A two-in-one molybdenum disulfide-chitosan nanoparticles system for activating plant defense mechanisms and reactive oxygen species to treat Citrus Huanglongbing.

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

Genomic analysis of "Candidatus Liberibacter africanus" strain from Zimbabwe reveals unique virulence and prophage characteristics compared to "Ca. Liberibacter asiaticus".

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

Effector CLas0185 targets methionine sulphoxide reductase B1 of Citrus sinensis to promote multiplication of 'Candidatus Liberibacter asiaticus' via enhancing enzymatic activity of ascorbate peroxidase 1.

Citrus huanglongbing (HLB) has been causing enormous damage to the global citrus industry. As the main causal agent, 'Candidatus Liberibacter asiaticus' (CLas) delivers a set of effectors to modulate host responses, while the modes of action adopted remain largely unclear. Here, we demonstrated that CLIBASIA_00185 (CLas0185) could attenuate reactive oxygen species (ROS)-mediated cell death in Nicotiana benthamiana. Transgenic expression of CLas0185 in Citrus sinensis 'Wanjincheng' enhanced plant susceptibility to CLas. We found that methionine sulphoxide reductase B1 (CsMsrB1) was targeted by the effector, and its abundance was elevated in CLas0185-transgenic citrus plants. Their interaction promoted CLas proliferation. We then determined that CsMsrB1 sustained redox state and enzymatic activity of ascorbate peroxidase 1 (CsAPX1) under oxidative stress. The latter reduced H2O2 accumulation and was associated with host susceptibility to CLas infection. Consistently, citrus plants expressing CLas0185 and CsMsrB1 conferred enhanced APX activity and decreased H2O2 content. Taken together, these findings revealed how CLas0185 benefits CLas colonization by targeting CsMsrB1, which facilitated the antioxidant activity and depressed ROS during pathogen infection.

A Detection Algorithm for Citrus Huanglongbing Disease Based on an Improved YOLOv8n.

Given the severe impact of Citrus Huanglongbing on orchard production, accurate detection of the disease is crucial in orchard management. In the natural environments, due to factors such as varying light intensities, mutual occlusion of citrus leaves, the extremely small size of Huanglongbing leaves, and the high similarity between Huanglongbing and other citrus diseases, there remains an issue of low detection accuracy when using existing mainstream object detection models for the detection of citrus Huanglongbing. To address this issue, we propose YOLO-EAF (You Only Look Once-Efficient Asymptotic Fusion), an improved model based on YOLOv8n. Firstly, the Efficient Multi-Scale Attention Module with cross-spatial learning (EMA) is integrated into the backbone feature extraction network to enhance the feature extraction and integration capabilities of the model. Secondly, the adaptive spatial feature fusion (ASFF) module is used to enhance the feature fusion ability of different levels of the model so as to improve the generalization ability of the model. Finally, the focal and efficient intersection over union (Focal-EIOU) is utilized as the loss function, which accelerates the convergence process of the model and improves the regression precision and robustness of the model. In order to verify the performance of the YOLO-EAF method, we tested it on the self-built citrus Huanglongbing image dataset. The experimental results showed that YOLO-EAF achieved an 8.4% higher precision than YOLOv8n on the self-built dataset, reaching 82.7%. The F1-score increased by 3.33% to 77.83%, and the mAP (0.5) increased by 3.3% to 84.7%. Through experimental comparisons, the YOLO-EAF model proposed in this paper offers a new technical route for the monitoring and management of Huanglongbing in smart orange orchards.

Balancing metabolism and reproduction.

The bacterium responsible for a disease that infects citrus plants across Asia facilitates its own proliferation by increasing the fecundity of its host insect.

Identification and Characterization of Polyamine Metabolism in Citrus in Response to 'Candidatus Liberibacter asiaticus' Infection.

Citrus Huanglongbing, one of the most devastating citrus diseases, is caused by 'Candidatus Liberibacter asiaticus' (CLas). Polyamines are aliphatic nitrogen-containing compounds that play important roles in disease resistance and are synthesized primarily by two pathways: an arginine decarboxylation pathway and an ornithine decarboxylation pathway. However, it is unclear whether polyamines play a role in the tolerance of citrus to infection by CLas and, if so, whether one or both of the core polyamine metabolic pathways are important. We used high-performance liquid chromatography and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry to detect the contents of nine polyamine metabolism-related compounds in six citrus cultivars with varying levels of tolerance to CLas. We also systematically detected the changes in polyamine metabolism-related compounds and H2O2 contents and compared the gene expression levels and the activities of enzymes involved in the polyamine metabolic pathway among healthy, asymptomatic, and symptomatic leaves of Newhall navel oranges infected with CLas. The tolerant and moderately tolerant varieties showed higher polyamine metabolism-related compound levels than those of susceptible varieties. Compared with the healthy group, the symptomatic group showed significantly increased contents of arginine, ornithine, γ-aminobutyric acid, and putrescine by approximately 180, 19, 1.5, and 0.2 times, respectively, and upregulated expression of biosynthetic genes. Arginase and ornithine decarboxylase enzyme activities were the highest in the symptomatic group, whereas arginine decarboxylase and agmatine deiminase enzyme activities were the highest in the asymptomatic group. The two polyamine biosynthetic pathways showed different trends with the increase of the CLas titer, indicating that polyamines were mainly synthesized through the arginine decarboxylase pathway in the asymptomatic leaves and were synthesized via the ornithine decarboxylase pathway in symptomatic leaves. These findings provide new insight into the changes in polyamine metabolism in citrus infected with CLas.

FlgI Is a Sec-Dependent Effector of Candidatus Liberibacter asiaticus That Can Be Blocked by Small Molecules Identified Using a Yeast Screen.

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The phloem-restricted bacterium Candidatus Liberibacter asiaticus (CLas) is considered to be the main pathogen responsible for HLB. There is currently no effective practical strategy for the control of HLB. Our understanding of how pathogens cause HLB is limited because CLas has not been artificially cultured. In this study, 15 potential virulence factors were predicted from the proteome of CLas through DeepVF and PHI-base searches. One among them, FlgI, was found to inhibit yeast growth when expressed in Saccharomyces cerevisiae. The expression of the signal peptide of FlgI fused with PhoA in Escherichia coli resulted in the discovery that FlgI was a novel Sec-dependent secretory protein. We further found that the carboxyl-terminal HA-tagged FlgI was secreted via outer membrane vesicles in Sinorhizobium meliloti. Fluoresence localization of transient expression FlgI-GFP in Nicotiana benthamiana revealed that FlgI is mainly localized in the cytoplasm, cell periphery, and nuclear periphery of tobacco cells. In addition, our experimental results suggest that FlgI has a strong ability to induce callose deposition and cell necrosis in N. benthamiana. Finally, by screening a large library of compounds in a high-throughput format, we found that cyclosporin A restored the growth of FlgI-expressing yeast. These results confirm that FlgI is a novel Sec-dependent effector, enriching our understanding of CLas pathogenicity and helping to develop new and more effective strategies to manage HLB.

Genetic diversity and population structure of the Asian citrus psyllid in China.

The Asian citrus psyllid (ACP) is the main vector of Citrus Huanglongbing, the most damaging citrus disease, causing significant financial losses in the citrus industry. Global warming has expanded the habitat of this pest, allowing it to continue its northward migration to China. Population genetic information of ACP is fundamentally essential for species management. This study investigated the genetic diversity and population structure of Chinese ACP using the mitochondrial cytochrome oxidase subunit I gene by dataset comprised 721 sequences from 27 geographic sites in China. Low haplotype diversity (0.323 ±â€…0.022) and low nucleotide diversity (0.00071 ±â€…0.00007) were observed in the entire population, which may indicate recent founder events. Twenty-three haplotypes were identified and clustered into 2 haplogroups: haplogroup I and haplogroup II. Haplogroup II included only 2 unique haplotypes, which occurred exclusively in the Southwest China ACP population. Genetic differentiation analyses were also indicative of Southwest China population was significantly differentiated from the remaining populations. Demographic history analysis showed that ACP population in China has experienced demographic expansion. Our results provided a better understanding of the genetic distribution patterns and structures of ACP populations in China.

A Sec-dependent effector, CLIBASIA_04425, contributes to virulence in 'Candidatus Liberibater asiaticus'.

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, CLIBASIA_04425 (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.

Screening Method and Antibacterial Activity of 1,3,4-Oxadiazole Sulfone Compounds against Citrus Huanglongbing.

Citrus Huanglongbing (HLB) is one of the most destructive diseases in the citrus industry. At present, Candidatus Liberibacter asiaticus (CLas) cannot be cultured in vitro, and there is a lack of rapid methods to test antibacterial activity, which greatly hinders the discovery of new antibacterial agents against HLB. To establish a rapid screening method for antibacterial agents against HLB with simple operation, a short cycle, and a large number of tests, the CLas contents in leaves from different citrus branches, different leaves from the same citrus branch, and two halves of the same citrus leaf were detected. Compared with the leaves on different branches and different leaves on the same branch, the difference in CLas content of the left and right halves of the same leaf was small; the difference was basically between 0.7 and 1.3. A rapid and efficient method for primary screening agents against HLB termed the "half-leaf method" was established through our long-term optimization and improvement. To verify the stability and reliability of the activity data measured using this method, 6-chloropurine riboside, which is highly soluble in water, was used as the test agent, and its antibacterial activity against HLB was tested 45 times. The results of the antibacterial activity test showed little difference in the mean values of each data group, indicating that this method could be used as a rapid method for screening agents against HLB. We used this method to test the antibacterial activity of compounds synthesized by our research group against HLB and found that some of the compounds showed good activity.

Classification of Citrus Huanglongbing Degree Based on CBAM-MobileNetV2 and Transfer Learning.

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.

Environmental Factors Affect the Bacterial Community in Diaphorina citri, an Important Vector of "Candidatus Liberibacter asiaticus".

Insects are associated with diverse microbial communities that can have substantial effects on hosts. Here, we characterized the bacterial communities in the Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae), a major vector of the devastating pathogen "Candidatus Liberibacter asiaticus," which causes citrus Huanglongbing (HLB). In total, 256 ACP individuals across 15 field sites and one laboratory population in China were sequenced. The results showed that the bacterial community diversity was the highest in the Guilin population (average Shannon index, 1.27), and the highest value for richness was found in the Chenzhou population (average Chao1 index, 298). The bacterial community structures of the field-collected populations were significantly different, and all of them harbored Wolbachia, which was assigned to strain ST-173. Structural equation models revealed that the dominant Wolbachia strain had a significantly negative correlation with the annual mean temperature. In addition, the results obtained with populations infected with "Ca. Liberibacter asiaticus" indicated that in total, 140 bacteria could be involved in interactions with this bacterium. The ACP field populations harbored a more diverse bacterial community than the laboratory population, and the relative occurrences of some symbionts differed significantly. However, the bacterial community of the ACP laboratory colony was connected in a more complex network structure (average degree, 54.83) than that of the field populations (average degree, 10.62). Our results provide evidence that environmental factors can influence the bacterial community structure and bacterial relative abundance in ACP populations. This is likely due to the adaptation of ACPs to local environments. IMPORTANCE The Asian citrus psyllid (ACP) is an important vector of the HLB pathogen, which is a major threat to citrus production around the world. Bacterial communities harbored by insects could be affected by different environmental factors. Understanding these factors that affect the bacterial community of the ACP could be important for the better management of HLB transmission. This work surveyed ACP field populations in mainland China in order to explore the bacterial community diversity of different populations and the potential relationships between environmental factors and predominant symbionts. We have assessed the differences in ACP bacterial communities and identified the prevalent Wolbachia strains in the field. In addition, we compared the bacterial communities of ACP field-collected and laboratory populations. Comparing populations subjected to contrasting conditions could help us to better understand how the ACP adapts to local environmental conditions. Our study provides new insights into how environmental factors influence the bacterial community of the ACP.

Role of long non-coding RNA in regulatory network response to Candidatus Liberibacter asiaticus in citrus.

Long non-coding RNAs (lncRNAs) serve as crucial regulators in plant response to various diseases, while none have been systematically identified and characterized in response to citrus Huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) bacteria. Here, we comprehensively investigated the transcriptional and regulatory dynamics of the lncRNAs in response to CLas. Samples were collected from leaf midribs of CLas- and mock-inoculated HLB-tolerant rough lemon (Citrus jambhiri) and HLB-sensitive sweet orange (C. sinensis) at week 0, 7, 17, and 34 following inoculation using CLas+ budwood of three biological replicates in the greenhouse. A total of 8,742 lncRNAs, including 2,529 novel lncRNAs, were identified from RNA-seq data with rRNA-removed from strand-specific libraries. Genomic variation analyses of conserved lncRNAs from 38 citrus accessions showed that 26 single nucleotide polymorphisms (SNPs) were significantly correlated with HLB. In addition, lncRNA-mRNA weighted gene co-expression network analysis (WGCNA) showed a significant module correlated with CLas-inoculation in rough lemon. Notably, the most significant LNC_28805 and multiple co-expressed genes related to plant defense in the module were targeted by miRNA5021, suggesting that LNC28805 might compete with endogenous miR5021 to maintain the homeostasis of immune gene expression levels. Candidate WRKY33 and SYP121 genes targeted by miRNA5021 were identified as two key hub genes interacting with bacteria pathogen response genes based on the prediction of protein-protein interaction (PPI) network. These two genes were also found within HLB-associated QTL in linkage group 6. Overall, our findings provide a reference for a better understanding of the role of lncRNAs involved in citrus HLB regulation.

A prophage-encoded effector from "Candidatus Liberibacter asiaticus" targets ASCORBATE PEROXIDASE6 in citrus to facilitate bacterial infection.

Citrus huanglongbing (HLB), associated with the unculturable phloem-limited bacterium "Candidatus Liberibacter asiaticus" (CLas), is the most devastating disease in the citrus industry worldwide. However, the pathogenicity of CLas remains poorly understood. In this study, we show that AGH17488, a secreted protein encoded by the prophage region of the CLas genome, suppresses plant immunity via targeting the host ASCORBATE PEROXIDASE6 (APX6) protein in Nicotiana benthamiana and Citrus sinensis. The transient expression of AGH17488 reduced the chloroplast localization of APX6 and its enzyme activity, inhibited the accumulation of reactive oxygen species (H2 O2 and O2 - ) and the lipid oxidation endproduct malondialdehyde in plants, and promoted the proliferation of Pseudomonas syringae pv. tomato DC3000 and Xanthomonas citri subsp. citri. This study reveals a novel mechanism underlying how CLas uses a prophage-encoded effector, AGH17488, to target a reactive oxygen species accumulation-related gene, APX6, in the host to facilitate its infection.

Physiological Variables Influenced by 'Candidatus Liberibacter asiaticus' Infection in Two Citrus Species.

'Candidatus Liberibacter asiaticus' is the bacterium associated with the citrus disease known as huanglongbing (HLB). This study evaluated the influence of 'Ca. L. asiaticus' infection on a number of key plant physiological variables concerning photosynthesis, cell integrity, reactive oxygen species scavengers' activity, and osmoregulation of two different species of citrus-the pomelo Citrus maxima and the mandarin C. reticulata 'Tankan'-relative to their measured 'Ca. L. asiaticus' infection load. Results indicated that all measured physiological variables except soluble sugar were affected by increased 'Ca. L. asiaticus' infection titers, wherein the variety C. maxima proved overall more resistant than C. reticulata. 'Ca. L. asiaticus' infection was linked in both plants to decrease in chlorophyll concentration, cell membrane permeability, and malondialdehyde, as well as increased free proline and starch contents. Chlorophyll fluorescence measurements taken 9 months after grafting the mandarin C. reticulata with 'Ca. L. asiaticus' scions revealed a significant decrease in the photosynthesis variables maximum photochemical quantum yield of photosystem II (PSII), effective photochemical quantum yield of PSII, and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae, whereas nonphotochemical fluorescence quenching increased significantly; C. maxima plants, on the other hand, did not show significant differences until the 12th month from infection exposure. The variables superoxide dismutase, catalase, peroxidase, and soluble protein initially increased and later decreased. In addition, progression of 'Ca. L. asiaticus' replication in both citrus species was accompanied by rapid changes in three reactive oxygen species scavenging enzymes in C. maxima, while the pattern was different in C. reticulata. We hypothesize that the observed interspecific differences in physiological change are related to their relative resistance against 'Ca. L. asiaticus' infection. These results provide a scaffold for better describing the pathogenesis, selecting the most resistant breeds, or even validating pertaining omics research; ultimately, these detailed observations can facilitate the diagnosis of 'Ca. L. asiaticus' infection.

Citrus Huanglongbing correlated with incidence of Diaphorina citri carrying Candidatus Liberibacter asiaticus and citrus phyllosphere microbiome.

In China, citrus Huanglongbing (HLB) disease is caused by the Candidatus Liberibacter asiaticus bacterium, which is carried by the Asian citrus psyllid Diaphorina citri Kuwayama. It was hypothesized that the epidemic of the HLB may related with the rate of bacterium presence in the insect vector and bacterium content in plant tissues, as well as the phyllosphere microbe communities changes. This study systematically analyzed the presence or absence of Ca. L. asiaticus in citrus tree leaves and in the insect vector D. citri over a 6-year period using real-time PCR. In addition, changes in the number of bacteria carried by D. citri over 12 months were quantified, as well as the relationship between the proportion of D. citri carrying Ca. L. asiaticus and the proportion of plants infected with Ca. L. asiaticus were analyzed. Results showed that the proportion of D. citri carrying bacteria was stable and relatively low from January to September. The bacteria in citrus leaves relatively low in spring and summer, then peaked in December. The proportion of D. citri carrying bacteria gradually declined from 2014 to 2019. The proportion of D. citri carrying Ca. L. asiaticus showed a significant positive correlation with the proportion of diseased citrus. The phyllosphere bacterial and fungal communities on the healthy citrus leaf were significantly different with the disease leaf in April and December. Pathogenic invasions change the citrus phyllosphere microbial community structure. It could be summarized that citrus Huanglongbing correlated with incidence of Diaphorina citri carrying Candidatus Liberibacter asiaticus and citrus phyllosphere microbiome.

Potential of citrus endophyte Bacillus subtilis L1-21 in the control of Candidatus Liberibacter asiaticus in Asian citrus psyllid, Diaphorina citri.

BACKGROUND: Asian citrus psyllid (ACP), also known as Diaphorina citri, is the natural vector of Candidatus Liberibacter asiaticus (CLas), which is responsible for Huanglongbing (HLB), a devastating citrus disease. Previously, the pathogen was successfully excluded from diseased citrus plants by using the indigenous endophyte Bacillus subtilis L1-21. However, the pathogen elimination and colonization potential of B. subtilis L1-21 in the carrier vector ACP, as well as the recruitment of native microbial communities of psyllid in the presence of endophytes, are still unknown. RESULTS: Initially, we suggested that endophyte L1-21 reduced the CLas copies in ACP from 6.58 × 106 to 5.04 × 104 per insect after 48 h, however, the pathogen copies remained stable in the negative control. The endophyte was stable for 48 h after application. Among the bacterial genera those highlighted in ACP were Candidatus Liberibacter, Pseudomonas, Candidatus Profftella, Methylobacterium-Methylorubrum, Pantoea, Curtobacterium, Wolbachia, Actinomycetospora, and Bacillus. Interestingly, B. subtilis L1-21 easily colonizes the midgut of ACP but cannot be detected in eggs. When ACP with endophyte L1-21 was allowed to feed on new citrus leaves, the highest colonization was observed. We also found that psyllids carrying endophyte L1-21 after feeding on citrus leaves reduced the CLas copies in leaves on the 0, 3rd and 5th day from 8.18 × 10,4 2.6 × 10,3 and 0 pathogen copies/g fresh midvein, respectively. CONCLUSIONS: We propose that B. subtilis L1-21 is a native endophyte in citrus and psyllid, which efficiently reduces the CLas pathogen in both citrus and psyllids, provides a more protective effect by increasing the number of cultivable endophytes, and successfully colonizes the midgut of ACP. © 2022 Society of Chemical Industry.

Transcriptome Profiling of 'Candidatus Liberibacter asiaticus' in Citrus and Psyllids.

'Candidatus Liberibacter asiaticus' (Las) is an emergent bacterial pathogen that is associated with the devastating citrus huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful, and dual-transcriptome analyses aiming to profile gene expression in both Las and its hosts have a low coverage of the Las genome because of the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, a lack of understanding of the Las transcriptome remains a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited high expression in citrus include transporters, ferritin, outer membrane porins, specific pilins, and genes involved in phage-related functions, cell wall modification, and stress responses. We also found 106 genes to be differentially expressed in citrus versus Asian citrus psyllids. Genes related to transcription or translation and resilience to host defense response were upregulated in citrus, whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. Finally, we determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.

Impact of Huanglongbing Pathogen Infection on the Amino Acid Composition in Both Citrus Plants and the Asian Citrus Psyllid.

The Asian citrus psyllid (ACP) Diaphorina citri is the main vector of the pathogen Candidatus Liberibacter asiaticus (CLas), which is the causal agent of citrus Huanglongbing disease. Feeding by both ACP nymphs and adults on host plants allows them to obtain nutrition. Therefore, the nutritional content within the plant phloem is of much importance for the development and reproduction of ACP. The infection by pathogenic microbiomes may affect the amino acid contents of their host plants and then indirectly affect the biology of sap-feeding insects. In this study, we investigated the amino acid contents and their proportions in both CLas-infected and CLas-free citrus plants, ACP adults, and also in honeydew produced by ACP nymphs. Results showed that infection by CLas had a large impact on the amino acid species and proportion in all the tested target plants, ACP adults, and in the honeydew of ACP nymphs. The content of total amino acids in CLas-infected citrus was much higher than that of CLas-free citrus. However, CLas infection significantly reduced the proportion of essential amino acids (EAAs) in these plants. When feeding on CLas-infected citrus plants, ACP adults absorbed less total amino acids than those adults feeding on healthy plants, but the proportion of EAAs was significantly higher when they fed on CLas-infected citrus plants. The proportion of EAAs also significantly increased in the honeydew secreted by ACP nymphs that fed on CLas-infected citrus plants. However, EAA detection in the honeydew of ACP nymphs indicated that the utilization rate of EAAs by CLas positive ACP nymphs was reduced. Our study has revealed that CLas infection significantly affects the contents, proportion, and utilization efficiency of different amino acids in citrus plants, ACP adults, and nymphs, leading to a developmental pattern of ACP that is more conducive to CLas transmission.

Controlling Citrus Huanglongbing: Green Sustainable Development Route Is the Future.

Huanglongbing (HLB) is the most severe bacterial disease of citrus crops caused by Candidatus Liberibacter spp. It causes a reduction in fruit yield, poor fruit quality, and even plants death. Due to the lack of effective medicine, HLB is also called citrus "AIDS." Currently, it is essential for the prevention and control of HLB to use antibiotics and pesticides while reducing the spread of HLB by cultivating pathogen-free seedlings, removing disease trees, and killing Asian citrus psyllid (ACP). New compounds [e.g., antimicrobial peptides (AMPs) and nanoemulsions] with higher effectiveness and less toxicity were also found and they have made significant achievements. However, further evaluation is required before these new antimicrobial agents can be used commercially. In this review, we mainly introduced the current strategies from the aspects of physical, chemical, and biological and discussed their environmental impacts. We also proposed a green and ecological strategy for controlling HLB basing on the existing methods and previous research results.