The promoting role of an isolate of dark-septate fungus on its host plant Saussurea involucrata Kar. et Kir.

A dark-septate endophytic (DSE) fungus EF-37 was isolated from the roots of Saussurea involucrata Kar. et Kir., an endangered Chinese medicinal plant. The molecular identification of the fungus was based on internal transcribed spacer regions and the result showed that EF-37 was congeneric to Mycocentrospora. This study was conducted to clarify the influence of the root endophyte EF-37 on the host plant S. involucrata using material grown in a sterile culture bottle. After cultivation for 40 days, fungal hyphae were found to be branching repeatedly and forming “hyphae nets” in the epidermal layers. Significant differences were detected between the study groups in plant dry weight, plant height, root dry weight, shoot dry weight, and the number of hair root tips. There was a positive effect of endophyte EF-37 on plant root development, with results showing that cortical cells dissolved and formed aerate structures. There was a positive effect of endophyte EF-37 on plant growth, but chlorophyll fluorescence analysis showed that there were no significant differences between the study groups. In addition, analysis of the chemical composition of seedlings showed that the level of rutin was higher in plants cultivated with the EF-37 fungus compared to the controls. This study helps to establish a basis for germplasm conservation and for further investigation of the interaction between dark-septate fungi and this alpine plant.     

Formation of structures resembling ericoid mycorrhizas by the root endophytic fungus Heteroconium chaetospira within roots of Rhododendron obtusum var. kaempferi

A resynthesis study was conducted to clarify the relationship between the root endophyte, Heteroconium chaetospira and the ericaceous plant, Rhododendron obtusum var. kaempferi. The host plant roots were recovered 2 months after inoculation, and the infection process and colonization pattern of the fungus were observed under a microscope. The hyphae of H. chaetospira developed structures resembling ericoid mycorrhizas, such as hyphal coils within the host epidermal cells. These structures were morphologically the same as previously reported ericoid mycorrhizal structures. The frequencies of hyphal coils within the epidermal cells of host roots ranged from 13 to 20%. H. chaetospira did not promote or reduce host plant growth. This is the first reported study that H. chaetospira is able to form structures resembling mycorrhizas within the roots of ericaceous plants.     

A New Fungal Endophyte,Scolecobasidium humicola,Promotes Tomato Growth under Organic Nitrogen Conditions

A new fungal endophyte, Scolecobasidium humicola, was identified as a common dark septate endophytic fungal (DSE) species under both natural and agricultural conditions. This fungus was found to grow endophylically in the roots of tomato seedlings. Light microscopy of cross-sections of colonized tomato roots showed that the intercellular, pigmented hyphae of the fungus were mostly limited to the epidermal layer and formed outer mantle-like structures. Two isolates of S. humicola, H2-2 and F1-3, have shown the ability to increase plant biomass with an organic nitrogen source. This finding is the first report of S. humicola as an endophyte and could help to improve plant growth with organic nitrogen sources.     

<Emphasis Type="Italic">Mycena</Emphasis> sp., a mycorrhizal fungus of the orchid <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

An endophytic fungus, F-23, was isolated from the roots of Dendrobium officinale Kimura et Migo, an endangered Chinese medicinal plant. The sequence of the ITS region indicated that the isolate belongs to the genus Mycena. After 4 months of inoculation, the root systems of D. officinale that were inoculated with F-23 fungus were much larger than the control’s root systems. We also observed that the hyphae of F-23 penetrated the epidermal cells within the host’s roots and spread from cell to cell. A large number of pelotons existed in the root cortical cells of D. officinale inoculated with F-23 fungus. Intracellular hyphae crossing through the host walls were also observed using SEM (scanning electron microscopy). In contrast, light microscopy and SEM showed that the transverse sections of the roots of control plants remained uncolonized. Therefore, the F-23 fungus can form mycorrhizal associations with the roots of its host plant, D. officinale, and enhance the growth of seedlings and roots. In brief, Mycena sp. was identified and shown to be a mycorrhizal fungus of the epiphytic orchid, D. officinale. This might be of potential use to the mass cultivation of D. officinale under artificial conditions.     

Effects of a dark-septate endophytic isolate LBF-2 on the medicinal plant <Emphasis Type="Italic">Lycium barbarum</Emphasis> L.

Dark septate endophytes (DSE) are ubiquitous root associated fungi; however, our understanding of their ecological function remains unclear. Here, we investigated the positive effect of a DSE fungus on its host plant Lycium barbarum L. A DSE isolate, LBF-2, isolated from the roots of L. barbarum, was inoculated onto the roots of plants, which were grown under greenhouse conditions for five weeks. The result of molecular analyses of internal transcribed spacer regions indicated that LBF-2 was 96% similar to Paraphoma chrysanthemicola. Melanized septate hyphae were observed in the root cortical cells of L. barbarum using a light microscope. Inoculation with LBF-2 increased the total biomass by 39.2% and also enhanced chlorophyll fluorescence. Inoculation increased the concentration of total chlorophyll by 22.8% and of chlorophyll a by 21.3%, relative to uninoculated controls. These data indicate that the LBF-2 isolate might be used to facilitate the cultivation of L. barbarum, which has medicinal applications.     

Anatomical study on the interaction between the root endophytic fungus <Emphasis Type="Italic">Heteroconium chaetospira</Emphasis> and Chinese cabbage

Chinese cabbage roots colonized by the dematiaceous fungal taxon Heteroconium chaetospira were previously found to become highly resistant to clubroot and Verticillium yellows. The dematiaceous fungus possesses an endophytic nature, but no detailed anatomical studies on endophyte–host plant interactions have so far been provided. Light and electron microscopy revealed that hyphae of H. chaetospira were abundant on and inside the root epidermal cells by 3 weeks following inoculation. The penetration pegs easily breached into epidermal cells, and the infection hyphae penetrated into cortical cells. Some appressorium-like swollen structures formed from intracellular hyphae, but no visible degradation of the host cell walls was evident where the hyphae contacted. No visible signs of host reactions and no invagination of the host plasma membrane around the hyphae were seen in the host cells. By 8 weeks following inoculation, masses of closely packed fungal cells had been formed in some cells of the epidermis and cortical layers, but further hyphal ingress was halted, mostly in the inner cortical cell layer. Thus, root vascular cylinders remained intact.     

Colonisation patterns of root tissues byPhytophthora nicotianae var.parasitica related to reduced disease in mycorrhizal tomato

Tomato plants pre-colonised by the arbuscular mycorrhizal fungusGlomus mosseae showed decreased root damage by the pathogenPhytophthora nicotianae var.parasitica. In analyses of the cellular bases of their bioprotective effect, a prerequisite for cytological investigations of tissue interactions betweenG. mosseae andP. nicotianae v.parasitica was to discriminate between the hyphae of the two fungi within root tissues. We report the use of antibodies as useful tools, in the absence of an appropriate stain for distinguishing hyphae ofP. nicotianae v.parasitica from those ofG. mosseae inside roots, and present observations on the colonisation patterns by the pathogenic fungus alone or during interactions in mycorrhizal roots. Infection intensity of the pathogen, estimated using an immunoenzyme labelling technique on whole root fragments, was lower in mycorrhizal roots. Immunogold labelling ofP. nicotianae v.parasitica on cross-sections of infected tomato roots showed that inter or intracellular hyphae developed mainly in the cortex, and their presence induced necrosis of host cells, the wall and contents of which showed a strong autofluorescence in reaction to the pathogen. In dual fungal infections of tomato root systems, hyphae of the symbiont and the pathogen were in most cases in different root regions, but they could also be observed in the same root tissues. The number ofP. nicotianae v.parasitica hyphae growing in the root cortex was greatly reduced in mycorrhizal root systems, and in mycorrhizal tissues infected by the pathogen, arbuscule-containing cells surrounded by intercellularP. nicotianae v.parasitica hyphae did not necrose and only a weak autofluorescence was associated with the host cells. Results are discussed in relation to possible processes involved in the phenomenon of bioprotection in arbuscular mycorrhizal plants.     

Septate endophyte colonization and host responses of grasses and forbs native to a tallgrass prairie

Native tallgrass prairies support distinct dark septate endophyte (DSE) communities exemplified by Periconia macrospinosa and Microdochium sp. that were recently identified as common root symbionts in this system. Since these DSE fungi were repeatedly isolated from grasses and forbs, we aimed to test their abilities to colonize different hosts. One Microdochium and three Periconia strains were screened for colonization and growth responses using five native grasses and six forbs in an in vitro system. Previously published data for an additional grass (Andropogon gerardii) were included and reanalyzed. Presence of indicative inter- and intracellular structures (melanized hyphae, microsclerotia, and chlamydospores) demonstrated that all plant species were colonized by the DSE isolates albeit to varying degrees. Microscopic observations suggested that, compared to forbs, grasses were colonized to a greater degree in vitro. Host biomass responses varied among the host species. In broad comparisons, more grass species than forbs tended to respond positively to colonization, whereas more forb species tended to be non-responsive. Based on the suspected differences in the levels of colonization, we predicted that tallgrass prairie grasses would support greater DSE colonization than forbs in the field. A survey of field-collected roots from 15 native species supported this hypothesis. Our study supports the “broad host range” of DSE fungi, although the differences in the rates of colonization in the laboratory and in the field suggest a greater compatibility between grasses and DSE fungi. Furthermore, host responses to DSE range from mutualism to parasitism, suggesting a genotype-level interplay between the fungi and their hosts that determines the outcome of this symbiosis.     

Dark Septate Root Endophytic Fungus <Emphasis Type="Italic">Nectria haematococca</Emphasis> Improves Tomato Growth Under Water Limiting Conditions

The ascomycetous dark septate endophytic (DSE) fungi characterized by their melanized hyphae can confer abiotic stress tolerance in their associated plants in addition to improving plant growth and health. In this study inoculation of the DSE fungus Nectria haematococca Berk. & Broome significantly improved all the plant growth parameters like the plant height, stem girth, leaf characteristics and plant biomass of drought-stressed tomato. Root characters like the total root length, primary root diameter, 2nd order root number and diameter, root hair number and length were also significantly influenced by the fungal inoculation. Nevertheless, N. haematococca inoculation did not affect root colonization by native arbuscular mycorrhizal (AM) fungi and no significant correlation existed between the AM and DSE fungal variables examined. The proline accumulation in shoots of N. haematococca inoculated plants was significantly higher than uninoculated plants. The present study clearly indicates for the first time the ability of the DSE fungus, N. haematococca in inducing the drought stress tolerance and promoting the growth of the host plant under water stress.     

<Emphasis Type="Italic">Heteroconium chaetospira</Emphasis>, a dark septate root endophyte allied to the Herpotrichiellaceae (Chaetothyriales) obtained from some forest soil samples in Canada using bait plants

During an extended search in Western Canada for fungal root endophytes useful as biocontrol agents against soil-borne pathogens, we isolated Heteroconium chaetospira, as well as Phialocephala fortinii or similar taxa, from seven samples of forest soil using herbaceous seedlings of four different species (i.e., barley, Chinese cabbage, eggplant, and melon) as bait plants. Our results support a previous observation that eggplant is a particularly effective species for baiting H. chaetospira from soil and confirm the ability of this fungus to grow as an endophyte in the roots of axenically reared host plants. Cultural characters show that this species is similar to P. fortinii and other melanized fungi in the dark septate endophyte (DSE) group (e.g., Leptodontidium orchidicola, P. sphaeroides, and Cadophora finlandica) in that it produces darkly pigmented colonies on agar media. Heteroconium chaetospira differs from P. fortinii and other melanized members of the Leotiomycetes in the DSE group in that its conidia are fusiform and develop in blastic acropetal chains. Heteroconium chaetospira is phylogenetically distant from most DSE taxa because DNA sequences for the nuclear small subunit (SSU) ribosomal RNA gene (rDNA) indicate that the taxon is affiliated with the Herpotrichiellaceae of the Chaetothyriales rather than with the Leotiomycetes.     

Effects of the mycorrhizal fungus Glomus intraradices on uranium uptake and accumulation by Medicago truncatula L. from uranium-contaminated soil

Phytostabilization strategies may be suitable to reduce the dispersion of uranium (U) and the overall environmental risks of U-contaminated soils. The role of Glomus intraradices, an arbuscular mycorrhizal (AM) fungus, in such phytostabilization of U was investigated with a compartmented plant cultivation system facilitating the specific measurement of U uptake by roots, AM roots and extraradical hyphae of AM fungi and the measurement of U partitioning between root and shoot. A soil-filled plastic pot constituted the main root compartment (C_A) which contained a plastic vial filled with U-contaminated soil amended with 0, 50 or 200 mg KH₂PO₄−P kg^–1soil (C_B). The vial was sealed by coarse or fine nylon mesh, permitting the penetration of both roots and hyphae or of just hyphae. Medicago truncatula plants grown in C_A were inoculated with G. intraradices or remained uninoculated. Dry weight of shoots and roots in C_A was significantly increased by G. intraradices, but was unaffected by mesh size or by P application in C_B. The P amendments decreased root colonization in C_B, and increased P content and dry weight of those roots. Glomus intraradices increased root U concentration and content in C_A, but decreased shoot U concentrations. Root U concentrations and contents were significantly higher when only hyphae could access U inside C_B than when roots could also directly access this U pool. The proportion of plant U content partitioned to shoots was decreased by root exclusion from C_B and by mycorrhizas (M) in the order: no M, roots in C_B > no M, no roots in C_B > M, roots in C_B > M, no roots in C_B. Such mycorrhiza-induced retention of U in plant roots may contribute to the phytostabilization of U contaminated environments.     

Positive association between mycorrhiza and foliar endophytes in Poa bonariensis, a native grass

The interaction between mycorrhiza and leaf endophytes (Neotyphodium sp.) was studied in three Poa bonariensis populations, a native grass, differing significantly in endophyte infection. The association between endophytes and mycorrhizal fungi colonisation was assessed by analysing plant roots collected from the field. We found that roots from endophyte-infected populations showed a significantly higher frequency of colonisation by mycorrhizal fungi and that soil parameters were not related to endophyte infection or mycorrhiza colonization. In addition, we did not observe significant differences in the number of AM propagules in soils of the three populations sites. We also report the simultaneous development of Paris-type and Arum-type mycorrhiza morphology within the same root systems of P. bonariensis. The co-occurrence of both colonisation types in one and the same root system found in the three populations, which differed in Neotyphodium infection, suggests that foliar endophytes do not determine AM morphology. The percentage of root length colonised by different types of fungal structures (coils, arbuscules, longitudinal hyphae and vesicles) showed significant and positive differences in arbuscular frequency associated with endophyte infection, whereas the much smaller amounts of vesicles and hyphal coils did not differ significantly.     

Effects of anthracene on development of an arbuscular mycorrhizal fungus and contribution of the symbiotic association to pollutant dissipation

The influence of anthracene, a low molecular weight polycyclic aromatic hydrocarbon (PAH), on chicory root colonization by Glomus intraradices and the effect of the root colonization on PAH degradation were investigated in vitro. The fungus presented a reduced development of extraradical mycelium and a decrease in sporulation, root colonization, and spore germination when exposed to anthracene. Mycorrhization improved the growth of the roots in the medium supplemented containing 140 mg l⁻¹ anthracene, suggesting a positive contribution of G. intraradices to the PAH tolerance of roots. Anthracene disappearance from the culture medium was quantified; results suggested that nonmycorrhizal chicory roots growing in vitro were able to contribute to anthracene dissipation, and in addition, that mycorrhization significantly enhanced anthracene dissipation. These monoxenic experiments demonstrated a positive contribution of the symbiotic association to anthracene dissipation in the absence of other microorganisms. In addition to anthracene dissipation, intracellular accumulation of anthracene was detected in lipid bodies of plant cells and fungal hyphae, indicating intracellular storage capacity of the pollutant by the roots and the mycorrhizal fungus.     

A comparison between hairy root cultures and wild plants of Saussurea involucrata in phenylpropanoids production

Saussurea involucrata is an important medicinal plant that produces a few bioactive secondary metabolites, such as hispidulin, rutin, and syringin. Previously, we established a hairy root culture system for this species through Agrobacterium-mediated transformation. The present study addressed the issue as how hairy root cultures perform in phenylpronoid accumulation. From the ethanolic extract of a hairy root culture established for Saussurea involucrata, syringin, rutin and hispidulin, were isolated and their chemical structures were confirmed by HPLC-ESI-MS. A quantitative study of the compounds showed great levels of syringin and hispidulin (being 43.5±1.13 and 0.34±0.023 mg g⁻¹ dry weight, respectively), about 40 and 3 times, respectively, higher than those from wild plants. But, the levels of rutin from hairy roots were much lower (0.71±0.043 vs. 6.59±0.56 mg g⁻¹ dry weight). Compared with untransformed root cultures, syringin and hispidulin levels were also higher. An experiment on culture media showed that MS was superior to others for phenylpropanoids accumulation in hairy roots, a 28-day culture produced 405 mg l⁻¹ syringin.     

Nitrogen form influences the response of Deschampsia antarctica to dark septate root endophytes

Fungi with dematiaceous septate hyphae, termed dark septate endophytes (DSE), are common in plant roots, particularly in cold-stressed habitats, but their effects on their host plants remain obscure. Here, we report a study that assessed the effects of six DSE on the growth and nutrient balance of Deschampsia antarctica when plants were supplied with the same amount of nitrogen in organic (casein hydrolysate) or inorganic (ammonium sulphate) form under controlled conditions. After 60 days, the DSE, that had each been isolated from D. antarctica and which analyses of internal transcribed spacer and large subunit regions indicated were similar to members of the Helotiales (Oculimacula yallundae, Mollisia and Tapesia spp.) and unassigned anamorphic ascomycetes, typically had no effect on, or reduced by 33–71%, shoot and root dry weights relative to uninoculated controls when plants had been supplied with nitrogen in inorganic form. In contrast, the DSE usually enhanced shoot and root dry weights by 51–247% when plants had been supplied with organic nitrogen. In the presence of inorganic nitrogen, only sporadic effects of DSE were recorded on shoot and root nitrogen or phosphorus concentrations, whereas in the presence of organic nitrogen, three to six of the DSE isolates increased shoot and root nitrogen and phosphorus contents. Most of the isolates decreased the phosphorus concentrations of shoots and roots when plants had been supplied with nitrogen in organic form. Our data suggest that DSE are able to mineralise peptides and amino acids in the rhizosphere, making nitrogen more freely available to roots.     

濒危药用植物桃儿七根的显微结构及其菌根真菌分布研究

本文研究了桃儿七Sinopodophyllum hexandrum根的显微结构及其真菌分布。结果表明,桃儿七的根为根状茎,节状,不定根形成的须根系发达。根的结构主要由表皮、皮层、维管柱三部分构成,其中,皮层所占比例最大,超过80%。根的木质部有四原型和五原型两种类型,五原型较为常见;四原型的根和五原型的根在皮层细胞形态上存在一定差异。在桃儿七的不定根和其上的侧根观察到真菌菌丝分布,其数量和种类与根的直径有关,在不定根较细(先端)的部位真菌以暗色有隔内生真菌(DSE真菌)为主,侵染率为77.9%;而较粗根中真菌菌丝为无隔菌丝为主,分布很少且仅存在于皮层细胞的一至二层,不侵染皮层深部和维管柱。不定根侧根中真菌以丛枝菌根真菌为主,丛枝菌根常常占据大部分的皮层细胞,侵染率高达90%以上。桃儿七根中没有发现根毛存在,因此,侧根中共生的丛枝菌根真菌可能是桃儿七养分和水分吸收的主要途径。     

Arbuscular mycorrhizal morphology and dark septate fungal associations in medicinal and aromatic plants of Western Ghats, Southern India

We investigated roots of 107 medicinal and aromatic plants (MAPs) in the Western Ghats region of Southern India for arbuscular mycorrhizal (AM) and dark septate endophyte (DSE) associations. Of the 107 MAPs belonging to 98 genera in 52 families examined, 79 were AM and 38 harbored a DSE association. Typical Arum- and Paris-type mycorrhizas are first reported in the presumed nonmycorrhizal family Amaranthaceae. Similarly, DSE associations are recorded for the first time in nine plant families and 37 plant species. Thirty MAPs had both AM and DSE associations. The number of MAPs having Arum-type mycorrhiza was greater than those having Paris-type. This was more prominent among herbaceous plants than in trees where the Paris-type was predominant. Similarly, the Arum-type was more prevalent in annuals than in perennials. DSE associations were more frequent in herbs and perennials compared to other MAPs.     

Litter N retention over winter for a low and a high phenolic species in the alpine tundra

Mycorrhizal fungus colonization of roots may modify plant metal acquisition and tolerance. In the present study, the contribution of the extraradical mycelium of an arbuscular mycorrhizal (AM) fungus, Glomus mosseae (BEG 107), to the uptake of metal cations (Cu, Zn, Cd and Ni) by cucumber (Cucumis sativus) plants was determined. The influence of the amount of P supplied to the hyphae on the acquisition and partitioning of metal cations in the mycorrhizal plants was also investigated. Pots with three compartments were used to separate root and root-free hyphal growing zones. The shoot concentration of Cd and Ni was decreased in mycorrhizal plants compared to non-mycorrhizal plants. In contrast, shoot Zn and Cu concentrations were increased in mycorrhizal plants. High P supply to hyphae resulted in decreased root Cu concentrations and shoot Cd and Ni concentrations in mycorrhizal plants. These results confirm that some elements required for plant growth (P, Zn, Cu) are taken up by mycorrhizal hyphae and are then transported to the plants. Conversely, Cd and Ni were transported in much smaller amounts by hyphae to the plant, so that arbuscular mycorrhizal fungus colonization could partly protect plants from toxic effects of these elements. Selective uptake and transport of plant essential elements over non-essential elements by AM hyphae, increased growth of mycorrhizal plants, and metal accumulation in the root may all contribute to the successful growth of mycorrhizal plants on metal-rich substrates. These effects are stimulated when hyphae can access sufficient P in soil.     

Fungal endophytes in Astromyelon-type (Sphenophyta,Equisetales, Calamitaceae) roots from the Upper Pennsylvanian of France

A distinctive fungal endophyte, Cashhickia acuminata nov. gen. et sp., is described from permineralized calamite roots from the Upper Pennsylvanian Grand-Croix cherts of France. Heavily infected roots contain numerous intracellular hyphae in the outer cortex that arise from a meshwork-like mycelium extending between cortical cells. All intracellular hyphae are oriented toward the root center; none occur on the inner periclinal host cell walls. Other roots of the same type show localized infection by this fungus in which isolated cortical cells contain or give rise to intracellular fungal growth. Within the cortical cells are host responses in the form of callosities that indicate the roots were alive at the time of infection. Other endophytes are present in the same host tissue but are less frequent. The discovery of this association provides the first detailed account on the morphology of a Carboniferous fungal root endophyte, as well as the spatial distribution within the host, and infection pathways within the cortical tissues.     

Experiments on competitiveness of three endomycorrhizal fungi

Summary This paper examines competition between three VA endophytes places respectively as “inoculant” fungus in an inner compartment (300 ml) below the germinating seed, and as “indigenous” fungus in an outer compartment (900 ml). Previously prepared infested soil was used as source of the endophytes. Inner and outer compartment roots were harvested separately and percentage infection determined for each fungus, using diagnostic anatomical features of each fungus. Changes in colonisation patterns were followed up to 4 and 5 months. Treatments with non-infested soil in the outer compartment were used to compare infectivity and spread of the different endophytes. All three endophytes were able to invade soil already colonised by either of the other two, butGlomus fasciculatum (E₃) was more invasive thanGlomus tenue. This pattern appeared to be related to rates of spread of the fungi in the non-infested soil. The pattern was not affected by host, clover or sorghum, or by position, immediately below the seed or in the outer compartment. It seemed that the endophyte in the inner compartment had overriding and persistent effects on plant growth, unrelated to its dominance pattern or share of overall root infection at the later harvests.