Taxonomic characterization of Xanthomonas species

Author(s)

Mariana Ferreira Tonin

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2012-06-03

Examining board members

Suzete Aparecida Lanza Destéfano; Fabiana Fantinatti Garboggini; Julio Rodrigues Neto; Ivan Paulo Bedendo; Ricardo Harakava

• Advisor: Suzete Aparecida Lanza Destéfano

Abstract

Xanthomonas species are responsible for diseases causing economic losses in many crops. These phytopathogens have been subject of several taxonomic studies resulting in significant changes at interespecific or infraspecific level. This study aimed the taxonomic characterization of Xanthomonas species including: (1) differentiation and phylogenetic analysis of Xanthomonas species; (2) clarification of taxonomic position of strains classified as Xanthomonas sp.; (3) differentiation of the pathovars of X. campestris; ( 4) development of specific primers for X. campestris, X. translucens, X. cucurbitae and X. melonis. The rpoB2F/rpoB3R primers, designed from rpoB gene sequences, were employed in amplification experiments using DNAs from 26 species of Xanthomonas genus and the products (800 bp) were digested with different restriction enzymes. Profiles using Hae III allowed to differentiate the most species of the genus, including pathogens the affect the same plant host as X. albilineans e X. sacchari (pathogenic to sugarcane); X. cucurbitae e X. melonis (pathogenic to melon); X. vesicatoria, X. gardneri and X. euvesicatoria/X. perforans (pathogenic to tomato). Amplification products of the rpoB gene were sequenced and the phylpgenetic tree constructed from these sequences also allowed the differentiation of the Xanthomonas species, indicating that the rpoB gene can be used as an efficient molecular marker for phylogenetic relationships studies within the Xanthomonas genus. DNA of the strains classified as Xanthomonas sp. were also analysed by the amplification experiments using specific primers for X. axonopodis species, DNA-DNA hybridization and multilocus sequence analysis of the genes rpoB, rpoA, atpA, recA and intergenic spacer region 16S-23S rDNA. Results showed that the strains of X. sp. pv. viticola, X. sp. pv. betae and X. sp. pv. paulliniae belong to X. axonopodis species, however, it was not possible to define the taxonomic position of X. sp. pv. arracaciae, X. sp. pv. esculenti and X. sp. pv. eucalypti, although different approaches have been used. Thus, further studies should be conducted in order to clarify their classification. In this study X. campestris, X. translucens, X. cucurbitae and X. melonis were also analyzed. In order to differentiate the pathovars of the X. campestris specie, the DNAs of these strains were submitted to PCR-RFLP analysis of the rpoB gene and the double digestions using Cfo I/Mbo I allowed the differentiation of the pathovars X. c. pv. raphani, X.c. pv. barbariae, X.c. pv. incanae, X.c. pv. armoraciae and X.c. pv. campestris/ X.c. pv. aberrans. Also, the data obtained in the rpoB gene analysis allowed the development of specific primers for the species X. campestris, pathogenic to crucifers (rpoB2F/xcamR) and X. translucens, pathogenic to grasses and cereals (trans1F/trans2R). Furthermore, intergenic spacer 16S-23S rDNA sequences analysis enabled the development of the mecF/mecR primers, specific for X cucurbitae and X melonis, both pathogenic to melon. The specificity of these primers was confirmed by amplification experiments using DNAs from bacteria belonging to different genera pathogenic to the same plant hosts. Sensitivity level of the PCR technique using the primers developed was 0,1 pg for rpoB/xcam, 0,01 ng for trans1F/trans2R and 1 pg for mecF/mecR (AU)