220 
Journal of Agricultural Research 
Vol. VIII, No. 6 
areas, they may, in the thinner leaved species, involve the entire leaf. 
(PL 68, A, B.) These areas are olivaceous black, 1 often somewhat 
lighter colored in the center, and they present a water-logged appearance. 
Although often present at first, the reddish border usually disappears 
with the rapid inroads upon the leaf made by the later stages. Finally 
in the thin-leaved species the entire blade may become a dark, olivaceous- 
black sodden mass of tissue which falls apart at the slightest touch. In 
the thick-leaved species, and especially in the case of older leaves, the 
spots, after attaining a diameter of 15 to 20 mm., may show no further 
increase for a considerable time. Such leaves, however, usually suc¬ 
cumb in the end. Under conditions favorable for the parasite, the length 
of life of the host depends only on its capacity repeatedly to send up new 
leaves. 
ISOLATION OF THE CAUSAL FUNGUS 
In order to ascertain the nature of the parasite, several leaves of the 
pond lily (Nymphaea odorata) with initial stages of infection were col¬ 
lected from the open ponds at Kenilworth, D. C., in May, 1913. A few 
of these leaves were washed for three minutes in a 1 to 1,000 solution of 
mercuric chlorid to kill surface organisms, so far as possible. They were 
then given three washings in sterile distilled water and placed in damp 
chambers. Another lot of leaves was placed directly in damp cham¬ 
bers without the previous sterilizing and washing. Freehand sections 
of young leafspots were examined under the microscope and showed 
sparingly a brownish, septate mycelium in the diseased leaf tissues. The 
leaves placed directly in damp chambers without washing developed 
various mold fungi, such as species of Penicillium and Botrytis, which 
soon covered the surface with a sporulating mass of hyphse. Although 
these fungi were evidently saprophytic surface organisms which were 
outgrowing the real parasite, pure cultures were started for inoculation 
tests. In the meantime, after four or five days, signs of a fruiting fungus 
were noted on many of the leaves sterilized before being placed in damp 
chambers. Small tawny and somewhat powdery areas were seen on the 
surface of some of the leaf spots, and a microscopical examination showed 
these small masses to consist of large, many-septate, curved spores borne 
in clusters. These conidia were larger at the basal end, tapering and 
more or less helicoid at the apical end. This form alone appeared on the 
majority of the sporulating spots and gave promise of a causal relation. 
Com-meal-agar plate cultures were poured, using these conidia and also 
using small pieces of leaf tissue from the younger spots. From the 
conidia single spore cultures were started by locating germinating conidia 
in the agar plates and transferring these to other plates of solidified sterile 
corn-meal agar, where the growth could be followed under the microscope. 
Colonies developing from these single spores were designated as isolation 
1 Ridgway, Robert, Color Standards and Color Nomenclature. 43 p., 53 col. pi. Washington, D. C„ 1912. 
