Reação de variedades e clones de laranjas a Xylella fastidiosa

A clorose variegada dos citros (CVC) é uma doença grave, causada por Xylella fastidiosa. As medidas usuais de controle mostram-se pouco eficientes ou práticas e com alto custo. Dessa forma, o uso de variedades resistentes e/ou tolerantes desponta como a alternativa mais eficiente, razão pela qual se julgou oportuna a realização deste trabalho. O presente estudo teve como objetivo avaliar o comportamento de variedades e clones de laranjas introduzidas em relação a X. fastidiosa. Foram estudados 59 variedades e clones de laranjas doces e 2 de laranjas azedas introduzidos da França, Itália e Portugal. O delineamento experimental foi o de blocos casualizados (DBC), com 62 tratamentos e 4 repetições, incluindo a variedade 'Pêra', como padrão. Cada parcela continha duas plantas, sendo uma inoculada e a outra sem inoculação. Para a inoculação do patógeno, foi empregado o método de encostia, utilizando-se de mudas infectadas. Para a avaliação da incidência da doença, utilizou-se de dados qualitativos, positivos ou negativos, enquanto para severidade empregou-se escala de notas, que foi estabelecida baseando-se nos sintomas de CVC, confirmados através dos testes de PCR. As variedades de laranjas azedas Beja e Sr. Pinto e as laranjas doces Navelina ISA 315, Navelina SRA 332 e Newhall Navel SRA 343 não apresentaram sintomas em folhas até 27 meses após a inoculação.

Ingestão de seiva do xilema de laranjeiras 'Pêra' e 'Valência' (Citrus sinensis (L.) Osbeck) sadias e infectadas por Xylella fastidiosa, pelas cigarrinhas vetoras Oncometopia facialis e Dilobopterus costalimai (Hemiptera: Cicadellidae)

Estudou-se o efeito da infecção pela bactéria Xylella fastidiosa, agente causal da Clorose Variegada dos Citros (CVC), sobre a taxa de ingestão de seiva do xilema de plantas cítricas por duas espécies de cigarrinhas vetoras (Hemiptera: Cicadellidae). Foram utilizados pés-francos de laranjeira-doce (Citrus sinensis) das variedades 'Pêra' e 'Valência', infectadas por X. fastidiosa da linhagem 9a5c, por meio de inoculação mecânica. Os insetos utilizados nos experimentos foram coletados em campo, sendo um representante da Tribo Cicadellini (Dilobopterus costalimai) e um da Proconiini (Oncometopia facialis). A taxa de ingestão de seiva do xilema por O. facialis foi quantificada nos ramos das plantas e a de D. costalimai nas folhas e ramos, por meio da avaliação do volume do líquido (honeydew) excretado por unidade de tempo. O consumo pela cigarrinha O. facialis nas plantas doentes foi menor do que nas plantas sadias. Na variedade 'Pêra' doente, o consumo foi baixo, não permitindo a quantificação da seiva eliminada. Na 'Pêra' sadia e na 'Valência' doente e sadia, O. facialis apresentou valores expressivos de excreção, com maior alimentação no período diurno. Nas plantas sadias das duas variedades, o consumo pela cigarrinha D. costalimai foi maior do que nas plantas com CVC. Comparando-se as variedades, o consumo foi superior na variedade 'Valência', e, em relação às partes da planta, folha e ramo, a taxa de ingestão foi maior no ramo das duas variedades, apresentando consumo maior no período diurno.

Evaluation of Xylella fastidiosa genetic diversity by fAFPL markers

The first phytopathogenic bacterium with its DNA entirely sequenced is being detected and isolated from different host plants in several geographic regions. Although it causes diseases in cultures of economic importance, such as citrus, coffee, and grapevine little is known about the genetic relationships among different strains. Actually, all strains are grouped as a single species, Xylella fastidiosa, despite colonizing different hosts, developing symptoms, and different physiological and microbiological observed conditions. The existence of genetic diversity among X. fastidiosa strains was detected by different methodological techniques, since cultural to molecular methods. However, little is know about the phylogenetic relationships developed by Brazilian strains obtained from coffee and citrus plants. In order to evaluate it, fAFLP markers were used to verify genetic diversity and phylogenetic relationships developed by Brazilian and strange strains. fAFLP is an efficient technique, with high reproducibility that is currently used for bacterial typing and classification. The obtained results showed that Brazilian strains present genetic diversity and that the strains from this study were grouped distinctly according host and geographical origin like citrus-coffee, temecula-grapevine-mulberry and plum-elm.

Locais e período de alimentação da cigarrinha vetora de Xylella fastidiosa, Bucephalogonia xanthophis (Berg) (Hemiptera: Cicadellidae), em mudas cítricas

A cigarrinha Bucephalogonia xanthophis (Berg) (Hemiptera: Cicadellidae) é um importante vetor da bactéria Xylella fastidiosa, agente causal da clorose variegada dos citros. Este trabalho teve como objetivo identificar o local preferido de alimentação e o período de maior atividade alimentar desta cigarrinha em citros, no sentido de elucidar o comportamento alimentar relacionado à transmissão da bactéria. O local de alimentação foi estudado em ensaio de escolha, no qual 30 insetos adultos foram liberados em gaiolas de observação (n = 10) contendo uma muda de laranja-doce [Citrus sinensis (L.) Osbeck]. Após 1; 15; 21; 25; 39; 45 e 49 h da liberação, contaram-se os insetos na parte superior (ramos com brotações) e inferior (haste principal, até H"40 cm de altura) da muda. Nos ramos da parte superior, avaliou-se a preferência entre a haste, o pecíolo e o limbo foliar. Em um segundo ensaio, 20 machos e 20 fêmeas de B. xanthophis foram confinados individualmente sobre a haste de 'seedlings' de laranja-doce para determinar os períodos de alimentação, quantificando-se a excreção de 'honeydew' (medida indireta da ingestão) em períodos sucessivos de dia e noite, durante 48 h. A maioria dos indivíduos de B. xanthophis preferiu a haste dos ramos novos (62%), na parte superior da muda cítrica (91%). Nos 'seedlings', observou-se maior volume de excreção e proporção de indivíduos excretando durante a fotofase, independentemente do sexo. Portanto, em estudos de transmissão de X. fastidiosa, deve-se considerar a preferência de B. xanthophis pela haste de brotações cítricas e sua maior atividade alimentar durante a fotofase.

Seqüenciamento e variabilidade do fragmento genômico de Xylella fastidiosa amplificado pelos iniciadores RST31/33

Xylella fastidiosa é agente causal de diversas doenças de importância econômica como a clorose variegada dos citros (Citrus spp.) (CVC), mal de Pierce da videira (Vitis vinifera), escaldadura da ameixeira (Prunus salicina) e requeima do cafeeiro (Coffea arabica). A seqüência nucleotídica do fragmento genômico, específico de X. fastidiosa, amplificado pelo par de iniciadores RST31/33 foi determinada para 38 isolados de citros e para isolados de videira, cafeeiro e ameixeira objetivando avaliar o nível de polimorfismo entre isolados e a identidade genômica do fragmento. Não foi observado polimorfismo de seqüência nucleotídica entre isolados de citros, mas foi detectado polimorfismo entre isolados de citros e de videira, cafeeiro e ameixeira. A presença do sítio de clivagem RsaI, que distingue isolados de citros e videira de isolados de ameixeira e outras espécies arbóreas, foi identificada em um isolado de ameixeira proveniente dos EUA mas não em outro proveniente do Brasil.

Detecção de Xylella fastidiosa em germoplasma de cafeeiro

A bactéria Xylella fastidiosa possui uma ampla gama de plantas hospedeiras que inclui espécies de pelo menos 28 famílias de mono e dicotiledôneas. Em cafeeiro (Coffea spp.), a ocorrência dessa bactéria foi relatada previamente em cultivares da espécie Coffea arabica. Estudos foram realizados para determinar a presença de X. fastidiosa em diferentes espécies e híbridos interespecíficos de cafeeiro. As amostragens foram realizadas em dois anos consecutivos. As espécies de cafeeiro examinadas foram: C. kapakata, C. canephora, C. racemosa, C. arabica, C. dewevrei, C. stenophylla e C. eugenioides. Também foram incluídos neste estudo híbridos interespecíficos de C. arabica: C. arabica x C. dewevrei, C. arabica x C. eugenioides, C. arabica x C. racemosa e C. arabica x C. robusta. Foram coletadas amostras de ramos plagiotrópicos de diferentes plantas para cada espécie e híbrido. A detecção de X. fastidiosa nas amostras foi realizada utilizando os testes serológicos de DAS-ELISA e imunofluorescência indireta. A bactéria foi detectada nas sete espécies e nos quatro híbridos de cafeeiro examinados. Entretanto, as plantas aparentemente não apresentavam sintomas de infecção por X. fastidiosa. A espécie C. arabica apresentou a maior proporção de amostras positivas e maiores valores de absorbância no teste de DAS-ELISA. Em contraste, as espécies C. racemosa e C. dewevrei foram as que apresentaram menores proporções de amostras positivas para presença de X. fastidiosa, como também menores valores de absorbância no teste de DAS-ELISA.

Development of a scar marker for Pierce's Disease strains of Xylella fastidiosa

The objective of this research was to develop a primer for a polymerase chain reaction specific for Xylella fastidiosa strains that cause Pierce's Disease (PD) in grapes (Vitis vinifera). The DNA amplification of 23 different strains of X. fastidiosa, using a set of primers REP1-R (5'-IIIICGICGIATCCIGGC-3') and REP 2 (5'-ICGICTTATCIGGCCTAC-3') using the following program: 94 ºC/2 min; 35 X (94 ºC/1 min, 45 ºC/1 min and 72 ºC/1 min and 30 s) 72 ºC/5 min, produced a fragment of 630 bp that differentiated the strains that cause disease in grapes from the other strains. However, REP banding patterns could not be considered reliable for detection because the REP1-R and REP 2 primers correspond to repetitive sequences, which are found throughout the bacterial genome. The amplified product of 630 bp was eluted from the agarose gel, purified and sequenced. The nucleotide sequence information was used to identify and synthesize an specific oligonucleotide for X. fastidiosa strains that cause Pierce's Disease denominated Xf-1 (5'-CGGGGGTGTAGGAGGGGTTGT-3') which was used jointly with the REP-2 primer at the following conditions: 94 ºC/2 min; 35 X (94 ºC/1 min, 62 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/10 min. The DNAs isolated from strains of X. fastidiosa from other hosts [almond (Prumus amygdalus), citrus (Citrus spp.), coffee (Coffea arabica), elm (Ulmus americana), mulberry (Morus rubra), oak (Quercus rubra), periwinkle wilt (Catharantus roseus), plums (Prunus salicina) and ragweed (Ambrosia artemisiifolia)] and also from other Gram negative and positive bacteria were submitted to amplification with a pair of primers Xf-1/REP 2 to verify its specificity. A fragment, about 350 bp, was amplified only when the DNA from strains of X. fastidiosa isolated from grapes was employed.

Genomics and X-ray microanalysis indicate that Ca2+ and thiols mediate the aggregation and adhesion of Xylella fastidiosa

The availability of the genome sequence of the bacterial plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, is accelerating important investigations concerning its pathogenicity. Plant vessel occlusion is critical for symptom development. The objective of the present study was to search for information that would help to explain the adhesion of X. fastidiosa cells to the xylem. Scanning electron microscopy revealed that adhesion may occur without the fastidium gum, an exopolysaccharide produced by X. fastidiosa, and X-ray microanalysis demonstrated the presence of elemental sulfur both in cells grown in vitro and in cells found inside plant vessels, indicating that the sulfur signal is generated by the pathogen surface. Calcium and magnesium peaks were detected in association with sulfur in occluded vessels. We propose an explanation for the adhesion and aggregation process. Thiol groups, maintained by the enzyme peptide methionine sulfoxide reductase, could be active on the surface of the bacteria and appear to promote cell-cell aggregation by forming disulfide bonds with thiol groups on the surface of adjacent cells. The enzyme methionine sulfoxide reductase has been shown to be an auxiliary component in the adhesiveness of some human pathogens. The negative charge conferred by the ionized thiol group could of itself constitute a mechanism of adhesion by allowing the formation of divalent cation bridges between the negatively charged bacteria and predominantly negatively charged xylem walls.

Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c

The features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3) located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00° values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.

Structural and Biochemical Characterization of Peroxiredoxin Q beta from Xylella fastidiosa CATALYTIC MECHANISM AND HIGH REACTIVITY

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 107 and 106 M(-1) s(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie similar to 12.3 angstrom apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Cloning, expression, purification and characterization of recombinant glutathione-S-transferase from Xylella fastidiosa

Xylella fastidiosa is an important pathogen bacterium transmitted by xylem-feedings leafhoppers that colonizes the xylem of plants and causes diseases on several important crops including citrus variegated chlorosis (CVC) in orange and lime trees. Glutathione-S-transferases (GST) form a group of multifunctional isoenzymes that catalyzes both glutathione (GSH)-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GSTs are the major detoxification enzymes found in the intracellular space and mainly in the cytosol from prokaryotes to mammals, and may be involved in the regulation of stress-activated signals by suppressing apoptosis signal-regulating kinase 1. In this study, we describe the cloning of the glutathione-S-transferase from X. fastidiosa into pET-28a(+) vector, its expression in Escherichia coli, purification and initial structural characterization. The purification of recombinant xfGST (rxfGST) to near homogeneity was achieved using affinity chromatography and size-exclusion chromatography (SEC). SEC demonstrated that rxfGST is a homodimer in solution. The secondary and tertiary structures of recombinant protein were analyzed by circular dichroism and fluorescence spectroscopy, respectively. The enzyme was assayed for activity and the results taken together indicated that rxfGST is a stable molecule, correctly folded, and highly active. Several members of the GST family have been extensively studied. However, xfGST is part of a less-studied subfamily which yet has not been structurally and biochemically characterized. In addition, these studies should provide a useful basis for future studies and biotechnological approaches of rxfGST. (C) 2008 Elsevier Inc. All rights reserved.

The iron stimulon of Xylella fastidiosa includes genes for type IV pilus and colicin V-like bacteriocins

Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2`-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 mu M ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.