Bacteria associated with spores of the arbuscular mycorrhizal fungi Glomus geosporum and Glomus constrictum

Spores of the arbuscular mycorrhizal fungi (AMF) Glomus geosporum and Glomus constrictum were harvested from single-spore-derived pot cultures with either Plantago lanceolata or Hieracium pilosella as host plants. PCR-denaturing gradient gel electrophoresis analysis revealed that the bacterial communities associated with the spores depended more on AMF than host plant identity. The composition of the bacterial populations linked to the spores could be predominantly influenced by a specific spore wall composition or AMF exudate rather than by specific root exudates. The majority of the bacterial sequences that were common to both G. geosporum and G. constrictum spores were affiliated with taxonomic groups known to degrade biopolymers (Cellvibrio, Chondromyces, Flexibacter, Lysobacter, and Pseudomonas). Scanning electron microscopy of G. geosporum spores revealed that these bacteria are possibly feeding on the outer hyaline spore layer. The process of maturation and eventual germination of AMF spores might then benefit from the activity of the surface microorganisms degrading the outer hyaline wall layer.     

Bacteria Associated with Spores of the Arbuscular Mycorrhizal Fungi Glomus geosporum and Glomus constrictum

Spores of the arbuscular mycorrhizal fungi (AMF) Glomus geosporum and Glomus constrictum were harvested from single-spore-derived pot cultures with either Plantago lanceolata or Hieracium pilosella as host plants. PCR-denaturing gradient gel electrophoresis analysis revealed that the bacterial communities associated with the spores depended more on AMF than host plant identity. The composition of the bacterial populations linked to the spores could be predominantly influenced by a specific spore wall composition or AMF exudate rather than by specific root exudates. The majority of the bacterial sequences that were common to both G. geosporum and G. constrictum spores were affiliated with taxonomic groups known to degrade biopolymers (Cellvibrio, Chondromyces, Flexibacter, Lysobacter, and Pseudomonas). Scanning electron microscopy of G. geosporum spores revealed that these bacteria are possibly feeding on the outer hyaline spore layer. The process of maturation and eventual germination of AMF spores might then benefit from the activity of the surface microorganisms degrading the outer hyaline wall layer.     

金沙江干热河谷(元谋段)丛枝菌根真菌多样性研究

从金沙江干热河谷(元谋段)75种植物的根际土壤中分离鉴定了44种丛枝菌根真菌, 分属无梗囊霉属Acaulospora、古孢霉属Archaeospora、内养囊霉属Entrophospora、巨孢囊霉属Gigaspora、球囊霉属Glomus和盾巨孢囊霉属Scutellospora, 其中,球囊霉属和无梗囊霉属为金沙江干热河谷中丛枝菌根真菌的优势属。齿状无梗囊霉A. denticulata、刺状无梗囊霉A. spinosa、瘤状无梗囊A. tuberculata,近明球囊霉Glomus claroideum、明球囊霉G. clarum、根内球囊霉G. intraradices、单孢球囊霉G. monosporum、弯丝球囊霉G. sinuosa是金沙江干热河谷(元谋段)的优势种。金沙江干热河谷土壤中丛枝菌根真菌的孢子密度为5~6400个/100g土壤,平均1504;每个根际土壤中丛枝菌根真菌的物种丰富度1~18种,平均9种。     

金沙江干热河谷(元谋段)丛枝菌根真菌多样性研究*

从金沙江干热河谷(元谋段)75种植物的根际土壤中分离鉴定了44种丛枝菌根真菌, 分属无梗囊霉属Acaulospora、古孢霉属Archaeospora、内养囊霉属Entrophospora、巨孢囊霉属Gigaspora、球囊霉属Glomus和盾巨孢囊霉属Scutellospora, 其中,球囊霉属和无梗囊霉属为金沙江干热河谷中丛枝菌根真菌的优势属。齿状无梗囊霉A. denticulata、刺状无梗囊霉A. spinosa、瘤状无梗囊A. tuberculata,近明球囊霉Glomus claroideum、明球囊霉G. clarum、根内球囊霉G. intraradices、单孢球囊霉G. monosporum、弯丝球囊霉G. sinuosa是金沙江干热河谷(元谋段)的优势种。金沙江干热河谷土壤中丛枝菌根真菌的孢子密度为5~6400个/100g土壤,平均1504;每个根际土壤中丛枝菌根真菌的物种丰富度1~18种,平均9种。     

Heavy-metal stress and developmental patterns of arbuscular mycorrhizal fungi

The rate of global deposition of Cd, Pb, and Zn has decreased over the past few decades, but heavy metals already in the soil may be mobilized by local and global changes in soil conditions and exert toxic effects on soil microorganisms. We examined in vitro effects of Cd, Pb, and Zn on critical life stages in metal-sensitive ecotypes of arbuscular mycorrhizal (AM) fungi, including spore germination, presymbiotic hyphal extension, presymbiotic sporulation, symbiotic extraradical mycelium expansion, and symbiotic sporulation. Despite long-term culturing under the same low-metal conditions, two species, Glomus etunicatum and Glomus intraradices, had different levels of sensitivity to metal stress. G. etunicatum was more sensitive to all three metals than was G. intraradices. A unique response of increased presymbiotic hyphal extension occurred in G. intraradices exposed to Cd and Pb. Presymbiotic hyphae of G. intraradices formed presymbiotic spores, whose initiation was more affected by heavy metals than was presymbiotic hyphal extension. In G. intraradices grown in compartmentalized habitats with only a portion of the extraradical mycelium exposed to metal stress, inhibitory effects of elevated metal concentrations on symbiotic mycelial expansion and symbiotic sporulation were limited to the metal-enriched compartment. Symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. Patterns exhibited by G. intraradices spore germination, presymbiotic hyphal extension, symbiotic extraradical mycelium expansion, and sporulation under elevated metal concentrations suggest that AM fungi may be able to survive in heavy metal-contaminated environments by using a metal avoidance strategy.     

Towards growth of arbuscular mycorrhizal fungi independent of a plant host

When surface-sterilized spores of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices Sy167 were germinated on agar plates in the slightly modified minimum mineral medium described by G. Bécard and J. A. Fortin (New Phytol. 108:211-218, 1988), slime-forming bacteria, identified as Paenibacillus validus, frequently grew up. These bacteria were able to support growth of the fungus on the agar plates. In the presence of P. validus, hyphae branched profusely and formed coiled structures. These were much more densely packed than the so-called arbuscule-like structures which are formed by AMF grown in coculture with carrot roots transformed with T-DNA from Agrobacterium rhizogenes. The presence of P. validus alone also enabled G. intraradices to form new spores, mainly at the densely packed hyphal coils. The new spores were not as abundant as and were smaller than those formed by AMF in the monoxenic culture with carrot root tissues, but they also contained lipid droplets and a large number of nuclei. In these experiments P. validus could not be replaced by bacteria such as Escherichia coli K-12 or Azospirillum brasilense Sp7. Although no conditions under which the daughter spores regerminate and colonize plants have been found yet, and no factor(s) from P. validus which stimulates fungal growth has been identified, the present findings might be a significant step forward toward growth of AMF independent of any plant host.     

西藏高原天然长芒草地丛枝菌根真菌接种效应

采用草地均匀打孔方法,就草地土壤未消毒条件下接种丛枝菌根（AM）真菌对长芒草(Stipa bungeana)的侵染效应以及对植物生长、吸磷效率、土壤微生物区系等的影响进行研究.结果表明,1)接种处理、不接种处理的菌根效应存在着明显的差异,多数接种处理根围土壤AM真菌孢子密度、菌根侵染率和侵染强度显著提高,但对丛枝丰度的影响相对较低.2)接种后AM真菌孢子密度对菌根侵染率具有极显著影响(r=0.7679**);随菌根侵染率的增加,植株总干物重和吸磷总量均呈极显著提高,r值分别为0.7556**、0.8018**.3)与植株地上部相比,接种AM真菌对提高根系干物重、根系吸磷量和含磷量的促进作用相对较大.4)多数接种处理根际土壤酸性磷酸酶、碱性磷酸酶活性均呈一定程度的提高,根际土壤细菌数量显著增加,真菌、放线菌的数量变化则不甚明显.5)各接种处理对寄主植物的综合侵染效应在总体上呈Glomus mosseae+G. intraradices+Scutellospora calospora＞G. mosseae+G. aggregatum＞Glomus sp.＞G. mosseae＞G. mosseae+ G. etunicatum+G. intraradices+S. erythropa＞G. geosporum的趋势.     

Anastomosis formation and nuclear and protoplasmic exchange in arbuscular mycorrhizal fungi

We observed anastomosis between hyphae originating from the same spore and from different spores of the same isolate of the arbuscular mycorrhizal fungi Glomus mosseae, Glomus caledonium, and Glomus intraradices. The percentage of contacts leading to anastomosis ranged from 35 to 69% in hyphae from the same germling and from 34 to 90% in hyphae from different germlings. The number of anastomoses ranged from 0.6 to 1.3 per cm (length) of hyphae in mycelia originating from the same spore. No anastomoses were observed between hyphae from the same or different germlings of Gigaspora rosea and Scutellospora castanea; no interspecific or intergeneric hyphal fusions were observed. We monitored anastomosis formation with time-lapse and video-enhanced light microscopy. We observed complete fusion of hyphal walls and the migration of a mass of particles in both directions within the hyphal bridges. In hyphal bridges of G. caledonium, light-opaque particles moved at the speed of 1.8 +/- 0.06 microm/s. We observed nuclear migration between hyphae of the same germling and between hyphae belonging to different germlings of the same isolate of three Glomus species. Our work suggests that genetic exchange may occur through intermingling of nuclei during anastomosis formation and opens the way to studies of vegetative compatibility in natural populations of arbuscular mycorrhizal fungi.     

磷和镉对根内球囊霉Glomus intraradices孢子萌发、菌丝生长和外生菌丝内聚磷酸累积的影响

丛枝菌根真菌(AMF)能够显著提高植物对重金属的抗性,菌丝内聚磷酸(Polyphosphate,PolyP)可能参与了这种抗性的形成。试验以Glomus intraradices孢子为试材,对灭菌条件进行了优化,并进一步研究了不同P和Cd2+水平对孢子萌发、菌丝生长、分支和外生菌丝中聚磷酸含量的影响。结果表明,孢子在1%氯胺T+0.02%链霉素+0.01%庆大霉素+1/100(V/V)吐温-20中灭菌5min的效果最好。孢子萌发率、菌丝分支和菌丝长度随着Cd2+浓度的增加不断降低;当Cd2+浓度达到0.1mmol/L时,孢子萌发率降低为0%,表明Glomus intraradices的孢子萌发对Cd2+的耐受极限为0.1mmol/L;1mmol/L的P促进菌丝分支增加,却降低了萌发率,但对菌丝生长没有影响;在培养23d以后,三者基本不再变化。外生菌丝内的聚磷酸含量随着P的升高而增加;在Cd2+胁迫作用下,聚磷酸的含量降低而菌丝密度随着聚磷酸的升高而升高,表明聚磷酸在减弱重金属毒性方面起了重要作用。     

Breaking dormancy in spores of the arbuscular mycorrhizal fungus Glomus intraradices: a critical cold-storage period

To elucidate the effect of cold storage on spore dormancy in the arbuscular mycorrhizal (AM) fungus Glomus intraradices, spores were cold stratified at 4 degrees C, for either 0, 3, 7, 14, 90 or 120 days, prior to germination tests at 25 degrees C. The results showed that cold stratification longer than 14 days significantly increased spore germination. Moreover, the longer cold storage periods clearly reduced spore mortality from 90% to 50% and considerably altered the hyphal growth pattern. Long polarized hyphae were only observed after cold stratification periods longer than 14 days, involving consequences for root infectivity. The results clearly show that environmental factors, e.g., coldness, can affect the physiology of AM fungal spores.     

Genetic variation of morphological characters within a single isolate of the endomycorrhizal fungus Glomus clarum (Glomaceae)

The nature of variation in morphological characters in spores of arbuscular endomycorrhizal fungi (Order Glomales, Class Zygomycetes) has received little attention, despite the importance of these characters in modern taxonomy of the order. We tested the hypothesis that genetic variation exists in spore size and color (presumably important taxonomic characters) within a single isolate of the glomalean fungus Glomus clarum. Phenotypic variation in size and color of spores was determined from a pot culture population (designated P). A 10% selection pressure was imposed on replicate pot cultures of the first progeny culture generation (G1) by selecting the smallest, largest, yellowest, and whitest spores from the P generation and inoculating Sorghum bicolor plants. The experiment was repeated for another generation (G2), but with a 5% selection pressure. In both the G1 and G2 generations, significant differences in spore size and color were observed among the various treatments, indicating substantial genetic variation in these characters. Despite efforts to keep the physical environment constant across generations, we observed variation in the overall means of spore size and color among the generations (regardless of treatment), indicating a strong nongenetic influence on character expression. This study provides empirical evidence that will help delimit species boundaries among isolates of Glomus clarum and similar morphospecies. It also demonstrates a promising method to help elucidate the nature of character diversity in obligately asexual fungi.     

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.     

Assessing the diversity of AM fungi in arid gypsophilous plant communities

In the present study, we used PCR-Single-Stranded Conformation Polymorphism (SSCP) techniques to analyse arbuscular mycorrhizal fungi (AMF) communities in four sites within a 10 km² gypsum area in Southern Spain. Four common plant species from these ecosystems were selected. The AM fungal small-subunit (SSU) rRNA genes were subjected to PCR, cloning, SSCP analysis, sequencing and phylogenetic analyses. A total of 1443 SSU rRNA sequences were analysed, for 21 AM fungal types: 19 belonged to the genus Glomus , 1 to the genus Diversispora and 1 to the Scutellospora. Four sequence groups were identified, which showed high similarity to sequences of known glomalean species or isolates: Glo G18 to Glomus constrictum , Glo G1 to Glomus intraradices , Glo G16 to Glomus clarum , Scut to Scutellospora dipurpurescens and Div to one new genus in the family Diversisporaceae identified recently as Otospora bareai . There were three sequence groups that received strong support in the phylogenetic analysis, and did not seem to be related to any sequences of AM fungi in culture or previously found in the database; thus, they could be novel taxa within the genus Glomus : Glo G4, Glo G2 and Glo G14. We have detected the presence of both generalist and potential specialist AMF in gypsum ecosystems. The AMF communities were different in the plant studied suggesting some degree of preference in the interactions between these symbionts.     

The bacterium Paenibacillus validus stimulates growth of the arbuscular mycorrhizal fungus Glomus intraradices up to the formation of fertile spores

Two isolates of Paenibacillus validus (DSM ID617 and ID618) stimulated growth of the arbuscular mycorrhizal fungus Glomus intraradices Sy167 up to the formation of fertile spores, which recolonize carrot roots. Thus, the fungus was capable of completing its life cycle in the absence of plant roots, but relied instead on the simultaneous growth of bacteria. The supernatant of a mixed batch culture of the two P. validus isolates contained raffinose and another, unidentified trisaccharide. Among the oligosaccharides tested, raffinose was most effective in stimulating hyphal mass formation on plates but could not promote growth to produce fertile spores. A suppressive subtractive hybridization library followed by reverse Northern analyses indicated that several genes with products involved in signal transduction are differentially expressed in G. intraradices SY 167 when grown in coculture with P. validus (DSM 3037). The present investigation, while likely representing a significant step forward in understanding the arbuscular mycorrhizal fungus symbioses, also confirms that its optimal establishing and functioning might rely on many, as yet unidentified factors.     

Effect of the fungicide benomyl on spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae.

The fungicide benomyl inhibited spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae when applied at doses of 21.25 microg/ml (agronomic dose), 10.62 microg/ml and 10 microg/ml. G. mosseae was able to germinate in the presence of 2.12 microg/ml of benomyl, and the percentage of spore germination was unaffected by dosis of 0.1, 0.01 and 0.001 microg/ml of the fungicide. However, all doses of fungicide tested in this study decreased the hyphal length. When ungerminated G. mosseae spores previously exposed to benomyl were transferred to water-agar medium without benomyl, the maximum germination was 16%. Small spores of G. mosseae were more resistant to benomyl than the larger ones. Our results show some of the factors which can explain the variability of the effect of benomyl on arbuscular mycorrhizal fungi.     

南方红豆杉丛枝菌根(AM)的研究

研究了南方红豆杉根部丛枝菌根真菌(AMF)侵染情况、菌根形态结构以及根际土中AMF孢子的种类与数量.结果显示:南方红豆杉可与AMF形成典型的丛枝-泡囊型菌根,侵染率在71.2%~94.4%,但是历山、蟒河自然保护区的侵染强度优于人工栽培区;在南方红豆杉根际土中共分离鉴定出5种AMF,无梗囊霉属1种、球囊霉属4种,分别是:光壁无梗囊霉、地表球囊霉、地球囊霉、缩球囊霉、明球囊霉,其中光壁无梗囊霉为优势种;南方红豆杉的根由表皮、外皮层、内皮层、中柱组成,AMF只侵染表皮层、内皮层,不能侵染中柱.这为将来利用AMF接种技术进行南方红豆杉的繁殖、移植栽培以及紫杉醇的积累等研究提供了理论依据.     

Evidence for specificity of cultivable bacteria associated with arbuscular mycorrhizal fungal spores

Bacteria associated with arbuscular mycorrhizal (AM) fungal spores may play functional roles in interactions between AM fungi, plant hosts and defence against plant pathogens. To study AM fungal spore-associated bacteria (AMB) with regard to diversity, source effects (AM fungal species, plant host) and antagonistic properties, we isolated AMB from surface-decontaminated spores of Glomus intraradices and Glomus mosseae extracted from field rhizospheres of Festuca ovina and Leucanthemum vulgare. Analysis of 385 AMB was carried out by fatty acid methyl ester (FAME) profile analysis, and some also identified using 16S rRNA gene sequence analysis. The AMB were tested for capacity to inhibit growth in vitro of Rhizoctonia solani and production of fluorescent siderophores. Half of the AMB isolates could be identified to species (similarity index 0.6) within 16 genera and 36 species. AMB were most abundant in the genera Arthrobacter and Pseudomonas and in a cluster of unidentified isolates related to Stenotrophomonas. The AMB composition was affected by AM fungal species and to some extent by plant species. The occurrence of antagonistic isolates depended on AM fungal species, but not plant host, and originated from G. intraradices spores. AM fungal spores appear to host certain sets of AMB, of which some can contribute to resistance by AM fungi against plant pathogens.     

Phosphorus availability influences elemental uptake in the mycorrhizal fungus Glomus intraradices, as revealed by particle-induced X-ray emission analysis

We investigated element accumulation in the arbuscular mycorrhizal fungus Glomus intraradices. Fungal spores and mycelia growing in monoxenic cultures were analyzed. The elemental composition was quantified using particle-induced X-ray emission (PIXE) in combination with scanning transmission ion microscopy. In the spores, Ca and Fe were associated mainly with the spore wall, while P and K showed patchy distributions and their concentrations were correlated. Excess of P in the hyphal growth medium increased the P and Si concentrations in spores and increased the K/Ca ratio in spores. Increased P availability decreased the concentration of Zn and Mn in spores. We concluded that the availability of P influences the uptake and accumulation of several elements in spores. It is demonstrated that PIXE analysis is a powerful tool for quantitative analysis of elemental accumulation in fungal mycelia.     

Functional diversity of arbuscular mycorrhizal fungal isolates in relation to extraradical mycelial networks

We investigated the functional significance of extraradical mycorrhizal networks produced by geographically different isolates of the arbuscular mycorrhizal fungal (AMF) species Glomus mosseae and Glomus intraradices. A two-dimensional experimental system was used to visualize and quantify intact extraradical mycelium (ERM) spreading from Medicago sativa roots. Growth, phosphorus (P) and nitrogen (N) nutrition were assessed in M. sativa plants grown in microcosms. The AMF isolates were characterized by differences in extent and interconnectedness of ERM. Phenotypic fungal variables, such as total hyphal length, hyphal density, hyphal length per mm of total or colonized root length, were positively correlated with M. sativa growth response variables, such as total shoot biomass and plant P content. The utilization of an experimental system in which size, growth rate, viability and interconnectedness of ERM extending from mycorrhizal roots are easily quantified under realistic conditions allows the simultaneous evaluation of different isolates and provides data with a predictive value for selection of efficient AMF.     

野生植物根围的丛枝菌根真菌Ⅱ

本文主要报道了野生植物根围Glomus属的17个种,聚球囊霉G.aggregatumSchenck＆Smith,苏格兰球囊霉G.caledonium（Nicol.＆Gerd.）Trappe＆Gerd,近明球囊霉G．claroideumSchenck＆Smith,明球囊霉G．clarumNicolson＆Schenck,缩球囊霉G．constrictumTrappe,透光球囊霉G．diaphanumMorton,幼套球囊霉G．etunicatumBecker＆Gerdemann,集球囊霉G．fasciculatum（Thaxter）Gerd．＆Trappe,何氏球囊霉G．hoiBerch＆Trappe,地球囊霉G.geosporum（Nicol．＆Derd.）Warker,根内球囊霉G．intraradicesSchenck＆Smith,摩西球囊霉G．mosseae（Nicol．＆Gerd.）Gerd.＆Trappe,隐球囊霉G.occultumWalker,网状球囊霉G.reticulatumBhattcharjee＆Mukerji,地表球囊霉G.versiforme（Karsten）Berch,台湾球囊霉G．formosanumWu＆Chen,悬钩子球囊霉G．rubiformeGerdemann＆Trappe）Almeida＆Schenck;内养囊霉属1个种,稀有内养囊霉Entrophosporainfrequens（Hall）Ames＆Schenider。其中,网状球囊霉为我国新记录种。