Sept, ad, 1933 
Bacterial Leaf spot of Clovers 
485 
might be indentical. Infections were readily secured upon red clover, 
but the inoculations of soybeans were unsuccessful. Other isolations 
including strain 1919-I, made in the summer of 1919, were successfully 
employed in infecting red clover in another series of experiments. Again, 
in 1920, a more extended series of tests of pathogenicity was instituted. 
These trials included the four clover species, Trifolium pratense f T . 
medium , T. repens , T. hybridum , and the white sweet clover, Atelilotus 
alba . Four recently isolated strains of the organism, three from Trifolium 
pratense and one from T. medium , were used in these series. Water sus¬ 
pensions of 3- to 4-day-old potato-agar cultures were applied with an 
atomizer, after which the wetted leaflets were gently rubbed between the 
fingers. The inoculated plants were then covered from 48 to 72 hours 
with bell jars when the sky was clear, and were left uncovered if cloudy 
weather prevailed. Inoculated plants were sprayed once daily with 
sterile water to favor infection. Plants both out of doors and in 
the greenhouse were thus inoculated. After a period of incubation of 6 
to 10 days, lesions appeared uniformly on all inoculated plants of Tri¬ 
folium pratense and T. medium . Inoculated white clover, alsike clover* 
and white sweet clover, however, remained free from infection. The 
failures to secure infection of these species receive support from the field 
observations made in Wisconsin, since no cases of natural infection of 
these species or of alfalfa have been discovered, although all have been 
found growing closely intermingled with diseased red clover. 
Several series of inoculation experiments have also been conducted at 
Raleigh, N. C., during the several seasons in which the investigationa 
have been in progress. One of these, in the summer of 1922, is repre¬ 
sentative in all respects of the others and is, therefore, briefly described. 
Cultures from each of the clovers, red, white, and alsike, which had been 
grown for 48 hours on bacto-agar were used as an inoculum. The 
growth was washed off with sterile water and the bacterial suspension 
poured into Petri dishes. On the morning of July 22 inoculations were 
effected by immersing the leaves in these suspensions. The strain 
from any one of the host species was used to inoculate plants of that 
species and also the other two species. All inoculated plants were 
grown in the greenhouse and after inoculation were shaded lightly for 
48 hours with newspapers. On July 29 small translucent areas, evi¬ 
dent only on the lower leaf surface, were present in abundance on all 
inoculated plants. Three days later these lesions had developed into 
small, blackish brown spots characteristic of the disease in nature. 
The same strains were also used in North Carolina to inoculate plants 
of soybean, but no evidence of infection appeared. 
At Washington both greenhouse and out-of-door plants were used in 
the inoculation experiments, which extended through two years. Bac¬ 
teria from 1- to 4-day-old-agar cultures were washed off in sterile water 
and this bacterial suspension was then sprayed on the plants with an 
atomizer. Usually the plants were covered with bell jars or placed in 
moist chambers for 24 to 48 hours after inoculation, then returned to 
normal conditions. 
The first evidence of infection was noted in from 6 to 10 days as tiny, 
translucent areas which enlarged in a few days into the nearly black 
spots with translucent borders. Successful but slower infections resulted 
if the plants were not kept unusually moist for a period following inocu¬ 
lation. Check plants invariably remained free from infection. 
