6种外生菌根真菌对西南桦幼苗的接种效应

【背景】西南桦是兼具内生、外生菌根的典型菌根营养型树种,菌根化育苗是其壮苗培育的有效措施。【目的】揭示外生菌根真菌对西南桦无性系幼苗生长和养分含量的影响,为其菌根化育苗筛选优良外生菌根真菌提供科学依据。【方法】以BY1、FB4、FB4+和A5等4个西南桦优良无性系为研究对象,选用土生空团菌(Cenococcumgeophilum)、松乳菇(Lactariusdeliciosus)、黄硬皮马勃(Scleroderma flavidum)、多根硬皮马勃(S. polyrhizum)、褐环乳牛肝菌(Suillus luteus)和红绒盖牛肝菌(Xerocomuschrysenteron)6个外生菌根真菌进行盆栽接种试验,分析接种处理间及无性系间苗高、地径、生物量以及养分含量差异。【结果】6个菌种均能与西南桦无性系幼苗形成外生菌根共生体,接种多根硬皮马勃与黄硬皮马勃显著促进了幼苗生长和养分吸收(P0.05),说明其与幼苗的亲和力明显优于其它菌种。尽管菌根侵染率在4个无性系之间无显著差异(P≥0.05),但各菌种对FB4、BY1幼苗生长的促进作用显著强于其它2个无性系。【结论】多根硬皮马勃和黄硬皮马勃可作为西南桦菌根化育苗的优选菌种。     

两株溶磷真菌的筛选、鉴定及溶磷效果的评价

【目的】从作物根围土壤中筛选高效溶磷菌株。【方法】结合溶磷圈筛选法和钼锑抗比色法评价菌株的溶磷能力;利用菌株的形态学特性、培养性状和微管蛋白β-tubulin基因序列分析方法进行菌株的鉴定;采用气相色谱-质谱法(GC-MS)对溶磷菌的产酸物质进行分析;并用平板亲和性实验测定菌株间的兼容性。【结果】筛选得到2株高效溶磷菌株P1-1、P2-2;经鉴定,菌株P1-1为黑曲霉(Aspergillus niger),P2-2为塔宾曲霉(A.tubingensis)。2株溶磷菌株的产酸物质相同,均为草酸、葡萄糖酸、乳酸和甘油酸。这2株溶磷菌与杀线虫功能菌株淡紫拟青霉(Purpureocillium lilacinum)、橄榄色链霉菌(Streptomyces olivaceus)和苍白杆菌(Ochrobactrum pseudogrignonense)兼容性好。2菌株分别在Ca_3(PO_4)_2、Zn_3(PO_4)_2、羟基磷灰石为磷源的无机磷固体培养基中25°C培养5 d,测定溶磷圈的直径(D)与菌落直径(d),通过计算其比值D/d的大小对比,以及在Ca_3(PO_4)_2、Zn_3(PO_4)_2、羟基磷灰石为磷源的液体培养基中培养5 d,测定发酵液中有效磷含量进行比较后判定,这2株溶磷菌溶解磷的能力强且效果相当。【结论】获得了2株高效的溶磷真菌。它们能活化多种难溶性磷源,同时伴随挥发性酸性物质的产生;2个菌株与1组杀根结线虫微生物菌群兼容性均良好。     

磷与信号抑制剂对外生菌根真菌分泌草酸的调控作用

摘要:【目的】磷是树木生长的必需营养元素之一,磷素营养丰缺条件下,研究外生菌根真菌的草酸分泌及调控有益于揭示它们活化利用土壤无机磷的机理。【方法】试验设置低、正常、高3种不同磷浓度,液体培养外生菌根真菌,研究了磷和Ca2+信号/阴离子通道抑制剂对草酸分泌的调控作用。【结果】外生菌根真菌能分泌大量的氢离子和草酸、乙酸、苹果酸、柠檬酸和丁二酸等多种有机酸,对溶解难溶性无机磷有重要作用。在外生菌根真菌分泌的有机酸中,草酸占15.14%－36.01%;低磷促进草酸分泌,正常和高磷则产生抑制作用;培养液磷浓度和菌丝含磷量分别与供试菌种的草酸分泌速率呈显著或极显著负相关,相关系数依次为r=－0.264*和r=－0.349＊＊,n=60,*表示显著(P0.05),＊＊表示极显著(P 0.01),说明磷能调控外生菌根真菌分泌草酸。在低磷胁迫下,钙调蛋白抑制剂、Ca2+通道抑制剂、Ca2+内膜通道抑制剂和阴离子通道抑制剂显著抑制外生菌根真菌分泌草酸。但是,在正常和高磷条件下,草酸分泌速率低,未响应Ca2+信号/阴离子通道抑制剂。【结论】供试外生菌根真菌能分泌大量的氢离子和有机酸(尤其是草酸),有益于溶解土壤无机磷,改善寄主植物的磷营养;供磷水平调控草酸分泌速率;在低磷胁迫下,Ca2+信号是介导外生菌根真菌分泌草酸的信号因子。     

溶磷菌对4种难溶性磷酸盐溶解能力的初步研究

以4种难溶性磷酸盐为培养基,发现供试菌株溶解这些磷酸盐的特性差异很大,真菌溶磷能力普遍比细菌要高得多。以NO3-为氮源时的溶磷量通常高于以NH4+为氮源时的溶磷量,只有2TCiF2对氟磷灰石及4TCiF6对磷酸铝的溶解能力以NH4+为氮源时较高。大多数菌株较易溶解CaP(氟磷灰石和磷矿粉),其次为AlP(AlPO4),而溶解FeP(FePO4·4H2O的能力都比较弱,只有曲霉2TCiF2具有较强的溶解FeP能力,尤其是当供给NO3-时,溶解FeP的活性比供给NH4+时大幅度提高。欧文氏菌4TCRi22和肠杆菌1TCRi15能大量地溶解氟磷灰石,而两株节杆菌对磷矿粉的溶解能力最强。供试菌株的溶磷作用可能是由于分泌的有机酸与金属离子络合或螯合作用所致,欧文氏菌和肠杆菌溶解难溶性磷过程中,非有机酸物质可能在起主要作用。     

外生菌根真菌对土壤钾的活化作用

【目的】外生菌根真菌是森林生态系统中的重要组成成分,与木本植物的根系形成菌根,参与树木养分吸收。【方法】试验在液培条件下,以土壤为钾源,利用我国西南地区分离的牛肝菌(Boletnus sp.,Bo 07)、松乳菇(Lactarius delicious,Ld 03)、彩色豆马勃(Pisolithus tinctorius,Pt 715)和内蒙古大青山分离的土生空团菌(Cenococcum geophilum,Cg 04)为材料,研究了它们的生长、钾吸收、氢离子和有机酸分泌,以及土壤钾的变化。【结果】结果表明,Bo 07、Ld 03和Pt 715的生物量、含钾量和吸收量显著高于Cg 04,说明在长期缺钾的环境中,外生菌根真菌经过"物竞天择"可能进化出适应低钾和较强的吸钾能力。以土壤为钾源培养菌根真菌,培养液中的钾浓度显著提高,故外生菌根真菌可促进土壤钾的溶解。在外生菌根真菌的培养液中,分别检测到苹果酸、丁二酸和柠檬酸,均检测到草酸和乙酸。Bo 07、Ld 03和Pt 715显著提高土壤交换性钾含量,Bo 07和Ld 03还显著降低矿物结构钾含量,说明外生菌根真菌不同程度地活化了土壤无效钾,分离自南方的菌株Bo 07、Ld 03和Pt 715总体上大于分离自北方的菌株Cg 04。此外,土壤矿物结构钾分别与总有机酸和草酸分泌量呈极显著负相关(r有机酸=-0.989**,r草酸=-0.950*,n=5),与培养液pH值呈显著正相关(r=0.916*,n=5),故外生菌根真菌分泌的氢离子和有机酸尤其是草酸可能活化土壤无效钾。【结论】供试外生菌根真菌能不同程度的活化土壤无效钾,其活化能力可能与分泌氢离子和有机酸尤其是草酸密切相关。     

秦岭辛家山林区落叶松外生菌根真菌多样性

【目的】以秦岭辛家山林区落叶松为研究对象,观察鉴定与其共生的外生菌根真菌。【方法】通过野外调查结合形态学和分子生物学鉴定方法。【结果】鉴定出31种外生菌根真菌,分属2门4目11科11属,分别有毛革菌属(Tomentella)、丝盖伞属(Inocybe)、红菇属(Russula)、Amphinema、蜡蘑属(Laccaria)、蜡壳耳属(Sebacina)、鹅膏菌属(Amanita)、牛肝菌属(Boletus)、丝膜菌属(Cortinarius)、乳菇属(Lactarius)和硬皮马勃属(Scleroderma),丝盖伞属是优势类群。阳坡外生菌根真菌多样性高于阴坡。对菌根根际土与非根际土化学性质分析表明,pH值显著低于非根际,速效磷、速效钾含量显著高于非根际。对根际土化学性质与外生菌根侵染率相关性分析表明,落叶松外生菌根侵染率与土壤pH值呈显著正相关;与速效钾呈极显著正相关;与全氮、速效磷呈显著负相关。【结论】落叶松外生菌根真菌多样性丰富,且受坡向影响。外生菌根真菌可降低根际pH,提高根际有机质、全氮、速效磷、速效钾、水溶性钙和水溶性镁的含量。外生菌根侵染率受土壤pH值、全氮、速效磷、速效钾的影响。     

盐胁迫下外生菌根真菌与根际有益细菌互作对杨树光合特性的影响

以美洲黑杨幼苗为对象,利用盆栽试验,在100 mmol/L NaCl胁迫条件下研究单接种和双接种外生菌根真菌红绒盖牛肝菌(Xerocamus chrysenteron,简称Xc)、溶磷细菌恶臭假单胞菌(Pseudomonas putida,简称JW-SX1)和菌根辅助细菌蜡状芽孢杆菌(Bacillus cereus,简称HB59)对杨树幼苗的气体交换参数、叶绿素荧光和光响应曲线参数的影响。结果表明:盐胁迫下,接种Xc、JW-SX1及HB59的杨树幼苗净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)显著增强,且均显著高于对照,而单接JW-SX1和双接Xc+JW-SX1处理的胞间CO2浓度(Ci)低于对照,但与对照相比差异不显著;各接种处理显著增强了杨树的最大光化学效率(Fv/Fm),但处理之间差异不显著性;不同接种处理杨树的实际光化学效率(ΦPSⅡ)与对照(CK)差异不明显,其中接种HB59和Xc+JW-SX1处理分别高于对照14.36%和19.33%;同时接种处理还明显提高了宿主的最大光合速率(Pmax)、光饱和点(LSP)、表观有效利用效率(YAQ),降低了光补偿点(LCP)和暗呼吸速率(RD),且双接种菌根真菌Xc和JW-SX1处理效果更为明显。研究发现,外生菌根真菌、溶磷细菌和菌根辅助细菌可通过改善叶片叶绿素荧光和光合参数、光响应参数来减轻盐胁迫对宿主造成的伤害,从而提高杨树的耐盐性能力。     

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。     

早期真菌与本土和外来松的菌根合成

为探讨早期真菌与本土和外来松树的共生特性,选用3种早期外生菌根真菌(环褐乳牛肝菌Suillus luteus、虎皮乳牛肝菌S. phylopictus和酒红蜡蘑Laccaria vinaceoavellanea)接种2种本地松(云南松Pinus yunnanensis、华山松P. armandii)和2种外来松(P. greggii、P. maximartinensis),并对接种后的侵染率、菌根特征和松苗株高等进行测量和分析,结果显示：6个月后,2种乳牛肝菌与4种松均能形成菌根,华山松与2种乳牛肝菌的亲和性最好,酒红蜡蘑仅能与2种外来松P. greggii和P. maximartinensis形成菌根,且菌根合成成功率仅为14.3%。此次合成的10种菌根组合均为首次报道,其中同一种真菌与不同松形成的菌根在形态和解剖特征上较为接近。3种真菌对宿主生长的促进作用因树种而异,整体上外来松苗的生长速率要快于本土松苗;华山松苗虽然菌根感染率最高,但生长效应却均不明显。研究认为：孢子接种对乳牛肝菌、蜡蘑等早期真菌的菌根合成研究是一种经济有效的手段;外生菌根真菌可能对外来树种具有更为重要的作用,与本土树种相比,外来树种与外生菌根真菌在能否形成菌根、菌根形成时间以及对宿主的生长效应等方面存在差异,需要开展进一步深入的相关研究。     

外生菌根真菌对杉木的接种效应

对商南县2年生杉木苗木进行乳牛肝菌(Suillus bovines)、褐环乳牛肝菌(Suillus luteus)和褐黄牛肝菌(Boletus luridus)3种外生菌根真菌的接种试验.结果显示,外生菌根真菌对杉木苗木成活率、苗高和地径生长均有明显的促进作用,能明显提高苗木的菌根化水平,增加苗木体内叶绿素含量,促进其游离脯氨酸含量的累积,降低丙二醛含量.对不同坡向杉木苗木各项生长指标的分析显示,在杉木人工造林中,阳坡的综合效应明显优于阴坡.乳牛肝菌和褐黄牛肝菌可作为一种生物肥料,在杉木人工造林中应用.     

过量钠盐胁迫对3种外生菌根真菌生长和钠元素积累的影响*

通过测定3种外生菌根真菌-美味牛肝菌(Boletus edulis)、红绒盖牛肝(Xerocomus chtysenterort)和铆钉菇(Gomphidius viscidus)在Na2SO4和NaCl处理下的菌斑增长特性、生物量积累和菌丝中钠元素积累量显示,2种盐都没有影响菌丝的生长模式。但在0．1mol／L浓度下,NaCl显抑制了X．chrysertterort和G．viscidus的干重增长;在Na2SO4处理下,X．chrysenteron生物增长量比对照显增加,G．viscidus则降低,而2种盐处理对B．edtdis的生物量积累没有显影响。3供试菌种在2种盐处理下,其对钠元素的吸收和积累量差异显。NaCl处理下,X．chtysenteron菌丝积累的钠量最多,而在Na2SO4条件下,B．edulis菌丝中钠积累量显高于其他2菌种。     

微生物溶解磷矿粉能力与pH及分泌有机酸的关系

从玉米根际和非根际土壤中分离得到的 74株溶解磷矿粉的微生物 ,发现它们的溶磷能力差异很大 ,主要决定于菌株本身的特性 ,与其来源无关 ,真菌普遍比细菌要强。真菌的溶磷能力与其培养介质的 pH之间呈显著的负相关 ,但细菌的这种关系非常弱。二者都产生多种有机酸 ,真菌主要分泌草酸、丙二酸和乳酸 ,而细菌主要分泌草酸、酒石酸、丙二酸、乳酸和乙酸 ,不同菌株分泌有机酸的数量和种类差异很大 ,但溶磷量与有机酸总量或单个有机酸浓度之间 ,没有发现显著的相关性 ,唯独柠檬酸与真菌的溶磷量之间存在显著的相关性。说明不同菌株有完全不同的溶磷机理 ,可能多种机理并存。     

The effect of drought on mycorrhizas of beech (Fagus sylvatica L.): changes in community structure,and the content of carbohydrates and nitrogen storage bodies of the fungi

In a water-exclusion experiment, five different ecotypes of beech (Fagus sylvatica L.; representing regions of different environmental and climatic conditions in Baden-Württemberg, Germany) were subjected to drought conditions of different severity between July and September of two consecutive years. Drought stress as characterised by the water content and the pre-dawn water potential of the leaves was related to the degree of mycorrhization, the type of ectomycorrhiza, and the physiological properties of individual fungus/plant interactions at the fine roots of different beech ecotypes. Our data show that decreased soil water availability did not significantly change either the degree of fungal colonisation of beech roots (measured by the amount of ergosterol) or the number of ectomycorrhizal types per root system. Drought did, however, have an influence on the composition of the ectomycorrhizal community, and different mycorrhizal types responded to drought differently in terms of their patterns of occurrence/abundance. While the abundance of the dominant mycorrhizal types, formed with Byssocorticium atrovirens and Lactarius subdulcis, was not affected, drought increased the abundance of mycorrhiza formed between beech and Xerocomus chrysenteron. A detailed analysis of plant and fungal carbohydrates in mycorrhizas indicated that different drought intensities led to distinguishable responses. In plants exhibiting a pre-dawn water potential of down to -1.96 MPa, drought caused the accumulation of sucrose, glucose and fructose, and of fungus-specific compounds such as mannitol and arabitol in mycorrhizal roots at the expense of, e.g. trehalose. The accumulation of sugar alcohols, which constitute compatible solutes known to counteract drought stress, was species-specific. Mycorrhizas with X. chrysenteron formed large amounts of arabitol, while those with L. subdulcis accumulated mannitol. Sustained partitioning of carbon towards the mycorrhizal fungi under drought was also reflected by an increase of nitrogen storage in the fungal vacuoles. In treatments where the pre-dawn water potential reached values of as low as -2.4 MPa, such alterations were no longer found. In such plants, the starch and soluble sugars content was generally reduced, which also resulted in a lack of increase in protective, fungus-specific sugar alcohols. In summary, the data show that, within certain limits, an increase in drought causes a shift in plant/fungus communities. The shift in the pattern of fungus-specific compounds could possibly be used as a sensitive measure of physiological stress imposed on this symbiosis.     

Factors determining rock phosphate solubilization by microorganisms isolated from soil

Forty two soil isolates (31 bacteria and 11 fungi) were studied for their ability to solubilize rock phosphate and calcium phosphate in culture medium. Eight bacteria and 8 fungi possessed solubilizing ability. Pseudomonas cepacia and Penicillium purpurogenum showed the highest activity. There was a correlation between final pH value and titratable acidity (r=–0.29 to –0.87) and between titratable acidity and soluble phosphate (r=0.22 to 0.99). Correlation values were functions of insoluble phosphate and of the group of microorganisms considered. A high correlation was observed between final pH and soluble phosphate only for the rock phosphates inoculated with the highest concentration of solubilizing bacteria (r=–0.73 to –0.98).     

A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.     

杨树根际解无机磷细菌Mp1-Ha4的鉴定及其解磷机理

解无机磷细菌能够溶解土壤中的难溶性磷酸盐,增加土壤有效磷含量,促进植物生长。以一株杨树根际土壤中筛选得到的解无机磷细菌Mp1-Ha4为研究对象,利用分子生物学的方法对该菌株进行鉴定,测定了其对磷酸钙、磷酸铝和磷酸铁的解磷能力,并对该菌株9 d内的磷酸钙溶解动力学进行了研究。结果表明,解无机磷细菌Mp1-Ha4为一株西地西菌Cedecea sp.,其对磷酸钙的溶解能力远强于对磷酸铝和磷酸铁的溶解能力。在NBRIP液体培养基中,该菌株对磷酸钙的溶解能力达到了497.4 mg/L,在其对磷酸钙解磷过程中,培养基pH及可滴定酸度与解磷量分别呈显著负、正相关。高效液相色谱分析显示,该菌株在解磷过程中分泌了大量有机酸,主要包括α-酮戊二酸,酒石酸和苹果酸。分泌有机酸,降低环境pH可能是解无机磷细菌西地西菌(Cedecea sp.)Mp1-Ha4溶解难溶性磷酸盐的主要机制,同时该菌株对磷酸钙的高效溶解作用使其具有较大的研究和应用前景。     

Bio-production of compost with low pH and high soluble phosphorus from sugar cane bagasse enriched with rock phosphate

Summary Aspergillus niger and Trichoderma viride strains were used together as a fungal activator in the presence or absence of farmyard manure (FM) for composting of bagasse enriched with rock phosphate. Quality of the composts produced was compared with that obtained from non-inoculated bagasse. The composts were evaluated as organic phosphatic fertilizers, for broad bean plants. The results showed that composting of bagasse without microbial inoculation or FM addition was not complete after 105 days of fermentation. An excellent decomposition in a relatively short time however was obtained with the use of A. niger and T. viride as inoculant agents with or without FM. The inoculation with A. niger + T. viridewith or without FM, also represented the most suitable conditions for phosphate solubilization. Acidic conditions (pH 4–5) at the end of the experiment were obtained in all piles receiving Aspergillus niger and there was a correlation between the amounts of soluble phosphorus and the reduction in pH values in the compost piles. There were no phosphate-dissolving fungi present in any composted piles except those treated with Aspergillus niger and Trichoderma viride. The number of phosphate-dissolving bacteria increased only in the treatments that were treated with FM. The non-fertilized sandy soil and the non-inoculated bagasse compost did not provide broad bean plants with phosphorus while the composts produced by inoculation with A. niger + T. viride provided the plants with the highest amounts of phosphorus.     

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。