Screening and Identification of Endophytic Bacteria with Nematicidal Activity Against Bursaphelenchus xylophilus in Pinus massoniana

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

Abstract

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

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.

Figures/Tables 8

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

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

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

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

Fig.4 Growth curve of strain NZM13-11

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	+

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

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

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