This study evaluated the antifungal activity of varying concentrations of watersoluble extracts from native plants (Vitex rotundifolia, Tetragonia tetragonoides, Artemisia capillaris, Hibiscus hamabo and Ficus carica) against Stemphylium vesicarium, Penicillium italicum, Sclerotinia sclerotiorum, Pythium ultimum, Botrytis cinerea, Rhizoctonia solani and Colletotrichum gloeosporioides. Mycelium growth of pathogenic bacteria generally decreased in a concentration-dependent manner following treatment with the water extracts from donor plants. Closer analyses indicate varying inhibitory capacities depending on the type of donor plant and pathogenic bacteria. Specifically, mycelium growth of S. vesicarium varied depending on the concentration of the water extracts from T. tetragonoides (r = -0.857, p<0.01) and A. capillarys (r = -0.868, p<0.01). Also, P. italicum and V. rotundifolia (r = -0.833, p<0.01), S. sclerotiorum and V. rotundifolia (r = -0.862, p<0.01), A. capillaris (r = -0.902, p<0.01), B. cinerea and T. tetragonoides (r = -0.896, p<0.01) showed an inverse relationship. The rate of mycelial growth inhibition of pathogenic bacteria analysed are as follows: P. ultimum 94%, B. cinerea 50%, C. gloeosporioides 80% in 100% treatment of T. teragonoides. A. capillaris inhibited S. vesicarium by 43%, P. ultimum by 90%; H. hamabo inhibited P. italicum by 50%, S. sclerotiorum by 26%, and F. carica inhibited R. solani by 74%. Total phenol content with antifungal activities are as follows: A. capillaris 16.15 mg/g, F. carica 7.81 mg/g, V. rotundifolia 6.18 mg/g, H. hamabo 5.25 mg/g, T. tetragonoides 4.41 mg/g, and total flavonoid content is as follows: A. capillaris 27.57 mg/g, V. rotundifolia 12.49 mg/g, F. carica 11.45 mg/g, H. hamabo 5.77 mg/g, T. tetragonoides 5.08 mg/g.

Pink Rot on melon and White Stain Symptom on grape are caused by Trichothecium roseum, one of the most important diseases of grape and melon. These diseases have been occurred in national-wide in Korea and causes irreversible damage on the grape and the melon at harvest season. This research presents the evaluation of the capacity of Bacillus subtillis HK2 to protect both melon and grape against T. reseum and establishes its role as a biocontrol agent. In this study, we isolated a Bacillus strain HK2 from rhizosphere soil, identified it as Bacillus subtillis by 16S rRNA analysis and demonstrated its antifungal activity against T. roseum. Under I-plate assay it was observed that the effect of hyphal growth inhibition was not due to production of volatile compounds. The optimum culture condition of HK2 was found at 30℃ and initial pH of 7.0. Application of HK2 culture suspension reduced 90.2% of white stain symptom on grape as compared to control, resulting in greater protection to grape against T. roseum infestation. Butanol extract of HK2 culture purified using flash column chromatography. The antifungal material was a polar substance as it showed antifungal activity in polar elute. Therefore, our results indicated a clear potential of B. subtilis HK2 to be used for biocontrol of Pink rot in melon and white stain symptom on grape caused by T. roseum.

The study researched germination of the plants and growth of Fungus according to concentration of aqueous extracts in order to provide basic data for developing natural agricultural resources by using Persicaria longiseta. The seed germination of Amaranthus spinosus was inhibited at 25% P. longiseta extract, while Agrostis stolonifera ssp. palustris was not affected at all concentrations tested. Especially, the seed germination rate and fresh weight of Trifolium incarnatum at 20% P. blumei extract were higher than those of control plot. The early growth of most receptor plant seedlings was promoted at 25% and 50% of P. blumei extracts, but the radicle growth of all receptor plants was significantly inhibited at > 25% of P. longiseta extract. The response of receptor plants to P. longiseta extract was different according to the plant species and the plant parts. The growth of plant pathogenic fungus in PDA medium showed an increasing inhibition tendency with increasing concentrations of P. longiseta extract. Especially, P. longiseta extract showed the greatest antimicrobial activity against Phytophthora infestans, Phythium graminicola, and Pythium venterpoolii. The content of total phenolic compound in P. longiseta was higher in leaves (1082.3 mg/L) but lower in roots (228.6 mg/L) and stems (207.8 mg/L), which is an allelopathic chemical. As these results are summarized, P. longiseta have competitive advantage because they release phenolic compounds with allelopathic effect and affect on germination, growth and fungi growth on underground flora compared to native plants and they have eligibility for natural herbicide and germicide.

Small RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), play crucial roles in post-transcriptional gene silencing (PTGS) in eukaryotes. Small RNAs function cell-autonomously as well as non-cell-autonomously. It has been well characterized that pathogenic fungi secrete some effector molecules facilitating their infection into plants. However, it is unclear whether molecules produced in plant cells are able to move into fungal cells during infection. To test if small RNAs generated from plant cells can move to fungal cells during infection, we generated transgenic Arabidopsis and rice plants expressing siRNAs targeting GFP gene generated from double-stranded RNA interference (dsRNAi) constructs for GFP gene. And then these transgenic plants were inoculated with transgenic rice blast fungus, Magnaporthe oryzae, expressing GFP transgene. Here, we showed that ectopic expression of siRNAs targeting GFP gene in transgenic plants significantly suppressed GFP expression in rice blast fungi inoculated, indicating that small RNA molecules generated in plant cells can move into infected fungal cells and efficiently degrade fungal GFP transcripts. Our results would provide a new small RNA-based strategy for the development of resistant crops against fungal pathogens.

Zoysiagrass are damaged by fungi diseases such as large patch, dollar spot, pythium blight and brown patch. Large patch is one of the major diseases caused by Rhizoctonia solani AG2-2 on zoysiagrass fields e.g. golf courses. Plant chitinases have been known PR (Pathogen related)-protein. In this study, we isolated two chitinase genes (Zjchi1 and Zjchi2) from zoysiagrass. Antifungal activity analysis revealed that Zjchi2 protein inhibited mycelium extension of fungi. A further study, we cloned 5` upstream region from two chitinase genes for investigating transcription regulatory mechanism that inducing of two chitinase genes dependent R. solani. -818 bp and -799 of upstream region from Zjchi1 and Zjchi2 successfully isolated using in vitro LA (Long and Accurate) PCR system. And then, we generated promoter-GUS reporter constructs with deletion construct based on W-boxes. Constructs were introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation for stable expression of GUS reporter gene.

본 연구는 귀화식물인 Solidago altissima, Amaranthus retroflexus, Sida spinosa 등을 이용하여 친환경 농자재로 개발하기 위한 기초자료를 제공하기 위해 수용성 추출액의 농도에 따른 수용체 식물의 발아 및 유식물 생장과 실험 병원균의 생장을 조사하였다. 공여체식물에 따른 수용성 추출액 농도가 증가됨에 따라 대부분 검정식물의 상대발아율은 감소하는 경향을 보이는데 S. spinosa(r = -0.540, p＜0.01), Physalis wrightii(r = -0.693, p＜ 0.01), A. retroflexus(r = -0.724, p＜0.01), S. altissima(r = -0.728, p＜0.01), Eclipta prostrata(r = -0.779, p＜0.01) 순으로 감소하는 경향이 큰 것으로 조사되었고 평균발아일수도 처리구 농도가 증가함에 따라 발아하는데 소요되는 시간이 증가 되었으며(r=0.769, p＜0.01) 공여체식물과 검정식물에 따라 약간의 정도 차이를 보였다. 또한 공여체식물의 수용성 추출액 농도가 증가함에 따라 유식물의 지상부의 길이(r = -0.587, p＜0.01), 지하부의 길이(r = -0.741, p＜0.01), 생체량(r = -0.574, p＜0.01)과 뿌리털의 발생도 감소하였다. 한편 공여체식물의 수용성추출액 농도 증가에 따른 검정 병원균의 생장은 Botrytis cinerea(r = -0.266, p＜0.05), Diaporthe citri(r = -0.323 p＜0.01), Colletotrichum gloeosporioides(r = -0.512, p＜0.01), Pythium ultimum(r = -0.581, p＜0.01), Rhizoctonia solani(r = -0.806, p＜0.01) 순으로 생장이 억제되었다. 제초 및 항균활성을 보이는 수용체식물의 총 페놀 함량은 S. altissima 17.3±0.5mg/g, A. retroflexus 13.1±0.3mg/g, P. wrightii 12.0±0.4mg/g, S. spinosa 9.5±0.1mg/g, E. prostrata 4.1±0.1mg/g 순으로 분석되었다. 이들 결과를 종합하면 귀화식물인 수용체식물들은 자생식물과의 경쟁을 함에 있어 알레로패시 효과를 나타내는 페놀 화합물 등이 수관 내 토양으로 방출하여 하부식생에 대한 발아 및 생장과 토양미생물 생장 등에 영향을 주기 때문에 경쟁적 우위를 점하고 있으며, 천연제초제 · 살균제로서의 활용 가능성을 가지고 있는 것으로 판단된다.

The antifungal activity against cheese fungi by lactic acid bacteria isolated from kimchi was investigated. Eight fungi were isolated from cheese in the cheese ripening room. Two of them were identified as Penicillium and Cladosporium via ITS-5.8S rDNA analysis. Twenty-two species of lactic acid bacteria with antifungal activity were isolated from kimchi. Two of them were identified as Lactobacillus and Pediococcus via 16S rRNA sequence analysis. Of the 22 lactic acid bacteria species, six were selected (L. sakei subsp. ALJ011, L. sakei subsp. ALI033, L. sakei subsp. ALGy039, P. pentosaceus ALJ015, P. pentosaceus ALJ024 and P. pentosaceus ALJ026) due to their higher activity against the eight fungi isolated from cheese in the cheese ripening room; and among the six species, the P. pentosaceus ALJ015 and P. pentosaceus ALJ024 isolates from the Jeonju area kimchi and the L. sakei subsp. ALI033 isolate from the Iimsil area kimchi had higher antifungal activity than the other lactic acid bacteria. The minimum inhibitory concentration (MIC) of L. sakei subsp. ALI033 against the eight fungi isolated from cheese in the cheese ripening room was 62.5 ㎍/mL.

Small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs) play crucial roles in post-transcriptional gene silencing (PTGS) in eukaryotes. Small RNAs function cell-autonomously as well as non-cell-autonomously. It has been well characterized that pathogenic fungi secrete some effector molecules, which facilitate their infection into plants. However, it is not clear whether molecules in plant cells are able to move into fungal cells during infection. To test if small RNAs generated from plant cells can also move to fungal cells during infection, we generated transgenic Arabidopsis and rice plants ectopically expressing either double-stranded RNA interference (dsRNAi) or artificial miRNA (amiRNA) constructs targeting GFP gene. And then these transgenic plants were inoculated with transgenic rice blast fungus, Magnaporthe oryzae, expressing GFP transgene. Here, we showed that ectopic expression of both dsRNAi and amiRNA targeting GFP gene in transgenic plants significantly suppressed GFP expression in rice blast fungi inoculated, indicating that small RNA molecules generated in plant cells can move into infected fungal cells and efficiently degrade fungal GFP transcripts. Our results would provide a new small RNA-based strategy for the development of resistant crops against fungal pathogens.

This study was performed to investigate the four-week repeated-dose toxicity of the crude antifungal compounds produced by Lactobacillus plantarum AF1 (Lb. plantarum AF1), a lactic acid bacterium isolated from kimchi, in male and female rats. Sprague-Dawley male and female rats were divided into four groups, with 10 animals in each group. The test article was administered once daily by gavage to rats at dosage levels of 0, 500, 1,000, and 2,000 mg/kg/day for four weeks. There were no test-article-related deaths or abnormal clinical signs in both the male and female rats during the observation period. Furthermore, no differences in the body weight changes, food intake and water consumption levels of the control and treatment groups were found. The hematological parameters, serum biochemical analysis results, histopathological examination results and all other findings also showed no significant or dose-dependent changes. There were also no changes in the organ weights upon the administration of the crude antifungal compounds produced by Lb. plantarum AF1. These results suggest that the oral administration of the crude antifungal compounds produced by Lb. plantarum AF1 had no adverse effects up to a dosage level of 2,000 mg/kg in both male and female rats.

The SSH100-10 bacterial strain, which exhibits strong antifungal (anti-mold and anti-yeast) activity, was isolated from traditional korean soysauce aged 100 years. The strain was identified as Bacillus velezensis based on Gram-staining, the biochemical properties and 16S rRNA gene sequence determination. B. velezensis SSH100-10 showed strong proteinase activity and NaCl tolerance, but did not produce enterotoxin. Two-antifungal compounds from B. velezensis SSH100-10 were purified using SPE, preparative HPLC, and reverse phase-HPLC. The purified antifungal compounds were identified as C14 and C15 iturin through MALDI-TOF-MS and amino acid composition analysis. The stability characteristics of the antifungal compounds after temperature, pH, and enzyme treatments suggested that B. velezensis SSH100-10 produced more than two antifungal compounds; pH-stable C14 iturin A and C15 iturin A, and unidentified pH-unstable compounds. The results suggested that B. velezensis SSH100-10 can be used in soybean fermentation as a starter. Moreover it has potential as a biopreservative in the food and feed industry and as a biocontrol agent in the field of agriculture.

This study was carried out to research microorganisms having the antifungal activity against ginseng Alternaria blight pathogen Alternaria panax and ginseng anthracnose pathogen Colletotrichum gloeosporioides. Eleven Bacillus strains. were isolated from Korean traditional soybean paste and Kimchi. Among the 11 isolates, DJ5, KC1, KC2 and KC4 showing antagonistic activity on the mycelial growth of A. panax and C. gloeosporioides in pairing culture were finally selected as the antagonistic microorganisms. Based on 16s rRNA sequence and phylogenetic tree analysis, they were identified as Bacillus spp.. The selected microorganisms were investigated antagonistic activity by measured leaf-segment colonization in pot test. When Bacillus sp. were injected after A. panax treatment, KC1, KC2 and KC4 showed similar effect to chemical pesticides treated control. To measure preventive effect of Bacillus sp, antagonistic microorganisms were injected and C. gloeosporioides was treated in pot. When measuring the effectiveness for the prevention of Anthracnose, All Bacillus spp. showed approximately 83~90 % degree of superior preventive effect. In general, The four Bacillus spp. isolated from Korean traditional fermented foods showed therapeutic effect of Alternaria blight and preventive effect of Anthracnose.

Conventional Thiamine Dilauryl Sulfate (TDS) powder has a low stability. In order to solve this problem, this study was performed to improve the solubility of TDS. The process for enhance solubility of TDS was nano grinding mill and ultrasonic dispersion process. TDS paticle was manufactured to nano size through nano grinding mill process. The size of TDS nanoparticle was measured as average 220 nm by DLS. And The TDS nanoparticle in water solution manufactured through ultrasonic dispersion process. The TDS nanoparticle in water solution was showed the highest solubility with 40% ethanol. These results was increased the concentration of TDS from 200 ppm to 240 ppm in water solution. The TDS nanoparticle in water solution showed diameter of Colletotrichum gloeosporioides growth with smaller than about 1.56 cm compared to the TDS paticle in water solution at same concentration. Also, TDS nanoparticle in water solution showed growth inhibition activity as 59.2% with higher than about 10% compared to the TDS paticle water solution in same concentration. Finally, TDS nanoparticle in water solution was increased solubility through nano grinding mill and ultrasonic dispersion process. Also, the increase of concentration in TDS nanopaticle in water solution according to solubility enhancement lead to an result enhancement of antifungal activity. Consequently, we suggested that the TDS nanoparticle in water solution was more effective than TDS particle in water solution owing to the sub-cellular particle size, ability to persistence and targeting to cell membrane of Colletotrichum gloeosporioides. Furthermore we expected the applicating possibility with bio pesticide.

This study was performed to enhance antifungal activity of anthracnose in chili pepper by nanopaticles of thiamine di-lauryl sulfate (TDS) through high pressure homogenization process. Yield of TDS was 79.14% by reaction of thiamine hydrochloride and sodium lauryl sulfate. TDS nanopaticle solution was manufactured through high pressure homogenization process. The turbidity of nanoparticles solution was increased with increasing the concentration of TDS, and nanoparticles solution of 100 ppm was showed the highest turbidity with absorbance of 3.212. The size of nanoparticles solution was measured as average 258.6 nm by DLS. Nanoparticles solution of 100 ppm showed growth inhibition activity with higher than about 80% compared to the control group against Colletotrichum gloeosporioides. Finally, nanoparticles solution was increased effectively the penetration of the TDS nanopaticles on attached cell membrane of hyphae and started to destruct the cells under microscope observation. Consequently, we suggested that the TDS nanoparticle solution by high pressure homogenization process might be suitable biochemical pesticides for improving the antifungal activities against anthracnose in pepper.

This study was conducted to find out environment-friendly disease control method to Alternaria blightcaused by Alternaria panax of ginseng. For this study, 150 methanol extracts from medicinal plants were evaluated and theextract of Impatiens balsamina showed most strong antifungal activity against A. panax. The methanol extract of I. balsam-ina showed also stable antifungal activity against other ginseng pathogens such as Botrytis cinerea and Fusarium sp., whentreated by heat or pH. In vivo, Alternaria blight incidence rate was low of 13% with the treatment of I. balsamina methanolextract compared to 35% of the non-treatment. The antifungal compound of I. balsamina was purified and identified byusing a silica gel column chromatography, TLC and ESI-LC/MS/MS analysis. The compound which showed strong antifun-gal activity was identified as 2-methoxy-1,4-naphtoquinone (C11H8O3).

본 연구에서는 제주도 해안에 자생하는 45종의 해양식물 추출물에 대한 항균 활성을 조사하였다. 해양식물 추출물은 80 % 메탄올에서 유효 성분을 추출하여 시료화 하였고 항균활성을 검증하였다. 그 결과, 45종의 해조류 중에서 넓패, 패, 구멍갈파래 등을 포함한 6종의 해양식물이 미생물 생육을 억제하는 결과를 보였다. 항균활성을 갖는 해양식물 중 패, 감태 2종은 항산화 효능도 동시에 가지고 있음을 확인하였다. 이러한 결과를 통해, 본 실험에서 확보된 추출물이 항균 물질로 사용될 수 있는 가능성을 확인하였다.

천연물은 예로부터 건강의 증진 및 질병의 치료를 위하여 다양하게 이용되어 왔다. 특히 천연물 및 식품류에 포함되어 있는 폴리페놀성 화합물들은 항산화, 암세포 성장억제, 항돌연변이 등 다양한 생리활성을 나타내어 심혈관계 질환 및 암의 치료와 예방제로서의 활용 가능성을 검토하는 많은 연구가 이루어지고 있다. 따라서 본 연구에서는 동백나무의 기능성 물질 확보와 가공을 통한 고부가가치 창출을 목표로 국내에서 자생하는 동백나무를 부위별로 추출물을 제조하여 항산화 및 항미생물 활성을 평가하고자 하였다. 항산화효과는 항산화력 성분인 총페놀 화합물의 함량과 BPPH radical 소거법을 이용하여 항산화 활성을 측정하였는데 총페놀 화합물의 함량은 동백나무 부위별 추출물의 건조중량 100 g에 대하여 어린잎, 꽃봉오리 및 꽃 추출물에 각각 74.24mg , 65.02mg, 그리고 62.42mg로 나타났다. Free radical소거활성을 공존시킨 DPPH의 50%를 환원시키는데 필요한 시료의 양 (RC50)은 어린잎, 꽃봉오리 그리고 꽃의 추출물에서 각각 7.16 μg/ml, 14.45 μg/ml 및 18.4 μg/ml의 낮은 농도로 활성을 나타내어 기존의 합성 항산화제인 BHT의 61.23 μg/ml보다 높은 항산화 효과를 나타내어 천연 항산화제로서 이용 가능성을 보여주었다. 또한 B. subitillis, S. fradiae, S. aureus, E. coli, P. aeruginosa, Enterbacter spp. C1036 및 S. typhimurium 7종의 시험균을 사용하여 동백나무 부위별 추출물의 항균활성을 측정 하였는데 어린잎의 경우 15 μg/ml의 농도에서 9~13mm의 투명환을 보여 비교적 높은 항미생물 활성을 나타내었고, 꽃봉오리 추출물을 첨가하였을 때 P. aeruginosa와 Enterbacter spp. C1036 균주에서 가장 장한 억제활성을 나타내었다. 7종의 시험균에 대한 항균력이 가장 높았던 어린일 추출물의 최소저해농도 (MIC)는 1~15으로 나타났으며, 그람양성균이 그람음성균보다 각 농도에서 최소저해농도의 효과가 더 높은 것으로 나타났다.

16과 20종의 공시 식물체 중 양파(Allium cepa), 대황(Rheum undulatum) 및 황련(Coptis japonica) 등의 추출액이 인삼 탄저병균(C. gloeosporioides)에 대해 균사생장 억제 효과가 우수하였다. 양파와 대황은 배지내의 추출액 농도가 1.0% 이상일 때 균사 생장억제 효과가 인정 되었고, 황련의 추출액은 배지 내의 농도가 0.5% 이상일 때에도 균사 생장 억제 효과가 50% 이상으로 나타나 공시된 식물체 추출액 중에서 항균 활성이 가장 우수하였다. 양파의 추출액은 10.0% 희석농도에서는 60% 이상의 발아 억제 효과가 있었고, 대황의 추출액은 2.0% 그리고 황련의 추출액은 1.0%의 희석 농도에서도 포자 발아억제율이 90% 이상으로 나타나 황련추출액이 가장 우수함을 알 수 있었다. 양파와 대황의 추출액은 2.0%의 희석농도에서는 어느 정도 방제 효과가 있었으나 1.0% 이하의 희석 농도에서는 효과가 많이 떨어졌고, 황련의 추출액은 1.0%의 희석농도에서도 50%이상의 방제 효과가 있었으나, 3가지 식물체 추출액 모두 대조약제인 Mancozebwp에 비해서는 그 효과가 상당히 떨어지는 경향이 었다. 세가지 식물체 추출액 모두 10.0%의 희석농도에서는 인삼 잎의 엽육이나 엽맥이 갈변하는 등의 약해의 징후가 나타났고, 황련의 추출액은 2.0%의 희석 농도에서도 약간의 약해 징후가 관찰되었다.

50종의 약용식물 추출액을 공시하여 생체외(in vitro) 및 생체내 (in vivo)에서 몇 가지 주요 종자전염성병 억제를 위한 항균활성식물을 선발하고 선발된 식물에 대한 항균활성을 검정한 결과는 다음과 같다. 가) 공시된 50종의 약용식물추출액을 공시병원균(벼도열병균, 참깨검은무늬병균, 고추탄저병균, 무·배추검은무늬병균)을 접종한 종자에 대한 항균활성을 습지법과 물한천법으로 정정한 결과 마늘, 주목, 대황, 무슬, 감초, 달맞이꽃 등에서 항균활성이 우수한 것으로 나타났다. 나) 1차 선발된 식물추출액을 종자에 희석 처리하여 감자한천법으로 검정한 결과, 마늘 추출액이 10배에서도 항균활성을 보였고 종자침적 시간에 따른 효과를 보면 긴 시간 침적이 가장 높은 항균활성을 나타냈으며 종자침적온도에 따른 항균효과를 보면 25℃ 처리가 대체적으로 우수했다. 다) 식물 추출액의 종자발아및 초기생육에 미치는 영향은 마늘 추출액의 경우 무처리에 비하여 종자발아에 차이가 없었고 초기생육은 오히려 양호하였다. 주목의 추출액은 무와 배추에서 약간의 영향이 있었고 우슬, 감초, 달맞이꽃 등에서는 종자 발아 및 초기 생육을 심하게 저해했다.