Tolerance of some wood-decomposing basidiomycetes to
aromatic compounds related to lignin degradation
Veikko Hintikka
Finnish Forest Research Institute, Unioninkatu 40 A, Helsinki 17, Finland
Abstract. The tolerance of 46 wood-decomposing Hymenomycetes to 23 phenolic and
rehted aromatic compounds was inveSit:igMed by <~Jding
the compoudns to Hagem agar
after autoclaving and cooling it to 70-80°C, and measuring the radial growth on these
substrates after 8- 1'0 days (Table 1). Considerable differences in tolerance were found
between the species. The mos1t wlerant were brown-rot fungi, whiJte-rot fungi being on the
average more sensi:tive. When nMura!l substrates of these fungi were treated with FeCI!~
soluthe presence of . phenols, a posi·tive reaction was genera!lly obtained wivh the
tion to rev<~Jl
brown-rot, but not with the white-rot fungi. The results sugges•t that phenolic compounds
may be important in the ecology of wood-decomposing fungi, e.g. in connection with the
drying of wood.
Introduction
Considerable attention has been paid in
recent years to the roles played by phenolic
compounds in the degradation of lignin, hostparasite interactions and as constituents of
higher plants (PRIDHAM 1960,HARBORNE 1964,
K URSANOV & ZAPIWMETOV 1968) . In trees,
phenolic compound's may have several different origins: i) they may occur as normal
constituents of living wood and bark, either
free or as glycosides; ii) they may be produced by the tree as a reaction to invading
pathogens (SHAIN 1967); iii) they may accumulate as lignin degradation products owing
to the activity of fungi; or iv) they may
be synthetized by fungi or other wood-inhab-
iting micro-organisms (PowER et a!. 1965,
ARMAND & THIVEND 1965) .
Wood decay in living and dead wood is generally caused a number of basi-diomycetes, many of which are effective lignin decomposers. Many earlier investigations (e.g.
RENNERFELT & NACHT 1955, GADD 1957)
have indicated that phenolic compounds
exert a toxic influence on the growth of these
fungi . This paper attempts, by studying
the tolerance of some common wood-decomposing basidiomycetes to phenolic compounds
known to occur in nature, to obtain
preliminary information on the possible effects of these compounds in fungal ecology.
Methods
Fungus strains preserved in the Forest
Biology Laboratory, Finnish Forest Research
Institute, were used. The strains were kept
on Hagem agar ( 5 g glucose, 5 g malt extract,
0,5 g KH 2 P0 4 , 0.5 g NH4 CJ, 0.5 g Mg
46
S0 4 , 0.5 ml FeCl 3 1 % solution, 15 g agar,
1000 ml dist. H 20) at +5°C with approximately two transfers a year. Most of the
strains were isolated in the years 1966- 68.
The following compounds were used: ani-
sic acid Fluka puriss., benzaldehyde reag.
qual., benzoic acid BDH anal. reag., caffeic
acid Fluka pur., cinnamic acid Merck, pcresol Fluka puriss., 2,5~dihyroxbenzc
acid Fluka puriss., ferulic acid Fluka purum,
gallic acid, 4-hydroxybenzoic acid Fluka
puriss., 4-hydroxybenzaldehyde Koch-Light,
pure, guaiacol Rhone Poulenc, 3-methoxybenzoic acid Fluka purum, phenol BDH
anal. reag., protocatechuic acid Sigma, pyrocatechol Merck pro anal., pyrogallol Merck
pro anal ., resorcinol BDH anal. reag., salicylic
acid BDH anal. reag., syringic acid Fluka
purum, tannic acid Baker analyzed reag., 3,4,
5-trirnethoxybenzoic acid Fluka pururn, vanillic acid Fluka pururn, thymol tech., vanillin
BDH anal. reag., veratric acid Fluka puriss.
The tolerance was studied on Hagern
agar, to which weighed amounts of phenolics were added after it had been autoclaved
and cooled to 80--70°C, and which was
poured into 10 ern petri dishes. Although
the phenols were not sterilized, infection
by molds and bacteria proved to be negligible. Radial growth on the agar was measured after 6--10 days. The results are given in Table 1.
Results
If we suppose that a natural wood substrate
contains certain phenolic compounds, we
can see from Table 1 that the activity and
rapidity of growth of the wood-decomposing
basidiomycetes investigated will differ in
many respects from that observed on standard media. The species listed below ar those
shown by their radial growth to be the
most active at high concentrations of the
respective compounds.
Anisic acid: Laetiporus suphureus, Gloeophyllum sepiarium, Lentinus lepideus, Panellus serotinus, Fomitopsis annosa, Pleurotus
ostreatus.
Benzoic acid: Gymnopilus penetrans, Stereum sanguinolentum; tolerance of other
species fairly uniform.
Caffeic acid: Fomitopsis pinicola, Laetiporus sulphureus, Daedalea quercina, Piptoporus betulin us, I no notus rheades.
Cinnarnic acid: Stereum purpureum, Corio! us hirsutus, Panellus serotinus, Phellinus
pini, Pholiota alnicola.
p-cresol: I no notus rheades, Lentinus lepideus, Stereum purpureum, Polyporus brumalis, Phellinus pini, Fomitopsis pinicola.
2,5-dihydroxybenzoic acid: Daedalea quercina, Fomitopsis pinicola, Laetiporus sulphureus, Coriolus vaporarius, Coriolellus serialis,
Inonotus rheades.
Ferulic acid: Stereum sanguinolentum, Coriolus hirsutus, Hirschioporus abietinus, Phellinus pini, Pycnoporus cinnabarinus, Pleurotus ostreatus, Gloeophyllum sepiarium.
Gallic acid :F omito psis pinicola, Daedalea
quercina, Inonotus rheades, Laetiporus sulp-
hureus, Piptoporus betulinus, Coriolellus serialis.
Guaiacol: I no notus rheades, Lentinus lepideus, Phcllinus pini.
4-Hydroxybenzaldehyde : Gloeophyllum sepiarium, Phellinus pini, Xeromphalina campanella, Stereum sanguinolentum, Phellinus
tremulae, Psathyrella spadicea.
3-Methoxybenzoic acrd: Gymnopilus penetrans, Phellinus tremulae, Stereum purpureum, Polyporus brumalis, Coriolus zonatus,
Pholiota aurivella.
Phenol: Gloeophyllum sepiarium, Fomitopsis pinicola, Phellinus pini, P. tremulae,
Stereum purpureum, Coriolus hirsutus.
Protocatechuic acid: did not inhibit the
growth rate of most species. Inhibition distinct in Pycnoporus cinnabarinus, Pleurotus
ostreatus, and in Flammulina velutipes.
Pyrocatechol: Phellinus pini, Daedalea
quercina, St ereum purpureum, Polyporus
brumalis, Fomito psis pinicola.
Pyrogallol: Polyporus brumalis, Stereum
sanguinolentum, Gloeoporus dichrous, Lentinellus omphalodes, Kuehneromyces mutabilis, Fames fomentarius.
Resorcinol: Coriolus hirsutus, Daedalea
quercina, Coriolus zonatus, Inonotus rheades, Panellus serotinus, Polypilus frondosu s,
Fomitopsis pinicola.
Salicylic acid: Gloeophyllum sepiarium,
Laetiporus suphureus, Lentinus lepideus,
Daedalea quercina, Polypilus frondosus, Panellus serotinus.
Syringic acid: Laetiporus sulphureus, Fomitopsis pinicola, Coriolus vaporarius, Pipto47
~
BENZOIC .I.CID
ANISIC .I.CID
-·----
CAFFEIC ACID
p-CRESOL 2. 5- DIHYDROXYBENZOIC ACID
CINNAI1IC ACID
~-
CONCENTRATION 0/oo
Aporpium. semisupinum
Armillariella mellea
Coriolellus eerialis
Coriolus hirsutus
c. vaporari us
c. zonatus
Daedalea quercina
Flammulina velutipea
Fames fomentariua
Fomitopsis annosa
F. pinicola
Gloeophyllum sepiarium
Gloeoporus dichrous
Gymnopilus penetrans
Hirschioporus abietinua
Hypholoma capnoides
Inonotus radiatus
I. rheades
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pH
17
34
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14 14 13
18 13 8
12 11 8
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4 3 7
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45 26 21
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P. pini
P. punctatus
P. tremulae
Pholiota alnicola
P. aurivella
F. squarrose.
Phyllotopsis nidulans
0
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3 12 8 0 0
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26221300
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Phellinus conchatua
0
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Table 1. Radial growth of certain wood-decomposing fungi at different concentrations
( 1953) and MosER ( 1967) .
BoNDARTSEV
0
GUAIACOL
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FERULIC ACID
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%
6
of phenolic compounds. The nomenclature follows
0 0
6 0
0 0
0 0
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0 0
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PHENOL
CONCENTRATIO!f
0
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PROTOCATECHUIC
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0 0 0 11 11 10
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42 25 16 ·5 + 0 47 40
29 · 5 :2 o o o 38 40
12 ·16 8 . 4 0 0 17 19
20 .15 9 · 2 0 0 32 32
22 14 . 5 0 0 0 37 38
3913 8 oo ·o2545
Fo11itopsis annosa
F. pinicola
Gloeophyllum sepiarium
Glosoporus dichrous
G;ymnopilus penetrans
Hirschioporus abietinus
Hypholom.a capnoides
lnonotus radiatus
I. · rheades ·
36. 20 3
41 • 16 11
321715
30 25 10
16 . 5 · 0
12 12 . ·3
6 0 0
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9 10 2
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Laetiporus sulphursus .
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32 6
gg
.ArmillB.riella· mellea
Coriolellus serialis
Coriolus hirsutus
C. vaporarius
.c. ·zonatus · · ·
Daedalea quercin8.
Flammulina velutipgs
~n
Lentinellus omphalodes
Lentiilua lepideus
Panellus mitis
P. aerotinus
P. stypticus.
:Panus conchatus ·
Fhellinua conchatus
~
Phellinus igniarius
·p. pini
P. punctatus
P. tremulae
Pholiota alnicola
P. auri vella
P. squarrosa
Phyllotopsis nidulans
.Piptoporus .betulinus
Pleurotus dryinus
P. ostreatus
Polyporus brumalis
Polypilua .f'rondoaus
Paatbyrella spadicea
Pycnoporus cinnabarinus
Stereum birautum.
s. purpureum
S. eanguinolentWI
.
Xeromphalin~
campanella
pH
6
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3 44 . 3 1 0 45 22 10 4 38 23 15 10 0 37 18 15 9 5
03710 . 5 1342714 .. 73018151202819 93 +
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0402171252010
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Table 1 (continued). Radial growth of certain wood-decomposing fungi at different concentrations ( 0/oo)
phenolic compounds.
<.0
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9 7 2 1 + 0 14 12 9 8 5 15 9 11 11 6 3 2 11 6 + + 0 11 12 . ? 5 + 9 6 4 2 7 . 3 7 5 3 +
12 7 0 . 000181510 9 022 7 5 1 0 - - 1 5 1 1+01512 9 301612? 010 +10 ?·1 +
3 . 5 . 3 1 + 0 12 .9 5 5 2 ? 3• 3 3· 1 + 0 2 . + 0 0 0 2 9 3 2 + 3 2 1 0 7 . 2 8 . 5 3 2
16 15 12 2 0 0 22 22 15 15 2 19. 7 ? 4 1 0 - 20 0 0 0 0 20 1? 10 ? 0 20 17 15 5 15 0 13. 0 0 0
15 5 2 00026252220 0211412 2 000241514.. 002416. 12 · 1001515. 10 122 02211 6 +
11 0 0 0 0 0 4 . 5 4 3 .0 12 ·+ 0 0 0 - - 18 15 0 0 0 18 19 11 3 0 13 1? 14 0 1? 0 9 . 9 . . 3 +
15 2 . 0 0 0 0 21 24 21 17 . 0 26 5 3 0 0 - - 20 0 0 0 0 20 11 2 1 0 18 18 12 . 0 13" 3 14 0 0 0
4010 510045454545 .45453624 . 8 +0034 0 0.003423161204030 . 25 . 52912252015?
20 18 3 o o o
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6 0 19 .16 0 0 21 0 23 10 ? 1 + 33 23 19 0 19 15 0 0 23 12 10 0 0 22
3 o 33 30 30 18 9 30 25 26.32 ·37 34 14 o o ·. o 33 37 20 14 25 35 29 24 3 10
1
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o o o 45 45 45 45 2 45 27 11 8 5 - ·- 50 30 · 21 ·+ o 50
15 ·9· o 34 35 2<i. 4 35 1 22 ·22 6 o
7 • + 0 46 46 46 46 · 36 45 45 48 29 10 4 0 45 8 2 0 0 45 40 28 21 2 45 19 15 0 35 16 38 20 12 ? ·
830454242371243383835 .1 63040 3 00 ·0403014 9034272618251130 3 1 0
+ o o 46 46 42 37 5 45 38 15 2 + - · - 34 24 15 8 o 34 9 1 o o 38 37 :26 o 25 3 30 o o ·o
0 0 01919181.5 + 17. 1? 11 6 2 - -20 3 0 0 0 2012 .3 + 0 21 . 13 ? 015 015 2 3 0
1 0 0 39 . 29 5 3 . 0 31 8 ? . 0 0 - - 21 13 50 0 21 14 . 8 50 21 17 8 + 28 0 14 3 0 0
0 0 0 5 6 1 1 0 3 . 0 0 0 . 0 - - 9 ? + 0 0 9 6 3 1 0 5 5 .+ 0 8 0 8 8 .2 0
0 0 0 15 .13 13 12 3 27 10 2 0 0 - - 1? ? 0 0 0 1? 10 .6 3 0 11 .16 12 0 10 3 14.10 10· ?.
1 0 0 22 22 18 18 18 26 21 21 12 8 ~ - 1? . 10 . .. o 0 1? 11 12 9 3 15 17 16 · 2 10 10 13 10 8 .2 .
+ 0 0 0 24 18 10 9 . 0 2? 8 10 0
5 . ·o o o 37 43 45 45 41 39 38 ·34 20
0 16
~
0 0
~ 1~
1~
1~
1~
~ . ~
2~
~ ~
1~ 1{ ~ ~
21 5 50 31 21 0 40 6 43 23 13 10 0 42 29 26 20 50 24 10
13 · o 35 26 12 o 30 16 27 1 1 o · o 40 o o o 35 29 17
11 3 13 19 14 3 16 7 8 . 2 .1 '() 0 22 7 ·2 0 13 11 7
·1 5 5 24 23 20 12 16 11 18 16 15 17 16 26 4 0 0 24 18 : 8
13 0 29 15 14 1 26 2 30 4 + 0 0 - - - - 29 8 1
4035 . 2014 035 o4o :3 o o o341313143511 +
§
VANILLIC
ACID
I'IETHOXYBENZOIC ACID
C!
o
3,4,5-TRI
THYMOL
~
6 1 1
o o 0
;0
2
2 + 0
0
0
1 1 +
0 0 0
n
0
+
0
+0
3 1
00
1 +
+ 0
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5 4
0 0
20
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23 5 0 0 0
30 19 9 4 0
24 2 + o 0
4 1 + + +
20 ? 3 1 0
25 11 2 0 0
50 2? 14 3 0
27 3 1 1 0
5 2 1 1
1.0
.n
1/\
~
11\.::t
ti...r.. ~
"'
porus betulinus, Gloeophyllurn sepiariurn,
Daedalea quercina.
Tannic acid: Laetiporus sulphureus and
Daedalea quercina grew well in 0. 75 % solution, Stereurn hirsuturn and Phellinus igniaTius significantly less well.
Trimethoxybenzoic acid: LactipoTUs sulphureus, Coriolus vaporarius, Gloeophyllum
sepiariurn, Coriolus hirsutus, Lentinus lepideus, Polyporus brurnalis.
Thymol: Stereurn purpureurn, Coriolus
hirsutus, Lentinellus ornphalodes, Fornes furnentarius, Hirschioporus abietinus.
Vanillic acid: Laetiporus sulphureus, Daedalea quercina, Lentinellus ornphalodes, Polypilus frondosus, Hirschioporus abictinus,
Gloeophyllurn separiurn.
Vanillin: Coriolus hirsutus, Fornitopsis p£nicola, Coriolellus serialis, Inonotus rheades,
Lentinus lepideus.
Discussion
The tox1c1ty of phenols is in many cases
highly dependent on environmental conditions, especially on pH (CocHRANE 1958,
CRUICKSHANK & PERRIN 1964). As seen from
Table 1, the acidity of the medium was in
several cases considerably increased when
the phenolic compounds were added. The
acidity of wood attacked by wood-rotting fungi varies within fairly wide limits, pH values
from 2.8 to 4.0 being common, and for this
reason strongly buttered media were not used.
In addition, the reaction of the fungus to
phenolic compounds can be modified by altering the composition of the medium in regard to its nitrogenous compounds, especially
amino acids (FwoD & KIRKHAM 1960). Also
the age of the isolated culture seems to some
extent to influence the results, especially in
pathogenic species.
Among the species investigated, it is possible to distinguish a group of species which
ha,·e in general a high tolerance to the compounds studied, viz.: Fornotopsis pinicola, Lentin us lepideus, Daedalea quercina, Laetiporus
sulphurcus, Stereurn sanguinolenturn, Gloeophyllurn sepiariurn, and Phellinus pini. Of
these fungi, Lmtinus is known to be fairly
resistant to phenolic compounds used in
wood protection, and Phellinus pini grows i'1
nature in pine heartwood, the fungitoxicity
of which depends largely on its content of
pinosylvin. Except for Stereurn and Phellinus, the species mentioned are pronounced
brown-rot fungi (CARTWRIGHT & FINDLAY
1958, K"ii.RIK 1965 ).
According to CsERJESI ( 1969), brown-rot
fungi are more tolerant to dihydroquercetin,
which occurs in bark of Pseudotsuga and Larix, than white-rot fungi.
In connection with the present investiga-
50
tion, observations on the occurrence of phenolic compounds in nature were made by
applying a solution of 1 % FeCl 3 in 5 % ethanol to wood near the basidiocarps of ca.
30 wood-rotting fungi. This reagent reacts
with orto-diphenols producing a blackish color. Sound wood does not react except near
the cambium. The natural substrates of the
following species gave a strong reaction with
FeCl 3 : Fornitopsis pinicola, Gloeophyllum
sepiariurn, PiptopoTUs betulinus, Daedalea
quercina, and Lactiporus sulphureus. When
these species and Fornitopsis annosa were
grown aseptically on pieces of birch wood for
3 months, a positive reaction with FeCl 3 was
obtained, but not with 14 white-rot fungi .
Thus there seems to exist a certain correlation between the tolerance of brown-rot fungi
to phenolic compounds and the occurrence
o.f ferric-chloride positive substances in wood
decomposed by the fungi in question.
It is interesting to note that certain species
which are in most cases highly tolerant to
most of the compounds investigated are in
some cases rather sensitive. For instance, cinnamic acid inhibited the growth of Lentinu.s
lepideus more than that of other brown-rot
fungi. Caffeic acid, which is known from pine bark (OKSANEN 1961), and pyrocatechol
proved to be slightly more toxic to Fornito psis annosa than to other rapidly growing species, and this was also the case with salicylic
acid and Pleurotus ostreatus and Kuehneromyces rnutabilis. Among species which often
grow together in birch trunks, Fornitopsis pinicola is in general most tolerant to most
compounds, but at high concentrations of
py.rogallol Fornes forn entarius, Gloeoporus
dichrous, Lentinellus ornphalodes, and Kuehnerornyces rnutabilis seem to be more active
Fig. 1. A piece of spruce stump decomposed by
Fomitopsis pinicola, treated with FeCla (above)
and diazotized sulfanilic acid (below) to reveal
phenolic compounds.
than F. pinicola. According to SIEGLE
(1967 ), pyrogallol occurs as a glycoside in
the heartwood of Betula papyrifera. In the
present material ,r emarkably many of the
species most active in pyrogallol medium
occur in nature in birch wood.
Our information concerning the occurrence
and absence of the investigated compounds
in natural wood and in wood decomposed
by micro-organisms is not comprehensive
enough to allow comparisons between the tolerances of decomposing species and the substrate in all cases. Oak wood is, however,
known to contain considerable amounts of
tannic acid (KARRER 1958) and the species
of the present material most tolerant to this
compound, Laetiporus sulphureus and Daedalea quercina, occur commonly in oak wood.
Ferulic acid occurs, according to ERDTMAN
(1958) in healing wounds of Pinus, and
among the species having the highest tolerance to it were Stereum sanguinolentum and
Phellinus pini, which occur as wound parasites of Pinus, and Hirschioporus abietinus,
which is one of the first colonizers of fallen
spruce. On the other hand, RoBBINs & HERVEY ( 1965) have found that this acid stimulates the growth of certain wood-destroying
fungus species, which are not included in
this investigation.
Lignin from the wood of deciduous trees
is known to yield more syringyl groups than
lignin from ·Conifer wood. In the present
experiments, syringic acid and syringa1dehyde
were not significantly more favorable to the
growth of species occurring in nature in deciduous wood than to that of species found in
conifer wood.
In general, species which are more or less
primary wood decomposers in nature, were
by and large the most resistant to the phenols,
and species occurring in wood at a more advanced stage of decay were more sensitive,
e.g. Xeromphalina campanella, but there were many exceptions.
Among fungi growing in aspen wood I nonotus rhcades was especially resistant to phenols, e.g. to p-cresol, to which it was more
tolerant than Lentinus lepideus.
When wood dries, its moisture content decreases from 150% to ca. 30 %, and the
concentration of the solutions in the wood
should consequently increase at least 5-fold,
near the surface possibly even more owing
to the transpiration stream. It is interesting
to note that some of the most resistant species often grow in wood exposed to drying,
as Gloeophyllum sepiarium, Coriolus vaporarius, and Lentinus lepideus.
On the basis of tolerance alone, it is not
possible to assess exactly the role of the compounds investigated in the ecology of wooddecomposing fungi, as in the changing conditions in natural wood account must be taken
of the ability to utilize and detoxify these
compounds (LvR 1962 ) as well as of the possible adaptation of mycelia to these compounds. In certain cases, the metabolic products may be more toxic than the original
compound (GsERJESI 1969). In addition, many mold species are known to utilize these
compounds in nature (HENDERSON 1965, JoNES and FARMER 1967).
51
Acknowledgements
This investigation has been carried out in
the Finnish Forest Research Institute on a
grant from Finnish State Board of Ag:ci-
culture and Forestry. The author is indebted
to Miss RrTVA KLEMETTI for technical assistance.
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~
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.
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52
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·
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Veikko Hintikka