番茄青枯病拮抗菌的定向筛选及其抗病促生机制研究

Isolation and identification of antagonistic bacteria against tomato bacterial wilt and the mechanisms in disease prevention and plant growth promotion

摘要:

[目的] 从抑病型番茄根际土壤中筛选青枯病的高效拮抗促生菌，阐明其防病促生机制。[方法] 以番茄青枯雷尔氏菌（Ralstonia solanacearum）为靶病原菌，采用平板抑菌圈法，筛选拮抗菌；通过BOX-PCR指纹图谱鉴定菌株多样性，以平板透明圈法评价其产酶活性，并针对抑菌能力强、产酶种类多的拮抗菌开展16S rRNA基因系统发育分析；通过温室试验评价拮抗菌的防病促生能力，并在此基础上通过实时荧光定量PCR研究生防细菌对番茄青枯病的防病促生机制。[结果] 从番茄根际土壤分离获得29株细菌，其中15株对青枯菌具有拮抗功能，进一步通过BOX-PCR指纹图谱、酶活分析获得4株具有潜在防治番茄青枯病、促进生长的功能菌（B2、B5、B20、B23），通过16S rRNA系统发育分析鉴定B2拮抗菌为解淀粉芽孢杆菌（Bacillus amyloliquefaciens），B5和B20拮抗菌为枯草芽孢杆菌（Bacillus subtilis），B23拮抗菌为贝莱斯芽孢杆菌（Bacillus velezensis）；温室试验表明，B2、B5、B20、B23拮抗菌的抑病效果分别为35.59%、8.47%、32.20%、96.61%，并且均能显著增加番茄生物量和生理性状，如地上部鲜重、总叶绿素含量、地下部根尖数等。B2、B5、B23拮抗菌显著促进番茄株高和根长，B2、B20、B23拮抗菌显著增加茎粗；而B23拮抗菌显著增加根系分叉数；实时荧光定量分析表明，B2、B20、B23拮抗菌株可促进抗病相关功能基因PR1α、POD1的表达量，B2、B5、B23拮抗菌促进吲哚乙酸（IAA）信号通路应答关键基因ctd1的表达量，B2、B5、B20、B23拮抗菌均降低乙烯（ETH）信号通路应答关键基因ERF2的表达量。[结论] 本研究分离筛选获得4株对番茄青枯病具有显著防治效果以及促进番茄生长的PGPR菌株，可为定向筛选植物促生防病菌提供理论依据。

Abstract:

[Objective] Isolation plant growth promoting rhizobacteria (PGPR) from disease-resistant tomato rhizosphere soil to control bacterial wilt caused by Ralstonia solanacearum and improve tomato growth, and explored the mechanisms.[Methods] PGPR bacteria with antibacterial activity was screened from tomato rhizosphere soil by inhibition zone method using a moderately pathogenic Ralstonia solanacearum HN4 as target; The highly similar strains were identified by BOX-PCR fingerprints, and the enzyme-producing activity of different genotype strains was evaluated by the plate transparent circle method; The 16S rRNA phylogenetic analysis was applied to identify the antagonistic strains with strong antibacterial ability and many kinds of enzyme production; The potential antagonistic bacteria were assessed for plant disease prevention and growth promotion in greenhouse, and the plant disease prevention and growth promotion related genes expression level were tested by real-time PCR.[Results] 29 strains were isolated and purified from tomato rhizosphere soil, of which 15 strains had antagonistic function against Ralstonia solanacearum. Moreover, four strains involved in controlling tomato bacterial wilt and promoting growth, were verified by the analysis of BOX-PCR fingerprinting and enzyme activity. In the result of 16S rRNA phylogenetic analysis, strain B2 was identified as Bacillus amyloliquefaciens. Strain B5 and B20 were identified as Bacillus subtilis, respectively. Strain B23 was identified as Bacillus velezensis. In greenhouse experiment, strain B2, B5, B20 and B23 inhibited Ralstonia solanacearum effectively, with the efficiency of 35.59%, 8.47%, 32.20% and 96.61%, respectively. Furthermore, all of them significantly increased the plant fresh weight, the total chlorophyll content and the number of root tips. The strain B2, B5 and B23 efficiently promoted plant height and root length of tomato. The strain B2, B20 and B23 efficiently promoted stem thickness of tomato. And strain B23 efficiently promoted the number of root forks. In the result of real-time PCR, strain B2, B20 and B23 upregulated the expression of PR1α and POD1 gene. Strain B2, B5 and B23 upregulated the expression of ctd1 gene. And strain B2, B5, B20 and B23 downregulated the expression of ERF2 gene.[Conclusion]] In this study, four biocontrol strains were isolated and identified to control tomato bacterial wilt and promote tomato growth, and can provide a theoretical basis for the targeted screening of plant growth-promoting and disease preventing bacteria.