Nematicidal anthranilic acid derivatives from Laccaria species.

Three undescribed natural products, the anthranilic acid derivatives laccanthrilic acids A, B, and C, as well as the known (3S)-1,2,3,4-tetrahydro-3-ß-carboline-3-carboxylic acid were isolated from fruiting bodies of Laccaria laccata. The structures were established by 1D and 2D NMR spectroscopy, HR-(+)-ESIMS and chemical synthesis. The absolute configuration of laccanthrilic acids A and B was determined by GC-MS after hydrolytic cleavage and derivatisation of the resulting glutamic acid with methanol and Mosher's reagent and subsequent comparison with authentic synthetic samples of known absolute configuration. The absolute configuration of laccanthrilic acid C was determined by comparison of the CD spectra of laccanthrilic acids B and C with each other. Metabolic profiling of related species showed that the compounds are common in the genus Laccaria. Laccanthrilic acid B exhibited moderate nematicidal effects against Caenorhabditis elegans, which might explain to some degree the beneficial role of these fungi for the growth and survival of their host plants.

ÁCIDOS GRASOS, ÉSTERES Y ESTEROLES DEL CUERPO FRUCTÍFERO DEL HONGO Laccaria laccata / FATTY ACIDS, ESTERS AND STEROLS OF THE Laccaria laccata MUSHROOM / ÁCIDOS GRAXOS, ÉSTERES E ESTERÓIS DO CORPO FRUTÍFERO DO FUNGO Laccaria laccata

Del extracto en acetato de etilo del hongo comestible Laccaria laccata se aislaron 3 ácidos grasos, 6 esteres etílicos, 5 esteroles y un triterpeno ergostánico. Los compuestos se identificaron por EM como ácido palmítico, ácido linoléico y ácido oléico, hexadecanoato de etilo, 8-octadecenoato de etilo, 9-octadecenoato de etilo, 9,12-octadecadienoato de etilo, estearato de etilo, eicosanoato de etilo, ergosta- 2,5,7,9(11),22-pentaeno, ergosta- 5,7,22-trien-3β (ergosterol), ergosta- 7,22-dien-3β, ergosta-7-en-3β, ergosta- 5,7,9(11),22-tetraen-3β-ol y estigmast- 5-en-3β. Tanto los ésteres como los dos últimos compuestos se reportan por primera vez en la especie laccata.

From the extract in ethyl acetate of the eatable fungus Laccaria laccata were isolated three fatty acids, 6 ethylic esters, 5 sterols and an ergostanic triterpene. The compounds were identified by M.S. as palmitic, linoleic and oleic acid, ethyl Hexadecanoate, ethyl 8-octadecenoate, ethyl 9-octadecenoate; 9,12-ethyl octadecadienoate, ethyl estearate, ethyl eicosanoate, ergost-2,5,7,9(11),22-pentaene, ergost-5,7,22-triene-3β-ol (ergosterol), ergost-7-22-diene-3β-ol, ergost-7- ene-3β-ol, ergost-5,7,9 (11),22-tetraen- 3β-ol and estigmast-5-ene-3β-ol. The esters and the two latter compounds are reported by first time in the Laccata mushroom.

Do extrato em acetato de etilo do fungo comestível Laccaria laccata, isolaram-se 3 ácidos graxos, 6 ésteres etílicos, 5 esteróis e um triterpeno ergostânico. Os compostos identificaram-se por E.M. como ácido palmítico, ácido linoléico e ácido oléico, hexadecanoato de etilo, 8-octadecenoato de etilo, 9-octadecenoato de etilo, 9,12-octadecadienoato de etilo, estearato de etilo, eicosanoato de etilo, ergosta- 2,5,7,9(11),22-pentaeno, ergosta- 5,7,22-trien-3β-ol (ergosterol), ergosta- 7,22-dien-3β-ol, ergosta- 7-en-3β-ol, ergosta- 5,7,9(11),22-tetraen-3-ol y estigmast- 5-en-3β-ol. Os ésteres e os dois últimos esteróis reportaram- se pelo primeira vez na espécie laccata.

Survival of an introduced ectomycorrhizal Laccaria bicolor strain in a European forest plantation monitored by mitochondrial ribosomal DNA analysis.

Mitochondrial and nuclear genes have different inheritance, thus studies of fungal populations should use both mitochondrial and nuclear markers. Using nuclear markers, the S238N strain of the ectomycorrhizal basidiomycete Laccaria bicolor ((Maire) Orton) has been previously shown to persist for at least 10 yr after outplanting in a plantation of Douglas fir (Pseudotsuga menziesii (Mir.) Franco) inoculated with this strain. In the present study, we have sampled 539 sporophores of Laccaria spp. from this plantation, some of which had the S238N nuclear genotype, to study mitochondrial DNA polymorphism and persistence of the inoculated S238N mitochondrial genome. Length polymorphism in fragments of the large subunit of mitochondrial ribosomal DNA (LrDNA) allowed distinction of the haplotypes present in the plantation at the species level. In addition, heteroduplex analysis and sequencing revealed intraspecific polymorphism of LrDNA among the L. bicolor sporophores and enabled specific identification of S238N LrDNA. This haplotype was only retained in sporophores carrying the S238N nuclear genome, confirming the survival of this introduced strain in a natural population.

Copper-binding proteins in ectomycorrhizal fungi.

The presence of copper-binding proteins produced in response to added copper was examined in isolates of Laccaria laccata (Scop, ex Fr.) Cooke and Paxillus involutus (Batsch ex Fr.) Fr. taken from copper-contaminated and uncontaminated sites, and in a single isolate of Scleroderma citrinum Pers. from a contaminated site. Two isolates of Laccaria (GLac4 and ELacl) grew better in 1-5 miu and 2-5 niM copper than a third (Lac3G) and were considered to be more tolerant. Amongst five isolates of P. involutus, three (WJPax2R, GPaxRSp2 and Pax4) were capable of growth in media containing 4-0 mM copper and were regarded as tolerant. All isolates of both Laccaria and Paxillus were capable of some growth in 2-5 mM copper, but S. citrinum was much more copper-sensitive and the concentration had to be reduced at least 10-fold before any growth occurred. Tolerance of isolates was not related to whether they were taken from copper-contaminated or uncontaminated sites. Copper-binding proteins were detected in response to copper in the culture media in the two tolerant isolates of Laccaria (GLac4 and ELacl) but not in the least tolerant isolate. In Paxillus, similar proteins were found in two tolerant isolates (GPaxRSp2 and Pax4) but not in WJPax2R, which was also regarded as tolerant, nor in any of the less tolerant isolates. Copper-binding proteins were not detected in S. citrinum. The copper-binding protein purified from the Laccaria isolate ELacl appeared as a single band in modified SDS-PAGE electrophoresis. Its molecular mass and spectral characteristics were consistent with it being a metallothionein.

Effects of cadmium on non-mycorrhizal and mycorrhizal Norway spruce seedlings [Picea abies (L.) Karst.] and its ectomycorrhizal fungus Laccaria laccata (Scop, ex Fr.) Bk. & Br.: Sulphate reduction, thiols and distribution of the heavy metal.

The effect of cadmium on assimilatory sulphate reduction and thiol content was studied in non-mycorrhizal and mycorrhizal Norway spruce seedlings (Picea abies) and its ectomycorrhtzal fungus Laccaria laccata. The distribution of cadmium was also investigated. Isotope dilution experiments indicated that the fungus reduced sulphate via adenosine 3'-phosphate 5'-phosphosulphate sulphotransferase, whereas Norway spruce seedlings assimilated sulphate via adenosine 5'-phosphosulphate sulphotransferase in both roots and needles. In mycorrhizal roots only the plant sulphotransferase activity could be measured. Mycorrhizal and non-mycorrhizal roots and the mycelium of Laccaria laccata contained increased activities of sulphotransferase and more acid-soluble thiols when cultivated with cadmium. The increase in acid-soluble thiols was due to phytochelatins in roots and to glutathione in Laccaria laccata, where neither phytochelatins nor metallothioneins could be detected. Even though the cadmium content of mycorrhizal roots was slightly higher than that of non-mycorrhizal roots, concentrations of phytochelatin were only half as high as in non-mycorrhizal roots. Cadmium content of needles of mycorrhizal plants was significantly lower than that of non-mycorrhizal plants. Most of the cadmium in Laccaria laccata was associated with the cell walls and could be exchanged with Ni2+ .

Effects of high soil moisture on formation of ectomycorrhizas and growth of karri (Eucalyptus diversicolor) seedlings inoculated with Descolea maculata, Pisolithus tinctorius and Laccaria laccata.

Seedlings of Eucalyptus diversicolor P. Muell. I noculated with the ectomycorrhizal fungi Descolea maculata Bougher (two isolates), Pisolithus tinctorius (Pers.) Coker & Couch and Laccaria laccata (Scop, ex Fr.) Berk. & Br. were raised under glasshouse conditions in a yellow sand at a gradient of four soil moisture levels ranging from above field capacity to near waterlogged. All fungi enhanced growth of seedlings above that of uninoculated seedlings, but in soils near saturation there was no response to inoculation. Reduced mycorrhizal formation in relation to increasing soil moisture occurred to various degrees for all fungi. This was particularly marked with Pisolithus tinctorius. In contrast, Laccaria laccata maintained a relatively high number of mycorrhizal roots at all moisture levels applied, except at the wettest soil treatment. An isolate of D. maculata from a swamp environment did not produce a greater number of mycorrhizal roots at high soil moisture than an isolate of this species from a forest environment.

Growth and phosphorus acquisition of karri (Eucalyptus diversicolor F. Muell.) seedlings inoculated with ectomycorrhizal fungi in relation to phosphorus supply.

Growth and phosphorus acquisition of pot-grown seedlings of karri (Eucalyptus diversicolor F. Muell.) were examined following inoculation with four ectomycorrhizal fungi -Descolea maculata Bougher (two isolates), Pisolithus tinctorius (Pers.) Coker & Couch, and Laccaria laccata (Scop, ex Fr.) Berk. & Br. Seedlings were raised in steam-sterilized sand to which 13 rates of phosphorus (0.100 mg P kg-1 soil) were applied. All fungi except P. tinctorius produced a plant growth response. L. laccata produced the largest growth response. Responses were greatest at low rates of application of P to soil. There was no effect of the fungi on growth at levels of P application above 28 mg P kg-1 soil. A threshold effect (no increase in growth with increasing additions of P) characteristic of non-mycorrhizal seedlings was eliminated by mycorrhizal infection. Mycorrhizal inoculation increased P content of plant tissues at sub-optimal levels of P supply. The effect of mycorrhizas on seedling P status diminished with increasing soil P. One isolate of D. maculata often had greater rates of P accumulation and produced higher concentrations of P in plant tissues than L. laccata, but did not produce greater plant biomass. Frequency of infection for all fungi was low in soils with no additional P, and greatest with the addition of 2 mg P kg-1 soil (L. laccata and D. maculata isolate A), or 4 mg P kg-1 soil (D. maculata isolate B). Infection was reduced with increasing soil P, and not evident at 36 mg P kg-1 soil or higher levels of soil P. L. laccata had higher infection frequency and mycorrhizal root length at all levels of soil P than the D. maculata isolates. Two fungi produced basidiomes. This occurred at levels of soil P application ranging from 4 to 28 mg P kg-1 soil for D. maculata (isolate B), and at 4 to 28 mg P kg-1 soil for L. laccata.

Ultrastructural localization of ATPase activity in the Pinus sylvestris/Laccaria laccata ectomycorrhizal association.

Ultrastructural localization of the plasmalemmal ATPase activity has been studied in basidiomycete ectomycorrhizas synthesized between Pinus sylvestris L, and Laccaria laccata Scop, ex Fries. The cortical cells showed a strong plasmalemma-bound ATPase activity. The activity was also associated with the fungal plasmalemma in the external hyphae, sheath and Hartig net. The plasmalemmal ATPase activity disappeared in degenerating cortical cells. In the Hartig net, only the parts of the plasmalemma adjacent to other hyphae were active. When the Hartig net hyphae adjoined on one side a living cortical cell and, on the other side, a dead cortical cell, the ATPase activity of fungus plasmalemma was asymmetrically distributed. These observations are discussed in relation to the possible function of ATPase in ectomycorrhizas.

PHENYLALANINE AMMONIA LYASE AND PEROXIDASE ACTIVITY IN MYCORRHIZAL AND NONMYCORRHIZAL SHORT ROOTS OF SCOTS PINE, PINUS SYLVESTRIS L.

Phenylalanine ammonia lyase was characterized in roots of Pinus sylvestris L. The Km for the pine root enzyme with phenylalanine as a substrate was l.2 ± 0.4 X 10-4 M. The enzyme had a pH activity optimum of 9 and the subunit molecular weight was 70 to 72 kD as determined by Western blotting. Enzyme activity could be inhibited by D,L-2-aminooxy 3 phenylpropionic acid at 1 µ. Treatments with zymosan, pectinase, light or kinetin and naphthylacetic acid did not induce higher phenylalanine ammonia lyase or peroxidase activity in pine roots. No significant differences were observed in phenylalanine ammonia lyase or peroxidase activity in mycorrhizal and nonmycorrhizal short roots in the P. sylvestris-L. laccata symbiosis 15 weeks after cultivation.