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Microbial degradation of mefenoxam in rhizosphere of Zinnia angustifolia
Chemosphere 44 (2001) 577±582
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Microbial degradation of mefenoxam in rhizosphere of Zinnia angustifolia Seema G. Pai a, Melissa B. Riley b, N.D. Camper a
b,*
Department of Microbiology and Molecular Medicine, Clemson University,Clemson, SC 29634-0377, USA b Department of Plant Pathology and Physiology, Clemson University, Clemson, SC 29634-0377, USA Received 14 June 2000; accepted 24 August 2000
Abstract The fate of the fungicide mefenoxam was studied in a containerized rhizosphere system. The rhizosphere system used Zinnia angustifolia (Tropic Snow) in a bark/sand potting mix and was compared to bulk potting mix (no plants). Rhizosphere microbial populations were allowed to establish for 3 weeks prior to fungicide addition (20 lg per g mix). Mefenoxam and degradation product concentrations were determined by High HPLC or capillary electrophoresis after extraction. Seventy eight percent of the fungicide originally applied to the rhizosphere was degraded after 21 days compared to 44% in bulk system (no plant). The primary degradation product was the free acid N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine, which accounted for 71% of the applied parent chemical after 30 days. N-(2,6dimethylphenyl)-acetamide was also detected, but in lesser amounts. Bacterial populations in the rhizosphere increased during the 30-day period, which correlated with an increase in degradation of the parent compound. Pure cultures of Pseudomonas ¯uorescens and Chrysobacterium indologenes isolated from the rhizosphere system could degrade the applied fungicide 10 lg=ml almost completely to the free acid within 54 h. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Fungicide; Rhizosphere; Zinna; Degradation
1. Introduction The rhizosphere is a zone of increased microbial activity and biomass at the root-soil interface ®rst described by Hiltner in 1904 (Curl and Truelove, 1986). Thus, it contains higher levels of microbial activity, diversity and biomass (Knaebel and Vestal, 1994). This increase in microbial growth and activity may be involved in increased xenobiotic degradation rates (Anderson et al., 1993). Microbial transformation of these organic compounds in the rhizosphere has focused
*
Corresponding author. Tel.: +1-864-656-5743; fax: +1-864656-0274. E-mail address: [email protected] (N.D. Camper).
mainly on agricultural chemicals, and a number of researchers have described increased pesticide degradation in the rhizospheres of a variety of plant species (see Table 1 in Anderson et al., 1993). The structural diversity represented in these studies suggest the involvement of microbial consortia rather than an individual community member or particular species of bacterium. Seibert et al. (1981) detected increased atrazine degradation in the rhizosphere of corn which was correlated with higher dehydrogenase activity in the rhizosphere soil. Enhanced atrazine degradation was observed corn in a containerized system (Costa, 1998). Enhanced degradation of 14 C-parathion in rice rhizosphere was reported by Reddy and Sethunathan (1983). More recent studies have shown accelerated degradation of nonagricultural chemicals in the soil rhizosphere of a variety of plants;
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 0 ) 0 0 3 6 8 - 4
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S.G. Pai et al. / Chemosphere 44 (2001) 577±582
Table 1 Mefenoxam degradation and metabolite levels in rhizosphere of Z. angustifoliaa
a b
No. days
Mefenoxam Concn (lg=ml)
%
CGA-62828b Concn (lg=ml)
%
CGA-42447b Concn (lg=ml)
%
0 6 14 21 30
233.7 4.1 193.2 4.1 105.7 18.2 40.2 1.5 8.2 0.9
100 82.7 45.2 17.2 3.5
nd 4.0 2.7 77.7 9.6 134.6 7.7 165.6 5.2
±
nd nd 6.2 0.03 8.9 1.1 10.5 0.8
± ± 2.7 3.8 4.5
1.7 33.2 57.6 70.9
Data given as lg/ml S.D. and % of control; nd none detected. CGA-62826 N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine and CGA-42447 N-(2,6-dimethylphenyl)-acetamide.
e.g., enhanced degradation of 1,1,2-trichloroethylene in rhizosphere soils (Walton and Anderson, 1990). Mefenoxam (R- isomer of metalaxyl) is an acylanilide fungicide with residual and systemic activity against plant pathogens. It is widely used for control of diseases caused by foliar and soil-borne oomycetous fungi of the order Peronosporales. Degradation of metalaxyl has been reported in agricultural soils of tobacco, citrus, avocado and corn (Droby and Coey, 1991). In agricultural soils, organisms such as Trichoderma, Fusarium, Pseudomonas and Bacillus species were capable of degrading the fungicide (Bailey and Coey, 1986). There is, however, no information available on the in¯uence of the rhizosphere on its degradation. No studies have been reported on the fate of metalaxyl (mefenoxam) in a containerized (potted) system, although it is commonly used in nursery operations in the ornamental industry. This study focused on the fate of mefenoxam in a containerized system with the ornamental plant, Zinnia angustifolia. Objectives were to: (a) determine degradation of mefenoxam (including identi®cation and quanti®cation of degradation products) in the rhizosphere compared to bulk potting mix (no plant); and (b) determine the ability of isolated bacteria to degrade the fungicide.
2. Materials and methods 2.1. Chemicals Analytical grade mefenoxam [(R)-N-(2,6-dimethylphenyl)-(methoxyacetylamino) propionic acid methyl ester] and its principal degradation products were provided by Novartis Crop Protection, Greensboro, NC. Solvents used in extractions and HPLC analysis were HPLC grade (Burdick and Jackson, Muskegon, MI). Bacteriological media (Tryptic Soy Broth-TSB and Tryptic Soy Agar-TSA; DIFCO Laboratories, Detroit, MI) were used for monitoring soil sterility, for isolation and culture of soil bacteria, maintenance of pure cultures.
2.2. Potting Matrix and Plants The potting mix used was a 45:55 mix of sand and bark (Metro-Mix 300 Growing Medium, supplied by Carolina Nurseries, Moncks Corner, SC), pH 5:6 0:1. Inorganic nutrients (Osmocote-Vegetable and Bedding Plant Food and Peters All-Purpose Plant Food) were added at the time of planting. Sterile and bulk potting mix were used as controls. Potting mix (6 kg) was sterilized by autoclaving for 90 min on three consecutive days at 121°C and 1:05 kg=cm2 . Sterility of potting mix was determined by the dilution plate technique. A 5 g sample was mixed with 100 ml sterile water and placed on the shaker at 200 rpm for 30 min. Serial dilutions (10 3 , 10 4 and 10 5 ) were made in sterile water and 0.1 ml of each dilution was inoculated onto sterile TSA plates by the spread plate method (three replicates for each dilution). Agar medium was sterilized by autoclaving for 45 min at 121°C and 1:05 kg=cm2 . Plates were observed for growth of colonies after 24 and 48 h of incubation at 28°C. The annual dicotyledon, Z. angustifolia, was grown from seed in a growth chamber maintained at 28°C and a 16-h photoperiod (light intensity 20 lmol. 2.3. Rhizosphere Studies Plastic containers (14 cm diameter 10 cm height) were used to grow experimental plants. When plants reached a height of about 17 cm, healthy plants with well-developed roots were selected for experiments. Plant roots were washed with sterile, deionized water prior to transfer of plants to pots containing 300 g of potting mix. Rhizosphere conditions were allowed to develop for 3 weeks in the pots. Pots were then amended with mefenoxam to give a ®nal concentration of 20 lg/g of potting mix. Bulk and sterile potting mix (300 g each) served as controls. Bacteria were then isolated from the rhizosphere and bacterial populations were quantitated. Predominant bacterial types were identi®ed by gas chromatography (GC) of the fatty acid methyl esters (FAME Pro®le; Miller and Berger, 1985) and microbial identi®cation software (MIDI, Inc., Newark, DE).
S.G. Pai et al. / Chemosphere 44 (2001) 577±582
2.4. Microbial Analysis Samples were collected in triplicate on days 0, 3, 6, 14, 21 and 30 for determination of rhizosphere bacterial ¯ora. Pots were used only once for analysis. The plant was gently uprooted and loosely bound potting mix was removed from the root surface. The tightly adhering matrix was used for analysis. Root and rhizosphere potting mix were mixed with 10 ml of sterile water and placed on a shaker for 30 min at 200 rpm. Samples diluted in sterile water (10 3 , 10 4 and 10 5 dilutions) were inoculated onto agar plates by the spread plate technique (three replicates per dilution). Bacterial colonies were recorded after 24 h of incubation at 28°C and expressed as log of colony forming units (CFU) per gram of dry weight of potting mix. Dry weight was determined by drying the bulk potting mix until a constant weight was obtained. Colonies were isolated from the 10 4 dilution plates and sub-cultured. Microorganisms were identi®ed by analysis of fatty acid pro®les (FAME Pro®le analysis). Cultures were preserved by inoculating 750 ll of TSB and 20% glycerol, and storing at 20°C. 2.5. Chemical Analysis Potting mix was sampled in triplicate on days 0, 6, 14, 21 and 30 for extraction of mefenoxam and its degradation products. Samples (10 g) removed from each pot were placed in a conical ¯ask wrapped in aluminum foil. They were stored in at 4°C until processed. Samples were extracted with aqueous methanol (90%) solution. Solvent (30 ml) was added to potting mix samples and placed on a rotary shaker at 300 rpm for 1 h. The extract was ®ltered through Whatman Filter paper No. 1 followed by 0:45 lm membrane ®lter (Millipore Corp., Bedford, MA), then concentrated to approximately 2 ml in a ¯ash evaporator and dissolved in 20 ml of water containing 200 ll of 0.2M HCl. The fungicide was extracted by solid-phase extraction (500 mg C18 octadecyl column; Burdick and Jackson, Muskegon, MI). Columns were conditioned with 3 ml of ethyl acetate, followed by 2 ml of methanol and 1 ml of water under vacuum. Water (1 ml) was then added without vacuum. The solution was ®ltered through the column; the column was then rinsed with 1 ml of water and allowed to dry. Mefenoxam was eluted using HPLC grade acetonitrile (7.5 ml). Eluted extract was concentrated to 2 ml under nitrogen and ®ltered through a 0.2 l nylon disc (Gelman Sciences, Ann Arbor, MI) into an amber autosampler vial. Samples were stored at 20°C prior to analysis. Analysis was via by HPLC (Varian Star-9000. Varian Associates, Sugar Land, TX) on a reverse phase C18 HPLC column (15 cm 4:6 mm, Regis Inc., Morton Grove, IL). The solvent phase consisted of a gradient of acetonitrile and water (from 25% acetonitrile to 40%
579
during 30-min). Detection wavelength was set at 232 nm. Retention times of mefenoxam, the acid metabolite and acetamide derivative were 15.5, 3.2 and 4.2, respectively. 2.6. Pure Culture Studies Pure cultures were grown in TSA medium (50 ml) amended with mefenoxam (10 lg/ml). Inoculated ¯asks were wrapped in aluminum foil and placed on a rotary shaker at 300 rpm. Samples (1 ml) were removed in triplicate at 0, 1, 2, 26 and 54 h. Samples were ®ltered through a 0.2 lm membrane (Millipore Corp., Bedford, MA) and transferred to autosampler vials. Standards of mefenoxam (1 ml) of 20 lg/ml concentration prepared in TSB, and a culture blank (2-h culture ®ltrate without mefenoxam), were used as controls. Detection and quantitation of mefenoxam and degradation products was done by capillary electrophoresis (Hewlett Packard Instruments, Palo Alto, CA). The capillary column was conditioned by ¯ushing 1 M NaOH for 20 min followed by 0.1 M NaOH for 20 min and buer (50 mM borate, 100 mM SDS, pH 9.3) for 15 min. Viability of the culture was determined by plating on TSA medium at each sampling time. 2.7. Data analysis All experiments contained three replicates and were repeated. No signi®cant variation <5% was observed between replicates; therefore, data from replicate experiments were pooled for analysis.. Statistical analysis was carried out using SAS (SAS Institute, Cary, NC). 3. Results and discussion Degradation of mefenoxam in the rhizosphere of Z. angustifolia was signi®cantly greater than in bulk potting mix (Fig. 1). Degradation in the rhizosphere was about 78% of the applied chemical on day 21 and 96% on day 30 as compared to 44% and 55%, respectively, in bulk potting mix. Half-life of the fungicide was 14 days in the rhizosphere, while it was almost 30 days in bulk potting mix. Minimal loss of the parent fungicide was observed in sterile potting mix, which may be attributed to photolysis and/or leaching of chemical from the container. Previous soil studies with tobacco, citrus, avocado and corn showed a half-life of 6 ±12 days with the acid derivative as the main degradation product (Droby and Coey, 1991). The principal degradation product observed was the acid metabolite N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine, which accounted for nearly 71% of the applied chemical in the rhizosphere after 30 days (Table 1). The decrease in parent compound corresponded to an increase in the principal degradation
580
S.G. Pai et al. / Chemosphere 44 (2001) 577±582
Fig. 1. Mefenoxam degradation in rhizosphere of Z. angustifolia, bulk and sterile potting mix (sand/bark). Data presented as concentration of mefenoxam (ppm) as a function of incubation time in days. Error bars represent one S.D.
product. The second metabolite detected was N-(2,6dimethylphenyl)-acetamide; it accounted for 4.5% of the applied chemical in the rhizosphere on day 30 (Table 1) . An increase in bacterial populations correlated with the increase in rate of degradation. Rhizosphere bacterial numbers increased twelve fold in 30 days increasing from 5:41 0:1 log c.f.u./g to 6:49 0:02 log c.f.u./g after 30 days (Fig. 2). The increased bacterial population
correlated with increased degradation of mefenoxam, implicating the bacterial ¯ora of the rhizosphere in active degradation of the fungicide. There was an increase in the bacterial population in bulk potting mix as well, although not to the same extent as in the rhizosphere, increasing from 5:04 0:02 log c.f.u./g to 5:50 0:03 log c.f.u./g in 30 days (Fig. 2). Despite this increase, the breakdown of mefenoxam in bulk potting mix was
Fig. 2. Bacterial populations in Z. angustifolia rhizosphere compared to bulk potting mix. Data presented as log colony forming units (cfu) per g potting mix as a function of incubation time in days. Error bars represent one S.D.
S.G. Pai et al. / Chemosphere 44 (2001) 577±582
about 55% of that in the rhizosphere after 30 days. This may imply that the plant rhizosphere ecosystem favored the propagation of bacterial ¯ora, which enhanced degradation of the chemical, while other bacterial populations remained constant or changed to a lesser extent. Sixteen dierent bacterial isolates, which appeared repeatedly in the rhizosphere and whose numbers were either constant or increased in the 30-day period were tested for their ability to degrade mefenoxam in pure culture (Table 2). In all cases, the Similarity Index for the FAME Pro®le Identi®cation program was >0.3; for the isolates identi®ed as Pseudomonas ¯uorescens and Chrysobacterium indologenes, the Similarity Index was 0.79 and 0.65, respectively. Several of these cultures degraded the fungicide slightly (2±32% of the applied chemical) over a period of 96 h. Two of the isolates, Pseudomonas ¯uorescens and Chrysobacterium indolog-
581
enes were capable of nearly 100% degradation. At the end of a 54-h experiment, 96% of applied mefenoxam was degraded in the Pseudomonas culture and 97% was degraded in the Chrysobacterium culture (Table 3). The acid metabolite CGA-62826 was the only degradation product detected in these cultures and accounted for approximately 69% of the applied chemical in the Chrysobacterium culture and 66% in the Pseudomonas culture at the end of 54 h (Table 3). These cultures may either not possess the mechanisms to degrade the acid derivative to the acetamide derivative, or the latter may have been formed in very low concentrations, which were not detectable. The enhanced rate of degradation may be due to both qualitative and quantitative changes in bacterial consortia in the rhizosphere in response to the chemical. The signi®cant increase in bacterial numbers correlates
Table 2 Mefenoxam degradation by pure cultures of rhizosphere bacterial isolatesa
a
Bacterial isolate
Concn at 0 h lg=ml
Concn at 96 h lg=ml
% degradation
Arthrobacter ilicis Bacillus brevis Bacillus cereus Bacillus coagulans Bacillus laterosporus Bacillus megaterium Bacillus pumilus Bacillus thuringiensis Burkhokderia cepacia Celluloemonas ¯avigena Chrysrobacterium indologenes Pseudomonas ¯uorescens Biotype C Pseudomonas ¯uorescens Biotype F Pseudomonas putida Pseudomonas syringae
14.9 1.5 11.2 2.3 9.3 0.9 16.0 2.6 11.8 0.5 12.6 2.4 7.1 1.2 8.9 0.7 12.7 0.3 15.3 2.5 16.7 2.0 7.5 0.4 6.6 1.3 14.3 1.5 13.1 0.2
14.2 1.2 10.1 1.1 8.9 0.3 14.7 0.8 11.5 0.1 10.3 1.8 7.0 0.6 6.2 1.2 9.6 0.6 14.0 1.9 nd 5.1 0.2 nd 11.7 1.1 11.4 0.8
4.7 9.8 4.3 8.1 2.5 18.3 1.4 30.3 24.4 8.5 100 32 100 18.2 12.9
Data presented for concentration of mefenoxam at 0 and 96 h of incubation ( S.D.; nd none detected).
Table 3 Fate of mefenoxam in pure cultures of Chrysobacteium indologenes and Pseudomonas ¯uorescens isolated from rhizosphere of Z. angustifoliaa Bacterial isolate
Sample time (h)
Mefenoxam concn lg=ml
%
CGA-62826 concn lg=ml
%
Chrysobacterium indologenes
0 1 2 26 54
16.7 2.0 16.2 0.8 16.1 0.9 1.2 0.01 0.57 0.02
100 97.1 96.6 7.2 3.4
nd nd nd 10.5 1.3 1.48 2.3
± ± ± 62.4 68.7
Pseudomonas ¯uorescens
0 1 2 26 54
6.57 1.3 5.8 0.1 5.8 0.8 0.73 0.01 0.26 0.04
100 88.3 88.2 11.1 3.9
nd nd nd 3.89 0.9 4.31 1.5
± ± ± 59.2 65.6
a Data presented as concentration of mefenoxam and CGA-62826 [N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine] as a function of sampling times ( S.D.; nd none detected).
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S.G. Pai et al. / Chemosphere 44 (2001) 577±582
with increased breakdown of mefenoxam. Some of the bacterial species isolated from the rhizosphere throughout the experiment were capable of degrading the chemical in pure culture. This provides evidence that rhizosphere bacterial ¯ora help to transform the fungicide. Sandmann and Loos (1984) found an increase in the number of 2,4-D-degrading bacteria in rhizosphere soils of previously untreated sugarcane. This study has laid the ground work for further research of the rhizosphere in¯uences on the fate of other chemicals in a containerized or pot system. The rhizosphere mix in containerized systems can be used for studies on fate of other pesticides used in ornamental nursery plant operations. Remediation of pesticides or nonagricultral chemicals in contaminated soils was not the focus of this study. However, demonstration of enhanced degradation of a pesticide in a containerized system (using a potting mix of sand and bark) could be used in remediation studies, or in decontamination of spray-tank rinsates or contanimated water. Rhizosphere degradation oers promise for in situ bioremediation (Shann and Boyle, 1994; Walton et al., 1994a,b). Acknowledgements Technical contribution No. 4485 of the South Carolina Agricultural Experiment Station, Clemson University. Funding supplied in part by SCAES Competitive Grant program. Mefenoxam and reference standards for degradation products were generously provided by Novartis Crop Protection, Inc. References Andreson, T.A., Guthrie, E.A., Walton, B.T., 1993. Bioremediation in the rhizosphere. Environ. Sci. Technol. 27, 2630± 2636. Bailey, A.M., Coey, M.D., 1986. Characterization of microorganisms involved in accelerated biodegradation of metalaxyl and metolachlor in soils. Can. J. Microbiol. 32, 562±569.
Costa, R.M., 1998. Herbicide-Microbe Interactions in the Rhizosphere. M.S. Thesis. Clemson University, Clemson, SC, p. 68. Curl, E.A., Truelove, B., 1986. The Rhizosphere, Springer, Berlin, p. 288. Droby, S., Coey, M.D., 1991. Biodegradation process and the nature of metabolism of metalaxyl in soil. Ann. Appl. Biol. 118, 543±553. Knaebel, D.B., Vestal, J.R., 1994. Intact rhizosphere microbial communities used to study microbial biodegradation in agricultural and natural soils. In: Anderson, T.A., Coats, J.R. (Eds.), Bioremediation through rhizosphere technology, ACS Symposium Series No. 563, pp. 56±70. Miller, L., Berger, T., 1985. Bacteria identi®cation by gas chromatography of whole fatty acids. Gas Chromatography Application Note 228-41. Hewlett-Packard Co., Microbial ID Inc., Newark, DE. Reddy, B.R., Sethunathan, N., 1983. Mineralization of parathion in the rice rhizosphere. Appl. Environ. Microbiol. 45, 826±829. Sandmann, E.R.I.C., Loos, M.A., 1984. Enumeration of 2,4± D±degrading microorganisms using indicator media: High populations associated with sugarcane Saccharum ocinarum. Chemosphere 13, 1073±1084. Seibert, K., Fuehr, F., Cheng, H.H., 1981. In: Theory and Practical Use of Soil-Applied Herbicides Symposium. European Weed Resource Society, Paris, pp. 137±146. Shann, J.R., Boyle, J.J., 1994. In¯uence of plant species on in situ rhizosphere degradation. In: Anderson, T.A., Coats, J.R. (Eds.), Bioremediation through Rhizosphere Technology. ACS Symposium Series No. 563, pp. 70±81. Walton, B.T., Anderson, T.A., 1990. Microbial mineralization of TCE in the rhizosphere; potential application to biological remediation of waste sites. Appl. Environ. Microbiol. 56, 1012±1016. Walton, B.T., Hoylman, A.M., Perez, M.M., Anderson, T.A., Johnson, T.R., Guthrie, E.A., Christman, R.F., 1994a. Rhizosphere microbial communities as a plant defense against toxic substances in soils. In: Anderson, T.A., Coats, J.R. (Eds.), Bioremediation through Rhizosphere Technology. ACS. Symposium Series No. 563, pp. 82±93. Walton, B.T., Guthrie, E.A., Hoylman, A.M., 1994b. Toxicant degradation in the rhizosphere. In: Anderson, T.A., Coats, J.R. (Eds.), Bioremediation through Rhizosphere Technology. ACS. Symposium Series, No. 563, pp. 11±25.
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Microbial degradation of mefenoxam in rhizosphere of Zinnia angustifolia Seema G. Pai a, Melissa B. Riley b, N.D. Camper a
b,*
Department of Microbiology and Molecular Medicine, Clemson University,Clemson, SC 29634-0377, USA b Department of Plant Pathology and Physiology, Clemson University, Clemson, SC 29634-0377, USA Received 14 June 2000; accepted 24 August 2000
Abstract The fate of the fungicide mefenoxam was studied in a containerized rhizosphere system. The rhizosphere system used Zinnia angustifolia (Tropic Snow) in a bark/sand potting mix and was compared to bulk potting mix (no plants). Rhizosphere microbial populations were allowed to establish for 3 weeks prior to fungicide addition (20 lg per g mix). Mefenoxam and degradation product concentrations were determined by High HPLC or capillary electrophoresis after extraction. Seventy eight percent of the fungicide originally applied to the rhizosphere was degraded after 21 days compared to 44% in bulk system (no plant). The primary degradation product was the free acid N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine, which accounted for 71% of the applied parent chemical after 30 days. N-(2,6dimethylphenyl)-acetamide was also detected, but in lesser amounts. Bacterial populations in the rhizosphere increased during the 30-day period, which correlated with an increase in degradation of the parent compound. Pure cultures of Pseudomonas ¯uorescens and Chrysobacterium indologenes isolated from the rhizosphere system could degrade the applied fungicide 10 lg=ml almost completely to the free acid within 54 h. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Fungicide; Rhizosphere; Zinna; Degradation
1. Introduction The rhizosphere is a zone of increased microbial activity and biomass at the root-soil interface ®rst described by Hiltner in 1904 (Curl and Truelove, 1986). Thus, it contains higher levels of microbial activity, diversity and biomass (Knaebel and Vestal, 1994). This increase in microbial growth and activity may be involved in increased xenobiotic degradation rates (Anderson et al., 1993). Microbial transformation of these organic compounds in the rhizosphere has focused
*
Corresponding author. Tel.: +1-864-656-5743; fax: +1-864656-0274. E-mail address: [email protected] (N.D. Camper).
mainly on agricultural chemicals, and a number of researchers have described increased pesticide degradation in the rhizospheres of a variety of plant species (see Table 1 in Anderson et al., 1993). The structural diversity represented in these studies suggest the involvement of microbial consortia rather than an individual community member or particular species of bacterium. Seibert et al. (1981) detected increased atrazine degradation in the rhizosphere of corn which was correlated with higher dehydrogenase activity in the rhizosphere soil. Enhanced atrazine degradation was observed corn in a containerized system (Costa, 1998). Enhanced degradation of 14 C-parathion in rice rhizosphere was reported by Reddy and Sethunathan (1983). More recent studies have shown accelerated degradation of nonagricultural chemicals in the soil rhizosphere of a variety of plants;
0045-6535/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 0 ) 0 0 3 6 8 - 4
578
S.G. Pai et al. / Chemosphere 44 (2001) 577±582
Table 1 Mefenoxam degradation and metabolite levels in rhizosphere of Z. angustifoliaa
a b
No. days
Mefenoxam Concn (lg=ml)
%
CGA-62828b Concn (lg=ml)
%
CGA-42447b Concn (lg=ml)
%
0 6 14 21 30
233.7 4.1 193.2 4.1 105.7 18.2 40.2 1.5 8.2 0.9
100 82.7 45.2 17.2 3.5
nd 4.0 2.7 77.7 9.6 134.6 7.7 165.6 5.2
±
nd nd 6.2 0.03 8.9 1.1 10.5 0.8
± ± 2.7 3.8 4.5
1.7 33.2 57.6 70.9
Data given as lg/ml S.D. and % of control; nd none detected. CGA-62826 N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine and CGA-42447 N-(2,6-dimethylphenyl)-acetamide.
e.g., enhanced degradation of 1,1,2-trichloroethylene in rhizosphere soils (Walton and Anderson, 1990). Mefenoxam (R- isomer of metalaxyl) is an acylanilide fungicide with residual and systemic activity against plant pathogens. It is widely used for control of diseases caused by foliar and soil-borne oomycetous fungi of the order Peronosporales. Degradation of metalaxyl has been reported in agricultural soils of tobacco, citrus, avocado and corn (Droby and Coey, 1991). In agricultural soils, organisms such as Trichoderma, Fusarium, Pseudomonas and Bacillus species were capable of degrading the fungicide (Bailey and Coey, 1986). There is, however, no information available on the in¯uence of the rhizosphere on its degradation. No studies have been reported on the fate of metalaxyl (mefenoxam) in a containerized (potted) system, although it is commonly used in nursery operations in the ornamental industry. This study focused on the fate of mefenoxam in a containerized system with the ornamental plant, Zinnia angustifolia. Objectives were to: (a) determine degradation of mefenoxam (including identi®cation and quanti®cation of degradation products) in the rhizosphere compared to bulk potting mix (no plant); and (b) determine the ability of isolated bacteria to degrade the fungicide.
2. Materials and methods 2.1. Chemicals Analytical grade mefenoxam [(R)-N-(2,6-dimethylphenyl)-(methoxyacetylamino) propionic acid methyl ester] and its principal degradation products were provided by Novartis Crop Protection, Greensboro, NC. Solvents used in extractions and HPLC analysis were HPLC grade (Burdick and Jackson, Muskegon, MI). Bacteriological media (Tryptic Soy Broth-TSB and Tryptic Soy Agar-TSA; DIFCO Laboratories, Detroit, MI) were used for monitoring soil sterility, for isolation and culture of soil bacteria, maintenance of pure cultures.
2.2. Potting Matrix and Plants The potting mix used was a 45:55 mix of sand and bark (Metro-Mix 300 Growing Medium, supplied by Carolina Nurseries, Moncks Corner, SC), pH 5:6 0:1. Inorganic nutrients (Osmocote-Vegetable and Bedding Plant Food and Peters All-Purpose Plant Food) were added at the time of planting. Sterile and bulk potting mix were used as controls. Potting mix (6 kg) was sterilized by autoclaving for 90 min on three consecutive days at 121°C and 1:05 kg=cm2 . Sterility of potting mix was determined by the dilution plate technique. A 5 g sample was mixed with 100 ml sterile water and placed on the shaker at 200 rpm for 30 min. Serial dilutions (10 3 , 10 4 and 10 5 ) were made in sterile water and 0.1 ml of each dilution was inoculated onto sterile TSA plates by the spread plate method (three replicates for each dilution). Agar medium was sterilized by autoclaving for 45 min at 121°C and 1:05 kg=cm2 . Plates were observed for growth of colonies after 24 and 48 h of incubation at 28°C. The annual dicotyledon, Z. angustifolia, was grown from seed in a growth chamber maintained at 28°C and a 16-h photoperiod (light intensity 20 lmol. 2.3. Rhizosphere Studies Plastic containers (14 cm diameter 10 cm height) were used to grow experimental plants. When plants reached a height of about 17 cm, healthy plants with well-developed roots were selected for experiments. Plant roots were washed with sterile, deionized water prior to transfer of plants to pots containing 300 g of potting mix. Rhizosphere conditions were allowed to develop for 3 weeks in the pots. Pots were then amended with mefenoxam to give a ®nal concentration of 20 lg/g of potting mix. Bulk and sterile potting mix (300 g each) served as controls. Bacteria were then isolated from the rhizosphere and bacterial populations were quantitated. Predominant bacterial types were identi®ed by gas chromatography (GC) of the fatty acid methyl esters (FAME Pro®le; Miller and Berger, 1985) and microbial identi®cation software (MIDI, Inc., Newark, DE).
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2.4. Microbial Analysis Samples were collected in triplicate on days 0, 3, 6, 14, 21 and 30 for determination of rhizosphere bacterial ¯ora. Pots were used only once for analysis. The plant was gently uprooted and loosely bound potting mix was removed from the root surface. The tightly adhering matrix was used for analysis. Root and rhizosphere potting mix were mixed with 10 ml of sterile water and placed on a shaker for 30 min at 200 rpm. Samples diluted in sterile water (10 3 , 10 4 and 10 5 dilutions) were inoculated onto agar plates by the spread plate technique (three replicates per dilution). Bacterial colonies were recorded after 24 h of incubation at 28°C and expressed as log of colony forming units (CFU) per gram of dry weight of potting mix. Dry weight was determined by drying the bulk potting mix until a constant weight was obtained. Colonies were isolated from the 10 4 dilution plates and sub-cultured. Microorganisms were identi®ed by analysis of fatty acid pro®les (FAME Pro®le analysis). Cultures were preserved by inoculating 750 ll of TSB and 20% glycerol, and storing at 20°C. 2.5. Chemical Analysis Potting mix was sampled in triplicate on days 0, 6, 14, 21 and 30 for extraction of mefenoxam and its degradation products. Samples (10 g) removed from each pot were placed in a conical ¯ask wrapped in aluminum foil. They were stored in at 4°C until processed. Samples were extracted with aqueous methanol (90%) solution. Solvent (30 ml) was added to potting mix samples and placed on a rotary shaker at 300 rpm for 1 h. The extract was ®ltered through Whatman Filter paper No. 1 followed by 0:45 lm membrane ®lter (Millipore Corp., Bedford, MA), then concentrated to approximately 2 ml in a ¯ash evaporator and dissolved in 20 ml of water containing 200 ll of 0.2M HCl. The fungicide was extracted by solid-phase extraction (500 mg C18 octadecyl column; Burdick and Jackson, Muskegon, MI). Columns were conditioned with 3 ml of ethyl acetate, followed by 2 ml of methanol and 1 ml of water under vacuum. Water (1 ml) was then added without vacuum. The solution was ®ltered through the column; the column was then rinsed with 1 ml of water and allowed to dry. Mefenoxam was eluted using HPLC grade acetonitrile (7.5 ml). Eluted extract was concentrated to 2 ml under nitrogen and ®ltered through a 0.2 l nylon disc (Gelman Sciences, Ann Arbor, MI) into an amber autosampler vial. Samples were stored at 20°C prior to analysis. Analysis was via by HPLC (Varian Star-9000. Varian Associates, Sugar Land, TX) on a reverse phase C18 HPLC column (15 cm 4:6 mm, Regis Inc., Morton Grove, IL). The solvent phase consisted of a gradient of acetonitrile and water (from 25% acetonitrile to 40%
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during 30-min). Detection wavelength was set at 232 nm. Retention times of mefenoxam, the acid metabolite and acetamide derivative were 15.5, 3.2 and 4.2, respectively. 2.6. Pure Culture Studies Pure cultures were grown in TSA medium (50 ml) amended with mefenoxam (10 lg/ml). Inoculated ¯asks were wrapped in aluminum foil and placed on a rotary shaker at 300 rpm. Samples (1 ml) were removed in triplicate at 0, 1, 2, 26 and 54 h. Samples were ®ltered through a 0.2 lm membrane (Millipore Corp., Bedford, MA) and transferred to autosampler vials. Standards of mefenoxam (1 ml) of 20 lg/ml concentration prepared in TSB, and a culture blank (2-h culture ®ltrate without mefenoxam), were used as controls. Detection and quantitation of mefenoxam and degradation products was done by capillary electrophoresis (Hewlett Packard Instruments, Palo Alto, CA). The capillary column was conditioned by ¯ushing 1 M NaOH for 20 min followed by 0.1 M NaOH for 20 min and buer (50 mM borate, 100 mM SDS, pH 9.3) for 15 min. Viability of the culture was determined by plating on TSA medium at each sampling time. 2.7. Data analysis All experiments contained three replicates and were repeated. No signi®cant variation <5% was observed between replicates; therefore, data from replicate experiments were pooled for analysis.. Statistical analysis was carried out using SAS (SAS Institute, Cary, NC). 3. Results and discussion Degradation of mefenoxam in the rhizosphere of Z. angustifolia was signi®cantly greater than in bulk potting mix (Fig. 1). Degradation in the rhizosphere was about 78% of the applied chemical on day 21 and 96% on day 30 as compared to 44% and 55%, respectively, in bulk potting mix. Half-life of the fungicide was 14 days in the rhizosphere, while it was almost 30 days in bulk potting mix. Minimal loss of the parent fungicide was observed in sterile potting mix, which may be attributed to photolysis and/or leaching of chemical from the container. Previous soil studies with tobacco, citrus, avocado and corn showed a half-life of 6 ±12 days with the acid derivative as the main degradation product (Droby and Coey, 1991). The principal degradation product observed was the acid metabolite N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine, which accounted for nearly 71% of the applied chemical in the rhizosphere after 30 days (Table 1). The decrease in parent compound corresponded to an increase in the principal degradation
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Fig. 1. Mefenoxam degradation in rhizosphere of Z. angustifolia, bulk and sterile potting mix (sand/bark). Data presented as concentration of mefenoxam (ppm) as a function of incubation time in days. Error bars represent one S.D.
product. The second metabolite detected was N-(2,6dimethylphenyl)-acetamide; it accounted for 4.5% of the applied chemical in the rhizosphere on day 30 (Table 1) . An increase in bacterial populations correlated with the increase in rate of degradation. Rhizosphere bacterial numbers increased twelve fold in 30 days increasing from 5:41 0:1 log c.f.u./g to 6:49 0:02 log c.f.u./g after 30 days (Fig. 2). The increased bacterial population
correlated with increased degradation of mefenoxam, implicating the bacterial ¯ora of the rhizosphere in active degradation of the fungicide. There was an increase in the bacterial population in bulk potting mix as well, although not to the same extent as in the rhizosphere, increasing from 5:04 0:02 log c.f.u./g to 5:50 0:03 log c.f.u./g in 30 days (Fig. 2). Despite this increase, the breakdown of mefenoxam in bulk potting mix was
Fig. 2. Bacterial populations in Z. angustifolia rhizosphere compared to bulk potting mix. Data presented as log colony forming units (cfu) per g potting mix as a function of incubation time in days. Error bars represent one S.D.
S.G. Pai et al. / Chemosphere 44 (2001) 577±582
about 55% of that in the rhizosphere after 30 days. This may imply that the plant rhizosphere ecosystem favored the propagation of bacterial ¯ora, which enhanced degradation of the chemical, while other bacterial populations remained constant or changed to a lesser extent. Sixteen dierent bacterial isolates, which appeared repeatedly in the rhizosphere and whose numbers were either constant or increased in the 30-day period were tested for their ability to degrade mefenoxam in pure culture (Table 2). In all cases, the Similarity Index for the FAME Pro®le Identi®cation program was >0.3; for the isolates identi®ed as Pseudomonas ¯uorescens and Chrysobacterium indologenes, the Similarity Index was 0.79 and 0.65, respectively. Several of these cultures degraded the fungicide slightly (2±32% of the applied chemical) over a period of 96 h. Two of the isolates, Pseudomonas ¯uorescens and Chrysobacterium indolog-
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enes were capable of nearly 100% degradation. At the end of a 54-h experiment, 96% of applied mefenoxam was degraded in the Pseudomonas culture and 97% was degraded in the Chrysobacterium culture (Table 3). The acid metabolite CGA-62826 was the only degradation product detected in these cultures and accounted for approximately 69% of the applied chemical in the Chrysobacterium culture and 66% in the Pseudomonas culture at the end of 54 h (Table 3). These cultures may either not possess the mechanisms to degrade the acid derivative to the acetamide derivative, or the latter may have been formed in very low concentrations, which were not detectable. The enhanced rate of degradation may be due to both qualitative and quantitative changes in bacterial consortia in the rhizosphere in response to the chemical. The signi®cant increase in bacterial numbers correlates
Table 2 Mefenoxam degradation by pure cultures of rhizosphere bacterial isolatesa
a
Bacterial isolate
Concn at 0 h lg=ml
Concn at 96 h lg=ml
% degradation
Arthrobacter ilicis Bacillus brevis Bacillus cereus Bacillus coagulans Bacillus laterosporus Bacillus megaterium Bacillus pumilus Bacillus thuringiensis Burkhokderia cepacia Celluloemonas ¯avigena Chrysrobacterium indologenes Pseudomonas ¯uorescens Biotype C Pseudomonas ¯uorescens Biotype F Pseudomonas putida Pseudomonas syringae
14.9 1.5 11.2 2.3 9.3 0.9 16.0 2.6 11.8 0.5 12.6 2.4 7.1 1.2 8.9 0.7 12.7 0.3 15.3 2.5 16.7 2.0 7.5 0.4 6.6 1.3 14.3 1.5 13.1 0.2
14.2 1.2 10.1 1.1 8.9 0.3 14.7 0.8 11.5 0.1 10.3 1.8 7.0 0.6 6.2 1.2 9.6 0.6 14.0 1.9 nd 5.1 0.2 nd 11.7 1.1 11.4 0.8
4.7 9.8 4.3 8.1 2.5 18.3 1.4 30.3 24.4 8.5 100 32 100 18.2 12.9
Data presented for concentration of mefenoxam at 0 and 96 h of incubation ( S.D.; nd none detected).
Table 3 Fate of mefenoxam in pure cultures of Chrysobacteium indologenes and Pseudomonas ¯uorescens isolated from rhizosphere of Z. angustifoliaa Bacterial isolate
Sample time (h)
Mefenoxam concn lg=ml
%
CGA-62826 concn lg=ml
%
Chrysobacterium indologenes
0 1 2 26 54
16.7 2.0 16.2 0.8 16.1 0.9 1.2 0.01 0.57 0.02
100 97.1 96.6 7.2 3.4
nd nd nd 10.5 1.3 1.48 2.3
± ± ± 62.4 68.7
Pseudomonas ¯uorescens
0 1 2 26 54
6.57 1.3 5.8 0.1 5.8 0.8 0.73 0.01 0.26 0.04
100 88.3 88.2 11.1 3.9
nd nd nd 3.89 0.9 4.31 1.5
± ± ± 59.2 65.6
a Data presented as concentration of mefenoxam and CGA-62826 [N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-DL-alanine] as a function of sampling times ( S.D.; nd none detected).
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with increased breakdown of mefenoxam. Some of the bacterial species isolated from the rhizosphere throughout the experiment were capable of degrading the chemical in pure culture. This provides evidence that rhizosphere bacterial ¯ora help to transform the fungicide. Sandmann and Loos (1984) found an increase in the number of 2,4-D-degrading bacteria in rhizosphere soils of previously untreated sugarcane. This study has laid the ground work for further research of the rhizosphere in¯uences on the fate of other chemicals in a containerized or pot system. The rhizosphere mix in containerized systems can be used for studies on fate of other pesticides used in ornamental nursery plant operations. Remediation of pesticides or nonagricultral chemicals in contaminated soils was not the focus of this study. However, demonstration of enhanced degradation of a pesticide in a containerized system (using a potting mix of sand and bark) could be used in remediation studies, or in decontamination of spray-tank rinsates or contanimated water. Rhizosphere degradation oers promise for in situ bioremediation (Shann and Boyle, 1994; Walton et al., 1994a,b). Acknowledgements Technical contribution No. 4485 of the South Carolina Agricultural Experiment Station, Clemson University. Funding supplied in part by SCAES Competitive Grant program. Mefenoxam and reference standards for degradation products were generously provided by Novartis Crop Protection, Inc. References Andreson, T.A., Guthrie, E.A., Walton, B.T., 1993. Bioremediation in the rhizosphere. Environ. Sci. Technol. 27, 2630± 2636. Bailey, A.M., Coey, M.D., 1986. Characterization of microorganisms involved in accelerated biodegradation of metalaxyl and metolachlor in soils. Can. J. Microbiol. 32, 562±569.
Costa, R.M., 1998. Herbicide-Microbe Interactions in the Rhizosphere. M.S. Thesis. Clemson University, Clemson, SC, p. 68. Curl, E.A., Truelove, B., 1986. The Rhizosphere, Springer, Berlin, p. 288. Droby, S., Coey, M.D., 1991. Biodegradation process and the nature of metabolism of metalaxyl in soil. Ann. Appl. Biol. 118, 543±553. Knaebel, D.B., Vestal, J.R., 1994. Intact rhizosphere microbial communities used to study microbial biodegradation in agricultural and natural soils. In: Anderson, T.A., Coats, J.R. (Eds.), Bioremediation through rhizosphere technology, ACS Symposium Series No. 563, pp. 56±70. Miller, L., Berger, T., 1985. Bacteria identi®cation by gas chromatography of whole fatty acids. Gas Chromatography Application Note 228-41. Hewlett-Packard Co., Microbial ID Inc., Newark, DE. Reddy, B.R., Sethunathan, N., 1983. Mineralization of parathion in the rice rhizosphere. Appl. Environ. Microbiol. 45, 826±829. Sandmann, E.R.I.C., Loos, M.A., 1984. Enumeration of 2,4± D±degrading microorganisms using indicator media: High populations associated with sugarcane Saccharum ocinarum. Chemosphere 13, 1073±1084. Seibert, K., Fuehr, F., Cheng, H.H., 1981. In: Theory and Practical Use of Soil-Applied Herbicides Symposium. European Weed Resource Society, Paris, pp. 137±146. Shann, J.R., Boyle, J.J., 1994. In¯uence of plant species on in situ rhizosphere degradation. In: Anderson, T.A., Coats, J.R. (Eds.), Bioremediation through Rhizosphere Technology. ACS Symposium Series No. 563, pp. 70±81. Walton, B.T., Anderson, T.A., 1990. Microbial mineralization of TCE in the rhizosphere; potential application to biological remediation of waste sites. Appl. Environ. Microbiol. 56, 1012±1016. Walton, B.T., Hoylman, A.M., Perez, M.M., Anderson, T.A., Johnson, T.R., Guthrie, E.A., Christman, R.F., 1994a. Rhizosphere microbial communities as a plant defense against toxic substances in soils. In: Anderson, T.A., Coats, J.R. (Eds.), Bioremediation through Rhizosphere Technology. ACS. Symposium Series No. 563, pp. 82±93. Walton, B.T., Guthrie, E.A., Hoylman, A.M., 1994b. Toxicant degradation in the rhizosphere. In: Anderson, T.A., Coats, J.R. (Eds.), Bioremediation through Rhizosphere Technology. ACS. Symposium Series, No. 563, pp. 11±25.