June, 1955 
each flask. After 2 weeks at 25 degrees, 
one-half to three-fourths of each wood 
piece was found to be covered with a 
dense mat of mycelium free from the 
broth, fig. 7. In this laboratory test, 
methods of field sampling and_ storing 
were duplicated as nearly as_ possible. 
Disks of mycelium were taken from some 
pieces with a cork borer and transferred 
to moist filter paper in sterilized 4-ounce 
bottles. Whole pieces were placed in bags 
of plastic film and closed tightly with rub- 
ber bands. Some bottles and bags were 
then subjected to a temperature of 0 de- 
gree and some to a temperature of 16 de- 
grees. For checks, germination counts 
were made of spores taken from some of 
the mats before treatment. 
After 7 days, germination counts were 
made of spores in the test material. It 
was found that storage at 0 and at 16 de- 
grees had no harmful effects on the spores. 
The germination rates were slightly 
higher for material stored at either of the 
two test temperatures than for the checks. 
Since the storage temperatures for field 
material were always within the range of 
0 to 16 degrees for a period of time not 
exceeding 5 days, no serious fault could 
be seen in the storage methods used. 
Germination of Pad Cells 
Zuckerman & Curl (1953) showed 
that single cells isolated from laboratory- 
grown mycelial pads of Endoconidiophora 
fagacearum are capable of producing typ- 
ical cultures of the fungus on potato dex- 
trose agar. Their finding suggested the 
possibility that pads on naturally occur- 
ring mats might constitute one of the 
sources of inoculum. 
During the summer and autumn of 
1953, pad cells from mycelial mats that 
had been collected from wilt-killed oaks 
were used in making germination tests. 
The surface of each pad was cut away 
with a flamed scalpel, and a small portion 
of the interior was removed and crushed 
thoroughly in a sterile water blank to form 
a suspension of separated cells. Drops of 
the suspension were placed on the surface 
of 2 per cent water agar in plates and in- 
cubated at 25 degrees. 
Good germination was always obtained 
with cells from the pads of immature 
Curt: Oak Witt INOCULA 291 
(class I) mats in 30 hours. Commonly 
one or two germ tubes, and frequently 
four or five, were produced on a single 
cell, fig. 8C. The germ tubes branched 
prolifically, forming typical conidiophores 
which produced endoconidia abundantly, 
fig. 84, D. Cells of all sizes, from 12 by 
15 to 45 by 60 microns, germinated read- 
ily. The germination rate was in some 
cases as high as 87 per cent. 
Germination of pad cells from mature 
(class IL) mats was obtained only occa- 
sionally, and no germination of pad cells 
was obtained from mats in the more ad- 
vanced stages of decline. The number of 
pad cells that appeared to be collapsed or 
plasmolyzed was greater in pads of ad- 
vanced condition classes than in pads of 
classes I and II. Most deteriorating pads 
consisted of mycelial fragments, bacteria, 
and spores and hyphae of fungi other than 
Endoconidiophora fagacearum. 
The temperature requirements for the 
germination of pad cells in vitro were de- 
termined and compared with similar re- 
quirements for germination of conidia and 
ascospores. A suspension of cells was pre- 
pared from the interior portion of a pad 
taken in September from an immature 
mat on a wilt-killed oak. Drops of the 
suspension were planted on the surface of 
2 per cent water agar in Petri dishes and 
each dish subjected to one of five incuba- 
tion temperatures: 8, 15, 22, 28, and 34 
degrees. Counts of 200 cells for each 
temperature were made at intervals of 12 
hours. 
The highest germination rate of cells 
was 71 per cent at 22 degrees after 60 
hours, table 5. Good germination oc- 
curred at 15 and 28 degrees, fig. 9. 
Among cells incubated at 8 degrees, ger- 
mination did not begin for 36 hours. 
Among cells subjected to 34 degrees of 
temperature, germination did not take 
place during the 60-hour period, nor later 
when the temperature was changed to 22 
degrees. Production of conidia by the 
branches, or conidiophores, of the germ 
tubes occurred in 12 hours at 22 degrees, 
fig. 8B, and 28 degrees, and in 36 hours 
at 15 degrees. Sporulation seemed to be 
most abundant at 28 degrees, fig. 8D. 
The results of the foregoing tests indi- 
cate that pad cells of Endoconidiophora 
fagacearum have a slightly lower opti- 
