6 i6 
Journal of Agricultural Research 
Vol. XXI, No. 9 
were frequently sterile, which at once suggested the possibility that there 
was disintegration of the tissue or “action in advance” of the growth of 
the hyphae. Cultures made a little more remote from the healthy tissue 
usually gave a pure culture of Rhizopus. Following up the clue sug¬ 
gested by the plate plantings, preliminary macerating experiments were 
made with the solution on which the fungus grew and with extracts of 
the hyphae, both of which resulted in the complete disintegration of raw 
sweet-potato disks within a few hours. 
Literally hundreds of macerating experiments have been carried out 
with the enzym contained in the hyphae and with that exuded into the 
substratum. Because of the large number of experiments detailed 
elsewhere dealing with special phases of the subject and which contribute 
additional evidence of the occurrence of pectinase in the mycelium and 
in the solution on which it grew, only a resume of the process of macera¬ 
tion will be described here. The raw sweet-potato disks, usually five, 
of uniform size (i mm. thick by 1.5 cm. in diameter), were dropped into 
the solution or into the liyphal suspension, as the case might be, both of 
which were prepared according to methods already described. The 
incubation was usually earned out at 37.5 0 C., although higher or lower 
temperatures, depending upon the object of the experiment, were some¬ 
times used. In some of the more powerful preparations a change in the 
appearance of the disks frequently took place in from 10 to 15 minutes 
after they were added to the solution. The disks became somewhat 
cupped or twisted in shape and slightly crisp, brownish in color, probably 
due to oxidation. Approximately hour thereafter, depending some¬ 
what upon the macerating power of the enzym, the disks became flaccid. 
This was followed by softening of the tissue at the surfaces and edge. 
The progress of the softening continued gradually until the disks were 
softened throughout so that they offered no resistance to pull. A mi¬ 
croscopic examination of completely macerated tissue shows that the 
cells are separated along the line of the middle lamellae but that the 
cells themselves remain intact. 
The time required to macerate the disks completely varied from about 
1 to 5 hours. Several factors were found to influence the rate of macera¬ 
tion. No two preparations could be regarded as exactly alike, although 
the cultures were grown and the solutions and hyphae treated thereafter 
under as nearly identical conditions as possible. Again it was found 
that the end point of maceration varied with the different disks in the 
same system, although they were all cut from the same sweet potato and 
from the same portion of the potato. It was found, therefore, that 
something must be left to the judgment of the observer. However, 
after considerable practice in the determination of the end point it is 
possible to determine it within relatively narrow limits. These controlled 
and carefully conducted experiments show that Rhizopus tritici produces 
a powerful intracellular and extracellular pectinase which can dissolve 
