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Background: Citrus canker caused by the bacterium Xanthomonas citri (Hasse 1915) is known as a dangerous disease that has serious economic impacts on citrus production in many tropical and subtropical countries. In Iran, Citrus canker disease has caused significant damage to various citrus fruits, particularly limes. Nowadays, employing antagonistic bacteria as an effective and environmentally friendly method for plant disease management has gained special importance. Results: Citrus leaf samples were collected from different regions of Iran, and after isolating epiphytic bacteria, ex planta and in planta investigations concerning antagonistic activities of the isolates against Xanthomonas citri pathotypes A and A* were performed. A total of 94 epiphytic bacterial isolates were isolated from citrus aerial parts. Based on biochemical, physiological, morphological, and genotypic tests, it was determined that these bacteria belong to the Bacillus and Staphylococcus genera. The highest inhibition activity against the pathogenic bacterium was related to isolates D4 and D5. Using the molecular method and the resulting dendrogram, it was found that these isolates were most similar to Bacillus amyloliquefaciens. The present findings demonstrated that pathogenicity test on key lime leaves infected with X. citri pathotypes A and A*, along with the application of the biocontrol strain B. amyloliquefaciens, resulted in a significant reduction in the number of canker lesions. Conclusion: The results strongly suggested that the identified antagonistic bacterial isolates hold promising potential as biocontrol agents for managing citrus bacterial canker disease. [ABSTRACT FROM AUTHOR]

Plant pathogens can alter the behavior of their insect vectors as well as their survival and reproduction. The African psyllid, Trioza erytreae, is one of the vectors of Huanglongbing, a citrus disease caused mainly by "Candidatus Liberibacter asiaticus" (CLas). The purpose of this study was to characterize the effects of CLas on the psyllid, T. erytreae using Citrus volkamerina plants as the study system. The study focused more specifically on the CLas effects prior to and after its acquisition by the psyllid T. erytreae. Our results did not support the hypothesis that CLas effects psyllid probing behavior prior to acquisition; few differences were observed between uninfected T. erytrea feeding on CLas‐infected versus control plants. On the other hand, compared to psyllids that had completed their development on control plants, the ones that had completed their development on a CLas‐infected plant exhibited changes in their behavior (greater velocity), physiology (smaller mass) and biochemistry (lower water and lipid content). Altogether, our results confirm the existence of a marked postacquisition effect on the vector locomotor behavior and a minor preacquisition effect of CLas on the vector behavior, which can be partially explained by physiological and biochemical changes. [ABSTRACT FROM AUTHOR]

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri, is one of the main threats to citrus fruit production. Several phenolic compounds active against X. citri have been described in recent years. Benzene‐1,2,4‐triol is a bio‐based phenolic compound that has shown high potential as a scaffold for the synthesis of new anti‐X. citri compounds. However, benzene‐1,2,4‐triol is prone to oxidative dimerization. We evaluated the antibacterial activity of benzene‐1,2,4‐triol, its oxidized dimers, and analogous compounds. Benzene‐1,2,4‐triol has a low inhibitory concentration against X. citri (0.05 mM) and is also active against other bacterial species. Spontaneous formation of benzenetriol dimers (e. g. by contact with oxygen in aqueous solution) reduced the antimicrobial activity of benzenetriol solutions. Dimers themselves displayed lower antibacterial activity and where shown to be more stable in solution. Unlike many other phenolic compounds with anti‐X. citri activity, benzene‐1,2,4‐triol does not act by membrane permeabilization, but seems to limit the availability of iron to cells. Benzene‐1,2,4‐triol is widely recognized as toxic – our results indicate that the toxicity of benzene‐1,2,4‐triol is largely due to spontaneously formed dimers. Stabilization of benzene‐1,2,4‐triol will be required to allow the safe use of this compound. [ABSTRACT FROM AUTHOR]

Background: Citrus canker is a significant bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that severely impedes the healthy development of the citrus industry. Especially when citrus fruit is infected by Xcc, it will reduce or even lost its commercial value. However, due to the prolonged fruiting cycle and intricate structure, much less research progress had been made in canker disease on fruit than on leaf. In fact, limited understanding has been achieved on canker development and the response to Xcc infection in fruit. Results: Herein, the progression of canker disease on sweet orange fruit was tracked in the field. Results indicated that typical lesions initially appear on the sepal, style residue, nectary disk, epicarp, and peduncle of young fruits after petal fall. The susceptibility of fruits to Xcc infection diminished as the fruit developed, with no new lesions forming at the ripening stage. The establishment of an efficient method for inoculating Xcc on fruit as well as the artificial inoculation throughout the fruit's developmental cycle clarified this infection pattern. Additionally, microscopic observations during the infection process revealed that Xcc invasion caused structural changes on the surface and cross-section of the fruit. Conclusions: An efficient system for inoculation on citrus fruit with Xcc was established, by which it can serve for the evaluation of citrus germplasm for canker disease resistance and systematic research on the interactions between Xcc and citrus fruits. [ABSTRACT FROM AUTHOR]

Pakistan's economy greatly benefits from citrus production since these fruits are sold and consumed all over the world. Although citrus fruits are easy to cultivate, they are susceptible to diseases caused by bacteria, viruses, and fungi. These challenges, as well as difficulties in obtaining the proper nutrients, might negatively impact fruit yields and quality. Citrus canker is another complicated problem caused by the germ Xanthomonas axonopodis. This germ affects many types of citrus fruits all over the world. This study looked closely at how citrus canker affects the leaves and the quality of the fruit in places like Sargodha, Bhalwal, Kotmomin, and Silanwali, which are big areas for growing citrus in the Sargodha district. What we found was that plants without the disease had more chlorophyll in their leaves compared to the sick plants. Also, the healthy plants had better amounts of important minerals like calcium, magnesium, potassium, and phosphorus in their fruits. But the fruits with the disease had too much sodium, and the iron levels were a bit different. The fruits with the disease also didn't have as much of something that protects them called antioxidants, which made them more likely to get sick. This study helps us understand how citrus canker affects plants and fruit, so we can think of ways to deal with it. [ABSTRACT FROM AUTHOR]

Wolbachia, a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Diaphorina citri Kuwayama, the vector of Candidatus Liberibacter asiaticus (CLas) that causes citrus greening disease, is naturally infected with Wolbachia (wDi). However, the interaction between wDi and D. citri remains poorly understood. In this study, we performed a pan-genome analysis using 65 wDi genomes to gain a comprehensive understanding of wDi. Based on average nucleotide identity (ANI) analysis, we classified the wDi strains into Asia and North America strains. The ANI analysis, principal coordinates analysis (PCoA), and phylogenetic tree analysis supported that the D. citri in Florida did not originate from China. Furthermore, we found that a significant number of core genes were associated with metabolic pathways. Pathways such as thiamine metabolism, type I secretion system, biotin transport, and phospholipid transport were highly conserved across all analyzed wDi genomes. The variation analysis between Asia and North America wDi showed that there were 39,625 single-nucleotide polymorphisms (SNPs), 2153 indels, 10 inversions, 29 translocations, 65 duplications, 10 SV-based insertions, and 4 SV-based deletions. The SV-based insertions and deletions involved genes encoding transposase, phage tail tube protein, ankyrin repeat (ANK) protein, and group II intron-encoded protein. Pan-genome analysis of wDi contributes to our understanding of the geographical population of wDi, the origin of hosts of D. citri, and the interaction between wDi and its host, thus facilitating the development of strategies to control the insects and huanglongbing (HLB). [ABSTRACT FROM AUTHOR]

Citrus canker is a disease caused by the gram-negative bacterium Xanthomonas citri subp. citri (X. citri), which affects all commercially important varieties of citrus and can lead to significant losses. Fruit sanitization with products such as chlorine-based ones can reduce the spread of the disease. While effective, their use raises concerns about safety of the workers. This work proposes essential oils (EOs) as viable alternatives for fruit sanitization. EOs from Cymbopogon species were evaluated as to their antibacterial activity, their effect on the bacterial membrane, and their ability to sanitize citrus fruit. The in vitro assays revealed that the EOs from C. schoenanthus and C. citratus had a lower bactericidal concentration at 312 mg L−1, followed by 625 mg L−1 for C. martini and C. winterianus. Microscopy assay revealed that the bacterial cell membranes were disrupted after 15 min of contact with all EOs tested. Regarding the sanitizing potential, the EOs with higher proportions of geraniol were more effective in sanitizing acid limes. Fruit treated with C. shoenanthus and C. martini showed a reduction of ∼68% in the recovery of viable bacterial cells. Therefore, these EOs can be used as viable natural alternatives in citrus fruit disinfection. [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]

Aims Citrus canker caused by Xanthomonas citri subsp. citri (X. citri) is a disease of economic importance. Control of this disease includes the use of metallic copper, which is harmful to the environment and human health. Previous studies showed that the crude extract from the fungus Pseudogymnoascus sp. LAMAI 2784 isolated from Antarctic soil had in vitro antibacterial action against X. citri. The aim of the present study was to expand the applications of this extract. Methods and results In greenhouse assays, the crude extract was able to reduce bacterial infection on citrus leaves from 1.55 lesions/cm2 (untreated plants) to 0.04 lesions/cm2. Bisdechlorogeodin was identified as the main compound of the bioactive fraction produced by Pseudogymnoascus sp. LAMAI 2784, which inhibited bacterial growth in vitro (IC90 ≈ 156 µg ml−1) and permeated 80% of X. citri cells, indicating that the membrane is the primary target. Conclusion The present results showed that the bioactive fraction of the extract is mainly composed of the compound bisdechlorogeodin, which is likely responsible for the biological activity against X. citri , and the main mechanism of action is the targeting of the cell membrane. This study indicates that bisdechlorogeodin has valuable potential for the control of X. citri. [ABSTRACT FROM AUTHOR]

Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm2) than in the plants producing scFvH6 (≤ 0.5 lesions/cm2) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease. [ABSTRACT FROM AUTHOR]

Virus-induced gene silencing (VIGS) is a powerful technology for molecular characterization of gene functions in plants and has the potential to prevent and control plant diseases. Unfortunately, VIGS in many woody perennials such as citrus are severely hindered because they have a long juvenile period and are recalcitrant to infection compared to herbaceous plants. Here, we report the development of a simple and effective VIGS system based on citrus leaf blotch virus (CLBV), a virus endemic in most citrus-producing countries but showing no symptoms in most citrus varieties. By Agrobacterium-mediated vacuum-infiltration (AVI), pCLBV201-su245 and pCLBV201-pds391 triggered efficient gene silencing (85%) and silencing phenotypes began to appear at 30 days post infiltration/inoculation (dpi), a similar period for achieving that on herbaceous plants. Moreover, the silencing phenotypes could still be observed four years post inoculation. Further, pCLBV201-lob369 was constructed to silence the gene lateral organ boundaries1 (lob1), encoding a key factor for susceptibility to citrus canker that caused by Xanthomonas citri ssp. citri (Xcc). The resistance evaluation results showed that the lesion area, disease index, and bacterial content in the pCLBV201-lob369 treatment group were decreased by 64%, 14%, and 67%, respectively. This work provides a simple and effective VIGS system for citrus which has the potential to be used for diseases control. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker (CBC) is a severe bacterial infection caused by Xanthomonas citri subsp. citri (Xcc), which continues to adversely impact citrus production worldwide. Members of the GATA family are important regulators of plant development and regulate plant responses to particular stressors. This report aimed to systematically elucidate the Citrus sinensis genome to identify and annotate genes that encode GATAs and evaluate the functional importance of these CsGATAs as regulators of CBC resistance. In total, 24 CsGATAs were identified and classified into four subfamilies. Furthermore, the phylogenetic relationships, chromosomal locations, collinear relationships, gene structures, and conserved domains for each of these GATA family members were also evaluated. It was observed that Xcc infection induced some CsGATAs, among which CsGATA12 was chosen for further functional validation. CsGATA12 was found to be localized in the nucleus and was differentially upregulated in the CBC-resistant and CBC-sensitive Kumquat and Wanjincheng citrus varieties. When transiently overexpressed, CsGATA12 significantly reduced CBC resistance with a corresponding increase in abscisic acid, jasmonic acid, and antioxidant enzyme levels. These alterations were consistent with lower levels of salicylic acid, ethylene, and reactive oxygen species. Moreover, the bacteria-induced CsGATA12 gene silencing yielded the opposite phenotypic outcomes. This investigation highlights the important role of CsGATA12 in regulating CBC resistance, underscoring its potential utility as a target for breeding citrus varieties with superior phytopathogen resistance. [ABSTRACT FROM AUTHOR]

The development of efficient and environmentally friendly plant protection systems is one of the major challenges for a sustainable agriculture of the future. Citrus canker caused by the pathogen Xanthomonas citri subsp. citri (X. citri) affects all cultivated citrus species worldwide and is responsible for enormous economic losses and restrictions in international trade. Currently used commercial copper‐based formulations are the origin of contamination for soil and groundwater, affecting local ecosystems and human health. A copper‐free sustainable microgel‐based plant protection system able to efficiently combat X. citri is developed. Microgels decorated with anchor peptides exhibit strong non‐covalent attachment to the surface of orange leaves and have the ability to release hexyl gallate, which inhibits the growth and spreading of pathogens. The tailored design of the microgel network allows high loadings of hexyl gallate (up to 40 wt.%) and a controlled release of gallates from microgels that provide long‐term protection. The antibacterial activity of the hexyl gallate‐loaded microgels is demonstrated by various in vitro assays as well as on orange plants in greenhouse settings. The experimental results suggest that the developed sustainable microgel‐based plant protection system for citrus trees allows efficient abatement of X. citri and reduces environmental pollution caused by copper formulations. [ABSTRACT FROM AUTHOR]

Citrus canker is an infectious bacterial disease and one of the major threats to the orange juice industry, a multibillion-dollar market that generates hundreds of thousands of jobs worldwide. This disease is caused by the Gram-negative bacterium Xanthomonas citri subsp. citri. In Brazil, the largest producer and exporter of concentrate orange juice, the control of citrus canker is exerted by integrated management practices, in which cupric solutions are intensively used in the orchards to refrain bacterial spreading. Copper ions accumulate and are as heavy metals toxic to the environment. Therefore, the aim of the present work was to evaluate bifunctional fusion proteins (BiFuProts) as novel and bio-/peptide-based alternatives to copper formulations to control citrus canker. BiFuProts are composed of an anchor peptide able to bind to citrus leaves, and an antimicrobial "killer" peptide to protect against bacterial infections of plants. The selected BiFuProt (Mel-CgDEF) was bactericidal against X. citri at 125 μg mL−1, targeting the bacterial cytoplasmic membrane within the first minutes of contact. The results in the greenhouse assays proved that Mel-CgDEF at 250 μg mL−1 provided protection against X. citri infection on the leaves, significantly reducing the number of lesions by area when compared with the controls. Overall, the present work showed that the BiFuProt Mel-CgDEF is a biobased and biodegradable possible alternative for substitute cupric formulations. Key points: • The bifunctional fusion protein Mel-CgDEF was effective against Xanthomonas citri. • Mel-CgDEF action mechanism was the disruption of the cytoplasmic membrane. • Mel-CgDEF protected citrus leaves against citrus canker disease. [ABSTRACT FROM AUTHOR]

Poplar is a major afforestation species which is widely planted worldwide. With the continuous expansion of poplar plant areas, the occurrence of poplar canker disease increased rapidly, causing serious damage to the trees. Currently, the major controlling method for poplar canker is chemical agents, but side effects such as environmental pollution and pathogen resistance are becoming more and more serious. There is an urgent need for developing effective non-polluting means to control the canker disease. By using microbiomes as a research tool, beneficial microorganisms that enhance plant disease resistance can be obtained. These antagonistic bacteria can inhibit the growth of canker pathogens without environmental pollution, and have good biological-control potential. This review mainly explores the role and mechanisms of antagonistic bacteria in the control of poplar canker, including inhibiting the growth of pathogenic bacteria through nutrient and water competition, secreting bacteriostatic substances, improving plant disease resistance to resist pathogen infection, and improving the host's own reactive oxygen species tolerance, in order to provide a theoretical basis for the development of efficient and specific bacterial agents, and scientific prevention and control technology. [ABSTRACT FROM AUTHOR]

Xanthomonas citri pv. citri (Xcc) causes the devastating citrus canker disease. Xcc is known to have been introduced into Florida, USA in at least three different events in 1915, 1986 and 1995 with the first two claimed to be eradicated. It was questioned whether the Xcc introduction in 1986 has been successfully eradicated. Furthermore, it is unknown how Xcc has spread throughout the citrus groves in Florida. In this study, we investigated the population structure of Xcc to address these questions. We sequenced the whole genome of 343 Xcc strains collected from Florida groves between 1997 and 2016. Our analysis revealed two distinct clusters of Xcc. Our data strongly indicate that the claimed eradication of the 1986 Xcc introduction was not successful and Xcc strains from 1986 introduction were present in samples from at least 8 counties collected after 1994. Importantly, our data revealed that the Cluster 2 strains, which are present in all 20 citrus-producing counties sampled in Florida, originated from the Xcc introduction event in the Miami area in 1995. Our data suggest that Polk County is the epicenter of the dispersal of Cluster 2 Xcc strains, which is consistent with the fact that three major hurricanes passed through Polk County in 2004. As copper-based products have been extensively used to control citrus canker, we also investigated whether Xcc strains have developed resistance to copper. Notably, none of the 343 strains contained known copper resistance genes. Twenty randomly selected Xcc strains displayed sensitivity to copper. Overall, this study provides valuable insights into the introduction, eradication, spread, and copper resistance of Xcc in Florida. Author summary: Sequencing and analyzing the genomes of 343 Xcc strains collected from 1997 to 2016 have enabled us to gain a deeper understanding of its introduction, spread, and eradication. This information is critical for optimizing strategies for responding to the invasion of quarantine pathogens. We have conclusively demonstrated that the Xcc introduction in 1986 was not successfully eradicated, three major hurricanes in 2004 play a critical role in spreading Xcc throughout Florida, and Polk County is the epicenter of Xcc dispersal. Additionally, none of the tested Xcc strains displayed resistance to copper, consistent with the absence of copper resistance genes in their genome. [ABSTRACT FROM AUTHOR]

In Mexico, Huanglongbing infection caused 42 and 33% decreases in orange and lemon production, respectively. Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a vector of Candidatus Liberibacter bacterium that causes the disease. Commercial orchards were searched to identify entomopathogenic fungi of D. citri in the citrus zone of Huasteca Potosina, San Luis Potosi, and Llera, Tamaulipas, Mexico. Entomopathogenic fungi associated with Asian citrus psyllid adults are in three genera: Hirsutella citriformis (88%), Beauveria bassiana (2%), and Sporothrix insectorum (10%). This is the first report of Sporothrix insectorum parasitizing H. citriformis in México. [ABSTRACT FROM AUTHOR]

This document, titled "PP 1/122 (3) Phytophthora nicotianae var. parasitica and other Phytophthora species causing canker on citrus," provides guidelines for conducting trials to evaluate the efficacy of fungicides against Phytophthora species that cause canker on citrus trees. The document outlines the experimental conditions, trial conditions, design and layout of the trial, application of treatments, mode of assessment, recording and measurements, and reporting of results. It emphasizes the importance of following good experimental practices and provides references to other relevant standards. The document does not provide specific results or conclusions, but rather serves as a guide for researchers conducting trials on this topic. [Extracted from the article]

Several studies have shown that spray-induced gene silencing (SIGS) in which double-stranded RNAs (dsRNAs) and small RNAs duplexes (sRNAs) are sprayed on plant surfaces to target pathogens genes or plant genes causing selective knockdown and provide effective crop protection. However, using SIGS to control bacterial diseases in plants has not been developed because the silencing phenomenon known as RNAi (RNA interference) is not present in bacteria. Here, we mainly focus on the applicability to use SIGS as an alternative eco-friendly strategy to control citrus canker caused by Xanthomonas; in parallel with citrus leaf miner control as a part of RNA-based citrus canker controlling strategy. We suggest several promising genes to be targeted or knocked down in citrus, citrus leaf miner and Xanthomonas, in addition to the application strategies of RNA molecules to enhance their uptake. Since the molecular mechanism of SIGS may be controlled by RNAi-like silencing machinery in Xanthomonas, it is important to study SIGS pathway components to better understand Xanthomonas virulence. Therefore, we propose a hypothetical and possible SIGS pathway; but this should be confirmed experimentally to insure the successful application of SIGS against citrus canker and other bacterial diseases. [ABSTRACT FROM AUTHOR]

Summary: Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), causes dramatic losses to the citrus industry worldwide. Transcription activator‐like effectors (TALEs), which bind to effector binding elements (EBEs) in host promoters and activate transcription of downstream host genes, contribute significantly to Xcc virulence. The discovery of the biochemical context for the binding of TALEs to matching EBE motifs, an interaction commonly referred to as the TALE code, enabled the in silico prediction of EBEs for each TALE protein. Using the TALE code, we engineered a synthetic resistance (R) gene, called the Xcc‐TALE‐trap, in which 14 tandemly arranged EBEs, each capable of autonomously recognizing a particular Xcc TALE, drive the expression of Xanthomonas avrGf2, which encodes a bacterial effector that induces plant cell death. Analysis of a corresponding transgenic Duncan grapefruit showed that transcription of the cell death‐inducing executor gene, avrGf2, was strictly TALE‐dependent and could be activated by several different Xcc TALE proteins. Evaluation of Xcc strains from different continents showed that the Xcc‐TALE‐trap mediates resistance to this global panel of Xcc isolates. We also studied in planta‐evolved TALEs (eTALEs) with novel DNA‐binding domains and found that these eTALEs also activate the Xcc‐TALE‐trap, suggesting that the Xcc‐TALE‐trap is likely to confer durable resistance to Xcc. Finally, we show that the Xcc‐TALE‐trap confers resistance not only in laboratory infection assays but also in more agriculturally relevant field studies. In conclusion, transgenic plants containing the Xcc‐TALE‐trap offer a promising sustainable approach to control CBC. [ABSTRACT FROM AUTHOR]

Autophagy functions in plant host immunity responses to pathogen infection. The molecular mechanisms and functions used by the citrus Huanglongbing (HLB)-associated intracellular bacterium ' Candidatus Liberibacter asiaticus' (C Las) to manipulate autophagy are unknown. We identified a C Las effector, SDE4405 (CLIBASIA_04405), which contributes to HLB progression. 'Wanjincheng' orange (Citrus sinensi s) transgenic plants expressing SDE4405 promotes C Las proliferation and symptom expression via suppressing host immunity responses. SDE4405 interacts with the ATG8-family of proteins (ATG8s), and their interactions activate autophagy in Nicotiana benthamiana. The occurrence of autophagy is also significantly enhanced in SDE4405-transgenic citrus plants. Interrupting NbATG8s-SDE4405 interaction by silencing of NbATG8c reduces Pseudomonas syringae pv. tomato strain DC3000 ΔhopQ1-1 (Pst DC3000 ΔhopQ1-1) proliferation in N. benthamiana , and transient overexpression of CsATG8c and SDE4405 in citrus promotes Xanthomonas citri subsp. citri (Xcc) multiplication, suggesting that SDE4405-ATG8s interaction negatively regulates plant defense. These results demonstrate the role of the C Las effector protein in manipulating autophagy, and provide new molecular insights into the interaction between C Las and citrus hosts. [ABSTRACT FROM AUTHOR]

The ETHYLENE INSENSITIVE3-LIKE (EIL) family is one of the most important transcription factor (TF) families in plants and is involved in diverse plant physiological and biochemical processes. In this study, ten EIL transcription factors (CsEILs) in sweet orange were systematically characterized via whole-genome analysis. The CsEIL genes were unevenly distributed across the four sweet orange chromosomes. Putative cis-acting regulatory elements (CREs) associated with CsEIL were found to be involved in plant development, as well as responses to biotic and abiotic stress. Notably, quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that CsEIL genes were widely expressed in different organs of sweet orange and responded to both high and low temperature, NaCl treatment, and to ethylene-dependent induction of transcription, while eight additionally responded to Xanthomonas citri pv. Citri (Xcc) infection, which causes citrus canker. Among these, CsEIL2, CsEIL5 and CsEIL10 showed pronounced upregulation. Moreover, nine genes exhibited differential expression in response to Candidatus Liberibacter asiaticus (CLas) infection, which causes Citrus Huanglongbing (HLB). The genome-wide characterization and expression profile analysis of CsEIL genes provide insights into the potential functions of the CsEIL family in disease resistance. [ABSTRACT FROM AUTHOR]

Plant–pathogen interaction is influenced by multiple environmental factors, including temperature and light. Recent works have shown that light modulates not only the defense response of plants but also the pathogens virulence. Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker, an important plant disease worldwide. The Xcc genome presents four genes encoding putative photoreceptors: one bacteriophytochrome and three blue light photoreceptors, one LOV and two BLUFs (bluf1: XAC2120 and bluf2: XAC3278). The presence of two BLUFs proteins is an outstanding feature of Xcc. In this work we show that the bluf2 gene is functional. The mutant strain, XccΔbluf2, was constructed demonstrating that BLUF2 regulates swimming-type motility, adhesion to leaves, exopolysaccharide production and biofilm formation, features involved in the Xcc virulence processes. An important aspect during the plant–pathogen interaction is the oxidative response of the host and the consequent reaction of the pathogen. We observed that ROS detoxification is regulated by Xcc bluf2 gene. The phenotypes of disease in orange plants produced by WT and XccΔbluf2 strains were evaluated, observing different phenotypes. Altogether, these results show that BLUF2 negatively regulates virulence during citrus canker. This work constitutes the first report on BLUF-like receptors in plant pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker (CBC) has become one of the most important diseases in many citrus cultivation areas, including Saudi Arabia (SA). This work aimed to characterize the genetic diversity of Xanthomonas citri pv. citri (Xcc) in SA by microsatellite and minisatellite analysis based on CRISPR typing. Our data revealed a high genetic infra‐pathovar diversity of Xcc in SA, consistent with a recent analysis targeting tandem repeats (MLVA). Our finding showed that SA hosted 15 spoligotypes out of 63 spoligotypes recorded worldwide. Interestingly, we noted 10 new spoligotypes, two detected in the western region and eight in the south‐western region. Two new spoligotypes were detected in Yemen. This finding suggests that the south‐western region of SA was probably the source of Xcc variations in SA. Sequencing of the CRISPR regions confirmed the presence of Xcc Pathotype A recently identified in SA based on tandem repeat genotyping. Globally, our study supported the application of CRISPR‐based sequence analysis for molecular epidemiology studies of Xcc and the usefulness of CRISPR typing to complement other typing tools, such as minisatellite typing. [ABSTRACT FROM AUTHOR]

Citrus canker, caused by the bacterium Xanthomonas citri (Xcc), is one of the most devastating diseases for the citrus industry. Xylose is a constituent of the cell wall of plants, and the ability of Xcc to use this carbohydrate may play a role in virulence. Xcc has two genes codifying for xylose isomerase (XI), a bifunctional enzyme that interconverts D-xylose into D-xylulose and D-glucose into D-fructose. The aim of this work was to investigate the functional role of the two putative XI ORFs, XAC1776 (xylA1) and XAC4225 (xylA2), in Xcc pathogenicity. XI-coding genes of Xcc were deleted, and the single mutants (XccΔxylA1 or XccΔxylA2) or the double mutant (XccΔxylA1ΔxylA2) remained viable. The deletion of one or both XI genes (xylA1 and/or xylA2) increased the aggressiveness of the mutants, causing disease symptoms. RT-qPCR analysis of wild strain and xylA deletion mutants grown in vivo and in vitro revealed that the highest expression level of hrpX and xylR was observed in vivo for the double mutant. The results indicate that XI depletion increases the expression of the hrp regulatory genes in Xcc. We concluded that the intracellular accumulation of xylose enhances Xcc virulence. [ABSTRACT FROM AUTHOR]

An economical, simple, rapid, and culture independent method was developed for routine analyses and detection of Xanthomonas axonopodis pv. punicae (Xap) that causes bacterial blight in pomegranate. Five DNA release buffers (B1-B5) were optimized for extracting bacterial genomic DNA (gDNA) directly from (a)symptomatic pomegranate leaves followed by conventional polymerase chain reaction (PCR)-based detection of Xap. B1, B3, and B4 were found suitable to release gDNA, which was subjected to PCR using universal primers for 16S rRNA and rpsL genes, and pathogen specific xopQ primers. DNA released from B1 and B4 successfully produced amplicons of expected sizes. Additional analyses found that DNA released using B4 buffer was stable up to 45 days at −20 °C/−80 °C and 35 days at 4 °C and 8-800 pg DNA could be detected by the PCR-based assay. B4 was further validated for versatility by extracting DNA of Xanthomonas spp. causing citrus canker and sorghum shoot stripe disease. [ABSTRACT FROM AUTHOR]

Transcription activator-like effectors are key virulence factors of Xanthomonas. They are secreted into host plant cells and mimic transcription factors inducing the expression of host susceptibility (S) genes. In citrus, CsLOBl is a direct target of PthA4, the primary effector associated with citrus canker symptoms. CsLOBl is a transcription factor, and its expression is required for canker symptoms induced by Xanthomonas citri subsp. citri. Several genes are up-regulated by PthA4; however, only CsLOBl was described as an S gene induced by PthA4. Here, we investigated whether other up-regulated genes could be direct targets of PthA4 or CsLOB 1. Seven up-regulated genes by PthA4 were investigated; however, an expansin-coding gene was more induced than CsLOBl. In Nicotiana benthamiana transient expression experiments, we demonstrate that the expansin-coding gene, referred here to as CsLOBl-INDUCED EXPANSIN 1 (CsLIEXPl), is not a direct target of PthA4, but CsLOB 1. Interestingly, CsLIEXPl was induced by CsLOBl even without the predicted CsLOBl binding site, which suggested that CsLOB 1 has other unknown binding sites. We also investigated the minimum promoter regulated by CsLOBl, and this region and LOB1 domain were conserved among citrus species and relatives, which suggests that the interaction PthM-CsLOB 1'-CsLIEXPl is conserved in citrus species and relatives. This is the first study that experimentally demonstrated a CsLOBl downstream target and lays the foundation to identify other new targets. In addition, we demonstrated that the CsLIEXPl is a putative S gene indirectly induced by PthA4, which may serve as the target for genome editing to generate citrus canker-resistant varieties. [ABSTRACT FROM AUTHOR]

The citrus canker pathogen Xanthomonas axonopodis has caused severe damage to citrus crops worldwide, resulting in significant economic losses for the citrus industry. To address this, a green synthesis method was used to develop silver nanoparticles with the leaf extract of Phyllanthus niruri (GS-AgNP-LEPN). This method replaces the need for toxic reagents, as the LEPN acts as a reducing and capping agent. To further enhance their effectiveness, the GS-AgNP-LEPN were encapsulated in extracellular vesicles (EVs), nanovesicles with a diameter of approximately 30–1000 nm naturally released from different sources, including plant and mammalian cells, and found in the apoplastic fluid (APF) of leaves. When compared to a regular antibiotic (ampicillin), the delivery of APF-EV-GS-AgNP-LEPN and GS-AgNP-LEPN to X. axonopodis pv. was shown to have more significant antimicrobial activity. Our analysis showed the presence of phyllanthin and nirurinetin in the LEPN and found evidence that both could be responsible for antimicrobial activity against X. axonopodis pv. Ferredoxin-NADP+ reductase (FAD-FNR) and the effector protein XopAI play a crucial role in the survival and virulence of X. axonopodis pv. Our molecular docking studies showed that nirurinetin could bind to FAD-FNR and XopAI with high binding energies (−10.32 kcal/mol and −6.13 kcal/mol, respectively) as compared to phyllanthin (−6.42 kcal/mol and −2.93 kcal/mol, respectively), which was also supported by the western blot experiment. We conclude that (a) the hybrid of APF-EV and GS-NP could be an effective treatment for citrus canker, and (b) it works via the nirurinetin-dependent inhibition of FAD-FNR and XopAI in X. axonopodis pv. [ABSTRACT FROM AUTHOR]

Diaphorina citri Kuwayama (D. citri) is an insect vector of phloem-limited 'Candidatus Liberibacter asiatus' (CLas), the presumed pathogen of citrus Huanglongbing (HLB). Recently, our lab has preliminarily found it acquired and transmitted Citrus tristeza virus (CTV), which was previously suggested to be vectored by species of aphids. However, the influences of one of the pathogens on the acquisition and transmission efficiency of the other pathogen remain unknown. In this study, CLas and CTV acquisition and transmission by D. citri at different development stages under field and laboratory conditions were determined. CTV could be detected from the nymphs, adults, and honeydew of D. citri but not from the eggs and exuviates of them. CLas in plants might inhibit CTV acquisition by D. citri as lower CTV–positive rates and CTV titers were detected in D. citri collected from HLB-affected trees compared to those from CLas–free trees. D. citri were more likely to obtain CTV than CLas from host plants co-infected with the two pathogens. Intriguingly, CTV in D. citri facilitated the acquisition and transmission of CLas, but CLas carried by D. citri had no significant effect on the transmission of CTV by the same vector. Molecular detection and microscopy methods confirmed the enrichment of CTV in the midgut after a 72-h acquisition access period. Collectively, these results raise essential scientific questions for further research on the molecular mechanism of pathogen transmission by D. citri and provide new ideas for the comprehensive prevention and control of HLB and CTV. [ABSTRACT FROM AUTHOR]

Citrus canker (CC), caused by one of the most destructive subfamilies of the bacterial phytopathogen Xanthomonas citri subsp. Citri (Xcc), poses a serious threat to the significantly important citrus fruit crop grown worldwide. This has been the subject of ongoing epidemiological and disease management research. Currently, five different forms have been identified of CC, in which Canker A (Xanthomonas citri subsp. citri) being the most harmful and infecting the majority of citrus cultivars. Severe infection symptoms include leaf loss, premature fruit drop, dieback, severe fruit blemishing or discoloration, and a decrease in fruit quality. The infection spreads rapidly through wind, rain splash, and warm and humid climates. The study of the chromosomal and plasmid DNA of bacterium has revealed the evolutionary pattern among the pathovars, and research on the Xcc genome has advanced our understanding of how the bacteria specifically recognize and infect plants, spread within the host, and propagates itself. Quarantine or exclusion programs, which prohibit the introduction of infected citrus plant material into existing stock, are still in use. Other measures include eliminating sources of inoculum, using resistant hosts, applying copper spray for protection, and implementing windbreak systems. The main focus of this study is to highlight the most recent developments in the fields of Xcc pathogenesis, epidemiology, symptoms, detection and identification, host range, spread, susceptibility, and management. Additionally, it presents an analysis of the economic impact of this disease on the citrus industry and suggests strategies to reduce its spread, including the need for international collaboration and research to reduce the impact of this disease on the global citrus industry. [ABSTRACT FROM AUTHOR]

Citrus cancer, caused by strains of Xanthomonas citri (Xc) and Xanthomonas aurantifolii (Xa), is one of the most economically important citrus diseases. Although our understanding of the molecular mechanisms underlying citrus canker development has advanced remarkably in recent years, exactly how citrus plants fight against these pathogens remains largely unclear. Using a Xa pathotype C strain that infects Mexican lime only and sweet oranges as a pathosystem to study the immune response triggered by this bacterium in these hosts, we herein report that the Xa flagellin C protein (XaFliC) acts as a potent defence elicitor in sweet oranges. Just as Xa blocked canker formation when coinfiltrated with Xc in sweet orange leaves, two polymorphic XaFliC peptides designated flgIII‐20 and flgIII‐27, not related to flg22 or flgII‐28 but found in many Xanthomonas species, were sufficient to protect sweet orange plants from Xc infection. Accordingly, ectopic expression of XaFliC in a Xc FliC‐defective mutant completely abolished the ability of this mutant to grow and cause canker in sweet orange but not Mexican lime plants. Because XaFliC and flgIII‐27 also specifically induced the expression of several defence‐related genes, our data suggest that XaFliC acts as a main immune response determinant in sweet orange plants. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker (CBC) is a serious bacterial disease affecting citrus plantations and the citrus industry all over the world. We have previously shown that an apetala 2/ethylene response factor in Citrus sinensis , CsAP2-09, positively regulated resistance to CBC, although the regulatory mechanisms remained undetermined. Here, we demonstrated that CsAP2-09 positively and sustainably controlled resistance to CBC in three-year transgenic plants. CsAP2-09 was found to be a transcriptional activator, and qRT-PCR and dual luciferase assays showed that it controlled the expression CsGH3.1L. CsAP2-09 bound directly to the promotor of CsGH3.1L, shown by yeast one-hybrid assay, with the binding site confirmed by electrophoretic mobility shift assay. Biochemical assays showed that CsAP2-09 negatively regulated the biosynthesis of indole acetic acid (IAA) and positively regulated that of salicylic acid (SA) and ethylene, verified with transient overexpression of CsGH3.1L. The combination of these results with those of previous reports indicated that SA, ethylene, and IAA can directly regulate CBC resistance. Overall, we revealed a pathway whereby CsAP2-09 conferred CBC resistance by direct binding to the CsGH3.1L promoter, activating its expression and modulating IAA, SA, and ethylene biosynthesis. Our study indicates the potential value of manipulating CsAP2-09 and CsGH3.1L in the breeding of CBC-resistant citrus. [ABSTRACT FROM AUTHOR]

Tahiti limes were an important fruit tree crop in South Florida until the late 1990s. Several factors contributed to the demise of the Florida lime industry, namely the impact of hurricane Andrew in 1992 and the arrival of Citrus Canker (CC) in 1995. The Florida lime industry was then eradicated in the early 2000s to protect the main commercial citrus industry from CC. This article focuses on the financial viability of reintroducing Tahiti lime production in South Florida. A total of 20 Tahiti lime scion/rootstock combinations were evaluated under endemic CC and Citrus Greening (CG) conditions. We created a deterministic and a stochastic budget to evaluate the feasibility of reintroducing Tahiti limes in South Florida that incorporated yields from experimental plots. We also implemented a financial analysis, that included net present value (NPV) and internal rate of return (IRR) estimations, for a 20-year period with the Tahiti lime/citrus macrophylla (TL/CM), the best performing scion/rootstock combination. Our findings indicated that Tahiti lime production in South Florida was not financially feasible; we obtained a negative NPV from both the deterministic and stochastic budgets. We conclude with a discussion of potential scenarios for the Tahiti lime industry in the US and on lessons for other crops that face similar production challenges. [ABSTRACT FROM AUTHOR]

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xcc), is a disease that causes serious problems to the global citrus industry. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has been used in human medicine to diagnose various diseases caused by both fungi and bacteria. In agriculture, this technique has potential for the diagnosis of diseases due to the low cost of large-scale analysis and quickness. This study showed that MALDI-TOF MS combined with chemometric analysis was effective for differentiating the macromolecule profile of orange leaves with canker lesions, healthy leaves, and leaves with phytotoxicity symptoms, proving that this technique may be used for the rapid diagnosis of citrus canker. [ABSTRACT FROM AUTHOR]

Xanthomonas axonopodis pv. citri (Xac) belongs to the Gram-negative species, causing citrus canker that seriously affects the fruit yield and quality of many rutaceae plants. Herein, we found that compound 2-(butyldisulfanyl) quinazolin-4(3H)-one exhibited remarkable anti-Xac activity in vitro with a half effective concentration (EC50) of 2.6 μg/mL, while the positive controls thiodiazole-copper with 57 μg/mL and bismerthiazol with 68 μg/mL and this compound showed great anti-citrus canker activity in vivo. This active compound also was confirmed to reduce biofilm formation, increase the level of reactive oxygen species, damage the morphological structure of the bacteria, and cause bacterial death. Proteomics and RT-qPCR analysis results indicated that this compound down-regulated the expression of enzymes in the MEP (2-methyl-D-erythritol 4-phosphate) pathway and might achieve destructive ability of Xac. Overall, this study indicates that such derivatives could be a promising scaffold to develop novel bactericides to control citrus canker. [ABSTRACT FROM AUTHOR]

Background: As one of the most widely planted fruit trees in southern China, citrus occupies an important position in the agriculture field and forestry economy in China. There are many kinds of citrus diseases. If citrus infected with diseases cannot be controlled in time, it easily seriously affects citrus production and causes large economic losses. Timely monitoring of disease characteristics in the citrus growth process is important for implementing timely control measures. Citrus images are easily disturbed by environmental factors such as dust, low light, clouds or leaf shadows. This makes it easy for some disease spot features in citrus pictures to be obscured. Occluded lesions cannot be effectively extracted and recognized. Second, similar characteristics of different diseases also make it difficult to distinguish the different types of diseases. However, the existing machine vision technology for identifying citrus diseases still has some difficulties in dealing with the above problems. Results: This paper proposes a new citrus disease identification framework. First, a citrus image enhancement algorithm based on the MSR-AMSR algorithm is proposed, which can enhance the image and highlight the disease characteristic information. The AMSR algorithm can also greatly alleviate the interference of clouds and low light on image lesions, making the image features clearer. Second, an MF-RANet network is proposed to recognize citrus disease images. MF-RANet is composed of a main feature frame and a detail feature frame. The main feature frame uses the cross stacking structure of ResNet50 and RAM to extract the main features in the citrus image dataset. RAM is used to extract the attention weight in the feature layer, which enables RAM to give higher weight to disease features. The detailed feature frame path uses AugFPN to extract features from multiple scales and fuse the main feature frame path. AugFPN enables the network to retain more detailed features, so it can effectively distinguish similar features in different diseases. In addition, we use the ELU activation function not only to solve the problem of gradient explosion and gradient disappearance but also to effectively use the negative input of the network. Finally, we use the label smoothing regularization method to prevent overfitting the network in the classification process. Finally, the experimental results show that the highest detection accuracy of the network for Huanglong disease, Corynespora blight of citrus, fat spot macular disease, citrus scab, citrus canker and healthy citrus is 96.77%, 96.22%, 95.96%, 95.93%, 94.04% and 97.55%, respectively. Conclusions: The citrus disease algorithm based on AMSR and MF-RANet can effectively perform the disease detection function. It has a high recognition rate for different kinds of citrus diseases. With the addition of AMSR preprocessing, RAM, AugFPN, ELU activation function and other structures, the MF-RANet network performance improves. [ABSTRACT FROM AUTHOR]

Molecular epidemiology studies are essential to refine our understanding of migrations of phytopathogenic bacteria, the major determining factor in their emergence, and to understand the factors that shape their population structure. Microsatellite and minisatellite typing are useful techniques for deciphering the population structure of Xanthomonas citri pv. citri, the causal agent of Asiatic citrus canker. This paper presents a molecular epidemiology study, which has improved our understanding of the history of the pathogen's introductions into the Arabian Peninsula, since it was first reported in the 1980s. An unexpectedly high genetic diversity of the pathogen was revealed. The four distinct genetic lineages within X. citri pv. citri, which have been reported throughout the world, were identified in the Arabian Peninsula, most likely as the result of multiple introductions. No copper‐resistant X. citri pv. citri strains were identified. The pathogen's population structure on Mexican lime (their shared host species) was closely examined in two countries, Saudi Arabia and Yemen. We highlighted the marked prevalence of specialist pathotype A* strains in both countries, which suggests that specialist strains of X. citri pv. citri may perform better than generalist strains when they occur concomitantly in this environment. Subclade 4.2 was the prevailing lineage identified. Several analyses (genetic structure deciphered by discriminant analysis of principal components, RST‐based genetic differentiation, geographic structure) congruently suggested the role of human activities in the pathogen's spread. We discuss the implications of these results on the management of Asiatic citrus canker in the region. [ABSTRACT FROM AUTHOR]

Bordeaux mixture (Bm) is a copper (Cu)-based pesticide that has been widely used for controlling citrus scab and citrus canker. However, frequent spraying of Bm is toxic to citrus. To our knowledge, few studies are available that discuss how the photosynthetic characteristics and chloroplast ultrastructure of citrus leaves are affected by Cu toxicity induced by excessive Bm. In the study, two-year-old seedlings of Citrus grandis (C. grandis) and Citrus sinensis (C. sinensis), which were precultured in pots, were foliar-sprayed with deionized water (as control) or Bm diluted 500-fold at intervals of 7 days for 6 times (4 times as recommended by the manufacturer) to investigate the leaf Cu absorption, photosynthesis, chloroplast ultrastructure and antioxidant enzymatic activities. Bm foliar-sprayed 6 times on citrus seedlings increased the leaf Cu content, decreased the photosynthetic pigments content and destroyed the chloroplast ultrastructure, which induced leaf chlorosis and photosynthetic inhibition. A lower Cu absorption, a higher light photon-electron transfer efficiency, a relative integrity of chloroplast ultrastructure and a promoted antioxidant protection contributed to a higher photosynthetic activity of C. grandis than C. sinensis under excessive spraying of Bm. The present study provides crucial references for screening and selecting citrus species with a higher tolerance to Cu toxicity induced by excessive Bm. [ABSTRACT FROM AUTHOR]

Background: Cold is a major abiotic stress and Huanglongbing and citrus canker disease are two devastating bacterial diseases for citrus. The Ca2+-CBL-CIPK network is known to regulate different types of stress signalling in plants. How do CBL–CIPK signalling networks function in response to cold and infection by CLas or Xcc in citrus? Results: Eight calcineurin B-like proteins (CBLs) and seventeen CBL-interacting protein kinases (CIPKs) were identified from the cold-tolerant satsuma mandarin 'Guijing2501' (Citrus. unshiu) and CLas/Xcc-sensitive sweet orange (C. sinensis). Phylogenetic analysis revealed that both CBL and CIPK family members in citrus were classified into an ancient and a recent clade according to their conserved domain characteristics and/or intron/exon structures. Genome duplication analysis suggested that both tandem and segmental duplications contributed to the amplification of the CBL and CIPK gene families in citrus under intense purifying selection, and the duplication events only existed in the recent clades. Expression comparison of the duplicated gene pairs indicated that the duplicated CBL and CIPK genes underwent functional differentiation. Further expression analysis identified that CBL1, 5, 6, and 8 and CIPK2, 8, 12, 15, 16, and 17 were significantly regulated by multiple stresses, including cold, Xcc infection and/or CLas infection, in citrus, whereas CBL2/7 and CIPK1/4/5/11/13/14 were independently highly regulated by cold and CIPK3 was uniquely responsive to Xcc infection. The combination analyses of targeted Y2H assay and expression analysis revealed that CBL6-CIPK8 was the common signalling network in response to cold and Xcc infection, while CBL6/CBL8-CIPK14 was uniquely responsive to cold in citrus. Further stable transformation and cold tolerance assay indicated that overexpression of CuCIPK16 enhanced the cold tolerance of transgenic Arabidopsis with higher POD activity and lower MDA content. Conclusions: In this study, evolution, gene expression and protein‒protein interaction analyses of citrus CBLs and CIPKs were comprehensively conducted over a genome-wide range. The results will facilitate future functional characterization of individual citrus CBLs and CIPKs under specific stresses and provide clues for the clarification of cold tolerance and disease susceptibility mechanisms in corresponding citrus cultivars. [ABSTRACT FROM AUTHOR]

PhoX is a high-affinity phosphate binding protein, present in Xanthomonas citri , a phytopathogen responsible for the citrus canker disease. Performing molecular dynamics simulations and different types of computational analyses, we study the molecular mechanisms at play in relation to phosphate binding, revealing the global functioning of the protein: PhoX naturally oscillates along its global normal modes, which allow it to explore both bound and unbound conformations, eventually attracting a nearby negative phosphate ion to the highly positive electrostatic potential on its surface, particularly close to the binding pocket. There, several hydrogen bonds are formed with the two main domains of the structure. Phosphate creates, in this way, a strong bridge that connects the domains, keeping itself between them, in a tight closed conformation, explaining its high binding affinity. • Both, phosphate-bound and phosphate-unbound PhoX can explore the entire configurational space of the protein. • This is contrary to the usually assumed correspondence between a closed/open conformation and bound/unbound state. • PhoX posseses an mostly positive electrostatic potential, which gives way to a strong attraction of the phosphate ion. • A patch containing S62, G63, G64, K199, with high positive potential, appears to be the one initiating the anion's binding. • Binding pocket's S34 and S62 from DI, and R159, S163 and G164 from DII, simultaneously form H-bonds with the anion. • These bridges connect and stabilize the interaction between domains, keeping PhoX in a highly stable and closed conformation. [ABSTRACT FROM AUTHOR]

Bacterial infections of plants are widespread and highly virulent, causing serious economic losses in agricultural production worldwide. These infections become particularly prevalent during rainy weather, characterized by high humidity and low light intensity (about 2–10% of a sunny day). Here we identified a class of photosensitizers that were effective in inhibiting plant bacterial infections under rainy weather with dim light. One of these compounds, compound 1 , demonstrated exceptional efficacy by eliminating more than 4 logs (99.99%) of either Xanthomonas perforans or Xanthomonas citri subsp. citri (Xcc) strain at a concentration of 6.25 μM under rainy weather conditions with a light exposure of 28.51 J/cm2. For Clavibacter michiganensis (Cm), compound 1 demonstrated a comparable bactericidal effect at a lower concentration of 3.13 μM. Furthermore, compound 1 was effective in controlling plant leaf infections during rainy weather. Moreover, compound 1 exhibited a potent inhibitory effect on the biofilms pertinent to tomato spot disease and citrus canker under natural illumination on rainy days. Additionally, compound 1 displayed remarkable resilience to rain-wash on plant leaves, retaining 41.20% of its initial concentration following exposure to three simulated rain events. These findings provide a new strategy for plant pathogen disease management highlight its feasibility even during rainy weather. [Display omitted] • Bacterial infections in plants are more prevalent during overcast or rainy weather. • The limited illumination during overcast or rainy days restricts the application of photodynamic strategy in controlling bacterial infection. • A range of photosensitizers were synthesized. Compound 1 effectively controls infections in plants during overcast or rainy days. • Compound 1 also inhibits the formation of bacterial biofilms on plant surfaces. • Compound 1 exhibits rain resistance, maintaining a 41.2% efficacy even after rain wash. [ABSTRACT FROM AUTHOR]

Citrus bacterial canker (CBC) is a harmful bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) , negatively impacting citrus production worldwide. The basic helix-loop-helix (bHLH) transcription factor family plays crucial roles in plant development and stress responses. This study aimed to identify and annotate bHLH proteins encoded in the Citrus sinensis genome and explore their involvement and functional importance in regulating CBC resistance. A total of 135 putative CsbHLHs TFs were identified and categorized into 16 subfamilies. Their chromosomal locations, collinearity, and phylogenetic relationships were comprehensively analyzed. Upon Xcc strain YN1 infection, certain CsbHLHs were differentially regulated in CBC-resistant and CBC-sensitive citrus varieties. Among these, CsbHLH085 was selected for further functional characterization. CsbHLH085 was upregulated in the CBC-resistant citrus variety, was localized in the nucleus, and had a transcriptional activation activity. CsbHLH085 overexpression in Citrus significantly enhanced CBC resistance, accompanied by increased levels of salicylic acid (SA), jasmonic acid (JA), reactive oxygen species (ROS), and decreased levels of abscisic acid (ABA) and antioxidant enzymes. Conversely, CsbHLH085 virus-induced gene silencing resulted in opposite phenotypic and biochemical responses. CsbHLH085 silencing also affected the expression of phytohormone biosynthesis and signaling genes involved in SA, JA, and ABA signaling. These findings highlight the crucial role of CsbHLH085 in regulating CBC resistance, suggesting its potential as a target for biotechnological-assisted breeding citrus varieties with improved resistance against phytopathogens. [ABSTRACT FROM AUTHOR]

The bacterium Xanthomonas citri ssp. citri (Xcc), causing citrus canker disease, poses a severe threat to citrus production worldwide. New approaches for managing the disease are required because of the limited number of bactericides available, reported pathogen resistance to copper pesticides, and the side effects of such compounds on citrus trees and the environment. In this study, the bacterium was isolated from a Mexican lime tree showing typical disease symptoms and identified as Xcc using conventional biochemical tests and specific PCR. The antibacterial activity of the recombinant peptide CAP18 (rCAP18) against the Xcc bacterium was assessed. Measuring the MIC parameter showed that purified rCAP18 caused bacterial growth inhibition by 90% at a concentration of 4.5 μM. Furthermore, a heterogeneous membrane system consisting of POPE:POPG:TOCL (5:1:3) was created using molecular dynamics simulation. The interaction effect of the oligomeric structure of rCAP18 (rCAP18OLIGO) on the components of a lipid bilayer membrane was determined by measuring the membrane thickness, the order parameter of the acyl chains and the area of each phospholipid. The results obtained from simulation and measurement of order parameters showed that, among the three phospholipids, rCAP18OLIGO more affected the orientation of carbon atoms in POPG. Fluctuations of average area per lipid for each phospholipid showed that rCAP18OLIGO had more interactions with POPG > TOCL > POPE, respectively, in the outer half of the membrane. The results obtained from in silico studies confirmed that the penetration of rCAP18OLIGO into the lipid bilayer membrane reduced the membrane thickness and caused its deformation during the simulation time. This mechanism is possibly the leading factor of Xcc growth inhibition by rCAP18 in vitro. Therefore, the rCAP18 peptide as an antibacterial agent can provide a way to develop a new approach to control the citrus canker disease. [ABSTRACT FROM AUTHOR]

Biologically synthesized silver nanoparticles are emerging as attractive alternatives to chemical pesticides due to the ease of their synthesis, safety and antimicrobial activities in lower possible concentrations. In the present study, we have synthesized silver nanoparticles (AgNPs) using the aqueous extract of the medicinal plant Euphorbia wallichii and tested them against the plant pathogenic bacterium Xanthomonas axonopodis, the causative agent of citrus canker, via an in vitro experiment. The synthesized silver nanoparticles were characterized by techniques such as UV-Vis spectroscopy, Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction analysis and transmission electron microscopy. Moreover, the plant species were investigated for phenolics, flavonoids and antioxidant activity. The antioxidant potential of the extract was determined against a DPPH radical. The extract was also evaluated for phenolic compounds using the HPLC technique. The results confirmed the synthesis of centered cubic, spherical-shaped and crystalline nanoparticles by employing standard characterization techniques. A qualitative and quantitative phytochemical analysis revealed the presence of phenolics (41.52 mg GAE/g), flavonoids (14.2 mg QE/g) and other metabolites of medicinal importance. Different concentrations (1000 µg/mL to 15.62 µg/mL—2 fold dilutions) of AgNPs and plant extract (PE) alone, and both in combination (AgNPs-PE), exhibited a differential inhibition of X. axanopodis in a high throughput antibacterial assay. Overall, AgNPs-PE was superior in terms of displaying significant antibacterial activity, followed by AgNPs alone. An appreciable antioxidant potential was recorded as well. The observed antibacterial and antioxidant potential may be attributed to eight phenolic compounds identified in the extract. The Euphorbia wallichii leaf-extract-induced synthesized AgNPs exhibited strong antibacterial activity against X. axanopodis, which could be exploited as effective alternative preparations against citrus canker in planta in a controlled environment. In addition, as a good source of phenolic compounds, the plant could be further exploited for potent antioxidants. [ABSTRACT FROM AUTHOR]

Background: Rangpur lime (RL) is a common rootstock cultivar used worldwide. However, it is known to have a high susceptibility to citrus canker (CC). To meet the increasing demand for healthy citrus seedlings, this research aimed to study the potency of RL endophytic bacteria as a biocontrol agent of the CC pathogen Xanthomonas citri subsp. citri. The isolates were collected from healthy RL leaves and subjected to in vitro and in planta antagonistic tests against XCC, alongside its cell-free supernatant (CFS). Potential isolates were identified according to their 16S rDNA sequence similarities. Results: As many as 21 isolates were obtained from the leaves of healthy RL trees. Two (B1 and C8) isolates demonstrated promising inhibitory activity against XCC. Based on the in vitro assays, the cell suspensions (CS) of these isolates could effectively inhibit the growth of XCC, with an optimum clear zone diameter of 8.41 and 7.51 mm, respectively. Consistent with CS, their CFSs also displayed similar antagonistic potencies against XCC with the highest clear zone recorded being 7.23 mm and 6.22 mm, respectively. Further investigations revealed that the minimum inhibitory concentration of B1 and C8 CFSs was 25 µg/ml. It was also found that both CFSs were sensitive to high heat and very low pH, but stable in the presence of proteinase-K. In the in vivo assay, both CS and CFS treatments of B1 and C8 could effectively protect the Rangpur lime from CC. Treated plants had significantly lower disease incidences and developed less severe symptoms than the control plants. Based on their 16S rDNA sequence, B1 and C8 were identified as Staphylococcus pasteuri and Staphylococcus warneri, respectively. Conclusion: It was concluded that Staphylococcus pasteuri and Staphylococcus warneri could be promising biocontrol agents of XCC. These bacterial isolates can be used as an alternative prevention measure to CC as opposed to commonly used chemicals. [ABSTRACT FROM AUTHOR]

[Objective] This study intended to explore and verify the acaricidal mechanism of the strain Wl at the molecular level by constructing the mutant of strain Wl and detecting its acaricidal and antibacterial activities, so as to provide a new microbial resource and control strategy for the prevention and control of two-spotted spider mites, which would lay a foundation for the subsequent development and utilization of more microbial resources, and also provide a theoretical basis for the development and application of new microbial acaricides. [Method] The active compounds of secondary metabolites of B. amyloliquejaciens Wl were studied in the early stage, and it was confirmed that the acaricidal active substances were diketopiperazines, macrolides, bacimethrins and other compounds. Macrolactin A was the main acaricidal active substance in macrolide compounds, and synthesized by the mln gene cluster. By constructing knockout integration vector pBS-mlnBkanR and transforming it into natural competent cells of the Wl strain, the macrolactin A biosynthesis-defective mutant Wl timlnB : : kanR was obtained. The successful construction of the knockout mutant was confirmed by PCR amplification, product sequencing and HPLC-MS/ MS technology, and its acaricidal and bacteriostatic activities were detected. [ Result] The knockout of the mlnB gene not only resulted in the inability of stain Wl to biosynthesize macrolactin A, but also weakened its antagonistic activity against pathogens causing soft rot of cabbage, citrus canker, black rot of cabbage, and bacterial wilt of Solanaceae. Compared with 73 . 33 % - 77. 22 % of the leathality of the Wl wildtype strain, the acaricidal activity of the macrolactin A biosynthesis defective mutant was decreased by 46. 66% - SO. 55% . [Conclusion] Macrolactin A synthesized by B. amyloliquefaciens Wl strain had the activity of killing two-spotted mites, and can inhibit the activity of pathogenic bacteria of Erwinia carotovora subsp. carotovora,Xanthomonas citri subsp. citri, Xanthomonas campestris pv. campestri and Ralstonia solanacearum. After the synthesis of macrolactin A was blocked, the activity of Wl strain was obviously weakened. [ABSTRACT FROM AUTHOR]

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 subsp. citri, a causative agent of the citrus canker (CC) disease, belongs to one of the essential groups of the bacterial phytopathogen family, Xanthomonadaceae. It has been a potential threat to the globally significant citrus fruit crop, which has remained under investigation for disease management and epidemiology since the 1980s. In Pakistan, the average yield of citrus is 11 t/ha, which is lower than other countries, including China, Brazil, and India, having average productions of 27, 26, and 22 tons/hectare, respectively. Citrus canker is one of the most devastating diseases, posing a significant threat to crop yield and fruit quality. To date, five distinct types (or forms) of the citrus canker have been recognized; the Asiatic (Canker A) form is most destructive and affects most citrus cultivars. Severe infection outcomes include dieback, defoliation, severely blemished fruit, premature fruit drop, and reduced fruit quality. The infection increases under humid, warm, cloudy climate, wind, and heavy rainfall. The analysis of plasmid and chromosomal DNA of X. citri subsp. citri depicted an evolutionary relationship among pathovars of Xanthomonas. The extensive study on the genome of X. citri subsp. citri has contributed to the current knowledge of plant host recognition of pathogens, host specificities, dissemination, and propagation. Regulatory programs, i.e., quarantine or exclusion, continued to be practiced, prohibiting infected citrus plant material into the existing stock. Other measures include removal of inoculums sources, resistant hosts, protective copper-containing sprays, and windbreak systems. In this review, we explored the latest trends in the areas of epidemiology, pathogenome, detection, host–pathogen interaction, biofilm formation, and management of X. citri subsp. citri. [ABSTRACT FROM AUTHOR]

Citrus canker is one of the main bacterial diseases that affect citrus crops and is caused by Xanthomonas citri which affects all citrus species worldwide. New strategies to control citrus canker are necessary and the use of bacteriophages as biocontrol agent could be an alternative. Phages that infect Xanthomonas species have been studied, such as XacN1, a myovirus that infects X. citri. Here we report the isolation and characterization of a new jumbo phage, vb_XciM_LucasX, which infects X. citri and X. fuscans. Transmission electron microscopy allowed classification of LucasX in the Myoviridae family, which was corroborated by its genomic sequencing, annotation, and proteome clustering. LucasX has a 305,651 bp-long dsDNA genome. ORF prediction and annotation revealed 157 genes encoding putative structural proteins such as capsid and tail related proteins and phage assembly associated proteins, however, for most of the structural proteins it was not possible assign specific functions. Its genome encodes several proteins related to DNA replication and nucleotide metabolism, five putative RNA polymerases, at least one homing endonuclease mobile element, a terminase large subunit (TerL), an endolysin and many proteins classified as beneficial to the host. Proteome clustering and phylogeny analyses showed that LucasX is a new jumbo phage having as its closest neighbor the Xanthomonas jumbo phage Xoo-sp14. LucasX presented a burst size of 40 PFU/infected cell of X. citri 306, was completely inactivated at temperatures above 50°C, presented survival lower than 25% after 80 s of exposition to artificial UV light and had practically no tolerance to concentrations above 2.5 g/L NaCl or 40% ethanol. LucasX presented optimum pH at 7 and a broad range of Xanthomonas hosts, infecting twenty-one of the twenty-three strains tested. Finally, the LucasX yield was dependent on the host strain utilized, resulting one order of magnitude higher in X. fuscans C 752 than in X. citri 306, which points out to the possibility of phage yield improvement, an usual challenge for biocontrol purposes. [ABSTRACT FROM AUTHOR]

Climatic conditions play a significant role in the development of citrus canker caused by Xanthomonas citri pv. citri (Xcc). Citrus canker is regarded as one of the major threats being faced by citrus industry in citrus growing countries of the world. Climatic factors exert significant impacts on growth stage, host susceptibility, succulence, vigor, survival, multiplication rate, pathogen dispersion, spore penetration rate, and spore germination. Predicting the impacts of climatic factors on these traits could aid in the development of effective management strategies against the disease. This study predicted the impacts of environmental variables, i.e., temperature, relative humidity, rainfall, and wind speed the development of citrus canker through multiple regression. These environmental variables were correlated with the development of canker on thirty (30) citrus varieties during 2017 to 2020. Significant positive correlations were noted among environment variables and disease development modeled through multiple regression model (Y = +24.02 + 0.5585 X1 + 0.2997 X2 + 0.3534 X3 + 3.590 X4 + 1.639 X5). Goodness of fit of the model was signified by coefficient determination value (97.5%). Results revealed the optimum values of environmental variables, i.e., maximum temperature (37°C), minimum temperature (27°C), relative humidity (55%), rainfall (4.7–7.1 mm) and wind speed (8 Km/h), which were conducive for the development of citrus canker. Current study would help researchers in designing better management strategies against citrus canker disease under changing climatic conditions in the future. [ABSTRACT FROM AUTHOR]