Distribution of N-Acylhomoserine Lactone-Producing Fluorescent Pseudomonads in the Phyllosphere and Rhizosphere of Potato (Solanum Tuberosum L.)
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Microbes Environ. Vol. 24, No. 4, 305–314, 2009 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME09155 Distribution of N-Acylhomoserine Lactone-Producing Fluorescent Pseudomonads in the Phyllosphere and Rhizosphere of Potato (Solanum tuberosum L.) NOBUTAKA SOMEYA1*, TOMOHIRO MOROHOSHI2, NOBUYA OKANO2, EIKO OTSU1, KAZUEI USUKI1, MITSURU SAYAMA1, HIROYUKI SEKIGUCHI1, TSUKASA IKEDA2, and SHIGEKI ISHIDA1 1National Agricultural Research Center for Hokkaido Region (NARCH), National Agriculture and Food Research Organization (NARO), 9–4 Shinsei-minami, Memuro-cho, Kasai-gun, Hokkaido 082–0081, Japan; and 2Department of Applied Chemistry, Utsunomiya University, 7–1–2 Yoto, Utsunomiya 321–8585, Japan (Received August 17, 2009—Accepted October 3, 2009—Published online October 30, 2009) Four hundred and fifty nine isolates of fluorescent pseudomonads were obtained from the leaves and roots of potato plants. Of these, 20 leaf isolates and 28 root isolates induced violacein production in two N-acylhomoserine lactone (AHL)-reporter strains—Chromobacterium violaceum CV026 and VIR24. VIR24 is a new reporter strain for long N- acyl-chain-homoserine lactones, which can not be detected by CV026. Thin-layer chromatography revealed that the isolates produced multiple AHL molecules. We compared the 16S rRNA gene sequences of these isolates with sequences from a known database, and examined phylogenetic relationships. The AHL-producing isolates generally separated into three groups. Group I was mostly composed of leaf isolates, and group III, root isolates. Group II com- prised both leaf and root isolates. There was a correlation between the phylogenetic cluster and the AHL molecules produced and some phenotypic characteristics. Our study confirmed that AHL-producing fluorescent pseudomonads could be distinguished in the phyllosphere and rhizosphere of potato plants. Key words: fluorescent pseudomonad, quorum sensing, N-acylhomoserine lactone, Solanum tuberosum L., 16S rRNA gene Potato (Solanum tuberosum L.) is one of the world’s most phenotypic characteristics of AHL-producing bacteria, espe- important crops. In Japan, many commercial cultivars are cially fluorescent pseudomonads present around potato susceptible to phytopathogens and require high-nutrient con- plants, have not. ditions for growth. Repeated pesticide applications and The objective of this study was to examine the composi- excessive fertilization may result in fungicide resistance, soil tion and production of AHL signal molecules produced by contamination, or harm to non-target organisms. We are try- fluorescent pseudomonad isolates within the total fluorescent ing to identify alternative disease management and fertiliza- pseudomonad communities present in the phyllosphere and tion strategies that are both environmentally friendly and rhizosphere of potato using a new long-acyl-chain-AHL inexpensive. One approach is the use of beneficial plant- reporter strain. We also examined the phenotypic character- related microorganisms such as biocontrol microorganisms istics such as biofilm formation, motility, antibiotics produc- and plant growth-promoting microorganisms. tion and virulence to potato plants and phylogenetic relation- Fluorescent pseudomonads are globally distributed as ships of collected isolates. judged by their frequent isolation from diverse environ- ments, including the plant rhizosphere and phylloplane (24). Materials and Methods It has been reported that many isolates of pseudomonad Chemicals species from the phylloplane and roots are useful biocontrol N-Butyryl-DL-homoserine thiolactone (C4HCTL), C4HSL, N- and plant growth-promoting agents (13, 34, 35). Fluorescent butyryl-DL-homoserine lactone (C4HSL), N-(β-ketocaproyl)-L- pseudomonads are known to have several biocontrol and homoserine lactone (3OC6HSL), N-hexanoyl-DL-homoserine plant growth-promoting traits such as antibiotic production, lactone (C6HSL), N-heptanoyl-DL-homoserine lactone (C7HSL), competition, production of plant growth-promoting sub- N-(3-oxooctanoyl)-L-homoserine lactone (3OC8HSL), N-octanoyl- DL DL stances and induction of resistance to host plants (3, 32). -homoserine lactone (C8HSL), N-decanoyl- -homoserine lactone (C10HSL), N-dodecanoyl-DL-homoserine lactone (C12HSL), These traits are regulated by complex factors in each bacte- and N-tetradecanoyl-DL-homoserine lactone (C14HSL) were used rium. Recently, it was shown that many of the beneficial as synthetic AHL-standards. All chemicals were purchased from traits of fluorescent pseudomonads are regulated by the Sigma-Aldrich (St. Louis, MO, USA). quorum-sensing system that functions via N-acylhomoserine Synthetic phenazine-1-carboxylic acid (PCA; Pharmeks, Mos- lactone (AHL) signal molecules (42). Although the diversity cow, Russia) was used as the control in the thin-layer chromatogra- of AHL-producing bacteria in some plant rhizospheres and phy (TLC) assay for the detection of PCA produced by the isolates. phylloplanes has been reported (9, 45), the distribution and Bacterial strains and culture conditions Chromobacterium violaceum ATCC 31532 produces C6HSL as * Corresponding author. E-mail: [email protected]; Tel: +81– a signal molecule for the quorum-sensing system. C. violaceum 155–62–9280; Fax: +81–155–61–2127. CV017 is a spontaneous streptomycin-resistant mutant of ATCC 306 SOMEYA et al. 31532. C. violaceum CV026, a reporter strain, is a non-C6HSL units (CFU) mL−1 of strain CV026 or VIR24 was streaked onto LB producer derived from CV017 by a mini-Tn5 insertion in the cviI agar medium. Each fluorescent pseudomonad isolate obtained from gene encoding the AHL synthase, LuxI homolog CviI (16). The potato plants was inoculated onto LB agar plates using a sterile C. violaceum type strain ATCC 12472 was used for the devel- toothpick. The tested bacterium was positioned 10 mm from the opment of a new reporter strain for long N-acyl-chain-homoserine streak of the reporter strains. The plates were incubated for 72 h at lactones. Both strains of C. violaceum were grown at 28°C in Luria- 25°C. After incubation, violacein (purple pigment) biosynthesis was Bertani (LB; Sigma-Aldrich) medium. examined at the boundary of each reporter strain with the tested iso- Fluorescent pseudomonad isolates were cultured on King’s B lates. medium (KB; Eiken Chemical, Tokyo, Japan). To examine biofilm To examine the violacein-inducing activity of each isolate, 10 formation by these isolates, a glucose-supplied basal salts medium mg of cells from the reporter strains used in the above assay was (GBS) was used, which had the following composition: (NH4)2SO4, placed in a 1.5-mL microtube. The cells were lysed by adding 100 1.1 g; K2HPO4, 2.29 g; KH2PO4, 0.9 g; MgSO4·7H2O, 5 mg; 5 mmol μL of 10% (w/v) sodium dodecylsulfate (SDS) and mixing for 10 s of glucose; and sterilized distilled water (SDW), 1 L (46). on a vortex mixer. Violacein was extracted from the cell lysate by For the potato tuber putrefaction assay, a pathogenic bacterium adding 900 μL of water-saturated butanol, vortexing for 5 s, and Pectobacterium carotovorum subsp. carotovorum NBRC 3830 was centrifuging for 5 min. Subsequently, 150 μL of each extract was used as the positive control. Strain NBRC 3830 was cultured on LB analyzed at 585 nm using a DU7400 spectrophotometer (Beckman agar medium. Coulter, CA, USA). Violacein-inducing activity was classified on the basis of A585 values, as follows: −, smaller than 0.01; ±, 0.01 to Development of a new reporter strain for long N-acyl-chain- 0.05; +, 0.05 to 0.5; ++, 0.5 to 1.0; and +++, greater than 1.0. Each homoserine lactones experiment was repeated three times. The violacein-inducing iso- The C. violaceum type strain ATCC 12472 produces several lates of fluorescent pseudomonads were selected as AHL-producers AHLs such as N-(3-hydroxydecanoyl)-L-homoserine lactone (20). and used for further analysis. An in-frame deletion mutant of the cviI gene encoding AHL synthase in ATCC 12472 was generated by homologous recombi- Extraction, detection and characterization of AHLs nation with sacB selection. DNA flanking sequences upstream and A cell suspension (1 μL) containing each AHL-producing isolate downstream of the cviI gene were amplified using genomic DNA at 108 CFU mL−1 was incubated in 5 mL of liquid LB medium on a from ATCC 12472 as the template and the following primer sets that reciprocal shaker (140 strokes min−1) for 72 h. The bacterial cells contained MluI, BglII, or SalI restriction sites (underlined): 5'-ACG- were collected by centrifugation (5,100×g for 10 min), and the CGTACTGAGCCGACTTGTCTATTTCCG-3' and 5'-AGATCT- supernatant was filtered through an 0.20-μm filter. Culture filtrates AGTACCAGTCCACCTTGTTGCAGC-3' for the upstream region, were subsequently extracted three times with an equivalent volume and 5'-AGATCTCGAAAACCGAGCTTATCCGTTCAC-3' and of ethyl acetate. After air-drying at room temperature, each extract 5'-GTCGACACCAGTACTCCCAGGAAAACTTCG-3' for the was re-dissolved in 50 μL of dimethyl sulfoxide. The sample (10 downstream region. The two PCR products were cloned into the μL, which contains extracts from 1 mL of culture filtrate) was spot- pGEM-T easy cloning vector (Promega, Madison, WI, USA) to ted onto a C18 reverse-phase TLC plate (KC18 Silica Gel 60A, construct pCV1 and pCV2. The regions upstream and downstream Whatman International, Maidstone, UK) and developed with a of the cviI gene were released from MluI-BglII-treated pCV1 and methanol-water solution (60:40, v/v). After elution, the plates were SalI-BglII-treated pCV2, respectively. Both DNA fragments were overlaid with 0.7% (w/v) agar containing 108 CFU mL−1 of inserted into the MluI-SalI-treated plasmid pGP704Sac38 (17) to the reporter strain, either CV026 or VIR24. The AHLs were char- create pGP-CV1, which contains a novel BglII site at the site of acterized on the basis of the Rf values and shapes of the spots. joining and a copy of a truncated cviI that lacks 80% of the coding Ten different AHLs were used as the standards. These were region. The 1.3-kbp Kanr cassette from PstI-digested pKPR11 (30) C4HCTL, C4HSL, 3OC6HSL, C6HSL, C7HSL, 3OC8HSL, was also inserted into the PstI site of pGP-CV1 to create pGP-CV2.