• Korean Journal of Environmental Agriculture
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• v.29 no.2
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• pp.165-175
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• 2010

A gas chromatographic (GC) method was developed to determine residues of captan, folpet, captafol, and chlorothalonil, known as broad-spectrum protective fungicides for the official purpose. All the fungicide residues were extracted with acetone containing 3% phosphoric acid from representative samples of five agricultural products which comprised rice, soybean, apple, pepper, and cabbage. The extract was diluted with saline, and dichloromethane partition was followed to recover the fungicides from the aqueous phase. Florisil column chromatography was additionally employed for final cleanup of the extracts. The analytes were then determined by gas chromatography using a DB-1 capillary column with electron capture detection. Reproducibility in quantitation was largely enhanced by minimization of adsorption or thermal degradation of analytes during GLC analysis. Mean recoveries generated from each crop sample fortified at two levels in triplicate ranged from 89.0~113.7%. Relative standard deviations (RSD) were all less than 10%, irrespective sample types and fortification levels. As no interference was found in any samples, limit of quantitation (LOQ) was estimated to be 0.008 mg/kg for the analytes except showing higher sensitivity of 0.002 mg/kg for chlorothalonil. GC/Mass spectrometric method using selected-ion monitoring technique was also provided to confirm the suspected residues. The proposed method was reproducible and sensitive enough to determine the residues of captan, folpet, captafol, and chlorothalonil in agricultural commodities for routine analysis.

• The Plant Pathology Journal
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• v.17 no.3
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• pp.149-153
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• 2001

A total of 2109 isolates of Botrytis cinerea were collected from infected plants fo strawberry, tomato, and cucumber in Korea from 1994 to 1996. Based on in virtotests for mycelial growth on potato-dextrose agar containing fungicides, the esolates were classified into six phenotypic groups : SSR, SRR, RSS, RRS, RSR, and RRR, representing sensitivity (S) or resistance (R) to carbendazim, procymidone, and diethofencarb. In that order the isolation frequencies of the SSR, SRR, RSS, RRS, RSR, and RRR phenotypes were 28.7, 1.1, 28.8, 39.4, 1.0, and 0.9%, respectively. Three isolates from each SSR, SRR, RSS, RRS, and RSR and an isolate of RRR phenotype were selected and evaluated for their fitness-related characteristics such as pathogenic aggressiveness, mycelial growth rate, sporulation, and sclerotial formation. Competitive abilities of the SSR, SRR, RSS, RRS, and RSR phenotypes were also compared by inculating mixtures of conidial suspensions of two phenotypes to cucumber plant, and then determining re-isolation frequencies from lesions. In general, significant differences in fitness-related characteristics, except pathogenic aggressiveness, were found not only between but also within phenotype groups. In the competitiveness tests, carbendazim-sinsitive phenotypes (SSR and SRR) were found to be more competitive than the resistant ones (RSS and RSR), whereas, the procymidone-resistant phenotypes (SRR and RRS) appeared to be more competitive than the sensitive ones (SSR, RSS, and RSR). There was no consistent dominance in competitiveness between the diethofencarb-resistant and sensitive phenotypes. The RSR phenotype was the least competitive among the five phenotypes.

• Research in Plant Disease
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• v.9 no.3
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• pp.131-136
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• 2003

The effects of temperature, duration of wetness period, and fungicides on the spore germination, appressorium formation, acervulus formation and lesion development by Colletotrichum spp., cause of soybean anthracnose, and their pathogenicity were assessed in controlled environment. C. gloeosporioides was highly pathogenic on inoculated soybean seeds as high as C. truncatum, whereas remarkably low on the soybean leaves. Spore germination, appressorium formation and mycelial growth of C. gloeosporioides were best at $25^{\circ}C$, but C. truncatum was best at $30^{\circ}C$. It has also done at $15^{\circ}C$, even though it was much retarded. C. truncatum showed high sensitivity to the fungicides, fluazinam and benomyl, meanshile C. gloeosporioides showed to fluazinam and triflumizole. At least 8 hrs. of wetness period was requird for the pathogen to develop lesions at $30^{\circ}C$. When the wetness period was 32 hrs. lesion size of was larger at $25^{\circ}C$ than $30^{\circ}C$, however it was traceable at $20^{\circ}C$. Different sensitivity of Colletotrichum spp. to fungicides suggests that proper fungicide is required to effective control of soybean anthracnose ingected multiply with Colletotrichum spp.

• Proceedings of the Microbiological Society of Korea Conference
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• 2007.05a
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• pp.120-122
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• 2007

All living organisms use numerous signal-transduction pathways to sense and respond to their environments and thereby survive and proliferate in a range of biological niches. Molecular dissection of these signalling networks has increased our understanding of these communication processes and provides a platform for therapeutic intervention when these pathways malfunction in disease states, including infection. Owing to the expanding availability of sequenced genomes, a wealth of genetic and molecular tools and the conservation of signalling networks, members of the fungal kingdom serve as excellent model systems for more complex, multicellular organisms. Here, we employed Cryptococcus neoformans as a model system to understand how fungal-signalling circuits operate at the molecular level to sense and respond to a plethora of environmental stresses, including osmoticshock, UV, high temperature, oxidative stress and toxic drugs/metabolites. The stress-activated p38/Hog1 MAPK pathway is structurally conserved in many organisms as diverse as yeast and mammals, but its regulation is uniquely specialized in a majority of clinical Cryptococcus neoformans serotype A and D strains to control differentiation and virulence factor regulation. C. neoformans Hog1 MAPK is controlled by Pbs2 MAPK kinase (MAPKK). The Pbs2-Hog1 MAPK cascade is controlled by the fungal "two-component" system that is composed of a response regulator, Ssk1, and multiple sensor kinases, including two-component.like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. We also identified and characterized the Ssk2 MAPKKK upstream of the MAPKK Pbs2 and the MAPK Hog1 in C. neoformans. The SSK2 gene was identified as a potential component responsible for differential Hog1 regulation between the serotype D sibling f1 strains B3501 and B3502 through comparative analysis of their meiotic map with the meiotic segregation of Hog1-dependent sensitivity to the fungicide fludioxonil. Ssk2 is the only polymorphic component in the Hog1 MAPK module, including two coding sequence changes between the SSK2 alleles in B3501 and B3502 strains. To further support this finding, the SSK2 allele exchange completely swapped Hog1-related phenotypes between B3501 and B3502 strains. In the serotype A strain H99, disruption of the SSK2 gene dramatically enhanced capsule biosynthesis and mating efficiency, similar to pbs2 and hog1 mutations. Furthermore, ssk2, pbs2, and hog1 mutants are all hypersensitive to a variety of stresses and completely resistant to fludioxonil. Taken together, these findings indicate that Ssk2 is the critical interface protein connecting the two-component system and the Pbs2-Hog1 pathway in C. neoformans.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.15-22
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• 2008

Four series (S, M, R, and W) of Alternaria longipes isolates were obtained based on consecutive selection with Dimethachlon (Dim) and ultraviolet irradiation. These isolates were then characterized according to their tolerance to Dim, sensitivity to osmotic stress, and phenotypic properties. All the selected Dim-resistant isolates showed a higher osmosensitivity than the parental strains, and the last generation was more resistant than the first generation in the M, R, and W series. In addition, the changes in the Dim resistance and osmotic sensitivity were not found to be directly correlated, and no distinct morphologic characteristics were found among the resistant and sensitive isolates, with the exception of the resistant isolate K-11. Thus, to investigate the molecular basis of the fungicide resistance, a group III two-component histidine kinase (HK) gene, AlHK1, was cloned from nineteen A. longipes isolates. AlHK1p was found to be comprised of a six 92-amino-acid repeat domain (AARD), HK domain, and response regulator domain, similar to the Os-1p from Neurospora crassa. A comparison of the nucleotide sequences of the AlHK1 gene from the Dim-sensitive and -resistant isolates revealed that all the resistant isolates contained a single-point mutation in the AARD of AlHK1p, with the exception of isolate K-11, where the AlHK1p contained a deletion of 107 amino acids. Moreover, the AlHK1p mutations in the isolates of each respective series involved the same amino acid substitution at the same site, although the resistance levels differed significantly in each series. Therefore, these findings suggested that a mutation in the AARD of AlHK1p was not the sole factor responsible for A. longipes resistance to dicarboximide fungicides.

• The Korean Journal of Pesticide Science
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• v.13 no.4
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• pp.283-289
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• 2009

Isolation frequency of resistant isolates of Phytophthora capsici to metalaxyl was reported to be 38.9% through the resistance monitoring for metalaxyl in P. capsici causing pepper Phytophthora blight. Metalaxyl was very effective to mycelium growth, while not to zoosporangium germination and zoospore release. $EC_{50}$ values of metalaxyl in the inhibition of mycelium growth were 0.204, 0.151, 0.379, and $0.215\;{\mu}g\;mL^{-1}$ against each isolate sensitive to the fungicide as P. capsici 06-119, 06-143, P08-7, and P08-31, respectively, whilst those were 5.242, 5.724, 6.621, and $5.377\;{\mu}g\;mL^{-1}$ in P. capsici 06-125, 06-155, P08-50, and P08-60. For the field fitness, several factors, which were mycelium growth, zoosporangium germination, zoospore release, virulence to pepper plants, and the zoosporangium and the oospore production, were investigated with 4 sensitive isolates and 4 resistant isolates. Between 2 groups differentiated by the sensitivity of metalaxyl, there was no significance in mycelium growth, zoosporangium germination, zoospore release, and virulence to pepper plants. However, the zoosporangium and the oospore production in each resistant isolate, which were related to survival of P. capsici in fields, were superior to those of sensitive isolates. Based on results of this study, it was suggested that the increase of the percentage of resistant isolates to metalaxyl resulted from the high capacities of the zoosporangium and the oospore production.

• Research in Plant Disease
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• v.8 no.4
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• pp.239-244
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• 2002

Among 34 isolates of Colletotrichum spp., 20 from red pepper and 14 from apple, only one isolate 2001-45 was identified as the isolate resistant to sterol biosynthesis inhibiting fungicides. EC$_{50}$ values of the isolate 2001-45 were 153.5, 42.7, 34.0, and 17.1 $\mu\textrm{g}$/ml for myclobutanil, tebuconazole, hexaconazole and nuarimol, respectively, The resistance factor of the isolate 2001-45 against the other isolate 2001-44 to 4 above fungicides was ranged from 17 to 57. However, EC$_{50}$ value of the 2001-45 for prochloraz was 0.07 $\mu\textrm{g}$/ml, which was lower than those of the 2001-44 and the isolate JC24. For the fitness test of the 2001-45, mycelial growth, sporulation on PDA and pathogenicity on fruits were investigated. No difference in mycelial growth was found between 2001-45 and 2001-44, but great difference in sporulation. No symptom was developed by 2001-45 even by wound inoculation of pepper fruit. Therefore, this study indicated that the isolate 2001-45 was inferior to the other isolates in the fitness, although the isolate 2001-45 was highly resistant to sterol biosynthesis inhibiting fungicides.

• Research in Plant Disease
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• v.26 no.1
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• pp.8-18
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• 2020

Red pepper, one of the major economic crops in Korea, is being affected by anthracnose disease caused by Colletotrichum acutatum. To control this disease, an antagonistic bacterial strain, Bacillus subtilis YGB36 identified by 16S rDNA sequencing, physiological and biochemical analyses is used as a biological control agent. In vitro screening revealed that the strain YGB36 possess strong antifungal activity against the pathogen Cylindrocarpon destructans. The strain exhibited cellulase, protease, amylase, siderophore production and phosphate solubility. In vitro conidial germination of C. acutatum was most drastically inhibited by YGB36 cell suspensions (10⁶ cfu/ml) or culture filtrate. Development of anthracnose symptoms was reduced on detached immature green pepper fruits by treatment with cell suspensions, and its control value was recorded as 65.7%. The YGB36 bacterial suspension treatment enhanced the germination rate of red pepper seeds and promoted root development and growth under greenhouse conditions. The in vitro screening of fungicide and insecticide sensitivity test against YGB36 revealed that the bacterial growth was not affected by any of the insecticides, and 11 fungicides out of 21 used. Collectively, our results clearly suggest that the strain YGB36 is considered as one of the potential biocontrol agents against anthracnose disease in red pepper.

• Korean Journal of Environmental Agriculture
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• v.31 no.3
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• pp.248-254
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• 2012

BACKGROUND: In order to elucidate residual characteristics of difenoconazole and thiamethoxam by treatment to sweet persimmons for one year and to generate the data for the maximum residue limit (MRL) establishment for those pesticides in or on sweet persimmon. METHODS AND RESULTS: Systemic fungicide difenoconazole WP (10% a.i.) and systemic insecticide thiamethoxam WG (10% a.i.) were sprayed onto 12~25-years-old sweet persimmons according to its preharvest interval (PHI), respectively, and then fresh sweet persimmons were harvested at 0, 1, 3, 7, 14, 21 days after treatment from pesticide-sprayed plots at each 3 sites. The analytical methods were evaluated to limit of quantification, linearity, specificity, reproducibility and recoveries. The crop samples were extracted with acetone and performed dichloromethane partition process. The extracted samples of difenoconazole were analyzed by GC-ECD and the thiamethoxam extracted samples were analyzed by HPLC with good sensitivity and selectivity of the method. The average recoveries of difenoconazole ranged from 87.5 to 99.5% with the percentage of coefficient variation in the range 4.1~7.6% at three different spiking levels(0.02, 0.2 and 2.0 mg/kg). And the average recoveries of thiamethoxam and clothianidin ranged from 88.8 to 98.9% and 83.2 to 96.6% with the percentage of coefficient variation in the range 3.6~5.0% and 3.8~9.4% at three different spiking levels(0.02, 0.2 and 2.0 mg/kg), respectively. The residue amounts ranges of difenoconazole were 0.2~0.56 mg/kg and the residue amount was decreased below the MRL level, 1.0 mg/kg, after 1 day harvest. The residue amounts ranges of thiamethoxam were 0.08~0.28 mg/kg and the residue amount was decreased below the MRL level, 0.5 mg/kg, after 1 day harvest. And the residue amount of clothianidin was below then 0.03 mg/kg for only one test site of 14 and 28 day samples. CONCLUSION: As a result, the residual amounts of difenoconazole and thiamethoxam were not exceeded the MRL of established criteria for sweet persimmon. The biological half-lives of difenoconazole and thiamethoxam were 13.6, 19.4, 16.3 and 10.0, 15.3, 14.0 days at each three test sites, respectively.

• Korean Journal of Environmental Agriculture
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• v.40 no.2
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• pp.108-117
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• 2021

BACKGROUND: Dichlobentiazox is a newly registered pesticide in Korea as a triazole fungicide and requires establishment of an official analysis method for the safety management. Therefore, the aim of this study was to determine the residual analysis method of dichlobentiazox for the five representative agricultural products. METHODS AND RESULTS: Three QuEChERS methods were applied to establish the extraction method, and the EN method was finally selected through the recovery test. In addition, various adsorbent agents were applied to establish the clean-up method. As a result, it was found that the recovery of the tested pesticide was reduced when using the d-SPE method with PSA and GCB, but C₁₈ showed an excellent recovery. Therefore this method was established as the final analysis method. For the analysis, LC-MS/MS was used with consideration of the selectivity and sensitivity of the target pesticide and was operated in MRM mode. The results of the recovery test using the established analysis method and inter laboratory validation showed a valid range of 70-120%, with standard deviation and coefficient of variation of less than 3.0% and 11.6%, respectively. CONCLUSION: Dichlobentiazox could be analyzed with a modified QuEChERS method, and the method determined would be widely available to ensure the safety of residual pesticides in Korea.