Júlio Rodrigues Neto

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii

BackgroundCitrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C.ResultsWe have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein.ConclusionWe have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.

High-throughput genomic sequencing of cassava bacterial blight strains identifies conserved effectors to target for durable resistance

Cassava bacterial blight (CBB), incited by Xanthomonas axonopodis pv. manihotis (Xam), is the most important bacterial disease of cassava, a staple food source for millions of people in developing countries. Here we present a widely applicable strategy for elucidating the virulence components of a pathogen population. We report Illumina-based draft genomes for 65 Xam strains and deduce the phylogenetic relatedness of Xam across the areas where cassava is grown. Using an extensive database of effector proteins from animal and plant pathogens, we identify the effector repertoire for each sequenced strain and use a comparative sequence analysis to deduce the least polymorphic of the conserved effectors. These highly conserved effectors have been maintained over 11 countries, three continents, and 70 y of evolution and as such represent ideal targets for developing resistance strategies.

Diversity of a Xylella fastidiosa Population Isolated from Citrus sinensis Affected by Citrus Variegated Chlorosis in Brazil

Summary Diversity of a population of Xylella fastidiosa isolated from sweet orange plants showing citrus variegated chlorosis (CVC) symptoms was assessed by PCR-based techniques. Thirty-seven strains were isolated throughout the 1997 year in the orange belt of Sao Paulo State, Brazil. Strains isolated from coffee, grape, oleander and plum were also included as outgroup reference strains. PCR amplification of the spacer sequence between the 16-23S rDNA yielded one fragment of 1.2 kb. Digestion with restriction enzymes, Dde I, Hinf I or Sau3 AI generated identical RFLP patterns for citrus and coffee strains, which could be distinguished from the strains isolated from the other hosts. Eight RAPD primers were also used and the results showed similarity from 80 to 100% within the CVC population. Three prevalent haplotypes comprising 24 CVC strains showed a high level of similarity (95%). Strains from the other hosts clustered apart from CVC strains, forming distinct groups.

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.

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.

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.

Agressividade de linhagens de Xanthomonas axonopodis pv. aurantifolii Tipo C em lima ácida 'Galego'

Aggressiveness of Xanthomonas axonopodis pv. aurantifolii Type C strains in ‘Mexican’ Lime The purpose of this research was to provide the information about the aggressiveness of strains of Xanthomonas axonopodis pv. aurantifolii – strains C (Xaa-C), producer (PP) or not of dark pigment (NP) in culture medium in comparison with X. axonopodis pv. citri – strains A (Xac). A total of 14 strains were inoculated by needle wounds in ‘Mexican’ Lime (Citrus aurantifolia) leaves, with bacterial cell suspension (10 7 CFU/mL): seven were Xaa-C PP, five were not producers and two were Xac strains. Ten replications of one plant each were used in each treatment. The plants were kept in greenhouse during the experiment. The strains differed on incubation period, lesion size and population growth. Xaa NP was more aggressive than PP. Symptoms induced by some strains varied in terms of water-soaked, chlorotic halos and necrosis. Additional keywords: citrus canker, pathogenicity, host-pathogen interaction.

Characterization of Xanthomonas spp. strains by bacteriocins

Twenty-five strains of Xanthomonas axonopodis pv. citri and 14 strains of Xanthomonas spp. were tested for bacteriocin production. X. axonopodis pv. passiflorae strains were sensitive to the bacteriocins produced by the 25 X. axonopodis pv. citri strains evaluated in this study while strains of X. axonopodis pv. manihotis and X. campestris pv. campestris showed variable sensitivity. Only five of the 25 X. axonopodis pv. citri strains were not inhibited by the bacteriocins produced by the two X. axonopodis pv. passiflorae strains. The bacteriocins produced by the Xanthomonas axonopodis pv. citri (FDC-806) and X. axonopodis pv. passiflorae (Mar-2850 A) strains were thermolabile, resistant to lysozyme and sensitive to DNAse. The bacteriocin produced by X. axonopodis pv. passiflorae was resistant to the action of proteinase K, trypsin and RNAse while the bacteriocin produced by X. axonopodis pv. citri was sensitive to these enzymes. The bacteriocins produced by X. axonopodis pv. passiflorae and X. axonopodis pv. citri were called passifloricin and citricin, respectively.

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.

Mancha bacteriana em Ruscus sp. causada por Burkholderia andropogonis no Brasil

Plants of ruscus (Ruscus sp.) showing symptoms of leaf spot were received for analysis in April 2008 from a field located in the region of Santo Antonio de Posse, state of Sao Paulo, Brazil. These spots were small, rounded, with 5 to 8 mm in diameter, dark brown in color, showing necrotic center and surrounded by chlorotic haloes. Slow-growing, cream-colored bacterial colonies were consistently obtained. The bacterium was Gram-negative, oxidative and not fluorescent. Artificial inoculations on healthy ruscus seedlings reproduced the symptoms observed in natural infections; the pathogen was re-isolated from the lesions. Biochemical, cultural, physiological and serological tests of the isolates identified the causal agent as Burkholderia andropogonis (former Pseudomonas andropogonis). This is the first report of this pathogen in ruscus in Brazil. Bacterial strains were deposited in the Phytobacteria Culture Collection of the Instituto Biologico (IBSBF) under numbers 2594 and 2595.