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Alternaria brassicicola inoculation on Arabidopsis

Author: Jiali Wang, updated date: , view: 15, Q&A: 0

Procedure

 

1.      -80度冰箱取出20%甘油保存的Alternaria brassicicola菌块,接到V8培养基平板上培养;

2.      培养7点后,刮一小片带有菌丝和孢子的培养基,转接到一块新的V8培养基平板上;(最好不要用从-80度冰箱拿出来的第一代菌,第二代到第12代的菌都可以用)

3.      选取适量培养14d左右的Alternaria brassicicola50ml离心管中

4.      用双蒸水配制0.1% non-fat milk,加入适量于离心管中,通过涡旋仪震荡将孢子洗脱,并用纱布过滤,收集孢子过滤液;

5.      摇匀菌液后吸取适量菌液点在血球计数板上,进行镜检计数,然后将菌液稀释至2.5*105个孢子/ml(可适量提高);

6.      将菌液倒入小烧杯中放在磁力搅拌器上混匀备用;(由于孢子容易沉底,在吸取菌液接种植物时,搅拌器不能停)

7.      选择生长35d左右的新鲜拟南芥叶片放入保湿环境良好的容器中,各吸取8ul菌液分别点在叶片左边的中央,然后盖上塑料透明盖子,用透明胶带封闭容器放入拟南芥培养箱(22 °C,12小时光照/12小时黑暗)中5-7d后观察;

8.      测量病斑的直接(横向),并拍照确定表型;


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