Inoculation of <Emphasis Type="Italic">Pinus halepensis</Emphasis> Mill. with selected ectomycorrhizal fungi improves seedling establishment 2 years after planting in a degraded gypsum soil

Vegetative inoculum of Amanita ovoidea (Bull.) Link and three isolates of Suillus collinitus (Fr.) Kuntze, as well as spore inoculum of Rhizopogon roseolus (Corda) Th. M. Fr. and S. collinitus, were evaluated for the production of Pinus halepensis Mill. in nursery and for the establishment of seedlings in a degraded gypsum soil. In nursery, most of the fungi significantly improved the height of seedlings and modified the accumulation of nutrients in needles. The percentage of ectomycorrhizas (ESR) per seedling ranged from 25 to 78%, depending on the fungi. One and 2 years after planting in the field, the survival of seedlings was significantly improved by inoculation with two isolates of S. collinitus and with spores of the same fungus. Inoculation with A. ovoidea had no significant effect on seedling survival, whilst R. roseolus caused a significant mortality of seedlings. Seedling height was significantly improved by inoculation with all fungi except R. roseolus and isolate CCMA-1 of S. collinitus. One year after planting, mycorrhization of control seedlings was negligible, and percentages of ESR were under 38% for the rest of treatments. In spring of the second year, seedlings in all treatments, including the control, became highly mycorrhizal (60–77% of ESR). Low ectomycorrhizal diversity (five morphotypes described) and seasonal variation on morphotype composition were detected 2 years after plantation. From a perspective of soil restoration management under limiting environmental conditions, nursery inoculation with selected fungi can be a key advantage for tree seedlings to surmount the initial transplant stress, assuring their establishment in the field. Our results emphasise the importance of selecting compatible fungal–host species combinations for nursery inoculation and sources of inoculum adapted to the environmental conditions of the transplantation site.     

Effects of pH,water stress and temperature on in vitro cultures of ectomycorrhizal fungi from Mediterranean forests

A comparative study of the growth of eight species of ectomycorrhizal fungi [Hebeloma edurum Métrod ex Bon, Lactarius deliciosus (L.: Fr.) S.F. Gray., Rhizopogon roseolus (Corda) Th.M.Fr., Suillus collinitus (Fr.) Kuntze, Suillus granulatus (L.: Fr.) Roussel, Suillus luteus (L.: Fr.) Roussel, Tricholoma focale (Fr.) Ricken, Tricholoma striatum (Schaeff.) Sacc.] in pure culture at different pH values, water potentials and temperatures was carried out. The biomass production and mycelial growth of each strain were assessed and characterised morphologically. H. edurum and L. deliciosus showed a clear affinity for neutral or slightly basic media. Most species had the lowest colony diameters and biomass production at pH 2.5. Only H. edurum, R. roseolus and S. collinitus grew at the highest water stress levels (−13.85 and −15.45 bar). All species grew at the lowest values of temperature whereas only R. roseolus and S. collinitus did at 30 °C.     

The effect of ectomycorrhizal fungi on Zn uptake and distribution in seedlings of Pinus sylvestris L

Seedlings of pine, infected with two different ectomycorrhizal fungi, Suillus bovinus (Fr.) O. Kuntze and an unidentified isolate (BP), were exposed to various external Zn concentrations. An additional strain of S. bovinus, cultured on a Zn-enriched medium, was also used. The effect of mycorrhizal associations on Zn uptake and distribution within the plant was determined by AAS.The results show that nonmycorrhizal seedlings have the capability to control the uptake and translocation of Zn to the shoot until the external Zn concentration reaches a threshold level, where no limitation of uptake is possible. Excess Zn is accumulated in the root system to protect the shoot against toxic tissue concentrations. The effect of an ectomycorrhizal infection on Zn uptake and distribution depends on (1) the fungal species (2) the external concentration and (3) the Zn content of the fungal culture medium. Under conditions of low external Zn supply, especially a mycorrhizal infection with S. bovinus led to an increased Zn uptake in root and needles of Pinus sylvestris. Under high external conditions the mycobionts varied considerably in their capability to reduce the transport of Zn to the shoot. Only by an infection with S. bovinus the plant was able to maintain the shoot tissue concentration on a low level. This effect can be enhanced by pretreatment of S. bovinus with high Zn concentrations.     

Suitability of mycorrhiza-defective mutant/wildtype plant pairs (Solanum lycopersicum L. cv Micro-Tom) to address questions in mycorrhizal soil ecology

The ectomycorrhizal (ECM) fungi associated with Pinus thunbergii seedlings grown on sand dune were identified by molecular method, and the diversity of bacteria associated with ECM and Extraradical mycelium were examined by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified 16S rDNA. The mycorrhizal formation rate of 1-year old P. thunbergii seedlings was more than 95%. Cenococcum geophilum was the most dominant ECM fungus, followed by T01, RFLP-8, Russula spp., and Suillus sp. Bacterial community was most diverse with C. geophilum- and RFLP-8-mycorrhiza. Sequencing analysis showed that Burkholderia spp. and Bradyrhizobium spp. were on the surface of ECM short root of seven ECM. The fungi detected as extraradical mycelium using DGGE of 18S rDNA were Suillus bovinus and RFLP-8-mycorrhiza. Bacterial community on the extraradical mycelium was more diverse than those on ECM root tip. Burkholderia spp. and Bradyrhizobium spp. were found also on extraradical mycelium.     

Phylogenetic Analysis of a Dataset of Fungal 5.8S rDNA Sequences Shows That Highly Divergent Copies of Internal Transcribed Spacers Reported from Scutellospora castanea Are of Ascomycete Origin

Using a dataset comprising 5.8S rDNA sequences from a wide range of fungi, we show that some sequences reported recently from the arbuscular mycorrhizal (AM) fungus Scutellospora castanea most likely originate from Ascomycetes. Other ITS and 5.8S sequences which were previously reported are confirmed as being clearly of mycorrhizal origin and are variable within one isolate of S. castanea. However, these results mean that previous conclusions which were drawn regarding the heterokaryotic status of AM fungal spores remain unproven. We provide an enlarged 5.8S rDNA dataset that can be used to check ITS sequences for conflicts with well-established phylogenies of the organisms that they were obtained from.     

Allergic contact dermatitis caused by mushrooms

The first author is allergic to skin contact with mushrooms of Suillus americanus, S. granulatus, S. grevillei, S. luteus, or S. neoalbidipes. Symptoms develop between one and two days after contact and last for approximately a week, disappearing completely without treatment. Symptoms consist of reddening, swelling, and itching, at the sites of contact with pileus cuticle mucilage of all five species. Pore layer tissues (tested for S. americanus and S. luteus) also produced strong reactions, as did pileus trama (tested for S. luteus). Spores from spore prints (tested for S. americanus and S. luteus) produced no reaction. The reaction can be avoided by wearing gloves when handling allergenic species and by washing hands promptly after working with these species. Similar cases, reported from North America, Europe, and Russia, involve Agaricus, Boletus, Lactarius, Paxillus, Ramaria, and Suillus species. Several cases involve allergy to multiple species or genera. Symptom severity varies, presumably with intensity of exposure. In one case, symptoms were renewed following ingestion. Most cases demonstrate delayed allergic contact sensitivity.     

Morphological analysis of early contacts between pine roots and two ectomycorrhizal Suillus strains

 Selection of ectomycorrhizal strains for application in forestry is mostly based on the evaluation of symbiotic performance in small-scale experiments. Two Suillus collinitus strains isolated from a Mediterranean and an alpine area were inoculated onto two pine tree species (Pinus pinea and P. nigra ssp. laricio var. corsicana) typical of these two environments. The early events during contact between the cell surfaces of plant and fungal partners were analysed morphologically using ultrastructural and immunocytochemical techniques. All four plant-fungus combinations led to a similar degree of mycorrhizal infection and to a similar colonization pattern. The first contact of fungal hyphae with root cap cells usually involved breakdown of the outermost electron-opaque layer of the plant cell walls. Hyphae further developed between this layer and the underlying wall strata. Ultrastructural observations revealed that S. collinitus strain J3-15-24, isolated from a Mediterranean area, induced a defence reaction in the roots of P. nigra, which grows typically in alpine areas. These observations suggest functional differences between the two fungal strains in their mycorrhizal capabilities. Accepted: 3 April 1998     

白及内生真菌多样性研究

白及( Bletilla striata)是兰科地生型多年生植物,也是我国传统中药材之一。利用菌根技术进行白及的保护和人工栽培,需要获得白及可培养的内生真菌。该研究以广西野生的白及根和叶为材料,采用分离培养法分离内生真菌,并结合真菌形态特征,及其核糖体的转录间隔区( ITS)序列分析,确定内生真菌的分类地位。结果表明：从2株白及植物90块组织中分离获得37株内生真菌,鉴定为15个分类单元,由9个属组成,分属于2门4纲7目8科,包括锤舌菌纲( Leotiomycetes)、座囊菌纲( Dothideomycetes)和粪壳菌纲( Sordariomy-cetes),伞菌纲( Agaricomycetes)。从根中分离获得内生真菌12种,蜡壳菌属为优势属;从叶中分离获得内生真菌3种,刺盘孢属为优势属;刺盘孢菌属( Colletotrichum)和蜡壳菌属( Sebacina)真菌的相对多度值均达到20％;4株担子菌均分布于根中,叶组织中未有分布。根组织中内生真菌的多样性指数(H＝1.863)高于叶组织(1.098)。该研究结果及其所分离培养的担子菌类真菌,为更好地利用菌根技术进行白及等兰科植物资源的保护与可持续利用奠定了基础。     

Intra-isolate heterogeneity of the ITS region of rDNA in Pythium helicoides

Heterogeneity of the rDNA ITS region in Pythium helicoides and the phylogenetic relationship between P. helicoides and closely related species were investigated. In PCR-RFLP analysis of the rDNA ITS region of six P. helicoides isolates investigated, including the type culture, intraspecific variation was found at the HhaI site. The total length of fragments was longer than before cutting, indicating sequence heterogeneity within isolates. Digestion of the cloned rDNA ITS region derived from seven isolates with HhaI revealed polymorphisms among and within single zoospore isolates, and variability of the region was also present among the clones derived from the same isolate. To test whether the rDNA ITS region of closely related species and other regions in the genome of P. helicoides are also variable, the rDNA ITS region of P. ultimum and the cytochrome oxydase II (cox II) gene encoded in mitochondria were sequenced. P. ultimum had little variation in the rDNA ITS region. The cox II gene sequences of both species revealed only a low intraspecific variability and no intra-isolate variation. In the phylogenic tree based on the rDNA ITS sequences, all clones of P. helicoides formed one large clade that was distinct from the clades comprising morphologically similar species, such as P. oedochilum and P. ostracodes, and was closely related to P. chamaehyphon rather than the other species.     

Copper-Adapted Suillus luteus, a Symbiotic Solution for Pines Colonizing Cu Mine Spoils

Natural populations thriving in heavy-metal-contaminated ecosystems are often subjected to selective pressures for increased resistance to toxic metals. In the present study we describe a population of the ectomycorrhizal fungus Suillus luteus that colonized a toxic Cu mine spoil in Norway. We hypothesized that this population had developed adaptive Cu tolerance and was able to protect pine trees against Cu toxicity. We also tested for the existence of cotolerance to Cu and Zn in S. luteus. Isolates from Cu-polluted, Zn-polluted, and nonpolluted sites were grown in vitro on Cu- or Zn-supplemented medium. The Cu mine isolates exhibited high Cu tolerance, whereas the Zn-tolerant isolates were shown to be Cu sensitive, and vice versa. This indicates the evolution of metal-specific tolerance mechanisms is strongly triggered by the pollution in the local environment. Cotolerance does not occur in the S. luteus isolates studied. In a dose-response experiment, the Cu sensitivity of nonmycorrhizal Pinus sylvestris seedlings was compared to the sensitivity of mycorrhizal seedlings colonized either by a Cu-sensitive or Cu-tolerant S. luteus isolate. In nonmycorrhizal plants and plants colonized by the Cu-sensitive isolate, root growth and nutrient uptake were strongly inhibited under Cu stress conditions. In contrast, plants colonized by the Cu-tolerant isolate were hardly affected. The Cu-adapted S. luteus isolate provided excellent insurance against Cu toxicity in pine seedlings exposed to elevated Cu levels. Such a metal-adapted Suillus-Pinus combination might be suitable for large-scale land reclamation at phytotoxic metalliferous and industrial sites.     

Preparation and regeneration of protoplasts of three fungi of Boletaceae

Protoplasts of three fungi of Boletaceae,Suillus luteus, S. grevillei, andBoletinus cavipes, were prepared with yields of 45, 8.0, and 1.8×10⁷/g fresh mycelia under the optimal conditions, respectively. Nucleate protoplasts accounted for 42% of the whole preparation ofS. luteus and 32% of that ofS. grevillei, and 21% of the nucleate protoplasts ofS. luteus and 35% of those ofS. grevillei possesed two nuclei. Regeneration efficiency of protoplasts was 0.4% forS. luteus and 0.05% forS. grevillei. The regeneration ofB. cavipes protoplasts was also confirmed. Optimal conditions for regeneration were determined. Addition of gellan gum instead of agar to the medium and activated charcoal treatment of agar medium increased the regeneration efficiency significantly.     

外生菌根菌对油松幼苗抗氧化酶活性及根系构型的影响

研究盆栽条件下,接种褐环乳牛肝菌(Suillus luteus)和红汁乳菇(Lactarius hatsudake)对油松(Pinus tabulaeformis)苗木生长,抗氧化酶活性和根系构型的影响。结果表明:(1)褐环乳牛肝菌和红汁乳菇均可与油松合成外生菌根,并显著提高苗木的苗高、地径、鲜重和干重。(2)接种褐环乳牛肝菌苗木针叶抗氧化酶(SOD、CAT、POD)活性分别比接种红汁乳菇苗木高14.77%、20.77%、34.68%;接种褐环乳牛肝菌苗木根系抗氧化酶(SOD、CAT、POD)活性分别比接种红汁乳菇苗木高8.54%,4.34%,33.31%;接种处理苗木抗氧化酶活性均显著高于不接种处理。接种处理苗木根长、表面积、体积、平均直径、根尖数、分叉数显著高于不接种处理;接种处理间苗木只有根尖数存在显著差异;接种褐环乳牛肝菌的油松根系分支成80°—90°的一级侧根数占23.81%,显著多于接种红汁乳菇和对照苗木。研究表明,接种褐环乳牛肝菌和红汁乳菇均能促进油松苗木生长,提高油松体内抗氧化酶活性,扩大苗木根系的吸收范围,其中褐环乳牛肝菌各方面的促进效果要优于红汁乳菇。     

Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China

To investigate the diversity of root endophytes in Rhododendron fortunei, fungal strains were isolated from the hair roots of plants from four habitats in subtropical forests of China. In total, 220 slow-growing fungal isolates were isolated from the hair roots of R. fortunei. The isolates were initially grouped into 17 types based on the results of internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with known sequences of ericoid mycorrhizal endophytes and selected ascomycetes or basidiomycetes. Based on phylogenetic analysis of the ITS sequences in GenBank, 15 RFLP types were confirmed as ascomycetes, and two as basidiomycetes; nine of these were shown to be ericoid mycorrhizal endophytes in experimental cultures. The only common endophytes of R. fortunei were identified as Oidiodendron maius at four sites, although the isolation frequency (3–65%) differed sharply according to habitat. Phialocephala fortinii strains were isolated most abundantly from two habitats which related to the more acidic soil and pine mixed forests. A number of less common mycorrhizal RFLP types were isolated from R. fortunei at three, two, or one of the sites. Most of these appeared to have strong affinities for some unidentified root endophytes from Ericaceae hosts in Australian forests. We concluded that the endophyte population isolated from R. fortunei is composed mainly of ascomycete, as well as a few basidiomycete strains. In addition, one basidiomycete strain was confirmed as a putative ericoid mycorrhizal fungus.     

Influence of ectomycorrhizal fungi on the response of Sitka spruce and Japanese larch to forms of phosphorus

Ectomycorrhizal fungi (Paxillus involutus, Suillus grevillei and two unidentified basidiomycetes from excised Sitka spruce mycorrhizas) were isolated from stands of Sitka spruce either in monoculture or in a mixture with Japanese larch in an Irish conifer plantation. The growth of these fungi and their mycorrhizal formation in Sitka spruce and Japanese larch were examined after incubation in modified Melin-Norkrans medium containing either KH₂PO₄, Ca₃(PO₄)₂ or Fe phytate as the phosphorus (P) source. P. involutus and S. grevillei utilized all three P sources. The unidentified basidiomycetes had limited ability to utilize Fe phytate. Basidiomycete 1 showed poor growth on KH₂PO₄ whereas growth of basidiomycete 2 was low on Ca₃(PO₄)₂. Pure culture synthesis studies confirmed that P. involutus and the two basidiomycetes formed mycorrhizas with both tree species but S. grevillei was mycorrhizal only on Japanese larch. P. involutus formed more mycorrhizas in both conifers than the other fungi. Following inoculation with each of the four fungi, shoot and root dry mass of both Sitka spruce and Japanese larch seedlings was enhanced compared with uninoculated/nonmycorrhizal controls. On Fe phytate, Paxillus-inoculated Sitka spruce seedlings had the lowest primary root length and on KH₂PO₄, Suillus-inoculated Japanese larch had the greatest number of short roots. The only differences when Sitka spruce seedlings were grown in either monoculture or in a mixture with Japanese larch mycorrhizal with S. grevillei were primary root length and number of short roots after growth on media containing Fe phytate.     

Molecular phylogenetic and morphological analyses of Oidium heveae, a powdery mildew of rubber tree

Powdery mildew of rubber tree caused by Oidium heveae is an important disease of rubber plantations worldwide. Identification and classification of this fungus is still uncertain because there is no authoritative report of its morphology and no record of its teleomorphic stage. In this study, we compared five specimens of the rubber powdery mildew fungus collected in Malaysia, Thailand, and Brazil based on morphological and molecular characteristics. Morphological results showed that the fungus on rubber tree belongs to Oidium subgen. Pseudoidium. Nucleotide sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region and the large subunit rRNA gene (28S rDNA) were conducted to determine the relationships of the rubber powdery mildew fungus and to link this anamorphic fungus with its allied teleomorph. The results showed that the rDNA sequences of the two specimens from Malaysia were identical to a specimen from Thailand, whereas they differed by three bases from the two Brazilian isolates: one nucleotide position in the ITS2 and two positions in the 28S sequences. The ITS sequences of the two Brazilian isolates were identical to sequences of Erysiphe sp. on Quercus phillyraeoides collected in Japan, although the 28S sequences differed at one base from sequences of this fungus. Phylogenetic trees of both rDNA regions constructed by the distance and parsimony methods showed that the rubber powdery mildew fungus grouped with Erysiphe sp. on Q. phillyraeoides with 100% bootstrap support. Comparisons of the anamorph of two isolates of Erysiphe sp. from Q. phillyraeoides with the rubber mildew did not reveal any obvious differences between the two powdery mildew taxa, which suggests that O. heveae may be an anamorph of Erysiphe sp. on Q. phillyraeoides. Cross-inoculation tests are required to substantiate this conclusion.     

Isolation and identification of mycorrhizal fungi associating with an achlorophyllous plant, Epipogium roseum (Orchidaceae)

The identity of mycorrhizal fungi associated with the achlorophyllous orchid Epipogium roseum was investigated by DNA analysis. The fungi were isolated from each coiled hypha (peloton), and the ITS region of nuclear rDNA was sequenced. Phylogenetic analysis based on the neighbor-joining method showed that all the isolates clustered with fungi belonging to Psathyrella or Coprinus in Coprinaceae. Those fungi are known as saprobes, using dead organic materials for a nutritive source. Large colonies of this orchid were frequently found around tree stumps or fallen logs. In such colonies, these decaying wood materials would be used as a large and persistent carbon source for the growth of this orchid. This is the first report of Coprinaceae as an orchid mycorrhizal fungi.     

Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation

Mycorrhization helper bacteria (MHB), isolated from phylogenetically distinct ectomycorrhizal symbioses involving Lactarius rufus, Laccaria bicolor or Suillus luteus, were tested for fungus specificity to enhance L. rufus–Pinus sylvestris or L. bicolor–P. sylvestris mycorrhiza formation. As MHB isolated from the L. rufus and S. luteus mycorrhiza were originally characterised using a microcosm system, we assessed their ability to enhance mycorrhiza formation in a glasshouse system in order to determine the extent to which MHB are system-specific. Paenibacillus sp. EJP73, an MHB for L. rufus in the microcosm, significantly enhanced L. bicolor mycorrhiza formation in the glasshouse, demonstrating that the MHB effect of this bacterium is neither fungus-specific nor limited to the original experimental system. Although the five MHB strains studied were unable to significantly enhance L. rufus mycorrhiza formation, two of them did have a significant effect on dichotomous short root branching by L. rufus. The effect was specific to Paenibacillus sp. EJP73 and Burkholderia sp. EJP67, the two strains isolated from L. rufus mycorrhiza, and was not associated with auxin production. Altered mycorrhiza architecture rather than absolute number of mycorrhizal roots may be an important previously overlooked parameter for defining MHB effects.     

Ribosomal DNA variation within and among individuals ofLisianthius (Gentianaceae) populations

Restriction endonuclease fragment analysis of nuclear ribosomal DNA (rDNA) was completed on 25 individuals each from seven populations of theLisianthius skinneri (Gentianaceae) species complex in Panama. Seven restriction enzymes were used to determine the amount and type of rDNA variation within and among individuals of the populations. No restriction site variation was seen within populations or individuals although site differences were seen among populations. Spacer length variation within and among individuals of populations was mapped to the internal transcribed spacer (ITS) region between the 18S and 5.8S rRNA genes, a region inLisianthius rDNA that previously was shown to exhibit length differences among populations. This is the first reported case of such variation within and among individuals of populations for the ITS region. Presence or absence of ITS spacer length variation is not correlated with levels of isozymic heterozygosity within populations. No detectable length variation within individuals or populations was seen in the larger intergenic spacer (IGS). Although populations varied with respect to IGS length, all individuals of a given population had a single and equivalent IGS length.     

Isolation and identification of mycorrhizal fungi associated with <Emphasis Type="Italic">Stigmatodactylus sikokianus</Emphasis> (Maxim. ex Makino) Rauschert (Orchidaceae)

The mycorrhizal fungi of Stigmatodactylus sikokianus (Orchidaceae) were isolated and identified to be nearly related to Sebacina spp. in Sebacinaceae (Basidiomycota) by a neighbor-joining phylogenetic analysis based on the sequences of the ITS region of nuclear rDNA. In spite of the geographically separated samplings, high sequence similarity was found among the obtained DNA sequences, which suggested that S. sikokianus might be highly specialized to the group of fungi. It is known that Sebacina spp. are saprobes or ectomycorrhiza-forming fungi. The mycorrhizal fungi of S. sikokianus were regarded to be saprobic from the environment of their habitats.