| Carbendazim (C9H9N3O2; methyl-2-benzimidazole carbamate; MBC) is widely used as an agricultural and horticultural fungicide around the world .It is a systemic benzimidazole fungicide that plays a very important role in plant disease control. Carbendazim is used to control a broad spectrum of diseases on arable crops (cereals, oilseed rape), fruits, vegetables and ornamentals. It is also used in post-harvest food storage, and used as a seed pre-planting treatment. Carbendazim works by inhibiting the development of fungi probably by interfering with spindle formation at mitosis (cell division). Carbendazim is a fungicide of major concern due to its suspected hormone disrupting effects. Carbendazim has a half-life (time taken for half the sample to decay) of 6-12 months on bare soil and 3-6 months on turf and is mainly decomposed by microorganisms. Microbial degradation is considered to be an essential approach in their decontamination .So it is necessary to discover and study the carbendazim degradation microbes.The green fluorescent protein (GFP) is a unique tool that permits the monitoring of gene expression and protein location in living cells. Green fluorescent protein is stable, does not require cofactors for activity and can be functionally expressed in different bacterial species. Because of GFP unique properties, it can be used as a reporter of gene expression, dynamic processes during bacterial development, and the behavior of single bacteria in complex environments.Based on the above-mentioned consideration, the author using a carbendazim-degrading bacterial strain NY97-1, isolated by Shanxi Key Laboratory of Pesticide Science, conducted a study on characteristics of a carbendazim degrading bacterium strain and GFP marking .The main contents and conclusions are as follows:1. Carbendazim-degrading bacterium NY97-1 the 16S rDNA was amplified by PCR then cloned into pMD18-T vector and sequenced. Using BLAST software, comparison results showed isolated NY97-1 should be identified as Bacillus puntil us.2. In order to construct the promoter gene libraries of B.pumilus, digested fragments of total DNA of NY97-I that was digested by Sau3AI was ligated to pUC19-gfp that was digested by BamH I , and the product was transformed to the E.coli DH5a competent cells. Selected two strong positive clones, subcloned B.pumilus promoter functional fragments F4, F5,and then constructed Escherichia coli-Bacillus shuttle vector pNW33N-F4-gfp,pNW33N-F5-gfp. They were transformed into carbendazim-degrading bacterium Bacillus pumilum NY97-1 by electroporation. They were expressed well in NY97-1 through fluorescence microscope. The functional F4 F5 showed constitutive promoter elements. The results provided fundamental data and materials for the further study on the ecological behavior of Carbendazim-degrading bacterium NY97-1.3. Carbendazim analysis: Carbendazim in culture medium was analyzed by high-performance liquid chromatography (HPLC) with an uv detector. Sample preparation: Culture medium were sampled after rearing for 24 hrs, decanted and centrifuged at 12,000g for 5 min at 4℃,and then supernatant fluid of 1 .0mL was cleaned up through SPE-C18 column (500mg), which was conditioned with 4.0mL actone, methanol and distilled water respectively in advance. Adding 1.0 mL sample and eluted with ca. 2mL methanol, the volume adjusted to 3.0mL with methanol. Calibration curve : Carbendazim standard was first soluted with drops diluted hydrochloric acid solution (1:11, v/v) and then stock solution of carbendazim was prepared in methanol at 1000mg/L.Working solutions of pertinent concentrations (1,10,12.5,25,30mg/L) were made by a serial dilution of stock solutions with methanol. All standard and stock solutions were stored in glass-stopper bottles at 4℃.The spiked sample prepared by adding known amount of carbendazim stock solutions to culture medium .The calibration curve was made by injecting known concentrations of working solutions of carbendazim. Carbendazim was qualified through comparing the retention time of standards and sample peaks. The concentration was calculated using the standard calibration curve of carbendazim. HPLC condition: The chromatographic conditions were as follows: Kromasil C18 5μm(250x4.6mm)(i.d. 5μ). The mobile phase was methanol: water (90:10 v/v) at flow rate of 0.8mL/min. The injection volume was 20μL and uv detector worked at 284nm.4. The influence of environmental factors on the performance of carbendazim degradation by Baillus pumilus strain NY97-1 was investigated. For this purpose, experiments were conducted under aerobic conditions, and carbendazim degradation percentage in culture medium was characterized by high performance liquid chromatography (HPLC) with uv detector. An initial carbendazim of 10, 30, 50, 100, 300mg/Lwere fortified in mineral salt medium(pH 7.0)with the degradation rate of 42.44, 48.97, 77.19, 78.66 and 90.07% were obtained in 24 hrs rearing at 30℃. Supplemention with little amount of organic nitrogenous source had a significant effect on the carbendazim degradation, while the inorganic nitrogenous source had negative effect. Carbendazim degradation rate varied form 61.55% to 87.76% when pH value within the range of 4-10 after addition of organic nitrogen, the lowest carbendazim degradation percentage of 61.55% was obtained on the pH 4. Carbendazim degradation ability was enhanced with temperature increasing. Future characterization of strain NY79-1 provides an opportunity for its development in bioremediation of carbendazim contamination.5. The effect of NY97-1 fermentation on toxicity of carbendazim to different pathogens was measured. The relative inhibition rates of the NY97-1 against Fusarium oxysporum (Schl.)f. sp.Lycopersici (Sacc.) Snyder et Hansen, Fusarium oxysporum (Schl.) f.sp. vasinfectum (Snyd. et Hans), Fusarium oxysporum f.sp. niveum (E.F.Smith) Snyder et Hansen, Fusarium sp, Fusarium sp, Fusarium oxysporum (Schl.) f.sp. cucumerinum Owen were 50.8%, 50.8%, 54.1%, 37.5%, 31.6%, 42.5%.6. Degradation ability of carbendazim and biocontrol efficacy against six Fusarium wilt diseases of multifunctional bacterial strain NY97-1 was estimated. The EC50 estimates of 10% NY97-1 fermentation-carbendazim mixtures increased at 3d and 7d compared to those in the treatments with carbendazim alone,showed a decreased effect to the pathogens.7. Activity of anti-oxidative enzymes of cucumber (Cucumis sativus L.) when carbendazim was applied as soil drench at a dosage of 0, 5, 50 and 100 mg kg-1 was determined. The changes in the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) enzymes were measured in the roots, stems and leaves of cucumber. The plants were sampled at the cotyledon phase (14 days) and the florescence phase (56 days). A strong correlation between antioxidant content and carbendazim concentration was observed. As concentration increased, the activities of SOD and CAT increased in roots and leaves. In stems, SOD and CAT activities were increased in the florescence phase and then decreased in the cotyledon phase. The content of GPX increased both in roots and stems, and decreased in leaves. Higher levels of SOD, CAT and GPX were observed in cucumber cotyledons than in the older leaves. The present study suggests that carbendazim treatments had different effects on the cucumber antioxidant system in different tissues. It was concluded that cotyledons might play an important role in stress adaptation as the carbendazim concentration increased, and the ability of mature cucumber to maintain a balance between the formation and detoxification of activated oxygen species seemed enhanced. On the basis of our observations, we conclude that increased SOD, CAT and GPX activities provide plants with increased carbendazim stress tolerance.
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