CLas Infection Research Articles

Spatial chemistry of citrus reveals molecules bactericidal to Candidatus Liberibacter asiaticus

Huanglongbing (HLB), associated with the psyllid-vectored phloem-limited bacterium, Candidatus Liberibacter asiaticus (CLas), is a disease threat to all citrus production worldwide. Currently, there are no sustainable curative or prophylactic treatments available. In this study, we utilized mass spectrometry (MS)-based metabolomics in combination with 3D molecular mapping to visualize complex chemistries within plant tissues to explore how these chemistries change in vivo in HLB-infected trees. We demonstrate how spatial information from molecular maps of branches and single leaves yields insight into the biology not accessible otherwise. In particular, we found evidence that flavonoid biosynthesis is disrupted in HLB-infected trees, and an increase in the polyamine, feruloylputrescine, is highly correlated with an increase in disease severity. Based on mechanistic details revealed by these molecular maps, followed by metabolic modeling, we formulated and tested the hypothesis that CLas infection either directly or indirectly converts the precursor compound, ferulic acid, to feruloylputrescine to suppress the antimicrobial effects of ferulic acid and biosynthetically downstream flavonoids. Using in vitro bioassays, we demonstrated that ferulic acid and bioflavonoids are indeed highly bactericidal to CLas, with the activity on par with a reference antibiotic, oxytetracycline, recently approved for HLB management. We propose these compounds should be evaluated as therapeutics alternatives to the antibiotics for HLB treatment. Overall, the utilized 3D metabolic mapping approach provides a promising methodological framework to identify pathogen-specific inhibitory compounds in planta for potential prophylactic or therapeutic applications.

Effector CLas0185 targets methionine sulphoxide reductase B1 of Citrus sinensis to promote multiplication of 'Candidatus Liberibacter asiaticus' via enhancing enzymatic activity of ascorbate peroxidase 1.

Citrus huanglongbing (HLB) has been causing enormous damage to the global citrus industry. As the main causal agent, 'Candidatus Liberibacter asiaticus' (CLas) delivers a set of effectors to modulate host responses, while the modes of action adopted remain largely unclear. Here, we demonstrated that CLIBASIA_00185 (CLas0185) could attenuate reactive oxygen species (ROS)-mediated cell death in Nicotiana benthamiana. Transgenic expression of CLas0185 in Citrus sinensis 'Wanjincheng' enhanced plant susceptibility to CLas. We found that methionine sulphoxide reductase B1 (CsMsrB1) was targeted by the effector, and its abundance was elevated in CLas0185-transgenic citrus plants. Their interaction promoted CLas proliferation. We then determined that CsMsrB1 sustained redox state and enzymatic activity of ascorbate peroxidase 1 (CsAPX1) under oxidative stress. The latter reduced H2O2 accumulation and was associated with host susceptibility to CLas infection. Consistently, citrus plants expressing CLas0185 and CsMsrB1 conferred enhanced APX activity and decreased H2O2 content. Taken together, these findings revealed how CLas0185 benefits CLas colonization by targeting CsMsrB1, which facilitated the antioxidant activity and depressed ROS during pathogen infection.

Individual Protective Covers Improve Yield and Quality of Citrus Fruit under Endemic Huanglongbing.

The use of individual protective covers (IPCs) to protect newly planted citrus trees from Huanglongbing (HLB) infection is being widely adopted in Florida, an HLB-endemic citrus-producing area. It is known that IPCs positively influence most horticultural traits, increasing tree growth, flush expansion, and leaf size, enabling trees to sustain balanced carbohydrate metabolism by preventing Candidatus Liberibacter asiaticus (CLas) infection, and inducing higher leaf chlorophyll levels. This may result in more productive trees. However, as the tree grows, IPCs eventually are removed, typically between 2 and 3 years after their initial installation. Once IPCs are removed, trees become exposed to the Asian citrus psyllid (ACPs) and ultimately become infected. In this work, we covered Valencia sweet orange trees with IPCs for 30 months, until the trees entered fruit-bearing age. We investigated how the IPC protection of newly planted trees for 30 months influenced the fruit quality and yield of "Valencia" trees for three consecutive seasons after IPC removal compared to non-covered trees. The use of IPCs kick-started the newly planted citrus trees, resulting in higher yields and fruits with better internal and external quality. After 30 months of IPC protection, tree canopies were larger and denser, supporting more fruit per tree than non-protected trees for three consecutive seasons, even though by the end of the first season after IPC removal, the trees were HLB-positive. Tree height, scion diameter, canopy volume, and leaf area were significantly improved compared to non-covered trees. Additionally, fruit quality was significantly improved in the three seasons following IPC removal compared to non-covered trees. However, a decline in quality was measurable in fruit from IPC trees after the second harvesting season, with trees affected by HLB. Based on the results from this study, we conclude that the benefits from IPC protection may last for at least three consecutive seasons once trees enter the productive age, despite CLas infection within 12 months after IPC removal.

Adipokinetic hormone signaling mediates the enhanced fecundity of Diaphorina citri infected by ‘Candidatus Liberibacter asiaticus’

Diaphorina citri serves as the primary vector for ‘Candidatus Liberibacter asiaticus (CLas),’ the bacterium associated with the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts and require extra energy expenditure. Therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. In this study, we found adipokinetic hormone (DcAKH) and its receptor (DcAKHR) were essential for increasing lipid metabolism and fecundity in response to CLas infection in D. citri. Knockdown of DcAKH and DcAKHR not only resulted in the accumulation of triacylglycerol and a decline of glycogen, but also significantly decreased fecundity and CLas titer in ovaries. Combined in vivo and in vitro experiments showed that miR-34 suppresses DcAKHR expression by binding to its 3’ untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and CLas titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes. Additionally, knockdown of DcAKH and DcAKHR significantly reduced juvenile hormone (JH) titer and JH signaling pathway genes in fat bodies and ovaries, including the JH receptor, methoprene-tolerant (DcMet), and the transcription factor, Krüppel homolog 1 (DcKr-h1), that acts downstream of it, as well as the egg development related genes vitellogenin 1-like (DcVg-1-like), vitellogenin A1-like (DcVg-A1-like) and the vitellogenin receptor (DcVgR). As a result, CLas hijacks AKH/AKHR-miR-34-JH signaling to improve D. citri lipid metabolism and fecundity, while simultaneously increasing the replication of CLas, suggesting a mutualistic interaction between CLas and D. citri ovaries.

Adipokinetic hormone signaling mediates the enhanced fecundity of Diaphorina citri infected by 'Candidatus Liberibacter asiaticus'.

Diaphorina citri serves as the primary vector for 'Candidatus Liberibacter asiaticus (CLas),' the bacterium associated with the severe Asian form of huanglongbing. CLas-positive D. citri are more fecund than their CLas-negative counterparts and require extra energy expenditure. Therefore, understanding the molecular mechanisms linking metabolism and reproduction is of particular importance. In this study, we found adipokinetic hormone (DcAKH) and its receptor (DcAKHR) were essential for increasing lipid metabolism and fecundity in response to CLas infection in D. citri. Knockdown of DcAKH and DcAKHR not only resulted in the accumulation of triacylglycerol and a decline of glycogen, but also significantly decreased fecundity and CLas titer in ovaries. Combined in vivo and in vitro experiments showed that miR-34 suppresses DcAKHR expression by binding to its 3' untranslated region, whilst overexpression of miR-34 resulted in a decline of DcAKHR expression and CLas titer in ovaries and caused defects that mimicked DcAKHR knockdown phenotypes. Additionally, knockdown of DcAKH and DcAKHR significantly reduced juvenile hormone (JH) titer and JH signaling pathway genes in fat bodies and ovaries, including the JH receptor, methoprene-tolerant (DcMet), and the transcription factor, Krüppel homolog 1 (DcKr-h1), that acts downstream of it, as well as the egg development related genes vitellogenin 1-like (DcVg-1-like), vitellogenin A1-like (DcVg-A1-like) and the vitellogenin receptor (DcVgR). As a result, CLas hijacks AKH/AKHR-miR-34-JH signaling to improve D. citri lipid metabolism and fecundity, while simultaneously increasing the replication of CLas, suggesting a mutualistic interaction between CLas and D. citri ovaries.

Identification and analysis of bZIP family genes in Citrus sinensis and the role of CsbZIP24 in response to Huanglongbing

Citrus Huanglongbing (HLB) is one of the most destructive citrus diseases worldwide, mainly caused by ‘Candidatus Liberibacter asiaticus’ (CLas). HLB plants commonly present zinc deficiency chlorosis, which is evidenced with decreased zinc content and upregulation of genes encoding zinc transport proteins (ZIPs). However, the mechanism of how ZIPs are activated in response to CLas infection is unknown. Herein, the phylogenetic analysis was done for ZIPs and its potential upstream regulator bZIPs on the genome-wide level. Fifty-six bZIP transcription factor members classified into thirteen groups and twelve ZIP members were identified from C. sinensis. CsbZIP23 and CsbZIP24 harboring Zinc-sensor motif (ZSM) were characterized as the homologues of Arabidopsis thaliana bZIP19/23 in regulation of zinc homeostasis, and both were highly expressed in leaves compared to stems and roots. CsbZIP24 was further demonstrated in activation of CsZIP3 gene expression by binding to its promoter containing Zinc Deficiency Response Elements (ZDRE) rather than CsbZIP23. Additionally, the induction of both CsbZIP24 and CsZIP3 was observed in HLB diseased plants. Taken together, zinc sensor members were firstly identified in citrus and CsbZIP24 was proposed as the key factor in regulation of CsZIP3 gene expression to accommodate zinc-deficient conditions that caused by CLas colonization.

Comparative Transcriptome and sRNAome Analysis Suggest Coordinated Citrus Immune Responses against Huanglongbing Disease.

Citrus Huanglongbing (HLB), caused by the phloem-inhibiting bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease, intimidating citrus production worldwide. Although commercially cultivated citrus cultivars are vulnerable to CLas infection, HLB-tolerant attributes have, however, been observed in certain citrus varieties, suggesting a possible pathway for identifying innate defense regulators that mitigate HLB. By adopting transcriptome and small RNAome analysis, the current study compares the responses of HLB-tolerant lemon (Citrus limon L.) with HLB-susceptible Shatangju mandarin (Citrus reticulata Blanco cv. Shatangju) against CLas infection. Transcriptome analysis revealed significant differences in gene expression between lemon and Shatangju. A total of 1751 and 3076 significantly differentially expressed genes were identified in Shatangju and lemon, respectively. Specifically, CLas infected lemon tissues demonstrated higher expressions of genes involved in antioxidant enzyme activity, protein phosphorylation, carbohydrate, cell wall, and lipid metabolism than Shatangju. Wet-lab experiments further validated these findings, demonstrating increased antioxidant enzyme activity in lemon: APX (35%), SOD (30%), and CAT (64%) than Shatangju. Conversely, Shatangju plants exhibited higher levels of oxidative stress markers like H2O2 (44.5%) and MDA content (65.2%), alongside pronounced ion leakage (11.85%), than lemon. Moreover, microscopic investigations revealed that CLas infected Shatangju phloem exhibits significantly more starch and callose accumulation than lemon. Furthermore, comparative sRNA profiles revealed the potential defensive regulators for HLB tolerance. In Shatangju, increased expression of csi-miR166 suppresses the expression of disease-resistant proteins, leading to inadequate defense against CLas. Conversely, reduced expression of csi-miR166 in lemon plants enables them to combat HLB by activating disease-resistance proteins. The above findings indicate that when infected with CLas, lemon exhibits stronger antioxidative activity and higher expression of disease-resistant genes, contributing to its enhanced tolerance to HLB. In contrast, Shatangju shows lower antioxidative activity, reduced expression of disease-resistant genes, significant ion leakage, and extensive callose deposition, possibly related to damage to plant cell structure and blockage of phloem sieve tubes, thereby promoting the development of HLB symptoms.

The beta pore-forming bacterial pesticidal protein Tpp78Aa1 is toxic to the Asian citrus psyllid vector of the citrus greening bacterium

The Asian citrus psyllid (ACP) Diaphorina citri transmits the causative agent of huanglongbing, or citrus greening disease, that has decimated global citrus production. Pesticidal proteins derived from bacteria such as Bacillus thuringiensis (Bt) can provide effective and environmentally friendly alternatives for management of D. citri, but few with sufficient toxicity to D. citri have been identified. Here, we report on the toxicity of 14 Bt-derived pesticidal proteins from five different structural groups against D. citri. These proteins were selected based on previously reported toxicity to other hemipteran species and on pesticidal protein availability. Most of the proteins were expressed in Escherichia coli and purified from inclusion bodies or His-tag affinity purification, while App6Aa2 was expressed in Bt and purified from spore/crystal mixtures. Pesticidal proteins were initially screened by feeding psyllids on a single dose, and lethal concentration (LC50) then determined for proteins with significantly greater mortality than the buffer control. The impact of CLas infection of D. citri on toxicity was assessed for selected proteins via topical feeding. The Bt protein Tpp78Aa1 was toxic to D. citri adults with an LC50 of approximately 204 µg/mL. Nymphs were more susceptible to Tpp78Aa1 than adults but no significant difference in susceptibility was observed between healthy and CLas-infected nymphs or adults. Tpp78Aa1 and other reported D. citri-active proteins may provide valuable tools for suppression of D. citri populations.

Root System Reductions of Grafted ‘Valencia’ Orange Trees Are More Extensive Than Aboveground Reductions after Natural Infection with Candidatus Liberibacter Asiaticus

Huanglongbing (HLB), which is associated with the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), is a devastating disease that affects citrus trees worldwide. Because of the pervasiveness of the bacteria and psyllid vector, the disease is considered endemic in Florida. Although the effects of CLas on tree growth and physiology have been investigated for decades, most studies compared infected and noninfected trees under greenhouse conditions. This study used newly planted field-grown ‘Valencia’ sweet orange (Citrus sinensis) trees on two different rootstocks to monitor the distribution and accumulation of CLas in aboveground and belowground tissues following natural psyllid colonization and assess tree physiological responses and biomass reductions under HLB-endemic conditions. Trees were transplanted into the field with individual protective covers (IPCs), which are used to exclude psyllids and prevent infection. Openings were cut in the IPCs of half of the trees; to promote infection, these IPCs were temporarily removed during the main vegetative flushing period when psyllid populations were high. All trees that were exposed to psyllids became infected and displayed the symptoms typically associated with HLB. Throughout the study, higher levels of CLas were detected in the leaves compared with those in the fibrous roots. Trees that were not exposed to psyllids remained noninfected and healthy. After 18 months, a subset of trees was excavated to assess biomass differences between infected and noninfected trees. Infected trees had root system reductions of 37% and shoot system reductions of 20%, thereby significantly reducing the belowground-to-aboveground biomass ratio. Fibrous root loss was 49% and more severe than the loss of the rest of the root tissue. This study is the first to demonstrate the full extent of damage caused by CLas infection under natural HLB-endemic conditions. The results confirm previous observations that suggested fibrous root loss as one of the major consequences of infection and colonization with CLas. They also reinforce the benefits of using IPCs to prevent infection of young citrus trees during the first years of growth in the field.

Transcriptomic response of citrus psyllid salivary glands to the infection of citrus Huanglongbing pathogen.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the key vector insect transmitting the Candidatus Liberibacter asiaticus (CLas) bacterium that causes the devastating citrus greening disease (Huanglongbing, HLB) worldwide. The D. citri salivary glands (SG) exhibit an important barrier against the transmission of HLB pathogen. However, knowledge on the molecular mechanism of SG defence against CLas infection is still limited. In the present study, we compared the SG transcriptomic response of CLas-free and CLas-infected D. citri using an illumine paired-end RNA sequencing. In total of 861 differentially expressed genes (DEGs) in the SG upon CLas infection, including 202 upregulated DEGs and 659 downregulated DEGs were identified. Functional annotation analysis showed that most of the DEGs were associated with cellular processes, metabolic processes, and the immune response. Gene ontology and Kyoto Encyclopaedia of Genes and Genomes enrichment analyses revealed that these DEGs were enriched in pathways involving carbohydrate metabolism, amino acid metabolism, the immune system, the digestive system, the lysosome, and endocytosis. A total of 16 DEGs were randomly selected to further validate the accuracy of RNA-Seq dataset by reverse-transcription quantitative polymerase chain reaction. This study provides substantial transcriptomic information regarding the SG of D. citri in response to CLas infection, which may shed light on the molecular interaction between D. citri and CLas, and provides new ideas for the prevention and control of citrus psyllid.

Integrated bacterial transcriptome and host metabolome analysis reveals insights into "Candidatus Liberibacter asiaticus" population dynamics in the fruit pith of three citrus cultivars with different tolerance.

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

Vesicular transport-related genes in Diaphorina citri are involved in the process of Candidatus Liberibacter asiaticus infection

Asian citrus psyllid (ACP, Diaphorina citri) is the major vector of Candidatus Liberibacter asiaticus (CLas), which is a bacterial pathogen causing the devastating citrus Huanglongbing (HLB) disease. Diaphorina citri is known to carry CLas in a persistent and propagative manner. Some studies have suggested that CLas may use the vesicular structures of D. citri cells as its propagation organelles. However, the mechanisms by which CLas enters the D. citri cells and how vesicle-mediated trafficking is involved remain unclear. In this study, we monitored the titer change of CLas in D. citri nymphs during the process of CLas acquisition from feeding on infected citrus plants. We found that the titer of CLas increased with the acquisition access period. After infection, there was a significant upregulation in the expression of several vesicular transport-related genes in D. citri. The titer of CLas was significantly reduced in the midgut and whole insect body when endocytosis and the endosome network in D. citri were inhibited. Furthermore, silencing the D. citri clathrin-heavy chain gene also led to a reduction in the CLas titer in D. citri. These results suggest that CLas infection upregulates the genes related to vesicular transport in D. citri, which facilitates the invasion of endocytosis-dependent pathogens.

Longitudinal Transcriptomic, Proteomic, and Metabolomic Response of Citrus sinensis to Diaphorina citri Inoculation of Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) is a fatal citrus disease that is currently threatening citrus varieties worldwide. One putative causative agent, Candidatus Liberibacter asiaticus (CLas), is vectored by Diaphorina citri, known as the Asian citrus psyllid (ACP). Understanding the details of CLas infection in HLB disease has been hindered by its Candidatus nature and the inability to confidently detect it in diseased trees during the asymptomatic stage. To identify early changes in citrus metabolism in response to inoculation of CLas using its natural psyllid vector, leaves from Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees were exposed to CLas-positive ACP or CLas-negative ACP and longitudinally analyzed using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry; data available in Dryad: 10.25338/B83H1Z), and metabolomics (proton nuclear magnetic resonance). At 4 weeks postexposure (wpe) to psyllids, the initial HLB plant response was primarily to the ACP and, to a lesser extent, the presence or absence of CLas. Additionally, analysis of 4, 8, 12, and 16 wpe identified 17 genes and one protein as consistently differentially expressed between leaves exposed to CLas-positive ACP versus CLas-negative ACP. This study informs identification of early detection molecular targets and contributes to a broader understanding of vector-transmitted plant pathogen interactions.

Simplified extraction protocol of citrus tissues for rapid detection of ‘Candidatus Liberibacter asiaticus’ using isothermal recombinase polymerase amplification assay and its application for prevalence studies

Huanglongbing disease caused by ‘Candidatus Liberibacter asiaticus’ is a serious threat to citrus production at global level. Its management largely relies on accurate detection and taking preventive measures to check its spread through sensitive and reliable diagnosis. The available standard nucleic acid-based detection assays rely on use of purified DNA as template and are cumbersome. This study reports a simplified extraction protocol of citrus phloem tissues for rapid recombinase polymerase amplification (RPA) assay-based detection of ‘Candidatus Liberibacter asiaticus’ (CLas)-associated with huanglongbing (HLB). Simplified crude plant extract prepared in phosphate buffer and NaOH:EDTA (using mortar pestle or extraction bag without the use of liquid nitrogen) was successfully employed in the RPA assay. Developed assay targeted specific outer membrane protein (OMP) and ribosomal protein (RP) genomic regions of CLas and could detect its infection up to 10−9 to 10−11 dilution of plant extract and up to 1−10 ag µl−1 of DNA of CLas positive citrus tissues and plasmid DNA having respective gene insert, thus exhibiting sufficient sensitivity. Assay was highly specific and showed no cross-reactivity with the common pathogens infecting citrus exhibiting high reliability. Developed RPA assay was validated using 447 citrus samples collected across the Indian citrus belts, where 79.19 % were detected CLas positive and was more sensitive as compared to PCR wherein 0.45 % to 3.80 % samples were detected false negative. The developed RPA assay rapidly detected CLas infection in simply prepared crude plant extract in cost-effective manner and can be performed at low isothermal temperature in resource-poor laboratories for prevalence studies, bud wood certification and production of CLas-free planting materials of citrus.

The greening-causing agent alters the behavioral and electrophysiological responses of the Asian citrus psyllid to a putative sex pheromone

The Asian Citrus Psyllid (ACP), Diaphorina citri, is a vector of the pathological bacterium Candidatus Liberibacter asiaticus (CLas), which causes the most devastating disease to the citrus industry worldwide, known as greening or huanglongbing (HLB). Earlier field tests with an acetic acid-based lure in greening-free, ‘Valencia’ citrus orange groves in California showed promising results. The same type of lures tested in São Paulo, Brazil, showed unsettling results. During the unsuccessful trials, we noticed a relatively large proportion of females in the field, ultimately leading us to test field-collected males and females for Wolbachia and CLas. The results showed high rates of Wolbachia and CLas infection in field populations. We then compared the olfactory responses of laboratory-raised, CLas-free, and CLas-infected males to acetic acid. As previously reported, CLas-uninfected males responded to acetic acid at 1 µg. Surprisingly, CLas-infected males required 50 × higher doses of the putative sex pheromone, thus explaining the failure to capture CLas-infected males in the field. CLas infection was also manifested in electrophysiological responses. Electroantennogram responses from CLas-infected ACP males were significantly higher than those obtained with uninfected males. To the best of our knowledge, this is the first report of a pathogen infection affecting a vector’s response to a sex attractant.

ABA-CsABI5-CsCalS11 module upregulates Callose deposition of citrus infected with Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) primarily caused by Candidatus Liberibacter asiaticus (CLas) has been threatening citrus production globally. Under HLB conditions, an excessive accumulation of the polysaccharide callose in citrus phloem occurs, leading to phloem blockage and starch accumulation in leaves. The callose production is controlled by callose synthases (CalS), which have multiple members within plants. However, the knowledge of callose production in the citrus upon infection with CLas is limited. In this study, we firstly identified 11 CalSs in the Citrus sinensis genome through bioinformatics and found the expression pattern of CsCalS11 exhibited a positive correlation with callose deposition in CLas-infected leaves (correlation coefficient of 0.77, P ≤ 0.05). Knockdown of CsCalS11 resulted in a reduction of callose deposition and starch accumulation in CLas-infected citrus. Interestingly, we observed significantly higher concentrations of abscisic acid (ABA) in HLB-infected citrus leaves compared to uninfected ones. Furthermore, the expressions of CsABI5, CsPYR, and CsSnRK2 in the ABA pathway substantially increased in citrus leaves upon CLas infection. Additionally, the expression of CsCalS11 was significantly upregulated in citrus leaves following the application of exogenous ABA. We confirmed that CsABI5, a pivotal component of the ABA signaling pathway, regulates CsCalS11 expression by binding to its promoter using yeast one-hybrid assay, dual luciferase assay, and transient expression in citrus leaves. In conclusion, our findings strongly suggest that the CsABI5-CsCalS11 module plays a crucial role in regulating callose deposition through the ABA signaling pathway during CLas infection. The results also revealed new function of the ABA signaling pathway in plants under biotic stress.

Transcriptome analysis of Citrus sinensis reveals potential responsive events triggered by Candidatus Liberibacter asiaticus.

Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is a devastating immune-mediated disorder that has a detrimental effect on the citrus industry, with the distinguishing feature being an eruption of reactive oxygen species (ROS). This study explored the alterations in antioxidant enzyme activity, transcriptome, and RNA editing events of organelles in C. sinensis during CLas infection. Results indicated that there were fluctuations in the performance of antioxidant enzymes, such as ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), peroxidase (POD), and superoxide dismutase (SOD), in plants affected by HLB. Transcriptome analysis revealed 3604 genes with altered expression patterns between CLas-infected and healthy samples, including those associated with photosynthesis, biotic interactions, and phytohormones. Samples infected with CLas showed a decrease in the expression of most genes associated with photosynthesis and gibberellin metabolism. It was discovered that RNA editing frequency and the expression level of various genes in the chloroplast and mitochondrion genomes were affected by CLas infection. Our findings provide insights into the inhibition of photosynthesis, gibberellin metabolism, and antioxidant enzymes during CLas infection in C. sinensis.

Effect of Individual Protective Covers on Young ‘Valencia’ Orange (Citrus sinensis) Tree Physiology

Huanglongbing (HLB), an important citrus disease, causes many physiological and anatomical changes such as phloem dysfunction, imbalance in carbohydrate partitioning, decrease in leaf chlorophyll, and nutritional imbalances in the affected trees, ultimately resulting in tree decline. In Florida, HLB is associated with phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), and it is vectored by the Asian citrus psyllid (Diaphorina citri). No cure for HLB has been found, and most of the HLB management efforts have been focused on vector control or exclusion, improved nutrient management, and the use of HLB-tolerant rootstocks. Individual protective covers (IPCs) are a type of psyllid exclusion tool that is increasingly used by growers for HLB management of newly planted citrus trees. However, no studies have evaluated their influence on citrus tree physiology. This study investigated the effect of IPCs and different rates of insecticides on CLas infection and different physiological attributes, including soluble (glucose, fructose, and sucrose) and nonsoluble (starch) carbohydrates, leaf chlorophyll, and leaf macronutrients and micronutrients over 2.5 years of field growth. The treatments (tree cover and insecticides rate) were applied in newly planted ‘Valencia’ sweet orange (Citrus sinensis) trees grafted on ‘Cleopatra’ (C. reticulata) rootstock. The IPCs prevented CLas transmission and accumulation of foliar starch, sucrose, and glucose commonly associated with HLB. IPC-covered trees had more leaf chlorophyll-a and chlorophyll-b than noncovered trees and more leaf nitrogen (N) and zinc (Zn). Our findings suggest that IPCs effectively prevent CLas infection and maintain the physiological health of young citrus trees under heavy HLB pressure. Therefore, IPCs are recommended as an important component of integrated pest management for this devastating disease.

Effects of vector‐borne plant pathogen on the interactions between Diaphorina citri and its natural enemy, Harmonia axyridis

AbstractThe Asian citrus psyllid, Diaphorina citri, is a vector of the bacterial pathogen, Candidatus Liberibacter asiaticus (CLas) that is believed to cause huanglongbing (HLB), also known as citrus greening disease. This phytopathogen can manipulate its vectors directly by modifying behaviour and altering fitness and indirectly by reducing host plant quality, which may affect the predation efficiency of natural enemies. Little is known about how this phytopathogen may affect third tropic level predators. The purpose of this investigation was to determine if the CLas pathogen modifies interactions between D. citri and its predators. We tested whether CLas infection of psyllids indirectly affects fitness of a key predator, the lady beetle Harmonia axyridis. We provided ladybeetles with 10 infected or uninfected adult psyllids in choice bioassays and measured the consumption rate over 24 h. Similarly, 15 infected or uninfected adult psyllids were provided to ladybeetles in no‐choice bioassays and feeding rates and net weight gain of male and female beetles were measured over 5 consecutive days. Fecundity and fertility of lady beetles feeding on CLas‐infected D. citri was reduced compared with those feeding on uninfected D. citri, and oocyte development appeared diminished in those beetles feeding on infected compared with uninfected psyllids. However, in choice assays, beetles did not distinguish between CLas‐infected and uninfected psyllids. We postulate that CLas may reduce the nutritional quality of psyllids diminishing fecundity and fertility of their predatory beetles. Our results lead us to speculate that under conditions of near 100% HLB infection, as occurs endemically in Florida, effectiveness of biological control for D. citri may benefit from provisioning ladybeetle predators with alternative hosts of higher nutritional quality or augmentative releases of laboratory‐reared predators.

Small RNAs contribute to citrus Huanglongbing tolerance by manipulating methyl salicylate signaling and exogenous methyl salicylate primes citrus groves from emerging infection.

Citrus production is severely threatened by the devastating Huanglongbing (HLB) disease globally. By studying and analyzing the defensive behaviors of an HLB-tolerant citrus cultivar 'Shatangju', we discovered that citrus can sense Candidatus Liberibacter asiaticus (CLas) infection and induce immune responses against HLB, which can be further strengthened by both endogenously produced and exogenously applied methyl salicylate (MeSA). This immune circuit is turned on by an miR2977-SAMT (encoding a citrus Salicylate [SA] O-methyltransferase) cascade, by which CLas infection leads to more in planta MeSA production and aerial emission. We provided both transgenic and multi-year trail evidences that MeSA is an effective community immune signal. Ambient MeSA accumulation and foliage application can effectively induce defense gene expression and significantly boost citrus performance. We also found that miRNAs are battle fields between citrus and CLas, and about 30% of the differential gene expression upon CLas infection are regulated by miRNAs. Furthermore, CLas hijacks host key processes by manipulating key citrus miRNAs, and citrus employs miRNAs that coordinately regulate defense-related genes. Based on our results, we proposed that miRNAs and associated components are key targets for engineering or breeding resistant citrus varieties. We anticipate that MeSA-based management, either induced expression or external application, would be a promising tool for HLB control.