Citrus Huanglongbing (HLB) or citrus greening, is the most destructive disease for citrus worldwide. It is caused by the psyllid-transmitted, phloem-limited bacteria “CandidatusLiberibacter asiaticus” (CLas). To date, there are still no effective practical strategies for curing citrus HLB. Understanding the mechanisms againstCLas can contribute to the development of effective approaches for combatting HLB. However, the unculturable nature ofCLas has hindered elucidating mechanisms againstCLas. In this review, we summarize the main aspects that contribute to the understanding about the mechanisms againstCLas, including (1)CLas virulence targets, focusing on inhibition of virulence genes; (2) activation of citrus host defense genes and metabolites of HLB-tolerant citrus triggered byCLas, and by agents; and (3) we also review the role of citrus microbiome in combattingCLas. Finally, we discuss novel strategies to continue studying mechanisms againstCLas and the relationship of above aspects.

Nano-emulsion is a promising delivery system for increasing pesticide use and enhancing the therapeutic efficiency against pathogens. The pathogen Candidatus Liberibacter asiaticus (Las) that causes destructive citrus huanglongbing (HLB) resides in citrus phloem, which makes it difficult to treat with chemicals. Based on various physiochemical characteristics of oils, surfactants, and organic solvents, an oil-in-water (O/W) nano-emulsion formulation was developed and optimized to combat citrus HLB. The nano-emulsion was formulated through a spontaneous emulsification method for efficient delivery of ampicillin into the citrus phloem using bark application. The nano-emulsion that was prepared from Cremophor EL (viscous oil), acetone (water miscibility organic solvent), and Span 80/Tween 80 (surfactant) formed a small droplet size (17.33 ± 0.52 nm) and exhibited an improved absorption rate. Peak concentration was detected at 2 days posttreatment and the maximum concentration (C_max) and relative bioavailability (RBA) of ampicillin in HLB-affected citrus were 71.86 ± 35.38 ng/g and 267.25% ± 44.1%, respectively. The peak concentration of Amp appeared at 6 days posttreatment in the citrus trees that were treated with Amp alone and their C_max and RBA were 56.44 ± 32.59 ng/g and 100%, respectively. The same nano-emulsion was used to deliver five different antimicrobials to control citrus HLB through bark application. We found that the droplet size of the antimicrobials in the nano-emulsion was significantly reduced and the nano-emulsion also enhanced the therapeutic efficiency of validoxylamine A alone and in combination with actidione as well as sulfadimoethoxine sodium against Las. Therefore, this study provides an efficient bark application nano-emulsion formulation for citrus HLB control.

Citrus huanglongbing (HLB) is a devastating disease for the citrus industry. The previous studies demonstrated that oxytetracycline and penicillin are effective antibiotics against Candidatus Liberibacter asiaticus (CLas). However, since CLas is uncultured, the mechanisms of action of antibiotics against CLas are still unclear. It was recently reported that the endophytic microbial communities are associated with the progression of citrus HLB after oxytetracycline and penicillin treatment. Therefore, we hypothesize that penicillin has greater antibacterial activity against CLas than oxytetracycline, which may be associated with the alteration of the structure and function of endophytic microbial communities in HLB-affected citrus in response to these antibiotics. To test this hypothesis, the microbiome of HLB-affected citrus leaves treated with these two antibiotics was analyzed using a metagenomic method. Our results indicate that the microbial structure and function in HLB-affected citrus were altered by these two antibiotics. The relative abundance of beneficial bacterial species, including Streptomyces avermitilis and Bradyrhizobium, was higher in penicillin-treated plants compared to those treated with oxytetracycline, and the relative abundance of the bacterial species (such as Propionibacterium acnes and Synechocystis sp PCC 6803) associated with CLas survival was lower for penicillin-treated plants compared to oxytetracycline-treated plants. These results indicate that penicillin has greater antibacterial activity against CLas. Based on the metagenomic analysis, this study elucidated the mechanism for the observed increase in antibacterial activity of penicillin against CLas. The data presented here are not only invaluable for developing eco-friendly and effective biocontrol strategies to combat citrus HLB, but also provide a method for revealing mechanism of antimicrobial against uncultured bacteria in host.

Huanglongbing (HLB), also known as citrus greening disease, is a devastating disease of citrus. However, there is no known cure so far. Recently, under Section 24(c) of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), a special local need label was approved that allows the trunk injection of antimicrobials such as oxytetracycline (OTC) for HLB management in Florida. The objectives of this study were to use UAS-based remote sensing to assess the effectiveness of OTC on the HLB-affected citrus trees in Texas and to differentiate the levels of HLB severity and canopy health. We also leveraged UAS-based features, along with machine learning, for HLB severity classification. The results show that UAS-based vegetation indices (VIs) were not sufficiently able to differentiate the effects of OTC treatments of HLB-affected citrus in Texas. Yet, several UAS-based features were able to determine the severity levels of HLB and canopy parameters. Among several UAS-based features, the red-edge chlorophyll index (CI) was outstanding in distinguishing HLB severity levels and canopy color, while canopy cover (CC) was the best indicator in recognizing the different levels of canopy density. For HLB severity classification, a fusion of VIs and textural features (TFs) showed the highest accuracy for all models. Furthermore, random forest and eXtreme gradient boosting were promising algorithms in classifying the levels of HLB severity. Our results highlight the potential of using UAS-based features in assessing the severity of HLB-affected citrus.

Irrigation is important in many crop production systems. However, irrigation water can be a carrier of plant pathogens that can enter the system and spread to fields, resulting in crop damage and yield losses. The Lower Rio Grande Valley of South Texas is an important area for agricultural production which depends on the Rio Grande River as a source of water for irrigation. Thus, the presence of plant pathogens in the Rio Grande River could have important implications for crop productivity in the region. Cultured-based methods and molecular identification methods are used for monitoring plant pathogens in irrigation water. However, these methods are labor-intensive and just detect targeted pathogens. To overcome these limitations, in this study, the ITS2 amplicon metagenomic method was applied for evaluating the fungal diversity, composition, and presence of fungal plant pathogens in irrigation water from the Rio Grande River as it leaves the water reservoir (WR) and it arrives at an irrigation valve at a farm (FA). Results from the Shannon (WR = 4.6 ± 0.043, FA = 3.63 ± 0.13) and Simpson indices (WR = 4.6 ± 0.043, FA = 3.63 ± 0.13) showed that there are significant differences in the fungal diversity and community structure between the two locations and the PCA analysis showed a clear differentiation between both fungal communities. Several OTUs identified in both locations included potential plant pathogens from diverse genera including Cladosporium, Exserohilum, and Nigrospora, while others such as Colletotrichum and Plectosphaerella were found only in one of the two locations assessed. This work indicates that microbes, including plant pathogens, may enter or exit throughout the irrigation-water distribution system, thereby modifying the microbial community composition along the way. Understanding the dynamics of plant pathogen movement in irrigation water systems can help growers identify risk factors to develop measures to mitigate those risks. This study also shows the usefulness of the metagenomic approach for detecting and monitoring plant pathogen in irrigation water.

Citrus Huanglongbing (HLB) is a serious disease for the citrus industry. Earlier studies showed that ampicillin (Amp) can reduce titers of the pathogen which causes HLB and the bacteria Candidatus Liberibacter asiaticus (CLas) in HLB-affected citrus. CLas has not yet been cultured, so the mechanisms of Amp against CLas are unclear. Some chemicals were demonstrated to trigger citrus defense systems against CLas. Therefore, we hypothesize that Amp may induce citrus defenses against CLas. Here we applied three nano-formulations of varying droplet sizes to HLB-affected citrus to achieve different accumulated concentrations of Amp (high, medium, low) in the plants. We then used RNA-seq to analyze induction of gene expression of citrus defense systems against CLas in response to different concentrations of Amp. The results indicated that at all accumulated concentrations of Amp can significantly suppress CLas titer and mitigate HLB symptoms. Transcriptomic analyses showed that Amp treatment induced expression of heat shock proteins (Hsps) in HLB-affected citrus, and these Hsps were significantly related to several defense genes encoding R proteins, transcription factors, splicing factors, RNA-binding proteins, RNA-dependent RNA polymerase, Gibberellic acid methyltransferase 2, L-ascorbate peroxidase 2, and ferruginol synthase that confer tolerance to CLas in citrus plants. Taken together, these results suggest that Amp treatment of citrus plants can trigger expression of Hsps and related defense genes to respond to CLas infection. These findings are valuable for developing novel strategies to combat citrus HLB.