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The genus Xanthomonas has been primarily studied for pathogenic interactions with plants. However, besides host and tissue-specific pathogenic strains, this genus also comprises nonpathogenic strains isolated from a broad range of hosts, sometimes in association with pathogenic strains, and other environments, including rainwater. Based on their incapacity or limited capacity to cause symptoms on the host of isolation, nonpathogenic xanthomonads can be further characterized as commensal and weakly pathogenic. This study aimed to understand the diversity and evolution of nonpathogenic xanthomonads compared to their pathogenic counterparts based on their cooccurrence and phylogenetic relationship and to identify genomic traits that form the basis of a life history framework that groups xanthomonads by ecological strategies. We sequenced genomes of 83 strains spanning the genus phylogeny and identified eight novel species, indicating unexplored diversity. While some nonpathogenic species have experienced a recent loss of a type III secretion system, specifically the hrp2 cluster, we observed an apparent lack of association of the hrp2 cluster with lifestyles of diverse species. We performed association analysis on a large data set of 337 Xanthomonas strains to explain how xanthomonads may have established association with the plants across the continuum of lifestyles from commensals to weak pathogens to pathogens. Presence of distinct transcriptional regulators, distinct nutrient utilization and assimilation genes, transcriptional regulators, and chemotaxis genes may explain lifestyle-specific adaptations of xanthomonads.

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21. These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield.

Bacterial blight, incited by Xanthomonas citri pv. malvacearum (Xcm), is historically one of the most devastating diseases of cotton globally. In India, the initial documentation of the disease dates back to 1918 and it assumed a great magnitude after 1947, primarily triggered by cultivation of tetraploid cotton, as the earlier cultivated indigenous diploid cotton was naturally resistant to the pathogen, and became widespread during the 1970s. Among 20 races of bacterial blight pathogen documented at global level, Race 18 is the most virulent one and prominent in India. The pathogen inflicts damage at all crop stages, beginning with seedlings. The disease caused regular yield losses of 30%–35% in India and had become a formidable adversary to cotton cultivation in the country; however, over the years its appearance diminished. Much larger gains were apparently achieved when disease was managed with a combination of seed treatment, foliar sprays and bio‐agents. Furthermore, extensive breeding programmes led to development of resistant cultivars, which proved instrumental in managing the disease. The disease exhibited a sharp decline from the beginning of the 21st century and is now no longer a chronic problem in India. This comprehensive review attempts to provide insight into the dynamic nature of bacterial blight threatening cotton cultivation in India during the last century and the underpinning multifaceted research efforts that paved the way for countering the disease. An attempt is made to discuss the qualitative and quantitative studies on genetics of bacterial blight for facilitating the breeding programmes. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight in rice. Polyhydroxyalkanoates (PHAs) consitute a diverse group of biopolyesters synthesized by bacteria under nutrient-limited conditions. The phaC gene is important for PHA polymerization. We investigated the effects of phaC gene mutagensis in Xoo strain PXO99A. The phaC gene knock-out mutant exhibited reduced swarming ability relative to that of the wild-type. Under conditions where glucose was the sole sugar source, extracellular polysaccharide (EPS) production by ΔphaC declined by 44.8%. ΔphaC showed weak hypersensitive response (HR) induction in the leaves of non-host Nicotiana tabacum, concomitant with downregulation of hpa1 gene expression. When inoculated in rice leaves by the leaf-clipping method, ΔphaC displayed reduced virulence in terms of lesion length compared with the wild-type strain. The complemented strain showed no significant difference from the wild-type strain, suggesting that the deletion of phaC in Xoo induces significant alterations in various physiological and biological processes. These include bacterial swarming ability, EPS production, transcription of hrp genes, and glucose metabolism. These changes are intricately linked to the energy utilization and virulence of Xoo during plant infection. These findings revealed involvement of phaC in Xoo is in the maintaining carbon metabolism by functioning in the PHA metabolic pathway. [ABSTRACT FROM AUTHOR]

Introduction: Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease worldwide. Previously, we successfully generated cankerresistant Citrus sinensis cv. Hamlin lines in the T0 generation. This was achieved through the transformation of embryogenic protoplasts using the ribonucleoprotein (RNP) containing Cas12a and one crRNA to edit the canker susceptibility gene, CsLOB1, which led to small indels. Methods: Here, we transformed embryogenic protoplasts of Hamlin with RNP containing Cas12a and three crRNAs. Results: Among the 10 transgene-free genome-edited lines, long deletions were obtained in five lines. Additionally, inversionswere observed in three of the five edited lines with long deletions, but not in any edited lines with short indel mutations, suggesting long deletions maybe required for inversions. Biallelic mutations were observed for each of the three target sites in four of the 10 edited lines when three crRNAs were used, demonstrating that transformation of embryogenic citrus protoplasts with Cas12a and three crRNAs RNP can be very efficient for multiplex editing. Our analysis revealed the absence of off-target mutations in the edited lines. These cslob1 mutant lines were canker-resistant and no canker symptoms were observed after inoculation with Xcc and Xcc growth was significantly reduced in the cslob1 mutant lines compared to the wild type plants. Discussion: Taken together, RNP (Cas12a and three crRNAs) transformation of embryogenic protoplasts of citrus provides a promising solution for transgene-free multiplex genome editing with high efficiency and for deletion of long fragments. [ABSTRACT FROM AUTHOR]

Background: Bacteria of the genus Xanthomonas cause economically significant diseases in various crops. Their virulence is dependent on the translocation of type III effectors (T3Es) into plant cells by the type III secretion system (T3SS), a process regulated by the master response regulator HrpG. Although HrpG has been studied for over two decades, its regulon across diverse Xanthomonas species, particularly beyond type III secretion, remains understudied. Results: In this study, we conducted transcriptome sequencing to explore the HrpG regulons of 17 Xanthomonas strains, encompassing six species and nine pathovars, each exhibiting distinct host and tissue specificities. We employed constitutive expression of plasmid-borne hrpG*, which encodes a constitutively active form of HrpG, to induce the regulon. Our findings reveal substantial inter- and intra-specific diversity in the HrpG* regulons across the strains. Besides 21 genes directly involved in the biosynthesis of the T3SS, the core HrpG* regulon is limited to only five additional genes encoding the transcriptional activator HrpX, the two T3E proteins XopR and XopL, a major facility superfamily (MFS) transporter, and the phosphatase PhoC. Interestingly, genes involved in chemotaxis and genes encoding enzymes with carbohydrate-active and proteolytic activities are variably regulated by HrpG*. Conclusions: The diversity in the HrpG* regulon suggests that HrpG-dependent virulence in Xanthomonas might be achieved through several distinct strain-specific strategies, potentially reflecting adaptation to diverse ecological niches. These findings enhance our understanding of the complex role of HrpG in regulating various virulence and adaptive pathways, extending beyond T3Es and the T3SS. [ABSTRACT FROM AUTHOR]

Xanthomonas species infect many important crops and cause huge yield loss. These pathogens deliver transcription activator-like (TAL) effectors into the cytoplasm of plant cells. TAL effectors move to host nuclei, directly bind to the promoters of host susceptible genes, and activate their transcription. However, the molecular mechanisms by which TAL effectors induce host transcription remain unclear. We herein demonstrated that TAL effectors interacted with the SIMILAR TO RCD ONE (SRO) family proteins OsSRO1a and OsSRO1b in nuclei. A transactivation assay using rice protoplasts indicated that OsSRO1a and OsSRO1b enhanced the activation of the OsSWEET14 promoter by the TAL effector AvrXa7. The AvrXa7-mediated expression of OsSWEET14 was significantly reduced in ossro1a mutants. However, the overexpression of OsSRO1a increased disease resistance by up-regulating the expression of defense-related genes, such as WRKY62 and PBZ1. This was attributed to OsSRO1a and OsSRO1b also enhancing the transcriptional activity of WRKY45, a direct regulator of WRKY62 expression. Therefore, OsSRO1a and OsSRO1b appear to positively contribute to transcription mediated by bacterial TAL effectors and rice transcription factors. [ABSTRACT FROM AUTHOR]

Fruits play an important role in human life on our planet, since they supply a variety of essential services. One of the paramount crops in Pakistan is Citrus reticulata (Kinnow), which plays a vital role in the country's economy. The citrus crops are confronted with various challenges such as fungi, bacteria, nematodes, and viruses, all of which have adverse effects on the quality and yield of the fruits. Citrus canker, in particular, stands as the most fatal disease, affecting numerous citrus species worldwide, inflicting devastating consequences. The objective of this study was to evaluate the impact of citrus canker on the morphology, physiology of leaves, and the quality of citrus fruits. The research was conducted in four major citrus-producing tehsils of the Sargodha district. The study found significant differences in morphological and physiological traits between healthy fruits and those infected with citrus canker. Healthy fruits exhibited higher values in fruit weight (FW) of 149.02 g, peel thickness (PT) of 3.76 g, rag weight (RW) of 35.95 g, leaf area (LA) of 22.49 cm2, and juice weight compared to the citrus canker-infected fruits. The significant variations in fruit weight, juice weight, chlorophyll content, vitamin C content were present between healthy and diseased fruits and leaves. A biochemical study revealed that healthy fruits had greater levels of total soluble solids (TSS), TSS-acid ratio, vitamin C, and reducing power (RP), but citrus canker-infected fruits had a higher acidity percentage. The significant decreases in important morphological and physicochemical characteristics, emphasizing the necessity for immediate disease control techniques to safeguard the citrus sector and maintain food security. [ABSTRACT FROM AUTHOR]

Although seed represents an important means of plant pathogen dispersion, the seed–pathogen dialogue remains largely unexplored. A multiomic approach was performed at different seed developmental stages of common bean (Phaseolus vulgaris L.) during asymptomatic colonization by Xanthomonas citri pv. fuscans (Xcf), At the early seed developmental stages, we observed high transcriptional changes both in seeds with bacterial recognition and defense signal transduction genes, and in bacteria with up‐regulation of the bacterial type 3 secretion system. This high transcriptional activity of defense genes in Xcf‐colonized seeds during maturation refutes the widely diffused assumption considering seeds as passive carriers of microbes. At later seed maturation stages, few transcriptome changes indicated a less intense molecular dialogue between the host and the pathogen, but marked by changes in DNA methylation of plant defense genes, in response to Xcf colonization. We showed examples of pathogen‐specific DNA methylations in colonized seeds acting as plant defense silencing to repress plant immune response during the germination process. Finally, we propose a novel plant–pathogen interaction model, specific to the seed tissues, highlighting the existence of distinct phases during seed–pathogen interaction with seeds being actively interacting with colonizing pathogens, then both belligerents switching to more passive mode at later stages. [ABSTRACT FROM AUTHOR]

Background: Bacterial spot of pepper (BSP), caused by four different Xanthomonas species, primarily X. euvesicatoria (Xe), poses a significant challenge in pepper cultivation. Host resistance is considered the most important approach for BSP control, offering long-term protection and sustainability. While breeding for resistance to BSP for many years focused on dominant R genes, introgression of recessive resistance has been a more recent focus of breeding programs. The molecular interactions underlying recessive resistance remain poorly understood. Results: In this study, transcriptomic analyses were performed to elucidate defense responses triggered by Xe race P6 infection by two distinct pepper lines: the Xe-resistant line ECW50R containing bs5, a recessive resistance gene that confers resistance to all pepper Xe races, and the Xe-susceptible line ECW. The results revealed a total of 3357 upregulated and 4091 downregulated genes at 0, 1, 2, and 4 days post-inoculation (dpi), with the highest number of differentially expressed genes (DEGs) observed at 2 dpi. Pathway analysis highlighted DEGs in key pathways such as plant-pathogen interaction, MAPK signaling pathway, plant hormone signal transduction, and photosynthesis – antenna proteins, along with cysteine and methionine metabolism. Notably, upregulation of genes associated with PAMP-Triggered Immunity (PTI) was observed, including components like FLS2, Ca-dependent pathways, Rboh, and reactive oxygen species (ROS) generation. In support of these results, infiltration of ECW50R leaves with bacterial suspension of Xe led to observable hydrogen peroxide accumulation without a rapid increase in electrolyte leakage, suggestive of the absence of Effector-Triggered Immunity (ETI). Furthermore, the study confirmed that bs5 does not disrupt the effector delivery system, as evidenced by incompatible interactions between avirulence genes and their corresponding dominant resistant genes in the bs5 background. Conclusion: Overall, these findings provide insights into the molecular mechanisms underlying bs5-mediated resistance in pepper against Xe and suggest a robust defense mechanism in ECW50R, primarily mediated through PTI. Given that bs5 provides early strong response for resistance, combining this resistance with other dominant resistance genes will enhance the durability of resistance to BSP. [ABSTRACT FROM AUTHOR]

LuxR-type regulators play pivotal roles in regulating numerous bacterial processes, including bacterial motility and virulence, thereby exerting a significant influence on bacterial behavior and pathogenicity. Xanthomonas oryzae pv. oryzicola, a rice pathogen, causes bacterial leaf streak. Our research has identified VmsR, which is a response regulator of the two-component system (TCS) that belongs to the LuxR family. These findings of the experiment reveal that VmsR plays a crucial role in regulating pathogenicity, motility, biofilm formation, and the production of extracellular polysaccharides (EPSs) in Xoc GX01. Notably, our study shows that the vmsR mutant exhibits a reduced swimming motility but an enhanced swarming motility. Furthermore, this mutant displays decreased virulence while significantly increasing EPS production and biofilm formation. We have uncovered that VmsR directly interacts with the promoter regions of fliC and fliS, promoting their expression. In contrast, VmsR specifically binds to the promoter of gumB, resulting in its downregulation. These findings indicate that the knockout of vmsR has profound effects on virulence, motility, biofilm formation, and EPS production in Xoc GX01, providing insights into the intricate regulatory network of Xoc. [ABSTRACT FROM AUTHOR]

Sugarcane is one of Australia's major agricultural industries, with approximately 95% of the crop being grown in Queensland and the remainder in northern New South Wales. In the last decade of the nineteenth century, cane growers in northern New South Wales started to see a new disease that resulted not only in the death of plants but also in difficulties in the extraction of sugar. Theories about the cause abounded, but investigations by the New South Wales vegetable pathologist Nathan Cobb revealed that the disease, previously unknown to the world, was caused by a microbe in the creamy 'gum' that could be commonly found in the vascular tissues of affected stalks. He named the organism Bacillus vascularum (now known as Xanthomonas axonopodis pv. vasculorum). For some time after, the disease was known as 'Cobb's gumming disease of sugarcane'. The Australian bacteriologist Robert Greig-Smith was not convinced that Cobb had conclusively demonstrated that B. vascularum was the culprit, mainly because he did not satisfy Koch's Postulates. However, the American bacteriologist Erwin Frink Smith came to Cobb's rescue when he proved beyond doubt that B. vascularum was to blame. The disease is now known simply as 'gumming disease of sugarcane'. At the start of the decade of 1890, sugarcane growers in southern Queensland and northern New South Wales began to notice a serious disease affecting their crops. American-born Nathan Cobb, who was the New South Wales Government Vegetable Pathologist, discovered that a bacterium, now known as Xanthomonas axonopodis pv. vasculorum , was the cause of the disease. Although others were not convinced that Cobb had conclusively proved that the bacterium was the causal agent, it was for many years known as 'Cobb's gumming disease of cane'. [ABSTRACT FROM AUTHOR]

This study was conducted on Xanthomonas axonopodis pv, which causes significant economic losses in the agricultural sector. Here, we study a common bacterial blight disease caused by the phaseoli (XaP) bacterial pathogen on Üstün42 and Akbulut bean genera. In this study, a total of 4000 images, healthy and diseased, were used for both bean breeds. These images were classified by AlexNet, VGG16, and VGG19 models. Later, reclassification was performed by applying pre-processing to the raw images. According to the results obtained, the accuracy rates of the pre-processed images classified by the VGG19, VGG16 and AlexNet models were determined as 0.9213, 0.9125 and 0.8950, respectively. The models were then hybridized with LSTM and BiLSTM for raw and pre-processed images and new models were created. When the performance of these hybrid models was evaluated, it was found that the models hybridized with LSTM were more successful than the simple models, while the models hybridized with BiLSTM gave better results than the models hybridized with LSTM. In particular, the VGG19+BiLSTM model attracted attention by achieving 94.25% classification accuracy with pre-processed images. This study emphasizes the effectiveness of image processing techniques in agriculture in the field of disease detection and is important as a new dataset in the literature for evaluating the performance of hybridized models. [ABSTRACT FROM AUTHOR]

Bacterial leaf blight (BLB) caused by Xanthomonas spp. is an important threat for eucalypt production. Planting of resistant genotypes is the best strategy for eucalypt BLB control. However, selection of resistant clones is challenging since the disease is caused by at least three Xanthomonas species that can be sympatric and cause a range of foliar symptoms. So far, standard area diagrams (SADs) to assess eucalypt BLB severity caused by different Xanthomonas species have not been reported. Here, SADs for eucalypt BLB severity assessment were developed. Plants of five eucalypt genotypes were individually inoculated with different Xanthomonas species and leaves exhibiting lesions varying in colour, shape, and size were digitally scanned for disease assessment. Among 150 scanned leaves, the actual leaf affected area ranged from 0.01% to 55%. Based on these minimum and maximum values and the frequency of severity distribution, the 10 leaves that best represented the distribution of affected leaf area were selected to assemble the SADs. Groups of experienced and inexperienced raters estimated BLB severity on a subset of 50 out of the 150 original leaves without and then with the use of the SADs. Correlation concordance analyses revealed that SADs provided a significant improvement in accuracy, precision and reliability of disease estimates by both rater groups, with higher gains observed for the inexperienced group. The SADs developed in this study will be useful for selection of eucalypt genotypes resistant to BLB under controlled conditions when knowledge on the Xanthomonas species composition is lacking or when local populations are comprised of several species. [ABSTRACT FROM AUTHOR]

Xanthomonas citri is a plant-pathogenic bacterium associated with a diverse range of host plant species. It has undergone substantial reclassification and currently consists of 14 different subspecies or pathovars that are responsible for a wide range of plant diseases. Wholc-genome sequencing (WGS) provides a cutting-edge advantage over other diagnostic techniques in epidemiological and evolutionary studies of X. citri because it has a higher discriminatory power and is replicable across laboratories. WGS also allows for the improvement of multilocus sequence typing (MLST) schemes. In this study, we used genome sequences of Xanthomonas isolates from the NCBI RefSeq database to develop a sevengene MLST scheme that yielded 19 sequence types (STs) that correlated with phylogenetic clades of X. citri subspecies or pathovars. Using this MLST scheme, we examined 2,911 Xanthomonas species assemblies from NCBI GcnBank and identified 15 novel STs from 37 isolates that were misclassified in NCBI. In total, we identified 545 X. citri assemblies from GenBank with 95% average nucleotide identity to the X. citri type strain, and all were classified as one of the 34 STs. Ail MLST classifications correlated with a phylogenetic position inferred from alignments using 92 conserved genes. We observed several instances where strains from different pathovars formed closely related monophyletic clades and shared the same ST. indicating that further investigation of the validity of these pathovars is required. Our MLST scheme described here is a robust tool for rapid classification of X. citri pathovars using WGS and a powerful method for further comprehensive taxonomic revision of X. cirri pathovars. [ABSTRACT FROM AUTHOR]

In this review, we highlight studies in which whole-genome sequencing, comparative genomics, and population genomics have provided unprecedented insights into past and ongoing pathogen evolution. These include new understandings of the adaptive evolution of secretion systems and their effectors. We focus on Xanthonionas pathosystems that have seen intensive study and improved our understanding of pathogen emergence and evolution, particularly in the context of host specialization: citrus canker, bacterial blight of rice, and bacterial spot of tomato and pepper. Across pathosystems, pathogens appear to follow a pattern of bursts of evolution and diversification that impact host adaptation. There remains a need for studies on the mechanisms of host range evolution and genetic exchange among closely related but differentially host-specialized species and to start moving beyond the study of specific strain and host cultivar pairwise interactions to thinking about these pathosystems in a community context. [ABSTRACT FROM AUTHOR]

Bacterial spot is a serious disease caused by several species of Xanthomonas affecting pepper and tomato production worldwide. Since the strategies employed for disease management have been inefficient and pose a threat for environmental and human health, the development of alternative methods is gaining relevance. The aim of this study is to isolate and characterize lytic phages against Xanthomonas pathogens. Here, we isolate two jumbo phages, named XaC1 and XbC2, from water obtained from agricultural irrigation channels by the enrichment technique using X. vesicatoria as a host. We determined that both phages were specific for inducing the lysis of X. vesicatoria strains, but not of other xanthomonads. The XaC1 and XbC2 phages showed a myovirus morphology and were classified as jumbo phages due to their genomes being larger than 200 kb. Phylogenetic and comparative analysis suggests that XaC1 and XbC2 represent both different and novel genera of phages, where XaC1 possesses a low similarity to other phage genomes reported before. Finally, XaC1 and XbC2 exhibited thermal stability up to 45 °C and pH stability from 5 to 9. All these results indicate that the isolated phages are promising candidates for the development of formulations against bacterial spot, although further characterization is required. [ABSTRACT FROM AUTHOR]

It became necessary to employ safer and more efficient alternatives to plant pathogenic bacteria and land snails control due to the growing issues these snails and bacteria were causing on various crops. Green nanoparticles have been used because of their stability, low-cost feasibility and antimicrobial characteristics and snails. The current work intends to investigate the effectiveness of green synthetic silver and iron oxide nanoparticles of an aqueous extract of Acacia nilotica pods Ag-NPs (AEANP-AgNPs) and FeO-NPs (AEANP-FeONPs) against two identified plant pathogenic bacterial strains (Xanthomonas euvesicatoria and Pseudomonas syringae pv. tomato) and to land snails (Monacha cartusiana and Eobania vermiculata) collected from sandy loam soil. AEANP-AgNPs and AEANP-FeONPs were made and characterized using a TEM, Zeta potential, FTIR and EDX. Different concentrations of AEANP-AgNPs (1700, 3400, and 5100 ppm) and AEANP-FeONPs (2700, 5400, and 8100 ppm) were tested. The concentration (5100 ppm) of Ag-NPs was higher in the inhibition against P. syringae pv. tomato while (5400 ppm) and (8100 ppm) concentrations of FeO-NPs were equal to inhibition against X. euvesicatoria. In planta, the treated strain of X. euvesicatoria exhibited low severity while the treated strain of P. syringae pv. tomato didn't exhibit a significant difference severity. So, M. cartusiana snails were more sensitive to the toxic effect of Ag-NPs and FeO-NPs than adult snails, E. vermiculata. The mortality for adult snails was increased with increasing concentration of both nanoparticles. The LC50 concentrations of Ag-NPs for adult snails M. cartusiana and E. vermiculata were (1.885×103and 7.618×10³) and (4.066×10³ ppm and 10.776×10³ ppm) for FeO-NPs respectively. [ABSTRACT FROM AUTHOR]

Abstract Background Bacteria of the genus Xanthomonas cause economically significant diseases in various crops. Their virulence is dependent on the translocation of type III effectors (T3Es) into plant cells by the type III secretion system (T3SS), a process regulated by the master response regulator HrpG. Although HrpG has been studied for over two decades, its regulon across diverse Xanthomonas species, particularly beyond type III secretion, remains understudied. Results In this study, we conducted transcriptome sequencing to explore the HrpG regulons of 17 Xanthomonas strains, encompassing six species and nine pathovars, each exhibiting distinct host and tissue specificities. We employed constitutive expression of plasmid-borne hrpG*, which encodes a constitutively active form of HrpG, to induce the regulon. Our findings reveal substantial inter- and intra-specific diversity in the HrpG* regulons across the strains. Besides 21 genes directly involved in the biosynthesis of the T3SS, the core HrpG* regulon is limited to only five additional genes encoding the transcriptional activator HrpX, the two T3E proteins XopR and XopL, a major facility superfamily (MFS) transporter, and the phosphatase PhoC. Interestingly, genes involved in chemotaxis and genes encoding enzymes with carbohydrate-active and proteolytic activities are variably regulated by HrpG*. Conclusions The diversity in the HrpG* regulon suggests that HrpG-dependent virulence in Xanthomonas might be achieved through several distinct strain-specific strategies, potentially reflecting adaptation to diverse ecological niches. These findings enhance our understanding of the complex role of HrpG in regulating various virulence and adaptive pathways, extending beyond T3Es and the T3SS.

Abstract Background Bacterial spot of pepper (BSP), caused by four different Xanthomonas species, primarily X. euvesicatoria (Xe), poses a significant challenge in pepper cultivation. Host resistance is considered the most important approach for BSP control, offering long-term protection and sustainability. While breeding for resistance to BSP for many years focused on dominant R genes, introgression of recessive resistance has been a more recent focus of breeding programs. The molecular interactions underlying recessive resistance remain poorly understood. Results In this study, transcriptomic analyses were performed to elucidate defense responses triggered by Xe race P6 infection by two distinct pepper lines: the Xe-resistant line ECW50R containing bs5, a recessive resistance gene that confers resistance to all pepper Xe races, and the Xe-susceptible line ECW. The results revealed a total of 3357 upregulated and 4091 downregulated genes at 0, 1, 2, and 4 days post-inoculation (dpi), with the highest number of differentially expressed genes (DEGs) observed at 2 dpi. Pathway analysis highlighted DEGs in key pathways such as plant-pathogen interaction, MAPK signaling pathway, plant hormone signal transduction, and photosynthesis – antenna proteins, along with cysteine and methionine metabolism. Notably, upregulation of genes associated with PAMP-Triggered Immunity (PTI) was observed, including components like FLS2, Ca-dependent pathways, Rboh, and reactive oxygen species (ROS) generation. In support of these results, infiltration of ECW50R leaves with bacterial suspension of Xe led to observable hydrogen peroxide accumulation without a rapid increase in electrolyte leakage, suggestive of the absence of Effector-Triggered Immunity (ETI). Furthermore, the study confirmed that bs5 does not disrupt the effector delivery system, as evidenced by incompatible interactions between avirulence genes and their corresponding dominant resistant genes in the bs5 background. Conclusion Overall, these findings provide insights into the molecular mechanisms underlying bs5-mediated resistance in pepper against Xe and suggest a robust defense mechanism in ECW50R, primarily mediated through PTI. Given that bs5 provides early strong response for resistance, combining this resistance with other dominant resistance genes will enhance the durability of resistance to BSP.

Xanthomonas spp. cause severe bacterial diseases. However, effective strategies for prevention and management of these diseases are scarce. Thus, it is necessary to improve the efficiency of control of diseases caused by Xanthomonas. In this study, Xanthomonas oryzae pv. oryzae (Xoo), which causes rice bacterial leaf blight, has been studied as a representative. A transposon insertion library of Xoo, comprising approximately 200,000 individual insertion mutants, was generated. Transposon sequencing data indicated that the mariner C9 transposase mapped at 35.7–36.4% of all potential insertion sites, revealing 491 essential genes required for the growth of Xoo in rich media. The results show that, compared to the functions of essential genes of other bacteria, the functions of some essential genes of Xoo are unknown, 25 genes might be dangerous for the Xanthomonas group, and 3 are specific to Xanthomonas. High-priority candidates for developing broad-spectrum, Xanthomonas-specific, and environment-friendly bactericides were identified in this study. In addition, this study revealed the possible targets of dioctyldiethylenetriamine using surface plasmon resonance (SPR) in combination with high performance liquid chromatography–mass spectrometry (HPLC–MS). The study also provided references for the research of some certain bactericides with unknown anti-bacterial mode of action. In conclusion, this study urged a better understanding of Xanthomonas, provided meaningful data for the management of bacterial leaf blight, and disclosed selected targets of a novel bactericide.

The microbiomes of two important rice cultivars in Vietnam which differ by their susceptibility to the bacterial leaf blight (BLB) disease were analyzed through 16S rRNA amplicon technology. A higher number of operational taxonomic units and alpha-diversity indices were shown in the BLB-resistant LA cultivar than in the BLB-susceptible TB cultivar. The BLB pathogen Xanthomonas was scantly found (0.003%) in the LA cultivar, whereas was in a significantly higher ratio in the TB cultivar (1.82%), reflecting the susceptibility to BLB of these cultivars. Of special interest was the genus Acholeplasma presented in the BLB-resistant LA cultivar at a high relative abundance (22.32%), however, was minor in the BLB-sensitive TB cultivar (0.09%), raising a question about its roles in controlling the Xanthomonas low in the LA cultivar. It is proposed that Acholeplasma once entered the host plant would hamper other phytopathogens, i.e. Xanthomonas, by yet unknown mechanisms, of which the triggering of the host plants to produce secondary metabolites against pathogens could be a testable hypothesis.

Bacterial spot disease of cucurbits, caused by Xanthomonas cucurbitae, is a major problem in cucurbit‐growing areas worldwide. In the Midwestern region of the United States, pumpkin and squash fields can have greater than 90% infected fruits, leading to high yield losses. While reference genomes are available for this bacterial species, the genetic diversity between different strains and populations is unknown. After performing restriction‐site associated DNA sequencing (RAD‐seq) analysis of X. cucurbitae isolates collected from the Midwestern region, we selected five representative isolates for further characterization, which included whole‐genome sequencing and in vitro enzyme and in planta virulence assays. Our results suggest that minimal genetic diversity exists between these isolates, and that the isolates have differential virulence on different cucurbit host plants. This study contributes to our understanding of X. cucurbitae population dynamics in the Midwestern region and may assist with developing additional management strategies for controlling bacterial spot disease of cucurbits. [ABSTRACT FROM AUTHOR]

Carioca and black bean groups play a significant role in Latin American common bean (Phaseolus vulgaris) consumption, serving as excellent sources of nutrients. One of the most significant threats for common bean production is common bacterial blight (CBB), a disease caused by the gram‐negative bacteria Xanthomonas phaseoli pv. phaseoli (Xpp) and Xanthomonas citri pv. fuscans (Xcf). An effective method for managing this disease is the use of plant genetic resistance. However, it is not known whether the same or overlapping sets of genes are associated with resistance to both pathogens. This study aimed to identify a repertoire of genes potentially associated with resistance to Xpp and compare it with that previously reported for Xcf. Eighty carioca and black common bean cultivars that had previously been challenged with Xcf were inoculated with a strain of Xpp, and a genome‐wide association study (GWAS) using single‐nucleotide polymorphisms (SNPs) and disease severity data was conducted. Three carioca and three black bean cultivars exhibited high levels of resistance to Xpp. Five SNPs with the highest −log10(p) values unveiled genes coding for diverse putative biochemical functions, including a light‐regulated Lir1 protein, a proton‐dependent oligopeptide transporter family protein, a metallo‐dependent phosphatase‐like protein, a WPP domain‐interacting tail‐anchored protein 2 and a pectin lyase‐like superfamily protein. No overlap between this repertoire of genes with that reported for Xcf was found. These findings provide important insights into genes potentially involved in the P. vulgaris resistance to Xpp and the complexity of the resistance response to CBB. [ABSTRACT FROM AUTHOR]

The present study was focused on exploring the efficient inhibitors of closed state (form) of type III effector Xanthomonas outer protein Q (XopQ) (PDB: 4P5F) from the 44 phytochemicals of Picrasma quassioides using cutting-edge computational analysis. Among them, Kumudine B showed excellent binding energy (−11.0 kcal/mol), followed by Picrasamide A, Quassidine I and Quassidine J with the targeted closed state of XopQ protein compared to the reference standard drug (Streptomycin). The molecular dynamics (MD) simulations performed at 300 ns validated the stability of top lead ligands (Kumudine B, Picrasamide A, and Quassidine I)-bound XopQ protein complex with slightly lower fluctuation than Streptomycin. The MM-PBSA calculation confirmed the strong interactions of top lead ligands (Kumudine B and QuassidineI) with XopQ protein, as they offered the least binding energy. The results of absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis confirmed that Quassidine I, Kumudine B and Picrasamide A were found to qualify most of the drug-likeness rules with excellent bioavailability scores compared to Streptomycin. Results of the computational studies suggested that Kumudine B, Picrasamide A, and Quassidine I could be considered potential compounds to design novel antibacterial drugs against X. oryzae infection. Further in vitro and in vivo antibacterial activities of Kumudine B, Picrasamide A, and Quassidine I are required to confirm their therapeutic potentiality in controlling the X. oryzae infection. [ABSTRACT FROM AUTHOR]

The introduction of plant pathogens can quickly reshape disease dynamics in island agro-ecologies, representing a continuous challenge for local crop management strategies. Xanthomonas pathogens causing tomato bacterial spot were probably introduced in Taiwan several decades ago, creating a unique opportunity to study the genetic makeup and adaptive response of this alien population. We examined the phenotypic and genotypic identity of 669 pathogen entries collected across different regions of Taiwan in the last three decades. The analysis detected a major population shift, where X. euvesicatoria and X. vesicatoria races T1 and T2 were replaced by new races of X. perforans. After its introduction, race T4 quickly became dominant in all tomato-growing areas of the island. The genomic analysis of 317 global genomes indicates that the Xanthomonas population in Taiwan has a narrow genetic background, most likely resulting from a small number of colonization events. However, despite the apparent genetic uniformity, X. perforans race T4 shows multiple phenotypic responses in tomato lines. Additionally, an in-depth analysis of effector composition suggests diversification in response to local adaptation. These include unique mutations on avrXv3 which might allow the pathogen to overcome Xv3/Rx4 resistance gene. The findings underscore the dynamic evolution of a pathogen when introduced in a semi-isolated environment and provide insights into the potential management strategies for this important disease of tomato. [ABSTRACT FROM AUTHOR]

Bacterial black spot (BBS) of mango, caused by the bacterium Xanthomonas citri pv. mangiferaeindicae, is an emerging disease affecting mango‐producing areas in Asia, Africa, Oceania, and the Americas. The disease spreads primarily through wind‐driven rain and farm implements, and its host range is limited, affecting mango, cashew, and pepper plants. BBS is characterized by dark, angular lesions with elevated borders on infected leaves and fruits in the lower and upper parts of the tree canopy. Infected plants display numerous tiny water‐soaked black lesions on leaves, fruits, and stems. Early symptoms on leaves include tiny, veined, water‐soaked patches with angular black dots and occasionally chlorotic spots. Managing BBS presents challenges, but various control measures have shown effectiveness in different regions. Integrated disease control measures, such as cultural methods and removing diseased plant parts, could further reduce BBS prevalence in the field. This paper reviews the detrimental effects associated with the disease, while also offering a concise overview of the existing body of research concerning BBS and its implications for mango cultivation. [ABSTRACT FROM AUTHOR]

The use of resistant varieties is an important strategy for managing tomato bacterial spot. The objective of this study was to evaluate the reaction of tomato genotypes to Xanthomonas euvesicatoria pv. perforans. The experiment was conducted in a randomized block design with 10 genotypes and four repetitions. The genotypes consisted of hybrids (UFU-1, UFU-2, UFU-3), wild resistant accession Solanum pennellii, commercial susceptible cultivar Santa Clara and homozygous lines (UFU-5, UFU-6, UFU-11, UFU-12, UFU-15). The UFU B8 isolate of X. euvesicatoria pv. perforans was used. The bacterial suspension was prepared and adjusted in a spectrophotometer OD550 = 0.5 (1 × 109 CFU mL−1). Inoculation occurred 10 days after transplantation. Disease severity was assessed at 3, 6, 9, 12 and 15 days after inoculation, and the area under the disease progress curve (AUDPC) was calculated. There was a significant difference between the genotypes regarding the severity of the disease at 3, 6 and 12 days after inoculation. Lineages 5, 6 and 12, the hybrid UFU-1 and the wild accession S. pennellii showed the lowest severity of the disease, being promising for promoting genetic improvement programs aimed at resistance to the bacteria. [ABSTRACT FROM AUTHOR]

In this study, we investigated native soil antagonists and the exotic Bacillus thuringiensis (Bt) in their effectiveness against chili bacterial pathogens, specifically Pseudomonas syringae and Xanthomonas vesicatoria. Putative native soil antagonists were isolated through plating heat-shocked (60 °C) serial dilutions on nutrient agar plates. Among these isolates, two (SK, CM) were further characterized based on phenotypic and biochemical traits, revealing gram-positive and catalase-positive characteristics, while being negative for urease, oxidase, and arginine dihydrolase. To evaluate the efficacy of SK, CM, and exotic Bt against P. syringae and X. vesicatoria, a dual culture confrontational assay was conducted. SK, CM, and Bt were found to restrict the growth of X. vesicatoria, not P. syringae. Consequently, further tests were exclusively performed with X. vesicatoria. Chemical compatibility with copper sulfate, topsin M, and sodium benzoate was assessed using the poisoned food technique, revealing inhibitory effects on microbial growth except for topsin M. Antibiotic sensitivity testing (erythromycin, streptomycin, tetracycline, penicillin) of X. vesicatoria, SK, CM, and Bt was carried out using the disk diffusion method. Results indicated a minimum zone of inhibition (0 mm) against penicillin and a maximum (29 mm) against streptomycin. The efficacy of antagonists was also tested in planta using detached leaf and seedling inoculation methods. Pre-inoculating leaves of a susceptible chili cultivar with antagonists and subsequently re-inoculating with X. vesicatoria demonstrated that, except for the positive control, leaves remained asymptomatic after 7 days post-inoculation. A pot experiment involving soil drenching and foliar spray inoculation further confirmed that plants initially treated with antagonists exhibited resistance against subsequent pathogen application compared to the positive control. In conclusion, this pilot study revealed that native putative Bacillus spp. isolates (SK, CM) and commercial exotic Bt have the potential to counteract X. vesicatoria in both local and distant tissues. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pv. oryzicola (Xoc) is a notorious plant pathogen. Like most bacterial pathogens, Xoc has evolved a complex regulatory network to modulate the expression of various genes related to pathogenicity. Here, we have identified TfmR, a transcriptional regulator belonging to the TetR family, as a key player in the virulence mechanisms of this phytopathogenic bacterium. We have demonstrated genetically that tfmR is involved in the hypersensitive response (HR), pathogenicity, motility and extracellular polysaccharide production of this phytopathogenic bacterium. Our investigations extended to exploring TfmR's interaction with RpfG and HrpX, two prominent virulence regulators in Xanthomonas species. We found that TfmR directly binds to the promoter region of RpfG, thereby positively regulating its expression. Notably, constitutive expression of RpfG partly reinstates the pathogenicity compromised by TfmR-deletion mutants. Furthermore, our studies revealed that TfmR also exerts direct positive regulation on the expression of the T3SS regulator HrpX. Similar to RpfG, sustained expression of HrpX partially restores the pathogenicity of TfmR-deletion mutants. These findings underscore TfmR's multifaceted role as a central regulator governing key virulence pathways in Xoc. Importantly, our research sheds light on the intricate molecular mechanisms underlying the regulation of pathogenicity in this plant pathogen. [ABSTRACT FROM AUTHOR]

The aim of the present study was nanoencapsulation of thymol to improve its poor water solubility and preservation of encapsulated thymol against environmental conditions. Another goal of the current investigation was to assess the antibacterial activity of thymol nanoemulsion as a sustainable biopesticide to control the bacterial blight of cluster bean. An oil-in-water (o/w) nanoemulsion containing thymol was prepared by a high-energy emulsification method using gum acacia and soya lecithin as natural emulsifiers/surfactants. The characterization of thymol nanoemulsion was carried out using dynamic light scattering (DLS), transmission electron microscope (TEM) and Fourier transform infrared spectroscopy (FTIR). A mean particle size of about 83.38 nm was recorded within 10 min of sonication. The stability analysis of optimized nanoemulsion showed kinetic stability up to two months of storage at room temperature. The thymol nanoemulsion was found to be spherical with a size ranging from 80–200 nm in diameter using transmission electron microscopy. Fourier transform infrared spectroscopy was used to study the molecular interaction between emulsifier/surfactant and thymol. The antibacterial studies of thymol nanoemulsion (0.01–0.06%, v/v) by growth inhibition analysis showed a potential antibacterial effect against Xanthomonas axonopodis pv. cyamopsidis (18–0.1 log CFU/ml). Further, in field experiments, foliar spray of the different concentration of thymol nanoemulsion (0.01–0.06%, v/v) significantly increased the percent efficiency of disease control (25.06–94.48%) and reduced the disease intensity (67.33–4.25%) of bacterial blight in cluster bean. [ABSTRACT FROM AUTHOR]

BACKGROUND: Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious diseases of rice, and there is a lack of bactericides for controlling this disease. We previously found parthenolide (PTL) is a potential lead for developing bactericides against Xoo, and subunit F of respiratory chain complex I (NuoF) is an important target protein of PTL. However, the binding modes of PTL with NuoF need further elucidation. RESULTS: In this study, we obtained the crystal structure of Xoo NuoEF (complex of subunit E and F of respiratory chain complex I) with a resolution of 2.36 Å, which is the first report on the protein structure of NuoEF in plant‐pathogenic bacteria. The possible binding sites of PTL with NuoF (Cys105 and Cys187) were predicted with molecular docking and mutated into alanine using a base mismatch method. The mutated proteins were expressed in Escherichia coli and purified with affinity chromatography. The binding abilities of PTL with mutated proteins were investigated via pull‐down assay and BIAcore analysis, which revealed that double mutation of Cys105 and Cys187 in NuoF severely affected the binding ability of PTL with NuoF. In addition, the binding modes were further simulated with combined quantum mechanical/molecular mechanical calculations, and the results indicated that PTL may have a stronger binding with Cys105 than Cys187. CONCLUSION: NuoEF protein structure of Xoo was resolved, and Cys105 and Cys187 in NuoF are important binding sites of PTL. This study further clarified the action mechanism of PTL against Xoo, and will promote the innovation of bactericides targeting Xoo complex I. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

High‐temperature‐requirement protein A (HtrA) family proteins play important roles in controlling protein quality and are recognized as virulence factors in numerous animal and human bacterial pathogens. The role of HtrA family proteins in plant pathogens remains largely unexplored. Here, we investigated the HtrA family protein, DegQ, in the crucifer black rot pathogen Xanthomonas campestris pathovar campestris (Xcc). DegQ is essential for bacterial attachment and full virulence of Xcc. Moreover, the degQ mutant strain showed increased sensitivity to heat treatment and sodium dodecyl sulfate. Expressing the intact degQ gene in trans in the degQ mutant could reverse the observed phenotypic changes. In addition, we demonstrated that the DegQ protein exhibited chaperone‐like activity. Transcriptional analysis displayed that degQ expression was induced under heat treatment. Our results contribute to understanding the function and expression of DegQ of Xcc for the first time and provide a novel perspective about HtrA family proteins in plant pathogen. [ABSTRACT FROM AUTHOR]

Key message: OsICS1 but not OsICS1-L mediates the rice response to Xoo inoculation, with its overexpression increasing resistance against this pathogen. OsICS1 but not OsICS-L is directly upregulated by OsWRKY6. Rice (Oryza sativa) is a staple crop for about half of the global population and is particularly important in the diets of people living in Asia, Latin America, and Africa. This crop is continually threatened by bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo), which drastically reduces yields; therefore, it is needed to elucidate the plant's resistance mechanisms against Xoo. Isochorismate synthase (ICS1) generates salicylic acid (SA) and increases resistance against bacterial disease. The OsICS1 is differently annotated in rice genome databases and has not yet been functionally characterized in the context of Xoo infection. Here, we report that the expression of the OsICS1 is directly regulated by OsWRKY6 and increases plant resistance against Xoo. Inoculation with Xoo increased the expression of OsICS1 but not that of the long variant of OsICS1 (OsICS1-L). OsWRKY6 directly activated the OsICS1 promoter but not the OsICS1-L promoter. OsICS1 overexpression in rice increased resistance against Xoo through the induction of SA-dependent bacterial defense genes. These data show that OsICS1 promotes resistance against Xoo infection. [ABSTRACT FROM AUTHOR]

Crop production plays a crucial role in ensuring global food security and maintaining economic stability. The presence of bacterial phytopathogens, particularly Xanthomonas species (a key focus of this review), poses significant threats to crops, leading to substantial economic losses. Current control strategies, such as the use of chemicals and antibiotics, face challenges such as environmental impact and the development of antimicrobial resistance. This review discusses the potential of bacteriocins, bacterial-derived proteinaceous antimicrobials and bacteriophages, viruses that target bacteria as sustainable alternatives for effectively managing Xanthomonas diseases. We focus on the diversity of bacteriocins found within xanthomonads by identifying and predicting the structures of candidate bacteriocin genes from publicly available genome sequences using BAGEL4 and AlphaFold. Harnessing the power of bacteriocins and bacteriophages has great potential as an eco-friendly and sustainable approach for precision control of Xanthomonas diseases in agriculture. However, realising the full potential of these natural antimicrobials requires continued research, field trials and collaboration among scientists, regulators and farmers. This collective effort is crucial to establishing these alternatives as promising substitutes for traditional disease management methods. [ABSTRACT FROM AUTHOR]

Phytopathogenic microbes use secreted effector proteins to increase their virulence in planta. If these effectors or the results of their activity are detected by the plant cell, the plant will mount an immune response which applies evolutionary pressure by reducing growth and success of the pathogen. Bacterial effector proteins in the AvrRps4 family (AvrRps4, HopK1, and XopO) have commonly been used as tools to investigate plant immune components. At the same time, the in planta functions of this family of effectors have yet to be fully characterized. In this minireview we summarize current knowledge about the AvrRps4 effector family with emphasis on properties of the proteins themselves. We hypothesize that the HopK1 C-terminus and the AvrRps4 Cterminus, though unrelated in sequence and structure, are broadly related in functions that counteract plant defense responses. [ABSTRACT FROM AUTHOR]

Extensive microbial interactions occur within insect hosts. However, the interactions between the Huanglongbing (HLB) pathogen and endosymbiotic bacteria within the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) in wild populations remain elusive. Thus, this study aimed to detect the infection rates of HLB in the ACP across five localities in China, with a widespread prevalence in Ruijin (RJ, 58%), Huidong (HD, 28%), and Lingui (LG, 15%) populations. Next, microbial communities of RJ and LG populations collected from citrus were analyzed via 16S rRNA amplicon sequencing. The results revealed a markedly higher microbial diversity in the RJ population compared to the LG population. Moreover, the PCoA analysis identified significant differences in microbial communities between the two populations. Considering that the inter-population differences of Bray–Curtis dissimilarity in the RJ population exceeded those between populations, separate analyses were performed. Our findings indicated an increased abundance of Enterobacteriaceae in individuals infected with HLB in both populations. Random forest analysis also identified Enterobacteriaceae as a crucial indicator of HLB infection. Furthermore, the phylogenetic analysis suggested a potential regulatory role of ASV4017 in Enterobacteriaceae for ACP, suggesting its possible attractant activity. This research contributes to expanding the understanding of microbial communities associated with HLB infection, holding significant implications for HLB prevention and treatment. [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]

Background: Two-tiered plant immune responses involve cross-talk among defense-responsive (DR) genes involved in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), effector-triggered immunity (ETI) and effector-triggered susceptibility (ETS). Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an important bacterial disease that causes serious threats to rice yield and quality. Transcriptomic profiling provides an effective approach for the comprehensive and large-scale detection of DR genes that participate in the interactions between rice and Xoc. Results: In this study, we used RNA-seq to analyze the differentially expressed genes (DEGs) in susceptible rice after inoculation with two naturally pathogenic Xoc strains, a hypervirulent strain, HGA4, and a relatively hypovirulent strain, RS105. First, bacterial growth curve and biomass quantification revealed that differential growth occurred beginning at 1 day post inoculation (dpi) and became more significant at 3 dpi. Additionally, we analyzed the DEGs at 12 h and 3 days post inoculation with two strains, representing the DR genes involved in the PTI and ETI/ETS responses, respectively. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed on the common DEGs, which included 4380 upregulated and 4019 downregulated genes and 930 upregulated and 1383 downregulated genes identified for the two strains at 12 h post inoculation (hpi) and 3 dpi, respectively. Compared to those at 12 hpi, at 3 dpi the number of common DEGs decreased, while the degree of differential expression was intensified. In addition, more disease-related GO pathways were enriched, and more transcription activator-like effector (TALE) putative target genes were upregulated in plants inoculated with HGA4 than in those inoculated with RS105 at 3 dpi. Then, four DRs were randomly selected for the BLS resistance assay. We found that CDP3.10, LOC_Os11g03820, and OsDSR2 positively regulated rice resistance to Xoc, while OsSPX3 negatively regulated rice resistance. Conclusions: By using an enrichment method for RNA-seq, we identified a group of DEGs related to the two stages of response to the Xoc strain, which included four functionally identified DR genes. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pv. oryzae (Xoo) injects major transcription activator-like effectors (TALEs) into plant cells to activate susceptibility (S) genes for promoting bacterial leaf blight in rice. Numerous resistance (R) genes have been used to construct differential cultivars of rice to identify races of Xoo, but the S genes were rarely considered. Different edited lines of rice cv. Kitaake were constructed using CRISPR/Cas9 gene-editing, including single, double and triple edits in the effector-binding elements (EBEs) located in the promoters of rice S genes OsSWEET11a, OsSWEET13 and OsSWEET14. The near-isogenic lines (NILs) were used as tracers to detect major TALEs (PthXo1, PthXo2, PthXo3 and their variants) in 50 Xoo strains. The pathotypes produced on the tracers determined six major TALE types in the 50 Xoo strains. The presence of the major TALEs in Xoo strains was consistent with the expression of S genes in the tracers, and it was also by known genome sequences. The EBE editing had little effect on agronomic traits, which was conducive to balancing yield and resistance. The rice-tracers generated here provide a valuable tool to track major TALEs of Xoo in Asia which then shows what rice cultivars are needed to combat Xoo in the field. [ABSTRACT FROM AUTHOR]

Xanthomonas and Stenotrophomonas are closely related genera in the family Lysobacteraceae. In our previous study of aroid-associated bacterial strains, most strains isolated from anthurium and other aroids were reclassified as X. phaseoli and other Xanthomonas species. However, two strains isolated from Spathiphyllum and Colocasia were phylogenetically distant from other strains in the Xanthomonas clade and two strains isolated from Anthurium clustered within the Stenotrophomonas clade. Phylogenetic trees based on 16S rRNA and nine housekeeping genes placed the former strains with the type strain of X. sacchari from sugarcane and the latter strains with the type strain of S. bentonitica from bentonite. In pairwise comparisons with type strains, the overall genomic relatedness indices required delineation of new species; digital DNA-DNA hybridization and average nucleotide identity values were lower than 70 and 95%, respectively. Hence, three new species are proposed: S. aracearum sp. nov. and S. oahuensis sp. nov. for two strains from anthurium and X. hawaiiensis sp. nov. for the strains from spathiphyllum and colocasia, respectively. The genome size of X. hawaiiensis sp. nov. is ~4.88 Mbp and higher than S. aracearum sp. nov. (4.33 Mbp) and S. oahuensis sp. nov. (4.68 Mbp). Gene content analysis revealed 425 and 576 core genes present in 40 xanthomonads and 25 stenotrophomonads, respectively. The average number of unique genes in Stenotrophomonas spp. was higher than in Xanthomonas spp., implying higher genetic diversity in Stenotrophomonas. [ABSTRACT FROM AUTHOR]

Citrus fruits, revered for their nutritional value, face significant threats from diseases like citrus canker, particularly impacting global citrus cultivation, notably in Pakistan. This study delves into the critical role of NPR1-like genes, the true receptors for salicylic acid (SA), in the defense mechanisms of citrus against Xanthomonas axonopodis pv. citri (Xcc). By conducting a comprehensive genome-wide analysis and phylogenetic study, the evolutionary dynamics of Citrus limon genes across diverse citrus cultivars are elucidated. Structural predictions unveil conserved domains, such as the BTB domain and ankyrin repeat domains, crucial for the defense mechanism. Motif analysis reveals essential conserved patterns, while cis-regulatory elements indicate their involvement in transcription, growth, response to phytohormones, and stress. The predominantly cytoplasmic and nuclear localization of NPR1-like genes underscores their pivotal role in conferring resistance to various citrus species. Analysis of the Ks/Ka ratio indicates a purifying selection of NPR1-like genes, emphasizing their importance in different species. Synteny and chromosomal mapping provide insights into duplication events and orthologous links among citrus species. Notably, Xac infection stimulates the expression of NPR1-like genes, revealing their responsiveness to pathogenic challenges. Interestingly, qRT-PCR profiling post-Xac infection reveals cultivar-specific alterations in expression within susceptible and resistant citrus varieties. Beyond genetic factors, physiological parameters like peroxidase, total soluble protein, and secondary metabolites respond to SA-dependent PR genes, influencing plant characteristics. Examining the impact of defense genes (NPR1) and plant characteristics on disease resistance in citrus, this study marks the inaugural investigation into the correlation between NPR1-associated genes and various plant traits in both susceptible and resistant citrus varieties to citrus bacterial canker. [ABSTRACT FROM AUTHOR]

Citrus canker disease affects citrus production. This disease is caused by Xanthomonas citri subsp. citri (Xcc). Previous studies confirmed that during Xcc infection, PthA4, a transcriptional activator like effector (TALE), is translocated from the pathogen to host plant cells. PthA4 binds to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 (EBEPthA4-LOBP) to activate its expression and subsequently cause canker symptoms. Previously, the Cas12a/CBE co-editing method was employed to disrupt EBEPthA4-LOBP of pummelo, which is highly homozygous. However, most commercial citrus cultivars are heterozygous hybrids and more difficult to generate homozygous/biallelic mutants. Here, we employed Cas12a/CBE co-editing method to edit EBEPthA4-LOBP of Hamlin (Citrus sinensis), a commercial heterozygous hybrid citrus cultivar grown worldwide. Binary vector GFPp1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1 was constructed and shown to be functional via Xcc-facilitated agroinfiltration in Hamlin leaves. This construct allows the selection of transgene-free regenerants via GFP, edits ALS to generate chlorsulfuron-resistant regenerants as a selection marker for genome editing resulting from transient expression of the T-DNA via nCas9-mPBE:ALS2:ALS1, and edits gene(s) of interest (i.e., EBEPthA4-LOBP in this study) through ttLbCas12a, thus creating transgene-free citrus. Totally, 77 plantlets were produced. Among them, 8 plantlets were transgenic plants (#HamGFP1 - #HamGFP8), 4 plantlets were transgene-free (#HamNoGFP1 - #HamNoGFP4), and the rest were wild type. Among 4 transgene-free plantlets, three lines (#HamNoGFP1, #HamNoGFP2 and #HamNoGFP3) contained biallelic mutations in EBEpthA4, and one line (#HamNoGFP 4) had homozygous mutations in EBEpthA4. We achieved 5.2% transgene-free homozygous/biallelic mutation efficiency for EBEPthA4-LOBP in C. sinensis cv. Hamlin, compared to 1.9% mutation efficiency for pummelo in a previous study. Importantly, the four transgenefree plantlets and 3 transgenic plantlets that survived were resistant against citrus canker. Taken together, Cas12a/CBE co-editing method has been successfully used to generate transgene-free canker-resistant C. sinensis cv. Hamlin in the T0 generation via biallelic/homozygous editing of EBEpthA4 of the canker susceptibility gene LOB1. [ABSTRACT FROM AUTHOR]

Background: Bacterial leaf blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases affecting rice production worldwide. Resistance breeding is considered a durable, effective and eco-friendly approach to control BB. Several BB resistance (R) genes have been identified that confer resistance against various strains of Xoo. The dynamic nature of this pathogen needs proper knowledge of pathotype composition and its virulence among the diverse Xoo strains which is imperative for designing a resistance breeding program. Methods: In this study, we conducted an extensive survey to collect BB infected samples from diverse rice-growing regions of Tamil Nadu. Further, the samples were identified as Xoo through morphological and molecular identification methods. The pathotyping and virulence profiling of Xoo isolates was carried out on a set of rice differentials consisting of 22 near-isogenic lines (NILs) of IR24 possessing different BB resistance genes under glasshouse conditions during Kharif 2021-2022. Result: Based on the disease reaction in 22 rice BB differentials (IRBB lines), the Xoo isolates were grouped into six pathotypes (I II, III, IV, V and VI). Further, the virulence analysis revealed that the near-isogenic lines possessing single R gene and gene pyramids were susceptible to about 20-88% and 4-20% respectively to Xoo isolates. R genes and their pyramids such as (Xa7, Xa21, Xa4+xa5+Xa21, Xa4+xa5+xa13, Xa4+xa13+Xa21, xa5+xa13+Xa21 and Xa4+Xa21+xa5+Xa21) were found to be effective against all the pathotypes in Tamil Nadu. The pathotype III was predominantly distributed (40%) over the Tamil Nadu. The isolates found in the pathotypes I and II are more virulent and in the pathotypes V, VI are less virulent. The current study identified the virulence nature of diverse Xoo isolates and the effectiveness of resistance (R) genes against the Xoo isolates. Therefore, the effective R genes identified in this study could be an effective source for resistance breeding program against BB in Tamil Nadu. [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]

Outbreaks of bacterial leaf blight (BLB) frequently affect eucalypt plants under nursery and field conditions in several countries. Although research has been conducted to unveil the causal agent, different bacterial species have been associated with similar disease symptoms in different countries. In order to determine the causal agent of BLB in Brazil, a survey was conducted in nine states to recover bacterial isolates from eucalypt plants exhibiting typical BLB symptoms. A total of 41 yellow‐colony isolates with varying aggressiveness towards a susceptible eucalypt clone were obtained, with 16S rDNA sequences indicating that they belong to the Xanthomonas genus. Rep‐PCR analysis separated the Xanthomonas population affecting eucalypt into six distinct groups revealing its high genetic diversity. The same population formed three clusters together with reference strains of X. citri, X. euvesicatoria and X. phaseoli in a phylogenetic tree constructed with partial dnaK, fyuA, gyrB and rpoD gene sequences. Clustering in the phylogenetic tree was clearly related to grouping based on rep‐PCR. Genome sequence comparisons of representative eucalypt isolates with type strains of validly published Xanthomonas species confirmed that the population consisted of X. citri, X. euvesicatoria and X. phaseoli. Inoculation of tomato, common bean, castor bean and eucalypt plants showed that the representative eucalypt isolates can cause disease in these plant species. Based on the results, the transfer of Xanthomonas axonopodis pv. eucalyptorum to Xanthomonas citri is proposed. These results are relevant for eucalypt BLB management under nursery and field conditions, including selection and deployment of effective plant resistance. [ABSTRACT FROM AUTHOR]

Walnut bacterial blight caused by Xanthomonas arboricola pv. juglandis (Xaj) is one of the most prevalent diseases of walnut (Juglans spp.), causing significant reductions in nut yield and important losses in economy. Enoyl-acyl carrier protein (ACP) reductase (ENR) is one of the key enzymes involved in the biosynthesis of bacterial fatty acids. In this study, we identified a single ENR-encoding gene, RS10040, in the genome of the XajDW3F3 strain. Sequence alignment analysis suggested RS10040 as a candidate fabV gene in Xaj. Expression of XajfabV restored the growth of the Escherichia coli fabI temperature-sensitive mutant under a nonpermissive growth condition. In vitro assays demonstrated that XajFabV catalyzed enoyl-ACPs of various chain lengths to acyl-ACPs, demonstrating its role in de novo fatty acid biosynthesis. Furthermore, we confirmed that XajfabV is an essential gene for growth, as no XajfabV deletion mutant could be obtained, although XajfabV in the chromosome could be deleted after compensating with a functional ENR-encoding gene via an exogenous plasmid. The fabV replacement mutants showed similar growth characteristic and fatty acid compositions. Our data further identified that fabV conferred Xaj with tolerance to various environmental stresses. Although XajFabV conferred Xaj with triclosan resistance, the resistance of Xaj was weaker than that found for Pseudomonas aeruginosa. Moreover, triclosan exhibited a control effect against infection of the ΔfabV/EcfabI to its host walnut. This study revealed the function of XajFabV and laid a theoretical foundation for the fatty acid synthesis mechanism of Xaj. [ABSTRACT FROM AUTHOR]

Bacterial leaf spot is a serious disease of chili pepper (Capsicum spp.) caused by Xanthomonas euvesicatoria pv. euvesicatoria. Conventional resistance screening is time and resource intensive. It was considered that a quick and simple determination of cultivar susceptibility could be achieved through estimating bacterial titers of inoculated plants. A SYBR quantitative polymerase chain reaction (qPCR)-based assay was compared with conventional PCR, then used to detect and enumerate pathogen titers in serial dilutions and DNA extracted from infected plant leaves. The qPCR detection limit was approximately 1 CFU µl−1, 10 times more sensitive than conventional PCR. A linear correlation (R² = 0.994) was obtained from the standard curve comparing plate-truthed serial dilutions of the pathogen with the qPCR cycle threshold. Six strains were used to inoculate cultivars Hugo and Warlock. One strain, X. euvesicatoria pv. euvesicatoria BRIP62403, was consistently the most virulent based on visual symptoms and pathogen titers in planta inferred by qPCR performed on DNA extracted from infected leaves 2 and 6 weeks postinoculation. Visual observations 6 weeks after inoculation were highly correlated (R² = 0.8254) to pathogen titers. The qPCR method was used to categorize 20 chili pepper cultivars 2 weeks after inoculation. A high positive correlation (R² = 0.6826) was observed between visual scoring and pathogen titers from 20 chili pepper cultivars, facilitating categorization of susceptible, intermediate, and resistant cultivars. The qPCR approach developed here facilitates susceptibility screening of chili pepper cultivars at an early stage of selection and could be readily adapted to a range of other pathosystems. [ABSTRACT FROM AUTHOR]

Xanthomonas species, including active axonopodis pv. malvacearum (X.a.m.), Xanthomonas axonopodis pv. Taum (X.a.p.), and Xanthomonas campestris pv. vesicatoria (X. c. v.), are used to make various products and fractions of seven medicinal plants, including Aristolochi thyme and Aristolochia methanol extracts shown substantial antibacterial activity against all harmful microorganisms when tested on seven different plants. The solvent extracts' antibacterial qualities are more prominent when compared to the entire plant's aqueous extract, suggesting that the antibacterial activity's active components are more soluble in organic solvents. Further testing of the methanolic extract of thyme and aristolochia is shown by comparing the inhibitory activity of the extracts with bacteriomycin 2000 and streptomycin. According to a phytochemical investigation of the plant's leaves, phenolic chemicals are what give the plant its antibacterial qualities. According to the findings, thyme and aristolochia are plants that can manage Xanthomonas. [ABSTRACT FROM AUTHOR]

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