초록 ▼

□ 과제명 : 병해충 • 잡초 약제저항성 모니터링 및 안전관리시스템 구축 연구
▶ 연구목적 : 국내 병해충 • 잡초의 약제 저항성 실태를 모니터링하고, 저항성 기작 구명을 통하여 보다 빠르고 정밀한 모니터링 시스템 구축을 통한 저항성 관리 방안 구축과 약제의 오남용으로 위협 받는 안전한 농산물 관리 시스템 구축
▶ 주요연구내용 :
○ 1세부과제명 : 시설재배 주요해충의 약제저항성 모니터링및 관리기술개발
- 점박이응애에 우수한 효과를 나태내는 약제는 아바멕틴, 아퀴세노실, 비페나자이드, 프로파자이드, 디코폴, 클

□ 과제명 : 병해충 • 잡초 약제저항성 모니터링 및 안전관리시스템 구축 연구
▶ 연구목적 : 국내 병해충 • 잡초의 약제 저항성 실태를 모니터링하고, 저항성 기작 구명을 통하여 보다 빠르고 정밀한 모니터링 시스템 구축을 통한 저항성 관리 방안 구축과 약제의 오남용으로 위협 받는 안전한 농산물 관리 시스템 구축
▶ 주요연구내용 :
○ 1세부과제명 : 시설재배 주요해충의 약제저항성 모니터링및 관리기술개발
- 점박이응애에 우수한 효과를 나태내는 약제는 아바멕틴, 아퀴세노실, 비페나자이드, 프로파자이드, 디코폴, 클로르페나피르 등이며, 기타 약제들은 지역간 편차가 큼
- 담배가루이에 효과적인 약제: 알 (스피로메시펜, 디노테푸란, 피라다벤 등), 약충 (에마멕틴벤조에이트, 스피로메시펜, 디노테푸란, 피리다벤 등), 성충 (에마멕틴벤조에이트, 디노테푸란, 피리다벤)
○ 2세부과제명 : 노지 양채류 미소 해충 약제저항성 모니터링 및 기작 구명
- 복숭아혹진딧물에 효과적인 약제는 이미다클로프리드, 아세타미프리드, 티아메톡삼, 티아클로프리드, 클로티아니딘 등이나 지역 간 편차가 있으며, 비펜스린 저항성에 관여하는 나트륨 채널의 MtoL 돌연변이 발견, QS (quantitative sequencing)을 통하여 분자 진단 가능
- nAChR beta subunit 내 RtoT 돌연변이를 발견하였으며 이는 신속한 분자 진단에 이용 가능, NTE, GSTs2, ABCC9, nAChR alpha 5 등의 유전자가 목화진딧물 이미다클로프리드 저항성에 관여하는 것으로 밝혀짐
○ 3세부과제명 : 약제저항성 유발 병원균의 살균제 저항성 기작 구명
- 저항성병원균은 약제의 농도에 따라 세포벽의 두께를 증가시킴으로써 약제에 저항성을 획득하며, 각 병원균의 약제에 대한 반응성의 차이는 efflux pump의 발현 유무 또는 발현 능력의 차이에서 오는 것을 밝혀짐
- 현재 시판되는 살균제를 사용하여 종자를 소독하는 방법으로는 볍씨 내에 존재하는 병원균의 완벽한 방제가 어려울 수 있으며, 벼 키다리병원균의 약제저항성 극복 대안으로서 병원균 세포벽의 합성을 저해하는 약제를 혼합하여 처리하거나 약제에 대한 efflux transporter의 작용을 저해하는 약제를 혼합하여 처리하는 방법을 제시
○ 4세부과제명 : 약제저항성 관련 유전자의 지역별 빈도분석
- 점박이응애 ace 유전자의 돌연변이를 근간으로 한 저항성 마커를 활용한 유전형질 분석을 위한 QS 회귀식 산출 및 QS를 이용한 집단 유전 형질 분석이 가능한 방법 개발
- 초위성체 마커를 활용한 개체군 군집분석 방법 개발하였는데, 일부 마커를 제외한 대부분의 마커가 Hardy-Weinberg Equilibrium test에 적합한 것으로 나타났음
○ 5세부과제명 : 아릴옥시 페녹시계 제초제 저항성 피 관리기술개발
- ACCase 저항성 피를 효율적으로 관리하기 위해서는 저항성을 조기에 검진할 수 있는 간이 신속검정법 확립
- 피 발생 전 제초제 처리 후 방제 효과가 높은 약제는 뷰타클로르 33% 유탁제, 옥사디아존 12% 유제, 펜톡사존 5% 액상수화제, 옥사디아길 1.7% 유제, 펜트라자마이드·옥사디아길 3.3% 유제 등의 5종만 95% 이상 방제
- 저항성 일년생잡초 발생이 심한 논과 저항성 다년생잡초 발생이 심한 논에 따른 방제 매뉴얼 작성
○ 6세부과제명 : 아릴옥시페녹시계 제초제 저항성 피 발생 메커니즘 규명 및 시,공간적 확산예측
- 충남 서산간척지에서 저항성 피를 국내 최초로 확인
- 아릴옥시페녹시계 제초제 저항성 물피와 강피의 저항성원인은 작용점인 ACCase는 물론 대사에 의한 원인 구명 및 저항성 조기 진단 기술 개발
- 저항성 피의 분포 및 세대진전에 따른 시간적 확산 평가를 통한 저항성 피 확산 예측 모델 개발
○ 7세부과제명 : 벼 병원균의 약제저항성 모니터링 및 최적관리기술 개발
- 벼키다리 병원균 (F . fujikuroi) 분리균의 저항성 판단 기준 농도 설정 및 약제 별 저항성 정도 분석 및 이중 저항성 패턴 분석
- 국내 벼 키다리병균 CYP51 유전자 염기서열 비교분석

Abstract ▼

Two spotted spider mites (TSSM) were caught in 9 sites of strawberry cultivating area, Geochang, Goryeong, Gokseong, Nonsan, Damyang, Milyang, Sancheong, Wanju, Oegwan. Local TSSMs showed different susceptibility to same insecticides. Abamectin, Aquicenocyl, Milbemectin, Bifenazate, Propargite, Azpc

Two spotted spider mites (TSSM) were caught in 9 sites of strawberry cultivating area, Geochang, Goryeong, Gokseong, Nonsan, Damyang, Milyang, Sancheong, Wanju, Oegwan. Local TSSMs showed different susceptibility to same insecticides. Abamectin, Aquicenocyl, Milbemectin, Bifenazate, Propargite, Azpcyclotin, Dicofol, Tebufenpyrad, Chlorfenapyr were still effective to TSSM control. TSSM showed high resistance to Monocrotophos and Fenpropathrin, also to Pyridaben and Spiromesifen. According to response of TSSM to insecticides, we could group the insecticides for managing insecticide resistance of TSSM. We can choose insecticides for TSSM control by rotating insecticide groups. Monocrotophos, Pyridaben, Tebufenpyrad, Fenpropathrin belonged to first group and were not so effective to TSSM. Second group is consist of Spiromesifen and Penproxymate, and also not so effective to TSSM. Third group Abamectin, Milbemectin, Propargite and Azocyclotin. The third group showed good control effect to TSSM. Ganemite, Aquicenocyl, Bifenazate, Fenbutanoxide, Dicofol and Chlorfenapyr belonged to fourth group. This group was also effective to TSSM. In the control test of insecticides to whitefly (Bemesia tabacci), Spiromesifen, Dinotefuran and Pyridaben were very effectivie to the eggs of whitefly. Neo nicotinoids which were frequently used for adult whitefly, did not showed any effect to eggs of whitefly. Emamectin benzoate. Spiromesife, Dinotefura and Pyridaben were effective to nymphs of whitefly. The adult whitefly was susceptible to Emamectin benzoate, Dinotefuran and Pyribaben.
The green peach aphid, M yzus persicae (Sulzer), is one of the most serious pest in cabbage cultivation. Field survey was carried out to know the insecticide resistance levels in five main cabbage cultivation regions (Pyeong-chang, Hong-cheon, Bong-wha, Mu-ju and Je-ju) during 2009 to 2011. M . persicae can resist a wide range of insecticides in five surveyed local populations. Among the nine tested insecticides, four chemicals (methomyl, bifenthrin, pymetrozine and flonicarmid) generally showed less than 60% mortality in the recommended concentration in all populations. Multi resistant (MR) strain was selected from these populations and esterase over-expression, modified AChE (MACE) and mutation(s) in para-type sodium channel were analysed using native IEF and quantitative sequencing with five local populations. Esterase over-expression and MACE (StoF mutation) were observed in all populations including MR strain. LtoF mutation is well known as a kdr mutation in para-type sodium channel. However, even though LC₅₀ values of MR strain noted over 2,000 times higher than that of susceptible strain against bifenthrin, LtoF mutation was not detected in para type sodium channel and also local populations. We found a novel mutation (MtoL) in para and that mutation highly correlated between mutation ratio and bioassay data. For preliminary resistance monitoring, we developed quantitative sequencing (QS) to detect the frequencies of point mutation in partial fragment of ace1 and papa as a population genotyping. These methods can apply to manage M . persicae resistant populations in field.
The cotton aphid, Aphis gossypii (Glover), is also one of the most serious pest in various vegetables. Neonicotinoid insecticides exhibit niceish insecticidal activity in M . persicae. However, some field populations of A. gossypii showed high resistant against neonicotinoid. The imidacloprid-resistant strain (IR) selected from that populations and that was 278 folds more resistant to imidacloprid compared to a susceptible strain (S) as judged by LC₅₀ values. The IR showed cross resistances to other neonicotinoid insecticides. IEF and 2DE analyses revealed that general esterase isozyme patterns in IR were almost identical to those of S. Nevertheless, a significantly overexpressed protein spot was detected in IR. To identify differentially expressed genes in IR, comparative transcriptome analyses based on GS-FLX were conducted using total RNAs extracted from both IR and S strains, which generated ca. 290 Mb reads for each strain. Generally, most nicotinic acetylcholine receptor subunit genes, such as alpha 2 and beta 1, were more transcribed in S than in IR. In contrast, only alpha 5 subunit gene was 1.8 fold more expressed in IR. Seven ATP-binding cassette (ABC) transporter genes were newly identified in A. gossypii, among which only ABCC9 gene was highly expressed in IR.
Therefore, this ABCC subfamily, a member of the MRP subfamily which is involved in multi-drug resistance, could be one of the factors associated with imidacloprid resistance in A. gossypii. Comparison of the nucleotide sequence of six nicotinic acetylcholine receptor (nAChR) subunit (alpha 1-5, beta 1) genes from S and IR strain revealed a point mutation in the loop D region of the nAChR beta 1 subunit of the IR, causing an arginine to threonine substitution (R81T). These mechanism also reported in M . persicae and previous studies have shown that the amino acid at this position within loop D is a key determinant of neonicotinoid binding to nAChRs and this amino acid change confers a vertebrate-like character to the insect nAChR receptor and results in reduced sensitivity to neonicotinoids. This is the first investigations of imidacloprid resistance mechanism in A. gossypii and also can apply to manage A. gossypii resistant populations in field.
In Korea, a number of reports on the emergence of rice bakanae pathogens with resistance against the fungicide prochloraz have increased since 2004. The pathogens cause severe problems such as production of empty grains and decrease in yield of rice crop. Understanding the resistance mechanism is of essentials to develop alternatives in order not to have fungicide resistance. The aim of the present study was to investigate resistance mechanisms of F usarium fujikuroi strains, rice bakanae pathogens, against prochloraz. Examination of fungal sensitivity to prochloraz showed that the strain F. fujikuroi CF106 was sensitive while the strains F . fujikuroi CF245 and F . fujikuroi CF337 were resistant to prochloraz. The resistant strains degraded prochloraz to N-[2-(2,4,6-trichlorophenoxy)ethyl]propan-1-amine and increased their cell wall thickness after prochloraz treatment as resistance mechanisms. They were also capable of removing prochloraz from their cells through ABC transporter, which was evidenced by a significant inhibition of fungl growth in the presence of prochloraz and membrane electron transfer inhibitors. These results suggest that cell wall thickening and efflux ransporters could be mechanisms responsible for prochloraz resistance of rice bakanae pathogens.
Resistances to monocrotophos, fenpropathrin and abamectin in the TSSM are primarily conferred by reduced sensitivity of respective target sites (i.e., TuAChE, TuVSSC and TuGluCl), which is due to point mutations (G228S and F439W in TuAChE; L1022V in TuVSSC; G323D in TuGluCl). As a population-based genotyping technique, quantitative sequencing (QS) protocol was developed for the detection of these four mutations putatively responsible for resistance in the TSSM. To generate standard prediction equations, the resistant nucleotide signal ratios for each mutation were plotted against the corresponding resistance allele frequencies and fitted to linear and quadratic equations. This process resulted in high correlation coefficients (r² = 0.993~0.999), demonstrating that the resistant nucleotide signal ratio is highly proportional to the resistance allele frequencies. The lower detection limits for the five resistance mutations were 3.7~13.4% (7.7 ± 3.7%), suggesting that QS can be employed as a preliminary resistance monitoring tool for the detection of resistance allele frequencies higher than ca. 3.7% at the 95% confidence level. The QS was successfully employed for the determination of resistance allele frequencies in 9 field-collected TSSM samples. The two mutations responsible monocrotophos resistance was almost saturated in most field populations. The L1022V mutation tentatively responsible for fenpropathrin resistance was also found in 8 field populations. However, the G323D mutation putatively associated with a high level of abamectin resistance was found only in one field population, suggesting that the high level abamectin resistance is not widespread yet. The QS protocol, as an alternative to traditional bioassays, will greatly facilitate resistance monitoring of the TSSM.
Altogether the allele frequency analysis, the genetic relatedness of local population was analyzed by using microsatellite markers. The eight population was divided by three clades by PCA analysis. However, it was not correlated with geographical distance, suggesting the artificial factors such as transporter or human beings might be associated the distribution of TSSM.
Two biotypes of E. crus-galli var. crus-galli were collected from Seosan Hyundai farm, Seosan(resistant biotype) and Suwon(susceptible biotype) in 2007 and tested with cyhalofop-butyl, to establish method diagnosing rapidly if E. crus-galli seeds corrected were resistant to cyhalofop-butyl EC. The results are summarized as follows. Germination of new shoot in the a part cut 24 hours after treatment with cyhalofop-butyl EC was responded differently by biotypes of E. crus-galli collected. In case of Suwon biotypes, the shoot in the cutting parts was not almost germinated in the treatment above standard amount whereas it was germinated from 2 days after cutting in most treatment of Seosan biotypes(R). R/S ratios by Dose-responses of Seosan(R) and Suwon biotypes(S) in the lower cutting parts after treatment with cyhalofop-butyl showed values of 3.4 and 5.7 at 4 and 5 leaf stage, respectively. Accordingly, judgement by response of shoot in the parts cutting 24 hours after treatment was considered to be effective as method to diagnose rapidly if E. crus-galli were resistant to cyhalofop-butyl.
The effects are investigated by herbicides and phases to enhance integrated weed management(IWM) of herbicide-resistant E. crus-galli. Soil-treatment herbicides for resistant E. crus-galli has proven that oxadizon 12% EC, oxadiargyl 1.7% EC and fentrazamide·oxadiargal 3.3% EC have been 100% effective, and pentaxazon 5% SC has been over 98% effective in spite of E. crus-galli broke out after 31 days from application. By leaf ages, 5 herbicides, mefenacet·pyrazosulfuron-ethyl 3.57% GR, bensulfuron-methyl· fentrazamide 7% SC, azimsulfuron·carfenstole 1.05% GR, bensulfuron-methyl·benzobicyclone· mefenacet 24.52% SC and bensulfuron-methyl·mefenacet·oxadiargyl 21.6% SC have been 100% effective to 1.0∼2.5 leaf/leaves ages. Two herbicides, mefenacet·pyrazosulfuron-ethyl 3.5% GR and benzobicyclone·mefenacet·penoxulam 21.5% SC, can be effective until 3.0 leaves age. It seems that the choices of pre-sprouting, early-stage and mid-stage herbicides and their systemized applications are important for control of E. crus-galli resistant to ACCase and ALS herbicide.
Echinochloa species accessions collected in Seosan, Iksan and Gimje showed resistance to aryloxyphenoxy propionate herbicides (ACCase inhibitors) and some of them also showed resistance to ALS inhibitors such as sulfonylurea herbicides. Our study revealed that those resistant Echinochloa species appeared to be wide-spread, particularly in western costal area where herbicide use in rice cropping has intensely been made.
Herbicide resistance mechanism study showed that metabolism seems to be more responsible for such resistances than target sites such as ACCase and ALS. We also developed a rapid bioassay method for diagnosing ACCase and ALS inhibitor resistance in Echinochloa species using the growth pouch. Herbicide dose-responses by the growth pouch method were highly correlated with those from whole plant test, suggesting that the growth pouch method can be utilized to diagnose herbicide resistance in Ecinochloa species with significant time and cost-savings as compared with the whole plant test. To control these resistant Echinochloa species, herbicides with different modes of action were also tested against herbicide resistant Echinochloa species. As a result, herbicides belonging to PPO and VLCFA inhibitors showed good efficacy against these resistant Echinochloa species, suggesting that they can be used for herbicide resistant Echinochloa management. However, their application window, only up to 2 leaf stage, is narrow, indicating that alternative herbicides which can control resistant Echinochloa greater than 3 leaf stage need to be developed and more integrated weed management strategy including sequential herbicide application system and reformation of herbicide regulatory and recommendation system is strongly required. Although this study is successfully completed, further works are strongly recommended to tackle wide-spread of herbicide resistance in Echinochloa species by regular resistance monitoring, herbicide baseline study and so on.
Rice bakanae disease caused by F usarium fujikuroi is one of the most serious rice diseases in Korea. From 2006 to 2009, F . fujikuroi isolates were collected from various regions of rice fields in Korea. Resistance assay of F . fujikuroi isolates to prochloraz, tebuconazole, and benomyl, were performed using agar dilution method. To investigate inhibitory effects of the fungicides, minimum inhibitory concentration of mycelial growth (MIC) and effective concentration inhibiting mycelial growth by 50% (EC50) for the F. fujikuroi isolates were calculated using Sigmaplot 8.02 (Antro, SPSS UK, Ltd). Based on the means of EC50 values, baseline resistance values were determined as 0.5 μg mL^-1 for prochloraz, 5.0 μg mL^-1 for tebuconazole, 8.0 μg mL^-1 for hexaconazole and 2.5 μg mL^-1 for benomyl. Number of resistant isolates to each fungicide was 24, 2, 28 and 57 for prochloraz, hexaconazole, tebuconazole and benomyl, respectively. Furthermore, 4 isolates showed the double resistance to both prochloraz and tebuconazole, 15 isolates to prochloraz and benomyl, and 6 isolates to tebuconazole and benomyl. Isolates CF366 and LF335 isolated from Gyeongbuk province were resistant to the three fungicides tested, prochloraz, tebuconazole and benomyl.

목차 Contents

• 제 출 문 ... 1
• 요 약 문 ... 3
• SUMMARY ... 4
• 제 1 장 서 론 ... 9
• 제 2 장 국내외 기술개발 현황 ... 13
• 제 3 장 연구개발 수행 내용 및 결과 ... 14
• 제1절 : 시설재배 주요해충의 약제저항성 모니터링및 관리기술개발 ... 14
• 제2절 : 노지 양채류 미소 해충 약제저항성 모니터링 및 기작 구명 ... 24
• 제 3 절 약제저항성 유발 병원균의 살균제 저항성 기작 구명 ... 44
• 제 4 절 약제저항성 관련 유전자의 지역별 빈도분석 ... 66
• 제 5 절 아릴옥시 페녹시계 제초제 저항성 피 관리 기술 개발 ... 73
• 제 6 절 아릴옥시페녹시계 제초제 저항성 피 발생 메커니즘 규명 및 시,공간적 확산예측 ... 90
• 제 7 절 벼 병원균의 약제저항성 모니터링 및 최적관리기술 개발 ... 106
• 제 4 장 연구개발 목표 달성도 및 대외기여도 ... 116
• 제 1 절 목표대비 대외달성도 ... 116
• 제 2 절 정량적 성과(논문게재, 특허출원, 기타)를 기술 ... 118
• 제 5 장 연구개발결과의 활용계획 ... 118
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 121
• 제 7 장 기타 중요 변동사항 ... 123
• 제 8 장 국가과학기술종합정보시스템에 등록한 연구장비 현황 ... 123
• 제 9 장 참고문헌 ... 124
• 주요 결과 요약서 ... 135