High-Throughput Screening Assays to Identify Plant Natural Products with Antifungal Properties Against Fusarium oxysporum

Abstract

Fusarium oxysporum is a cross-kingdom fungal pathogen that not only causes devastating plant vascular diseases but can also opportunistically infect humans. Here we describe two high-throughput screening assays, a resazurin cell viability assay and an optical density assay, to screen natural products from cultured plant cells with antifungal properties against a clinical isolate of F. oxysporum. After elicitation by applying methyl jasmonate or by co-culture with F. oxysporum, as an abiotic elicitor and a biotic elicitor, respectively, we identified three cell lines that produce materials that inhibit fungal growth. Our procedure validates the powerful potential of combining high-throughput methods for the discovery of novel anti-pathogenic leads.

1. Introduction

Fusarium oxysporum is a cross-kingdom fungal pathogen that is widely distributed in diverse environments, including soil, indoors, and aquatic habitats [1]. Members of F. oxysporum not only cause devastating plant vascular diseases but also infect immunocompromised individuals, resulting in localized or even life-threatening disseminated infections [2, 3]. Plants are known to produce a large repertoire of specialized metabolites [4, 5], offering great potential in discovering new drugs with distinct modes of action for developing new antifungal drugs [6, 7]. The Plant Cell Culture Library (PCCL) at UMass Amherst contains over thousand plant cell cultures representing ecologically and taxonomically diverse plant species, offering a rich resource of phytochemicals for the discovery of novel compounds with distinct modes of action to control fungal diseases.

In this chapter, we present protocols established for screening plant cell extracts to identify those with antifungal activity, including high-throughput screening assays to evaluate effectiveness of potential antifungals and for obtaining extracts from plant cell lines. We report results of evaluating these protocols against F. oxysporum NRRL 32931, a clinical isolate responsible for a disseminated fusariosis [3].

2. Materials

1. Fungal Strain: F. oxysporum NRRL 32931.

2. Murashige–Skoog (MS) medium: Add 3 g Gamborg’s B5 salts, 30 g sucrose, 4.33 g MS salts, and 15 g agar to 800 mL ddH₂O, and stir until dissolved. Adjust pH to 6.4 and bring to a final volume of 1 L with dd H₂O. Sterilize by autoclaving (see Note 1).

3. Chemicals

   Antifungals: the stock solutions of each commercial anti-fungal were prepared by dissolving weighted material in dimethyl sulfoxide (DMSO).

   1. Amphotericin B stock solution (20 mg/mL): Dissolve 200 mg amphotericin B in 10 mL DMSO, and sterilize by filtration and store at −20 °C.
   2. Fluconazole stock solution (100 mg/mL): Dissolve 1 g fluconazole in 10 mL DMSO, and sterilize by filtration and store at −20 °C.
   3. Nystatin stock solution (3.2 mg/mL): Dissolve 32 mg nystatin in 10 mL DMSO, and sterilize by filtration and store at −20 °C.
   4. Phenamacril stock solution (0.4 mg/mL): Dissolve 4 mg phenamacril in 10 mL DMSO, and sterilize by filtration and store at −20 °C.
   5. Resazurin solution (0.02%, w/v): Dissolve 0.02 g resazurin sodium salt in 100 mL of sterile distilled water, and sterilize by filtration. All the experiments are performed using 0.002% (w/v) as the final concentration of the dye (see Note 2).
   6. 1 mM methyl jasmonate (MJ) elicitation medium: Add 2.22 μL of MJ to 9.998 mL MS liquid medium.

4. Plant Cell Cultures (https://www.umass.edu/ials/pccl-database).

5. Consumables and Other Supplies.

   1. 96-well microplates.
   2. Spectrophotometer for 96-well plates.
   3. Orbital shaker for plates.
   4. Light box.
   5. Microcentrifuge tubes.
   6. Methanol.
   7. 2 mm metal beads.
   8. Tissue homogenizer.
   9. Vacuum centrifuge.
   10. Vortex.
   11. DMSO-compatible sealing foil for microplates.
   12. Bi-chamber transwell plates (with 0.4 μm polycarbonate membranes) (Corning Inc., Corning, NY).

3. Methods

3.1. Cultivation and Preparation of Fungi

1. Inoculate a MS agar plate with a thawed glycerol stock of F. oxysporum from −80 °C freezer on a MS agar plate and incubated at 28 °C for 3 days.

2. Transfer a small piece of the fungal agar into MS liquid media, and place on shaker at 28 °C for 150 rpm to grow biomass.

3. Collect spore suspension by filtering the fungal culture through three layers of miracloth. Concentration of fungal spores is determined by counting the conidia in a hemocytometer counting chamber. All the following experiments are performed using 1 × 10⁶ spores/mL as the final concentration of fungi (see Note 3).

3.2. Developing High-Throughput Screening Assays

We have adapted and validated two high-throughput screening assays, a resazurin cell viability assay, and an optical density assay both of which assess fungal cell viability and activities to investigate antifungal activities against F. oxysporum. Both methods are simple, straightforward, and capable of producing rapid results at a low cost. Four commercial antifungals, amphotericin B (AmB), fluconazole (Flu), nystatin (Nys), and phenamacril (Phe), were used to standardize the assay conditions in positive control wells. Each 96-well plate always includes positive control wells, negative control wells, and treatment wells with replicates. The negative control includes 178 μL MS, 20 μL of 1 × 10⁷ spores/mL F. oxysporum in MS and 2 μL of DMSO (see Note 4).

3.2.1. Resazurin Cell Viability Assay

Resazurin, a water-soluble, non-toxic, non-fluorescent blue dye, has been used as a redox indicator of metabolic activities and viability of cells. This feature enabled us to develop a robust screening assay to assess antifungal property of plant-derived materials against F. oxysporum.

Figure 1 shows two replicates of four commercial antifungal treatment wells. Each well contains 178 μL of MS, 20 μL of 1 × 10⁷ spores/mL F. oxysporum in MS, 2 μL of 100 × antifungal at each concentration (1% v/v) to make the following concentrations: a) Amphotericin B (AmB): 0.064, 0.32, 1.6, 8, 40, 200 μg/mL; b) Fluconazole (Flu): 0.32, 1.6, 8, 40, 200, 1000 μg/mL; c) Nystatin (Nys): 1, 2, 4, 8, 16, 32 μg/mL; and d) Phenamacril (Phe): 0.125, 25, 0.5, 1, 2, 4 μg/mL.

Fig. 1.

Resazurin cell viability assay. After 16-h inoculation, resazurin was added to the assay plate. The image was taken after a 20-min incubation with shaking

The top wells without antifungals are negative controls. The bottom wells are positive controls that lack fungal spores and contain the highest concentrations of antifungal solutions (AmB, 200 μg/mL; Flu, 1000 μg/mL; Nys, 32 μg/mL; Phe, 4 μg/mL)(see Note 5).

The protocol includes the following steps:

1. Incubate the above 96-well plates for 16 h at room temperature with gentile agitation (150 rpm on an orbital shaker).

2. Add ~20 μL of the 0.008% resazurin solution into each well.

3. Wrap the plate with aluminum foil, and incubate on a shaker at 150 rpm for 20 min.

4. Observe the extent of blue resazurin reduction to pink resorufin on the light box (Fig. 1), and make a photographic record.

3.2.2. Optical Density Assay

Monitoring growth using a spectrophotometer is one of the simplest methods to measure microbial growth by tracking changes in the optical density (OD) over time. Critical parameters for the success of this assay include initial spore concentration, culture medium, temperature, and sampling time points. Figure 2 shows three replicates of four commercial fungicide treatments. Each well contains 178 μL MS, 20 μL of 1 × 10⁷ spores/mL F. oxysporum in MS, 2 μL of 100 × antifungal at each concentration (1%, v/v) to make the screening concentration as: a) Amphotericin B (AmB): 0.064, 0.32, 1.6, 8, 40, 200 μg/mL; b) Fluconazole (Flu): 0.32, 1.6, 8, 40, 200, 1000 μg/mL; c) Nystatin (Nys): 1, 2, 4, 8, 16, 32 μg/mL; d) Phenamacril (Phe): 0.125, 0.25, 0.5, 1, 2, 4 μg/mL.

Fig. 2.

Results of optical density assay. Growth curves (optical density, OD = 600 nm) of F. oxysporum on different concentrations of commercial fungicides. Each value was calculated using mean of triplicates for each testing

The protocol includes the following steps:

1. Place the 96-well plate from Subheading 3.2.1 in the spectrophotometer.

2. Set the plate reader to read optical density at 600 nm, at intervals of 1 h.

3. Set temperature control to 25 °C.

4. Incubate without shaking for 16 h.

Results from both the resazurin cell viability and the optical density assays are confirmed using microscopic observation of F. oxysporum viability and potential growth (Fig. 3). Briefly, take 50 μL of the fungal culture under the same treatment as described above from each well onto a glass slide; before loading, mix each well thoroughly to ensure an evenly distributed mixture.

Fig. 3.

Morphological validation. F. oxysporum treated with fungicides: 16-h post inoculation, at 200 × magnification of light microscope

Control: F. oxysporum without antifungal treatment

3.3. Screening Plant Cell Lines for Compounds with Antifungal Properties

In general, plant secondary metabolites are produced at a relatively low level, if any, in cultured cell lines, as primary metabolite precursors are primarily used for biomass accumulation. One of the most effective strategies for enhancing the production of secondary metabolites is elicitation, using agents that act as a signal to stimulate the biosynthesis and accumulation of secondary metabolites [8-10]. We tested the induction of plant secondary metabolites with potential antifungal properties against F. oxysporum by applying methyl jasmonate or live F. oxysporum as the abiotic and biotic elicitors, respectively (see Note 6).

3.3.1. Abiotic Elicitation Using Methyl Jasmonate

Here we report the results of treating randomly selected PCCL cell lines by methyl jasmonate (MJ) (see Note 7).

Preparation of MJ-elicited Plant Callus

1. Transfer 100 mg of plant callus culture into a 2 mL microfuge tube in duplicate (two microfuge tubes per species).

2. Add 1 mL of MJ elicitation medium into one tube and 1 mL MS medium into the other tube.

3. Incubate samples on shaker at 150 rpm for 24 h at room temperature.

Preparation of Cellular Extracts

• 4.Centrifuge all tubes at 5000 × g for 10 min and remove the supernatant.
• 5.Wash the plant cells by adding 1 mL sterile water into elicited/non-elicited tube, and agitate by gentle vortexing of the tubes to break up the cell pellet.
• 6.Spin down the samples at 1000 rpm for 10 min and discard the washings. Repeat the centrifugation step, if necessary, to remove as much liquid as possible.
• 7.Combine the resulting cell pellets by adding 1 mL of methanol and two 2 mm ball bearings to each tube. Homogenize the sample in for 1.5 min at 25 Hz.
• 8.Spin down the samples at 12,000 rpm for 10 min, and transfer the supernatant into a pre-tared microfuge tube.
• 9.Evaporate all volatile components in a vacuum centrifuge.
• 10.Record the weight of residues and re-dissolve in DMSO to a concentration of 20 mg/mL. Vortex and/or sonicate to ensure complete solubilization.
• 11.Centrifuge or filter the resulting solution and transfer to wells of a cryo-compatible multiwell plate; cover the plate with DMSO-compatible sealing foil and store at −20 °C.

Preparing the Plate for Screening

• 12.Mix negative control wells containing 20 μL 1 × 10⁷ spores/mL of F. oxysporum spores in MS, 4 μL DMSO, and 176 μL MS media.
• 13.Mix positive control wells containing 20 μL 1 × 10⁷ spores/mL of F. oxysporum spores in MS, 4 μL 400 μg/mL nystatin, and 176 μL MS media.
• 14.For the treatment wells of the extract(s) to be tested, mix 4 μL of MJ-elicited /non-elicited callus extracts, 20 μL 1 × 10⁷ spores/mL of F. oxysporum spores in MS, and 176 μL of MS media.
• 15.After incubation for 16 h at room temperature, ~20 μL of resazurin solution is applied to each well, and the plate is imaged after a 20 min incubation at room temperature.

3.3.2. Biotic Elicitation Using F. oxysporum in a Bi-chamber Transwell Plate

Here we describe a high-throughput screening method for antifungal plant cell extracts using 96-well bi-chamber transwell plates. In this format, a plant cell culture and a biotic elicitor can be separated by a porous membrane. The pore size ranges from 0.4 to 8.0 μm in diameter for variable permeability. The pores on the membrane are small enough to prevent the physical cell to cell contact across the membrane, but allow for signaling molecules secreted from either cell cultures to diffuse through the membrane enabling bi-directional exchange between chambers (see Notes 7 and 8).

In our experimental setup (Fig. 4), the plant cell cultures are loaded on the top chamber of the transwell plate, and spores of F. oxysporum NRRL 32931 are added to the bottom chamber, with both wells suspended in MS liquid medium. This setup permits diffusion of fungal elicitors, their interaction with the plant cells, and induction of biosynthesis and release of phytochemical compounds with antifungal properties. The antifungal compounds produced by plant cell can be extracted from bottom wells and subjected to metabolomics studies to identify molecules responsible for the antifungal activities.

Fig. 4.

The transwell system. (a) A schematic representation of suspension of microbial cells (blue) in a microwell plate (grey) is combined with an insert containing a porous (0.4 μm) membrane at the bottom of each well (dashed lines). (b) Aliquots of plant cultures are suspended in medium within the insert wells (beige)

Loading Plant Cells in Upper Wells

1. Retrieve plant cell lines from the PCCL.

2. Harvest 0.05 ± 0.005 g of biomass (in three replicates) from each PCCL cell line culture plate, and load into the upper wells of a bi-chamber transwell plate (0.4 μm polycarbonate membrane) in a biosafety cabinet.

3. Each plant cell line has three replicates.

Loading Treatments in Lower Wells

• 4.Set up negative control wells containing 20 μL 1 × 10⁷ spores/ mL of F. oxysporum in MS, 4 μL DMSO, and 176 μL MS media.
• 5.The positive control wells are mixed by adding 20 μL 1 × 10⁷ spores/mL of F. oxysporum in MS, 4 μL 400 μg/mL nystatin in DMSO, and 176 μL MS media.
• 6.For assessment of each tested cell line, add 4 μL DMSO, with 20 μL 1 × 10⁷ spores/mL of F. oxysporum in MS and 76 μL of MS media to the cell line well.

Co-culture Incubation

• 7.The preloaded upper wells are inserted into the plate on top of the bottom wells, and 100 μL of MS media is added onto the upper sample wells to fully submerge the cells (Fig. 4).
• 8.Following the addition of F. oxysporum and the MS media, the transwell plate is covered by the lid, wrapped in aluminum foil, and placed on an orbital shaker set at 100 rpm, for 24 h at room temperature.

Evaluate Antifungal Activity

• 9.After 24 h of co-culture incubation, upper wells containing the plant cells are separated from the bottom wells in a biosafety cabinet.
• 10.Then ~20 μL of resazurin solution is applied to each of the bottom wells, and color changes are imaged (see Note 8).

4. Notes

1. Many available media can be used for growing fungi. However, potato dextrose agar (PDA) medium is too rich to be used in high-throughput screening assays.

2. Due to resazurin being light-sensitive, the resazurin solution should be wrapped in aluminum foil and stored at 4 °C. The resazurin solution is usually made as a 10× stock solution, and diluted before use. To make the experiment efficient and accurate, we suggest preparing a fresh resazurin solution every month.

3. Before loading the sample onto hemacytometer, mix spore suspension thoroughly, and obtain an evenly distributed mixture. Fungal spore suspension is usually made as 1 × 10⁷ spores/mL, and diluted to a final concentration of 1 × 10⁶ spores/mL in the assays.

4. A concentration gradient has been tested to show the effect of DMSO on fungal growth. DMSO (v/v, ≤4%) does not affect fungal growth. The fungal inhibition in the experiment is, therefore, directly caused by the antifungal compounds.

5. Sometimes resazurin changes color due to the reaction with compounds in solution (i.e., antioxidants). Adding compounds with resazurin without any F. oxysporum as control can be used to check for a color change due to assay compounds. If resazurin changes to resorufin (pink) without F. oxysporum, this indicates that the compounds interfere with resazurin, giving false positive results. Hence, it is beneficial to test the compounds separately with resazurin as a control. The optical density assay could be used for such samples.

6. Our preliminary results demonstrate both the validity of the high-throughput screening methods and the great potential of plant natural products in identifying novel antifungals. Both MJ and bi-chamber transwell co-culture tactics were effective in eliciting plant cells to produce phytochemicals with antifungal properties. Such discoveries are much needed to manage the alarming combination of rapidly emerging multidrug-resistant microorganisms, an increasing population of immunocompromised individuals, and cross-border travel in the globalized world, whereby previously manageable infectious diseases could get out of control and become pandemic [11, 12].

7. The consistency of resazurin cell viability assay and optical density assay confirms that these rapid, simple, and robust high-throughput assays can be used to screen for antifungal activities using both commercially available small molecular libraries and novel natural products. Revealed by the MJ elicitation, 10 out of 40 plant cell lines produced a certain level of inhibition of fungal growth. The extracts from MJ-elicited cells showed growth inhibition, as detected by resazurin processing in sample and control wells, with some of them approaching the effect seen in positive control (8 μg/mL nystatin) wells, suggesting that plant secondary metabolite biosynthesis genes can be induced by subjecting plant cells to stress, which are able to produce anti-fusarial compounds (Fig. 5). We also tested 14 plant cell lines using the transwell assay by co-culturing these plant cells with F. oxysporum in 96-well transwell plates. Five hits were identified with anti-fusarial activities (Fig. 6). Interestingly, three of them—IC, Ipomoea costata; EM, Enterolobium multiflorum; and SC, Strychnos colubrine—are identified by both assays. The former two, IC and EM, have displayed the highest anti-fusarial activities, as detected by the resazurin viability assay. These three plant species are reported for the first time to possess components with antifungal properties, while the families to which the three plants belong do contain species that produce antifungal compounds [13-15].

8. In contrast to other elicitation methods, in situ co-culture elicitation is more easily operated than cell suspension cultures because the plant cells are self-immobilized and retained within the culture plate. The antifungal activity is very straightforward, as indicated in Fig. 7. The exact incubation time of resazurin and the assay solutions is dependent on the color change of the negative control. The optimal time is when the initially blue resazurin is fully converted to pink resorufin.

Fig. 5.

Results after abiotic elicitation using methyl jasmonate. High-throughput screening of antifungal drug activity using 40 plant cell lines selected from the PCCL. The first and the last columns are negative (−) and positive (+) controls, respectively; wells in the first four rows of columns 2–11 were loaded with MJ-elicited extracts; wells in the bottom four rows of columns 2–11 were loaded with extracts from the corresponding cell lines without the elicitation. Ten extracts with antifungal property are highlighted with plant cell line IDs: AH, Allocasuarina helmsii; CO, Catalpa ovata; PF, Peperomia fraseri; CT, Costus tappenbeckianus; EM, Enterolobium multiflorum; AC, Agrostis castellana; ES, Eucalyptus saligna; HW, Hadrodemas warszewiczianum; IC, Ipomoea costata; SC, Strychnos colubrine

Fig. 6.

Antifungal screening using transwell co-culture technique. The first column is negative control without antifungals; the last column is positive control with 8 μg/mL nystatin. Each plant species has three replicates. Five hits were found with anti-fungal activities. The strength of the plant species with anti-fusarial activities can be ranked as: IC > EM > SC > HV > EB. IC, Ipomoea costata; EM, Enterolobium multiflorum; SC, Strychnos colubrine; HV, Hordeum vulgare; EB, Eucalyptus bosistoana

Fig. 7.

In situ co-culture elicitation. F. oxysporum strain NRRL 32931 is inoculated in the center of each plate, and different plant cell lines are on the side, showing both top and bottom views