MICHIGAN ACADEMY OF SCIENCE. 313 



This comparative test of the two cultures was repeated under more 

 exact conditions as follows : Two pots of red clover, each containing 

 numerous plants, all planted at the same time, from the same lot of seed 

 were used. One pot of plants was sprayed thoroughly with a spore 

 suspension from the old culture, and the other was sprayed with a suspen- 

 sion of spores from the new culture. In both cases, the quantity of 

 spores sprayed upon each plant, was made as nearly as possible the 

 same. Each pot was kept under a separate bell jar, and the atmosphere 

 maintained in a saturated condition. The plants sprayed with spores 

 from the new culture began to develop the disease upon the fifth day 

 after inoculation. After 10 days, these plants were heavily infected, 

 while those inoculated with spores from the old culture showed 

 only a few isolated cases of poorly developed spots. Examination of 

 some of the uninfected leaflets of the latter plants revealed many spores 

 on the surface which had not entered the host. The difference between 

 the degree of infection caused by the. old culture and the new culture is 

 illustrated by Plates XV and XVI which were taken after 10 days.* 



From the results of such a comparative experiment, we cannot but 

 conclude that the old culture has lost much, if not all, of its virulence. 

 This attenuation may be due to the prolonged saprophytic habit which 

 the fungus has been subject to while growing on culture media. It is 

 commonly asserted that parasitic fungi lose their virulence when kept 

 in culture for a long time, but exact experimental evidence is not at 

 hand. 



In view of the apparent attenuation of the fungus virulence, the 

 coincident failure of the filtrate from culture liquid to have any effect upon 

 the leaf tissue, may be explained. Time has not permitted a compara- 

 tive study of the filtrate from both the old and the new cultures. Sucli 

 a test would be the only way of determining definitely the relation be- 

 tween the loss in virulence and the toxic activity of the by-product of 

 the fungus. 



Perhaps conclusions are not justified on the basis of a few experiments 

 which are only preliminary. The following is a summary of the experi- 

 ments : 



1. The fungus secretes a substance in the culture medium, which 



discolors and finally kills the wounded leaves. 



2. This toxic substance is inactivated by boiling. 



3. The ammonia present in the culture liquid is not, at least by 



itself, the toxic principle. 



*Dr. Coons informs me tluit lie lias since repeated the experiment with similar results. 



