PHYTOCHEMICAL ANALYSIS

Phytochem. Anal. 11, 137–147, (2000)

Testing of Antifungal Natural Products:

Methodologies, Comparability of Results and
Assay Choice

Franz Hadacek* and Harald Greger

Comparative Phytochemistry Department, Institute of Botany, University of Vienna, A-1030 Vienna, Austria

Antifungal testing of filamentous fungi generally suffers from incomparability of results. In order to
illustrate this problem, the polyacetylene falcarindiol and the naphthoquinone juglone, two known
antifungal natural products, were assayed in various dilution and diffusion bioassays against three selected
microfungi, Botrytis cinerea, Cladosporium herbarum and Fusarium avenaceum. Broth microdilution, based
on the 96-well microtitre plate format, can be scored by various direct and biochemical methods. As
examples, direct observation with image analysis and the fluorescein diacetate scoring method are
compared. Of the other methodologies used, results obtained by thin-layer bioautography and the radial
growth rate method clearly deviated. Disk diffusion results, however, matched microdilution. In conclusion,
microdilution offers the greatest potential of all bioassays to become the future general standard
methodology. Copyright # 2000 John Wiley & Sons, Ltd.
Keywords: Antifungal bioassay; microdilution; radial growth; bioautography on thin-layer plates; disk diffusion;
falcarindiol; juglone; Botrytis cinerea; Cladosporium herbarum; Fusarium avenaceum.

INTRODUCTION Shephard (1987) estimated the cost to discover and

Large demands for fungicides exist in agriculture, food
protection and medicine. Plant pathogens are estimated to
cause yield reductions in crops of almost 20% worldwide
(Oerke et al., 1994). In order to cope with the needs of the
fast-growing world population, yields must be improved
by optimizing inputs, including fungicides (Knight et al.,
1997). Moreover, food quality has to be guaranteed by
controlling fungi that produce mycotoxins. Filamentous
fungi can also cause opportunistic systemic mycoses,
associated primarily with patients with AIDS or those
receiving treatment with immunosuppressive agents.
Antifungal chemotherapy relies heavily on fungicides
and many efforts have been made to standardize test
procedures in order to increase reproducibility between
laboratories (Cormican and Pfaller, 1996). As a result the
National Committee for Clinical Laboratory Standards
(NCCLS) proposed in 1997 a standardized antifungal
susceptibility test method for yeasts (M27-A; National
Committee for Clinical Laboratory Standards, 1997)
containing guidelines for broth macrodilution and
microdilution. A modification of that method (M38) for
filamentous fungi appears promising and its standardiza-
tion is in progress (Espinel-Ingroff et al., 1995, 1997;
Pujol et al., 1996; Espinel-Ingroff, 1998). However,
because filamentous fungi do not grow as single cells,
standardization is more challenging than with unicellular
yeasts and bacteria (Gadd, 1986).
* Correspondence to: F. Hadacek, Comparative Phytochemistry Department,
Institute of Botany, University of Vienna, A-1030 Vienna, Austria.
E-mail: franz.hadacek@univie.ac.at
Contract/grant sponsor: “Hochschuljubiläumsstiftung der Stadt Wien”.
Contract/grant sponsor: Austrian National Committee for the Intergovern-
mental Program “Man and Biosphere”.
develop a new fungicide at around 30 million US dollars.
Consequently, research is mainly carried out by leading
pesticide companies in Europe, Japan and the United
States. Automated screens, targeting specific modes of
action, have now been widely adopted (Major, 1995), all
based on the microtitre plate format, to allow screening of
10,000 or more compounds per week. The majority of
them are cell-based assays; however, their comparability
to conventional in vivo screens is much debated (Knight
et al., 1997).
Other groups of researchers, such as university-based
scientists or those working in smaller companies,
interested in detecting antifungal activity, cannot nor-
mally afford high-throughput-screening equipment. Of-
ten, lack of appropriate facilities or the small amounts of
test compounds available do not permit in vivo assays
which typically involve concentrations (100–500 mg/L)
of high volume sprays being applied to living plants
previously inoculated with pathogenic fungi; in vivo
assays have been extensively reviewed by Kato (1986),
Shephard (1987), Heaney and Knight (1994), and Bartley
and Youle (1996). Instead, in vitro assays are the method
of choice for many pharmacists, biologists, plant
pathologists and chemists interested in the biological
activity of natural products isolated in their laboratories.
Antimicrobial in vitro screening methods have pre-
viously been reviewed by Gadd (1986), Rios et al.
(1988), Paxton (1991), Mayfield (1993), and Cole (1994).
The present article presents a comparative evaluation of
various dilution and diffusion bioassays illustrated by
application to two antifungal natural products. The
polyacetylene falcarindiol and the naphthoquinone ju-
glone were assayed against three different microfungi,
Botrytis cinerea, Cladosporium herbarum, and Fusarium
avenaceum. Falcarindiol occurs in various genera within

Received 24 April 1999

Copyright # 2000 John Wiley & Sons, Ltd. Revised 1 September 1999
Accepted 1 September 1999
138 F. HADACEK AND H. GREGER
the Apiaceae and Araliaceae (Kemp, 1978; Kern and
Cardellina, 1982; Muir et al., 1982; Villegas et al., 1987),
and has even been identified as a phytoalexin in infected
tomato leaves (Elgersma et al., 1984); it was chosen to
represent aliphatic lipophilic compounds. Juglone can be
found in the genera Carya (Hedin et al., 1980) and
Juglans (Krajci and Lynch, 1977) of the Juglandaceae,
and is responsible for the allelopathic effects caused by
the walnut tree Juglans regia (Bode, 1958); it serves as an
example of a polar, aromatic natural product. As results
varied considerably we feature broth microdilution (used
in clinical research) as the most promising antifungal in
vitro bioassay. Its use should improve consistency
between those laboratories involved in natural product
research.
EXPERIMENTAL
Fungal inocula. Botrytis cinerea (Vienna Institute of
Applied Microbiology no. MA1510), Cladosporium
herbarum (Vienna Institute of Applied Microbiology
no. MA1511), and Fusarium avenaceum (Vienna In-
stitute of Applied Microbiology no. MA1512) were
cultured on 4% (w/v) malt extract–agar medium (Merck,
Darmstadt, Germany) or 4% (w/v) potato dextrose–agar
medium (Merck) in 9 cm Petri dishes at room tempera-
ture and in darkness. In order to obtain conidia, fungi
were cultured for 3–10 days. Harvesting was carried out
by suspending conidia in 1% (w/v) sodium chloride
solution containing 5% (v/v) dimethylsulphoxide
(DMSO) or Tween 80. A sterilized Drigalski spatula
was used to separate spores from hyphae. The spore
suspension was filtered (pore width 53 or 105 mm,
depending on spore size), transferred into 1.8 mL
cryotubes and stored at ÿ20 °C. Colony forming unit
(CFU) values were determined by plating 20 mL of 10-
times serially diluted suspensions and counting germi-
nated spores/dilution concentration.
Test compounds. Falcarindiol was isolated from roots of
Peucedanum cervaria (L.) Lapeyr. by repeated chroma-
tography on silica gel 60 (0.025–0.040 mm; Merck) with
hexane:ethyl acetate (85:12, v/v) as the mobile phase and
with UV detection at 254 nm. Chromatographic and
spectroscopic data were identical to published data.
Synthetic juglone was obtained from Professor Hartmut
Laatsch (Institute of Organic Chemistry, University of
Göttingen, Germany).
Bioautography on thin-layer plates. Test compounds
(10 mg) were dissolved in 500 mL acetone and twice
diluted from 0.03 to 200 mg/mL. Test solutions (10 mL)
were applied as small spots on TLC plates (Si 60;
0.25 mm; Merck) to give a concentration series of 0.1–
200 mg/application zone; the organic solvent was evapo-
rated by a stream of air. TLC plates were homogeneously
sprayed with about 30 mL of 4% (w/v) malt extract broth
containing conidia (105 CFU/mL). Plates were incubated
for 3 days in a moist chamber at 25 °C in the dark and the
appearance of blank zones in the mycelium layer
indicated antifungal activity. TLC plates were digitised
by a flatbed scanner. Minimum inhibitory concentrations
(MIC) were determined from the lowest test compound
concentration causing recognisable mycelium-free zones.
Copyright # 2000 John Wiley & Sons, Ltd.
Exposure (15–30 min) of fungi with unpigmented
mycelia and spores to iodine vapours significantly
enhanced contrast in order to detect inhibition zones.
Paper disk diffusion. Test compounds (6 mg) were
dissolved in 600 mL acetone and two-times diluted from
0.7 to 10.0 mg/mL; 20 mL aliquots were applied to 9 mm
diameter paper disks (no. 2668/2; Schleicher and Schuell,
Dassel, Germany). After evaporating the organic solvent,
disks were placed in the centre of 3 cm diameter agar
plates previously inoculated with 0.5 mL spore suspen-
sion (104 CFU/mL). After 3 days, growth was measured
(mm) with a ruler. A Super-VHS video camera was used
to capture still video images onto a hard disk. MIC was
determined as the lowest test compound concentration
causing mycelium-free zones.
Broth microdilution. Test compounds were dissolved in
acetone (2 mg/mL) and mixed with 4% (w/v) malt extract
broth, to which 0.2% (v/v) Tween 80 was added to
improve emulsification, to give a concentration of
400 mg/mL in stock solution. Microdilution was per-
formed in sterile disposable microtitre plates (96 U-
bottomed wells). Aliquots (50 mL) of 4% (w/v) malt
extract broth were dispensed in every well except the
first. Stock solutions were two-times serially diluted
using a 50 mL multichannel pipette. Compounds were
tested in duplicate. Stock solutions of the negative control
were prepared with pure organic solvent. Stock inoculum
suspensions were adjusted to 104 CFU/mL; 50 mL
aliquots were dispensed into each well. The final
concentrations were 0.1–200 mg/mL for each compound.
Microtitre plates were incubated 18 h at 25 °C in dark-
ness.
Germ-tube inhibition was determined by two methods:
direct observation and fluorometric measurement of
esterase activity. In order to determine germ-tube size,
10 mL aliquots of lactophenol blue (Merck) were
dispensed into each well (to stop growth and to enhance
contrast) and 10 germ-tube images per concentration
were captured to hard disk (magnification 140-times);
sizes were calculated in pixels (NIH Image 1.60 software;
National Institute of Health, Bethesda, MD, USA).
Esterase activity was determined following the method
of Chand et al. (1994), the verification of esterase
production being based on the conversion of fluorescein
diacetate (FDA) to the fluorescent dye fluorescein, the
concentration of which may be determined by emission at
635 nm with excitation at 485 nm. Fluorescence readings
in the microwells were performed using a Labsystems
(Helsinki, Finland) Fluoroskan Ascent FL and a Perkin-
Elmer (Wellesley, MA, USA) HTS7000 bioassay reader.
Prior to emission reading, 5 mL aliquots of a 0.25% (w/v)
solution of fluorescein diacetate (Sigma, St Louis, MO,
USA) in acetone were dispensed into each microwell; the
microtitre plate was then incubated for 90 min at 37 °C to
facilitate hydrolysis. The percentage of inhibition
compared to the control was determined from the
calculation:
‰…sample ÿ blank†=…control ÿ blank†Š 100
where the sample was the serially diluted test compound
incubated for 18 h, the control was the stock solution with
pure organic solvent, and the blank was the control with
ungerminated conidia suspension. Means of three emis-
Phytochem. Anal. 11: 137–147 (2000)
 ANTIFUNGAL ACTIVITIES TESTS
sion readings of sample and control, and two readings of
blank were used, all within the same microtitre plate.
MIC was determined as the lowest test compound
concentration completely inhibiting spore germination.
Radial growth rate. Test compounds (12 mg) were
dissolved in 2 mL acetone and twice diluted from 0.03 to
6 mg/mL; 1 mL acetone was added to 30 mL of 4% (w/v)
malt extract agar to give 1.5–200 mg/mL agar media.
Aliquots (2 mL) of medium were dispensed into 3 cm
diameter Petri dishes (Nunc, Roskilde, Denmark) and
stored for 24 h at room temperature to solidify (during the
whole process the agar medium was maintained at 50 °C).
Conidiospore suspensions (10 mL; 105 CFU/mL) were
used to inoculate the centre of the agar plate. After
incubation for 3 days, diameters of visible colonies were
measured with a ruler and percentage inhibition was
calculated from means of three replicates compared to
controls. A Super-VHS video camera was used to capture
still video images onto hard disk. MIC was determined as
the lowest test compound concentration completely
inhibiting spore germination.
Statistics. Broth microdilution and radial growth rates
are quantitative bioassays. EC50 and EC90 were calcu-
lated by probit-log analysis as described for quantitative
bioassays by Finney (1971, 1978) and Roberts and Boyce
(1972). The Mann–Whitney U-test was used to compare
distributions of control and sample group values.
Calculations were performed using SPSS 6.1 software
(SPSS Corp., Chicago, IL, USA).
RESULTS AND DISCUSSION
In all bioassays, juglone proved to be a more efficient
antifungal natural product than falcarindiol against
B. cinerea, C. herbarum, and F. avenaceum, results
generally in agreement with previously published data
(e.g. Ikekawa et al., 1967; Kemp, 1978; Villegas et al.,
1987; Rahalison et al., 1994). EC50 and EC90 have been
calculated for broth microdilution and radial growth
rates, but not for paper disk diffusion and bioautography
on TLC plates. The latter two bioassay techniques depend
on diffusion of the test compound into the medium, and
thus concentration within the medium is not homoge-
neous in contrast to the dilution methods. MIC values
were determined visually as the lowest concentration
causing an effect. Table 1 presents a summary of the
results.
MIC values obtained by the radial growth rate method
were significantly higher for all compounds tested
compared to those obtained using the broth microdilu-
tion. The different scoring methods of broth microdilu-
tion did not provide identical results, however,
differences fell within an acceptable range. Disk diffu-
sion MICs, despite the different nature of the bioassay,
compared rather well to broth microdilution MICs. Thin-
layer bioautography, of all assays, provided the lowest
MIC values and thus turned out to be the most sensitive
method of all.
In dilution bioassays, fiducial limits of EC50 and EC90
provide additional information about the nature of the
inhibition effect. Falcarindiol showed very broad fiducial
limits and a significant increase in the EC50:EC90 ratio in
Copyright # 2000 John Wiley & Sons, Ltd.
139
microdilution, well visible in both scoring methods. This
effect is caused by a trailing endpoint of the inhibition
effect. The situation was quite different with respect to
radial growth rates. Values were not only significantly
higher, but the EC50:EC90 ratio was reversed with
juglone, showing the broader fiducial limits. These facts
illustrate the differences between the two dilution assays,
the former relying on submerged growth and the latter on
surface growth of the test fungus.
MIC values of antifungal compounds
In the literature, antifungal activities are often reported as
MIC values, which typically denote the lowest concen-
tration of test compound which inhibits growth. Although
reproducible and yielding values expressed in mg/mL,
MICs are still a function of the conditions set by the tester
(Rex et al., 1997). This also applies to MIC values
presented in Table 1. To complicate matters, the
definition of MIC value may be ambiguous. In diffusion
bioassays, MIC is defined as the lowest test compound
concentration causing a visible inhibition effect, whilst in
dilution methods, MIC denotes the lowest test compound
concentration totally inhibiting growth: strictly speaking,
these values are not comparable.
Conidia suspensions
One of the major requirements to carry out most
antifungal bioassays is to obtain conidia from the chosen
test fungus. If the desired fungus does not sporulate then a
radial growth assay remains the method of choice. Plant
pathology manuals generally contain many recipes for
axenic culture media suitable to induce sporulation in
various microfungi (e.g. Singleton et al., 1992; Dhingra
and Sinclair, 1995). A great number of perthotrophic and
necrotrophic fungi generally produce conidia when
cultured on artificial media, contrary to biotrophic fungi.
The latter group can only be tested with in vivo assays
using clamydospores or teliospores (Dhingra and Sin-
clair, 1995). In bioassays with bacteria and yeasts,
especially in clinical studies, culture collection type
strains are used preferentially. This procedure is not as
commonly employed with filamentous fungi. As a
general rule, however, fungal strains included in
bioassays should be deposited in a culture collection
specifically to enable other researchers to obtain the same
isolate for comparative purposes. Methods for preserving
conidia and mycelia have been extensively reviewed by
Dhingra and Sinclair (1995). However, the most suitable
preservation method for a particular fungus must often be
optimised empirically. Procedures described in the
experimental section should apply to the majority of
microfungi.
Another relevant issue is inoculum size. Gehrt et al.
(1995) demonstrated the species-dependent propensity of
some microfungi to show lower susceptibility in broth
microdilutions starting at 105 CFU/mL. Similar effects
have previously been observed for some bacteria (Strat-
ton, 1993) and yeasts (Rex et al., 1993). The authors
correlate these effects with the mode of action of the test
compound: drugs attacking enzymes are especially
affected. Increasing numbers of microbial targets may
exceed the given amount of a test compound to inhibit
totally the growth of the micro-organism population in the
system. Similarly, as the number of fungal propagules is
Phytochem. Anal. 11: 137–147 (2000)
Copyright # 2000 John Wiley & Sons, Ltd.

140
Table 1. EC50,a EC90a and MIC values (mg/mL) of falcarindiol and juglone against Botrytis cinerea, Cladosporium herbarum and Fusarium avenaceum determined by various
bioassay techniques
Botrytis cinerea Cladosporium herbarum Fusarium avenaceum
b
EC50 (95% FL ) EC90 (95% FL) MIC EC50 (95% FL) EC90 (95% FL) MIC EC50 (95% FL) EC90 (95% FL) MIC

Radial growth ratec

Falcarindiol 86 (77±96) 219 (184±273) >200 98 (81±123) 213 (162±348) >200 52 (39±70) 139 (96±273) 200
Juglone 20 (12±35) 153 (75±592) 200 61 (29±135) 147 (82±2172) 100 12 (8±19) 57 (33±147) 50
Broth microdilution (germ-tube growth by image analysis)d
Falcarindiol 21 (8±108) >200 >200 11 (2±589) >200 >200 4 (2±9) 176 (61±1148) 200
Juglone 0.5 (0.2±1) 2 (0.9±16) 1.5 0.5 (0.2±0.5) 2 (1±6) 3 0.2 (0.1±0.2) 1 (0.7±1.6) 0.8

F. HADACEK AND H. GREGER

Broth microdilution (FDA hydrolysis)e
Falcarindiol 0.7 (0.2±1.6) >200 >200 0.3 (0.01±1) >200 >200 3 (1±7) 49 (20±305) >200
Juglone 0.3 (0.2±0.4) 0.8 (0.6±1.5) 3 1.6 (1.1±2.4) 3 (2±9) 3 0.05 (0.01±0.09) 0.3 (0.2±0.6) 1.5
Paper disk diffusionf
Falcarindiol 100 50 >200
Juglone 1.5 1.5 1.5
Thin-layer bioautographyg
Falcarindiol 25 12 50
Juglone 0.1 0.1 0.1
a
EC50 and EC90 were determined by probit-log analysis.
b
FL = ®ducial limits.
c
See Experimental section for protocol Ð growth zone diameters were compared with those of colonies growing on pure medium: MIC de®ned as the lowest concentration of the
dilution series which completely inhibited fungal growth.
d
See Experimental section for protocol Ð after 18 h growth at room temperature, 10 mL lactophenol blue was added to stop growth, and germ-tube size was determined by pixel
counts after capturing images of 10 germinated spores/concentration: MIC de®ned as the lowest concentration totally inhibiting spore germination.
Phytochem. Anal. 11: 137–147 (2000)

e
See Experimental section for protocol Ð percentage inhibition was determined by calculating [(sample ÿ blank)/(control ÿ blank)]1  100; MIC de®ned as the lowest concentration
totally inhibiting spore germination.
f
See Experimental section for protocol: MIC de®ned as the lowest concentration/disk causing a visible inhibition zone.
g
See Experimental section for protocol: MIC de®ned as the lowest concentration/spot causing mycelium-free zones.
 ANTIFUNGAL ACTIVITIES TESTS 141

Figure 1. Visual comparison of results of antifungal activity of juglone determined by the disk diffusion
method (top rows in each panel), by bioautography on thin-layer plates (middle rows), and by radial growth
rates (bottom rows) against the ®lamentous fungi Botrytis cinerea (top panel), Cladosporium herbarum
(middle panel), and Fusarium avenaceum (bottom panel). In each case the concentration of the test
compound was in the range 1±200 mg/mL.

growing, populations of resistant individuals may expand.
However, in the case of microfungi with conidia difficult
to separate from the mycelium, it is necessary to work with
inoculum sizes even lower than 104 CFU/mL. Besides
quantitative plating, Espinel-Ingroff et al. (1991) have
described a spectrophotometric method to determine
inoculum density.
Comparing fungal inocula with yeasts and bacteria,
one significant difference has to be kept in mind:
guidelines recommend taking inocula from bacterial
and yeast cultures during the logarithmic growth phase,
since this is when the culture is most homogeneous
(Howcroft et al., 1987). Fungi are multicellular; their
conidia, although they are not always unicellular,
represent most closely a unicellular life stage. However,
the conidia have to germinate first to grow, and
germination will not always be 100%; spore quality will
not be uniform throughout the suspension and between
suspensions. Consequently, standardization of bioassays
for filamentous fungi presents a greater challenge than for
yeasts and bacteria (Cormican and Pfaller, 1996).
Diffusion bioassay: bioautography on thin-layer
plates
Homans and Fuchs (1970) and Betina (1973) have

Copyright # 2000 John Wiley & Sons, Ltd. Phytochem. Anal. 11: 137–147 (2000)
142 F. HADACEK AND H. GREGER
described bioautography on TLC plates (Fig. 1) as a
quick and versatile method to detect antifungal natural
products of plant or microbial origin. In comparison to
other bioautographic methods, such as paper chroma-
tography (Paxton and Wilson, 1965), and agar overlaying
(Rahalison et al., 1991), TLC plates have been most
widely accepted. De Bolle et al. (1991) described how to
carry out bioautography on polyacrylamide gels in order
to detect antifungal proteins. The high possibility of
compound decomposition during the assay is certainly a
disadvantage of this method (Paxton, 1991). Conversely,
ease of performance and minimal requirements of
microbiological equipment make bioautography on
TLC plates useful in laboratories lacking sophisticated
equipment.
Diffusion bioassays, whilst not suitable for the com-
parison of activities of compounds, do allow the
identification of active compounds in crude extracts.
The high sensitivity of these assays is illustrated by the
MIC values of falcarindiol and juglone (Table 1), which
were the lowest obtained. The ability to separate complex
mixtures with subsequent identification of antifungal
compounds carried out on the same TLC plate distin-
guishes bioautography from all other antifungal bioassay
techniques discussed in this work. If this assay is chosen
to compare the antifungal potential of different com-
pounds, the following issues which have not explicitly
been discussed by previous reviewers of this method
must be considered: testing pure compounds in dilution
series, as demonstrated in Fig. 1, will provide concentra-
tion dependent effects of active compounds. However,
besides actual activity, diffusion of the test compound
and organic solvent concentration can also affect the size
of the inhibition zone. Solvent concentrations should be
equivalent, and as low as possible for all test compounds.
Generally, 10 mL aliquots are used as reported in the
Experimental section. Thorough evaporation of organic
solvent before spraying conidia is crucial. Inoculum size
in the spore suspension should be as high as possible,
usually greater than 105 CFU/mL in order to ensure the
formation of an uniform mycelium layer. Suitable
instruments for spraying spores are either glass sprayers
designed to apply chemical derivatisation agents to TLC
plates or airbrushes for colour and varnish application.
Both instruments are easy to sterilize.
Diffusion bioassay: paper disk diffusion
The paper disk diffusion bioassay was first developed for
bacteria (De Beer and Sherwood, 1945) and later
modified for filamentous fungi. The technique was
standardized by Bauer et al. (1966) and Ericson and
Sherris (1971) and later changed in a report of the World
Health Organisation (1977). Agar well diffusion assays
are also similar (Rios et al., 1988), including the hole–
plate assay method and the cylinder method. Disk
methods comprise the placing of filter paper disks
containing test compounds on agar plate surfaces
previously inoculated with the test organism. The
chemical then diffuses into the agar and inhibits
germination and growth of the test fungus. The hole–
plate method depends upon diffusion of the aqueous test
compound solution from a vertical hole in the agar layer,
while the cylinder method utilizes stainless steel or
porcelain cylinders. After incubation, mean diameters of
growth inhibition zones are recorded. Diffusion assays
Copyright # 2000 John Wiley & Sons, Ltd.
are recommended more for polar rather than non-polar
compounds such as essential oils (Janssen et al., 1987).
The dipping of filter paper disks into a test compound
solution should be avoided. Instead, it is more accurate to
dispense the compound dissolved in a small amount of
organic solvent directly on to the filter paper disk.
Rigorous drying of organic solvent is crucial. Negative
controls should be included in order to detect solvent
effects. Generally, 9 cm Petri dishes are recommended.
In contrast, we prefer to use 3 cm Petri dishes for
economical and practical reasons, each dish containing
just one disk.
Figure 1 illustrates effects caused by various concen-
trations of juglone against B. cinerea, C. herbarum, and
F. avenaceum. Results obtained from both diffusion
methods, thin-layer bioautography and paper disks,
compare well. However, the inhibition shown by radial
growth rates, a dilution bioassay with homogeneous test
compound concentration in the medium, is significantly
lower than in the diffusion assays. This example
illustrates the problem of incomparability of results.
Paper disk assays and agar well techniques account,
nevertheless, for the majority of applied antifungal assays
(Cole, 1994). Because it is impossible to measure the
amount of the test substance diffused into the agar
medium, diffusion assays are less suitable to determine
MIC values for compound activity comparisons.
Dilution bioassay: broth microdilution
Dilution bioassays have one major advantage over
diffusion bioassays, namely the test compound concen-
tration in the medium is defined. Consequently, dilution
assays are regarded as the method of choice to compare
MIC values (Gadd, 1986; Rios et al., 1988; Paxton, 1991;
Mayfield, 1993; Cole, 1994). The advent of microtitre
plates has led to significant reductions in test compound
concentrations; furthermore, in combination with spec-
trophotometric or fluorometric plate readers it is possible
to scale up sample throughput enormously. However, as
previously mentioned, filamentous fungi are not single-
cell organisms whose growth is as easy to quantify as
with bacteria and unicellular yeasts where turbidity
indicates growth. Turbidity can be assessed either
visually, as is the convention in clinical studies
(Cormican and Pfaller, 1996; Pfaller et al., 1997), or
spectrophotometrically at wavelengths in the range of
600 nm (Paxton, 1991; Pfaller et al., 1995). Gadd (1986)
and Mayfield (1993) elaborately review various methods
to determine growth rates, biomass and cell numbers of
bacteria, including continuous culture, enzyme activity,
bioluminescence, respirometry, microcalorimetry, moti-
lity, fluorescent antibody and gene probe techniques, the
latter two mostly applied in natural ecosystems. The more
uniform growth behaviour of unicellular bacteria and
yeasts has certainly contributed to methodology devel-
opment and its subsequent standardization (Mayfield,
1993). The differentiation into filamentous or mycelial
fungi and yeasts has been less rigorously applied in the
recent literature. One reason is the high degree of overlap
between the two groups, with mycelial fungi showing
yeast-like growth stages and some traditional yeasts
producing mycelia; the other reason is the more useful
development of antifungal test systems with unicellular
yeasts.
Originally, liquid broth assays for filamentous fungi
Phytochem. Anal. 11: 137–147 (2000)
 ANTIFUNGAL ACTIVITIES TESTS
Figure 2. Scoring methods in broth microdilution: (a)
illustration of the direct observation of germ-tube length by
image analysis (IMG); (b) reaction of ¯uorescein diacetate
(FDA) employed for the determination of growth by the
¯uorometric method; (c) graph showing the agreement of
results of both scoring methods.
were carried out in 20 mL flasks, e.g. as described by
Weidenbörner et al. (1989). After a 7 day growth period,
fungal biomass was determined after filtering and drying
to constant biomass. This assay had the major disadvan-
tage of requiring large amounts of test compound. The
standardized broth macrodilution method recommends
the use of 12 plastic test tubes (75  12 mm i.d.)
containing 1 mL test suspension per concentration. Broth
microdilutions carried out in 96-well U-bottomed micro-
titre plates reduce the medium amount to 100 mL
(National Committee for Clinical Laboratory Standards,
1997). Consequently, the microtitre plate format will
become the future standard. Wilson et al. (1997)
described how to assay volatile essential oils in microtitre
plates by sealing the single wells with dental wax to
prevent cross-contamination.
Visual MIC determination is the most commonly used
method to describe effects of natural or synthetic
chemicals on filamentous fungi. Direct observation of
developed germ-tubes also allows detection of morpho-
logical deformations, such as swelling, curling and bead
shape (Brownlee et al., 1990; Kobayashi et al., 1996).
Scoring inhibitory effects can be carried out with the help
Copyright # 2000 John Wiley & Sons, Ltd.
143
of photographic standards to determine 0, 25, 50 and
100% of drug-free growth (Espinel-Ingroff et al., 1995)
or the indices ÿ, ‡, 2‡, 3‡ (Kobayashi et al., 1996). In
order to score microdilution plates, a reading mirror can
be used (Espinel-Ingroff et al., 1995); otherwise, exact
determination of the germ-tube length with a micrometer
scale in a microscope is a cumbersome task. However,
this has greatly stimulated the search for alternative
methods. The possibility of digitizing images from the
microscopic has led to the utilization of image analysis to
determine germ-tube sizes (Hilber and Schüepp 1992; Oh
et al., 1996). In our laboratory a less sophisticated
method is used to quantify germ-tube growth, as
described in detail in the Experimental section and
illustrated in Fig. 2(a).
Paxton (1991) recommended that turbidimetric scoring
of fungal growth be used with caution because of the
dispersed growth behaviour of the many filamentous
fungi and the possible formation of degradation products
absorbing in the turbidity wavelength. Conversely,
Broekaert et al. (1990) demonstrated a straight-line
relationship of turbidimetric scoring at 595 nm to dry
weight of the developed mycelium and thus present
turbidity measurements with a microplate reader as a
possible tool to detect antifungal activity with an
incentive perspective for automatization. In order to
exclude disturbing test compound absorption, medium or
conidia, blank measurements have to be carried out
before sampling. Yeasts are generally scored around
490 nm (Barchiesi et al., 1993; Del Poeta et al., 1994;
Pfaller et al., 1995) or 630 nm (Rodrı́guez-Tudela et al.,
1996).
In addition to turbidity, various other elegant methods
have been developed to quantify fungal growth colori-
metrically or spectrofluorometrically. Mitochondrial
dehydrogenases of metabolically active cells reduce
yellow 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
tetrazolium bromide (MTT) to a blue formazan which
can be measured photometrically (Mosmann, 1983;
Hansen et al., 1989). This method has been adapted to
quantify antifungal growth by Jahn et al. (1995), Clancy
and Nguyen (1997), and Adam et al. (1998). Alterna-
tively, Espinel-Ingroff et al. (1997) and Pelloux-Prayer et
al. (1998) used alamarBlue2 which consists of a
reduction–oxidation indicator yielding a colorimetric
change and fluorescent signal in response to metabolic
changes (Pagé et al., 1993). Fluorescent stains such as
Celluflor (Hector and Braun, 1986), Fungiqual (Coleman
et al., 1989), and fluorescein diacetate (Chand et al.,
1994) are also applicable.
We have compared two scoring methods in broth
microdilutions for falcarindiol and juglone, namely a
simple image analysis method (IMG) and staining with
fluorescein diacetate [FDA; Fig. 2(a) and (b)]. The
agreement of EC50, EC90 and MIC values between
scoring methods is acceptable [Table 1; Fig. 2(c)], taking
into consideration their different nature. However, the
trailing endpoint of activity of falcarindiol, and the sharp
endpoint of activity of juglone, are clearly evident in the
results gained from both methodologies. The most
important issue in microplate reading is the correct
design of the control and blank. The use of organic
solvents and emulsifiers, such as Tween 20 or Tween 80,
are unavoidable in order to incorporate test compounds
into the liquid media successfully, especially in the case
of the more lipophilic compounds (Rios et al., 1988;
Phytochem. Anal. 11: 137–147 (2000)
144 F. HADACEK AND H. GREGER
Figure 3. Example illustrating the inhibition effects of
falcarindiol and juglone against Botrytis cinerea in broth
microdilution scored by image analysis: (a) structures of the
test compounds, (b) inhibition (% of control growth) of tested
concentrations, endpoints and estimated EC50 and EC90
values.
Cole, 1994). Any inhibitory or stimulating effects of such
additives on fungal growth have to be considered during
scoring.
Either graphic presentation or regression analysis can
be used to describe effects caused by chemicals on fungal
growth: Fig. 3(b) illustrates both types of presentation.
Broth microdilution is a quantitative bioassay (Roberts
and Boyce, 1972), but concentration–response curves are
not always sigmoidal as can be seen for juglone:
falcarindiol shows a pronounced trailing endpoint, hence
the curve is non-sigmoidal. Although measurement
distributions between sample and control in the range
from 6.3 to 200 mg/mL are highly significant (p < 0.05),
the effect stagnates around 50% of control growth.
Falcarindiol and juglone exemplify the two common
activity responses one is liable to encounter in this type of
bioassay. Graphs are useful to compare two or three
compounds, but results for three or more substances are
generally clearer in tabular form. Regression analyses
such as probit-log are conventionally used for statistical
analysis of concentration–response experiments (Finney,
1971, 1978; Roberts and Boyce, 1972; Nyholm et al.,
1992; Forbes, 1993); they can be carried out using
various statistical packages containing the appropriate
module. EC50 values are presented in order to compare
compounds, whilst EC90 values additionally inform about
endpoints to be expected. Fiducial limits define the
precision of the estimated EC50 or EC90 values. These
parameters are more descriptive than an MIC because the
latter just denotes the lowest concentration causing 100%
of the inhibition effect and it does not reveal anything
about the nature of the concentration–response relation-
ship. Additionally, if the endpoint trails as in falcarindiol,
which is more the rule than exception, unequivocal MIC
Copyright # 2000 John Wiley & Sons, Ltd.
determination is difficult: broad fiducial limits character-
ise this response behaviour. Conversely, EC50 and EC90
with narrow fiducial limits typify highly active com-
pounds, such as juglone (Table 1; Fig. 3). EC50 and EC90
can thus inform in more detail about the nature of the
observed inhibition effect in activity comparisons if 95%
fiducial limits are also presented.
Radial growth rate
Contrary to previously described methods, agar dilution
methods in Petri dishes do not require conidiospore
suspensions. Instead, mycelial plugs or disks excised
from the edges of actively growing colonies are used
(Gadd, 1986; Cole, 1994). However, in case of fungi
readily producing masses of easily disengageable coni-
dia, e.g. Penicillium or Aspergillus, inoculation with a
spore suspension is recommended instead. The high
temperatures (50–60 °C) of the molten agar into which
test compounds are mixed may be disadvantageous if
assaying thermolabile compounds. In comparison to agar
diffusion bioassays, however, the concentration in the
medium is well defined and, consequently, estimating
EC50 and EC90 is possible. Scoring inhibition effects is
generally carried out by comparing colony diameters,
which are easy to measure on a mm scale. Figure 1
illustrates the effects caused by juglone.
Compared to growth in liquid media, growth on solid
media differs in one essential aspect: submerged hyphal
growth takes place under low oxygen concentrations,
whereas fungi growing on agar surfaces are less subject
to this constraint. Moreover, a fungus growing on an agar
surface does not come into contact with a possible toxic
compound to the same extent as when growing in
submerged culture. These circumstances are clearly
reflected in the results, where probit-log estimates differ
considerably from those obtained from broth dilution
assays by showing significantly higher values (Table 1).
Comparability of results
As recently as a decade ago, antifungal susceptibility
testing was used only occasionally (Rex et al., 1997). One
study found inter-laboratory variations in absolute MIC
values of up to 50,000-fold employing arbitrary test
methods (Galgiani et al., 1987), whilst another found that
variations up to 512-fold still remained when laboratories
used a single published, but non-standardized, methodol-
ogy (Calhoun et al., 1986). In response to these findings,
the NCCLS established in 1982 a subcommittee for
antifungal susceptibility testing which developed a
standardized broth dilution method to test antifungal
susceptibility of yeasts. The standard version was
published in 1997. As previously mentioned, the
unicellular character of yeasts has greatly facilitated the
standardization process, but this is not the case for
filamentous fungi. However, as a next step, broth
microdilution for filamentous fungi must be standardized
(Espinel-Ingroff et al., 1995, 1997; Pujol et al., 1996,
1997a, b; Espinel-Ingroff, 1998), at least for clinical
studies. The problem of standardization of antifungal
bioassays in order to improve inter-laboratory compari-
sons of results not only applies to clinical screenings but
also to natural product research, especially if one bears in
mind that antifungal susceptibilities together with other
biological activities are increasingly incorporated in
Phytochem. Anal. 11: 137–147 (2000)
 ANTIFUNGAL ACTIVITIES TESTS
Figure 4. MIC ratios of juglone to falcarindiol obtained by
radial growth inhibition (RGR), broth microdilution scored
with image analysis of germ-tubes (IMG), and staining with
¯uorescein diacetate (FDA), disk diffusion (DDF), and thin
layer chromatography (TBA) against B. cinerea, C. herbarum
and F. avenaceum.
natural product bibliographies and databases. Incompar-
ability of results greatly diminishes data quality and
renders compilation efforts useless. Thus, standardization
should not be confined to clinical research alone; it
should be extended to all other research fields employing
antifungal susceptibility testing including natural product
research, ecotoxicology and phytopathology.
The susceptibilities of B. cinerea, C. herbarum, and F.
avenaceum to falcarindiol and juglone presented here
confirm the above outlined problem (Table 1). Figure 4
displays the MIC ratio, which is the only parameter that
allows a raw comparison of diffusion and dilution
bioassays; comparing the activity ratio of falcarindiol to
juglone overcomes the problem of non-uniform MIC
definitions in the various bioassays. Still, even on that
level, Fig. 4 clearly illustrates an incomparability of
results and the need to introduce procedure standardiza-
tion, in analogy to the situation in clinical studies
(Calhoun et al., 1986; Galgiani et al., 1987). Ratios
obtained in disk diffusion and broth microdilution are
more or less in accordance with each other, correlation of
these two susceptibility methods having already been
observed (Trancassini et al., 1986). However, the
falcarindiol to juglone ratios in the simple radial growth
rates and thin-layer bioautography assays do not compare
at all. Comparing the susceptibility behaviour of the three
different microfungi in the various bioassays, however,
F. avenacum always shows the greatest ratio (Fig. 4). In
conclusion, these results suggest some congruence in the
relative susceptibility of fungal species to different
chemicals, but they lack comparability in the extent of
the inhibition effect that manifests itself in the MIC and
the estimated EC50 and EC90 values.
The development of standardized procedures for
bioassays against filamentous fungi, which is most
advanced in clinical research, should not be confined to
one research area; biological activities are of interdisci-
plinary interest, to biologists, ecologists, pharmacists,
Copyright # 2000 John Wiley & Sons, Ltd.
145
phytopathologists and ecotoxicologists. Consequently,
ideally, the standardization discussion should involve
scientists of all these disciplines to guarantee the broadest
possible inter-laboratory comparability of results, es-
pecially in consideration of the cost- and labour-inten-
sive processes to isolate natural products from plants or
micro-organisms, costs for drug synthesis and, finally,
costs to perform the tests themselves. The standardization
of the broth microdilution methodology is promising, not
only for clinical research (Espinel-Ingroff et al., 1995,
1997; Pujol et al., 1996, 1997a, b; Espinel-Ingroff, 1998),
but the methodology is also applicable in phytopathology
(e.g. Mischke, 1997; Wilson et al., 1997) and ecotoxi-
cology (Mayfield, 1993). Scoring methods are manifold
and comprise simple indexing, biochemical and physio-
logical methodologies. The alamarBlue colorimetric
method has probably the most potential to become
widely accepted in the near future. Scoring can be carried
out either visually or more exactly with a fluorometric
microplate reader (e.g. Espinel-Ingroff et al., 1997;
Pelloux-Prayer et al., 1998). Comparisons of conven-
tional and colorimetric evaluations have demonstrated
excellent agreement (Espinel-Ingroff et al. 1997), similar
to the results of germ-tube measurements and FDA
staining presented here. The use of the totally synthetic
medium RPMI 1640 may hold advantages over semi-
synthetic malt extract or potato-dextrose broth usually
employed for axenic culture of filamentous fungi
(Dhingra and Sinclair, 1995). A temperature of 35 °C is
generally used for testing human pathogens, including
filamentous fungi. Pujol et al. (1997b), however,
identified temperature as one of the less influential
parameters to affect filamentous fungal susceptibility.
To natural product researchers, microdilution in
microtitre plates is attractive. Generally, quantities of
test compounds represent the limiting factor in bioassays.
Natural products are mostly thermolabile; high tempera-
tures are unavoidable in the incorporation of compounds
into agar media but not into liquid media. The
methodology offers potential for high sample throughput
by automatization on the one hand, and allows direct
observation to detect morphological anomalies on the
other. Additionally, concentration–response effects can
be studied by regression analysis. EC50 and EC90
represent ideal parameters to compare activities in
contrast to MIC values. A standardized broth microdilu-
tion methodology will most certainly meet the demands
of all researchers involved in antifungal susceptibility
testing of filamentous fungi, the gained inter-laboratory
result comparability can contribute to decrease costs by
eliminating unnecessary parallel experiments. Radial
growth rate determination and thin-layer bioautography
have their merits, but also their limitations concerning
result comparability.
Acknowledgements
The authors are indebted to Professor Hartmut Laatsch (University of
Göttingen, Germany) for the donation of synthetic juglone, to Doris
Engelmeier for providing conidia suspensions, to Perkin Elmer
(Austria) and Bartelt GmbH for supplying fluorometric microplate
reader demonstration units, and to two anonymous reviewers for
improving the manuscript. Parts of the work were supported by the
‘Hochschuljubiläumsstiftung der Stadt Wien’ and the Austrian
National Committee for the Intergovernmental Program ‘Man and
Biosphere’.
Phytochem. Anal. 11: 137–147 (2000)
146 F. HADACEK AND H. GREGER
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