Destruxins (Dtxs) are insecticidal cyclic hexadepsipeptides produced by the entomopathogenic fungus Metarhizium anisopliae. Media composition for dtxs production was optimized with industrial grade media. Glycerol and casein peptone were selected as a carbon source and a nitrogen source, respectively. Dtxs production varied with C/N ratios. High yields of dtxs were observed at C/N ratios ranging 0.3 to 1.5, with concentrations mostly higher than 800 mg/L. Low yields were caused by high C/N ratio ranging from 3.0 to 8.0, resulting in less than 500 mg/L. The highest yield of Dtxs was obtained with 2% glycerol and 3% casein peptone, showing 192.2 mg/L of dtx A, 911.1 mg/L of dtx B, and 113.3 mg/L of dtx E, respectively. These results indicated that dtxs production is highly influenced by C/N ratio, especially the content of nitrogen source.

Mass breeding of Protaetia brevitarsis seluensis results in the entomopathogenic fungal infection, usually Metarhizium anisoplaie. A mixture of microorganisms (Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae) delayed fungal infection by M. anisopliae, which infected fewer P. b. seluensis when the microorganism mixture was added to sawdust as feed for P. b. seluensis for 30d, their mortality rate was approximately 35% less than that of the control group, which was fed sawdust without the EM. In addition, the growth of M. anisopliae on agar media spread with each bacterium as inhibited by up to 80%.

The entomopathogenic fungus Metarhizium anisopliae is one of potent biological control agents against a variety ofinsect pests. In this study, we investigated enzyme production of M. anisopliae strains A and B. They produced extracellularenzymes for degrading the epidermis of Monochamus alternatus, a crucial mediator of the pinewood nematode Bursaphelenchusxylophilus. With Q-TOF MS/MS analysis, 29 kDa protein, a major band on a SDS-PAGE gel, was identified as subtilisin-likeserine protease PR1A. M. anisopliae A produced an extracellular enzyme more efficiently than M. anisopliae B: however,enzyme activities targeted for the cuticle were comparable. Our results suggest that the two strains of M. anisopliae havethe biological potential against M. alternatus with insecticidal protease production.

The entomopathogenic fungus Metarhizium anisopliae B is a powerful biological control agent against Monochamusalternatus, a crucial mediator of the pinewood nematode Bursaphelenchus xylophilus. In this study, production of destruxins(dtxs), insecticidal cyclic hexadepsipeptides, was monitored in the submerged culture of M. anisopliae B. Three typesof dtxs, i.e., destruxin A, B, and E, were produced during the culture. Among the three dtxs, the production yield ofdestruxin A was best, followed by destruxin B and E. Destruxin A production was increased when pH was controlledat 6.0, whereas production of destruxin E was not affected by the pH control. The highest yield of dtxs A, B, and Ewere 16.4, 7.3, and 6.1 mg L-1, respectively. Considering that process for dtxs production has not been optimized, M.anisopliae B has more powerful implication as a biocontrol agent.

The purpose of this study was to identify the new fungal disease of Scopendra subspinipes mutilans (Chilopoda: Scolopendridae) from Jeju island in Korea. Scopendra subspinipes mutilans are also known the Chinese red head and average 20cm in length. It is used for traditional medicine for skin-disease and tumor and S. s. mutilans is recently raised in Jeju island to use healing properties. We isolated fungi from surface of infected S. s. mutilans and incubated in SDBA at 24℃ for 14days, and then identified the morphology of the fungi by light and electron microscopy. For the specific diagnosis of Metarhizium spp. in S. s. mutilans, 18srRNA of fungi was amplified by primers ITS4_MF and LR3_MR and sequenced by using the BLAST server at the National Center for Biotechnology Information. The result of the 18srRNA sequencing alignment was Metarhizium anisopliae (99.9%). The entomopathogenic fungi, M. anisopliae would be fatal cause of higher mortality to S. s. mutilans, and it is needed in vivo assay to confirm the pathogenic activities of M. anisopliae to S. s. mutilans.

Beet armyworm, Spodoptera exigua, diamondback moth, Plutella xylostella and tobacco cutworm, Spodoptera litura are the three most serious pests of many economically important crops such as cruciferous crops, various vegetables and ornamental plants. Because these pests are known to be resistant to lots of chemical insecticides, integrated control using both or either entomophathogen and/or natural enemy is thought to be an attractive alternative for effective control. One of the obstacles using and expanding mycopesticide is narrow host ranges. At commercial farms, cultivating crops are seriously damaged by various Lepidopteran pests. Farmers want to use a microbial control agent which can control various host insects to reduce cost and labor. In previous study, we selected two entomopathogenic fungi, Metarhizium anisopliae and Paecilomyces fumosoroseus, which shown high virulence against beet armyworm. For wide use of the isolates in farm, we tested its host ranges, especially to diamondback moth and tobacco cutworm, which also are serious pest in Korea. The two isolates were shown a good control effect in leaf disc bioassy.

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.

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.

In agricultural fields, the entomopathogenic fungal species have been investigated for their potential as the biological control agents due to their role of natural enemies for insects. To address the requirements of a potential South Korea based biocontrol effort using entomopathogenic fungi, we investigated the occurrence of various entomopathogenic fungi in 1080 soil samples representing from various area and locations in South Korea. Entomopathogenic fungi were isolated from soils using semiselective medium SDA-D50 contained saboraund dextrose agar, 50 ug/ml dodine, 100 ug/ml chloramphenicol and 50 ug/ml streptomycin. The isolated putative fungi were identified by the determination of internal transcribed spacer (ITS) region sequences of the nuclear ribosomal analysis. As a result, entomopathogenic fungi were found to occur in 30.8% of the soil samples studied. The most abundant species were Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metschn.) Sorok. Isolates of B. brongniartii, Cordyceps sp., Lecanicillium sp., Isaria sp. and Tolypocladium cylindrosporum were also found. The occurrence of entomopathogenic fungi was analyzed by the area and soil types. These positive entomopathogenic fungi may have potential against variety pests in agriculture and forest

곤충자원의 대량사육을 위한 병 발생 예방과 해충의 효과적인 방제를 위하여 흰점박이꽃무지 이병충으로부터 곤충병원 사상균을 분리하였다. 전자현미경 관찰 결과 분리균주 KMA-1은 Metharizium속의 전형적인 쇠사슬형의 분생자를 paliside-like masse에 형성하였다. 따라서, 정확한 동정을 위하여 28S rRNA와 ITS염기 서열을 바탕으로 제작한 특이 프라이머쌍을 사용하여 PCR 반응을 수행하였다. 각각의 프라이머쌍을 사용한 PCR반응으로부터 특이 밴드가 검출되었으며 이 증폭 산물들의 염기 서열을 결정, 비교하였다. 분리 균주 KMA-1의 PCR산물인 28S rRNA와 ITS DNA염기서열을 GenBank데이터베이스에 등록된 염기서열 정보와의 상동성을 검색한 결과, 모두 Metarhizium anisopliae와 가장 높은 서열 상동성을 보였다. 이상의 결과로서 본 실험에서 분리 명명된 KMA-1는 M. anisopliae로 동정되었다.

농약이 한국산 곤충병원성곰팡이 Beauveria bassiana와 Metarhizium anisopliae에 미치는 영향을 알아본 결과, SMAY배지상에서의 농약농도에 따른 곰팡이 포자형성수는 metalaxyl과 tolclofos-methyl 처리에서는 대조구와 큰 차이가 없었으나. fenitrothion과 mepronil 처리에서는 권장량의 배 처리에서도 포자형성이 미약하였다. 농약과의 동시 처리가 곤충병원성 곰팡이에 미치는 영향을 알아보기 위하여 농약희석액에 병원성 곰팡이를 1-5시간 동안 침지시켜 병원성을 알아본 결과 전 약종에서 100%의 병원성을 나타내었다. 배추밭에서 농약전처리가 M. anisopliae의 병원성에 끼치는 영향을 알아본 결과는 Bt나 teflubenzuron, metalaxyl, carbofuran 처리구는 대조구와 병원성에서 차이를 보이지 않았으나 alachlol 처리구에서는 병원성이 80% 이하로 떨어졌다. 잔디밭에서 농약의 후처리가 병원성곰팡이의 병원성과 지속성에 미치는 영향을 알아본 바 iprodione+thiram과 tolclofos-methyl 처리구에서는 4달째까지 병원성이 지속되었고 fenitrothion처리구에서는 3개월 후부터 B. bassiana와 M. anisopliae의 병원성이 상실되었으며 mepronil처리구에서는 M. anisopliae가 병원성을 상실하였다.

우리나라의 곤충병원성곰팡이 분포를 조사하기 위하여 5개도와 3개 광역시에서 200개소의 토양 sample을 채집하여 꿀벌부채명나방 유충을 이용하여 분리하였다. 8종의 곤충 사체로 부터는 직접 분리하였다. 그 결과 40개소(20.0%)의 토양과 8종의 곤충 사체에서 곰팡이가 분리되었는데, 토양에서 분리된 균종별로는 Bequveria가 21개소(10.5%), Metarhizium이 19개소(9.5%)이었다. 그외 7종의 곤충에서 Beauvria가 분리되었다. 서식처별로는 산림 110개소중 25개소(22.7%), 경작지 20개소중 2개소(11.0%), 휴한지 11개소중 6개소(54.5%), 하천 제방 19개소중 7개소(36.8%)에서 곤충병원성곰팡이가 검출되었다. 한편, 곤충 사체에서는 긴수염 대벌레에서 Metarhizium이 분리된 것을 제외하고는 모두 Beauveria가 분리되었다.