Bao, Minli

ABSTRACT Huanglongbing (HLB) is a severe citrus disease in China caused by Candidatus Liberibacter asiaticus ( C Las). Since its initial identification, the pathogen has spread to 10 mainland provinces in China and caused devastating loss. Three distinct prophage types have been identified in C Las; however, their distribution and diversity in China remain inadequately understood. In this study, we collected 500 C Las samples from 10 provinces in China, employing three specific genomic loci to identify prophage types. Subsequently, Sanger sequencing was employed to analyze the genetic diversity of prophage within populations of C Las in China. In addition, the MaxEnt model optimized by the ENMeval software package, was used to predict the habitat suitability of populations of C Las and assess the potential impact of future climate change on its distribution in China. Our analysis revealed that type 2 prophage is the most prevalent, accounting for 55% in China. Among the 10 provinces tested, C Las populations in Yunnan and Sichuan demonstrated higher genetic diversity. Further analysis reveals that C Las populations harboring type 1 prophage remain relatively stable, whereas those carrying type 2 and type 3 prophages undergo population expansion. Furthermore, our predictive models indicate that the presently suitable habitat for C Las populations is concentrated in the southern and certain central regions of China, with an anticipated expansion under future climate change conditions. Presently, the center of populations of C Las characterized by favorable living conditions is situated in Zunyi City, Guizhou Province. Nevertheless, a projected trend indicates a shift toward the northeast, particularly targeting Tongren City in the foreseeable future. IMPORTANCE This study offers significant insights into the distribution and genetic diversity of three types of prophages associated with Candidatus Liberibacter asiaticus ( C Las) in China. Our predictions underscore the implications of climate change on the future distribution of C Las. These findings contribute to a better understanding of Huanglongbing management strategies and can facilitate the development of effective measures to control the spread of this devastating disease within the citrus industry.

Huanglongbing (HLB) is a devastating citrus disease caused by Candidatus Liberibacter asiaticus (CLas). Since its initial outbreak in Guangdong Province, China, it has spread to 10 provinces and caused significant economic losses. Hence, assessing CLas genetic diversity and demographic history is crucial for HLB epidemic prevention and control. In this study, we collected 500 leaf samples of CLas-infected plants from 10 provinces. We performed multi-loci sequence analysis on four gene fragments (omp, DnaA, GroEL, and SDE1) to explore the genetic differentiation and diversity of CLas in China. Our results indicated low nucleotide diversity (0.00005 ± 0.00001) in CLas, with the absence of significant systematic geographic structure in its distribution. Molecular variance analysis revealed predominant (81.7%) genetic variations within the population, with a minor variation (18.3%) occurring between populations as well as Yunnan provinces. In the Fujian population, significant gene exchange occurred with the other nine populations. Significant negative values in Tajima’s D and Fu’s FS neutrality tests indicated historical population expansions. The nucleotide mismatch distribution curve exhibits a single peak pattern, further supporting the expansion events. Our findings hold potential for advancing epidemiological research and providing suggestions for effective strategies to mitigate the spread of CLas and control HLB.

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.

‘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.

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.