Effect of peat on mycorrhizal colonization and effectiveness of the arbuscular mycorrhizal fungus Gigaspora margarita

Abstract

The influence of the addition of Chinese peat and Canadian peat on arbuscular mycorrhizal colonization, mycorrhizal effectiveness and host-plant growth was investigated in a pot experiment. Chinese peat or Canadian peat was mixed with Masa soil (weathered granite soil) at different levels (0, 25, 50, 100, 150 or 200 g kg^?1) into which an arbuscular mycorrhizal fungus (AMF) Gigaspora margarita Becker & Hall was inoculated, and seedlings of Miscanthus sinensis Anderess were planted. There was a significant increase in plant growth with increasing amounts of Chinese peat. The growth-promoting effect of the AMF on the host was enhanced when the addition of Chinese peat was increased from 25 to 100 g kg^?1. Root colonization and the number of spores proliferating increased with increases at low levels of Chinese peat (from 25 to 100 g kg^?1), and decreased gradually with higher Chinese peat increments. Although plant growth and root colonization with the addition of Canadian peat increased slightly, Canadian peat suppressed mycorrhizal effectiveness. In contrast to Canadian peat, the addition of Chinese peat improved considerably the physical and chemical properties of the soil, which might result in the promotion of AM formation and mycorrhizal effectiveness.     

Intraradical hyphae phosphatase of the arbuscular mycorrhizal fungus, Gigaspora margarita

An alkaline phosphatase in the intraradical hyphae of arbuscular mycorrhizal fungi was found to be closely related to an improvement of plant growth. To detect the phosphatase activity in a crude extract of mycorrhizal roots, phosphatase isozymes in mycorrhizal and non-mycorrhizal onion roots were compared with those in Gigaspora margarita by electrophoresis. A mycorrhiza-specific band was found when the phosphatase was stained under alkaline conditions. To clarify the origin of this phosphatase, the phosphatase extracted from intraradical hyphae was also compared with the phosphatase from mycorrhizal roots by electrophoresis. The intraradical hyphae was isolated from mycorrhizal roots by enzyme digestion followed by Percoll gradient centrifugation. The soluble protein was extracted from the hyphae by ultra-sonication after treatment with chitinase. A phosphatase in the hyphal soluble protein showed a similar, but slightly higher, relative mobility on the gel, compared with the mycorrhiza-specific phosphatase from roots. By adding the hyphal extract to the root extract, the relative mobility of the mycorrhiza-specific phosphatase was slightly changed and became identical to that of the phosphatase in the hyphae. This indicated that the specific band of phosphatase found in the crude extract from mycorrhizal roots was of intraradical hyphal origin. Received: 16 April 1997     

Estimation of the root colonization of soybean by an arbuscular mycorrhizal fungus,Gigaspora rosea,based on specific fatty acid profiles

Root colonization by arbuscular mycorrhizal (AM) fungi has traditionally been analyzed by microscopy. However, this method is time consuming and it is often difficult to distinguish between AM and non-AM fungi. In this study, we analyzed the fatty acid profiles in soybean roots colonized by AM fungi to determine if specific fatty acids derived from AM fungi can be used as markers for the intensity of the AM fungal colonization. The wild-type Enrei and hypernodulating Kanto100 soybean cultivars were inoculated with an AM fungus (Gigaspora rosea) alone or with Bradyrhizobium diazoefficiens, which nodulates soybean roots. Fatty acids 20:1ω9, 20:4ω6, and 20:5ω3 were specifically detected in the lateral roots of AM fungus-inoculated and dual-inoculated soybean plants. In the second lateral roots, the percentage of AM-specific fatty acids (i.e., 20:1ω9, 20:4ω6, and 20:5ω3) derived from AM fungi was closely correlated with the intensity of the AM fungal colonization. We propose that the AM-specific fatty acids represent useful markers for estimating the degree of AM fungal colonization. The percentage of AM-specific fatty acids was more than twofold higher in the second lateral roots than in the first lateral roots. Thus, the degree of AM fungal colonization is probably twofold higher in the second lateral roots than in the first lateral roots.     

三种土壤上六种丛枝菌根真菌生长特征和接种效应

以分离于华北、华中和华南3个生态区及法国引进的丛枝菌根真菌为试验菌株,采用三室根箱培养的方法,研究了它们在华北、华中和华南3种典型土壤褐土、棕壤和红壤上的菌根形成、接种效应、磷吸收贡献和根外菌物量情况。结果表明,6种菌株在上述指标上存在显著的种间或生态型差异,土壤与菌株间存在显著的交互作用。6种菌株在3种土壤上都能与玉米形成菌根,在褐土和棕壤上大多数菌株在分离地所在地区土壤类型上的菌根侵染率较高,说明其对该土壤条件的适应性较强;在红壤强酸性土壤条件抑制了菌根真菌的侵染。菌株BEG168、BEG167、BEG151、BEG221和BEG141在褐土上,BEG151和BEG221在棕壤上,BEG168和BEG150在红壤上显著提高了玉米的生物量。在褐土和棕壤上,除BEG150外,BEG168、BEG167、BEG151、BEG221和BEG141能显著促进宿主吸磷;而在红壤上,BEG168和BEG141显著促进了宿主吸磷。若以真菌的根外菌物量作为衡量AM真菌菌株土壤生态适应性的指标,BEG141和BEG167是生态适应性强的菌株,为广幅生态型菌株;菌株BEG168和BEG151次之,前者在棕壤上适应性高,后者在红壤上适应性高。BEG150和BEG221生态适应性较窄,仅适应红壤或褐土,为窄幅生态型菌株。Glomus.etunicatum的两个生态型BEG168和BEG221在土壤生态适应性上差异很大,前者在两种土壤上收集到菌物量,而后者只在褐土上收集到菌物量。土壤条件可以决定丛枝菌根真菌的生长状况和功能。     

Application of a Molecular Method for the Identification of a Gigaspora margarita Isolate Released in a Field

A molecular technique for the identification of the Gigaspora margarita isolate CK based on the detection of a DNA sequence of 235 bp as its diagnostic marker was evaluated to investigate the survival and establishment of introduced arbuscular mycorrizal fungi (AMF) in a field ecosystem. In March 2001, roots and rhizosphere soil of Eragrostis curvula and Miscanthus sinensis were collected from the Mizunashi River at Mt. Fugendake (Nagasaki Prefecture, Japan), where plant seeds and AMF including G. margarita CK had been introduced for reforestation after the occurrence of repeated pyroclastic flows. We detected the marker sequence from DNA preparations of E. curvula roots and Gigaspora spores in the rhizosphere. This clearly showed that the isolate occurred at both hyphal and sporal stages. It was shown that the isolate survived and developed a life cycle in the revegetation area for 4 years. It was confirmed that the method was effective for tracing the isolate in samples collected from field ecosystems.     

Response of a wild-type and modern cowpea cultivars to arbuscular mycorrhizal inoculation in sterilized and non-sterilized soil

Cowpea is an important crop that serves as a legume and vegetable source to many smallholder farmers in sub-Saharan Africa. Soil fertility is a significant limitation to its production thus; inoculation with beneficial soil biota such as arbuscular mycorrhizal fungi (AMF) could improve its performance. However, plant–AMF interaction could vary based on crop cultivar hence affecting overall crop production. The present study aimed at determining the effect of AMF inoculation and soil sterilization on root colonization and growth of a wild-type and three modern cowpea cultivars grown by smallholder farmers in Kenya. Potted cowpea plants were inoculated with a commercial AMF inoculum comprising of Rhizophagus irregularis, Funneliformis mosseae, Glomus aggregatum and Glomus etunicatum and maintained in a greenhouse for 40 days. After harvesting, mycorrhizal colonization, nodule number and dry weight, root and shoot dry weights, nitrogen (N,) phosphorus (P) and potassium (K) content were determined. Interestingly, the modern cultivars showed significantly (p < 0.001) higher root colonization, nodulation, shoot P and N compared to the wild-type cultivar. Moreover, a strong positive correlation between AMF root colonization and shoot P (r² = 0.73, 0.90, p < 0.001), AMF root colonization and shoot N (r² = 0.78; 0.89, p < 0.001) was observed in both sterilized and non-sterilized soil, respectively. Soil sterilization affected root colonization and growth parameters with plants grown in non-sterilized soil performing better than those grown in sterilized soil. This study provides major evidence that modern cowpea cultivars are still responsive to mycorrhizal inoculation suggesting that modern breeding programs are not deleterious AMF symbiosis.     

大豆丛枝菌根共生结构和多聚磷累积双定位方法

【目的】多聚磷是丛枝菌根内磷的主要贮存形式,定性、定量观察多聚磷对于解析菌根中磷代谢具有重要意义。随着植物体内越来越多的参与菌根真菌与寄主植物之间营养交换过程的基因被鉴定,迫切需要进一步提高根内菌根共生结构和多聚磷累积的染色和定位分析技术。【方法】本研究利用丛枝菌根真菌Glomus mosseae侵染的大豆植株,采集新鲜根样制片,一部分薄根片利用低浓度荧光染料麦胚凝集素,室温染色30 min,在波长488 nm的蓝光激发下使用荧光显微镜观察拍照;另一部分薄根片利用荧光染料4’,6-二脒基-2-苯基吲哚二盐酸盐(DAPI)进行染色,在波长405 nm紫外光激发下观察并拍照;进一步取新鲜制备的薄根片,先后用以上两种荧光染料进行染色,分别在波长405 nm和488 nm的激发光下观察并拍照,完成了菌根共生结构和多聚磷的共定位。【结果】1)使用荧光染料麦胚凝集素,大豆丛枝菌根真菌侵染结构的荧光标记活性染色法,可以清晰地检测到大豆丛枝菌根中所有的共生结构,包括丛枝,泡囊和根内菌丝等。2)在丛枝菌根真菌侵染的根中,各种共生结构都呈现出黄色荧光,为DAPI与多聚磷结合在紫外光激发下的呈色。根段中部分细胞内的蓝白色斑点为DAPI与细胞核中DNA结合的显色结果。在含有成熟丛枝结构的细胞中,也可观察到大部分丛枝呈蓝白色,主要是丛枝膜质结构的呈色。因此,利用荧光染料4’,6-二脒基-2-苯基吲哚二盐酸盐染色法定位多聚磷,能很好地区分多聚磷酸盐、DNA和膜质。3)在以上研究的基础上,通过荧光光路的切换,可以同时观察到菌根共生结构和多聚磷的共定位。处于发育阶段的整个丛枝中多聚磷累积的亮黄色清晰可见。在成熟的丛枝中,由于膜质结构发达,对累积在丛枝结构中的多聚磷的染色观察产生了一定影响,导致仅仅局部的多聚磷累积清晰可见。【结论】本研究建立的大豆菌根共生结构与多聚磷累积的双定位分析系统,能够直观观察植物与丛枝菌根真菌的养分交换,清晰地对丛枝菌根共生结构中多聚磷的累积进行定位分析,可作为从组织和细胞水平研究菌根共生体的重要技术手段。     

Effect of Concentration of Phosphate on Spore Germination and Hyphal Growth of Arbuscular Mycorrhizal Fungus,Gigaspora margarita Becker & Hall

Extract

Arbuscular mycorrhizae are symbiotic associations between plant roots and soil fungi that belong to the order Glomales. They enhance the uptake of phosphate and improve plant growth (Mos.se 1973). Those functions have not fully been utilized in Japan, where a large amount of phosphate fertilizer is usually applied, because arbuscular mycorrhizal colonisation in plant is inhibited by a large application of phosphate (Mosse 1973; Abbott et al. 1984; Tawaraya et al. 1985; Schubert and Hayman 1986; Thomson et al. 1986; Amijee et al. 1989). It is important to analyse the conditions for symbiotic association between fungi and host plant to use arbuscular mycorrhiza.     

接种丛枝菌根真菌对脱毒马铃薯微型薯生长及产量的影响

在大田条件下采用混合菌种(Glomus mosseas+Glomus intraradices)作为接种剂,研究了接种丛枝菌根真菌对脱毒马铃薯微型薯菌根侵染、磷吸收和产量的影响。结果表明,接种菌根真菌,马铃薯菌根侵染率增加73.3%,植株吸磷量增加15.4%,块茎产量增加8.0%。上述结果证明,在大田条件下,接种菌根真菌能侵染马铃薯根部,促进植株对磷的吸收,从而增加产量。     

钼污染对丛枝菌根和球囊霉素的影响

丛枝菌根作为地球上最广泛的共生体,在各种逆境环境中发挥着重要作用.基于不同钼污染程度的钼矿区,分析了钼污染对丛枝菌根真菌侵染、繁殖及其分泌球囊霉素能力的影响.结果 表明,在4个不同钼污染级别的土壤中,丛枝菌根真菌与植物共生关系的形成未受到钼污染的影响,平均菌根侵染频度为50.64％.洛阳钼矿区土壤的平均孢子密度为19....     

Root exudate-stimulated RNA accumulation in the arbuscular mycorrhizal fungus Gigaspora rosea

Plant root exudates induce the transition from asymbiosis to presymbiosis in arbuscular mycorrhizal fungi. In order to get an insight into this developmental switch, two libraries of Gigaspora rosea and one library of Gigaspora gigantea were screened for fragments of genes that show enhanced RNA accumulation 1 h after addition of a semi-purified exudate fraction of carrot roots. Among 150 clones, 40 seemed to contain inserts of root exudate-induced genes. One of the genes, GrosRbp1, putatively encoding an RNA binding protein involved in developmental control showed RNA accumulation which correlates to the extent of stimulation of presymbiotic hyphal branching.     

丛枝菌根真菌(Glomus caledonium)对铜污染土壤生物修复机理初探

利用盆栽试验,研究了丛枝菌根真菌(Glomus.caledonium)在不同程度铜污染土壤上对玉米苗期生长的影响。结果表明,即使在土壤施铜量达150mg/kg时,菌根真菌对玉米仍有近55%的侵染率;接种菌根真菌,能显著促进玉米根系的生长。菌根玉米的根系生物量和根系长度,平均较未接种处理分别提高108.4%和58.8%;接种处理的植株地上部生物量达到每盆(3株)10.58g,显著高于不施铜的非菌根玉米。这些结果表明,丛枝菌根真菌对铜污染具有较好的抗性;并且由于菌根的形成,使宿主植物明显地改善了对磷的吸收和运输,并能通过抑制土壤酸化、降低土壤可溶态铜的浓度等机制,增强宿主植物对铜污染的抗(耐)性。在150mg/kg施铜水平时,与非菌根玉米相比,菌根玉米地上部和根系铜浓度分别降低24.3%和24.1%,吸铜量分别提高了28.2%和60.0%,表明菌根植物对铜污染土壤具有一定的生物修复作用。     

Assessing the progress of colonization by arbuscular mycorrhiza of four plant species under different temperature regimes

To capitalize on the benefits offered by arbuscular mycorrhizal fungi (AMF) in agricultural systems, the effect of low soil temperature has to be taken into consideration over large areas of the planet. However, the effect of suboptimal root‐zone temperatures on AMF colonization is poorly understood. It has been suggested that it depends on the host plant species. We hypothesized that this interdependence is a function of the parameter used to assess the presence of AMF in the roots. In a pot experiment with non‐sterilized soil, we investigated the influence of three soil temperatures (10, 15, and 20°C) on the progress of root colonization of four host plant species (Ornithopus compressus, Lolium rigidum, Triticum aestivum, and Zea mays) by indigenous AMF. Plant root density, arbuscular colonization rate (AC) and colonized root density (CRD) were assessed 14, 28, and 42 d after sowing. Based on CRD, the effect of temperature on the progress of root colonization by AMF was independent of the host plant species. The apparent influence of the host plant species was only due to the species‐specific effect of soil temperature on root growth and therefore on AC. The host plant species only determined the minimum temperature for the AM colonization initiation, possibly due to species‐specific response of root growth and exudation to cool temperatures.     

供硫和丛枝菌根真菌对洋葱生长和品质的影响

以珍珠岩为植物的生长基质盆栽试验,分别供给0.1、1.75和4 mmol/L三个不同硫水平的Long Ashton营养液,研究接种丛枝菌根真菌Glomus versiform对洋葱（Allium cepa L.）生长和品质的影响。结果表明,接种丛枝菌根真菌显著的改善了宿主植物的磷营养水平,促进了洋葱的生长;而硫处理对洋葱生长的影响差异不显著,但随着供硫水平的提高植株地上部全硫含量和有机硫含量显著增加。接种菌根真菌对洋葱硫营养的影响受外界供硫水平的影响,在供硫0.1 mmol/L时降低了洋葱植株的硫含量;而在供硫1.75和4 mmol/L时显著改善了洋葱的硫营养状况,宿主植物的酶解丙酮酸（enzyme produced pyruvic acid, EPY）的含量也显著增加。说明丛枝菌根真菌能够帮助宿主植物吸收外界环境中硫营养成分,改善洋葱的硫营养状况及品质。     

低磷环境下接种丛枝菌根真菌促进紫花苜蓿生长和磷素吸收的机理

[目的]磷极易被土壤吸附和固定,导致土壤中磷有效性较低.研究接种丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)和低磷处理两者交互对紫花苜蓿生长和磷吸收的影响,为提高碱性土壤中磷肥利用率提供理论依据.[方法]以黄绵土和紫花苜蓿(Medicago sativa)为试验材料进行盆栽试验.在施...     

Technique for visual demonstration of germinating arbuscular mycorrhizal spores and their multiplication in pots

We describe a simple technique for the germination of arbuscular mycorrhizal (AM)–fungal spores and their multiplication in pots. Glomus fasciculatum, G. mosseae, and Gigaspora margarita were used. A single wheat seedling was tied to a glass slide, previously covered with filter paper with the help of thread. One single surface‐sterilized AM‐fungal spore was placed on the middle portion of the root of the wheat seedling using a sterilized syringe. The slide was placed vertically in a 100 mL glass beaker filled with 25 mL of root exudates–water (1:4, v/v) solution, which was collected by growing twenty wheat seedlings in a 150 mL beaker filled with 100 mL sterilized distilled water for 7 d. The slide was observed daily using a compound microscope to follow the time course of germination. In this technique, the spore is directly in contact with the host root, and a visualization of spore germination, hyphal development, and appressorium formation is possible without disrupting fungal growth or the establishment of the symbiosis. The method allows to document the germination events and to assess hyphal‐elongation rates by photographing the same spore on consecutive days. The inoculated seedling was used to initiate single‐spore multiplication in a sterilized (autoclave on 3 alternate days at 120°C for 120 min at 1.05 kg cm^–2 pressure) potted sandy soil (150 mL volume) into which the slide with the inoculated seedling was inserted carefully through a previously made slit. The wheat seedlings in all pots (4 treatments and 15 replications) became colonized by mycorrhiza, confirming that the establishment of the AM‐fungal symbiosis is highly reproducible. Our technique permits the relatively undisturbed growth of the symbiotic partners, the visualization of germinating AM‐fungal spores, and their multiplication in pots. This simple and low‐cost method facilitates the production of pure lines of AM fungi from single spores, allowing for the study of intraspecific variation and potentiality for cytological, biochemical, physiological, and taxonomical studies.     

接种丛枝菌根真菌对小麦根内氮转运蛋白基因表达的影响

通过盆栽试验,研究了在低氮(不施氮)和高氮(施氮0.2 g·kg~(-1))水平下接种不同种类丛枝菌根(AM)真菌[Funneliformis mosseae(BGC-NM03D)、Claroideoglomus etunicatum(BGC-NM01B)和Rhizophagus intraradices(BJ09)]对小麦生长、氮吸收及根内4个硝态氮转运蛋白(NRT)基因、1个辅助蛋白(NAR)基因和2个铵态氮转运蛋白(AMT)基因表达的影响。结果表明,3种AM真菌均能够侵染小麦根系,以R.intraradices菌根的侵染率最高;接种R.intraradices或C.etunicatum能够显著提高小麦的生物量或地上部氮吸收量;无论是高氮还是低氮处理,接种AM真菌后均显著下调了小麦根内NRT、NAR和AMT基因的表达水平,且不同AM真菌调控小麦根内氮转运蛋白基因表达的能力具有明显差异。