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tified as ClO, C20 and C30 (10, 20 and 30 cm sampling depths, respectively), while samples from paddy soil S are identified as Sb, S20 and S30, respectively.
Annals of Microbiology, 57 (3)

299-306 (2007)

The phylogeny of fluorescent pseudomonads in an unflooded rice paddy soil Hang-YeOfl WEON 1 , Robert S. DUNGAN 2 , Soon-Wo KWON 3 , Jong-Shik KIM'* 'Applied Microbiology Division, National Institute of Agricultural Science and Technology, Rural Development Administration (RDA), Suwon 441-707, Korea; 2 USDA-ARS, Environmental Management & Byproduct Utilization Laboratory, Beltsville, MD 20705, USA; 3 Korean Agricultural Culture Collection (K.ACC), Genetic Resources Division, National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea; 4 University of California, Department of Environmental Sciences, Riverside, CA 92521, USA Received 8 January 2007/ Accepted 18 June 2007

Abstract - The purpose of this research was to determine the diversity and distribution of fluorescent pseudomonads in an unflooded rice paddy soil. A region of the 16S ribosomal RNA gene from isolates was amplified using PCR and subsequently analysed by sequence analysis for bacterial identification and phylogenetic classification. A total of 117 fluorescent pseudomonads, representing between 10 and 21 species, were isolated from two sampling sites within the same paddy (designated as soils C and 5). The isolates were found to be ^ 96°Io homologous with known sequences, and were most closely related to the following Pseudomonas species: P. antarctica, P. costantini, P. extremorien ta/is, P. frederiksbergensis, P. ki/onensis, P. koreensis, P. I/ni, P. mande/ii, P. poae, P. rhodesiae, and P. veronü. Of these matches, the bulk of the isolates (49%) were affiliated with P. mandelii. In soils C and 5, phylogenetic analysis revealed that 35 and 82 isolates co-clustered with 39 and 59 010 of 66 fluorescent pseudomonad type strains, respectively. Key words: diversity, fluorescent pseudomonads, phylogeny, 16S rRNA gene, rice paddy soil.

INTRODUCTION Rice is one of the World's most important agronomic crops, with over 100 million hectares under cultivation globally. To date, microbial community studies in rice paddy soils have only focused on anoxic flooded systems (Ludemann and Liesack, 2000; Weber et a!., 2001; Noll et a!., 2005), but none have investigated the diversity of microbial populations, specifically fluorescent pseudomonads, under oxic conditions during the dry season. Fluorescent pseudomonads are an ecologically important group of soil bacteria and one of best studied bacterial groups due to the ability of some species to colonise the root zone and improve plant health and increase yield (O'Sullivan and O'Gara, 1992; Glick eta!., 1999; Lugtenberg et a!., 2001; Hass and Keel, 2003; Dey et a!., 2004; Costa et a!., 2006; Janvier et a!., 2007). Given the relative ease by which fluorescent pseudomonads can be cultured and their common association with plant roots, there has been considerable interest in identifying their genotypic and phenotypic diversity in agricultural soils (Glick et a!. 1999; Ellis et a!., 2000; Cornelis and Matthijs, 2002; Misko and Germida, 2002; Costa eta!., 2006). The purpose of this research was to determine the diversity and distribution of fluorescent pseudomonads in *

Corresponding author: Phone: (951) 827-2582; Fax: (951) 827-3993; E-mail: [email protected]

an unflooded rice paddy soil from South Korea. The fluorescent pseudomonads isolates were identified by sequencing a region of the 16S ribosomal RNA (rRNA) gene, which was subsequently used for bacterial identification and phylogenetic classification. Considering the ecological importance of fluorescent pseudomonads, further investigation of their significance in rice paddy soils is highly recommended.

MATERIALS AND METHODS Site description and soil sampling. The unflooded rice paddy soil was located in the city of Samchuck, Gangwon Province, which is in the east-central region of South Korea. The area has an average temperature of 12.5 °C and receives about 120 cm of rainfall annually, 50% of which falls during June and July. The Samchuck soil is a sandy loam and select chemical properties are presented in Table 1. Soil samples were taken from two sites (designated as soils C and S), located within the same rice paddy, that were 50 m apart from each other. Triplicate soil samples were aseptically collected from 10, 20, and 30 cm below the soil surface. Samples from paddy soil C are identified as ClO, C20 and C30 (10, 20 and 30 cm sampling depths, respectively), while samples from paddy soil S are identified as Sb, S20 and S30, respectively. After collection, the triplicate soil samples were passed through a 2mm sieve, homogenised, and then preserved at 4 °C for 72 h prior to microbial and chemical analyses.

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TABLE 1 - Soil chemical properties Sample pH tECt

OM

Heavy metals#

Exchangeable cations* Ca

K

Mg

Na

Cd

Cr

Cu

Ni

Pb

Zn

ClO C20 C30

6.8 6.9 7.2

0.4 0.2 0.1

42 38 24

7.8 7.0 5.8

0.2 0.2 0.2

0.8 0.7 0.7

0.3 0.3 0.3

0.3 0.2 0.1

0.2 0.1 0.1

4.1 3.4 2.4

1.5 1.2 0.6

11.2 7.3 4.7

17.0 10.0 3.8

SlO S20 S30

6.9 6.9 6.8

0.4 0.3 0.3

23 65 66

6.8 7.5 7.5

0.2 0.2 0.1

1.4 2.0 1.4

0.3 0.3 0.3

0.3 1.0 1.3

0.1 0.1 0.2

4.4 11.8 14.0

0.4 1.3 1.9

10.4 13.9 14.5

16.2 34.2 42.8

Soil to deionized water, 1:5; 4 Electrical conductivity, dS rn- 1 ; § Organic matter, g kg-1; * crnol kg- 1 ; 1 mg kg-1.

Enumeration of bacterial populations. Dilution plating on various media was conducted to determine microbial counts of fluorescent pseudornonads, heterotrophic bacteria, and Gram-negative bacteria. Fluorescent pseudomonads were cultured on P1 agar as described by Kato and Itoh, 1983. Heterotrophic bacteria were cultured on yeastglucose (YG) agar (yeast extract, 3 g; K 2 HPO4 , 0.3 g; KH 2 PO4 , 0.2 g; MgSO4 7H 20, 0.2 g; cyclohexamide, 0.05 g; agar, 15 g; distilled water, 1 L; pH 6.8). Gram-negative bacteria were cultured by adding 5 mL of 0.1% crystal violet to 1 L of YG. Average colony forming units (CFUs) were obtained by triplicate plate counts. Isolation of fluorescent pseudomonads and sequence analysis. A detailed method for the isolation of fluorescent pseudomonads and sequencing is described by Kwon et a!. (2005). In short, 117 fluorescent pseudomonads were isolated from the rice paddy soils using P1 agar medium (Kato and Itoh, 1983). Pure cultures were obtained by streaking the organisms several times on P1 medium. For sequencing, the 16S rRNA gene from the isolates was amplified by PCR using the universal primer fDl and rP2 (Weisburg et a!., 1991). Inserts into pGEM-T easy vector (Promega, Madison, WI, USA) were automatically sequenced on an Applied Biosystems 377 sequencer (Foster City, CA, USA). Putative chimeric sequences were identified using Bellerophon (Huber et al., 2004) and the nucleotide sequences were aligned using the NAST aligner at the Greengenes web site (DeSantis eta!., 2006). The phylogenetic tree was drawn using MEGA3 (Kumar et a!., 2004). Bootstrap analyses of the neighbor-joining data, based on 1000 samples, were used to assess the stability of relationships. The nucleotide sequence data reported in this paper will appear in GenBank/EMBL/DDB] nucleotide sequence databases with the accession number(s) DQ910376-DQ910490 and EF201980-EF201982.

Diversity indices and statistical analysis. The computer program DOTUR was used to estimate fluorescent

pseudomonad diversity and species richness (Schloss and Handelsman, 2005).

RESULTS AND DISCUSSION Fluorescent pseudomonads are a group of bacteria known to play important ecological roles in soil habitats (Misko and Germida, 2002; Costa et a!., 2006; Watt et a!., 2006), but little is known about their diversity in rice paddy soils, especially under unflooded conditions during the dry season. A comparison of total cultivable numbers of fluourescent pseudomonads, heterotrophic bacteria, and Gram-negative bacteria is shown in Table 2. The data revealed that the population size of fluorescent pseudomonads in paddy soils C and S were substantially smaller than that of the heterotrophic and Gram-negative bacteria. In fact, the pseudomonad population was, on average, 99.9% and 98.5% smaller than the other respective populations. To determine the distribution of the fluorescent pseudomonads among known species, a phylogenetic analysis of 16S rRNA gene sequences from 117 fluorescent pseudomonad isolates was compared to the same sequence region of 66 previously described type strains. A total of 35 isolates from paddy soil C and 82 from paddy soil S were examined (Tables 3 and 4, respectively). As shown in Figs. 1 and 2, the fluorescent Pseudomonas isolates from each soil clustered into several distinct taxonomic groups. In soil C, the 35 isolates co-clustered with 26 of the type strains, while the 82 isolates from soil S co-clustered with 37 type strains. All of the isolates were ^ 96% homologous with known 16S rRNA gene sequences. The isolates from soil C were most closely related with the following fluorescent Pseudomonas: P. antarctica, P. extremorienta/is, P. frederiksbergensis, P. ki!onensis, P. koreensis, P. mande!ii, P. poae, and P. veronii. The largest cluster in the phylogenetic tree was associated with P. mandelii, followed by P. frederiksbergensis, P. antarctica, and P. koreensis, which encompassed 40, 17, 14, and 9%

TABLE 2 - Population sizes of fluorescent pseudomonads, heterotrophic bacteria, and Gram-negative bacteria Population

ClO

C20

C30

SlO

S20

S30

Fluorescent pseudomonads Heterotrophic bacteria Gram-negative bacteria

3.6* 7.0 5.3

2.5 6.4 4.6

2.8 6.3 5.2

3.7 6.4 5.5

3.1 6.9 5.1

3.5 6.7 5.0

*(Log jo CFU/g soil)

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Ann. Microbiol, 57 (3), 299-306

TABLE 3 - Phylogenetic affiliations of the isolates from paddy soil C Match Accession % No.

Isolate

Closest database match

C10-01 cio-03 C10-08 C10-09 CIO-10 ClO-il C10-12 C10-13 C10-14 C10-15 C10-16 C10-18 C10-21 C10-23 C10-25 C10-30 C10-31 C10-32 C10-33 C10-35 C10-38 C20-02 C20-07 C20-10 C20-19 C20-23 C20-29 C30-01 C30-04 C30-11 C30-21 C30-29 C30-41 C30-45 C30-48

99 Pseudomonas sp. c313 100 Pseudomonas sp. Circle 100 Pseudomonas sp. Circle 100 Pseudomonas sp. Circle 100 Pseudomonas sp. LAB-23 100 Pseudomonas sp. Circle 100 Pseudomonas lini PD 11 100 Pseudomonas sp. Circle 100 Pseudomonas mandelii 99 Pseudomonas poae BIHB 730 Pseudomonas extremorientalis KMM 3447T 98 99 Pseudomonas lini PD 11 99 Pseudomonas sp. c313 100 Pseudomonas sp. c313 100 Pseudomonas poae BIHB 730 99 Pseudomonas sp. LAB-23 100 Pseudomonas mandelii 97 Pseudomonas sp. LAB-23 99 Pseudomonas sp. EK1 99 Pseudomonas lini PD 11 98 Pseudomonas I/ni PD 11 98 Pseudomonas sp. c313 100 Pseudomonas mandelii 100 Pseudomonas sp. LAB-23 99 Pseudomonas putida IA2XCDB 99 Pseudomonas veronii CIP 104663T 99 Pseudomonas sp. NZ039 96 Pseudomonas veronhi CIP 104663T 96 Pseudomonas veronii CIP 1046631 99 Pseudomonas sp. c313 100 Pseudomonas sp. LAB-23 99 Pseudomonas sp. c313 100 Pseudomonas sp. LAB-OS 100 Pseudomonas sp. LAB-23 100 Pseudomonas I/ni PD 11

AB167 182 AJ417370 A1417370 A3417370 AB051699 A)417370 DQ377752 A1417370 AYi79326 DQ536513 AF405328 DQ377752 AB167 182 AB167 182 DQ536513 ABO5 1699 AY 179 326 ABO5 1699 PJ237965 DQ377752 DQ377752 AB167 182 AY 1793 26 ABO5 1699 AY5 12613 AF064460 AY014808 AF064460 AF064460 AB167 182 AB051699 AB167 182 ABO5 1692 ABO5 1699 DQ377752

Source

Most closely related species

P. frederiksbergensis Phenol enrichment, Japan P. antarctica Synthetic pyrethroid degrading, UK P. antarctica Synthetic pyrethroid degrading, UK P. antarctica Synthetic pyrethroid degrading, UK P. mandelü Phenol bioremediation site, Japan P. antarctica Synthetic pyrethroid degrading, UK P. mandelii Potato cropping soil, Canada P. antarctica Synthetic pyrethroid degrading, UK P. mande/ii Nonylphenol biodegradation, Sweden Rhizosphere of Hippophae rhamnoides, India P. poae P. extremorientalis Drinking water reservoir, Japan P. mandelh Potato cropping soil, Canada P. frederiksbergensis Phenol enrichment, Japan P. frederiksbergensis Phenol enrichment, Japan Rhizosphere of Hippophae rhamnoides, India P. poae P. mande/ii Phenol bioreniediation site, Japan P. mandelfi Nonylphenol biodegradation, Sweden P. mande/ii Phenol bioremediation site, Japan P. mandelii Industrial waste water, Germany P. mandelii Potato cropping soil, Canada P. mandelii Potato cropping soil, Canada P. frederiksbergensis Phenol enrichment, Japan P. mandelu Nonylphenol biodegradation, Sweden P. koreensis Phenol bioremediation site, Japan BTEX contaminated industrial site, Belgium P. kilonensis P. veronii Natural mineral waters, France P. koreensis Agaricus bisporus, New Zealand P. veronii Natural mineral waters, France P. veronii Natural mineral waters, France P. frederiksbergensis Phenol enrichment, Japan P. mandelu Phenol bioremediation site, Japan P. frederiksbergensis Phenol enrichment, Japan P. koreensis Phenol bioremediation site, Japan P. mande/ii Phenol bioremediation site, Japan P. mande/ii Potato cropping soil, Canada

TABLE 4 - Phylogenetic affiliations of the isolates from paddy soil S Isolate

Closest database match

Match Accession % No.

Source

Most closely related species

Slo-Ol S10-04 S 10-08 S 10-09 S10-11 S10-12 Sb- 16 S10-17 S10-18 S10-19 S10-20 S10-24 S10-27 S10-28 S10-29 S10-30 S10-31

Pseudomonas I/ni Pseudomonas lurida DSM 158351 Pseudomonas mandelii Pseudomonas lurida DSM 15835T Pseudomonas lurida DSM 15835T Pseudomonas sp. NZ052 Pseudomonas lurida DSM 15835T Pseudomonas lurida DSM 15835T Pseudomonas sp. LAB-23 Pseudomonas sp. NZ052. Pseudomonas sp. LAB-23 Pseudomonas I/ni Pseudomonas lurida DSM 15835T Pseudomonas lurida DSM 15835T Pseudomonas sp. ps8-29 Pseudomonas sp. LAB-23 Pseudomonas sp. NZ052

100 DQ377752 99 AJ581999 100 AY179326 100 A3581999 100 P3581999 100 AYO 148 11 100 AJ581999 99 AJ581999 100 AB051699 100 AYO 148 11 98 AB051699 99 0Q377752 100 AJ581999 100 AJ581999 98 AY303325 100 AB051699 98 AVO 148 11

Potato cropping soil, Canada Grass, phyllosphere, Germany Nonylphenol biodegradation, Sweden Grass, phyllosphere, Germany Grass, phyllosphere, Germany Agaricus bisporus, New Zealand Grass, phyllosphere, Germany Grass, phyllosphere, Germany Phenol bioremediation site, Japan Agaricus bisporus, New Zealand Phenol bioremediation site, Japan Potato cropping soil, Canada Grass, phyllosphere, Germany Grass, phyllosphere, Germany Rice paddy soil, Korea Phenol bioremediation site, Japan Agaricus bisporus, New Zealand

P. mandelii P. costantinii P. mandelii P. costantinil P. costantinii P. poae P. costantinii P. costantinii P. mandelii P. poae P. mandelii P. mandelii P. costantinii P. costantinii P. frederiksbergensis P. mandelil P. poae (Follow)

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H-Y. Wean et a!.

TABLE 4 - Phylogenetic affiliations of the isolates from paddy soil S (Continued) Isolate S10-32 S 10-33 S10-34 S10-35 S10-39 S10-40 S10-41 S10-45 S 10-50 S20-02 S20-03 S20-04 S20-05 S20-06 S20-07 S20-08 S20-09 S20-10 S20-14 S20-19 S20-20 S20-21 S20-25 S20-27 S20-28 S20-29 S20-30 S20-31 S20-33 S20-41 S20-43 S20-44 S20-46 S20-48 S30-01 S30-02 S30-03 S30-04 S30-05 S30-11 S30-12 S30-13 S30-14 S30-15 S30-16 S30-17 S30-18 S30-19 S30-20 S30-21 S30-23 S30-24 S30-26 S30-30 530-32 S30-34 S30-38 S30-39 S30-40 S30-42 S30-44 S30-51 S30-53 S30-54 S30-55

Closest database match

Match Accession % No.

Pseudomonas lini 100 Pseudomonas sp. NZ052 100 Pseudomonas sp. LAB-23 100 Pseudomonas lurida DSM 15835T 100 Pseudomonas sp. LAB-23 98 Pseudomonas lurida DSM 15835T 100 Pseudomonas lurida DSM 158351 100 Pseudomonas sp. LAB-23 99 Pseudomonas I/ni 98 Pseudomonas I/ni 99 Pseudomonas mandelii 100 Pseudomonas mandel/i 100 Pseudomonas lini 100 Pseudomonas mandelii 100 Pseudomonas mandelii 100 Pseudomonas I/ni 100 Pseudomonas sp. HF3/S21027 99 Pseudomonas mandelii 100 Pseudomonas sp. 6C_10 100 Pseudomonas sp. LAB-23 100 Pseudomonas sp. c313 99 Pseudomonas sp. Circle 100 Pseudomonas lini 99 Pseudomonas lini 100 Pseudomonas sp. LAB-23 100 Pseudomonas sp. CLi21 100 Pseudomonas mandelii CIP 1052731 98 Pseudomonas sp. 6C_10 99 Pseudomonas mandelii 100 Pseudomonas sp. LAB-23 100 Pseudomonas sp. S-H52 98 Pseudomonas sp. LAB-23 99 Pseudomonas sp. LAB-23 100 Pseudomonas mandelii 99 Pseudomonas sp. IBUN S1901 98 Pseudomonas poae BIHB 730 100 Pseudomonas sp. Circle 98 Pseudomonas sp. NZ039 99 Pseudomonas extremorientalis KMM 3447T 98 Pseudomonas mandelii 99 Pseudomonas extremorientalis KMM 3447T 99 Pseudomonas extremorientalis KMM 3447T 97 Pseudomonas sp. LAB-23 99 Pseudomonas sp. NZ052 99 Pseudomonas sp. 6C_10 99 Pseudomonas sp. 6C_10 99 Pseudomonas sp. Circle 100 Pseudomonas sp. ARK9995 98 Pseudomonas extremorientalis KMM 3447T 99 Pseudomonas sp. LAB-23 100 Pseudomonas sp. BEldil 99 Pseudomonas sp. MGi 98 Pseudomonas sp. Circle 100 Pseudomonas mandelii 100 Pseudomonas mandelii 100 Pseudomonas I/ni PD 11 100 Pseudomonas sp. pSlO-20 99 Pseudomonas sp. 6C_10 100 Pseudomonas extremorientalis KMM 3447T 99 Pseudomonas lini PD 11 99 Pseudomonas mandelii 100 Pseudomonas mandelii 100 Pseudomonas sp. LAB-23 100 Pseudomonas sp. HF3/S21027 100 Pseudomonas lini PD 11 100

DQ377752 AYO 14811 AB051699 AJ58 1999 AB051699 AJ581999 AJ58 1999 AB051699 DQ377752 DQ377752 AY179326 AY179326 DQ377752 AY179326 AY179326 DQ377752 AY337597 AY179326 AY689050 AB051699 AB167182 A3417370 DQ377752 DQ377752 ABO5 1699 AF529319 AF058286 AY689050 AY179326 ABO5 1699 AY622270 AB051699 AB051699 AY179326 DQ8 13328 DQ536513 A3417370 AYO 14808 AF405328 AY179326 AF405328 AF405328 AB051699 AYO 14811 AY689050 AY689050 A1417370 AF468447 AF405328 AB051699 AY263471 AF326378 AJ417370 AY179326 AY179326 DQ377752 AY303261 AY689050 AF405328 DQ377752 AY179326 AY179326 AB051699 AY337591 DQ377752

Source Potato cropping soil, Canada Agar/cus b/sporus, New Zealand Phenol bioremediation site, Japan Grass, phyllosphere, Germany Phenol bioremediation site, Japan Grass, phyllosphere, Germany Grass, phyllosphere, Germany Phenol bioremediation site, Japan Potato cropping soil, Canada Potato cropping soil, Canada Nonylphenol biodegradation, Sweden Nonylphenol biodegradation, Sweden Potato cropping soil, Canada Nonylphenol biodegradation, Sweden Nonylphenol biodegradation, Sweden Potato cropping soil, Canada Rice paddy soil, Japan Nonylphenol biodegradation, Sweden Lake water, Korea Phenol bioremediation site, Japan Phenol enrichment, Japan Synthetic pyrethroid degrading, UK Potato cropping soil, Canada Potato cropping soil, Canada Phenol bioremediation site, Japan PCE-contaminated site, USA Mineral water, France Lake water, Korea Nonylphenol biodegradation, Sweden Phenol bioremediation site, Japan Subsurface soil, USA Phenol bioremediation site, Japan Phenol bioremediation site, Japan Nonylphenol biodegradation, Sweden Sugar cane cropping soil, Colombia Rhizosphere of Hippophae rhamnoides, India Synthetic pyrethroid degrading, UK Agaricus bisporus, New Zealand Drinking water reservoir, Japan Nonylphenol biodegradation, Sweden Drinking water reservoir, Japan Drinking water reservoir, Japan Phenol bioremediation site, Japan Agar/cus bisporus, New Zealand Lake water, Korea Lake water, Korea Synthetic pyrethroid degrading, UK Arctic sea ice-melt pond Drinking water reservoir, Japan Phenol bioremediation site, Japan Alpine soil, USA Mn(II)-oxidizing strain, USA Synthetic pyrethroid degrading, UK Nonylphenol biodegradation, Sweden Nonylphenol biodegradation, Sweden Potato cropping soil, Canada Rice paddy soil, Korea Lake water, Korea Drinking water reservoir, Japan Potato cropping soil, Canada Nonylphenol biodegradation, Sweden Nonylphenol biodegradation, Sweden Phenol bioremediation site, Japan Rice paddy soil, Japan Potato cropping soil, Canada

Most closely related species P. mandelii P. poae P. mandel// P. costant/nii P. mandelii P. costantin// P. costant/nii P. mandel// P. mandel/i P. mandelii P. mandelii P. mandelii P. mandel/i P. mandelii P. mandel// P. mandel/i P. mandelii P. mandelii P. koreens/s P. mandelii P. frederiksbergens/s P. antarctica P. mandelii P. mandelii P. mandelii P. koreensis P. mandelii P. koreens/s P. mandel// P. mandelii P. rhodes/ae P. mandelii P. mandel/i P. mandel// P. freder/ksbergensis P. poae P. antarctica P. koreensis P. extremor/entalis P. mandeli/ P. extremorientalis P. extremorientalis P. mandelii P. oae P. koreensis P. koreensis P. antarctica P. koreensis P. extremor/entalis P. mandelii P. veronii P. I/ni P. antarctica P. mandelii P. mandel// P. mandel/i P. extremorientalis P. koreensis P. extremorientalis P. mandel/i P. mandelii P. mandelii P. mandelii P. mandel/i P. mandel//

Ann. Microbiol., 57 (3), 299-306 (2007)

303

C10_18 C10-12 C20-07 C30-48 CIO-32 9___ C10-38 P. borealis NB6 C30-45 Cl 0-10 I 010-30 I C30-21 C1P105470 IqP.P.migulae corrugate ATCC29736 P. brassicacearum DBK1 1 L. P. thivervalensis SBK26 C 10-33 P. frederiksbergensis JAJ28 P. mandelii CI P105273 C10-31 C 10-14 P. tolaasii ATCC33618 P. veron,i C1P104663 P. marginalis ATCC1O844 P. brenneri CFML97-3 C30-11 C20-02 C30-29 C10_01 010-23 1 0-21 C30-04 C30-01 C10-15 odesiae104664 F P—rhCIP C 10-16 LO-25 meridiana CMS38 P. fluorescens ATCC1 3525 P. rimonti, CFML97-514 C, - 13 C10_09 C 10-08 C10-03 010-11 020-23 P. orientalis CFML961 70 P. cedrina CFML96-198 P. mucidolens AM 12406 P. synxantha IAM1 2356 P. libanensis C1P105460 P. gessard,, C1P105469 P. ezotoformans lAM 1603 020-19 P. umsongensis KACC1 0846 C30-4 1 P jessenii C1P10527 020-10 P. koreensis KACC10848 81 020-29 P. pavonaceae ]AM ll55 P. aurentiace ATCC33663 P. chlororaphis DSM50083 P. ficuserectee JCM2400 caricapapayae ATCC33615 eliae MAFF301463 syringae ATCC1931O P. amygdali ATCC33614 LMC301 90 P. lundensis ATCC49968 P. fragi 1F03458T P taetrolens 1AM 165 P. graminls JPL-8 P. agarici ATCC25941 P. v,r,d,flava LMG2352 avellanae P90 84'P. P. cichodiATCC10857 P. fuscovaginae MAFF301 177 [P. asplenii ATCC 23835 P. plecoglossicida FPC951 JL_. P. oryzihabitans LAM 1 568 I P. monteiliiClPlO4883 P. p utida 1AM1236 P. fulva 1AM1529 D8 P. straminea [AM 1 598 P. flavescens B62 P. alcaligenes IAM1 2411 —1P. alcaliphila ALl 5-21 mendocina ATCC2541 1 86 P.P.pseudoalcaligenes DSM5001 P. balearica SP1402 - P. pohangensis H3-R1 P. aeruginosa LMC2042 P. resinovorans ATCC1 3235 - P. anguiiiseptica NCMB1944 _________ P. oleovorans 1AM1508 P. luteola IAM1 3000 P. citronellolis ATCC13674 P. nitroreducens lAM 1439 ________ P. pertucinogena 1F014163 P. denitrificans lAM 12023

P. mandelii (VI)

P. frederiksbergensis (IX) P. veronii(IX) P. pope P. extremorientalis P. poae

(IV)

P. antarctica P. veronll

P. kionensis P.

koreensis (VIII)

0.02

FIG. 1 - Phylogenetic tree of fluorescent pseudomonads from paddy soil C at 10, 20, and 30 cm depths, including 35 isolates and 66 reference species. Clusters identified with Roman numerals are similar to the clusters identified by Kwon et al (2005) in upland Korean agricultural soils. of the isolates, respectively (Fig. 1 and Table 3). Isolates mandelji and P. frederiksbergensis clusters were found at all soil depths (i.e. 10, 20 and 30 cm), while the isolates within the other smaller clusters were generally found at only one sampling depth, except P. koreensis at 20 and 30 cm. As with soil C, the isolates from soil S were from both P.

closely related to P. antarctica, P. extremorientalis, P. frederiksbergensjs, P. koreensis, P. mandelil, P. poae, and P. veronii, but did not match with P. kilonensis (Fig. 2 and

Table 4). In addition, 12 isolates from soil S were found to

match closely with P. costantini (10), P. fin! (1), and P. rhodesiae (1). The P. fin/isolate (530-24) was found among the cluster dominated by P. mandeli!. Once again, the only isolates found at all sampling depths were those that mandeli! and P. frederiksbergensis. While only 3 isolates were affiliated with P. frederiksbergensis, 43 of 82 isolates matched with P. mandelii, which represents the largest cluster in the phylogenetic tree (Fig. 2). Table 5 shows the diversity and species richness estimates based on the 16S rRNA gene from the flourescent matched with P.

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-S30-14 S30-53 S30-54 S10-34 S10-18 S20-19 S20-41 S20-46 S20-28 S30-21 P. rnigu/ae C1P105470 - P corrugate ATCC29736 S30 - 24 P. brass/cacearum DBK1 1 P. thivervalenois SBK26

P.

mande///(VI)

-P.P.borealis frederikobergensis JAJ28 NB6 .S10-30 S30-11 P. manSe/il CIP105273

veroroi C/P 104663 P.to/aaon ATCC33618

P. antarctica 3 0-13 Ji—S S0-12 Ii S10-31 S10-19 S30-15 S 10-33 I S10-12 P. f/uoresceno ATCC1 3520 P. mendiana CMS38 0-21 P. Or/month CFML97-514 P. orienta/is CFML96170 -L P. cednna CFML96-198 1 S30-01 1T520-20 S10-29 S30-23 S20-43 .S30-02 - P. rhodes/ac C/P 104664 • P marginal;s ATCC10844 P. brenneri CFML973 P. mucidolens /AM12406 P Synxant//a IAM12356 /ibanensis C/P105460 F pPf.es1sardifc/p105469 S7 S10-04 IS1O-11 15 10-35 IS1O-27 - Si0-16 S10-28 S10-41 S1040 S 10-09 P. aurantiaca ATCC33663 P. ch/ororaphis DSM50083 P. amvgda//ATCC33614 P corona/isojens LMC30I90 P. oynngae ATM 9310 P. me/lee MAFF301463 • P. CanCapapayae ATCC33615 P ficuserectae JCM2400 P. cichon, ATCC1Q8O7 P. v,r,d,flave LMG2352 --P avellanae P90 P9moongensis KACC1 0846 P. jessen;i C1P10527 P graminis JPL-8 F 6SS—20jL

P. koreens,s KACC1 0848 S30-04 P. pavonaeae IAMI 155

P. extremorienta/is P. poae

(VI)

P. antarctica P. frederiksbergensis (IX) P. rhodesjae P. veronii(IV)

P. costantini(IV)

P. koreensis (VIII)

0030-19

P.

P. agarici ATCC25941 P. fuscovaqjnae MAFF301 177 P. asp/coil ATCC 23835 P plecoglosoicida FPC951 P. oryzihab,tans 1AM1568 Ponte,Sj C/ P104883 P. putida /AM1236 Lm P. fu/va 1AM15299 P. straminea 1AM1598 P. f/avescens B62 P. a/ca//genes /AM1241 1 P. a/caliph/Ta AL15-21 P. mendocina ATCC2541 1 84 P. pseudoalca/igenes DSM5001 azotofv,rmano /0 IA1Rni

P. o/eovorans 1AM1508 a/a 1AM130/30 P. citrone//o/js ATCC13674 ducens 1AM1439 P. pertucino6ena I F014163 P denitr6cans 1A 12023 II 0.02

FIG. 2 - Phylogenetic tree of fluorescent pseudomonads from paddy soil S at 10, 20, and 30 cm depths, including 82 isolates and 66 reference species. Clusters identified with Roman numerals are similar to the clusters identified by Kwon et al. (2005) in upland Korean agricultural soils.

305

Ann. MicrobioL, 57 (3), 299-306 (200

TABLE 5 - Diversity and richness estimates based on the 16S rRNA gene of fluorescent pseudomonas isolates from the unflooded rice paddy soils Richness* Diversity Sample rarefaction jacknife Chaol bootstrap ACE Simpson (lID) Shannon (H) Paddy soil C

1.90

6.25

15

12

12

14

10

Paddy soil S

1.75

3.57

21

15

21

20

13

* Number of observed OTUs

PseudomoflaS isolates. According to the Shannon and Simpson diversity indices, diversity was greater in soil C than in soil S. To estimate species richness, we used the abundance-based coverage estimator (ACE) and bootstrap, Chaol, and jacknife estimators. Based on these estimators, the species richness was conclusively greater in soil S, but this is likely a result of the larger sample size taken from soil S. The number of operational taxonomic units (OTU5) observed in the ACE and Chaol richness estimates were 21 in soil S and 15 and 12 OTUs, respectively, in soil C (Table 5). A similar number of OTUs was observed in the jacknife richness estimate. In the rarefaction estimate, 10 OTU5 were observed in the 35 sequences from soil C, while 13 OTU5 were observed in the 82 sequences from soil S. Overall, these results suggest that soils C and S contain between 10 and 21 unique species of fluorescent Pseudomonas. Kwon et a!. (2005) recently analysed 160 fluorescent pseudomonad isolates, representing 53 genotypes, from 7 upland agricultural soils in Korea. Paddy soil isolate clusters identified with Roman numerals IV, VI, VIII, and IX in Figs. 1 and 2 were similar to those found in the agricultural soil study, although Kwon et a!. (2005) only collected samples from 10 cm below the soil surface. The fact that these phylogenetic clusters were present in both the paddy and upland soils suggest that this could be a common ecological attribute of fluorescent pseudomonads. Only P. kilonensis (C20-19) from soil C and P. fin! (S30-24) and P. rhodesiae (S20-43) from soil S were identified as being unique to the rice paddy soils. These pseudomonads may have developed the ability to survive in the paddy soil, but not in the upland soil. However, Cho and Tiedje (2000) reported that fluorescent pseudomonads from undisturbed sites are not globally distributed, which suggests that there still might be some endemic species in these disturbed paddy soils. In summary, phylogenetic analysis revealed that the fluorescent pseudomonads from unflooded rice paddy soils clustered into taxonomic groups that were similar to those found in upland Korean agricultural soils. While P. mandelii was identified as the most abundant isolate, only three isolates were identified as being unique to the unflooded rice paddy soils. A potential limitation with this study, however, was that the diversity and distribution of fluorescent pseudomonads was based upon their ability to be cultivated. Regardless, these results contribute to better our understanding of the ecology of fluorescent pseudomonads, which may ultimately offer important insights into the m anagement of paddy soils.

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