Mayi5. I9IS Seedling Diseases of Stigar Beets 137 



which remained healthy, but he makes no mention of treating the seed 

 to insure the eUmination of Phoma hetae, vfhich. he regarded as absent 

 (10, p. 344). 



THE SECURING OF CULTURES 



The first steps in the present work were naturally the securing of cul- 

 tures, which were obtained in various ways. Beet seed was placed in 

 sterilized filter papers in sterile moist chambers, and transfers were made 

 from the colonies of fungi which developed during the progress of ger- 

 mination. This method is uncertain and yields a large number of 

 harmless saprophytes. Seed was sown in soil which had been sterilized 

 in an autoclave at 1 2 pounds' pressure for from four to six hours on two 

 successive days. These were watered with sterile water and protected 

 from outside sources of infection. Whenever damping-off appeared, 

 the diseased seedlings were removed and treated for one minute with 

 more or less shaking in a solution (1:1 ,000) of bichlorid of mercury in water 

 or in similar bichlorid solutions containing either i gm. of ammonium 

 chlorid or % c. c. of concentrated hydrochloric acid per liter. They 

 were then rinsed in sterilized water and dropped upon suitable nutrient- 

 agar medium in Petri dishes (PI. XVI and XVII). The agar most 

 commonly used is sufficiently acid to materially check the development of 

 bacteria and is at the same time a very satisfactory medium for the 

 cultivation of most fungi. It has the following composition: 



Dextrose 100 Dipotassium hydrogen 



Peptone 5 phosphate 2. 5 



Ammonium nitrate 10 Calcium chlorid .1 



Potassium nitrate 5 Water i, 000 



Magnesium sulphate. ... 2. 5 Agar 20 



As soon as growth appeared from the seedlings, isolation transfers were 

 made. Cultures obtained by these two methods were regarded as origi- 

 nating from the seed. 



Cultures were made from the soil indirectly by means of seedlings in 

 the following manner: Beet seed, treated by a method to be discussed 

 later, so as to insure the absence of parasitic fungi, was sown in unster- 

 ilized soil in pots that were thoroughly sterilized before using. When 

 damping-off occurred, isolations were made in the manner already 

 described. Cultures were secured from decayed beets by cutting out 

 with a sterile knife small portions of material on the border line between 

 healthy and diseased tissue. These blocks were placed upon a suitable 

 medium, and isolations were made from the developing colonies. Another 

 method employed was to sow treated beet seed in sterilized soil, subse- 

 quently infected with fragments of decaying beets. Isolations were then 

 made from the seedlings when disease developed. Large numbers of 

 isolations were made from sugar-beet seedlings grown in commercial 

 fields, and a considerable number of cultures were courteously contrib- 

 uted by various workers from time to time. 



