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Abstract The sustainable development of the citrus industry is greatly affected by citrus canker, an important bacterial disease. To explore the transcriptional regulatory mechanism of citrus resistance to canker disease, this study used the susceptible Citrus sinensis cv. ‘Newhall’ and its citrus canker-resistant bud mutation variety ‘Longhuitian’ (LHT) as materials. Through analysing the variances in leaf phenotypes between Newhall and LHT, as well as the variations in their transcriptional expression under Xanthomonas citri subsp. citri (Xcc) inoculation, our study concluded that LHT displays markedly greater resistance to Xcc compared to Newhall. Additionally, the spongy parenchyma of LHT leaves is significantly thicker than that of Newhall, and the stomatal number is significantly higher in LHT leaves, while the length and width of individual stomata in LHT leaves are significantly smaller than those in Newhall. RNA-seq analysis indicates that the differentially expressed genes between LHT and Newhall are involved in biotic stress-related biological processes, secondary metabolite biosynthesis, as well as phytohormone signalling pathways. Furthermore, significant differences were observed in reactive oxygen metabolism and phenylalanine metabolism pathways. The findings of our study provide data support for a deeper understanding of the citrus-Xcc interactions and offer valuable clues for unravelling citrus resistance to citrus canker.

The sustainable development of the citrus industry is greatly affected by citrus canker, an important bacterial disease. To explore the transcriptional regulatory mechanism of citrus resistance to canker disease, this study used the susceptible Citrus sinensis cv. 'Newhall' and its citrus canker-resistant bud mutation variety 'Longhuitian' (LHT) as materials. Through analysing the variances in leaf phenotypes between Newhall and LHT, as well as the variations in their transcriptional expression under Xanthomonas citri subsp. citri (Xcc) inoculation, our study concluded that LHT displays markedly greater resistance to Xcc compared to Newhall. Additionally, the spongy parenchyma of LHT leaves is significantly thicker than that of Newhall, and the stomatal number is significantly higher in LHT leaves, while the length and width of individual stomata in LHT leaves are significantly smaller than those in Newhall. RNA-seq analysis indicates that the differentially expressed genes between LHT and Newhall are involved in biotic stress-related biological processes, secondary metabolite biosynthesis, as well as phytohormone signalling pathways. Furthermore, significant differences were observed in reactive oxygen metabolism and phenylalanine metabolism pathways. The findings of our study provide data support for a deeper understanding of the citrus-Xcc interactions and offer valuable clues for unravelling citrus resistance to citrus canker. [ABSTRACT FROM AUTHOR]

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

SUMMARY: Citrus bacterial canker (CBC) is a serious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that adversely impacts the global citrus industry. In a previous study, we demonstrated that overexpression of an Xcc‐inducible apetala 2/ethylene response factor encoded by Citrus sinensis, CsAP2‐09, enhances CBC resistance. The mechanism responsible for this effect, however, is not known. In the present study, we showed that CsAP2‐09 targeted the promoter of the Xcc‐inducible WRKY transcription factor coding gene CsWRKY25 directly, activating its transcription. CsWRKY25 was found to localize to the nucleus and to activate transcriptional activity. Plants overexpressing CsWRKY25 were more resistant to CBC and showed higher expression of the respiratory burst oxidase homolog (RBOH) CsRBOH2, in addition to exhibiting increased RBOH activity. Transient overexpression assays in citrus confirmed that CsWRKY25 and CsRBOH2 participated in the generation of reactive oxygen species (ROS) bursts, which were able to restore the ROS degradation caused by CsAP2‐09 knockdown. Moreover, CsWRKY25 was found to bind directly to W‐box elements within the CsRBOH2 promoter. Notably, CsRBOH2 knockdown had been reported previously to reduce the CBC resistance, while demonstrated in this study, CsRBOH2 transient overexpression can enhance the CBC resistance. Overall, our results outline a pathway through which CsAP2‐09‐CsWRKY25 transcriptionally reprograms CsRBOH2‐mediated ROS homeostasis in a manner conducive to CBC resistance. These data offer new insight into the mechanisms and regulatory pathways through which CsAP2‐09 regulates CBC resistance, highlighting its potential utility as a target for the breeding of CBC‐resistant citrus varieties. Significant Statement: In the present study, we identify a pathway in which CsAP2‐09 and CsWRKY25 form a transcription factor cascade, transcriptionally reprogramming CsRBOH2‐mediated reactive oxygen species homeostasis and citrus bacterial canker (CBC) resistance. Our study explores new insights into the mechanistic role of CsAP2‐09 and the associated regulatory network, extending the potential value of CsAP2‐09 in the breeding of CBC‐tolerant citrus plants. [ABSTRACT FROM AUTHOR]

Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker, elicits canker symptoms in citrus plants because of the transcriptional activator-like (TAL) effector PthA4, which activates the expression of the citrus susceptibility gene CsLOB1. This study reports the regulation of the putative carbohydrate-binding protein gene Cs9g12620 by PthA4-mediated induction of CsLOB1 during Xcc infection. We found that the transcription of Cs9g12620 was induced by infection with Xcc in a PthA4-dependent manner. Even though it specifically bound to a putative TAL effector-binding element in the Cs9g12620 promoter, PthA4 exerted a suppressive effect on the promoter activity. In contrast, CsLOB1 bound to the Cs9g12620 promoter to activate its expression. The silencing of CsLOB1 significantly reduced the level of expression of Cs9g12620, which demonstrated that Cs9g12620 was directly regulated by CsLOB1. Intriguingly, PhtA4 interacted with CsLOB1 and exerted feedback control that suppressed the induction of expression of Cs9g12620 by CsLOB1. Transient overexpression and gene silencing revealed that Cs9g12620 was required for the optimal development of canker symptoms. These results support the hypothesis that the expression of Cs9g12620 is dynamically directed by PthA4 for canker formation through the PthA4-mediated induction of CsLOB1.

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

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca), causal agents of citrus bacterial canker, are both regulated by the European Union to prevent their introduction. Xcc is responsible for severe outbreaks of citrus production worldwide, therefore, a prompt and reliable detection is advisable for the early detection of this bacterium either in symptomatic or asymptomatic plant material. The current EPPO (European and Mediterranean Plant Protection Organization) diagnostic protocol, PM 7/44(1), includes several diagnostic tests even if new assays have been developed in the latter years for which validation data are needed. Recently, a test performance study was organized within the Valitest EU Project to validate Xcc diagnostic methods and provide evidence on the most reliable assays; however, the influence of DNA extraction methods (DEM) on the reliability of the detection has never been assessed. In this study we evaluate four different DEM, by following two different approaches: (i) a comparison by real-time PCR standard curves of bacterial DNA versus bacterial DNA added to plant DNA (lemon, leaves and fruit; orange fruit); and (ii) the evaluation of performance criteria of spiked samples (plant extract added with ten-fold diluted bacterial suspensions at known concentrations). Droplet digital PCR is developed and compared with real-time PCR, as the detection method. [ABSTRACT FROM AUTHOR]

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca) are causal agents of Citrus Bacterial Canker (CBC), a devastating disease that severely affects citrus plants. They are harmful organisms not reported in Europe or the Mediterranean Basin. Host plants are in the Rutaceae family, including the genera Citrus, Poncirus, and Fortunella, and their hybrids. In addition, other genera of ornamental interest are reported as susceptible, but results are not uniform and sometimes incongruent. We evaluated the susceptibility of 32 ornamental accessions of the Rutaceae family belonging to the genera Citrus, Fortunella, Atalantia, Clausena, Eremocitrus, Glycosmis, Microcitrus, Murraya, Casimiroa, Calodendrum, and Aegle, and three hybrids to seven strains of Xcc and Xca. Pathotyping evaluation was assessed by scoring the symptomatic reactions on detached leaves. High variability in symptoms and bacterial population was shown among the different strains in the different hosts, indicative of complex host–pathogen interactions. The results are mostly consistent with past findings, with the few discrepancies probably due to our more complete experimental approach using multiple strains of the pathogen and multiple hosts. Our work supports the need to regulate non-citrus Rutaceae plant introductions into areas, like the EU and Mediterranean, that are currently free of this economically important pathogen. [ABSTRACT FROM AUTHOR]

Microorganisms with biocontrol capabilities against plant pathogens are considered as one of the most promising approaches for healthy crop management. In this study, ethyl acetate extracts of 25 Bacillus strains were investigated for their antagonistic effect on Xanthomonas citri subsp. citri (Xcc), which causes the citrus bacterial canker (CBC) disease. Among them, 21 strains exerted antibacterial activity against wild-type Xcc strains. Based on the strength of the antibacterial activity, nine Bacillus strains were selected for 16S rRNA analysis. 16S rRNA sequence homology revealed that several strains were closely related to B. velezensis , where strains with no antibacterial activity grouped as the soil-associated community of B. amyloliquefaciens. B. velezensis Bv-21 exhibited the highest antibacterial activity against wild type and streptomycin resistant Xcc with inhibition zones of 22.91 ± 0.45 and 20.28 ± 0.53, respectively. Furthermore, B. velezensis Bv-21 strain was tested for biocontrol activity against a streptomycin-resistant Xcc M4 in detached susceptible citrus leaves. The strain reduced the incidence of CBC by 26.30% and pathogen density of Xcc M4 by 81.68% over control. The results of the study strongly suggest that B. velezensis can be used as an effective and eco-friendly biocontrol agent either by itself or as an active compound, against both, the wild-type and streptomycin-resistant Xcc. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major disease of citrus plants, causing a significant loss in the citrus industry. The pthA is a bacterial effector protein mediates protein–protein and protein-DNA interactions and modulates host transcription. Injection of pthA effector protein into the host cell induces the expression of the susceptibility gene CsLOB1 which is required for citrus canker disease development. In this study, we described in planta expression of a specific anti-pthA single-chain variable fragment (scFv) recombinant antibody, scFvG8, and assessed its function using molecular docking, immunoblotting, and indirect enzyme-linked immunosorbent assay (ELISA). Based on the results, homology-based molecular docking suggested that at least eight intermolecular hydrogen bonds are involved in pthA-scFvG8 interactions. Immunoblotting and indirect ELISA results reconfirmed specific binding of scFvG8 to pthA protein. Moreover, gene fragment encoding scFvG8 was cloned into plant expression vector and transiently expressed in leaves of Nicotiana tabacum cv. Samson by agroinfiltration method. Transient expression of scFvG8 (at the expected size of 35 kDa) in N. tabacum leaves was confirmed by western blotting. Also, immunoblotting and indirect ELISA showed that the plant-derived scFvG8 had similar activity to purified scFvG8 antibody in detecting pthA. Additionally, in scFvG8-expressing tobacco leaves challenged with Xcc, a reduction (for up to 70%) of hypersensitive response (HR) possibly via direct interaction with pthA, was observed in the necrotic leaf area compared to control plants infected with empty vector. The results obtained in this study confirm that scFvG8 can suppress the function of pthA effector protein within plant cells, thus the induction of stable expression of scFvG8 in lime trees can be considered as an appropriate approach to confer resistance to Xcc. [ABSTRACT FROM AUTHOR]

The Xanthomonas citri pv. citri (Xcc) is causal agent of bacterial citrus canker which is major disease of citrus throughout the world. The pthA bacterial effector protein is presented within the infected plants and indispensable of canker. The scFv antibodies are valuable tools for diagnosis and suppression of pathogens within plants. The present article describes developing and characterization of specific recombinant monoclonal scFv antibodies against pthA effector protein. For this aim, the gene encoding pthA protein was heterologously expressed in Escherichia coli and used for screening of Tomlinson phage display antibody library to pinpoint specific single chain variable fragment (scFv). In each round of panning, the affinity of phage towards pthA was checked by enzyme linked immunosorbent assay (ELISA). The data was indicative of about 50% of the monoclonal phages to be reactive strongly against pthA protein. Among the positive clones, 5 samples (A12, B8, C1, H8 and G8) were capable of detecting Xcc-infected plant samples and recombinant pthA protein. Restriction fragment length polymorphism showed similar banding pattern for all 5 scFvs as renamed to pthA-scFG8. HB2151 E. coli cells were infected by the phage bearing pthA-scFG8, and the expression of the peptide was induced by IPTG to produce a 30 kDa recombinant molecule. I-TASSER was used for homology modeling of both scFv and pthA and docking was carried out by Hex program. The latter demonstrated binding energy of −784 kcal/mol in scFv-pthA.

Citrus Bacterial Canker disease, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most devastating diseases that attacks citrus, especially in the southern areas of Iran. The objective of this investigation was to analyze several characteristics involved in Xcc virulence in relation to strain aggressiveness. To achieve this, swarming motility, biofilm formation, resistance to H2O2, and production of xanthan were evaluated considering 44 local strains causing citrus bacterial canker disease in Iran. All strains showed differential swarming motilities, biofilm formation abilities, and xanthan production. The most virulent strain, Xcc-KVXCC1, exhibited the greatest capability to form biofilm on solid surfaces, xanthan production, and pathogenicity on detached leaves of Citrus aurantifolia as well as positive chemotaxis toward C. aurantifolia extract. In this study, a total of 14 common and commercial citrus genotypes were evaluated for resistance to Xcc-KVXCC1. Genotypes were categorized into susceptible and resistant groups based on lesion number per inoculation site. Based on our results, C. aurantifolia was the most susceptible citrus cultivar to Xcc-KVXCC1. In contrast, C. aurantium, C. jumbhori, and C. reticulata cv. ponkan demonstrated high and moderately high resistance against Xcc-KVXCC1. [ABSTRACT FROM AUTHOR]

Xanthomonas citri pv. citri (Xcc) and X. citri pv. aurantifolii (Xca) are causal agents of Citrus Bacterial Canker (CBC), a devastating disease that severely affects citrus plants. They are harmful organisms not reported in Europe or the Mediterranean Basin. Host plants are in the Rutaceae family, including the genera Citrus, Poncirus, and Fortunella, and their hybrids. In addition, other genera of ornamental interest are reported as susceptible, but results are not uniform and sometimes incongruent. We evaluated the susceptibility of 32 ornamental accessions of the Rutaceae family belonging to the genera Citrus, Fortunella, Atalantia, Clausena, Eremocitrus, Glycosmis, Microcitrus, Murraya, Casimiroa, Calodendrum, and Aegle, and three hybrids to seven strains of Xcc and Xca. Pathotyping evaluation was assessed by scoring the symptomatic reactions on detached leaves. High variability in symptoms and bacterial population was shown among the different strains in the different hosts, indicative of complex host–pathogen interactions. The results are mostly consistent with past findings, with the few discrepancies probably due to our more complete experimental approach using multiple strains of the pathogen and multiple hosts. Our work supports the need to regulate non-citrus Rutaceae plant introductions into areas, like the EU and Mediterranean, that are currently free of this economically important pathogen.

Citrus bacterial canker is a disease caused by Xanthomonas citri subsp. citri (Xcc). The bacterial type III secretion system (TTSS) consist of an extracellular filamentous structure mediating transfer of bacterial proteins into the plant cytosols. The main part of the pilus is pilin protein (HrpE) that is encoded by the HrpE gene. Production of specific antibodies against the pilin protein can be implemented for detection of infected plants as well as to develop a source of genetic resistance against disease. Inhibition of HrpE protein through antibody therapy leads to suppression of disease in the plant. The objective of the present study was to isolate, clone and express the HrpE gene. To do this, the HrpE gene was amplified by PCR using gene-specific primers and cloned to the pTZ57R/T vector. Then, it was cloned to the pET28a(+) bacterial expression vector and expressed in the E. coli strain Rosetta as host. The protein production procedure was optimized for incubation time and temperature as well as the concentration of inducer. The greatest amount of the recombinant protein was yielded at 1 mM IPTG at 30° C for 16 h. Purification of HrpE recombinant protein was done via metal affinity chromatography. The results of both SDS-PAGE and Western blotting assays confirmed the expression accuracy and purity of recombinant pilin protein. The recombinant protein can be used as an antigen to develop monoclonal and polyclonal antibodies.

Class III peroxidase (CIII prx) is a plant-specific multigene family that regulates the physiological and stress responses. This research aimed to exhaustively annotate and analyse the CIII prx family in sweet orange and to explore the regulated expression profiles by Xanthomonas citri subsp. citri (Xcc) and plant hormones. We further assessed the relationship between CIII prxs and citrus bacterial canker. The phylogeny, gene structure, conserved motifs, gene duplications and microsynteny of the CIII prx family were analysed. Expression profiles of specific CsPrxs induced by Xanthomonas citri subsp. citri and plant hormones were detected by quantitative reverse transcription-polymerase chain reaction. Subcellular localization was analysed through transient expression assessments. A total of 72 CIII prx members were identified from the genomes of sweet orange. In all chromosomes of sweet orange, the CsPrxs could be detected except in chromosome 8. In addition, three segmental duplications, four tandem duplications and 11 whole-genome duplications occurred among the CsPrxs, contributing to the family size expansion. From the Ka/Ks ratios, 15 of 18 duplicated CsPrxs pairs have experienced purifying selection process. A total of 15 conserved motifs were detected in CsPrxs, four of which were detected in all complete CsPrxs. A total of 12 expressed genes were identified from the EST database. The expression trends of 12 CsPrxs were differently expressed at different stages of infection by Xcc, five of which were potential candidate genes involved in Xcc resistance. These genes could be induced by salicylic acid and methyl jasmonate, and were extracellular proteins. These results further support our understanding of CIII prxs in citrus, particularly in citrus bacterial canker studies. [ABSTRACT FROM AUTHOR]

Bacterial citrus canker, caused by Xanthomonas citri subsp. citri, is a major disease of citrus throughout the world. The pthA bacterial effector protein, as an essential element for pathogenesis, is present within the infected plants. Here, development of a specific antibody raised against pthA as an early diagnostic tool is reported. For this aim, partial sequence at the C-terminus of pthA gene (606 bp), was PCR-amplified and cloned. The coding region of truncated pthA, t-pthA, was inserted into the pET28a(+) bacterial expression vector. The recombinant t-pthA was expressed in Escherichia coli strain Rosetta (DE3) and purified via affinity chromatography. The purity and integrity of expressed protein was confirmed by SDS-PAGE followed by Western blot analysis using anti-His antibody. The purified recombinant t-pthA was used for immunization of rabbits. Immunoglobulin was purified using protein A Sepharose. The binding ability of prepared IgG was confirmed via a suppression assay performed by direct injection of purified IgG into tobacco leaves followed by infiltration of purified t-pthA protein into leaves. This assay revealed that the presence of anti-pthA IgG prevents hypersensivity response (HR) of t-pthA within the infiltrated leaves. Further in vitro assays such as PTA-ELISA, Dot Immunobinding Assay (DIBA), and Western blot analysis proved the ability of prepared antibody for efficient detection of infected citrus plants. To the best of our knowledge, this is the first report for applying bacterial effector protein, such as pthA, as an antigen for developing a diagnostic approach against a plant bacterial disease. [ABSTRACT FROM AUTHOR]

The relative susceptibility of six Rutaceae family genotypes to bacterial citrus canker (CC) caused by Xanthomonas citri subsp. citri (Xcc) was assessed by measuring and comparing in planta bacterial populations over time following inoculation using a minimally destructive inoculation. These genotypes included lime (Citrus aurantifolia), grapefruit (C. paradisi), sweet orange (C. sinensis), calamondin (C. reticulata X C. japonica), kumquat (C. japonica), and orange jessamine (Murraya paniculata). Internal Xcc populations in orange jessamine plateaued early and remained significantly lower than other genotypes. Xcc populations increased rapidly in all other genotypes, decreasing precipitously and significantly over time in kumquat and calamondin as compared to the more susceptible genotypes sweet orange, grapefruit, and lime. Xcc populations in calamondin peaked by 7 DPI then began to fall significantly relative to the more susceptible genotypes sweet orange, grapefruit, and lime. Given the steep decline in populations in calamondin and kumquat, we compared water-soluble extracts from healthy leaf tissue from all previously investigated Citrus genotypes to determine if the extracts had any inhibitory effect on Xcc, implicating them in this abrupt population decline late in the infection process. Only extracts from calamondin leaf tissue significantly inhibited Xcc growth within 24 h. Potentially, constitutively produced inhibitory compounds in calamondin may result in less severe CC symptoms as a result of the rapid decline in Xcc populations. [ABSTRACT FROM AUTHOR]

Genetic engineering approaches offer an alternative method to the citrus canker resistance breeding. The ethylene response factor (ERF) family is a member of families of transcription factors that are particular to plants and contribute significantly to biotic stress response and to plant growth. CsAP2-09 belongs to the citrus AP2/ERF transcription factor family. Initially, we proved the induction of CsAP2-09 in wild-types by Xcc and some hormones involved in pathogen response. We successfully cloned the CsAP2-09 and proved that CsAP2-09 protein is targeted to the nucleus. The CsAP2-09 was functionally characterized with over-expression and RNAi silencing strategy. In the overexpression lines, the diseased lesions and disease index were significantly decreased while in RNAi lines of CsAP2-09 the diseased lesions and disease index were significantly enhanced. Thus, the over-expression conferred Xcc resistance to transgenic citrus while silencing of CsAP2-09 in sweet orange leads to Xcc susceptibility. When the transcriptomes of WT and overexpression transcriptomes were compared, they revealed that some genes involved in phenylpropanoid biosynthesis, pathogen responses, transcript regulation etc. were modified. Our results provide a possibility for improving citrus canker disease resistance by over-expression of CsAP2s. Furthermore, various functions of CsAP2-09 provide significant information about the role of AP2/ERFs in plant disease resistance and stress tolerance. • The expression of transcription factor CsAP2-09 was induced by some exogenous hormone and Xanthomonas citri subsp. Citri. • Overexpression of CsAP2-09 conferred Xcc resistance to transgenic citrus while Silencing of CsAP2-09 leads to Xcc susceptibility. • CsAP2-09 involved in phenylpropanoid biosynthesis, Plant-pathogen interaction and plant hormone signal transduction. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker (CBC) occurs due to Xanthomonas axonopodis pv. Citri (Xac) is a pathogenic bacteria. It was first reported in Japan, USA and many areas of India in 19th century. Synchronized by the spread of the causative agent the diversity of labelled pathogen continues to spread with new strains appearing in many other regions of the world. However, its diagnosis is quite difficult due to its symptoms similarity with other bacterial strains. This piece of study was designed to identify Xanthomonas axonopodis using polymerase chain reaction (PCR). PCR is more accurate, precise and fast method which is used for detection and quantification of many bacterial species. Two primers Xac01 and Xac02 were used for PCR based CBC detection. These primers were based on rpf gene region which give very specific results in Xac identification and can separate two much similar bacterial strains. There is another Primer pair designed on the pthA gene which act as universal primer and usually used for detection of all varieties of citrus canker bacteria. Leaves sample from different infected Citrus paradisi plants were collected for further analysis. Results confirmed that three samples among five have CBC. It is concluded that using PCR we can detect citrus canker disease more specifically which will help in discovering new eradication methods for its control. [ABSTRACT FROM AUTHOR]

The citrus bacterial canker is among the most important diseases in Mexican lime gardens in southern area of Iran. The disease is caused by Xanthomonas citri subsp. citri (Xcc). The PthA bacterial protein, as an effector protein, is crucial in pathogenesis pathway. Inhibition of its functions through antibody could lead to suppression of disease in plant. The present study was performed for isolation and expression of pthA gene and purification of PthA recombinant protein. For this aim the specific primers were designed for isolation of 606 bp of hydroxyl terminal of this gene followed by PCR amplification and cloning into pTZ57R/T vector. The coding sequence of truncated pthA was inserted to pET28a bacterial expression vector as a C-terminal fusion to 6XHis tag. Production of recombinant protein was performed in BL21(DE3) strain of E. coli. The purification was done under native condition through affinity chromatography on nickel column. Total yield was assessed by SDS-PAGE and western blotting analysis. The results confirmed production of PthA recombinant protein with molecular weight around 26 kDa. The purified recombinant protein could be used as an antigen for production of recombinant monoclonal antibodies.

Abstract Following a request from the European Commission, the European Food Safety Authority (EFSA) Plant Health (PLH) Panel analysed a dossier submitted by the Japanese authorities in order to clarify the host status of Citrus junos with regard to Xanthomonas citri pv. citri and Xanthomonas citri pv. aurantifolii, causal agents of citrus bacterial canker, and to indicate whether C. junos fruit could represent a pathway for the introduction of citrus bacterial canker into the European Union. In a previous opinion in the year 2014, the EFSA PLH Panel concluded that commercial fresh citrus fruit is generally pathway and that no commercially important Citrus species or variety can be considered as immune to citrus bacterial canker. In the current assessment, the EFSA PLH Panel analysed the two scientific papers provided by the Japanese authorities, as well as 16 additional papers identified through a systematic literature review. The PLH Panel considered that the conclusions of its previous opinion remain valid and that convergent lines of evidence provide sufficient demonstration that C. junos is a host of X. citri pv. citri and X. citri pv. aurantifolii. Therefore, there is no reason to consider the C. junos fruit differently from other citrus species. Consequently, the assessment of the general citrus fruit pathway from the 2014 opinion still applies. Uncertainties on these conclusions are a result of the scarce scientific evidence published on this subject in addition to the methodological and reporting limitations of the published papers.

Citrus bacterial canker (CBC), caused by the plant pathogenic bacterium Xanthomonas citri subsp. citri (Xcc), is a devastating disease in many commercial citrus cultivars. Every year, CBC causes a substantial reduction in fruit quality and quantity that corresponds to significant economic losses worldwide. Endophytic microorganisms produce numerous bioactive secondary metabolites that can control plant pathogens. We investigated the antagonistic activities of 66 endophytic bacteria isolated from nine citrus cultivars to control streptomycin-resistant Xcc. The suspension of Endophytic Bacteria-39 (EB-39), identified as Bacillus velezensis, exhibited the highest antibacterial activity against three wild-type and six streptomycin-resistant Xcc strains, with the inhibition zones between 39.47 ± 1.6 and 45.31 ± 1.6 mm. The ethyl acetate extract of EB-39 also controlled both wild-type and streptomycin-resistant Xcc strains, with the inhibition zones between 29.28 ± 0.6 and 33.88 ± 1.3 mm. Scanning electron microscopy indicated the ethyl acetate extract of EB-39-induced membrane damage and lysis. The experiments using the detached leaves of a susceptible Citrus species showed that EB-39 significantly reduced the incidence of canker on the infected leaves by 38%. These results strongly suggest that our newly isolated EB-39 is a novel biocontrol agent against CBC caused by wild-type and streptomycin-resistant Xcc strains.

The lateral organ boundary domain (LBD) genes encode a group of plant-specific proteins that function as transcription factors in the regulation of plant growth and development. Citrus sinensis lateral organ boundary 1 ( CsLOB1) is a member of the LBD family and functions as a disease susceptibility gene in citrus bacterial canker (CBC). Thirty-four LBD members have been identified from the Citrus sinensis genome. We assessed the potential for additional members of LBD genes in citrus to function as surrogates for CsLOB1 in CBC, and compared host gene expression on induction of different LBD genes. Using custom-designed transcription activator-like (TAL) effectors, two members of the same clade as CsLOB1, named CsLOB2 and CsLOB3, were found to be capable of functioning similarly to CsLOB1 in CBC. RNA sequencing and quantitative reverse transcription-polymerase chain reaction analyses revealed a set of cell wall metabolic genes that are associated with CsLOB1, CsLOB2 and CsLOB3 expression and may represent downstream genes involved in CBC. [ABSTRACT FROM AUTHOR]

Following a request from the European Commission, the European Food Safety Authority (EFSA) Plant Health (PLH) Panel analysed a dossier submitted by the Japanese authorities in order to clarify the host status of Citrus junos with regard to Xanthomonas citri pv. citri and Xanthomonas citri pv. aurantifolii, causal agents of citrus bacterial canker, and to indicate whether C. junos fruit could represent a pathway for the introduction of citrus bacterial canker into the European Union. In a previous opinion in the year 2014, the EFSA PLH Panel concluded that commercial fresh citrus fruit is generally pathway and that no commercially important Citrus species or variety can be considered as immune to citrus bacterial canker. In the current assessment, the EFSA PLH Panel analysed the two scientific papers provided by the Japanese authorities, as well as 16 additional papers identified through a systematic literature review. The PLH Panel considered that the conclusions of its previous opinion remain valid and that convergent lines of evidence provide sufficient demonstration that C. junos is a host of X. citri pv. citri and X. citri pv. aurantifolii. Therefore, there is no reason to consider the C. junos fruit differently from other citrus species. Consequently, the assessment of the general citrus fruit pathway from the 2014 opinion still applies. Uncertainties on these conclusions are a result of the scarce scientific evidence published on this subject in addition to the methodological and reporting limitations of the published papers. [ABSTRACT FROM AUTHOR]

Asiatic citrus canker caused by Xanthomonas citri subsp. citri is a bacterial disease with economic importance in tropical and subtropical citrus-producing areas. One of the potential reaction of plants to pathogens attack is an increased levels of pathogenesis-related proteins such as chitinase and β - 1,3 -glucanase. The objective of present study was to investigate the expression of the PR proteins; β-1,3-glucanase and chitinase in Washington Navel orange (Citrus sinensis) inoculated with Xanthomonas citri sub sp.citri. Quantitative reverse transcription-polymerase chain reaction was used to quantify the mRNA level of chitinase and ß- 1,3 -glucanase genes in early stage of plant defense response. For this purpose, total RNA was extracted from inoculated and non inoculated citrus plants at different times post-inoculation. Higher amounts of transcripts were detected 24 hours after pathogen inoculation of citrus leaves and four-fold more than the control citrus plant. The transcript levels of the two genes were reduced with the time after inoculation, and decreased significantly at 96 hours post inoculation. This study indicated that the role of chitinase gene is more than glucanase in early stage of citrus canker disease.

The individual and combined effects ofPseudomonas fluorescens(Pf) and salicylic acid (SA) were investigated for control of citrus bacterial canker (CBC). Both treated plants with copper hydroxide and untreated ones were used as controls. Mexican lime (Citrus aurantifolia) seedlings were treated with SA at 10 mM, Pf and distilled water. Plants were initially inoculated withXanthomonas citrisubspcitri72 h post treatments. Results indicated that the Pf and SA treatment controlled CBC more effectively compared to separately applying Pf or SA. The application of Pf in combination with SA significantly reduced lesion number per leaf (72%) and disease severity (84%). Significant changes in the activities of peroxidase and catalase were found. In conclusion, the integration of Pf with SA complements each other and can be applied to manage citrus canker disease in conjunction with other control programmes. [ABSTRACT FROM PUBLISHER]

The goal of this study was to determine whether bacterial antagonists could be used to control Xanthomonas citri subsp citri (Xcc), the causal agent of bacterial citrus canker disease. A total of 22 potentially bacterial antagonists isolated as epiphytes from the phylloplane of healthy citrus trees were screened for their in vitro biological control capability against Xcc. These strains were identified as Pseudomonas fluorescens on the basis of biochemical and physiological tests and 16S rDNA. Out of these 22 potentially bacterial antagonists, five strains (KSA1, KSA9, KSA14, KSA17, and KSA20) showed high potential growth inhibition capabilities against Xcc. The KSA1 strain was selected for further studies to test its in vivo capability to control bacterial citrus canker pathogen. It was sprayed in a suspension of 107 CFU ml-1 on citrus leaves which were subsequently inoculated after 72 h with 108 CFU ml-1 suspension of Xcc strain JQ890095. According to the in vivo biocontrol tests, the putative antagonist KSA1 significantly reduced the symptoms on the leaves of Mexican lime seedlings compared with untreated inoculated plants. [ABSTRACT FROM AUTHOR]

Citrus canker [caused by Xanthomonas citri subsp. citri ( Xcc)] can cause yield loss of susceptible citrus and result in trade restrictions of fresh fruit. For both regulatory purposes and epidemiological studies, accurate detection and quantification of viable inoculum are critical. Two accepted methods used to detect and quantify Xcc are injection-infiltration bioassay and culture, but these two methods have not been directly compared using field-obtained samples. The two methods were compared using washates of lesions taken from fruit, leaves and shoots in a commercial orchard in Florida in 2009-2010 and 2010-2011, with bioassay being the assumed standard. Despite some misclassifications, true positives (sensitivity) and true negatives (specificity) were the dominant classes using culture. False positives for lesions from shoots ranged from 13.1 to 21.4% in 2009-2010 and 2010-2011, respectively, and false positives for lesions from fruit and leaves ranged from 4.3 to 15.7%, in the two seasons, respectively. The false positive rate for culture compared with injection-infiltration bioassay was highest (0.16-0.55), due to more frequent recovery of Xcc by culture at ≤103 colony-forming units ( CFU) Xcc per ml. The false negative rate was consistently lower (0.02-0.21), confirming that in only a few cases did culture fail to detect Xcc when it was present. The area under the curve for receiver operator characteristic analysis ranged from 0.80 to 0.97, confirming that culture provided an accurate diagnosis in most cases. There was a higher frequency of lesions from shoots with a CFU ≤103 Xcc compared with lesions from fruit or leaves, making culture more effective at detecting these. The data demonstrate that culture is a reliable way to detect and quantify Xcc compared with injection-infiltration bioassay, particularly when the CFU is ≤103 Xcc per ml. [ABSTRACT FROM AUTHOR]

Molecular typing was applied and optimized for genetic characterization for three pathogenic variants of Xanthomonas axonopodis pv. citri ( Xac) from Taiwan. These three novel variants of atypical symptom-producing X. axonopodis pv. citri were designated as Xac-Af, Xac-Ap and Xac-Ar. Based on polymerase chain reaction (PCR) with primers specific to X. axonopodis pv. citri, leucine-responsive regulatory protein ( lrp) gene assay and DNA fingerprintings generated by repetitive-sequence PCR (rep-PCR) and amplified fragment length polymorphism (AFLP) were used to compare strains including the three types of atypical symptom-producing strains Xac-Af, Xac-Ap and Xac-Ar, and additional reference strains from pathotypes Xac-A, Xac-A*, Xac-Aw, X. axonopodis pv. auruantifolii and X. axonopodis pv . citrumelo. These three types of X. axonopodis pv. citri variants can be detected with six sets of primer specific for X. axonopodis pv. citri. Cluster analyses by lrp sequence assay, AFLP and combing the band patterns of rep-PCR clearly grouped the atypical symptom-producing variants in types Xac- Af, Xac-Ar and Xac-Ap into the same cluster with typical symptom-producing strains in pathotype Xac-A. These three types of X. axonopodis pv. citri variants could be excluded from strains of Xac-A* and Xac-Aw in these genotypic analyses. Strains of Xac-A* and Xac-Aw were closely related to Xac-A strains in our results. No Taiwan isolate was related to X. axonopodis pv. auruantifolii or X. axonopodis pv . citrumelo. The results further confirmed the atypical symptom-producing variants of X. axonopodis pv. citri in Taiwan belong to pathotype Xac-A. [ABSTRACT FROM AUTHOR]

The purpose of this study was to assess the stability of mRNA and rRNA for evaluation of viability for Xanthomonas citri subsp. citri (Xcc). Total RNA from Xcc suspensions subjected to different stress treatments (high temperature or chemical treatment with sodium orthophenylphenate at different concentrations) was extracted at different time periods post-treatment (0, 3, 24 and 48 h) and analysed by quantitative real-time reverse transcription PCR (Q-RT-PCR). Primers were designed from selected fragments of rRNA and mRNA from genes involved in bacterial fitness, virulence or general metabolic mechanisms ( gumD, rpfB, avrBs2 and gyrB). After stress treatment, only a 445-bp fragment from the gumD mRNA was detected in live Xcc cells specifically, whereas other RNA fragments, as well as DNA targets, were detected in both viable and nonviable cells. Statistical analyses demonstrated that the amount of some transcripts from genes involved in xanthan synthesis, pathogenicity factor regulation and DNA processing was significantly reduced after lethal treatments. The amplification of the 445-bp product from gumD mRNA was demonstrated to be useful for the detection of viable Xcc; the product was detected specifically from viable bacteria on leaf and citrus fruit surfaces and in citrus canker lesions. Instability of long RNA fragments can be used as a practical tool for the study of survival of citrus canker bacteria or for diagnostic purposes when the presence of viable bacteria needs to be confirmed. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker (CBC) is one of the most important diseases of citrus. It is caused by Xanthomonas citri subsp. citri (Xcc). To investigate the variability of Xcc, a collection of twenty-five strains were isolated from the Fars, Hormozgan, Kerman and Sistan-va-Baluchestan provinces of Iran. The twenty-five strains were assessed phenotypically and genetically. These strains had similar biochemical properties. Based on host range determination, the strains were divided into two groups; the first group was pathogenic on Mexican lime (Citrus aurantifolia), citrumelo (Poncirus trifoliata x C. paradisi), citrange (C. sinensis x P. trifoliata) and sour orange (C. aurantium) varieties. The second group was pathogenic on Mexican lime only. Profile of cellular soluble proteins analyzed by sodium dodecyl sulphate-polyacryamid gel electrophoresis (SDS-PAGE) did not reveal any considerable differences among strains. Genetic diversity analyses were performed using two marker systems; repetitive polymerase chain reaction (rep-PCR) and random amplified polymorphic DNA (RAPD). The results of this research showed that two primers, ERIC 1R and 232, with the highest marker index, resulted in the most genetic variability among strains. Cluster analysis by band patterns showed that strains from the Sistan-va-Baluchestan province were a different group, so it was concluded that geographical origin of strains from the Sistan-va-Baluchestan province is different than the geographical origin of strains isolated from other provinces. [ABSTRACT FROM AUTHOR]

The typical citrus canker lesions produced by Xanthomonas axonopodis pv. citri are erumpent, callus-like, with water-soaked margins. Three novel atypical symptom-producing variants of X. axonopodis pv. citri were described recently in Taiwan. Only the variant designated as A type produces typical erumpent canker lesions on Mexican lime ( Citrus aurantifolia) but induces flat necrotic with water-soaked margin lesions on grapefruit leaves ( C. paradisi). Two homologous pthA were cloned and characterized from strains XW19 (a typical canker lesion producing strain) and XW47 (a strain of A type). The pthA homolog from XW19 was transformed into XW47. The transformant of XW47 induced typical erumpent canker lesions on grapefruit leaves. Sequence analyses of transformants XW19 and XW47 revealed over 99% homology in nucleotide and deduced amino acid sequences compared with pthA homologs deposited in GenBank. The amino acid residues located at positions 49, 286, 742 and 767 of PthA were different between XW47 and XW19. The PthA mutants with a single amino acid substitution at each of these four positions were constructed by site-directed mutagenesis. Modified PthA (S286P) from XW47 in transformant 47SP induced erumpent canker lesions on grapefruit leaves, whereas another modified PthA (P286S) from XW19 in transformant 47PS only induced flat necrotic lesions. These results suggested that a single amino acid substitution from either serine to proline or proline to serine at position 286 of PthA can alter canker formation by X. axonopodis pv. citri on grapefruit leaves. [ABSTRACT FROM AUTHOR]

Nucleic acid sequence based amplification (NASBA) is a method of amplifying RNA, for the detection of RNA viruses and human pathogenic bacteria. Recently, NASBA has also been employed for the detection of plant diseases caused by viruses and quarantine bacteria. A major citrus pathogen, Xanthomonas citri subsp. c itri (Xcc) , causal agent of citrus bacterial canker, is being studied in depth due to its economic importance, with recent focus concentrating on its viability and survival under different stress conditions and control treatments. In this work, a NASBA protocol using primers for gumD mRNA has been developed to assess the viability of this pathogen under different bacteriocidal treatments. This method is rapid, specific and sensitive, and is able to detect viable bacterial cells, using a hybridization device which allows the visualization of the results in only 30 min. The usefulness of the method has been confirmed with bacterial suspensions subjected to different heat treatments and to sodium orthophenylphenate. [ABSTRACT FROM AUTHOR]

Twenty-four strains of Xanthomonas axonopodis pv. citri (Xac), the causal agent of bacterial canker of citrus, isolated from Mexican lime (Citrus aurantifolia) and lemon (Citrus limon) in southern Iran, were characterized phenotypically. Strains were all pathogenic on C. aurantifolia. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed slight differences in soluble protein profiles among the strains. Based on host range specificity and phenotypic characteristics, representative strains were differentiated into two groups of Asiatic (A) and atypical Asiatic (aA) forms. DNA fingerprinting analysis using EcoRI as the restriction endonuclease showed a negligible difference in restriction pattern between the two groups. On the basis of isozymic analysis, the two groups were distinct with respect to superoxide dismutase (SOD) and esterase (EST) banding patterns. Plasmid DNA profile analysis showed that the bacterial strains were different from each other in terms of plasmid number and molecular weight. Phage typing study revealed that most of group A strains were susceptible to Cp1 and/or Cp2 and some were resistant to both phage types including the strain in aA group. Bacteriocin production test indicated that there was a variation among Xac strains using different indicators for each bacteriocin producer. It is concluded that the Iranian strains of Xac are heterogeneous and constitute a subgroup(s) within the pathotype A. [ABSTRACT FROM AUTHOR]

Strains of Xanthomonas axonopodis pv. citri were isolated from Mexican lime (Citrus aurantifolia) trees in several countries in southwest Asia. These strains produced typical erumpent bacterial canker lesions on Mexican lime but not on grapefruit (C. paradisi). Lesions on grapefruit were watersoaked and blister-like in contrast to the typical erumpent lesions seen after artificial inoculation with all described pathotypes of X. axonopodis pv. citri. This group of strains hydrolysed gelatin and casein and grew in the presence of 3% NaCl as is typical of X. axonopodis pv. citri pathotype A. RFLP analyses and DNA probe hybridization assays also gave results consistent with X. axonopodis pv. citri pathotype A. Metabolic fingerprints prepared with the Biolog® system showed similarities as well as differences to X. axonopodis pv. citri pathotype A. In spite of the physiological and genetic similarities to pathotype A of X. axonopodis pv. citri, these strains had no or very little affinity for polyclonal antiserum prepared against any of the reference strains of X. axonopodis pv. citri and also did not react with monoclonal antibody A1, an antibody that detects all strains of pathotype A of X. axonopodis pv. citri. These strains were also insensitive to bacteriophage Cp3 like X. axonopodis pv. citri pathotype A and unlike X. axonopodis pv. citri pathotype B. We conclude that these strains, designated Xcc-A*, represent a variant of X. axonopodis pv. citri pathotype-A with pathogenicity limited to C. aurantifolia. The existence of extensive genotypic and phenotypic variation within pathotype A of X. axonopodis pv. citri was unexpected and further complicates the systematics of this species. [ABSTRACT FROM AUTHOR]

A kind of citrus fruit with special flavor, Citrus sudachi harvested in Japan, are exported to various countries. However, the Citrus sudachi needs to be sterilized using aqueous solution of sodium hypochlorite because there is a possibility of the adhesion of citrus bacterial canker (CBC) which is not found in Europe. Due to the sterilization with time-consuming work, a more effective decontamination technique is required. A decontamination method using ultraviolet (UV) light irradiation is thus anticipated. Especially, the use of light emitting diodes (LEDs) wi UV light has many advantages in terms of energy consumption, lifetime, and compactness； although an appropriate method is yet to be established. In this study, we evaluate the fundamental effectiveness of UV-LED decontamination on the basis of the bactericidal ability on CBC in petri dishes, using six kinds of UV-LEDs (265, 280, 285, 300, 310, and 365 nm) . For each irradiation, the resultant bactericidal abilities (BAs) were evaluated precisely taking into account the differences in their optical absorptions. In addition, BAs per unit photon number were also estimated, as a fundamental wavelength-dependence of BA. As a result, the effectiveness of UV-LED irradiation with relatively short wavelengths was demonstrated clearly.

• Nine CsLYK genes were confirmed from the genome of sweet orange. • CsLYKs could be induced by Xanthomonas citri subsp. citri and some phytohormones. • CsLYKs were involved in citrus bacterial canker development. Lysin motif receptor-like kinases (LYKs) plays a crucial role in plant-microbe interaction. During microbial infection, LYK recognizes the microbial entry into the host cell and triggers plant defense responses against pathogens. The citrus LYK family and its roles in the citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc) remains uncertain. In this study, we have performed a systematic annotation of CsLYK family and analyzed the altered expression patterns of genes induced by Xcc and biotic stress-related phytohormones (abscisic acid, methyl-jasmonate, and salicylic acid). We found that the Citrus sinensis genome harbors 9 LYK genes, which were further divided into 3 subgroups, and localized on 5 chromosomes and the unassembled scaffolds. They were characterized by the classic lysM and a Pkinase domain, and were found to be primarily localized on the plasma membrane. Most of the CsLYK family members were involved in a putative protein-protein interaction network. Expression profiles showed that CsLYKs could be induced by Xcc infection and some phytohormones. This highlights the correlation between the LYKs and the CBC resistance. These findings extend our understanding of LYKs in citrus, notably their roles in CBC studies. Besides, this study highlights the potential role of plant LYKs involved in pathogen resistance. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker (CBC), caused by the plant pathogenic bacterium Xanthomonas citri subsp. citri (Xcc), is a devastating disease in many commercial citrus cultivars. Every year, CBC causes a substantial reduction in fruit quality and quantity that corresponds to significant economic losses worldwide. Endophytic microorganisms produce numerous bioactive secondary metabolites that can control plant pathogens. We investigated the antagonistic activities of 66 endophytic bacteria isolated from nine citrus cultivars to control streptomycin-resistant Xcc. The suspension of Endophytic Bacteria-39 (EB-39), identified as Bacillus velezensis, exhibited the highest antibacterial activity against three wild-type and six streptomycin-resistant Xcc strains, with the inhibition zones between 39.47 ± 1.6 and 45.31 ± 1.6 mm. The ethyl acetate extract of EB-39 also controlled both wild-type and streptomycin-resistant Xcc strains, with the inhibition zones between 29.28 ± 0.6 and 33.88 ± 1.3 mm. Scanning electron microscopy indicated the ethyl acetate extract of EB-39-induced membrane damage and lysis. The experiments using the detached leaves of a susceptible Citrus species showed that EB-39 significantly reduced the incidence of canker on the infected leaves by 38%. These results strongly suggest that our newly isolated EB-39 is a novel biocontrol agent against CBC caused by wild-type and streptomycin-resistant Xcc strains. [ABSTRACT FROM AUTHOR]