Zheng, Zheng

Huanglongbing, a highly destructive disease of citrus, is associated with the non-culturable phloem-limited α-proteobacterium “Candidatus Liberibacter asiaticus” (CLas). The distribution patterns of CLas in infected plant are variable and not consistent, which make the CLas detection and characterization more challenging. Here, we performed a systemic analysis of CLas distribution in citrus branches and fruits of 14 cultivars. A significantly high concentration of CLas was detected in fruit pith (dorsal vascular bundle) of 14 citrus cultivars collected at fruit maturity season. A 2-year monitoring assay of CLas population in citrus branches of “Shatangju” mandarin (Citrus reticulata Blanco “Shatangju”) revealed that CLas population already exhibited a high level even before the appearance of visual symptoms in the fruit rind. Quantitative analyses of CLas in serial 1.5-cm segments of fruit piths showed the CLas was unevenly distributed within fruit pith and tended to colonize in the middle or distal (stylar end) regions of pith. The use of CLas-abundant fruit pith for dual RNA-seq generated higher-resolution CLas transcriptome data compared with the leaf samples. CLas genes involved in transport system, flagellar assembly, lipopolysaccharide biosynthesis, virulence, stress response, and cell surface structure, as well as host genes involved in biosynthesis of antimicrobial-associated secondary metabolites, was up-regulated in leaf midribs compared with fruit pith. In addition, CLas infection caused the severe collapse in phloem and callose deposition in the plasmodesmata of fruit pith. The ability of fruit pith to support multiplication of CLas to high levels makes it an ideal host tissue for morphological studies and in planta transcriptome analyses of CLas–host interactions.

Abstract Whole‐genome sequencing of “ Candidatus Liberibacter asiaticus” (Las) indicated some polymorphic gene regions enabling the molecular characterization of this bacterium. Although the population diversity of Las in China has been previously studied, no reports have used a combination of a prophage region and short tandem repeat (STR) loci for phylogenetic relationship characterization. In this study, we investigated the genetic diversity and structure of 667 Las strains from nine provinces in southern China using multiple genetic loci including three type‐specific prophage loci, two STR loci, and a miniature inverted‐repeat transposable element (MITE) region. The results indicated that the genetic diversity varied according to the gene loci used. The prophage regions, including the MITEs, revealed significant genetic differences of Las populations in Yunnan and Guizhou from other provinces, while the STR loci only indicated a difference of Las population in Guizhou from those of other provinces. In particular, the Las population shown to be diverse in Jiangxi at the CLIBASIA_01215_STR locus was not diverse when measured using the other loci. Considering all loci, the Las populations from Yunnan and Guizhou were different, those from Guangdong and Guangxi were complex, while the Las population in Jiangxi was comparatively simple. Las populations from five neighbouring provinces, including Guangdong, Guangxi, Hunan, Fujian, and Jiangxi were grouped in a big cluster. The Las population structure changed with age according to the prophage type. This study evaluated the method used for studying the molecular diversity of Las populations and provided more detailed information on the geographical origin and epidemic trend of Chinese Las populations.

‘Candidatus Liberibacter asiaticus,’ 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 ‘Ca. L. asiaticus’ strain Myan16 from an 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 a ‘Ca. L. asiaticus’ strain from Myanmar, which will enrich the current ‘Ca. L. asiaticus’ genome sequence database and facilitate HLB epidemiology research in Asia and world.

Summary ‘ Candidatus Liberibacter asiaticus’ (CLas) is a phloem‐limited non‐culturable α‐proteobacterium associated with citrus Huanglongbing, a highly destructive disease threatening global citrus industry. Research on CLas is challenging due to the current inability to culture CLas in vitro and the low CLas titre in citrus plant. Here, we develop a CLas enrichment system using the holoparasitic dodder plant ( Cuscuta campestris ) as an amenable host to acquire and enrich CLas from CLas‐infected citrus shoots maintained hydroponically. Forty‐eight out of fifty‐five (87%) dodder plants successfully parasitized CLas‐infected citrus shoots with detectable CLas by PCR. Among 48 dodders cultures, 30 showed two‐ to 419‐fold CLas titre increase as compared to the corresponding citrus hosts. The CLas population rapidly increased and reached the highest level in dodder tendrils at 15 days after parasitizing citrus shoot. Genome sequencing and assembly derived from CLas‐enriched dodder DNA samples generated a higher resolution than those obtained for CLas from citrus hosts. No genomic variation was detected in CLas after transmission from citrus to dodder during short‐term parasitism. Dual RNA‐Seq experiments showed similar CLas gene expression profiles in dodder and citrus samples, yet dodder samples generated a higher resolution of CLas transcriptome data. The ability of dodder to support CLas multiplication to high levels, as well as its advantage in CLas genomic and transcriptomic analyses, make it an optimal model for further studies on CLas–host interaction.

‘Candidatus Liberibacter asiaticus’ (Las) is an unculturable α-proteobacterium associated with citrus huanglongbing (HLB), a devastating disease currently threatening the citrus industry worldwide. Here, we present the genome sequence of Las strain TaiYZ2 from an HLB-affected pomelo tree in Hat Yai district, Songkhla Province, Thailand. The TaiYZ2 genome is composed of 1,230,623 bp with G+C content of 36.4%. This is the first Las genome sequence from Thailand, which will enrich current Las genome resource and facilitate HLB research and management.

‘Candidatus Liberibacter asiaticus’ (CLas) is an unculturable α-proteobacterium associated with citrus Huanglongbing (HLB; yellow shoot disease). PCR procedures that accurately confirm or exclude CLas infection in citrus tissue/Asian citrus psyllid (ACP) samples are critical for HLB management. When CLas was described in 1994, a 23-bp signature oligonucleotide sequence (OI1) in the 16S rRNA gene (rrs, three genomic copies) was identified based on Sanger sequencing. OI1 contains single nucleotide polymorphisms (SNPs) distinguishing CLas from non-CLas species. The SNPs were used to design the primer HLBas, a key primer for a commonly used TaqMan PCR system (HLBas-PCR) for CLas detection. Recent developments in next-generation sequencing technology have led to the identification of 15 CLas whole genome sequence strains (WGSs). Analyses of CLas WGSs have generated a significant amount of biological information that could help to improve CLas detection. Utilizing the WGS information, this study re-evaluated the sequence integrity of OI1/HLBas and identified and/or confirmed a missing nucleotide G in the two primers. Replacement primers for OI1 and HLBas are proposed. At low cycle threshold (Ct) values (e.g., <30), HLBas-PCR remained reliable in CLas determination. At high Ct values (e.g., >30), HLBas-PCR alone was unreliable in differentiating whether samples contain low CLas titers or whether CLas is not present. The availability of ribonucleotide reductase (RNR)-PCR derived from the five-copy nrdB gene helped to resolve this problem. To further enhance low CLas titer detection, a 4CP-PCR system, based on a four-copy genomic locus, was developed. Evaluation of 107 HLB samples (94 citrus and 13 ACP) showed that 4CP-PCR was more sensitive than HLBas-PCR and shared similar sensitivity with RNR-PCR.

The draft genome sequence of “ Candidatus Liberibacter asiaticus” strain YNJS7C, isolated from a navel orange tree in Yunnan Province, China, is presented here. The YNJS7C strain has a genome size of 1,258,986 bp, with a G+C content of 36.6%, 1,174 predicted open reading frames, and 53 RNA genes.

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.

ABSTRACT We report here the draft genome sequence of “ Candidatus Liberibacter asiaticus” strain TX2351, collected from Asian citrus psyllids in south Texas, USA. The TX2351 genome has a size of 1,252,043 bp, a G+C content of 36.5%, 1,184 predicted open reading frames, and 52 RNA genes.