점박이응애(Tetranychus urticae)는 250과 1400여종의 식물에 피해를 주고 있는 해충으로 높은 번식력, 근친 교배, 무성생식, 짧은 수명주기 로 년간 발생회수가 많기 때문에 약제에 대한 저항성이 빠르게 발달하는 종이다. 저항성 기작은 AChE 저해, sodium channel modulator, glutamate gated chloride channels allosteric, mite growth inhibitors, inhibitors of acetyl CoA carboxylase, mitochondrial ATP synthase 저 해, mitochondrial complex I, II, III, 그리고 electron transport 저해 등이 있으며 약제에 따라 작용기작이 다르므로 약제살포 시 확인이 필요하 다. 약제저항성 발달을 평가하는 독성시험방법으로 slide dip, leaf dip, leaf disc, topical application, vial leaf dip, spray potter tower, leaf vial dipping 등이 있으며 최근에는 분자학적 진단법 등도 개발되어 있으나 이들 방법은 해충 종이나 발육상태에 약제에 따라 정확도가 달라지므로 현장 에 가장 맞는 방법을 찾아야 할 것이다. 저항성 점박이응애를 효과적으로 관리하기 위한 방법으로 저항성 위험이 낮은 살응애제(chemical acaricide) 약제 선발, 천적인 칠레이리응애, 사막이리응애를 이용한 생물적 방제, 님, 제충국, 등 식물추출물이나 정유를 활용한 친환경적인 방법 등이 제시되 고 있으며 종합관리를 위한 다양한 요인을 찾아 약제 사용을 줄여 나가야 할 것이다.

본 연구는 닭 진드기 구제를 목적으로 개발된 살비제인 와구잡이 II® (WGJB, 편백정유 : 계피정유 = 20 : 56)에 대하여 토끼와 기니픽을 이용하여 피부 자극성 및 감작성 평가를 각각 수행하였다. 일차피부자극시험에서 토끼의 피부에 WGJB를 24시간 동안 처리한 후 피부 자극성을 확인한 결과, WGJB는 비찰과 부위에서 홍반과 부종과 같은 어떠한 부작용도 일으키지 않았으나, 몇몇 토끼의 찰과 부위에서 매우 약한 홍반과 부종을 나타내어 WGJB의 1차 피부자극 지수는 0.625이었다. 따라서 WGJB는 약한 자극성이 있는 물질로 분류되었다. 피부감작성 시험에서 기니픽에 0.1 mL의 WGJB을 피내주사한 후 24시간 동안 감작시켰다. 감작 1주일 후 WGJB를 함유한 패취를 주사 부위에 부착하여 48시간 동안 처리한 다음, 2주 후에 WGJB 를 함유한 패취를 부착하여 감작을 야기시켰다. WGJB는 어떠한 알러지 반응도 나타내지 않았다. 따라서, 본 연구를 통하여 WGJB는 약한 피부 자극성을 가지며 감작성을 야기하지 않는 물질로 평가되었다.

Rapid resistance detection of resistance level is most important step to manage the resistant population in Tetranychus urticae in Korea. Residual contact vial bioassay (RCV) and quantitative sequencing (QS) methods were employed to determine the resistance level and applied to measure the resistance level of 46 field population collected from rose, strawberry and apple trees. Most populations exhibited multiple resistance pattern to various types of acaricides. Especially, the resistance levels in mites from rose cultivation area were higher than those from strawberry and apple cultivation areas. The resistance level detection would provide a useful parameter enabling the proactive action for a proper resistant population management for T. urticae.

The two spotted mite, Tetranychus urticae Koch, is a global pest, and has developed severe resistance to several types of acaricides. This study compared the development in susceptible (S) and acequinocyl, bifenazate, pyridaben, abamectin and etoxazole-resistant (AcR, BR, PR, AbR and ER) strains of T. urticae by X-ray irradiation. When eggs were irradiated with 100 Gy, the egg hatching was completely inhibited in all strains. When nymphs were irradiated with 200 Gy, their hatching was completely inhibited in all strains. When adults were irradiated, the fecundity and egg hatching was decreased at 150 Gy and above, and hatchability of F1 generation was completely inhibited at 300 Gy in all strains. Adult longevity was slightly increase at 150 Gy and above. In addition, we performed quantitative real-time PCR on several genes. To examine the difference of all strains on radiation stress-induced gene expression, we performed quantitative RT-PCR (qPCR) of several known stress-induced genes.

The two-spotted spider (Tetranychus urticae) is one of the most serious pests worldwide and has developed resistance to almost all types of acaricides. Various mutations on acaricide target and detoxification genes and their duplication (including overexpression) have been identified since the completion of T. urticae genome analysis. The mutations are mainly observed in functionally important domains (i.e. transmembrane, cellular loops and catalytic triad, etc.), which likely confer acaricide resistance directly or indirectly. Gene duplication was found on major detoxification and insecticide target enzymes, such as cytochrome P450, glutathione S-transferase, ABC-transporter, UDP-glycosyltranferase and acetylcholinesterase (AChE). Interestingly, co-occurrence of both mutation and gene duplication (especially, single gene amplification) was found in AChE, which possibly explains the compensatory role of gene duplication to minimize the fitness cost mediated by point mutations. Such mutation and duplication traits associated with resistance can be utilized as molecular markers for the determination of resistance levels based on the quantitative sequencing and real-time PCR.

The detoxification enzymes activities were investigated to Tetranychus urticae Koch using five acaricide-resistant strains. Activities of detoxification enzymes which are glutathione S-transferase (GST), general esterases (α-naphthyl acetate and β-naphthyl acetate), and cytochrome C oxidase were determined to each resistant strain mite. Acequinocyl-resistant strain and bifenazate-resistant strain of T. urticae were showed 2.1 folds and 1.6 folds higher relative activity (RA) level of GST than susceptible strain. Other three resistant strain mites were not significant different to susceptible strain mite. General esterases and cytochrome C oxidase were not significant to all the strain of T. urticae. Acaricidal activities of acequinocylresistant strain and bifenazate-resistant strain of T. urticae showed cross-resistant both acequinocyl and bifenazate. However, other strain mites were showed susceptible acaricidal activities to two acaricides. In this result suggests that resistance of two acaricides (acequinocyl and bifenazate) might be influenced by glutathione S-tansferase activity.

Emergence of resistant two-spotted spider mite (TSSM) can induce the over usage of standard amount of acaricides and result in various side effects. Rapid resistance monitoring is essential step for the efficient management of resistant populations by enabling the selection of appropriate acaricides. Here, we evaluated the 19 acaricides to determine its suitability for residual contact vial bioassay (RCV) by using PyriF strain as a reference. Twelve acaricides (Amitraz Abamectin, Bifenthrin, Bifenazate, Chlorfenapyr, Cyenopyrafen, Cyflumetofen, Endosulfan, Fenothiocarb, Monocrotophos, Omethoate and Tebufenpyrad) revealed the dose-dependent mortality within 8 h, whereas other remaining acaricides (Dicofol, Etoxazole, Fenbutatin oxide, Fenpyroxymate, Flufenoxuron, Spiromesifen and Pyridaben) did not. This finding suggests that the application of RCV method is limited depending on the mode of action and physicochemical properties of each acaricide. Resistance levels to 12 acaricides were determined for four field populations of TSSM by using RCV diagnostic kit. All TSSM populations showed the highest sensitivity to cyflumetofen, indicating that it would be most effective in controling field populations. RCV diagnostic kit would enable to provide crucial information for choosing the most appropriate acaricides in the field.

Acquisition of a reference Tetranychusstrains that are completely susceptible to acaricides and retain identical genetic backgrounds to acaricide-resistant strains is an essential step in elucidating mechanisms of resistance. To establish both completely susceptible and acaricide-resistant strains for this purpose, I collected Tetranychus mite populations from various regions in South Korea including both heavily cultivated and remote regions. Suitability as a susceptible or resistant reference strain was tested by determining species identity as Tetranychus urticae along with baseline susceptibility to common acaricides. The majority of mite populations collected from cultivated areas belonged to a monophyletic group of the previously reported green-type T. urticae as determined by mtCOI- and ITS2-based phylogenetic analysis. These strains showed relatively reduced levels of susceptibility to the acaricides tested, suggestive of the development of resistance. Among them, the AbaR strain was classified as a minor group in the T. urticae complex. The UD strain, originally collected from a remote island region, was found to be susceptible to the acaricides tested and generated an independent phylogenetic branch. The UD strain was also considered a minor group in the T. urticae complex. Phenotypic analysis based on morphological characters confirmed that both the AbaR and UD strains were statistically undistinguishable from the major green-type T. urticae. Taken together, I propose that the UD strain be used as a susceptible reference strain as it provides baseline susceptibility to acaricides and a wild-type genetic background for the resistance studies.

The toxicity of 10 plant essential oils to adults of acaricide-susceptible, chlorfenapyr-resistant (CRT-53), fenpropathrin-resistant (FRT-53), pyridaben-resistant (PRT-53), and abamectin-resistant (ART-53) strains of Tetranychus urticae Koch and to female Neoseiulus californicus McGregor was examined using spray or vapor-phase mortality bioassays. In bioassay with the susceptible adults, strong toxicity was produced by lemon eucalyptus, peppermint, citronella Java, thyme red, caraway seed, pennyroyal, and clove leaf essential oils (LC50, 0.0193-0.0327 mg/cm3). The toxicity of these essential oils was almost identical against adults from either of the susceptible and resistant strains, even though CRT-53, FRT-53, PRT-53, and ART-53 adults exhibited high levels of resistance to chlorfenapyr [resistanc ratio (RR), >9140], fenpropathrin (RR, 94), pyridaben (RR, >390), and abamectin (RR,85), respectively. Against female N. californicus was 1.0 to 1.9 times more tolerant than T. urticae to the test essential oils. Thus, these essential oils merit further study as potential acaricides for the control of acaricide-resistant T. urticae populations as fumigants because of their lower toxicity to N. californicus.

This study was performed to investigate the bacterial diversity isolated from the twospotted spider mite and to interpret their correlation between insect bacteria and acaricide resistance. Twospotted spider mite, Tetranychus urticae was used the resistance strains, which developed over eight years to the six acaricides such as abamectin, acequinocyl, bifenozate, etoxazole, fenpropathrin, and pyridaben, respectively. After cultivating the bacteria from body maceration, bacterial colony was selected and identified through 16S rRNA gene sequences. We are identified six genus from Pyridaben resistant strain, five genus from acequinocyl, three genus from abamectin, bifenozate, etoxazole, and two genus from fenpropathrin. However, we could not found correlation between bacterial density and diversity (phylotypes) among these resistant strains. By analyzing the diversity of population microorganisms, fenpropathrin was showed 40% of Cs value (Similarity coefficient) with susceptible strain, however, abamectin and pyridaben were perfectly different (0%) with susceptible strain. It remains to be learned about how microorganisms co-evolutionary developed with their host insect correlating to the resistance and how microorganisms play role in acaricide resistant mite.

To establish a rapid diagnosis method for the monitoring of acaricide resistance in the two-spotted spider mite, Tetranychus urticae, we evaluated the performance of residual contact vial (RCV) method as a routine bioassay for T. urticae by using two widely used acaricides, abamectin and tebupenpyrad. Appropriate concentrations of test acaricides were dissolved in acetone and evenly coated (100 μl) onto the inside wall of a 4-ml glass vial using a rolling machine. The average survival times in untreated control vial was longer than 12 hrs in the absence of food or water regardless of cap being closed or open. Webbing behavior of mites inside the vial, which may interfere with maximum chemical contact, began to be observed from 8 hrs post treatment. The minimum concentrations causing 100% mortality within 8 hrs posttreatment in a susceptible strain of T. urticae were determined to be 30 and 60 ppm in abamectin and tebupenpyrad, respectively. Dose-response curve was significantly affected by temperature in both acaricides, in which the knockdown rate increased greatly as temperature increased. The endpoint mortality at 6-8 hrs posttreatment, however, was not significantly affected by temperature. Nymphal stage of mites showed more rapid intoxication response than adults but endpoint mortality at 6-8 hrs posttreatment was not substantially different between developmental stages. When compared with the results from conventional spray method, RCV method showed moderate to high correlation coefficients (r=0.51~0.98), suggesting that it is a reliable in determining susceptibility of T. urticae. The vial-coated pesticides were stable at least one year when stored at -20°C as determined by LC-MS/MS analysis. Moreover, there was no significant difference in the bioassay results in repeated experiments with three different persons, indicative of high reproducibility of RCV. The RCV diagnostic kit, when used by farmers on site, should provide crucial and essential information for the selection of most suitable acaricides for different field populations of T. urticae.

Population density of the citrus red mite, Panonychus citri (McGregor), in Japanese pear orchards remained low until mid-August, even after inoculation of pear leaves with a considerable number of adult female P. citri from May onwards. This raised the possibility that pear leaves contain a natural compound that suppresses an increase of P. citri populations. The rate of development from larva to adult was significantly lower on leaves collected in July than on leaves collected earlier or later, in several years. The population suppression was caused by molting inhibition and ovicidal activity, according to our close observation in the laboratory [Gotoh and Kubota (1997) Exp. Appl. Acarol. 21: 343-356]. To clarify whether a natural pear compound caused this molting inhibition, a methanol crude extract of pear leaves was isolated and added to a newly developed artificial diet, consisting of sodium caseinate, sucrose, levulose, glucose and inositol. The compound extracted from pear leaves resulted in the molting inhibition as observed on pear leaves. Based on infrared and NMR spectral analysis, the compound extracted from pear leaves closely resembled the synthetic acaricide hexythiazox. Furthermore, the LC50-values of the compound extracted from pear leaves for ovicidal activity of P. citri eggs and for inhibition of molting to protonymphs were similar to those of hexythiazox. These results strongly suggest that the molting deterrent extracted from pear leaves was in fact hexythiazox, an acaricide in use on pear trees, rather than a natural product. This suggestion becomes even stronger, considering that the molting inhibition was observed in a hexythiazox-spray year, but not in a non-spray year.

남부지방의 5개 차재배지에서 차응애(Tetranychus kanzawai Kishida)를 채？하여 7종 살비제에 대한 저항성 수준을 leaf spray 방법으로 검정한 결과 살비제의 종류 또는 지역 계통별로 상당한 감수성 차이를 나타내었다. 성전과 제주 계통은 azocylotin, tebufenpyrad, fenpytoxi-mate, propargite 와 fenpropathrin에 대해 , 해남 계통은 propargite와 fenyroximate에 대하여 , 보성 계통은 propargite에 대해, 영암계통은 pyidaben에 대하여 저항성비 20이상의 높은 수준을 나타내었다. 따라서 성전과 제주계통에 있어서는 다른 대체살비제들의 사용이 요망된다. 그러나 해남, 보성, 영암 계통들에 대해서는 적절한 살비제의 선택적 이용으로 차응애를 효과적으로 방제할 수 있을 것으로 생각된다.

전국 6개 지역의 사과원에서 점박이응애를 채집하여, 15종의 살비제에 대한 성충과 알의 감수성을 비교하였다. 공시살비제에 대한 성충과 알의 약제감수성은 서로 비슷한 경향을 나타내었다. 약제의 종류별 감수성은 지역에 따라 다향하게 나타났는데, 지역 계통별 살비제에 대한 저항성비가 20이상인 약제들을 성충과 알로 나누어 정리하며, 성충의 경우, 수원 계통은 azocyclotin, cyhexatin, fenbutatin-oxide에 대하여, 대전 계통은 dicofol, fenbutatin-oxide에 대하여, 청주 계통은 dicofol에 대하여, 진주 계통은 cyhexatin, dicofol, fenbutatin-oxide에 대하여 높은 저항성을 나타내었다. 알의 경우는 수원 계통은 bifenthrin, clofentezine, hexythiazox, tetradifon에 대하여, 군위, 청주, 광주 계통들은 bifenthrin에 대하여, 대전 계통은 amitraz, bifenthrin에 대하여, 진주 계통은 amitraz, bifenthrin, clofentezine, dicofol, tetradifon에 대하여 높은 저항성을 나타내었다. 그러나, abamectin, chlorfenson, fenpyroximate에 대한 약제감수성은 채집 지역에 관계없이 실내 감수성 계통과 차이가 없었다.

전국 8개 지역별 각 사과원에서 채집된 점박이응애(Tetranychus urticae Koch)에 대한 저항성 정도를 일본 감수성 계통과 비교한 결과 지역별 현저한 감수성 차이를 보였다. Azocyclotin, fenpropathrin, propargite 및 abamectin에 대해서는 낮거나 중간 정도의 저항성을, dicofol, fenpyroximate 및 pyridaben에 대해서는 높은 저항성을 나타내었다. 이들 계통은 한종 또는 두종 이상의 약제에 대해 감수성을 보여 특정 지역에 대해서는 적당한 살비제의 선택적 이용으로 점박이응애를 효과적으로 방제할 수 있을 것으로 사료된다.

야외계통 점박이응애에 대한 amitraz와 bifenthrin, propargite, fenbutatin oxide, dicofol을 여러 비율로 혼합하여 연합독작용을 시험한 결과, 살비제 혼합에 의한 협력작용은 살비제 조합 또는 그들의 혼합비율에 따라 큰 차이가 있었다. 모든 약제조합에서 협력작용이 나타나, amitraz와 bifenthrin의 혼합에서 2:8, amitraz와 fenbutatin oxide의 조합에서 8:2, amitraz와 propagite의 혼합에서 4:6 그리고 amitraz와 dicofol의 조합에서는 6:4의 비율에서 최대의 협력 효과를 나타내었다.

정박이응애(Tetranychus urticae koch)의 감수성계통에 대하여 유기찬계 살충제인 carbophenothion, ethion과 유기소계인 dicofol, 유기주석계인 cyhexatin 및 합성 pyrethroid계인 biphenthrin등으로 단제루대도태를 실시하여 각 살충제의 저항성발달 차이와 약제상호간의 교우저항성의 유무 및 정도를 시험.조사하였다. carbophenothion과 ethion은 22세대와 24세대 도태에 서 각각 156배와 64.1배의 높은 저항성을 나타내었다. dicofol은 28세대 도태에서 39.7배로 biphenthrin은 24세대도태에서 13배로 화학적계통이나 동일계통내에서도 약종에 따라 저항성유발 정도에 차이가 있었다. Carbophenothion 도태계통은 도태에 관치하지 않은 ethion에 대하여 높은 수준의 교우저항성을 나타내었으나 dicofol과 biphenthrin에는 낮은 수준의 교우저항성을 나타내었고, ethion 도태계통은 carbophenothion에 대해서 고도의 교우저항성을 나타내었다. Dicofol이과 biphenthrin 도태계통들은 모두 carbophenothion과 ethion에 대해서 중도의 교우저항성을 보였으며, cyhexatin 도태계통은 ethion과 carbophenothion에 대하여 고도의 교우저항성을 나타내 었다.