Zheng, Zheng

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.

“Candidatus Liberibacter asiaticus” (CLas) is an unculturable phloem-limited α-proteobacterium associated with citrus Huanglongbing (HLB; yellow shoot disease). HLB is currently threatening citrus production worldwide. Understanding the CLas biology is critical for HLB management. In this study, a novel single-stranded DNA (ssDNA) phage, CLasMV1, was identified in a CLas strain GDHZ11 from Guangdong Province of China through a metagenomic analysis. The CLasMV1 phage had a circular genome of 8,869 bp with eight open reading frames (ORFs). While six ORFs remain uncharacterized, ORF6 encoded a replication initiation protein (RIP), and ORF8 encoded a major capsid protein (MCP). Based on BLASTp search against GenBank database, amino acid sequences of both MCP and RIP shared similarities (coverage > 50% and identity > 25%) to those of phages in Microviridae, an ssDNA phage family. Phylogenetic analysis revealed that CLasMV1 MCP and RIP sequences were clustered with genes from CLas and “Ca. L. solanacearum” (CLso) genomes and formed a unique phylogenetic lineage, designated as a new subfamily Libervirinae, distinct to other members in Microviridae family. No complete integration form but partial sequence (∼1.9 kb) of CLasMV1 was found in the chromosome of strain GDHZ11. Read-mapping analyses on additional 15 HiSeq data sets of CLas strains showed that eight strains harbored complete CLasMV1 sequence with variations in single-nucleotide polymorphisms (SNPs) and small sequence insertions/deletions (In/Dels). PCR tests using CLasMV1-specific primer sets detected CLasMV1 in 577 out of 1,006 CLas strains (57%) from southern China. This is the first report of Microviridae phage associated with CLas, which expands our understanding of phage diversity in CLas and facilitates current research in HLB.

Citrus Huanglongbing (HLB; yellow shoot disease) is associated with an unculturable α-proteobacterium “Candidatus Liberibacter asiaticus” (CLas). HLB was found in southern California in 2012, and the current management strategy is based on suppression of the Asian citrus psyllid (Diaphorina citri) that transmits CLas and removal of confirmed CLas-positive trees. Little is known about Asian citrus psyllid-associated bacteria and citrus-associated bacteria in the HLB system. Such information is important in HLB management, particularly for accurate detection of CLas. Recent advancements in next-generation sequencing technology provide new opportunities to study HLB through genomic DNA sequence analyses (metagenomics). In this study, HLB-related bacteria in Asian citrus psyllid and citrus (represented by leaf midrib tissues) samples from southern California were analyzed. A metagenomic pipeline was developed to serve as a prototype for future bacteriomic research. This pipeline included steps of next-generation sequencing in Illumina platform, de novo assembly of Illumina reads, sequence classification using the Kaiju tool, acquisition of bacterial draft genome sequences, and taxonomic validation and diversity evaluation using average nucleotide identity. The identified bacteria in Asian citrus psyllids and citrus together included Bradyrhizobium, Buchnera, Burkholderia, “Candidatus Profftella armature,” “Candidatus Carsonella ruddii,” CLas, Mesorhizobium, Paraburkholderia, Pseudomonas, and Wolbachia. The whole genome of a CLas strain recently found in San Bernardino County was sequenced and classified into prophage typing group 1 (PTG-1), one of the five known CLas groups in California. Based on sequence similarity, Bradyrhizobium and Mesorhizobium were identified as possible source that could interfere with CLas detection using the 16S rRNA gene-based PCR commonly used for HLB diagnosis, particularly at low or zero CLas titer situation.

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.

‘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.

‘Candidatus Liberibacter asiaticus’, an unculturable α-proteobacterium, is associated with citrus huanglongbing (HLB), a devastating disease threatening citrus production in Brazil and worldwide. In this study, a draft whole-genome sequence of ‘Ca. L. asiaticus’ strain 9PA from a sweet orange (cultivar Pera) tree collected in São Paulo State, Brazil, is reported. The 9PA genome is 1,231,881 bp, including two prophages, with G+C content of 36.7%. This is the first report of a whole-genome sequence of ‘Ca. L. asiaticus’ from Brazil or South America. The 9PA genome sequence will enrich ‘Ca. L. asiaticus’ genome resources and facilitate HLB research and control in Brazil and the world.

‘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.