12 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 
taking advantage of the fact that when the ascospores are dis- 
charged from the ascus they are thrown several millimeters verti- 
cally. Repeated trials showed that if fresh leaves were used, spores 
could sometimes be obtained in considerable numbers on an agar 
surface placed over the apothecia without bacterial or fungous con- 
tamination. This work of collecting spores was best carried out in 
a Petri dish in which a layer of very clear agar had been poured. 
The dish was inverted and the leaf bearing the apothecia supported 
2 or 3 millimeters below the agar. After a short period the area over 
the apothecia was marked, the dish turned, and examination made 
with the low power of the microscope to determine whether the 
requisite number of spores were present. The difficulty would have 
been lessened had a few spores been sufficient for the development 
of a culture, but experience soon showed that a large number of 
fungous colonies crowded together developed better than a few. 
When several areas on the plate had been scattered with spores, the 
leaf bearing the apothecia was transferred to another plate. By using 
the utmost care and exposing a large number of plates a few could 
be obtained without contamination or with so small a number of 
foreign organisms that they could be cut out with a sterile needle. 
After a plate had been observed until it appeared certain that no 
foreign organism was present, it was found advisable in order to 
prevent dr} T ing to cut out the area bearing the developing fungous 
colonies and transfer them to agar slopes in test tubes. 
In the course of experiments with cultures made in this way the 
first culture of Pseudopeziza from alfalfa to produce apothecia was 
obtained. The spores were discharged on an alfalfa-agar plate on 
October 5, and the agar was transferred to a water-agar slope on 
October 22. The ascospores were being produced on November 6. 
At first it was assumed that the fungus had been starved into fruiting 
by this process, but later work does not indicate that this was the 
case. Fruiting cultures can be obtained most readily by transferring 
the developing fungous colonies as soon as they become macroscopic 
from the water-agar plates to oatmeal-agar slopes. In this way the 
fungus was isolated six times in the autumn of 1914 and once in the 
autumn of 1916. 
Pseudopeziza was isolated from red clover in the same way as 
from alfalfa. Two isolations were made of this fungus in 1915, one 
from clover leaves collected by Prof. H. H. Whetzel at Ithaca, N". Y., 
and one from clover collected in Door County, Wis. 
A later successful reisolation of this fungus from plants inoculated 
in the greenhouse suggests that it may not always be necessary to 
employ this tedious process. In the instance referred to, diseased 
leaf fragments were cut from clover leaves two weeks after inocula- 
tion. The fragments, each bearing from one to three infections, were 
