Nonspecific adhesion of Bipolaris sorokiniana sporelings
Nonspecific adhesion of Bipolaris sorokiniana sporelings
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Bipolaris sorokiniana 孢子体的非特异性粘附
DOI：
10.1080/07060668109501396
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发表时间：
1981
期刊：
影响因子：
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通讯作者：
R. B. Pringle
中科院分区：
文献类型：
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作者：
R. B. Pringle
材料和方法 Cochliobolus sativus (Ito & Kurib.) Drechsl 的十四个分离株。使用来自毒素研究 (6) 的前 Dastur 鉴定为 Helminthosporium sativum Pamm.、King & Bakke 或 Bipolaris sorokiniana(Sacc. in Sorok) Shoem.。 SAF 制剂的孢子和涂片用高碘酸希夫染色 (PAS)(2) 进行染色。该程序的一个变体用于对凝胶电泳板进行染色 (10)。通过将半个玻璃显微镜载玻片（纵向切割）放入 125 mL Delong 烧瓶中的 35 mL Fries 改良培养基 No. 3 (7) 中，使其靠在壁上，部分浸没，对玻璃上的真菌附着进行定性检查。高压灭菌后，将 1 mL 分生孢子悬浮液（约 10 个孢子）接种到冷却的烧瓶中，并在摇床上于 25°C 下培养，摇床缓慢旋转，使孢子沿载玻片长度上下冲洗。经过不同时间段后，将载玻片从烧瓶中取出，用新鲜无菌Fries'3号培养基冲洗并进行显微镜检查。所有玻璃器皿在使用前均用H2SO4-重铬酸盐溶液清洗并用玻璃蒸馏水彻底冲洗。通过修改 Reissig 等人 (8) 开发的程序，对粘附在玻璃上的孢子数量进行定量测量。在实验开始时和不同时间长度后，通过在库尔特计数器（Coulter Electronics，Inc.，Hialeah Fl 33010）中计数来确定悬浮液中的孢子数。悬浮液中孢子数量的减少代表通过紧固到载玻片和烧瓶壁上而去除的孢子数量。 为了生产 SAF，将各种真菌分离株在 1800 mL Fernbach 烧瓶中的 1800 mL Fernbach 烧瓶中的 1000 mL 批 Fries' 3 基础培养基中以 25±IC5 浓度在 60 rpm 旋转的摇床上生长 5 天。通过布氏漏斗上的 Miracloth 过滤除去菌丝体，用 100 mL 份的水洗涤 3 次，并在 105°C 的热风烘箱中干燥至恒重。将等体积的冷甲醇添加到冷却的培养物滤液中，并将所得粘性纤维状沉淀物与连续 2 mL 份的 1.0 M NaCl 一起研磨。这改变了部分材料，首先变成凝胶，然后变成粘稠溶液。根据Sevaget等人(11)的方法，将通常约10mL的混合物与等体积的CHCl3-异戊醇(24:1)一起在分液漏斗中摇动多次以除去蛋白质。除去不溶于 1.0 M NaCl 的物质后，通过添加 1.3 体积的异丙醇第二次沉淀 SAF。通过离心收集该沉淀物，用1mL冰水洗涤3次，冻干并称重。在 Visking 套管中透析后，该套管保留了分子量为 10 000 或
Materials and methodsFourteen isolates of Cochliobolus sativus (Ito & Kurib.) Drechsl. ex Dastur identified as Helminthosporium sativum Pamm., King & Bakke or as Bipolaris sorokiniana(Sacc. in Sorok) Shoem., from toxin studies (6) were used. Sporelings and smears of SAF preparations were stained with periodic acid-Schiff stain (PAS)(2). A variation of this procedure was used to stain gel electrophoresis slabs (10). The fungus attachment to glass was examined qualitatively by placing half a glass microscope slide (cut lengthwise) into 35 mL of Fries' modified medium No. 3 (7) in a 125 mL Delong flask so that it leaned against the wall, partly submerged. After autoclaving, the cooled flask was inoculated with 1 mL of a conidial suspension in Fries' No. 3 medium (about 10'spores) and incubated at 25 C on a shaker, which was rotated slowly so that the spores were washed up and down the length of the slide. After various periods of time, the slide was removed from the flask, rinsed with fresh sterile Fries' No. 3 medium and examined microscopically. All glassware was cleaned with H2 S04-dichromate solution and rinsed thoroughly with glass-distilled water before use. Quantitative measurements of the number of sporelings adhering to glass were made by modification of the procedure developed by Reissig et al.(8). The number of sporelings in suspension was determined by counting in a Coulter counter (Coulter Electronics, Inc., Hialeah Fl 33010) at the beginning of the experiment and after various lengths of time. The decrease in the number of sporelings in suspension represented those removed by fastening to the slide and to the walls of the flask.For production of SAF, the various fungal isolates were grown at 25±IC in 1000 mL lots of Fries' No. 3 basal medium in 1800 mL Fernbach flasks on a shaker rotating at 60 rpm for 5 days. Mycelium was removed by filtration through Miracloth on a buchner funnel, washed three times with 100 mL portions of water and dried to constant weight in a hot air oven at 105 C. An equal volume of cold methanol was added to the chilled culture filtrate and the resulting sticky, fibrous precipitate was triturated with successive 2 mL portions of 1.0 M NaCl. This changed a portion of the material, first to a gel then to a viscous solution. The mixture, usually about 10 mL, was shaken in a separatory funnel a number of times with equal volumes of CHCl3-isoamyl alcohol (24: 1) to remove protein according to the method of Sevaget al.(11). After removing material insoluble in 1.0 M NaCl, SAF was precipitated a second time by the addition of 1.3 volumes of isopropanol. This precipitate was collected by centrifugation, washed 3 times with 1 mL of ice water, lyophilized and weighed. After dialyzing in a Visking casing, which retains substances of molecular weight 10 000 or