278 
Journal of Agricultural Research 
Vol. XXIX, No. 6 
described showed no signs of infection. 
Further examinations were made on 
the fourth day and some of the seed¬ 
lings were found to be heavily in¬ 
fected by these hyphae (PI. 9, a, b , e, 
g, k, and m). There was no mistake 
as to the identity of the fungus. Similar 
cultures were made in Petri dishes on 
sterile paper with the same result. 
The seedlings were easy to remove for 
study and, as with the tube cultures, 
no washing was necessary before the 
examination was made. In this way 
the spores were not disturbed and 
could be found attached to the hyphae. 
Infections of seedlings of the following 
varieties were found by this method: 
Tennessee Winter, Han River, Nakano 
Wase, and Smyrna (C. I. 195), a two- 
rowed barley. In one case the first 
two leaves of a seedling of Han River 
were found to be heavily infected 
(PI. 9, h and n). Nakano Wase, the 
variety which has not shown any 
smut at maturity, apparently was in¬ 
fected as easily as the susceptible 
varieties (PI. 9, k and m). One hull¬ 
less barley, C. I. 2222 was sown in the 
dish cultures and examined to a 
limited extent but no infections were 
found. Reports indicate that the 
naked barleys smut heavily in nature. 
In many cases the mycelium was 
found entering the epidermis at or near 
the wall between two cells (PI. 9, b , e, g , 
j ). This was not always true, however, 
as the hyphae seemed to be able to 
penetrate the cell wall at any point (PI. 
9, a, d, /, f). The fact that the fungus 
is able to infect the coleoptile and, in 
cases, the first leaves, is not proof that 
it will reach the growing point of the 
seedling and finally result in a smutted 
plant. However, the injury caused to 
the seedling (PL 3, 4, 7, and 8) is a very 
good indication that its activities are 
not confined to the coleoptile and the 
superficial tissues of the plant. Fur¬ 
thermore, the high percentages of loose- 
smut occurring in the plants of suscep¬ 
tible varieties grown from inoculated 
seed is definite proof that the fungus 
reaches the growing point of the seed¬ 
ling and keeps pace with its develop¬ 
ment. With Nakano Wase the fungus 
evidently penetrates beyond the super¬ 
ficial layers, as evidenced by severe 
seedling injury (PI. 3 and 8) but is 
unable for some reason to keep pace 
with the development of the plant. 
Consequently, the surviving plants are 
smut-free at maturity. The seedling 
injury no doubt is due to heavy infec¬ 
tion and invasion of the tissues of the 
plant by the fungus mycelium. If a 
toxin actually is responsible for the 
injury it evidently is produced within 
the tissues of the host as invasion takes 
place. 
DISCUSSION 
Floral infection has been recognized as 
the only means of infection of barley by 
the loose-smut fungus, Ustilago nuda , for 
many years. No doubt, if Jensen (12) 
had continued his infection studies 
with dehulled seed and included the 
loose-smut fungus after discovering it 
he would have found that it could 
infect the seedlings of barley. Maddox 
(16) through whose work floral infec¬ 
tion was first established, apparently 
overlooked the possibility of the spores 
remaining viable between the glumes 
and the kernel when the flower is inocu¬ 
lated. He failed to consider the fact 
that the hulls furnish a very effective 
obstruction to infection. This is true 
for both barley smuts. Since the time 
of Maddox other investigators have fol¬ 
lowed practically the same methods of 
inoculation used by him, with the 
exception of Brefeld (3) in a part of his 
investigations. Brefeld not only inocu¬ 
lated the flowers but also inoculated 
young seedlings with germinated spores. 
With the latter method he secured 1 
per cent of infection in one experiment. 
This might have been due to natural 
infection or perhaps to contamination r 
but there is a chance that it was due to 
the inoculation. Perhaps Brefeld’s 
method of inoculation or else the treat¬ 
ment of the spores, or seedlings, or 
both, previous to inoculation had upset 
their normal functions. The spores 
were first germinated in water and then 
finely divided in a nutrient solution 
before spraying on the seedlings. There 
no doubt are many reasons why they 
might not have functioned normally. 
Other investigators have inoculated 
the flowers at various stages of develop¬ 
ment and have found that more in¬ 
fection takes place if the flower is 
inoculated in one stage than if it is 
inoculated in another. It seems en¬ 
tirely possible that the weather condi- 
EXPLANATORY LEGEND FOR PLATE 7 
Effects of viable and devitalized spores of Ustilago nuda on Han River barley. C and D were shown in 
Plate 4. 
A. —Seed dehulled and inoculated. Plants 91.89 per cent smutted. 
B. —Seed dehulled and uninoculated. No smut. 
C. —Seed dehulled and inoculated. Plants 100 per cent smutted. 
D. —Seed hulled and inoculated with devitalized spores. No smut. 
