Involvement of fungi and bacteria in enhanced and ...

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Involvement of fungi and bacteria in enhanced and nonenhanced biodegradation of carbendazim and other benzimidazole compounds in soil 0. YARDEN' Department of Plant Pathology and Microbiology, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel

R. SALOMON Department of Virology, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel

J.

KATAN~

Department of Plant Pathology and Microbiology, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot 76100, Israel AND

N. AHARONSON Department of Chemistry of Pesticides and Natural Products, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel Received July 17, 1989 Accepted September 25, 1989 YARDEN,O., SALOMON, R., KATAN,J., and AHARONSON, N. 1990. Involvement of fungi and bacteria in enhanced and nonenhanced biodegradation of carbendazim and other benzimidazole compounds in soil. Can. J. Microbiol. 36: 15-23. The relationship between chemical structure and the enhancement of microbial degradation of three benzimidazole compounds in soil was determined. Preapplication of methyl benzimidazole-2-ylcarbamate (carbendazim or MBC), 2-aminobenzimidazole (2AB), and benzimidazole enhanced their degradation upon repeated application (self-enhanced degradation). MBC and 2AB cross-enhanced the degradation of each of these two compounds, whereas benzimidazole did not enhance the degradation of MBC. Thiabendazole (TBZ) did not enhance its own degradation or cross-enhance the degradahon of MBC. No increase in h e number of MBC-degrading fungi or in the capacity of soilborne fungi to d e p d e MBC was detected in soil exhibiting enhanced MBC degradation (MBC-history). A sharp increase in esterolytic activity in the microsomal fraction of Altermria a/ternatacapable of degrading MBC in culture was induced by the presence of MBC in the growth medium. 2AB was the main metabolite of MBC that accumulated in A. alternata cultures and in cell-free preparations. MBC was degraded much faster by mixed bacterial cultures that originated from MBC-history soil than in cultures from MBC-nonhistory soil. Fluctuations in the MBC degrading capacity of mixed bacterial cultures occurred during repeated subculturing of the mixed culture. Inoculation of nonhistory soil with mixed bacterial cultures resulted in enhanced MBC degradation, whereas inoculation with A. alternata did not enhance MBC degradation. It is suggested that while fungi contribute to MBC dissipation in soil, bacteria have a greater role in enhanced biodegradation of MBC in soil. Key words: accelerated degradation, fungicide, problem soils.

N. 1990. Involvement of fungi and bacteria in enhanced and YARDEN,O., SALOMON,R., KATAN,J., et AHARONSON, nonenhanced biodegradation of carbendazim and other benzimidazole compounds in soil. Can. J. Microbiol. 36 : 15-23. La relation entre la structure chimique et le rehaussement de la dkgradation microbienne de trois composks benzimidazoliques dans les sols a kt6 dkterminte. Une pkapplication des composks mkthyl benzimidazole-2-yl carbamate (carbendazim ou MBC), 2-aminobenzimidazole (2AB) et benzimidazole a induit un rehaussement de la dkgradation de ces substances au cours de rkpktitions des applications, soit un auto-rehaussement de la degradation. Le MBC et le 2AB ont rehauss6 la dkgradation de chacun de ces deux composks de f a ~ o ncroiske, tandis que le benimidazole n'a pas rehausd la d6gradation du MBC. Le thiabendazole (TBZ) n'a pas augment6 sa propre dkgradation, ni le rehaussement croise de la dkgradation du MBC. Dans les sols qui dkgradent le MBC et dans lesquels cette dkgradation ktait augment6 (comportant donc un historique du MBC), aucune iugmentation du nombre de champignons ou de leur capacitk h d&ader le MBC n'a 6tk dkcelke. Un accroissement marquk de I'activitk estkrolytique, qui est pksente dans la fraction microsomique de I'Alternaria altermfaet qui est capable de dkgrader le MBC, a kt6 induite par la presence de MBC dans le milieu de croissance. Le 2AB a kt6 dktermin6 cornme ktant le principal mktabolite du MBC dans les cultures de 1'A. alternata et dans les prkparations acellulaires. Le MBC a kt6 dkgradk beaucoup plus rapidement dans les cultures baceriennes mixtes provenant de sols comportant un historique du MBC que dans les cultures de sols sans un tel historique. Dans les cultures mixtes de bactkries, des fluctuations dans la capacit6 de degrader le MBC ont kt6 not& au cows des r6p6titions de sous-cultures de ces cultures mixtes. L'inoculation de sols non liks B I'historique du MBC avec des cultures bactkriennes mixtes s'est traduite par un rehaussement de la dkgradation du MBC, contrairement 21 l'inoculation avec 1'A. alternata. La suggestion est alors avancke que les champignons contribuent, d'une part, 2I disperser le MBC dans les sols alors que les bactkries ont, d'autre part, un r6le plus prononck dans I'accroissement de la biodkgradation du MBC. Mots elks : dkgradation accklkr6e, fongicide, sols problkmatiques. [Traduit par la revue] 'Current address: Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, U.S.A. 'Autnor to whom all correspondence should be addressed.

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CAN. J. MICROBIOL. VOL. 36. 1990


Introduction Reports concerning the enhanced microbial degradation of a diverse variety of soil-applied pesticides have accumulated throughout the past decade (Katan and Aharonson 1989; Kaufman et al. 1985; Roeth 1986). This phenomenon has been generally linked to previous applications of the pesticide (self-enhancement) or of a structurally related compound (cross-enhancement) to the soil (Racke and Coats 1987, 1988a, 1988b; Roeth 1986; Wilson 1984). Isolation of microorganisms involved in degradation of pesticides has been widely described in the literature. Less is known about microorganisms and the enzymatic reactions related to enhanced degradation. In a few cases, a predominant microorganism that is likely to have a key role in the enhanced degradation process has been isolated (Racke and Coats 1987). In other instances, such as in the case of the widely studied herbicide S-ethyl N, N,dipropylthiocarbamate (EPTC), a variety of EPTC degraders (bacteria as well as fungi) have been isolated (Lee 1984; Tam et al. 1987), yet their significance in degrading the compound in soils where enhanced degradation prevails has not been fully elucidated. Benomyl (methyl 1-(butyl-carbamoy1)benzimidazole-2-ylcarbamate) and other benzimidazole fungicides have had a great impact on chemical control of a variety of fungal diseases of vegetable, fruit, ornamental, and stored crops (Fry 1982). Helweg (1977) studied the microbial degradation of the fungicide benomyl and its fungitoxic hydrolysis product methyl benzimidazole-2-ylcarbamate(carbendazim or MBC) in soil. He concluded that MBC was a poor carbon source and suggested that the biodegradation of MBC is a cometabolic process. Several reports demonstrate the recalcitrance of benzimidazole rings to microbial degradation (Fuchs and DeVries 1978a, 19786; Sisler 1982) as opposed to the relative ease in which the aliphatic moiety is hydrolysed (Fleeker et al. 1974). In the mentioned reports, several MBC-degrading bacteria that have been isolated from soil-perfusion or enrichment culture systems have been described. Soilborne fungi capable of degrading MBC have also been isolated (Yarden et al. 1985b). The enhanced biodegradation of MBC in soil has been characterized in a previous report (Yarden et al. 1987). Accelerated degradation of MBC can be self-enhanced or conferred upon soil with no history of MBC applications (nonhistory) by minute quantities of soil preconditioned for enhanced degradation by prior MBC application (MBC-history). The purpose of this work was to further elucidate the role of microorganisms and the mechanisms involved in the enhanced degradation of MBC and some other benzirnidazole derivatives in soil.

Materials and methods

soil (100 g/L) (CB) was used to grow mixed bacterial cultures. MBC was added at 10 or 100 (~g/mL,as indicated. Laboratory studies of degradation of benzimidazoles

Acetone solutions of compounds were freshly prepared, added to soil, incubated in biometer flasks (Bartha Pramer 1965), and sampled as previously described (Yarden et al. 1987). Unless otherwise stated, initial fungicide or metabolite application rates were 10 pg/g soil. When added to liquid media, 10 pg/mL of MBC was applied after autoclaving the medium. The warm flasks were left open for 15 min under sterile conditions to allow solvent evaporation. When 14C-labeled MBC was used, 50 pg (0.26 pCi) of the radioactive compound was added to the total soil or liquid volume. In such experiments, both openings of the biometer flasks were sealed with rubber stoppers. Ten millilitres of 0.5 M KOH, which was added to the side arm of the flask as a C02trap, was sampled and replaced periodically. All radioactivity measurements were carried out with Instagell I1 (Packard) as a scintillation fluid and measured with a Kontron Betamatic scintillation counter. Extraction of benzimidazoles and aniline was as previously described (Yarden et al. 1987). Analysis was carried out either by C-18 reversed phase HPLC (1 mL/min isocratic flow of MeOH-H20 (6040) amended with 10 drops per litre of concentrated NH40H) and fluorescent spectrophotometry (282 nm excitation and 307 nm analysis) or by TLC on Alufolien Kieselgel 60 FZs4(Merck) with ethyl acetate - acetic acid - chloroform (5551) as described by Davidse (1976). Alternatively, fungitoxic residues in soil or liquid samples were determined by the soil-agar pellet bioassay (Yarden etal. 1985a) which is based on the inhibition of a test fungus by fungitoxic residues diffusing from a soil-agar pellet placed in the center of a petri dish preinoculated with the test organism. To enable the quantitative determinatin of liquid samples, the agar component of the pellet was prepared at 40 g/L (rather than 30 g/L used to prepare the soil-agar pellets). The ability of fungal isolates to degrade MBC was determined by growing the tested isolate on MBC-amended medium and determining fungitoxic residues in the medium by bioassay, as previously described (Yarden et al. 1985b). Isolates capable of degrading at least 10%of the MBC in the growth medium were considered MBC degraders. To assess bacterial degradation of the pesticide (measured as pesticide degradation units (PDUs)), soil minidilutions were prepared in CB. Ten microlitres of the appropriatedilution (in 5 replicates) was plated in each well of a standard 96-well microtiter dish containing 150 pL solid CB amended with 2 pg/mL MBC. Moist filter paper was placed adjacent to the dish cover to maintain humidity during the incubation period (5 days at 27OC). Following incubation, 10-pL aliquots of a Penicillum digitatum Sacc. (sensitive to benzimidazole fungicides) conidial suspension ( 1 6 conidia/mL PDB containing 250 mg/L chloramphenicol) were added to each well. The covered dishes were incubated for an additional 2 days, by which time growth or inhibition of growth could be determined. Wells that had remained clear (i.e., no growth of the fungus) indicated the presence of fungitoxic residues.

Chemicals, soil, and media

Preparation of fungal microsomal @action, polyacrylamide gel electrophoresis (PAGE), and determination of esterolytic activity

Analytical-grade ethyl acetate and methanol were glass-redistilled before use. All other solvents and chemicals were analytical grade. Analytical, as well as [14C]MBC (labeled at position 2; 1 mCi/mM specific activity (1 Ci = 37 GBq)) were a gift of E.I. du Pont de Nemours & Co. Inc. 2-Aminobenzimidazole(2AB) and benzimidazole were purchased from Sigma. Aniline was purchased from Merck. Soil used was from Rehovot (sandy loam; 0.6% organic matter; 3.8% clay; pH 7.9; field capacity, 9%, v/w). Storage, when required, was at 5OC. Soil sterilization, when required, was by autoclaving (121°C at 1013.2 millibar for 1 h on 2 successive days (1 bar = 100 kPa)). Difco's potato dextrose agar (PDA) or broth (PDB) was used for maintenance and growth of all fungi in this study. Martin's medium (Gherna et al. 1980) amended with 30 pg/mL rose bengal and 10 pg/mL MBC (MRB) was used to isolate fungi from soil. Minimal salts medium amended with

Log phase fungal mycelium was harvested, rinsed with 0.1 M phosphate buffer (pH 7.6 and containing 0.15 M NaCl), and filtered (Whatman no. 1). Fifteen grams (fresh weight) was suspended in 50 mL (filter sterilized) of the mentioned buffer containing 10% mannitol and 500 mg Novozym 234 (Novo Industries, Denmark) and shaken (100 rpm at 30°C) for 1 h for protoplast preparation. The protoplasts were rinsed twice with cold phosphate-NaC1 buffer and harvested by centrifugation (10 min at 300 x g). Sterile distilled water was added to lyse the protoplasts, and after 5 min, a one-tenth volume of 1 M phosphate- 1.5 M NaCl buffer was added. The cell mixture was gently homogenized with an emphasis on slow rotation of the teflon homogenizer. Following a 10-rnin centrifugation (10000 X g), mimsomes were isolated as described (Shoun et al. 1985). SDS-PAGE was carried out on a 8-20% polyacrylarnide gradient. Native PAGE

YARDEN ET AL.

-

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ALLENGE COMPOUND: MBC

0 '" 10 (r


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z

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BENZIMIDAZOCE

2AB

MBG

NUNE

PREAPPLIED COMPQUND

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CHALLENGE COMPOUND: 2AB

10

I

NOT

DETERMINED

f NONE

/ MBG

/I

, 2AB

BENZlMlDAZOLE

PREAPPLlED COMPOUND

FIG. 1. HPLC reversed phase separation and relative fluorescent emission of three benzimidazoles and aniline. Elution sequence of the various compounds is aniline, benzimidazole, 2-aminobenzimidazole (2AB), and methylbenzimidazole-2-ylcarbamate(MBC).

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was carried out on 7% polyacrylamide. Following native PAGE, gels were either maintained on ice or immediately transferred to sodiumpotassium phosphate buffer (50 mM, pH 6.5) containing 1 mM a-napthyl acetate and 0.6 mg/mL fast blue RR (both chemicals by Sigma) for determination of esterolytic activity (Rosenberg et al. 1975). To determine the esterolytic degradation of MBC and thiabendazole (TBZ) by proteins separated by native PAGE, unstained bands from lanes adjacent to the reactive bands were cut out and immersed in 400 p,L of phosphate-NaC1 buffer containing 2.5 mg/L MBC or TBZ. The mixture was incubated for 2 h at 30°C. To inhibit serine-esterase activity, a reaction buffer containing 10 p,M diisopropylfluorophosphate (Dm,Fluka) was used.

Results

Qualitative and quantitative analysis of four possible metabolites of MBC extractedfrom soil A method, using HPLC and fluorescence detection, was developed for the chemical analysis of MBC and of three of its possibie metabolites (Fig. 1). of MBC, 2AB, benzimidazole, and aniline from soil or liquid medium was 86-92, 63-69, 80-88, and 50-60%, respectively.

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Cross-enhanced degradation of benzimidazoles in soil The relationship of structure to persistence of MBC and two of its potential metabolites (Helweg 1977; Rouchaud et al. 1974, 1977) was studied. As determined by HPLC analysis of

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CHALLENGE COMPOUND: BENZIMIDAZOLE

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MBC

2AB

BENZlMlOAZOLE

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FIG. 2. Degradation of (A) MBC, (B) 2AB, and (C) benzimidazole (challenge compound) in soil pretreated with these compounds. Preapplication was with 10 p,g/g of the compound tested; the challenge compound (10 p,g/g) was applied 10 days after it was determined that no residues of compounds preapplied to the soil remained.

soil sampled from triplicate sets of biometer flasks, MBC was rapidly degraded in soils previously treated with either MBC or 2AB (Fig. 2A). A slight reduction in the persistence of MBC in soil pretreated with benzimidazole was observed, suggesting a possible effect of this pretreatment. The nature of this benzimidazole-inducednonenhanced MBC degradation has yet to be elucidated. 2AB dissipated more rapidly from soils

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CAN. J. MICROBI(3L. VOL. 36. 1990

TABLE1. Frequency of MBC-degradingfungi randomly isolated from soil with (history) or without (nonhistory) prior application of MBC


MBC application history Identified isolates

Nonhistory

MBC-history

Alternaria alternata Bipolaris tetramera Ulocladium sp. Acremonium falciforme Total

49(70.O) 13(18.6) 8(11.4) O(O.0) 70

42(64.6) 16(24.6) 6(9.2) l(1.5) 60

NOTE:Values in parentheses are the number of identified fungal cfu expressed as a percentage of the total number of MBC-degrading fungi randomly isolated from each soil. 4

previously treated with either MBC or 2AB (Fig. 2B). No significant change in benzimidazole persistence was observed in soils preconditioned for accelerated degradation of MBC. Benzimidazole only enhanced its own degradation (Fig. 2C), suggesting that a different metabolic or enzymatic reaction than that common to MBC and 2AB was involved. No significant increase in the rate of TBZ degradation was observed in soil that had been preconditoned with MBC. After 34 days of incubation, 82,85, and 80% of the applied TBZ was recovered (by soil-agar pellet bioassay) from nonhistory, MBC-history, and TBZ-history soils, respectively. Hence, TBZ was the only benzimidazole compound tested that, when reapplied, was not degraded at an enhanced rate (self-enhancement).

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12 24 36 48 60 72 84 96 108 120

INCUBATION PERIOD (hours) FIG. 3. MBC degradation and growth of Alternaria alternata in potato dextrose broth. MBC was applied at 10 pg/rnL medium. Inoculum for this experiment was grown either in the presence (MBC-history) or absence (nonhistory) of MBC. Each point represents a mean of three replicates.

Isolation and characterization offungal populations capable of degrading MBC Soil fungi tolerant to MBC were isolated from MBC-history and nonhistory soils on a medium containing MBC. The number of tolerant isolates in MBC-history soil (9 X lo2 cfu/g) was significantly lower than in the nonhistory soil (1.4 x lo3cfu/g). Among the 80 isolates tested from each of the two soils, 70 from MBC-history and 65 from nonhistory soil were capable of degrading the fungicide. Among the various fungal isolates capable of degrading MBC, approximately two-thirds were identified as Alternaria alternata (Table 1). Nested statistical analysis based on the ability of each isolate to degrade the fungicide showed that there was no significant difference (P = 0.05) between the populations isolated from either MBChistory or nonhistory soils. Degradation ofMBC by A. alternata in culture and in a cell-free system When grown in PDB, A. alternata degraded MBC (Fig. 3). Growing the fungus in the presence of MBC (10 pg/mL) prior to inoculating it into fresh MBC-amended medium (used for this MBC degradation experiment) had no significant effect on the rate of MBC degradation or on fungal biomass accumulation. Analysis of benzimidazole residues in the culture medium showed an accumulation of 2AB throughout the growth period (1.8 and 3.9 pg/mL following 3 and 7 days of incubation, respectively). To further characterize the mechanism involved in fungal degradation of MBC, an active cell-free extract capable of degrading the compound in vitro was prepared. Crude fungal extracts prepared by mechanical cell disruption or homogenization of protoplasts did not degrade the compound. MBC degradation was observed only in the isolated fungal microsomal fraction. This activity was detected only in micro-

0

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30

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FIG. 4. Degradation of MBC as determined by reversed phase HPLC of extracts prepared from a microsomal fraction of Alternaria alteranta grown in the (A) absence or (B) presence of MBC. The microsomes were incubated for 2 h with 4 p,g/mL MBC prior to extraction.


MBC

MBC+DFP

TBZ

PESTICIDE IN BUFFER NONINDUCED

m i d

FIG. 5. Esterolytic activity of a native PAGE separated rnicrosomal protein preparation, as determined by a-naphthyl acetate hydrolysis. Microsomal protein (40 p g in each lane) was prepared from Alternaria alternata grown in the (A) absence or (B) presence of MBC.

somal extracts prepared from A. alternata that had grown in the presence of MBC, and the major accumulated metabolite detected was 2AB (Fig. 4). When microsomal proteins were separated by native PAGE, an intense band of esterolytically active protein was observed in extracts derived from the MBC-treated fungal preparation, in contrast to a much lighter band present in extracts prepared from fungus grown in MBC-free medium (Fig. 5). When the intensive band was extracted from the native gel and re-run as a denatured preparation, an intensified polypeptide (estimated molecular mass, 65 kDa) was the major band observed. This polypeptide comigrates with to an intensified polypeptide observed in microsomal extracts from the MBC-treated fungus. Incubation of the active, native gel separated area, as based on the esterolysis assay of an adjacent lane, for 2 h in phosphate buffer containing MBC resulted in degradation of the fungicide (Fig. 6). This activity was inhibited by the serin-esterase inhibitor DFP, thus reconfirming the esterolytic nature of the reaction, while further characterizing this activity as serine esterolytic. TBZ, as expected, was not degraded by this PAGE-isolated microsomal fraction (even though A. alternata will degrade TBZ in culture (Yarden et al. 1985b)), since the compound does not have an ester bond. Bacterial degradation of MBC in soil and liquid culture The number of MBC-tolerant bacterial cfu as isolated on CB was higher in MBC-history soil than in nonhistory soil (2.6 x lo4 and 1.6 X 104/g, respectively). An increase in the number of active bacterial PDUs was observed in soil conditioned for enhanced MBC degradation (as determined by soil minidilu-

@ MBC-INDUCED

FIG. 6. Degradation of MBC in the absence (MBC) or in the presence of diisopropyl-fluorophosphate (MBC DFP) and thiabendazole (TBZ) by an esterolitically active microsomal protein preparation obtained from Altenaria alternata cells cultured in the presence (MBC-INDUCED) or absence (NONINDUCED) of MBC. After separation by native PAGE and identification of active bands on adjacent lanes, the active gel volume was transferred to MBCcontaining buffer (2 pg/mL) for 2 h, followed by determination of fungitoxic residues.

+

tions). When the dilution factor was taken into account, an increase of at least one order of magnitude in the number of PDUs was observed in the MBC-history soil when compared with the nonhistory contol (lo4 and lo3 PDUs/g, respectively). Among 250 individual isolates obtained from the various dilutions in the PDU assay, none were capable of degrading MBC. Further isolation of individual MBC-degrading bacteria was attempted by several methods. Over 300 colonies were randomly isolated from soil dilution plates or from dilution plates from an MBC-degrader enrichment culture, with no success in obtaining a pure isolate capable of degrading the fungicide in culture. The involvement of bacteria in the enhanced MBC degradation was studied further by comparing the rate of fungicide degradation by mixed cultures favouring bacterial activity. These were prepared by inoculating liquid media with either MBC-history or nonhistory soils. In the mixed culture obtained from the MBC-history soil, much faster dissipationof MBC was observed, when compared with the MBC degradation rate in the nonhistory soil inoculated medium (Fig. 7). The half-life of MBC in MBC-history soil mixed cultures was 3-5 days, whereas the half-life of MBC in the nonhistory cultures was about 21 days. Three antibacterial agents inhibited MBC degradation by the mixed bacterial cultures. Thus, 50 pg/mL of either chloramphenicol or streptomycin or 50 units/mL penicillin inhibited the degradation of MBC in MBC-history soil mixed cultures by 65,85, and 70%, respectively, as determined 48 h after inoculation. To determine the stability of the degradation capacity upon subculturing of the original MBC-history soil inoculated culture, MBC degradation was determined after repeated reinoculation of a fresh medium with 5 mL of the preceding culture. The degradation capacity of the subcultures derived from reinoculations at 5-day intervals is shown in Fig. 8. In cultures grown in concentrations of either 10 (Fig. 8A) or 100 pg/mL (Fig. 8B) MBC, a fluctuation in MBC-degrading capacity was observed. In the higher MBC concentration, the fluctuations were more frequent.


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CAN. J. MICROBIIOL. VOL. 36, 1990

0

5

10

76

20

TIME (days)

TEZANSFER NUMBER

FIG. 7. MBC degradation by mixed bacterial cultures obtained from soil with or without a history of MBC application. The inoculant consisted of 5 g of the indicated soil, which was added to 45 mL minimal salts medium containing 10 ~ g / m LMBC. Each point represents the mean of three replicates.

Induction of enhanced MBC degradation in soil by a mixed bacterial culture inoculum or by A. alternata Bacteria harvested from an active mixed bacterial culture (as in transfer no. 4 demonstrated in Fig. 8) were added to MBC nonhistory soil. Following a 10-day incubation period, when the soil was challenged with MBC, enhanced degradation of the fungicide was evident (Fig. 9). In contrast, no enhanced degradation in the nonhistory soil was induced when the soil was inoculated with bacteria from a nonactive mixed culture (transfer no. 11). The same nonactive mixed culture inoculum did not affect the enhanced degradation of MBC in an MBC-history soil. In parallel experiments, soil was inoculated with preparations of A. alternata (5 X lo5 cfu/g). The persistence of MBC and TBZ was not affected by the fungal amendments. When similar preparations were used to inoculate autoclaved (sterile) soil, a significant, yet not enhanced, dissipation of MBC from the soil was observed (58 and 14% degradation in inoculated and noninoculated soils, respectively, following 60 days of incubation). A similar trend was observed in a comparison of TBZ degradation in A. alternata inoculated and noninoculated autoclaved soil (93 and 71% degradation, respectively, following 60 days of incubation). Viability counts of fungal cfu in the preautoclaved and subsequently inoculated soils throughout the experiments showed no significant reduction in the n-~mberof cfu during the incubation period. This indicates that the nonenhanced degradation in these inoculated soils was not due to a reduction in the fungal population in the soil throughout the experiment. Qualitative comparison of [14C]MBC degradation between soil, mixed bacterial cultures, and A. alternata culture 14c-labeledMBC was used to determine the metabolic fate of the fungicide in the mentioned degradation systems. Recovery of 14C in this series of experiments ranged between 84.6 and 90.5% of the applied labeled carbon. The distribution of the 14C degradation products among the various fractions is summarized in Fig. 10. The distribution of the labeled carbon recovered from the fungal extracts differed from the other four extracts. This was mainly reflected by the lack of MBC mineralization (as expressed by the accumulation of 2AB (27%) and no detectable quantities of 14C02).The accumulation of 2AB in the fungal

TRANSFER NUMBER

FIG. 8. Effect of repeated transfer (at 5-day intervals) on the stability of the MBC degradation capacity of mixed bacterial cultures obtained from soil exhibiting enhanced MBC degradation. Cultures were grown in the presence of either (A) 10 or (B) 100 ~ g / m LMBC.

INCUBATION TIME (days)

FIG. 9. Effect of two bacterial inoculants on MBC degradation in MBC-history (H) and nonhistory (NH) soils. "Active" bacterial inoculum was prepared from transfer no. 4 and "nonactive" inoculum was prepared from transfer no. 11 of a mixed bacterial cultue with an initial MBC-degrading capacity (see Fig. 8A). Inoculants consisted of 3 x lo6 cfu/g. Bacteria-amended soils were incubated for 10 days prior to adding MBC (10 pg/g).

culture revalidates earlier findings obtained with the fungal cell-free preparation (Fig. 4). In the MBC-history and nonhistory soils, labeled 2AB was not detected and the relative amounts of 14C02 released and radioactive residues in the aqueous and organic extracts were similar. Analysis of the

YARDEN ET AL.

MIXED


SOIL

AQUEOUS PHASE

NH

H

A . alternate

H

NH

EXPERIMENTAL SYSTEM

FIG. 10. Distribution of 14Cderived from 14c-labeledMBC in three microbial degradation systems tested in the laboratory in biometer flasks. Nonhistory (NH) soil was sampled after a 3-week incubation period. MBC-history (H) soil was sampled after 4 days of incubation. Mixed bacterial cultures prepared from H soil were sampled after 4 days of incubation. Mixed bacterial cultures prepared from NH soil were sampled after 2 weeks of incubation. A culture of Alternaria alteranata (A.a.) was sampled after 3 days of incubation. The distribution of 14cin the extracts is presented as percent of total I4C recovered, which ranged between 84.6 and 90.5% of the 14Capplied.

media from the two mixed bacterial cultures revealed a similar distributon of 14C. TWOdifferences in the distribution pattern between the soil and mixed culture extracts were evident. The concentration of the unidentified 14cresidues in the organic phase was higher in the mixed culture extracts (22-25%), as compared with the soil extracts (3-4%). Secondarily, small amounts (2-6%) of 2AB were detected only in the mixed cultures.

Discussion This study suggests that the relationship between structure and persistence of pesticides can be utilized to elucidate the mechanisms that govern the compound's persistence in soil. A study of the negative or positive cross-enhanced degradation effects among various benzimidazoles was carried out as an initial step to determine the structure-persistence relationships linked to enhanced degradation. The fact that among the benzimidazoles tested, only those substituted via an amino group at position 2 (2AB or MBC) cross-enhanced degradation of either compound indicates that this substitution is needed to induce enhanced degradation of MBC. This is further emphasized by the observations that benzimidazole is a self-enhancer but does not cross-enhance MBC degradation and the MBChistory soil does not cross-enhance benzimidazole degradation. The cyclic moiety linked to the benzimidazole, as in TBZ, does not cross-enhance MBC degradation. Self-enhanced benzimidazole degradation provides evidence that another mechanism, possibly ring cleavage, occurs in the absence of substitution on the benzimidazole heterocyclic ring at position 2. It is evident that the combination of the benzimidazole and the thiolic heterocyclic rings contribute to the relatively longer

persistence of TBZ in soil. This may be analogous to the persistence of cycloate in soil as compared with other thiocarbamate herbicides (Wilson 1984). Among MBC-degrading fungi isolated from MBC-treated soil, A. alternata was the most abundant and therefore was chosen for further study. Initial experiments (in vivo) did not show an induction of MBC degradation. This was mainly due to the fact that the time course of the assay used was sufficiently long to mask the relatively rapid induction process. Once an active cell-free extract had been isolated, a clear inductive degradation process could be defined. An indication of the similarity of the two degradation processes (in vivo and in vitro) was provided by the observation that the main degradation product (2AB) accumulating in both systems was identical. The increase in esterolytic activity observed in microsomes prepared from MBC-induced fungus and the fact that the gel-separated enzyme(s) was active in MBC degradation established at least one of the metabolic mechanisms by which A. alternata degrades MBC. This mechanism does not rule out the possibility of alternative degradation pathways (inducible or constituative) that might be active in the fungal culture. Apart from the esterolytic reaction, conjugation or oxidative detoxification should also be considered as processes likely to be present (Matsumura and Boush 1966; Zbozinek 1984). The basis for this assumption is strengthened by the fact that another fungitoxicbenzimidazole, TBZ, is degraded by the same fungus (Yarden et al. 1985b). The source of inoculum in mixed culture degradation experiments was a key factor in determining the pesticide degradation rate. Thus, initial inoculation of MBC-amended medium with soil from either MBC-history or nonhistory soils


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CAN. J. MICROBIOL. VOL. 36, 1990

resulted in enhanced or nonenhanced degradation of the fungicide, respectively. Repetitive reinoculation experiments revealed fluctuations in the mixed bacterial culture's capacity to degrade MBC. Even though a sharp fluctuation in the degradation capacicy of cultures was observed, the MBC-degradation capacity eventually recovered. Since the repetitive transfer of the culture to fresh medium minimizes the possibility of nutrient depletion as a cause for the observed fluctuations, it is tempting to speculate that a biological factor antagonistic to MBC degraders is involved. Indeed, induction of enhanced degradation in a nonhistory soil could not be achieved when a nonactive mixed culture was used (Fig. 9). Various lines of evidence regarding the predominant role of bacteria, as compared with A. alternata, in the enhanced degradation process, could be shown. The fact that a mixed bacterial culture that was isolated from soil, grown in the absence of its possible inducer (MBC), introduced to a nonhistory soil that was later challenged with MBC, subsequently induced enhanced degradation strongly suggests that bacteria have a key role in the enhanced degradation of MBC in soil. This was further emphasized by the observation that no increase in fungal PDUs was observed in MBC-history soil. The fungal culture, though capable of degrading MBC in sterile soil (but not at an enhanced rate), did not enhance the dissipation of the fungicide in natural soil. Furthermore, the fate and distribution of 14C-labeled MBC is strikingly different, both in detoxification rates as well as in qualitative metabolite accumulation, between the fungal culture and soil or mixed bacterial cultures. It appears, therefore, that a microbial shift favoring bacterial degradation takes place when a nonhistory soil acquires enhanced degradation capacity. Thus, enhanced degradation may involve both quantitative and qualitative changes in the specific activity of microbial species and in the concomitant pesticide degradation process in soil. The impact of transfemng microorganisms from a culture to the hostile soil environment is a crucial factor that must be taken into consideration when soil inoculation experiments are conducted. Goldstein et al. (1985) reported some of the reasons for failure of inoculation to enhance biodegradation. The fact that inoculation of either MBC-history or nonhistory soils with a mixed bacterial culture from the inactive phase in the fluctuating retransfered culture had no effect on the MBC degradation rate may be attributed to some of the reasons mentioned. The same reasons may apply to inoculations with the fungal preparations; however, these would not include a diminishing of the fungal population in the soil, since this parameter was monitored. Probing the processes involved in the fate of pesticides in soil and determining the mechanisms that govern these processes are prerequisites for proper exploitation of the increasingly diminishing arsenal of agrochemicals at the disposal of modem agriculture. The knowledge accumulating in this field should be utilized to develop measures to control the persistence of xenobiotic compounds in the environment. Properly managed, it might also allow for better control with reduced application rates. This would insure that the required pesticide activity would be available when needed, while possessing the valuable option of enhancing the dissipation of toxic residues once they pose a threat.

Acknowledgements This work was supported by The Seagram Fund for Research Development and Training in Soil and Water. O.Y. was

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