Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Biodegradation of sulfosulphuron in agricultural soil by Trichoderma sp. U. Yadav1 and P.P. Choudhury2 1 Govt. M. H. Autonomous College of Home Science and Science for Women, Rani Durgavati Vishwavidayalaya, Jabalpur, MP, India 2 Directorate of Weed Science Research, Jabalpur, MP, India
Significance and Impact of the Study: The degradation of sulfosulphuron by any individual fungus is being reported for the first time. Trichoderma sp. isolated from wheat-rhizospheric soil could survive in minimal broth rich in sulfosulphuron. Previous reports have described the complete degradation of any sulfonyl urea herbicides by micro-organisms only after the pH-dependent chemical hydrolysis of the sulfonyl urea bridge of the herbicide. This study demonstrates the novel result that the Trichoderma sp. utilized the sulfosulphuron as a sole carbon source and degraded it by cleaving sulfonyl urea bridge and sulfonylamide linkage. Thus, the application of Trichoderma sp., which is nonphytopathogenic, has the potential to decontaminate agricultural soil from sulfosulphuron load.
Keywords biodegradation, bioremediation, sulfonylurea herbicide, sulfosulphuron, Trichoderma sp. Correspondence Partha P. Choudhury, Directorate of Weed Science Research (ICAR), Maharajpur, Jabalpur, MP 482 004, India. E-mail: [email protected] 2014/0388: received 24 February 2014, revised 5 June 2014 and accepted 4 July 2014 doi:10.1111/lam.12306
Abstract Sulfosulphuron-degrading fungus was isolated by enrichment technique from the sulfosulphuron-contaminated soil of wheat rhizosphere. To assess the biodegradation potential of isolated Trichoderma sp., minimal potato dextrose agar broth with different levels of sulfosulphuron (up to 2 g l 1) was evaluated in the growth and biotransformation experiments. ESI LC-MS/MS analysis revealed the presence of degradation products 2-amino-4,6dimethoxypyrimidine (I) and 2-ethylsulfonyl imidazo{1,2-a} pyridine-3sulfonamide-2-ethylsulfonyl imidazo{1,2-a} pyridine-3-sulfonamide (II) indicating the cleavage of the urea bridge and the presence of the by-product N(4,6-dimethoxypyrimidin-2-yl)urea (III) indicating the degradation of sulfonylamide linkage. Two other metabolites, N-(4,6-dimethoxypyrimidin-2yl)-N’-hydroxyurea (IV) and N, N’-bis(4,6-dimethoxypyrimidin-2-yl)urea (V), were also identified. From the previous reports, it was found that the degradation of sulfonyl urea herbicides took place through the chemical degradation of the sulfonylurea bridge followed by microbial degradation. During this investigation, Trichoderma sp. grew well with and degraded sulfosulphuron via both the decarboxylation on the sulphonyl urea bridge and the hydrolytic cleavage of the sulfonylamide linkage as demonstrated by the formation of metabolites. Trichoderma is nonphytopathogenic in nature, and some species of it restrict the growth of soil-dwelling phytopathogens. Therefore, it is a promising candidate for the decontamination of soil from sulfosulphuron residues.
Introduction Sulfosulphuron, 1-(4,6-dimethoxypyrimidin-2-yl)-3-(2-ethylsulfonylimidazo [1,2-a]pyridine-3-yl) sulfonylurea, is a selective and systemic pyrimidinylsulfonylurea herbicide. It is absorbed through roots as well as leaves and transloLetters in Applied Microbiology © 2014 The Society for Applied Microbiology
cated throughout the plant. Biochemically, it acts as an inhibitor of the acetolactate synthase enzymes responsible for branched-chain amino acid biosynthesis, hence stopping cell division and plant growth. Sulfosulphuron controls grassy and broad leaf weeds in wheat at an application rate of 20 g ha 1 (Parrish et al. 1995). It controls 1
Biodegradation of sulfosulphuron
weeds in wheat like Phalaris minor, Avena ludoviciana, and Rumex retroflex, resulting in higher grain yield (Banga et al. 2003). Barley (Hordeum vulgare), oats (Avena sativa), maize (Zea mays), sunflower (Helianthus annuus L.) and sorghum (Sorghum bicolor L.) are among those crops which are sensitive to sulfosulphuron (Shinn et al. 1998; Miller et al. 1999; Kelly and Peeper 2003). Carry-over effect of sulfosulphuron was noticed in the growth of some crops like maize and sorghum (Chhokar et al. 2006). The half-life (DT50) of it in general is within the range of 20–60 days. Sulfosulphuron residue was found to be below detectable limit after 15 days of spraying @ 25 g ai ha 1 in wheat on sandy-loam soil, the half-life was within 53–64 days (Saha et al. 2003; Singh and Kulshrestha 2007), and no residue was detected in the Inceptisol soil after harvest of wheat crop (Atmakuru and Maheswari 2003). The disappearance of the herbicide from soil takes place either through the physical displacement, viz., leaching down, runoff, etc. or through the degradation of the compound. Members of the sulfonyl urea family undergo pH-dependent chemical hydrolysis, the rate increases with the decreasing soil pH, and thereafter, the hydrolysed products undergo microbial degradation (Beyer et al. 1988). Under alkaline condition, sulfosulphuron yields 1-(2-ethylsulfonylimidazo[1,2-a] pyridin-3-yl-3-(4,6-dimethoxypyrimidin-2-yl) amine, and under acidic condition, it degrades to 1-(2-ethylsulfonylimidazo[1,2-a] pyridin)-3-sulfonamide and 4,6-dimethoxy-2-aminopyrimidine (Saha and Kulshrestha 2002). But in the agronomic pH, the degradation of herbicides is mainly caused enzymatically by soil-dwelling microbes. The present investigation was undertaken to isolate sulfosulphuron-degrading fungi from the black soil and, thereafter, to judge its suitability in the bioremediation of sulfosulphuron-contaminated soil. Results and discussion Isolation and characterization of sulfosulphurondegrading fungus Fungi, isolated from rice rhizospheric soil, were allowed to grow in the minimal media having sulfosulphuron as carbon and nitrogen source. Trichoderma sp., a widely distributed fungus in soil, survived and grew in that media with sulfosulphuron at the level as high as 200 mg l 1. The organism was characterized by its morphological characters (Fig. 1). Colonies of this filamentous fungus were found at first white and gradually becoming either dull white or light green to yellowish green or deep shades with age and grew rapidly and mature in 5 days at 25°C. Vegetative hyphae were fertile. Conidiophores were erect developing from side branch, 2
U. Yadav and P.P. Choudhury
Figure 1 Microscopy of Trichoderma sp. isolated from wheat rhizospheric soil.
branching usually opposite, and bore terminal conidial heads. Conidia were ovate to elliptic or glabrous and smooth. Species ‘viride’ was characterized by microscopic observations. Colonies were typically fast growing at 25–30°C, but the growth was inhibited at 35°C. Conidia were formed typically within 1 week in compact or loose tufts in shades of green or yellow or less frequently white. Conidiophores were highly branched and terminated in one or a few phialides, which were enlarged in the middle. But the confirmation of this species as ‘viride’ is subjected to further investigation. Degradation of sulfosulphuron by Trichoderma sp. in sterilized soil The incubation of Trichoderma in minimal media as well as soil led to major degradation of the compound. Five key metabolites, which give the direction of degradation pathway, are confirmed by mass spectra and synthesis of the metabolites, and related literatures. Metabolite-I From the mass spectrum (Fig. 2a) of this compound, which shows the M + H peak at m/z 156, the molecular mass is determined as 155. Peaks at m/z 140, 139 and 124 suggest the loss of one methyl (-CH3), one amino (-NH2) and one methoxy (-OCH3) group of the parent molecule. From this, the structure of the metabolite was assigned as 2-amino4,6-dimethoxypyrimidine (I), which is further confirmed by the previously reported spectrum of the compound (Saha and Kulshrestha 2002; Atmakuru et al. 2007). Metabolite-II The parent ion peak is at m/z 290 (Fig. 2b) indicating the molecular weight of the compound as 289. The peaks Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
U. Yadav and P.P. Choudhury
8·9e5
Biodegradation of sulfosulphuron
100·1
(a)
8·5e5
H3CO
8·0e5
+ •
H
7·5e5
H3CO
7·0e5 6·5e5 6·0e5
H3CO
Intensity, cps
5·5e5 5·0e5
156·2
N
4·5e5 4·0e5 57·2
H3CO
+ • NH2
N
3·5e5
N
H3CO
82·1 2·5e5
H3CO
+ • NH
N
N
3·0e5 68·1
N
+ •
124·2
N
H3CO
1·5e5
140·0
1·0e5
112·1
5·0e4
58·1
43·0 0·0 40
67·1
55·1 50
60
82·9 70·2
70
92·3
80
90
139·3
114·1
97·9
84·2
81·0
100
110
123·3
120 m/z, Da
130
140
150
160
170
180
+ •
(b)
+ •
3·6e4
200
273·1 290·1
N
O2S
N
3·2e4
190
N 125·1
3·4e4
N
SO2CH2CH3 + •
3·0e4 181·3 2·8e4
N
2·6e4
+ •
2·4e4 Intensity, cps
+ • NH2
N
H3CO
N
2·0e5
3·8e4
+ •
N
105·0
SO2CH2CH3
117·1
2·2e4
N
2·0e4
HC
N
H2N O2S + •
N N
1·8e4
N
1·6e4
SO2CH3 + •
1·4e4 165·3
1·2e4
78·4
1·0e4
N SO2CH3
137·3 152·1 149·1
4000·0
0·0 40
N
153·1
96·3
6000·0
61·1 70·177·4 60
80
90·2
198·2 180·1
118·1 123·4 135·2 107·1 100
+ •
N
133·4
8000·0
2000·0
197·1
121·2
199·0 211·1
167·9 120
140
160
180
200
N SO2CH2CH3
225·0 220
244·8 240
262·1 260
275·0 280
300
m/z, Da
Figure 2 Mass fragmentation pattern of products formed through decarboxylation of sulfonylurea bridge; (a) 2-amino-4,6-dimethoxypyrimidine (I) and (b) 2-ethylsulfonyl imidazo {1,2-a} pyridine-3-sulfonamide (II).
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
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Biodegradation of sulfosulphuron
U. Yadav and P.P. Choudhury
produces a fragment of m/z 182. The possible fragment for H2ON is -NH-OH (hydroxyl amine group). The presence of the peaks at m/z 155 and 139 indicates the presence of dimethoxy pyrimidinyl ring. Hence, the peak at 182 is due to one carbonyl group added to dimethoxy pyrimidinyl amine. Considering this fragmentation pattern, the structure of the compound is assigned as N- (4,6-dimethoxypyrimidin-2-yl)-N’hydroxyurea (IV).
at 117 and 211 confirm the presence of ethyl sulfonyl imidazo pyridinyl moiety of sulfosulphuron. The addition of the SO2NH2 group to this moiety imparts the complete structure of this metabolite, and hence, the structure is assigned as 2-ethylsulfonyl imidazo {1,2-a} pyridine-3sulfonamide (II). This structure is further confirmed by the previously reported spectrum (Saha and Kulshrestha 2002; Atmakuru et al. 2007). Metabolite-III The peak at m/z 199 (Fig. 3) indicates the molecular mass of 198. The parent molecule loses one amino group (16 amu) to give a peak at m/z 182, which on losing one carbonyl group (28 amu) produces a daughter ion (155 amu) at m/z 156. Peaks at m/z 140, 139 and 124 suggest the loss of one methyl (-CH3), one amino (-NH2) and one methoxy (-OCH3) group from the daughter ion of 155 amu. From this fragmentation pattern, the structure is assigned as N-(4,6-dimethoxypyrimidin-2-yl)urea (III). This is further confirmed by the mass spectrum of similar urea derivative formed from chlorimuron ethyl (Sharma et al. 2012).
Metabolite-V Peaks at m/z 139 and 156 in the spectrum (Fig. 4b) indicate the presence of 4,6-dimethoxypyrimidinyl-2-amino group, which on adding a carbonyl group produces a fragment of m/z 182. The molecular ion peak is found at m/z 337. There is no peak found in between m/z 337 and 182 and no major peak other than peaks at m/z 139, 156 and 182. It is possible if there is any repetition of the fragment 4,6-dimethoxypyrimidinyl group. Thus, the compound is assigned as N, N’-bis(4,6-dimethoxypyrimidin-2-yl)urea (V). It is a demonstrated fact that the first degradation step of any sulfonyl urea herbicide is the cleavage of the sulfonyl urea bridge, which is a pH-dependent response. The pH should be moderately to highly acidic. But in the present experiment, the pHs of the media and the
Metabolite-IV The molecular ion peak appears at m/z 214 (Fig. 4a). A loss of 22 amu (H2ON) from the molecular ion 83·3
750
H3CO
700
H3CO
650
68·1
+
H3CO
N 100·1
H3CO
550
N
+
N
•
NH2
500 Intensity, cps
•
•
N
57·2
600
+
N
N
82·2
H3CO
450
+
N
H3CO
•
NH
400
H3CO
N
+
N
350
O NH C
•
N 300
140·0
69·3
250 200
H3CO
93·3
H3CO
55·2 43·2
112·3
139·1
N
67·3
150
92·3 98·0 58·0 56·0
100 50
71·0
41·1 53·3 59·2 65·1
0 40
50
60
77·3
81·2
79·1 80·5 72·5
70
80
111·3
124·1 109·4 94·2 99·4 103·8 114·0 128·1 97·0
90
O
+ •
NH C NH2
70·4
H3CO
N
156·0 141·1
182·3
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 m/z, Da
Figure 3 Mass fragmentation pattern of N-(4,6-dimethoxypyrimidin-2-yl)urea (III) formed through the cleavage of sulfonylamide linkage.
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Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
U. Yadav and P.P. Choudhury
Biodegradation of sulfosulphuron
182·1 4·6e4
(a)
4·4e4 4·2e4
H3CO
4·0e4
+ •
N
3·8e4
NH2 N
3·6e4
H3CO
3·4e4
H3CO
O NH C
N
3·2e4 3·0e4
H3CO
Intensity, cps
2·8e4
+ •
N
+ •
N
H3CO
2·6e4
N
2·4e4 2·2e4
H3CO
83·3
2·0e4
H3CO
139·2
1·8e4
+ O • NH C NHOH
N
1·6e4 1·4e4
H3CO
1·2e4
N
1·0e4 214·2
8000·0 6000·0
157·1
4000·0 2000·0
71·1
0·0 40
60
82·0
93·2
80
100·2 109·3
138·2
120
100
140
200
180 m/z, Da
220
240
260
280
300
156·2
8·3e4 8·0e4
160
(b)
H3CO
7·5e4
N
7·0e4
N
+ • NH2
H3CO
6·5e4 6·0e4 5·5e4
Intensity, cps
5·0e4
H3CO
N
4·5e4 4·0e4
O NH C
+ •
N
H3CO
3·5e4 3·0e4
182·4
2·5e4
H3CO
2·0e4 1·5e4
H3CO
1·0e4 5000·0 0·0
40
H3CO
80
100
+ •
O NH C NH
N
N
H3CO
N
+ •
OCH3
N 337·0 139·3
100·3
60
N
N
OCH3
120
140
160
180
200 m/z, Da
220
240
260
280
300
320
340
Figure 4 Mass fragmentation pattern (a) N-(4,6-dimethoxypyrimidin-2-yl)-N’-hydroxyurea (IV); (b) N, N’-bis(4,6-dimethoxypyrimidin-2-yl)urea (V).
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
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Biodegradation of sulfosulphuron
U. Yadav and P.P. Choudhury
soil were neutral. The incubation of the sulfosulphurontreated media and sterilized soil without Trichoderma did show hardly any degradation of the herbicide. But in the presence of the fungus Trichoderma, both in the media and in the soil, two metabolites, viz., 2-amino-4,6dimethoxypyrimidine (I) and 2-ethylsulfonyl imidazo {1,2-a} pyridine-3-sulfonamide (II), are formed. The formation of the couple of products, I and II, is possible due to the cleavage of the sulfonyl urea bridge. It strongly suggests that this cleavage is not always pH dependent; enzyme can also do the same. This is basically a decarboxylation reaction of the sulfonylurea bridge, and a decarboxylase type of enzyme is involved here to catalyse the reaction. But, interestingly, the presence of metabolite, N-(4,6-dimethoxypyrimidin-2-yl)urea (III), suggests a different mode of degradation. The formation of this metabolite is possible through the degradation of the sulfonyl amide linkage. This is a hydrolysis reaction at the sulfonyl amide bond, and probably a hydrolase type of enzyme was involved to catalyse the reaction. The metabolite, N-(4,6-dimethoxypyrimidin-2yl)-N’-hydroxyurea (IV), is formed due to oxidative hydroxylation on the nitrogen of urea bridge. Merely that whether it is on the terminal nitrogen of 2-amino4,6-dimethoxypyrimidine or it is on the urea nitrogen of sulfosulphuron is indistinct from the present outcome.
H3CO H3CO
N N
H3CO
N N
O C N N H H
Materials and methods Chemicals Samples of technical sulfosulphuron (905%) and analytical sulfosulphuron (999%) were obtained from Indofil
O O N C N S N N H H O SO CH CH 2 2 3 Cleavage of sulfonyl urea bridge
Cleavage of sulfonyl amide linkage
H3CO
Perhaps, the monooxygenase-mediated oxidation of the terminal amino group caused the formation of the metabolite-IV. This particular step occurred not only in the media, but also in the soil. The metabolite, N, N’-bis (4,6-dimethoxypyrimidin-2-yl)urea (V), is probably formed by the union of two radicals, Ф-NH-C=O and NH- Ф, which are produced during the initial steps of microbial metabolism of sulfosulphuron. A scheme on the Trichoderma-assisted degradation pathways of sulfosulphuron in soil has been proposed (Fig. 5). Sulfosulphuron seems to have the ability to inhibit the growth of some fungi present in soil as it did not allow many of them to grow in media and soil incubated with the herbicide. But the high tolerance of Trichoderma sp. towards sulfosulphuron and its ability to utilize the herbicide as a sole source of carbon indicate to consider the fungus as a promising candidate for the bioremediation of sulfosulphuroncontaminated soil.
H3CO
O N N S O SO CH CH 2 2 3
H3CO
O C
O N N N S N H O SO CH CH 2 2 3 Decarboxylation N
N H
HOH H3CO H3CO
N N III
O NH C NH2
H3CO H3CO
N N
H3CO
N N
N N
H2N O2S
SO2CH2CH3 I
H3CO
NH2
II
O NH C NHOH IV
Figure 5 Proposed pathways for the degradation of sulfosulphuron by Trichoderma sp.
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Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
U. Yadav and P.P. Choudhury
Chemicals Company, Mumbai. Technical sulfosulphuron was further purified by recrystallization until a constant melting point of 201°C was achieved. It is a white amorphous solid. The purity was checked by thin layer chromatography (TLC) (solvent system—chloroform:: acetonitrile: 2 : 3, v/v; Rf = 035). Laboratory grade reagents and solvents were procured locally. All solvents were dried and distilled before use. HPLC-grade solvents and reagents were used during chromatographic and spectroscopic analysis. Deionized water was obtained from the Milli-Q SP Reagent water system (Millipore, Bedford, MA). Soil Soil samples were collected from different locations of wheat field of the research farm in the directorate. The field was having the history of the sulfosulphuron application for consecutive 5 years. Soils were collected in sterile plastic bags, air-dried at room temperature and sieved (2 mm mesh). Fresh soil was used for the isolation of fungi. The soil used was a typical black soil (vertisol) with the chemical status as follows: organic matter 096% (w/w); pH (water 1 : 25) 708; EC 048 dS 1; and CEC 338 Cmol (p+)kg 1. Isolation and identification of sulfosulphuron-degrading fungus The collected soil was enriched with sulfosulphuron (5 mg in 100 g of soil) and incubated for a week at 30°C. For a selection of fungi as a suitable sulfosulphurondegrading agent, serial dilution following agar plating of incubated soil was done. Fungi that appeared on potato dextrose agar (PDA) plates (prepared from 200 g of potato, 20 g of dextrose, 20 g of agar and 1000 ml of water) after 5 days of incubation were further plated for obtaining pure cultures. The fungi screened from the sulfosulphuron-enriched soil were again incubated for 7 days in minimal PDA broth (prepared from 10 g of potato, 20 g of dextrose and 1000 ml of water) containing different levels of sulfosulphuron, viz., 25, 50, 100 and 200 mg per 100 ml of broth. The most efficient fungus was screened out on the basis of their growth and was further inoculated on PDA plates. After 2 days of incubation, colony morphology of the isolate was examined. On the basis of colony morphology and microscopy of spore structures, the fungus was characterized. Degradation of sulfosulphuron by Trichoderma sp For the degradation studies, 25 mg of sulfosulphuron was added to 100 ml of sterile dextrose-minimal broth Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Biodegradation of sulfosulphuron
(prepared from 100 g of potato, 10 g of dextrose and 1000 ml of water) in 250-mL flask. The sulfosulphuron was allowed to dissolve overnight on shaker. Twenty such flasks were incubated with isolated Trichoderma sp. in the dark at 25°C for 27 days in BOD incubator. Three flasks with minimal broth and sulfosulphuron and without the incubation with Trichoderma sp. were kept in the dark as a control. Degraded products were extracted by partitioning in chloroform from the broth sampled at different time intervals, viz., 3, 9, 16 and 27 days of incubation. The solvent was then evaporated under low pressure in the rotary vacuum evaporator to obtain a crude mixture of products. The products were purified by the preparative TLC and characterized by the spectroscopic techniques. Liquid chromatography–mass spectroscopy An API 3200 Qtrap mass spectrometer hyphenated to Shimadzu Ultra Fast Liquid Chromatography was used for the mass characterization of degraded products. Mass spectrometry analysis was performed with electrospray ionization (ESI) in positive (5500 eV) mode for each sample. The nebulizer gas and heater gases were adjusted at 30 psi and 55 psi, respectively. The ion source temperature was set at 500°C. Each sample was injected by infusion technique at the rate of 10 lls 1. Preparation of major metabolites Acid hydrolysis of sulfosulphuron Sulfosulphuron (200 mg) was added to distilled water (100 ml). The pH of the solution was adjusted to 25 by the addition of concentrated hydrochloric acid (2 ml). The solution was stirred magnetically at 32°C, and the reaction was monitored by TLC and continued until the disappearance of the spot of sulfosulphuron, which took 48 h. The products formed were separated by preparative TLC, purified by crystallization from benzene and characterized by spectroscopic methods. Compounds were structurally assigned as 2-amino-4,6-dimethoxypyrimidine (I) and 2-ethylsulfonyl imidazo {1,2-a} pyridine-3-sulfonamide (II) with the help of mass spectra (Figs 2 and 3). Acknowledgements The authors are grateful to the Director, Directorate of Weed Science Research (ICAR), Jabalpur, MP, India, for providing the research facilities to complete this PG dissertation work of U.Y., Govt. M. H. Autonomous College of Home Science and Science for Women, Rani Durgavati Vishwavidayalaya, Jabalpur (M.P.). 7
Biodegradation of sulfosulphuron
Conflict of interest We hereby declare that no conflict of interest exists between us. References Atmakuru, R. and Maheswari, S.T. (2003) Dissipation of sulfosulfuron in soil and wheat plant under predominant cropping conditions and in a simulated model ecosystem. J Agric Food Chem 51, 3396–3400. Atmakuru, R., Thirugnanam, P.E. and Sathiyanarayanan, S. (2007) Identification of residues of sulfosulfuron and its metabolites in subsoil-dissipation kinetics and factors influencing the stability and degradation of residues from topsoil to subsoil under predominant cropping conditions. Environ Monit Assess 130, 519–528. Banga, R.S., Yadav, A. and Malik, R.K. (2003) Bioefficacy of flufenacet and sulfosulfuron alone and in combination against weed flora in wheat. Indian J Weed Sci 35, 179–182. Beyer, E.M. Jr, Duffy, M.J., Hay, J.V. and Schlueter, D.D. (1988) Sulfonylureas. In Herbicides: Chemistry, Degradation, and Mode of Action, ed. Kearney, P.C. and Kaufman, D.D. pp. 117–189. New York, NY: Dekker. Chhokar, R.S., Sharma, R.K., Chauhan, D.S. and Mongia, A.D. (2006) Evaluation of herbicides against Phalaris minor in wheat in north-western Indian plains. Weed Res 46, 40–49.
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Kelly, J.P. and Peeper, T.F. (2003) Mon 37500 application timing affects cheat (Bromus secalinus) control and winter wheat. Weed Sci 51, 231–236. Miller, P.A., Westra, P. and Nissen, S.J. (1999) The influence of surfactant and nitrogen on foliar absorption of Mon 37500. Weed Sci 47, 270–274. Parrish, S.K., Kaufmann, J.E. and Croon, K.A. (1995) MON 37500: a new selective herbicide to control annual and perennial weeds in wheat. In Proc Brighton Crop Protect Conf-Weeds. pp. 57–63. Surrey: BCPC Farnham. Saha, S. and Kulshrestha, G. (2002) Degradation of sulfosulfuron, a sulfonylurea herbicide, as influenced by abiotic factors. J Agric Food Chem 50, 4572–4575. Saha, S., Yaduraju, N.T. and Kulshrestha, G. (2003) Residue studies and efficacy of sulfosulfuron in wheat crop. Pestic Res J 15, 173–175. Sharma, S., Banerjee, K. and Choudhury, P.P. (2012) Degradation of chlorimuron-ethyl by Aspergillus niger isolated from agricultural soil. FEMS Microbiol Lett 337, 18–24. Shinn, S.L., Thill, D.C., Price, W.J. and Ball, D.A. (1998) Response of downy brome (Bromus tectorum) and rotational crops to Mon 37500. Weed Technol 12, 690–698. Singh, S.B. and Kulshrestha, G. (2007) Determination of sulfosulfuron residues in soil under wheat crop by a novel and cost-effective method and evaluation of its carryover effect. J Environ Sci Health B 42, 27–31.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
ORIGINAL ARTICLE
Biodegradation of sulfosulphuron in agricultural soil by Trichoderma sp. U. Yadav1 and P.P. Choudhury2 1 Govt. M. H. Autonomous College of Home Science and Science for Women, Rani Durgavati Vishwavidayalaya, Jabalpur, MP, India 2 Directorate of Weed Science Research, Jabalpur, MP, India
Significance and Impact of the Study: The degradation of sulfosulphuron by any individual fungus is being reported for the first time. Trichoderma sp. isolated from wheat-rhizospheric soil could survive in minimal broth rich in sulfosulphuron. Previous reports have described the complete degradation of any sulfonyl urea herbicides by micro-organisms only after the pH-dependent chemical hydrolysis of the sulfonyl urea bridge of the herbicide. This study demonstrates the novel result that the Trichoderma sp. utilized the sulfosulphuron as a sole carbon source and degraded it by cleaving sulfonyl urea bridge and sulfonylamide linkage. Thus, the application of Trichoderma sp., which is nonphytopathogenic, has the potential to decontaminate agricultural soil from sulfosulphuron load.
Keywords biodegradation, bioremediation, sulfonylurea herbicide, sulfosulphuron, Trichoderma sp. Correspondence Partha P. Choudhury, Directorate of Weed Science Research (ICAR), Maharajpur, Jabalpur, MP 482 004, India. E-mail: [email protected] 2014/0388: received 24 February 2014, revised 5 June 2014 and accepted 4 July 2014 doi:10.1111/lam.12306
Abstract Sulfosulphuron-degrading fungus was isolated by enrichment technique from the sulfosulphuron-contaminated soil of wheat rhizosphere. To assess the biodegradation potential of isolated Trichoderma sp., minimal potato dextrose agar broth with different levels of sulfosulphuron (up to 2 g l 1) was evaluated in the growth and biotransformation experiments. ESI LC-MS/MS analysis revealed the presence of degradation products 2-amino-4,6dimethoxypyrimidine (I) and 2-ethylsulfonyl imidazo{1,2-a} pyridine-3sulfonamide-2-ethylsulfonyl imidazo{1,2-a} pyridine-3-sulfonamide (II) indicating the cleavage of the urea bridge and the presence of the by-product N(4,6-dimethoxypyrimidin-2-yl)urea (III) indicating the degradation of sulfonylamide linkage. Two other metabolites, N-(4,6-dimethoxypyrimidin-2yl)-N’-hydroxyurea (IV) and N, N’-bis(4,6-dimethoxypyrimidin-2-yl)urea (V), were also identified. From the previous reports, it was found that the degradation of sulfonyl urea herbicides took place through the chemical degradation of the sulfonylurea bridge followed by microbial degradation. During this investigation, Trichoderma sp. grew well with and degraded sulfosulphuron via both the decarboxylation on the sulphonyl urea bridge and the hydrolytic cleavage of the sulfonylamide linkage as demonstrated by the formation of metabolites. Trichoderma is nonphytopathogenic in nature, and some species of it restrict the growth of soil-dwelling phytopathogens. Therefore, it is a promising candidate for the decontamination of soil from sulfosulphuron residues.
Introduction Sulfosulphuron, 1-(4,6-dimethoxypyrimidin-2-yl)-3-(2-ethylsulfonylimidazo [1,2-a]pyridine-3-yl) sulfonylurea, is a selective and systemic pyrimidinylsulfonylurea herbicide. It is absorbed through roots as well as leaves and transloLetters in Applied Microbiology © 2014 The Society for Applied Microbiology
cated throughout the plant. Biochemically, it acts as an inhibitor of the acetolactate synthase enzymes responsible for branched-chain amino acid biosynthesis, hence stopping cell division and plant growth. Sulfosulphuron controls grassy and broad leaf weeds in wheat at an application rate of 20 g ha 1 (Parrish et al. 1995). It controls 1
Biodegradation of sulfosulphuron
weeds in wheat like Phalaris minor, Avena ludoviciana, and Rumex retroflex, resulting in higher grain yield (Banga et al. 2003). Barley (Hordeum vulgare), oats (Avena sativa), maize (Zea mays), sunflower (Helianthus annuus L.) and sorghum (Sorghum bicolor L.) are among those crops which are sensitive to sulfosulphuron (Shinn et al. 1998; Miller et al. 1999; Kelly and Peeper 2003). Carry-over effect of sulfosulphuron was noticed in the growth of some crops like maize and sorghum (Chhokar et al. 2006). The half-life (DT50) of it in general is within the range of 20–60 days. Sulfosulphuron residue was found to be below detectable limit after 15 days of spraying @ 25 g ai ha 1 in wheat on sandy-loam soil, the half-life was within 53–64 days (Saha et al. 2003; Singh and Kulshrestha 2007), and no residue was detected in the Inceptisol soil after harvest of wheat crop (Atmakuru and Maheswari 2003). The disappearance of the herbicide from soil takes place either through the physical displacement, viz., leaching down, runoff, etc. or through the degradation of the compound. Members of the sulfonyl urea family undergo pH-dependent chemical hydrolysis, the rate increases with the decreasing soil pH, and thereafter, the hydrolysed products undergo microbial degradation (Beyer et al. 1988). Under alkaline condition, sulfosulphuron yields 1-(2-ethylsulfonylimidazo[1,2-a] pyridin-3-yl-3-(4,6-dimethoxypyrimidin-2-yl) amine, and under acidic condition, it degrades to 1-(2-ethylsulfonylimidazo[1,2-a] pyridin)-3-sulfonamide and 4,6-dimethoxy-2-aminopyrimidine (Saha and Kulshrestha 2002). But in the agronomic pH, the degradation of herbicides is mainly caused enzymatically by soil-dwelling microbes. The present investigation was undertaken to isolate sulfosulphuron-degrading fungi from the black soil and, thereafter, to judge its suitability in the bioremediation of sulfosulphuron-contaminated soil. Results and discussion Isolation and characterization of sulfosulphurondegrading fungus Fungi, isolated from rice rhizospheric soil, were allowed to grow in the minimal media having sulfosulphuron as carbon and nitrogen source. Trichoderma sp., a widely distributed fungus in soil, survived and grew in that media with sulfosulphuron at the level as high as 200 mg l 1. The organism was characterized by its morphological characters (Fig. 1). Colonies of this filamentous fungus were found at first white and gradually becoming either dull white or light green to yellowish green or deep shades with age and grew rapidly and mature in 5 days at 25°C. Vegetative hyphae were fertile. Conidiophores were erect developing from side branch, 2
U. Yadav and P.P. Choudhury
Figure 1 Microscopy of Trichoderma sp. isolated from wheat rhizospheric soil.
branching usually opposite, and bore terminal conidial heads. Conidia were ovate to elliptic or glabrous and smooth. Species ‘viride’ was characterized by microscopic observations. Colonies were typically fast growing at 25–30°C, but the growth was inhibited at 35°C. Conidia were formed typically within 1 week in compact or loose tufts in shades of green or yellow or less frequently white. Conidiophores were highly branched and terminated in one or a few phialides, which were enlarged in the middle. But the confirmation of this species as ‘viride’ is subjected to further investigation. Degradation of sulfosulphuron by Trichoderma sp. in sterilized soil The incubation of Trichoderma in minimal media as well as soil led to major degradation of the compound. Five key metabolites, which give the direction of degradation pathway, are confirmed by mass spectra and synthesis of the metabolites, and related literatures. Metabolite-I From the mass spectrum (Fig. 2a) of this compound, which shows the M + H peak at m/z 156, the molecular mass is determined as 155. Peaks at m/z 140, 139 and 124 suggest the loss of one methyl (-CH3), one amino (-NH2) and one methoxy (-OCH3) group of the parent molecule. From this, the structure of the metabolite was assigned as 2-amino4,6-dimethoxypyrimidine (I), which is further confirmed by the previously reported spectrum of the compound (Saha and Kulshrestha 2002; Atmakuru et al. 2007). Metabolite-II The parent ion peak is at m/z 290 (Fig. 2b) indicating the molecular weight of the compound as 289. The peaks Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
U. Yadav and P.P. Choudhury
8·9e5
Biodegradation of sulfosulphuron
100·1
(a)
8·5e5
H3CO
8·0e5
+ •
H
7·5e5
H3CO
7·0e5 6·5e5 6·0e5
H3CO
Intensity, cps
5·5e5 5·0e5
156·2
N
4·5e5 4·0e5 57·2
H3CO
+ • NH2
N
3·5e5
N
H3CO
82·1 2·5e5
H3CO
+ • NH
N
N
3·0e5 68·1
N
+ •
124·2
N
H3CO
1·5e5
140·0
1·0e5
112·1
5·0e4
58·1
43·0 0·0 40
67·1
55·1 50
60
82·9 70·2
70
92·3
80
90
139·3
114·1
97·9
84·2
81·0
100
110
123·3
120 m/z, Da
130
140
150
160
170
180
+ •
(b)
+ •
3·6e4
200
273·1 290·1
N
O2S
N
3·2e4
190
N 125·1
3·4e4
N
SO2CH2CH3 + •
3·0e4 181·3 2·8e4
N
2·6e4
+ •
2·4e4 Intensity, cps
+ • NH2
N
H3CO
N
2·0e5
3·8e4
+ •
N
105·0
SO2CH2CH3
117·1
2·2e4
N
2·0e4
HC
N
H2N O2S + •
N N
1·8e4
N
1·6e4
SO2CH3 + •
1·4e4 165·3
1·2e4
78·4
1·0e4
N SO2CH3
137·3 152·1 149·1
4000·0
0·0 40
N
153·1
96·3
6000·0
61·1 70·177·4 60
80
90·2
198·2 180·1
118·1 123·4 135·2 107·1 100
+ •
N
133·4
8000·0
2000·0
197·1
121·2
199·0 211·1
167·9 120
140
160
180
200
N SO2CH2CH3
225·0 220
244·8 240
262·1 260
275·0 280
300
m/z, Da
Figure 2 Mass fragmentation pattern of products formed through decarboxylation of sulfonylurea bridge; (a) 2-amino-4,6-dimethoxypyrimidine (I) and (b) 2-ethylsulfonyl imidazo {1,2-a} pyridine-3-sulfonamide (II).
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U. Yadav and P.P. Choudhury
produces a fragment of m/z 182. The possible fragment for H2ON is -NH-OH (hydroxyl amine group). The presence of the peaks at m/z 155 and 139 indicates the presence of dimethoxy pyrimidinyl ring. Hence, the peak at 182 is due to one carbonyl group added to dimethoxy pyrimidinyl amine. Considering this fragmentation pattern, the structure of the compound is assigned as N- (4,6-dimethoxypyrimidin-2-yl)-N’hydroxyurea (IV).
at 117 and 211 confirm the presence of ethyl sulfonyl imidazo pyridinyl moiety of sulfosulphuron. The addition of the SO2NH2 group to this moiety imparts the complete structure of this metabolite, and hence, the structure is assigned as 2-ethylsulfonyl imidazo {1,2-a} pyridine-3sulfonamide (II). This structure is further confirmed by the previously reported spectrum (Saha and Kulshrestha 2002; Atmakuru et al. 2007). Metabolite-III The peak at m/z 199 (Fig. 3) indicates the molecular mass of 198. The parent molecule loses one amino group (16 amu) to give a peak at m/z 182, which on losing one carbonyl group (28 amu) produces a daughter ion (155 amu) at m/z 156. Peaks at m/z 140, 139 and 124 suggest the loss of one methyl (-CH3), one amino (-NH2) and one methoxy (-OCH3) group from the daughter ion of 155 amu. From this fragmentation pattern, the structure is assigned as N-(4,6-dimethoxypyrimidin-2-yl)urea (III). This is further confirmed by the mass spectrum of similar urea derivative formed from chlorimuron ethyl (Sharma et al. 2012).
Metabolite-V Peaks at m/z 139 and 156 in the spectrum (Fig. 4b) indicate the presence of 4,6-dimethoxypyrimidinyl-2-amino group, which on adding a carbonyl group produces a fragment of m/z 182. The molecular ion peak is found at m/z 337. There is no peak found in between m/z 337 and 182 and no major peak other than peaks at m/z 139, 156 and 182. It is possible if there is any repetition of the fragment 4,6-dimethoxypyrimidinyl group. Thus, the compound is assigned as N, N’-bis(4,6-dimethoxypyrimidin-2-yl)urea (V). It is a demonstrated fact that the first degradation step of any sulfonyl urea herbicide is the cleavage of the sulfonyl urea bridge, which is a pH-dependent response. The pH should be moderately to highly acidic. But in the present experiment, the pHs of the media and the
Metabolite-IV The molecular ion peak appears at m/z 214 (Fig. 4a). A loss of 22 amu (H2ON) from the molecular ion 83·3
750
H3CO
700
H3CO
650
68·1
+
H3CO
N 100·1
H3CO
550
N
+
N
•
NH2
500 Intensity, cps
•
•
N
57·2
600
+
N
N
82·2
H3CO
450
+
N
H3CO
•
NH
400
H3CO
N
+
N
350
O NH C
•
N 300
140·0
69·3
250 200
H3CO
93·3
H3CO
55·2 43·2
112·3
139·1
N
67·3
150
92·3 98·0 58·0 56·0
100 50
71·0
41·1 53·3 59·2 65·1
0 40
50
60
77·3
81·2
79·1 80·5 72·5
70
80
111·3
124·1 109·4 94·2 99·4 103·8 114·0 128·1 97·0
90
O
+ •
NH C NH2
70·4
H3CO
N
156·0 141·1
182·3
100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 m/z, Da
Figure 3 Mass fragmentation pattern of N-(4,6-dimethoxypyrimidin-2-yl)urea (III) formed through the cleavage of sulfonylamide linkage.
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Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
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Biodegradation of sulfosulphuron
182·1 4·6e4
(a)
4·4e4 4·2e4
H3CO
4·0e4
+ •
N
3·8e4
NH2 N
3·6e4
H3CO
3·4e4
H3CO
O NH C
N
3·2e4 3·0e4
H3CO
Intensity, cps
2·8e4
+ •
N
+ •
N
H3CO
2·6e4
N
2·4e4 2·2e4
H3CO
83·3
2·0e4
H3CO
139·2
1·8e4
+ O • NH C NHOH
N
1·6e4 1·4e4
H3CO
1·2e4
N
1·0e4 214·2
8000·0 6000·0
157·1
4000·0 2000·0
71·1
0·0 40
60
82·0
93·2
80
100·2 109·3
138·2
120
100
140
200
180 m/z, Da
220
240
260
280
300
156·2
8·3e4 8·0e4
160
(b)
H3CO
7·5e4
N
7·0e4
N
+ • NH2
H3CO
6·5e4 6·0e4 5·5e4
Intensity, cps
5·0e4
H3CO
N
4·5e4 4·0e4
O NH C
+ •
N
H3CO
3·5e4 3·0e4
182·4
2·5e4
H3CO
2·0e4 1·5e4
H3CO
1·0e4 5000·0 0·0
40
H3CO
80
100
+ •
O NH C NH
N
N
H3CO
N
+ •
OCH3
N 337·0 139·3
100·3
60
N
N
OCH3
120
140
160
180
200 m/z, Da
220
240
260
280
300
320
340
Figure 4 Mass fragmentation pattern (a) N-(4,6-dimethoxypyrimidin-2-yl)-N’-hydroxyurea (IV); (b) N, N’-bis(4,6-dimethoxypyrimidin-2-yl)urea (V).
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
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Biodegradation of sulfosulphuron
U. Yadav and P.P. Choudhury
soil were neutral. The incubation of the sulfosulphurontreated media and sterilized soil without Trichoderma did show hardly any degradation of the herbicide. But in the presence of the fungus Trichoderma, both in the media and in the soil, two metabolites, viz., 2-amino-4,6dimethoxypyrimidine (I) and 2-ethylsulfonyl imidazo {1,2-a} pyridine-3-sulfonamide (II), are formed. The formation of the couple of products, I and II, is possible due to the cleavage of the sulfonyl urea bridge. It strongly suggests that this cleavage is not always pH dependent; enzyme can also do the same. This is basically a decarboxylation reaction of the sulfonylurea bridge, and a decarboxylase type of enzyme is involved here to catalyse the reaction. But, interestingly, the presence of metabolite, N-(4,6-dimethoxypyrimidin-2-yl)urea (III), suggests a different mode of degradation. The formation of this metabolite is possible through the degradation of the sulfonyl amide linkage. This is a hydrolysis reaction at the sulfonyl amide bond, and probably a hydrolase type of enzyme was involved to catalyse the reaction. The metabolite, N-(4,6-dimethoxypyrimidin-2yl)-N’-hydroxyurea (IV), is formed due to oxidative hydroxylation on the nitrogen of urea bridge. Merely that whether it is on the terminal nitrogen of 2-amino4,6-dimethoxypyrimidine or it is on the urea nitrogen of sulfosulphuron is indistinct from the present outcome.
H3CO H3CO
N N
H3CO
N N
O C N N H H
Materials and methods Chemicals Samples of technical sulfosulphuron (905%) and analytical sulfosulphuron (999%) were obtained from Indofil
O O N C N S N N H H O SO CH CH 2 2 3 Cleavage of sulfonyl urea bridge
Cleavage of sulfonyl amide linkage
H3CO
Perhaps, the monooxygenase-mediated oxidation of the terminal amino group caused the formation of the metabolite-IV. This particular step occurred not only in the media, but also in the soil. The metabolite, N, N’-bis (4,6-dimethoxypyrimidin-2-yl)urea (V), is probably formed by the union of two radicals, Ф-NH-C=O and NH- Ф, which are produced during the initial steps of microbial metabolism of sulfosulphuron. A scheme on the Trichoderma-assisted degradation pathways of sulfosulphuron in soil has been proposed (Fig. 5). Sulfosulphuron seems to have the ability to inhibit the growth of some fungi present in soil as it did not allow many of them to grow in media and soil incubated with the herbicide. But the high tolerance of Trichoderma sp. towards sulfosulphuron and its ability to utilize the herbicide as a sole source of carbon indicate to consider the fungus as a promising candidate for the bioremediation of sulfosulphuroncontaminated soil.
H3CO
O N N S O SO CH CH 2 2 3
H3CO
O C
O N N N S N H O SO CH CH 2 2 3 Decarboxylation N
N H
HOH H3CO H3CO
N N III
O NH C NH2
H3CO H3CO
N N
H3CO
N N
N N
H2N O2S
SO2CH2CH3 I
H3CO
NH2
II
O NH C NHOH IV
Figure 5 Proposed pathways for the degradation of sulfosulphuron by Trichoderma sp.
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Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
U. Yadav and P.P. Choudhury
Chemicals Company, Mumbai. Technical sulfosulphuron was further purified by recrystallization until a constant melting point of 201°C was achieved. It is a white amorphous solid. The purity was checked by thin layer chromatography (TLC) (solvent system—chloroform:: acetonitrile: 2 : 3, v/v; Rf = 035). Laboratory grade reagents and solvents were procured locally. All solvents were dried and distilled before use. HPLC-grade solvents and reagents were used during chromatographic and spectroscopic analysis. Deionized water was obtained from the Milli-Q SP Reagent water system (Millipore, Bedford, MA). Soil Soil samples were collected from different locations of wheat field of the research farm in the directorate. The field was having the history of the sulfosulphuron application for consecutive 5 years. Soils were collected in sterile plastic bags, air-dried at room temperature and sieved (2 mm mesh). Fresh soil was used for the isolation of fungi. The soil used was a typical black soil (vertisol) with the chemical status as follows: organic matter 096% (w/w); pH (water 1 : 25) 708; EC 048 dS 1; and CEC 338 Cmol (p+)kg 1. Isolation and identification of sulfosulphuron-degrading fungus The collected soil was enriched with sulfosulphuron (5 mg in 100 g of soil) and incubated for a week at 30°C. For a selection of fungi as a suitable sulfosulphurondegrading agent, serial dilution following agar plating of incubated soil was done. Fungi that appeared on potato dextrose agar (PDA) plates (prepared from 200 g of potato, 20 g of dextrose, 20 g of agar and 1000 ml of water) after 5 days of incubation were further plated for obtaining pure cultures. The fungi screened from the sulfosulphuron-enriched soil were again incubated for 7 days in minimal PDA broth (prepared from 10 g of potato, 20 g of dextrose and 1000 ml of water) containing different levels of sulfosulphuron, viz., 25, 50, 100 and 200 mg per 100 ml of broth. The most efficient fungus was screened out on the basis of their growth and was further inoculated on PDA plates. After 2 days of incubation, colony morphology of the isolate was examined. On the basis of colony morphology and microscopy of spore structures, the fungus was characterized. Degradation of sulfosulphuron by Trichoderma sp For the degradation studies, 25 mg of sulfosulphuron was added to 100 ml of sterile dextrose-minimal broth Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Biodegradation of sulfosulphuron
(prepared from 100 g of potato, 10 g of dextrose and 1000 ml of water) in 250-mL flask. The sulfosulphuron was allowed to dissolve overnight on shaker. Twenty such flasks were incubated with isolated Trichoderma sp. in the dark at 25°C for 27 days in BOD incubator. Three flasks with minimal broth and sulfosulphuron and without the incubation with Trichoderma sp. were kept in the dark as a control. Degraded products were extracted by partitioning in chloroform from the broth sampled at different time intervals, viz., 3, 9, 16 and 27 days of incubation. The solvent was then evaporated under low pressure in the rotary vacuum evaporator to obtain a crude mixture of products. The products were purified by the preparative TLC and characterized by the spectroscopic techniques. Liquid chromatography–mass spectroscopy An API 3200 Qtrap mass spectrometer hyphenated to Shimadzu Ultra Fast Liquid Chromatography was used for the mass characterization of degraded products. Mass spectrometry analysis was performed with electrospray ionization (ESI) in positive (5500 eV) mode for each sample. The nebulizer gas and heater gases were adjusted at 30 psi and 55 psi, respectively. The ion source temperature was set at 500°C. Each sample was injected by infusion technique at the rate of 10 lls 1. Preparation of major metabolites Acid hydrolysis of sulfosulphuron Sulfosulphuron (200 mg) was added to distilled water (100 ml). The pH of the solution was adjusted to 25 by the addition of concentrated hydrochloric acid (2 ml). The solution was stirred magnetically at 32°C, and the reaction was monitored by TLC and continued until the disappearance of the spot of sulfosulphuron, which took 48 h. The products formed were separated by preparative TLC, purified by crystallization from benzene and characterized by spectroscopic methods. Compounds were structurally assigned as 2-amino-4,6-dimethoxypyrimidine (I) and 2-ethylsulfonyl imidazo {1,2-a} pyridine-3-sulfonamide (II) with the help of mass spectra (Figs 2 and 3). Acknowledgements The authors are grateful to the Director, Directorate of Weed Science Research (ICAR), Jabalpur, MP, India, for providing the research facilities to complete this PG dissertation work of U.Y., Govt. M. H. Autonomous College of Home Science and Science for Women, Rani Durgavati Vishwavidayalaya, Jabalpur (M.P.). 7
Biodegradation of sulfosulphuron
Conflict of interest We hereby declare that no conflict of interest exists between us. References Atmakuru, R. and Maheswari, S.T. (2003) Dissipation of sulfosulfuron in soil and wheat plant under predominant cropping conditions and in a simulated model ecosystem. J Agric Food Chem 51, 3396–3400. Atmakuru, R., Thirugnanam, P.E. and Sathiyanarayanan, S. (2007) Identification of residues of sulfosulfuron and its metabolites in subsoil-dissipation kinetics and factors influencing the stability and degradation of residues from topsoil to subsoil under predominant cropping conditions. Environ Monit Assess 130, 519–528. Banga, R.S., Yadav, A. and Malik, R.K. (2003) Bioefficacy of flufenacet and sulfosulfuron alone and in combination against weed flora in wheat. Indian J Weed Sci 35, 179–182. Beyer, E.M. Jr, Duffy, M.J., Hay, J.V. and Schlueter, D.D. (1988) Sulfonylureas. In Herbicides: Chemistry, Degradation, and Mode of Action, ed. Kearney, P.C. and Kaufman, D.D. pp. 117–189. New York, NY: Dekker. Chhokar, R.S., Sharma, R.K., Chauhan, D.S. and Mongia, A.D. (2006) Evaluation of herbicides against Phalaris minor in wheat in north-western Indian plains. Weed Res 46, 40–49.
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Kelly, J.P. and Peeper, T.F. (2003) Mon 37500 application timing affects cheat (Bromus secalinus) control and winter wheat. Weed Sci 51, 231–236. Miller, P.A., Westra, P. and Nissen, S.J. (1999) The influence of surfactant and nitrogen on foliar absorption of Mon 37500. Weed Sci 47, 270–274. Parrish, S.K., Kaufmann, J.E. and Croon, K.A. (1995) MON 37500: a new selective herbicide to control annual and perennial weeds in wheat. In Proc Brighton Crop Protect Conf-Weeds. pp. 57–63. Surrey: BCPC Farnham. Saha, S. and Kulshrestha, G. (2002) Degradation of sulfosulfuron, a sulfonylurea herbicide, as influenced by abiotic factors. J Agric Food Chem 50, 4572–4575. Saha, S., Yaduraju, N.T. and Kulshrestha, G. (2003) Residue studies and efficacy of sulfosulfuron in wheat crop. Pestic Res J 15, 173–175. Sharma, S., Banerjee, K. and Choudhury, P.P. (2012) Degradation of chlorimuron-ethyl by Aspergillus niger isolated from agricultural soil. FEMS Microbiol Lett 337, 18–24. Shinn, S.L., Thill, D.C., Price, W.J. and Ball, D.A. (1998) Response of downy brome (Bromus tectorum) and rotational crops to Mon 37500. Weed Technol 12, 690–698. Singh, S.B. and Kulshrestha, G. (2007) Determination of sulfosulfuron residues in soil under wheat crop by a novel and cost-effective method and evaluation of its carryover effect. J Environ Sci Health B 42, 27–31.
Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
