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

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.

scholarly journals Targeted Early Detection of Citrus Huanglongbing Causal Agent ‘Candidatus Liberibacter asiaticus’ Before Symptom Expression

2019 ◽  
Vol 109 (6) ◽  
pp. 952-959 ◽  
Author(s):  
Sheo Shankar Pandey ◽  
Nian Wang
Keyword(s):  
Early Detection ◽  
Early Diagnosis ◽  
Disease Incidence ◽  
Blue Staining ◽  
Insect Vector ◽  
Symptom Expression ◽  
Candidatus Liberibacter Asiaticus ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus

Citrus Huanglongbing (HLB) is the most severe disease of citrus plants caused by ‘Candidatus Liberibacter asiaticus’ and transmitted by the insect vector Asian citrus psyllid (ACP). No effective curative measure is available against HLB. For citrus production areas without HLB or with low HLB disease incidence, removal of ‘Ca. L. asiaticus’ inoculum is critical to prevent HLB spread. Such a strategy requires robust early diagnosis of HLB for inoculum removal to prevent ACP acquisition and transmission of ‘Ca. L. asiaticus’. However, early diagnosis of HLB is challenging, because the citrus trees remain asymptomatic for several months to years after ‘Ca. L. asiaticus’ transmission by ACP. In this study, we report a new method for targeted early detection of ‘Ca. L. asiaticus’ in cultivar Valencia sweet orange (Citrus sinensis) before HLB symptom expression. We take advantage of the fact that ‘Ca. L. asiaticus’ remains around the ACP feeding site immediately after transmission into the young flush and before flush maturation. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect ‘Ca. L. asiaticus’ in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting ‘Ca. L. asiaticus’ inoculum removal and preventing HLB from spreading.

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

2014 ◽  
Vol 32 (3) ◽  
pp. 377-389 ◽  
Author(s):  
Jorge Evelio Ángel ◽  
Erick Geovanni Hernández ◽  
Néstor Andrés Herrera ◽  
Linda Yhiset Gómez ◽  
Ángela Patricia Castro ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Candidatus Liberibacter Asiaticus ◽  
Plant Materials ◽  
Citrus Plant ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus ◽  
Reliability And Robustness

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.

scholarly journals Home Detection Kit for Candidatus Liberibacter asiaticus (LAS) Associated with Citrus Huanglongbing from Psyllids

2014 ◽  
Vol 1 (1) ◽  
Author(s):  
Manjunath L. Keremane ◽  
Chandrika Ramadugu ◽  
Ryo Kubota ◽  
Yongping Duan ◽  
David Hall ◽  
...  
Keyword(s):  
Candidatus Liberibacter Asiaticus ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus

Regional Spatial-Temporal Spread of Citrus Huanglongbing Is Affected by Rain in Florida

2018 ◽  
Vol 108 (12) ◽  
pp. 1420-1428 ◽  
Author(s):  
M. M. Shimwela ◽  
T. S. Schubert ◽  
M. Albritton ◽  
S. E. Halbert ◽  
D. J. Jones ◽  
...  
Keyword(s):  
Large Scale ◽  
Disease Incidence ◽  
Kernel Density ◽  
Candidatus Liberibacter Asiaticus ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Density Analysis ◽  
Liberibacter Asiaticus ◽  
Kernel Density Analysis ◽  
Temporal Spread

Citrus huanglongbing (HLB), associated with ‘Candidatus Liberibacter asiaticus’ (Las), disseminated by Asian citrus psyllid (ACP), has devastated citrus in Florida since 2005. Data on HLB occurrence were stored in databases (2005 to 2012). Cumulative HLB-positive citrus blocks were subjected to kernel density analysis and kriging. Relative disease incidence per county was calculated by dividing HLB numbers by relative tree numbers and maximum incidence. Spatiotemporal HLB distributions were correlated with weather. Relative HLB incidence correlated positively with rainfall. The focus expansion rate was 1626 m month−1, similar to that in Brazil. Relative HLB incidence in counties with primarily large groves increased at a lower rate (0.24 year−1) than in counties with smaller groves in hotspot areas (0.67 year−1), confirming reports that large-scale HLB management may slow epidemic progress.

scholarly journals Historical Perspectives, Management, and Current Research of Citrus HLB in Guangdong Province of China, Where the Disease has been Endemic for Over a Hundred Years

2018 ◽  
Vol 108 (11) ◽  
pp. 1224-1236 ◽  
Author(s):  
Zheng Zheng ◽  
Jianchi Chen ◽  
Xiaoling Deng
Keyword(s):  
Southern China ◽  
Candidatus Liberibacter Asiaticus ◽  
Sequencing Technology ◽  
Next Generation Sequencing Technology ◽  
Historical Perspectives ◽  
Citrus Production ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus ◽  
Generation Sequencing

Citrus huanglongbing (HLB) is a highly destructive disease currently threatening citrus production worldwide. In China, the disease is exclusively associated with ‘Candidatus Liberibacter asiaticus’, a nonculturable proteobacterium. HLB was observed in Guangdong of China over a hundred years ago. Researchers and citrus growers have been battling with the disease through vigorous research and have exercised various control practices. Much of the early work was not well known outside China. This review is intended to fill in gaps of historical information by reviewing selected literature records. Along the way, the HLB system within southern China was evaluated. Emphases were on comparison of symptomatology, evolution of etiology, control practices, and impacts of using next-generation sequencing technology for ‘Ca. L. asiaticus’ research and detection.

scholarly journals First report on molecular evidence of Candidatus Liberibacter asiaticus associated with citrus Huanglongbing in Venezuela

2020 ◽  
Vol 102 (4) ◽  
pp. 1333-1333
Author(s):  
Edgloris Marys ◽  
Eduardo Rodríguez-Román ◽  
Rafael Mejías ◽  
Alexander Mejías ◽  
Mailyn Mago ◽  
...  
Keyword(s):  
Molecular Evidence ◽  
Candidatus Liberibacter Asiaticus ◽  
First Report ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus

scholarly journals Latency and Persistence of ‘Candidatus Liberibacter asiaticus’ in Its Psyllid Vector, Diaphorina citri (Hemiptera: Liviidae)

2017 ◽  
Vol 107 (3) ◽  
pp. 264-272 ◽  
Author(s):  
Maria Cristina Canale ◽  
Arthur Fernando Tomaseto ◽  
Marineia de Lara Haddad ◽  
Helvécio Della Coletta-Filho ◽  
João Roberto Spotti Lopes
Keyword(s):  
Latent Period ◽  
Acquisition Time ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Old Adults ◽  
Sequential Inoculation ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus ◽  
Series Of Experiments

Although ‘Candidatus Liberibacter asiaticus’ (Las) is a major pathogen associated with citrus huanglongbing (HLB), some characteristics of transmission by the psyllid vector Diaphorina citri are not fully understood. We examined the latent period and persistence of transmission of Las by D. citri in a series of experiments at 25°C, in which third-instar psyllid nymphs and 1-week-old adults were confined on infected citrus for an acquisition access period (AAP), and submitted to sequential inoculation access periods (IAPs) on healthy citrus seedlings. The median latent period (LP50, i.e., acquisition time after which 50% of the individuals can inoculate) of 16.8 and 17.8 days for psyllids that acquired Las as nymphs and adults, respectively, was determined by transferring single individuals in 48-h IAPs. Inoculation events were intermittent and randomly distributed over the IAPs, but were more frequent after acquisition by nymphs. A minimum latent period of 7 to 10 days was observed by transferring groups of 10 psyllids in 48-h IAPs, after a 96-h AAP by nymphs. Psyllids transmitted for up to 5 weeks, when submitted to sequential 1-week IAPs after a 14-day AAP as nymphs. The long latent period and persistence of transmission are indirect evidences of circulative propagation of Las in D. citri.

scholarly journals Development on Infected Citrus over Generations Increases Vector Infection by ‘Candidatus Liberibacter Asiaticus in Diaphorina citri’

Insects ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 469
Author(s):  
Inaiara de Souza Pacheco ◽  
Diogo Manzano Galdeano ◽  
João Roberto Spotti Lopes ◽  
Marcos Antonio Machado
Keyword(s):  
Management Strategies ◽  
Fourth Generation ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus ◽  
Citrus Orchards ◽  
Vector Efficiency ◽  
Control And Eradication

‘Candidatus Liberibacter asiaticus’ (CLas) is a major causal agent of citrus Huanglongbing (HLB), which is transmitted by Asian citrus psyllid (ACP), Diaphorina citri, causing severe losses in various regions of the world. Vector efficiency is higher when acquisition occurs by ACP immature stages and over longer feeding periods. In this context, our goal was to evaluate the progression of CLas population and infection rate over four ACP generations that continuously developed on infected citrus plants. We showed that the frequency of CLas-positive adult samples increased from 42% in the parental generation to 100% in the fourth generation developing on CLas-infected citrus. The bacterial population in the vector also increased over generations. This information reinforces the importance of HLB management strategies, such as vector control and eradication of diseased citrus trees, to avoid the development of CLas-infected ACP generations with higher bacterial loads and, likely, a higher probability of spreading the pathogen in citrus orchards.

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