Isolation of Microorganisms from a Texas Soil Capable of DegradingUrea Derivative Herbicides1

C. E. LOPEZ AND JAMES I. KiRKWooo2

ABSTRACTSamples of Hockley fine sandy loam were perfused with 500

tag/ml concentrations of monuron 3-(p-chlorophenyl)-l, 1-di-methylurea or diuron 3-(3,4-dichlorophenyl)-l, 1-dimethylureafor 2 weeks to isolate microorganisms tolerant to these concen-trations. A fungus of the genus Fusarium and two unidentifiedbacteria were isolated by plating out small samples of thetreated soil on Czapek's solution agar without sugar with 20ppm of both herbicides added. The three isolates were testedfor their ability to grow in solid and liquid media with dif-ferent concentrations of the herbicides as sources of carbon.The same optimal growth of the fungus on Czapek's solutionagar without sugar with 1 g/liter concentration of yeast ex-tract was attained at 10 and 20 ppm monuron. When the yeastextract was excluded, best growth was attained at 2 ppmmonuron. In Czapek's solution agar without sugar, and with 50mg/liter of yeast extract, the fungus produced the highestamount of dry mycelia with 20 ppm monuron.

Cultures of the three isolates in Czapek's solution agarwithout sugar containing 20 ppm diuron, including 0.5 ppm ofcarbonyl lj*C-diuron, were incubated for 2 weeks. The orga-nisms grew profusely, but no breakdown products of diuronwere detected from the extracts of these cultures as analyzedby TLC (thin layer chromatography) and strip scanning tech-niques. Approximately 3.5% of the added diuron was collectedas 14CC>2 from soil samples incubated for 80 days with carbonyllabeled 14C-diuron and a mixture of the three isolates. Periodicanalysis of soil samples from these experiments did not indi-cate any breakdown products of diuron apparently due toinsensitivity of the instrumentation and procedures. However,data from the fungal growth and COZ evolution experimentssuggest that these organisms can use monuron and diuronas a source of carbon.

Additional Index Words: Fusarium, monuron, diuron, herbi-cides degradation, soil microorganisms.

SINCE THEIR DISCOVERY more than two decades ago, ureaderivative herbicides have made an increasing contribu-

tion to weed control. More than 20 urea derivatives arenow available. Their versatility ranges from soil steri-lants to selective weed control for some crops. Monuron

3-(p-chlorophenyl)-l, 1-dimethylurea and diuron 3-(3,4-dichlorophenyl)-!, 1-dimethylurea were two of the firstcommercial products (3). These two have a greater inherentphytotoxicity and tend to persist more than most otherherbicides in their class (4). While their persistence makesthem good soil sterilants at high rates (above 22 kg/ha) where no vegetation is desired, their selective use incrops, even at very low rates (0.8 kg/ha), has been haz-ardous in some cases for subsequent crops (12). Investiga-tions have shown that degradation by soil microorganismsis one of the most important mechanisms of deactivationof these compounds in soils (8, 9, 14). Hill et al. (5)isolated a bacterium of the genus Pseudomonas from a soilin the eastern part of the US which could use monuron asan energy source in agar medium. Bacteria species of Ba-cillus, Sarcina, and Xanthomanas, and fungi species of As-pergillus and Penicillum were subsequently isolated whenyeast extract was added to the medium (5). Contrary tothese findings, Doxtader reported little or no microbialbreakdown of diuron in three western US soils after 4months of incubation (K. B. Doxtader, 1969. Soil, pesti-cides and quality of water. Unpublished report). This em-phasizes the need for more research on the breakdown ofthese compounds especially in arid region soils where resi-due problems are often most severe. This investigation wasdesigned to isolate microorganisms from a soil of the GulfCoast Prairie of Texas capable of using monuron and di-uron as sources of carbon which degrade these compounds.

MATERIALS AND METHODSSamples from the top 152 mm (6 inches) of a Hockley fine

sandy loam soil having the following characteristics—pH 5.8

310 SOIL SCI. SOC. AMER. PROC., VOL. 38, 1974

(1:1, soil/water); organic matter, 2%; CEC, 14.3 meq/100 gobtained from the Prairie View A. and M. College farm weremixed and about 25-g portions were perfused using a soilpercolator (13) and Czapek's solution (12) without sugar butwith monuron and diuron added to the solution at a concentra-tion of 500 jug/ml, respectively. After 2 weeks of perfusion,small amounts of treated soil were transferred to a culture me-dium consisting of Czapek's solution agar without sugar, con-taining 20 ppm of monuron and diuron, respectively. After 1week of incubation at 28C, a fungus of Fusarium and two un-identified bacterial species were isolated from the medium. Thethree isolates were then tested for their ability to grow in mediacontaining different concentrations of the herbicides.

Experiment 1. Growth of the Fusarium sp. in Agar in thePresence of Monuron and Diuron—The fungus was grown onCzapek's solution agar without sugar with 1 g/liter yeast ex-tract, and concentrations of monuron or diuron of 0, 5, 10, 20,50, 75, and 100 ppm. Four replicates of each concentrationwere inoculated with a 5-mm disc of a 2-day old culture of thefungus and were incubated at 28C. The diameters of the colo-nies were measured at 24-hour intervals and used as indices ofgrowth. A similar trial was conducted using the same mediumwith no yeast extract but at herbicide concentrations of 0, 2, 5,10, 20, and 40 ppm. Measurements were taken at 12-hour inter-vals. Streptomycin at 30 ppm was used to inhibit bacterialgrowth in both trials.

Experiment 2. Growth of Fusarium sp. in Liquid Mediumwith Different Monuron Concentrations—The fungus wasgrown in Czapek's solution without sugar with 50 mg/liter yeastextract and monuron concentrations of 0, 5, 10, 15, 20, 40, 60,and 80 ppm. Enough medium was prepared to make four repli-cations of 50 ml each for each concentration. The flasks with themedium were autoclaved as described above. When cool, 1 mlof a 1-week old liquid culture of the fungus was dispensed toeach flask and incubated at 28C for 8-days. The flasks wereshaken by hand every 6 to 8 hours. The fungal crop was har-vested by passing the content of each flask through a filterpaper which had been previously dried in a hot air oven at105C for 24 hours. The mycelia plus filter paper were driedand weighed as described above. The dry weight of the myceliawas calculated by difference.

Experiment 3. Analyses of the Liquid Cultures of the ThreeIsolates—The three organisms were grown individually in 50ml of Czapek's solution without sugar and 20 ppm diuron. Non-radioactive diuron and carbonyl labeled 14C-diuron were usedin a ratio of 19:1. A stock solution containing 24.44 /ig/ml witha specific activity of 209,709 dpm/ml was the source of theradioactive diuron. The medium was sterilized by filtration, andeach flask inoculated with 1 ml of 1-week old liquid culture ofthe respective organisms. The flasks were incubated for 2 weekswith continuous rotatory shaking at 200 rpm and SOC.

Analysis of Cultures—The cultures were extracted twice with25 ml of ether and the extracts reduced to dryness by flaskevaporation at 40—45C. The residues were taken up in 2 ml ofacetone and examined using TLC and strip scanning techniques.The residues were spotted on precoated sheets of Baker FlexSilica gel type S, and developed for 40 min in a solvent systemhaving benzene/acetone in the ratio of 2:1 (v/v). Nonradioac-tive standards of diuron, monomethyldiuron 3- (3,4-dichloro-phenyl) urea were spotted with the residues. Their Rf valueswere determined as seen under ultraviolet light and used toidentify the residue spots. Then the sheets were cut into stripsand run through a radiochromatogram strip scanner to deter-mine the distribution of the radioactivity for the extract of eachculture.

Experiment 4. 14CO2 Measurement from Incubated Soil withDiuron—Ten-gram samples of the soil (oven dry) were placedinto twelve 125-ml conical flasks. Eight of the flasks wereinoculated with 2 ml of a suspension of the three isolates,supplementary carbon and nitrogen sources, and diuron pre-pared by combining 25 mg/ml of soluble starch, 25 mg/ml of(NH2)2SO4, and 98.8 mg/ml of nonradioactive diuron micro-bial isolates and distilled water. The microorganisms used in

this experiment were grown individually for 1 week in 50 ml ofCzapek's solution without sugar with 20 ppm of nonradioactivediuron under constant rotatory shaking at 200 ppm and 30C.The cultures were centrifuged for 15 min at 10,000 rpm, thesupernatant liquid was discarded, and the residues of each cul-ture were added to the suspension. At this point 1.1 ml of the14C-diuron stock solution was added to each of the eight flasks.The final concentration of diuron in each flask was 22.45 ug/gof soil, including 2.69 ug/g of radioactive diuron. An additionalfour flasks were sterilized twice at 24-hour intervals by auto-claving at 121C and 1 kg/cm2 steam pressure, before adding2 ml of the suspension containing the carbon and nitrogensources, and diuron, but with no microorganisms. Again 1.1 mlof the radioactive 14C-diuron stock solution were added to eachof these four flasks.

All of the flasks were connected to an assembly especiallydesigned to measure CO2 evolution from soil (3). HumidifiedCO2-free air was flushed through the system and the gases com-ing out of the flask were bubbled through 10-ml mixture of 1:2ethanolamine/2-methoxyethanol to absorb any evolved CO2from the system (6). Every 5 days this CO2 trapping mixturewas changed and 3 ml aliquots were mixed with 15 ml of aliquid scintillation solution containing 7 g of 2.5-diphenyl-oxa-zole (PPO), 0.3 g of l,4-bis-2(4-methyl-5-phenyloxazoly)-bene-zene (dimethyl POPOP), and 100 g of naphthalene, dissolvedin 1 liter of 1,4-dioxane. The radioactivity in each sample wasdetermined using a liquid scintillation spectrometer and the per-cent of applied diuron evolved as 14CO2 calculated. Air comingthrough the system was bubbled directly through 10 ml of theCO2 trapping mixture and used as radioactivity backgroundcheck. The experiment was conducted for 80 days at 23-29C.

Soil Analysis—At 3, 6, 9, and 12 weeks, two of the inocu-lated flasks were taken off the assembly, and the soil was ex-tracted with 100 ml of acetone for 6 hours using a Soxhletapparatus. The acetone extract was concentrated to 2 ml byflask evaporation and analyzed for the herbicides and residueusing TLC and strip scanning as described above. At 6 and 12weeks, two of the noninoculated flasks were taken off the as-sembly and received the same treatment as the inoculatedflasks.

RESULTS AND DISCUSSION

Experiment 1—Growth of the fungus under differentconcentration of monuron and diuron are shown in Fig. 1,2, and 3. In Fig. 1, the fungus grew best when the mediumcontained up to 20 ppm monuron with yeast as comparedwith the check. Beyond the 20 ppm level of monuronwith yeast, retardation of growth was observed.

Utilizing diuron as a test herbicide indicates that thebest growth occurred at 10 ppm level of diuron. No in-crease in growth was measured at any concentrations abovethis level in which yeast extract was added to the medium.At the 48-hour reading, there was an inexplicable signifi-cant retardation of growth at the 10 ppm level of diuron.

Without yeast, the results were significantly different(Fig. 2 and 3). Best growth of the fungus was attainedbetween 2 and 5 ppm for monuron and diuron. At allother levels growth was retarded below that of the check.Under all treatments, growth progressively increased up to108 hours of incubation time.

The ability of the fungus to grow best when yeast extractwas added to the medium substantiates the findings of Hillet al. (5) that some essential growth factors may be presentin yeast extract which are not supplied by the medium. Itis important to notice that the yeast extract apparently en-abled the fungus to tolerate a higher concentration of thechemical than when yeast was omitted. This agrees with

LOPEZ & KIRKWOOD: MICROORGANISMS CAPABLE OF DEGRADING UREA DERIVATIVE HERBICIDES 311

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Concentrations ol Herbicides (ppm)Fig. 1—Effect of different concentrations of monuron and

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the explanation of Sheets (13) that the organisms degrad-ing urea-derivative herbicides may utilize these chemicalsas a source of energy, but not preferentially, and the highergrowth may also be due in part to the organisms using yeastextract as a carbon source.

Experiment 2—The effect of different concentrations ofmonuron on the amount of dry mycelium produced by theFusarium sp. is shown in Fig. 4. Best growth occurred at20 ppm monuron. This concentration produced a signifi-cant increase in fungal dry matter over any other concen-tration. Apparently, the fungus grew better at the 20 ppmbecause of its ability to use monuron as a carbon sourceand monuron was not toxic at this level.

Experiment 3—Profuse growth of the three microorga-nisms in liquid cultures of Czapek's solution with diuronas the only carbon source were observed by turbidity in theflask after 1 week of incubation. This qualitative observa-tion further attests to the stimulatory effect of urea herbi-cides on the growth of these microorganisms.

The Rf values corresponding to diuron, monomethyldiu-ron, and dimethylated diuron in the liquid culture referredto above were found to be 0.88, 0.73 and 0.41, respec-tively, as seen under short wave UV light. The curves ob-tained from the spotted extracts of the fungus and the twobacteria by strip scanning do not show any evidence ofmetabolic products of diuron. Only one prominent peakwas seen in the curves, that corresponding to diuron (Rf0.88).

Experiment 4—Figure 5 shows that the total amount ofdiuron degraded to CO2 in noninoculated liquid culturewas greater than expected. The fact that the suspensioncontaining supplementary carbon and nitrogen sources, as

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well as the solution containing the herbicides, was notsterilized before addition to sterilized soil may have per-mitted contamination of the culture resulting in a subse-quent multiplication of foreign microorganisms in theflasks which can degrade diuron.

312 SOIL SCI. SOC. AMER. PROC., VOL. 38, 1974

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The degradation of diuron in soil systems has beenshown to start by successive removal of the methyl groupsfollowed by hydrolysis of the urea to dichloroaniline andCO2 (2). There are two important mechanisms by whichurea derivative herbicides can be broken down; one, meta-bolic action by microorganisms and two, photochemicaldecomposition (1, 7). However, photochemical decompo-sition was believed not to have taken place in this experi-ment since it was carried out under minimum amount oflight. It was supposed that the breakdown of diuron wasprimarily due to microbial action. Hill et al. (5) reported10% degradation of monuron in a similar experiment,using methyl labeled 14C-diuron.

Soil Analysis—The TLC plates and radiochromatogramsof the soil extracts at different time intervals during theexperiments did not show the presence of any compoundsother than diuron. In some cases, peaks which may be dueto the presence of monomethylated and dimethylated di-uron could be seen, but these were too small for a reliableidentification.

The inability to obtain any biodegradable products ofdiuron may have been due to the techniques used for ex-tracting diuron and derivatives from the liquid cultures andsoil. Dalton et al. (2) however were able to isolate andidentify three diuron residues from fields which had beentreated with 5.6 kg/ha of diuron 4 to 7 years previously.

The ability of the fungus to grow better in media con-taining some monuron as compared with media containingnone, as shown by results of the CO2 evolution suggests

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that these organisms may be able to use monuron and di-uron as carbon sources. The data also indicates that bio-logical breakdown of these herbicides in this soil couldoccur at a slow rate, depending on the specific field condi-tions.