Dec. 10, 1913 
Disease of Sugar-Beet and Nasturtium Leaves 
205 
summer at room temperature, 24 0 to 30° C. Beef-agar cultures live 
from 4 to 10 months, depending upon the temperature under which they 
are grown. Those cultures which die in from four to five months are 
grown at temperatures of 24 0 to 30° C. 
LOSS OF VIRULENCE 
No loss of virulence was noticed in the organism isolated from nastur¬ 
tium until April, 1910 (two years after the first isolation), when inocula¬ 
tions were made into nasturtium and bean plants growing in the green¬ 
house. Five days after inoculation no apparent discoloration of the 
tissue could be observed. This result was unusual, since in all past inoc¬ 
ulations the diseased spots had been readily produced. After repeated 
inoculations had been made from cultures of the bacterium grown in 
beef bouillon upon agar slants and potato cylinders it became evident 
that the organism, which had been growing on artificial media for two 
years, had lost its virulence. 
In the case of the organism isolated from the sugar-beet leaf, no loss of 
virulence was noticed until about three years after obtaining the organism, 
and up to that time practically every needle-prick inoculation into sugar- 
beet leaves proved infectious. After three years the percentage of posi¬ 
tive results from inoculations fell off considerably, as only the youngest 
leaves, growing under the proper conditions of moisture and temperature, 
became diseased. Efforts were made in the summer of 1911 to obtain a 
new strain of the organism from the field, but they were unsuccessful. 
Later, string-bean agar was tried and proved to be a rejuvenator of the 
organism isolated from both hosts. After growing on this medium, the 
organism was almost as infectious to sugar-beet leaves and nasturtium 
leaves as when it was first isolated. This virulence, however, was not 
permanent, for in the course of a year it became much reduced. 
BACTERIA IN CELL TISSUE 
Diseased tissue produced in both hosts by inoculation was fixed, em¬ 
bedded in paraffin, sectioned, and stained in carbol fuchsin. Microscopic 
examinations of these sections showed the presence of bacteria in large 
quantities within the cells of the diseased tissue (fig. 5). In sections cut 
through the central portion of the diseased spots the walls appeared 
ruptured or collapsed. The cells at the margins of these ruptured places 
show that the bacteria are in the cells, although most of the bacteria 
were seen in the broken-down tissues adjacent to the sound cells. 
NATURAL INFECTION AND CONTROL 
Since practically all of the work has been done under laboratory and 
greenhouse conditions, there has been no opportunity to investigate the 
complete life cycle of this organism or to follow out the natural means of 
