Fungus Paxillus Involutus Research Articles

Protein biosynthesis changes during mycorrhiza formation in roots of micropropagated birch

Summary Birch (Betula pendula Roth) micropropagated plantlets were inoculated with 7 different isolates of the mycorrhizal fungus Paxillus involutus Batsch. Based on the level of fungal ergosterol measured in roots at the end of the “mycorrhiza formation stage”, strain P0 was chosen as the reference strain. Electrophoretic analysis of in vivo labeled proteins extracted from mycorrhizal roots 96 h post-inoculation with this strain, noninoculated roots, and free-living mycelium, revealed that specific polypeptides were synthesized during ectomycorrhiza formation. To examine hypothetical similarity between some of these polypeptides and defense proteins, parts of corresponding putative genes of birch were isolated. Partial sequencing of one clone have shown that it contained a portion of the gene for phenylalanine ammonialyase.

The role of ectomycorrhizal inoculations in landfill site restoration programmes

Greenhouse landfill models were used to examine the efficacy of the mycorrhizal fungus Paxillus involutus to promote the growth of Betula pendula (Silver Birch) seedlings in different site restoration strategies. Direct planting into refuse resulted in death of the fungal symbiont whilst with topsoil and subsoil no positive growth promotions, compared with uninoculated controls, resulted. With sterile seedlings, mycorrhizal associations formed within 24 weeks in both subsoil and topsoil. The need for mycorrhizal tree seedlings in site restoration programmes is, therefore, questioned.

Ectomycorrhizal seedling response to selected components of landfill leachate

The threshold toxicity concentrations of seven landfill leachate components (acetic, propionic, butyric, isobutyric, valeric, isovaleric and hexanoic acids) were determined with respect to the growth of three ectomycorrhizal fungi (Laccaria proxima, Hebeloma crustuliniforme and Paxillus involutus) and to the survival of mycorrhizal and mycorrhizal-free Betula pendula seedlings. Inoculation increased tree seedling tolerance to the acids, with the exception of the susceptibilities of L. proxima and H. crustuliniforme to valerate and isovalerate. Interacting microbial associations catabolically active against the acids were isolated from three different source materials (refuse, subsoil, topsoil). Minimum inhibitory acid concentrations exceeded the threshold toxicity concentrations to B. pendula seedlings, fungal isolates and mycorrhizal associations. Thus, provided that the acid concentrations were not toxic to the microbial associations and the hydraulic loading rates were lower than the maximum specific growth rates of the associations, concentrations should be reduced by microbial intervention during vertical migration. The potential for the routine use of mycorrhizal tree seedlings in landfill site restoration is discussed in the context of site-specific variables.

Production of acid is responsible for antibiosis by some ectomycorrhizal fungi

The interaction of Pythium ultimum with a variety of ectomycorrhizal fungi was investigated in vitro. Out of 19 isolates of ectomycorrhizal fungi tested for inhibition of the growth of P. ultimum, 16 caused swelling and bursting of the tips of leading hyphae. These effects were eliminated by maintaining the culture medium at pH 6 with a biological buffer. This indicated that acidification of the medium was the cause of the inhibition. Hyphal growth of P. ultimum was inhibited below pH 3. In liquid culture, the ectomycorrhizal fungus Paxillus involutus reduced the pH of the medium to less than 3 before it reached stationary phase. It is suggested that the antibiotic effect exhibited by some ectomycorrhizal fungi is due to acid production. HPLC analyses of culture filtrates of Pax. involutus were shown not to contain significant amounts of oxalic acid, but other acids were present in large quantities.

Effect of nitrogen level on acid phosphatase activity of eight isolates of ectomycorrhizal fungus Paxillus involutus cultured in vitro

The higher levels of nitrogen in ammonium form stimulated the growth of mycelia and increased the accessible as well as the total acid phosphatase activity of Paxillus involutus (Batsch) Fr. isolates grown in pure culture. Rates of mycelia growth and acid phosphatase activities varied widely from one isolate to another. Scots pine (Pinus sylvestris L.) seedlings were inoculated with different P. involutus isolates in axenic conditions. Shoots of pine seedlings with mycorrhizae contained more phosphorus than shoots of non-mycorrhizal seedlings. The relations between growth and phosphatase activity of P. involutus isolates and their efficiency in supplying the host plant with phosphate are discussed.

Release of a reducing substance by the ectomycorrhizal fungi Pisolithus tinctorius and Paxillus involutus

Culture solutions of the ectomycorrhizal fungi Pisolithus tinctorius and Paxillus involutus are shown to reduce a high valency oxide of manganese at a range of pH values from 3.0 to 13.0. Manganese reduction in unmoculated culture media was confined to pH values below 5.0 and above 10.0. The results are consistent with the release of a reducing substance from the fungal mycelium.

Later events in suppression of Fusarium root rot of red pine seedlings by the ectomycorrhizal fungus Paxillus involutus

Colonization of primary roots of red pine (Pinus resinosa Ait.) seedlings by two isolates of the fungal pathogen Fusarium oxysporum Schlecht. emend Snyd. & Hans. f.sp. pini in a sterile, soil-free system and a nonsterile rooting medium was suppressed for 2 months if the roots were exposed to the ectomycorrhizal fungus Paxillus involutus (Batsch.) Fr. for 1 week before being inoculated with the pathogen. Roots of protected seedlings harboured only a few hyphae of the pathogen. These were either vacuolated or filled with slightly electron-dense substances and did not penetrate host cells. In contrast, roots of seedlings not inoculated by P. involutus were extensively colonized by F. oxysporum, and host tissue vacuolation and disorganization occurred. Papillae, consisting of either small protuberances or elongate projections, were infrequently formed in response to infection. Chlamydospores of F. oxysporum that formed in the sterile system had numerous lipid deposits and amorphous thick walls but few mitochondria and little endoplasmic reticulum. Key words: biological control, ectomycorrhiza, Fusarium, Pinus resinosa, disease suppression.

Induction of protoplast formation in the ectomycorrhizal fungus Paxillus involutus by the root rot pathogen Fusarium oxysporum

SUMMARYCo‐culture of the pathogenic fungus Fusarium oxysporum f. sp. pini Schlecht. emend Snyd. & Hans, with the ectomycorrhizal fungus Paxillus involutus Fr. on potato dextrose agar caused no visible morphological changes in the fungi. Co‐culture on modified Melin Norkrans medium in petri plates yielded, however, protoplast formation at or near tips of P. involutus hyphae. No changes were visible in F. oxysporum as a result of co‐culture and the two fungi grew in close association with each other without inhibition. Staining with Calcofluor White M2R New and fluorescein diacetate displayed that, at this stage in development, a wall surrounding the protoplast was absent but a plasma membrane sheathed by an extracellular mucilage was present. Protoplasts lacking definite form adhered closely to adjoining hyphae while those with the characteristic spherical form were easily dislodged from hyphae. Average size of spherical protoplasts was 17–20 μm. Breaks were present in the hyphal wall in regions adjacent to protoplast formation and these may have been pathways for cytoplasmic leakage. Transmission electron microscopy revealed these breaks more clearly, and provided additional evidence of a plasma membrane around protoplast cytoplasm. Protoplasts contained few organelles except vesicles, larger electron dense bodies and occasional nuclei. Hyphae forming protoplasts often lacked cytoplasmic integrity although those not forming protoplasts appeared healthy.

Integrated control of Fusarium damping-off in red pine seedlings with the ectomycorrhizal fungus Paxillusinvolutus and fungicides

Damping-off caused by Fusariumoxysporum Schlecht. in red pine (Pinusresinosa Ait.) was significantly reduced when seedlings were treated with either of two fungicides (benomyl or oxine benzoate) concomitantly with the ectomycorrhizal fungus Paxillusinvolutus (Batsch.) Fr. compared with inoculation with P. involutus alone. Both fungicides at concentrations of 10 ppm and above significantly reduced spore germination and germ-tube length of F. oxysporuminvitro. On the other hand, invitro growth of P. involutus mycelium was not affected by the fungicides at concentrations up to 1000 ppm. The growth of P. involutus was, however, significantly reduced by the two fungicides at 5000 and 10 000 ppm. Inoculation of red pine seedlings with P. involutus was effective under growth chamber and greenhouse conditions in reducing the incidence of damping-off induced by F. oxysporum. When benomyl and oxine benzoate were applied in combination with P. involutus, seedling mortality was not observed. Oxine benzoate was phytotoxic and inhibited mycorrhiza formation by P. involutus at the recommended dose. Benomyl, on the other hand, had no inhibitory effect on seedling growth and stimulated ectomycorrhiza formation either by a naturally occurring mycorrhizal fungus or P. involutus in the greenhouse. The concept of incorporating integrated biological and chemical treatment for controlling Fusarium damping-off of red pine seedlings is suggested.

Early effects of the ectomycorrhizal fungus Paxillus involutus on the root rot organism Fusarium associated with Pinus resinosa

Infection of Pinus resinosa Ait. seedlings in a sterile test tube culture system by Fusarium oxysporum f.sp. pini Schlecht. emend. Snyd. & Hans., which was determined microscopically by the presence of the fungus within root tissues, was significantly reduced within the first 6 days of infection after prior inoculation with the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. Hyphae and conidia of the pathogen were swollen and filled with osmiophilic material when P. involutus was also present in the rhizosphere of P. resinosa seedlings. Germination of F. oxysporum conidia was suppressed when they were placed in holes in P. involutus cultures on modified Melin Norkrans medium in Petri plates but was restored with removal of conidia from plates. Suppressed, ungerminated conidia had thick cell walls and large cytoplasmic inclusions. Staining using Calcofluor White M2R New revealed changes in the binding properties of septal wall material. Key words: Paxillus, Fusarium, ectomycorrhiza, disease suppression, anatomy.

Degradation of 14C-labelled lignin and dehydropolymer of coniferyl alcohol by ericoid and ectomycorrhizal fungi

The ability of ericoid and ectomycorrhizal fungi to utilize 14C-labelled lignin and O14CH3-labelled dehydropolymer of coniferyl alcohol as sole C sources has been assessed in pure culture studies. The results indicate that ericoid mycorrhizal fungi are more effective in degrading lignin than ectomycorrhizal fungi. Amongst the ectomycorrhizal fungi the facultative mycorrhizal fungus Paxillus involutus degraded lignin more readily than those which are normally considered to be obligately mycorrhizal fungi such as Suillus bovinus and Rhizopogon roseolus. The importance of these lignin degrading capabilities is discussed in relation to the predominance of specific mycorrhiza forms along a gradient of increasing organic matter and hence lignin content of soil.

The effects of SO 2 pollution on mycorrhizal and non-mycorrhizal seedlings of [formula omitted

The effects of air pollution in the form of 6 concentrationsof SO 2 (ranging from 0 – 520 μg/m 3) on 9 week-old seedlings of Pinus sylvestris L. were studied. Seedlings were uninoculated or inoculated with the mycorrhizal fungi Paxillus involutus or Laccaria proxima . Exposure to SO 2 for 7 weeks decreased the photosynthesis and the cold sensitivity of the plants significantly. Mycorrhizal formation of L. proxima was not affected by the treatments, whilst that of P. involutus decreased significantly from 120 μg/m 3 SO 2 onwards.

Non‐target effect of herbicides: II. The influence of glyphosate on ectomycorrhizal symbiosis of red pine (Pinus resinosa) under greenhouse and field conditions

AbstractThe effects of the herbicide glyphosate [N‐(phosphonomethyl)glycine]on seedling growth of Pinus resinosa and on the ectomycorrhizal development of the symbiotic fungus Paxillus involutus were investigated under greenhouse and field conditions. Glyphosate at 0–54 and 3–23 kg ha−1 did not reduce seedling growth or ectomycorrhizal development under greenhouse conditions. There was no seedling mortality due to glyphosate treatment at either rate. Under field conditions, seedling growth of P. resinosa and ectomycorrhizal development of P. involutus were not affected by the above rates. Seedlings (46–48%) failed to survive in non‐glyphosate‐treated plots, presumably because of weed competition, whereas all survived in glyphosate‐treated plots. All the non‐mycorrhizal seedlings were colonized bv indigenous mvcorrhizal fungi within 2 months after planting in both control and glyphosate‐treated plots. The injection rates varied from 74–86 %.

The effects of aluminium and Paxillus involutus Fr. on the growth of Norway spruce [Picea abies (L.) Karst.

SUMMARYNorway spruce [Picea aides (L.) Karst.] seed lots were obtained from populations growing on an acid soil in the Black Forest, West Germany (acid), and a calcareous soil in the Schwabische Alb, West Germany (calc). Seedlings were grown in sterile perlite culture containing 0–6 mM aluminium. Hypocotyl extension was inhibited by aluminium in the calcareous seedlings, hut not in the acidic seedlings. In a longer term experiment acidic and calcareous plants were grown for 10 weeks in perlite. Some of the tubes were inoculated with the fungus Paxillus involutus Fr. (designated F +) and some were not (F ‐). Aluminium sulphate solutions were then added to the tubes to raise the aluminium concentrations to 0–6 mM. Plants were harvested after a further 10 weeks. Fungus was associated with the roots in F+ plants, but mycorrhizas did not form. Growth of acid(F ‐) was somewhat stimulated by aluminium treatment, but that of calc(F ‐) was greatly reduced, and the plants were chlorotic. The presence of a rhizospherie fungus (F +) enhanced the growth of the calcareous plants, but had little effect on the acidic plants. Shoot analyses suggested that the greater aluminium sensitivity of the calcareous plants involves an inability to exclude aluminium or to maintain normal levels of calcium and magnesium uptake in its presence. The presence of rhizospheric fungi reduced the effects of aluminium.

Uptake, translocation and assimilation of nitrogen from 15N‐labelled ammonium and nitrate sources by intact ectomycorrhizal systems of Fagus sylvatica infected with Paxillus involutus

SUMMARYThe uptake and assimilation of nitrogen from 15N‐labelled ammonium and nitrate sources was followed in intact ectomycorrhizal systems containing Fagus sylvatica L. plants infected with the fungus Paxillus involutus (Mich. ex Pers.) Cohen & Couch. Plants were grown in flat perspex observation chambers containing non‐sterile peat; the fungal mycelium growing from a host plant was allowed to cross a barrier and to colonize an area of peat from which roots had been excluded. Labelled ammonium chloride or sodium nitrate was fed to the mycelium, and the shoot, root and mycelial tissues analysed for total and 15N‐labelled amino acid contents after a feeding period of 72 h. Both free and protein‐incorporated amino acids were analysed. Labelled nitrogen was incorporated into a range of free amino acids, the principal sinks for assimilation being alanine, aspartate/asparagine and glutamate/glutamine. The spectrum of labelling in protein‐incorporated amino acids was wider with significant incorporation into the above compounds but additional assimilation of nitrogen as glycine, valine, serine, leucine, isoleucine and arginine. In total 78% of the nitrogen assimilated was incorporated into proteinaceous material. Label was incorporated from both nitrogen sources but the levels of enrichment in most free and protein‐bound amino acids were usually higher in systems supplied with ammonium than in those supplied with nitrate. The mean amount of nitrogen assimilated From nitrate was only 62% of that assimilated from ammonium.

Disease suppression by the ectomycorrhizal fungus Paxillus involutus: contribution of oxalic acid

Seedlings of Pinus resinosa Ait. grown in test tubes were inoculated with the ectomycorrhizal fungus Paxillus involutus Fr. Oxalic acid was identified as one of the ethanol-soluble fungistatic and (or) fungitoxic components of the rhizosphere after fractionation by high performance liquid chromatography, paper chromatography, and gel filtration. Simultaneous inoculation of P. resinosa seedlings with authentic oxalic acid and a spore suspension of Fusarium oxysporum f.sp. pini protected the seedlings against Fusarium root rot and decreased the sporulation of F. oxysporum in the rhizosphere when compared with controls lacking oxalic acid. Quantitation of oxalic acid showed a five fold increase in production by Pax. involutus in tubes containing P. resinosa seedlings when compared with tubes lacking seedlings. The synthesis of oxalic acid by Pax. involutus is, therefore, stimulated by P. resinosa root exudate.

The time-course of disease suppression and antibiosis by the ectomycorrhizal fungus Paxillus involutus.

Seedlings of Pinus resinosa Ait. grown in test tubes were inoculated with the ectomycorrhizal fungus Paxillus involutus Fr. and, one day later, with a spore suspension of the root pathogenic fungus Fusarium oxysporum Schlecht emend, Sny. & Hans f.sp. pini. Controls consisted of seedlings inoculated with plugs of sterile modified Melin Norkrans medium and F. oxysporum. Seedling survival was counted, sporulation of F. oxysporum was measured, and ethanol extractions of the rhizosphere were made, 1, 2, 3, 4, 5, 6, 8, 10 and 14 d after inoculation with the pathogen. The ethanol extractives were concentrated and assayed for fungitoxic activity by measuring the germination of F. oxysporum microconidia. Dead seedlings were first observed 3 d after inoculation with the pathogen. Sporulation of F. oxysporum was reduced significantly 3 d after seedling inoculation with P. involutus when compared with controls lacking P. involutus. The fungitoxic activity of the rhizosphere of the seedlings inoculated with P. involutus was greater than the fungitoxic activity of the rhizosphere of control seedlings, increasing strongly 2.4 d after inoculation with the pathogen. Suppression of Fusarium root rot by P. involutus may, therefore, be the result of antibiosis by this ectomycorrhizal fungus.

Protein synthesis inPinus resinosa and the ectomycorrhizal fungusPaxillus involutus prior to ectomycorrhiza formation

Seedlings ofPinus resinosa Ait. in test tubes were inoculated with the ectomycorrhizal fungusPaxillus involutus Fr. or with discs of sterile modified Melin-Norkrans (MMN) medium.Paxillus involutus was also inoculated to control tubes in the absence ofPinus resinosa seedlings. In vivo labelling of proteins inPinus resinosa roots and inPaxillus involutus mycelium was carried out using35Sl-methionine 1, 2, 3, 4, 5 and 7 days after inoculation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE) of the protein extracts from the four treatments and autoradiography demonstrated that the presence of root exudates altered protein synthesis inPaxillus involutus as three major bands disappeared whenPaxillus involutus was exposed to root exudates. Protein synthesis inPinus resinosa was also altered whenPaxillus involutus was introduced into the tubes, since at least two bands were more intense when seedlings were inoculated withPaxillus involutus, as compared to control roots. No difference was observed in the growth and the label incorporation ofPaxillus involutus growing with or without root exudates. Ectomycorrhizal roots were not formed during this experiment. Gene regulation in this ectomycorrhizal association occurs, therefore, prior to the formation of ectomycorrhizal roots.

Oxalate synthesis from soil bicarbonate by the mycorrhizal fungusPaxillus involutus

The ability of the ectomycorrhizal fungusPaxillus involutus (Batsch. ex Fr.) Fr. to utilize bicarbonate ions from solution, for oxalate synthesis was demonstrated using NaH14CO3 in a nitrate-nitrogen medium. Fifteen percent of the carbonate ions incorporated was used for the synthesis of oxalic acid either directly from oxalacetate, or via citrate, isocitrate, and glyoxylate.

Pine root exudate stimulates the synthesis of antifungal compounds by the ectomycorrhizal fungus Paxillus involutus

SUMMARYThe ectomycorrhizal fungus Paxillus involutus Fr. was grown in the presence of root exudate of Pinus resinosa Ait, for two weeks. The effect of root exudate on the synthesis of ethanol‐soluble antifungal compounds by P. involutus was determined by a bioassay based on microconidial germination of Fusarium oxysporum Schlecht emend, Sny. & Hans f. sp. pini. Cultivation of P. involutus on root exudate led to a greater than two‐fold increase in fungitoxic activity when compared to cultivation on modified Melin Norkrans medium in Petri dishes. Inoculation of P. resinosa root exudate with both P. involutus and a spore suspension of F. oxysporum led to an 80 % suppression of the sporulation of F. oxysporum when compared to controls lacking P. involutus. The increased resistance of P. resinosa P. involutus associations to Fusarium root rot may, therefore, be the result of biosynthesis of antifungal compounds by P. involutus which is stimulated by P. resinosa root exudate.