ABSTRACT
Plant pathogenic bacteria often have a narrow host range, which can vary among different isolates within a population. Here, we investigated the host range of the tomato pathogen Clavibacter michiganensis (Cm). We determined the genome sequences of 40 tomato Cm isolates and screened them for pathogenicity on tomato and eggplant. Our screen revealed that out of the tested isolates, five were unable to cause disease on any of the hosts, 33 were exclusively pathogenic on tomato, and two were capable of infecting both tomato and eggplant. Through comparative genomic analyses, we identified that the five non-pathogenic isolates lacked the chp/tomA pathogenicity island, which has previously been associated with virulence in tomato. In addition, we found that the two eggplant-pathogenic isolates encode a unique allelic variant of the putative serine hydrolase chpG (chpGC), an effector that is recognized in eggplant. Introduction of chpGC into a chpG inactivation mutant in the eggplant-non-pathogenic strain Cm101, failed to complement the mutant, which retained its ability to cause disease in eggplant and failed to elicit hypersensitive response (HR). Conversely, introduction of the chpG variant from Cm101 into an eggplant pathogenic Cm isolate (C48), eliminated its pathogenicity on eggplant, and enabled C48 to elicit HR. Our study demonstrates that allelic variation in the chpG effector gene is a key determinant of host range plasticity within Cm populations.
Subject(s)
Alleles , Clavibacter , Host Specificity , Plant Diseases , Solanum lycopersicum , Plant Diseases/microbiology , Solanum lycopersicum/microbiology , Clavibacter/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Solanum melongena/microbiology , Solanum melongena/genetics , Virulence/genetics , Genetic VariationABSTRACT
BACKGROUND: Xanthomonas translucens pv. graminis (Xtg) is a major bacterial pathogen of economically important forage grasses, causing severe yield losses. So far, genomic resources for this pathovar consisted mostly of draft genome sequences, and only one complete genome sequence was available, preventing comprehensive comparative genomic analyses. Such comparative analyses are essential in understanding the mechanisms involved in the virulence of pathogens and to identify virulence factors involved in pathogenicity. RESULTS: In this study, we produced high-quality, complete genome sequences of four strains of Xtg, complementing the recently obtained complete genome sequence of the Xtg pathotype strain. These genomic resources allowed for a comprehensive comparative analysis, which revealed a high genomic plasticity with many chromosomal rearrangements, although the strains were highly related. A high number of transposases were exclusively found in Xtg and corresponded to 413 to 457 insertion/excision transposable elements per strain. These mobile genetic elements are likely to be involved in the observed genomic plasticity and may play an important role in the adaptation of Xtg. The pathovar was found to lack a type IV secretion system, and it possessed the smallest set of type III effectors in the species. However, three XopE and XopX family effectors were found, while in the other pathovars of the species two or less were present. Additional genes that were specific to the pathovar were identified, including a unique set of minor pilins of the type IV pilus, 17 TonB-dependent receptors (TBDRs), and 11 plant cell wall degradative enzymes. CONCLUSION: These results suggest a high adaptability of Xtg, conferred by the abundance of mobile genetic elements, which could play a crucial role in pathogen adaptation. The large amount of such elements in Xtg compared to other pathovars of the species could, at least partially, explain its high virulence and broad host range. Conserved features that were specific to Xtg were identified, and further investigation will help to determine genes that are essential to pathogenicity and host adaptation of Xtg.
Subject(s)
Genome, Bacterial , Xanthomonas , Genomics/methods , Xanthomonas/genetics , Poaceae/genetics , Plant Diseases/microbiology , PhylogenyABSTRACT
Xanthomonas translucens pv. translucens (Xtt) is a global barley patho-gen and a concern for resistance breeding and regulation. Long-read whole genome sequences allow in-depth understanding of pathogen diversity. We have completed long-read PacBio sequencing of two Minnesotan Xtt strains and an in-depth analysis of available Xtt genomes. We found that average nucleotide identity (ANI)-based approaches organize Xtt strains different from the previous standard multilocus sequencing analysis approach. According to ANI, Xtt forms a separate clade from X. translucens pv. undulosa and consists of three main groups which are represented on multiple continents. Some virulence factors, such as 17 Type III-secreted effectors, are highly conserved and offer potential targets for the elicitation of broad resistance. However, there is a high degree of variation in virulence factors, meaning that germplasm should be screened for resistance with a diverse panel of Xtt.
Subject(s)
Hordeum , Xanthomonas , Virulence Factors/genetics , Plant Diseases , Plant Breeding , Genomics , PhylogenyABSTRACT
Bacterial leaf streak (BLS) of wheat (Triticum aestivum), caused by Xanthomonas translucens pv. undulosa, is a disease of major concern in the Northern Great Plains. The host range for X. translucens pv. undulosa is relatively broad, including several small grains and perennial grasses. In Minnesota, X. translucens pv. undulosa was isolated from weedy grasses in and around wheat fields that exhibited BLS symptoms and from cultivated wild rice (Zizania palustris) with symptomatic leaf tissue. Currently, no genomic resources are available for X. translucens pv. undulosa strains isolated from non-wheat hosts. In this study, we sequenced and assembled the complete genomes of five strains isolated from weedy grass hosts, foxtail barley (Hordeum jubatum), green foxtail (Setaria viridis), and wild oat (Avena fatua), and from cultivated wild rice and wheat. These five genomes were compared with the publicly available genomes of seven X. translucens pv. undulosa strains originating from wheat and one genome of an X. translucens pv. secalis strain originating from rye (Secale cereale). Global alignments of the genomes revealed little variation in genomic structures. Average nucleotide identity-based phylogeny and life identification numbers revealed that the strains share ≥99.25% identity. We noted differences in the presence of Type III secreted effectors, including transcription activator-like effectors. Despite differences between strains, we did not identify unique features distinguishing strains isolated from wheat and non-wheat hosts. This study contributes to the availability of genomic data for X. translucens pv. undulosa from non-wheat hosts, thus increasing our understanding of the diversity within the pathogen population.
Subject(s)
Hordeum , Oryza , Xanthomonas , Poaceae , Plant Diseases/microbiology , Genomics , Hordeum/microbiology , Triticum/microbiologyABSTRACT
Bacterial leaf streak (BLS) is a disease of monocot plants caused by Xanthomonas translucens on small grains, X. vasicola on maize and sorghum, and X. oryzae on rice. These three pathogens cause remarkably similar symptomology in their host plants. Despite causing similar symptoms, BLS pathogens are dispersed throughout the larger Xanthomonas phylogeny. Each aforementioned species includes strain groups that do not cause BLS and instead cause vascular disease. In this commentary, we hypothesize that strains of X. translucens, X. vasicola, and X. oryzae convergently evolved to cause BLS due to shared evolutionary pressures. We examined the diversity of secreted effectors, which may be important virulence factors for BLS pathogens and their evolution. We discuss evidence that differences in gene regulation and abilities to manipulate plant hormones may also separate BLS pathogens from other Xanthomonas species or pathovars. BLS is becoming an increasing issue across the three pathosystems. Overall, we hope that a better understanding of conserved mechanisms used by BLS pathogens will enable researchers to translate findings across production systems and guide approaches to control this (re)emerging threat.
Subject(s)
Oryza , Xanthomonas , Plant Diseases/microbiology , Xanthomonas/genetics , Virulence Factors , Oryza/microbiology , PhylogenyABSTRACT
Evolutionarily, early-branching xanthomonads, also referred to as clade-1 xanthomonads, include major plant pathogens, most of which colonize monocotyledonous plants. Seven species have been validly described, among them the two sugarcane pathogens Xanthomonas albilineans and Xanthomonas sacchari, as well as Xanthomonas translucens, which infects small-grain cereals and diverse grasses but also asparagus and pistachio trees. Single-gene sequencing and genomic approaches have indicated that this clade likely contains more, yet-undescribed species. In this study, we sequenced representative strains of three novel species using long-read sequencing technology. Xanthomonas campestris pv. phormiicola strain CFBP 8444 causes bacterial streak on New Zealand flax, another monocotyledonous plant. Xanthomonas sp. strain CFBP 8443 has been isolated from common bean, and Xanthomonas sp. strain CFBP 8445 originated from banana. Complete assemblies of the chromosomes confirmed their unique phylogenetic position within clade 1 of Xanthomonas. Genome mining revealed novel genetic features, hitherto undescribed in other members of the Xanthomonas genus. In strain CFBP 8444, we identified genes related to the synthesis of coronatine-like compounds, a phytotoxin produced by several pseudomonads, which raises interesting questions about the evolution and pathogenicity of this pathogen. Furthermore, strain CFBP 8444 was found to contain a second, atypical flagellar gene cluster in addition to the canonical flagellar gene cluster. Overall, this research represents an important step toward better understanding the evolutionary history and biology of early-branching xanthomonads.
Subject(s)
Flagellin , Xanthomonas , Flagellin/genetics , Phylogeny , Plant Diseases/microbiology , Whole Genome SequencingABSTRACT
Bacterial leaf streak, bacterial blight, and black chaff caused by Xanthomonas translucens pathovars are major diseases affecting small grains. Xanthomonas translucens pv. translucens and X. translucens pv. undulosa are seedborne pathogens that cause similar symptoms on barley, but only X. translucens pv. undulosa causes bacterial leaf streak of wheat. Recent outbreaks of X. translucens have been a concern for wheat and barley growers in the Northern Great Plains; however, there are limited diagnostic tools for pathovar differentiation. We developed a multiplex PCR based on whole-genome differences to distinguish X. translucens pv. translucens and X. translucens pv. undulosa. We validated the primers across different Xanthomonas and non-Xanthomonas strains. To our knowledge, this is the first multiplex PCR to distinguish X. translucens pv. translucens and X. translucens pv. undulosa. These molecular tools will support disease management strategies enabling detection and pathovar incidence analysis of X. translucens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Hordeum , Xanthomonas , Edible Grain , Plant Diseases/microbiology , Hordeum/microbiology , Xanthomonas/genetics , Triticum/microbiologyABSTRACT
Strains of Xanthomonas citri pv. malvacearum cause bacterial blight of cotton, a potentially serious threat to cotton production worldwide, including in sub-Saharan countries. Development of disease symptoms, such as water soaking, has been linked to the activity of a class of type 3 effectors, called transcription activator-like (TAL) effectors, which induce susceptibility genes in the host's cells. To gain further insight into the global diversity of the pathogen, to elucidate their repertoires of TAL effector genes, and to better understand the evolution of these genes in the cotton-pathogenic xanthomonads, we sequenced the genomes of three African strains of X. citri pv. malvacearum using nanopore technology. We show that the cotton-pathogenic pathovar of X. citri is a monophyletic lineage containing at least three distinct genetic subclades, which appear to be mirrored by their repertoires of TAL effectors. We observed an atypical level of TAL effector gene pseudogenization, which might be related to resistance genes that are deployed to control the disease. Our work thus contributes to a better understanding of the conservation and importance of TAL effectors in the interaction with the host plant, which can inform strategies for improving resistance against bacterial blight in cotton.
ABSTRACT
Bacterial leaf streak (BLS) of barley is caused by the Gram-negative bacterial pathogen Xanthomonas translucens (Sapkota et al. 2020). In 2021, we observed multiple hill plots with BLS symptomatic plants in a barley stripe rust nursery in Vancouver, BC, Canada. We collected 29 leaf samples showing typical BLS symptoms (e.g. necrotic lesions; Fig. S1) and stored at 4 oC until bacterial isolation. Samples were surface-sterilized in 10% NaOCl for 20 sec and rinsed twice. About 1 cm2 of leaf tissue containing BLS characteristic lesions was macerated in 200 µL sterile H2O on a petri dish, incubated for 15 min, and 10 µl of the homogenates was streaked onto Wilbrink's - Boric Acid - Cephalexin (WBC) agar medium. Plates were incubated at 28-30 oC for 48 hrs. Four single colonies were obtained: BC10-1-2a (USask BC10-2a), BC10-1-2b (USask BC10-2b), UBC026 and UBC028. Colonies were grown in WBC broth and gDNA was extracted using E.Z.N.A. Bacterial DNA Kit (Omega Bio-Tek) or DNeasy Plant Pro Kit® (Qiagen) following manufacturer protocols. Genus-level identification was achieved using 16S rRNA sequencing with 27F/1492R primers (Lane 1991) of UBC026 (1,399 bp; NCBI # OP327375) and UBC028 (1,415 bp; NCBI #OP327376). Complete 16S rRNA sequences (1,533bp) of BC10-2a and BC10-2b (1,533 bp) were extracted from the draft whole-genome sequences (WGS) generated in this study. The 16S rRNA sequence homology values of 99.0-100% were recorded between the 4 strains. BLAST analyses of the 16S rRNA sequences to GenBank entries exhibited 99.5-100% similarity values (100% coverage) with the pathotype strains of Xtt DSM 18974T (LT604072) and X. translucens pv. undulosa (Xtu) CFBP 2055 (CP074361). Whole genomes of BC10-2a (JANUQY01) and BC10-2b (JANUQZ01) were sequenced (150-bp; reads 33.1 million; mean coverage 2125x) using NovaSeq Illumina, assembled (Unicycler v0.4.8; Wick et al. 2017) and analyzed to identify the strains to the species-level (Tambong et al. 2021). WGS of strains USask BC10-2a and USask BC10-2b exhibited genome-based DNA-DNA hybridization (dDDH; Meier-Kolthoff et al. 2013) and BLAST-based average nucleotide identity (ANIb; Richter et al. 2015) of 100%. The two strains also showed dDDH and ANIb of 90.4% (species-leel cut-off of 70%) and 98.780% and 98.80% (cut-off of 96%), respectively, with Xtt DSM 18974T (LT604072). In contrast, the WGS of BC10-2a and BC10-2b exhibited only 78.2% dDDH homology values with Xtu CFBP 2055T, suggesting that the strains are genetically more similar to Xtt. The assignment of these strains to Xtt is corroborated by phylogenomic analysis (Fig. S2; Meier-Kolthoff and Göker 2019) that showed the two strains clustering together (100% bootstrap) with the type strain DSM 18974T. These data suggest that these strains are taxonomically members of Xtt. Identification was also confirmed to the genus-level by LAMP assay using published X. translucens primers (Langlois et al. 2017). Pathovar-level identification was confirmed using a cbsA and S8.pep multiplex PCR diagnostic assay (Roman-Reyna et al. 2022). Koch's postulates were verified by greenhouse inoculation via leaf infiltration of UBC026 and UBC028 on 21-day old barley plants (line HB522) using an inoculum of 108 CFU ml-1 followed by re-isolation of the bacteria on WBC. The inoculated plants showed typical BLS symptoms similar to those observed in the field (Fig. S1). Water-inoculated plants had no symptoms. To our knowledge, this is the first published report of BLS of barley in British Columbia.
ABSTRACT
Winterberries (Ilex verticillata and hybrids) are deciduous species of holly whose branches bearing colorful fruit are cut in late Fall to be used for seasonal decorations. The annual wholesale value of the woody cuts is $1.5 million nationally (NASS, 2019). In June 2021, approximately 80% of the 45 Ilex verticillata 'Maryland Beauty' potted plants, which were maintained in a container yard at The Ohio State University research farm in Columbus, OH, presented leaves with irregular necrotic lesions surrounded by a chlorotic halo. No other symptoms were present on the plants. Bacterial streaming was observed from the lesions using a compound microscope and isolations were performed after surface disinfesting small sections of leaf tissue from the border of the lesions by soaking in 10% bleach for 30 sec, rinsing twice in sterile water, macerating in sterile water, and streaking the suspension on nutrient broth yeast extract agar. Creamy white, circular, smooth, and convex colonies were recovered after incubation at 28°C for 48 h. Bacterial identification of one representative isolate was initially pursued from single colonies of a purified culture using five discriminative phenotypic tests (i.e., LOPAT: "L", levan production; "O", oxidase activity; "P", pectinolytic activity; "A", arginine dehydrolase production; "T", tobacco hypersensitive reaction), which resulted in the L+ O- P- A- T+ profile consistent with the description of Pseudomonas syringae (Lelliott et al. 1996). Molecular identification was performed based on rpoD marker amplification and sequencing using primers PsrpoD FNP1/PsrpoDnprpcr1 (Parkison et al. 2011). NCBI GenBank BLASTn comparison of the rpoD sequence (GenBank Acc. No. OP221440) shared 99.12% identity to P. syringae pv. passiflorae (AB163366.1). Whole genome sequence analysis was conducted to strengthen the classification of the isolate species. To this extent, DNA was sequenced with an iSeq 100 Illumina benchtop sequencer using Illumina DNA Prep kit and iSeq 100 i1 Reagent v2 (Illumina, Inc, REF: 20060060 and 20031371). Illumina Local Run Manager software was used for base calling, demultiplexing, and trimming of the raw reads. Unicycler v0.5.0 was used for de novo assembly of the genome (Wick et al. 2017). The assembled genome size was 5.9 Mb with 959 contigs and 10× coverage (NCBI GenBank Biosample No. SAMN30281368; Acc. No. JANQCB010000000). Average nucleotide identity (ANI) analysis was performed on the server MiGA online (Rodriguez-R et al. 2018). Subgroup identification was inconclusive (p>0.05), positioning this isolate between P. syringae pv. actinidiae (96.45% ANI) and pv. viburni (96.65% ANI) (Rodriguez-R & Konstantinidis, 2016). Both these pathovars cause leaf spots on woody plants such as kiwi and viburnum (Donati et al. 2020; Garibaldi et al. 2005). To confirm pathogenicity, three separate branches on each of two I. verticillata 'Maryland Beauty' potted plants were selected, and 5-7 individual young leaves (>2 weeks from emergence) on each branch were infiltrated with a bacterial suspension (108 CFU/mL) in sterile water (SW) using a needleless syringe by delivering 30-50 µL of suspension per infiltration point. One additional branch per plant was infiltrated with SW to serve as control. Plants were covered with a plastic bag for two days post-inoculation (DPI) and maintained in the laboratory at an average of 23°C. All inoculated leaves showed necrotic lesions two DPI while control leaves remained asymptomatic. To fulfill Koch's postulates, the bacterium was re-isolated from the symptomatic leaves six DPI and confirmed to be identical to the original isolate based on rpoD gene sequencing. To the best of our knowledge, this report signifies the first instance of P. syringae causing bacterial leaf spot on winterberry worldwide. Ornamental plant sales are based primarily on visual appeal; therefore, identification and monitoring of emerging pathogens is essential to ensure the health of the industry.
ABSTRACT
Ralstonia solanacearum phylotype II sequevar 1 (RsII-1, formerly race 3 biovar 2) causes tomato bacterial wilt, potato brown rot, and Southern wilt of geranium. Strains in RsII-1 cause wilting in potato and tomato at cooler temperatures than tropical lowland R. solanacearum strains. Although periodically introduced, RsII-1 has not established in the United States. This pathogen is of quarantine concern and listed as a Federal Select Agent. We report a rapidly sequenced (<2 days) draft genome of UW848, a RsII-1 isolate introduced to the United States in geranium cuttings in spring 2020. UW848 belongs to the near-clonal cluster of RsII-1 global pandemic strains.
Subject(s)
Geranium , Ralstonia solanacearum , Solanum lycopersicum , Solanum tuberosum , Geranium/genetics , Plant Diseases , Ralstonia solanacearum/genetics , United StatesABSTRACT
Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani Kühn, continues to be an important and challenging rice disease worldwide. Here, we used genome-wide association studies over a high-density rice array to facilitate the identification of potential novel genes and quantitative trait loci related to sheath blight resistance. We identified multiple regions that significantly associated with independent disease components in chromosomes 1, 4, and 11 under controlled condition. In particular, we investigated qLN1128, a quantitative trait locus enriched with defense-related genes that reduce disease lesions in a near-isogenic line. RNA profiling of the line carrying qLN1128 showed a number of differentially expressed genes related to the reactive oxygen species (ROS)-redox pathway. Histochemical staining revealed less ROS accumulation on the resistant line, suggesting efficient ROS deregulation that delays pathogen colonization. The detection of genomic regions controlling multiple mechanisms of resistance to sheath blight will provide tools to design effective breeding interventions in rice.
Subject(s)
Disease Resistance , Genome-Wide Association Study , Oryza , Reactive Oxygen Species , Rhizoctonia , Disease Resistance/genetics , Gene Expression Profiling , Oryza/genetics , Oryza/microbiology , Reactive Oxygen Species/metabolism , Rhizoctonia/physiologyABSTRACT
In July 2018, a sample of lavender var. Grosso (Lavandula × intermedia 'Grosso') from Miami County, OH was received by The Ohio State University Vegetable Pathology Laboratory in Wooster. Lavender plants were field-grown in sandy clay soil with plastic mulch under drip irrigation. Disease incidence ranged from 0 to 32% depending on variety. Leaves and stems showed dark necrotic lesions that varied from roughly circular (ca. 0.3 to 0.5 mm diameter) to large coalesced necrotic areas surrounded by a water-soaked halo. Bacterial streaming from lesions was observed microscopically. Leaf tissue pieces (~0.5 cm2) were surface sterilized in 70% ethanol for 30 seconds and rinsed in sterile deionized water. The tissue was sliced aseptically into smaller sections in 100 µl sterile water and the bacterial suspension was streaked on yeast dextrose calcium carbonate agar medium. Ten yellow Xanthomonas-like colonies were selected after 72 hours of incubation at 28ºC in the dark. Strains were gram negative, oxidase negative and caused hypersensitive reactions on Nicotiana benthamiana (L.). All strains were genotyped after whole-cell DNA extraction by BOX-PCR (Louws et al. 1999) and had the same banding profile. Four 8-wk-old lavender plants (Lavandula dentata and Lavandula × ginginsii 'Goodwin Creek Gray') were spray-inoculated with a 106 CFU/ml suspension of strain SM175-2018 in sterile water. Control plants were sprayed with sterile water. Plants were kept in plastic bags for the first 48 h at 28°C with a 14-h photoperiod. Water-soaked necrotic lesions appeared 14 days after inoculation with SM175-2018, whereas mock-inoculated plants did not show symptoms. Bacterial isolation from symptomatic leaf tissue was carried out as described above. The BOX-PCR profile of the re-isolated strain was identical to that of SM175-2018. Multilocus sequence analysis of the housekeeping genes fuyA, gyrB, and rpoD was performed (Accession numbers: MT764834 - MT764836). The resulting concatenated data set was used to perform a phylogenetic analysis using maximum likelihood criteria to evaluate relationships with closely related Xanthomonas spp. using published reference sequences (Young et al. 2008). SM175-2018 was assigned to the X. hortorum clade (Moriniere et al. 2020) with strong bootstrap support. The strain was subjected to whole genome analysis. Genomic DNA was extracted using a QIAGEN Genomic DNA buffer set with genomic-tip 100/G following manufacturer's protocol and sequenced using the iSeq-100 Illumina platform with the Nextera DNA Flex Library Prep protocol kit and Nextera DNA CD indexes. Average nucleotide identity (ANI) analysis was performed with the ANI-Matrix software Enveomics tool (Rodriguez-R and Konstantinidis 2016) using the sequenced genome (NCBI GenBank Biosample no. SAMN11831455) and those of other X. hortorum (Vauterin et al. 1995) bacteria (pvs. hederae, carotae, vitians). SM175-2018 shared a 96% ANI with other X. hortorum strains. X. hortorum is associated with bacterial leaf spot of carrot (Scott and Dung, 2020) and also reported on ornamental plants (Mirik et al. 2010, Oliver et al. 2012, Roberts and Parkinson 2014, Klass et al. 2019), however additional research is needed to establish the host specificity of lavender strains. To our knowledge this is the first report of X. hortorum causing bacterial leaf spot of lavender in Ohio. The disease may negatively impact the yield and quality of flowers used in production of lavender oils and essences.
ABSTRACT
Xanthomonas translucens pv. translucens causes bacterial leaf streak and bacterial blight diseases of barley. This pathogen limits barley production globally but remains understudied, with limited genomic resources. To better understand the biology of this X. translucens subgroup, we sequenced the complete genome of the X. translucens pv. translucens strain UPB886.
Subject(s)
Genome, Bacterial , Xanthomonas , Genome, Bacterial/genetics , Genomics , Hordeum/microbiology , Plant Diseases/microbiology , Xanthomonas/geneticsABSTRACT
Chemistry in eukaryotic intercellular spaces is shaped by both hosts and symbiotic microorganisms such as bacteria. Pathogenic microorganisms like barley-associated Xanthomonas translucens (Xt) swiftly overtake the inner leaf tissue becoming the dominant microbial community member during disease development. The dynamic metabolic changes due to Xt pathogenesis in the mesophyll spaces remain unknown. Genomic group I of Xt consists of two barley-infecting lineages: pathovar translucens (Xtt) and pathovar undulosa (Xtu). Xtu and Xtt, although genomically distinct, cause similar water-soaked lesions. To define the metabolic signals associated with inner leaf colonization, we used untargeted metabolomics to characterize Xtu and Xtt metabolism signatures associated with mesophyll growth. We found that mesophyll apoplast fluid from infected tissue yielded a distinct metabolic profile and shift from catabolic to anabolic processes over time compared to water-infiltrated control. The pathways with the most differentially expressed metabolites by time were glycolysis, tricarboxylic acid cycle, sucrose metabolism, pentose interconversion, amino acids, galactose, and purine metabolism. Hierarchical clustering and principal component analysis showed that metabolic changes were more affected by the time point rather than the individual colonization of the inner leaves by Xtt compared to Xtu. Overall, in this study, we identified metabolic pathways that explain carbon and nitrogen usage during host-bacterial interactions over time for mesophyll tissue colonization. This foundational research provides initial insights into shared metabolic strategies of inner leaf colonization niche occupation by related but phylogenetically distinct phyllosphere bacteria. IMPORTANCE: The phyllosphere is a habitat for microorganisms including pathogenic bacteria. Metabolic shifts in the inner leaf spaces for most plant-microbe interactions are unknown, especially for Xanthomonas species in understudied plants like barley (Hordeum vulgare). Xanthomonas translucens pv. translucens (Xtt) and Xanthomonas translucens pv. undulosa (Xtu) are phylogenomically distinct, but both colonize barley leaves for pathogenesis. In this study, we used untargeted metabolomics to shed light on Xtu and Xtt metabolic signatures. Our findings revealed a dynamic metabolic landscape that changes over time, rather than exhibiting a pattern associated with individual pathovars. These results provide initial insights into the metabolic mechanisms of X. translucens inner leaf pathogenesis.
Subject(s)
Hordeum , Xanthomonas , Hordeum/microbiology , Xanthomonas/genetics , Plant Leaves , WaterABSTRACT
Quickly understanding the genomic changes that lead to pathogen emergence is necessary to launch mitigation efforts and reduce harm. In this study, we tracked in real time a 2022 bacterial plant disease outbreak in U.S. geraniums (Pelargonium × hortorum) caused by Xhp2022, a novel lineage of Xanthomonas hortorum. Genomes from 31 Xhp2022 isolates from seven states showed limited chromosomal variation and all contained a single plasmid (p93). Time tree and single nucleotide polymorphism whole-genome analysis estimated that Xhp2022 emerged within the last decade. The phylogenomic analysis determined that p93 resulted from the cointegration of three plasmids (p31, p45, and p66) found sporadically across isolates from previous outbreaks. Although p93 had a 49 kb nucleotide reduction, it retained putative fitness genes, which became predominant in the 2022 outbreak. Overall, we demonstrated, through rapid whole-genome sequencing and analysis, a recent, traceable event of genome reduction for niche adaptation typically observed over millennia in obligate and fastidious pathogens.IMPORTANCEThe geranium industry, valued at $4 million annually, faces an ongoing Xanthomonas hortorum pv. pelargonii (Xhp) pathogen outbreak. To track and describe the outbreak, we compared the genome structure across historical and globally distributed isolates. Our research revealed Xhp population has not had chromosome rearrangements since 1974 and has three distinct plasmids. In 2012, we found all three plasmids in individual Xhp isolates. However, in 2022, the three plasmids co-integrated into one plasmid named p93. p93 retained putative fitness genes but lost extraneous genomic material. Our findings show that the 2022 strain group of the bacterial plant pathogen Xanthomonas hortorum underwent a plasmid reduction. We also observed several Xanthomonas species from different years, hosts, and continents have similar plasmids to p93, possibly due to shared agricultural settings. We noticed parallels between genome efficiency and reduction that we see across millennia with obligate parasites with increased niche specificity.
Subject(s)
Xanthomonas , Plasmids/genetics , Xanthomonas/genetics , Genomics , Disease OutbreaksABSTRACT
Changes in Xanthomonas race and species composition causing bacterial spot of tomato have occurred throughout the world and are often associated with epidemics. Knowledge of bacterial population structure is key for resistance discovery and deployment. We surveyed Xanthomonas spp. composition from processing tomato fields in the Midwestern United States over a 4-year period between 2017 and 2020, compared these to strains collected previously, and found that X. perforans is currently the most prevalent species. We characterized 564 X. perforans isolates for sequence variation in avrXv3 to distinguish between race T3 and T4 and validated race designation using hypersensitive response (HR) assays for 106 isolates. Race T4 accounted for over 95% of X. perforans isolates collected in the Midwest between 2017 and 2020. Whole genome sequencing, Average Nucleotide Identity (ANI) analysis, core genome alignment and single nucleotide polymorphism (SNP) detection relative to a reference strain, and phylogenomic analysis suggest that the majority of Midwestern X. perforans strains collected between 2017 and 2020 were nearly identical, with greater than 99.99% ANI to X. perforans isolates collected from Collier County, Florida in 2012. These isolates shared a common SNP variant resulting an a premature stop codon in avrXv3. One sequenced isolate was identified with a deletion of avrXv3 and shared 99.99% ANI with a strain collected in Collier Co., Florida in 2006. A population shift to X. perforans T4 occurred in the absence of widely deployed resistance, with only 7% of tomato varieties tested having the resistant allele at the Xv3/Rx-4 locus. The persistence of nearly identical strains over multiple years suggests that migration led to the establishment of an endemic population. Our findings validate a genomics-based framework to track shifts in X. perforans populations due to migration, mutation, drift, or selection based on comparisons to 146 genomes.
ABSTRACT
The Xanthomonas translucens species comprises phytopathogenic bacteria that can cause serious damage to cereals and to forage grasses. So far, the genomic resources for X. translucens were limited, which hindered further understanding of the host-pathogen interactions at the molecular level and the development of disease-resistant cultivars. To this end, we complemented the available complete genome sequence of the X. translucens pv. translucens pathotype strain DSM 18974 by sequencing the genomes of all the other 10 X. translucens pathotype strains using PacBio long-read technology and assembled complete genome sequences. Phylogeny based on average nucleotide identity (ANI) revealed three distinct clades within the species, which we propose to classify as clades Xt-I, Xt-II, and Xt-III. In addition to 2,181 core X. translucens genes, a total of 190, 588, and 168 genes were found to be exclusive to each clade, respectively. Moreover, 29 non-transcription activator-like effector (TALE) and 21 TALE type III effector classes were found, and clade- or strain-specific effectors were identified. Further investigation of these genes could help to identify genes that are critically involved in pathogenicity and/or host adaptation, setting the grounds for the development of new resistant cultivars.
ABSTRACT
Xylella fastidiosa (Xf) is a globally distributed plant-pathogenic bacterium. The primary control strategy for Xf diseases is eradicating infected plants; therefore, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Conventional Xf diagnostics primarily relies on quantitative PCR (qPCR) assays. However, these methods do not consider new or emerging variants due to pathogen genetic recombination and sensitivity limitations. We developed and tested a metagenomics pipeline using in-house short-read sequencing as a complementary approach for affordable, fast, and highly accurate Xf detection. We used metagenomics to identify Xf to the strain level in single- and mixed-infected plant samples at concentrations as low as 1 pg of bacterial DNA per gram of tissue. We also tested naturally infected samples from various plant species originating from Europe and the United States. We identified Xf subspecies in samples previously considered inconclusive with real-time PCR (quantification cycle [Cq], >35). Overall, we showed the versatility of the pipeline by using different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional information for Xf diagnostics without extensive knowledge of the disease. This pipeline demonstrates that metagenomics can be used for early detection of Xf and incorporated as a tool to inform disease management strategies. IMPORTANCE Destructive Xylella fastidiosa (Xf) outbreaks in Europe highlight this pathogen's capacity to expand its host range and geographical distribution. The current disease diagnostic approaches are limited by a multiple-step process, biases to known sequences, and detection limits. We developed a low-cost, user-friendly metagenomic sequencing tool for Xf detection. In less than 3 days, we were able to identify Xf subspecies and strains in field-collected samples. Overall, our pipeline is a diagnostics tool that could be easily extended to other plant-pathogen interactions and implemented for emerging plant threat surveillance.