This study was conducted to prevent and control charcoal rot damage as the occurrence of charcoal rot increased in Korea due to high temperature and dry climate change. Soybeans withered from the soybean cultivation field were collected to investigate the isolation pattern of the pathogens their characteristics. Macrophomina phaseolina was more frequently isolated at 35℃ than at 25℃, and was mainly isolated from upland field soybean plantations than from paddy field soybean plantations. Mycelial growth of M. phaseolina at 35℃ was similar to or faster than at 25℃ in PDA medium. Pathogenicity was conducted by inoculating the fungal mycelia on soybean and the result showed that M. phaseolina was highly pathogenic at 35℃. In September 2021, severe wilting and blighting symptoms of Aramkong occurred in Gujwa-eup, Jeju-do. As a result of isolating pathogens from dried dead Aram-kong, M. phaseolina was isolated by 20-50% in each of the 5 regions of Jeju-si, but not at all in Andeok-myeon, Seogwipo-si. Pathogenicity was conducted on two soybean cultivars, Daewon-kong and Aram-kong and the disease incidence was compared. The incidence on Daewon-kong was 50 to 80% as copmared to Aram-kong, which was less than 10%.
A pathogenic assay method for charcoal rot was established. Divided into indoor and greenhouse, sclerotium inoculation and mycelium inoculation using Petri dishes and conical tubes were used in vitro, and toothpick inoculation and treatment inoculation with small sclerotium seed powder were used for pathogenicity testing in greenhouses. Microsclerotium and hyphae were used as inoculate to cause disease in soybeans, and pathogenicity was confirmed.
In this experiment, M. phaseolina isolated from diseased soybean and sesame plants respectively were used to investigate the genetic diversity, mycological characteristics, and pathogenicity to the host. Based on the result of multigene phylogenetic analysis using the nucleotide sequence of the ITS region, TEF-1α, β-tubulin (TUB2), and actin (ACT) genes of the pathogen, the total isolates were classified into two groups; soy-group and ses-group. The mycelial growth was faster in M. phaseolina soy-group than ses-group isolates at temperature between 25℃ and 37.5℃. The mycelial growth of the ses-group isolates was faster than that of the soy-group isolates even in the medium to which potassium chlorate was added, indicating strong resistance to salt of the ses-group. In addition, the ses-group isolates had stronger pathogenicity to soybean and sesame than the soy-group isolates.
M. phaseolina species-specific primers amplified only M. phaseolina gDNA due to their high species specificity. A more rapid diagnosis was possible when species-specific primers were used to diagnose soybeans showing suspicious symptoms of charcoal rot in the field. Species-specific primers could be used for diagnosis not only in soybean but also in other hosts such as watermelon and sesame.
Using the agar dilution method, benomyl, prochloraz, tebuconazole, metconazole, pyraclostrobin, fluazinam, and fludioxonil were selected as experimental fungicides for the control of charcoal rot. M. phaseolina was isolated from 2016 to 2018, and in 2020 and 2021, respectively, and their sensitivity to 7 fungicides was compared by using an agar dilution method. Compared with the former (2016 - 2018), the mean EC50 value of benomyl, fludioxonil, metconazole, and tebuconazole increased more than twice in the latter (2020 - 2021). When comparing the EC50 values of the isolates (n=20) collected in Jeju in September 2021 and the isolates (n=13) collected outside of Jeju in 2021, the Jeju local group had a lower EC50 value than the non-Jeju group in the five fungicides except for benomyl and pyraclostrobin. Comparing the coefficient of determination between each fungicide, it was found that there was high cross-resistance between tebuconazole, metconazole, and prochloraz.
As a result of testing the effectiveness of seed dressing in vitro and in greenhouses with benomyl, prochloraz+tebuconazole, metconazole, pyraclostrobin, fluazinam, and fludioxonil fungicides, benomyl, pyraclostrobin, and fludioxonil were 100% effective in vitro. In the greenhouse, the control of benomyl was the most effective with more than 73%. Prochloraz+tebuconazole and metconazole inhibited soybean growth. In the field in 2021, the control value of all six fungicides was low, but the yield survey results of benomyl, pyraclostrobin, and fludioxonil were excellent. As a result of re-examination of the seed dressing effect in the field in 2022 with these three fungicides, the control value of benomyl was 76.3%, which was the most effective, and the yield result was also excellent for benomyl. The effect of foliar treatment was tested using benomyl, metconazole, pyraclostrobin, and fludioxonil, but foliar treatment was not effective against charcoal rot. The seed dressing effect of benomyl was the best in all in vitro, greenhouse, and field experiments.
The effect of soil irrigation and seed dressing with fungicides on the control of soybean charcoal rot was investigated in a greenhouse. The charcoal rot control effect by soil irrigation was tested by dividing the number of irrigation treatments into 1 time/day and 1 time/3 days using three greenhouse pathogenicity assays. In all three methods, the incidence of disease decreased when irrigation was performed for 1 time/day. Soil irrigation and treatment with fungicide seed dressing were carried out simultaneously. The disease control effect of benomyl, pyraclostrobin, and fludioxonil was investigated according to the frequency of soil irrigation. The number of irrigation was divided into 1 time/day and 1 time/3 days. In the once daily irrigation treatment, the disease incidence of the uninoculated control was 11.1%, and no disease occurred in all the treatments in which the seeds were treated with chemical fungicides. However, in the case where irrigation was supplied once in three days, the disease incidence of the uninoculated control was 66.7%, and the control values of benomyl, pyraclostrobin, and fludioxonil were 83.3, 75.0, and 50.0% respectively. The results of this study showed that it is necessary to irrigate the soil in the soybean cultivation field in order to increase the controlling effect of benomyl, pyraclostrobin, and fludioxonil.