Ultrastructural and cytochemical aspects of the interaction between the ectomycorrhizal fungus Laccaria laccata and the saprotrophic fungi, Trichoderma harzianum and T. virens

Different interactions between soil fungi competing in the rhizosphere with each other are necessary to understand their influence on plant growth and health. The interactions between the ectomycorrhizal (ECM) fungus Laccaria laccata and soil saprotrophic fungi (T. harzianum, T. virens) were studied by transmission electron microscopy, and by gold cytochemistry to assess the potential role of cell wall lytic enzymes in mycoparasitism. Anti-β-1,3-glucan antibody, WGA/ovomucoid-gold complex and PATAg test were used to localize β-1,3-glucan, chitin and polysaccharides. Cytoplasm disorganisation of the saprotrophic fungi occurred concurrently with dissolution of β-1,3-glucan in walls of hyphae and conidia of the saprotrophic fungi. Then digestion of polysaccharides and chitin of colonised fungal structures occurred. The studies suggest sequential contribution of cell wall lytic enzymes and importance of disturbing the host's cell integrity during mycoparasitism. We conclude that the ECM fungus can parasitise on the saprotrophic fungi not only in dual culture on artificial medium but also in the rhizosphere of Scots pine.     

Solarization in a forest nursery: effect on ectomycorrhizal soil infectivity and soil receptiveness to inoculation with Laccaria bicolor

 Field experiments were carried out in a forest nursery during the summer of 1994 to examine the effect of soil solarization on ectomycorrhizal soil infectivity (ESI) and soil receptiveness to inoculation with Laccaria bicolor. Soil samples from solarized, steamed, fumigated and untreated plots were periodically collected and assayed for ESI. Untreated soil exhibited high ESI. Solarization was as effective as steaming or fumigation in reducing ESI in the uppermost layer. Solarization with a double layer of polyethylene film and fumigation were the only treatments which reduced ESI deeper in the soil. During July, the temperature of covered beds reached 50  °C at a soil depth of 5 cm. Ectomycorrhizal fungi were among the soil-borne fungi most sensitive to solar heating. Soil solarization provides an effective disinfection method for controlled mycorrhization in forest nurseries. Accepted: 10 April 1997     

From field sampling to pneumatic bioreactor mycelia production of the ectomycorrhizal mushroom Laccaria trichodermophora

In order to increase survival rates of greenhouse seedlings destined for restoration and conservation programs, successful mycorrhization of the seedlings is necessary. To reforest forest ecosystems, host trees must be inoculated with ectomycorrhizal fungi and, in order to guarantee a sufficient supply of ectomycorrhizal inoculum, it is necessary to develop technologies for the mass production of ectomycorrhizal fungi mycelia. We selected the ectomycorrhizal fungus Laccaria trichodermophora, due to its ecological traits and feasible mycelia production in asymbiotic conditions. Here, we report the field sampling of genetic resources, as well as the highly productive nutritional media and cultivation parameters in solid cultures. Furthermore, in order to achieve high mycelial production, we used strain screening and evaluated pH, carbon source concentration, and culture conditions of submerged cultures in normal and baffled shake flasks. The higher productivity culture conditions in shake flasks were selected for evaluation in a pneumatic bioreactor, using modified BAF media with a 10 g/L glucose, pH 5.5, 25 °C, and a volumetric oxygen transfer coefficient (K_La) of 36 h⁻¹. Under those conditions less biomass (12–37 %) was produced in the pneumatic bioreactor compared with the baffled shake flasks. This approach shows that L. trichodermophora can generate a large biomass concentration and constitute the biotechnological foundation of its mycelia mass production.     

Sulfur uptake in the ectomycorrhizal fungus Laccaria bicolor S238N

The importance of the ectomycorrhiza symbiosis for plant acquisition of phosphorus and nitrogen is well established whereas its contribution to sulfur nutrition is only marginally understood. In a first step to investigate the role of ectomycorrhiza in plant sulfur nutrition, we characterized sulfate and glutathione uptake in Laccaria bicolor. By studying the regulation of sulfate uptake in this ectomycorrhizal fungus, we found that in contrast to bacteria, yeast, and plants, sulfate uptake in L. bicolor was not feedback-inhibited by glutathione. On the other hand, sulfate uptake was increased by sulfur starvation as in other organisms. The activity of 3′-phosphoadenosine 5′-phosphosulfate reductase, the key enzyme of the assimilatory sulfate reduction pathway in fungi, was increased by sulfur starvation and decreased after treatment with glutathione revealing an uncoupling of sulfate uptake and reduction in the presence of reduced sulfur compounds. These results support the hypothesis that L. bicolor increases sulfate supply to the plant by extended sulfate uptake and the plant provides the ectomycorrhizal fungus with reduced sulfur.     

Temporal variation in temperature and rainfall differentially affects ectomycorrhizal colonization at two contrasting sites

We examined the roles that seasonal shifts in precipitation and temperature played in the ectomycorrhizal (ECM) colonization of pinyon pine ( Pinus edulis Engelm.) at two contrasting sites in northern Arizona. Pinyons growing in ash and cinder soils experienced much greater water and nutrient stress than pinyons growing nearby in sandy-loam soils. Over a one year period, we obtained monthly measurements of ECM colonization, root zone soil moisture and temperature, and air temperature and precipitation. Four major patterns emerged. Firstly, although climate as measured by ambient temperature and precipitation did not vary between the two sites, soil temperature was significantly higher and soil moisture significantly lower at the cinder site than at the sandy-loam site. Secondly, ECM colonization was significantly higher at the cinder site for 5 of 12 months. Thirdly, although nearly 70% of the variation in ECM colonization of pinyons growing in cinder soil was predicted by a combination of soil moisture and soil temperature, these same variables had little predictive power for pinyons growing in sandy-loam soils. Air temperature and precipitation were also significantly correlated with ECM colonization at the cinder site but not the sandy-loam site. Fourthly, a watering experiment showed that ECM colonization significantly increased with supplemental water at the cinder site, but not at the sandy-loam site. Thus, in two sites that did not differ in plant community or climate, ectomycorrhizas in cinder soils were far more sensitive to changes in moisture and temperature than ectomycorrhizas in sandy-loam soils.     

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.     

磷对外生菌根真菌松乳菇和双色蜡蘑草酸、氢离子和磷酸酶分泌的影响

在液体培养基中设置无磷、低磷、中磷和高磷四种磷浓度,离体培养松乳菇Lactarius deliciosus的3个株系Ld-01、Ld-02、Ld-03和双色蜡蘑Laccaria bicolor S238N,分别测定外生菌根真菌的生长量、草酸、氢离子和酸性磷酸酶分泌速率。结果表明,在中磷的条件下,外生菌根真菌的生长最好,无磷、低磷或高磷对它们的生长产生抑制作用,抑制程度因菌种(株)不同而异。草酸、氢离子和酸性磷酸酶的分泌速率顺序为:无磷(0gNaH2PO4/L)>低磷(0.229gNaH2PO4/L)>中磷(1.147gNaH2PO4/L)>高磷(5.735gNaH2PO4/L),表现出明显的种(株)间差异,看来外源磷的供应状况可能有调节外生菌根真菌活化土壤无机磷和有机磷的作用。在缺磷和低磷条件下,外生菌根真菌具有较强的活化作用,在足磷的条件下活化作用减小,由此有效地利用土壤中的磷。     

磷对外生菌根真菌松乳菇和双色蜡蘑草酸、氢离子和磷酸酶分泌的影响

在液体培养基中设置无磷、低磷、中磷和高磷四种磷浓度,离体培养松乳菇Lactarius deliciosus的3个株系Ld-01、Ld-02、Ld-03和双色蜡蘑Laccaria bicolor S238N,分别测定外生菌根真菌的生长量、草酸、氢离子和酸性磷酸酶分泌速率。结果表明,在中磷的条件下,外生菌根真菌的生长最好,无磷、低磷或高磷对它们的生长产生抑制作用,抑制程度因菌种（株）不同而异。草酸、氢离子和酸性磷酸酶的分泌速率顺序为：无磷（0g NaH2PO4/L）> 低磷（0.229g NaH2PO4/L）     

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.     

Spatial structure and diversity of woody plants and ectomycorrhizal fungus sporocarps in a natural subtropical forest

Spatial patterns of ectomycorrhizal fungi, ectomycorrhizal plants, and non-ectomycorrhizal plants were investigated in a natural subtropical forest using second-order analysis. The results of spatial pattern analysis showed that the degree of clumping of woody plants and ectomycorrhizal sporocarps were correlated. There was a significantly positive correlation of relative aggregation indices between ectomycorrhizal fungi and both non-ectomycorrhizal trees and ectomycorrhizal saplings. Correlations between percentage of ectomycorrhizal trees and sporocarp occurrence of ectomycorrhizal fungi and between diversities of woody plants and ectomycorrhizal fungi were distance-dependent or scale-related. A significantly high percentage of ectomycorrhizal trees was found only at relatively short distance from ectomycorrhizal fungal sporocarps, and significantly positive correlation of the diversity between woody plants and ectomycorrhizal fungi was found only at relative long distance, which implied that ectomycorrhizal sporocarps prefer ectomycorrhizal-tree-dominant micro-sites at near distances and at relatively large scales, diverse ectomycorrhizal sporocarps could be found in woodlands with high diversity of woody plants. Important factors affecting the spatial distribution, occurrence, and diversity of ectomycorrhizal fungi include spatial pattern of ectomycorrhizal plants and non-ectomycorrhizal plants, percentage of ectomycorrhizal plants, and plant diversity in a natural forest.     

Recovery of the ectomycorrhizal community after termination of long-term nitrogen fertilisation of a boreal Norway spruce forest

Ectomycorrhizal fungi (ECM) are a fundamental component of boreal forests promoting tree growth and participating in soil nutrient cycling. Increased nitrogen (N) input is known to largely influence ECM communities but their potential recovery is not well understood. Therefore, we studied the effects of long-term N-fertilisation on ECM communities, and their recovery after termination of N treatment. Fungal ITS sequencing data indicated that N-fertilisation (34 kg N ha⁻¹ y⁻¹) for 46 y decreased the relative abundance of ECM species in the fungal community and suppressed originally dominating medium-distance fringe exploration types adapted to N-limited conditions, while the ECM diversity remained unaffected. In other plots, 23 y after termination of fertilisation at 73 kg N ha⁻¹ y⁻¹ for 23 y, the relative abundance of ECM species shifted closer to, but did not reach, control levels. These observations indicate only slow recovery of ECM community, likely due to a high soil N retention capacity.     

Strong differences in Quercus robur-associated ectomycorrhizal fungal communities along a forest-city soil sealing gradient

Ectomycorrhizal fungi (EM) that associate with tree roots in a symbiotic relationship may be crucial in mediating tree health in urban environments, but research on the effects of urbanization on EM communities is very limited so far. Here, we compared EM communities of adult pedunculate oaks (Quercus robur) between urban and forest environments, and assessed the effect of soil sealing around the trees on their EM community composition and EM diversity. We sampled 32 oak individuals across 4 sampling classes (Street, Lane, Park and Forest), and we characterized their EM communities using 454 amplicon pyrosequencing. The EM communities were not nested but they were significantly different between all sampling classes, with a very strong community differentiation between forest and urban trees. There were indications that EM richness declined with increasing sealed soil surface, with a significant effect of sampling class on estimated EM richness and diversity. We also identified indicator EM of the different sampling classes. The most important soil factor affecting EM community composition was pH, followed by plant available phosphorus, total nitrogen content and organic matter. Our results may have important implications when developing EM inocula for managing tree health in urban environments.     

The function of melanin in the ectomycorrhizal fungus Cenococcum geophilum under water stress

Despite the ubiquity and importance of ectomycorrhizal fungi to ecosystem function, our understanding of their functional ecology remains poor. The highly melanized and common ectomycorrhizal fungus, Cenococcum geophilum, is drought tolerant and abundant in water-stressed habitats, yet the responsible functional traits have not been identified. The production of melanin, a class of complex dark polymers found in fungal cell walls, may be a key functional trait to water stress tolerance. To test this hypothesis, we devised a series of experiments determining the effect of the melanin biosynthesis inhibitor, tricyclazole, on response to osmotic and desiccation stresses. Melanin inhibition only had negative effects on growth when C. geophilum isolates were subjected to osmotic and desiccation stress (?1.7 MPa and desiccated) but not under control conditions (?0.01 MPa and non-desiccated). This suggests that melanin production is an important functional trait that contributes to water stress tolerance of this cosmopolitan ectomycorrhizal fungus.     

Small‐scale spatial variability in the distribution of ectomycorrhizal fungi affects plant performance and fungal diversity

The effects of spatial heterogeneity in negative biological interactions on individual performance and species diversity have been studied extensively. However, little is known about the respective effects involving positive biological interactions, including the symbiosis between plants and ectomycorrhizal (EM) fungi. Using a greenhouse bioassay, we explored how spatial heterogeneity of natural soil inoculum influences the performance of pine seedlings and composition of their root‐associated EM fungi. When the inoculum was homogenously distributed, a single EM fungal taxon dominated the roots of most pine seedlings, reducing the diversity of EM fungi at the treatment level, while substantially improving pine seedling performance. In contrast, clumped inoculum allowed the proliferation of several different EM fungi, increasing the overall EM fungal diversity. The most dominant EM fungal taxon detected in the homogeneous treatment was also a highly beneficial mutualist, implying that the trade‐off between competitive ability and mutualistic capacity does not always exist.