贝莱斯芽孢杆菌Ba-0321胞外蛋白的抑菌特性及其诱导的烟草抗病性

Characteristics of extracellular antimicrobial proteins from Bacillus velezensis Ba-0321 and their induced disease resistance in tobacco

摘要:

背景 贝莱斯芽孢杆菌(Bacillus velezensis) Ba-0321是前期筛选获得的一株具有促生防病作用的生防菌株，能够产生多种抑菌活性物质。目的 获得新型广谱有效的抑菌蛋白，解析其抑菌机制，明确其对烟草病害的防治效果，为开发新型抗菌蛋白制剂奠定基础。方法 采用硫酸铵分级沉淀处理菌株Ba-0321发酵上清液，经离心和透析获得胞外粗蛋白，采用牛津杯法测定其抑菌活性，并进行高温和酸碱度敏感性分析。利用液相色谱串联质谱(LC-MS/MS)进一步分析胞外粗蛋白中的抑菌成分，综合运用组织化学染色及抗病相关基因 qPCR分析等技术，解析菌株Ba-0321胞外粗蛋白诱导的烟草抗病性，通过接种试验验证其对烟草镰刀菌根腐病和赤星病的防治效果。结果 当硫酸铵的饱和度为40%时，从菌株Ba-0321发酵上清液沉淀获得的胞外粗蛋白对烟草尖孢镰刀菌(Fusarium oxysporum)的抑菌活性最强，抑菌率可达49.16%，并且在40-90 ℃和pH 7.0-11.0条件下保持稳定，同时对茄病镰刀菌(Fusarium solani)、疫霉菌(Phytophthora nicotianae)、立枯丝核菌(Rhizoctonia solani)、链格孢菌(Alternaria alternata)和间座壳属(Diaporthe)真菌等其他5种烟草病原菌均具有抑菌活性。经LC-MS/MS鉴定，菌株Ba-0321胞外粗蛋白包含β-葡聚糖酶、壳聚糖酶、α-淀粉酶、枯草杆菌蛋白酶、丝氨酸蛋白酶、几丁质结合蛋白、抗菌肽LCI等多种抑菌相关蛋白，同时还鉴定出7个未知蛋白。该粗蛋白可触发烟草产生过敏反应(hypersensitivity reaction, HR)和过氧化氢(H₂O₂)积累，激活水杨酸(salicylic acid, SA)、乙烯(ethylene, ET)和茉莉酸(jasmonic acid, JA)信号通路关键基因(NtNPR1, NtPR1a, NtEIN2, NtEIN3, NtJAR1, NtPDF1.2)表达从而诱导烟草的系统抗病性，对尖孢镰刀菌(F. oxysporum)引起的烟草根腐病和链格孢菌(A. alternata)引起的烟草赤星病的相对防效分别为42.88%和68.87%。结论 菌株Ba-0321胞外粗蛋白可作为生物农药或植物免疫诱抗剂应用于烟草病害绿色防控。

Abstract:

Background Bacillus velezensis Ba-0321 obtained previously is a biocontrol strain with plant growth-promoting and disease-preventing effects, capable of producing a variety of antimicrobial substances.Objective This study screened novel broad-spectrum and effective antimicrobial proteins, analyzed their antimicrobial mechanisms, and determined their control efficacy against tobacco diseases, aiming to lay a foundation for developing new antimicrobial agents.Methods Extracellular crude proteins from Ba-0321 were isolated through ammonium sulfate precipitation at varying saturation levels, followed by centrifugation and dialysis. The Oxford cup method was employed to assess the antimicrobial activity, and the protein stability was evaluated under different temperature and pH conditions. The antimicrobial components within the crude protein extracts were further characterized by LC-MS/MS. Histochemical staining was combined with qPCR analysis of disease resistance-related genes to reveal the mechanisms of tobacco resistance induced by the crude proteins. Inoculation assays were conducted to validate the efficacy of these proteins against Fusarium root rot and brown spot disease in tobacco.Results When ammonium sulfate saturation was 40%, the extracellular crude proteins precipitated from the fermentation supernatant of Ba-0321 exhibited the strongest antifungal activity against Fusarium oxysporum, with an inhibition rate of 49.16%. The proteins remained stable at 40-90 ℃ and pH 7.0-11.0. Moreover, they exhibited inhibitory activities against other tobacco pathogenic fungi including Fusarium solani, Phytophthora nicotianae, Rhizoctonia solani, Alternaria alternata, and Diaporthe. LC-MS/MS analysis identified multiple antimicrobial proteins in the extracellular crude protein fraction of Ba-0321, which included β-glucanase, chitosanase, α-amylase, subtilisin, serine protease, chitin-binding protein, and the antimicrobial peptide LCI, along with seven unidentified proteins. The crude proteins triggered hypersensitive response (HR) and hydrogen peroxide (H₂O₂) accumulation in tobacco, activated the expression of key genes (including NtNPR1, NtPR1a, NtEIN2, NtEIN3, NtJAR1, and NtPDF1.2) involved in salicylic acid (SA), ethylene (ET), and jasmonic acid (JA) signaling pathways, thereby inducing systemic disease resistance in tobacco. The control efficacies of extracellular crude proteins from Ba-0321 against the root rot caused by F. oxysporum and the brown spot disease caused by A. alternata in tobacco reached 42.88% and 68.87%, respectively.Conclusion The extracellular crude proteins of strain Ba-0321 can be applied as biopesticides or plant immunity inducers for the green control of tobacco diseases.