Chapter 17 Antifungal natural products: assays and applications

Rai and Carpinella (eds.) Naturally Occurring Bioactive Compounds r 2006 Elsevier B.V. All rights reserved.

423

CHAPTER 17

Antifungal natural products: assays and applications DORIS ENGELMEIER, FRANZ HADACEK

Introduction Natural products or plant secondary metabolites comprise low-molecular-weight compounds that are regarded as dispensable for sustaining life, but as indispensable for the survival of the producing organism (Hartmann, 1996; Hadacek, 2002). For ages, plants have provided mankind with medicines and food-conserving additives. Still, plant- or microbial-derived compounds are regarded as a substantial source for novel lead structures to develop medicines and biocides. Recently, especially huge expectations were directed to those from hitherto uncharacterized microorganisms that live as endophytes in plants or occur in deep-sea habitats (Clardy and Walsh, 2004). Furthermore, the application of combinatorial chemistry in generating structural diversity has somehow affected the significance of natural products in biological activity screening. However, especially concerning the identification of novel mode of actions, natural products are still regarded as a valuable pool for lead structures. Further, for the release of novel antimycotic drugs and fungicides to the market, costs have increased dramatically during the last decades due to additionally imposed conditions to elucidate mode of actions and side effects; an increasingly chemistry-critical public has also fuelled this process. As a result, cost estimates for the development of a new drug or biocide as well as the formulation of their application amount to around 150 millions US Dollar today. These facts may somehow impose a constraint on the development, all the more as some representatives from the industry regard the quality of existing biocides more or less as sufficient to combat the recognized pest organisms (Stetter and Lieb, 2000). Contrary to this view, other authors voice increasing concern regarding the rapid emergence of resistance phenomena in pathogens toward specific applied fungicides (Knight et al., 1997; Henningsen, 2003). In antimycotic therapy, resistance also constitutes a recognized problem (Baddly and Moser, 2004). The tremendous pathotype diversity of some pathogenic fungi may additionally complicate the development of efficient control mechanisms as, e.g., shown for the rice blast fungus Pyricularia ( ¼ teleomorph Magnaporthe) grisea (Kareiva, 1999). Further, a broad

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successful introduction of transgenic disease resistant crop cultivars is not expected to take place before 2015 (Henningsen, 2003). In a concomitant development of increased alertness toward the application of synthetic chemicals in agricultural practice, the implementation of integrated pest management programs has increased (Yang and del Rio, 2002). This approach generally favors fungicides that affect non-target mechanisms less than their synthetic analogues, and here natural products certainly constitute potential candidates. Consequently, there still exists interest in screening low-molecular-weight compounds for antifungal activities within various research approaches that reflect themselves in the choice of assays. So far, key areas for the application of antifungal bioassays include control of crop pathogens in phytopathology and human pathogenic fungi in antimycotic chemotherapy. Further, the authors opine that resource utilization and susceptibility to secondary metabolites will attract more attention in future to address basic ecological questions, such as the functioning and structuring of plant communities. In this context, the impact of microbial symbionts and pathogens will attract more attention as was hitherto been paid to. Antifungal modes of actions have been primarily elucidated for antimycotic drugs. The available antimycotic drugs show various modes of actions and, as a result of the dire consequences, resistance phenomena are much more attended to than in previous times, for a review see Baddly and Moser (2004). Polyenes, such as amphotericin B (1 in Figure 1), are in use since the 1950s and bind to ergosterol units of the fungal cell membrane (Figure 1). Changes in the sterol content decrease binding of the drug and contribute to resistance. Further, an alteration in cell wall 1,3-b-D-glucan restricts the ability of this drug to reach its target site.

OH OH OH

O

HO HOOC HO

OH OH O

O

OH O

O

HO

1

OH NH2 N O

O

OH N N

Cl

O Cl

N O

N

F

F

N N N

N O

N

2

3

Fig. 1. Antimycotics active at the cytoplasmic membrane. 1, amphotericin B; 2, the azole ketoconazole; 3, the triazole fluconazole.

Antifungal natural products: assays and applications

425

Azoles constitute less toxic and effective alternatives to the former antimycotic. These compounds do not react with but inhibit the biosynthesis of ergosterol. Ketoconazole (2 in Figure 1) contains two nitrogen atoms in the five-membered azole ring whereas fluconazole (3 in Figure 1) contains three. Resistance to the azoles may be caused by several mechanisms: (1) enhanced efflux by up-regulation of multidrug efflux reporter genes (ABC transporter genes); (2) amino acid substitutions in the 14-a-demethylase (catalyzing the demethylation of 24-methylendihydrolanosterin); (3) up-regulation of the 14-a-demethylase gene ERG11; or (4) alterations in the ergosterol biosynthetic pathway (Figure 2). Echinocandins are semi-synthetic lipopeptide antimycotics developed from naturally occurring polypeptides that were originally isolated from Aspergillus nidulans var. echinulatus (Nyfeler and Keller-Schierlein, 1974). They act on the fungal cell wall by inhibiting the synthesis of 1,3-b-D-glucan, which leads to osmotic instability and finally to lysis of the cell wall. Caspofungin is the only commercially available derivative (Figure 3). Resistance can result from mutations of FKS1 and FKS2, genes that encode subunits of the 1,3-b-D-glucan synthase. The mode of action of flucytosine (1 in Figure 4) is based on antimetabolite properties. After uptake by fungi, it is converted by intercellular deamination into 5fluorouracil (2 in Figure 4). The converted 5-fluorouridine triphosphate (3 in Figure 4) incorporates into fungal RNA and inhibits protein synthesis. 5-Fluorouracil is also converted into fluorodeoyxuridine monophosphate (4 in Figure 4), which interferes with DNA synthesis by inhibiting thymidylate synthetase. Various mechanisms of resistance exist: (1) mutation of the enzymes resulting in decreased uptake or conversion of the drug; (2) loss of activity of uracil phosphoribosyltransferase; and (3) increased synthesis of pyrimidines that compete with the fluorinated antimicrobials (Figure 3). However, not the whole world population benefits from antimycotic drugs. Especially in developing countries, people rely and still have to rely on traditional medicines from plant sources. This fact, the emerging resistance phenomena, the increased occurrence of fungal strains with multiple antibiotic resistance, and new emerging fungal diseases still fuel interest in screening studies for antimycotic natural products (Ficker et al., 2004). Consequently, plants from countries, such as India (Vonshak et al., 2003), Latin America (Freixa et al., 1998), and Africa (Cos et al., 2002), or even Canada (Jones et al., 2000), are still under investigation, in most cases guided by an ethnopharmacological background.

Fungicides in agriculture A compendium of pesticide common names can be found under the following hyperlink: http://www.hclrss.demon.co.uk/class_fungicides.html. Anderson et al. (2004) have listed various emerging fungal diseases in agricultural ecosystems: Phytophthora infestans, potato blight in south America; Pyricularia grisea, rice blast in all rice-producing areas including USA; Tilletia indica, carnal bunt on gramineous crop plants originally in India, in the last decade in South Africa and USA; and Puccinia kuehnli, sugar cane orange rust which reduced sugar cane production in Australia by 25%. Compared to our knowledge of crop plant emerging infectious

Naturally occurring bioactive compounds

426

X

A HO

HO

HO 3

2

1

X

B

X

C

HO

HO

HO

6

B

5

4

X

HO

HO

HO 9

8

7

A

B

C

N N

Cl

N Cl

O O O N F

HO

H N O

Cl epiconazole

spiroxamines

fenhexamide

Fig. 2. Steroid biosynthesis and exemplary structures of inhibitors (A, B, and C); inhibited reactions are marked with X. 1, lanosterol; 2, 2,4-methylendihydrolanosterin; 3, 4,4-dimethylergosta-8,14,24-triene-3b-ol; 4, 4,4-dimethylzymosterol; 5, 4-methylzymosterol; 6, zymosterol; 7, fecosterol; 8, episteriol; 9, ergosterol.

diseases, we know definitely less about those of wild plants. Examples of studied cases include Ophiostoma ulmi, the Dutch elm disease, Cryphonectria parasitica, the chestnut blight in America, Phytophthora cinnamoni, an emerging root rot disease in Proteaceae, Fabaceae, Mimosaceae, and Epacridaceae in Australia, Pestalotiopsis microspora, Floradia torreya mycosis, Discula destructiva, dogwood anthracnose on

Antifungal natural products: assays and applications

427

H 2N NH

OH

O

O

HO

NH

NH O

N

H 2N

HN

O HO

O

NH H N

O

OH

N OH

O

HO

OH

OH

Fig. 3. Caspofungin, an echinocandin polypeptide antimycotic acting on fungal cell walls.

H N

O N

NH2

O

H N

HN

F

F O

1

2

H N

O

O HO P HO O

O

N

F O

OH

HO

3

H N

O

O HO

O HO P O P O P O O HO OH

O

N

F O

OH

HO

4

Fig. 4. Mode of action of flucytosine (1): uptake and desamination to 5-fluorouracil (2), conversion to fluorouridine triphosphate (3) that inhibits protein synthesis and to fluorodeoxyuridine monophosphate (4) that inhibits DNA synthesis.

Cornus florida in USA, and Phytophthora ramorum, sudden oak death syndrome in England and Poland. Fungicides do not exclusively target true fungi. Although Phytophthora is classified as a fungus in many instances, it is not treated as a true fungus by modern classifications systems. True fungi are characterized by a number of specific traits, many of which they have in common with insects, with whom they are classified as

428

Naturally occurring bioactive compounds

ophistokonts, whereas oomycetes belong to the heterokonts including diatoms and brown algae and share more characters with plants (Table 1). However, both true fungi and oomycetes produce highly similar disease symptoms. Their pathogenicity is often caused or tremendously enhanced by the production of huge numbers of asexual conidia, another reason for their often-to-be observed joint treatment. As many classic fungicides target the biosynthesis of ergosterol, they fail to affect oomycete fungal pathogens because their cell walls are made of cellulose, such as those of higher plants (Deacon, 1997). One of the first pathogenic organisms that caused substantial effects on human crops was potato blight, Phytophthora infestans. Potatoes were introduced to Europe during the 16th century. About 300 years later, in 1845, the first severe outbreak of potato blight in Ireland caused severe famine in the following years and induced many people to leave the country forever. This incident is often used as marketing argument for the application of fungicides by the agrochemical industry. Large (1940) published a most recommendable survey on the development of phytopathology as a scientific discipline and recapitulates how we became aware of many fungal pathogens and devised control mechanisms, the development of which was not always as straightforward as it seems today. According to up-to-date estimates, about 10–20% of today’s production of staple foods and cash crops are destroyed by plant pathogens, always depending on the crop and the region. Despite the existence of disease-tolerant cultivars, crop rotation, and sanitation practices, the use of fungicides is still regarded as indispensable to maximize yields (Knight et al., 1997; Hewitt, 2000; Henningsen, 2003). In attempts to lower biocide input, attention has shifted from discovering novel fungicides with known mode of actions to the identification of novel modes of fungicidal activities. In this context, we have also to consider the meaning of the term ‘‘fungicide.’’ The designation of fungicide to a specific compound defines the ability of this compound to kill fungal hyphae or its propagules. Usually, most so-called antifungal compounds are fungistatic, i.e., they inhibit or delay conidia and spore germination or hyphal growth to a certain extent. Even if some compounds can be identified as fungicides in in vitro and in vivo glasshouse experiments, the thus determined effects may be totally different when the candidate fungicide is actually applied in the field. The more complex the environment of the application becomes, the more difficult it is to predict the actual nature and strength of a biocide’s efficiency. And nature is very complex. In order to become a pathogen, conidia or spores have to germinate on leaf or root surfaces. Especially on leaves, in most cases the development of specific infection structures, so-called appressoria, is required that allow the fungus to establish itself in the leaf’s tissues (Dean, 1997). Once this is accomplished, it starts attempting to penetrate cell walls. Plant cells contain specific receptors to recognize cell wall fragments caused by fungal invasions, glucans from oomycetes, chitosans from true fungi, polygalacturonids from their own cell walls, or extracellular fungal proteins. Efficient recognition of these so-called ellicitors triggers a signal cascade initiating the hypersensitive reaction process (HR) that includes the expression of pathogenesis-related proteins (PR-proteins) and genes for phytoalexin biosynthesis, and ultimately may lead to local cell death in the affected tissue (Knogge, 1996; Baker et al., 1997; Morel and Dangl, 1997; Dixon, 2001; Lam et al., 2001).

True fungi

Animals

Oomycota

Plants

Growth habit

Hyphal, tip growth

Not hyphal

Hyphal, tip growth

Not hyphal

Nutrition

Heterotrophic, absorptive

Heterotrophic, ingestive

Heterotrophic, absorptive

Autotrophic

Cell wall

Chitin

Chitin in exoskeleton cellulose

Cellulose

Cellulose

Nuclei

Haploid, membrane persists during division; spindle pole bodies do not have a centriolar arrangement

Typically diploid; typical centrioles

Diploid; typical centrioles

Typically diploid; typical centrioles

Microtubules

Sensitive to benzimidazoles and griseofulvin

Sensitive to colchicine

Sensitive to colchicine

Sensitive to colchicine

Golgi cisternae

Unstacked, tubular

Stacked

Stacked

Stacked

Mitochondria

Plate- or disc-like cisternae

Plate- or disc-like cisternae

Tubular

Tubular

Lysine

Synthesized by aaminoadipic acid pathway

Not synthesized

Synthesized by diaminopimelic acid pathway

Synthesized by diaminopimelic acid pathway

Translocable carbohydrates

Polyols, trehalose

Trehalose in insects

Glucose, sucrose, etc.

Glucose, sucrose, etc.

Storage compounds

Glycogen, lipids, trehalose

Glycogen, lipids, trehalose in some

Mycolaminarin

Starch, lipids, sucrose

Mitochondrial UGA codon usage

Tryptophan

Tryptophan

Chain termination

Chain termination

Sterols

Ergosterol

Cholesterol

Sitosterol

Sitosterol

Taxonomy

Ophistokonts

Ophistokonts

Heterokonts

Plants

429

Character

Antifungal natural products: assays and applications

Table 1 Some major characteristics of the chitin-walled (true) fungi compared to animals, oomycetes, and plants, after Deacon (1997), modified

Naturally occurring bioactive compounds

430

Pathogenic fungi may show manifold mechanisms of disease development (Agrios, 1997), and thus the successful use of a fungicide usually also requires the dissemination of its correct application procedure. The development of germ tubes from conidia and spores represents on the most sensitive developmental stages in the life of a pathogenic fungus and thus offers itself as target for a preventive fungicide. Once the fungus has entered the plant’s tissues, a curative systemic fungicide is required. However, the increased insights into plant resistance mechanisms, the hypersensitive response (see previous paragraph), has stimulated the exploitation of respective signal molecules, such as salicylic and jasmonic acid and their mimics, as inductors of plant resistance in disease control (Feys and Parker, 2000; Hewitt, 2000; Conrath et al., 2001; Terry and Joyce, 2004). Consequently, recent formulations of fungicides comprise various compounds with preventive, curative, or resistance-eliciting and potentiating effects (e.g., Labourdette and Latorse, 2003). The quest to discover novel modes of actions has significantly spurred and is still the major driving force in the search for natural, semi-natural, and synthetic compounds (Lyr, 1995; Hewitt, 2000; Henningsen, 2003). One of the classical modes of actions of fungicides that affect true fungi is the inhibition of the steroid biosynthesis leading to ergosterol, an important building block of the fungal cell wall, such as shown by diazoles and triazoles, spiroxamines and morpholins (Figure 2). As a matter of fact, numerous cases of resistance are known in the literature. Strobilurins and oudemansins (Figure 5) have been discovered in culture filtrates of basidiomycetes to colonize dead wood and produce these antifungal compounds to gain advantages toward other fungi competing for the same nutrient source. These

Lead structures OCH3

OCH3

O

OCH3

O O

O

1

2

Stabilization with aromatic rings N

N

O N

O

OCH3

O

CN

O

O 3

OCH3

O

4

Fig. 5. Strobilurin and oudemansin-based fungicides inhibit enzymes of the respiratory chain. 1, Strobilurin A; 2, oudemansin A; 3, kresoxym-methyl; 4, azoxystrobin.

Antifungal natural products: assays and applications

431

compounds inhibit mitochondria by blocking an ubichinone receptor in the respiratory chain, an hitherto unknown mechanism of inhibition (Kraiczy et al., 1996). As a consequence, these compounds inhibit both oomycetes and true chitin fungi and thus offered themselves as promising lead structures for broadband fungicides; today, azoxystrobin is commercialized by Zeneca and kresoxim-methyl by BASF, both being less photolabile than the original natural products (Figure 5; Sauter et al., 1999; Henningsen, 2003). However, resistant strains surfaced within Erysiphe graminis (Chin et al., 2001; Hollomon, 2001) and Podosphaera fusca as well as Pseudoperonospora cubensis (Ishii et al., 2001). Monsanto has introduced a fungicide, with silthiopham as active compound (Figure 6), against the take-all fungus, Gaeumannomyces graminis, which specifically inhibits ATP transporter molecules. In case of the rice blast fungus, Pyricularia grisea, various compounds were found that inhibit the melanin biosynthesis that is required for the development of efficient appressoria facilitating the penetration of the leaf epidermis by the hyphal tip (Figure 7). Flumorph, dimethomorph, iprovalicarb, and benthiavalicarb also inhibit cell wall biosynthesis of various oomycetes and true fungi; however their mode of action has not been elucidated yet (Figure 8). Though the introduction of these fungicides occurred only recently, tolerance was already noted; Phytophthora capsici to flumorph (Huang et al., 2004), Phytophthora infestans to dimethomorph (Stein and Kirk, 2003), and Plasmopara viticola as well as Phytophthora infestans to iprovalicarb (Suty and Stenzel, 1999; Kast, 2004). Other fungicides with a rather broad activity against true fungi and oomycetes include various inhibitors of mitosis, such as zoxamides (Figure 9); quinoxifens affects the G proteins of the fungal hyphae, which are essential for successful signal transduction (Figure 9). After treatment, the hyphae of Plasmopara viticola failed to penetrate the leaf epidermis of vine. However, recently resistant strains have surfaced (Anonymous, 2004). Despite of the preponderance of semi-synthetic and synthetic compounds in the control of postharvest diseases, natural products are still viewed as potential alternatives. The application of volatile compounds, such as monoterpenes and glucosinolates, as fumes may gain importance in the future. Besides, the application of resistance-inducing elicitors, such as plant and fungal cell wall components, and signal compounds, such as salicylic and jasmonic acid, also shows potential. As a consequence, consumers will be less subjected to the cancerogenic and teratogenic properties besides the high and acute residual toxicity known for most non-volatile synthetic fungicides (Tripathi and Dubey, 2004).

S

Si H N O

silthiopham

Fig. 6. Silthiopham, an inhibitor of ATP transporter molecules.

Naturally occurring bioactive compounds

432

Co

a

leaf epidermis

App Hy

OH OH acetate

pentaketide HO

OH 1

b OH OH

OH O

A, B, C X

HO

HO

OH

3

O

2

OH

O

OH

A, B, C melanin

X HO 5

4

A

B

C

O N H Cl

O Cl

N H

Cl Cl

capropamid

Cl

CN

diclocymet

O N H

Cl

CN

fenoxanil

Fig. 7. (a) Appressorium exemplified by the rice blast fungus Pyricularia grisea; App, appressorium; Co, conidium; Hy, hyphae. (b) Melanin biosynthesis in the appressorium and structures of inhibitors; inhibited catalytic reactions are marked with X; 1, 1,3,6,8,-tetrahydronaphthalene; 2, scylatone; 3, 1,3,8-trihydronaphthalene; 4,vermelone; 5, 1,8-dihydronaphthalene.

Antifungal molecules in biotic interactions The majority of authors consent that fungal communities are defined by bottom-up factors, i.e., by competition rather than by predators (top-down control, Wardle, 2002). This notion agrees with the presence of numerous antibiotic natural products that are produced by fungi. The strobilurins, one of the most successful discoveries of

Antifungal natural products: assays and applications

433

F

Cl

O

O N

N O

H3CO

O

H3CO

OCH3

OCH3 2

1

O

O O

N H

N

O

N H S

O

O

F 4

3

Fig. 8. Inhibitors of fungal cell wall biosynthesis with yet unknown mode of action. 1, flumorph; 2, dimethomorph; 3, iprovalicarb; 4, benthiavalicarb. F

O Cl

Cl

Cl

O

O Cl

Cl 1

N 2

Fig. 9. Zoxamide (1) inhibits mitosis in true fungi and oomycetes; Quinoxyfen (2) affects G proteins in fungal hyphae and thus inhibits signal transduction.

antifungal natural products within the last decade, originate from a wood-decaying basidiomycete (Sauter et al., 1999). However, the production of antibiotic natural products is restricted to the true fungi, the oomycetes being less talented producers. Especially those fungi that are classified as necrotrophic pathogens rely on the phytotoxicity of their secondary metabolites (Prell and Day, 2001). This ability to produce secondary metabolites is usually lost when isolates are cultured on axenic media for longer periods. From efforts to optimize culture conditions in biofermenters we know today that fungi require nutrient shortage to produce these toxic secondary metabolites (Demain, 1996). Such situations are also caused by a competitive environment and thus these traits concur with the hypothesized bottom-up definition of their communities. Many mycotoxin-producing fungi are thought to originate from lichen-associated fungi (Lutzoni et al., 2001). In this form of symbiosis, the ability to produce toxic

434

Naturally occurring bioactive compounds

secondary metabolites in a competitive environment may have contributed to defense against herbivore predators. Today, several fungi that colonize crop plants may often severely affect human health by production of mycotoxins, e.g., the trichothecenes from Fusarium on wheat. Mycotoxins constitute an additional reason why fungal control on crop plants is mandatory (Cardwell et al., 2001). A fungus may not only be confronted by secondary metabolites from competitors. As a plant saprophyte and pathogen it has to tolerate the constitutive defenses of a plant, i.e., diverse secondary metabolites stored in adapted tissue compartments, such as resin ducts and idioblasts. Further, during the colonization process, induced secondary metabolites, also called phytoalexins in many instances, may suppress the infection process (see Hadacek, 2002). Ecologists have attempted to develop models that try to explain species diversity and agreement exists that microbial biodiversity is closely linked to plant biodiversity. A mathematical exploration of a feedback model between plants, their competitor plants, and the microbial communities of both concludes that negative feedbacks promote the coexistence of species (Bever, 2003). In this context, the evaluation of antifungal properties of natural products may constitute an important tool to test the proposed hypotheses in exploring extant interactions of plant and fungi in a community scenario. As a consequence, bioassays determining susceptibility of fungal isolates to specific secondary metabolites may become a key technology in this field, all the more as belowground plant defense may affect spatiotemporal processes in plant communities (van der Putten, 2003). As disease outbreaks also occur in natural plant communities (Burdon, 1993), a parallel exploration of agricultural and natural ecosystems has to be carried out to develop new and optimize existing sustainable control mechanisms. In recent years, reports emerged suggesting that complex symbiotic communities, such as the fungal species composition of fungus gardens of leaf cutting ants, may be determined by antifungal compounds produced by ant-associated bacteria belonging to genera Streptomyces (Currie et al., 1999) and Burkholderia (Santos et al., 2004). These reports somehow illustrate the complexity of the issue. These examples also suggest that antifungal susceptibility testing constitutes an essential methodology in dissecting scenarios of biotic interactions. Properly set-up bioassays combined with adequate statistics will probably help to obtain better insights into complex biological phenomena. For secondary metabolites, the more or less anthropocentrically coined function of pure chemical defense metabolites will have to be revised, and, most certainly, in this process, antifungal susceptibility assays will constitute an important tool.

Assay procedures Going through the numerous publications dealing with the assessment of susceptibility effects of specific fungi to specific antifungal natural products or synthetic compounds, the interested reader becomes confronted with numerous methodologies, always depending on the specific aim of the published investigation and, of course, the type of fungus the focus is set on. Consequently, in many instances, the choice of assay constitutes the first arising difficulty. One of the most inherent

Antifungal natural products: assays and applications

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problems is that we have to be aware that the single methodologies do not really produce comparable results (Hadacek and Greger, 2000). Further, when assayed against the identical chemical compound, results may vary using various fungal strains of the same species and the identical isolate cultivated under different regimes in the same assay. Researchers involved in antimycotic chemotherapy are especially aware of this problem of interlaboratory result comparability (e.g., Calhoun et al., 1986; Rex et al., 1993; Cormican and Pfaller, 1996). This concern has led to the development of guidelines as how to perform antifungal assays for clinical susceptibility assays with yeasts and filamentous fungi (Pfaller et al., 1997, 2002a, 2002b; Sheehan et al., 2004), and for recommendations to use specific strains from culture collections. To all interested in performing clinical-related assays we recommend to consult these guidelines. Besides, there exist various publications that aim to aid with this issue (Rios et al., 1988; Paxton, 1991; Chand et al., 1994; Cole, 1994; Kerwin and Semon, 1999; Hadacek and Greger, 2000).

Culture of fungi in the laboratory Fungi can colonize a broad range of substrates of living and dead tissues of organisms. Their propagules can be found in the air and on surfaces of nearly everything. The variety of substrate sources for fungi is too diverse to be reviewed. In the ongoing text we will predominantly focus on filamentous fungi and, in selected cases, also on yeasts from filamentous fungi. As our research concentrates on fungi associated with plants we will only superficially treat fungi that are associated with human diseases. Fungi that attack humans have specific requirements that have to be taken into consideration. Isolates may be recovered from nails, hairs, sputum, pus, cerebrospinal fluids, blood, urine, etc. For those interested in introductory information about culture techniques media, microscopy techniques, and stains as well as specimen collection and processing, we recommend to visit the Mycology Online homepage (http://www.mycology.adelaide.edu.au/). However, many principles of what we state is also valid for those fungi. Further, the experimental procedures are much more standardized in this area of antifungal activity research and we recommend consulting the published NCCLS guidelines (Pfaller et al., 1997, 2002a, 2002b; Sheehan et al., 2004). One fundamental requirement in obtaining and maintaining a monoxenic culture of a fungus is to exclude bacteria. For this purpose, a broad range of antibiotics is available that may be included into the culture medium of the Petri dishes. As the media have to be autoclaved, the thermal stability of the antibiotic has to be considered. In case of thermally labile additives, a sterile filtered solution of the respective compound may also be added after autoclaving. Other annoying contaminants are mites that may be introduced with samples or with impure cultures obtained from elsewhere (Figure 10). In this case, all strains have to be destroyed and further culture is only possible on agar media with insecticides incorporated; Bills (1996) recommends dieldrin that has no side effects on fungal growth and, furthermore, does not decompose during autoclaving. We have also observed that tobacco taken from cigarettes serves as an efficient repellent to mites when deposited at the place of storage.

Naturally occurring bioactive compounds

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Fig. 10. Mite in a fungal Petri dish culture (140
).

Virtually, there exists no limitation in modifying the media composition. The largest biodiversity of fungi occurs in soil, and the majority of fungi is considered as unculturable (Bridge and Spooner, 2001). In attempts to find the appropriate recipe, we have to consider that structures of fungal communities are usually regulated by bottom-up effects, i.e., by the quality of resource. As a conclusion, to successfully establish a culture of a particular fungal strain, a specific set of available nutrients that more or less resembles those present in its natural environment has to be offered. These circumstances reflect themselves in numerous recipes for selective media for phytopathological and endophytic fungi from plants (e.g., Singleton et al., 1992; Dhingra and Sinclair, 1995; Bills, 1996). As natural carbon sources usually contain mixtures of carbohydrates, they have to be also offered in respective artificial media. Figure 11 illustrates the extent how plant root carbohydrate mixtures may stimulate the growth of rhizosphere microfungi compared to offered single sugars. Obtaining conidia or spores With the exception of biotrophic plant pathogenic fungi, either conidia or spores may be obtained from quite a large number of fungi grown in axenic cultures, in Petri dishes, within manageable efforts. In some instances, however, certain isolates may be reluctant to conidiate or sporulate. The literature is full with various recommendations to overcome this problem. However, we opine that modification of the culture medium yields the best results anyhow. Conidia are directly produced on conidiophores, and it is comparatively simple to collect them by overlaying the Petri dish culture by 0.9% aqueous NaCl with an addition of 5% DMSO (to reduce

Antifungal natural products: assays and applications Penicillium citrinum

Doratomyces stemonitis

Relative growth to control (%)

437

100

Cylindrocarpum destructans

Glucose

90 80 70 60 50 40 30 20 10 0 5000

2500

1250

625

313

156

78

39

20

10

Relative growth to control (%)

Conc (µg/mL)

Sucrose

100 90 80 70 60 50 40 30 20 10 0 5000

2500

1250

625

313

156

78

39

20

10

39

20

10

Relative growth to control (%)

Conc (µg/mL)

Host plant carbohydrates

100 90 80 70 60 50 40 30 20 10 0 5000

2500

1250

625

313

156

78

Conc (µg/mL)

Fig. 11. Utilization of carbohydrates by rhizosphere soil fungi of a calcareous grassland in central Europe. Doratomyces stemonitis and Penicillium citrinum occur in the rhizosphere of the umbellifer Peucedanum alsaticum and Cylindrocarpum destructans in the rhizosphere of Peucedanum cervaria. Glucose and sucrose were obtained commercially whereas the watersoluble fraction of the methanolic root extract represented the host plant carbohydrates. GC–MS analysis revealed that besides glucose and sucrose, fructose and mannitol were accumulated as major carbohydrates apart from various trace compounds (Hadacek and Kraus, 2002). Growth rates considerably vary among isolates, and thus the relative growth was determined as percentage of the maximum growth that a fungus could achieve within the assayed period in an offered substrate in the dilution series. The assay was performed in microwell plates. The duration varied until growth was sufficient for scoring.

Naturally occurring bioactive compounds

438 Conidia attached to hyphae x 400

Filter using a sieve large enough not to retain the conida or spores

Add liquid and loose attached conidia by scraping with a Drigalski spatula, and ...

Determine CFU number and store until further use at low temperatures

Concentrate suspension by centrifugation at 3000 g

Fig. 12. Preparation of a stock suspension for the conidia or spore inoculum. Petri dish cultures containing spore- or conidia-producing mycelia are overlaid with 0.9% aqueous NaCl and scraped off with a Drigalski spatula. The suspension is filtered and centrifuged for concentration.

surface tension of the liquid) and scraping off the conidia with a sterilized Drigalski spatula (Figure 12). For storage, the suspension has to be centrifuged to concentrate the liquid. Small cryovials are ideal for storage at –201C. The quality of the propagule suspension has to be assessed and the ideal storage temperature has to be determined for each isolate. The described conditions may suffice in most instances, but in case of failure the proper set of modifications has to be found. A number of issues have to be considered: for successful storage, the suspension medium has to penetrate the propagules, and this takes time. Thus, immediate transfer to the low temperature may compromise the conservation process. It is advisable to store the vials at 41C and then to transfer them to lower temperatures. However, if the described set of condition fails altogether, you may change the regime and use an aqueous solution of sucrose (140 g/L) and peptone (10 g/L) as medium instead (Hadacek and Kraus, 2002). In some cases, storage may only be possible at temperatures above zero. The viability of the spore or conidia suspension can be determined by the method as illustrated in Figure 13. As a matter of fact, to obtain reliable CFU (colony forming units) numbers, a series of replicates has to be performed. Compared to just counting the number of spores or conidia, determining the CFU number is better suited for adjusting the right inoculum’s size, which is around 105 CFU/mL for the majority of applications in assaying fungi. In

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8

1

Agar plate 180 µL aqueous NaCl (12 g/L)

7

2

6

3

....

100 µL spore suspension

5 ....

4

CFU = 50 n X 10 x After 2 days:

20 µL

20 µL

20 µL

....

8

20 µL

1

n = germinated conidia x = order of dilution

Agar plate

x= 1

2

3

4

....

8

7

2

6

3

Microwell plate

5

4

7

1.5 x 10 CFU/mL

Fig. 13. Determination of colony forming units (CFU) of conidia and spore suspensions, 10fold dilutions of the suspension are performed as illustrated. Twenty microliters of each dilution are plated on the agar medium and the number of germinated propagules is assessed. The number of CFUs is calculated using the given equation.

certain instances, automation by using calibrated optical density measurements in microwell plates is also applicable (Espinel-Ingroff et al., 1991). Gehrt et al. (1995) demonstrated the species-dependent propensity of some filamentous fungi to show lower susceptibility above CFUs of 105. Similar effects were also reported for yeasts (Rex et al., 1993). The authors interpret these phenomena with the fact that an increasing amount of microbial targets may exceed the number of available molecules and, as a consequence, advance the development of comparatively more resistant genotypes among the inoculum population. However, in some situations, CFU numbers lower than 105 will have to be utilized due to various difficulties obtaining enough propagules, which are either culture-related or fungal species-related, or caused by low tendency of conidia to free themselves from the conidiophores. Conidia may be obtained from both oomycetes and true fungi, and are especially characteristic for the majority of pathogenic fungi because they facilitate a quick and efficient asexual propagation of the pathogenic genotypes. Especially in the case of phytopathogenic fungi, direct isolates from the infected plant organs are advantageous in every respect: they represent the proper pathotype and usually copiously produce conidia, definitely much more readily than the majority of isolates available from culture collections. The latter, in many cases, at least in our experience, seems

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to have been re-inoculated too often on axenic media, and thus usually fail to compare to freshly isolated strains. Concerning biotrophic fungi, such as arbuscular mycorrhizal fungi and powdery mildews, spores and conidia may be obtained by recovering the propagules from soil (mycorrhiza, Daniels and Skipper, 1982) or culturing the fungus on sterilized plant tissue, respectively (e.g., powdery mildews). In the latter case difficulties may arise to obtain suspensions that are completely free of bacterial contaminants. If the focus is on the discovery of compounds that are to be applied in antifungal chemotherapy, culture collections such as ATCC (American Type Culture Collection; http://www.lgcpromochem.com/atcc/), BCCM (Belgium Coordinated Collections of Microorganisms; http://www.belspo.be/bccm/), CBS (Centraalbureau voor Schimmelcultures; http://www.cbs.knaw.nl/), or JCM (Japan Collection of Microorganisms; http://www.jcm.riken.go.jp/) constitute recommendable sources. Conversely, they may be also used to deposit strains for documentary purposes. What to do when there are no conidia or spores available Spores and conidia are characterized by the inherent advantage that they present a defined starting point for the development of fungal growth. Hyphae are characterized by apical tip growth (Deacon, 1997), which means that they are not growing uniformly. The antifungal assays focus on the inhibition of the germ tube that is usually most sensitive to the presence of chemicals (Guarro et al., 1997). Germ tubes are easily obtained from conidia and spores, but what to do if neither is available. This will be the case with those isolates that fail to produce spores or conidia or yeasts that propagate by budding (Figure 14). Reliable germination of the inoculum represents a fundamental requirement to obtain reproducible results. This presents no problem with conidia or spores, but affects all assays that have to be performed with developed mycelia or yeasts. The budding yeasts resemble bacteria, and growth curves have to be assessed to determine the optimal time for inoculation, which usually occurs during the acceleration phase and first stage of exponential growth phase (Figure 15). Conventionally, if filamentous fungi fail to produce conidia, agar plugs taken from the actively growing border of the colony may be used for inoculation. Here, control of the quality of the inoculum usually depends more on the care and expertise of the experimenter than on objectively assessable parameters. Recovery of strains and preservation Figure 16 illustrates a simple isolation procedure for the recovery of culturable fungi colonizing leaf tissues including surface sterilization, incubation of the source tissue in a moist chamber, and transfer of conidia formed on the colonized tissue to a Petri dish medium. Underground organs, such as stolons and roots also contain endophytic fungi, symptomless colonizers of plant tissues. Surface sterilization methods have to be adjusted to the type of plant tissue; structured surfaces have to be treated differently than smooth leaf cuticles; instead of the commonly used hydrogen peroxide, 70% ethanol and subsequent flaming proves more efficient. Sieber (2002) represents an excellent source for suggestions on how to proceed. If roots represent

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Fig. 14. Budding yeast (Taphrina sp.) (H.J. Prillinger, with permission).

Inoculation Stationary phase

Declining phase

Log cell number

Deceleration phase

Exponential phase

Acceleration phase

Lag phase 0

1

2

3

4

5

6 7 Time

8

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Fig. 15. Growth phases of yeast.

11

12

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(a)

(b)

2 min H20

2 min H202

2 min H20

Surface sterilization

Infected plant organs

(c)

Development in moist chamber

(d)

Transfer to agar plate

Fig. 16. Recovery of a filamentous fungus (Drechslera sp.) from a plant leaf. (a) Dark spots or lesions often indicate fungal infections on plant tissues, aerial parts, or roots; (b) to isolate the pathogen or endophyte of interest the plant tissue has to surface sterilized, e.g., with hydrogen peroxide; (c) incubation of the plant tissue in moist chambers (Petri dishes with moistened filter paper); (d) Petri dish culture with conidia.

the tissue under investigation, another fact has to be considered: plant roots usually accumulate distinctly larger amounts of secondary metabolites than aerial tissues. In attempts to isolate a fungus, the tissue has to be damaged and this action also affects the compartmented secondary metabolites. Many plant roots may contain a sticky resin or latex that quickly polymerizes into a callous layer representing an impenetrable barrier for the endophyte. In this case, covering the plant tissue with agar medium is advantageous because (1) the secondary metabolites diffuse into the agar and dilute themselves and (2) the formation of a callous crust on the surface of the plant tissue is avoided. The largest biodiversity of fungi occurs in soil. Gams and Domsch (1967) have devised a combination of sieves that is used to wash soil samples. The advantage is that fungi occurring as dormant spores or conidia can be differentiated from those that have developed a mycelium. The latter are retained by the sieves whereas the

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former can be found in the water used for the washing procedure. As interest of ecologists is increasingly focused on rhizosphere biology, this classical regime to assess the biodiversity may re-attract attention as complementary direct accession methodology to the currently favored indirect molecular methods, such as DGGE and T-RFLP (Anderson and Cairney, 2004). If special care is taken with the choice of the sieve pore sizes, the direct approach also yields rewarding results. We found a combination of sieves with a pore size of 0.8, 0.4, 0.2, and 0.05 mm as efficient. Particles retained by the 0.2 and 0.05 mm sieve were collected and transferred to agar media (Hadacek and Kraus, 2002). More likely, small particles contain only mycelial fragments of a single fungal strain. The obtained suspensions can be diluted accordingly and, upon transfer on agar media, develop into cultures containing a single strain. If the washed particles contain more than one fungus, the more vigorous strain may suppress the other, which, as a consequence, may not be recovered. The problem of antagonism reducing the diversity of strain recovery is well known and some researches even attack vigorous isolates, which emerged in the Petri dishes, with a soldering iron (Dreyfuss, 1986). Once a fungal strain is isolated, it has to be preserved with its given characteristics. Culture collections usually cryopreserve their strains at –801C, or even better at –1301C (American Type Culture Collection, 1991; Espinel-Ingroff et al., 2004); for a review of the actual standard methodologies see Smith and Onions (1994). The primary objective of culture collections is to ensure that the specific characteristics of a given strain remain stable throughout storage. However, Ryan et al. (2001) showed that secondary metabolite profiles, extracellular enzyme production, and DNA polymorphisms may be affected by non-optimal cooling and thawing regimes. If regeneration rates decline, improvements of the regime are mandatory. Insights from preserving fungal cultures do also apply to the preservation of inoculum suspensions and suggestions for its improvement of the viability may also be gleaned from published efforts to improve strain preservation. Still, the regime has to be adapted to the fungus of interest. This can be quite laborious sometimes. However, there exist alternatives to the cryopreservation, such as the filter paper technique (Dhingra and Sinclair, 1995; Fong et al., 2000). Sterile filter paper disks of 5–10 mm diameter – paper disks as used for antibiotic disk diffusion assays offer themselves – are placed into actively growing fungal cultures. After a couple of days, when they are completely covered by fungal mycelia, the disks are removed, dried, and preserved at –201C. In our laboratory, thus preserved strains could still be regenerated after 6 years. Figure 17 illustrates this technique. Preparation of microscopic slides The shape of spores, sporangia, conidia, and condiophores represent relevant characters for the proper identification of filamentous fungi. Figure 18 illustrates the ways to obtain permanent slides adapted from Kreisel and Schauer (1987). As mounting medium, LPCB (lactophenol cotton blue) or methylene blue in lactic acid meet most of the requirements. Potassium hydroxide (KOH) can be used to improve the visibility of fungi in pigmented structures. If the above-described procedures do not yield satisfactory results we recommend searching species-specific literature for suggestions. For fixation, the coverslip is usually sealed with nail polish.

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Fig. 17. Filter paper technique to preserve fungal strains, a simple regime that may work in many cases: (a) add filter paper discs to freshly inoculated fungal cultures; (b) wait until fungal hyphae penetrate the discs; (c) remove discs; (d) store them in glass vials.

The various types of antifungal assays The choice of bioassays we introduce here is biased by those techniques that are established in our laboratory, and the focus will be more on filamentous fungi colonizing living or dead plant tissues. However, many of the stated principles and exemplified techniques are also valid when focusing on fungi from other sources or yeasts. Most of the assay methodologies will be described in detail to allow even those, which are less familiar with microbiological protocols, to follow instructions. However, several issues are valid for all techniques: (1) the test organisms should grow exponentially, (2) positive controls are to be presented to facilitate more or less a comparison with published data, and (3) negative controls are to be set up to assess any affects caused by added organic solvents and surfactants. Bioautography on thin-layer plates This technique was introduced by Homans and Fuchs (1970) and is preferably carried out on thin-layer plates (TLC), but is also applicable on polyacrylamide gels (De Bolle et al., 1991). TLC has an enormous potential for separating mixtures of low-molecularweight compounds. Egon Stahl (1967), one of the pioneers in this field, reviews numerous methods to be applied for a various compound classes. However, advances in LC–DAD and LC–MS technologies have ousted TLC as analytical tool in the majority of applications. Still, TLC has merits for bioautography as it constitutes the only directly

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Fig. 18. Permanent slide technique.

combined application of an analytical chemical method with an in situ bioassay that allows a rapid identification of the active compound or compounds in a complex mixture. Accordingly, it is very popular among those researches that are also experienced in the application of chromatography techniques, such as organic chemists and pharmacognosists. As only few journals nowadays accept reports that focus only on

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Naturally occurring bioactive compounds

novel structures alone, this rather easy-to-perform assay is very popular among articles dealing with natural product chemistry (e.g., Hostettmann et al., 2000). Autobiography on TLC plates facilitates the submission of a wide range of filamentous fungi to antifungal testing. Preference is given to those fungi that are characterized by pigmented hyphae, spores, or conidia. If contrast is poor, it can be enhanced by treatments with iodine vapor (Gerlach, 1977). Similarly, this visualization method is also applicable for yeasts (Hadacek, unpublished). Figure 19 illustrates the procedure. Total evaporation of the organic solvent is mandatory to obtain a mycelial layer on the surface of the plate. In some applications, the choice of solvents will have to be constrained to more volatile ones, such as diethyl ether and hexane. We successfully employ an air brush to spray the inoculum suspension onto the thin-layer plate. The suspension usually consists of malt extract broth or glucose medium with mineral salts added (Homans and Fuchs, 1970). However, the nutrient medium composition may have to be adjusted to the specific requirements of each test fungus. The application procedure of the inoculum is difficult to standardize and this may affect the comparability of results with other laboratories, besides a wide range of other factors. Figure 20 illustrates the testing of extract fractions obtained from column chromatography of a crude extract of underground organs of tarragon, Artemisia dracunculus, which contains antifungal polyacetylenes and isocoumarins (Engelmeier et al., 2004). Apart from the advantages of rapidly detecting active compounds in mixtures, the depicted bioautography also points to a potential disadvantage of this diffusion assay. The more lipophilic the compounds are, the more they show diffusion effects. These diffusion effects may significantly hamper a comparison of activities between different compounds with differing chemical properties. Another factor that may also affect results is the stability of the compound on the TLC plate as the duration of the assay may last for several days and exact quantization of the amounts of the compound that survived on the TLC plate are rarely performed due to the amount of effort required. The usually applied method of evaluation is assessing the MIC (minimum inhibitory concentration) that designates the lowest concentration in a dilution series that still yields a detectable effect. Undoubtedly, this assay has its merits for identifying active compounds in complex mixtures. Wedge and Nagle (2000) published the application of 2D-TLC as efficient approach to obtain improved separation of compounds with a concomitant gain in sensitivity of the assay. For exploring the efficacy of specific compounds in comparison to positive controls, we rate this assay as less apt due to previously discussed limitations. Diffusion assays are – despite their quick and versatile application – generally less suitable to assess the quality of the antifungal activity in comparison to positive controls. Disk diffusion This technique belongs to one of the most widely employed antifungal screening methodologies, and it is primarily used to determine if a compound or a compound mixture, such as crude extracts, possesses any activity at all. One of the major shortcomings is that, as for all diffusion assays, the concentration of the test compound or test compound mixture is unknown. The simplicity of the procedure (Cole, 1994) and

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Prepare the appropiate diluten of the conidia suspension

Spray homogenously on the developed TLC plate, e.g. as illustrated here with an air brush, and transfer into a bioassay chamber; incubatel until fungal growth becomes visible

Bioassay chamber (25 x 25 cm)

Moistened filter paper

U-shaped glass rod

Fig. 19. Bioautography on TLC plates.

the relative good comparability to another widely employed method, broth microdilutions (Trancassini et al., 1986), have considerably contributed to its wide distribution; a guideline has even been published recently for antimicrobial chemotherapy purposes (Sheehan et al., 2004). Figure 21 illustrates this procedure affording a minimum of equipment. Antibiotic paper disks and stars that facilitate the application of the test compound or mixture (Figure 22) are commercially available. Conventionally, diameters of inhibition zones are presented to document the observed antifungal activity. In

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Naturally occurring bioactive compounds

Fig. 20. TLC bioautography of the fractionated lipophilic extract of underground organs of Artemisia dracunculus, the tarragon with Cladosporium herbarum as tested fungus. White zones in the layer of mycelium indicate active compounds, polyacetylenes and isocoumarins (Engelmeier et al., 2004), that inhibit the development of dark pigmented hyphae and conidia. Note that diffusion of compounds increases with lipophilicity. The TLC plate was developed in a mixture of diethyl ether and hexane (4:6, v/v).

interpreting these diameters, the fact has to be considered that variable diffusion properties of the test compound may affect the outcome, especially if results from this assay are used to compare MIC values of different compounds. There exist modifications of this method, such as the agar well diffusion, including the hole-plate (diffusion of the aqueous test compound solution into the agar medium from a vertical hole in the agar layer) and the cylinder method (stainless-steel or ceramic cylinders placed on top of the agar medium) (Rios et al., 1988). These two modifications have their merits when the test compound shows good solubility in aqueous solvents. However, as the majority of active compounds are better soluble in organic solvents, the addition of a specific portion of organic solvent to obtain an aqueous suspension of the test compound is required – in all instances, avoid adding more than 5% of organic solvent (v/v). This modification has the evident advantage that pure organic solvent can be used for the stock solution, which gets completely lost during the preparation of the disks after efficient drying. Figure 23 exemplifies the effects of an active compound, the naphtochinone juglone. Applying the compound

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Fig. 21. Disk diffusion test.

with a pipette dissolved in a micro-volume of solvent allows the incorporation of the compound into the medium in high concentrations even in situations when test compound amounts are limited. The inhibition zones are usually distorted as this application procedure does not guarantee the test compound to be evenly distributed across the disk. However, if the solvent has not been removed properly and causes inhibition effects by itself, the zones are highly concentric to the disk. The peculiarity of this phenomenon facilitates the experienced researcher to become aware of the deficiency in his work. Microdilution For sure, diffusion assays have their merits as preliminary screening methods, but subsistence with minimal test compound amounts and concomitant provision of a wide range of concentrations together with the hydrophobic nature of the majority of the candidate compounds turns broth dilutions assays, especially those that are carried out in microwell plates, into one of the most attractive and concomitantly efficient methodologies to characterize antifungal activity of defined compounds (Wedge and Kuhajek, 1998; Kerwin and Semon, 1999; Hadacek and Greger, 2000). Recommendations of NCCLS for antifungal susceptibility testing focus also on this technique (Pflaller et al., 1997, 2002a, 2002b). Figure 24 illustrates the procedure required to test a potential antifungal hydrophobic compound – such as is the nature of the majority of the candidate compounds. In this case, the use of organic solvents and surfactants is mandatory to obtain a viable suspension for the assay. The concentration of organic solvents should not exceed 5% of

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Naturally occurring bioactive compounds

Fig. 22. Antibiotic test disks and stars. Copyright and provided courtesy of MACHEREYNAGEL. All rights reserved.

the volume of the stock solution. Organic solvent (250 mL) is used to dissolve 2 mg of test compound; this solution is added to 4750 mL of medium – the quotient of organic solvent is 5%. Usually, to obtain a practicable emulsion a surfactant also needs to be added. The amount of the latter should be kept to a manageable minimum. The viscosity of the surfactant complicates the application of its dosage. Variable combinations of organic solvents and surfactants may cause different effects of the growth of the test fungus. In some cases, even significant inhibition effects may occur. Thus, all of these additives have to be present in the stock solutions of the control as well. During the dilution procedure, concentrations of the additives become lower, which usually improves the analysis of the results obtained from the diluted concentrations of the stock solution. Partially, the dilution attenuates this unwanted effect as it proceeds. Depending on the fungus, concentrations of organic solvents, such as DMSO, acetone, or ethanol, not exceeding 5% per well could also be useful if test compounds exhibit very low solubility in water and therefore precipitate when added to the aqueous culture medium. This phenomenon may seem to be idiosyncratic but, once its merit is recognized, it proves as helpful. However, the optimum relation of applied concentrations, combinations of organic solvents, and surfactants has to be individually determined for each test scenario.

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Fig. 23. Disk diffusion test of the naphtochinone juglone. Test fungus Cladosporium herbarum; 100 mg (upper left), 50 mg (upper right), 25 mg (lower right), control (lower left, treated with the same amount of organic solvent). Incubation for 3 days at room temperature.

Growth rates of fungi may vary depending on the strain and the quality of the medium used and the optimal point of time to evaluate the growth has to be determined for each test strain. The dilution assays represent typical quantitative bioassays, which will be discussed later in more detail. Characteristically, they allow to estimate effective concentrations (EC50, Finney, 1972; Roberts and Boyce, 1972). The inclusion of a wide range of concentrations to obtain values that range between 10% and 90% is conditional to obtain a sound estimate. Roughly, there exist three types of evaluation methodologies that are characterized by both advantages and disadvantages: in case of yeasts, turbidity of the culture broth indicates growth and this phenomenon can be easily scored with the naked eye, especially when the assay is targeted at assessing the MIC of the tested compound. This procedure is widespread in the evaluation of efficacy of various antimycotic drugs (Cormican and Pfaller, 1996). This approach is also applicable for yeasts. However, the filamentous growth of the majority of fungi prevents its broad application because their growth cannot be recognized by the naked eye unambiguously. A certain amount of magnification is required to determine if germ-tube formation has occurred or not and to what extent it differs from the control. The advent of digitized micrographs enormously contributed to increased throughput rates in determining germ-tube growth (Figure 25), similarly as microwell plates facilitated microdilutions that considerably reduced the amounts of required test compounds (Figure 24, Hadacek and Greger, 2000).

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Naturally occurring bioactive compounds

Fig. 24. Broth dilution in microwell plates, the method of choice to perform assays with minimal amounts of test compounds in a wide range of test concentrations to facilitate an estimate of the EC50.

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For comparative analyses, one approach recommends the usage of indices that represent different degrees of growth inhibition (Kobayashi et al., 1996). The evaluation of micrographs has a distinctive advantage: morphological deformations that are caused by the test compound, such as curling (see Figure 26), swelling, hyperdivergency, and beads shape can be detected (Kobayashi et al., 1996), even in case that no significant growth reduction occurs. Although means for automation have been suggested (e.g., Hilber and Schu¨epp, 1992; Oh et al., 1996), its broad application for various groups of fungi has never been established. Appressorium formation (Figure 27) constitutes another important morphological character that is characteristic for many plant pathogenic fungi infecting leaves (Dean, 1997). Thines and co-workers (Thines et al., 1997) developed an assay system that not only allowed assessing inhibitory effects of chemicals but also pointed to the affected signal pathways. The authors used either Parafilm ‘M’ (American National Can) or GelBond (Sigma-Aldrich Chemical Company, St Louis, MO)-coated slides as hydrophobic surfaces and various chemicals, among the cAMP (Sigma-Aldrich Chemical Company) and 1,16-hexadecanediol (Sigma-Aldrich Chemical Company) to induce appressorium formation on hydrophilic surfaces (microwell plates).

Fig. 25. Principle procedure in applying image analysis as tool to assess germ-tube size (magnification 140
).

Fig. 26. Morphological deformations, such as curling (depicted here, fungus Pyricularia grisea, magnification 140
), may be missed in microplate reader-based evaluations. Consequently, in certain instances, preference may be given to morphological analysis. (A) control; (B) curling effect.

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Fig. 27. Appressorium formation of germ tubes (magnification 140
). Certain fungi, such as the rice blast fungus Pyricularia grisea, may develop infection structures of leaf surfaces that are decisive for phytopathogenicity. (A) control; (B) appressoria (a).

For automation, other methodologies, such as those developed for the assessment of cell proliferation, have been applied to fungi: in metabolically active cells, mitochondrial dehydrogenases reduce the soluble tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; BD Biosciences) and turns it into a blue formazan, which can be measured photometrically (Clancy and Nguyen, 1997; Adam et al., 1998); redox-based indicator dyes, such as Alamar Blue (Sigma BD Biosciences) or similar products, indicate cell growth by a colorimetric change and fluorescent signal (e.g., Espinel-Ingroff et al., 1997; Pelloux-Prayer et al., 1998). Other reagents that are turned into fluorescent products include FungiqualTM (Coleman et al., 1989) and fluorescein diacetate (Chand et al., 1994; Hadacek and Greger, 2000). In a comparative study of assessing fungal biomass by weighing the dried mycelia and turbidity around 600 nm, Broekaert et al. (1990) demonstrated the applicability of the latter method. However, for all these microwell plate-based assays the following issues should be taken into consideration: positive controls are to be included in any case to allow extrapolation of the chosen test methodology, and also to facilitate practicable comparisons between different assay methodologies. The positive controls should consist of efficient drugs and fungicides and share one trait: ready availability from a commercial source to facilitate comparison of assays systems. Further, matrix effects from the diluted test compound or mixture of compounds may affect the readings of the microwell plates. Thus, a blank of the test compound is mandatory to assess the actual strength of the inhibitory effect. Inhibition of radial growth Dilution assays can be performed in liquids or in agar medium. The main difference is that in liquids growth is submersed, and on agar media superficial. As a result, susceptibilities of the same fungus may vary considerably if tested by microdilution or radial surface growth methodologies (Hadacek and Greger, 2000). However, the latter assay may prove useful in certain questions, such as in screening fungicides to preserve surfaces. Figure 28 illustrates the procedure of this assay. As alternative to inoculations with micro-volumes of conidia or spore suspensions, plugs from the actively growing zones of Petri dish cultures may also be used. Here, the advantage lies in the fact that fungi that fail to produce conidia or spores may be used as tested

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Fig. 28. Inhibition of radial growth on media supplemented with the test compound.

organism. Conversely, the inoculation procedure may be disadvantageous in case of profusely sporulating fungi with the unpleasant consequence that emerging stray colonies may contaminate the assay. Disk diffusion and radial growth have one characteristic in common: they are easy to perform and also require minimal laboratory equipment. Thus, radial growth assays are often employed in ecological studies (more lipophilic compounds) similarly as disk diffusion assays (more hydrophilic compounds) are used in clinical studies. The inclusion of positive controls is highly recommended since this test

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system is less sensitive than others. The fungal mycelium that develops on the surface of the agar is less confronted to the test compound’s effects compared to the submersed growth in the microdilution assay (Hadacek and Greger, 2000).

Statistics In this final chapter we recommend some statistical procedures that we think to be useful for the discussed antifungal bioassays. It is limited to those assays where the concentration of the test compound in the medium is known. Accordingly, this excludes all diffusion bioassays. In this case, the report of the MIC or, in case of disk diffusion assays, the diameter of the inhibition zone is sufficient. We have to bear in mind that the development of an endpoint is specific for each compound and does not inform about when the effect starts. Consequently, this value is biased as it favors compounds with an inhibition effect that (1) is limited to few dilution steps and (2) is clearly shown. The inherent problems associated with ‘‘trailing endpoints’’ are extensively discussed by Rex et al. (1997). A good example for this is the inhibition of the rice blast fungus Pyricularia grisea (Figure 29) by the commercially available fungicide BenlateTM (50% benomyl). All assays that operate with known concentrations in the medium can be evaluated with statistical procedures recommended for a quantitative biological assay (Roberts and Boyce, 1972). In those cases where the dose–response relation is not sigmoid, no estimates are probable, and this applies more often to quantitative than to quantal assays. We chose the naphtochinone juglone and the polyacetylene falcarindiol (Figure 30) to illustrate the statistical procedures on specific examples; for juglone, the dose–response relationship follows a sigmoid curve and for falcarindiol it does not. Moreover, we compared two scoring methodologies: germ-tube size assessment on one hand and turbidity at 620 nm on the other hand. Table 2 lists the germ-tube sizes in pixels obtained while assaying juglone and falcarindiol against Botrytis cinerea. Usually, two rows were prepared for each test compound and the control. Ten representative germ tubes were selected for measurements. The means were used for calculation of the percentages of control growth for each dilution step. This procedure helps to minimize potential effects of the organic solvent and added surfactants, such as Tween 80. The inhibitory effects of the latter can be noted in the reduced growth of the control at higher dilutions. Table 3 presents the readings for juglone obtained from the microplate reader at 620 nm. A blank is measured about 1 h postinoculation after the conidia suspension has settled. Turbidity that may be caused by higher concentrations of the test compounds is thus taken into account. In our experience, the assessment of the difference in turbidity increase between 24 and 48 h yields practicable results. Both readings were previously corrected by deducting the blank. In higher concentrations of juglone, precipitation of the originally dissolved test compound occurred which affected the readings. As juglone is a highly active compound and inhibition already occurs at lower concentrations, the readings at higher concentrations can more or less be omitted. Alternatively, the dilution series can be performed in molten agar medium that adds temperature stress to, but prevents precipitation of the test compound or mixture.

Antifungal natural products: assays and applications 1008

457

BenlateTM Control

1006 1004

pixels

1002 1000 800 600 400 200 endpoint

0 100

50

25

13

6

3 µg/mL

1.5

0.8

0.4

0.2

0.1

Fig. 29. Inhibition of BenlateTM to the rice blast fungus Pyricularia grisea (microdilution in U-bottomed microplates; CFU 104, medium 4% malt extract; germ-tube size assessed after 16 h growth in darkness at 251C, magnification 120
; pixel counts by Scion Image 4.02).

OH

O

OH O 1

OH 2

Fig. 30. Juglone (1) and falcarindiol (2) serve as examples for the statistical evaluation of microdilution assay scores by germ-tube image analysis and turbidimetry at 620 nm.

Analogously, Table 4 lists the readings for falcarindiol. Test compound and control are run in four replicates each in the identical microwell plate. Figure 31 compares the results from both assays. In both assay methodologies, the inhibitions of juglone are comparable; the test compound caused a sigmoidal dose–response relation. The 95% fiducial limits do not differ significantly, the EC50 estimates differ by 20%, but this is negligible in a two-fold dilution series. Both EC50 values fall within the fiducial limits of the other assay. Consequently, the assays can be regarded as highly reproducible and their results as comparable. Some authors report the mean estimates of several replicates and present the variation as standard variation or error. Such a procedure treats the estimates as measurements and ignores the asymmetry of the fiducial limits. These values contain valuable additional information about the accuracy of the measurement and the inherent raw data and should thus not be ignored.

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Table 2 Microdilution of juglone and falcarindiol in U-bottomed microwell plates, test fungus Botrytis cinerea, CFU 104, medium 4% malt extract, assessment of germ-tube size after 16 h growth at 251C by digitizing micrographs at 140
magnification; number of pixels determined by Scion Image 4.02 Dilution Concentration (mg/mL)

1 200 100

2 50

3 4 5 25 12.5 6.3

6 3.1

7 1.6

8 0.8

9 0.4

10 0.2

11 0.1

622 1289 1091 1007 860 721 1475 1921 907 913 1081 388 0.6 0.87

1078 852 1169 623 505 646 699 526 900 793 779 223 0.44 0.99

590 1257 706 804 607 509 525 683 495 912 709 235 0.65 0.8

835 933 806 686 435 774 818 594 1085 909 788 183 0.54 0.93

471 488 508 694 383 715 768 619 829 559 603 145 0.46 0.98

Control

Mean Standard deviation Kolmogorov-Smirnov Z Asymptotic significance Juglone

370 682 1143 803 281 663 331 546 733 409 513 1113 529 660 568 640 757 388 581 782 967 523 657 268 259 529 1064 461 345 442 491 575 735 160 166 321 0.33 0.6 0.52 1 0.87 0.95

Mean Standard deviation Kolmogorov-Smirnov Z Asymptotic significance % of control p (Mann– Whitney U-test) Falcarindiol 150 111 253 208 127 155 330 109 204 80

1517 1026 770 1020 727 765 816 413 623 512 819 313 0.64 0.8

662 1203 683 913 1024 713 712 776 598 432 1077 814 813 816 601 756 896 484 525 1020 725 871 846 1143 923 424 408 1243 749 360 785 883 653 228 217 225 0.55 0.53 0.55 0.93 0.94 0.92

-

-

-

-

-

196 339 264 95 125 92 274 173 182 338

393 218 361 274 411 621 103 127 118 184

512 558 410 160 355 285 261 216 947 414

89 99 459 511 522 185 365 573 611 235

226 693 228 339 772 440 310 705 483 644

-

173 171 210 644 904 205 317 505 774 1133 165 228 453 391 1090 208 174 768 237 720 309 268 464 421 1248 131 197 573 419 612 183 182 713 656 199 162 523 812 586 729 90 178 499 589 344 80 210 254 326 435 171 245 525 504 741 65 108 200 170 353 0.58 0.83 0.51 0.6 0.44 0.89 0.5 0.96 0.87 0.99 16 31 74 64 123 0 0 0.089 0.001 0.393

938 465 670 768 953 682 808 777 403 207 536 335 281 595 639 245 648 531 600 1183 650 866 474 554 976 514 483 311 484 736

342 728 373 563 945 581 839 670 504 802

875 480 472 863 618 380 842 609 501 366 660 640 282 358 552 1109 376 818 503 675

Antifungal natural products: assays and applications

459

Table 2 (continued ) Dilution Concentration (mg/mL)

1 200 100

2 50

3 4 5 25 12.5 6.3

Mean 173 208 281 412 Standard deviation 77 92 166 226 Kolmogorov-Smirnov Z 0.61 0.48 0.47 0.62 Asymptotic significance 0.85 0.98 0.98 0.84 % of control 35 36 38 50 p (Mann– Whitney U-test) 0 0 0.002 0.002

365 199 0.58 0.89 46 0

484 207 0.57 0.9 55 0

6 3.1

7 1.6

8 0.8

9 0.4

10 0.2

11 0.1

600 663 568 635 568 630 310 240 131 199 188 247 0.71 0.71 0.65 0.34 0.43 0.46 0.69 0.69 0.79 1 0.99 0.99 92 61 73 90 72 104 0.853 0.007 0.052 0.631 0.035 0.912

Table 3 Microdilution of juglone in U-bottomed microwell plates, test fungus Botrytis cinerea, CFU 104, medium 4% malt extract, measurement of turbidity at 620 nm at the beginning, after 24 and 48 h Juglone Dilution Concentration (mg/mL) Blank Juglone

Control

Absorbance_24 h Juglone

Control

Absorbance_48 h Juglone

Control

(abs_48 h-blank)–(abs_24 h-blank) Juglone

200

1 100

0.14 0.17 0.19 0.17 0.04 0.05 0.04 0.04

0.15 0.15 0.08 0.07 0.04 0.04 0.05 0.05

0.27 0.31 0.19 0.21 0.23 0.24 0.23 0.23

2 50.0

3 4 5 25.0 12.5 6.3

6 3.1

7 1.6

8 0.8

9 0.4

10 0.2

11 0.1

0.08 0.08 0.08 0.07 0.04 0.05 0.04 0.04

0.06 0.06 0.06 0.06 0.05 0.05 0.05 0.04

0.06 0.06 0.06 0.06 0.04 0.04 0.04 0.04

0.06 0.06 0.05 0.06 0.05 0.05 0.05 0.05

0.06 0.06 0.05 0.06 0.05 0.05 0.05 0.05

0.05 0.05 0.05 0.06 0.04 0.05 0.05 0.05

0.05 0.05 0.05 0.05 0.05 0.05 0.04 0.04

0.05 0.06 0.05 0.06 0.05 0.05 0.05 0.05

0.05 0.06 0.05 0.06 0.05 0.05 0.05 0.05

0.06 0.06 0.05 0.06 0.05 0.05 0.05 0.05

0.22 0.29 0.20 0.19 0.21 0.22 0.22 0.23

0.26 0.22 0.22 0.25 0.21 0.20 0.21 0.21

0.11 0.11 0.10 0.12 0.19 0.19 0.19 0.20

0.10 0.09 0.09 0.11 0.17 0.17 0.17 0.19

0.10 0.09 0.08 0.09 0.17 0.17 0.17 0.18

0.08 0.07 0.07 0.08 0.17 0.17 0.17 0.17

0.07 0.06 0.07 0.07 0.14 0.16 0.15 0.16

0.07 0.09 0.11 0.11 0.15 0.15 0.15 0.15

0.13 0.13 0.16 0.17 0.13 0.13 0.13 0.14

0.18 0.18 0.20 0.22 0.11 0.12 0.12 0.14

0.21 0.20 0.21 0.21 0.12 0.06 0.07 0.13

0.35 0.46 0.37 0.36 0.62 0.66 0.73 0.75

0.26 0.41 0.32 0.31 0.58 0.58 0.56 0.65

0.26 0.20 0.21 0.23 0.56 0.58 0.53 0.58

0.12 0.11 0.10 0.12 0.48 0.46 0.45 0.50

0.10 0.10 0.10 0.11 0.46 0.42 0.52 0.56

0.10 0.09 0.09 0.10 0.43 0.60 0.58 0.54

0.09 0.08 0.08 0.09 0.41 0.41 0.42 0.56

0.08 0.07 0.11 0.09 0.41 0.45 0.41 0.56

0.17 0.23 0.32 0.28 0.39 0.43 0.42 0.50

0.32 0.29 0.34 0.36 0.39 0.44 0.44 0.59

0.45 0.38 0.45 0.42 0.44 0.42 0.44 0.50

0.59 0.50 0.51 0.53 0.47 0.47 0.47 0.55

0.08 0.16 0.18 0.15

0.04 0.00 0.01 0.00 0.13 0.01 0.00 0.00 0.12 0.01 0.00 0.00 0.12 0.02 0.00 0.00

0.00 0.00 0.00 0.01

0.01 0.01 0.01 0.01

0.01 0.01 0.04 0.02

0.10 0.14 0.21 0.17

0.20 0.17 0.17 0.19

0.27 0.20 0.25 0.20

0.38 0.30 0.31 0.32

Naturally occurring bioactive compounds

460 Table 3 (continued ) Dilution Concentration (mg/mL) Mean Standard deviation Kolmogorov-Smirnov Z Asymptotic significance % of control p (Mann– Whitney U-test) Control

Mean Standard deviation Kolmogorov-Smirnov Z Asymptotic significance

200

1 100

0.14 0.10 0.04 0.04 0.64 0.82 0.81 0.51 29 27 0.02 0.02 0.39 0.37 0.42 0.36 0.50 0.35 0.52 0.42 0.48 0.38 0.06 0.03 0.50 0.63 0.96 0.83

2 50.0 0.00 0.01 0.50 0.96 0 0.02 0.35 0.37 0.32 0.38 0.36 0.03 0.43 0.99

3 4 5 25.0 12.5 6.3

6 3.1

7 1.6

0.00 0 0.88 0.42 1 0.02 0.28 0.27 0.26 0.29 0.28 0.01 0.30 1.00

0.01 0 3 0.01 0.25 0.24 0.25 0.40 0.30 0.08 0.85 0.46

0.02 0.16 0.18 0.23 0.32 0.01 0.05 0.01 0.03 0.04 0.52 0.26 0.60 0.60 0.67 0.95 1.00 0.87 0.86 0.77 6 52 51 71 79 0.02 0.02 0.02 0.02 0.04 0.27 0.24 0.26 0.33 0.35 0.30 0.28 0.30 0.29 0.41 0.25 0.27 0.32 0.32 0.40 0.41 0.35 0.45 0.36 0.42 0.32 0.30 0.36 0.32 0.41 0.07 0.05 0.08 0.03 0.03 0.58 0.59 0.62 0.36 0.63 0.89 0.89 0.84 0.99 0.83

0.00 0 0 0.01 0.29 0.25 0.35 0.37 0.32 0.05 0.48 0.98

0.00 0 0.88 0.42 1 0.02 0.26 0.43 0.41 0.37 0.40 0.08 0.53 0.95

8 0.8

9 0.4

10 0.2

11 0.1

Test compound precipitated as crystals that influence the readings of the microplate reader.

Falcarindiol (Figure 31) is inhibited in both assays, but the effect does not follow a sigmoidal dose–response effect. In our experience, such results are generally more the rule than the exception. The development of the visible inhibition in both methodologies is comparable, but the EC50 values differ more, and moreover the fiducial limits differ significantly. The endpoint in the turbidity scoring is much lower than in the germ-tube size assessment. One explanation might be that the inhibitory effect of the test compound reduces the number of germinating conidia, an effect that is more accurately assessed by the turbidity measurements than by the selected germ-tube measurements. In this context, one may point out that counting germinated versus non-germinated spores or conidia might facilitate an approach to solve this problem. Such a scoring procedure is possible but cumbersome in case of small size of the propagules and often complicated by incomplete germination of the control inoculum. In this aspect, turbidity certainly holds advantages compared to germ-tube size assessment. However, in the latter we can directly monitor the actual inhibition whereas in the former we have to trust the readings of the microplate reader. In specific cases one might be tempted to explore synergism in the activities of compound mixtures. Here we recommend to consult Tallarida (2000).

Conclusions The research outset and goals ultimately determine the assay to be actually used. On one hand different kinds of researchers prefer different assays, and on the other hand lacking facilities may exclude the application of some specific assays. Generally we would welcome to see the standardizations of procedures, as it is done for screenings in antifungal chemotherapy, to be extended to other fields. However, the incorporation of positive controls may help to facilitate comparison of results to some extent. Antifungal assays will continue to be applied in a wide range of basic and applied research problems, from antifungal chemotherapy to agrochemistry in applied sciences. Biotic interactions are

Antifungal natural products: assays and applications

461

Table 4 Microdilution of falcarindiol in U-bottomed microwell plates, test fungus Botrytis cinerea, CFU 104, medium 4% malt extract, measurement of turbidity at 620 nm at the beginning, after 24 and 48 h Falcarindiol Dilution Concentration (mg/mL) Blank Falcarindiol

Control

Absorbance_24 h Falcarindiol

Control

Absorbance_48 h Falcarindiol

Control

200

1 100

0.11 0.11 0.11 0.11 0.04 0.05 0.04 0.05

0.08 0.07 0.07 0.07 0.05 0.05 0.05 0.04

0.06 0.06 0.06 0.06 0.04 0.05 0.05 0.04

0.05 0.06 0.06 0.06 0.04 0.05 0.05 0.05

0.20 0.20 0.21 0.22 0.23 0.24 0.23 0.23

0.19 0.20 0.19 0.21 0.21 0.22 0.22 0.23

0.21 0.20 0.19 0.20 0.21 0.20 0.21 0.21

0.13 0.14 0.14 0.17 0.47 0.47 0.47 0.55

0.13 0.12 0.13 0.18 0.44 0.42 0.44 0.50 0.00 0.00 0.00 0.00 0.00 0.00 0 0.014 0.23 0.2 0.22 0.27 0.228 0.03 0.52 0.95

(abs_48 h–blank)–(abs_24 h–blank) Falcarindiol 0.00 0.00 0.00 0.00 Mean 0.00 Standard deviation 0.00 Kolmogorov-Smirnov Z Asymptotic significance % of control 0 p (Mann– Whitney U-test) 0.014 Control 0.24 0.23 0.24 0.32 Mean 0.264 Standard deviation 0.04 Kolmogorov-Smirnov Z 0.76 Asymptotic significance 0.61

2 3 4 50.0 25.0 12.5

5 6.3

6 3.1

7 1.6

8 0.8

9 0.4

10 0.2

11 0.1

0.06 0.06 0.06 0.06 0.05 0.05 0.05 0.07

0.05 0.06 0.05 0.06 0.04 0.05 0.05 0.05

0.05 0.05 0.05 0.06 0.05 0.05 0.05 0.06

0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.07

0.05 0.05 0.06 0.05 0.05 0.05 0.05 0.05

0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05

0.06 0.05 0.06 0.05 0.05 0.04 0.05 0.05

0.05 0.06 0.06 0.06 0.04 0.05 0.05 0.05

0.21 0.20 0.20 0.22 0.19 0.19 0.19 0.20

0.22 0.20 0.22 0.20 0.17 0.17 0.17 0.19

0.21 0.19 0.20 0.22 0.17 0.17 0.17 0.18

0.22 0.21 0.21 0.23 0.17 0.17 0.17 0.17

0.21 0.19 0.22 0.23 0.14 0.16 0.15 0.16

0.13 0.16 0.16 0.18 0.15 0.15 0.15 0.15

0.10 0.13 0.11 0.11 0.13 0.13 0.13 0.14

0.10 0.10 0.11 0.11 0.11 0.12 0.12 0.14

0.10 0.12 0.12 0.13 0.12 0.06 0.07 0.13

0.18 0.22 0.19 0.19 0.39 0.44 0.44 0.59

0.26 0.30 0.31 0.33 0.39 0.43 0.42 0.50

0.36 0.35 0.38 0.39 0.41 0.45 0.41 0.56

0.45 0.37 0.36 0.40 0.41 0.41 0.42 0.56

0.40 0.38 0.37 0.41 0.43 0.60 0.58 0.54

0.48 0.39 0.40 0.39 0.46 0.42 0.52 0.56

0.50 0.37 0.43 0.42 0.48 0.46 0.45 0.50

0.54 0.40 0.41 0.41 0.56 0.58 0.53 0.58

0.47 0.37 0.43 0.49 0.58 0.58 0.56 0.65

0.59 0.57 0.55 0.58 0.62 0.66 0.73 0.75

0.00 0.02 0.00 0.00 0.006 0.01 0.88 0.42 0 0.018 0.18 0.23 0.23 0.38 0.282 0.09 0.71 0.69

0.05 0.10 0.11 0.11 0.093 0.03 0.71 0.70 37 0.020 0.2 0.24 0.23 0.3 0.255 0.04 0.57 0.91

0.14 0.15 0.16 0.19 0.159 0.02 0.50 0.96 54 0.012 0.24 0.28 0.23 0.38 0.297 0.07 0.52 0.95

0.23 0.18 0.16 0.18 0.189 0.03 0.70 0.71 64 0.020 0.24 0.24 0.25 0.39 0.292 0.07 0.84 0.48

0.18 0.17 0.15 0.17 0.167 0.01 0.66 0.78 41 0.020 0.26 0.43 0.41 0.38 0.405 0.08 0.59 0.88

0.27 0.20 0.18 0.15 0.201 0.05 0.50 0.96 58 0.043 0.32 0.27 0.37 0.4 0.344 0.06 0.38 0.99

0.37 0.21 0.27 0.24 0.272 0.07 0.53 0.94 84 0.248 0.33 0.32 0.31 0.34 0.323 0.01 0.37 0.99

0.44 0.27 0.30 0.30 0.329 0.07 0.78 0.58 76 0.080 0.43 0.44 0.41 0.44 0.43 0.02 0.53 0.95

0.37 0.27 0.33 0.38 0.334 0.05 0.49 0.97 71 0.021 0.47 0.45 0.44 0.52 0.471 0.03 0.52 0.95

0.48 0.45 0.43 0.45 0.452 0.02 0.60 0.87 72 0.020 0.49 0.60 0.66 0.62 0.624 0.07 0.59 0.88

Naturally occurring bioactive compounds

462

control juglone falcarindiol

Assessment of germ-tube size, n=10 1600

EC50 = 0.5 (0.2–1) EC50 = 21 (6–141)

1400 1200

a

pixels

1000 800

a

a a

a

600 400

a

a

c

0.4

0.2

a

a a a

c

c

c

c

c

a

c

c

200

a

a

a a

a

a

a

c

0 200

100

50

25

13

6

3

1.6

0.8

0.1

µg/mL control juglone falcarindiol

Difference of turbidity (48h–24h), n=4

0.8

EC50 = 0.4 (0.2–0.7) EC50 = 4 (1–39)

0.7

a

absorbance

0.6 0.5

a

a

0.4

a

a a

0.3

a

a

a

a

0

b

b b b 200

100

50

a

b 25

13

6

a ab

a

b

a b b

b b

b

b

b

a

a

b

b

b 0.1

a

a

a

a

a

a

0.2

a

a

b

a

3

1.6

0.8

0.4

0.2

0.1

µg/mL juglone (germ-tube size)/juglone (turbidity):p = 0.268 (Mann-Whitney U test ) falcarindiol (germ-tube size)/falcaridiol (turbidity):p < 0.001 (Mann-Whitney U test)

Fig. 31. Result comparison of two scoring methodologies: germ-tube size (10 germ tubes, control in the same microwell) and turbidity at 620 nm (four replicates; controls in each microwell), for juglone and falcarindiol; test fungus Botrytis cinerea, microdilutions in 4% malt extract, CFU 105; probit-log estimates by SPSS 10.0.7; bars indicate mean values+standard deviation; significance levels: a, p>0.05; b, po0.05; and c, po0.005.

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gaining more attention in community ecology. Natural products, as pharmacognosists call them, or allelochemicals or secondary metabolites, as biologists call them, will thus be evaluated for their antifungal activities by many researchers from different disciplines in the future. In many cases, optimization of high-throughput procedures will be a major requirement. This can be achieved by using robot pipetting and automated scoring systems. Despite the enthusiasm we might develop for the gain in results, we have to be aware that we lose awareness of what is actually going on in the single well of the microtiter plate, and thus we might fail to interpret our assays correctly. Further, another methodology is offering itself to be applied for antifungal testing: metabolomics, sometimes also called metabonomics or metabolic profiling (Hall, 2006). Here, NMR- or MS-based methods may allow not only to determine the antifungal effect alone but also to obtain insights into its mode of action. An example how such an approach might work is given by Aliferis and Chrysati-Tokousbalides (2006), who studied the effects of naturally occurring phytotoxins and synthetic herbicides on oat seedlings.

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