[논문]Resazurin 기반 호흡 측정법을 이용한 고추탄저병균에 대한 살균제의 효과 검정

박수빈; 김흥태

doi:10.5423/rpd.2023.29.1.11

Resazurin 기반 호흡 측정법을 이용한 고추탄저병균에 대한 살균제의 효과 검정
Evaluation of Acitivity of QoI Fungicide against Colletotrichum acutatum s. lat. Causing Pepper Anthracnose Using Resazurin-Based Respiration Assay 원문보기

Research in plant disease = 식물병연구, v.29 no.1, 2023년, pp.11 - 22

박수빈 (충북대학교 농업생명환경대학 식물의학과) , 김흥태 (충북대학교 농업생명환경대학 식물의학과)

초록
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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.

Abstract ▼ AI-Helper

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.

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표/그림 (11)

표 Table 1. Fungicides and their concentrations used in each test method
그림 Fig. 2. Effects of media and spore densities on relative fluorescence unit of Colletotrichum acutatum s. lat. 20JDS8 causing pepper anthracnose by lapse of incubation time. In this experiment, as media were used potato dextrose broth (A) and Czapek Dox broth (B).
그림 Fig. 1. The influence of incubation media and period on relative fluorescence units of PrestoBlue reagent used to monitor the growth of Colletotrichum acutatum s. lat. 20JDS8 causing pepper anthracnose. Data were collected from experiments with 3 replication and expressed as mean±standard error. The readings of relative fluorescence unit were made in fluorescence units at 530/590 nm.
그림 Fig. 3. The growth of Colletotrichum acutatum s. lat. 20JDS8 (A) observed over time after inoculating the pathogen into the media as potato dextrose broth (PDB) and Czapek Dox broth (CDB), and the color change of media (B) treated with PrestoBlue reagent according to incubation time. The spore density of the pathogen inoculated into PDB and CDB, where spore germination and mycelial growth of pathogens were observed under a microscope, was 1×10⁶ spores/ml.
그림 Fig. 4. Inhibitory efficacy of pyraclostrobin against Colletotrichum acutatum s. lat. 20JDS8 causing pepper anthracnose according to the application time. Pyraclostrobin was treated with each indicated concentration at each growth stage of the pathogen such as spore stage, germinating stage of spore, hyphal growth stage and constructing stage of mycelia. The spore stage was the time when the spores were inoculated into the potato dextrose broth (PDB), and the germinating stage of spores was the time when the spores inoculated in the PDB were cultured for 6 hr and more than 70% of the spores germinated. The hyphal growth stage was the period in which germ tubes grew into long hyphae after culturing for 12 hr, and the constructing stage of mycelia was the period when the wells of the microtiter plate were filled with mycelia. Respiration of the pathogen was measured after being treated with a fungicide and then incubated for another 24 hr. Relative fluorescence unit was measured by adjusting the excitation and emission wavelengths to 530 nm and 590 nm, 1 hr after treatment with PrestoBlue reagent.
그림 Fig. 5. Inhibitory efficacy of dithianon (A), isopyrazam (B), and fluazinam (C) against Colletotrichum acutatum s. lat. 20JDS8 causing pepper anthracnose according to the application time. Fungicides were treated with each indicated concentration at each growth stage such as spore stage, germinating stage of spore, hyphal growth stage and constructing stage of mycelia. Respiration of the pathogen was measured after being treated with a fungicide and then incubated for another 24 hr. One hour after treatment with PrestoBlue reagent, relative fluorescence unit was measured by adjusting the excitation and emission wavelengths to 530 nm and 590 nm.
그림 Fig. 6. Inhibitory efficacy of tebuconazole (A) and prochloraz (B) against Colletotrichum acutatum s. lat. 20JDS8 causing pepper anthracnose according to the application time. Fungicides were treated with each indicated concentration at each growth stage such as spore stage, germinating stage of spore, hyphal growth stage and constructing stage of mycelia. Respiration of the pathogen was measured after being treated with a fungicide and then incubated for another 24 hr. One hour after treatment with PrestoBlue reagent, relative fluorescence unit was measured by adjusting the excitation and emission wavelengths to 530 nm and 590 nm.
그림 Fig. 7. Inhibitory effect of fungicides on mycelial growth of Colletotrichum acutatum s. lat. 20JDS8 sensitive to pyraclostrobin and 20CDJ6 resistant to pyraclostrobin on potato dextrose agar amended with each fungicide. Inhibition ratio (%) of mycelial growth was evaluated by using an agar dilution method. (A) Pyraclostrobin. (B) Isopyrazam. (C) Fluazinam. (D) Dithianon. (E) Tebuconazole. (F) Prochloraz.
그림 Fig. 8. Response of Colletotrichum acutatum causing pepper anthracnose to pyraclostrobin according to the assay method. In this experiment, C. acutatum 20JDS8 sensitive to pyraclostrobin and 20CDJ6 resistant to the fungicide, were used. Numbers in each assay represent the concentrations (μg/ml) of pyraclostrobin. (A) Agar dilution method investigating the mycelial growth inhibitory effect of pyraclostrobin. (B) Respiration assay method using PrestoBlue reagent. (C) Microscopic observation for investigating the change of pathogen morphology. In the agar dilution method, pathogens were inoculated on potato dextrose agar medium amended with fungicide at indicated concentrations, and cultured at 25°C for 5 days. Then the diameter of the mycelial colonies were measured. In the respiration assay method, the pathogens were treated with pyraclostrobin by indicated concentrations at that time when the pathogens were inoculated into potato dextrose broth. After culturing the pathogen for 24 hr, the PrestoBlue reagent was treated for 1 hr and fluorescence was measured. In light microscopy, it was observed after treatment with pyraclostrobin and incubation for 24 hr.
그림 Fig. 9. Respiratory inhibition effect of Colletotrichum acutatum s. lat. 20JDS8 (A, B) 20CDJ6 (C, D) to pyraclostrobin using PrestoBlue assay. C. acutatum s. lat. 20JDS8 was sensitive to pyraclostrobin, and 20CDJ6 resistant the fungicide. Pyraclostrobin was treated at indicated concentrations at the same time as the spores of the pathogen were inoculated into the potato dextrose broth (A, C) and after 12 hr of incubation after the spores were inoculated (B, D). The pathogen was cultured for 24 hr after treatment with the fungicide. For measuring relative fluorescence unit (RFU), PrestoBlue reagent was treated after 24 hr of incubation, and RFU was measured 1 hr after treatment with the reagent. The respiratory inhibition effect was calculated by comparing the RFU treated with the fungicide to that of the untreated control.
그림 Fig. 10. Inhibitory effect (%) of strobilurin fungicides included into quinone outside inhibitors on the cell respiration of Colletotrichum acutatum 20JDS8 (A-C) and 20CDJ6 (D-F). Fungicides were used in this experiment as azoxystrobin (A, D), trifloxysytrobin (B, E), and kresoximmethyl (C, F). Isolates of the pepper pathogen used in this experiment were C. acutatum s. lat. 20JDS8, which is sensitive to pyraclostrobin, and C. acutatum s. lat. 20CDJ6, which is resistant.

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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