400 
Journal o f Agricultural Research 
Vol. XXIX, No. 8 
through the release of centrifugal pres¬ 
sure on the epidermal layers, and are 
rapidly hollowed out by a soft, wet rot. 
The cutinized epidermis, the underlying 
cork layers when present, and the 
lignified elements of the fibrovascular 
system are not included in this decay 
but form a hollow double cylinder, the 
outer (epidermal) firm and continuous, 
the inner (vascular) shredded and 
fibrous. Rotting of cortex and pith 
finally stops at the point of demarca¬ 
tion. Leaf scars included in the dis¬ 
eased area are hollowed out, leaving 
holes in the epidermal cylinder, through 
which a black exudate from the decay¬ 
ing tissues may be squeezed out. 
Microscopic examination reveals hya¬ 
line, nonseptate hyphae ramifying within 
and between the cells of the discolored 
tissues. Oospores with the peculiar 
antheridia characteristic of this or¬ 
ganism are found within the infected 
cells just before their turgidity is de¬ 
stroyed, and are particularly abundant 
in the crushed cells of the later stage. 
Bacteria and nematodes are often pres¬ 
ent in later stages in the decaying tis¬ 
sue, but not in the turgid advancing 
brown area in which the coenocytic 
hyphse may be observed reaching into 
the healthy cells beyond. 
ISOLATION OF THE CASUAL OR¬ 
GANISM 
The causal organism was first isolated 
in November, 1919, from two out of 
six blackened geranium stems collected 
in the agricultural greenhouses. The 
remainder yielded Pythium de baryanum , 
which had been isolated earlier in the 
work and appears to be the pathogen 
most frequently associated with the 
stemrot. The fungus under discussion 
has since been repeatedly obtained and 
recognized in platings from naturally 
infected cuttings from Washington and 
from collections made at Enid, Okla. 
(1923). 
Stems from which isolations were to 
be made were first scrubbed free of 
adhering soil, then rinsed in alcohol 
and immediately flamed. They were 
then bisected longitudinally down¬ 
ward, beginning at the healthy portion 
above the advancing margin. A pre¬ 
liminary split was made with a sterile 
scalpel and the two halves were pulled 
apart with sterile forceps, thus ex¬ 
posing infected internal tissue un¬ 
touched by any outer agent. Bits of 
tissue at the infection margin were 
quickly excised with a flamed scalpel 
and planted on corn-meal agar plates. 
Isolations were also made by searing 
the surface of the cleaned stem with a 
hot knife and digging underneath with 
a flamed scalpel or needle. The first 
method was usually found more satis¬ 
factory, since one could see and select 
transplant material sufficiently remote 
from the badly decayed interior to 
minimize contamination by secondary 
organisms. 
Isolations on corn-meal agar from 
slightly discolored advancing areas 
usually yielded colonies which for this 
organism were characterized by a 
compact prostrate growth of closely 
parallel silky hyphse, later forming an 
abundance of oospores immediately 
around the transplant material. The 
fungus was obtained with difficulty 
from stems which were hollowed out 
up to the demarcation; the plates 
were often overrun with bacteria, 
nematodes, and soil fungi. In the 
absence of other fungi, the Pythium 
could be freed from bacteria by plant¬ 
ing part of the growth in the center 
of another corn-meal agar plate, to 
which a drop of 50 per cent lactic acid 
had been added. Transfers were 
finally made to oatmeal agar tubes for 
use as stock cultures, after purity had 
been assured by the absence of bacterial 
growth in transfers on beef agar plates. 
INFECTION EXPERIMENTS 
Inoculations have been repeated at 
various intervals during the past four 
years, and have shown that the typical 
blackening and decay can be pro¬ 
duced in healthy geranium cuttings 
when a portion of a pure culture is 
placed in contact with any wounded 
part of the stem. Mycelium from 
oatmeal agar or corn-meal agar cul¬ 
tures was generally used. Inoculations 
without previous injury were not 
successful. Since natural infections 
appeared to progress from the base, 
it was concluded that the freshly cut 
base of the stem placed in the soil was 
the usual means of entry of the organism. 
Cuttings placed in sterilized soil and 
inoculated at the base showed within 
24 hours a sinking in of the pith and a 
EXPLANATORY LEGEND FOR PLATE 1 
A.—Geranium cutting eight days after artificial inoculation. Note demarcation at node, 
shriveled black area below, turgid healthy tissues above 
B—Colony on corn-meal agar plate, natural size, 48 hours at 25° C. 
C. —Carrot agar cultures one week-old. Front and side views 
D. —Potato dextrose agar culture one week old. Side view 
E — Decayed base, advanced state; hollowed out, epidermis and F. V. B. remaining 
