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Pathogens have evolved mechanisms to manipulate their hosts to facilitate effective colonization and infection. One such mechanism involves the secretion of effectors that interfere with the host immune response. Effectors are typically secreted by dedicated secretion machinery that delivers the protein to the host cell. Liberibacters are intracellular bacterial pathogens that do not encode the typical secretion systems employed to secrete effectors; therefore, other mechanisms might be at play to allow Liberibacters to infect their hosts, such as the secretion of effectors via the sec-secretion system or via non-classical secretion systems. In this study, we datamined the genomes of five Liberibacter pathogens and identified from 66 to 102 putative non-classical secreted proteins encoded. Then, we focused on two predicted non-classical secreted proteins encoded by ‘Candidatus Liberibacter solanacearum’ haplotype B, CKC₀5770 and CKC₀0930, which showed similarities to non-classical secreted effectors from other Liberibacter species. We evaluated their secretion using alkaline phosphatase assays, if they suppressed programmed cell death or reactive oxygen species accumulation in Nicotiana benthamiana upon transient expression, and whether they could interact with tomato ascorbate peroxidases. We also evaluated if CKC₀5770 had a peroxidase activity. Our results suggest that CKC₀5770 interacted with tomato ascorbate peroxidases in two in vivo assays but not in vitro. Further, CKC₀5770 did not suppress plant immunity nor did it have a peroxidase activity as the ‘Candidatus Liberibacter asiaticus’ homologs did. Therefore, Liberibacter pathogens encode non-classical secreted proteins that could be effectors, but the roles of these proteins need to be validated in each pathosystem.

Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrus australiasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr. Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application.

Early detection and rapid response are crucial to avoid severe epidemics of exotic pathogens. However, most detection methods (molecular, serological, chemical) are logistically limited for large-scale survey of outbreaks due to intrinsic sampling issues and laboratory throughput. Evaluation of 10 canines trained for detection of a severe exotic phytobacterial arboreal pathogen, Candidatus Liberibacter asiaticus (CLas), demonstrated 0.9905 accuracy, 0.8579 sensitivity, and 0.9961 specificity. In a longitudinal study, cryptic CLas infections that remained subclinical visually were detected within 2 wk postinfection compared with 1 to 32 mo for qPCR. When allowed to interrogate a diverse range of in vivo pathogens infecting an international citrus pathogen collection, canines only reacted to Liberibacter pathogens of citrus and not to other bacterial, viral, or spiroplasma pathogens. Canines trained to detect CLas-infected citrus also alerted on CLas-infected tobacco and periwinkle, CLas-bearing psyllid insect vectors, and CLas cocultured with other bacteria but at CLas titers below the level of molecular detection. All of these observations suggest that canines can detect CLas directly rather than only host volatiles produced by the infection. Detection in orchards and residential properties was real time, ~2 s per tree. Spatiotemporal epidemic simulations demonstrated that control of pathogen prevalence was possible and economically sustainable when canine detection was followed by intervention (i.e., culling infected individuals), whereas current methods of molecular (qPCR) and visual detection failed to contribute to the suppression of an exponential trajectory of infection.

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an invasive species in America and is the main vector of the pathogen associated with Huanglongbing, a deadly disease of citrus plants in the world. The management of such a problem includes the intensive use of insecticides to reduce vector populations and risk of pathogen transmission. As an alternative to synthetic insecticides to control D. citri, the present study determined the chemical composition of diverse plant essential oils and assessed the toxicity and repellency of oil extracts against D. citri. Their chemical composition and abundance were determined by gas chromatography coupled to mass spectrometry. Adults and nymphs were exposed to leaf citrus discs treated by spraying or immersion with different oil extract concentrations. Repellency was assessed by exposing adults to treated leaves in experimental arenas and determining the number of insects remaining on the leaf after different time periods compared with the control. The main oil compounds in the tested plants were anethole, verbenone, 4-ethyl-4-methyl-1-hexene, 4-allylanisole, and trans-tagetone. Oils from Rosmarinus officinalis L. and Schinus molle L. caused no repellent or insecticide effects on D. citri. In contrast, oil extracts from the Foeniculum vulgare Mill. and Tagetes species were toxic and/or repellent for both adults and nymphs. There was a positive relationship between toxicity and concentration. Oil extracts from Tagetes lucida , T. coronopifoliai, and T. terniflora were repellent (> 92%) at 40 mg mL-1; this was correlated with the concentration and decreased over time. Essential oils extracts from the Tagetes species could represent a potential defense that could be integrated into the management of D. citri.

Since the arrival of the Asian citrus psyllid (ACP), Diaphorina citri , in the United States, citrus crops have faced the ongoing threat of citrus greening disease (HLB). This disease affects all settings across Florida, especially backyard citrus production. To address this, educational outreach efforts are being tailored for Florida residents to reduce the spread of ACP and HLB in urban areas. In 2023 to 2024, workshops were held in six North Florida counties (Gadsden, Jefferson, Leon, Bay, Columbia, and Santa Rosa) to help residents learn to identify, monitor, and control ACP and HLB. The outreach also introduced individual protective trees and reflective mulch as strategies to manage psyllid populations. A total of 71 homeowners participated in the post-training assessment, with 93% reporting increased knowledge of ACP identification. We found that many homeowners were not able to recognize ACP in situ, whereas they were able to identify it in a picture. From the survey, 86% of the responders expressed willingness to manage ACP and 89% agreed to remove infected trees. However, only 20% and 35% were willing to adopt individual protective tree covers and reflective mulch, respectively. Feedback suggests that public awareness and education on best management practices should be prioritized.

Citrus is a globally consumed fruit with great popularity. Mandarin (Citrus reticulata cv. ‘Shatangju’) is a local variety, and its planting area and yield are the greatest regarding fruit tree planting in Guangdong Province, China. However, its resistance to Huanglongbing (HLB) is weak. After infection by HLB, the fruits cannot develop normally. In this study, four kinds of fruits were classified as HBG, XQG, ZQG, and DHG, according to the color of their peels. The metabolomes of the three abnormally colored groups (HBG, XQG, and ZQG) and the normally colored group (DHG) were compared using a UPLC-QQQ-MS-based metabolomics approach. In total, 913 metabolites were identified and classified into 23 different categories, including phenylpropanoids and flavonoids; among them, 215 (HBG, 177; XQG, 124; and ZQG, 62) metabolites showed differential accumulation in the three comparison groups (HBG/XQG/ZQG versus DHG). A total of 2 unique metabolites, O-caffeoyl maltotriose and myricetin were detected only in DHG samples. When comparing HBG with DHG, there were 109 decreased and 68 increased metabolites; comparing XQG with DHG, there were 88 decreased and 36 increased metabolites; comparing ZQG with DHG, 41 metabolites were decreased, and 21 metabolites were increased. Metabolic pathway enrichment analysis of these differential metabolites showed significant enrichment of the “phenylpropanoid biosynthesis” pathway in all comparison groups. The hierarchical cluster analysis of the differential metabolites of the four groups showed a clear grouping patterns. The relative contents of three phenylpropanoids, four flavonoids, two alkaloids, one anthocyanin, and two other metabolites were significantly different between each comparison group. This study might provide fundamental insight for the isolation and identification of functional compounds from the peels of citrus fruit infected with HLB and for in-depth research on the effect of HLB on the formation of fruits pigment and the development of HLB-resistant citrus varieties.

Citrus has become a pivotal industry for the rapid development of agriculture and increasing farmers’ incomes in the main production areas of southern China. Knowing how to diagnose and control citrus huanglongbing has always been a challenge for fruit farmers. To promptly recognize the diagnosis of citrus huanglongbing, a new classification model of citrus huanglongbing was established based on MobileNetV2 with a convolutional block attention module (CBAM-MobileNetV2) and transfer learning. First, the convolution features were extracted using convolution modules to capture high-level object-based information. Second, an attention module was utilized to capture interesting semantic information. Third, the convolution module and attention module were combined to fuse these two types of information. Last, a new fully connected layer and a softmax layer were established. The collected 751 citrus huanglongbing images, with sizes of 3648 × 2736, were divided into early, middle, and late leaf images with different disease degrees, and were enhanced to 6008 leaf images with sizes of 512 × 512, including 2360 early citrus huanglongbing images, 2024 middle citrus huanglongbing images, and 1624 late citrus huanglongbing images. In total, 80% and 20% of the collected citrus huanglongbing images were assigned to the training set and the test set, respectively. The effects of different transfer learning methods, different model training effects, and initial learning rates on model performance were analyzed. The results show that with the same model and initial learning rate, the transfer learning method of parameter fine tuning was obviously better than the transfer learning method of parameter freezing, and that the recognition accuracy of the test set improved by 1.02~13.6%. The recognition accuracy of the citrus huanglongbing image recognition model based on CBAM-MobileNetV2 and transfer learning was 98.75% at an initial learning rate of 0.001, and the loss value was 0.0748. The accuracy rates of the MobileNetV2, Xception, and InceptionV3 network models were 98.14%, 96.96%, and 97.55%, respectively, and the effect was not as significant as that of CBAM-MobileNetV2. Therefore, based on CBAM-MobileNetV2 and transfer learning, an image recognition model of citrus huanglongbing images with high recognition accuracy could be constructed.

Citrus Huanglongbing (HLB) is the most destructive citrus disease worldwide, mainly caused by ‘Candidatus Liberibacter asiaticus’ (CLas). It encodes a large number of Sec-dependent effectors that contribute to HLB progression. In this study, an elicitor triggering ROS burst and cell death in Nicotiana benthamiana, CLIBASIA₀4425 (CLas4425), was identified. Of particular interest, its cell death-inducing activity is associated with its subcellular localization and the cytoplasmic receptor Botrytis-induced kinase 1 (BIK1). Compared with CLas infected psyllids, CLas4425 showed higher expression level in planta. The transient expression of CLas4425 in N. benthamiana and its overexpression in Citrus sinensis enhanced plant susceptibility to Pseudomonas syringae pv. tomato DC3000 ΔhopQ1-1 and CLas, respectively. Furthermore, the salicylic acid (SA) level along with the expression of genes NPR1/EDS1/NDR1/PRs in SA signal transduction was repressed in CLas4425 transgenic citrus plants. Taken together, CLas4425 is a virulence factor that promotes CLas proliferation, likely by interfering with SA-mediated plant immunity. The results obtained facilitate our understanding of CLas pathogenesis.

Huanglongbing (HLB) is a bacterial infection of citrus trees transmitted by the Asian citrus psyllid Diaphorina citri . Mitigation of HLB has focused on spraying of insecticides to reduce the psyllid population and removal of trees when they first show symptoms of the disease. These interventions have been only marginally effective, because symptoms of HLB do not appear on leaves for months to years after initial infection. Limited knowledge about disease spread during the asymptomatic phase is exemplified by the heretofore unknown length of time from initial infection of newly developing cluster of young leaves, called flush, by adult psyllids until the flush become infectious. We present experimental evidence showing that young flush become infectious within 15 d after receiving an inoculum of Candidatus Liberibacter asiaticus (bacteria). Using this critical fact, we specify a microsimulation model of asymptomatic disease spread and intensity in a grove of citrus trees. We apply a range of psyllid introduction scenarios to show that entire groves can become infected with up to 12,000 psyllids per tree in less than 1 y, before most of the trees show any symptoms. We also show that intervention strategies that reduce the psyllid population by 75% during the flushing periods can delay infection of a full grove, and thereby reduce the amount of insecticide used throughout a year. This result implies that psyllid surveillance and control, using a variety of recently available technologies, should be used from the initial detection of invasion and throughout the asymptomatic period. Significance Huanglongbing (HLB) is a vector-transmitted bacterial infection of citrus trees that poses a major threat to the citrus industry in Florida, Texas, and California. Current control strategies that focus on the vector, the Asian citrus psyllid Diaphorina citri , are usually initiated when the trees become symptomatic, anywhere from 10 mo to several years after initial infection. We show, experimentally, that newly infected young leaves can become infectious within 10–15 d after receiving an inoculum of bacteria from an adult psyllid. We then show by microsimulation of the asymptomatic spread of HLB through a grove under different invasion scenarios and control strategies that reduction of up to 75% of adult psyllids and nymphs can enhance citrus production.

Citrus Huanglongbing (HLB) is the most catastrophic citrus disease worldwide. SEC-dependent effectors (SDEs) play prominent roles in HLB pathogenesis, and 86 were predicted from the genome of Candidatus Liberibacter asiaticus. Nevertheless, little is known about the comprehensive picture of effector action mechanisms. In this study, RNA-seq was performed to explore the gene expression profiling of transgenic citrus plants expressing a CLas SDE, CLIBASIA₀0185 (0185-OE). A total of 6,506 differentially expressed genes (DEGs) were identified between 0185-OEs and the wild-type (WT) control through RNA-seq. Gene ontology (GO) analysis indicated that molecular functions associated with cellular process, cell part, binding, and catalysis activity were affected. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that DEGs were enriched in pathways including primary and secondary metabolisms. Further analysis demonstrated that DEGs implicated in sugar metabolism, phenylpropanoid biosynthesis, and endocytosis pathway were markedly induced in 0185-OE group compared to those in WTs. Ten genes were chosen to verify RNA-seq data with qRT-PCR, and their expression patterns were in good agreement with those of RNA-seq. The transcriptomic analysis demonstrated that CLIBASIA₀0185 (CLas0185) governed sugar metabolism, phenylpropanoid biosynthesis and endocytosis for pathogen survival and development. The results determined a prominent role of CLas0185 in HLB pathogenicity that could be a target for disease management.