미세방울 디지털 PCR(Droplet digital PCR, ddPCR) 법은 정량 PCR 법과 같이 형광물질을 사용하여 정량분석이 가능하다는 점은 유사하지만, PCR 반응액을 만든 후 이 반응액을 바로 PCR에 사용하지 않고 수 만 개의 동일한 크기의 미세방울(droplet)로 구획(partitioning)한 후에 PCR 반응을 수행하는 점이 기존의 PCR 법과 가장 큰 차이라고 볼 수 있다. 이렇게 구획된 나노리터 크기의 미세방울 중에서 형광신호가 검출되는 미세방울과 검출되지 않은 미세방울의 수를 계수하여 프아송 분포(Poisson distribution) 계산에 적용하면 표준검량선 없이 목적 유전자의 절대정량 분석이 가능하다. 곤충이 매개하는 질병 바이러스의 경우 소량의 바이러스 감염 여부를 확인하기 위해서 다수의 곤충 유전자를 확인해야 하는데, 이와 같이 미세방울 형태의 ddPCR을 이용하면 기존 Real-time PCR 법에 비해 극소량의 목적 유전자를 높은 민감도로 검출할 수 있으며, PCR 저해요소(inhibitor)에도 큰 영향을 받지 않는다는 장점이 있다. 또한 미세방울 방식의 디지털 PCR을 이용하면 다중 PCR 분석이 가능하여 1개의 시료에서 다양한 질병매개 바이러스를 검출할 수 있다.

소나무재선충은 소나무에이즈라고 불리는 소나무해충으로서 전국적으로 큰 피해를 끼치고 있다. 최근 소나무재선충 피해는 경북지역의 경주 및 포항지역에 많이 발생하고 있다. 특히 경주지역에는 역사적인 유적지 및 고분공원이 많은데 이 지역의 소나무 숲에 재선충 피해가 확산되고 있어서 역사적 가치뿐만 아니라 관광산업에 큰 피해가 예상된다. 재선충 피해를 줄이기 위하여 재선충 감염목에 대한 신속한 제거작업이 필요한데 이에 대한 진단이 어려운 실정이다. 본 연구에서는 LAMP-PCR 진단키트를 이용하여 경주 고적지 지역의 재선충 감염이 의심되는 소나무를 진단하였다. 경주남산 및 불국사부근 7개 지역에서 재선충 감염 의심목으로부터 시료를 채취하여 조사해 본 결과 약 50%의 소나무에서 양성반응을 나타내었다. 본 연구를 통하여 경주 고적지의 재선충 감염여부를 신속하게 진단할 수 있었고 경주 유적지의 재선충 피해를 억제하는데 기여할 수 있을 것으로 판단한다.

Increasing numbers of insecticide resistant insect is one of the main problem in current agriculture. To manage the insecticide resistance, IRAC suggest some tips for farmers : First, sequence or alteration of insecticide based on MOA. Second, choosing the effective insecticide. In this point raised following question : How to distinguish the effective or non-effective insecticide. Until now, bioassay is the right answer. However that is time-consuming, labor-intensive and costly process. So we have been steadily advancing molecular based insecticide resistance diagnosis for supporting and/or substitution of classical bioassay. It can summarized following steps : First, identification of insecticide resistance mechanism. Second, marker development. Third, development molecular diagnosis such as PASA, LAMP etc. Forth, application on field collected insects. Here we discuss some case of these study particular several major pests in highland agriculture.

최근 국내 포도원에서 과실 표면에 구름모양의 흰색 얼룩이 자주 발견되고 있으나, 그 원인에 대해 명확히 알려진 바가 없어 방제대책을 세우지 못하고 있는 실정이다. 흰색의 달무리 반점은 포도 과실 표면에 부정형으로 퍼져 있으며, 그 중앙에는 총채벌레가 산란할 때 만들어진 작은 구멍의 상처가 남아 있다. 이 상처부위는 시간이 지나면서 코르크화되고 표피세포와 분리되어 딱지로 남거나 떨어져 나가게 된다. 이러한 증상은 총채벌레의 섭식이나 노린재의 흡즙에 의한 상처와는 구별된다. 산란구멍에서 발견한 총채벌레 알껍질에서 DNA를 추출하여 ITS2 부위의 염기 서열을 PCR-RFLP 방법으로 분자동정을 실시한 결과 꽃노랑총채벌레의 것으로 확인되었다. 미토콘드리아 COI 염기서열은 이러한 분자 동정 결과를 재확인하여 주었다. 본 연구결과는 포도 과피에 꽃노랑총채벌레 산란에 의해 유발되는 독특한 피해 증상에 대한 정확한 정보를 제공하며 포도원에서 이 해충에 대한 방제전략 수립에 도움이 될 것으로 여겨진다.

복숭아순나방붙이(Grapholita dimorpha Komai)는 동북아시아에 주로 발생하는 과수 해충으로, 국내에서는 2009년에 사과에 피해를 준다는 것이 보고되었으나, 그 이외의 과수에서는 직접적인 피해가 명확히 알려지지 않았다. 본 연구는 핵과류인 복숭아와 자두를 대상으로 복숭아 순나방류의 피해로 보이는 피해순과 피해과를 채집하여, 복숭아순나방붙이와 그 유사종인 복숭아순나방(Grapholita molesta Busck)의 피해율을 비교 조사하였다. 두 유사종의 정확한 구별을 위해 우선 종 특이적 프라이머와 PCR 반응조건을 이용한 분자동정법을 개발하였다. 복숭아와 자두의 신초와 과실을 가해하는 종을 야외에서 채집하여 분자동정법으로 확인한 결과, 복숭아의 신초와 과실은 거의 모두 복숭아순나방이 가해하였으며, 자두는 신초의 경우 복숭아순나방이, 과실의 경우 복숭아순나방붙이가 주로 가해하는 것으로 나타났다. 즉, 복숭아와 자두에서 조사한 신초는 모두(100%) 복숭아순나방에 의해 피해를 받았으나, 복숭아 과실은 대부분(92.5%) 복숭아순나방이, 자두 과실은 대부분(97.0%) 복숭아순나방붙이가 가해하는 것으로 나타났다. 본 연구결과는 복숭아순나방붙이가 복숭아 보다는 자두 과실에 주로 피해를 준다는 것을 보여주며, 특히 자두나무에서도 신초와 과실에 따라 각기 다른 종이 피해를 준다는 점을 보여준다. 이렇게, 기주식물에 따라 두 유사종의 가해특성이 다른 것은 복숭아와 자두과원에서 두 종의 발생을 예찰하는데 중요한 정보를 제공할 것으로 여겨진다.

Recently, a rapid movement of agricultural products with an extensive international trading and climate change have led to an increased attention to nematodes as having regulatory significance. With the increase of global dispersal of pests, new diagnosis methods are required for a rapid and reliable species and/or biotype identification to restrict introduction of the pests. Recently, novel molecular diagnostic techniques provide clues to solve taxonomic problems associated with conventional species identification. In this articles, we proposed various molecular diagnostic techniques to complement the limitation of morphological taxonomy.

The green peach aphid (Myzus persicae) is a cosmopolitan pest of agricultural and horticultural crops and causes serious economic damages. M. persica has rapidly developed resistance to a wide variety of insecticides, including pyrethroids. Target site insensitivity mechanism mediated by two mutations (L1014F and M918T) on the para-type voltage-sensitive sodium channel (vssc) is mainly responsible for pyrethroid resistance. To predict the vssc resistance allele frequency, quantitative sequencing (QS) protocol was established. Frequency prediction equations generated from the plots of signal ratios and amplification critical time showed a high correlation coefficient (r2>0.993), indicating its high accuracy in prediction. QS results revealed that the kdr-type L1014F mutation is only present in Pyeongchang strain. No field strains of M. persicae possessed the super-kdr type M918T mutation. However, a novel M918L mutation was found by genotyping approach. The allele frequencies of M918L and L1014F were 0% to 53% in populations examined, and the level of M918L mutation frequency was closely related with pyrethroid resistance. Therefore, QS-based detection of M918L mutation frequency should faciltate the monitoring of pyrethroid resistance in the field.

Larval stages of Callipogon relictus (Semenov) (Coleoptera: Cerambycidae), a gigantic longhorn beetle designated as a natural monument of Korea, has never been studied as it is hardly discovered in nature. The DNA barcoding gene, mt-COI, was used to identify a dead larva found in the Gwangneung forest of the Korea National Arboretum. Based on the result, we provide the morphology of the immature stage, with the illustrations of diagnostic characteristics.

Species of root knot nematodes, Meloidogyne spp. were identified through the PCR amplification and sequence analysis of mitochondrial DNA (mtDNA) region between COII and lrRNA genes. Root knot nematodes were extracted from 5 plant samples [kiwi (Japan), rhododendron (Japan), cornus (Japan), ficus (China) and jasmine (Vietnam)] that were collected from Plant Quarantine Station in Korea. At first, species was identified using morphological characters, such as the length and shape of stylet, the tail length and the perineal patterns. Secondary, single individuals of either female and juvenile collected from plant tissues were used for PCR and further sequencing analysis. The results showed that cornus, jasmine and ficus plants were infected by M. incognita, and kiwi and rhododendron plants were infected by M. interolobii.

Spider mites are one of major pests in cultivations of various ornamental plants and also important in plant quarantine service. Due to the very small body size and high similarity within the Genus the identification of species is difficult even at the microscopic observation. To identify 5 major species (Tetranychus cinnabarinus, T. urticae, T. phaselus, T. kanzawai and T. truncatus) in the Genus Tetranychus at the molecular level, we designed 4 species-specific primer sets using nucleotide sequences of the internal transcribed spacer (ITS2) region in the nuclear ribosomal repeat unit. At the PCR diagnosis of extracted genomic DNAs of 5 species using each primer set, specific primers of both T. phaselus and T. truncates were species-specific to their own species samples. However, specific primer set of T. urticae detected T. cinnabarinus as well as T. urticae. Specific primer set of T. kanzawai detected T. truncates as well as T. kanzawai, even though detection intensities were lower in non-target species.

The green peach aphid, Myzus 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) in 2009. The green peach aphid can resist a wide range of insecticides in five surveyed local populations. Among the nine tested insecticides, four chemicals (methomyl, bifenthrin, pymetrozine and flonicarmid) showed less than 50% 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, any mutation was not detected in para type sodium channel and also local populations. These results suggested that unusual case could be existed in pyrethroid resistance mechanism in green peach aphid.

Insensitive acetylcholinesterase (AChE) was determined to be basically involved in a EPN-resistant (ER) strain and ER contaminated susceptible (CS) strain of diamondback moth (DBM, Plutella xylostella L.), as estimated by the AChE inhibition assay using DDVP as a inhibitor in nondenaturing electrophoresis gel. ER strain exhibited very high insensitivity revel, high resistance ratio and two point mutations (G227A, A201S) in ace1. CS strain showed intermediate insensitivity level, low resistance ratio and a point mutation (G227A). This finding suggests that the A201S mutation along with reported G227A mutation (Baek et al, 2005) can be main player to develop the organophosphate resistance. Additionally, surveyed seven local population DBMs saturated G227A mutation and A201S mutation mixed in some population. Also A441G mutation was detected in some population, but there was no significant correlation. These results suggest that A201S mutation could be one of the good candidate for molecular diagnosis marker of resistance monitoring.

Root knot nematode species, such as Meloidogyne hapla, M. incognita, M. arenaria and M. javanica are economically most notorious nematode pests, causing serious damage to the various crops throughout world. In this study, DNA sequence analyses of the D1-D3 expansion segments of the 28S gene in the ribosomal DNA were conducted to characterize genetic variation of the four Meloidogyne species obtained from Korea and United States. PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism), SCAR (Sequence Characterized Amplified Region) marker and RAPD (Random Amplification of Polymorphic DNA) also were used to develop the methods for exact and rapid species identification. In the sequence analysis of the D1-D3 expansion segments, only a few nucleotide sequence variation were detected among M. incognita, M. arenaria, and M. javanica, except for M. hapla. The PCR-RFLP analysis that involves amplification of the mitochondrial COII and lrRNA region yielded one distinct amplicon for M. hapla at 500 bp, enabling us to distinguish M. hapla from M. incognita, M. arenaria, M. javanica reproduced by obligate mitotic parthenogenesis. SCAR markers successfully identified the four root knot nematode species tested. We are under development of RAPD primers specific to the three root knot nematodes found in Korea.