Genome-wide screening antifungal genes in <i>Streptomyces griseus</i> S4-7, a Fusarium wilt disease suppressive microbial agent

Hong, Sung Won,Kim, Da-Ran,Kwon, Youn Sang,Kwak, Youn-Sig Published by Elsevier/North Holland on behalf of t 2019 FEMS microbiology letters Vol.366 No.12

<P> <I>Streptomyces</I> is a widely studied bacterial genus, particularly with regard to secondary metabolites and antibiotics production. <I>Streptomyces griseus</I> S4-7 was isolated from a strawberry Fusarium wilt disease suppressive soil, and its biological control ability has been well established. However, the antifungal mechanism of strain S4-7 is not yet fully understood at the molecular and biochemical level. Therefore, in this study we created a random mutant library for strain S4-7 with the Tn5 transposon element to investigate antifungal traits on a genome-wide scale. In total 4646 individual mutant strains were created and 13 mutants were selected based on loss of antifungal activity. The knockout genes were identified as electron transfer oxidoreductase (eto),sigma factor-70(sig70) and nrps by Inverse PCR (I-PCR). eto regulates the <I>geranylgeranyl</I> reductase gene, which is involved in terpenoid-quinone biosynthesis, an important factor in cell fitness. In the <I>△eto</I> strain, expression of <I>wbl,</I> a master regulator of the production of secondary metabolites, was significantly reduced. sig70 is responsible for the cell differentiation sensing mechanism in genus <I>Streptomyces. △nrps</I> showed decreased production of hybrid peptide-polyketide siderophores. These results suggest that <I>S. griseus</I> S4-7 may have various antifungal mechanisms, and each mechanism is essential to maximal antifungal activity. <P>

Different oxidative burst patterns occur during host and nonhost resistance responses triggered by Xanthomonas campestris in pepper

Kwak, Youn-Sig,Han, Ki-Soo,Lee, Jung-Han,Lee, Kyung-Hee,Chung, Woo-Sik,Mysore, Kirankumar S.,Kwon, Young-Sang,Kim, Hee-Kyu,Bae, Dong-Won The Korean Society of Plant Biotechnology 2009 식물생명공학회지 Vol.36 No.3

The hypersensitive reaction (HR) is the most common plant defense reaction against pathogens. HR is produced during both host- and nonhost-incompatible interactions. Several reports suggest that similarities exist between host and nonhost resistances. We assayed the pattern of generation of reactive oxygen species (ROS) and scavenging enzyme activities during nonhost pathogen-plant interactions (Xanthomonas campestris pv. campestris/Capsicum annuum L.) and incompatible host pathogen-plant interactions (Xanthomonas campestris pv. vesicatoria race1/Capsicum annuum L.). Both ${O_2}^-\;and\;H_2O_2 $ accumulated much faster during nonhost resistance when compared to the host resistance. The scavenging enzyme activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX) were also different during the host- and nonhost-incompatible interactions. CAT activity was much higher during nonhost resistance, and several new isozymes of SOD and POX were detected during nonhost resistance when compared to the host resistance. Lipoxygenase (LOX) activity was higher in host resistance than nonhost resistance during the early stages of infection. Interestingly, the nitric oxide (NO) radical accumulated equal amounts during both host and nonhost resistance at early stages of infection. Further studies are needed to determine the specific pathways underlying these differences between host and nonhost resistance responses.

Crop Health Management through understanding Microbiota Community Ecology

Youn-Sig Kwak(곽연식) 한국농약과학회 2022 한국농약과학회 학술발표대회 논문집 Vol.2022 No.11

Plants lack genetic resistance to most necrotrophic pathogens. To compensate for this disadvantage, plants recruit antagonistic members of microbiota to defend themselves against pathogens and other pests. Pseudomonas, Bacillus, and Trichoderma are well-known plant probiotic agents, which can protect plants against various biotic and abiotic stresses. In 2015, the Phytobiome initiative was launched by the White House. Phytobiomes consist of crops, the environment, and associated communities of microbes. Interkingdom interaction within the microbiome is dynamic and critical affecting crops and agroecosystem healthiness. In this presentation, a new probiotic strain Streptomyces, plant and environment factorial interaction, and the impact of the microbe in the agroecosystem will be introduced. Streptomyces is a gram-positive bacterium and more than two third of antibiotic compounds, currently used in the human clinic, are originated from the bacterium. However, in interaction with plant and agrosystem, the role and weight of Streptomyces is undervalued. Therefore, we recently start “STreptomyce in Agriculture Research” project with various cropping systems. Of special interest is that rhizosphere and anthosphere microbial community structures and shifting have been analyzed using massive pyrosequencing platforms. Based on the results, three putative probiotic Streptomyces strains have been isolated and characterized. The strains were well colonized in the rhizosphere, anthosphere, and phyllosphere. Unexpectedly, the three independent stains were revealed they had identical genome sequences. The following questions arise why the identical stains exist in totally different habitats and what is the function of the strains in such a system? In conclusion, Streptomyces is ubiquitous and unique in the system to provide a beneficial effect to both plant and it phytobiome communities. The STAR project will provide more opportunities and possibilities in agriculture.

Take-all of Wheat and Natural Disease Suppression: A Review

Kwak, Youn-Sig,Weller, David M. The Korean Society of Plant Pathology 2013 Plant Pathology Journal Vol.29 No.2

In agro-ecosystems worldwide, some of the most important and devastating diseases are caused by soil-borne necrotrophic fungal pathogens, against which crop plants generally lack genetic resistance. However, plants have evolved approaches to protect themselves against pathogens by stimulating and supporting specific groups of beneficial microorganisms that have the ability to protect either by direct inhibition of the pathogen or by inducing resistance mechanisms in the plant. One of the best examples of protection of plant roots by antagonistic microbes occurs in soils that are suppressive to take-all disease of wheat. Take-all, caused by Gaeumannomyces graminis var. tritici, is the most economically important root disease of wheat worldwide. Take-all decline (TAD) is the spontaneous decline in incidence and severity of disease after a severe outbreak of take-all during continuous wheat or barley monoculture. TAD occurs worldwide, and in the United States and The Netherlands it results from a build-up of populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp. during wheat monoculture. The antibiotic 2,4-DAPG has a broad spectrum of activity and is especially active against the take-all pathogen. Based on genotype analysis by repetitive sequence-based-PCR analysis and restriction fragment length polymorphism of phlD, a key 2,4-DAPG biosynthesis gene, at least 22 genotypes of 2,4-DAPG producing fluorescent Pseudomonas spp. have been described worldwide. In this review, we provide an overview of G. graminis var. tritici, the take-all disease, Pseudomonas biocontrol agents, and mechanism of disease suppression.

미생물 군집의 이해를 통한 유기농업 작물 건강관리

곽연식 ( Youn-sig Kwak ) 한국환경농학회 2023 한국환경농학회 학술대회집 Vol.2023 No.0

식물은 대부분의 괴사성 병원체에 대해 유전적 저항력을 갖지 못한다. 이를 보완하기 위해, 식물은 미생물 군집에서 유익한 미생물을 동원하여 병원체와 해충으로부터 자신을 방어한다. Pseudomonas, Bacillus, 그리고 Trichoderma는 식물을 다양한 스트레스로부터 보호하는 잘 알려진 식물 프로바이오틱스이다. 2015년 식물마이크로바이옴 이니셔티브는 작물 건강과 생태계 균형에서 식물-미생물 군집의 중요성이 강조되었다. 본 발표에서는 Streptomyces라는 새로운 프로바이오틱스 균주, 식물 및 환경과의 상호작용, 그리고 농업 생태계에 미치는 영향을 소개하고자 한다. 인간이 사용하는 항생물질의 70% 이상이 Streptomyces에서 유래되었다. 그러나 Streptomyces의 식물 및 농업 생태계 상호작용에서의 역할과 중요성은 심도 있는 접근이 이루어지지 않았다. 이를 해결하기 위해 우리는 “농업 연구에서의 Streptomyces (STAR)” 프로젝트를 시작하였다. 이 프로젝트는 다양한 작물 재배 체계에 초점을 맞추었다. 차세대 파이로시퀀싱 플랫폼을 사용하여 작물의 근권, 꽃권, 그리고 내권의 미생물 군집을 분석하였다. 연구를 통해 식물의 뿌리, 꽃, 그리고 내부조직에서 서식하는 세 가지의 가상 프로바이오틱스 Streptomyces 균주를 분리 및 특성화했다. 놀랍게도, 이 세 균주는 서로 다른 서식지에서 발견되었음에도 동일한 유전체 서열을 보였다. 이는 서로 다른 시스템에서 동일한 균주의 존재와 기능에 관한 의문을 제기하였다. 결론적으로, Streptomyces는 작물 생태계에 유익한 효과를 제공하는 널리 분포하는 독특한 미생물이다. STAR 프로젝트는 유기농 농업을 위한 유망한 기회와 가능성을 가지고 있다. Plants lack genetic resistance to most necrotrophic pathogens. To compensate for this, they enlist beneficial microbes from their microbiota to defend against pathogens and pests. Pseudomonas, Bacillus, and Trichoderma are well-known plant probiotics that safeguard plants from various stresses. The Phytobiome initiative, launched in 2015, recognizes the importance of plant-microbe communities in crop health and ecosystem balance. In this presentation, we introduce a new probiotic strain called Streptomyces, its interactions with plants and the environment, and its impact on the agroecosystem. Streptomyces, a gram-positive bacterium, is the source of over two-thirds of antibiotics used in human medicine, yet its role and significance in plant and agroecosystem interactions are underestimated. To address this, we initiated the “STreptomyce in Agriculture Research” (STAR) project, focusing on various cropping systems. We employed advanced pyrosequencing platforms to analyze the microbial communities in the rhizosphere and anthosphere. Through this study, we isolated and characterized three putative probiotic Streptomyces strains that were found to colonize the rhizosphere, anthosphere, and phyllosphere. Surprisingly, these three strains exhibited identical genome sequences despite inhabiting different habitats. This raises questions about the existence and function of identical strains in distinct systems. In conclusion, Streptomyces is a ubiquitous and unique microbe that provides beneficial effects to plants and their phytobiome communities. The STAR project holds promising opportunities and possibilities for organic agriculture.

Investigation of soybean sprout rot and the elimination of improper seeds for quality control on soybean seedlots

Jung Han Lee,Youn-Sig Kwak 경상대학교 농업생명과학연구원 2016 농업생명과학연구 Vol.50 No.1

Soybean sprouts have been a considered a nutrient-rich vegetable for hundreds of years. To evaluate the seedlot quality of soybean sprouts grown, and to evaluate a method for reducing the presence of improper seeds in soybean seedlots, microbes associated with soybean sprout rot were isolated from samples collected. Morphological characteristics and gas chromatography profiles of the cultured fungal and bacterial strains were identified. Eight types of improper seeds were identified: purple stain(Ps), black rot(Br), seed coat black spot(Cb), wrinkled seed(Ws), brown hilum(Bh), seed coat fracture(Cf), unripe seed(Us), and brown seed coat(Bc). The improper seeds were also dipped into 15%, 20%, and 25% NaCl solutions, as well as a saturated solution of NaCl, for 1min. As the NaCl concentration increased, the number of floating improper seeds increased as well. The highest floating rates were observed for the Cf seeds.

Variations in Kiwifruit Microbiota across Cultivars and Tissues during Developmental Stages

Su-Hyeon Kim(Su-Hyeon Kim),Da-Ran Kim(Da-Ran Kim),Youn-Sig Kwak(Youn-Sig Kwak) 한국식물병리학회 2023 Plant Pathology Journal Vol.39 No.3

The plant microbiota plays a crucial role in promot-ing plant health by facilitating the nutrient acquisition, abiotic stress tolerance, biotic stress resilience, and host immune regulation. Despite decades of research efforts, the precise relationship and function between plants and microorganisms remain unclear. Kiwifruit (Actinidia spp.) is a widely cultivated horticultural crop known for its high vitamin C, potassium, and phytochemical content. In this study, we investigated the microbial communities of kiwifruit across different cultivars (cvs. Deliwoong and Sweetgold) and tissues at various developmental stages. Our results showed that the microbiota community similarity was confirmed be-tween the cultivars using principal coordinates analysis. Network analysis using both degree and eigenvector centrality indicated similar network forms between the cultivars. Furthermore, Streptomycetaceae was identi-fied in the endosphere of cv. Deliwoong by analyzing amplicon sequence variants corresponding to tissues with an eigenvector centrality value of 0.6 or higher. Our findings provide a foundation for maintaining ki-wifruit health through the analysis of its microbial com-munity.

Investigation of relationship between antifungal activity of caryolan-1-ol and ceramide synthesis in fungi

Gyeongjun Cho,Youn-Sig Kwak 한국농약과학회 2018 한국농약과학회 학술발표대회 논문집 Vol.2018 No.10

Genetic Variation of Strawberry Fusarium Wilt Pathogen Population in Korea

Cho Gyeongjun,Kwak Youn-Sig 한국균학회 2022 Mycobiology Vol.50 No.1

Strawberries are a popular economic crop, and one of the major plantations and exporting countries is Korea in the world. The Fusarium oxysporum species complex (FOSC) is a soil-borne pathogen with genetic diversity, resulting in wilt disease in various crops. In Korea, strawberries wilt disease was first reported in the 1980s due to the infection of FOSC, causing significant economic damage every year. The causal agent, F. oxysporum f. sp. fragariae,isasoil-borne pathogen with a characteristic of FOSC that is difficult to control chemically and mutates easily. This study obtained genetic polymorphism information that was based on AFLP, of F. oxyspo- rum f. sp. fragariae 91 strains, which were isolated from strawberry cultivation sites in Gyeongsangnam-do and Chungcheongnam-do, and compared strains information, which was the isolated location, host variety, response to chemical fungicide, and antagonistic bacteria, andmyceliumphenotype.Asaresult,AFLPphylogenyfoundthattwogroupsweremainlypre- sent, and group B was present at a high frequency in Gyeongsangnam-do. Group B proved less sensitive to tebuconazole than group A through Student’s t-test. In addition, the fractions pat- tern of AFLP was calculated by comparing the strain information using PCA and PERMANOVA, and the main criteria were separated localization and strawberry varieties (PERMANOVA; p < 0.05). And tebuconazole was different with weak confidence (PERMANOVA; p < 0.10). This study suggests that the F. oxysporum f. sp. fragariae should be continuously monitored and managed, including group B, which is less chemically effective.

A Genome-Wide Analysis of Antibiotic Producing Genes in Streptomyces globisporus SP6C4

Da-Ran Kim,Youn-Sig Kwak 한국식물병리학회 2021 Plant Pathology Journal Vol.37 No.4

Soil is the major source of plant-associated microbes. Several fungal and bacterial species live within plant tissues. Actinomycetes are well known for producing a variety of antibiotics, and they contribute to improving plant health. In our previous report, Streptomyces glo- bisporus SP6C4 colonized plant tissues and was able to move to other tissues from the initially colonized ones. This strain has excellent antifungal and antibacterial activities and provides a suppressive effect upon various plant diseases. Here, we report the genome-wide analysis of antibiotic producing genes in S. globisporus SP6C4. A total of 15 secondary metabolite biosynthetic gene clusters were predicted using antiSMASH. We used the CRISPR/Cas9 mutagenesis system, and each biosynthetic gene was predicted via protein basic local alignment search tool (BLAST) and rapid annotation using subsystems technology (RAST) server. Three gene clusters were shown to exhibit antifungal or anti- bacterial activity, viz. cluster 16 (lasso peptide), cluster 17 (thiopeptide-lantipeptide), and cluster 20 (lanti- peptide). The results of the current study showed that SP6C4 has a variety of antimicrobial activities, and this strain is beneficial in agriculture.