1044 Jour nal of Agricultural Research v 0 i. xxx, No. 11 
complectens , where a cork layer laid 
down in advance of infection prevents 
further progress of the hyphae after 
nfection has already extended some 
distance from the point of inoculation. 
The rate of progress of infection is 
lways less rapid where comparatively 
mature plants are involved (pi. 2), and 
may be localized when the epidermal 
cylinder is thickly cutinized and the 
basal tissues are old and woody. 
The hyaline, coenocytic hyphae of 
these fungi may be observed in and 
between cells of the less discolored ad¬ 
vancing area, particularly as long 
strands along the fibrovascular system 
(pi. 3, A). Farther back the fruiting 
bodies are formed, including oospores 
and conidia in the case of B ( P . de¬ 
baryanum) ; conidia only were formed 
in tissues by the other two Pythium 
spp. Bacteria were frequently ob¬ 
served in the more decayed tissues, 
particularly in xylem cells^ but not in 
the less infected regions where advanc¬ 
ing hyphae could^be seen. 
ISOLATION 
P. debaryanum (B) and the large 
conidia form A were first isolated, oc¬ 
curring together in a plate colony 
made from a single diseased geranium 
from the Washington greenhouses. 
They were separated by making single¬ 
spore cultures from conidia, those of 
A being by far the larger and readily 
recognizable. Form D was obtained 
alone in a later isolation. Repeated 
isolations from naturally blackened 
and diseased geranium cuttings in the 
Department of Agriculture greenhouses 
have yielded these fungi, occurring 
sometimes together, usually singly. 
The methods of isolation used have 
been described in the paper on P. com¬ 
plectens (4) • Cultural studies from 
single-spore isolations soon showed 
that these fungi are not identical, nor 
corresponding to previously de¬ 
scribed species of Pythium with the 
exception of B, the P. debaryanum 
isolation. 
PATHOGENICITY 
Controlled inoculation experiments 
involving some 230 cuttings in 17 
inoculation series since isolation have 
shown that the disease can be re¬ 
produced with great uniformity w T hen 
the inoculum is placed at the cut base 
of fresh Pelargonium cuttings in 
sterilized soil, using any of the three 
isolations. Progressive discoloration 
and rot occurred as in naturally in¬ 
fected cuttings from the severed end 
up; check plants formed a callus as 
a rule and rooted normally. No 
differences in the symptoms produced 
could be observed with either isolation, 
except in the rate of progress; Pythium 
debaryanum (B) and isolation A were 
the most severe. Death ensued with¬ 
in 6 to 10 days, depending on the 
organism used. Material fixed in 
Flemming’s strong solution and in 
Merckel’s fluid and sectioned showed 
the hyphae within the tissues, conidia 
in the case of A, conidia and oospores 
in the case of B. Each was readily 
reisolated from advancing margins of 
the discoloration. Repeated reisola¬ 
tions with subsequent inoculation 
have shown their ability to attack 
geranium cuttings. Cross-inoculations 
on coleus, begonia, cucumber seed¬ 
lings, and radish seedlings result in 
soft rots in the first two hosts and 
typical damping off in the seedlings, 
form B causing by far the most severe 
infection. A and D required a longer 
incubation period in the case of the 
seedlings and caused slower progress 
of the disease. In one experiment 
where cuttings were placed in soil 
which had previously contained plants 
artificially infected with Pythium 
species, four out of five were infected 
where B had been used, two were 
killed and three showed basal blacken¬ 
ing'when A had been used, and four 
out of five showed infection when D 
had been used, indicating the role of 
the soil as a carrier. After four or 
more years in culture, there is a 
distinct reduction of virulence evident 
in a slower progress of discoloration 
and lower percentages of infection 
compared with the uniformly com¬ 
plete infections obtained early in the 
work. 
The comparative rates of discolora¬ 
tion caused by each of the organisms 
are shown below in data from one of 
the inoculation experiments (Septem¬ 
ber 15, 1921): 
Isolation A.—Ten plants inoculated at the 
base. Discoloration averaged 35.9 mm. up the 
stem by the second day; 59.4 mm. by the fifth day, 
with five plants collapsed. Eight were dead the 
sixth day, and the discoloration on the remainder 
averaged 67.2 mm. 
Isolation B.—Ten plants. Discoloration reached 
an average of 32.6 mm. up the stem by the second 
day; eight had collapsed by the fifth day, the 
remainder averaging 63.7 mm. All were dead 
and on the ground the sixth day. 
Isolation D.—Ten plants. Discoloration reached 
an average of 21.6 mm. the second day, 32.7 the 
fifth day, 38.5 the sixth day. None were dead till 
the tenth day. All had collapsed four days later. 
Ten checks remained healthy, formed 
a callus and rooted long before in¬ 
fected plants began to collapse. 
