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     

七株外生菌根真菌与三种松苗菌根的形成能力

松树外生菌根菌资源丰富,但实际应用的种类不多。为筛选出与松苗形成菌根能力较强的菌种,采用播种接菌和芽苗截根移栽接菌两种方法,对7株外生菌根真菌与马尾松、湿地松和黑松3种松苗的菌根合成进行了研究。结果表明：形成的菌根以二叉分枝状为主,棒状菌根相对较少,多叉状菌根以马尾松较多。PC2形成的菌根表面菌丝厚且紧密,504、EG、Pt,形成的菌根表面菌丝紧密程度中等,而505、ZJ和HX形成的菌根其表面菌丝则比较稀疏;Pt1、Pt2、EG形成的菌根外延菌丝较长,而505、HX形成的菌根外延菌丝极短。截根接菌时的感染率和感染指数要高于播种接菌。504形成菌根的能力最强,在3种松苗上的菌根感染率都达100％,感染指数最高可达90,最低也达70;Pt2和EG与马尾松和黑松形成菌根的能力较强;而505和HX仅与马尾松形成菌根的能力较强;Pt1形成菌根的能力较差,在3种松苗上菌根感染率和感染指数都较低。在3种松苗中,马尾松的菌根化状况最好,其次为黑松,湿地松的菌根化状况较差。     

Controlled mycorrhizal initiation as a means to improve root development in somatic embryo plantlets of hybrid larch (Larix × eurolepis)

This study represents the first report of in vitro mycorrhization of somatic embryo-derived plantlets. Effects of fungi on the rooting of plantlets as well as on their ultra-structure were studied. Embryo-derived plantlets of a hybrid larch ( Larix × eurolepis Henry) were grown aseptically in the presence of four ectomycorrhizal fungi. One month after inoculation with Laccaria laccata, Hebeloma cylindrosporum and Pisolithus tinctorius , the number of plantlets which had developed a well-growing root system was significantly improved compared with controls. Furthermore, root branching was strongly stimulated by L. laccata and H. cylindrosporum , which were more efficient species than P. tinctorius and Suillus grevillei . Development of the root system was concomitantly accompanied by an enhancement of shoot growth, except for plantlets inoculated with S. grevillei , which died after three to six months. At the ultrastructural level, cross-sections of lateral roots made six months after inoculation, showed the presence of a Hartig net in the case of L. laccata and H. cylindrosporum , whereas no such net was observed with the two other fungi. With S. grevillei , some perforating hyphae penetrated the cortical cells and resulted in cell death. Evidence of a relationship between structure and efficiency of the association was found.     

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.     

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.     

Surface-bound phosphatase activity in living hyphae of ectomycorrhizal fungi of Nothofagus obliqua

We determined the location and the activity of surface-bound phosphomonoesterase (SBP) of five ectomycorrhizal (EM) fungi of Nothofagus oblique. EM fungal mycelium of Paxillus involutus, Austropaxillus boletinoides, Descolea antartica, Cenococcum geophilum and Pisolithus tinctorius was grown in media with varying concentrations of dissolved phosphorus. SBP activity was detected at different pH values (3-7) under each growth regimen. SBP activity was assessed using a colorimetric method based on the hydrolysis of p-nitrophenyl phosphate (pNPP) to p-nitrophenol phosphate (pNP) + P. A new technique involving confocal laser-scanning microscopy (LSM) was used to locate and quantify SBP activity on the hyphal surface. EM fungi showed two fundamentally different patterns of SBP activity in relation to varying environmental conditions (P-concentrations and pH). In the cases of D. antartica, A. boletinoides and C. geophilum, changes in SBP activity were induced primarily by changes in the number of SBP-active centers on the hyphae. In the cases of P. tinctorius and P. involutus, the number of SBP-active centers per μm hyphal length changed much less than the intensity of the SBP-active centers on the hyphae. Our findings not only contribute to the discussion about the role of SBP-active centers in EM fungi but also introduce LSM as a valuable method for studying EM fungi.     

Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities

Drought stress conditions (DC) reduce plant growth and nutrition, restraining the sustainable reestablishment of Nothofagus dombeyi in temperate south Chilean forest ecosystems. Ectomycorrhizal symbioses have been documented to enhance plant nitrogen (N) and phosphorus (P) uptake under drought, but the regulation of involved assimilative enzymes remains unclear. We studied 1-year-old N. dombeyi (Mirb.) Oerst. plants in association with the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch. and Descolea antartica Sing. In greenhouse experiments, shoot and root dry weights, mycorrhizal colonization, foliar N and P concentrations, and root enzyme activities [glutamate synthase (glutamine oxoglutarate aminotransferase (GOGAT), EC 1.4.1.13-14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), nitrate reductase (NR, EC 1.6.6.1), and acid phosphomonoesterase (PME, EC 3.1.3.1-2)] were determined as a function of soil-water content. Inoculation of N. dombeyi with P. tinctorius and D. antartica significantly stimulated plant growth and increased plant foliar N and P concentrations, especially under DC. Ectomycorrhizal inoculation increased the activity of all studied enzymes relative to non-mycorrhizal plants under drought. We speculate that GDH is a key enzyme involved in the enhancement of ectomycorrhizal carbon (C) availability by fuelling the tricarboxylic acid (TCA) cycle under conditions of drought-induced carbon deficit. All studied assimilative enzymes of the ectomycorrhizal associations, involved in C, N, and P transfers, are closely interlinked and interdependent. The up-regulation of assimilative enzyme activities by ectomycorrhizal fungal root colonizers acts as a functional mechanism to increase seedling endurance to drought. We insist upon incorporating ectomycorrhizal inoculation in existing Chilean afforestation programs.     

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。     

黑松菌根共生体中真菌液泡形态构架及其活力

 为探讨菌根共生体中菌根菌液泡结构及其活力变化对宿主生长的影响, 对3种外生菌根菌黄色须腹菌(Rhizopogen luteous, 简称Rl)、彩色豆马勃(Pisolithus tinctorius, 简称 Pt₂)、美味牛肝菌 (Boletus edulis, 简称Be)离体菌丝及与黑松(Pinus thunbergii)形成菌根共生体的冰冻切片进行6-羧基二乙酸荧光素(6-CFDA)染色, 观察了真菌液泡形态构架及其活力的变化。结果表明, 在Pt₂与Rl离体菌丝中既可见单个分散的大液泡, 又可见相邻液泡间由细管状连接而成的管状液泡系统, Be离体菌丝的液泡则呈细小颗粒状, 密集分布于菌丝体的各个部分; Be离体菌丝的活性强于Pt₂和Rl。上述3种菌根菌与黑松形成菌根共生体后, Pt₂与Rl对黑松的促生效果好于Be, 菌根共生体中菌套和哈氏网菌丝的活性明显强于Be, 且仍可见数个液泡连接成管状系统; 而在Be菌根中菌丝液泡系统不形成管状, 活性较低。结果暗示, 菌根共生体中真菌的管状液泡系统及其活力与对黑松的促生作用似存在一定的相关性。该研究对于进一步了解菌根的促生作用机理及菌根真菌与宿主植物的协同进化具有一定的意义。     

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.     

Iron ore weathering potentials of ectomycorrhizal plants

Plants in association with soil microorganisms play an important role in mineral weathering. Studies have shown that plants in symbiosis with ectomycorrhizal (ECM) fungi have the potential to increase the uptake of mineral-derived nutrients. However, it is usually difficult to study many of the different factors that influence ectomycorrhizal weathering in a single experiment. In the present study, we carried out a pot experiment where Pinus patula seedlings were grown with or without ECM fungi in the presence of iron ore minerals. The ECM fungi used included Pisolithus tinctorius, Paxillus involutus, Laccaria bicolor and Suillus tomentosus. After 24?weeks, harvesting of the plants was carried out. The concentration of organic acids released into the soil, as well as potassium and phosphorus released from the iron ore were measured. The results suggest that different roles of ectomycorrhizal fungi in mineral weathering such as nutrient absorption and transfer, improving the health of plants and ensuring nutrient circulation in the ecosystem, are species specific, and both mycorrhizal roots and non-mycorrhizal roots can participate in the weathering process of iron ore minerals.     

黄山地区松树林外生菌根菌资源及生态分布

1998～2003年,对黄山地区松树林下外生菌根菌资源及生态分布进行了调查,鉴定出外生菌根菌43种,隶属于10科17属.其中马尾松林下有43种、黄山松林下有12种.马尾松幼林下的外生菌根菌种类单一,彩色豆马勃、硬皮马勃类为优势菌种;马尾松成熟林下外生菌根菌种类丰富,其优势种类为红菇科、鹅膏菌科、牛肝菌科和鸡油菌科的一些种类.并分析了树种植被对外生菌根菌分布的影响,菌根真菌的分布与温度、湿度及土壤条件的关系.     

Ectomycorrhizal weathering of the soil minerals muscovite and hornblende

Ectomycorrhizal fungi are hypothesized to enhance mineral weathering in forest soils. Several studies have shown an increased uptake of mineral-derived nutrients by trees when in symbiosis with ectomycorrhizal fungi. However, it is difficult to determine from these studies if the improved nutrient uptake is the result of increased weathering or better exploitation of the substrate by the ectomycorrhizal fungi. In a pot experiment, Pinus sylvestris (Scots pine) seedlings were grown with or without ectomycorrhizal fungi, and with or without the mineral muscovite as the only potassium (K) source or the mineral hornblende as the only magnesium (Mg) source. After 27 wk, all pools of non-mineral-bound K or Mg were determined. The ectomycorrhizal fungus Paxillus involutus increased weathering of muscovite but not hornblende. The other ectomycorrhizal fungi tested, Piloderma croceum and Suillus bovinus, did not increase weathering of either muscovite or hornblende compared with the nonmycorrhizal trees. The P. involutus-mediated mobilization of K from muscovite resulted in increased K content of root plus adhering hyphae, but not of shoots. In conclusion, ectomycorrhizal fungi may increase weathering of minerals in response to nutrient deficiencies, but this response is species specific.     

Pure culture growth of ectomycorrhizal fungi on inorganic nitrogen sources

Four ectomycorrhizal fungi were tested for their ability to grow (i.e., mycelial mat radial extension and fungal biomass) on nutrient media either supplemented with ammonium-nitrogen or nitrate-nitrogen or in the absence of an inorganic nitrogen source.Pisolithus tinctorius, Cenococcum geophilum andThelephora terrestris exhibited greater growth on ammonium-nitrogen.Suillus granulatus grew better on the nitrate-nitrogen nutrient medium. Regardless of inorganic nitrogen form preference (i.e., ammonium-nitrogen or nitrate-nitrogen), all 4 species showed some growth on each of the 3 nutrient media. Growth rate maxima varied by fungal species as well as by inorganic nitrogen source. Maximum growth rate forT. terrestris exceeded rates exhibited by the other 3 fungi by 2–5 times.     

Apatite as a P source in mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings

The objectives of the study are firstly to test the ability of ectomycorrhizal pine seedlings to use apatite as a P source in comparison with non-mycorrhizal pine seedlings and secondly, to determine if there is a relation between exudation of organic acids and the ability to use apatite as a P source. Non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 4 different isolates of ectomycorrhizal fungi were grown for 220 days in sand/peat filled pots with apatite (Ca₅(F,OH)(PO₄)₃) as the sole P source. In an additional experiment, non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 2 different isolates of ectomycorrhizal fungi were grown without any P source for 250 days. All other nutrients were supplied in a balanced nutrient solution.Ectomycorrhizal seedlings grew less than non-mycorrhizal seedlings but ectomycorrhizal seedlings produced a large external mycelium not included in the biomass estimates. All seedlings in the present study had low shoot:root ratios compared to seedlings growing under optimal conditions. All seedlings grown with apatite as P source had higher foliar P concentrations (0.71–2.11 mg/g) than seedlings growing without any P source (0.57–0.75 mg/g) indicating a significant ability to use apatite as a P source. Seedlings colonized by Suillus variegatus and Paxillus involutus had higher concentrations and total contents of P in shoots compared with non-mycorrhizal seedlings, indicating significant improvement of P uptake by these fungi in comparison with non-mycorrhizal seedlings or seedlings colonized Piloderma croceum.No clear relationship between exudation of organic acids and uptake of P was found. Seedlings colonized by S. variegatus reduced the pH of the soil more than seedlings colonized by P. involutus or non-mycorrhizal seedlings. It is suggested that S. variegatus colonization improves the P uptake by reducing the pH of the soil while P. involutus improves P uptake by having a greater ability to absorb dissolved phosphate than non-mycorrhizal roots or roots colonized by the other fungi used in the study.     

4种外生菌根真菌对难溶性磷酸盐的溶解能力

以红绒盖牛肝菌(Xerocomus chrysenteron)、美味牛肝菌(Boletus edulis)、黄须腹(Rhizopogen luteous)和劣味乳菇(Lactarius insulsus)4种外生菌根真菌为对象,在纯培养条件下比较它们对4种难溶性磷酸盐[Ca_3(PO_4)_2、Ca_(10)(PO_4)_6(OH)_2、FePO_4·4H_2O和AlPO_4]的溶解能力,以探讨外生菌根真菌对难溶性磷酸盐的溶解作用及其影响因素.结果表明:(1)4种外生菌根真菌均能在Ca_3(PO_4)_2为唯一磷源的NBRIP平板上生长,但生长速率明显不同,且红绒盖牛肝菌生长速率最快.(2) 4种菌根真菌对4种磷酸盐的溶解能力存在显著差异,红绒盖牛肝菌发酵液可溶性磷含量大多显著高于对照和其他菌根真菌,对磷酸盐的溶解能力最强.(3)4种菌根真菌发酵液可滴定酸度(2.30～46.00 mmol·L~(-1))和pH(4.57～6.99)与其溶磷量分别呈极显著正相关(r=0.991**)和极显著负相关(r=-0.939**).(4)4种外生菌根真菌均能分泌乙酸、柠檬酸和反丁烯二酸,且它们分泌有机酸总量(14.14～1 537.46 mg·L~(-1))与其溶磷量呈显著正相关(r=0.998*).研究发现, 4种外生菌根真菌发酵液可滴定酸度、pH和有机酸的种类与含量是影响其溶解Ca_3(PO_4)_2能力的主要因素;红绒盖牛肝菌的溶磷能力较强,具有应用于中国土壤缺磷地区造林的潜力.     

The Multifunctional Role of Ectomycorrhizal Associations in Forest Ecosystem Processes

Belowground biological interactions that occur among plant roots, microorganisms and animals are dynamic and substantially influence ecosystem processes. Among these interactions, the ectomycorrhizal (ECM) symbiosis is remarkable but unfortunately these associations have mainly been considered within the rather narrow perspective of their effects on the uptake of dissolved mineral nutrients by individual plants. More recent research has placed emphasis on a wider, multifunctional perspective, including the effects of ectomycorrhizal symbiosis on plant and microbial communities, and on ecosystem processes. This includes mobilization of N and P from organic polymers, release of nutrients from mineral particles or rock surfaces via weathering, effects on carbon cycling, interactions with mycoheterotrophic plants, mediation of plant responses to stress factors such as drought, soil acidification, toxic metals, and plant pathogens, rehabilitation and regeneration of degraded forest ecosystems, as well as a range of possible interactions with groups of other soil microorganisms. Ectomycorrhizas are almost invariably characterized by a Hartig net composed of highly branched hyphae which entirely surround the outer root cortical cells. The Hartig net is the place of massive bidirectional exchanges of nutrients between the host and the fungus. Through these branched hyphae ectomycorrhizal fungi connect their plant hosts to the heterogeneously distributed nutrients required for their growth, enabling the flow of energy-rich compounds required for nutrient mobilization whilst simultaneously providing conduits for the translocation of mobilized products back to their hosts. In addition to increasing the nutrient absorptive surface area of their host plant root systems, the extraradical mycelium of ectomycorrhizal fungi provides a direct pathway for translocation of photosynthetically derived carbon from their hosts to microsites in the soil and a large surface area for interaction with other soil micro-organisms. The detailed functioning and regulation of these mycorrhizosphere processes is still poorly understood and needs detailed molecular approach to study these mycorrhizosphere processes but recent progress in ectomycorrhizal associations is reviewed and potential benefits of improved understanding of mycorrhizosphere interactions are discussed.     

Hypaphorine from the ectomycorrhizal fungus Pisolithus tinctorius counteracts activities of indole-3-acetic acid and ethylene but not synthetic auxins in eucalypt seedlings

Very little is known about the molecules regulating the interaction between plants and ectomycorrhizal fungi during root colonization. The role of fungal auxin in ectomycorrhiza has repeatedly been suggested and questioned, suggesting that, if fungal auxin controls some steps of colonized root development, its activity might be tightly controlled in time and in space by plant and/or fungal regulatory mechanisms. We demonstrate that fungal hypaphorine, the betaine of tryptophan, counteracts the activity of indole-3-acetic acid (IAA) on eucalypt tap root elongation but does not affect the activity of the IAA analogs 2,4-D ((2,4-dichlorophenoxy)acetic acid) or NAA (1-naphthaleneacetic acid). These data suggest that IAA and hypaphorine interact during the very early steps of the IAA perception or signal transduction pathway. Furthermore, while seedling treatment with 1-amincocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, results in formation of a hypocotyl apical hook, hypaphorine application as well as root colonization by Pisolithus tinctorius, a hypaphorine-accumulating ectomycorrhizal fungus, stimulated hook opening. Hypaphorine counteraction with ACC is likely a consequence of hypaphorine interaction with IAA. In most plant-microbe interactions studied, the interactions result in increased auxin synthesis or auxin accumulation in plant tissues. The P. tinctorius / eucalypt interaction is intriguing because in this interaction the microbe down-regulates the auxin activity in the host plant. Hypaphorine might be the first specific IAA antagonist identified.     

Chitinase activities are induced in Eucalyptus globulus roots by ectomycorrhizal or pathogenic fungi, during early colonization

A comparative study of root chitinase activities induced by the ectomycorrhizal basidimycete Pisolithus tinctorius Coker and Couch and the pathogenic fungus Phytophthora cinnamomi , has been carried out. Two chitinases were constitutively present in Eucalyptus globulus ssp. bicostata (Maid et al.) Kirkp. roots. When 7-day-old seedlings were challenged with the ectomycorrhizal fungus, root chitinase activity was stimulated already after 6 h, during the very early stages of ectomycorrhizal colonization. Comparing chitinase electrophoretic patterns induced by symbiotic strains more or less compatible with Eucalyptus , a strong stimulation of chitinase activity indicated a successful interaction, which evolved quickly towards root infection and mature mycorrhizae formation. Root chitinase activity remained constant over 7 days during the establishment of the symbiosis. No new chitinase band was induced by the pathogen, when compared with the symbiotic fungi. Chitinase activity was only stimulated quantitatively after pathogenic infection. Root chitinase activities were also stimulated by fungal cell extracts applied in vitro. Such stimulation mimicked precisely the stimulation by living fungi. The intensity of the plant response to fungal extracts was related to fungal strain aggressiveness.