Abietic acid, and activator of basidiospore germination in ectomycorrhizal species of the genus Suillus (Boletaceae)

EXPERIMENTAL MYCOLOGY 11, 360-363 (1987)

BRIEF NOTE Abietic Acid, an Activator of Basidiospore Germination in Ectomycorrhizal Species of the Genus Suillus (Boletaceae) NILS FRIES,* KLAUS SERCK-HANSSEN,* LENA HALL DIMBERG,? ANDOLOFTHEANDER~ “Institute of Physiological Botany, University of Uppsala, Box 540, S-751 21 Upps&, Sweden,and fDepartment of Chemistry and Molecular Biology, Swedish University of Agricultural Sciences, Box 7015, S-750 07 Uppsala, Sweden

Accepted for publication June 22, 1987 FRIES, N., SERCK-HANSSEN, K., DIMBERG, L. H., AND THEANDER, 0. 1987. Abietic acid, an activator of basidiospore germination in ectomycorrhizal species of the genus Suillus (Boletaceae). Experimental Mycology 11, 360-363. Germination of basidiospores from four ectomycorrhizaforming Suillus species (S. granulatus, S. grevillei, S. luteus, and S. variegatus) was induced by exposing the spores to extracts or exudates from roots of Scotch pine (Pinus sylvestris L.). An active compound was contained in the lipophilic phase obtained by extraction of pine roots with chloroform/methanol. It was isolated and identified as abietic acid, a diterpene resin acid. With S. granulatus, the bioassay species, abietic acid caused germination at concentrations down to lo-’ M. 0 1987 Academic

Press, Inc.

INDEX DESCRIPTORS: Suillus granulatus; S. grevillei; S. luteus; S. variegatus; abietic acid; diterpene resin acid; basidiospore germination; germination induction; ectomycorrhiza.

Gray, and S. variegatus (Schwartz ex Fr.) 0. Kuntze. Basidiospores were obtained from basidiocarps collected during the fall of 1986 in the forests around Uppsala, Sweden. They were stored in sterile, plastic petri dishes at 4°C in darkness. Germination, although always scanty, had earlier been observed in these species under various circumstances. Sometimes spontaneous germination occurred in some spore collections and could be increased by adding an amino acid mixture to the medium (Fries, 1976) or by coculturing colonies of Rhodotorula yeast near the spores on an agar plate (Fries, 1941). However, after the composition of the nutrient medium had been modified and an inhibitory factor in the agar removed (Bjurman, 1984), it became evident that extracts and exudates from tree roots were the most efficient material available for germination induction in the four Suillus species (Fig. 1).

The basidiospores of most ectomycorrhiza-forming basidiomycetes require special environmental conditions for germination. In most species these conditions are still unknown. It has been found, however, that in some species the presence of other living organisms near the fungal spores on an agar plate-or any other appropriate substrate -may induce germination, although usually at very low levels. Generally, this effect is due to exudates from various microorganisms or to root exudates of seed plants. These exudates thus contain compounds possessing the capacity to trigger spore germination. The present report describes the following case in which such a compound, acting on the spores of ectomycorrhizal fungi, has been isolated and chemically identified. The four fungal species studied all belong to the genus Suillus (Boletaceae): S. granuZatus (L. ex Fr.) 0. Kuntze, S. grevillei (Klotzsch) Sing., S. luteus (L. ex Fr.) S. E 360 0147-5975187

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SPORE GERMINATION

INDUCED

All of these Suillus species, except S. grevillei, are strongly associated with Pinus spp. In Sweden the native Scotch pine (Pinus sylvestris) is their major host tree. S. grevillei only forms basidiocarps as an ectomycorrhiza former with larch (Larix decidua), but spore germination can be induced with pine root exudates and extracts (Fries, unpublished). Hence, root systems of 2-year-old plantlets of Scotch pine, with ca. 30-cm-long shoots, were used as the material for attempts to identify the germination-inducing factor. Since root exudate could not be obtained in sufficient quantities, extracts of roots which contained the active factor were prepared. Each step in the fractionation procedure for the extract was accompanied by bioassays to determine the absence or presence of the active factor in each new fraction. S. granulatus was used as the test species. The method was the same as the agar strip technique reported earlier in a study of spore germination in Leccinum

FXG. 1. The effect of root exudate on spore germination in Suillus luteus. Young mycelia are developing from germinated basidiospores on a nutrient agar plate in the vicinity of a pine seedling root. The root, most of the mycelia, and part of the agar medium are covered by a circular cellophane film.

BY ABIETK

ACID

(Bjurman and Fries, 1984). The first ger hyphae appeared on the agar strip surface 3 days after the start. Germination reached its highest and final value between the 7th and 10th days. Percentage germination was estimated by counting the number of germinated spores among loo-200 spores on photomicrographs of the agar strips Fresh pine roots (25 g) were grou fine powder in liquid nitrogen a stirred for 1 h at room temperature with methanol/chloroform (100 ml, I/1) under nitrogen. After filtration the filter cake was washed with the same solvent ml) followed by chloroform ( the combined filtrates were water (100 ml). Only the residue (ca. mg) from evaporation of the resulting lo lipophihc phase was bioactive. Extraction of this residue with boiling left an inactive solid (ca. solved. Further concentration tive material could be achieved by §~ak~~g the pentane soIution with a saturated aqueous solution of sodium carbonate (10 mg) followed by water (IO ml) and then extracting the combined aqueous pha.ses with ethyl acetate (2 x 100 ml). This proced. isolated the active material as so dissolved in ethyl acetate. The were isolated by adding concen drochloric acid (10 drops) to the v~go~o~s~~ stirred ethyl acetate solution of the sodium salts, followed by addition of anhydrous sodium sulfate, filtration, and evaporation, leaving a tough, dark-colored residue (ca. 250 mg) with high biological a layer chromatography reveale components. This active material (500 mg, from two runs) was now fractionated by chromatography on a silica-gel column (3.5 X 24 cm) by elution with methanol/chloroform (1125). An intermediate bioactive fraction ( eluting after ca. 140 ml) was rechs graphed on another silica-gel column (2.5 x 40 cm) with m~t~ano~/ch~orofor~~ (II3 as eluanb. A very bioactive tail fraction (I8

362

FRIES ET AL.

GQ “‘COOH Abietic

Acid

C20H3002

MW 302 FIG. 2. Structural formula of abietic acid.

mg eluting after ca. 150 ml) had a uv absorption maximum at 241 nm, a possible molecular ion peak at 302 m/z, and a resinacid-like ‘H NMR spectrum. This suggested the possible presence of abietic acid (Fig. 2). In fact, authentic abietic acid gave a strong germination-inducing effect. The active root factor further purified by reversed-phase preparative HPLC (C,,, 5 pm, 10 x 250 mg; 80% aqueous methanol as eluant; detection at 254 nm) and methylated with diazomethane gave a ‘H NMR spectrum which agreed closely with that of methyl abietate published by Zinkel et al. (1971). Subsequently, access to highly purified abietic acid made it possible to evaluate more accurately the activity of this compound as a germination activator (Table 1). A response was still noticeable between 10e6 and 10e7 M (ca. 0.1 mg/liter) with S. TABLE 1 Induction of Spore Germination in Suiiius granulatus with Different Concentrations of Abietic Acid Concentration of abietic acid” (mg/liter)

Germinated sporesb (%I

1000 100 10 1 0.1

28 47 28 7 3

n Concentrations of the solutions administered the agar strips in amounts of 0.1 ml per strip. b Incubation time 10 days.

granulatus as the test fungus. Spore germination in S. luteus reached the same level as in S. granulatus but started later. The other two Suillus species germinated more

slowly and at a lower percentage. It came as a surprise that a diterpene resin acid was capable of inducing spore germination. This group of compounds, which occurs mainly in coniferous trees, is chiefly known for its adverse effects on various organisms, e.g, being toxic to animals (Leach and Thakore, 1975; Kubik and Jackson, 1981), inhibiting fungal growth (Henriks et al., 1979), and repressing photosynthetic CO, fixation in plants (Martin et al., 1984). The presence of these acids in tree tissues suggests that they protect the tree against attacks by parasitic fungi and herbivorous insects. Abietic acid was also tested for germination induction in Hebeloma mesophaeum, Paxillus involutus, and Thelephora terrestris, three ectomycorrhizal fungus species which all respond to tree root exudates. None of them was brought to germination by this treatment. Some specificity in the activity of abietic acid is thus indicated. A consequence of this simplified procedure for inducing spore germination in Suillus is that homokaryons of these species can be easily produced and isolated on nutrient agar plates containing abietic acid. This may promote the development of new techniques, which will finally enable genetic studies in these common and ecologically important ectomycorrhizal fungi. ACKNOWLEDGMENTS

to

This work was supported by grants from the Swedish Natural Science Research Council and Consul Faxe’s Fund. We are indebted to the staff of the Swedish National Forest Enterprise at the forestry nursery, “Lugnet,” for having generously furnished us with uniform pine plantlets for the root extractions. We also thank Lennart Lundgren for running the NMR analyses and Gunilla Swedjemark and Helena Hedhall for valuable assistance in the early stages of this investigation.

SPORE GERMINATION

INDUCED

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BY ABIETIC

ACID

Y, M., AND JACKSON, L. L. 1981. Embryo resorptions in mice induced by diterpene resin acids of Pinus ponderosa needles. Cornei/ Vet. 71: 34-42.

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ZINKEL, D. .I?., ZANK, L. C., AND WESOLOWSKI, M. F. 197 1. Diterpene Resin Acids-A Compilation of Infrared, MQSS. Nuclear Magnetic Resonunce, Ultraviolet Spectra and Gas Ghromatographic Retention Data. USDA Forest Service, Forest

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