초록 ▼

Ⅳ. 연구개발결과
직접적인 항생작용이 아닌 미생물에 의해 식물의 면역반응을 증가 시키는 방법을 통하여 식물의 저항성을 증진 시키는 방법인 식물 전신 유도 저항성 (ISR, induced systemic resistance) 방법을 이용하여, 식물생장촉진근권세균에 의해 유도 되어지는 유도저항성에 대한 저항성 기작을 확인하였다. ISR 실험을 위하여 고추에서 유도저항성을 유도하는 Bacillus thuringiensis strain BS107을 유도저항성 스크린을 통하여 선별하였다. BS107은 다양한 조건에서 일정한 유도저항성

Ⅳ. 연구개발결과
직접적인 항생작용이 아닌 미생물에 의해 식물의 면역반응을 증가 시키는 방법을 통하여 식물의 저항성을 증진 시키는 방법인 식물 전신 유도 저항성 (ISR, induced systemic resistance) 방법을 이용하여, 식물생장촉진근권세균에 의해 유도 되어지는 유도저항성에 대한 저항성 기작을 확인하였다. ISR 실험을 위하여 고추에서 유도저항성을 유도하는 Bacillus thuringiensis strain BS107을 유도저항성 스크린을 통하여 선별하였다. BS107은 다양한 조건에서 일정한 유도저항성 능력을 보여 주었으며, 포장과 온실 검정에서도 강력한 유도저항성 능력을 보여 상품화를 위한 시제품화 작업을 진행 하였다. 고추에서 유도 저항성 기작을 알기 위해 우리는 Bacillus thuringiensis strain BS107를 사용하여 고추 잎에서 Xanthomonas axonopodis pv. vesicatoria 의해 발생하는 고추 궤양병에 대한 유도 저항성 실험을 수행하였다. 특히 고추에서 B. thuringiensis strain BS107 에 의해 유도저항성 반응이 유도되어질 때 priming되는 유전자에 대한 연구를 집중적으로 관찰하였다. 기존에 고추에서 선행 연구 되어졌던 저항성 관련 유전자중 CaPR4와 CaPR1 유전자는 B. thuringiensis strain BS107 균주의 뿌리에 처리함으로 인해 전신적으로 저항성이 촉진되어짐을 정량적 유전자 증폭 방법(quantitative-reverse transcriptase PCR)을 이용하여 확인하였다. 또한 에틸렌에 의해 반응하는 유전자인 CaBPR1, CaTin1, 그리고 CaTin1-2 유전자도 Bacillus thuringiensis strain BS107에 의해 빠르고 강하게 발현되는 것을 확인하였다. 더 많은 저항성 반응 촉진 유전자를 찾기 위하여 microarray 분석을 수행하였다. 총 345,000 유전자가 포함된 칩에서 604개의 유전자가 BS107처리 후 X. axonopodis pv. vesicatoria 처리시 과발현되었으며, 62개 유전자가 BS107을 처리하지 않은 고추에 비하여 적게 발현되었다. 대부분 과발현된 유전자들은 물질 대사와 저항성 반응 신호 전달에 관련된 유전자들이 과 발현되어 있었다. 온실 실험을 토대로 선별된 B. thuringiensis strain BS107 를 이용하여 포장실험을 수행하였다. 그 결과 Bacillus thuringiensis strain BS107 처리 후 10일째에 X. axonopodis pv. vesicatoria에 대하여 유도 저항성이 있음을 확인하였다. 또한 포장에서 자연적으로 발생하는 고추 궤양병과 바이러스 병에 대하여서도 저항성이 유도됨을 확인하였다. 포장에서 B. thuringiensis strain BS107에 의해 유저 저항성이 유도되는 동안 빠르고 강하게 발현되는 저항성 관련 유전자의 발현을 조사한 결과 CaPR4 유전자가 온실 실험 동일하게 강하게 발현됨을 확인하였다. 포장에서 B.thuringiensis strain BS107에 의해 유도 저항성은 이루어지나 온실 실험과 같이 식물 생장 촉진은 관찰할 수 없었다. 최종적으로 BS107에서 고추에 저항성 반응을 유도하는 유도결정인자를 선별하였다. fractionation을 통한 선발과정을 통하여 2-aminobenzoic acid을 선별할 수 있었다. 2-aminobenzoic acid는 고추에서 X.axonopodis pv. vesicatoria에 대하여 저항성을 유도하고 담배에서 Erwinia carotovora subsp. crotovora에 대하여도 저항성을 유도하였다. 결론적으로 식물근권세균 중에서 내생포자를 만들 수 있는 바실러스종의 BS107균주를 선발하였으며 이렇한 유도저항성이 식물의 저항성유도에 의한 작용인 것을 분자생물학적 방법에 의해 증명하였으며, 그 결정인자로 2-aminobenzoic acid를 선별하여, 시제품을 만들어 상품화 할 수 있게 되었다. 본 연구를 통하여 유도저항성 기본의 생물적 방제 방법과 미생물과 미생물 유래의 물질을 실용화 할 수 있는 전기를 마련하였다.

Abstract ▼

1. Induced systemic resistance in pepper plant by Bacillus spp.
Within an ecosystem, many organisms exist in perpetual inter-and intra-species relationships that can be either positive or negative, depending on the needs of each organism. A group of beneficial plant bacteria, which includes Bacil

1. Induced systemic resistance in pepper plant by Bacillus spp.
Within an ecosystem, many organisms exist in perpetual inter-and intra-species relationships that can be either positive or negative, depending on the needs of each organism. A group of beneficial plant bacteria, which includes Bacillus spp., has been shown to increase crop growth referring to as plant growth-promoting rhizobacteria (PGPR). PGPR can decrease plant disease directly, through the production of antagonistic compounds, and indirectly, through the elicitation of a plant defense response termed induced systemic resistance (ISR). While the mechanism of ISR elicited in response to PGPR has been studied extensively in the model plant Arabidopsis thaliana, it is less well characterized in crop plants such as tomato and pepper. In an effort to better understand the mechanism of ISR in crop plants, we investigated the induction of ISR to bacterial spot disease caused by Xanthomonas axonopodis pv. vesicatoria in pepper leaves by Bacillus cereus strain BS107. We focused on the priming effect of B. cereus strain BS107 on plant defense genes as an ISR mechanism. Of ten known pepper defense genes that were previously reported to be involved in pathogen defense signaling, the expression of Capsicum annum pathogenesis-protein 4 (CaPR4) and CaPR1 was systemically primed by the application of strain BS107 onto pepper roots confirming by quantitative-reverse transcriptase PCR. Additionally ethylene signaling-dependent genes (CaBPR1, CaTin1, and CaTin1-2) appeared to be primed by strain BS107. To find more priming genes during ISR by BS107 in pepper, we employed microarray methods. A method of microarray analysis using oligo-genechip probe representing approximately 345,000 genes showed 604 genes to be up regulated and 62 genes down regulated at least two fold by X. axonopodis pv. veiscatoria infiltration after treated BS107 compare to non-treatments. Most up regulated genes were involved in metabolism and defense responses.
Bacillus thuringiensis strain BS107 was selected through in vitro and field experiment in order to be increased ISR capacity and plant growth promotion. The results of ISR capacity of B. thuringiensis BS107 was accomplished only at 10 day after treated Bacillus thuringiensis strain BS107 under field condtions. For validation of ISR capacity elicited by B. thuringiensis BS107 and ISR chemical inducers (BTH and BABA) against naturally occurred diseases in the field, we measured disease severity of naturally occurred diseases including bacterial spot disease and viral diseases at day 60 after treatment of B. thuringiensis BS107 and ISR chemical inducer. To understand mechanism on strain BS107-mediated ISR on pepper, we focused on priming effect of the defense related genes after pathogen challenge. We assessed CaPR4 expression as a marker gene for induction of plant defense after pathogen challenge with X. axonopodis pv. vesicatoria. To confirm these results, we used qRT-PCR to analyze the priming of CaPR4 expression by strain BS107 at day 10. During the ISR by Bacillus thuringiensis strain BS107 in the field, plant growth promotion by Bacillus thuringiensis strain BS107 was not observed. For investigation of pepper growth promotion ability by B. thuringiensis BS107 treatment, we harvest pepper fruits through in twice. However, the yield of pepper fruits treated by B. thuringiensis BS107 was not differ compared with control and chemical inducer treatments. Finally, 2-aminobenzoic acid was identified as an ISR determinant secreted from BS107. 2-aminobenzoic acid itself could be induced resistance against X. axonopodis pv. veiscatoria in pepper and Erwinia carotovora subsp carotovora in tobacco. Our results provide novel biological mechanism for biological control in order to be applied in the field with endospore-forming rhizobacterium to manage broad spectrum plant pathogens. In aiddition to practical application of rhizobacterium, we also exploited genetic evidence of the priming effect of a rhizobacterium on the expression of pepper defense genes involved in ISR as well as provide 2-aminobenzoic acid as new ISR elicitor in pepper.
2 .Management of rice diseases by induced systemic resistance
II. Importance and objectives of the research
Rice disease control methods aimed at preventing the disease outbreaks through the use of chemical pesticides. However, current research focuses on environmentally-friendly agricultural practices and materials are required to produce pesticide-free agricultural products. Biological control tactics have become an major approach to establishing sustainable agriculture. However, most of biological control methods are not thought of as acceptable alternative for chemical pesticides because of the lack of the mode of action and consistent performance. The beneficial rhizobacteria has been known to provoke the elicitation of a plant defense response termed induced systemic resistance (ISR).
The purpose of this research was to develop technical and scientific knowledge in control of rice diseases by induced systemic resistance.
III. Research content and scope
In order to get rice genes associated with induced systemic resistance upon root colonization of the rhizobacteria, Pseudomonas chlororaphis O6, Affymetrix rice 57K microarray analysis were performed. The microarray analysis did lead to be resulted in the selection of ISR-related genes such as WRKY45,terpene synthase and galactinol synthase. The O6-pretreated rice plants showed priming effects on their selected genes. In addition, the selected genes were regulated by pathogen-responsive MAPK cascade in rice. The selected genes were also characterized molecular biologically and transformed into rice to produce the disease resistant rice plants. The GolST4 transgenic rice plants were examined disease resistance against rice disease and drought tolerance as well. Although crop carrying stress-resistant gene is produced, its function is effective at green house but not in field with hard environmental condition. Thus, GolST4 transformed rice was studied its function in both green house and field with the raising seedling and confirmed whether it could be the practical molecular breeding resource or not.
3. Isolation of ISR determinant from rhizobacteria
In order to characterize the indueced systeminc resistance (ISR)-determinants of Paenibacillus polymyxa E681, the extracellular molecules from bacterial culture supernatant were successively extracted with organic solvents and fractionated by silica gel chromatography column. Among the fractions tested, sub-fraction No. 7-8 triggered ISR activity. Collectively, the two bacterial metabolites, acetoin and 2,3-butanediol, extracted from P. polymyxa E681 were ISR inducing determinants in this study.
And in order to detect ISR-determinants of Ochrobactrum lupini KUDC1013, a novel strain isolated from Dokdo, extracelluar components were extracted with organic solvents. Of four fractions tested for ISR activity on tobacco, hexane and butanol fractions showed ISR activity on tobacco seedlings. Further analyzes such as HPLC and TLC were needed to identify the ISR-determinants of O. lupini KUDC1013.
To find of new strains with high ISR ability, roots, stem and leaves of pepper and rice cultivated in the southern part of Korea were collected in 2006 and wild plant family Solanum nigrum L. plants in Dokdo collected in 2007. From samples collected in 2006, eighty four nitrogen-fixing bacteria and 16 spore-forming bacteria, mostly Bacillus species, were isolated. The isolates were partially identified by 16S rDNA sequence analysis. Among those isolates tested for ISR ability, Xanthomonas axonopodis KNUC393, Bacillus megaterium KNUC251 and Paenibacillus polymyxa KNUC265, induced systemic resistance in pepper and tobacco. The three treatments reduced disease severity compared with negative control and significantly increased pepper fresh and dry weight.
These results suggest that these 3 isolates might be considered as useful rhizobacteria capacities of inducing systemic resistance and promoting plant growth.
From samples collected rhizosphere of Solanium nigrum L. plants in Dokdo, fourty four nitrogen-fixing or spore forming bacteria were isolated. These isolates were tested for their ability to elicit ISR and to promote growth in pepper and tobacco. Among them, O. lupini KUDC1013, Bacillus megaterium KUDC1015 and Novosphingobium panipatense KUDC1065 elicited ISR against Xanthomonas axonopodis pv. vesicatoria as well as promoted growth of pepper and tobacco seedlings.
To investigate whether these 6 isolates can play as biological control agents, we applied the isolates to pepper seedlings transplanted to greenhouse.
KUDC1013 and KUDC1065 were useful for the suppression of pathogen when applied as bacterial suspensions at various growth stages of pepper. It was therefore expected that the mixture of two isolates will protect crop from phytopathogen.
In addition, field trial was conducted in order to determine the effect of treatment with single, mixture of two strains and two ISR inducers on growth of pepper and their capacity as biological controls. Acetoin treatment showed higher ISR activity compared with 2,3-butanediol treatment. Although there was no greater disease suppression of mixtures compared to single strains, there were a trend in disease reduction. Among the three mixtures, mixtures of E681 and KUDC1013 showed the highest fresh and dry weight. There was a trend to significant enhancement of fresh and dry weight of pepper with mixtures compared when applied singly. Collectively, the application on pepper of mixtures of two stains and two ISR-inducers has potential as biocontrol agents under greenhouse and field conditions.
4. Management of pepper diseases under greenhouse and field conditions Biological management of pepper anthracnose (Colletotrichum acutatum) and bacterial leaf spot diseases (Xanthomonas campestris. pv. vesicatoria, Xcv) were using rhizosphere bacteria (444 isolates) isolated during 2006 from Jeonbuk and Chungnam pepper fields. Three isolates of X . campestris pv. vesicatoria KACC11157, KACC11158 and 1523 were obtained from various sources and inoculated on pepper plants at three leaves stage leaf stage at various concentrations (OD at 600nm). The isolate 1523 was identified as a virulent pathogen based on pathogenicity tests at a concentration of OD600 0.4 and used for our further studies.
Since, rhizosphere bacteria found to induce systemic resistance in plant system against the pathogen; we have used acibenzolar-S-methyl in combination with mancozeb as a standard chemical ISR inducer. Under greenhouse conditions, all the 444 isolates were screened against bacterial leafs pot by soil drenching methods (5 ㎖/plant) and selected 16 isolates for our studies. Owing to high number isolates, they were further screened against the bacterial pathogen. Roots of pepper seedlings at 2 leaves stage were dipped in Bacillus spp. cell suspension for one hour and transplanted in the pots and inoculated with Xcv cell suspension (OD600=0.4) at one week and two weeks later. Based on the results it was noticed that the pathogen inoculation one week after the biocontrol agent showed good results. We have selected 9 isolated out of 16 for our studies. Among 9, 5 isolated selected in random and again tested on pepper plants grown in green house were given with sequential treatments of root drenching at two leaves stage, seedling soaking at six leaves stage and spraying at different stages of crop viz., flowering, green pepper and red pepper formation. The isolates 21-13 and 22-5 were showed 100% efficacy when the plants were inoculated 2 days after Bacillus spp. spray. The selected isolates were tested for their effectiveness against Alternaria solani and Pestalotiopsis longiseta which revealed that the isolate 21-13 was effective against the former while the isolates 22-7 and 25-3 were against the later. The isolate 25-2 was equalyl effective against the pepper bacterial pathogen Xcv also. Among the different treatments including pre and post inoculation of anthracnose pathogen on pepper fruits, the pre inoculation (2 days before the pathogen inoculation) and post inoculation of Bacillus spp. isolate 22-5 at 12 hrs after pathogen inoculation showed potential disease reduction activity and recorded 100% control value, while the post inoculations of Bacillus spp. at one and two days after pathogen inoculation were not effective. The same isolate 22-5 showed 76% C. acutatum spore germination and very low number of appresorium formation (3%). During 2007, 12 isolated were tested under field conditions for the management of anthracnose disease. Application of the isolate 22-5 for four times (root dipping, soil drenching two times followed by two spray) effectively reduced the disease incidence and showed 93% control value and found on par with acibenzolar-S-methyl + mancozeb (100%) followed by the isolate 22-9 (64%) at last observation.
In the year 2008, three isolates namely 22-5, 21-13 and 25-2 were tested under field conditions to select the best one for commercial exploitation. Pepper plants were given with sequential application of bacterial isolates viz., soaking, drenching (two times), spraying (three times) revealed that the isolate 25-2 found effective than the other isolates and recorded 100% control value on first and second observation, while the same showed 79, 61 and 13% on 3rd, 4th and 5th observations. Based on the aforesaid experiments, we have selected two isolates (22-5 and 25-2) for commercial exploitation and experimented for their growth parameters. Both the isolates were best grown in a media with tryptone as nitrogen source at a pH of 6-7 and incubated at a temperature of 24 to 28°C for 36-48 hours.
On overall performance, we have sequenced the 16s rDNA of the isolates 22-5, 25-2 and 21-13 and compared with other bacteria in NCBI website. The phylogenetic tree generated using the software PHYDIT by neighbour joining method revealed that the isolates 21-13, 22-5 and 25-2 showed 99% similarity and homology with B. samanii, B. megaterium and B cereus, respectively.
5. Commercialization of induced systemic resistance microbes and its subs tances
Recently, well-being life became a main issue among us. As peoples interest on eco-friendly agriculture, farmers concerned about biological agent to prevent crop disease and insects. One of the best agent to alternate chemical pesticide is microbial biopesticide. But commercialized microbial biopesticides are less applied because its low efficacy and difficulty of application. The purpose of this research was to develop plant scale fermentation and commercial formulation in order to commercialize induced systemic resistance microbes and its substances.
To develop induced systemic resistance microbes into microbial biopesticide, we've searched for the effective microorganisms of induced systemic resistance. Bacillus cereus BS107 and other microbes were selected as target microorganism. Target microbes were tested on plant disease in vitro. And finally Bacillus cereus BS107 was selected to develop microbial biopesticide. Before developing microbial biopesticide, plant scale fermentation design and formulation design of target microbe have to be proceeded. Lab scale, pilot scale and plant scale fermentation design were designed in order. 15 hours after seed inoculation, spore counts were slightly increased, and at 22 hours after inoculation, spore counts showed highest increasing rate. With designed plant scale fermentation method, formulation design of BS107 was proceeded. Fermented broth was formulated as liquid form and spray dried powder form with profit carriers and additives.
In vivo efficacy on plant disease was tested at hot pepper field in Hwasung, Gyeonggi Province. BS107 was treated on hot pepper with root soaking method before planting. As a result, both powder form and liquid form have more than 60% efficacy on hot pepper anthracnose. This result shows that Bacillus cereus BS107 can be developed as microbial biopesticide.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• SUMMARY ... 18
• 제 1 장 연구 개발과제의 개요 ... 25
• 제 1 절 연구개발의 필요성 ... 25
• 제 2 절 연구개발과제의 개요 ... 26
• 제 2 장 국내외 기술 개발 현황 ... 30
• 제 1 절 연구개발대상 기술의 국내ㆍ외 현황 ... 30
• 제 2 절 국내외 기술개발 현황 ... 33
• 제 3 절 제작성 ... 34
• 제 4 절 국내외 기술개발 현황 ... 35
• 제 3 장 연구개발 수행 내용 및 결과 ... 37
• 제 1 절 전신유도저항성(induced systemic resistance)을 이용한 고추병 방제 기술 개발 ... 37
• 제 2 절 ISR을 이용한 벼 병해 방제 ... 103
• 제 3 절 세균으로부터 ISR 결정 인자 탐색 및 개발 (협동2) ... 120
• 제 4 절 ISR 유도물질 및 미생물의 미생물제제의 산업화 연구(협동4) ... 168
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 191
• 제 1절 ... 191
• 제 2 절 목표달성도 및 관련분야에의 기여도 ... 192
• 제 3 절 ... 193
• 제 4 절 목표달성도 및 관련분야에의 기여도 ... 194
• 제 5 절 ... 195
• 제 5 장 연구개발 성과 및 성과활용 계획 ... 196
• 제 1절 ... 196
• 제 2절 ... 196
• 제 3절 ... 197
• 제 4절 ... 203
• 제5절 ... 205
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 208
• 제 1절 ... 208
• 제 2절 ... 208
• 제 3절 ... 208
• 제 4절 ... 208
• 제 5절 ... 208
• 제 7 장 참고문헌 ... 208
• 제 1절 ... 208
• 제 2절 ... 214
• 제 3절 ... 214
• 제 4절 ... 217
• 제 5절 ... 221
• 끝페이지 ... 222