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Cationic amino acid binding protein (CLasArgBP), one of the two amino acid binding receptor in Candidatus Liberibacter asiaticus (CLas), is predominately expressed in citrus psyllids as a part of ATP-binding cassette transport system. The present study describes characterization of CLasArgBP by various biophysical techniques and in silico study, to identify potential inhibitor molecules against CLasArgBP through virtual screening and MD simulations. Further, in planta study was carried out to assess the effect of selected inhibitors on Huanglongbing infected Mosambi plants. The results showed that CLasArgBP exhibits pronounced specificity for arginine, histidine and lysine. Surface plasmon resonance (SPR) study reports highest binding affinity for arginine (Kd, 0.14 µM), compared to histidine and lysine (Kd, 15 µΜ and 26 µΜ, respectively). Likewise, Differential Scanning Calorimetry (DSC) study showed higher stability of CLasArgBP for arginine, compared to histidine and lysine. N(omega)-nitro-L-arginine, Gamma-hydroxy-L-arginine and Gigartinine emerged as lead compounds through in silico study displaying higher binding energy and stability compared to arginine. SPR reports elevated binding affinities for N(omega)-nitro-L-arginine and Gamma-hydroxy-L-arginine (Kd, 0.038 µΜ and 0.061 µΜ, respectively) relative to arginine. DSC studies showed enhanced thermal stability for CLasArgBP in complex with selected inhibitors. Circular dichroism and fluorescence studies showed pronounced conformational changes in CLasArgBP with selected inhibitors than with arginine. In planta study demonstrated a substantial decrease in CLas titer in treated plants as compared to control plants. Overall, the study provides the first comprehensive characterization of cationic amino acid binding protein from CLas, as a potential drug target to manage HLB disease.

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Citrus huanglongbing (HLB), which is caused by the phloem-colonizing bacteria Candidatus Liberibacter asiaticus (CLas), poses a significant threat to citrus production worldwide. The pathogenicity mechanism of HLB remains poorly understood. SEC-dependent effectors (SDEs) have been suggested to play critical roles in the interaction between citrus and CLas. Here, we explored the function of CLIBASIA_05320 (SDE19), a core SDE from CLas, and its interaction with its host target. Our data revealed that SDE19 is expressed at higher level during infection of citrus than that during infection of the Asian citrus psyllid. Subcellular localization assays showed that SDE19 is localized in the nucleus and cytoplasm and is capable of moving from cell to cell in Nicotiana benthamiana. To investigate whether SDE19 facilitates pathogen infection, we generated transgenic Arabidopsis thaliana and citrus plants overexpressing SDE19. Transgenic A. thaliana and citrus plants were more susceptible to Pseudomonas syringae pv. tomato (Pst) and Xanthomonas citri subsp. citri (Xcc), respectively. In addition, RNA-seq analysis demonstrated that overexpression of SDE19 resulted in a reprogramming of expression of genes related to biotic stimulus responses. SDE19 interacts with Citrus sinensis Sec12, a guanine nucleotide exchange factor responsible for the assembly of plant COPII (coat protein II)-coated vesicles, which mediate vesicle trafficking from the ER to the Golgi. SDE19 colocalizes with Sec12 in the ER by binding to its N-terminal catalytic region, affecting the stability of Sec12 through the 26S proteasome. This interaction hinders the secretion of apoplastic defense-related proteins such as PR1, P69B, GmGIP1, and RCR3. Furthermore, the secretion of PR1 and callose deposition is decreased in SDE19-transgenic A. thaliana. Taken together, SDE19 is a novel virulent SDE secreted by CLas that interacts with Sec12 to disrupt vesicle trafficking, inhibit defense-related proteins secretion, and promote bacterial infection. This study sheds light on how CLas manipulates the host vesicle trafficking pathway to suppress the secretion of defense-related proteins and interfere with plant immunity.

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Citrus Huanglongbing (HLB) poses an enormous challenge to Citrus cultivation worldwide, necessitating groundbreaking interventions beyond conventional pharmaceutical methods. In this study, we propose molybdenum disulfide-chitosan nanoparticles (MoS2-CS NPs) through electrostatic adsorption, preserving the plant-beneficial properties of molybdenum disulfide (MoS2), while enhancing its antibacterial effectiveness through chitosan modification. MoS2-CS NPs exhibited significant antibacterial efficacy against RM1021, and the closest relatives to Candidatus Liberibacter asiaticus (CLas), Erwinia carotovora, and Xanthomonas citri achieved survival rates of 7.40 % ± 1.74 %, 8.94 % ± 1.40 %, and 6.41 % ± 0.56 %, respectively. In vivo results showed, CLas survival rate of 10.42 % ± 3.51 %. Furthermore, treatment with MoS2-CS NPs resulted in an increase in chlorophyll and carotenoid content. Concomitantly, a significant reduction in malondialdehyde (MDA), soluble sugar, hydrogen peroxide (H2O2), and starch contents was also observed. Mechanistically, MoS2-CS NPs enhanced the activity of antioxidant-related enzymes by upregulating the expression of antioxidant genes, thereby galvanizing the antioxidant system to alleviate oxidative stress. Collectively, this dual functionality-combining direct antibacterial action with the activation of plant defense mechanisms-marks a promising strategy for managing Citrus Huanglongbing and suggests potential agricultural applications for MoS2-based antibacterial treatments.

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"Candidatus Liberibacter asiaticus" (CLas) is a phloem-limited alpha-proteobacteria causing Citrus Huanglongbing, the destructive disease currently threatening global citrus industry. Genomic analyses of CLas provide insights into its evolution and biology. Here, we sequenced and assembled whole genomes of 135 CLas strains originally from 20 citrus cultivars collected at ten citrus-growing provinces in China. The resulting dataset comprised 135 CLas genomes ranging from 1,221,309 bp to 1,308,521 bp, with an average coverage of 675X. Prophage typing showed that 44 strains contained Type 1 prophage, 89 strains contained Type 2 prophage, 44 strains contained Type 3 prophage, and 34 of them contained more than one type of prophage/phage. The SNP calling identified a total of 5,090 SNPs. Genome-based phylogenetic analysis revealed two major clades among CLas strains, with Clade I dominated by CLas strains containing Type 1 prophage (79/95) and Clade II dominated by CLas strains containing Type 1 or Type 3 prophage (80/95). This CLas genome dataset provides valuable resources for studying genetic diversity and evolutionary pattern of CLas strains.

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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.

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Imidacloprid (IDP) is an active ingredient of the Admire brand pesticide used to control the vector (Asian citrus psyllid) that transmits the causative organism Candidatus Liberibacter asiaticus (CLas) for citrus greening or huanglongbing disease. Imidacloprid products are applied via soil drench where citrus roots are mostly concentrated which is between 0 and 60 cm depth. These soil depths exhibit different characteristics that may affect IDP leaching beyond the rooting zone. Representative soil samples were collected from Entisols and Ultisols, which are the dominant soil orders under citrus production in central Florida, at 15 cm increments up to 60 cm to estimate and understand the batch sorption, kinetics, equilibria, and degradation of IDP. Results showed that the equilibrium time for IDP at 0-15 cm depth (10 hours) was 2 times faster than at 15-60 cm (20 hours) for the Entisol. Nevertheless, all depths reached equilibrium within 24 hours for the Entisol. The 0-30 cm depth adsorbed 2 times more IDP than the 30-60 cm depth for both soils. Nevertheless, the adsorption coefficient was approximately ≤ 1 mL g-1 for both soils. The half-life of IDP in both soils ranged from 10 to 17 days. The Entisol showed higher adsorption than the Ultisol at both depths, probably due to relatively lower organic carbon (OC) content in the Ultisol compared to the Entisol. Thus, the Ultisol showed high IDP leaching vulnerability compared to the Entisol. Movement of IDP is affected by the amount of OC in the citrus critical zone.

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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.

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Citrus Huanglongbing (HLB) is caused by the phloem-limited α-proteobacterium "Candidatus Liberibacter spp.", among which "Ca. Liberibacter africanus" (CLaf) have posed a significant threat to citrus production in Africa near a century. CLaf is closely related to the globally prevalent "Ca. Liberibacter asiaticus" (CLas), whereas little is known about the virulence of CLaf, primarily due to limited genome resources. In this study, we completed the whole-genome assembly and annotation of CLaf strain Zim (from Zimbabwe). Compared to CLas, a total of 102 CLaf unique genes were identified, including 14 potential Sec-dependent effectors (SDEs) genes, 29 phage-associated genes, and 59 genes with hypothetical function. Among 14 SDEs, V9J15_03810 was able to induce a significant hypersensitive response (HR) in Nicotiana benthamiana, indicating its potential as a virulence factor for CLaf. Genome analysis showed that CLaf strain Zim genome harbored a complete prophage region (named P-Zim-1, 42,208 bp). P-Zim-1 retained two immunosuppressive peroxidase genes (V9J15_02125 and V9J15_02130) homologous to CLas prophage SC1/SC2, whereas the lysogen-associated genes encoding integrase (V9J15_01970) and repressor (V9J15_02080) were homologous to the prophage of "Ca. Liberibacter solanacearum", the causal agent of potato zebra chip disease. In addition, P-Zim-1 carried a novel CRISPR/Cas system, including a CRISPR array (located within V9J15_02040, ranging from 443,643 to 443,897) and five CRISPR-related Cas proteins (V9J15_02005, 02010, 02015, 02025 and 02035). This study first characterized the unique genomic feature of CLaf related to virulence and prophage, which will facilitate future research on CLaf biology and African HLB management.

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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.

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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.

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The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama is the leading vector of Candidatus Liberibacter asiaticus (CLas), the causative agent of citrus Huanglongbing (HLB) disease. The distribution and dynamics of CLas within ACP are critical to understanding how the transmission, spread and infection of CLas occurs within its host vector in nature. In this study, the distribution and titer changes of CLas in various tissues of ACP 5th instar nymphs and adults were examined by fluorescence in situ hybridization (FISH) and real-time quantitative PCR (qPCR) techniques. Results demonstrated that 100% of ACP 5th instar nymphs and adults were infected with CLas following feeding on infected plants, and that CLas had widespread distribution in most of the tissues of ACP. The titers of CLas within the midgut, salivary glands and hemolymph tissues were the highest in both 5th instar nymphs and adults. When compared with adults, the titers of CLas in these three tissues of 5th instar nymphs were significantly higher, while in the mycetome, ovary and testes they were significantly lower than those of adults. FISH visualization further confirmed these findings. Dynamic analysis of CLas demonstrated that it was present across all the developmental ages of ACP adults. There was a discernible upward trend in the presence of CLas with advancing age in most tissues of ACP adults, including the midgut, hemolymph, salivary glands, foot, head, cuticula and muscle. Our findings have significant implications for the comprehensive understanding of the transmission, dissemination and infestation of CLas, which is of much importance for developing novel strategies to halt the spread of CLas, and therefore contribute to the efficient prevention and control of HLB.

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Citrus Huanglongbing (HLB) is known as the cancer of citrus, where Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain causing HLB. In this study, we report a novel electrochemiluminescence (ECL) biosensor for the highly sensitive detection of the CLas outer membrane protein (Omp) gene by coupling rolling circle amplification (RCA) with a CRISPR/Cas12a-responsive smart DNA hydrogel. In the presence of the target, a large number of amplicons are generated through RCA. The amplicons activate the trans-cleavage activity of CRISPR/Cas12a through hybridizing with crRNA, triggering the response of smart DNA hydrogel to release the encapsulated AuAg nanoclusters (AuAg NCs) on the electrode and therefore leading to a decreased ECL signal. The ECL intensity change (I0 - I) is positively correlated with the concentration of the target in the range 50 fM to 5 nM, with a limit of detection of 40 fM. The performance of the sensor has also been evaluated with 10 samples of live citrus leaves (five HLB negative and five HLB positive), and the result is in excellent agreement with the gold standard qPCR result. The sensing strategy has expanded the ECL versatility for detecting varying levels of dsDNA or ssDNA in plants with high sensitivity.

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Insecticide susceptibility is mainly determined by the insect host, but symbiotic bacteria are also an important affecting factor. In this study, we investigate the relationship between the structure of gut bacterial symbionts and insecticide susceptibility in Diaphorina citri, the important carrier of Candidatus Liberibacter asiaticus (CLas), the causal agent of Huanglongbing (HLB). Our results indicated that antibiotic treatment significantly increased the susceptibility of D. citri to bifenthrin and thiamethoxam, and significantly decreased the relative abundance of Wolbachia and Profftella, enzyme activities of CarEs, and expression level of multiple CarE genes. The relative loads of Wolbachia and Profftella were positively correlated with DcitCCE13, DcitCCE14, DcitCCE15, and DcitCCE16. RNAi and prokaryotic expression revealed that DcitCCE15 is associated with bifenthrin metabolism. These results revealed that bacterial symbionts might regulate DcitCCE15 expression, which is involved in the susceptibility of D. citri to bifenthrin.

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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.

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The Asian citrus psyllid, Diaphorina citri, is the primary vector of the HLB pathogen, Candidatus Liberibacter asiaticus (CLas). The acquisition of CLas shortens the developmental period of nymphs, accelerating the emergence into adulthood and thereby facilitating the spread of CLas. Cuticular proteins (CPs) are involved in insect emergence. In this study, we investigated the molecular mechanisms underlying CLas-promoted emergence in D. citri via CP mediation. Here, a total of 159 CP genes were first identified in the D. citri genome. Chromosomal location analysis revealed an uneven distribution of these CP genes across the 13 D. citri chromosomes. Proteomic analysis identified 54 differentially expressed CPs during D. citri emergence, with 14 CPs exhibiting significant differential expression after CLas acquisition. Five key genes, Dc18aa-1, Dc18aa-2, DcCPR-24, DcCPR-38 and DcCPR-58, were screened from the proteome and CLas acquisition. The silencing of these 5 genes through a modified feeding method significantly reduced the emergence rate and caused various abnormal phenotypes, indicating the crucial role that these genes play in D. citri emergence. This study provides a comprehensive overview of the role of CPs in D. citri and reveals that CLas can influence the emergence process of D. citri by regulating the expression of CPs. These key CPs may serve as potential targets for future research on controlling huanglongbing (HLB) transmission.

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Huanglongbing (HLB) is a systemic plant disease caused by 'Candidatus Liberibacter asiaticus (CLas)' and transmitted by Diaphorina citri. D. citri acquires the CLas bacteria in the nymph stage and transmits it in the adult stage, indicating that molting from the nymph to adult stages is crucial for HLB transmission. However, the available D. citri reference genomes are incomplete, and gene function studies have been limited to date. In the current research, PacBio single-molecule real-time (SMRT) and Illumina sequencing were performed to investigate the transcriptome of D. citri nymphs and adults. In total, 10,641 full-length, non-redundant transcripts (FLNRTs), 594 alternative splicing (AS) events, 4522 simple sequence repeats (SSRs), 1086 long-coding RNAs (lncRNAs), 281 transcription factors (TFs), and 4459 APA sites were identified. Furthermore, 3746 differentially expressed genes (DEGs) between nymphs and adults were identified, among which 30 DEGs involved in the Hippo signaling pathway were found. Reverse transcription-quantitative PCR (RT-qPCR) further validated the expression levels of 12 DEGs and showed a positive correlation with transcriptome data. Finally, the spatiotemporal expression pattern of genes involved in the Hippo signaling pathway exhibited high expression in the D. citri testis, ovary, and egg. Silencing of the D. citri transcriptional co-activator (DcYki) gene significantly increased D. citri mortality and decreased the cumulative molting. Our results provide useful information and a reliable data resource for gene function research of D. citri.

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The fabrication of the first label-free electrochemical DNA probe biosensor for highly sensitive detection of Candidatus Liberibacter asiaticus (CLas), as the causal agent of citrus huanglongbing disease, is conducted here. An OMP probe was designed based on the hybridization with its target-specific sequence in the outer membrane protein (OMP) gene of CLas. The characterization of the steps of biosensor fabrication and hybridization process between the immobilized OMP-DNA probe and the target ssDNA oligonucleotides (OMP-complementary and three mismatches OMP or OMP-mutation) was monitored using cyclic voltammetry and electrochemical impedance spectroscopy based on increasing or decreasing in the electron transfer in [Fe (CN)6]3-/4- on the modified gold electrode surface. The biosensor sensitivity indicated that the peak currents were linear over ranges from 20 to 100 nM for OMP-complementary with the detection limit of 0.026 nM (S/N = 3). The absence of any cross-interference with other biological DNA sequences confirmed a high selectivity of fabricated biosensor. Likewise, it showed good specificity in discriminating the mutation oligonucleotides from complementary target DNAs. The functional performance of optimized biosensor was achieved via the hybridization of OMP-DNA probe with extracted DNA from citrus plant infected with CLas. Therefore, fabricated biosensor indicates promise for sensitivity and early detection of citrus huanglongbing disease.

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Huanglongbing (HLB), a devastating citrus disease caused by Candidatus Liberibacter asiaticus, is efficiently vectored by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). Tamarixia radiata (Waterston) plays a crucial role as an ectoparasitoid, preying on D. citri nymphs. By collecting and identifying headspace volatiles from fifth instar nymphs of D. citri using a gas chromatograph-mass spectrometer (GC-MS), we obtained a collection of 9 volatile compounds. These compounds were subsequently chosen to investigate the electrophysiological and behavioral responses of female T. radiata. At a concentration of 10 µg/µl, 9 compounds were compared with cis-3-hexen-1-ol (control), resulting in trans-2-nonenal inducing the highest relative electroantennogram (EAG) value, followed by hexanal, heptanal, n-heptadecane, tetradecanal, n-tetradecane, n-pentadecane, 1-tetradecanol, and 1-dodecanol. The top 5 EAG responses of female T. radiata to these compounds were further investigated through EAG dose-response experiments. The results showed positive dose-responses as concentrations increased from 0.01 to 10 µg/µl. In Y-tube olfactometer bioassays, female T. radiata exhibited a preference for specific compounds. They were significantly attracted to tetradecanal at a concentration of 10 µg/µl and trans-2-nonenal at 0.01 µg/µl, while no significant attraction was observed toward hexanal, heptanal, or n-heptadecane. Our report is the first to demonstrate that volatiles produced by D. citri nymphs attract T. radiata, which suggests that this parasitoid may utilize nymph volatiles to locate its host.

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São Paulo, Brazil, and Florida, USA, were the two major orange production areas in the world until Huanglongbing (HLB) was discovered in São Paulo in 2004 and Florida in 2005. In the absence of resistant citrus varieties, HLB is the most destructive citrus disease known because of the lack of effective tools to reduce spread of the vector, Diaphorina citri (Asian citrus psyllid), and transmission of the associated pathogen, Candidatus Liberibacter asiaticus. In both countries, a three-pronged management approach was recommended and begun: planting only disease-free nursery trees, effective psyllid control, and removal of all symptomatic trees. In Brazil, these management procedures were continued and improved and resulted in relatively little overall loss of production. In contrast, in Florida the citrus industry has been devastated with annual production reduced by approximately 80%. This review compares and contrasts various cultural and pest management strategies that have been used to reduce infection by the pathogen and increase tolerance of HLB in the main orange-growing regions in the world.

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Wolbachia, a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Diaphorina citri Kuwayama, the vector of Candidatus Liberibacter asiaticus (CLas) that causes citrus greening disease, is naturally infected with Wolbachia (wDi). However, the interaction between wDi and D. citri remains poorly understood. In this study, we performed a pan-genome analysis using 65 wDi genomes to gain a comprehensive understanding of wDi. Based on average nucleotide identity (ANI) analysis, we classified the wDi strains into Asia and North America strains. The ANI analysis, principal coordinates analysis (PCoA), and phylogenetic tree analysis supported that the D. citri in Florida did not originate from China. Furthermore, we found that a significant number of core genes were associated with metabolic pathways. Pathways such as thiamine metabolism, type I secretion system, biotin transport, and phospholipid transport were highly conserved across all analyzed wDi genomes. The variation analysis between Asia and North America wDi showed that there were 39,625 single-nucleotide polymorphisms (SNPs), 2153 indels, 10 inversions, 29 translocations, 65 duplications, 10 SV-based insertions, and 4 SV-based deletions. The SV-based insertions and deletions involved genes encoding transposase, phage tail tube protein, ankyrin repeat (ANK) protein, and group II intron-encoded protein. Pan-genome analysis of wDi contributes to our understanding of the geographical population of wDi, the origin of hosts of D. citri, and the interaction between wDi and its host, thus facilitating the development of strategies to control the insects and huanglongbing (HLB).

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