scholarly journals Genetic Diversity of ‘Candidatus Liberibacter asiaticus’ Revealed by Short Tandem Repeats and Prophage Typing Indicates Population Homogeneity in Brazil

2019 ◽  
Vol 109 (6) ◽  
pp. 960-971 ◽  
Author(s):  
Priscila Alves da Silva ◽  
Camila Giacomo Fassini ◽  
Laís Simões Sampaio ◽  
Gabriel Dequigiovanni ◽  
Maria Imaculada Zucchi ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Genomic Organization ◽  
The United States ◽  
Candidatus Liberibacter Asiaticus ◽  
Homogeneous Population ◽  
South Central ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Short Tandem

‘Candidatus Liberibacter asiaticus’ is the most common huanglongbing-associated bacteria, being present in Asia, South, Central, and North America. Genomic approaches enabled sequencing of ‘Ca. L. asiaticus’ genomes, allowing for a broader assessment of its genetic variability with the application of polymerase chain reaction (PCR)-based tools such as microsatellite or short tandem repeat (STR) analysis. Although these tools contributed to a detailed analysis of strains from Japan, China, and the United States, Brazilian strains were analyzed in either too few samples with several STRs or in several strains with only a single microsatellite and a single PCR marker. We used 573 ‘Ca. L. asiaticus’ strains, mainly collected from São Paulo State (SPS), in our genetic analyses, employing three STRs and several prophage PCR markers. STR revealed a homogeneous population regardless of sampling year or geographic regions of SPS. Thirty-eight haplotypes were recognized with a predominance of VNTR_005 higher than 10 repeats, with VNTR_002 and VNTR_077 containing 11 and 8 repeats, respectively. This haplotype is indicated as class HE, which comprised 80.28% of strains. Classes HA and HB, predominant in Florida, were not found. A new genomic organization in the junction of prophages SC2 and SC1 is prevalent in Brazilian strains, indicating gene rearrangement and a widespread occurrence of a type 1 prophage as well as the presence of a type 2-like prophage. Our results indicate that ‘Ca. L. asiaticus’ populations are homogeneous and harbor a new genomic organization in prophages type 1 and 2.

scholarly journals Prophage Diversity of ‘Candidatus Liberibacter asiaticus’ Strains in California

2019 ◽  
Vol 109 (4) ◽  
pp. 551-559 ◽  
Author(s):  
Z. Dai ◽  
F. Wu ◽  
Z. Zheng ◽  
R. Yokomi ◽  
L. Kumagai ◽  
...  
Keyword(s):  
Los Angeles ◽  
Draft Genome ◽  
Illumina Miseq ◽  
The United States ◽  
Genomic Diversity ◽  
Candidatus Liberibacter Asiaticus ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Huanglongbing (HLB) is a highly destructive citrus disease and is associated with a nonculturable bacterium, ‘Candidatus Liberibacter asiaticus’. ‘Ca. L. asiaticus’ in the United States was first found in Florida in 2005 and is now endemic there. In California, ‘Ca. L. asiaticus’ was first detected in Hacienda Heights in Los Angeles County in 2012 and has now been detected in multiple urban locations in southern California. Knowledge of ‘Ca. L. asiaticus’ strain diversity in California is important for HLB management. In this study, genomic diversity among 10 ‘Ca. L. asiaticus’ strains from six California locations were analyzed using a next-generation sequencing (NGS) (Illumina MiSeq and HiSeq) approach. Draft genome sequences of ‘Ca. L. asiaticus’ strains were assembled. Sequences of the 16S ribosomal RNA gene and nrdB confirmed ‘Ca. L. asiaticus’ identity. Prophages were detected in all ‘Ca. L. asiaticus’ strains. The California ‘Ca. L. asiaticus’ strains formed four prophage typing groups (PTGs): PTG1, with type 1 prophage only (strains from Anaheim, San Gabriel, and Riverside); PTG2, with type 2 prophage only (strains from Hacienda Heights); PTG1-3, with both type 1 and 3 prophages (a strain from Cerritos); and PTG1-2, with both type 1 and type 2 prophages (a strain from La Habra). Analyses of the terL sequence showed that all California ‘Ca. L. asiaticus’ strains were not introduced from Florida but likely from locations in Asia. Miniature inverted-repeat transposable elements were found in all ‘Ca. L. asiaticus’ strains, yet, a jumping-out event was detected in the ‘Ca. L. asiaticus’ strain from Cerritos. Altogether, this study demonstrated that the NGS approach focusing on prophage variation was sensitive and effective in revealing diversity of ‘Ca. L. asiaticus’ strains in California.

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.

scholarly journals First Report of ‘Candidatus Liberibacter asiaticus’ Associated with Huanglongbing on Citrus latifolia in Martinique and Guadeloupe, French West Indies

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 683-683 ◽  
Author(s):  
G. Cellier ◽  
A. Moreau ◽  
N. Cassam ◽  
B. Hostachy ◽  
P. Ryckewaert ◽  
...  
Keyword(s):  
West Indies ◽  
Plant Protection ◽  
The United States ◽  
Nutrient Deficiencies ◽  
Candidatus Liberibacter Asiaticus ◽  
Protein Subunit ◽  
French West Indies ◽  
First Report ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Huanglongbing is an unculturable vascular citrus pathogen transmitted from infected to healthy plants through grafting or by citrus psyllids, Diaphorina citri mainly in Asia and America and Trioza erytreae in Africa. This phloem limited gram-negative bacterium causes dramatic yield losses and is classified into three species based on 16S rDNA sequence analysis (2): (i) ‘Candidatus Liberibacter asiaticus’ (Las), the most epidemiologically active, widespread and heat tolerant species; (ii) ‘Ca. L. africanus’ (Laf), only found in Africa; and (iii) the newly described ‘Ca. L. americanus’ (Lam), which appeared in 2005 in Brazil (5). Considered as a quarantine organism in America and Europe, Las is actively affecting North America and Asia, and research is leading toward psyllid management and resistance breeding. Despite the fact that Reunion Island has successfully controlled Las by introducing a psyllid parasitoid, Tamarixia radiata (1), this strategy was less effective or reproducible within other territories. D. citri was first detected in Guadeloupe in 1998, where the control of the the psyllid population has been effective with T. radiata (3); and was first detected in Martinique in 2012. Following the outbreak in the United States and the Caribbean, and also supported by reports of symptoms in citrus orchards, local National Plant Protection Organizations (NPPO) organized a detection survey across both islands to verify the occurrence of Huanglongbing. Since 2012, 450 sites were prospected each year in Martinique and Guadeloupe, where 20 leaves from 10 to 30 trees were analyzed. DNA extraction was performed (DNeasy Plant Mini Kit, Qiagen) on fresh or dried leaf midribs, along with negative control midribs (Citrus paradisi ‘Star Rubis’) and PCR amplification was done with the species-specific primers A2/J5 (4) and GB1/GB3 (5). Only Las-specific 703-bp amplicons were obtained (n = 43) and 20 were sequenced (Beckman Coulter Genomics, United Kingdom; sequences available through GenBank Accession Nos. KF699074 to KF699093) and blasted against the National Center for Biotechnology Information non-redondant database (NCBI-nr). BLAST analysis revealed 100% identity with the 50S ribosomal protein subunit L1 (rplA) and L10 (rplJ) of ‘Ca. L. asiaticus’ (all strains), and no significant homology to other organisms. Additionally, sequence assembly on a reference genome (NC_012985) showed 100% homology. Huanglongbing was detected in Guadeloupe on March 2012 at Le Moule (East coast) in a Tahiti lime orchard (C. latifolia) and crossed the island in 6 months. Las was detected in Martinique on May 2013 on Tahiti lime (C. latifolia) at Bellefontaine (Northwest) in a private garden and at Le Lorrain (Northeast) in an orchard. Other species from the Rutaceae family were affected by HLB (C. reticulat and C. sinensis) on both islands; however, few of the positive samples showed HLB symptoms (blotchy mottle patterns and green islands on leaves), but presented symptoms similar to nutrient deficiencies. Despite the former presence of T. radiata in Guadeloupe and its detection in Martinique a few weeks after the detection of D. citri, where it had a mean parasitism rate of 70%, an outbreak of HLB spread across both islands. These analyses confirm the presence of HLB in Martinique and Guadeloupe and to our knowledge represent the first report of Las in the French West Indies. Introduction events remain unclear, but this report raises the importance of plant certification, psyllid population control, and surveillance of territories close to the French West Indies, with regards to the risk that HLB presents to citrus production worldwide. References: (1) B. Aubert et al. Fruits. 38, 1983. (2) J. M. Bové. J. Plant Pathol. 88:1, 2006. (3) J. Etienne et al. Fruits. 56:05, 2001. (4) A. Hocquellet et al. Mol. Cell. Probes 13:5, 1999. (5) D. C. Teixeira et al. Mol. Cell. Probes 19:3, 2005.

scholarly journals Guangdong and Florida Populations of ‘Candidatus Liberibacter asiaticus’ Distinguished by a Genomic Locus with Short Tandem Repeats

2010 ◽  
Vol 100 (6) ◽  
pp. 567-572 ◽  
Author(s):  
J. Chen ◽  
X. Deng ◽  
X. Sun ◽  
D. Jones ◽  
M. Irey ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Candidatus Liberibacter Asiaticus ◽  
Bacterial Strains ◽  
Genomic Locus ◽  
Global Epidemiology ◽  
Geographical Regions ◽  
The World ◽  
Citrus Production ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Huanglongbing (HLB) (yellow shoot disease) is a highly destructive disease that threatens citrus production worldwide. The disease was first observed in Guangdong, P.R. China, over 100 years ago, and was found in Florida, United States, in 2005. ‘Candidatus Liberibacter asiaticus’ has been associated with HLB in many citrus-growing regions around the world, including Guangdong and Florida. The global epidemiology of HLB, as well as management of the disease, relies on knowledge of ‘Ca. L. asiaticus’ populations in different geographical regions around the world. In this study, we identified a genetic marker containing small tandem repeats in the genome of ‘Ca. L. asiaticus’ and comparatively analyzed the tandem repeat numbers (TRNs) in ‘Ca. L. asiaticus’ populations from Guangdong and Florida. Analyses of TRNs showed that the bacterial population in Guangdong was different from that in Florida. The Guangdong population consisted predominately of strains with a TRN of 7 (TRN7) at a frequency of 47.6%. The Florida population consisted predominately of strains with a TRN of 5 (TRN5) at a frequency of 84.4%. TRNs ranged from 3 to 16. The apparent absence of TRNs of 9, 10, 11, and 12 separated the bacterial strains into two groups: TRNs < 10 (TRN<10) and TRNs > 10 (TRN>10). In Florida, TRN<10 strains (103/109, or 94.5%) were widely distributed in all HLB-affected counties. TRN>10 strains (6/109, or 5.5%) were found in central Florida. This is the first report documenting the differentiation of ‘Ca. L. asiaticus’ populations between Asia and North America and the possible presence of two differentially distributed genotypes of ‘Ca. L. asiaticus’ in Florida.

scholarly journals Genetic Diversity of the Indian Populations of ‘Candidatus Liberibacter asiaticus’ Based on the Tandem Repeat Variability in a Genomic Locus

2015 ◽  
Vol 105 (8) ◽  
pp. 1043-1049 ◽  
Author(s):  
Dilip Kumar Ghosh ◽  
Sumit Bhose ◽  
Manali Motghare ◽  
Ashish Warghane ◽  
Krishanu Mukherjee ◽  
...  
Keyword(s):  
Tandem Repeat ◽  
Tandem Repeats ◽  
Economic Loss ◽  
Class Iii ◽  
Candidatus Liberibacter Asiaticus ◽  
Genomic Locus ◽  
Citrus Greening Disease ◽  
Geographical Regions ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Citrus huanglongbing (HLB, citrus greening disease) is an extremely destructive disease affecting citrus and causes severe economic loss to the crop yield worldwide. The disease is caused by a phloem-limited, noncultured, gram-negative bacteria Candidatus Liberibacter spp., the widely present and most destructive species being ‘Candidatus Liberibacter asiaticus’. Although the disease has been reported from almost all citrus growing regions of India, knowledge on the molecular variability of the pathogen ‘Ca. L. asiaticus’ populations from different geographical regions and cultivars is limited. In the present study, variability of the Indian ‘Ca. L. asiaticus’ based on the tandem repeats at the genomic locus CLIBASIA_01645 was characterized and categorized into four classes based on the tandem repeat number (TRN); Class I (TRN ≤ 5), Class II (TRN > 5 ≤ 10), Class III (TRN > 10 ≤ 15), and Class IV (TRN > 15). The study revealed that the Indian population of ‘Ca. L. asiaticus’ is more diverse than reported for Florida and Guangdong populations, which showed less diversity. While Florida and Guangdong populations were dominated by a TRN5 and TRN7 genotype, respectively, the Indian ‘Ca. L. asiaticus’ populations with TRN copy numbers 9, 10, 11, 12, and 13 were widely distributed throughout the country. Additionally, TRN2 and TRN17 genotypes were also observed among the Indian ‘Ca. L. asiaticus’ populations. The predominant ‘Ca. L. asiaticus’ genotypes from the northeastern region of India were TRN6 and TRN7 (53.12%) and surprisingly similar to neighboring South China populations. Preliminary results showed absence of preference of citrus cultivars to any specific ‘Ca. L. asiaticus’ genotype.

scholarly journals Investigation of Citrus HLB Symptom Variations Associated with “Candidatus Liberibacter asiaticus” Strains Harboring Different Phages in Southern China

Agronomy ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 2262
Author(s):  
Minli Bao ◽  
Zheng Zheng ◽  
Jianchi Chen ◽  
Xiaoling Deng
Keyword(s):  
Southern China ◽  
Phage Type ◽  
Genomic Research ◽  
Citrus Limon ◽  
Symptom Development ◽  
Candidatus Liberibacter Asiaticus ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Huanglongbing (HLB) is a devastating disease affecting citrus production worldwide. In China, the disease is associated with an unculturable alpha-proteobacterium, “Candidatus Liberibacter asiaticus” (CLas). Phages/prophages of CLas have recently been identified through intensive genomic research. The phage information has facilitated research on CLas biology such as population diversity and virulence gene identification. However, little is known about the roles of CLas phages in HLB symptom development. Such research is challenging due to the unculturable nature of CLas and the lack of laboratory strains that carry a single phage. In this study, CLas strains singly carrying Type 1 phage (Type 1 CLas) and Type 2 phage (Type 2 CLas) were identified and maintained in an experimental screenhouse in southern China. The strains were characterized through next-generation sequencing (NGS). Then, each CLas strain was inoculated into seedlings of three different citrus cultivars/species through graft transmission in a screenhouse in Guangdong, China. Symptom developments were recorded. All CLas-infected cultivars showed HLB symptoms in seven months. In cultivar Nianju (Citrus reticulata), Type 1 CLas caused pronounced yellowing symptoms and severe defoliation, whereas Type 2 CLas caused typical Zn-deficiency-like symptoms. In contrast, symptoms from the two CLas strains’ infections on cultivars Shatianyu (C. maxima), and Eureka lemon (Citrus limon) were more difficult to differentiate. Results from this study provide baseline information for future research to investigate the roles of CLas phages in HLB symptom development.

scholarly journals Distribution of Candidatus Liberibacter asiaticus in Citrus and the Asian Citrus Psyllid in Texas Over a Decade

Plant Disease ◽  
2020 ◽  
Vol 104 (4) ◽  
pp. 1118-1126 ◽  
Author(s):  
Mamoudou Sétamou ◽  
Olufemi J. Alabi ◽  
Madhurababu Kunta ◽  
Jon Dale ◽  
John V. da Graça
Keyword(s):  
Leaf Tissue ◽  
The United States ◽  
Asian Citrus Psyllid ◽  
Candidatus Liberibacter Asiaticus ◽  
Residential Areas ◽  
Survey Period ◽  
Tissue Samples ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Trees

Huanglongbing (HLB, citrus greening disease) in the major citrus-producing states of the United States is associated with Candidatus Liberibacter asiaticus (CLas), which is vectored by the Asian citrus psyllid (ACP). Surveys were conducted in Texas from 2007 to 2017 to assess the prevalence and titer of CLas in ACPs and citrus trees. ACP and citrus leaf tissue samples were collected from suspect trees in residential areas and commercial groves (orchards) and assayed for CLas by quantitative PCR. CLas detection in ACPs (2011) preceded that of citrus trees (2012) by several months. Annual incidences of CLas-positive ACPs and leaf tissue followed an exponential growth pattern over the survey period, varying from 0.03 to 28.7% in ACPs and 0.6 to 36.5% in citrus trees. There was a significant and positive relationship between the monthly incidences of CLas-positive ACP and leaf tissue samples. The proportion of HLB detection sites also increased with time, reaching 26 and 40% of commercial groves and residential sites, respectively, by 2017. Seasonal variations were observed in the incidences of CLas-positive ACPs and citrus trees such that significantly more CLas-positive ACPs and trees were recorded during the fall and winter of a given year relative to the hot summer. A temporal analysis of the class distribution of cycle threshold values revealed a trend of increased bacterial accumulation in ACPs and trees over time, with the trend more pronounced for the former than the latter host type. These findings provide a comprehensive insight into the ongoing CLas/HLB epidemic in Texas, with potential lessons for California and other citrus-producing areas where the disease is not yet established.

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