Frontiers in Plant Science

Introduction Huanglongbing (HLB) is a devastating disease impacting citrus orchards worldwide. Identifying candidate genes and/or genomic regions that significantly influence plants' response to HLB is essential for breeding programs. Nevertheless, this remains a challenge, especially during the early stages of the citrus-pathogen interaction, when phenotypically distinguishing tolerant genotypes is not trivial. This work focused on identifying genomic regions and candidate genes associated with citrus response to Candidatus Liberibacter asiaticus ( C Las). Methods Quantitative trait loci (QTL) mapping was performed in a citrandarin population (278 individuals) derived from a fullsib cross between susceptible Citrus sunki (CS) and tolerant Poncirus trifoliata (PT). The mapping population was genotyped using DArTseq markers and the SNPs obtained from them (DArTseq-derived SNPs), as well as the EMBRAPA Multispecies 65K Illumina Infinium® chip. Phenotyping was based on four individual trials (two in a greenhouse and two in field conditions). QTL mapping was based on composite interval mapping implemented in the fullsibQTL software, with the cycle threshold (Ct) of quantitative PCR (qPCR) as the trait of interest for assessing C Las quantification in infected tissues, which can be evaluated in plants at any developmental stage. The biological function of QTLs was investigated by gene annotation using the chromosome-level phased Citrus sinensis genome (assembly DVS_A1.0) as the reference. The candidate genes were functionally annotated using Blast2Go software. Results A predominance of markers in a 1:1 ratio was observed, resulting in two maps, one for each parent. The linkage map had 5464 cM (1872 markers) for CS and 4794 cM (1340 markers) for PT. A total of 44 QTLs were mapped: 25 for the CS map (13 for field and 12 for greenhouse conditions), of which 17 were located at gene sites; and 19 were mapped for the PT (nine for field and 10 for greenhouse conditions), of which 16 were located at gene sites. Discussion Several genes associated with the identified QTLs are involved in stress responses, making them potential targets for further functional validation. These genomic regions and candidate genes provide valuable targets for future incorporation into citrus breeding programs aimed at developing HLB tolerance.

Understanding plant-vector-pathogen interactions is essential for explaining the persistence of stolbur diseases associated with the plant pathogenic bacterium ‘ Candidatus Phytoplasma solani’ (CaPsol) in agroecosystems. This study investigates the role of crop-associated Reptalus planthoppers (Insecta: Hemiptera: Cixiidae) in shaping CaPsol epidemiology within a four-crop rotation system (sunflower, wheat, sugar beet, and maize) in Serbia under field and experimental conditions. Over a three-year period (2023–2025), nymphal surveys, adult monitoring, emergence-cage experiments, molecular identification of planthoppers, assessment of CaPsol in crops, and vector transmission assays were conducted, with a particular focus on the sunflower-wheat sequence. Sunflower was identified as a developmental host for Reptalus quinquecostatus and Reptalus panzeri , with high CaPsol infection rates detected in both the host plant and associated vectors. In contrast, wheat appeared to primarily support nymphal overwintering, while its significance as a CaPsol reservoir remains inconclusive. Furthermore, the maize-wheat sequence supported the development of R. quinquecostatus while confirming its role in the life cycle of R. panzeri . Experimental trials demonstrated efficient CaPsol transmission to sunflower but limited transmission to wheat. Despite high infection levels under field conditions, sunflower remained asymptomatic, suggesting its role as a cryptic pathogen reservoir. The findings indicate that the sunflower-wheat sequence may sustain CaPsol-infected Reptalus spp. populations across seasons, facilitating pathogen persistence within cropping systems. The study highlights the importance of integrating vector life cycle dynamics into epidemiological frameworks of stolbur diseases, particularly in crop rotations where major cultivated hosts may support in-field CaPsol pathosystems.

A field investigation was conducted during the winter seasons of 2021–22 and 2022–23 at the Jawaharlal Nehru Krishi Vishwa Vidyalaya (JNKVV), Jabalpur, Central India, to identify and characterize the phytoplasma strains associated with niger (var. JNC6) and sesame (var. RT315) exhibiting severe phyllody symptoms. Disease incidence ranged from 0-3%, and 3 to 15% were recorded in niger and sesame crops during the study period. Nested PCR assays using universal phytoplasma primer pairs P1/P7 followed by R16F2n/R16R2 consistently amplified ~1.25 kb fragments of the 16S rRNA gene from both the symptomatic plants, confirming phytoplasma association. Additional amplification of phytoplasma-specific sec A (~600 bp) and sec Y (~1.7 kb) gene fragments further validated the phytoplasma infection in both crops. Two predominant leafhopper species, Amrasca biguttula and Orosius albicinctus , colonizing and feeding niger and sesame fields were identified. Notably, only Amrasca biguttula was tested positive for phytoplasma using the similar PCR assays mentioned above, implicating it as a putative vector. Sequence identity, phylogenetic analysis, and in silico RFLP profiling of 16S rRNA, sec A, and sec Y gene sequences from both the symptomatic plants and leafhoppers confirmed the presence of a ‘ Ca. P. asiaticum’ related strain (16SrII-C subgroup). This study represents the first global report of 16SrII-C subgroup phytoplasma infection in niger, highlighting a new host record. The detection of an identical phytoplasma strain in niger, sesame, and Amrasca biguttula suggests a possible epidemiological link and underscores the role of these natural hosts and the leafhopper in the dissemination of ‘ Ca. P. asiaticum’-related phytoplasma strains in Central India.

Among citrus diseases, Huanglongbing (HLB) is recognized as the most destructive and economically damaging worldwide. It is mainly associated with ‘ Candidatus Liberibacter asiaticus’ ( C Las) being transmitted by Diaphorina citri . There are no curative treatments or commercial citrus varieties resistant to C Las. Wild Aurantioideae species have been widely screened and, recently, Oceanian genotypes graft- and sexually compatible with Citrus were identified as HLB-resistant; however, there is no information regarding their use as interstocks of commercial varieties yet. Under greenhouse conditions, six HLB-resistant genotypes were evaluated as interstocks between ‘Valencia’ sweet orange scion and ‘Rangpur’ lime rootstock, both susceptible to HLB, with ‘Valencia’ interstock as the control. Rootstocks were nucellar seedlings, and the scion was a commercial accession preimmunized with a mild CTV strain. Plants were assessed for C Las infection and titer in leaves, stem bark, and roots up to 12–24 months after graft-inoculation in the scion and compared to non-inoculated controls. Furthermore, molecular, anatomical, and biometric variables were investigated. As expected, the scion variety was colonized by C Las regardless of the genotype evaluated as interstock. Although bacteria were detected in the roots of most C Las-inoculated plants, C Las movement from the scion to the roots was blocked in 42% and 86% of composite plants when using a F1 hybrid of C. australis × C. inodora or an admixture hybrid of C. glauca , C. australis , and C. australasica as interstocks. Overall, C Las titers were similar in infected plant tissues among the evaluated genotypes, but titers were lower in interstock bark tissues compared to scion and rootstock ones. After one-two years of C Las + CTV infection (experiments I and II, respectively), the dry weight of the root system decreased by 50% in infected trees compared to control trees for most genotypes, and C Las + CTV infection was associated with changes in the sieve phloem and gene expression. These findings suggest that, despite CTV infection, interstocks derived from some hybrids of Australian citrus types have the potential to restrict the movement of C Las from the scion into the roots of infected citrus trees. Long-term evaluation of composite plants in field conditions is necessary to assess tree performance and, ultimately, the impact of C Las blockage by interstocking on HLB disease damage.

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is the most destructive citrus disease worldwide, severely reducing yield and fruit quality. Although no naturally resistant cultivars are available, citrus plants overexpressing Arabidopsis NPR1 ( At NPR1) display enhanced tolerance, yet the molecular mechanisms underlying this phenotype remain insufficiently understood. To uncover early transcriptional events associated with tolerance, we performed comparative RNA-seq and phytohormone profiling of susceptible wild-type (WT) and tolerant At NPR1-overexpressing ( At NPR1-OE) Duncan grapefruit at 0, 6, and 24 hours post infection (hpi). Before infection, At NPR1 plants downregulated genes involved in cytoskeleton organization, cell wall biogenesis, and receptor signaling, suggesting a primed basal defense state. After CLas exposure, At NPR1 plants exhibited stronger and earlier transcriptional reprogramming, with substantially more differentially expressed genes at 6 hpi than WT. At 24 hpi, At NPR1 plants showed suppression of callose synthase genes and selective induction of β-1,3-glucanases, indicating more controlled phloem callose regulation. Concurrently, attenuated expression of respiratory burst oxidase homologs and ROS-associated genes suggested a moderated and less damaging oxidative burst. At NPR1 plants maintained stable levels of salicylic acid, and gibberellins while preventing the CLas-induced induction of abscisic acid observed in WT. Collectively, these findings reveal that At NPR1 overexpression enhances HLB tolerance by integrating early transcriptional reprogramming with balanced structural, oxidative, and hormonal responses. This study provides a mechanistic framework for understanding NPR1-mediated tolerance to CLas during the initial stages of infection.

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.