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     

Induced defence responses limit Hartig net formation in ectomycorrhizal birch roots

Roots of clonal birches ( Betula pendula ) were inoculated with the ectomycorrhizal fungi Paxillus involutus (isolates P0 and Mi) and Hebeloma cylindrosporum (strains D1 and D105). These fungi showed different rates of mycorrhiza formation in vitro . Mature mycorrhizas were obtained after only 2–4 d with H. cylindrosporum , whereas 6–8 d were necessary with P. involutus isolate P0, and P. involutus isolate Mi was not able to form mature mycorrhiza during the 10 d of the experiment. Temporal changes in PAL activity and the expression of genes encoding intracellular pathogenesis-related proteins were followed after inoculating birch roots with these fungi. Transient increase of PAL activity, and transient induction of expression of the wound-inducible Bet v1-SC1 gene, were observed in roots challenged with both H. cylindrosporum strains and the P. involutus isolate P0. These changes were found to coincide with hyphal penetration between root cells during Hartig net formation, and were never observed in roots inoculated with the poorly aggressive P. involutus isolate Mi. Examination of mycorrhizal root sections under u.v. light indicated the presence of phenolic compounds in the host cell walls at the vicinity of the Hartig net. These results strongly suggest that hyphal penetration between the root cells triggers a transient defence response which, in turn, could limit Hartig net formation to the outer layer of the root cortex.     

The auxin-inducible GH3 homologue Pp-GH3.16 is downregulated in Pinus pinaster root systems on ectomycorrhizal symbiosis establishment

In an attempt to determine whether auxin-regulated plant genes play a role in ectomycorrhizal symbiosis establishment, we screened a Pinus pinaster root cDNA library for auxin-upregulated genes. This allowed the identification of a cDNA, Pp-GH3.16, which encodes a polypeptide sharing extensive homologies with GH3 proteins of different plants. Pp-GH3.16 was specifically upregulated by auxins and was not affected by cytokinin, gibberellin, abscisic acid or ethylene, or by heat shock, water stress or anoxia. Pp-GH3.16 mRNAs were quantified in pine roots inoculated with two ectomycorrhizal fungi, Hebeloma cylindrosporum and Rhizopogon roseolus. Surprisingly, Pp-GH3.16 was downregulated following inoculation with both fungal species. The downregulation was most rapid on establishment of symbiosis with an indole-3-acetic acid (IAA)-overproducing mutant of H. cylindrosporum, which overproduced mycorrhizas characterized by a hypertrophic Hartig net. This indicates that, despite being auxin-inducible, Pp-GH3.16 can be downregulated on establishment of symbiosis with a fungus that releases auxin. By contrast, Pp-GH3.16 was not downregulated in pine root systems inoculated with a nonmycorrhizal mutant of H. cylindrosporum, suggesting that the downregulation we observed in mycorrhizal root systems was a component of the molecular cross-talk between symbiotic partners at the origin of differentiation of symbiotic structures.     

The feeding preference of mycophagous Collembola varies with the ectomycorrhizal symbiont

The interactions of the collembolan insect Proisotoma minuta with ectomycorrhizal and/or pathogenic fungi was examined in three experiments: (1) in vitro analysis of feeding patterns, (2) in vitro food preference test, and (3) in situ analysis of ectomycorrhizal colonization in relation to population density. The ectomycorrhizal fungi Laccaria laccata, Pisolithus tinctorius, Suillus luteus, Thelephora terrestris and the pathogenic fungi Rhizoctonia solani were employed in all experiments. In vitro and in situ experiments revealed that Pr. minuta consumed all the ectomycorrhizal fungi tested but the feeding pattern and consumption varied with each isolate. In a comparative in vitro feeding preference test, where Pr. minuta was given a choice, R. solani was grazed more heavily than the ectomycorrhizal fungi. Among the ectomycorrhizal fungi examined, Pi. tinctorius was consumed significantly less than L. laccata, S. luteus or T. terrestris in the presence of R. solani. A 10-week in situ analysis of loblolly pine (Pinus taeda L.) seedling root systems inoculated with Pr. minuta revealed that ectomycorrhizal colonization was significantly less than that of control plants (without Pr. minuta). Collectively, these data suggest that mycophagous Collembola may play a major role in the distribution and biomass of ectomycorrhizal fungi in the rhizosphere of tree seedlings.     

Effect of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of arbuscular mycorrhiza-forming Prunus avium and Prunus cerasus

 The effect of different genotypes of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of Prunus avium and P. cerasus was studied in an attempt to determine whether ectomycorrhizal fungi could enhance in vitro adventitious root formation in plants which form arbuscular endomycorrhizas. The rooting percentage of P. avium cuttings was approximately 16% in the absence of hormonal treatment; it increased up to 30% in the presence of 5.7 μM IAA which was the most favourable auxin concentration. The rooting percentage of cuttings cultivated in the absence of IAA was enhanced by all the studied strains of H. cylindrosporum. It ranged from 50 to 60% with the IAA-overproducing mutant D 111 or the wild-type dikaryon D1, to 100% in the presence of the mutants 331 or D 117. The cuttings of P. cerasus showed a higher rooting ability than those of P. avium since approximately 40% of them were able to root in the absence of hormonal treatment. Except for the mutant D117, their rooting percentage was not significantly improved by H. cylindrosporum. Fungal inoculation also affected the survival of cuttings at acclimatization: 50% of the uninoculated P. avium cuttings survived whereas the survival percentage of inoculated cuttings ranged from 30 to 100% depending on the fungal genotype. With P. cerasus, the percentage of survival of uninoculated cuttings ranged from 85 to 100% and fungi either did not significantly improve it or lowered it. At acclimatization fungal hyphae could be observed in close contact with adventitious roots, but they did not establish mycorrhizal association. The shoot height of P. avium plantlets obtained from inoculated cuttings was not significantly different from that of plantlets originating from uninoculated ones. By contrast, fungal inoculation generally depressed the growth of acclimatized P. cerasus plantlets. The possibility of using ectomycorrhizal fungi as a tool to enhance rooting of micropropagated cuttings of plants which do not form ectomycorrhizas is discussed. Received: 25 November 1996 / Accepted: 2 June 1997     

Indole 3-acetic acid production by ectomycorrhizal fungi.

Ability of 8 ectomycorrhizal fungi to synthesise indole 3-acetic acid from L-tryptophan and their growth rate were studied. Differences in the levels of IAA synthesis and biomass production among the 8 mycorrhizal fungi were observed. A positive correlation was recorded between IAA level and mycelial growth. The synthesis of IAA and mycelial biomass were maximum on 30th day after incubation. Pisolithus tinctorius and Laccaria laccata exhibited higher amounts of IAA production than other fungi, whereas Amanita muscaria and Rhizopogon luteolus showed least quantity of IAA.     

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.     

模拟氮沉降和不同外生菌根真菌侵染对樟子松幼苗生长的影响

盆栽条件下,采用单接种Suillus bovines(以下简称SB)和混合接种(Suillus luteus、Suillus grevillea、Tricholoma fulvum、Boletinus grisellus、Suillus bovinus、Leucocortinarius bulbiger、Rhizopogon luteolus、Pisolithus tinctorius 8个菌种的等量混合菌剂接种,以下简称HJ)方法对樟子松(Pinus sylvestris var.mongolica)2个月幼苗进行接种处理,幼苗生长6个月形成菌根后对樟子松幼苗进行模拟氮沉降实验,5个月后通过测定幼苗生长指标、根系形态结构和菌根侵染率,旨在探讨模拟氮沉降对樟子松不同接种处理幼苗生长的影响。结果表明:①随着氮浓度的增加,单接种乳牛肝(SB)和混合接种(HJ)菌根侵染率均呈下降趋势,且氮沉降对SB处理的菌根侵染率影响更显著。②接种显著促进幼苗地上和地下生物量积累,且混合接菌效果更好;同一接菌处理中,氮浓度增加对地上生物量的积累有抑制作用;接种和氮浓度对地径和地上生物量有显著交互作用,但对苗高和地下生物量无交互作用。③HJ处理中不施氮(CKN)和1倍氮浓度(CN)较不接菌(CK)和SB处理显著促进幼苗根系的平均直径、总根长和总表面积;氮浓度增加显著促进CK和SB处理的幼苗根系平均直径、总根长和总表面积,但是对HJ处理的根系各形态结构指标均产生抑制作用。接种和氮浓度以及两者的交互作用均极显著影响幼苗根系形态结构。     

外生菌根真菌对‘NL-895杨’光合作用的影响

以‘NL-895杨’(Populus×euramericanacv.Nanlin 895)幼苗为材料,研究6种外生菌根真菌接种对其幼苗光合作用的影响.结果显示:接种60 d后‘NL-895杨’叶片的叶绿素a和b含量、PSⅡ最大光能转化效率(Fv/Fm)比对照极显著提高,其中叶绿素a和b的增加幅度分别为28.16%～68.03%和69.29%～138.26%,并以接种劣味乳菇的‘NL-895杨’Fv/Fm最高,且与对照差异极显著.各处理菌根苗培养150 d时‘NL-895杨’的净光合速率表现为美味牛肝菌>红绒盖牛肝菌>劣味乳菇>彩色豆马勃2号>紫金蜡蘑>彩色豆马勃1号>对照;菌根真菌处理对‘NL-895杨’叶片胞间CO2浓度影响较小,但其蒸腾速率显著高于对照,而且能促进总糖的合成与积累,并以接种彩色豆马勃2号的‘NL-895杨’苗总糖含量最高.研究表明,接种菌根真菌可以有效提高‘NL-895杨’对光能资源的利用率,增加其光合产物积累,显著促进其生长.     

The Ecological Relation Between Pinus taiwanensis and Fungi in the Process of Ectomycorrhizae Synthesis

The root samples of Pinus taiwanensis from seedlings to large trees of Mt. Huangsban were studied under light microscope(LM) and scanning electron microscope(SEN). It is obvious that ectomycorrhizae from extensively between the pine roots and the fungi. Isolation and pure culture of the samples of sporocarps were carried out, of which six strains were inoculated to the dissinfected seedlings were under laboratory condition. Pisolithus tinctorius was introduced to inoculate the pine seedlings too. Studies on the development of dichotomous mycorrhizae and the early stage of ectomycorrhizae formation were specially emphasized. Three types of the process of hyphae infection and the ontogeny of ectomycorrhizae synthesis, development and subsequent degeneration were described. Some ecological effects of ectomycorrhizae, which increase the growth of pine trees were also mentioned. Finally the authors sugge. st that the success inoculation of Pisolithus tinctorius to the pine trees will offer a new device for the formation and development of the ectomycorrhizae in terms to promote their growth.     

In vitro evidence of mycoparasitism of the ectomycorrhizal fungus <Emphasis Type="Italic">Laccaria laccata</Emphasis> against <Emphasis Type="Italic">Mucor hiemalis</Emphasis> in the rhizosphere of <Emphasis Type="Italic">Pinus sylvestris</Emphasis>

Interactions between the ectomycorrhizal fungus Laccaria laccata and the soil fungus Mucor hiemalis f. hiemalis in co-culture, and in the rhizosphere of in vitro-grown Pinus sylvestris seedlings were investigated by light- and scanning electron-microscopy. In co-culture, mycelial growth away from the L. laccata colony reduced the number of aerial hyphae at the contact zone and increased the density and compactness of the mycelium-characterized gross morphology of the saprobic fungus. Although the growth of M. hiemalis was suppressed, no penetration of M. hiemalis hyphae after the colony was entered by L. laccata was observed. Instead, dense coiling of L. laccata hyphae around sporangiophores, overpowering them and causing them to disappear, was quite common. On nonmycorrhizal roots, sporangiospores germinated heavily and formed long hyphae for 2 days post inoculation, whereas their germination was totally inhibited on mycorrhizal roots. At 3 days after inoculation, only sporangia were seen with mycelial mats firmly attached to the roots by the mantle hyphae, whereas some remnants of sporangiophores, ruptured sporangial walls and degraded hyphae of M. hiemalis were overgrown by the mantle hyphae. During the next 3 days, the mantle-hyphae-invading sporangia formed short, thin branches that grew directly towards individual spores, tapering off upon contact.     

Rooting and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris are enhanced by the ectomycorrhizal fungus Hebeloma cylindrosporum

The effect of different strains of the ectomycorrhizal fungus Hebeloma cylindrosporum on rooting in vitro and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris was studied. Two clones of P. pinaster and one of P. sylvestris were unable to root in the absence of auxin, but were induced to root on a medium devoid of auxin by all the fungal strains. Wild-type and indoleacetic acid (IAA)-overproducing mutant strains of the fungus stimulated rooting of clones showing a good reactivity to auxin to the same extent. In contrast, with a clone of P. sylvestris that showed low reactivity to auxin, IAA-overproduction by the fungus was advantageous for the induction of rooting of cuttings. Adventitious roots formed in the presence of a fungal strain were completely surrounded by a loosely packed network of hyphae which formed mycorrhizas as soon as roots grew outside the agar medium. During acclimatization, fungal inoculation improved the survival of rooted cuttings. At the end of acclimatization, fungal mycelia could be easily detected in the culture substrate of cuttings inoculated with dikaryotic strains and most of the pines' short roots were mycorrhizal. Monokaryotic mycelia, which have a lower growth rate and a lower infectivity, displayed poor ability to colonize the substrate and to form mycorrhizas. Two months after the end of acclimatization, fungal inoculation frequently depressed the growth of acclimatized cuttings of the clone J of P. pinaster . No depressive effect was observed with clone 78 and growth stimulation could even be observed with the infective dikaryon D1 which formed numerous mycorrhizas. From these studies, it was concluded that ectomycorrhizal fungi could be a suitable tool for improving rooting in vitro and survival at acclimatization of micropropagated conifer cuttings.     

Cell wall proteins of the ectomycorrhizal basidiomycete Pisolithus tinctorius: identification, function, and expression in symbiosis.

Specific cell-cell and cell-substrate interactions direct the growth of ectomycorrhizal fungi to their host root targets. These elaborate mechanisms lead to the differentiation of distinct multihyphal structures, the mantle, and the Hartig net. In the ectomycorrhizal basidiomycete Pisolithus tinctorius, the use of two-dimensional gel electrophoresis, immunocytochemical microscopy, and RNA blot analysis has demonstrated the differential expression of cell wall proteins (CWPs), such as hydrophobins, adhesins, and mannoproteins, during symbiotic interaction. In other fungi, these CWPs have been suggested to play a role in hyphae aggregation, intracellular signaling cascades, and cytoskeletal changes. The recent cloning of the genes for several of these CWPs in P. tinctorius allows us to address their function in symbiosis. This review summarizes our knowledge of CWPs in P. tinctorius and considers parallels with other biotrophic fungi as a possible framework for future work.     

Comparative structural study ofQuercus serrata andQ. acutissima formed byPisolithus tinctorius andHebeloma cylindrosporum

Ectomycorrhizas were synthesized in pots and growth pouches betweenQuercus serrata, Q. acutissima, and two ectomycorrhizal fungi,Pisolithus tinctorius andHebeloma cylindrosporum. Root morphology and the structure of the mantle and Hartig net were compared using light, fluorescence, scanning and transmission electron microscopy.P. tinctorius initially colonized root cap cells, and eventually produced a highly branched lateral root system with a complete mantle, whereasH. cylindrosporum promoted root elongation with few hyphae on the root apex surface indicating that interaction between roots differs with fungal species. Hartig net structure and hyphal inclusions varied between all the combinations tested. There were structural differences between mycorrhizas ofH. cylindrosporum/Q. acutissima grown in soil and growth pouches, which indicate that the growth pouch environment can induce artefacts in roots. Fruit bodies ofH. cylindrosporum developed in pots withQ. acutissima. AlthoughP. tinctorius has been used to inoculate oak seedlings in the nursery, results of this study indicate thatH. cylindrosporum may also be an effective ectomycorrhizal fungus forQ. serrata andQ. acutissima.     

Effect of two vesicular-arbuscular mycorrhizal fungi on the growth of micropropagated potato plantlets and on the extent of disease caused by <Emphasis Type="Italic">Rhizoctonia</Emphasis><Emphasis Type="Italic">solani</Emphasis>

Two micropropagated potato cultivars, Goldrush and LP89221, were inoculated into sowing trays with either Glomus etunicatum or G. intraradices in a greenhouse. After 2 weeks, plantlets were transplanted into pots and roots were challenged 7 days later with Rhizoctonia solani. At different times after R. solani infection, disease severity, mortality rate, root colonization levels, various growth parameters, and shoot mineral content were evaluated. In Goldrush, only inoculation with G. etunicatum led to a significant reduction in disease severity, ranging between 60.2% and 71.2%, on both shoot and crown. This decrease was not observed in LP89221. Compared with the control plantlets, inoculation of Goldrush with G. etunicatum or G. intraradices reduced significantly the mortality rate by 77% and 26%, respectively, whereas vesicular-arbuscular mycorrhizal (VAM) fungi did not significantly influence the mortality rate in LP89221. In Goldrush, inoculation with G. etunicatum significantly increased shoot fresh weight, root dry weight and the number of tubers produced per plant, whereas G. intraradices only significantly increased the number of tubers. Tuber and root fresh weights of both potato cultivars were significantly reduced by R. solani infection. However, R. solani-infected plantlets of both Goldrush and LP89221, inoculated with G. etunicatum, produced significantly greater tuber fresh weight than non-VAM plantlets. In R. solani-infected plantlets of Goldrush but not LP89221, G. etunicatum and G. intraradices increased root fresh weight by approximately 140.3% and 76.5%, respectively, compared with non-VAM plants. The potato cultivars Goldrush and LP89221 responded differently to VAM fungal inoculation and to R. solani infection in terms of shoot mineral content.     

菌丝桥在日本落叶松幼苗间磷传递和植株生长中的作用

 应用四室隔网系统研究了菌丝桥在日本落叶松(Larix kaempferi)幼苗间传递磷的作用。结果表明,供体接种卷缘桩菇(Paxillus involutus)和彩色豆马勃（Pisolithus tinctorius）后,其外延菌丝可以穿过隔离层侵染受体落叶松,在供体和受体落叶松间形成了菌丝桥。供体植株接种菌根真菌后生物量明显增加,但是对受体植株没有显著的影响。菌根真菌侵染的供体和受体植株的根、地上部吸磷量均分别显著高于对照,而且供体植株根、地上部吸磷量增加的程度明显高于受体。被卷缘桩菇和彩色豆马勃侵染的受体植株体内32P的放射性强度分别是对照的10倍和6倍,两者形成菌丝桥后传递到受体植株的32P分别为供体植株体内32P的1.10%和0.22%。供体植株吸收的32P可以通过菌丝桥传递给受体,但是绝对数量十分有限,对受体植株磷营养没有产生显著的影响,但P. involutus和P. tinctorius侵染受体植株后,促进了受体落叶松对磷的吸收,这是菌丝桥形成后,真菌帮助受体植株吸收磷引起的。     

Mycorrhizal Fungi Prevent Disease in Stressed Pine Seedlings

The common rhizospheric fungus Cylindrocarpon destructans caused severe root damage leading to mortality in light-starved, stunted seedlings of Pinus sylvestris. This effect was, however, almost fully prevented by the presence of mycorrhizal fungi; i.e. Laccaria laccata, Pisolithus tinctorius, or unidentified species present in small amounts of conifer soil. Protection took place even before visible mycorrhizae were found on the roots. This is the first time that protection ofstressed plants against non-parasitic (non-invasive) pathogens has been reported. The results indicate that root protection by mycorrhizal fungi may not only be effective inside the root but probably also extends out into the rhizoplane or rhizosphere.     

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.     

Control of nodule number by the phytohormone abscisic Acid in the roots of two leguminous species

The effects of the phytohormone abscisic acid (ABA) on plant growth and root nodule formation were analyzed in Trifolium repense (white clover) and Lotus japonicus, which form indeterminate and determinate nodules, respectively. In T. repense, although the number of nodules formed after inoculation with Rhizobium leguminosarum bv. trifolii strain 4S (wild type) was slightly affected by exogenous ABA, those formed by strain H1(pC4S8), which forms ineffective nodules, were dramatically reduced 28 days after inoculation (DAI). At 14 and 21 DAI, the number of nodules formed with the wild-type strain was decreased by exogenous ABA. In L. japonicus, the number of nodules was also reduced by ABA treatment. Thus, exogenous ABA inhibits root nodule formation after inoculation with rhizobia. Observation of root hair deformation revealed that ABA blocked the step between root hair swelling and curling. When the ABA concentration in plants was decreased by using abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, the number of nodules on lateral roots of abamine-treated L. japonicus increased dramatically, indicating that lower-than-normal concentrations of endogenous ABA enhance nodule formation. We hypothesize that the ABA concentration controls the number of root nodules.