纳米银材料对植物病原细菌抑制作用的研究
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摘要
本研究测定了纳米银材料对8种供试植物病原细菌的抑制活性,初步探讨了纳米银材料对植物病原细菌的作用机理以及防病效果,为进一步开发纳米银植物病原细菌防治剂提供依据。
分别采用抑菌圈法、最小抑制浓度法(MIC)和平板菌落计数法,测定了纳米银对供试的8种植物病原细菌的抑制效果。结果表明,纳米银对8种供试细菌都有一定的抑制作用,说明纳米银作为新型杀菌剂具有较为广谱的杀菌效果。平板菌落计数法毒力测定结果显示,纳米银对黄单胞菌属(Xanthomonas)的4种细菌和茄青枯病菌(Ralstonia solanacearum)有较强的抑制效果,其中,对柑橘溃疡病菌(Xanthomonas axonopodis pv.citri)、稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)、稻细菌性条斑病菌(Xanthomonas oryae pv.oryzicola)、甘蓝黑腐病菌(Xanthomonas campestris pv.campestris)和茄青枯病菌的EC_(50)分别为0.0820μg·mL~(-1)、0.1889μg·mL~(-1)、0.1779μg·mL~(-1)、0.1817μg·mL~(-1)和0.1820μg·mL~(-1);而纳米银对其它属的供试植物病原细菌的抑制效果则较弱,其中对玉米细菌性条斑病菌(Acidovorax avenae subsp.avenae)的EC_(50)为0.4042μg·mL~(-1)、对白菜软腐病菌(Pectobacterium carotovora subsp.carotovora)的EC_(50)为0.2654μg·mL~(-1)、对西瓜细菌性果斑病菌(Acidovorax enae subsp.citrulli)的EC_(50)为0.2370μg·mL~(-1)。
纳米银对甘蓝黑腐病菌菌体生长抑制作用的结果表明,剂量为6μg·mL~(-1)的纳米银与菌体接触0.5 h后细菌的群体数量有明显的减少,且随着时间的推移,细菌菌体数量呈下降趋势。
纳米银能显著地抑制甘蓝黑腐病菌的氧呼吸作用,使细菌的氧呼吸量明显变小,且随着纳米银对菌体作用时间的延长,细菌氧呼吸作用的受抑制程度增加,用剂量为9μg·mL~(-1)的纳米银处理75 min后,细菌的呼吸作用基本停止。
采用电导率法测定纳米银作用后甘蓝黑腐病菌菌悬液的电导率,以研究细菌细胞膜透性的变化。结果表明,经纳米银处理后细菌悬液的电导率快速上升,表明了纳米银具有使细菌细胞膜功能受损、胞内电解质快速外渗的作用。
胞外纤维素酶活性和胞外多糖产量与植物病原细菌的致病性关系密切。对甘蓝黑腐病菌胞外纤维素酶活性和胞外多糖产量的研究表明,在纳米银的作用下,甘蓝黑腐病菌胞外纤维素酶活性比对照下降了约34%,说明纳米银具有降低胞外纤维素酶活性的作用,但纳米银并不影响甘蓝黑腐病菌胞外多糖的产量。
通过盆栽试验发现,纳米银对十字花科蔬菜黑腐病有较好的防治效果。用纳米银浓度为30μg·mL~(-1)的悬浮液处理后,对黑腐病的防治效果达67.67%;纳米银对供试植物的安全性观察结果表明,30μg·mL~(-1)的纳米银药液对小白菜苗的生长无不良的影响。
In this thesis,the inhibitory activities of nano-silver to eight tested bacterium were bioassayed.The antibacterial mechanisms and control effect of nano-silver to plant bacteria disease were studied.It could provide some theoretical basis for research and application of nano-silver bactericide
The nano-silver for its inhibitory effects to eight plant pathogenic bacterium were evaluated by the bacteriostatic circle,minimum inhibitory concentration(MIC) and plate cultural count.The results showed that the nano-silver had obviously inhibitory effect on the bacterium of Xanthomonas spp.and Ralstonia solanacearum.The EC_(50) values of nano-silver were 0.0820,0.1889,0.1779,0.1817 and 0.1820μg·mL~(-1) on X. axonopodis pv.citri,X.oryzae pv.oryzae,X.oryzae pv.Oryzicola,X.campestris pv. campestris and R.solanacearum,respectively.While the nano-silver showed less inhibitory effects on the other three bacterium:Acidovorax avenae subsp,avenae (EC_(50)=0.4042μg·mL~(-1)),Pectobacterium.carotovora subsp,carotovora(EC_(50) =0.2654μg·mL~(-1)) and Acidovorax avenae subsp,citrulli(EC_(50)=0.2370μg·mL~(-1)).
The study of inhibitory effect of nano-silver on the growth of X.campestris pv. campestris showed under the dosage 6μg·mL~(-1) of nano-silver,the population of bacteria had reduced significantly after exposed to nano-silver for 0.5 h.
Nano-silver could obviously suppress the respiration of X.campestris pv. campestris.The bacteria oxygen respiration was suppressed intensively in the process of time.After exposure for 75 min,the respiration of bacteria basic stopped.
The cells membrane damage of X.campestris pv.campestris was evaluated to analyze the change of the tested bacteria cells membrane infiltration by conductance method.The result indicated that the conductivity of X.campestris pv.campestris increased fast,showing nano-silver could destroy bacteria cells membrane function and cause cells electrolyte exosmose fast.
The pathogenicity of plant bacterium was related to the cellulose enzyme activity and extracellular polysaccharides.The study of the cellulose enzyme activity and extracellular polysaccharides output of X.campestris pv.campestris showed that the cellulose enzyme activity of bacteria decreased about 34%compared with control, but there were little effects on the output of extracellular polysaccharides of X. campestris pv.campestris.
The result of the pot experiment evaluated nano-silver had control effect on cabbage black rot.At the concentration of 30μg·mL~(-1),the control effect was 67.67% on cabbage black rot.The security observation of nano-silver indicated the nano-silver showed no harmful to the growth of chinese cabbage.
分别采用抑菌圈法、最小抑制浓度法(MIC)和平板菌落计数法,测定了纳米银对供试的8种植物病原细菌的抑制效果。结果表明,纳米银对8种供试细菌都有一定的抑制作用,说明纳米银作为新型杀菌剂具有较为广谱的杀菌效果。平板菌落计数法毒力测定结果显示,纳米银对黄单胞菌属(Xanthomonas)的4种细菌和茄青枯病菌(Ralstonia solanacearum)有较强的抑制效果,其中,对柑橘溃疡病菌(Xanthomonas axonopodis pv.citri)、稻白叶枯病菌(Xanthomonas oryzae pv.oryzae)、稻细菌性条斑病菌(Xanthomonas oryae pv.oryzicola)、甘蓝黑腐病菌(Xanthomonas campestris pv.campestris)和茄青枯病菌的EC_(50)分别为0.0820μg·mL~(-1)、0.1889μg·mL~(-1)、0.1779μg·mL~(-1)、0.1817μg·mL~(-1)和0.1820μg·mL~(-1);而纳米银对其它属的供试植物病原细菌的抑制效果则较弱,其中对玉米细菌性条斑病菌(Acidovorax avenae subsp.avenae)的EC_(50)为0.4042μg·mL~(-1)、对白菜软腐病菌(Pectobacterium carotovora subsp.carotovora)的EC_(50)为0.2654μg·mL~(-1)、对西瓜细菌性果斑病菌(Acidovorax enae subsp.citrulli)的EC_(50)为0.2370μg·mL~(-1)。
纳米银对甘蓝黑腐病菌菌体生长抑制作用的结果表明,剂量为6μg·mL~(-1)的纳米银与菌体接触0.5 h后细菌的群体数量有明显的减少,且随着时间的推移,细菌菌体数量呈下降趋势。
纳米银能显著地抑制甘蓝黑腐病菌的氧呼吸作用,使细菌的氧呼吸量明显变小,且随着纳米银对菌体作用时间的延长,细菌氧呼吸作用的受抑制程度增加,用剂量为9μg·mL~(-1)的纳米银处理75 min后,细菌的呼吸作用基本停止。
采用电导率法测定纳米银作用后甘蓝黑腐病菌菌悬液的电导率,以研究细菌细胞膜透性的变化。结果表明,经纳米银处理后细菌悬液的电导率快速上升,表明了纳米银具有使细菌细胞膜功能受损、胞内电解质快速外渗的作用。
胞外纤维素酶活性和胞外多糖产量与植物病原细菌的致病性关系密切。对甘蓝黑腐病菌胞外纤维素酶活性和胞外多糖产量的研究表明,在纳米银的作用下,甘蓝黑腐病菌胞外纤维素酶活性比对照下降了约34%,说明纳米银具有降低胞外纤维素酶活性的作用,但纳米银并不影响甘蓝黑腐病菌胞外多糖的产量。
通过盆栽试验发现,纳米银对十字花科蔬菜黑腐病有较好的防治效果。用纳米银浓度为30μg·mL~(-1)的悬浮液处理后,对黑腐病的防治效果达67.67%;纳米银对供试植物的安全性观察结果表明,30μg·mL~(-1)的纳米银药液对小白菜苗的生长无不良的影响。
In this thesis,the inhibitory activities of nano-silver to eight tested bacterium were bioassayed.The antibacterial mechanisms and control effect of nano-silver to plant bacteria disease were studied.It could provide some theoretical basis for research and application of nano-silver bactericide
The nano-silver for its inhibitory effects to eight plant pathogenic bacterium were evaluated by the bacteriostatic circle,minimum inhibitory concentration(MIC) and plate cultural count.The results showed that the nano-silver had obviously inhibitory effect on the bacterium of Xanthomonas spp.and Ralstonia solanacearum.The EC_(50) values of nano-silver were 0.0820,0.1889,0.1779,0.1817 and 0.1820μg·mL~(-1) on X. axonopodis pv.citri,X.oryzae pv.oryzae,X.oryzae pv.Oryzicola,X.campestris pv. campestris and R.solanacearum,respectively.While the nano-silver showed less inhibitory effects on the other three bacterium:Acidovorax avenae subsp,avenae (EC_(50)=0.4042μg·mL~(-1)),Pectobacterium.carotovora subsp,carotovora(EC_(50) =0.2654μg·mL~(-1)) and Acidovorax avenae subsp,citrulli(EC_(50)=0.2370μg·mL~(-1)).
The study of inhibitory effect of nano-silver on the growth of X.campestris pv. campestris showed under the dosage 6μg·mL~(-1) of nano-silver,the population of bacteria had reduced significantly after exposed to nano-silver for 0.5 h.
Nano-silver could obviously suppress the respiration of X.campestris pv. campestris.The bacteria oxygen respiration was suppressed intensively in the process of time.After exposure for 75 min,the respiration of bacteria basic stopped.
The cells membrane damage of X.campestris pv.campestris was evaluated to analyze the change of the tested bacteria cells membrane infiltration by conductance method.The result indicated that the conductivity of X.campestris pv.campestris increased fast,showing nano-silver could destroy bacteria cells membrane function and cause cells electrolyte exosmose fast.
The pathogenicity of plant bacterium was related to the cellulose enzyme activity and extracellular polysaccharides.The study of the cellulose enzyme activity and extracellular polysaccharides output of X.campestris pv.campestris showed that the cellulose enzyme activity of bacteria decreased about 34%compared with control, but there were little effects on the output of extracellular polysaccharides of X. campestris pv.campestris.
The result of the pot experiment evaluated nano-silver had control effect on cabbage black rot.At the concentration of 30μg·mL~(-1),the control effect was 67.67% on cabbage black rot.The security observation of nano-silver indicated the nano-silver showed no harmful to the growth of chinese cabbage.
引文
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