【作者】 廖春燕；

【导师】 马国瑞；

【作者基本信息】 浙江大学 ， 植物营养学， 2002， 博士

【摘要】 1．在马铃薯蔗糖培养基(PDA)上测定了两种酸溶性壳聚糖(C-1和C-2)和一种水溶性壳聚糖(C-3)对17种植物病原真菌的拮抗作用，初步探索了壳聚糖分子量及浓度对其抗菌性能的影响，水溶性壳聚糖C-3的抗菌能力最佳且随着浓度增大而增强，酸溶性壳聚糖C-1和C-2的抑菌强度差异不显著，但两者的最佳抑菌浓度对不同病原真菌表现不一致。不同分子量的壳聚糖对病原真菌的抗性机制不同。 2．用含毒介质法测定了壳聚糖对番茄枯萎病菌、早疫病菌菌丝生长和孢子萌发的抑制作用，浓度大于1mg／ml的壳聚糖能明显地抑制菌丝生长，各设定浓度均可在一定程度上抑制孢子萌发，而且抑制效果随着处理浓度的增大而增强；浓度大于0.1mg／ml的壳聚糖处理可诱导初生菌丝发生菌丝肿胀、分枝增多且菌体分隔增加及体细胞变短等形态学变化。壳聚糖的抑菌作用机制与其增加菌丝细胞膜的透性有关。 3．不同浓度壳聚糖喷施对番茄幼苗株高、茎粗、叶绿素含量、地上部鲜重、地上部干重、地下部鲜重及磷的含量均无明显影响，而显著的增加地下部干重、根冠比及地上和地下部氮钾含量，适当的壳聚糖浓度(1mg／ml)还可调节番茄幼苗氮磷钾比例。根冠比值的增加有利于提高幼苗的抗逆能力，植株体内钾含量的提高与抗病能力有关。 4．在番茄四叶期用1mg／ml的壳聚糖进行诱导接种，可诱导番茄植株产生对早疫病的抗病性。经壳聚糖诱导后，番茄高抗、中抗、敏感品种的病叶率和病情指数均显著地低于接种对照，相对防效分别为39.8％、49.9％和56.4％；壳聚糖诱导番茄叶片过氧化物酶(POD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)、几丁质酶、β-1，3葡聚糖酶活性提高，诱导活性在不同抗性品种中表现不同，不同酶的时序变化也有所不同。 5．POD和PPO在接种早疫病病菌或喷施壳聚糖后，接种早疫病病菌组的POD和PPO活性均高于壳聚糖处理组。壳聚糖对PAL、几丁质酶和β-1，3-葡聚糖酶活性的诱导能力更强，在处理早期，壳聚糖处理组的活性均高于接种早疫病病菌组。喷施壳聚糖提高植株POD和PPO的活性可能是植株的应激反 应，而提高 PAL、几丁质酶和 P1，3－葡聚糖酶的活性是壳聚糖的直接诱导机 制。 6．壳聚糖处理、早疫病病菌接种处理下番茄叶片＊OD同工酶带谱与未接种基本 相同，只是带的强弱与测定的酶活一致，此结果表明早疫病和壳聚糖诱导 POD活性主要是增加原有POD同工酶的酶分子数日，而不是诱导新的同工 酶的表达。 7t 根扼同源序列设计特异性引物，以CTS处理后for、4hr、61。r的p A混合样 品作为反转录的膜板，合成CDNA，再以此CDNA作为PCR扩增的膜板，通 过RT PCR扩增出番仙卜1，｝葡聚糖酶＊in c＊＊A的一个片段，测序结果在 NCBI网站上进行blastn比较，其序列与番茄p刁，3－葡聚糖酶某囚同源性很高， 表明通过RTPCR扩增出的片段为番茄pl，3－葡聚糖酶GI［J CDNA的一个片 段。通过NOrthefflblottiflg分析表明，壳聚糖确实能够诱导卜1，3－葡聚糖酶的 快速表达，在壳聚糖处理l’J山，6－1，3－葡聚糖酶的表达很快增强，并在12 小时内保持在较高水平，接种处理卜1，3－葡聚糖酶的表达与对照相比略有提 高，但远低于壳聚糖诱导处理。可见壳聚糖诱导番茄对’I疫病的抗性机制是 在转率水平诱导了抗性基1习的表达，而非组成性酶的活性增强。 8．不同分于量不同浓度的壳聚糖在接种iiJ一天D－l。面喷雾处理对诱导玉米小斑病 的抗性均有一定效果，其中以低分子量壳聚糖 C上 mg／ml浓度最好。壳聚糖 ＊－1和＊－2随着处理浓度的增人，其防病效果呈下降趋势。接种后一天、接 种后三天壳聚栅喷施处理，川W1队效叨显下1籽，说叨）’c聚糖仪只有较灯的 防病效果，而非抗病效果。 9．壳聚糖处理番茄种子后接种枯萎病菌与对照相比发芽数增多，成活率提高， 发病数减少。在同一接种量条件下，壳聚糖对枯萎病菌引起的番茄苗期狩倒 病具有较好的防效，特别是较大接种量时，成活率差异更加明显但随着病原 菌接种最的增大，壳聚糖防病的总体趋势逐渐下降。 川．壳聚糖对玫瑰白粉病的防效低于甲基拖布津，高于对照和氨基寡糖。施用甲 基拖介津后三大基本控制住了白粉病的发病率，府情指数与对照相比有极显 著地下降，喷施后第10天川对阶效达79．5％。壳聚糖施J－IJ后玫瑰白粉病的发 病率及病情指数均显著地低于对照，而且处理后第10天发病率厂始下降，但 ＊ ！2 所要求的浓度较大，成本过高，直接大规模应川于生产有待于进一?更多还原

【Abstract】 1. The effects of three kinds of chitosan on radial growth seventeen phytopathogenic fungi was investigated in vitro. Except 2 fungi, the radial growth of 15 fungi tested was inhibited by three kinds of chitosan. The inhibit effect to fungi of water-soluble chitosan C-3 was the most effective, and it’s antifungal activity was increased when it’s concentration increased. The antifungal activity of chitosan C-l and chitosan C-2 had no significant difference, but the most effective concentration was related to the kind of phytopathogenic fungi. The main antifungal mechanism of different molecular weight chitosan was different.2. Chitosan was effective in inhibiting radial growth and sporogensis of F. oxysporum. f. sp. lycopersici and Alternaria solani(E. et M.) Jans et Grout in vitro. The inhibiting activity of chitosan was increased when its concentration increased. Light microscope observations showed that chitosan at concentration greater than Img/ml induced morphological changes, including hyphal swelling, distortion, excessive branching, shortening of hyphal segments in new hyphal of F. oxysporum. f. sp. Lycopersii and Alternaria solani(E. et M.) Jans et Grout. The resistance mechanism of chitosan is related to its ability to increase the permeability of the fungal plasma membrane.3. It’s no significant relationship between seedling weight, stem diameter, chlorophyll content, FW of top, DW of top, FW of root and P content in tomatoes with spraying different concentrations of chitosan. It could make tomato’s DW of root, root/top, and N, K content of top and root to increase significantly. Img/ml chitosan can adjust the ratio of N: P: K in top of tomato. The plant’s response to stress will increase with the increased root/ top. The increasing K content in plant has the relationship to resistance to disease.4. It could make the tomato plants resistance to early blight when the tomato seedlings were induced by chitosan (Img/ml) at the stage of the forth leaf. Chitosan treatment of tomato leaves before inoculation reduced the rates of diseased leaf and disease index, the rates of diseased leaf of resistant and susceptible tomato cultivars were 35.9% > 34.2%$I 34.9% respectively and lower than inoculation control significantly. The relative immunization efficiencies of the three tomato cultivars were 39.8%, 49.9%fll 56.4%, respectively. Chitosan-treated leaves induced an increase in POD, PPO, PAL, chitinase, (3-1,3- glucanase activities. The activity of these enzymes induced by chitosan in difference tomato cultivars showed different. Different enzymes vary differently after chitosan.5. Both of activities of POD and PPO in the tomato inoculated with Alternaria solani(E. et M.) Jam et Grout arc higher than those treated with chitosan. However, chitosan can induce the activities of PAL, chitinase andfi-1,3- glucanase more strongly. At early stage of treatment, these activities in tomato treated with chitosan are higher than those in plants inoculated with Alternaria solani(E. et M.) Jans et Grout. It shows that the inducation of POD and PPO by chitosan is the plant’s response to stress, hut that chitosan can induce of PAL, chitinase and p-1 ,3- glucanase specially.6. Enzyme activity staining showed that no new isoforms of peroxidase were found in the leaves of plants treated with chitosan or inoculated with Alternaria so!ani(E. et M.) Jans et Grout. But the densities of bands are relevance with the results of enzyme activities. Our data indicates that early blight and chitosan do not induce new isoforms of POD, but improve the level of POD protein.7. We designed the PCR primer according to the homology sequence of P~l,3- glucanase induced by fungi and amplified a cDNA fragment by RT-PCR. The blast result showed that it was a gene P-1,3- glucanase of tomato. Chitosan can induce the expression of this gene strongly. In an hour of treatment of chitosan, the expression of P-1,3- glucanase was improved by sever更多还原