OUP user menu

Inducible hydroxylation and demethylation of the herbicide isoproturon by Cunninghamella elegans

Martin Hangler, Bo Jensen, Stig Rønhede, Sebastian R. Sørensen
DOI: http://dx.doi.org/10.1111/j.1574-6968.2006.00599.x 254-260 First published online: 1 March 2007

Abstract

A screening of 27 fungal strains for degradation of the phenylurea herbicide isoproturon was performed and yielded 15 strains capable of converting the herbicide to polar metabolites. The zygomycete fungus Cunninghamella elegans strain JS/2 isolated from an agricultural soil converted isoproturon to several known hydroxylated metabolites. In addition, unknown metabolites were produced in minor amounts. Inducible degradation was indicated by comparing resting cells pregrown with or without isoproturon. This shows that strain JS/2 is capable of partially degrading isoproturon and that one or more of the enzymes involved are inducible upon isoproturon exposure.

Keywords
  • pesticide bioconversion
  • physiological response
  • fungi

Introduction

Isoproturon [N-(4-isopropylphenyl)-N′, N′-dimethylurea], IPU, is a phenylurea herbicide used for pre and postemergence control of annual grasses and broad-leaved weeds in winter cereals. The compound has been banned in several European countries, due to the widespread detection of IPU in surface and groundwater in concentrations exceeding the European Union limit for drinking water (Spliid & Køppen, 1998; Sørensen et al., 2003). As a consequence, IPU has been included on a list of priority hazardous substances compiled by the EC in 2001 (European Commission, 2001). However, it is still extensively used in cereal production in for example the United Kingdom and France. Toxicological data suggest that the compound is carcinogenic (European Commission, 2002). Even though the acute toxicity of IPU is rather low, degradation products can be more toxic than the parent compound (Mansour et al., 1999). IPU has been detected in soil at least 3 years after application (Johnson et al., 2001) and mineralization is highly variable throughout fields (Bending et al., 2003).

A potential for microbial degradation of phenylurea herbicides has been found in a variety of microorganisms (Vroumsia et al., 1996; Cullington & Walker, 1999; Turnbull et al., 2001). Three bacterial strains capable of mineralizing 14C-ring-labeled IPU have been isolated from previously IPU-treated soils (Sørensen et al., 2001; Bending et al., 2003; Sebai et al., 2004), and degradation pathways involving successive demethylations or direct hydrolysis of the dimethylurea side-chain to 4-isopropyl-aniline have been proposed (Sørensen et al., 2003). Recently, a study in our laboratory showed that the degradation mechanism of IPU for five fungi isolated from agricultural soil included hydroxylation and to a lesser extent demethylation (Rønhede et al., 2005). Such hydroxylated IPU metabolites have only been detected following fungal degradation and no studies have so far described bacterial hydroxylation of IPU. Field studies have reported detection of hydroxylated metabolites in soil and nearby water resources following IPU treatment (Mudd et al., 1983; Schuelein et al., 1996). This suggests that soil fungi may have a role in the environmental fate of IPU by producing hydroxylated metabolites.

In contrast to the IPU-mineralizing bacteria that generally utilize the herbicide as a source for carbon, nitrogen and energy (Sørensen et al., 2003), the physiological function of fungal degradation of IPU is unknown. Therefore, the aim of this study is to examine the mechanism of IPU degradation in a broad range of fungi and secondly to investigate physiological traits related to degradative activity in one selected strain performing fast degradation.

Materials and methods

Chemicals

IPU, purity 99% (CAS no. 34123-59-6), MDIPU, [N-(4-isopropylphenyl)-N′-methylurea], purity 99.4% (CAS no. 34123-57-4), DDIPU [N-(4-isopropylphenyl)-urea], 98.5% (CAS no. 56046-17-4), and 4-IA, (4-isopropyl-aniline), purity 99.5% (CAS no. 99-88-7), were purchased from Dr Ehrenstorfer GmbH (Augsberg, Germany). The [phenyl-U-14C]-labeled compounds, 14C-IPU (22.7 mCi mmol−1), 14C-MDIPU (22.57 mCi mmol−1) and 14C-4IA (29.9 mCi mmol−1) were all purchased from Institute of Isotopes (Budapest, Hungary) and had a purity of 98%. Hydroxylated reference compounds for qualitative analysis of N-4-(2-hydroxyisopropylphenyl)-N′, N′-dimethylurea (2-OH-IPU), N-4-(1-hydroxyisopropylphenyl)-N′, N′-dimethylurea (1-OH-IPU) and N-4-(1-hydroxyisopropylphenyl)-N′-methylurea (1-OH-MDIPU) were biosynthesized in our laboratory as described previously (Rønhede et al., 2005).

Media and fungal strains

GC medium (per liter): 15 g glucose, 3 g (NH4)2SO4, 0.2 g MgSO4·7H2O, 4.8 g K2HPO4, 1.5 g NaCl.1 mL filter-sterilized FeCl3·6H2O solution (5.14 mg L−1) were added after autoclaving and cooling. E3 medium (per liter): 15 g glucose, 3 g NH4Cl, 18.69 g KH2PO4, 11.16 g Na2HPO4·2 H2O, 0.75 g MgSO4·7H2O. Malt extract agar (MEA) (per liter): 10 g malt extract broth (Lab M, Lancashire, UK) and 20 g agar (Difco, Kansas City, MI).

Twenty-seven fungal strains from the culture collection at the Department of Microbiology at the University of Copenhagen were screened for their ability to degrade IPU. The strains were stored in 20% glycerol at −80°C and 2 weeks before the initiation of the experiments, they were revived on MEA. Depending on the fungal species, inocula were prepared as suspensions of either mycelia or spores in 3 mL distilled water containing 1 g L−1 Tween80 and 8.5 g L−1 NaCl. One milliliter of these suspensions were used for inoculation.

Dissipation of IPU

IPU dissipation was measured by an isocratic HPLC method using an HPLC system (1050 HP, Hewlett Packard, Waldbronn, Germany) with a UV/VIS detector and a 250 × 2 mm Hypersil 5 µm-C18 column (Phenomenex, Cheshire, UK) (Juhler et al., 2001). Analytical-grade IPU dissolved in acetonitrile was added to 100-mL bottles to a final concentration of 15 mg L−1 and after evaporation, 30 mL GC media were added and the bottles were inoculated. Samples for HPLC were filtered through a Titan syringe filter containing a 0.20 µm PTFE membrane (Scientific Resources. Eatontown, NJ) into HPLC glass vials and kept frozen at −18°C until analysis. Fungi degrading IPU were chosen for an additional experiment aimed at detecting the degradation products.

Detection of degradation products

Analytical-grade IPU and 14C-IPU were added to a final concentration of 15 mg L−1 and a specific activity of 50 000 DPM mL−1. After evaporation, 30 mL GC media were added and IPU was allowed to dissolve in the media for 1 day before inoculation. Cultures were grown in the dark on a rotary shaker at 20°C and 150 r.p.m. for 49 days. Degradation of IPU was analyzed using a thin-layer chromatography (TLC) method developed by Rasmussen & Jacobsen (2005) with a silica60 plate (Merck, Darmstadt, Germany) as the stationary phase and hexane/isopropanol/acetic acid (70/30/0.1) as the mobile phase. 0.7 mL samples were collected with Pasteur pipettes, transferred to HPLC vials and kept frozen at −18°C until analysis. 100-µL subsamples were suspended in 10 mL OptiSafe HiSafe 3 scintillation liquid (Turku, Finland), and radioactivity was measured on a Wallac 1409 Liquid Scintillation Counter (Wallac, Turku, Finland). Uninoculated flasks with 15 mg L−1 IPU served as abiotic controls.

Degradation of IPU by strain JS/2

A more detailed study was performed with Cunninghamella elegans strain JS/2 originally isolated from an agricultural field with no history of IPU treatment. Degradation was monitored during growth in GC and E3 media. In 100 mL redcap bottles, 50 mL media-added IPU corresponding to final concentrations of 20 mg L−1 were prepared as described above. Cultures were grown in the dark on a rotary shaker at 25°C and 200 r.p.m. Dissipation of IPU was analyzed using a slightly modified HPLC-method. The method used the same HPLC system as previously described (Juhler et al., 2001) but the flow rate and acetonitrile:water ratio were altered to 0.5 µL min−1 and 35 : 65% v. Inoculated flasks without IPU were included to verify that the detected metabolites originated from IPU. Abiotic controls were also included. pH was monitored using strips during the experiment (Merck, Darmstadt, Germany).

Resting cells

Cultures of strain JS/2 were grown in liquid E3 media containing 20 mg L−1 IPU (treated, T) or with no IPU (untreated, U) and the growth conditions were the same as those used in the degradation experiment. The media were transferred to 50-mL centrifuge tubes after 4 days of incubation and centrifuged for 5 min at 700 g. The supernatant was discarded and 10 mL fresh E3 media without glucose were added to the tubes. This was repeated twice to wash the mycelia. Flasks prepared with 20 mg L−1 IPU in 50 mL E3 media without glucose were inoculated with the washed mycelia and incubated under the same conditions. HPLC analysis of samples from resting cell cultures was performed with a gradient HPLC method as described earlier (Rønhede et al., 2005). Control flasks without IPU were inoculated with washed mycelia from the T treatment to verify that no IPU or metabolites were transferred. At the end of the experiment, the mean dry weights of the treated samples (T) and the untreated (U) samples were 0.115 g and 0.120 g, respectively.

Results

Dissipation of IPU and formation of degradation products by fungal strains

IPU was degraded by 15 of 27 tested strains, of which nine transformed more than 20% of the initially added compound (Table 1). The Cunninghamella species, defined as group A, converted IPU to two unknown polar metabolites with Rf values of 0.4 and 0.5, respectively, while Mucor sp. JS/7, Syncephalastrum racemosum 554, Zygorrhyncus sp. JS/9, Acremonium sp. JS/4, Phoma sp. 67, Talaromyces flavus 358, Rhizoctonia solani and Trametes versicolor, defined as groups B and C, converted IPU to one polar metabolite with an Rf value 0.4. MDIPU was also detected in some of the samples and group D represents fungi degrading <20% (Table 1). 12 fungi showed no IPU degradation: Trichoderma sp. 583, Phialophora cyclamens 653, Chaetomium globosum 357, Alternaria sp. 874, Pleospora herbarum 33, Acremonium strictum 500, Paecilomyces carneus 726, Volutella sp. 288, Acremonium cerealis 475, Apiosordaria verruculosa 15, Arthrobotrys sp. 74 and Cylindrocarpon destructans 794. The 14C-amount in the liquid media was constant throughout the experiment with all strains. It was not possible to quantify 14C-4IA using the TLC approach as no distinct 14C spot was apparent. As the greatest extent of degradation was achieved by Cunninghamella elegans strain JS/2, this fungus was selected for further studies.

View this table:
Table 1

Overview of fungi capable of isoproturon (IPU) degradation, grouped according to degradation activity and mode of action

Polar metabolites (%)
FungiIDMDIPU formation (%)Rf 0.4Rf 0.5% IPU conversionTaxonomic divisionGroup
Cunninghamella elegansJS/2<225.964.695.4ZygomycotaA
Cunninghamella sp.JS/3<217.267.493.1ZygomycotaA
Mucor sp.JS/7<240.041.0ZygomycotaB
Syncephalastrum racemosum554<221.023.2ZygomycotaB
Zygorrhyncus sp.JS/9<215.322.6ZygomycotaB
Phoma sp.67<281.387.1AscomycotaB
Talaromyces flavus358<225.026.6AscomycotaB
Rhizoctonia solaniJS/1019.921.9BasidiomycotaC
Trametes versicolorJS/620.320.3BasidiomycotaC
Acremonium sp.JS/415.617.1AscomycotaD
Alternaria sp.JS/84.17.815.5AscomycotaD
Cladorrhinum bulbillosum6247.57.9AscomycotaD
Cladosporium herbarum377.07.0AscomycotaD
Scopulariopsis brevicaulis33612.712.8AscomycotaD
Wardomyces sp.7153.23.6AscomycotaD
  • Metabolite not detected.

  • Detection of the metabolite N-(4-isopropylphenyl)-N′-methylurea (MDIPU) and the two polar metabolites is shown.

Degradation of IPU by strain JS/2

85% of the initially added IPU was degraded in GC media after c. 200 h. The degradation rate was slower in E3 media, with only 25% degraded after 200 h (Fig. 1).

Figure 1

Degradation of isoproturon (IPU) by Cunninghamella elegans strain JS/2. The left y-axis shows the concentration of IPU (mg L−1). ▲, E3 media with constant pH 6.5; •, GC media; ▪, pH for the GC media (values are given on the right y-axis). The data, except pH values, are mean values (n=3), and error bars indicate the SDs.

Resting cells

In the T cultures, 6% of the initially added IPU was degraded at the end of the experiment. In the U cultures, less than one percent was degraded (Fig. 2). HPLC analysis revealed several hydroxylated compounds (Fig. 2). These compounds did not appear in the abiotic controls or in the controls with washed IPU-treated mycelia without IPU. The hydroxylated metabolites 1-OH-IPU and 2-OH-IPU appeared c. 80 h sooner in the T samples compared with the U samples (Fig. 2). The demethylated metabolite MDIPU was detected in both T and U samples already after 20 h. The formation of MDIPU however, seemed faster for the T samples in comparison with the U samples (Fig. 2). The unknown metabolite with a retention time of 18.9 min showed a pattern similar to the hydroxylated metabolites 1-OH-IPU and 2-OH-IPU. The amounts of the compounds are shown as integrated areas as no analytical standards were available for the unknown and hydroxylated metabolites. However, the compounds had UV spectra comparable to IPU and MDIPU, and the total integrated area of all compounds remained constant throughout the experiment.

Figure 2

Degradation of isoproturon (IPU) and formation of the metabolites N-(4-isopropylphenyl)-N′-methylurea (MDIPU), N-(4-(2-hydroxyisopropylphenyl)-N′, N′-dimethylurea (2-OH-IPU), N-(4-(1-hydroxyisopropylphenyl)-N′, N′-dimethylurea (1-OH-IPU) and an unknown metabolite with a retention time of 18.9 min for resting cells of Cunninghamella elegans JS/2 in E3-buffer without glucose. ▪, Resting cells originating from mycelia grown for 4 days in E3 media with IPU (T) at an initial concentration of 20 mg L−1. ○, Resting cells originating from mycelia grown for 4 days in E3-media without IPU (U). The data are mean values (n=3), and the error bars indicate the SDs.

Discussion

Fungal IPU degradation has previously been proved by screening a large number of strains (Vroumsia et al., 1996; Khadrani et al., 1999). However, these studies did not include detection of metabolites or examined whether mineralization or sorption took place. Here, it is shown that 14C originating from 14C-ring-labeled IPU following fungal degradation is readily extractable, and that the 14C amount in the culture media is constant throughout the incubation, proving that no mineralization takes place and that sorption to fungal biomass is negligible. Recently, it has been shown that five natural soil fungi isolated from an IPU-treated agricultural field degraded IPU to metabolites identified as various hydroxylated and demethylated derivatives (Rønhede et al., 2005). This study combines a screening of 27 fungi with identification of the degradation mechanism and shows that the dissipation of IPU is due to biotransformation, probably mainly hydroxylation, suggesting that this is the explanation for the dissipation seen in earlier studies. In field and lysimeter experiments, leachates collected following IPU application have been shown to contain both hydroxylated and demethylated metabolites (Schuelein et al., 1996; Beulke et al., 2004). So far, none of the isolated IPU-degrading soil bacteria have been shown to produce these hydroxylated metabolites (Cullington & Walker, 1999; Turnbull et al., 2001; Bending et al., 2003; Sørensen et al., 2003; Sebai et al., 2004), suggesting that soil fungi play a role in the environmental fate of IPU.

The degradative fungi were divided into different groups based on their degradation activity and mode of action. These groups (A, B, C and D in Table 1) may mirror the phylogeny of the included fungi. Apparently, only some zygomycetes belong to group A, while group B consists of both zygomycetes and ascomycetes. Group C consists solely of basidiomycetes and group D of different ascomycetes with limited bioconversion activity. The conversion of IPU to different metabolites may involve certain species-specific enzymes. However, care should be exercised as, for example, the media composition may affect the growth rate of individual species and bias the direct comparison between the different strains. Cunninghamella elegans strain JS/2 was the fastest IPU degrader under these experimental conditions and it was therefore chosen for further experiments.

The difference in the rate of transformation of IPU by Cunninghamella elegans strain JS/2 between the GC and E3 media may be explained by differences in the ratio of glucose to available nitrogen. This has been shown to have a significant influence on the hydroxylation of pyrene to 1-pyrenol by Pencillium janthinellum (Launen et al., 1999). The enhanced dissipation of IPU in the GC media is not caused by abiotic degradation, as IPU is stable from pH 4 to 10 (European Commission, 2002), where most of the degradation occured. However, a pH decline may have an effect as was shown for two algal species (Mostafa & Helling, 2001). In that study, the observed difference was explained by increased metabolism under more acidic conditions. In order to assure comparability in the resting cell experiment, pH of the growth media and the carbon-free media should be the same. Therefore, the E3 media were chosen even though it did not yield the highest degradation rate.

Studies on fungal IPU degradation with the basidiomycete Rhizoctonia solani have suggested a constitutive expression of the degradative enzymes based on the apparent lack of a lag phase within a 20-day study (Vroumsia et al., 1996). However, these results show that a lag phase may be difficult to detect, if only the disappearance of IPU is measured (Fig. 2). The difference between the formation of the hydroxylated and demethylated metabolites suggests that different enzymes are involved in these processes. It can be argued that the demethylating process is up-regulated in the T samples but is not absent in the U-sample, as MDIPU appears after 20 h in both treatments (Fig. 2), while the hydroxylating process is detectable in the T samples after 20 h but absent in the U samples until about 100 h after the transfer. The degradation of naphthalene by a Cunninghamella elegans has also been shown to be inducible in rich media (Faber et al., 2001). However, the first degradation step was only inducible in early growth phase mycelia, while the fungus apparently constitutively expressed the activity in latter phases.

As the 14C is constant in the culture media throughout the experiment, the fungus is not utilizing the aromatic ring of IPU as a carbon or energy source. The metabolites formed are similar to compounds formed during phase I detoxification in mammals (Zhang et al., 1996). Therefore, the inducibility may reflect a detoxifying response. Cunninghamella elegans has been shown to be involved in the metabolism and biotransformation of other xenobiotics and has been used as a model organism for mammalian detoxification metabolism (Wang et al., 2000). The IPU degradation capability of our strain complements a previous degradation study, where a Cunninghamella elegans degraded more than 95% of the added IPU in 5 days (Vroumsia et al., 1996). Combined with the fact that our strain was isolated from an agricultural field without prior IPU treatment, this suggests that Cunninghamella elegans ability to degrade IPU is not a result of adaptation.

An unknown metabolite with a retention time of 18.9 min was detected following the degradation of IPU by strain JS/2. This retention time is closer to the retention time of IPU than MDIPU or any other of the known metabolites. Glaβgen (1999) observed a metabolite with a retention time similar to our unknown metabolite using a comparable HPLC method and subsequently identified it as isopropenyl-IPU. Therefore, isopropenyl-IPU could be a candidate for the identity of the compound at 18.9 min produced by strain JS/2 in this study.

In conclusion, it is proposed that the degradation of IPU by Cunninghamella elegans JS/2 is induced by the herbicide and that more than one enzyme is involved. The fungus can in this way respond specifically to the presence of IPU and hydroxylate it.

Footnotes

  • Editor: Clive Edwards

References

View Abstract