31P NMR for the study of P metabolism and translocation in arbuscular mycorrhizal fungi

³¹P nuclear magnetic resonance (NMR) spectroscopy was used to study phosphate (P) metabolism in mycorrhizal and nonmycorrhizal roots of cucumber (Cucumis sativus L) and in external mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith. The in vivo NMR method allows biological systems to be studied non-invasively and non-destructively. ³¹P NMR experiments provide information about cytoplasmic and vacuolar pH, based on the pH-dependent chemical shifts of the signals arising from the inorganic P (P_i) located in the two compartments. Similarly, the resonances arising from α, β and γ phosphates of nucleoside triphosphates (NTP) and nucleoside diphosphates (NDP) supply knowledge about the metabolic activity and the energetic status of the tissue. In addition, the kinetic behaviour of P uptake and storage can be determined with this method. The ³¹P NMR spectra of excised AM fungi and mycorrhizal roots contained signals from polyphosphate (PolyP), which were absent in the spectra of nonmycorrhizal roots. This demonstrated that the P_i taken up by the fungus was transformed into PolyP with a short chain length. The spectra of excised AM fungi revealed only a small signal from the cytoplasmic P_i, suggesting a low cytoplasmic volume in this AM fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Augmentation of AM fungi fails to ameliorate the adverse effects of temporal resource variation on a lettuce crop

While agricultural research has traditionally focused on average environmental conditions, environmental variability, independent of the mean, can also have biological consequences. Using lettuce (Lactuca sativa) as a model system, we tested two hypotheses: (1) increased temporal variability in water supply impacts plant growth, yield, photosynthesis, water relations and nutrition and (2) arbuscular mycorrhizal AM fungal associations benefit this agricultural crop, especially when plants experience temporal variability in water supply. The experiment used a randomized complete block design with two blocks and three variables (each with two levels): ± mycorrhizal inoculation, high or low variability in watering intervals, and high or low total watering volume. Temporal variability in water supply, at a time scale similar to what is common in agricultural practices, had negative effects on lettuce production. Inoculation treatments were successful in doubling the extent of AM fungal infection in lettuce roots. There were no main effects of mycorrhizal inoculation on any measured variable, but augmented mycorrhizal associations interacted with variability in water supply to increase root/shoot ratios and decrease tissue concentrations of N and P. Successful application of AM fungi to sustainable agriculture probably requires a general theoretical framework for predicting when effects on plants will be beneficial versus neutral or even detrimental.     

接种不同AM真菌对滇重楼幼苗功能基因表达的影响

为探究接种不同外源性从枝菌根(Arbuscular mycorrhizae,AM)真菌对滇重楼幼苗基因表达的影响,本研究以灭菌土壤为生长基质,将滇重楼(Paris polyphylla var. yunnanensis)新鲜种子与28种AM真菌于室温盆栽条件下共培养,采用实时荧光定量PCR的方法检测鲨烯环氧酶基因(Squalene epoxidase,SE)、共生受体类似激酶基因(Symbiosis-receptor-like kinase,SYMRK)、产生钙离子振荡的通道蛋白基因(Doesn’t making fections 1,DMI1)、钙/钙调依赖性蛋白激酶基因(Calcium/calmodulin-dependent protein kinase,CCa MK)4个功能基因在滇重楼幼苗的差异表达情况。结果表明:28株AM菌株可不同程度的影响SE、SYMRK、DMI1、CCa MK 4个功能基因的表达,其中薄壁两性囊霉(Ambisporaleptoticha,Ale)和崔氏原囊霉(Archaeospora trappei,Atr)可以显著增加4个功能基因的表达量。隐类球囊霉(Paraglo-mus occultum,Po)和透明盾巨孢囊霉(Scutellospora pellucida,Spe)可以增加SE、SYMRK的表达,细凹无梗囊霉(Acau-lospora scrobiculata,Asc),亮色盾巨孢囊霉(Racocetra fulgida,Rfu),哥伦比亚内养囊霉(Entrophospora colombiana,Ec),明球囊霉(Rhizophagus clarus,Rcl),根内球囊霉(Rhizophagus intraradices,Rin)可以增加DMI1的表达。综上所述,接种的28株AM真菌菌株中Ale和Atr能显著的提高4种功能基因的表达,结合前期与滇重楼无菌播种幼苗进行共生培养分析AM真菌对种子萌发及幼苗化学成分的影响,推测这两株菌可望能作为培育滇重楼菌根化苗的理想菌株,人工接种AM真菌可为保护和提高滇重楼开辟了一条新的途径。     

盐胁迫下接种AM真菌对玉米耐盐性的影响

以玉米品种陕单16号幼苗为材料,用盆栽法研究了不同含盐量(0、0.5、1.0、1.5和2.0 NaCl g/kg)土壤接种AM真菌(Glomus mosseae)对玉米幼苗生物量、盐害级数,以及叶片中电解质透出率、丙二醛、O·2-、H2O2含量和保护酶活性的影响.结果表明:在盐胁迫下,接种AM真菌增加了玉米植株生物量,降低了玉米的盐害级数;菌根植株叶片中过氧化氢酶的活性高于非菌根植株,而过氧化物酶、抗坏血酸氧化酶和多酚氧化酶的活性则为非菌根植株高于菌根植株;超氧化物歧化酶的活性在NaCl浓度为0、0.5和1.0 g/kg时为非菌根植株高于菌根植株,而在NaCl浓度为1.5和2.0 g/kg时则为菌根植株高于非菌根植株;菌根植株叶片中电解质透出率、丙二醛、O·2-和H2O2的含量低于非菌根植株.可见,AM真菌的侵染提高了玉米的耐盐性,缓解了由盐胁迫引起的过氧化胁迫对玉米植株的伤害,但这一缓解作用并不只是通过提高保护酶活性来实现的,可能还存在一些非酶促的调节机制.     

Evidence that differences in phosphate metabolism in mycorrhizas formed by species of Glomus and Gigaspora might be related to their life-cycle strategies

A glasshouse experiment was done to assess the development and phosphate metabolism of mycorrhizas formed by species of arbuscular mycorrhizal fungi (AMF) from two different genera, Gigaspora and Glomus on Desmodium ovalifolium plants at three concentrations of a phosphate source. The addition of phosphate (0–100 mg P kg⁻¹) had no effect on the alkaline phosphatase activity, stained histochemically, in the intra-radical mycelium of Gigaspora rosea (BEG111), but decreased that of Glomus manihotis (BEG112) over a 10-wk period. The alkaline phosphatase activity of the extra-radical mycelium was unaffected by increasing phosphate addition (0–100 mg P kg⁻¹) in both species of AMF over a 10-wk period. The extra-radical mycelium of Gi. rosea (BEG111) accumulated polyphosphate, determined by staining with 4',6-diamidino-2-phenylindole, whereas polyphosphate was not detected in the extra-radical mycelium of G. manihotis (BEG112). This work indicates differences in the mechanisms of phosphate metabolism in the mycelium of AMF from different genera on a tropical host. This might be determined by the life-cycle strategies of these fungi, in particular the formation of auxiliary cells in Gigaspora . The possibility of a negative-feedback mechanism between alkaline phosphatase and polyphosphate in the extra-radical mycelium of Gi. rosea (BEG111) and the role of polyphosphate in the symbiosis are discussed.     

Influence of arbuscular mycorrhizal fungi on soil structure and aggregate stability of a vertisol

The influence of arbuscular mycorrhizal (AM) fungi on aggregate stability of a semi-arid Indian vertisol was studied in a pot experiment in which Sorghum bicolor (L.) was grown as test plant for 10 weeks. Pasteurized soil inoculated with AM fungi was studied with pasteurized and unpasteurized soils as references. A part of the soil in each pot was placed in nylon mesh bags to separate effects of roots and hyphae. The sorghum plants were planted outside the mesh bags which permitted AM hyphae to enter while excluding roots. Aggregate stability of the soil was determined by wet-sieving and turbidimetric measurements. Development of the AM fungi was quantified as colonized root length and external hyphal length. Soil exposed to growth of roots and hyphae (outside mesh bags) showed aggregates with larger geometric mean diameter (GMD) in pasteurized soil inoculated with AM fungi than in pasteurized uninoculated soil. There was no significant difference in GMD of the inoculated, pasteurized soil and the unpasteurized soil. No significant effects of inoculation or plant growth were found in pasteurized soil exposed to hyphal growth only (inside the mesh bags). However, the unpasteurized soil had significantly higher GMD than the pasteurized soil, irrespective of plants and inoculum. Turbidimetric measurements of soil exposed to roots and hyphae (outside mesh bags) showed the highest aggregate stability for the inoculated pasteurized soil. These results demonstrate that AM fungi contribute to the stabilization of soil aggregates in a vertisol, and that the effect is significant after only one growing season. The effect was associated with both AM hyphae and the stimulation of root growth by AM fungi. The contribution from plant roots and AM hyphae to aggregate stability of different size fractions is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Phylogenetic trait conservatism and the evolution of functional trade-offs in arbuscular mycorrhizal fungi

The diversity of functional and life-history traits of organisms depends on adaptation as well as the legacy of shared ancestry. Although the evolution of traits in macro-organisms is well studied, relatively little is known about character evolution in micro-organisms. Here, we surveyed an ancient and ecologically important group of microbial plant symbionts, the arbuscular mycorrhizal (AM) fungi, and tested hypotheses about the evolution of functional and life-history traits. Variation in the extent of root and soil colonization by AM fungi is constrained to a few nodes basal to the most diverse groups within the phylum, with relatively little variation associated with recent divergences. We found no evidence for a trade-off in biomass allocated to root versus soil colonization in three published glasshouse experiments; rather these traits were positively correlated. Partial support was observed for correlated evolution between fungal colonization strategies and functional benefits of the symbiosis to host plants. The evolution of increased soil colonization was positively correlated with total plant biomass and shoot phosphorus content. Although the effect of AM fungi on infection by root pathogens was phylogenetically conserved, there was no evidence for correlated evolution between the extent of AM fungal root colonization and pathogen infection. Variability in colonization strategies evolved early in the diversification of AM fungi, and we propose that these strategies were influenced by functional interactions with host plants, resulting in an evolutionary stasis resembling trait conservatism.     

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。     

The effectivity of arbuscular mycorrhizal fungi from high input conventional and organic grassland and grass-arable rotations

The effectivity of arbuscular mycorrhizal spores in promoting growth of Allium ameloprasum L. cv. Musselburgh and Trifolium repens L. cv. Menna was tested for inocula from three soil series under long term organic or intensive, conventional grass and grass-arable rotations. For two soil series, Allium responses to inocula from soils recently converted to organic fanning were also assessed. Finally, Trifolium root fragments were used to inoculate Allium so as to evaluate responses to this inoculation procedure. Plants were sown into previously sterilised, matched soils from organic farms with no nutrient input. Mycorrhizal treatments generally increased growth for Allium. However, for Trifolium, infection decreased growth in the most fertile soil and gave an increase only in the least fertile. In the least fertile soil, inocula from organic farms were more effective than those from conventional farms. For Trifolium (all soils) and for Allium (least fertile soil), there was evidence of more efficient uptake of phosphorus in plants inoculated with spores from organic farms. The pattern of Allium response to inoculation with spores from conventional, conversion and organic sources was not consistent between soil type, but there was evidence of lower root infection for conversion compared with organic inocula and of a trend towards higher infectivity as the time period under organic management increased. Inoculating Allium with AMF root fragments produced a plant response similar to that obtained when spores were used, confirming that spore viability was not the sole factor influencing AMF effectivity in earlier experiments. Intensive farming practices may reduce the effectiveness of indigenous arbuscular mycorrhizal populations, particularly where fertiliser inputs are high and inherent fertility is low. This could have practical implications where high input farms are converted to organic management.     

Screening of arbuscular mycorrhizal fungi for the revegetation of eroded red soils in subtropical China

Some acidic red soils in hilly regions of subtropical China were degraded as a result of slope erosion following the removal of natural vegetation, primarily for fuel. Revegetation is important for the recovery of the degraded ecosystem, but plant growth is limited by the low fertility of eroded sites. One factor contributing to the low fertility may be low inoculum density of arbuscular mycorrhizal (AM) fungi. Compared to red soils under natural vegetation or in agricultural production, substrates on eroded sites had significantly lower AM fungal propagule densities. Thus, the management and/or application of AM fungi may increase plant growth and accelerate revegetation. Thirteen species of AM fungi were identified in red soils by spore morphology. Scutellospora heterogama, Glomus manihotis, Gigaspora margarita, Glomus aggregatum and Acaulospora laevis were among the most common according to spore numbers. Pot cultures were used to isolate and propagate 14 isolates of AM fungi indigenous to red soil. The effectiveness of each fungus in promotion of growth of mungbean was evaluated in red soil. For comparison, three isolates from northern China, known to be highly effective in neutral soils, and two isolates from Australia, known to be from acidic soil were used. Effectiveness was positively related to root infection (r ² = 0.601). For two of these isolates, Glomus caledonium (isolated from northern China) and Glomus manihotis (an isolate indigenous to red soil), the applied P concentration giving the highest infection and response to infection was approximately 17.5 mg P kg^–1 soil. In field experiments in which this concentration of P was applied, the five most effective isolates were tested on mungbean. The Glomus caledonium isolate from northern China was the most effective, followed by the indigenous Glomus manihotis isolate. The Glomus caledonium isolate was also shown to be effective on Lespedeza formosa, which is commonly used in revegetation efforts. We conclude that inoculation of plants with selected isolates of AM fungi may aid in revegetation efforts on eroded red soils in subtropical China.     

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.     

Parasitism of arbuscular mycorrhizal fungi: reviewing the evidence.

In order to understand the functioning of mycorrhizal fungi in ecosystems it is necessary to consider the full suite of possible biotic interactions in the soil. While a number of such interactions have recently been shown to be crucially important, parasitism is a highly neglected feature in the ecology of arbuscular mycorrhizal fungi (AMF). A number of studies have classified some interactions between populations of bacteria and fungi with AMF as parasitism, generating discussion about its consequences at both 'parasite' and host population levels. This paper reviews these various publications, and based on a set of criteria that are necessary to demonstrate parasitism, it was concluded that parasitism has not been conclusively shown to exist in AMF, even though some data are highly suggestive of such a relationship. The difficulties in gathering data supportive of parasitism were discussed, and hypotheses for defense were offered. This paper concludes by presenting potential consequences of AMF parasitism at the population/community levels and by discussing applied aspects.     

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.