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Keywords:

  • White rot fungus;
  • Pesticide degradation;
  • Ligninolytic potential;
  • Poly R-478;
  • Bioremediation

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

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.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

White rot fungi are defined by their physiological capacity to degrade lignin[1]. The peroxidase enzyme systems employed are non-specific, 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 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 potential of white rot fungi to degrade pesticides in organic substrates typical of on-farm pesticide remediation systems.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Fungal 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.

Table 1.  Fungal isolates and Poly R-478 decolouration
  1. aBRE, Building Research Establishment, Garston, UK.

  2. bPoly R-478 decolouration after 42 days (103 (absorbance 520 nm/absorbance 350 nm)).

FungusOrderCulture originaPoly R-478 decolourationb
Agrocybe semiorbicularis (Bull. Ex Fr.) Fay.BolbitaceaeR122 BRE7
Auricularia auricola (Hook). Under.AuriculariaceaeR252 BRE12
Coriolus versicolor (L.) Quel.TricholomataceaeR11 BRE45
Dichotomitus squalens (Quel.) Dom. and Orl.PolyporaceaeR26 BRE205
Flammulina velupites (Curt. Ex Fr.) Sing.PolyporaceaeR28 BRE196
Hypholoma fasciculare (Huds. Ex Fr.) KummerStrophariaceaeOxley Wood, Warwickshire, UK30
Phanerochaete velutina (DC.) ParmasatoCortiaceaeDepartment of Microbiology, University of Sheffield, UK58
Pleurotus ostreatus (Jacq). KummerPolyporaceaeR155 BRE91
Stereum hirsutum (Willd) S.F. GrayStereaceaeR97 BRE150
Uninoculated  640
LSD (P=0.05)  39

2.2Presumptive 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 filter-sterilised, and added to basal liquid nutrient solution[10] to give a concentration of 20 mg l−1. 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 Nescofilm, and incubated in the dark at 25°C. After 42 days, decolouration of Poly R-478 was determined according to Glenn and Gold[4].

2.3Degradation 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 14C ring-labelled compound (DuPont Agrochemicals, Wilmington, DE, USA) was added to give approximately 300 Bq ml−1. Once the methanol had evaporated completely, 500 ml nutrient solution was added, providing a pesticide concentration of 10 mg l−1, 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.

Table 2.  Pesticide characteristics
PesticideTypeKow log P[16]Koc[17]
MetalaxylPhenylamide fungicide1.850
AtrazineTriazine herbicide2.5100
DiuronPhenylurea herbicide2.9480
IprodioneDicarboximide fungicide3.0700
TerbuthylazineTriazine herbicide3.2 
ChlorpyrifosOrganophosphorus insecticide4.76070

After 42 days, pesticide remaining in the dishes was determined. 1 ml of culture liquid was spun at 400×g 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 (250×4.6 mm, Merck). The pesticides were eluted using a mobile phase of acetonitrile:water:orthophosphoric acid of 75:25:0.25, at a flow rate of 1 ml min−1, 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.

2.4Degradation 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 μg pesticide g−1 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–H2O (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.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Degradation 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 effective isolates with over 95% decolouration of Poly R-478 after 42 days. In the case of Dichotomitus squalens, Flammulina velutipes and S. hirsutum, there had been less than 77% Poly R-478 decolouration at this time.

3.2Pesticide 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 quantified.

Table 3.  Pesticides remaining in liquid culture after 42 days
  1. ND, not determined (interfering metabolite).

Fungus% Pesticide remaining
 MetalaxylTerbuthylazineAtrazineDiuron% Diuron ring 14C remaining
Agrocybe semiorbicularisND40.858.130.381.7
Auricularia auricolaND62.983.489.377.1
Coriolus versicolor56.236.713.80.653.4
Dichotomitus squalens89.948.074.478.685.7
Flammulina velupites100.069.0100.093.587.1
Hypholoma fasciculareND3.242.128.983.7
Phanerochaete velutina96.146.179.796.489.9
Pleurotus ostreatus89.969.084.587.685.9
Stereum hirsutum35.411.642.119.659.8
LSD (P=0.05)7.35.58.65.44.2

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 effective 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 14C-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 significant correlations between degradation of Poly R-478 and any of the pesticides (Table 4). However, there were significant correlations between the degradation of all of the pesticides.

Table 4.  Relationships between degradation of Poly R-478 and the pesticides
  1. *P<0.05; **P<0.01.

CompoundMetalaxylTerbuthylazineAtrazineDiuron
Poly R-4780.1080.1690.3260.212
Metalaxyl 0.865*0.811*0.916**
Terbuthylazine  0.767*0.751*
Atrazine   0.934**

3.3Degradation 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 differences 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 significant amounts of the dicarboximide fungicide iprodione, with S. hirsutum a more effective degrader of the compound that the other fungi. In the case of chlorpyrifos, the fungi generally had lower abilities to degrade 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.

Table 5.  Pesticide remaining biobed matrix after 42 days
  1. ND, not determined (interfering metabolite).

Fungus% Pesticide remaining
 MetalaxylTerbuthylazineAtrazineDiuronIprodioneChlorpyrifos
Coriolus versicolor60.160.148.947.742.163.8
Hypholoma fasciculareND63.038.784.152.671.0
Stereum hirsutum46.121.442.725.937.793.9
LSD (P=0.05)3.639.536.221.316.25.4

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

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 different 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 detoxification 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 differences in their abilities to degrade model synthetic lignin polymers, arising from different enzyme production patterns.

The three members of the Polyporaceae used in our study showed differences 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 different 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 effective degraders of natural lignin and synthetic polymers, the nature of the ligninolytic enzyme as well as the detoxification 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 differences 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 indicate 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

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

We thank Sonia Cavanna and Mercedes Franey for assistance, Mike Challen (HRI) and Jonathan Leake (University of Sheffield) for many of the fungal cultures, and the Department of Environment, Food and Rural Affairs, and the European Community for financial support.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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