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.     

Cryopreservation of entrapped monoxenically produced spores of an arbuscular mycorrhizal fungus

A study was conducted to quantify the ability of entrapped, monoxenically produced spores of an arbuscular mycorrhizal fungus to germinate and reproduce the fungal life cycle after cryopreservation. No germination was obtained after incubation of entrapped spores in glycerol and mannitol and subsequent cryopreservation at −70 °C, regardless of the concentration of cryoprotectants and duration of incubation. Incubation for 1 d in 0.5 M sucrose, and for 1 and 2 d in 0.5 M trehalose, led to spore germination after cryopreservation at −70 °C. Lower cryopreservation temperatures were tested with entrapped spores incubated for 1 d in 0.5 M trehalose. The highest germination rate, estimated by the percentage of potentially infective beads (%PIB), was obtained at −100 °C. A %PIB of 95% (water agar medium) to 100% (Strullu–Romand medium) was obtained at this temperature. Thereafter, %PIB rapidly decreased at −140 and −180 °C. Heavy sporulation and high internal root colonization were obtained after re-association of the entrapped spores, incubated for 1 d in 0.5 M trehalose and subsequently cryopreserved at −100 °C, with transformed carrot roots. This demonstrates the ability of entrapped spores to reproduce the fungal life cycle following cold treatment.     

Ultrastructure of rapidly frozen and freeze-substituted germ tubes of an arbuscular mycorrhizal fungus and localization of polyphosphate

In arbuscular mycorrhizas (AM), the supply of phosphorus from the fungi is one of the most important benefits to the host plant. Here we describe for the first time the ultrastructure and polyphosphate (poly P) distribution in rapidly frozen and freeze-substituted germ tubes of the AM fungus Gigaspora margarita. At the ultrastructural level, phosphorus distribution was analysed using energy-filtering transmission electron microscopy, and poly P was detected using an enzyme-affinity method. Semithin sections and live cells were also stained with 4',6-diamidino-2-phenylindole, which is not specific but fluoresces yellow when viewed under UV irradiation by binding with poly P. The cryotechnique method showed that extensive elongate ellipsoid vacuoles containing a uniform electron-opaque material occupied most of the cell volume. Combining the results of multiple methods revealed that poly P was localized in a dispersed form in vacuoles and in the outer fungal cell wall. These results show the significant potential of AM fungi for phosphorus storage based on its localization in the extensive complement of vacuoles in thick hyphae. The mechanism of translocation of poly P in tubular vacuoles, and the role of poly P in the cell wall, need to be elucidated.     

Transmembrane H + and Ca 2+ fluxes through extraradical hyphae of arbuscular mycorrhizal fungi in response to drought stress

丛枝菌根真菌(AMF)能够和大多数陆地植物形成共生体系, 对于植物生长发育和适应各种逆境胁迫具有重要作用。很多研究表明干旱胁迫下AMF能够促进宿主植物对水分的吸收从而增强植物抗旱能力, 但目前针对AMF根外菌丝响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究并不多。该研究利用AMF Rhizophagus irregularis和胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净根外菌丝, 向培养基添加聚乙二醇(PEG)模拟干旱胁迫, 运用场发射扫描电子显微镜(FE-SEM-EDS)观察干旱胁迫对AMF根外菌丝形态的影响, 同时采用非损伤微测技术(NMT)观测根外菌丝跨膜H ⁺和Ca ²⁺离子流变化。结果发现, PEG处理1 h后菌丝尖端和侧面发生H ⁺外流和强烈的Ca ²⁺内流, 荧光探针分析也显示菌丝胞内pH值显著上升、Ca ²⁺浓度增加; PEG处理24 h后菌丝形态发生明显变化, 培养基pH值降低, P、Ca、Fe等元素在菌丝际积累。这些试验结果表明, 干旱胁迫下AMF根外菌丝跨膜H ⁺和Ca ²⁺流发生变化, 促进了菌丝与环境之间的物质交换。菌丝酸化生长环境有利于养分吸收, 并促进AMF与宿主植物之间的信号交流以增强植物的耐旱性。     

Polyphosphates in intraradical and extraradical hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita

The amount of polyphosphate in the intraradical and extraradical hyphae of Gigaspora margarita was estimated from successive extractions with trichloroacetic acid (TCA), EDTA, and phenol-chloroform (PC). In the intraradical hyphae, most of the polyphosphate was present in TCA- and EDTA-soluble (short-chain and long-chain) fractions, whereas most of the polyphosphate in the extraradical hyphae was present in EDTA- and PC-soluble (long-chain and granular) fractions.     

脱落酸对丛枝菌根真菌侵染和产孢的调控效应研究

[目的]揭示脱落酸(ABA)对丛枝菌根(AM)真菌侵染和产孢的影响,建立利用外源ABA促进孢子产量的高效菌剂扩繁方法.[方法]利用番茄毛状根和AM真菌Rhizophagus irregularis DAOM 197198建立双重培养体系,通过外源施用ABA、赤霉素(GA)或者使用ABA、GA的缺陷突变体,染色观察菌根侵...     

Copper sorption and accumulation by the extraradical mycelium of different Glomus spp. (arbuscular mycorrhizal fungi) isolated from the same polluted soil

The form and localisation of Cu accumulation in the extraradical mycelium (ERM) of three arbuscular mycorrhizal fungi (AMF), isolated from the same polluted soil contaminated with the Cu and Arsenate, was studied. There were differences in the capacity of the ERM of the three AMF to sorb and accumulate Cu. Glomus caledonium BEG133 had a significantly lower Cu-sorption capacity than Glomus mosseae BEG132 and Glomus claroideum BEG134 isolated from the polluted soil as well as an isolate of G. mosseae BEG25 from a non-polluted soil. This was directly related to the cation exchange capacity (CEC) of the ERM of these fungi. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) linked to an energy dispersive X-ray spectrometer (EDAX) gave more detailed information, showing that the ERM of AMF from the polluted soil was able to accumulate Cu in the mucilaginous outer hyphal wall zone, cell wall and inside the hyphal cytoplasm. The EDAX spectra showed that the accumulated Cu was mainly associated with Fe in the mucilaginous outer hyphal wall zone and in the cell wall. Cu was associated with traces of arsenate inside the cytoplasm of the ERM of Glomus mosseae BEG132 but this was not visible inside the ERM of Glomus caledonium BEG133 or Glomus claroideum BEG134. This work suggests that the ERM of AMF is able to sorb and accumulate Cu, but different tolerance mechanisms exist between the three AMF isolated from the same polluted soil providing further evidence for functional diversity within populations of AMF in soils.     

Nitrogen assimilation in Lolium perenne colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum

To investigate nitrogen assimilation in Lolium perenne L. colonized by the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum (Thax. sensu Gerd.), nitrate uptake, key enzyme activities, and ¹⁵N incorporation into free amino acids were measured. After a 4-h labelling period with [¹⁵N]nitrate, ¹⁵N content was higher in roots and shoots of AM-plants than in those of control plants. Glutamine synthetase (GS) and nitrate reductase (NR) activities were increased in shoots of AM-plants, but not in roots. More label was incorporated into amino acids in shoots of AM plants. Glutamine, glutamate, alanine and γ-aminobutyric acid were the major sinks for ¹⁵N in roots and shoots of control and AM plants. Interactions between mycorrhizal colonization, phosphate and nitrate nutrition and NR activity were investigated in plants which received different amounts of phosphate or nitrate. In shoots of control plants, NR activity was not stimulated by high levels of phosphate nutrition but was stimulated by high levels of nitrate. At 4 m M nitrate in the nutrient solution, NR activity was similar in control and AM plants. We concluded that mycorrhizal effects on nitrate assimilation are not mediated via improved phosphate nutrition, but could be due to improved nitrogen uptake and translocation.     

Time-course study and partial characterization of a protein on hyphae of arbuscular mycorrhizal fungi during active colonization of roots

Material on the surface of hyphal walls of arbuscular mycorrhizal fungi (AMF) during active colonization of plant roots was detected by a monoclonal antibody. Pot-cultured isolates of Glomus, Acaulospora, Gigaspora, Scutellospora, and Entrophospora had immunofluorescent material (IM) on younger, thinner, intact hyphae, but IM was scant to absent on thicker, melanized or lysing hyphae. Colonization of corn (Zea mays L.), Sudangrass (Sorghum sudanense (Piper) Staph.) or red clover (Trifolium pratense L.) was examined during 5 months of plant growth by removing cores and performing an indirect immunoassay on roots with attached hyphae. Fresh spores of some Glomus spp. had IM on the outer layer of the spore wall. Abundant IM was seen on root hairs of plants colonized by some isolates, and some IM was detected on root surfaces of all plants examined even during early colonization. After cultures were dried, hyphae, roots and spores had little to no IM. Uninoculated control roots had very rare, small patches of IM. An immunoreactive protein was extracted from hyphae of Gigaspora and Glomus isolates by using 20mM citrate (pH 7.0) at 121°C for 90 min. Gel electrophoresis profiles indicated that all isolates tested had the same banding patterns. Lectin-binding of extracted protein is suggestive of a glycoprotein. The immunofluorescence assay can be used to examine root sections for active colonization by AMF, and the potential use of the protein to quantify AMF activity in soil is discussed.     

Heritable variation and mechanisms of inheritance of spore shape within a population of Scutellospora pellucida,an arbuscular mycorrhizal fungus

Substantial variation was found among single-spore cultures established from a single population of the arbuscular mycorrhizal fungus Scutellospora pellucida. A common environment experiment demonstrated that five single-spore cultures differed in their average spore shape (as measured by length:width ratios) and size (volume) with interisolate heritabilities of offspring mean values of 0.96 and 0.87, respectively (0.66 and 0.43 for the shape and size of individual spores). The distribution of offspring spore shapes also differed in levels of variance, skewness, and kurtosis. In fact, these aspects of the distributions shifted with mean spore shape as predicted by the binomial distribution—the distribution expected due to the segregation of genetically diverse nuclei through dividing hyphae. Thus, the original parental spores generating these cultures appear to have contained genetically variable nuclei, which then segregate into the offspring spores to generate consistent differences in the mean, variance, skewness, and kurtosis of the distribution of offspring spore shapes. This nuclear segregation may be followed by the assemblage of novel combinations of nuclei through hyphal fusion. Together these processes are rarely considered mechanisms for the creation of novel genetic combinations and may contribute to the maintenance of the high level of heritable variation observed in this study.     

Comparison of phosphatase localization in the intraradical hyphae of arbuscular mycorrhizal fungi,Glomus spp. and Gigaspora spp.

The localization of acid and alkaline phosphatases in the intraradical hyphae of the arbuscular mycorrhizal fungi, Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (Gm), Gl. etunicatum Becker and Gerd. (Ge) and Gigaspora rosea Nicol. and Schenck (Gir) were compared. Marigold (Tagetes patula L.) and leek (Allium porrum L.) were inoculated with each of the three fungi. The mycorrhizal roots were harvested at 3, 4, 5 and 6 weeks after sowing (WAS), treated with a digestion solution containing cellulase and pectinase, and then stained for phosphatase activities at pH 5.0 and pH 8.5. The development of fungal structures in the host root was also examined. Gm formed fine-branched (mature) arbuscules only at the early phase of infection (3 to 4 WAS). Mature arbuscules of Ge and Gir were observed from the early phase (4 WAS) up to the end of experiment. At pH 5.0, the localization of the phosphatase activities of the three fungi were similar irrespective to host plant species. The activity appeared in mature arbuscules and intercellular hyphae, whereas the collapsed arbuscules were inactive. Ten millimolar NaF, an acid phosphatase inhibitor, inhibited the phosphatase activities of Gm and Ge but did not affect that of Gir. At pH 8.5, a difference among the fungal species was found in the localization of phosphatase activity while that between host species was not. The mature arbuscules were also the active sites in all three species. Only Gir showed the activity in the intercellular hyphae while the two Glomus spp. did not. Five millimolar EDTA inhibited the activity of Gir at pH 8.5 while the activities of Ge and Gm were not affected by either 5 mM EDTA or 10 mM KCN (both are alkaline phosphatase inhibitors).     

丛枝菌根提高宿主植物抗旱性分子机制研究进展

丛枝菌根(arbuscular mycorrhiza, AM)对于植物适应各种逆境胁迫具有重要生态学意义。有关菌根共生体对植物抵御干旱胁迫的积极作用已有较多文献报道:无论在植物个体层面——AM调节植物水分生理,还是在生态层面——干旱条件下菌根真菌和宿主植物之间的互动关系,人们都已有一定的认识。然而,目前对于菌根植物适应干旱胁迫的生理和分子机制还缺乏系统深入的研究。综述了近年来相关研究成果,从干旱胁迫相关植物基因入手,讨论了AM对晚期胚胎富集蛋白(LEA)、脯氨酸合成限速酶△¹-吡咯啉-5-羧酸合成酶(P5CS)、水孔蛋白(MIPs),及脱落酸合成途径重要酶9-顺式-环氧类胡萝卜素双加氧酶(NCED)编码基因的可能调控机制,旨在揭示AM共生体提高植物抗旱性的分子基础和实质贡献,同时通过分析当前研究工作薄弱之处及未来研究热点,期望推动相关研究进展。     

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.     

Microchambers and video-enhanced light microscopy for monitoring cellular events in living hyphae of arbuscular mycorrhizal fungi

Mycelial elongation and protoplasmic flow rate in vitro were monitored for germinated spores of Gigaspora rosea and Glomus caledonium respectively, growing on membranes in microchambers, by using a combination of time-lapse and video-enhanced light microscopy and image analysis. The microchambers allowed continuous observation of living mycelium over a period of several hours during which protoplasm flow and bidirectional movements of cellular organelles and particles were monitored in individual hyphae. Growth rate of G. rosea hyphae, calculated 8 days after germination, was 2.64 μm/min. Protoplasmic flow rate, measured on the basis of the movement of particles, ranged from 2.98 to 4.27 μm/s in living hyphae of G. caledonium. We showed that G. rosea, when growing in axenic culture in the absence of the host, ceased growth within 8 days of germination and underwent a process of protoplasm retraction from hyphal tips, leading to the formation of empty mycelial segments. A process of resource reallocation was inferred in spores of G. rosea showing multiple germination. Detailed developmental studies of living hyphae by using microchambers could provide useful information on spatio-temporal dimensions of cellular events occurring in arbuscular mycorrhizal fungi. This revised version was published online in June 2006 with corrections to the Cover Date.     

Quantification of polyphosphate: different sensitivities to short-chain polyphosphate using enzymatic and colorimetric methods as revealed by ion chromatography

Polyphosphate is ubiquitous and has a variety of biochemical functions. Among polyphosphate quantification methods, an enzymatic assay using Escherichia coli polyphosphate kinase (PPK), in which polyphosphate is converted to adenosine 5'-triphosphate and quantified by luciferase assay, is the most specific and most sensitive. However, chain-length specificity of the assay has not been analyzed in detail so far. Ion chromatography equipped with an on-line hydroxide eluent generator enabled us to analyze polyphosphate up to 50 inorganic phosphate (P(i)) residues, and we employed this method to investigate the chain-length specificity of PPK in this study. Several fractions of short-chain polyphosphate were prepared by electrophoresis, and the chain-length distribution was analyzed before and after 1-6 h PPK reaction by ion chromatography. Polyphosphates longer than 23 P(i) residues were processed by PPK completely after 1 h incubation, but complete processing of those between 11 and 22 P(i) residues required 6h incubation. Limited processing of polyphosphates of 10 P(i) residues or shorter were observed even after 6h incubation. Metachromasy of Toluidine blue O, an alternative method for polyphosphate quantification, showed broader chain-length specificity although it was not as sensitive as the enzymatic assay. Combination of these two methods would be practically applicable to analysis of polyphosphate dynamics in living organisms.     

Effects of long-term NP-fertilization on abundance and diversity of arbuscular mycorrhizal fungi under a maize cropping system

Diversity of arbuscular mycorrhizal fungi (AMF) in 27-year long-term NP-fertilization plots under a maize cropping system in Thailand was studied through spore morphological characterization. The plots received 0–0, 60–60, 120–120 and 180–180 kg N-P₂O₅ ha^–1 year^–1 as ammonium sulfate and triple superphosphate. The plots were sampled monthly for one year, the AMF spores were counted and morphotyped, and taxa were identified after morphotyping and monospecific pot culture. Spore number g^–1 soil, relative spore abundance and Shannon-Wiener indexes were calculated. Sixteen putative taxa were recorded from the field of which nine sporulated on maize roots in pot culture. The long-term fertilization caused decreases in AMF total spore numbers and variation in species diversity depended on sampling time. Effects of fertilization on spore number and also relative spore abundance varied with species and sampling time. Among the nine species sporulating under maize, only Acaulospora sp.1 showed no change (P > 0.003 after Bonferroni correction) in spore number with fertilization in the field; and was therefore classified as an AMF species insensitive to fertilization. Spores of Entrophospora schenckii, Glomus mosseae, Glomus sp.1, Glomus geosporum-like and Scutellospora fulgida, though they decreased in absolute numbers in response to fertilization, showed no change (P > 0.003 after Bonferroni correction) in relative abundance; these species were classified as AMF species slightly sensitive to fertilization. Three unidentified species of Glomus, though they decreased in absolute numbers in response to fertilization, showed decreases (P < 0.003 after Bonferroni correction) in relative abundance; these species were classified as AMF species highly sensitive to fertilization.