Beauveria bassiana JEF-007 with strong virulence against Riptortus pedestiris was selected for the Agrobacerium tumefaciens-mediated transformation(AtMT). AtMT generated two transformants, B1-06 and C1-49, showed significantly reduced virulence against R. pedestris. To identify the virulence-related genes, thermal asymmetric interlaced(TAIL) PCR and flanking region analysis were performed. From the analysis, two genes, Complex I intermediated-associated protein 30(CIA30) and Autophagy protein 22(Atg22), possibly related virulence in B. bassiana JEF-007. For the analysis of two putative virulence-related genes in JEF-007, hairpin RNA interference (hpRNAi) is under consideration. This work can provide the functional roles of the virulence-related genes in B. bassiana JEF-007.

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.

Bean bug, Riptortus pedestris (Hemiptera: Alydidae) is an agriculturally serious pest in East Asian countries. Chemical pesticides have been 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. Herein entomopathogenic Beauveria bassiana JEF isolates were collected, identified and assayed against bean bugs in laboratory conditions. Some isolates showed >80% virulence by contact-exposure and spray methods. The Agrobacterium tumefaciens-mediated transformation of B. bassiana JEF-007 generated random transformants and some mutants showed reduced virulence against Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and R. pedestris nymph. Compared to the wild-type, the two transformants showed remarkably different morphology, conidial production, and thermotolerance. To figure out pathogenicity-related genes, thermal asymmetric interlaced (TAIL) PCR of the random transformants was performed and possibly some virulence-related genes were predicted. This work can be a strong platform for the functional genetics of bean bug-pathogenic B. bassiana.

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.

S. aureus is reported as a major cause of nosocomial infections after dental care and involved in endocarditis, bacteremia, osteomyelitis, peritonitis, and soft tissues etc. It is very important to identify the distribution and the diversity of toxin gene associated with the S. aureus expression in dental care patients with periodontitis directly for an effective prevention and treatment of dental diseases. Fifty four strains of S. aureus were isolated from the saliva of 129 patients who were diagnosed with periodontitis at dental clinics and hospitals located in Seoul. The distribution of the virulence gene and the genetic diversity of the strains were studied using the polymerase chain reaction with isolated strains. The enterotoxin test showed Seb was the most frequent gene with 88.9%. The hemolysin gene of Hla, Hib and Hld were the most frequently gene with 98.1% (53 strains), leukocidins gene of lukM showed 90.7% (49 strains), and laminin binding protein gene of Eno showed 100% (54 strains), respectively. The diversity of the enterotoxin gen was held as Seb-Seg-Sei gene of 35.2% (19 strains), the diversity of hemolysin gene of Hla-Hlb-Hld gene was 98.1% (53 strains) and the diversity of leukocidins gene of LukD-LukM were 88.9% (48 strains), respectively. Patients with dental disease showed somehow high toxin gene expression so that S. aureus in dental care area is judged to show very highly pathogen with a high and infection rate. In the future, additional studies for these toxin genes seem to be required.

The aim of study was to investigate the virulence profile of Escherichia coli O157:H7 bacteriophages isolated from sewage and livestock stools. Among 23 E. coli O157:H7 bacteriophages, 14 strains were isolated from sewage and 9 were from animal stools collected from 10 livestock farms in Korea. For each bacteriophage DNA sample, the presence of stx1, stx2, eae, aafII, ial, elt, estI, estII, astA, afa, and cnf was examined by polymerase chain reaction. The detection rate of eae, stx2, estI, astA, and ial was 100%, 69.6%, 13.0%, 13.0%, 8.7%, respectively. While all E. coli O157:H7 bacteriophages isolated from stools carried eae+stx2, stx2+eae, eae+astA, eae, stx2+eae+estI, eae+estI, stx2+eae+ial, and eae+ial were observed in bacteriophages isolated from sewage. As several plasmid-carrying virulence factors (estI, astA, and ial) were found in E. coli O157:H7 bacteriophages obtained from sewage and stools, the microbial safety of bacteriophages should be investigated in further study.

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.

We previously developed a novel attenuated Salmonella Typhimurium (S.Typhimurium) △lon△cpxR vaccine. This study was conducted in order to examine whether this vaccine could effectively protect growing piglets against Salmonella infection. Pregnant sows in group A were primed and boosted with the vaccine, whereas pregnant sows in group B received sterile PBS-sucrose. After farrowing, newborn piglets in groups A and B were challenged with a wild type virulent S. Typhimurium at three weeks of age. During the study, serum IgG titers of piglets in group A were significantly higher than those of piglets in group B (P<0.001). In addition, clinical signs were observed in 5.9% of piglets in group A during the entire experimental period after the challenge, while diarrhea was observed in 81.6% of piglets in group B. These results indicate that vaccination of the pregnant sows resulted in effective protection in piglets against Salmonella infection.

Beet armyworm, Spodoptera exigua are difficult to control using chemical insecticides because of the development of insecticide resistance. For eco-friendly beet armyworm managements, various control agents are required. Entomopathogenic fungus is one of promise control agents as an alternative to chemical control agent. We isolated entomopathogenic fungi from soil samples of suwon by insect-bait method using Galleria mellonella and conducted bioassay to larva of beet armyworm. As a result of bioassay isolate FG274, FG340, FG344 had high virulence as 100% against second instar larva of S. exigua. To identify the fungus isolates, their’s morphological characteristic was observed and ITS of 18srRNA was sequenced. ITS sequence of FT274, 340, 344 were highly matched (100%) to that of Beauveria bassiana, Paecilomyces fumosoroseus, Metarhizium anisopliae. To investigate the optimal concentration, three isolates were sprayed at three different concentration(1×106 ,107 and 108 conidia/㎖) in laboratory conditions. 나타내었다.

본 연구는 새로운 뿌리 혹병균 균주의 병원성 차이와 배추와 무의 저항성 차이를 구명하고자 수행하였다. Williams 판별 기주 4종을 이용한 접종 균주들의 race검정에서 기보유 균주는 발병률 64.1-90.6%이 었고, PC91214 균주는 발병률 98.4-100%, 발병도 4.2-4.6를 보여 두 균주 모두 race 4이지만 PC91214 균주가 더 높은 병원성을 보이는 것으로 나타났다. 각 균주별 배추와 무의 시판 품종의 저항성을 조사하였다. 기보유 균주 접종 처리에서 발병률과 발병도를 조사하였는데, 역광 100.0%와 3.1, 매력 100%와 3.9, 노랑봄 100%와 4.8, 춘광 100%와 3.9, 정상 100%와 4.8을 보여 총 5품종에서 강하게 발병하였다. 반면 무는 청대봄무만 발병률 35.9%와 발병도1.0의 극약으로 발병하였고 공시한 무 품종 대부분이 기보유균주에 대하여 저항성인 것으로 확인되었다. 하지만 PC91214 균주 접종 처리에서 모든 배추에서 발병률100.0%와 발병도 3.7–5.0을 보였고, 무의 발병률과 발병도는 강성무 60.9%와 1.5, 관동여름무 98.4%와 3.2, 백봉무 100%와 4.1, 청대봄무 100%와 4.7을 보여 PC91214 균주가 기보유 균주보다 더 강한 병원성과 시판 품종간 차이를 극명하게 나타내었다. 이상의 결과로부터 고랭지 주산지인 평창군에서 기존의 Plasmodiophora brassicae보다 더 강한 병원성을 가진 PC91214 균주가 확인되었다. 또한 배추는 CR 저항성 품종을 포함한 모든 품종이 이병되어 PC91214 균주에 대한 저항성 품종 육성이 시급한 것으로 나타났다. 무는 배추와 달리 PC91214 균주에 대하여 품종간 저항성 수준이 극명한 것으로 나타나 배추와 다른 유전자 및 유전 양식을 가지는 것으로 나타났다.

Beet armyworm, Spodoptera exigua are difficult to control using chemical insecticides because of the development of insecticide resistance. For eco-friendly beet armyworm managements, various control agents are required. Entomopathogenic fungus is one of promise control agents as an alternative to chemcal control agent. We conducted bioassays with entomopathogenic fungi to select high virulence isolate to larva of beet armyworm. The bioassay was used 150 entomopathogenic fungal isolates which were isolated from soil samples of nine provinces by insect-bait method using Galleria mellonella and Tenebrio molitor. . Three isolates, Metarhizium spp. FT83, FT89 and FT90 had high virulence as 84.6%, 100% and 100%, respectively, against S. exigua. The medial lethal time(LT50) of conidia of three isolates was 5.01, 2.99 and 2.92days respectively.

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

Insect-killing (entomopathogenic) 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 fungal transformant also shows mammal fibrinolytic activity, by which the transformant can be used pharmaceutically. 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.

Hemorrhagic Escherichia coli (E. coli) is one of the most important agent of diarrhea in piglet. The aim of this study was to investigate the characteristics of virulence genes and genetic diversity in hemorrhagic E. coli isolated from piglets with diarrhea. Among 122 hemorrhagic E. coli, 62 isolates carried single toxin gene like heat-stable toxin (ST), heatlabile toxin (LT), verotoxin 1 (VT1), verotoxin 2 (VT2), spa and eae. The most prevalent toxin gene carried by isolates was ST gene (23 isolates), while the most common association was ST/LT (14 isolates) and ST/LT/VT2 (13 isolates). In pulsed field gel electrophoresis, isolates were not classified as one cluster by epidemiological information including isolated area, isolated year and possessed toxins.

The polyhedrin is responsible to form polyhedra of nucleopolyhedrovirus(NPV) and highly conserved in most completely sequenced in lepidopteran NPVs. Previously, we have reported that the substitution of polyhedrin of Autographa californica NPV(AcNPV) with that of Spodoptera exigua NPV(SeNPV) or Bombyx mori NPV(BmNPV) result the change of polyhedra morphology. In this study, we investigated the influence of changed polyhedra morphology to the virulence of AcNPV. The recombinant AcNPVs were propagated in Spodoptera frugiperda clone 9, 21 cells and S. exigua larvae. Each collected recombinant polyhedra were used in bioassays using S. exigua larvae. The recombinant AcNPVs show that difference virulence according to the polyhedra morphologies. Internal and external morphological features of each recombinant AcNPV were also compared on the electron microscope. Our results suggest that the morphology of polyhedra influence the virulence of NPV and is well worth considering for the development viral insecticide.

To investigate the epidemiological characteristics of 68 Listeria monocytogenes isolates, including 11 reference strains and 57 isolates from imported US beef, domestic meats (beef, pork, chicken meat), raw milk, and milk plants. L. monocytogenes was to evaluate the production of virulence proteins, such as hemolysin (LLO) and lecithinase (LCP), the adsorption of Congo red (CRA), and to detect virulence genes using the polymerase chain reaction (PCR). In the study of virulence protein production, 68(100%), 62(91.2%), and 54(79.4%) of the 68 L. monocytogenes strains were positive for LLO production, the LCP test, and the CRA test, respectively, while strains of other species, such as L. innocua, L. gray, L. murrayi, and L. welshimeri, were not. There were no significant differences between L. monocytogenes serotypes and the ability to produce LLO or LCP. L. monocytogenesstrains had very high hemolytic titers (2 to 16 fold), while the other Listeria species, other than L. ivanovii and L. seeligeri, did not. The hemolysin activities of L. monocytogenes, L. ivanovii, and L. seeligeri usually exceeded 1.0 HU/mg, while those of other Listeria spp. were less than 0.04 HU/mg. In the PCR assay, all of the L. monocytogenes strains contained the hlyA, plcA, plcB, inlA, and inlB virulence genes and produced a product of the expected size. In the PCR of the actA gene, the expected 385-bp product was seen in 39(57.4%) L. monocytogenesstrains, while an unexpected 268-bp product was seen in 29(42.6%) strains. Most L. monocytogenes strains isolated from Hanwoo beef produced the 385- bp actA gene product, while strains of imported US beef usually produced the 268-bp actA gene product. By contrast, no virulence gene products were amplified in the other Listeria spp.