Development and application of molecular‐based diagnosis for ‘<i>Candidatus</i> Liberibacter asiaticus’, the causal pathogen of citrus huanglongbing

Wang, Z.; Yin, Yanni; Hu, Hanjie; Yuan, Qing; Peng, Guoxiong; Xia, Yunni

doi:10.1111/j.1365-3059.2006.01438.x

Cited by 118 publications

(79 citation statements)

References 15 publications

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“…The amount of DNA obtained from the lysis was estimated using electrophoresis, with known concentrations of standard fragments of the vector pGEM ® 3Zf (+) (supplied by Applied Biosystems) as a control. The quantification of CLas based on copy number (CN) of the target gene was done as described by Wang et al (2006). The plasmid solution was serially diluted ten-fold from 10 ngµl −1 (2.1×10 9 CN) to 0.01 fgµl −1 (7×10 1 CN), and the Q-PCR assay was run by adding at each dilution point 100 ng of total DNA from healthy citrus plants.…”

Section: Standard Curvementioning

confidence: 99%

“…Li et al (2006) reported a Q-PCR assay capable of detecting and quantifying the three species of Liberibacter (africanus, asiaticus, and americanus). Wang et al (2006) reported a Q-PCR assay detecting CLas and a method for estimating the copy number of a cloned Liberibacter gene as an indirect measure of bacterial populations in plants. We have also previously developed a Q-PCR assay based on the TaqMan system and the 16S rDNA genomic sequences of CLas as a template (Carlos et al 2006).…”

Section: Introductionmentioning

confidence: 99%

“…A third experiment was conduced to test bacterial transmission to new trees using budwood infected with different amounts of bacteria. We used our published Q-PCR protocol (Carlos et al 2006) for CLas detection and an adaptation of the Wang et al (2006) method for population estimation.…”

Section: Introductionmentioning

confidence: 99%

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In planta multiplication and graft transmission of ‘Candidatus Liberibacter asiaticus’ revealed by Real-Time PCR

Coletta-Filho¹,

Carlos

Alves³

et al. 2009

Eur J Plant Pathol

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The bacterium Candidatus Liberibacter asiaticus (CLas) is associated with huanglongbing (HLB) in citrus in many countries. Despite the fact that many characteristics of the disease are known, the rate of multiplication of the bacterium within an infected tree is still poorly understood. To study this feature, we used the quantitative Real-Time polymerase chain reaction (Q-PCR) assay to follow and to quantify the multiplication of CLas in grafted infected young sweet orange plants. The rate of infection by grafting reached 100% at 120 days post-inoculation (dpi) showing that grafting could easily transmit CLas. A well-adjusted linear regression equation describing the bacterial growth in planta was obtained independently with measurements taken using repeated sampling in the same plant or different plants through the analysed period. The bacterial population, measured as copy number (CN) of the 16S rDNA target gene g −1 of tissue, increased 10,000 times from 10 3 at 30 dpi to approximately 10 8 CN at 240 dpi indicating that CLas multiplication was fastest in young citrus plants. We observed a direct relationship between the concentration of pathogen and the expression of symptoms. Yellowed leaves or shoots, are commonly the first observed symptom of HLB, and were present in trees with a low amount of bacteria (10 5 CN g −1 ). Blotchy mottle symptoms were observed in trees with 10 7 CN g −1 of bacteria after 180 dpi. Buds taken from infected, but nonsymptomatic branches were grafted on Rangpur lime and resulted in transmission rates ranging from 10 to 60%.

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Section: Standard Curvementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In planta multiplication and graft transmission of ‘Candidatus Liberibacter asiaticus’ revealed by Real-Time PCR

Coletta-Filho¹,

Carlos

Alves³

et al. 2009

Eur J Plant Pathol

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“…Transcriptome analysis has been used to successfully identify how Las infection influences gene expression in citrus plants on a global scale. 9,32,33 In particular, extensive changes in gene expression were identified for major biological processes such as stress responses, signal transduction, transport, cell organization and carbohydrate metabolism. 34 From a bacterial prospective, several proteins have been identified as important for virulence and growth.…”

Section: Introductionmentioning

confidence: 99%

Molecular mechanisms behind the accumulation of ATP and H2O2 in citrus plants in response to ‘Candidatus Liberibacter asiaticus’ infection

2017

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Candidatus Liberibacter asiaticus (Las) is a fastidious, phloem-restricted pathogen with a significantly reduced genome, and attacks all citrus species with no immune cultivars documented to date. Like other plant bacterial pathogens, Las deploys effector proteins into the organelles of plant cells, such as mitochondria and chloroplasts to manipulate host immunity and physiology. These organelles are responsible for the synthesis of adenosine triphosphate (ATP) and have a critical role in plant immune signaling during hydrogen peroxide (H 2 O 2 ) production. In this study, we investigated H 2 O 2 and ATP accumulation in relation to citrus huanglongbing (HLB) in addition to revealing the expression profiles of genes critical for the production and detoxification of H 2 O 2 and ATP synthesis. We also found that as ATP and H 2 O 2 concentrations increased in the leaf, so did the severity of the HLB symptoms, a trend that remained consistent among the four different citrus varieties tested. Furthermore, the upregulation of ATP synthase, a key enzyme for energy conversion, may contribute to the accumulation of ATP in infected tissues, whereas downregulation of the H 2 O 2 detoxification system may cause oxidative damage to plant macromolecules and cell structures. This may explain the cause of some of the HLB symptoms such as chlorosis or leaf discoloration. The findings in this study highlight important molecular and physiological mechanisms involved in the host plants' response to Las infection and provide new targets for interrupting the disease cycle.

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“…Quantification of the plasmid DNA based on the copy number (CN) of the target gene was determined as previously described by Wang et al (2006). The plasmid solution was serially diluted from 10 8 CN to 1 CN.…”

Section: Dna Sample and Plasmid Preparationmentioning

confidence: 99%

Novel set of real-time PCR primers for simultaneous detection of Liberibacter species associated with citrus Huanglongbing

Orce¹,

Sendín²,

Maraño

et al. 2015

Sci. agric. (Piracicaba, Braz.)

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Huanglongbing (HLB), a devastating citrus disease caused by the bacterium "Candidatus Liberibacter spp.", is now responsible for significant economic losses worldwide. Yet, no effective disease control has been found, and the non-cultivability of the bacterium has severely hampered studies on the pathogen. The 16S rDNA gene is a well-characterized sequence, essential for cell survival, and is used for bacterial identification or assignment of close relationships at the genus and species levels. Quantitative Real-Time PCR (qPCR) assays based on 16S rDNA genes are widely used in the detection of "Ca. Liberibacter spp." in multiplex reactions. We have developed for the first time a set of qPCR primers based on the conserved 16S rDNA gene, which specifically and simultaneously detects in a singleplex reaction, all three bacterial species associated with HLB, and can differentiate Ca. Liberibacter asiaticus or africanus from americanus by their characteristic melting curves. The assay is very sensitive, and it was possible to amplify expected DNA fragments with an efficiency of 98 % using the Syber Green system and a Ct value lower than tested methods for HLB diagnosis. The application of this fast, simple and efficient detection methodology could also be important in the detection of all species of HLB-associated Liberibacters and could contribute to early pathogen detection, a crucial step in the development of preventive strategies aimed at avoiding the dissemination of this devastating disease in HLB-free areas.

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Development and application of molecular‐based diagnosis for ‘Candidatus Liberibacter asiaticus’, the causal pathogen of citrus huanglongbing

Cited by 118 publications

References 15 publications

In planta multiplication and graft transmission of ‘Candidatus Liberibacter asiaticus’ revealed by Real-Time PCR

In planta multiplication and graft transmission of ‘Candidatus Liberibacter asiaticus’ revealed by Real-Time PCR

Molecular mechanisms behind the accumulation of ATP and H2O2 in citrus plants in response to ‘Candidatus Liberibacter asiaticus’ infection

Novel set of real-time PCR primers for simultaneous detection of Liberibacter species associated with citrus Huanglongbing

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