Little consideration has been given to the insect epizootiology in pest management. High production cost of fungalinsecticides might be a technical barrier. Application of the fungal insecticides to unfavorable conditions might result inlower performance in pest management. Herein this work, we suggest ecological biocontrol considering long-term colonizationin nature rather than quick pest control. Fungal biopesticides should be frequently combined with chemical pesticides infields, particualrly spatial combination strategy rather than temporal combination. Additionally other important technicalaspects need to be strongly considered, such as economic downstream process, effective control and environmentally safe,so finally proposing e-biopesticide. Lastly R&D system needs to be clearly and efficiently organized for high performance.Week-based summary and reporting system, weekly report and project file are main system in our laboratory which hasour lab work more effectively and efficiently, consequently resulting in tech-transfer to an industry and re-investment.

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.

The hard tick, Haemaphysalis longicornis (Ixodida: Ixodidae) is one of the vectors of severe fever with thrombocytopeniasyndrome virus (SFTSV) causing in humans, however little consideration was given to the biological control of the pest.We evaluated three bioassay methods, such as spray, dipping and dropping. As a result, the dipping method was themost efficient way to measure the fungal virulence. Finally, we constructed a Haemaphysalis longicornis pathogenic fungallibrary to further facilitate the resources to be used as potential biological control agents. A conidial suspension of 1×107conidia/ml was exposed to the ticks by dipping. We confirmed that some isolates were highly virulent to the ticks. Thisresults indicated that some fungi could be used to effectively control the Haemaphysalis longicornis.

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.

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.

Agrobacterium tumefaciens-mediated transformation (AtMT) has been widely used for generation of fungal transformants and recently applied to Beauveria bassiana. In this study to comprehend how the AtMT promoter influences on the expression of selection marker (hygromycin B resistance gene; hph), two different Ti-Plasmids were constructed: pCeg (gpdA promoter-based) and pCambia-egfp (CaMV 35S promoter-based). Putative transformants were subjected to the PCR, RT-PCR and qRT-PCR to inspect the T-DNA insertion rate and gene expression level. In conclusion, more than 80% of the colonies succeeded in AtMT transformation and the hph expression level of AtMT/pCeg colonies was higher than that of AtMT/pCambia-egfp colonies. This result can provide useful information on the AtMT of B. bassiana, especially antibiotics susceptibility and promoter-dependant expression level.

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

Entomopathogenic fungi are potential pesticides as biological control agents. However, many previous studies have focused on basic researches to control pest using insect-pathogenic fungi. Herein, the molecular characteristics of insect defense mechanisms against fungal pathogens and fungal pathogenicity to insects were investigated. The whole genome of Beauveria bassiana (Bb) JEF-007 was sequenced (36,538,394 bp) using Pacbio sequencing. Based on this data, the transcriptomes of bean bug and fungus were analyzed using RNA-seq. In the transcriptome analyses, six and five genes were validated to be involved in the fungal pathogenicity and insect defense, respectively. This work can provide an understanding of the insect-pathogen interactions

Myeloid differentiation factor 88 (MyD88) is an intracellular adaptor protein involved in Toll 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. To better understand the roles of Toll pathway in mealworm immune system, TmGPR and TmMyD88 was knocked down by RNAi silencing. Target gene expressions were decreased 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 TmGPR 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 dsGPR. These results indicate that TmMyD88 is required in mealworms for survival against B. bassiana JEF-007.

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.

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.

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.

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.

Western flower thrips, (Pergande) (Thysanoptera: Thripidae) have been recognized as serious pests worldwide. Commercially available chemical pesticides challenges against the resistance of the pest and safety to consumers. Herein we investigated the usability of soil-treatment of entomopathogenic Beauveria bassiana (Bb) ERL isolates in tomatoes. Bb-egfp was highly virulent to western flower thrips in laboratory conditions and colonized in soil, which provided a chance for western flower thrips larvae to contact the colonized fungal mass. In a glasshouse trial, Bb ERL836 and Bb ERL1578 granule (GR) were applied to soil surface and mixed well, and five females were infested to each pot of tomato 3 days after the application. The two treatments significantly reduced thrips population in tomatoes 20 and 40 days post applications, comparable to the control efficacy of clothianidin granule (GR) formulation. In the following field trial, the two ERL fungal treatments showed 69.2% and 61.5% of control efficacy, which were similar to that of clothianidin GR (76.9%). This work suggests that applied B. bassiana colonized in soil well and thrips larvae easily contacted the fungal mass, which possibly enabled the control strategy to be alternative way to the chemical control.