甲酸乙酯的熏蒸应用研究与毒性相关蛋白质的筛选
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摘要
磷化氢和溴甲烷是现有两种使用最为广泛的熏蒸剂,在粮谷熏蒸,土壤消毒,检疫处理,交通工具消毒等领域都发挥了很大的效果。但由于对臭氧层的破坏作用,溴甲烷被列入《关于消耗臭氧层蒙特利尔议定书》修正案的受控物质名单,由限用逐渐到禁用。磷化氢作为溴甲烷的替代品,在长期及不合理使用的情况下已经导致多种害虫在全球范围内产生了对磷化氢的抗性。为应对溴甲烷从限用到禁用对我国除害处理行业的影响,本研究对甲酸乙酯作为熏蒸剂在除害处理方面的应用价值进行评估。采用甲酸乙酯对赤拟谷盗、四纹豆象、松材线虫和桔小实蝇及其相应的寄主(面粉、绿豆、松木及五种水果)进行了熏蒸处理实验,并对面粉和绿豆的品质和熏蒸后残留进行了检测以综合评估甲酸乙酯作为熏蒸剂的应用价值。同时以赤拟谷盗为实验对象,从分子水平上对甲酸乙酯处理前后赤拟谷盗的蛋白表达差异进行了初步研究,为甲酸乙酯进一步应用提供基础。主要研究结果如下:
1.甲酸乙酯对面粉中赤拟谷盗的熏蒸效果及残留研究
甲酸乙酯熏蒸能有效防治面粉中的赤拟谷盗各虫态,赤拟谷盗不同虫态对甲酸乙酯的敏感性强弱依次为:卵>低龄幼虫>成虫>高龄幼虫>蛹。在23℃,6 h和12 h的处理条件下对面粉中赤拟谷盗的防除指标(LCt99)分别为448.42 mg h L~(-1)和599.34 mg h L~(-1)。熏蒸后面粉中的甲酸乙酯降解速率较慢,15 d后依然能够检测出面粉中的甲酸乙酯残留。
2.甲酸乙酯对绿豆中四纹豆象的熏蒸效果及熏蒸后绿豆的甲酸乙酯残留与品质研究
甲酸乙酯熏蒸能够有效控制绿豆中的四纹豆象各虫态,四纹豆象不同虫态对甲酸乙酯的敏感性强弱依次为:成虫>卵>低龄幼虫>高龄幼虫>蛹。在23℃,6、12、24 h处理条件下,甲酸乙酯对绿豆中四纹豆象的防除指标分别为511.43 mg h L~(-1)、760.84 mg h L~(-1)和822.98 mg h L~(-1)。熏蒸后在绿豆甲酸乙酯的残留在3 d后就低于检出限,同时甲酸乙酯熏蒸对绿豆的外观及发芽率均无显著影响。
3.甲酸乙酯对松材线虫的熏蒸效果研究
甲酸乙酯对松材线虫表现出较高的杀线活性。在3、6、12、24、48 h的处理时间下,松材线虫的LC50分别为2.63 mg L~(-1)、1.60 mg L~(-1)、0.99 mg L~(-1)、0.41 mg L~(-1)和0.20 mg L~(-1)。温度对毒力有显著影响,甲酸乙酯在较低温度下对松材线虫表现出比较高温度下更高的毒力。同时甲酸乙酯熏蒸在12 h内就能完全杀灭木段中的松材线虫。甲酸乙酯在23℃,6 h和12 h的处理时间下对木段中松材线虫的防除指标分别为453.94 mg h L~(-1)和424.14 mg h L~(-1)。
4.甲酸乙酯对水果中桔小实蝇的熏蒸效果及熏蒸后水果的品质研究
甲酸乙酯对桔小实蝇有一定的熏蒸毒性,并且作用较迅速,甲酸乙酯对桔小实蝇的LC50在3、6、12、24 h的处理下分别为54.68 mg L~(-1)、14.30 mg L~(-1)、12.54 mg L~(-1)、12.54 mg L~(-1)。同时甲酸乙酯对水果中的实蝇幼虫也能发挥较好的处理效果,桔小实蝇果内虫期对甲酸乙酯的敏感性强弱依次为卵>1龄幼虫>3龄幼虫,甲酸乙酯在23℃,6 h和12 h和24 h下对水果中桔小实蝇的防除指标分别为880.90 mg h L~(-1)、553.63 mg h L~(-1)和798.49 mg h L~(-1)。经甲酸乙酯处理后的芒果、杨桃、火龙果、番石榴、木瓜的外观和品质均出现了较严重的药害。
5.甲酸乙酯毒理机制的比较蛋白组学初步研究
本研究建立了甲酸乙酯熏蒸中毒的赤拟谷盗模型,应用双向电泳技术分离赤拟谷盗经甲酸乙酯熏蒸后差异表达的蛋白。结果表明昆虫虫体粗提总蛋白经过丙酮沉淀后进行双向电泳才能得到较理想的分离效果,而通过比较蛋白质组学分析发现经甲酸乙酯熏蒸的赤拟谷盗总蛋白中有8个蛋白出现了差异表达。其中7个蛋白出现了上调,1个蛋白出现了下调,可能是损伤技能的执行分子或损伤作用的产物。
The methyl bromide and phosphine are widely applied fumigants which played a critical role in cereal fumigation, soil disinfection, quarantine treatment and vehicle sterilization. However, the methyl bromide has been listed in Montreal Protocol due to its property of ozone depleting. As an alternative of methyl bromide, phosphine, has caused severe resistance development of several insects because of irrational and long-term application. For coping the adverse effect to the phytosanitary industry by phasing out of methyl bromide, this study investigated the value of ethyl formate on fumigation application. Four pests (Tribolium castaneum, Callosobruchus maculates, Bursaphelenchus xylophilus and Bactrocera dorsalis) and corresponding hosts are choosen for fumigation experiments. The quality and residue of ethyl formate in flour and mung beans after fumigation were evaluated for the purpose of comprehensive evaluation of ethyl formate as a fumigant. Besides, the prelimary toxicology mechanism of ethyl formate was studied by detecting the difference of proteins expression after treatment on T. castaneum to provide evidence for further application of ethyl formate. The results are following:
1. The fumigation effect of ethyl formate towards T. castaneum in flour and residue analysis.
The purpose of this research is to evaluate the fumigation activity of ethyl formate against T. castaneum and residue in flour after fumigation. The results indicate that ethyl formate is effective in disinfecting all life stage of T. castaneum in flour, and the sensitivities of life stages of T. castaneum to ethyl formate, which arranged from strongest to weakest, were egg, low instar larva, adult, high instar larva and pupa. The LCt99 for disinfecting all life stages of T. castaneum in 6 h and 12 h at 23℃are 448.42 mg h L~(-1) and 599.34 mg h L~(-1), respectively. The ethyl formate residue could be detected 15 days after fumigation which desmonstrates ethyl formate degraded slowly in flour after fumigation.
2. Toxicity, residue and impact on quality of ethyl formate fumigation towards C. maculatus and mung bean.
The toxicity of ethyl formate to C. maculatus and its impacts to mung bean were evaluated. The results indicate C. maculatus were eradicated by ethyl formate fumigation within 24 h exposure and the sensitivities of life stages of C. maculatus to ethyl formate, which arranged from strongest to weakest, are adult, egg, low instar larva, high instar larva and pupa. The LCt99 for disinfecting all life stages of C. maculatus in 6 h, 12 h and 24 h at 23℃are 511.43 mg h L~(-1), 760.84 mg h L~(-1) and 822.98 mg h L~(-1) respectively. The residue of ethyl formate was declined to undetecable level within 3 d after exposure. And ethyl formate fumigation showed no influence on the appearance and germination percentage of mung bean.
3. Nematicidal activity of ethyl formate towards B. xylophilus
The nematicidal activity of ethyl formate towards B. xylophilus was evaluated. Results of bio-assay indicated that B. xylophilus is sensitive to ethyl formate. The LC50 of B. xylophilus to ethyl formate in 3 h, 6 h. 12 h, 24 h and 48 h under 25℃are 2.63 mg L~(-1), 1.60 mg L~(-1), 0.99 mg L~(-1), 0.41 mg L~(-1) and 0.20 mg L~(-1), respectively. The B. xylophilus showed higher mortality under relative low temperature and lower mortality was observed under relatively higher temperature which indicates that the nematicidal activity of ethyl formate is significantly affected by temperature. The disinfection experiments of nematodes infected woods were carried out in plexiglass bins and the fumigant concentration was detected by portable GC during the exposure for computing the Ct product. The results show that B.xylophilus were eradicated from wood in 12 h treatment and the LCt99 of ethyl formate to B. xylophilus in 6 h and 12 h are 453.94 mg h L~(-1) and 424.14 mg h L~(-1), respectively.
4. The study of fumigation toxicity of ethyl formate to B. dorsalis and phytotoxicity to fruits.
The fumigation activity of ethyl formate to B. dorsalis and phytotoxicity was studied. The LC50 of high instar larva of B. dorsalis to ethyl formate at 3 h, 6 h. 12 h and 24 h are 54.68 mg L~(-1), 14.30 mg L~(-1), 12.54 mg L~(-1), 12.54 mg L~(-1). The lavae of B. dorsalis inside fruits showed fair mortality after ethyl formate fumigation. The The LCt99 of ethyl formate to B. dorsalis at 6 h, 12 h and 24 h are 880.90 mg h L~(-1), 553.63 mg h L~(-1), and 798.49 mg h L~(-1), respectively. The sensitivities of larva and egg stages of B. dorsalis to ethyl formate, which arranged from strongest to weakest, are egg, low instar larva and high instar. However, the fruits showed relatively severe phytotoxicity after exposure.
5. The preliminary research of compared proteomics on toxicology mechanism of ethyl formate.
The T. castaneum model of ethyl formate fumigation was established and the 2-D PAGE was used to separate the proteins differently expressed after fumigation. The 2-D result indicates a perfect 2-D image was achieved while the rude protein of T. castaneum was precipitated by acetone. And out of total 8 differently expressed proteins, 7 proteins were escalated after ethyl formate fumigation and 1 proteins was down-regulation which may be the executors or products of injury mechanism.
1.甲酸乙酯对面粉中赤拟谷盗的熏蒸效果及残留研究
甲酸乙酯熏蒸能有效防治面粉中的赤拟谷盗各虫态,赤拟谷盗不同虫态对甲酸乙酯的敏感性强弱依次为:卵>低龄幼虫>成虫>高龄幼虫>蛹。在23℃,6 h和12 h的处理条件下对面粉中赤拟谷盗的防除指标(LCt99)分别为448.42 mg h L~(-1)和599.34 mg h L~(-1)。熏蒸后面粉中的甲酸乙酯降解速率较慢,15 d后依然能够检测出面粉中的甲酸乙酯残留。
2.甲酸乙酯对绿豆中四纹豆象的熏蒸效果及熏蒸后绿豆的甲酸乙酯残留与品质研究
甲酸乙酯熏蒸能够有效控制绿豆中的四纹豆象各虫态,四纹豆象不同虫态对甲酸乙酯的敏感性强弱依次为:成虫>卵>低龄幼虫>高龄幼虫>蛹。在23℃,6、12、24 h处理条件下,甲酸乙酯对绿豆中四纹豆象的防除指标分别为511.43 mg h L~(-1)、760.84 mg h L~(-1)和822.98 mg h L~(-1)。熏蒸后在绿豆甲酸乙酯的残留在3 d后就低于检出限,同时甲酸乙酯熏蒸对绿豆的外观及发芽率均无显著影响。
3.甲酸乙酯对松材线虫的熏蒸效果研究
甲酸乙酯对松材线虫表现出较高的杀线活性。在3、6、12、24、48 h的处理时间下,松材线虫的LC50分别为2.63 mg L~(-1)、1.60 mg L~(-1)、0.99 mg L~(-1)、0.41 mg L~(-1)和0.20 mg L~(-1)。温度对毒力有显著影响,甲酸乙酯在较低温度下对松材线虫表现出比较高温度下更高的毒力。同时甲酸乙酯熏蒸在12 h内就能完全杀灭木段中的松材线虫。甲酸乙酯在23℃,6 h和12 h的处理时间下对木段中松材线虫的防除指标分别为453.94 mg h L~(-1)和424.14 mg h L~(-1)。
4.甲酸乙酯对水果中桔小实蝇的熏蒸效果及熏蒸后水果的品质研究
甲酸乙酯对桔小实蝇有一定的熏蒸毒性,并且作用较迅速,甲酸乙酯对桔小实蝇的LC50在3、6、12、24 h的处理下分别为54.68 mg L~(-1)、14.30 mg L~(-1)、12.54 mg L~(-1)、12.54 mg L~(-1)。同时甲酸乙酯对水果中的实蝇幼虫也能发挥较好的处理效果,桔小实蝇果内虫期对甲酸乙酯的敏感性强弱依次为卵>1龄幼虫>3龄幼虫,甲酸乙酯在23℃,6 h和12 h和24 h下对水果中桔小实蝇的防除指标分别为880.90 mg h L~(-1)、553.63 mg h L~(-1)和798.49 mg h L~(-1)。经甲酸乙酯处理后的芒果、杨桃、火龙果、番石榴、木瓜的外观和品质均出现了较严重的药害。
5.甲酸乙酯毒理机制的比较蛋白组学初步研究
本研究建立了甲酸乙酯熏蒸中毒的赤拟谷盗模型,应用双向电泳技术分离赤拟谷盗经甲酸乙酯熏蒸后差异表达的蛋白。结果表明昆虫虫体粗提总蛋白经过丙酮沉淀后进行双向电泳才能得到较理想的分离效果,而通过比较蛋白质组学分析发现经甲酸乙酯熏蒸的赤拟谷盗总蛋白中有8个蛋白出现了差异表达。其中7个蛋白出现了上调,1个蛋白出现了下调,可能是损伤技能的执行分子或损伤作用的产物。
The methyl bromide and phosphine are widely applied fumigants which played a critical role in cereal fumigation, soil disinfection, quarantine treatment and vehicle sterilization. However, the methyl bromide has been listed in Montreal Protocol due to its property of ozone depleting. As an alternative of methyl bromide, phosphine, has caused severe resistance development of several insects because of irrational and long-term application. For coping the adverse effect to the phytosanitary industry by phasing out of methyl bromide, this study investigated the value of ethyl formate on fumigation application. Four pests (Tribolium castaneum, Callosobruchus maculates, Bursaphelenchus xylophilus and Bactrocera dorsalis) and corresponding hosts are choosen for fumigation experiments. The quality and residue of ethyl formate in flour and mung beans after fumigation were evaluated for the purpose of comprehensive evaluation of ethyl formate as a fumigant. Besides, the prelimary toxicology mechanism of ethyl formate was studied by detecting the difference of proteins expression after treatment on T. castaneum to provide evidence for further application of ethyl formate. The results are following:
1. The fumigation effect of ethyl formate towards T. castaneum in flour and residue analysis.
The purpose of this research is to evaluate the fumigation activity of ethyl formate against T. castaneum and residue in flour after fumigation. The results indicate that ethyl formate is effective in disinfecting all life stage of T. castaneum in flour, and the sensitivities of life stages of T. castaneum to ethyl formate, which arranged from strongest to weakest, were egg, low instar larva, adult, high instar larva and pupa. The LCt99 for disinfecting all life stages of T. castaneum in 6 h and 12 h at 23℃are 448.42 mg h L~(-1) and 599.34 mg h L~(-1), respectively. The ethyl formate residue could be detected 15 days after fumigation which desmonstrates ethyl formate degraded slowly in flour after fumigation.
2. Toxicity, residue and impact on quality of ethyl formate fumigation towards C. maculatus and mung bean.
The toxicity of ethyl formate to C. maculatus and its impacts to mung bean were evaluated. The results indicate C. maculatus were eradicated by ethyl formate fumigation within 24 h exposure and the sensitivities of life stages of C. maculatus to ethyl formate, which arranged from strongest to weakest, are adult, egg, low instar larva, high instar larva and pupa. The LCt99 for disinfecting all life stages of C. maculatus in 6 h, 12 h and 24 h at 23℃are 511.43 mg h L~(-1), 760.84 mg h L~(-1) and 822.98 mg h L~(-1) respectively. The residue of ethyl formate was declined to undetecable level within 3 d after exposure. And ethyl formate fumigation showed no influence on the appearance and germination percentage of mung bean.
3. Nematicidal activity of ethyl formate towards B. xylophilus
The nematicidal activity of ethyl formate towards B. xylophilus was evaluated. Results of bio-assay indicated that B. xylophilus is sensitive to ethyl formate. The LC50 of B. xylophilus to ethyl formate in 3 h, 6 h. 12 h, 24 h and 48 h under 25℃are 2.63 mg L~(-1), 1.60 mg L~(-1), 0.99 mg L~(-1), 0.41 mg L~(-1) and 0.20 mg L~(-1), respectively. The B. xylophilus showed higher mortality under relative low temperature and lower mortality was observed under relatively higher temperature which indicates that the nematicidal activity of ethyl formate is significantly affected by temperature. The disinfection experiments of nematodes infected woods were carried out in plexiglass bins and the fumigant concentration was detected by portable GC during the exposure for computing the Ct product. The results show that B.xylophilus were eradicated from wood in 12 h treatment and the LCt99 of ethyl formate to B. xylophilus in 6 h and 12 h are 453.94 mg h L~(-1) and 424.14 mg h L~(-1), respectively.
4. The study of fumigation toxicity of ethyl formate to B. dorsalis and phytotoxicity to fruits.
The fumigation activity of ethyl formate to B. dorsalis and phytotoxicity was studied. The LC50 of high instar larva of B. dorsalis to ethyl formate at 3 h, 6 h. 12 h and 24 h are 54.68 mg L~(-1), 14.30 mg L~(-1), 12.54 mg L~(-1), 12.54 mg L~(-1). The lavae of B. dorsalis inside fruits showed fair mortality after ethyl formate fumigation. The The LCt99 of ethyl formate to B. dorsalis at 6 h, 12 h and 24 h are 880.90 mg h L~(-1), 553.63 mg h L~(-1), and 798.49 mg h L~(-1), respectively. The sensitivities of larva and egg stages of B. dorsalis to ethyl formate, which arranged from strongest to weakest, are egg, low instar larva and high instar. However, the fruits showed relatively severe phytotoxicity after exposure.
5. The preliminary research of compared proteomics on toxicology mechanism of ethyl formate.
The T. castaneum model of ethyl formate fumigation was established and the 2-D PAGE was used to separate the proteins differently expressed after fumigation. The 2-D result indicates a perfect 2-D image was achieved while the rude protein of T. castaneum was precipitated by acetone. And out of total 8 differently expressed proteins, 7 proteins were escalated after ethyl formate fumigation and 1 proteins was down-regulation which may be the executors or products of injury mechanism.
引文
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