【作者】 陈雨；

【导师】 周明国；

【作者基本信息】 南京农业大学 ， 农药学， 2009， 博士

【摘要】 氰烯菌酯(2-氰基-3-氨基-3-苯基丙烯酸乙酯,试验号：JS399-19)是由国家南方农药创制中心江苏基地最新合成的一种对镰孢菌具有较高活性的化合物,特别是对禾谷镰孢菌(Fusarium graminearum)菌丝生长具有强烈的抑制活性,显示了该化合物具有防治麦类赤霉病的应用前景。在新型杀菌剂进入市场之前研究其作用方式及抗药性遗传,对科学制定该杀菌剂的开发和应用策略具有重要意义。离体条件下,采用菌丝生长测定法测定该药剂对禾谷镰孢菌抗性菌株和敏感菌株的生长抑制活性；同时采用孢子萌芽测定法测定了其对禾谷镰孢菌分生孢子萌芽的影响。结果表明,氰烯菌酯能够强烈地抑制禾谷镰孢菌敏感菌株菌丝的生长,ECso值分布在0.092～0.141μg/mL之间；并可降低敏感菌株分生孢子的萌发速率,以及影响其萌发的方式,使芽管从分生孢子基部和中间细胞萌发的比率增加；同时氰烯菌酯使敏感菌株分生孢子膨大畸形,并使其芽管肿胀、扭曲,明显抑制其芽管的伸长生长,但对抗性菌株的抑制作用和致畸作用不明显。氰烯菌酯对禾谷镰孢菌菌体呼吸的抑制作用较弱,同时也几乎不影响菌体丙酮酸的合成量,说明该药剂很可能不直接作用于禾谷镰孢菌的呼吸代谢。将3个抗氰烯菌酯的禾谷镰孢菌(Fusarium graminearum Schw.)菌株分别在含氯酸盐的MMC培养基上培养,共获得了50个硝酸盐利用缺陷突变体(nit)。比较了抗性菌株的nit突变体与亲本在无性和有性阶段的主要生物学性状。抗性菌株的nit突变体与亲本在菌落生长速率、培养性状和致病性方面没有显著差异；但某些突变体中,产孢量和产子囊壳能力方面存在一定差异。此外,禾谷镰孢菌对氯酸盐和氰烯菌酯之间没有交互抗药性,且抗性可以稳定遗传。因此,可以将nit作为遗传标记来研究禾谷镰孢菌对氰烯菌酯的抗药性遗传学。离体条件下,通过紫外光诱变和药剂驯化从5个野生敏感的亲本菌株中共获得了86个对氰烯菌酯的抗性菌株,紫外光诱变产生抗性的频率在1.25×10-7-2.29×10-7之间,而通过药剂驯化产生抗性的频率在2.5-6.0%之间,说明发生抗性的频率偏高,且大部分抗性菌株都属于中等抗性水平(MR)或高等抗性水平(HR)。虽然禾谷镰孢菌对氰烯菌酯发生抗药性以后,不会改变其营养亲和能力,但是抗药性基因不能通过菌丝融合传递给另一个菌株或发生的概率极低,这将不利于对氰烯菌酯的抗性群体的发展。因此,菌丝融合在禾谷镰孢菌对氰烯菌酯的抗药性群体发展中的作用较小。选择了43个单孢分离的田间野生敏感菌株和45个实验室诱导的对氰烯菌酯的抗性菌株,测定这些菌株对氰烯菌酯的敏感性,将其划分为敏感(S)、中抗(MR)和高抗(HR)3个水平(根据其对氰烯菌酯的敏感性)。所选的3个禾谷镰孢菌(2043,Y2021B和YNT,分别代表S,MR和HR)对氰烯菌酯的敏感性在自交和无性繁殖过程中可以稳定遗传。从这些菌株中,随机选取8个代表这三个敏感性水平的菌株用于抗药性的遗传研究(F1代法)。并以硝酸盐营养缺陷型突变体(nit)作为遗传标记,来确认来自杂交的单个子囊壳。按照S×S、S×HR、MR×HR、HR×HR、MR×S等共设计了6个杂交组合,对各杂交后代对氰烯菌酯的敏感性测定发现, S×HR、MR×HR和MR×S的杂交后代出现了1：1的分离比例。在杂交组合S×S和HR×HR的后代中均未出现除双亲表现型以外的重组性个体(即未出现抗性水平的分离)。因此,笔者认为,禾谷镰孢菌对氰烯菌酯的抗药性是由单个基因控制的,该基因的不同突变类型很可能导致不同水平的抗药性(MR和HR),抗药性不受修饰基因或细胞质遗传因子的影响。以硝酸盐营养缺陷型突变体(nit)和多菌灵抗性为遗传标记,在6个所选菌株中设计了3个禾谷镰孢菌(Furarium graminearum)的室内杂交组合和三个田间杂交组合,使各菌株之间进行杂交,从而研究有性重组。在每个田间杂交组合的稻桩表面随机挑取100个以上杂交或自交的单个子囊壳,检测结果表明三个杂交组合的杂交频率为5.7-20.9%,从而确认了在田间条件下发生了有性重组。从各杂交组合的后代中任意挑选出3个有性重组体,比较了这些有性重组体与其亲本在无性和有性阶段的主要生物学性状。结果表明,禾谷镰孢菌中的nit基因及对杀菌剂多菌灵的抗药性基因可以通过有性杂交的方式重组,即发生了有性重组。有性重组体与其亲本在菌落生长、培养性状和致病性方面没有显著差异；但某些有性重组体中,产孢量和产子囊壳能力方面存在一定差异。总体看来,有性重组体仍然保持了较高的适合度。因此,可以认为有性重组在禾谷镰孢菌群体对多菌灵抗药性发生发展以及群体遗传进化中可能起着重要的作用。为了评估田间对多菌灵已经产生抗药性的禾谷镰孢菌对氰烯菌酯产生抗性的风险,选择了5个菌株(对多菌灵抗性或敏感),并被鉴定为对多菌灵敏感(S)、中抗(MR)和高抗(HR),并用来在含10μg/mL氰烯菌酯的PSA培养基平板上诱导对氰烯菌酯的抗药性。总共获得了24个对氰烯菌酯抗性的突变体这些抗药性突变体能在PSA培养基平板上转代培养8次后和在4℃冰箱里保存60天后仍然保持着对氰烯菌酯和(／或)多菌灵的抗药性。可以推测,对多菌灵的抗药性和对氰烯菌酯的抗药性在两个亲和菌株的菌丝融合过程中不能发生抗药性物质的交换,这可能会在一定程度上能够延缓田间菌株对氰烯菌酯产生抗药性。在某些抗药性突变体中,菌丝生长和产孢能力出现下降,说明禾谷镰孢菌对氰烯菌酯产生抗药性突变也可能导致适合度的降低。然而,大多数突变体都显示了与其亲本相似的有性繁殖能力和致病性。而且,总体看来,大多数突变体都拥有着与亲本相似的适合度。多菌灵和氰烯菌酯的防效试验与室内活性测定的结果一致。氰烯菌酯对田间抗多菌灵的菌株引起的赤霉病有着较好的防效,而对抗氰烯菌酯的菌株及双抗菌株的防效较差。而且同时使用多菌灵和氰烯菌酯对双抗菌株的防效显著低于对单抗菌株或敏感菌株的防效。以上结果表明,氰烯菌酯和多菌灵同时使用,在防治小麦赤霉病时具有较高的抗药性风险,而且对这两种药剂的抗药性很可能会产生并且成为实际生产中一个很严重的问题。为了避免对氰烯菌酯抗药性的产生以及维持氰烯菌酯的使用价值,应当尽早采取抗药性治理措施,防患于未然。更多还原

【Abstract】 JS399-19,2-cyano-3-amino-3-phenylancryic acetate is a novel cyanoacrylate fungicide introduced by Jiangsu Branch of National Pesticide Research & Department South Center. This fungicide was demonstrated to have activity against Fusarium. spp, especially for Fusarium graminearum. In order to develop a sound recommendation for the use and exploitation of a new crop fungicide, sufficient biological mode of action and genetic analysis of its resistance should be studied.In vitro, the inhibitory activity of the fungicide against mycelial growth of both sensitive and resistant isolates of F. graminearum was measured and the influence of the fungicide on conidial germination of F. graminearum was determined. The results showed that JS399-19 could strongly inhibit the mycelial growth of a sensitive isolate of F. graminearum with the EC50 values 0.092-0.141μg/mL. JS399-19 decreased the speed of conidial germination of the sensitive isolate of F. graminearum, strongly inhibited the germtube growth of the conidia, and affected the conidia germination by causing the ratio of the tubes germinated from the basal and that from the middle parts of the conidia increased. Moreover, JS399-19 could cause abnormality of conidia and the tubes of the sensitive isolate, by inducing the conidia to swell, and inducing the tubes to swell and contort, respectively. However, this fungicide weakly affected the JS399-19-resistant isolates. JS399-19 could hardly inhibit the respiration of and pyruvic acid production of F. graminearum, suggesting that JS399-19 might not directly affect the respiration pathway of this fungus.Fifty nitrate nonutilizing mutants (nit) were obtained by transferring the 3 JS399-19-resistant mutants of F. graminearum on MMC media. The results showed that there were no significant differences in mycelial growth rate, cultural characters and pathogenicity between JS399-19-resistant nit mutants and their parental isolates. But the conidiophore production and perithecigerous capacity changed more or less in some mutants. Results also indicated that there were no cross-resistance toward chlorate and fungicide JS399-19 in F. graminearum, and the resistance to both chlorate and JS399-19 could be transferred by asexual reproduction steadily. Therefore, the nit can be used as a genetic marker for genetic studies of F. graminearum resistant to fungicide JS399-19.In vitro, a total of 86 JS399-19-resistant mutants were recovered from 5 wild-type strain by ultra-violet (UV) irradiating and fungicide training with the high frequency 1.25×10-7-2.29×10-7 for UV and 2.5-6.0% for fungicide training, respectively. The results also revealed that most of the resistant mutants belong to MR or HR. Although JS399-19-resistance mutation did not change the vegetative compatibility of F. graminearum, nevertheless, JS399-19-resistance could not be transferred by hyphal fusion or could be transferred with very low chance between two vegetatively compatible isolates. Therefore, hyphal fusion presumably took very little part in the development of JS399-19-resistant population in F. graminearum. Forty-three isolates sensitive to fungicide JS399-19 were collected from three commercial wheat fields of China. Forty-five isolates resistant to JS399-19 which had already been recovered from five sensitive isolates by selection for resistance to the fungicide JS399-19 were selected. Three sensitivity levels were identified:sensitive (S), moderately resistant (MR) and highly resistant (HR) to JS399-19 based on their sensitivity to JS399-19. All the conidia and ascospore progeny of three representative isolates (2043, Y2021B and YNT, representing S, MR and HR) exhibited unchanged resistance level to JS399-19, indicating resistance stability in asexual and self-crossed reproduction. Eight isolates representing three sensitivity level phenotypes were randomly selected for a study on the inheritance of JS399-19 resistance by analyzing the sensitivity of hybrid F1 progeny. The nitrate nonutilizing mutant (nit) was used as a genetic marker to confirm that individual perithecia were the result of out-crossing. Six crosses were assessed:S×S, S×HR, MR×HR, HR×HR, and MR×S. In crosses between the parents with different sensitivity levels, such as S×HR, MR×HR and MR×S, the progeny fit a 1:1 segregation ratio of the two parental phenotypes. No segregation was observed in the crosses of S×S and HR×HR. We concluded that the MR and HR phenotypes in F. graminearum were presumably conferred by different allelic mutations within the same locus. In these isolates, resistance to JS399-19 was not affected by modifying genes or cytoplasmic components.In order to study sexual recombination of F. graminearum, six selected isolates were adopted as parents and crossed in three designed pairs under laboratory conditions and under field conditions, respectively, by using nitrate non-utilizing (nit) mutants and carbendazim-resistance as genetic markers. Three sexual recombinants from each of the three combinations were randomly selected to compare the major biological properties with their parental isolates. The results showed that the nit gene and carbendazim-resistance gene could be recombined by sexual crosses. There were no significant differences in mycelial growth, traits of culture and pathogenicity between the sexual recombinants and their parental isolates. Sporulation and perithecigerous capacities, however, changed more or less in some sexual recombinants. The fitness of the sexual recombinants was comparable to the parents in general. Over 100 putative self-crossing or outcrossing perithecia for each cross were randomly sampled on the surface of the haulms of dead rice for each pair of the two parents and the results showed that about 5.7-20.9% outcrossing frequency occurred in the three crosses and confirmed sexual recombination under field conditions. Therefore, it is concluded that sexual recombination may play an important role in carbendazim resistance development and genetic evolution of F. graminearum populations.To evaluate the potential risk of resistance development in MBC-resistant F. graminearum isolates to the new fungicide JS399-19, five isolates (MBC-resistant or-sensitive) which were classified into three different sensitivity phenotypes, such as sensitive (S), moderately resistant (MR), and highly resistant (HR) to MBC, were selected to induce JS399-19-resistant mutants by selection for resistance on potato sucrose agar (PSA) plates amended with 10μg/mL JS399-19. Totally, twenty-four JS399-19-resistant mutants were obtained by selection for resistance to the new fungicide from the selected MBC-resistant or -sensitive isolates. All of the resistant mutants maintained their resistance to JS399-19 and/or MBC through 8 transfers on PSA plates for 40 days and when stored on PSA slants at 4℃for 60 days. It was hypothesized that MBC resistance and JS399-19 resistance could not be exchanged by mycelial fusion or there is a small chance to be exchanged between two compatible isolates, which might delay the development of JS399-19 resistance in field MBC-resistant F. graminearum isolates. The mycelial growth and conidial production capacity were decreased in some resistant mutants, indicating that a fitness cost was associated with JS399-19 resistant phenotypes of F. graminearum isolates. However, most of the mutants resistant to both MBC and JS399-19, exhibited high sexual reproduction capacity and pathogenicity as their parental isolates. Nevertheless, most of these mutants possessed fitness levels comparable to their parents. The results on the efficacy of the two fungicides for controlling FHB incited by the fungicide-resistant mutants were generally in consistence with that of in vitro sensitivity tests. JS399-19 was effective in controlling FHB caused by MBC-resistant isolates under field conditions, while it was not effective in controlling FHB caused by the JS399-19 resistant isolates and the special isolates resistant to both MBC and JS399-19. Moreover, the efficacy of MBC+JS399-19 was also significantly lower when controlling FHB caused by the special isolates resistant to both MBC and JS399-19 than when controlling disease caused by the sensitive isolates, the MBC-resistant isolates or JS399-19-resistant isolates. All these results indicated that JS399-19 possessed a high risk in the development of resistance in MBC-resistant and -sensitive F. graminearum isolates, and double resistance to the two fungicides could presumable emerge and become a practical problem when both of the fungicides were extensively used. Appropriate precautions against JS399-19 resistance development in natural populations should be taken into account to avoid unexpected control failures and to sustain the usefulness of MBC and the new product JS399-19.更多还原