870 
Journal of Agricultural Research 
Vol. XXIV, No. lo 
which two glass tubes passed, one of which was long enough to extend 
nearly to the bottom of the flask. The liquid was drawn out through 
the long tube. The other tube, through which the air entered the flask, 
was stoppered with cotton to prevent the entrance of contaminating 
organisms with the inflowing air. The flasks so prepared were auto¬ 
claved for 20 minutes at 15 pounds pressure. 
The culture flasks, after being finally prepared, were allowed to stand 
for two days before inoculation in order to determine which if any of 
them were contaminated. In spite of all the possible precautions, 
some of the flasks in every experiment became contaminated. These 
were discarded and a portion of the remaining flasks were inoculated 
with a spore suspension of Rhizopus tritici. Some of the flasks were 
held as controls. The cultures were incubated in the dark at a constant 
temperature of 35° C. 
The duration of the experiment varied from 5 to 8 days. At the close 
of the experiment a portion of the mycelium and of the solution on 
which the fungus grew was prepared according to methods already 
described and used for macerating tests. Another portion of the my¬ 
celium was used in obtaining the dry weight of the fungous material 
produced. A portion of the solution was used to determine the hydrogen- 
ion concentration and the amount of dextrose present in Czapek’s 
solution only. The sugar was determined by means of a Fric sacchari- 
meter. The hydrogen-ion concentration and sugar content of the 
controls and inoculated flasks were determined on the day the experiment 
was terminated. 
Fifteen flasks of each hydrogen-ion concentration were prepared, 10 
of which were inoculated. The remaining 5 were held as controls. 
EXPERIMENTAL DATA 
It has already been shown that pectinase was not produced in Czapek’s 
nutrient solution when dextrose was used as a source of carbon. It 
was found, however, that when pectin was employed in the substrate 
as the only source of carbon in Czapek’s solution a powerful middle 
lamellae dissolving enzym was secreted. When pectin was combined 
with dextrose as a source of energy pectinase was produced, but its 
action was much slower, being presumably secreted in a much smaller 
amount. It was likewise pointed out that Rhizopus tritici produced a 
macerating substance on several vegetable decoctions (turnip, sweet 
potato, Irish potato, carrot, and bean) but not on synthetic media 
(Pfeffer’s, Richard’s, and Czapek’s solution and beef bouillon). Further¬ 
more, it has been demonstrated that some substance which causes 
maceration of raw sweet-potato disks is produced when Rhizopus tritici 
is grown on Czapek’s nutrient solution. That this substance was an 
acid seems likely as some of the experiments to be detailed below will 
show. In view of the fact that Czapek’s nutrient solution became 
rapidly acid when R, tritici was grown on it, it was suspected that the 
production of the acid interfered with the secretion of pectinase. It is 
also interesting to note in the way of comparison that when sweet-potato 
decoction in which pectinase was abundantly produced was used as a 
substrate the final acidity as measured in Ph, although increased, was 
never as high as in Czapek’s solution. 
A series of experiments were therefore outlined to determine what 
effect the original hydrogen-ion concentration of the substrate (Czapek’s 
