Basidiomycete Hebeloma Cylindrosporum Research Articles

The ectomycorrhizal basidiomycete Hebeloma cylindrosporum undergoes early waves of transcriptional reprogramming prior to symbiotic structures differentiation.

To clarify the early molecular interaction between ectomycorrhizal partners, we performed a RNA-Seq study of transcriptome reprogramming of the basidiomycete Hebeloma cylindrosporum before symbiotic structure differentiation with Pinus pinaster. Mycorrhiza transcriptome was studied for comparison. By reference to asymbiotic mycelium, 47 and 46 genes were specifically upregulated over fivefold (p ≤ 0.05) upon rhizosphere colonization and root adhesion respectively. Other 45 were upregulated throughout the symbiotic interaction, from rhizosphere colonization to differentiated mycorrhizas, whereas 274 were specifically upregulated in mycorrhizas. Although exoproteome represents 5.6% of H. cylindrosporum proteome, 38.5% of the genes upregulated upon pre-infectious root colonization encoded extracellular proteins. The proportion decreased to 23.5% in mycorrhizas. At all studied time points, mycorrhiza-induced small secreted proteins (MiSSPs), representing potential effectors, were over-represented among upregulated genes. This was also the case for carbohydrate-active enzymes (CAZymes). Several CAZymes were upregulated at all studied stages of the interaction. Consistent with a role in fungal morphogenesis and symbiotic interface differentiation, CAZymes over-expressed before and upon root attachment targeted fungal and both fungal and plant polysaccharides respectively. Different hydrophobins were upregulated upon early root adhesion, in mycorrhizas or throughout interaction. The functional classification of genes upregulated only in mycorrhizas pointed to intense metabolic activity and nutritional exchanges.

A fungal conserved gene from the basidiomycete Hebeloma cylindrosporum is essential for efficient ectomycorrhiza formation.

We used Agrobacterium-mediated insertional mutagenesis to identify genes in the ectomycorrhizal fungus Hebeloma cylindrosporum that are essential for efficient mycorrhiza formation. One of the mutants presented a dramatically reduced ability to form ectomycorrhizas when grown in the presence of Pinus pinaster. It failed to form mycorrhizas in the presence of glucose at 0.5 g liter(-1), a condition favorable for mycorrhiza formation by the wild-type strain. However, it formed few mycorrhizas when glucose was replaced by fructose or when glucose concentration was increased to 1 g liter(-1). Scanning electron microscopy examination of these mycorrhizas revealed that this mutant was unable to differentiate true fungal sheath and Hartig net. Molecular analyses showed that the single-copy disrupting T-DNA was integrated 6,884 bp downstream from the start codon, of an open reading frame potentially encoding a 3,096-amino-acid-long protein. This gene, which we named HcMycE1, has orthologs in numerous fungi as well as different other eukaryotic microorganisms. RNAi inactivation of HcMycE1 in the wild-type strain also led to a mycorrhizal defect, demonstrating that the nonmycorrhizal phenotype of the mutant was due to mutagenic T-DNA integration in HcMycE1. In the wild-type strain colonizing P. pinaster roots, HcMycE1 was transiently upregulated before symbiotic structure differentiation. Together with the inability of the mutant to differentiate these structures, this suggests that HcMycE1 plays a crucial role upstream of the fungal sheath and Hartig net differentiation. This study provides the first characterization of a fungal mutant altered in mycorrhizal ability.

Phosphorus acquisition from phytate depends on efficient bacterial grazing, irrespective of the mycorrhizal status of Pinus pinaster

Phosphorus from phytate, although constituting the main proportion of organic soil P, is unavailable to plants. Despite the well-known effects of rhizosphere trophic relationships on N mineralization, no work has been done yet on P mineralization. We hypothesized that the interactions between phytate-mineralizing bacteria, mycorrhizal fungi and bacterial grazer nematodes are able to improve plant P use from phytate. We tested this hypothesis by growing Pinus pinaster seedlings in agar containing phytate as P source. The plants, whether or not ectomycorrhizal with the basidiomycete Hebeloma cylindrosporum, were grown alone or with a phytase-producing bacteria Bacillus subtilis and two bacterial-feeder nematodes, Rhabditis sp. and Acrobeloides sp. The bacteria and the nematodes were isolated from ectomycorrhizal roots and soil from P. pinaster plantations. Only the grazing of bacteria by nematodes enhanced plant P accumulation. Although plants increased the density of phytase-producing bacteria, these bacteria alone did not improve plant P nutrition. The seedlings, whether ectomycorrhizal or not, displayed a low capacity to use P from phytate. In this experiment, the bacteria locked up the phosphorus, which was delivered to plant only by bacterial grazers like nematodes. Our results open an alternative route for better utilization of poorly available organic P by plants.

Hydrophobins in ectomycorrhizas: heterologous transcription of the Pisolithus HydPt-1 gene in yeast and Hebeloma cylindrosporum.

Hydrophobins are fungal cell wall proteins involved in aggregation of hyphae. Upon the development of the ectomycorrhizal symbiosis between tree roots and fungal hyphae, the transcripts of hydrophobin genes markedly accumulated. As the precise role of these proteins in symbiosis is not yet known, we develop heterologous expression system of the Pisolithus hydrophobin HYDPt-1. This gene has been introduced in Saccharomyces cerevisiae and in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. Introns were required for hydPt-1 transcript accumulation in the basidiomycete H. cylindrosporum. Heterologous transcript accumulation did not alter the phenotype of either species. The lack of altered phenotype resulted from the absence of HYDPt-1 polypeptide accumulation in transformed strains.

Two differentially regulated phosphate transporters from the symbiotic fungus Hebeloma cylindrosporum and phosphorus acquisition by ectomycorrhizal Pinus pinaster

Ectomycorrhizal symbiosis markedly improves plant phosphate uptake, but the molecular mechanisms underlying this benefit are still poorly understood. We identified two ESTs in a cDNA library prepared from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum with significant similarities to phosphate transporters from the endomycorrhizal fungus Glomus versiforme and from non-mycorrhizal fungi. The full-length cDNAs corresponding to these two ESTs complemented a yeast phosphate transport mutant (Deltapho84). Measurements of (33)P-phosphate influx into yeast expressing either cDNA demonstrated that the encoded proteins, named HcPT1 and HcPT2, were able to mediate Pi:H(+) symport with different affinities for Pi (K(m) values of 55 and 4 mum, respectively). Real-time RT-PCR showed that Pi starvation increased the levels of HcPT1 transcripts in H. cylindrosporum hyphae grown in pure culture. Transcript levels of HcPT2 were less dependent on Pi availability. The two transporters were expressed in H. cylindrosporum associated with its natural host plant, Pinus pinaster, grown under low or high P conditions. The presence of ectomycorrhizae increased net Pi uptake rates into intact Pinus pinaster roots at low or high soil P levels. The expression patterns of HcPT1 and HcPT2 indicate that the two fungal phosphate transporters may be involved in uptake of phosphate from the soil solution under the two soil P availability conditions used.

Expression of the nitrate transporter nrt2 gene from the symbiotic basidiomycete Hebeloma cylindrosporum is affected by host plant and carbon sources

Although the function of the extramatrical mycelium of ectomycorrhizal fungi is considered essential for the acquisition of nitrogen by forest trees, gene regulation in this fungal compartment is poorly characterized. In this study, the expression of the nitrate transporter gene nrt2 from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was shown to be regulated by plant host and carbon sources. In the presence of a low fructose concentration, nrt2 expression could not be detected in the free-living mycelium but was high in the extramatrical symbiotic mycelium associated to the host plant Pinus pinaster. In the absence of nitrogen or in the presence of nitrate, high sugar concentrations in the medium were able to enhance nrt2 expression. Nevertheless, in the presence of high fructose concentration, high ammonium concentration still completely repressed nrt2 expression indicating that the nitrogen repression overrides sugar stimulation. This is the first report revealing an effect of host plant and of carbon sources on the expression of a fungal nitrate transporter-encoding gene.

Green fluorescent protein expression in the symbiotic basidiomycete fungusHebeloma cylindrosporum

The symbiotic basidiomycete Hebeloma cylindrosporum is a model fungal species used to study ectomycorrhizal symbiosis at the molecular level. In order to have a vital marker, we developed a green fluorescent protein (GFP) reporter system efficiently expressed in H. cylindrosporum using the sgfp coding region bordered by two introns fused to the saprophytic basidiomycete Coprinopsis cinerea cgl1 promoter. Expression of this reporter system was tested under different environmental conditions in two transformants, and glucose was shown to repress gfp expression. Such a reporter system will be used in plant-fungus interaction to evaluate sugar supply by the plant to the compatible mycorrhizal symbiont and to compare the expression of various genes of interest in the free-living mycelia, in the symbiotic (mycorrhizas) and the reproductive (fruit bodies) structures formed by H. cylindrosporum.

Hebeloma cylindrosporum- a model species to study ectomycorrhizal symbiosis from gene to ecosystem.

The basidiomycete Hebeloma cylindrosporum has been extensively studied with respect to mycorrhiza differentiation and metabolism and also to population dynamics. Its life cycle can be reproduced in vitro and it can be genetically transformed. Combined biochemical, cytological, genetical and molecular approaches led to the characterisation of mutant strains affected in mycorrhiza formation. These studies demonstrated the role of fungal auxin as a signal molecule in mycorrhiza formation and should allow the characterisation of essential fungal genes necessary to achieve a compatible symbiotic interaction. Random sequencing of cDNAs has identified numerous key functional genes which allowed dissection of essential nitrogen assimilation pathways. H. cylindrosporum also proved to be a remarkable model species to uncover the dynamics of natural populations of ectomycorrhizal fungi and the way in which they respond and adapt to anthropogenic disturbance of the forest ecosystem. Although studies on mycorrhiza differentiation and functioning and those on the population dynamics of H. cylindrosporum have been carried out independently, they are likely to converge in a renewed molecular ecophysiology which will envisage how ectomycorrhizal symbiosis functions under varying field conditions. Contents Summary 481 I. Introduction 482 II. Taxonomy, distribution, autecology, and host range of H. cylindrosporum 482 III. The Hebeloma cylindrosporum toolbox 483 IV. Mycorrhiza differentiation 486 V. Nutritional interactions 488 VI. Genetic diversity and dynamics of H. cylindrosporum populations in P. pinaster forest ecosystems 491 VII. Future directions 494 Acknowledgements 494 References 494.

Dynamics of ectomycorrhizal mycelial growth and P transfer to the host plant in response to low and high soil P availability

The aim of this work was to investigate the quantitative relationships between the rates of fungal soil exploration and their effects on plant growth and phosphorus (P) nutrition in soil with varying P availability. Ectomycorrhizal associations were established between Pinus pinaster and the basidiomycete Hebeloma cylindrosporum. Plants were grown for 4 and 6 months in mini-rhizoboxes filled with a 0.5 mm soil layer with two contrasting P levels (−P and +P soils), containing 3 or 50 mg of bicarbonate extractable P per kg of dry soil, respectively. Surface areas of the soil layers colonised by the roots and the hyphae were estimated using image analysis. High P availability decreased the rates of fungal soil colonisation, calculated as 0.92 ± 0.19 cm 2 day −1 plant −1 in the −P soil and 0.42 ± 0.1 cm 2 day −1 plant −1 in the +P soil over the 4–6 months period. Four-month old mycorrhizal plants accumulated lower amounts of biomass and total P than non-mycorrhizal plants, regardless the level of P availability. By contrast, 6-month old mycorrhizal plants were larger and contained more P than non-mycorrhizal plants, especially in the +P soil. However, mycorrhizal plants were always different from non-mycorrhizal P-deficient plants, which had an increased root surface and root P allocation. To explain these contradictory results, we propose that P accumulation by mycorrhizal plants derives mainly from fungal P uptake. The net P transfer from the fungus to the plant was estimated as 0.36 and 0.66 μmol of P per cm 2 of mycelium in −P and +P soil, respectively. Our data demonstrated that, despite the inhibitory effect of the high soil P availability on the rates of fungal soil colonisation, the ectomycorrhizal symbiosis was more efficient to improve host plant P nutrition in these conditions.

An expression cDNA library for suppression cloning in yeast mutants, complementation of a yeast his4 mutant, and EST analysis from the symbiotic basidiomycete Hebeloma cylindrosporum.

An oriented expression library was constructed from the mycelia of the symbiotic model fungus Hebeloma cylindrosporum in the high-level yeast expression vector pDR196. DNA sequencing of approximately 500 expressed sequence tags (ESTs) showed that 15% correspond to known genes, two thirds contain sequences with unknown function, andthe remaining 20% showed no significant similarity to any known genes. The ESTs had a GC content between 44 and 56%, with most of them having a GC content of 52-54%, which could be correlated with GC contents of fungal genes. The library was successfully used to identify the Hebeloma HIS4 gene by functional complementation of a yeast his4 mutant. Thus, the library may serve as a powerful tool for identification and characterization of mycorrhizal genes by EST analysis and for the identification of ectomycorrhizal genes by means of suppression cloning.

Characterisation and expression analysis of a nitrate transporter and nitrite reductase genes, two members of a gene cluster for nitrate assimilation from the symbiotic basidiomycete Hebeloma cylindrosporum

Symbiotic ectomycorrhizal fungi contribute to the nitrogen nutrition of their host-plants but little information is available on the molecular control of their nitrogen metabolism. We cloned and characterised genes encoding a nitrite reductase and a nitrate transporter in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. These two genes are divergently transcribed and linked to a previously cloned nitrate reductase gene, thus demonstrating that nitrate assimilation gene clusters occur in homobasidiomycetes. The nitrate transporter polypeptide (NRT2) is characterised by 12 transmembrane domains and presents both a long putative intracellular loop and a short C-terminal tail, two structural features which distinguish fungal high-affinity transporters from their plant homologues. In different wild-type genetic backgrounds, transcription of the two genes was repressed by ammonium and was strongly stimulated not only in the presence of nitrate but also in the presence of organic nitrogen sources or under nitrogen deficiency.

Spatial distribution of the below-ground mycelia of an ectomycorrhizal fungus inferred from specific quantification of its DNA in soil samples

In natural forest ecosystems several ectomycorrhizal fungal species cohabit on host plant root systems. To evaluate the ecological and functional impact of each species, it is necessary to appreciate the distribution and abundance of its mycelia in the soil. We developed a competitive PCR (cPCR) method for the basidiomycete Hebeloma cylindrosporum that allows quantification of its DNA in complex DNA mixtures extracted directly from soil samples. The target sequence chosen for the cPCR analysis was a 533-bp fragment of the nuclear ribosomal intergenic spacer, amplified using two species-specific primers. The detection threshold of the cPCR protocol developed was 0.03 pg of genomic DNA. This method was applied to soil samples collected from beneath and at various distances from a group of fruit bodies in a Pinus pinaster forest stand. The results revealed that H. cylindrosporum below-ground biomass was concentrated directly underneath the fruit bodies or very close to them, while no DNA of this species could be detected in soil samples collected at more than 50 cm away. In the vicinity of fruit bodies, H. cylindrosporum soil DNA concentration varied considerably (between 10 and 0.07 ng g soil(-1)) and decreased sharply with increased distance from the fruit bodies. This work demonstrates the potential of competitive quantitative PCR for the study of the distribution, abundance and persistence of the mycelia of an ectomycorrhizal fungal species in soil.

Spatial distribution of the below-ground mycelia of an ectomycorrhizal fungus inferred from specific quantification of its DNA in soil samples

In natural forest ecosystems several ectomycorrhizal fungal species cohabit on host plant root systems. To evaluate the ecological and functional impact of each species, it is necessary to appreciate the distribution and abundance of its mycelia in the soil. We developed a competitive PCR (cPCR) method for the basidiomycete Hebeloma cylindrosporum that allows quantification of its DNA in complex DNA mixtures extracted directly from soil samples. The target sequence chosen for the cPCR analysis was a 533-bp fragment of the nuclear ribosomal intergenic spacer, amplified using two species-specific primers. The detection threshold of the cPCR protocol developed was 0.03 pg of genomic DNA. This method was applied to soil samples collected from beneath and at various distances from a group of fruit bodies in a Pinus pinaster forest stand. The results revealed that H. cylindrosporum below-ground biomass was concentrated directly underneath the fruit bodies or very close to them, while no DNA of this species could be detected in soil samples collected at more than 50 cm away. In the vicinity of fruit bodies, H. cylindrosporum soil DNA concentration varied considerably (between 10 and 0.07 ng g soil −1) and decreased sharply with increased distance from the fruit bodies. This work demonstrates the potential of competitive quantitative PCR for the study of the distribution, abundance and persistence of the mycelia of an ectomycorrhizal fungal species in soil.

Forest habitat characteristics affect balance between sexual reproduction and clonal propagation of the ectomycorrhizal mushroom Hebeloma cylindrosporum

We investigated how habitat characteristics and in particular the level of disturbance was affecting colonisation processes and population dynamics of an ectomycorrhizal fungus. The basidiomycete Hebeloma cylindrosporum associated with Pinus pinaster trees occurs in two distinct habitats within the forest dune ecosystem along the Atlantic coast of France. The ‘dune habitat’ (D habitat) is characterised by scattered trees growing in almost pure sand at the extreme fringe of the forest marking the transition with the bare dune. The ‘forest habitat’ (F habitat) is within the mature forest in places of high human frequentation which prevents litter and humus accumulation. We studied over several years (up to ten) populations of H. cylindrosporum present in three geographic sites. Within each site we studied two areas corresponding each to one of the two possible habitats. In all sampled areas fruit bodies occurred within discrete patches of ground. Although an interaction between habitat and geographic site existed, habitat had significant effects on patch sizes and patch internal densities. Compared to the F habitat, patches in the D habitat were significantly larger and presented a lower density of fruit bodies. Patches very seldom occurred at the same place from one year to the next in the F habitat when in most cases they were perennial and increased in size in the D habitat. Fruit body size, which reflects reproductive output of the genets, differed between the two habitats (means of the cap diameters of 25.6 and 40.4 mm in respectively the F and D habitats). Almost all patches in the F habitat were made of several annual genets each producing on average 1.6 fruit bodies while most patches in the D habitat were dominated by one perennial genet which could simultaneously produce up to 60 fruit bodies. Under‐representation of small sized genets in the D habitat suggests a low level of sexual recruitment in this habitat despite the large number of fruit bodies (and therefore of spores) produced by each of the existing genets. We therefore suggest that H. cylindrosporum could be considered as a fugitive species. Genets of this species survive in the D habitat in which few ectomycorrhizal fungal competitors occur. This habitat could represent the optimal habitat for this species. In the F habitat frequent recruitment could occur in ‘empty patches’ resulting from the local and transient elimination of competitors induced by human disturbance.

Transcription of a nitrate reductase gene isolated from the symbiotic basidiomycete fungus Hebeloma cylindrosporum does not require induction by nitrate.

Ectomycorrhizal fungi contribute to the nitrogen nutrition of their host plants, but no information is available on the molecular control of their nitrogen metabolism. The cloning and pattern of transcriptional regulation of two nitrite reductase genes of the symbiotic basidiomycete Hebeloma cylindrosporum are presented. The genomic copy of one of these genes (nar1) was entirely sequenced; the coding region is interrupted by 12 introns. The nar1 gene, which is transcribed and codes for a putative 908-amino acid polypeptide complemented nitrate reductase-deficient mutants of H. cylindrosporum upon transformation, thus demonstrating that the gene is functional. The second gene (nar2), for which no mRNA transcripts were detected, is considered to be an ancestral, non-functional duplication of nar1. In a 462-nt partial sequence of nar2 two introns were identified at positions identical to those of introns 8 and 9 of nar1, although their respective nucleotide sequences were highly divergent; the exon sequences were much more conserved. In wild-type strains, transcription of nar1 is repressed in the presence of a high concentration of ammonium. High levels of transcription are observed in the presence of either very low nitrogen concentrations or high concentrations of nitrate or organic N sources such as urea, glycine or serine. This indicates that in H. cylindrosporum, in contrast to all nitrophilous organisms studied so far, an exogenous supply of nitrate is not required to induce transcription of a nitrate reductase gene. In contrast, repression by ammonium suggests the existence of a wide-domain regulatory gene, as already characterized in ascomycete species.

Some potential inaccuracies of the p -nitrophenyl phosphomonoesterase assay in the study of the phosphorus nutrition of soil borne fungi

The p-nitrophenol phosphomonoesterase assay (pNPPase) is commonly used to measure cell-wall-associated and extracellular phosphatase activity of soil fungi. pNPPases are usually assayed in the context of fungal nutrition, where inorganic P supply might be enhanced by the mineralisation of organic P sources in the soil. We report here on a series of experiments with the ectomycorrhizal basidiomycete Hebeloma cylindrosporum that highlight components of accepted methodology that might impinge on the reliability of the assay. These include the loss of pNPPase after filtration, inaccuracies in measuring wall-associated enzyme and the ample pool of intracellular pNPPase can be mistakenly measured as external pNPPase if cells are accidentally damaged.

Isolation and characterization of nitrate reductase deficient mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum

To clarify the role of the fungal nitrate assimilation pathway in nitrate reduction by mycorrhizal plants, nitrate reductase (NR)-deficient (NR- ) mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi have been selected. These mutants were produced by u.v. mutagenesis on protoplasts originating from homokaryotic mycelia belonging to complementary mating types of this heterothallic tetrapolar species. Chlorate-resistant mutants were first selected in the presence of different nitrogen (N) sources in the culture medium. Among 1495 chlorate resistant mycelia, 30 failed to grow on nitrate and lacked a detectable NR activity. Growth tests on different N sources suggested that the NR activity of all the different mutants is specifically impaired as a result of mutations in either the gene coding for NR apoprotein or genes controlling the synthesis of the molybdenum cofactor. Furthermore, restoration of NR activity in some of the dikaryons obtained after crosses between the different mutant mycelia suggested that not all the selected mutations mapped in the same gene. Utilization of N on a NH4 15 NO3 medium was studied for two mutant strains and their corresponding wild-type homokaryons. None of the mutants could use nitrate whereas 15 N enrichment values indicated that 13-27% of N present in 13-d-old wild-type mycelia originated from nitrate. Apparently, the mutant mycelia do not compensate their inability to use nitrate by a more efficient use of ammonium. These different NR mutants still form mycorrhizas with the habitual host plant, Pinus pinaster (Ait.), making them suitable for study of the contribution of the fungal nitrate assimilation pathway to nitrate assimilation by mycorrhizal plants.

Nitrate uptake in maritime pine (Pinus pinaster) and the ectomycorrhizal fungus Hebeloma cylindrosporum: effect of ectomycorrhizal symbiosis

Nitrate uptake rates were determined for nonmycorrhizal and mycorrhizal maritime pine (Pinus pinaster) associated with the ectomycorrhizal basidiomycete Hebeloma cylindrosporum and for this fungal species grown in vitro. Different [NO3−] (0–1.3 and 10 mM) were supplied to nonmycorrhizal or mycorrhizal P. pinaster plants grown axenically in test tubes for 21 weeks. Uptake of NO3− by the plants was determined by measuring NO3− depletion from the medium that was added to the culture tubes without root pertubation. For all applied [NO3−] the uptake rates for NO3− in mycorrhizal plants were increased about twofold compared with nonmycorrhizal plants. The same range of [NO3−] was supplied to the fungus in pure culture, and uptake rates were calculated either from the measurement of NO3− depletion from the medium or by 15N accumulation in the mycelium. For the same external nitrate concentrations the results obtained with these two methods were not significantly different. The amount of fungal biomass in mycorrhizal root systems was determined with the chitin assay. The contribution of each partner to nitrate uptake in the mycorrhizal plant was calculated from the kinetic parameters of nitrate uptake by the fungus and host plant alone given by the Lineweaver–Burk plots. For external [NO3−] higher than 0.1 mM the measured uptake rates for NO3− in mycorrhizal plants were greater than the calculated sum of the individual symbiotic partners. The significance of this excess nitrate uptake is discussed in relation to the role of ectomycorrhizae in nitrogen accumulation. Key words: NO3− uptake rates, Km, Vmax, ectomycorrhizal symbiosis, Hebeloma cylindrosporum, Pinus pinaster.

Genotype/nutrition interactions in the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnesi

SUMMARYIntraspecific variability in mycelial growth of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi has been studied within 20 sib‐monokaryons, each being the progeny of one wild dikaryon fruiting in vitro, and the 50 synthesized dikaryons obtained from all the compatible fusions of these monokaryons. The interaction between fungal diversity and nutrition was also studied.The range of variation in mycelial growth (expressed as mg protein per culture) among the sib‐monokaryons was the same as that recorded among the synthesized dikaryons. The average growth of both mono‐ and dikaryons was highest on a synthetic medium containing nitrate as the sole nitrogen source, but showed the greatest variation. Fungal growth was approximately halved when nitrate was replaced by ammonium, and was intermediate on an organic nutrient‐rich medium. A decrease from 24 mM to 24 /M concentration of phosphorus reduced the average growth of the mycelia 40‐fold.The variation in mycelial growth in the synthesized dikaryons involved additive and non‐additive genetic components. Their ratio was variable depending on the nutrient conditions. In all cases but one, the interactive component was higher than the additive one.From an ecological point of view, these results emphasize the tremendous effect of nutrition on the phenotypic expression of fungal growth. Their possible significance for the dynamics of natural populations of ectomycorrhizal fungi is discussed.

Isolation and preliminary characterization of 5‐fluoroindole‐resistant and IAA‐overproducer mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnesi

summaryIAA‐overproducer mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi were isolated in an attempt to clarify the role of fungal IAA in ectomycorrhiza formation. By contrast to the wild type, mutants are able to metabolize endogenous Trp to IAA. They were isolated using a two‐step method. One hundred and forty‐four mutants with altered regulation of the Trp branch of the shikimate pathway were isolated from two compatible monokaryons by selecting for resistance to the indole analogue, 5‐fluoroindole. These mutants are Trp‐overproducers and most of them accumulate free Trp. Seventeen mutants able to synthesize IAA in the absence of exogenous precursor (tryptophan or intermediates of the tryptophan biosynthetic pathway) were identified by screening for the ability to metabolize endogenous Trp into IAA. By studying the activity of key enzymes of the Trp biosynthetic pathway (i.e. anthranilate synthase and Trp synthase), it was evident that different mechanisms led to IAA‐overproduction. A correlation was detected between the level of intracellular Trp accumulation and the ability of the mutants to metabolize endogenous Trp to IAA. This indicates that, irrespective of the mutation, intracellular Trp accumulation is a prerequisite for IAA overproduction. A positive correlation was also detected between, on the one hand, resistance to fluoroindole and, on the other hand, intracellular Trp accumulation and, subsequently, IAA‐overproduction. Thus, resistance to high fluoroindole concentrations appeared to provide a method for the direct selection of IAA‐overproducer mutants. In order to be able to compare these mutants with the wild parental dikaryon and to perform genetic analyses, the mutants were fused with a compatible wild monokaryon. The resulting dikaryons were able to form mycorrhizas with the normal host plant, Pinus pinaster, and to fruit under controlled culture conditions, thus allowing stabilization and purification of the mutations. IAA‐overproducer monokaryons were detected within the progeny of all the mutants. They are now being used to study the role of fungal IAA in ectomycorrhizas.