Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi (AMF) are important symbionts of plants that improve plant nutrient acquisition and promote plant diversity. Although within-species genetic differences among AMF have been shown to differentially affect plant growth, very little is actually known about the degree of genetic diversity in AMF populations. This is largely because of difficulties in isolation and cultivation of the fungi in a clean system allowing reliable genotyping to be performed. A population of the arbuscular mycorrhizal fungus Glomus intraradices growing in an in vitro cultivation system was studied using newly developed simple sequence repeat (SSR), nuclear gene intron and mitochondrial ribosomal gene intron markers. The markers revealed a strong differentiation at the nuclear and mitochondrial level among isolates. Genotypes were nonrandomly distributed among four plots showing genetic subdivisions in the field. Meanwhile, identical genotypes were found in geographically distant locations. AMF genotypes showed significant preferences to different host plant species (Glycine max, Helianthus annuus and Allium porrum) used before the fungal in vitro culture establishment. Host plants in a field could provide a heterogeneous environment favouring certain genotypes. Such preferences may partly explain within-population patterns of genetic diversity.     

Biolistic transformation of arbuscular mycorrhizal fungi

Gene transfer systems have proved effective for the transformation of a range of organisms for both fundamental and applied studies. Biolistic transformation is a powerful method for the gene transfer into various organisms and tissues that have proved recalcitrant to more conventional means. For fungi, the biolistic approach is particularly effective where protoplasts are difficult to obtain and/or the organisms are difficult to culture. This is particularly applicable to arbuscular mycorrhizal (AM) fungi, being as they are obligate symbionts that can only be propagated in association with intact plants or root explants. Furthermore, these fungi are aseptate and protoplasts cannot be released. Recent advancements in gene transformation systems have enabled the use of biolistic technology to introduce foreign DNA linked to molecular markers into these fungi. In this review we discuss the development of transformation strategies for AM fungi by biolistics and highlight the areas of this technology which require further development for the stable transformation of these elusive organisms.     

The mycorrhizal dependence of subordinates determines the effect of arbuscular mycorrhizal fungi on plant diversity

It is widely recognized that arbuscular mycorrhizal fungi (AMF) influence plant community structure. Several studies in the past few years have focused on the effect of AMF on plant diversity, showing contradictory results. On the basis of studies carried out in grasslands, it has been proposed that these results may not be in conflict but rather depend on mycorrhizal dependence of dominant and subordinate species in the plant community. Here, we propose a simple conceptual model to explain the effect of AMF on plant diversity and dominance hierarchy. Our model suggests that the effect of AMF on plant diversity can be explained mainly by mycorrhizal dependence of subordinates. It also gives predictions for a wider range of community types, and helps in identifying the systems in which empirical data are most critically needed.     

Genetic processes in arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi (Glomeromycota) colonize roots of the majority of land plants and facilitate their mineral nutrient uptake. Consequently, AM fungi play an important role in terrestrial ecosystems and are becoming a component of sustainable land management practices. The absence of sexual reproductive structures in modern Glomeromycota combined with their long evolutionary history suggest that these fungi may represent an ancient asexual lineage of great potential interest to evolutionary biology. However, many aspects of basic AM fungal biology, including genome structure, within-individual genetic variation, and reproductive mode are poorly understood. These knowledge gaps hinder research on the mechanisms of AM fungal interactions with individual plants and plant communities, and utilization of AM fungi in agricultural practices. I present here the current state of research on the reproduction in AM fungi and indicate what new findings can be expected in the future.     

Different growth response of five co-existing stoloniferous plant species to inoculation with native arbuscular mycorrhizal fungi

Five species of stoloniferous plants originating from the same field site (Galeobdolon montanum, Glechoma hederacea, Potentilla anserina, Ranunculus repens and Trifolium repens) were studied with respect to their interaction with arbuscular mycorrhizal (AM) fungi. More specifically, the question was addressed whether mycorrhizal growth response of host plant species could be related to their vegetative mobility. The roots of all the species examined were colonised with AM fungi in the field, with the percentage of colonisation varying among species from approximately 40% to 90%. In a subsequent pot experiment, plants of all the species were either left non-inoculated or were inoculated with a mixture of three native AM fungi isolated from the site of plant origin (Glomus mosseae, G. intraradices and G. microaggregatum). AM fungi increased phosphorus uptake in all the plant species; however, plant growth response to inoculation varied widely from negative to positive. In addition to the biomass response, AM inoculation led to a change in clonal growth traits such as stolon number and length or ramet number in some species. Possible causes of the observed differences in mycorrhizal growth response of various stoloniferous plants are discussed.     

丛枝菌根真菌对入侵植物南美蟛蜞菊生长及竞争力的影响

【背景】丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)能促进植物的养分吸收及生长发育。入侵中国华南地区的外来入侵植物南美蟛蜞菊(Wedelia trilobata)常分布于养分匮乏的荒地,却能迅速生长并排挤本地植物而快速扩张领地。【目的】探究丛枝菌根真菌是否促进南美蟛蜞菊的生长与竞争能力。【方法】采用南美蟛蜞菊及其同属本地植物蟛蜞菊(Wedelia chinensis)的盆栽对比控制试验,并设置接种及不接种AMF变形球囊霉(Glomus versiforme)、不同磷营养水平以及单种或混种的种植方式3种处理对两种植物的生长及竞争能力进行比较。【结果】AMF均能侵染上述两种植物,并且AMF对南美蟛蜞菊根系的侵染率显著高于其对蟛蜞菊根系的侵染,尤其是在低磷水平下南美蟛蜞菊的菌根侵染率更高,而且AMF的侵染显著促进了低磷水平下南美蟛蜞菊的生长及其对蟛蜞菊的竞争能力。【结论】丛枝菌根真菌能够促进南美蟛蜞菊的生长,增强其对本地植物的竞争优势,该效应很可能对外来植物南美蟛蜞菊的成功入侵产生一定的作用。     

喀斯特地区丛枝菌根真菌遗传多样性

为探明喀斯特地区丛枝菌根真菌( AMF)的遗传多样性特征,利用巢式PCR和DGGE相结合的分子生物学方法对茂兰喀斯特多个植被类型下的AMF遗传多样性进行了研究.结果表明,喀斯特地区AMF遗传多样性指数和物种丰富度分别平均为3.50和41,远高于非喀斯特对照样地的2.68和17,分析表明,喀斯特地区较高的AMF多样性与此地区丰富的植物多样性以及特殊的生态环境有关,是与喀斯特生态系统长期相互选择的结果.不同植被类型下的AMF多样性差异显著,相似性指数最高为0.34,喀斯特地区AMF的群落结构随着植被类型的改变发生显著变化;基因测序显示,喀斯特地区AMF的优势菌属是生态适应性很强的球囊霉属,在喀斯特石漠化生态恢复中具有较强的利用潜力.     

不同喀斯特小生境中土壤丛枝菌根真菌的遗传多样性

为探明西南喀斯特地区小生境土壤中丛枝菌根真菌(AMF)的遗传多样性特征, 利用巢式PCR和变性梯度凝胶电泳相结合的分子生物学方法, 对茂兰3种植被类型下的小生境(石缝、石沟、土面) AMF遗传多样性进行了研究。结果发现: 各类小生境都含有丰富的AMF遗传多样性, 灌木林土面的多样性指数和物种丰富度最高, 为4.06和68; 次生林石缝的最低, 为3.16和29, 所研究的9个小生境多样性指数和物种丰富度的平均值分别高达3.67和48, 高于同类研究在其他地区的结果, 这可能主要与喀斯特生态系统复杂的结构和较高的植物多样性有关; 聚类分析显示各类小生境间的AMF群落结构差异显著, 相似性指数最高仅为0.45, 说明小生境所带来的空间异质性对AMF的遗传多样性产生了显著影响; 基因测序显示球囊霉属(Glomus)极有可能是喀斯特地区AMF的优势菌属, 在以后筛选喀斯特地区的高效生态恢复菌种时可重点考虑球囊霉属的一些菌种。     

丛枝菌根真菌物种多样性研究进展

丛枝菌根真菌(Arbuscular mycorrhizal fungi,AMF)在不同生态系统均发挥至关重要的作用,研究其多样性能够为AMF物种资源的保护和利用提供科学依据。AMF不能被离体纯培养以及自身的高变异性等因素严重阻碍了对其进行深入研究,随着研究方法的不断改进,尤其是新一代测序技术的运用,极大加速了人们对AMF物种多样性的认识。本文主要从AMF分类系统、不同宿主植物和不同生境中的AMF物种多样性及AMF物种多样性研究方法(包括形态鉴定、Sanger测序和高通量测序)方面介绍AMF物种多样性研究进展,并且探讨AMF物种多样性研究中存在的主要问题,认为在今后AMF物种多样性研究中不仅要注重运用新的研究手段,还应该着重解决AMF不能离体纯培养的问题。     

Distribution of dominant arbuscular mycorrhizal fungi among five plant species in undisturbed vegetation of a coastal grassland

Most plant species in mixed grassland vegetation are colonized by arbuscular mycorrhizal (AM) fungi. Previous studies have reported differences in host preferences among AM fungi, although the fungi are known to lack host specificity. In the present study, the distribution of phylogenetic groups of AM fungi belonging to a clade of Glomus species was studied in five plant species from a coastal grassland in Denmark. The occurrence of the fungi was determined by PCR analyses of fungal large subunit ribosomal DNA sequences amplified from root fragments using a specific primer set. The results showed that the dominant Glomus species were able to colonize all the studied plant species, supporting the view that the AM fungi represent a large underground interconnecting mycelial network.     

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.     

Functional diversity of arbuscular mycorrhizal fungal isolates in relation to extraradical mycelial networks

We investigated the functional significance of extraradical mycorrhizal networks produced by geographically different isolates of the arbuscular mycorrhizal fungal (AMF) species Glomus mosseae and Glomus intraradices. A two-dimensional experimental system was used to visualize and quantify intact extraradical mycelium (ERM) spreading from Medicago sativa roots. Growth, phosphorus (P) and nitrogen (N) nutrition were assessed in M. sativa plants grown in microcosms. The AMF isolates were characterized by differences in extent and interconnectedness of ERM. Phenotypic fungal variables, such as total hyphal length, hyphal density, hyphal length per mm of total or colonized root length, were positively correlated with M. sativa growth response variables, such as total shoot biomass and plant P content. The utilization of an experimental system in which size, growth rate, viability and interconnectedness of ERM extending from mycorrhizal roots are easily quantified under realistic conditions allows the simultaneous evaluation of different isolates and provides data with a predictive value for selection of efficient AMF.     

Indigenous and introduced arbuscular mycorrhizal fungi contribute to plant growth in two agricultural soils from south-western Australia

Arbuscular mycorrhizal (AM) fungi occur in all agricultural soils but it is not easy to assess the contribution they make to plant growth under field conditions. Several approaches have been used to investigate this, including the comparison of plant growth in the presence or absence of naturally occurring AM fungi following soil fumigation or application of fungicides. However, treatments such as these may change soil characteristics other than factors directly involving AM fungi and lead to difficulties in identifying the reason for changes in plant growth. In a glasshouse experiment, we assessed the contribution of indigenous AM fungi to growth of subterranean clover in undisturbed cores of soil from two agricultural field sites (a cropped agricultural field at South Carrabin and a low input pasture at Westdale). We used the approach of estimating the benefit of AM fungi by comparing the curvature coefficients ( C) of the Mitscherlich equation for subterranean clover grown in untreated field soil, in field soil into which inoculum of Glomus invermaium was added and in soil fumigated with methyl bromide. It was only possible to estimate the benefit of mycorrhizas using this approach for one soil (Westdale) because it was the only soil for which a Mitscherlich response to the application of a range of P levels was obtained. The mycorrhizal benefit ( C of mycorrhizal vs. non-mycorrhizal plants or C of inoculated vs. uninoculated plants) of the indigenous fungi corresponded with a requirement for phosphate by plants that were colonised by AM fungi already present in the soil equivalent to half that required by non-mycorrhizal plants. This benefit was independent of the plant-available P in the soil. There was no additional benefit of inoculation on plant growth other than that due to increased P uptake. Indigenous AM fungi were present in both soils and colonised a high proportion of roots in both soils. There was a higher diversity of morphotypes of mycorrhizal fungi in roots of plants grown in the Westdale soil than in the South Carrabin soil that had a history of high phosphate fertilizer use in the field. Inoculation with G. invermaium did not increase the level of colonisation of roots by mycorrhizal fungi in either soil, but it replaced approximately 20% of the root length colonised by the indigenous fungi in Westdale soil at all levels of applied P. The proportion of colonised root length replaced by G. invermaium in South Carrabin soil varied with the level of application of P to the soil; it was higher at intermediate levels of recently added soil P.     

丛枝菌根真菌影响作物非生物胁迫耐受性的研究进展

土壤中存在着大量不同种类的微生物资源,土壤微生物能够与自然界中的大多数植物密切合作,其中丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)起着十分重要的作用。近年来,对于AMF的研究越来越多。AMF是存在于土壤中的重要真菌之一,是土壤中的菌根真菌菌丝与高等植物营养根系形成的一种联合体。AMF可以与陆地上90%左右的植物根系形成共生体,通过在植物根系形成重要的“丛枝菌根”结构而为植物提供更多的养分。为了了解接种AMF对作物生长过程中耐受一些非生物胁迫(如干旱、极端温度、重金属污染、盐分、不利的土壤pH变化等)性能方面的影响,基于之前接种AMF对养分胁迫下玉米生长影响的研究,在扩大作物品种的基础上,通过查阅大量文献,结合试验研究及对前人和近年来关于AMF的一些最新研究进展,获得了具有实践性意义的新发现：AMF与植物共生有助于植物生长,可以改善植物的营养状况,并且可以保护植物免受各种非生物环境胁迫的影响。由此可以得出结论：AMF通过各种机制改善植物生长状况,提高作物抗逆性,为作物增产、农民增收创造了福利,并且避免了由于肥料过量施用导致的一些污染环境问题。本文主要综述了接种AMF在各种非生物环境胁迫(干旱、极端温度、重金属污染、盐分、不利的土壤pH变化等)条件下对植株生长和发育的有利影响,并对目前存在的不足和今后研究的重点提出几点建议。     

Different native arbuscular mycorrhizal fungi influence the coexistence of two plant species in a highly alkaline anthropogenic sediment

Different species of arbuscular mycorrhizal fungi (AMF) can produce different amounts of extraradical mycelium (ERM) with differing architectures. They also have different efficiencies in gathering phosphate from the soil. These differences in phosphate uptake and ERM length or architecture may contribute to differential growth responses of plants and this may be an important contributor to plant species coexistence. The effects of the development of the ERM of AMF on the coexistence of two co-occurring plant species were investigated in root-free hyphal chambers in a rhizobox experimental unit. The dominant shrub (Salix atrocinerea Brot.) and herbaceous (Conyza bilbaoana J. Rémy) plant species found in a highly alkaline anthropogenic sediment were studied in symbiosis with four native AMF species (Glomus intraradices BEG163, Glomus mosseae BEG198, Glomus geosporum BEG199 and Glomus claroideum BEG210) that were the most abundant members of the AMF community found in the sediment. Different AMF species did not influence total plant productivity (sum of the biomass of C. bilbaoana and S. atrocinerea), but had a great impact on the individual biomass of each plant species. The AMF species with greater extracted ERM lengths (G. mosseae BEG198, G. claroideum BEG210 and the four mixed AMF) preferentially benefited the plant species with a high mycorrhizal dependency (C. bilbaoana), while the AMF species with the smallest ERM length (G. geosporum BEG199) benefited the plant species with a low mycorrhizal dependency (S. atrocinerea). Seed production of C. bilbaoana was only observed in plants inoculated with G. mosseae BEG198, G. claroideum BEG210 or the mixture of the four AMF. Our results show that AMF play an important role in the reproduction of C. bilbaoana coexisting with S. atrocinerea in the alkaline sediment and have the potential to stimulate or completely inhibit seed production. The community composition of native AMF and the length of the mycelium they produce spreading from roots into the surrounding soil can be determinant of the coexistence of naturally co-occurring plant species.