Sept. 22, 19.13 
Bacterial Leafspot of Clovers 
475 
Infection of petioles, stems, stipules, and flower pedicels are of less 
common occurrence than infection of the leaflets, and the lesions are 
less characteristic. Those of the petiole and stem appear as dark, elon¬ 
gated, slightly sunken spots. On stipules of Trifolium pannonicum , long, 
inky-black lines were produced (PI. 3, D). The translucent margin is 
less pronounced than on leaf lesions. 
CAUSAL ORGANISM 
ISOLATION 
Bacteria are abundant in the lesions, and at each of the three labora¬ 
tories, Madison, Raleigh, and Washington, the identical organism has 
been isolated repeatedly. In some cases (Wisconsin, Washington) 
isolation has been preceded by surface rinsing of the tissues with alcohol 
or mercuric chlorid solution; in others (North Carolina) direct macera¬ 
tion was made of young lesions in sterile water. One of the early Wis¬ 
consin isolations from red clover, termed 1916-II, was first proved to 
be pathogenic, then used in the earlier detailed cultural studies, and 
more recently has been compared with 
the subsequent isolations from Madi¬ 
son red clovers, termed 1919-I and 
1920-III. These three have been 
carried critically through comparative 
cultural studies at Madison and have 
throughout the experiments shown 
essentially identical characters. 
At Raleigh several like strains were 
isolated from red, white, and alsike 
clovers and their pathogenicity proved 
by successful inoculation and reisola¬ 
tion. One strain from each of these 
host species was used for cultural lyU 
studies at Roleigh in 1922. Other \—| 
Strains from isalations of previous years Fig . ,_^ ctaiam causing leafspot of red 
were also used in comparative studies clover. Casares-Gil flagella stain, 
with the Wisconsin strain 1920-III. 
At Washington isolations were made from natural infections on the 
red, the common white, the large-leaved white (var. latum) and alsike 
clovers, also from Trifolium medium and T. pannonicum. All the 
strains were compared and found to be practically identical in morpho¬ 
logical and cultural characters and in ability to reproduce the disease 
not only on the original host but also on other clover species. Three 
strains were selected for the further studies: Strain 3 from white clover, 
strain 4 from red clover, and strain M, a reisolation from red clover 
inoculated with strain 3. 
MORPHOLOGY 
The pathogen is a small rod with rounded ends, usually occurring 
singly but tending in bouillon to form short chains. It stains readily 
with Ziehl’s carbol fuchsin, anilin gentian violet, and Loeffier’s methylene 
blue. When stained from 24-hour potato-agar cultures with methylene 
blue, the cells are 1.7 by o.6.u with extremes in length from 1.2 to 3.0 
and in width from 0.4 to 1.0/x. 
