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United States
Department of
Agriculture

Forest Service

Pacific Northwest
Forest and Range
Experiment Station

Research Note
PNW-428
October 1985

Abstract

Maintaining Cultures
of Wood-Rotting Fungi

i
PSW FOREST AND RANGE
EXPERIMENT STATION

E.E. Nelson and H.A. Fay JAN 2 91965

STATION LIBRARY COPY |

Phellinus weirii cultures were stored successfully for 10 years in small alder (Alnus rubra
Bong.) disks at 2 °C. The six isolates tested appeared morphologically identical and after
10 years varied little in growth rate from those stored on malt agar slants. Long-term
storage on alder disks reduces the time required for maintaining cultures and the risk of
contamination inherent in frequent transfers necessary in storage on agar slants.

Keywords: Root rot, Phellinus weirii, cultures, laboratory methods, storage methods,
cold storage.

Mycologists, plant pathologists, and others have often wished that cultures of fungi could
be kept indefinitely in the laboratory ready to use, with little or no maintenance, and
without altering their physiology or structure. Until recently, our culture collection, mainly
isolates of Phellinus weirii (Murr.) Gilbertson, was kept at 2-5 °C on malt agar slants and
transferred to fresh media annually. Isolates appeared normal 15 or more years, but the
procedure was time consuming and risk of contamination of the cultures was greater
than seemed necessary.

We propose an alternative that requires infrequent attention. Five isolates of P. weirii
from roots of mountain hemlock (Tsuga mertensiana (Bong.) Carr.) growing at high
elevations in the Cascade Range in Oregon, and five isolates from roots of Douglas-fir
(Pseudotsuga menziesii (Mirb.) Franco) growing at low elevations in western Oregon
and Washington were compared for their abilities to survive over a range of temperatures
(Nelson and Fay 1975). With the same procedures used in that study, we have main-
tained cultures of P. weirii for more than 10 years at 2 °C. Thin sections of red alder
(A/nus rubra Bong.) stemwood were placed over gravel and water in loose-capped,
60-ml-capacity French Square bottles; they were autoclaved and seeded with a small
agar plug of the desired fungal isolate. Once the fungus had colonized the disks, bottles
were tightly capped and maintained at a constant temperature. We successfully isolated
P. weirii from all bottles of 6 of the original 10 isolates maintained at 2 °C for 10 years. We
used malt agar containing 1 p/m benomy| as our isolation medium.

E.E. NELSON is a research plant pathologist and H.A. FAY isa
biological laboratory technician at the Pacific Northwest Forest and
Range Experiment Station, Forestry Sciences Laboratory, 3200
Jefferson Way, Corvallis, Oregon 97331.

After 10 years storage, we compared the growth rate and general appearance of the jglis
colonies of the six isolates maintained on alder disks with the growth rate and general
appearance of the colonies of the same isolates maintained over the same period on malt

agar slants. Subcultures of each isolate from both methods of storage were grown in 1.5
percent malt agar in individual petri plates incubated in the dark at 5, 10, 15, 20, 25, and rf
30 °C. Four replicates of the six isolates from both storage methods were measured
periodically, daily in most cases. Colony diameter was plotted over time for each
temperature, and slope of maximum growth was determined. Isolates stored in alder

disks generally had slightly slower rates of growth than those stored on malt agar slants

(fig. 1). Analysis of variance determined that these differences, though small, were

significant (P = 0.05), as were differences in growth among isolates. We noted no
differences in morphology of an isolate stored on disks vs. those stored on slants. No
pathogenicity tests were made.

We have not used this means of storing cultures of other fungi; however, there is reason
to expect the technique to be applicable to cultures of other species of wood-rotting fungi.
We suggest that others explore this alternative to frequent periodic maintenance of their
culture collections of wood-rotting fungi.

OD Transferred annually on malt agar slants
B Stored 10 years on alder disks

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Temperature (°C)

Figure 1.—Maximum rates of growth of Phellinus weirii isolates by
two storage methods; three isolates are from a high elevation mixed
conifer forest (W), and three are from a lower elevation Douglas-fir
forest (T).

ist Equivalents 1 milliliter (ml) = 0.001056 quart

1 millimeter (mm) = 0.0394 inch

SC = 7S/9 (oF - 32)

a’ ure Cited Nelson, E.E.; Fay, H.A. Effect of temperature on growth and survival of high- and

low-elevation isolates of Phellinus (Poria) weirii. Northwest Science. 49: 119-121;
1975.

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