白星花金龟虫砂-GelMA水凝胶载菌体系优化及对棉花黄萎病菌抑制率的影响

Optimization of Protaetia brevitarsis Frass-GelMA Hydrogel Bacteria-Loading System and Its Effect on the Inhibition Rate of Verticillium dahliae in Cotton

摘要: 目的：制备白星花金龟虫砂与载菌水凝胶(B-GelMA)复合体系，评价其对棉花黄萎病菌的抑制效果。方法：以解淀粉芽孢杆菌为目标菌，建立OD₆₀₀与菌量标准曲线；优化B-GelMA的载菌条件；通过释放动力学实验考察温度、pH对解淀粉芽孢杆释放的影响；采用平板对峙法测定虫砂、B-GelMA及复合体系对大丽轮枝菌的抑制率。结果：菌液浓度与OD₆₀₀在0~0.35范围内线性良好，R² = 0.9882。最佳载菌条件：浸泡时间8 h、温度31℃、pH 6~7、凝胶质量2.5 g、菌液体积30 mL、初始浓度8 × 10⁸ cfu/mL，载菌量达2.16 × 10⁹ cfu/g。释放动力学显示：温度 ≤ 31℃时为不规则扩散，≥34℃转为Fick扩散；pH 6~9时均为不规则扩散。平板对峙结果表明，B-GelMA及复合体系的抑菌率分别达61.72%和60.00%显著高于白星花金龟虫砂，二者之间无显著差异。结论：白星花金龟虫砂具有一定抑菌作用，在体外抑菌测试中，B-GelMA展现了比虫砂更强的抑制活性，其在复合体系中起主导作用；虫砂的综合作用及协同潜力有待土壤环境中的进一步验证。该载菌水凝胶在适宜条件下负载效率高、释放可控，具备防控棉花黄萎病的应用潜力。

Abstract: Objective: To prepare a composite system of Protaetia brevitarsis frass and bacteria-loaded hydrogel (B-GelMA) and evaluate its inhibitory effect against Verticillium dahliae in cotton. Method: Using Bacillus amyloliquefaciens as the target biocontrol bacterium, a standard curve between OD₆₀₀ and bacterial count was established. The bacteria-loading conditions of B-GelMA were optimized; the effects of temperature and pH on bacterial release were investigated through release kinetics experiments. The inhibition rates of frass, B-GelMA, and the composite system (frass + B-GelMA) against Verticillium dahliae were determined using the plate confrontation method. Result: The bacterial concentration showed a good linear relationship with OD₆₀₀ in the range of 0~0.35, with R² = 0.9882. The optimal loading conditions were soaking time 8 h, temperature 31˚C, pH 6~7, hydrogel mass 2.5 g, bacterial solution volume 30 mL, and initial concentration 8 × 10⁸ cfu/mL, achieving a bacterial loading capacity of 2.16 × 10⁹ cfu/g. Release kinetics indicated that bacterial release followed anomalous diffusion at temperatures ≤ 31˚C and switched to Fickian diffusion at ≥34˚C. At pH 6~9, the release mechanism was anomalous diffusion in all cases. Plate confrontation results showed that the inhibition rates of B-GelMA and the composite system reached 61.72% and 60.00%, respectively, which were significantly higher than that of Protaetia brevitarsis frass alone, with no significant difference between the two. Conclusion: Protaetia brevitarsis frass possesses certain antifungal activity. In in vitro antibacterial tests, B-GelMA exhibited stronger inhibitory activity than Chongsha, playing a dominant role in the composite system. The comprehensive effect and synergistic potential of Chongsha require further validation in the soil environment. This bacterial-loaded hydrogel demonstrates high loading efficiency and controllable release under suitable conditions, showing application potential for the prevention and control of cotton Verticillium wilt.