166 Journal of Agricultural Research y 0 i. xxx, no. 2 
Table I .—Isolation and infection results obtained from several collections of the 
corn smut organism , Ustilago zeae , in experiments conducted at Manhattan , 
Kans ., 1916 to 1919, inclusive 
Year 
Collections 
Inoculations 
Source 
Total 
number 
U. zeae in 
culture 
plate a 
Number 
of isola¬ 
tions (cul¬ 
tures) 
used 
Number 
of trials 
Infections 
produced 
Cultures 
found 
virulent 
1916_ 
Soil b ___ 
15 
1 
Lost. 
1916. ... 
Air... . . 
43 
24 
3 
6 
1(?) 
1(?) 
1917_ 
_do... 
73 
18 
2 
10 
r 
1 
1918.- _ 
... do.. 
301 
87 
44 
165 
14 
12 
1919_ 
_ ___do_ 
150 
58 
32 
369 
70 
24 
1916. 
Plant leaf axil 
71 
37 
5 
27 
9 
1 
1917_ 
do_ 
■ 84 
39 
13 
120 
11 
6 
1918. 
__ do. . ' . 
162 
53 
111 
369 
41 
37 
1919_ 
_do___ 
103 
25 
20 
213 
15 
8 
• Some of these microscopic identifications are necessarily uncertain, because of the occurrence of yeast - 
like cultures and their similarity to those of the smut fungus and the impossibility of isolation for adequate 
study because of contaminations. 
* Isolations from soil proved very difficult because of the large number of other organisms capable of more 
rapid growth. In more recent work isolations from the soil have been successful. 
Culture No. L 44c was isolated on 
June 14, 1916, when the plant was 
about 1 foot high. With a specially 
made sterile pipette, about 1 cc. of 
liquid was drawn from the axil of one 
of the upper leaves of plant No. 44 in 
row “L” of the experimental plat. 
Immediately after drawing the liquid 
from the leaf axil of a plant it was placed 
in a sterilized tube, taken to the labo- 
ratorj^, and plated out in carrot agar. 
These plates were kept at from 20° to 
23° C. Five days later, three colonies 
of Ustilago zeae (designated hereafter 
as L 44a, L 44b, and L 44c) were noted 
in the plate in the midst of other 
fungous and bacterial growth. These 
colonies had been identified a few days 
earlier under the microscope (low 
magnification) by the characteristic 
“snowflake” appearance. Plate 3, A 
and B, shows a photomicrograph of 
two colonies from culture plate No. L 
44c, with similar colonies, C and D, 
from a known culture of corn smut for 
comparison. Because of the slow de¬ 
velopment of the smut colonies as 
compared with the contaminations in 
the culture plate, it was necessary to 
remove them as soon as possible. 
This was done by ringing the colonies, 
waiting a few days until they became 
visible to the naked eye (about 0.1 to 
0.2 mm. in diameter), and then re¬ 
moving the colony by means of a flat 
platinum wire (9 ), taking pains to 
leave a bit of the agar surrounding 
the fungous growth. 
The characteristic gray-white color 
and “stringy” consistency of the 
fungous mass in the culture medium 
in the early stages of growth made it 
possible in subsequent studies to iso¬ 
late the colonies without preliminary 
microscopic identification (pi. 3, D). 
It was found also that contamination 
by bacteria could be largely eliminated 
by adding two ot three drops of a 5 
per cent solution of lactic acid to 10 
cc. of the carrot agar. This did not 
inhibit the growth of the smut fungus. 
Cultures of Ustilago zeae show a 
greater tendency to hyphal or mold¬ 
like growth than do those of head 
smut of maize and sorghum, Sorospo- 
rium reilianum. In old cultures this 
frequently appears as a whitish pubes¬ 
cence over the surface of the growth, or 
the white hyphae are noticeable at the 
margins of the cultures. This character¬ 
istic does not seem to be dependent en¬ 
tirely upon the age of the culture after 
transfer but, to some extent, at least, up¬ 
on its age from the time of spore germina¬ 
tion. This has been most commonly 
noted on carrot agar. Generally after 
transfer the colony darkens gradually 
through tan, brown, or olive, unt 1 
almost black, the exact appearance 
being affected by the hyphal develop¬ 
ment which seems inclined to occur in 
spots over the usually rugose surface 
of the growth. In old cultures the 
consistency becomes almost leathery; 
somewhat like the fruiting body in 
the Hymenomycetes. 
Having determined by the cultural 
characteristics above described that 
the three isolations, a, b, and c, ob¬ 
tained from the plating of No. L 44, 
corresponded to cultures of corn smut, 
an effort was made to verify this 
identification by using them in inocula¬ 
tions. Transfers were made to tubes 
