【作者】 沙月霞；

【导师】 肖崇刚； 简桂良；

【作者基本信息】 西南农业大学 ， 植物病理学， 2005， 硕士

【摘要】 棉花是我国重要经济作物,而棉花黄萎病是当前影响我国棉花产量与品质的主要病害之一,是由大丽轮枝菌（Vertillium dahliae Kleb.）引起的一种危害性极大的维管束系统病害。国内外在棉花黄萎病防治及抗病育种方面虽然取得了一些进展,但进展缓慢。这是因为棉花黄萎病是一种土传病害,利用物理化学手段进行黄萎病的防治效果甚微。培育棉花抗黄萎病品种是解决黄萎病危害的最为经济有效的手段,然而我国陆地棉缺乏免疫抗源,使传统育种困难重重;利用基因工程手段培育棉花抗黄萎病品种已成为防治棉花黄萎病的新途径。 本试验选用11个不同致病型的黄萎病菌株,以黄萎病菌株V₉₉₁为抗原制作的抗血清进行ELISA检测。结果发现,所有菌株相同的培养条件下24h后就能在培养滤液中检测到毒素,这比生物测定要早3天;而且不同致病型的菌株产毒能力随培养时间的增加存在差异,落叶型菌株平均产毒量在前2天低于非落叶型菌株,从第3天开始落叶型菌株平均产毒量明显高于非落叶型菌株:其中5个供试菌株(V₉₉₁、V_43-1、T₉、V₂₅₀、V₁₂₃)的产毒量在培养至9天时达到高峰,9天后菌株产毒量逐渐下降;而另外5个供试菌株(V₂₃₂、V_B、V₅₆、V₁₂₀、V₅₉)的产毒量却在第11天才达到高峰;V_JV11在第7天毒素含量即达到了高峰。 选用感黄萎病棉花品种军棉1号为材料,采用不同浓度的黄萎病菌V₉₉₁毒素液对棉苗进行试管法测定。结果发现:不同浓度的黄萎病菌毒素对棉苗均有致萎作用,黄萎病菌毒素在一定范围内,浓度越高对棉苗的致萎力越强,1/8倍毒素液为最佳测定浓度。另选用4种不同致病型的黄萎病菌(V₉₉₁、V₅₆、V₂₃₂、V₅₉),采用1/8倍毒素液浸根处理棉苗。结果显示,不同菌株之间致萎力有明显差异,说明室内毒素检测能反应出田间不同黄萎病菌对棉株的致病性。在一定时间范围内,毒素处理时间越长萎蔫指数越大,处理36～48h落叶型和非落叶型菌株萎蔫指数之间的差异最明显,落叶型菌株在致萎力上高于非落叶型菌株。 选取5个不同抗感棉花品种文五、X033、海岛棉7124、9456D和军棉1号,采用黄萎病菌株V₉₉₁ 1/8倍毒素液浸根,结果发现,不同抗感品种的萎蔫指数均随处理时间的延长而增大。感病品种较抗病品种萎蔫指数增加速率大,处理24h抗感品种间的萎蔫指数差异在22.77～24.77之间,处理36h差异增大到34.53～43.81,但一般处理时间范围应在36～48h。试验结果显示,不同抗感品种对毒素的萎蔫指数存在明显差异。因此,可以利用黄萎病菌毒素来鉴别不同抗感品种。 比较了4种不同的缓冲液提取棉叶总蛋白的方法和3种不同的SDS-PAGE电泳的方法及3种不同浓度的分离胶方法。结果显示,25 mmol·L^-1,pH8.0 Tris-HCl缓冲液提取的棉叶总蛋白,不仅效率高,而且蛋白条带多;经过改良的丙酮沉降法效果最佳,电泳结果条带多而且清晰;10%～12%的分离胶最适合棉花总蛋白的SDS-PAGE电泳;试验表明,温度与脱色效果有关,在一定的温度范围内,温度越高脱色时间越短。 在不同抗感品种文五、9456D、军棉1号被黄萎病菌诱导后对棉花叶片总蛋白SDS-PAGE更多还原

【Abstract】 Cotton is one of the most important economic crop in China. However, Verticillium wilt of cotton, which is one of the most dangerous fascicular disease caused by Verticillium dahliae Kleb., is the major diseases which seriously affect the yield and quality of cotton. Although much progress has been made in prevention and control of Verticillium wilt of cotton, the progess is slow. Because Verticillium dahliae is soil-borned fungal pathogen, the effect of prevention and contrail is very little using the physical and chemical method. It is strongly consided that developing resistant and tolerant cultivar is the most economic and effective ways to resolve this problem. But the basic research about the pathogenic mechanism of Verticillium dahliae shows that developing resistance and tolerant cultivar by conventional breeding is far from enough because lack of resistant resources.A specific polyclonal antiserum was prepared again a purified phytotoxin from Verticillium dahliae of cotton, and an indirect EL1SA procedure was developed for detection of Verticillium dahliae Phytotoxin (PLPC) in culture filtrates. Results showed that the PLPC in culture filtrates was detectable when 11 strains were grown in Czapek’s medium under shaking condition at 25 ℃. only for 24 hours, which is 3 days earlier than that of the bioassay. The ability that different character strain produced phytotoxin was different. The average phytotoxin concentration of defoliating strain was lower than that of strain which was not defoliating in the other two days. But from the third day, the ability of that defoliating strain produced phytotoxin was higher than it of strain which was not defoliating. The phytotoxin concentration of five strains (V991, V43.1, T9, V250, V123) were the highest in the ninth day; other five strains (V232, VB, V56, V120, V59) produced phytotoxin the most in the eleventh day; the phytotoxin concentration of VJVII was the highest in the seventh day.Using the susceptive cultivar (Junmian 1) and different concentration phytotoxin of V991 to detect cotton seedling depending on the method of root dipping in phytotoxin, the result was that all of the different concentration phytotoxin of Verticillium dahliae could cause cotton wilt; in a certain degree, the higher concentration phytotoxin could cause cotton wilt more seriously. 1/8 times of phytotoxin was fit for early diagnosis of Verticillium wilt of cotton and it was used to detect the wilt difference among the strains (V991, V56, V232, V59) . As a result, we found that the phytotoxin of Verticillium dahliae could be used to judge the character of cultivar in diagnosis and the wilt difference of Verticillium dahliae in experiment. In a certain period, the longer the experiment time was, the higher the wilt index was. When cotton seedling was treated 36h~48h, the difference of every strain was clear and the wilt index of defoliating strain was higher than that of strain which was not defoliating.Through method of root dipping in phytotoxin, 1/8 times of phytotoxin was used to detect the character of Wenwu, 9456D, X033 (Gossypium hirsutium) , searisland cotton 7124 (G.. barbadense) , Junmian 1 in experiment. It was found that the wilt index of different cultivar would change as the experimental time increasing. When experimental time was 24 h, the wilt index difference of cultivars was 22.77~24.77. When experimental time was 36 h, the difference was 34.53~43.81. But experimental time should be 36~48 h. The experimental result showed that the wilt index of different cultivar was apparently different. Whats’more, it could utlize the phytotoxin of Verticillium dahliae to differentiate the different cultivar.The four methods of total protein extraction in cotton leaves, three methods of SDS-PAGE and three different concentration detaining were compared and improved. The results showed that it could make better to utilize 25 mmol L~-1, pH8.0 Tris-HCl on total protein extraction in cotton leaves. Using this method, the protein was extracted completely and efficiently. Moreover, satisfied results were achieved in SDS-PAGE. The method of modified pyruvic subside in SDS-PAGE in cotton leaves could be used to test toothful and the clear electrophoresis result could be obtained. When it was used modified pyruvic subside in SDS-PAGE, confecting different consistent separate gel, the consisitence of 10 %~12 % fit for SDS-PAGE of total protein in cotton leaves. Through SDS-PAGE, A conclusion was found that the temperature related to destaining: the higher temperature, the shorter time of destaining, within some range temperature. The experimental cultivars were the ones which were resistance cultivars: Wenwu, 9456D, X033 (Gossypium hirsutium) , sea-island cotton 7124 (G. barbadense) and susceptive cultivar: Junmian 1 and which were induced by Verticillium dahilae, another ones which were planted without pathogeny, such as Wenwu, 9456D, X033, sea-island cotton 7124, Junmianl. Depending on SDS-PAGE, it was found there was one more trap in the protein induced by Verticillium更多还原