氧硫化碳在检疫处理梨黑斑病菌中的应用及其分子机理研究
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摘要
作为溴甲烷的潜在替代熏蒸剂,氧硫化碳以其杀虫效果好,穿透能力强,低残留,天然环保等优点在仓储害虫熏蒸方面受到广泛关注,但是氧硫化碳对鲜活农产品及其真菌病害的熏蒸效果研究还相对较少,氧硫化碳的毒理机理研究方面目前则更是空白。
本研究针对梨黑斑病病原菌链格孢菌(Alternaria.Alternata(Fr.)Keissler),在不同熏蒸温度和时间条件下进行了室内毒力测定,结果表明,温度越高,氧硫化碳对链格孢菌的抑制效果越好,氧硫化碳链格孢菌处理在15℃以上较为适宜;随着熏蒸时间的延长,氧硫化碳对链格孢菌的毒力显著提高,说明长时间熏蒸有助于提高处理效果。对人工接种伤梨的熏蒸试验表明,氧硫化碳熏蒸对梨黑斑病的发病有明显的抑制作用,梨黑斑病的发病率随着熏蒸剂量升高而降低。装载量50%左右剂量为200g/m~3COS熏蒸4h,几乎可以完全抑制梨黑斑病的发病。
氧硫化碳熏蒸对鸭梨品质的影响同样是我们的关注点,为此本文开展了25℃不同剂量氧硫化碳熏蒸对鸭梨失重,硬度,可溶性糖含量,总酸含量以及呼吸强度的影响的研究。试验结果表明,氧硫化碳熏蒸对鸭梨处理期间的呼吸作用有一定程度的增强,可以抑制鸭梨在贮藏中的失重,对储藏期间水果硬度、可溶性糖和总酸度均无明显影响,但是装载量50%左右剂量60g/m~3以上COS熏蒸4h处理会使鸭梨表皮出现药害。
为了探明氧硫化碳熏蒸杀灭链格孢菌的分子机理,本研究采用了抑制消减杂交技术(Suppression subtractive hybridization,SSH)成功构建了处理前后的cDNA差异表达文库,寻找到了氧硫化碳熏蒸后差异表达的基因。序列分析表明,链格孢菌中几个参与蛋白磷酸化,钙离子应答等与信号转导过程相关的基因,其表达量在处理0.5h后迅速升高,几个参与细胞蛋白合成,分裂增殖过程相关的基因,其表达量在1至2h后降低;说明COS可能通过迅速激活一系列信号转导过程抑制了链格孢菌的蛋白合成,细胞分裂和增殖,进而达到杀灭效果;另一方面,几个参与硫化氢和半胱氨酸代谢的基因表达量也在处理0.5h后迅速升高,说明COS还可能同时直接侵入细胞并代谢为硫化氢进而达到杀灭效果。
The toxicity of carbonyl sulfide (COS) to stored products pests has been well studied as a potential alternative of methyl bromide for insect disinfestations, however, little is known about the efficacy of COS for control of fungal pathogens causing postharvest diseases of the fresh products.. The molecular mechanism of its toxicity is currently empty.
Fumigation of pear fruit with COS at several concentrations, times and temperatures was studied for control of Alternaria alternata (Fr.) Keissler and to determine the effects on the quality of treated fruits. Our results showed that the fungitoxicity of COS became higher as the treatment temperature and time were increased, COS concentrations of 72.7 g/m~3,117.8 g/m~3 and 174.5 g/m~3 resulted in 50%, 90% and 99% inhibition in vitro after four hours fumigation at 25℃, respectively. Furthermore, four hours COS fumigation repressed A. alternata in wounded pears at the dosage of 200 g/m~3 at 25℃, indicating that control of this fungal pathogen with COS is achievable.
COS fumigation was performed at different doses at 25℃on ya pears for 4hours and the quality parameters of pears were measured after stored at 25℃in order to study the influence of COS fumigation on the quality of ya pears. The results showed that COS fumigation could promote respiration during the treatment, reduced the water loss, While the quality parameters of flesh firmness, total soluble sugar and total acidity were not significant affected during storage at 25℃. When loading rate was about 50%, carbonyl sulfide fumigation at the dosage of 60 g/m~3 for 4 hours can cause slight surface injury.
In order to investigate the molecular mechanism of the fungitoxicity of COS, suppression subtractive hybridization (Suppression subtractive hybridization, SSH) was successfully proformed to construct the subtracted cDNA libraries and find the differentially expressed genes after COS fumigation. Sequence analysis showed that expression of several signal transduction-related genes involved in protein phosphorylation, calcium ion response were rapidly increased after 0.5 hours fumigation with COS , expression of several genes involved in cell protein synthesis, the process of proliferation-related genes were decreased after 1 hours fumigation with COS, indicating COS may activate a series of signal transduction to inhibit protein synthesis, cell division and proliferation of Alternaria alternata. Furthermore, expression of several genes involved in the metabolism of hydrogen sulfide and cysteine are rapidly increased after 0.5 hours fumigation with COS, indicating COS may form H_2S in the cells and kill Alternaria alternate directly.
本研究针对梨黑斑病病原菌链格孢菌(Alternaria.Alternata(Fr.)Keissler),在不同熏蒸温度和时间条件下进行了室内毒力测定,结果表明,温度越高,氧硫化碳对链格孢菌的抑制效果越好,氧硫化碳链格孢菌处理在15℃以上较为适宜;随着熏蒸时间的延长,氧硫化碳对链格孢菌的毒力显著提高,说明长时间熏蒸有助于提高处理效果。对人工接种伤梨的熏蒸试验表明,氧硫化碳熏蒸对梨黑斑病的发病有明显的抑制作用,梨黑斑病的发病率随着熏蒸剂量升高而降低。装载量50%左右剂量为200g/m~3COS熏蒸4h,几乎可以完全抑制梨黑斑病的发病。
氧硫化碳熏蒸对鸭梨品质的影响同样是我们的关注点,为此本文开展了25℃不同剂量氧硫化碳熏蒸对鸭梨失重,硬度,可溶性糖含量,总酸含量以及呼吸强度的影响的研究。试验结果表明,氧硫化碳熏蒸对鸭梨处理期间的呼吸作用有一定程度的增强,可以抑制鸭梨在贮藏中的失重,对储藏期间水果硬度、可溶性糖和总酸度均无明显影响,但是装载量50%左右剂量60g/m~3以上COS熏蒸4h处理会使鸭梨表皮出现药害。
为了探明氧硫化碳熏蒸杀灭链格孢菌的分子机理,本研究采用了抑制消减杂交技术(Suppression subtractive hybridization,SSH)成功构建了处理前后的cDNA差异表达文库,寻找到了氧硫化碳熏蒸后差异表达的基因。序列分析表明,链格孢菌中几个参与蛋白磷酸化,钙离子应答等与信号转导过程相关的基因,其表达量在处理0.5h后迅速升高,几个参与细胞蛋白合成,分裂增殖过程相关的基因,其表达量在1至2h后降低;说明COS可能通过迅速激活一系列信号转导过程抑制了链格孢菌的蛋白合成,细胞分裂和增殖,进而达到杀灭效果;另一方面,几个参与硫化氢和半胱氨酸代谢的基因表达量也在处理0.5h后迅速升高,说明COS还可能同时直接侵入细胞并代谢为硫化氢进而达到杀灭效果。
The toxicity of carbonyl sulfide (COS) to stored products pests has been well studied as a potential alternative of methyl bromide for insect disinfestations, however, little is known about the efficacy of COS for control of fungal pathogens causing postharvest diseases of the fresh products.. The molecular mechanism of its toxicity is currently empty.
Fumigation of pear fruit with COS at several concentrations, times and temperatures was studied for control of Alternaria alternata (Fr.) Keissler and to determine the effects on the quality of treated fruits. Our results showed that the fungitoxicity of COS became higher as the treatment temperature and time were increased, COS concentrations of 72.7 g/m~3,117.8 g/m~3 and 174.5 g/m~3 resulted in 50%, 90% and 99% inhibition in vitro after four hours fumigation at 25℃, respectively. Furthermore, four hours COS fumigation repressed A. alternata in wounded pears at the dosage of 200 g/m~3 at 25℃, indicating that control of this fungal pathogen with COS is achievable.
COS fumigation was performed at different doses at 25℃on ya pears for 4hours and the quality parameters of pears were measured after stored at 25℃in order to study the influence of COS fumigation on the quality of ya pears. The results showed that COS fumigation could promote respiration during the treatment, reduced the water loss, While the quality parameters of flesh firmness, total soluble sugar and total acidity were not significant affected during storage at 25℃. When loading rate was about 50%, carbonyl sulfide fumigation at the dosage of 60 g/m~3 for 4 hours can cause slight surface injury.
In order to investigate the molecular mechanism of the fungitoxicity of COS, suppression subtractive hybridization (Suppression subtractive hybridization, SSH) was successfully proformed to construct the subtracted cDNA libraries and find the differentially expressed genes after COS fumigation. Sequence analysis showed that expression of several signal transduction-related genes involved in protein phosphorylation, calcium ion response were rapidly increased after 0.5 hours fumigation with COS , expression of several genes involved in cell protein synthesis, the process of proliferation-related genes were decreased after 1 hours fumigation with COS, indicating COS may activate a series of signal transduction to inhibit protein synthesis, cell division and proliferation of Alternaria alternata. Furthermore, expression of several genes involved in the metabolism of hydrogen sulfide and cysteine are rapidly increased after 0.5 hours fumigation with COS, indicating COS may form H_2S in the cells and kill Alternaria alternate directly.
引文
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