江苏省核盘菌（Sclerotinia sclerotiorum）的抗药性监测及对菌核病的防治研究
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摘要
油菜菌核病是一种世界性植物病害,严重影响油菜的产量和品质。应用杀菌剂是防治油菜菌核病的主要手段。20世纪80年代以来,多菌灵是我国防治油菜菌核病的主要杀菌剂。由于油菜菌核病菌对多菌灵出现了抗药性,二甲酰亚胺类杀菌剂如扑海因、速克灵、乙烯菌核利、菌核净等在本世纪开始用于油菜菌核病防治。针对江苏省防治油菜菌核病的用药情况,本文监测了油菜菌核病菌对多菌灵、菌核净的抗药性,并研究比较了抗性菌株和敏感菌株的菌丝生长量、产菌核能力、致病力等生物学性状。结果表明抗多菌灵的油菜菌核病菌在自然界已经形成优势群体,导致多菌灵失去使用价值；通过油菜菌核病菌对菌核净的抗药性监测,首次发现江苏已经出现抗药性菌株,存在菌核净抗性风险；筛选出对菌核病菌具有极高抗菌活性的新型杀菌剂啶酰菌胺,并建立了油菜菌核病菌对该药剂的敏感性基线。
2006～2008年从江苏省采集分离1786个油菜菌核病菌菌株,以5μg/mL和100μg/mL为区分剂量,监测油菜菌核病菌对多菌灵的抗药性。监测结果表明其对多菌灵的抗性频率为29.5%。不同地区抗性频率不同,抗性范围为3.1%～54.9%。抗性菌株和敏感菌株在菌丝生长、产菌核能力和致病力方面没有显著性差异,说明田间抗性菌株和敏感菌株在田间具有相似的寄主适合度。
测定2006～2008年分离的1786个菌株对菌核净的敏感性。其中大部分对菌核净是敏感的,只有2007年从盐城地区和常州地区采集的5个菌株能够在5μg/mL正常生长,抗性倍数是敏感菌株的10倍。通过药剂驯化方法从10个出发菌株获得25个抗性突变体,抗性倍数为198～484,这些突变体的抗性水平可以认为是极高抗的菌株。因此推断油菜菌核病对菌核净的抗药性机制至少应该有两类：一类是田间发现的低抗菌株,另一类是实验室诱导的极高抗菌株。菌核净对其它的二甲酰亚胺类杀菌剂如扑海因、速克灵等有明显的正交互抗药性。田间抗性菌株和实验室诱导抗性菌株,与亲本菌株比较,菌丝生长减慢,菌核干重下降,致病力没有明显差异。所测定的实验室诱导菌株在含有2%、4%、6%、8%(w/v) NaCl的PDA培养基上的生长速率都比亲本菌株慢,田间抗性菌株和敏感菌株生长速率则没有显著差异。
120个核盘菌(Sclerotinia sclerotiorum)菌株分别采自江苏南通、盐城、泰州、扬州、宿迁、镇江、苏州等地。采用菌丝生长速率法在PDA培养基上测定各菌株对啶酰菌胺的敏感性,测定结果表明,自然界野生敏感菌株对啶酰菌胺的敏感性存在差异,120个核盘菌对啶酰菌胺的EC50值变化范围是0.028～0.408μg/mL,平均EC50为0.170±0.085μg/mL,最低抑制浓度(MIC)≤20μg/mL。表明该杀菌剂对核盘菌的菌丝生长有较强的的抑制活性。通过测定啶酰菌胺、多菌灵和菌核净的敏感性,结果表明：这三类药剂没有交互抗药性。
Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan fungal pathogen which attacks more than 400 species of higher plants, Sclerotinia stem rot continues to impose serious limitations on oilseed rape production all over the world, application of fungicides is the principal tool in most oilseed rape crops for managing Sclerotinia stem rot. A benzimidazole fungicide, carbendazim (MBC), was widely used to control this disease routinely during the 1980s in China, but wide spread control failures attributed to development of MBC resistance in S. sclerotiorum were reported in 2001 and 2002.The dicarboximide fungicides (e.g., iprodione, procymidone, vinclozolin, dimethachlon) have also been used for control of plant diseases incited by S. sclerotiorum, S. homoeocarpa and S. minor for 5 to 6 years. We detected the resistance of Sclerotinia sclerotiorum to carbendazim and dimethachlon, compared the mycelial growth, sclerotial production and pathogenicity, and determinded the cross-resistance patterns. The results indicated the wide spread of MBC resistance and occurrence of dimethachlon insensitivity in S. sclerotiorum in the oilseed rape crops of Jiangsu Province. The baseline sensitivity to boscalid was established, and the results showed that boscalid exhibited excellent activity against S. sclerotiorum and might became a new fungicide for the control of Sclerotinia stem rot.
The MBC resistance in S. sclerotiorum populations was widely spread throughout Jiangsu Province with the resistance frequency 29.5% among the 1786 collected isolates during the growing seasons of oilseed rapes between 2006 and 2008. The resistance frequencies were different among sampled cities, ranging from 3.1%-54.9%. The field MBC-resistant isolates showed comparable mycelial growth, sclerotial production and pathogenicity to the wild-type sensitive isolates, which suggested that the field MBC-resistant isolates might have sufficient parasitic fitness to compete with the field sensitive isolates in the field.
Fungal populations were collected throughout Jiangsu Province between 2006 and 2008 in order to determine their sensitivity to dimethachlon. A total of 1786 single-sclerotium isolates of S. sclerotiorum were collected and most of the isolates were considered sensitive to dimethachlon. Five isolates collected in Yancheng and Changzhou showed normal growth at 5μg/ml dimethachlon with the resistance factor≈10 compared to the sensitive isolates. Through in vitro selection for resistance to the fungicide,25 dimethachlon-resistant mutants were derived from 10 wild-type isolates of S. sclerotiorum. The resistance factors for the isolates ranged from 198 to 484, and the isolates were considered highly resistant to dimethachlon. Therefore, at least two different mechanisms of resistance seem to be involved:one that may provide a moderate resistance (insensitivity) and a second that may give a high resistance level under laboratory conditions. There was positive cross-resistance between dimethachlon and other dicarboximide fungicides, such as iprodione and procymidone in these S. sclerotiorum isolates. The field dimethachlon-insensitive and the laboratory-induced dimethachlon-resistant isolates appeared to have mycelial growth, sclerotial production and pathogenicity to comparable their wild-type parental isolates. Also, results of osmotic tests showed that there were no significant difference in mycelial radial growth between the field dimethachlon-sensitive and field dimethachlon-insensitive isolates on potato dextrose agar (PDA) plates amended with 2,4,6, or 8%(w/v) NaCl, but the laboratory-induced dimethachlon-resistant isolates grew significantly slower than their wild-type sensitive parents under all concentrations of NaCl.
120 isolates of Sclerotinia sclerotiorum were isolated from oilseed samples from Nantong, Yancheng, Taizhou, Yangzhou, Suqian, Zhenjiang and Suzhou of Jiangsu Province, where boscalid had never been used before. The sensitivities to boscalid were determined by the method of mycelial growth test on PDA medium. The results that the sensitivity of wild-type population in the nature against boscalid exhibit a certain extend diversity and the EC50 values of the compound inhibiting growth of S. sclerotiorum was ranged from 0.028～0.408μg/mL and with the mean value of 0.170±0.085μg/mL. The MIC were and 0.712±0.255μg/mL, respectively. It showed that boscalid inhibited strongly mycelial growth of S. sclerotiorum. Though the sensitivity of carbendazim, dimethachlon and boscalid, it showed that there was no cross resistance between these fungicides.
2006～2008年从江苏省采集分离1786个油菜菌核病菌菌株,以5μg/mL和100μg/mL为区分剂量,监测油菜菌核病菌对多菌灵的抗药性。监测结果表明其对多菌灵的抗性频率为29.5%。不同地区抗性频率不同,抗性范围为3.1%～54.9%。抗性菌株和敏感菌株在菌丝生长、产菌核能力和致病力方面没有显著性差异,说明田间抗性菌株和敏感菌株在田间具有相似的寄主适合度。
测定2006～2008年分离的1786个菌株对菌核净的敏感性。其中大部分对菌核净是敏感的,只有2007年从盐城地区和常州地区采集的5个菌株能够在5μg/mL正常生长,抗性倍数是敏感菌株的10倍。通过药剂驯化方法从10个出发菌株获得25个抗性突变体,抗性倍数为198～484,这些突变体的抗性水平可以认为是极高抗的菌株。因此推断油菜菌核病对菌核净的抗药性机制至少应该有两类：一类是田间发现的低抗菌株,另一类是实验室诱导的极高抗菌株。菌核净对其它的二甲酰亚胺类杀菌剂如扑海因、速克灵等有明显的正交互抗药性。田间抗性菌株和实验室诱导抗性菌株,与亲本菌株比较,菌丝生长减慢,菌核干重下降,致病力没有明显差异。所测定的实验室诱导菌株在含有2%、4%、6%、8%(w/v) NaCl的PDA培养基上的生长速率都比亲本菌株慢,田间抗性菌株和敏感菌株生长速率则没有显著差异。
120个核盘菌(Sclerotinia sclerotiorum)菌株分别采自江苏南通、盐城、泰州、扬州、宿迁、镇江、苏州等地。采用菌丝生长速率法在PDA培养基上测定各菌株对啶酰菌胺的敏感性,测定结果表明,自然界野生敏感菌株对啶酰菌胺的敏感性存在差异,120个核盘菌对啶酰菌胺的EC50值变化范围是0.028～0.408μg/mL,平均EC50为0.170±0.085μg/mL,最低抑制浓度(MIC)≤20μg/mL。表明该杀菌剂对核盘菌的菌丝生长有较强的的抑制活性。通过测定啶酰菌胺、多菌灵和菌核净的敏感性,结果表明：这三类药剂没有交互抗药性。
Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan fungal pathogen which attacks more than 400 species of higher plants, Sclerotinia stem rot continues to impose serious limitations on oilseed rape production all over the world, application of fungicides is the principal tool in most oilseed rape crops for managing Sclerotinia stem rot. A benzimidazole fungicide, carbendazim (MBC), was widely used to control this disease routinely during the 1980s in China, but wide spread control failures attributed to development of MBC resistance in S. sclerotiorum were reported in 2001 and 2002.The dicarboximide fungicides (e.g., iprodione, procymidone, vinclozolin, dimethachlon) have also been used for control of plant diseases incited by S. sclerotiorum, S. homoeocarpa and S. minor for 5 to 6 years. We detected the resistance of Sclerotinia sclerotiorum to carbendazim and dimethachlon, compared the mycelial growth, sclerotial production and pathogenicity, and determinded the cross-resistance patterns. The results indicated the wide spread of MBC resistance and occurrence of dimethachlon insensitivity in S. sclerotiorum in the oilseed rape crops of Jiangsu Province. The baseline sensitivity to boscalid was established, and the results showed that boscalid exhibited excellent activity against S. sclerotiorum and might became a new fungicide for the control of Sclerotinia stem rot.
The MBC resistance in S. sclerotiorum populations was widely spread throughout Jiangsu Province with the resistance frequency 29.5% among the 1786 collected isolates during the growing seasons of oilseed rapes between 2006 and 2008. The resistance frequencies were different among sampled cities, ranging from 3.1%-54.9%. The field MBC-resistant isolates showed comparable mycelial growth, sclerotial production and pathogenicity to the wild-type sensitive isolates, which suggested that the field MBC-resistant isolates might have sufficient parasitic fitness to compete with the field sensitive isolates in the field.
Fungal populations were collected throughout Jiangsu Province between 2006 and 2008 in order to determine their sensitivity to dimethachlon. A total of 1786 single-sclerotium isolates of S. sclerotiorum were collected and most of the isolates were considered sensitive to dimethachlon. Five isolates collected in Yancheng and Changzhou showed normal growth at 5μg/ml dimethachlon with the resistance factor≈10 compared to the sensitive isolates. Through in vitro selection for resistance to the fungicide,25 dimethachlon-resistant mutants were derived from 10 wild-type isolates of S. sclerotiorum. The resistance factors for the isolates ranged from 198 to 484, and the isolates were considered highly resistant to dimethachlon. Therefore, at least two different mechanisms of resistance seem to be involved:one that may provide a moderate resistance (insensitivity) and a second that may give a high resistance level under laboratory conditions. There was positive cross-resistance between dimethachlon and other dicarboximide fungicides, such as iprodione and procymidone in these S. sclerotiorum isolates. The field dimethachlon-insensitive and the laboratory-induced dimethachlon-resistant isolates appeared to have mycelial growth, sclerotial production and pathogenicity to comparable their wild-type parental isolates. Also, results of osmotic tests showed that there were no significant difference in mycelial radial growth between the field dimethachlon-sensitive and field dimethachlon-insensitive isolates on potato dextrose agar (PDA) plates amended with 2,4,6, or 8%(w/v) NaCl, but the laboratory-induced dimethachlon-resistant isolates grew significantly slower than their wild-type sensitive parents under all concentrations of NaCl.
120 isolates of Sclerotinia sclerotiorum were isolated from oilseed samples from Nantong, Yancheng, Taizhou, Yangzhou, Suqian, Zhenjiang and Suzhou of Jiangsu Province, where boscalid had never been used before. The sensitivities to boscalid were determined by the method of mycelial growth test on PDA medium. The results that the sensitivity of wild-type population in the nature against boscalid exhibit a certain extend diversity and the EC50 values of the compound inhibiting growth of S. sclerotiorum was ranged from 0.028～0.408μg/mL and with the mean value of 0.170±0.085μg/mL. The MIC were and 0.712±0.255μg/mL, respectively. It showed that boscalid inhibited strongly mycelial growth of S. sclerotiorum. Though the sensitivity of carbendazim, dimethachlon and boscalid, it showed that there was no cross resistance between these fungicides.
引文
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