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. BIODIVERSITY OF PLANT GROWTH-PROMOTING RHIZOBACTERIA 779 (µg/ml) Indole production production Siderophore fixation Nitrogen Phosphate solubilization 99% Weed99% Weed99% Barley +99%99% Green onion + Onion + - - + + +99% - + Green onion +99%8.945 ± 71.60 Onion + +99% + + Onion - +99% + +99% Green onion - - 99% Green onion1.761 ± 46.96 -99% Green onion99% Barley + + -99% Barley +99% Barley + -0.850 ± 42.31 98% Barley - -99% Barley - + Chinese cabbage - - + ------7.116 35.80 ± - + -1.509 ± 13.28 - - - + - - - - - + +19.460 ± 65.18 - - 2.756 ± 7.73 - - - 98%98% Weed Barley ------1.949 ± 18.23 0.840 ± 6.14 100% Barley100% Weed100% Green onion +100% +100% Barley -100% Barley -100% Barley100% Barley + - Onion + +100% - Barley + -100% - +3.341 ± 2.99 Onion - -100% -1.907 ± 19.15 0.298 ± 17.237 100% Green onion + + Green onion - + + + + + + + + + + - +0.147 ± 51.94 + - + -0.565 ± 8.25 - -100% -5.048 ± 20.61 Chinese cabbage -0.244 ± 2.56 + - - + + 85.68 ± 9.343 Pseudomonas lini Paenibacillus polymyxa Pseudomonas fluorescens Burkholderia cepacia Paenibacillus polymyxa Bacillus pumilus Pseudomonas fluorescens Pseudomonas fluorescens Paenibacillus polymyxa Paenibacillus polymyxa Pseudomonas fluorescens Paenibacillus polymyxa Pseudomonas putida ananatis Pantoea Pseudomonas putida Acinetobacter calcoaceticus Acinetobacter calcoaceticus cereus Bacillus Paenibacillus polymyxa Paenibacillus polymyxa megateriumBacillus niacini Bacillus Paenibacillus terrae Paenibacillus terrae simplexBacillus Paenibacillus polymyxa Paenibacillus polymyxa Bacillus aquimaris Paenibacillus polymyxa Pseudomonas fluorescens Paenibacillus terrae Bacillus megateriumBacillus Identification of 90 PGPR and characterization of their plant growth-promoting traits. Index Strain Year Accession No. Best matchedspecies Identityplant Host 12 L223 G1574 Mc07 20045 2004 M45 EF672047 6 EF672048 2004 I277 EF672049 2004 B2-138 EF672050 4-39 2004 2004 4-10 EF672051 10 EF672052 43-811 2004 88-7-2 2004 EF690395 12 141-9 EF690397 2004 200413 159-9 EF690398 EF690399 14 2004 A7-10 EF690400 15 2004 B1-9 2004 EF690401 16 B4-11 EF690402 17 2004 B8-22 200418 EF690403 B10-4 EF690409 200419 B11-11 EF690411 200420 CSH5-1 2004 EF690413 21 CSH7-5 2004 EF694696 22 EF694697 CRH11-1 200423 2004 EF694698 CSH12-4 EF694699 24 2004 CSH12-525 EF694700 2004 EH23-526 EF694701 EH27-8 200427 EH44-1 EF694702 200428 EH50-13 EF694703 200429 2004 EH52-3 EF694704 30 EF694705 16-2 2004 35-10 EF694706 2004 2004 EF694707 EF694708 32 M1-4 2005 EF690404 31 LH15-4 2005 EF690396 Table 2. 780 Kim et al. (µg/ml) Indole production production Siderophore fixation Nitrogen Phosphate solubilization 94%98% Weed Weed - + - - - -0.492 ± 9.44 1.318 ± 11.04 99%99% Barley98% Barley98% Barley99% Barley98% Barley -99% Barley -96% Barley -98% Barley -98% Weed - -98% Weed - -99% Weed - -99% Weed - - -99% Weed - - -99% Barley - - -98% Weed -3.008 ± 14.28 + -99% Weed +5.863 ± 17.73 + -99% Weed -7.718 ± 53.23 - -99% Weed - + +96% Weed -2.646 ± 20.04 - +99% Weed + - 15.280 ± 38.20 -99% + Weed - +5.678 ± 9.21 +98% Weed -5.751 ± 28.76 +99% - Weed -0.604 ± 14.85 - -97% Weed - -97% + Weed - +0.779 ± 0.999 +99% Weed - - + -0.323 ± 6.66 99% Weed + +99% Weed -0.000 ± 53.72 - + +99% Weed - - 5.715 ± 18.54 + - Weed - - - +1.511 ± 1.80 9.419 ± 18.22 + - + +2.733 ± 8.85 - - - + -10.247 ± 48.60 + + ------0.528 ± 2.19 - - - 3.111 19.40 ± - +14.490 ± 17.49 - - 0.323 ± 17.55 -1.278 ± 5.10 3.359 ± 57.41 - Microbacterium terrae Microbacterium proteobacterium Methylotrophic Bacillus simplex Bacillus megaterium Bacillus simplex Bacillus drentensis Bacillus simplex Bacillus simplex Bacillus cereus Rhodococcus erythropolis nicotianae Arthrobacter arenosi Viridibacillus Paenibacillus amylolyticus Bacillus acidiceler arenosi Viridibacillus maltophilia Stenotrophomonas Bacillus cereus Paenibacillus telluris Bacillus cereus Bacillus cereus sphaericus Lysinibacillus Paenibacillus castaneae Paenibacillus taichungensis fusiformis Lysinibacillus brevis Brevibacillus Rhodococcus erythropolis Paenibacillus odorifer Bacillus simplex Bacillus weihenstephanensis Bacillus thuringiensis Bacillus simplex Paenibacillus amylolyticus Continued. Index Strain Year Accession No. Best matched species Identity Host plant 63 K10-1 2006 EU095363 64 K10-2 2006 EU095364 3334 M3-435 M4-236 2005 M5-3 EF690433 37 2005 M5-6 EF690405 38 2005 M7-2 EF690406 39 2005 M14-10 EF690434 40 2005 M16-1 2005 EF690407 41 M17-3 EF690435 200542 M18-2 EF690408 200543 M18-3 EF690410 200544 M18-5 EF690412 200545 M18-7 EF690414 200546 M19-5 EF690415 200547 M17-5 EF690416 200548 N2-4 EF690417 200549 N8-3 EF690418 50 2005 NH11-251 EF690419 2005 NH5-1 200552 EF690420 EF690421 NH7-1 200553 M10-6 EF690422 200554 M11-2 EF690423 200555 M11-5 EF690424 200556 M13-7 EF690425 200557 M14-4 EF690426 200558 M9-2 EF690427 200559 M9-3 EF690428 60 2005 NH12-2 EF690429 61 2005 NH14-1 2005 EF690430 62 EF690431 K3-2 2005 EF690432 K8-4 2006 EU095361 2006 EU095362 Table 2. BIODIVERSITY OF PLANT GROWTH-PROMOTING RHIZOBACTERIA 781 , - (µg/ml) Indole production production Siderophore fixation Nitrogen nt growth-promoting functions of the selected PGPR, phosphate ere deposited in the National Center for Biotechnology Information Biotechnology for Center National the in deposited ere Phosphate solubilization thods. The presence and absence of a particular trait is indicated by + and 99%99% Weed99% Weed99% Weed95% Weed96% Weed +98% Weed -98% Weed +98% Weed + +98% Weed + +99% Weed + -99% Weed - - Weed - + -99% - - -99% Weed + + -99% Weed - + -0.215 35.25 ± 99% Weed - -1.504 ± 22.95 + +99% Weed - 12.899 37.46 ± +99% Weed - - +99% Garlic - + +99% Leaf mustard - - - +98% Leaf mustard - + +98% Leaf mustard - - 10.198 73.80 ± + + -98% Onion -4.302 ± 20.00 - + + +99% Green onion 10.198 73.80 ± + -99% Barley -2.337 ± 14.39 - - Barley - - - - - +2.355 ± 5.19 - -2.563 ± 12.88 + + - - - + + +8.839 22.26 ± - -0.049 19.73 ± - - - - + - - - + - - - 8.273 17.445 ± 0.77 ± 11.479 100% Weed + + -2.657 ± 5.14 Bacillus weihenstephanensis Ensifer adhaerens nicotinovorans Arthrobacter Bacillus megaterium oxydans Microbacterium Bacillus luciferensis koreensis Variovorax Curtobacterium flaccumfaciens Pseudomonas fluorescens Pseudomonas tolaasii Rahnella aquatilis macmurdoensis Sporosarcina Bacillus pumilus Bacillus aquimaris foliorum Microbacterium Acetobacter pasteurianus Bacillus megaterium Bacillus cereus Bacillus megaterium niigatensis Arthrobacter Bacillus cereus oxydans Microbacterium Pseudomonas synxantha Serratia proteamaculans Bacillus cereus nitroguajacolicus Arthrobacter Continued. Index Strain Year Accession No. Best matchedspecies Identityplant Host 6566 K10-367 K10-6 200668 K16-6 EU095365 200669 K16-8 EU095366 200670 K18-7 EU095367 200671 K21-5 EU102268 200672 K26-1 EU102269 200673 K26-5 EU102270 200674 K30-2 EU102271 200675 K31-3 EU102272 200676 K32-6 EU102273 200677 K33-7 EU102274 200678 CN1-3 EU102275 200679 CN1-16 EU102276 200680 CN2-6 2006 EU102277 81 EU102278 CN4-5 200682 CN6-3 EU102279 200683 JPH20-2 EU102280 200684 2006 Q7-2 EU102281 EU102282 85 Q17-12 200686 Q23-15 2006 EU102283 87 Q34-4 EU102284 200688 Q40-14 EU104731 200689 Q42-3 2006 EU104732 90 Q48-1 EU104733 2006 Q49-2 EU104734 2006 EU104735 2006 EU104736 Table 2. PGPR w selected 90 the of sequences The determined. were PGPR of each sequences rRNA 16S partial species, bacterial identify To (NCBI)’s GenBank. Collection year, species names, sequence accession number, and host plant are listed. To characterize the pla the characterize To listed. are plant host and number, accession names, sequence species year, Collection GenBank. (NCBI)’s solubilization, nitrogen fixation, siderophore production, and indole production were examined as described in Materials and Me respectively. Values are the means of three replicates ± standard deviation. deviation. ± standard replicates means of three the are Values respectively. 782 Kim et al. measured 2 weeks after bacterial inoculation. After three independent 2.5 mM 1:1 mixed dNTPs, 5 µl of 10× Taq DNA polymerase experiments, plant growth-promoting bacteria were selected. buffer, 1 unit of Taq DNA polymerase (1 U/µl) (Takara, Shiga, Japan), and 34 µl of sterile water. PCR was performed as follows: Statistical Analysis 94oC for 5 min, 32 cycles of 94oC for 1 min, 55oC for 1 min, and The obtained results were subjected to analysis of variance (ANOVA) 72oC for 1 min, and 72oC for 10 min. Amplified products were using SAS software (SAS Institute, Inc., Cary, NC, USA). The electrophoretically separated and visualized on 0.8% agarose gels significance of the effect of bacterial treatment was determined stained with ethidium bromide. Bands of interest were excised from according to the magnitude of the F-value at P=0.05. When a gels and purified with a QIAquick Gel Extraction Kit (QIAGEN, significant F-value was obtained, a separation of the means was Hilden, Germany). Finally, purified products were ligated into the accomplished using Fisher’s protected least significant difference pGEM-T Easy Vector (Promega, Madison, WI, USA) and ligation (LSD) at P=0.05. products were transformed into E. coli DH5α competent cells. White transformed colonies were randomly selected and bacterial Determination of Plant Growth-Promoting Traits of 90 Selected plasmids were extracted with a QIAprep Spin Miniprep Kit (QIAGEN). PGPR Sequencing was done by Macrogen Co., Ltd. (Seoul, Korea). To test mineral phosphate solubilization activity, 5 µl of individual PGPR was spotted onto Pikovskaya’s agar medium [32] with 0.5% Nucleotide Sequence Accession Numbers tricalcium phosphate (TCP) as the inorganic phosphate source. The The 16S rRNA gene sequences of all 90 PGPR were deposited in plates were incubated at 30oC for 4 days and TCP solubilization was the National Center for Biotechnology Information (NCBI) nucleotide characterized by the presence of a clear zone around the bacterial sequence database under the accession numbers EF672047-EF672052, colony. The ability to fix nitrogen was evaluated using semi-solid EF690395-EF690435, EF694696-EF694708, EU095361-EU095367, nitrogen-free NFb medium [10]. The presence of a characteristic EU102268-EU102284, and EU104731-EU104736. pellicle formation within the medium indicated nitrogen fixation. Siderophore synthesis was investigated by following the method Phylogenetic Analysis described by Schwyn and Neilands [45]. Ten µl of bacterial Full 16S rRNA gene sequences were compiled after sequencing suspension was spotted on CAS plates. Development of a deep yellow with SeqMan software (DNASTAR Inc., Madison, WI, USA) and to orange color was indicative of a positive result for siderophore 16S rRNA gene sequences of test strains were edited with BioEdit production. software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Multiple Production of indoles in the culture supernatant was measured alignments were performed with ClustalW software (http://www.ebi.ac.uk/ after incubation of PGPR for 4 days at 30oC in liquid DF minimal clustalw/). Each sequence type was compared by BLAST search salts medium [11] supplemented with 100 µg/ml tryptophan. A 5 ml with those available in GenBank (http://www.ncbi.nlm.nih.gov) to portion of the culture was centrifuged at 8,000 ×g for 15 min and determine approximate phylogenetic affiliations. Evolutionary distances 2 ml of supernatant was transferred to a fresh tube to which 100 µl were calculated using the Kimura two-parameter model. The of 10 mM orthophosphoric acid and 4 ml of Salkowski reagent phylogenic tree was constructed with a neighbor-joining method (1 ml of 0.5 M FeCl3 in 50 ml of 35% HClO4) were added. The using MEGA 4.0 software [53]. mixture was incubated at room temperature for 25 min and the absorbance of the resulting pink solution was read at 530 nm on a UV spectrophotometer (UV-1601, Shimadzu, Japan). The IAA concentration RESULTS in the culture was determined by comparison with a calibration curve constructed using pure IAA as a standard [12]. Isolation and Selection of PGPR from Diverse Amplified Ribosomal DNA Restriction Analysis (ARDRA) Rhizobacteria PCR products amplified with the eubacterial primers 27f and 1492r To isolate PGPR, we collected samples, mostly from were double-digested with HaeIII and HhaI enzymes (New England rhizospheres of plants, with high growth-promoting capabilities. Biolabs, Hitchin, UK). Restriction fragment length polymorphism (RFLP) To increase the diversity of this group, we collected patterns were analyzed on 2.5% MetaPhore agarose (BioWhittaker, samples from a variety of plants and geographical regions. Walkersville, USA) gels with a 100 bp ladder (New England Biolabs). The samples were collected in the period 2004-2006 in Gels were photographed under UV illumination. Redundant rhizobacteria South Korea. Detailed information on sampling locations, were removed from the dataset based on their RFLP patterns. which included fields, burned forests, high mountains, islands, and reclaimed lands, is presented in Table 1. We DNA Extraction, PCR Amplification, Cloning, and DNA isolated 7,638 native rhizobacteria that were divided into Sequencing three groups by collection year: group A, containing 3,577 Crude genomic DNA from each isolate was prepared by boiling a isolates collected in 2004; group B, containing 1,921 small quantity of cell material extracted from colonies in 100 µl of 5% Chelex 100 solution (BioRad, Hercules, CA, USA) for 10 min. isolates collected in 2005; and group C, containing 2,143 Preparations were then centrifuged at 12,000 ×g for 2 min. The isolates in 2006 (Fig. 1). supernatant was used as the template for PCR. Amplification of the 16S rRNA gene (~1,500 base-pair product) was performed using the Selection of Growth-Promoting Rhizobacteria eubacterial primers 27f and 1492r [33]. The amplification reaction We used the cucumber plant for our assays since it included 10 pmol of each primer, 2 µl of genomic DNA, 4 µl of provides an ideal model for screening PGPR; relevant BIODIVERSITY OF PLANT GROWTH-PROMOTING RHIZOBACTERIA 783

selected for further analysis. To increase the sensitivity and accuracy of PGPR selection, we used three screening steps (Fig. 1). In each year of the study, approximately one thousand bacterial isolates were selected in the first screening on the basis of high plant growth promotion as compared with the control after inoculation of all isolates on cucumber seedlings. For the second and third screenings, selected isolates were again inoculated on cucumber seedlings. After all of the screening steps were complete Fig. 1. Three-step screening procedure for selection of the most and ARDRA was performed to remove redundant strains, effective PGPR. 30 potential PGPR were finally selected in each of the A total of 7,638 rhizobacteria were isolated from various rhizosphere three years of the study for a total of 90 rhizobacteria that regions in South Korea in the period 2004–2006. Plant growth-promoting greatly enhanced plant growth compared with control abilities of individual isolates were examined in three intensive screening E. coli steps. A total of 90 rhizobacteria were categorized as PGPR. plants (treated with or untreated) (Fig. 2). The plant growth-promoting rates of the selected strains based on plant fresh weight ranged from 10% to 40% compared attributes for this purpose include its good germination with control plants (data not shown). rate, fast growth, and high yield [62]. In total, 7,638 bacterial isolates were individually inoculated into cucumber Evaluation of Plant Growth-Promoting Ability of the seedlings. Only those rhizobacteria with a high growth- Selected PGPR promoting capacity in comparison with controls were We determined the treatment effects of the 90 selected PGPR in detail by measuring plant responses in terms of plant height, number, length and width of leaves, stem diameter, and fresh weight (data not shown). Although all of the PGPR were selected for their effects on plant fresh weight, there was no correlation among the six morphological traits. For example, only 59% of the identified PGPR increased cucumber plant height compared with controls. Treatment with nine of the selected PGPR had negative effects on cucumber height. Stem diameter was not altered significantly in comparison with fresh weight by most of the PGPR. Of the 90 selected PGPR, 11 increased plant fresh weight by at least 150% of that of controls. We selected 20 representative PGPR that remarkably enhanced plant growth in terms of height, stem diameter, and fresh weight (Table 3). Among these strains, the three strains 141-9, LH15-4, and K26-1 increased plant height by more than 20% of that of controls (Table 3). Treatment with strain B10-4 increased stem diameter significantly, as did K10-1 and Q7-2. Seven other strains increased stem diameter by >15% of that of controls (Table 3). Strains B10-4, LH15-4, and NH5-1 dramatically increased the fresh weight of cucumber seedlings by at least 50% (Table 3). Fig. 2 presents examples of growth promotion in cucumber seedling after treatment with 10 selected PGPR. Half of the 90 selected PGPR were isolated from various weeds, whereas the remaining strains were found in association with barley (24 PGPR), green onion (9), onion (6), leaf mustard (3), Chinese cabbage (2), and garlic (1) (Table 2). Based on this result, it seems that barley might be a favorable host plant for PGPR. Although we collected plant samples Fig. 2. Cucumber plant growth promotion by selected PGPR. Suspension cultures of individual PGPR or Escherichia coli were inoculated growing under various environmental conditions, we could into the rhizospheres of cucumber plants. Controls are plants without not find any correlation between the identity of isolates bacterial treatment. Images were captured 30 days after treatment. and soil types (data not shown). 784 Kim et al. LSD Standard Standard deviation ait are listed. Statistical tests including ANOVA and LSD were LSD and ANOVA tests including Statistical listed. ait are value Mean LSD Standard Standard deviation value Mean LSD Plant height (cm) diameter Stem (mm) fresh Plant weight (g) Standard Standard deviation value Mean 43-888-7-2ABCDEFG 1.04 21.48 141-9ABC 0.80 22.14 B1-9A 22.66 0.77 ABCDEFGHI 0.11 4.66 B10-4ABCDEF 1.31 21.60 CSH5-1ABCDEFG 1.38 21.40 ABCDEFGHIJKLMNOPQRSTUVW 0.32 4.30 20.94 1.26 CSH7-5BCDEFGHIJ 1.19 20.64 LH15-4GHIJKLMNOPQRSTU 3.42 0.18 4.36 3.67 0.58 CDEFGHIJKLM 0.17 ABABCDEFGHIJK 0.34 0.18 ABCDEF 4.90 4.62 LMNOPQRSTUVW 0.30 4.26 ABM16-1 1.43 22.52 3.43 0.36 CDEFGHIJKLMNO CDEFGHIJKLM 0.24 4.54 M17-3ABCDEFG 1.21 21.46 3.06 0.42 JKLMNOPQRSTUVWXYZA M19-5ABC 22.02 1.26 3.50 0.25 ABCDEFGHIJK 3.51 0.42 ABCDEFGHIJK NH5-1ABC 22.20 1.24 CDEFGHIJKLMNO 0.31 4.54 ABCDEFGHIJKLMM9-2 0.23 4.58 ABCDE 21.64 1.79 K3-2 3.97 3.31 ABCDEFG 0.36 1.34 21.44 0.72 A EFGHIJKLMNOPQ ABCDEFGHI0.18 4.66 3.84 K10-1 0.41 ABC BCDEFGHIJ 20.68 2.16 JKLMNOPQRSTUVW0.24 4.30 K10-2ABCDEFG0.07 4.70 ABCDEFGHI 20.98 0.77 ABCDEF 0.28 4.74 K10-3 3.64 ABCDEFG 21.50 1.74 0.58 ABCDEFG0.20 ABCDEFG 4.70 ABCDE0.26 K26-1 4.82 ABCDEFGH 3.25 1.24 21.18 0.55 FGHIJKLMNOPQRS Q7-2A 22.68 1.54 FGHIJKLMNOPQRS0.23 4.40 Q49-2 3.56 STUVWXYZA 0.19 4.08 0.24 ABCDEFGHIJ BCDEFGHIJ 1.06 20.66 Control 3.82 ABCDEFGHI 3.62 0.22 21.08 0.77 ABCD 0.25 3.62 ABCDEFGH 0.54 ABCDEFGH KLMNOPQRSTU 18.60 0.70 A 0.33 4.92 3.60 OPQRSTUVWXYZ0.34 4.20 0.40 ABCDEFGHI JKLMNOPQRSTUVW0.31 4.30 3.37 0.35 CDEFGHIJKLMN GHIJKLMNOPQRSTU0.18 4.36 3.36 0.43 CDEFGHIJKLMN 2.42 0.52 3.79 FG 0.14 ABCDE 3.38 0.58 CDEFGHIJKLMN Strain Table 3. Plant growth promotionTable by twenty selected PGPR based on three parameters. screen PGPR,To we used cucumber seedlings. The mean values and standard deviation of five replicates for each morphological tr performed for plant height, stem diameter, and plant fresh weight. BIODIVERSITY OF PLANT GROWTH-PROMOTING RHIZOBACTERIA 785

Phylogenetic Relationships of the Selected PGPR For the phylogenetic analysis, we first divided the 90 PGPR into two groups, namely, 68 Gram-positive and 22 Gram-negative bacteria, on the basis of the BLAST results (Table 2). The 16S rRNA sequences of these PGPRs and taxonomically similar bacterial strains were aligned (Fig. 4). For phylogenetic analysis of Gram-positive PGPR, the 16S rRNA sequences of 11 Gram-positive bacteria were obtained from GenBank: Bacillus cereus CWBI B1432 (EU128486), B. simplex OSS 24 (EU124559), B. megaterium B15 (EU169176), B. sphaericus KSC SF3b (DQ870695), Sporosarcina globisporus KFC-52 (EF459543), Brevibacillus formosus LMG 16101 (AF378234), Paenibacillus polymyxa GS01 (DQ365577), Rhodococcus erythropolis H212 Fig. 3. Distribution of the number of PGPR showing plant- (EF204442), Arthrobacter nitroguajacolicus CCM4924T, growth promoting traits. The Venn diagram illustrates the number of PGPR showing the PGP traits Curtobacterium albidum DSM 20512T, and Microbacterium phosphate solubilization, nitrogen fixation, siderophore production, and lacticum DSM 20427 (Fig. 4A). Many of the selected indole production. PGPR were identified as Bacillus cereus, including NH11- 2 and NH5-1. Strain 43-8 had high homology with Functional Characterization of Selected PGPR Associated Paenibacillus polymyxa. Four PGPR strains, M17-3, Q49- with Plant Growth-Promoting Traits 2, K26-5, and K10-1, were identified as Rhodococcus In order to characterize the functions of the selected PGPR, erythropolis, Arthrobacter nitroguajacolicus, Curtobacterium we examined four different plant growth-promoting traits flaccumfaciens, and Microbacterium terrae, respectively. in vitro: phosphate solubilization, nitrogen fixation, For the phylogenetic analysis of Gram-negative bacteria, siderophore production, and indole production. Of the 90 we obtained 16S rRNA sequences of eight Gram-negative PGPR selected, 83 have at least one trait related to plant bacteria from GenBank: Pseudomonas fluorescens SCAM growth promotion (Fig. 3 and Table 2). For instance, a BA 1 (AM900685), Acinetobacter calcoaceticus AGL 17 majority of the isolated PGPR can solubilize phosphate (50 (EU121781), Enterobacteriaceae bacterium A46 (AB081580), strains) and produce indole (55). In addition, 36 PGPR Pantoea ananatis BD 622 (DQ195523), Stenotrophomonas produce siderophores, whereas 29 are able to fix nitrogen. maltophilia ISSDS-50 (EF620453), Variovorax koreensis Fifty-one of the selected PGPR were identified as having GH 9-3 (DQ432053), Methylotrophic proteobacterium loc440 multiple plant growth-promoting traits. Interestingly, nine (AF250404), and Ensifer adhaerens CCBAU 01077 of them are capable of solubilizing phosphate, fixing (EU170545) (Fig. 4B). Six of the PGPR, Mc07, 4-3, 4-10, nitrogen, and producing siderophores and indole: L22 141-9, 35-10, and K30-2, were identified as Pseudomonas (Pseudomonas lini), M45 (Burkholderia cepacia), 4-10 fluorescens. Two strains, B10-4 and CN4-5, were closely (Pseudomonas fluorescens), B1-9 (Pantoea ananatis), B4-11 related to Acinetobacter calcoaceticus and Acetobacter (Pseudomonas putida), M17-5 (Stenotrophomonas maltophilia), pasteurianus, respectively. In addition, three proteobacteria, M13-7 (Brevibacillus brevis), 35-10 (Pseudomonas fluorescens), K26-1, K10-2, and K10-6, were homologous with Variovorax and K31-3 (Pseudomonas tolaasii). koreensis, Methylotrophic proteobacterium, and Ensifer adhaerens, respectively. Identification of Selected PGPR on the Basis of Partial 16S rRNA Sequences Genetic Diversity of the Selected PGPR To identify the 90 selected PGPR, we performed colony BLAST searches and phylogenetic analysis showed that PCR using primers specific for 16S rRNA sequences. Each the 90 selected PGPR belonged to 21 different genera amplified PCR product was cloned and used for sequencing. (Table 2). The largest generic grouping (32 PGPR) belonged The 16S ribosomal RNA sequence of each of the selected to Bacillus (Table 2), whereas 19 strains belonged to PGPRs was deposited in GenBank under a unique accession Paenibacillus and 11 to Pseudomonas. We identified a number (Table 2). To determine the genetic identity of range of other genera among the PGPR: Acetobacter, individual PGPR, we performed BLASTN searches on the Arthrobacter, Brevibacillus, Burkholderia, Curtobacterium, obtained sequences against the nucleotide database. Ninety Ensifer, Lysinibacillus, Methylotrophic, Microbacterium, PGPR were identified to the genus level by this procedure. Rahnella, Rhodococcus, Sporosarcina, Variovorax, and For phylogenetic analysis, the 16S rRNA sequences of Viridibacillus. A large proportion (63%) of the PGPR bacterial species closely related to the 90 selected PGPR formed a cluster of low G+C Gram-positive bacteria; γ- were retrieved from the GenBank database. proteobacteria (17%) and actinobacteria (12%) were also 786 Kim et al.

Fig. 4. Phylogenetic relationship of 90 identified PGPR. The 90 identified PGPR were categorized into two groups, (A) Gram-positive (68 PGPR) and (B) Gram-negative (22 PGPR), on the basis of partial 16S rRNA gene sequences. 16S rRNA sequences of PGPR and closely related bacteria were aligned using ClustalW software with manual modification, and a phylogenetic tree was constructed with a neighbor-joining algorithm using MEGA4 software. One thousand bootstrap replicates were performed. Methanosarcina barkeri DSM800T was used as an outgroup. The scale bar is equivalent to 0.05 changes per nucleotide position. well represented (Fig. 4 and Table 2). Alpha- and β- species: P. amylolyticus, P. castaneae, P. odorifer, P. proteobacteria made up the smallest groups, each containing polymyxa, P. taichungensis, P. telluris, and P. terrae. one PGPR. The majority of Gram-positive PGPR fell into two groups depending on their G+C ratios. For instance, Bacillus, Sporosarcina, Brevibacillus, and Paenibacillus are DISCUSSION proteobacteria with low G+C ratios. In contrast, Rhodococcus, Arthrobacter, Curtobacterium, and Microbacterium form a The composition and colonization patterns of rhizobacteria group of actinobacteria with high G+C ratios. closely associated with plant hosts may vary by plant In detail, each PGPR genus could be further divided into species and environmental conditions, including soil type, many species. For example, PGPR in the genus Bacillus climate, humidity, and temperature. Recently, a wide range included 11 species: B. acidiceler, B. aquimaris, B. cereus, of PGPR was identified from various environmental B. drentensis, B. luciferensis, B. megaterium, B. niacin, B. conditions and plant species [5, 25]. However, in many pumilus, B. simplex, B. thuringiensis, and B. weihenstephanensis. cases, PGPR have been identified from single plant species In addition, PGPR in the genus Paenibacillus included 7 or under a limited set of environmental conditions. With BIODIVERSITY OF PLANT GROWTH-PROMOTING RHIZOBACTERIA 787 such restricted sampling, PGPR have often been assigned resistance to cucumber mosaic virus in tomato and to a small range of bacterial genera whose growth- Arabidopsis plants [29, 40]. It is obvious that members of promoting capabilities were tested on a small number of the genus Bacillus are not the dominant bacteria in the plant species. For example, a previous study identified rhizosphere of various plants; however, the proportion of several PGPR in samples restricted to the rhizosphere soil PGPR belonging to Bacillus in this study (32/90) suggests of wheat plants [17]. PGPR have occasionally been they are highly competent in the rhizosphere compared screened under high salinity and water stress [23, 31]. Our with those of other genera. Although previous studies approach was much broader as we aimed to identify the reported numerous PGPR belonging to the genus Bacillus, greatest diversity of PGPR. To do this, we sampled across they were mostly restricted to certain species. The three a range of environmental conditions such as burned Bacillus species identified in our study, B. cereus (8 forests, high mountains, and reclaimed lands. We collected PGPR), B. simplex (7), and B. megaterium (6), were the from a range of host plants, including wild weed species. most representative PGPR. However, we identified an Our 3-year sampling program produced 7,638 isolates. It is additional eight Bacillus species: B. aquimaris, B. pumilus, noteworthy that the culture medium and conditions play B. weihenstephanensis, B. acidiceler, B. drentensis, B. important roles in the isolation of rhizobacteria. In our luciferensis, B. niacin, and B. thuringiensis. These results study, we used TSB as the selection medium. If we had indicate the novelty of our study, revealing a high diversity used another culture medium instead, the identity of the of Bacillus PGPR species. isolated rhizobacteria might have been different. Finally, Paenibacillus was the second largest PGPR group in we used a three-step screening process to identify 90 this study. The 19 isolates in this genus had high plant PGPR that enhanced the growth of cucumber seedlings. growth-promoting properties. The genetic diversity of The proportion of PGPR among the isolated rhizobacteria PGPR in this genus was not as high as those in Bacillus. was only 1.1%, indicating the rigor of our screening steps. However, a total of seven Paenibacillus species were Surprisingly, the 90 PGPR isolated had a high genetic identified. Among them, P. polymyxa (10 PGPR) was the diversity with 21 genera and 47 species, a result which most dominant followed by P. te rr ae (3) and P. amylolyticus may hold great promise for future applications in agriculture. (2). In particular, the growth-promoting capacity of P. It might be interesting to see the relationship between polymyxa has been intensively characterized in various the diversity of PGPR and host plants. Half of the PGPR in plant species [7, 8, 41]. In addition, four other species, P. this study were identified from the rhizosphere of different castaneae, P. odorifer, P. taichungensis, and P. telluris, weeds. However, we could not present the species name were identified; these species might be mostly unreported of each weed plant owing to the large sampling size. PGPR. The most interesting point is that many Paenibacillus Interestingly, the majority of the PGPR derived from species can play a key role in biocontrol by producing weeds were determined to be members of Bacillus (16 peptide antibiotics, such as polymyxins and mattacin, that PGPR), Paenibacillus (6), and Pseudomonas (4), suggesting are antagonistic factors [27, 47]. Moreover, many Paenibacillus that these three genera represent the dominant PGPR in the strains are capable of metabolizing starch, which may be rhizosphere of various weed plants. In addition, we also significant in the establishment of rhizobacteria in the identified 24 PGPR from barley that could largely be vicinity of plant roots [46]. Overall, we propose that the divided into the two groups Bacillus (10 PGPR) and identified Paenibacillus species are one group of beneficial Paenibacillus (9). Furthermore, a recent study by real-time rhizobacteria that could be applied in the field owing to PCR confirmed that a large number of Paenibacillus their abilities as PGPR and biocontrol agents. polymyxa cells were present in the rhizosphere of wild A third group of PGPR belonged to the genus barley [54]. Based on such data, we propose that members Pseudomonas. Among the known PGPR, those belonging of Paenibacillus may be dominant PGPR, especially in to Pseudomonas are probably the best characterized. association with barley plants. Several taxa of Pseudomonas increase the growth of maize, Among the isolates obtained in this study, the genera asparagus, bean, wheat, pepper, Catharanthus roseus, and most often identified were Bacillus, Paenibacillus, and tomato [4, 14, 15, 23, 30, 48, 59]. Of the Pseudomonas Pseudomonas. Members of Bacillus are ubiquitous bacteria species identified as PGPR in this study, P. fluorescens that include both free-living PGPR and pathogenic species. strains (6 PGPR) were frequently identified. Furthermore, Hence, it was not surprising that we identified Bacillus with the exception of P. putida, four Pseudomonas species, PGPR in most of the plants sampled, which included P. putida, P. l ini , P. synxantha, and P. tolaasii, were identified weeds, barley, onion, green onion, leaf mustard, Chinese as PGPR for the first time herein, lending significance to cabbage, and garlic (Table 2). PGPR belonging to Bacillus our study. Interestingly, many previous studies have shown have been reported to enhance the growth of several plants that Pseudomonas PGPR are highly resistant to various such as Pinus, wheat, tomato, sugar beet, sorghum, peanut, environmental stresses. For instance, the effects of the and onion [7, 34, 60]. Several Bacillus strains also induce PGPR Pseudomonas fluorescens MSP-393 are not altered 788 Kim et al. in highly saline soils such as those on coastlines [31]. cadmium toxicity [26]. Recently, a PGPR in the genus Furthermore, production of 1-aminocyclopropane-1-carboxylic Pantoea was shown to promote the growth of pepper acid (ACC) deaminase by Pseudomonas fluorescens plants [15]. PGPR in the genus Serratia are able to increases the resistance of plants to salt stress [44]. A produce IAA and siderophores [19]. They are able to recent study also subjected diverse Pseudomonas species increase growth in Zea mays, especially in heavy-metal- to osmotic stress, demonstrating that stress-adaptable contaminated soil [19]. In addition, the isolate Serratia PGPR are good candidates for selecting stress-tolerant plymuthica A21-4 increases the efficacy of biocontrol strains [43]. Therefore, it might be interesting to test the against Phytophthora blight of pepper [49]. Although plant resistance of the PGPR identified in this study to various growth promotion by Sporosarcina has not yet been environmental stresses in the future. reported, it is able to produce glucan by converting L- Besides members of these three major genera, the arabinose [64]. One member of the genus Variovorax remaining PGPR belonged to 24 species in 18 genera. containing ACC deaminase increases the growth and yield Such PGPR biodiversity could be due to the large number of pea by reducing abscisic acid-mediated signaling [3]. of locations and plants sampled. However, had we only Taking all of these diverse PGPR characteristics into collected plant samples from fields, it is possible that a account, it is clear that the 90 identified isolates have great similarly diverse set of PGPR would have been revealed. potential for future research. Previous studies of closely Our efforts to study the biodiversity of PGPR may provide related PGPR species show that these bacteria play critical information to help distinguish the proper PGPR for roles not only in plant growth promotion but also in certain purposes. biological control, biofertilization, and phytoremediation. Other PGPR belonged to the genera Arthrobacter, Hence, future studies should focus on the functional Burkholderia, and Microbacterium. Arthrobacter taxa are characterization of PGPR for practical applications in the usually found in soil, and some function in bioremediation. field. Growth promotion in diverse field-grown plants, For instance, Arthrobacter chlorophenolicus has the ability epiphytic and endophytic colonization abilities, and metabolite to degrade high concentrations of 4-chlorophenol, which is production should be examined in these PGPR. Genome- a recalcitrant toxic compound in contaminated soils [58]. wide approaches such as transcriptomics, proteomics, Several Burkholderia species promote the growth of genomics, and metabolomics will facilitate elucidation of potatoes, vegetables, grapes, and wheat by producing ACC the functional interactions between plants and the PGPR deaminase [9, 48]. Some Microbacterium species function identified in this study. in phytoextraction by assimilating heavy metals such as Pb and Ni; they also increase the growth of apple and rape [16, 50]. Acknowledgments PGPR members of the genus Acinetobacter promote wheat, pea, chickpea, maize, and barley production through This study was carried out with the support of the nitrogen fixation, siderophore production, and mineral “Cooperative Research Program for Agricultural Science solubilization [13, 42]. The genus Stenotrophomonas consists and Technology Development (Project No. PJ007612), of at least eight species that are strongly associated with Rural Development Administration, Republic of Korea to plants. Among them, the endophyte Stenotrophomonas W.I.K. and the National Research Foundation of Korea maltophilia promotes plant growth and plays roles in (NRF) grant funded by the Korea government (MEST) bioremediation and phytoremediation [38]. Moreover, (No. 2011-0010027) to W.K.C. members of the genus Rhodococcus are able to reduce chromate in metal-contaminated soil and promote plant REFERENCES growth at low temperatures when the bacteria are endophytic [35, 55]. One member of the genus Brevibacillus has 1. Adesemoye, A. O. and J. W. Kloepper. 2009. Plant-microbes strong symbiotic interactions with arbuscular mycorrhizal interactions in enhanced fertilizer-use efficiency. Appl. Microbiol. fungi that promote plant growth in lead-contaminated soil Biotechnol. 85: 1-12. [57]. A bacterial strain belonging to Curtobacterium and 2. Barea, J. M., M. J. Pozo, R. Azcon, and C. Azcon-Aguilar. other PGPR control multiple cucumber pathogens [36]. 2005. Microbial co-operation in the rhizosphere. J. Exp. Bot. 56: - Members of Ensifer occur as legume root-nodule bacteria 1761 1778. 3. Belimov, A. A., I. C. Dodd, N. Hontzeas, J. C. Theobald, V. I. that function in a nitrogen-fixing symbiosis [22]. The Safronova, and W. J. Davies. 2009. Rhizosphere bacteria containing family Enterobacteriaceae contains PGPR members with 1-aminocyclopropane-1-carboxylate deaminase increase yield of diverse abilities, including nitrogenase activity, phosphate plants grown in drying soil via both local and systemic solubilization, IAA production, and antifungal activity hormone signalling. New Phytol. 181: 413-423. against Fusarium solani [51]. Methylotrophic species 4. Belimov, A. A., I. C. Dodd, V. I. Safronova, N. Hontzeas, and promote the growth of tomato while reducing nickel and W. J. Davies. 2007. Pseudomonas brassicacearum strain Am3 BIODIVERSITY OF PLANT GROWTH-PROMOTING RHIZOBACTERIA 789

containing 1-aminocyclopropane-1-carboxylate deaminase can 20. Kuklinsky-Sobral, J., W. L. Araujo, R. Mendes, I. O. Geraldi, show both pathogenic and growth-promoting properties in its A. A. Pizzirani-Kleiner, and J. L. Azevedo. 2004. Isolation and interaction with tomato. J. Exp. Bot. 58: 1485-1495. characterization of soybean-associated bacteria and their potential 5. Bloemberg, G. V. and B. J. Lugtenberg. 2001. Molecular basis for plant growth promotion. Environ. Microbiol. 6: 1244-1251. of plant growth promotion and biocontrol by rhizobacteria. 21. Lebeau, T., A. Braud, and K. Jézéquel. 2008. Performance of Curr. Opin. Plant Biol. 4: 343-350. bioaugmentation-assisted phytoextraction applied to metal 6. Caballero-Mellado, J., J. Onofre-Lemus, P. Estrada-de Los contaminated soils: A review. Environ. Pollut. 153: 497-522. Santos, and L. Martinez-Aguilar. 2007. The tomato rhizosphere, 22. Leon-Barrios, M., M. J. Lorite, J. Donate-Correa, and J. Sanjuan. an environment rich in nitrogen-fixing Burkholderia species 2009. Ensifer meliloti bv. lancerottense establishes nitrogen- with capabilities of interest for agriculture and bioremediation. fixing symbiosis with Lotus endemic to the Canary Islands and Appl. Environ. Microbiol. 73: 5308-5319. shows distinctive symbiotic genotypes and host range. Syst. 7. Caceres, E. A. R., G. G. Anta, J. R. Lopex, C. A. Di Ciocco, J. Appl. Microbiol. 32: 413-420. C. P. Basurco, and J. L. Parada. 1996. Response of field-grown 23. Liddycoat, S. M., B. M. Greenberg, and D. J. Wolyn. 2009. The wheat to inoculation with Azospirillum brasilense and Bacillus effect of plant growth-promoting rhizobacteria on asparagus polymyxa in the semiarid region of Argentina. Arid Soil Res. seedlings and germinating seeds subjected to water stress under Rehab. 10: 13-20. greenhouse conditions. Can. J. Microbiol. 55: 388-394. 8. Choi, O., J. Kim, C. Ryu, and C. S. Park. 2004. Colonization 24. Lucy, M., E. Reed, and B. R. Glick. 2004. Applications of free and population changes of a biocontrol agent, Paenibacillus living plant growth-promoting rhizobacteria. Antonie Van polymyxa E681, in seeds and roots. Plant Pathol. J. 20: 97-102. Leeuwenhoek 86: 1-25. 9. Compant, S., B. Reiter, A. Sessitsch, J. Nowak, C. Clement, and 25. Lugtenberg, B. and F. Kamilova. 2009. Plant-growth-promoting E. Ait Barka. 2005. Endophytic colonization of Vitis vinifera L. rhizobacteria. Annu. Rev. Microbiol. 63: 541-556. by plant growth-promoting bacterium Burkholderia sp. strain 26. Madhaiyan, M., S. Poonguzhali, and T. Sa. 2007. Metal PsJN. Appl. Environ. Microbiol. 71: 1685-1693. tolerating methylotrophic bacteria reduces nickel and cadmium 10. Dobereiner, J., S. Urquiaga, and R. M. Boddey. 1995. Alternatives toxicity and promotes plant growth of tomato (Lycopersicon for nitrogen nutrition of crops in tropical agriculture. Fertilizer esculentum L.). Chemosphere 69: 220-228. Res. 42: 339-346. 27. Martin, N. I., H. Hu, M. M. Moake, J. J. Churey, R. Whittal, R. 11. Dworkin, M. and J. Roster. 1958. Experiments with some W. Worobo, and J. C. Vederas. 2003. Isolation, structural microorganisms which utilize ethane and hydrogen. J. Bacteriol. characterization, and properties of mattacin (polymyxin M), a 75: 592-601. cyclic peptide antibiotic produced by Paenibacillus kobensis M. 12. Gordon, S. A. and R. P. Weber. 1951. Colorimetric estimation J. Biol. Chem. 278: 13124-13132. of indole acetic acid. Plant Physiol. 26: 192-195. 28. Montesinos, E., A. Bonaterra, E. Badosa, J. Frances, J. Alemany, 13. Gulati, A., P. Vyas, P. Rahi, and R. C. Kasana. 2009. Plant I. Llorente, and C. Moragrega. 2002. Plant-microbe interactions growth-promoting and rhizosphere-competent Acinetobacter and the new biotechnological methods of plant disease control. rhizosphaerae strain BIHB 723 from the cold deserts of the Int. Microbiol. 5: 169-175. Himalayas. Curr. Microbiol. 58: 371-377. 29. Murphy, J. F., M. S. Reddy, C. M. Ryu, J. W. Kloepper, and R. 14. Jaleel, C. A., P. Manivannan, B. Sankar, A. Kishorekumar, R. Li. 2003. Rhizobacteria-mediated growth promotion of tomato Gopi, R. Somasundaram, and R. Panneerselvam. 2007. Pseudomonas leads to protection against Cucumber Mosaic Virus. Phytopathology fluorescens enhances biomass yield and ajmalicine production 93: 1301-1307. in Catharanthus roseus under water deficit stress. Colloids Surf. 30. Nezarat, S. and A. Gholami. 2009. Screening plant growth B Biointerfaces 60: 7-11. promoting rhizobacteria for improving seed germination, seedling 15. Kang, S. H., H. S. Cho, H. Cheong, C. M. Ryu, J. F. Kim, and growth and yield of maize. Pak. J. Biol. Sci. 12: 26-32. S. H. Park. 2007. Two bacterial entophytes eliciting both plant 31. Paul, D. and S. Nair. 2008. Stress adaptations in a plant growth growth promotion and plant defense on pepper (Capsicum promoting rhizobacterium (PGPR) with increasing salinity in annuum L.). J. Microbiol. Biotechnol. 17: 96-103. the coastal agricultural soils. J. Basic Microbiol. 48: 378-384. 16. Karlidag, H., A. Esitken, M. Turan, and F. Sahin. 2007. Effects 32. Pikovskaya, R. I. 1948. Mobilization of phosphorous in soil in of root inoculation of plant growth promoting rhizobacteria connection with the vital activity of some microbial species. (PGPR) on yield, growth and nutrient element contents of Mikrobiologiya 17: 362-370. leaves of apple. Sci. Hortic. 114: 16-20. 33. Polz, M. F. and C. M. Cavanaugh. 1998. Bias in template-to- 17. Khalid, A., M. Arshad, and Z. A. Zahir. 2004. Screening plant product ratios in multitemplate PCR. Appl. Environ. Microbiol. growth-promoting rhizobacteria for improving growth and yield 64: 3724-3730. of wheat. J. Appl. Microbiol. 96: 473-480. 34. Probanza, A., J. L. Mateos, J. A. Lucas Garcia, B. Ramos, M. 18. Kloepper, J. W., R. M. Zablotowicz, E. M. Tipping, and R. R. De Felipe, and F. J. Gutierrez Manero. 2001. Effects of Lifshitz. 1991. Plant growth promotion mediated by bacterial inoculation with PGPR Bacillus and Pisolithus tinctorius on rhizosphere colonizers, pp. 315-326. In D. L. Keister and P. B. Pinus pinea L. growth, bacterial rhizosphere colonization, and Cregan (eds.). The Rhizosphere and Plant Growth. Kluwer mycorrhizal infection. Microb. Ecol. 41: 140-148. Academic Publishers, Dordrecht, The Netherlands. 35. Rasche, F., R. Trondl, C. Naglreiter, T. G. Reichenauer, and A. 19. Koo, S. Y. and K. S. Cho. 2009. Isolation and characterization Sessitsch. 2006. Chilling and cultivar type affect the diversity of of a plant growth-promoting rhizobacterium, Serratia sp. SY5. bacterial endophytes colonizing sweet pepper (Capsicum anuum J. Microbiol. Biotechnol. 19: 1431-1438. L.). Can. J. Microbiol. 52: 1036-1045. 790 Kim et al.

36. Raupach, G. S. and J. W. Kloepper. 1998. Mixtures of plant bacteria from rape (Brassica napus) roots and their potential in growth-promoting rhizobacteria enhance biological control of promoting the growth and lead accumulation of rape. Environ. multiple cucumber pathogens. Phytopathology 88: 1158-1164. Pollut. 156: 1164-1170. 37. Rodriguez, H. and R. Fraga. 1999. Phosphate solubilizing bacteria 51. Shoebitz, M., C. M. Ribaudo, M. A. Pardo, M. L. Cantore, L. and their role in plant growth promotion. Biotechnol. Adv. 17: Ciampi, and J. A. Curá. 2009. Plant growth promoting properties 319-339. of a strain of Enterobacter ludwigii isolated from Lolium 38. Ryan, R. P., S. Monchy, M. Cardinale, S. Taghavi, L. Crossman, perenne rhizosphere. Soil Biol. Biochem. 41: 1768-1774. M. B. Avison, G. Berg, D. van der Lelie, and J. M. Dow. 2009. 52. Silby, M. W., A. M. Cerdeno-Tarraga, G. S. Vernikos, S. R. The versatility and adaptation of bacteria from the genus Giddens, R. W. Jackson, G. M. Preston, et al. 2009. Genomic Stenotrophomonas. Nat. Rev. Microbiol. 7: 514-525. and genetic analyses of diversity and plant interactions of 39. Ryan, R. P., K. Germaine, A. Franks, D. J. Ryan, and D. N. Pseudomonas fluorescens. Genome Biol. 10: R51. Dowling. 2008. Bacterial endophytes: Recent developments and 53. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: applications. FEMS Microbiol. Lett. 278: 1-9. Molecular evolutionary genetics analysis (MEGA) software 40. Ryu, C. M., J. F. Murphy, K. S. Mysore, and J. W. Kloepper. version 4.0. Mol. Biol. Evol. 24: 1596-1599. 2004. Plant growth-promoting rhizobacteria systemically protect 54. Timmusk, S., V. Paalme, U. Lagercrantz, and E. Nevo. 2009. Arabidopsis thaliana against Cucumber Mosaic Virus by a Detection and quantification of Paenibacillus polymyxa in the salicylic acid and NPR1-independent and jasmonic acid- rhizosphere of wild barley (Hordeum spontaneum) with real- dependent signaling pathway. Plant J. 39: 381-392. time PCR. J. Appl. Microbiol. 107: 736-745. 41. Ryu, C. M., J. Kim, O. Choi, S. H. Kim, and C. S. Park. 2006. 55. Trivedi, P., A. Pandey, and T. Sa. 2007. Chromate reducing and Improvement of biological control capacity of Paenibacillus plant growth promoting activities of psychrotrophic Rhodococcus polymyxa E681 by seed pelleting on sesame. Biol. Control 39: erythropolis MtCC 7905. J. Basic Microbiol. 47: 513-517. 282-289. 56. Van Wees, S. C., S. Van der Ent, and C. M. Pieterse. 2008. 42. Sachdev, D., P. Nema, P. Dhakephalkar, S. Zinjarde, and B. Plant immune responses triggered by beneficial microbes. Curr. Chopade. 2010. Assessment of 16S rRNA gene-based phylogenetic Opin. Plant Biol. 11: 443-448. diversity and promising plant growth-promoting traits of 57. Vivas, A., R. Azcon, B. Biro, J. M. Barea, and J. M. Ruiz- Acinetobacter community from the rhizosphere of wheat. Lozano. 2003. Influence of bacterial strains isolated from lead- Microbiol. Res. 165: 627-638. polluted soil and their interactions with arbuscular mycorrhizae 43. Sandhya, V., S. Z. Ali, B. Venkateswarlu, G. Reddy, and M. on the growth of Trifolium pratense L. under lead toxicity. Can. Grover. 2010. Effect of osmotic stress on plant growth promoting J. Microbiol. 49: 577-588. Pseudomonas spp. Arch. Microbiol. 192: 867-876. 58. Westerberg, K., A. M. Elvang, E. Stackebrandt, and J. K. 44. Saravanan, V. S., J. Osborne, M. Madhaiyan, L. Mathew, J. Jansson. 2000. Arthrobacter chlorophenolicus sp. nov., a new Chung, K. Ahn, and T. Sa. 2007. Zinc metal solubilization by species capable of degrading high concentrations of 4- Gluconacetobacter diazotrophicus and induction of pleomorphic chlorophenol. Int. J. Syst. Evol. Microbiol. 50: 2083-2092. cells. J. Microbiol. Biotechnol. 17: 1477-1482. 59. Yadegari, M., H. A. Rahmani, G. Noormohammadi, and A. 45. Schwyn, B. and J. B. Neilands. 1987. Universal chemical assay Ayneband. 2008. Evaluation of bean (Phaseolus vulgaris) seeds for the detection and determination of siderophores. Anal. inoculation with Rhizobium phaseoli and plant growth promoting Biochem. 160: 47-56. rhizobacteria on yield and yield components. Pak. J. Biol. Sci. 46. Seldin, L., A. S. Rosado, D. W. da Cruz, A. Nobrega, J. D. van 11: 1935-1939. Elsas, and E. Paiva. 1998. Comparison of Paenibacillus 60. Yan, Z., M. S. Reddy, and J. W. Kloepper. 2003. Survival and azotofixans strains isolated from rhizoplane, rhizosphere, and colonization of rhizobacteria in a tomato transplant system. Can. non-root-associated soil from maize planted in two different J. Microbiol. 49: 383-389. Brazilian soils. Appl. Environ. Microbiol. 64: 3860-3868. 61. Yang, J., J. W. Kloepper, and C. M. Ryu. 2009. Rhizosphere 47. Selim, S., J. Negrel, C. Govaerts, S. Gianinazzi, and D. van bacteria help plants tolerate abiotic stress. Trends Plant Sci. 14: Tuinen. 2005. Isolation and partial characterization of antagonistic 1-4. peptides produced by Paenibacillus sp. strain B2 isolated from 62. Yeoung, S. B., S. J. Seung, S. P. Chang, and K. K. Hee. 1995. the sorghum mycorrhizosphere. Appl. Environ. Microbiol. 71: In vitro and greenhouse evaluation of cucumber growth 6501-6507. enhanced by rhizosphere microorganisms. Plant Pathol. J. 11: 48. Shaharoona, B., G. M. Jamro, Z. A. Zahir, M. Arshad, and K. S. 292-297. Memon. 2007. Effectiveness of various Pseudomonas spp. and 63. Zaidi, A., M. S. Khan, M. Ahemad, and M. Oves. 2009. Plant Burkholderia caryophylli containing ACC-deaminase for improving growth promotion by phosphate solubilizing bacteria. Acta growth and yield of wheat (Triticum aestivum L.). J. Microbiol. Microbiol. Immunol. Hung. 56: 263-284. Biotechnol. 17: 1300-1307. 64. Zhang, Z., S. Srichuwong, T. Kobayashi, M. Arakane, J. Y. 49. Shen, Shun-Shan, Sin-Hyo Park, and Chang-Seuk Park. 2005. Park, and K. Tokuyasu. 2010. Bioconversion of L-arabinose and Enhancement of biocontrol efficacy of Serratia plymuthica other carbohydrates from plant cell walls to alpha-glucan by a A21-4 against phytophthora blight of pepper by improvement of soil bacterium, Sporosarcina sp. N52. Bioresour. Technol. 101: inoculation buffer solution. Plant Pathol. J. 21: 68-72. 9734-9741. 50. Sheng, X. F., J. J. Xia, C. Y. Jiang, L. Y. He, and M. Qian. 2008. Characterization of heavy metal-resistant endophytic