ONION SMUT FUNGUS. 107 



Fresh Spores in Soil Decoction Agar. — After the role playerl by air was 

 determined, it seemed that the writer's previous failure to induce germina- 

 tion in soil decoction might have been due to exclusion of air. Therefore a 

 medium was prepared by adding 2 per cent of agar to the soil decoction. 

 Tests were made as with the onion agar, using soil decoction, however, 

 for floating the spores over the surface. After five days, germination of 

 1 to 2 per cent was observed. With each day, however, more of them 

 germinated, and this continued for several weeks until the plates became 

 too dry or were exhausted. We may conclude from these experiments 

 that (1) the soil contains all the essential sthnulating elements for ger- 

 mination, and (2) not all the spores germinate at once, but there is a pro- 

 gressive preparation. 



Fresh Spores on Dung Decoction Agar. — This medium was prepared by 

 adding 2 per cent of agar to the dung decoction mentioned above. Since 

 the soil used in making the soil decoction had been heavily manured dur- 

 ing the previous season, it was thought that some element in the manure 

 might furnish the stimulus and a higher percentage of germination would 

 be secured. The percentage of germination in this medium, however, was 

 scarcely as high as for the soil decoction. Here is proof, however, that 

 stable manure contains some substance which is capable of inducing 

 germination. 



Effect of Freezing the Spores. — ■ It has been previously mentioned that 

 Thaxter froze smutted mature onions in the soil and then found the spores 

 capable of germination. This experiment was duplicated as nearly as 

 possible by the writer, but he was entirely unable to get the spores free 

 from bacteria and other fungi, and abandoned the method rather than 

 work ^\'ith contaminated cultures. 



An attempt was next made to freeze the spores under sterile conditions. 

 Smutted seedlings were sterihzed with mercuric chloride 1 to 1,000, washed 

 in sterile water, and sealed in sterile test tubes with a drop of water in the 

 bottom of each tube. After being exposed for nine days during December, 

 during which there were some light freezes, they were tested in onion 

 decoction. There was a germination of about 2 per cent. In similar tests 

 during January, in which they were frozen soUd for ten days or more, 

 buried under the snow in zero weather, the spores were apparently killed. 

 No germination at all was observed, although tried on or in the various 

 media described above. In \dew of the fact that the mycelium in culture 

 is not killed by freezing, these results are difficult to explain. In a later 

 series of tests smutted seedUngs, sterihzed on the surface, were buried in 

 sterile soil in test tubes and then frozen out of doors for eight weeks. On 

 onion decoction agar plates, varying percentages of germination were then 

 secured, but it was never as high as for spores which had been kept in damp 

 soil during the same length of time, but not frozen. The conclusion seems 

 warranted that freezing does not kill spores in the soil, but it does not 

 render them more capable of germination, and is not necessary. 



Effect of a Period of Rest in Damp Earth. — Seedlings with unopened 

 lesions were sterilized and buried in sterile soil in test tubes. The tubes 



