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）     

过量铜对4种外生菌根真菌的生长、碳氮和铜积累的影响

为了了解外生菌根真菌在过量铜胁迫下的生长和物质积累特点,揭示外生菌根真菌对过量铜胁迫的抵抗能力,研究了四种外生菌根真菌—美味牛肝菌(Boletus edulis)、铆钉菇(Gomphidius viscidus)、厚环乳牛肝菌(Suillus grevillei)和红绒盖牛肝(Xerocomus chrysenteron)在过量铜胁迫条件下菌丝中铜积累量、菌丝生长特性以及碳氮积累速率。四种测试菌种菌丝中的铜积累量,随营养液中铜浓度的增加而增加,在46mg/L铜培养基下生长15d,四种菌种菌丝内铜浓度分别是对照的40～60倍。B.edulis和X.chrysenteron菌丝中铜浓度与培养基中铜浓度呈直线相关,S.grevillei和G.viscidus为指数相关。菌丝在铜胁迫下依然呈S曲线增长,但初始生长推迟,指数增长期比对照晚1～2d。菌丝生物量和碳氮积累随铜浓度增加而显著降低。综合所有试验结果显示,四种测试菌种对过量铜的抗性强度为：B.edulis＞G.viscidus＞S.grevillei＞X.chrysenteron。     

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。     

几种外生菌根真菌培养特性的研究

对 4种外生菌根真菌的培养特性进行了研究。结果表明 ,PDMA和PD培养基是培养外生菌根真菌的较适合的培养基 ;木屑、麸皮、米糠、玉米粉、蛭石是培养菌根菌菌剂的较适宜的基质 ;用MMN营养液拌料以及适宜的水分供给都有利于外生菌根真菌的生长。     

铜胁迫下4种外生菌根真菌的耐受性比较

外生菌根对重金属具有较强的耐受性,通过生物固定对重金属污染土壤有一定修复能力。本试验选择4种外生菌根真菌红绒盖牛肝菌(Xerocomus chrysenteron)、双色蜡蘑(Laccaria bicolor)、铆钉菇(Gomphidius viscidus)和彩色豆马勃(Pisolithus tinctorius)进行离体培养,通过分析不同浓度铜(0、10、25、35、50和90 mg/L)胁迫下外生菌根真菌生长、铜积累和分泌耐热蛋白的变化,以探索4种外生菌根真菌的重金属耐受性及其机制。研究结果发现,铜胁迫处理组X.chrysenteron、P.tinctorius的菌丝干重比对照组分别仅减少7.8%~33.6%、0.2%~11.5%,而单位菌丝铜积累量显著低于L.bicolor和G.viscidus(P0.05),在生长方面表现出对过量铜胁迫的较强耐受性;从单位菌丝耐热蛋白分泌量来看,G.visciduss(X.chrysenteron,P.tinctorius)L.bicolor,但由于X.chrysenteron、P.tinctorius生物量干重较大,耐热蛋白分泌总量足够多,从而降低培养环境中的Cu2+,X.chrysenteron、P.tinctorius通过分泌耐热蛋白耐受铜胁迫能力更强;通过SDS-PAGE图谱发现铜胁迫诱导X.chrysenteron、G.viscidus和L.bicolor分泌出分子量在31~43 k D之间的多种新耐热蛋白成分。     

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（0.229gNaH2PO4/L）〉中磷（1．147gNaH2PO4／L）〉高磷（5.735g NaH2PO4／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。     

辽东栎幼苗的外生菌根合成

 在温室花盆中播种辽东栎（Quercus liaotungensis）种子获得辽东栎幼苗,并对幼苗接种外生菌根真菌进行菌根合成试验。所用的外生菌根真菌有：铆钉菇（Gomphidius viscidus）、臭红菇（Russula foetens）、厚环乳牛肝菌（Suillus grevillei）、褐环乳牛肝菌（S. luteus）、彩色豆马勃（Pisolithus tinctorius）、美味牛肝菌（Boletus edulis）、赭丝膜菌（Cortinarius russus）、土生空团菌（Cenococ     

荧光蛋白在双色蜡蘑中的构建与表达

菌根是真菌与植物之间形成的互惠互利的营养共生体,对生态环境有重大的意义。外生菌根真菌与植物互作机制以及真菌基因功能的深入研究都需要对菌根真菌进行遗传转化,本研究以外生菌根真菌模式生物双色蜡蘑(Laccaria bicolor)为研究对象,选择细胞核中的核小体蛋白H₂B基因为目的基因,以pCEBN为表达载体,融合红色荧光蛋白,最终构建在真菌中表达的双元载体,使用根瘤农杆菌介导转化法转化双色蜡蘑菌丝,随后利用PCR对真菌转化子进行验证后,通过激光共聚焦显微镜观察到菌丝细胞核中的红色荧光,成功将融合荧光蛋白转化菌根真菌,为后续研究菌根真菌中基因的亚细胞定位提供了实验平台。结果表明,利用双元载体和农杆菌转化方法,建立了高效的双色蜡蘑转化体系(93.33%),在激光共聚焦显微镜下观察到菌丝细胞核中红色荧光信号,验证了融合荧光蛋白在双色蜡蘑中的成功表达。本研究成功地构建了菌根真菌中的核小体蛋白和红色荧光蛋白融合表达的真菌转化体系。     

温度、pH及干旱胁迫对沙地樟子松外生菌根菌生长影响

通过对采集于沙地樟子松人工林内的3种外生菌根真菌（牛肝菌Boletus sp.、乳菇Lactariussp.、高环柄菇Macrolepiota procera）进行纯培养,在不同温度、pH值和PEG模拟干旱胁迫条件下,观测了外生菌根菌的生长发育状况.结果表明,3种外生菌根菌生物量在不同pH范围（3～7）内存在着显著差异（P＜0.05）;在低于5℃和高于37℃时,3种外生菌根菌均不能生长,其最适生长温度分别为：牛肝菌,25℃高环柄菇,25～28℃,乳菇,25～30℃;在低浓度PEG胁迫处理条件下（10%,-0.20MPa）,3种外生菌根菌的生长均有所增加,当PEG胁迫达到30%（-1.53 MPa）时,3种外生菌根菌的生长均受到抑制.与上述结果比较,沙地樟子松人工林地土壤的实际pH值在外生菌根菌的最适生长范围内;林地最高气温值远远超过3种外生菌根菌的最适生长值的时间在每年的生长季都会出现;在极端干旱年份（如1996年）,林地土壤的实际含水量在生长季节（5～9月）远远低于抑制外生菌根菌生长的土壤含水量,可能影响到外生菌根菌的生存.因此,可以推断,沙地樟子松人工林地的外生菌根菌的生长与发育,在干旱胁迫及高温作用下受到很大程度的影响,如果仅从微生物角度分析,外生菌根菌在干旱和高温条件下不能生长或已经死亡是导致沙地樟子松人工林衰退的一个原因.     

有机碳和无机碳对3种真菌胞外酸性磷酸酶和蛋白酶活性的影响

多数研究表明外生菌根真菌能够促进植物养分吸收并提高植物生长,但是对其发生的原因研究较少。本文在室内控制条件下,研究了真菌菌丝分泌N、P相关胞外酶及其受土壤有机碳（胡敏酸）和无机碳（碳酸钙）添加的影响,结果表明：1）3种真菌——松乳菇(Lactarius deliciosus)、变色红菇（Russula integra）、铆钉菇（Gomphidius viscidus）菌丝均能够分泌酸性磷酸酶和蛋白酶,而且多数情况下,MMN培养基培养14 d时,各个酶活性较高,而不同菌的胞外酶活性存在较大的差异,平均值来看铆钉菇酸性磷酸酶活性最低而蛋白酶活性最高,其它2个真菌菌丝的胞外酶活性差异不大;2）添加胡敏酸后,3种菌丝的酸性磷酸酶活性都是随着胡敏酸添加量的增加而逐渐增加;但蛋白酶活性存在差异：松乳菇的蛋白酶活性随着胡敏酸添加量的增加而逐渐增加;变色红菇的蛋白酶活性对胡敏酸不敏感,受其影响不大;铆钉菇的蛋白酶活力在少量的胡敏酸作用下最强,但浓度过高反而抑制其蛋白酶的活性。3）添加碳酸钙后,总体来看,3种菌丝胞外酸性磷酸酶和蛋白酶活性都是添加少量碳酸钙时酶活性最强,随着浓度的增加(如0.1 g),其酶活性开始受到抑制。综上所述,真菌菌丝能够分泌酸性磷酸酶和蛋白酶,这可能是因为这些外生菌根真菌能够促进植物养分吸收和快速生长的原因;有机碳和无机碳的加入可以直接影响真菌菌丝胞外酶的分泌,进而影响土壤内有机磷和有机氮化合物的分解,显示其在土壤碳循环中的作用。     

四种外生菌根真菌对油松幼苗的抗猝倒病和促生作用

为了筛选拮抗油松猝倒病菌及对油松苗木具有促生作用的外生菌根（ectomycorrhizal,ECM）真菌,通过对盆栽油松幼苗进行人工接种,测定了ECM真菌对油松根的侵染率、对猝倒病的防治效果以及对油松株高和地径的影响。结果表明,供试ECM真菌中,绒粘盖牛肝菌Suillus tomentosus、灰环粘盖牛肝菌Suillus laricinus、灰鹅膏菌Amaniata vaginata和血红铆钉菇Gomphidius viscidus可在盆栽条件下与油松合成菌根。灰鹅膏菌对油松猝倒病防治效果最好,为43%     

铝对外生菌根真菌草酸分泌及磷、钾、铝吸收的影响

试验研究了在铝胁迫条件下,6种(株)外生菌根真菌(ECMF)的生长、草酸分泌,以及磷、钾、铝的吸收状况。结果表明,铝对抗(耐)型菌种Pt715、HrSp、CgSIV的生长无抑制作用,但显著抑制敏感型菌株LbS238N、LbS238A和Lb270的生长,说明ECMF对铝胁迫的生长反应可能是筛选抗(耐)铝的指标之一。在铝胁迫条件下,无论是抗(耐)型还是敏感型菌种(株),都会发生一系列有益于抗(耐)铝的生化反应,包括草酸分泌量、菌丝磷和钾含量增加,H+分泌改变等。在培养液中,草酸电离产生的H+仅占H+总浓度的少数,说明溶液中H+的主要来源不是ECMF所分泌的草酸,而是菌丝细胞为保持吸收阳离子的电荷平衡排出的H+或分泌的其它有机酸。     

Effects of bacteria on mycorrhizal development and growth of container grown Eucalyptus diversicolor F. Muell. seedlings

The development of ectomycorrhizas on inoculated eucalypt seedlings in commercial nurseries is often slow so that only a small percentage of roots are mycorrhizal at the time of outplanting. If mycorrhizal formation could be enhanced by co-inoculation with bacteria which promote rapid root colonisation by specific ectomycorrhizal fungi, as demonstrated by certain bacteria in the Douglas fir-Laccaria bicolor association, this would be of advantage to the eucalypt forest industry. Two bacterial isolates with a demonstrated Mycorrhization Helper Bacteria (MHB) effect on ectomycorrhiza formation between Pseudotsuga menziesii and Laccaria bicolor (S238), and seven Western Australian bacterial isolates from Laccaria fraterna sporocarps or ectomycorrhizas were tested in isolation for their effect on ectomycorrhizal development by three Laccaria spp. with Eucalyptus diversicolor seedlings. Mycorrhizal formation by L. fraterna (E710) as measured by percentage infected root tips, increased significantly (p < 0.05) by up to 296% in treatments coinoculated with MHB isolates from France (Pseudomonas fluorescens Bbc6 or Bacillus subtilis MB3), or indigenous isolates (Bacillus sp. Elf28 or a pseudomonad Elf29). In treatments coinoculated with L. laccata (E766) and the MHB isolate P. fluorescens (Bbc6) mycorrhizal development was significantly inhibited (p < 0.05). A significant Plant Growth Promoting Rhizobacteria (PGPR) effect was observed where the mean shoot d.w. of seedlings inoculated only with an unidentified bacterium (Elf21), was 49% greater than the mean of uninoculated controls (-fungus, -bacterium). Mean shoot d.w. of seedlings coinoculated with L. bicolor (S-238), which did not form ectomycorrhizas with E. diversicolor, and an unidentified bacterium (Slf14) or Bacillus sp. (Elf28) were significantly higher than uninoculated seedlings or seedlings inoculated with L. bicolor (S-238) alone. This is the first time that an MHB effect has been demonstrated in a eucalypt-ectomycorrhizal fungus association. These organisms have the potential to improve ectomycorrhizal development on eucalypts under nursery conditions and this is particularly important for fast growing eucalypt species where the retention time of seedlings in the nursery is of short duration (2–3 months).     

Induced hydrolytic enzymes of ectomycorrhizal fungi against pathogen Rhizoctonia solani

Interactions between ectomycorrhizal fungi (Suillus laricinus, S. tomentosus, Amanita vaginata and Gomphidius viscidus) and the pathogen Rhizoctonia solani in co-culture were studied using both light and scanning electron microscopy. S. laricinus, S. tomentosus and A. vaginata inhibited the growth of the pathogen. Moreover, A. vaginata exhibited coiling around and penetration of the hyphae into R. solani was observed in the interaction zone. Furthermore, the production of chitinases, beta-1,3-glucanases and beta-glucosidases by these ectomycorrhizal fungi on colloidal chitin or cell walls of R. solani was evaluated: chitinases were not induced by colloidal chitin but all three enzymes were induced by R. solani cell walls. No correlation between inhibition rate and production of lytic enzymes was found.     

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.     

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.     

Gene organization of the mating type regions in the ectomycorrhizal fungus Laccaria bicolor reveals distinct evolution between the two mating type loci

In natural conditions, basidiomycete ectomycorrhizal fungi such as Laccaria bicolor are typically in the dikaryotic state when forming symbioses with trees, meaning that two genetically different individuals have to fuse or 'mate'. Nevertheless, nothing is known about the molecular mechanisms of mating in these ecologically important fungi. Here, advantage was taken of the first sequenced genome of the ectomycorrhizal fungus, Laccaria bicolor, to determine the genes that govern the establishment of cell-type identity and orchestrate mating. The L. bicolor mating type loci were identified through genomic screening. The evolutionary history of the genomic regions that contained them was determined by genome-wide comparison of L. bicolor sequences with those of known tetrapolar and bipolar basidiomycete species, and by phylogenetic reconstruction of gene family history. It is shown that the genes of the two mating type loci, A and B, are conserved across the Agaricales, but they are contained in regions of the genome with different evolutionary histories. The A locus is in a region where the gene order is under strong selection across the Agaricales. By contrast, the B locus is in a region where the gene order is likely under a low selection pressure but where gene duplication, translocation and transposon insertion are frequent.     

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.