番茄根际土尚未培养细菌的分离培养方法探究  

Development of the isolation and cultivation approaches of uncultured bacteria in tomato rhizosphere soil

作　　者：吴晨晨 李怡君 徐双 马荣 韦善君[1] 张晓霞[2] WU Chen-chen;LI Yi-jun;XV Shuang;MA Rong;WEI Shan-jun;ZHANG Xiao-xia(College of Life and Environmental Science,Minzu University of China,Beijing 100081;Institute of Agricultural Resources and Regional Planning,Chinese Academy of Agricultural Sciences/Key Laboratory of Microbial Resources Collection and Preservation,Ministry of Agriculture and Rural Affairs,Beijing 100081)

机构地区：[1]中央民族大学生命与环境科学学院,北京100081 [2]中国农业科学院农业资源与农业区划研究所/农业农村部农业微生物资源收集与保藏重点实验室,北京100081

出　　处：《中国土壤与肥料》2023年第8期233-242,共10页Soil and Fertilizer Sciences in China

基　　金：国家重点研发计划(2019YFD1002000);中国农业科学院科技创新工程(CAAS-ZDRW202201);北京市乡村振兴科技项目(BJXCZX2022);中央民族大学大学生创新训练计划项目(URTP2021110091)。

摘　　要：为探究获得土壤中尚未培养细菌资源的方法,以MS无机盐为基础养分,设置1/100×TSB、1/50×R2A和土壤浸提液(SE)3种低有机营养类型,凝固剂用琼脂(A)或结冷胶(G),得到一组培养基MS+TSG、MS+TSA、MS+R2A和MS+SE,以及在此基础上添加1.5 g/L丙酮酸钠作为抗氧化剂的第二组培养基。用上述8种培养基分离培养健康番茄根际土壤中的细菌,并对各种培养基平板上代表性菌落进行16S rRNA基因分析。结果表明,在MS无机盐和低有机营养组合的培养基平板上,在28℃下培养28 d后可检测到菌落数达1×10^(7)CFU/g土,4种培养基平板上检测到的菌落数量由多到少顺序为MS+SE>MS+TSG>MS+R2A>MS+TSA;在相同的低有机营养条件下,用结冷胶作为凝固剂可获得更多菌落,添加丙酮酸钠为抗氧化剂会显著降低菌落的多样性。鉴定的84个代表性菌落属于34个属的52个种,其中7个(占检测菌株数的8.33%)为可能新种,不同培养基上种丰富度表现为MS+TSA>MS+R2A>MS+TSG>MS+SE。结果说明,采用低有机营养的MS无机盐培养基有利于分离获得土壤中的一些尚未培养细菌。In order to develop effective approaches to obtain uncultured bacteria in soil,four culture media containing MS mineral salt and reduced organic nutrition named as MS+TSG,MS+TSA,MS+R2A and MS+SE respectively,were designed by adding 1/100×TSB,1/50×R2A or soil extraction(SE)into the MS basic medium with agar(A)or gellan gum(G)as coagulant.A content of 1.5 g/L sodium propionate was added into the media mention above to obtain a group of antioxidant culture media.Bacteria in the rhizosphere soil of healthy tomato plants were cultured on the eight media mentioned above,and the representative colonies selected randomly from different media were identified by 16S rRNA gene sequencing.The results indicated that,on the four media containing MS mineral salt plus reduced organic nutrition,more than 1×10^(7)cfu were obtained from per gram of soil after being cultured at 28℃for 28 d.The order of colony account on media from high to low was MS+SE>MS+TSG>MS+R2A>MS+TSA.In the condition of mineral salt plus low level of organic nutrients,gellan gum could help to obtain more colonies compared to agar,but the supplement of sodium propionate dramatically reduced the diversity of colonies.Among the 84 representative colonies identified,a total of 52 species from 34 genera were included,of which 7(8.33%of detected colonies)are possible new species.The order of species diversity on media from high to low was MS+TSA>MS+R2A>MS+TSG>MS+SE.The results suggest that media consisting of MS mineral salt and low level of organic nutrients are effective to isolate uncultured bacteria and new antagonism resource to plant pathogens from soil.

关 键 词：尚未培养微生物 分离培养 番茄 根际土壤 

分 类 号：S154.3[农业科学—土壤学]