Xanthomonas Species Research Articles

Isolation of atypical wheat-associated xanthomonads in Algeria

Bacterial leaf streak and black chaff are important bacterial diseases of wheat, which have been reported to be caused by Xanthomonas translucens. In 2016, symptoms of bacterial leaf streak and black chaff were observed in Algeria, at experimental wheat breeding stations and in farmers' fields under sprinkler irrigation on two wheat cultivars, 'Hiddab' and 'Simeto'. Yellow Xanthomonas-like bacterial colonies were isolated from plant material, including leaves, spikes and post-harvest crop residues. Initial characterisation using biochemical, physiological and pathogenicity tests identified the bacteria as Xanthomonas. Diagnostic PCR targeting the 16S-23S rRNA intergenic region indicated that the strains were X. translucens, a clade-1 xanthomonad. However, partial DNA sequences of the housekeeping genes gyrB and rpoD revealed that the strains belong to clade 1, but likely represent a new Xanthomonas species that has not been previously described on wheat or other Gramineae. The most closely related strain, NCPPB 2654, was isolated from a bean plant in the United Kingdom in 1974. Further characterization is required to clarify the taxonomic status of the Algerian Xanthomonas isolates from wheat, and to determine their host ranges and impacts on plant cultivation.

Characterization of Lysobacter spp. strains and their potential use as biocontrol agents against pear anthracnose

Colletotrichum fructicola, is an important fungal pathogen that has been reported to cause pear (Pyrus) anthracnose in China, resulting in substantial economic losses due to severe defoliation and decreased fruit quality and yield. In the search for novel strategies to control pear anthracnose, Lysobacter strains have drawn a great deal of attention due to their high-level production of extracellular enzymes and bioactive metabolites. In the present study, we compared four Lysobacter strains including Lysobacter enzymogenes OH11, Lysobacter antibioticus OH13, Lysobacter gummosus OH17 and Lysobacter brunescens OH23 with respect to their characteristics and activity against pear anthracnose caused by C. fructicola. The results showed that the evaluated Lysobacter species presented various colony morphologies when cultured on different media and were proficient in producing protease, chitinase, cellulase and glucanase, with L. enzymogenes OH11 showing typical twitching motility. L. enzymogenes OH11 and L. gummosus OH17 showed potent activity against the tested fungi and oomycetes. L. gummosus OH17 produced HSAF (heat-stable antifungal factor) which was demonstrated to be a major antifungal factor in L. enzymogenes OH11 and C3. Furthermore, L. antibioticus OH13 and L. brunescens OH23 exhibited strong antibacterial activity, especially against Xanthomonas species. Cultures of L. enzymogenes OH11 protected pear against anthracnose caused by C. fructicola, and the in vivo results indicated that treatment with an L. enzymogenes OH11 culture could decrease the diameter of lesions in pears by 35 % and reduce the severity of rot symptoms compared to that observed in the control. In the present study, we systemically compared four Lysobacter strains and demonstrated that they have strong antagonistic activity against a range of pathogens, demonstrating their promise in the development of biological control agents.

Novel Xanthomonas Species From the Perennial Ryegrass Seed Microbiome - Assessing the Bioprotection Activity of Non-pathogenic Relatives of Pathogens.

The productivity of the Australian dairy industry is underpinned by pasture grasses, and importantly perennial ryegrass. The performance of these pasture grasses is supported by the fungal endophyte Epichloë spp. that has bioprotection activities, however, the broader microbiome is not well characterized. In this study, we characterized a novel bioprotectant Xanthomonas species isolated from perennial ryegrass (Lolium perenne L. cv. Alto). In vitro and in planta bioassays against key fungal pathogens of grasses (Sclerotium rolfsii, Drechslera brizae and Microdochium nivale) indicated strong bioprotection activities. A complete circular chromosome of ∼5.2 Mb was generated for three strains of the novel Xanthomonas sp. Based on the 16S ribosomal RNA gene, the strains were closely related to the plant pathogen Xanthomonas translucens, however, comparative genomics of 22 closely related xanthomonad strains indicated that these strains were a novel species. The comparative genomics analysis also identified two unique gene clusters associated with the production of bioprotectant secondary metabolites including one associated with a novel nonribosomal peptide synthetase and another with a siderophore. The analysis also identified genes associated with an endophytic lifestyle (e.g., Type VI secretion system), while no genes associated with pathogenicity were identified (e.g., Type III secretion system and effectors). Overall, these results indicate that these strains represent a novel, bioactive, non-pathogenic species of the genus Xanthomonas. Strain GW was the designated type strain of this novel Xanthomonas sp.

Genome-Scale Metabolic Model of Xanthomonas phaseoli pv. manihotis: An Approach to Elucidate Pathogenicity at the Metabolic Level.

Xanthomonas phaseoli pv. manihotis (Xpm) is the causal agent of cassava bacterial blight, the most important bacterial disease in this crop. There is a paucity of knowledge about the metabolism of Xanthomonas and its relevance in the pathogenic process, with the exception of the elucidation of the xanthan biosynthesis route. Here we report the reconstruction of the genome-scale model of Xpm metabolism and the insights it provides into plant–pathogen interactions. The model, iXpm1556, displayed 1,556 reactions, 1,527 compounds, and 890 genes. Metabolic maps of central amino acid and carbohydrate metabolism, as well as xanthan biosynthesis of Xpm, were reconstructed using Escher (https://escher.github.io/) to guide the curation process and for further analyses. The model was constrained using the RNA-seq data of a mutant of Xpm for quorum sensing (QS), and these data were used to construct context-specific models (CSMs) of the metabolism of the two strains (wild type and QS mutant). The CSMs and flux balance analysis were used to get insights into pathogenicity, xanthan biosynthesis, and QS mechanisms. Between the CSMs, 653 reactions were shared; unique reactions belong to purine, pyrimidine, and amino acid metabolism. Alternative objective functions were used to demonstrate a trade-off between xanthan biosynthesis and growth and the re-allocation of resources in the process of biosynthesis. Important features altered by QS included carbohydrate metabolism, NAD(P)+ balance, and fatty acid elongation. In this work, we modeled the xanthan biosynthesis and the QS process and their impact on the metabolism of the bacterium. This model will be useful for researchers studying host–pathogen interactions and will provide insights into the mechanisms of infection used by this and other Xanthomonas species.

Transcriptome-Based Analysis of Tomato Genotypes Resistant to Bacterial Spot (Xanthomonas perforans) Race T4.

Bacterial spot (BS) is one of the most devastating foliar bacterial diseases of tomato and is caused by multiple species of Xanthomonas. We performed the RNA sequencing (RNA-Seq) analysis of three tomato lines with different levels of resistance to Xanthomonas perforans race T4 to study the differentially expressed genes (DEGs) and transcript-based sequence variations. Analysis between inoculated and control samples revealed that resistant genotype Solanum pimpinellifolium accession PI 270443 had more DEGs (834), followed by susceptible genotype tomato (S. lycopersicum L) breeding line NC 714 (373), and intermediate genotype tomato breeding line NC 1CELBR (154). Gene ontology (GO) terms revealed that more GO terms (51) were enriched for upregulated DEGs in the resistant genotype PI 270443, and more downregulated DEGs (67) were enriched in the susceptible genotype NC 714. DEGs in the biotic stress pathway showed more upregulated biotic stress pathway DEGs (67) for PI 270443 compared to more downregulated DEGs (125) for the susceptible NC 714 genotype. Resistant genotype PI 270443 has three upregulated DEGs for pathogenesis-related (PR) proteins, and susceptible genotype NC 714 has one downregulated R gene. Sequence variations called from RNA-Seq reads against the reference genome of susceptible Heinz 1706 showed that chr11, which has multiple reported resistance quantitative trait loci (QTLs) to BS race T4, is identical between two resistant lines, PI 270443 and NC 1CELBR, suggesting that these two lines share the same resistance QTLs on this chromosome. Several loci for PR resistance proteins with sequence variation between the resistant and susceptible tomato lines were near the known Rx4 resistance gene on chr11, and additional biotic stress associated DEGs near to the known Rx4 resistance gene were also identified from the susceptible NC 714 line.

A SAM-I riboswitch with the ability to sense and respond to uncharged initiator tRNA

All known riboswitches use their aptamer to senese one metabolite signal and their expression platform to regulate gene expression. Here, we characterize a SAM-I riboswitch (SAM-IXcc) from the Xanthomonas campestris that regulates methionine synthesis via the met operon. In vitro and in vivo experiments show that SAM-IXcc controls the met operon primarily at the translational level in response to cellular S-adenosylmethionine (SAM) levels. Biochemical and genetic data demonstrate that SAM-IXcc expression platform not only can repress gene expression in response to SAM binding to SAM-IXcc aptamer but also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shine-Dalgarno (SD) sequence and freeing the SD for translation initiation. These findings identify a SAM-I riboswitch with a dual functioning expression platform that regulates methionine synthesis through a previously unrecognized mechanism and discover a natural tRNA-sensing RNA element. This SAM-I riboswitch appears to be highly conserved in Xanthomonas species.

Crystal structure of β-L-arabinobiosidase belonging to glycoside hydrolase family 121.

Enzymes acting on α-L-arabinofuranosides have been extensively studied; however, the structures and functions of β-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of β-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular β-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing β-1,2-linked arabinofuranose disaccharide (β-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 Å resolution. The HypBA2 structure consists of a central catalytic (α/α)6 barrel domain and two flanking (N- and C-terminal) β-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the β-Ara2 disaccharide. Three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of structural homologs in other GH families: GH63 α-glycosidase, GH94 chitobiose phosphorylase, GH142 β-L-arabinofuranosidase, GH78 α-L-rhamnosidase, and GH37 α,α-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. β-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the β-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens.

Bactericidal type IV secretion system homeostasis in Xanthomonas citri.

Several Xanthomonas species have a type IV secretion system (T4SS) that injects a cocktail of antibacterial proteins into neighbouring Gram-negative bacteria, often leading to rapid lysis upon cell contact. This capability represents an obvious fitness benefit since it can eliminate competition while the liberated contents of the lysed bacteria could provide an increase in the local availability of nutrients. However, the production of this Mega Dalton-sized molecular machine, with over a hundred subunits, also imposes a significant metabolic cost. Here we show that the chromosomal virB operon, which encodes the structural genes of this T4SS in X. citri, is regulated by the conserved global regulator CsrA. Relieving CsrA repression from the virB operon produced a greater number of T4SSs in the cell envelope and an increased efficiency in contact-dependent lysis of target cells. However, this was also accompanied by a physiological cost leading to reduced fitness when in co-culture with wild-type X. citri. We show that T4SS production is constitutive despite being downregulated by CsrA. Cells subjected to a wide range of rich and poor growth conditions maintain a constant density of T4SSs in the cell envelope and concomitant interbacterial competitiveness. These results show that CsrA provides a constant though partial repression on the virB operon, independent of the tested growth conditions, in this way controlling T4SS-related costs while at the same time maintaining X. citri's aggressive posture when confronted by competitors.

Weeds as alternate hosts of Xanthomonas euvesicatoria pv. euvesicatoria and X. campestris pv. campestris in vegetable-growing fields in the state of Pernambuco, Brazil

Plant pathogenic Xanthomonas species attack a wide range of agriculture crops and is capable of surviving on weeds. In this study, Xanthomonas strains obtained from weeds grown withing vegetable crop fields in the state of Pernambuco, Northeast Brazil, were identified. Bacterial colonies resembling Xanthomonas were obtained from Aeollanthus suaveolens (strain CCRMXe03), Amaranthus lividus (CCRMXe01 and CCRMXe02), Sida glomerata (CCRMXe04), and Emilia fosbergii (CCRMXe04). All weeds but E. fosbergii exhibited lesions on the leaves. Phylogenetic analysis of the gyrB and rpoD genes and PCR-specific assays allowed to identify X. euvesicatoria pv. euvesicatoria (CCRMXe01, CCRMXe02, CCRMXe03, and CCRMXe04) and X. campestris pv. campestris (CCRMXcc371). All strains but CCRMXe03 were pathogenic to its host of origin. All X. euvesicatoria pv. euvesicatoria strains were pathogenic to leaves and fruits of tomato and bell pepper. Two strains, CCRMXe03 and CCRMXe04, were capable of inducing soft rot in fruits. Amylolytic activity was found in all strains and two strains (CCRMXe03 and CCRMXe04) degraded pectate. Strain CCRMXcc371 was pathogenic to cabbage, kale, cauliflower, and broccoli. This work provides new knowledge of hosts for two important plant pathogenic bacteria for vegetable crops.

The Immune Receptor Roq1 Confers Resistance to the Bacterial Pathogens Xanthomonas, Pseudomonas syringae, and Ralstonia in Tomato.

Xanthomonas species, Pseudomonas syringae and Ralstonia species are bacterial plant pathogens that cause significant yield loss in many crop species. Generating disease-resistant crop varieties can provide a more sustainable solution to control yield loss compared to chemical methods. Plant immune receptors encoded by nucleotide−binding, leucine−rich repeat (NLR) genes typically confer resistance to pathogens that produce a cognate elicitor, often an effector protein secreted by the pathogen to promote virulence. The diverse sequence and presence/absence variation of pathogen effector proteins within and between pathogen species usually limits the utility of a single NLR gene to protecting a plant from a single pathogen species or particular strains. The NLR protein Recognition of XopQ 1 (Roq1) was recently identified from the plant Nicotiana benthamiana and mediates perception of the effector proteins XopQ and HopQ1 from Xanthomonas and P. syringae respectively. Unlike most recognized effectors, alleles of XopQ/HopQ1 are highly conserved and present in most plant pathogenic strains of Xanthomonas and P. syringae. A homolog of XopQ/HopQ1, named RipB, is present in most Ralstonia strains. We found that Roq1 confers immunity to Xanthomonas, P. syringae, and Ralstonia when expressed in tomato. Strong resistance to Xanthomonas perforans was observed in three seasons of field trials with both natural and artificial inoculation. The Roq1 gene can therefore be used to provide safe, economical, and effective control of these pathogens in tomato and other crop species and reduce or eliminate the need for traditional chemical controls.

Complete genome sequence of Xanthomonas phage RiverRider, a novel N4-like bacteriophage that infects the strawberry pathogen Xanthomonas fragariae.

Xanthomonas phage RiverRider is a novel N4-like bacteriophage and the first phage isolated from the plant pathogen Xanthomonas fragariae. Electron microscopy revealed a Podoviridae morphology consisting of isometric heads and short noncontractile tails. The complete genome of RiverRider is 76,355bp in length, with 90 open reading frames and seven tRNAs. The genome is characteristic of N4-like bacteriophages in both content and organization, having predicted proteins characterized into the functional groups of transcription, DNA metabolism, DNA replication, lysis, lysis inhibition, structure and DNA packaging. Amino acid sequence comparisons for proteins in these categories showed highest similarities to well-characterized N4-like bacteriophages isolated from Achromobacter xylosoxidans and Erwinia amylovora. However, the tail fiber proteins of RiverRider are clearly distinct from those of other N4-like phages. RiverRider was able to infect seven different strains of X. fragariae and none of the other species of Xanthomonas tested.

Chemical Structure, Biological Roles, Biosynthesis and Regulation of the Yellow Xanthomonadin Pigments in the Phytopathogenic Genus Xanthomonas.

Xanthomonadins are membrane-bound yellow pigments that are typically produced by phytopathogenic bacterial Xanthomonas spp., Xylella fastidiosa, and Pseudoxanthomonas spp. They are also produced by a diversity of environmental bacterial species. Considerable research has revealed that they are a unique group of halogenated, aryl-polyene, water-insoluble pigments. Xanthomonadins have been shown to play important roles in epiphytic survival and host-pathogen interactions in the phytopathogen Xanthomonas campestris pv. campestris, which is the causal agent of black rot in crucifers. Here, we review recent advances in the understanding of xanthomonadin chemical structures, physiological roles, biosynthetic pathways, regulatory mechanisms, and crosstalk with other signaling pathways. The aim of the present review is to provide clues for further in-depth research on xanthomonadins from Xanthomonas and other related bacterial species.

Phenolic Acid-Degrading Consortia Increase Fusarium Wilt Disease Resistance of Chrysanthemum

Soil microbial community changes imposed by the cumulative effects of root-secreted phenolic acids (PAs) promote soil-borne pathogen establishment and invasion under monoculture systems, but the disease-suppressive soil often exhibits less soil-borne pathogens compared with the conducive soil. So far, it remains poorly understood whether soil disease suppressiveness is associated with the alleviated negative effects of PAs, involving microbial degradation. Here, the long-term monoculture particularly shaped the rhizosphere microbial community, for example by the enrichment of beneficial Pseudomonas species in the suppressive soil and thus enhanced disease-suppressive capacity, however this was not observed for the conducive soil. In vitro PA-degradation assays revealed that the antagonistic Pseudomonas species, together with the Xanthomonas and Rhizobium species, significantly increased the efficiency of PA degradation compared to single species, at least partially explaining how the suppressive soil accumulated lower PA levels than the conducive soil. Pot experiments further showed that this consortium harboring the antagonistic Pseudomonas species can not only lower PA accumulation in the 15-year conducive soils, but also confer stronger Fusarium wilt disease suppression compared with a single inoculum with the antagonistic bacteria. Our findings demonstrated that understanding microbial community functions, beyond the single direct antagonism, facilitated the construction of active consortia for preventing soil-borne pathogens under intensive monoculture.

Advances and Challenges in Bacterial Spot Resistance Breeding in Tomato (Solanum lycopersicum L.).

Bacterial spot is a serious disease of tomato caused by at least four species of Xanthomonas. These include X. euvesicatoria (race T1), X. vesicatoria (race T2), X. perforans (races T3 and T4), and X. gardneri, with the distinct geographical distribution of each group. Currently, X. gardneri and X. perforans are two major bacterial pathogens of tomato in North America, with X. perforans (race T4) dominating in east-coast while X. gardneri dominating in the Midwest. The disease causes up to 66% yield loss. Management of this disease is challenging due to the lack of useful chemical control measures and commercial resistant cultivars. Although major genes for resistance (R) and quantitative resistance have been identified, breeding tomato for resistance to bacterial spot has been impeded by multiple factors including the emergence of new races of the pathogen that overcome the resistance, multigenic control of the resistance, linkage drag, non-additive components of the resistance and a low correlation between seedling assays and field resistance. Transgenic tomato with Bs2 and EFR genes was effective against multiple races of Xanthomonas. However, it has not been commercialized because of public concerns and complex regulatory processes. The genomics-assisted breeding, effectors-based genomics breeding, and genome editing technology could be novel approaches to achieve durable resistance to bacterial spot in tomato. The main goal of this paper is to understand the current status of bacterial spot of tomato including its distribution and pathogen diversity, challenges in disease management, disease resistance sources, resistance genetics and breeding, and future prospectives with novel breeding approaches.

Strain-Level Identification of Bacterial Tomato Pathogens Directly from Metagenomic Sequences

Routine strain-level identification of plant pathogens directly from symptomatic tissue could significantly improve plant disease control and prevention. Here we tested the Oxford Nanopore Technologies (ONT) MinION sequencer for metagenomic sequencing of tomato plants either artificially inoculated with a known strain of the bacterial speck pathogen Pseudomonas syringae pv. tomato or collected in the field and showing bacterial spot symptoms caused by one of four Xanthomonas species. After species-level identification via ONT's WIMP software and the third-party tools Sourmash and MetaMaps, we used Sourmash and MetaMaps with a custom database of representative genomes of bacterial tomato pathogens to attempt strain-level identification. In parallel, each metagenome was assembled and the longest contigs were used as query with the genome-based microbial identification Web service LINbase. Both the read-based and assembly-based approaches correctly identified P. syringae pv. tomato strain T1 in the artificially inoculated samples. The pathogen strain in most field samples was identified as a member of Xanthomonas perforans group 2. This result was confirmed by whole genome sequencing of colonies isolated from one of the samples. Although in our case metagenome-based pathogen identification at the strain level was achieved, caution still must be exercised in interpreting strain-level results because of the challenges inherent to assigning reads to specific strains and the error rate of nanopore sequencing.

High-Quality Genome Resource of Xanthomonas hyacinthi Generated via Long-Read Sequencing.

The bacterial plant pathogen Xanthomonas hyacinthi is the causal agent of yellow disease of Hyacinthus and other ornamental plant genera. There is no available complete genome for X. hyacinthi, limiting basic research for this pathogen. Here, we release a high-quality complete genome sequence for the X. hyacinthi type strain, CFBP 1156. Single-molecule real-time (SMRT) sequencing with a mean coverage of 306× revealed two contigs of 4,918,645 and 44,381 bp in size. This was the first characterized plant-disease-causing species of Xanthomonas and this genome provides a resource to better understand the biology of yellow disease of hyacinth.

Crystal Structure of an Anti-CRISPR Protein, AcrIF2, and its Interaction With Type I-F Cas Proteins

CRISPRs and Cas proteins form a microbial adaptive immune system against phage infection. To counteract this defense mechanism, phages evolved anti-CRISPR (Acr) proteins that inhibit the CRISPR-Cas systems. Here, we report the crystal structure of AcrIF2, an Acr protein inactivating the type I-F CRISPR-Cas system in Pseudomonas aeruginosa. In the type I-F systems, multiple Cas proteins (Csy1-4) constitute a surveillance complex with a guide RNA for target recognition. We demonstrated that AcrIF2 binds strongly to Csy1-Csy2 subcomplexes from two Xanthomonas species.

Species of the genus Xanthomonas infecting cereals and oilseeds in the Russian Federation and its diagnostics

Plant pathogenic bacteria of the genus Xanthomonas display high levels of genetic diversity and cause remarkable damage to about 400 plant species. In 2001–2008, a new group of strains of Xanthomonas arboricola has been found as pathogens on novel host plants such as wheat, rye, barley, tomato, sunflower, and brassicas in Russia. Physiological tests and multilocus sequence typing (MLST) analysis confirmed their position within the Xanthomonas arboricola species. The obtained draft genome sequence of Xanthomonas arboricola strain 3004 from barley plants, also virulent to sunflower, brassicas, and chestnut, has demonstrated an evidence for the lateral gene transfer (LGT) of the virulence genes. It can be suggested that the virE and other genes of T4SS, obtained due to LGT, may contribute to the host range extension. Thus, T4SS genes can be used as the target for group-specific PCR analysis of this emerging pathogen of cereals and oilseeds. We propose to use virB3, virB4, and virB9 genes to design a detection system.

Evaluating Quantitative Trait Locus Resistance in Tomato to Multiple Xanthomonas spp.

Bacterial spot of tomato is a foliar disease caused by four Xanthomonas species. Identifying genetic resistance in wild tomatoes and subsequent breeding of varieties has been a strategy to reduce the loss from this disease because control using pesticides has been ineffective. Three independent sources of resistance have been identified with quantitative trait loci (QTL) mapping to the centromeric region on chromosome 11. These sources are derived from Hawaii 7998 (QTL-11A), PI 114490 (QTL-11B), and LA2533 (QTL-11C). To determine which QTL introgression from chromosome 11 provides the greatest resistance to multiple species, we developed near-isogenic lines (NILs) using marker-assisted backcrossing. In parallel, we developed an NIL that contains Rx-4/Xv3, which provides major gene resistance to Xanthomonas perforans. Additionally, we combined Rx-4/Xv3 resistance with QTL-11A. These sources of resistance were independently introduced into the susceptible parent, OH88119. During a 3-year period from 2016 to 2018, we evaluated backcross-derived families and NILs from each source in independent field trials inoculated with X. perforans, X. euvesicatoria, or X. gardneri. Our results suggest that both QTL-11C and QTL-11A combined with Rx-4/Xv3 provide effective genetic resistance against multiple Xanthomonas species. In addition, we provide evidence for additive to dominant genetic action for the QTL introgressions.

Marker-assisted development and characterization of near-isogenic lines carrying the Rx4 gene for hypersensitive resistance to Xanthomonas euvesicatoria pv. perforans race T3 in tomato

Bacterial spot caused by several species of Xanthomonas is a devastating disease resulting in severe fruit yield loss and quality decline in tomato production areas worldwide. Lack of resistant commercial varieties is among the most critical factors limiting the application of integrated management to control the disease. Although sources for resistance to all species and races have been discovered over years, breeding for resistance to the disease is slow because the resistance is quantitatively inherited and sometimes linked to poor horticultural traits. Development of near-isogenic lines (NILs) by introgressing the shortest DNA fragment of the resistance gene region from the donor into elite cultivars is a promising approach to eliminating the negative effect on horticultural traits. In the current study, a strategy of combining traditional backcross with molecular marker-assisted foreground and background selection was adopted to generate NILs carrying the Rx4 gene for resistance to Xanthomonas euvesicatoria pv. perforans race T3 strain. The donor parent is a wild species Solanum pimpinellifolium accession PI 128216, and the recurrent parent is an elite processing tomato breeding line OH88119. Disease evaluation was performed in F1, BC4F1, BC6F1, BC6F2, BC6F3, and BC6F4 generations by infiltration of race T3 strain Xv829 to leaves of plants. Meanwhile, fruit traits including fruit weight, fruit color parameters, soluble solid content, pH, and firmness were collected from replicated field trials in BC6F4 and BC6F5. Five NILs with 159–1901 kb DNA fragments in the Rx4 gene region were obtained. They all showed high level of hypersensitive resistance to race T3, and their fruit traits were not significantly different from the recurrent parent OH88119. However, fruit weight of one NIL Rx4–1812 carrying the largest DNA fragment of 1901 kb in the Rx4 gene region was 9.5–11.8% lower than OH8819 though the difference was not statistically significant. This might be due to the linkage drag of FW11.2, a QTL for fruit weight in the same region. Overall, the NILs created here can be used as sources for developing resistant varieties and deciphering the mechanism of resistance to bacterial spot in tomato.