荧光假单胞菌ZX对采后锦橙绿霉病的防治及其抑菌机制

Biocontrol of green mold decay in Jincheng citrus fruits by Pseudomonas fluorescens ZX

摘要:

[目的]采后柑橘极易受指状青霉（Penicillium digitatum）侵染而发生严重的绿霉病腐烂，生物防治因具有安全、有效、环保等特点近年来备受关注。论文旨在研究荧光假单胞菌（Pseudomonas fluorescens）ZX对采后柑橘绿霉病的防治效果，揭示P.fluorescens ZX对P.digitatum可能存在的作用机制。[方法]以"北碚447"锦橙果实为试材，先分别接种20 μL拮抗菌培养液、滤液（培养液离心后，上清经0.22 μm滤膜过滤）、菌悬液（培养液离心后，菌体用无菌水反复洗涤并用无菌水重悬）和热杀死液（培养液高温高压灭菌），2 h后接种20 μL P.digitatum孢子悬浮液（1×10⁴ spores/mL），所有果实于20℃、90%相对湿度环境下恒温恒湿培养8 d后，测定果实的发病率和病斑直径；制备柑橘皮培养基，进行平板抑菌试验，探索P.fluorescens ZX对P.digitatum孢子发芽情况的影响；采用两板对扣法和生物熏蒸法研究P.fluorescens ZX挥发性次级代谢产物的抑菌作用；利用插入式细胞培养皿等分析P.fluorescens ZX和P.digitatum之间竞争的营养物质；同时，测定P.fluorescens ZX的生长曲线，利用结晶紫染色法评估P.fluorescens ZX的生物膜形成能力。[结果]P.fluorescens ZX不同处理液之间对采后锦橙绿霉病的作用效果差异显著，菌悬液抑菌效果最好，经菌悬液处理的果实，发病率和病斑直径分别仅为40.83%和1.78 cm；不论是在柑橘皮固体培养基上对峙培养还是在液体培养基中混合培养，菌悬液和原液的作用效果较好，固体平板上，相对抑制率达到了35%-45%，液体培养基中，P.digitatum孢子12 h后的发芽率不超过27%；P.fluorescens ZX产生的挥发性物质具有抑菌作用，经P.fluorescens ZX熏蒸处理的锦橙果实，发病率和病斑直径都显著降低；营养竞争试验结果表明，P.fluorescens ZX能更快速有效地消耗柑橘皮培养基中的营养，并和P.digitatum竞争葡萄糖、果糖、蔗糖、天冬氨酸、苏氨酸、丝氨酸、亮氨酸、精氨酸和脯氨酸等营养物质；同时，P.fluorescens ZX生命力强，培养4 h后即进入对数生长期，约24 h后形成成熟的生物膜。[结论]P.fluorescens ZX可能通过抑制P.digitatum孢子发芽、营养与空间竞争、形成生物膜、产生抑菌物质等方式抑制P.digitatum的生长繁殖，有效防治采后锦橙绿霉病。

关键词:荧光假单胞菌;绿霉病;生物防治;锦橙;采后病害;拮抗机制

Abstract:

[Objective] Researches were carried out for biocontrol of green mold decay in citrus by Pseudomonas fluorescens ZX. [Methods] The biocontrol effectiveness in fruits and effects on Penicillium digitatum growth were evaluated along with the antifungal activity of volatile organic compounds (VOCs). Meanwhile, assays of competitive grow between Pseudomonas fluorescens ZX and Penicillium digitatum were conducted, as well as the growth curve and biofilm formation ability of Pseudomonas fluorescens ZX. [Results] Pseudomonas fluorescens ZX suspension showed potent ability for the depression of Penicillium digitatum, so did the VOCs. In addition, Pseudomonas fluorescens ZX made better use of nutrients than Penicillium digitatum, and it entered the logarithmic growth stage quickly (4 h) and formed an effective biofilm after 24 h. [Conclusion] The application of Pseudomonas fluorescens ZX was effective to control green mold, and the potential mechanisms included inhibition of spore germination, competition for nutrients and space, and formation of biofilm.

Key words:Pseudomonas fluorescens;green mold decay;biocontrol;Citrus sinensis osbeck (Jincheng);postharvest diseases;antagonistic mechanism