Rathayibacter Agropyri (Non O’Gara, 1916) Comb

TAXONOMIC DESCRIPTION Schroeder et al., Int J Syst Evol Microbiol DOI 10.1099/ijsem.0.002708

Rathayibacter agropyri (non O’Gara, 1916) comb. nov., nom. rev., isolated from western wheatgrass (Pascopyrum smithii)

Brenda K. Schroeder,1 William L. Schneider,2 Douglas G. Luster,2 Aaron Sechler2 and Timothy D. Murray3,*

Abstract Aplanobacter agropyri was first described in 1915 by O’Gara and later transferred to the genus Corynebacterium by Burkholder in 1948 but it was not included in the Approved Lists of Bacterial Names in 1980 and, consequently, is not recognized as a validly published species. In the 1980s, bacteria resembling Corynebacterium agropyri were isolated from plant samples stored at the Washington State Mycological Herbarium and from a diseased wheatgrass plant collected in Cardwell, Montana, USA. In the framework of this study, eight additional isolates were recovered from the same herbarium plant samples in 2011. The isolates are slow-growing, yellow-pigmented, Gram-stain-positive and coryneform. The peptidoglycan is type B2g containing diaminobutyric acid, alanine, glycine and glutamic acid, the cell-wall sugars are rhamnose and mannose, the major respiratory quinone is MK-10, and the major fatty acids are anteiso-15 : 0, anteiso 17 : 0 and iso-16 : 0, all of which are typical of the genus Rathayibacter. Phylogenetic analysis of 16S rRNA gene sequences placed the strains in the genus Rathayibacter and distinguished them from the six other described species of Rathayibacter. DNA– DNA hybridization confirmed that the strains were members of a novel species. Based on phenotypic, chemotaxonomic and phylogenetic characterization, it appears that strains CA-1 to CA-12 represent a novel species, and the name Rathayibacter agropyri (non O’Gara, 1916) comb. nov., nom. rev. is proposed; the type strain is CA-4T (=DSM 104101T;=ATCC TSD-78T).

The genus Rathayibacter belongs to the family Microbacter- phosphatidylglycerol and diphosphatidylglycerol as the iaceae in the order Actinomycetales and the phylum Actino- major phospholipids, saturated anteiso-15 : 0, anteiso-17 : 0 bacteria. Historically, species in genus Rathayibacter were and iso-16 : 0 as the major branched fatty acids, and rham- classified as members of the genus Corynebacterium [1], nose and mannose as the major cell-wall sugars, to the and more recently of the genus Clavibacter [2]. Originally, genus Rathayibacter. Sasaki et al. [13] subsequently pro- plant pathogenic species in the genus Corynebacterium con- posed Rathayibacter toxicus comb. nov. based primarily on sisted of Gram-stain-positive, non-spore-forming, rod- composition of its cell wall peptidoglycan content, phyloge- shaped bacteria [1]; however, this was a heterogeneous netic analyses of 16S rRNA gene and menaquinone profiles, genus [3–9] and Davis et al. [2] proposed establishing the as well as other phenotypic characteristics. Currently, the genus Clavibacter comprising the Gram-stain-positive, genus Rathayibacter contains six validly described coryneform bacteria containing 2,4-diaminobutyric acid species including Rathayibacter caricis [14], Rathayibacter (DAB) in their cell walls. At the time, plant pathogenicity festucae [14], Rathayibacter iranicus, Rathayibacter rathayi was considered a distinguishing characteristic of the bacter- (type species), Rathayibacter tritici [12] and Rathayibacter ia; however, the morphological and physiological character- toxicus [13, 15]. In addition, there are 13 genome istics, along with differences in menaquinone composition sequences deposited in NCBI GenBank including those of and phylogenetic analyses led to the discrimination of sev- R. rathayi (NZ_OCNL00000000.1), R. toxicus eral coryneform species in separate genera [1, 10, 11]. Con- (NZ_CP013292.1, NZ_CP010848.1, NZ_AUDF00000000.1, sequently, Zgurskaya et al. [12] proposed moving all of the NZ_LBFI01000000), R. tritici (NZ_CP015515.1) and one species that contain B2g peptidoglycan with DAB in the likely new species [16, 17], VKM Ac-2596 cell wall, have MK-10 as the major menaquinone, (NZ_CP015515.1), which has been provisionally named

Author affiliations: 1Department of Entomology, Plant Pathology and Nematology, University of Idaho, Moscow, ID 83844, USA; 2United States Department of Agriculture, Agricultural Research Service, Foreign Disease-Weed Science Research Unit, Ft. Detrick, MD 21702, USA; 3Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA. *Correspondence: Timothy D. Murray, [email protected] Keywords: Aplanobacter agropyri; Corynebacterium agropyri; Rathayibacter toxicus; gummosis; coryneform. Abbreviations: DAB, diaminobutyric acid; DDH, DNA-DNA hybridization; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; MP, maximum-parisomony; NBY, nutrient broth yeast extract agar; NCBI, National Center for Biotechnology Information; NCPPB, National Collection of Plant Pathogenic Bacteria; PPNS, Plant Pathology New Series; SEM, scanning electron microscopy; USDA, United States Department of Agricul- ture. Two supplementary tables are available with the online version of this article.

002708

1 Schroeder et al., Int J Syst Evol Microbiol

‘Rathayibacter tanaceti’ [16]. The other six genomes belong used in this study were obtained from the same plant spe- to unnamed species; Rathayibacter sp. Leaf294 cies, with symptoms similar to those in the original descrip- (NZ_LMNO00000000.1), Rathayibacter sp. Leaf299 tion, and from the same general geographic region as those (NZ_LMNT00000000.1), Rathayibacter sp. Leaf185 made by O’Gara, we chose to use the specific epithet from (NZ_LMPP00000000.1), Rathayibacter sp. Leaf296 O’Gara’s original work. (NZ_LMNR00000000.1), Rathayibacter sp. VKM Ac-2121 Bacterial strains CA-5 to CA-12 were isolated on nutrient (NZ_FXBM00000000.1) and Rathayibacter sp. VKM Ac- broth yeast extract (NBY) [24] agar as previously described 2630 (MUKN00000000), which is most closely related to R. for CA-1 to CA-3 [23] from seed heads labelled as Agro- caricis [17]. pyron species obtained from the Washington State Univer- Aplanobacter agropyri [18] was first isolated from Agro- sity Mycological Herbarium exhibiting signs of dried T pyron smithii (now Pascopyrum smithii) collected in the Salt bacterial ooze. Strain CA-4 was isolated from a fresh sam- Lake Valley, Utah, USA in 1915 and described as Aplano- ple of Pascopyrum smithii in the same way and later selected bacter agropyri by O’Gara [18–20]. In 1923, Bergey et al. as the type strain since it is the only isolate in this study [20] transferred A. agropyri to Phytomonas agropyri; Bur- obtained from freshly collected plant material and its char- kholder [21] subsequently transferred it to the genus Cory- acteristics are representative of other strains tested. No signs nebacterium as Corynebacterium agropyri. However, in of nematodes were observed. Gram-stain reaction was 1980 when the Approved Lists of Bacterial Names [22] was determined on 48-hr-old bacterial cells as previously published, neither A. agropyri, P. agropyri nor C. agropyri described [23] using the method described by Schaad [25]. were included, because extant cultures were not available Strains CA-1, CA-2 and CA-3 were cultivated on NBY agar, and C. agropyri and C. rathayi could not be distinguished incubated at 28 C and 48-hr-old bacterial cells were visual- on the basis of their original descriptions. Four bacterial ized by scanning electron microscopy (SEM). For SEM, cells T strains resembling C. agropyri, CA-1 to CA-4 , were iso- were harvested by scraping colonies from the agar surface, lated in the 1980s (Table 1) [23]. Eight additional bacterial suspending them in water and filtering the suspension strains, CA-5 to CA-12 (Table 1), resembling those isolated through Whatman 0.5 µm nucleopore filters (GE Healthcare by Murray [23] subsequently were isolated from Pascopy- Life Sciences). The filters containing bacterial cells were rum smithii in 2011. Traditional and molecular techniques fixed in Karnovsky’s fixative, dehydrated through a graded were used to differentiate bacterial species and there is now ethanol series and critical-point dried in a Bomer critical- sufficient evidence, presented here, to propose a new point bomb using CO2. Pieces of the filter were then placed Rathayibacter species. Cultures of the bacteria isolated and on aluminum stubs, sputter-coated with gold and viewed described by O’Gara are not available for comparison and, with a Etec auto scan scanning electron microscope (Etec therefore, it is not possible to conclude that they represent Systems). Strains were Gram-stain-positive and on NBY the same species. However, because the bacterial isolates agar colonies were slow-growing, yellow-pigmented, round,

Table 1. Bacterial isolates used in this study

Strain Host Reference

Rathayibacter agropyri CA-1 Agropyron smithii* (now Pascopyrum smithii) [23] Rathayibacter agropyri CA-2 Agropyron trachycaulum* (now Elymus trachycaulus) [23] Rathayibacter agropyri CA-3 Agropyron riparium* (now Elymus lanceolatus) [23] Rathayibacter agropyri CA-4T (DSM 104101T; ATCC TSD-78T) Pascopyrum smithii This study Rathayibacter agropyri CA-5 Pascopyrum smithii This study Rathayibacter agropyri CA-6 Pascopyrum smithii This study Rathayibacter agropyri CA-7 Pascopyrum smithii This study Rathayibacter agropyri CA-8 Pascopyrum smithii This study Rathayibacter agropyri CA-9 Pascopyrum smithii This study Rathayibacter agropyri CA-10 Pascopyrum smithii This study Rathayibacter agropyri CA-11 Pascopyrum smithii This study Rathayibacter agropyri CA-12 Pascopyrum smithii This study Rathayibacter iranicus NCPPB 2253T Triticum aestivum [4] Rathayibacter rathayi NCPPB 2980T Dactylis glomerata [4] Rathayibacter toxicus FH 79T (NCPPB 3552T) Lolium rigidum [33] Rathayibacter toxicus FH 232 (FH 100) Polypogon monspeliensis [32] Rathayibacter tritici NCPPB 18570T Triticum aestivum [4]

*Name given on original herbarium collection.

2 Schroeder et al., Int J Syst Evol Microbiol smooth and non-mucoid. The cells are non-motile, non- the order Actinomycetales (Table S1). CA strains having spore-forming coryneform rods with a size range of 0.36– identical sequences were placed into haplotypes for analysis 0.69Â0.65–1.57 µm (Fig. 1). (Table S1). Neighbour-joining (NJ) and maximum-parisomony (MP) algorithms were completed in Geneious using Phylogenetic analyses were completed using 16S rRNA gene PAUP* and MrBayes. Bootstrap analysis was completed sequences to confirm placement of bacterial strains CA-1 to with 1000 replicates. Trees generated by NJ and MP analy- CA-12 within the the Actinomycetales (Table S1, available ses were congruent and determined that strains CA-1 to in the online version of this article; Fig. 2), and subsequently CA-12 fell in a clade with Rathayibacter rathayi, separate in the Rathayibacter genus using 16S rRNA genes sequences from other genera in the Actinomycetales and supported by of the type strain of the type species of the type genus of high posterior probabilities (0.56 or greater) (Fig. 2). Within representative families. Efforts were also made to include the Rathayibacter clade, strains CA-1 to CA-12 formed two sequences that were used in a previous phylogenetic analy- clades separate from R. rathayi, which suggests that the CA ses to solidify the phylogenetic placement of the Rathayi- strains constitute a unique species. The 16S rRNA gene bacter strains (26, 27) The 16S rRNA gene sequences were sequences of all CA strains, except CA-2 and CA-9, were amplified from genomic DNA isolated using the Gram- identical and formed a monophyletic clade, supported by positive protocol for Promega Wizard using primers 27f high posterior probabilities (Fig. 3). The 16S rRNA gene (5¢-AGAGTTTGATCMTGGCTCAG-3¢) [28] and 1492 r(l) ¢ ¢ sequences of strains CA-2 and CA-9 differed from the other (5 -CCTTGTTACGACTTC-3 ) [29] from strains CA-1 to CA strains by one and two base pairs, respectively, and NJ CA-12 (Table 1) and sequenced by Elim Biopharmaceutical and MP analyses placed them within the Rathayibacter (Hayward, CA) using the GC rich protocol. 16S rRNA clade but separate from the other CA strains (Fig. 2). NJ sequences for each strain obtained from two separate ampli- and MP analyses of the 16S rRNA gene sequences from the cons were assembled using Geneious software (Biomatters) CA strains and other representative Rathayibacter species and aligned with 16S rRNA gene sequences from bacteria in demonstrated that all CA strains fell into a strongly supported clade that included R. iranicus (Fig. 3); however, only strain CA-9 was in a subclade with R. iranicus. This is likely the result of the four nucleotide differences in the 16S rRNA gene sequences observed between R. iranicus and CA haplotype 1, CA-2 and CA-9 strains that are at the same nucleotide positions, as well as the one additional nucleotide position where CA-9 and R. iranicus are the same and different from CA-2 and CA haplotype 1 strains. A preliminary multi-locus sequence analysis using rpoB, recA, gyrB and ppK gene sequences further supports the delineation of CA-1 to CA-12 strains from previously described Rathayibacter species (data not shown). Finally, DNA–DNA hybridization (DDH) was performed at DSMZ for strain CA-4T against Rathayibacter species strains R. rathayi (NCPPB 2980T), R. tritici (NCPPB 1857T), R. iranicus (NCPPB 2253T), R. caricis (DSM 15933T) and R. festucae (DSM 15932T) to confirm placement of the CA strains in the genus Rathayibacter. Based on DDH analysis, strain CA-4T exhibited 43.1±1.9% DNA relatedness with R. rathayi, 38.3±3.8 % with R. tritici, 65.3±4.1 % with R. iranicus, 40.0±5.3 % with R. caricis, and 44.0±1.1 % with R. festucae. Clearly, the placement of the CA strains as a separate Rathayibacter species is supported since all were below the 70 % DDH threshold for unique species. Bacterial strains CA-1 to CA-12 were evaluated for utilization and fermentation of specific carbon sources using the APICoryne, API20NE, APIZYMA and API 50 CH test strips (bioMerieux) with modifications to the protocols for API20NE and APICH test strips that included standardiza- tion of suspensions to 6 McFarland units, 1 ml of the sus- Fig. 1. Scanning electron micrograph of Rathayibacter agropyri cells pension being added to the API 50 CHB/E medium, and showing coryneform shape. Magnification is Â20 000; bar, 1 µm. results were recorded every 24 h for 4 days. Test results for strains CA-1 to CA-12 are recorded in Tables 2 and S2.

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Clostridium acetobutylicum Rubrobacter xylanophilus Acidothermus cellulolyticus Frankia alni 0.96 Salinispora tropica 0.99 Mycobacterium smegmatis 0.71 0.99 Nocardia farcinica 1.0 Corynebacterium efﬁciens Propionibacterium acnes

0.89 1.0 Rathayibacter rathayi Haplotype 1 0.93 Agromyces cerinus subsp. cerinus 0.67 Leifsonia poae 0.94 Agrococcus jenensis 1.0 0.94 Okibacterium fritillariae 0.89 1.0 Agreia biocolorata Subtercola boreus Cryobacterium psychrophium 0.56 0.58 Frigorbacterium faeni 0.98 Mycetocola saprophilus Clavibacter michiganensis subsp. michiganensis 1.0 Curtobacterium flaccumfaciens subsp. flaccumfaciens Janibacter sp. 0.99 1.0 Renibacterium salmoninarum 0.91 Bifidobacterium adolescentis Kineococcus radiotolerans Streptomyces avermitilis

Fig. 2. Phylogenetic placement of strains CA-1 to CA-12 in the Order Actinomycetales based on sequence similarity of the 16S rRNA gene. Tree was created in MrBayes using the HKY model with 100 000 generations. Branch labels indicate posterior probability.

Bacterial strains CA-1 and CA-4T were evaluated for sensi- performance liquid chromatography. The major respiratory tivity to iranicin using the bacteriocin assay of Gross and quinones present for strains CA-1 and CA-4T were MK-10 Vidaver [30]. Strains were evaluated in triplicate against R. (67 %), MK-9(H8) (14 %), and MK-11 (6 %). Two dimen- iranicus (NCPPB 2253T) and the assay was repeated four sional-TLC determined that strains CA-1 and CA-4T con- times (Table 2). tained three major polar lipids: diphosphatidylglycerol, phosphatidylglycerol and glycolipid. Fatty acids were Analyses of the peptidoglycan, cell-wall sugars, respiratory extracted following the method by Sasser [31] and analysed quinones and polar lipids were performed by the Identifica- using gas chromatography with a temperature ramping pro- tion Service, Leibniz-Institute DSMZ-German Collection of gram (170 C increasing 5 C every minute until 270 C was Microorganisms and Cell Cultures (DSMZ; Braunschweig, reached) for strains CA-1 to CA-12, as well at R. iranicus, R. Germany) for strains CA-1 and CA-4T. Thin layer chroma- rathayi and R. tritici (Table 1) grown for 48 h on tryptic soy tography (TLC) and two-dimensional silica gel thin-layer broth agar at 28 C. Sherlock MIS Software [31] was used to chromatography (2D-TLC) were used to determine that the identify three prevalent fatty acids including anteiso-15 : 0 peptidoglycan contained 2,4-diaminobutyric acid (DAB), (approx. 50 % of the total fatty acids), anteiso 17 : 0 (approx. alanine, glycine and glutamic acid. TLC was used to deter- 23 %) and iso-16 : 0 (approx. 15 %) characteristic of the mine that whole cell-wall sugars of strains CA-1 and CA-4T genus Rathayibacter. contained rhamnose and mannose with lesser amounts of glucose (CA-1) and glucose and galactose (CA-4T). TLC on Therefore, based on this phenotypic, chemotaxonomic and silica gel was used to separate the respiratory lipoquinones phylogenetic study it is proposed that strains CA-1 to CA- into different classes. The bands corresponding to the differ- 12 should be placed in the genus Rathayibacter as a novel ent classes of quinones were removed and analysed by high- and unique species with strain CA-4T serving as the type

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Fig. 3. Phylogenetic relationship of strains CA-1 to CA-12 with all known Rathayibacter species based on sequence similarity of the 16S rRNA gene. Tree was created in MrBayes using the HKY+I model with 10 000 000 generations; branch labels indicate posterior probability.

strain. Since neither Aplanobacter agropyri nor Corynebacte- recognize the pioneering work of O’Gara on bacterial plant rium agropyri were included in the Approved Lists [22], pathogens and to indicate that this species is not the same they were not validly published. Furthermore, because cul- as that described by O’Gara [18]. tures from O’Gara’s work are not available for comparison and because the description provided by O’Gara differs in DESCRIPTION OF RATHAYIBACTER AGROPYRI Gram-stain reaction (negative) and cell shape, we cannot (NON O’GARA, 1916) COMB. NOV., NOM. REV. conclude that the bacterium described in this study repre- sents the same species described by O’Gara [18]. Regarding Rathayibacter agropyri (a.gro.py¢ri) N.L. gen. n. agropyri cell-shape and Gram-stain reaction, O’Gara noted in his from Agropyron, former generic name of western wheat- original description [18] that ‘The organism is a short rod grass, the plant from which the type strain was isolated. ’ ’ with rounded ends and Many efforts at staining were made Basonym: Aplanobacter agropyri O’Gara 1916 [18] with Gram’s method but the results hardly warrant a con- clusion. As a rule the stain was never completely lost nor The description for Rathayibacter agropyri comb. nov., was it ever retained in the original intensity…The mounts nom. rev. differs from the original description notably in washed in alcohol lost a great deal of their intensity as com- describing a coryneform, Gram-stain-positive bacterium. pared with those not treated with alcohol and consequently Several characteristics beyond those in the original descrip- the organism must be regarded as Gram-negative’, respec- tion [18] and Burkholder’s [21] are provided. tively. In Bergey’s sixth edition, Burkholder [21] describes Colonies on NBY agar are slow-growing, yellow-pigmented, Corynebacterium agropyri as ‘Gram variable’. Considering round, smooth and non-mucoid. The Grain-stain-positive that the bacteria used in this study were isolated from the cells are non-spore-forming, non-motile, coryneform rods same plant species (some of which were collected over with a size range of 0.36–0.69Â0.65–1.57 µm [23]. Reduced 60 years ago), with symptoms similar to those in O’Gara’s nitrates to nitrogen, oxidase-negative and catalase-positive. description [19], and collected from the same general geo- Utilizes glucose, arabinose, mannose, mannitol, maltose, graphic region, we propose naming this species Rathayi- potassium gluconate and malate as a sole carbon source. N- bacter agropyri comb. nov., nom. rev. (non O’Gara, 1916) to acetyl-glucosamine, capric acid, adipic acid, trisodium citrate

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Table 2. Phenotypic characteristics that distinguish Rathayibacter agropyri comb. nov., nom. rev. from other Rathayibacter species

+, Positive; –, negative; W, weak; ND, no data; V, variable positive and negative reaction; Strains: CA-1, Rathayibacter agropyri (CA-1); CA-2, Rathayi- bacter agropyri (CA-2); CA-3, Rathayibacter agropyri (CA-3); CA-4T, Rathayibacter agropyri (CA-4T); RR, R. rathayi (NCPPB 2980T); RT, R. tritici (NCPPB 18570T); RI, R. iranicus (NCPPB 2253T); RC, R. caricis (DSM 15933T); RF, R. festucae (DSM 15932T). Strain

Characteristic CA-1 CA-2 CA-3 CA-4T RR RT RI RC RF RTx* RTx†

a Oxidase‡ ÀÀÀÀÀÀÀÀÀ ND W Hydrolysis of: b Gelatin V ÀÀÀÀÀÀ + V ND À Utilization of: Inulin ÀÀ +|| +|| À + ÀÀ + ÀÀa Melibiose ÀÀÀÀÀÀÀ + + ÀÀa Mannose + + + + À + + + + À ND Trisodium citrate ÀÀÀÀ + À + ÀÀÀ ND Fermentation of: Maltose + + + + V + À + + V†† ND Lactose ÀÀÀ + + À V + + À ND Erythritol ÀÀÀÀÀÀÀÀ + ÀÀb Methyl b-D-xylopyranoside + + + + + + + ÀÀÀ ND b D-Galactose + + + + + + + ÀÀÀ + b D-Mannose + À + + À + + + + + + L-Rhamnose ÀÀÀÀÀÀÀ + + À ND Methyl a-D-glycopyranoside ÀÀÀ +¶ ÀÀÀ + + À ND Amygdalin ÀÀÀÀÀÀÀ + + À ND Arbutin ÀÀÀ +# ÀÀ + + + À ND Melobiose ÀÀÀÀÀÀÀ + + À ND Trehalose V + + À + + À + ÀÀ ND Potassium gluconate ÀÀÀÀÀÀÀ + ÀÀ ND Potassium 5-ketoglucoate À + ÀÀ + ÀÀÀÀÀ ND Sensitivity to iranicin§ + + + + +** À** ND ND ND ND ND *RTx, R. toxicus (FH 79T [NCPPB 3552T] and FH 232). †R. toxicus data from a, Dorofeeva et al. [14]; b, Riley and Ophel [15]. ‡Data was obtained following Kovacs [33]. §Iranicin is the bacteriocin of R. iranicus Gross and Vidaver [30] strains CA-5, CA-6, CA-7, CA-8, CA-9, CA-10, CA-11, CA-12 are also sensitive to iranicin. ||Eight of twelve CA strains evaluated were positive (Table S2). ¶Ten of twelve CA strains evaluated were negative (Table S2). #Nine of twelve CA strains evaluated were positive (Table S2). **Data obtained from Zgurskaya et al. [12]. ††R. toxicus strain FH 79T was positive and strain FH 232 was variable. Shaded areas indicate physiological responses unique to different species of Rathayibacter.

and phenylacetic acid are not utilized as a carbon source. alanine, glycine and glutamic acid. Cell-wall sugars are Acid is produced from D-glucose, D-xylose, D-mannitol, rhamnose and mannose and to a lesser amount glucose. The maltose, sucrose, glycerol, L-arabinose, methyl b-D-xylopyr- major respiratory quinone is MK-10. The major fatty acids anoside, D-galactose, D-fructose, D-mannose, arbutin, salicin, are anteiso-15 : 0, anteiso 17 : 0 and iso-16 : 0. cellobiose and inulin. Aesculin is hydrolysed, but not gelatin The type strain CA-4T (=DSM104101T;=ATCC TSD-78T) or urease. Indole production is positive but not arginine was isolated from Pascopyrum smithii collected near Card- dihydrolase. Positive for pyrazinamidase, alkaline phospha- well, MT (N 45.749, W 111.849) in 1986. tase, a-glucosidase and b-galactosidase enzymatic activity. No enzymatic activity of pyrolidonyl arylamidase, b-glucu- Funding information ronidase, b-galactosidase and N-acetyl-b-glucosaminidase. We gratefully acknowledge financial support through grants funded by The cell-wall peptidoglycan is type B2g and contains DAB, the 2008 Farm Bill, Section 10 201 administered through the United

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States Department of Agriculture (USDA) Animal and Plant Health 14. Dorofeeva LV, Evtushenko LI, Krausova VI, Karpov AV, Subbotin Inspection Service (13-8130-0247-CA and 14-8130-0367-CA) and the SA et al. Rathayibacter caricis sp. nov. and Rathayibacter festucae Emerging Research Issues Program funded by the Washington State sp. nov., isolated from the phyllosphere of Carex sp. and the leaf University Agricultural Research Centre. PPNS #0726 Department of gall induced by the nematode Anguina graminis on Festuca rubra Plant Pathology, College of Agricultural, Human, and Natural Resource L., respectively. Int J Syst Evol Microbiol 2002;52:1917–1923. Sciences, Agricultural Research Centre, Hatch Project No. WNP00670, 15. Riley IT, Ophel KM. Clavibacter toxicus sp. nov., the bacterium Washington State University, Pullman, WA 99164-6430, USA. responsible for annual ryegrass toxicity in Australia. Int J Syst Acknowledgements Bacteriol 1992;42:64–68. The technical assistance of Michael J. Adams in obtaining SEM photo- 16. Vasilenko OV, Starodumova IP, Tarlachkov SV, Dorofeeva LV, graphs and the laboratory support of Stacy Mauzey is gratefully Avtukh AN et al. Draft genome sequence of "Rathayibacter tana- acknowledged. Special thanks to Dr. Ann Kennedy for her help in the ceti" strain VKM Ac-2596 isolated from Tanacetum vulgare infested completion of the fatty acid analysis. by a foliar nematode. Genome Announc 2016;4:e00512-16. Mention of trade names or commercial products in this publication is 17. Starodumova IP, Tarlachkov SV, Prisyazhnaya NV, Dorofeeva LV, solely for the purpose of providing specific information and does not Ariskina EV et al. Draft genome sequence of Rathayibacter sp. imply recommendation or endorsement by the US Department of Agri- strain VKM Ac-2630 isolated from leaf gall induced by the knap- culture. USDA is an equal opportunity provider and employer. weed nematode Mesoanguina picridis on Acroptilon repens. Conflicts of interest Genome Announc 2017;5:e00650-17. The authors declare that there are no conflicts of interest. 18. O’Gara PJ. A bacterial disease of western wheat grass, Agropyron smithiii. Phytopathol 1916;6:341–350. References 19. O’Gara PJ. A bacterial disease of western wheat-grass. First 1. Collins MD, Bradbury JF. Plant pathogenic species of Corynebacte- account of the occurrence of a new type of bacterial disease in rium. In: Sneath PHA, Mair NS, Sharpe ME and Holt JG (editors). America. Science 1915;42:616–617. Bergey’s Manual of Systematic Bacteriology, vol. 2. Baltimore, MD: 20. Bergey DH, Harrison FC, Breed RS, Hammer BW, Huntoon FM The Williams & Wilkins Co; 1986. pp. 1276–1284. et al. Genus VIII. Phytomonas gen. nov. In: Bergey’s Manual of 2. Davis MJ, Gillaspie AG, Vidaver AK, Harris RW. Clavibacter: a new Determinative Bacteriology: A Key for the Identification of Organisms genus containing some phytopathogenic coryneform bacteria, of the Class Schizomycetes. Baltimore, MD: The Williams & Wilkins including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. and Clavi- Co; 1923. pp. 174–193. bacter xyli subsp. cynodontis subsp. nov., pathogens that cause 21. Burkholder WH. Family VIII Corynebacteriaceae Lehman and Neu- ratoon stunting disease of sugarcane and bermudagrass stunting mann. In: Breed RS, Murray EGD and Hitchens A (editors). disease. Int J Syst Bacteriol 1984;34:107–117. Bergey’s Manual of Determinative Bacteriology, 6th ed. Baltimore, 3. Bousfield IJ, Smith GL, Dando TR, Hobbs G. Numerical analysis of MD: The Williams and Wilkins Co; 1948. pp. 381–401. total fatty acid profiles in the identification of coryneform, nocar- 22. Skerman VBD, Sneath PHA, McGowan V. Approved lists of bacte- dioform and some other bacteria. Microbiology 1983;129:375–394. rial names. Int J Syst Evol Microbiol 1980;30:225–420. 4. Carlson RR, Vidaver AK. Taxonomy of Corynebacterium plant 23. Murray TD. Isolation of Corynebacterium agropyri from 30- to 40- pathogens, including a new pathogen of wheat, based on poly- year-old herbarium specimens of Agropyron species. Plant acrylamide gel electrophoresis of cellular proteins. Int J Syst Disease 1986;70:378–380. Bacteriol 1982;32:315–326. 24. Vidaver AK. Synthetic and complex media for the rapid detection 5. Collins MD. Cell wall peptidoglycan and lipid composition of the of fluorescence of phytopathogenic pseudomonads: effect of the phytopathogen Corynebacterium rathayi (smith). Syst Appl Microbiol carbon source. Appl Microbiol 1967;15:1523–1524. 1983;4:193–198. 25. Schaad WW. Identification schemes. In: Schaad NW (editor). 6. Collins MD, Jones D. Lipids in the classification and identification Laboratory Guide for Identification of Plant Pathogenic Bacteria. St. of coryneform bacteria containing peptidoglycans based on 2, 4- – diaminobutyric acid. J Appl Bacteriol 1980;48:459–470. Paul, MN: American Phytopathological Society; 1980. pp. 1 11. 7. Döpfer H, Stackebrandt E, Fiedler F. Nucleic acid hybridization 26. Stackebrandt E, Brambilla E, Richert K. Gene sequence phyloge- – studies on Microbacterium, Curtobacterium, Agromyces and related nies of the family Microbacteriaceae. Curr Microbiol 2007;55:42 46. taxa. J Gen Microbiol 1982;128:1697–1708. 27. Zhi XY, Li WJ, Stackebrandt E. An update of the structure and 16S 8. Dye DW, Kemp WJ. A taxonomic study of plant pathogenic Coryne- rRNA gene sequence-based definition of higher ranks of the class bacterium species. N ZJ Agric Res 1977;20:563–582. Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher 9. Starr MP, Mandel M, Murata N. The phytopathogenic coryneform taxa. Int J Syst Evol Microbiol 2009;59:589–608. bacteria in the light of dna base composition and DNA–DNA seg- mental homology. J Gen Appl Microbiol 1975;21:13–26. 28. Lane DJ. 16S/23S rRNA sequencing. In: Stakebrandt E and Good- fellow M (editors). Nucleic Acid Techniques in Bacterial Systematics. 10. Collins MD, Brown J, Jones D. Brachybacterium faecium gen. nov., London, UK: Wiley; 1991. pp. 115–176. sp. nov., a coryneform bacterium from poultry deep litter. Int J Syst Bacteriol 1988;38:45–48. 29. Fessehaie A, de Boer SH, Levesque CA. Molecular characterization of DNA encoding 16S-23S rRNA intergenic spacer regions 11. Stackebrandt E, Smida JAN, Collins MD. Evidence of phylogenetic and 16S rRNA of pectolytic Erwinia species. Can J Microbiol 2002; heterogeneity within the genus Rhodococcus: revival of the genus 48:387–398. Gordona (Tsukamura). J Gen Appl Microbiol 1988;34:341–348. 30. Gross DC, Vidaver AK. Bacteriocins of phytopathogenic Corynebac- 12. Zgurskaya HI, Evtushenko LI, Akimov VN, Kalakoutskii LV. terium species. Can J Microbiol 1979;25:367–374. Rathayibacter gen. nov., including the species Rathayibacter rathayi comb. nov., Rathayibacter tritici comb. nov., Rathayibacter iranicus 31. Sasser M. Identification of Bacteria by Gas Chromatography of comb. nov., and six strains from annual grasses. Int J Syst Cellular, Technical note 101. North Newark, Del: MIDI, Inc; 2001. Bacteriol 1993;43:143–149. 32. Agarkova IV, Vidaver AK, Postnikova EN, Riley IT, Schaad NW. 13. Sasaki J, Chijimatsu M, Suzuki K. Taxonomic significance of 2,4- Genetic characterization and diversity of Rathayibacter toxicus. diaminobutyric acid isomers in the cell wall peptidoglycan of acti- Phytopathology 2006;96:1270–1277. nomycetes and reclassification of Clavibacter toxicus as Rathayi- 33. Kovacs N. Identification of Pseudomonas pyocyanea by the oxidase bacter toxicus comb. nov. Int J Syst Bacteriol 1998;48:403–410. reaction. Nature 1956;178:703.