scholarly journals A New Diagnostic System for Detection of ‘Candidatus Liberibacter asiaticus’ Infection in Citrus

Plant Disease ◽  
2013 ◽  
Vol 97 (10) ◽  
pp. 1295-1300 ◽  
Author(s):  
Lianming Lu ◽  
Baoping Cheng ◽  
Jinai Yao ◽  
Aitian Peng ◽  
Danchao Du ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Polyclonal Antibodies ◽  
Diagnostic System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Candidatus Liberibacter Asiaticus ◽  
Negative Results ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

In this study, two polyclonal antibodies were produced against the Omp protein of ‘Candidatus Liberibacter asiaticus’. First, omp genes were sequenced to exhibit 99.9% identity among 137 isolates collected from different geographical origins. Then, two peptides containing the hydrophobic polypeptide-transport-associated (POTRA) domain and β-barrel domain, respectively, were identified on Omp protein. After that, these two peptides were overexpressed in Escherichia coli and purified by affinity chromatography to immunize the white rabbits. Finally, the antiserum was purified by affinity chromatography. The two Omp antibodies gave positive results (0.454 to 0.633, 1:1,600 dilution) in enzyme-linked immunosorbent assay against ‘Ca. L. asiaticus’-infected samples collected from different geographical origins but revealed negative results against other pathogen-infected, nutrient-deficient and healthy samples. The antibody against the POTRA domain of Omp protein could detect ‘Ca. L. asiaticus’ in 45.7% of the symptomatic samples compared with a 56.2% detection rate with a polymerase chain reaction assay. These new antibodies will provide a very useful supplement to the current approaches to ‘Ca. L. asiaticus’ detection and also provide powerful research tools for tracking distribution of this pathogen in vivo.

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

2008 ◽  
Vol 98 (5) ◽  
pp. 592-599 ◽  
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.

scholarly journals Reaction of sweet orange cultivars expressing the attacin A gene to 'Candidatus Liberibacter asiaticus' infection

2013 ◽  
Vol 48 (11) ◽  
pp. 1440-1448 ◽  
Author(s):  
Rafaella Teles Arantes Felipe ◽  
Francisco de Assis Alves Mourão Filho ◽  
Silvio Aparecido Lopes ◽  
Beatriz Madalena Januzzi Mendes ◽  
Maurel Behling ◽  
...  
Keyword(s):  
Sweet Orange ◽  
Pcr Analysis ◽  
Candidatus Liberibacter Asiaticus ◽  
Randomized Design ◽  
Transgenic Lines ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain ◽  
Attacin A ◽  
Leaf Symptoms

The objective of this work was to evaluate the reaction of four sweet orange cultivars expressing the attacin A gene to 'Candidatus Liberibacter asiaticus' (Las) infection, a bacterium associated to huanglongbing (HLB) disease. Transgenic sweet orange plants of Hamlin, Natal, Pêra, and Valência cultivars, as well as nontransgenic controls received inocula by grafting budwood sections of HLB-infected branches. Disease progression was evaluated through observations of leaf symptoms and by polymerase chain reaction (PCR) analysis, eight months after inoculation. A completely randomized design was used, with four experiments (one for each cultivar) performed simultaneously. Bacteria title was estimated by quantitative PCR (qPCR). HLB symptoms and Las titers were present in nontransgenic and transgenic plants expressing the attacin A gene of the four sweet orange cultivars, eight months after bacteria inoculation. Five transgenic lines (transformation events) of 'Pêra' sweet orange expressing the attacin A gene have significantly lower Las titers in comparison with nontransgenic plants of this cultivar.

scholarly journals Seasonal Variation of ‘Candidatus Liberibacter asiaticus’ Titers in New Shoots of Citrus in Distinct Environments

Plant Disease ◽  
2017 ◽  
Vol 101 (4) ◽  
pp. 583-590 ◽  
Author(s):  
Silvio A. Lopes ◽  
Fernanda Q. B. F. Luiz ◽  
Hermes T. Oliveira ◽  
Juan C. Cifuentes-Arenas ◽  
Laudecir L. Raiol-Junior
Keyword(s):  
Sweet Orange ◽  
Quantitative Polymerase Chain Reaction ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Cumulative Rainfall ◽  
Data Loggers ◽  
Minas Gerais State ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

The major citrus area of Brazil occupies near 450,000 ha between the Triângulo Mineiro (TM) region of Minas Gerais State and the south of São Paulo State (SPS). Significant climatic variation occurs between regions which could affect huanglongbing (HLB) progress, which is lower in TM. To investigate this possibility, young sweet orange shoots were sampled periodically over 2 years to determine ‘Candidatus Liberibacter asiaticus’ titers in naturally infected trees in orchards in Analândia, central SPS, and Frutal and Comendador Gomes, within TM. Data-loggers recorded local temperature and relative humidity hourly. In the laboratory, five ‘Ca. L. asiaticus’-free Diaphorina citri adults were placed on each sampled shoot for 48 h to feed and acquire the pathogen. Shoots and insects were individually analyzed by quantitative polymerase chain reaction to determine ‘Ca. L. asiaticus’ titers. The incidence of ‘Ca. L. asiaticus’-positive shoots, ‘Ca. L. asiaticus’ titers, and acquisition rates were lower for shoots from Comendador Gomes than those from Frutal or Analândia. Stronger association was observed between ‘Ca. L. asiaticus’ titers and the number of hours below 15°C (h < 15°C) or above 30°C (h > 30°C), and cumulative rainfall registered during the 15 days prior to sampling of shoots on each occasion. ‘Ca. L. asiaticus’ titers associated positively with h < 15°C and rainfall and negatively with h > 30°C. The slower spread and lower incidence of HLB in TM may be related to lower incidences of ‘Ca. L. asiaticus’-positive young shoots and lower titers of ‘Ca. L. asiaticus’ in the same shoots as a consequence of the warmer and drier conditions.

scholarly journals Heat Treatment Eliminates ‘Candidatus Liberibacter asiaticus’ from Infected Citrus Trees Under Controlled Conditions

2013 ◽  
Vol 103 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Michele T. Hoffman ◽  
Melissa S. Doud ◽  
Lisa Williams ◽  
Mu-Qing Zhang ◽  
Fang Ding ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Thermal Exposure ◽  
Candidatus Liberibacter Asiaticus ◽  
Time Duration ◽  
Bacterial Titer ◽  
Heat Treated ◽  
Candidatus Liberibacter ◽  
Vigorous Growth ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide. The three known causal agents of HLB are species of α-proteobacteria: ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’. Previous studies have found distinct variations in temperature sensitivity and tolerance among these species. Here, we describe the use of controlled heat treatments to cure HLB caused by ‘Ca. L. asiaticus’, the most prevalent and heat-tolerant species. Using temperature-controlled growth chambers, we evaluated the time duration and temperature required to suppress or eliminate the ‘Ca. L. asiaticus’ bacterium in citrus, using various temperature treatments for time periods ranging from 2 days to 4 months. Results of quantitative polymerase chain reaction (qPCR) after treatment illustrate significant decreases in the ‘Ca. L. asiaticus’ bacterial titer, combined with healthy vigorous growth by all surviving trees. Repeated qPCR testing confirmed that previously infected, heat-treated plants showed no detectable levels of ‘Ca. L. asiaticus’, while untreated control plants remained highly infected. Continuous thermal exposure to 40 to 42°C for a minimum of 48 h was sufficient to significantly reduce titer or eliminate ‘Ca. L. asiaticus’ bacteria entirely in HLB-affected citrus seedlings. This method may be useful for the control of ‘Ca. Liberibacter’-infected plants in nursery and greenhouse settings.

scholarly journals Murraya paniculata and Related Species as Potential Hosts and Inoculum Reservoirs of ‘Candidatus Liberibacter asiaticus’, Causal Agent of Huanglongbing

Plant Disease ◽  
2010 ◽  
Vol 94 (5) ◽  
pp. 528-533 ◽  
Author(s):  
V. D. Damsteegt ◽  
E. N. Postnikova ◽  
A. L. Stone ◽  
M. Kuhlmann ◽  
C. Wilson ◽  
...  
Keyword(s):  
Bacterial Disease ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
Infection Rates ◽  
Disease Symptoms ◽  
Murraya Paniculata ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain ◽  
Southern Florida

Huanglongbing (HLB), considered to be the most serious insect-vectored bacterial disease of citrus, is transmitted in nature by the Asian citrus psyllid Diaphorina citri and the African citrus psyllid Trioza erytreae. D. citri was discovered in southern Florida in 1998 and the HLB disease in 2005. Both have become established throughout citrus-producing areas of Florida. Murraya species are widely grown in southern Florida as ornamental hedges and are readily colonized by D. citri vectors. Colonies of D. citri, isolates of ‘Candidatus Liberibacter asiaticus’ from Taiwan and Florida, and the Murraya species were established in the BSL-3 biosecurity facility at Fort Detrick. In controlled inoculation experiments, D. citri transmitted ‘Ca. L. asiaticus’ into M. paniculata (34/36 plants) and M. exotica (22/23 plants), but not into Bergera (Murraya) koenigii. Disease symptoms rarely developed in Murraya plants; however, positive infections were determined by conventional and real-time polymerase chain reaction (PCR). Back-inoculations of ‘Ca. L. asiaticus’ from M. paniculata to Madam Vinous sweet orange resulted in disease development in 25% of the inoculated plants. Considerable variability was observed in infection rates, titer, and persistence of ‘Ca. L. asiaticus’ in infected Murraya.

scholarly journals Development of a Tandem Repeat-Based Polymerase Chain Displacement Reaction Method for Highly Sensitive Detection of ‘Candidatus Liberibacter asiaticus’

2018 ◽  
Vol 108 (2) ◽  
pp. 292-298 ◽  
Author(s):  
Binghai Lou ◽  
Yaqin Song ◽  
Moytri RoyChowdhury ◽  
Chongling Deng ◽  
Ying Niu ◽  
...  
Keyword(s):  
Tandem Repeat ◽  
High Sensitivity ◽  
Cost Effective ◽  
Detection Methods ◽  
Displacement Reaction ◽  
Candidatus Liberibacter Asiaticus ◽  
Highly Sensitive ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

Huanglongbing (HLB) is one of the most destructive diseases in citrus production worldwide. Early detection of HLB pathogens can facilitate timely removal of infected citrus trees in the field. However, low titer and uneven distribution of HLB pathogens in host plants make reliable detection challenging. Therefore, the development of effective detection methods with high sensitivity is imperative. This study reports the development of a novel method, tandem repeat-based polymerase chain displacement reaction (TR-PCDR), for the detection of ‘Candidatus Liberibacter asiaticus’, a widely distributed HLB-associated bacterium. A uniquely designed primer set (TR2-PCDR-F/TR2-PCDR-1R) and a thermostable Taq DNA polymerase mutant with strand displacement activity were used for TR-PCDR amplification. Performed in a regular thermal cycler, TR-PCDR could produce more than two amplicons after each amplification cycle. Sensitivity of the developed TR-PCDR was 10 copies of target DNA fragment. The sensitive level was proven to be 100× higher than conventional PCR and similar to real-time PCR. Data from the detection of ‘Ca. L. asiaticus’ with filed samples using the above three methods also showed similar results. No false-positive TR-PCDR amplification was observed from healthy citrus samples and water controls. These results thereby illustrated that the developed TR-PCDR method can be applied to the reliable, highly sensitive, and cost-effective detection of ‘Ca. L. asiaticus’.

scholarly journals DETEKSI MENGGUNAKAN PCR (POLYMERASE CHAIN REACTION) CANDIDATUS LIBERIBACTER ASIATICUS, PENYEBAB HUANGLONGBING PADA JERUK SIEM DENGAN BEBERAPA TIPE GEJALA PADA DAUN

2010 ◽  
Vol 10 (2) ◽  
pp. 178-183
Author(s):  
Achmad Himawan ◽  
Y.B Sumardiyono ◽  
Susamto Somowiyarjo ◽  
Y. Andi Trisyono ◽  
Andrew Beattie
Keyword(s):  
Polymerase Chain Reaction ◽  
Candidatus Liberibacter Asiaticus ◽  
Chain Reaction ◽  
Causal Organism ◽  
Pcr Polymerase Chain Reaction ◽  
Different Types ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain ◽  
Leaf Symptoms

Detection using PCR (Polymerase Chain Reaction) Candidatus Liberibacter asiaticus, Huanglongbing causal Organism on Siem Mandarin with different types of symptoms.  Huanglongbing (HLB) or Citrus Vein Phloem Degeration (CVPD) is one of major diseases on Siem mandarin in Indonesia. HLB is caused by bacteria Candidatus liberibacter asiatus (LAS). The bacteria only live in the phloem cells of host tree and only recently it was reported to be successfully cultured on agar medium. Early detection method of LAS is needed to support healthy Siem mandarin cultivation program. This research was conducted to detect LAS in different types of HLB leaf symptoms based on Polymerase Chain Reaction (PCR) method with specific primer forward MHO 353 and reverse MHO 354.  The results suggested that 8 types of HLB leaf symptoms were found on the samples used in this experiment. LAS was detected at 60% on the leaves without any symptom followed by the leaves with completely chlorosis symptom at 66%. The leaves with unevenly yellow showing higher percentage of LAS detection ranged from 80-86%. PCR technique successfully amplified DNA of LAS with the size target of 600 bp.

scholarly journals Co-cultivation of ‘Candidatus Liberibacter asiaticus’ with Actinobacteria from Citrus with Huanglongbing

Plant Disease ◽  
2008 ◽  
Vol 92 (11) ◽  
pp. 1547-1550 ◽  
Author(s):  
Michael J. Davis ◽  
Sachindra N. Mondal ◽  
Huiqin Chen ◽  
Michael E. Rogers ◽  
Ronald H. Brlansky
Keyword(s):  
16S Rrna ◽  
Rrna Gene ◽  
Insect Vector ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Greening Disease ◽  
Transmission Electron ◽  
Pcr Products ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Polymerase Chain

Huanglongbing (HLB), also known as citrus greening disease, is a devastating disease of citrus caused by phloem-limited bacteria that have not been grown in culture. Three species, ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’, are known. ‘Ca. L. asiaticus’ and its insect vector, the psyllid Diaphorina citri, have been recently introduced into Florida. We attempted to isolate ‘Ca. L. asiaticus’ using media formulations developed in response to the growth of another bacterium that appears to be related to the liberibacters based on 16S rRNA gene identities. Cultures were obtained that were polymerase chain reaction (PCR) positive for ‘Ca. L. asiaticus’. However, transmission electron microscope examination of the culture, PCR using generic primers, and sequencing of the PCR products revealed the presence of other bacteria in the cultures. These were actinobacteria related to Propionibacterium acnes based on 16S rRNA identities. The co-cultures remained after attempts to purify the cultures by single-colony isolation, suggesting that the bacteria might be mutually beneficial to each other in culture. The co-cultures have survived more than 10 weekly passages to fresh medium. PCR using P. acnes-specific primers indicated that actinobacteria are common inhabitants of citrus and psyllids, whether or not ‘Ca. L. asiaticus’ is present.

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