276 
Journal o f Agricultural Research 
Vol. XXIX, No. 6 
The data in Table V show that in¬ 
jury to the seedlings was not caused by 
spores of Ustilago nuda devitalized in hot 
water. They show that neither viable 
nor devitalized spores had any notice¬ 
ably injurious effect on seedlings of 
Red Wave wheat. The viable spores 
had the same injurious effect on barley 
seedlings which has been shown in 
previous experiments (PI. 4, G and 
PI. 8, E). The surviving plants of 
Nakano Wase from seed dehulled and 
inoculated with viable spores were 
smut-free, while those of Han River 
were 100 per cent smutted (PI. 7, C). 
This agrees with the results of previous 
experiments. 
CYTOLOGIC EVIDENCE OF SEEDLING IN¬ 
FECTION 
After noting the injurious effects of 
Ustilago nuda on the seedlings of barley, 
it was decided to make in addition to 
the studies of possible toxicity, a micro¬ 
scopic study of seedling infection to 
obtain actual proof of infection and to 
learn the possible relationship of the 
amount of infection to seedling injury. 
Seedlings that failed to emerge from 
inoculated seed in some of the previous 
experiments were dug up for this study. 
Dehulled seed was inoculated and sown 
in greenhouse flats to obtain further 
material for this examination. The 
abnormal seedlings were carefully re¬ 
moved from the soil by taking a clump 
of dirt with the roots. They were then 
carefully washed to remove as much of 
the dirt as possible without any rub¬ 
bing. The coleoptile was removed 
from the young seedling with a scalpel 
and placed on a microscopic slide with 
the inner surface downward. A few 
drops of water were then placed on the 
specimen and pains taken to spread 
the coleoptile so that it did not fold 
when the cover glass was gently pressed 
over it. Glycerin was then added, a 
drop occasionally, and the slides re¬ 
mained in good condition for study for 
several days. These slides were'studied 
carefully under the microscope. The 
coleoptile is sufficiently translucent so 
that the cellular structure is easily 
visible with the proper manipulation of 
the microscope. After considerable 
search a germinated spore was found 
adhering to the epidermis of the cole¬ 
optile, and the mycelium had pene¬ 
trated the epidermal cell wall and 
passed through two or three cells 
beneath (Pl.9,j). The spore wall was 
sufficiently intact so that it could be 
identified as a spore of U . nuda by the 
echinulations on its surface. Many 
cases of penetration were detected by 
this method on the seedlings of differ¬ 
ent varieties of barley including Nakano 
Wase which has never produced smut¬ 
ted heads in the writers’ experiments 
(PI. 9, c, d, f, i, j, and l ). A more direct 
method was then employed for study¬ 
ing penetration, one in which the pos¬ 
sibility of contamination was reduced to 
a minimum and in which no washing 
of the seedlings was necessary before 
the examination was made. A method 
similar to the one employed by Tisdale 
(IS) in his studies of the penetration of 
the root hairs of flax seedlings by 
Fusarium Uni was used. As the smuts 
infect the seedlings of small grains so 
early in their development, this method 
was considered satisfactory. The seed¬ 
ling is not far enough advanced to be 
drawing much, if any, of its food from 
the soil at the time when infection 
takes place. The seedlings apparently 
are in normal condition in the tube 
cultures at this age. 
Strips of paper towel were rolled and 
inserted into test tubes, the rolls being 
one-third the length of the tubes, and 
enough water added to wet the paper 
thoroughly and to fill the tube up to 
about one-Tialf the depth of the paper 
roll. The tubes were sterilized in an 
autoclave. Dehulled and hulled barley 
seeds were treated by the modified 
hot-water method to kill any smut 
spores or other fungi on or in them. 
A part of the seed was.then inoculated 
with spores of Ustilago nuda which had 
been collected from Tennessee Winter 
barley the previous year and kept in a 
well-corked bottle after drying. This 
inoculated seed and a similar lot of 
uninoculated seed were sown in these 
sterile tubes. The tubes were placed 
in a dark closet at room temperature 
(about 21° C.) and left for two days, 
at the end of which time the inoculated 
seeds which were germinating had a 
frosty appearance. When examined 
under the microscope this whitish sub¬ 
stance proved to be the mycelium of 
germinated spores of U. nuda. Cole- 
optiles of these seedlings examined ac¬ 
cording to the method previously 
EXPLANATORY LEGEND FOR PLATE 6 
Infection of Han River barley, from dehulled seed, by Ustilago nuda. Same plants shown in Plate 4, A to D. 
A. —Seed inoculated. Plants 82.34 per cent smutted. 
B. —Seed uninoculated. No smut. 
C. —Seed inoculated. Plants 100 per cent smutted. 
D. —Seed uninoculated. No smut. 
