高渗胁迫对玉米大斑病菌生长发育的影响及菌丝细胞中渗透调节物质的分析

【目的】了解不同高渗胁迫条件对玉米大斑病菌(Setosphaeria turcica)菌丝发育及胞内黑色素含量的影响,明确在病菌菌丝细胞中起主要作用的渗透调节物质的种类及这些物质在不同高渗胁迫条件下的变化规律。【方法】采用3种不同浓度梯度(0.4、0.8、1.2 mol·L-1 Na Cl)的高渗胁迫条件处理玉米大斑病菌,分析高渗胁迫对菌落生长、菌丝发育及菌丝胞内黑色素含量的影响;利用高效液相色谱(high performance liquid chromatography,HPLC)技术测定3种高渗浓度处理下菌丝细胞中甘油、赤藓醇、葡萄糖、甘露醇、海藻糖5种多羟基醇的含量并分析其随时间变化的规律。【结果】与在普通PDA培养基上的病菌相比,在高渗胁迫条件下病菌菌落生长速率明显降低,菌丝细胞间隔变短、细胞显著膨大,且随胁迫浓度增加细胞膨大程度增强;高渗胁迫下的菌株在各个时间点的胞内黑色素含量与对照相比有明显差异,其中高渗处理12 h之前样品中黑色素的含量均比对照组低,但不同处理之间含量均很接近;而在24、48 h两个时间点不同处理浓度对黑色素的影响不同,1.2 mol·L-1Na Cl处理下与对照相比黑色素含量均显著增加,0.8 mol·L-1 Na Cl处理下与对照相比黑色素含量降低,0.4 mol·L-1Na Cl处理下样品的黑色素含量与对照相比在24 h时显著降低,但48 h时与对照基本一致;在不同的高渗胁迫条件下(0.4、0.8、1.2 mol·L-1 Na Cl)菌丝细胞中甘露醇的含量均有随处理时间延长而增加的趋势,在处理时间为36 h时甘露醇含量较对照增加最为明显,具有显著性差异;菌丝中甘油含量的变化与甘露醇的变化规律相似,且在高渗胁迫处理下甘油含量增加幅度更加明显,并在处理24 h后甘油含量较对照显著增加;菌丝中海藻糖含量总体上有随时间延长而降低的趋势,且渗透胁迫的强度越高该趋势越明显,不同浓度Na Cl处理的菌丝中海藻糖含量均在36 h与对照相比显著降低;而赤藓醇、葡萄糖的含量与对照相比没有显著变化;在普通PD培养基或高渗处理条件下,菌丝培养滤液中均没有检测到甘露醇、海藻糖和甘油的存在,而培养基中葡萄糖的含量随高渗胁迫浓度的增加而减少。【结论】高渗胁迫抑制了玉米大斑病菌菌落的生长,使菌丝细胞膨大、间隔变短;高渗胁迫处理病菌12 h之前对菌丝胞内黑色素含量均有明显的抑制作用;甘露醇、甘油为病菌菌丝细胞中的主要渗透调节物质,海藻糖也参与了病菌的高渗胁迫反应。

Effect of Hyperosmotic Stress on Growth and Development of Setosphaeria turcica and Determination of Osmolytes in the Mycelium Cells of the Pathogen

【Objective】The objective of this study is to understand the effect of hyperosmotic stress on the growth and development, melanin content in mycelium cells, determine the probable osmolytes in the mycelium cells and to clarify the variation rule of these substances under different hyperosmotic stress conditions in Setosphaeria turcica. 【Method】The effects of hyperosmotic stress on colony growth rate, mycelium morphological characteristics and melanin content in S. turcica mycelium cell were analyzed under different hyperosmotic stress media, in which 0.4, 0.8, and 1.2 mol·L^-1 NaCl were added in PDA medium. High performance liquid chromatography (HPLC) technology was employed to detect the content of polyhydroxy-alcohol including glycerol, erythrol, glucose, mannitol, trehalose and analyzed the change profiles of these substances as time increased. 【Result】Compared to the strain cultured on PDA medium, the strain cultured under hyperosmotic stress treatments appeared with decreased in colony growth rate, shortened septum and swollen cells. Melanin content in mycelium cells cultured under hyperosmotic stress treatments in all time points showed a significant difference compared to the control group, in which melanin content in the samples treated before 12 h were lower than that in control, but there were no obvious differences between the samples treated. Moreover, there were different effects under different times (24, 48 h) and different concentration treatments. Compared to the control group, melanin content in the samples treated with 1.2 mol·L^-1 NaCl was significantly increased, melanin content treated with 0.8 mol·L^-1 NaCl was significantly decreased, melanin content in samples treated with 0.4 mol·L^-1 NaCl was significantly decreased in 24 h, but the content in the samples after 48 h treatment was almost the same with the control. Mannitol contents in mycelium cell under different hyperosmotic stress treatments were increased with the increase in time, especially the mannitol content significantly increased after 36 h treatment. There was a similar change tendency in glycerol and mannitol contents in mycelium cells under different treatments. Glycerol content increased most obviously compared with the control group and the content was significantly increased after 24 h treatment. Trehalose content in mycelium cells showed an increasing tendency as time increased, and the tendency increased with the increase of stress intensity, however, trehalose content after 36 h treatment was significantly lower than that in control group, while no significant difference was found in the changes of erythrol and glucose contents. No mannitol, trehalose and glycerol were detected in culture filtrate, while glucose content had no significant change no matter the fact that the mycelium was cultured in PDA medium or under hyperosmotic stress treatments.【Conclusion】 The colony growth rate was inhibited, mycelium cell was swollen, and septum was shortened under hyperosmotic stress. There was a significant inhibition on mannitol content before 12 h treatment. Mannitol and glycerol were main osmolytes in mycelium cells. Trehalose was also involved in hyperosmotic stress reaction in S. turcica.