International Journal of Systematic and Evolutionary Microbiology (2015), 65, 4335–4340 DOI 10.1099/Ijsem.0.000581

International Journal of Systematic and Evolutionary Microbiology (2015), 65, 4335–4340 DOI 10.1099/ijsem.0.000581

Variovorax gossypii sp. nov., isolated from Gossypium hirsutum

Peter Ka¨mpfer,1 Hans-Ju¨rgen Busse,2 John A. McInroy3 and Stefanie P. Glaeser1

Correspondence 1Institut fu¨r Angewandte Mikrobiologie, Universita¨t Giessen, Giessen, Germany Peter Ka¨mpfer 2Institut fu¨r Mikrobiologie, Veterina¨rmedizinische Universita¨t, A-1210 Wien, Austria peter.kaempfer@umwelt. 3 uni-giessen.de Department of Entomology and Plant Pathology, Auburn University, AL 36849, USA

A beige-pigmented bacterial strain (JM-310T), isolated from the healthy internal root tissue of 4-week-old cotton (Gossypium hirsutum, cultivar ‘DES-119’) in Tallassee (Macon county), Alabama, USA, was studied taxonomically. The isolate produced small rod-shaped cells, which showed a Gram-negative staining behaviour. A comparison of the 16S rRNA gene sequence of the isolate revealed 99.2, 98.8, 98.7, 98.7, 98.1 and 97.6 % similarity to the 16S rRNA gene sequences of the type strains of Variovorax paradoxus, Variovorax boronicumulans, Variovorax ginsengisoli, Variovorax soli, Variovorax deﬂuvii and Variovorax dokdonensis, respectively. In phylogenetic trees based on 16S rRNA gene sequences, strain JM-301T was placed within the monophyletic cluster of Variovorax species. The fatty acid proﬁle of strain JM-310T

consisted mainly of the major fatty acids C16 : 0,C10 : 0 3-OH and summed feature 4 (iso- T C15 : 0 2-OH/C16 : 1v7c/t). The quinone system of strain JM-310 contained predominantly ubiquinone Q-8 and lesser amounts of Q-7 and Q-9. The major polyamine was putrescine and the diagnostic polyamine 2-hydroxyputrescine was detected as well. The polar lipid proﬁle consisted of the major lipids phosphatidylethanolamine, phosphatidylglycerol, diphospatidylglycerol and several unidentiﬁed lipids. DNA–DNA hybridization experiments with V. paradoxus LMG 1797T, V. boronicumulans 1.22T, V. soli KACC 11579T and V. ginsengisoli 3165T gave levels of relatedness of ,70 %. These DNA–DNA hybridization results in addition to differential biochemical properties indicate clearly that strain JM-310T is a member of a novel species, for which the name Variovorax gossypii sp. nov. is proposed. The type strain is JM-310T (5LMG 28869T5CIP 110912T5CCM 8614T).

The genus Variovorax was initially proposed along with the 2006), V. boronicumulans (Miwa et al., 2008), V. ginsengisoli reclassification of Alcaligenes paradoxus as Variovorax para- (Im et al., 2010) and V. defluvii (Jin et al., 2012). doxus (Willems et al., 1991). Phylogenetic analyses based Strain JM-310T was isolated in 1990 as an endophyte from on 16S rRNA gene sequences revealed that this genus was the healthy internal root tissue of 4-week-old cotton grouped within the family Comamonadaceae in the class (Gossypium hirsutum, cultivar ‘DES-119’) grown at the Plant Betaproteobacteria (Anzai et al., 2000). The name refers to Breeding Unit facility at the E.V. Smith Research Center in the organotrophic and facultatively chemolithotrophic Tallassee (Macon county), Alabama, USA. metabolism of species of the genus (Willems et al., 1991), and species are characterized by their versatility in using a Initial isolation and the testing of cultural and mor- wide variety of organic acids, including aminoacids. The phological characteristics were determined from fresh major fatty acids are palmitoleic acid (C16 : 1), palmitic cultures grown on trypticase soy agar (TSA; Oxoid) at acid (C16 : 0)andcis-vaccenic acid (C18 : l). The majority of 28 8C. For Gram staining, a slightly modified method the described species have been isolated from soil, waste- of that of Gerhardt et al. (1994) was used and the motility water and plants. The genus harbours, at the time of test was done under light microscopy (Axiophot2; Zeiss) writing, six recognized species, Variovorax paradoxus, at 10006 on cells grown for 3 days in trypticase soy V. dokdonensis (Yoon et al., 2006), V. soli (Kim et al., broth (TSB; Oxoid) at 28 8C. Temperature-dependent growth was tested at 4, 10, 15, 20, 30, 36 and 45 8Con nutrient agar (NA; Oxoid). NaCl tolerance was investi- The GenBank/EMBL/DDBJ accession number of the 16S rRNA gene gated at different concentrations of NaCl [0.5–8.0 % sequence of strain JM-310T is KR349468. (w/v)] in TSB.

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Table 1. Differential physiological characteristics between strain JM-310T and the type strains of recognized Variovorax species

Strains: 1, JM-310T;2,V. ginsengisoli Gsoli 3165T;3,V. paradoxus DSM 66T;4,V. soli GH9-3T;5,V. boronicumulans BAM-48T;6,V. dokdonensis DS-43T. All data (except for V. dokdonensis) are from this study. All strains were Gram-stain-negative, motile, rod-shaped and positive for catalase and oxidase. +, Positive; 2, negative; w, weakly positive.

Characteristic 1 2 3 4 5 6

Cell shape Rods Rods Rods Rods Rods Oval or rods Cell width (mm) 0.3–0.5 0.3–0.5 0.5–0.6 0.5–0.7 0.5–0.7 0.3–0.5 Cell length (mm) 1.5–3.0 1.5–3.0 1.2–3.0 1.0–1.5 1.0–2.0 0.7–2.8 a-Glucosidase 2 ++22W Utilization of substrates: D-Glucose + 2* +* +* +D + L-Arabinose ++* +* +* W* 2 D-Mannose 2 +* +* 2* 22 D-Mannitol 2 +* +* +* W* 2 Gluconate + 2* +* +* 2* + Adipate + 2* +* +* 2* + Citrate 22* 2* 2* 2D 2 Phenylacetate 2 +* +* +D 2* 2 D-Sorbitol + 2* +* +* 2* 2

*Results in congruence with those reported by Im et al. (2010) obtained with the API systems. D Results not in congruence with those reported by Im et al. (2010) obtained with the API systems.

The strain stained Gram-negative and produced visible analysis against the EzTaxon type strain database (Kim (diameter about 2 mm) beige colonies within 48 h at et al., 2012). Phylogenetic trees were calculated in the soft- 28 8C. No growth was observed at 4 or 45 8C. The strain ware package ARB release 5.2 (Ludwig et al., 2004) and the grew at 10–36 8C and at NaCl concentrations of 1–3 % (w/v). ‘All-Species Living Tree’ Project (LTP; Yarza et al., 2008) database release LTPs119 (November 2014). The sequence Colonies of the isolate showed a beige colour with entire T edges. Oxidase activity was positive when tested with of strain JM-310 and all other sequences not included in Oxidase reagent (bioMe´rieux). Cells were rod-shaped the database were aligned using the SINA online tool (0.5–0.8 mm in width and 1.5–2.0 mminlength).Nospores (v.1.2.11; Pruesse et al., 2012) and implemented into the were detected. Good growth of strain JM-310T was observed LTP database. The alignment of sequences used for tree on NA, brain heart infusion agar, R2A agar and TSA and also reconstruction was checked manually. Pairwise 16S rRNA on MacConkey agar (Oxoid). gene sequence similarities were calculated using the ARB neighbour-joining tool without an evolutionary substi- Physiological/biochemical characterization (96-well plate tution model. Phylogenetic trees were reconstructed with test system) and additional biochemical tests were per- different treeing methods, including the maximum-likeli- formed as described previously (Ka¨mpfer et al., 1991). hood method using RAxML v.7.04 (Stamatakis, 2006) The biochemical/physiological data are given in Table 1 with GTR-GAMMA and rapid bootstrap analysis and the and in the species description. maximum-parsimony method using DNAPARS v.3.6 (Felsen- Isolate JM-310T was maintained and subcultivated on NA stein, 2005). Both trees were calculated with 100 resamplings at 30 8C for 48 h and subsequently analysed for its 16S (bootstrap analysis; Felsenstein, 1985) and based on 16S rRNA gene sequence, fatty acid methyl ester composition rRNA gene sequence positions between gene termini 75 of the whole-cell hydrolysate, quinone, polyamine and and 1407 (according to the E. coli rrnB numbering; Brosius polar lipid patterns, further phenotypic characteristics, et al., 1981). and DNA–DNA relatedness to those species most closely Phylogenetic calculations placed strain JM-310T in the genus related on the basis of 16S rRNA gene sequence similarities. Variovorax within the family Comamonadaceae, clustering Phylogenetic analysis was based on nearly full-length 16S most closely with the type strains of V. paradoxus, rRNA gene sequences. The sequenced 16S rRNA gene frag- V. boronicumulans and V. ginsengisoli within the monophy- ment of strain JM-310T was a continuous stretch of 1437 nt letic cluster of the genus Variovorax (Fig. 1). Pairwise spanning gene positions 19–1466 according to the Escheri- calculations revealed highest 16S rRNA gene sequence chia coli rrnB numbering (Brosius et al., 1981). A ﬁrst phy- similarities to the type strains of V. paradoxus (99.2 %), logenetic placement and pairwise sequence similarities to V. boronicumulans (98.8 %), and V. ginsengisoli and V. soli the closest related type strains were obtained by BLAST (both 98.7 %). Sequence similarities to all other type strains

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100 5 Giesbergeria 0.10 YY287T (EF015884) 91 Comamonas composti 4 Delftia T Comamonas terrae A3-3 (GQ4972444) T Comamonas faecalis FF42 (KF509984) 73 2 Brachymonas IAM 14890T (AB089485) Lampropedia hyalina T 96 Comamonas serinivorans SP-35 (JN604116) C Dant 3-8T (DQ453128) omamonas odontotermitis T 100 Comamonas thiooxydans S23 (DQ322069) Comamonas testosteroni ATCC 11996T (M11224) Comamonas guangdongensis CY01T (EU515237) KCTC 12005T (AF275377) Comamonas koreensis T Comamonas granuli Ko03 (AB187586) 3 Diaphorobacter K601T (AJ418042) Alicycliphilus denitrificans T Melaminivora alkalimesophila CY1 (JQ676982) IAM 14880T (AB077037) Macromonas bipunctata T Tepidicella xavieri TU-16 (DQ295805) 84 Malikia granosa P1T (AJ627188) Malikia spinosa IAM 14918T (AB077038) Hydrogenophaga bisanensis K102T (EF532793) ATCC 33668T (AF078770) Hydrogenophaga pseudoflava T 100 Hydrogenophaga flava CCUG 1658 (AF078771) CCUG 20334T (AF078769) Hydrogenophaga palleronii T Hydrogenophaga taeniospiralis ATCC 49743 (AF07876) S1T (AF019037) Hydrogenophaga intermedia T Hydrogenophaga atypica BSB 41.8 (AJ585992) 98 BSB 9.5T (AJ585993) Hydrogenophaga defluvii T Hydrogenophaga caeni EMB71 (DQ372983) CC-21T (EF469609) Pseudacidovorax intermedius T Acidovorax cattleyae ICMP 2826 (AF137504) 74 ICMP 7500T (AF137506) Acidovorax citrulli T Acidovorax avenae ICMP 3183 (AF137505) Acidovorax oryzae FC-143T (DQ360414) CFBP 3232T (AJ007013) Acidovorax anthurii T Acidovorax konjaci DSM 7481 (AJ420325) NF 1078T (JQ946365) Acidovorax wautersii T Acidovorax valerianellae CFBP 4730 (AJ431731) BSB411T (Y18616) Acidovorax defluvii T Acidovorax radicis N35 (HM027578) CCUG 2113T (AF078765) Acidovorax facilis T 100 Verminephrobacter eiseniae EF01-2 (DQ327663) At4T (FJ214186) Verminephrobacter aporrectodeaeT Acidovorax delafieldii ATCC 17505 (AF078764) CCUG 11779T (AF078766) Acidovorax temperansT Acidovorax soli BL21 (FJ599672) T Acidovorax caeni R-24608 (AM084006) 100 7 Polaromonas 74 ANT.BRT (GU233447) Rhodoferax antarcticus T 93 Rhodoferax fermentans FR2 (D16211) DSM 15236T (CP000267) Albidiferax ferrireducens T Rhodoferax saidenbachensis ED16 (FJ755906) T 100 Curvibacter gracilis 7-1 (AB109889) ATCC 14669T (AB021390) 86 Curvibacter lanceolatus 4 Limnohabitans AQ9T (AB120963) Curvibacter fontanus T Curvibacter delicatus LMG 4328 (AF078756) 4 Ramlibacter 96 3 Pseudorhodoferax T Variovorax boronicumulans BAM-48 (AB300597) DSM 66T (AJ420329) Variovorax paradoxus T Variovorax gossypii JM-310 (KR349468) T Variovorax ginsengisoli Gsoil 3165 (AB245358) 78 Variovorax dokdonensis DS-43T (DQ178978) Variovorax defluvii 2C1-bT (HQ385753) 87 Variovorax soli GH9-3T (DQ432053) Caenimonas koreensis EMB320T (DQ349098) Caenimonas terrae SGM1-15T (GU181268) 93 3 Simplicispira T Xylophilus ampelinus DSM 7250 (AJ420330) 3 Xenophilus 3 Ottowia ATCC 19624T (AF078753) Hylemonella gracilis T Comamonas kerstersii LMG 3475 (AJ430347) Comamonas terrigena IMI 359870T (AF078772) YW1T (JQ941713) Comamonas jiangduensis T Comamonas aquatica LMG 2370 (AJ430344) 100 123T (AF233877) Comamonas denitrificans T Comamonas nitrativorans 23310 (AJ251577) 96 Comamonas humi GAU11T (AB907700) BF-3T (GQ245981) Comamonas zonglianii T 100 Comamonas badia IAM 14839 (AB164432) Extensimonas vulgaris S4T (HQ596491) 100 Paucibacter toxinivorans 2C20T (AY515390) T Pelomonas saccharophila DSM 654 (AB021407)

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Fig. 1. Maximum-parsimony tree based on nearly full-length 16S rRNA gene sequences showing the phylogenetic placement of T strain JM-310 within the genus Variovorax of the family Comamonadaceae. The tree was generated in ARB using DNAPARS v.3.6 and based on 16S rRNA gene positions 75–1407 (Brosius et al., 1981). Numbers in the ‘boxes’ represent numbers of strains (sequences) considered in that ‘box’. Numbers at nodes represent bootstrap values .70 % (100 replications). Nodes marked with filled circles were also present in the maximum-likelihood tree calculated in parallel. Larger circles mark those nodes that were also supported by high bootstrap values (.70 %) in the maximum-likelihood tree. The type strains of Paucibacter toxinivorans and Pelomonas saccharophila were used to root the tree (outgroup). Bar, 0.10 nt substitutions per nucleotide position. were below 98.5 %, the recommended threshold for the extracted as described by Busse & Auling (1988). HPLC differentiation of two species (Kim et al., 2014). Sequence analysis was carried out as described by Busse et al. similarities to the type strains of the other two recognized (1997) using the equipment reported by Stolz et al. Variovorax species, V. defluvii and V. dokdonensis,were (2007). Biomass for analyses of quinones and polar lipids 98.1 and 97.6 %, respectively. was harvested at the stationary growth phase. Cellular fatty acid analysis was performed for strain JM-310T Quinones and polar lipids were extracted and analysed apply- and with the same method for V. ginsengisoli Gsoli 3165T, ing an integrated procedure (Tindall, 1990a, b; Altenburger V. paradoxus LMG 1797T, V. soli KACC 11579T and et al., 1996; Stolz et al., 2007). The quinone system was V. boronicumulans 1.22T after extraction of whole-cell composed of ubiquinones Q-8 (69 %), Q-7 (29 %) and hydrolysates according to Ka¨mpfer & Kroppenstedt (1996) Q-9 (2 %). Similar quinone systems have also been reported using the Sherlock Microbial Identification System (Sherlock for other species of the genus Variovorax, including the MIDI software v.2.11 and a TSBA peak naming table v.4.1). type species of the genus, V. paradoxus (Oyaizu-Masuchi & Komagata, 1988). The polar lipid profile (Fig. 2), here The fatty acid profile of isolate JM-310T was characterized analysed for the first time for a strain of the genus Variovorax, by large amounts of C and summed feature 4 (iso- 16 : 0 consisted of the major lipids phosphatidylethanolamine, C 2-OH/C v7c/t), which is in agreement with the 15 : 0 16 : 1 phosphatidylglycerol, diphospatidylglycerol and minor results obtained by Im et al. (2010). The detailed fatty amounts of four unidentified glycolipids (GL1–4), one uni- acid pattern is given in Table 2. dentified phospholipid (PL1), one unidentified aminolipid For analysis of the polyamines, respiratory quinones and (AL1) and one unidentified lipid (L1) not containing an polar lipids, strain JM-310T was grown on PYE medium amino residue, a phosphate residue or a sugar moiety. [0.3 % (w/v) peptone from casein and 0.3 % (w/v) yeast Major polyamines were putrescine [66.1 mmol (g dry 2 extract, pH 7.2]. Biomass subjected to polyamine analysis weight) 1] and the diagnostic polyamine 2-hydroxyputres- 2 was harvested at the late exponential growth phase and cine [11.1 mmol (g dry weight) 1]. Minor amounts of

Table 2. Cellular fatty acid proﬁles of strain JM-310T and the type strains of recognized Variovorax species

Strains: 1, JM-310T;2,V. ginsengisoli Gsoli 3165T;3,V. paradoxus DSM 66T;4,V. soli GH9-3T (Kim et al., 2006); 5, V. boronicumulans BAM-48T;6, V. dokdonensis DS-43T. All data except those in parentheses are from this study; all strains were grown on TSA for 2 days. Data in parentheses are from Im et al. (2010), with all strains grown on R2A agar for 2 days.

Fatty acid 1 2 3 4 5 6

C10 : 0 22(0.5) 2 (2) 0.5 (0.6) 2 (2) (0.6) C12 : 0 3.7 3.4 (3.6) 3.5 (3.9) 2.8 (3.3) 3.7 (3.5) (6.3) C14 : 0 2 4.2 (4.9) 2 (1.4) (0.8) 2 (1.1) (1.2) C15 : 0 22(0.5) 2.9 (7.1) 3.4 (1.2) 2 (2)(2) C16 : 0 27.7 34.4 (39.1) 26.3 (31.6) 30.4 (38.1) 32.7 (36.1) (34.9) C17 : 0 22(2) 2.3 (2.7) (1.2) 2 (2)(2) C8:03-OH 22(2) 1.0 (2)(2) 1.2 (2)(2) C10 : 0 3-OH 12.8 4.1 (3.5) 3.6 (2.9) 3.9 (4.8) 4.3 (2.9) (3.3) C14 : 0 2-OH 2.5 2 (2) 2.6 (2)(2) 2.9 (2)(2) C17 : 0 cyclo 3.9 13.6 (18.8) 2.3 (23.8) 20.6 (22.5) 7.1 (19.0) (23.1) C15 : 1v6c 22(2) 1.2 (2) 1.6 (2) 2 (2)(2) Summed feature 4* 29.8 24.4 (19.7) 23.1 (17.0) 21.6 (21.5) 33.8 (21.4) (23.4) Summed feature 7* 19.6 16.0 (9.3) 17.9 (11.9) 12.0 (5.1) 14.3 (11.3) (7.1)

*Summed features are groups of two or three fatty acids which cannot be separated by GLC with the MIDI system. Summed feature 4 comprised C16 : 1v7c/iso-C15 : 0 2-OH; summed feature 7 comprised C18 : 1v7c/v9t/v12t.

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GL1 Description of Variovorax gossypii sp. nov. 9 GL2 Variovorax gossypii (gos.sy pi.i. N.L. gen. n. gossypii of Gossypium hirsutum). DPG GL3 Cells are Gram-stain-negative, non-spore forming rods, m m GL4 approx. 0.3–0.5 m in width and 1.5–3.0 minlength. PL1 Aerobic, oxidase- and catalase-positive. Good growth PE occurs after 48 h on NA, R2A agar and TSA (all Oxoid) at 11–36 uC. Unable to grow below 4 uCorabove45uC. Colonies on NA are smooth, beige, circular and translucent. D L PG Acid is very weakly produced from -glucose, -arabinose, D-mannitol and adonitol. Acid is not produced from maltose, L-rhamnose, sucrose, trehalose, cellobiose, lactose, AL1 D-arabitol, dulcitol, erythritol, i-inositol, melibiose, methyl a-D-glucoside, raffinose, salicin, D-sorbitol or D-xylose. Aesculin hydrolysis is positive and b-galactosidase activity (ONPG) is negative. The following compounds are utilized as sole source of carbon: N-acetyl-D-glucosamine, L-arabinose, L1 D-fructose, D-glucose, gluconate, D-mannose, a-melibiose, D- ribose, D-xylose, D-mannitol, D-sorbitol, acetate, propionate, 4-aminobutyrate, adipate, azelate, fumarate, glutarate, DL-3- hydroxybutyrate, 2-oxoglutarate, pyruvate, L-alanine, b-ala- Fig. 2. Total polar lipid profile of strain JM-310T after staining nine, L-ornithine, L-aspartate, L-histidine, L-leucine, L-serine, with molybdatophosphoric acid. PE, phosphatidylethanolamine; L-proline and L-tryptophan. The following compounds are PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; GL1–4, not utilized as sole source of carbon: N-acetyl-D-galactosa- unidentified glycolipids; L1, unidentified polar lipid; PL1, unidenti- mine, L-arbutin, cellobiose, maltose, D-galactose, glycerol, fied phospholipid; AL1, unidentified aminolipid. maltitol, L-rhamnose, sucrose, salicin, trehalose, adonitol, i- inositol, putrescine, cis-aconitate, trans-aconitate, itaconate, suberate,citrate,mesaconate,L-phenylalanine, 3-hydroxybenzoate, 4-hydroxybenzoate and phenylacetate. The 2 cadaverine [0.2 mmol (g dry weight) 1] and traces of spermi- chromogenic substrates p-nitrophenyl-b-D-glucopyranoside, 2 dine and spermine [,0.1 mmol (g dry weight) 1]werealso p-nitrophenyl-b-D-galactopyranoside, p-nitrophenyl-a-D- found. This polyamine profile is in good agreement with glucopyranoside, bis-p-nitrophenyl-phosphate, bis-p-nitro- those of the majority of betaproteobacteria, including phenyl-phenyl-phosphonate, bis-p-nitrophenyl-phosphoryl- V. paradoxus (Busse & Auling, 1988; Hamana et al.,2000, choline, 2-deoxythymidine-29-p-nitrophenyl-phosphate, L- 2006). alanine-p-nitroanilide, c-L-glutamate-p-nitroanilide and L- proline-p-nitroanilide are hydrolysed but p-nitrophenyl- DNA–DNA hybridization experiments were performed b-D-xylopyranoside and p-nitrophenyl-b-D-glucuronide are between strain JM-310T and the type strains of the four clo- not hydrolysed. The major cellular fatty acids are C16 : 0, sest related Variovorax species, namely V. ginsengisoli Gsoli C10 : 0 3-OH and summed feature 4 (iso-C15 : 0 2-OH/ 3165T, V. paradoxus LMG 1797T, V. soli KACC 11579T and v T C16 : 1 7c/t). The major menaquinone is ubiquinone Q-8 V. boronicumulans 1.22 , according to the method of with lesser amounts of Q-7 and Q-9. The major polar lipids Ziemke et al. (1998) (except that for nick translation are phosphatidylethanolamine, phosphatidylglycerol and m 2 g of DNA was labelled during 3 h of incubation at diphospatidylglycerol; minor amounts of four unknown 15 8C). These experiments with V. paradoxus LMG T T T glycolipids are also present. The polyamine pattern contains 1797 , V. boronicumulans 1.22 , V. soli KACC 11579 major amounts of putrescine and 2-hydroxyputrescine. and V. ginsengisoli 3165T resulted in levels of DNA–DNA T T relatedness of 39 % (reciprocal 64 %), 40 % (reciprocal The type strain is JM-310 (5LMG 28869 5CIP T T 52 %), 24 % (reciprocal 47 %) and 17 % (reciprocal 110912 5CCM 8614 ), isolated as an endophyte from 10 %), respectively. the healthy internal root tissue of 4-week-old cotton (Gos- sypium hirsutum, cultivar ‘DES-119’) grown at the Plant Although the chemotaxonomic data demonstrate close T Breeding Unit facility at the E.V. Smith Research Center similarity between strain JM-310 with the type strains of in Tallassee (Macon county), Alabama, USA. species of the genus Variovorax, the hybridization experiments, and physiological and chemotaxonomic data allow its clear separation. On the basis of the reported results, we suggest that strain JM-310T represents a novel species Acknowledgements of the genus Variovorax, for which the name Variovorax We are grateful to Gundula Will, Maria Sowinsky and Katja Grebing gossypii sp. nov. is proposed. for excellent technical assistance.

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