Zika virus, mosquito-borne disease, caused by mosquitoes has been increased the importance. From March to September, twice a month from 7 different points (3 residential areas, 3 migratory bird sanctuary and 1 cattle shed) were collected using BG trap and BL trap. After identifying the mosquitoes collected, we confirmed the virus infection. Total 26,531 mosquitoes (6 genus 9 species) were collected, virus has been detected from the 3 species (Aedes vexans, Cuilex tritaeniorhynchus, and Amigeres subalbatus) of mosquitoes of them. It showed the highest peak in August, and then gradually decreased. The most common mosquito species was collected Aedes vexan (16,637) in the cattle shed.

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.

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.

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.

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 JEF004, and produced in three grains, such as sorghum, millet and Italian millet as substrates for solid cultures, followed by thermotolerance assays to compare the potential of the three substrates for thermotolerance. 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 as carriers of an oil-based formulation. 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.

Entomopathogenic fungi infect some arthropod pest and have been used for biological control. Some entomopathogenic fungi have high potential in insect pest management worldwide, and most of researches were given to Hypocreales order of Ascomycota, but little in Zygomycota, such as Entomophthorales in our country. We have identified some species belonging to Entomophthorales and investigated cultural features and ecology of Entomophaga aulicae. E. aulicae in sweet potato fields, where Aedia leucomelas was a dominant pest, had caused epizootics from 2002–2005. E. aulicae-infected A. leucomelas larva were mostly found from August to October and its occurrence was significantly related to the precipitation. E. aulicae were mainly observed in dead A. leucomelas larval populations (infection rate = 41.3% in 2002), rather than other lepidopteran pests. E. aulicae was morphologically and genetically identified and its virulence was characterized in laboratory conditions. Additionally, pest infection by Zoophthora radicans, Neozygites floridana and some Entomophthorales have been identified. On the basis with this information, we need research to predict the prevalence and to develop biological control agent using Entomophthorales including E. aulicae that contribute to regulation of host populations.

Mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae) has high and safe protein contents, which enables it to be animal feed. However, occurrence of entomopathogenic fungi in mealworms is one of the limitations for mass production. In this work, we investigated relationships between abiotic conditions and occurrence of fungal pathogens and established an effective control method using fungicides. In virulence assay, third instar mealworm larvae were sprayed by six entomopathogenic Beauveria bassiana isolates and kept under high relative humidity; B. bassiana ERL1575 isolate had highest virulence. Under normal humidity, ERL1575 conidia showed different virulence between spray (~0% virulence) and digestion (~80% virulence) method. Furthermore, mealworms, which digested conidia, were exposed to various temperature (20-35°C) and humidity (1-3 ml distilled water spray/35 mm diam. dish) conditions for 5 days. All the treatments showed ~90% virulence except 35°C incubations (~20% virulence), but irrespective to the humidity conditions. Forty chemical fungicides were assayed against conidial germination and hyphal growth of ERL1575. Fluazinam and mancozeb showed strong inhibition of conidial germination at standard application dose (SD), 1/2 SD and 1/5 SD; besides, fluazinam showed strong inhibition of hyphal growth. When fluazinam and mancozeb were applied to the fungal conidia-inoculated wheat bran, most of mealworms were alive after 3 days post application. However, high mortality rate (~100%) were observed in the conidia-inoculated wheat bran without any fungicides. In conclusion, this work suggests that B. bassiana isolates could be pathogens at <30°C when they were digested by mealworms, and fluazinam and mancozeb would be used as effective control agents against the pathogen.

The legume family (Fabaceae) is the third largest family of flowering plants worldwide and often damaged by stink bugs, particularly Riptortus clavatus Thunberg (Heteroptera: Alydidae). This insect selectively feeds and oviposits on these plants, resulting in >50% of damage to soybeans harvested in autumn. The most common control methods for this insect are periodic application of chemical pesticides, pheromone traps, and bait crops, but still they encounters residual problem, insect resistance or low control efficacy. The current management system needs alternative control methods, which possibly combined with the present methods. Some entomopathogenic fungi have high virulence against the nymphs and adults of R. clavatus in a favorable conditions, reaching ∼80% mortality with mycosis in 3∼5 days. The genera of Beauveria and Metarhizium are possible control agents for effective and safe management of the serious pest. Fungal infection in R. clavatus was observed using an egfp-expressing fungal transformant. Thermotolerant entomopathogenic fungal conidia can be produced in cereal substrates, which enables conidia to be stable for long times. Our considerations need to be given to the combinations with other control methods, such as chemical pesticides or pheromone trap.

Antimicrobial peptides (AMPs) can be produced in mealworms. In this work, we integrated Bombyx mori (Bm) AMP, cecropin A to Beauveria bassiana ERL1170 by restriction enzyme-mediated integration method, which was confirmed by RT-PCR and an antibacterial activity assay. For the extracellular secretion of Bm cecropin A protein, the active domain of the cecropin A gene was tailed to the signal sequence of B. bassiana chitinase (Bbs). To exchange Bbs-cecropin A gene with egfp gene in pBARKS1-egfp, Bbs-cecropin A fragment was cut from pGEMBbs-cecropin A using XbaI/blunted and BamHI and ligated with cut pBARKS1-egfp using NcoI/blunted and BamHI, designated as pBARKS1-Bbs-cecropin A. After the transformation, transformants were grown on Czapek’s solution agar containing 600 μg ml-1PPT. Expression of Bm cecropin A was confirmed by RT-PCR. Strong clear zone was observed in the co-culture of the transformant D-6 and Bacillus subtilis on fourth strength Sabouraud dextrose agar 1 day after the culture at 25°C, whereas the wild type had no clear zone. This work suggests that Bm cecropin A can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry.

Bean bug, Riptortus clavatus Thunberg (Heteroptera: Alydidae) causes serious damage to Leguminosae. Herein an entomopathogenic fungal virulence assay system against bean bugs was established to construct a fungal database which can be used in integrated pest management (IPM). First to obtain as many bean bugs as possible at the same stage, host plant-preference and developmental synchronization of bean bugs were investigated. In the preference assay, five pairs of adults were infested in a plastic cage, where a pot of green bean, pea or cowpea was previously placed. The highest fecundity and the fastest development of bean bug was observed in the green bean cage. Secondly, in the synchronization experiment, eggs were collected from the cage of adults in 1, 3, 5 and 7 days after oviposition and transferred to a fresh cage with green beans. From the every 4 days of survey, similar stages of bean bugs were found in the cages with the oviposition for 1 and 3 days, rather than the longer times of oviposition. A fungal bioassay against bean bugs was conducted using the bean bugs from the above insect rearing system. Ten Beauveria bassiana isolates were cultured on quarter-strength Sabouraud dextrose agar (¼SDA) for 7 days at 25°C. Ten 4th instar of nymphs were placed on a cultured plate for 1 hour and tranferred to a fresh moisturized plate with grains of green bean. ERL836 isolate treatment showed the highest virulence and fungal mycosis was observed on the bean bugs. In conclusion, these results can be useful to establish an entomopathogenic fungal database for IPM.

Antimicrobial peptides (AMPs) can be produced in mealworms, currently being used as animal feeds, by the infection of genetically engineered-entomopathogenic fungi. In this work, we integrated Bombyx mori (Bm) AMP, cecropin A to Beauveria bassiana ERL1170 by restriction enzyme-mediated integration method, which was confirmed by RT-PCR and an antibacterial activity assay. For the extracellular secretion of Bm cecropin A protein, the active domain of the cecropin A gene was tailed to the signal sequence of B. bassiana chitinase (Bbs). To exchange Bbs-cecropin A gene with egfp gene in pBARKS1-egfp, Bbs-cecropin A fragment was cut from pGEM-Bbs-cecropin A using XbaI/blunted and BamHI and ligated with cut pBARKS1-egfp using NcoI/blunted and BamHI, designated as pBARKS1-Bbscecropin A. After the transformation, transformants were grown on Czapek’s solution agar containing 600 μg ml-1PPT. Expression of Bm cecropin A was confirmed by RT-PCR. Strong clear zone was observed in the co-culture of the transformant D-6 and Bacillus subtilis on fourth strength Sabouraud dextrose agar 1 day after the culture at 25°C, whereas the wild type had no clear zone. This work suggests that Bm cecropin A can be efficiently produced in this mealworm-based fungal expression platform, thereby increasing the value of mealworms in the animal feed additive industry.

Entomopathogenic fungi formulated as wettable powders and suspension concentrates have been sprayed to crop pests for pest management. However, the use of fungal granules to control paddy field pests has not been fully explored. Herein, several Beauveria bassiana isolates (ERL 1170, 1578 and 836) were produced as granules using a millet-based solid culture. The granules were applied to the rice nursery 3 days before transplanting and their control efficacy against rice water weevils was determined in paddy fields. The solid cultures produced ~1×108conidiag-1ofmilletgrains10daysaftertheinoculation. The granules were applied to the soil in the rice nursery 3 days before the rice seedlings were transplanted in the paddy fields. Rice in plots with granules of ERL1578 had 17.3% leaf damage (74% control efficacy) 14 days post application, whereas rice plants in the non-treated control had 66.5% damage. Rice plants treated in the nursery with ERL1170 and ERL836 had 52~54% damage. In the rice plots previously treated with ERL1578 the smallest numbers of larvae and adults were observed 38 days post application. In laboratory conditions, ERL1578-treated larvae were tuned pink and covered with mycelial mass. Applications of millet-based B. bassiana granules on rice nursery soil can be an effective and efficient biological control strategy for the management of rice water weevils. This method is relatively inexpensive and requires less labor compared to practices involving the spraying of fungi directly on rice in paddy fields.

Brown planthopper, Nilaparvata lugens is giving enormous damage to rice production. In this work, virulence of four Beauveria bassiana isolates against brown planthoppers was investigated by applying fungal granules on the water of pots, and we further examined the growth of hypha on the rice plants from the water surcae to explain the insecticidal mode of action. We used Beauveria bassiana (Bb) ERL 836, 1170, 1575 and 1578 isolates, which produced ~2×108 conidia/g of millet grain in a solid culture. Rice seeding was grown in breeding boxes at 28±2℃ for 5 days. Mycotized millet grains were treated on the water in a box at 1 g/box and the rice seeding was infested with 18~25 brown planthopper adults per box. A chemical pesticide and water treatment served as controls. Among the treatments, Bb ERL 836-treated plants had the lowest damage, rather than the other fungal treatments in laboratory assays. Hyphal growth on the stem of rice plants was observed in Petridish conditions under a fluorescent microscopy. This work suggests a possible control of brown planthoppers using entomopathogenic fungi.

Beauveria bassiana isolates have been used in integrated pest management, but little consideration has been given to the studies on fungal gene expressions and their functions. In this work, to determine the functions of genes, B. bassiana ERL1170 was transformed by restriction enzyme-mediated integration method, where pABeG with bar gene was used as a transformation vector. Among seven hundred of transformants, morphologically different ERL1170-pABeG-#160 transformant, particularly dysfunctional in conidiogenesis. The transformant had yellow hyphal growth on fourth strength Sabouraud dextrose agar (SDA/4) and produced very small amount of conidia (<1.0×105 conidia/cm2 agar) in 7 days, whereas wild type had white mycelial growth and significantly greater conidia (3.6×106 conidia/cm2 agar). Additionally under microscopic observation, hyphae of #160 seemed like indian club, compared to the straight forms of wild type hyphae. The next work is figure out possible genes contributing the conidiogenesis of B. bassiana.

Entomopathogenic fungus, Beauveria bassiana can be used in integrated crop management and pharmaceutical applications. Recently some efforts have been given to the fungus to improve its biological performances, but low fungal transformation efficiency is one of the limitations. In this work, B. bassiana ERL1170 isolate was used for fungal transformation by restriction enzyme-mediated integration, where pBARKS1-Bbs-cecropinA was linearied using HindIII. The fungal transformation comprised two steps, preparation of competent blastospores and integration of the plasmid into the cells. To prepare competent blastospores, 2-d cultured blastospores were individually treated with 0.1, 0.2, 0.4 and 0.8 M lithium acetate (LiAc). Secondly in the integration step, concentration of LiAc and calcium chloride (CaCl2), and time period of heat shock were investigated as follows: LiAc, 1, 2 and 4 M; CaCl2, 50, 100 and 200 mM; and heat shock at 42℃, 20, 40 and 60 min. Consequently, combination of 0.2 M LiAc in preparing competent blastospores and 2 M LiAc and 200 mM CaCl2 in the second step showed the highest transformation efficiency. This work would be helpful in the fungal transformation of B. bassiana.

To identify subspecies and stocks of minke whale meats purchased from Korean markets during 2005-2007, we first obtained their complete sequences of mitochondrial DNA cytochrome b and control region sequences, and compared these sequences to the corresponding sequences of the common minke whale (Balaenoptera acutorostrata), obtained from GenBank. From analyses with partial cytochrome b sequences (383 bp) and non-coding, partial control region sequences (463 bp), Korean mink whale meats are identified as products from the North Pacific minke whale (B. a. scammoni). In addition, the sequences of the partial control region from these meats showed G at site no. 298 and G or A at site no. 463, and the meats appeared to originate from the J stock within this subspecies. Thus, because the J stock has been protected since 1986, implementation of strict regulation measures to reduce their accidental fisheries by catch seems urgent. In addition, B. a. scammoni is distinct from B. a. acutorostrata, with an average Jukes-Cantor distance of 2.21% in the complete control region sequence analysis (935 bp) and 1.31% in the complete cytochrome b gene sequence analysis; the current results support the current subspecies classification, although further sequencing analyses with nuclear genes are necessary.