Deng, Xiaoling

BackgroundHuanglongbing (HLB, yellow shoot disease) is a highly destructive citrus disease associated with a nonculturable bacterium, “Candidatus Liberibacter asiaticus” (CLas), which is transmitted by Asian citrus psyllid (ACP, Diaphorina citri). In Mexico, HLB was first reported in Tizimin, Yucatán, in 2009 and is now endemic in 351 municipalities of 25 states. Understanding the population diversity of CLas is critical for HLB management. Current CLas diversity research is exclusively based on analysis of the bacterial genome, which composed two regions, chromosome (> 1,000 genes) and prophage (about 40 genes).Methods and resultsIn this study, 40 CLas-infected ACP samples from 20 states in Mexico were collected. CLas was detected and confirmed by PCR assays. A prophage gene(terL)-based typing system (TTS) divided the Mexican CLas strains into two groups: Term-G including four strains from Yucatán and Chiapas, as well as strain psy62 from Florida, USA, and Term-A included all other 36 Mexican strains, as well as strain AHCA1 from California, USA. CLas diversity was further evaluated to include all chromosomal and prophage genes assisted by using machine learning (ML) tools to resolve multidimensional data handling issues. A Term-G strain (YTMX) and a Term-A strain (BCSMX) were sequenced and analyzed. The two Mexican genome sequences along with the CLas genome sequences available in GenBank were studied. An unsupervised ML was implemented through principal component analysis (PCA) on average nucleotide identities (ANIs) of CLas whole genome sequences; And a supervised ML was implemented through sparse partial least squares discriminant analysis (sPLS-DA) on single nucleotide polymorphisms (SNPs) of coding genes of CLas guided by the TTS. Two CLas Geno-groups, Geno-group 1 that extended Term-A and Geno-group 2 that extended Term-G, were established.ConclusionsThis study concluded that: 1) there were at least two different introductions of CLas into Mexico; 2) CLas strains between Mexico and USA are closely related; and 3) The two Geno-groups provide the basis for future CLas subspecies research.

“Candidatus Liberibacter asiaticus” (CLas) is a phloem-restricted α-proteobacterium that is associated with citrus huanglongbing (HLB), which is the most destructive disease that affects all varieties of citrus. Although midrib is usually used as a material for CLas detection, we recently found that the bacterium was enriched in fruits, especially in the fruit pith. However, no study has revealed the molecular basis of these two parts in responding to CLas infection. Therefore, we performed transcriptome and UHPLC–MS-based targeted and untargeted metabolomics analyses in order to organize the essential genes and metabolites that are involved. Transcriptome and metabolome characterized 4834 differentially expressed genes (DEGs) and 383 differentially accumulated metabolites (DAMs) between the two materials, wherein 179 DEGs and 44 DAMs were affected by HLB in both of the tissues, involving the pathways of phenylpropanoid biosynthesis, phytohormone signaling transduction, starch and sucrose metabolism, and photosynthesis. Notably, we discovered that the gene expression that is related to beta-glucosidase and endoglucanase was up-regulated in fruits. In addition, defense-related gene expression and metabolite accumulation were significantly down-regulated in infected fruits. Taken together, the decreased amount of jasmonic acid, coupled with the reduced accumulation of phenylpropanoid and the increased proliferation of indole-3-acetic acid, salicylic acid, and abscisic acid, compared to leaf midribs, may contribute largely to the enrichment of CLas in fruit piths, resulting in disorders of photosynthesis and starch and sucrose metabolism.

“Candidatus Liberibacter asiaticus” (CLas) is the causal agent of citrus Huanglongbing (HLB, also called citrus greening disease), a highly destructive disease threatening citrus production worldwide. A novel Microviridae phage (named CLasMV1) has been found to infect CLas, providing a potential therapeutic strategy for CLas/HLB control. However, little is known about the CLasMV1 biology. In this study, we analyzed the population dynamics of CLasMV1 between the insect vector of CLas, the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) and the holoparasitic dodder plant (Cuscuta campestris Yunck.); both acquired CLasMV1-infected CLas from an HLB citrus. All CLas-positive dodder samples were CLasMV1-positive, whereas only 32% of CLas-positive ACP samples were identified as CLasMV1-positive. Quantitative analyses showed a similar distribution pattern of CLasMV1 phage and CLas among eight citrus cultivars by presenting at highest abundance in the fruit pith and/or the center axis of the fruit. Transcriptome analyses revealed the possible lytic activity of CLasMV1 on CLas in fruit pith as evidenced by high-level expressions of CLasMV1 genes, and CLas genes related to cell wall biogenesis and remodeling to maintain the CLas cell envelope integrity. The up-regulation of CLas genes were involved in restriction–modification system that could involve possible phage resistance for CLas during CLasMV1 infection. In addition, the regulation of CLas genes involved in cell surface components and Sec pathway by CLasMV1 phage could be beneficial for phage infection. This study expanded our knowledge of CLasMV1 phage that will benefit further CLas phage research and HLB control.

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.