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水溶性甲壳素羧基衍生物的制备及其对根结线虫的防控研究
战将
学位类型博士
导师李鹏程
2022-05-10
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
关键词根结线虫病害 甲壳素 6-羧基甲壳素 淡紫拟青霉 生物防治
摘要
植物寄生线虫是植物侵染性病害的重要病原体之一,每年可造成的经济损失高达 1570 亿美元,其中以根结线虫的危害最为严重。现有杀线剂多存在高毒、抗药性严重、稳定性较差等问题,在应用上受到很大限制。安全、高效杀线剂的严重缺乏已成为制约农业绿色发展的瓶颈。因此,如何有效的开展根结线虫的生物防治已成为目前线虫防治的热点。在根结线虫的生物防治领域,目前研究较多的是利用线虫拮抗微生物来控制线虫病害的发生,由于拮抗微生物所生存的土壤环境极其复杂,使得其在土壤中难以定殖并稳定地分泌杀线活性物质,导致田间的防治效果并不理想,难以在实际生产中得到广泛的应用。近年来以甲壳素/聚糖等为代表的天然活性多糖因具有良好的生物相容性、生物可降解性以及广谱的土壤生防菌诱导能力,在线虫防治领域具备优越条件和潜力,备受关注。然而甲壳素因水溶性差、用量大,在实际应用中存在很大的局限性。因此为了增加甲壳素的生物利用度,发掘更有应用潜力的甲壳素线虫防控制剂,本论文对甲壳素进行结构修饰,在保留甲壳素核心骨架基础上,定位引入亲水的羧酸活性基团,制备了水溶性好的甲壳素羧基衍生物(6-oxychitin),对其制备条件进行了优化并评价了其线虫防控潜力。结果表明:
  1、根据原料的粒径、预处理方式以及反应时间对 6-oxychitin 的制备进行了优化,实验结果发现采用粒径小于 150 μm 的甲壳素原料,预处理方法选用DMAC-LiCl 法,氧化反应时间控制在 120 min,更有利于获得氧化度高,水溶性好的 6-oxychitin
  2、在直接防控线虫病害方面,6-oxychitin 与甲壳素相比并未有明显提高,但是具有明显的生防菌诱导增效功能以及植物诱抗能力。6-Oxychitin 可作为碳源诱导生防菌提高体外杀线能力且具有明显的广谱性;还可促进黄瓜种子的萌发及生长,提高植物体内β-1,3 葡聚糖酶、几丁质酶以及过氧化氢酶防御酶活活性。
  36-Oxychitin 诱导培养淡紫拟青霉后,可使杀线活性增加 42%。机理研究表明,杀线的主要作用机制是通过抑制二龄幼虫的运动能力以及乙酰胆碱酯酶酶活,造成神经紊乱,影响幼虫的正常运动行为;干扰正常生理代谢活动,进而使甲壳素羧基衍生物的制备及其对根结线虫的防控研究幼虫死亡;通过调控脂肪酶以及几丁质酶酶活,抑制线虫卵孵化。
  46-Oxychitin 诱导的淡紫拟青霉代谢产物对植物根结线虫病害具有明显的防控效果。凝胶实验中浓度为 10%时便能够明显抑制植物根结的产生,抑制率高 99%。盆栽实验中,30 d 对黄瓜根结的相对抑制率达到 68.22%。机理研究发6-oxychitin 一方面可通过促进光合色素的合成,增强根系活力,诱导植物体内CAT 酶、POD 酶以及几丁质酶等生理生化指标间接提高植物对线虫的诱导抗性,促进植物生长,减轻淡紫拟青霉引起的植物毒副作用。另一方面能够通过提高土壤微生物多样性,改变线虫侵染土壤的微生物组成,增加有益微生物丰度,降低某些致病菌的丰度等方式提高对线虫的防控能力。
  本文首次将 6-oxychitin 应用到农业防治根结线虫领域,通过一系列的体外、体内实验发现 6-oxychitin 在保留甲壳素原有防控线虫的活性基础上,改善了甲壳素的应用缺点,具有广谱的诱导生防菌杀线增效、促植物生长等活性。当6-oxychitin 作为诱导因子培养淡紫拟青霉时防控效果最佳,不仅能显著提高淡紫拟青霉的杀线能力,而且能缓解淡紫拟青霉的毒副作用。因此,6-oxychitin 在根结线虫病防治中具有广阔的应用前景。本研究不仅扩大了 6-oxychitin 的应用范围,而且为开发绿色、高效、环境友好的杀线剂提供了新的思路。
其他摘要
Plant parasitic nematodes (PPNs) are one of the important plant infecting pathogens, which can cause economic losses up to $ 157 billion yearly, among them root-knot nematodes (Meloidogyne sp.) are the most harmful. As the existing nematicides have problems such as high toxicity, serious drug resistance and poor stability, which greatly limit their application. The serious shortage of safe and efficient pesticides has become a bottleneck restricting the development of green agriculture. Therefore, how to effectively carry out the biological control has become the focus of nematode control. Concerning the biological control of nematodes, the current research is mostly based on antagonistic microorganisms to control the occurrence of nematode disease. However, the complex soil environment makes it difficult for biocontrol microorganisms to colonize, resulting in poorly control effects that are difficult to apply widely in actual agriculture field. Recently, natural active polysaccharides such as chitin and chitosan have aroused wide concern with good biocompatibility and biodegradability and broad-spectrum inducibility, but their application is limited due to poor solubility which caused large dosage. Therefore, in order to increase the bioavailability of chitin, we prepared the carboxyl chitin derivative (6-oxychitin) by introducing carboxylic acid groups based on retaining the core skeleton of chitin which improved the water solubility of chitin. In our paper, the  preparation conditions of 6-oxychitin were optimized and the potential for nematode control was evaluated. The main results are as follows:
  1.The experiment optimized the preparation conditions of 6-oxychitin from the particle size of the raw material, the pretreatment method and the reaction time. Our results show that the particle size is less than 150 μm, the DMAC-LiCl pretreatment method, the oxidation reaction time of 120 min, which is more conducive to obtain 6-oxychitin with high oxidation degree and good water solubility.
  2.Although the direct control effect of 6-oxychitin on nematode diseases is not good and has not been significantly improved compared with chitin, it has obvious induction ability of biocontrol induction nematicidal ability and plant disease resistance. 6-Oxychitin can be used as sole carbon source to induce biocontrol microorganisms to improve the nematicidal ability in vitro and with an obvious broad-spectrum induction. On the other hand, it can promote the germination and growth of cucumber seeds, and increase the β-1,3 glucanase, chitinase and catalase defense enzymatic activity in plants.
  3.6-Oxychitin can increase the nematicidal activity of P.lilacinus by 42%. The main mechanism is that it can inhibit the movement ability and acetylcholinesterase activity of J2s, causing neurological disorders and affecting their normal motor behavior, by interfering with their normal physiological metabolic activities, thereby causing the larvae to die. In addition, it can regulate lipase and chitinase activity to inhibit the egg hatching of nematode.
  4.The fermentation product of P. lilacinus induced by 6-oxychitin (OTC-PLF) has a certain control effect on plant root-knot nematode diseases. When the concentration was 10%, the relative inhibition rate of root-knot was as high as 99% in Pluronic gel assays. In the pot experiment, the relative inhibition rate reached 68.22% at 30 days. On the one hand, we found that it can indirectly improve the induction resistance of plants to nematodes by promoting the synthesis of photosynthetic pigments, enhancing its root activity, inducing CAT enzymes, POD enzymes and chitinases in plants, promoting plant growth, and alleviating the phytotoxicity of P.lilacinus alone. On the other hand, it can improve soil microbial diversity, change the microbial composition of nematode-infected soil, by increasing the abundance of beneficial microorganisms and reducing he abundance of some pathogenic microorganisms to a certain extent, and to improve the ability to control nematodes.
  In this paper, for the first time, 6-oxychitin was applied to the field of agricultural control of root-knot nematodes. We found that 6-oxychitin can improve the application shortcomings of the original chitin on the basis of retaining the original nematode control activity and has a broad spectrum of inducing activities such as by enhancing nematicidal activity of biocontrol microorganisms and promoting plant growth. When it was used as an inducer of P. lilacinus, the nematode control effect was the best, which not only significantly improved the nematicidal ability of P. lilacinus, but also alleviated the toxicity of P.lilacinus alone. Thus, 6-oxychitin has great potential in the control of nematode diseases. The study not only expanded the application range of 6-oxychitin but also provided new insights for the development of green, environment-friendly and efficient nematicides.
学科门类理学
语种中文
目录
第一章 绪论
1 根结线虫
1.1 根结线虫概述
1.2 根结线虫危害
1.3 根结线虫的防治
2 根结线虫的生物防治
2.1 生防细菌
2.2 放线菌
2.3 生防真菌
2.4 生物防治应用中的问题及对策
3 甲壳素及其衍生物的农用活性
3.1 甲壳素概述
3.2 甲壳素及其衍生物在农业上的应用
3.3 甲壳素及其衍生物防治线虫研究.
4 选题意义及思路
5 研究技术路线与内容
第二章 水溶性 6-羧基甲壳素的制备与表征
第三章 水溶性 6-羧基甲壳素防治线虫效果研究
第一节 6-羧基甲壳素防治黄瓜根结线虫的研究
第二节 6-羧基甲壳素对常见生防菌诱导杀线效果研究
第三节 6-羧基甲壳素的植物诱抗活性研究
第四节 本章小结
第四章 6-羧基甲壳素诱导淡紫拟青霉抗根结线虫研究
第一节 6-羧基甲壳素对淡紫拟青霉生物学特性以及土壤定殖影响
第二节 6-羧基甲壳素对淡紫拟青霉发酵产物杀线活性影响
第三节 黄瓜根结线虫病害防效评价
第四节 土壤微生物群落分析
第五章 结论与创新点
参考文献
附 录
致 谢
作者简历及攻读学位期间发表的学术论文与研究成果
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/178316
专题实验海洋生物学重点实验室
推荐引用方式
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战将. 水溶性甲壳素羧基衍生物的制备及其对根结线虫的防控研究[D]. 中国科学院海洋研究所. 中国科学院大学,2022.
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