Molecular diversity of fungi from ericoid mycorrhizal roots

In order to investigate the diversity of fungal endophytes in ericoid mycorrhizal roots, about 150 mycelia were isolated from surface-sterilized roots of 10 plants of Calluna vulgaris. Each mycelium was reinoculated to C. vulgaris seedlings under axenic conditions, and the phenotype of the plant-fungus association assessed by light and electron microscopy. Many isolates that were able in vitro to produce typical ericoid mycorrhizae did not form reproductive structures under our culture conditions, whereas others could be identified as belonging to the species Oidiodendron maius. Morphological and molecular analysis of the fungal isolates showed that the root system of a single plant of C. vulgaris is a complex mosaic of several populations of mycorrhizal and non mycorrhizal fungi. PCR-RFLP techniques, used to investigate the mycorrhizal endophytes, revealed up to four groups of fungi with different PCR-RFLP patterns of the ITS ribosomal region from a single plant. Some of the mycorrhizal fungi sharing the same PCR-RFLP pattern showed high degree of genetic polymorphism when analysed with the more sensitive RAPD technique; this technique may prove a useful tool to trace the spread of individual mycorrhizal mycelia, as it has allowed us to identify isolates with identical RAPD fingerprints on different plants.     

Molecular detection,community structure and phylogeny of ericoid mycorrhizal fungi

Through traditional culturing and molecular characterization, we have determined that five putative species and 2 polyphyletic assemblages of fungi produce ericoid mycorrhizae in Gaultheria shallon, other Ericaceae and Epacridaceae. Using phylogenetic analysis of ITS2 sequences in GenBank, we have confirmed that most of these fungi occur in North America, Europe, and Australia. The low recovery rate of culturable ericoid mycorrhizal fungi from Gaultheria shallon may partly be explained by the fact that most mycorrhizal root segments contain an unculturable basidiomycete, revealed by direct amplification, cloning, and sequencing of LSU fungal DNA from root. Molecular characterization and phylogenetic analysis are powerful tools in revealing the geographic distribution and identity of ericoid mycorrhizal fungi.     

银叶杜鹃和繁花杜鹃根部真菌的多样性

植物根系与真菌形成菌根, 在自然生态系统的物质能量循环中具有重要的生态功能。作者在四川省的中国杜鹃园选取银叶杜鹃(Rhododendron argyrophyllum)和繁花杜鹃(R. floribundum), 通过直接扩增杜鹃花根部真菌rDNA-ITS区片段, 来揭示该地区杜鹃花属植物根部真菌的多样性。ITS序列分析结果表明: 从两种杜鹃的根部共检测到41个真菌分类单元, 分别属于子囊菌纲的柔膜菌目(Helotiales)、散囊菌目(Eurotiales)、盘菌目(Pezizales)、假球壳目(Pleosporales)和担子菌纲的蜡壳耳目(Sebacinales)、伞菌目(Agaricales)、Erythrobasidiales、线黑粉菌目(Filobasidiales)。银叶杜鹃和繁花杜鹃根部真菌种类丰富, 包括了杜鹃花类菌根真菌、外生菌根真菌和其他类型真菌, 其中担子菌纲的蜡壳耳目和子囊菌纲的柔膜菌目占有较大比例。     

Diversity of fungi in hair roots of Ericaceae varies along a vegetation gradient

Ericaceous dwarf shrubs including Calluna vulgaris and Vaccinium spp. occur both in open heathland communities and in forest ecosystems as understory vegetation. Ericaceous shrubs were once thought to form ericoid mycorrhizal associations with a relatively narrow range of ascomycetous fungi closely related to, and including, Rhizoscyphus ericae. However, perceptions have recently changed since the realization that a broader range of ascomycete fungi, and in some cases basidiomycete fungi, can also form associations with the roots of ericaceous plants. We used a combination of molecular approaches, including denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism, cloning and sequencing, to investigate the diversity of fungi associated with C. vulgaris roots collected across a heathland/native Scots pine forest vegetation gradient. We also determined differences in fungal community composition between roots of co-occurring C. vulgaris and Vaccinium myrtillus in the forest understory. Collectively, the data show that a large diversity of potentially ericoid mycorrhizal fungal taxa associate with roots of C. vulgaris and V. myrtillus, and that ascomycetes were about 2.5 times more frequent than basidiomycetes. The assemblages of fungi associated with C. vulgaris and V. myrtillus were different. In addition, the community of fungi associated with C. vulgaris hair roots was different for samples collected from the forest, open heathland and a transition zone between the two. This separation was partly, but not entirely, due to the occurrence of typical ectomycorrhizal basidiomycetes associated with the hair roots of C. vulgaris in the forest understory. These data demonstrate that forest understory ericaceous shrubs associate with a diverse range of ascomycete and basidiomycete taxa, including typical ectomycorrhizal basidiomycetes.     

Diversity of ericoid mycorrhizal fungi isolated from hair roots of Rhododendron obtusum var. kaempferi in a Japanese red pine forest

 The diversity of ericoid mycorrhizal fungi of Rhododendron obtusum var. kaempferi was examined in a stand of Pinus densiflora at Tsukuba, Japan. In total, 153 slow-growing fungal isolates were obtained from roots of R. obtusum var. kaempferi, in which 113 isolates formed an ericoid mycorrhizal structure in vitro. Among them, 53 isolates were morphologically identified as Oidiodendron maius, but the others were not identified due to their sterilities. PCR-RFLP analysis in the rDNA-ITS region divided them into four different RFLP types. Phylogenetic analysis from sequence data of the region suggested that the four RFLP types belonging to distinct taxa and one sterile type are considered to be Hymenoscyphus ericae. This study is the first report of ericoid mycorrhizal fungi in a natural habitat in Japan. Received: August 23, 2002 / Accepted: December 11, 2002 Acknowledgments We thank Dr. K. Narisawa, Plant Biotechnology Institute, Ibaraki Agricultural Center, and Dr. R.S. Currah, Department of Biological Science, University of Alberta, for their helpful advice. Contribution no. 176, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan Correspondence to:M. Kakishima     

杜鹃花科植物菌根的研究进展

杜鹃花科Ericaceae植物可与土壤真菌形成杜鹃花类菌根ericoid mycorrhizas (ERM)共生体,且广泛分布于全球不同的陆地生态系统,特别是在贫瘠、酸性等严酷的环境中占优势.杜鹃花科植物菌根类型多样,绝大多数宿主具有ERM,还有少量宿主具有其他类型的菌根结构,且常与暗隔内生菌(dark septate...     

Inoculation of cranberry (Vaccinium macrocarpon) with the ericoid mycorrhizal fungus Rhizoscyphus ericae increases nitrate influx

Despite the ubiquitous presence of ericoid mycorrhizal (ERM) fungi in cranberry (Vaccinium macrocarpon), no prior studies have examined the effect of ERM colonization on NO(3)(-) influx kinetics. Here, (15)NO(3)(-) influx was measured in nonmycorrhizal and mycorrhizal cranberry in hydroponics. Mycorrhizal cranberry were inoculated with the ERM fungus Rhizoscyphus (syn. Hymenoscyphus) ericae. (15)NO(3)(-) influx by R. ericae in solution culture was also measured. Rhizoscyphus ericae NO(3)(-) influx kinetics were linear when mycelium was exposed for 24 h to 3.8 mm NH(4)(+), and saturable when pretreated with 3.8 mm NO(3)(-), 50 microm NO(3)(-), or 50 microm NH(4)(+). Both low-N pretreatments induced greater NO(3)(-) influx than either of the high-N pretreatments. Nonmycorrhizal cranberry exhibited linear NO(3)(-) influx kinetics. By contrast, mycorrhizal cranberry had saturable NO(3)(-) influx kinetics, with c. eightfold greater NO(3)(-) influx than nonmycorrhizal cranberry at NO(3)(-) concentrations from 20 microm to 2 mm. There was no influence of pretreatments on cranberry NO(3)(-) influx kinetics, regardless of mycorrhizal status. Inoculation with R. ericae increased the capacity of cranberry to utilize NO(3)(-)-N. This finding is significant both for understanding the potential nutrient niche breadth of cranberry and for management of cultivated cranberry when irrigation water sources contain nitrate.     

Isolation and identification of hydroxamate siderophores of ericoid mycorrhizal fungi

Three ericoid mycorrhizal fungi were grown in pure culture under iron deprivation: (i) the ascomyceteHymenoscyphus ericae, a characteristic endophyte of ericaceous plants on acid soils; (ii) the hyphomyceteOidiodendron griseum, an ericoid mycorrhizal fungus which is also a soil-borne fungus able to colonize wood; and (iii) an endophyte of the calciculous ericaceous plantRhodothamnus chamaecistus. All three fungi produced several hydroxamate siderophores which were isolated in the ferric form by adsorption to Amberlite XAD-2, gel chromatography on Sephadex LH20 and by HPLC on a C₁₈ reversed-phase column. Siderophores were identified by (i) co-chromatography with known fungal siderophores, (ii) ion spray mass spectrometry after semi-preparative HPLC and (iii) analyzing their electrophoretic behavior. WhileH. ericae andO. griseum were similar in producing ferricrocin as their principal siderophore, the endophyte ofR. chamaecistus produced mainly fusigen.     

Depth-dependent effects of ericoid mycorrhizal shrubs on soil carbon and nitrogen pools are accentuated under arbuscular mycorrhizal trees

Plant mycorrhizal associations influence the accumulation and persistence of soil organic matter and could therefore shape ecosystem biogeochemical responses to global changes that are altering forest composition. For instance, arbuscular mycorrhizal (AM) tree dominance is increasing in temperate forests, and ericoid mycorrhizal (ErM) shrubs can respond positively to canopy disturbances. Yet how shifts in the co-occurrence of trees and shrubs with different mycorrhizal associations will affect soil organic matter pools remains largely unknown. We examine the effects of ErM shrubs on soil carbon and nitrogen stocks and indicators of microbial activity at different depths across gradients of AM versus ectomycorrhizal (EcM) tree dominance in three temperate forest sites. We find that ErM shrubs strongly modulate tree mycorrhizal dominance effects. In surface soils, ErM shrubs increase particulate organic matter accumulation and weaken the positive relationship between soil organic matter stocks and indicators of microbial activity. These effects are strongest under AM trees that lack fungal symbionts that can degrade organic matter. In subsurface soil organic matter pools, by contrast, tree mycorrhizal dominance effects are stronger than those of ErM shrubs. Ectomycorrhizal tree dominance has a negative influence on particulate and mineral-associated soil organic matter pools, and these effects are stronger for nitrogen than for carbon stocks. Our findings suggest that increasing co-occurrence of ErM shrubs and AM trees will enhance particulate organic matter accumulation in surface soils by suppressing microbial activity while having little influence on mineral-associated organic matter in subsurface soils. Our study highlights the importance of considering interactions between co-occurring plant mycorrhizal types, as well as their depth-dependent effects, for projecting changes in soil carbon and nitrogen stocks in response to compositional shifts in temperate forests driven by disturbances and global change.     

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.     

Rapid and reliable DNA extraction techniques from trypan-blue-stained mycorrhizal roots: comparison of two methods

Two improved DNA extraction techniques from trypan-blue-stained root fragments were developed and compared for rapid and reliable analyses. In Method A, 1 cm trypan-blue-stained mycorrhizal root fragments were individually isolated, crushed by bead beating, and purified with Chelex-100 (Bio-Rad). In Method B, DNA extraction was carried out using an UltraClean microbial DNA isolation kit (MoBio Laboratories). DNA was extracted from the mycorrhizal roots of four plant species, quantified by UV absorbance, and PCR-amplified with primers specific to arbuscular mycorrhizal fungi. Although PCR inhibitors might still exist when using Method A, appropriate dilution and employment of nested-PCR overcame this problem. Method B removed PCR inhibitors, but sometimes, depending on the mycorrhizal colonization within the root fragments, it also required nested PCR. In conclusion, both methods enabled us to handle many samples in a short time. Method B provided greater reliability and Method A provided better cost performance. Both techniques can be useful for PCR-based applications to identify species and estimate species composition after measuring mycorrhizal colonization rate with trypan blue staining.     

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.     

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.     

Molecular diversity within and between ericoid endophytes from the Ericaceae and Epacridaceae

This study investigated the relationships between ericoid mycorrhizal endophytes of the Ericaceae (Northern Hemisphere) and the Epacridaceae (Australia). Over 200 fungi were isolated from the roots of two species of Epacridaceae from Victoria, Australia. The isolates were divided into 12 groups by morphology on quarter-strength potato dextrose agar. All were slow-growing and most were dematiaceous, but groups varied from white through pink to dark olive. The ITS1–5.8S–ITS2 ribosomal DNA was amplified and sequenced from eight isolates, forming typical ericoid mycorrhizal morphology in Epacris impressa and one nonmycorrhizal isolate. Sequences were compared, by using similarities and maximum-parsimony analysis, with those of Hymenoscyphus ericae (Leotiales) and Oidiodendron species (Hyphomycetes), the most common endophytes of the Ericaceae. Maximum-parsimony analysis produced four clusters: (1) all Oidiodendron species (at least 90% similarity); (2) all five Victorian dark grey-olive isolates (at least 96% similarity); (3) one Victorian isolate and Cistella grevillei (88% similarity); (4) two light-coloured Victorian isolates and H. ericae (81% similarity). This suggests that these isolates from the Epacridaceae do not belong to the same species as those forming ericoid mycorrhiza in the Ericaceae.     

环境因子对AM真菌多样性的影响

土壤,气候和地理因子等环境因子对丝枝菌根（AM）真菌多样性有重要影响,本文系统地综述了最近10年来AM真菌生态学在该方面的最新研究成果,分析当前研究中所存在的问题和动向。     

Assemblages of ericoid mycorrhizal and other root-associated fungi from Epacris pulchella (Ericaceae) as determined by culturing and direct DNA extraction from roots

Ericoid mycorrhizal fungal endophytes form mycorrhizal associations with Ericaceae plant taxa and are regarded as essential to the ecological fitness of the plants in extremely nutrient-poor soils worldwide. We isolated fungi from roots of Epacris pulchella (Ericaceae) in a south-eastern Australian sclerophyll forest and compared rDNA internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs) and sequences for the cultured isolate assemblage with fungi identified in DNA extracted directly from the same root systems by cloning or denaturing gradient gel electrophoresis (DGGE). The most abundant RFLP types in the cultured isolate assemblage were identified as putative ericoid mycorrhizal ascomycete endophytes, and these also represented the most abundant RFLP types in the cloned assemblage and the most intense bands in DGGE profiles. Each method identified unique taxa, notably putative basidiomycetes in the DNA extracted directly from E. pulchella roots. However, the relative abundance of these was low.     

Utilisation of carbon substrates by orchid and ericoid mycorrhizal fungi from Australian dry sclerophyll forests

The utilisation of a range of cell-wall-related and aromatic carbon substrates by multiple genotypes of three ericoid mycorrhizal fungal taxa was compared with two orchid mycorrhizal fungal taxa. Both groups of fungi catabolised most common substrates, though significant inter- and intraspecific variability was observed in the use of a few carbon substrates. Orchid mycorrhizal fungi had limited access to tannic acid as a carbon source and did not use phenylalanine, while the ericoid mycorrhizal fungi used both. Utilisation of tryptophan was limited to single genotypes of each of the orchid mycorrhizal fungi, and to only two of the three ericoid mycorrhizal fungi examined. Although broadly similar, some significant differences apparently exist in carbon catabolism of ericoid and orchid mycorrhizal fungi from the same habitat. Functional and ecological implications of these observations are discussed.