具有杀线活性马尾松内生细菌的筛选与鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2021-1393

摘要/Abstract

摘要：

为探究马尾松内生细菌的多样性,寻找具有杀松材线虫活性的内生细菌。以前期从南京中山陵健康马尾松上分离的58株内生细菌为研究对象,利用浸渍法对内生细菌发酵滤液的杀线活性进行测定,采用形态学观察、生理生化特征测定、16S rDNA序列以及系统发育树等方法对筛选出的具有杀线活性的内生细菌进行种类鉴定。从中筛选出一株具有明显杀线功能的内生细菌NZM13-11。内生细菌NZM13-11发酵滤液在与松材线虫混合培养12 h后,校正死亡率达到92.2%,36 h后,校正死亡率达到100%。在12 h时,松材线虫的虫体被消解,消解率达85%,在培养48 h后,消解率达到100%,在显微镜视野下已经没有完整的虫体。对发酵滤液进行不同倍数稀释后,在8倍稀释倍数处理36 h,死亡率依然高达72.9%。通过对NZM13-11进行形态学观察、生理生化特征测定、16S rDNA序列以及系统发育树分析,将菌株NZM13-11鉴定为铜绿假单胞菌（Pseudomonas aeruginosa）。研究表明,在松树体内筛选出一株内生细菌NZM13-11能够高效地杀死松材线虫,该菌株被鉴定为铜绿假单胞菌（Pseudomonas aeruginosa）。该研究结果可以进一步深入,以探究利用马尾松内生细菌资源来防控松材线虫病的可能性。

关键词: 内生细菌, 铜绿假单胞菌, 松材线虫, 马尾松

Abstract:

The purpose of this study is to explore the diversity of endophytic bacteria in Pinus massoniana,and to search for endophytic bacteria with nematicidal activity against Bursaphelenchus xylophilus. With 58 endophytic bacteria isolated from healthy P. massoniana in Nanjing Zhongshan Mausoleum as the research object,and the nematicidal activity against B. xylophilus of endophytic bacteria in fermentation filtrate was determined by immersion method. The species of endophytic bacteria with high nematicidal activity against B. xylophilus were identified by morphological observation,physiological and biochemical characteristics,16S rDNA sequence and phylogenetic tree methods. An endophytic bacterium NZM13-11 was selected with obvious nematicidal activity against B. xylophilus. The adjusted mortality rate of B. xylophilus reached 92.2% after mixing the fermented filtrate of NZM13-11 with B. xylophilus for 12 h,and reached 100% after 36 h. In addition,the body of B. xylophilus was decomposed at 12 h,the decomposition rate reached 85%. After 48 h,the decomposition rate reached 100%,and there was no complete body under the microscope. After the fermentation filtrate was diluted at different multiples,the mortality rate was still as high as 72.9% after 36 h of 8-fold dilution treatment.The strain NZM13-11 was identified as Pseudomonas aeruginosa by morphological observation,physiological and biochemical characterization,16S rDNA sequence and phylogenetic tree analysis. This study shows that an endophytic bacterium NZM13-11 screened from p P. massoniana trees can efficiently kill B. xylophilus,and the strain is identified as Pseudomonas aeruginosa. This research results can be further deepened to explore the possibility of using endophytic bacteria resources of P. massoniana to prevent and control pine wilt diseases.

Key words: endophytic bacteria, Pseudomonas aeruginosa, Bursaphelenchus xylophilus, Pinus massoniana

贺丽娜, 冯源, 石慧敏, 叶建仁. 具有杀线活性马尾松内生细菌的筛选与鉴定[J]. 生物技术通报, 2022, 38(8): 159-166.

HE Li-na, FENG Yuan, SHI Hui-min, YE Jian-ren. Screening and Identification of Endophytic Bacteria with Nematicidal Activity Against Bursaphelenchus xylophilus in Pinus massoniana[J]. Biotechnology Bulletin, 2022, 38(8): 159-166.

图/表 8

表1 菌株NZM13-11对松材线虫的杀线活性

Table 1 Nematicidal activities of the strain NZM13-11 against B. xylophilus

菌株编号 Strain No.	校正死亡率Adjusted mortality/%				消解率Decomposition rate/%
菌株编号 Strain No.	12 h	24 h	36 h	48 h	12 h	24 h	36 h	48 h
NZM 13-11	92.2	97.2	100	100	85.0	86.6	90.0	100

图1 不同稀释倍数下菌株NZM13-11的杀线活性

Fig.1 Nematicidal activities of the strain NZM13-11 against B. xylophilus under different dilution times

图2 NZM 13-11菌株发酵滤液处理松材线虫后的虫体形态观察 A：CK;B：线虫体内内容物流出;C：线虫虫体断裂,内含物外渗;D：线虫体表消解;E：处理24 h后在50倍镜下的视野;F：处理36 h后在50倍镜下的视野

Fig.2 Morphological observation of B.xylophilus treated with fermentation filtrate of NZM 13-11 strain A：CK;B：the contents of nematodes flowed out;C：the body of nematodes was broken and the contents were extravasated;D：the body wall of the nematodes was dissolved;E：the field of vision under 50 times microscopy after 24 h of treatment;F：the field of vision under 50 times microscopy after 36 h treatment

图3 菌株NZM13-11的菌落形态（A）和革兰氏染色（B）

Fig. 3 Colony morphology of strain NZM13-11（A）and Gram staining（B）

图4 菌株NZM13-11的生长曲线

Fig.4 Growth curve of strain NZM13-11

表2 菌株NZM13-11的生理生化特征

Table 2 Physiological and biochemical characteristics of strain NZM13-11

生理生化试验 Physiological and biochemical test	鉴定结果 Results
葡萄糖 Glucose	+
果糖 Fructose	+
接触酶 Catalase	+
氧化酶 Oxidase	+
甲基红试验 Methyl red test	-
VP（乙酰甲基甲醇）VP（Acetyl methyl methanol）	-
淀粉水解 Starch hydrolysis	-
硝酸盐还原 Nitrate reduction	+
亚硝酸盐还原 Nitrite reduction	+
反硝化 Denitrification	+
明胶水解 Elatin hydrolysis	+
精氨酸双水解酶 Arginine dihydrolase	+

图5 菌株NZM13-11的基因组DNA（A）以及16S rDNA的PCR产物电泳结果（B） M：Maker;1,2：菌株NZM13-11

Fig.5 Electrophoresis results of genomic DNA（A）and 16S rDNA PCR products（B）of strain NZM13-11 M：Maker. 1,2：Strain NZM13-11

图6 基于16S rDNA基因序列构建的菌株NZM13-11的系统进化树

Fig. 6 Phylogenetic tree of strain NZM13-11 based on 16S rDNA gene sequence

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