Trans. Br. mycol. Soc. 75 (3), 455-459 (1980).

[ 455 ]

Printed in Great Britain

STIMULATION OF LITTER-DECOMPOSINGBASIDIOMYCETES BY FLAVONOIDS

By G. LINDEBERG AND M. LINDEBERG

Division of Forest Mycology and Pathology

AND L. LUNDGREN, T. POPOFF AND 0. THEANDER

Department of Chemistry, Swedish University of Agricultural Sciences, S-75007 Uppsala

The stimulating effect of extractives from pine needles on the growth of litter-decomposingbasidiomycetes in synthetic nutrient .nedia was partly due to z-butanone soluble compon-ents. Taxifolin glucoside (dihydroquercetin-jv/i-glucoside) which constitutes 1-2 % of thedry weight of the needles was separated and in pure form, significantly enhanced the growthrate of Marasmius androsaceus at a concentration of 5 p.p.m, Other flavonoids, belongingto the catechins, dihydrochalcones, flavanones, flavonols, and anthocyanidins, as well asgallic acid, had a similar effect. Collybia peronataand Micromphale perforans which like M.androsaceus are needle-decomposing fungi, were also stimulated by taxifolin glucoside.

The growth of litter-decomposing basidiomycetesin synthetic media is strongly stimulated byaddition of cold-water extracts of leaf litter ofvarious forest trees (Lindeberg, 1944; Melin,1946; Olsen, Odham & Lindeberg, 1971). Fungiwhich behave in this way were found within thegenera Marasmius, Collybia, Mycena, and Clavaria.The growth-stimulating principle was found to bethermostable and dialyzable (Melin, 1946) andafter extraction of water extracts of litter with ethylacetate, the stimulatory factor remained in thewater phase (Olsen et al., 1971).

The chemical composition of coniferous leaveshas been studied by Theander and co-workers(Popoff & Theander, 1976). We have used theirmethods to separate and identify stimulatory sub-stances present in the extracts of the green needlesof Pinus sylvestris L. Marasmius androsaceus Fr.,which is a very common decomposer of celluloseand lignin in pine litter in Scandinavia, was usedas the main test organism (Lindeberg, 1944).

MATERIALS AND METHODS

Pure strains of the fungi studied were isolatedfrom basidiocarps and ascocarps by spore or tissuecultures.

Green needles from Pinus sylvestris werecollected from the trees in the autumn.

Fresh needles (200 g), 49 % dry weight, wereextracted three times for 40 min with 1400 ern"boiling acetone. After cooling, precipitating waxeswere discarded, the solution filtered off, and theneedles collected, dried, milled and extracted in anultrasonic bath with 350 em" acetone for 30 min

followed by extraction twice with 350 em"acetone-water (1: 1) for 30 min. All the acetone extractswere combined and the acetone evaporated offunder vacuum. The remaining water phase(containing soluble and suspended material) wasdiluted with water to 0'5 1and extracted consecu-tively with o·5 1 of chloroform three times, with0'5 I of ethyl acetate three times, and with 0'5 I ofz-butanone, saturated with water, three times. Thethree organic extracts and the remaining waterphase were evaporated to dryness in vacuum andthe dry weight of the fractions determined. Thesewere 7'4 g, 4'0 g, 2'5 g, and 18'9 g respectively.

The basal medium for the bioassay work was asfollows: Glucose 20'0 or 5'0 g, NH4-tartrate 5'0 g,KH2P0 4 1'0 g, MgS04. 7H20 0'5 g, NaCI 0'5 g,Fe3+ (as ferric citrate) 1 mg, Zn +2 (as ZnS04.7H20) 1 mg, MnS04.4H20 5 mg, CaS04.2H201 mg, Hoagland's A-Z solution 1 em", thiamine50 pg, biotin 1 pg, distilled water to 1000 em",

The fungi were grown in 100 em" conical flasks,each containing 20 em" of the basal medium.

Parts of the extracts, or equimolar amounts ofvarious phenolic compounds, were dissolved in96 % ethanol and four volumes of distilled wateradded. These solutions were added at the rate of0' 5 em 3 to each flask of the basal medium with thecontrol series receiving 0' 5 em 3 20 % ethanol perflask. After autoclaving, the flasks were inoculatedwith 3 x 3 mm pieces cut from the outer part ofmycelial mats of the test fungi, grown on Hagem'smalt-extract agar (Modess, 1941). The inocula ofthe Suillus species were preinoculated on freshmalt extract agar for 48 h before the inoculation ofthe flasks (Norkrans, 1950). Care was taken that

0007-1536/80/2828-6850 $01.00 © 1980 The British Mycological Society

Stimulation by flavonoids

Table 1. Effect of the z-butanone and water phases fro m green pine needles extract on the growth ofM. androsaceus

Added Mycelial Increase (%)extractives dry weight per mgof

(mg per flask) (mg) added extractivesControl 6'8± o'3Butanonephase 2'3 24'8 ± 1,6 115

4·6 11'6± o'7 159'2 13'4 ±1 'O 11

Water phase 16,6 54'5± 1'1 4233'3 68'o ±o'6 2766'6 82'5 ±4'6 17

30

'""' 20eo

.5-@,';:;3»0

10

C But I II III IV V VI VII VIII IX X I- X

Fig. 1. Effect of z-butanone extract from pine needles on growth of M. androsaceus (18 days). C, control;But, butanone extract, I to X, fractions from a silicic acid column; I-X, all fractions combined. Eachsample added in 3 concentrations (1:2 :4), the amount added per flask in the second one correspondingto the amount of extractivesfrom 350mg needles. Glucose concn.20 g 1-'.

the inocula remained floating on the surface of theliquid. Each series usually comprised five parallelflasks which were incubated at 22 °C. Growth wasassessed as mycelial dry weight after 18 to 20 daysunless otherwise stated.

RE SUL TS

The effect of the z-butanone and water phases onthe growth of M. androsaceus is shown in Table 1.

The growth-stimulating effect of the water phasewas very strong with the effect of the butanonephase somewhat weaker. However, if the amountof extracted material present in each extract isconsidered, the highest relative activity was exer-

ted by the butanone phase. Therefore subsequentwork was primarily concentrated on the butanonesoluble fraction.

The strongest effect of this fract ion on thegrowth of M. androsaceus was observed at thelowest concentration (T able 1), indicating thatsome kind of inhibitory substance was present inaddition to the stimulatory princ iple . Fractiona-tion of th e butanone phase on silicic acid andtesting the fractions for their effect on the growthof M . androsaceus gave the results shown in Fig. 1.

The highest activity was found in fractions I-III.When all the fractions were combined, the effectobtained was even higher than that of the non-fractionated butanone phase, possibly due to

G. Lindeberg, M. Lindeberg, L. Lundgren, T. Popoff and O. Theander 457

Table 2. Effect of various flavonoids and simple phenols (50 pM) on growth of M. androsaceus(mg dry weight)

No addition 9-8± 1-0Simple phenols Flavanones

Catechol 9-0±0-S Cryptostrobin 1S-8±1-8Pyrogallol 7-8±o'4 Naringenin 2S-8±1'1Phloroglucinol 7-6±0-2 Hesperitin 22·6±1·4

Aromadendrin 2S -4±1'9Phenolic acids Dihydrofisetin 21-0±0-9

Gallic acid 16-4±1-7 Taxifolin 20-2±1-1

Catechins FlavonolsCathechin 19'2 ± 1-7 Morin 13'9±1-0Epicatechin 21'4±0'7 Quercetin 1S'6±1-0Gallocatechin 16'0 ±0-9 Rhamnetin 17-1 ±1-0

Dihydrochalcones AnthocyanidinsPhloretin 1S'6±0-8 Luteolinidin 17-7±2'1

30

0:0 20

-5E,!:Po~c-,...

0

10

30050 100 200ppm TFG

Fig. 2. Effect of different concentrations of taxifolin glucoside on growth of M. androsaceus (18 days).Glucose concn, 20 g 1-1,

trapping of the inhibitory components on thesilica column.

Thin layer chromatography of the fractionsshowed that fraction III contained only onecompound, the flavanonol, taxifolin glucoside(dihydroquercetin-j-j3-glucoside).

Addition of different amounts of purified taxi-folin glucoside to the basal medium resulted in adoubling of the growth rate at a concentration of

only 5 p.p.m. (ca 10 pM) (Fig. 2). Increasingconcentrations caused a further increase in growthbut at a slower rate and at concentrations as highas 2000 pM the effect was only slightly higher thanat 10 pM.

Taxifolin glucoside occurs in pine needles inamounts corresponding to 1 to 2 % of the dryweight. Related flavonoid glycosides and aglyconeshave also been isolated (Popoff & Theander,

458 Stimulation by flavonoids

Table 3. Effect of taxi/olin glucoside (TFG) (25 11M) on growth of various fungi

Mycelial dry weight (mg)A

Control 25 p,M TFG

6'3±O'622'6±1'417'l±o,g

5'4±o'534'l±o,g14'o±l'410'7±o,g

Litter decomposers:Collybia peronata Sing.Marasmius graminum Berk,Marasmius scorodonius Fr.Micromphale perforans Sing.

Mycorrhizal fungi:Suillusbovinus KzeSuillusluteus GraySuillusvariegatus Kze

White rot:Polyporus zonatusFr.

Parasites:Lophodermium pinastriChev. 14'3 ±o'6Gremmeniella abietina (Lagerb.) Morelet 47'3 ± 1'6

TFG = Taxifolin glucoside

g'6±o'334'7±2'213'5±1'423'6±o'6

7'9±O'421'2±O'719'4±l'O

10'3±O'3

16'1 ±o,g46'1 ± 1'5

7060502010 30 40Days

Fig. 3, Growth of M. androsaceus with (.-.) and without (0-0) the addition of taxifolin glucoside(25 p,M). Glucose concn. 5 g 1-1.

1976). Therefore it was of interest to investigatethe effect of various other flavonoids and someother phenolic compounds on the growth of M.androsaceus. The results are presented in Table 2.

Simple phenols had no effect on growth.However, gallic acid, the catechins, the dihydro-chalcone phloretin, the flavanones and flavonolstested, as well as the anthocyanidin luteolinidin,all enhanced the growth rate of the fungus.Obviously, the glycoside forms of these substancesare not necessary for the growth-stimulating effect.

Fig. 3 shows the growth curves for M. andro-saceus cultured in the basal medium with andwithout the addition of taxifolin glucoside at aconcentration of 25 11M.

As with other basidiomycetes (Pedersen &Lindeberg, 1970) the growth of M. androsaceus insurface culture first goes through an accelerationphase, followed by an almost linear growth phase.The addition of taxifolin glucoside shortened theacceleration phase compared with the control. Themaximum yield attained after the carbon sourceW<lS exhausted was not affected by the addition oftaxifolin glucoside to any significant degree.

The influence of taxifolin glucoside on thegrowth of some other fungi, including litter-decomposing and mycorrhiza-forming basidio-mycetes as well as some parasitic ascomycetes, isshown in Table 3, It is notable that a stimulatingeffect was found only in Micromphale perforans

G. Lindeberg, M. Lindeberg, L. Lundgren, T. Popoffand O. Theander 459

Sing. and Collybia peronata Sing . These species,like M. androsaceus, are typical decomposers ofconiferous litter.

DISCUSSION

The growth-stimulating and growth-inhibitingeffects of water soluble organic compounds presentin the environment probably to a great extentdetermine the distribution and activity of differentlitter-decomposing fungi. Phenolic compoundsdue to their widespread occurrence and strongbiological effects, are probably of special im-portance.

While it is generally accepted that phenolicsubstances in the living plants may contribute tothe defence against various parasitic fungi, only afew cases of the stimulatory effects of phenolicson the growth of saprophytic basidiomycetes havebeen reported. Gallic acid caused an increasedmycelial production in the white rot fungusPolyporuszonatus Fr. (Lindeberg, 1949) and in thelitter-decomposing species Marasmius foetidus Fr.(Lindeberg & Korjus, 1949). Fries (1950) foundthat aniline promoted growth of certain Polyporusspecies and of Tricholoma fumosum Fr.; all arelignin and cellulose decomposers. Nishida, Kondo& Funaoka (1951) found that low concentrationsof taxifolin (distylin) had a weak stimulatory effecton the radial growth of Po/ystictus sanguineus Fr.,cultured on a synthetic agar medium. Aniline andp-hydroxybenzoic acid stimulated growth ofPolyporus versicolor Fr. and some other laccase-producing basidiomycetes (Fahraeus, 1962).

In the present work, a significant stimulatoryeffect was found for taxifolin glucoside, taxifolinand some other fiavonoids on the growth of certainbasidiomycetes (M. androsaceus, M. perforansandC. peronata) which occur on coniferous litter.Some other litter-decomposing species, restrictedto other substrates, as well as some ectomycorr-hizal fungi and fungal pathogens of pine andspruce, were apparently unaffected by thefiavonoids.

Taxifolin glucoside was found to be the majorphenolic glucoside in green pine needles andconstituted 0'3 % of dry weight of brown needlesjust before needle cast (Theander, 1977). Sinceonly low concentrations of taxifolin glucoside arerequired for growth-stimulation of the litter-decomposing fungi, the data indicate that theamount of the glucoside present even in brown

pine needles is probably high enough to stimulatethe colonizat ion of the litter by specialized ligninand cellulose decomposing basidiomycetes.

The study was supported by a grant from theSwedish Council for Forestry and AgriculturalResearch. The authors are indebted to Mr GunnarGlad for skilled technical assistance.

REFERENCES

FRIES, N. (1950). Precursors of tryptophan in thenutrition of Lentinus omphalodes Fr. and some otherhymenomycetes. Physiologia Plantarum 3, 185-196.

FXHRAEUS, G. (1962). Aromatic compounds as growthsubstances for laccase-producing rot fungi. Physic-logia Plantarum 15, 572-580.

LINDEBERG, G. (1944). Uber die Physiologie ligninab-bauender Bodenhymenomyzeten. Symbolae Botani-cae Upsalienses8,1-183.

LINDEBERG, G. (1949). Influence of enzymaticallyoxidizedgallicacid on the growth of some hyrneno-mycetes. Svensk Botanisk Tidskrift 43, 438-447.

LINDEBERG, G. & KORJus, M. (1949). Gallic acid andgrowth of Marasmius foetidus, Physiologia Plantarum2, 103-113 .

MELIN, E. (1946). Der Einfluss von Waldstreuextrak-ten auf das Wachstum von Bodenpilzen, mit beson-derer Berucksichtigung der Wurzelpilze vonBaumen. Symbolae Botanicae Upsalienses 8, 1-113.

MODESS, O. (1941). Zur Kenntnis der Mykorrhiza-bildner von Kiefer und Fichte. Symbolae BotanicaeUpsalienses 5,1-146.

NISHIDA, K., KONDO, T . & FUNAOKA, K. (1951).Growth-regulating activity of certain heartwood-pigments on wood-destroying fungi. Journal of theJapanese Forest Society 33, 390-393 .

NORKRANS, B. (1950). Studies in growth and cellulo-lytic enzymes of Tr icholoma. Symbolae BotanicaeUpsalienses 11, 1-126.

OLSEN, R., ODHAM, G. & LINDEBERG, G. (1971).Aromatic substances in leaves of Populu s tremula asinhibitors of mycorrhizalfungi. Physiologia Plantar-um 25,122-129.

PEDERSEN, T. A. & LINDEBERG, G. (1970). Growth ofBoletus uariegatus in surface and shake cultures.Physiologia Plantarum 23, 1110-1118.

POPOFF, T. & THEANDER, O. (1976). Phenolic glyco-sides from Pinus sy luestris L. Applied PolymerSymposium 28, 1341-1347.

THEANDER, O. (1977). Chemistry and microbiology ofthe litter of some forest trees. In Proceedings of theTAPPI fore st biology-wood chemistry conference1977, Madison, USA.

(Receivedfor publication 4 January 1980)