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

‘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.

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Prophage Diversity of ‘Candidatus Liberibacter asiaticus’ Strains in California

Phytopathology ◽

10.1094/phyto-06-18-0185-r ◽

2019 ◽

Vol 109 (4) ◽

pp. 551-559 ◽

Cited By ~ 10

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.

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

Agronomy ◽

10.3390/agronomy11112262 ◽

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.

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A Type 3 Prophage of ‘Candidatus Liberibacter asiaticus’ Carrying a Restriction-Modification System

Phytopathology ◽

10.1094/phyto-08-17-0282-r ◽

2018 ◽

Vol 108 (4) ◽

pp. 454-461 ◽

Cited By ~ 21

Author(s):

Zheng Zheng ◽

Minli Bao ◽

Fengnian Wu ◽

Christopher Van Horn ◽

Jianchi Chen ◽

...

Keyword(s):

Southern China ◽

Lytic Cycle ◽

Candidatus Liberibacter Asiaticus ◽

Specific Pcr ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus ◽

Type 3 ◽

Restriction Modification

Prophages, the lysogenic form of bacterial phages, are important genetic entities of ‘Candidatus Liberibacter asiaticus’ (CLas), a nonculturable α-proteobacterium associated with citrus Huanglongbing. Two CLas prophages have been described, SC1 (NC_019549.1, Type 1) and SC2 (NC_019550.1, Type 2), which involve the lytic cycle and the lysogenic cycle, respectively. To explore the prophage repertoire, 523 CLas DNA samples extracted from leaf petioles of CLas-infected citrus were collected from southern China and surveyed for Type 1 and Type 2 prophages by specific PCR. Eighteen samples were found lacking both prophages. One sample, JXGC, sequenced using Illumina HiSeq, generated >100 million short sequence reads (150 bp per read). Read mapping to known prophage sequences showed a sequence coverage of 46% to SC1 and 50% to SC2. BLAST search using SC1 and SC2 as queries identified three contigs from the JXGC de novo assembly that form a circular P-JXGC-3 (31,449 bp), designated as a new Type 3 prophage. Chromosomal integration of P-JXGC-3 was detected to occur within a helicase gene, resulting in a duplication of this gene. P-JXGC-3 had 36 open reading frames (ORFs), 10 of which were not found in Type 1 or Type 2 prophages, including four genes that encoded a restriction-modification (R-M) system (hsdR, hsdS, hsdM1, and hsdM2). Typed by prophage-specific PCR, the CLas strains in southern China contained all combinations of the three prophage types with the exception of a Type 2−Type 3 combination, suggesting active ongoing prophage−phage interactions. Based on gene annotation, P-JXGC-3 is not capable of reproduction via the lytic cycle. The R-M system was speculated to play a role against Type 1 prophage−phage invasion.

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Historical Perspectives, Management, and Current Research of Citrus HLB in Guangdong Province of China, Where the Disease has been Endemic for Over a Hundred Years

Phytopathology ◽

10.1094/phyto-07-18-0255-ia ◽

2018 ◽

Vol 108 (11) ◽

pp. 1224-1236 ◽

Cited By ~ 16

Author(s):

Zheng Zheng ◽

Jianchi Chen ◽

Xiaoling Deng

Keyword(s):

Southern China ◽

Candidatus Liberibacter Asiaticus ◽

Sequencing Technology ◽

Next Generation Sequencing Technology ◽

Historical Perspectives ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus ◽

Generation Sequencing

Citrus huanglongbing (HLB) is a highly destructive disease currently threatening citrus production worldwide. In China, the disease is exclusively associated with ‘Candidatus Liberibacter asiaticus’, a nonculturable proteobacterium. HLB was observed in Guangdong of China over a hundred years ago. Researchers and citrus growers have been battling with the disease through vigorous research and have exercised various control practices. Much of the early work was not well known outside China. This review is intended to fill in gaps of historical information by reviewing selected literature records. Along the way, the HLB system within southern China was evaluated. Emphases were on comparison of symptomatology, evolution of etiology, control practices, and impacts of using next-generation sequencing technology for ‘Ca. L. asiaticus’ research and detection.

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Genome Sequence Resource of “Candidatus Liberibacter asiaticus” strain Myan16 from Myanmar

Plant Disease ◽

10.1094/pdis-08-20-1841-a ◽

2020 ◽

Author(s):

Yongqin Zheng ◽

jun guo ◽

Xiaoling Deng ◽

Zheng Zheng

Keyword(s):

Genome Sequence ◽

Draft Genome ◽

Sequence Database ◽

Candidatus Liberibacter Asiaticus ◽

Genome Sequence Database ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus

“Candidatus Liberibacter asiaticus” (CaLas), an uncultured α-proteobacterium, is associated with citrus Huanglongbing (HLB, yellow shoot disease), a destructive disease threatening citrus production worldwide. Here, we reported the draft genome sequence of CaLas strain Myan16 from a HLB-affected lime tree in Myitkyina, Kachin State, Myanmar. The strain Myan16 genome is 1,229,102 bp with an average G+C content of 36.4%, along with a circular prophage: P-Myan16-2 (36,303 bp, Type 2). This is the first genome sequence of CaLas strain from Myanmar, which will enrich the current CaLas genome sequence database and facilitate HLB epidemiology research in Asia and world.

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Genome Sequence Resources of Two ‘Candidatus Liberibacter asiaticus’ Strains from Pakistan

Plant Disease ◽

10.1094/pdis-12-19-2648-a ◽

2020 ◽

Vol 104 (8) ◽

pp. 2048-2050 ◽

Cited By ~ 1

Author(s):

Kehong Liu ◽

Sagheer Atta ◽

Xuejin Cui ◽

Chunhua Zeng ◽

Jianchi Chen ◽

...

Keyword(s):

Genome Sequence ◽

Gc Content ◽

Sequence Database ◽

Candidatus Liberibacter Asiaticus ◽

Genome Sequences ◽

Citrus Production ◽

Candidatus Liberibacter ◽

Citrus Huanglongbing ◽

Liberibacter Asiaticus

‘Candidatus Liberibacter asiaticus’ (CLas) is an unculturable, phloem-restricted αProteobacteria, associated with citrus Huanglongbing (HLB), which is one of the most destructive diseases in citrus production worldwide. Here, we present the genome sequences of CLas strains PA19 and PA20 from HLB-affected kinnow trees in Multan, Punjab Province, Pakistan. The CLas genomes of PA19 and PA20 comprise 1,224,156 bp and 1,226,225 bp, respectively, with an average GC content of 36.4%. Both harbored the Type 2 prophage. In this study, we report two CLas genomes from Pakistan, which extends the sequence database of CLas and will contribute to CLas biology and HLB management.

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First Report of Candidatus Liberibacter asiaticus Associated with Citrus Huanglongbing in California

Plant Disease ◽

10.1094/pdis-09-12-0845-pdn ◽

2013 ◽

Vol 97 (2) ◽

pp. 283-283 ◽

Cited By ~ 59

Author(s):

L. B. Kumagai ◽

C. S. LeVesque ◽

C. L. Blomquist ◽

K. Madishetty ◽

Y. Guo ◽

...

Keyword(s):

Los Angeles ◽

Real Time ◽

16S Rdna ◽

Real Time Pcr ◽

Bacterial Species ◽

The United States ◽

Candidatus Liberibacter Asiaticus ◽

Citrus Diseases ◽

Candidatus Liberibacter ◽

Liberibacter Asiaticus

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive citrus diseases worldwide and is seen as a major threat to the multimillion dollar citrus industry in California. The vector of the two bacterial species associated with this disease, Candidatus Liberibacter asiaticus and Ca. L. americanus, is the Asian citrus psyllid (ACP), Diaphorina citri (4). ACP was detected in California in August of 2008 and has since been detected in nine counties in southern California. As part of a long term survey and testing program for the ACP carrying the HLB associated bacteria, groups of ACP nymphs and adults were submitted to the Jerry Dimitman Citrus Research Board/Citrus Pest and Disease Prevention Program Laboratory in Riverside, CA. In March 2012, DNA extracted using the Qiagen MagAttract 96 DNA plant kit (QIAGEN Inc., 27220 Turnberry Lane, Suite 200, Valencia, CA 91355) from a group of three ACP adults tested positive for Ca. L. asiaticus with the real-time PCR assay developed by Li et al. (4). ACP adults were collected from a residential citrus tree located in the Hacienda Heights area of Los Angeles County, California. The approximately 1.8 meter tall lemon tree had 23 graft unions, primarily of lemon (Citrus × meyeri) and pomelo (Citrus maxima) varieties. The tree was unthrifty, with yellow shoots and chlorotic leaves. Symptoms on the lemon and pomelo leaves included asymmetrical blotchy mottling, yellowing, and corking of the leaf veins, with the blotchy mottle more prominent in the pomelo leaves. Pomelo leaves appeared crinkled along the thickened veins. Lemon leaves had yellow veins and a few had islands of green tissue completely surrounded by yellow tissue. The entire tree was removed, cut into sections, bagged, and transported to the CDFA Plant Pest Diagnostics Lab for analysis. Two hundred milligrams of petiole and midrib tissue from leaves apical to each graft union was collected, and DNA from each sample was extracted using the Qiagen DNeasy plant mini kit. DNA extracted from both lemon and pomelo leaves tested positive for Ca. L. asiaticus using real-time PCR (4). A 1,160-bp fragment of the 16S ribosomal RNA gene was amplified from the insect and plant DNA extracts using conventional PCR with primers Ol1 and OI2c (2). A 703-bp fragment of the β-operon gene was amplified from the insect and plant extracts with primers A2 and J5 (1). The 16S rDNA fragments from the insect and plant respectively (GenBank Accession Nos. JX430434 and JX455745) and the β-operon fragments (JX430435 and JX455746) showed 100% identity with the corresponding regions of Ca. L. asiaticus (CP001677) strain psy 62. Our 16S rDNA sequence showed 98% identity with Ca. L. africanus (EU921620), 97% identity with Ca. L. solanacearum (HM246509), and 96% with Ca. L. americanus (FJ036892). In response to the detection of HLB, a 241 km2 quarantine area around the detection site was established. Surveys for ACP and symptomatic host plants within the HLB quarantine area are ongoing. To date, there have been no additional positive detections. In the United States, HLB was first detected in Florida in 2005 (4) and in Texas in January of 2012 (3). To our knowledge, this is the first confirmed report of Ca. L. asiaticus associated with HLB in California. References: (1) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) M. Kunta et al. Phytopathology 102:S4.66, 2012. (4) W. Li et al. J. Microbiol. Methods 66:104, 2006.

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

Phytopathology ◽

10.1094/phyto-08-18-0295-r ◽

2019 ◽

Vol 109 (6) ◽

pp. 960-971 ◽

Cited By ~ 2

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.

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