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.     

Saprobic characteristics of three fungal taxa from ericalean roots and their association with the roots of Rhododendron groenlandicum and Picea mariana in culture

Simultaneous associations among ectotrophic and ericoid mycorrhizal hosts and their mycorrhizal fungi are expected in boreal bogs where ericaceous shrubs and conifers coexist rooted in an organic matrix dominated by Sphagnum mosses. We were thus prompted to examine, in vitro, the abilities of three ericoid mycorrhizal fungi [ Hymenoscyphus ericae, Oidiodendron maius, and Variable White Taxon (VWT)] to associate with Picea mariana (Pinaceae), with both P. mariana and Rhododendron groenlandicum (Ericaceae) simultaneously, and to decompose Sphagnum fuscum. Hymenoscyphus ericae and VWT developed an intracellular association with roots of P. mariana and with roots of R. groenlandicum. Two strains of O. maius did not form typical infection units in R. groenlandicum, nor did they colonize the root cells of P. mariana. Mass losses incurred by sterilized S. fuscum plants inoculated with these three taxa indicated that O. maius could be more efficient as a free-living saprophyte on this material than either H. ericae or VWT and may in part explain why atypical associations with the roots of ericaceous hosts were formed.     

Ericoid mycorrhizal colonization and associated fungal communities along a wetland gradient in the Acadian forest of Eastern Canada

Wetlands provide numerous ecosystem services, and ericaceous plants are important components of these habitats. However, the ecology of fungi associated with ericaceous roots in these habitats is poorly known. To investigate fungi associated with ericaceous roots in wetlands, ericoid mycorrhizal colonization was quantified, and fungal communities were characterized on the roots of Gaultheria hispidula and Kalmia angustifolia along two upland – forested wetland transects in spring and fall. Ericoid mycorrhizal colonization was significantly higher in the wetlands for both plant species. Both upland and wetland habitats supported distinct assemblages of ericaceous root associated fungi including habitat specific members of the genus Serendipita. Habitat was a stronger driver of ericoid mycorrhizal colonization and ericaceous root associated community composition than host or sampling season, with differences related to soil water content, soil nutrient content, or both. Our results indicate that ericaceous plant roots in forested wetlands are heavily colonized by habitat specific symbionts.     

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.     

Salinity and flooding stress effects on mycorrhizal and non-mycorrhizal citrus rootstock seedlings

Seedlings of the rootstocks Pineapple sweet orange (SwO), Carrizo citrange (CC), and sour orange (SO) were grown in low phosphorus (P) sandy soil and either inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus,Glomus intraradices, or were non-mycorrhizal (NM) and fertilized with P. VAM and NM seedings of similar shoot size and adequate P-status were selected for study of salinity and flooding stress. One-third of each of the VAM and NM plants were given 150 mM NaCl for a period of 24 days. One-third of the plants were placed into plastic bags and flooded for 21 days while the remaining third were non-stressed controls. In general, neither stress treatment affected mycorrhizal colonization. Salinity stress reduced the hydraulic conductivity of roots, leaf water potential, stomatal conductance and net assimilation of CO₂ (ACO₂) of mycorrhizal and non-mycorrhizal seedlings to a similar extent. VAM plants of CC and SO accumulated more Cl in leaves than NM plants. Cl was higher in non-mycorrhizal roots of SwO and CC than in mycorrhizal roots. Flooding the root zone for 3 weeks did not produce visible symptoms in the shoot but did influence plant water relations and reduce ACO₂ of all 3 rootstocks. VAM and NM plants of each rootstock were affected similarly by flooding. Comparable reduction in nitrogen and P content of both mycorrhizal and non-mycorrhizal plants suggested that flooding stress was primarily affecting root rather than hyphal nutrient uptake. Florida Agricultural Experimental Station Journal Series No. 7773.     

Chitinase in roots of mycorrhizal Allium porrum: regulation and localization

Chitinase (EC 3.2.1.14) activity was measured in roots of Allium prorrum L. (leek) during development of a vesicular-arbuscular mycorrhizal symbiosis with Glomus versiforme (Karst.) Berch. During the early stages of infection, between 10 and 20 d after inoculation, the specific activity of chitinase was higher in mycorrhizal roots than in the uninfected controls. However, 60–90 d after inoculation, when the symbiosis was fully established, the mycorrhizal roots contained much less chitinase than control roots. Chitinase was purified from A. porrum roots. An antiserum against beanleaf chitinase was found to cross-react specifically with chitinase in the extracts from non-mycorrhizal and mycorrhizal A. porrum roots. This antiserum was used for the immunocytochemical localization of the enzyme with fluorescent and gold-labelled probes. Chitinase was localized in the vacuoles and in the extracellular spaces of non-mycorrhizal and mycorrhizal roots. There was no immunolabelling on the fungal cell walls in the intercellular or the intracellular phases. It is concluded that the chitin in the fungal walls is inaccessible to plant chitinase. This casts doubts on the possible involvement of this hydrolase in the development of the mycorrhizal fungus. However, fungal penetration does appear to cause a typical defense response in the first stages that is later depressed.     

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.     

Ericoid mycorrhizal fungi: some new perspectives on old acquaintances

Many ericaceous species colonize as pioneer plants substrates ranging from arid sandy soils to moist mor humus, in association with their mycorrhizal fungi. Thanks to the symbiosis with ericoid mycorrhizal fungi, ericaceous plants are also able to grow in highly polluted environments, where metal ions can reach toxic levels in the soil substrate. For a long time this mycorrhizal type has been regarded as an example of a highly specific interaction between plants and fungi. More recent studies have been challenging this view because some ericoid mycorrhizal endophytes seem also able to colonise plants from very distant taxa. A molecular approach has allowed the investigation of genetic diversity and molecular ecology of ericoid mycorrhizal fungi, and has revealed that ericaceous plants can be very promiscuous, with multiple occupancy of their thin roots. The molecular analysis of sterile morphotypes involved in this symbiosis has also led to deeper understanding of the species diversity of ericoid fungi. Genetic polymorphism of ericoid fungi is wider than previously thought, and often increased by the presence of Group I introns in the nuclear small subunit rDNA.     

Molecular diversity of ericoid mycorrhizal endophytes isolated from Woollsia pungens

One hundred and sixty-eight sterile endophytic mycelia were isolated from roots of four Woollsia pungens (Cav.) F. Muell. (Epacridaceae) plants collected from a field site in New South Wales, Australia. All isolates formed typical ericoid mycorrhizal structures when inoculated onto roots of Vaccinium macrocarpon Ait. (Ericaceae). Microsatellite-primed PCR fingerprints generated using the primers (GTG)₅ and (GACA)₄ indicated that considerable genetic diversity exists within the endophyte population. It was estimated that a minimum of 43 genetically distinct mycelial genets were present in the root systems of the sampled W. pungens population, with most genets confined to individual plants. Two genets, however, were present within the root systems of two adjacent plants. While most genets were represented by less than eight isolates, three genets contained up to 41 isolates, suggesting that root system colonization by some endophytic mycelia might be extensive.     

The Structure and Function of the Ericoid Mycorrhizal Root

The uniformity of structure of the anatomically simple ericoidmycorrhizal hair root across many plant families, includingEpacridaceae, that are diagnostic of heathland, and the characteristicrestriction of its occurrence to nutrient impoverished soils,are both emphasized. The extent to which the predominantly ascomycetousfungal endophytes of these roots are taxonomically related isdiscussed. In functional terms, the role of the mycorrhiza innutrient mobilization is evaluated on the basis of experimentswith ericaceous plants. The considerable saprotrophic potentialof endophytes such asHymenoscyphus ericae is demonstrated andthe significance of this for nitrogen (N) and phosphorus (P)nutrition of plants growing in sclerophyllous litter of highC:N and C:P ratios is discussed. The need to carry out experimentsusing epacrid hosts is stressed. It is considered that the selectiveprovision, by ericoid mycorrhizal fungi, of access to recalcitrantorganic sources of N and P facilitates niche differentiationand so contributes to the maintenance of species diversity whichis a feature of heaths with a significant component of epacridor ericaceous plants particularly in the southern hemisphere. Ericoid mycorrhiza; hair root; nitrogen mobilization; heathland; Epacridaceae     

Exclusion of grass roots from soil organic layers by Calluna: the role of ericoid mycorrhizas

The role of ericoid mycorrhizal colonization in competition between the dwarf shrub Calluna vulgaris and coarse grass Nardus stricta was investigated. Nardus was grown alone, or in competition with Calluna, in a layered organic/sand substrate with and without inoculation with the ericoid mycorrhizal endophyte Hymenoscyphus ericae, and with and without the addition of nitrogen. Root length and allocation between different substrate layers was assessed along with plant biomass, nutrient uptake and mycorrhizal colonization. Calluna was the superior competitor for nutrients, probably because of its ability to concentrate root growth in the upper organic layer. In the presence of Calluna both the absolute amount and proportion of Nardus root length in the organic layer were reduced, and this reduction was greatest when Calluna was mycorrhizal. The presence of ericoid mycorrhizal colonization did not reduce Nardus shoot nutrient content or concentration, suggesting that ericoid mycorrhizal suppression of Nardus growth was not due to nutrient competition: alternative mechanisms of interference are discussed.     

Widespread association between the ericoid mycorrhizal fungus Rhizoscyphus ericae and a leafy liverwort in the maritime and sub-Antarctic

A recent study identified a fungal isolate from the Antarctic leafy liverwort Cephaloziella varians as the ericoid mycorrhizal associate Rhizoscyphus ericae. However, nothing is known about the wider Antarctic distribution of R. ericae in C. varians, and inoculation experiments confirming the ability of the fungus to form coils in the liverwort are lacking. Using direct isolation and baiting with Vaccinium macrocarpon seedlings, fungi were isolated from C. varians sampled from eight sites across a 1875-km transect through sub- and maritime Antarctica. The ability of an isolate to form coils in aseptically grown C. varians was also tested. Fungi with 98-99% sequence identity to R. ericae internal transcribed spacer (ITS) region and partial large subunit ribosomal (r)DNA sequences were frequently isolated from C. varians at all sites sampled. The EF4/Fung5 primer set did not amplify small subunit rDNA from three of five R. ericae isolates, probably accounting for the reported absence of the fungus from C. varians in a previous study. Rhizoscyphus ericae was found to colonize aseptically-grown C. varians intracellularly, forming hyphal coils. This study shows that the association between R. ericae and C. varians is apparently widespread in Antarctica, and confirms that R. ericae is at least in part responsible for the formation of the coils observed in rhizoids of field-collected C. varians.     

Nutrient transfer from soil nematodes to plants: a direct pathway provided by the mycorrhizal mycelial network

A pathway for the transfer of nutrients from dead nematodes to mycorrhizal plants is described for the first time. Plants of Betula pendula were grown in transparent microcosms in the mycorrhizal (M) or non‐mycorrhizal (NM) condition, either with or without nematode necromass of known nitrogen (N) and phosphorus (P) contents as the major potential source of these elements. Plants colonized by the mycorrhizal fungus Paxillus involutus produced greater yields and had larger N and P contents in the presence of nematodes than did their NM counterparts. The symbiotic systems were shown to exploit the N and P originally contained in necromass more effectively, and to transfer the nutrients to the plants in quantities approximately double those seen in NM systems. Even so, NM plants obtained sufficient N and P from dead nematodes to enable some enhancement of growth. Our observations confirm that mycorrhizal fungi provide the potential for the recycling of nutrients contained in this quantitatively important component of the soil mesofauna and demonstrate that the symbiotic pathway is considerably more effective than that provided by saprotrophs alone. The consequences of this nutrient transfer pathway for nutrient recycling in temperate forest ecosystems are considered.     

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     

Genetic diversity of root-associated fungal endophytes from Calluna vulgaris at contrasting field sites

A total of 107 putative ericoid mycorrhizal endophytes were isolated from hair roots of Calluna vulgaris from two abandoned arsenic/copper mine sites and a natural heathland site in southwest England. The endophytes were initially grouped as 14 RFLP types, based on the results of ITS-RFLP analysis using the restriction endonucleases Hin f I, Rsa I and Hae III. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with sequences for known ericoid mycorrhizal endophytes and selected ascomycetes. The majority of endophyte isolates (62–92%) from each site were identified as Hymenoscyphus ericae , but a number of other less common mycorrhizal RFLP types were also identified, all of which appear to have strong affinities with the order Leotiales. None of the less common RFLP types was isolated from C. vulgaris at more than one field site. Neighbour-joining analysis indicated similarities between the endophytes from C. vulgaris and mycorrhizal endophytes isolated from other Ericaceae and Epacridaceae hosts in North America and Australia.     

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.     

Soil persistence and biodiversity of ericoid mycorrhizal fungi in the absence of the host plant in a Mediterranean ecosystem

The occurrence of suitable mycorrhizal inocula may be an important factor affecting the dynamics of plant communities. We investigated the persistence and diversity of ericoid mycorrhizal fungi in the soil of a mature Quercus ilex forest where ericaceous hosts were absent. Erica arborea was used as a bait plant and results were compared to soil samples from experimental plots where cuttings had allowed reappearance of this ericaceous species. Fungal endophytes were isolated and tested in mycorrhiza resynthesis trials. Sterile mycorrhizal endophytes were assigned to morphotypes whose consistency was confirmed by ITS-RFLP. The ITS region of a representative of each morphotype was sequenced. BLAST searches and Neighbour-Joining analysis indicated taxonomic affinities with different classes within Ascomycota. Our results indicate that ericoid mycorrhizal fungi persist and maintain mycorrhizal ability in habitats lacking the ericaceous host. Their persistence could favour the establishment of E. arborea seedlings in pure Q. ilex forests after disturbance phenomena.     

Evidence that soil nutrient stoichiometry controls the competitive abilities of arbuscular mycorrhizal vs. root-borne non-mycorrhizal fungi

A majority of plant species has roots that are colonized by both arbuscular mycorrhizal (AM) and non-mycorrhizal (NM) fungi. The latter group may include plant mutualists, commensals, parasites and pathogens. The co-occurrence of these two broad groups may translate into competition for root volume as well as for plant-derived carbon (C). Here we provide evidence that the relative availability of soil nitrogen (N) and phosphorus (P) (i.e., soil nutrient stoichiometry) controls the competitive balance between these two fungal guilds. A decrease in the soil available N:P ratio resulted in a lower abundance of AM fungi and a corresponding increase in NM fungi. However, when the same fertilization treatments were applied in a soil in which AM fungi were absent, lowering the soil available N:P ratio did not affect NM fungal abundance. Taken collectively, our results suggest that the increase in NM fungal abundance was not a direct response to soil nutrient stoichiometry, but rather a competitive release from AM fungi responding negatively to higher soil P. We briefly discuss the mechanisms that may be responsible for this competitive release.     

Distribution of ericoid mycorrhizal endophytes and root-associated fungi in neighbouring Ericaceae plants in the field

Three hundred and twenty-seven fungal endophyte isolates were obtained from hair roots of neighbouring Woollsia pungens Cav. (Muell.) and Leucopogon parviflorus (Andr.) Lindl. (both Ericaceae) plants at an Australian dry sclerophyll forest site and mapped according to the root segments from which they were obtained. Restriction fragment length polymorphism (RFLP) analysis of the rDNA internal transcribed spacer (ITS) region indicated that the isolate assemblage comprised 21 RFLP-types (= putative taxa), five of which were shown in gnotobiotic culture experiments to be ericoid mycorrhizal endophytes. While two mycorrhizal RFLP-types were exclusive to either W. pungens or L. parviflorus, RFLP-type VI was isolated from both hosts. This putative taxon had strong ITS sequence identity with Helotiales ericoid mycorrhizal ascomycetes, comprised ca. 75% of all isolates from each plant and was spatially widespread in both root systems. Inter-simple sequence repeat PCR analysis indicated that two and four genotypes of RFLP-type VI were present in the W. pungens and L. parviflorus root systems respectively, however single genotypes appeared to dominate each root system. One genotype was present in both root systems. The data suggest that assemblages of ericoid mycorrhizal fungi from hair roots of individual Ericaceae plants in dry sclerophyll forest habitats are characterised by relatively low genetic diversity.     

Intracellular colonization of Rhododendron and Vaccinium roots by Cenococcum geophilum, Geomyces pannorum and Meliniomyces variabilis

Four in vitro experiments were set up to verify the colonization potential of ectomycorrhizal (EcM) Cenococcum geophilum FR. (strain CGE-4), saprotrophic Geomyces pannorum (LINK) SIGLER & CARMICHAEL (GPA-1) and a frequent root-associated, potentially ericoid mycorrhiza (ErM)-forming Meliniomyces variabilis Hambleton & Sigler (MVA-1) in roots of Rhododendron and Vaccinium. A typical ErM fungus, Rhizoscyphus ericae (Read) Zhuang & Korf (RER-1), was included for comparison. All fungal strains intracellularly colonized rooted Vaccinium microcuttings: GPA-1 occasionally produced hyphal loops similar to ErM, MVA-1 and RER-1 exhibited a typical ErM colonization pattern. CGE-4 hyphae grew vigorously on and around newly formed roots and rarely penetrated turgescent rhizodermal cells forming intracellular loose loops. Rooting of Rhododendron sp. microcuttings was not promoted by any fungal strain except CGE-4, which also promoted the most vigorous growth of Rhododendron ponticum L. seedlings. The widespread EcM fungus C. geophilum has a potential to colonize non-EcM roots and support their development which may influence overall growth of ericaceous plants. As shown for G. pannorum, structures resembling ErM may be formed by fungi that are to date not regarded as ericoid mycorrhizal.