Size and complexity of the nuclear genome of the ectomycorrhizal fungus Paxillus involutus

The basidiomycete Paxillus involutus is forming ectomycorrhizal symbiosis with a broad range of forest trees. Reassociation kinetics on P. involutus nuclear DNA indicated a haploid genome size of 23 Mb including 11% of repetitive DNA. A similar genome size (20 Mb) was estimated by genomic reconstruction analysis using three single copy genes. To assess the gene density in the P. involutus genome, a cosmid containing a 33-kb fragment of genomic DNA was sequenced and used to identify putative open reading frames (ORFs). Twelve potential ORFs were predicted, eight displayed significant sequence similarities to known proteins found in other organisms and notably, several homologues to the Podospora anserina vegetative incompatibility protein (HetE1) were found. By extrapolation, we estimate the total number of genes in the P. involutus haploid genome to approximately 7700.     

The mycorrhizal fungus Paxillus involutus transports magnesium to Norway spruce seedlings. Evidence from stable isotope labeling

Although it is well established that ectomycorrhizas improve the mineral nutrition of forest trees, there has been little evidence that they mediate uptake of divalent cations such as Mg. We grew nonmycorrhizal seedlings and seedlings mycorrhizal with Paxillus involutus Batsch in a sand culture system with two compartments separated by a 45-μm Nylon mesh. Hyphae, but not roots, can penetrate this net. Labeling the compartment only accessible to hyphae with ²⁵Mg showed that hyphae of the ectomycorrhizal fungus Paxillus involutus transported Mg to their host plant. No label was found in nonmycorrhizal control plants. Our data support the idea that ectomycorrhizas are important for the Mg nutrition of forest trees. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of heavy metals on the glutathione status in different ectomycorrhizal <Emphasis Type="Italic">Paxillus involutus</Emphasis> strains

The glutathione (GSH) status and heavy metal tolerance were investigated in four Paxillus involutus strains isolated from different heavy-metal-polluted and non-polluted regions of Europe. The heavy metal burden in the habitats did not affect significantly either the heavy metal (Cr₂O₇²⁻, Cd²⁺, Hg²⁺, Pb²⁺, Zn²⁺, Cu²⁺) tolerance and accumulation or the GSH production of the strains tested. Exposures to heavy metals increased the intracellular GSH concentrations in 12 from 24 experimental arrangements (four strains exposed to six heavy metals) independently of the habitats of the strains. The importance of GSH in heavy metal tolerance (high MIC values, ability to accumulate heavy metals and to grow in the presence of heavy metals) was thus demonstrated in this ectomycorrhizal fungus.     

Nitrogen and phosphorus acquisition by the mycelium of the ectomycorrhizal fungus Paxillus involutus and its effect on host nutrition

The contribution of the extramatrical mycelium to N and P nutrition of mycorrhizal Norway spruce ( Picea abies (L.) Karst.) was investigated. Seedlings either inoculated with Paxillus involutus (Batsch) Fr. or non-mycorrhizal were grown in a two compartment sand culture system where hyphae were separated from roots by a 45 μm nylon net. Nutrient solution of the hyphal compartment contained either 1.8 m m NH₄⁺ and 0.18 m m H₂PO₄⁻ or no N and P. Aluminium added to the hyphal compartment as a tracer of mass flow was not detected in the plant compartment, indicating that measurements of N and P transfer by the mycelium were not biased by solute movement across the nylon net.
The addition of N and P to the hyphal compartment markedly increased dry weight, N and P concentration and N and P content of mycorrhizal plants. Calculating uptake from the difference in input and output of nutrient in solution confirmed a hyphal contribution of 73% and 76% to total N and P uptake, respectively. Hyphal growth was increased at the site of nutrient solution input.     

Cross-colonization of Scots pine (Pinus sylvestris) seedlings by the ectomycorrhizal fungus Paxillus involutus in the presence of inhibitory levels of Cd and Zn

The effects of Cd and Zn on cross-colonization by Paxillus involutus of Scots pine seedlings was examined by using pairs of ectomycorrhizal (ECM) and non-mycorrhizal (NM) seedlings grown in the same vessel. This was done to assess, first, the ability of P. involutus to colonize NM Scots pine seedlings by growth from colonized roots of other Scots pine seedlings in the presence of Cd or Zn, and, second whether ECM colonization of Scots pine by P. involutus provided a competitive advantage over NM seedlings. Ectomycorrhizal colonization of Scots pine was shown to be more sensitive than Scots pine itself to Cd and Zn, but prior colonization did provide a competitive advantage with respect to biomass production. This beneficial effect over NM seedlings was, however, equal in the control, Cd and Zn treatments, and was due simply to growth stimulation in the presence of ECM colonization. Cross-colonization from an ECM to a NM seedling was reduced but not prevented by Cd and Zn. Cd had a more negative effect on cross-colonization than on initial colonization of seedlings, whereas Zn had an equally inhibitory effect on both parameters. These results have important implications for plant establishment on metal-contaminated sites. If cross-colonization between plants is reduced by toxic metals, plant establishment on contaminated sites might be retarded.     

Lignosulfonate promotes the interaction between Scots pine and an ectomycorrhizal fungus Pisolithus tinctorius in vitro

Lignosulfonate (LS) is a lignin-based polymer obtained as a by-product from paper industry, which may have potential as an amendment with macronutrients. We studied effects of LS on the interaction between Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings and the ectomycorrhizal (ECM) fungusPisolithus tinctorius (Pers.) Coker and Couch. The experiments were performed in vitroon the MMN agar medium containing Fe–LS chelate at the concentrations of 0, 5, 10 and 25 mg/L. Inoculation with P. tinctoriusincreased root growth of the seedlings. Fe–LS enhanced P. tinctorius induced formation of lateral roots and had a dose-dependent positive effect on the establishment of mycorrhizas on the seedlings. The growth of the fungal mycelium was improved by Fe–LS, which might cause faster and more intensive contact with the roots and, thus, better root growth and mycorrhiza formation. P.tinctorius enhanced also adventitious root formation and subsequent root growth of the hypocotyl cuttings but without any synergistic effect with Fe–LS. Our study with P. tinctorius and Scots pine in vitro indicates that a low-cost by-product Fe–LS, obtained from paper industry, may be a potential tool to improve the efficiency of fungal inoculations, thus, facilitating the early interaction between an ECM fungus and host seedling.     

In vitro weathering of phlogopite by ectomycorrhizal fungi

The ways in which ectomycorrhizal fungi benefit tree growth and nutrition have not been fully elucidated. Whilst it is most probably due to improved soil colonization, it is also likely that ectomycorrhizal fungi could be directly involved in nutrient cycling of soil reserves. This study assessed access by two species of ectomycorrhizal fungi to soil nonexchangeable K⁺ reserves. The incubation of ectomycorrhizal fungi in bi-compartment Petri dishes with phlogopite led to cation exchange reactions and to crystal lattice weathering. Paxillus involutus COU led to irreversible phlogopite transformations, while Pisolithus tinctorius 441 led to reversible ones. Simultaneous depletion in K⁺ and Mg²⁺ led to an enhanced weathering of phlogopite by P. tinctorius 441. The observation of phlogopite evolution shows that some specific Al³⁺ immobilization occurred under P. tinctorius 441. The data suggest that these bio-weathering mechanisms could be related to the release of fungal organic acids or other complex forming molecules.     

外生菌根菌对樟子松苗木生长的影响

以内蒙古大兴安林樟子松分布区较典型的6个乡土菌种与本实验室从法国引进的宿主范围较广的2个菌种及内蒙古地区分布广泛的土生空团菌为研究对象,采用固体和液体两种菌剂类型,对出苗30d左右的樟子松实生苗进行外生菌根菌接种试验,接种130d后观察菌根侵染情况及生长特性,同时对菌根化的樟子松苗木进行自然干旱胁迫,研究外生菌根菌接种对樟子松苗木生长及抗逆性的影响。结果显示:(1)5个乡土菌种和1个法国引进菌种均能够与樟子松实生苗共生菌根,菌根化苗木较对照的苗高增长10.5%～111.4%、地茎增长4.55%～92.8%、苗木干重增加6.8%～184.5%,且固体菌剂效果优于液体菌剂。(2)在干旱胁迫下,菌根化苗木叶绿素含量较对照增加5.82%～36.92%、净光合速率提高120.2%～217.03%,叶片推迟10.3～25h出现萎蔫,临界致死时间推迟13.8～38.2h。(3)接种菌种厚环乳牛肝菌和点柄乳牛肝菌的苗木菌根侵染率、苗木生长量、干旱胁迫下各项生理指标均显著优于其他菌种和对照。研究表明,樟子松实生苗菌根化对苗木生长有明显的促进作用,能够提高苗木对干旱胁迫的抵御能力;厚环乳牛肝菌和点柄乳牛肝菌是有潜在研究和应用价值的优良菌株,这两个菌株的固体菌剂可用于樟子松菌苗生产。     

Spermidine and the ectomycorrhizal fungus Pisolithus tinctorius synergistically induce maturation of Scots pine embryogenic cultures

Exogenous spermidine (Spd) and the ectomycorrhizal (ECM) fungus Pisolithus tinctorius (Pers.) Coker and Couch had a synergistic effect on the maturation of Scots pine (Pinus sylvestris L.) somatic embryos. Induced maturation was expressed as a higher number of cell masses able to form embryos and a greater number of embryos formed per cell mass. In contrast, treatment with P. tinctorius alone on the hormone-free medium resulted in the lowest embryo-forming capacity. Retarded proliferation growth appeared to be required for maturation, but did not explain the synergistic effect of the fungus and exogenous Spd. Simultaneous treatment did not result in lower concentrations of putrescine (Put), Spd or spermine (Spm) in the embryogenic cell masses relative to the separate treatments. Our study is the first report on the use of a specific ECM fungus to induce maturation of somatic embryos, and it indicates that P. tinctorius was able to modify the maturation media in a way that, together with exogenous Spd, positively affected embryogenic cultures of Scots pine. Our study also shows that it is possible to enhance plant development other than root formation by using specific ECM fungi.     

Root Distribution and Nutrient Status of Mycorrhizal and Non-mycorrhizal Pinus sylvestris L. Seedlings Growing in a Sandy Substrate with Lignite Fragments

Reclaimed mine soils of the Lusatian mining district are characterised by small-scale heterogeneous distribution of lignite fragments of varying size embedded in a matrix of Tertiary and Quaternary sandy material. Despite amelioration with basic fly ashes, ongoing pyrite oxidation and the subsequent acidification generate a high physical and chemical heterogeneity within the substrate, which could negatively affect root proliferation. We hypothesised that this limitation for the root system may be compensated for by intensive exploration of the porous lignite fragments by roots and/or mycorrhizal hyphae to access water and nutrients stored in these fragments. To test this hypothesis, we compared growth, shoot nutrient content, and root distribution of mycorrhizal and non-mycorrhizal Pinus sylvestris L. seedlings in lignite-containing and lignite-free sandy substrate. Rhizotrons used for this experiment were filled with a sandy matrix with 6–9 evenly distributed spots of lignite fragments. Treatments included different levels of water and nutrient availability. After 8 months of growth, root tip vitality as well as growth and shoot nutrient concentration of the plants was higher for treatments with lignite spots in the sandy substrate than for sandy substrate without such amendments. Compared to the non-mycorrhizal plants, the seedlings inoculated with Paxillus involutus (Batsch) Fr. had a higher root dry mass, an increased number of root tips and a higher root length. These results confirm our hypothesis that the lignite fragments are an important nutrient and water reservoir for plants in these mine soils and they indicate that mycorrhizal colonisation may allow an intensive exploration of porous lignite fragments by mycorrhizal hyphae.     

Variations in ergosterol content and ornithine decarboxylase activity of ectomycorrhizal root-soil systems

Influences of soil P fertilization on temporal changes in ergosterol content and ornithine decarboxylase (E.C. 4.1.1.17, ODC) activity were monitored in rhizosphere soil, non-rhizosphere soil and Pinus contorta roots ectomycorrhizal with Hebeloma crustuliniforme grown in a loamy sand. With addition of mycorrhizal inoculum to loamy sand, ODC activity mg^-1 root increased between 10% and 2 fold within 21 weeks of pianting. Inoculation also decreased root mass per seedling. Inoculation increased mycelia mass per root mass by up to 2 fold but no differences were observed for total seedling mass until 35 weeks. Intramatrical mycelia were detrimental to early plant growth, but inoculated seedlings had 1.7 times more root mass and 1.3 times more shoot mass at 35 weeks. Rhizosphere soil contained up to 5 times more mycelia and up to 6 times greater ODC activity than non-rhizosphere soil. Inoculation increased rhizophere metabolic activity and intramatrical mycelia mass. Their sensitivity to fungal inoculation, P fertilization and temporal trends may make the methods useful in studies of rhizosphere ecology and root-microbe relationships.     

Spatial patterns of gene expression in the extramatrical mycelium and mycorrhizal root tips formed by the ectomycorrhizal fungus Paxillus involutus in association with birch (Betula pendula) seedlings in soil microcosms

Functional compartmentation of the extramatrical mycelium of ectomycorrhizal (ECM) fungi is considered important for the operation of ECM associations, although the molecular basis is poorly characterized. Global gene expression profiles of mycelium colonizing an ammonium sulphate ((NH4)2SO4) nutrient patch, rhizomorphs and ECM root tips of the Betula pendula-Paxillus involutus association were compared by cDNA microarray analysis. The expression profiles of rhizomorphs and nutrient patch mycelium were similar to each other but distinctly different from that of mycorrhizal tips. Statistical analyses revealed 337 of 1075 fungal genes differentially regulated among these three tissues. Clusters of genes exhibiting distinct expression patterns within specific tissues were identified. Genes implicated in the glutamine synthetase/glutamate synthase (GS/GOGAT) and urea cycles, and the provision of carbon skeletons for ammonium assimilation via beta-oxidation and the glyoxylate cycle, were highly expressed in rhizomorph and nutrient patch mycelium. Genes implicated in vesicular transport, cytoskeleton organization and morphogenesis and protein degradation were also differentially expressed. Differential expression of genes among the extramatrical mycelium and mycorrhizal tips indicates functional specialization of tissues forming ECM associations.     

Development,persistence and regeneration of foraging ectomycorrhizal mycelial systems in soil microcosms

Development of extraradical mycelia of two strains each of Paxillus involutus and Suillus bovinus in ectomycorrhizal association with Pinus sylvestris seedlings was studied in two dimensions in non-sterile soil microcosms. There were significant inter- and intra-specific differences in extraradical mycelial growth and morphology. The mycelial systems of both strains of P. involutus were diffuse and extended more rapidly than those of S. bovinus. Depending on the strain, P. involutus mycelia were either highly plane filled, with high mass fractal dimension (a measure of space filling) or sparse, low mass fractal dimension systems. Older mycelial systems persisted as linear cords interlinking ectomycorrhizal tips. S. bovinus produced either a mycelium with a mixture of mycelial cords and diffuse fans that rapidly filled explorable area, or a predominately corded mycelium of minimal area cover. In the soil microcosms, mass fractal dimension and mycelial cover tended to increase with time, mycelia encountering litter having significantly greater values. Results are discussed in terms of the ecology of these fungi, their foraging activities and functional importance in forest ecosystems.     

Influence of ectomycorrhizal fungi on the response of Sitka spruce and Japanese larch to forms of phosphorus

Ectomycorrhizal fungi (Paxillus involutus, Suillus grevillei and two unidentified basidiomycetes from excised Sitka spruce mycorrhizas) were isolated from stands of Sitka spruce either in monoculture or in a mixture with Japanese larch in an Irish conifer plantation. The growth of these fungi and their mycorrhizal formation in Sitka spruce and Japanese larch were examined after incubation in modified Melin-Norkrans medium containing either KH₂PO₄, Ca₃(PO₄)₂ or Fe phytate as the phosphorus (P) source. P. involutus and S. grevillei utilized all three P sources. The unidentified basidiomycetes had limited ability to utilize Fe phytate. Basidiomycete 1 showed poor growth on KH₂PO₄ whereas growth of basidiomycete 2 was low on Ca₃(PO₄)₂. Pure culture synthesis studies confirmed that P. involutus and the two basidiomycetes formed mycorrhizas with both tree species but S. grevillei was mycorrhizal only on Japanese larch. P. involutus formed more mycorrhizas in both conifers than the other fungi. Following inoculation with each of the four fungi, shoot and root dry mass of both Sitka spruce and Japanese larch seedlings was enhanced compared with uninoculated/nonmycorrhizal controls. On Fe phytate, Paxillus-inoculated Sitka spruce seedlings had the lowest primary root length and on KH₂PO₄, Suillus-inoculated Japanese larch had the greatest number of short roots. The only differences when Sitka spruce seedlings were grown in either monoculture or in a mixture with Japanese larch mycorrhizal with S. grevillei were primary root length and number of short roots after growth on media containing Fe phytate.     

Carbon and nitrogen utilization and acid production by mycelia of the ectomycorrhizal fungusTricholoma bakamatsutake in vitro

Mycelial growth of an isolate ofT. bakamatsutake was tested in media with C/N ratio ranging from 0 to 50 and with 32 carbon and 12 nitrogen sources. The isolate grew best at the C/N ratio of 30. It utilized the monosaccharidesd-glucose,d-mannose, andd-fructose, the disaccharide trehalose, and polysaccharide pectin among the carbon sources; and yeast extract,l-glutamic acid, and ammonium compounds among the nitrogen sources. The growth of ten isolates and secretion of gluconic and oxalic acids were compared ind-glucose, trehalose, and pectin media. The utilization ofd-glucose, trehalose, and pectin differed among the ten isolates, but all the isolates secreted gluconic acid in thed-glucose media and oxalic acid in the pectin media.     

大兴安岭北部天然针叶林土壤氮矿化特征

采用顶盖埋管法对大兴安岭地区天然针叶林(樟子松林、樟子松-兴安落叶松混交林和兴安落叶松林)土壤铵态氮(NH~+_4-N)、硝态氮(NO~-_3-N)、净氮矿化速率进行研究,并探索土壤理化性质与氮矿化之间的相关性,为大兴安岭地区森林生态系统土壤养分管理及森林经营提供帮助。结果表明:观测期内(5—10月)3种林型土壤无机氮变化范围为31.51—70.42 mg/kg,以NH~+_4-N形式存在为主,占比达90%以上,且与纯林相比混交林土壤无机氮含量较高。3种林型土壤净氮矿化、净氨化、净硝化速率月变化趋势呈V型,7、8月表现为负值,其他月份为正值。净氮矿化速率变化范围樟子松林为-0.54—1.28 mg kg~(-1) d~(-1)、樟子松-兴安落叶松混交林为-0.13—0.55 mg kg~(-1) d~(-1)、兴安落叶松林为-0.80—1.05 mg kg~(-1) d~(-1)。土壤净氨化过程在土壤氮矿化中占主要地位,占比达60%以上。3种林型土壤净氮矿化、净氨化及净硝化速率垂直差异显著,0—10 cm土层矿化作用明显高于10—20 cm土层(P0.05)。土壤氮矿化速率与土壤含水量、土壤有机碳含量、土壤C/N、枯落物全氮含量和枯落物C/N均存在显著相关性。不同类型的森林土壤及枯落物的质量也存在差异,进而影响土壤氮矿化特征。     

模拟氮沉降和接种外生菌根真菌对马尾松(Pinus massoniana)幼苗营养元素的影响

量化植物地上部和地下部元素含量对于理解和预测植物养分平衡如何响应大气氮沉降的变化至关重要。通过盆栽试验研究了氮沉降增加背景下外生菌根真菌对马尾松幼苗营养元素的影响。对马尾松幼苗进行了接种两种外生菌根真菌：（彩色豆马勃（Pisolithus tinctorius,Pt）与厚环乳牛肝菌（Suillus grevillei,Sg））以及4种氮素浓度添加：0 kg N hm^-2a^-1（N0）、正常氮沉降30 kg N hm^-2a^-1（N30）、中度氮沉降60 kg N hm^-2a^-1（N60）、重度氮沉降90 kg N hm^-2a^-1（N90）,共12个处理,测定了马尾松地上部和地下部大量元素和微量元素的含量。结果表明：施氮改变了营养元素在马尾松幼苗地上部和地下部的含量,马尾松幼苗磷（P）、钙（Ca）、铁（Fe）、锰（Mn）等元素均在N60时达到临界值,而当输入的量超过了马尾松对氮的需求时,氮沉降会使马尾松营养元素含量较最适浓度时降低,地上部碳（C）随施氮浓度的升高先升高后降低,N随施氮浓度的升高而升高,根系和叶片钾（K）、Ca、镁（Mg）均随施氮浓度的升高而降低,施氮也降低了根系C及微量元素的含量。但在同一施氮浓度下,接种外生菌根真菌（EMF）后能够提高大多数元素的含量,N90时接种厚环乳牛肝菌（Sg）和彩色豆马勃（Pt）的叶片N含量与对照相比分别提高112.6%和138.6%,根系N含量分别提高73.1%、71.6%;N60时接种Sg和Pt的植株叶片P含量比不施氮未接种对照分别提高了166.3%、132.9%,根系P含量分别提高了40.8%、38.5%。EMF能够维持植物养分平衡,从而降低高施氮量对植物的影响效果。这为未来气候变化情景中氮沉降增加下接种EMF可以调节植物元素含量,从而达到更适应环境的元素平衡来促进生长提供理论依据。