Modelling ‘Candidatus Liberibacter asiaticus’ movement within citrus plants

2021 ◽  
Author(s):  
Laudecir Lemos Raiol Junior ◽  
Juan Camilo Cifuentes-Arenas ◽  
Nik J Cunniffe ◽  
Robert Turgeon ◽  
Silvio Aparecido Lopes
Keyword(s):  
Plant Colonization ◽  
Symptom Expression ◽  
Candidatus Liberibacter Asiaticus ◽  
Potted Plants ◽  
Inoculation Site ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Growth Chambers ◽  
Citrus Disease ◽  
Over Time

The phloem-limited ‘Candidatus Liberibacter asiaticus’ (Las) causes huanglongbing (HLB), a destructive citrus disease. Graft-inoculated potted plants were used to assess Las speed of movement in phloem in greenhouse (GH), and the impacts of temperature on plant colonization in growth chambers (GC) experiments. For assessment of Las speed, plants were inoculated at the main stem and assessed over time by qPCR or symptom at various distances from the inoculum. For colonization, the plants were inoculated in one of two opposite top branches, were maintained at 8 to 20, 18 to 30 or 24 to 38ºC daily range, and assessed by qPCR of samples taken from non-inoculated shoots. For all experiments, frequencies of Las-positive sites were submitted to ANOVA and binomial GLM and logistic regression analysis. Probabilities of detecting Las in GH plants were functions of time and distance from the inoculation site, which resulted in 2.9 and 3.8 cm day-1 average speed of movement. In GC, the temperature impacted plant colonization by Las, new shoot emission and symptom expression. After a 7-month exposure time, Las was absent in all new shoot in the cooler environment (avg 3 per plant), and present in 70% at the milder (6 shoots, severe symptoms) and 25% in the warmer (8 shoots, no visible symptoms) environments. Temperature of 25.7ºC was the optimum condition for plant colonization. This explains the higher impact and incidence of HLB during the winter months or regions of milder climates in Brazil.

scholarly journals Ultrastructural Changes and Putative Phage Particles Observed in Sweet Orange Leaves Infected with ‘Candidatus Liberibacter asiaticus’

Plant Disease ◽  
2015 ◽  
Vol 99 (3) ◽  
pp. 320-324 ◽  
Author(s):  
S. M. Fu ◽  
John Hartung ◽  
C. Y. Zhou ◽  
H. N. Su ◽  
J. Tan ◽  
...  
Keyword(s):  
Sweet Orange ◽  
Light And Electron Microscopy ◽  
Ultrastructural Changes ◽  
Candidatus Liberibacter Asiaticus ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Orange Trees ◽  
First Time ◽  
Citrus Disease ◽  
Putative Phage

Huanglongbing (HLB), also known as citrus greening, is currently the most destructive citrus disease. Anatomical analyses of HLB-affected sweet orange were carried out by light and electron microscopy. As compared with healthy citrus, the phloem plasmodesmata were plugged with callose, and in some samples the phloem was collapsed. Chloroplast structures were deformed. Prophage sequences occupy a significant portion of the genome of ‘Candidatus Liberibacter asiaticus’ and have been used to distinguish strains from Yunnan and Guangdong provinces in China and Florida. Interestingly, a large number of possible putative phage particles were observed attached on the surface of ‘Ca. L. asiaticus’ cells in plants inoculated with strain FJ3 from Fujian Province, China. Phage particles have been observed previously only in periwinkle plants artificially inoculated in Florida with ‘Ca. L. asiaticus’ that carried the SC1-type prophage. PCR assays verified the presence of the SC1-type prophage sequences previously described from this bacterium in Florida in the FJ3 isolate. This is the first time that suspected phage particles have been observed in sweet orange trees infected with ‘Ca. L. asiaticus.’

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 First Report of the Huanglongbing Bacterium ‘Candidatus Liberibacter asiaticus’ Infecting Satkara (Citrus macroptera) in India

Plant Disease ◽  
2010 ◽  
Vol 94 (3) ◽  
pp. 375-375 ◽  
Author(s):  
A. K. Das ◽  
A. Kumar
Keyword(s):  
Sequence Comparison ◽  
Ribosomal Proteins ◽  
Rdna Locus ◽  
Candidatus Liberibacter Asiaticus ◽  
First Report ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Total Dna ◽  
Primer Sets ◽  
Citrus Disease

In India, satkara (Citrus macroptera) is found in the northeastern regions of Shella and Dowki near Cherrapunji in Meghalaya, the Jampui Hills of Tripura and Mizoram, and the Chendel District of Manipur. Locally, it is called satkara because ‘sat’ refers to multiples of seven and the fruit generally contains fourteen segments. The fruit is used in the preparation of pickles and its oil is used in the perfume industry. In January 2007, in Behliangchhip, Jampui Hills, Tripura, India, we noticed seven satkara trees of 26 showing leaf mottling and yellowing in young shoots, symptoms typical of huanglongbing (HLB). HLB is a destructive citrus disease caused by a nonculturable, phloem-limited alpha-proteobacterium of the genus ‘Candidatus Liberibacter’. Among the three known ‘Ca. Liberibacter’ species (Ca. Liberibacter asiaticus, Ca. L. africanus, and Ca. L. americanus), only Ca. L. asiaticus has been reported in India (1) but not from satkara. To identify the pathogen, total DNA was extracted and purified from 200 mg of leaf midribs from five symptomatic and five symptomless trees with the DNeasy Plant Mini Kit (Qiagen, Gmbh, Hilden, Germany) as per the manufacturer's instructions. Samples were tested for ‘Ca. L. asiaticus’ by PCR with primer sets OI1/OI2c targeting the 16S rDNA locus (3) and A2/J5 targeting the beta-operon locus of ribosomal proteins (2). DNA samples from all symptomatic leaves were positive with both primer sets. The OI1/OI2c primers generated an amplicon of approximately 1,160 bp and digestion of the amplicon by XbaI yielded two DNA fragments of approximately 640 bp and 520 bp, suggesting the presence of ‘Ca. L. asiaticus’(3). The A2/J5 primer set generated an ~703-bp amplicon, indicating ‘Ca. L. africanus’ was not present. No DNA was amplified from the asymptomatic satkara trees. One each of the OI1/OI2c and A2/J5 amplicons was purified, cloned (pGEM-T Easy vector, Promega, Hampshire, UK) and sequenced (Bangalore Genei, Bengaluru, Karnataka, India). The derived sequences, 1167 bp for the OI1/OI2c amplicon and 703 bp for the A2/J5 amplicon, have been deposited in the GenBank database under Accession Nos. GQ369792 and GU074017, respectively. Sequence comparison revealed that GQ369792 and GU074017 shared >99% identity to the corresponding regions of ‘Ca. L. asiaticus’ in the GenBank database. No DNA was amplified by PCR with primer set GB1/GB2 (4) specific for ‘Ca. L. americanus’. To our knowledge, this is the first report of molecular identification of ‘Ca. L. asiaticus’ infecting satkara in India. References: (1) A. K. Das. Curr. Sci. 87:1183, 2004. (2) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (3) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (4) D. C. Teixeira et al. Mol. Cell. Probes 19:173, 2005.

scholarly journals Evidence That ‘Candidatus Liberibacter asiaticus’ Moves Predominantly Toward New Tissue Growth in Citrus Plants

Plant Disease ◽  
2021 ◽  
Vol 105 (1) ◽  
pp. 34-42 ◽  
Author(s):  
Laudecir L. Raiol-Junior ◽  
Juan C. Cifuentes-Arenas ◽  
Everton V. de Carvalho ◽  
Eduardo A. Girardi ◽  
Silvio A. Lopes
Keyword(s):  
Sweet Orange ◽  
Force Production ◽  
Tissue Growth ◽  
Candidatus Liberibacter Asiaticus ◽  
In Planta ◽  
Potted Plants ◽  
Candidatus Liberibacter ◽  
Leaf Tissues ◽  
Basal Portion ◽  
Liberibacter Asiaticus

‘Candidatus Liberibacter asiaticus’ (Las) is an unculturable, phloem-limited, insect-transmitted bacterium associated with the Asiatic form of huanglongbing (HLB), the most destructive citrus disease. In Asia and the Americas, it is transmitted by the Asian citrus psyllid (Diaphorina citri Kuwavama). Despite considerable research, little is known about the processes involved in plant infection and colonization by Las. This study was conducted to determine whether the basal portion (below girdling) of the plant is an important route for Las to move laterally from a point of inoculation on a branch to pathogen-free branches elsewhere in the canopy, and to quantify the influence of actively growing tissues on vertical upward (acropetally) or downward (basipetally) movement of Las. Nongirdled and fully or partially girdled stems of potted plants of ‘Pera’ sweet orange, graft-inoculated above or below girdling, were sampled in distinct regions and assessed by qPCR, 6 months postinoculation. Las invaded all regions of partially and nongirdled plants but remained restricted to the inoculated regions of fully girdled plants, evidence that in planta bacterium movement is limited to the phloem. In fully girdled plants, starch accumulated above the girdling site, probably because of changes in flow of phloem sap. To study the influence of actively growing tissues, inoculated ‘Valencia’ sweet orange plants were kept intact or were top- or root-pruned to force production of new tissues, and sampled at 15-day intervals. Las migrated rapidly and most predominantly toward newly developing root and leaf tissues. The rapid and predominant movement of Las to newly developed shoots and roots would explain failures of canopy heat treatments and pruning to cure HLB-affected trees, and reinforces the need to protect rapidly growing new shoots from feeding by D. citri in order to minimize transmission and spread of the pathogen by the vector within and between orchards.

scholarly journals Two ‘Candidatus Liberibacter asiaticus’ Strains Recently Found in California Harbor Different Prophages

2017 ◽  
Vol 107 (6) ◽  
pp. 662-668 ◽  
Author(s):  
Z. Zheng ◽  
F. Wu ◽  
L. B. Kumagai ◽  
M. Polek ◽  
X. Deng ◽  
...  
Keyword(s):  
Los Angeles ◽  
Geographic Region ◽  
Mapping Technique ◽  
Polymorphism Analysis ◽  
Candidatus Liberibacter Asiaticus ◽  
Next Generation Sequencing Technology ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

‘Candidatus Liberibacter asiaticus’ (CLas), an α-proteobacterium, is associated with citrus Huanglongbing (HLB; yellow shoot disease). In California, two cases of CLas have been detected in Los Angeles County, one in Hacienda Heights in 2012 and the other in San Gabriel in 2015. Although all infected trees were destroyed in compliance with a state mandate, citrus industry stakeholder concerns about HLB in California are high. Little is known about the biology of CLas, particularly the California strains, hindering effective HLB management efforts. In this study, next-generation sequencing technology (Illumina MiSeq) was employed to characterize the California CLas strains. Data sets containing >4 billion (Giga) bp of sequence were generated from each CLas sample. Two prophages (P-HHCA1-2 and P-SGCA5-1) were identified by the MiSeq read mapping technique referenced to two known Florida CLas prophage sequences, SC1 and SC2. P-HHCA1-2 was an SC2-like or Type 2 prophage of 38,989 bp in size. P-SGCA5-1 was an SC1-like or Type 1 prophage of 37,487 bp in size. Phylogenetic analysis revealed that P-HHCA1-2 was part of an Asiatic lineage within the Type 2 prophage group. Similarly, P-SGCA5-1 was part of an Asiatic lineage within Type 1 prophage group. The Asiatic relatedness of both P-HHCA1-2 and P-SGCA5-1 was further presented by single nucleotide polymorphism analysis at terL (encoding prophage terminase) that has been established for CLas strain differentiation. The presence of different prophages suggests that the two California CLas strains could have been introduced from different sources. An alternative explanation is that there was a mixed CLas population containing the two types of prophages, and limited sampling in a geographic region may not accurately depict the true CLas diversity. More accurate pathway analysis may be achieved by including more strains collected from the regions.

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.

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