[논문]콩균핵마름병균에 대한 병원성 검정법 확립

안소현; 김흥태

doi:10.5423/rpd.2023.29.1.1

콩균핵마름병균에 대한 병원성 검정법 확립
Establishment of Pathogenicity Test Method for Macrophomina phaseolina Causing Soybean Charcoal Rot 원문보기

Research in plant disease = 식물병연구, v.29 no.1, 2023년, pp.1 - 10

안소현 (충북대학교 농업생명환경대학 식물의학과) , 김흥태 (충북대학교 농업생명환경대학 식물의학과)

초록
AI-Helper

콩 균핵마름병을 일으키는 Macrophomina phaseolina의 병원성 평가를 위하여 실험실과 온실 검정법을 확립하였다. 실험실 검정에서는 소립균핵과 균사를 접종원으로 사용하였다. 소립균핵을 접종원으로 사용한 실험실 검정에서 M. phaseolina NSW17-108과 HSM17-034의 발병도는 25℃보다 35℃에서 더 높았다. NSW17-108과 HSM17-034 중에서 참깨에서 분리된 HSM17-034의 발병도가 콩에서 분리된 NSW17-108보다 높았다. M. phaseolina의 균사를 접종원으로 사용한 경우, NSW17-108과 HSM17-034는 35℃에서의 발병도가 접종 5일 만에 80%를 상회하였다. HSM17-034는 25℃에서의 발병도가 접종 5일 후에 80%를 상회하였다. 온실의 병원성 검정에는 소립균핵이 형성된 이쑤시개 또는 potato dextrose agar 배지에서 수확한 소립균핵 자체를 접종원으로 사용하였다. 모든 온실 검정에서 M. phaseolina NSW17-10과 HSM17-034는 접종 방법에 따라 병원균을 접종하고 35-65일 후에 40-60%의 발병도를 보였다. 두 균주 중에서 HSM17-034의 병원성이 NSW17-108보다 강했으며, 이 결과는 실험실 검정 결과와 일치하였다. 본 연구에서 확립한 실험실 및 온실 검정법은 각 방법에 따라 장단점이 있기 때문에, 연구의 목적에 부합할 수 있는 시험법을 선택하여 사용하는 것이 필요하다.

Abstract ▼ AI-Helper

The establishment of a laboratory assay and a greenhouse assay was conducted for evaluating the pathogenicity of Macrophomina phaseolina causing soybean charcoal rot established. In the laboratory assay, microsclerotia and hyphae were used as inoculum. In the laboratory assays using microsclerotia as an inoculum, disease incidences of M. phaseolina NSW17-108 and HSM17-034 were higher at 35℃ than 25℃. Of the two isolates NSW17-108 and HSM17-034, the disease incidence of HSM17-034 isolated from diseased sesame is higher than that of NSW17-108 isolated from diseased soybean. When the mycelia of M. phaseolina were used as an inoculum, the disease incidence of NSW17-108 and HSM17-034 at 35℃ exceeded 80% even after only 5 days of inoculation. Even at 25℃, furthermore, that of HSM17-034 exceeded 80% 5 days later. In the pathogenicity assays at a greenhouse, toothpicks where microsclerotia were produced or microsclerotia harvested from potato dextrose agar medium were used as an inoculum. In all greenhouse assays, M. phaseolina NSW17-108 and HSM17-034 showed 40-60% of disease incidences 35-65 days after inoculation with the pathogen, depending on the inoculation method. Between the two isolates, the pathogenicity of HSM17-034 was stronger than that of NSW17-108, and this result was consistent with laboratory assay results. Since the laboratory and greenhouse test methods tested in this study have different advantages and disadvantages depending on each test method, it is thought that the test method that can meet the purpose of the study should be selected and used.

주제어

표/그림 (13)

그림 Fig. 1. Inoculum used in laboratory and greenhouse assay. (A) Microsclerotia formed after culturing for 7 days in a potato dextrose agar (PDA) medium at 30°C. (B) Mycelial colonies cultured in PDA medium at 30°C for 3 days. (C) Sterilized toothpicks placed on a PDA medium for culturing Macrophomina phaseolina to prepare the inoculum used in the greenhouse test. (D) Toothpicks with microsclerotia used as an inoculum in the greenhouse test. Red arrows are toothpicks with microsclerotia.
그림 Fig. 2. Inoculation location of microsclerotia of Macrophomina phaseolina in a pathogenicity assay through a microsclerotia inoculation in a plant culture dish. (A) Upper layer. (B) Lower layer. (C) Middle layer. (D) Non-inoculated control. Number 1 shows a 0.8% water agar medium with the addition of microsclerotia, and number 2 shows a water agar medium without the addition of microsclerotia.
그림 Fig. 3. Inoculation location of microsclerotia of Macrophomina phaseolina in a pathogenicity assay through a microsclerotia inoculation in a 50 ml conical tube. The location of the inoculum was the same layer as the seed (A), the lower layer of the seed (B), the stem of the soybean (C), and the non-inoculated control (D).
그림 Fig. 4. Disease index to evaluate the disease incidence of soybean charcoal rot caused by Macrophomina phaseolina. 0, healthy; 1, reddish or browning root 0%≤x<50%, no cotyledon red or browning; 2, reddish or browning of root 0%≤x<50%, red or browning of cotyledons 0

그림 Fig. 5. A disease index used in the assessment of charcoal rot development caused by Macrophomina phaseolina on a soybean plant. 0, healthy; 1, wilting of above ground parts; 2, wilting and browning of above ground parts; 3, wilting and browning of above ground parts and formation of microscopic sclerotia on roots; 4, wilting and browning of above ground parts and formation of microscopic sclerotia on roots and crown.

그림 Fig. 6. Pathogenicity assay using the microsclerotia inoculation method for Macrophomina phaseolina causing soybean charcoal rot. (A) Disease incidence of M. phaseolina NSW17-108 investigated by using microsclerotia inoculation method in plant culture dishes. (B) The results of an experiment investigating the disease incidence of M. phaseolina NSW17-108 in a plant culture dish by the microsclerotia inoculation method. (C) Disease incidence of M. phaseolina NSW17-108 by using microsclerotia inoculation method in plant culture dishes. (D) The results in a conical tube. The numbers indicate the method of inoculating the pathogen. In A and B, no. 1 represents the treatment in which water agar (WA) medium containing microsclerotia was added to the top, no. 2 represents the treatment poured at the bottom, and no. 3 represents the treatment poured in the center. In C and D, no. 1 refers to the treatment in which WA medium was added to the seed placed on the WA medium without microsclerotia, and No. 2 refers to the treatment in which microsclerotia was added to the WA medium on which the seed was placed. And no. 3 refers to the treatment in which WA medium with the inoculum was added to the soybean seedling at the lower part of the stem. CK represents the control not inoculated with pathogens. All treatment means were separated using Duncan's multiple range test according to the fungicide used in the experiment. Treatments with the same letters are not statistically different at P≤0.05.

그림 Fig. 7. In vitro pathogenicity test using three inoculation methods. (A) Microsclerotia inoculation method in a plant culture dish. (B) Microsclerotia inoculation method in a conical tube. (C) Mycelial inoculation method. All pictures were taken 7, 10, and 7 days after inoculation with Macrophomina phaseolina NSW17-108 and HSM17-034 isolated from diseased soybean and sesame, respectively.

그림 Fig. 8. Disease incidence of Macrophomina phaseolina NSW17-108 and HSM17-034 in in vitro pathogenicity tests using three inoculation methods. (A) Microsclerotia inoculation method using a plant culture dish. (B) Microsclerotia inoculation method using conical tube. (C) Mycelial inoculation method. M. phaseolina NSW17-108 was a pathogen isolated from diseased soybean plant, and HSM17-034 was a pathogen isolated from diseased sesame plant. All treatment means were separated using Duncan's multiple range test according to the fungicide used in the experiment. Treatments with the same letters are not statistically different at P≤0.05.

그림 Fig. 9. Incidence of Macrophomina phaseolina NSW17-108 and HSM17-034 in greenhouse pathogenicity tests using toothpick inoculation method. The difference between the two isolates was statistically analyzed by t-test. *Significant at P<0.05.

그림 Fig. 10. Incidence of Macrophomina phaseolina NSW17-108 and HSM17-034 in greenhouse pathogenicity tests using toothpick inoculation method. The difference between the two isolates was statistically analyzed by t-test.

그림 Fig. 11. Disease incidence of Macrophomina phaseolina NSW17- 108 causing soybean charcoal rot according to the amount of microsclerotia in the seed dressing inoculation method with microsclerotia. All treatment means were separated using Duncan's multiple range test according to the fungicide used in the experiment. Treatments with the same letters are not statistically different at P≤0.05.

그림 Fig. 12. Incidence of pathogenicity tests in a greenhouse using microsclerotia soil drench inoculation method. (A) Percentage incidence of charcoal rot on soybean inoculated with soybean and sesame isolates. (B) Symptoms of charcoal rot on soybean. The difference between the two isolates was statistically analyzed by ttest. *Significant at P<0.05.

표 Table 1. Comparison of advantages and disadvantages of assay methods for investigating the pathogenicity and fungicide efficacy on Macrophomina phaseolina causing soybean charcoal rot

표/그림 (13)

그림 Fig. 1. Inoculum used in laboratory and greenhouse assay. (A) Microsclerotia formed after culturing for 7 days in a potato dextrose agar (PDA) medium at 30°C. (B) Mycelial colonies cultured in PDA medium at 30°C for 3 days. (C) Sterilized toothpicks placed on a PDA medium for culturing Macrophomina phaseolina to prepare the inoculum used in the greenhouse test. (D) Toothpicks with microsclerotia used as an inoculum in the greenhouse test. Red arrows are toothpicks with microsclerotia.

그림 Fig. 2. Inoculation location of microsclerotia of Macrophomina phaseolina in a pathogenicity assay through a microsclerotia inoculation in a plant culture dish. (A) Upper layer. (B) Lower layer. (C) Middle layer. (D) Non-inoculated control. Number 1 shows a 0.8% water agar medium with the addition of microsclerotia, and number 2 shows a water agar medium without the addition of microsclerotia.

그림 Fig. 3. Inoculation location of microsclerotia of Macrophomina phaseolina in a pathogenicity assay through a microsclerotia inoculation in a 50 ml conical tube. The location of the inoculum was the same layer as the seed (A), the lower layer of the seed (B), the stem of the soybean (C), and the non-inoculated control (D).

그림 Fig. 4. Disease index to evaluate the disease incidence of soybean charcoal rot caused by Macrophomina phaseolina. 0, healthy; 1, reddish or browning root 0%≤x<50%, no cotyledon red or browning; 2, reddish or browning of root 0%≤x<50%, red or browning of cotyledons 0

그림 Fig. 5. A disease index used in the assessment of charcoal rot development caused by Macrophomina phaseolina on a soybean plant. 0, healthy; 1, wilting of above ground parts; 2, wilting and browning of above ground parts; 3, wilting and browning of above ground parts and formation of microscopic sclerotia on roots; 4, wilting and browning of above ground parts and formation of microscopic sclerotia on roots and crown.

그림 Fig. 6. Pathogenicity assay using the microsclerotia inoculation method for Macrophomina phaseolina causing soybean charcoal rot. (A) Disease incidence of M. phaseolina NSW17-108 investigated by using microsclerotia inoculation method in plant culture dishes. (B) The results of an experiment investigating the disease incidence of M. phaseolina NSW17-108 in a plant culture dish by the microsclerotia inoculation method. (C) Disease incidence of M. phaseolina NSW17-108 by using microsclerotia inoculation method in plant culture dishes. (D) The results in a conical tube. The numbers indicate the method of inoculating the pathogen. In A and B, no. 1 represents the treatment in which water agar (WA) medium containing microsclerotia was added to the top, no. 2 represents the treatment poured at the bottom, and no. 3 represents the treatment poured in the center. In C and D, no. 1 refers to the treatment in which WA medium was added to the seed placed on the WA medium without microsclerotia, and No. 2 refers to the treatment in which microsclerotia was added to the WA medium on which the seed was placed. And no. 3 refers to the treatment in which WA medium with the inoculum was added to the soybean seedling at the lower part of the stem. CK represents the control not inoculated with pathogens. All treatment means were separated using Duncan's multiple range test according to the fungicide used in the experiment. Treatments with the same letters are not statistically different at P≤0.05.

그림 Fig. 7. In vitro pathogenicity test using three inoculation methods. (A) Microsclerotia inoculation method in a plant culture dish. (B) Microsclerotia inoculation method in a conical tube. (C) Mycelial inoculation method. All pictures were taken 7, 10, and 7 days after inoculation with Macrophomina phaseolina NSW17-108 and HSM17-034 isolated from diseased soybean and sesame, respectively.

그림 Fig. 8. Disease incidence of Macrophomina phaseolina NSW17-108 and HSM17-034 in in vitro pathogenicity tests using three inoculation methods. (A) Microsclerotia inoculation method using a plant culture dish. (B) Microsclerotia inoculation method using conical tube. (C) Mycelial inoculation method. M. phaseolina NSW17-108 was a pathogen isolated from diseased soybean plant, and HSM17-034 was a pathogen isolated from diseased sesame plant. All treatment means were separated using Duncan's multiple range test according to the fungicide used in the experiment. Treatments with the same letters are not statistically different at P≤0.05.

그림 Fig. 9. Incidence of Macrophomina phaseolina NSW17-108 and HSM17-034 in greenhouse pathogenicity tests using toothpick inoculation method. The difference between the two isolates was statistically analyzed by t-test. *Significant at P<0.05.

그림 Fig. 10. Incidence of Macrophomina phaseolina NSW17-108 and HSM17-034 in greenhouse pathogenicity tests using toothpick inoculation method. The difference between the two isolates was statistically analyzed by t-test.

그림 Fig. 11. Disease incidence of Macrophomina phaseolina NSW17- 108 causing soybean charcoal rot according to the amount of microsclerotia in the seed dressing inoculation method with microsclerotia. All treatment means were separated using Duncan's multiple range test according to the fungicide used in the experiment. Treatments with the same letters are not statistically different at P≤0.05.

그림 Fig. 12. Incidence of pathogenicity tests in a greenhouse using microsclerotia soil drench inoculation method. (A) Percentage incidence of charcoal rot on soybean inoculated with soybean and sesame isolates. (B) Symptoms of charcoal rot on soybean. The difference between the two isolates was statistically analyzed by ttest. *Significant at P<0.05.

표 Table 1. Comparison of advantages and disadvantages of assay methods for investigating the pathogenicity and fungicide efficacy on Macrophomina phaseolina causing soybean charcoal rot

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