Sphingomonas melonis sp. nov., a novel pathogen that causes brown spots on yellow Spanish melon...

International Journal of Systematic and Evolutionary Microbiology (2002), 52, 2081–2087 DOI: 10.1099/ijs.0.02063-0

NOTESphingomonas melonis sp. nov., a novelpathogen that causes brown spots on yellowSpanish melon fruits

1 Dipartimento diArboricoltura e Protezionedelle Piante, Universita' diPerugia, Borgo XX Giugno,74, 06121 Perugia, Italy

2 GENISTA srlCentro di Ricerca – Azienda‘Luigi Maresca’, Stat.Flacca Km. 9,5 Loc.Capratica, 04022 Fondi(Latina), Italy

3 Japan Collection ofMicroorganisms, RIKEN,Wako, Saitama 351-0198,Japan

4 Department of HostDefense, Osaka CityUniversity Graduate Schoolof Medicine. 1-4-3 Asahi-machi, Abeno-ku, Osaka545-8585, Japan

5 Department ofMicrobiology, GifuUniversity School ofMedicine, Tsukasa-machi40, Gifu 500-8705, Japan

Roberto Buonaurio,1 Vittorio M. Stravato,2 Yoshimasa Kosako,3

Nagatoshi Fujiwara,4† Takashi Naka,4 Kazuo Kobayashi,4

Curgonio Cappelli1 and Eiko Yabuuchi5

Author for correspondence: Roberto Buonaurio. Tel : 39 075 585 6461. Fax: 39 075 585 6482.e-mail : buonaurio!unipg.it

A polyphasic taxonomic study was performed on the phytopathogenicbacterial strains DAPP-PG 224T and DAPP-PG 228, which cause brown spot onyellow Spanish melon (Cucumis melo var. inodorus) fruits. Based on thepresence of glucuronosyl ceramide (SGL-1) in cellular lipids, the results of fattyacid analysis and 16S rDNA sequence comparison, the strains had beenidentified as belonging to the genus Sphingomonas and as phylogeneticallyrelated to Sphingomonas mali, Sphingomonas pruni and Sphingomonasasaccharolytica. The levels of 16S rDNA sequence similarity of these threespecies to strain DAPP-PG 224T were respectively 98<0, 98<0 and 97<4%.DNA–DNA hybridization experiments between strains pathogenic on melonfruit and S. mali, S. pruni and S. asaccharolytica revealed a16% relatedness.Based on these results, the two isolates studied are regarded as independentfrom the type strains of the three species mentioned above. Sphingomonasstrains from melon fruits are recognized as forming a genetically andphenotypically discrete species and to be differentiated by phenotypiccharacteristics from all 29 named species of the genus. Thus, the nameSphingomonas melonis sp. nov. is proposed for the isolates from diseasedmelon fruits. The type strain is DAPP-PG 224T (¯LMG 19484T ¯DSM 14444T). TheGMC content of DNA of the type strain is 65<0 mol%.

Keywords : brown spot on melon fruit, Cucumis melo var. inodorus, polyphasictaxonomy, Sphingomonas melonis sp. nov.

Brown spots caused by Sphingomonas sp. have beenreported on yellow Spanish melons (Cucumis melo var.inodorus Naud.) cultivated in greenhouses in Almeria,Spain (Buonaurio et al., 2001). When yellow Spanishmelon fruits and leaves were inoculated with thebacterium, only fruits showed disease symptoms. 16SrDNA sequence analysis performed on one bacterialisolate revealed that it was phylogenetically closelyrelated to Sphingomonas mali, Sphingomonas pruni

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Published online ahead of print on 17 May 2002 as DOI 10.1099/ijs.0.02063-0.

†Present address: Department of Microbiology and Immunology, AlbertEinstein College of Medicine, Bronx, New York, NY 10461, USA.

The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence ofstrain DAPP-PG 224T is AB055563.

and Sphingomonas asaccharolytica (Buonaurio et al.,2001).

In this paper, we have determined the taxonomicposition of two strains associated with brown spotdisease of yellow Spanish melons by a polyphasictaxonomic study. All the species transferred to threenew genera proposed by the splitting of the genusSphingomonas (Takeuchi et al., 2001) are recognizedas junior subjective synonyms of the correspondingSphingomonas species (Yabuuchi & Kosako, 2003).Thus, we show here that our two strains are membersof a novel species in the genus Sphingomonas, for whichthe name Sphingomonas melonis sp. nov. is proposed.

Phenotypic and genotypic characterization was carriedout on two isolates from melon fruits, DAPP-PG 224T

(¯LMG 19484T¯DSM 14444T) and DAPP-PG 228

02063 # 2002 IUMS Printed in Great Britain 2081

R. Buonaurio and others

Table 1. Physiological and biological characteristics of Sphingomonas melonis sp. nov. and the type strains of fourother Sphingomonas species.................................................................................................................................................................................................................................................................................................................

Strains are identified as: 1, S. melonis sp. nov. DAPP-PG 224T ; 2, S. melonis sp. nov. DAPP-PG 228; 3, S. pruni EY 4228T ; 4, S.mali EY 4341T ; 5, S. asaccharolytica EY 4229T ; 6, S. paucimobilis EY 2395T (type species of the genus). Characteristics are scoredas: , positive reaction within 3 days ; (), positive reaction after more than 4 days; ®, negative reaction, , no growth. Inbasic microbiological tests, all six strains were Gram-negative, rod-shaped, positive for hydrolysis of aesculin and negative foralcapton production, lysine and ornithine decarboxylases, arginine dihydrolase base Moeller and urease in Christensen’s medium.In the API 20NE system, all strains were positive for aesculin hydrolysis, p-nitrophenyl β--galactopyranoside and assimilation ofglucose, -arabinose, N-acetyl -glucosamine and maltose and negative for reduction of nitrate, indole from tryptophan, glucosefermentation, arginine dihydrolase, urease, gelatinase and assimilation of -mannitol and n-caprate. In oxidative acid productiontests, all six strains produced acid oxidatively from -arabinose, cellobiose, fructose, galactose, lactose, maltose, mannose,trehalose and xylose and none of the strains produced acid oxidatively from adonitol or -ribose. In Biotype 100 assimilationtests, all six strains assimilated α-()-glucose, ()-trehalose, ()-mannose, sucrose, ()-cellobiose, aesculin, fumarate and()-galactose and none of the strains assimilated ()-sorbose, (®)-ribose, ()-arabitol, (®)-arabitol, dulcitol, -tagatose,myo-inositol, -sorbitol, adonitol, hydroxyquinoline β-glucuronide, -lyxose, i-erythritol, 3-o-methyl -glucopyranose, ()-tartrate, (®)-tartrate, 2-keto--gluconate, 5-keto--gluconate, -tryptophan, (®)-quinate, m-hydroxybenzoate, benzoate, 3-phenylpropionate, m-coumarate, trigonelline, histamine, -histidine, -α-amino-n-valerate, ethanolamine, tryptamine, -glucosamine, malonate or 2-oxoglutarate.

Characteristic 1 2 3 4 5 6

Basic microbiological tests

Motility* ® ® Colony colour†

Oxidase, Kovacs’ ®Growth in the presence of 3% NaCl ®Hydrolysis of :

Gelatin ® ® ® ® ® Starch ® ® * ® Tween 80 ® ®

Citrate, Simmons’ ® ® ® ® ® DNase ®Phenylalanine deaminase ® ® ® ® ® Acylamidase ® ® ® ®Malonate () () () () ®API 20NE system tests

Assimilation of:

-Mannose ® -Malate ® ® ® Phenylacetate ® ® ® ®Gluconate ® ® ® ® Adipate ® ® ® ® ® Sodium citrate ® ® ® ® ®

Oxidase ® ®Oxidative acid production from:

Glucose () ® Rhamnose ® ®Melezitose () ® ® ® Sucrose ® ® ® -Arabinose ® ® ® Ethanol ® ® ® Melibiose ® () ® ® ® ()

Dulcitol ® ® ® ®Glycerol ® ® ® ® ®Inositol ® ® ® ® ®Mannitol ® ® ® ® ®Raffinose ® ® ® ® ® ()

Sorbitol ® ® () ® ® ®Salicin ® ® ® ® ()

Inulin ® ® ® ® ®

2082 International Journal of Systematic and Evolutionary Microbiology 52

Sphingomonas melonis sp. nov.

Table 1 (cont.)

Characteristic 1 2 3 4 5 6

Assimilation tests (Biotype 100)

Maltotriose, -glutamate ® Maltose, ()-xylose ® (®)-Malate ® α-()-Melibiose ® ® β-()-Fructose, ()-arabinose, N-acetyl

-glucosamine, -lactate, succinate

® ®

-Aspartate ® ® ® Phenylacetate, -proline ® ® ®α--Rhamnose ® ® ® ®-Galacturonate ® ® ® ®Paratinose, ()-raffinose, α-(®)-fucose,

()-melezitose, maltitol, ()-turanose,

1-o-methyl α--glucopyranoside, cis-aconitate,

citrate, glutarate, -β-hydroxybutyrate,

-tyrosine, -alanine, -serine, gentisate

® ® ® ®

1-o-Methyl β--glucopyranoside, trans-aconitate,

α-lactose, lactulose, 1-o-methyl β-galactopyranoside,

1-o-methyl α-galactopyranoside, protocatechuate,

p-hydroxybenzoate, betaine

® ® ® ® ®

β-Gentiobiose, -saccharate, -mannitol,

xylitol, glycerol, mucate, meso-tartrate,

()-malate, tricarballylate, -glucuronate,

putrescine, -gluconate, -α-amino-n-butyrate,

caprate, caprylate, -glycerate, itaconate,

-alanine, propionate

® ® ® ® ®

*Spreading growth on semi-solid agar (Gard) plate.

†, Deep yellow; , lemon yellow.

(¯LMG 19485¯DSM 14445), in comparison withSphingomonas paucimobilis EY 2395T, S. mali EY4341T, S. pruni EY 4228T and S. asaccharolytica EY4229T.

The morphology of the isolates was observed by Gramstaining and under the transmission electron micro-scope (Philips EM 400) after negative staining of thecells with an aqueous solution of 1% sodium phospho-tungstate and 0±4% sucrose (pH 6±5). Phenotypic testslisted in Table 1, referred to as ‘basic microbiologicaltests ’, were performed as reported previously (Yabuu-chi et al., 1990). The API 20NE system (bioMe! rieux)was used according to the manufacturer’s instructions.Oxidative acid production was tested by using Bacto-OF basal medium (Difco) supplemented with a total of26 carbohydrates, sugar alcohols (1%) and ethanol(3%). Biotype 100 strips (bioMe! rieux) were used toestimate the nutritional profiles of bacteria followingthe manufacturer’s instructions.

Antimicrobial susceptibility of the organisms wastested using 36 kinds of Sensi-discs and ready-to-useMueller–Hinton II medium (Becton-Dickinson), asdescribed previously (Yabuuchi et al., 1999).

For the analyses of cellular lipids and fatty acids, cellsgrown on agar media were harvested and cellular lipids

were extracted twice with chloroform}methanol, first2:1, then 1:3 (v}v). Extracted lipids were hydrolysedwith 0±5 M NaOH, as described previously (Yabuuchiet al., 1990, 1999). The total extractable lipids and theiralkaline hydrolysates were analysed by TLC with anacidic solvent system composed of chloroform}metha-nol}acetic acid}water (100:20:12:5, by vol.). Purifiedglucuronosyl ceramide (SGL-1) of S. paucimobilis EY2395T was used as the standard. Cellular fatty acidanalysis was performed as described previously(Yabuuchi et al., 1990, 1999).

Ubiquinones were purified from lipid extracts on silicagel TLC plates (Kiesel gel 60 F254, 20¬20 cm;Merck), developed with benzene and separated byreverse-phase TLC (HPTLC RP-18 F254; Merck),developed with acetone}acetonitrile (80:20, v}v) andvisualized under UV light (254 nm) (Collins & Jones,1981). The ubiquinone type was confirmed by com-paring R

fvalues with standard ubiquinones (Collins &

Jones, 1981; Komagata & Suzuki, 1987), including Q-7, Q-9 and Q-10 (Sigma) and Q-8 from Ralstoniapickettii EY 3254T (Yabuuchi et al., 1995).

Genomic DNA was prepared according to the pro-cedure of Wilson (1987). The GC content of DNAwas determined by the HPLC method described by

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Fig. 1. Electron micrograph of a negatively stained cell ofSphingomonas melonis sp. nov. strain DAPP-PG 224T. Cells werestained with a solution of 1% sodium phosphotungstate and0±4% sucrose (pH 6±5) and observed under a Philips EM 400transmission electron microscope. Plait-like structures can beobserved on the cell surface. Bar, 0±5 µm.

Tamaoka & Komagata (1984). DNA–DNAhybridiza-tion was performed at 50 °C according to the methoddescribed by Ezaki et al. (1989).

The 16S rDNA sequence of strain DAPP-PG 224T hasbeen deposited in the DDBJ under accession no.AB055563. As reference sequences, the sequences ofthe type strains of 29 validly named Sphingomonasspecies and Acetobacter aceti were obtained from theNCBI (National Center of Biotechnology Informa-tion; http:}}www.ncbi.nlm.nih.gov}). (Thompson et al., 1994) was used to align multiplesequences and to calculate nucleotide substitutionrates (K

nucvalues ; Kimura, 1980). The neighbour-

joining method (Saitou & Nei, 1987) was used toreconstruct a phylogenetic tree from the distancematrices by (written by Manolo Gouy, Labora-toire de Biome! trie, Universite! Claude Bernard – Lyon1, Villeurbanne, France). To draw our phylogenetictree, A. aceti DSM 3508T (X94066) was used as theoutgroup. Alignment positions that included gapsand}or unidentified bases were not taken into con-sideration for calculations. To evaluate the topologyof our phylogenetic tree, the sequence data weresampled 1000 times for bootstrap analysis (Felsenstein,1985).

On nutrient agar, colonies of DAPP-PG 224T andDAPP-PG 228 were 0±4–0±5 mm in diameter, circular,entire, domed, deep-yellow-pigmented with a smoothsurface and entire margins after 2 days of incubation at27 °C. Cells of the two strains were Gram-negative,non-spore-forming, rod-shaped (0±68–0±85¬1±2–1±9 µm), non-motile without flagella. However, plait-like structures were observed on the cell surface byelectron microscopy (Fig. 1).

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Fig. 2. Phylogenetic position of Sphingomonas melonis sp. nov.strain DAPP-PG 224T among the type strains of 29 validly namedSphingomonas species. Sequence accession numbers are givenin parentheses.

Results for strains DAPP-PG 224T and DAPP-PG 228in basic microbiological and API 20NE system testsare given in Table 1. Both strains produced acidoxidatively from 13 of 26 carbohydrates, sugaralcohols and ethanol in OF basal medium. Results thatdiffered for the two strains were that strain DAPP-PG 224T produced acid from glycerol while strainDAPP-PG 228 produced acid from melibiose(Table 1).

The Biotype 100 system revealed that both strainsassimilated 21 compounds as sole carbon sources. Incontrast to the other four Sphingomonas speciescompared, they assimilated -galacturonate and notα-()-melibiose (Table 1).

Both strains DAPP-PG 224T and DAPP-PG 228 wereresistant to amoxicillin, penicillin, ampicillin, pipera-cillin, cefoperazon, moxalactam, cefaclor, ceftazidime,flomoxef, cefazolin, cefmetazole, cefotaxime, aztreo-nam, carumonam, panipenem, polymyxin B and tri-methoprim. Strain DAPP-PG 228 was also resistant toroxithromycin, norfloxacin and sparfloxacin.

TLC analysis developed with an acidic solvent systemrevealed the presence of glucuronosyl ceramide (SGL-1) in extractable lipids of both melon strains as well asin extracts from the other four Sphingomonas typestrains examined.

Octadecenoic acid (C18:1) was the major componentof total fatty acids and 2-hydroxytetradecanoic acid(2-OH C14:0) was the major 2-hydroxy acid in allstrains tested except for S. asaccharolytica EY 4229T.In the type strain of S. asaccharolytica, C

"(-cyclopro-

panoic acid (C17:0 ∆) and 2-hydroxypentadecanoic



Table 2. Phenotypic characteristics of Sphingomonas melonis sp. nov. and 11 phylogenetically related Sphingomonasspecies.................................................................................................................................................................................................................................................................................................................

Species are identified as: 1, S. melonis sp. nov. ; 2, S. paucimobilis (data from Yabuuchi et al., 1990) ; 3, S. adhaesiva (Yabuuchi etal., 1990) ; 4, S. asaccharolytica (Takeuchi et al., 1995) ; 5, S. echinoides (Denner et al., 1999) ; 6, S. mali (Takeuchi et al., 1995) ; 7,S. parapaucimobilis (Yabuuchi et al., 1990) ; 8, S. pruni (Takeuchi et al., 1995) ; 9, S. roseiflava (Yun et al., 2000) ; 10, S. sanguinis(Takeuchi et al., 1993) ; 11, S. suberifaciens (van Bruggen et al., 1990) ; 12, S. trueperi (Ka$ mpfer et al., 1997). Assimilation datawere obtained from the API 20 NE, API 50 CH or Biotype 100 systems. , Positive or most strains positive when n" 1; ®,negative or most strains negative when n" 1; , not determined.

Substrate or test 1 2 3 4 5 6 7 8 9 10 11 12

Number of strains examined 2 7 1 1 1 3 4 1 5 1 12 1

Yellow or deep-yellow colonies ® ® ® ® ® ® ®Phenylalanine deaminase ® ® ® ®* ® ® ® ®* ®*

Nitrite from nitrate ® ® ® ® * ® ® ®* ®Hydrolysis of gelatin ® ® ® ® ® ® ® ® Acid produced from:

Glycerol ® ® ® ® ® ® ®* ® ®*

Rhamnose ® ® ® ® ®* ® ®Salicin ® ® ® ® ® ® ®* ®* ®

Assimilation of:

Gluconate ® ® ® ® ® ® ®Adipate ® ® ® ® ®* ® ® ®* ®Malate ® ® ® ® ® Citrate ® ® ® ® ® ® ®* ®Fructose ® ® * Galactose ® ® ®* * Melezitose ® ® ® ®* ® ® ®* *

*Data taken from Yabuuchi & Kosako (2003).

acid (2-OH C15:0) were respectively the major compo-nent of the total fatty acids and the major 2-hydroxyacid. No 3-hydroxy acid was detected in any strainstested.

The major ubiquinone of both isolates from melonfruit was Q-10, with Q-8 and Q-9 as minor compo-nents. The ubiquinone composition was consistentwith that of S. paucimobilis.

The 16S rDNA sequence of the type strain, DAPP-PG224T (1439 nt determined), revealed high similarity toS. mali (98%), S. pruni (98%), S. asaccharolytica(97±4%), Sphingomonas adhaesiva (96±7%), Sphingo-monas sanguinis (95±8%), Sphingomonas parapauci-mobilis (95±6%), Sphingomonas trueperi (95±4%),Sphingomonas echinoides (95±4%), Sphingomonas rose-iflava (95±2%), Sphingomonas suberifaciens (94±5%)and S. paucimobilis (94±1%). Fig. 2 shows theposition of strain DAPP-PG 224T within the radiationof species of the genus Sphingomonas on the basis of aneighbour-joining analysis of 16S rDNA sequences.The GC content of the DNA was respectively 65±0and 64±9 mol% for strainsDAPP-PG 224T and DAPP-PG 228. The level of DNA–DNA reassociation be-tween strains DAPP-PG 224Tand DAPP-PG 228 was97%, while it was respectively 14, 11 and 11% betweenDAPP-PG 224T and the type strains of S. mali, S. pruniand S. asaccharolytica.

Differential phenotypic characteristics for the strainsfrom melon fruit and 11 Sphingomonas species with thehigh values (94±1–98%) of 16S rDNA sequence simi-larity are listed in Table 2.

Among the members of the genus Sphingomonas, asdescribed in the next volume of the new edition ofBergey’s Manual of Systematic Bacteriology byYabuuchi & Kosako (2003), five species have beenreported to be associated with plants. Sphingomonasrosa, S. pruni, S. asaccharolytica and S. mali havebeen isolated from the roots of different plants(Takeuchi et al., 1995), while S. roseiflava has beenisolated from the ears of some Gramineae (Yun et al.,2000). Whilst these five species are not known asphytopathogenic, S. suberifaciens (originally placederroneously in the rejected genus ‘Rhizomonas ’ as‘Rhizomonas suberifaciens ’) causes corky root diseaseof lettuce (van Bruggen et al., 1990). A phytopatho-genic Sphingomonas species has been reported to causebrown spot on yellow Spanish melon (Cucumis melovar. inodorus) fruit (Buonaurio et al., 2001). Since ithas been reported that a large population of Sphingo-monas species grows on the surface of many plants(Kim et al., 1998), it could be hypothesized thatthis Sphingomonas species grows as an epiphyte onmelon fruit and that this capacity increases the like-lihood of fruit infection.



The 16S rDNA sequence of the melon fruit isolates has97% or more similarity to those of S. mali, S. pruniand S. asaccharolytica. The DNA–DNA hybridizationvalues (% 16%) obtained in the present study betweenthe two isolates from melon fruit and three phylo-genetically related Sphingomonas species indicateclearly that the two isolates represent a novel species inthe genus. Thus, we propose the name Sphingomonasmelonis sp. nov. for these isolates.

The plait-like structures on the S. melonis cell surfacewere similar to those described by Hisano et al. (1996)for Sphingomonas strain A1. They also reported thatthese structures became larger (mouth-like) when thebacterium was grown in the presence of alginate.Hashimoto et al. (1999) suggested that Sphingomonasstrain A1 incorporated alginate directly throughmouth-like pit structures using a novel ATP-bindingcassette transporter (ABC transporter).

Description of Sphingomonas melonis sp. nov.

Sphingomonas melonis [me.lo«nis. L. gen. n. melonis ofmelon (Cucumis melo var. inodorus, Spanish melon),referring to the fruit of the plant for which theorganism is pathogenic].

Colonies are pinpoint after 2 days incubation and arenever larger than 0±7–0±8 mm in diameter on sub-sequent days. They are circular and domed with asmooth surface and entire margins and are deep-yellow-pigmented. Cells are Gram-negative, rod-shaped (0±68–0±85 µm wide, 1±2–1±9 µm long), non-spore-forming and non-motile and present plait-likestructures on the cell surface. Oxidase-positive. Meta-bolism is respiratory, never fermentative or photosyn-thetic. Hydrolyses aesculin and alkalinizes malonatebroth, but does not alkalinize Simmons’ citrate me-dium. Does not hydrolyse gelatin, starch or Tween 80.Grows in the presence of 3% NaCl and is positive forp-nitrophenyl β--galactopyranoside. Assimilates glu-cose, -arabinose, N-acetyl -glucosamine, maltose, -mannose, -malate and phenyl acetate. Other proper-ties are given in Table 1. The major component ofalkali-stable cellular lipids is SGL-1. The major fattyacid of cellular lipid is C18:1; the only hydroxy acid is2-OH C14:0. No 3-OH acid are present. The majorisoprenoid quinone is ubiquinone Q-10. Susceptible toaminoglycosides, imipenem, tetracyclins and newquinolones, while resistant to penicillins and cephalo-sporins. Isolated from the brown spots of yellowSpanish melon fruits (Cucumis melo var. inodorus) inAlmeria (Spain) as the causative agent of the disease.The type strain is DAPP-PG 224T (¯LMG 19484T¯DSM 14444T). The GC content of DNA is65±0 mol%.

Acknowledgements

The work was supported partly by grants from CNR‘Diversita' di microrganismi in ambiente agroambientale-alimentare. Indagini sulla biodiversita' di batteri fitopato-

geni ’ to R.B. and partly by the Ministry of Health, Labourand Welfare in Japan (Research in Environmental Health,H-11-Seikatsu-015, Research on Emerging and Re-emergingInfectious Diseases, Health Sciences Research Grants), theMinistry of the Environment in Japan (Global EnvironmentResearch Fund) and the United States–Japan CooperativeMedical Science Program against Tuberculosis and Leprosy.

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Sphingomonas melonis sp. nov., a novel pathogen that causes brown spots on yellow Spanish melon...

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