致病疫霉对缬菌胺敏感基线的建立及抗性风险评估

为建立致病疫霉Phytophthora infestans (Mont.) de Bary对缬菌胺的敏感基线，采用菌丝生长速率法测定了从河北省、黑龙江省、内蒙古自治区、贵州省和四川省未使用过缬菌胺的地区采集分离的105个致病疫霉菌株对缬菌胺的敏感性；为明确致病疫霉对缬菌胺产生抗性突变体的难易程度，进行了紫外诱导和药剂驯化试验；为明确缬菌胺与常用药剂之间的交互抗性，测定了8个抗缬菌胺突变体及其6个亲本敏感菌株对6种常用杀菌剂的敏感性。结果表明:105株致病疫霉对缬菌胺的EC₅₀值范围为0.0594~0.159 mg/L，平均EC₅₀值为(0.102 ± 0.024) mg/L，不同敏感性菌株的频率呈连续单峰曲线分布，未发现敏感性下降的亚群体，因此可将缬菌胺对105株致病疫霉的平均EC₅₀值作为致病疫霉对缬菌胺的敏感基线；通过紫外诱变敏感菌株菌丝体获得了4个抗缬菌胺的突变体，其抗性水平介于 3.1~14.9倍之间，突变频率为0.54%，通过紫外照射敏感菌株孢子囊悬浮液获得了2个抗性水平分别为8.1倍和8.2倍的抗性突变体，突变频率为1.33 × 10?7；通过在含缬菌胺的黑麦蔗糖琼脂培养基上继代培养敏感菌株11代，获得2个抗性水平分别为3.1倍和9.4倍的抗性突变体。缬菌胺与烯酰吗啉和双炔酰菌胺存在交互抗性，与氟吡菌胺、嘧菌酯、甲霜灵和霜脲氰不存在交互抗性。初步推测致病疫霉对缬菌胺具有低到中等抗性风险，建议在生产上将缬菌胺与其他类型杀菌剂交替或混合使用，以延缓致病疫霉对缬菌胺抗性的产生。     

辣椒疫霉产毒缺陷与抗药性突变体筛选及其遗传特性

采用MNNG诱变与自交（S1－S3）纯合的方法,从辣椒疫霉的游动孢子群体中,筛选出1株带有抗霜脲氰标记的产毒缺陷突变体、2株抗甲霜灵的产毒突变体。产毒缺陷菌体和产毒菌株均对茄门甜椒致病,但产毒缺陷菌株的致病力降低一半,证明辣椒疫霉的致病显症过程与毒素作用有关,病菌毒素是重要的致病因子。此外,突变菌株的抗药性和产毒缺陷表型在无性游动孢子后代和有性孵孢子后代群体中均可稳定遗传。在姐妹配对F2代卵孢子群体中,辣椒疫霉毒素产生和对甲霜灵的抗性为不完全显性基因所控制,而对霜脲氰的抗性为完全显性基因所控制。     

致病疫霉抗药性、交配型和适合度

 在离体条件下,我国马铃薯和番茄上62株致病疫霉中对甲霜灵和恶霜灵抗性菌株占11.29%,平均抗性水平15022倍和24733倍,未发现抗霜脲氰菌株和抗烯酰吗啉菌株。在活体条件下,比利时马铃薯上66株致病疫霉中对甲霜灵和恶霜灵抗性菌株占27.3%,河北省围场、崇礼马铃薯上217株中对甲霜灵和恶霜灵抗性菌株分别占29.0%和32.7%,河北省徐水番茄上88株中仅发现1株抗甲霜灵和恶霜灵。比利时马铃薯上的66株中4株为A2交配型,占6.1%,1株交配型为A1A2,抗甲霜灵。围场、崇礼马铃薯上的73株中6株为A2交配型,占8.2%,6株A2交配型中3株抗甲霜灵和恶霜灵。徐水番茄上的4 4株中3株A2交配型占6.8%。致病疫霉对甲霜灵和恶霜灵具有交互抗性,而2药与霜脲氰或烯酰吗啉之间无交互抗性关系。由于抗性菌株的产孢能力明显较高,田间抗甲霜灵和恶霜灵菌株明显比中间型菌株和敏感菌株的适合度高。     

“霜霉威(Propamocarb)”“甲霜灵(Metalaxyl)”对我国辣椒疫霉菌作用方式比较及交互抗性的研究

通过霜霉威(Propamocarb商品名普力克)、甲霜灵(Metalaxyl商品名瑞毒霉)对辣椒疫霉菌(Phytophthora capsici)6个菌株作用方式的体外测定结果表明,霜霉威浓度在2000ppm时对P. capsici孢子囊和卵孢子的形成,游动孢子的释放、游动及休止孢萌发、菌丝体的生长没有明显的抑制作用。而甲霜灵对P. capsici的各种孢子的产生及菌丝体的生长有较强的抑制作用,对菌丝体生长抑制的Ec_(50)和Ec_(95)分别为0.5596ppm和2.1511ppm;对孢子囊形成抑制的Ec_(50)和Ec_(95)分别为0.1520ppm和15.0032ppm。0.1ppm的甲霜灵可显著抑制卵孢子的生成。但500ppm的甲霜灵对于游动孢子的释放和休止孢萌发没有明显的抑制作用。对霜霉威和甲霜灵的体内活性试验结果表明,800ppm的霜霉威对辣椒疫霉菌的内吸保护防效达94％,1.0ppm的甲霜灵对该病的防效达100％。室内初步试验结果表明,甲霜灵与霜霉威对P. capsici没有交互抗性。     

疫霉菌对甲霜灵抗性的研究现状

疫霉菌对甲霜灵抗性的研究现状王源超，郑小波，陆家云（南京农业大学植保系２１００９５）甲霜灵（ｍｅｔａｌａｘｙｌ）属酰基丙氨酸类杀菌剂，１９７７年由瑞士汽巴一嘉基公司生产，用以防治包括疫霉菌在内的卵菌所致植物病害，具强内吸性和治疗、保护作用。该药剂最早...     

“霜霉威(Propamocarb)”“甲霜灵(Metalaxyl)”对我国辣椒疫霉菌作用方式比较及交互抗性的研究

 通过霜霉威(Propamocarb商品名普力克)、甲霜灵(Metalaxyl商品名瑞毒霉)对辣椒疫霉菌(Phytophthora capsici)6个菌株作用方式的体外测定结果表明,霜霉威浓度在2000ppm时对P. capsici孢子囊和卵孢子的形成,游动孢子的释放、游动及休止孢萌发、菌丝体的生长没有明显的抑制作用。而甲霜灵对P. capsici的各种孢子的产生及菌丝体的生长有较强的抑制作用,对菌丝体生长抑制的Ec₅₀和Ec₉₅分别为0.5596ppm和2.1511ppm;对孢子囊形成抑制的Ec₅₀和Ec₉₅分别为0.1520ppm和15.0032ppm。0.1ppm的甲霜灵可显著抑制卵孢子的生成。但500ppm的甲霜灵对于游动孢子的释放和休止孢萌发没有明显的抑制作用。对霜霉威和甲霜灵的体内活性试验结果表明,800ppm的霜霉威对辣椒疫霉菌的内吸保护防效达94%,1.0ppm的甲霜灵对该病的防效达100%。室内初步试验结果表明,甲霜灵与霜霉威对P. capsici没有交互抗性。     

江西省辣椒疫霉病菌的交配型及对霜脲氰敏感性的测定

由辣椒疫霉病菌Phytophthora capsici引起的辣椒疫病是辣椒生产上一种重要病害,在我国各地均有发生.辣椒疫霉是一种异宗配合的卵菌.异宗配合的疫霉菌的不同交配型菌株成对培养时,可以形成有性器官并发生基因重组,使该种病菌获得更强的生存能力、致病力和更广泛的寄主范围.因此,研究疫霉交配型在理论和实践上均具有重要意义.霜脲氰(cymoxanil)由美国杜邦公司开发,用于卵菌纲病害的防治.自投入市场使用,至今未发现在自然条件下关于疫霉菌对霜脲氰抗药性报道.     

重庆地区辣椒疫霉对氟吡菌胺的敏感性及抗性突变体的生物学性状

为明确重庆地区辣椒疫霉对氟吡菌胺的抗性风险,采用菌丝生长速率法测定了采自重庆未使用过氟吡菌胺地区的110株辣椒疫霉菌株对氟吡菌胺的敏感性,并对辣椒疫霉抗氟吡菌胺突变体的诱导方法及抗性突变体的主要生物学性状进行了研究。结果表明:110株辣椒疫霉对氟吡菌胺的EC₅₀平均值为(0.32 ± 0.11) μg/mL,不同菌株的敏感性频率呈连续单峰曲线分布,未出现敏感性明显下降的亚群体,因此可将该EC₅₀平均值作为重庆地区辣椒疫霉对氟吡菌胺田间抗性监测的敏感基线。通过紫外诱导菌丝体的方法,共获得3株可稳定遗传的抗氟吡菌胺突变体,抗性倍数介于69.5~98.5之间,突变频率为0.86%;抗性突变体BS11-5-1与亲本菌株BS11-5在菌丝生长速率、温度适应性、产孢子囊能力及致病力方面均无显著差异,而抗性突变体JLP11-4-2和JLP11-4-3在菌丝生长速率、温度适应性、产孢子囊能力及致病力方面均显著低于亲本菌株JLP11-4;不同抗性突变体对渗透压的敏感性与亲本菌株之间均存在不同程度差异;3个抗性突变体对Biolog PM1中95种碳源的利用情况与亲本菌株基本相似。交互抗性测定表明,辣椒疫霉抗氟吡菌胺突变体对甲霜灵、霜脲氰、烯酰吗啉、丁吡吗啉及嘧菌酯之间均不存在交互抗性,建议可将氟吡菌胺与上述几种杀菌剂交替或混合使用。     

葡萄霜霉菌对仿生杀菌剂缬霉威的敏感基线及其抗性突变体生物学性状的研究

 本文对葡萄霜霉菌对缬霉威的抗性风险评估进行了研究。采集尚未使用过缬霉威的105个葡萄霜霉菌株,建立其对缬霉威的敏感基线。葡萄霜霉菌对缬霉威的有效抑制中浓度EC₅₀为0.04～0.60 μg·mL^-1,其敏感性频率符合正态分布,表明这105株葡萄霜霉菌对缬霉威均表现敏感。取其中10株敏感菌的孢子囊经紫外线(UV)诱导,获得4个缬霉威抗性突变体,突变频率为6.8×10^-7。将这10株葡萄霜霉菌孢子囊在叶片上进行缬霉威药剂驯化处理获得7个抗性突变体。11个抗性突变体的EC₅₀值为4.37～14.11 μg·mL^-1,抗性水平17.4～71.6。在未用药处理的健康葡萄叶片上继代培养10代后,这11个抗性突变体抗性水平保持在17.1～71.0。其中药剂驯化获得的7个抗性突变体和1个紫外诱导获得的抗性突变体的抗性水平均表现稳定遗传。但是,其余3个紫外诱导获得的抗性突变体抗性水平明显下降。与各亲本菌株进行比较,11个抗性突变体的潜育期、侵染率、病斑扩展速度和产孢能力都有显著不同。其中2个药剂驯化获得的抗性突变体和3个紫外诱导获得的抗性突变体表现潜育期变长,2个药剂驯化抗性突变体和1个紫外诱导抗性突变体表现侵染率下降。表明在一些情况下,葡萄霜霉菌抗性突变体的潜伏期和侵染率会发生改变。药剂驯化获得的抗性突变体产孢能力存在显著差异,但与抗性突变与否无关。缬霉威与嘧菌酯、甲霜灵、烯酰吗啉和霜脲氰之间无交互抗药性。推测:葡萄霜霉菌对缬霉威为中到高等抗性风险,今后应定期对葡萄霜霉菌对缬霉威的抗性发生动态进行监测。     

掘氏疫霉抗土菌灵突变体的诱导及其特性研究

掘氏疫霉Phytophthora drechsleri是引起黄瓜疫病的主要病原菌。采用紫外线诱导获得了20株掘氏疫霉抗土菌灵的突变体,并对其生物学特性进行了研究。结果表明,掘氏疫霉对土菌灵 的抗性突变频率高于对甲霜灵的抗性突变频率,但其抗性水平低于抗甲霜灵突变体,在1.3~4.3倍 之间;掘氏疫霉对土菌灵的抗性在继代培养和常温保存过程中稳定,突变体的生长速率与亲本敏感 菌株相比发生了不同程度的变化;此外,掘氏疫霉抗土菌灵突变体对甲霜灵和烯酰吗啉无交互抗性。     

稻瘟病菌对稻瘟灵抗性遗传研究

在离体条件下就稻瘟病菌对稻瘟灵抗性的诱导、抗性水平和遗传进行了研究。结果表明,供试３个小种（ＺＡ_４９、ＺＦ_１和ＺＤ_１）４个菌株分别经稻瘟灵５０μｇ／ｍｌ、１００μｇ／ｍｌ和稻瘟灵１００μｇ／ｍｌ＋亚硝基胍０.５μｇ／ｍｌ的３种处理诱变,均得到了抗稻瘟灵突变株,在含稻瘟灵的培养基中加入诱变剂亚硝基胍可显著提高稻瘟病菌对稻瘟灵抗性的突变率。上述３个不同处理获得的突变株的抗性水平相似,为野生型亲本的２.８～８.８倍。突变株对稻瘟灵的抗性在单分生孢子无性系后代可以稳定遗传。     

马铃薯晚疫病菌对甲霜灵和霜脲氰的敏感性检测

2003 ~2004年从内蒙古和黑龙江省共采集分离了127株马铃薯晚疫病菌Phytophthora infestans,并在离体条件下测定了这些菌株对甲霜灵和霜脲氰的敏感性。结果表明:在2003年分离的内蒙菌株、2003年及2004年分离的黑龙江菌株中对甲霜灵敏感(MS)的菌株频率分别为86.2%、13.8%、17.8%;虽然霜脲氰在这些地区使用近10年,但病菌群体对该药剂的敏感性没有下降,EC50值分布于0.10 ~0.35 μg/mL之间,平均EC50值为0.20(±0.06)μg/mL,不同敏感性的菌株频率呈连续的单峰曲线分布,未出现敏感性下降的抗药性亚群体,可作为马铃薯晚疫病菌对霜脲氰抗药性监测的敏感性基线;此外,马铃薯晚疫病菌对甲霜灵和霜脲氰之间没有交互抗药性,霜脲氰对抗甲霜灵和野生敏感菌株具有相同的活性。     

棉铃疫病菌对甲霜灵的抗药性风险研究

在实验室条件下,对分离自安徽省芜湖、无为、宣城、望江、肥东、寿县、滁州等7个地区的棉铃疫病菌(Phytophthora boehmeriae Sawada)对甲霜灵的抗药性进行了测定。结果显示,甲霜灵对各供试菌株的EC50分布为0.008 3～0.079 1μg／mL,平均为0.026 1 μg／mL,远小于1 μg／mL,且供试菌株在含甲霜灵质量浓度为1μg／mL的LBA平板上菌丝生长几乎完全被抑制,表明各供试菌株对甲霜灵十分敏感,但其敏感程度地区间存在较大差异。通过室内药剂直接诱变,获得抗甲霜灵菌株,经抗性测定结果表明,抗性菌株的抗性水平较其敏感亲本菌株高13 000倍以上,提示安徽省棉铃疫病菌对甲霜灵具有潜在的抗药性风险。     

抗氟吡菌胺辣椒疫霉菌株的诱导及其生物学特性的研究

 采用菌丝生长速率法, 测定了采自泰安、平谷、杭州和昆明4个地区的42株辣椒疫霉病菌对氟吡菌胺的敏感性, 结果表明, 其EC₅₀值在0.618 ~0.927 μg·mL^-1之间, 平均EC₅₀ =(0.743±0.067 7) μg·mL^-1。 42个菌株对氟吡菌胺的敏感性分布呈单峰曲线, 未出现抗性的病原菌亚群体, 可将其单峰曲线作为辣椒疫霉对氟吡菌胺的敏感性基线。采用药剂驯化和紫外照射对辣椒疫霉敏感菌株TA进行诱变处理, 获得了2株辣椒疫霉抗氟吡菌胺菌株;利用菌落直径法测定抗氟吡菌胺菌株的遗传稳定性、菌丝生长速率及对其它杀菌剂的交互抗性;通过活体叶盘法测定抗氟吡菌胺菌株和敏感菌株的致病力及产孢子囊能力, 分析抗性和敏感菌株之间生物学特性差异。结果表明, 敏感菌株TA经过氟吡菌胺 48代连续汰选, 其抗性达58.0倍, 获得中抗菌株TA-R;紫外照射获得了抗性达260.6倍的高抗菌株TA-UV, 且其抗性均能稳定遗传;TA-R和TA-UV对甲霜灵、霜脲氰表现出正交互抗性, 对烯酰吗啉、百菌清、代森锰锌和丙森锌无交互抗性;抗氟吡菌胺菌株TA-R、TA-UV与敏感菌株在活体叶盘上的致病力、离体产孢能力均差异不显著(P＜0.05), 但其菌丝生长速率、菌丝干重均低于敏感菌株。     

Biological and genetic characterization of Phytophthora capsici mutants resistant to flumorph

Baseline sensitivity to flumorph, a carboxylic acid amide (CAA) fungicide used to control some oomycetes, was examined using 83 Phytophthora capsici isolates, resulting in a unimodal distribution of effective concentration for 50% inhibition of mycelial growth ranging from 0·716 to 1·363, with a mean of 1·033 ± 0·129 μg mL^?1. To assess the potential risk of developing flumorph resistance, 13 flumorph‐resistant mutants of P. capsici were obtained using ultraviolet irradiation. Most of these mutants and their progeny had high levels of fitness, including mycelial growth, sporulation and virulence. The resistance to flumorph changed slightly, either increasing or decreasing, after 10 transfers on agar media. Cross‐resistance was found between flumorph and other CAA fungicides (dimethomorph and iprovalicarb), but not between flumorph and non‐CAA fungicides (cymoxanil, metalaxyl, azoxystrobin and cyazofamid). To investigate the genetics of the flumorph resistance, 619 progeny were obtained by self‐crossing and sexual hybridization. Segregation of sensitivity to fungicide was measured as a ratio of sensitive (S) to resistant (R) isolates. Segregation of the progeny, from self‐crossed isolate PCAS1 (flumorph resistant), was 1:15 in the first generation; and 0:1 or 1:15 in the second generation. In sexual hybridization, segregation of progeny was 0:1 and 1:7 for R × R hybridization; and 1:3 for R × S hybridization. Therefore, the resistance of P. capsici against flumorph was controlled by two dominant genes.     

Laboratory studies to assess the risk of development of resistance to zoxamide

Laboratory studies were conducted to evaluate the risk of developing field resistance to zoxamide, a new Oomycete fungicide which acts on microtubules. Zoxamide, metalaxyl and dimethomorph were compared with respect to the ease with which fungicide‐resistant mutants could be isolated and their level of resistance. Attempts to generate mutants of Phytophthora capsici and P infestans with resistance to zoxamide by mycelial adaptation on fungicide‐amended medium were unsuccessful. Similarly, changes in sensitivity to zoxamide were small (resistance factors ≤2.2) in mutants of P capsici isolated by chemical mutagenesis of zoospore cysts. In parallel experiments with metalaxyl, highly resistant mutants were obtained using both adaptation (P capsici or P infestans) and chemical mutagenesis (P capsici). For dimethomorph, chemical mutagenesis (P capsici) yielded moderately resistant mutants (maximum resistance factor = 20.9), and adaptation (P capsici or P infestans) did not induce resistance. It is proposed that failure to isolate mutants resistant to zoxamide results from the diploid nature of Oomycete fungi and the likelihood that target‐site mutations would produce a recessive phenotype. Our studies suggest that the risk of a highly resistant pathogen population developing rapidly in the field is much lower for zoxamide than for metalaxyl. However, as with any site‐specific fungicide, appropriate precautions against resistance development should be taken. © 2001 Society of Chemical Industry     

Resistance of Phytophthora capsici to metalaxyl in plastic‐house capsicum crops in southern Italy*

In Calabria (southern Italy), control of crown and root rot of capsicum caused by Phytophthora capsici has relied primarily on soil drenches of metalaxyl. However, severe outbreaks occur every year in glasshouse crops, in which the practice of using plastic mulch and furrow irrigation favours the disease. Single‐hypha isolates of P. capsici collected in Calabria in 1992/1998 were tested in vitro for their level of sensitivity to metalaxyl. Isolates of other species of Phytophthora were used as reference. Fungicide sensitivity was determined by plating mycelial plugs onto potato dextrose agar amended with metalaxyl, at final concentrations ranging from 0.1 to 1000μg mL^?1 a.s. Inhibition of radial growth (%) was determined when colonies on unamended medium had covered approximately two‐thirds of the plate. The ED₅₀ values for inhibition of mycelial growth of P. capsici isolates ranged from 1.41 to44.6μg mL^?1 a.s. More than 80% of the P. capsici isolates from commercial plastic‐house crops in Calabria showed a moderate level of resistance as they were inhibited less than 60% at 5 μg mL^?1 but more than 60% at 100μg mL^?1     

防治大豆疫霉根腐病的药剂筛选

在离体条件下测定了 8种内吸性杀菌剂对大豆疫霉菌菌丝生长的抑制作用。甲霜灵、甲霜灵锰锌、安克锰锌、杀毒矾、霜脲锰锌、克露和加瑞农为有效杀菌剂 ,霜霉威不具有抑菌效果。甲霜灵的 EC50 值为0 .4 870 μg/ m L,抑菌效果最好 ,安克锰锌、甲霜灵锰锌的 EC50 值分别为 1.4 2 6 1μg/ m L和 1.76 97μg/ m L,效果次之。在活体条件下 ,甲霜灵和甲霜灵锰锌能完全抑制大豆疫霉菌对感病大豆品种的侵入 ,并具有较长的药效期 ,安克锰锌、杀毒矾和霜霉威虽然具有明显的防治效果但药效期很短。大豆疫霉菌菌株间对甲霜灵锰锌的敏感性存在差异 ,但这种差异与菌株来源没有相关性 ,EC50 平均值为 1.830 2μg/ m L ,没有发现耐药性菌株。在我国黑龙江省目前缺乏有效抗病品种的情况下 ,建议应用甲霜灵锰锌防治大豆疫霉根腐病。     

Sensitivity ofFusarium graminearum to fungicide JS399-19:In vitro determination of baseline sensitivity and the risk of developing fungicide resistance

The baseline sensitivity ofFusarium graminearum Schwade [teleomorph =Gibberella zeae (Schweinitz) Petch] to the fungicide JS399-19 (development code no.) [2-cyano-3-amino-3-phenylacrylic acetate] and the assessment of risk to JS399-19 resistancein vitro are presented. The mean EC₅₀ values for JS399-19 inhibiting mycelial growth of three populations of wild-typeF. graminearum isolates were 0.102±0.048, 0.113±0.035 and 0.110±0.036 μg ml⁻¹, respectively. Through UV irradiation and selection for resistance to the fungicide, we obtained a total of 76 resistant mutants derived from five wild-type isolates ofF. graminearum with an average frequency of 1.71 × 10⁻⁷% and 3.5%, respectively. These mutants could be divided into three categories of resistant phenotypes with low (LR), moderate (MR) and high (HR) level of resistance, determined by the EC₅₀ values of 1.5–15.0 μg ml⁻¹, 15.1–75.0 μg ml⁻¹ and more than 75.0 μg ml⁻¹, respectively. There was no positive cross-resistance between JS399-19 and fungicides belonging to other chemical classes, such as benzimidazoles, ergosterol biosynthesis inhibitors and strobilurins, suggesting that JS399-19 presumably has a new biochemical mode of action. Although the resistant mutants appeared to have comparable pathogenicity to their wild-type parental isolates, they showed decreased mycelial growth on potato-sucrose-agar plates and decreased sporulation capacity in mung bean broth. Nevertheless, most of the resistant mutants possessed fitness levels comparable to their parents and had MR or HR levels of resistance. As these studies yielded a high frequency of laboratory resistance inF. graminearum, appropriate precautions against resistance development in natural populations should be taken into account. http://www.phytoparasitica.org posting August 7, 2008.