A viral genome was assembled de novo from next-generation sequencing (NGS) data from bean bugs, Riptortus pedestris, infected with an entomopathogenic fungus, Beauveria bassiana (Bb), and was further confirmed via the RACE method. Based on the phylogenetic analysis of the RdRp sequences, RiPV-1 was clustered in the unassigned insect RNA viruses with two other viruses, APV and KFV. These three viruses were suggested to constitute a new group of insect RNA viruses. Interestingly, RiPV-1 replication was increased dramatically in bean bugs 2 to 6 days after fungal infection. In conclusion, a novel insect RNA virus was found by NGS data assembly. This virus can provide further insight into the interaction between virus, fungus and the host.

Chemical pesticides have been used to control persimmon pests, however the overuse of the pesticides caused insect resistance. Herein we investigate the potential of eco-friendly organic pesticides (EFOP) on the control persimmon pests, Stathmopoda masinissa and Riptortus pedestris. Eleven commercially available EFOP were sprayed on the target insects in laboratory conditions. The chemical pesticide, buprofezin+dinotefuran wettable powder served as a positive control. In the bioassay against persimmon fruit moth, the mixture of Chinese scholar tree, goosefoot and subtripinnata extracts (EFOP-2) treatment showed the highest control efficacy: 27.7% (5 days) of survival rates. In the bioassay against bean bugs, the EFOP-2 showed the highest control efficacy: 20.0% (5 days) of survival rates. These results suggest that the mixture of three plants extracts (EFOP-2) has high and multiple potential in the management of the persimmon pests.

Alternative control agents, hopefully overcoming the present issues and problems in agrochemicals, should be considered and finally applied to the vector controls. Entomopathogenic fungi can used as one of the possible novel control agents and great considerations are given to the control of soil- or water-dwelling stages of vectors, such as mosquitoes and ticks. In our research group, the entomopathogenic fungal library has been constructed using the mealworm-based isolation system, which showed a variety of opportunities of their use in vector control. Important key production technologies including granular formulation have been developed to increase their industrialization. Some entomopathogenic fungal isolates showed high biological performance in the control of mosquitoes and ticks in field stands. R&D of down-stream process should be seriously considered as the key step.

Monochamus saltuarius is one of the insect vector of pine wilt disease with Monochamus alternatus. The present study aimed to investigate the dose to induce sterile and the effect of sterility according to the gender of M. saltuarius. As the electron beam irradiation dose increased, the hatchability, adult eclosion rate and longevity of the former mid and late maturation feeding stage of adults was slightly reduced. However, there was no difference between these two adults. In addition, female adults were more sensitive to the electron beam than the male adults. Hatchability of the F1 generation of ♂T × ♀N o r♀T × ♂N (T: 200 Gy electron beam irradiation, N: 0 Gy) was completely suppressed. As electron beam irradiation dose increased, the fecundity of female adults were slightly decreased. Results by comet assay showed that electron beam irradiation induced as dose increased DNA damage in M. saltuarius adults. These results suggest that SIT using electron beam may be useful for control of M. saltuarius.

Composite resins are developed as restorative materials to improve esthetics and mechanical properties. To improve the physical properties of resin material, resin filler have to be added. However, no imaging method is adopted for resin filler distribution. Optical coherence tomography (OCT) is a optical imaging technique to delineate microscopic structures within biological tissue. The OCT application to dental composites resin and its filler is not described yet. So, this new and advanced optical method is needed for clinical application for evaluation of dental composite resin. To analyze the spatial distribution of dental composite resin and to evaluate the resin restoration in cavity, frequency domain optical coherence tomography (FD-OCT) was used for their analysis. Resin restored tooth was prepared. For morphological observation, serially sectioned teeth, conventional X-ray taking and micro computed-tomography (CT) images were compared with OCT images. The experiment has done to evaluate the success of the resin restoration using 3 dimensional structure OCT image. In this research, OCT is evaluated as a new technique to image resin restoration. The evaluation of resin restored tooth was performed by OCT. Inappropriate restoration such as marginal adaptation, large porosities, internal integrity and poor contour could be detected. Resin filler also could be checked by OCT. The distribution, number, regularity and size of resin filler can be differentiated from several commercial products. Considering the characteristics of the OCT, it can be used to evaluate the defects of resin restoration, resin filler distribution, and internal integrity between resin material and tooth structure. The OCT can be considered to be a new and advanced method for the evaluation of resin restorations.

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.

In Korea, six mealybug species have been reported on pears. This study investigated the occurrence of mealybugs in 19 pear orchards from 2013 to 2014. Two species, Crisicoccus matsumotoi (Siraiwa) (Hemiptera: Pseudococcidae) and Pseudococcus comstocki (Kuwana) were mainly found. The dominant species was C. matsumotoi identified on 73% of infested fruit. Toxicities of 14 registered insecticides (7 single formulations and 7 mixed formulations) commonly used to control P. comstocki were evaluated to C. matsumotoi nymphs and adults at the recommended concentration. As a results, all insecticides exhibited strong insecticidal activity with 100% mortality of both nymphs and adults. These results indicate that the 14 insecticides can be used in control for C. matsumotoi in field.

Mealybugs (Hemiptera: Pseudococcidae) are major pests of a wide range of crops and ornamental plants worldwide, and represented by 56 species in Korea. Occurrence of, or damage by, the longtailed mealybug, Pseudococcus longispinus Targioni-Tozzetti has not been reported since 2002. However, for the first time in many years, P. longispinus was reported at Phalaenopsis orchids by Kim et al in 2015. Therefore, the present study aimed to investigate the distribution of P. longispinus which is usually found inside a greenhouse or a building where the host plants are. We have collected 129 mealybug samples at 254 sites from Korea. Among these, 27 of P. longispinus and 62 of an unidentified Pseudococcus species were found mainly in ornamental plants such as Heteropanax fragrans, commonly known as happy trees in Korea. Morphological characteristics of the unidentified mealybug were very similar to those of P. longispinus. However, the genetic variability in COI sequence comparison was significantly different between the two species. Further studies are needed to clarify the valid name for this unidentified species, and we are currently working on it.

A novel insect-infecting positive sense single-stranded RNA virus, Riptortus pedestris virus-1 (RiPV-1), was found in the Riptortus pedestris transcriptome data by de novo assembly and further confirmed by RACE method. The genome of RiPV-1 consists of 10,554 nucleotides (nt) excluding the poly(A) tail and contains a single large open reading frame (ORF) of 10,371 nt encoding a 3,456 aa polyprotein and flanked by 71 and 112 nt 5' and 3' noncoding regions, respectively. RiPV-1 genome contains the consensus genome organization of picorna-like RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) array in that order from the 5' to the 3' end. From the phylogenetic analysis, RiPV-1 was clustered with unassigned insect RNA viruses, APV and KFV, which suggests that these three insect picorna-like viruses might constitute a novel group of insect-infecting RNA viruses. Tissue tropism analysis revealed that RiPV-1 was relatively abundant in the thorax, abdomen, midgut and fat body. Interestingly, RiPV-1 replication was enhanced by Beauveria bassiana JEF-007 infection that was quantified using qRT-PCR. This study identified a novel insect-infecting virus and provided further insight into the relationship between virus, fungus and host.

Beauveria bassiana (Bb) is an entomopathogenic fungus with a wide host range, and is commonly used as an environment-friendly biopesticide. However, the molecular mechanisms of Bb-host interactions are not well understood. Here, RNA isolated from a highly virulent strain of B. bassiana (Bb JEF-007) and Riptortus pedestris (Hemiptera: Alydidae) (bean bug) infected with this strain were subjected to high throughput next generation sequencing (NGS) to analyze and compare transcriptomes. Differentially expressed gene (DEG) analysis showed that 2,381 genes were up-regulated and 2,303 genes were down-regulated upon infection. Most DEGs were classified into the categories of single-organism, cellular and metabolism processes by gene ontology (GO) analysis. Carbon metabolism-related enzymes in the glyoxylate cycle were significantly up-regulated, suggesting a possible role for them in Bb growth in the host. This work provides insight into how entomopathogenic B. bassiana occupies agriculturally harmful bean bug at the late stage, which might be essential during fungal infection.

Bean bug, Riptortus pedestris (Hemiptera: Alydidae) is an agriculturally serious pest in East Asian countries. Chemical pesticides have been contributed to the management of the pest, but nowadays insect resistance limits the use of chemical pesticides, thus alternatively new pesticides with different mode of actions such as entomopathogenic fungi are considered. Herein entomopathogenic Beauveria bassiana JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by contact-exposure and spray methods. The Agrobacterium tumefaciens-mediated transformation of B. bassiana JEF-007 generated random transformants and some mutants showed reduced virulence against Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and R. pedestris nymph. Compared to the wild-type, the two transformants showed remarkably different morphology, conidial production, and thermotolerance. To figure out pathogenicity-related genes, thermal asymmetric interlaced (TAIL) PCR of the random transformants was performed and possibly some virulence-related genes were predicted. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in Toll signaling pathway. In this study, we monitored the response of 4 key genes of the insect immune system against Beauveria bassiana JEF-007 in Tenebrio molitor using RT-PCR. TmGPR, antimicrobial peptide Tenecin 1 and Tenecin 2 were up-regulated after fungal infection. To better understand the roles of Toll signaling pathway in mealworm immune system, TmGRP and TmMyD88 was knocked down by RNAi silencing. Target gene expressions were decreased at 2 days post-dsRNA injection, and dramatically reduced at 6 days post-dsRNA injection. Therefore, mealworms were compromised by B. bassiana JEF-007 at 6 days post-dsRNA injection. Silencing of the TmMyD88 and TmGRP resulted in reducing the resistance of the host to fungal infection. However, only dsTmMyD88 showed significant difference with dsEGFP by statistical analysis, which may be due to partial gene knock down of dsGRP. These results indicate that TmMyD88 is required in mealworms for survival against B. bassiana JEF-007.

Entomopathogenic fungi are facultative microorganisms, dwelling in soil or infecting host insects, and some of the genera have been used as biological control agents worldwide. Collection of fungal isolates should be a platform for the development of highly effective resources, thus in this work we constructed a fungal library using a mealworm (Tenebrio molitor) pathogenicity-based fungal collection method and further characterized some isolates with high virulence. A phylogenetic three was generated, and of the isolates 17 isolates’ biological features were characterized, such as morphology, spectrum of virulence, cultural characteristics, thermo-stability of fungi, production of biologically active materials, such as enzymes. This work reports an attractive entomopathogenic fungal library including the information of effective isolates in pest management.

Although expression of foreign genes in a crop species has been target techniques for powerful protection against insect pests, classical breeding programs using varietal resistance of a crop are still being processed. It is, however, frequently difficult to find key products expressed from resistance-related genes in the variety, and those action mechanism. Here, an unterminated story about seed of a mungbean (Vigna radiata) variety with high resistance against the adzuki bean weevil and the bean bug is introduced. The two insects cannot survive on seeds of the variety, although they well develop on susceptible ones. Molecular markers linked to the resistance were selected through BAC library screening and near isogenic lines, and finally a bruchid resistance gene was suggested after map-based cloning approaches. Starvation and chemicals were suggested for a resistant mechanism and a related factor through feeding and behavior experiments, respectively. The seed flour was extracted with organic solvents, and isolated into several fractions on chromatography. Several peaks on HPLC in a fraction were related with the high mortality of the bean bug. A partial structure in an isolated chemical was observed before full identification.

본 연구는 다양한 냉동과 해동처리에 따른 계육 가슴살에 natural microflora, 접종된 L. monocytogenes와 C. jejuni 수와 미세구조 변화 구명을 위하여 연구를 수행하였다. −20℃ 송풍식 냉동처리구의 총 호기성 세균과 C. jejuni 수는 4.06 log CFU/g과 4.09 log CFU/g으로 대조구와 비교하여 각각 약 0.7 log CFU/g과 1.0 log CFU/g의 감소를 보였다. 한편, 계육 가슴살 접종된 L. monocytogenes 수는냉동방법과 냉동온도에 따라 유의적인 차이를 보이지 않았다. 4℃와 25℃ 송풍식 해동처리구의 총 호기성 세균수는 3.70 log CFU/g과 4.02 log CFU/g으로 측정되어 대조구와 비교하여 각각 0.72 log CFU/g과 0.40 log CFU/g 감소한 반면 25℃ 유수식 해동처리구의 총 호기성 세균 수는 해동과정에서 균수가 급격히 증가하여 5.78 log CFU/g으로 관찰되었다. 해동 중 C. jejuni 수 변화는 해동방법보다 해동온도에 영향을 받는 것으로 나타났다. 냉동-해동반복 5회 후 총 호기성 세균과 효모 및 곰팡이 수는 감소하여 각각 4.15 log CFU/g과 2.30 log CFU/g을 보였다. 계육 가슴살에 접종된 L. monocytogenes 수는 냉동-해동 반복처리에 유의적인 영향이 없었지만 C. jejuni 수는 냉동-해동 반복 횟수가 증가함에 따라 감소하는 경향을 보였다. 냉동-해동 반복이 증가함에 따라 근섬유 조직을 재 손상시켰으며 특히 냉동-해동 반복 5회 후에는 계육 가슴살 시료의 조직세포 외부뿐만 아니라 내부에서도 공간이 발생하였으며 불균일하게 찢어진 세포가 관찰되었다.

This study was conducted to develop and evaluate an appropriate control device for a purge type controlled atmosphere (CA) storage in Korea. To determine ideal performance, oxygen and carbon dioxide control capability and airtightness were analyzed according to the postharvest management manual of CA storage of Fuji apples. In shortened experiments for CA storage, the condition was delayed CA at 0-0.5oC for three days and stored at 0.1-0.5% carbon dioxide levels for 3 days and then further stored 6 days under 1% carbon dioxide. As a result, the temperature control range of a developed CA container was 0.0-0.5oC, and the relative humidity was more than 90%, except for the defrosting step for the freezer during the storage period. The rate of pressure reduction for the CA container in the negative and positive pressure states was 0.45 and 0.21 mmH2O/min, respectively, and it was twofolds higher than standard airtightness for CA storage. After nitrogen injection, oxygen concentration was achieved at 2%, and carbon dioxide concentration was maintained at 0.1-0.5% for 6 days. Afterwards, carbon dioxide levels were tightly controlled between 0.1-1.0%. These results suggest that a developed purge type CA container could be effective in commercially maintaining the quality of agricultural products.

Hypocreales entomopathogenic Beauveria bassiana and Metarhizium anisopliae are well-known biological control agents worldwide and have high potential in industrialization. However their thermo-susceptibility limits long-term storage under high temperature conditions and high insecticidal activity after application to target pests. Herein we isolated highly virulent isolates, B. bassiana JEF006 and JEF007 and M. anisopliae JEF003 and 004, and produced in three grains, as substrates for solid cultures, followed by thermotolerance assays to compare the potential of the three substrates. The JEF isolates were exposed to dry and wet heat at 50°C and overall conidia were more stable under dry heat condition rather than wet heat. Of the three grains, Italian millet was superior to the other grains in the production of thermotolerant conidia. Additionally Italian millet did not severely aggregated, which enabled air to penetrate into the substrate well compared to the sorghum and millet. JEF isolates were more thermotolerant when they were kept in oil conditions. The conidia of JEF 007 and 003 wild type and AtMT-based generating random mutants were subjected to SDS-PAGE. A significant relationship between conidial thermotoelrance and detected proteins was observed. This work suggests that Italian millet can be used as an effective substrate to produce more thermotolerant conidia, thus maintaining viability for long times under unfavorable environment and biological activity against target pests.

Bean bug, Riptortus pedestris is an agriculturally serious pest in north eastern Asian countries, damaging to several legumes and fruit trees. Chemical pesticides have been largely used to control the pest but it encounters insecticide-resistance and environmental toxicity issues. Alternatively different mode of action and environmentally sound pest management system can be found in entomopathogenic fungal insecticides. Herein we developed a platform to optimize the fungal production to express their maximum virulence against bean bug, by focusing on solid culture system for thermotolerance, formulation to select effective surfactants to carry the fungal conidia on the cuticles, and relationship between environmental abiotic factors and fungal mortality. First to produce highly thermotolerance fungal conidia, Beauveria bassiana and Metarhizium anisopliae isolates were cultured on several granular cereal substrates, which could be subjected to formulation process. Among the tested media, four media (millet, non-glutinous italian foxtail millet, glutinous italian foxtail millet, brown rice) were superior to the other grains in the spore production and thermotolerance. Next to efficiently deliver the fungal conidia on the cuticles of bean bug, total of six surfactant (CO-2.5, CO-12, LE-7, PE-61, TED-3 and Siloxane) was used to experiment. CO-12 was superior to the other surfactant in mortality of 100 ppm consistence. This work suggests that solid culture system and formulation and application should be seriously considered to reach an optimal level of mortality by inducing their maximum virulence.

Entomopathogenic fungi are facultative microorganisms, dwelling in soil or infecting host insects, and some of the genera have been used as biological control agents worldwide. Collection of fungal isolates should be a platform for the development of highly effective resources, thus in this work we constructed a fungal library using a mealworm pathogenecity-based fungal collection method and further characterized some isolates with high virulence. A phylogenetic three was generated, and of the isolates 17 isolates’ biological features were characterized, such as morphology, spectrum of virulence, cultural characteristics, thermo-stability of fungi, production of biologically active materials, such as enzymes. This work reports an attractive entomopathogenic fungal library including the information of effective isolates in pest management.

ocust, Locusta migratoria (Orthoptera: Acrididae) is one of the outbreaking pests worldwide and such big occurrence was recorded in 2014, Korea, however little consideration was given to the management strategy of the pest. Herein we established a indoor locust-rearing system and constructed a locust-pathogenic fungal library to further facilitate the resources to be used as possible biological control agents. A locust colony was provided from the National Institute of Agricultural Science and Technology and reared in corn or barley plants at artificially manipulated rooms. The critical developmental stages, such as oviposition, hatching and mating were successfully proceeded. Entomopathogenic fungal granules were treated to the locust (2 g/rearing box), and in 5~7 days mycosis was observed in the membranous cuticles of head, abdomen and legs. In particular JEF-003 (Metarhizium anisopliae), JEF-186 (M. lepidiotae) and JEF-187 (Clonostachys rogersoniana) showed high virulence against the locust. A population of locust was exposed to the entomopathogenic fungal conidia-incorporated soil to investigate the possibility of the fungal isolation from natural soil, which resulted in the pathogenesis in 7~10 days in laboratory conditions. More than 80% of control efficacy was observed in the greenhouse trial of fungal granular application. This work suggests that locust rearing system was successfully established and entomopathogenic fungi can be used to control the migratory locust.