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.

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.

본 연구에서는 다양한 식품유해미생물에 대한 길항작용 및 항산화활성이 우수한 B. subtilis SRCM102046 균주를 식품보존 소재로서 이용하기 위해 반응표면분석법을 이용하여 균체 증량을 통한 항균활성 및 항산화활성 증대를 도모하고자 하였다. SRCM102046의 산업적 활용을 위한 성장조건을 최적화하기 위해 배양시간에 따른 균체 성장을 조사하였으며, 통계학적 기법인 반응표면분석법을 사용하였다. SRCM102046의 최적 성장을 위한 배지 성분을 선별하기 위해 Plackett-Burman design을 이용하였으며, PBD 결과 선별된 배지 성분으로 molasses, sucrose, peptone으로 예측되었다. 각 배지 성분의 최적농도를 결정하기 위한 방법으로 central composite design을 이용하였으며, 최종적으로 예측된 각 배지 성분의 농도는 molasses 7 g/L, sucrose 7 g/L, peptone 2 g/L로 예측되었다. 이때의 균체량은 22.03±1.30 g/L로 예측되었으며, 통계분석을 통해 실험모델의 적합성을 확인하였다. 또한, 실험 모델을 수행하여 건조균체량을 측정한 결과 22.02±0.35 g/L로 측정되어 실험모델에 의해 예측된 값이 오차범위 내에 존재하여 모델의 신뢰성이 매우 높음을 확인하였으며, 이는 실험모델에 의해 예측된 최적배지 사용시 최적화 이전의 LB 배지에서의 균체량(2.47±0.03 g/L)대비 약 9배의 균체량이 증가함을 확인하였다. 또한 최적배지에서 B. subtilis SRCM102046 배양 시 항균활성은 대조구로 사용된 LB 배지에서의 항균활성 대비 최대 140% 향상되었으며, 항산화활성은 약 100.41% 증가함을 확인하였다. 본 연구를 통하여 식품보존제로서B. subtilis SRCM102046의 산업화를 위한 배지최적화를 수행하였으며, 추후 박테리오신의 정제 및 특성 등에 대한 세부적인 연구가 필요하지만 본 연구를 통해 확립된 배양조건을 기반으로 식품보존 소재의 측면에서 매우 유용한 자료로 활용될 수 있을 것으로 기대된다.

When used as a starter in the manufacture of Meju, it is expected that the quality of the soup products can be improved. In this study, we isolated Lactobacillus strain having possible safety and food-industrial benefits as a starter. Four hundred and seven isolates were screened from Meju, and chemically characterized for their antibacterial activity against Bacillus cereus, non-productivity of biogenic amine, and hemolysis. Eight of the isolates were selected upon chemical characterization, and their antioxidant and β-glucosidase activity was measured. Finally, we selected, and measured its enzyme activity and antibiotic resistance. Next, we investigated its cell growth, showed maximum biomass of 3.5 g/L after 28 h of culture. The ingredients of the medium to improve biomass were selected using the Plackett-Burman design (PBD) and central composite design (CCD). The results obtained using PBD revealed molasses, yeast extract, and maltose to be significant factors determining the biomass of the L. brevis SCML 432 strain. The CCD was then applied with three variables found from PBD and the optimum values were predicted to be 5.5% molasses, 1.5% yeast extract, and 2.0% maltose, and the maximum biomass was predicted to be 11.2 g/L. Through model verification, we confirmed that the predicted and actual results were similar, with about 3.2-fold improvement in the biomass from 3.5 g/L to 11.3 g/L when compared to that obtained in basal medium. These results suggest that SCML 432 has high potential in the food industry as a starter.

In this study, we tried to screen the Bacillus strain having safety probability by isolation of strains from traditional fermented food, measurement of probiotic properties, and the fermentative characteristics of Cheonggukjang. We isolated 400 Bacillus-like isolates from traditional fermented foods. Selected strains examined on the prevalent characteristic such as extracellular enzyme and antibacterial activities, and their safety probability was confirmed by biogenic amine productivity, hemolytic, and harmful substances and enzyme productivity. We selected the 5 strains by analysis of biogenic amine, antibacterial and B. cereus toxic associated gene. Five selected strains were examined on cell surface hydrophobicity, and bile and acid tolerance, and we selected the SRCM100730 as the final strain. SRCM100730 was confirmed B. amyloliquefaciens by 16S rRNA sequencing, and named the B. amyloloquefaciens SRCM100730 (KCCM11966P). Finally, we manufactured Cheonggukjang using SRCM100730 for confirmation of fermentation properties. Manufactured Cheonggukjang did not contain B. cereus, and showed that γ-PGA and extracellular enzyme activities were superior to commercial Chunggukjang. Amino nitrogen content was 544.02 mg% and 26 free amino acid were detected, and the bitterness-related amino acid content was lower than commercial Cheonggukjang. Especially, the amount of GABA was 3 fold higher than commercial Cheonggukjang. These results suggest that SRCM100730 have high availability in commercial probiotics market and fermented food industry.