Introduction

　Cyclamen (Cyclamen persicum Miller) is a popular plant for horticulture, and for many years, sales of seeds and saplings have benefited the economy of the Ena-Nakatsugawa district, Gifu Prefecture, Japan. This success can be attributed, in part, to the close at-tention that gardeners pay to diseases affecting the production of seeds and saplings. Several diseases of

cyclamen are caused by fungi and bacteria, such as anthracnose (Colletotrichum gloeosporioides), bacte-rial bud blight (Pseudomonas marginalis pv. margina-lis), fusarium wilt (Fusarium oxysporum Schlechten-dahl f. sp. cyclaminis), gray mold (Botrytis cinerea), bacterial soft rot (Erwinia carotovora subsp. carotovo-ra), and bacterial leaf blight (Pantoea agglomerans) (Database of Plant Diseases in Japan, NIAS GenBank, https://www.gene.affrc.go.jp/databases-micro_pl_dis-eases_en.php). In particular, anthracnose and bacte-rial leaf blight frequently occur in Gifu.　Among several bacterial species with fungicidal ac-tivity, Bacillus subtilis is well characterized (Weller, 1988). Most fungicidal B. subtilis secrete iturin group compounds, which consist of linear alkyl fatty acids and circular depsipeptides, including D-amino acids, as a highly active fungicidal component (Maget-Dana

J. Gen. Appl. Microbiol., 59, 89‒95 (2013)

Bacterial strain possessing both bacteriostatic and fungistatic activity (biocontrol activity) against pathogens of cyclamen (Cyclamen sp.) was isolated from the soil in Gifu Prefecture, Japan, and characterized with respect to its taxonomic and biocontrol properties. The sequence of its 16S rRNA gene, morphology, biochemistry, and fatty acid composition demonstrated that it is a strain most closely related to Alcaligenes faecalis subsp. faecalis LMG 1229T. The isolate was named A. faecalis strain AD15. A. faecalis AD15 produced hydroxylamine at maximum yields of 33.3±1.7 mg/L after 16 h cultivation in LB medium and 19.0±0.44 mg/L after 19 h cultivation in synthetic medium. Moreover, minimum inhibitory concentrations of hydroxylamine against the cyclamen pathogens Pantoea agglomerans and Colletotrichum gloeosporioides were 4.20±0.98 and 16.5±0.67 mg/L. These results indicated that the biocontrol activity of strain AD15 might be attributed to hydroxylamine, a metabolite in the culture medium, and it had the potential for biopes-ticide application.

Key Words—Alcaligenes faecalis; biocontrol; hydroxylamine; plant pathogen; taxonomy

　* Corresponding author: Dr. Shin-ichiro Yokoyama, De-partment of Environmental and Chemical Research, Industrial Technology Center, Gifu Prefectural Government, 47 Kitaoyobi, Kasamatsu, Hashima, Gifu 501‒6064, Japan.　Tel: +81‒58‒388‒3151　　Fax: +81‒58‒388‒3155　E-mail: yokoyama-shinichiro@pref.gifu.lg.jp　The DDBJ/GenBank/EMBL accession number for the 16S rRNA gene sequence of isolate AD15 is AB741081.

Full Paper

Characterization of Alcaligenes faecalis strain AD15 indicating biocontrol activity against plant pathogens

Shin-ichiro Yokoyama,1,* Yoshitomi Adachi,1 Shuichi Asakura,1 and Erina Kohyama2

1 Department of Environmental and Chemical Research, Industrial Technology Center, Gifu Prefectural Government, Hashima, Gifu 501‒6064, Japan

2 Gifu Prefectural Research Institute for Health and Environmental Sciences, Kakamigahara, Gifu 504‒0838, Japan

(Received September 4, 2012; Accepted November 15, 2012)

90 Vol. 59YOKOYAMA et al.

and Peypoux, 1994). Several kinds of biological agro-chemicals incorporating B. subtilis are registered as commercial pesticides against fungal-induced plant diseases in Japan. These agrochemicals are becom-ing increasingly popular as biopesticides. For exam-ple, Botokiller® (Arysta LifeScience Co., Tokyo, Japan) is used for the control of B. cinerea and antibacterial biopesticides using non-pathogenic E. carotovora are commercially available as the brands Ecomate® (Ku-miai Chemical Industry Co., Ltd., Tokyo, Japan) or Bio-keeper® (Arysta LifeScience). However, there is no available pesticide that has both antifungal and anti-bacterial activities. In the present study, we isolated and characterized a bacterial strain possessing bacte-riostatic and fungistatic activity against multiple patho-gens of cyclamen.

Materials and Methods

　Reagents and microorganisms.　Luria-Bertani (LB) broth (Invitrogen, Carlsbad, CA, USA) and synthetic medium (SM) consisting of 14 g K2HPO4, 6 g KH2PO4, 2 g (NH4)2SO4, 0.2 g MgSO4･7H2O, and 1 g trisodium citrate dihydrate per liter (Honda et al., 1998), were used for bacterial culture media. Potato dextrose agar and sensitivity test broth for determining the minimum inhibitory concentration (MIC) of bacteria were ob-tained from Nissui Pharmaceuticals (Tokyo, Japan). Potato dextrose broth was purchased from Becton, Dickinson and Company (Sparks, MD, USA). Other chemicals were purchased from Wako Pure Chemical Industries, Ltd, (Osaka, Japan). Escherichia coli NBRC 3301 (K-12) was used as a siderophore-positive and hydrogen cyanide-negative strain. Pantoea agglomer-ans NBRC 102470T and Colletotrichum gloeosporioi-des MAFF 237943 were used as pathogens of cycla-men.　Mixed culture assay.　Isolation of the bacterial strain with biocontrol activity was conducted as follows: The soil samples were collected at a depth of 10‒20 cm from the ground surface in various locations in Gifu Prefecture, Japan. The soil was suspended in steril-ized saline by shaking, and the soil suspension was serially diluted with sterilized saline. The diluent was spread onto an LB agar plate, which was pre-streaked with P. agglomerans NBRC 102470T or C. gloeosporioi-des MAFF 237943. The bacterial colony causing the zone of growth inhibition was picked after 3 days of incubation at 25°C. This procedure was repeated at

least three times for clonal purification of the bacteri-um.　Morphological and biochemical analyses.　The iso-lated bacteria were grown on an LB agar plate, and growth and colony formation were observed. Colonies were stained with Favor G Nissui (Nissui Pharmaceuti-cals) and observed using light microscopy (Olympus model BX50F4, Tokyo, Japan). Cultures were grown aerobically using a reciprocal shaker (10 cm stroke, 100 oscillations/min) at 30°C in LB broth upon reach-ing logarithmic phase, and a 20 µl droplet of the cul-ture on a mica disk was prepared for atomic force mi-croscopy (AFM) imaging. AFM phase images were recorded using an SPM-9600 scanning probe micro-scope (Shimadzu Corporation, Kyoto, Japan) in tap-ping mode at room temperature. Images were taken using a Type NCHR-10 PointProbe® (NanoWorld AG, Neuchâtel, Switzerland) with a spring constant of 42 N/m at a scan speed of 0.8 Hz.　The biochemical characteristics of the isolate were determined using an API system 20NE and an ID32GN API kit (bioMérieux, Lyon, France). To investigate sub-strate utilization, different supplementary carbohy-drates were added at a final concentration of 0.5% (w/v) to the medium supplemented with an ID32GN API kit and the utility was estimated.　Genetic analysis.　Genomic DNA was extracted from the isolate using a Wizard® Genomic DNA Purifi-cation kit (Promega, Madison, WI, USA). The following oligonucleotides were synthesized as primers for am-plification of the bacterial 16S rRNA gene: 9F, 5′-GAG TTTGATCCTGGCTCAG-3′; and 1510R, 5′-GGCTACC TTGTTACGA-3′. Sequencing of the 16S rRNA gene fragments was performed using an ABI PRISM 3100 Genetic Analyzer System (Applied Biosystems, Fos-ter City, CA, USA). Sequence analysis of the 16S rRNA gene was performed using BLAST (Altschul et al., 1997) at the Ribosomal Database Project (RDP, http://rdp.cme.msu.edu/). A phylogenetic tree was constructed using the neighbor-joining method with the CLUSTAL W program (Thompson et al., 1994) and MEGA (ver. 3.1) software (Kumar et al., 2004).　Analysis of cellular fatty acid content.　Extraction of bacterial fatty acids and the determination of their composition followed the procedures described in the manual (Version 6) for the Sherlock® Microbial Identifi-cation System (Version 4.5) (MIDI, Inc., Newark, DE, USA).　Determination of the G+C content of bacterial DNA.

2013 91Characterization of Alcaligenes faecalis AD15

　We used an HPLC (Model LC-10; Shimadzu) to ana-lyze enzymatically digested DNA (Katayama-Fujimura et al., 1984). An equimolar mixture of four deoxyribo-nucleotides (GC kit, Seikagaku Kogyo, Tokyo, Japan) was used as the quantitative standard.　Analyses of biocontrol compounds.　Purification of bactericidal polyketides followed the method of Kami-giri et al. (1996). In brief, culture supernatant was sub-jected to Diaion HP-20 (Mitsubishi Chemical Co., To-kyo, Japan) column chromatography (water/acetone), extracted with ethyl acetate, and purified by silica gel column chromatography (chloroform/methanol). Fifty microliters of the collected fraction was soaked into steril-ized paper disc (8 mm in diameter) on the plate pre-streaked with P. agglomerans, and the inhibitory zone was observed after 24 h incubation at 30°C. Assay for volatile organic compounds (VOCs) produced by the bacterium was performed using gas chromatography-mass spectrometer (GC-MS). Strain AD15 was cultivated in LB broth for 24 h at 30°C. Ten milliliters of the culture was sampled in Shimadzu vial (27 ml volume, Shimadzu Co., Ltd., Kyoto, Japan), capped, and incubated for 5 min at 40°C. Five hundred microliters of the head-space was subjected to GC-17A GCMS-QP5000 (Shi-madzu). GC-MS was performed as follows: DB1 (30 m × 0.25 mm I.D. × 0.25 µm thickness, Agilent Tech-nologies, Inc., Santa Clara, CA, USA) was used for GC column. The temperature of GC oven was 40°C for 3 min, 40‒250°C at 10°C/min, and held at 250°C for 5 min. MS scan range was 45 to 300 (m/z). Siderophore production was estimated using a Chrome Azurol S Agar plate assay (Schwyn and Neilands, 1987). For detection of secreted bacterial β-glucanase and chitin-ase, LB agar plates containing 0.1% (w/v) lichenan (Walsh et al., 1995) and 0.1% (w/v) colloidal chitin (Garbeva et al., 2004) were used, and the formation of halo was observed. Hydrogen cyanide was detected using cyanide indicator paper (Cyan-Test, Wako). The hydroxylamine concentration in medium was mea-sured according to the method of Frear and Burrell (1955). To measure hydroxylamine production, a 1：1,000 dilution of an overnight culture (OD600 = 0.16) was inoculated into the media and cultivated at 30°C with aeration using a reciprocal shaker (10 cm stroke, 100 osc/min). These experiments were performed in triplicate on separate occasions.　Hydroxylamine susceptibility.　The susceptibility of P. agglomerans to hydroxylamine was evaluated as MIC. In brief, 50 µl of P. agglomerans culture (OD600 =

0.16) was mixed with 12 ml of sensitivity test broth, and 20 µl of the mixture was inoculated into wells of a 96-well Multiwell™ plate (Becton-Dickinson) containing 180 µl of the test medium and hydroxylamine (0‒50 mg/L). The MIC for C. gloeosporioides was evaluated according to the method of Honda et al. (1998). The MIC was determined as residual concentration of hydroxylamine in the test culture broth after incuba-tion for 24 h (P. agglomerans) or 48 h (C. gloeosporioi-des) at 30°C. These tests were performed in triplicate with freshly prepared media on separate occasions.

Results

Isolation of a bacterium that inhibits the growth of bac-terial pathogens of cyclamen　In the mixed culture assay, 12 strains obtained from the soil in Gifu Prefecture, Japan, inhibited the growth of C. gloeosporioides MAFF 237943, which was the cause of anthracnose in cyclamen. Among these iso-lates, four strains also inhibited the growth of P. agglo-merans NBRC 102470T, which caused bacterial leaf blight in cyclamen. One of these, strain AD15, pro-duced large inhibitory zones (over 5 mm in width) with both microorganisms. Therefore, we chose strain AD15 for our studies described here.

Morphological properties of strain AD15 　The morphological properties of strain AD15 were as follows: strain AD15 grew on LB agar plates as smooth-surfaced, light yellow colonies with a diameter of less than 1.0 mm after aerobic incubation for 48 h at 30°C. Strain AD15 grew at temperatures ranging from 25°C to 45°C. Light microscopy revealed that the cells were Gram-negative bacilli and non-sporulating. Us-ing AFM, we determined that the cells were approxi-mately 0.6‒0.7 µm wide and 1.0‒1.2 µm long with pe-ritrichous flagella (3‒5 µm long and 8 nm in diameter) (Fig. 1). Strain AD15 formed clumps consisting of sev-eral cells.

Genetic analysis of strain AD15　The 1,414 base-long nucleotide sequence of the 16S rRNA gene of strain AD15 was determined and deposited in DDBJ/GenBank/EMBL under accession number AB741081. The identity of strain AD15 was de-termined by comparing the 16S rRNA gene sequence with those in GenBank. The sequence of the isolate is 99.1%, 98.9%, 98.5%, and 97.3% identical to those of

92 Vol. 59YOKOYAMA et al.

Alcaligenes faecalis subsp. faecalis LMG 1229T (acces-sion no. D88008), A. aquatilis LMG 22996T (AJ937889), A. faecalis subsp. phenolicus JT (AY296718), and A. faecalis subsp. parafaecalis GT (AJ242986), respec-tively (Fig. 2). Therefore, strain AD15 was identified as A. faecalis (and referred to as such in the text that fol-lows), a subspecies of A. faecalis.

Biochemical characteristics of A. faecalis AD15 　The cells were found to be non-sporulating and mo-tile aerobes. Catalase and oxidase assays were posi-tive, and nitrate reduction and indole production were negative. A. faecalis AD15 utilized n-capric acid, DL-malic acid, citrate, phenyl acetate, malonate, acetate, lactate, L-alanine, propionate, n-valerate, L-histidine, 3-hydroxybutyrate, L-proline, and L-tryptophan as car-bon sources, but did not utilize other carbon sources including D-glucose, L-arabinose, D-mannose, D-manni-tol, N-acetyl-D-glucosamine, maltose, gluconate, adipic acid, L-rhamnose, D-ribose, inositol, saccharose, ita-conic acid, suberic acid, 5-ketogluconic acid, glyco-gen, m-hydroxybenzoic acid, L-serine, salicin, D-melibi-ose, L-fucose, D-sorbitol, L-arabinose, 2-keto-D-gluconic acid, and p-hydroxybenzoic acid. Further, the strain hydrolyzed neither esculin, gelatin, nor starch. A. fae-

calis AD15 produced cytochrome oxidase, but not ar-ginine dihydrolase, urease, β-galactosidase, or lipase. The major fatty acids were C16:0 (32.8%), C17:0 cyclo (25.7%), and C18:1 w7c (9.86%). The G+C content of A. faecalis AD15 genomic DNA was 57.8%.　The biochemical properties of the four strains that are closely related to strain AD15 as shown by the phylogenetic tree were summarized in Table 1 (Reh-fuss and Urban, 2005; Schroll et al., 2001; van Trap-pen et al., 2005). Except for assimilation of L-serine, valerate, and α-hydroxybutyric acid as well as nitrite reduction, strain AD15 was quite similar to A. faecalis subsp. faecalis LMG 1229T. Moreover, its fatty acid composition also closely resembled that of strain LMG 1229T with respect to the major compounds (C16:0, C17:0 cyclo and C18:1 w7c) (Table 2) (Rehfuss and Ur-ban, 2005; Schroll et al., 2001; van Trappen et al., 2005).

Identification of a biocontrol compound from A. faeca-lis AD15 　Through the purification process of bactericidal polyketide, the biocontrol activity was rapidly lost, and no inhibitory zone was observed in any fraction from the silica gel column chromatography process. The results showed that a biocontrol compound produced from A. faecalis AD15 was unstable. No ion fragment corresponding to fungistatic VOC candidates like acet-

　Fig. 1.　AFM image of strain AD15.　The cells were scanned on freshly cleaved mica after air-dry-ing and observed by phase-imaging in tapping mode using an Si probe (force contact = 42 N/m).

　Fig. 2.　Comparison of 16S rRNA gene sequences from strain AD15 with other Betaproteobacteria.　The phylogenetic tree was constructed using the FigTree pro-gram ver. 1.3.1 (http://tree.bio.ed.ac.uk/). The super-imposed T represents the type strains. Numbers at the branch points are bootstrap values based on 1,000 samples. Database accession numbers are enclosed in parentheses. Burkholderia cepacia ATCC25416T was used as the outgroup. The scale bar repre-sents the genetic distance (operational taxonomic unit).

2013 93Characterization of Alcaligenes faecalis AD15

amide, benzothiazole, phenylacetaldehyde, 1-decene, methanamine, 1-butanamine, or benzaldehyde (Zou et al., 2007) was detected by GC-MS. Tests for sidero-phore, β-glucanase, chitinase, and hydrogen cyanide production were also negative. Only hydroxylamine was positive.　We next examined the time course of production of hydroxylamine by A. faecalis AD15 cultivated in LB or

SM (Fig. 3). The maximum yield of hydroxylamine was 33.3±1.7 mg/L after 16 h cultivation in LB and 19.0±0.44 mg/L after 19 h cultivation in SM.

Biocontrol activity of hydroxylamine against pathogens of cyclamen 　In the hydroxylamine susceptibility test, we found that hydroxylamine was easy to decompose through

Table 1.　Characteristics that differentiate strain AD15 from other closely related genus Alcaligenes.

Characteristic Strain AD15A. faecalis

A. aquatilissubsp. faecalis subsp. phenolicus subsp. parafaecalis

Growth at 42°C + + + - -Nitrite reduction - + + - +Assimilation of:　Glycogen - - - w +　L-Histidine + + + - +　L-Proline + + + + -　L-Tryptophan + + + - -　L-Serine - + - - +　Malonate + + + + -　Propionate + + + + -　Suberate - - ND ND +　Valerate + - ND + +α-Hydroxybutyric acid + - - + -α-Ketoglutaric acid - - + - -Degradation of gelatin - - ND + -GC content (%) 57.8 56‒59 54.8 56 56

　w: weak, ND: not determined.

Table 2.　Fatty acid compositions of Alcaligenes species.

Fatty acid Strain AD15A. faecalis

A. aquatilissubsp. faecalis subsp. phenolicus subsp. parafaecalis

C10:0 tr 1.8 － - 2.30C12:0 3.58 1.5 3.2 tr trC12:0 2-OH 1.57 2.6 2.2 1.9 2.60C14:0 3.24 1.1 tr 2.2 trC14:0 2-OH - - 8.1 8.6 -C16:0 32.8 30.6 35.8 31.4 32.7C16:1 - - － 30.4 -C17:0 cyclo 25.7 27.7 9.5 16.3 13.3C18:0 1.60 1.1 1.1 tr 1.00C18:1 w7c 9.86 11.2 1.1 7.5 9.10Summed　feature 2

9.49 11.0 8.4 - 11.3

Summed　feature 3

8.91 10.2 29.3 - 24.6

　Mean percentage of total fatty acid is given for strain AD15. Data are from Rehfuss and Urban (2005), Schroll et al. (2001), van Trappen et al. (2005), and this study.　tr: trace amounts (＜ 1% of total), - : not detected.

94 Vol. 59YOKOYAMA et al.

the incubation time in the medium. P. agglomerans and C. gloeosporioides indicated growth inhibition, when the hyclroxlamine was added above 50 mg/L and 500 mg/L as reagent base, respectively. However, the residual concentration evaluated in the sensitivity test broth (50 mg/L) was 25.1±0.22 mg/L at the time of addition (0 h) and 4.20±0.98 mg/L after 24 h incuba-tion. The residual concentrations in potato dextrose broth (500 mg/L) were 133±11 mg/L at the time of ad-dition, 20.5±0.55 mg/L after 24 h incubation, and 16.5±0.67 mg/L after 48 h incubation. Therefore, the MICs of hydroxylamine for P. agglomerans and C. gloeo-sporioides were 4.20±0.98 mg/L and 16.5±0.67 mg/L.

Discussion

　We report here the isolation and characterization of a strain of A. faecalis designated AD15 that exhibited bacteriostatic and fungistatic activities. The identity of AD15 was revealed by the sequence of its 16S rRNA gene, which was 99.1‒97.3% identical to those of Alca-ligenes species, including A. faecalis and A. aquatilis. The phylogenetic tree indicates that strain AD15 be-longs to the same cluster as other A. faecalis subspe-cies (Fig. 2).　The morphological and biochemical properties (Ta-bles 1 and 2) indicate that strain AD15 is most closely related to A. faecalis subsp. faecalis.　Alcaligenes sp. YL-02632S (Kamigiri et al., 1996; Toku-naga et al., 1996), Pseudomonas batumici (Smirnov et al., 2000), and P. fluorescens (Mattheus et al., 2010) produce polyketides kalimantacin/batumin that are bacteriocidal for Staphylococcus aureus. Zou et al. (2007) reported that A. faecalis MHS033 and MHS013

produces fungistatic VOCs, such as methanamine, 1-butanamine, and/or benzaldehyde. It was also re-ported that A. faecalis BCCM 2374 excretes the fungi-cidal siderophore bavistin (Sayyed and Chincholkar, 2009). Moreover, Kavroulakis et al. (2010) reported that Alcaligenes sp. AE1.16 produce β-glucanase, chi-tinase and hydrogen cyanide as biocontrol agents.　Bactericidal polyketides, fungistatic VOCs, β-glucanase, chitinase, hydrogen cyanide, and siderophore were not detected from A. faecalis AD15, but hydroxylamine was detected. A. faecalis No. 4 was introduced as a fun-gistatic bacterium, because it produces hydroxylamine (Honda et al., 1998; Joo et al., 2005). There is no taxo-nomical data for A. faecalis No. 4 available for compari-son with that of A. faecalis AD15.　It is difficult to evaluate the biocontrol activity of hy-droxylamine because it is unstable, particularly in the presence of iron and high temperature (Iwata and Koseki, 2003) and would be gradually consumed in culture medium. Honda et al. (1998) estimated the bio-control activity of hydroxylamine against F. oxysporum and determined the MIC value as 10 mg/L. In the pres-ent study, the MIC values were 4.20±0.98 mg/L (against P. agglomerans) and 16.5±0.67 mg/L (against C. gloeosporioides). Considering the level of hydroxyl-amine production (33.3±1.7 mg/L after 16 h cultiva-tion in LB medium and 19.0±0.44 mg/L after 19 h cul-tivation in synthetic medium), A. faecalis AD15 would produce enough hydroxylamine to exert biocontrol activity against these pathogens in the culture medium, whereas the biocontrol activity of hydroxylamine was gradually lost upon extended cultivation, even if hydrox-ylamine was added over the MIC. Hence, hydroxyl-amine was bacteriostatic and fungistatic but not bacte-ricidal or fungicidal.　In conclusion, we demonstrate here that A. faecalis AD15, which was isolated from soil in Gifu Prefecture, exerts biocontrol activity against pathogens of cycla-men. Taxonomic analyses indicated that strain AD15 belongs to A. faecalis. A. faecalis AD15 produces hy-droxylamine as a biocontrol compound and has the potential to be a good candidate as a low persistent biopesticide. We are now investigating the suppres-sive effect of strain AD15 on the damping-off of cycla-men caused by P. agglomerans and C. gloeosporioi-des in soil and plant systems.

　Fig. 3.　Growth and hydroxylamine production by A. faecalis strain AD15 in LB and synthetic media.　Symbols: circles, LB-culture; triangles, synthetic medium-culture. Open symbols and closed symbols indicate the OD600 and hydroxylamine concentration. Values are means of tripli-cate values ± standard deviation.

2013 95Characterization of Alcaligenes faecalis AD15

Acknowledgments

　This work was supported in part by a Grant-in-Aid for Encour-agement of Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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