22 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 
eased alfalfa plants have never shown a trace of this leaf-spot during the two 
years that these diseases have been under observation. 
Trifolium pratense with Pseudopeziza trifoliit.—infection from inoculation 
with spores from pure cultures has been at all times easy under greenhouse 
conditions. Frequently such abundant infection has been obtained that the 
leaves are killed before the spots develop to a fruiting stage. ‘The incuba- 
tion period of the disease appears to be slightly longer than that of Pseudope- 
ciza medicaginis, varying somewhat with the abundance of the infection. If a 
leaf is heavily infected, the individual infections appear to develop more 
rapidly and produce small characteristic killed areas earlier than if infections 
are few. Numerous infections appear in 11 or 12 days, and in the greenhouse the 
leaf frequently dies a few days later. Fruiting bodies have not been observed 
on such leaves, but a reisolation has been made from infections two weeks after 
inoculation. f 
Less numerous infections may not show for two weeks or even longer, but 
at the end of three weeks or more they may produce typical apothecia if the 
air of the greenhouse is sufficiently moist. These apothecia have been found 
on leaves nearly or quite dead. In this respect the fungus behaves differently 
than does Pseudopeziza medicaginis. Apothecia on dead leaves are very diffi- 
cult to discover unless the leaf has been in a moist atmosphere for some time, 
when they appear as dark-amber gelatinous masses on the leaf surface. The 
exposure of these gelatinous masses of asci to dry air for even a few minutes 
causes them to discharge the larger part of the spores present and to shrink 
to a minute mass only a little darker in color than the leaf and therefore 
difficult to identify. The best development of apothecia has been obtained by 
placing infected plants outside the greenhouse during protracted periods of rainy 
weather. 
Trifolium hybridum with Pseudopeziza medicaginis.—No success in obtaining 
visible infections has been attained. 
Trifolium hybridum with Pseudopeziza trifolii.mOf the several plants inocu- 
lated only one survived in a vigorous condition for a sufficient period to show 
infection. This showed an abundant spotting, which was in every way charac- 
teristic of the Pseudopeziza spot on red clover except that the spot appeared to 
be somewhat limited in development by the veins, thus showing a slight ten- 
dency to become angular. No fruiting bodies were produced. Apparently 
infection takes place only under the most favorable circumstances. This plant 
appears to be a much less congenial host than red clover. 
GERMINATION OF THE SPORES ON THE LEAF, 
While study was being made of the leaf-spot fungus in the host 
tissue it was found that the mode of penetration could be observed 
very readily by decolorizing the leaf soon after inoculation was 
made. This method of study was used, not only to determine the nor- 
mal penetration of these fungi into their own hosts, but to determine 
the relation of these parasites to other closely related plants reputed 
to be hosts of these fungi but upon which infection had not been 
obtained. In case preliminary inoculations failed to give visible 
results it was more simple and rapid to determine whether the spores 
of that fungus could penetrate the host in question and develop after 
penetration had taken place than to conduct other extensive inocula- 
tions. Thus, a study of penetration has formed a part of all inocu- 
