20 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 
practicable method for using them has been developed. Since no 
evidence has been found indicating that they occur in nature or that 
they germinate, no great importance attaches to them. 
In the first inoculations an attempt was made to obtain the asco- 
spores in water suspension, but when the apothecia were crushed in 
water usually only a few spores became separated from the ascus. 
The few attempts to make inoculations with such meager spore sus- 
pensions failed. The method finally adopted and used with minor 
modifications in all inoculations reported takes advantage of the 
natural discharge of spores from cultures. If a Pseudopeziza cul- 
ture is removed from the test tube carefully it may be cut into frag- 
ments and placed on a support, where it will continue to discharge 
spores for several days provided it is not exposed to direct sunlight 
or high temperatures. A culture may thus be removed from a test 
tube and placed over a plant upon which the spores will fall. If the 
whole plant is to be inoculated, the culture may be placed in the top 
of a bell jar which is set over the plant and turned from time to 
time to insure a uniform distribution of the spores. 
If single leaves are to be inoculated, the culture or fragments of the 
culture may be placed for a short time over these leaves in succession. 
A more uniform discharge of spores for long periods is obtained in a 
dark room at 16° to 20° C. If inoculations are made in the field, they 
should be made at night or on a cloudy day. The plants may be wet 
with a fine spray before the spores are discharged, or if time permits 
this may be deferred until the spores are on the leaf. In the latter 
case, a larger number of infections are usually secured, due appar- 
ently to the fact that spores falling on large drops of water are held 
from sinking by surface tension and germinate too far from the leaf 
surface to effect penetration. After the inoculated plants are sprayed, 
they should be kept in a moist chamber for at least 12 hours. 
This method has been employed in all inoculations made, unless 
otherwise stated. 
CONDITIONS UNDER WHICH INOCULATIONS WERE MADE, 
If inoculations are to be entirely conclusive in result, the control 
plants must remain free from the disease. It has been found impos- 
sible to keep plants free from leaf-spot for infection experiments 
during the summer at Madison, Wis., where the work was done. This 
has been due to the fact that alfalfa fields are located so close to the 
greenhouse that spores are easily blown in through the ventilators. 
But it has been found that if all diseased alfalfa foliage was removed 
from the greenhouses in the autumn after the ground outside froze, it 
was possible to keep alfalfa plants free from infection with leaf-spot 
during the winter and spring. Therefore all inoculations have been ~ 
made or at least repeated during the winter months. This precaution 
