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Inhibition of fungi by gallic acid in relation to growth on leaves and litter
[ 329 ] Trans. Br. mycol. Soc. 73 (2) 329-336 (1979)
Printed in Great Britain
INHIBITION OF FUNGI BY GALLIC ACID IN RELATION TO GROWTH ON LEAVES AND LITTER By N.
J.
DIX
Biology Department, The University, Stirling, Scotland
Gallic acid was inhibitory to many fungi from leaves and soil. Notable exceptions included some Penicillium spp. which grew in solutions containing the acid as a sole source of carbon. Inhibition was correlated with polyphenoloxidase activity and the accumulation of the products of incomplete oxidation in the medium. These products had no effect on Penicillium spp. Several substances from the oxidation of the acid by two fungi have been separated and described. Most of these were inhibitory and in some cases toxic in bioassay tests. Two at least were relatively stable and were only slowly broken down by two bacterium species. Stable substances such as these may play an important rOle in restricting growth of some species of fungi on leaves and in litter containing the acid. Decomposition of this type of litter may be restricted to tolerant fungi such as some Penicillium spp. Gallic acid, trihydroxybenzoic acid, is widely distributed in woody plants and their litter as a component of hydrolysable tannin (jurd, 1962). Tannins decompose slowly in soil and have, with some exceptions, generally been found to be inhibitory to fungi (Cook & Taubenhaus, 1911) and although hydrolysable tannins decompose more readily than condensed tannins (Musgrave, 1948; Lewis & Starkey, 1968) gallic acid has been shown to be inhibitory to some basidiomycetes and microfungi from leaf surfaces (Lindeberg, 1949; Dix, 1974). Lindeberg (1949) observed that only those basidiomycete species with high catecholase (polyphenoloxidase) activity were inhibited in the presence of gallic acid and linked inhibition with the oxidation products forming in the enzyme catalysed reaction. Characteristically the end products of reactions of this type are conspicuous brown polymers which accumulate externally, staining the medium and allowing the reaction to be seen. Hydrolysable tannin is abundant in nature and the biological oxidation of gallic acid may affect development of phylloplane populations and decomposition of litter by fungal species. Some aspects of this problem have been investigated further in relation to certain common microfungi associated with leaves and litter in the phylloplane and in soil. MATERIALS AND METHODS
Standard inocula were cut from actively growing mycelia and transferred to Petri dishes containing
0'5 % (w/v) gallic acid incorporated into CzapekDox agar. Increase in mycelial diameter was recorded after 4 days, for fast growing species, and after 8 days for others. The cultures were examined at the end of the experiment for the development of brown pigmentation in the medium. Reduction in linear growth for each species was calculated from a comparison of the mean with that of a no acid control. Measurements were made on two replicates for each species after incubation at 20°. The acid agar was prepared from an appropriate concentration of acid in solution sterilized by Millipore filtration. The sterile acid solutions were then added to molten cooled Czapek-Dox agar and immediately poured into Petri dishes. The ED 50 (the effective dose of acid causing 50 % inhibition) for each species was determined by measuring the percentage germination of spore suspensions placed on to the surface of acid-agar disks, incubated at 20° in damp chambers. A range of concentrations of acid-agar was prepared by sterilizing the acid solutions separately, as described above, and adding to cooled molten 4 % agar in distilled water. A calculated straight line was fitted to a plot of the results from which the ED 50 % acid (wIv) for each species was read. Growth in liquid culture was studied in media containing 0'5 % (w/v) acid as a sole source of carbon and the following mineral salts: (NH.)2S04, 2'0 g; KCl, 0'5 g; MgSO•. 7H 20, 0'5 g; KH 2PO., 0'5 g; Fe 3 +, trace; distilled water 1 1; pH adjusted to 5"5 with NaOH. The medium was sterilized by Millipore filtration as 200 em" in 1 1 flasks. Inoculation was by spores and visible growth was
0007-1536/79/2828-5410 $01.00 © 1979The British Mycological Society
33°
Inhibition by gallic acid
estimated after 6'5 days on an orbital incubator at a speed of 100 rev/min at 25°. Acid removal from the cultures was measured after 2'5 days by spectrophotometric analysis at 270 nm using a standard acid curve. Two flasks were set up for each fungus and three readings taken for each flask. Close agreement was obtained between results and the final figures were reduced to a mean and compared with the uninoculated control. Polyphenoloxidase activity was estimated during the incubation of the fungus mycelium in o·5 % acid solution in distilled water, pH adjusted to 5'5, for 24 h at 20°. The fungi were cultured on liquid Czapek-Dox until a large enough mycelial mat had formed which was collected, thoroughly washed with sterile water, under aseptic conditions, before introduction into about 200 ern" of the acid solution. Readings were taken periodically at 320 or 340 nm, according to species, on a SP 1800 Pye-Unicam spectrophotometer against uninoculated acid blanks. After the experiment the mycelium was collected and the dry weight determined. Activity was expressed as absorbance change per mg dry weight h- l • Oxidation of acid by Setosphaeria rostrata and Ulocladium sp. was in 0'25 % acid solutions, pH 5'5, as described above. The products of acid oxidation by these species were extracted with chloroform, concentrated at 30° by rotary evaporation under vacuum and partially separated on silica gel preparation plates developed in mixtures of chloroform: methanol, 3: 1. Final separation was on silica gel t.l.c. plates developed in chloroform. T.l.c. plates were examined at 254 and 366 nm for fluorescing and absorbing bands. The bands were scraped from the plates and eluted with methanol or methanol: acetone mixtures. Absorption spectra were determined in methanol solutions. Bioassay tests were carried out on the material using Cladosporium herbarum spores. These were made into suspension in Czapek-Dox solution and sprayed on to developed t.l.c. plates to detect inhibitory bands (Bailey, 1971). Percentage germination of spores and germ tube growth was estimated on water agar in small sterile plastic bottle tops to which 100 pI of each eluate in sterile solution, in methanol or water, had been added separately. The material was allowed to soak into the gel before the spores, in suspension in 100 pI of distilled water, were added. After air drying, under aseptic conditions, the spores were incubated at 20° in a damp chamber and the percentage germination calculated after counting about 400 spores on each of two disks. Mean germ tube growth in arbitrary units was calculated from the measurement of about 100 germ tubes using a projection microscope and a map measurer.
Bioassay tests were carried out on several separately prepared samples and the controls were similarly prepared methanol elutions from t.l.c. plates from the solvent front area which had been in contact with chloroform but not the oxidized materials. Bioassay tests were also carried out using some of these materials and spores pregerminated in Czapek-Dox liquid medium on sterile slides prior to application of the eluted material. After preincubation the medium was removed and replaced with water. Some slides were stained at this point to obtain the initial germ tube length. After the removal of the water from the others, 20 pI of the sterile test material in water or 20 pI of sterile distilled water was added, the latter acting as controls. After incubation at 20° half the test slides were stained, measured, and compared with the controls, the other half were washed as above and incubated for a further period on the slide in 2 % liquid malt extract. The oxidized material was first partially purified by passing through Sephedex LH20 and collected in 3 ern" fractions. In all bioassay tests the absorbance of the solutions was measured at an appropriate wavelength on the spectrophotometer prior to use. The solutions were sterilized with Millipore filters and stored at 4°. The material was used as quickly as possible after extraction and separation, since some of these materials were found to be unstable at room temperature (especially A and E which appear to give rise to B after a while). It must be recognized however that some chemical change probably took place prior to and during the course of the experiments with some materials.
RESULTS
Species inhibited
The linear growth of the majority of the test fungi from the soil and the phylloplane was considerably reduced on Czapek-Dox-gallic acid agar and most species showed a correspondingly high sensitivity to the presence of the acid in spore germination tests (Table 1). Growth was usually accompanied by the accumulation of brown pigments in the culture medium indicating substantial oxidation of the acid. Uninhibited species were almost exclusively Penicillium spp. but also included Aureobasidium pullulans and Aspergillus fumigatus. Significantly, the growth of these species in the presence of the acid did not lead to any noticeable accumulation of pigment in the medium. When cultured in mineral salt solutions where gallic acid was supplied as a sole source of carbon, most species removed or transformed considerable
N.J. Dix quantities of acid in a relatively short space of time but only in the case of Penicillium spp, and Aspergillus fumigatus was this accompanied by any appreciable visible signs of growth (Table 2). The washed mycelium of most species when introduced into 0"5 % acid solution in water, pH 5"5, absorbing at 270 nm, caused a darkening of the Table
33 1
solution and gave rise to an additional peak of absorbance at 320 or 340 nm according to species within 24 h, This was usually accompanied by another peak appearing at 390 or 395 nm, Little change in absorbance was observed at these wavelengths in inoculated water controls and these changes in the pattern of absorbance have been
1. Inhibition of fungal species by gallic acid
Reduction in linear growth (%) ED so spores (% acid) Cladosporium herbarum (Pers.) Lk ex S. F. Gray Aureobasidium pullulans (De Bary) Am, Epicoccum purpurascens Ehrenb. ex Schlecht. Alternaria alternata (Fr.) Keissl. Ulocladium sp. Gliocladium roseum Bainier Setosphaeria rostrata Leonard Trichocladium asperum Harz Cylindrocarpon radicicola Wollenw. Fusarium oxysporum Schlecht. Aspergillus fumigatus Fres. Penicillium funiculosum Thorn P. janthinellum Biourge P. nigricans Bainier ex Thorn P. expansum Lk ex S. F. Gray P. frequentans Westling Mucor hiemalis Wehrner Pythium debaryanum Hesse
,-----"---.
0"34
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12 48 29 30
0'37 0'21 0"21 0"21 0'24 0'29 0"18 0'28
31 56 100 44 34
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6 24
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+13 +27 10 13
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26 89 100
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22 9
+6 o +8
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Browning* E S E E E E E E E E N N N N N N S S
* E, Extensive; S, slight; N, none.
Table
2. Behaviour of fungal species in solutions and cultures containing gallic acid
Polyphenoloxidase activity* Cladosporium herbarum Aureobasidium pullulans Epicoccum purpurascens Alternaria alternata Ulocladium sp. Gliocladium roseum Setosphaeria rostrata Trichocladium asperum Cylindrocarpon radicicola Fusarium oxysporum Aspergillus fumigatus Penicillium funiculosum P. janthinellum P. nigricans P. expansum P, frequentans Mucor hiemalis Pythium debaryanum
2"07
o
Visible growth'[
+
0'01
Acid lost (g I-I) 0"9 1"1
2"5
0'25
2"4
0'11
3'1 3'3
0"008 ~oo
~7
0"17 0'42 0'01 0'14 0'77
0'3 2'7 2"3 1'9 3'1
+ + + + + +
++
o o o
++
o
+ + +
+ + +
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2'9 2"3 2'1 5"0 1"0 0'3
0'3
* Units of absorbance at 320 or 340 nrn (mg"? dry wt) h- 1• t +, Slight; + +, appreciable; + + +, considerable.
332
Inhibition by gallic acid
assumed to be due to the oxidation of the acid by polyphenoloxidases. Enzyme activity was found to vary among species grown under standard conditions (T able 2). Certain Penicillium spp. proved exceptional and no new peaks of absorbance were detected in acid solutions inoculated with these. In mineral salt solutions where acid was supplied as the sole source of carbon, A . pullulans removed little acid compared with the control and spectrophotometric analysis of 0'5 % acid solutions in water in which the fungu s had been inoculated showed no detectable accumulation of oxidation products, suggesting that the acid was not being oxidized or metabolized. Some other species also removed little acid from the liquid culture medium. In contrast to A. pullulans however, these species were inhibited on Czapek-Doxgallic acid agar plates. In this test Cladosporium herbarum and Trichocladium asperum caused extensive browning but with Pythium debaryanum and Mucor hiemalis browning was slight and confined to the mycelium and the agar of the inoculum plug. M. hiemalis oxidation products were detected in 0'5 % acid solutions, but only after a lapse of 4 days. None appeared in P. debaryanum cultures. There was a single new peak of absorbance at 350 om for M. hiemalis. The spores of all the Penicillium spp. and those of A.fumigatus germinated readily and germination was not depressed in a mineral salt solution containing products of the oxidation of gallic acid by Setosphaeria rostrata. Spores of Alternaria alternata and Uloc/adium sp. however showed 37 and 43 % reduction in germination respectively in the same solution.
Inh ibitory substances
Chloroform extracts of gallic acid solutions which had been incubated with the mycelia of Ulocladium sp. or S. rostrata were prepared as described in the methods. Spectrophotometric analysis of the crude extract showed that it absorbed strongly between 230260 om with a major peak at 246 om and a minor peak at about 330 nm. There was little absorbance by the solution beyond 360 nm and those oxidation products absorbing at 390-395 nm , which had been noted in the unextracted oxidized acid solutions, were evidentl y not extracted by the technique and were not considered further. Concentrated samples of the chloroform extracts of the oxidized acid from S. rostrata cultures usually separated into six components on t .l.c, plates developed with chloroform, leaving a considerable residue at the base line. Similar extracts from Ulocladium sp. cultures usually separated into eight components. Some components from the two extracts had similar properties but others were evidently different (Table 3). Purpurogallin has been mentioned as forming during the oxidation of gallic acid by mushroom polyphenoloxidase (Flaig & Haider, 1961). None of the bands separated here bore any similarity with this substance. Purpurogallin does not run in chloroform and its absorption spectrum did not match up with any of the separated materials. Preliminary screening for inhibitory activity was carried out by spraying suspensions of C. herbarum spores in liquid Czapek-Dox directly on to developed t.l.c , plates (Bailey, 1971). With S. rostrata material no inhibitory activity could be
Table 3. Chara cteristics of bands eluted from thin layer chromatography plates Major absorbance t.l.c. band Rp peaks (run) Setosphaeria products Ai Not measured Occasionally present, fluorescing, white 0'09 Absorbing, red 0'1 A 274, 558, 630 Fluorescing,violet B 246,274 0'37 Weak,absorbing, blue C 248,274 0'45 Not measured Weak, visible, yellow D 0'5 1 Absorbing, red 248 E 0'57 Absorbing, blue F 0·65 24 8 Ulocladium products Not measured Fluorescing, white a1 0'08 Not measured Occasionally present, absorbing, red al 0'13 Not measured a3 Fluorescing, white 0'20 Not measured b Absorbing, blue 0'3 0 Not measured Fluorescing, violet c 0'44 Not measured d Weak, absorbing, blue 0'5 1 e Weak, absorbing, red 248 0'55 Absorbing, blue 0·61 265 f g Absorbing, blue 0·68 265
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detected by this method, except in the residue at the base line, but with material from Ulocladium sp. the growth of C. herbarum on the plates was inhibited around areas corresponding to bands e, j, and g as well as the basal residue. More detailed analysis of the biological properties of some of these substances was investigated in germination tests using C. herbarum spores and material eluted from the t.l.c. plates . All the major components from the extracts of S. rostrata material except band C and band D, which was not tested because of the low concentration available, reduced the germination of C. herbarum spores in these tests (Table 4). Considerable reduction in germination occurred with band A and F material and less with bands Band E. The effect on germ tube growth was similar; most of the bands tested inhibited germ tube growth and tests were significantly different from controls (d test on standard deviation) except in the case of bands C and F (Table 4). Bands j and g separated from Ulocladium sp. oxidized-acid solutions strongly inhibited further germ tube growth in pregerminated spores of C. herbarum. These materials were toxic, as treated germ tubes appeared dead when examined microscopically and took up no cotton blue lactophenol stain. Germ tubes treated with band g material made no recovery after treatment when the material was removed and replaced with 2 % liquid malt extract (no significant difference before and after treatment in the d test). Sufficient germ tubes remained alive after treatment with bandj material and some recovery was evident after treatment with liquid malt extract (significant difference before and after treatment in the d test) (T able 5). The inhibitory activity of band g material from solutions oxidized by Ulocladium sp. was maintained for up to 3 weeks when stored in sterile solution in water at 4°; under similar conditions bandj material remained active up to 1 week but lost appreciable activity by the end of 3 weeks (no significant difference from controls in d test) (Table 6). After 1 week storage under these conditions band g material showed no change in absorbance at 265 nm compared with that of the solution at the beginn ing of the experiment whereas band j material showed a fall in absorbance at 265 nm and a change in the spectrum of absorbance. After 3 weeks the absorbance at 265 om of band g material had dropped to about half that of the original solution and t.l.c. analysis showed that the solution now conta ined a significant quantity of material which remained on the base line when run in chloroform. The inhibitory activity of bands j and g material was tested on two coccoid bacteria isolated from
Inhibition by gallic acid
334
Table 5, Effect of some components of Ulocladium acid oxidation on C, herb arum germ tube growth Mean germ-tube length* After 48 h
Expt + malt
Absorbance t.l.c, band f LH 20 fraction 3 g LH 20 fraction 3
(265 nm) 3,69
Initial
Control
Expt
(24 h)
32'8± 12'2
31'4±11,6
1'47
32'8± 12'2
58'9± 13'3 58'9 ± 13'3
40'9± 17'4 31'3±9'7
38'O± 12'7
* Arbitrary units with standard deviations.
Table 6. Stability of some components of Ulocladium acid oxidation after storage at 4
0
Mean germ-tube length* After 46 h incubation After 1 week
t.l.c, band
Initial
Control
Expt
f LH 20 fraction 3
15'8 ± 10'1 15'8 ± 10'1
66'4±26'4 66'4±26'4
20,6± 12'9 18'4± 12'1
g
LH 20 fraction 3
After 18 h incubation After 3 weeks
Initial
Control
Expt
f
8'8±3' 6
29'2±9'4
g
5'5±3'1
22'4±8'o 28'6±9'2 6'O±2'2 6'2±2·6
* Arbitrary units with standard deviations. the phyllophane of ornamental cherry (Serrulata sp.), Both were gram negative, one cream in colour on nutrient agar, flagellate, the other yellow and probably a flavobacterium. Negative results were obtained using antibiotic disks impregnated with the test material on nutrient agar plates seeded with the bacteria, The material was used at a concentration of absorbance 4'56 for f and 6'16 for gat 265 nm. When the bacteria were inoculated separately into sterile solutions off and g in water at the above concentrations loss of absorbance at 265 om was detected in 2 em" of the solution incubated at 29 in small tubes (12'5 x 1'5 em). At the end of 1 week the yellow bacterium reduced the level of absorbance in solution f by 39 % and in solution g by 40 %. Over the same period the white bacterium reduced the level ing by 38 % but the absorbance in f increased at this wavelength. Thin layer chromatographic analysis of chloroform extracts of these solutions indicated that the breakdown of both f and g had occurred in the presence of the bacteria although whereas f had disappeared completely some g remained. These changes were accompanied by the appearance of 0
residual base line material and some other different substances. DISCUSSION
In general these results follow closely the observation made by Lindeberg (1949) that the inhibition of fungi by gallic acid is due to the accumulation of the products of acidic oxidation by polyphenoloxidases. It has been confirmed that where no inhibition occurs polyphenoloxidase activity is low or absent, Even in the case of Pythium debaryanum and Mucor hiemalis where the accumulation of oxidation products in the medium was not high, some signs of oxidation were detected and growth was very much reduced, indicating that these species may merely be extremely sensitive. The oxidation of simple polyhydroxyphenols by the catalytic action of polyphenoloxidases is a wellknown reaction which leads through intermediates to the formation of quinone compounds which polymerize to form characteristic and conspicuous brown pigments, This has led to the speculation that quinones are responsible for the inhibitory
N.J. Dix action (Lindeberg, 1949) since some quinones inhibit respiration in micro-organisms (Webb, 1966). However, this has never been investigated and, in view of the number of different inhibitory substances which have now been shown to be produced in this reaction, it is probable that this is an oversimplification. Since polyphenoloxidases can be extracellular this reaction has some potential ecological significance in soil and on leaves of woody plants where gallic acid may leak onto the surface (Dix, 1974). It is impossible however to speculate which of the inhibitory substances detected in these experiments is likely to be of greatest ecological significance. They have not been identified and since their concentration in these experiments is unknown their relative activity cannot be determined. Of the material tested, that from band g at least would appear to be important. It is toxic, stable at low temperatures and may be less readily broken down by bacteria. The potential inhibitory effect of these substances seems likely to be most important in relation to the growth of common leaf-inhabiting fungi on leaf surfaces and in litter. Harrison (1971) reported the inhibition of Cladosporium herbarum and Epicoccum nigrum (= E. purpurascens) by tannins from oak litter and there is evidence that the colonization of the leaves of woody plant species may be restricted whilst free gallic acid exists in the phylloplane (Irvine et at., 1978). This makes it important to understand better the role of these species in the decomposition of litter. Although it has been demonstrated that C. herbarum will spread from inocula over the surface of sterilized leaves placed in the Land F layers of a forest soil (Hering, 1963) and that leafinhabiting species are commonly isolated from tree litter and some develop their perfect stages there (Hogg & Hudson, 1966) we know little about the role of these species in the decomposition of tree litter, where growth may be restricted due to the release of gallic acid and possibly other hydroxyphenols from the breakdown of aromatic polymers during decomposition. Among phylloplane species Aureobasidium pullulans is especially interesting. It was not inhibited by the acid in these experiments, and previously Harrison (1971) noted that it was not inhibited by tannin from oak litter. These results suggest an adaptation to growth on leaf surfaces which contain tannins and related phenolics such as gallic acid and may explain the ubiquitous distribution of this species on the leaf surfaces of plants of many species. A number of soil-inhabiting species were also found to be inhibited by the acid. This is in
335
agreement with the results of Cowley & Whittingham (1961) and Harrison (1971) who found that several soil fungi including species from the genus Mucor and Fusarium were inhibited by tannins. Any effects of these inhibitory substances on the growth of fungi in litter will however depend upon a number of variable factors affecting concentration. Whilst some free acid may exist in the litter immediately following leaf fall, the level is probably very low (Irvine et al., 1978) and most will arise subsequently from the microbiological breakdown of tannins. The substrate concentration will therefore depend upon the rate of breakdown of tannin and the rate of removal of the acid by oxidation in alternative metabolic pathways. Other important factors affecting concentration are polyphenoloxidase activity, which varies with species (Table 1), the possibility of the removal of the inhibitory products through microbiological breakdown and a tendency for them to form polymers which may render them innocuous. Concentration in the soil is therefore unpredictable and in fact may be so low that the real effect of these substances may be minimal. Penicillium species from soil were found to be generally tolerant of the acid. This appeared to be correlated with low polyphenoloxidase activity and growth in the presence of the acid. Oxidation of the acid appears to be complete and to proceed via ring fusion. Lewis & Starkey (1969) recorded that when hydrolysable tannin was decomposed by some Penicillium spp. gallic acid was liberated which then decomposed without the formation of phenolics in the medium. Of the species tested here none were inhibited when exposed to the products of incomplete oxidation of the acid. These observations are consistent with the view that some Penicillium species are especially important in the decomposition of tree litter. Some species have long been recognized as being generally tolerant of tannins (Cook & Taubenhaus, 1911) and readily decompose some natural tannins when these are supplied as sources of carbon in culture (Knudson, 1913; Cowley & Whittingham, 1961; Lewis & Starkey, 1969).
REFERENCES
BAILEY, J. A. (1971). Production of antifungal compounds in cow pea (Vigna sinensis) and pea (Pisum sativum) after virus infection. Journal of General Microbiology 75, 119-123. COOK, M. T. & TAUBENHAUS,
J. J. (1911). The relation of parasiticfungi to the contents of the cellsof host plants. I. The toxicity of tannin. Bulletin Delaware College Agricultural Experimental Station 91, 1-77.
Inhibition by gallic acid COWLEY, G. T. & WHITTINGHAM, W. F. (1961). The effect of tannin on the growth of selected soil microfungi in culture. Mycologia 53, 539-542. DIX, N. J. (1974). Identification of a water-soluble fungal inhibitor in the leaves of Acer platanoides L. Annals of Botany 38, 505-514. FLAIG, W. & HAIDER, K. (1961). Reaktionen mit oxydierenden enzymen aus mikroorganismen. Planta Medica 9,123-14°. HARRISON, A. F. (1971). The inhibitory effect of oak leaf litter tannins on the growth of fungi in relation to litter decomposition. Soil Biology and Biochemistry 3, 167-172. HERING, T. F. (1963). Growth of litter fungi in a forest soil. In Soil Organisms (ed. J. Doeksen & J. Van der Drift), pp. 183-189. Amsterdam: NorthHolland. HOGG, B. M. & HUDSON, H. J. (1966). Microfungi on leaves of Fagus syloatica. I. The microfungal succession. Transactions of the British Mycological Society 49, 185-192. IRVINE, J. A., DIX, N. J. & WARREN, R. C. (1978). Inhibitory substances in Acer platanoides leaves.
Seasonal activity and effects on growth ofphylloplane fungi. Transactions of the British Mycological Society 70, 363-371. JURD, L. (1962). The hydrolysable tannins. In Wood Extractives (ed. W. E. Hillis). London: Academic Press. KNUDSON, L. (1913). Tannic acid fermentation. Journal of Biological Chemistry 14, 159-184. LEWIS, J. A. & STARKEY, R. L. (1968). Vegetable tannins: their decomposition and effects on decomposition of some organic compounds. Soil Science 106,241-247. LEWIS, J. A. & STARKEY, R. L. (1969). Decomposition of plant tannins by some microorganisms. Soil Science 107, 235-241. LINDEBERG, G. (1949). Influence of enzymatically oxidised gallic acid on the growth of some hymenomycetes. Svensk Botanisk Tidskrift 43, 438-447. MUSGRAVE, A. J. (1948). The mycology of the leather industry. Journal of the Society of Leather Trades Chemists 32, 3-18. WEBB, J. L. (1966). Enzymes and Metabolic Inhibitors 3,421-594. London: Academic Press.
(Accepted/or publication 25 January 1979)
Printed in Great Britain
INHIBITION OF FUNGI BY GALLIC ACID IN RELATION TO GROWTH ON LEAVES AND LITTER By N.
J.
DIX
Biology Department, The University, Stirling, Scotland
Gallic acid was inhibitory to many fungi from leaves and soil. Notable exceptions included some Penicillium spp. which grew in solutions containing the acid as a sole source of carbon. Inhibition was correlated with polyphenoloxidase activity and the accumulation of the products of incomplete oxidation in the medium. These products had no effect on Penicillium spp. Several substances from the oxidation of the acid by two fungi have been separated and described. Most of these were inhibitory and in some cases toxic in bioassay tests. Two at least were relatively stable and were only slowly broken down by two bacterium species. Stable substances such as these may play an important rOle in restricting growth of some species of fungi on leaves and in litter containing the acid. Decomposition of this type of litter may be restricted to tolerant fungi such as some Penicillium spp. Gallic acid, trihydroxybenzoic acid, is widely distributed in woody plants and their litter as a component of hydrolysable tannin (jurd, 1962). Tannins decompose slowly in soil and have, with some exceptions, generally been found to be inhibitory to fungi (Cook & Taubenhaus, 1911) and although hydrolysable tannins decompose more readily than condensed tannins (Musgrave, 1948; Lewis & Starkey, 1968) gallic acid has been shown to be inhibitory to some basidiomycetes and microfungi from leaf surfaces (Lindeberg, 1949; Dix, 1974). Lindeberg (1949) observed that only those basidiomycete species with high catecholase (polyphenoloxidase) activity were inhibited in the presence of gallic acid and linked inhibition with the oxidation products forming in the enzyme catalysed reaction. Characteristically the end products of reactions of this type are conspicuous brown polymers which accumulate externally, staining the medium and allowing the reaction to be seen. Hydrolysable tannin is abundant in nature and the biological oxidation of gallic acid may affect development of phylloplane populations and decomposition of litter by fungal species. Some aspects of this problem have been investigated further in relation to certain common microfungi associated with leaves and litter in the phylloplane and in soil. MATERIALS AND METHODS
Standard inocula were cut from actively growing mycelia and transferred to Petri dishes containing
0'5 % (w/v) gallic acid incorporated into CzapekDox agar. Increase in mycelial diameter was recorded after 4 days, for fast growing species, and after 8 days for others. The cultures were examined at the end of the experiment for the development of brown pigmentation in the medium. Reduction in linear growth for each species was calculated from a comparison of the mean with that of a no acid control. Measurements were made on two replicates for each species after incubation at 20°. The acid agar was prepared from an appropriate concentration of acid in solution sterilized by Millipore filtration. The sterile acid solutions were then added to molten cooled Czapek-Dox agar and immediately poured into Petri dishes. The ED 50 (the effective dose of acid causing 50 % inhibition) for each species was determined by measuring the percentage germination of spore suspensions placed on to the surface of acid-agar disks, incubated at 20° in damp chambers. A range of concentrations of acid-agar was prepared by sterilizing the acid solutions separately, as described above, and adding to cooled molten 4 % agar in distilled water. A calculated straight line was fitted to a plot of the results from which the ED 50 % acid (wIv) for each species was read. Growth in liquid culture was studied in media containing 0'5 % (w/v) acid as a sole source of carbon and the following mineral salts: (NH.)2S04, 2'0 g; KCl, 0'5 g; MgSO•. 7H 20, 0'5 g; KH 2PO., 0'5 g; Fe 3 +, trace; distilled water 1 1; pH adjusted to 5"5 with NaOH. The medium was sterilized by Millipore filtration as 200 em" in 1 1 flasks. Inoculation was by spores and visible growth was
0007-1536/79/2828-5410 $01.00 © 1979The British Mycological Society
33°
Inhibition by gallic acid
estimated after 6'5 days on an orbital incubator at a speed of 100 rev/min at 25°. Acid removal from the cultures was measured after 2'5 days by spectrophotometric analysis at 270 nm using a standard acid curve. Two flasks were set up for each fungus and three readings taken for each flask. Close agreement was obtained between results and the final figures were reduced to a mean and compared with the uninoculated control. Polyphenoloxidase activity was estimated during the incubation of the fungus mycelium in o·5 % acid solution in distilled water, pH adjusted to 5'5, for 24 h at 20°. The fungi were cultured on liquid Czapek-Dox until a large enough mycelial mat had formed which was collected, thoroughly washed with sterile water, under aseptic conditions, before introduction into about 200 ern" of the acid solution. Readings were taken periodically at 320 or 340 nm, according to species, on a SP 1800 Pye-Unicam spectrophotometer against uninoculated acid blanks. After the experiment the mycelium was collected and the dry weight determined. Activity was expressed as absorbance change per mg dry weight h- l • Oxidation of acid by Setosphaeria rostrata and Ulocladium sp. was in 0'25 % acid solutions, pH 5'5, as described above. The products of acid oxidation by these species were extracted with chloroform, concentrated at 30° by rotary evaporation under vacuum and partially separated on silica gel preparation plates developed in mixtures of chloroform: methanol, 3: 1. Final separation was on silica gel t.l.c. plates developed in chloroform. T.l.c. plates were examined at 254 and 366 nm for fluorescing and absorbing bands. The bands were scraped from the plates and eluted with methanol or methanol: acetone mixtures. Absorption spectra were determined in methanol solutions. Bioassay tests were carried out on the material using Cladosporium herbarum spores. These were made into suspension in Czapek-Dox solution and sprayed on to developed t.l.c. plates to detect inhibitory bands (Bailey, 1971). Percentage germination of spores and germ tube growth was estimated on water agar in small sterile plastic bottle tops to which 100 pI of each eluate in sterile solution, in methanol or water, had been added separately. The material was allowed to soak into the gel before the spores, in suspension in 100 pI of distilled water, were added. After air drying, under aseptic conditions, the spores were incubated at 20° in a damp chamber and the percentage germination calculated after counting about 400 spores on each of two disks. Mean germ tube growth in arbitrary units was calculated from the measurement of about 100 germ tubes using a projection microscope and a map measurer.
Bioassay tests were carried out on several separately prepared samples and the controls were similarly prepared methanol elutions from t.l.c. plates from the solvent front area which had been in contact with chloroform but not the oxidized materials. Bioassay tests were also carried out using some of these materials and spores pregerminated in Czapek-Dox liquid medium on sterile slides prior to application of the eluted material. After preincubation the medium was removed and replaced with water. Some slides were stained at this point to obtain the initial germ tube length. After the removal of the water from the others, 20 pI of the sterile test material in water or 20 pI of sterile distilled water was added, the latter acting as controls. After incubation at 20° half the test slides were stained, measured, and compared with the controls, the other half were washed as above and incubated for a further period on the slide in 2 % liquid malt extract. The oxidized material was first partially purified by passing through Sephedex LH20 and collected in 3 ern" fractions. In all bioassay tests the absorbance of the solutions was measured at an appropriate wavelength on the spectrophotometer prior to use. The solutions were sterilized with Millipore filters and stored at 4°. The material was used as quickly as possible after extraction and separation, since some of these materials were found to be unstable at room temperature (especially A and E which appear to give rise to B after a while). It must be recognized however that some chemical change probably took place prior to and during the course of the experiments with some materials.
RESULTS
Species inhibited
The linear growth of the majority of the test fungi from the soil and the phylloplane was considerably reduced on Czapek-Dox-gallic acid agar and most species showed a correspondingly high sensitivity to the presence of the acid in spore germination tests (Table 1). Growth was usually accompanied by the accumulation of brown pigments in the culture medium indicating substantial oxidation of the acid. Uninhibited species were almost exclusively Penicillium spp. but also included Aureobasidium pullulans and Aspergillus fumigatus. Significantly, the growth of these species in the presence of the acid did not lead to any noticeable accumulation of pigment in the medium. When cultured in mineral salt solutions where gallic acid was supplied as a sole source of carbon, most species removed or transformed considerable
N.J. Dix quantities of acid in a relatively short space of time but only in the case of Penicillium spp, and Aspergillus fumigatus was this accompanied by any appreciable visible signs of growth (Table 2). The washed mycelium of most species when introduced into 0"5 % acid solution in water, pH 5"5, absorbing at 270 nm, caused a darkening of the Table
33 1
solution and gave rise to an additional peak of absorbance at 320 or 340 nm according to species within 24 h, This was usually accompanied by another peak appearing at 390 or 395 nm, Little change in absorbance was observed at these wavelengths in inoculated water controls and these changes in the pattern of absorbance have been
1. Inhibition of fungal species by gallic acid
Reduction in linear growth (%) ED so spores (% acid) Cladosporium herbarum (Pers.) Lk ex S. F. Gray Aureobasidium pullulans (De Bary) Am, Epicoccum purpurascens Ehrenb. ex Schlecht. Alternaria alternata (Fr.) Keissl. Ulocladium sp. Gliocladium roseum Bainier Setosphaeria rostrata Leonard Trichocladium asperum Harz Cylindrocarpon radicicola Wollenw. Fusarium oxysporum Schlecht. Aspergillus fumigatus Fres. Penicillium funiculosum Thorn P. janthinellum Biourge P. nigricans Bainier ex Thorn P. expansum Lk ex S. F. Gray P. frequentans Westling Mucor hiemalis Wehrner Pythium debaryanum Hesse
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26 89 100
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0'26
74 100
22 9
+6 o +8
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Browning* E S E E E E E E E E N N N N N N S S
* E, Extensive; S, slight; N, none.
Table
2. Behaviour of fungal species in solutions and cultures containing gallic acid
Polyphenoloxidase activity* Cladosporium herbarum Aureobasidium pullulans Epicoccum purpurascens Alternaria alternata Ulocladium sp. Gliocladium roseum Setosphaeria rostrata Trichocladium asperum Cylindrocarpon radicicola Fusarium oxysporum Aspergillus fumigatus Penicillium funiculosum P. janthinellum P. nigricans P. expansum P, frequentans Mucor hiemalis Pythium debaryanum
2"07
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+
0'01
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2"4
0'11
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0'3
* Units of absorbance at 320 or 340 nrn (mg"? dry wt) h- 1• t +, Slight; + +, appreciable; + + +, considerable.
332
Inhibition by gallic acid
assumed to be due to the oxidation of the acid by polyphenoloxidases. Enzyme activity was found to vary among species grown under standard conditions (T able 2). Certain Penicillium spp. proved exceptional and no new peaks of absorbance were detected in acid solutions inoculated with these. In mineral salt solutions where acid was supplied as the sole source of carbon, A . pullulans removed little acid compared with the control and spectrophotometric analysis of 0'5 % acid solutions in water in which the fungu s had been inoculated showed no detectable accumulation of oxidation products, suggesting that the acid was not being oxidized or metabolized. Some other species also removed little acid from the liquid culture medium. In contrast to A. pullulans however, these species were inhibited on Czapek-Doxgallic acid agar plates. In this test Cladosporium herbarum and Trichocladium asperum caused extensive browning but with Pythium debaryanum and Mucor hiemalis browning was slight and confined to the mycelium and the agar of the inoculum plug. M. hiemalis oxidation products were detected in 0'5 % acid solutions, but only after a lapse of 4 days. None appeared in P. debaryanum cultures. There was a single new peak of absorbance at 350 om for M. hiemalis. The spores of all the Penicillium spp. and those of A.fumigatus germinated readily and germination was not depressed in a mineral salt solution containing products of the oxidation of gallic acid by Setosphaeria rostrata. Spores of Alternaria alternata and Uloc/adium sp. however showed 37 and 43 % reduction in germination respectively in the same solution.
Inh ibitory substances
Chloroform extracts of gallic acid solutions which had been incubated with the mycelia of Ulocladium sp. or S. rostrata were prepared as described in the methods. Spectrophotometric analysis of the crude extract showed that it absorbed strongly between 230260 om with a major peak at 246 om and a minor peak at about 330 nm. There was little absorbance by the solution beyond 360 nm and those oxidation products absorbing at 390-395 nm , which had been noted in the unextracted oxidized acid solutions, were evidentl y not extracted by the technique and were not considered further. Concentrated samples of the chloroform extracts of the oxidized acid from S. rostrata cultures usually separated into six components on t .l.c, plates developed with chloroform, leaving a considerable residue at the base line. Similar extracts from Ulocladium sp. cultures usually separated into eight components. Some components from the two extracts had similar properties but others were evidently different (Table 3). Purpurogallin has been mentioned as forming during the oxidation of gallic acid by mushroom polyphenoloxidase (Flaig & Haider, 1961). None of the bands separated here bore any similarity with this substance. Purpurogallin does not run in chloroform and its absorption spectrum did not match up with any of the separated materials. Preliminary screening for inhibitory activity was carried out by spraying suspensions of C. herbarum spores in liquid Czapek-Dox directly on to developed t.l.c , plates (Bailey, 1971). With S. rostrata material no inhibitory activity could be
Table 3. Chara cteristics of bands eluted from thin layer chromatography plates Major absorbance t.l.c. band Rp peaks (run) Setosphaeria products Ai Not measured Occasionally present, fluorescing, white 0'09 Absorbing, red 0'1 A 274, 558, 630 Fluorescing,violet B 246,274 0'37 Weak,absorbing, blue C 248,274 0'45 Not measured Weak, visible, yellow D 0'5 1 Absorbing, red 248 E 0'57 Absorbing, blue F 0·65 24 8 Ulocladium products Not measured Fluorescing, white a1 0'08 Not measured Occasionally present, absorbing, red al 0'13 Not measured a3 Fluorescing, white 0'20 Not measured b Absorbing, blue 0'3 0 Not measured Fluorescing, violet c 0'44 Not measured d Weak, absorbing, blue 0'5 1 e Weak, absorbing, red 248 0'55 Absorbing, blue 0·61 265 f g Absorbing, blue 0·68 265
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detected by this method, except in the residue at the base line, but with material from Ulocladium sp. the growth of C. herbarum on the plates was inhibited around areas corresponding to bands e, j, and g as well as the basal residue. More detailed analysis of the biological properties of some of these substances was investigated in germination tests using C. herbarum spores and material eluted from the t.l.c. plates . All the major components from the extracts of S. rostrata material except band C and band D, which was not tested because of the low concentration available, reduced the germination of C. herbarum spores in these tests (Table 4). Considerable reduction in germination occurred with band A and F material and less with bands Band E. The effect on germ tube growth was similar; most of the bands tested inhibited germ tube growth and tests were significantly different from controls (d test on standard deviation) except in the case of bands C and F (Table 4). Bands j and g separated from Ulocladium sp. oxidized-acid solutions strongly inhibited further germ tube growth in pregerminated spores of C. herbarum. These materials were toxic, as treated germ tubes appeared dead when examined microscopically and took up no cotton blue lactophenol stain. Germ tubes treated with band g material made no recovery after treatment when the material was removed and replaced with 2 % liquid malt extract (no significant difference before and after treatment in the d test). Sufficient germ tubes remained alive after treatment with bandj material and some recovery was evident after treatment with liquid malt extract (significant difference before and after treatment in the d test) (T able 5). The inhibitory activity of band g material from solutions oxidized by Ulocladium sp. was maintained for up to 3 weeks when stored in sterile solution in water at 4°; under similar conditions bandj material remained active up to 1 week but lost appreciable activity by the end of 3 weeks (no significant difference from controls in d test) (Table 6). After 1 week storage under these conditions band g material showed no change in absorbance at 265 nm compared with that of the solution at the beginn ing of the experiment whereas band j material showed a fall in absorbance at 265 nm and a change in the spectrum of absorbance. After 3 weeks the absorbance at 265 om of band g material had dropped to about half that of the original solution and t.l.c. analysis showed that the solution now conta ined a significant quantity of material which remained on the base line when run in chloroform. The inhibitory activity of bands j and g material was tested on two coccoid bacteria isolated from
Inhibition by gallic acid
334
Table 5, Effect of some components of Ulocladium acid oxidation on C, herb arum germ tube growth Mean germ-tube length* After 48 h
Expt + malt
Absorbance t.l.c, band f LH 20 fraction 3 g LH 20 fraction 3
(265 nm) 3,69
Initial
Control
Expt
(24 h)
32'8± 12'2
31'4±11,6
1'47
32'8± 12'2
58'9± 13'3 58'9 ± 13'3
40'9± 17'4 31'3±9'7
38'O± 12'7
* Arbitrary units with standard deviations.
Table 6. Stability of some components of Ulocladium acid oxidation after storage at 4
0
Mean germ-tube length* After 46 h incubation After 1 week
t.l.c, band
Initial
Control
Expt
f LH 20 fraction 3
15'8 ± 10'1 15'8 ± 10'1
66'4±26'4 66'4±26'4
20,6± 12'9 18'4± 12'1
g
LH 20 fraction 3
After 18 h incubation After 3 weeks
Initial
Control
Expt
f
8'8±3' 6
29'2±9'4
g
5'5±3'1
22'4±8'o 28'6±9'2 6'O±2'2 6'2±2·6
* Arbitrary units with standard deviations. the phyllophane of ornamental cherry (Serrulata sp.), Both were gram negative, one cream in colour on nutrient agar, flagellate, the other yellow and probably a flavobacterium. Negative results were obtained using antibiotic disks impregnated with the test material on nutrient agar plates seeded with the bacteria, The material was used at a concentration of absorbance 4'56 for f and 6'16 for gat 265 nm. When the bacteria were inoculated separately into sterile solutions off and g in water at the above concentrations loss of absorbance at 265 om was detected in 2 em" of the solution incubated at 29 in small tubes (12'5 x 1'5 em). At the end of 1 week the yellow bacterium reduced the level of absorbance in solution f by 39 % and in solution g by 40 %. Over the same period the white bacterium reduced the level ing by 38 % but the absorbance in f increased at this wavelength. Thin layer chromatographic analysis of chloroform extracts of these solutions indicated that the breakdown of both f and g had occurred in the presence of the bacteria although whereas f had disappeared completely some g remained. These changes were accompanied by the appearance of 0
residual base line material and some other different substances. DISCUSSION
In general these results follow closely the observation made by Lindeberg (1949) that the inhibition of fungi by gallic acid is due to the accumulation of the products of acidic oxidation by polyphenoloxidases. It has been confirmed that where no inhibition occurs polyphenoloxidase activity is low or absent, Even in the case of Pythium debaryanum and Mucor hiemalis where the accumulation of oxidation products in the medium was not high, some signs of oxidation were detected and growth was very much reduced, indicating that these species may merely be extremely sensitive. The oxidation of simple polyhydroxyphenols by the catalytic action of polyphenoloxidases is a wellknown reaction which leads through intermediates to the formation of quinone compounds which polymerize to form characteristic and conspicuous brown pigments, This has led to the speculation that quinones are responsible for the inhibitory
N.J. Dix action (Lindeberg, 1949) since some quinones inhibit respiration in micro-organisms (Webb, 1966). However, this has never been investigated and, in view of the number of different inhibitory substances which have now been shown to be produced in this reaction, it is probable that this is an oversimplification. Since polyphenoloxidases can be extracellular this reaction has some potential ecological significance in soil and on leaves of woody plants where gallic acid may leak onto the surface (Dix, 1974). It is impossible however to speculate which of the inhibitory substances detected in these experiments is likely to be of greatest ecological significance. They have not been identified and since their concentration in these experiments is unknown their relative activity cannot be determined. Of the material tested, that from band g at least would appear to be important. It is toxic, stable at low temperatures and may be less readily broken down by bacteria. The potential inhibitory effect of these substances seems likely to be most important in relation to the growth of common leaf-inhabiting fungi on leaf surfaces and in litter. Harrison (1971) reported the inhibition of Cladosporium herbarum and Epicoccum nigrum (= E. purpurascens) by tannins from oak litter and there is evidence that the colonization of the leaves of woody plant species may be restricted whilst free gallic acid exists in the phylloplane (Irvine et at., 1978). This makes it important to understand better the role of these species in the decomposition of litter. Although it has been demonstrated that C. herbarum will spread from inocula over the surface of sterilized leaves placed in the Land F layers of a forest soil (Hering, 1963) and that leafinhabiting species are commonly isolated from tree litter and some develop their perfect stages there (Hogg & Hudson, 1966) we know little about the role of these species in the decomposition of tree litter, where growth may be restricted due to the release of gallic acid and possibly other hydroxyphenols from the breakdown of aromatic polymers during decomposition. Among phylloplane species Aureobasidium pullulans is especially interesting. It was not inhibited by the acid in these experiments, and previously Harrison (1971) noted that it was not inhibited by tannin from oak litter. These results suggest an adaptation to growth on leaf surfaces which contain tannins and related phenolics such as gallic acid and may explain the ubiquitous distribution of this species on the leaf surfaces of plants of many species. A number of soil-inhabiting species were also found to be inhibited by the acid. This is in
335
agreement with the results of Cowley & Whittingham (1961) and Harrison (1971) who found that several soil fungi including species from the genus Mucor and Fusarium were inhibited by tannins. Any effects of these inhibitory substances on the growth of fungi in litter will however depend upon a number of variable factors affecting concentration. Whilst some free acid may exist in the litter immediately following leaf fall, the level is probably very low (Irvine et al., 1978) and most will arise subsequently from the microbiological breakdown of tannins. The substrate concentration will therefore depend upon the rate of breakdown of tannin and the rate of removal of the acid by oxidation in alternative metabolic pathways. Other important factors affecting concentration are polyphenoloxidase activity, which varies with species (Table 1), the possibility of the removal of the inhibitory products through microbiological breakdown and a tendency for them to form polymers which may render them innocuous. Concentration in the soil is therefore unpredictable and in fact may be so low that the real effect of these substances may be minimal. Penicillium species from soil were found to be generally tolerant of the acid. This appeared to be correlated with low polyphenoloxidase activity and growth in the presence of the acid. Oxidation of the acid appears to be complete and to proceed via ring fusion. Lewis & Starkey (1969) recorded that when hydrolysable tannin was decomposed by some Penicillium spp. gallic acid was liberated which then decomposed without the formation of phenolics in the medium. Of the species tested here none were inhibited when exposed to the products of incomplete oxidation of the acid. These observations are consistent with the view that some Penicillium species are especially important in the decomposition of tree litter. Some species have long been recognized as being generally tolerant of tannins (Cook & Taubenhaus, 1911) and readily decompose some natural tannins when these are supplied as sources of carbon in culture (Knudson, 1913; Cowley & Whittingham, 1961; Lewis & Starkey, 1969).
REFERENCES
BAILEY, J. A. (1971). Production of antifungal compounds in cow pea (Vigna sinensis) and pea (Pisum sativum) after virus infection. Journal of General Microbiology 75, 119-123. COOK, M. T. & TAUBENHAUS,
J. J. (1911). The relation of parasiticfungi to the contents of the cellsof host plants. I. The toxicity of tannin. Bulletin Delaware College Agricultural Experimental Station 91, 1-77.
Inhibition by gallic acid COWLEY, G. T. & WHITTINGHAM, W. F. (1961). The effect of tannin on the growth of selected soil microfungi in culture. Mycologia 53, 539-542. DIX, N. J. (1974). Identification of a water-soluble fungal inhibitor in the leaves of Acer platanoides L. Annals of Botany 38, 505-514. FLAIG, W. & HAIDER, K. (1961). Reaktionen mit oxydierenden enzymen aus mikroorganismen. Planta Medica 9,123-14°. HARRISON, A. F. (1971). The inhibitory effect of oak leaf litter tannins on the growth of fungi in relation to litter decomposition. Soil Biology and Biochemistry 3, 167-172. HERING, T. F. (1963). Growth of litter fungi in a forest soil. In Soil Organisms (ed. J. Doeksen & J. Van der Drift), pp. 183-189. Amsterdam: NorthHolland. HOGG, B. M. & HUDSON, H. J. (1966). Microfungi on leaves of Fagus syloatica. I. The microfungal succession. Transactions of the British Mycological Society 49, 185-192. IRVINE, J. A., DIX, N. J. & WARREN, R. C. (1978). Inhibitory substances in Acer platanoides leaves.
Seasonal activity and effects on growth ofphylloplane fungi. Transactions of the British Mycological Society 70, 363-371. JURD, L. (1962). The hydrolysable tannins. In Wood Extractives (ed. W. E. Hillis). London: Academic Press. KNUDSON, L. (1913). Tannic acid fermentation. Journal of Biological Chemistry 14, 159-184. LEWIS, J. A. & STARKEY, R. L. (1968). Vegetable tannins: their decomposition and effects on decomposition of some organic compounds. Soil Science 106,241-247. LEWIS, J. A. & STARKEY, R. L. (1969). Decomposition of plant tannins by some microorganisms. Soil Science 107, 235-241. LINDEBERG, G. (1949). Influence of enzymatically oxidised gallic acid on the growth of some hymenomycetes. Svensk Botanisk Tidskrift 43, 438-447. MUSGRAVE, A. J. (1948). The mycology of the leather industry. Journal of the Society of Leather Trades Chemists 32, 3-18. WEBB, J. L. (1966). Enzymes and Metabolic Inhibitors 3,421-594. London: Academic Press.
(Accepted/or publication 25 January 1979)