Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery-grown Douglas fir by PCR of the rDNA intergenic spacer

The large-scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR-amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large-scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated-Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large-scale microbial application.     

Structure and dynamics of experimentally introduced and naturally occurring laccaria sp. Discrete genotypes in a douglas fir plantation

Ectomycorrhizal fungi have been introduced in forest nurseries to improve seedling growth. Outplanting of inoculated seedlings to forest plantations raises the questions about inoculant persistence and its effects on indigenous fungal populations. We previously showed (M.-A. Selosse et al. Mol. Ecol. 7:561-573, 1998) that the American strain Laccaria bicolor S238N persisted 10 years after outplanting in a French Douglas fir plantation, without introgression or selfing and without fruiting on uninoculated adjacent plots. In the present study, the relevance of those results to sympatric strains was assessed for another part of the plantation, planted in 1985 with seedlings inoculated with the French strain L. bicolor 81306 or left uninoculated. About 720 Laccaria sp. sporophores, collected from 1994 to 1997, were typed by using randomly amplified polymorphic DNA markers and PCR amplification of the mitochondrial and nuclear ribosomal DNAs. All plots were colonized by small spontaneous discrete genotypes (genets). The inoculant strain 81306 abundantly fruited beneath inoculated trees, with possible introgression in indigenous Laccaria populations but without selfing. In contrast to our previous survey of L. bicolor S238N, L. bicolor 81306 colonized a plot of uninoculated trees. Meiotic segregation analysis verified that the invading genet was strain 81306 (P < 0.00058), implying a vegetative growth of 1.1 m. year-1. This plot was also invaded in 1998 by strain S238N used to inoculate other trees of the plantation. Five other uninoculated plots were free of these inoculant strains. The fate of inoculant strains thus depends less on their geographic origin than on unknown local factors.     

The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) inhibits the stimulation of in vitro lateral root formation and the colonization of the tap-root cortex of Norway spruce (Picea abies) seedlings by the ectomycorrhizal fungus Laccaria bicolor

Norway spruce ( Picea abies (L.) Karst.) seedlings were inoculated with the ectomycorrhizal fungus Laccaria bicolor ((Marie) Orton), strain S238 N, in axenic conditions. The presence of the fungus slowed tap–root elongation by 26% during the first 15 d after inoculation and then stimulated it by 136%. In addition, it multiplied in vitro lateral root formation by 4.3, the epicotyl growth of the seedlings by 8.4 and the number of needles by 2. These effects were maintained when the fungus was separated from the roots by a cellophane membrane preventing symbiosis establishment, thus suggesting that the fungus acted by non-nutritional effects. We tested the hypothesis that IAA produced by L. bicolor S238 N would be responsible for the stimulation of fungal induced rhizogenesis. We showed in previous work that L. bicolor S238 N can synthesize IAA in pure culture. Exogenous IAA supplies (100 and 500 μ m ) reproduced the stimulating effect of the fungus on root branching but inhibited root elongation. The presence of 2,3,5-triiodobenzoic acid (TIBA) in the culture medium significantly depressed lateral root formation of inoculated seedlings. As TIBA had no significant effect on IAA released in the medium by L. bicolor S238 N, but counteracted the stimulation of lateral rhizogenesis induced by an exogenous supply of IAA, we suggest that TIBA inhibited the transport of fungal IAA in the root. Furthermore TIBA blocked the colonization of the main root cortex by L. bicolor S238 N and the formation of the Hartig net. These results specified the role of fungal IAA in the stimulation of lateral rhizogenesis and in ectomycorrhizal symbiosis establishment.     

Effect of dual inoculation of Douglas fir with the ectomycorrhizal fungus Laccaria laccata and mycorrhization helper bacteria (MHB) in two bare-root forest nurseries

Douglas fir (Pseudotsuga menziesii) seedlings in two bare-root forest nurseries were inoculated with the ectomycorrhizal fungus Laccaria laccata, together or not with one of five mycorrhization helper bacteria isolated from L. laccata sporocarps or mycorrhizas and previously selected by in vitro and glasshouse screenings. With the most efficient MHB isolates, when compared to the control with no bacteria, the percent of mycorrhizal short roots was increased from 60 to 90 or from 80 to 100, depending on the nursery, with inoculation doses as low as 10⁶ living cells per m². A dual inoculum made of calcium alginate beads containing the two microorganisms appears to be a valuable technique for increasing the efficiency of ectomycorrhizal inoculation of planting stocks in forest nurseries.     

Mycorrhiza formation and growth of Eucalyptus globulus seedlings inoculated with spores of various ectomycorrhizal fungi

 As many eucalypts in commercial plantations are poorly ectomycorrhizal there is a need to develop inoculation programs for forest nurseries. The use of fungal spores as inoculum is a viable proposition for low technology nurseries currently producing eucalypts for outplanting in developing countries. Forty-three collections of ectomycorrhizal fungi from southwestern Australia and two from China, representing 18 genera, were tested for their effectiveness as spore inoculum on Eucalyptus globulus Labill. seedlings. Seven-day-old seedlings were inoculated with 25 mg air-dry spores in a water suspension. Ectomycorrhizal development was assessed in soil cores 65 and 110 days after inoculation. By day 65, about 50% of the treatments had formed ectomycorrhizas. By day 110, inoculated seedlings were generally ectomycorrhizal, but in many cases the percentage of roots colonized was low (<10%). Species of Laccaria, Hydnangium, Descolea, Descomyces, Scleroderma and Pisolithus formed more ectomycorrhizas than the other fungi. Species of Russula, Boletus, Lactarius and Hysterangium did not form ectomycorrhizas. The dry weights of inoculated seedlings ranged from 90% to 225% of the uninoculated seedlings by day 110. Although plants with extensively colonized roots generally had increased seedling growth, the overall mycorrhizal colonization levels were poorly correlated to seedling growth. Species of Laccaria, Descolea, Scleroderma and Pisolithus are proposed as potential candidate fungi for nursery inoculation programs for eucalypts. Accepted: 7 May 1998     

Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete Laccaria bicolor in a French forest plantation

Selected strains of ectomycorrhizal fungi, such as the basidiomycete Laccaria bicolor , are currently being used as inoculants in nurseries to improve growth of forest trees after outplanting. Information is needed on the survival of these introduced strains in forests and their impact on indigenous biodiversity. Dissemination and persistence of an American strain, L. bicolor S238N, were studied 10 years after outplanting in a Douglas fir plantation located at Saint-Brisson (Morvan, France). About 430 Laccaria spp. sporophores were collected over 3 years. Inheritance of nuclear ribosomal DNA, as well as RAPD markers, was characterized in L. bicolor S238N, using a haploid progeny set of 91 monokaryons. More than 50 markers were identified (19 heterozygous and 33 homozygous or cytoplasmic markers), which unambiguously confirmed that the introduced strain was still present in the inoculated plots. Neither selfing ( P < 0.0008) nor introgression with indigenous strains was detected although in vitro interfertility between the American strain and indigenous L. bicolor was identified. No ingress of the introduced genet into adjacent uninoculated plots colonized by various local Laccaria genets was detected. It is proposed that the spatial distributions identified have developed through mycelial propagation of the introduced strain and intraspecific competition with native genets. Although longer-term data is still lacking, the stability of the inoculant strain and the limited disturbance to indigenous populations described support large-scale nursery production of this host-fungal combination.     

A comparison of arbuscular and ectomycorrhizal Eucalyptus coccifera: growth response, phosphorus uptake efficiency and external hyphal production

Eucalyptus coccifera Hook., a plant capable of forming both arbuscular mycorrhizas and ectomycorrhizas, was used to compare the effects of the two mycorrhizal types on phosphorus uptake and C allocation. Seedlings were grown in a P-deficient soil/sand mixture inoculated with peat/vermiculite spawn of Laccaria bicolor (Maire) Orton or Thelephora terrestris (Ehrh.) Fr.; or with 250-μm sievings from leek colonized by Glomus caledonium (Nicol. & Gerd.) Trappe & Gerde., Glomus sp. type E3 or Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe or with autoclaved spawn (non-mycorrhizal control). Before the 89-d harvest, a subset of the harvested plants was labelled with ¹⁴C (45–60-min pulse, 202-h chase). Growth promotion and the increase in seedling P content was largest in the two ectomycorrhizal treatments. Production of fluorescein diacetate-stained external hyphae was three to seven times higher by ectomycorrhizal (ECM) fungi compared with arbuscular mycorrhizal (AM) fungi and was highly correlated with P uptake and shoot weight. Phosphorus inflow rates of ECM and AM seedlings were 3·8 times, and 2·0–2·7 times those of non-mycorrhizal seedlings. Phosphorus acquisition efficiencies were similar (11·2 and 10·0 μmol P mmol⁻¹C for T. terrestris and Glomus E3 plants, respectively) for the two mycorrhizal types, and appeared to be greater than in uninoculated plants (7.2 μmol P mmol⁻¹C) grown at the same P level.     

Effect of poplar genotypes on mycorrhizal infection and secreted enzyme activities in mycorrhizal and non-mycorrhizal roots

The impact of ectomycorrhiza formation on the secretion of exoenzymes by the host plant and the symbiont is unknown. Thirty-eight F(1) individuals from an interspecific Populus deltoides (Bartr.)×Populus trichocarpa (Torr. & A. Gray) controlled cross were inoculated with the ectomycorrhizal fungus Laccaria bicolor. The colonization of poplar roots by L. bicolor dramatically modified their ability to secrete enzymes involved in organic matter breakdown or organic phosphorus mobilization, such as N-acetylglucosaminidase, β-glucuronidase, cellobiohydrolase, β-glucosidase, β-xylosidase, laccase, and acid phosphatase. The expression of genes coding for laccase, N-acetylglucosaminidase, and acid phosphatase was studied in mycorrhizal and non-mycorrhizal root tips. Depending on the genes, their expression was regulated upon symbiosis development. Moreover, it appears that poplar laccases or phosphatases contribute poorly to ectomycorrhiza metabolic activity. Enzymes secreted by poplar roots were added to or substituted by enzymes secreted by L. bicolor. The enzymatic activities expressed in mycorrhizal roots differed significantly between the two parents, while it did not differ in non-mycorrhizal roots. Significant differences were found between poplar genotypes for all enzymatic activities measured on ectomycorrhizas except for laccases activity. In contrast, no significant differences were found between poplar genotypes for enzymatic activities of non-mycorrhizal root tips except for acid phosphatase activity. The level of enzymes secreted by the ectomycorrhizal root tips is under the genetic control of the host. Moreover, poplar heterosis was expressed through the enzymatic activities of the fungal partner.     

Survival of an introduced ectomycorrhizal Laccaria bicolor strain in a European forest plantation monitored by mitochondrial ribosomal DNA analysis

Mitochondrial and nuclear genes have different inheritance, thus studies of fungal populations should use both mitochondrial and nuclear markers. Using nuclear markers, the S238N strain of the ectomycorrhizal basidiomycete Laccaria bicolor ((Maire) Orton) has been previously shown to persist for at least 10 yr after outplanting in a plantation of Douglas fir ( Pseudotsuga menziesii (Mir.) Franco) inoculated with this strain. In the present study, we have sampled 539 sporophores of Laccaria spp. from this plantation, some of which had the S238N nuclear genotype, to study mitochondrial DNA polymorphism and persistence of the inoculated S238N mitochondrial genome. Length polymorphism in fragments of the large subunit of mitochondrial ribosomal DNA (LrDNA) allowed distinction of the haplotypes present in the plantation at the species level. In addition, heteroduplex analysis and sequencing revealed intraspecific polymorphism of LrDNA among the L. bicolor sporophores and enabled specific identification of S238N LrDNA. This haplotype was only retained in sporophores carrying the S238N nuclear genome, confirming the survival of this introduced strain in a natural population.     

Location and Survival of Mycorrhiza Helper Pseudomonas fluorescens during Establishment of Ectomycorrhizal Symbiosis between Laccaria bicolor and Douglas Fir

The mycorrhiza helper bacterium Pseudomonas fluorescens BBc6, isolated from a Laccaria bicolor sporocarp, consistently promotes L. bicolor-Douglas fir (Pseudotsuga menziesii) ectomycorrhizal formation, even with low doses of bacterial inoculum. In order to describe this phenomenon more accurately, we have looked at the location and survival of the introduced bacterial strain in the soil and in the rhizosphere during the establishment of mycorrhizal symbiosis in glasshouse and nursery experiments. Bacterial populations were quantified with a spontaneous, stable, rifampin-resistant mutant, BBc6R8, which phenotypically conformed to the parental strain. BBc6R8 populations declined rapidly, reaching the detection limit after 19 weeks, and did not increase either when L. bicolor sporocarps were forming in autumn or when Douglas fir roots resumed growing in spring. BBc6R8 was neither an endophyte nor a rhizobacterium. Furthermore, it was not particularly associated with either mycorrhizas of Douglas fir-L. bicolor or L. bicolor sporocarps. Surprisingly, a significant mycorrhiza helper effect was observed when the inoculated BBc6R8 population had dropped as low as 30 CFU g of dry matter(sup-1) in the soil. This study raises questions concerning the bacterial concentration in the soil which is effective for promotion of mycorrhizal establishment and the timing of the bacterial effect. It allows us to develop working hypotheses, which can be tested experimentally, to identify the mechanisms of the mycorrhiza helper effect.     

The ability of 16 ectomycorrhizal fungi to increase growth and phosphorus uptake of Eucalyptus globulus Labill. and E. diversicolor F. Muell.

The effectiveness of 16 fungal isolates in forming ectomycorrhizas and increasing the growth and phosphorus uptake of Eucalyptus globulus Labill. and E. diversicolor F. Muell. seedlings was examined in the glasshouse. Seedlings were grown in yellow sand at 2 phosphorus levels (4 and 12 mg P kg^-1 sand). At the time of harvest (100 days), the non-inoculated seedlings and seedlings inoculated with Paxillus muelleri (Berk.) Sacc. and Cortinarius globuliformis Bougher had a low level of contamination from an unknown mycorrhizal fungi. Seedlings inoculated with Thaxterogaster sp. nov. and Hysterangium inflatum Rodway had developed mycorrhizas of the superficial type whereas Hydnangium carneum Wallr. in Dietr., Hymenogaster viscidus Massee & Rodway, Hymenogaster zeylanicus Petch, Setchelliogaster sp. nov., Laccaria laccata (Scop. ex. Fr.) Berk., Scleroderma verrucosum (Vaillant) Pers., Amanita xanthocephala (Berk.) Reid & Hilton, Descolea maculata Bougher and Malajczuk and Pisolithus tinctorius (Pers.) Coker & Couch formed typical pyramidal ectomycorrhizas. The dry weight of non-inoculated and inoculated E. globulus seedlings at 12 mg P kg^-1 sand did not differ, whereas several isolates caused growth depression of E. diversicolor. By contrast, at 4 mg P kg^-1 sand growth increases ranged from 0–13 times above that of non-inoculated seedlings. P. tinctorius produced the largest growth increase on both eucalypt species. In general, isolates which developed more extensive mycorrhizas on roots produced the largest growth responses to inoculation. Isolates which increased plant growth also increased phosphorus uptake by the plant. Seedlings inoculated with L. laccata and S. verrucosum retained more phosphorus in their roots than plants inoculated with the other fungal isolates.     

SCAR markers to detect mycorrhizas of an American Laccaria bicolor strain inoculated in European Douglas-fir plantations

The American strain S238N of the ectomycorrhizal fungus Laccaria bicolor (Maire) Orton has been used to inoculate Douglas-fir [Pseudotsuga menziesii (Mir.) Franco] plantations in France over the last two decades. Laccaria fruit bodies are scarce in mature plantations, which precludes further assessment of its persistence by fruit body surveys. Our objective was to develop new markers to identify this strain and its eventual non-fruiting progeny on root tips. We converted nine random amplified polymorphic DNA markers into sequence characterized amplified region (SCAR) markers. Two of these SCAR markers enabled us to detect S238N on roots of seedlings and mature trees. No amplification of non-fungal (host plant, bacterial, etc.) DNA was observed. Moreover, both SCARs were amplified from Laccaria-like mycorrhizas in a Douglas-fir plantation inoculated 14 years ago, demonstrating the long-term persistence of the inoculant strain. We also obtained a SCAR marker to detect one strain of European origin (L. bicolor 81306), indicating that SCARs are potential markers to type the naturally occurring genets. Thus, SCAR markers are of great value in studying the persistence of inoculant strains and the effects on local populations of introducing foreign strains.     

Ectomycorrhizal symbiosis affects functional diversity of rhizosphere fluorescent pseudomonads

Here we characterized the effect of the ectomycorrhizal symbiosis on the genotypic and functional diversity of soil Pseudomonas fluorescens populations and analysed its possible consequences in terms of plant nutrition, development and health. Sixty strains of P. fluorescens were isolated from the bulk soil of a forest nursery, the ectomycorrhizosphere and the ectomycorrhizas of the Douglas fir (Pseudostuga menziesii) seedlings-Laccaria bicolor S238N. They were characterized in vitro with the following criteria: ARDRA, phosphate solubilization, siderophore, HCN and AIA production, genes of N2-fixation and antibiotic synthesis, in vitro confrontation with a range of phytopathogenic and ectomycorrhizal fungi, effect on the Douglas fir-L. bicolor symbiosis. For most of these criteria, we demonstrated that the ectomycorrhizosphere significantly structures the P. fluorescens populations and selects strains potentially beneficial to the symbiosis and to the plant. This prompts us to propose the ectomycorrhizal symbiosis as a true microbial complex where multitrophic interactions take place. Moreover it underlines the fact that this symbiosis has an indirect positive effect on plant growth, via its selective pressure on bacterial communities, in addition to its known direct positive effect.     

Mycorrhizal association of isolates from sporocarps and ectomycorrhizas withPinus densiflora seedlings

Thirty-two isolates from sporocarps of 27 species of macromycetes, 43 isolates from ectomycorrhizas ofPinus densiflora (Japanese red pine) and 1 isolate from an ectomycorrhiza ofQuercus myrsinaefolia were tested for the ability to form mycorrhizas withP. densiflora seedlings in glass tubes. Ten isolates from sporocarps ofHebeloma sp.,Laccaria bicolor, Lactarius chrysorrheus, Suillus granulatus, Scleroderma areolatum, Russula mariae andR. nigricans had formed ectomycorrhizas by 8 months after transplantation. Twenty isolates taken from mycorrhizas including ofCenococcum geophilum, R. mariae andR. nigricans formed ectomycorrhizas. The synthesized mycorrhizas were classified based on morphological characteristics such as hyphal arrangement of their fungal sheath, and appearance of cystidia and emanating hyphae. Twenty-one mycorrhizal types were recognized.Contribution No. 122, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

In situ identification of intracellular bacteria related to Paenibacillus spp. in the mycelium of the ectomycorrhizal fungus Laccaria bicolor S238N

Bacterial proliferations have recurrently been observed for the past 15 years in fermentor cultures of the ectomycorrhizal fungus Laccaria bicolor S238N, suggesting the presence of cryptic bacteria in the collection culture of this fungus. In this study, intracellular bacteria were detected by fluorescence in situ hybridization in combination with confocal laser scanning microscopy in several collection subcultures of L. bicolor S238N. They were small (0.5 micro m in diameter), rare, and heterogeneously distributed in the mycelium and were identified as Paenibacillus spp. by using a 16S rRNA-directed oligonucleotide probe initially designed for bacteria isolated from a fermentor culture of L. bicolor S238N.     

Cation exchange capacity and lead sorption in ectomycorrhizal fungi

Two ectomycorrhizal fungi, Paxillus involutus 533 and Laccaria bicolor S238, differing greatly in their mycelial characteristics, were investigated with regard to their cation exchange capacity and Pb-binding capacity in vitro after growth with either NO₃ ^- or NH₄ ⁺ as N source. The CECs of 800–1200 mol g-1 dry weight for Paxillus involutus 533 and 2000–3000 mol g-1 dry weight for Laccaria bicolor S238, were high compared to plant roots. The fungal mycelium also had a high Pb sorption capacity. It was higher in Laccaria bicolor S238 than in Paxillus involutus 533 and higher after pregrowth in NO₃ ^- compared to NH₄ ⁺. Both the higher CEC and the higher Pb sorption capacity of Laccaria bicolor S238 compared to Paxillus involutus 533 might have been the result of the hydrophilic nature of the of Laccaria bicolor S238 mycelium. It would have absorbed the solutions better than the hydrophobic mycelium of Paxillus involutus 533. X-ray microanalysis of the cell walls revealed that the Pb content of the cell walls was higher in Paxillus involutus 533 than in Laccaria bicolor S238. Nevertheless, electron dense deposits in the cell walls of Laccaria bicolor S238 contained large amounts of Pb, P and S. Thus, while Pb was evenly distributed in the cell walls of Paxillus involutus 533, Pb was accumulated in electron dense deposits in Laccaria bicolor S238. The results are discussed in view of their significance for the mycorrhizal symbiosis.     

The potential role of ectomycorrhizal fungi in determining Douglas-fir resistance to defoliation by the western spruce budworm (Lepidoptera: Tortricidae)

There is phenotypic variation among individual trees of interior Douglas-fir (Pseudotsuga menziesii var. glauca [Beissn.] Franco) in their resistance to defoliation by the western spruce budworm (Choristoneura occidentalis Freeman). We evaluated the potential role of ectomycorrhizal fungi in determining this resistance using half-sib seedlings derived from parent trees that are resistant versus susceptible to budworm defoliation in the field. The seedlings were inoculated with Laccaria bicolor ectomycorrhizal fungi, fertilized, or untreated. Approximately 48 d after treatment, late-instar larvae from a nondiapausing laboratory colony of C. occidentalis were allowed to feed on pairs of resistant versus susceptible seedlings for 1 wk. Chemical analyses of current-year shoots for nitrogen (N), phosphorus (P), magnesium (Mg), and zinc (Zn) indicated that the fungus increased foliar concentrations of P and Mg in resistant seedlings, but it did not increase their growth rate. However, L. bicolor had no effect on foliar concentrations of P or Mg in susceptible seedlings, even though seedling growth rates increased slightly in response to the inoculation. L. bicolor had no effect on foliar levels of N or Zn in any of the seedlings. As expected, fertilization increased levels of N and P in the foliage of both resistant and susceptible seedlings, but it did not affect levels of Mg and Zn. Surprisingly, the fertilizer treatment had no effect on seedling growth rates. Despite these differences, late-instar budworms showed no feeding preference among untreated, mycorrhizal, or fertilized seedlings. The fact that seedlings from resistant versus susceptible Douglas-firs responded differently to the L. bicolor treatment lends preliminary support to the hypothesis that ecotmycorrhizae might play a role in Douglas-fir resistance to damage from the western spruce budworm. Finally, differences in foliar concentrations of N and P among untreated seedlings from different maternal trees suggested that foliar nutritional chemistry is influenced by the tree's genotype.     

Phylogenetic, genomic organization and expression analysis of hydrophobin genes in the ectomycorrhizal basidiomycete Laccaria bicolor

Hydrophobins are morphogenetic, small secreted hydrophobic fungal proteins produced in response to changing development and environmental conditions. These proteins are important in the interaction between certain fungi and their hosts. In mutualistic ectomycorrhizal fungi several hydrophobins form a subclass of mycorrhizal-induced small secreted proteins that are likely to be critical in the formation of the symbiotic interface with host root cells. In this study, two genomes of the ectomycorrhizal basidiomycete Laccaria bicolor strains S238N-H82 (from North America) and 81306 (from Europe) were surveyed to construct a comprehensive genome-wide inventory of hydrophobins and to explore their characteristics and roles during host colonization. The S238N-H82 L. bicolor hydrophobin gene family is composed of 12 genes while the 81306 strain encodes nine hydrophobins, all corresponding to class I hydrophobins. The three extra hydrophobin genes encoded by the S238N-H82 genome likely arose via gene duplication and are bordered by transposon rich regions. Expression profiles of the hydrophobin genes of L. bicolor varied greatly depending on life stage (e.g. free living mycelium vs. root colonization) and on the host root environment. We conclude from this study that the complex diversity and range of expression profiles of the Laccaria hydrophobin multi-gene family have likely been a selective advantage for this mutualist in colonizing a wide range of host plants.