| Phthalate,referred to as PAEs,is a kind of very important organic compounds,which is widely used in plastics,cosmetics,spices and coatings and other industrial production.Due to the wide application of PAEs,a certain concentration of PAEs has been detected in soil,air and water in most areas of China,di-n-butyl phthalate(DBP)pollution is particularly serious.Because the PAEs in soil in some areas of serious pollution exceed the standard,therefore the plant body absorbed a large amount of plasticizer in the process of plant growth.For example,there have been reports that more than 70% green vegetables those commonly eaten contained excessive levels of PAEs,which may do great harm to agricultural products.Endophytes are the intercellular spaces or intracellular bacteria in various tissues and organs of healthy plants at certain or all stages of their life history.It can be effective colonization in plants and is not easily affected by external environment.Therefore,endophytes with DBP degradation ability can be used to accelerate the metabolism and degradation of DBP in green vegetables,it is important for the prevention and control of the plasticizer in the future.In this study,Chinese chives was the supplied experimental plants which collected from jiangsu province academy of agricultural sciences' test field.we applied the surface sterilization and grinding method,to successive screen and isolate the endophytes that capable of degrading DBP from the Chinese chives,using DBP as the sole carbon source of the inorganic salt medium for screening DBP degradation and culturing endophytic bacteria.The characteristics of DBP degradation in vitro and in vivo were analyzed,respectively.The endophytic bacteria were identified by physiological and biochemical analysis combined with 16 SrDNA sequence analysis.At the same time,the degradation pathway in vitro and in vivo was discussed and the general way to degrade DBP was obtained.To further study the degradation mechanism of persistent organic pollutants such as PAEs residues in plants by endophytic bacteria,then a certain foundation was established.The results of this research can provide some theoretical guidance for the use of plasticizer endophyte regulation of agricultural residues,and provide some new ideas.The main results are as follows:1.A strain of Endophytic Bacillus JR20(Bacillus amyloliquefaciens subsp.)was isolated from Chinese chive.The degradation rate of DBP in 10 days with initial concentration of 5 mg/L was 98.06%.Strain JR20 had a broad-spectrum tolerance to temperature.In the range of 30~40 ?,the degradation rate of DBP was almost no difference,and the degradation rate of DBP was greater when the p H in the range of 7.0~8.0 than in 6.0.Therefore,the optimal culture condition of degrading DBP by strain JR20 was 37 ?,pH was about 7.0,and the initial concentration was 5 mg/L.By mass spectrometry,phthalic acid(PA)was detected in the degradation products of DBP.With the decreased of DBP concentration,the concentration of PA increased continuously,on the fifth day PA reached the maximum amount.Thus,the specific pathway for the degradation of DBP by strain JR20 was that the esters were hydrolyzed to the corresponding monoester,and then the monoester were hydrolyzed to the corresponding alcohols.2.The endophytic strain JR20 was isolated from the root of Chinese chive,in addition to the degradation characteristics of DBP in vitro,it can also be transferred into vegetables through colonization to promote the degradation of DBP in green vegetables.By gradient acclimation,gradually increase the concentration of rifampicin.Finally the strain that can grow in the media containing 100 mg/L rifampicin was selected.Through observation and detection,the strain was the rifampin resistant marker JR20.Rifampin resistant marker strain JR20 can be colonization to the green vegetables through the vegetables absorb the nutrient solution,and reached the peak number of colonization in about eighth days,later,due to the green vegetables growth in late stage,the organization began to aging,so the number of strains in roots,stems,leaves decreased,until finally zero.The results also show that DBP can be self degraded in nutrient solution,and within 20 days can be completely degraded when DBP initial concentration was 1 mg/L.3.After rifampicin resistance marker strain JR20 colonization into green vegetables,the results showed that the concentration of DBP in roots,stems and leaves showed a downward trend,but the degradation rate of the experimental group was significantly higher than that of the control group without strain JR20.At about 20 th day,the degradation rate of DBP in the root of the experimental group was 95.13%,while the DBP degradation rate in the control group was 89.52%,the degradation rate of DBP in the stems and leaves of the experimental group was about 90.45%,and the DBP degradation rate in the control group was 80.61%,the DBP concentration in the experimental group was significantly lower than that in the control group.Therefore,strain JR20 could accelerate the degradation rate of DBP in the green vegetables,and its concentration was significantly lower than that of the same period without strains JR20.The concentration of DBP in the nutrient solution of the experimental group and the control group also showed a downward trend,at twentieth days or so,DBP in the nutrient solution of the experimental group and the control group was close to zero.Therefore,due to the rapid degradation rate of DBP,in order to achieve balance,the green vegetables with JR20 prompt it to quickly absorb DBP in the environment.Strain JR20 could degrade DBP in roots,stems and leaves of green vegetables,and the final degradation product of DBP was PA,the degradation pathway was the same as that in inorganic salt medium.4.Colonization of strain JR20 had little effect on transfer factor of DBP in green vegetables and adsorption factor of DBP in nutrient solution by roots in the early stage of green vegetables,but in the middle and late will promote the adsorption of DBP by vegetables. |
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