Measurement of the viability of arbuscular-mycorrhizal fungi using three different stains; relation to growth and metabolic activities of soybean plants

Histochemical staining of alkaline phosphatase (ALP) and succinate dehydrogenase (SDH) activities in four arbuscular mycorrhizal fungi (Glomus intraradices, G. fasciculatum, G. monosporum and G. mosseae) and their relation to growth and metabolic activities of soybean plants were investigated in a greenhouse experiment. In general, mycorrhizal inoculation significantly increased the growth responses, phosphorus and nitrogen contents, acid and alkaline phosphatases as well as total soluble protein of soybean compared to non-mycorrhizal plants. Stimulation was related to the viability of each mycorrhizal fungus. The localization of succinate dehydrogenase (as a vital stain of metabolically active fungus) and alkaline phosphatase activity (as a potential marker of efficiency of the symbiosis) in the arbuscular mycorrhizal fungi were variable. The activity appeared in young arbuscles and intercellular hyphae, whereas the collapsed arbuscules were inactive. The histochemical staining results demonstrated that the activity of alkaline phosphatase fungi was lower than succinate dehydrogenase. The use of nitroblue tetrazolium chloride as a vital stain for SDH activity showed that all mycorrhizal infection revealed by trypan blue staining was not physiologically active. Thus, the possible utilization of these enzymes to assess the activity of mycorrhizal fungi and its relation with effectively for plant growth and mineral contents is discussed.     

Effects of defoliation and symbiosis on polyamine levels in pine and birch

 We report the effect of ectomycorrhizal fungi (Suillus variegatus, Paxillus involutus) and defoliation on polyamine concentrations in pine (Pinus silvestris) and birch (Betula pendula) foliage and roots. Symbiotic root tips showed consistently higher concentrations of putrescine than non-symbiotic roots. Partial defoliation had no effect on the polyamine levels in mycorrhizal pine or birch roots. The foliage of mycorrhizal pine seedlings had lower putrescine concentrations and higher spermidine than foliage of non-mycorrhizal plants, and defoliation reversed this pattern. The response to partial defoliation differed in birch foliage: mycorrhizal status had no effect and all new growth after defoliation had higher spermidine levels than in non-defoliated birch. The potential role of polyamines in mycorrhizal symbiosis is discussed. Accepted: 26 February 1997     

Specialized cheating of the ectomycorrhizal symbiosis by an epiparasitic liverwort

Many non-photosynthetic vascular plants in 10 diverse families obtain all of their carbon from fungi, but in most cases the fungi and the ultimate sources of carbon are unknown. In a few cases, such plants have been shown to be epiparasitic because they obtain carbon from neighbouring green plants through shared mycorrhizal fungi. In all such cases, the epiparasitic plants have been found to specialize upon narrow lineages of ecto- or arbuscular mycorrhizal fungi. Here we show that a non-vascular plant, the non-photosynthetic liverwort Cryptothallus mirabilis, is epiparasitic and is specialized on Tulasnella species that form ectomycorrhizae with surrounding trees at four locations in England, France and Portugal. By using microcosm experiments we show that the interaction with Tulasnella is necessary for growth of Cryptothallus, and by using labelling experiments we show that (14)CO(2) provided to birch seedlings is transferred to Cryptothallus by Tulasnella. This is one of the first documented cases of epiparasitism by a non-vascular plant and of ectomycorrhizal formation by Tulasnella. These results broaden the emerging association between epiparasitism and mycorrhizal specialization into a new class of plants and a new order of fungi.     

Changes in carbon allocation and expression of carbon transporter genes in Betula pendula Roth. colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr.

Comparative analyses of aspects of the carbon (C) physiology and the expression of C transporter genes in birch (Betula pendula Roth.) colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. were performed using mycorrhizal (M) and non‐mycorrhizal (NM) plants of similar foliar nutrient status. After six months of growth, the biomass of M plants was significantly lower than that of NM plants. Diurnal C budgets of both sets of plants revealed that M plants exhibited higher rates of photosynthesis and root respiration expressed per unit dry weight. However, the diurnal net C gain of M and NM plants remained similar. Ectomycorrhizal roots contained higher soluble carbohydrate pools and increased activity of cell wall invertase, suggesting that additional C was allocated to these roots and their ectomycorrhizal fungi consistent with an increased sink demand for C due to the presence of the mycobiont. In M roots, the expression of two hexose and one sucrose transporter genes of birch were reduced to less than one‐third of the expression level observed in NM roots. Analysis using a probe against the birch ribosomal internal transcribed spacer region revealed that M roots contained 22% less plant RNA than NM roots. As the expression of birch hexose and sucrose transporter genes was reduced to a much greater extent, this suggests that these specific genes were down‐regulated in response to alterations in C metabolism within M roots.     

Correction to: Performance of cowpea as influenced by native strain of rhizobia,lime and phosphorus in Samaru,Nigeria

Garden thyme (Thymus vulgaris L., Lamiaceae) is an important aromatic herb used for its medicinal values including antioxidant and antimicrobial properties. The present study was performed to analyze the changes in natural antioxidants after inoculation of in vitro propagated garden thyme plants with arbuscular mycorrhizal fungi (AMF). An efficient and low-cost protocol for large-scale multiplication of this aromatic plant was developed. The explants were cultured on full and half strength Murashige and Skoog (MS) medium containing indole-3-butyric acid (IBA). The maximum number of shoots and roots was obtained on ½ MS medium supplemented with 0.1 mg L^?1 IBA after 4 weeks of culture. The successfully adapted in vitro plants (survival rate 95%) were inoculated with arbuscular mycorrhizal fungi (Claroideoglomus claroideum, ref. EEZ 54). Plants were then transferred into field conditions. Mycorrhizal fungi enhanced the activity of some soil enzymes, acid and alkaline phosphatase, urease as well as the levels of extractable glomalin-related proteins in plant rhizosphere. Arbuscular mycorrhizal associations with higher plants promote the accumulation of antioxidant metabolites such as phenols and flavonoids and increase the activity of antioxidant enzymes. The results from the present study suggest enhanced antioxidant capacity of the inoculated T. vulgaris plants which was due mainly to increased accumulation of phenolic compounds (total phenols and flavonoids) together with stimulation of the activity of superoxide dismutase (SOD) and guaiacol peroxidase (GPO).     

Arbuscular mycorrhizal fungal application to improve growth and tolerance of wheat (Triticum aestivum L.) plants grown in saline soil

A pot experiment was conducted to examine the effects of three different arbuscular mycorrhizal fungi, Glomus mosseae, G. deserticola and Gigaspora gergaria, on growth and nutrition of wheat (Triticum aestivium L. cv. Henta) plants grown in saline soil. Under saline condition, mycorrhizal inoculation significantly increased growth responses, nutrient contents, acid and alkaline phosphatases, proline and total soluble protein of wheat plants compared to non-mycorrhizal ones. Those stimulations were related to the metabolic activity of the each mycorrhizal fungus. The localization of succinate dehydrogenase “SDH” (as a vital stain for the metabolically active fungus) in the arbuscular mycorrhizal fungi was variable. In general, mycorrhizal shoot plant tissues had significantly higher concentrations of P, N, K and Mg but lower Na concentration than those of non-mycorrhizal plants. In saline soil, growth and nutrition of wheat plants showed a high degree of dependency on mycorrhizal fungi (especially G. mosseae). The use of the nitroblue tetrazolium chloride method as a vital stain for SDH activity showed that all the structures of mycorrhizal infections in the wheat plant estimated by the trypan blue staining (non-vital stain) were not metabolically active. Interestingly, the reduction in Na uptake along with associated increases in P, N and Mg absorption and high proline, phosphatase activities and chlorophyll content in the mycorrhizal plants could be important for salt alleviation in plants growing in saline soils.     

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.     

No significant contribution of arbuscular mycorrhizal fungi to transfer of radiocesium from soil to plants

The diffuse pollution by fission and activation products following nuclear accidents and weapons testing is of major public concern. Among the nuclides that pose a serious risk if they enter the human food chain are the cesium isotopes 137Cs and 134Cs (with half-lives of 30 and 2 years, respectively). The biogeochemical cycling of these isotopes in forest ecosystems is strongly affected by their preferential absorption in a range of ectomycorrhiza-forming basidiomycetes. An even more widely distributed group of symbiotic fungi are the arbuscular mycorrhizal fungi, which colonize most herbaceous plants, including many agricultural crops. These fungi are known to be more efficient than ectomycorrhizas in transporting mineral elements from soil to plants. Their role in the biogeochemical cycling of Cs is poorly known, in spite of the consequences that fungal Cs transport may have for transfer of Cs into the human food chain. This report presents the first data on transport of Cs by these fungi by use of radiotracers and compartmented growth systems where uptake by roots and mycorrhizal hyphae is distinguished. Independent experiments in three laboratories that used different combinations of fungi and host plants all demonstrated that these fungi do not contribute significantly to plant uptake of Cs. The implications of these findings for the bioavailability of radiocesium in different terrestrial ecosystems are discussed.     

Carbon and nitrogen metabolism in mycorrhizal networks and mycoheterotrophic plants of tropical forests: a stable isotope analysis

Most achlorophyllous mycoheterotrophic (MH) plants obtain carbon (C) from mycorrhizal networks and indirectly exploit nearby autotrophic plants. We compared overlooked tropical rainforest MH plants associating with arbuscular mycorrhizal fungi (AMF) to well-reported temperate MH plants associating with ectomycorrhizal basidiomycetes. We investigated (13)C and (15)N abundances of MH plants, green plants, and AMF spores in Caribbean rainforests. Whereas temperate MH plants and fungi have higher δ(13)C than canopy trees, these organisms displayed similar δ(13)C values in rainforests, suggesting differences in C exchanges. Although temperate green and MH plants differ in δ(15)N, they display similar (15)N abundances, and likely nitrogen (N) sources, in rainforests. Contrasting with the high N concentrations shared by temperate MH plants and their fungi, rainforest MH plants had lower N concentrations than AMF, suggesting differences in C/N of exchanged nutrients. We provide a framework for isotopic studies on AMF networks and suggest that MH plants in tropical and temperate regions evolved different physiologies to adapt in diverging environments.     

Phosphatase activity of extra-radical arbuscular mycorrhizal hyphae: A review

Phosphatase activity of arbuscular mycorrhizal (AM) fungi has attracted attention in three fairly distinct domains: intracellular enzymes with defined metabolic functions that have been studied in intraradical hyphae, histochemical staining of alkaline phosphatase as an indicator of fungal activity measured both intra- and extraradically, and extracellular activity related to mineralization of organic P (P_o) compounds that may enhance mycorrhizal utilization of an important nutrient pool in soil. This review focuses on the latter subjects with emphasis on extraradical mycelium (ERM), while it draws on selected data from the vast material available concerning phosphatases of other organisms. We conclude that histochemical staining of alkaline phosphatase is a sensitive and suitable method for monitoring the effect of adverse conditions encountered by ERM both as a symbiotically functional entity in soil, and in vitro without modifying interference of soil or other solid substrates. Furthermore, the quantitative importance of extracellular enzymes for P nutrition of AM plants is estimated to be insignificant. This is concluded from the low quantitative contribution extracellular hyphae of AM fungi give to the total phosphatase activity in soil, and from estimations of which processes that may be rate limiting in organic P mineralization. Maximum values for the former is in the order of a few percent. As for the latter, solubilization of P_o seems to be far more important than P_o hydrolysis for utilization of P_o by AM fungi and plants, as both endogenous soil phosphatase activity and phosphatases of other soil organisms are ubiquitous and abundant. Our discussion of mycorrhizal phosphatases supports the view that extracellular phosphatases of roots and micro-organisms are to a large extent released incidentally into soil, and that the source has limited benefit from its activity. This revised version was published online in June 2006 with corrections to the Cover Date.     

No Significant Contribution of Arbuscular Mycorrhizal Fungi to Transfer of Radiocesium from Soil to Plants

The diffuse pollution by fission and activation products following nuclear accidents and weapons testing is of major public concern. Among the nuclides that pose a serious risk if they enter the human food chain are the cesium isotopes ¹³⁷Cs and ¹³⁴Cs (with half-lives of 30 and 2 years, respectively). The biogeochemical cycling of these isotopes in forest ecosystems is strongly affected by their preferential absorption in a range of ectomycorrhiza-forming basidiomycetes. An even more widely distributed group of symbiotic fungi are the arbuscular mycorrhizal fungi, which colonize most herbaceous plants, including many agricultural crops. These fungi are known to be more efficient than ectomycorrhizas in transporting mineral elements from soil to plants. Their role in the biogeochemical cycling of Cs is poorly known, in spite of the consequences that fungal Cs transport may have for transfer of Cs into the human food chain. This report presents the first data on transport of Cs by these fungi by use of radiotracers and compartmented growth systems where uptake by roots and mycorrhizal hyphae is distinguished. Independent experiments in three laboratories that used different combinations of fungi and host plants all demonstrated that these fungi do not contribute significantly to plant uptake of Cs. The implications of these findings for the bioavailability of radiocesium in different terrestrial ecosystems are discussed.     

Fungi associated with terrestrial orchid mycorrhizas,seeds and protocorms

The identity and ecological role of fungi in the mycorrhizal roots of 25 species of mature terrestrial orchids and in 17 species of field incubated orchid seedlings were examined. Isolates of symbiotic fungi from mature orchid mycorrhizas were basidiomycetes primarily in the generaCeratorhiza, Epulorhiza andMoniliopsis; a few unidentified taxa with clamped hyphae were also recovered. More than one taxon of peloton-forming fungus was often observed in the cleared and stained mycorrhizas. AlthoughCeratorhiza andEpulorhiza strains were isolated from the developing protocorms, pelotons of clamped hyphae were often presents in the cleared protocorms of several orchid species. These basidiomycetes are difficult to isolate and may be symbionts of ectotrophic plants. The higher proportion of endophytes bearing clamp connections in developing seeds than in the mycorrhizas is attributed to differences in the nutritional requirements of the fully mycotrophic protocorms and partially autotrophic plants. Most isolates ofCeratorhiza differed enzymatically fromEpulorhiza in producing polyphenol oxidases. Dual cultures with thirteen orchid isolates and five non-orchid hosts showed that some taxa can form harmless associations with non-orchid hosts. It is suggested that most terrestrial orchid mycorrhizas are relatively non-specific and that the mycobionts can be saprophytes, parasites or mycorrhizal associates of other plants.     

Root surface phosphatase activities and uptake of 32P-labelled inositol phosphate in field-collected gray birch and red maple roots

 This study examined select, naturally-occurring tree mycorrhizae for differences related to efficiency of organic phosphorus hydrolysis in forest soils. We investigated the activity of several phosphatases and root respiration in field-collected ectomycorrhizae of American beech and gray birch and VAM of red maple. Root materials were collected in the early and late growing season from a common soil type. American beech occurred in a late-successional stand, whereas gray birch and red maple grew in a mid-successional stand. All of the root types examined had phosphatase activities with p-nitrophenyl phosphate, bis-p-nitrophenyl phosphate and phytic acid and thus the potential to mineralize monoester and diester forms of organic phosphorus. Rates of hydrolysis at pH 5.0 were greatest with p-nitrophenyl phosphate. Although enzyme activity varied with season and ectomycorrhizal morphotype, VAM roots of red maple consistently had the lowest enzyme activities on a length and dry weight basis. Comparison of ³²P uptake from inositol phosphate by gray birch and red maple roots suggested that phosphomonoesterase activity was linked to P uptake from this source. Differences between species in oxygen consumption rates were less pronounced than those observed for enzymatic activities, suggesting similar short-term energy demands by the root types examined. The quantitative differences observed between plants growing on a common soil potentially relate to differences in host demand or reflect differences in basic morphology and/or physiology of associated mycobionts. Further study is necessary to understand the importance of these enzymes in the functional ecology of mycorrhizal fungi. Accepted: 20 December 1996     

菌根真菌提高植物抗旱性机制的研究回顾与展望

菌根真菌与全世界约97％的维管植物具有广泛的共生关系.大量研究结果显示菌根植物相比于非菌根植物对于干旱胁迫具有更高的耐受性,说明菌根真菌在植物抗旱过程中发挥着重要作用.本文对近年来国内外在菌根真菌协助植物抵御干旱作用机制方面的研究进行了归纳和总结,主要包括在干旱胁迫下菌根真菌对植物生理学特性的影响机制、菌根真菌提高植物...     

菌根真菌与植物共生营养交换机制研究进展

菌根是陆地生态系统普遍存在的、由土壤中的菌根真菌侵染宿主植物根系形成的联合共生体.菌根的建立是以共生体双方的营养交换为基础的:菌根真菌从土壤中吸收氮、磷等营养物质并转运给宿主植物,供其生长;作为交换,植物则以脂质或糖的形式向菌根真菌提供其生长所必需的碳水化合物.近年来,菌根真菌与宿主植物间的营养交换机制一直是研究的热点,国内外对菌根真菌介导的植物营养物质吸收和转运机制的研究也取得了巨大进展.本文综述了丛枝和外生两种菌根真菌与宿主植物间营养交换的最新研究进展,尤其是碳、氮、磷等几种重要营养物质的吸收与双向转运机制,以及营养交换在菌根形成中的潜在调控作用,并对目前存在的关键问题和未来研究方向进行了分析和展望,这对菌根模型的建立及菌根效益的优化具有重要意义.     

Boron uptake by ectomycorrhizas of silver birch

Boron (B) is an essential micronutrient for plants but it is thought not to be essential for fungi. We studied whether the extraradical mycelia of Paxillus involutus in symbiosis with silver birch (Betula pendula) take up B and transport it to the host plant. We grew mycorrhizal plants in flat microcosms with a partitioning wall, below which there was only extraradical mycelium. A boric acid solution enriched in ¹⁰B was applied to these mycelia. Increased ¹⁰B/¹¹B isotope ratios were subsequently measured in birch leaves, stems, and roots plus mycorrhizas in the upper compartment. Boron was therefore taken up by the mycorrhizal mycelia and transported to the host plant in this species combination.     

Compatible and Incompatible Interactions Between Callus Tissue and Mycorrhizal or Pathogenic Fungi

Tissue cultures of Nicotiana tabacum were utilized to investigate the mechanisms associated with host specificity and non-host incompatibility in mycorrhizal and pathogenic fungi. They were tested for expression of resistance to different species of mycorrhizal fungi and to a fungal pathogen of tobacco, Thielaviopsis basicola , by monitoring the production of callose, phenolic compounds and peroxidases in dual cultures. Tobacco cells reacted to the presence of all the mycorrhizal fungi with callose deposits, whereas callose was nearly always absent in tobacco cells inoculated with their pathogen T. basicola. The broad-host range ectomycorrhizal fungi Hebeloma crustuliniforme, Lac-caria laccata and Suilhis granulatus elicited less intense responses than did Hymenoscyphus ericae. The results obtained for phenolic production and peroxidase activity were consistently similar to those obtained for callose deposition. They showed that H. ericae , an endomycorrhizal symbiont of Ericaceae, was highly incompatible with tobacco cells and that the tobacco pathogen T. basicola did not elicit strong reactions in the cells of its host. In this paper, the possibility of utilizing callus cultures as a simple model system to study both the different degrees of compatibility and the early events of recognition between mycorrhizal fungi and their host or non-host plants is discussed.     

In vitro degradation of the moss Hylocomium splendens by three pleosporalean fungi

Three darkly pigmented species of conidial fungi of the family Pleosporaceae isolated from plants colonizing the Saskatchewan Glacier forefield were examined for potential roles in the degradation of moss gametophytes. Curvularia inaequalis and Ulocladium atrum isolated from bryophytes Ditrichum flexicaule and Tortella tortuosa , respectively, and Chalastospora gossypii from Saxifraga oppositifolia were inoculated onto autoclaved gametophytes of the moss Hylocomium splendens. All three species of fungi caused mass losses of the moss gametophytes. In vitro enzymatic tests revealed that all three fungi degraded cellulose, while none degraded insoluble polyphenols. When this material was examined by scanning electron microscopy, it was evident that the fungi had eroded the outer wall layer of the moss leaf cells to some extent but not the inner layer containing more lignin-like compounds. Once the outer wall layer was removed, the cells easily disarticulated. It is proposed that accumulations of these phenolics-rich leaf fragments subsequently ameliorate the rooting environment for vascular plants and have the potential to support the growth of basidiomycetes and other fungi, potentially mycorrhizal with pioneer vascular plants.     

Molecular identification of mycorrhizal fungi in <Emphasis Type="Italic">Dactylorhiza sambucina</Emphasis> (Orchidaceae)

We amplified and sequenced internal transcribed spacer (ITS) region of the nuclear ribosomal repeat from fungi in roots of Dactylorhiza sambucina (Orchidaceae) and used the extended database to identify the mycorrhizal fungi. We molecularly identified three ITS recovered from D. sambucina roots, one belonging to Rhizoctonia group, and two to ascomycetes, for the first time in Orchidoidae. In many cases, two sequence types were found from the same orchid root, providing that two taxa may be involved in mycorrhizal formation (multiple mycobiont colonization). Moreover, we demonstrated that D. sambucina plants, irrespective of their colour polymorphism, possess roots containing several fungi belonging to both asco- and basidiomycetes.     

Comparative study of key phosphorus and nitrogen metabolizing enzymes in mycorrhizal and non-mycorrhizal plants of Dendrobium chrysanthum Wall. ex Lindl.

The role of mycorrhizal fungi in overcoming nutrient limitation to plant growth by enhancing nutrient acquisition, especially phosphorus (P) and nitrogen (N), is well documented. However, in orchids, the importance of mycorrhizal fungi in nutrient acquisition is not as extensively studied as in other plants. Therefore, an in vitro culture system to study the effects of mycorrhizal association on P and N metabolizing enzymes, viz., acid phosphatase, alkaline phosphatase, nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) in Dendrobium chrysanthum was developed. After 90 days of mycorrhizal treatment, activities of acid phosphatase, alkaline phosphatase, NR, NiR and GS were higher in mycorrhizal plantlets than in control plantlets. The hardened plantlets that were initially treated with mycorrhiza under in vitro conditions also showed higher activities of the enzymes investigated. These mycorrhizal plantlets showed higher survival (96.33 %), shoot length (3.7 cm) and shoot fresh weight (0.359 g) as compared to control after 120 days of hardening. The results presented in this study suggest that mycorrhizal association might have increased the assimilation of P and N in D. chrysanthum plantlets, indicating the importance of mycorrhiza in orchids.