Zheng, Zheng

A Microviridae phage, CLasMV1, was recently identified in ‘ Candidatus Liberibacter asiaticus’ (CLas), the bacterium associated with citrus Huanglongbing (HLB). The CLasMV1 strain GDHZ11D (CP045566) exhibited a circular genome of 8,869 bp containing eight open reading frames (ORFs). Phages of CLas play crucial roles in regulating bacterial population diversity that have potential use for HLB control. However, knowledge of CLasMV1 population diversity is currently limited. In this study, 1,520 CLas samples were collected from 10 citrus-growing provinces in China. PCR screening revealed that 1,042 samples (69%) harbored CLasMV1. Complete genome sequences of 43 CLasMV1 strains were obtained through Illumina HiSeq sequencing with genome sizes ranging from 8,696 to 8,881 bp. Variations were mostly related to single-nucleotide polymorphisms and sequence insertions/deletions. Notably, two hypervariable regions (HVRs) were identified. Two new ORFs were detected, ORF-A (144 bp) in HVR-1 and ORF-B (342 bp) in HVR-2. Whole-genome phylogenetic analysis using 43 strains from this study, 9 previously reported Chinese strains, and 2 Florida strains (CP040636 and MZ382797) delineated five genomic groups. Chinese strains dominated Groups 1 to 4, and Group 5 contained the Florida strains. Further analyses of HVR-1 across all 1,042 CLasMV1 samples detected a 173-bp sequence with suspected mobile activity. Analysis of a PacBio long-read assembly published previously detected a concatemer of the CLasMV1 sequence, suggesting that the phage used the rolling circle mechanism for its replication. These findings significantly enhance our understanding of CLasMV1 genomic diversity and will facilitate future research on CLasMV1 classification and HLB biology.

Citrus Huanglongbing (HLB), caused by “Candidatus Liberibacter asiaticus” (CLas), is a destructive disease threatening global citrus industry. Although citrus cultivars differ in HLB sensitivity, how infection alters endophytic bacterial communities in cultivars with contrasting susceptibility remains unclear. Here, we compared endophytic microbiome shifts in leaf and root tissue of HLB-susceptible Shatangju mandarin (C. reticulata cv. Shatangju) and HLB-tolerant Shatian pomelo (C. maxima cv. Shatian) at 10 months post grafting (MPG) with CLas-infected buds. Infected Shatangju mandarin showed severe symptoms and higher CLas levels in leaf than root, while infected Shatian pomelo remained asymptomatic with higher CLas in root than leaf. High-throughput 16S rRNA gene sequencing revealed that CLas infection restructured endophytic microbial community in tissue-specific manner. CLas-infected Shatian pomelo leaf displayed increased microbial diversity and network complexity, while CLas-infected Shatangju mandarin root harbored more disease-suppressive taxa like Streptomyces. Across both cultivars, network complexity correlated inversely with CLas abundance. Key genera (e.g., Pseudomonas, Streptomyces, Prevotella) correlated positively with CLas, suggesting roles in mitigating pathogen effects. Predicted functional profiling indicated activated pathways (amino acid metabolism, terpenoid/polyketide biosynthesis, xenobiotic biodegradation) potentially supporting host defense in infected tissues. The HLB tolerance of Shatian pomelo appeared linked to tissue-specific bacterial restructuring, particularly recruitment of antibiotic-producing bacteria and enhanced metabolic resilience. These findings elucidated cultivar-specific microbial strategies against CLas, offering insights for microbiome-mediated HLB management.

Citrus Huanglongbing (HLB) is caused by the phloem-limited α-proteobacterium ‘Candidatus Liberibacter spp.’, among which ‘Ca. L. africanus’ (CLaf) has posed a significant threat to citrus production in Africa for nearly a century. CLaf is closely related to the globally prevalent ‘Ca. L. asiaticus’ (CLas), whereas little is known about the virulence of CLaf, primarily because of limited genome resources. In this study, we completed the whole-genome assembly and annotation of the CLaf strain Zim (from Zimbabwe). Compared with CLas, a total of 102 CLaf unique genes were identified, including 14 potential Sec-dependent effector (SDE) genes, 29 phage-associated genes, and 59 genes with hypothetical function. Among 14 SDEs, V9J15_03810 was able to induce a significant hypersensitive response in Nicotiana benthamiana, indicating its potential as a virulence factor for CLaf. Genome analysis showed that the CLaf strain Zim genome harbored a complete prophage region (named P-Zim-1, 42,208 bp). P-Zim-1 retained two immunosuppressive peroxidase genes (V9J15_02125 and V9J15_02130) homologous to CLas prophage SC1/SC2, whereas the lysogen-associated genes encoding integrase (V9J15_01970) and repressor (V9J15_02080) were homologous to the prophage of ‘Ca. L. solanacearum’, the causal agent of potato zebra chip disease. In addition, P-Zim-1 carried a novel CRISPR/Cas system, including a CRISPR array (located within V9J15_02040, ranging from 443,643 to 443,897) and five CRISPR-related Cas proteins (V9J15_02005, V9J15_02010, V9J15_02015, V9J15_02025, and V9J15_02035). This study first characterized the unique genomic feature of CLaf related to virulence and prophage, which will facilitate future research on CLaf biology and African HLB management.

The ‘Cuimi’ kumquat is a unique citrus cultivar known for its thin, crisp pulp and sweet, aromatic flavor. In addition to its use in fresh consumption and processing, this variety exhibits certain medicinal properties. This study aims to investigate the genetic diversity of the Huanglongbing (HLB) bacterium across different tissues of the ‘Cuimi’ kumquat, offering a theoretical basis for understanding the HLB epidemic in Dechang County, Sichuan. The research focuses on the absolute quantification of the HLB bacterium in seven specific tissues of the ‘Cuimi’ kumquat, including new leaves, upper phloem of branches, fruit peduncle, pith, fruit axis, old leaves, and lower phloem of branches. Additionally, the types and contents of prophages were identified in these tissues. In the same diseased branch group, Candidatus Liberibacter asiaticus (CLas) exhibited an uneven distribution, with the highest concentration detected in the pith, significantly surpassing levels found in the stem and leaf tissues (new leaves, upper phloem of branches, old leaves, lower phloem of branches). Infected fruit peduncles and pith slices showed noticeable shrinkage and collapse in the phloem. Prophage analysis indicated that multiple types of prophages could be simultaneously detected within the same infected ‘Cuimi’ kumquat branch. New shoot tissues contained both Type 2 and Type 4 prophages, with a relatively higher abundance of Type 4 and a lower abundance of Type 2. The relative abundance of Type 1 prophage in the fruit tissues was generally higher than in other tissues. CLas primarily accumulates in the fruit tissues of the ‘Cuimi’ kumquat, and the situation in Dechang County suggests that individual trees may be infected with multiple prophage strains simultaneously.

ABSTRACT “ Candidatus Liberibacter asiaticus” (CLas), the causal agent of citrus Huanglongbing (HLB), is able to multiply to a high abundance in citrus fruit pith. However, little is known about the biological processes and phytochemical substances that are vital for CLas colonization and growth in fruit pith. In this study, CLas-infected fruit pith of three citrus cultivars (“Shatangju” mandarin, “Guanxi” pomelo, and “Shatian” pomelo) exhibiting different tolerance to CLas were collected and used for dual RNA-Seq and untargeted metabolome analysis. Comparative transcriptome analysis found that the activation of the CLas noncyclic TCA pathway and pathogenic-related effectors could contribute to the colonization and growth of CLas in fruit pith. The pre-established Type 2 prophage in the CLas genome and the induction of its CRISPR/ cas system could enhance the phage resistance of CLas and, in turn, facilitate CLas population growth in fruit pith. CLas infection caused the accumulation of amino acids that were correlated with tolerance to CLas. The accumulation of most sugars and organic acids in CLas-infected fruit pith, which could be due to the phloem blockage caused by CLas infection, was thought to be beneficial for CLas growth in localized phloem tissue. The higher levels of flavonoids and terpenoids in the fruit pith of CLas-tolerant cultivars, particularly those known for their antimicrobial properties, could hinder the growth of CLas. This study advances our understanding of CLas multiplication in fruit pith and offers novel insight into metabolites that could be responsible for tolerance to CLas or essential to CLas population growth. IMPORTANCE Citrus Huanglongbing (HLB, also called citrus greening disease) is a highly destructive disease currently threatening citrus production worldwide. HLB is caused by an unculturable bacterial pathogen, “ Candidatus Liberibacter asiaticus” (CLas). However, the mechanism of CLas colonization and growth in citrus hosts is poorly understood. In this study, we utilized the fruit pith tissue, which was able to maintain the CLas at a high abundance, as the materials for dual RNA-Seq and untargeted metabolome analysis, aiming to reveal the biological processes and phytochemical substances that are vital for CLas colonization and growth. We provided a genome-wide CLas transcriptome landscape in the fruit pith of three citrus cultivars with different tolerance and identified the important genes/pathways that contribute to CLas colonization and growth in the fruit pith. Metabolome profiling identified the key metabolites, which were mainly affected by CLas infection and influenced the population dynamic of CLas in fruit pith.

Citri Reticulatae Pericarpium, especially the pericarp of Citrus reticulata Blanco cv. Chachiensis (PCRC), is an important edible and medicinal ingredient for health and pharmacological properties. Citrus Huanglongbing, a devastating disease that currently threatens the citrus industry worldwide, is caused by a phloem-limited alpha-proteobacterium, “Candidatus Liberibacter asiaticus” (CLas). The industry of cultivar Chachiensis has been suffering from HLB. Although HLB affected the quality of citrus fruit, whether the quality of PCRC was affected by HLB remains unclear. In this study, we compared the metabolite profiles between HLB-affected and healthy PCRC from three sources: fresh, 6-month-old, and 9-year-old PCRC, through the untargeted LC–MS method. Compared to healthy controls, various types of bioactive compounds, mainly flavonoids, terpenoids, alkaloids, coumarins, polysaccharides, and phenolic acids, accumulated in HLB-affected PCRC, especially in the HLB-affected 9-year PCRC. In particular, isorhamnetin, isoliquiritigenin, luteolin 7-O-beta-D-glucoside, limonin, geniposide, pyrimidodiazepine, scoparone, chitobiose, m-coumaric acid, malonate, and pantothenic acid, which contributed to the pharmacological activity and health care effects of PCRC, were highly accumulated in HLB-affected 9-year-old PCRC compared to the healthy control. Multibioassay analyses revealed that HLB-affected 9-year-old PCRC had a higher content of total flavonoids and total polyphenols and exhibited similar antioxidant capacity as compared to healthy controls. The results of this study provided detailed information on the quality of HLB-affected PCRC.

Abstract Objectives “Candidatus Liberibacter asiaticus” (CLas) is an un-culturable α-proteobacterium that caused citrus Huanglongbing (HLB), a destructive disease threatening citrus production worldwide. In China, the presence of HLB was first reported in Chaoshan region of Guangdong province, China around a century ago. Thus, whole genome information of CLas strains from Chaoshan area become the most important resource to understand the population diversity and evaluation of CLas in China. Data description CLas strain GDCZ was originally from Chaozhou city (Chaoshan area) and sequenced using PacBio Sequel long-read sequencing and Illumina short-read sequencing. The genome of strain GDCZ comprised of 1,230,507 bp with an average G + C content of 36.4%, along with a circular CLasMV1 phage: CLasMV1_GDCZ (8,869 bp). The CLas strain GDCZ contained a Type 2 prophage (37,452 bp) and encoded a total of 1,057 open reading frames and 53 RNA genes. The whole genome sequence of CLas strain GDCZ from the historical HLB endemic region in China will serve as a useful resource for further analyses of CLas evolution and HLB epidemiology in China and world.

Abstract Objectives: “Candidatus Liberibacter asiaticus” (CLas) is an un-culturable α-proteobacterium that caused citrus Huanglongbing (HLB), a destructive disease threatening citrus production worldwide. In China, the presence of HLB was first reported in Chaoshan region of Guangdong province, China around a century ago. Thus, whole genome information of CLas strains from Chaoshan area become the most important resource to understand the population diversity and evaluation of CLas in China. Data description: CLas strain GDCZ was originally from Chaozhou city (Chaoshan area) and sequenced using Pacbio Sequel long-read sequencing and Illumina short-read sequencing. The genome of strain GDCZ comprised of 1,230,507 bp with an average G+C content of 36.4%, along with a circular CLasMV1 phage: CLasMV1_GDCZ (8,869 bp). The CLas strain GDCZ contained a Type 2 prophage (37,452 bp) and encoded a total of 1,057 open reading frames and 53 RNA genes. The whole genome sequence of CLas strain GDCZ from the historical HLB endemic region in China will serve as a useful resource for further analyses of CLas evolution and HLB epidemiology in China and world.