Growth optimization procedures for the phytopathogen Xylella fastidiosa

For the first time, growth curves are shown for the phytopathogen Xylella fastidiosa on traditional growth media such as PW (periwinkle wilt), BCYE (buffered charcoal yeast extract), and on new ones such as GYE (glutamate yeast extract) and PYE (phosphate yeast extract) that were developed in this work. The optimal growth conditions on solid and liquid media as well as their measurements are presented, by using total protein content and turbidity determinations. The results demonstrated that yeast extract provided sufficient nutrients for X. fastidiosa, since the cells grew well on PYE medium.

Proteome of the phytopathogen Xanthomonas citri subsp citri: a global expression profile

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monitoramento de raças de Bremia lactucae em 2010 e 2011 no Estado de São Paulo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Whole-genome expression profiling of Xylella fastidiosa in response to growth on glucose

Xylella fastidiosa is the etiologic agent of diseases in a wide range of economically important crops including citrus variegated chlorosis, a major threat to the Brazilian citrus industry. The genomes of several strains of this phytopathogen have been completely sequenced enabling large-scale functional studies. In this work we used whole-genome DNA microarrays to investigate the transcription profile of X. fastidiosa grown in defined media with different glucose concentrations. Our analysis revealed that while transcripts related to fastidian gum production were unaffected, colicin-V-like and fimbria precursors were induced in high glucose medium. Based on these results, we suggest a model for colicin-defense mechanism in X. fastidiosa.

Genomics-based design of defined growth media for the plant pathogen Xylella fastidiosa

Based on the genetic analysis of the phytopathogen Xylella fastidiosa genome, five media with defined composition were developed and the growth abilities of this fastidious prokaryote were evaluated in liquid media and on solid plates. All media had a common salt composition and included the same amounts of glucose and vitamins but differed in their amino acid content. XDM1 medium contained amino acids threonine, serine, glycine, alanine, aspartic acid and glutamic acid, for which complete degradation pathways occur in X fastidiosa; XDM2 included serine and methionine, amino acids for which biosynthetic enzymes are absent, plus asparagine and glutamine, which are abundant in the xylem sap; XDM3 had the same composition as XDM2 but with asparagine replaced by aspartic acid due to the presence of complete degradation pathway for aspartic acid; XDM4 was a minimal medium with glutamine as a sole nitrogen source; XDM5 had the same composition as XDM4, plus methionine. The liquid and solidified XDM2 and XDM3 media were the most effective for the growth of X. fastidiosa. This work opens the opportunity for the in silico design of bacterial defined media once their genome is sequenced. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier B.V. B.V. All rights reserved.

Solarização em microcosmo: efeito de materiais vegetais na sobrevivência de fitopatógenos de solo e na produção de voláteis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative analysis of differentially expressed sequence tags of sweet orange and mandarin infected with Xylella fastidiosa

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Recombinant expression and characterization of a Xylella fastidiosa cysteine protease differentially expressed in a nonpathogenic strain

Xylella fastidiosa is a xylem-limited, Gram-negative bacterium responsible for citrus variegated chlorosis (CVC) in sweet oranges. In the present study, we present the recombinant expression, purification and characterization of an X. fastidiosa cysteine protease (dubbed Xylellain). The recombinant Xylellain ((HIS)Xylellain) was able to hydrolyze carbobenzoxy-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) and carbobenzoxy-Arg-Arg-7-amido-4-methylcoumarin (Z-RR-MCA) with similar catalytic efficiencies, suggesting that this enzyme presents substrate specificity requirements similar to cathepsin B. The immunization of mice with (HIS)Xylellain provided us with antibodies, which recognized a protein of c. 31 kDa in the X. fastidiosa pathogenic strains 9a5c, and X. fastidiosa isolated from coffee plants. However, these antibodies recognized no protein in the nonpathogenic X. fastidiosa J1a12, suggesting the absence or low expression of this protein in the strain. These findings enabled us to identify Xylellain as a putative target for combating CVC and other diseases caused by X. fastidiosa strains.

Copper resistance of biofilm cells of the plant pathogen Xylella fastidiosa

Xylella fastidiosa is a phytopathogen that causes diseases in different plant species. The development of disease symptoms is associated to the blockage of the xylem vessels caused by biofilm formation. In this study, we evaluated the sensitivity of biofilm and planktonic cells to copper, one of the most important antimicrobial agents used in agriculture. We measured the exopolysaccharides (EPS) content in biofilm and planktonic cells and used real-time reverse transcription polymerase chain reaction to evaluate the expression of the genes encoding proteins involved in cation/multidrug extrusion (acrA/B, mexE/czcA, and metI) and others associated with different copper resistance mechanisms (copB, cutA1, cutA2, and cutC) in the X. fastidiosa biofilm formed in two different media. We confirmed that biofilms are less susceptible to copper than planktonic cells. The amount of EPS seems to be directly related to the resistance and it varies according to the media where the cells are grown. The same was observed for gene expression. Nevertheless, some genes seem to have a greater importance in biofilm cells resistance to copper. Our results suggest a synergistic effect between diffusion barriers and other mechanisms associated with bacterial resistance in this phytopathogen. These mechanisms are important for a bacterium that is constantly under stress conditions in the host.

Semi-selective culture medium for Xanthomonas axonopodis pv. malvacearum detection in cotton seeds (Gossypium hirsutum L.)

The cotton disease known as angular leaf spot, caused by Xanthomonas axonopodis pv. malvacearum (Xam) has been causing cotton losses in several producing regions around the world. Xam is transmitted by seeds, which may be infected both externally and internally. Infected seeds constitute the main long-distance dissemination mode of the pathogen. In view of this, the use of healthy seeds is a must. To accomplish that, detection methodologies for the bacteria must be developed be used in seed health analysis laboratories. This study aimed to develop a semi-selective medium for Xam detection in cotton seeds. The semi-selective culture medium was named MSSXAN and it was consisted of peptone (5.0 g), beef extract (3 g), sucrose (5 g), soluble starch (10 g), agar (15 g), CaCl 2 (0.25 g), Tween 80 (10 mL), distilled water (1,000 mL), crystal violet solution at 1% (150 μL), cephalexin (50 mg 1*), methyl thyophanate (10 mg*) and chlorothalonil (10 mg*) - *added after culture medium autoclaving. This MSSXAN medium shows low repressiveness to Xam and it be used for isolation of this bacteria in cotton seeds health analysis. © 2009 Academic Journals Inc.

Avaliação de microrganismos antagônicos, Saccharomyces cerevisiae e Bacillus subtilis para o controle de Penicillium Digitatum

Citrus fruits are affected by diverse diseases, mainly the fungal infections, which affect productivity and quality, especially when it targets the market of fresh fruit. Among the fungal diseases that occur in postharvest, there is the green mold caused by Penicillium digitatum. The control measures are based mainly in the treatment of fruits with different combinations of fungicides in packing-house. Due to restrictions on the presence of residues of fungicides in citrus fruits and the increasing development of resistant strains of pathogens to the fungicide used, it is necessary to search for control alternatives such as biological control. Therefore, this study aimed to: (i) verify the antagonistic effect of biological control agents (BCA), being 13 isolates of Bacillus subtilis and 06 isolates of Saccharomyces cerevisiae against P. digitatum, (ii) study in vitro interactions between pathogen and BCA (iii) determine the effect of integration of antagonists with sodium bicarbonate and carnauba wax in the control of green mold. The results showed that the majority of the isolates, and all yeast isolates inhibited the mycelial growth of the phytopathogen. Only one isolate of B. subtilis (ACB-84) was able to inhibit the germination of P. digitatum (72% of inhibition), whereas ACB-K1 and ACB-CR1 (S. cerevisiae) were the most effective with inhibition from 78 and 85.7% respectively. The addition of sucrose (0.5%) favored the inhibition of conidia germination by the yeast isolates. The results from the in vivo control showed the viability of S. cerevisiae ACB-K1 and ACB-CR1 to control P. digitatum in 'Tahiti' lime fruits and orange 'Hamlin' fruits, respectively. The combination of sodium bicarbonate and biocontrol agents did not result in improvements in the curative control of the green mold. Carnauba wax (18% of TSS) favored the antagonistic activity of S. cerevisiae, and this effect depended on the variety of fruits in the study and of the yeast isolate used for the biocontrol.

Distribuição de Candidatus Liberibacter americanus e Candidatus Liberibacter asiaticus em plantas cítricas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Predição computacional de promotores em Xanthomonas axonopodis pv. citri

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo de isoenzimas de metabolismo de carboidratos e clonagem e expressão da enolase de Xylella fastidiosa

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV