May 15,1925 
965 
Study of Two Sweet Potato Storage-Rot Fungi 
Diplodia tubericola. —The enzyme 
raffinase, secreted by this organism, 
was able to hydrolyze more than twice 
as much of the sugar as that of Mucor 
racemosus in the same length of time. 
Only 0.71 mgm. of reducing sugars was 
obtained from the control, while an 
average of 31.28 mgm. was obtained 
per 10 c. c. of solution in the prepara¬ 
tion containing the active enzyme. 
CYTASE 
Diplodia tubericola. —It was pre¬ 
viously pointed out that Diplodia tuberi¬ 
cola caused a slow dry-rot of the sweet 
potato, the tissue finally becoming hard 
and mummified. The cells of the de¬ 
cayed tissue do not separate in the 
same way as when the rot is caused by 
Mucor racemosus or Rhizopus nigricans. 
It was also shown that the enzyme pec- 
tinase, which is abundantly produced 
by Mucor and Rhizopus, is not pro¬ 
duced by D . tubericola, at least within 
the limits of these experiments. These 
results, together with the fact that 
hyphae have been observed within the 
cells, suggested the possibility that the 
enzyme cytase may be produced. 
The first attempt to demonstrate the 
production of cytase was made with 
mycelium grown on sweet potato de¬ 
coction. The mycelium was treated 
in the usual way and added to a sus¬ 
pension of cellulose in water. The 
cellulose was prepared from filter paper 
according to the method of Scales {12). 
One gram of powdered mycelium was 
added to 50 c. c. of the cellulose sus¬ 
pension and incubated at 35° C. At 
the end of 48 hours the system was 
tested for reducing sugars and none 
were found. Another method used 
successfully by Keller man (10) to dem¬ 
onstrate the production of cytase by 
Penicillium pinophilum was tried. A 
beef agar with cellulose added was used 
as a medium. This preparation in test 
tubes was inoculated with Diplodia 
tubericola on the surface and incubated 
at a constant temperature of 30°. 
These cultures were grown for several 
weeks, but there was no clearing of the 
agar. On the other hand, Sclerotium 
rolfsii, used as a control, cleared the 
agar almost to the bottom of the tube 
,in the same length of time. The ex¬ 
periment was repeated, using carrot 
and sweet potato agar with cellulose 
added. After inoculation the cultures 
were held for a number of weeks at 30°, 
but there was no evidence of the pro¬ 
duction of cytase. In this experiment 
water was placed in the incubator in 
order to prevent the media from drying 
out too rapidly. 
Mucor racemosus. —This fungus pro¬ 
duced no cytase when tested according 
to the above method. 
INFLUENCE OF SUBSTRATE ON PECTINASE 
PRODUCTION, DRY WEIGHT, AND HY¬ 
DROGEN-ION CONCENTRATION 
Essentially the same methods were 
employed for both Mucor racemosus and 
Diplodia tubericola, some variations 
being necessary to meet the different 
habits of the two fungi. M. racemosus 
was incubated at 25° C. and D. tuberi¬ 
cola, being a higher temperature form 
was incubated at 30°. Since the latter 
organism develops much more slowly, 
it was grown for a longer time. 
The following culture media were 
employed: Prune, sweet potato, potato, 
carrot, turnip, and bean decoctions, 
beef bouillon, and Czapek’s, Pfeifer’s, 
and Richard’s solutions. Czapek’s so¬ 
lution was modified by the substitution 
of ammonium nitrate for sodium ni¬ 
trate. Enough of each of the stock 
solutions was prepared at one time to 
carry out the duplicated experiments. 
The experiments were set up as follows: 
Fifteen 100 c. c. flasks were used for 
each organism, 50 and 35 c. c. of the 
medium being used in each flask for 
Mucor racemosus and Diplodia tuberi¬ 
cola, respectively. As soon as the cul¬ 
ture media were pipetted into the flasks 
they were autoclaved for 20 minutes at 
15 pounds pressure. They were then 
held for a few days in the laboratory at 
room temperature. As soon as it was 
evident that none of the flasks were 
contaminated, 10 of each medium were 
inoculated and incubated at tempera¬ 
tures given above, the remaining 5 
being covered with oiled paper to pre¬ 
vent evaporation and held as controls, 
that is, for hvdrogen-ion determina¬ 
tions. 
Mucor racemosus. —At the end of 
five days’ growth three flasks of each 
medium were removed and the con¬ 
tents of each made into one compound 
sample. Two flasks were then pre¬ 
pared from this solution, one of which, 
after steaming 10 minutes to inactivate 
the enzyme, served as a control. Raw 
sweet potato disks were added to the 
solution and a record made from time 
to time of the progress of maceration. 
None of the disks in the steamed con¬ 
trols were macerated. The produc¬ 
tion of pectinase was not entirely what 
might be expected. It was abundantly 
secreted on sweet-potato and string- 
bean decoctions after 23 hours, on car¬ 
rot decoctions after 30 hours; and well 
advanced on turnip decoction after 30 
52205—25f-6 
