2019-2022년 국내에서 분리한 Erwinia amylovora균의 옥시테트라사이클린과 옥솔린산 항생제에 대한 저항성 평가

함현희,오가람,이방울,이용환,이용훈 한국농약과학회 2023 농약과학회지 Vol.27 No.4

Fire blight, a destructive plant disease causing great threats to the apple and pear industries, was first occurred in South Korea in 2015. Various pesticides have been registered and used for the control of fire blight. Among them, antibiotics are known as very effective agents, but developed countries including Europe have been avoiding their use due to concerns about the emergence of antibiotic resistance and ecosystem disturbance. Antibiotics have been used for fire blight control since 2015 in Korea, however, antibiotic resistance of the pathogen has not been systematically investigated yet. Therefore, in this study, we surveyed the minimal inhibition concentration (MIC) of Erwinia amylovora, a causal agent of fire blight, against the oxytetracycline and oxolinic acid using a total of 258 isolates that are collected from overall areas of South Korea from 2019 to 2022 to assess the presence of antibiotic resistance. The MIC of oxytetracycline was in a range of 0.25-2 µg/ml, and that of oxolinic acid was less than 0.25 µg/ml. In addition, the MIC of oxytetracycline was increased in 2022 compared to 2019. However, no significant differences in MIC existed between isolated regions or hosts. Taken together, the results indicate that oxytetracycline and oxolinic acid resistance may not occur in E. amylovora of Korea. This study also provides fundamental information to the assessment of resistance against antibiotics registered for the control of fire blight in Korea.

UHPLC를 이용한 볏짚 사일리지와 동계사료작물의 오크라톡신과 제랄레논 분석법 최적화

함현희,문혜연,이경아,이수형,홍성기,이데레사,류재기,Ham, Hyeonheui,Mun, Hye Yeon,Lee, Kyung Ah,Lee, Soohyung,Hong, Sung Kee,Lee, Theresa,Ryu, Jae-Gee 한국초지조사료학회 2016 한국초지조사료학회지 Vol.36 No.4

조사료에서 오크라톡신 A (OTA) 및 제랄레논(ZEA)의 단성분 분석법을 최적화하기 위하여 곰팡이 독소에 오염되지 않은 볏짚 사일리지, 이탈리안 라이그라스, 청보리, 호밀을 대상으로 각 시료 별로 OTA는 50, 250, $5,000{\mu}g/kg$, ZEA는 300, 1,500, $3,000{\mu}g/kg$의 농도로 스파이킹하고 면역친화성 컬럼을 이용하여 독소를 정제한 후 UHPLC-FLD를 이용하여 분석하였다. 분석 결과 표준용액의 검량선은 OTA가 결정계수 0.9999, ZEA가 0.9995로 높은 직선성을 나타냈으며, 검출 및 정량한계는 OTA가 각각 $0.1{\mu}g/kg$, $0.3{\mu}g/kg$, ZEA는 각각 $5{\mu}g/kg$, $16.7{\mu}g/kg$을 나타냈다. 유효성 및 정밀성 검정결과 OTA는 볏짚이 회수율 84.23~95.33%, 상대표준편차 (RSD) 2.59~4.77%, 이탈리안 라이그라스는 회수율 79.02~95%, RSD 0.86~5.83%, 청보리는 회수율 74.93~97%, RSD 0.85~9.19%, 호밀은 회수율 77.99~96.67%, RSD 0.33~6.26%를 나타냈으며, ZEA는 볏짚이 회수율 109.6~114.22%, RSD 0.67~7.15%, 이탈리안 라이그라스는 회수율 98.01~109.44%, RSD 1.65~4.81%, 청보리는 회수율 98~113.53%, RSD 0.25~5.85%, 호밀은 회수율 90.44~108.56%, RSD 2.5~4.66%를 나타내어 유럽연합에서 제시한 기준을 만족하였다. 따라서 본 연구에서 제시한 분석법은 국내의 볏짚 등 조사료 4종에서 OTA와 ZEA의 분석에 활용할 수 있음을 시사하였다. The objective of this study was to optimize analytical methods for ochratoxin A (OTA) and zearalenone (ZEA) in rice straw silage and winter forage crops using ultra-high performance liquid chromatography (UHPLC). Samples free of mycotoxins were spiked with $50{\mu}g/kg$, $250{\mu}g/kg$, or $500{\mu}g/kg$ of OTA and $300{\mu}g/kg$, $1500{\mu}g/kg$, or $3000{\mu}g/kg$ of ZEA. OTA and ZEA were extracted by acetonitrile and cleaned-up using an immunoaffinity column. They were then subjected to analysis with UHPLC equipped with a fluorescence detector. The correlation coefficients of calibration curves showed high linearity ($R^2{\geq_-}0.9999$ for OTA and $R^2{\geq_-}0.9995$ for ZEA). The limit of detection and quantification were $0.1{\mu}g/kg$ and $0.3{\mu}g/kg$, respectively, for OTA and $5{\mu}g/kg$ and $16.7{\mu}g/kg$, respectively, for ZEA. The recovery and relative standard deviation (RSD) of OTA were as follows: rice straw = 84.23~95.33%, 2.59~4.77%; Italian ryegrass = 79.02~95%, 0.86~5.83%; barley = 74.93~97%, 0.85~9.19%; rye = 77.99~96.67%, 0.33~6.26%. The recovery and RSD of ZEA were: rice straw = 109.6~114.22%, 0.67~7.15%; Italian ryegrass = 98.01~109.44%, 1.65~4.81%; barley = 98~113.53%, 0.25~5.85%; rye = 90.44~108.56%, 2.5~4.66%. They both satisfied the standards of European Commission criteria (EC 401-2006) for quantitative analysis. These results showed that the optimized methods could be used for mycotoxin analysis of forages.

국내 과수화상병 등록 약제의 사과나무 개화기 방제 효과

함현희,이미현,노은정,이우형,최효원,양미숙,이용환 한국농약과학회 2023 농약과학회지 Vol.27 No.4

Since first report of fire blight, caused by Erwinia amylovora, in Korea in 2015, enormous economic losses of apple and pears have been reported in Korea. E. amylovora has been registered as a prohibited quarantine pathogen in Korea, so fire blight control tests in the open fields are prohibited. Accordingly, the control agents (CAs) that are being used abroad to control fire blight and that are registered domestically for other plant diseases of apples and pears are being registered as the control agents for fire blight disease in Korea. In this study, the field screening of registered control agents on fire blight disease of apple during flowering period were conducted in an isolated house built in accordance with the LMO safety management regulations operated by the Rural Development Administration in 2021 and 2023. In 2021, one flower per tree (Fuji variety 15-year-old) was inoculated with bacterial suspension at full bloom, and then natural infection was induced to other flowers using bees (Bombus agrorum). CAs were applied twice at three-day intervals, on the 1st and 4th days after inoculation, and the flower infection rate was examined on the 21st day after inoculation. In 2023, all flowers were inoculated at full bloom for 3-year-old Hong-ro varieties. CAs were sprayed twice at three-day intervals, 1 day before and 2 days after inoculation, and the flower infection rate was examined on the 21st and 28th days after inoculation. Most CAs containing streptomycin, oxytetracycline, and oxolinic acid were highly effective, with control values exceeding 70%. On the other hand, biocontrol agents were not effective, with a control value of 47.3-59.3%.

겉보리의 저장온도와 수분함량이 붉은곰팡이병균과 곰팡이독소 오염에 미치는 영향

함현희,이경아,이데레사,한상현,홍성기,이수형,류재기 한국식품위생안전성학회 2017 한국식품위생안전성학회지 Vol.32 No.4

Fusarium graminearum is a mycotoxigenic plant pathogen, which could remain in harvested barley grains and produces mycotoxins at preferable conditions during storage. To elucidate the factors affecting contamination of Fusarium and Fusariotoxin in hulled barley during storage, three hulled barley grain samples were collected from Jeolla province. Moisture content of each sample was adjusted to 14% and 20%, respectively, then stored in two warehouses where temperature was controlled differently: one controlled below 12oC, and the other with no control. While monitoring temperature and relative humidity of warehouses hourly, grain moisture content, Fusarium occurrence, and mycotoxin level was analyzed at 1, 3, 6, and 12 month after storage. The average monthly temperature and relative humidity ranged 3~29oC, and 58~70% in warehouse without temperature control, whereas 3~13oC and 62~ 74% in warehouse controlled below 12oC. Grain moisture content of the samples decreased in both warehouses except 14% samples which increased in the warehouse with temperature control. Fusarium frequency of the contaminated grains decreased continuously in the warehouse without temperature control. But in the warehouse below 12oC, Fusarium decreasing rate was slower because of high grain moisture content. In most samples, nivalenol was detected more in the warehouse without temperature control after 12 month but there was little difference after 1, 3, and 6 month. Therefore, it will be efficient to store hulled barley in the warehouse controlled below 12oC to reduce Fusarium contamination when the barley is not dried properly. In addition, when storage period exceeds 12 month, it is recommended to store hulled barley in a warehouse controlled below 12oC to reduce nivalenol contamination.

저장 중 겉보리에 발생하는 곰팡이와 곰팡이독소 변화

함현희,백지선,이미정,이데레사,홍성기,이승돈 한국식품저장유통학회 2017 한국식품저장유통학회지 Vol. No.

To establish good storage practices for hulled barley against mycotoxin contamination, we measured occurrence of fungi and mycotoxin in hulled barley grains under various storage conditions. Hulled barley grains collected from two places were stored in five different warehouses: 1) two without temperature control, 2) one with temperature controlled at 12℃, 3) a chamber set at 15℃/65% relative humidity, and 4) one seed storage set at 10℃. The samples were stored for six month with temperature and relative humidity monitored regularly. Every stored samples were retrieved after 0, 1, 3, and 6 month to investigate fungal and mycotoxin contamination. From the stored grains, Fusarium, Epicoccum, Alternaria, and Drechslera spp. were frequently detected. In the warehouses without temperature control, Fusarium and Alternaria spp. constantly decreased, whereas Drechslera spp. increased along with storage period. In the other warehouses with temperature controlled, Fusarium spp. decreased slowly and more than 2.5 log CFU/g of Fusarium spp. were detected after 6 month storage. The level of nivalenol was maintained during 0-3 month but increased after 6 month storage. There was no difference in the nivalenol levels between the warehouses. Therefore reducing storage period less than 6 months could be more effective to control nivalenol contamination in hulled barley grains.

저장 중 겉보리에 발생하는 곰팡이와 곰팡이독소 변화

함현희,백지선,이미정,이데레사,홍성기,이승돈,Ham, Hyeonheui,Baek, Jiseon,Lee, Mijeong,Lee, Theresa,Hong, Sung-Kee,Lee, Seungdon 한국식품저장유통학회 2017 한국식품저장유통학회지 Vol.24 No.6

본 연구는 곰팡이독소에 안전한 보리 저장법을 수립하기 위해 다양한 저장환경 중 겉보리에 발생하는 곰팡이와 곰팡이독소를 측정하였다. 두 지역에서 수집한 겉보리를 상온 창고 2곳, 저온창고, $15^{\circ}C/65%$ 항온항습실 및 종자저장고에 6개월간 저장하면서 장소별 온도, 상대습도 및 곡물수분함량을 측정하였으며 0, 1, 3 및 6개월 후 겉보리에 오염된 곰팡이와 곰팡이독소를 조사하였다. 저장 중 보리에 오염된 주요 곰팡이 속은 Fusarium, Epicoccum, Alternaria 및 Drechslera 이었다. 상온창고는 저장 후기로 갈수록 Fusarium 과 Alternaria 속 곰팡이가 큰 폭으로 감소한 반면 Drechslera는 증가하였다. 저온창고, 항온항습실 및 종자저장고에서는 Fusarium 오염립의 감소폭이 적었고 저장 6개월 후에도 2.5 log CFU/g 이상이 검출되었다. 니발레놀 오염은 한 시료에서 저장 0-3개월 간 비슷한 수준을 유지하다가 6개월 후 증가하였으며, 저장소 간 차이는 나타나지 않았다. 따라서 겉보리 저장 중 니발레놀 오염 예방을 위해 저장 기간을 6개월 미만으로 줄이는 것이 효율적일 것으로 보인다. To establish good storage practices for hulled barley against mycotoxin contamination, we measured occurrence of fungi and mycotoxin in hulled barley grains under various storage conditions. Hulled barley grains collected from two places were stored in five different warehouses: 1) two without temperature control, 2) one with temperature controlled at $12^{\circ}C$, 3) a chamber set at $15^{\circ}C/65%$ relative humidity, and 4) one seed storage set at $10^{\circ}C$. The samples were stored for six month with temperature and relative humidity monitored regularly. Every stored samples were retrieved after 0, 1, 3, and 6 month to investigate fungal and mycotoxin contamination. From the stored grains, Fusarium, Epicoccum, Alternaria, and Drechslera spp. were frequently detected. In the warehouses without temperature control, Fusarium and Alternaria spp. constantly decreased, whereas Drechslera spp. increased along with storage period. In the other warehouses with temperature controlled, Fusarium spp. decreased slowly and more than 2.5 log CFU/g of Fusarium spp. were detected after 6 month storage. The level of nivalenol was maintained during 0-3 month but increased after 6 month storage. There was no difference in the nivalenol levels between the warehouses. Therefore reducing storage period less than 6 months could be more effective to control nivalenol contamination in hulled barley grains.

Survey of Oxolinic Acid-Resistant Erwinia amylovora in Korean Apple and Pear Orchards, and the Fitness Impact of Constructed Mutants

함현희,오가람,Dong Suk Park,이용훈 한국식물병리학회 2022 Plant Pathology Journal Vol.38 No.5

Fire blight caused by Erwinia amylovora (Ea) is a devastating disease in apple and pear trees. Oxolinic acid (OA), a quinolone family antibiotic that inhibits DNA gyrase, has been employed to control fire blight in South Korea since 2015. The continuous use of this bactericide has resulted in the emergence of OA-resistant strains in bacterial pathogens in other countries. To investigate the occurrence of OA-resistant Ea strains in South Korea, we collected a total of 516 Ea isolates from diseased apple and pear trees in 2020-2021 and assessed their sensitivities to OA. We found that all isolates were susceptible to OA. To explore the possibility of emerging OA-resistant Ea by continuous application of OA, we exposed Ea stains to a range of OA concentrations and constructed OA-resistant mutant strains. Resistance was associated with mutations in the GyrA at codons 81 and 83, which result in glycine to cysteine and serine to arginine amino acid substitutions, respectively. The in vitro growth of the mutants in nutrient media and their virulence in immature apple fruits were lower than those of wild-type. Our results suggest that OA-resistance decreases the fitness of Ea. Future work should clarify the mechanisms by which OA-resistance decreases virulence of this plant pathogen. Continuous monitoring of OA-resistance in Ea is required to maintain the efficacy of this potent bactericide.

2015‒2019년 국내 과수 화상병 발생

함현희(Hyeonheui Ham),이영기(Young-Kee Lee),공현기(Hyun Gi Kong),홍성준(Seong Jun Hong),이경재(Kyong Jae Lee),오가람(Ga-Ram Oh),이미현(Mi-Hyun Lee),이용환(Yong Hwan Lee) 한국식물병리학회 2020 식물병연구 Vol.26 No.4

과수 화상병을 일으키는 Erwinia amylovora는 국내에서 금지병원균으로 지정되어 화상병 발생 시, 중앙 정부의 진단을 근거로 기주를 매몰하는 공적 방제가 실시되고 있다. 국내 과수 화상병은 2015년 안성, 천안 및 제천의 43농가에서 발생하여 42.9 ha를 매몰한 것을 시작으로, 2019년 발생 지역이 11개 시군으로확산되었으며, 총 348농가 260.4 ha가 매몰되었다. 배나무 화상병은 주로 경기남부와 충남에서 발생되었고, 발생 건수가 연평균 29±9.2건으로 매년 비교적 고르게 발생되었으며 20-30년생 과수에서 발병 비율이 가장 높았다. 반면, 사과나무 화상병은 주로 경기북부, 강원, 충북에서 발생되었고, 발생 건수가 연평균 41±57.6건으로 2018‒2019년 발생건수가 크게 증가하였으며, 20년 이하의 과수의 발병 비율이 높았다. 국내 과수 화상병은 어린 사과나무에서 병의 확산이 빠르므로, 특히 미성숙과수가 식재된 과원에서는 화상병이 발병하지 않도록 약제를적기에 살포하는 등 예방을 철저히 하고, 발병 시 신속히 방제해야 한다. Erwinia amylovora, a causal bacterium of fire blight disease, is registered as a prohibited quarantine pathogen in Korea. To control the disease, the government should diagnose the disease, dig and bury the host trees when fire blight occurs. Fire blight was the first reported in 43 orchards of Anseong, Cheonan, and Jecheon in 2015, and 42.9 ha of host trees were eradicated. However, the disease spread to eleven cities, so that 348 orchards and 260.4 ha of host trees were eradicated until 2019. Fire blight of Asian pear occurred mainly in the southern part of Gyeonggi, and Chungnam province, on average of 29±9.2 orchards per year. And the age of the infected trees were mostly 20–30 years old. In apple trees, the disease occurred mainly in the northern part of Gyeonggi, Gangwon, and Chungbuk province, on average of 41±57.6 orchards per year, increased highly in 2018 and 2019. The age of infected apple trees were under 20 years old. Therefore, because the disease spread rapidly in young apple trees, spraying control agents to the trees in a timely manner and removing infected trees quickly are important to prevent the spread of fire blight in the orchard of immature trees.

Discrimination and Detection of Erwinia amylovora and Erwinia pyrifoliae with a Single Primer Set

함현희(Hyeonheui Ham),Kyongnim Kim,양수인(Suin Yang),공현기(Hyun Gi Kong),이미현(Mi-Hyun Lee),Yong Ju Jin,박동석(Dong Suk Park) 한국식물병리학회 2022 Plant Pathology Journal Vol.38 No.3

Erwinia amylovora and Erwinia pyrifoliae cause fire blight and black-shoot blight, respectively, in apples and pears. E. pyrifoliae is less pathogenic and has a narrower host range than that of E. amylovora. Fire blight and black-shoot blight exhibit similar symptoms, making it difficult to distinguish one bacterial disease from the other. Molecular tools that differentiate fire blight from black-shoot blight could guide in the implementation of appropriate management strategies to control both diseases. In this study, a primer set was developed to detect and distinguish E. amylovora from E. pyrifoliae by conventional polymerase chain reaction (PCR). The primers produced amplicons of different sizes that were specific to each bacterial species. PCR products from E. amylovora and E. pyrifoliae cells at concentrations of 104 cfu/ml and 107 cfu/ml, respectively, were amplified, which demonstrated sufficient primer detection sensitivity. This primer set provides a simple molecular tool to distinguish between two types of bacterial diseases with similar symptoms.

2019년 국내 사과와 배 화상병 대발생과 그 특징

함현희(Hyeonheui Ham),이경재(Kyong Jae Lee),홍성준(Seong Jun Hong),공현기(Hyun Gi Kong),이미현(Mi-Hyun Lee),김현란(Hyun-Ran Kim),이용환(Yong Hwan Lee) 한국식물병리학회 2020 식물병연구 Vol.26 No.4

2019년 국내의 사과와 배에 화상병이 크게 발생한 원인을 파악하기 위하여 화상병 발생한 30개 과원을 대상으로 각각의 발생 상황과 농가 면담을 통해 경종적 특징 등을 조사하였다. 화상병은 이미 감염된 지 2년 이상 오래 된 과원에서 대부분 발생하였는데, 이런 원인은 (1) 농가가 병 증상을 정확히 알지 못하여 농작업과 방화곤충 등을 통해 과원 내에서 퍼지게 되고, (2) 방화곤충이나 농작업자 등에 의해 처음 발생 과원에서 주변과수원으로 확산되었고, (3) 동일 경작자 또는 공동 농작업자에 의해 근거리 또는 원거리로 확산된 것이라고 추정할 수 있다. (4) 이런 일련의 과정이 새롭게 확산된 지역에서 반복되다가 농가들이 화상병을 알게 되면서 신고가 증가한 것이 2019년 화상병 대발생의 일련의 원인이라고 추정할 수 있었다. 국내에서 화상병 확산을 최소화하기 위해서는 조기진단을 위한 철저한 농업인 교육과 무병징 식물체에서도 화상병균 진단이 가능한 정량적 검출기술이 요구되고 있다. 또한 큰 열매를 주로 생산하는국내 재배법에 적합한 약제방제 체계 개발이 필요하다. 화상병방제에서 가장 중요한 가지의 궤양 증상, 묘목, 양봉장 등의 전염원 관리를 위해서 과원별 병원균의 분자역학연구를 통해 정확한 확산경로를 구명할 것을 제안한다. To find out the cause of the fire blight outbreak in apples and pears of Korea in 2019, we investigated disease appearing situation of thirty fire blight infected orchards, and interviewed farmers to determine the cultivation characteristics. Fire blight occurred mostly in orchards that had infected more than 2 years before. The cause of this were as follows: farmers did not know the symptoms of the disease properly. It is presumed that it has spread from the first occurrence to the surrounding orchards by flower-visiting insects or farmers and to a short distance or a long distance by the same cultivator or co-farmer. These series of processes repeated in the newly spreading area, and then disease reports increased as farmers became aware of fire blight. To minimize the spread of fire blight in Korea, it suggested that thorough education of farmers for early diagnosis and quantitative detection technology that can diagnose even in no symptom showing plants. And chemical or biological spraying systems suitable for domestic cultivation methods, which are producing large fruits, and molecular epidemiological studies of pathogens.