FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J. 2001; 16: 245–248

DOI: 10.1002/ffj.987

Structure–fungitoxicity relationships of some volatile

flavour constituents of the edible mushrooms Agaricus
bisporus and Pleurotus florida
Eugene Sebastian J. Nidiry∗
Indian Institute of Horticultural Research, Hessaraghatta Lake (PO), Bangalore 560089, India
Received 27 June 2000
Revised 3 January 2001
Accepted 9 January 2001

ABSTRACT: The fungitoxicity of the diethyl ether extracts of two basidiomycete mushrooms, Agaricus bisporus
and Pleurotus florida, and 14 flavour constituents present in these mushrooms is being reported. Median
effective molar concentrations (EC50 ) of the compounds for the mycelial growth inhibition of Colletotrichum
gloeosporioides on potato–dextrose–agar (PDA) medium were computed and compared. Among the constituents
tested for fungitoxicity, 1-octanol exhibited the highest activity. Structure–activity relationship studies of the
constituents revealed that high hydrophobicity of the alkyl moiety, the presence of the primary alcoholic group
and the absence of branching of the alkyl group are responsible for the high activity of 1-octanol. Copyright 
2001 John Wiley & Sons, Ltd.
KEY WORDS: fungitoxicity; volatile mushroom constituents; structure–activity relationships; 1-octanol; Col-
letotrichum gloeosporioides; Agaricus bisporus; Pleurotus florida; Basidiomycetes

Introduction However, less attention has been paid to the struc-

Investigations on the chemical nature of antifungal com-
pounds produced by fungi, mushrooms, bacteria and
higher plants and their structure–activity relationships
have received a great deal of interest for two reasons.
First, these studies provide important insights into the
chemical defence mechanisms evolved by these organ-
isms against parasitic fungi. Second, the structural fea-
tures of these molecules provide strategies for the synthe-
sis of compounds that can be used as commercial fungi-
cides. Although many fungi and mushrooms are known
to produce antifungal compounds, those belonging to
the Basidiomycetes have received special attention after
the isolation and characterization of naturally occurring
strobilurin A and oudamansin from the basidiomycete
fungi Strobilurus tenacellus and Oudamansiella mucida,
respectively.1,2 It may be noted that it is based on
the structure of these naturally occurring antifungal
compounds that the commercially important fungicides,
azoxystrobin and kresoxim methyl (collectively known
as synthetic strobilurins) were developed.2 – 3
Survey of the literature shows several reports on
the antifungal activity of metabolites of mushrooms
and fungi.4 – 10 There are several reports on the chem-
ical nature of flavour constituents of mushrooms.11 – 12
*Correspondence to: E. S. J. Nidiry, Indian Institute of Horticultural
Research, Hessaraghatta Lake (PO), Bangalore 560089, India.
ture–fungitoxicity relationship studies on the volatile
constituents of mushrooms. A recent study from this
Institute reported about the chemical nature of the
flavour constituents of the basidiomycete mushrooms
Agaricus bisporus and Pleurotus florida.13 The present
study investigates the fungitoxicity of diethyl ether
extracts of A. bisporus and P. florida and 14 flavour con-
stituents of these mushrooms. Median effective molar
concentrations of these constituents for the mycelial
growth inhibition of Colletotrichum gloeosporioides on
potato–dextrose–agar (PDA) medium were computed
and compared. The structure–activity relationships of
these compounds are discussed. It may be noted that,
although Mumpuni et al.10 had reported the antifungal
activity of the metabolites of A. bisporus, they had not
reported the chemical nature of the metabolites.
Experimental
Extraction
P. florida was grown on paddy straw and A. bisporus
was grown on compost at the Indian Institute of Hor-
ticultural Research, Bangalore, India. On the peak day
of flush, mushrooms of uniform size and maturity were
harvested. About 250 g each were taken in batches from

Copyright  2001 John Wiley & Sons, Ltd.

246 E. S. J. NIDIRY

2 kg of fresh mushrooms, cut into pieces and homog- where C is the mycelial diameter of the control and
enized in a Waring blender at low speed with 600 ml T that of the treated ones. This was calculated after
distilled water for 2 min. The homogenate was left for giving due adjustment for the initial diameter of the
15 min to maximize the enzymatic production of the mycelial discs (0.8 cm) used for the inoculation. Each
compounds, as reported by Hong et al.14 The simul- treatment was replicated twice and the averages of the
taneous distillation and extraction method adopted by two values, along with standard deviations, were calcu-
Venkateshwarlu et al.13 gave a very small amount of lated and are presented in Table 1. The percentages were
flavour concentrate, which was insufficient for the fun- converted to probits and the median effective concentra-
gitoxicity assay. Therefore, the diethyl ether extract tions (EC50 ) were computed from the linear relationship
(diethyl ether soluble fraction, from which the solvent between logarithms of concentrations and probits.16 For
was completely removed) was directly taken for the
bioassay. It may be noted that the diethyl ether extract,
apart from flavour constituents, contain lipids also.13 Table 1. Fungitoxicity of diethyl ether extracts of the
mushrooms and their volatile constituents
The homogenate was partitioned three times with diethyl
ether (100 ml each) in a separating funnel. The ether- Extract/ Concentration MGI (%) EC50 pEC50
soluble fractions were combined, dried with anhydrous compound (mg/l) Mean(šSD) (mg/l) (moles/l)
sodium sulphate and the ether was completely distilled Extract of 100 9.5(š 0.0) 575 —
out at low temperature (<40 ° C) over a water bath. The Agaricus 250 26.7(š 0.6)
bisporus 500 46.1(š 0.2)
extract remaining in the flask had a typical mushroom 750 58.0(š 0.2)
flavour but no odour of diethyl ether. 1000 66.3(š 0.5)
There was a substantial difference in the amounts of Extract of 250 8.3(š 0.0) >1000 —
extracts obtained from the two mushrooms. In the case Pleurotus 500 13.3(š 0.8)
florida Ł 1000 25.0(š 2.1)
of P. florida, the extract was 0.072% of the fresh weight
p-Anisaldehyde 300 21.7(š 0.0) 712 2.28
of the mushroom and in the case of A. bisporus, it was 500 30.4(š 0.4)
only 0.012% of the fresh weight. 700 47.6(š 1.5)
800 50.7(š 0.7)
1000 70.1(š 2.0)
Benzaldehyde 500 16.0(š 1.6) 927 2.06
Bioassay 600 24.9(š 0.2)
800 38.8(š 0.4)
1000 55.6(š 2.3)
The extracts obtained by the above process and the 1200 66.3(š 1.6)
commercially available standards of 2-methyl propanol, Benzyl acetate 200 13.2(š 0.8) 999 2.18
pentanol, 3-methyl butanol, 1-hexanol, benzyl alcohol 500 29.4(š 1.5)
and 1-octen-3-ol (Aldrich, USA), phenyl ethyl alcohol, 800 33.0(š 0.2)
1000 53.0(š 0.3)
hexanal and benzaldehyde (Sigma, USA), 1-butanol and 1200 61.3(š 1.0)
1-octanol (Spectrochim, India), p-anisaldehyde and ben- Benzyl alcohol 500 24.8(š 0.2) 1181 1.96
zyl acetate (S. D. Fine, India) were used for the bioassay. 1000 49.4(š 0.1)
It may be noted that all constituents tested for fungitox- 1200 50.5(š 0.4)
1500 56.8(š 0.0)
icity in the present study were reported to be present in 1700 58.7(š 1.8)
either of the two mushrooms by Venkateshwarlu et al.13 1-Butanol 1000 6.3(š 0.2) 3694 1.30
Mycelial growth inhibition of Colletotrichum gloeospo- 2500 31.6(š 0.0)
rioides on PDA medium was determined by the poisoned 5000 65.5(š 0.5)
7500 80.5(š 1.6)
food technique.15 Appropriate amounts of the extracts 10 000 86.8(š 2.4)
and standard compounds were dissolved in 0.25 ml ace- 1-Hexanal 1000 12.5(š 0.2) 2363 1.63
tone and were added to 30 ml sterilized medium to 2000 20.4(š 1.0)
obtain a medium of required concentration, the same 2200 52.7(š 0.8)
2400 56.8(š 1.5)
amount of acetone being added in the control also. Main- 2500 59.2(š 2.0)
tenance of acetone-free controls revealed that this con- 1-Hexanol 1000 31.7(š 0.0) 1271 1.92
centration of acetone did not inhibit the mycelial growth 1200 52.0(š 0.5)
of the fungus. Mycelial discs of C. gloeosporioides were 1500 57.8(š 0.8)
1700 69.2(š 2.0)
transferred aseptically to the centres of Petri plates, 2000 78.7(š 2.4)
which were incubated at 27 š 2 ° C for 5 days. The per- Linalool ŁŁ ŁŁ 1090 2.15
centage mycelial growth inhibition (P) at each concen- 3-Methyl 1000 15.0(š 0.2) 3713 1.38
tration with respect to the control was calculated by the 1-butanol 2000 18.5(š 0.3)
formula, 3000 42.1(š 1.5)
C  T 4000 48.0(š 1.2)
PD ð 100 5000 69.8(š 1.0)
C

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 245–248
 STRUCTURE–FUNGITOXICITY RELATIONSHIPS IN EDIBLE MUSHROOMS 247

Table 1. Continued It may be noted that increase in the hydrophobicity

Extract/ Concentration MGI (%) EC50 pEC50 of the alkyl moiety also follows the same trend. The
compound (mg/l) Mean(šSD) (mg/l) (moles/l) most abundant flavour constituent, 1-octen-3-ol, which
2-Methyl 2000 13.3(š 1.8) 8205 0.96 provides typical mushroom flavour,13 shows less activ-
1-propanol 3000 18.6(š 1.2) ity than 1-octanol, although both are C8 alcohols. This
5000 26.2(š 0.0) difference in fungitoxicity could be due to the differ-
7500 46.8(š 0.6)
10 000 62.2(š 1.3) ence in the nature of the alcoholic groups present in
1-Octanol 100 9.4(š 0.2) 298 2.64 these compounds. In 1-octanol, the alcoholic group is
200 20.9(š 0.2) primary, while in 1-octen-3-ol it is secondary. An ear-
300 50.1(š 1.5) lier study17 from this Institute on monoterpenoids also
400 71.6(š 2.0)
500 74.9(š 2.4) showed that primary alcoholic group is preferred over
1-Octen-3-ol 1000 29.7(š 0.6) 1286 2.00 secondary and tertiary alcoholic groups for fungitox-
1200 47.5(š 1.0) icity. It can be observed that 1-butanol shows higher
1300 50.2(š 1.7) fungitoxicity than 2-methyl propanol, although both are
1400 56.1(š 2.0)
1500 62.1(š 2.0) C4 primary alcohols. Similarly, 1-pentanol shows higher
1-Pentanol 1000 15.9(š 0.5) 2129 1.62 activity than 3-methyl butanol, although both are C5 pri-
2000 40.1(š 0.8) mary alcohols. These observations show that branching
3000 59.7(š 1.0) of the alkyl group has an adverse effect on fungitoxicity.
3500 76.8(š 2.0)
4000 89.1(š 2.3) All these facts show that the remarkable hydrophobicity
Phenyl ethyl 500 25.8(š 1.4) 1000 2.09 of the alkyl moiety, presence of primary alcoholic group
alcohol 800 39.0(š 0.6) and absence of branching of the alkyl moiety contribute
1000 47.4(š 2.2) to the significant activity of 1-octanol. These observa-
1200 60.6(š 0.0)
1500 65.8(š 1.2) tions further indicate that the high activity of monoter-
pene alcohols, such as citronellol and geraniol, reported
MGI D mycelial growth inhibition.
Ł
earlier,17 is not because they are terpenoids, but because
In the case of extract of P. florida, concentrations higher than 1000 mg/l
could not be tried because of the poor solubility of the extract in acetone. they are C10 aliphatic primary alcohols with remarkable
ŁŁ
Mycelial growth inhibition (%) at different concentrations, previously hydrophobicity of the alkyl moiety. Among aldehydes,
reported in reference 17. the aromatic aldehydes, benzaldehyde and anisaldehyde
exhibit higher activity than the aliphatic aldehyde, 1-
structure–activity relationship studies, pEC50 , which is hexanal. Higher activity of 1-hexanol compared to 1-
equal to log EC50 , where EC50 is the median effective hexanal is consistent with the earlier observation18 of
molar concentration, was computed for each compound Kurita et al. that aliphatic alcohols exhibit higher fungi-
and these values are also given in Table 1. It may be toxicity than their corresponding aldehydes.
noted that pEC50 is a positive measure of fungitoxicity Acknowledgements—The author expresses his gratitude to Dr P. P.
and is normally used for the quantitative comparison of Reddy, Director, IIHR, for providing facilities and to Mr C. S. Bujji
the efficacies of fungitoxic compounds.17 Babu and Mrs H. Shanthamma for technical assistance.

Results and Discussion References

The results presented in Table 1 show that the diethyl
ether extract of A. bisporus exhibits much higher activity
than that of P. florida. In the case of P. florida, the
maximum concentration tried was 1000 mg/l. Higher
concentrations could not be tried because of the poor
solubility of the extract in acetone.
Table 1 gives the fungitoxicity of 14 constituents
at different concentrations with their computed EC50
values. The constituents comprise of 10 alcohols, three
aldehydes and one ester. Among the constituents tested
for fungitoxicity, 1-octanol exhibited the highest activity.
All other constituents showed moderate or poor activity.
Comparison of the fungitoxicity of the following primary
alcohols show a definite increasing trend in activity:
1-butanol < 1-pentanol < 1-hexanol < 1-octanol
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