烟草内生细菌分离方法的优化研究

Research on separation optimization of endophytic bacteria in tobacco

用研磨组织稀释分离法与表面消毒有效性评价相结合的方法确定了适宜烟草组织的表面消毒方法以及内生细菌分离培养基。采用1%次氯酸钠浸泡5 min,再用75%酒精浸泡2分钟能够100%去除表面附生菌,并且分离得到更多的内生细菌。研究比较了TSA、NA、PDA、LB四种常用的细菌分离培养基在分离烟草内生细菌中的差异,其中TSA和NA培养基分离得到的内生细菌数量较高,TSA培养基从烟草根、茎、叶中分离出的内生细菌数量分别为5.18 log cfu/g.fw、2.17 log cfu/g.fw、4.69 log cfu/g.fw;NA培养基分别为5.01 log cfu/g.fw、1.81 log cfu/g.fw、4.93 log cfu/g.fw。对分离得到的127株内生细菌进行16S rDNA序列测定表明,TSA和NA培养基分离得到的内生细菌种类最多,其中TSA培养基分别从根、茎、叶中分离到9、7、13种内生细菌;其次是NA培养基,分别从根、茎、叶中分离到8、6、10种内生细菌。结果表明,采用TSA和NA培养基分离烟草内生细菌,可以达到较为理想的分离效果。

The best surface sterilization method and isolation medium of endophytic bacteria in tobacco were determined by grinding separation and surface sterilization efficacy check. Results showed that isolation and disinfection effect of soaking by 1%NaClO （5 min）＋75%alcohol （2 min） was better in that surface epiphytic bacteria can be 100%eliminated and more bacteria can be separated. Four isolating media including Nutrient Agar （NA）, Tryptic Soy Agar （TSA）, Potato Dextrose Agar （PDA）, Luria-Bertani medium （LB） were utlized to isolate endophytic bacteria from tobacco, aiming at comparing difference of isolation effect between them. Results showed that the isolation effect of TSA and NA was better than that of other media, while LB and PDA showed the lowest isolation effect. The amount of bacteria isolated from root, stem, leaf by TSA medium were 5.18 log cfu/g.fw, 2.17 log cfu/g.fw, 4.69 log cfu/g.fw respectively. The amount of bacteria isolated from root, stem, leaf by NA medium are 5.01 log cfu/g.fw, 1.81 log cfu/g.fw, 4.93 log cfu/g.fw respectively. The 16S rDNA of 127 endophytic strains were sequenced. Nine, seven and 13 different kinds of bacteria were isolated from root, stem, leaf by TSA medium respectively while only 8, 6 and 10 different kinds of bacteria isolated from root, stem, leaf by NA medium. These results indicated that TSA and NA medium were suitable for isolating endophytic bacteria in tobacco by which ideal separating effect can be obtained.