EnpotTu1a CANKER OF CHESTNUT 569 
has not been explained satisfactorily. It would suggest that possibly 
the gelatinous substance which binds the spores together in the tendrils 
may serve also as a protective covering. Gardner (1914), however, 
finds that when washed down into the soil at the base of the tree the spores 
retain their viability in every case, in large numbers, until the next rain. 
Even when such soil is taken into the laboratory and kept dry, he has 
found the spores still alive at the end of one hundred and nineteen days. 
Ascospores.— These were also collected and tested during each month 
of the winter, but apparently winter conditions had no effect on their 
viability. Their longevity also seems to vary with the amount of aggre- 
gation and exposure. In one series of .experiments, ascospores ejected 
from perithecia were caught on slides and stored in a dry place. Tests 
showed that the percentage of viable spores decreased each month, until, 
at the end of five months and six days, none of the spores could be made 
to germinate. In Bulletin 9 of the Pennsylvania Chestnut Tree Blight 
Commission, however, it is stated that when the ascospores are washed 
and then dried they are very sensitive to desiccation, and that ‘‘ drying 
alone has been found to kill as many as ninety-four per cent in certain 
tests.” In another series of experiments made by the writers of this 
bulletin, bark containing stromata with mature perithecia was stored 
in a dry place and germination tests were made from spores taken from 
these perithecia each month. At the end of one year very little diminu- 
tion in the percentage of germination could be seen. In another series 
of experiments, a large percentage of the ascospores similarly stored 
germinated after eighteen months, while ascospores from material kept 
for thirty-one months produced only primary and secondary buds and 
no germ tubes nor mycelium. 
Inoculation and infection 
The inoculum.— The disease may be produced on healthy trees by intro- 
ducing into the bark (1) pieces of diseased bark or wood from other trees, 
(2) bits of agar or other culture media containing the mycelium in pure 
culture, (3) pycnospores, either as dry spore horns or suspended in water, 
(4) ascospores suspended in water or dry. The writers have used all of 
these in producing hundreds of cankers. The highest percentage of infec- 
tion was obtained from the first, the next highest from the second; very 
little difference was noticed in the last two, which have been used more 
extensively and which best imitate the process as it occurs in nature. 
Necessity of a wound.— Murrill (1906 a) failed to secure infection as 
long as “ the thin brown layer of cortex remained intact,” and was there- 
fore of the opinion that wounds are necessary; but he also suggested 
the possibility of lenticels being channels of entrance. Metcalf and 
Collins (1910) state that ‘“‘ the parasite can enter without visible breaks 
