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.

토마토 재배에 사용되는 농자재를 이용하여 곤충 병원성 곰팡이인 Beauveria bassiana의 병원성에 끼치는 영향을 조사하였다. Chlorothalonil 등 13종의 살균제 첨가 배지에서는 B. bassiana 균사가 전혀 생장하지 못하였고 chlorothalonil 등 12종의 살균제 첨가 배지에서균총이 형성되지 않았다. 살충제와 친환경자재를 첨가한 모든 배지에서 균사가 생장하고 포자가 형성되었지만 대부분 배지에서 무처리와 비교했을 때 유의차를 보였다. 온실가루이에 대한 살충력은 B. bassiana 단독처리일 때보다 화학살균제(polyoxin B, mandipropamid)와 친환경농자재(sulfur) 혼합시 떨어졌으며 화학살충제(pyridaben, dinotefuran)와 식물추출물 유래 친환경농자재(pyrethrum) 혼합시에는 유의차가 없었으며 온실에서 살균제인 polyoxin B의 혼합으로 온실가루이에 대한 B. bassiana의 감염력이 떨어지는 것을 확인하였다.

Beauveria bassiana is one of universal insect pathogenic fungi that have been used for biocontrol agent against insect pests. This fungus has also been studied for medicinal use. To meet for commercial use, the artificial production of the fruit body of this fungus has been established by the Mushtech Co in Korea. This study was carried out to define the morphological features of the fruit body of B. bassiana developed through artificial cultivation. For the observation of mycelia growth, B. bassiana was cultured on the Sabouraud Dextrose agar plus Yeast Extract(SDAY), nut-supplemented medium, and Fe ion-supplemented SDAY at 25℃ for 15 days. The variation of colony color was observed between the different media. Strong pigmentation was observed on Fe ion-supplemented SDAY. To investigate morphological characteristics of fruit body, geminating ascospores and vegetative hyphae were observed though light microscopy and scanning microscope. During seven weeks of cultivation period, the development process of apical fertile part of stromata can be separated by the development stage of perithecia. To understand the developing process of fruit body at the transcript level, investigating process of distinct gene expression according to cultural condition and developmental stage was discussed.

Low thermotolerance in entomopathogenic fungi is the main impediment to their industrialization. This research, for the first time, describes the generation of a thermotolerant colony by pairing and subculturing two Beauveria bassiana isolates without sexual reproduction. A mixture of B. bassiana ERL1578 and ERL1576 was inoculated on 1/4SDAY. The paired culture was subcultured three times, followed by a heat treatment as a selection pressure. Two morphologically different colonies (BbHet1 and BbHet2) were isolated from the pairing. BbHet2 had the most rapid mycelial growth and produced sponge-like mycelial masses (the others were flat), and its conidia were darker than the non-paired colonies under a microscope. BbHet2 conidia had 60.7% germination after exposure to 45°C for 60 min (the others had <15%) without significant loss of virulence against western flower thrips, but a slight decrease in conidial yield was found. Sequencing of Bloc locus and 2-D electrophoresis were done to further clarify the phenomenon. The new phenotypes formed suggested that a genetic variation happened as a result of heterokaryosis and/or recombination, more than environmental adaptation, when mixing different conidia. This methodology seems to be very useful for enhancing thermotolerance in fungi.

뽕나무병원성세균에 대한 Beaveria bassiana 6균주의 항균활성을 paper disc법으로 조사하였고 최소성장저해농도(MIC)를 액체배지희석법으로 조사하였다. 항균활성은 B. bassiana J200에서만 나타났으며, Erwinia rhapontici KACC 10407에서는 13mm, Pseudomonas syringae KACC 10390와 Xanthomonas campestris KACC 12134에서는 각각 17 mm의 항균활성이 나타났다. MIC 측정 결과 E. rhapontici KACC 10407, P. syringae KACC 10390 및 X. campestris KACC 12134 모두 4.0%에서 MIC를 보였다. 이상의 결과로 B. bassiana의 뽕나무병원성세균에 대한 생물학적 방재제로서의 이용 가능성을 확인하였다.

Two-spotted spider mite, Tetranychus urticae (Koch) is one of the serious pests in economically important crops such as strawberry and cucumber and so on. Acaricides have been used as the main control agents. This study was conducted to test the synergistic effects of the Beauveria bassiana GHA, which has been registered for whitefly and thrips, and five pesticides (abamectin, acrinathrin, bifenthrin imidacloprid, dinotefuran, and indoxacarb, which are commonly used pesticides in strawberry in Korea), on the two-spotted spider mite T. urticae. Five tested pesticides did not inhibit spore germination and mycelial growth of B. bassiana. Pesticides were applied to potted strawberry plants at four different treatments (recommended concentration, 1/5 recommended concentration, 1/5 recommended concentration + GHA(108 conidia/ml), and only GHA(108 conidia/ml). Mortality in larvae of two-spotted spider mite was 12% in GHA 5 day after treatment. Mortality in abamectin treated T. urticae was 98.5% and 100% at 1/5 recommended concentration and recommended concentration, respectively, 3 days after treatment. Acrinathrin, indoxacarbe, dinotefuran and difenthrin imidacloprid caused 60%, 14%, 16% and 91% mortality at recommended concentration 5 day after treatment. The tested five pesticides and B. bassiana GHA had no synergistic effect.

Beauveria bassiana 등의 곤충병원성곰팡이를 해충방제에 적극적으로 활용하 기 위해서는 효과적인 대량배양방법의 개발이 필수적이다. 이에 대한 한 가지 방 안으로 다음의 방법을 제시하고자한다. 곰팡이의 배양 시 곡류는 탄소원을 다량 함유하고 있으나 질소원이 부족한 반면, 기장은 탄소원은 물론이거니와 질소원 역 시 다량 함유하고 있어서 포자 생산과 균사생장에 있어서 매우 매력적인 재료이므 로 기장을 이용하여 시험을 수행하였다. 우선 B. bassiana 균주를 PDB 배지에 2 5℃, 5일간 배양하여 종균으로 사용하였다. 고체 배지원인 기장은 물에 불리는 시 간을 달리하고(10분, 30분, 60분) 버섯재배용기에 넣고 121℃, 30분간 멸균후 B. bassiana접종하여 배양하였다. 배양온도는 25℃±0.5, 습도 30%±5, 암조건에서 배 양하였으며, 배양 15일 후, 1.9×109cfu/g, 2.9×109 cfu/g, 3.0×109 cfu/g의 포자를 생 산하였다. 최초 접종량은 5ml, 10ml로 달리하여 배양하였으며, 초기 배양성은 30 분, 60분 불리고, 10ml접종한 것이 좋았으나, 배양 20일 후에는 차이를 보이지 않았다.

Entomopathogenic fungi are widely available as biological control agents for controlling insect pests in agriculture and forestry. The fungal culture broth contains various pathogenesis-related components such as blastospores, mycelium and insecticidal enzymes such as chitinase, Pr1- and Pr2-proteases, which have been reported to play an important role in penetrating insect cuticles. In this study, we tried to evaluate the utility of culture broth from Beauveria bassiana SFB-205 to control lepidopteran pests. High level of insecticidal activity correspond to over 90% of mortality were observed when the culture broth of B. bassiana SFB-205 was inoculated to the Spodoptera litura larvae together with the B. thuringiensis K1. The freeze-dried culture broth showed synergistic effects in insecticidal activity against larvae of S. exigua and S. litura when treated with corresponding baculoviruses, SeNPV and SlNPV. Active ingredient of the B. bassiana SFB-205 culture broth was identified to chitinase, which have truncated form by insertional mutation compared to previously reported chitinases.

This study was conducted to investigate optimal conditions for formation of synnemata from Beauveria bassiana. The strain of B. bassiana was isolated from a host of Oncotympana fuscata in Halla mountain of Jeju island. The yield of synnemata was the highest at application of brown rice (90%) and silkworm pupae (10%) media. On the other hand, the other media did not result in formation of synnemata. The highest formation of synnemata was achieved at conditions of moderate air inflow and 20℃. The optimal moisture and light intensity for formation of synnemata were 60% and 300 lux, respectively. In addition, inoculation of the liquid spawn resulted in higher yield than that of asexual spore.

Entomopathogenic fungi were isolated directly from a cadaver of adult M. saltuarius (infected with white fungi) supporting fungal sporulation, to develop biological control of pine wilt disease vector, M. saltuarius which was the most abundant in the middle to northern part of Korea and caused enormous damage to native pine tree in Korea, Japan and other regions of Asia. Pathogenicity of each fungus was tested using oak longicorn beetle, Moechotypa diphysis, as substitutive insect. As the result, only one of them showed pathogenic to adults of M. diphysis, with up to 100% mortality within 13 days of inoculation. Selected fungus was named as MsW1 and identified by Beauveria bassiana using microscopic examination, B. bassiana-specific PCR primers and genetic sequencing of the ITS region analysis. Pathogenicity test were conducted with various concentration of conidial suspensions of this isolate on M. saltuarius (3rd instar larvae and adults). Mortality rates varied from 57.1% to 100.0% and from 16.7% to 100.0% of M. saltuarius (3rd instar larvae and adults), respectively at 30 days. This is the first report of natural infection of M. saltuarius by B. bassiana.

Among the cultured products of Beauveria bassiana SFB-205 (KCCM 10892P), the supernatant showed the highest insecticidal activity against 2nd instars of Aphis gossypii (Aphididae) nymphs under glasshouse condition. The enzymes in the supernatant were confirmed as active materials, and the chitinase was finally selected as a QC factor for commercial production. However, the chitinase activity in the supernatant decreased by 11-folds due to the thermal stress at 50℃ for 2 h. To obtain thermal stability, the chitinase in the supernatant was adsorbed to a precipitable material and the pellet was freeze-dried (PCT/KR2007/005886). The adsorbent-A showed about 92.7% of harvesting efficiency which was higher than the other candidates. The chitinase activity of the freeze-dried powder was kept up about 82.0% of initial activity for the same thermal stress. Subsequently, an optimal formulation recipe was established to maximize long-term storage stability and efficacy. SFB-205 oil-based formulation was stable up to 18 months at room temperature. It showed 96.1% efficacy against 2nd instars of A. gossypii nymphs at 1 day after the treatment in the glasshouse. This novel approach can be a promising method to develop competitive biopesticies in the entomopathogenic fungi, even though it needs to be intensively studied.

Autographa californica 핵다각체병 바이러스(AcNPV)의 다각체 단백질과 Bacillus thuringiensis(Bt) cryIA(c) 내독소 단백질의 융합단백질을 생산하는 새로운 재조합 바이러스를 제작하고, 곤충세포주(Spodoptera frugiperda 9)에서 발현된 융합단백질의 특성을 분석하였다. Bt kurstaki HD-73의 cryIA(c) 내독소 단백질 유전자의 N-발단 AcNPV의 완전한 다각체 단백질 유전자의 앞쪽에 융합함에 의하여 또는 다닥체 단백질 유전자내의 제한효소 HindII부위에 삽입함에 의하여 다각체 단백질 유전자의 프로모터 조절하에 도입하였다. 이렇게 작성된 재조합 바이러스를 각각 Btrusl 또는 BtrusII라고 명명하였다. BtrusI은 분명히 단일 전사체를 보임에도 92kDa의 융합 단백질과 다각체 단백질의 두 단백질을 생산하였다. 또한 Btrusl에 의해 만들어진 융합 단백질은 다각체를 형성하지 않았다. 한편, BtrusII에 의해 감염된 곤충세포주에서는 33kDa의 다각체 단백질은 보이지 않았고 단지 융합 단백질만 생산하였으나 다각체는 형성하지 않았다. 따라서 Btrusl에 의해 생산된 융합 단백질의 독성을 조사하기 위하여, Btrusl으로 감염된 곤충세포주를 2령 누에(Bombyx mori)에 접종한 결과 융합 단백질에 의한 독성이 관찰되었다. 결론적으로 다각체 단백질과 Bt cryIA(c) 내독소 단백질에 의한 융합 단백질이 독성을 가지고 있음을 확인하였다.

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.

산림청으로부터 분양받은 백강균의 균주 F101, F587, FJ8의 포자형성력과 균사와 포자의 esterase 활성도를 비교한 결과 F101이 가장 우수한 것으로 나타났다. 균의 포자를 도말한 후 조건에서 보관하면 5~7일부터 충이 치사하여 24~28일 후에는 충의 표면에 균사와 포자가 분출한다. 그러나 보관조건에서는 뚜렷한 병적 증상이 나타나지 않았고 50일 이상이 경과하여도 충밖으로 균사나 포자가 분출되는 것을 관찰할 수 없다. 백강균의 포자를 충체에 주입하였을 경우에는 충의 치사시기가 빨라지며 4보다 에 보관하면서 지방체, 표피, 혈림프로 구분하여 전기영동법으로 조사한 결과 각 조직의 일반단백질과 esterase는 충 치사시기인 3일(60시간)을 기점으로 상당수가 사라지는 현상을 보인다. 백강균은 기주에 재차감염을 일으킬 수 있는 확율을 최대한으로 하기 위하여 공기중에 있을 때는 포자를, 토중에서는 길고 가는 균사를 형성하는 생리적인 적응을 한다.

목화진딧물과 복숭아혹진딧물은 채소를 포함한 다양한 작물에 큰 피해를 주는 해충이다. 이들 해충의 친환경 방제를 위해 진딧물에 병원성이 높은 곰팡이(B. bassiana) 유래 대사물 질을 이용한 방제 연구를 수행하였다. 이 균주의 배양여액의 살충율은 목화진딧물에 처리 3일 후 100%, 복숭아혹진딧물에 처리 5일 후 99%였다. 이 추출물을 CHCl3 : MeOH 비율을 달리하여 silica gel column chromatography 한 결과, 50:1 (CHCl3 : MeOH)의 추출물 분획의 살충률이 목화진딧물과 복숭아혹진딧물에 각 80%와 75.4%로 다른 분획에 비해 현저하게 높았다. 또 각 용매비율별 추출물을 1:1로 혼합하여 처리한 결과, 분획 추출물 혼합에 의한 상승효과는 보이지 않았으며 두 진딧물에 모두 50:1 (CHCl3 : MeOH) 단독처리에서 살충률 (목화진딧물 77.3%, 복숭아혹진딧물 76.4%)이 가장 높았으며 50:1 + 70:1이 두 번째로 높았 다(목화진딧물 72%, 복숭아혹진딧물 70.2%). 앞으로 안정적인 진딧물 방제를 위해 50:1 분 획 추출물 내 진딧물 살충성 물질을 분리 동정 및 대량생산 연구를 진행할 예정이다.

This study was carried out to development the environment-friendly control method of Myzus persicae using Beauveria bassiana and Environment-friendly agricultural materials (EFAM). Germination rate the dried conidia of B. bassiana was 90 percent at 24-27 ˚C temperature condition. Mortalíty of M. persícae was 56. 7% at 1 X1 07 cfu/ml conidia concentration. Four EFAM such as Pachungtan, Wangjungwang, Newbiodakill, Muginchon were low toxicity to B. bassiana growth. Among them, Muginchon (derris extract) and Wangjungwang (neem extract) could have synergistic effect to aphid mortality when mixing treat with B. bassiana, which protection value was 99% in 3 days after treat.