【作者】 李睿；

【导师】 张安强；

【作者基本信息】 华南理工大学 ， 材料科学与工程， 2024， 硕士

【摘要】 香蕉枯萎病是一种由尖孢镰刀菌古巴专化型（Fusarium oxysporum f.sp.cubense,Foc）引起的的土传植物病害,Foc孢子具有在土壤中长期休眠的能力,故土壤中腐生的高浓度Foc孢子是诱发香蕉枯萎病的主要原因,也是香蕉枯萎病难以防治的重要原因。如果可以实现对Foc的现场快速检测,则可有针对性地开展土壤消毒等防控措施,对防控香蕉枯萎病有重要意义。目前主要使用平板涂布、聚合酶链式反应（PCR）、拉曼光谱等对土壤中Foc孢子进行检测,但上述三种检测方法难以兼顾快速、便携和现场检测。通过将微生物吸附在表面印迹聚合物（Surface imprinted polymer,SIP）表面,继而通过荧光染色,从而实现可肉眼直接半定量观察,是一种极具挑战性的方法,主要受速度、准确性和定量精度的限制。因此,本文将SIP技术与Foc孢子印迹、计数、染色及定量计算相结合,提出了一种基于微接触压印法制备Foc孢子表面印迹聚合物（Foc SIP）的方法,可初步实现半定量和快速肉眼检测,并探索了其在快速半定量检测土壤中Foc孢子浓度中的应用,主要工作包括:（1）基于ITO导电层,使用6V DC的电沉积法制备出约9000 mm^-2位点密度的Foc4印章,继而使用无溶剂法铺展PDMS预聚物,制备出具有高印迹位点密度的聚二甲基硅氧烷表面印迹聚合物膜（Foc4-PDMS SIP）。（2）通过32V DC的电吸附,首次完整记录了孢子吸附的全过程。镜下计数表明,45至60 min时SIP膜层达到接饱和吸附状态,且该状态的孢子吸附数在孢子悬浮液浓度处于10⁴～10⁷个/m L时呈现强相关关系,经染色后可实现数量级级别的肉眼半定量检测。对不同微生物的吸附测试表明,该SIP膜仅对Foc具有完全的识别,对相似的尖孢镰刀菌冬瓜专化型（Fob）识别效率仅仅为Foc系列的9.88%。重复使用测试表明了SIP膜层具有一定的可重复利用性,其无损最大使用次数达到了3次。结果表明,Foc4-PDMS SIP具有现场快速、半定量检测土壤中Foc4浓度的能力,可为香蕉栽培提供良好的支持,这种方法在检测其他真菌疾病方面也有潜在的应用。更多还原

【Abstract】 Banana wilt is a soil borne plant disease caused by Fusarium oxysporum f.sp.Cubense（Foc）,whose spores have the ability to dormant in the soil for long time,so the high concentration of Foc spores in soil is the main cause of banana wilt disease,and it is also an important reason for the difficulty in preventing and controlling banana wilt disease.If on-site rapid detection of Foc can be achieved,targeted soil disinfection and other prevention and control measures can be carried out,which is of great significance for the prevention and control of banana wilt disease.At present,the main methods for detecting Foc spores in soil are plate coating,polymerase Chain Reaction（PCR）,and Raman spectrum,but the three detection methods mentioned can hardly achieve fast,on site and portable at the same time.It is a highly challenging method to adsorb microbe on surface imprinted polymers（SIP）.Subsequently,fluorescence staining is used to achieve direct semi quantitative observation with the naked eye,which is a highly challenging method mainly limited by speed,accuracy,and semi quantitative accuracy.Therefore,a method based on micro contact imprinting method is proposed to prepare Foc spore surface imprinted polymer（Foc SIP）by combining SIP technology with Foc spore imprinting,counting,staining,and quantitative calculating was propsed in this article,which can preliminarily achieve semi-quantitative and rapid visual detection,and explore its application in rapid semi-quantitative detection of Foc spore concentration in soil.The main work includes:（1）Based on the ITO conductive layer and 6 V DC,Foc4 stamp with about 9000 mm^-2 site density was prepared.Then,PDMS prepolymer was spread using a solvent-free method to prepare a polydimethylsiloxane surface imprinted polymer film（Foc4-PDMS SIP）with high imprinting site density.（2）With 32 V DC,the entire process of spore adsorption was fully recorded for the first time.dsorption statistics show that the SIP film reached saturated adsorption state at 45 to 60minutes,and the spore adsorption number in this state showed a strong correlation when the spore suspension concentration is between 10⁴-10⁷ m L^-1.And after staining,it could achieve semi-quantitative detection with the naked eye at an order of magnitude level.The adsorption tests on different microorganisms showed that the SIP membrane only had complete recognition of Foc,and the recognition efficiency for similar Fusarium oxysporum f.sp.benincasae（Fob）was only 9.88%of the Foc series.Reuse tests showed that the SIP film layer has a certain degree of reusability,with a maximum of 3 non-destructive uses.As a result,Foc4-PDMS SIP detection has:good detection accuracy,good naked-eye detection,and there is no need to carry professional detection equipment.Foc4-PDMS-SIP has ability to rapidly on-site and semi quantitatively detect concentration of Foc in soil,which can provide good support for banana cultivation.This method also has potential applications in the detection of other fungal diseases.更多还原