大丽轮枝菌拮抗细菌8-52菌株的筛选与鉴定

为筛选对棉花黄萎病病原菌大丽轮枝菌(Verticillium dahliae)具有拮抗作用的芽孢细菌,采用改良的琼脂平板扩散法,通过初筛从各地的土样中分离到83株拮抗细菌菌株,对其中的69株进行了复筛,得到一株具有较强的抑菌活性的拮抗细菌8-52菌株。对该菌株进行了形态特征的观察、生理生化试验和16S rDNA的序列分析。将该菌株的形态特征和生理生化特性鉴定的结果与相应种、属模式菌的性状相对照,认为菌株8-52与芽孢杆菌(Bacillus)的相应性状很相近。根据16S rDNA序列相似性分析,此菌株与Bacillus velezensis标准菌株AB245422的16S rDNA序列相似度达99.78%,因此鉴定拮抗菌株8-52为Bacillus velezensis。     

Bacillus malacitensis Z-5菌株合成抑菌物质培养基优化及成分鉴定

【目的】Bacillus malacitensis Z-5菌株对棉花黄萎病有显著防治效果,产生抑菌物质是其发挥防治作用的主要因素。本研究旨在优化Z-5菌株产生抑菌物质的培养基组成,分析抑菌物质的成分及其合成相关基因。【方法】由单因素试验确定最佳碳源、氮源及无机盐,利用最速上升法确定碳源、氮源以及无机盐最优含量区域,根据Box-Behnken试验设计,以发酵上清液对棉花黄萎病菌大丽轮枝菌(Verticillium dahlia Kleb.)的拮抗活性为指标,筛选Z-5菌株产生抑菌物质的最优培养基组成。利用液相色谱-质谱联用(Liquid Chromatography-Mass Spectrometer,LC-MS)法鉴定抑菌物质,并对抑菌物质合成相关基因进行检测。【结果】Z-5菌株产生抑菌物质最佳培养基组成为2.68%(质量分数,下同)玉米粉、1.45%酵母粉、0.03%K_2SO_4、0.8%Na_2HPO_4·12H_2O、0.2% NaH_2PO_4·2H_2O(pH 7.2～7.4),抑菌圈直径为1.34 cm,与预测值相近,证实该模型可靠有效。抑菌物质主要为脂肽抗生素C14表面活性素B,C14～C17伊枯草菌素A,C14、C15、C17伊枯草菌素B等。聚合酶链式反应检测结果显示,Z-5菌株基因组含有合成脂肽抗生素的基因srf AB、ituC和ituD。【结论】本研究优化了Z-5菌株产生抑菌物质的培养基组成,明确了其抑菌物质为脂肽抗生素,为利用该抑菌物质进行棉花黄萎病菌的防治提供了理论参考和物质基础。     

Optimization of the Fermentation Medium and Analysis of the Compositions of Antifungal Compounds from Bacillus malacitensis Z-5

[Objective] Bacillus malacitensis Z-5 has a significant biocontrol effect on cotton Verticillium wilt mainly through its production of antifungal substances. This study aimed to optimize the culture medium’s composition. Additionally, it aimed to identify the genes that produce the antifungal substances and determine the components of these antifungal substances. [Methods] Single factor tests were used to determine the optimal carbon and nitrogen sources, as well as the inorganic salt level. The most rapid rise method was used to determine the carbon and nitrogen sources and the inorganic salt’s optimal content area. Box–Behnken response surface methodology was applied, and the composition of the medium that produced the optimum level of antifungal substances was determined using antifungal activity as the screening index. The composition of the antifungal substances and the related genes from the Z-5 strain were identified using liquid chromatography-mass spectrometry and polymerase chain reaction, respectively. [Results] The optimal medium for the generation of antifungal substances by the Z-5 strain consisted of 2.68% (mass fraction) corn flour, 1.45% yeast powder, 0.03% K₂SO₄, 0.8% Na₂HPO₄·12H₂O, and 0.2% NaH2PO4·2H₂O at pH 7.2–7.4. The antifungal zone was 1.34 cm in diameter, which was close to the predicted value, confirming that the model was reliable and effective. Antifungal substances were mainly lipopeptide antibiotics C14 surfactin B, C14–C17 iturin A, and C14, C15, and C17 iturin B. polymerase chain reaction revealed that srfAB, ituC, and ituD occur in the Z-5 genome. [Conclusion] This study optimized the culture medium for producing antifungal substances, which are composed of lipopeptide antibiotics, from the B. malacitensis Z-5 strain. The results provide a theoretical reference and material basis for the prevention of cotton Verticillium wilt using these antifungal substances.