Fatty acid composition of various ectomycorrhizal fungi and ectomycorrhizas of Norway spruce

Whole cell fatty acid (WCFA) compositions of three different structures of ectomycorrhizal (ECM) fungi: sporocarps, pure culture mycelia and ectomycorrhizas were analysed to evaluate the potential use of fatty acid profiles as biomarkers for ECM fungi and ectomycorrhiza-associated bacteria. Sporocarps of Amanita muscaria, Amanita rubescens, Lactarius rufus, Lactarius thejogalus, Leccinum scabrum, Paxillus involutus, Russula foetens, Russula rosea, Russula vesca, Suillus grevillei, Tylopilus felleus, Xerocomus badius, Xerocomus subtomentosus, pure cultures of A. muscaria, P. involutus, X. badius, X. subtomentosus, Suillus bovinus Suillus luteus and seven ectomycorrhizal morphotypes of Norway spruce were examined. Our results revealed species-specific composition of fatty acids of fungal sporocarps and pure culture mycelia. Ectomycorrhizal morphotypes distinguished and identified by morphological and molecular methods (PCR-RLFP and sequencing) created specific fatty acid profiles. The dominating fatty acids in pure cultures and sporocarps were 18:2ω6,9, 18:1ω9 and 16:0, whereas ectomycorrhizas also contained plant and bacterial specific fatty acids. Especially, fatty acids specific to Gram-positive bacteria 15:0 anteiso and 17:0 anteiso were present in relatively high amounts and suggested that these bacteria are dominating in the examined Norway spruce mycorrhizosphere. In conclusion, our results show that fatty acid based methods can be useful in studies of ectomycorrhizal fungi, both as a quick method for differentiation of fungal species and also in studies of mycorrhiza-associated microorganisms in the field.     

Interactions between the external mycelium of the mycorrhizal fungus Glomus intraradices and other soil microorganisms as affected by organic matter

The influence of organic matter on the interactions between external mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, the bacterium Burkholderia cepacia and other soil microorganisms was studied in a root-free sand environment. Organic matter amendment, in terms of ground barley leaves, markedly increased the growth of the external mycelium of G. intraradices as estimated both with the fatty acid biomarker 16:1ω5 and hyphal length measurements. Mycelial proliferation of G. intraradices in sand with organic matter was unaffected by both inoculation with B. cepacia and a soil filtrate containing a mixed population of indigenous microorganisms. On the other hand, in the absence of organic matter, both inoculation with B. cepacia and the soil filtrate reduced the growth of G. intraradices, as estimated with measurements of 16:1ω5. In contrast, B. cepacia inoculation increased hyphal length density of G. intraradices in the absence of organic matter. Overall, the presence of external mycelium of G. intraradices increased the bacterial biomass and counteracted a suppressive effect of B. cepacia on the growth of saprotrophic fungi.     

利用变性梯度凝胶电泳分析正红菇菌根围土壤真菌群落多样性

以正红菇（Russula griseocarnosa）菌根围土壤为研究对象,通过提取土壤基因组DNA,以通用引物扩增真菌18S rRNA基因V1+V2区,将PCR产物进行变性梯度凝胶电泳（Denaturing Gradient Gel Electrophoresis）,获得土壤微生物群落的DNA特征指纹图谱,并对图谱中的优势条带回收测序,通过Blast进行同源性比对并构建系统发育树,进而分析正红菇菌根围真菌群落组成及多样性。同源性比对结果表明,在回收测序的19条DGGE条带中,4条为非真菌真核生物序列,系统发育分析显示全部序列可以分为4类菌群,GroupⅠ主要为担子菌门（Basidiomycota）真菌,GroupⅡ主要为子囊菌门（Ascomycota）真菌,GroupⅢ为未知真菌,GroupⅣ主要为节肢动物门生物（Arthropoda）。     

Interactions between mycorrhizal fungi and mycorrhizosphere bacteria during mineral weathering: Budget analysis and bacterial quantification

The impact of ectomycorrhizal fungi or rhizosphere bacteria on tree seedling growth and nutrient uptake is well known. However, few studies have combined those microorganisms in one experiment to clarify their relative contribution and interactions in nutrient acquisition. Here, we monitored the respective contributions of pine roots, two ubiquitous forest ectomycorrhizal fungi Scleroderma citrinum and Laccaria bicolor, and two S. citrinum-mycorrhizosphere bacterial strains of Burkholderia glathei and Collimonas sp., on mineral weathering, nutrient uptake, and plant growth. Pinus sylvestris plants were grown on quartz–biotite substrate and inoculated or not with combinations of mycorrhizal fungi and/or bacterial strains. Magnesium and potassium fluxes were measured and nutrient budgets were calculated. Both ectomycorrhizal fungi significantly increased Mg plant uptake. No significant effects of the two bacterial strains were detected on the K and Mg budgets, but co-inoculating the mycorrhizal fungus S. citrinum and the efficient mineral-weathering B. glathei bacterial strain significantly improved the Mg budget. Similarly, co-inoculating S. citrinum with the Collimonas sp. bacterial strain significantly improved the pine biomass compared to non-inoculated pine plants. In order to better understand this process, we monitored the survival of the inoculated bacterial strains in the quartz–biotite substrate, the pine rhizosphere, and the mycorrhizal niche. The results showed that the two bacterial strains harboured different colonization behaviours both of which depended on the presence of the ectomycorrhizal partner. The populations of the Burkholderia strain were maintained in all these environments with a significantly higher density in the mycorrhizal niche, especially of S. citrinum. In contrast the population of the Collimonas strain reached the detection level except in the treatment inoculated with S. citrinum. These results highlight the need for taking into account the ecology of the microorganisms, and more specifically the fungal–bacterial interactions, when studying mineral weathering and plant nutrition.     

Long term impact of mineral amendment on the distribution of the mineral weathering associated bacterial communities from the beech Scleroderma citrinum ectomycorrhizosphere

Liming is a known forest management procedure used to amend nutrient-poor soils such as soils of acidic forests to rectify cation deficiencies and to restore soil pH. However, although this procedure is well known for its beneficial effect on the forest trees, its relative impact on the functional and taxonomic diversity of the soil bacterial communities has been poorly investigated. In this study, we characterized the ability of the soil bacteria to weather soil minerals and to hydrolyze chitin. A collection of 80 bacterial strains was isolated from the Scleroderma citrinum ectomycorrhizosphere and the adjacent bulk soil in two stands of mature beeches (Fagus sylvatica) developed on very acidic soil and presenting two levels of calcium (Ca) availability: a control plot as well as a plot amended with Ca in 1973. All the bacterial isolates were identified by partial 16S rRNA gene sequence analysis as members of the genera Burkholderia, Bacillus, Dyella, Kitasatospora, Micrococcus, Paenibacillus, Pseudomonas, and Rhodanobacter. Using a microplate assay for quantifying the production of protons and the quantity of iron released from biotite, we demonstrated that the bacterial strains from the amended plot harbored a significant higher mineral weathering potential that the ones isolated from the control plot. Notably, the weathering efficacy of the ectomycorrhizosphere bacterial isolates was significantly greater than that of the bulk soil isolates in the control treatment but not in the amended plot. These data reveal that forest management, here mineral amendment, can strongly affect the structure of bacterial communities even over the long term.     

Baiting of rhizosphere bacteria with hyphae of common soil fungi reveals a diverse group of potentially mycophagous secondary consumers

Fungi and bacteria are primary consumers of plant-derived organic compounds and therefore considered as basal members of soil food webs. Trophic interactions among these microorganisms could, however, induce shifts in food web energy flows. Given increasing evidence for a prominent role of saprotrophic fungi as primary consumers of root-derived carbon, we propose that fungus-derived carbon may be an important resource for rhizosphere bacteria. To test this assumption, two common saprotrophic, rhizosphere-inhabiting fungi, Trichoderma harzianum and Mucor hiemalis, were confronted in a microcosm system with bacterial communities extracted from the rhizospheres of a grass and sedge species, Carex arenaria and Festuca rubra. This showed a widespread ability of rhizosphere bacteria to attach to and feed on living hyphae of saprotrophic fungi. The identity of the fungi had a strong effect on the composition of these potentially mycophagous bacteria, whereas plant species identity was less important. Based on our results, we suggest that food web models should account for bacterial secondary consumption since this has important consequences for carbon fluxes with more carbon dioxide released by microbes and less microbial carbon available for the soil animal food web.     

Suppression of other soil microorganisms by mycelium of arbuscular mycorrhizal fungi in root-free soil

The influence of mycelium of two arbuscular mycorrhizal (AM) fungi, Glomus intraradices and Glomus mosseae, on other soil microorganisms, was examined in root-free soil with and without organic substrate amendment in terms of cellulose. The AM fungi were grown in symbiosis with cucumber in a compartmented growth system, which allowed AM fungal external mycelium to grow into root-free compartments. The fungicide Benomyl was applied to the root-free compartments to create an alternative non-mycorrhizal control treatment. Whole cell biomarker fatty acids were employed to quantify different groups of soil microorganisms including the two AM fungi. Abundance of most microbial groups were reduced by external mycelium of both AM fungi, though differential effects on the microbial community composition were observed between the two AM fungi as revealed from principal component analysis. Inhibition of other soil microorganisms was more pronounced in root-free soil with mycelium of G. mosseae than with mycelium of G. intraradices. In general, cellulose increased the amount of biomarker fatty acids of most groups of soil microorganisms, but cellulose did not affect the influence of AM fungi on other soil microorganisms. Benomyl suppressed growth of the external mycelium of the two AM fungi and had limited non-target effects on other microbial groups. In conclusion, our results show differential effects of external mycelium of AM fungi on other soil microbial communities, though both AM fungi included in the study overall inhibited most microbial groups as examined using whole cell biomarker fatty acids.     

The bacterial community of tomato rhizosphere is modified by inoculation with arbuscular mycorrhizal fungi but unaffected by soil enrichment with mycorrhizal root exudates or inoculation with Phytophthora nicotianae

Arbuscular mycorrhizal (AM) fungi have been shown to induce the biocontrol of soilborne diseases, to change the composition of root exudates and to modify the bacterial community structure of the rhizosphere, leading to the formation of the mycorrhizosphere. Tomato plants were grown in a compartmentalized soil system and were either submitted to direct mycorrhizal colonization or to enrichment of the soil with exudates collected from mycorrhizal tomato plants, with the corresponding negative controls. Three weeks after planting, the plants were inoculated or not with the soilborne pathogen Phytophthora nicotianae growing through a membrane from an adjacent infected compartment. At harvest, a PCR-Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments amplified from the total DNA extracted from each plant rhizosphere was performed. Root colonization with the AM fungi Glomus intraradices or Glomus mosseae induced significant changes in the bacterial community structure of tomato rhizosphere, compared to non-mycorrhizal plants, while enrichment with root exudates collected from mycorrhizal or non-mycorrhizal plants had no effect. Our results support that the effect of AM fungi on rhizosphere bacteria would not be mediated by compounds present in root exudates of mycorrhizal plants but rather by physical or chemical factors associated with the mycelium, volatiles and/or root surface bound substrates. Moreover, infection of mycorrhizal or non-mycorrhizal plants with P. nicotianae did not significantly affect the bacterial community structure suggesting that rhizosphere bacteria would be less sensitive to the pathogen invasion than to mycorrhizal colonization. Of 96 unique sequences detected in the tomato rhizosphere, eight were specific to mycorrhizal fungi, including two Pseudomonas, a Bacillus simplex, an Herbaspirilium and an Acidobacterium. One Verrucomicrobium was common to rhizospheres of mycorrhizal plants and of plants watered with mycorrhizal root exudates.     

Efficient mineral weathering is a distinctive functional trait of the bacterial genus Collimonas

The mineral weathering ability of 45 bacterial strains belonging to the genus Collimonas and coming from various terrestrial environments was compared to that of 5 representatives from the closely related genera Herbaspirillum and Janthinobacterium. Using glucose as the sole carbon source in a microplate assay for quantifying the release of iron and protons from biotite, all Collimonas strains proved to be very efficient weathering agents, in contrast to the Herbaspirillum and Janthinobacterium strains. The weathering phenotype was also evident during growth of collimonads on mannitol and trehalose, but not on gluconic acid. All Collimonas strains were able to solubilize inorganic phosphorus and produce gluconic acid from glucose, suggesting that acidification is one of the main mechanisms used by these bacteria for mineral weathering. The production of siderophores may also be involved, but this trait, measured as the ability of collimonads to mobilize iron, was shared with Herbaspirillum and Janthinobacterium strains. These findings are discussed in an ecological context that recognizes collimonads as mycophagous (fungal-eating) and efficient mineral weathering bacteria and suggests that this ability has evolved as an adaptation to nutrient-poor conditions, possibly as part of a mutualistic relationship with mycorrhizal fungi.     

Effects of mycorrhizal roots and extraradical hyphae on N uptake from vineyard cover crop litter and the soil microbial community

The objectives of this study were to evaluate the contribution of arbuscular mycorrhizal (AM) fungal hyphae to ¹⁵N uptake from vineyard cover crop litter (Medicago polymorpha), and to examine the soil microbial community under the influence of mycorrhizal roots and extraradical hyphae. Mycorrhizal grapevines (Vitis vinifera) were grown in specially designed containers, within which a polyvinyl chloride (PVC) mesh core was inserted. Different sizes of mesh allowed mycorrhizal roots (mycorrhizosphere treatment) or extraradical hyphae (hyphosphere treatment) to access dual labeled ¹⁵N and ¹³C cover crop litter that was placed inside the cores after 4 months of grapevine growth. Mesh cores in the bulk soil treatment, which served as a negative control, had the same mesh size as the hyphosphere treatment, but frequent rotation prevented extraradical hyphae from accessing the litter. Grapevines and soils were harvested 0, 7, 14, and 28 days after addition of the cover crop litter and examined for the presence of ¹⁵N. Soil microbial biomass and the soil microbial community inside the mesh cores were examined using phospholipid fatty acid analysis. ¹⁵N concentrations in grapevines in the hyphosphere treatment were twice that of grapevines in the bulk soil treatment, suggesting that extraradical hyphae extending from mycorrhizal grapevine roots may have a role in nutrient utilization from decomposing vineyard cover crops in the field. Nonetheless, grapevines in the mycorrhizosphere treatment had the highest ¹⁵N concentrations, thus highlighting the importance of a healthy grapevine root system in nutrient uptake. We detected similar peaks in soil microbial biomass in the mycorrhizosphere and hyphosphere treatments after addition of the litter, despite significantly lower microbial biomass in the hyphosphere treatment initially. Our results suggest that although grapevine roots play a dominant role in the uptake of nutrients from a decomposing cover crop, AM hyphae may have a more important role in maintaining soil microbial communities associated with nutrient cycling.