In Planta Distribution of ‘<i>Candidatus</i> Liberibacter asiaticus’ as Revealed by Polymerase Chain Reaction (PCR) and Real-Time PCR

Satyanarayana, T.; Sagaram, Uma Shankar; Gowda, Siddarame; Robertson, Cecile J.; Dawson, William O.; Iwanami, Toru; Wang, Nian

doi:10.1094/phyto-98-5-0592

Cited by 246 publications

(221 citation statements)

References 19 publications

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“…HLB bacterial concentration was higher in leaves than in roots. Previous reports have also indicated an uneven distribution of Las cells in different tissues of infected sweet orange trees (Tatineni et al 2008). The present observation is also consistent with the findings for phytoplasma (Christensen et al 2004).…”

Section: Dynamic Development Of Las Concentration In Plantamentioning

confidence: 80%

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: 80%

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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“…The copy numbers for the target PCR products were calculated from the standard curves as previously described and based on the assumption of the average weight of a base pair equal to 650 Daltons (Broberg et al, 2003, Bustin et al, 2009, Fey et al, 2004, Gruss and Sauer, 1975, Tatineni et al, 2008.…”

Section: Accepted M Manuscriptmentioning

confidence: 99%

The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology

Sohrabi

Nair

Samaranayake

et al. 2016

Journal of Microbiological Methods

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Q., The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology, Journal of Microbiological Methods (2016Methods ( ), doi: 10.1016Methods ( /j.mimet.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A C C E P T E DM A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Highlights Lysozyme-achromopeptidase cell lysis yielded the highest gDNA level  Lysozyme-zirconium beads cell lysis extracted the highest level of bacterial DNA  Lysozyme-zirconium beads cell lysis yielded the highest level of Firmicutes A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT ABSTRACTRecent culture-independent studies have enabled detailed mapping of human microbiome that has not been hitherto achievable by culture-based methods. DNA extraction is a key element of bacterial culture-independent studies that critically impacts on the outcome of the detected microbial profile. Despite the variations in DNA extraction methods described in the literature, no standardized technique is available for the purpose of microbiome profiling.Hence, standardization of DNA extraction methods is urgently needed to yield comparable data from different studies. We examined the effect of eight different cell lysis protocols on the yield and quality of the extracted DNA from oral rinse samples. These samples were exposed to cell lysis techniques based on enzymatic, mechanical, and a combination of enzymatic-mechanical methods. The outcome measures evaluated were total bacterial population, Firmicutes levels and human DNA contamination (in terms of surrogate GAPDH levels). We noted that all three parameters were significantly affected by the method of cell lysis employed. Although the highest yield of gDNA was obtained using lysozymeachromopeptidase method, the lysozyme-zirconium beads method yielded the peak quantity of total bacterial DNA and Firmicutes with a lower degree of GAPDH contamination compared with the other methods. Taken together our data clearly points to an urgent need for a consensus, standardized DNA extraction technique to evaluate the oral microbiome using oral rinse samples. Further, if Firmicutes levels are the focus of investigation in oral rinse microbiome analyses then the lysozyme-zirconium bead method would be the method of choice in preference to others.

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“…Moreover, variations in feeding behaviour among species may also contribute to the existing differential transmission efficiency of vectors (Turell et al 2001;Daugherty and Almeida 2009) through governing the degree to which they are exposed to a pathogen. Differential exposure may result from the heterogeneous distribution of pathogens, either temporally or spatially, among and/or within host species (Dekker et al 1998;Saracco et al 2005;Shriram et al 2005;Kilpatrick et al 2006;Tatineni et al 2008). Furthermore, while feeding behaviour may affect vector exposure to pathogens, it also affects an individual's exposure to would-be predators present in the surrounding environment.…”

Section: Introductionmentioning

confidence: 99%

Background matching behaviour and pathogen acquisition: plant site preference does not predict the bacterial acquisition efficiency of vectors

Rashed

Killiny

Kwan

et al. 2010

Arthropod-Plant Interactions

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Many insect-borne pathogens are heterogeneously distributed within their hosts: therefore, a vector's within-plant distribution may be a predictor of its exposure to pathogens. In this study, we set out to quantify plant site preference, in the context of background matching, and investigated its effect on acquisition of a bacterial pathogen by its leafhopper vectors. The two green-coloured species, Graphocephala atropunctata and Draeculacephala minerva, preferred green plant tissue and artificial backgrounds whereas the brown-coloured Homalodisca vitripennis preferred brown stem tissue and backgrounds. Within-plant feeding site did not predict either the acquisition success or the number of plant-pathogenic Xylella fastidiosa cells acquired by the vectors; an 86% mortality for G. atropunctata was reported on the lignified stem tissue. Overall, H. vitripennis acquired significantly more cells than G. atropunctata. A novel artificial diet-based transmission system was used to further illustrate that the observed between-species difference in the number of cells acquired was independent of vector-host plant interactions. H. vitripennis, a less efficient vector of the bacterium X. fastidiosa on grapevines, acquired more bacterial cells than G. atropunctata, possibly due to its larger size. Contrary to previous assumptions, pathogen acquisition efficiency by the vectors did not explain their reported differences in inoculation. Vector interactions with the host during the inoculation stage should be evaluated as another determinant of X. fastidiosa transmission efficiency.

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In Planta Distribution of ‘Candidatus Liberibacter asiaticus’ as Revealed by Polymerase Chain Reaction (PCR) and Real-Time PCR

Cited by 246 publications

References 19 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)

The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology

Background matching behaviour and pathogen acquisition: plant site preference does not predict the bacterial acquisition efficiency of vectors

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