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Citrus Huanglongbing (HLB) caused by ' Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. CLas induces systemic and chronic reactive oxygen species (ROS) production, which has been suggested to be a primary cause of cell death in phloem tissues and subsequent HLB symptoms. Mitigating oxidative stress caused by CLas using horticultural approaches has been suggested as a useful strategy to reduce HLB damages. To provide information regarding the application timing to mitigate ROS, we investigated monthly dynamics of CLas concentration, CLas-triggered ROS, and phloem cell death in the bark tissues of asymptomatic and symptomatic branches of HLB-positive Hamlin and Valencia sweet orange trees in the field. Healthy branches in the screenhouse were used as controls. CLas concentration exhibited significant variations over the course of the year, with two distinct peaks observed in Florida citrus groves-late spring/early summer and late fall. Within both Hamlin and Valencia asymptomatic tissues, CLas concentration demonstrated a negative correlation with the deviation between the monthly average mean temperature and the optimal temperature for CLas colonization in plants (25.7°C). However, such a correlation was not evident in symptomatic tissues of Hamlin or Valencia sweet oranges. ROS levels were consistently higher in symptomatic or asymptomatic branches than in healthy branches in most months. ROS concentrations were higher in symptomatic branches than in asymptomatic branches in most months. CLas triggered significant increases in ion leakage in most months for asymptomatic and symptomatic branches compared with healthy controls. In asymptomatic branches of Hamlin, a positive correlation was observed between CLas concentration and ROS concentrations, CLas concentration and ion leakage levels, as well as ROS and ion leakage. Intriguingly, such a relationship was not observed in Valencia asymptomatic branches or in the symptomatic branches of Hamlin and Valencia. This study sheds light on the pathogenicity of CLas by providing useful information on the temporal dynamics of ROS production, phloem cell death, and CLas growth, as well as provides useful information in determining the timing for application of antioxidants and antimicrobial agents to control HLB., Competing Interests: The author(s) declare no conflict of interest.

The bacterium Candidatus Liberibacter asiaticus (CLas) causes citrus Huanglongbing disease. Our understanding of the pathogenicity and biology of this microorganism remains limited because CLas has not yet been cultivated in artificial media. Its genome is relatively small and encodes approximately 1136 proteins, of which 415 have unknown functions. Here, we use a high-throughput yeast-two-hybrid (Y2H) screen to identify interactions between CLas proteins, thus providing insights into their potential functions. We identify 4245 interactions between 542 proteins, after screening 916 bait and 936 prey proteins. The false positive rate of the Y2H assay is estimated to be 2.9%. Pull-down assays for nine protein-protein interactions (PPIs) likely involved in flagellar function support the robustness of the Y2H results. The average number of PPIs per node in the CLas interactome is 15.6, which is higher than the numbers previously reported for interactomes of free-living bacteria, suggesting that CLas genome reduction has been accompanied by increased protein multi-functionality. We propose potential functions for 171 uncharacterized proteins, based on the PPI results, guilt-by-association analyses, and comparison with data from other bacterial species. We identify 40 hub-node proteins, including quinone oxidoreductase and LysR, which are known to protect other bacteria against oxidative stress and might be important for CLas survival in the phloem. We expect our PPI database to facilitate research on CLas biology and pathogenicity mechanisms., (© 2023. The Author(s).)

' Candidatus Liberibacter asiaticus' (CLas) is associated with the devastating citrus disease Huanglongbing (HLB). Young flushes are the center of the HLB pathosystem due to their roles in the psyllid life cycle and in the acquisition and transmission of CLas. However, the early events of CLas infection and how CLas modulates young flush physiology remain poorly understood. Here, transmission electron microscopy analysis showed that the mean diameter of the sieve pores decreased in young leaves of HLB-positive trees after CLas infection, consistent with CLas-triggered callose deposition. RNA-seq-based global expression analysis of young leaves of HLB-positive sweet orange with (CLas-Pos) and without (CLas-Neg) detectable CLas demonstrated a significant impact on gene expression in young leaves, including on the expression of genes involved in host immunity, stress response, and plant hormone biosynthesis and signaling. CLas-Pos and CLas-Neg expression data displayed distinct patterns. The number of upregulated genes was higher than that of the downregulated genes in CLas-Pos for plant-pathogen interactions, glutathione metabolism, peroxisome, and calcium signaling, which are commonly associated with pathogen infections, compared with the healthy control. On the contrary, the number of upregulated genes was lower than that of the downregulated genes in CLas-Neg for genes involved in plant-pathogen interactions and peroxisome biogenesis/metabolism. Additionally, a time-course quantitative reverse transcription-PCR-based expression analysis visualized the induced expression of companion cell-specific genes, phloem protein 2 genes, and sucrose transport genes in young flushes triggered by CLas. This study advances our understanding of early events during CLas infection of citrus young flushes., Competing Interests: The author(s) declare no conflict of interest.

Citrus huanglongbing (HLB) caused by ' Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. Most commercial citrus cultivars are susceptible to HLB, with a few more tolerant exceptions such as 'LB8-9' Sugar Belle mandarin. Transcriptomic analyses have been widely used to investigate the potential mechanisms for disease susceptibility, resistance, or tolerance. Previous transcriptomic studies related to HLB mostly focused on single time point data collection. We hypothesize that changes in day length and temperature throughout the seasons have profound effects on citrus-CLas interactions. Here, we conducted RNA-seq analyses on HLB-susceptible Valencia sweet orange and HLB-tolerant mandarin 'LB8-9' in winter, spring, summer, and fall. Significant variations in differentially expressed genes (DEGs) related to HLB were observed among the four seasons. For both cultivars, the highest number of DEGs were found in the spring. CLas infection stimulates the expression of immune-related genes such as NBS-LRR, RLK, RLCK, CDPK, MAPK pathway, reactive oxygen species (ROS), and PR genes in both cultivars, consistent with the model that HLB is a pathogen-triggered immune disease. HLB-positive mandarin 'LB8-9' trees contained higher concentrations of maltose and sucrose, which are known to scavenge ROS. In addition, mandarin 'LB8-9' showed higher expression of genes involved in phloem regeneration, which might contribute to its HLB tolerance. This study shed light on the pathogenicity mechanism of the HLB pathosystem and the tolerance mechanism against HLB, providing valuable insights into HLB management.

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A simple method for rapid and portable Huanglongbing (HLB) diagnosis is presented here that combines recombinase polymerase amplification and a fluorescent reporter utilizing the nuclease activity of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a (CRISPR-Cas12a) system. The sensitivity of this technique is much higher than PCR. Furthermore, this method showed similar results to qPCR when leaf samples were used. Compared with conventional CLas detection methods, the detection method presented here can be completed in 90 min and works in an isothermal condition that does not require the use of PCR machines. In addition, the results can be visualized through a handheld fluorescent detection device in the field.

Candidatus Liberibacter spp. are fastidious α-proteobacteria that cause multiple diseases on plant hosts of economic importance, including the most devastating citrus disease: Huanglongbing (HLB). HLB was reported in Asia a century ago but has since spread worldwide. Understanding the pathogenesis of Candidatus Liberibacter spp. remains challenging as they are yet to be cultured in artificial media and infect the phloem, a sophisticated environment that is difficult to manipulate. Despite those challenges, tremendous progress has been made on Ca . Liberibacter pathosystems. Here, we first reviewed recent studies on genetic information of flagellar and type IV pili biosynthesis, their expression profiles, and movement of Ca. Liberibacter spp. inside the plant and insect hosts. Next, we reviewed the transcriptomic, proteomic, and metabolomic studies of susceptible and tolerant plant genotypes to Ca . Liberibacter spp. infection and how Ca . Liberibacter spp. adapt in plants. Analyses of the interactions between plants and Ca . Liberibacter spp. imply the involvement of immune response in the Ca . Liberibacter pathosystems. Lastly, we reviewed how Ca . Liberibacter spp. movement inside and interactions with plants lead to symptom development.

Streptomycin (STR) has been used to control citrus huanglongbing (HLB) caused by ' Candidatus Liberibacter asiaticus' (CLas) via foliar spray. Here, we studied the residue dynamics of STR and its effect on CLas titers in planta applied by foliar spray and trunk injection of 3-year-old citrus trees that were naturally infected by CLas in the field. After foliar spray, STR levels in leaves peaked at 2 to 7 days postapplication (dpa) and gradually declined thereafter. The STR spray did not significantly affect CLas titers in leaves of treated plants as determined by quantitative PCR. After trunk injection, peak levels of STR were observed 7 to 14 dpa in the leaf and root tissues, and near-peak levels were sustained for another 14 days before significantly declining. At 12 months after injection, moderate to low or undetectable levels of STR were observed in the leaf, root, and fruit, depending on the doses of STR injected, with a residue level of 0.28 µg/g in harvested fruit at the highest injection concentration of 2.0 µg/tree. CLas titers in leaves were significantly reduced by trunk injection of STR at 1.0 or 2.0 g/tree, starting from 7 dpa and throughout the experimental period. The reduction of CLas titers was positively correlated with STR residue levels in leaves. The in planta minimum effective concentration of STR needed to suppress the CLas titer to an undetectable level (cycle threshold ≥36.0) was 1.92 µg/g fresh weight. Determination of the in planta minimum effective concentration of STR against CLas and its spatiotemporal residue levels in planta provides the guidance to use STR for HLB management.

' Candidatus Liberibacter asiaticus' (CLas) is the predominant causal agent of citrus huanglongbing, the most devastating citrus disease worldwide. CLas colonizes phloem tissue and causes phloem dysfunction. The pathogen population size in local tissues and in the whole plant is critical for the development of disease symptoms by determining the load of pathogenicity factors and metabolic burden to the host. However, the total population size of CLas in a whole plant and the ratio of CLas to citrus cells in local tissues have not been addressed previously. The total CLas population size for 2.5-year-old 'Valencia' sweet orange on 'Kuharske' citrange rootstock trees was quantified using quantitative PCR to be approximately 1.74 × 10 9 cells/tree, whereas 7- and 20-year-old sweet orange trees were estimated to be 4.3 × 10 10 cells/tree, and 6.0 × 10 10 cells/tree, respectively. The majority of CLas cells were distributed in leaf tissues (55.58%), followed by those in branch (36.78%), feeder root (4.75%), trunk (2.39%), and structural root (0.51%) tissues. The ratios of citrus cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples were within approximately 39 to 79, 44 to 124, 153 to 1,355, 191 to 1,054, and 561 to 3,760, respectively, representing the metabolic burden of CLas in different organs. It was estimated that the ratios of phloem cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples are approximately 0.39 to 0.79, 0.44 to 1.24, 1.53 to 13.55, 1.91 to 10.54, and 5.61 to 37.60, respectively. Approximately 0.01% of the total citrus phloem volume was estimated to be occupied by CLas, explaining the difficulty to observe CLas in most tissues under transmission electron microscopy. The CLas titer inside the leaf was estimated to be approximately 1.64 × 10 6 cells/leaf or 9.2 × 10 4 cells cm -2 in leaves, approximately 10 4 times less than that of typical apoplastic bacterial pathogens. This study provides quantitative estimates of phloem colonization by bacterial pathogens and furthers the understanding of the biology and virulence mechanisms of CLas.

' Candidatus Liberibacter asiaticus' (CLas), the causal agent of citrus huanglongbing (HLB), colonizes inside the phloem and is naturally transmitted by the Asian citrus psyllid (ACP). Here, we investigated spatiotemporal CLas colonization in different tissues after ACP transmission. Of the nine plants successfully infected via ACP transmission, CLas was detected in the roots of all trees at 75 days postremoval of ACPs (DPR) but in the mature leaf of only one tree; this finding is consistent with the model that CLas moves passively from source to sink tissues. At 75 and 365 DPR, CLas was detected in 11.1 and 43.1% of mature leaves not fed on by ACPs during transmission, respectively, unveiling active movement to the source tissue. The difference in colonization timing of sink and source tissues indicates that CLas is capable of both passive and active movement, with passive movement being dominant. At 225 DPR, leaves fed on by ACPs during the young stage showed the highest ratio of HLB symptomatic leaves and the highest CLas titer, followed by leaves that emerged after ACP removal and mature leaves not fed on by ACPs. Importantly, our data showed that ACPs were unable to transmit CLas via feeding on mature leaves. It is estimated that it takes 3 years at most for CLas to infect the whole tree. Overall, spatiotemporal detection of CLas in different tissues after ACP transmission helps visualize the infection process of CLas in planta and subsequent HLB symptom development and provides evidence showing that young leaves should be the focus of HLB management.

Competing Interests: The author declares no competing interest.

Huanglongbing (HLB) or greening currently is the most devastating citrus disease worldwide. The fastidious phloem-colonizing bacterium ' Candidatus Liberibacter asiaticus' (CLas) is the causal agent of citrus HLB in Florida. Bactericides containing the active ingredient oxytetracycline (OTC) have been used in foliar spray to control citrus HLB in Florida since 2016. However, the minimum concentration of OTC required to suppress CLas in planta remains unknown. We developed a new method for evaluating the effects of OTC treatment on CLas titers in infected plants and determined the relationship between OTC residue levels and control levels achieved for CLas using mathematical modeling in greenhouse and field experiments. In both greenhouse and field, OTC spray did not reduce the titers of CLas, and it produced undetectable or mild levels of OTC residue in leaves within 7 days post-application (DPA). In greenhouse, OTC injection at 0.05 g per tree decreased CLas titers to an undetectable level (cycle threshold value ≥ 36.0) from 7 to 30 DPA and produced a residue level of OTC at 0.68 to 0.73 µg/g of fresh tissue over this period. In the field, OTC injection at 0.50 g per tree resulted in the decline of CLas titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27 to 0.33 µg/g of fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for CLas achieved. The results suggested that the minimum concentrations of OTC required to suppress CLas populations in planta to below the detection limit are 0.68 and 0.86 µg/g and that the minimum concentrations of OTC required for initial inhibition of CLas growth in planta are ∼0.17 and ∼0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target CLas in infected plants for effective control of citrus HLB.

Citrus huanglongbing (HLB) is one of the most destructive diseases affecting citrus plants. ' Candidatus Liberibacter asiaticus', an uncultivated α-proteobacteria, is the most widely spread causal agent of HLB and is transmitted by the Asian citrus psyllid Diaphorina citri . ' Ca . L. asiaticus' attachment to the psyllid midgut is believed to be critical to further infect other organs, including the salivary gland. In this study, the type IVc tight adherence (Tad) pilus locus encoded by ' Ca. L. asiaticus' was characterized. The Tad loci are conserved among members of Rhizobiaceae, including ' Ca. L. asiaticus' and Agrobacterium spp. Ectopic expression of the ' Ca. L. asiaticus' cpaF gene, an ATPase essential for the biogenesis and secretion of the Tad pilus, restored the adherence phenotype in cpaF mutant of A. tumefaciens , indicating CpaF of ' Ca. L. asiaticus' was functional and critical for bacterial adherence mediated by Tad pilus. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that ' Ca. L. asiaticus' Tad pilus-encoding genes and ' Ca. L. asiaticus' pilin gene flp3 were upregulated in psyllids compared with in planta. A bacterial one-hybrid assay showed that ' Ca. L. asiaticus' VisN and VisR, members of the LuxR transcriptional factor family, were bound to the flp3 promoter. VisNR regulate flp3 . Negative regulation of the flp3 promoter by both VisN and VisR was demonstrated using a shuttle strategy, with analysis of the phenotypes and immunoblotting together with quantification of the expression of the flp3 promoter fused to the β-galactosidase reporter gene. Comparative expression analysis confirmed that ' Ca. L. asiaticus' visNR was less expressed in the psyllid than in the plant host. Further, motility and biofilm phenotypes of the visNR mutant of A. tumefaciens were fully complemented by expressing ' Ca. L. asiaticus' visNR together. The physical interaction between VisN and VisR was confirmed by pull-down and stability assays. The interaction of the flp3 promoter with VisR was verified by electrophoretic mobility shift assay. Taken together, the results revealed the contribution of the Tad pilus apparatus in the colonization of the insect vector by ' Ca. L. asiaticus' and shed light on the involvement of VisNR in regulation of the Tad locus.

Citrus Huanglongbing (HLB) is the most severe disease of citrus plants caused by ' Candidatus Liberibacter asiaticus' and transmitted by the insect vector Asian citrus psyllid (ACP). No effective curative measure is available against HLB. For citrus production areas without HLB or with low HLB disease incidence, removal of ' Ca. L. asiaticus' inoculum is critical to prevent HLB spread. Such a strategy requires robust early diagnosis of HLB for inoculum removal to prevent ACP acquisition and transmission of ' Ca. L. asiaticus'. However, early diagnosis of HLB is challenging, because the citrus trees remain asymptomatic for several months to years after ' Ca. L. asiaticus' transmission by ACP. In this study, we report a new method for targeted early detection of ' Ca. L. asiaticus' in cultivar Valencia sweet orange ( Citrus sinensis ) before HLB symptom expression. We take advantage of the fact that ' Ca. L. asiaticus' remains around the ACP feeding site immediately after transmission into the young flush and before flush maturation. ACPs secrete salivary sheaths at their feeding sites, which can be visualized using Coomassie brilliant blue staining owing to the presence of salivary sheaths secreted by ACP. Epifluorescence and confocal microscopy indicate the presence of salivary sheaths beneath the blue spots on ACP-fed leaves. Quantitative real-time polymerase chain reaction (PCR) and conventional PCR assays are able to detect ' Ca. L. asiaticus' in the ACP feeding surrounding areas as early as 2 to 20 days after ACP feeding. This finding lays a foundation to develop much-needed tools for early diagnosis of HLB before symptom expression, thus assisting ' Ca. L. asiaticus' inoculum removal and preventing HLB from spreading.

Citrus Huanglongbing (HLB), also known as greening, is a destructive disease caused by the fastidious, phloem-colonizing bacteria Candidatus Liberibacter spp.; 'Ca. Liberibacter asiaticus' (Las) is the most prevalent of the species causing HLB. The Asian citrus psyllid (ACP, Diaphorina citri) transmits Las. HLB is threatening citrus production worldwide, and there is no cure for infected trees. Management strategies targeting diseased trees at different stages of colonization by Las are needed for sustainable citrus production in HLB-endemic regions. We evaluated the effect of the combinations of plant defense elicitors, nitrogen (N) fertilizer, and compost on mildly diseased trees. We tested thermotherapy on severely diseased trees and assessed tree protectors to prevent feeding by ACP, thus preventing Las from being transmitted to new plantings that replaced HLB-moribund trees. After four applications over two consecutive growing seasons we found that the combination of compost, urea, and plant defense elicitors β-aminobutyric acid, plus ascorbic acid and potassium phosphite with or without salicylic acid, slowed down the progression of HLB and reduced disease severity by approximately 18%, compared with the untreated control. Our data showed no decline in fruit yield, indeed treatment resulted in a higher yield compared with the untreated control. Thermotherapy treatment (55°C for 2 min) exhibited a suppressive effect on growth of Las and progress of HLB in severely diseased trees for 2 to 3 months after treatment. The tree protectors prevented feeding by ACP, and therefore young replant trees remained healthy and free from infection by Las over the 2-year duration of the experiment. Taken together, these results may contribute to a basis for developing a targeted approach to control HLB based on stage of host colonization, application of plant defense elicitors, N fertilizer, compost, thermotherapy, and tree protectors. There is potential to implement these strategies in conjunction with other disease control measures to contribute to sustainable citrus production in HLB-endemic regions.

The citrus industry is facing an unprecedented challenge from Huanglongbing (HLB). All cultivars can be affected by the HLB-associated bacterium 'Candidatus Liberibacter asiaticus' (CLas) and there is no known resistance. Insight into HLB pathogenesis is urgently needed in order to develop effective management strategies. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all CLas isolates, as a molecular probe to understand CLas virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in CLas-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1-expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants.

"Candidatus Liberibacter" species are associated with economically devastating diseases of citrus, potato, and many other crops. The importance of these diseases as well as the proliferation of new diseases on a wider host range is likely to increase as the insects vectoring the "Ca. Liberibacter" species expand their territories worldwide. Here, we review the progress on understanding pathogenesis mechanisms of "Ca. Liberibacter" species and the control approaches for diseases they cause. We discuss the Liberibacter virulence traits, including secretion systems, putative effectors, and lipopolysaccharides (LPSs), as well as other important traits likely to contribute to disease development, e.g., flagella, prophages, and salicylic acid hydroxylase. The pathogenesis mechanisms of Liberibacters are discussed. Liberibacters secrete Sec-dependent effectors (SDEs) or other virulence factors into the phloem elements or companion cells to interfere with host targets (e.g., proteins or genes), which cause cell death, necrosis, or other phenotypes of phloem elements or companion cells, leading to localized cell responses and systemic malfunction of phloem. Receptors on the remaining organelles in the phloem, such as plastid, vacuole, mitochondrion, or endoplasmic reticulum, interact with secreted SDEs and/or other virulence factors secreted or located on the Liberibacter outer membrane to trigger cell responses. Some of the host genes or proteins targeted by SDEs or other virulence factors of Liberibacters serve as susceptibility genes that facilitate compatibility (e.g., promoting pathogen growth or suppressing immune responses) or disease development. In addition, Liberibacters trigger plant immunity response via pathogen-associated molecular patterns (PAMPs, such as lipopolysaccharides), which leads to premature cell death, callose deposition, or phloem protein accumulation, causing a localized response and/or systemic effect on phloem transportation. Physical presence of Liberibacters and their metabolic activities may disturb the function of phloem, via disrupting osmotic gradients, or the integrity of phloem conductivity. We also review disease management strategies, including promising new technologies. Citrus production in the presence of Huanglongbing is possible if the most promising management approaches are integrated. HLB management is discussed in the context of local, area-wide, and regional Huanglongbing/Asian Citrus Psyllid epidemiological zones. For zebra chip disease control, aggressive psyllid management enables potato production, although insecticide resistance is becoming an issue. Meanwhile, new technologies such as clustered regularly interspaced short palindromic repeat (CRISPR)-derived genome editing provide an unprecedented opportunity to provide long-term solutions.

Pathogens from the fastidious, phloem-restricted 'Candidatus Liberibacter' species cause the devastating Huanglongbing (HLB) disease in citrus worldwide and cause diseases on many solanaceous crops and plants in the Apiaceae family. However, little is known about the pathogenic mechanisms due to the difficulty in culturing the corresponding 'Ca. Liberibacter' species. Here, we report that the citrus HLB pathogen 'Ca. L. asiaticus' uses an active salicylate hydroxylase SahA to degrade salicylic acid (SA) and suppress plant defenses. Purified SahA protein displays strong enzymatic activity to degrade SA and its derivatives. Overexpression of SahA in transgenic tobacco plants abolishes SA accumulation and hypersensitive response (HR) induced by nonhost pathogen infection. By degrading SA, 'Ca. L. asiaticus' not only enhances the susceptibility of citrus plants to both nonpathogenic and pathogenic Xanthomonas citri but also attenuates the responses of citrus plants to exogenous SA. In addition, foliar spraying of 2,1,3-benzothiadiazole and 2,6-dichloroisonicotinic acid, SA functional analogs not degradable by SahA, displays comparable (and even better) effectiveness with SA in suppressing 'Ca. L. asiaticus' population growth and HLB disease progression in infected citrus trees under field conditions. This study demonstrates one or more pathogens suppress plant defenses by degrading SA and establish clues for developing novel SA derivatives-based management approaches to control the associated plant diseases.

The Huanglongbing (HLB) disease pyramid is composed of Liberibacters, psyllid vectors, citrus hosts, and the environment. The epidemiological outcomes for Liberibacter-associated plant diseases are collectively determined by the inherent relationships among plant-Liberibacters-psyllids, and how various environmental factors affect plant-Liberibacter-psyllid interactions. Citrus-Liberibacter-psyllid interactions occur in a complex microbiome system. In this review, we focus on the progress in understanding the HLB disease pyramid, and how the microbiome affects the HLB disease pyramid including the interaction between HLB and the citrus microbiome; the interaction between Liberibacters and psyllids; the interaction between Liberibacters and gut microbiota in psyllids; and the effect of HLB on selected above- and belowground citrus pathogens. Their implications for HLB management are also discussed.

Citrus huanglongbing (HLB) or greening is a devastating bacterial disease that has destroyed millions of trees and is associated with phloem-residing 'Candidatus Liberibacter asiaticus' (Las) in Florida. In this study, we evaluated the spatiotemporal dynamics of oxytetracycline in planta and its control effect against HLB via trunk injection. Las-infected 'Hamlin' sweet orange trees on 'Swingle' citrumelo rootstock at the early stage of decline were treated with oxytetracycline hydrochloride (OTC) using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by high-performance liquid chromatography method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root system was achieved 2 days postinjection. High levels of OTC (>850 µg/kg) were maintained in leaf and root for at least 1 month and moderate OTC (>500 µg/kg) persisted for more than 9 months. Reduction of Las populations in root system and leaves of OTC-treated trees were over 95% and 99% (i.e., 1.76 and 2.19 log reduction) between 2 and 28 days postinjection. Conditions of trees receiving OTC treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB.

Background: Huanglongbing (HLB) or citrus greening is a devastating disease of citrus. The gram-negative bacterium Candidatus Liberibacter asiaticus (Las) belonging to the α-proteobacteria is responsible for HLB in North America as well as in Asia. Currently, there is no cure for this disease. Early detection and quarantine of Las-infected trees are important management strategies used to prevent HLB from invading HLB-free citrus producing regions. Quantitative real-time PCR (qRT-PCR) based molecular diagnostic assays have been routinely used in the detection and diagnosis of Las. The oligonucleotide primer pairs based on conserved genes or regions, which include 16S rDNA and the β-operon, have been widely employed in the detection of Las by qRT-PCR. The availability of whole genome sequence of Las now allows the design of primers beyond the conserved regions for the detection of Las explicitly., Results: We took a complimentary approach by systematically screening the genes in a genome-wide fashion, to identify the unique signatures that are only present in Las by an exhaustive sequence based similarity search against the nucleotide sequence database. Our search resulted in 34 probable unique signatures. Furthermore, by designing the primer pair specific to the identified signatures, we showed that most of our primer sets are able to detect Las from the infected plant and psyllid materials collected from the USA and China by qRT-PCR. Overall, 18 primer pairs of the 34 are found to be highly specific to Las with no cross reactivity to the closely related species Ca. L. americanus (Lam) and Ca. L. africanus (Laf)., Conclusions: We have designed qRT-PCR primers based on Las specific genes. Among them, 18 are suitable for the detection of Las from Las-infected plant and psyllid samples. The repertoire of primers that we have developed and characterized in this study enhanced the qRT-PCR based molecular diagnosis of HLB.

Citrus huanglongbing (HLB) is one of the oldest citrus diseases and has been known for over a century. HLB is caused by 'Candidatus Liberibacter' spp. that are phloem-limited, fastidious α-proteobacteria and infect hosts in different Kingdoms (i.e., Animalia and Plantae). When compared with well-characterized, cultivatable plant-pathogenic Gram-negative bacteria, the interactions of uncultured insect-vectored plant-pathogenic bacteria, including 'Ca. Liberibacter' spp., with their hosts remain poorly understood. 'Ca. Liberibacter' spp. have been known to cause HLB, which has been rapidly spreading worldwide, resulting in dramatic economic losses. HLB presents an unprecedented challenge to citrus production. In this review, we focus on the most recent research on citrus, 'Candidatus Liberibacter asiaticus', and psyllid interactions, specifically considering the following topics: evolutionary relationships among 'Ca. Liberibacter' spp., genetic diversity, host range, genome analysis, transmission, virulence mechanisms, and the ecological importance of HLB. Currently, no efficient management strategy is available to control HLB, although some promising progress has been made. Further studies are needed to understand citrus, 'Ca. L. asiaticus', and psyllid interactions to design innovative management strategies. Although HLB has been problematic for over a century, we can only win the battle against HLB with a coordinated and deliberate effort by the citrus industry, citrus growers, researchers, legislatures, and governments.

Huanglongbing (HLB) or citrus greening disease is a destructive disease of citrus worldwide, which is associated with Candidatus Liberibacter asiaticus. This phloem-limited fastidious pathogen is transmitted by the Asian citrus psyllid, Diaphorina citri, and appears to be an intracellular pathogen that maintains an intimate association with the psyllid or the plant throughout its life cycle. The molecular basis of the interaction of this pathogen with its hosts is not well understood. We hypothesized that, during infection, Ca. L. asiaticus differentially expresses the genes critical for its survival and/or pathogenicity in either host. To test this hypothesis, quantitative reverse transcription-polymerase chain reaction was performed to compare the gene expression of Ca. L. asiaticus in planta and in psyllid. Overall, 381 genes were analysed for their gene expression in planta and in psyllid. Among them, 182 genes were up-regulated in planta compared with in psyllid (P < 0.05), 16 genes were up-regulated in psyllid (P < 0.05) and 183 genes showed no statistically significant difference (P ≥ 0.05) in expression between in planta and in psyllid. Our study indicates that the expression of the Ca. L. asiaticus genes involved in transcriptional regulation, transport system, secretion system, flagella assembly, metabolic pathway and stress resistance are changed significantly in a host-specific manner to adapt to the distinct environments of plant and insect. To our knowledge, this is the first large-scale study to evaluate the differential expression of Ca. L. asiaticus genes in a plant host and its insect vector., (© 2013 BSPP AND BLACKWELL PUBLISHING LTD.)

Huanglongbing is the most devastating disease of citrus caused by Candidatus Liberibacter asiaticus (Las). In the present study, we report the discovery of novel small molecule inhibitors against SecA ATPase of Las by using structure based design methods. We built the homology model of SecA protein structure of Las based on the SecA of Escherichia coli. The model was used for in-silico screening of commercially available compounds from ZINC database. Using the glide flexible molecular docking method, twenty structures were chosen for in vitro studies. Five compounds were found to inhibit the ATPase activity of SecA of Las at nano molar concentrations and showed antimicrobial activities against Agrobacterium tumefaciens with MBC ranging from 128 to 256 μg/mL. These compounds appear to be suitable as lead compounds for further development of antimicrobial compounds against Las., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Candidatus Liberibacter asiaticus is the causal agent of Huanglongbing (HLB) disease of citrus. Current management practices have not been able to control HLB and stop the spread of HLB. The current study is focused on screening small molecule inhibitors against SecA protein of Ca. L. asiaticus. Homology modeling, structure based virtual screening and molecular docking methods have been used to find the novel inhibitory compounds against SecA activity at ATP binding region. At 20μm 17 compounds showed >50% inhibition and four compounds had more than 65% inhibition. The most active compound has IC(50) value of 2.5μM. The differences between the activities of the compounds are explained by their inter-molecular interactions at ATP binding site., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Citrus greening or huanglongbing (HLB) is a devastating disease of citrus. HLB is associated with the phloem-limited fastidious prokaryotic alpha-proteobacterium 'Candidatus Liberibacter spp.' In this report, we used sweet orange (Citrus sinensis) leaf tissue infected with 'Ca. Liberibacter asiaticus' and compared this with healthy controls. Investigation of the host response was examined with citrus microarray hybridization based on 33,879 expressed sequence tag sequences from several citrus species and hybrids. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes whose encoded proteins were categorized according to function. The categories included genes associated with sugar metabolism, plant defense, phytohormone, and cell wall metabolism, as well as 14 other gene categories. The anatomical analyses indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. The up-regulation of three key starch biosynthetic genes including ADP-glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB-affected leaves. The HLB-associated phloem blockage resulted from the plugged sieve pores rather than the HLB bacterial aggregates since 'Ca. Liberibacter asiaticus' does not form aggregate in citrus. The up-regulation of pp2 gene is related to callose deposition to plug the sieve pores in HLB-affected plants.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic alpha-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of 'Candidatus Liberibacter asiaticus,' respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that 'Ca. Liberibacter asiaticus' was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/mug of total DNA in different tissues. A relatively high concentration of 'Ca. Liberibacter asiaticus' was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that 'Ca. Liberibacter asiaticus' was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.

'Candidatus Liberibacter' species are insect-transmitted, phloem-limited α-Proteobacteria in the order of Rhizobiales. The citrus industry is facing significant challenges due to huanglongbing, associated with infection from 'Candidatus Liberibacter asiaticus' (Las). In order to gain greater insight into 'Ca. Liberibacter' biology and genetic diversity, we have performed genome sequencing and comparative analyses of diverse 'Ca. Liberibacter' species, including those that can infect citrus. Our phylogenetic analysis differentiates 'Ca. Liberibacter' species and Rhizobiales in separate clades and suggests stepwise evolution from a common ancestor splitting first into nonpathogenic Liberibacter crescens followed by diversification of pathogenic 'Ca. Liberibacter' species. Further analysis of Las genomes from different geographical locations revealed diversity among isolates from the United States. Our phylogenetic study also indicates multiple Las introduction events in California and spread of the pathogen from Florida to Texas. Texan Las isolates were closely related, while Florida and Asian isolates exhibited the most genetic variation. We have identified conserved Sec translocon (SEC)-dependent effectors likely involved in bacterial survival and virulence of Las and analysed their expression in their plant host (citrus) and insect vector (Diaphorina citri). Individual SEC-dependent effectors exhibited differential expression patterns between host and vector, indicating that Las uses its effector repertoire to differentially modulate diverse organisms. Collectively, this work provides insights into the evolution of 'Ca. Liberibacter' species, the introduction of Las in the United States and identifies promising Las targets for disease management.

Summary: Citrus huanglongbing (HLB) is the most devastating citrus disease worldwide. 'Candidatus Liberibacter asiaticus' (Las) is the most prevalent HLB causal agent that is yet to be cultured. Here, we analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin‐encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin‐encoding genes. Only flgJAtu but not flgJLas restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull‐down assays demonstrated that FlgJLas interacts with FlgB but not with FliE. Ectopic expression of flaALas in A. tumefaciens mutants restored the swimming motility of ∆flaA mutant and ∆flaAD mutant, but not that of the null mutant ∆flaABCD. No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin‐specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control. [ABSTRACT FROM AUTHOR]