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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.

Ascorbate peroxidases (APXs) are essential for plants as they act as hydrogen peroxide-scavenging enzymes, providing protection against oxidative damage. Using bioinformatic methods, five APX genes were discovered in the genome of Citrus sinensis in this study. APX genes of C. sinensis (CsAPXs) encode polypeptides between 250 and 436 residues in length, with molecular weights that range from 27.56 to 47.34 kDa. Additionally, the isoelectric point of CsAPXs varies from 5.64 to 8.63. The predicted locations of CsAPXs are peroxisome, chloroplast, and mitochondrion, with an uneven distribution across four chromosomes and eight orthologous gene pairs with Arabidopsis thaliana. A phylogenetic analysis revealed that the CsAPXs were divided into three clades. The CsAPXs all contained a conserved APX domain and six common motifs. Upon promoter analysis, it was found that CsAPXs could respond to abscisic acid, auxin, ethylene, gibberellin, methyl jasmonate, salicylic acid, and wounding stress treatments. In addition, the analysis of expression patterns revealed that the presence of Candidatus Liberibacter asiaticus (CLas) has a dynamic impact on the expression of CsAPXs, with CsAPX2 showing significant inhibition in response to CLas infection. These findings will provide novel insights for the forthcoming functional investigations of CsAPXs within the process of citrus-CLas interactions. [ABSTRACT FROM AUTHOR]

BACKGROUND: Citrus huanglongbing (HLB) is a devastating disease caused by Candidatus Liberibacter asiaticus (CLas) that affects the citrus industry. In nature, CLas relies primarily on Diaphorina citri Kuwayama as its vector for dissemination. After D. citri ingests CLas‐infected citrus, the pathogen infiltrates the insect's body, where it thrives, reproduces, and exerts regulatory control over the growth and metabolism of D. citri. Previous studies have shown that CLas alters the composition of proteins in the saliva of D. citri, but the functions of these proteins remain largely unknown. RESULTS: In this study, we detected two proteins (DcitSGP1 and DcitSGP3) with high expression levels in CLas‐infected D. citri. Quantitative PCR and Western blotting analysis showed that the two proteins were highly expressed in the salivary glands and delivered into the host plant during feeding. Silencing the two genes significantly decreased the survival rate for D. citri, reduced phloem nutrition sucking and promoted jasmonic acid (JA) defenses in citrus. By contrast, after overexpressing the two genes in citrus, the expression levels of JA pathway‐associated genes decreased. CONCLUSION: Our results suggest that CLas can indirectly suppress the defenses of citrus and support feeding by D. citri via increasing the levels of effectors in the insect's saliva. This discovery facilitates further research into the interaction between insect vectors and pathogens. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a major pest of citrus due to its role as the vector of the bacterium that causes huanglongbing. In commercial citrus, ACP control currently relies on the application of insecticides, which may not be sustainable long-term, nor practical in urban areas. The sterile insect technique (SIT) is an alternative strategy in which large numbers of pests are reared, sterilized using radiation, and then released into the field to compete with wild individuals for matings, suppressing population growth. As a fundamental step toward the development of SIT for ACP, this study sought to identify the optimum radiation dose required to sterilize ACP without affecting their survival and mating capacity. Virgin adult ACP of both sexes were subjected to doses of X-ray irradiation ranging from 40 to 480 Gy, then paired with a nonirradiated mate and allowed to produce offspring. Fecundity was estimated as the number of eggs laid, and fertility as the proportion of those eggs that hatched. Females were more radio-sensitive than males, exhibiting a major drop in fecundity at even the lowest dose and 100% sterility at 80 Gy. In contrast, a fivefold higher dose (400 Gy) did not achieve complete sterility in males, with around 5% offspring survival. However, F1 progeny of males exposed to 320 Gy or higher were subsequently found to be 100% sterile. This confirmation of inherited sterility suggests that balancing the sterilizing effects of radiation against its mortality-inducing effects may warrant further evaluation. [ABSTRACT FROM AUTHOR]

Targeted precise management of intravascular diseases such as citrus Huanglongbing (HLB) involves the application of therapeutics via trunk injection. Although trunk injection has proven more effective than foliar spray and soil drenching, multiple studies have highlighted limitations for large-scale commercial tree crop applications because of the manual process involved, emphasizing the need for an automated injection system. The design of a trunk injection mechanism to efficiently dispense aqueous therapeutic materials to the trunk of HLB-affected citrus trees requires the characterization of the hydraulic properties of the trunk and the determination of the penetration force of a dispensing needle. This study developed an equation for estimating penetration force at maximum depth based on the needle diameter, depth of penetration, and specific gravity of the wood tissue, and presents a method for estimating injection pump pressure. Trunk hydraulic conductance was measured using a hydraulic conductance flow meter, and needle insertion and removal force were measured using a force gauge in an Instron universal testing machine. The average maximum force was 2517, 3739, and 5387 N for dowel pins 4.76, 6.35, and 7.93 mm in diameter at a 12 mm penetration depth. Average specific conductivity was 2.45, 0.34, and 0.15 kgs-1m-1MPa-1for trunk segments 0-12.7, 12.7-26.7, and 26.7-40.6 cm above the ground, with a corresponding estimated pump pressure of 0.14, 0.99, and 2.31 MPa, respectively. [ABSTRACT FROM AUTHOR]

Highlights An AI-enabled web application was developed to classify insects on sticky traps. The AI models were trained on images exposed to real environmental conditions. Automated insect identification can enhance integrated pest management. This technology could be used in other environmental applications. The Huanglongbing (HLB) disease has had a devastating impact on several citrus production areas across the world. The disease is associated with three phloem-limited bacteria of the Candidatus Liberibacter group, which can be vectored by citrus-infesting psyllids. In Florida, it is vectored by Diaphorina citri, also known as Asian citrus psyllid (ACP), and in South Africa, by a native species, Trioza erytreae, or African citrus triozid (ACT). For better disease management, continued monitoring of these vectors is highly recommended. Furthermore, it is important to keep track of other related psyllids that reside in citrus environments (e.g., the South African native Diaphorina specimens DN, such as D. virgata, D. punctulata, and D. zebrana), as well as natural predators of psyllids (e.g., lady beetles, LB). Among the tools used to monitor pests in the field, sticky traps are one of the most common and widely used. Analyzing the traps and identifying the specimens of interest usually require specialized personnel, which can be costly and time-consuming. In an effort to automate this process, a web application called “Psyllid Detector” was developed. Three YOLOv7-based object detection algorithms (YOLOv7, YOLOv7-ResNet50, and YOLOv7-ResNeXt50) were selected to train six models using two datasets (South Africa and Florida datasets) composed of yellow sticky trap pictures taken from several citrus orchards. Three models were trained to detect ACP and LB using the Florida dataset, and three were trained to detect DN and ACT in the South Africa dataset. The models trained with the original YOLOv7 for both datasets showed the best overall results. For example, for the ACT detection in the South Africa dataset, precision, recall, and F1 scores of 90%, 70%, and 79% were achieved, respectively. By automating the process of insect detection, farmers can save time and resources and make informed decisions about pest control measures. [ABSTRACT FROM AUTHOR]

Abstract 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.

Citrus Huanglongbing (HLB), which is caused by ‘Candidatus Liberibacter asiaticus’ (CLas), is one of the most destructive citrus diseases worldwide, and defense-related Citrus sinensis gene resources remain largely unexplored. Calcium signaling plays an important role in diverse biological processes. In plants, a few calcium-dependent protein kinases (CDPKs/CPKs) have been shown to contribute to defense against pathogenic microbes. The genome of C. sinensis encodes dozens of CPKs. In this study, the role of C. sinensis calcium-dependent protein kinases (CsCPKs) in C. sinensis defense was investigated. Silencing of CsCPK6 compromised the induction of defense-related genes in C. sinensis. Expression of a constitutively active form of CsCPK6 (CsCPK6CA) triggered the activation of defense-related genes in C. sinensis. Complementation of CsCPK6 rescued the defense-related gene induction in an Arabidopsis thaliana cpk4/11 mutant, indicating that CsCPK6 carries CPK activity and is capable of functioning as a CPK in Arabidopsis. Moreover, an effector derived from CLas inhibits defense induced by the expression of CsCPK6CA and autophosphorylation of CsCPK6, which suggests the involvement of CsCPK6 and calcium signaling in defense. These results support a positive role for CsCPK6 in C. sinensis defense against CLas, and the autoinhibitory regulation of CsCPK6 provides a potential genome-editing target for improving C. sinensis defense. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

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. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is caused by “Candidatus Liberibacter asiaticus” and is spread by citrus psyllids. It is a highly damaging and infectious disease of citrus for which there is no reliable treatment. Timely detection and removal of diseased trees is an effective strategy to control HLB. Various citrus cultivars infected with HLB exhibit distinct symptoms, posing a challenge to generalize HLB detection methods across multiple cultivars. Volatile organic compounds (VOCs) in HLB‐infected leaves from cultivars grown in different regions were comprehensively analysed by gas chromatography–mass spectrometry (GC‐MS) and gas chromatography‐ion mobility spectrometry (GC‐IMS), aiming to establish a new generalized HLB detection method applicable to a range of citrus cultivars. The changes in VOCs were analysed in leaves of four citrus cultivars after infection with HLB. The results showed that there was a similar response to HLB infection in different citrus cultivars, and this response was reflected in both the concentration and type of VOCs. By combining GC‐MS and GC‐IMS with orthogonal partial least squares discriminant analysis (OPLS‐DA) model, the accurate identification of the HLB infection status of different citrus cultivars was achieved, with the prediction indices reaching 0.994 for GC‐MS and 0.972 for GC‐IMS. In addition, 27 compounds were identified that were significantly different between citrus cultivars affected by HLB and healthy plants. This study provides valuable insights into the changes in VOCs in citrus cultivars after HLB infection and lays the theoretical foundation for VOC‐based HLB detection strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plant pathogens can alter the behavior of their insect vectors as well as their survival and reproduction. The African psyllid, Trioza erytreae, is one of the vectors of Huanglongbing, a citrus disease caused mainly by "Candidatus Liberibacter asiaticus" (CLas). The purpose of this study was to characterize the effects of CLas on the psyllid, T. erytreae using Citrus volkamerina plants as the study system. The study focused more specifically on the CLas effects prior to and after its acquisition by the psyllid T. erytreae. Our results did not support the hypothesis that CLas effects psyllid probing behavior prior to acquisition; few differences were observed between uninfected T. erytrea feeding on CLas‐infected versus control plants. On the other hand, compared to psyllids that had completed their development on control plants, the ones that had completed their development on a CLas‐infected plant exhibited changes in their behavior (greater velocity), physiology (smaller mass) and biochemistry (lower water and lipid content). Altogether, our results confirm the existence of a marked postacquisition effect on the vector locomotor behavior and a minor preacquisition effect of CLas on the vector behavior, which can be partially explained by physiological and biochemical changes. [ABSTRACT FROM AUTHOR]

Increased preharvest fruit drop rates and reduced fruit sizes are common symptoms associated with Huanglongbing (HLB) in sweet orange. Small fruit may be more likely to drop during preharvest fruit drop. The objective of the current study was to determine whether fruit size could be used as an indicator of the preharvest fruit drop likelihood. Nearly 1400 fruit were surveyed over the following three timepoints across two years: 11 Mar 2022, 15 Apr 2022, and 22 Mar 2023. Each fruit was evaluated to determine the equator and peduncle diameter, fruit detachment force (FDF), type of detachment (mechanically broken or abscission), and lopsidedness. The total soluble solids content was determined for a subset of fruit. The FDF was consistently lower in abscising fruit than in mechanically broken fruit, suggesting that the physiological process of abscission had begun in these fruit and that they were more likely to drop. The fruit diameter was significantly smaller in abscising fruit than in mechanically broken fruit on 11 Mar 2022 and 22 Mar 2023, but not on 15 Apr 2022. Similarly, the fruit diameter and FDF were also significantly and positively correlated, but this relationship was weaker at time points late in the season (closer to harvest). These findings suggest that small fruit are indeed more likely to drop early in the preharvest period. The abscising fruit had total soluble solids contents similar to those of the mechanically broken fruit. Therefore, we hypothesized that the smaller fruit ceased growing and are able to respond to abscission signals earlier than the larger fruit. Therefore, any strategies to mitigate fruit drop, such as the use of plant growth regulators, should be applied early in the season when the fruit are still actively growing. When growth ceases, the fruit are vulnerable to drop. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is a devastating citrus disease that causes significant financial losses in the citrus industry. However, there is no cure by current control strategies. Damaged soils were observed in citrus orchards; root lesions and nematodes were observed in HLB-affected citrus tree roots. Bacillus subtilis (B. subtilis) makes a great contribution to pathogen control and has the potential to control HLB. Purpureocillium lilacinum (P. lilacinum) and Trichoderma harzianum (T. harzianum) can kill pathogenic organisms such as nematodes and Phytophthora spp. to protect roots. Restoration of the damaged soils and improvement of citrus root growth through the introduction of soil amendments and biocontrol agents (containing B. subtilis, P. lilacinum, and T. harzianum) may provide an efficient approach to controlling HLB. Results revealed that soil properties such as soil pH and organic matter content were improved. After three months of combination treatment of soil amendment and biocontrol agent through root drenches, the citrus grew new roots and its leaves changed from yellow to green. The percentage of HLB-positive citrus trees—Orah, Tangerine, and Navel Orange—decreased from 90 to 0%, 87.5% to 21.88%, and 81.25% to 0% over three years, respectively. Furthermore, the productivity of HLB-affected orchards was restored, and both production and quality saw significant improvement. These results suggested that combining soil remediation and biocontrol improved soil quality and protected citrus root growth, thus effectively controlling HLB. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is a severe citrus disease worldwide. Wild Australian limes like Citrus australasica, C. inodora, and C. glauca possess beneficial HLB resistance traits. Individual trees of the three taxa were extensively used in a breeding program for over a decade to introgress resistance traits into commercial-quality citrus germplasm. We generated high-quality, phased, de novo genome assemblies of the three Australian limes using PacBio long-read sequencing. The genome assembly sizes of the primary and alternate haplotypes were determined for C. australasica (337 Mb/335 Mb), C. inodora (304 Mb/299 Mb), and C. glauca (376 Mb/379 Mb). The nine chromosome-scale scaffolds included 86–91% of the genome sequences generated. The integrity and completeness of the assembled genomes were estimated to be at 97.2–98.8%. Gene annotation studies identified 25,461 genes in C. australasica, 27,665 in C. inodora, and 30,067 in C. glauca. Genes belonging to 118 orthogroups were specific to Australian lime genomes compared to other citrus genomes analyzed. Significantly fewer canonical resistance (R) genes were found in C. inodora and C. glauca (319 and 449, respectively) compared to C. australasica (576), C. clementina (579), and C. sinensis (651). Similar patterns were observed for other gene families associated with potential HLB resistance, including Phloem protein 2 (PP2) and Callose synthase (CalS) genes predicted in the Australian lime genomes. The genomic information on Australian limes developed in the present study will help understand the genetic basis of HLB resistance. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are pivotal in signal transduction processes in plant–pathogen interactions. The ROS signaling pathways involved in Candidatus Liberibacter asiaticus (CLas) and Xanthomonas citri subspecies citri (Xcc) infections in Citrus sinensis (sweet orange) are unclear. In this study, we comprehensively identified ROS metabolism-associated genes, including 9 NADPH oxidase (RBOH), 14 superoxide dismutase (SOD), 1 catalase (CAT), 9 peroxiredoxin (PrxR), 5 ascorbate peroxidase (APX), 4 glutathione peroxidase (GPX), 3 monodehydroascorbate reductase (MDAR), 2 dehydroascorbate reductase (DHAR), 2 glutathione reductase (GR), 24 thioredoxin (Trx), and 18 glutaredoxin (GLR) genes in C. sinensis. An analysis revealed variable gene structures but conserved motifs and domains in ROS subfamilies. A comparative synteny analysis with Arabidopsis thaliana and Vitis vinifera indicated evolutionary conservation of most ROS metabolism-associated genes, with some originating from gene duplication events post-species divergence in C. sinensis. Expression profiling revealed five up-regulated genes and four down-regulated genes during both CLas and Xcc infections. Promoter analysis revealed numerous stress-responsive elements in the promoter of ROS metabolism-associated genes. Protein–protein interaction network analysis highlighted the involvement of ROS metabolism in various biological processes. A comparison of ROS metabolism-associated genes between C. sinensis and Poncirus trifoliata indicated multiple gene gain and loss events within ROS subfamilies of C. sinensis. This study enhances our understanding of ROS metabolism in C. sinensis and sheds light on citrus–pathogen interactions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Extensive microbial interactions occur within insect hosts. However, the interactions between the Huanglongbing (HLB) pathogen and endosymbiotic bacteria within the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) in wild populations remain elusive. Thus, this study aimed to detect the infection rates of HLB in the ACP across five localities in China, with a widespread prevalence in Ruijin (RJ, 58%), Huidong (HD, 28%), and Lingui (LG, 15%) populations. Next, microbial communities of RJ and LG populations collected from citrus were analyzed via 16S rRNA amplicon sequencing. The results revealed a markedly higher microbial diversity in the RJ population compared to the LG population. Moreover, the PCoA analysis identified significant differences in microbial communities between the two populations. Considering that the inter-population differences of Bray–Curtis dissimilarity in the RJ population exceeded those between populations, separate analyses were performed. Our findings indicated an increased abundance of Enterobacteriaceae in individuals infected with HLB in both populations. Random forest analysis also identified Enterobacteriaceae as a crucial indicator of HLB infection. Furthermore, the phylogenetic analysis suggested a potential regulatory role of ASV4017 in Enterobacteriaceae for ACP, suggesting its possible attractant activity. This research contributes to expanding the understanding of microbial communities associated with HLB infection, holding significant implications for HLB prevention and treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: Huanglongbing (HLB) is the most devastating citrus disease across Africa, America and Asia. It is associated with three species of bacteria belonging to the genus 'Candidatus Liberibacter', transmitted by the psyllid vector Trioza erytreae, among others. This insect has spread throughout part of the Iberian Peninsula, threatening citrus orchards with the possible arrival and transmission of HLB. Currently, there are no curative methods of control for the disease, so finding plant material less suitable for psyllids can reduce the risk of spreading. We have evaluated how the citrus combination, rootstocks (Flying dragon, 'Cleopatra' mandarin, Carrizo citrange, Forner-Alcaide no.5, Forner-Alcaide no.517 and Citrus macrophylla) and scion cultivars ('Star Ruby', 'Clemenules', 'Navelina', 'Valencia Late', 'Fino 49' and 'Ortanique') determine the biology of T. erytreae. Our results show that T. erytreae behaves differently depending on the combination, and is the least suitable for its survival with 'Ortanique' on Forner-Alcaide no. 517 and 'Valencia Late' on Carrizo citrange. The psyllid preferred 'Fino 49' grafted onto Carrizo citrange for egg laying rather than Flying Dragon. Trioza erytreae (Del Guercio, 1918) (Hemiptera: Triozidae) is a citrus pest which produces gall symptoms on leaves and transmits bacteria associated with the citrus disease Huanglongbing, 'Candidatus Liberibacter' spp. In the present work, the biology and behaviour of T. erytreae were studied in different rootstock–cultivar combinations. Six rootstocks were used, Flying dragon (FD), 'Cleopatra' mandarin (CL), Carrizo citrange (CC), Forner-Alcaide no.5 (FA5), Forner-Alcaide no.517 (FA517) and Citrus macrophylla (CM), and six scion cultivars: 'Star Ruby', 'Clemenules', 'Navelina', 'Valencia Late', 'Fino 49' and 'Ortanique'. Survival and oviposition were evaluated in a no-choice trial, and preference in a choice trial, all of them under greenhouse conditions. Trioza erytreae did not show a clear settle preference for any citrus combination. However, it was able to lay more eggs in 'Fino 49' grafted on CC than on FD. In terms of survival, 'Ortanique' grafted onto FA5 was more suitable than when grafted onto FA517, and in the case of 'Valencia Late', when it was grafted onto CM rather than CC. Our results showed that T. erytreae behave differently depending on the citrus combination. [ABSTRACT FROM AUTHOR]

Nodule-specific cysteine-rich (NCR) peptides, encoded in the genome of the Mediterranean legume Medicago truncatula (barrelclover), are known to regulate plant-microbe interactions. A subset of computationally derived 20-mer peptide fragments from 182 NCR peptides was synthesized to identify those with activity against the unculturable vascular pathogen associated with citrus greening disease, 'Candidatus Liberibacter asiaticus' (CLas). Grounded in a design of experiments framework, we evaluated the peptides in a screening pipeline involving three distinct assays: a bacterial culture assay with Liberibacter crescens, a CLas-infected excised citrus leaf assay, and an assay to evaluate effects on bacterial acquisition by the nymphal stage of hemipteran vector Diaphorina citri. A subset of the 20-mer NCR peptide fragments inhibits both CLas growth in citrus leaves and CLas acquisition by D. citri. Two peptides induced higher levels of D. citri mortality. These findings reveal 20-mer NCR peptides as a new class of plant-derived biopesticide molecules to control citrus greening disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: Heavy metal-associated isoprenylated plant proteins (HIPPs) play vital roles in maintaining heavy metal balance and responding to both biotic and abiotic stresses in vascular plants. However, the role of HIPPs in the response to Huanglongbing (HLB), a harmful disease of citrus caused by the phloemcolonizing bacterium Candidatus Liberibacter asiaticus (CLas), has not been examined. Methods and results: In this study, a total of 26 HIPP genes were identified in Citrus sinensis, and they were grouped into 5 clades. The CsHIPP genes are distributed on 8 chromosomes and exhibited considerable synteny with HIPPs found in Arabidopsis thaliana. Additionally, we analyzed the gene structure, conserved motifs and domains of the CsHIPPs. Various cis-acting elements related to plant hormones and stress responses were identified in the promoters of CsHIPPs. Public transcriptome data and RT-qPCR analysis showed that the expression level of CsHIPP03 was significantly reduced in samples infected by CLas and Xanthomonas citri ssp. citri (Xcc). Furthermore, silencing the homologous gene of CsHIPP03 in Nicotiana benthamiana increased the disease resistance of plants to bacteria. Discussion: Our results provide a basis for functional studies of HIPP gene family in C. sinensis, highlighting their functions in bacterial resistance, and improve our understanding to the susceptibility mechanism of HLB. [ABSTRACT FROM AUTHOR]

Aims Huanglongbing (citrus greening) is a plant disease putatively caused by the unculturable Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), and it has caused severe damage to citrus plantations worldwide. There are no definitive treatments for this disease, and conventional disease control techniques have shown limited efficacy. This work presents an in silico evaluation of using specifically targeting anti-microbial peptides (STAMPs) consisting of a targeting segment and an antimicrobial segment to inhibit citrus greening by inhibiting the BamA protein of CLas, which is an outer membrane protein crucial for bacterial viability. Methods and results Initially, a set of peptides with a high affinity toward BamA protein were screened and evaluated via molecular docking and molecular dynamics simulations and were verified in vitro via bio-layer interferometry (BLI). In silico studies and BLI experiments indicated that two peptides, HASP2 and HASP3, showed stable binding to BamA. Protein structures for STAMPs were created by fusing known anti-microbial peptides (AMPs) with the selected short peptides. The binding of STAMPs to BamA was assessed using molecular docking and binding energy calculations. The attachment of high-affinity short peptides significantly reduced the free energy of binding for AMPs, suggesting that it would make it easier for the STAMPs to bind to BamA. Efficacy testing in vitro using a closely related CLas surrogate bacterium showed that STAMPs had greater inhibitory activity than AMP alone. Conclusions In silico and in vitro results indicate that the STAMPs can inhibit CLas surrogate Rhizobium grahamii more effectively compared to AMPs, suggesting that STAMPs can achieve better inhibition of CLas, potentially via enhancing the site specificity of AMPs. [ABSTRACT FROM AUTHOR]

Trunk injection of chemical compounds is a target-specific method of applying therapeutics that increases applied compound efficiency without compromising environmental safety and has proven to be an effective method to control citrus Huanglongbing (HLB). However, conventional (manual) trunk injection methods are labour-intensive and time-consuming, resulting in high application costs and posing implementation challenges for commercial citrus growers. For this reason, an automated, drill-free system for injecting therapeutic materials into citrus trees to control HLB has been developed. The injection mechanism consists of an end effector and a retractable telescoping positioning arm mountable to a farm vehicle. The system employs a metering pump with adjustable maximum pressure and flow rate for injecting therapeutic materials into tree trunks. This novel technology was evaluated in commercial citrus orchards; the applied volume, injection rate, and injection pressure were measured, and differences based on the time of injection, trunk diameter at the injection point, and irrigation status were examined. The injection was performed until the flow stopped; the injection duration ranged from 30 s to 72 s for all trees. The minimum and maximum volume injected was 53.9 mL and 243.6 mL, respectively, at 44 s and 68 s. The average injection rate per tree ranged from 1.23 mL s−1 to 3.58 mL s−1, and cut-off pressure ranged from 0.97 MPa to 2.01 MPa. • An automated drill-free mechanism for rapid trunk injection has been developed. • The device is mountable to farm vehicles to supply therapeutic materials to trees. • This novel technology was evaluated in commercial citrus orchards. • This is the first automated trunk injection system developed for tree crops. [ABSTRACT FROM AUTHOR]

The citrus root and rhizosphere microbiomes have been relatively well described in the literature, especially in the context of Huanglonbing disease. Yet questions addressing the assembly of root microbial endophytes have remained unanswered. In the above ground tree tissues, leaves and stems have been the research focus point, while flush and flower microbiomes, two important tissues in the vegetative and reproductive cycles of the tree, are not well described. In this study, the fungal and bacterial taxa in five biocompartments (bulk soil, rhizosphere, root endosphere, flower and flush) of citrus trees grown in a single California orchard were profiled using an amplicon-based metagenomic Illumina sequencing approach. Trees with no observable signs of abiotic or biotic stresses were sampled for two consecutive years during the floral development phase. The rhizosphere was the most biodiverse compartment compared to bulk soil, root endosphere, flower and flush microbiomes. In addition, the belowground bacteriome was more diverse than the mycobiome. Microbial richness decreased significantly from the root exosphere to the endosphere and was overall low in the above ground tissues. Root endophytic microbial community composition shared strong similarities to the rhizosphere but also contained few taxa from above ground tissues. Our data indicated compartmentalization of the microbiome with distinct profiles between above and below ground microbial communities. However, several taxa were present across all compartments suggesting the existence of a core citrus microbiota. These findings highlight key microbial taxa that could be engineered as biopesticides and biofertilizers for citriculture.

Citri Reticulatae Pericarpium, especially the pericarp of Citrus reticulata Blanco cv. Chachiensis (PCRC), is an important edible and medicinal ingredient for health and pharmacological properties. Citrus Huanglongbing, a devastating disease that currently threatens the citrus industry worldwide, is caused by a phloem-limited alpha-proteobacterium, "Candidatus Liberibacter asiaticus" (CLas). The industry of cultivar Chachiensis has been suffering from HLB. Although HLB affected the quality of citrus fruit, whether the quality of PCRC was affected by HLB remains unclear. In this study, we compared the metabolite profiles between HLB-affected and healthy PCRC from three sources: fresh, 6-month-old, and 9-year-old PCRC, through the untargeted LC–MS method. Compared to healthy controls, various types of bioactive compounds, mainly flavonoids, terpenoids, alkaloids, coumarins, polysaccharides, and phenolic acids, accumulated in HLB-affected PCRC, especially in the HLB-affected 9-year PCRC. In particular, isorhamnetin, isoliquiritigenin, luteolin 7-O-beta-D-glucoside, limonin, geniposide, pyrimidodiazepine, scoparone, chitobiose, m-coumaric acid, malonate, and pantothenic acid, which contributed to the pharmacological activity and health care effects of PCRC, were highly accumulated in HLB-affected 9-year-old PCRC compared to the healthy control. Multibioassay analyses revealed that HLB-affected 9-year-old PCRC had a higher content of total flavonoids and total polyphenols and exhibited similar antioxidant capacity as compared to healthy controls. The results of this study provided detailed information on the quality of HLB-affected PCRC. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is a major disease of citrus associated with phloemlimited bacteria in the genus Candidatus Liberibacter that affects all known citrus species and relatives, with many commercial cultivars being greatly damaged. Testing cultivar tolerance to HLB in field conditions is difficult because of the erratic spread of the bacteria, scion and rootstock interactions, and influence of many biotic and abiotic factors on the tree response to the disease. This study aimed to validate the effect of CLas infection on different citrus species and hybrids thought to have different levels of tolerance to the disease using CLas graft inoculation under controlled greenhouse conditions. Young potted seedlings from 12 different citrus germplasm selections were graft-inoculated with CLas or mock-inoculated. Plants were monitored periodically during 18 months for canopy growth, HLB and nutritional leaf symptoms, and leaf CLas titers. The leaf nutrient content was measured at the end of the experiment. Roots were also assessed at 18 months after inoculation (mai) for CLas titers and biomass distribution. There were significant differences in most analyzed variables of healthy and infected plants. Some plants of all cultivars were successfully infected; however, overall, the CLas transmission rate was low and inconsistent. Ct values of roots were generally higher than those in leaves at 18 mai. HLB symptoms were not observed on seedlings until 1 year after inoculation; at 18 mai, infected trees of all cultivars were HLB symptomatic. Significant shoot and root biomass reductions (44%-75%) in infected 'Cleopatra', 'Duncan', 'Olinda Valencia', 'Sunburst', and 'Valencia 1-14-19', considered susceptible to HLB, were measured. These cultivars also showed more severe HLB symptoms than the presumed tolerant cultivars such as Microcitrus inodora, Rich 16-6 trifoliate orange, and US-897. This study provides new knowledge of the efficacy and value of greenhouse screening of citrus germplasm for response to HLB to support the development of new cultivars with improved HLB tolerance or resistance. [ABSTRACT FROM AUTHOR]

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

Diaphorina citri is a global citrus pest. As a vector insect, it can transmit the causative agents of citrus huanglongbing, causing irreversible losses to the citrus industry. The acquisition of genomic information can provide a molecular genetic basis for effective control of D. citri. Here, the DNBSEQ™, Oxford Nanopore Technologies, and Hi‐C technologies are applied to generate a high‐quality chromosome‐level genome of D. citri. The genome size of D. citri was 523.78 Mb with a scaffold N50 of 47.05 Mb distributed on 13 chromosomes. A total of 250.64 Mb (47.85%) repeat sequences and 24 048 protein‐coding genes were predicted. Genome resequencing of female and male individuals indicated that the sex chromosome system of D. citri is XO. Phylogenetic analysis demonstrated that D. citri and Pachypsylla venusta, which separated from their most recent common ancestor about 336.62 million years ago, were the most closely related. Additionally, we identified genes potentially involved in detoxification metabolism, pathogen transmission, and honeydew secretion for further investigation. The high‐quality genome provides an important reference for developing effective management strategies of D. citri. [ABSTRACT FROM AUTHOR]

• A protocol for simplified crude citrus plant extract prepared in phosphate and NaOH:EDTA buffer. • A simplified RPA-based detection of Candidatus Liberibacter asiaticus (CLas). • Developed assay could detect the CLas infection with sufficient sensitivity. • Developed RPA assay was field and was more sensitive compared to standard PCR. 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. [ABSTRACT FROM AUTHOR]

[Objective] This study aims to investigate the rhizosphere soil bacterial community of Citrus reticulata plants in the Nanping region of Fujian province, China. The objective is to analyze the impact of Huanglongbing（HLB）disease on the bacterial community and provide a scientific basis for HLB management strategies in the Nanping region.[Method] We collected root-zone soil samples from both healthy and HLB-affected C. reticulata plants using the five-point sampling method. We used amplicon sequencing technology and bioinformatics methods to investigate the changes in diversity and composition of the bacterial community in the root-zone soil of HLB-affected C. reticulata plants. Additionally, we analyzed the correlation between soil physicochemical factors and the bacterial community. [Result] The findings of the study demonstrate that the diversity of the bacterial community in the root-zone soil of C. reticulata plants affected by HLB was slightly higher when compared to the microbiota of healthy plants. Specifically, there was a significant increase in the relative abundance of the Proteobacteria, and a significant decrease in the relative abundance of the Actinobacteriota in the root-zone soil of HLB-affected C. reticulata plants. At the genus level, it was observed that the relative abundance of the Roseiarcus and Acidiphilium significantly increased in the bacterial community of HLB-affected C. reticulata plants. Furthermore, these genera presented a significant negative correlation with available potassium, available phosphorus, and organic matter, while showing a significantly positive correlation with pH values. Conversely, the relative abundance of the Conexibacter and Chujaibacter were significantly lower in HLB-affected plants than healthy ones. Additionally, these genera demonstrated a significantly positive correlation with available potassium, available phosphorus, and organic matter, but a significant negative correlation with pH values.[Conclusion] The infection of the HLB pathogen altered the physicochemical properties of citrus soil and significantly reduced the abundance of beneficial bacteria in the rhizosphere soil. This, in turn, leads to considerable changes in both the diversity and composition of the rhizosphere bacterial community in C. reticulata plants. Concurrently, HLB-infected C. reticulata plants appear to counteract pathogen invasion by recruiting beneficial bacteria that not only fix nitrogen but also promote plant growth in the rhizosphere. [ABSTRACT FROM AUTHOR]

Although huanglongbing (HLB) is a devastating citrus disease, improved tolerant cultivars, such as Sugar Belle (SB) mandarin, have been identified. To understand the responses that HLB-affected SB undergoes, we compared 14CO2 fixation, carbohydrate export, phloem callose accumulation, relative expression of plant defense activators, and anatomical changes between healthy and infected SB trees versus susceptible Pineapple (PA) sweet orange. Eight- to ten-week-old leaves of infected SB showed a 2.5-fold increase in 14CO2 fixation and a 13% decrease in 14C-carbohydrate export, whereas HLB-affected PA presented a decrease of 33 and 50%, respectively. The mean distance of a callose deposit to its closest neighbor was 36% smaller in infected SB versus healthy, whereas in HLB-affected PA, it was 33% higher. Expression of papain-like cysteine proteases (PLCPs) was upregulated in SB but downregulated in PA. Infected SB showed minor alterations in the number of xylem vessels, a 16% larger xylem vessel lumen area, and a 14% increase in the proportional area of the xylem. In contrast, PA showed a 2.4-fold increase in the xylem vessel number and a 2% increase in the proportional xylem area. Three complementary mechanisms of tolerance in SB are hypothesized: (i) increased carbohydrate availability induced by greater CO2 fixation, mild effect in carbohydrate export, and local accumulation of callose in the phloem; (ii) activation of defense response via upregulation of PLCPs, and (iii) increased investment in the xylem structure. Thus, phloem and xylem modifications seem to be involved in SB tolerance. [ABSTRACT FROM AUTHOR]

Citrus Huanglongbing (HLB) is one of the most serious bacterial diseases in the citrus industry. Starch and flavonoids are two organic materials that are associated with HLB affection. Thermotherapy can be effective in eliminating the "Candidatus Ldiberibacter asiaticus" (CLas), the putative pathogen of HLB. However, the previous thermotherapy cannot fully meet the large-scale production, due to the low efficiency, high energy consumption, or with high cost. In this study, a microwave heat treatment was proposed to control the citrus HLB. A systematic investigation was implemented to explore the inhibitory effect of microwave heat treatment on the physiological materials of HLB-affected periwinkle plants. Firstly, a batch of 30-day-old periwinkles seedlings were cultivated and then graft-inoculated in an insect-proof screen house. Secondly, a microwave thermotherapy platform was built preliminarily. A single-factor test was conducted on three microwave parameters, including the rotating speed of the turntable (RST), microwave power (MP), and number of magnetrons (NM). The parameter levels were determined after the orthogonal test, according to the degree of overheating. Thirdly, the orthogonal test of L9(34) was carried out. Range and analysis of variance (ANOVA) were performed on the three indicators i.e., plant overheating degree, temperature range, and activity evaluation. The optimal combination of parameters was determined using the comprehensive balance. Finally, the optimized microwave treatment was used for thermotherapy on HLB-affected periwinkle plants. CLas titers were determined by real-time quantitative PCR. The starch and flavonoid concentrations of the leaves from the treated and untreated plants were detected within 90 days after heat treatment. The results show that: 1) The factor values for the L9(34) orthogonal test were obtained after single factor testing: RST of 10, 15, and 20 r/min, MP of 150, 200, and 250 W, and NM of 2, 3, and 4. The range analysis showed that the MP was the most important influencing factor on heating, followed by NMs, and finally RSP. 2) Combined with the ANOVA, the optimal combination was determined to be MP of 150 W with 3 magnetrons, where the value range of RST was from 15 r/min. The Ct value of infected samples increased from 17.01±0.97 before treatment to 31.91±2.35 at 90 days post-treatment, with the titers of CLas decreased by 99.98% after treatment with the optimized. 3) The previously CLas-infected plants were recovered after heat treatment, compared with the untreated plants, with more new leaves and lateral branches. The contents of starch and flavonoid were gradually recovered to the levels in the healthy control plants. Comparatively, the starch content of healthy samples increased significantly on the day of heating but gradually recovered to normal level. While there was no significant difference in the total flavonoid content. The flavonoid content in the untreated HLB-affected control was lower than that of the healthy control. The flavonoid content continued to increase until died, as time went by. In conclusion, 1) most CLas in periwinkle plants were eliminated to recover the growth conditions of plants into the healthy control level after microwave heat treatment. 2) Microwave heat treatment gradually restored the starch and flavonoid contents of infected samples, compared with the healthy control and the untreated diseased plants. 3) The platform of microwave heat treatment can be effective for pot-growing periwinkle seedlings. The microwave heat treatment is also required for long-term verification with the different conditions in citrus trees in the future. 4) Microwave heat treatment performed the better inhibitory effect on CLas, with the low cost, short treatment cycle, low consumption and high efficiency. It is also expected to achieve large-scale promotion in the field. [ABSTRACT FROM AUTHOR]

Citrus is affected by many diseases, and hence, the movement of citrus propagative materials is highly regulated in the USA. Currently used regulatory pathogen detection methods include biological and laboratory-based technologies, which are time-consuming, expensive, and have many limitations. There is an urgent need to develop alternate, rapid, economical, and reliable testing methods for safe germplasm exchange. Citrus huanglongbing (HLB) has devastated citrus industries leading to an increased need for germplasm exchanges between citrus growing regions for evaluating many potentially valuable hybrids for both HLB resistance and multilocational performance. In the present study, Next-Generation Sequencing (NGS) methods were used to sequence the transcriptomes of 21 test samples, including 15 well-characterized pathogen-positive plants. A workflow was designed in the CLC Genomics Workbench software, v 21.0.5 for bioinformatics analysis of the sequence data for the detection of pathogens. NGS was rapid and found to be a valuable technique for the detection of viral and bacterial pathogens, and for the discovery of new citrus viruses, complementary to the existing array of biological and laboratory assays. Using NGS methods, we detected beet western yellows virus, a newly reported citrus virus, and a variant of the citrus yellow vein-associated virus associated with the "fatal yellows" disease. [ABSTRACT FROM AUTHOR]

Objective: The objective of this investigation is to identify huanglongbing (HLB) in the field and know their control altenatives. Design/methodology/approach: Was investigated the introduction of the phytopathogen Candidatus Liberibacter, symptoms and control of HLB. The generally observed symptoms of this disease are irregular yellowing in the leaves and reversed ripening on the fruit. Results: According to various authors, an integrated control of the disease must be carried out, such as pest control, production of seedlings in certified nurseries, a suitable nutrition management and working together with educational and research institutions. Limitations on study/implications: HLB or citrus greening is the most devastating disease of citrus trees which has caused millions of dollars in losses worldwide. However, although there are several research, there is still no established cure. Findings/conclusions: Research such as that carried out by geneticist Hailing Jin at the University of California Riverside can give hope for the cure or control of this disease, however, there are limitations to this product since it is not yet commercialized. [ABSTRACT FROM AUTHOR]

The evolution of insect vector‐pathogen relationships has long been of interest in the field of molecular ecology. One system of special relevance, due to its economic impacts, is that between Diaphorina citri and 'Candidatus Liberibacter asiaticus' (CLas), the cause of the severe Asian form of huanglongbing. CLas‐positive D. citri are more fecund than their CLas‐negative counterparts, boosting opportunities for pathogens to acquire new vector hosts. The molecular mechanism behind this life‐history shift remains unclear. Here, we found that CLas promoted ovarian development and increased the expression of the vitellogenin receptor (DcVgR) in ovaries. DcVgR RNAi significantly decreased fecundity and CLas titer in ovaries, extended the preoviposition period, shortened the oviposition period and blocked ovarian development. Given their importance in gene regulation, we explored the role of miRNAs in shaping these phenotypes and their molecular triggers. Our results showed that one miRNA, miR‐275, suppressed DcVgR expression by binding to its 3' UTR. Overexpression of miR‐275 knocked down DcVgR expression and CLas titer in ovaries, causing reproductive defects that mimicked DcVgR knockdown phenotypes. We focused, further, on roles of the Juvenile Hormone (JH) pathway in shaping the observed fecundity phenotype, given its known impacts on ovarian development. After CLas infection, this pathway was upregulated, thereby increasing DcVgR expression. From these combined results, we conclude that CLas hijacks the JH signalling pathway and miR‐275, thereby targeting DcVgR to increase D. citri fecundity. These changes simultaneously increase CLas replication, suggesting a pathogen‐vector host mutualism, or a seemingly helpful, but cryptically costly life‐history manipulation. [ABSTRACT FROM AUTHOR]

Citrus greening disease, or Huanglongbing (HLB), has devastated citrus crops globally in recent years. The causal bacterium, 'Candidatus Liberibacter asiaticus', presents a sampling issue for qPCR diagnostics and results in a high false negative rate. In this work, we compared six metabolomics assays to identify HLB-infected citrus trees from leaf tissue extracted from 30 control and 30 HLB-infected trees. A liquid chromatography-mass spectrometry-based assay was most accurate. A final partial least squares-discriminant analysis (PLS-DA) model was trained and validated on 690 leaf samples with corresponding qPCR measures from three citrus varieties (Rio Red grapefruit, Hamlin sweet orange, and Valencia sweet orange) from orchards in Florida and Texas. Trees were naturally infected with HLB transmitted by the insect vector Diaphorina citri. In a randomized validation set, the assay was 99.9% accurate to classify diseased from nondiseased samples. This model was applied to samples from trees receiving plant defense-inducer compounds or biological treatments to prevent or cure HLB infection. From two trials, HLB-related metabolite abundances and PLS-DA scores were tracked longitudinally and compared with those of control trees. We demonstrate how our assay can assess tree health and the efficacy of HLB treatments and conclude that no trialed treatment was efficacious. [ABSTRACT FROM AUTHOR]

Candidatus Liberibacter asiaticus (CLas) is associated with Citrus Huanglongbing (HLB), a devastating disease in the US. Previously, we conducted a two-year-long monthly HLB survey by quantitative real-time PCR using root DNA fractions prepared from 112 field grapefruit trees grafted on sour orange rootstock. Approximately 10% of the trees remained CLas-free during the entire survey period. This study conducted 16S metagenomics using the time-series root DNA fractions, monthly prepared during twenty-four consecutive months, followed by microbial co-occurrence network analysis to investigate the microbial factors contributing to the CLas-free phenotype of the aforementioned trees. Based on the HLB status and the time when the trees were first diagnosed as CLas-positive during the survey, the samples were divided into four groups, Stage H (healthy), Stage I (early), II (mid), and III (late) samples. The 16S metagenomics data using Silva 16S database v132 revealed that HLB compromised the diversity of rhizosphere microbiota. At the phylum level, Actinobacteria and Proteobacteria were the predominant bacterial phyla, comprising >93% of total bacterial phyla, irrespective of HLB status. In addition, a temporal change in the rhizosphere microbe population was observed during a two-year-long survey, from which we confirmed that some bacterial families differently responded to HLB disease status. The clustering of the bacterial co-occurrence network data revealed the presence of a subnetwork composed of Streptomycetaceae and bacterial families with plant growth-promoting activity in Stage H and III samples. These data implicated that the Streptomycetaceae subnetwork may act as a functional unit against HLB. [ABSTRACT FROM AUTHOR]

Due to human activities and changes in land use, the spatiotemporal pattern of citrus has undergone significant changes after the outbreak of Huanglongbing (HLB). We selected time-series Sentinel-2 images to delineate citrus orchard areas following the onset of HLB. This was conducted to extract citrus orchards in Xunwu County between 2017 and 2022. The spatial and temporal patterns and their influencing factors were investigated by spatial analysis. Results show (1) a notable decline in total citrus orchard area by 216.70 km2, primarily witnessed in orchards without insect-proof screens (IPS), shifting towards cropland, bush, and IPS areas. Contrastingly, citrus orchards with IPS exhibited a modest increase from 7.82 km2 to 111.39 km2, predominantly converting from areas lacking IPS, cropland, and bare land. (2) Spatial distribution patterns revealing a "cold in the south and hot in the north" trend. Orchards without IPS are concentrated in central and northern regions, while those with IPS are clustered predominantly in the north, with a recent shift towards the northeast. (3) Landscape analysis indicating a trend of fragmentation of citrus orchards, while a gradual dispersion of orchards without IPS and those with IPS showcased enhanced concentration and aggregation. (4) Orchards with IPS predominantly occupy regions characterized by an elevation ranging between 300 m and 400 m, primarily in the southeast, southwest, and southern directions. These areas exhibit slopes averaging between 10° and 15°, with surface temperatures ranging from 18 °C to 26 °C. Additionally, these orchards tend to be situated in proximity to impervious surfaces and roads. [ABSTRACT FROM AUTHOR]

The arrival (2005) and rapid establishment in Florida of the most devastating disease known to citriculture, namely Huanglongbing or Citrus Greening Disease, has caused dramatic changes to the long-term citrus rootstock breeding program at the University of Florida's Citrus Research and Education Center (UF-CREC). Since all commercial scion/rootstock combinations have proven to be susceptible to the disease, there is an urgent need to quickly develop improved rootstocks with good horticultural attributes that can mitigate the disease in trees grafted with commercial scions. This report describes our efforts to achieve this goal. Our approach is to rapidly develop and screen a large population of genetically-diverse hybrid rootstock candidates. Multiple available sources of potential rootstock germplasm are being exploited in our efforts. We have identified several good rootstock candidates that are showing improved HLB tolerance. These are now being propagated either by seed or micropropagation as needed for advanced multi-site trials with multiple scions. This research offers potential for the ultimate solution to HLB, leading to sustainable and profitable citriculture in HLB-endemic areas. [ABSTRACT FROM AUTHOR]

Citrus is one of the most genetically diverse fruit trees used by humans, and yet few rootstock breeders have ventured beyond a narrow range of parents. For the last 25 years we have explored wider graft and sexual compatibility within the citrus subfamily (Aurantioideae) than any previous attempts, and have identified new genera and species for rootstock breeding. Whilst the vast majority of this work has met with failure, it has identified factors such as sexual incompatibility, late-acting lethality and poor field adaptation as the reasons why many citrus relatives are not currently amenable to rootstock breeding. A major breakthrough early in the program was the discovery that Oceania citrus species are extremely sensitive to Citrus tristeza virus (CTV) when used as rootstocks, and the realisation that resistance must be introgressed if we are to ever discover useful traits masked by this disease sensitivity. Bridging hybrids were required to transfer this virus resistance and a newly discovered species (Citrus wakonai) was employed to speed-up the process. After three generations of crossing, our "extreme hybrids" with Oceania parentage now show commercial performance equivalent to industry standards. By providing citrus growers with a range of high-performance rootstocks from extremely complex genetic backgrounds we can increase the biological diversity of orchards without compromising production. Our hope is that these "extreme hybrids" may help in the battle against Huanglongbing (HLB) disease. [ABSTRACT FROM AUTHOR]

Increased preharvest fruit drop rates and reduced fruit sizes are common symptoms associated with Huanglongbing (HLB) in sweet orange. Small fruit may be more likely to drop during preharvest fruit drop. The objective of the current study was to determine whether fruit size could be used as an indicator of the preharvest fruit drop likelihood. Nearly 1400 fruit were surveyed over the following three timepoints across two years: 11 Mar 2022, 15 Apr 2022, and 22 Mar 2023. Each fruit was evaluated to determine the equator and peduncle diameter, fruit detachment force (FDF), type of detachment (mechanically broken or abscission), and lopsidedness. The total soluble solids content was determined for a subset of fruit. The FDF was consistently lower in abscising fruit than in mechanically broken fruit, suggesting that the physiological process of abscission had begun in these fruit and that they were more likely to drop. The fruit diameter was significantly smaller in abscising fruit than in mechanically broken fruit on 11 Mar 2022 and 22 Mar 2023, but not on 15 Apr 2022. Similarly, the fruit diameter and FDF were also significantly and positively correlated, but this relationship was weaker at time points late in the season (closer to harvest). These findings suggest that small fruit are indeed more likely to drop early in the preharvest period. The abscising fruit had total soluble solids contents similar to those of the mechanically broken fruit. Therefore, we hypothesized that the smaller fruit ceased growing and are able to respond to abscission signals earlier than the larger fruit. Therefore, any strategies to mitigate fruit drop, such as the use of plant growth regulators, should be applied early in the season when the fruit are still actively growing. When growth ceases, the fruit are vulnerable to drop.

ABSTRACT The ‘Tahiti’ acid lime and orange trees are hosts of ‘Candidatus Liberibacter asiaticus’ (CLas), the pathogen associated with the severe Asian form of huanglongbing (HLB), the most devasting citrus disease. They are also hosts of the vector of CLas, the Asian citrus psyllid (ACP) Diaphorina citri Kuwayama. Relatively small numbers of lime trees occur in gardens and small orchards near large commercial ‘Valencia’ sweet orange orchards in Brazil. Applications of insecticides to suppress populations of ACP on the lime trees are usually nil or less frequent than in the orange orchards. Abundance of the psyllid on lime trees may therefore increase the risk of CLas spreading to the orchards. Because the abundance of the psyllid is influenced by the suitability of the trees as hosts, we compared reproductive potential of the insect on the two hosts in a controlled environment chamber (CEC) and in a greenhouse (GH). Daily temperature and relative humidity averaged 22ºC and 60% inside the CEC and 24°C and 70% inside the GH. Two pairs of adult male and female psyllids were caged for 3 days on new shoots and the fecundity and durations of development and survival of eggs and nymphs evaluated. Overall, acid ‘Tahiti’ was 3.5 times less suitable to ACP than ‘Valencia’. Fecundity and survival of nymphs were 27% and 59% lower, and the life cycle 34% longer on ‘Tahiti’ than on ‘Valencia’. Potential impacts of the results on CLas spread and HLB control are discussed.

Abstract Introduction The application of host‐derived antibacterial peptides has been highlighted as a potential efficacious and safe tool for the treatment of Huanglongbing (HLB), the most devastating disease of citrus. However, pathogenic bacteria such as HLB‐causing Candidatus liberibacter asiaticus (CLas) often develop resistance against the host antibacterial peptides. We showed that chimeras containing two different host antibacterial peptides not only retain antibacterial activity but also overcome bacterial resistance and enhance plant defence responses. Also, chimeric peptides can have an off‐target impact on the structure and function of plant‐associated microbiomes. However, there is a lack of understanding of the impact of the chimeric peptide therapy on the microbial structure in the citrus phyllosphere while reducing the CLas titre. Here, we aim to evaluate the efficacy of a chimeric peptide (UGK17) to reduce CLas titre, inducing plant defence response and impacting the microbiome associated with the citrus phyllosphere. Material and Method Leaf samples were collected from orange and grapefruit trees in Texas and identified as old and young leaves according to their maturity. We collected three different types of leaves based on their infection and symptoms: healthy, symptomatic (infected with typical symptoms), and asymptomatic (infected without symptoms). In planta assay was performed by dipping the leaves in the 0, 5 and 25 μM of UGK17 solutions for 48 h. The quantifications of CLas titre and pathogenesis‐related (PR) gene expression were done by quantitative polymerase chain reaction (qPCR) and reverse transcription‐qPCR, respectively. Amplicon sequencing was done to evaluate the impact of UGK17 on individual bacterial community structures. In addition, we performed an ex planta assay to assess the effect of UGK17 on the growth of bacterial isolates including Liberibacter crescens instead of unculturable CLas predominant in the phyllosphere. Result The UGK17 treatment reduced the CLas titre in both asymptomatic and symptomatic citrus leaves, regardless of the age of the leaves. The UGK17 application augmented the PR gene expression. In ex planta assay, the growth of L. crescens along with four other strains belonging to the family Rhizobiaceae, was significantly inhibited by the UGK17 while the growth of 74 strains were unaffected. Additionally, there was no statistically significant changes in the microbial community structure with UGK17 treatment. Conclusion Our results suggest that the chimeric peptide therapy is a promising solution to combat HLB by targeting mainly Gram‐negative pathogens and enhancing the plant immune responses without impairing the indigenous microbial community in the citrus phyllosphere.

Abstract Objectives “Candidatus Liberibacter asiaticus” (CLas) is an un-culturable α-proteobacterium that caused citrus Huanglongbing (HLB), a destructive disease threatening citrus production worldwide. In China, the presence of HLB was first reported in Chaoshan region of Guangdong province, China around a century ago. Thus, whole genome information of CLas strains from Chaoshan area become the most important resource to understand the population diversity and evaluation of CLas in China. Data description CLas strain GDCZ was originally from Chaozhou city (Chaoshan area) and sequenced using PacBio Sequel long-read sequencing and Illumina short-read sequencing. The genome of strain GDCZ comprised of 1,230,507 bp with an average G + C content of 36.4%, along with a circular CLasMV1 phage: CLasMV1_GDCZ (8,869 bp). The CLas strain GDCZ contained a Type 2 prophage (37,452 bp) and encoded a total of 1,057 open reading frames and 53 RNA genes. The whole genome sequence of CLas strain GDCZ from the historical HLB endemic region in China will serve as a useful resource for further analyses of CLas evolution and HLB epidemiology in China and world.