Functional Analysis of ganA in the Nematocidal of Enterobacter ludwigii against Bursaphelenchus xylophilus

doi:10.16409/j.cnki.2095-039x.2022.11.020

Abstract

Abstract: Biocontrol microorganisms have been used to control Bursaphelenchus xylophilus disease, but the molecular mechanism of nematicidal is not clear. This study found that the corrected mortality of B. xylophilus treated with E. ludwigii AA4 was more than 85%, and strain AA4 could significantly reduce B. xylophilus infection of pine seedlings. In order to explore the molecular mechanism of AA4 against B. xylophilus, the DNA homologous recombination technique was used to construct the β-galactosidase gene ganA, and the function of ganA in AA4 nematode inhibition was analyzed. The results showed that the B. xylophilus killing activity and B. xylophilus disease prevention in a ganA knockout mutant strain decreased significantly, and the mobility and ability to colonize nematodes of the mutant strain decreased significantly. In addition, compared with the wild-type strain, the ability of the mutant strain to restrict the movement of nematodes was significantly reduced. Therefore, ganA may participate in the process of AA4 killing B. xylophilus by regulating AA4’s ability to colonize B. xylophilus and restrict nematode movement.

Key words: ganA, Enterobacter ludwigii, Bursaphelenchus xylophilus, nematocidal activity, colonization

CLC Number:

S476

WANG Haoyu, ZHANG Hongtao, ZHANG Ying, ZHAO Yu, WANG Chuanzhen, ZHANG Lixia, WANG Qi, WU Di, YU Cuifang, LI Haiyong, WANG Shuang, NIU Ben. Functional Analysis of ganA in the Nematocidal of Enterobacter ludwigii against Bursaphelenchus xylophilus[J]. Chinese Journal of Biological Control, 2023, 39(5): 1094-1103.

References

[1] 国家林业和草原局. 国家林业和草原局公告2020年第4号[R]. 北京: 国家林业和草原局, 2020.
[2] 郭进, 葛明华, 顾晓峰. 我国松材线虫病的主要防治技术探析[J]. 现代园艺, 2017, 21(11): 139-140.
[3] 许培容. 松材线虫病的危害与综合防治对策[J]. 世界热带农业信息, 2021, 12(12): 43.
[4] 张弘弢, 赵宇, 牛犇, 等. 生防细菌杀松材线虫的作用机制及应用[J]. 中国森林病虫, 2021, 40(4): 26-33.
[5] Cheng C, Qin J, Wu C, et al. Suppressing a plant-parasitic nematode with fungivorous behavior by fungal transformation of a Bt cry gene[J]. Microbial Cell Factories, 2018, 17(1): 116.
[6] Wang Y, Mei L, Wu J, et al. Detection and characterisation of a Bacillus thuringiensis crystal protein with nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus[J]. Biocontrol Science and Technology, 2012, 22(10): 1143-1153.
[7] Phani V, Somvanshi V S, Rao U. Silencing of a Meloidogyne incognita selenium-binding protein alters the cuticular adhesion of Pasteuria penetrans endospores[J]. Gene, 2018, 677(8): 289-298.
[8] Niu Q, Tian Y, Zhang L, et al. Overexpression of the key virulence proteases Bace16 and Bae16 in Bacillus nematocida B16 to improve its nematocidal activity[J]. Journal of Molecular Microbiology and Biotechnology, 2011, 21(3/4): 130-137.
[9] Huang X, Tian B, Niu Q, et al. An extracellular protease from Brevibacillus laterosporus G4 without parasporal crystals can serve as a pathogenic factor in infection of nematodes[J]. Research in Microbiology, 2005, 156(5/6): 719-727.
[10] Tian B, Li N, Lian L, et al. Cloning, expression and deletion of the cuticle-degrading protease BLG4 from nematophagous bacterium Brevibacillus laterosporus G4[J]. Archives of Microbiology, 2006, 186(4): 297-305.
[11] Chan S Y, Liu S Y, Seng Z, et al. Biofilm matrix disrupts nematode motility and predatory behavior[J]. The ISME Journal, 2021, 15(1): 260-269.
[12] Liang L, Liu Z, Liu L, et al. The nitrate assimilation pathway is involved in the trap formation of Arthrobotrys oligospora, a nematode-trapping fungus[J]. Fungal Genetics and Biology, 2016, 92(5): 33-39.
[13] Zhao Y, Yuan Z, Wang H, et al. Gene sdaB is involved in the nematocidal activity of Enterobacter ludwigii AA4 against the pine wood nematode Bursaphelenchus xylophilus[J]. Frontiers in Microbiology, 2022, 13: 870519.
[14] Nantana S, Varunya S, Pumin N. Production of indole-3-acetic acid by Enterobacter sp. DMKU-RP206 using sweet whey as a low-cost feed stock[J]. Journal of Microbiology and Biotechnology, 2018, 28(9): 1511-1516.
[15] Habibi S, Djedidi S, Ohkama-Ohtsu N, et al. Isolation and screening of indigenous plant growth-promoting rhizobacteria from different rice cultivars in afghanistan soils[J]. Microbes and Environments, 2019, 34(4): 347-355.
[16] Oh M, Han J W, Lee C, et al. Nematicidal and plant growth-promoting activity of Enterobacter asburiae HK169: genome analysis provides insight into its biological activities[J]. Journal of Microbiology and Biotechnology, 2018, 28(6): 968-975.
[17] Munif A, Hallmann J, Sikora A R. Evaluation of the biocontrol activity of endophytic bacteria from tomato against Meloidogyne incignita[J]. Mededelingen Faculteit Landbouwkundige En Toegepaste Biologische Wetenschappen, 2000, 65(8): 471-480.
[18] 周晓辉. β-D-半乳糖苷酶活性测定方法的研究[J]. 河北工业科技, 2004, 21(5): 16-18.
[19] 张凤玉. 2型猪链球菌β-半乳糖苷酶功能的初步研究[D].南京: 南京医科大学, 2014.
[20] Siddiqui I A, Shaukat S S, Khan A. Differential impact of some Aspergillus species on Meloidogyne javanica biocontrol by Pseudomonas fluorescens strain CHA0[J]. Letters in Applied Microbiology, 2004, 39(1): 74-83.
[21] Niu B, Paulson J N, Zheng X, et al. Simplified and representative bacterial community of maize roots[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(12): 2450-2459.
[22] 张弘弢. 路德维希肠杆菌AA4杀松材线虫相关基因的挖掘及其功能的初步解析[D]. 哈尔滨: 东北林业大学, 2021.
[23] 牛犇, 史亚新, 赵宇, 等. 一株防治松材线虫病的寡养单胞菌LC00168及其应用. 中国: CN112877240B [P]. 2021-09-28.
[24] 李鑫, 李亚芯, 戴建君. Red两步同源重组法在大肠杆菌基因敲除中的应用[J]. 中国畜牧兽医, 2017, 44(7): 1934-1940.
[25] Doublet B, Douard G, Targant H, et al. Antibiotic marker modifications of lambda Red and FLP helper plasmids, pKD46 and pCP20, for inactivation of chromosomal genes using PCR products in multidrug-resistant strains[J]. Journal of Microbiology Method, 2008, 75(2): 359-361.
[26] Datsenko A K, Wanner L B. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J]. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(12): 6640-6645.
[27] 王方. 杀松材线虫细菌的分离、鉴定及其活性成分研究[D]. 青岛: 青岛大学, 2019.
[28] 张婉君, 吴小芹, 王亚会. 松材线虫拮抗细菌的杀线活性及其发酵培养特性[J]. 生物技术通报, 2019, 35(7): 76-82.
[29] 朱澂, 林辰涛. 一种新型的DNA荧光染料—DAPI的光学特性及其应用[J]. 武汉植物学研究, 1986, 4(1): 91-102.
[30] 刘晓燕. 樟子松容器育苗技术[J]. 农业科技与信息, 2017, 5(4): 104-105.
[31] 贾艳才. 樟子松容器苗播种繁育技术要点[J]. 江西农业, 2020, 13(4): 52-53.
[32] 贲爱玲. 松材线虫体表优势细菌的分离及定殖能力研究[D]. 南京: 南京林业大学, 2010.
[33] Cunda D P, Iribarnegaray V, Papa-Ezdra R, et al. Characterization of the different stages of biofilm formation and antibiotic susceptibility in a clinical Acinetobacter baumannii strain[J]. Microbial Drug Resistance, 2020, 26(6): 569-575.
[34] 丁睿清, 徐双媛, 马贞锦疆, 等. 肠炎沙门菌双组份系统qseC基因缺失株的构建及其生物学特性分析[J]. 中国家禽, 2021, 43(11): 20-27.
[35] 段强德. F18+大肠杆菌鞭毛与其致病相关性的研究[D]. 扬州: 扬州大学, 2012.
[36] 丁晓磊. 基于高通量测序的松材线虫致病机理与毒力差异研究[D]. 南京: 南京林业大学, 2016.
[37] 王慧琦. 野油菜黄单胞菌β-半乳糖苷酶缺失菌株的构建及双组分系统RavA/RavR调控鞭毛合成通路的初步研究[D]. 海口: 海南大学, 2019.
[38] Hua C, Li C, Jiang Y, et al. Response of soybean cyst nematode (Heterodera glycines) and root-knot nematodes (Meloidogyne spp.) to gradients of pH and inorganic salts[J]. Plant and Soil, 2020, 7(5): 305-318.