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Growth of Phanerochaete chrysosporium in soil and its ability to degrade the fungicide benomyl
Bioresource Technology 49 (1994) 197-201 Elsevier Science Limited Printed in Great Britain 0260-8774/94/$7.00 0960-8524(94)00018-2
ELSEVIER
GROWTH OF PHANEROCHAETE CHR YSOSPORIUM IN SOIL A N D ITS ABILITY TO D E G R A D E THE FUNGICIDE B E N O M Y L Tasneem Adam Ali & M. Wainwright Department of Molecular Biology and Biotechnology, Universityof Sheffield, Sheffield, S I O2TN, UK (Received 29 March 1994; revised version received 4 May 1994; accepted 7 May 1994)
Abstract
Following inoculuation Phanerochaete chrysosporium grew faster across the surface of an agricultural loam soil at 37°C than when incubated at 25°C. Starch or peptone, alone or in combination, failed to stimulate growth of the fungus, while the addition of peptone or peptone-plus-starch to soil incubated at 25°C (but not at 37°C) stimulated a bacterium antagonistic to P. chrysosporium. The bacterium produced an antifungal agent in vitro, which inhibited the growth of filamentous fungi including P. chrysosporium and the yeast Candida tropicalis. The assumption that the addition of nutrients to soils necessarily stimulates growth and soil colonization by an inoculated fungus is thereby questioned. Soil incubation with a spore suspension of P. chrysosporium caused a 60% reduction in the time taken to completely degrade 56"25 #g of benomyl g- i soil and at least a 30%-plus reduction in the time taken to completely degrade higher concentrations. These results are discussed in relation to practical approaches to the bioremediation of benomyl-treated soils. Key words: Bioremediation, white rot fungi, fungicide, degradation, soil microbiology.
INTRODUCTION
Attempts have been made recently to increase the rate of degradation of pesticides and other environmental pollutants in soil by enhancing microbial activity (Racke & Coats, 1990). Such bioremediation can be achieved by adding a specific microbial inoculant known to degrade the pollutant, or by increasing overall soil microbial activity by adding nutrients. The inoculant may be a single, unaltered or genetically modified microorganism, or a mixture isolated from the contaminated soil and then re-inoculated as a cocktail (i.e. the so-called 'vanguard approach', Bridges 1991 ). Since the wood-decomposing fungus Phanerochaete chrysosporium can degrade a wide variety of pollutants it has been evaluated widely as a means of enhancing pollutant degradation in soils (Lamar et al., 1990).
Effective bioremediation using microorganisms such as P. chrysosporium depends upon uniform, rapid and sustained growth of the inoculant in the polluted soil. Such growth is difficult to achieve, particularly in the field. As a result, alternatives have been tried; for example polluted soil can be removed and exposed to the bioremediating microorganism in rotating drums, in which optimum environmental conditions for microbial growth and activity can be maintained. Before soil bioremediation with P. chrysosporium can be achieved, the environmental conditions which enable it to grow in soils and treatment systems must first be determined. Studies on the growth and bioremediating activity of P. chrysosporium using both autoclaved and non-sterilized soils have been reported. Although fungi can be grown easily in autoclaved soil, this form of sterilization has been avoided because it is uneconomic for use in the field and in large scale treatment systems. Thus non-sterile soil mimics more closely conditions in vivo. Our aim was to determine: (a) what temperature conditions best promoted rapid growth of P. chrysosporium in soils; (b) whether the addition of nutrients to soil (as starch and peptone alone Ol~combined) stimulated the growth and pollutant-degrading activity of P. chrysosporium; and (c) the effect of temperature and nutrient addition on the ability of this fungus to degrade a specific organic pollutant (the fungicide benomyl) in soil. Although benomyl is not a particular refractory compound it is assayed easily in soil and can therefore provide an effective model to study the ability of P. chrysosporium to degrade organic compounds in soil. METHODS
Phanerochaete chrysosporium was maintained on malt extract agar (Oxoid). Two 20-cm cotton strips were first soaked in liquid malt extract (10 g in 1000 ml, sterilized at 120°C for 20 min) and then in spore suspension of P. chrysosporium. Next, they were transferred to a sterile beaker, covered with cling film and incubated at 37°C for 7 days. The inoculated strips were then used in soil colonization studies. 197
198
T. Adam Ali, M. Wainwright
An agricultural loam soil was used throughout these experiments (organic C, 3"9% w/w; total N, 0-6% w/w; pH 7.3). The soil was not sieved, but large roots and stones were removed. After adjusting the moisture content of the soil to - 0.75 MPa, the soil was placed in square (10 cm 2) or round (14 cm diameter) Petri dishes to a depth of approximately 1"5 cm. Inoculated cotton strips (fully colonized with mycelium) were placed on the centre of the soil surface and the Petri dishes were incubated at either 25°C or 37°C. Soil samples were incubated without amendment (control) or following treatment with a solution of soluble starch (2% w/v solution, BDH, Analar); peptone (2%, w/v Oxoid) or starch plus peptone (both 2% w/v). The treatments were set up in triplicate and fungal colonization of the soil surface was assessed by measuring the extent of white surface mycelial growth along two axes at 2-day intervals for 30 days. Studies on a bacterium antagonistic to P. chrysosporium A bacterium which forms white, mucoid colonies and is antagonistic to the growth of P. chrysosporium was isolated from the soil using Czapek Dox medium. The antifungal properties of the isolate were tested by growing it in shaking culture in Czapek Dox liquid medium (Oxoid) at 25°C. After 5 days, the medium was membrane filtered (first through a 0"45-/zm and then through a 0.2-/zm cellulose-nitrate filter). The antifungal activity of the filtrate was then determined by exposing a test fungus growing on Czapek Dox agar. Spore suspensions (0"5 ml) were spread on the surface of the medium and a well was cut (using a sterile cork borer) into which the filtrate was pipetted. Growth of the following fungi was tested: Aspergillus niger;
Candida albicans; Fusarium solani; Phanerochaete chrysosporium; Penicillium notatum; Rhizopus sp.; Trichoderma harzianum. Tests were made to determine the presence of intracellular antifungal agents after sonication or after French press treatment of the bacterial cells. Prior to French pressing, the cells were centrifuged at 100 rpm for 30 min, washed three times with sterile distilled water and then re-suspended in distilled water. Sonication was performed at maximum frequency on cells in 100 ml of medium, cooled in ice; treatment was in 30 bursts over 5 min, with approximately 10-s intervals in between. After sonication, the disrupted cellular material was extracted with ethyl ether in a separating column. The ether-soluble fraction was then allowed to evaporate and the residue was resuspended in ether. Water- and ether-soluble fractions were then assayed for antifungal activity. Effect of Phanerochaete chrysosporium on benomyl degradation in soil Phanerochaete chrysosporium was grown and maintained on malt extract agar (Oxoid). Samples (200 g) of the agricultural loam soil were treated with benomyl
and incubated in triplicate in polythene bags at 25°C. Benomyl (2.25 g) was dissolved in 1 litre of water at a concentration of 0.225% (w/v). The fungicide formulation was added in 45-ml portions to 200 g of soil to achieve final concentrations of 225, 112-5 and 56"25 /~g g- ~ soil. The soil was sieved (less than 2 ram) before use. Controls using deionized water were also added; triplicates were used throughout. Soils were adjusted to a water potential of - 0 . 7 5 MPa (22.5% w/v, soil/ water) and maintained at this level throughout the incubation period. Samples of soil were then removed at intervals, and the concentration of benomyl was determined using the bioassay method of Munnecke (1958). The presence of P. chrysosporium in the inoculated soil was detected by plating samples onto the selective isolation medium, as described by Dietrich and Lamar (1990). Bioassay technique The Munnecke (1958) technique was used to bioassay benomyl concentrations in soil, using a benomyl-sensitive strain of Trichoderma viride as the indicator organism. Each of the triplicate soil samples was assayed once.
RESULTS AND DISCUSSION
Phanerochaete chrysosporium readily grew from the cotton strip inoculum to colonize the surface of nonsterile soil (Figs l(a) and 2), with measurable growth appearing after 4 days at both 25°C and 37°C (Fig. 2). Growth of the inoculant over non-sterile soil was slower at 25°C than at 37°C, with only 30% cover of the surface occurring after 30 days at 250C, compared to 45% at 37°C (Fig. 2). Starch addition had no effect on surface growth at 25°C, but stimulated fungal growth at 37°C (Fig. 2). However, peptone alone (Fig. 3) or in combination with starch (Fig. 4) generally inhibited the surface growth of the fungus at both incubation temperatures. When peptone alone was added, surface growth was very slow, approximately one-tenth of the control value, and stopped after 14 days (Fig. 3). In the case of starch plus peptone, added at 25°C, mycelial extension progressed slightly ahead of the rate seen in untreated soil up to day 14 of the incubation period and then declined markedly (Fig. 4). Following incubation at 25°C with peptone and peptone-starch, respectively, there was a dramatic decrease in mycelial growth, followed by complete degradation of surface mycelium after 14 and 22 days, respectively (Figs l(b) and (c), 3 and 4); any mycelium that had previously grown disappeared from the soil surface to leave bare soil. This effect was not seen at 37°C (Figs 3(a) and 4(a)), or in soils treated with starch alone at either 25°C or 37°C (Fig. 2). The complete disappearance of mycelium at 25°C following peptone and peptone-starch treatment was rapid, occurrring 2-4 days after the initial decline in growth was observed.
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This remarkable destruction of fungal mycelium at 25°C in the presence of peptone or peptone-starch was investigated further. Mycelial destruction occurred at 37°C but not at 25°C, and was not initiated by starch alone at 25°C. The most marked effect occurred with peptone alone, while the addition of starch to the peptone delayed the onset of the mycelial destruction. This shows that the mycelial destruction phenomenon was temperature-dependent and was induced by treating soil with peptone. The most likely explanation for this phenomenon is that a proteolytic microorganism or population capable of lysing the mycelium is induced in the soil following peptone treatment at 25°C, but not at 37°C. Potential antagonists were therefore isolated from the soil. The most frequently isolated microorganisms from peptone treated soils were fungi (species of Fusarium, Rhisopus and Mucor) and a bacterium producing mucoid, white colonies. None of the fungi were antagonistic towards P. chrysosporium, while the bacterium in contrast was highly antagonistic, as shown by its inhibitory effect on growth. The bacterium was Gram negative, grew in the form of short rods, and when grown for 4-5 days in a liquid Czapek Dox medium it produced a fluorescent yellow-green pigment, characteristics which suggest that it was a fluorescent pseudomonad. Although the bacterium grew well in vitro at 37°C and was stimulated by peptone, when inoculated onto the surface of pre-grown mycelium of P. chrysosporium incubated at 25°C, it failed to lyse the mycelium in the way observed above. Crude growth medium from 5-6 day old cultures of the mucoid bacterium did, however, inhibit P. chrysosporium, Apsergillus niger, Rhizopus sp., Penicillium notatum, Trichoderma harzianum, Fusarium solani and Candida tropicalis. These fungi were also inhibited when grown
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Effect of starch (2% w/w) on growth of P. chrysosporium across soil surface. Incubated at (a) 37°C; (b) 25°C: tz, soil plus P. chrysosporium; o, soil plus P. chrysosporium plus starch. Means of triplicates _+standard deviation. Fig. 2.
together with the bacterium for 5 days on Czapek Dox agar. When the crude filtrate from the growth medium was passed through a 0"2-pm membrane filter, antibiotic activity was lost. However, no similar loss of activity was seen following filtration through a 0"45-pm filter, suggesting that antifungal activity was due to the presence of bacterial cells in the filtrate. Long-term sonication (40 min) was necessary to disrupt the cells, while the use of a French press was not successful. When the sonicated fraction was extracted with ethyl ether, the antifungal activity was found in the extract, while water-soluble fractions showed little activity. Maximum antifungal activity occurred over the range pH 5-6, being most marked at pH 5"5. Antifungal activity was lost when the ether extract was allowed to evaporate
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Fig. 3. Effect of peptone (2% w/w) on growth of P. chrysosporium across soil surface. Incubated at (a) 37°C; (b) 25°C: % soil plus P. chrysosporium; e, soil plus P. chrysosporium plus peptone. Means of triplicates + standard deviation.
and was then re-dissolved in fresh ether. Neither chloroform nor alcohol could solubilize these fractions which, although insoluble in water, were active against C. tropicalis in water suspension. Following repeated sub-culturing (4-5 times), the bacterium lost its antifungal activity, making it necessary to use fresh isolates. The effects of P. chrysosporium inoculation on benomyl degradation in terms of percentage time taken for total degradation are shown in Fig. 5. In this case a spore suspension of the funghs was used as inoculant, rather than the inoculated cotton strips used in the above experiments. Inoculation of P. chrysosporium, led to a 60% reduction in the time taken for the lowest concentration of benomyl to degrade. The addition of P. chrysosporium to soil treated with 112"5 /ag and 56"25 /ag benomyl g-1 soil immediately stimulated benomyl breakdown (Fig. 5). In contrast, the break-
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Fig. 4. Effect of starch plus peptone (2% w/w) on growth of P. chrysosporium across soil surface. Incubated at (a) 37°C; (b) 25°C; t3, soil plus P. chrysosporium; e, soil plus P. chrysosporium plus starch plus peptone. Means of triplicates + standard deviation.
down of the highest concentration of benomyl (225 pg g- 1 soil) did not begin for 40-50 days after the addition of the inoculum. Complete degradation of the lowest concentration of benomyl occurred after 46 days, while the two other concentrations were completely degraded after 85 days. This compares to control values of 115, 127 and 130 days for 56"25, 112"5 and 225/~g, respectively. Growth of the inoculant was confirmed by the use of the specific isolation medium of Dietrich and Lamar (1990). Culture medium alone was included as a control in these experiments; here benomyl was still detected after 130 days, i.e. beyond the point at which the fungicide persisted in soil in the absence of a medium. The medium itself was therefore not responsible for
Benomyl degradation in soil
degrader organism can be achieved, growing in close contact with the pesticide undergoing degradation. Laboratory studies do, however, enable the potential of various bioremediation approaches to be evaluated, since if an organism fails to enhance pollutant degradation under laboratory conditions then it is obviously unlikely to do so in the field. The results of this study demonstrate the potential for using P. chrysosporium to increase the rate of breakdown in soil with high concentrations of benomyl, and presumably other, more recalcitrant, organic compounds. However, while the potential clearly exists for using a single fungal inoculant like P. chrysosporium as a practical means of bioremediating fungicide-polluted soils, experience gained from similar attempts to use mycorrhizal inoculants to improve plant growth, and fungal inoculants for biocontrol suggest that the problems involved will be considerable. The addition of a mixed inoculant of various microorganisms may therefore prove equally or more effective than a single species such as P. chrysosporium, in terms of bioremediation (Adam Ali, 1992).
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Fig. 5. Effect of P. chrysosporium inoculation on benomyl degradation in soils. Benomyl concentration: o, 56/~g g-1; ,,, 112/tg g-l; • 225 ~g g-L Means of triplicates +standard deviation. any stimulation in benomyl breakdown resulting from the addition of inoculants. Phanerochaete chrysosporium has been widely evaluated as an inoculant in soil bioremediation studies (Bumpus, 1993), mainly because it produces a number of non-specific ligninases which can degrade a wide variety of pollutants (Zurer, 1987). It is generally grown on wood chips which are then introduced into the soil. Our results show that liquid cultures of P. chrysosporium, are effective in stimulating benomyl degradation. It seems, therefore, that in this case effective soil inoculation of the fungus can be achieved without first needing to grow it on wood chips or other solid substrates; using liquid substrates is obviously less time-consuming and costly than the use of wood chip-based inoculants. Laboratory studies such as these, in which optimal conditions for microbial growth are provided, can only indicate the potential success of any bioremediation strategy. In laboratory incubated soils, as opposed to field conditions, the inoculum can be thoroughly mixed in the soil and a large inoculum potential of the
ACKNOWLEDGEMENT T.A.A. wishes to thank the Government of Pakistan for provision of a research scholarship.
REFERENCES
Adam Ali, T. (1993). Physiology and biotechnological potential of the white-rot fungus Phanerochaete chrysosporium. PhD thesis, University of Sheffield, UK. Bridges, E. M. (1991 ). Dealing with contaminated soils. Soil Use Mgment., 7, 151-8. Bumpus, J. A. (1993). White rot fungi and their potential use in soft bioremediation. Soil Biochem., 8, 65-100. Dietrich, D. M. & Lamar, R. T. (1990). Selective medium for isolating Phanerochaete chrysosporium from soft. Appl. Environ. Microbiol., 56, 3088-92. Lamar, R. T., Glaser, J. A. & Kirk, T. K. (1990). Fate of pentachlorophenol (PCP) in sterile soils inoculated with the white-rot basidiomycete Phanerochaete chrysosporium: Mineralization, volatilization and depletion of PCP. Soil Biol. Biochem., 22,433-40. Munnecke, D. E. (1985). A biological assay of non-volatile, diffusible fungicides in soft. Phytopathol., 48, 61-3. Racke, K. D. & Coats, J. R. (1990). Enhanced biodegradation of pesticides in the environment. Am. Chem. Soc., Washington, DC. Zurer, P. S. (1987). Fungus shows promise in hazardous waste treatment. Chem. Eng. News, 17-19.
ELSEVIER
GROWTH OF PHANEROCHAETE CHR YSOSPORIUM IN SOIL A N D ITS ABILITY TO D E G R A D E THE FUNGICIDE B E N O M Y L Tasneem Adam Ali & M. Wainwright Department of Molecular Biology and Biotechnology, Universityof Sheffield, Sheffield, S I O2TN, UK (Received 29 March 1994; revised version received 4 May 1994; accepted 7 May 1994)
Abstract
Following inoculuation Phanerochaete chrysosporium grew faster across the surface of an agricultural loam soil at 37°C than when incubated at 25°C. Starch or peptone, alone or in combination, failed to stimulate growth of the fungus, while the addition of peptone or peptone-plus-starch to soil incubated at 25°C (but not at 37°C) stimulated a bacterium antagonistic to P. chrysosporium. The bacterium produced an antifungal agent in vitro, which inhibited the growth of filamentous fungi including P. chrysosporium and the yeast Candida tropicalis. The assumption that the addition of nutrients to soils necessarily stimulates growth and soil colonization by an inoculated fungus is thereby questioned. Soil incubation with a spore suspension of P. chrysosporium caused a 60% reduction in the time taken to completely degrade 56"25 #g of benomyl g- i soil and at least a 30%-plus reduction in the time taken to completely degrade higher concentrations. These results are discussed in relation to practical approaches to the bioremediation of benomyl-treated soils. Key words: Bioremediation, white rot fungi, fungicide, degradation, soil microbiology.
INTRODUCTION
Attempts have been made recently to increase the rate of degradation of pesticides and other environmental pollutants in soil by enhancing microbial activity (Racke & Coats, 1990). Such bioremediation can be achieved by adding a specific microbial inoculant known to degrade the pollutant, or by increasing overall soil microbial activity by adding nutrients. The inoculant may be a single, unaltered or genetically modified microorganism, or a mixture isolated from the contaminated soil and then re-inoculated as a cocktail (i.e. the so-called 'vanguard approach', Bridges 1991 ). Since the wood-decomposing fungus Phanerochaete chrysosporium can degrade a wide variety of pollutants it has been evaluated widely as a means of enhancing pollutant degradation in soils (Lamar et al., 1990).
Effective bioremediation using microorganisms such as P. chrysosporium depends upon uniform, rapid and sustained growth of the inoculant in the polluted soil. Such growth is difficult to achieve, particularly in the field. As a result, alternatives have been tried; for example polluted soil can be removed and exposed to the bioremediating microorganism in rotating drums, in which optimum environmental conditions for microbial growth and activity can be maintained. Before soil bioremediation with P. chrysosporium can be achieved, the environmental conditions which enable it to grow in soils and treatment systems must first be determined. Studies on the growth and bioremediating activity of P. chrysosporium using both autoclaved and non-sterilized soils have been reported. Although fungi can be grown easily in autoclaved soil, this form of sterilization has been avoided because it is uneconomic for use in the field and in large scale treatment systems. Thus non-sterile soil mimics more closely conditions in vivo. Our aim was to determine: (a) what temperature conditions best promoted rapid growth of P. chrysosporium in soils; (b) whether the addition of nutrients to soil (as starch and peptone alone Ol~combined) stimulated the growth and pollutant-degrading activity of P. chrysosporium; and (c) the effect of temperature and nutrient addition on the ability of this fungus to degrade a specific organic pollutant (the fungicide benomyl) in soil. Although benomyl is not a particular refractory compound it is assayed easily in soil and can therefore provide an effective model to study the ability of P. chrysosporium to degrade organic compounds in soil. METHODS
Phanerochaete chrysosporium was maintained on malt extract agar (Oxoid). Two 20-cm cotton strips were first soaked in liquid malt extract (10 g in 1000 ml, sterilized at 120°C for 20 min) and then in spore suspension of P. chrysosporium. Next, they were transferred to a sterile beaker, covered with cling film and incubated at 37°C for 7 days. The inoculated strips were then used in soil colonization studies. 197
198
T. Adam Ali, M. Wainwright
An agricultural loam soil was used throughout these experiments (organic C, 3"9% w/w; total N, 0-6% w/w; pH 7.3). The soil was not sieved, but large roots and stones were removed. After adjusting the moisture content of the soil to - 0.75 MPa, the soil was placed in square (10 cm 2) or round (14 cm diameter) Petri dishes to a depth of approximately 1"5 cm. Inoculated cotton strips (fully colonized with mycelium) were placed on the centre of the soil surface and the Petri dishes were incubated at either 25°C or 37°C. Soil samples were incubated without amendment (control) or following treatment with a solution of soluble starch (2% w/v solution, BDH, Analar); peptone (2%, w/v Oxoid) or starch plus peptone (both 2% w/v). The treatments were set up in triplicate and fungal colonization of the soil surface was assessed by measuring the extent of white surface mycelial growth along two axes at 2-day intervals for 30 days. Studies on a bacterium antagonistic to P. chrysosporium A bacterium which forms white, mucoid colonies and is antagonistic to the growth of P. chrysosporium was isolated from the soil using Czapek Dox medium. The antifungal properties of the isolate were tested by growing it in shaking culture in Czapek Dox liquid medium (Oxoid) at 25°C. After 5 days, the medium was membrane filtered (first through a 0"45-/zm and then through a 0.2-/zm cellulose-nitrate filter). The antifungal activity of the filtrate was then determined by exposing a test fungus growing on Czapek Dox agar. Spore suspensions (0"5 ml) were spread on the surface of the medium and a well was cut (using a sterile cork borer) into which the filtrate was pipetted. Growth of the following fungi was tested: Aspergillus niger;
Candida albicans; Fusarium solani; Phanerochaete chrysosporium; Penicillium notatum; Rhizopus sp.; Trichoderma harzianum. Tests were made to determine the presence of intracellular antifungal agents after sonication or after French press treatment of the bacterial cells. Prior to French pressing, the cells were centrifuged at 100 rpm for 30 min, washed three times with sterile distilled water and then re-suspended in distilled water. Sonication was performed at maximum frequency on cells in 100 ml of medium, cooled in ice; treatment was in 30 bursts over 5 min, with approximately 10-s intervals in between. After sonication, the disrupted cellular material was extracted with ethyl ether in a separating column. The ether-soluble fraction was then allowed to evaporate and the residue was resuspended in ether. Water- and ether-soluble fractions were then assayed for antifungal activity. Effect of Phanerochaete chrysosporium on benomyl degradation in soil Phanerochaete chrysosporium was grown and maintained on malt extract agar (Oxoid). Samples (200 g) of the agricultural loam soil were treated with benomyl
and incubated in triplicate in polythene bags at 25°C. Benomyl (2.25 g) was dissolved in 1 litre of water at a concentration of 0.225% (w/v). The fungicide formulation was added in 45-ml portions to 200 g of soil to achieve final concentrations of 225, 112-5 and 56"25 /~g g- ~ soil. The soil was sieved (less than 2 ram) before use. Controls using deionized water were also added; triplicates were used throughout. Soils were adjusted to a water potential of - 0 . 7 5 MPa (22.5% w/v, soil/ water) and maintained at this level throughout the incubation period. Samples of soil were then removed at intervals, and the concentration of benomyl was determined using the bioassay method of Munnecke (1958). The presence of P. chrysosporium in the inoculated soil was detected by plating samples onto the selective isolation medium, as described by Dietrich and Lamar (1990). Bioassay technique The Munnecke (1958) technique was used to bioassay benomyl concentrations in soil, using a benomyl-sensitive strain of Trichoderma viride as the indicator organism. Each of the triplicate soil samples was assayed once.
RESULTS AND DISCUSSION
Phanerochaete chrysosporium readily grew from the cotton strip inoculum to colonize the surface of nonsterile soil (Figs l(a) and 2), with measurable growth appearing after 4 days at both 25°C and 37°C (Fig. 2). Growth of the inoculant over non-sterile soil was slower at 25°C than at 37°C, with only 30% cover of the surface occurring after 30 days at 250C, compared to 45% at 37°C (Fig. 2). Starch addition had no effect on surface growth at 25°C, but stimulated fungal growth at 37°C (Fig. 2). However, peptone alone (Fig. 3) or in combination with starch (Fig. 4) generally inhibited the surface growth of the fungus at both incubation temperatures. When peptone alone was added, surface growth was very slow, approximately one-tenth of the control value, and stopped after 14 days (Fig. 3). In the case of starch plus peptone, added at 25°C, mycelial extension progressed slightly ahead of the rate seen in untreated soil up to day 14 of the incubation period and then declined markedly (Fig. 4). Following incubation at 25°C with peptone and peptone-starch, respectively, there was a dramatic decrease in mycelial growth, followed by complete degradation of surface mycelium after 14 and 22 days, respectively (Figs l(b) and (c), 3 and 4); any mycelium that had previously grown disappeared from the soil surface to leave bare soil. This effect was not seen at 37°C (Figs 3(a) and 4(a)), or in soils treated with starch alone at either 25°C or 37°C (Fig. 2). The complete disappearance of mycelium at 25°C following peptone and peptone-starch treatment was rapid, occurrring 2-4 days after the initial decline in growth was observed.
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This remarkable destruction of fungal mycelium at 25°C in the presence of peptone or peptone-starch was investigated further. Mycelial destruction occurred at 37°C but not at 25°C, and was not initiated by starch alone at 25°C. The most marked effect occurred with peptone alone, while the addition of starch to the peptone delayed the onset of the mycelial destruction. This shows that the mycelial destruction phenomenon was temperature-dependent and was induced by treating soil with peptone. The most likely explanation for this phenomenon is that a proteolytic microorganism or population capable of lysing the mycelium is induced in the soil following peptone treatment at 25°C, but not at 37°C. Potential antagonists were therefore isolated from the soil. The most frequently isolated microorganisms from peptone treated soils were fungi (species of Fusarium, Rhisopus and Mucor) and a bacterium producing mucoid, white colonies. None of the fungi were antagonistic towards P. chrysosporium, while the bacterium in contrast was highly antagonistic, as shown by its inhibitory effect on growth. The bacterium was Gram negative, grew in the form of short rods, and when grown for 4-5 days in a liquid Czapek Dox medium it produced a fluorescent yellow-green pigment, characteristics which suggest that it was a fluorescent pseudomonad. Although the bacterium grew well in vitro at 37°C and was stimulated by peptone, when inoculated onto the surface of pre-grown mycelium of P. chrysosporium incubated at 25°C, it failed to lyse the mycelium in the way observed above. Crude growth medium from 5-6 day old cultures of the mucoid bacterium did, however, inhibit P. chrysosporium, Apsergillus niger, Rhizopus sp., Penicillium notatum, Trichoderma harzianum, Fusarium solani and Candida tropicalis. These fungi were also inhibited when grown
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together with the bacterium for 5 days on Czapek Dox agar. When the crude filtrate from the growth medium was passed through a 0"2-pm membrane filter, antibiotic activity was lost. However, no similar loss of activity was seen following filtration through a 0"45-pm filter, suggesting that antifungal activity was due to the presence of bacterial cells in the filtrate. Long-term sonication (40 min) was necessary to disrupt the cells, while the use of a French press was not successful. When the sonicated fraction was extracted with ethyl ether, the antifungal activity was found in the extract, while water-soluble fractions showed little activity. Maximum antifungal activity occurred over the range pH 5-6, being most marked at pH 5"5. Antifungal activity was lost when the ether extract was allowed to evaporate
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and was then re-dissolved in fresh ether. Neither chloroform nor alcohol could solubilize these fractions which, although insoluble in water, were active against C. tropicalis in water suspension. Following repeated sub-culturing (4-5 times), the bacterium lost its antifungal activity, making it necessary to use fresh isolates. The effects of P. chrysosporium inoculation on benomyl degradation in terms of percentage time taken for total degradation are shown in Fig. 5. In this case a spore suspension of the funghs was used as inoculant, rather than the inoculated cotton strips used in the above experiments. Inoculation of P. chrysosporium, led to a 60% reduction in the time taken for the lowest concentration of benomyl to degrade. The addition of P. chrysosporium to soil treated with 112"5 /ag and 56"25 /ag benomyl g-1 soil immediately stimulated benomyl breakdown (Fig. 5). In contrast, the break-
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Fig. 4. Effect of starch plus peptone (2% w/w) on growth of P. chrysosporium across soil surface. Incubated at (a) 37°C; (b) 25°C; t3, soil plus P. chrysosporium; e, soil plus P. chrysosporium plus starch plus peptone. Means of triplicates + standard deviation.
down of the highest concentration of benomyl (225 pg g- 1 soil) did not begin for 40-50 days after the addition of the inoculum. Complete degradation of the lowest concentration of benomyl occurred after 46 days, while the two other concentrations were completely degraded after 85 days. This compares to control values of 115, 127 and 130 days for 56"25, 112"5 and 225/~g, respectively. Growth of the inoculant was confirmed by the use of the specific isolation medium of Dietrich and Lamar (1990). Culture medium alone was included as a control in these experiments; here benomyl was still detected after 130 days, i.e. beyond the point at which the fungicide persisted in soil in the absence of a medium. The medium itself was therefore not responsible for
Benomyl degradation in soil
degrader organism can be achieved, growing in close contact with the pesticide undergoing degradation. Laboratory studies do, however, enable the potential of various bioremediation approaches to be evaluated, since if an organism fails to enhance pollutant degradation under laboratory conditions then it is obviously unlikely to do so in the field. The results of this study demonstrate the potential for using P. chrysosporium to increase the rate of breakdown in soil with high concentrations of benomyl, and presumably other, more recalcitrant, organic compounds. However, while the potential clearly exists for using a single fungal inoculant like P. chrysosporium as a practical means of bioremediating fungicide-polluted soils, experience gained from similar attempts to use mycorrhizal inoculants to improve plant growth, and fungal inoculants for biocontrol suggest that the problems involved will be considerable. The addition of a mixed inoculant of various microorganisms may therefore prove equally or more effective than a single species such as P. chrysosporium, in terms of bioremediation (Adam Ali, 1992).
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Fig. 5. Effect of P. chrysosporium inoculation on benomyl degradation in soils. Benomyl concentration: o, 56/~g g-1; ,,, 112/tg g-l; • 225 ~g g-L Means of triplicates +standard deviation. any stimulation in benomyl breakdown resulting from the addition of inoculants. Phanerochaete chrysosporium has been widely evaluated as an inoculant in soil bioremediation studies (Bumpus, 1993), mainly because it produces a number of non-specific ligninases which can degrade a wide variety of pollutants (Zurer, 1987). It is generally grown on wood chips which are then introduced into the soil. Our results show that liquid cultures of P. chrysosporium, are effective in stimulating benomyl degradation. It seems, therefore, that in this case effective soil inoculation of the fungus can be achieved without first needing to grow it on wood chips or other solid substrates; using liquid substrates is obviously less time-consuming and costly than the use of wood chip-based inoculants. Laboratory studies such as these, in which optimal conditions for microbial growth are provided, can only indicate the potential success of any bioremediation strategy. In laboratory incubated soils, as opposed to field conditions, the inoculum can be thoroughly mixed in the soil and a large inoculum potential of the
ACKNOWLEDGEMENT T.A.A. wishes to thank the Government of Pakistan for provision of a research scholarship.
REFERENCES
Adam Ali, T. (1993). Physiology and biotechnological potential of the white-rot fungus Phanerochaete chrysosporium. PhD thesis, University of Sheffield, UK. Bridges, E. M. (1991 ). Dealing with contaminated soils. Soil Use Mgment., 7, 151-8. Bumpus, J. A. (1993). White rot fungi and their potential use in soft bioremediation. Soil Biochem., 8, 65-100. Dietrich, D. M. & Lamar, R. T. (1990). Selective medium for isolating Phanerochaete chrysosporium from soft. Appl. Environ. Microbiol., 56, 3088-92. Lamar, R. T., Glaser, J. A. & Kirk, T. K. (1990). Fate of pentachlorophenol (PCP) in sterile soils inoculated with the white-rot basidiomycete Phanerochaete chrysosporium: Mineralization, volatilization and depletion of PCP. Soil Biol. Biochem., 22,433-40. Munnecke, D. E. (1985). A biological assay of non-volatile, diffusible fungicides in soft. Phytopathol., 48, 61-3. Racke, K. D. & Coats, J. R. (1990). Enhanced biodegradation of pesticides in the environment. Am. Chem. Soc., Washington, DC. Zurer, P. S. (1987). Fungus shows promise in hazardous waste treatment. Chem. Eng. News, 17-19.