32 BULLETIN 759, U. S. DEPARTMENT OF AGRICULTURE. 
fungous colonies were found developing upon the seed coats, whether 
they remained attached to the cotyledon leaves or fell to the agar. 
In no case were the cotyledon leaves attacked. Subsequent attempts 
to infect cotyledon leaves of seedlings did not produce macroscopic 
lesions. The seed coats bearing the minute fungous colonies were 
subsequently transferred to fresh agar slopes to keep them moist, and 
finally they developed minute apothecia. This development was so 
slow, however, that it is doubtful whether it could proceed so far 
under field conditions, where periods of drying would occur and com- 
petition with other fungi would be encountered. 
Efforts to grow the fungus on soil sterilized or unsterilized have 
been entirely unsuccessful. 
If the fungus occurs on the outside of the seed or in débris it can 
easily be destroyed by the surface sterilization of the seed. If such 
treated seed can then be grown under conditions which will exclude 
other sources of infection and which will also be favorable for the 
development of the fungus, the occurrence of the disease will indi- 
cate that the fungus is carried within the seed. - 
A satisfactory method of seed sterilization has been worked out 
by Mr. A. H. Gilbert (in an unpublished manuscript). He found 
that treatment with a solution of 1 part of bichlorid of mercury in 
1,000 parts of water for five minutes rendered the seed sterile, while 
treatment for 10 minutes injured the seed. These treatments were 
repeated, and it was found that treatment for eight minutes was 
more than sufficient to render the seeds sterile without injury, pro- 
vided they were washed promptly after treatment. All sterilized 
seed mentioned in the experiments here described were treated in 
this way. 
Suitable conditions for growing the treated seed were difficult to 
obtain. Two places were tried—in the greenhouse during the winter 
months and in the open field in localities as remote as possible from 
other alfalfa. Experience in the greenhouse in the winter of 1915 
showed that unless great care was taken with infected plants, the 
fungus was likely to occur occasionally on other alfalfa plants in the 
same house. During the following two winters all inoculated plants 
were cared for so thoroughly that in not a single instance did the leaf- 
spot develop upon any other plant in the houses until the disease 
appeared in the fields outside in the spring. Alfalfa plants grown 
close to red clover infested with Pseudopeziza trifolit remained free 
from leaf-spot. The following greenhouse-plat trials were made: 
(1) Four grams of sterilized alfalfa seed were exposed to a discharge of 
ascospores of Pseudopeziza medicaginis for three days before sowing in the 
garden greenhouse on March 3, 1916. Thousands of viable spores must have 
been attached to the seeds at the time they were sown. By April 15 the plants 
were 6 inches tall and very vigorous. When the experiment was discontinued 
