200 Journal of Agricultural Research voi. xiii, no. 3 



ISOLATION OF THE CAUSAL ORGANISM 



Numerous free-hand sections of diseased leaves were made from what 

 appeared to be the early stages of the disease — that is, the small browa 

 spots and the adjacent portion of leaf tissue. In a few of these sections 

 a delicate, frequently-branching mycelium was obser^-ed. Failure to 

 find the fungus in a larger portion of the sections studied was undoubt- 

 edly due to the fact that it is not found far beyond the browned areas, 

 the reduction of the chlorophyll beyond these areas being due possibly 

 to toxic products of the fungus growth. 



About a dozen of the diseased leaves in all stages of infection were 

 placed without surface sterilization on moist filter paper in petri dishes, 

 which were kept imder large bell jars. Another set of dishes was 

 arranged in the same way, except that the leaves were surface-sterilized 

 by immersing them for one to five minutes in i to 1,000 mercuric- 

 chlorid solution. The petri dishes and filter papers used were also 

 sterilized. After a few days the leaves which had not been sterilized 

 were covered vvith dense mycelial growths of a variety of fungi : Species 

 of Altemaria, Pestalozzia, Cephalotheciuni, Aspergillus, etc. Numerous 

 pycnidia were developing on a few of the leaves. Some of these were 

 found to be the fruiting bodies of Phyllosticta spp. However, the major- 

 ity did not appear to belong to this genus, but they were at this time 

 too immature for examination. After about two weeks it was evident 

 that they were not the pycnidia of Phyllosticta spp. Yellow spore masses 

 in the form of cirri were extruding from them. These cirri were found 

 to consist of spores of two kinds: An oval, hyalin, i -celled spore with 

 two or more definite oil drops, and a much longer, filiform, curved or 

 hooked spore, also hyalin and i -celled. 



In the meantime those leaves which had been sterilized before being 

 placed in the sterile moist petri dishes showed none of the abundant 

 fungus mycelium present on the unsterilized leaves, but did show numer- 

 ous pycnidia of the same type as some of those found on the latter, and 

 they were also extruding the same kind of long, yellow cirri. These 

 cirri were found to contain the two kinds of spores just described. 



Petri-dish poured plates were made in the usual way — that is, the 

 leaves were first rinsed in alcohol, then immersed in a i to 1,000 mer- 

 curic-chlorid solution. After rinsing in sterile water, small pieces of leaf 

 tissue representing the various stages of infection were cut out and 

 placed in the dishes, over which was poured corn-meal agar and syn- 

 thetic agar. In both media there appeared after three to four days a 

 delicate, white mycelial growth. Transfers were made to corn-meal agar, 

 potato, etc. After about two weeks those transfers made on potato 

 cylinders showed numerous, dark-brown fruiting bodies which were 

 beginning to extrude cirri in the same way as the pycnidia on the leaves. 

 Microscopic examination showed these spores to be of the same character 

 as those found on the leaves placed in damp chambers. 



