Lupin pyranoisoflavones inhibiting hyphal development in arbuscular mycorrhizal fungi

White lupin (Lupinus albus L.), a non-host plant for arbuscular mycorrhizal (AM) fungi in the typically mycotrophic family Fabaceae, has been investigated for root metabolites that inhibit hyphal development in AM fungi. Four known pyranoisoflavones, licoisoflavone B (1), sophoraisoflavone A (2), alpinumisoflavone (3) and 3′-hydroxy-4′-O-methylalpinumisoflavone (4), together with three previously unknown pyranoisoflavones, lupindipyranoisoflavone A (5), 10′-hydroxylicoisoflavone B (6) and 10′-hydroxysophoraisoflavone A (7) were isolated from the root exudates of white lupin as an inhibitor of germ tube growth in the AM fungus Gigaspora margarita. Pyranoisoflavones 1, 2 and 3 strongly inhibited germ tube growth at 0.63, 1.25 and 0.63 μg/disc, respectively. The remaining compounds 4–7 were either moderate or weak inhibitors that inhibited germ tube growth at concentrations higher than 10 μg/disc. Licoisoflavone B (1) and sophoraisoflavone A (2) completely inhibited hyphal branching induced by a lupin strigolactone, orobanchyl acetate, in G. margarita at 0.16 and 0.63 μg/disc, respectively.     

Different nitrogen sources modulate activity but not expression of glutamine synthetase in arbuscular mycorrhizal fungi

Glutamine synthetase (GS) is a central enzyme of nitrogen metabolism that allows assimilation of nitrogen and biosynthesis of glutamine. We isolated the cDNA encoding GS from two arbuscular mycorrhizal fungi, Glomus mosseae (GmGln1) and Glomus intraradices (GiGln1). The deduced protein orthologues have a high degree of similarity (92%) with each other as well as with GSs from other fungi. GmGln1 was constitutively expressed during all stages of the fungal life cycle, i.e., spore germination, intraradical and extraradical mycelium. Feeding experiments with different nitrogen sources did not induce any change in the mRNA level of both genes independent of the symbiotic status of the fungus. However, GS activity of extraradical hypahe in G. intraradices was considerably modulated in response to different nitrogen sources. Thus, in a N re-supplementation time-course experiment, GS activity responded quickly to addition of nitrate, ammonium or glutamine. Re-feeding with ammonium produced a general increase in GS activity when compared with hyphae grown in nitrate as a sole N source.     

Quantification of arbuscular mycorrhizal fungi activity by the glomalin concentration on hyphal traps

 Strips of horticultural film (16–32 cm²) were used to trap extraradical hyphae emanating from roots of sudangrass [Sorghum sudanense (Piper) Staph] enclosed in 40-μm mesh bags and colonized by Gigaspora rosea FL 224-1, Glomus intraradices EY 113/114, or Glomus caledonium UK 301-1. Strips of film were placed at opposite sides of 17–21 replicate sand culture pots for each isolate and were removed after 12–14 weeks of plant growth. To extract glomalin, a strip was cut into small pieces and submerged in 2 ml of 20 mM citrate, pH 7.0 and then autoclaved for 60 min. A quantitative enzyme-linked immunosorbent assay (ELISA) detected 0.005–0.04 μg glomalin in the volume of extract tested. The Bradford protein assay detected 1.25–5 μg of protein in the volume of extract tested. Both assays gave results ranging from 5–40 μg glomalin/cm² of film. Protein assay values were correlated with ELISA values (r=0.6091, P≤0.001, n=118). Analysis of variance indicated that isolates differed in Bradford protein values (P=0.001), but not ELISA values (P=0.154). Spatial variability of glomalin deposition ca. 7 cm from roots on opposite sides of pots was indicated by significant paired T tests (P<0.05) for protein values for each of the three isolates and ELISA for two isolates. These results indicate that hyphal traps, Bradford protein assay and ELISA are useful to assess hyphal activity over a growing season. Accepted: 11 October 1998     

Evolutionary maintenance of genomic diversity within arbuscular mycorrhizal fungi

Most organisms are built from a single genome. In striking contrast, arbuscular mycorrhizal fungi appear to maintain genomic variation within an individual fungal network. Arbuscular mycorrhizal fungi dwell in the soil, form mutualistic networks with plants, and bear multiple, potentially genetically diverse nuclei within a network. We explore, from a theoretical perspective, why such genetic diversity might be maintained within individuals. We consider selection acting within and between individual fungal networks. We show that genetic diversity could provide a benefit at the level of the individual, by improving growth in variable environments, and that this can stabilize genetic diversity even in the presence of nuclear conflict. Arbuscular mycorrhizal fungi complicate our understanding of organismality, but our findings offer a way of understanding such biological anomalies.     

3种丛枝菌根真菌与3种寄主植物的共生关系

【目的】利用丛枝菌根(Arbuscular mycorrhiza,AM)真菌对寄主植物的偏好性和不同寄主植物的功能互补作用,建立AM真菌的高效繁殖体系。【方法】以玉米(Zea may L.)、高粱[Sorghum bicolor(L.)Moench]和白车轴草(Trifolium repens L.)为寄主植物,采用寄主植物单作和间作的盆栽培养法,研究不同栽培模式对光壁无梗囊霉(Acaulospora laevis)、单孢球囊霉(Glomus monosporum)和根内球囊霉(G.intraradices)3种AM真菌繁殖能力的影响,通过地上部分生物量的分配分析,探索C3和C4植物对AM真菌共生关系的"功能互补"效应及机制。【结果】间作模式下,寄主植物地上部分总生物量和3种AM真菌的孢子密度均显著高于单作(P0.05);单作和间作栽培模式下,3种AM真菌对玉米地上部分生物量响应无显著差异(P0.05),但单孢球囊霉和根内球囊霉对高粱地上部分生物量产生显著影响(P0.05);两种间作栽培模式下,根内球囊霉对白车轴草地上部分生物量也产生了显著影响(P0.05)。【结论】3种AM真菌对3种寄主植物的共生偏好性不同,且C3和C4植物对AM真菌共生关系存在一定的"功能互补"效应,利用AM真菌的寄主植物偏好性和不同植物间的功能互补关系,增加AM真菌的孢子产量,有利于AM真菌高效繁殖体系的建立。     

Traits related to differences in function among three arbuscular mycorrhizal fungi

Diversity in phosphorus (P) acquisition strategies was assessed among three species of arbuscular mycorrhizal fungi (AMF) isolated from a single field in Switzerland. Medicago truncatula was used as a test plant. It was grown in a compartmented system with root and root-free zones separated by a fine mesh. Dual radioisotope labeling (³²P and ³³P) was employed in the root-free zone as follows: ³³P labeling determined hyphal P uptake from different distances from roots over the entire growth period, whereas ³²P labeling investigated hyphal P uptake close to the roots over the 48 hours immediately prior to harvest. Glomus intraradices, Glomus claroideum and Gigaspora margarita were able to take up and deliver P to the plants from maximal distances of 10, 6 and 1 cm from the roots, respectively. Glomus intraradices most rapidly colonized the available substrate and transported significant amounts of P towards the roots, but provided the same growth benefit as compared to Glomus claroideum, whose mycelium was less efficient in soil exploration and in P uptake and delivery to the roots. These differences are probably related to different carbon requirements by these different Glomus species. Gigaspora margarita provided low P benefits to the plants and formed dense mycelium networks close to the roots where P was probably transiently immobilized. Numerical modeling identified possible mechanisms underlying the observed differences in patterns of mycelium growth. High external hyphal production at the root-fungus interface together with rapid hyphal turnover were pointed out as important factors governing hyphal network development by Gigaspora, whereas nonlinearity in apical branching and hyphal anastomoses were key features for G. intraradices and G. claroideum, respectively.     

Influence of two legume species on hyphal production and activity of two arbuscular mycorrhizal fungi

 Two arbuscular mycorrhizal (AM) fungi (Glomus mosseae and G. intraradices) were compared for abundance of intraradical and soil-borne hyphae in association with Astragalus sinicum, a small-seeded, and Glycine max, a large-seeded legume. A. sinicum was more responsive than G. max to mycorrhizal formation, especially at early growth stages. Biomass allocation was greater in roots than shoots for mycorrhizal A. sinicum, while the opposite was true for G. max. Hyphal development in root and soil compartments was estimated by trypan blue staining and after staining for succinate dehydrogenase (SDH) or alkaline phosphatase (ALP) activity. Total fungal abundance increased steadily in roots and soil with time to a maximum 8 weeks after planting. SDH- and ALP-active AM hyphae increased in roots during plant growth but decreased in soil at later harvests. Mycorrhizal root mass in A. sinicum and G. max increased about 14-fold and 2.5-fold, respectively, but total length of soil hyphae produced per plant differed little, so that the pattern of AM soil to root abundance of the two fungi varied considerably with the host plant. Accepted: 23 July 1997     

Specific PCR primers to identify arbuscular mycorrhizal fungi within colonized roots

 A set of PCR primers targeted at five major phylogenetic subgroups of arbuscular mycorrhizal fungi (Glomales) was designed to facilitate specific amplification of internal transcribed spacers and 18 S rRNA gene fragments from colonized roots in the absence of spores. The subgroups include the recently discovered deeply divergent lineages of Glomales, which could not be detected by previously reported PCR primers, and the former genus Sclerocystis. Restriction fragment length polymorphism patterns presented allow identification of presently known members of these groups. The resulting PCR products can be used to identify the fungal symbionts at the genus or species level by restriction digests or DNA sequencing. A novel DNA extraction method allows visual control of the analyzed roots by staining procedures after analysis by PCR. Accepted: 2 April 2000     

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.     

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.     

丛枝菌根真菌生物地理学研究进展

丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)普遍存在于陆地生态系统中,能与绝大多数高等植物形成菌根共生体系。AMF能够促进植物对矿质养分的吸收,增强植物的抗逆能力,在维持生态系统稳定性和生产力中发挥着重要作用。AMF生物地理学主要研究AMF的生物地理分布格局和群落构建机制,对于理解AMF在不同生态系统中的重要性至关重要。总结了AMF生物地理学最新研究进展及研究方法,提出了AMF生物地理学研究理论框架。认为AMF在自然界中并非简单随机分布,宿主植物、地理因子、气候因子和土壤因子共同决定AMF的群落结构,不同尺度下的AMF群落构建符合生态位-中性连续统假说,但在不同尺度下这些驱动因子的相对重要性不同。在全球尺度和区域尺度下,AMF的地理分布格局主要受地理距离和气候因子的影响,中性理论的作用大于生态位理论。随着空间尺度的缩减,宿主植物和环境因子对AMF群落的影响胜过地理距离和扩散限制的作用,生态位理论取代中性理论在AMF群落构建中的主导地位。此外,很多研究发现,同一生境中AMF的群落构建机制并非一成不变,会随环境的变化而发生改变。在未来的研究中,应在野外调查和公共数据库的基础上加强整合分析和数据挖掘工作,从而进一步丰富和完善AMF生物地理学理论。     

Response of 11 eucalyptus species to inoculation with three arbuscular mycorrhizal fungi

 Numerous publications have reported growth stimulation of Eucalyptus following ectomycorrhizal inoculation in nursery or field conditions. Although Eucalyptus species can also form arbuscular mycorrhiza, their dependency on this type of mycorrhiza is still debatable. This paper presents information on the effect of inoculation of arbuscular mycorrhizal fungi on eucalypt growth. Twenty weeks after mycorrhizal inoculation, Eucalyptus seedlings' stem dry weight could be increased up to 49% compared to non-inoculated control plants. Intensity of root colonization by a given fungus depended on the host species, but it was not related to a plant growth response. Leaf phosphorus concentration of non-inoculated Eucalyptus seedlings varied greatly between species. Increases in leaf phosphorus concentration following mycorrhizal infection were not necessarily associated with plant growth stimulation. The most mycorrhiza-dependent Eucalyptus species tended to be those having the highest leaf phosphorus concentration in the absence of a fungal symbiont. These mycorrhiza-dependent Eucalyptus species seem to have greater phosphorus requirements and consequently to rely more on the symbiotic association. Accepted: 1 September 1995     

Competition and substrate colonization strategies of three polyxenically grown arbuscular mycorrhizal fungi

Intra- and extraradical colonization competition and hyphal interactions among arbuscular mycorrhizal fungi (AMF) Glomus intraradices, Glomus proliferum and Gigaspora margarita were investigated in two in vitro experimental systems. AMF were polyxenically cultured with a Ri T-DNA transformed carrot root organ culture (ROC) in either big Petri plates containing three culture compartments and a common hyphal compartment (i.e. an independent host root for each AMF) or two by two in the culture compartment of regular bicompartmented Petri dishes (i.e. a common host root and a common hyphal compartment). Maps of the extraradical mycelial development of the three AMF were obtained. Two distinct substrate colonization strategies (Glomus-type and Gigaspora-type) were identified, reflecting intrinsic differences among AMF genera/families. Our data reveal a general lack of antagonism between the isolates when extraradical hyphae explore and exploit the substrate outside the root influence zone; however certain growth restrictions were imposed by Gi. margarita extraradical mycelium when developing near the host root and by G. proliferum intraradical hyphae. This work highlights once more the appropriateness of AM in vitro culture systems to perform in vivo studies on the biology of this symbiosis and opens new avenues to the formulation of in vitro AMF inoculants.     

Root and hyphal interactions influence N transfer by arbuscular mycorrhizal fungi in soybean/maize intercropping systems

In maize-soybean intercropping systems, the transfer of N from soybean to maize gives the intercropping system the advantage of improved N utilization and higher yields. Mycorrhiza acts as an important pathway for N transfer, providing a constant supply of N to sustain the growth and development of maize in its early stages. However, it is not clear how arbuscular mycorrhizal fungi (AMF) drive the transfer of N from soybean to maize in the intercropping system. Therefore, we quantified the amount of N transferred from soybean to maize under low and high N levels in the intercropping system, and the abundance and diversity of AMF involved in N transfer (¹⁵N-AMF) under different conditions by ¹⁵N leaf marker and DNA-SIP technology. We found that the interaction between roots and reducing the application of N fertilizer increased the amount of N transfer from soybean to maize. Compared with plastic plate separation (PS), no separation (NS) and mesh separation (MS) significantly increased the N fixation rate (from 14.33% to 39.09%), and the amount of N transfer under NS was 1.95–3.48 times that under MS. N transfer from soybean to maize ranged from 9.7 to 43.42 mg per pot in the no N treatment, while the addition of N fertilizer reduced N transfer by 14.12–66.28%. This is due to root interaction and reduced N fertilization increased the abundance and diversity of the ¹⁵N-AMF community, thereby promoting AMF colonization of maize and soybean roots. AMF colonization in soybean and maize roots under NS treatment was 6.47–17.24% higher than under MS treatment in all three levels of N addition. The increase of mycorrhiza in root system increased the N transfer from soybean to maize significantly. These results suggest that reduced N fertilizer in maize-soybean intercropping systems can increase N transfer by the mycorrhizal pathway, meeting maize N requirements and reducing chemical N fertilizer, which is important for sustainable agricultural development.