Development and Validation of a HTS Platform for the Discovery of New Antifungal Agents against Four Relevant Fungal Phytopathogens
Abstract
:1. Introduction
2. Materials and Methods
2.1. Fungal Strains and Conidia Production
2.2. Agar-Based Assays against Fungal Phytopathogens
2.3. Liquid-Based Assays against Fungal Phytopathogens
2.4. Validation of Assay Conditions
2.5. Data Analysis
3. Results and Discussion
3.1. Induction of the Sporulation of Fungal Phytopathogens
3.2. Botrytis cinerea Agar-Based Assay
3.3. Botrytis cinerea Microdilution Assay
3.4. Colletotrichum acutatum Microdilution Assay
3.5. Fusarium proliferatum Microdilution Assay
3.6. Magnaporthe grisea Microdilution Assay
3.7. Sensitivity and Resistance to Antifungal Standards
3.8. Antifungal Potential of Microbial Extract Libraries
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Culture Media | Concentration (conidia/mL) | |||
---|---|---|---|---|
B. cinerea | C. acutatum | F. proliferatum | M. grisea | |
Solid Culture Media | ||||
CMA | - | - | 2.4 × 107 ± 1.3 | 2.7 × 108 ± 1.0 |
MEA | 5.9 × 105 ± 1.9 | 1.3 × 106 ± 0.9 | 1.2 × 108 ± 1.0 | 9.3 × 107 ± 1.2 |
OAT | 3.7 × 107 ± 1.5 | 4.6 × 107 ± 3.4 | 1.9 × 107 ± 0.6 | - |
PDA | 6.7 × 105 ± 2.9 | 3.3 × 107 ± 1.7 | 1.3 × 108 ± 0.5 | 1.3 × 108 ± 0.8 |
YM | 8.3 × 105 ± 2.9 | 8.8 × 107 ± 1.7 | 1.4 × 108 ± 0.8 | 1.9 × 108 ± 0.9 |
Liquid Culture Media | ||||
SDB | - | 5.3 × 106 ± 3.3 | 9.0 × 107 ± 1.4 | 9.3 × 107 ± 2.3 |
SMY | - | 7.8 × 107 ± 1.9 | 2.5 × 108 ± 0.4 | 1.9 × 108 ± 1.1 |
PDB | - | 2.0 × 106 ± 0.4 | 8.7 × 107 ± 1.0 | 9.7 × 107 ± 2.4 |
GMS | - | 8.7 × 106 ± 2.1 | 8.7 × 107 ± 1.3 | 1.5 × 108 ± 0.6 |
VME | - | 2.9 × 107 ± 1.3 | 1.4 × 108 ± 0.5 | 2.3 × 108 ± 0.9 |
Parameters | Solid Medium | Liquid Medium |
---|---|---|
Time of incubation | 10–21 days | 24–72 h |
Volume of inoculum | 200 mL (five Petri plates) | 16 mL (one test tube) |
Time to obtain conidia solution | 1 h (Scraping + Filtration) | 30 min (Filtration) |
Concentration of Inoculum | Absorbance | Fluorescence | ||
---|---|---|---|---|
S/B | RZ’ | S/B | RZ’ | |
5 × 105 conidia/mL | 15.31 | 0.76 | 11.74 | 0.76 |
1 × 106 conidia/mL | 37.40 | 0.77 | 23.19 | 0.90 |
5 × 106 conidia/mL | 2.33 | <0 | 7.65 | 0.55 |
Parameters | Botrytis cinerea B05.10 | Colletotrichum acutatum CF-137177 | Fusarium proliferatum CBS 115.97 | Magnaporthe grisea CF-105765 |
---|---|---|---|---|
Sporulation condition | OAT plates | SMY tube | SMY tube | VME tube |
Time for sporulation | 14–28 days | 3 days | 2 days | 3 days |
Assay medium | RPMI-1640 | RPMI-1640 | RPMI-1640 | RPMI-1640 |
Concentration of conidia | 1 × 106 conidia/mL | 1 × 106 conidia/mL | 1 × 106 conidia/mL | 1 × 106 conidia/mL |
Tolerance to DMSO | 2% | 2% | 4% | 2% |
Growth inhibition assay (absorbance readout) | ||||
Incubation time | 72 h | 40 h | 20 h | 48 h |
RZ’ (n = 240 wells) | 0.80 ± 0.05 | 0.75 ± 0.01 | 0.77 ± 0.05 | 0.81 ± 0.03 |
S/B (n = 240 wells) | 48.95 ± 10.10 | 53.05 ± 5.50 | 57.71 ± 7.04 | 54.21 ± 5.89 |
IC50 AmB (µg/mL) | 0.45 (0.33–0.60) | 1.25 (1.12–1.33) | 6.77 (6.11–7.51) | 4.51 (4.33–4.69) |
Viability inhibition assay (fluorescence readout) | ||||
Incubation time (resazurin) | 5–15 min | 3 h | 2 h | 1 h 30 min |
RZ’ (n = 240 wells) | 0.94 ± 0.03 | 0.91 ± 0.01 | 0.94 ± 0.01 | 0.95 ± 0.01 |
S/B (n = 240 wells) | 24.98 ± 4.90 | 17.01 ± 1.35 | 17.54 ± 1.76 | 13.62 ± 1.65 |
IC50 of AmB (µg/mL) | 0.27 (0.25–0.30) | 1.21 (1.19–1.23) | 6.75 (6.41–7.12) | 5.38 (5.30–5.46) |
Chemical Class | Compound | Mode of Action/ Target Site | Botrytis cinerea | Colletotrichum acutatum | Fusarium proliferatum | Magnaporthe grisea | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Agar | Abs | Flu | Agar | Abs | Flu | Agar | Abs | Flu | Agar | Abs | Flu | |||
MIC | IC50 | IC50 | MIC | IC50 | IC50 | MIC | IC50 | IC50 | MIC | IC50 | IC50 | |||
Acylalanines | benalaxyl | nucleic acids metabolism/RNA polymerase I | i | i | i | i | i | i | i | i | i | i | i | i |
metalaxyl | i | i | i | i | i | i | i | i | i | i | i | i | ||
Anilino- pyrimidines | cyprodinil | protein synthesis | 7.81 | 0.06 (0.05–0.07) | 0.29 (0.24–0.35) | 15.63 | >20 | 14.99 (11.69–19.20) | i | 8.28 (7.12–9.62) | 14.33 (13.88–15.35) | i | 16.02 (13.11–19.57) | >20 |
Benzimidazoles | benomyl | cytoskeleton and motor proteins/tubulin | 62.5 | 0.06 (0.05–0.07) | 0.06 (0.05–0.06) | 125 | 0.35 (0.33–0.38) | 0.46 (0.43–0.49) | 500 | 0.98 (0.88–1.09) | 0.95 (0.88–1.03) | 1000 | 1.64 (1.51–1.78) | 1.33 (1.29–1.37) |
thiabendazole | 250 | 0.24 (0.21–0.28) | 0.25 (0.21–0.30) | 62.5 | 10.71 (9.66–11.87) | >20 | 1000 | 2.15 (1.95–2.36) | 1.99 (1.84–2.15) | - | 18.57 (17.86–19.31) | 16.39 (16.04–16.75) | ||
Cinnamic acid amides | dimethomorph | cell wall biosynthesis/ cellulose synthase | i | i | i | i | i | i | i | i | i | i | i | i |
Cyano-methylene thiazolidines | flutianil | unknown | i | i | i | i | i | i | i | i | i | i | i | i |
Cyclic nonribosomal peptides | cyclosporin A | signal transduction | 125 | 1.27 (0.92–1.74) | 2.20 (1.66–2.93 | i | 3.87 (3.198–4.70) | 10.84 (10.27–11.44) | i | i | i | i | i | i |
Dicarboximide | cycloheximide | protein synthesis | 250 | 8.96 (7.27–11.05) | 4.91 (4.05–5.96) | 250 | 0.57 (0.49–0.66) | 1.67 (1.53–1.82) | 125 | 1.29 (1.07–1.56) | 1.21 (1.11–1.31) | 250 | 7.94 (7.44–8.48) | 5.07 (4.90–5.25) |
Dithio-carbamates | ferbam | multi-site contact | 250 | 0.30 (0.28–0.32) | 0.37 (0.36–0.38) | 62.5 | 0.24 (0.22–0.25) | 0.66 (0.63–0.70) | 125 | 1.23 (1.08–1.41) | 1.84 (1.75–1.94) | i | 0.45 (0.43–0.47) | 0.28 (0.28–0.29) |
Ethylamino- thiazolecarboxamide | ethaboxam | cytoskeleton and motor proteins/tubulin | i | i | i | i | i | i | i | i | i | i | i | i |
Glucopyranosyl antibiotics | validamycin | unknown/trehalase | i | i | i | i | i | i | i | i | i | i | i | i |
Imidazolone | triflumizole | sterol biosynthesis/ C14-demethylase | 62.5 | <0.04 | <0.04 | 15.63 | <0.04 | <0.04 | 7.81 | 0.26 (0.22–0.32) | 0.28 (0.23–0.35) | 31.25 | 0.05 (0.04–0.06) | <0.04 |
fenamidone | respiration/complex III: cyt bc1 at Qo site | i | i | i | 500 | 11.33 (10.25–12.51) | >20 | i | i | i | 500 | 9.45 (8.79–10.16) | 1.94 (1.85–2.02) | |
Macrocyclic | radicicol | histidine kinase/Hsp90 | i | i | i | i | i | i | i | i | i | i | i | i |
Mandelic acid amides | mandipropamid | cell wall biosynthesis/ cellulose synthase | i | i | i | i | i | i | i | i | i | i | i | i |
Monocarboxylic acid amide | cerulenin | fatty acid biosynthesis/ b-ketoacyl-acyl carrier protein synthase | i | i | i | i | 6.70 (5.64–7.96) | 11.98 (11.47–12.51) | i | i | i | i | i | i |
Mycotoxin | cytochalasin B | cell division | i | i | i | i | i | i | i | i | i | i | i | i |
N-methoxy- (phenylethyl)- pyrazolecarboxamides | pydiflumetofen | respiration/complex II: succinate-dehydrogenase | 15.63 | 0.07 (0.07–0.08) | 0.12 (0.11–0.13) | i | 6.32 (5.32–7.50) | 2.08 (1.89–2.29) | 15.63 | <0.04 | <0.04 | 15.62 | 0.14 (0.11–0.18) | <0.04 |
Nucleoside | tubercidin | protein synthesis | i | i | i | i | 12.25 (9.69–15.49) | >20 | i | i | i | i | i | i |
tunicamycin | 125 | 2.62 (1.79–3.84) | 2.55 (2.34–2.77) | 500 | 4.05 (3.55–4.63) | 6.71 (5.26–7.20) | i | i | i | i | i | i | ||
Phenyl-oxo- ethylthiophene amide | isofetamid | respiration/complex II: succinate-dehydrogenase | 31.25 | 0.16 (0.13–0.19) | 0.40 (0.34–0.45) | i | i | i | i | 14.00 (10.95–17.91) | 19.94 (16.92–23.50) | 62.5 | 1.49 (1.26–1.77) | 0.23 (0.21–0.26) |
Phenylpyrroles | fludioxonil | signal transduction/ MAP/Histidine Kinase | 62.5 | <0.04 | 0.07 (0.07–0.07) | 31.25 | 0.07 (0.07–0.07) | 0.07 (0.07–0.07) | 31.25 | 0.09 (0.09–0.11) | <0.04 | 125 | 0.22 (0.21–0.24) | 0.09 (0.09–0.09) |
Picolinamides | fenpicoxamid | respiration/complex III: cyt bc1 at Qi site | 62.5 | 0.07 (0.06–0.08) | 0.43 (0.32–0.57) | 15.63 | i | i | i | i | i | 62.5 | 2.04 (1.61–2.59) | 0.40 (0.39–0.42) |
Pyridinecarboxamides | boscalid | respiration/complex II: succinate-dehydrogenase | 125 | 0.33 (0.30–0.36) | 0.70 (0.65–0.74) | i | i | i | i | i | i | i | i | i |
Pyridinyl- ethylbenzamides | fluopyram | 125 | 0.28 (0.25–0.32) | 1.27 (1.17–1.37) | i | i | i | i | 5.77 (4.08–8.15) | 12.24 (10.49–14.28) | i | >20 | 17.28 (16.02–18.64) | |
Strobirulin | azoxystrobin | respiration/complex III: cyt bc1 at Qo site | 250 | 0.06 (0.06–0.07) | 0.06 (0.05–0.07) | 62.5 | 0.05 (0.04–0.05) | 0.09 (0.09–0.09) | 31.25 | 0.39 (0.32–0.49) | 0.22 (0.19–0.26) | 62.5 | 0.10 (0.09–0.11) | 0.08 (0.07–0.08) |
mandestrobin | 250 | <0.04 | 0.78 (0.69–0.87) | 250 | 0.12 (0.11–0.13) | 0.08 (0.08–0.09) | 31.25 | 0.12 (0.11–0.13) | 0.04 (0.04–0.05) | 500 | 2.27 (2.07–2.50) | 0.82 (0.79–0.85) | ||
pyraclostrobin | 31.25 | <0.04 | 0.07 (0.05–0.09) | 3.91 | <0.04 | <0.04 | 7.81 | 0.06 (0.05–0.07) | <0.04 | 1.95 | <0.04 | <0.04 | ||
trifloxystrobin | 31.25 | 0.88 (0.51–1.50) | 1.99 (1.66–2.40) | 7.81 | 6.41 (5.83–7.05) | 6.43 (6.06–6.81) | 7.81 | 7.23 (6.44–8.12) | 7.79 (6.59–9.21) | 3.90 | 1.83 (1.52–2.20) | 1.59 (1.49–1.70) | ||
cyproconazole | sterol biosynthesis/ C14-demethylase | 500 | 0.34 (0.27–0.43) | 0.39 (0.35–0.44) | 62.5 | 0.68 (0.61–0.76) | 2.08 (1.93–2.22) | i | 1.76 (1.17–2.64) | 1.58 (1.37–1.81) | i | i | i | |
Triazoles | difenoconazole | 250 | 0.44 (0.39–0.49) | 0.81 (0.67–0.97) | 250 | 0.16 (0.14–0.17) | 0.61 (0.56–0.67) | 125 | 0.70 (0.52–0.96) | 0.38 (0.32–0.45) | 1000 | 11.89 (11.34–12.47) | 7.78 (7.34–8.25) | |
fenbuconazole | 500 | 0.20 (0.18–0.22) | 0.43 (0.38–0.49) | i | 0.23 (0.20–0.27) | 0.58 (0.52–0.64) | i | 1.82 (1.41–2.37) | 1.39 (1.15–1.67) | i | i | i | ||
itraconazole | 62.5 | 0.31 (0.19–0.51) | 0.31 (0.29–0.33) | 62.5 | 0.12 (0.11–0.13) | 0.63 (0.58–0.70) | 62.5 | 0.53 (0.41–0.68) | 0.74 (0.62–0.87) | i | i | i | ||
mefentrifluconazole | 250 | 0.07 (0.06–0.08) | 0.11 (0.10–0.12) | 62.5 | 0.11 (0.09–0.12) | 0.28 (0.23–0.34) | 1000 | 0.55 (0.42–0.71) | 0.22 (0.20–0.25) | 1000 | i | i | ||
metconazole | 250 | 0.06 (0.05–0.05) | 0.10 (0.09–0.11) | 62.5 | <0.04 | 0.05 (0.05–0.06) | 31.25 | <0.04 | <0.04 | 500 | 4.15 (3.86–4.47) | 4.16 (3.91–4.41) | ||
tetraconazole | i | 12.52 (7.73–20.29) | 5.40 (4.96–5.88) | i | 1.30 (0.97–1.75) | 3.16 (2.89–3.46) | i | 8.02 (6.26–10.29) | 9.07 (8.29–9.92) | i | i | i | ||
flutriafol | i | 11.02 (9.69–12.55) | >20 | i | 6.82 (6.26–7.43) | 6.48 (6.16–6.82) | i | 3.19 (2.79–3.64) | 3.38 (3.09–3.70) | i | i | i | ||
Valinamide carbamates | iprovalicarb | cell wall biosynthesis/ cellulose synthase | i | i | i | i | i | i | i | i | i | i | i | i |
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Serrano, R.; González-Menéndez, V.; Tormo, J.R.; Genilloud, O. Development and Validation of a HTS Platform for the Discovery of New Antifungal Agents against Four Relevant Fungal Phytopathogens. J. Fungi 2023, 9, 883. https://doi.org/10.3390/jof9090883
Serrano R, González-Menéndez V, Tormo JR, Genilloud O. Development and Validation of a HTS Platform for the Discovery of New Antifungal Agents against Four Relevant Fungal Phytopathogens. Journal of Fungi. 2023; 9(9):883. https://doi.org/10.3390/jof9090883
Chicago/Turabian StyleSerrano, Rachel, Víctor González-Menéndez, José R. Tormo, and Olga Genilloud. 2023. "Development and Validation of a HTS Platform for the Discovery of New Antifungal Agents against Four Relevant Fungal Phytopathogens" Journal of Fungi 9, no. 9: 883. https://doi.org/10.3390/jof9090883