Punina N.V 1 . , Ignatov A.N. 1,2 , Matveeva E.V. 2 , Schaad N.W. 3

1. GENETIC VARIABILITY AMONG BACTERIA OF GENUS XATHOMONAS AND DEVELOPMENT OF MULTIPLEX MOLECULAR DIAGNOSTIC METHODS Punina N.V1., Ignatov A.N.1,2, Matveeva E.V.2, Schaad N.W.3 1 Center “Bioengineering” RAS, Prospect 60-let Octyabrya, 7/1 1173121 Moscow, Russia, hin-enkelte@yandex.ru ; 2Russian Research Institute of Phytopathology, Moscow region, 143080, Russia; 3USDA-ARS, Foreign Disease-Weed Science Research Unit, Ft. Detrick, MD 21702, USA ABSTRACT Genus Xanthomonas includes plant-associated proteobacteria responsible for diseases of important agricultural crop. These bacteria are disseminated with seeds and a planting stock worldwide, and identification of bacteria becomes more and more critical problem. Routine diagnostic methods are identifying the strains on species taxonomic level, and application of them is limited to only known phytopathogenic bacteria. Strains of xanthomonads may belong to either natural populations with high genetic diversity or to "epidemic" clonal group with narrow genetic base and clear difference between any two groups. So taxonomic markers for a various level must be included at PCR assay. Collection of Xanthomonas strains representative for X. campestris (Xсс), X. oryzae, X. malvacearum, X. vesicatoria, etc., was used. The genes gyrB, operon Xcc0006- Xcc0007 (annot.at genome of Xсс ATCC 33913), cytP450 were investigated. Comparison of the gyrB sequences revealed high variability for intra- and inter-specific level. Further analysis shows strains of X. vesicatoria, X. malvacearum, X. citri, X. phaseoli, X. oryzae but Xcc had an 140bp insert within the IR Xcc0006-Xcc0007. The cluster analysis of the Xcc0006-Xcc0007 nucleotide sequences had revealed 3 major groups (A, B and C) of Xcc authentic at 95 % bootstrap value, and 8 ones - at above 65 %. "Epidemic" strains from the different countries, from natural populations, and virulent on several host plant were placed in separate groups. It was kept for cytP450 gene. Table 1. List of Xanthomonas sp. strains used in the work RESULTS Strains. Used strains of xanthomonads infecting cruciferous, compositeous, solanaceous, and gramineous plants were collected from over the World and represent considerable part of genetic variability within the genus Xanthomonas (Table 1).. Pathogenicity andpathovarsstructure. All cultures were tested on corresponded host plants and produced either systemic or leaf spot lesions under glasshouse conditions. Genetic diversity.108gyrBgene fragments with full length 2395 b.p. (97.96% of whole gene)display clearintraspecies differences for Xcc, X.vesicatoria, X.oryzae,X.axonopodis, including8groupsХсс, 2groups belonging to nonidentified species, affected crucifers and solanacées (Fig. 1a). 74fragmentsof Xcc0006-Xcc0007operon, length 1971b.p. (86.30% per whole length) display clearintraspecies differences for Xcc, X.vesicatoria(1), X.oryzae(2), X.axonopodis(2), dealing with presents/absents of insertions 25 b.p.(1) и 127 (2)b.p., respectively (Figs. 1b and 2). 37gene cytP450 frugments, length 655b.p. (26.84% per whole length) display intermediate intraspeciesvariationfor Xcc, X.c. pv. gardneri and considerable nucleotide and amino acid differences between species (Figs. 1c. and 3). Totally, 5 consensus genotypes were recognised for Xcc: Genotype 1 (41 strain) was distinct from others by gyrB and Xcc0006-7 ITS sequences with bootstrap over 98%, and included type strains NCPPB528T and strains collected in different countries from plants with epidemic development of black rot. Genotype 2 (6 strains, bootstrap 97%) includes strains from Russian South (Krasnodar region) collected at 2006. Genotype 3 includes Xcc strains of epidemic origin (PHW117 and others) and X. c. pv. raphani NCPPB1946T. Genotype 4 was found in Moscow region (strain 26) and share similarity with xanthomonads isolated from sunflower. Genotype 5 was found in Krasnodar region on cabbage variety. Genotypes 4 and 5 were clearly distinct from other Xcc and most similar to strains of X. vesicatoria. Primers designed for gyrB, ITS Xcc006-7 and cytP450 were tested on wide range of xanthomonads for specificity of identification of Xcc in brassicas seeds. The best results were obtained for combination of ITS Xcc006-7 and cytP450 (Fig. 3 and 4). INTRODUCTION X. campestris pv. campestris (Xcc), the causal agent of black rot of crucifers, infects cruciferous plants, including agriculturally important crops and many weeds. These bacteria are disseminated with seeds and a planting stock worldwide, and identification of bacteria becomes more and more critical problem. Routine diagnostic methods are identifying the strains on species taxonomic level, and application of them is limited to only known phytopathogenic bacteria. Strains of Xanthomonas may belong to either natural populations with high genetic diversity or to "epidemic" clonal group with narrow genetic base and clear difference between any two groups. So taxonomic markers for a various level must be included at PCR assay. The genes gyrB, (annot.at genome of Xсс ATCC 33913), were investigatedas potencial ones. gyrBor topoisomerase IV (Topo IV). Composed of two pairs of A and B subunits. It catalyzes ATP-dependent negative supercoiling of DNA and involved in DNA replication, recombination, transcription. operon Xcc0006- Xcc0007. Xcc0006 – tranlation factor: degradation of proteins, peptides, and glycopeptides; Xcc0007- transcriptional regulator (for tonB genes and whole genome) with tetratricopeptide repeats (TPR) (COG0457). cytP450 (abbreviated CYP, P450, infrequently CYP450).. Cytochrome P450 is haem-thiolate proteins involved in the oxidative degradation of various compounds. It’s particularly well known for their role in the degradation of environmental toxins and mutagens,secondary metabolites biosynthesis, transport, and catabolism. MATERIALS AND METHODS Collections of original strains and known cultures. The extensive collection of 108 Xanthomonas sp., including species X. сampestris (Xсс), X oryzae, X malvacearum, X. vesicatoria, X. albilineans, X. citri, X. phaseoli, was used in this study (Table 1). Genetic analysis.Bacteria were grown on King’s B medium for 72 hours. Total genomic DNA was extracted by using Birnboim & Doly (1979) protocol in own modifications for the bateria with rich polysaccharide production and purified with the wizard Genomic DNA purification system (Promega, США).Primers for genes gyrB, Xcc0006, Xcc0007 (annotation of genome Xсс ATCC 33913), cytP450 and intergenic region Xcc0006-Xcc0007 were designed with program Oligo v. 6.1 (Wojciech& Rychlik,2000). The genes were amplified, purified, PCR fragments were sequensed(Sanger et al, 1977)and aligned using the Clustal Wv.1.75 (Thompson et al., 1994). Phylogenetic trees were constructed with Mega v. 3.1 (Kumar & Nei, 2006), using the neighbor-joiningmethod(Saitoua. Nei, 1987)with genetic distances computed by Kimura’s 2-parameter model (Kimura, 1980) CONCLUSIONS Comparison of the gyrB sequences revealed high variability for intra- and inter-specific level. Further analysis shows strains of X. vesicatoria, X. malvacearum, X. citri, X. phaseoli, X. oryzae but Xcc had an 140bp insert within the IR Xcc0006-Xcc0007. The cluster analysis of the Xcc0006-Xcc0007 nucleotide sequences had revealed 3 major groups (A, B and C) of Xcc authentic at 95 % bootstrap value, and 8 ones - at above 65 %. "Epidemic" strains from the different countries, from natural populations, and virulent on several host plant were placed in separate groups. It was kept for cytP450 gene. Primers designed for genes gyrB and cytP450 were useful for diagnostics of xanthomonads on species and pathovar levels. REFERENCES Edmilson R., Goncalves E.R., Rosato Y.B. Phylogenetic analysis of Xanthomonas species based upon 16S-23S rDNA intergenic spacer sequences // IJSEM. 2002. V. 52. P. 355-361. Hauben L., Vauterin L., Swings J., Moore E.R.S. Comparison of 16S ribosomal DNA sequences of all Xanthomonas species // Int. J. Syst. Bacteriology. 1997. V. 47. P. 328-335. Huang W. M. Bacterial diversity based on type II DNA topoisomerase genes // annu. Rev. Genet. 1996; 30:79-107. Nei.M. and S. Kumar. 2000. Molecular Evolution and Phylogenetics. Oxford University Press, New York. Vauterin L., Hoste B., Kersters K., Swings J. Reclasification of Xanthomonas // Int. J. Syst. Bacteriology. 1995. V. 45. P. 472-489. Fig.2. Presence / absence of insertsfor Xcc0006-0007intergenic region in Xanthomonas sp. a c b 655b.p. 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 18 19 20 21 22 23 Fig.3. PCR amplification of Xanthomonas sp.with primers cytP450(F1/R1). 1-X. campestris pv. campestris Sm10; 2-X. vesicatoria pv. vesicatoria 3–X. campestris pv. campestris 1344; 4–X. campestris pv. campestris 1349; 5–X. campestris pv. campestris F-4; 6–X. campestris pv. campestris 1385; 7–X. campestris pv. campestris 1386; 8-X. campestris pv. campestris 1387; 9–X. campestris pv. campestris 1388; 10–X. campestris pv. campestris 1389; 11-X. campestris pv. campestris 1390; 12-X. campestris pv. campestris 1391; 13-X. vesicatoria pv. vesicatoria NCPPB422t; 14-X. vesicatoria pv. vesicatoria NCPPB422t Amp+; 15-X. vesicatoria pv. vesicatoria K-444; 16-X. campestris pv. campestris 1392; 17-X. campestris pv. campestris 1393; 18-X. campestris pv. campestris 1394; 19-X. campestris pv. campestris 1395; 20-X. campestris pv. campestris 1396; 21-X. campestris pv. campestris 1397; 22-control; 23-markerGeneRuler 100bp DNA ladder plus (Fermentas) 1294 b.p. 1152 b.p. 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 Fig.4. PCR amplification of Xanthomonas sp.with primers Xcc0006-0007 1- control; 2- X. vesicatoria pv. vesicatoriaK-441; 3- X. campestris pv. campestris 1396; 4- X. campestris pv. campestris 1397; 5– X. campestris pv. campestris NCPPB528t; 6- X. vesicatoria pv. vesicatoria NCPPB422t; 7-. vesicatoria pv. vesicatoria 5001; 8- X. axonopodis pv. malvacearum#10; 9- X. axonopodis pv. malvacearum#17; 10- X. axonopodis pv. phaseoli var. fuscans HRI924a; 11- X. transluceans pv. transluceansО-3028; 12- X. oryzae pv. oryzicola NCPPB1585T; 13- X. vesicatoria pv. gardneri GA2; 14- X. oryzae pv. oryzae NCPPB3002t; 15- X. axonopodis pv. malvacearum NCPPB633T; 16- X. vascular 1a 117- markerGeneRuler 100bp DNA ladder plus (Fermentas) • Fig.1. Phylogenetic trees constructed with Mega v. 3.1 (Kumar & Nei, 2006), using the neighbor-joiningmethod (Saitou a. Nei, 1987) with genetic distances computed by Kimura’s 2-parameter model (Kimura, 1980) forXanthomonas sp. genes • genegyrB • operon Xcc0006-0007 • gene cytP450