Frontiers in Plant Science

Phloem feeders, such as the psyllid Diaphorina citri ( D. citri ), feed on plants by inserting their stylet bundle followed by probing the apoplast before reaching the phloem. The psyllids secrete watery saliva containing various proteins into the phloem, which can act as effectors to facilitate their feeding or modulate host defense responses. Concomitantly, feeding is the main mode of transmitting the Candidatus Liberibacter asiaticus ( C Las) bacteria to the phloem. C Las produces several effectors that have been hypothesized to contribute to Huanglongbing (HLB) virulence. Here, we aimed to identify putative effector proteins from both D. citri and C Las. To achieve this, we used different omics techniques on different tissues and organs from both plants and insects. More specifically, we performed transcriptomics on the heads of healthy and C Las-infected D. citri and proteomics of artificial diet and of phloem of four different plant species fed on by healthy and C Las-infected D. citri. Subsequently, we used various criteria and bioinformatics tools to predict putative effectors. This resulted in the identification of four proteins from D. citri [ferritin, prisilkin, CG31997-PA, and pterin-4-alpha-carbinolamine dehydratase-like protein (PCBD)] and two Sec-dependent effectors from C Las, CLIBASIA_04560 and CLIBASIA_05320, that were used for further functional studies. The expression of these six proteins in Nicotiana benthamiana modified the ROS burst triggered by flagellin, indicating that they can indeed function as effector proteins in planta . In addition, expression of the psyllid effectors in planta significantly reduced the growth of Pseudomonas syringae pv. tabaci ( Pta ).

Huanglongbing (HLB), or citrus greening, remains one of the most destructive diseases affecting citrus production globally. Associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus ( C Las) and vectored by Diaphorina citri , HLB leads to canopy decline, fibrous root loss, and reductions in fruit yield and quality. Recently, the systemic delivery of oxytetracycline (OTC) via trunk injection was approved in Florida as a targeted therapy to reduce C Las titers and improve tree health. In parallel, Individual Protective Covers (IPCs) have been adopted to delay C Las infection in newly planted citrus trees by vector exclusion. This study investigates the combined use of IPCs and trunk injection of OTC for post-IPC therapy. ‘Valencia’ sweet orange trees grafted on US-812 and US-942 rootstocks were planted in December 2020 under HLB-endemic conditions in southwest Florida. IPCs were installed at planting and removed after 18 months. The first OTC injection was performed in May 2023, 10 months after IPC removal. A second injection was performed in May 2024. A 2 × 2 × 2 factorial experimental design evaluated the effects of infection history (early-infected and late-infected), rootstock cultivar (US-812 and US-942), and injection treatment (OTC-injected and non-injected) on tree responses over two consecutive production seasons. In year 1, infection history significantly influenced tree size, fruit yield, total soluble solids (TSS), TSS/titratable acidity ratio, and peel color. Late-infected trees outperformed early-infected trees, regardless of injection treatment and rootstock cultivar. In year 2, OTC-injected trees exhibited significantly higher yields, improved juice quality, and enhanced canopy health regardless of infection history and rootstock cultivar. Fibrous root microbiome analyses based on 16S rRNA sequencing revealed no significant effects of OTC injection on bacterial alpha or beta diversity, with stable community structure observed across treatments and time points. This suggests that targeted vascular delivery of OTC may not cause any major disruption to the root endorhizosphere microbiome. Together, the results from this study demonstrate the efficacy of integrating preventative (use of IPCs) and therapeutic (OTC vascular delivery) strategies for sustainable HLB management while preserving microbial integrity and offering a model for citrus production in parts of the world where HLB is prevalent.

Huanglongbing (HLB) is a devastating disease of citrus plants caused by the non-culturable phloem-inhabiting bacterium Candidatus Liberibacter ssp., being Ca. Liberibacter asiaticus (CLas) the most aggressive species. CLas is vectored by the psyllid Diaphorina citri and introduced into sieve cells, establishing a successful infection in all Citrus species. Partial or complete resistance has been documented in the distant relatives Murraya paniculata and Bergera koenigii, respectively, providing excellent systems to investigate the molecular basis of HLB-resistance. It has been shown previously that the first weeks after bacterial release into the phloem are critical for the establishment of the bacterium. In this study, a thorough transcriptomic analysis of young flushes exposed to CLas-positive and negative psyllids has been performed in Citrus × sinensis, as well as in the aforementioned resistant species, along the first eight weeks after exposure. Our results indicate that the resistant species do not deploy a classical immunity response upon CLas recognition. Instead, transcriptome changes are scarce and only a few genes are differentially expressed when flushes exposed to CLas-positive and negative psyllid are compared. Functional analysis suggests that primary metabolism and other basic cellular functions could be rewired in the resistant species to limit infection. Transcriptomes of young flushes of the three species are very different, supporting the existence of distinct biochemical niches for the bacterium. These findings suggest that both intrinsic metabolic inadequacies to CLas survival, as well as inducible reprogramming of physiological functions upon CLas recognition, could orchestrate together restriction of bacterial multiplication in these resistant hosts.

Citrus Huanglongbing (HLB) represents a significant threat to the citrus industry, mainly caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas). In this review, we summarize recent advances in understanding the relationship between citrus and CLas, particularly examining the functions of Sec-dependent effectors (SDEs) and non-classically secreted proteins (ncSPs) in virulence, as well as their targeted interactions with citrus. We further investigate the impact of SDEs on various physiological processes, including systemic acquired resistance (SAR), reactive oxygen species (ROS) accumulation, vesicle trafficking, callose deposition, cell death, autophagy, chlorosis and flowering. Additionally, we focus on the functional research on specific disease-resistant genes in citrus and the molecular mechanisms underlying disease resistance. Finally, we discuss the existing gaps and unresolved questions regarding citrus-CLas interactions, proposing potential solutions to facilitate the development of HLB-resistant citrus varieties.

The global citrus industry faces a great threat from Huanglongbing (HLB), a destructive disease caused by ‘Candidatus Liberibacter asiaticus’ (CLas) that induces significant economic losses without any known cure. Understanding how citrus plants defend against HLB, particularly at the early stages of infection, is crucial for developing long-term solutions. This study investigated the earliest metabolic responses of fresh citrus leaves to CLas infection using untargeted metabolomics and machine learning models. HLB-tolerant and HLB-sensitive cultivars were compared to analyze their biochemical reactions within 48 hours post-infection. HESI/Q-Orbitrap MS analysis identified temporal differential metabolites, revealing distinct metabolic pathways activated in response to CLas infection. Both cultivars responded by increasing specific metabolite concentrations, such as flavonoids, within 2 hours post-infection, but the HLB-tolerant cultivar maintained higher levels throughout the 48-hour period. This early metabolic activity could influence long-term plant health by enhancing disease resistance and reducing pathogen impact. These findings provide potential biomarkers for breeding HLB-resistant cultivars and offer valuable insights for developing sustainable management strategies to mitigate the impact of HLB on the citrus industry, ensuring its long-term productivity and economic viability.

The tomato-potato psyllid, Bactericera cockerelli (Šulc), belonging to the Hemiptera order, is an insect pest of solanaceous crops and vectors a fastidious bacterium, Candidatus Liberibacter solanacearum (CLso), the presumptive causal agent of zebra chip and vein greening diseases in potatoes and tomatoes, respectively. The genome of B. cockerelli has been sequenced recently, providing new avenues to elucidate mechanistic insights into pathogenesis in vegetable crops. In this study, we performed RNA-sequencing of the critical psyllid organs (salivary glands and ovaries) involved in CLso pathology and transmission to host plants. Transcriptome analysis revealed differentially expressed genes and organ-specific enrichment of gene ontology (GO) terms related to metabolic processes, response to stress/stimulus, phagocytosis, proteolysis, endocytosis, and provided candidate genes encoding transcription factors (TFs). To examine gene regulatory networks across the psyllid organs under CLso(-) and CLso(+) conditions, we performed weighted gene co-expression network analysis (WGCNA), and unique modules differentiating the psyllid organs were identified. A comparative GO analysis of the unique gene modules revealed functional terms enriched in response to stress, gene regulation, and cell division processes in the ovaries. In contrast, respiration, transport, and neuronal transmission-related GO terms were enriched in the salivary glands. Altogether, this study reveals new insights into tissue-specific expression of the psyllid organs in the absence or presence of CLso bacterium. This knowledge can be leveraged to develop new pest and disease management strategies by delineating the regulatory networks involved in the psyllid-CLso interaction.

IntroductionHuanglongbing (HLB), a disease that’s ubiquitous worldwide, wreaks havoc on the citrus industry. The primary culprit of HLB is the gram-negative bacterium Candidatus Liberibacter asiaticus (CLas) that infects the phloem, but its damaging mechanism is yet to be fully understood.Methods and resultsIn this study, a multitude of tools including weighted correlation network analysis (WGCNA), protein-protein interaction (PPI) network analysis and gene expression profiling are employed to unravel the intricacies of its pathogenesis. The investigation pinpoints various central genes, such as the ethylene-responsive transcription factor 9 (ERF9) and thioredoxin reductase 1 (TrxR1), that are associated with CLas invasion and resultant disturbances in numerous biological operations. Additionally, the study uncovers a range of responses through the detection of differential expressed genes (DEGs) across different experiments. The discovery of core DEGs leads to the identification of pivotal genes such as the sieve element occlusion (SEO) and the wall-associated receptor kinase-like 15 (WAKL15). PPI network analysis highlights potential vital proteins, while GO and KEGG pathway enrichment analysis illustrate a significant impact on multiple defensive and metabolic pathways. Gene set enrichment analysis (GSEA) indicates significant alterations in biological processes such as leaf senescence and response to biotic stimuli.DiscussionThis all-encompassing approach extends valuable understanding into the pathogenesis of CLas, potentially aiding future research and therapeutic strategies for HLB.

The strains of the genus Microbacterium, with more than 150 species, inhabit diverse environments; plant-associated bacteria reveal their plant growth-promoting activities due to a number of beneficial characteristics. Through the performance of diverse techniques and methods, including isolation of a novel Microbacterium strain from the aerial roots of leafless epiphytic orchid, Chiloschista parishii Seidenf., its morphological and biochemical characterization, chemotaxonomy, phylogenetic and genome analysis, as well as bioassays and estimation of its auxin production capacity, a novel strain of ET2T is described. Despite that it shared 16S rRNA gene sequence similarity of 99.79% with Microbacterium kunmingense JXJ CY 27-2T, so they formed a monophyletic group on phylogenetic trees, the two strains showed clear divergence of their genome sequences. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values of ET2T differed greatly from phylogenetically close JXJ CY 27-2T. Based on the differences being below the threshold for species similarity, together with the unique chemotaxonomic characteristics, strain ET2T represents a novel species of the genus Microbacterium. Several genes, putatively involved in auxin biosynthesis were predicted. This strain revealed obvious plant growth-promoting activities, including diazotrophy and biosynthesis of tryptophan-dependent auxins (indole-3-acetic and indole-3-pyruvic acids). Microbial auxins directly stimulated the rhizogenesis, so that the ET2T-inoculated seeds of wheat, cucumber and garden cress showed evident promotion in their growth and development, both under optimal and under cold stress conditions. Based on phenotypic, chemotypic and genotypic evidences, the strain ET2T belongs to the genus Microbacterium, order Micrococcales, class Actinomycetes, and it represents a novel species, for which the name Microbacterium albopurpureum sp. nov. is proposed, with strain ET2T (VKPM Ac-2212, VKM Ас-2998) as the type strain.

IntroductionAsian citrus psyllid (ACP, Diaphorina citri) is an important transmission vector of “Candidatus Liberibacter asiaticus” (CLas), the causal agent of Huanglongbing (HLB), the most destructive citrus disease in the world. As there are currently no HLB-resistant rootstocks or varieties, the control of ACP is an important way to prevent HLB. Some viruses of insect vectors can be used as genetically engineered materials to control insect vectors.MethodsTo gain knowledge on viruses in ACP in China, the prevalence of five RNA and DNA viruses was successfully determined by optimizing reverse transcription polymerase chain reaction (RT-PCR) in individual adult ACPs. The five ACP-associated viruses were identified as follows: diaphorina citri bunyavirus 2, which was newly identified by high-throughput sequencing in our lab, diaphorina citri reovirus (DcRV), diaphorina citri picorna-like virus (DcPLV), diaphorina citri bunyavirus (DcBV), and diaphorina citri densovirus-like virus (DcDV).ResultsDcPLV was the most prevalent and widespread ACP-associated virus, followed by DcBV, and it was detected in more than 50% of all samples tested. DcPLV was also demonstrated to propagate vertically and found more in salivary glands among different tissues. Approximately 60% of all adult insect samples were co-infected with more than one insect pathogen, including the five ACP-associated viruses and CLas.DiscussionThis is the first time these viruses, including the newly identified ACP-associated virus, have been detected in individual adult ACPs from natural populations in China’s five major citrus-producing provinces. These results provide valuable information about the prevalence of ACP-associated viruses in China, some of which have the potential to be used as biocontrol agents. In addition, analysis of the change in prevalence of pathogens in a single insect vector is the basis for understanding the interactions between CLas, ACP, and insect viruses.

IntroductionCandidatus Liberibacter solanacearum (CLso) is a regulated plant pathogen in European and some Asian countries, associated with severe diseases in economically important Apiaceous and Solanaceous crops, including potato, tomato, and carrot. Eleven haplotypes of CLso have been identified based on the difference in rRNA and conserved genes and host and pathogenicity. Although it is pathogenic to a wide range of plants, the mechanisms of plant response and functional decline of host plants are not well defined. This study aims to describe the underlying mechanism of the functional decline of tomato plants infected by CLso by analyzing the transcriptomic response of tomato plants to CLso haplotypes A and B.MethodsNext-generation sequencing (NGS) data were generated from total RNA of tomato plants infected by CLso haplotypes A and B, and uninfected tomato plants, while qPCR analysis was used to validate the in-silico expression analysis. Gene Ontology and KEGG pathways were enriched using differentially expressed genes.ResultsPlants infected with CLso haplotype B saw 229 genes upregulated when compared to uninfected plants, while 1,135 were downregulated. Healthy tomato plants and plants infected by haplotype A had similar expression levels, which is consistent with the fact that CLso haplotype A does not show apparent symptoms in tomato plants. Photosynthesis and starch biosynthesis were impaired while starch amylolysis was promoted in plants infected by CLso haplotype B compared with uninfected plants. The changes in pathway gene expression suggest that carbohydrate consumption in infected plants was more extensive than accumulation. In addition, cell-wall-related genes, including steroid biosynthesis pathways, were downregulated in plants infected with CLso haplotype B suggesting a reduction in membrane fluidity, cell signaling, and defense against bacteria. In addition, genes in phenylpropanoid metabolism and DNA replication were generally suppressed by CLso infection, affecting plant growth and defense.DiscussionThis study provides insights into plants’ defense and functional decline due to pathogenic CLso using whole transcriptome sequencing and qPCR validation. Our results show how tomato plants react in metabolic pathways during the deterioration caused by pathogenic CLso. Understanding the underlying mechanisms can enhance disease control and create opportunities for breeding resistant or tolerant varieties.