Effects Of Repeated Carbendazim Applications On Soil Microbial Community And Its Bioremediation

Carbendazim is a broad-spectrum fungicide widely used for the control of plant fungal diseases on arable crops, fruits, vegetables and ornamentals which results in potential environmental contamination. To evaluate the safety of carbendazim applications, studies were conducted concerning its influences on soil microbial functional diversity and genetic structure. The soil functional resilience and structural stability exposed to carbendazim were also investigated. One bacterial consortium capable of degrading carbendazim was isolated. The bioaugmentation of carbendazim in soil by this bacterial consortium, its effect on microbial functional diversity and genetic structure were further studied. The results were summarized as follows:1. Microbial functional diversity and functional resilience in soil after repeated carbendazim applications were estimated with BIOLOG Ecoplates. An obvious inhibitory effect was observed in microbial utilization of carbon substrates and diversity after the first introduction of carbendazim. With subsequent carbendazim applications, this effect disappeared gradually. Soil bacterial diversity in all treatments recovered to the control level 15 d after the fourth applications. AWCD and diversity index, such as Simpson, McIntosh and Shannon indexes can be used to indicate the soil functional resilience to carbendazim. The best results can be obtained by using AWCD and Simpson index to indicate the functional resilience. Soil functional resilience to carbendazim appeared after the second application and formed completely after the third application.2. The effects of repeated applications of carbendazim on bacterial community structure as well as the structural stability exposed to carbendazim was studied under field conditions using TGGE and partial sequence analysis of PCR-amplified 16S rRNA genes. The results showed that bacterial community structure was not changed after the first carbendazim application, but gradually altered after the second application. With the application frequency and carbendazim concentration increased, the effect on bacterial community structure became much more obvious. At the same time, an application-responsive band from the carbendazim treatments was sequenced and aligned. The sequence of this band was related to members of theγsubdivision, showing 98% similarity with an uncultured gamma proteobacterium clone, CRE-PA17.Soil microbial structural stability was weak after the second carbendazim application and became stronger after the third and fourth applications. However, the microbial community structure in the carbendazim-treated soil was different from that in the control soil after the third and fourth applications. After one year, the bacterial community in carbendazim-treated soil recovered to that in the control.3. A bacterial consortium capable of utilizing carbendazim as sole carbon and energy sources was isolated from Huajiachi campus, Zhejiang University, Hangzhou, China. This bacterial consortium was mainly composed of Bacillus subtilis,Paracoccus sp.,Flavobacterium omnivorum and Pseudomonas sp. The ability of the bacterial consortium to degrade carbendazim in pure cultures depends on pesticide concentration, pH and temperature. The degradation of carbendazim at concentrations of 1, 10 and 100 mg l^-1 within 1 d by this consortium were measured to be 64.25%,20.59% and 17.47%, respectively. The degradation rates of carbendazim were affected by pH and temperature following an order of pH 9.0 > pH 7.0 > pH 5.0 and 35℃> 25℃> 15℃, respectively.Degradation of carbendazim in soil was significantly accelerated by the inoculation with the carbendazim-degrading bacterial consortium. Compared with the uninoculated soil, the degradation rates of carbendazim in the soil inoculated one, two and three times 11d after the carbendazim application were accelerated by 1.07, 1.33 and 1.53 times, respectively.4. The effect of repeatedly inoculating a soil with the carbendazim-degrading bacterial consortium on the soil microbial functional diversity was studied by BIOLOG method. The results showed that soil microbial functional diversity was not affected by the inoculation with the carbendazim-degrading bacterial consortium before carbendazim application, but influenced after carbendazim application. Microbial utilization of carbon substrates and Simpson index were stimulated in inoculated soil, especially in the soil inoculated three times with the carbendazim-degrading bacterial consortium 11d after carbendazim application. After initial variations, soil microbial diversity recovered to similar levels of the controls 18 d after carbendazim application.5. TGGE method was applied to determine the relative genetic complexity of microbial communities in uninoculated and inoculated soils. The result showed that soil microbial community structure was not changed by the inoculation with the carbendazim-degrading bacterial consortium before carbendazim application, but was altered 11 d after carbendazim application. Microbial community structure of inoculated soil, especially the soil inoculated only one time recoverd to the control level 18 d after carbendazim application.