红皮云杉外生菌根菌单接种及混合接种对苗木生长的影响

以提高外生菌根真菌对红皮云杉苗木促生长效果为目的,在前期研究的基础上,采用菌株配对培养的方法对获得的云杉外生菌根真菌进行混合接种菌株组合筛选;采用沟施菌剂方法,对3年生红皮云杉移床苗进行外生菌根菌菌株组合野外混合接种试验,同时以各菌株野外单接种作为对照,研究外生菌根真菌单接种及混合接种对红皮云杉苗木生长的影响。试验结果表明:供试菌株及菌株组合对红皮云杉苗木生长均有一定的促进作用。菌株L15促生长效果最佳,接种100d苗木高生长高于对照30.88%,地径高于对照15.29%。组合L15/025与L15/009促苗木生长效果低于单接种L15的效果,高于单接种009和025的效果。接种菌株010和组合L15/025的苗木与对照相比,叶绿素含量提高差异显著,叶绿素a水平分别高于对照59.15%、54.61%,叶绿素b水平分别高于对照76.34%和67.78%。除接种菌株010的苗木外,其他处理苗木其过氧化氢酶活性与对照相比均有所降低。所有处理苗木的根系活力均低于对照。综上,外生菌根菌高效菌株与一般菌株混合接种会弱化高效菌株自身接种效果;苗木过氧化氢酶活性、苗木根系活力与苗木的生物量间无相关性。     

不同生境外生菌根真菌对铝胁迫的响应

以南北方不同生境下的10株外生菌根真菌为研究对象,采用液体培养的方法,研究了铝对不同菌根真菌的生物量、有机酸分泌及养分含量的影响,以期筛选出抗铝性强的优良菌株,并探讨其抗铝机理。结果表明:外生菌根真菌Sl 08抗铝性最强;Pt 715、Ld 03、Bo 11、Sl 01、Bo 15也具有不同程度的耐铝性;Sl 14、Gc 99、Cg 04抗铝性较差;Sg 11抗铝性最差。来自南方酸性森林土壤的菌株总体抗铝性强于来自北方石灰性土壤的菌株,这表明外生菌根真菌的铝耐受能力与其原始生境有着密切的联系。外生菌根真菌能分泌多种有机酸,且不同菌株分泌的有机酸种类不同。其中,受铝胁迫分泌量增加最多的是草酸。研究中,铝胁迫能增加大多数铝抗性菌株的草酸分泌量,其中铝抗性最强的Sl 08表现最为明显。但铝胁迫并没有促进具备一定铝抗性的Bo 11和Sl 01草酸的分泌量,同时在铝敏感的菌株中均观察到了草酸分泌量的增加。这表明分泌草酸可能并不是外生菌根真菌抵抗铝毒的唯一途径。对各菌株铝胁迫下对氮,磷及钾的吸收研究表明,除铝敏感菌株Sl 14外,铝胁迫均能促进各供试菌株对氮,磷或钾的吸收。综上,在一定铝浓度下,一些外生菌根真菌可通过增加草酸分泌来抵御铝毒。此外,铝胁迫下外生菌根真菌还可通过调控氮、磷、钾等营养元素的吸收来抵抗铝毒,即通过增加对营养元素的吸收来增强其在铝胁迫下的生存能力,这可能是其抵御铝胁迫的应激反应之一。     

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。     

外生菌根菌不同接种方法对樟子松苗木生长的影响

 以提高外生菌根真菌对樟子松(Pinus sylvestris var. mongolica)苗木促生长效果为目的, 在前期研究的基础上, 采用菌株配对培养的方法对获得的樟子松外生菌根真菌进行混合接种菌株组合筛选; 采用苗木截根-菌液浸根和沟施接种方法分别对2年生和3年生樟子松苗木进行野外单接种及混合接种。研究外生菌根真菌不同接种方法、菌株单接种及混合接种对樟子松苗木生长的影响。试验结果表明: 供试菌株及菌株组合对樟子松苗木生长均有一定的促进作用。菌株GT005和菌株035为供试菌株中对樟子松苗木促生长效果最佳的菌株。采用苗木截根-菌液浸根方法接种2年生樟子松苗木130 d, GT005接种的苗木高生长提高54%, 地径生长提高15%, 过氧化氢酶活性提高48%, 而根系活力降低3%; 菌株035接种的苗木高生长提高42%, 地径生长提高56%, 过氧化氢酶活性提高47%, 根系活力提高11%。沟施接种方法接种3年生樟子松苗木100 d, GT005接种苗木高生长提高10%, 地径生长提高15%, 过氧化氢酶活性提高90%, 而根系活力降低34%; 菌株035接种苗木高生长提高7%, 地径生长提高9%, 过氧化氢酶活性提高6%, 而根系活力降低46%; 菌株组合044/GT001和GT001/GT005 接种的苗木高生长仅比对照提高3.47%和2.07%, 而菌株组合 044/025 和044/009 接种的苗木高生长低于对照; 混合接种的苗木其地径生长高于对照0.16%～7.98%。综上所述, 苗木截根-菌液浸根接种方法对苗木的促生长效果显著高于沟施接种方法; 外生菌根菌高效菌株与一般菌株混合接种会弱化高效菌株自身接种效果; 苗木过氧化氢酶活性、苗木根系活力与苗木的生物量间无相关性。     

红皮云杉外生菌根菌生物学特性的研究

通过对红皮云杉 (PiceakoraiensisNakai) 4种外生菌根菌的生物学特性的研究 ,确定 4种外生菌根菌的生长所需最适温度、最适碳源、最适氮源以及最适pH值 ,以便应用于菌种的大量生产。试验结果表明 :林地蘑菇 (AgaricussilvaticusSchaeff.)和菌株L15生长最适温度相对较高 ,均为 30℃ ,属于耐高温菌 ;而菌株 0 31和菌株 0 0 9生长最适温度分别为 2 5℃和 2 0℃ ,属于中温菌。各菌株生长所需最适pH值均为 6 5。林地蘑菇生长所需最佳碳源为麦芽糖和蔗糖 ;菌株 0 0 9生长所需最佳碳源为蔗糖 ;菌株L15和菌株 0 31生长所需最佳碳源为葡萄糖。除菌株 0 0 9生长所需最佳氮源为硝酸钠外 ,其余菌株生长所需最佳氮源均为天门冬酰氨。     

外生菌根菌对樟子松苗木生长的影响

通过菌种野外采集、分离培养、纯化及室内人工接种试验,获得9株樟子松外生菌根菌。采用苗木截根—菌剂浸根方法,对2年生樟子松移床苗进行外生菌根菌野外单接种试验,以接种无菌液体培养基的苗木作为对照,研究外生菌根菌单接种对樟子松苗木生长的影响。野外单接种试验表明,9株外生菌根菌对樟子松苗木均有一定的促生长效果。接种130 d,除菌株010外,其他菌株均可提高樟子松苗高23%以上。其中,接种菌株GT005的苗高提高54.24%;接种菌株035、009、LH004及GT001的苗高分别提高41.53%、36.44%、35.59%和35.59%。菌株035、LH004、025、010和GT001可使苗木地径提高20%以上,其中菌株035提高地径56.31%,菌株LH0040、25分别提高39.93%和29.01%。除接种菌株010的苗木外,接种其他菌株的樟子松苗木过氧化氢酶活性均高于对照30.23%~48.37%。接种菌株004的苗木根系活力最强,高于对照39.17%;接种菌株GT005和010的苗木,根系活力低于对照;接种其他菌株的苗木根系活力高于对照2.5%~16.67%。接种菌株GT005的苗木的叶绿素含量最高,高于对照20%以上。9个菌株均可用于樟子松苗木生产中。樟子松苗木的过氧化氢酶活性、根系活力与苗木的生物量间无相关性。     

钾与信号抑制剂对外生菌根真菌分泌乙酸的调控作用

外生菌根真菌是森林生态系统中的重要成分,参与树木养分的吸收利用。试验液体培养外生菌根真菌,设置不同供钾水平,添加钙信号抑制剂,研究了它们的有机酸和氢离子分泌,以及乙酸分泌对供钾和信号抑制剂的响应。结果表明,供试菌株的生长,氮、磷、钾含量和吸收量因菌株不同而异,生物量变化于52.91—121.72 mg/瓶之间,相差1倍以上。外生菌根真菌吸收养分的差异可能与它们对土壤养分环境的长期适应、进化、选择有关。在外生菌根真菌的培养液中,分别检测出草酸、乙酸、苹果酸、柠檬酸和丁二酸等,以及大量的氢离子,说明菌根真菌能分解土壤含钾矿物,释放钾离子,改善寄主植物的钾营养。其中,乙酸分泌量较大,具有普遍性,低钾刺激分泌乙酸,高钾时分泌减少,其分泌速率与供钾浓度和菌丝吸钾量之间呈显著负相关(r=-0.734,r=-0.617,n=60)。钾对菌根真菌分泌乙酸的调控作用具有改善森林钾素营养,防止土壤养分淋失的生理和生态意义。此外,在低钾条件下,阴离子通道和钙信号抑制剂抑制外生菌根真菌分泌乙酸。说明钙信号和阴离子通道参与了乙酸分泌,缺钾可能是刺激乙酸分泌的原初信号,通过信号转导和一系列级联反应促进乙酸分泌。     

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.     

接种彩色豆马勃对模拟酸沉降下马尾松幼苗生物量的影响

外生菌根能够提高宿主植物对外界环境胁迫的抵抗力,促进植物的生长,本文试图揭示外生菌根对酸雨胁迫下马尾松生长的保护作用。本研究采用盆栽试验,共设置四个处理：酸雨对照处理（Control check (CK), 约pH值5.5）不接种,酸雨对照处理接种,酸雨pH值3.5处理不接种,酸雨pH值3.5处理接种。pH值3.5的酸雨处理降低马尾松的生物量,在试验前期降低根冠比,试验中后期则提高根冠比,在试验初期增加了叶面积,但中后期显著降低了叶面积。接种外生菌根菌有利于马尾松幼苗的生长,pH值3.5处理下接种外生菌根菌能提高马尾松幼苗的生物量,外生菌根菌对生物量分配和叶面积的影响与酸雨胁迫的影响是相反的,即外生菌根菌抵消了酸雨胁迫对马尾松的影响。     

外生菌根蘑菇分离技术折DNA鉴定方法

外生菌根真菌中存在一些食用蘑菇,由于其食药用价值极高而自然资源稀少,人们正努力探索驯化栽培的可能途径,作为供试菌种的各种分离培养物的真伪鉴定就成了研究者关注的焦点.评述了国内外近年来发展的几种外生菌根蘑菇分离菌株的DNA鉴定方法,并对它们进行了分析讨论.     

Production of gibberellin-like substances by fungi isolated from mycorrhizae of pine (Pinus silvestris/L.).

Gibberellin-like substances were found to be produced by fungi isolated from mycorrhizae of pine. Among the isolates studied gibberellin-like substances producers were most numerous among the Basidiomycetes forming ectotrophic mycorrhiza and among non-sporulating fungi forming no mycorrhiza. In most strains the gibberellin-like subtances were elaborated after 10--20 days of incubation. The Rf values of the gibberellin-like compounds were different in various strains, but in most of them substances showing gibberellin activity appeared on the chromatograms run with benzene, acetic acid (10:3 v/v) at Rf 0.2--0.6.     

三种松树内生真菌的分离鉴定及其对红脂大小蠹伴生真菌的拮抗作用

【背景】松树的内生真菌会影响红脂大小蠹及其伴生真菌的生长及扩散,从而影响红脂大小蠹的入侵。【目的】掌握赤峰地区红脂大小蠹寄主树种内生真菌的物种多样性,筛选对其伴生菌有拮抗作用的菌株,为红脂大小蠹的生物防治提供资源。【方法】采用组织分离法、形态学鉴定和internal transcribed spacer (ITS)序列分析相结合的方法对红脂大小蠹寄主树种油松、樟子松和潜在寄主落叶松进行内生真菌多样性研究,并用两点对峙法进行拮抗菌株筛选。【结果】松树内生真菌鉴定为2门6纲10目18科19属,其中落叶松韧皮部分离到的内生真菌数量最多,为39株,隶属于9属12种,真菌检出率为43.33%;油松次之,为30株,隶属于7属8种,真菌检出率为33.33%;樟子松最少,为29株,隶属于10属13种,真菌检出率为32.22%。3个树种的内生真菌相似性较低,无共有的菌种,青霉属(Penicillium)和篮状菌属(Talaromyces)是唯二的共有菌属,且青霉属在3个树种中均为优势菌属。平板对峙结果表明90%以上的树木内生真菌均能够与伴生真菌形成稳定的对峙,抑制率在50%-86%之间,且Phialocephala sp.和Pochonia bulbillosa对伴生菌的抑制率能高达93.7%。【结论】松树韧皮部的内生真菌具有较高的生防潜力,Phialocephala sp.和P. bulbillosa对红脂大小蠹伴生菌有较好的抑制效果,可作为红脂大小蠹的生防资源。     

The effects of toxic metals,content of nutrients and inoculation with mycorrhizal fungi on the level of phenolics in roots and growth of Scots pine seedlings

The effects of two substrates and several strains of mycorrhizal fungi on the content of soluble phenolics in roots and growth of Scots pine seedlings was investigated. The first substrate was fertile and contaminated with copper, zinc and lead, whereas the second one displayed nutrient deficiency. The dry weights of needles, trunks, roots and the total biomass were higher in groups of seedlings inoculated with mycorrhizal fungi on fertile and polluted substrate. Inoculation of pine seedlings on this substrate resulted in a decrease in concentration of phenolics in roots and except for seedlings inoculated with Laccaria laccata negatively influenced the above-ground part: root ratio.     

Enzymes of ammonium metabolism in ectendomycorrhizal and ectomycorrhizal symbionts of pine

Ammonium assimilation enzymes from several strains of ectendo- and ectomycorrhizal fungi were assayed after three weeks culture on a buffered synthetic medium containing ammonium as sole nitrogen source. Activity of NADP-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) of ectomycorrhizal strains was very low despite excellent mycelial growth. Only ectendomycorrhizal fungus MrgX isolated from roots of Pinus sylvestris showed high GDH activity. Similar results were obtained when the enzyme extracts were subjected to starch gel electrophoresis. Growth of the fungi, except ectendomycorrhizal MrgX, was arrested when inhibitors of glutamine synthetase (GS, EC 6.3.1.2) or glutamate synthase (GOGAT. EC 1.4.7.1) (methionine sulphoximine or albizine, respectively) were included in the culture medium. Glutamine synthetase activity was found in all fungi tested. The results suggest that the GS pathway for ammonium assimilation is potentially operative in ectomycorrhizal fungi and imply only a minor role for GDH in ammonium assimilation by the studied ectomycorrhizal symbionts of pine. Some physiological and ecological implications of these results are discussed.     

Tolerance and biodegradation of m-toluate by Scots pine, a mycorrhizal fungus and fluorescent pseudomonads individually and under associative conditions

The tolerance to, and degradation of m-toluate by Scots pine (Pinus sylvestris), a symbiotic mycorrhizal fungus (Suillus bovinus) and Pseudomonas fluorescens strains, with or without m-toluate-degrading capacity, was determined individually and in all symbiotic/associative plant-microbe combinations. Fungal survival on medium with m-toluate was increased in co-culture with the degradative bacterial strains on agar plates (up to 0.02%, w/v). When fungi were grown in mycorrhizal association with Scots pine seedlings in test-tube microcosms containing expanded clay pellets and growth media, the fungus was able to withstand m-toluate concentrations up to 2.0%, w/v in all treatments. The seedling tolerance remained unaltered regardless of the presence or absence of mycorrhizal fungi or biodegradative bacteria. Reduction in m-toluate levels was only detected in treatments inoculated with bacterial strains harbouring TOL catabolic plasmids. The plant and fungus, alone or in mycorrhizal symbiosis, were unable to cleave m-toluate. The presence of easily available plant-derived carbon sources did not impede m-toluate degradation by the bacteria in the mycorrhizosphere.     

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。     

Root-associated bacteria contribute to mineral weathering and to mineral nutrition in trees: a budgeting analysis

The principal nutrient source for forest trees derives from the weathering of soil minerals which results from water circulation and from plant and microbial activity. The main objectives of this work were to quantify the respective effects of plant- and root-associated bacteria on mineral weathering and their consequences on tree seedling growth and nutrition. That is why we carried out two column experiments with a quartz-biotite substrate. The columns were planted with or without pine seedlings and inoculated or not with three ectomycorrhizosphere bacterial strains to quantify biotite weathering and pine growth and to determine how bacteria improve pine growth. We showed that the pine roots significantly increased biotite weathering by a factor of 1.3 for magnesium and 1.7 for potassium. We also demonstrated that the inoculation of Burkholderia glathei PML1(12) significantly increased biotite weathering by a factor of 1.4 for magnesium and 1.5 for potassium in comparison with the pine alone. In addition, we observed a significant positive effect of B. glathei PMB1(7) and PML1(12) on pine growth and on root morphology (number of lateral roots and root hairs). We demonstrated that PML1(12) improved pine growth when the seedlings were supplied with a nutrient solution which did not contain the nutrients present in the biotite. No improvement of pine growth was observed when the seedlings were supplied with all the nutrients necessary for pine growth. We therefore propose that the growth-promoting effect of B. glathei PML1(12) mainly resulted from the improved plant nutrition via increased mineral weathering.     

Root-Associated Bacteria Contribute to Mineral Weathering and to Mineral Nutrition in Trees: a Budgeting Analysis

The principal nutrient source for forest trees derives from the weathering of soil minerals which results from water circulation and from plant and microbial activity. The main objectives of this work were to quantify the respective effects of plant- and root-associated bacteria on mineral weathering and their consequences on tree seedling growth and nutrition. That is why we carried out two column experiments with a quartz-biotite substrate. The columns were planted with or without pine seedlings and inoculated or not with three ectomycorrhizosphere bacterial strains to quantify biotite weathering and pine growth and to determine how bacteria improve pine growth. We showed that the pine roots significantly increased biotite weathering by a factor of 1.3 for magnesium and 1.7 for potassium. We also demonstrated that the inoculation of Burkholderia glathei PML1(12) significantly increased biotite weathering by a factor of 1.4 for magnesium and 1.5 for potassium in comparison with the pine alone. In addition, we observed a significant positive effect of B. glathei PMB1(7) and PML1(12) on pine growth and on root morphology (number of lateral roots and root hairs). We demonstrated that PML1(12) improved pine growth when the seedlings were supplied with a nutrient solution which did not contain the nutrients present in the biotite. No improvement of pine growth was observed when the seedlings were supplied with all the nutrients necessary for pine growth. We therefore propose that the growth-promoting effect of B. glathei PML1(12) mainly resulted from the improved plant nutrition via increased mineral weathering.     

Rapid identification and detection of pine pathogenic fungi associated with mountain pine beetles by padlock probes

Fifteen million hectares of pine forests in western Canada have been attacked by the mountain pine beetle (Dendroctonus ponderosae; MPB), leading to devastating economic losses. Grosmannia clavigera and Leptographium longiclavatum, are two fungi intimately associated with the beetles, and are crucial components of the epidemic. To detect and discriminate these two closely related pathogens, we utilized a method based on ligase-mediated nucleotide discrimination with padlock probe technology, and signal amplification by hyperbranched rolling circle amplification (HRCA). Two padlock probes were designed to target species-specific single nucleotide polymorphisms (SNPs) located at the inter-generic spacer 2 region and large subunit of the rRNA respectively, which allows discrimination between the two species. Thirty-four strains of G. clavigera and twenty-five strains of L. longiclavatum representing a broad geographic origin were tested with this assay. The HRCA results were largely in agreement with the conventional identification based on morphology or DNA-based methods. Both probes can also efficiently distinguish the two MPB-associated fungi from other fungi in the MPB, as well as other related fungi in the order Ophiostomatales. We also tested this diagnostic method for the direct detection of these fungi from the DNA of MPB. A nested PCR approach was used to enrich amplicons for signal detection. The results confirmed the presence of these two fungi in MPB. Thus, the padlock probe assay coupled with HRCA is a rapid, sensitive and reproducible method for the identification and detection of these ophiostomatoid fungi.