2 wk. At the end of the experiment 100% of Glomus inoculum cubes had germinated (i.e. presented growing hyphae exiting from them) (TABLE I). In contrast only 67% of Gi. margarita spores germinated in dixenic culture with G. intraradices; this proportion dropped to 60% when in dixenics with G. proliferum (TABLE I). In the Gi. margarita/G. intraradices dixenic culture all Gigaspora cultures in which spores had germinated continued growing (i.e. 100% of germinated), and half of these developed in the HC after root colonization. For Gi. margarita/G. proliferum dixenic cultures just 20% of the cases in which Gigaspora had produced germ-tubes kept growing after germination, all of these reaching the HC after root colonization and developing within it (Table I).
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). A generic Petri plate of G. proliferum/G. intraradices culture is depicted; all the other nine replicates exactly following this same pattern of growth. Both AMF freely developed all over the CC and HC, apparently taking no notice of the presence of the other species (FIG. 4, top). When considering the two Glomus/Gigaspora combinations things were slightly different: When one of the fungi developed extensively in the CC, the partner usually was less invasive (e.g. FIG. 4, plates 2 and 4). In certain occasions a kind of status quo was reached by the two AMF, developing altogether all over the Petri plate (FIG. 4, Plate 1, 2 and 7). In those cases a slight reduction in spore/auxiliary cell production was noted in both fungi. In general G. proliferum appeared to be less aggressive extraradically than G. intraradices, which was clearly the most invasive strain of the three studied (in both experiments).
AM fungal competition within the host root.— –
Because the real sink for nutrients in arbuscular mycorrhizas is within the host root where AMF acquire carbohydrates for life, a competition for colonization of the best root’s sites could be expected. To test this hypothesis roots of dixenic Glomus/Gigaspora cocultures from Experiment 2 were analyzed for intraradical colonization. We analyzed only two fungal combinations (i.e. G. intraradices/Gi. margarita and G. proliferum/Gi. margarita) because of the feasibility of distinguishing between their intraradical structures (FIG. 5). For each fungal coculture two sites of the Petri plate were tested, each one closer to one of the inoculum sources (FIG. 4, plates 1 and 7, sites A, B, C and D).
The results after quantification of intraradical colonization of the above-mentioned samples are shown (TABLE II). These correspond to one representative Petri plate for each fungal combination. Photomicrographs of different root sites, colonized by one or two of the AMF, are shown (FIG. 5). For the G. proliferum + Gi. margarita + DC2 combination (TABLE II), 100% of the root segments close to the G. proliferum inoculum zone (FIG. 4, Plate 7A) presented colonization, of which the majority corresponded to G. proliferum-only colonization (52%). No root pieces in this area appeared colonized by Gi. margarita alone; nevertheless cocolonization by the two AMF was frequent (48% of root pieces), although dixenically colonized root pieces held a much more intense G. proliferum (25.5%) than Gi. margarita (2.7%) colonization. Ninety-six percent of root pieces extracted from the Gi. margarita inoculum zone (FIG. 4, Plate 7B) were mycorrhizal, of which just 8% were colonized by both AM fungi. In this case 20% of the root segments were colonized by Gi. margarita only and 68% by G. proliferum only, despite the distance (ca. 6 cm) separating the inoculum source of this fungus from that zone. Most surprising, colonization intensity reached only 0.8% for Gi. margarita, whereas it was a 17% in the case of G. proliferum.
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), just half (52%) of the root segment closer to the G. intraradices inoculum zone (FIG. 4, Plate 1C) presented colonization, of which 22% corresponded to G. intraradices–only colonization and 19% to Gi. margarita–only colonization. Cocolonization by the two AMF reached 11%. Colonization intensity was low for both AMF, being 1% for G. intraradices and 0.9% for Gi. margarita. Sixty-five percent of root pieces extracted from the Gi. margarita inoculum zone (FIG. 4, Plate 1D) were mycorrhizal, of which 10% were colonized by both AM fungi. The highest proportion of root segment colonized by Gi. margarita only was reached here (30%). This was the only occasion in which Gigaspora beat Glomus (only 25% of root pieces were colonized by G. intraradices alone). Colonization intensity for Gi. margarita was slightly higher but within the range of the other analyzed sites (3.9%); it remained low (2.4%) in the case of G. intraradices.
At any rate morphological changes in hyphae indicating signs of antagonism for site colonization were noticed when observing intraradical structures of roots in dixenic culture (FIG. 5); however when growing within the same root site Gigaspora and Glomus maintained a certain distance, occupying different cell layers and never touching each other.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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). This phenomenon has been divided commonly into "interference competition" and "exploitation competition"; however because competition for nutrients is usually brought about by competition for space, the former division is not appropriate (Boddy 2000). In saprotrophic microorganisms such as wood-decaying higher fungi (Boddy 2000) competition could be observed as a two-phase event. The first step ("primary resource capture") is characterized by such factors as good dispersal, rapid spore germination, rapid mycelial extension and the ability to use compounds available in previously uncolonized substrates. On the second step ("secondary resource capture") the fungus must hold and defend the conquered territory, either by means of exudation of antibiotic compounds, which would avoid direct contact with any prospective challenger, or by contacting the invader hyphae causing such reactions as gross mycelium contact, hyphal interference or even mycoparasitism (Boddy 2000). Results of fungal interactions depend on such factors as environmental conditions (e.g. substrate temperature, pH and atmospheric CO2 pressure), the physiological/nutritional status of the isolates involved and most important the intrinsic nature of the species/isolates involved (i.e. species combativity/aggresivity). Indeed there is a full range in hierarchy for species competitiveness, from those showing strong competitive effects (i.e. they are good at suppressing other’s resources to compete) to those showing good competitive responses (i.e. they are good at defending the acquired benefits against new invaders). The picture becomes more complicated when considering that the presence of a third competing fungus may modify responses of two previously competing species and that mycelial interactions are generally dynamic (Schoeman et al 1996); these interactions may result in deadlock or mutual or partial or total replacement by means of different mechanisms (overgrowth and lytic events). As a consequence of species competition morphological/developmental changes in fungal colonies are observed, often due to changes in enzyme production, but sometimes also due to nutrient re-allocation within competing mycelia. Overall the results presented here suggest that little competition exists among AM fungal strains for exploring and exploiting both mineral (extraradical) and host-derived (intraradical) nutrients. Nevertheless some effects were noted.
AM fungal competition for extraradical sites.— – Our results strongly suggest the existence of at least two distinct substrate colonization strategies in AMF. Glomus-type developmental strategy consists of an invasive behavior, in which the main goal for extraradical hyphae is to colonize distant substrate zones, profusely sporulating on these, rather than remaining close to the mother host root. Fungi presenting this colonization strategy seem to cohabit well with any other extraradical mycelia present in the distant zones invaded; no antagonistic behavior is noted anyway. When the encountered hyphae are of Glomus type no hindrance of mycelial development is noted. If hyphae are of the second substrate-colonization strategy type (i.e. Gigaspora-type) growing near their mother host root, a certain growth restriction of the Glomus-type hyphae occurs and a certain segregation of both hyphal types within the substrate is noted (meaning both hyphal types developing at different substrate layers).
In the second type of substrate-colonization strategy presented by AMF (Gigaspora-type) extraradical hyphae develop preferentially near mother host roots, within the host influence area. Such strategy seems not to be a consequence of Gigaspora hyphae being unable to jump over any physical barrier (indeed they can do so) but rather of an intrinsic affinity of Gigaspora ERM to host roots. It is tempting to speculate that certain root exudates somehow retain Gigaspora extraradical hyphae, thus restricting their development far from the root. AM in vitro polyxenic culture systems, such as those presented here, could be of use in the study of the fascinating yet elusive issue of the effect of root exudates on AMF presymbiotic and symbiotic behavior (Buée et al 2000, Gadkar et al 2003). In addition a certain exclusion of any foreign invader hyphae is noted in zones already colonized by a Gigaspora-type fungus; further research is needed to reveal the bases of such exclusion.
Some Gi. margarita extraradical hyphae present negative geotropism, which occasionally makes them develop aerially within the roots and at the bottom of the Petri plate lid, where they coil and extensively form auxiliary cells. This is, no doubt, a further indication of the intrinsic developmental strategy of this AM fungus, perhaps of the whole Gigaspora genus, and is in agreement with the well known negative geotropism of Gigaspora germ-tubes (Bécard and Fortin 1988). In contrast Glomus extraradical hyphae usually develop within the culture medium, almost never becoming aerial. We also might speculate that such behaviors are related to the distinct ecological niches usually occupied by Gigaspora and Glomus; whereas the former is frequently associated with well oxygenated substrates (e.g. sand dunes), the latter is more ubiquitous and frequently associated with clayey soils with lower aeration.
Using a single-compartment culture box Tiwari and Adholeya (2002) claimed that no antagonism (i.e. no true competition) between a Gigaspora and a Glomus isolate was observed; nevertheless the authors noted a certain distribution of both fungal colonies in layers (i.e. Gigaspora developing mostly on the medium surface, Glomus growing within the culture medium). The authors attributed these results to a side effect mediated by the negative geotropism of Gigaspora. However our results indicate that, aside from such an effect, there is indeed some kind of deleterious interaction when a Glomus and a Gigaspora species develop in the same root compartment. This effect translates to a reduction of spore germination and spore formation by both fungi. In contrast no clear antagonism/interaction occurs when two Glomus species develop in the same compartment, either in or out of the influence zone of the mother host root. Further research should be carried out to test the possibility of a certain antagonism among species within the Gigaspora genus, as was reported by Douds and Bécard (1993). If confirmed this could be considered a characteristic behavior intrinsic to members of the Gigaspora genus, rather than a general mechanism for competition in AM fungi. In any case our results suggest that formulation of AMF species for in vitro inoculants is possible without undesired collateral negative interactions among isolates, although some attention must be paid to the compartments where the different AMF are initially cultured.
AM fungal competition for intraradical niches.— –
Considering AM fungal development on sites where carbon resources are acquired (intraradical colonization), our results indicate an avoidance strategy rather than a true competition among isolates. No interactions or even hyphal contact seem to occur; the colonized root seems to be divided into fungi growth zones. G. intraradices, which extraradically was the most invasive strain, appears to be much less aggressive intraradically. Indeed only 33% of the root segments contained this fungus within its own inoculum zone. This percentage was maintained at 35% in roots sampled close to the Gi. margarita inoculum zone, indicating that intraradically these two fungi can develop closely with no apparent interaction. G. intraradices intraradical colonization intensity was low in all cases (<4%), however more than 20 000 spores were formed by the extraradical mycelium of this fungus in all the replicate plates. The low intraradical colonization percentage obtained for G. intraradices in vitro is common, and it clearly indicates that the capacity for AMF to develop an extraradical mycelium is independent of their capacity for root colonization (Graham et al 1982) but closely related to the lipid translocation ability of the fungus (Bago et al 2002). G. proliferum seem to be the best intraradical colonizer of the three species, both in terms of percentage of roots colonized by this fungus (100% within its inoculum zone, 76% in the Gigaspora inoculum zone) and in terms of colonization intensity (25.5% within its inoculum zone, 17% in the Gigaspora inoculum zone). More than 30% of roots appeared colonized by Gi. margarita in all the sampled sites, and this fungus seems to willingly share the root with the Glomus species. Of note, no root segments were colonized by Gi. margarita alone at the G. proliferum inoculum zone; this reinforce the apparent aggressivity shown by this fungus intraradically, which is further supported by the 68% root pieces colonized by this fungus only at its inoculum zone.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONLITERATURE CITED |
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, 1989) and Frankia (Sempavalan et al 1995) have been reported when colonizing common host plants. The mechanisms at work in these responses could be at the base of the mutualism/saprophytism/parasitism continuum (Smith and Smith 1996, Johnson et al 1997) and certainly would merit further studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. Email: abago{at}eez.csic.es
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LITERATURE CITED |
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———, Cano C. 2005. Breaking myths in AM in vitro biology. In: Declerck S, Strullu DG, Fortin A, eds. In vitro culture of mycorrhizas. Soil Biology Series. Springer-Verlag Berlin Heidelberg. p 111–138.
———, ———, Azcón-Aguilar C, Samson J, Coughlan AP, Piché Y. 2004. Differential morphogenesis of the extraradical mycelium of an arbuscular mycorrhizal fungus grown monoxenically on spatially heterogeneous culture media. Mycologia 96:452–462.
———, Vierheilig H, Piché Y, Azcón-Aguilar C. 1996. Nitrate depletion and pH changes induced by the extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic culture. New Phytol 133:273–280.[CrossRef]
———, Zipfel W, Williams RC, Jun J, Arreola R, Pfeffer PE, Lammers PJ, Shachar-Hill Y. 2002. Translocation and utilization of fungal lipid in the arbuscular mycorrhizal symbiosis. Plant Physiol 128:108–124.
Barea JM, Azcón-Aguilar C, Azcón R. 1987. Vesicular-arbuscular mycorrhizas improve both symbiotic N2-fixation and N uptake from soil as assessed with a 15N technique under field conditions. New Phytol 106:717–725.[CrossRef]
———, ———, ———. 1992. Vesicular-arbuscular mycorrhizal fungi in nitrogen-fixing systems. Methods Microbiol 24:391–416.
Bécard G, Fortin A. 1988. Early events of vesicular-arbuscular mycorrhiza formation on Ri T-DNA transformed roots. New Phytol 108:211–218.[CrossRef]
Boddy L. 2000. Interspecific combative interactions between wood-decaying basidiomycetes. FEMS Microbiol Ecol 31:185–194.[CrossRef][Medline]
Buee M, Rossignol M, Jauneau A, Ranjeva R, Bécard G. 2000. The pre-symbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates. MPMI 13:693–698.
Chabot S, Bécard G, Piché Y. 1992. Life cycle of Glomus intraradix in root organ culture. Mycologia 84:315–321.[CrossRef]
Declerck S, Cranenbrouck S, Dalpé Y, Séguin S, Grandmougin-Ferjani A, Fontaine J, Sancholle M. 2000. Glomus proliferum sp. nov.: a description based on morphological, biochemical, molecular and monoxenic cultivation data. Mycologia 92:1178–1187.[CrossRef]
———, Strullu DG, Fortin A, eds. 2005. In vitro biology of mycorrhizal symbiosis. Soil Biology Series, Berlin Heidelberg: Springer-Verlag. 388 p.
Douds DD, Bécard G. 1993. Competitive interaction between Gigaspora margarita and Gigaspora gigantea in vitro. Proceedings of the Ninth North American Conference on Mycorrhizas. Guelph, Ontario, Canada.
Fortin JA, Bécard G, Declerck S, Dalpé Y, St-Arnaud M, Coughlan AP, Piché Y. 2002. Arbuscular mycorrhiza on root-organ cultures. Can J Bot 80:1–20.[CrossRef]
Gadkar V, David-Schwartz R, Nagahashi G, Douds DD, Wininger S, Kapulnik Y. 2003. Root exudate of pmi tomato mutant M161 reduces AM fungal proliferation in vitro. FEMS Microbiol Letters. 223:193–198.[CrossRef][Medline]
Graham JH, Linderman RG, Menge JA. 1982. Development of external hyphae by different isolates of mycorrhizal Glomus spp. in relation to root colonization of troyer citrange. New Phytol 91:183–189.[CrossRef]
Johnson NC, Graham JH, Smith FA. 1997. Functioning of mycorrhizal associations along the mutualism-parasitism continuum. New Phytol 135:575–585.[CrossRef]
Keddy P. 1989. Competition. New York, USA: Chapman and Hall.
Mosse B, Hepper CM. 1975. Vesicular-arbuscular infections in root organ cultures. Physiol Plant Pathol 5:215–223.
Mugnier J, Mosse B. 1987. Vesicular-arbuscular infections in transformed root-inducing T-DNA roots grown axenically. Phytopathology 77:1045–1050.[CrossRef]
Phillips JM, Hayman DS. 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycol Soc 55:158–160.
Schoeman MW, Webber JF, Dickinson DJ. 1996. The effect of diffusible metabolites of Trichoderma harzianum on in vitro interactions between basidiomycete isolates at two different temperature regimes. Mycol Res 100:1454–1458.
Sempavalan J, Wheeler CT, Hooker JE. 1995. Lack of competition between Frankia and Glomus for infection and colonization of roots of Casuarina equisetifolia (L.). New Phytol 130:429–436.[CrossRef]
Smith FA, Smith SE. 1996. Mutualism and parasitism: biodiversity in function and structure in the ‘arbuscular’ (VA) mycorrhizal symbiosis. Adv Bot Res 22:1–43.
St-Arnaud M, Hamel C, Vimard B, Caron M, Fortin JA. 1996. Enhanced hyphal growth and spore production of the arbuscular mycorrhizal fungus G. intraradices in an in vitro system in the absence of host roots. Mycol Res 100:328–332.
Tiwari P, Adholeya A. 2002. In vitro co-culture of two AMF isolates Gigaspora margarita and Glomus intraradices on Ri T-DNA transformed roots. FEMS Microbiol Letters 206:39–43.[CrossRef][Medline]
Trouvelot A, Kough JL, Gianinazzi-Pearson V. 1986. Mesure du taux de mycorhyzation VA d’un système radiculaire. Recherche de methods d’estimation ayant une signification fonctionelle. In: Gianinazzi-Pearson V, Gianinazzi S, eds. Physiological and genetical aspects of mycorrhizae. Paris: INRA. p 217–221.
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