朱砂叶螨对丁氟螨酯的抗性形式遗传及抗性稳定性研究

发布时间：2018-03-12 10:28

本文选题：朱砂叶螨　切入点：丁氟螨酯　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：朱砂叶螨(Tetranychus cinnabarinus)是一种重要的农业害螨,可为害豇豆、茄子、花卉等百余种农业、经济作物。该螨具有繁殖力强、世代周期短、活动范围小等生物学特点,加之杀螨剂大量不科学的使用,使其抗药性问题尤为严重。丁氟螨酯是一种新型杀螨剂,在我国上市晚,还未广泛使用。该药剂杀螨活性高,对非靶标生物安全,具有成为未来主流杀螨剂的潜力。本论文通过长期在室内进行朱砂叶螨对丁氟螨酯的抗性筛选,分析了朱砂叶螨丁氟螨酯抗性品系的抗性遗传方式、适合度、抗性稳定性,并评估了丁氟螨酯的抗性风险,研究结果为丁氟螨酯的合理使用以及抗性治理的有效开展奠定了理论基础。本文主要研究内容与结果如下:1.以敏感品系(SS)为源头,室内经过97代筛选获得丁氟螨酯抗性品系(CyR),其LC50值由2.19mg/L上升到58.80mg/L,相对于室内敏感品系抗性倍数为26.85倍。2.利用丁氟螨酯抗性品系与敏感品系进行杂交和回交,明确其抗药性遗传形式,结果表明:F1代正交(CyR_80♀×SS♂)及反交(SS♀×CyR_80♂)所得显性度DRS和DSR分别为-0.24和-0.32,表明朱砂叶螨对丁氟螨酯的抗性为不完全隐性遗传;t测验得出正反交所得D值的95%置信限重叠,不存在显著性差异,表明抗性基因位于常染色体上;回交F2代期望值与实际LD-P曲线及χ2值存在明显差异,表明抗性受多对等位基因控制。3.通过构建抗性品系(CyR)与敏感品系(SS)的生命表,逐日统计其生长发育情况,计算生物学参数,发现CyR品系的生殖力及存活率均低于SS品系,相对适合度(RR/SS)Rf=0.881,表明CyR品系存在一定的抗性适合度代价。4.通过从CyR_85中分出一支亚系,去除其药剂筛选压,研究了朱砂叶螨对丁氟螨酯的抗性稳定性。主要以下三方面来评价:首先在药剂敏感性方面,抗性亚系(CyR_D)在停止药剂筛选后仅过了5代,其LC50从47.34mg/L下降到11.35mg/L,抗性倍数从21.6倍下降到5.2倍,12代后抗性倍数又下降至3.8倍,表明去除药剂压力后,CyR_D对丁氟螨酯的药剂敏感性逐渐恢复。其次从生理生化方面,CyR_D在停止药剂筛选的情况下仅过了5代,三大解毒酶GSTs、MFO、CarE酶活性均发生显著性下降,12代后MFO和CarE酶活性下降到敏感水平。表明去除药剂压力后,CyR_D解毒酶活性会逐渐下降。分子生物学方面,结合高通量测序及qPCR验证的结果,鉴定出10条可能与丁氟螨酯抗性相关的解毒酶基因,在停止筛选后的不同阶段测定其mRNA表达量,发现TCGST-M2、TCGST-M3、TCGST-M4,TCCarE-4、TCCarE-14,CYP389C11、CYP392D6、CYP385C2,8条原本显著性上调表达的基因在停止筛选12代后表达量较原表达水显著性下降,2条显著性下调的基因TCCarE-12、TCCarE-23较原表达水平显著上升,表明去除药剂压力后,CyR_D解毒酶基因mRNA表达量呈现不稳定性,会逐渐变化至与敏感品系相当的水平。药剂敏感性,解毒酶活性及相关解毒酶基因mRNA表达量三方面的结果均表明朱砂叶螨对丁氟螨酯的抗药性不稳定。5.应用数量遗传学方法估算出朱砂叶螨丁氟螨酯抗性品系的抗性现实遗传力h2=0.005,表明其对丁氟螨酯的抗性发展较为缓慢。抗性风险评估表明,在70%-90%致死率的药剂筛选压力下,朱砂叶螨对丁氟螨酯产生10倍抗性需13-36代。通过测定朱砂叶螨丁氟螨酯抗性品系与5种不同作用杀虫杀螨剂的交互抗性,发现丁氟螨酯与甲氰菊酯、阿维菌素、联苯肼酯、炔螨特之间无交互抗性,而与哒螨灵会产生明显的交互抗性,在农业害螨防治中应当避免丁氟螨酯与哒螨灵的混用、轮用等。本论文研究结果表明:朱砂叶螨对丁氟螨酯的抗性发展较为缓慢,去除药剂压力后,已产生的抗药性会迅速衰退;多基因、不完全隐性的抗性遗传形式及适合度代价使得抗药性基因在种群间传播不利,抗性种群发展慢。综上所述,丁氟螨酯在防治朱砂叶螨时抗性风险较小。
[Abstract]:Tetranychuscinnabarinus (Tetranychus cinnabarinus) is one of the most important agricultural pest mites, can infect cowpea, eggplant, flowers and other hundreds of agricultural crops. The mites have high fecundity, short generation cycle, the scope of activities of small biological characteristics, coupled with the use of a large number of acaricides is not scientific, the drug resistance problem is serious. Ding fluorine mites ester is a new acaricide, listed in late in China, is not widely used. The chemical acaricidal activity and high safety to non target organisms, has become the mainstream of acaricide potential. This paper through indoors in tetranychuscinnabarinus screening of resistance to fluorine Ding chlorfenson, analyzes the inheritance mode, tetranychuscinnabarinus Ding fluorine mites ester resistant strains for resistance, stability, and to assess the resistance risk of Ding fluorine mites ester, the results for the rational use of Ding Fu and chlorfenson resistance management has laid a theoretical development effect The foundation. In this paper, the main research contents and results are as follows: 1. sensitive strains (SS) as the source of indoor, after 97 generations of screened mite resistant strains of Ding fluorine ester (CyR), the LC50 value increased from 2.19mg/L to 58.80mg/L, compared to the resistance ratio indoor sensitive strains was 26.85 times.2. using fluorine Ding chlorfenson resistance and susceptible strains of hybridization and backcross, the resistance genetic form, the results showed that the F1 generation (CyR_80 * SS male female) and reciprocal (SS * CyR_80 male female) the dominant degree of DRS and DSR were -0.24 and -0.32, indicated that the resistance of tetranychuscinnabarinus Ding fluorine mites ester is not completely recessive; t tests out the reciprocal values of the 95% confidence limit of the D overlap, there is no significant difference, indicated that the resistance gene is located on chromosome; and the actual LD-P curve and 2 were significantly different backcross F2 generation expectations, indicated that the resistance by multiple alleles.3. controlled by structure Construction of resistant strain (CyR) and susceptible strain (SS) of the life table, the daily statistics of its growth and development, computational biology parameters, found CyR strains of fecundity and survival rate were lower than that of the SS strain, relative fitness (RR/SS) Rf=0.881, showed that the CyR strain had some resistance to fitness costs.4. from the CyR_85 in the separation of a sub line, remove the chemical screening pressure, study the stability of resistance to Ding Zhu Shaye mite fluorine mites ester. Mainly the following three aspects to evaluate: first in the susceptibility to insecticides, resistance subline (CyR_D) in chemical screening after only 5 generations, the LC50 from 47.34mg/L down to 11.35mg/L, the resistance ratio from 21.6 times down to 5.2 times, after 12 generations of resistance ratio decreased to 3.8 times, showed that the removal agent pressure, drug sensitivity of CyR_D Ding Fu mite ester gradually resume. Secondly from the physiological and biochemical aspects of CyR_D in the case of only stop drug screening After 5 generations, three GSTs MFO, CarE enzyme and enzyme activity were significantly decreased after 12 generations of MFO and CarE activity declined to sensitive level. After that the removal agent pressure, detoxification enzyme activity of CyR_D decreased. The molecular biology, combined with high-throughput sequencing and qPCR verification results, identification 10 may be associated with resistance to fluoride Ding chlorfenson detoxification enzyme gene in different stages, stop after screening to determine the expression of mRNA, TCGST-M3, TCGST-M4, TCGST-M2, TCCarE-4, TCCarE-14, CYP389C11, CYP392D6, CYP385C2,8 had a significant increase in the expression of genes to stop screening than the original expression significantly the reduced expression of TCCarE-12 gene after 12 generations, 2 significantly reduced, compared with the original TCCarE-23 expression level increased significantly, suggesting that removal of chemical pressure, the expression of CyR_D gene mRNA detoxifying enzymes is not stable, will gradually change to sensitive strains The level of mRNA gene. Drug sensitivity, detoxification enzyme activity and detoxification expression in three aspects of the results showed that the mite resistance to mite ester unstable fluorine Ding.5. application method of quantitative genetics to estimate the resistance of reality tetranychuscinnabarinus Ding fluoride resistant strains of chlorfenson genetic h2=0.005, showed that the the Ding fluorine mites ester resistance development is relatively slow. Assessment showed the risk of resistance in 70%-90%, mortality rate of chemical screening under pressure, tetranychuscinnabarinus produces 10 times the 13-36 generation of fluoride resistant mites. Through the determination of ester Ding Ding tetranychuscinnabarinus fluoride resistant strains of mite ester and 5 kinds of different insecticidal acaricide cross resistance, Ding found fluorine mites ester and fenpropathrin, abamectin, bifenazate, no cross resistance between propargite, and pyridaben will produce significant cross resistance in agriculture, control of pest mite should avoid mixing Ding fluorine mites ester and pyridaben, Wheel etc. in this paper, the results show that the development of tetranychuscinnabarinus resistant Ding ester fluorine mites is slow, removal agent pressure, has produced resistance will rapidly decline; multi gene, genetic resistance is not completely recessive and the fitness costs of resistance gene in population spread bad resistance the population development is slow. To sum up, Ding fluorine mite ester resistance in control of t.cinnabarinus when the risk is small.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433.7

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