【作者】 吴志华；

【导师】 柏连阳；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2011， 博士

【摘要】 水稻稻瘟病是由Magnaporthe oryzae引起的一种病害,是世界上重要的水稻病害之一,与纹枯病、白叶枯病被列为水稻三大病害。本研究采用菌落生长速率法,测定了2008年从湖南、湖北和广西三省水稻主产区典型病株上分离得到的50个水稻稻瘟病菌菌株对六种杀菌剂的抗药性,建立了水稻稻瘟病菌对六种杀菌剂的敏感性基线,经诱导得到抗稻瘟灵的高、低抗突变株,对参试药剂各抗性菌株抗药性的水平进行测定,对低、高抗菌株及敏感菌株中菌落生长速率进行测定和比较得到较好结果；在测定低、高抗菌株及敏感菌株致病力时采用活体接种法,比较了低、高抗菌株及敏感菌株的适合度和不同浓度的稻瘟灵处理后每个样品中的溶液用电导仪测定电导率的不同；分析高低抗、敏感菌株的可溶性蛋白质的聚丙烯酰胺凝胶电泳图谱和测定相关酶的活性进行及其五种药剂的交互抗性。经过本试验得到以下结果：1.稻瘟灵、三环唑、异稻瘟净、多菌灵、稻瘟酰胺和春雷霉素对40个菌株的EC50值分布在1.08-22.54、0.68-11.53、10.67-92.74、50.34-196.55、2.12-7.09和2.29-178.77μg/mL之间,均值为7.96、3.51、43.10、116.93、4.91和43.38μg/mL。2.稻瘟灵、三环唑、异稻瘟净、多菌灵、稻瘟酰胺和春雷霉素对均来自敏感群体、组成近正态连续抗性频次分布的40个菌株的EC50均值0.7487、0.4259、6.5729、3.4521、0.8532和1.8532μg/mL可分别作为水稻稻瘟病菌的敏感基线。3.经过实验室培养稻瘟灵药剂诱导产生了低抗突变株十个和高抗突变株一个。在浓度范围为0-400μg/mL中抗性突变株发生频率与稻瘟灵浓度呈正比,突变株的发生频率与诱导药剂浓度的对数之间存在高度正相关。4.抗突变株的抗药性能遗传下去。水稻稻瘟病菌株在第F2、F4、F6代逐渐稳定。5.稻瘟灵对十个低抗突变株EC50值在16.02-56.02μg/mL范围之内,EC50均值为36.00μg/mL,抗性水平分布在4.26-14.90范围之内,平均抗性大约为9.57；为稻瘟灵药剂对高抗突变株200M-008的EC50值(1832.10μg/mL)最大,其抗性水平(487.26)最高；没有出现中抗突变株。抗稻瘟灵可能属于质量性状的突变。6.供试敏感菌株LX003在的离、活体适合度指数和综合适合度指数方面高于抗性突变株；在抗突变株在离、活体适合度指数和综合适合度指数方面中最高的是高抗突变株200M-008。7.稻瘟灵能对水稻稻瘟病菌的细胞膜造成伤害,敏感菌株LX003和低抗菌株200M-005和高抗菌株200M-008三个菌株中,前两个菌株的细胞膜透性持续升高；高抗菌株相对来说受到的影响最小且这种能力与稻瘟灵药剂的浓度呈反比。8.水稻稻瘟病菌对稻瘟灵的敏感菌株LX003、高、低抗菌株200M-008、200M-005的相关酶在聚丙烯酰胺凝胶电泳图谱上有很大的不同。敏感菌株LX003的特征带为Rf0.716和Rf0.835酶带,高抗菌株200M-008的特征带为Rf0.452酶带。水稻稻瘟病敏感菌株LX003的Rf0.716和Rf0.835酶带低于高抗菌株200M-008和低抗菌株200M-005的Rf0.033酶带很多的量。9.水稻稻瘟病菌对稻瘟灵的敏感菌株LX003、高抗菌株200M-008与低抗菌株200M-005三个菌株在可溶性蛋白质电泳图谱上发现仅有极小的特异性不同,但抗性菌株高于敏感菌株在图谱上的表达。10.抗稻瘟灵水稻稻病菌菌株对内吸性杀菌剂异稻瘟净有负交互抗性现象,而对保护性杀菌剂三环唑、内吸性杀菌剂多菌灵、稻瘟酰胺和春雷霉素之间则无交互抗性。更多还原

【Abstract】 Rice blast which is caused by Magnaporthe oryzae is one of the most serious diseases in the world.Rice blast,rice sheath Blight and rice bacterial leaf blight are listed as rice three diseases.According to the research,the plants resistances to tric-yclazole which is protective fungicide and five systemic fungicides of isoprothio- lane, iprobenfos, carbendazim, fenoxanil, kasumin of the 50 strains of Magnaporthegrisea (Herbert) Barr gathered from rice of plants in Hunan province,Hubei province,Guangxi probince in the south of China in 2008 were determined by the mean of bacterial colonial growth-rate.By analyzi-ng the frequence, the baselines-sensitivity to above six fungicides of Magnaporthe grisea(Herbert)Barr were established. And the low and high resistam-metalaxy 1 strains of Magnaporthe grisea (Herbert) Barr have been gained with the method of isoprothiolane inducement, as well as the hereditary stabilities of all the resistant-isoprothiolane strains and the resistance levels of all the resistant- isoprothiolane strains. Besides, in the research of the colonial growth-rate,a lot of sporangium, the pathogenigity with comformity of the high and low resistant-isoprothiolane strains and the sensitive strain were compared with the path of colonial diameter,The method of count, the method of inoculation in vivo and the mean of computation, respectively. The conformity of the low and high resistant-isoprothiolane strains and the sensitive strain and the conductivity changing of isoprothiolane solution by dealing with different concentrations were analysed. As well as the polyacrylamide gel electrophoretical patterns of the esterases and the soluble proteins of the low and high resistant- isoprothiolane strains and the sensitive strain and the interactive resistances of the five systemic fungicides of resistant-isoprothiolane strains in Magnaporthe grisea (Herbert) Barr.The test results were as below:l.The EC50 values of isoprothiolane,tricyclazole,iprobenfos,carbendazim,fenoxanil and kasumin to the 40 strains of Magnaporthe grisea (Herbert) Barr ranged from 1.08μg/mL to 22.54μg/mL,0.68μg/mL to 11.53μg/mL,10.67μg/mL to 92.74μg/mL, 50.34μg/mL to 96.55μg/mL,2.12μg/mL to 7.09μg/mL and 2.29μg/mL to 178.77μg/ mL.The average values of EC50 to the 40 strains of above six fungicides were 7.9 6μg/mL,3.51μg/mL,43.10μg/mL,116.93μg/mL,4.91μg/mL and 43.38μg/mL.2.The mean EC50 values of isoprothiolane, tricyclazole,iprobenfos,carbendazim, fenoxanil and kasumin to the 40 strains,which were all from sensitive colony and composed the continuous and approximately normal distributions-resistance, respec-ti vely were 0.7487μg/mL,0.4259μg/mL,6.5729μg/mL,3.4521μg/mL,0.8532μg/mL and 1. 8532μg/mL.They could be used as the baselines-sensitivity of Magnapor the grisea (Herbert) Barr.3.10 lower resistance strains with 1 higher resistance strains were engendered by isoprothiolane experiment. when isoprothiolane focus increased within the range of from 0μg/mL to 400μg/mL, The occurrence frequency of resistant strains increased. The mutation frequency of resistant strains and the logarithm of isoprothiolane concentration is highly positive correlation.4. Resistance mutations of resistance can genetic down. Rice blast strains in F2, F4, F6 generation is stable and genetic down gradually.5. Ten low resistant strains’ EC50 of isoprothiolane ranged from 16.02μg/mL to 56.02μg/mL, and the average EC50 was 36.00μg/mL; the resistance levels of 10 low resistance strain ranged from 4.26 to 14.90 and the average resistance level were about 9.57.The EC50 of isoprothiolane of mutations 200M-008 EC50 value (1832.10μg/mL) is the largest, its resistance level (487.26) highest; Did not appear of resistant mutations. Resistant-isoprothiolane could belong to quality traits mutations.6. The vitro,vivo and compound fitness indexes of the sensitive strain,LX003 were higher than the tested resistant strains,and the highest index specie was high resistant strain,200M-008. But the three fitness indexes had no any correlationship with resistances of Magnaporthe grisea.7.The membrane of Magnaporthe grisea (Herbert) Barr could be damaged by Isoprothiolane.During the sensitive strain,LX003, the low resistant strain,200M-005and the high resistant strain,200M-008,The membrane permeability of first two st rains has increased after isoprothiolane treatment. While the high resistant strain, 200M-008 had stronger ability to resist to isoprothiolane. And this ability has the inverse ratio with the concentration of isoprothiolane.8. Some Magnaporthe grisea hot the spirit of rice LX003, low antibacterial strains of the sensitive plant 200M-005 and 200M-008 strains resistant to the esterase on electrophoresis there exist obvious differences between the specificity,LX003,the low resistant-isoprothiolane strain,200M-005,and the high resistant-isoprothiolane strain,200M-008, in Magnaporthe grisea (Herbert) Barr. Besides the sensitive strain,LX003, had the esterase-gonos of Rf0.716 and Rf0.835 while the highresistant strain 200M-008 only had the esterase-zone of Rf0.452. The quantity of the esterasezone of Rf 0.033 in the high resistant strain,200M-008, and in the low resistant strain, 200M-005,was higher than that in the sensitive strain,LX003.9.These are little significant differences in the polyacrylamide gel electrophoretical patterns of the soluble proteins between the spirit of rice LX003 strains resistant strains sensitive,200M-008and low antibacterial strains of 200-M005 3 strains in Magnaporthe grisea(Herbert)Barr.But the expression on the picture of resistant-isoprothiolane strains were higher than sensitives.10. The resistant-isoprothiolane strains in Magnaporthe grisea (Herbert) Barr had negative cross-resistance to iprobenfos,which belongs to the systemic fungicide and had no cross-resistance to tricyclazole,which belongs to the unsystemic fungicide,to carbendazim, which belongs to the systemic fungicide,to fenoxanil,which belongs to the systemic fungicide,and to kasumin, which belongs to the systemic fungicide.更多还原