【作者】 王丽；

【导师】 王文桥； 刘颖超；

【作者基本信息】 河北农业大学 ， 农药学， 2010， 硕士

【摘要】 植物病原菌抗药性是植物病害化学防治中的突出问题。甲霜灵是目前防治马铃薯晚疫病中应用最为普遍的杀菌剂。本文通过检测马铃薯晚疫病菌对包括甲霜灵在内的6种内吸性杀菌剂的抗性;筛选防治晚疫病高效、与甲霜灵之间无交抗关系及抗药性风险较低的药剂;筛选对抗甲霜灵菌株增效的药剂组合;研究药剂交替使用后对晚疫病菌对甲霜灵抗性发展的影响,制订了马铃薯晚疫病菌对甲霜灵抗性治理的用药策略。主要研究结果如下:1.对薯片夹叶分离法进行了改进,使分离成功率升至100%。测定了马铃薯晚疫病菌在RSA①、RSA②和CA三种培养基上的菌落扩展和产孢能力显示,晚疫病菌在RSA①和CA上生长较快,RSA②次之;产孢量依次为RSA①>CA>RSA②,RSA①较适合马铃薯晚疫病菌的培养。晚疫病菌在CA上的菌落扩展速度与在RSA①上相当,且CA原料易得、制作简便,可用于测定药剂对晚疫病菌菌丝生长的抑制作用及离体条件下检测马铃薯晚疫病菌对杀菌剂敏感性。2.采用菌丝生长速率法(在含区分剂量药剂的CA平板上)和叶盘漂浮法分别检测了2007～2009年采自我国北方五省的马铃薯晚疫病菌对甲霜灵的抗性(分别为380个菌株和227个菌株),发现抗性频率分别为80%和74%,表明上述地区晚疫病菌对甲霜灵已普遍产生了抗性。3.采用叶盘漂浮法检测2007～2009年采自我国北方五省的马铃薯晚疫病菌对精甲霜灵、霜脲氰、烯酰吗啉、氟吗啉和嘧菌酯的敏感性,并建立了晚疫病菌对霜脲氰、烯酰吗啉、氟吗啉和嘧菌酯的敏感基线,结果显示:(1)精甲霜灵对96株晚疫病菌的EC50值为0.002 9～395.867 5μg/mL,其中抗性菌株占54.2%,中间型菌株占44.8%,敏感菌株仅占1.0%。(2)晚疫病菌对霜脲氰的敏感基线为(0.673 6±0.090 2)μg/mL,110株晚疫病菌中70%的菌株已对霜脲氰产生了抗药性,其中高抗菌株占1.8%,抗性水平最高达1 024倍。(3)晚疫病菌对烯酰吗啉、氟吗啉和嘧菌酯的敏感基线分别为(0.215 5±0.011 9)μg/mL、(0.337 4±0.019 9)μg/mL和(0.218 8±0.011 1)μg/mL,并且敏感菌株占优势,频率分别为92.2%、93.7%和88.4%,仅有少量低抗菌株存在,未检测到中抗及高抗菌株。(4)田间存在对甲霜灵和霜脲氰的双抗菌株(MRCRDSAS型)、对甲霜灵和嘧菌酯的双抗菌株(MRCSDSAR型)及对甲霜灵、霜脲氰和嘧菌酯的三抗菌株(MRCRDSAR型)频率分别为78.0%、1.1%和3.3%。4.采用叶盘法测定了19种杀菌剂对抗甲霜灵菌株的毒力,结果表明嘧菌酯?苯醚甲环唑、嘧菌酯、烯酰吗啉、双炔酰菌胺、吡唑醚菌酯、吡唑醚菌酯?代森联和唑胺菌酯有较强的抑制作用,其EC90值均小于10μg a.i/mL,其次为氟菌?霜霉威、嘧菌酯?百菌清、烯酰?锰锌、噁唑菌酮?锰锌、代森锰锌、霜脲?锰锌、噁唑菌酮?霜脲氰、百菌清和氢氧化铜,其EC90值在10～100μg a.i/mL之间,氟啶胺、霜霉威和丙森锌的相对防效较差,其EC90值均大于100μg a.i/mL。5.在河北围场县克勒沟镇围字村进行的田间药效试验结果表明,氟菌?霜霉威和双炔酰菌胺对马铃薯晚疫病具有良好的防效(88.9%和82.7%),显著高于霜脲?锰锌、烯酰吗啉、嘧菌酯、代森锰锌和百菌清的防效;在河北崇礼县狮子沟原种场进行的田间药效试验结果表明,氟菌?霜霉威对马铃薯晚疫病具有较好的防效(93.8%),其次为霜脲?锰锌和烯酰吗啉(84.7%和80.2%),且显著高于代森锰锌、嘧菌酯和百菌清的防效。6.初步评估了马铃薯晚疫病菌对双炔酰菌胺和唑胺菌酯的抗药性风险:(1)早期田间抗性检测结果显示,232株晚疫病菌中对双炔酰菌胺敏感的菌株占94.4%,低抗菌株占5.6%,未检测到中抗及高抗菌株;双炔酰菌胺与甲霜灵、霜脲氰和嘧菌酯之间不存在交互抗药性关系,但与烯酰吗啉和氟吗啉之间存在交互抗药性关系;田间抗双炔酰菌胺菌株的抗药性不能稳定遗传,且其适合度指数显著低于敏感菌株,表明晚疫病菌对双炔酰菌胺具有较低的抗性风险。(2)早期田间抗性检测结果表明,127株晚疫病菌中对唑胺菌酯敏感的菌株占91.3%,低抗菌株占8.7%,未检测到中抗及高抗菌株;唑胺菌酯与甲霜灵、霜脲氰和烯酰吗啉之间不存在交互抗药性关系,但与嘧菌酯之间存在交互抗药性关系;田间抗唑胺菌酯菌株的抗药性能稳定遗传,其适合度指数与敏感菌株相当,表明晚疫病菌对唑胺菌酯具有较高的抗性风险。7.采用脱叶法检测药剂混配的联合毒力,以Horsfall法与Wadley法评价药剂混用是否具有增效作用,结果表明,化合物A与吡唑醚菌酯、百菌清分别以5:5和4:6混配,对抗甲霜灵菌株有明显的增效作用,增效系数分别2.8和2.0;化合物B与百菌清、代森锰锌分别以9:1和3:7混配,对抗甲霜灵菌株亦有增效作用,增效系数分别为1.6和2.2。8.田间交替喷施72%霜脲?锰锌WP(1 080 g a.i./hm2)、68%精甲霜?锰锌WG(1 224 g a.i./hm2)、68.75%氟菌?霜霉威(SC 1 031 g a.i./hm2)和50%烯酰吗啉WP(375 g a.i./hm2)对马铃薯晚疫病具有良好的防控作用(防效90%以上)。检测交替用药地区晚疫病菌对甲霜灵及嘧菌酯的敏感性,发现交替用药可延缓晚疫病菌群体对甲霜灵和嘧菌酯的抗性发展。9.根据田间抗药性检测、替代甲霜灵的高效低抗药性风险药剂筛选、对甲霜灵抗性菌株增效药剂组合筛选、交替用药对甲霜灵抗性发展影响的研究结果,制定了马铃薯晚疫病菌对甲霜灵抗性治理的用药策略,即选用双炔酰菌胺、烯酰吗啉、唑胺菌酯、嘧菌酯等与甲霜灵之间无交抗关系的药剂以及氟菌?霜霉威交替使用,或将化合物A与吡唑醚菌酯、百菌清混用,化合物B与百菌清、代森锰锌混用;限制每个生长季节甲霜灵?锰锌或精甲霜灵?锰锌使用次数不超过2次。更多还原

【Abstract】 Resistance to fungicides in plant pathogens is outstanding problem in chemical control of palnt diseases. Metalaxyl is now the fungicide used most widely for control potato late blight. In the thesis, the management strategy of resistance in Phytophthora infestans was established through testing sensitivity to 6 systemic fungicides including metalaxyl, screening the fungicides with high control efficacy to potato late blight, no positively correlated resistance with metalaxyl and low risk of rensistance, screening the fungicide combinations synergistic against metalaxyl-resistant isolates of P. infestans, and studies on effects on the development of resistance to metalaxyl in P. infestans. The major result were reviewed as followed.1. The isolation method of potato slices claping leaves infected by late blight was improved, which made the ratio of successfully isolation up to 100%. The colonial spreading and sporulation of P. infestans on 3 media (RSA①, RSA②and CA) was tested. The result showed that P. infestans grows faster on paltes of RSA①and CA than on plates of RSA②. The sporulation capacity is biggest on plates of RSA①than on plates of CA and RSA②. So, RSA①is more suitable for cultivation of P. infestans. Besides, speed of colonial extension of P. infestans on CA is almost the same as that on RSA①, and the raw material of CA is easily acquired, so CA can be used for testing inhibitory activity of fungicides against mycelial growth and in vitro testing sensitivity to fungicides in P. infestans.2. Sensitivity to metalaxyl in 380 isolates and 227 isolates collected from five provinces of northern China during 2007~2009 with mycelaial growth rate on CA amended with metalaxyl of discriminatory concentration and leaf disc floating test, respectively, the result showed that the percentages of metalaxyl-resistance isolates of P. infestans was 80% and 74%, respectively. It suggests that metalaxyl-resistance of P. infestans occurred widely in the detected areas.3. Sensitivity to mefenoxam, cymoxanil, dimethomorph, flumorph and azoxystrobin of P. infestans collected from five provinces of northern China during 2007~2009 was tested through leaf disc floating test. The result showed that (1) EC50 values of mefenoxam in 96 field isolates of P. infestans were 0.002 9~395.867 5μg/mL, mefenoxam-resistant isolates accounts for 54.2%, intermediate isolates accounts for 44.8%, only 1.0% is sensitive isolates. (2) The baseline-sensitivity to cymoxanil of P. infestans was (0.673 6±0.090 2)μg/mL. 70% were resistant to cymoxanil among in 110 field isolates, 1.8% is high-resistant to cymoxanil with resistance level up to 1 024 folds. (3) The baseline-sensitivity of dimethomorph, flumorph and azoxystrobin to P. infestans was (0.215 5±0.011 9)μg/mL, (0.337 4±0.019 9)μg/mL and (0.218 8±0.011 1)μg/mL, respectively. Isolates sensitive to dimethomorph, isolates sensitive to flumorph and isolates sensitive to azoxystrobin were dominated in the population of P. infestans, account for 92.2%, 93.7% and 88.4%, respectively. A few isolates lowly resistant to the three fungicides exist, no moderately or highly resistant isolates were detected. (4) Isolates double resistant to metalaxyl and cymoxanil (MRCRDSAS) , or double resistant to metalaxyl and azoxystrobin (MRCSDSAR) or triple resistant to metalaxyl, cymoxanil and azoxystrobin (MRCRDSAR) exist in the field, accounting for 78.0%, 1.1% and 3.3%, respectively.4. The fungitoxicity of 19 fungicides to P. infestans was assessed by a leaf disc spray test. The result showed that azoxystrobin-difenoconazole, azoxystrobin, dimethomorph, mandipropamid, pyraclostrobin, pyraclostrobin-metiram and pyrametostrobin showed higher toxicity to metalaxyl-resistant isolates, Theirs EC90 values were lower than 10μg/mL. EC90 values of fluopicolide-propamocarb, azoxystrobin-chlorothalonil, dimethomorph-mancozeb, famoxadone-mancozeb, mancozeb, cymoxanil-mancozeb, famoxadone-cymoxanil, chlorothalonil and cupric hydroxide were 10~100μg/mL. And fluazinam, propamocarb, propineb showed a lower toxicity to metalaxyl-resistance isolates, theirs EC90 values were higher than 100μg/mL.5. The result of the trial on control efficacy done in Weichong showed that: control efficacy of fluopicolide-propamocarb and mandipropamid to potato late blight in field was 88.9% and 82.7%, respectively, which was significantly higher that of cymoxanil-mancozeb, dimethomorph, azoxystrobin, mancozeb and chlorothalonil. The result of the trial on control efficacy done in Chongli showed that control efficacy of fluopicolide-propamocarb to potato late blight in field was 93.8%, control efficacy of cymoxanil-mancozeb and dimethomorph was 84.7% and 80.2%, respectively, which was significantly higher that of mancozeb, azoxystrobin and chlorothalonil.6. The resistance risk of mandipropamid and pyrametostrobin to P. infestans was preliminarily assessed. (1) The result of early detection of resistance to mandipropamid showed that among 232 isolates, sensitive isolates account for 94.4%, lowly resistant isolates account for 5.6%, no moderately resistant or highly reisitant isolates were found. No positively correlated cross resistance between mandipropamid, metalaxyl and cymoxanil, but positively correlated cross resistance between mandipropamid, dimethomorph and flumorph. Resistance to mandipropamid of resistant isolates from the fields not stable and their fitness index were significantly lower than the sensitive isolates. These suggests that P. infestans has a lower risk of resistance to mandipropamid. (2) The result of early detection of resistance to pyrametostrobin showed that among 127 isolates, sensitive isolates account for 91.3%, 8.7% is lowly resistant isolates, no moderately resistant or highly reisitant isolates are found. No positively correlated cross resistance exists between pyrametostrobin, metalaxyl, cymoxanil and dimethomorph, but positively correlated cross resistance exists between pyrametostrobin and azoxystrobin. Resistance to pyrametostrobin can keep stable of resistant isolates, and their fitness index were not significantly different from that of the sensitive isolates. It suggested that P. infestans has a high risk of resistance to pyrametostrobin.7. The joint fungitoxicity of fungicides was tested by detached leaf spray test and whether the synergistic action exists between fungicides was judged by Horsfall formula and Wadley formula. The results showed that synergistic effects (SR 2.8 and 2.0) against metalaxyl isolates exist between compound A and pyraclostrobin and between compound A and chlorothalonil as mixing suspension of compound A and pyraclostrobin at ratio of 5:5, and the mixing suspension of compound A and chlorothalonil at ratio of 4:6 were applied to the detached leaves of potato. Synergistic action (SR 1.6 and 2.2) to metalaxyl-resistant isolates exists while mixing suspension of compound B and chlorothalonil at ratio of 9:1 and mixing suspension of compound B and mancozeb at ratio of 3:7 were applied to the detached leaves of potato.8. Alternate application of cymoxanil-mancozeb 72 WP (1 080 g a.i./hm2) , mefenoxam-mancozeb WG (1 224 g a.i./hm2), fluopicolide-propamocarb 68.75 SC (1 031 g a.i./hm2) and dimethomorph 50 WP (375 g a.i./hm2) in demonstration plots exhibited good control efficacy (>90%). And development of resistance to metalaxyl or azoxystrobin in the population of P. infestans was delayed through detecting the sensitivity to metalaxyl or azoxystrobin in the alternate application areas in the population of P. infestans. 9 Based on the results of detection of field resistance to fungicides, screening of fungicides with high control efficacy, low risk of resistance to the novel fungicides, screening of fungicide combinations synergistic to metalaxyl-resistant isolates, and studies on effects of alternate application of fungicides with different modes of action on development of resistance to fungicides, a strategy of management strategy of resistance to metalaxyl in P. infestans was established, that is, selecting and applying novel fungicides (such as mandipropamid, dimethomorph, pyrametostrobin, azoxystrobin, fluopicolide-propamocarb) without positive correlated cross resistance with metalaxyl in alternation, or applying the mixing suspension of compound A with pyraclostrobin or chlorothalonil, or the mixing suspension of compound B with chlorothalonil or mancozeb;limiting metalaxyl-mancozeb or mefenoxam-mancozeb within the 2 applications per season of growth.更多还原