scholarly journals Evaluation of Prophage Gene Revealed Population Variation of ‘Candidatus Liberibacter Asiaticus’: Bacterial Pathogen of Citrus Huanglongbing (HLB) in Northern Thailand

2021 ◽  
Author(s):  
Jutamas Kongjak ◽  
Angsana Akarapisan
Keyword(s):  
Bacterial Pathogen ◽  
Lytic Cycle ◽  
Population Variation ◽  
Northern Thailand ◽  
Fourth Group ◽  
Citrus Greening Disease ◽  
Prophage Sequence ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Key Lime

Abstract ‘Candidatus Liberibacter asiaticus’ is a non-culturable bacterial pathogen, the causal agent of Huanglongbing (HLB, yellow shoot disease, also known as citrus greening disease), a highly destructive disease of citrus (Rutaceae). The pathogen is transmitted by the Asian citrus psyllid: Diaphorina citri Kuwayama. Recent studies, have shown that the HLB pathogen has two prophages, SC1 that has a lytic cycle and SC2 associated with bacterial virulence. This study aimed to search for SC1 and SC2 prophages of HLB in mandarin orange, sweet orange, bitter orange, kumquat, key lime, citron, caviar lime, kaffir lime, pomelo and orange jasmine from five provinces in Northern Thailand. A total of 216 samples collected from Northern Thailand during 2019 and 2020 were studied. The results revealed that 62.04% (134/216) citrus samples were infected with the ‘Ca. L. asiaticus’ the bacterial pathogen associated with citrus HLB. The prophage particles are important genetic elements of bacterial genomes that are involved in lateral gene transfer, pathogenicity, environmental adaptation and interstrain genetic variability. Prophage particles were evaluated in the terminase gene of SC1 and SC2-type prophages. The results of the analysis of the prophage terminase genes of SC1 and SC2 revealed four groups of samples in Northern Thailand.The first group was the population of ‘Ca. L. asiaticus’ which was non-prophage; the frequency was 7.46% (10/134) from the total infected citrus samples. The second and third groups contained one prophage sequence with 1.49% (2/134) containing SC1-type prophage and 64.93% (87/134) containing SC2-type prophage. The fourth group contained both prophage sequence with 26.12% (35/134) containing SC1 and SC2-type prophages.Samples of leaves showing various HLB symptoms were collected from infected citrus trees which correlated with the prophages. In the first group (non-prophage) the symptoms were much less severe than other groups which had prophages. On the other hand, the third group containing the SC2-type prophage and fourth group containing SC1 and SC2-type prophages showed the most severe symptoms. The evaluation of the prophage effect on HLB-induced leaf symptoms showed that isolates with the SC2-type prophage particles produced the most severe symptoms.

scholarly journals Annotation of Putative Circadian Rhythm-Associated Genes in Diaphorina citri (Hemiptera : Liviidae)

2021 ◽  
Author(s):  
Max Reynolds ◽  
Lucas de Oliveira ◽  
Thompson Paris ◽  
Chad Vosburg ◽  
Crissy Massimino ◽  
...  
Keyword(s):  
Circadian Rhythm ◽  
Crop Yields ◽  
Bacterial Pathogen ◽  
Danaus Plexippus ◽  
Diaphorina Citri ◽  
Citrus Greening Disease ◽  
Manual Curation ◽  
Molecular Therapeutics ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

The circadian rhythm is a process involving multiple genes that generates an internal molecular clock, allowing organisms to anticipate environmental conditions produced by the earth's rotation on its axis. This report presents the results of the manual curation of twenty-seven genes likely associated with circadian rhythm in the genome of Diaphorina citri, the Asian citrus psyllid. This insect acts as the vector of the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease (Huanglongbing). This disease is the most severe detriment to citrus industries and has drastically decreased crop yields worldwide. Based on the genes identified in the psyllid genome, namely cry1 and cry2, D. citri likely possesses a circadian model similar to that of the lepidopteran butterfly, Danaus plexippus. Manual annotation of these genes will allow future molecular therapeutics to be developed that can disrupt the psyllid biology.

scholarly journals Effect of Adjuvants on Oxytetracycline Uptake upon Foliar Application in Citrus

Antibiotics ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 677
Author(s):  
Nabil Killiny ◽  
Faraj Hijaz ◽  
Pedro Gonzalez-Blanco ◽  
Shelley E. Jones ◽  
Myrtho O. Pierre ◽  
...  
Keyword(s):  
Foliar Application ◽  
Foliar Spray ◽  
Bacterial Pathogen ◽  
Water Addition ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Liberibacter Asiaticus ◽  
Trunk Injection ◽  
Citrus Leaves ◽  
Target Effects

Recently in Florida, foliar treatments using products with the antibiotics oxytetracycline and streptomycin have been approved for the treatment of citrus Huanglongbing (HLB), which is caused by the putative bacterial pathogen ‘Candidatus Liberibacter asiaticus’. Herein, we assessed the levels of oxytetracycline and ‘Ca. L. asiaticus’ titers in citrus trees upon foliar applications with and without a variety of commercial penetrant adjuvants and upon trunk injection. The level of oxytetracycline in citrus leaves was measured using an oxytetracycline ELISA kit and ‘Ca. L. asiaticus’ titer was measured using quantitative PCR. Low levels of oxytetracycline were taken up by citrus leaves after foliar sprays of oxytetracycline in water. Addition of various adjuvants to the oxytetracycline solution showed minimal effects on its uptake by citrus leaves. The level of oxytetracycline in leaves from trunk-injected trees was higher than those treated with all foliar applications. The titer of ‘Ca. L. asiaticus’ in the midrib of leaves from trees receiving oxytetracycline by foliar application was not affected after four days and thirty days of application, whereas the titer was significantly reduced in oxytetracycline-injected trees thirty days after treatment. Investigation of citrus leaves using microscopy showed that they are covered by a thick lipidized cuticle. Perforation of citrus leaf cuticle with a laser significantly increased the uptake of oxytetracycline, decreasing the titer of ‘Ca. L. asiaticus’ in citrus leaves upon foliar application. Taken together, our findings indicate that trunk injection is more efficient than foliar spray even after the use of adjuvants. Our conclusion could help in setting useful recommendations for the application of oxytetracycline in citrus to improve tree health, minimize the amount of applied antibiotic, reduce environmental exposure, and limit off-target effects.

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’.

scholarly journals Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing

Metabolites ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 477
Author(s):  
Nabil Killiny ◽  
Shelley E. Jones ◽  
Faraj Hijaz ◽  
Abdelaziz Kishk ◽  
Yulica Santos-Ortega ◽  
...  
Keyword(s):  
Leaf Morphology ◽  
Bacterial Pathogen ◽  
Gas Chromatography Mass Spectrometry ◽  
Insect Vector ◽  
Diaphorina Citri ◽  
Metabolomic Analysis ◽  
Citrus Industry ◽  
Candidatus Liberibacter ◽  
Citrus Maxima ◽  
Liberibacter Asiaticus

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen “Candidatus Liberibacter asiaticus” and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

scholarly journals Identification and Characterization of the Huanglongbing Bacterium in Pummelo from Multiple Locations in Guangdong, P. R. China

Plant Disease ◽  
2008 ◽  
Vol 92 (4) ◽  
pp. 513-518 ◽  
Author(s):  
X. Deng ◽  
J. Chen ◽  
Z. Feng ◽  
Z. Shan ◽  
H. Guo ◽  
...  
Keyword(s):  
Sweet Orange ◽  
Nucleotide Polymorphisms ◽  
Sequence Comparisons ◽  
Research Attention ◽  
Republic Of China ◽  
Citrus Greening Disease ◽  
Citrus Production ◽  
History Of ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Huanglongbing (HLB, yellow shoot disease, ex. citrus greening disease), caused by Candidatus Liberibacter spp., is highly destructive to citrus production in Asia, Africa, and South America. Although primarily affecting sweet orange and mandarin, HLB has long been observed in pummelo in Guangdong Province, People's Republic of China; however, the disease in pummelo has received little research attention. Accordingly, it remains unclear how closely related the strains of Ca. Liberibacter in pummelo are to those in other citrus species. In this study, the loci of 16S rDNA, rplAJ (β-operon of ribosomal protein), and an outer membrane protein (omp) gene were analyzed and characterized among strains of Ca. Liberibacter in pummelo samples from six different locations in Guangdong. Sequence comparisons indicated that ‘Candidatus Liberibacter asiaticus’, but not ‘Ca. Liberibacter africanus’ or ‘Ca. Liberibacter americanus’, was exclusively associated with HLB symptoms in pummelo. The pummelo strains of ‘Ca. Liberibacter asiaticus’ from Guangdong were highly homogeneous. Analyses of single-nucleotide polymorphisms in the omp locus showed that the Guangdong pummelo strains grouped with ‘Ca. Liberibacter asiaticus’ strains from Thailand, Nepal, and an unspecified location in China but differed from the Philippine and China-Behai strains. Based on the sequence homogeneity at the omp locus, the history of pummelo culture and the means by which HLB is known to be spread, we believe that, likely, the pummelo strain of ‘Ca. Liberibacter asiaticus’ recently was spread to pummelo in the study areas from infected sweet orange or mandarin trees by insect vectors or by propagation of pummelo infected elsewhere.

scholarly journals Analysis of a Prophage Gene Frequency Revealed Population Variation of ‘Candidatus Liberibacter asiaticus’ from Two Citrus-Growing Provinces in China

Plant Disease ◽  
2011 ◽  
Vol 95 (4) ◽  
pp. 431-435 ◽  
Author(s):  
Rui Liu ◽  
Pei Zhang ◽  
Xuelian Pu ◽  
Xiaoqian Xing ◽  
Jianchi Chen ◽  
...  
Keyword(s):  
Population Variation ◽  
Candidatus Liberibacter Asiaticus ◽  
Bacterial Strains ◽  
Geographical Regions ◽  
Candidatus Liberibacter ◽  
Citrus Huanglongbing ◽  
Recent Origin ◽  
Liberibacter Asiaticus ◽  
Environmental Adaptations ◽  
The Difference

Prophages are important genetic elements of bacterial genomes and are involved in lateral gene transfer, pathogenicity, environmental adaptations, and interstrain genetic variability. In this study, the sequence of a prophage terminase gene of ‘Candidatus Liberibacter asiaticus’, a bacterium associated with citrus Huanglongbing (HLB), was selected as a molecular marker to assess the genetic variation in two ‘Ca. L. asiaticus’ populations from geographically distinct provinces (Guangdong and Yunnan) in China. The frequency of the prophage terminase gene was 15.8% (19/120) in Guangdong (altitude <500 m) and 97.4% (38/39) in Yunnan (altitude >2,000 m). The difference was highly significant (P < 0.0001) based on χ2 analysis. However, the partial prophage terminase gene sequences obtained from 10 Guangdong strains and 6 Yunnan strains were identical or highly similar, suggesting that at least some bacterial strains in the two locations shared a common recent origin. This is the first report on population variation of ‘Ca. L. asiaticus’ in China, where HLB was first described. The population variation of ‘Ca. L. asiaticus’ in the two geographical regions and the related HLB epidemiology were discussed.

scholarly journals ‘Ca. Liberibacter asiaticus’ Carries an Excision Plasmid Prophage and a Chromosomally Integrated Prophage That Becomes Lytic in Plant Infections

2011 ◽  
Vol 24 (4) ◽  
pp. 458-468 ◽  
Author(s):  
Shujian Zhang ◽  
Zomary Flores-Cruz ◽  
Lijuan Zhou ◽  
Byung-Ho Kang ◽  
Laura A. Fleites ◽  
...  
Keyword(s):  
Lytic Cycle ◽  
Citrus Greening ◽  
In Planta ◽  
Dna Library ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Lysogenic Conversion ◽  
Florida Citrus ◽  
Alpha Proteobacteria

Huanglongbing (HLB), also known as citrus greening, is a lethal disease of citrus caused by several species of ‘Candidatus Liberibacter’, a psyllid-transmitted, phloem-limited, alpha proteobacteria. ‘Ca. Liberibacter asiaticus’ is widespread in Florida citrus. The recently published ‘Ca. L. asiaticus’ psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with ‘Ca. L. asiaticus’ to date. In the present study, shotgun sequencing and a fosmid DNA library of curated ‘Ca. L. asiaticus’ UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in ‘Ca. L. asiaticus’ and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with ‘Ca. L. asiaticus’ were found in the phloem of infected periwinkles by transmission electron microscopy. In psyllids, both SC1 and SC2 were found only as prophage.

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