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.

scholarly journals Visualization of ‘Candidatus Liberibacter asiaticus’ Cells in the Vascular Bundle of Citrus Seed Coats with Fluorescence In Situ Hybridization and Transmission Electron Microscopy

2013 ◽  
Vol 103 (6) ◽  
pp. 545-554 ◽  
Author(s):  
Mark E. Hilf ◽  
Kenneth R. Sims ◽  
Svetlana Y. Folimonova ◽  
Diann S. Achor
Keyword(s):  
16S Rrna ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Candidatus Liberibacter Asiaticus ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Seed ◽  
Seed Coats

‘Candidatus Liberibacter asiaticus’ is the bacterium implicated as a causal agent of the economically damaging disease of citrus called huanglongbing (HLB). Vertical transmission of the organism through seed to the seedling has not been demonstrated. Previous studies using real-time polymerase chain reaction assays indicated abundant bacterial 16S rRNA sequences in seed coats of citrus seed but the presence of intact bacterial cells was not demonstrated. We used microscopy to verify that intact bacterial cells were present in citrus seed coats. Bacterial cells with the morphology and physical dimensions appropriate for ‘Ca. L. asiaticus’ were seen in phloem sieve elements in the vascular bundle of grapefruit seed coats using transmission electron microscopy (TEM). Fluorescence in situ hybridization (FISH) analyses utilizing probes complementary to the ‘Ca. L. asiaticus’ 16S rRNA gene revealed bacterial cells in the vascular tissue of intact seed coats of grapefruit and pummelo and in fragmented vascular bundles excised from grapefruit seed coats. The physical measurements and the morphology of individual bacterial cells were consistent with those ascribed in the literature to ‘Ca. L. asiaticus’. No bacterial cells were observed in preparations of seed from fruit from noninfected trees. A small library of clones amplified from seed coats from a noninfected tree using degenerate primers targeting prokaryote 16S rRNA gene sequences contained no ‘Ca. L. asiaticus’ sequences, whereas 95% of the sequences in a similar library from DNA from seed coats from an infected tree were identified as ‘Ca. L. asiaticus’, providing molecular genetic corroboration that the bacterial cells observed by TEM and FISH in seed coats from infected trees were ‘Ca. L. asiaticus’.

scholarly journals A New ‘Candidatus Liberibacter’ Species Associated with Diseases of Solanaceous Crops

Plant Disease ◽  
2009 ◽  
Vol 93 (3) ◽  
pp. 208-214 ◽  
Author(s):  
Lia W. Liefting ◽  
Paul W. Sutherland ◽  
Lisa I. Ward ◽  
Kerry L. Paice ◽  
Bevan S. Weir ◽  
...  
Keyword(s):  
16S Rrna ◽  
Pcr Primers ◽  
23S Rrna ◽  
Rrna Gene ◽  
High Identity ◽  
Specific Pcr ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Polymerase Chain ◽  
Specific Pcr Primer

A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curling or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR) primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacterium shared high identity with ‘Candidatus Liberibacter’ species. Phylogenetic analysis showed that the bacterium is distinct from the three citrus liberibacter species previously described and has been named ‘Candidatus Liberibacter solanacearum’. This is the first report of a liberibacter naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was developed for its detection.

scholarly journals Understanding Citrus Greening Disease and Its Possible Management Strategies in Nepal

2021 ◽  
Vol 9 (4) ◽  
pp. 227-234
Author(s):  
Sameer Pokhrel ◽  
Swikriti Pandey ◽  
Ashish Ghimire ◽  
Savyata Kandel
Keyword(s):  
Management Strategies ◽  
Citrus Greening ◽  
Insect Vector ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Greening Disease ◽  
The World ◽  
Citrus Production ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Orchards

Huanglongbing (HLB), also known as citrus greening, is a devastating disease of citrus that has decimated several citrus orchards throughout the world. The disease is associated with three species of unculturable and phloem-limited bacteriae, Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. The most common species of bacteria found in Nepal is Candidatus Liberibacter asiaticus which is transmitted by an insect vector, Asian citrus psyllid (Diaphorina citri). This disease has been detected in several economically important citrus production areas of Nepal, which resulted in heavy yield loss. No cure for the disease has been discovered yet and it is essential to practice proper management strategies to maintain citrus health and sustain citrus production under HLB pressure. Several disease management approaches such as pathogen-free nursery establishment, use of disease tolerant rootstock cultivars, proper irrigation and nutrient supply, removal of HLB affected trees, and control of psyllid with frequent insecticide application are widely practiced throughout the world. This review article highlights the characteristics of the citrus greening disease and its insect vector and gives insights into their management techniques. Several technologically advanced options available to minimize the HLB infection might not be feasible currently in Nepal due to economic and topographic constraints. This article also aims to bring into focus the cost-effective methods that growers in Nepal can practice to mitigate the impact of HLB disease in their citrus orchards. Int. J. Appl. Sci. Biotechnol. Vol 9(4): 227-238.

Detection of Liberibacter asiaticus causing Citrus Vein Phloem Degeneration from Siam Citrus leaves (Citrus nobilis var. microcarpa) in Singkawang City plantation, Pontianak, West Kalimantan

2020 ◽  
Vol 14 (3) ◽  
pp. 2
Author(s):  
RAHMAWATI RAHMAWATI ◽  
ILIANA ILIANA ◽  
AGUS RACHMAT ◽  
LATIFFAH ZAKARIA ◽  
MUKARLINA MUKARLINA
Keyword(s):  
16S Rrna ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Rrna Gene ◽  
Candidatus Liberibacter Asiaticus ◽  
Specific Primers ◽  
West Kalimantan ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Leaves

The objective of the present study was to detect presence of pathogenic fastidious bacterium, Candidatus Liberibacter asiaticus using PCR from leaves of Siam citrus showing Citrus Vein Phloem Degeneration (CVPD) symptoms in Singkawang City plantation, Pontianak, West Kalimantan, Indonesia. Citrus leaf samples were collected based on visual observation of symptoms showing CVPD infection. Typical symptoms of CVPD include leaf yellowing (chlorosis), vein banding, leaves become stiff, thicker and smaller in size. The pathogenic bacterium, Candidatus Liberibacter asiaticus was detected using two specific primers, namely, OI1/ OI2c amplified 16S rRNA gene and A2/J5, amplified ribosomal  protein gene of the rplKAJL-rpoBC operon (β-operon). PCR amplification detected the presence of 1100 bp band using OI1/ OI2c primers, and 703 bp band using A2/J5 primers from symptomatic Siam citrus leaves. PCR products were not detected from healthy plants serve as control. By using two sets of specific primers to amplify 16S rRNA gene and ribosomal protein gene, Candidatus Liberibacter asiaticus was detected in symptomatic Siam citrus leaves in Singkawang City, Pontianak, Indonesia. Detection of the bacterial pathogen causing CVPD is important to prevent spreading of the disease which could affect production of citrus fruits.

scholarly journals Susceptibility of Sixteen Citrus Genotypes to ‘Candidatus Liberibacter asiaticus’

Plant Disease ◽  
2016 ◽  
Vol 100 (6) ◽  
pp. 1080-1086 ◽  
Author(s):  
Greg McCollum ◽  
Mark Hilf ◽  
Mike Irey ◽  
Weiqi Luo ◽  
Tim Gottwald
Keyword(s):  
The United States ◽  
Insect Vector ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Germplasm ◽  
Citrus Production ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Florida Citrus ◽  
Citrus Macrophylla ◽  
Free Ranging

Huanglongbing (HLB) disease is the most serious threat to citrus production worldwide and, in the last decade, has devastated the Florida citrus industry. In the United States, HLB is associated with the phloem-limited α-proteobacterium ‘Candidatus Liberibacter asiaticus’ and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri). Significant effort is being put forth to develop novel citrus germplasm that has a lower propensity to succumb to HLB than do currently available varieties. Effective methods of screening citrus germplasm for susceptibility to HLB are essential. In this study, we exposed small, grafted trees of 16 citrus types to free-ranging ACP vectors and ‘Ca. L. asiaticus’ inoculum in the greenhouse. During 45 weeks of exposure to ACP, the cumulative incidence of ‘Ca. L. asiaticus’ infection was 70%. Trees of Citrus macrophylla and C. medica were most susceptible to ‘Ca. L. asiaticus’, with 100% infection by the end of the test period in three trials, while the complex genetic hybrids ‘US 1-4-59’ and ‘Fallglo’ consistently were least susceptible, with approximately 30% infection. Results obtained in this greenhouse experiment showed good agreement with trends observed in the orchard, supporting the validity of our approach for screening citrus germplasm for susceptibility to HLB.

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 Viability of ‘Candidatus Liberibacter asiaticus’ Prolonged by Addition of Citrus Juice to Culture Medium

2014 ◽  
Vol 104 (1) ◽  
pp. 15-26 ◽  
Author(s):  
Jennifer K. Parker ◽  
Sarah R. Wisotsky ◽  
Evan G. Johnson ◽  
Faraj M. Hijaz ◽  
Nabil Killiny ◽  
...  
Keyword(s):  
Grapefruit Juice ◽  
Culture Medium ◽  
Quantitative Polymerase Chain Reaction ◽  
Chemical Characterization ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Greening Disease ◽  
Candidatus Liberibacter ◽  
The Media ◽  
Liberibacter Asiaticus

Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium ‘Candidatus Liberibacter asiaticus’. Infection with ‘Ca. L. asiaticus’ is incurable; therefore, knowledge regarding ‘Ca. L. asiaticus’ biology and pathogenesis is essential to develop a treatment. However, ‘Ca. L. asiaticus’ cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of ‘Ca. L. asiaticus’ in vitro, ‘Ca. L. asiaticus’ inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima ‘Mato Buntan’) was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air–liquid interface of juice cultures contained ‘Ca. L. asiaticus’ cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining ‘Ca. L. asiaticus’ viability in vitro, which will contribute to future development of a culture medium for ‘Ca. L. asiaticus’.

Probing the application of OmpA-derived peptides to disrupt the acquisition of ‘Candidatus Liberibacter asiaticus’ by Diaphorina citri

2021 ◽  
Author(s):  
Marcus Vinicius Merfa e Silva ◽  
Eduarda Regina Fischer ◽  
Mariana de Souza e Silva ◽  
Carolina Sardinha Francisco ◽  
Helvécio Coletta-Filho ◽  
...  
Keyword(s):  
Control Strategies ◽  
Insect Vector ◽  
Diaphorina Citri ◽  
Candidatus Liberibacter Asiaticus ◽  
In Planta ◽  
Host Interaction ◽  
Vector Population ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Bacterial Outer Membrane

Huanglongbing (HLB) is currently the most devastating disease of citrus worldwide. Both bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) and ‘Ca. Liberibacter americanus’ (CLam) are associated with HLB in Brazil, but with a strong prevalence of CLas over CLam. Conventionally, HLB management focuses on controlling the insect vector population (Diaphorina citri; also known as Asian citrus psyllid – ACP) by spraying insecticides, an approach demonstrated to be mostly ineffective. Thus, development of novel more efficient HLB control strategies is required. The multifunctional bacterial outer membrane protein OmpA is involved in several molecular processes between bacteria and their hosts and has been suggested as a target for bacterial control. Curiously, OmpA is absent in CLam in comparison to CLas, suggesting a possible role on host-interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides aiming to evaluate the acquisition of CLas by the insect vector. Treatment of psyllids with 5 µM of Pep1, Pep3, Pep5 and Pep6 in artificial diet significantly reduced the acquisition of CLas, while increasing the concentration of Pep5 and Pep6 to 50 µM abolished this process. In addition, in planta treatment with 50 µM of Pep6 also significantly decreased the acquisition of CLas and sweet orange plants stably absorbed and maintained this peptide for as long as three months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.

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