31 6 Journal of Agricultural Research voi. xiii, no. 6 



diseased fragments of the leaf after surf ace sterilization were unsuccessful 

 at that time because bacteria or saprophytic fungi, which had entered the 

 diseased tissue at a very early stage, quickly overran the slow-growing 

 pathogen in culture. However, in October, 191 5, the disease appeared 

 on clean, vigorous plants in the greenhouse. Platings from these leaves 

 gave the first success in isolation that was achieved. The fungus thus 

 obtained grew very slowly, and produced conidia indistinguishable from 

 those found on the diseased leaves. Like the conidia found on diseased 

 leaves, these failed to germinate, and did not produce the disease when 

 sprayed on alfalfa plants. From this time on nearly all the successful 

 isolations have been made from plants in the greenhouse. However, 

 late in the autumn of 1916, successful isolations were made from vigorous 

 plants in a field near the laboratory where the progress of infection 



Fig. 4. — Pyrenopeziza medicaginis: Conidia-like structures which occasionally develop on mycelium from 



germinating ascospores. 



could be watched and isolations made at the time of the first visible 

 stages in the development of the disease. 



The method followed in making these isolations was as follows. The 

 diseased area at an early stage of development was cut from the leaf. 

 The fragments were small — not more than 5 mm. long — and they were 

 cut in such a way that the diseased area v/as not entirely surrounded 

 by healthy tissue. Much time is required by the fungus in culture to 

 cross even narrow bands of healthy tissue which may separate it from 

 the surface of the substratum. The leaf fragment was then dipped into 

 50 per cent alcohol, and sterilized on the surface in a mercuric chlorid 

 solution for from i to iK minutes. After washing, the fragments were 

 placed singly on slopes in test tubes. Petri dishes are unsatisfactory, 

 since they usually dry out before the end of the three weeks that are 

 required for the fungus to grow sufficiently to furnish transfers. The 

 culture medium most favorable for the development of mycelium appears 

 to be potato agar. 



After the ascogenous stage of the fungus had been found, efforts were 

 made to make isolations from ascospores. But no practicable method 



