The influence of ammonium nitrate on the root growth and ericoid mycorrhizal colonization of Calluna vulgaris (L.) Hull from a Danish heathland

Conversion of European heathlands to grassland has been reported as a response to increased nutrient availability, especially of nitrogen; a direct effect upon mycorrhizal colonization has been proposed as an likely explanation.This hypothesis was tested in a random block experiment with four blocks and four replicates on a Danish inland heath, Hjelm Hede. Ammonium nitrate was applied (0, 35, 50 and 70 kg N ha^–1 year^–1) to a stand of Calluna vulgaris (L.) Hull four times annually for 2 years. Calluna roots were sampled on four occasions in the 2nd year of the nitrogen treatment. The extent of ericoid mycorrhizal colonization was determined by direct observation of the roots using a line-intersection method. The nitrogen content of the current-year shoots of Calluna increased when they were treated with nitrogen. Nitrogen fertilization had no significant effects on ericoid mycorrhizal colonization of Calluna nor on root biomass. The seasonal variation in mycorrhizal colonization of the Calluna roots was highly significant. The spatial variability of mycorrhizal colonization, both in replicated plots and in the two contrasted soil horizons – the mor layer and the bleached sand – within the plots, were considerable. I conclude that heather decline under enhanced nitrogen input is unlikely to be caused by a direct impact on the ericoid mycorrhizae of Calluna. Received: 1 December 1998 / Accepted: 7 December 1999     

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     

Utilisation of organic nitrogen and phosphorus sources by mycorrhizal endophytes of Woollsia pungens (Cav.) F. Muell. (Epacridaceae)

 The ability of four ericoid mycorrhizal endophytes isolated from roots of Woollsia pungens (Cav.) F. Muell. (Epacridaceae) to utilise organic forms of nitrogen and phosphorus during growth in axenic culture was assessed. All isolates were able to utilise glutamine, arginine and bovine serum albumin (BSA), along with NH₄ ⁺ or NO₃ ^–, in most cases yielding at least as much biomass as the ericoid mycorrhizal endophyte Hymenoscyphus ericae (Read) Korf & Kernan. All isolated endophytes were able to utilise BSA, arginine and glutamine as sole sources of N and C. With the exception of a single isolate (C40), which showed little growth on glutamine, biomass yields on glutamine as the sole N and C source was significantly greater for all isolates than on either of the other two organic N sources. Two isolates from W. pungens (C40 and A43) utilised DNA and sodium inositol hexaphosphate as sole P sources, in each case yielding significantly more biomass than H. ericae. The results suggest that mycorrhizal endophytes from epacrid plant hosts and those from ericaceous hosts have similar abilities to utilise organic forms of N and P. Accepted: 4 September 1998     

Colonization and decomposition of salal (Gaultheria shallon) leaf litter by saprobic fungi in successional forests on coastal British Columbia

The colonization of leaf litter by saprobic fungi was studied in old-growth and post-harvest successional Douglas-fir forests on southeast Vancouver Island, British Columbia. This study focused on leaf litter of salal (Gaultheria shallon Pursh.), a dominant understory shrub in all stands. Salal litter is characterized by the occurrence of bleached portions attributable to fungal colonization of the litter and to the variable decomposition of recalcitrant compounds, such as lignin. Analyses of proximate chemical fractions, fungal assemblages on the bleached leaf area, and pure culture decomposition assays indicated that Marasmius sp. and Coccomyces sp. were responsible for rapid decomposition and bleaching of salal leaf litter. The bleached area accounted for 17%-22% of total area of salal leaf litter collected in immature (40-60 years old), mature (85-105 years old), and old-growth (more than 290 years old) stands, but for only 2% in regeneration (5-15 years old) stands. The reduction of bleached leaf area occupied by Marasmius sp. and Coccomyces sp. in regeneration stands could be due to the changes in microenvironmental conditions on the forest floor, in litter quality, or in food-web structure in soils. The decrease of fungi able to decay recalcitrant compounds may lead to a reduction of salal decomposition rates in clear-cut sites that would persist until canopy closure occurs.     

Tissue density and growth response of ectomycorrhizal fungi to nitrogen source and concentration

 Amanita rubescens Pers., Lactarius affinis Pk., Leccinum aurantiacum (Fr.) S.F. Gray, Tylopilus felleus (Bull. ex Fe.) Karsten, and two isolates of Suillus intermedius (Smith & Thiers) Smith & Thiers collected from an approximately 55-year-old Pinus resinosa Ait. plantation, and Pisolithus tinctorius (Pers.) Coker & Couch obtained from another source, were tested for their abilities to grow with protein as the primary source of nitrogen. Protein plates contained 63 mg l^–1 N as bovine serum albumen and 7 mg l^–1 N as arginine. Control plates contained only 7 mg l^–1 N as arginine. All isolates except Leccinum aurantiacum and one isolate of S. intermedius attained greater dry weight with protein as the primary source of N. Lactarius affinis, Leccinum aurantiacum, P. tinctorius, and both isolates of S. intermedius had higher tissue densities on protein medium. Amanita rubescens had lower tissue density. To determine if increase in tissue density was an effect of total N concentration or an effect of N source (protein versus arginine), we performed a second experiment in which arginine concentration was increased (7 mg l^–1 N versus 70 mg l^–1 N). The second experiment also included Cenococcum geophilum Fr. but excluded T. felleus. Higher tissue densities with increased nutrients were found in C. geophilum, Lactarius affinis, Leccinum aurantiacum, and both isolates of S. intermedius. Only A. rubescens and P. tinctorius did not have increased densities. The results suggest that these ectomycorrhizal fungi alter their growth forms according to N concentration. At low N concentrations, a growth form likely to promote exploitation of a large volume of medium for a given biomass is produced. At high concentrations, a growth form likely to promote exploitation of a rich source of N is produced. Whether ectomycorrhizal fungi growing in association with roots would act in a similar fashion is not known. Accepted: 30 July 1998     

Effect of host shoot clipping on carbon and nitrogen sources for arbuscular mycorrhizal fungi

The effect of clipping of the host-plant shoot on the sources of carbon and nitrogen for the arbuscular mycorrhizal (AM) fungus Gigaspora margarita was determined by measuring ¹³C in spores and hyphae in cocultures of C₃ and C₄ plants and by differential ¹⁵N labeling. C₃ and C₄ plants, which have different δ¹³C values, were grown in the same container separated by a series of hyphal compartments. The C₃ and C₄ plants were applied with ¹⁴N- and ¹⁵N-urea, respectively. After clipping of the C₃ shoots, spore δ ¹³C gradually approached that of the C₄ roots. Hyphal δ ¹³C paralleled that of spores. Spore % ¹⁵N was similar to that of mineral N in the C₄ plant compartment. Thus C in G. margarita coming from the clipped plants decreased with time. This demonstrates that C in AM fungi comes from living plants, whilst the N in spores comes mostly from the soil. Accepted: 28 November 2000     

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     

Studies of iron transport by arbuscular mycorrhizal hyphae from soil to peanut and sorghum plants

 The influence of an arbuscular mycorrhizal (AM) fungus on phosphorus (P) and iron (Fe) uptake of peanut (Arachis hypogea L.) and sorghum (Sorghum bicolor L.) plants was studied in a pot experiment under controlled environmental conditions. The plants were grown for 10 weeks in pots containing sterilised calcareous soil with two levels of Fe supply. The soil was inoculated with rhizosphere microorganisms only or with rhizosphere microorganisms together with an AM fungus (Glomus mosseae [Nicol. & Gerd.] Gerdemann & Trappe). An additional small soil compartment accessible to hyphae but not roots was added to each pot after 6 weeks of plant growth. Radiolabelled P and Fe were supplied to the hyphae compartment 2 weeks after addition of this compartment. After a further 2 weeks, plants were harvested and shoots were analysed for radiolabelled elements. In both plant species, P uptake from the labelled soil increased significantly more in shoots of mycorrhizal plants than non-mycorrhizal plants, thus confirming the well-known activity of the fungus in P uptake. Mycorrhizal inoculation had no significant influence on the concentration of labelled Fe in shoots of peanut plants. In contrast, ⁵⁹Fe increased in shoots of mycorrhizal sorghum plants. The uptake of Fe from labelled soil by sorghum was particularly high under conditions producing a low Fe nutritional status of the plants. These results are preliminary evidence that hyphae of an arbuscular mycorrhizal fungus can mobilise and/or take up Fe from soil and translocate it to the plant. Accepted: 6 March 1998     

菌根真菌促进植物吸收利用氮素机制的研究进展

作为自然界最为普遍的一种植物共生体,菌根能够极大地促进植物对氮素的吸收和利用,其中菌根真菌在共生结构功能中发挥了重要作用。本文分别从菌根解剖构造、生理生化和分子生物学方面系统总结了菌根真菌促进植物吸收和利用氮素的研究现状。重点介绍了菌根真菌可利用的氮素形态及影响其利用的主要因素、菌根真菌的氮代谢途径GS-GOGAT以及菌根真菌中存在的鸟氨酸循环途径,指出精氨酸是菌丝内氮转运的主要形式,NH3可能为菌根真菌和植物界面质外体的主要转运形式。     

Influence of organic amendments on arbuscular mycorrhizal fungi in relation to rice sheath blight disease

 The effect of various organic soil amendments on arbuscular myorrhizal (AM) fungal activity on rice plants was tested under greenhouse and field conditions with reference to sheath blight (ShB) disease caused by Rhizoctonia solani. AM spore density, per cent infection, and intensity of infection were increased by organic amendments, whilst ShB disease was decreased. Certain amendments, especially green leaf manure, stimulated arbuscule development in rice plants. Mycorrhiza formation and sporulation were higher with healthy rice plants than with rice plants infected with R. solani. Our results indicate the possibility of using selective organic amendments to enhance development of native AM fungi and thus reduce disease incidence. Accepted: 9 November 1995     

Cultivation of mushrooms of edible ectomycorrhizal fungi associated with Pinus densiflora by in vitro mycorrhizal synthesis

Edible mushroom fungi in the genera Lyophyllum, Tricholoma, Leucopaxillus, Suillus, Rhizopogon, Lactarius, and Morchella were tested for mycorrhization with Pinus densiflora in vitro. Most of the tested fungi in the genera Lyophyllum, Tricholoma, Suillus, Rhizopogon, and Lactarius formed ectomycorrhizas 2–4 months after fungal inoculation. Mycorrhizal seedlings were then acclimatized in open-pot soil under growth-chamber conditions. Almost all mycorrhizal seedlings sustained their symbiont and developed new mycorrhizas for 8–9 months after transplantation. Under these conditions, more than half of the tested species formed primordia and Tricholoma flavovirens, Rhizopogon rubescens, and Lactarius akahatsu developed basidiocarps with young host plants. Accepted: 28 November 2000     

Determination of the nitrogen source for arbuscular mycorrhizal fungi by 15N application to soil and plants

The source of nitrogen in the spores of arbuscular mycorrhizal (AM) fungi was quantified by a ¹⁵N-labeling technique. N was applied as coated urea to the soil and in solution to plant shoots. Soil-applied fertilizer had a significant effect on spore % ¹⁵N (P<0.01), with a 24–75% contribution to spore N. Fertilizer applied to either alfalfa shoots or bahia grass shoots had little effect on spore % ¹⁵N, accounting for 0–14% or 1–9% of spore N, respectively. These results indicate that AM fungi obtain spore N mostly from the soil. The small amount of spore N originating from shoot-applied N may have been obtained via root exudation. Accepted: 6 November 2000     

Effects of vesicular-arbuscular mycorrhizal inoculation at different soil P availabilities on growth and nutrient uptake of in vitro propagated coffee (Coffea arabica L.) plants

 In a pot experiment, the growth and the nutrient status of in vitro propagated coffee (Coffea arabica L.) microcuttings were investigated for 5 months following vesicular-arbuscular mycorrhizal (VAM) inoculation with either Acaulospora melleae or Glomus clarum at four soil P availabilities. Control plants remained P-deficient even at the highest soil P availability while mycorrhizal plants were P-sufficient at all soil P availabilities. Growth of control plants was only improved at the highest soil P availability. In P-deficient soil, neither of the two VAM species improved plant growth. Plant growth increased by 50% following inoculation with either A. melleae or G. clarum when P availability went from deficient to low. No further plant growth improvement was induced by either VAM species at intermediate and high soil P levels. Nevertheless, growth of plants inoculated with G. clarum was still significantly greater than that of non-mycorrhizal plants at the highest soil P availability. Root colonization by G. clarum increased with increasing soil P availability while root colonization by A. mellea decreased with soil P level increasing above low P availability. Soil P availability also affected Zn nutrition through its influence on VAM symbiosis. With increasing soil P availability, foliar Zn status increased with G. clarum or decreased with A. mellea in parallel to root colonization by VAM. This study demonstrates the beneficial effects of VAM inoculation on in vitro propagated Arabica coffee microcuttings, as shown previously for seedlings. This study also demonstrates differences in tolerance to soil P availability between VAM species, most likely resulting from their differing abilities to enhance coffee foliar P status. Accepted: 14 November 1996     

A comparison of AM fungi inoculants using Capsicum and Polianthes in marginal soil amended with organic matter

 Different types of nursery inocula formulations, namely mixed indigenous cultures and Glomus intraradices Schenck and Smith, were compared with commercially available inoculants of AM fungi in a pot experiment using two horticultural crops, Capsicum and Polianthes. Soil-based inocula and soil beads produced the highest response in both crops. Glomus intraradices resulted in the highest yield in both Polianthes (45% increase in spike length) and Capsicum (112% increase in fruit yield). Among the commercial inocula tested, only Mycorise enhanced spike length (33%) and fruit yield (11%) in the two hosts. Overall AM colonization was higher in Polianthes than in Capsicum. Sheared root inocula of G. intraradices resulted in high colonization (upto 68%) but the yield enhancement was lower than with soil-based formulations. The mixed indigenous culture produced the highest number of spores and propagules and commercial inocula the lowest. Accepted: 2 February 1998     

Effects of Pseudomonas fluorescens DF57 on growth and P uptake of two arbuscular mycorrhizal fungi in symbiosis with cucumber

 The effect of Pseudomonas fluorescens DF57 on growth and P uptake of two arbuscular mycorrhizal (AM) fungi in symbiosis with cucumber plants was studied in compartmentalised growth systems. Hyphae of Glomus intraradices Schenck & Smith (BEG87) or G. caledonium (Nicol. & Gerd.) Trappe & Gerdeman (BEG15) grew into lateral root-free compartments. Non-mycorrhizal plants served as control. The soil in half of the growth units of each mycorrhizal treatment was inoculated with P. fluorescens DF57. P. fluorescens DF57 enhanced hyphal length density of one of the AM fungi, G. caledonium, but this was not reflected in a higher hyphal transport of P from the root-free soil to the plant. The total P content was higher in plants grown in symbiosis with G. intraradices than in plants in the other treatments. G. caledonium and P. fluorescens DF57 had a synergistic effect in that total P content in plants inoculated with G. caledonium was higher in the presence than in the absence of P. fluorescens DF57. Accepted: 7 January 1999     

Effect of root exudate fractions from P-deficient and P-sufficient onion plants on root colonisation by the arbuscular mycorrhizal fungus Gigaspora margarita

 The effect of root exudates from P-deficient onion on root colonisation by an arbuscular mycorrhizal fungus was examined. Onions (Allium cepa L.) were grown in solution culture at phosphorus concentrations of 0 (P0) and 2 (P2) mg P l^–1. Root exudates were collected and fractionated with Amberlite XAD-4 resin to give EtOH and water soluble fractions. Onions inoculated with the arbuscular mycorrhizal fungus Gigaspora margarita Becker & Hall were grown with or without (control) root exudates and exudate fractions in a growth chamber. After 24 days, arbuscular mycorrhiza levels and appressoria formation had increased in plants treated with P0-root exudate or the P0-EtOH fraction when compared to corresponding P2 treatments or control plants. P0 and P2 water-soluble fractions did not significantly affect either aspect of fungal development. These results suggest that hydrophobic compounds found in root exudates from P-deficient onion increase appressorium formation and, therefore, enhance mycorrhiza development. Accepted: 2 June 1998     

Effect of heavy metal pollution on mycorrhizal colonization and function: physiological, ecological and applied aspects

 High concentrations of heavy metals in soil have an adverse effect on micro-organisms and microbial processes. Among soil microorganisms, mycorrhizal fungi are the only ones providing a direct link between soil and roots, and can therefore be of great importance in heavy metal availability and toxicity to plants. This review discusses various aspects of the interactions between heavy metals and mycorrhizal fungi, including the effects of heavy metals on the occurrence of mycorrhizal fungi, heavy metal tolerance in these micro-organisms, and their effect on metal uptake and transfer to plants. Mechanisms involved in metal tolerance, uptake and accumulation by mycorrhizal hyphae and by endo- or ectomycorrhizae are covered. The possible use of mycorrhizal fungi as bioremediation agents in polluted soils or as bioindicators of pollution is also discussed. Accepted: 23 June 1997     

Identification of arbuscular mycorrhizal fungi in soils and roots of plants colonizing zinc wastes in southern Poland

 Analysis of the community of arbuscular mycorrhizal (AM) fungi in roots of Fragaria vesca growing in a heavy metal contaminated site was carried out on a Zn waste site near Chrzanow (southern Poland). The waste substratum was characterized by high contents of Pb, Zn, Cd, Cu and As, and by low levels of N, P and organic matter. Spores of Glomales were isolated by wet sieving and DNA was isolated from individual spores. Nested polymerase chain reaction (PCR) with taxon-specific primers was used to identify the species Glomus mosseae, Glomus intraradices and Glomus claroideum. Spores of other fungi were morphologically characterized and new taxon-discriminating molecular probes were developed for two of them (Glomus sp. HM-CL4 and HM-CL5) based on variations in the large ribosomal subunit (25S rDNA). High sequence similarities were found between Glomus sp. HM-CL4 and Glomus gerdemanii, and between Glomus sp. HM-CL5 and Glomus occultum. The designed primers were used to characterize the population of AM fungi colonizing the roots of F. vesca collected from the Zn waste site. The analysis, carried out on roots stained with trypan blue, showed that the most effective colonizer was closely related to G. gerdemannii. G. claroideum and the G. occultum-like fungus were slightly less common whilst frequencies of G. intraradices and G. mosseae in roots were much lower. The analysis of mycorrhiza stained with rhodizoniate to localize heavy metal accumulation showed that the stain does not influence the PCR reaction. Seventy percent of the root samples containing positively stained fungal hyphae were found to be colonized by G. mosseae. The data obtained demonstrate the usefulness of nested PCR for studies carried out in polluted areas. It will enable selection of AM fungi which are able to colonize plant roots under heavy metal stress conditions, as well as the identification of fungi showing high in situ accumulation of potentially toxic elements. Accepted: 7 July 2000     

Effect of organic and inorganic nitrogen sources on endogenous polyamines and growth of ectomycorrhizal fungi in pure culture

 The abilities of three ectomycorrhizal fungi, Paxillus involutus, Suillus variegatus and Lactarius rufus, to utilize organic and inorganic nitrogen sources were determined by measuring the growth and endogenous free polyamines (putrescine, spermidine and spermine) of pure culture mycelium. Differences were found in the utilization of the nitrogen sources and in the polyamine concentrations between the fungal species and between isolates of L. rufus. All the fungi grew well on ammonium and on several amino acids. Endogenous polyamine levels varied with the nitrogen source. Spermidine was commonly the most abundant polyamine; however, more putrescine than spermidine was found in P. involutus growing on inorganic nitrogen or arginine. Low amounts of spermine were found in S. variegatus and some samples of L. rufus. None or only a trace of spermine was found in P. involutus mycelium. In all fungi, putrescine concentrations were higher with ammonium than with the nitrate treatment. The total nitrogen content of peat did not determine the ability of L. rufus strains isolated from peatland forest sites to utilize organic nitrogen. Accepted: 27 November 1998     

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