A virulent Pseudomonas viridiflava-related bacterium has been identified as a new pathogen of soybean, one of the most important crops worldwide. The bacterium was recovered from forage soybean leaves with dark-reddish spots, and damage on petioles and pods was also observed. In contrast, common bean was not affected

Fernández, A.M. (Ana)

González, A.J. (Ana)

González-Varela, Germán

Rodicio, M.R. (M.)

San, Mateo

González Fernández, Ana J.

Fernández, Ana M.

San José, Mateo

Rodicio, M. Rosario

Repositorio Institucional de Asturias (RIA)

  Published Ahead of Print 9 March 2012. 10.1128/AEM.00223-12. 2012, 78(10):3756. DOI:Appl. Environ. Microbiol. Germán González-Varela and M. Rosario RodicioAna J. González, Ana M. Fernández, Mateo San José, Disease in Glycine maxBacterium Causes a Dark-Reddish Spot A Pseudomonas viridiflava-Relatedhttp://aem.asm.org/content/78/10/3756Updated information and services can be found at: These include:REFERENCEShttp://aem.asm.org/content/78/10/3756#ref-list-1at: This article cites 16 articles, 10 of which can be accessed freeCONTENT ALERTS more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when newhttp://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:  on October 21, 2013 by UOV BIBLIOTECA UNIVERSITARIAhttp://aem.asm.org/Downloaded from  on October 21, 2013 by UOV BIBLIOTECA UNIVERSITARIAhttp://aem.asm.org/Downloaded from  on October 21, 2013 by UOV BIBLIOTECA UNIVERSITARIAhttp://aem.asm.org/Downloaded from  on October 21, 2013 by UOV BIBLIOTECA UNIVERSITARIAhttp://aem.asm.org/Downloaded from A Pseudomonas viridiflava-Related Bacterium Causes a Dark-ReddishSpot Disease in Glycine maxAna J. González,a Ana M. Fernández,a Mateo San José,b Germán González-Varela,a and M. Rosario RodiciobLaboratorio de Fitopatología, Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Villaviciosa, Asturias, Spain,a and Departamento de BiologíaFuncional, Área de Microbiología, Universidad de Oviedo, Oviedo, Asturias, SpainbA virulent Pseudomonas viridiflava-related bacterium has been identified as a new pathogen of soybean, one of the most impor-tant crops worldwide. The bacteriumwas recovered from forage soybean leaves with dark-reddish spots, and damage on petiolesand pods was also observed. In contrast, common bean was not affected.Soybean (Glycine max [L.] Mer. In), a subtropical leguminousplant native to southeastern Asia, is one of themost importantcrops for both human food and animal feed worldwide. Threebacterial species are recognized as soybean pathogens: Pseudomo-nas syringae pv. glycinea, Xanthomonas axonopodis pv. glycines,and Curtobacterium flaccumfaciens pv. flaccumfaciens, whichcause soybean bacterial blight, soybean bacterial pustule, and bac-terial tan spot, respectively (3, 7). In Spain, a nonproducer countryfor soybean, only the last species has been sporadically detected incommon bean (Phaseolus vulgaris L.) (5).Asturias is a farming region in the north of Spain where cattlebreeding for meat andmilk production is an important economicactivity. The Regional Service of Agrofood Research andDevelop-ment (SERIDA) has an ongoing project to assess the suitability ofdifferent varieties of forage plants as cattle feed. In one of theexperimental orchards, dark-reddish spots were noticed in theleaves of soybean, which was one of the plants under study. Withtime the lesions extended to the whole plant, and the intensity ofthe damage was such that the entire culture had to be removed.In order to identify the pathogen responsible for the disease,four lots of symptomatic leaves were processed for microbiologi-cal analysis, through tissue dilacerations followed by soaking ofthe material in sterile water during 2 h. After plating on King Bagar, fluorescent and nonfluorescent colonies were obtained andtested for Gram staining and biochemical traits. Isolates showingidentical features were considered a single strain. In total, threestrains, LPPA 221, LPPA 222, and LPPA 223, were differentiated.As common features, they were Gram-negative rods, oxidative inHugh-Leifson medium, positive for the tobacco hypersensitivity,esculin, casein, gelatin, betaine, trigonelline, L-lactate, quinate,mannitol, erythritol, and m-inositol tests, and negative for thelevan, oxidase, arginine dihydrolase, sucrose, adonitol, homoser-ine, D-tartrate, and sorbitol tests (Table 1). The three strains dif-fered only with regard to fluorescence in King B agar and pectino-lytic activity. On the basis of their overall characteristics, theycould be tentatively assigned to Pseudomonas, but their biochem-ical profiles did not precisely match those expected for any otherPseudomonas species, including previously reported soybeanand/or common bean pathogens, such as P. syringae pv. glycinea,P. syringae pv. phaseolicola, P. syringae pv. syringae, and P. viridi-flava (Table 1).The sequences of nearly complete 16S rRNA genes determinedfor the three soybean strains (4) proved to be identical (GenBankaccession number FM865870 for LPPA 221) and more closelyrelated (99% identity) to 16S rRNA gene sequences of P. viri-diflava (accession no. AM909660), Pseudomonas corrugata(AF348508), andPseudomonas tolaasii (AF348507).However, thesoybean strains shared a higher number of biochemical traits withP. viridiflava (Table 1) than with P. corrugata (a nonfluorescentbacterium, positive in the oxidase test and weakly positive forarginine dihydrolase activity) or with P. tolaasii (fluorescent, pos-itive for the oxidase and the arginine dyhidrolase reactions, andnegative for hypersensitivity in tobacco leaves).For a more precise identification of the new soybean patho-gens, multilocus sequence analysis (MLSA) based on their 16SrRNA, rpoD, gyrB, and gltA genes was also performed (8, 15).Sequences were edited and aligned using the software programMEGAversion 5 (18), and phylogenetic trees were constructed forthe concatenated sequences of the four genes (Fig. 1) and for eachindividual gene (not shown), using the neighbor-joining methodwith genetic distances computed by Kimura’s two-parametermodel (9). Homologous sequences obtained from databases orgenerated in the present work were included as controls. The se-lected sequences corresponded to P. viridiflava, strains of P. syrin-gae belonging to the five clades described for this species, P. to-laasii, and P. fluorescens (13, 19). In all trees, the sequences of thesoybean strains clustered together and appeared to bemore closelyrelated to P. viridiflava than to any other Pseudomonas species,including P. syringae pv. glycinea, P. syringae pv. phaseolicola, andP. syringae pv. syringae.The three soybean strainswere also analyzed by pulsed-field gelelectrophoresis (PFGE) performed with the restriction enzymesPmeI (11 U, 6 h, 37°C; TaKaRa Biomedicals) and SwaI (11 U, 6 h,30°C; TaKaRa Biomedicals) under the conditions described bySan José et al. (14). As shown in Fig. 2A, the PFGE profiles gener-ated with each of the two enzymes were indistinguishable andwere clearly different from those obtained for P. viridiflava CECT458 and P. syringae pv. syringae CECT 4429, included as controls.Similarly, the three strains could not be differentiated by ran-domly amplified polymorphic DNA (RAPD) typing performedReceived 24 January 2012 Accepted 2 March 2012Published ahead of print 9 March 2012Address correspondence to Ana J. González, anagf@serida.org.Copyright © 2012, American Society for Microbiology. All Rights Reserved.doi:10.1128/AEM.00223-123756 aem.asm.org 0099-2240/12/$12.00 Applied and Environmental Microbiology p. 3756–3758with primers A (AGCAGCGCCTCA), OPB-6 (TGCTCTGCCC),OPB-10 (CTGCTGGGAC), OPS-19 (GAGTCAGCAG), prim-er-21 (GTGAGCGTC) and primer-6 (GAAACAGCGG) (12, 11,6, 10, 1) (see Fig. 2B for primer-21, primer-6, and OPB-10).To demonstrate that the new disease was caused by the P.viridiflava-related bacteria, LPPA 221, LPPA 222, LPPA 223, andtwo control P. viridiflava strains (CECT 458 and LPPA 79) (4)were artificially inoculated on healthy seedlings of both forageTABLE 1 Biochemical features of Pseudomonas strains from forager soybeanStraina Species Original hostResult of biochemical testbF L O P A T ES C G S AD B H TR D-Ta L-La Q M ER SO ILPPA 221 Glycine max                     LPPA 222 Glycine max                     LPPA 223 Glycine max                     Controls P. syringae pv. glycinea Glycine max        unk   unk       v   CECT 321 P. syringae pv. phaseolicola Phaseolus vulgaris                     CECT 4429 P. syringae pv. syringae Syringa vulgaris                     CECT 458 P. viridiflava Phaseolus sp.                     LPPA 79 P. viridiflava Phaseolus vulgaris    v    v v            LPPA 522 P. tolaasii Agaricus bisporus                      P. corrugata Lycopersicon esculentum      v  unk     unk  v  unk    a LPPA, Laboratory of Phytopathology of the Principality of Asturias; CECT, Spanish Type Culture Collection; , results according to Schaad (16) for P. syringae pv. glycinea andaccording to Sutra et al. (17) and Catara et al. (2) for P. corrugata.b F, fluorescence; L, levan production; O, oxidase reaction; P, pectinolytic activity, A, arginine dihydrolase; T, tobacco hypersensitivity; ES, esculin; C, casein; G, gelatin; S, sucrose;AD, adonitol; B, betaine; H, homoserine; TR, trigonelline; D-Ta, D-tartrate; L-La, L-lactate; Q, quinate; M, mannitol; ER, erythritol; SO, sorbitol; I,m-inositol;, negative;, positive; unk, unknown; v, variable.FIG 1 Phylogenetic consensus tree of the soybean Pseudomonas strains basedon the nucleotide sequences of the 16S rRNA, rpoD, gyrB, and gltA genes. Thescale bar represents the number of substitutions per site. The number shownnext to each node indicates the percent bootstrap values of 1,000 replicates.Sequences from Erwinia amylovora were treated as the outgroup. The topo-logical characteristics of the phylogenetic trees produced for individual geneswere almost identical (data not shown). P.s., P. syringae.FIG 2 PFGE and RAPD analysis of soybean strains. (A) PFGE using PmeI andSwaI. Lane 1, Pseudomonas syringae pv. syringae CECT 4429; lane 2, LPPA 221;lane 3, LPPA 222; lane 4, LPPA 223; lane 5, Pseudomonas viridiflavaCECT 458;lane S, Salmonella enterica serovar BraenderupH9812 digested with XbaI; laneL, lambda ladder PFGmarker (New England BioLabs); the last two are used assize standards. (B) RAPD analysis performed with the indicated primers. LaneM, 50- to 2,000-bp BioMarker EXT size marker (Bioventures Inc., Murfrees-boro, TN); lane 1, Pseudomonas syringae pv. syringae CECT 4429; lane 2, LPPA221; lane 3, LPPA 222; lane 4, LPPA 223; lane 5, Pseudomonas viridiflavaCECT458.Pseudomonas viridiﬂava-Like Pathogen of Glycine maxMay 2012 Volume 78 Number 10 aem.asm.org 3757soybean and common bean. For each strain, 10 seedlings weresprayedwith a bacterial suspension (108 CFU/ml) in yeast extract-peptone-glucose broth. The seedlings were maintained at 22°Cduring 1 month, with a 16/8 photoperiod and 80% relative hu-midity. They were initially kept under transparent plastic bags for48 h, to saturating humidity. Seedlings treatedwith sterile distilledwater were included as controls, and the experiments were carriedout twice. The strains from forage soybean experimentally repro-duced the dark-reddish spots characteristic of the natural infec-tion in leaves, and damage in petioles and pods was also noticed(Fig. 3). The bacterium was reisolated from inoculated plants,hence fulfilling Koch’s postulates. In contrast, symptomswere notobserved either in leaves or in petioles or pods of common bean(not shown).To the best of our knowledge, the P. viridiflava-related bacte-rium described in this study has not been previously reported as asoybean pathogen. Considering the importance of soybean as hu-man food and in animal feed and the high virulence of the newpathogen, further surveillance should be performed.ACKNOWLEDGMENTSM. Antonia Cueto Ardavín gave us the symptomatic samples of foragesoybean.Ana M. Fernández and Germán González-Varela are recipients ofgrants from the Instituto Nacional de Investigación y Tecnología Agrariay Alimentaria.Mateo San José is a recipient of a grant from theMinistry ofScience and Innovation of Spain, cofunded by the European Social Fund(reference AP-2007-03173). This work was supported by projectsRTA2005-00076 and RTA2008-00019 from the Instituto Nacional de In-vestigación y Tecnología Agraria y Alimentaria.REFERENCES1. Anonymous. 2005–2012. RAPD-primer generator. http://www2.uni-jena.de/biologie/mikrobio/tipps/rapd.html. Accessed 18 January 2012.2. Catara V, et al. 2002. Phenotypic and genomic evidence for the revision ofPseudomonas corrugata and proposal of Pseudomonas mediterranea sp.nov. Int. J. Syst. Evol. Microbiol. 52:1749–1758.3. Dunleavy JM. 1982. Bacterial tan spot, a new foliar disease of soybeans.Crop Sci. 23:473–476.4. González AJ, Rodicio MR, Mendoza MC. 2003. Identification of anemergent and atypical Pseudomonas viridiflava lineage causing bacteriosisin plants of agronomic importance in a Spanish region. Appl. Environ.Microbiol. 69:2936–2941.5. González AJ, Tello JC, RodicioMR. 2005. Bacterial wilt of beans (Phaseo-lus vulgaris) caused by Curtobacterium flaccumfaciens in SoutheasternSpain. Plant Dis. 89:1361.6. Gwo-Fang Y, Chiung-Shu L, Chann-Chao C. 1995. Differentiation ofAspergillus parasiticus from Aspergillus sojae by random amplification ofpolymorphic DNA. Appl. Environ. Microbiol. 61:2384–2387.7. Horst RK. 2008. Westcott’s plant disease handbook, 7th ed. Springer,New York, NY.8. Hwang MSH, Morgan RL, Sarkar SF, Wang PW, Guttman DS. 2005.Phylogenetic characterization of virulence and resistence phenotypes ofPseudomonas syringae. Appl. Environ. Microbiol. 71:5182–5191.9. Kimura M. 1980. A simple method for estimating evolutionary rates ofbase substitutions through comparative studies of nucleotide sequences. J.Mol. Evol. 16:111–120.10. Laconcha I, et al. 1998. Phage typing combined with pulsed-field gelelectrophoresis and random amplified polymorphic DNA increases dis-crimination in the epidemiological analysis of Salmonella enteritidisstrains. Int. J. Food Microbiol. 40:27–34.11. Lin AW, Usera MA, Barnet TJ, Goldsby RA. 1996. Application ofrandom amplified polymorphic DNA analysis to differentiate strains ofSalmonella enteritidis. J. Clin. Microbiol. 34:870–876.12. Miyata M, Aoki T, Inglish V, Yoshida T, Endo M. 1995. RAPD analysisof Aeromonas salmonicida and Aeromonas hydrophila. J. Appl. Bacteriol.79:181–185.13. Mulet M, Lalucat J, García-Valdés E. 2010. DNA sequence-based analysisof the Pseudomonas species. Environ. Microbiol. 12:1513–1530.14. San José M, Rodicio MR, Argudín MA, Mendoza MC, González AJ.2010. Regional variations in the population structure of Pseudomonas sy-ringae pathovar phaseolicola from Spain are revealed by typing with PmeIpulsed-field gel electrophoresis, plasmid profiling and virulence genecomplement. Microbiology 156:1795–1804.15. Sarkar SF, Guttman DS. 2004. Evolution of the core genome of Pseu-domonas syringae, a highly clonal, endemic plant pathogen. Appl. Environ.Microbiol. 70:1999–2012.16. SchaadNW. 1988. Laboratory guide for identification of plant pathogenicbacteria, 2nd ed. APS Press, St. Paul, MN.17. Sutra L, et al. 1997. Taxonomy of Pseudomonas strains isolated fromtomato pith necrosis: emended description of Pseudomonas corrugata andproposal of three unnamedfluorescentPseudomonas genomospescies. Int.J. Syst. Bacteriol. 47:1020–1033.18. Tamura K, et al. 2011. MEGA5: molecular evolutionary genetics analysisusing maximum likelihood, evolutionary distance, and maximum parsi-mony methods. Mol. Biol. Evol. 28:2731–2739.19. Yamamoto S, et al. 2000. Phylogeny of the genus Pseudomonas: intrage-neric structure reconstructed from the nucleotide sequences of gyrB andrpoD genes. Microbiology 146:2385–2394.FIG 3 Bacterial dark-reddish spot disease in soybean. Healthy leaves inocu-lated with sterile water (A) and damage in leaves (B), petioles (C), or pods (D)caused by strain LPPA 223 are shown.González et al.3758 aem.asm.org Applied and Environmental Microbiology

Applied and Environmental Microbiology

A Pseudomonas viridiflava-Related Bacterium Causes a Dark-Reddish Spot Disease in Glycine max

A Pseudomonas viridiflava-Related Bacterium Causes a Dark-Reddish Spot Disease in Glycine max

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