340 Edwin F. Hopkins 



(1903 a: 24) demonstrated the pathogenicity of the organism by his investi- 

 gations, for he did not use pure-culture methods, but merely developed 

 Botrytis conidia on leaves in moist chambers and then dusted the conidia 

 on sliced bulbs, causing the bulbs thus treated to decay. 



Experimental methods 



The writer made isolations of the fungus from material obtained from 

 many localities in Holland, Germany, England, Canada, and the United 

 States. These isolations were made both from dry specimens (for it was 

 found that the fungus would retain its vitality for a long time without 

 moisture) and from recently infected plants. Moreover, they were made 

 from sclerotia, mycelium, and conidia from various parts of the host — 

 bulbs, stalks, leaves, buds, perianth, and stamens. 



Although Klebahn (1905:12) found it difficult to obtain pure cultures 

 from the sclerotia, the writer has experienced little difficulty in isolating 

 from sclerotia by the following method: The sclerotium is rubbed free 

 from all adhering material with a clean piece of cheesecloth, and is dipped 

 for instant in 95-per-cent alcohol to remove the surface film of air, that is, 

 to wet the surface. It is then placed in a 1 : 1000 mercuric-chloride solution 

 for about thirty seconds, after which it is quickly removed with sterilized 

 forceps and placed in a drop of sterilized water in a sterilized petri dish. 

 To thoroughly remove the mercuric chloride, the sclerotium is then rinsed 

 in several successive drops of sterilized water in the same petri dish. 

 Usually six washings are sufficient. The sclerotium, thus prepared, is 

 cut into four pieces and planted on a poured plate of potato-dextrose 

 agar. The whole operation should not take more than five minutes. The 

 writer has used this method in isolating several hundred Botrytis specimens 

 as well as specimens of other fungi, and rarely has a contamination occurred. 



Fungi may be isolated from leaf tissue in this way if care is taken not 

 to leave the material too long in the alcohol. In these experiments the 

 mycelium was usually isolated from the leaf tissue and the stems by 

 cleaning the epidermis with alcohol and then peeling it back, or often, 

 when using stems and bulbs, by breaking or splitting them open so as to 

 expose an uncontaminated surface. Small parts of the diseased tissue 

 were then picked out with a sterilized, sharp-pointed scalpel and planted 

 in agar. 



