Auxofuran, a novel metabolite that stimulates the growth of fly agaric, is produced by the mycorrhiza helper bacterium Streptomyces strain AcH 505

The mycorrhiza helper bacterium Streptomyces strain AcH 505 improves mycelial growth of ectomycorrhizal fungi and formation of ectomycorrhizas between Amanita muscaria and spruce but suppresses the growth of plant-pathogenic fungi, suggesting that it produces both fungal growth-stimulating and -suppressing compounds. The dominant fungal-growth-promoting substance produced by strain AcH 505, auxofuran, was isolated, and its effect on the levels of gene expression of A. muscaria was investigated. Auxofuran and its synthetic analogue 7-dehydroxy-auxofuran were most effective at a concentration of 15 microM, and application of these compounds led to increased lipid metabolism-related gene expression. Cocultivation of strain AcH 505 and A. muscaria stimulated auxofuran production by the streptomycete. The antifungal substances produced by strain AcH 505 were identified as the antibiotics WS-5995 B and C. WS-5995 B completely blocked mycelial growth at a concentration of 60 microM and caused a cell stress-related gene expression response in A. muscaria. Characterization of these compounds provides the foundation for molecular analysis of the fungus-bacterium interaction in the ectomycorrhizal symbiosis between fly agaric and spruce.     

WS-5995 B,an antifungal agent inducing differential gene expression in the conifer pathogen <Emphasis Type="Italic">Heterobasidion annosum</Emphasis> but not in <Emphasis Type="Italic">Heterobasidion abietinum</Emphasis>

The mycorrhization helper bacterium Streptomyces sp. AcH 505 inhibits Norway spruce root infection and colonisation by the root and butt rot fungus Heterobasidion annosum 005 but not by the congeneric strain Heterobasidion abietinum 331 because of higher sensitivity of H. annosum 005 towards the AcH 505-derived naphthoquinone antibiotic WS-5995 B. Differences in antibiotic sensitivity between two isolates belonging to two species, H. annosum 005 and H. abietinum 331, were investigated by comparative gene expression analysis using macroarrays and quantitative RT-PCR after WS-5995 B, structurally related mollisin and unrelated cycloheximide application. Treatment with 25 μM WS-5995 B for 2 h resulted in a significant up-regulation of expression of inosine-5′-monophosphate dehydrogenase, phosphoglucomutase and GTPase genes, while the expression of genes encoding for thioredoxin and glutathione dependent formaldehyde dehydrogenase was down-regulated in the sensitive fungal strain. No differential expression in the tolerant strain was detected. Application of WS-5995 B at higher concentrations over a time course experiment revealed that H. annosum 005 and H. abietinum 331 responded differently to WS-5995 B. The fungal gene expression levels depended on both the concentration of WS-5995 B and the duration of its application. The WS-5995 B-unrelated cycloheximide caused highly specific changes in patterns of gene expression. Our findings indicate considerable variations in response to bacterial metabolites by the isolates of the conifer pathogen.     

Suppression of plant defence response by a mycorrhiza helper bacterium

The aim of the present study was to determine whether the mycorrhiza helper bacterium Streptomyces sp. AcH 505 could serve as a biocontrol agent against Heterobasidion root and butt rot. Bacterial influence on mycelial growth of Heterobasidion sp. isolates, on the colonization of wood discs and Norway spruce (Picea abies) roots was determined. The effect of AcH 505 on plant photosynthesis, peroxidase activity and gene expression, and needle infections were investigated. AcH 505 was antagonistic to 11 of 12 tested fungal Heterobasidion isolates. The antagonism resulted in a suppression of fungal colonization of Norway spruce roots and wood discs. Mycelial growth rate of the 12th strain, Heterobasidion abietinum 331 was not affected by AcH 505, and colonization of roots by this fungal strain was promoted by AcH 505. Bacterial inoculation led to decreased peroxidase activities and gene expression levels in roots. AcH 505 promotes plant root colonization by Heterobasidion strains that are tolerant to antifungal metabolites produced by the bacterium. This may result from unknown bacterial factors that suppress the plant defence response.     

Extending the hyphal area of the ectomycorrhizal fungus Laccaria parva co-cultured with ectomycorrhizosphere bacteria on nutrient agar plate

The members of Proteobacteria have been frequently detected from ectomycorrhizal roots; however, their function in the mycelial growth of ectomycorrhizal fungi remains uncertain. This study examined the extension of the hyphal area of Laccaria parva co-cultured with the ectomycorrhizosphere bacteria. One mycelial disk of L. parva was placed on the center of a plastic dish containing diluted modified Melin-Norkrans agar media, and each bacterial strain was incubated 2?cm away from the mycelial disk at four orthogonal directions. Several strains of Rhizobiaceae, including those closely related to Bradyrhizobium, significantly increased the extension of the hyphal areas of several strains of L. parva; however, the majority of bacteria tended to decrease them. The effects of bacteria on the hyphal growth area differed according to the combination of strains of bacteria and L. parva in several cases, indicating that the interactions between bacteria and L. parva can be specific at the strain level.     

Growth of mycorrhizal jack pine (Pinus banksiana) and white spruce (Picea glauca) seedlings planted in oil sands reclaimed areas

The effectiveness of ectomycorrhizal inoculation at the tree nursery seedling production stage on growth and survival was examined in jack pine (Pinus banksiana) and white spruce (Picea glauca) planted in oil sands reclamation sites. The seedlings were inoculated with Hebeloma crustuliniforme strain # UAMH 5247, Suillus tomentosus strain # UAMH 6252, and Laccaria bicolor strain # UAMH 8232, as individual pure cultures and in combinations. These treatments were demonstrated to improve salinity resistance and water uptake in conifer seedlings. The field responses of seedlings to ectomycorrhizal inoculation varied between plant species, inoculation treatments, and measured parameters. Seedling inoculation resulted in higher ectomycorrhizal colonization rates compared with non-inoculated control, which had also a relatively small proportion of roots colonized by the nursery contaminant fungi identified as Amphinema byssoides and Thelephora americana. Seedling inoculation had overall a greater effect on relative height growth rates, dry biomass, and stem volumes in jack pine compared with white spruce. However, when examined after two growing seasons, inoculated white spruce seedlings showed up to 75 % higher survival rates than non-inoculated controls. The persistence of inoculated fungi in roots of planted seedlings was examined at the end of the second growing season. Although the inoculation with H. crustuliniforme triggered growth responses, the fungus was not found in the roots of seedlings at the end of the second growing season suggesting a possibility that the observed growth-promoting effect of H. crustuliniforme may be transient. The results suggest that the inoculation of conifer seedlings with ectomycorrhizal fungi could potentially be carried out on a large scale in tree nurseries to benefit postplanting performance in oil sands reclamation sites. However, these practices should take into consideration the differences in responses between the different plant species and fungal strains.     

The cultivable endophytic community of Norway spruce ectomycorrhizas from microhabitats lacking ericaceous hosts is dominated by ericoid mycorrhizal Meliniomyces variabilis

Most of the temperate conifers associate with ectomycorrhizal fungi, but their roots also harbour a wide range of endophytes. We focused on ascomycetes associating with basidiomycetous ectomycorrhizas of Norway spruce in a temperate montane forest in central Europe and found that the majority of the co-associated fungi belonged to the Rhizoscyphus ericae aggregate (REA), being dominated by Meliniomyces variabilis. We further tested the ability of representative isolates to colonize spruce root tips and European blueberry (Vaccinium myrtillus) hair roots in an agar system as well as their effect on blueberry growth in a peat-agar system. M. variabilis intracellularly colonized spruce (Picea abies) root tip cortex, formed ericoid mycorrhizas in blueberry and enhanced blueberry shoot and root growth in comparison with non-inoculated plants. Our findings suggest that spruce ectomycorrhizas may represent selective niches for ericoid mycorrhizal fungi in habitats lacking suitable ericaceous hosts.     

Changes in hyphal morphology and activity of phenoloxidases during interactions between selected ectomycorrhizal fungi and two species of <Emphasis Type="Italic">Trichoderma</Emphasis>

Patterns of phenoloxidase activity can be used to characterize fungi of different life styles, and changes in phenoloxidase synthesis were suspected to play a role in the interaction between ectomycorrhizal and two species of Trichoderma. Confrontation between the ectomycorrhizal fungi Amanita muscaria and Laccaria laccata with species of Trichoderma resulted in induction of laccase synthesis, and the laccase enzyme was bound to mycelia of ectomycorrhizal fungi. Tyrosinase release was noted only during interaction of L. laccata strains with Trichoderma harzianum and T. virens. Ectomycorrhizal fungi, especially strains of Suillus bovinus and S. luteus, inhibited growth of Trichoderma species and caused morphological changes in its colonies in the zone of interaction. In contrast, hyphal changes occurred less often in the ectomycorrhizal fungi tested. Species of Suillus are suggested to present a different mechanism in their interaction with other fungi than A. muscaria and L. laccata.     

Effect of Temperature,pH, and Media on the Mycelial Growth of Tuber koreanum

Members of the genus Tuber are ectomycorrhizal fungi; this genus includes more than 180 species worldwide. In the present study, the optimal pH, temperature, and medium suitable for the mycelial growth of the Korean truffle, Tuber koreanum, were determined. Mycelium of T. koreanum, isolated from fruiting bodies collected in Korea, was used to investigate the effects of these environmental factors. The results showed that malt extract agar and potato dextrose agar were the most suitable for the mycelial growth of T. koreanum when cultured at a pH of 6.0 at 25 °C for 30 days.     

Regulation of genes involved in nitrogen utilization on different C/N ratios and nitrogen sources in the model ectomycorrhizal fungus Hebeloma cylindrosporum

Nitrogen (N) utilization by ectomycorrhizal fungi is an essential aspect of their ecosystem function. N deposition changes both the N pools and the carbon/nitrogen (C/N) ratio of the substrates where ectomycorrhizal fungi are found, and it is important to understand how these changes affect the N forms used by ectomycorrhizal fungi. To overcome the difficulties of studying ectomycorrhizal fungi in situ, we investigated all known N genes in the model fungus, Hebeloma cylindrosporum in a culture study. In addition to studying the regulation of all known N utilization genes, we aimed to understand whether there are gene clusters that undergo similar regulation. Lastly we studied how C/N ratio, N transporter type, and N source affected relative gene expression levels. We grew the D2 strain of H. cylindrosporum on a range of inorganic and organic N sources under low, medium, and high C/N ratios. We found three gene clusters that were regulated in a similar pattern. Lastly, we found C/N ratio, N source and N transporter type all affected gene expression levels. Relative expression levels were highest on the high C/N ratio, BSA and diLeucine N sources, and inorganic N transporters were always expressed at higher levels than organic N transporters. These results suggest that inorganic N sources may always the default preference for H. cylindrosporum, regardless of both the N sources and the C/N ratio of the substrate.     

Escherichia coli mrsC Is an Allele of hflB, Encoding a Membrane-Associated ATPase and Protease That Is Required for mRNA Decay

The mrsC gene of Escherichia coli is required for mRNA turnover and cell growth, and strains containing the temperature-sensitive mrsC505 allele have longer half-lives than wild-type controls for total pulse-labeled and individual mRNAs (L. L. Granger et al., J. Bacteriol. 180:1920–1928, 1998). The cloned mrsC gene contains a long open reading frame beginning at an initiator UUG codon, confirmed by N-terminal amino acid sequencing, encoding a 70,996-Da protein with a consensus ATP-binding domain. mrsC is identical to the independently identified ftsH gene except for three additional amino acids at the N terminus (T. Tomoyasu et al., J. Bacteriol. 175:1344–1351, 1993). The purified protein had a K_m of 28 μM for ATP and a V_max of 21.2 nmol/μg/min. An amino-terminal glutathione S-transferase–MrsC fusion protein retained ATPase activity but was not biologically active. A glutamic acid replacement of the highly conserved lysine within the ATP-binding motif (mrsC201) abolished the complementation of the mrsC505 mutation, confirming that the ATPase activity is required for MrsC function in vivo. In addition, the mrsC505 allele conferred a temperature-sensitive HflB phenotype, while the hflB29 mutation promoted mRNA stability at both 30 and 44°C, suggesting that the inviability associated with the mrsC505 allele is not related to the defect in mRNA decay. The data presented provide the first direct evidence for the involvement of a membrane-bound protein in mRNA decay in E. coli.     

Fungal associates of the tree-killing bark beetle,Ips typographus,vary in virulence,ability to degrade conifer phenolics and influence bark beetle tunneling behavior

The bark beetle Ips typographus carries numerous fungi that could be assisting the beetle in colonizing live Norway spruce (Picea abies) trees. Phenolic defenses in spruce phloem are degraded by the beetle's major tree-killing fungus Endoconidiophora polonica, but it is unknown if other beetle associates can also catabolize these compounds. We compared the ability of five fungi commonly associated with I. typographus to degrade phenolic compounds in Norway spruce phloem. Grosmannia penicillata and Grosmannia europhioides were able to degrade stilbenes and flavonoids faster than E. polonica and grow on minimal growth medium with spruce bark constituents as the only nutrients. Furthermore, beetles avoided medium amended with phenolics but marginally preferred medium colonized by fungi. Taken together our results show that different bark beetle-associated fungi have complementary roles in degrading host metabolites and thus might improve this insect's persistence in well defended host tissues.     

Cadmium-Responsive Thiols in the Ectomycorrhizal Fungus Paxillus involutus

Molecular and cellular mechanisms underlying the sustained metal tolerance of ectomycorrhizal fungi are largely unknown. Some of the main mechanisms involved in metal detoxification appear to involve the chelation of metal ions in the cytosol with thiol-containing compounds, such as glutathione, phytochelatins, or metallothioneins. We used an improved high-performance liquid chromatography method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (γ-glutamylcysteine, glutathione) to higher-molecular-mass compounds (phytochelatins). We found that glutathione and γ-glutamylcysteine contents increased when the ectomycorrhizal fungus Paxillus involutus was exposed to cadmium. An additional compound with a 3-kDa molecular mass, most probably related to a metallothionein, increased drastically in mycelia exposed to cadmium. The relative lack of phytochelatins and the presence of a putative metallothionein suggest that ectomycorrhizal fungi may use a different means to tolerate heavy metals, such as Cd, than do their plant hosts.     

Substrate Utilization by an Oxalate-Consuming Spirillum Species in Relation to Its Growth in Ozonated Water

The nutritional versatility of a vibrio-shaped, oxalate-utilizing isolate, strain NOX, obtained from tap water supplied with low concentrations of formate, glyoxylate, and oxalate, was determined by growth experiments with low-molecular-weight carbon compounds at high (grams per liter) and very low (micrograms per liter) concentrations. The organism, which was identified as a Spirillum species, appeared to be specialized in the utilization of a number of carboxylic acids. Yields of 2.9 × 10⁶ CFU/μg of oxalate C and 1.2 × 10⁷ CFU/μg of acetate C were obtained from growth experiments in tap water supplied with various low amounts of either oxalate or acetate. A substrate saturation constant of 0.64 μM oxalate was calculated for strain NOX from the relationship between growth rate and concentration of added oxalate. Maximum colony counts of strain NOX grown in ozonated water (dosages of 2.0 to 3.2 mg of O₃ per liter) were 15 to 20 times larger than the maximum colony counts of strain NOX grown in water before ozonation. Based on the nutritional requirements of strain NOX, it was concluded that carboxylic acids were produced by ozonation. Oxalate concentrations were calculated from the maximum colony counts of strain NOX grown in samples of ozonated water in which a non-oxalate-utilizing strain of Pseudomonas fluorescens had already reached maximum growth. The oxalate concentrations obtained by this procedure ranged from 130 to 220 μg of C/liter.     

The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspension-cultured cells of its host Picea abies

A cell-wall fraction of the mycorrhizal fungus Amanita muscaria increased the chitinase activity in suspension-cultured cells of spruce (Picea abies (L.) Karst.) which is a frequent host of Amanita muscaria in nature. Chitinase activity was also increased in roots of spruce trees upon incubation with the fungal elicitor. Non-induced levels of chitinase activity in spruce were higher in suspension cells than in roots whereas the elicitorinduced increase of chitinase activity was higher in roots. Treatment of cells with hormones (auxins and cytokinin) resulted in a severalfold depression of enzyme activity. However, the chitinase activity of hormone-treated as well as hormone-free cells showed an elicitor-induced increase. Suspension cells of spruce secreted a large amount of enzyme into the medium. It is postulated that chitinases released from the host cells in an ectomycorrhizal system partly degrade the fungal cell walls, thus possibly facilitating the exchange of metabolites between the symbionts.     

Development,persistence and regeneration of foraging ectomycorrhizal mycelial systems in soil microcosms

Development of extraradical mycelia of two strains each of Paxillus involutus and Suillus bovinus in ectomycorrhizal association with Pinus sylvestris seedlings was studied in two dimensions in non-sterile soil microcosms. There were significant inter- and intra-specific differences in extraradical mycelial growth and morphology. The mycelial systems of both strains of P. involutus were diffuse and extended more rapidly than those of S. bovinus. Depending on the strain, P. involutus mycelia were either highly plane filled, with high mass fractal dimension (a measure of space filling) or sparse, low mass fractal dimension systems. Older mycelial systems persisted as linear cords interlinking ectomycorrhizal tips. S. bovinus produced either a mycelium with a mixture of mycelial cords and diffuse fans that rapidly filled explorable area, or a predominately corded mycelium of minimal area cover. In the soil microcosms, mass fractal dimension and mycelial cover tended to increase with time, mycelia encountering litter having significantly greater values. Results are discussed in terms of the ecology of these fungi, their foraging activities and functional importance in forest ecosystems.     

Purification and Characterization of Two Enantioselective α-Ketoglutarate-Dependent Dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH

α-Ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and α-ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His₆-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the α-ketoglutarate-dependent dioxygenases and share the specific motif HXDX₂₄TX₁₃₁HX₁₀R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent K_m values were 99 μM for (R)-mecoprop, 164 μM for (R)-dichlorprop, and 3 μM for α-ketoglutarate for RdpA and 132 μM for (S)-mecoprop, 495 μM for (S)-dichlorprop, and 20 μM for α-ketoglutarate for SdpA. Both enzymes had high apparent K_m values for oxygen; these values were 159 μM for SdpA and >230 μM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace α-ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdAα-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.     

红皮云杉外生菌根菌单接种及混合接种对苗木生长的影响

以提高外生菌根真菌对红皮云杉苗木促生长效果为目的,在前期研究的基础上,采用菌株配对培养的方法对获得的云杉外生菌根真菌进行混合接种菌株组合筛选;采用沟施菌剂方法,对3年生红皮云杉移床苗进行外生菌根菌菌株组合野外混合接种试验,同时以各菌株野外单接种作为对照,研究外生菌根真菌单接种及混合接种对红皮云杉苗木生长的影响。试验结果表明:供试菌株及菌株组合对红皮云杉苗木生长均有一定的促进作用。菌株L15促生长效果最佳,接种100d苗木高生长高于对照30.88%,地径高于对照15.29%。组合L15/025与L15/009促苗木生长效果低于单接种L15的效果,高于单接种009和025的效果。接种菌株010和组合L15/025的苗木与对照相比,叶绿素含量提高差异显著,叶绿素a水平分别高于对照59.15%、54.61%,叶绿素b水平分别高于对照76.34%和67.78%。除接种菌株010的苗木外,其他处理苗木其过氧化氢酶活性与对照相比均有所降低。所有处理苗木的根系活力均低于对照。综上,外生菌根菌高效菌株与一般菌株混合接种会弱化高效菌株自身接种效果;苗木过氧化氢酶活性、苗木根系活力与苗木的生物量间无相关性。