Monochamol is a male-produced aggregation pheromone of the Monochamus species, which are vectors of the pine wood nematode (PWN). This nematode is a fatal devastating pest, infecting pinewood trees and is one of the serious forest pest in Korea. The nematicidal activity of synthetic monochamol and its homologues (ROEtOH: R = C7-C13) were determinated to potential alternatives to the currently used PWN control agents. In this study, for elucidation of the lethal mechanism induced by monochamol, we performed Transcriptome analysis by next-generation sequencing (RNA-seq) from LC50 treated PWN and are characterizing some significant genes which were differentially expressed in PWN.

Bursaphelenchus xylophilus, commonly known as pine wood nematode (PWN) and pathogen of pine wilt disease, is transmitted to new host trees through Monochamus spp.. In this study, five points were set 1 km apart placing the interface of the damaged area in the middle to measure density of the beetles and confirm the presence of PWN in Chungcheongbuk-do, Republic of Korea. The vectors collected from five traps on each point were sorted on weekly basis until the 12th week. We confirmed the presence of PWN in each collected vector by PCR with species-specific ITS primers.

Pine wood nematode (Bursaphelenchus xylophilus), the pathogen of pine wilt disease (PWD), is transmitted to new host trees through Monochamus spp., vector insects. In Korea, aerial application has been allowed conventionally to eliminate the vectors in their flying season. In this study, thiacloprid was applied to four-year-old Pinus densiflora, which were fed to adult M. alternatus emerged from PWD infected logs for 72 h in the condition of 1 DAT (Days after treatment of thiacloprid) and 15 DAT, respectively. We confirmed the presence of nematodes in each beetle applied to experiment by Baermann funnel method and PCR with species-specific ITS primers. After 72 h of 1 DAT and 15 DAT, thiacloprid residues in pine trees were 0.59-16.32 and 0.043-0.131 mg/kg and the mortalities of the beetle were 100 and 99.2%, respectively. Detection rate of B. xylophilus was 51.3%. The incidence rate of PWD in 1 DAT and 15 DAT showed no mortality and 5.55% compared with 55.5% of control, respectively.

Pine wilt disease (PWD) induced by the pinewood nematode (PWN), Bursaphelenchus xylophilus, is a great threat to pine forests in Europe and East Asia. Identification of interrelation between Monochamus spp., the major vectors of PWD and PWN is the key factor for effective control of PWD because PWN is trasmitted to healthy pine trees only by Monochamus vectors. In this study, direct injection system is designed to apply PWN artificially to M. alternatus just before emergence to adults. After injection of PWN (300~500 nematodes in 10 μl of 1X PBS) using adapted glass pipets, the survival rate of M. alternatus and detection rate of live PWN were 80.0% and 68.8% at 168 h respectively. As a result, we demonstrated the artificial injection methods of nematode to Monochamus vectors for further study of coeffect between vectors and specific stage of PWN including 3rd stage dispersal juvenile.

Pine wood nematode (PWN), Bursaphelenchus xylophilus causes devastating damage to pines worldwide. To develop new agent for controlling PWN, alkyloxyalcohols(A-H) were synthesized and nematicidal activity was tested against PWN. As a control, monochamol, which reported to have nematicidal activity was also tested. The nematicidal activity was diffferent according to carbon chain length in compounds. LC50 values of F, G and H were as same as that of monochamol. However A-E which have shorter carbon length than F-H showed weak nematicidal activity.

It has been proved that agroinfiltration-based temporary expression of coatomer subunit alpha (COPA) gene from Tetranychus urticae hairpin RNA induces RNA interference (RNAi) and lethality to T. urticae. To establish detailed protocols for agroinfiltration, the efficiency of agroinfiltration to the soybean and kidney bean was determined with respect to different Agrobacterium delivery methods (sea sand, carborundum and syringe) and the spacial expression patterns of hairpin RNA was investigated following Agrobacterium delivery. Sea sand and syringe showed the highest expression level in soybean and kidney bean, respectively. Considering the resulting tissue damage, syringe appeared the best choice for agroinfiltration in both soybean and kidney bean. The apical region of a leaf showed more relative expression levels in both soybean and kidney bean compared to the basal region. Following agroinfiltration, adjacent untreated leaves were determined to express hairpin RNA though the expression level was low, suggesting that hairpin RNA can be translocated to other leaves. In conclusion, Agrobacterium delivery by syringe and use of whole leaf were recommended for T. urticae bioassay following agroinfiltration.

Recently, RNA interference (RNAi) technology has been emerged as a potent tool for pest control strategy. Based on the previous studies on RNAi via leaf disc-mediated systemic delivery of dsRNA and in planta expression of hairpin RNA by agroinfiltration, the coatomer subunit alpha (COPA) gene has been found to be a crucial target for RNAi against Tetranychus urticae. In current study, transgenic plants of Arabidopsis thaliana expressing COPA hairpin RNA were generated by the floral dip method. Putative transgenic plants were screened by PCR and positive transformants were subjected to bioassay using age-synchronized and host-adapted T. urticae. T. urticae feeding on plants expressing dsRNA/siRNA showed more than 80% mortality as compared to the mites feeding on control plants at 6 days post-infestation. Our data shows that in planta expression of hairpin gene such as COPA may serve as an effective way for the control of this important pest in ornamental and economically important plants.

In our previous study, COPA (coatomer subunit alpha) gene from the two-spotted spider mite, Tetranychus urticae, exhibited RNA interference (RNAi)-based lethality when its double-stranded RNA (dsRNA) was systemically delivered via multi-unit chambers or its hairpin RNA was in planta-expressed by agroinfiltration. The cumulative mortality of T. urtcae was 55.0 ±14.2% in soybean plants agroinfiltrated with COPA gene. To investigate the temporal expression profiles of hairpin RNA following agroinfiltration, the amount of hairpin RNA expressed in plants was quantified over time by quantitative real-time PCR. Relative transient expression levels of T. urticae COPA hairpin RNA was highest at 46 h post-agroinfiltration and the extent of COPA gene knockdown was lowest at 12 h post-infestation on soybean plants. To investigate small interference RNA (siRNA) profiling, northern blot assay is currently under progress.

RNA interference (RNAi) has been proven as an operative technique for efficient gene silencing in many organisms. In our study, Tetranychus urticae, an extremely polyphagous and rapidly resistance developing mite against acaricides, was screened by double-stranded RNA (dsRNA) delivery method using multi-unit chambers. Among several lethal genes of T.urticae, COPA (the coatomer subunit alpha), a gene involved in membrane transport between the endoplasmic reticulum (ER) and the Golgi complex, showed the highest mortality rate [median lethal time (LT50)=54h]. To investigate the effect of dsCOPA treatment to lysosome formation, we used the Lysotracker green DND26 dye, selective to acidic cellular compartments such as lysosome. The result revealed that the dsEGFP-treated T. urticae has 1.3-fold more of lysosome than that of dsCOPA treated, indicating that downregulation of COPA affected lysosomes function and autophagy, thereby resulting in lethality. To investigate the further detailed toxic mechanism of COPA knockdown, investigation on histological changes in T. urticae fed COPA dsRNA is currently on going.

Expression of hairpin RNA corresponding to the part of COPA transcript was done by agroinfiltration in soybean plants and was confirmed by qRT-PCR. In a pot experiment, T. urticae was infested on agroinfiltrated soybean plants and T. urticae mortality was observed and compared with control plants overtime. Significantly higher mortalities of T. urticae were observed in the COPA-agroinfiltrated soybean plants from post-infestation day 2 (15 ±5%), day 4 (50 ±10 %). At post-infestation day 6, mortality reached to (70 ± 15%). To validate the observed COPA silencing effect in T. urticae fed on the agroinfiltrated soybean plant expressing COPA hairpin RNAs, qRT-PCR analysis was performed. The transcript level of COPA gene was decreased in T. urticae fed on agroinfiltrated soybean plants expressing COPA hairpin RNA from post-infestation day 2. At post-infestation day 2, 4 and 6, COPA transcript levels were reduced by 23.8, 20.7 and 18.8 fold, respectively compared to post-infestation day 1 (control). The results obtained in this study also ruled that the plant mediated production and uptake of silencing (dsRNAs/siRNAs) is an effective way to trigger RNAi in the T. urticae.