生姜根腐病病原鉴定及拮抗贝莱斯芽孢杆菌筛选与发酵优化

Identification of pathogens causing ginger root rot and screening and fermentation optimization of a Bacillus velezensis strain for biocontrol

摘要:

背景 生姜根腐病是一种严重危害生姜生产的土传病害，对生姜的产量、品质及姜农经济造成重大损失。目的 系统分离与鉴定来自河北省石家庄市生姜主产区的根腐病病原菌，并筛选高效拮抗菌株，为绿色生物防治制剂开发提供优良菌种资源和技术支撑。方法 采用组织分离与稀释涂布法分离生姜根际病原与拮抗菌株，通过形态学观察、分子系统发育分析(ITS/16S rRNA基因序列)进行物种鉴定，利用平板对峙法评估拮抗菌株对病原真菌的抑菌活性，通过单因素试验、Plackett-Burman、最陡爬坡试验及Box-Behnken响应面法系统优化发酵条件，以拮抗菌贝莱斯芽孢杆菌(Bacillus velezensis) ND-J的活菌数为响应值，建立其发酵条件的二次回归模型。结果 共鉴定获得4株病原真菌，其中菌株J-4、J-5为红栖新赤壳菌(Neocosmospora rubicola)，菌株J-6、J-7为藤仓镰刀菌(Fusarium fujikuroi)；筛选获得3株芽孢杆菌属拮抗菌，其中贝莱斯芽孢杆菌(Bacillus velezensis) ND-J对上述病原菌抑菌效果最优。通过响应面分析建立了装液量(A)、碳源(B)与氮源(C)添加量对活菌数(Y)的二次回归模型：Y=89.80+0.62A+3.50B+1.88C-0.75AB+1.75BC-7.65A²-15.90B²-7.65C²。经优化后其最佳发酵条件为：蔗糖6.35 g/L，麸皮+豆粕(1:1) 21.41 g/L，NaCl 5.0 g/L，KH₂PO₄ 1.0 g/L，MnSO₄ 0.4 g/L，装液量80.71 mL/500 mL，温度35 ℃，初始pH 7.5，转速200 r/min，接种量2%。在此条件下验证活菌数达9.1×10⁹ CFU/mL，模型预测值与实测值高度吻合(R²=0.986 4)，较优化前提高了110.0%。结论 明确了河北省石家庄市生姜主产区根腐病病原菌组成，获得具有高效拮抗活性的贝莱斯芽孢杆菌ND-J，其高密度发酵工艺优化结果为后续产业化开发与生姜病害绿色防控提供了可靠依据。

Abstract:

Background Ginger root rot, a destructive soil-borne disease, poses a serious threat to ginger production, causing significant reductions in yield and quality and substantial economic losses to growers.Objective We systematically isolated and identified the pathogens causing ginger root rot in major ginger-producing areas of Shijiazhuang, Hebei and screened effective antagonistic strains, aiming to providing excellent bacterial resources and technical support for the development of green biocontrol agents.Methods The pathogens and antagonistic strains from the rhizosphere of ginger were isolated by tissue isolation and dilution plate methods. Species identification was performed through morphological observation and molecular phylogenetic analysis (ITS/16S rRNA gene sequences). The inhibitory activity of the antagonistic strain against the pathogenic fungi was assessed via a dual culture assay. The fermentation conditions were systematically optimized through single-factor experiments, Plackett-Burman design, steepest ascent experiment, and Box-Behnken response surface methodology. With the viable cell count of the antagonistic strain Bacillus velezensis ND-J as the response value, a quadratic regression model of its fermentation conditions was established.Results Four strains of pathogenic fungi were identified. J-4 and J-5 were Neocosmospora rubicola, and J-6 and J-7 were Fusarium fujikuroi. Three antagonistic Bacillus strains were screened out, with B. velezensis ND-J showing the most effective inhibition of the above pathogens. A quadratic regression model describing the effects of liquid volume (A), carbon source (B), and nitrogen source (C) on viable cell count (Y) was established based on response surface analysis: Y=89.80+0.62A+3.50B+1.88C-0.75AB+1.75BC-7.65A²-15.90B²-7.65C². The optimized fermentation conditions were 6.35 g/L sucrose, 21.41 g/L wheat bran+soybean meal (1:1), 5.0 g/L NaCl, 1.0 g/L KH₂PO₄, 0.4 g/L MnSO₄, liquid volume of 80.71 mL/500 mL, temperature of 35 °C, initial pH 7.5, shaking at 200 r/min, and inoculum concentration of 2%. Under these conditions, the viable cell count reached 9.1×10⁹ CFU/mL, with a high degree of agreement between the predicted and observed values (R²=0.986 4), a 110.0% increase compared to the pre-optimization value of 4.333×10⁹ CFU/mL.Conclusion We identified the pathogenic fungi causing ginger root rot in the main ginger-producing areas of Shijiazhuang, Hebei and obtained a B. velezensis strain ND-J with efficient antagonistic activity. The optimized high-density fermentation process provides a reliable basis for subsequent industrial development and eco-friendly control of ginger diseases.