Suzete Aparecida Lanza Destéfano

Differentiation of Xanthomonas species Pathogenic to Sugarcane by PCR-RFLP Analysis

The PCR-RFLP of the 16S-23S rDNA spacer region was used to differentiate Xanthomonas species pathogenic to sugarcane. Strains of X. albilineans, X. campestris pv. vasculorum Types A and B, X. sacchari and Xanthomonas sp. from Trinidad, South Africa and India were examined. The amplification products were digested with Alu I, Hae III, Hpa II and Mbo I and the results showed that the different groups of bacterial strains exhibited distinct RFLP patterns for each tested endonuclease, except X. albilineans and X. sacchari which could only be differentiated from each other by the digestion with Hpa II. The results also allowed the separation of X.c. pv. vasculorum Type A from X.c. pv. vasculorum Type B and strongly suggested that the analyzed Xanthomonas sp. strains belong to X. sacchari. Nine X. campestris (pv. not determined) strains included in this study showed identical profiles to X.c. pv. vasculorum Type A group and DNA–DNA hybridization experiments confirmed these results. PCR-RFLP of the 16S-23S rDNA spacer region could be applied as a reliable method for differentiating the xanthomonads pathogenic to sugarcane.

Reassessment of the taxonomic position of Burkholderia andropogonis and description of Robbsia andropogonis gen. nov., comb. nov.

Abstract The phylogenetic classification of the species Burkholderia andropogonis within the Burkholderia genus was reassessed using 16S rRNA gene phylogenetic analysis and multilocus sequence analysis (MLSA). Both phylogenetic trees revealed two main groups, named A and B, strongly supported by high bootstrap values (100%). Group A encompassed all of the Burkholderia species complex, whi.le Group B only comprised B. andropogonis species, with low percentage similarities with other species of the genus, from 92 to 95% for 16S rRNA gene sequences and 83% for conserved gene sequences. Average nucleotide identity (ANI), tetranucleotide signature frequency, and percentage of conserved proteins POCP analyses were also carried out, and in the three analyses B. andropogonis showed lower values when compared to the other Burkholderia species complex, near 71% for ANI, from 0.484 to 0.724 for tetranucleotide signature frequency, and around 50% for POCP, reinforcing the distance observed in the phylogenetic analyses. Our findings provide an important insight into the taxonomy of B. andropogonis. It is clear from the results that this bacterial species exhibits genotypic differences and represents a new genus described herein as Robbsia andropogonis gen. nov., comb. nov.

Phylogenetic analysis of Xanthomonas based on partial rpoB gene sequences and species differentiation by PCR-RFLP

The rpoB gene was evaluated as an alternative molecular marker for the differentiation of Xanthomonas species and in order to understand better the phylogenetic relationships within the genus. PCR-RFLP experiments using HaeIII allowed differentiation of Xanthomonas species, particularly those that affect the same plant host such as Xanthomonas albilineans and X. sacchari, pathogenic to sugar cane, Xanthomonas cucurbitae and X. melonis, which cause disease in melon, and Xanthomonas gardneri, X. vesicatoria and X. euvesicatoria/X. perforans, pathogenic to tomato. Phylogenetic relationships within the genus Xanthomonas were also examined by comparing partial rpoB gene sequences (612 nt) and the Xanthomonas species were separated into two main groups. Group I, well supported by bootstrap values of 99 %, comprised X. euvesicatoria, X. perforans, X. alfalfae, X. citri, X. dyei, X. axonopodis, X. oryzae, X. hortorum, X. bromi, X. vasicola, X. cynarae, X. gardneri, X. campestris, X. fragariae, X. arboricola, X. cassavae, X. cucurbitae, X. pisi, X. vesicatoria, X. codiaei and X. melonis. Group II, again well supported by bootstrap values of 99 %, comprised X. albilineans, X. sacchari, X. theicola, X. translucens and X. hyacinthi. The rpoB gene sequence similarity observed among the species in this study ranged from 87.8 to 99.7 %. The results of PCR-RFLP of the rpoB gene indicated that this technique can be used for diagnosis and identification of most Xanthomonas strains, including closely related species within the genus. However, species that showed identical profiles could be differentiated clearly only by sequence analysis. The results obtained in our phylogenetic analysis suggested that the rpoB gene can be used as an alternative molecular marker for genetic relatedness in the genus Xanthomonas. The results of PCR-RFLP of the rpoB gene indicate that this technique can be used for diagnosis and identification of closely related species within the genus, representing a rapid and inexpensive tool that can be easily standardized between laboratories.

Detection of a New Bacterium Related to Xanthomonas fuscans subsp aurantifolii Infecting Swingle Citrumelo in Brazil.

In March 2009, in a sweet orange orchard (Citrus sinensis) cv. Valencia grafted on Swingle citrumelo (C. paradisi Macf. × Poncirus trifoliata L. Raf.) rootstock in Severínia County, São Paulo State, Brazil, approximately 40 trees were detected with small, necrotic, dark brown leaf spots. These lesions occurred whether or not citrus leafminer (Phyllocnistis citrella) was present and they were only found on leaves from branches arising from the rootstock. Sweet orange foliage was not affected even when in contact with infected rootstock branches. Symptoms were unusual and distinct from typical citrus canker lesions because the lesions were smaller and did not have erumpent margins. Typical yellow Xanthomonas colonies were isolated from the lesions on nutrient agar. The isolates were aerobic, gram negative, rod shaped, and they produced a dark pigment, which is characteristic of some Xanthomonas fuscans subsp. aurantifolii strains. Two reference strains were tested for pathogenicity on not fully expanded leaves of sweet orange, Swingle citrumelo, and key/Mexican lime (C. aurantifolia) plants by wound inoculation with a sterile needle previously dipped in a bacterial suspension (approximately 106 ml-1). Two plants of each species were used for inoculations in greenhouse conditions and six leaves were inoculated per plant. Each inoculated leaf received six point inoculations. These tests confirmed that the host range of this pathogen was restricted to Swingle citrumelo. Symptoms similar to those in the orchard were observed 3 weeks after inoculation and Kochs postulates were completed by reisolation of the bacterium and comparing it with the original isolates. Molecular fingerprinting with PCR-restriction fragment length polymorphism (RFLP) of the 16S-23S spacer region polymorphism (1) and ERIC- and BOX-PCR (2) was used to compare the new strain with 26 reference strains of Xanthomonas citri subsp. citri types A, A* and Aw, X. fuscans subsp. aurantifolii types B and C, and X. alfalfae subsp. citrumelonis. PCR-RFLP and ERIC-PCR showed that this new pathogen had the same profile as X. fuscans subsp. aurantifolii (B and C types). In BOX-PCR, this new strain had a unique profile, but it was still most similar to X. fuscans subsp. aurantifolii and very distinct from X. citri subsp. citri (A, A*, and Aw) and X. alfalfae subsp. citrumelonis strains. During the rainy season in Brazil, this new Xanthomonas strain is less aggressive than X. citri subsp. citri on Swingle citrumelo, inducing fewer lesions without erumpent margins even in young leaves severely infested by the citrus leafminer. The disease only occurred on trees that were separated from each other by 3 to 20 m, suggesting that the bacterium is spread by windblown rain and/or cultural practices. Xanthomonads pathogenic to citrus are of great importance for regulatory purposes worldwide. X. fuscans subsp. aurantifolii is only known to be pathogenic on lemons and limes in the field, and until now, has only been reported to infect lemons and limes in Argentina and key/Mexican lime in São Paulo (Brazil) (3). To our knowledge, this is the first report of a strain of this subspecies that infects Swingle citrumelo but not key/Mexican lime. References: (1) S. A. L. Destéfano and J. Rodrigues Neto. Summa Phytopathol. 28:167, 2002. (2) F. J. Louws et al. Appl. Environ. Microbiol. 60:2286, 1994. (3) N. W. Schaad et al. Syst. Appl. Microbiol. 28:494, 2005.

Characterization of Ralstonia solanacearum strains from Brazil using molecular methods and pathogenicity tests.

SUMMARY Forty one Brazilian strains of Ralstonia solanacearum from ornamental plants (Begonia sp., Fuchsia sp., Oxalis sp., Pelargonium x hortorum and Tagetes sp.), eucalyptus (Eucalyptus sp.), Solanaceae (Nicotiana tabacum, Solanum jilo, S. lycopersicum, S. melongena, S. tuberosum), banana (Musa sp.) and heliconia (Heliconia sp.), belonging to three different races and five biovars, were characterized by ERIC and BOX-PCR and classified in phylotypes by sequence analysis of the endoglucanase (Egl) and MutS genes. The virulence of 22 strains was evaluated on tomato cv. Petomech VF, potato cv. Agata and tobacco var. Burley. In the ERIC and BOX-PCR analysis, strains were separated into two main clusters showing high genetic diversity among them, with no correlation concerning race, biovar or geographical origin. Phylogenetic analysis based on sequences of part of the Egl and MutS genes revealed the predominance of phylotype II in Brazil. With exception of strains belonging to race 2, which were not included in the experiments, pathogenicity tests carried out with different strains of R. solanacearum showed high virulence on tomato and potato, and lower virulence on tobacco, showing no relationship with race, biovar or host plant from which the tested strain were originally isolated.

Grapevine bacterial canker in the State of São Paulo, Brazil: detection and eradication

Symptoms of bacterial canker of grapevine in the variety Red Globe were observed in August 2009 in an orchard at Tupi Paulista, Sao Paulo State, Brazil, and the causal agent Xanthomonas campestris pv. viticola was identified by pathological and molecular tests. Eradication procedure was adopted and approximately 4,700 plants were destroyed. A survey was conducted on grape-producing regions in the state of Sao Paulo, which found no other contaminated orchard, and this bacterial species is considered absent in the state.

Marmoricola aquaticus sp. nov., an actinomycete isolated from a marine sponge

A novel marine actinomycete, designated B374(T), was isolated from a marine sponge, Glodia corticostylifera, which was collected from São Paulo, Brasil. The taxonomic position of B374(T) was established by using data derived from a polyphasic approach. The organism showed a combination of chemotaxonomic and morphological characteristics consistent with its classification in the genus Marmoricola and it formed a distinct phyletic line in the clade of the genus Marmoricola, based on 16S rRNA gene sequences. Strain B374(T) was most closely related to Marmoricola aequoreus SST-45(T) (98.5% 16S rRNA gene sequence similarity), but was distinguished from this strain and from the other type strains of species of the genus Marmoricola on the basis of a combination of phenotypic properties. The data obtained, therefore, indicates that isolate B374(T) ( = CBMAI 1089(T) = DSM 28169(T)) should be classified as a novel species of the genus Marmoricola, for which the name Marmoricola aquaticus sp. nov. is proposed.

Pseudomonas syringae pv. tabaci in papaya seedlings

The natural occurrence of Pseudomonas syringae pv. tabaci causing leaf spot symptoms in papaya seedlings is reported. The pathogen was identified through biochemical, physiological, serological, and molecular assays and artificial inoculations in papaya plants. It was also shown that the strains were pathogenic to bean and tobacco plants. The restriction patterns obtained with Afa I, Alu I, Dde I, Hae III, Hpa II, Hinf I, Sau 3A I and Taq I of the PCR-RFLP of 16S-23S DNAr were identical to the P. s. pv. tabaci patterns. Primers corresponding to hrpL gene of P. syringae were also tested and the results grouped the papaya strains with P s. pv. tabaci. Bacterial strains were deposited at Colecao de Culturas IBSBF, Instituto Biologico, Campinas, Brazil, under access numbers 1687 and 1822.

Draft Genome Sequence of Burkholderia andropogonis Type Strain ICMP2807, Isolated from Sorghum bicolor

ABSTRACT Here, we report the draft genome sequence of Burkholderia andropogonis ICMP2807, a phytopathogenic bacterium isolated from Sorghum bicolor plants in the United States.

Pathogenicity of Brazilian strains of Ralstonia solanacearum in Strelitzia reginae seedlings

Twenty four strains of Ralstonia solanacearum belonging to races 1, 2 and 3 of biovars I, II and III, isolated from various hosts were investigated for their ability to cause disease on Strelitzia seedlings through artificial inoculation. Results revealed that, with one exception, only strains isolated from plants of Musa or Heliconia (classified as race 2) caused wilt symptoms on Strelitzia, indicating their pathogenic potential to that plant species. Seedlings of Strelitzia could be used as test plants for presumptive diagnosis for banana Moko disease.