FEMS Microbiology Letters 212 (2002) 59^63
www.fems-microbiology.org
Degradation of contrasting pesticides by white rot fungi and its
relationship with ligninolytic potential
Gary D. Bending , Maxime Friloux, Allan Walker
Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK
Received 14 February 2002 ; received in revised form 16 April 2002 ; accepted 22 April 2002
First published online 28 May 2002
Abstract
The capacity of nine species of white rot fungus from a variety of basidiomycete orders to degrade contrasting mono-aromatic
pesticides was investigated. There was no relationship between degradation of the dye Poly R-478, a presumptive test for ligninolytic
potential, and degradation of the highly available pesticides diuron, metalaxyl, atrazine or terbuthylazine in liquid culture. However, there
were significant positive correlations between the rates of degradation of the different pesticides. Greatest degradation of all the pesticides
was achieved by Coriolus versicolor, Hypholoma fasciculare and Stereum hirsutum. After 42 days, maximum degradation of diuron,
atrazine and terbuthylazine was above 86%, but for metalaxyl less than 44%. When grown in the organic matrix of an on-farm ‘biobed’
pesticide remediation system, relative degradation rates of the highly available pesticides by C. versicolor, H. fasciculare and S. hirsutum
showed some differences to those in liquid culture. While H. fasciculare and C. versicolor were able to degrade about a third of the poorly
available compound chlorpyrifos in biobed matrix after 42 days, S. hirsutum, which was the most effective degrader of the available
pesticides, showed little capacity to degrade the compound. 3 2002 Federation of European Microbiological Societies. Published by
Elsevier Science B.V. All rights reserved.
Keywords : White rot fungus; Pesticide degradation ; Ligninolytic potential ; Poly R-478 ; Bioremediation
1. Introduction
White rot fungi are de¢ned by their physiological capacity to degrade lignin [1]. The peroxidase enzyme systems employed are non-speci¢c, and have been implicated
in the degradation by white rot fungi of a wide variety of
aromatic xenobiotics, including polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls, pentachlorophenol and various groups of pesticides [2].
The ligninolytic system has great complexity, with enzymes involved in the cleavage of a variety of carbon^
carbon and carbon^oxygen bonds, resulting in the depolymerisation of lignin, and the subsequent degradation of
aromatic and aliphatic fragments [3]. Degradation of a
number of polymeric dyes, including Remazol brilliant
blue and Poly R-478 correlates well with ligninolytic potential [4,5], and has been used as a presumptive test to
screen fungi for their potential abilities to degrade lignin
* Corresponding author. Tel. : +44 (1789) 470382;
Fax : +44 (1789) 470552.
E-mail address : gary.bending@hri.ac.uk (G.D. Bending).
and xenobiotics. Degradation of Poly R-478 has been
shown to correlate well with the capacity of diverse white
rot fungi to degrade 3^5-ring PAHs [6]. However, it is
unclear how ligninolytic activity relates to the degradation
by white rot fungi of mono-aromatic xenobiotics, including many pesticides, which do not require depolymerisation. In the case of the persistent insecticides lindane and
DDT, ligninolytic peroxidases were found to have no involvement in degradation by Phanerochaete chryosporium
[7,8].
While there are many reports in the literature of pesticide degradation by white rot fungi, these have focussed
on the degradation of single pesticides by one or a few
isolates [2,7,8,9]. It is unclear whether such fungi have
generic abilities to degrade pesticides, and whether similar
degradative abilities are ubiquitous among the white rot
fungi.
The aims of this study were to compare the abilities of
white rot fungi from a variety of basidiomycete orders to
degrade contrasting mono-aromatic pesticides, and to determine whether ligninolytic activity could be used as a
presumptive test to characterise the capacity of these fungi
to degrade such pesticides. We also investigated the poten-
0378-1097 / 02 / $22.00 3 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 7 1 0 - 3
FEMSLE 10507 12-6-02
60
G.D. Bending et al. / FEMS Microbiology Letters 212 (2002) 59^63
tial of white rot fungi to degrade pesticides in organic
substrates typical of on-farm pesticide remediation systems.
2. Materials and methods
2.1. Fungal isolates
A total of nine wood- or litter-inhabiting fungi with
‘white rot’ characteristics were included in the study
(Table 1). The fungi represented a variety of basidiomycete orders.
2.2. Presumptive ligninolytic activity ^ degradation of
Poly R-478
Degradation of Poly R-478 by the fungi was assessed in
liquid culture. Poly R-478 (Sigma, Poole, Dorset, UK) was
¢lter-sterilised, and added to basal liquid nutrient solution
[10] to give a concentration of 20 mg l31 . Aliquots of 16 ml
were transferred to 9-cm Petri dishes, which were inoculated with a 6-mm disc cut from the margin of an active
fungus culture growing on malt extract agar. Control uninoculated plates were also set up. There were three replicates for each treatment. The plates were sealed with Nesco¢lm, and incubated in the dark at 25‡C. After 42 days,
decolouration of Poly R-478 was determined according to
Glenn and Gold [4].
2.3. Degradation of pesticides in liquid culture
The capacity of the nine fungal isolates to degrade the
phenylamide fungicide metalaxyl, the triazine herbicides
atrazine and terbuthylazine and the phenylurea herbicide
diuron in liquid culture was determined. These are all
highly available compounds with relatively low potentials
to bind to organic substrates, including fungal hyphae
(Table 2) [11]. Analytical grades of each pesticide (Greyhound, Birkenhead, UK), were dissolved in methanol, and
1 ml of a stock solution dispensed into an empty 500-ml
Duran bottle. In the case of diuron, a further 0.1 ml methanol solution of 14 C ring-labelled compound (DuPont
Agrochemicals, Wilmington, DE, USA) was added to
give approximately 300 Bq ml31 . Once the methanol had
evaporated completely, 500 ml nutrient solution was
added, providing a pesticide concentration of 10 mg l31 ,
and the solution shaken until the pesticide had dissolved.
Aliquots of 16 ml were transferred to 9-cm Petri dishes,
which were inoculated with a 6-mm disc cut from the
margin of an active fungus culture, which had been incubated on a plate of malt extract agar overnight so that
active growth had commenced. Control uninoculated
plates were also set up for each pesticide. There were three
replicate dishes for each pesticide/fungus treatment and
control.
After 42 days, pesticide remaining in the dishes was
determined. 1 ml of culture liquid was spun at 400Ug
for 5 min to precipitate fungal mycelium. To 0.5 ml of
the supernatant, 0.5 ml of acetonitrile was added. Pesticide
concentrations were determined by HPLC using Kontron
Series 300 equipment with a Lichrosorb RP18 column
(250U4.6 mm, Merck). The pesticides were eluted using
a mobile phase of acetonitrile:water :orthophosphoric acid
of 75:25:0.25, at a £ow rate of 1 ml min31 , and were
detected by UV absorbance at 210 (metalaxyl), 225 (terbuthylazine and atrazine) and 240 (diuron) nm. In the
diuron treatment 0.2-ml aliquots of culture liquid supernatant was added to 10 ml scintillation liquid (Ecoscint,
National Diagnostics, Atlanta, GA, USA), and radioactivity measured using a Rackbeta 1215 liquid scintillation
counter.
2.4. Degradation of pesticides by fungi in biobed substrate
Biobeds are on-farm pesticide bioremediation systems
developed in Sweden to retain pesticides and facilitate natural attenuation, and are currently being evaluated in a
number of other European countries [12]. Biobed matrix
was prepared by mixing together on a w/w basis 50%
barley straw, 25% topsoil (Wick series sandy loam, 1%
organic C [13]) and 25% compost, according to Fogg
[14]. The matrix was incubated at room temperature for
90 days before being autoclave sterilised. 20-g aliquots of
matrix were added to 200-ml glass jars, and 0.1 ml of a
pesticide stock solution in methanol added to give a concentration of 20 Wg pesticide g31 biobed matrix. In addition to the four pesticides described above, the organophosphate insecticide chlorpyrifos and the dicarboximide
fungicide iprodione were included. In contrast to the other
compounds, chlorpyrifos is strongly sorbed to organic
matter, and would have had low solution-phase availability in the biobed matrix (Table 2). After mixing, a 6-mm
disc of inoculum of Stereum hirsutum, Hypholoma fasciculare or Coriolus versicolor, cut from the margin of an active culture, was added. For each pesticide/fungus treatment and control, three replicate jars were set up. The jars
were sealed with aluminium foil, and incubated in the dark
at 20‡C. After 42 days, 50 ml of acetonitrile^H2 O (90:10
v/v) was added to each jar, which was shaken for 1 h.
After the contents had settled, 1 ml of the extract was
analysed for pesticide residues, as described above.
3. Results
3.1. Degradation of Poly R-478
All of the fungi were able to decolour Poly R-478
(Table 1). Agrocybe semiorbicularis, Auricularia auricola,
and H. fasciculare were the most e¡ective isolates with
over 95% decolouration of Poly R-478 after 42 days. In
FEMSLE 10507 12-6-02
G.D. Bending et al. / FEMS Microbiology Letters 212 (2002) 59^63
61
Table 1
Fungal isolates and Poly R-478 decolouration
Fungus
Order
Culture origina
Poly R-478
decolourationb
Agrocybe semiorbicularis (Bull. Ex Fr.) Fay.
Auricularia auricola (Hook). Under.
Coriolus versicolor (L.) Quel.
Dichotomitus squalens (Quel.) Dom. and Orl.
Flammulina velupites (Curt. Ex Fr.) Sing.
Hypholoma fasciculare (Huds. Ex Fr.) Kummer
Phanerochaete velutina (DC.) Parmasato
Bolbitaceae
Auriculariaceae
Tricholomataceae
Polyporaceae
Polyporaceae
Strophariaceae
Cortiaceae
7
12
45
205
196
30
58
Pleurotus ostreatus (Jacq). Kummer
Stereum hirsutum (Willd) S.F. Gray
Uninoculated
LSD (P = 0.05)
Polyporaceae
Stereaceae
R122 BRE
R252 BRE
R11 BRE
R26 BRE
R28 BRE
Oxley Wood, Warwickshire, UK
Department of Microbiology, University
of She⁄eld, UK
R155 BRE
R97 BRE
a
b
91
150
640
39
BRE, Building Research Establishment, Garston, UK.
Poly R-478 decolouration after 42 days (103 (absorbance 520 nm/absorbance 350 nm)).
the case of Dichotomitus squalens, Flammulina velutipes
and S. hirsutum, there had been less than 77% Poly R478 decolouration at this time.
3.2. Pesticide removal from liquid culture
There was considerable variation between the fungi with
respect to their abilities to degrade the pesticides (Table 3).
C. versicolor and S. hirsutum were the only fungi able to
degrade appreciable amounts of metalaxyl, with 56 and
35% remaining respectively after 42 days. D. squalens,
Phanerochaete velutina and Pleurotus ostreatus were able
to degrade 10% or less of the pesticide, while F. velupites
showed no ability to degrade the compound. The three
remaining fungi produced a metabolite which interfered
with the measurement of metalaxyl, and degradation could
not be quanti¢ed.
Hypholoma fasciculare and S. hirsutum had degraded
over 88% of terbuthylazine by the end of the experiment.
All of the other fungi were able to induce some degradation of terbuthylazine, with between 31 and 63% of the
compound degraded. Relative to terbuthylazine, all of the
fungi had lower abilities to degrade the related triazine
compound atrazine. C. versicolor showed greatest ability
to degrade atrazine, with 86.2% degraded. With the exception of H. fasciculare and S. hirsutum, all of the other
fungi degraded less than 50% of the atrazine added.
C. versicolor induced almost complete degradation of
diuron. H. fasciculare, S. hirsutum and A. semiorbicularis
were also e¡ective degraders of diuron, with 70^80% of
the compound degraded. All of the other fungi had degraded less than 22% of the compound after 42 days.
Analysis of 14 C-labelled diuron ring residues in solution
at this time showed that less than 60% remained in cultures of C. versicolor and S. hirsutum. In cultures of the
remaining fungi, including H. fasciculare and A. semiorbicularis which degraded approximately 70% of the parent
compound, over 77% of the ring C remained in solution.
There were no signi¢cant correlations between degradation of Poly R-478 and any of the pesticides (Table 4).
However, there were signi¢cant correlations between the
degradation of all of the pesticides.
3.3. Degradation of pesticides by fungi in biobed substrate
All of the fungi were able to degrade the pesticides when
grown on sterile biobed matrix (Table 5). However, there
were di¡erences in degradation between the fungi. S. hirsutum generally degraded larger amounts of the pesticides
than C. versicolor and H. fasciculare, with over 50% of
metalaxyl and atrazine, and 70% of terbuthylazine and
diuron degraded after 42 days. The fungi were able to
degrade signi¢cant amounts of the dicarboximide fungicide iprodione, with S. hirsutum a more e¡ective degrader
of the compound that the other fungi. In the case of
chlorpyrifos, the fungi generally had lower abilities to de-
Table 2
Pesticide characteristics
Pesticide
Type
Kow log P [16]
Metalaxyl
Atrazine
Diuron
Iprodione
Terbuthylazine
Chlorpyrifos
Phenylamide fungicide
Triazine herbicide
Phenylurea herbicide
Dicarboximide fungicide
Triazine herbicide
Organophosphorus insecticide
1.8
2.5
2.9
3.0
3.2
4.7
FEMSLE 10507 12-6-02
Koc [17]
50
100
480
700
6070
62
G.D. Bending et al. / FEMS Microbiology Letters 212 (2002) 59^63
Table 3
Pesticides remaining in liquid culture after 42 days
Fungus
% Pesticide remaining
Agrocybe semiorbicularis
Auricularia auricola
Coriolus versicolor
Dichotomitus squalens
Flammulina velupites
Hypholoma fasciculare
Phanerochaete velutina
Pleurotus ostreatus
Stereum hirsutum
LSD (P = 0.05)
Metalaxyl
Terbuthylazine
Atrazine
Diuron
% Diuron ring
ND
ND
56.2
89.9
100.0
ND
96.1
89.9
35.4
7.3
40.8
62.9
36.7
48.0
69.0
3.2
46.1
69.0
11.6
5.5
58.1
83.4
13.8
74.4
100.0
42.1
79.7
84.5
42.1
8.6
30.3
89.3
0.6
78.6
93.5
28.9
96.4
87.6
19.6
5.4
81.7
77.1
53.4
85.7
87.1
83.7
89.9
85.9
59.8
4.2
14
C remaining
ND, not determined (interfering metabolite).
grade the compound relative to the other pesticides. While
C. versicolor and H. fasciculare degraded 36 and 29% of
the compound respectively, S. hirsutum was able to degrade less than 7% of the compound.
4. Discussion
There was considerable variation among the white rot
fungi in their ability to degrade the pesticides. Further,
there was no relationship between presumptive ligninolytic
activity and the degradation of any of the pesticides. However, there were clear relationships between the abilities of
the fungi to degrade the di¡erent pesticide classes, indicating that similar mechanisms were involved in degradation
of all of the compounds.
Most workers have assumed that degradation of xenobiotics by white rot fungi is mediated by ligninolytic peroxidases [2]. However, in the case of P. chryosporium, degradation of the pesticide lindane has been found to occur
via detoxi¢cation by a cytochrome P450 monooxygenase
system, independent of the production of ligninolytic peroxidase enzymes [8]. Further, white rot fungi have been
classed into several groups with respect to the nature of
the enzyme systems involved in lignin degradation [1].
Members of each of these groups show di¡erences in their
abilities to degrade model synthetic lignin polymers, arising from di¡erent enzyme production patterns.
The three members of the Polyporaceae used in our
study showed di¡erences in the extent to which they
were able to degrade the pesticides, with C. versicolor
showing strong degradative abilities, and D. squalens and
P. ostreatus limited degradative potential. Polyporaceae
fungi are known to produce di¡erent ligninolytic enzyme
systems, with C. versicolor producing lignin and manganese peroxidases, and D. squalens and P. ostreatus producing manganese peroxidase and laccase, but not lignin peroxidase [1]. While all of these fungi are very e¡ective
degraders of natural lignin and synthetic polymers, the
nature of the ligninolytic enzyme as well as the detoxi¢cation systems they produce, may determine their ability to
degrade xenobiotics.
When C. versicolor, H. fasciculare and S. hirsutum were
grown in biobed matrix, they were all able to degrade the
pesticides, although there were di¡erences in the relative
degradative capacities of the fungi in liquid and biobed
media. Boyle et al. [15] found that in the case of PAHs
there was little relationship between the capacity of white
rot fungi to degrade compounds in liquid culture and soil
systems. This was attributed to high adsorption of PAHs
by soil organic matter, reducing accessibility of the compounds to the fungi. The pesticides used in our liquid
culture experiment, together with iprodione show low
susceptibility to adsorption to organic matter, and
would have had high availabilities in the biobed matrix
(Table 2). However, the organophosphorus compound
chlorpyrifos is strongly sorbed to organic matter, and
would have been far less available to the fungi than the
other compounds. Despite its low availability, C. versicolor and H. fasciculare were able to degrade 36 and 29% of
the chlorpyrifos after 42 days. However, S. hirsutum,
which generally showed greater degradation of the highly
available pesticides than the other fungi, was capable of
only limited degradation of chlorpyrifos. This could indi-
Table 4
Relationships between degradation of Poly R-478 and the pesticides
Compound
Metalaxyl
Terbuthylazine
Atrazine
Diuron
Poly R-478
Metalaxyl
Terbuthylazine
Atrazine
0.108
0.169
0.865*
0.326
0.811*
0.767*
0.212
0.916**
0.751*
0.934**
*P 6 0.05; **P 6 0.01.
FEMSLE 10507 12-6-02
G.D. Bending et al. / FEMS Microbiology Letters 212 (2002) 59^63
63
Table 5
Pesticide remaining biobed matrix after 42 days
Fungus
Coriolus versicolor
Hypholoma fasciculare
Stereum hirsutum
LSD (P = 0.05)
% Pesticide remaining
Metalaxyl
Terbuthylazine
Atrazine
Diuron
Iprodione
Chlorpyrifos
60.1
ND
46.1
3.6
60.1
63.0
21.4
39.5
48.9
38.7
42.7
36.2
47.7
84.1
25.9
21.3
42.1
52.6
37.7
16.2
63.8
71.0
93.9
5.4
ND, not determined (interfering metabolite).
cate that white rot fungi show contrasting abilities to access poorly available substrates.
We conclude that white rot fungi have the capacity to
degrade contrasting groups of pesticide, although the
mechanisms involved are not clearly related to ligninolytic
potential. Selected white rot fungi could prove valuable in
on-farm pesticide bioremediation systems.
Acknowledgements
We thank Sonia Cavanna and Mercedes Franey for assistance, Mike Challen (HRI) and Jonathan Leake (University of She⁄eld) for many of the fungal cultures, and
the Department of Environment, Food and Rural A¡airs,
and the European Community for ¢nancial support.
References
[1] Hataaka, A. (1994) Lignin-modifying enzymes from selected whiterot fungi: production and role in lignin degradation. FEMS Microbiol. Rev. 13, 125^135.
[2] Higson, F.K. (1991) Degradation of xenobiotics by white rot fungi.
Rev. Environ. Contam. Toxicol. 122, 111^152.
[3] Hammell, K.E. (1997) Fungal degradation of lignin. In: Driven by
Nature, Plant Litter Quality and Decomposition (Cadisch, G. and
Giller, K.E., Eds.), pp. 33^45. CAB International, Wallingford.
[4] Glenn, J.K. and Gold, M.H. (1983) Decolourization of special polymeric dyes by the lignin degrading basidiomycete Phanerochaete
chryosporium. Appl. Environ. Microbiol. 45, 1741^1747.
[5] Freitag, M. and Morrell, J.J. (1992) Decolourization of the polymeric
dye Poly R-478 by wood inhabiting fungi. Can. J. Microbiol. 38,
811^822.
[6] Field, J.A., de Jong, E., Costa, G.F. and de Bont, J.A.M. (1992)
Biodegradation of polycyclic aromatic hydrocarbons by new isolates
of white rot fungi. Appl. Environ. Microbiol. 58, 2219^2226.
[7] Kohler, A., Jager, A., Wilershausen, H. and Graf, H. (1988)
Extracellular ligninase of Phanerochaete chryosporium Burdsall has
no role in the degradation of DDT. Appl. Microb. Biotechnol. 29,
618^620.
[8] Mougin, C., Pericaud, C., Malosse, C., Laugero, C. and Asther, M.
(1996) Biotransformation of the insecticide lindane by the white rot
basidiomycete Phanerochaete chryosporium. Pestic. Sci. 47, 51^59.
[9] Kullman, S.W. and Matsumura, F. (1996) Metabolic pathways utilized by Phanerochaete chryosporium for degradation of the cyclodiene pesticide endosulfan. Appl. Environ. Microbiol. 62, 593^600.
[10] Kirk, T.E., Schultz, E., Connors, W.J., Lorenz, L.F. and Zeikus, J.G.
(1978) In£uence of culture parameters on lignin metabolism by Phanerochaete chryosporium. Arch. Microbiol. 117, 277^285.
[11] Hance, R.J. (1965) Observations on the relationship between the
adsorption of diuron and the nature of the adsorbent. Weed Res.
5, 108^114.
[12] Torstensson, L. and Castillo, M.D. (1997) Use of biobeds in Sweden
to minimise environmental spillages from agricultural spray equipment. Pestic. Outlook 8, 24^27.
[13] Whit¢eld, W.A.D. (1974) The soils of the National Vegetable Research Station, Wellesbourne. Report of the National Vegetable Research Station for 1973, pp. 21^30.
[14] Fogg, P. (2001) Biobeds : safe disposal of pesticide waste and washings. In: Pesticide Behaviour in Soils and Water, British Crop Protection Council Symposium 78 (Walker, A., Ed.), pp. 217^222. British Crop Protection Council, Surrey.
[15] Boyle, D., Wiesner, C. and Richardson, A. (1998) Factors a¡ecting
the degradation of polyaromatic hydrocarbons in soil by white-rot
fungi. Soil Biol. Biochem. 30, 873^882.
[16] Tomlin, C.D.S. (2000) The Pesticide Manual, 11th edn. British Crop
Protection Council, Surrey.
[17] Ware, G.W. (1994) Rev. Environ. Contam. Toxicol. 137.
FEMSLE 10507 12-6-02