“寒富”苹果园土壤氨氧化细菌的筛选与鉴定

Screening and Identification of Soil Ammonia-Oxidizing Bacteria from "Hanfu" Apple Orchard

为研究氨氧化细菌的作用机理及其在预警和调控土壤微生态环境方面的作用,采集2011年5月、7月、9月和11月4个不同生育时期"寒富"苹果园根区土壤,应用富集培养结合硅胶平板方法分离土壤可培养氨氧化细菌;依据氨氧化活性检测,个体形态、培养特征及16S r DNA序列分析,对具有良好硝化能力的优势氨氧化细菌菌株进行筛选与鉴定。结果表明:苹果园土壤中共分离获得33株优势氨氧化细菌菌株,4个生育时期中,11月氨氧化细菌数量最多,7月最少,各时期分离菌株的氨氧化活性随生育时期而各异。从总体上看,11月分离菌株氨氧化活性较为明显。初筛与复筛结果表明:菌株S5-4和S11-10在不同p H值培养条件下均能表现出良好的氨氧化活性,初步鉴定二者分别为粘着剑菌(Ensifer adhaerens)和中华根瘤菌(Sinorhizobium sp.)。菌株S5-4和S11-10在无机与有机培养基中均能生长,表现出混合营养特性。

Ammonia oxidation, the first step in nitrification process, is crucial for the soil nitrogen cycle. Investigation of cultured ammonia-oxidizing bacteria and their activities would help to further understand their unique contribution to nitrification in the soil N cycle. The cultured soil ammonia-oxidizing bacteria in ＂Hanfu＂ apple orchard in north of China were isolated at different growth stages（May, July, September and November in 2011）, using the methods of enrichment cultures and silica gel plates. The strains with high ammonia oxidation capabilities were classified according to the determination of ammonia-oxidizing activities, the morphological and cultural characteristics, and 16 S r DNA sequence analysis. The results revealed that 33 strains of dominant ammonia-oxidizing bacteria were isolated from orchard soil at 4 different growth stages. The numbers of ammonia-oxidizing bacteria were the highest in November, while the lowest in July. The ammonia oxidation activities of strains isolated from different growth stages were also different, which was most obvious in November. Among the 33 strains, strains S5-4 and S11-10 always displayed the highest ammonia oxidation activities at different p H, and they were identified as Ensifer adhaerens and Sinorhizobium sp., respectively. In addition, strains S5-4 and S11-10 could be cultured on both inorganic and organic medium,displaying a characteristic of mixotrophy.