丛枝菌根真菌产球囊霉素研究进展

球囊霉素（Glomalin）是由丛枝菌根真菌（arbuscular mycorrhizal fungi,AMF）产生的一种含金属离子的糖蛋白,由于丛枝菌根真菌在自然和人工陆生生态系统中广泛分布,丛枝菌根真菌在生态系统中的生态学功能一直是菌根生物学研究中诱人的问题。自1996年球囊霉素被发现以来,球囊霉素在土壤生态系统中的生态学功能、生态学地位日益受到重视。本文对球囊霉素作为土壤主要有机源和超级胶的功能作了简介,综述了球囊霉素的研究现状,并对其研究前景作了展望。     

Seasonality of arbuscular mycorrhizal hyphae and glomalin in a western Montana grassland

In order to more fully understand the basic biology of arbuscular mycorrhizal fungi (AMF), and their role in natural ecosystems, it is necessary to document seasonal changes of various aspects of the life history of these fungi. Due to their unique position at the root-soil interface, AMF have been described as `keystone mutualists' in ecosystems. Despite the importance of AMF in ecosystems, few studies exist that examine the seasonality of external hyphae and their exuded products (e.g. glomalin), the AMF variables directly related to ecosystem function through their contributions to soil aggregation. This study examined seasonal dynamics of several soil variables, with a specific interest in the seasonality of external hyphae and glomalin, a glycoprotein produced by AMF, which is correlated with soil aggregate stability. Here we measured glomalin concentrations and external AMF and non-AMF hyphal length, as well as soil moisture, percent fungal root colonization (AMF and non-AMF), and root length in soil in an intermountain grassland in western Montana over one growing season (13 time points). Of the glomalin pools and hyphal lengths measured, significant seasonal changes occurred for total glomalin (TG; 24.5% change), immunoreactive easily extractable glomalin (IREEG; 53.8% change), and AM hyphal length (107% change). Prior studies on glomalin in natural systems have not considered seasonal effects. The small seasonal change in glomalin pools lends further support to the hypothesis that glomalin is relatively stable in soils, and suggests that one-time sampling may be sufficient to satisfactorily capture this response variable. However, the generality of this observation has yet to be tested in a wider range of ecosystems.     

Quantification of arbuscular mycorrhizal fungi activity by the glomalin concentration on hyphal traps

 Strips of horticultural film (16–32 cm²) were used to trap extraradical hyphae emanating from roots of sudangrass [Sorghum sudanense (Piper) Staph] enclosed in 40-μm mesh bags and colonized by Gigaspora rosea FL 224-1, Glomus intraradices EY 113/114, or Glomus caledonium UK 301-1. Strips of film were placed at opposite sides of 17–21 replicate sand culture pots for each isolate and were removed after 12–14 weeks of plant growth. To extract glomalin, a strip was cut into small pieces and submerged in 2 ml of 20 mM citrate, pH 7.0 and then autoclaved for 60 min. A quantitative enzyme-linked immunosorbent assay (ELISA) detected 0.005–0.04 μg glomalin in the volume of extract tested. The Bradford protein assay detected 1.25–5 μg of protein in the volume of extract tested. Both assays gave results ranging from 5–40 μg glomalin/cm² of film. Protein assay values were correlated with ELISA values (r=0.6091, P≤0.001, n=118). Analysis of variance indicated that isolates differed in Bradford protein values (P=0.001), but not ELISA values (P=0.154). Spatial variability of glomalin deposition ca. 7 cm from roots on opposite sides of pots was indicated by significant paired T tests (P<0.05) for protein values for each of the three isolates and ELISA for two isolates. These results indicate that hyphal traps, Bradford protein assay and ELISA are useful to assess hyphal activity over a growing season. Accepted: 11 October 1998     

The arbuscular mycorrhizal fungal protein glomalin is a putative homolog of heat shock protein 60

Work on glomalin-related soil protein produced by arbuscular mycorrhizal (AM) fungi (AMF) has been limited because of the unknown identity of the protein. A protein band cross-reactive with the glomalin-specific antibody MAb32B11 from the AM fungus Glomus intraradices was partially sequenced using tandem liquid chromatography-mass spectrometry. A 17 amino acid sequence showing similarity to heat shock protein 60 (hsp 60) was obtained. Based on degenerate PCR, a full-length cDNA of 1773 bp length encoding the hsp 60 gene was isolated from a G. intraradices cDNA library. The ORF was predicted to encode a protein of 590 amino acids. The protein sequence had three N-terminal glycosylation sites and a string of GGM motifs at the C-terminal end. The GiHsp 60 ORF had three introns of 67, 76 and 131 bp length. The GiHsp 60 was expressed using an in vitro translation system, and the protein was purified using the 6xHis-tag system. A dot-blot assay on the purified protein showed that it was highly cross-reactive with the glomalin-specific antibody MAb32B11. The present work provides the first evidence for the identity of the glomalin protein in the model AMF G. intraradices, thus facilitating further characterization of this protein, which is of great interest in soil ecology.     

The role of arbuscular mycorrhizal fungi and glomalin in soil aggregation: comparing effects of five plant species

Soil aggregation and soil structure are fundamental properties of natural and managed ecosystems. However, most of our knowledge on the role of plant species in soil aggregation is derived from work in agroecosystems or with agriculturally important plants. Here we examined the effects of five plant species on soil aggregate water stability. The five species (three grasses, one forb, and a legume) were from the same natural grassland, and were grown in monoculture plots in the field. Our first goal was to test if productivity-related or species-specific factors would prevail in determining soil aggregation. We also tested what the relative importance of the soil protein glomalin (produced by arbuscular mycorrhizal fungi, AMF) in soil aggregation is, compared to other factors, including AMF hyphal and root length and percent plant cover. We found significant differences in soil aggregate water stability (1–2 mm size class) for the five plant species examined, and corresponding differences in plant cover, root weight and length, AMF soil hyphal length, and glomalin concentrations. A structural equation modeling approach (path analysis) was used to distinguish direct from indirect effects of factors on soil aggregation based on covariance structures. Root length, soil glomalin, and percent cover contributed equally strong paths to water-stable aggregation. The direct effect of glomalin was much stronger than the direct effect of AMF hyphae themselves, suggesting that this protein is involved in a very important hypha-mediated mechanism of soil aggregate stabilization, at least for the 1–2-mm size class of aggregates.     

库布齐沙漠白沙蒿根际丛枝菌根及其球囊霉素的时空异质性

为探明流动沙地先峰植物白沙蒿(Artemisia sphaerocephala)根际丛枝菌根真菌产球囊霉素时空分布, 在库布齐沙漠选设白沙蒿样地, 于春、夏和秋季分0-10, 10-20, 20-30, 30-40和40-50 cm土层采集土壤样品, 测定其根际丛枝菌根真菌的菌丝侵染率、孢子密度、球囊霉素含量和土壤理化因子, 并系统分析了两两间的相互关系。结果表明: ①白沙蒿和丛枝菌根真菌具有良好的共生关系, 夏季和秋季的真菌菌丝侵染率略高于春季, 夏季平均为89.75%, 秋季平均达到92.37%, 两季的最大值都出现在20-30 cm土层。②白沙蒿根际丛枝菌根真菌活性有明显的空间异质性。真菌孢子密度为1.21-12.31 个·g土-1, 最大值出现在夏季的0-10 cm土层。孢子密度在不同季节有显著差异, 夏季>秋季>春季, 各季都随土层加深而递减。③白沙蒿根际土壤中总球囊霉素含量范围为0.37-1.27 mg·g-1, 易提取球囊霉素含量范围为0.19-0.81 mg·g-1, 两者在各季节最大值都出现在0-10cm土层, 呈现明显的表层土富集性。④球囊霉素与土壤中真菌孢子密度呈极显著正相关, 并和土壤养分及大多土壤酶活性呈显著正相关, 可作为评价土壤丛枝菌根真菌活性和土壤肥力的重要指标。     

不同丛枝菌根真菌侵染对土壤结构的影响

为了定量化比较研究接种丛枝菌根真菌后,根际、菌根际和菌丝际土壤结构的变化,采用四室分根装置,比较中性紫色土接种不同AM真菌后,菌根际、根际、菌丝际和非根际土壤平均重量直径(MWD)、几何平均直径(GMD)和大于0.25mm团聚体总量(R_0.25)的变化。结果表明:接种3个菌种后菌丝际EEG和有机质含量均呈高于菌根际的趋势。菌丝密度和易提取球囊霉素相关蛋白(EEG)与MWD、GMD和R_0.25呈显著正相关,菌根际和菌丝际土壤水稳性R_0.25与菌丝密度显著正相关,相关系数分别为0.777和0.671。接种G. mosseae的菌根际土壤R_0.25值显著高于其它分室土壤,而接种G.etunicatum的菌丝际土壤R_0.25值显著高于其它分室土壤。试验结果在一定程度上说明不同菌种对土壤结构均有不同程度的影响,反映了丛枝菌根真菌生态功能的多样性。     

Polyphosphate dynamics in mycorrhizal roots during colonization of an arbuscular mycorrhizal fungus

Inorganic polyphosphate (poly P) has been considered to be a translocatable form of phosphate (Pi) in arbuscular mycorrhizal fungi (AMF). Here we examined time-course changes in poly P content during the AMF colonization process. Onion (Allium cepa) plants were cultured with or without inoculation with Gigaspora margarita for 2-8 wk with periodic sampling. Poly P in the extracts, purified through gel filtration, was quantified by the reverse reaction of polyphosphate kinase. The length of poly P in mycorrhizal roots appeared to be shorter than in extraradical hyphae or in spores of the AMF, indicating that AMF depolymerize poly P before providing Pi to the host. The poly P content increased as colonization proceeded, and was highly correlated with the weight of the colonized roots. These results support the model that AMF supply Pi to the host through the poly P pool, and that the poly P content of a mycorrhizal root can be a good indicator of the Pi-supplying activity of AMF.     

Growth response of Atriplex nummularia to inoculation with arbuscular mycorrhizal fungi at different salinity levels

Chenopods are generally regarded as non-host plants for mycorrhizal fungi and are believed not to benefit from colonization by mycorrhizal fungi. Perennial Atriplex nummularia Lindl., growing under field conditions, showed a relatively high level of colonization by mycorrhizal fungi (10–30% of root length colonized) in spring and summer. Accordingly, two glasshouse experiments were designed to assess the effects of inoculation with mycorrhizal fungi (with a single species or a mixture of different species) on growth, nutrient uptake, and rhizosphere bacterial community composition of A. nummularia at high and low salinity levels (2.2 and 12 dSm^–1). Only low and patchy colonization by mycorrhizal fungi (1–2 of root length colonized) was detected in inoculated plants under glasshouse conditions which was unaffected by salinity. Despite the low colonization, inoculation increased plant growth and affected nutrient uptake at both salinity levels. The effects were higher at an early stage of plant development (6weeks) than at a later stage (9–10 weeks). Salinity affected the bacterial community composition in the rhizosphere as examined by ribosomal intergenic spacer amplification (RISA) of 16S rDNA, digitization of the band patterns and multivariate analysis. The effects of inoculation with mycorrhizal fungi on growth of A. nummularia may be attributed to (i) direct effects of mycorrhizal fungi on plant nutrient uptake and/or (ii) indirect effects via mycorrhizal-induced changes in the bacterial community composition.     

有机磷杀虫剂——灭克磷对丛枝菌根真菌Glomus mosseae生长的效应

采用多室隔网培养法,在宿主植物生长状况相同的条件下研究了土壤中不同浓度灭克磷对AM真菌生长的直接效应。与对照相比,0.5、1.5和3.5mg/kg灭克磷使Glomusmosseae菌丝生长量和具有琥珀酸脱氢酶活性的菌丝生长量明显地增加;随着灭克磷剂量的增加,菌丝总量的相对生长速率呈降低趋势,但在数值上仍然高于对照的;与此同时,高剂量(1.5和3.5mg/kg)灭克磷使Glomusmosseae活性菌丝量的相对增长速率明显低于对照,达到几乎停止的水平。上述结果说明,低剂量灭克磷(0.5mg/kg)对AM真菌生长和代谢活性都有一定刺激作用,但是在高剂量灭克磷(1.5和3.5mg/kg)下菌丝生物量的增加和周转速度的加快只是AM真菌对毒物的应激反应。     

接种时期对丛枝菌根喜树幼苗喜树碱含量的影响

喜树(Camptotheca acuminata)是我国特有的多年生亚热带落叶阔叶树种, 因其次生代谢产物喜树碱具有良好的抗肿瘤活性而受到人们的广泛关注。通过温室盆栽接种试验, 观察了喜树幼苗不同生长时期接种蜜色无梗囊霉(Acaulospora mellea)和根内球囊霉(Glomus intraradices)对喜树幼苗喜树碱积累的影响。结果表明接种两种丛枝菌根真菌均促进了喜树幼苗喜树碱的积累, 表现为喜树碱产量(单株幼苗所含的喜树碱量, 喜树碱含量与幼苗生物量的乘积)的显著提高。进一步分析发现, 接种丛枝菌根真菌导致幼喜树苗喜树碱产量的提高, 早期(幼苗出土20天)接种主要是源于喜树碱含量的提高, 特别是叶片喜树碱含量的提高, 而晚期(幼苗出土60天)接种则主要是源于幼苗生物量的增加。     

Anthropogenic disturbance equalizes diversity levels in arbuscular mycorrhizal fungal communities

The arbuscular mycorrhizal (AM) symbiosis is a key plant–microbe interaction in sustainable functioning ecosystems. Increasing anthropogenic disturbance poses a threat to AM fungal communities worldwide, but there is little empirical evidence about its potential negative consequences. In this global study, we sequenced AM fungal DNA in soil samples collected from pairs of natural (undisturbed) and anthropogenic (disturbed) plots in two ecosystem types (10 naturally wooded and six naturally unwooded ecosystems). We found that ecosystem type had stronger directional effects than anthropogenic disturbance on AM fungal alpha and beta diversity. However, disturbance increased alpha and beta diversity at sites where natural diversity was low and decreased diversity at sites where natural diversity was high. Cultured AM fungal taxa were more prevalent in anthropogenic than natural plots, probably due to their efficient colonization strategies and ability to recover from disturbance. We conclude that anthropogenic disturbance does not have a consistent directional effect on AM fungal diversity; rather, disturbance equalizes levels of diversity at large scales and causes changes in community functional structure.     

A survey of soils for aggregate stability and glomalin,a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi

Understanding the contributions of soil microorganisms to soil stabilization at the molecular level will lead to ways to enhance inputs for sustainable agricultural systems. Recent discoveries of copious production of glycoprotein (glomalin) by arbuscular mycorrhizal (AM) fungi and the apparent recalcitrance of this material in soils led to the comparison between concentration of glomalin and aggregate stability. Stability was measured on air-dried aggregates rewetted by capillary action and then subjected to wet sieving for 10 min. Thirty-seven samples from four geographic areas of the U.S. and one area of Scotland were tested. The monoclonal antibody used to discover glomalin on AM hyphae was employed to assess immunoreactive glomalin on aggregate surfaces by immunofluorescence and in extracts from aggregates by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was observed on at least some surfaces of aggregates from all soils examined, but was most evident on aggregates with high glomalin concentrations. Easily extractable glomalin (EEG) was solubilized by 20 mM citrate, pH 7.0 at 121 °C for 30 min, and total glomalin (TG) was solubilized with 50 mM citrate, pH 8.0 at 121 °C for 90 to 450 min. Some soils required up to seven sequential extractions to remove all of the glomalin. Aggregate stability was linearly correlated (p < 0.001) with all measures of glomalin (mg/g of aggregates) in these soils. The best predictor of aggregate stability (AS) was immunoreactive easily extractable glomalin (IREEG) according to the following relationship: AS = 42.7 +61.3 × log₁₀ IREEG (r₂ = 0.86; p <0.001, n = 37).     

丛枝菌根真菌对玉米秸秆降解的影响及其作用机制

为了比较菌根、菌丝、植物根系对玉米秸秆降解的影响,采用4室分根装置即土壤室(S)、根室(R)、菌根室(M)和菌丝室(H),分室间用400目尼龙网和有机板分隔,尼龙网袋包埋玉米秸秆于不同分室内,以玉米为宿主植物,接种丛枝菌根(AM)真菌Glomus mosseae。试验分别在移栽后第20、30、40、50、60天时取样,通过测定接种AM真菌后玉米秸秆的碳、氮释放,土壤中3种常见酶活性、微生物量碳和氮及土壤呼吸的动态变化,探讨AM真菌降解玉米秸秆可能的作用机制。研究结果表明:经60 d的培养后,与未接种S室相比,接种AM真菌的M室和H室玉米秸秆降解量提高了27.72%和8.07%;另外,M室玉米秸秆碳素释放显著增加,而氮素的释放减少,致使碳氮比显著低于其他3室,较初始值降幅达8.72%,有利于秸秆进一步降解。在试验条件下,M室中土壤酸性磷酸酶、蛋白酶、过氧化氢酶活性较其他3室都有显著提高,并增加了微生物量碳、氮和土壤呼吸作用,形成了明显有别于根际的微生物区系。这一系列影响都反映出AM直接或间接作用于玉米秸秆的降解过程,是导致玉米秸秆降解加快的重要原因。     

丛枝菌根真菌在寄主植物抵御生物和非生物胁迫中的作用

丛枝菌根真菌(arbuscular mycorrhiza fungi,AMF)是生态系统中普遍存在的土壤微生物,能与绝大多数植物形成共生关系,它在寄主植物抵御生物和非生物胁迫中所起的作用逐渐引起国内外学者的关注.论文综述了丛枝菌根真菌在植物抵御非生物胁迫(重金属污染、有机污染、盐胁迫和干旱胁迫)以及生物胁迫(致病菌和线虫侵染)中的作用,并在此基础上提出了未来该领域值得进一步研究的方向.     

几种生态环境中AM真菌多样性比较研究

对渤海湾的海岛林地(IFL),黄河三角洲盐碱地(SAS)、鲁西南煤矿(CMS)和内蒙古退化草原(DGL)等几种生态环境中丛枝菌根(Arbuscular mycorrhiza,AM)真菌的多样性进行了调查。结果表明,在所调查的样点中,AM真菌的物种多样性不同,IFL中的最高,其次是SAS,CMS和DGL的最低。各地AM真菌种的丰度、孢子密度、频度、相对多度等也差异较大。这与各生态环境中的生态因子的差异相关。在总体上,Glomus属在各采样点出现的频度和相对多度最高,其次是Acaulospora属。但不同生态环境之问又存在差异,例如CMS中Acaulospora属的频度和相对多度比Glomus属的高。在IFL中,Gigaspora属的相对多度比Acaulospora属的高。各生态环境中的生态优势种不同,如在CMS中是A．mellea,在IFL和SAS土壤中却分别是G．margarita和G．caledonium,而DGL中各个种的分布却较均衡。     

Detection of an arbuscular mycorrhizal fungus in roots of different plant species with the PCR.

PCR was used with the primer pair VANS1-NS21 to detect the arbuscular mycorrhizal fungus Glomus intraradices (commercial inoculum source) on roots of lettuce, zinnia, leek, pepper, and endive plants. The appropriate amplification product was obtained directly from roots without DNA extraction and purification.     

Interactive influence of light intensity and soil fertility on root-associated arbuscular mycorrhizal fungi

Background and aims

Soil nutrients and light have major effects on the economics of arbuscular mycorrhizal (AM) symbioses. This study tests the main and interactive effects of soil fertility and light on AM fungal community.

Methods

We conducted a 3 year mesocosm experiment with a full two factorial design: light (full light or shade) and soil fertility (unfertilized or fertilized), on the Qinghai-Tibetan Plateau. Plant traits, soil characteristics and the AM fungal communities inside roots and in soils were measured.

Results

Shade reduced AM colonization of roots, fertilization reduced the hyphal abundance in the soil, and both factors reduced species richness of AM fungi inside plant roots. Fertilization exacerbated the negative impacts of shade on AM fungal abundance and diversity. We observed 15 phylotypes of AM fungi inside roots and ten morphotypes of AM fungal spores in the soil. Taxa responded differently to shade and fertilization and there was little congruence between the responses of fungi inside the roots and in the spore community.

Conclusions

Our findings indicate that both shade and fertilization reduce the abundance of AM fungi, but the two factors have different effects on the quality of plant roots as habitat for AM fungi.