콩 종자에서 Xanthomonas axonopodis pv. glycines의 검출을 위한 Direct PCR 방법 개발

이용주,강미형,노태환,이두구,이건휘,김시주 한국식물병리학회 2009 식물병연구 Vol.15 No.2

Direct Polymerase Chain Reaction (PCR) method that combines biological and enzymatic amplification of PCR targets was developed for the detection of Xanthomonas axonopodis pv. glycines on soybeen seeds without DNA isolation. Primers Xag F1 and Xag R1 were designed to specifically amplify a 401 bp fragment of the glycinecin A gene of X. axonopodis pv. glycines. Xag F1 and Xag R1 were used to carry out the PCR analysis with genomic DNA from 45 different bacterial strains including phylogenetically related bacteria with X. axonopodis pv. glycines, and other bacterial strains of different genus and species. The PCR assay using this set of primers were able to detect X. axonopodis pv. glycines with DNA concentration as low as 200 fg and 1.8×103 cfu/ml. The Xag was detected from the seed samples incubated for 2 hrs with shaking and the intensity of the band was increase with the incubation time of seeds. The Direct PCR assay method without DNA isolation makes detection of X. axonopodis pv. glycines on soybean seeds easier and more sensitive than other conventional methods. The developed seed assay using direct PCR method will be useful for the specific detection of X. axonopodis pv. glycines in soybean seed samples. Direct Polymerase Chain Reaction (PCR) method that combines biological and enzymatic amplification of PCR targets was developed for the detection of Xanthomonas axonopodis pv. glycines on soybeen seeds without DNA isolation. Primers Xag F1 and Xag R1 were designed to specifically amplify a 401 bp fragment of the glycinecin A gene of X. axonopodis pv. glycines. Xag F1 and Xag R1 were used to carry out the PCR analysis with genomic DNA from 45 different bacterial strains including phylogenetically related bacteria with X. axonopodis pv. glycines, and other bacterial strains of different genus and species. The PCR assay using this set of primers were able to detect X. axonopodis pv. glycines with DNA concentration as low as 200 fg and 1.8×103 cfu/ml. The Xag was detected from the seed samples incubated for 2 hrs with shaking and the intensity of the band was increase with the incubation time of seeds. The Direct PCR assay method without DNA isolation makes detection of X. axonopodis pv. glycines on soybean seeds easier and more sensitive than other conventional methods. The developed seed assay using direct PCR method will be useful for the specific detection of X. axonopodis pv. glycines in soybean seed samples.

PCR 기법을 이용한 사탕무씨스트선충과 콩씨스트선충의 간이동정

고형래,김은화,김세종,이재국,이왕휴,Ko, Hyoung Rai,Kim, Eun Hwa,Kim, Se Jong,Lee, Jae Kook,Lee, Wang Hyu 한국식물병리학회 2017 식물병연구 Vol.23 No.3

강원도 고랭지배추 포장에서 검출된 사탕무씨스트선충(H. schachtii)과 콩씨스트선충(H. glycines)을 구별할 수 있는 신속진단법을 개발하고자 하였다. 이를 위해 mtDNA COI 유전자 영역의 계통분석으로 동정된 사탕무씨스트선충 GC147, GC408, PM001 개체군과 콩씨스트선충 YS224, DA142, BC115 개체군을 대상으로 PCR-RFLP와 본 연구에서 개발한 특이 프라이머 세트를 이용한 PCR을 수행하였다. 사탕무씨스트선충과 콩씨스트선충 각각 3개 개체군의 mtDNA COI 영역 PCR 증폭산물에 8종류의 제한효소를 처리하여 DNA 절편길이다형성을 확인하였으며, 2종류의 제한효소 RsaI과 HinfI을 처리하면 DNA 밴드 양상의 차이로 사탕무씨스트선충과 콩씨스트선충 두 종을 구별할 수 있었다. 또한, 본 연구에서 개발한 프라이머 세트(JBS1, JBG1과 JB3R)는 사탕무씨스트선충 mtDNA COI 영역의 277과 339 bp, 콩씨스트선충의 339 bp의 특정 DNA 단편을 증폭시켰으며, 뿌리혹선충 3종과 뿌리썩이선충 2종의 식물기생선충은 증폭시키지 않았다. 따라서, 본 연구에서 개발한 프라이머 세트를 이용하여 PCR을 수행하면 사탕무씨스트선충과 콩씨스트선충을 구별할 수 있었다. The purpose of this study was to develop rapid methods for distinguishing between Heterodera schachtii and H. glycines detected from chinese cabbage fields of highland in Gangwon, Korea. To do this, we performed PCR-RFLP and PCR with the primers set developed in this study for GC147, GC408 and PM001 population, H. schachtii, and YS224, DA142 and BC115 population, H. glycines. Eight restriction enzymes generated RFLP profiles of mtDNA COI region for populations of H. schachtii and H. glycines, repectively. As a result, treatment of two restriction enzymes, RsaI and HinfI, were allowed to distinguish H. schachtii from H. glycines based on the differences of DNA band patterns. The primer set, #JBS1, #JBG1 and #JB3R, amplified specific fragments with 277 and 339 bp of H. schachtii, 339 bp of H. glycines, respectively, while it did not amplify fragments from three root-knot nematodes and two root-lesion nematodes. Thus, the primer set developed in this study could be a good method, which is used to distinguish between H. schachtii and H. glycines.

콩씨스트선충(Heterodera glycines)의 밀도가 콩 수량에 미치는 영향

강헌일(Heonil Kang),박병용(Byeongyong Park),박세근(Sekeun Park),김은화(Eunwha Kim),박은형(Eunhyeong Park),고형래(Hyoung-Rai Ko) 한국자원식물학회 2021 한국자원식물학회지 Vol.34 No.4

본 연구는 토양 내 콩씨스트선충(Heterodera glycines)의 초기 밀도에 따른 콩의 수량구성요소 및 수량에 미치는 영향을 규명하기 위하여 실시하였다. 재배포를 35개의 구역을 나누고 콩씨스트선충의 파종 전 토양 내 씨스트 밀도와 알 밀도를 조사하였으며, 수확기 콩의 초장, 협수, 립수, 백립중을 비교 분석하였다. 초기 씨스트의 밀도보다 씨스트 내 알의 밀도가 수량에 더욱큰 영향을 미쳤으며, 초기 알 밀도가 높을수록협수와 수량이 초기 알 밀도가 높을수록 감소하는 경향을 보였고 토양 100 ㎤ 당알의 밀도가 2,000개 이상일 때부터 수량이 20% 이상 급격하게감소하였다. 본 연구결과를 통하여 콩 재배 시 콩씨스트선충에의한 피해를 예측할 수 있으며, 국내 콩 재배지의 약 40%가 감염되어 있는 콩씨스트선충에 대한 저항성 품종 육성을 포함한 다양한 방제방법 연구가 필요하다. This study was conducted to investigate the yield loss of soybean by initial density of Heterodera glycines in soil. A soybean cultivar named ‘Cheongja5ho’ was cultivated as test material in Dangjin city, Chungcheongnam-do, Korea. Each seed was planted with distance of 25 cm in 35 plots by initial density of egg of the cyst nematode on June 21st, and allowed to harvest on November 12st in 2019. As a results, the yield of soybean decreased sharply when as the initial density of eggs of cyst nematode increased. The initial density of eggs in soil affected largely on the decreased yields of the soybean by more than 20% in above 2,000 eggs, and 30% in above 6,000 eggs of the cyst. Beyond the 6,000 eggs, the yield loss was more slowly decreased compared with the above cases. Eggs of H. glycines are widely distributed in Korea, resulting in the serious soybean yield loss by H. glycines.

Development of microsatellite loci from the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae)

Hyojoong Kim,Min-Young Kim,Kim A. Hoelmer,Seunghwan Lee 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.05

Ten novel microsatellite loci were isolated and characterized from the soybean aphid, Aphis glycines. The soybean aphid was recently introduced into North America where it has become a serious pest of soybeans. This invasive pest has rapidly spread throughout the midwestern United States and southern Canada since 2000. The isolated loci were polymorphic, with two to 18 alleles in 20 individuals from a single population in Korea. The analyses revealed that 19 individuals had different multilocus genotypes, showing expected heterozygosity values ranging from 0.180 to 0.891. We report the development of microsatellite markers for A. glycines potentially suitable for further studies of population structure, dispersal, and host alternation.

콩 불마름병 방제를 위한 살균제 선발

홍성준,이봉춘,김용기,지형진,윤영남,박성태 한국식물병리학회 2010 식물병연구 Vol.16 No.3

Bacterial pustule of soybean (Glycines max) caused by Xanthomonas axonopodis pv. glycines is one of the most prevalent bacterial diseases of soybean. This bacterium shows strong pathogenicity to the plants and distributes throughout Korea. However, no good control measures including bactericides and resistant cultivars are available to control the disease in Korea. Therefore, this study was conducted to develop chemical control method against soybean bacterial pustule. The present study was undertaken to find out the growth inhibitory effect bactericides (8 antibiotics, 2 copper compounds, quinoline, 18 agro-chemicals) on bacterial pustule pathogen. Antibiotics test showed that tetracycline and streptomycin sulfate significantly suppressed the growth of bacterial pustule pathogen. Also, application of oxolinic acid was found to be effective for pathogen inhibition. However, vancomycin, polymyxin B sulfate and copper compounds did not show the positive suppressive effect on growth of the pathogen. Among the eighteen agro-chemicals,streptomycin sulfate + oxytetracyclin (18.8 + 1.5%) WP, oxytetracycline (17%) WP and oxolinic acid (20%)WP were found to be effective for the inhibition of the pathogen in vitro. The selected 5 agro-chemicals were also applied on soybean in field and their control effects against the soybean bacterial pustule were tested. The foliar application of streptomycin sulfate + oytetracyclin WP and oxytetracycline WP on the naturally infected soybean (Taekwangkong) showed high control value (above 70%). Therefore, it is concluded that the bactericides used in this study showed strong inhibitory effect to soybean bacterial pustule and they can be recommend to farmers to control the disease. Xanthomonas axonopodis pv. glycines에 의해 발병되는 콩 불마름병은 콩에 가장 많이 발생하는 중요한 세균성병해 중 하나이다. 게다가 병원균은 강한 병원성을 가지고 한국 전역에 분포하고 있지만, 한국에서는 불마름병 방제를 위한 약제나 저항성 품종이 개발되어 있지 못한 실정이다. 따라서 본 연구의 목적은 불마름병을 화학적 방법으로 방제할 수 있는 방법을 개발하기 위해 수행되었다. 콩 불마름병 병원균의 효과적인 생육억제 약제를 선발하기 위해 여러 가지 살세균제들(8개의 항생제, 2개의 구리화합물, 1개의 quinoline, 18개의 농약)을 이용하여 효과를 확인하였다. 실내 생장억제 효과조사 결과, tetracycline, streptomycin sulfate 처리에서 병원균의 생육이 크게 억제되었으며, 또한 oxolinic acid도 병원균의 생육억제 효과가 우수하였다. 하지만 vancomycin, polymyxin B sulfate and copper compounds 등은 불마름병 병원균의 생육억제효과가 확인되지 않았다. 18종의 농용약제 중에는 streptomycin sulfate + oxytetracycline(18.8 + 1.5%)WP, oxytetracycline(17%)WP, oxolinic acid(20%) 등이 실내에서 병원균의 생육억제 효과가 높은 것으로 조사되었다. 선발된 5종의 농용약제에 대하여 포장에서 방제효과 시험을 실시한 결과, streptomycin sulfate + oxytetracycline (18.8+1.5%) WP와 oxytetracycline(17%) WP가 자연 감염된 태광콩 포장에서 70% 이상의 높은 방제효과를 나타내었다. 그러므로 콩불마름병균에 대한 강한 억제효과를 가지는 이 약제들은 콩 불마름병의 화학적 방제에 실용성이 있을 것으로 생각된다.

콩 불마름병 발생정도가 수량에 미치는 영향

홍성준,이봉춘,김용기,지형진,심창기,김민정,박종호,한은정 한국식물병리학회 2011 식물병연구 Vol.17 No.3

Bacterial pustule of soybean (Glycine max) caused by Xanthomonas axonopodis pv. glycines is one of the most prevalent bacterial diseases of soybean in Korea, where it causes considerable yield loss. This study was carried out to develop yield prediction model for bacterial pustule by analyzing correlation between the percentage of diseased leaf area and yield. The severe disease incidence of soybean bacterial pustule caused yield losses by 19.8% in 2006 and 16.8% in 2007, respectively. Severity of bacterial pustule greatly affected on 100 seed weight and yield, but did not on stem length, number of branches per plant, number of pods per plant, number of seeds per plant. On the other hand, correlation coefficients between diseased leaf area and yield were −0.93*(‘06) and −0.77*(‘07), respectively. The regression equation obtained by analyzing correlation between the percentage of diseased leaf area and yield loss in 2006 and in 2007 was y = −3.2914x + 348.19(R^2 = 0.8603) and y = −2.9671x +302.08(R^2 = 0.9411), respectively. These results will be helpful in estimating losses on a field-scale and thereby predicting the production of soybean. Xanthomonas axonopodis pv. glycines에 의해 발병되는 콩 불마름병은 콩에 가장 많이 발생하는 중요한 세균성 병해 중 하나로 콩 수량에 심각한 손실을 가져온다. 본 연구는 불마름병 병반면적률에 따른 수량예측모델을 개발하기 위해 수행되었다. 이번 실험의 결과 불마름병이 심하게 발병된 처리구의 경우 수량이 19.8%(2006년), 16.8% (2007년) 감소되는 것으로 조사되었다. 또한 발병률의 차이에 따라 100립중 및 수량은 영향을 받으나 경장, 분지수, 협수, 립수는 유의성있는 차이를 나타내지 못하였다. 즉 수량구성요소 중 100립중의 감소가 수량감소에 직접적인 영향을 주는 것으로 조사되었다. 2006년도와 2007년도의 연차별 콩 불마름병 병반면적률과 수량과의 상관관계를 5% 유의수준에서 분석한 결과, -0.93(06년), -0.77(07년) 부의상관이 있었으며, 회귀식은 각각 y = -3.2914x + 348.19 (R^2= 0.8603), y = -2.9671x + 302.08 (R^2= 0.9411)로 나타낼 수 있었다. 이런 결과들은 포장에서 불마름병 발병에 따른 수량손실을 평가하고 콩 생산량을 예측하는데 도움을 줄 것으로 생각한다.

콩 유묘 부패를 일으키는 종자전염성 세균의 동시 검출을 위한 duplex PCR법 개발

강미형 ( Mi Hyung Kang ),노태환 ( Tae Whan Noh ),심형권 ( Hyeong Kwon Shim ),최만영 ( Man Young Choi ),이휘종 ( Hwi Jong Lee ),김형무 ( Hyung Moo Kim ) 전북대학교 농업과학기술연구소 2014 농업생명과학연구 Vol.45 No.2

콩 종자로부터 유래하여 유묘에 부패를 일으키는 콩불마름병(X. axonopodis pv. glycines, Xag)과 콩나물 무름병(Pectobacterium carotovorum subsp. carotovorum, Pcc)을 동시에 진단하는 PCR 방법을 개발하였다. 콩불마름병 진단을 위하여 X. axonopodis pv. glycines의 특이적인 유전자로 알려져 있는 glycinecin A로부터 증폭 크기가 401bp인 Xag F1와 Xag R1 프라이머를 고안하였으며, 콩나물 무름병균의 특이적인 진단을 위하여 Carotovoricine 유전자에서 227bp 크기로 증폭이 가능한 Pcc F와 Pcc R 프라이머를 고안하였다. 두 쌍의 프라이머들은 두 세균만을 증폭하고 다른 오염균들은 증폭하지 않았으며, 이들 프라이머는 Xag를 2pg까지, Pcc는 20pg까지 증폭이 가능하였다. 이들 프라이머를 이용한 검출 방법은 콩 및 유묘 부패의 원인균인 감염종자를 검정할 수 있는 방법으로 활용 가능할 것이다. Duplex PCR primers were developed for simultaneous detection of the important soybean seed pathogenic bacteria Xanthomonas axonopodis pv. glycines (Xag) and Pectobacterium carotovorum subsp. carotovorum (Pcc) which cause highly loss of yield and decreased quality of seeds in order to eliminate infected soybean seeds with these pathogen. Two specific primer pairs, Xag F1/R1 and Pcc F/R, were designed from the sequences of glycinecin A gene of Xag and carotovoricin gene of Pcc for certain identification of Xag and Pcc, respectively, in order to distinguish contaminated seeds with pathogens. A duplex PCR method for simultaneous identification and detection of Xag and Pcc was developed by amplifying both 401bp and 227bp bands. Identification of Xag and Pcc were done in the sensitivity of 2pg and 20pg, respectively. This approach can be used to exclude infected soybean seeds which act as soft rot inoculum of soybean seedling.

Microbiota Communities of Healthy and Bacterial Pustule Diseased Soybean

김다란,김수현,이수인,곽연식 한국식물병리학회 2022 Plant Pathology Journal Vol.38 No.4

Soybean is an important source of protein and for a wide range of agricultural, food, and industrial applications. Soybean is being affected by Xanthomonas citri pv. glycines, a causal pathogen of bacterial pustule disease, result in a reduction in yield and quality. Diverse microbial communities of plants are involved in various plant stresses is known. Therefore, we designed to investigate the microbial community differentiation depending on the infection of X. citri pv. glycines. The microbial community's abundance, diversity, and similarity showed a difference between infected and non-infected soybean. Microbiota community analysis, excluding X. citri pv. glycines, revealed that Pseudomonas spp. would increase the population of the infected soybean. Results of DESeq analyses suggested that energy metabolism, secondary metabolite, and TCA cycle metabolism were actively diverse in the non-infected soybeans. Additionally, Streptomyces bacillaris S8, an endophyte microbiota member, was nominated as a key microbe in the healthy soybeans. Genome analysis of S. bacillaris S8 presented that salinomycin may be the critical antibacterial metabolite. Our findings on the composition of soybean microbiota communities and the key strain information will contribute to developing biological control strategies against X. citri pv. glycines.

콩 잎에 서식하는 세균 및 콩나물 부패균의 밀도 변화

최재을,이은정,신철우 한국자원식물학회 1999 한국자원식물학회지 Vol.12 No.2

1. 콩잎의 세균밀도는 4.60$\times$$10^2$~ 9.10$\times$$10^{5}$CFU/$\textrm{cm}^2$으로, 생육단계가 진전됨에 따라 세균밀도가 증가하는 경향이었다. 2. 콩나물 부패 세균의 밀도는 콩잎에서 0~5.00$\times$$10^3$CFU/$\textrm{cm}^2$으로, 부패세균의 밀도는 생육단계에 관련이 없었으나 재배지역과는 관련이 있었다. 3. 나물 콩 품종과 콩나물 부패세균의 밀도는 품종과 관련이 적었으며 생육단계와 작물의 부위에 따라 변이가 심하였다. 4. 콩잎에서 분리된 콩나물 부패세균은 Erwinia cypripedii, E. carotovora subsp. carotovora, Xanthomonas campestris pv. glycines, Staphylococcus sp., Micrococcus sp. 이며, E. carotovora subsp. carotovora, X. campestris pv. glycines가 밀도가 높았다.다. Bacterial population density on soybean leaves was $10^2~10^5CFU/cm^2$. Bacterial population density was increased by progress of plant growth stage. Population density of soybean sprout rotting bacteria on soybean leaves was $0~10^3CFU/cm^2$. Population density of soybean sprouts rotting bacteria was related to cultivating area, but not related to plant growth stage. Cultivar and population density of soybean sprout rotting bacteria were less corelated, and varied by plant growth stages and plant parts. Erwina cypripedii, E. carotovora subsp. carotovora, Xanthomonas campestris pv. glycines, Staphylococcus sp., and Micrococcus sp. were identified as pathogenic bacteria causing soybean sprout rot. In generally population density of E. cypripedii, E. carotovora subsp. carotovora, Micrococcus sp., and X. campestris pv. glycines were high.

Heterodera glycines에 대한 콩 내병성과 관련된 거대세포의 형태적 특성

염규진,박은우,김영호 한국식물병리학회 1992 Plant Pathology Journal Vol.8 No.4