24 BULLETIN 1474, U. S. DEPARTMENT OF AGRICULTURE 
INOCULATION TESTS IN GROVES 
During the progress of the work covered in this bulletin outdoor 
inoculation experiments received far more attention than is usually 
considered necessary, not only because a relatively small amount of 
work had been done on this phase of the problem previously but also 
because Koch's postulates, the generally accepted proof of patho- 
genicity, can not be followed in two of the steps, at the beginning and 
end of the cycle. Fortunately these are the least important of the 
four postulates. For these tests a wide range of rutaceous plants 
growing under natural conditions on the laboratory grounds of the 
United States Citrus-Disease Field Laboratory at Orlando, Fla., 
were used. 
In general these experiments were designed (1) to gain further 
evidence on the pathogeneity of Phomopsis citri; (2) to determine 
the host range of the disease and the relative susceptibility of the 
various species; and (3) to determine the age or size when leaves 
and fruits of susceptible forms become immune to infection, in order 
to place the practical control of the disease on a more definite basis. 
For the most part pure cultures of the causal organism for inocula- 
tion purposes were obtained from fruits suffering from stem-end rot. 
These cultures were grown at room temperature on corn-meal agar, 
potato-dextrose agar, stems of pigeon peas, sweet clover, or sweet 
potatoes, and when sporulation became abundant the media were 
washed with sterile water in order to get the spores in suspension. 
Ordinarily the spores were plentiful enough in this wash water to 
cause it to become distinctly clouded. These same cultures were 
used repeatedly for spore production until they became contami- 
nated. Absorbent cotton was saturated with this spore-charged 
water, and liberal portions of the wetted cotton were placed on the 
parts to be inoculated, wrapped immediately with several layers of 
paraffin paper, and left on from 24 to 48 hours, depending upon the 
weather, before the wrappers and cotton swabs were removed. Ordi- 
narily, less than 15 minutes elapsed between washing the spores 
and applying the inoculum. No other method of inoculation was 
employed in any of the infection tests. 
The inoculations were made under a wide range of conditions of 
weather and tenderness of host parts. The tests were so extensive 
and involved such a wide range of hosts that it was obviously impos- 
sible to have tests on each host rounded out to the extent that 
scientific interests may demand, but they do seem sufficiently com- 
plete for a practical consideration of the problem. 
In addition to the large number of inoculations on the orange 
and the grapefruit, tests were made on miscellaneous citrus plants. 
At least 30 inoculations with a liberal number of checks were made 
on each host. The results of inoculations on rare forms that are not 
accounted for in the following tables are recorded in Table 1. 
There was a pronounced increase in the degree of infection from 
slight or medium in the early tests to very severe on the later ones, 
even on the same plant. This increase in intensity of infection is 
probably due to the use of a thicker swab with heavier spore charges 
in the later tests than was the case earlier. Such an explanation 
seems probable, since it is next to impossible to have the spore-laden 
wash water from cultures charged uniformly, and the natural tend- 
