MICHIGAN ACADEMY OF SCIENCE. 



300 



Table 10. 



Toxic filtrate on Clover Leaflets. 



Test with Wounded and Sound Leaves under Van Tieghem Cells. 



'This was taken from the same lot of culture medium which was used for the above 

 culture. 



It may be stated here that while contamination of the liquid in the 

 cells was to be expected, there was no evidence of fungus growth at the 

 end of the 56 hour period. A hanging drop of the sterile clover juice 

 used as a check showed numerous bacteria, but these apparently had no 

 effect even upon the wounded leaflets. The filtrate which caused the 

 discoloration contained but a few bacteria. It is therefore reasonable 

 to suppose that the contaminating organism played no part in the dis- 

 coloration of the tissue. Microscopic examination of the discolored 

 tissues did not reveal any fungous growth and no growth appeared on 

 the agar to which transfers had been made from the filtrate. Four days 

 after the first series of tests were started a similar series was set up, 

 using the same filtrate as before which in the meantime liad been kept 

 sterile in the filter flask. In this series, practically the same results as 

 before, Mere obtained. From this experiment there seems no doubt that 

 the clover pathogene produces a substance which can kill the wounded 

 clover leaves. 



The Nature of the Toxic Substance : 



The purpose of this experiment was, (1) to determine whether the 

 toxic substance is an enzyme; (2) whether any toxic substance is secreted 

 within the fungus itself; (3) a repetition of experiment 1; and (4) the 

 effect of heating upon toxicity. 



In this case a clover-juice culture 32 days old was used. The super- 

 ficial fungous growth was removed by passing through a filter paper, 

 thereby obtaining a filtrate of 80 cc. volume. To this was added 300 cc. 

 of 95% alcohol, and the precipitate thus formed allowed to settle for 

 one hour. This flocculent, brown precipitate was filtered off and more 



