May 6, 1918 



Yellow-Leaf blotch of Alfalfa 



317 



of obtaining cultures free from bacteria from ascospores from apothecia 

 on dead leaves was devised. When plated out in agar, the ascospores 

 refuse to germinate. "When discharged on an agar surface, germination 

 was not vigorous and bacteria were almost always present. A few 

 germinating single spores were obtained free from bacteria, but they 

 did not develop into cultures. In fact, no cultures from single spores 

 of this fungus have yet been obtained. 



Later, however, a method of obtaining apothecia under conditions of 

 pure culture made it possible to secure cultures from ascospores. While 

 isolations from the diseased plants in the greenhouse were being made 

 repeatedly to determine if the fungus which had been obtained was con- 

 stantly associated with the disease, various culture media were tried out. 

 In the course of these experiments it was found that when agar with no 



Fig. s. — Pyrenopeziza medicaginis: Semidiagrammatic section of an apothecium. The tissue of the leaf has 

 been largely replaced by the fungus hyphje and stroma. 



nutrient material added was used as a culture medium for isolation very 

 little mycelial growth developed outside the diseased leaf fragment, but 

 that in many cases apothecia were produced. A little less than half of 

 the leaf fragments thus treated usually developed apothecia in about 

 three weeks if kept at a temperature of 1 8 to 20° C. Above 22° apothecia 

 occur less frequently and are not so well developed. On many of these 

 leaf fragments conidia are not produced abundantly along with the 

 apothecia. Thus, by selection, apothecia can be obtained which will 

 discharge in great abundance spores free from conidia or the spores of 

 any foreign organism. From these spores discharged on an agar surface 

 cultures can easily be obtained. The peculiar behavior of these cultures 

 which distinguishes them from those obtained from mycelium in the leaf 

 tissue will be noted in the following section. 



