Beauveria bassiana (Bb) is a potential biopesticide against western flower thrips. To understand molecular characteristics of two Bb isolates (ERL836 and JEF-007), whole genome sequencing and RNA-seq were performed. The whole genome of Bb ERL836 was sequenced (3,934,801,443 bp) using Pacbio sequencing and 15 contigs were analyzed. To understand the molecular mechanisms of pathogenicity in two Bb genome (before infection and after infection), next generation sequencing (NGS) was analyzed to compare transcriptomes. A result of differentially expressed gene (DEG) showed that 2,689 contigs were up-regulated and 2,619 contigs were down-regulated. Randomly selected five genes were validated to be highly up-regulated. This work can provide an understanding of the interaction between Bb and westetn flower thrips.

Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae) in forest is considered as a serious forest pest in pine trees. In this study, we collected entomopathogenic fungi from soil and assessed their virulence against the adults of the inset using a spray method in laboratory condition. Two entomopathogeinc fungi isolates showed high virulence against the adults. In the concentration of 1.0 × 107conidia/ml, the mortality rate of the adults were 53% and 60%. In semi-field condition, one isolate showed a virulence of 60% against the adult. Consequently, we confirmed the possibility of the fungal isolates in controlling the beetles. In near the future, we will investigate several factors which is possibly related to the control of the forest insect pest using entomopathogenic fungi in field conditions, given the importance of fungal formulation and practical application methods.

The hard tick, Haemaphysalis longicornis (Ixodida: Ixodidae) is one of the vectors of severe fever with thrombocytopenia syndrome virus (SFTSV) causing in humans, however little consideration was given to the biological control of the pest. Therefore, this study provides a screening method to select entomopathogenic fungi, having high virulence against H. longicornis. The virulence test was conducted by dipping method with a conidial suspension (1×107 conidia/ml). As a result, cadavers of H. longicornis infected with entomopathogenic fungi were obtained by this method. Based on this results, we selected several isolates having high virulence to H. longicornis. We suggest that, selected isolates in this study can be used for the control of H. longicornis.

Entomopathogenic fungi have been known as promising candidates for biological control of insect pests. Recently, researchers consider the fungal thermotolerance in formulations and field applications. In this study, we investigated the production of thermotolerant Isaria javanica and I.fumosorosea conidia through grain-based solid cultures and exposure to light stress. As results, of the ten grain substrates, Italian millet, rice, perilla seed and sesame, rice, sorghum produced highly thermotolerant conidia in the strains. The two strains were exposed to a light stress and a heat stress. And they showed enhanced thermal stability compared to control, when exposed to 45°C for 2 hours. This work suggests that heat-resistant entomopathogenic fungal conidia can be produced by grain-based solid cultures and exposure to light stress.

Melon thrips, Thrips palmi Karny (Thysanoptera: Thripidae) is a serious insect pest of various crops in the world. The management of this pest mainly have relied on chemical agents. However, the overuse of chemical agents for long times causes insect resistance, and negative effects on environment. Therefore, alternative control methods, such as biological control, that are less harmful to the environment and have different mode of action are needed to control this pest. In this study, we isolated entomopathogenic fungi from Korean soil, and characterized them via morphological and molecular identifications and pathogenicity check against Tenebrio molitor larvae. The isolated fungi were subjected to virulence assay against T. Palmi with a conidial suspension of 1×107 conidia/ml in laboratory conditions. This entomopathogenic fungi library would be a good resource to select effective strains for the control of T. palmi in fields.

Entomopathogenic fungi play an important natural role in regulating their insect host populations, and their ecology was also associated with plant and soil. These microorganisms have been living by reacting to insect, plant and environmental factor. The advanced bioinformatics technology such as next-generation sequencing and RNA sequencing has revolutionized in understanding of entomopathogenic fungi. Recently studies provided a lot of information on evolutionary relationships and virulence-related characteristics. We are starting to know where these microorganisms from is, and how they live in nature. The bioinformatics technology will give us further our understanding of the natural roles of these fungi in nature.

The entomopathogneic fungal colonization has become an interesting issue in ecological pest management. Plant-orientedfungi induce plant growth promotion and act as natural control agents against insect pest. Herein this work, we evaluatedthe ability of fungal colonization on bark, branches and leaves of pine tree (Pinus densiflora) to select optimal candidatesfor controlling Japanese pine sawyer (Monochamus alternatus), which is the pine wilt disease vector in Korea. As a result,some Beauveria and Metarhizium isolates could colonize on the substrates of P. densiflora and showed pathogenicity againstM. alternatus. This result suggests that the selected entomopathogenic fungi are possibly candidates for long-term managementof M. alternatus in pine tree forest.

Japanese pine sawyer, Monochamus alternatus HOPE (Coleoptera: Cerambycidae) are considered as important pine forestinsect pests in several countries which transmits the pine wood nematode, Bursaphelenchus xylophylus. In this study, wetried to control Japanese pine sawyer using entomopathogenic fungi to prevent the spread of pine wilt disease in Korea.Several entomopathogenic fungi were exposed to the adults of Japanese pine sawyer and mycosis was observed in themembranous cuticles after 6 days. In particular, two isolates showed high virulence against the adults. The selected 2isolates were sprayed to the adults at 1×105, 1×106 and 1×107 conidia/ml. In the concentration of 1×107 conidia/ml, thesurvival rates of insects were 40% and 47%, respectively. Based on these results, the possibility of controlling the Japanesepine sawyer was confirmed.

Chemical pesticides have been used for persimmon pest control, however the overuse caused insect resistance andenvironmental toxicity. Herein this work, we investigated the biological potential of eco-friendly organic pesticides (biologicals)and entomopathogenic fungi in controlling persimmon pests, Stathmopoda masinisaa and Riptortus pedetris. A botanicalmixture of Chinese scholar tree, goosefoot and subtripinnata extract, Bacillus thuringesis NT0423 and one isolate of Beauveriabassiana showed high insecticidal activity against the pests in laboratory conditions. The selected botanical extract andmicroorganism were combined with a chemical pesticide (buprofezin+dinotefuran WP) and were assessed their controlefficacy in persimmon fields. Compared to the three times of chemical spray, the combination of more than one timeof chemical spray and the selected biologicals controlled the persimmon pests with an acceptable levels. Consequentlyone time of biological spray can be integrated to the spray program where only chemicals are strongly considered.

Many entomopathogenic fungal isolates received interests for controlling insect pests. But very few numbers of isolatesreached agricultural fields as commercial products. In this study, we investigated the feasibility of grain substrates forconidial production, focusing on conidial productivity and thermotolerance. Next to efficiently use the produced conidiafor spray application, conidia need to be separated from the grain substrates. Isaria javanica and I. fumosorosea conidiawere solid-cultured on ten kinds of cereal grains. Solid-cultured grains were packed in a screen bag and immersed inwater with a variety of surfactants at 100 ppm, such as Silwet, CO-2.5, CO-12, LE-7, PE-61, TDE-3, Tween 20 or Tween80. High levels of conidial release were observed on LE-7, TDE-3 and Tween80 treatments. This work suggests thatthe screen bag can be used to separated conidia from the cultured grains with the help of surfactants.

A genus of entomopathogenic Beaveria bassiana has been widely used in pest management, however little studies havebeen given to its virulence-related genes. To identify the roles of virulence genes, AtMT-induced random mutants weregenerated and followed by localization study with TAIL-PCR. Two genes, Complex I intermediated-associated protein30 (CIP30) and Autophagy protein 22 (Atg22), were predicted as virulence-related genes in B.bassiana JEF-007. To validatethe a possible relationship between two genes and fungal virulence, hpRNAi was performed. A hpRNAi plasmid wasconstructed as a model system to knock down of egfp gene in egfp-expressing B. bassiana transformant. A Real-TimePCR showed the knock down of egfp gene expression via hpRNAi. The CIP30 and Atg22 genes in B. bassiana willbe validated by the established RNAi technique. This work can be a strong platform for the functional genetics in B.bassiana.

Anthriscus sylvestris (L.) Hoffm. is a perennial herb found widely distributed in various regions of Korea, Europe, and New Zealand. The root of A. sylvestris have been extensively used in the treatment for antitussive, antipyretic, cough remedy in Oriental medicine, but the physiologically active function of the leaf of A. sylvestris is as yet unknown. In this study, we investigated the anti-cancer activity and the mechanism of cell death of water extracts of leaf of Anthriscus sylvestris (WELAS), on human FaDu hypopharyngeal squamous carcinoma cells. Our data showed that WELAS treatment inhibited cell viability in a concentration- and time-dependent manner. In addition, the treatment of WELAS markedly induced apoptosis in FaDu cells, as determined by the viability assay, DAPI stain and FACS analysis. WELAS also increased the proteolytic cleavage of procaspase-3, -9 and PARP (poly(ADP-ribose) polymerase). In addition, exposure to WELAS decreased the expression of Bcl-2 (an anti-apoptotic factor), but increased the expression of Bax (a pro-apoptotic factor), suggesting that mitochondria-dependent apoptotic pathways are mediated in WELAS-induced apoptosis. Taken together, these results indicate that water extracts of leaf of A. sylvestris inhibits cell growth and induces apoptosis via the mitochondrial-dependent apoptotic pathway in FaDu human hypopharyngeal squamous carcinoma cells. Therefore, we propose that the water extracts of leaf of A. sylvestris is a novel chemotherapeutic drug, having growth inhibitory properties and induction of apoptosis in human oral cancer cells.

Purpose: This study evaluates the clinical judgment levels of nursing school students in simulation practice of diabetic ketoacidosis and score differences in clinical judgment levels based on academic record grades. Methods: The clinical judgment levels of 149 nursing students (3rd grade) who received a lecture on simulation practice for diabetic ketoacidosis simulation were evaluated during the period from March 3 to June 20, 2014. Data were collected and measured for the general characteristics, clinical judgment levels of nursing students in simulation of diabetic ketoacidosis, and difference in scores of clinical judgment levels according to grades. Results: Clinical judgment levels of these test participants was 2.82 points on average out of a full score of 4 points, and it was represented in the order of noticing (3.01), interpretation (2.86), response (2.79), and reflection (2.60) for each area. In addition, score differences in clinical judgment levels based on academic record grades were significantly higher in the upper ranks than in the lower ranks in all the areas. Conclusion: In simulation practice of this study, through observation by rubric education method, an objective recognition could be assessed and the importance of nursing knowledge was implied.

Entomopathogenic fungi, widely distributed in nature, have been developed as biopesticides of agricultural pests. However, little is known about isolation method for obtaining a variety of highly virulent strains from the soil. In this work, the previously established method using mealworm (larvae of Tenebrio molitor) as a baiting system was used to efficiently isolate fungal strains from soil, and the isolated fungal library in Korea was established. To isolate entomopathogenic fungi, soil samples from 10 regions of Korea such as Iksan, Imsil, Gunsan, Yeosu, Suncheon, Gwangyang, Jinju, Hamyang, Boseong and Jangseong were collected. Of the collected soils, 21 and 34 isolates of entomopathogenic fungi were isolated from Iksan and Gunsan, respectively. The isolates were classified as genera of Metarhizium, Beauveria and Isaria. This work can suggest an effective insect pathogenic fungal isolation method

Entomopathogenic fungi have been known as promising candidates in biological control. However, a large number of conidia with higher stability should be produced for industrialization. In this work investigated the possibility of several grains for the production of thermotolerant Isaria javanica and I. fumosoroseus conidia. The fungal conidia were produced on ten grains for 14 days in Petri dish conditions. Of the ten granular substrates, millet, perilla seed and barley showed the high conidial production, 15.7 ± 8.4, 17.3 ± 5.2 and 13.5 ± 1.9 × 109 conida/g in I. javanica and 15.9 ± 0.6, 15.0 ± 0.5 and 15.0 ± 0.7 ×109 conida/g in I. fumosoroseus, respectively. However, the two strains showed relatively low thermal stability regardless of substrates, when exposed to 45℃ for 1, 2 and 3 hours. This work suggests a possible substrate for conidial production of entomopathogenic fungi with low cost.

Entomopathogenic fungi are expected to play a role as great biological control agents in the global bio-pesticide market in the future. The first step is the collection of fungal isolates and it should be a platform for the development of highly effective biopesticides. 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 tree was generated, and of the isolates 17 isolates’ biological features were characterized, such as morphology, virulence against several insect, stability of conidia for heat, 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.

Many turfgrass pests, Ectinohoplia rufipes, Exomala orientalis and Popillia quadriguttata, cause serious damage to grass fields in Korea. In this study, control of turfgrass pests using entomopathogenic fungi were investigated using Protaetia brevitarsis seulensis as an alternative insect. In a preliminary experiment, some Metathizium sp. showed ca. 60% virulence after 7 days exposure to the entomopathogenic fungi. Compared to the contact-exposure, overall a spray treatment showed lower control efficacy against P. brevitarsis seulensis. Furthermore, mortality of various developmental stages of P. brevitarsis seulensis was investigated under laboratory conditions. This work suggests that when treated with fungi mortality of P. brevitarsis seulensis was closely related with developmental stage. These results provide a basic information for the control of P. brevitarsis seulensis using entomopathogenic fungi

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the β-casein gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5’ arm region and 1.8 kb or 0.64 kb of 3’ arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of endostatin gene and inserted into exon 7 of the β-casein gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3’ arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine β-casein gene in the mammary gland.