LEAF-SPOT OF ALFALFA AND RED CLOVER. 23 
lation trials. The results of the two methods of attacking host rela- 
tionships should be considered together. 
METHOD OF STUDY. 
The most of the data given here have been obtained by the follow- 
ing simple procedure : Leaves which have just reached full develop- 
ment are selected for inoculation. A culture of Pseudopeziza known 
to be discharging spores abundantly is supported over the leaf or one 
of the leaflets so that the spores as they are discharged will all fall 
upon it. The leaf may be removed from the plant for studies which 
do not involve a period of more than two days, since results obtained 
from such leaves have always been found by comparison to agree 
with results obtained from leaves attached to the plant. The leaf 
may be sprayed with very fine spray before the spores are discharged 
upon it, but more abundant penetrations are usually obtained if the 
spores are allowed to stick to the leaf before it is wet. When the 
leaf has been kept moistened for at least 12 hours, usually longer, 
it is removed, dropped into a mixture of equal parts of acetic acid 
and alcohol and promptly heated to the boiling point. Leaves which 
are killed promptly in this fashion decolorize in better condition than 
when slower killing takes place in cold acetic alcohol. The acetic 
alcohol is changed until all color has been removed from the leaf. 
The leaf may then be mounted in this liquid on a slide under a 
cover glass and examined under the microscope. The epidermal cells 
should be perfectly clear, and the entire structure of the leaf to its 
very center should be visible. The spores remain attached to the leaf 
during the treatment, and the method of entry and the mycelium 
within the leaf can be clearly seen. No method of staining has been 
found to improve the visibility of the fungus. 
Although this method works best in the case of alfalfa leaves, it 
works well enough with the various clovers to give entirely satis- 
factory results. 
METHOD OF PENETRATION. 
In all of the hundreds of penetrations observed the method of 
entry has invariably been as here stated. The spore is found stuck 
fast to the leaf. The germ tube emerges from the spore either within 
or at the margin of the area of contact of the spore with the leaf and 
passes directly through the cuticle into the epidermal cell. Occa- 
sionally a spore sends out its germ tube along the surface of the 
leaf, but such a germ tube has never been observed to enter the leaf. 
Apparently the germ tube must enter the leaf at the moment of 
emergence from the spore, if at all. Ordinarily there is no per- 
ceptible thickening or alteration of the wall in consequence of this 
penetration. The actual opening appears to be extremely minute. 
