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.

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.

Western flower thrips (WFT), Franklinella occidentalis, is a major pest of ornamentals. Mycotized millet grains with entomopathogenic fungi applied to soil of potted marigold plants was tested to target pupating thrips. Two experimental fungal isolates, (Beauveria bassiana [ARS7060] and Metarhizium anisopliae [ERL1171]), were compared with the registered B.bassiana strain GHA and untreated controls in greenhouse caged trials. Mycotized millet grains were mixed into the upper surface of the potting soil in pots of flowering ‘Hero Yellow’ marigolds (4 g/pot). One week after application five mated WFT females were released onto each plant (four plants per cage). At 8 wks post-infestation, the mean total number of thrips per plant was 81 and 90% less in the ERL1171 and ARS 7060 treatments, respectively, than in the controls. The mean numbers of thrips per plant for the control and GHA treatments were not significantly different. Plant damage was 60% less on plants treated with the experimental fungi than the control and GHA treatments. At 10 wks post-application, 75-90% of WFT collected from the treatments were infected with the experimental isolates. These results demonstrate that soil applications of entomopathogenic fungi can reduce WFT populations significantly and prevent damage.

Entomopathogenic fungi have been studied to develop for biological control agents as an alternative to chemical control agents in insect pest management. Two Lepidopteran insects, Spodoptera exigua and Plutella xylostella, are serious insect pests infest various crops, but not effectively controlled by commercial chemical pesticides due to its high insecticide resistance. A fungal isolate was isolated from S. exigua larvae collected from green onion field in Andong, Korea. To identify the fungal isolate, 18srRNA sequence for internal transcribed spacer (ITS) and β-tubulin regions were sequenced. The ITS and β-tubulin sequence were highly matched to Beauveria bassiana and morphological characteristics also was fit to known B. bassiana. Finally, isolated fungus has identified as B. bassiana and named B. bassiana ANU1. The result of bioassay, median lethal concentrations were 2.7×103 and 0.9×103 conidia/ml and medial lethal times were 65.6 and 60.8 h to S. exigua and P. xylostella, respectively. B. bassiana ANU1 showed high pathogenicity to two insect pests from 20℃ to 30℃ at 50% relative humidity (RH) and more than 40% RH at 25℃ with 107 conidia/ml of concentration.

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.

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.

곤충 병원성 곰팡이 Metharizium anisopilae JEF 003, 004와 Beauveria bassiana JEF 006, 007의 대량생산배지 조건에 따른 열안정성을 평가하였다. 첫 번째로 millet 배지에서 배양된 포자의 열안정성 평가를 위하여 50℃ 조건에 0, 30, 60, 90, 120 min으로 포자 현탁액 상태와 grain상태로 노출한 결과 현탁액 상태에서 포자 의 열안정성이 더 많이 감소하는 것을 확인하였다. 다음으로 각기 다른 고체배지 (조: 1~5.×109 conidia/g, 수수: 1~2×109 conidia/g, 기장: 2~3×109 conidia/g) 조건에 서 생산된 포자의 열안정성 확인을 위하여 배양이 완료된 포자를 현탁액과 grain상 태에서 50℃ 조건에 0, 1, 2, 4, 8 hours동안 노출하여 열안정성을 평가하였다. 실험 결과 현탁액 상태보다 grain상태에서 포자의 열안정성이 더 높은 것을 확인하였으 며, 조 배지 조건에서 포자의 열안정성이 가장 높게 향상된 것을 확인하였다. 마지 막으로 포자의 열안정성의 추가 향상을 위하여 배양이 완료된 고체배지 포자에 cotton seed oil, coconut oil, soybean oil, castor oil, olive oil, mineral oil을 넣고, 5 0℃의 온도에 0, 1, 2, 4, 8 hours으로 열안정성을 평가하였다. 결과적으로 cotton seed oil, soybean oil, castor oil, olive oil을 처리한 포자에서 높은 열안정성을 확인 하였다. 따라서 곤충병원성 곰팡이의 열안정성 실험 결과로 확인 된 조를 이용하여 높은 열안전성을 확보할 수 있을 것으로 판단이 되며, 추가적인 열안정성 확보를 위 하여 식물성 오일을 제제에 이용할 수 있을 것으로 전망된다.

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.

To determine whether S-(-)-10,11-dihydroxyfarnesic acid methyl ester (DHFAME) produced by Beauveria bassiana CS1029 potentially causes acute skin irritation as a cosmetic ingredient, a skin toxicity test was conducted as recommended for compliance with Korea Food and Drug Administration regulations. New Zealand White rabbits were treated with 100 mg/dose of DHFAME according to standard guidelines. No significant skin lesions or inflammation was observed in the DHFAME-treated group. Furthermore, DHFAME did not appear to cause skin irritation, as assessed by clinical observation of the rabbits. Thus, when taken together, the present results suggest that DHFAME is a promising potential cosmetic ingredient that does not irritate the skin.

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.

백강잠(Bombycis corpus)은 흰가루병 또는 백강균(Beauveria bassiana)이라 불 리 우는 곰팡이균에 감염되어 죽은 누에(Bombyx mori)를 말하는 것으로, 최근 연 구결과에서는 항경련작용, 항균작용, 항암작용, 수면촉진작용, 미백작용, 부신피 질자극작용 및 당뇨병 등에 효과가 있는 것으로 보고가 되어 있다. 누에 유충이 백 강잠으로 되게 하는 원인균인 백강균은 곤충병원성 곰팡이의 일종으로 같은 종 (species)에 속하는 것들이라도 분리된 곰팡이마다 살충활성을 비록하여 서로 다 른 생물학적 특성을 지니고 있어 이용 목적에 따라 최적의 곰팡이 균주의 분리 및 선택이 매우 중요한 것으로 알려져 있으며, 백강균의 종류에 따라 다양한 백강잠의 생성과 그 효능이 다양할 수 있다. 현재까지의 연구에서는 백강균의 종류에 따라 생 성되는 백강잠의 다양성을 비롯하여 효능평가에 대한 연구는 거의 이루어지지 못 하고 있으므로 백강균의 종류에 따른 백강잠의 생성의 다양성 및 효능 평가에 대한 연구가 필요한 실정이다. 본 연구에서는 국내에서 분리 확보한 20종의 백강균을 이 용하여 백강잠을 제작하고, 생산된 백강잠에서 에탄올과 증류수를 사용하여 생리 활성 물질을 추출하였다. 추출된 생리활성 물질을 이용하여 항세균, 항산화, 항암 에 대한 활성평가를 실시하였다. 본 연구결과를 통하여 백강잠의 이용목적에 따라 적절한 백강균을 제시함으로써 보다 효유적인 백강잠의 생산이 가능할 것으로 기 대되어진다.

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.

Use of conidia or spores of entomopathogenic fungi are influenced by environmental conditions such as temperature and relative humidity and caused slow and fluctuation of mortality. In addition, although the fungi have the advantage of a restricted host range, this specificity is one of the limiting factors for their use. These factors are preventing wider application and use of these biocontrol agents. To mitigate such problems we selected an Beauveria bassiana Bb08 which kill green peach aphid with its liquid culture filtrate. In this study we conducted bioassay with the fungal culture filtrate and culture fluid to greenhouse pests such as cotton aphid, whitefly and thrips. Culture fluid showed high mortality against green peach aphid, as well as cotton aphid, sweet potato whitefly, and western flower thrips. However, control effect of culture filtrate varied with pests. Culture filtrate showed high mortality to cotton aphid. Mortality of western flower thrips with culture filtrate was slower than culture fluid including spores and sweet potato whitefly was much lower. These results indicated that the culture fluid of Beauveria bassiana Bb08 has potential to simultaneously control various greenhouse pests.

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.

본 연구는 곤충 병원성 균주 Beauveria bassiana 배양액을배양일 수에 따라 이화학적 특성, 항산화 활성, 항염증활성 및항암활성을 검정하여 천연 생리활성 물질 및 기능성 신소재개발을 위한 기초자료를 얻고자 하였다.1. 유리당은 Glucose, Maltose, Fructose 존재하였고 Glucose함량이 B. bassiana 15.2g·L−1으로 배양 3일 시 다량 함유되어 있었으며 배양기간이 길어질수록 함량이 감소하였다. 2. 유기산은 Citric acid, Malic acid, Formic acid, Lacticacid, Acetic acid, Succinic acid 총 6종의 성분이 동정되었고,Citric acid 성분이 다량 함유되었으며 균주 중 B. bassiana.3일 배양액이 276.4mg·L−1로 가장 많이 검출되었음.3. B. bassiana 균주 배양액은 총 16종의 아미노산으로 조성되어 있었으며 Glutamate 함량이 배양 3일 시 987.0pmol/µl으로 다량 함유되어 있었다.4. 무기성분은 Na이 다량 함유되어 있었으며 B. bassiana 15일 배양액이 179.5mg·L−1로 가장 많은 Na을 함유하고 있었다.5. B. bassiana 배양 5일 시 6.34±0.30ppm, 플라보노이드함량은 B. bassiana 배양 3일 시 1.09±0.05ppm, 배양 15일시 1.10±0.06ppm으로 높은 함량을 나타냈다.6. DPPH free radical 소거능은 B. bassiana 배양 15일 시263.4±13.3µg·ml−1, SOD 활성은 B. bassiana 배양 15일 시93.7±16.6µg·ml−1, ABTS 활성은 B. bassiana 배양 5일 시99.7±1.2µg·ml−1으로 높은 활성이 나타났다.7. Anticancer activity 결과 A549 cell lines은 B. bassiana5일 배양액(163.8±4.1µg·ml), HepG2 cell lines는 B. bassiana10일 배양액(127.8±2.3µg·ml)으로 항암활성이 높게 나타났으며, MCF-7 cell lines에 검정한 결과, B. bassiana 5일 배양액(129.1±6.8µg·ml)으로 높은 활성을 나타냈으나 대조구인Doxorubicin보다는 낮은 활성이 나타났다.

Insect-killing fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana ERL1170 induced melanization of yellow spotted longicorn beetles, Psacothea hilaris as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm, Spodopetra exigua larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment

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.