862 
• Journal of Agricultural Research 
Vol. XXIV, No. lo 
more striking. It was found that in the case of this solution steaming 
caused no change, the steamed solutions macerating as readily as the 
unsteamed. These results indicated that the enzym was not secreted, 
but that some other substance was produced which had the power to 
dissolve the middle lamellae. 
These results indicated two possibilities; first, that the enzym was 
not secreted when the fungus was grown on certain substrates or only in 
a small amount; and, second, that although the enzym was not secreted 
the organisms made the solution sufficiently acid to cause maceration of 
raw sweet-potato disks. In view of these facts, the authors undertook to 
make a study of the influence of the substrate on the production of 
pectinase, and of the changes in acidity produced by the fungus in 
different media as measured in terms of hydrogen-ion concentration. 
It will be sho'v\m in the discussion later that investigations have enabled 
the authors to explain some of the curious and puzzling results with 
respect to the parasitism of some of the species of Rhizopus causing soft- 
rot of the sweet potato. 
Since Rhizopus tritici has been employed in so many of the investiga¬ 
tions by the writers in this particular field, it was decided to use it in 
these studies. Rhizopus tritici grows readily in culture. It is parasitic 
on the sweet potato and produces an abundance of pectinase. Although 
not as common in storage as Rhizopus nigricans, it lends itself to physiolog¬ 
ical studies herein outlined more readily than does the latter species. 
INFLUENCE OF THE SUBSTRATE ON PECTINASE PRODUCTION 
methods of experimentation 
In order to determine what influence the substrate had on the pro¬ 
duction of pectinase, lo different media were employed as follows: Bean, 
prune, Irish-potato, carrot, turnip, and sweet-potato decoctions, beef 
bouillon and a modified Czapek's, Pfeffer's and Richard's solutions. 
The vegetable and fruit decoctions were prepared by using 500 gm. of 
the fruit or vegetable in i liter of water, cooking for one hour and then 
filtering. Measured portions of these decoctions were put in small 
flasks, which were then plugged and autoclaved for about 20 minutes 
at 15 pounds pressure. The beef bouillon was prepared according to the 
usual method for preparing this medium. Some modifications were 
made in Czapek's, Keffer’s, and Richard's solutions. For example, the 
NaNOg in Czapek's solution was replaced by an equal amount of NH4NO3. 
Glucose, 50 gm. per liter, was used as a source of carbon. The substitu¬ 
tion of NH4NO3 for NaNOg was made because previous experiments 
showed that Rhizopus tritici thrived better when the nitrogen was 
derived from ammonium nitrate. Investigations have also shown that 
the fungus can not utilize cane sugar, hence glucose was substituted 
for it. 
Pfeffer's and Richard's solutions were made according to the following 
formulae: 
Pfeffer's. Richard’s. 
KH2PO4. 5*0 gin. o. 5gm. 
KNO3. 4. o gm. 
MgS04. 2. 5 gm. o. 5 gm 
NH4NO3. 10. o gm. 10. ogm. 
FeS04. Trace. Trace 
Glucose. 100 gm. 60. o gm. 
Water. i, 000 cc. i 000 cc. 
