贝莱斯芽孢杆菌SH-1471发酵条件优化及其番茄枯萎病的防治效果

Bacillus velezensis SH-1471: optimization of fermentation conditions and evaluation of the biocontrol effect on tomato Fusarium wilt

摘要:

【目的】贝莱斯芽孢杆菌(Bacillus velezensis) SH-1471是一株兼具防控作物土传病害、促进土壤养分转化以及促进作物生长等功能的菌株(保藏编号：CCTCC No. M 2022923，专利号：ZL 2022 1 1479280.X)。挖掘其潜在的生物活性并探究其最适发酵条件，是推进该菌株产业化和商业化开发的有效手段之一。【方法】结合形态学、分子生物学、16S rRNA基因和gyrB基因对菌株SH-1471进行分类地位鉴定；利用PCR技术，对菌株抗生素合成基因检测；使用平板对峙试验和发酵液抑菌试验测定菌株抑菌广谱性；并测定其体外产酶、解磷、解钾、固氮及产铁载体能力；以菌株发酵液OD₆₀₀值和抑菌率为指标，通过设计单因素试验和响应面优化试验，探究菌株的最佳发酵配方和最佳发酵条件；采用室内盆栽试验测定优化前后菌株发酵液对番茄植株的促生效果及其对番茄枯萎病的防治效果。【结果】经鉴定，SH-1471为贝莱斯芽孢杆菌(Bacillus velezensis)，具有srfA、fenB、ituA、ituD和bymA等抗生素合成基因，对番茄枯萎病菌(Fusarium oxysporum)、烟草赤星病菌(Alternaria alternate)和玉米大斑病菌(Exserohilum turcicum)等8种病原微生物具有良好的拮抗效果；且具有产蛋白酶、纤维素酶、解磷、固氮和分泌铁载体的能力。菌株SH-1471最适培养配方为：蔗糖17.92 g/L、豆粉16.95 g/L、硫酸镁2.88 g/L、酵母膏5.0 g/L；最适发酵条件为pH 7.5、温度33 ℃、转速220 r/min、发酵时间20‒24 h；优化后，抑菌率提升至94.08%，提高15.6%，发酵液OD₆₀₀提升至3.28，提高36.7%，盆栽防效提升至93.8%；此外，菌株SH-1471可显著提高番茄幼苗的株高、茎围、根长、根重、地上部位鲜重和地上部位干重等农艺性状。【结论】贝莱斯芽孢杆菌SH-1471具有丰富的抗生素合成基因，同时具有产蛋白酶、纤维素酶、解磷、固氮和分泌铁载体等生物活性，对多种病原物具有良好的抑制效果，可显著降低番茄枯萎病的发病率，并对番茄幼苗农艺性状具有显著促进作用，在植物病害生物防治方面以及促进植物生长方面具有广阔的应用前景。

Abstract:

[Objective] Bacillus velezensis SH-1471 (CCTCC No. M 2022923, Patent No. ZL 2022 1 1479280.X) is a strain that can control soil-borne diseases of crops and promote soil nutrient conversion and crop growth. This study aims to explore its potential biological activity and the optimum fermentation conditions, so as to promote the industrialization and commercial development of this strain.[Methods] A strain SH-1471 was identified based on the morphological, physiological, and biochemical characteristics and the 16S rRNA gene and gyrB-based phylogenetic trees. PCR was carried out to detect antibiotic synthesis genes in the strain. The inhibitory spectrum of the strain was measured by the plate confrontation assay and the fermentation liquid inhibition test. The abilities of the strain to produce enzymes, solubilize phosphorus and potassium, fix nitrogen, and secrete siderophores in vitro were measured. The fermentation conditions were optimized by single factor tests and response surface methodology with OD₆₀₀ value and inhibition rate as indicators. The growth-promoting effect of the fermentation liquid on tomato plants and the control effect of the fermentation liquid on tomato Fusarium wilt before and after optimization were determined by indoor pot experiments. [Results] Strain SH-1471 was identified as B. velezensis, carrying the antibiotic synthesis genes srfA, fenB, ituA, ituD, and bymA. It had strong antagonistic effects on 8 pathogenic microorganisms such as Fusarium oxysporum, Alternaria alternate, and Exserohilum turcicum. Moreover, the strain was capable of producing protease and cellulase, solubilizing phosphorus, fixing nitrogen, and secreting siderophores. The optimal medium formula for the fermentation of SH-1471 was composed of 17.92 g/L sucrose, 16.95 g/L soybean powder, 2.88 g/L magnesium sulfate, and 5.0 g/L yeast extract. The optimum fermentation conditions were pH 7.5, 33 ℃, 220 r/min, and 20–24 h. After optimization, the inhibition rate, OD₆₀₀, and potted control efficiency of the fermentation liquid reached 94.08% (increasing by 15.6%), 3.28 (increasing by 36.7%), and 93.8%, respectively. Moreover, strain SH-1471 significantly improved plant height, stem circumference, root length, root weight, and fresh weight and dry weight of the aboveground part of tomato seedlings. [Conclusion] B. velezensis SH-1471 carries rich genes for antibiotic synthesis, has the abilities of producing protease and cellulase, solubilizing phosphorus, fixing nitrogen, and secreting siderophores, and demonstrates strong inhibitory effects on a variety of pathogens. It can significantly reduce the incidence of tomato Fusarium wilt and improve the agronomic traits of tomato seedlings. Therefore, the strain has broad application prospects in the biocontrol of plant diseases and the promotion of plant growth.