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Genetic Diversity of Cultivable Plant Growth-Promoting Rhizobacteria in Korea

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Genetic Diversity of Cultivable Plant Growth-Promoting Rhizobacteria in Korea J. Microbiol. Biotechnol. (2011), 21(8), 777–790 doi: 10.4014/jmb.1101.01031 First published online 13 June 2011 Genetic Diversity of Cultivable Plant Growth-Promoting Rhizobacteria in Korea Kim, Won-Il1†*, Won Kyong Cho2†, Su-Nam Kim3, Hyosub Chu4, Kyoung-Yul Ryu1, Jong-Chul Yun1, and Chang-Seuk Park5* 1Microbial Safety Division, National Academy of Agricultural Science, Rural Development Administration (RDA), Suwon 441-707, Korea 2Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea 3Organic Agriculture Division, National Academy of Agricultural Science, Rural Development Administration (RDA), Suwon 441-707, Korea 4Bioindustrial Process Center, Jeonbuk Branch Institute of Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeonbuk 580-185, Korea 5Department of Applied Biology and Environmental Sciences, Gyeongsang National University, Jinju 660-701, Korea Received: January 20, 2011 / Revised: May 11, 2011 / Accepted: May 12, 2011 To elucidate the biodiversity of plant growth-promoting Keywords: PGPR, plant growth promotion, diversity, rhizobacteria (PGPR) in Korea, 7,638 bacteria isolated rhizobacteria, cucumber, rhizosphere from the rhizosphere of plant species growing in many different regions were screened. A large number of PGPR were identified by testing the ability of each isolate Plant-microbe interactions may be beneficial or harmful, to promote the growth of cucumber seedlings. After depending on the characteristics of the microbes involved redundant rhizobacteria were removed via amplified and the ways in which they interact with plants. Among rDNA restriction analysis, 90 strains were finally selected such microbes, plant growth-promoting rhizobacteria as PGPR. On the basis of 16S ribosomal RNA sequences, (PGPR) are distributed on plant roots or in the surrounding 68 Gram-positive (76%) and 22 Gram-negative (24%) soil and have beneficial effects on plants [5, 18, 25]. PGPR isolates were assigned to 21 genera and 47 species. Of may promote plant growth, thus providing high crop these genera, Bacillus (32 species) made up the largest yields, and they also function as biocontrol agents against complement, followed by Paenibacillus (19) and Pseudomonas plant diseases caused by phytopathogenic microorganisms (11). Phylogenetic analysis showed that most of the Gram- [18, 24, 25]. PGPR are also important in bioaugmentation- positive PGPR fell into two categories: low- and high- assisted phytoextraction from soils contaminated with G+C (Actinobacteria) strains. The Gram-negative PGPR heavy metals [21]. Moreover, recent studies indicate that were distributed in three categories: α-proteobacteria, β- PGPR are able to boost plant tolerance to abiotic stresses proteobacteria, and γ-proteobacteria. To our knowledge, such as salt and drought [61]. This suite of benefits has led this is the largest screening study designed to isolate to the increasing application of PGPR in arable agriculture. diverse PGPR. The enlarged understanding of PGPR genetic The bacteria can be used to replace chemical fertilizers and diversity provided herein will expand the knowledge base pesticides that are agents of pollution [1, 28]. regarding beneficial plant-microbe interactions. The PGPR produce a wide range of metabolites that regulate outcome of this research may have a practical effect on cell content according to ambient biotic and abiotic stresses crop production methodologies. [56, 61]. For example, some produce hormones such as *Corresponding author indole acetic acids (IAAs), ethylene, and gibberellins that W.-I. Kim enhance plant growth, seed germination, and root growth. Phone: +82-31-290-0444; Fax: +82-31-290-0407; Recently, jasmonate and ethylene production by PGPR E-mail: [email protected] C.-S. Park was demonstrated [56]; these two compounds are involved Phone: +82-55-751-5442; Fax: +82-55-758-5110; in plant defense signaling pathways. Rhizosphere bacteria E-mail: [email protected] are also able to fix nitrogen symbiotically and to solubilize †These authors contributed equally to this work. mineral phosphates and other nutrients [6, 20, 37, 63]. 778 Kim et al. Plant growth promotion by PGPR requires a close result, we were able to compile a comprehensive list of 90 relationship between the bacteria and their host plants. PGPR in 21 genera and 47 species, providing fundamental This interaction may be recognized as rhizospheric or data and expanded knowledge regarding PGPR distribution endophytic [2, 39]. During the PGPR colonization process, and diversity. bacteria first occupy the rhizosphere. Endophytes are then able to enter plant tissues through the root zone, after which they penetrate plant cells, often conferring beneficial MATERIALS AND METHODS effects on hosts [2, 39]. Numerous PGPR strains belonging to several genera have Collection of Rhizobacteria from a Diversity of Plants and been identified in recent decades, including Azotobacter, Locations Arthrobacter, Bacillus, Clostridium, Hydrogenophaga, Roots of various plant species were collected in diverse areas of Korea Enterobacter, Serratia, and Azospirillum [24, 25]. Among (Table 1). Root samples of barley, Chinese cabbage, garlic, green onion, leaf mustard, onion, and weeds were washed (Table 2) and the taxa, Pseudomonas fluorescens has been the best homogenized using a sterile mortar and pestle. Homogenized samples characterized through detailed descriptions of species- were suspended in 0.1 M MgSO4 solution, and serially diluted specific properties [52]. The availability of PGPR genome suspensions were plated on 1/50 tryptic soy broth (TSB; BD Co., sequences will enhance the knowledge base at the genomic, USA). After 3 days of incubation, bacterial colonies grown on 1/50 transcriptomic, and proteomic levels [52]. TSB were selected on the basis of colony morphology, and individual Thus far, large numbers of PGPR have been identified bacterial colonies were stored in TSB medium with 20% glycerol at from soils and rhizospheres of diverse plant species. -72oC. However, information on the distribution and diversity of PGPR is more fragmentary. In this study, we undertook a Screening of Plant Growth-Promoting Bacteria Using a Cucumber Assay large-scale screening of PGPR, the purposes of which Cucumis sativus were to (1) isolate a wide range of rhizobacteria from Cucumber ( L. cv. Nongwoo) plants were grown to the cotyledon stage in a greenhouse. Fifty ml of suspension culture diverse environments and plant species, (2) identify plant 8 prepared for each bacterial isolate (10 cells/ml) was inoculated into growth-promoting bacteria using a cucumber seedling cucumber plant rhizospheres. Suspension cultures of E. coli were assay, and (3) describe PGPR diversity on the basis of 16S also prepared as reference material. Plant growth promotion was rRNA gene sequences and phylogenetic analyses. As a Table 1. Sampling locations for isolation of PGPR in the rhizosphere of various plants in South Korea. Index Location Name Description Latitude Longitude Year A Boseong Reclaimed land, field 34o 46' 17.24'' N 127o 4' 47.62'' O 2004-2006 B Chiak-San High mountain 37o 22' 21.08'' N 128o 3' 1.84'' O 2005 C Deogyu-San High mountain 35o 50' 38.93'' N 127o 44' 35.49'' O 2005 D Gangjin Reclaimed land 34o 38' 31.48'' N 126o 46' 2.14'' O 2005 E Gangneung Forest fire area 37o 45' 6.67'' N 128o 52' 33.81'' O 2004 F Goheung Reclaimed land, field 34o 36' 40.40'' N 127o 17' 5.92'' O 2004, 2006 G Haenam Reclaimed land, field 34o 34' 23.71'' N 126o 35' 56.14'' O 2004, 2006 H Jangheung Reclaimed land 34o 40' 54.07'' N 126o 54' 24.94'' O 2004, 2005 I Jin-Do Field, island 34o 29' 12.74'' N 126o 15' 48.55'' O 2004, 2006 J Jiri-San High mountain 35o 19' 59.33'' N 127o 37' 2.08'' O 2004 K Oenaro-Do Island 34o 26' 59.60'' N 127o 29' 48.51'' O 2004, 2005 L Pohang Forest fire area 36o 1' 8.46'' N 129o 20' 36.53'' O 2004 M Sacheon Reclaimed land 35o 0' 13.60'' N 128o 3' 51.07'' O 2004, 2005 N Samcheok Forest fire area 37o 26' 59.52'' N 129o 9' 54.74'' O 2004 O Seorak-San High mountain 38o 6' 40.78'' N 128o 25' 51.10'' O 2004 P Taebak-San High mountain 37o 5' 44.66'' N 128o 54' 54.86'' O 2004 P Uljin Forest fire area 36o 59' 35.04'' N 129o 24' 1.51'' O 2004 Q Ulleung-Do Island 37o 30' 22.92'' N 130o 51' 25.75'' O 2004, 2006 R Wan-Do Field, island 34o 21' 39.82'' N 126o 45' 21.19'' O 2004, 2006 S Worak-San High mountain 36o 53' 21.49'' N 128o 5' 27.47'' O 2005 T Yangyang Forest fire area 38o 4' 31.41'' N 128o 37' 7.86'' O 2005 U Yokji-Do Island 34o 38' 8.67'' N 128o 14' 57.84'' O 2005 Root samples were collected from diverse plants growing in fields, burned forests, high mountains, islands, and reclaimed lands. We randomly collected among plants with high growth-promoting responses. The majority of the sampling regions are located in the southern part of the Korean peninsula. Table 2. Identification of 90 PGPR and characterization of their plant growth-promoting traits. Phosphate Nitrogen Siderophore Indole production Index Strain Year Accession No. Best matched species Identity Host plant solubilization fixation production (µg/ml) 1 L22 2004 EF672047 Pseudomonas lini 99% Weed + + + 71.60 ± 8.945 2 G157 2004 EF672048 Paenibacillus polymyxa 100% Barley + - + 2.99 ± 3.341 3 Mc07 2004 EF672049 Pseudomonas fluorescens 99% Weed + + + - 4 M45 2004 EF672050 Burkholderia cepacia 100% Weed + + + 19.15 ± 1.907 5 I27 2004 EF672051 Paenibacillus polymyxa 99% Barley - - + - 6 B2-13 2004 EF672052 Bacillus pumilus 100% Green onion -
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