Produção e caracterização de anticorpos policlonais contra Xanthomonas campestris pv. viticola

Araujo, João Sebastião de Paula; Reis, Fábio Bueno dos; Cruz, Geraldo Baeta; Oliveira, Bruno Cardoso de; Robbs, C. F.; Ribeiro, Raul de Lucena Duarte; Polidoro, J. C.

doi:10.1590/s0100-204x2005000300015

Cited by 7 publications

(6 citation statements)

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“…Thus, it was possible to prove, based on the specificity of the AC4558 antiserum, the identities of the observed bacteria under the light microscope, with the formation of black precipitates in the Xcvi infection and colonization sites ( Figures 4D, E). These results are in conformity with the reports by Araujo et al (2005) and Tostes (2012). A detail of the methodological sequence to identify the bacterium is schematized in Figure 4C, based on studies by James et al (1994) and Olivares et al (1997), reporting immune-stained diazothrophic endophytic bacteria associated with wheat and sugar cane crops.…”

Section: Resultssupporting

confidence: 94%

“…Despite considerable progress in detecting Xcvi in asymptomatic samples (Araujo et al, 2005;Freitas, 2012), there remain several questions that should guide future research. The ecology of this bacterium is poorly understood and little is known about epiphytic growth, mechanisms of pathogenesis, modes of dissemination and survival.…”

Section: Resultsmentioning

confidence: 99%

“…Then, after a fast freshwater washing procedure, the primary AC4558 antiserum was applied (Araujo et al, 2005) at a 1:400 dilution. The material was incubated in a moist chamber for 1 hour at 25ºC.…”

Section: Scanning Electron Microscopymentioning

confidence: 99%

“…After reaching a favorable site, Xcvi can resist removal, which is a selective advantage responsible for increasing and stabilizing the resident population (Araujo, 2001;Nascimento & Mariano, 2004). Araujo et al (2005) developed antibodies against Xcvi. These allowed the authors to specifically detect Xcvi cells associated to the surface and the interior of symptomatic and asymptomatic plant tissues in grapevines.…”

Section: Introductionmentioning

confidence: 99%

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Detection and cellular localization of Xanthomonas campestris pv. viticola in seeds of commercial 'Red Globe' grapes

Tostes

Araujo

Farias

et al. 2014

Trop. plant pathol.

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Commercial grapevine fruit (Vitis vinifera) of the Red Globe variety were collected in vineyards from Vale do São Francisco lower basin, an area of occurrence of grapevine bacterial canker. Seeds were extracted, classified as symptomatic or asymptomatic and processed in order to be observed under light (LM) and scanning electron microscopy (SEM) along with silver-enhanced immunogold labeling, to allow bacterial detection using a policlonal antibody against Xanthomonas campestris pv. viticola (Xcvi), etiological agent of the disease. The seed samples showed bacterial aggregates associated to the tegument surface and to the first parenchymal layer beneath the seed tegument. Bacterial identity was confirmed by immunogold labeling. This appears to be the first report of Xcvi associated to asymptomatic seeds and berries, suggesting a systemic mechanism to spread and colonize different tissues and sites, driving attention to seeds, presenting them as an important niche for survival and dissemination of this pathogen. These results point towards the need of including seed-bearing fruit in studies regarding Xcvi epidemiology.

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Section: Resultssupporting

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Section: Scanning Electron Microscopymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Detection and cellular localization of Xanthomonas campestris pv. viticola in seeds of commercial 'Red Globe' grapes

Tostes

Araujo

Farias

et al. 2014

Trop. plant pathol.

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“…Adicionaram-se 100 mL/poço do substrato ABTS (2.2'-Amino-di-[3etil-benzotiazolinasulfonato(6)] sal de diamônio cristalizado, (Boehringer Mannheim Biochemica) na concentração de 1 mg/mL diluído em tampão ABTS (Boehringer Mannheim Biochemica -perborato de sódio, ácido cítrico e fosfato de sódio dibásico -1,67 g em 10 mL de água destilada A avaliação da sobrevivência do Azospirillum spp no inoculante turfoso (preparado como citado) foi feita através de contagens em meio de cultivo semissólido LGI pH 6,0-6,2, em 24 horas após a inoculação e a intervalos de 15 dias subsequentemente por um período de armazenamento de 60 dias utilizando o método do NMP (DÖBEREINER et al 1995) usando três repetições a cada coleta. Resultados similares foram constatados por ARAÚJO et al (2005) utilizando soros policlonais purificados em proteína A, cujo nível mínimo de detecção ficou em torno de 10 6 células mL -1 , para os isolados 4558 e 4560 ambos de Xanthomonas campestris pv. vitícola.…”

Section: Methodsunclassified

Produção, caracterização e aplicação de anticorpo policlonal contra Azospirillum amazonense estirpe Am15

Silva

Reis

2009

Bragantia

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RESUMOO uso de ferramentas moleculares e imunológicas permite a detecção e o monitoramento específico de microrganismos usados como inoculantes agrícolas. O maior exemplo de sucesso com uso de bactérias diazotróficas na agricultura é o caso da inoculação de soja no Brasil. Entretanto, a inoculação de bactérias diferentes de rizóbio não permite a localização de um sítio específico onde ocorra A redução do nitrogênio atmosférico, pois não há a formação de estruturas nodulares. Este trabalho foi realizado na EmbrapaAgrobiologia, e objetivou produzir e caracterizar um anticorpo policlonal a partir da inoculação de células intactas da estirpe Am15 pertencente à espécie Azospirillum amazonense (Anti-Am15) utilizando o método de ELISA indireto. O anticorpo foi usado em sementes peletizadas com turfa contendo a bactéria-alvo e avaliada sua sobrevivência durante 60 dias. Este anticorpo foi capaz de quantificar populações bacterianas em amostras cujo número mínimo celular foi superior a valores de 100.000 células por mL. De acordo com os resultados, o soro policlonal foi especifico contra o antígeno de interesse, sendo possível sua utilização em estudos de quantificação e monitoramento do Azospirillum sp em plantas de milho que receberam inóculo e no controle da qualidade de inoculante turfoso.Palavras-chave: imunologia, anticorpos, bactérias diazotróficas. ABSTRACT PRODUCTION, CHARACTERIZATION AND APPLICATION OF POLICLONAL ANTIBODIES AGAINST AZOSPIRILLUM AMAZONENSE STRAIN AM15Molecular and immunological approaches are useful for monitoring the localization of microrganism used as agricultural inoculants. A successful example is the use of a diazotrophic bacteria, as soybean in Brazil. However, as diazotrophic bacterias other than rizobium do not form nodular structures, the localization of the specific site where nitrogen reduction occurs is impossible. In this study, carried out at Embrapa Agrobiology, a polyclonal antibody against Azospirillum amazonense (AsAm15) was to produced and characterized through an indirect Enzyme Linked Immunosorbent Assay (ELISA). Maize seedscoated with peat containing the target bacterium were enoculated with the policlonal antibody and evaluated during 60 days. The antibody was able to quantify bacteria populations in samples where the minimal cell number were superior to 100,000 cells L -1 . According to these results, the policlonal antibody As-Am15 showed high specificity to the target antigen and was able to quantify or monitor Azospirillum strains and able to be used as a control of the quality of a peat inoculant product.

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Detection of Xanthomonas citri pv. viticola on grapevine by real-time PCR and BIO-PCR using primers designed from pathogenicity and xanthomonadin gene sequences

et al. 2019

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Grapevine bacterial canker caused byXanthomonas citri pv. viticola (X. campestris pv. viticola) (Xcv), was detected in Brazil in 1998 and is currently regarded as a quarantine disease with limited distribution in the country. To improve sensitivity and speed in the detection of Xcv in asymptomatic grapevines, two pairs of primers were designed, targeting sequences of a pathogenicity gene (hrpB) and the xanthomonadin coding cluster. Both pairs were tested in conventional PCR (cPCR) and real-time PCR (qPCR) formats. Primers targeting the hrpB gene showed cross reactions with other Xanthomonas spp. but were effective for use in both cPCR and qPCR, whereas primers for the xanthomonadin gene were highly specific for Xcv but showed low efficiency in qPCR. Enrichment of plant extracts in semi-selective medium before qPCR allowed a significant increase in sensitivity when compared to total DNA extraction, making it possible to detect as low as 10 1 CFU ml -1 . Under natural infection conditions, symptomatic and asymptomatic grapevines were tested by qPCR with hrpB primers and cPCR with xanthomonadin primers. In both cases, plant extracts were enriched for 36-72h. Xcv was detected in all symptomatic samples by qPCR and the result was confirmed by cPCR. For the asymptomatic samples, Xcv was detected in 93.4% with qPCR and in 89.5% with cPCR. These two methods offer advantages in terms of sensitivity and specificity, and they could be useful in quarantine programs, certification of grapevine propagating material and detection of inoculum sources in alternative hosts, contributing to the prevention of pathogen spread to disease-free areas.Keywords Grapevine bacterial canker . Vitis vinifera . Xanthomonas campestris pv. viticola. PCR-based diagnosis . qPCR

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Produção e caracterização de anticorpos policlonais contra Xanthomonas campestris pv. viticola

Cited by 7 publications

References 6 publications

Detection and cellular localization of Xanthomonas campestris pv. viticola in seeds of commercial 'Red Globe' grapes

Detection and cellular localization of Xanthomonas campestris pv. viticola in seeds of commercial 'Red Globe' grapes

Produção, caracterização e aplicação de anticorpo policlonal contra Azospirillum amazonense estirpe Am15

Detection of Xanthomonas citri pv. viticola on grapevine by real-time PCR and BIO-PCR using primers designed from pathogenicity and xanthomonadin gene sequences

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