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.

Bean bug, Riptortus pedestris is an agriculturally serious pest in East Asian countries, reducing the value of crop quality and loss of income in agribusiness. Chemical pesticides have 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. Beauveria bassiana and Metarhizium anisopliae JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by spray and contact-exposure methods. Supernatant showed different level of enzyme activity including chitinase, Pr1 protease and lipase. The Agrobacterium tumefaciens-mediated transformation generated random transformants and some mutants had reduced virulence. TAIL-PCR of the random transformants revealed virulence-related genes. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

The ascomycete fungus Beauveria bassiana is a wide host range entomo- pathogenic fungus, which is commonly used as an environmental friendly biopesticide. However, the molecular mechanisms of host-pathogen interaction of B. bassiana are not well understood. Here, the high throughput next generation sequencing was performed to analyze the transcriptome of B. bassiana JEF-007 infected bean bug (Riptorus pedestris). Differentially expressed gene (DEG) analysis results showed that total 4,684 genes including 2,381 up and 2,303 down regulated genes were identified. Most of the DEGs were classified into single- organism, cellular and metabolism processes by gene ontology (GO) analysis. Metabolism pathway was the most abound category of DEGs via KEGG pathway mapping. Several possible candidates of virulence factors were dramatically expressed after infection, such as cytotoxic lectin, bacterial-like toxin, and proteins related to cell wall, hyphal growth, nutrient uptake and halogenated compounds synthesis. Furthermore, we also found the highest expression of a novel small RNA virus in the infected bean bug, but the relationship between fungal virulence and the RNA virus was under determination. The functional roles of these possible virulence factors are remained unclear, but this work provides a new insight for further fungal studies. Our results reflect systemic impacts of fungal pathogenesis and these findings represent a significant advance in the fungal functional genomics.

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.

Bean bug, Riptortus pedestris is an agriculturally serious pest in East Asian countries, reducing the value of crop quality and loss of income in agribusiness. Chemical pesticides have 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. Beauveria bassiana and Metarhizium anisopliae JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by spray and contact-exposure methods. The isolates produced high levels of pathogenesis-related enzymes, such as chitinase, Pr1 protease and lipase. The Agrobacterium tumefaciens-mediated transformation generated random transformants and some mutants had reduced virulence against bean bugs, which provided some materials to figure out pathogenicity-related genes in the fungi. Now characterization of flanking region of the integrated fragment is underway and this work may reveal some important genes in the pathogenesis. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

The entomopathogenic fungus, Beauveria bassiana is widely used in integrated pest management (IPM), however its successful application is often limited by the little effort to explore its functions of unknown genes. In this work, egfp-expression cassette was randomly integrated into B. bassiana using Agrobacterium tumefaciensmediated transformation, and the general features of the mutants with unusual characteristics and the localization of the integrated genes were explored. To construct a transformation vector, egfp-expression cassette including gpdA promoter and trpC terminator was cut from pBARKS1-egfp using SacI and HindIII and integrated into pCAMBIA containing hygromycin B resistant hygR gene, designated as pCAMBIA-egfp. Transformed B. bassiana isolates were grown on quarter strength-Sabouraud dextrose agar containing 150 μg hygromycinB ml-1. Expression of egfp was investigated by RT-PCR and a fluorescent microscope (400×). Through the genome walking of the transformants using adaptor primers and gene specific primers, unique bands were detected on the egfp-expressing transformants, which were sequenced to figure out the flanking regions. This work provides a platform of methodology to figure out unknown functional genes of B .bassiana and possibly suggest an improved strategy to use the entomopathogen in IPM.

This study was conducted in order to examine relationship between stroke patients' degree of satisfaction with leisure and quality of life according to their leisure activity types and whether they conducted leisure activities. A direct survey was performed from April 8 to May 3, 2013 on 92 inpatients and outpatients who received occupational therapy at hospitals located in Jeonju and Gunsan. A frequency analysis was carried out in order to look at the general characteristics of the subjects, their degree of participation in leisure activities, and their degree of satisfaction with leisure activities. A Pearson's coefficient was used to examine relationship between their degree of satisfaction and quality of life according to participation in leisure activities. Their degree of satisfaction according to participation in leisure activities was significantly higher when they took part in leisure activities such as handicraft activities, sports activities, or travel or tourism activities than when they did not perform any leisure activities. Their quality of life according to participation in leisure activities was higher when they conducted sports activities, outing activities, or tourism activities than when they did not. There was significant correlation between their degree of satisfaction with leisure and quality of life according to leisure activities.

Enhanced green fluoresce protein gene (egfp) was expressed in Beauveria bassiana ERL836 based on the Agrobacterium tumefaciens-mediated transformation (AtMT) method in this study. The ERL836 transformants were generated with pCambia-egfp binary vector. Ten transformants were randomly selected and analyzed for the T-DNA insertion and gene expression. The results revealed that 60% of the fungal putative transformants were inserted by the T-DNA fragment. Of these transformants, 33.33% (2 transformants) expressed the egfp gene. The egfp transformants showed strong green fluorescence with different expression levels. The results of this study could provide a reference for foreign protein expression in B. bassiana by using the AtMT method.

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.

An easy and rapid resistance detection protocol for the Western flower thrips Frankliniella occidentalis was established based on the residual contact vial bioassay (RCV), in which insecticide resistance levels can be estimated at 8 h-post treatment of diagnostic doses. The RDA strain was used as a reference susceptible strain, which has been reared under laboratory conditions over 10 years without exposure to any insecticides. Seven insecticides were tested for the determination of diagnostic dose. Among them, five insecticides (chlorfenapyr, acrinathrin, spinosad, emmamectin benzoate and thiamethoxam, ranged as 0.03 ~ 0.42 μg-1cm2) were applicable to the RCV. However, two insecticides (omethoate and imidacloprid) were not able to be used for the RCV because the treated inner surface of glass vials by these insecticides were too viscous, causing non-specific mortality. The RCV detection kit was employed for the estimation of resistance levels for the five insecticides in five local populations. Almost field-collected populations revealed high levels of resistance to the four insecticides (acrinathrin, spinosad, emmamectin benzoate and thiamethoxam) by showing less than 50% mortality. The baseline resistance detection by RCV method will facilitate the selection of proper insecticides for farmers to manage insecticide resistant-populations of F. occidentalis.

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.

To clarify the role of stem cells in hepatocarcinogenesis, CD44 expression was investigated in mouse livers as well as embryonic cell lineages treated with diethylnitrosamine (DEN). Liver tumors induced by DEN were analyzed by immunohistochemisty for CD44. Liver tissues were sampled at 6, 24, and 48 hr after treatment with saline or DEN. Mouse embryonic stem cells (ESCs), hepatic progenitor cells (HPCs), and hepatocyte like cells (HCs), representing 0, 22, and 40 days of differentiation, respectively, were treated with DEN at four doses (0, 1, 5, and 15 mM, respectively) for 24 hr, after which CD44 expression levels were examined by relative quantitative real-time PCR. CD44 expression was weakly detected in tumor cells as well as in some hepatocytes surrounding the tumor cells. However, CD44 expression was not detected in liver tissue treated with DEN at early time points. The CD44 mRNA expression level was significantly different among cells treated with 5 mM DEN at day 22 (P<0.01) as well as 1, 5, and 15 mM DEN at day 40 (P<0.01) compared with control. Taken together, CD44 expression slightly increased in mouse DEN-induced tumors. Furthermore, expression of CD44 in embryonic cell lineages treated with various doses of DEN significantly differed among embryo stem cells and derived hepatic lineage cells. This suggests that CD44 expression may be modulated in the progeny of stem cells during their differentiation toward hepatocytes, and its expression may increase in the tumor stage but not during early carcinogenesis.

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.