【作者】 顾春波；

【导师】 王开运；

【作者基本信息】 山东农业大学 ， 植物检疫与农产品安全， 2010， 博士

【摘要】 本研究利用戊唑醇和多菌灵2种药剂对草莓枯萎病菌进行室内抗性选育,获得了对戊唑醇和多菌灵的高抗菌株;研究了各菌株的抗性发展规律;检测了各抗性菌株的抗药性遗传稳定性;并测定了各抗性菌株对几种常用药剂的交互抗性;系统研究了不同抗性菌株和敏感菌株在碳源、氮源、产孢能力等方面的生物学差异,不同抗性菌株和敏感菌株对不同pH值和温度的敏感性;比较了以不同浓度的戊唑醇和多菌灵处理后抗戊唑醇菌株、抗多菌灵菌株和敏感菌株的相对渗率变化情况;比较了抗戊唑醇菌株、抗多菌灵菌株和敏感菌株经不同浓度的药剂处理后,其苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)的活力随时间变化的差异;并对抗戊唑醇菌株和抗多菌灵菌株的生理生化特性进行了探讨。本研究取得了以下主要研究结果:1.草莓枯萎病菌对多菌灵和戊唑醇的抗性发展速度均较快,在含多菌灵的培养基上选育到45代,对多菌灵的抗性增加到53.91倍,在含戊唑醇的培养基上选育到36代,对戊唑醇的抗性增加到34.22倍。通过对各抗性菌株进行遗传稳定性测定,发现各菌株在无药培养基上连续转代培养8代以后,其生长速率变化较小,抗性稳定性较强,说明草莓枯萎病菌对多菌灵和戊唑醇的抗性能够通过无性繁殖稳定遗传。2.利用诱导获得的抗多菌灵和抗戊唑醇菌株,进行交互抗性试验,不同抗性菌株对其它药剂的交互抗性为:抗多菌灵菌株对甲基硫菌灵、福美双的抗性分别达29.98倍、27.58倍,交互抗性十分显著;对三唑酮、苯醚甲环唑、腈菌唑、丙环唑、戊唑醇的抗性分别达7.15倍、5.42倍、5.07倍、4.78倍、2.73倍,交互抗也较明显。抗戊唑醇菌株对三唑酮、丙环唑、烯唑醇、腈菌唑的交互抗性分别达22.21倍、14.12倍、11.91倍、10.52倍,交互抗性也十分显著;对甲基硫菌灵、苯醚甲环唑、多菌灵的抗性分别达6.96倍、4.55倍和2.12倍,交互抗性亦较明显,但总体而言,多菌灵和戊唑醇的双向交互抗性较小,戊唑醇可以作为多菌灵比较理想的轮用或替代药剂。3.采用菌落直径法和记录产孢量的方法,研究培养基中不同营养源、pH值和温度对抗性和敏感菌株的影响,以探明各抗性菌株和敏感菌株之间生理适合度的差异。发现在不同的碳源条件下,敏感菌株在蔗糖和甘油中生长最好,抗多菌灵菌株在蔗糖和麦芽糖中生长最好,抗戊唑醇菌株在甘油和麦芽糖中生长最好,敏感菌株和两抗性菌株均在蔗糖、淀粉、甘油中产孢量最大。在不同氮源条件下,敏抗菌株均在KNO3和NaNO3中生长最好,敏感菌株在酵母膏、蛋白胨、尿素中产孢量最高,抗多菌灵菌株在酵母膏、尿素、牛肉膏中产孢量最高,抗戊唑醇菌株在酵母膏、牛肉膏、蛋白胨中产孢量最高。两抗性菌株与敏感菌株对酸碱度和温度反应差异不明显。4.在葡萄糖的高渗环境和低渗环境下,两抗性菌株和敏感菌株的渗透压敏感性存在差异,抗多菌灵菌株对葡萄糖的高渗透压表现最为敏感,菌丝生长受到的抑制最为明显;在葡萄糖中渗环境下,抗戊唑醇表现最为敏感。在不同浓度的NaCl渗透环境下,两种抗性菌株和敏感菌株的生长均随着渗透压的增高而受到抑制,其中又以抗戊唑醇受到的抑制最为显著。5.以低浓度的多菌灵分别处理敏感菌株和抗多菌灵菌株,抗性菌株的相对渗率始终高于敏感菌株,而且相对渗率也随时间的延长而增大;以高浓度的多菌灵处理敏感菌株和抗多菌灵菌株后,抗性菌株和敏感菌株的相对渗率先下降后上升,并且最终抗性菌株的相对渗率要高于敏感菌株。当用不同浓度的戊唑醇处理敏感菌株和抗戊唑醇菌株后,敏感菌株始终能够释放更多的电解质,其相对渗率要高于抗性菌株。6.用多菌灵处理抗多菌灵菌株和敏感菌株,抗性菌株的苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)活性均高于敏感菌株,在药剂处理条件下,其PAL活性表现出先迅速上升后又迅速下降的趋势。用戊唑醇处理抗戊唑醇菌株和敏感菌株,抗性菌株体内的PAL活性高于敏感菌株,经药剂处理的抗性菌株和敏感菌株PAL活性也表现出先上升后下降的趋势。7.敏感菌株与抗性菌株的适应性存在差异:敏抗菌株对碳氮营养要求均不严格,离体适合度较高;多菌灵抗性菌株对渗透压的适应力、对药剂的耐受力和排泄能力较强,戊唑醇抗性菌株最差,敏感菌株居中;两抗性菌株体内的防御酶活力均高于敏感菌株。更多还原

【Abstract】 The purpose of this paper is to study the resistance of Fusarium oxysporum f.sp. fragariae to carbendazim and tebuconazole. In this study, the resistance selection of Fusarium oxysporum f.sp. fragariae to carbendazim and tebuconazole and the cross-resistance test of each resistant strain was carried out. The resistance rule of each strain was studied. The differences on some characterization such as the growth rate, carbon sources, nitrogen sources and the capacity of spore production were studied. The effect of different pH values and temperature on sensitive and resistant strain was also studied. After immersed into carbendazim solution and tebuconzaole solution with different concentrations, the conductivity and the activity of phenylalanine ammonia lyase (PAL) and peroxidase (POD) was compared. At last, the resistance mechanism was studied. And the resistance risk of Fusarium oxysporum f. sp. fragariae to carbendazim and tebuconazole was evaluated. The results could be summarized as follows:1. The studies of resistance selection showed that the resistant ratio of ZY-D which is resistant to carbendazim had reached 53.91 fold after 45-time selection, and the ZY-W which is resistant to tebuconazole reached 34.22 fold after 36-time treatment in the laboratory. After 8 successive incubations on the free-fungicide medium, the resistant strains could maintain their resistance.2. The cross-resistance of induced resistant strains to fungicides were as follows: the ZY-D had significant positive resistance to Thiophanatemethy and Thiram, the resistance ratio was 29.98 and 27.58 fold, respectively, while the resistance ratio of triadimefon, difenoconazole, myclobutanil, propiconazole and tebuconazole was 7.15, 5.42, 5.07, 4.78 and 2.73 fold; the ZY-W had significant positive resistance to triadimefon, propiconazole, diniconazole and myclobutanil, the resistance ratio was 22.21, 14.12, 11.91 and 10.52 fold, while the resistance to thiophanatemethy , difenoconazole and carbendazim was still remarkable, the resistance ratio was 6.96, 4.55 and 2.12 fold respectively. 3. By mycelia growth rate tests and counting the number of spore production, the effect of different carbon and nitrogen sources, pH value and temperature on the resistant strains and sensitive strain was determined. The results are as follows: in the different carbon sources, the sensitive strain grew fastest in glycerol and sucrose, while the ZY-D grew fastest in sucrose and maltose, and the ZY-W grew fastest in glycerol and maltose, the number of spore production of the resistant strains and the sensitive strain in sucrose, amylum and glycerol was higher than that in other carbon sources. In the different nitrogen sources, the resistant strains and the sensitive strain grew fastest in KNO3 and NaNO3. For the spore yield of sensitive strain, yeast extract, peptone and carbamide were the best nitrogen source; for the spore yield of ZY-D, yeast extract, carbamide and beef extract were the best nitrogen source; for the ZY-W, yeast extract, beef extract and peptone were the best. There was no significantly difference in the effect on the sensitive and resistant strain of pH value and temperature.4. In the medium with high and low concentration of dextrose, there was difference among ZY-D, ZY-W and sensitive strain. In the high concentration of dextrose, the ZY-D was inhibited more than other strains; while in the middle concentration of dextrose, the ZY-W was inhibited more than other strains. In different concentration of NaCl, the ZY-W was inhibited more than other strains. The colony diameter of the ZY-W had negative relationship with concentration of NaCl.5. After treated with middle and low concentration of carbendazim, the relative leakage of resistant strains is higher than that of sensitive strain. Moreover, the relative leakage had positive relation with treated time. After treated with high concentration of carbendazim, at the beginning, the relative leakage of ZY-D and sensitive strain was decreased, and then the relative leakage of both strains was increased. Furthermore, the sensitive strain could leak more electrolyte than the ZY-D in the short time. However, the relative leakage of ZY-D is higher than sensitive strain finally. After treated with different concentration of tebuconazole, the strain ZY-W could leak more electrolyte than the sensitive strain continuously.6. After treated with carbendazim, the activity of phenylalanine ammonia lyase(PAL) and peroxidase(POD) of the resistant strains was higher than that of the sensitive strain. After treated with carbendazim, the activity of PAL of the ZY-D and sensitive strain firstly increased, and then decreased. After treated with tebuconazole, the activity of PAL of the ZY-W was higher than that of sensitivity. After treated with tebuconzaole, the activity of PAL of the ZY-W and sensitive strain firstly increased, and then decreased.7. The fitness differences between sensitive and two resistant strains are as follows: the ZY-D work best in suiting NaCl and dextrose, and leaking fungicide; followed by the sensitive strain; and the ZY-W is the last one. Both resistant strains have more POD and PAL than the sensitive strain.更多还原