[보고서]밤나방류와 흰등멸구의 살충제 저항성 분자 진단 기술개발

권민

밤나방류와 흰등멸구의 살충제 저항성 분자 진단 기술개발
Development of molecular diagnostic technique for insecticide resistance from Noctuidae pests and White-backed planthopper 원문보기

보고서 정보
주관연구기관	국립식량과학원 National Institute of Crop Science
연구책임자	권민
참여연구자	김주일 , 남화연 , 최낙중 , 김현주
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2021-02
과제시작연도	2020
주관부처	농촌진흥청 Rural Development Administration(RDA)
등록번호	TRKO202100009988
과제고유번호	1395064390
사업명	작물시험연구(R&D)
DB 구축일자	2021-10-02
키워드	살충제 저항성.종합적 저항성 관리.분자 진단법.밤나방과.흰등멸구.Noctuidae.Insecticide resistance.Molecular diagnosis.Diagnosis.Resistance management.

초록 ▼

[연구개발 성과]
- 파밤나방 방제효과가 높은 살충제 6종 선발(Fluxametamide 등)
- 파밤나방 diamide 살충제 저항성 국내 분포도 작성(2014-2020)
- 생물검정을 이용한 파밤나방 diamide 살충제 저항성 특성 분석
- 생물검정을 이용한 담배거세미나방 살충제 저항성 특성 분석
- 파밤나방에 대한 diamide 살충제 저항성 기작 구명 및 유전변이 탐색
- 저항성 계통 특이적 유전적 변이 기반 진단 마커 개발, 활용
- 파밤나방의 diamide 살충제 저항성 분자진단법 개발 LAMP, PCR
- 왕담배나방의 pyrethroid계 살충제 저항성 분자진단법 개발 LAMP
- LAMP는 약 90분이면 저항성 진단이 가능하고 개체 수준의 진단 가능
- 국내 비래 흰등멸구 개체군의 확보 및 실내사육, 저항성 검정
- 에토펜프록스 등 저항성 발달이 높은 살충제 선발, 시기적, 지리적 분석
- 흰등멸구의 살충제 저항성 기작 구명, 저항성 관여 유전자 분석
※ 정량적 성과: 논문 SCI 1, 영농기술정보 4, 학술발표(국제) 6, 학술발표(국내) 4, 생명정보 등록 6, 농가현장기술지원 4

(출처 : 요약서 3p)

Abstract ▼

Purpose&Contents
○ Purpose of study: Insecticide resistance survey and molecular diagnostic technology development of Noctuid pests and Sogatella furcifera
○ Research content:
- Examine the resistance of registered insecticide through bioassay experiment of Noctuid pests and Sogatella furcifera by region
- Determine the insecticide resistance mechanism of Noctuid pests and Sogatella furcifera, investigate the resistance related gene and developemnt of molecular marker based on gene mutation
- Development of molecular diagnostic marker to diagnosis the insecticide resistance of Noctuid pests and Sogatella furcifera

Results
○ Selection of six high control efficacy insecticides in Spodoptera exigua (Fluxametamide etc)
○ Mapping the diamide insecticide resistance ofSpodoptera exigua in Korea (2014-2020)
○ Analysis the diamide insecticide resistance characteristic of Spodoptera exigua through bioassay experiment
○ Analysis the diamide insecticide resistance characteristic of Spodoptera litura through bioassay experiment
○ Determine the diamide insecticide resistance mechanism and investigate gene mutation in Spodoptera exigua
○ Development and application of molecular diagnostic marker to diagnosis the genetic variation in resistance strain
○ Development of insecticide resistance molecular diagnostic method (LAMP, PCR) of Spodoptera exigua
○ Development of pyrethroid insecticide resistance molecular diagnostic method (LAMP, PCR) of Spodoptera litura
○ Secure the migration population of Sogatella furcifera in Korea and proceed the indoor breeding and the bioassay experiment
○ Select the insecticides, such as etofenprox etc, based on high level of resistance development, and analysis timely and geographically
○ Determine the insecticide resistance of Sogatella furcifera, and analysis the resistance involvement gene

Expected Contribution
○ Insecticide resistance of Noctuid pest such as Spodoptera exigua is extremely serious, therefore, the research content of resistance mechanism in this project is interested in domestically and internationally which may utilize in resistance research through international collaboration
○ Local government and farm corporation may diagnosis insecticide resistance by using LAMP and this method may applied to skill instruction and utilized to farming education data
○ Research in insecticide resistance and selection of suitable insecticide constantly required from framers, therefore, this information may utilized to farming education data
○ Insecticide resistance and molecular diagnostic technology development of Sogatella furcifera research will be lined to future project which may utilized to establish advanced countermeasures in resistance management

(출처 : Summary 5p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
국 문 요 약 문 ... 4
Summary ... 5
목차 ... 7
제 1 장 연구 개발 과제의 개요 ... 8
제1절 연구 개발 목적 ... 8
제2절 연구 개발의 필요성 ... 9
제3절 연구 개발 범위 ... 10
제 2 장 연구 수행 내용 및 결과 ... 11
제 3 장 목표달성도 및 관련분야 기여도 ... 41
제1절 목표대비 달성도 ... 41
제2절 정량적 성과(논문게재, 특허출원, 기타)를 기술 ... 41
제 4 장 연구 결과의 활용 계획 ... 42
제 5 장 연구 개발 결과의 보안 등급 ... 43
제 6 장 연구시설·장비종합정보시스템에 등록한 연구시설·장비 현황 ... 44
제 7 장 연구개발과제의 대표적 연구실적 ... 45
제 8 장 기타사항 ... 46
제 9 장 참고문헌 ... 47
끝페이지 ... 52

표/그림 (34)

표 Susceptibility of a field population of Spodoptera exigua against 22 insecticides. 3rd instar larvae were used and insecticide were treated in the artificial diet. The same letter within a graph were not significantly different at P < 0.05 using Tukey’s range test (SAS 9.4)
표 Susceptibility of various populations of Spodoptera exigua against Chlorantraniliprole that one of the representative diamide insecticide at recommended concentration. The local populations of S. exigua were collected from potato, cabbage and green onion fields in each year (2014, 2017, 2018 and 2020). 2019 bioassay result documented separately as a table. 3rd instar larvae were used and insecticide were treated in the artificial diet
표 Susceptibility of six populations of Spodoptera exigua which collected in 2019 against two diamides (Chlorantraniliprole and Flubendiamide)
표 Susceptibility of diamide susceptible and resistant strain of Spodoptera exigua against two diamides (Chlorantraniliprole and Flubendiamide)
표 Diamide resistant and susceptible strain (A) F1 and (B) F2 hybrid screening results. (A) Chlorantraniliprole was used for F1 hybrid screening in recommended concentration. Five families of RS (R♂ × S ♀) and SR (S♂ × R♀) were used, respectively. (B) F2 hybrid was generated via F1 families mass mating and five diamide insecticides were tested. Third instar larvae were used with three replications. There were not significantly different at P < 0.05 using Tukey’s range test (SAS 9.4)
표 Susceptibility of a field population of Spodoptera litura against 16 insecticides. 3rd instar larvae were used and insecticide were treated in the artificial diet. The same letter within a graph were not significantly different at P < 0.05 using Tukey’s range test (SAS 9.4)
표 Mortality comparisons of four diamide and a meta-diamide insecticide to filed population of Spodoptera litura which collected in Miryang, 2019
표 Susceptibility of susceptible and multiple resistant strain as well as it’s F1 and F2 hybrids of Spodoptera litura against three insecticides. 3rd instar larvae were used and insecticide were treated using topical applicator. Technical grade insecticide were diluted in acetone. The same letter within a graph were not significantly different at P < 0.05 using Tukey’s range test (SAS 9.4)
표 Statistics of raw and trimmed data stats for RNAseq of diamide resistant (R), susceptible (S) strains and its F1 hybrid (RS)
표 Statistics of unigene and ORF prediction for RNAseq of diamide resistant, susceptible strains and its F1 hybrid
표 Heat map for hierarchical clustering using Z-score for normalized value (log2 based)
표 MDS (Multidimensional Scaling) plot result
표 Volcano plot between diamide resistant (BAW-R) and susceptible (BAW-S) strains
표 Heatmap for cytochrome P450 gene family expression level based on RNAseq of diamide resistant (R), susceptible (S) strains and its F1 hybrid (RS)
표 BLASTx result of partial Ryanodine receptor gene, particularly trans membrane region (A). Point mutation only found in ItoM (B) not in GtoE (C)
표 Representative chromatograms of direct sequencing for I4790M mutation genotyping in ryanodine receptors from (A) susceptible and diamide-resistant strains of Spodoptera exigua. Green and red boxes denote the I4790M mutation sites in susceptible and resistant strains, respectively. (B) Diamide-resistant strain-specific transcript variants and the existence of indels were confirmed via gel electrophoresis of PCR results using gDNA as a template (RyR_4790UF and RyR_I4790M_R primer set was used). (C) Resistant alleles were amplified from cDNA and gDNA templates of the diamide resistant strain and its F1 hybrids, SR (male susceptible × female resistant strain) and RS (male resistant × female susceptible strain) . F1 hybrids generated by single pair mating using susceptible and diamide-resistant strains. Diagnostic LAMP primer, BAW_RyR_F3 and BAW_RyR_B3 were used. Primer information is documented in Table 4
표 Location of primers and primer binding regions on partial sequence of in ryanodine receptor, Spodoptera exigua. Sequence alignment of six local populations with two susceptible and a diamide resistant strains. I4790M mutation was found in diamide resistant strain and six local populations. Amino acid sequence noted according to the DNA sequence. LAMP primers designed on intron region. Inner primer, FIP is consisted of F1c (complementary sequences of F1) and F2. Another inner primer, BIP is also composed of B1 and B2c (complementary sequences of B2). Essential four LAMP primers (F3, FIP, BIP and B3) generate the dumbbell structure and two loop primers, LF and LB accelerate the LAMP reaction. More detail used primer information is documented in the Table 4
표 Sensitivity of the LAMP assay results on three range of working temperature under (A) visible light, (B) ultraviolet light with Cyber Green and (C) for diamide resistant allele detection. Using four primer set (BAW_RyR_F3, B3, FIP and BIP), LAMP assay was tested in three incubation temperature, 65, 63 and 61℃ for 2 hours. The original pink color of the reaction mixture turned yellow in a positive reaction when product was formed but remained pink in negative reactions (A). NC: negative control, S: susceptible strain and Di-R: diamide resistant strain
표 The LAMP assay results with (A) 4 primers and (B) additional loop primers, (C) loop forward, LF or (D) loop backward, LB and LF with LB under visible light (left side) or ultraviolet light with Cyber Green (right side). LAMP assay was tested in an incubation temperature, 65℃ for 90 minutes. NC: negative control, S: susceptible strain, Di-R: diamide resistant strain, Sn: another susceptible stain from NAS, National Institute of Agricultural Sciences, SR and RS: F1 hybrid between S and Di-R
표 Identification of the detection limit of genomic DNA in the LAMP assay from 10ng to 1fg under (A) visible light, (B) ultraviolet light with Cyber Green and (C) gel electrophoresis. LAMP assay was tested in an incubation temperature, 65℃ for 90 minutes with six primers
표 Sensitivity of the LAMP assay results with the DNA releasing technique from insect tissue. (A) Around 10mg of larval tissue or adult leg (or antenna) were incubated in 95℃ for 5minutes. (B) LAMP products under visible light, (C) ultraviolet light with Cyber Green and (D) and gel electrophoresis. Abbreviations as in Fig. 7, except PC (positive control, isolated DNA from diamide resistant strain, Di-R)
표 Sensitivity of the LAMP assay results for resistant allele survey on filed collected local populations with susceptible and diamide resistant strains under (A) visible light, (B) ultraviolet light with Cyber Green and (C) gel electrophoresis. NC: negative control, S: susceptible strain, Di-R: diamide resistant strain, Sn: another susceptible stain from NAS, National Institute of Agricultural Sciences, SR and RS: F1 hybrid between S and Di-R. L1 ~ L11: field collected local populations, Anseong (AS), Cheongju (CJ), Gangneung (GN), Icheon (IC), Jindo (JD), Yeoju (YJ), Haenam (HN), Hoengseong (HS), Hongchen (HC), Miryang (MY) and Pyeongchang (PC)
표 Multiplex PCR results for (A) susceptible and resistant alleles diagnostic combination with positive control or (B) without positive control. Ace1 type acetylcholinesterase gene was used as a positive control. Alternatively, (C) resistant allele possibly amplified with positive control. Abbreviations as in Fig. 11
표 Statistics of raw and trimmed data stats for RNAseq of pyrethroid resistant (R), susceptible (S) strains of S. litura
표 Comparisons of deferentially expressed genes which related with detoxification in the S. litura. Detoxification reacted gene families such as P450 (cytochrome P450, UGT (UDP-Glucuronosyltransferase), ABC (ATP-binding cassette transporter), GST (Glutathione S-transferase), esterase were collected using InterProScan program. Some of deferentially expressed genes were selected from each detoxification reacted gene families
표 Phylogentic analysis of known pyrethroid resistant related genes in Helicoverpa armigera such as CYP321 and CYP337B3 with other cytochrome P450 form Noctuidae species including the S. litura (A). Three cytochrome P450 genes such as CYP6B5, CYP6a13 and CYP6B2-like were highly expressed in resistant starin (B)
표 Alignment of the DNA sequences of a CYP337B1, CYP337B2 and 10 different CYP337B3 alleles, a CYP337B3v1 and positions of 6 primers designed for specifically amplifying chimeric CYP337B3 by loop-mediated isothermal amplification (LAMP) reaction. A blue arrow head points to a crossing over site. Red arrows indicate primary priming regions of LAMP (F3 and B3). Purple arrows show the Forward internal primer (FIP, a combination of F1c and F2). Backward internal primer (BIP, a combination of B1c and B2) is shown in orange. CYP337B3v2 [MG744341]K and CYP337B3v7 [MG744342]K were cloned from Korean Helicoverpa armigera. Red letters indicate different sequence compared to that of CYP337B3v1
표 CBP337B3 detecting LAMP assay has a wide range of working temperature. To examine a range of working temperature for this LAMP assay, the LAMP mixtures were incubated in various temperatures from 61 to 65℃ at 2℃ intervals. Successfully amplified LAMP assays were detected under visible light (A1 and A2). The original pink color of the reaction mixture turned yellow in a positive reaction when product was formed but remained pink in negative reactions Amplified DNA ladders were clearly separated by 1.0% agarose gel electrophoresis (B1 and B2). In addition, field collected populations were tested using same procedure
표 2018년 흰등멸구의 채집량 및 채집 지역
표 2019년 흰등멸구 채집량 및 채집 지역
표 흰등멸구 살충제의 8종 살충제에 대한 저항성 검정 결과
표 흰등멸구 저항성 집단 탐색을 위한 11개 지역 개체의 살충제 검정 결과
표 흰등멸구의 연도별, 지역별 개체의 약제저항성 수준 진단 결과
표 살충제 저항성 흰등멸구의 개체간 전사체 분석 결과

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연합인증

밤나방류와 흰등멸구의 살충제 저항성 분자 진단 기술개발
Development of molecular diagnostic technique for insecticide resistance from Noctuidae pests and White-backed planthopper 원문보기