棉铃疫病菌孢子囊产生及游动孢子悬液稀释的方法

STUDIES OF METHOD OF THE SPORANGIA PRODUCTION AND DIL UTION OF THE ZOOSPORE SUSPENSIONS FROM PHYTOPHTHORA BOEHMERIAE

本文报道棉铃疫病菌即苎麻疫霉菌(Phytophthora boehmeriae)孢子囊的离体生产及游动孢子悬液的稀释方法。将疫病菌菌饼置于马铃薯葡萄糖培养液(PDL)或菜豆葡萄糖培养液(BDL)中,24C条件下暗培养48—72小时,然后将菌丝块在20-22℃条件下用矿质盐溶液(MSS)更换漂洗(4次)培养10-12小时并辅以光照(日光灯);最后除去MSS,继续光照培养12小时即可得到大量孢子囊。黑暗条件可抑制孢子囊的形成而促进卵孢子的产生。培养液采用BDL优于PDL,除了产孢量更高外,突出的特点表现在菌丝块的颜色变化与产孢同步,菌丝块由淡橙色转变为紫色,指示孢子大量产生。Tris-琥珀酸盐缓冲液(Tris-Succinate Buffer,TSB,pH6.8.5.0mmol/L)是效果很好的定量稀释液,可维持游动孢子较高的运动活性。

A simple method for in vitro production of sporangia by Phytophthora boehmeriae causing cotton boll blight in China was developed, and a method for dilution of zoospore suspensions was presented as well, Large amounts of sporangia were obtained when mycelial mats, produced from Potato extract-Dextrose Liquid (PDL) or Bean extract-Dextrose Liquid (BDL) at 24 ℃ for 48—72 hr ,were rinsed with Mineral Salt Solution (MSS) four times at 20—22℃ for 10—12 h with continuous fluorescent light (cool white), and incubated for additional 12 h after drained off MSS. Darkness favored oospore whereas suppressed sporangial production by P. boehmeriae. BDB is superior to PDB in the production of oospores and sporangia by P. boehmeriae as evidenced by the amount of sporangia or oospores and in particular the fact that the color change of mycelial mats from light orange to purple was synchronous with the sporulation of P. boehmeriae. Tris-succinate buffer(pH6.8, 5.0mmol/L, TSB) was the quantitatively excellent diluent for preserving motility of zoospores of P. boehmeriae in the dilution process.