Fd Chem. Toxic. Vol. 27, No. 6, pp.

393-397, 1989

0278-6915/89 $3.00 + 0.00

Copyright 1989 Pergamon Press plc

Printed in Great Britain. All rights reserved

ASSAYS OF THE BIOLOGICAL ACTIVITIES OF GUAIANE

SESQUITERPENOIDS ISOLATED FROM THE FRUIT
BODIES OF EDIBLE LACTARIUS SPECIES
H. ANKE
Department of Biotechnology, University of Kaiserslautern, D-6750 Kaiserslautern, Federal
Republic of Germany
and
O. BERGENDORFF and O. STERNER*
Department of Organic Chemistry. Lund Institute of Technology, POB 124, S-221 00 Lund, Sweden

(Received 14 November 1988; revisions received 20 March 1989)

Abstract--Three sesquiterpenoids that are found in the edible mushrooms Lactarius deliciosus,
L. deterrimus and L. sanguifluus, have been assayed for biological activity. The compounds tested were
a stearic acid ester of a sesquiterpene (I) and a sesquiterpene aldehyde (lactaroviolin, II) and alcohol
(deterrol, III). The assays used were for mutagenic activity in the Ames Salmonella assay, for antimicrobial
activity against bacterial fungi and algae, for cytotoxicity against Ehrlich ascitic tumour cells and L 1210
cells, and for phytotoxic activity against Lepidium sativum and Seteria italica. All three compounds showed
weak mutagenic activity in the Ames assay. Two compounds (II and III) were found to have moderate
cytotoxic activity and one (III) exhibited weak antibacterial activity. No compound revealed phytotoxic,
algicidal or antifungal activity.

INTRODUCTION
Fruit bodies of the Basidiomycetes Lactarius deliciosus Fr., L. deterrimus Gr6ger and L. sanguifluus
Paulet ex Fr., belonging to the section Dapetes of the
genus Lactarius, are generally considered to be edible
and delicious, and large amounts of these mushrooms
are consumed annually. However, as is often the case
for edible mushrooms, surprisingly little is known
about the biological activities and/or toxicity of
secondary metabolites present in them. The fruit
bodies of the three species mentioned above are easily
identified by their latex, which is strongly coloured by
the presence of a number of azulene sesquiterpenoids
with a guaiane skeleton, for instance the stearic acid
ester (Fig. 1, I; Schmitt, 1974; Vokac et al.; 1971),
lactaroviolin (Fig. 1, II; Heilbronner and Schmid
1953; Schmitt, 1974) and deterrol (Fig. 1, III; Bergendorff and Sterner, 1988). The nature of these sesquiterpenes has been studied for more than 100yr
(for a review see Schmitt, 1974), but except for a note
on the antibacterial activity of lactaroviolin (II)
against tubercle bacilli (Willstaedt and Zetterberg,
1946), to our knowledge, no reports on their biological activities have appeared. Recently it was
shown that the fruit bodies of L. deliciosus and
L. deterrimus originally contain only one sesquiterpene, existing in the mushroom as stearic acid (i.e.
compound I in Fig. 1) and linoleic acid esters, and
that these esters are enzymatically transformed into
several sesquiterpene alcohols and aldehydes, for
*To whom correspondence and requests for reprints should
be addressed.
Abbreviation: MICs = minimal inhibition concentrations.

instance, lactaroviolin (Fig. 1, II) and deterrol (Fig. 1,

III) when the fruit bodies are damaged (Bergendorff
and Sterner, 1988).
The fruit bodies of other Lactarius species, belonging for instance to the section Albati have a very
pungent taste and are generally not considered to be
edible. The pungency is caused by unsaturated dialdehyde sesquiterpenes with marasmane and lactarane
skeletons for example (Fig. 1, IV), which are formed
enzymatically from a fatty-acid-ester precursor when
these mushrooms are injured (Sterner et al., 1985).
The formation of such pungent unsaturated dialdehydes, which posses strong antimicrobial (Sterner
et aL, 1985), antifeedant (Camazine et al., 1983) and
mutagenic (Sterner et al., 1987) activities, probably
protects the mushrooms from parasites and these
compounds appear to be the active principles in a
chemical defence system (Sterner et al., 1985). The
sesquiterpenes investigated here, which are found in
the fruit bodies of L. deliciosus, L. deterrimus and
L. sanguifluus, do not contain an unsaturated dialdehyde moeity and are not especially pungent, but there
are still similarities between the two groups of Lactarius species in the conversions of sesquiterpenes in
injured fruit bodies. The fruit bodies of both groups
originally contain fatty acid esters of a single
sesquiterpene, which are enzymatically converted to
sesquiterpene aldehydes and alcohols if the fruit
bodies are injured.
In view of the potent biological activities of the
sesquiterpenes formed in the pungent Lactarius
species, we considered it to be important to investigate the biological activities of the guaiane sesquiterpenes isolated from edible Lactarius species. Because
of the chemical instability of several of these
393

H. ANKEet al.

394

oco

rt

CH2ON~
]E[

~,.CHO
~ ~ "CHO
T~

Fig. 1. The sesquiterpene ester (I), lactaroviolin (II) and

deterrol (II1) are formed in edible Lactarius species, while
isovelleral (IV) is formed in pungent Lactarius species.
sesquiterpenes, the investigation was limited to the
reasonably stable compounds (Fig. 1, I, II, and III).
We chose four assays, which together cover a wide
range of biological activities and for which the limited
amounts of the compounds that were available were
sufficient. The assays selected were to determine
mutagenic and antimicrobial activities and cyto- and
phytotoxicity.

M A T E R I A L S AND M E T H O D S

The three compounds assayed were isolated from

ground fruit bodies of Lactarius deliciosus and
L. deterrimus as described earlier (Bergendorff and
Sterner, 1988).
Mutagenicity assay. Mutagenic activity was investigated in the Ames Salmonella/mammalian-microsome assay (Ames et al., 1975) with the frameshift
mutant strain TA98 and the base-pair substitution
mutant strain TA100 (the strains were kindly provided by Professor B. Ames, University of California,
Berkeley, CA, USA). They were carried out both
without and with 3.3 or 10% S-9 mix, which was
prepared from the livers of SPD rats induced with
Arochlor 1254. The assays of the ester (I) and
lactaroviolin (II) were carried out with quintuplicate
plates but in the assay of deterrol (III) the plates were
triplicated because of the limited amounts of this
compound available. The solvent used throughout
was acetone. Before the assay was performed, the
compounds were tested for toxicity towards strain
TAI00 on nutrient agar plates (about 1000 bacteria/plate), and the concentration that caused a 50%
reduction in the number of colonies was taken as the
highest concentration to be tested. As a solvent
control, blanks were run with acetone only. The
average numbers of spontaneous revertants/plate
were as follows: for TA98--53 without S-9 mix, 53
with 3.3% S-9 mix, and 64 with 10% S-9 mix; for
TA100--151 without S-9 mix, 141 with 3.3% S-9
mix, and 151 with 10% S-9 mix. 1-[(Diethylaminoethyl)- amino]-4-(methansulphonyloxymethyl)thioxanthen-9-one, kindly provided by Winthrop
Ltd, New York, USA was used to determine the
sensitivity of the strains: 100#g of this compound/plate gave 620 colonies for TA98, and 690 for
TAI00 (inclusive of the spontaneous background, in
all cases the average of three plates). As a positive
control of the S-9 activation, 2-aminoanthracene was
tested in strain TAI00:1 pg/plate gave 147 colonies

in the absence of S-9 mix, 1818 colonies in the

presence of 3.3% S-9 mix, and 586 colonies in the
presence of 10% S-9 mix (in all cases the average of
three plates). The analysis by linear regression and
the calculation of the correlation coefficients were
carried out with the software 'Statview' for Apple
Macintosh computers.
Assay of antimicrobial activiO,. Antimicrobial activities against two Gram-negative and three Grampositive bacteria, three yeasts, four filamentous fungi
and one alga were tested in the plate diffusion assay
(Zfihner and Maas, 1972). Bacteria were grown on
nutrient agar (Difco, Detroit, USA), yeasts and other
fungi on agar containing (/litre): 4 g yeast extract;
10g malt extract; 4g glucose. The inoculum was
5 l0 s cells or spores/ml. Chlorella vulgaris was
grown under the conditions described by Noll (1987).
The size ofinoculum was 1 10 7 cells/ml. Filter discs
(6 mm diameter) bearing the compounds were placed
on agar plates seeded with the appropriate test organism, and the plates were incubated at 27 ~ or 3 7 C for
24 hr. The minimal inhibition concentrations (MICs)
for the most sensitive organisms were determined
with the broth dilution method (Ericsson and Sherris,
1971). The size ofinoculum w a s 10 6 cells/ml, while the
time of incubation, temperature and media (without
agar) were the same as in the plate diffusion assay.
Assay of cytotoxic acti~,iO'. For the cytotoxicity
tests Erlich ascitic tumour cells (ECA cells) and L
1210 cells (lymphocytic leukaemia mouse, ATCC
CCL 163) were used (Ekwall, 1983; Leonhard, 1987).
The cells were grown and tested as suspension cultures, as described by Leonhard (1987). The cell
densities were 6 l0 s cells (ECA) and 1 106 cells
(L 1210) per ml. Cytotoxic activity was evaluated
after 24 and 48 hr.
Assay of phytotoxic activity. Phytotoxic activity
was assayed according to the method of Yopp (1985),
except that Lepidium sativum (dicotyledon) and
Setaria italica (monocotyledon) were used instead of
Lactuca sativa. Filter discs (12-mm diameter) bearing
50 or 100/~g of the compounds were placed in plastic
vials (diameter, 13 mm, height 50 ram), and wetted
with 200 pl water. Six seeds of the plants were placed
on each disc, and the vials were incubated in a humid
chamber at 20~C in the dark for 72 hr, and then for
48 hr in daylight. The germination of the seeds and
the lengths of the seedlings and roots were measured
after 48, 72, 96 and 120 hr, and compared with a
negative control. The assays were carried out in
triplicate.
RESULTS

The results of the Ames Salmonella assays of the

ester (I), lactaroviolin (II) and deterrol (III) are
shown in Tables 1, 2, and 3, respectively. For all three
compounds the numbers of revertants of at least one
strain increased with the concentration, indicating
mutagenic activity. Assuming linear dose-response
relationships, the slopes and the correlation coefficients were calculated by linear regression of the
primary data given in Tables 1-3. The ester (I), which
was found to be the least toxic of the three compounds towards the tester strains, gave the strongest
response in strain TAI00 in the absence of S-9 mix

395

Biological activity o f sesquiterpenes

Table I. Results of testing the sesquiterpene ester (I, Fig. 1) in the
Ames Salmonella/mammalian-microsome mutagenicity assay
Compound I
(concn ~g/
plate)
1000

500

250

125

Salmonella
Strain
TA98
TA98
TA98
TA100
TA100
TAI00
TA98
TA98
TA98
TA100
TA100
TAI00
TA98
TA98
TA98
TA100
TA100
TA100
TA98
TA98
TA98
TAI00
TA100
TA100
TA98
TA98
TA98
TAI00
TAI00
TAI00

S-9
mix*

No. of revertants/plate
(including
spontaneous revertants)

-+
+
-+
+
-+
+
-+
+
-+
+
-+
+
-+
+
-+
+
-+
+
-+
+

70, 73, 77, 82, 82

63, 69, 73, 78, 86
67, 68, 76, 76, 81
337, 366, 372, 381,
336, 339, 349, 359,
290, 293, 301, 306,
53, 60, 70, 71, 72
54, 65, 70, 73, 77
55, 63, 71, 77, 79
246, 249, 275, 276,
243, 244, 2 5 1 , 2 6 1 ,
229, 233, 241, 253,
49, 51, 57, 59, 62
45, 55, 57, 62, 71
52, 57, 65, 71, 72
177~ 178, 193, 204,
175, 176, 184, 186,
144, 165, 169, 17t,
41, 48, 48, 58, 61
53, 57, 66, 67, 75
54, 61, 64, 70, 72
172, 173, 174, 183,
151, 164, 175, 177,
155, 159, 161, 163,
48, 50, 55, 55, 58
45, 50, 53, 57, 59
53, 59, 63, 69, 75
118, 146, 147, 171,
125, 130, 137, 145,
141, 145, 149, 157,

3.3
10
3.3
10
3.3
10
3.3
10
3.3
10
3,3
10
3.3
10
3.3
10
3.3
10
3.3
10

383
373
336
25
278
281
266
12.5
215
199
183
0
196
189
189

173
146
161

Table 2. Results of testing the sesquiterpene aldehyde, lactaroviolin

(II, Fig. 1) in the Ames Salmonella/mammalian-microsome m u t a genicity assay

200

100

50

25

Salmonella
strain
TA98
TA98
TA98
TAI00
TA100
TA100
TA98
TA98
TA98
TAI00
TAI00
TA100
TA98
TA98
TA98
TAI00
TAI00
TA100
TA98
TA98
TA98
TAI00
TA100
TAI00
TA98
TA98
TA98
TAI00
TA100
TAI00

Deterrol
(concn gg/
plate)
50

*Plates were made up without ( - ) or with 3.3% ( + 3.3) or 10%

( + 10) S-9 mix.

Lactaroviolin
(concn gg/
plate)

Table 3. Results of testing the sesquiterpene alcohol, deterrol (III,

Fig. I) in the Ames Salmonella/mammalian-microsome mutagenicity
assay

S-9
mix*

No. of revertants/plate
(including
spontaneous revertants)

-+ 3.3
+ 10
-+ 3.3
+ 10
-+ 3.3
+ 10
-+ 3.3
+ 10
-+ 3.3
+ 10
-+ 3.3
+ 10
-+ 3.3
+ 10
-+ 3.3
+ 10
-+ 3.3
+ 10
-+3.3
+ 10

42, 47, 54, 55, 65

92, 97, 103, 110, 119
95, t17, 122, 129, 130
151, 170, 176, 188, 192
201, 202, 216, 220, 231
289, 299, 306, 320, 343
45, 52, 55, 55, 61
101, 103, 106, 112, 129
95, 97, 99, 99, 110
162, 175, 177, 183, 203
165, 188, 189, 193, 201
179, 185, 203, 212, 217
37, 39, 47, 51, 60
83, 89, 96, 101, 107
77, 79, 82, 90, 91
151, 157, 166, 170, 179
155, 177, 183, 189, 190
169, 183, 187, 193, 201
49, 51, 56, 59, 62
60, 63, 70, 76, 80
62, 72, 79, 83, 90
143, 158, 162, 174, 176
136, 146, 152, t60, 166
124, 139, 145, 150, 155
48, 50, 55, 55, 58
45, 50, 53, 57, 59
53, 59, 63, 69, 75
118, 146, 147, 171, 173
125, 130, 137, 145, 146
141, 145, 149, 157, 161

*Plates were made up without ( - ) or with 3.3% ( + 3.3) or 10%

( + lO) S-9 mix.

Salmonella
strain
TA98
TA98
TA98
TA100
TA100
TA100
TA98
TA98
TA98
TA100
TA100
TA100
TA98
TA98
TA98
TAI00
TAI00
TAI00
TA98
TA98
TA98
TAI00
TAI00
TAI00

S-9
mix*

No. of revertants/plate
(including
spontaneous revertants)

-+ 3.3
+ 10
-+ 3.3
+ 10
-+ 3.3
+ 10
-+3.3
+ 10
-+ 3.3
+ 10
-+ 3.3
+ 10
-+ 3.3
+ 10
-+ 3.3
+ 10

86, 106, 120

162, 168, 204
142, 154, 160
164, 164, 165
175, 179, 200
249, 276, 283
83, 84, 96
82, 92, 97
96, 103, 125
148, 151, 157
154, 159, 179
187, 196, 209
67, 69, 90
81, 85, 88
75, 95, 110
142, 160, 177
176, 183, 207
175, 188, 193
48, 50, 55, 55,
45, 50, 53, 57,
53, 59, 63, 69,
118, 146, 147,
125, 130, 137,
141, 145, 149,

58
59
75
171, 173
145, 146
157, 161

*Plates were made up without ( - ) or with 3.3% ( + 3.3) or 10%

( + 10) S-9 mix.

(slope =0.22 revertants/#g/plate, correlation coefficient--0.95). The response was not significantly altered by the presence of 3.3% S-9 mix, while 10% S-9
mix reduced it substantially. Lactaroviolin (II) increased the number of revertants only in the presence
of S-9 mix. The strongest response was obtained in
strain TA100 in the presence of 10% S-9 mix
(slope=0.83 revertants//~g/plate, correlation coefficient = 0.90), while the responses in the same strain
in the presence of 3.3% S-9 mix, as well as in strain
TA98 in the presence of 3.3 or 10% S-9 mix, were
considerably lower. Deterrol (III) is the most toxic
compound towards the Salmonella tester strains,
50/~g/plate was found to be the highest non-toxic
concentration. Deterrol (llI) gave the strongest response in TA98 in the presence of 3.3% S-9 mix
( s l o p e = 2 . 4 revertants/~g/plate, correlation coefficient = 0.91), and in TA100 in the presence of 10%
S-9 mix (slope = 2.3 revertants/~g/plate, correlation
coefficient = 0.94).
As shown in Table 4, no antifungal activity could
be detected for either compound (up to 50/~g/disc).
Nor was Chlorella vulgaris affected (at 50#g/disc),
and no phytotoxic activity towards Lepidium sativum
and Setaria italica was detected (the highest concentration tested was 500#g/ml). Deterrol (III) is the
only compound to exhibit weak antibacterial activity,
and the most sensitive organism was found to be
Acinetobaeter calcoaceticus for which the MIC was
5/~g/ml in the broth dilution test. The MIC for
Micrococeus luteus and Proteus vulgaris was
30/~g/ml. Deterrol (III) also shows moderate cytotoxic activity towards ECA cells and weak toxicity
towards L 1210 cells (Table 5). Lactaroviolin (II)
exhibits a weak inhibitory effect towards ECA cells,
while the ester (I) did not impair either cell line at

50/~g/ml.

396

H. ANKE et al.
Table 4. Antimicrobial activity of three sesquiterpene compounds in the plate diffusion assay.
Inhibition zone (mm) after treatment with*:
Incubation
temperature
(C)

Test organism
Bacteria
Acinetobacter calcoaceticus
Bacillus brevis
B. subtilis
Micrococcus luteus
Proteus vulgaris

I
Compound conch
(#g/disc)...

II

III
.

10

50

10

50

10

50

27
37
37
37
37

0
0
0
0
0

0
0
0
0
0

0
0
0
0
0

0
0
0
0
0

12
0
I)
0
0

15
0
0
0
12

27
37
27
37
27
27
27

NT
NT
NT
NT
NT
NT
NT

0
0
0
0
0
0
0

NT
NT
NT
NT
NT
NT
NT

0
0
0
0
0
0
0

NT
NT
NT
NT
NT
NT
NT

0
0
0
0
0
0
0

NT

NT

NT

Yeasts and fungi

Fusarium oxysporum
Mucor miehei
Nematospora coo'li
Paecilomyces variotii
Penicillium notatum
Saccharomyces cerevisiae iS1
Saccharomyces cerevisiae S 288

Algae
Chlorella t'ulgaris

NT = not tested
*The three compounds tested are shown in Fig. I, and were a sesquiterpene ester (I) lactaroviolin (1I) and deterrol (Ill).
DISCUSSION
T h e r e were large differences b e t w e e n the biological
activities o f the s e s q u i t e r p e n e s isolated f r o m the fruit
b o d i e s o f edible L a c t a r i u s species (this investigation)
a n d t h o s e isolated f r o m the p u n g e n t L a c t a r i u s
species. T h e u n s a t u r a t e d d i a l d e h y d e s , such as isovelleral (IV), that are p r e s e n t in the p u n g e n t species,
are reactive c o m p o u n d s a n d give their effects
p r o m p t l y . This is d e m o n s t r a t e d by their intense p u n gent taste, their m u t a g e n i c i t y , a n d their s t r o n g antibiotic activity. F o r c o m p a r i s o n , isovelleral (IV) has
been s h o w n to induce 155 r e v e r t a n t s # g / p l a t e in
strain T A I 0 0 , in the a b s e n c e o f S-9 mix ( S t e r n e r et al.,
1987), a n d its M I C s a g a i n s t Acinetobaeter calcoaceticus, Micrococcus luteus a n d Proteus vulgaris are ten
times lower t h a n t h o s e r e p o r t e d here for d e t e r r o l (III;
A n k e et al., 1989). F u r t h e r m o r e , isovelleral (IV) has
been s h o w n to s u p p r e s s c o m p l e t e l y g e r m i n a t i o n o f
Lepidium sativum a n d Setaria italica at 50/~g/ml
( A n k e et al., 1989), a n d to inhibit the g r o w t h o f E C A
a n d L 1210 cells at 1-2 p g / m l ( A n k e et al., 1989). T h e
g u a i a n e s e s q u i t e r p e n e s a s s a y e d here a p p e a r to be less
p o t e n t , a n d are activated r a t h e r t h a n i n a c t i v a t e d by
rat-liver m i c r o s o m a l e n z y m e s (S-9) in the A m e s
S a l m o n e l l a assay. It was u n e x p e c t e d that d e t e r r o l
(III) is m o r e toxic t o w a r d s b o t h the A m e s tester
strains a n d the E C A a n d L 1210 cells t h a n lactaroviolin (II) was, since an a l d e h y d e f u n c t i o n a l i t y is
Table 5. Cytotoxic activity of three sesquiterpene compounds against
ECA and L 1210 cells
Concentration (pg/ml) needed for
growth inhibitions of
Cell line
ECA

Compound*
10-20%
50%
100%
I
> 50
> 50
> 50
II
10
20
50
III
5
10
20
L 1210
1
> 50
> 50
> 50
1I
50
> 50
> 50
III
10
50
> 50
*The three compounds tested are shown in Fig. I and were a
sesquiterpene ester (I), lactaroviolin (II) and deterrol (III).
+Concentrations (,ug/ml) needed for reduction in cell numbers in
comparison with untreated control cell cultures.

n o r m a l l y m o r e reactive a n d t h e r e b y m o r e toxic t h a n
the c o r r e s p o n d i n g alcohol functionality, a n d this
suggests that the azulene p o r t i o n o f these c o m p o u n d s
c o n t r i b u t e s significantly to the biological activities
assayed here.
E v e n if the o b s e r v e d activities are low, the weak
r e s p o n s e s in the A m e s S a l m o n e l l a assay o f all three
c o m p o u n d s investigated here, as well as the cytotoxicity o f deterrol (III) t o w a r d s E C A cells s h o u l d be
o f s o m e c o n c e r n . H o w e v e r , these c o m p o u n d s are
r a t h e r sensitive, a n d if left at r o o m t e m p e r a t u r e for
days o r h e a t e d to 50"C for h o u r s (for i n s t a n c e d u r i n g
e v a p o r a t i o n o f solvents) they are largely d e s t r o y e d .
W e t h e r e f o r e believe, a l t h o u g h we have n o t investigated this, that these c o m p o u n d s will d i s a p p e a r if the
m u s h r o o m s are t h o r o u g h l y c o o k e d p r i o r to ingestion.
thank A. Heifer for expert
technical assistance. This work was supported financially
by the Bundesministerium ffir Forschung und Technologie,
and the Swedish Natural Science Research Council. grant
No. K - K U 8570-300.
Acknowledgements--We

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