实验海洋生物学重点实验室知识库

    在现代农业生产过程中，由于高毒农药长期大量使用，使得农药残留带来的食品安全问题越来越严重。新型的绿色无毒、高效低残留的生物农药的研发成为了一个全球性的课题。我国是海洋生物资源非常丰富的国家，以虾蟹壳为原料制备的壳聚糖作为一类无毒无害可降解和生物相容性好的海洋生物多糖，具有抑制植物病原真菌、诱导农作物抗病和促生长等多种作用，被广泛应用于农业生产。利用壳聚糖对现代农药进行修饰，可以得到一类具有协同作用的新型壳聚糖农药偶合物，提高药效，降低毒副作用，为农药创制提供新的思路。

    本文就是在以上思路的指导下，在壳聚糖的分子结构基础上，通过接枝共聚等化学手段，将阿维菌素、恶霉灵和嘧霉胺三种农药接枝到壳聚糖的分子上，制备得到一系列壳聚糖与农药的偶合物。通过红外光谱、核磁氢谱、元素分析、X 射线衍射和高效液相色谱对壳聚糖偶合物进行了结构表征，研究了壳聚糖与它们的协同增效作用及影响因素。主要研究结果如下：

1.通过N,N-环己基二亚胺/4-甲氨基吡啶（DCC/DMAP）脱水体系反应制备了一系列壳聚糖与阿维菌素的偶合物。实验结果表明，偶合物对粘虫、苜蓿蚜、褐飞虱和朱砂叶螨均显示了较好的杀虫活性，同时偶合物的光稳定性相比阿维菌素原药有明显提升，证明壳聚糖与阿维菌素表现出了协同作用。通过分析影响协同作用因素发现，双位点羧甲基化的壳聚糖、高分子量的壳聚糖对于阿维菌素的协同效果更加明显；琥珀酰化壳聚糖对阿维菌素的协同效果高于马来酰化壳聚糖。

2. 通过以氯乙酰氯、3-氯丙酰氯和4-（氯甲基）氯甲酰氯三种酰氯为连接物，将恶霉灵分子整体连接到壳聚糖骨架中，制备了一系列壳聚糖与恶霉灵的偶合物。偶合物对立枯丝核菌、玉蜀黍赤霉、苹果炭疽病菌、水稻纹枯病菌等4株植物病原菌具有较好的抑菌活性。壳聚糖和恶霉灵表现出了较好的协同作用，并且偶合物的接枝率越高、空间位阻越小，壳聚糖与恶霉灵两者的协同效果越好。

3.将嘧霉胺通过定位接枝到了壳聚糖骨架中，制备了一系列壳聚糖与嘧霉胺的偶合物。抑菌试验显示偶合物对立枯丝核菌、玉蜀黍赤霉、苹果炭疽病菌3株真菌具有较好的抑制活性，说明壳聚糖与嘧霉胺表现出了协同效果，而且接枝率和空间位阻是影响壳聚糖与嘧霉胺偶合物的协同增效作用的主要因素。

    本论文首次制备了壳聚糖与阿维菌素、恶霉灵和嘧霉胺三种农药的偶合物，并对壳聚糖与三种农药偶合物的协同作用进行了系统研究，发现壳聚糖可以与阿维菌素、恶霉灵和嘧霉胺三类农药产生协同作用，从而通过提高农药的活性和改善理化性质来降低化学农药的使用量和提高农药的稳定性，因而使解决目前化学农药带来的环境问题和食品安全问题成为可能。这类具有协同效果的壳聚糖与农药偶合物为新型高效、低毒、低残留的绿色农药的开发提供了一条新的途径，具有巨大的应用潜力。

 

In the process of modern agricultural, agricultural agents including pesticides and fungicides are used in amount for a long period. The food security problem caused by agricultural agents residual become more and more serious. The research and development of novel agricultural agents with non-toxic, high efficiency and low residue properties is a global issue nowadays. Chitosan is a kind of bio-degradable and bio-compatible marine polysaccharide produced from shells of crabs and shrimps. Due to its antifungal and growth induction properties, it’s widely applied in agriculture. Novel agricultural agents with synergistic effect can be achieved by modification based on the structure of chitosan. This is a special strategy to develop new agricultural agents.

Under the guidance of the idea introduced above, avermectin, hymexazol and pyrimethanil were grafted onto the chitosan structure by chemical reactions. In this way, we achieved a serious of novel agricultural agents grafted chitosan derivatives. The structures of the derivatives were confirmed by infrared spectrum, nuclear magnetic hydrogen spectrum (NMR), elemental analysis, X-ray diffraction (XRD) and high performance liquid chromatography (HPLC). The synergism of chitosan and these agricultural agents was investigated by our research. The main results were as the followings:

1. Avermectin grafted chitosan derivatives were prepared in the DCC/DMAP reacting system. The derivatives showed enhanced insecticidal activity against armyworm, carmine spider mites, black bean aphids and brown plant hoppers. The photostability was greatly improved as well. The results showed that there existed synergism by grafting avermectin with chitosan. Double-sited carboxymethylation and higher molecular weight could enhance the synergism. The synergistic effect of succinylation is more obvious than maleylation.

2. Hymexazol grafted chitosan derivatives were synthesized by using chloroacetyl chloride, chloropropionyl chloride and 4-(chloromethyl) benzoyl chloride as linkers. The obtained derivatives showed great antifungal activity against Rhizoctonia solani, Gibberella zeae, Glomerella cingulate, and Thanatephorus cucumeris. Chitosan and hymexazol showed good synergistic effect with each other. The synergistic effect increased with higher grafting ratios and less steric hindrance.

3. Pyrimethanil was grafted onto chitosan structure. The pyrimethanil grafted chitosan derivatives performed enhanced antifungal activity against Rhizoctonia solani, Gibberella zeae, and Glomerella cingulate. The pyrimethanil grafted chitosan derivatives also showed synergistic effect. The results indicated that the main influencing factors of synergism were grafting ratios and steric hindrance.

In this study, three kinds of chitosan conjugates with avermectin, hymexazol and pyrimethanil were sythesised for the first time. It was found that chitosan showed synergistic effect with avermectin, hymexazol and pyrimethanil. With the synergism, the dosages of these agricultural agents could be greatly decreased by improving their efficiency and other physicochemical properties. In this way, we could possibly solve the environmental and food security problems introduced by agricultural agents. This synergistic agricultural agents grafted chitosan derivatives showed a totally different way to develop novel agricultural agents with non-toxic, high efficiency and low residue properties. This kind of agricultural agents showed great potential in application in the future.