Volume 14, Part 3, August 2000

A modified filter paper technique for long-term

preservation of some fungal cultures
Y. K. FONG\ S. ANUAR\ H. P. LIM\ F. Y. THAM2 & F. R. SANDERSON3
'National Parks Board, Singapore Botanic Gardens, Cluny Road, Singapore 259569;
2School of Science, Nanyang Technological University, Bukit Timah Road, Singapore 259569;

3PNG Oil Palm Research Association, P.O. Box 36, Alotau, Milne Bay Province, Papua New Guinea

A modified filter paper method was found to be very effective for preservation of Marasmiellus
inoderma, Ganoderma sp. and Fusarium oxysporum cultures. At -19°C, 75% of M. inoderma
isolates were found to be viable after two years of storage, 81% of Ganoderma isolates after five
months and 100% of F oxysporum isolates after four years. All recovered cultures were found to
be free from mite infestation. For successful recovery, fungal cultures must be incubated under
optimum growth conditions prior to preservation and mycelial mats on filter papers must be
properly dried. When compared to conventional freeze-drying methods, this method of
preservation is much simpler, less expensive and requires only a cheap vacuum system consisting
of a desiccator and a vacuum pressure station, and either a domestic refrigerator or domestic
freezer. For small-scale preservation of cultures in the laboratory, this modified method is also
less costly than using liquid nitrogen.

Introduction This communication reports the results of

The study of pathogenic fungi often involves use
of living cultures. Many fungi thrive on full
strength Potato Dextrose Agar (PDA) (Difco,
USA). This rich medium encourages vigorous
mycelial growth of fungi but may lead to eventual
loss of sporulation. A period of growth on a
starvation medium may be needed to encourage
sporulation (Smith & Onions, 1994; Dhingra &
Sinclair, 1995). Infectivity of propagules (and
quality of inoculum) is often related to nutritive
status. Repeated transfer of a pathogen on
artificial media usually results in the loss of
either its pathogenicity or sporulation or both
(Dhingra & Sinclair, 1995).
In studying pathogenic fungi, it is thus
important that cultures be kept viable, and if
they are of economic importance, they should be
maintained alive and unchanged for future
research. Several preservation methods are used.
Continuous growth methods to maintain isolates
can only be used for the short-term. Methods
that reduce rates of metabolism of
microorganisms are for long term storage (8 to 20
years) but not all isolates stored this way will
survive (Hawksworth et al., 1995). Methods that
halt or suspend metabolism of microorganisms
can be used to store cultures for longer terms or
indefinitely (Dhingra & Sinclair, 1995).
using a reliable, effective and simple method for
long-term preservation of Marasmiellus inoderma,
Ganoderma sp., and Fusarium oxysporum isolates
from different host plants and locations in
Singapore. The preservation method is modified
from the long-term storage method for Fusarium
used by the Crown Research Institute of NZ
(Rees-George, J., Crown Research Institute,
Auckland, personal communication). All three
fungi tested in this study are of economic
importance in Singapore as M. inoderma is the
causal agent of basal rot of the orchid, Oncidium
Goldiana (Fong, et al., 1996); Ganoderma, the
causal agent of basal rot of ornamental palms
(Yok King & Sanderson, 1994) and F oxysporum,
the causal agent of Angsana tree wilt disease
(Sanderson, Fong & Saiful Anuar, 1997; Sanderson
et al., 1997; Sanderson et al., 1998). A reliable and
effective method of preservation for these isolates
allows sufficient infective propagules for
inoculation in pathogenicity and virulence tests,
disease screening, fungicide screening and
population study experiments. The development
of a method to store the cultures in a viable, pure
and stable condition is also necessary for
comparing and noting variations in isolates
collected over time and for purposes of exchange
of study material between laboratories.

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 Volume 14, Part 3, August 2000
Materials and methods
The three fungi were preserved as described in
some detail below (all steps requiring sterile
conditions are carried out within a laminar flow
hood):
1. A piece of sterile 70 mm filter paper (No.1
Qualitative, Whatman, UK) was placed in the
centre of a 90 mm plastic Petri dish containing
full strength PDA at pH 5.6. Sterile filter
papers were prepared by placing filter papers
in an aluminium bag in an autoclave for 60 min
at 121°C and 15 p.s.i. (15 lb/in'), followed by
drying in an oven for three hours at 150°C.
2. A sterile scalpel was used to cut the mycelia at
the advancing margin of a seven-day old
actively growing fungal culture on full strength
PDA into small 3 mm' pieces. Six pieces were
placed face down on a Petri dish of PDA
(prepared as in step 1) at equal distances
apart, a few mm away from the circumference
of the filter paper (Fig la).
PDA
fungal
fungal plug mycelia
a desiccator had to be closed first before
Fig 1. (a) Full strength PDA plate inoculated with six
pieces of 3 rnm square fungal plugs, placed face down on
PDA a few mm away from the circumference of 70 mm
filter paper. (b) Seven days after inoculation of PDA with
fungal plugs at 28°C and incubated in darkness . Mycelia
of fungus growingout from plugs and radiating onto filter
paper.
silica gel - -_
filter paper ramified
with fungal mycelia
Fig 2. Vacuum desiccator with blue indicating silica gel at
the base. On the perforated plate are the 90 rnm plastic
Petri dishes with peeled filter papers for drying. Vacuum
desiccator is connected to a pressure vacuum station via
the outlet valve.
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3. The Petri dish was sealed with plastic film
(Glad wrap, USA) and incubated at 28°C under
total darkness in an incubator.
4. When the filter paper in the Petri dish was
completely covered by the mycelia of the
fungus (Fig lb) , it was carefully peeled off and
separated from the PDA using sterile forceps
inside a laminar flow hood. Most fungal
species take more than seven days to grow
over the filter paper. Care was taken to ensure
the filter paper was free of most of the PDA as
remains of media stuck to the paper can slow
down the drying process.
5. The filter pap er with the mycelial mat was
placed inside a sterile plastic 90 mm Petri dish
(one filter paper per Petri dish). This was then
placed on the perforated plate inside a simple
vacuum desiccator (Model 5311, Nalgene,
USA), completely filled with dry silica gel at
the base (Fig 2). The desiccator was connected
to a vacuum pressure station (Air Cadet , Cole-
Palmer Instrument Company, USA) and the
filter paper was dried under vacuum. During
this process, the silica gel may have to be
replaced as it becomes wet (turns from blue to
pink) ; this is especially so when there are
many Petri-dishes of filter papers with mycelia
to be dried. To replace the silica gel inside the
desiccator that was still attached to the
vacuum station, the outlet valve of the
switching off the vacuum station. When the
pressure of the station dropped to zero, the
desiccator was separated from the station and
placed inside a laminar flow hood. The
desiccator was slowly depressurised by
opening the outlet valve a little at a time. Once
completely depressurised, it was opened, and
the silica gel replaced. The desiccator was then
reconnected to the vacuum station. When the
filter papers appeared completely dry, the
process was stopped immediately. On average
it takes a minimum of 15 hours to dry 10 filter
papers. Over-drying may result in increased
cell death in the fungal mycelial mats .
However, it is very important that the filter
papers are completely dry before placing them
in the freezer for storage. This is because too
much moisture remaining in the filter papers
can affect the viability of the fungal cultures
destined for storage below freezing
temperatures.

6. To remove the dried filter papers, the
desiccator was placed inside the laminar flow
hood and depressurised as in step 5. Once
depressurised, the outlet valve was closed. It is
important, especially in the tropics, that the
desiccator be kept airtight at all times to
prevent the dry filter papers from absorbing
moisture from the humid atmosphere. The
Petri dishes with the dry filter papers were
removed from the desiccator one at a time and
the filter paper within each was subsequently
taken out with forceps and quickly cut into
small strips in the laminar flow hood using
scissors (forceps, scissors and other apparatus
used were sterile). The filter paper strips were
quickly placed inside a dry sterile 30 ml screw
cap bottle using forceps. If any filter paper
appeared to be not completely dry, it was put
back in the Petri dish and vacuum dried for
another few hours in the desiccator. Each
fungal isolate was stored in a separate bottle.
A few pieces of the filter paper strips from
every isolate were also placed on full strength
PDA at pH 5.6 and incubated at 28°C in total
darkness in an incubator. This was to check for
culture viability before storage in the freezer.
7. Care was taken to ensure the cap of the
storage bottle was screwed tightly. The dried
and bottled isolate was immediately placed
inside the freezer compartment of a normal
household refrigerator at -19°C for storage.
8. When an isolate was needed for an
experiment, the bottle was removed from the
freezer compartment and the desired number
of fungal paper strips were taken out in an
aseptic environment with sterile forceps. The
bottle was then quickly closed and replaced in
the freezer. Fungal strips for plating were
placed on full strength PDA and cultured as in
step 6. Cultures were recovered from these
fungal strips in approximately a week to 10
days.
In the Crown Research Institute of NZ, a more
elaborate Venturi System on a cold water tap was
used for drying the filter paper strips. We have
relied successfully on a small portable vacuum
pressure station instead to create the vacuum for
the drying process.
The drying process was used and optimised
for M. inaderma from Oncidium orchids,
Ganaderma sp. from ornamental palms and F
axysporum from Angsana (Pterocarpus indicus)
Volume 14, Part 3, August 2000
trees in the Plant Pathology laboratory at the
Singapore Botanic Gardens. To assess its
effectiveness and reliability, 40 isolates each of M.
ina derma, Ganaderma sp. and F axysparum,
stored as above, were randomly selected from the
freezer compartment. Approximately ten pieces
of fungal filter paper strips of each isolate were
taken out from each bottle, plated onto full
strength PDA at pH 5.6 and incubated at 28°C in
total darkness in an incubator. The viability of
isolates was assessed by observing fungal growth
from the filter paper strips placed on PDA after
10 days of incubation.
Results and discussions
The drying method was found to be very effective
in preserving and storing isolates of M. inaderma,
Ganaderma sp. and F axysparum. The results
indicated that after two years of storage at -19°C
in the freezer compartment of a domestic
refrigerator, 75% of the M. ina derma isolates
tested remained viable. In Ganaderma sp., the
viability was 81% after the fifth month of storage
and the viability for F oxysparum isolates were
100% after four years. All cultures preserved
using this method were found to be free from
mite infestation.
Cultures preserved using this protocol may be
as stable as those preserved using lyophilization
methods as both involve vacuum drying of the
fungi. However, in the lyophilization method,
dried cultures are sealed under vacuum before
storage in either room or freezer temperatures.
Pre-freezing of the cultures before vacuum drying
was also sometimes included. Removal of water
from fungal cells under vacuum is known to slow
down or suspend metabolism so that subsequent
storage in the refrigerator prolonged the longevity
of the cultures (Kondo, 1961; Hughes & Macer,
1964; Flor, 1967).
For successful storage of fungal cultures using
this method, the following criteria, in particular,
had to be strictly observed. Firstly, prior to
preservation, fungal cultures must be incubated
under optimum conditions for growth. This is
also one of the key factors for lyophilization to
be successful (Dhingra & Sinclair, 1995).
Secondly, the degree of dehydration of fungal
mycelial mats was also found to be critical for
culture survival. Too much moisture remaining
in the mycelial mats and filter papers can cause
the death of the cultures during the freezing and
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 Volume 14, Part 3, August 2000
thawing processes, and hence affect viability.
Over-drying of cultures by increasing the drying
time could also result in increase in cell death.
As with lyophilization, the drying process should
be stopped immediately after the culture
appeared dry (Dhingra & Sinclair, 1995). Thirdly,
bottles used for the storage of dry fungal paper
strips have to be thoroughly sterilised and dried
before use to prevent contamination and
introduction of unnecessary moisture to the dry
paper strips. Lastly, dried cultures on filter
papers must be cut into strips as soon as
possible after removal from the desiccator and
placed into storage bottles with tight screw
caps. This was necessary to prevent the dry
cultures from absorbing moisture from the
atmosphere.
Long-term preservation is useful as it
overcomes some of the disadvantages of
continually maintaining isolates in a state of
growth or using conventional storage methods.
Continuous growth methods often lead to loss of
pathogenicity, as well as changes in other
physiological or morphological characteristics in
cultures. Cultures are also subjected to airborne
mite contamination. Constant specialist
supervision is also necessary to ensure cultures
are not replaced by contaminants or sub-cultured
from atypical sectors (Smith & Onions, 1994).
When compared to conventional freeze-drying
methods, this method of preserving cultures of
M. inoderma, Ganoderma sp. and F oxysporum
cultures is simple, neat, inexpensive and required
only a cheap vacuum system consisting of a
desiccator and a vacuum pressure station, and
either a domestic refrigerator or domestic freezer.
For small-scale preservation of cultures in the
laboratory, this modified preservation method is
also less costly than using liquid nitrogen
methods of preservation. The latter requires
costly apparatus, regular supply of expensive
nitrogen gas and a well-ventilated room for the
storage of nitrogen gas. When the storage bottles
with the filter papers are tightly sealed with
plastic film, cultures can be easily and
conveniently transported to other laboratories for
exchange purposes.
Acknowledgements
YKF thanks Jonathan Rees-George and Brian
Hawthorne of Crown Research Institute at
Auckland, New Zealand, for sharing their filter
paper method for long-term preservation of
fungal cultures. The authors also thank National
Parks Board of Singapore and School of Science,
Nanyang Technological University of Singapore
for research funding.
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