Supplementary Materials and methods
Fatty acid methyl ester profiling
Total cellular fatty acid-methyl ester (FAME) analysis of strains CCR04 and CCR80
was performed by gas chromatography (Hewlett-Packard 5898A, GC system, Avondale,
PA, USA) with the Microbial Identification System (MIDI) (Newark, DE, USA)
according to the manufacturer’s instructions.
Morphological, biochemical and physiological identification
The shape, cell size and flagella of strains CCR04 and CCR80 were examined using
transmission electron microscopy (TEM) (LEO 912AB, LEO Electron Microscopy,
Cambridge, England) as described by Kim et al. (2012). The phenotypic characteristics
of strains CCR04 and CCR80 were compared with seven reference species of
Pseudomonas, which were obtained from the Korean Agricultural Culture Collection
(KACC) of the National Institute of Agricultural Biotechnology in the Rural
Development Administration (Suwon, Korea). Gram staining; hydrolysis of gelatin,
starch and Tween 80; production of H2S, catalase and acid from carbohydrate; growth
on trypticase soy agar and nutrient agar supplemented with 1, 3 and 5 % NaCl;
fluorescence production; glucose utilization were tested using the methods of Gerhardt
(1994). Degradation of urea was determined using the procedures of Bowman et al.
(1996).
16S rRNA gene sequence analysis
Total genomic DNA of strains CCR04 and CCR80 was isolated using a QIAGEN
Genomic-tip kit (Qiagen GmbhH, Hilden, Germany) according to the manufacturer’s
instructions. The 1403-bp and 1400-bp DNA fragments encompassing the 16S rRNA
genes of strains CCR04 and CCR80, respectively, were amplified by polymerase chain
reaction (PCR) using universal primers: fD1 (5-AGAGTTTGATCCTGGCTCAG-3)
and rP2 (5-ACGGCTACCTTGTTACGACTT-3) (Weisburg et al. 1991). The PCR
reaction mixture (100 μl) contained 5 U of Taq DNA polymerase (Roche, Mannheim,
Germany), 100 ng of template, 10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2,
250 μM dNTP and 0.4 μM of each primer. The mixture was first incubated for 5 min at
95 °C; then, 35 cycles were performed as follows: 4 min at 95 °C, 1 min at 58 °C and 2
1
min at 72 °C. The amplified fragments were purified from an 0.8 % agarose gel using a
gel extraction kit (iNtRON Biotechnology, Seongnam, Korea) and sequenced by
Cosmogenetech Sequencing Service (Cosmogenetech, Seoul, Korea). The 16S rRNA
sequence analysis was performed using BLAST network services at the National Center
for Biotechnology Information (NCBI). A phylogenetic tree was constructed with the
neighbor-joining method using Molecular Evolutionary Genetics Analysis (MEGA)
version 5.1 software package after performing multiple alignment of the sequences
using the CLUSTAL W program (Thompson et al. 1997). Bootstrap analysis with 1,000
replicates was conducted to evaluate the stability of the groups in the neighbor-joining
tree, using the same program (Felsenstein, 1985).
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