Huanglongbing Research Articles

Impact of Direct Contact and Ingestion of Selected Insecticides on the Predator Harmonia axyridis of Citrus Psyllids

The Asian citrus psyllid, Diaphorina citri, serves as the primary vector for Huanglongbing (HLB) by transmitting Candidatus Liberibacter asiaticus. Lambda-cyhalothrin and thiamethoxam are commonly employed for the control of D. citri. The multicolored Asian lady beetle, or harlequin ladybird, Harmonia axyridis, is an important predator of D. citri in both greenhouse and field settings. The effectiveness of integrated pest management (IPM) relies on the judicious use of selective insecticides that minimize harm to natural enemies. However, the effects of these insecticides on H. axyridis’ predation of D. citri remain thoroughly unexplored. In this study, we assessed the toxicity of lambda-cyhalothrin and thiamethoxam to H. axyridis and examined their impact on the functional response of this predator to D. citri using direct contact exposure methods. We also evaluated the indirect effects on predator voracity through ingestion exposure. Our results demonstrated that exposure to both insecticides at LC50 concentrations significantly prolonged the developmental durations of H. axyridis larval stages. The type-II functional response model effectively described the prey consumption patterns of H. axyridis, revealing a significant reduction in predation capacity across all life stages, particularly among second instar larvae, which experienced reductions of 85.30% and 88.58% following lambda-cyhalothrin and thiamethoxam treatments, respectively. Furthermore, H. axyridis’ predation significantly declined when feeding on D. citri contaminated at LC50 concentrations. These findings indicate that lambda-cyhalothrin and thiamethoxam adversely affect the predation of H. axyridis, both via direct contact and ingestion. Evaluating the potential impacts of these insecticides on H. axyridis is critical for the development of effective IPM strategies targeting D. citri.

Exploring the dual roles of sec-dependent effectors from Candidatus Liberibacter asiaticus in immunity of citrus plants.

The three SDEs of CLas were expressed in citrus leaves by AuNPs-PEI mediated transient expression system, and promoted the proliferation of CLas and inhibited citrus immunity. Huanglongbing (HLB) is the most severe bacterial disease of citrus caused by Candidatus Liberibacter asiaticus (CLas). CLas suppress host immune responses and promote infection by sec-dependent effectors (SDEs), thus insight into HLB pathogenesis is urgently needed to develop effective management strategies. In this study, we focused on the roles of SDE4310, SDE4435 and SDE4955 in citrus. We found that the expression of SDE4310, SDE4435 and SDE4955 to increase with increasing citrus immune genes CsPR1, CsPR2, CsPR5, CsNPR1, CsRBOHD, CsMAP3K and CsBIK1, suggesting that the level of citrus immunity could be judged by the expression of SDE. To further explore the relationship between these three SDEs and citrus immunity, we established a transient expression system in citrus leaves, using gold nanoparticle-polyethyleneimine (AuNPs-PEI) to deliver recombinant plasmid containing SDE4310, SDE4435 or SDE4955 respectively into citrus leaves. Results showed that SDE4310, SDE4435 and SDE4955 were successfully expressed in citrus leaves using this transient expression system, and found that SDE4310, SDE4435 and SDE4955 could promote the CLas proliferation by decreasing the immune gene expression of the citrus. Additionally, we used AuNPs-PEI to deliver siRNA4310 to citrus cells, significantly reducing the expression of SDE4310 within 3days. Although the suppression of SDE4310 expression did not inhibit the CLas proliferation, it increased the expression level of CsPR1, CsNPR1 and CsBIK1. This is also the first time that AuNPs-PEI has been found to be able to deliver exogenous plasmids into citrus cells and express the target protein, providing a new method for future studies on citrus HLB.

Revolutionizing Citrus Health: AI-Based Detection of Greening Disease and Nutrient Deficiencies in Sweet Orange

This paper explores the application of Artificial Intelligence (AI) techniques for detecting plant diseases, focusing on citrus greening disease and various foliar nutrient deficiencies in sweet orange. Agriculture faces numerous challenges from cultivation to harvesting, including significant yield losses due to disease infections and environmental hazards from excessive use of insecticides and fungicides. As the global population grows, the demand for food is surging, and traditional farming methods fall short in meeting this demand, often degrading soil health through intensive pesticide use. AI offers significant advantages over conventional techniques in disease detection. This study employs AI for visual detection of citrus greening disease and foliar nutrient deficiencies, achieving 87% accuracy on a test dataset of infected, nutrient-deficient, and healthy sweet orange leaves. Performance is evaluated using metrics such as Accuracy, Recall, Precision, and F1-Score. Specifically, Convolutional Neural Network (CNN) architectures, including Visual Geometry Group (VGG-16), are utilized for image-based detection and classification, demonstrating the potential of AI in enhancing plant health monitoring.

Development and Validation of an RPA Assay for Detecting Xanthomonas citri subsp. citri A, A* and Aw and X. fuscans subsp. aurantifolii B and C

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc) and X. fuscans subsp. aurantifolii (Xfa), is a destructive disease of citrus. It is sparsely distributed in the Caribbean, and effective biosecurity measures, including accurate pathogen detection systems, are essential to prevent further spread. Current in-field screening methods rely on visual symptom identification and serological tools like Agdia’s X. axonopodis subsp. citri (Xac) ImmunoStrips®. However, they failed to detect the Aw and C pathotypes in this study. Nucleic acid amplification is the gold standard for confirming detection but is resource-intensive and less accessible in Caribbean plant diagnostic laboratories and regulatory services. To address this, a rapid recombinase polymerase amplification (RPA) assay was developed using a portable fluorometer and primers designed to detect all canker-causing pathotypes. The RPA assay accurately detected all five citrus canker pathotypes and was selective against Xanthomonas euvesicatoria pv. citrumelonis, the fastidious citrus greening bacteria Candidatus Liberibacter asiaticus, and the xylem-limited bacterial pathogen Xylella fastidiosa. However, X. perforans, X. citri pv. glycines, and X. citri pv. phaseoli, pathogens unrelated to citrus and unlikely to be present in citrus, were detected. Real-time detection was completed in 25 minutes with high sensitivity levels of 0.001 pg/µl (Xcc A), 0.1 pg/µl (Xfa B), and 1 pg/µl (Xfa C) of DNA. This RPA assay offers a robust, in-field detection method, combining molecular sensitivity and specificity with the field-friendly simplicity of crude extraction and single-tube reactions.

Genetic Diversity of Diaphorina citri and Its Endosymbiont Across Diffusion Frontier and Epidemic Areas of Citrus Huanglongbing in China

Citrus huanglongbing (HLB) is one of the most severe diseases affecting the citrus industry, with Diaphorina citri (Hemiptera: Liviidae) serving as its primary natural vector. To understand the genetic diversity and population structure of D. citri in the context of HLB diffusion, we analyzed 13 populations from the HLB diffusion frontier and 25 populations from epidemic areas in China. The HLB diffusion frontier areas refer to the peripheral regions of HLB distribution in China, including the western Zhejiang, southern Jiangsu, northern Jiangxi, northern Hunan, and eastern Sichuan provinces. In contrast, the HLB epidemic areas represent regions in China where HLB is actively widespread and causing significant impacts. We utilized mitochondrial genes (COI, ND5, and Cytb) of D. citri and housekeeping genes (dnaQ, rpoC, and argH) of its endosymbiont Candidatus Carsonella ruddii (Ca. C. ruddii) for this analysis. Our findings revealed that the D. citri and Ca. C. ruddii in different regions showed low haplotype diversity and nucleotide diversity. While the genetic variation in D. citri populations primarily occurred within populations, the endosymbiont showed contrasting patterns in the HLB epidemic areas. We identified three dispersal paths: (1) migration of the Yunnan population to Sichuan, Guizhou, and Guangxi; (2) movement of the Guangdong population to Fujian, Jiangxi, and Zhejiang; and (3) dispersal of the Guangdong population to Hunan and Guangxi. Our study suggests that D. citri populations at the HLB diffusion frontier are predominantly transmitted from neighboring epidemic areas.

Swords and shields: the war between Candidatus Liberibacter asiaticus and citrus.

Citrus Huanglongbing (HLB) represents a significant threat to the citrus industry, mainly caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas). In this review, we summarize recent advances in understanding the relationship between citrus and CLas, particularly examining the functions of Sec-dependent effectors (SDEs) and non-classically secreted proteins (ncSPs) in virulence, as well as their targeted interactions with citrus. We further investigate the impact of SDEs on various physiological processes, including systemic acquired resistance (SAR), reactive oxygen species (ROS) accumulation, vesicle trafficking, callose deposition, cell death, autophagy, chlorosis and flowering. Additionally, we focus on the functional research on specific disease-resistant genes in citrus and the molecular mechanisms underlying disease resistance. Finally, we discuss the existing gaps and unresolved questions regarding citrus-CLas interactions, proposing potential solutions to facilitate the development of HLB-resistant citrus varieties.

A Simple Diagnostic Method for Citrus Greening Disease With Deep Learning

ABSTRACTCitrus greening disease (CG) is the most destructive disease of citrus, leading to branch dieback and plant death. Currently, there is no cure for CG, the early detection and removal of infected trees is important to prevent the spread of the disease. In recent years, there have been growing expectations for CG detection with digital images, especially deep learning techniques applied to digitized herbarium specimen image data. However, this approach faces challenges in practical applicability and detection efficiency. In this paper, we proposed a simple diagnostic method for CG using transfer learning with the Faster RCNN architecture. We collected in‐field images from a citrus orchard in Thailand where CG has caused significant damage. We compared the performance of two annotation methods with the in‐field leaf dataset and discussed their effects on pre‐trained VGG and Resnet models. Five‐fold cross‐validation was utilized for model training and evaluation, with average precision (AP) used as the performance metric. The results showed that the Resnet models performed better than the VGG models, with the Resnet152 model scoring the highest in this task. The annotation method which included annotations of healthy and other disease leaves achieved an AP of 84.13% lower than another one but indicated better performance in practical applications with more robustness. Additionally, we developed a web application that performs real‐time diagnosis using our trained models and verified the effectiveness of our system.

Genomic Analysis of 'Candidatus Liberibacter africanus' Strain from Zimbabwe Reveals Unique Virulence and Prophage Characteristics Compared with 'Ca. L. asiaticus'.

Citrus Huanglongbing (HLB) is caused by the phloem-limited α-proteobacterium 'Candidatus Liberibacter spp.', among which 'Ca. L. africanus' (CLaf) has posed a significant threat to citrus production in Africa for nearly a century. CLaf is closely related to the globally prevalent 'Ca. L. asiaticus' (CLas), whereas little is known about the virulence of CLaf, primarily because of limited genome resources. In this study, we completed the whole-genome assembly and annotation of the CLaf strain Zim (from Zimbabwe). Compared with CLas, a total of 102 CLaf unique genes were identified, including 14 potential Sec-dependent effector (SDE) genes, 29 phage-associated genes, and 59 genes with hypothetical function. Among 14 SDEs, V9J15_03810 was able to induce a significant hypersensitive response in Nicotiana benthamiana, indicating its potential as a virulence factor for CLaf. Genome analysis showed that the 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. L. 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, V9J15_02010, V9J15_02015, V9J15_02025, and V9J15_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.

Evaluation of Legume Cover Crop Species for Citrus Production in Southeast Florida

Because of the low fertility soils native to southeast Florida and the high nutrient demand of citrus trees under citrus greening—an endemic bacterial disease impacting nutrient uptake—growers are returning to the use of cover crops to improve soil fertility. Cover crops, specifically legumes, can improve soil nitrogen (N) availability because of their ability to fix N from the atmosphere. More citrus growers in southeast Florida are growing cover crops; however, there is a lack of recent research of suitable legume species and their impact on soil N cycling. To address this gap in the literature, six different treatments consisting of five legume species monocultures and one fallow plot (control) were organized into a completely randomized design. The experiment was conducted twice under warm and cool season conditions, and each treatment was replicated five times (n = 5), for a total of 30 experimental plots. Legume species were hairy indigo (Indigofera hirsuta), sunn hemp (Crotalaria juncea), cowpea (Vigna unguiculata), alfalfa (Medicago sativa), and aeschynomene (Aeschynomene americana). Biomass production, N concentration, and nodule characteristics as well as their impact on soil N were measured over a 1-year span in both warm and cool seasons. Overall, both cowpea and hairy indigo produced more biomass and, as a result, higher tissue N compared with those of the other legumes at both 60 and 150 days after planting. However, no impact on soil N was observed. Additionally, all legumes were unable to survive in the cool season, resulting in no measurable biomass at 150 days after planting. These results are relevant for citrus growers aiming to enhance soil fertility through cover crops in southeast Florida. While several legume options are available for the warm season (e.g., cowpea, hairy indigo, and sunn hemp), suitable legume species for the cool season have yet to be identified.

Analysis of the Distribution Pattern and Prophage Types in Candidatus Liberibacter Asiaticus 'Cuimi' Kumquat.

The 'Cuimi' kumquat is a unique citrus cultivar known for its thin, crisp pulp and sweet, aromatic flavor. In addition to its use in fresh consumption and processing, this variety exhibits certain medicinal properties. This study aims to investigate the genetic diversity of the Huanglongbing (HLB) bacterium across different tissues of the 'Cuimi' kumquat, offering a theoretical basis for understanding the HLB epidemic in Dechang County, Sichuan. The research focuses on the absolute quantification of the HLB bacterium in seven specific tissues of the 'Cuimi' kumquat, including new leaves, upper phloem of branches, fruit peduncle, pith, fruit axis, old leaves, and lower phloem of branches. Additionally, the types and contents of prophages were identified in these tissues. In the same diseased branch group, Candidatus Liberibacter asiaticus (CLas) exhibited an uneven distribution, with the highest concentration detected in the pith, significantly surpassing levels found in the stem and leaf tissues (new leaves, upper phloem of branches, old leaves, lower phloem of branches). Infected fruit peduncles and pith slices showed noticeable shrinkage and collapse in the phloem. Prophage analysis indicated that multiple types of prophages could be simultaneously detected within the same infected 'Cuimi' kumquat branch. New shoot tissues contained both Type 2 and Type 4 prophages, with a relatively higher abundance of Type 4 and a lower abundance of Type 2. The relative abundance of Type 1 prophage in the fruit tissues was generally higher than in other tissues. CLas primarily accumulates in the fruit tissues of the 'Cuimi' kumquat, and the situation in Dechang County suggests that individual trees may be infected with multiple prophage strains simultaneously.

Enhanced Control Efficacy of Different Insecticides Mixed with Mineral Oil Against Asian Citrus Psyllid, Diaphorina citri Kuwayama, Under Varying Climates.

Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Liviidae), is one of the most devastating pests in citrus orchards due to its role in transmitting Huanglongbing (HLB). Currently, chemical control remains the most effective strategy for ACP management. Mineral oils are commonly used as insecticides or adjuvants in integrated pest management (IPM) practices. To extend the product life of synthetic pesticides, we evaluated the toxicity of chlorpyrifos (CPF), thiamethoxam (THX), or pyriproxyfen (PPF) mixed with mineral oil Lvying (LY) against ACP nymphs under different weather conditions. Individual application of CPF, THX, and PPF effectively controlled against ACP nymphs under no rain conditions, with mortality rates varying from 20 to 100% during 1-5 d after treatment. The addition of LY at 1.0% or 0.5% rates to CPF, THX, and PPF significantly enhanced their control efficacy, achieving sustained mortality rates from 60 to 100% during the same period. Light rain had less impact on the control efficacy of these insecticide mixtures compared to individual insecticides. However, cumulative rainfall above 20 mm significantly reduced the control efficacy of individual insecticides and their mixtures. The addition of LY decreased both surface tension and contact angle of THX solution on citrus leaves, thereby enhancing the solution's wetting and increasing THX deposition. Moreover, the rainfastness of THX was improved after adding LY, leading to a greater retention on the leaves. LY at a rate of 1.0% exhibited excellent efficacy against ACP nymphs, and observations using scanning electron microscopy (SEM) showed that LY altered ACP mouthpart morphology and blocked spiracles, likely contributing to its insecticidal effects. This study revealed that mineral oils can serve as both insecticides to combine with synthetic pesticides for enhancing toxicity against ACP and as adjuvants to facilitate the deposition and rainfastness of synthetic pesticides on leaves, which could be recommended for sustainable ACP management in citrus orchards.

Integrated Transcriptome and Metabolome Analysis Reveals Insights into Flavone and Flavonol Biosynthesis in Salicylic Acid-Induced Citrus Huanglongbing Tolerance.

Salicylic acid (SA) exhibits positive effects against Citrus Huanglongbing (HLB), but how SA affects citrus resistance to HLB is currently unknown. This study conducted integrated transcriptome and metabolome analyses on SA-treated Citrus sinensis (HLB-sensitive) and Poncirus trifoliata (HLB-tolerant). The results indicated that the syntheses of flavones and flavonols were induced by SA, while the expression levels of associated genes and the contents of corresponding metabolites varied significantly between the two species after SA treatment or HLB infection. These differences may underpin the enhanced HLB management through SA treatment and the inherent HLB tolerance of P. trifoliata. Furthermore, two insertions of miniature inverted-repeat transposable element (MITE) were identified within the promoter of PtrF3'H in P. trifoliata, whereas none were found in the promoter of CsF3'H in C. sinensis. These MITE insertions notably enhanced the promoter activity of PtrF3'H in an SA-dependent manner. Our findings deepen the understanding of the correlation between SA response and HLB tolerance in Citrus.

An insect virus differentially alters gene expression among life stages of an insect vector and enhances bacterial phytopathogen transmission.

Huanglongbing (HLB), caused by fastidious bacteria from three Candidatus Liberibacter species, is the most damaging disease impacting the citrus industry worldwide. Spread by the Asian citrus psyllid (Diaphorina citri) in Asia and the Americas, HLB causes substantial financial losses, and has reduced citrus production in Florida by more than 90%. Although there are ongoing efforts to limit spread of the disease, effective HLB management remains elusive. Suppressing vector populations and decreasing CLas transmission are the two strategies that need to be urgently improved. Recently, a D. citri flavi-like virus (DcFLV) was characterized within its D. citri host, and it co-occurs intracellularly with CLas in psyllid populations. Here, we show that viruliferous nymphs exhibit higher CLas acquisition than non-viruliferous nymphs. Furthermore, both viruliferous adults and nymphs exhibit increased CLas transmission efficiency. We suggest the possibility of manipulating DcFLV in D. citri populations to reduce CLas transmission for HLB disease management.

Investigation of the anti-Huanglongbing effects using antimicrobial lipopeptide and phytohormone complex powder prepared from Bacillus amyloliquefaciens MG-2 fermentation

Global citrus production has been severely affected by citrus Huanglongbing (HLB) disease, caused by Candidatus Liberibacter asiaticus (Clas), and the development of effective control methods are crucial. This study employed antimicrobial lipopeptide and phytohormone complex powder (L1) prepared from the fermentation broth of the endophytic plant growth promoting bacterium (PGPB) of Bacillus amyloliquefaciens strain MG-2 to treat Candidatus Liberibacter asiaticus (CLas)-infected ‘Citrus reticulata ‘Chun Jian’ plants. Real-time fluorescence quantitative polymerase chain reaction (qPCR) and PCR were employed for disease detection. The results revealed that after 15 spray-drench treatments with L1 solution, the HLB infection rate decreased from 100 to 50%, the bacterial titer decreased by 51.9% compared with a 27.9% decrease in the control group. L1 treatment triggered the production of reactive oxygen species, increased lignin content, and increased defense enzyme activities (p < 0.05). Defense-related gene expression significantly increased within 12 h of treatment. In addition, L1 application also promoted plant growth, as evidenced by higher transpiration rates and net photosynthetic rates as well as increased leave or root density. Root flora analysis revealed that the abundances of Burkholderia_thailandensis, unclassified_g_Burkholderia-Caballeronia-Paraburkholderia, unclassified_g__Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, and Pseudomonas_mosselii were 1.64, 1.46, 5.84, and 6.93 times greater, respectively, than those in the control group. The levels of phenylpropanoids, polyketides, lipids, lipid-like molecules, organic acids, and derivatives, significantly increased following L1 treatment (FC > 2, p < 0.05). Additionally, salicylic acid, dihydrojasmonic acid, and isopentenyl adenosine levels in leaves markedly increased. High-performance liquid chromatography (HPLC) confirmed that L1 contained surfactin, iturin and fengycin cyclic-lipopeptides (CLPs) as well as indole-3-acetic acid (IAA), 3-indolebutyric acid (IBA), indole-3-carboxylic acid and indole-3-carboxaldehyde auxins, N6-entopentenyladenine and t-zeatin-riboside cytokinins, abscisic acid, 1-aminocyclicpanecarboxylic acid, salicylic acid, and gibberellin A1, A3 and A4 phytohormones. These findings provide insight into multiple mechanisms by which endophytic Bacillus PGPB L1 is able to combat HLB disease, to promote citrus plant growth, and to optimize the root flora for soil health which offering an innovative strategy for sustainable management of this severe disease and improving citrus plant growth and productivity

Optimizing control parameters for Huanglongbing disease in citrus orchards using SAIR-SI compartmental model, epidemic final size, and genetic algorithms

Huanglongbing (HLB) is a bacterial disease that affects citrus trees worldwide. We present an innovative approach for identifying optimal control and risk measures for HLB in citrus orchards. Our method is based on a mathematical model that incorporates the number of roguing trees and a logistic growth model for the dynamic of the Asian Citrus Psyllid (ACP), the primary vector for HLB transmission. We derive an expression for: (1) the basic reproduction number R0; (2) the final size for the number of roguing trees; and (3) the transmission risk. The above let us propose a difference map equation that assesses this final size with a low computational cost. We use this difference map in an evolutionary algorithm to identify the most effective combination of control parameter values for reducing HLB transmission, including the timing and frequency of roguing and the use of insecticides. In this sense, we propose two control strategies, which we called tree-centered and vector-centered.

Isolation and characterization of plant growth stimulating bacteria Serratia marcescens from the microbial community of Huanglongbing (HLB) escape citrus trees in Punjab, Pakistan

Beneficial bacteria play a crucial role in combating Huanglongbing (HLB) disease, a global concern for the citrus industry around the globe. To explore the role of beneficial bacteria and enhance the productivity and sustainability of citrus against HLB disease, the bacteria associated with citrus were investigated. Samples were collected from HLB diseased, symptomatic, and asymptomatic citrus trees from Sargodha, a citrus growing region of Pakistan. Plant growth-stimulating bacteria isolated and characterized from the rhizosphere of citrus were identified as Serratia marcescens KRED strain, based on their phenotypic features, biochemical nature, and 16S rRNA sequence analysis. Out of twenty samples, KRED was identified in four samples of HLB asymptomatic trees while no strain of KRED was identified in symptomatic trees. Bacterial isolates possessing plant-beneficial properties were dominant species among the isolated PGPBs. The isolated bacteria were studied for their plant growth-promoting traits like IAA production, nitrogen fixation ability, siderophore production and ­lactose fermentation. Serratia marcescens exhibits plant growth promotion ability, but its specific interaction with HLB disease is not explored. In this context, the findings of this study underscore the importance of beneficial bacteria from the citrus rhizosphere and innovative approaches in the fight against HLB in citrus plants.

ZnO quantum dots reduce acquisition of huanglongbing pathogen by Diaphorina citri.

Diaphorina citri Kuwayama is the main vector of the Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas). Therefore, reducing CLas acquisition by D. citri is crucial for controlling the spread of HLB. This study aimed to investigate the effect of ZnO quantum dots (ZnO QDs) on CLas acquisition by D. citri. The results showed that ZnO QDs were absorbed by citrus plants and D. citri, inducing reactive oxygen species generation in the intestine and causing intestinal cell apoptosis. The uptake rate of CLas was reduced to 75% and 22.3% after treatment with 2 and 5 mg ∙ mL-1 ZnO QDs, respectively, compared with 100% in the control group. Transcription analysis showed significant changes in the expression of D. citri genes related to apoptosis, oxidative stress, and DNA replication and repair. The results suggested that ZnO QDs could efficiently reduce CLas acquisition by D. citri, providing a safe and reliable tool for HLB prevention and control. © 2024 Society of Chemical Industry.

Genetic and physiological characteristics of CsNPR3 edited citrus and their impact on HLB tolerance.

Huanglongbing (HLB) disease, caused by Candidatus Liberibacter asiaticus (CaLas), severely impacts citrus production, and currently, there is no cure. Developing HLB-resistant or tolerant cultivars is crucial, with modifying defense-related genes being a promising approach to managing HLB. NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) is a positive regulator of systemic acquired resistance (SAR), which enhances resistance to pathogens, whereas NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 3 (NPR3) is a negative regulator of SAR. To unambiguously address the role of CsNPR3 in HLB, we introduced mutations into the CsNPR3 gene in sweet orange (Citrus sinensis L. Osbeck) through genome editing and assessed their effects on morphology, physiology, and resistance/tolerance to HLB. Several genome-edited 'Hamlin' sweet orange trees harboring frameshift-inducing insertions or deletions were identified. After confirming the genome editing using Sanger sequencing, selected lines were grafted onto C-146 trifoliate hybrid rootstocks for clonal propagation. The progenies were then infected with CaLas using a no-choice Asian Citrus Psyllid (ACP) feeding assay. Evaluation of the genetic and physiological characteristics of CsNPR3-edited citrus trees under greenhouse conditions revealed that the edited trees exhibited greater vigor than the wild-type trees, despite the lack of significant differences in CaLas titers. Although further field evaluation is needed, our findings indicate that CsNPR3 contributes to HLB-caused tree deterioration and demonstrate that editing CsNPR3 can enhance tolerance to HLB.

HHS-RT-DETR: A Method for the Detection of Citrus Greening Disease

Background: Given the severe economic burden that citrus greening disease imposes on fruit farmers and related industries, rapid and accurate disease detection is particularly crucial. This not only effectively curbs the spread of the disease, but also significantly reduces reliance on manual detection within extensive citrus planting areas. Objective: In response to this challenge, and to address the issues posed by resource-constrained platforms and complex backgrounds, this paper designs and proposes a novel method for the recognition and localization of citrus greening disease, named the HHS-RT-DETR model. The goal of this model is to achieve precise detection and localization of the disease while maintaining efficiency. Methods: Based on the RT-DETR-r18 model, the following improvements are made: the HS-FPN (high-level screening-feature pyramid network) is used to improve the feature fusion and feature selection part of the RT-DETR model, and the filtered feature information is merged with the high-level features by filtering out the low-level features, so as to enhance the feature selection ability and multi-level feature fusion ability of the model. In the feature fusion and feature selection sections, the HWD (hybrid wavelet-directional filter banks) downsampling operator is introduced to prevent the loss of effective information in the channel and reduce the computational complexity of the model. Through using the ShapeIoU loss function to enable the model to focus on the shape and scale of the bounding box itself, the prediction of the bounding box of the model will be more accurate. Conclusions and Results: This study has successfully developed an improved HHS-RT-DETR model which exhibits efficiency and accuracy on resource-constrained platforms and offers significant advantages for the automatic detection of citrus greening disease. Experimental results show that the improved model, when compared to the RT-DETR-r18 baseline model, has achieved significant improvements in several key performance metrics: the precision increased by 7.9%, the frame rate increased by 4 frames per second (f/s), the recall rose by 9.9%, and the average accuracy also increased by 7.5%, while the number of model parameters reduced by 0.137×107. Moreover, the improved model has demonstrated outstanding robustness in detecting occluded leaves within complex backgrounds. This provides strong technical support for the early detection and timely control of citrus greening disease. Additionally, the improved model has showcased advanced detection capabilities on the PASCAL VOC dataset. Discussions: Future research plans include expanding the dataset to encompass a broader range of citrus species and different stages of citrus greening disease. In addition, the plans involve incorporating leaf images under various lighting conditions and different weather scenarios to enhance the model’s generalization capabilities, ensuring the accurate localization and identification of citrus greening disease in diverse complex environments. Lastly, the integration of the improved model into an unmanned aerial vehicle (UAV) system is envisioned to enable the real-time, regional-level precise localization of citrus greening disease.

Analysis of Huanglongbing transmission model with vector preferences and heterogeneous environments

The world citrus industry is confronting an unprecedented challenge from citrus Huanglongbing (HLB). With no resistant commercial citrus varieties or curable chemicals currently available, HLB remains the top threat to the world citrus industry. In this paper, two dynamic models of citrus HLB are proposed based on the preference and diffusion of citrus psyllids. The first model is a single-patch model of HLB with vector preference. The basic reproduction number of the model is calculated, and dynamic properties of the single-patch model, including the existence and local stability of the disease-free equilibrium, are analyzed. By sensitivity analysis, the parameters that have a significant impact on the basic reproduction number are identified. Numerical simulations are conducted to demonstrate that the preference of citrus psyllids is not conducive to disease prevention. Considering the diffusion of citrus psyllids between two patches, a two-patch diffusion model of HLB with preference is formulated. The global basic reproduction number of the diffusion model is determined, and the global stability of the disease-free equilibrium is established under certain conditions. Finally, numerical simulation results explore how the preference behavior of the vector and the coupling strength between two patches affect the HLB transmission.