American foulbrood (AFB) is caused by the bacterium Paenibacillus larvae, which is highly contagious and often lethal to honeybee broods. To control AFB, rapid diagnostic tools including those based on immunological methods are required. We produced several specific mouse monoclonal antibodies (MAbs) against P. larvae. Interestingly, a few of the MAbs were revealed to be an IgM-type antibody. To ascertain the effects of adjuvants on immunoglobulin isotype switching, BALB/c mice were immunized with various adjuvants, i.e., Freund's adjuvant (FA), Alum adjuvant, and AddaVax™ followed by the generation of hybridoma that secreted monoclonal antibodies to P. larvae. In the case of AddaVax™, all screened hybridoma clones secreted IgG-type MAbs, whereas hybridomas generated by Alum and FA secreted 91.25% (7/80) and 66.67% (11/33) respectively, IgG-type MAbs. Although the mechanism of incomplete immunoglobulin isotype switching associated with the P. larvae antigen needs further study, our results indicate that the applied adjuvants can have a significant effect on immunoglobulin isotype switching results.

버섯배지의 발효를 효율적으로 행하면서 버섯의 재배 시 빈번하게 발생하는 푸른곰팡이 병의 원인 균주인 Trichoderma sp. 곰팡이 성장을 억제하는 세균의 분리를 행하였다. 균원시료로부터 1차 분리한 약 200여 균주 중에서 성장속도가 빠르고, SM, AM 및 CM의 평판배지 상에서 clear zone이 뚜렷한 6균주를 2차 분리하였다. 분리한 6균주 중 cellulase, amylase 및 protease의 효소활성이 높고 T. virens와 T. harzianum에 대하여 강한 항균활성을 나타낸 C-1균주를 최종 분리균주로 선정하였다. 분리한 C-1균주는 Bergey's Manual of Systematic Bacteriology에 의한 동정과 16S rDNA 염기서열 분석을 행한 결과 Paenibacillus polymyxa 밝혀져 P. polymyxa CK-1으로 명명하였다. P. polymyxa CK-1균주의 생육조건을 검토한 결과 최적배양온도는 45˚C, 생육을 위한 배지의 최적 pH는 6.0~7.0 범위로 나타났다. T. virens와 T. harzianum 곰팡이의 생육억제를 위한 P. polymyxa CK-1의 배양시간은 22~36시간이 적당하였다. 그리고 P. polymyxa CK-1균주의 24시간째 배양용액을 처리한 petri dish에 두 곰팡이를 각각 접종한 후 10일 동안 방치하여도 곰팡이의 생육은 관찰되지 않았다. P. polymyxa CK-1 균주가 생산한 길항물질의 열안정성을 검토한 결과 60˚C 및 100˚C로 20분 동안 처리한 시험구에서는 두 곰팡이의 균사성장이 전혀 관찰되지 않았지만 121˚C에서 20분 동안 처리한 시험구에서는 약간의 균사성장이 관찰되었다. P. polymyxa CK-1배양액이 버섯균사 생육에 미치는 영향을 검토한 결과 팽이버섯, 표고버섯 등의 다양한 버섯균사 생육에 전혀 영향을 미치지 않는 것으로 나타났다.

Paenibacillus elgii HOA73는 신규 개발된 미생물 제제로 작물생장 및 병원균 방 제 등 다양한 효능에 대한 연구가 수행중이다. 본 연구는 P. elgii 배양액의 배추좀 나방 살충률 제고와 시판농자재 사용량 감소를 통한 방제비용 절감을 위하여 시판 친환경 농자재(1000배 ~ 10,000배)와 P. elgii 농도별(5 × 107 ~ 9 × 107) 혼합 살포 에 따른 살충효과를 분석하고자 하였다. 속효성과 약효성이 높은 세 종류의 시판 농 자재를 선발 한 후 이들의 희석 배율과 P. elgii의 배양액 농도별 혼합 실험 결과, 시 판 농자재는 권장 희석액의 두 배 희석농도(5 × 107 농도의 P. elgii 배양액)에서도 세 종류 모두 72시간내 96% 이상의 살충력을 나타내었다. 이들에 대한 복숭아혹진 딧물과 점박이응애에 대한 혼합 살충력 분석결과, 복숭아혹진딧물의 경우 권장 희 석액의 두 배 희석농도(5 × 107 농도의 P. elgii 배양액) 역시 80% ~ 100%의 살충력 을 그리고 점박이응애의 경우 모두 100%의 살비력을 나타내었다. 이로써 시판 친 환경농자재를 P. elgii 배양액과 혼합하여 사용할 경우 친환경농자재 단독 처리시 보다 최소 반의 가격 절감효과가 있으며 하나의 약재를 이용하여 저작성 배추좀나 방과 흡즙성의 복숭아혹진딧물과 점박이응애를 동시에 방제할 수 있을 것으로 판 단된다. 향후 소포장 검정과 확장된 실내검정을 통해 작물생장 및 병원균 방제에 효 능을 갖고 있는 P. elgii의 적용범위에 대한 추가 연구를 수행하고자 한다.

We analyzed the viable population changes of Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium, on seeds and roots after bioformulation at varying time intervals during the storage. The viable population of E681 on tested crop seeds sustained log 4-5 cfu/seed after 300 days of seed treatment. The ability of root colonization and inhibition of fungal mycellial growth was not influenced even after 300 days of seed treatment. The seed-soaking treatment returned better results than powder formulation, in increasing the initial population of E681 on plant roots. Collectively, it was found that E681 is a durable and stable biological control agent for application to crop seeds.

Allomyrina dichotoma is a typical pet beetles in Korea. From 2010, similar symptoms of milky spore disease were found collectively in grubs of the species reared in insect farms. They shared a specific symptom that the skin of last instar larvae was changed softer with opaque white and infested grubs eventually died. To clarify the cause of the symptom, we collected the larvae of A. dichotoma from five farms and examined intestinal bacterial florae of them using pyrosequencing technique. From those results, a member of Paenibacillus was found only in the larvae showing the symptom of disease. Through PCR analysis using a Paenibacillus specific primer set, we obtained the partial 16S rRNA gene sequence and confirmed the microbe as Paenibacillus sp. For detailed characterizing, a whole guts was extracted from each larva showing the sign of the disease and incubated at 70℃ for 15 min to isolate spore forming bacteria. After then, each content of guts was cultured on MYPGPNAL agar medium (12.5 μg/ml of nalidixic acid) at 30℃. The 16S rRNA gene sequence analysis for six isolate showed that they were closely related to P. rigui (97.5~98.0% similarity) and to P. chinjuensis (95.9~96.6% similarity). Additional tests including API test and cellular fatty acid composition analysis were performed, but the strain couldn’t be identified at species level, suggesting it may represent novel species of the genus Paenibacillus.

The extracellular GH11 β-1,4-xylanase (XylY) gene (633-bp) of Paenibacillus sp. strain KYJ-16 was molecularly cloned by repeated DNA walking and nested PCR method. The xylY gene was predicted to encode an extracellular protein consisting of 611 amino acids with a nesuced molecular mass of 23 kDa and a calculated pI of 9.55. Protein blast search revealed that the enzyme consisted of a putative catalytic domain, which is homologous to a catalytic GH11 domain. The highest sequence identity (92%) was obtained as the catalytic GH11 domain of XylY was compared to that of Paenibacillus sp. strain HGF5 (GenBank accession number: EGG35584) that has not yet been characterized. Enzymatic properties of the recombinant His-tagged enzyme (rXylY) overexpressed in E. coli BL21 harboring pET-28a(+)/xylY will be also presented.

Root-knot nematodes, Meloidogyne spp., are the most important plant-parasitic nematodes, causing severe crop yield loss with an estimated 1000 billion dollars a year worldwide. The nematodes also cause disease complexes with other microbial pathogens, damaging plants more severely than each of the pathogens alone or their sum does and making control efficiencies weakened or nullified in disease complexes. In our study, the synergistic effect of the root-knot nematode Meloidogyne incognita was confirmed in the fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici, which showed greatly increased wilt symptoms in combined inoculations. Use of antifungal and nematicidal plant growth-promoting rhizobacteria (PGPR) were considered for controlling the disease complex because of its causal agents. Among forty PGPR strains tested, P. polymyxa G508, G462 and P. lentimorbus G158 showed strong antifungal and nematicidal activities against F. oxysporum f. sp. lycopersici and M. incognita second stage juveniles (J2), respectively. The Cultures of these bacterial strains inhibited the nematode egg hatching completely even at 1% diluted concentration. In pot experiments, treatment of the Paenibacillus strains reduced wilt severity of tomato with the control efficacy of about 90% ~ 98%. Their treatment also reduced gall formation by 64% - 88% compared to the untreated control. P. lentimorbus GBR158, which well established on seeds and hypocotyls at high population levels, reduced the disease complex greatly with the control value of about 98% when the tomato seeds were treated with the bacterial strain. Plant growth was also stimulated by the seed treatment of the bacterial strain. Scanning electron microscopy revealed alteration and distortion of hyphal cell walls of F. oxysporum and lysis of M. incognita egg shell by the bacterial treatment, showing direct antifungal and nematicidal action mechanisms. No extensive giant cell formation was observed near nematode in the tomato roots treated with the bacteria, indicating a systemic action mechanism. All of these results suggest that the Paenibacillus strains, especially G158 may have a high potential to be developed as biological agents for controlling the root-knot nematode and the disease complex.

새로운 항균물질의 탐색이 활발하다. 선행연구에서 새로운 항균활성물질을 생산하는 세균이 한국토양에서 분리되어 Paenibacillus polymyxa DY1으로 동정 및 명명되었으며, 다제내성 장내세균들에 대한 항균활성 특성이 규명되어 새로운 항생물질로서 잠재력을 보여주었다. 본 연구에서 배지의 탄소원, 무기질소원, 유기질소원, 아미노산, 무기염류 등의 영양 조성물과 물리화학적 생장조건이 P. polymyxa DY1 균체의 생장과 항균활성 생산에 미치는

From the course of screening of useful xylanase producing microorganism from a phytophagous longicorn beetle, we isolated an extra-cellular xylanase producing strain, Paenibacillus sp. HY-8 from the intestine of Moechotypa diphysis adult. On the basis of morphological, biochemical and phylogenetic studies of the new isolate was identified as a Paenibacillus species. Production of xylanase in this strain was strongly induced by adding xylan to the growth medium and repressed by glucose or xylose. The highest xylanase production was attained in the M9 media containing 1% yeast extract and 0.5% birchwood xylan when cultured at 25℃ for 24 hrs. HY-8 producing xylanase showed superior hydrolytic activities against various plant source feedstuff than control xylanase produced by Tricoderma sp. at pH 6.0.

Paenibacillus larvae는 Gram양성의 내생포자 형성 세균으로, 꿀벌의 질병 중 가장 심각한 피해를 입히는 미국부저병(AFB)의 원인균이다. 이 P. larvae를 발병전인 봉군 또는 애벌레에서 보다 빠르게 검출하기 위하여 기니아피그를 이용하여 이 병원균에 대한 다클론 항체를 제작하였고 또한 그 항체의 성능을 평가하였다 제작된 항체를 이용한 ELISA검색법을 개발한 후, 다수의 P. larvae균주를 검사하였다. 그 결과 P. larvae ATCC 25748의 균체를 사용하여 제작된 항체는 ATCC 9545 (대표균주), ATCC 25747, 국내 분리 균주인 SJl5등과 높은 항체 친화성을 나타내었으며. 다른 세균 종들과는 반응하지 아니하였다. 또한 이 ELISA검색법은 미국 부저병의 현장검사에 적용하기 위하여 사용되었으며, 그 결과로 본 연구에서 제시하는 항체는 벌집 내 또는 애벌레 내에 존재하는 P. larvae의 빠른 동정과 모니터 링에 매우 유용함을 보여주었다.