Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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Evaluation of Acitivity of QoI Fungicide against Colletotrichum acutatum s. lat. Causing Pepper Anthracnose Using Resazurin-Based Respiration Assay

Resazurin 기반 호흡 측정법을 이용한 고추탄저병균에 대한 살균제의 효과 검정

  • Subin Park (Department of Plant Medicine, College of Agriculture, Life & Environment Sciences, Chungbuk National University) ;
  • Heung Tae Kim (Department of Plant Medicine, College of Agriculture, Life & Environment Sciences, Chungbuk National University)
  • 박수빈 (충북대학교 농업생명환경대학 식물의학과) ;
  • 김흥태 (충북대학교 농업생명환경대학 식물의학과)
  • Received : 2023.02.02
  • Accepted : 2023.03.06
  • Published : 2023.03.31
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Abstract

Resazurin-based microtiter assay was used to evaluate the inhibitory effect of fungicides on the respiration of Colletotrichum acutatum s. lat. 20JDS8 sensitive and 20CDJ6 resistant to strobilurin fungicides. The spores of C. acutatum s. lat. 20JDS8 were inoculated into potato dextrose broth (PDB) at densities of 1x104, 1x105 and 1x106 spores/ml, respectively. The relative fluorescence unit (RFU) of all treatments inoculated at each spore density started to rise after 12 hr of incubation, and were 1,965.5, 5,412.5, and 10,061.0, respectively, after 24 hr of incubation. To evaluate the inhibitory effect of fungicide on the respiration of the pathogen, the spores of the pathogen were inoculated into the PDB and treated with the fungicides 0, 6, 12, and 24 hr after incubation, respectively. After keeping the pathogen culturing for another 24 hr, PrestoBlue reagent was treated into the PDB culturing the pathogen. The RFU of each treatment was examined 1 hr after the reagent was treated. When dithianon, isopyrazam, pyraclostrobin, and fluazinam were treated at high concentrations in the stages of spores (immediately after inoculation [0 hr]), spore germination (after incubation for 6 hr), and hyphal growth (after incubation for 12 hr), the respiration of pathogens was inhibited by 90-100%. When the fungicides were treated after culturing the pathogen for 24 hr, the respiratory inhibitory effects were greatly reduced. With pyraclostrobin-resistant C. acutatum s. lat. 20CDJ6, azxoystrobin, trifloxystrobin and kresoxim-methyl, which have the same mode of action, had very little or no respiratory inhibitory effect in all growth stages of pathogens. Based on the above results, it was thought that the resazurin-based microtiter assay could quickly and accurately evaluate the inhibitory efficacy of the fungicides that inhibited respiration.

Resazurin 기반 호흡 측정법으로 strobilurin계 살균제에 대해서 감수성인 고추탄저병균 Colletotrichum acutatum s. lat. 20JDS8에 대한 살균제의 호흡 억제 효과를 조사하였다. Potato dextrose broth에 병원균의 포자를 접종하고 배양하면서 시간별로 상대 형광값(relative fluorescence unit)을 조사한 결과, 12시간 후부터 상승하기 시작하여 24시간 후의 1×104, 1×105, 1×106 spores/ml의 포자 접종구에서 상대 형광값은 1,965.5, 5,412.5, 10,061.0이었다. 병원균을 0, 6, 12, 24시간씩 배양 후에 공시한 살균제를 처리하고, 24시간 후에 상대 형광값을 조사하였다. Dithianon, isopyrazam, pyraclostrobin, fluazinam을 병원균의 포자(0시간), 포자 발아(6시간), 균사 생장(12시간) 단계에 처리할 경우, 각 살균제의 고농도에서 호흡을 90-100% 억제하였다. 하지만 병원균을 24시간 배양한 후에 살균제를 처리할 경우에는 호흡 억제 효과가 크게 감소하였다. Pyraclostrobin 저항성인 C. acutatum s. lat. 20CDJ6에 대해서 pyraclostrobin, azxoystrobin, trifloxystrobin, kresoxim-methyl을 병원균의 모든 생장 단계에 각각 처리하였을 때, 호흡에 대한 억제 효과는 매우 미미하거나, 나타나지 않았다.

Keywords

Acknowledgement

This work was supported by the Korean Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through the Crop Viruses and Pests Response Industry Technology Development Program, funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; Grant No. 320042-5).

References

  1. Ayyagari, V. N., Hsieh, T.-H. J., Diaz-Sylvester, P. L. and Brard, L. 2017. Evaluation of the cytotoxicity of the Bithionol - cisplatin combination in a panel of human ovarian cancer cell lines. BMC Cancer 17: 49.
  2. Boncler, M., Rozalski, M., Krajewska, U., Podsedek, A. and Watala, C. 2014. Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells. J. Pharmacol. Toxicol. Methods 69: 9-16. https://doi.org/10.1016/j.vascn.2013.09.003
  3. Emter, R. and Natsch, A. 2015. A fast Resazurin-based live viability assay is equivalent to the MTT-test in the KeratinoSens assay. Toxicol. In Vitro 29: 688-693. https://doi.org/10.1016/j.tiv.2015.02.003
  4. Espinel-Ingroff, A. and Kerkering, T. M. 1991. Spectrophotometric method of inoculum preparation for the in vitro susceptibility testing of filamentous fungi. J. Clin. Microbiol. 29: 393-394. https://doi.org/10.1128/jcm.29.2.393-394.1991
  5. Hsieh, C.-H., Chung, W.-C., Chen, Y.-N. and Chung, W.-H. 2013. Phylogenetic diversity and sensitivity to MBI and QoI fungicides of Magnaporthe oryzae in Taiwan. J. Pestic. Sci. 38: 194-199. https://doi.org/10.1584/jpestics.D12-059
  6. Isa, D. A. and Kim, H. T. 2022. Cytochrome b gene-based assay for monitoring the resistance of Colletotrichum spp. to pyraclostrobin. Plant Pathol. J. 38: 616-628. https://doi.org/10.5423/PPJ.OA.06.2022.0081
  7. Kamiloglu S., Sari, G., Ozdal, T. and Capanoglu, E. 2020. Guidelines for cell viability assays. Food Front. 1: 332-349. https://doi.org/10.1002/fft2.44
  8. Kim, S., Min J. and Kim, H. T. 2019. Occurrence and mechanism of fungicide resistance in Colletotrichum acutatum causing pepper anthracnose against pyraclostrobin. Korean J. Pestic. Sci. 23: 202-211. https://doi.org/10.7585/kjps.2019.23.3.202
  9. Kim, Y.-S., Dixon, E. W., Vincelli, P. and Farman, M. L. 2003. Field resistance to strobilurin (QoI) fungicides in Pyricularia grisea caused by mutations in the mitochondrial cytochrome b gene. Phytopathology 93: 891-900. https://doi.org/10.1094/PHYTO.2003.93.7.891
  10. Lall, N., Henley-Simith, C. J., De Canha, M. N., Oosthuizen, C. B. and Berrington, D. 2013. Viability reagent, PrestoBlue, in comparison with other available reagents, utilized in cytotoxicity and antimicrobial assays. Int. J. Microbiol. 2013:420601.
  11. Lee, S. M., Jang, H. S. and Kim, H. T. 2014. In vitro fruit assay for the evaluation of fungicide activity against pepper anthracnose. Korean J. Pestic. Sci. 18: 115-121. (In Korean) https://doi.org/10.7585/kjps.2014.18.2.115
  12. Luzak, B. Siarkiewicz, P. and Boncler, M. 2022. An evaluation of a new high-sensitivity PrestoBlue assay for measuring cell viability and drug cytotoxicity using EA.hy926 endothelial cells. Toxicol. In Vitro 83: 105407.
  13. Ma, Z. and Michailides, T. J. 2005. Advances in understanding molecular mechanisms of fungicide resistance and molecular detection of resistant genotypes in phytopathogenic fungi. Crop Prot. 24: 853-863. https://doi.org/10.1016/j.cropro.2005.01.011
  14. Miret, S., De Groene, E. M. and Klaffke, W. 2006. Comparison of in vitro assays of cellular toxicity in the human hepatic cell line HepG2. J. Biomol. Screen. 11: 184-193. https://doi.org/10.1177/1087057105283787
  15. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55-63. https://doi.org/10.1016/0022-1759(83)90303-4
  16. Oosthuizen, C., Arbach, M., Meyer, D., Hamilton, C. and Lall, N. 2017. Diallyl polysulfides from Allium sativum as immunomodulators, hepatoprotectors, and antimycobacterial agents. J. Med. Food 20: 685-690. https://doi.org/10.1089/jmf.2016.0137
  17. Park, S. and Kim, H. T. 2022. Cross-resistance of Colletotrichum acutatum s. lat. to strobilurin fungicides and inhibitory effect of fungicides with other mechanisms on C. acutatum s. lat. resistant to pyraclostrobin. Res. Plant Dis. 28: 122-131. (In Korean) https://doi.org/10.5423/RPD.2022.28.3.122
  18. Park, S.-J., Lee, S.-M., Gwon, H.-W., Lee, H. and Kim, H. T. 2014. Control efficacy of Bordeaux mixture against pepper anthracnose. Korean J. Pestic. Sci. 18: 168-174. (In Korean) https://doi.org/10.7585/kjps.2014.18.3.168
  19. Pasche, J. S., Piche, L. M. and Gudmestad, N. C. 2005. Effect of the F129L mutation in Alternaria solani on fungicides affecting mitochondrial respiration. Plant Dis. 89: 269-278. https://doi.org/10.1094/PD-89-0269
  20. Pijls, C. F. N., Shaw, M. W. and Parker, A. 1994. A rapid test to evaluate in vitro sensitivity of Septoria tritici to flutriafol, using a microtitre plate reader. Plant Pathol. 43: 726-732. https://doi.org/10.1111/j.1365-3059.1994.tb01612.x
  21. Rampersad, S. N. and Teelucksingh, L. D. 2012. Differential responses of Colletotrichum gloeosporioides and C. truncatum isolates from different hosts to multiple fungicides based on two assays. Plant Dis. 96: 1526-1536. https://doi.org/10.1094/PDIS-10-11-0906-RE
  22. Slawecki, R. A., Ryan, E. P. and Young, D. H. 2022. Novel fungitoxicity assays for inhibition of germination-associated adhesion of Botrytis cinerea and Puccinia recondita spores. Appl. Environ. Microbiol. 68: 597-601. https://doi.org/10.1128/AEM.68.2.597-601.2002
  23. Spiegel, J. and Stammler, G. 2006. Baseline sensitivity of Monilinia laxa and M. fructigena to pyraclostrobin and boscalid. J. Plant Dis. Prot. 113: 199-206. https://doi.org/10.1007/BF03356181
  24. Vega, B., Liberti, D., Harmon, P. F. and Dewdney, M. M. 2012. A rapid resazurin-based microtiter assay to evaluate QoI sensitivity for Alternaria alternata isolates and their molecular characterization. Plant Dis. 96: 1262-1270. https://doi.org/10.1094/PDIS-12-11-1037-RE
  25. Vega-Avila, E. and Pugsley, M. K. 2011. An overview of colorimetric assay methods used to assess survival or proliferation of mammalian cells. Proc. West. Pharmacol. Soc. 54: 10-14.