Downloaded from www.microbiologyresearch.org by IP: 54.90.42.35 On: Wed, 22 Jun 2016 12:50:07 Clavibacter michiganensis subsp. phaseoli subsp. nov., pathogenic in bean Ana J. Gonza ´ lez and Estefanı ´a Trapiello Correspondence A. J. Gonza ´ lez anagf@serida.org Servicio Regional de Investigacio ´ n y Desarrollo Agroalimentario (SERIDA), Principado de Asturias, 33300 Villaviciosa, Spain A yellow Gram-reaction-positive bacterium isolated from bean seeds (Phaseolus vulgaris L.) was identified as Clavibacter michiganensis by 16S rRNA gene sequencing. Molecular methods were employed in order to identify the subspecies. Such methods included the amplification of specific sequences by PCR, 16S amplified rDNA restriction analysis (ARDRA), RFLP and multilocus sequence analysis as well as the analysis of biochemical and phenotypic traits including API 50CH and API ZYM results. The results showed that strain LPPA 982 T did not represent any known subspecies of C. michiganensis. Pathogenicity tests revealed that the strain is a bean pathogen causing a newly identified bacterial disease that we name bacterial bean leaf yellowing. On the basis of these results, strain LPPA 982 T is regarded as representing a novel subspecies for which the name Clavibacter michiganensis subsp. phaseoli subsp. nov. is proposed. The type strain is LPPA 982 T (5CECT 8144 T 5LMG 27667 T ). Clavibacter michiganensis causes serious bacterial diseases in a wide variety of agriculturally important plants leading to heavy economic losses (Francis et al., 2010). There are five subspecies according to analyses of phenotypic and biochemical features (Dye & Kemp, 1977; Davis et al., 1984; Vidaver & Davis, 1988), genetic markers (Metzler et al., 1997) and specific hosts (Jahr et al., 1999). Clavibacter michiganensis subsp. michiganensis causes tomato canker; Clavibacter michiganensis subsp. sepedonicus induces potato ring rot; Clavibacter michiganensis subsp. insidiosus causes wilting and stunting in alfalfa; Clavibacter michiganensis subsp. nebraskensis is responsible for Goss’s bacterial wilt and leaf blight in corn; and Clavibacter michiganensis subsp. tessellarius causes bacterial mosaic in wheat. Three of the five subspecies (C. michiganensis subsp. michiganensis, C. michiganensis subsp. sepedonicus and C. michiganensis subsp. insidiosus) are classified as quarantine organisms worldwide and are subject to international quarantine regulations. These are vascular pathogens causing systemic infections after entering the hosts through lesions. The occurrence of symptomless latent infections and the invasion of seeds by the bacteria are widespread (Eichenlaub et al., 2006). Infected seed is considered by several authors to be the primary source of outbreaks of C. michiganensis subsp. michiganensis (Tsiantos, 1987; Milijas ˇevic ´-Marcic ´ et al., 2012). In previous work conducted in our laboratory, a yellow- pigmented, Gram-reaction-positive coryneform bacterium was isolated from bean seeds preserved in a germplasm bank and subsequently identified as C. michiganensis by 16S rRNA gene sequence comparisons (accession number HE608962; Trapiello & Gonza ´lez, 2012). This bacterial isolate (LPPA 982 T ) turned out to be responsible for a novel bacterial disease and the aim of the present study was the accurate identification of this strain. Cells were Gram-reaction-positive (Ryu, 1938), coryneform and non-motile. Colonies grown at 25 u C on King B medium (King et al., 1954) were yellow, round and entire. Strain LPPA 982 T was aerobic and negative for fluorescence on King B medium, levan (Noval, 1991), cytochrome-c oxidase, pectinolytic activity on potato slides, arginine dihydrolase (Thornley, 1960), casein (Goszczynska & Serfontein, 1998) and gelatin hydrolysis (Noval, 1991), methyl red (Dye & Kemp, 1977) and utilization of carbon compounds such as adonitol, erythritol, sorbitol, homoserine, D-tartrate, trigo- nelline, betaine and quinate; and positive for Tween 80 (Goszczynska & Serfontein, 1998) and aesculin hydrolysis, growth on 2,3,5-triphenyltetrazolium chloride (TTC; Noval, 1991) and utilization of carbon compounds such as sucrose, mannitol, myo-inositol, L-lactate, D-xylose and lactose in Ayer’s medium. The Hugh–Leifson reaction (Hugh & Leifson, 1953) with glucose was oxidative. Standardized systems (API 50CH and API ZYM; bioMe ´rieux) were used for the study of the carbohydrate metabolism Abbreviations: ARDRA, amplified rDNA restriction analysis; TTC, 2,3,5- triphenyltetrazolium chloride. The GenBank/EMBL/DDBJ accession numbers for the recA, gyrB and rpoB gene sequences of LPPA 982 T are HF954356–HF954358, respectively. Five supplementary figures and a supplementary table are available with the online version of this paper. International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1752–1755 DOI 10.1099/ijs.0.058099-0 1752 058099 G 2014 IUMS Printed in Great Britain