香石竹切花茎末端细菌生物被膜的形成及其调控机制研究

Bacterial biofilm as the predominant form of bacteria in nature, its formation on the stem ends of cut flowers, is regarded as an important reason causing damage of ornamental quality and shortening of vase life of cut flowers. But so far, little research in bacteria biofilm of cut flowers, especially in its formation on stem ends of cut flowers and its regulation mechanism. Hence, the present project, using cut carnations as the main experimental materials, which is prone to be impaired by various bacteria, via in vitro and in vivo (namely the cut flowers during vase period and simulated transportation), based on physi-chemical analyses, histochemistry and microstructure investigation, and postharvest treatments (including bacterial biofilm inhibitors, low-frequency ultrasonic, change in pH or temperature, etc), to exploit and understand the structure, composition and spatial-temporal features of formation of bacteria biofilm on the stem end of cut carnations, and further to reveal the laws formation of bacteria biofilm on stem end of cut carnations during vase period and simulated transportation and its regulation mechanism, and then to seek a new idea to alleviate the negative effects of bacteria on ornamental quality of cut carnations, by means of inhibiting the bacterial biofilm formation and reducing its impairment. The research results from this project will help to reveal the formation and regulation mechanism of bacterial biofilm on cut carnations and even other cut flowers, and to address new approach and technologies to prevent the harmful effects of bacteria on cut flowers.

细菌生物被膜作为自然界细菌最主要的存在方式，其在切花茎末端的形成很可能是影响切花观赏品质和采后寿命的重要原因。但迄今有关切花细菌生物被膜，特别是切花茎末端细菌生物被膜的形成及其调控机制的研究还极少。为此，本项目拟选择易受细菌侵害的香石竹切花为材料，经由离体和活体（即瓶插和模拟贮运的切花）两条主线，并采用理化分析与显微观察等技术以及切花采后相关处理（如细菌生物被膜抑制处理、低频超声波处理、温度和pH等环境因子变化等），深入研究香石竹切花茎末端细菌生物被膜的结构与成分及其形成的时空特点，进而探明香石竹切花在瓶插及模拟贮运期间细菌生物被膜形成规律与调控机制，并探寻一条“控制细菌生物被膜的形成和危害=>减轻细菌对切花观赏品质的影响”的新思路。本项目研究成果，有助于揭示和阐明香石竹及其他切花采后细菌生物被膜的形成与调控机制，并可望为形成和提出更有效的控制切花采后细菌危害的新策略与处理技术提供科学依据

细菌生物被膜在切花茎末端的形成很可能是影响切花观赏品质和采后寿命的重要原因。本项目主要研究香石竹（Dianthus caryphyllus）切花茎末端细菌生物被膜的结构与成分及其形成的时空特点与调控机制。主要研究内容和结果如下：首先建立了细菌生物被膜在香石竹切花茎末端活体和离体研究观测系统，并发现，离体培养的香石竹茎末端细菌生物被膜在12 h后逐渐形成，24 h可以明显看到胞外多糖和菌体均匀分布。通过对几种常用的细菌生物被膜抑菌剂的理化性质和抑菌效果进行研究和比较发现，纳米银（Nano-silver，NS）既能够有效抑制细菌的繁殖又能抑制细菌又能抑制生物被膜的形成，并且NS可显著延长香石竹切花瓶插寿命和维持观赏品质，并显著改善瓶插期间花枝水分关系，是理想的调控细菌繁殖和细菌生物被膜形成抑制剂。利用石蜡切片、激光扫描共焦显微镜和扫描电镜观察研究香石竹切花采后瓶插期间茎末端微生物堵塞发生的时空特点，结果表明，香石竹切花采后均易发生细菌性堵塞，其堵塞机制主要为细菌大量繁衍并形成生物被膜堵塞切花茎末端切口处；用NS预处理香石竹切花可有效抑制茎末端细菌聚集及细菌生物被膜形成，进而延缓和减轻细菌性茎堵塞的发生；对香石竹切花茎末端分离得到的细菌进行离体生物被膜培养，添加NS时培养24 h只观察到少量菌体和胞外多糖，且未见明显的膜状物形成。通过香石竹切花细菌相关序列扩增多态性（SRAP）遗传多样性分析，以及基于16S rDNA利用高通量测序技术对香石竹切花茎末端细菌多样性分析，结果表明NS处理可显著降低香石竹切花瓶插期间茎末端细菌丰富度及α多样性，且明显改变香石竹切花采后茎末端细菌群落结构；不动杆菌属、泛菌属、肠杆菌属、梭菌属、果胶杆菌属、气单胞菌属、假单孢杆菌属和芽孢杆菌属等作为相对优势菌可能是引起香石竹切花采后细菌性堵塞的主要菌群。本项目研究所取得的有关成果有助于阐明切花茎基部细菌生物被膜的形成与调控机制，并有望为形成相应的切花采后保鲜与处理技术提供科学依据。