Citrus huanglongbing: validation of Real-Time PCR (qPCR) for the detection of Candidatus Liberibacter asiaticus and Candidatus Liberibacter americanus in Colombia

Citrus huanglongbing (HLB) is the most destructive citrus disease. Two of the three known HLB-associated CandidatusLiberibacter species were recently found to be present in the Americas. In this study, eggs, nymphs and adults of Diaphorinacitri Kuwayama (Hemiptera: Liviidae) and suspect citrus plant materials were collected in 25 municipalities in the departments of Cundinamarca, Santander, Valle del Cauca, Meta and Quindio (Colombia). The detection sensitivity, specificity and assay performance of the 16S rDNA-based real-time PCR (qPCR) were validated for the field survey of the disease in Colombia. The validation confirmed the reliability and robustness of the real-time PCR method for the detection of HLB bacteria in host citrus plant tissues and the vector D. citri. The diagnosis was performed for Candidatus Liberibacter asiaticus (Ca. L. asiaticus) and for Candidatus Liberibacter americanus (Ca. L. americanus) on 168 citrus plant material samples and 239 insect samples. Neither Ca. L. asiaticus nor Ca. L. americanus were detected in the host plants or insects vector, confirming the absence of the disease in the citrus-producing areas of Colombia.

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Resolution of the Identity of ‘Candidatus Liberibacter’ Species From Huanglongbing-Affected Citrus in East Africa

Plant Disease ◽

10.1094/pdis-11-16-1655-re ◽

2017 ◽

Vol 101 (8) ◽

pp. 1481-1488 ◽

Cited By ~ 13

Author(s):

Ronel Roberts ◽

Glynnis Cook ◽

Tim G. Grout ◽

Fathiya Khamis ◽

Ivan Rwomushana ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Housekeeping Genes ◽

Diaphorina Citri ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus ◽

Resolution Of The Identity

‘Candidatus Liberibacter asiaticus’, the bacterium associated with citrus Huanglongbing (HLB), was reported from Uganda and tentatively from Tanzania, posing a threat to citriculture in Africa. Two surveys of citrus expressing typical HLB symptoms were conducted in Uganda, Kenya, and Tanzania to verify reports of ‘Ca. L. asiaticus’ and to assess the overall threat of HLB to eastern and southern African citrus production. Samples were analyzed for the presence of ‘Candidatus Liberibacter’ species by real-time PCR and partial sequencing of three housekeeping genes, 16S rDNA, rplJ, and omp. ‘Ca. L. africanus’, the bacterium historically associated with HLB symptoms in Africa, was detected in several samples. However, samples positive in real-time PCR for ‘Ca. L. asiaticus’ were shown not to contain ‘Ca. L. asiaticus’ by sequencing. Sequences obtained from these samples were analogous to ‘Ca. L. africanus subsp. clausenae’, identified from an indigenous Rutaceae species in South Africa, and not to ‘Ca. L. asiaticus’. Results indicate a nontarget amplification of the real-time assay and suggest that previous reports of ‘Ca. L. asiaticus’ from Uganda and Tanzania may be mis-identifications of ‘Ca. L. africanus subsp. clausenae’. This subspecies was additionally detected in individual Diaphorina citri and Trioza erytreae specimens recovered from collection sites. This is the first report of ‘Ca. L. africanus subsp. clausenae’ infecting citrus and being associated with HLB symptoms in this host.

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Evaluation of DNA Amplification Methods for Improved Detection of “Candidatus Liberibacter Species” Associated with Citrus Huanglongbing

Plant Disease ◽

10.1094/pd-91-0051 ◽

2007 ◽

Vol 91 (1) ◽

pp. 51-58 ◽

Cited By ~ 49

Author(s):

Wenbin Li ◽

John S. Hartung ◽

Laurene Levy

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Dna Amplification ◽

Conventional Polymerase Chain Reaction ◽

Detection Sensitivity ◽

Conventional Pcr ◽

Plant Materials ◽

Standard Format ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing

Citrus huanglongbing (HLB), also known as citrus greening or citrus yellow shoot, is considered the most serious disease of citrus worldwide. The disease has Asian, African, and American forms caused by “Candidatus Liberibacter asiaticus”, “Ca. L. africanus”, and “Ca. L. americanus”, respectively, which can be spread efficiently by the psyllid vectors Diaphorina citri and Trioza erytreae and through contaminated plant materials. Infected citrus groves are usually destroyed or become unproductive in 5 to 8 years. The presumed low concentration and uneven distribution of the pathogens in citrus plants and vector insects make the phloem-limited bacterium difficult to detect consistently. In this study, we compared and validated four conventional polymerase chain reaction (PCR)-based protocols, one loop-mediated isothermal amplification (LAMP) protocol, and three TaqMan real-time PCR protocols. The detection sensitivity of the validated conventional PCR assays reported are improved compared with the original protocols. All of the validated conventional and the newly developed real-time methods were reliable for confirmatory tests for the presence of “Ca. Liberibacter spp.” in symptomatic samples. There were no differences in assay specificity among the standard format PCR-based methods evaluated. The TaqMan real-time PCR was 10- to 100-fold more sensitive than conventional PCR and LAMP, showing the potential to become a valuable tool for early detection and identification of “Ca. Liberibacter spp.” prior to the appearance of disease symptoms. The methods validated in this study will be very useful for regulatory response, effective management of infected trees, and development of a “Ca. Liberibacter spp.”-free nursery system.

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Highly sensitive and rapid detection of citrus Huanglongbing pathogen (Candidatus Liberibacter asiaticus) using Cas12a-based methods

Phytopathology ◽

10.1094/phyto-09-20-0443-r ◽

2021 ◽

Author(s):

Matthew Wheatley ◽

Yong-Ping Duan ◽

Yinong Yang

Keyword(s):

Early Detection ◽

Nucleic Acids ◽

Detection Sensitivity ◽

Sensitive Detection ◽

Detection Methods ◽

Candidatus Liberibacter Asiaticus ◽

Fluorescent Reporter ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus

Citrus Huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of its causal agent, Candidatus Liberibacter asiaticus (CLas), is critical for early diagnosis and successful management of HLB. However, current nucleic acid-based detection methods are often insufficient for the early detection of CLas from asymptomatic tissue, and unsuitable for high-throughput and field-deployable diagnosis of HLB. Here we report the development of the Cas12a-based DETECTR (DNA endonuclease-targeted CRISPR trans reporter) assay for highly specific and sensitive detection of CLas nucleic acids from infected samples. The DETECTR assay, which targets the five-copy nrdB gene specific to CLas, couples isothermal amplification with Cas12a trans-cleavage of fluorescent reporter oligos and enables detection of CLas nucleic acids at the attomolar level. The DETECTR assay was capable of specifically detecting the presence of CLas across different infected citrus, periwinkle and psyllid samples, and shown to be compatible with lateral flow assay technology for potential field-deployable diagnosis. The improvements in detection sensitivity and flexibility of the DETECTR technology position the assay as a potentially suitable tool for early detection of CLas in infected regions.

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Repertoire of novel sequence signatures for the detection of Candidatus Liberibacter asiaticus by quantitative real time-PCR

Journal of Citrus Pathology ◽

10.5070/c411024725 ◽

2014 ◽

Vol 1 (1) ◽

Author(s):

Sunitha Kogenaru ◽

Nian Wang

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Candidatus Liberibacter Asiaticus ◽

Quantitative Real Time Pcr ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Sequence Signatures

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

Phytopathology ◽

10.1094/phyto-98-5-0592 ◽

2008 ◽

Vol 98 (5) ◽

pp. 592-599 ◽

Cited By ~ 151

Author(s):

Satyanarayana Tatineni ◽

Uma Shankar Sagaram ◽

Siddarame Gowda ◽

Cecile J. Robertson ◽

William O. Dawson ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Real Time Pcr ◽

Citrus Tree ◽

Candidatus Liberibacter Asiaticus ◽

In Planta ◽

Chain Reaction ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Polymerase Chain

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic α-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of ‘Candidatus Liberibacter asiaticus,’ respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that ‘Ca. Liberibacter asiaticus’ was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/μg of total DNA in different tissues. A relatively high concentration of ‘Ca. Liberibacter asiaticus’ was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that ‘Ca. Liberibacter asiaticus’ was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.

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