Integrated Approaches for Detection of Plant Pathogenic Bacteria and Diagnosis of Bacterial Diseases

Alvarez, Anne M.

doi:10.1146/annurev.phyto.42.040803.140329

Cited by 173 publications

(144 citation statements)

References 123 publications

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“…These quantification methods significantly extrapolate the number of viable cells in mixed populations (Nocker and Camper 2006). Other methods for determination of viable cells such as those utilising RNA-based diagnostics are expensive and time-consuming (Alvarez 2004;Norton and Batt 1999). EMA allows selective amplification of DNA by QPCR from only viable and not dead cells, which substantially increases the utility of PCR for rapidly determining and quantifying the presence of targeted viable microorganisms in environmental samples without enrichment (Rudi et al 2005).…”

Section: Dynamic Development Of Las Concentration In Plantamentioning

confidence: 99%

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Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

et al. 2009

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Citrus Huanglongbing (HLB) is a devastating disease of citrus known to be associated with a fastidious, phloem-limited Gram-negative, yet to be cultured bacterium in the genus Candidatus Liberibacter. In the present study we have developed a method to quantify viable Candidatus Liberibacter asiaticus (Las) with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells. First, calibration curves were developed with the aid of quantitative real-time PCR (QPCR) by using a plasmid template consisting of a 703 bp DNA fragment of rplKAJL-rpoBC (β-operon) region. Standard equations were then developed to quantify Las genome equivalents in citrus, periwinkle, and Asian citrus psyllid, Diaphorina citri. To overcome the limitation of quantitative PCR in discriminating between live and dead bacterial cells, EMA was used to inhibit the amplification of DNA from the dead cells of Las in plant samples. By using the standard equations and EMA-QPCR methods developed in this study, we found that the proportion of viable cells in citrus and periwinkle ranged from 17-31% and 16-28%, respectively. It was determined that a minimum bacterial concentration is required for HLB symptom development by quantifying the population of Las in symptomatic and asymptomatic leaves. The EMA-QPCR methodology developed in the present study should provide an accurate assessment of viable HLB pathogen, providing a tool to investigate disease epidemiology and thus act as a crucial component for disease assessment and management.

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Section: Dynamic Development Of Las Concentration In Plantamentioning

confidence: 99%

“…Quantitative assays are useful for determining the virulence mechanism(s) of pathogens, infection ability of insect vectors, and development of efficient management strategies (Alvarez 2004;Bach et al 2002). Since the efforts to isolate Las in pure culture have been unsuccessful, accurate quantification remains difficult.…”

Section: Introductionmentioning

confidence: 99%

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

et al. 2009

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“…However, boiling the bacteria samples could improve the sensitivity of detection. Several previous reports have mention that using extraction buffer containing EDTA and lysozyme to removed cell surface component could improve the sensitivity of ELISA detection (Jones et al, 1997, Alvarez, 2004. However, EDTA was not used in this study but the sensitivity of detection was still high.…”

Section: Bacteria Isolation and Pathogenicity Testmentioning

confidence: 86%

“…Many studies reported that, the ELISA sensitivity 10 5~ 10 6 CFU/mL is sufficient for identification of bacteria pathogens from symptomatic plants and colonies on selective media (Jin et al, 2001;Alvarez, 2004;de Leon et al, 2008;Kokoskova and Mraz, 2008). However, boiling the bacteria samples could improve the sensitivity of detection.…”

Section: Bacteria Isolation and Pathogenicity Testmentioning

confidence: 99%

Comparison between Serological and Molecular Detection of Citrus Canker Pathogen (<i>Xanthomonas axonopodis</i> pv. <i>citri</i>)

Songwattana¹,

Kutudat-Cairns²

2011

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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The specificity and sensitivity of serological and molecular tools for the detection of citrus canker pathogen (Xanthomonas axonopodis pv. citri; Xac) were investigated and compared. Virulence Xac BP210 was used as antigen for antisera production. The sensitivity of 1:2,000 diluted antisera were at 10 6 CFU/mL for live cell and 10 5 CFU/mL for dead cells. The cross-reaction of the antisera was observed only with X. campestris pv. vesicatoria but not other Xanthomonas nor other unrelated bacteria tested. Molecular tool was performed using specific primer to the rpf gene on Xac. The PCR amplification indicated that, allXac isolates amplified a product of 581 bp which was not seen in other Xanthomonas sp. and unrelated bacteria tested. The sensitivity of these specific primers was down to at least 1 cell which was effective to detect the pathogen in both infected symptomatic and asymptomatic lime tissues. The serological tool was able to detect the pathogen only on infected leaves of day 4 post inoculation when the symptoms were already detected by eye. The serological tool can be used to detect and quantify the present of Xac to study the disease development on symptomatic tissues.

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“…Antibodies are commercially available for use in immunofluorescence and ELISA (Jones et al, 1997;Alvarez, 2004); however, published data on their specificity across X. euvesicatoria, X. gardneri, X. perforans and X. vesicatoria are not reported. Some monoclonal antibodies appear to be species-specific (Bouzar et al, 1994a).…”

Section: Serological Methodsmentioning

confidence: 99%

PM 7/110 (1) Xanthomonas spp. (Xanthomonas euvesicatoria, Xanthomonas gardneri, Xanthomonas perforans, Xanthomonas vesicatoria) causing bacterial spot of tomato and sweet pepper

2013

EPPO Bulletin

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Integrated Approaches for Detection of Plant Pathogenic Bacteria and Diagnosis of Bacterial Diseases

Cited by 173 publications

References 123 publications

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

Comparison between Serological and Molecular Detection of Citrus Canker Pathogen (<i>Xanthomonas axonopodis</i> pv. <i>citri</i>)

PM 7/110 (1) Xanthomonas spp. (Xanthomonas euvesicatoria, Xanthomonas gardneri, Xanthomonas perforans, Xanthomonas vesicatoria) causing bacterial spot of tomato and sweet pepper

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