Characterization of glomalin as a hyphal wall component of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) produce a protein, glomalin, quantified operationally in soils as glomalin-related soil protein (GRSP). GRSP concentrations in soil can range as high as several mg g⁻¹ soil, and GRSP is highly positively correlated with aggregate water stability. Given that AMF are obligate biotrophs (i.e. depending on host cells for their C supply), it is difficult to explain why apparently large amounts of glomalin would be produced and secreted actively into the soil, since the carbon could not be directly recaptured by the mycelium (and benefits to the AMF via increased soil structure would be diffuse and indirect). This apparent contradiction could be resolved by learning more about the pathway of delivery of glomalin into soil; namely, does this occur via secretion, or is glomalin tightly bound in the fungal walls and only released after hyphae are being degraded by the soil microbial community? In order to address this question, we grew the AMF Glomus intraradices in in vitro cultures and studied the release of glomalin from the mycelium and the accumulation of glomalin in the culture medium. Numerous protein-solubilizing treatments to release glomalin from the fungal mycelium were unsuccessful (including detergents, acid, base, solvents, and chaotropic agents), and the degree of harshness required to release the compound (autoclaving, enzymatic digestion) is consistent with the hypothesis that glomalin is tightly bound in hyphal and spore walls. Further, about 80% of glomalin (by weight) produced by the fungus was contained in hyphae and spores compared to that released into the culture medium, strongly suggesting that glomalin arrives mainly in soil via release from hyphae, and not primarily through secretion. These results point research on functions of glomalin and GRSP in a new direction, focusing on the contributions this protein makes to the living mycelium, rather than its role once it is released into the soil.     

Density-dependent grazing on the extraradical hyphal network of the arbuscular mycorrhizal fungus, Glomus intraradices, by the collembolan, Folsomia candida

The effect of microarthropod density on the relative palatibility of selected microfungi and on the integrity of extraradical arbuscular-mycorrhizal (AM) hyphal networks was examined. A series of microcosm experiments were conducted using two conidial fungi (Alternaria alternata and Trichoderma harzianum), one AM fungus (Glomus intraradices) and the microarthropod Folsomia candida (Collembola). We did not detect a significant effect of athropod density on food preference. Food preferences for conidial over mycorrhizal hyphae were consistent across a wide range of collembolan densities. Collembolan density did, however, have a significant effect on the numbers of extraradical AM hyphae which were severed from the plant root in vitro. Even in the presence of a preferred food source, numbers of severed AM hyphal networks increased with increasing collembolan density. Also, the fecundity of Folsomia candida increased in the presence of preferred food sources. We conclude that microarthropod population densities are influenced by the fungal species available and that the potential of microarthropods to impair the efficacy of AM symbioses is density-dependent. Received: 16 May 1997     

丛枝菌根真菌对玉米生长生态效应的影响

1996～1998年试验研究了大田不施肥条件下3种泡囊丛枝菌根真菌Arbuscular mycorrhizal fungi (AMF),即 Glomus mosseae(G.m)、Glomus versifome(G.v)、Sclerocystis sinousa(S.s)对玉米生长的生态效应.结果表明,AMF处理的玉米根系活力为对照的2.32～3.05倍,玉米N素吸收比对照增加24.14%～56.65%,玉米P素吸收比对照增加41.14%～78.29%,促进玉米生长发育显著;在灭菌条件下G.m、G.v、S.s处理分别比对照增产17.63%、22.91%和10.80%;而不灭菌条件下3个处理分别比对照增产10.35%、20.56%和4.53%,同时显著提高玉米的水分供应状况.     

Limitations to using benomyl in evaluating mycorrhizal functioning

Arbuscular mycorrhizal (AM) grasses compete for nutrients with ectomycorrhizal (EM) pine in the southeastern United States. Our objective was to determine if benomyl could be used to selectively inhibit the function of AM and thereby reduce grass competition in the field. The effects of Benlate (active ingredient: benomyl) in the greenhouse and field were evaluated. No effect was observed on pine inoculated with Pisolithus tinctorius in the greenhouse. Colonized root length of benomyl-treated Zea mays L. plants inoculated with Glomus sp. in the greenhouse remained static over time and the response was not dose dependent at concentrations of 0, 20, 60 and 150kg benomyl ha^–1 equivalent. In contrast, colonization of nontreated plants increased over time. In the field, a minimal reduction of grass colonization was observed following four applications of benomyl ranging from 5 to 20kgha^–1. We conclude that benomyl can successfully inhibit development of AM fungi under controlled conditions in the greenhouse with no inhibitory effects on the EM fungus P. tinctorius; however, in the field several factors may interfere with the effect of benomyl on AM fungi. These factors include: (a) the presence of ground cover which obstructs penetration of the fungicide to the soil, (b) timing of application in relation to mycorrhizal development, and (c) the application method of benomyl, a soil drench being preferable to a foliar spray. Received: 30 September 1996     

Soil type and land use intensity determine the composition of arbuscular mycorrhizal fungal communities

The objective of this study was to test whether soil types can be characterized by their arbuscular mycorrhizal fungal (AMF) communities. To answer this question, a well-defined study area in the temperate climatic zone of Central Europe was chosen with a large spectrum of soils and parent materials. Representative soil samples were taken from three soil types (Cambisol, Fluvisol and Leptosol) at in total 16 sites differing in agricultural land use intensity (9 grasslands and 7 arable lands). AMF spores were isolated and morphologically identified directly from field soils and after reproduction in trap cultures. AMF diversity and community composition strongly depended on soil type and land use intensity, and several AMF species were characteristic for a specific soil type or a specific land use type and hence had a specific niche. In contrast, other AM fungi could be considered as ‘generalists’ as they were present in each soil type investigated, irrespective of land use intensity. An estimated 53% of the 61 observed AMF species could be classified as ‘specialists’ as (almost) exclusively found in specific soil types and/or under specific land use intensities; 28% appeared to be ‘generalists’ and 19% could not be classified. Plant species compositions (either natural or planted) had only a subordinate influence on the AMF communities. In conclusion, land use intensity and soil type strongly affected AMF community composition as well as the presence and prevalence of many AM fungi. Future work should examine how the differences in AMF species compositions affect important ecosystem processes in different soils and to which extent the loss of specific groups of AM fungi affect soil quality.     

Take-all disease is systemically reduced in roots of mycorrhizal barley plants

The systemic effect of root colonization by the arbuscular mycorrhizal fungus Glomus mosseae on infection of barley by Gaeumannomyces graminis var. tritici (Ggt) was studied. In split-root systems of barley one side was inoculated with G. mosseae and the other side was inoculated with Ggt.Root infection by Ggt was systemically reduced when barley plants showed high degrees of mycorrhizal root colonization, whereas a low mycorrhizal root colonization exhibited no effect on Ggt infection. Our results show a clear systemic bioprotectional effect depending on the degree of root colonization by the mycorrhizal fungus. At a higher mycorrhizal colonization rate the concentration of salicylic acid (SA) was increased in roots colonized by the mycorrhizal fungus but no systemic increase of SA could be measured in non-mycorrhizal roots of mycorrhizal plants, indicating that the systemic bioprotectional effect against Ggt is not mediated by salicylic acid.     

Hyphal transport by a vesicular-arbuscular mycorrhizal fungus of N applied to the soil as ammonium or nitrate

Summary Transport of N by hyphae of a vesicular-arbuscular mycorrhizal fungus was studied under controlled experimental conditions. The N source was applied to the soil as ¹⁵NH _inf4 ^sup+ or ¹⁵NO _inf3 ^sup- . Cucumis sativus was grown for 25 days, either alone or in symbiosis with Glomus intraradices, in containers with a hyphal compartment separated from the root compartment by a fine nylon mesh. Mineral N was then applied to the hyphal compartment as ¹⁵NH _inf4 ^sup+ or ¹⁵NO _inf3 ^sup- at 5 cm distance from the root compartment. Soil samples were taken from the hyphal compartment at 1, 3 and 5 cm distance from the root compartment at 7 and 12 days after labelling, and the concentration of mineral N in the samples was measured from 2 M KCl extracts. Mycorrhizal colonization did not affect plant dry weight. The recovery of ¹⁵N in mycorrhizal plants was 38 or 40%, respectively, when ¹⁵NH _inf4 ^sup+ or ¹⁵NO _inf3 ^sup- was applied. The corresponding values for non-mycorrhizal plants were 7 and 16%. The higher ¹⁵N recovery observed in mycorrhizal plants than in non-mycorrhizal plants suggests that hyphal transport of N from the applied ¹⁵N sources towards the host plant had occurred. The concentration of mineral N in the soil of hyphal compartments was considerably less in mycorrhizal treatments than in controls, indicating that the hyphae were able to deplete the soil for mineral N.     

Differential growth response of Curculigo orchioides to native arbuscular mycorrhizal fungal (AMF) communities varying in number and fungal components

The study reports diversity of arbuscular mycorrhizal fungal (AMF) species in the rhizosphere of an endangered anticancerous herb – Curculigo orchioides Gaertn. in its natural habitat. A total of 18 species of AMF, belonging to three genera (Acaulospora, Glomus and Gigaspora) were recorded, with Glomus microcarpum being the most abundant species type. The AMF species composition across the study sites appeared to be influenced by soil pH rather than soil P and vegetation. Acaulospora laevis spores were restricted to sites where the soil pH was acidic. The effectiveness of these native AMF species on growth performance of C. orchioides plants was compared under experimental conditions. In general, the mycorrhizal plants were superior in most of the evaluated parameters, but the extent to which the growth of mycorrhizal plants was influenced varied with the inocula used. The plants inoculated with mixed consortia containing maximum AMF species richness exhibited improved growth in comparison to consortia containing lower AMF diversity and monospecies cultures. The variable plant responses observed with any two consortia having same species richness in the present study could be due to variable component AMF species and their relative abundance. These results emphasize the need to protect the below-ground diversity of AMF and recommend their usage for restoration practices.     

Salinity tolerance and mycorrhizal responsiveness of native xeroriparian plants in semi-arid western USA

Restoration of salt-affected soils is a global concern. In the western United States, restoration of salinized land, particularly in river valleys, often involves control of Tamarix, an introduced species with high salinity tolerance. Revegetation of hydrologically disconnected floodplains and terraces after Tamarix removal is often difficult because of limited knowledge regarding the salinity tolerance of candidate native species for revegetation. Additionally, Tamarix appears to be non-mycorrhizal. Extended occupation of Tamarix may deplete arbuscular mycorrhizal fungi in the soil, further decreasing the success of revegetation efforts. To address these issues, we screened 42 species, races, or ecotypes native to southwestern U.S. for salinity tolerance and mycorrhizal responsiveness. As expected, the taxa tested showed a wide range of responses to salinity and mycorrhizal fungi. This variation also occurred between ecotypes or races of the same species, indicating that seed collected from high-salinity reference systems is likely better adapted to harsh conditions than seed originating from less saline environments. All species tested had a positive or neutral response to mycorrhizal inoculation. We found no clear evidence that mycorrhizae increased salinity tolerance, but some species were so dependent on mycorrhizal fungi that they grew poorly at all salinity levels in pasteurized soil.     

Consumption of mycorrhizal and saprophytic fungi by Collembola in grassland soils

Although soil-dwelling Collembola can influence plant growth and nutrient cycling, their specific role in soil food webs is poorly understood. Soil-free microcosm studies suggest that Collembola are primarily fungivores where they feed preferentially on saprophytic fungi (SF) over other fungal types. We directly assessed collembolan consumption of arbuscular mycorrhizal fungi (AMF) and SF using plant-soil mesocosms and natural abundance stable carbon isotope techniques. Mycorrhizal Andropogon gerardii (C₄ grass) seedlings were placed in pots containing Collembola and soil from a C₃ plant dominated site, while mycorrhizal Pascopyrum smithii (C₃ grass) seedlings were placed in pots with Collembola and soil collected at a C₄ plant dominated site. After 6 weeks, collembolans assimilated carbon derived from C₃ and C₄ sources in both A. gerardii and P. smithii treatments. Comparing Collembola isotope values in AMF vs. AMF-suppressed treatments, our data show that both AMF and SF were consumed in these experimental soil environments.     

Fitting plants to soil through mycorrhizal fungi: Mycorrhiza effects on plant growth and soil organic matter

Summary Vesicular-arbuscular mycorrhizal (VAM) fungi affect diverse aspects of plant form and function. Since mycorrhiza-mediated changes in host-plant responses to root colonization by different VAM fungi vary widely, it is important to assess each endophyte for each specific effect it can elicit from its host as part of the screening process for effectiveness. Three species of VAM fungi and a mixture of species were compared with non-VAM controls for their effects on soil organic matter contents and on nutrition and morphology in two varieties (native and hybrid) of corn (Zea mays L.) and one of sunflower (Helianthus annuus L.) in P-sufficient and N-deficient soil in pot cultures. Differences in soil organic matter due to the fungal applications were highly significant with all host plants. Native corn responded more to VAM colonization than the hybrid did; differences in treatments were significant in leaf area, plant biomass, and root: shoot ratio in the former, but not in the latter. Responses in the sunflower were similar to those in the native corn. Significant VAM treatment-related differences in shoot N and P contents were not reflected in shoot biomass, which was invariant. Correlations between plant or soil parameters and the intensity of VAM colonization were found only in soil organic matter with the native corn, in specific leaf area in the hybrid corn, and in plant biomass in the sunflower. The presence of the different endophytes and not the intensity of colonization apparently elicited different host responses.     

Rhodotorulic acid enhances root colonization of tomato plants by arbuscular mycorrhizal (AM) fungi due to its stimulatory effect on the pre-symbiotic stages of the AM fungi

Exudates of Rhodotorula mucilaginosa, a yeast commonly found in the rhizosphere, increased hyphal length of the arbuscular mycorrhizal (AM) fungi Gigaspora rosea and Gigaspora margarita. Rhodotorulic acid (RA), a siderophore compound obtained from R. mucilaginosa exudates, increased hyphal length and branching. Thus, the increase in the number of entry points and the higher AM root colonization of tomato plants in the presence of RA can at least partially be explained by the positive effect of RA on the pre-symbiotic stages of the AM fungi.     

葫芦科蔬菜对丛枝菌根真菌依赖性的研究

在盆栽条件下试验研究了5种葫芦科(Cucurbitaceae)蔬菜对2种丛枝菌根(Arbuscular Mycorrhiza，AM)真菌的依赖性。结果表明，AM真菌Glomus mosseae(G.m)和Gigaspora rosea(Gi.r)均能有效地促进葫芦科蔬菜的生长，显著增加叶面积和植株干物质量，提高叶片的光合速率，降低气孔阻力。供试葫芦科蔬菜对菌根的依赖程度顺序为黄瓜＞西瓜＞苦瓜＞葫芦＞南瓜。5种葫芦科蔬菜对AM的依赖性与AM真菌对根系的浸染率呈正相关关系，符合直线回归方程：y=117.19+0.7468x。     

Network establishment of arbuscular mycorrhizal hyphae in the rhizospheres between citrus rootstocks and <Emphasis Type="Italic">Paspalum notatum</Emphasis> or <Emphasis Type="Italic">Vulpia myuros</Emphasis> grown in sand substrate

A greenhouse experiment was conducted to examine the favorable effects of sod culture system with bahiagrass (Paspalum notatum Flügge.) and Vulpia myuros (L.) C. C. Gmel. intercropped with citrus trees on the establishment of the network of arbuscular mycorrhizal (AM) fungus hyphae in their rhizospheres. Special acrylic root boxes with three compartments were used for the experiment. Four types of citrus rootstock seedlings, trifoliate orange (Poncirus trifoliata Raf.), sour orange (Citrus aurantium L.), rough lemon (Citrus jambhiri Lush.), and Citrus natsudaidai Hayata, were separately transplanted into one outer compartment in each box, and the seedlings of bahiagrass and V.myuros were separately transplanted into the other outer compartment. An AM fungus, Gigaspora margarita Becker and Hall, was inoculated in the center compartment of each box. Some boxes with both outer compartments without plants and with some plants in only one outer compartment were also prepared. The box with bare × bare had very low density of AM hyphae. There were a few hyphae in bare compartments in the boxes of trifoliate orange × bare, sour orange × bare, rough lemon × bare, and C. natsudaidai × bare. The density of hyphae in the compartments with citrus seedlings and grasses, however, was significantly higher than in every bare compartment, and the hyphae in the compartments with plants penetrated deeply into the sand. In particular, the density in the compartments of citrus seedlings increased when bahiagrass or V. myuros was transplanted as a neighboring plant. The percentage of AM fungus colonization in every plant root was high. New spore formation was observed in compartments with plants, whereas there were few spores in every bare compartment. In particular, the spore formation in bahiagrass compartments was superior to that in other compartments with plants. Our results suggest that the network system by AM hyphae is easily discernible in the rhizospheres between citrus rootstocks and bahiagrass or V. myuros, but bare ground severely inhibits the formation and development of AM hyphal network and reduces the number of AM spores in the soil.     

On the use of sodium hexametaphosphate to extract spores of arbuscular mycorrhizal fungi from soil

Extraction of arbuscular mycorrhizal fungal (AMF) spores from soil is widely used to assess AMF community structure and abundance. The most widely used protocol relies on a water-sucrose gradient flotation technique. Na-hexametaphosphate has also been used to deflocculate soil aggregates prior to spore extraction in order to optimize recovery, but its effect on spore viability remains unknown. Here, we report that Na-hexametaphosphate increases average spore yield in a high clay soil by about 15%, but decreases average spore viability by about 20%. Na-hexametaphosphate should therefore be used cautiously where the extracted spores are destined to be used as inoculum for subsequent studies.     

Differential decomposition of arbuscular mycorrhizal fungal hyphae and glomalin

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts of most higher plants. In addition to being a major component of soil microbial biomass, AMF hyphae produce glomalin, a recalcitrant glycoproteinaceous substance highly correlated with soil aggregate water stability. This study addresses the lack of knowledge concerning the decomposition of hyphae and glomalin. We used an experimental design that exploited the lack of saprobic capabilities of AMF hyphae by incubating field soil samples in the dark, and hence in the absence of plant or AMF hyphal growth. In 150 days, hyphal length decreased 60%, while glomalin, quantified by the Bradford protein assay, declined only 25%. Immuno-reactive glomalin decreased 46%. This study serves as a proof-of-concept for further examination of factors that influence decomposition of AMF hyphae using similar experimental designs.     

Effects of low temperature and shade on relationships between nodulation,vesicular-arbuscular mycorrhizal infection,and shoot growth of soybeans

The effects of low temperature and reduced light on a Glycine-Bradyrhizobium-Glomus spp. symbiosis were examined in pot experiments. Soybean plants, Glycine max L. Merr. cv. Tachiyutaka, were grown with N fertilization or inoculation with Bradyrhizobium japonicum plus P fertilization or inoculation with Glomus mosseae in the glasshouse. After the flowering stage, half the pots with soybean plants were subjected to low temperature (15°C 14h/13°C 10 h) with light reduced by shading. At 0, 7, 16, and 28 days after the application of the treatments, the growth, nodulation, vesicular-arbuscular mycorrhizal (VAM) infection and the N and P contents of the soybean plants were measured. In all symbiont-fertilization combinations, the low-temperature treatment reduced the production of dry matter by the soybeans. Nodulation (weight and number) was slightly reduced by this treatment but the proportion of larger nodules was increased. The root length infected by the VAM fungus was little affected by the low-temperature treatment. Both the nodule weight and the infected root length were linearly related to shoot dry weight regardless of treatment and of the symbiont-fertilization combination used. These results suggest that the growth of the symbionts on the root was in balance with the shoot growth of the host, irrespective of climatic conditions, and imply a considerable degree of host control. P inflows to root systems were greatly affected by low-temperature treatment regardless of the symbiont-fertilization combination. This suggests that a simple comparison of P inflows between mycorrhizal and non-mycorrhizal plants may give misleading information on the effects of low temperature or reduced light conditions on P uptake by mycorrhizal plants.     

Differential effect of arbuscular mycorrhizal fungal communities from ecosystems along management gradient on the growth of forest understorey plant species

Arbuscular mycorrhizal fungi (AMF) are important functional components of ecosystems. Although there is accumulating knowledge about AMF diversity in different ecosystems, the effect of forest management on diversity and functional characteristics of AMF communities has not been addressed. Here, we used soil inoculum representing three different AM fungal communities (from a young forest stand, an old forest stand and an arable field) in a greenhouse experiment to investigate their effect on the growth of three plant species with contrasting local distributions - Geum rivale, Trifolium pratense and Hypericum maculatum. AM fungal communities in plant roots were analysed using the terminal restriction fragment length polymorphism (T-RFLP) method. The effect of natural AMF communities from the old and young forest on the growth of studied plant species was similar. However, the AMF community from the contrasting arable ecosystems increased H. maculatum root and shoot biomass compared with forest inocula and T. pratense root biomass compared to sterile control. According to ordination analysis AMF inocula from old and young forest resulted in similar root AMF communities whilst plants grown with AM fungi from arable field hosted a different AMF community from those grown with old forest inocula. AMF richness in plant roots was not related to the origin of AMF inoculum. G. rivale hosted a significantly different AM fungal community to that of T. pratense and H. maculatum. We conclude that although the composition of AM fungal communities in intensively managed stands differed from that of old stands, the ecosystem can still offer the ‘symbiotic service’ necessary for the restoration of a characteristic old growth understorey plant community.     

Reduction of bacterial growth by a vesicular-arbuscular mycorrhizal fungus in the rhizosphere of cucumber (Cucumis sativus L.)

Summary Cucumber was grown in a partially sterilized sand-soil mixture with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum or left uninoculated. Fresh soil extract was places in polyvinyl chloride tubes without propagules of mycorrhizal fungi. Root tips and root segments with adhering soil, bulk soil, and soil from unplanted tubes were sampled after 4 weeks. Samples were labelled with [³H]-thymidine and bacteria in different size classes were measured after staining by acridine orange. The presence of VAM decreased the rate of bacterial DNA synthesis, decreased the bacterial biomass, and changed the spatial pattern of bacterial growth compared to non-mycorrhizal cucumbers. The [³H]-thymidine incorporation was significantly higher on root tips in the top of tubes, and on root segments and bulk soil in the center of tubes on non-mycorrhizal plants compared to mycorrhizal plants. At the bottom of the tubes, the [³H]-thymidine incorporation was significantly higher on root tips of mycorrhizal plants. Correspondingly, the bacterial biovolumes of rods with dimension 0.28–0.40×1.1–1.6 m, from the bulk soil in the center of tubes and from root segments in the center and top of tubes, and of cocci with a diameter of 0.55–0.78 m in the bulk soil in the center of tubes, were significantly reduced by VAM fungi. The extremely high bacterial biomass (1–7 mg C g^-1 dry weight soil) was significant reduced by mycorrhizal colonization on root segments and in bulk soil. The incorporation of [³H]-thymidine was around one order of magnitude lower compared to other rhizosphere measurements, probably because pseudomonads that did not incorporate [³H]-thymidine dominated the bacterial population. The VAM probably decreased the amount of plant root-derived organic matter available for bacterial growth, and increased bacterial spatial variability by competition. Thus VAM plants seem to be better adapted to compete with the saprophytic soil microflora for common nutrients, e.g., N and P, compared to non-mycorrhizal plants.