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Inducers, substrates, and inhibitors of a propanil-degrading amidase of Fusarium oxysporum
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
39, 240-250 (1991)
Inducers, Substrates, and Inhibitors of a Propanil-Degrading Amidase of Fusarium oxysporum1s2 HELENAREICHEL, **3 H.D. *Department
of Botany,
iFSoit Microbial
University
SISLER,* AND D.D.
of Maryland, USDA,
Systems Laboratory,
College Park, ARS, Bebville,
KAUFMAN? Maryland Maryland
20742, and 20705
Received August 28, 1990; accepted November 13, 1990 Ureas, carbamates, and amides were investigated as inducers, substrates, and inhibitors of propanil-degrading amidase activity in Fusarium oxysporum. Urea herbicides were potent inducers but not substrates of the amidase that degrades propanil. Linuron-induced cells degraded propanil approximately 67 times more rapidly than propanil-induced ceils. Noninduced cells did not degrade propanil. All urea inducers were also inhibitors of the linuron-induced propanil-degrading amidase activity in whole cells and cell-free extracts. The urea inducers linuron, diuron, and monuron gave respective ID, values of 19, 20 and 70 FM as inhibitors of amidase degradation of propanil by whole cells. The respective ID,, values for degradation by cell-free extracts were 12.0, 4.5 and 48 PM. The carbamates PPG-124 (p-chlorophenyl methylcarbamate) and mobam were powerful inhibitors of the amidase activity with respective ID,, values in whole cells of 0.33 and 4.4 FM. Respective values for cell-free extracts were 0.2 and 3.6 )LM. Mobam, CIPC (chlorpropham), and diallate were fair inducers of the amidase activity, but amidase induction by PPG-124 could not be demonstrated, possibly because it inactivated the enzyme. Acetanilide did not induce amidase activity for the breakdown of propanil. Likewise, propanil did not induce amidase activity for the breakdown of acetanilide. Different enzymes seem to be involved here. The K, for the linuron-induced, propanil-degrading amidase in cell-free extracts from freeze-dried cells was 7.1 x lo-‘M.
INTRODUCTION
Low pesticide efficacy and failure to control the target pest have been observed in soils in which pesticide has been applied frequently. Problem soils of this type have also been observed when different pesticides with a similar chemical structure have been applied (1). The involvement of microbial activity in the development of problem soils has been indicated by autoclaving (2) and by the use of antibiotics or y irradiation (3). Studies have shown that some pesticides ’ This research was supported by a grant from The National Pesticide Impact Assessment Program (NAPIAP), Cooperative States Research Service, USDA. * Scientific Publication 6064, Contribution 8226 of The University of Maryland Agricultural Experiment Station. 3 Present address: Centro Intemacional de Agricultura Tropical (CIAT), Apartado Aereo No. 6713, Cali, Colombia, SA. 240 0048-3575/91 $3.00
may act as inducers (or inhibitors) of enzymes in soil microorganisms for their own breakdown, as well as that of other pesticides (4). This ability of microorganisms to degrade pesticide molecules after adaptation to an original similar structure was first demonstrated by Audus in 1951 (5). In some cases, fungicides may affect the breakdown of other pesticides by inhibiting growth or enzyme activity of microorganisms involved in the biodegradation process (6).
Among the enzymes involved in pesticide degradation in the soil are the amidases produced by fungi and bacteria. Wallndfer (7) reported the degradation of urea herbicides by Bacillus sphaericus isolated from the soil. An amidase responsible for the breakdown of urea herbicides by this organism was described soon thereafter (8). The urea herbicide linuron induced amidase activity in B. sphaericus for the breakdown of linuron itself, monolinuron, and meto-
PROPANIL
DEGRADING
AMIDASE
bromuron. Working with this organism, Engelhardt et al. (9) demonstrated a low substrate specificity of this linuron-induced amidase. A large variety of herbicides and fungicides of the acylanilide and methoxysubstituted phenylurea type were degraded by this enzyme. Acylanilides were degraded at a rate at least 10 times higher than the methoxy-substituted phenylureas. This same amidase was found to have powerful activity for the breakdown of the amide herbicide propanil. However, propanil was not a good inducer of this linuron degrading amidase (10). The microbial transformation of propanil to the corresponding aniline and the disappearance of the parent compound had already been reported by Bartha et al., in 1967 (11). Subsequently, several microorganisms capable of degrading propanil were isolated from soils, among them Fusarium solani and Fusarium oxysporum (12, 13). In 1975, Blake and Kaufman demonstrated that propanil induces an acylamidase in F. oxysporum for its own degradation (14). Since F. oxysporum has been reported to occur in various problem soils, the following study was done to determine which pesticides might act as inducers, inhibitors, or substrates of amidase activity in this organism. The results of this research may aid in understanding some of the interactions between pesticides in the presence of microbial-degrading microorganisms and in preventing the formation of certain problem soils. MATERIALS
AND
METHODS
Chemicals. The common and chemical names, and the uses of pesticides included in this investigation are presented in Table 1. The following compounds were provided as gifts from the indicated sources: Propanil (99.1%), Rohm and Haas Co. (Philadelphia, PA); linuron (99%), diuron (95%), monuron (95%)) carbendazim, cymoxanil and thiram, DuPont de Nemours Co. (Wilmington, DE); metalaxyl (97.4%), Ciba Geigy
ACTIVITY
OF F. oxysporum
241
Corp. (Greensboro, NC); iprodione, RhonePoulenc (Lyon, France); carboxin, U.S. Rubber Co. (Naugatuk, CN). The following were provided by the Pesticide Degradation Laboratory, USDA (Beltsville, MD): CIPC (chlorpropham, 99%), PPG-124 (98%), Mobam (99.6%), propachlor (Monsanto, 99.9%), metolachlor (Ciba Geigy, 94.5%), alachlor (Monsanto, 98%), CDAA (Monsanto, 95%), bendiocarb (96%), sulfallate (Monsanto, 95%), EPTC (Stauffer, 98%), diallate (95%)) carbofuran (FMC , 95%)) diphenamid (Lilly, 96.5%) (pebulate, 97%), and metobromuron (99.6%). The following were provided by The U.S. Environmental Research Center (Research Triangle Park, NC): monolinuron (99.7%), chlorbromuron (98.9%), isoproturon (99.8%), metoxuron (99.3%), fluometuron (99.8%), chlortoluron (99.2%), dioxacarb (99.6%). Acetanilide, 3,4-dichlorophenylurea (3,4DCPU), 1,1,3,3-tetramethylurea, and lphenyl-2-thiourea were purchased from commercial sources. Stock solutions were prepared by dissolving the pesticides in acetone. Organisms and culture methods. F. oxysporum Schlecht, which was isolated from problem soils in which enhanced biodegradation of pesticides had been observed (14), was used as the test organism in this study. Stock cultures were grown on potato dextrose agar (PDA) at room temperature and then stored at 5°C as a source of inoculum for the liquid cultures. F. oxysporum cells for use in induction, substrate, and inhibition studies were grown in a liquid medium of the following composition: 0.2 g K,HP04, 0.6 g KH,PO,, 0.2 g NH4N03, 0.2 g MgSO, . 7H,O, 0.001 g FeSO, * 7H,O, 2.0 g sucrose, and 1.0 g yeast extract in 1 liter distilled H,O. The pH after autoclaving was 6.3. Cultures were initiated by transferring a fragment from a PDA culture into 100 ml of the liquid medium in a 250-ml Erlenmeyer flask and incubating the culture on a rotary
242
REICHEL,
SISLER,
AND
TABLE Common
and Chemical
Common name Alachlor Bendiocarb Carbendazim Carbofuran Carboxin CDAA Chlorbromuron Chlorpropham” Chlortoluron Cymoxanil Diallate Dioxacarb Diphenamid Diuron EPTC Flumeturon Iprodione Isoproturon Linuron Metalaxyl Metoxuron Metobromuron Metolachlor Mobam Monolinuron Monuron Pebulate PPG-124 Propachlor Propanil Sulfallate Thiram
Names
KAUFMAN
1
of Pesticides
Mentioned
in Text and Their
Chemical name 2-Chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide 2,2-Dimethyl-1,3-benzodioxol-4-yl methylcarbamate Methyl lH-benzimidazol-2-ylcarbamate 2,3-Dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate 5,6-Dihydro-2-methyl-N-phenyl1,4-oxathiin-3-carboxamide 2-ChIoro-N,N-di-a-propenylacetamide N’-(4-Bromo-3-chlorophenyl)-N-methoxy-N-methylurea I-Methylethyl-3-chlorophenylcarbamate N’-(3-chloro-4-methylphenyl)-N,N-dimethylurea 2-Cyano-N-[(ethylamino)carbonyl]-2-(methoxyi~no)acet~de S-(2,3-dichloro-2-propenyl) bis(l-methylethyl)carbamothioate O-l ,3-dioxolan-2-ylphenyl methylcarbamate N,N-dimethyl-a-phenylbenzeneacetamide N’-(3,4-dichlorophenyl)-N,N-dimethylurea S-Ethyl dipropylcarbamothioate N,N-dimethyl-N’-[3-(trifluoromethyl)phenyl]urea 3-(3,5-Dichlorophenyl)-N-(l-methylethyl)-2,4-dioxo-limidazolidinecarboximide N,N-Dimethyl-N’-[4-(1-methylethyl)phenyl]urea N’-(3,4-dichlorophenyl)-N-methoxy-N-methylurea Methyl N-(2,6-dimethylphenyl)-N-(2-methoxyacetyl)-DL-~~nate N’-(3-chloro-4-methoxyphenyl)N,N-dimethylurea N’-(4-bromophenyl)-N-methoxy-N-methylurea 2-Chloro-N-(2-ethyl-6methylphenyl)-N(2-methoxy-1-methylethyl)acetamide 4-Benzothienyl methylcarbamate N’-(4-chlorophenyl)-N-methoxy-N-methylurea N’-(4-chlorophenyl)-N, N-dimethylurea S-propyl butylethylcarbamothioate p-Chlorophenyl methylcarbamate 2-Chloro-N-(1-methylethyl)-N-phenylacetamide N-(3,4-dichlorophenyl)propanamide 2Chloroallyl diethyldithiocarbamate Tetramethylthioperoxydicarbonic diamide
Use
Use Herbicide Insecticide Fungicide Insecticide Fungicide Herbicide Herbicide Herbicide Herbicide Fungicide Herbicide Herbicide Herbicide Herbicide Herbicide Herbicide Fungicide Herbicide Herbicide Fungicide Herbicide Herbicide Herbicide Insecticide Herbicide Herbicide Herbicide Extender Herbicide Herbicide Herbicide Fungicide
n Referred to as CIPC in text.
shaker at 28°C for 3 days. Under these conditions F. oxysporum makes yeast-like growth consisting of microconidia and small hyphal fragments. For induction studies, 1 ml of this suspension was used to start new liquid cultures, which were then shaken for 3 days, at which time the inducer was added in acetone solution (1 ml or less acetone was added to 100 ml culture medium). After 3 days of additional growth with the inducer, conidia and hyphal fragments were collected by filtration through cheesecloth and
washed three times with distilled water and twice with 0.05 M phosphate buffer, pH 7.0. Control and induced cells were then resuspended in this buffer, and standardized to an optical density of 1.0 at 450 nm (0.7 mg fungal dry weight/ml). This suspension was used directly or concentrated or diluted to give the desired cell concentration (0.7, 1.4, or 0.07 mg fungal dry weight/ ml) for particular experiments. Measurement of amidase activity. A substrate in acetone solution was added to standardized suspensions of induced cells.
PROPANIL
DEGRADING
AMIDASE
The solvent did not exceed 1% in the final solution. Incubation was at 28°C on a rotary shaker. At various intervals, an aliquot of the suspension was removed and centrifuged and the supernatant used for colorimetric determination of aniline product released (15) as a measure of amidase activity. Duplicate samples were used for each measurement. Compounds tested as amidase inhibitors were added at the same time as the substrate in acetone solution. Preparation of crude cell-free extract. Induced or uninduced cells were grown in 2liter Erlenmeyer flasks containing 1 liter of medium under the same conditions described previously. After 6 days, cells were harvested by centrifugation at 4000g for 5 min at 5°C and rinsed twice in cold distilled water and twice in cold phosphate buffer (0.05 M, pH 7.0). The pellet was frozen in liquid nitrogen and subsequently lyophilized. Lyophilized material was stored at - 70°C. One gram of freeze-dried cells was homogenized at 4000 rpm during 2 min in 10 ml of cold homogenizing buffer (0.03 M Tris buffer, pH 80, containing 1 mM ethylenediaminetetraacetic acid and 0.5 mM DLdithiothreitol with 10 g of Pyrex glass beads (0.25 mm diameter), using a Braun cell homogenizer MSK cooled with a jet of CO*. After homogenization, 19 ml of homogenizing buffer were added and the homogenate was centrifuged at 10,OOOgfor 5 min at 4°C. The supernatant fraction was collected and a 1:l dilution was then made with the homogenizing buffer. Each milliliter of this solution contained the extract from 17.0 mg fungal dry weight. Aliquots of 1 ml were stored in glass vials at -70°C. Enzyme assays. For enzyme assays, crude cell-free extracts were thawed and brought to room temperature (26°C). In all tests using cell-free extracts from linuroninduced cells and propanil as a substrate, the amount of extract in the reaction mixture represented 0.34 mg/ml fungal dry
ACTIVITY
OF F. o.xysporum
243
weight. When acetanilide was used as a substrate, the extract represented 3.4 mg/ ml fungal dry weight. The substrate and/or inhibitor were added in acetone solution (volume not exceeding 1% in the final solution) to 3.0 ml of diluted cell-free extract. Appropriate solvent controls were included. The reaction mixture was incubated at 28°C and reactions were stopped by adding 5 ml of glacial acetic acid. K, determination. The K, of the linuroninduced amidase responsible for the degradation of propanil was determined using a cell-free extract from lyophilized cells. In this test, the extract represented 1.02 mg fungal weight, in a final volume of 3 .O ml of reaction mixture. Propanil was used as a substrate at four different final concentrations of 150 t&ml (6.9 x 1O-4 M), 100 pg/ml(4.6 x 10e4 M), 50 p&ml (2.3 x 10e4 M), and 25 pg/ml (1.1 x lop4 M). Amidase activity was measured after time intervals of 10,20, and 30 min at 28°C. The reactions were stopped by the addition of 5 ml of glacial acetic acid. For the calorimetric test, 0.4 ml of the reaction mixture were used. The K, was determined using a Lineweaver-Burk plot (16). Polyacrylamide gel electrophoresis. Gel electrophoresis was performed by the method of Davis (17) with a “Mighty Small” slab gel electrophoresis unit (Hoefer Scientific Instruments, San Francisco, CA), with 7.5% cross-linked gels (9). Samples (10 ~1) of crude cell-free extract representing 170 pg fungal dry weight were loaded in each slot, with 1 ~1 of sucrose dye solution. Electrophoresis was carried out at 5°C during 1 hr at a constant current of 10.0 mA. The gels were then sliced into l-mm segments and each segment was placed in 1 ml of the homogenizing buffer mentioned above. Propanil was added as a substrate in acetone solution. The final concentration of propanil was 100 kg/ml. After an incubation period of 90 min at 28°C 0.4 ml of the reaction mixture was used for 3,Cdichloro-
244
REICHEL,
SISLER, AND KAUFMAN
aniline (3,4-DCA) determination (15) to indicate relative amidase activity for each segment of gel. RESULTS
A number of amides, ureas, and carbamates (mainly pesticides) were tested as inducers of amidase activity in cells of F. oxysporum. Propanil at 100 pg/ml (0.46 mM) final concentration was the primary substrate used to assay the amidase activity; however, a number of other compounds were also tested as substrates of amidases in the induced cells. In standard assays using propanil as a substrate, the concentration of F. oxysporum cells was adjusted so that the rate of 3 ,CDCA released was linear during the measurement period which ranged from 0.5 to 4 hr in various experiments. Amide inducers. In propanil-induced cells, amidase activity resulting in propanil breakdown released an average of 16.7 kmol of 3,4-DCA per gram fungal dry weight per hour (Table 2). Uninduced cells had an amidase activity less than 0.6 pmol TABLE Induction oxysporum
Inducer (mM) None Propanil (0.11) Acetanilide (0.19) None Acetanilide (0.19) Propanil (0.11)
2
of Amidase
Activity in Fusarium Cells by Propanil and Acetanilide
Substrate (mM)
Amidase activity”
Propanil (0.46)
0
Propanil (0.46)
16.7
Propanil (0.46) Acetanilide (0.74)
0 71.0
Acetanilide (0.74)
55.9
Acetanilide (0.74)
51.6
a Micromoles of 3,4-DCA released from propanil per gram fungal dry weight per hour. Activity of less than 0.6 recorded as zero. ’ Micromoles of aniline released from acetanilide per gram fungal dry weight per hour. ’ Values are the means of duplicate cultures from two experiments.
of 3,4-DCA released from propanil per gram fungal dry weight per hour during the first hour, which was recorded as zero. In contrast to the lack of amidase activity in uninduced cells with propanil as a substrate, these cells showed appreciable amidase activity with acetanilide as a substrate (Table 2). This activity was not improved if cells had been induced for 3 days with 25 pg/ml (0.19 m&f) acetanilide or with 25 kg/ml of propanil (0.11 mA4). Surprisingly, cells induced with acetanilide did not show activity toward propanil. Propachlor (0.12 m&f), carboxin (0.11 mM), iprodione (0.08 mM), metalaxyl (0.36 mM), diphenamid (0.10 n-&f), and cymoxanil (0.13 mM) did not induce amidase activity for the degradation of propanil or acetanilide . Structure of amide compounds tested as inducers of propanil-degrading amidase activity are shown in Fig. 1. Urea inducers. Urea compounds which were tested as inducers of propanil-degrading amidase activity in F. oxysporum are shown in Fig. 2. Phenylureas were the most effective group of compounds tested as inducers of amidase activity for degradation of propanil. In two experiments, monolinuron-induced cells released an average of 1346 bmol (218 mg) of 3 ,CDCA from propanil per gram of fungal dry weight per hour (Table 3). In four experiments, linuroninduced cells released an average of 1111 pmol (180 mg) of 3,4-DCA from propanil per gram fungal dry weight per hour (Table 3). The rates of propanil degradation by monolinuron and linuron-induced cells were, respectively, 81 and 67 times that of propanil-induced cells (Tables 2 and 3). The propanil degraded by these cells in 6 hr was greater than the cell dry weight. The phenylurea compounds metabromuron, metoxuron, and chlorbromuron (Fig. 2) were moderately strong inducers of the amidase but much inferior to monolinuron or linuron (Table 3). Diuron and monuron
PROPANIL
DEGRADING
AMIDASE
ACTIVITY
Cl PROPANIL
Cl
MONOLINURON
ACETANILIDE
0
245
OF F. oxysporum
Br
LINURON
METOBROMURON
CH,
NH-%
PROPACHLOR
Cl
CH,O CAABOXIN
METOXURON
0 NH-&d
CHLORBROMURON
CH,
DIURON 0 NH-&d
CH, ‘CH,
Cl
Cl IPRODIONE
/\ Q-HC-C-N9 FH3
H8H8
ii-O-CH,
FIG. 1. Structures of amide inducers of propanil-degrading oxysporum cells.
Cl-$’
were the least active of the urea inducers of propanil-degrading amidase activity, but were better inducers than propanil. The following urea compounds did not induce amidase activity for the degradation of propanil: fluometuron (0.10 m&f), 3,4-dichlorophenylurea (3,bDCPU, 0.12 mM), and 1,1,3,3-tetramethylurea (0.21 mM). Since linuron was a good inducer of propanil-degrading amidase activity and was readily available in a high purity form, it was used as the primary inducer in these studies. Carbamate inducers. Mobam, CIPC (chlorpropham), and diallate (Fig. 3) were fair inducers of propanil-degrading amidase activity with respective values of 26.5, 13.0, and 12.3 pmol of 3,CDCA released from propanil per gram fungal dry weight per hour (Table 4). Bendiocarb, EPTC (Eptam), and carbofuran at 25 pg/ml (112-
\
N- 8 -N,
, CH, CH3
1,?.3,3,TETRAMETHYLUR)EA
CYMOXANIL
compounds tested as amidase activity in F.
FLUOMETURON
3.4,DCPU
CH,
C,H,-N-C-N-C-C-C:N
‘CH,
DIPHENAMID
MONURON
METALAXYL
2. Structures of urea compounds duction of propanil-degrading amidase oxysporum cells. FIG.
tested for inactivity in F.
132 $I@ final concentration or carbendazim at 8 t&ml (42 p&f) did not induce amidase activity for the breakdown of propanil. Although induction of propanil-degrading amidase activity by PPG-124 in F. oxysporum was reported by Blake et al. (14), amidase induction in these studies could not be demonstrated for PPG-124 at final concentrations of 134, 67, 5.4 and 0.54 pi%!. PPG124 is a potent inhibitor of amidase activity in F. oxysporum (14); therefore, it was presumed that an amidase induced by this compound might not be detected because of the inhibitory action of the inducer (18, 19). This possibility was indicated by experiments in which linuron-induced cells were treated with 54 t.& (10 p&ml) of PPG124 for 1 hr and then washed four times in phosphate buffer (0.05 M, pH 7.0). The cells were then assayed for propanildegrading amidase activity at various inter-
246
REICHEL.
SISLER,
AND
KAUFMAN
TABLE 3 Activity of Ureas as Inducers of Propanil-Degrading Amidase Activity in Fusarium oqsporum Cells Inducer (mM)
Amidase activity”
Relative inductive activityb
Monolinuron (0.11) Linuron (0.10) Metobromuron (0.10) Metoxuron (0.11) Chlorbromuron (0.09) Diuron (0.17) Monuron (0.11) None
1346 1111 130 123 99 49 19 0
81.0 67.0 7.8 7.4 6.0 2.9 1.1 0
u Micromoles of 3,4-DCA released from propanil per gram fungal dry weight per hour. Activity of less than 0.6 recorded as zero. Values for monolinuron are the mean of two experiments; values for the remaining compounds are the mean of four experiments. b Relative inductive activity with respect to propanil.
vals thereafter. When assayed after a I-hr incubation at 28°C amidase activity was completely absent. However, when these cells were washed two additional times in phosphate buffer and incubated in buffer at 15°C for 16 and 25 hr, with stirring, there was a 20% recovery of amidase activity after 16 hr and a 30% recovery after 25 hr. Substrates. None of the urea compounds tested as inducers (Fig. 2) were substrates of the linuron-induced propanil-degrading amidase as indicated by lack of measurable release of an aniline product. In addition to propanil, acetanilide was a good substrate for the amidase(s) induced by two methoxy-substituted phenylurea compounds, monolinuron, and linuron (Table 5). In one experiment, monolinuroninduced cells degraded acetanilide with an amidase activity of 301 kmol of aniline released per gram fungal dry weight per hour. In three experiments, acetanilide (0.74 mA4 final concentration) was degraded by linuron-induced cells with an average amidase activity of 699 pmol of aniline released per gram fungal dry weight per hour. Induction with the two methoxy urea compounds, monolinuron and linuron, mark-
0 NH-Z-O-dH
0
CH3
‘CH, Cl CIPC
KH,l,CH
0 ‘N-&SCH,CCl=CHCI
(cH,l,c~’ DIALLATE PPG- 124 OH Q-6N-CH3 CH,-CH,-S-C-N’
9
CHz-CHiCH, ‘cH,-CH,-CH,
CARBOFURAN
EPTC
0
BENDIOCARB
FIG. 3. Structures of some carbamate compounds tested as inducers of amidase activity for the degradation of propanil in F. oxysporum cells.
edly increased acetanilide-degrading activity over that present in uninduced cells. Other ureas such as diuron and monuron, which induced some propanil-degrading amidase activity, did not induce detectable increases in activity for acetanilide degraTABLE 4 Activity of Carbamates as Inducers of Propanil-Degrading Amidase Activity in Fusarium oxysporum Cells Inducer (mM)
Amidase activity”
Relative inductive activityb
Mobam (0.12) CIPC (0.12) Diallate (0.09) PPG-124 (0.07)
26.5 13.0 12.3 0
1.6 0.8 0.7 0
a Micromoles of 3,4-DCA released from propanil per gram fungal dry weight per hour. b Relative to that of propanil.
PROPANIL
DEGRADING
AMIDASE
TABLE 5 Acetanilide and CIPC as Substrates of Linuron or Mono&won-Induced Amidase in Cells of Fusarium oxysporum Inducer WW
Substrate (mM)
Amidase activity
None Linuron (0.1) Monolinuron (0.1) None Linuron (0.1)
Acetanilide (0.74)
70.0”
Acetanilide (0.74)
699.0
Acetanilide (0.74) CIPC (0.47)
301.0 Ob
a Micromoles per gram fungal b Micromoles per gram fungal
CIPC (0.47)
13.3
of aniline released from acetanilide dry weight per hour. of 3-chloroaniline released from CIPC dry weight per hour.
dation above that present in uninduced cells. The carbamate compound CIPC was a substrate for linuron-induced cells and was degraded with an amidase activity of 13.3 pmol of 3chloroaniline released per gram fungal dry weight per hour. Uninduced cells did not degrade CIPC (Table 5). Propanil was the only substrate degraded by the amidase induced by carbamates. Cells induced by the carbamates PPG-124, mobam, CIPC, bendiocarb, carbofuran, diallate, or carbendazim did not show amidase activity with linuron, monuron, diuron, and CIPC or increased amidase activity with acetanilide above that present in uninduced cells. Amidase activity
of cell-free extracts.
Duplicate samples from 12 experiments using three different sets of linuron-induced lyophilized cell gave an average rate of propanil-degrading amidase activity of 1889 p.mol3,4-DCA released per hour by extract representing 1 g of fungal dry weight. This rate of propanil degradation by cell-free extracts was somewhat greater than that by whole cells (Table 3). The data from duplicate samples of three experiments from two different sets of linuron-induced lyophilized cells in which pro-
ACTIVITY
OF F. oqsporum
247
panil and acetanilide were both included as substrates showed that the mean rate of propanil degradation by extract representing 1 g of fungal dry weight was 1549 pmol of 3,CDCA released per hour compared to 279 p.mol of aniline released from acetanilide per hour. In contrast to propanil, the rate of acetanilide degradation was more rapid in whole cells (Table 5) than in these cell-free extracts. Polyacrylamide gel electrophoresis. Proteins of a cell-free extract from linuroninduced cells were separated by a nondenaturing acrylamide gel electrophoresis (17). A lo-p.1 sample of cell-free extract from linuron-induced cells (representing 170 pg fungal dry weight) was loaded in each slot of the gel with 1 ~1 of sucrose dye solution (17). Electrophoresis was carried out at 5°C during 1 hr at a constant current of 10 mA. After separation, the gel was sliced into lmm segments and each segment was placed in 1 ml of homogenizing buffer. Propanil was added as a substrate to give a final concentration of 100 l&ml. After an incubation period of 90 min at 28°C 0.4 ml of the reaction mixture was analyzed for 3,4-DCA as a measure of amidase activity. In two experiments in which propanil was used as a substrate, there was one major band of activity occurring between 17 and 24 mm from the origin (Fig. 4). The minor activity that preceded the strong band may have been due to one or more bands of weak activity, or to trailing of the major band. When acetanilide was used as a substrate (final concentration, 100 pg/ml), amidase activity occurred in the same region as the strong propanil band (17-24 mm), but activity was weak in comparison with that when propanil was used as a substrate. K,,, determination. A Lineweaver-Burk plot was done using the amidase in a cellfree extract of linuron-induced cells and propanil as a substrate. The K, was estimated to be 7.1 x lo-’ M. Amide inhibitors. The following amide compounds were tested as inhibitors of
REICHEL,
SISLER, AND KAUFMAN TABLE
6
Urea Inhibitors of Linuron-Induced, Propanil-Degrading Amidase Activity in Whole Cells and Cell-Free Extracts of Fusarium oxysporum
% Inhibition at indicated concentration Whole cells
FIG. 4. Amidase activity (3,4-DCA released from propanil) after separation of proteins in a cell-free extract from linuron-induced cells by acrylamide gel electrophoresis. Activity was determined by assay of l-mm segments of gel between origin and front (40 mm). Amidase activity is expressed in umol of 3,4DCA x 100190 min.
linuron-induced, propanil-degrading amidase activity in cells of F. oxysporum, and were found to be ineffective at the indicated concentration (PM): propachlor (250), alachlor (190), metolachlor (180), CDAA (280), diphenamid (170), and metalaxyl (180). Only the amide cymoxanil with an IDS, value of 210 FM, was inhibitory to propanil degradation. In cell-free extracts of linuron-induced cells, cymoxanil was inhibitory to propanil degradation with an ID,, value of 90 pM, which was about 2.3 times lower than that for whole cells. Urea inhibitors. All the urea compounds which were inducers of propanil-degrading amidase activity were also inhibitors of the induced amidase activity in whole cells and cell-free extracts (Table 6). Linuron, diuron, monuron, and metoxuron gave somewhat lower IDSa values in cell-free extracts than in whole cells. Among these urea compounds, linuron and diuron were the most potent inhibitors in the cell-free extracts. The following ureas gave the indicated ID,, values (k&f) in cell-free extracts: isoproturon, 6.3; chlortoluron, 18; and fluometuron, 140. Isoproturon ranked with diuron and linuron (Table 6) as the most potent urea inhibitors of amidase activity in cell-free extracts. Other urea compounds tested were es-
Cell-free extracts
Inhibitor
%
PM
%
PM
Linuron Diuron Monouron Metoxuron Monolinuron Chlorbromuron Metobromuron
50 50 50 50 93 85 73
19 20 IO 130 140 loo 110
50 50 50 50 50 50 50
12.0 4.5 48.0 87.0 57.0 77.0 22.0
sentially ineffective as inhibitors. 1-Phenyl-Zthiourea showed a 5% inhibition of the amidase activity in the whole cells at 330 PM. Phenylurea, 3,4-DCPU, and 1,1,3,3-tetramethylurea did not inhibit the amidase activity in whole cells at respective concentrations of 370, 240 and 860 pM. Carbamate inhibitors. Some of the carbamates tested as inhibitors of linuroninduced, propanil-degrading amidase activity are listed in Table 7. The carbamates PPG-124 and mobam were powerful inhibitors of linuron-induced amidase activity in both whole cells and cell-free extracts of F. oxysporum (Table 7). The remaining carbamates were relatively poor inhibitors of the amidase activity both in whole cells and cell-free extracts. PPG-124 was the most potent inhibitor of amidase activity tested, with an ID,, value in the whole cells of 0.33 PM compared to 19 and 20 PM for linuron and diuron, the most potent urea inhibitors (Tables 6 and 7). DISCUSSION
This study showed that linuron, monolinuron, and several other phenylureas are far superior to propanil as inducers of a propanil-degrading amidase in F. oxysporum. In respect to amidase induction, F. oxyspo-
PROPANIL
TABLE
DEGRADING
AMIDASE
7
Carbarnate Inhibitors of Linuron-Znduced, Propanil-Degrading Amidase Activity in Cells and Cell-Free Extracts of Fusarium oxysporum
% Inhibition at indicated concentration Cell-free extracts
Whole cells Inhibitor
%
CLM
%
CLM
PPG-124 Mobam Bendiocarb Dioxacarb Diallate Carbendazim CIPC EPTC Pebulate SulfalIate Thiram Carbofuran
50 50 40 52 50 13 18 7 7 4 7 0
0.33 4.4 180.0 130.0 500.0 40.0 230.0 260.0 200.0 180.0 120.0 230.0
50 50 50 10 13
0.2 3.6 480.0 90.0 370.0 -a 530.0 90.0 450.0
24 8 11
0 Not tested.
rum responds like Bacillus sphaericus isolated by Engelhardt et al. (9) from problem soils. In this bacterium, as in F. oxysporum, linuron was more effective as an inducer of acylamidase activity than propanil (10). Moreover, the two organisms were similar in that phenylureas, phenylamides, and phenylcarbamates all induced the amidase activity. Although phenylamides of the secondary amine type were the preferred substrate of the amidase(s) in both organisms, the enzyme(s) of F. oxysporum was not observed to degrade methoxy-substituted phenylureas like linuron and monolinuron; whereas the enzyme from B. sphaericus did degrade these ureas (9). However, these methoxysubstituted phenylureas were extremely poor substrates for the B. sphaericus enzyme in comparison to the better phenylamide substrates. For example, the specific activity of the B. sphaericus amidase for degradation of the phenylamide, propanil, was 1245 times higher than for the methoxy-substituted phenylureas, linuron and monolinuron which were the best urea substrates tested (9, 10).
ACTIVITY
OF F. oxysporum
249
The present study shows that propanil induces amidase activity in F. oxysporum for its own breakdown but not for the degradation of acetanilide. Likewise, acetanilide does not induce amidase activity for the breakdown of propanil (thus different enzymes seem to be involved here). Apparently F. oxysporum has a low level of constitutive enzyme which degrades acetanilide. This finding resembles that reported by Lanzilotta and Pramer (20) in which uninduced cells of F. solani showed amidase activity for the degradation of acetanilide. Although induction of amidase activity by PPG-124 for the degradation of propanil by F. oxysporum has been reported (14), it could not be demonstrated in the present study. PPG-124 might induce a propanildegrading amidase, which it inhibits temporarily as is characteristic of some carbamate inhibitors which carbamylate enzymes (18, 19). Therefore, failure to detect amidase activity in the present study might not have been due to a lack of induction by PPG-124 but due rather to a failure of the PPG-124-inhibited enzyme to recover activity by decarbamylation. A somewhat higher rate of propanil degradation was demonstrated in cell-free extracts of linuron-induced cells of F. oxysporum than in linuron-induced whole cells. This suggests that the rate of degradation in whole cells might be limited by the rate of propanil penetration into the cells. On the other hand, the higher rate of acetanilide degradation in linuron-induced whole cells as compared to cell-free extracts might be explained by loss of enzyme activity during preparation of the cell-free extracts. Acetanilide degradation by linuron-induced whole cells probably involves a constitutive enzyme acting only on acetanilide and a linuron-induced enzyme acting on both acetanilide and propanil. The K, determined for propanil with linuron-induced amidase in this study was 7.1 x 10e5 M. This compares favorably to a value of 2.3 x 10T5 A4 determined by Blake et al. (14) for the propanil-induced
250
REICHEL,
SISLER,
enzyme in this organism. The linuron and the propanil-induced enzymes are believed to be the same, but further studies with purified enzymes would be required to determine this. The observations in the present study and those by Engelhardt et al. (10) in studies with B. sphaericus that methoxysubstituted phenylurea herbicides are powerful inducers of amidase acting on phenylamides suggest that this phenomenon may occur widely among soil microorganisms degrading soil applied pesticides. Thus, caution should be exercised when phenylamide and methoxy-substituted phenylurea herbicides are applied together in the soil. Whether induction of amidases by such phenylureas would lead to long-term enrichment of these amidase producing microorganisms in soil is unknown.
AND
KAUFMAN
5. L. J. Audus, The biological detoxification of hormone herbicides in soil, Plant Sod 3, 170 (1951). 6. H. D. Sisler, Biodegradation of agricultural fungicides, in “Biodegradation of Pesticides” (F. MatsumuraandC. R. K. Murti, Eds.), pp. 133155, Plenum, New York, 1982. 7. P. Wallnofer, The decomposition of urea herbicides by Bacillus sphaericus, isolated from soil, Weed Res. 9, 333 (1969). 8. P. R. Wallnofer and J. Bader, Degradation of urea herbicides by cell-free extracts of Bacillus sphaericus,
Appl.
Microbial.
19, 714 (1970).
9. G. Engelhardt, P. R. Wallnbfer, and R. Plapp, Degradation of linuron and some other herbicides and fungicides by a linuron-inducible enzyme obtained from Bacillus sphaericus, Appl. Microbial. 22, 284 (1971). 10. G. Engelhardt, P. R. Wallnofer, and R. Plapp, Purification and properties of an aryl acylarnidase of Bacillus sphaericus, catalyzing the hydrolysis of various phenylamide herbicides and fungicides, Appl. Microbial. 26, 709 (1973). 11. R. Bartha and D. Pramer, Pesticide transformation to aniline and azo compounds in soil, Science 156, 1617(1%7).
ACKNOWLEDGMENTS
We thank M. L. Gullino and T. Chida for their assistance in various aspects of this study. We thank J. E. Kurent for his interest and support. REFERENCES
D. D. Kaufman, Y. Katan, D. F. Edwards, and E. G. Jordan, Microbial adaptation and metabolism of pesticides, in “Beltsville Symposia in Agricultural Research. Agricultural Chemicals of the Future” (J. L. Hilton, Ed.), Vol. 8, pp. 437-451, Rowman and Allanheld, Totawa, NJ, 1983. 2. H. Geissbuhler, The substituted ureas, in “Degradation of Herbicides” (P. C. Kearney and D. D. Kaufman, Eds.), pp. 79-l 11, Marcel Dekker, New York, 1969. 3. F. M. Ashton, Persistence and biodegradation of herbicides, in “Biodegradation of Pesticides” (F. Matsumura and C. R. K. Murti, Eds.), pp. 117-131. Plenum Press, New York, 1982. 4. D. D. Kaufman and D. F. Edwards, Pesticide/ microbe interaction effects on persistence of pesticides in soil, in “Pesticide Chemistry, Human Welfare and The Environment. Pesticide Residues and Formulation Chemistry” (J. Miyamoto and P. C. Kearney, Eds.), Vol. 4, pp. 177-182, Pergamon, New York, 1983. 1.
12. R. P. Lanzilotta and D. Pramer, Herbicide transformation. I. Studies with whole cells of Fusarium sold,
Appl.
Microbial.
19, 301 (1970).
D. D. Kaufman and J. Blake, Microbial degradation of several acetamide, acylanilide, carbamate, toluidine and urea pesticides, Soil Biol. Biochem. 5, 297 (1973). 14. J. Blake and D. D. Kaufman, Characterization of acylanilide-hydrolyzing enzyme(s) from Fusarium oxysporum Schlecht, Pestic. Biochem. Physiol. 5, 305 (1975). 1.5. H. L. Pease, Separation and calorimetric determination of monuron and diuron residues, J. Ag13.
ric.
Food
Chem.
10, 279 (1962).
16. I. H. Segel, “Biochemical Calculations,” 2nd ed., p. 234, Wiley, New York, 1968. 17. B. J. Davis, Disc electrophoresis. II. Method and application to human serum proteins. Ann N. Y. Acad.
Sci. 121, 404 (1964).
R. D. O’Brien, “Insecticides. Action and Metabolism,” p. 88, Academic Press, New York, 1967. 19. K. A. Hassall, “The Chemistry of Pesticides. Their Metabolism, Mode of Action and Uses in Crop Protection,” p. 103, Verlag Chemie, Weinheim, 1982. 20. R. P. Lanzilotta and D. Pramer, Herbicide transformation. II. Studies with an acylamidase of 18.
Fusarium
(1970).
solani,
Appl.
Microbial.
19,
307
BIOCHEMISTRY
AND
PHYSIOLOGY
39, 240-250 (1991)
Inducers, Substrates, and Inhibitors of a Propanil-Degrading Amidase of Fusarium oxysporum1s2 HELENAREICHEL, **3 H.D. *Department
of Botany,
iFSoit Microbial
University
SISLER,* AND D.D.
of Maryland, USDA,
Systems Laboratory,
College Park, ARS, Bebville,
KAUFMAN? Maryland Maryland
20742, and 20705
Received August 28, 1990; accepted November 13, 1990 Ureas, carbamates, and amides were investigated as inducers, substrates, and inhibitors of propanil-degrading amidase activity in Fusarium oxysporum. Urea herbicides were potent inducers but not substrates of the amidase that degrades propanil. Linuron-induced cells degraded propanil approximately 67 times more rapidly than propanil-induced ceils. Noninduced cells did not degrade propanil. All urea inducers were also inhibitors of the linuron-induced propanil-degrading amidase activity in whole cells and cell-free extracts. The urea inducers linuron, diuron, and monuron gave respective ID, values of 19, 20 and 70 FM as inhibitors of amidase degradation of propanil by whole cells. The respective ID,, values for degradation by cell-free extracts were 12.0, 4.5 and 48 PM. The carbamates PPG-124 (p-chlorophenyl methylcarbamate) and mobam were powerful inhibitors of the amidase activity with respective ID,, values in whole cells of 0.33 and 4.4 FM. Respective values for cell-free extracts were 0.2 and 3.6 )LM. Mobam, CIPC (chlorpropham), and diallate were fair inducers of the amidase activity, but amidase induction by PPG-124 could not be demonstrated, possibly because it inactivated the enzyme. Acetanilide did not induce amidase activity for the breakdown of propanil. Likewise, propanil did not induce amidase activity for the breakdown of acetanilide. Different enzymes seem to be involved here. The K, for the linuron-induced, propanil-degrading amidase in cell-free extracts from freeze-dried cells was 7.1 x lo-‘M.
INTRODUCTION
Low pesticide efficacy and failure to control the target pest have been observed in soils in which pesticide has been applied frequently. Problem soils of this type have also been observed when different pesticides with a similar chemical structure have been applied (1). The involvement of microbial activity in the development of problem soils has been indicated by autoclaving (2) and by the use of antibiotics or y irradiation (3). Studies have shown that some pesticides ’ This research was supported by a grant from The National Pesticide Impact Assessment Program (NAPIAP), Cooperative States Research Service, USDA. * Scientific Publication 6064, Contribution 8226 of The University of Maryland Agricultural Experiment Station. 3 Present address: Centro Intemacional de Agricultura Tropical (CIAT), Apartado Aereo No. 6713, Cali, Colombia, SA. 240 0048-3575/91 $3.00
may act as inducers (or inhibitors) of enzymes in soil microorganisms for their own breakdown, as well as that of other pesticides (4). This ability of microorganisms to degrade pesticide molecules after adaptation to an original similar structure was first demonstrated by Audus in 1951 (5). In some cases, fungicides may affect the breakdown of other pesticides by inhibiting growth or enzyme activity of microorganisms involved in the biodegradation process (6).
Among the enzymes involved in pesticide degradation in the soil are the amidases produced by fungi and bacteria. Wallndfer (7) reported the degradation of urea herbicides by Bacillus sphaericus isolated from the soil. An amidase responsible for the breakdown of urea herbicides by this organism was described soon thereafter (8). The urea herbicide linuron induced amidase activity in B. sphaericus for the breakdown of linuron itself, monolinuron, and meto-
PROPANIL
DEGRADING
AMIDASE
bromuron. Working with this organism, Engelhardt et al. (9) demonstrated a low substrate specificity of this linuron-induced amidase. A large variety of herbicides and fungicides of the acylanilide and methoxysubstituted phenylurea type were degraded by this enzyme. Acylanilides were degraded at a rate at least 10 times higher than the methoxy-substituted phenylureas. This same amidase was found to have powerful activity for the breakdown of the amide herbicide propanil. However, propanil was not a good inducer of this linuron degrading amidase (10). The microbial transformation of propanil to the corresponding aniline and the disappearance of the parent compound had already been reported by Bartha et al., in 1967 (11). Subsequently, several microorganisms capable of degrading propanil were isolated from soils, among them Fusarium solani and Fusarium oxysporum (12, 13). In 1975, Blake and Kaufman demonstrated that propanil induces an acylamidase in F. oxysporum for its own degradation (14). Since F. oxysporum has been reported to occur in various problem soils, the following study was done to determine which pesticides might act as inducers, inhibitors, or substrates of amidase activity in this organism. The results of this research may aid in understanding some of the interactions between pesticides in the presence of microbial-degrading microorganisms and in preventing the formation of certain problem soils. MATERIALS
AND
METHODS
Chemicals. The common and chemical names, and the uses of pesticides included in this investigation are presented in Table 1. The following compounds were provided as gifts from the indicated sources: Propanil (99.1%), Rohm and Haas Co. (Philadelphia, PA); linuron (99%), diuron (95%), monuron (95%)) carbendazim, cymoxanil and thiram, DuPont de Nemours Co. (Wilmington, DE); metalaxyl (97.4%), Ciba Geigy
ACTIVITY
OF F. oxysporum
241
Corp. (Greensboro, NC); iprodione, RhonePoulenc (Lyon, France); carboxin, U.S. Rubber Co. (Naugatuk, CN). The following were provided by the Pesticide Degradation Laboratory, USDA (Beltsville, MD): CIPC (chlorpropham, 99%), PPG-124 (98%), Mobam (99.6%), propachlor (Monsanto, 99.9%), metolachlor (Ciba Geigy, 94.5%), alachlor (Monsanto, 98%), CDAA (Monsanto, 95%), bendiocarb (96%), sulfallate (Monsanto, 95%), EPTC (Stauffer, 98%), diallate (95%)) carbofuran (FMC , 95%)) diphenamid (Lilly, 96.5%) (pebulate, 97%), and metobromuron (99.6%). The following were provided by The U.S. Environmental Research Center (Research Triangle Park, NC): monolinuron (99.7%), chlorbromuron (98.9%), isoproturon (99.8%), metoxuron (99.3%), fluometuron (99.8%), chlortoluron (99.2%), dioxacarb (99.6%). Acetanilide, 3,4-dichlorophenylurea (3,4DCPU), 1,1,3,3-tetramethylurea, and lphenyl-2-thiourea were purchased from commercial sources. Stock solutions were prepared by dissolving the pesticides in acetone. Organisms and culture methods. F. oxysporum Schlecht, which was isolated from problem soils in which enhanced biodegradation of pesticides had been observed (14), was used as the test organism in this study. Stock cultures were grown on potato dextrose agar (PDA) at room temperature and then stored at 5°C as a source of inoculum for the liquid cultures. F. oxysporum cells for use in induction, substrate, and inhibition studies were grown in a liquid medium of the following composition: 0.2 g K,HP04, 0.6 g KH,PO,, 0.2 g NH4N03, 0.2 g MgSO, . 7H,O, 0.001 g FeSO, * 7H,O, 2.0 g sucrose, and 1.0 g yeast extract in 1 liter distilled H,O. The pH after autoclaving was 6.3. Cultures were initiated by transferring a fragment from a PDA culture into 100 ml of the liquid medium in a 250-ml Erlenmeyer flask and incubating the culture on a rotary
242
REICHEL,
SISLER,
AND
TABLE Common
and Chemical
Common name Alachlor Bendiocarb Carbendazim Carbofuran Carboxin CDAA Chlorbromuron Chlorpropham” Chlortoluron Cymoxanil Diallate Dioxacarb Diphenamid Diuron EPTC Flumeturon Iprodione Isoproturon Linuron Metalaxyl Metoxuron Metobromuron Metolachlor Mobam Monolinuron Monuron Pebulate PPG-124 Propachlor Propanil Sulfallate Thiram
Names
KAUFMAN
1
of Pesticides
Mentioned
in Text and Their
Chemical name 2-Chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide 2,2-Dimethyl-1,3-benzodioxol-4-yl methylcarbamate Methyl lH-benzimidazol-2-ylcarbamate 2,3-Dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate 5,6-Dihydro-2-methyl-N-phenyl1,4-oxathiin-3-carboxamide 2-ChIoro-N,N-di-a-propenylacetamide N’-(4-Bromo-3-chlorophenyl)-N-methoxy-N-methylurea I-Methylethyl-3-chlorophenylcarbamate N’-(3-chloro-4-methylphenyl)-N,N-dimethylurea 2-Cyano-N-[(ethylamino)carbonyl]-2-(methoxyi~no)acet~de S-(2,3-dichloro-2-propenyl) bis(l-methylethyl)carbamothioate O-l ,3-dioxolan-2-ylphenyl methylcarbamate N,N-dimethyl-a-phenylbenzeneacetamide N’-(3,4-dichlorophenyl)-N,N-dimethylurea S-Ethyl dipropylcarbamothioate N,N-dimethyl-N’-[3-(trifluoromethyl)phenyl]urea 3-(3,5-Dichlorophenyl)-N-(l-methylethyl)-2,4-dioxo-limidazolidinecarboximide N,N-Dimethyl-N’-[4-(1-methylethyl)phenyl]urea N’-(3,4-dichlorophenyl)-N-methoxy-N-methylurea Methyl N-(2,6-dimethylphenyl)-N-(2-methoxyacetyl)-DL-~~nate N’-(3-chloro-4-methoxyphenyl)N,N-dimethylurea N’-(4-bromophenyl)-N-methoxy-N-methylurea 2-Chloro-N-(2-ethyl-6methylphenyl)-N(2-methoxy-1-methylethyl)acetamide 4-Benzothienyl methylcarbamate N’-(4-chlorophenyl)-N-methoxy-N-methylurea N’-(4-chlorophenyl)-N, N-dimethylurea S-propyl butylethylcarbamothioate p-Chlorophenyl methylcarbamate 2-Chloro-N-(1-methylethyl)-N-phenylacetamide N-(3,4-dichlorophenyl)propanamide 2Chloroallyl diethyldithiocarbamate Tetramethylthioperoxydicarbonic diamide
Use
Use Herbicide Insecticide Fungicide Insecticide Fungicide Herbicide Herbicide Herbicide Herbicide Fungicide Herbicide Herbicide Herbicide Herbicide Herbicide Herbicide Fungicide Herbicide Herbicide Fungicide Herbicide Herbicide Herbicide Insecticide Herbicide Herbicide Herbicide Extender Herbicide Herbicide Herbicide Fungicide
n Referred to as CIPC in text.
shaker at 28°C for 3 days. Under these conditions F. oxysporum makes yeast-like growth consisting of microconidia and small hyphal fragments. For induction studies, 1 ml of this suspension was used to start new liquid cultures, which were then shaken for 3 days, at which time the inducer was added in acetone solution (1 ml or less acetone was added to 100 ml culture medium). After 3 days of additional growth with the inducer, conidia and hyphal fragments were collected by filtration through cheesecloth and
washed three times with distilled water and twice with 0.05 M phosphate buffer, pH 7.0. Control and induced cells were then resuspended in this buffer, and standardized to an optical density of 1.0 at 450 nm (0.7 mg fungal dry weight/ml). This suspension was used directly or concentrated or diluted to give the desired cell concentration (0.7, 1.4, or 0.07 mg fungal dry weight/ ml) for particular experiments. Measurement of amidase activity. A substrate in acetone solution was added to standardized suspensions of induced cells.
PROPANIL
DEGRADING
AMIDASE
The solvent did not exceed 1% in the final solution. Incubation was at 28°C on a rotary shaker. At various intervals, an aliquot of the suspension was removed and centrifuged and the supernatant used for colorimetric determination of aniline product released (15) as a measure of amidase activity. Duplicate samples were used for each measurement. Compounds tested as amidase inhibitors were added at the same time as the substrate in acetone solution. Preparation of crude cell-free extract. Induced or uninduced cells were grown in 2liter Erlenmeyer flasks containing 1 liter of medium under the same conditions described previously. After 6 days, cells were harvested by centrifugation at 4000g for 5 min at 5°C and rinsed twice in cold distilled water and twice in cold phosphate buffer (0.05 M, pH 7.0). The pellet was frozen in liquid nitrogen and subsequently lyophilized. Lyophilized material was stored at - 70°C. One gram of freeze-dried cells was homogenized at 4000 rpm during 2 min in 10 ml of cold homogenizing buffer (0.03 M Tris buffer, pH 80, containing 1 mM ethylenediaminetetraacetic acid and 0.5 mM DLdithiothreitol with 10 g of Pyrex glass beads (0.25 mm diameter), using a Braun cell homogenizer MSK cooled with a jet of CO*. After homogenization, 19 ml of homogenizing buffer were added and the homogenate was centrifuged at 10,OOOgfor 5 min at 4°C. The supernatant fraction was collected and a 1:l dilution was then made with the homogenizing buffer. Each milliliter of this solution contained the extract from 17.0 mg fungal dry weight. Aliquots of 1 ml were stored in glass vials at -70°C. Enzyme assays. For enzyme assays, crude cell-free extracts were thawed and brought to room temperature (26°C). In all tests using cell-free extracts from linuroninduced cells and propanil as a substrate, the amount of extract in the reaction mixture represented 0.34 mg/ml fungal dry
ACTIVITY
OF F. o.xysporum
243
weight. When acetanilide was used as a substrate, the extract represented 3.4 mg/ ml fungal dry weight. The substrate and/or inhibitor were added in acetone solution (volume not exceeding 1% in the final solution) to 3.0 ml of diluted cell-free extract. Appropriate solvent controls were included. The reaction mixture was incubated at 28°C and reactions were stopped by adding 5 ml of glacial acetic acid. K, determination. The K, of the linuroninduced amidase responsible for the degradation of propanil was determined using a cell-free extract from lyophilized cells. In this test, the extract represented 1.02 mg fungal weight, in a final volume of 3 .O ml of reaction mixture. Propanil was used as a substrate at four different final concentrations of 150 t&ml (6.9 x 1O-4 M), 100 pg/ml(4.6 x 10e4 M), 50 p&ml (2.3 x 10e4 M), and 25 pg/ml (1.1 x lop4 M). Amidase activity was measured after time intervals of 10,20, and 30 min at 28°C. The reactions were stopped by the addition of 5 ml of glacial acetic acid. For the calorimetric test, 0.4 ml of the reaction mixture were used. The K, was determined using a Lineweaver-Burk plot (16). Polyacrylamide gel electrophoresis. Gel electrophoresis was performed by the method of Davis (17) with a “Mighty Small” slab gel electrophoresis unit (Hoefer Scientific Instruments, San Francisco, CA), with 7.5% cross-linked gels (9). Samples (10 ~1) of crude cell-free extract representing 170 pg fungal dry weight were loaded in each slot, with 1 ~1 of sucrose dye solution. Electrophoresis was carried out at 5°C during 1 hr at a constant current of 10.0 mA. The gels were then sliced into l-mm segments and each segment was placed in 1 ml of the homogenizing buffer mentioned above. Propanil was added as a substrate in acetone solution. The final concentration of propanil was 100 kg/ml. After an incubation period of 90 min at 28°C 0.4 ml of the reaction mixture was used for 3,Cdichloro-
244
REICHEL,
SISLER, AND KAUFMAN
aniline (3,4-DCA) determination (15) to indicate relative amidase activity for each segment of gel. RESULTS
A number of amides, ureas, and carbamates (mainly pesticides) were tested as inducers of amidase activity in cells of F. oxysporum. Propanil at 100 pg/ml (0.46 mM) final concentration was the primary substrate used to assay the amidase activity; however, a number of other compounds were also tested as substrates of amidases in the induced cells. In standard assays using propanil as a substrate, the concentration of F. oxysporum cells was adjusted so that the rate of 3 ,CDCA released was linear during the measurement period which ranged from 0.5 to 4 hr in various experiments. Amide inducers. In propanil-induced cells, amidase activity resulting in propanil breakdown released an average of 16.7 kmol of 3,4-DCA per gram fungal dry weight per hour (Table 2). Uninduced cells had an amidase activity less than 0.6 pmol TABLE Induction oxysporum
Inducer (mM) None Propanil (0.11) Acetanilide (0.19) None Acetanilide (0.19) Propanil (0.11)
2
of Amidase
Activity in Fusarium Cells by Propanil and Acetanilide
Substrate (mM)
Amidase activity”
Propanil (0.46)
0
Propanil (0.46)
16.7
Propanil (0.46) Acetanilide (0.74)
0 71.0
Acetanilide (0.74)
55.9
Acetanilide (0.74)
51.6
a Micromoles of 3,4-DCA released from propanil per gram fungal dry weight per hour. Activity of less than 0.6 recorded as zero. ’ Micromoles of aniline released from acetanilide per gram fungal dry weight per hour. ’ Values are the means of duplicate cultures from two experiments.
of 3,4-DCA released from propanil per gram fungal dry weight per hour during the first hour, which was recorded as zero. In contrast to the lack of amidase activity in uninduced cells with propanil as a substrate, these cells showed appreciable amidase activity with acetanilide as a substrate (Table 2). This activity was not improved if cells had been induced for 3 days with 25 pg/ml (0.19 m&f) acetanilide or with 25 kg/ml of propanil (0.11 mA4). Surprisingly, cells induced with acetanilide did not show activity toward propanil. Propachlor (0.12 m&f), carboxin (0.11 mM), iprodione (0.08 mM), metalaxyl (0.36 mM), diphenamid (0.10 n-&f), and cymoxanil (0.13 mM) did not induce amidase activity for the degradation of propanil or acetanilide . Structure of amide compounds tested as inducers of propanil-degrading amidase activity are shown in Fig. 1. Urea inducers. Urea compounds which were tested as inducers of propanil-degrading amidase activity in F. oxysporum are shown in Fig. 2. Phenylureas were the most effective group of compounds tested as inducers of amidase activity for degradation of propanil. In two experiments, monolinuron-induced cells released an average of 1346 bmol (218 mg) of 3 ,CDCA from propanil per gram of fungal dry weight per hour (Table 3). In four experiments, linuroninduced cells released an average of 1111 pmol (180 mg) of 3,4-DCA from propanil per gram fungal dry weight per hour (Table 3). The rates of propanil degradation by monolinuron and linuron-induced cells were, respectively, 81 and 67 times that of propanil-induced cells (Tables 2 and 3). The propanil degraded by these cells in 6 hr was greater than the cell dry weight. The phenylurea compounds metabromuron, metoxuron, and chlorbromuron (Fig. 2) were moderately strong inducers of the amidase but much inferior to monolinuron or linuron (Table 3). Diuron and monuron
PROPANIL
DEGRADING
AMIDASE
ACTIVITY
Cl PROPANIL
Cl
MONOLINURON
ACETANILIDE
0
245
OF F. oxysporum
Br
LINURON
METOBROMURON
CH,
NH-%
PROPACHLOR
Cl
CH,O CAABOXIN
METOXURON
0 NH-&d
CHLORBROMURON
CH,
DIURON 0 NH-&d
CH, ‘CH,
Cl
Cl IPRODIONE
/\ Q-HC-C-N9 FH3
H8H8
ii-O-CH,
FIG. 1. Structures of amide inducers of propanil-degrading oxysporum cells.
Cl-$’
were the least active of the urea inducers of propanil-degrading amidase activity, but were better inducers than propanil. The following urea compounds did not induce amidase activity for the degradation of propanil: fluometuron (0.10 m&f), 3,4-dichlorophenylurea (3,bDCPU, 0.12 mM), and 1,1,3,3-tetramethylurea (0.21 mM). Since linuron was a good inducer of propanil-degrading amidase activity and was readily available in a high purity form, it was used as the primary inducer in these studies. Carbamate inducers. Mobam, CIPC (chlorpropham), and diallate (Fig. 3) were fair inducers of propanil-degrading amidase activity with respective values of 26.5, 13.0, and 12.3 pmol of 3,CDCA released from propanil per gram fungal dry weight per hour (Table 4). Bendiocarb, EPTC (Eptam), and carbofuran at 25 pg/ml (112-
\
N- 8 -N,
, CH, CH3
1,?.3,3,TETRAMETHYLUR)EA
CYMOXANIL
compounds tested as amidase activity in F.
FLUOMETURON
3.4,DCPU
CH,
C,H,-N-C-N-C-C-C:N
‘CH,
DIPHENAMID
MONURON
METALAXYL
2. Structures of urea compounds duction of propanil-degrading amidase oxysporum cells. FIG.
tested for inactivity in F.
132 $I@ final concentration or carbendazim at 8 t&ml (42 p&f) did not induce amidase activity for the breakdown of propanil. Although induction of propanil-degrading amidase activity by PPG-124 in F. oxysporum was reported by Blake et al. (14), amidase induction in these studies could not be demonstrated for PPG-124 at final concentrations of 134, 67, 5.4 and 0.54 pi%!. PPG124 is a potent inhibitor of amidase activity in F. oxysporum (14); therefore, it was presumed that an amidase induced by this compound might not be detected because of the inhibitory action of the inducer (18, 19). This possibility was indicated by experiments in which linuron-induced cells were treated with 54 t.& (10 p&ml) of PPG124 for 1 hr and then washed four times in phosphate buffer (0.05 M, pH 7.0). The cells were then assayed for propanildegrading amidase activity at various inter-
246
REICHEL.
SISLER,
AND
KAUFMAN
TABLE 3 Activity of Ureas as Inducers of Propanil-Degrading Amidase Activity in Fusarium oqsporum Cells Inducer (mM)
Amidase activity”
Relative inductive activityb
Monolinuron (0.11) Linuron (0.10) Metobromuron (0.10) Metoxuron (0.11) Chlorbromuron (0.09) Diuron (0.17) Monuron (0.11) None
1346 1111 130 123 99 49 19 0
81.0 67.0 7.8 7.4 6.0 2.9 1.1 0
u Micromoles of 3,4-DCA released from propanil per gram fungal dry weight per hour. Activity of less than 0.6 recorded as zero. Values for monolinuron are the mean of two experiments; values for the remaining compounds are the mean of four experiments. b Relative inductive activity with respect to propanil.
vals thereafter. When assayed after a I-hr incubation at 28°C amidase activity was completely absent. However, when these cells were washed two additional times in phosphate buffer and incubated in buffer at 15°C for 16 and 25 hr, with stirring, there was a 20% recovery of amidase activity after 16 hr and a 30% recovery after 25 hr. Substrates. None of the urea compounds tested as inducers (Fig. 2) were substrates of the linuron-induced propanil-degrading amidase as indicated by lack of measurable release of an aniline product. In addition to propanil, acetanilide was a good substrate for the amidase(s) induced by two methoxy-substituted phenylurea compounds, monolinuron, and linuron (Table 5). In one experiment, monolinuroninduced cells degraded acetanilide with an amidase activity of 301 kmol of aniline released per gram fungal dry weight per hour. In three experiments, acetanilide (0.74 mA4 final concentration) was degraded by linuron-induced cells with an average amidase activity of 699 pmol of aniline released per gram fungal dry weight per hour. Induction with the two methoxy urea compounds, monolinuron and linuron, mark-
0 NH-Z-O-dH
0
CH3
‘CH, Cl CIPC
KH,l,CH
0 ‘N-&SCH,CCl=CHCI
(cH,l,c~’ DIALLATE PPG- 124 OH Q-6N-CH3 CH,-CH,-S-C-N’
9
CHz-CHiCH, ‘cH,-CH,-CH,
CARBOFURAN
EPTC
0
BENDIOCARB
FIG. 3. Structures of some carbamate compounds tested as inducers of amidase activity for the degradation of propanil in F. oxysporum cells.
edly increased acetanilide-degrading activity over that present in uninduced cells. Other ureas such as diuron and monuron, which induced some propanil-degrading amidase activity, did not induce detectable increases in activity for acetanilide degraTABLE 4 Activity of Carbamates as Inducers of Propanil-Degrading Amidase Activity in Fusarium oxysporum Cells Inducer (mM)
Amidase activity”
Relative inductive activityb
Mobam (0.12) CIPC (0.12) Diallate (0.09) PPG-124 (0.07)
26.5 13.0 12.3 0
1.6 0.8 0.7 0
a Micromoles of 3,4-DCA released from propanil per gram fungal dry weight per hour. b Relative to that of propanil.
PROPANIL
DEGRADING
AMIDASE
TABLE 5 Acetanilide and CIPC as Substrates of Linuron or Mono&won-Induced Amidase in Cells of Fusarium oxysporum Inducer WW
Substrate (mM)
Amidase activity
None Linuron (0.1) Monolinuron (0.1) None Linuron (0.1)
Acetanilide (0.74)
70.0”
Acetanilide (0.74)
699.0
Acetanilide (0.74) CIPC (0.47)
301.0 Ob
a Micromoles per gram fungal b Micromoles per gram fungal
CIPC (0.47)
13.3
of aniline released from acetanilide dry weight per hour. of 3-chloroaniline released from CIPC dry weight per hour.
dation above that present in uninduced cells. The carbamate compound CIPC was a substrate for linuron-induced cells and was degraded with an amidase activity of 13.3 pmol of 3chloroaniline released per gram fungal dry weight per hour. Uninduced cells did not degrade CIPC (Table 5). Propanil was the only substrate degraded by the amidase induced by carbamates. Cells induced by the carbamates PPG-124, mobam, CIPC, bendiocarb, carbofuran, diallate, or carbendazim did not show amidase activity with linuron, monuron, diuron, and CIPC or increased amidase activity with acetanilide above that present in uninduced cells. Amidase activity
of cell-free extracts.
Duplicate samples from 12 experiments using three different sets of linuron-induced lyophilized cell gave an average rate of propanil-degrading amidase activity of 1889 p.mol3,4-DCA released per hour by extract representing 1 g of fungal dry weight. This rate of propanil degradation by cell-free extracts was somewhat greater than that by whole cells (Table 3). The data from duplicate samples of three experiments from two different sets of linuron-induced lyophilized cells in which pro-
ACTIVITY
OF F. oqsporum
247
panil and acetanilide were both included as substrates showed that the mean rate of propanil degradation by extract representing 1 g of fungal dry weight was 1549 pmol of 3,CDCA released per hour compared to 279 p.mol of aniline released from acetanilide per hour. In contrast to propanil, the rate of acetanilide degradation was more rapid in whole cells (Table 5) than in these cell-free extracts. Polyacrylamide gel electrophoresis. Proteins of a cell-free extract from linuroninduced cells were separated by a nondenaturing acrylamide gel electrophoresis (17). A lo-p.1 sample of cell-free extract from linuron-induced cells (representing 170 pg fungal dry weight) was loaded in each slot of the gel with 1 ~1 of sucrose dye solution (17). Electrophoresis was carried out at 5°C during 1 hr at a constant current of 10 mA. After separation, the gel was sliced into lmm segments and each segment was placed in 1 ml of homogenizing buffer. Propanil was added as a substrate to give a final concentration of 100 l&ml. After an incubation period of 90 min at 28°C 0.4 ml of the reaction mixture was analyzed for 3,4-DCA as a measure of amidase activity. In two experiments in which propanil was used as a substrate, there was one major band of activity occurring between 17 and 24 mm from the origin (Fig. 4). The minor activity that preceded the strong band may have been due to one or more bands of weak activity, or to trailing of the major band. When acetanilide was used as a substrate (final concentration, 100 pg/ml), amidase activity occurred in the same region as the strong propanil band (17-24 mm), but activity was weak in comparison with that when propanil was used as a substrate. K,,, determination. A Lineweaver-Burk plot was done using the amidase in a cellfree extract of linuron-induced cells and propanil as a substrate. The K, was estimated to be 7.1 x lo-’ M. Amide inhibitors. The following amide compounds were tested as inhibitors of
REICHEL,
SISLER, AND KAUFMAN TABLE
6
Urea Inhibitors of Linuron-Induced, Propanil-Degrading Amidase Activity in Whole Cells and Cell-Free Extracts of Fusarium oxysporum
% Inhibition at indicated concentration Whole cells
FIG. 4. Amidase activity (3,4-DCA released from propanil) after separation of proteins in a cell-free extract from linuron-induced cells by acrylamide gel electrophoresis. Activity was determined by assay of l-mm segments of gel between origin and front (40 mm). Amidase activity is expressed in umol of 3,4DCA x 100190 min.
linuron-induced, propanil-degrading amidase activity in cells of F. oxysporum, and were found to be ineffective at the indicated concentration (PM): propachlor (250), alachlor (190), metolachlor (180), CDAA (280), diphenamid (170), and metalaxyl (180). Only the amide cymoxanil with an IDS, value of 210 FM, was inhibitory to propanil degradation. In cell-free extracts of linuron-induced cells, cymoxanil was inhibitory to propanil degradation with an ID,, value of 90 pM, which was about 2.3 times lower than that for whole cells. Urea inhibitors. All the urea compounds which were inducers of propanil-degrading amidase activity were also inhibitors of the induced amidase activity in whole cells and cell-free extracts (Table 6). Linuron, diuron, monuron, and metoxuron gave somewhat lower IDSa values in cell-free extracts than in whole cells. Among these urea compounds, linuron and diuron were the most potent inhibitors in the cell-free extracts. The following ureas gave the indicated ID,, values (k&f) in cell-free extracts: isoproturon, 6.3; chlortoluron, 18; and fluometuron, 140. Isoproturon ranked with diuron and linuron (Table 6) as the most potent urea inhibitors of amidase activity in cell-free extracts. Other urea compounds tested were es-
Cell-free extracts
Inhibitor
%
PM
%
PM
Linuron Diuron Monouron Metoxuron Monolinuron Chlorbromuron Metobromuron
50 50 50 50 93 85 73
19 20 IO 130 140 loo 110
50 50 50 50 50 50 50
12.0 4.5 48.0 87.0 57.0 77.0 22.0
sentially ineffective as inhibitors. 1-Phenyl-Zthiourea showed a 5% inhibition of the amidase activity in the whole cells at 330 PM. Phenylurea, 3,4-DCPU, and 1,1,3,3-tetramethylurea did not inhibit the amidase activity in whole cells at respective concentrations of 370, 240 and 860 pM. Carbamate inhibitors. Some of the carbamates tested as inhibitors of linuroninduced, propanil-degrading amidase activity are listed in Table 7. The carbamates PPG-124 and mobam were powerful inhibitors of linuron-induced amidase activity in both whole cells and cell-free extracts of F. oxysporum (Table 7). The remaining carbamates were relatively poor inhibitors of the amidase activity both in whole cells and cell-free extracts. PPG-124 was the most potent inhibitor of amidase activity tested, with an ID,, value in the whole cells of 0.33 PM compared to 19 and 20 PM for linuron and diuron, the most potent urea inhibitors (Tables 6 and 7). DISCUSSION
This study showed that linuron, monolinuron, and several other phenylureas are far superior to propanil as inducers of a propanil-degrading amidase in F. oxysporum. In respect to amidase induction, F. oxyspo-
PROPANIL
TABLE
DEGRADING
AMIDASE
7
Carbarnate Inhibitors of Linuron-Znduced, Propanil-Degrading Amidase Activity in Cells and Cell-Free Extracts of Fusarium oxysporum
% Inhibition at indicated concentration Cell-free extracts
Whole cells Inhibitor
%
CLM
%
CLM
PPG-124 Mobam Bendiocarb Dioxacarb Diallate Carbendazim CIPC EPTC Pebulate SulfalIate Thiram Carbofuran
50 50 40 52 50 13 18 7 7 4 7 0
0.33 4.4 180.0 130.0 500.0 40.0 230.0 260.0 200.0 180.0 120.0 230.0
50 50 50 10 13
0.2 3.6 480.0 90.0 370.0 -a 530.0 90.0 450.0
24 8 11
0 Not tested.
rum responds like Bacillus sphaericus isolated by Engelhardt et al. (9) from problem soils. In this bacterium, as in F. oxysporum, linuron was more effective as an inducer of acylamidase activity than propanil (10). Moreover, the two organisms were similar in that phenylureas, phenylamides, and phenylcarbamates all induced the amidase activity. Although phenylamides of the secondary amine type were the preferred substrate of the amidase(s) in both organisms, the enzyme(s) of F. oxysporum was not observed to degrade methoxy-substituted phenylureas like linuron and monolinuron; whereas the enzyme from B. sphaericus did degrade these ureas (9). However, these methoxysubstituted phenylureas were extremely poor substrates for the B. sphaericus enzyme in comparison to the better phenylamide substrates. For example, the specific activity of the B. sphaericus amidase for degradation of the phenylamide, propanil, was 1245 times higher than for the methoxy-substituted phenylureas, linuron and monolinuron which were the best urea substrates tested (9, 10).
ACTIVITY
OF F. oxysporum
249
The present study shows that propanil induces amidase activity in F. oxysporum for its own breakdown but not for the degradation of acetanilide. Likewise, acetanilide does not induce amidase activity for the breakdown of propanil (thus different enzymes seem to be involved here). Apparently F. oxysporum has a low level of constitutive enzyme which degrades acetanilide. This finding resembles that reported by Lanzilotta and Pramer (20) in which uninduced cells of F. solani showed amidase activity for the degradation of acetanilide. Although induction of amidase activity by PPG-124 for the degradation of propanil by F. oxysporum has been reported (14), it could not be demonstrated in the present study. PPG-124 might induce a propanildegrading amidase, which it inhibits temporarily as is characteristic of some carbamate inhibitors which carbamylate enzymes (18, 19). Therefore, failure to detect amidase activity in the present study might not have been due to a lack of induction by PPG-124 but due rather to a failure of the PPG-124-inhibited enzyme to recover activity by decarbamylation. A somewhat higher rate of propanil degradation was demonstrated in cell-free extracts of linuron-induced cells of F. oxysporum than in linuron-induced whole cells. This suggests that the rate of degradation in whole cells might be limited by the rate of propanil penetration into the cells. On the other hand, the higher rate of acetanilide degradation in linuron-induced whole cells as compared to cell-free extracts might be explained by loss of enzyme activity during preparation of the cell-free extracts. Acetanilide degradation by linuron-induced whole cells probably involves a constitutive enzyme acting only on acetanilide and a linuron-induced enzyme acting on both acetanilide and propanil. The K, determined for propanil with linuron-induced amidase in this study was 7.1 x 10e5 M. This compares favorably to a value of 2.3 x 10T5 A4 determined by Blake et al. (14) for the propanil-induced
250
REICHEL,
SISLER,
enzyme in this organism. The linuron and the propanil-induced enzymes are believed to be the same, but further studies with purified enzymes would be required to determine this. The observations in the present study and those by Engelhardt et al. (10) in studies with B. sphaericus that methoxysubstituted phenylurea herbicides are powerful inducers of amidase acting on phenylamides suggest that this phenomenon may occur widely among soil microorganisms degrading soil applied pesticides. Thus, caution should be exercised when phenylamide and methoxy-substituted phenylurea herbicides are applied together in the soil. Whether induction of amidases by such phenylureas would lead to long-term enrichment of these amidase producing microorganisms in soil is unknown.
AND
KAUFMAN
5. L. J. Audus, The biological detoxification of hormone herbicides in soil, Plant Sod 3, 170 (1951). 6. H. D. Sisler, Biodegradation of agricultural fungicides, in “Biodegradation of Pesticides” (F. MatsumuraandC. R. K. Murti, Eds.), pp. 133155, Plenum, New York, 1982. 7. P. Wallnofer, The decomposition of urea herbicides by Bacillus sphaericus, isolated from soil, Weed Res. 9, 333 (1969). 8. P. R. Wallnofer and J. Bader, Degradation of urea herbicides by cell-free extracts of Bacillus sphaericus,
Appl.
Microbial.
19, 714 (1970).
9. G. Engelhardt, P. R. Wallnbfer, and R. Plapp, Degradation of linuron and some other herbicides and fungicides by a linuron-inducible enzyme obtained from Bacillus sphaericus, Appl. Microbial. 22, 284 (1971). 10. G. Engelhardt, P. R. Wallnofer, and R. Plapp, Purification and properties of an aryl acylarnidase of Bacillus sphaericus, catalyzing the hydrolysis of various phenylamide herbicides and fungicides, Appl. Microbial. 26, 709 (1973). 11. R. Bartha and D. Pramer, Pesticide transformation to aniline and azo compounds in soil, Science 156, 1617(1%7).
ACKNOWLEDGMENTS
We thank M. L. Gullino and T. Chida for their assistance in various aspects of this study. We thank J. E. Kurent for his interest and support. REFERENCES
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