312 



NINETEENTH REPORT. 



When the filtrate was titrated, it was found to be 9° alkaline (Fuller's 

 scale). A test with Nessler's solution gave indication of the presence of 

 ammonia, wliich upon quantitative analysis was determined as .013% 

 NHy. It was thought that the ammonia might be the cause of the dis- 

 coloration of the tissue. To determine this, leaves both wounded and 

 unwounded were subjected to the action of concentrations of ammonia 

 equaling .01%, .02%, and .05% respectively, using the same setup as 

 in the preceding experiments. No signs of discoloration appeared within 

 three days, proving that at least the ammonia alone is not the toxic prin- 

 ciple. 



This series of experiments was later repeated. For this purpose, clover 

 juice cultures similar to those tested before, were used. These were 

 inoculated October 11th, just six weeks after the cultures used in the 

 preceding experiment were inoculated. The filtrate was prepared as 

 before, at intervals when the cultures were four, five, and seven weeks 

 old. In no case, however, were the leaves in any way discolored as a 

 result of their contact with these filtrates. Likewise the filtrate from a 

 culture in Dunham's peptone solution had no effect upon the leaves, 

 although it contained considerable ammonia. 



A probable cause for this apparent loss in toxicity was later discovered. 

 It was observed that inoculations which were made from time to time 

 during the months of October and November failed to produce positive 

 infection. Inoculations were made repeatedly under a variety of condi- 

 tions but without success. It was therefore evident that the fungus had 

 lost much, if not all, of its virulence. In order to make certain of this, 

 a fresh strain of the fungus was isolated from material collected during 

 the summer. Four sets of red clover plants of the same stage of growth 

 were selected. These were then inoculated with both the old and the 

 new cultures as shown in the following table: 



Table 12. 



Test of Old and New Isolation Plants Under Bell Jars with Saturated 



Atmosphere. 



Culture. 



Set 



3 



4 



Old 



Old 



New 



New 



Treatment. 



Condition after 7 days. 



Sprayed with suspension of No infection 



spores 



Mass of fungus growth placed Discoloration only under in- 



upon leaves and covered with oculuma 



cotton _ 



Sprayed with suspension of Tlbundance of typical in- 



spores j fections 



Mass of fungus growth placed Abundant typical infections 

 upon leaves and covered with 

 cotton ! 



a Spores show germination sprouts, barely enter leaf, then stop growing. 



