Characterization of Emerging Xanthomonas campestris Isolates on the Nonnative Weed Garlic Mustard (Alliaria petiolata).

Nonnative plant infestations provide unique opportunities to investigate pathogen emergence with evolutionarily recent plant introduction events. The widespread distribution of invasive plants and their proximity to genetically related crops highlights the risks of nonnative plants acting as ancillary hosts and fostering microbial recombination and pathogen selection. Garlic mustard (Alliaria petiolata) is a widespread, nonnative cruciferous weed that grows throughout North America and along the forested edges of diverse agricultural fields. The recent identification of a novel Xanthomonas campestris pv. incanae strain isolated from a diseased A. petiolata population led to the current investigation of the distribution and diversity of X. campestris isolates from naturally infected A. petiolata. A total of 14 diseased A. petiolata sites were sampled across three states, leading to the identification of diverse X. campestris pathotypes and genotypes. Pathogenicity assays and multilocus sequence analyses identified pathogenic X. c. pv. incanae and X. c. pv. barbareae strains collected from disparate A. petiolata populations. Moreover, independently collected X. c. pv. incanae strains demonstrated a broad cruciferous host range by infecting cabbage (Brassica oleracea var. capitata), garden stock (Matthiola incana), and the cover crop yellow mustard (Guillenia flavescens). This study highlights the genetic variability and host potential of natural X. campestris populations and the potential risks to Brassica crops via widespread, dense garlic mustard reservoirs.

Cruciferous Weed Isolates of Xanthomonas campestris Yield Insight into Pathovar Genomic Relationships and Genetic Determinants of Host and Tissue Specificity.

Pathovars of Xanthomonas campestris cause distinct diseases on different brassicaceous hosts. The genomic relationships among pathovars as well as the genetic determinants of host range and tissue specificity remain poorly understood despite decades of research. Here, leveraging advances in multiplexed long-read technology, we fully sequenced the genomes of a collection of X. campestris strains isolated from cruciferous crops and weeds in New York and California as well as strains from global collections, to investigate pathovar relationships and candidate genes for host- and tissue-specificity. Pathogenicity assays and genomic comparisons across this collection and publicly available X. campestris genomes revealed a correlation between pathovar and genomic relatedness and provide support for X. campestris pv. barbareae, the validity of which had been questioned. Linking strain host range with type III effector repertoires identified AvrAC (also 'XopAC') as a candidate host-range determinant, preventing infection of Matthiola incana, and this was confirmed experimentally. Furthermore, the presence of a copy of the cellobiosidase gene cbsA with coding sequence for a signal peptide was found to correlate with the ability to infect vascular tissues, in agreement with a previous study of diverse Xanthomonas species; however, heterologous expression in strains lacking the gene gave mixed results, indicating that factors in addition to cbsA influence tissue specificity of X. campestris pathovars. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Cruciferous Weeds Do Not Act as Major Reservoirs of Inoculum for Black Rot Outbreaks in New York State.

Cruciferous weeds have been shown to harbor diverse Xanthomonas campestris pathovars, including the agronomically damaging black rot of cabbage pathogen, X. campestris pv. campestris. However, the importance of weeds as inoculum sources for X. campestris pv. campestris outbreaks in New York remains unknown. To determine if cruciferous weeds act as primary reservoirs for X. campestris pv. campestris, fields that were rotating between cabbage or had severe black rot outbreaks were chosen for evaluation. Over a consecutive 3-year period, 148 cruciferous and noncruciferous weed samples were collected at 34 unique sites located across five New York counties. Of the 148 weed samples analyzed, 48 X. campestris isolates were identified, with a subset characterized using multilocus sequence analysis. All X. campestris isolates originated from weeds belonging to the Brassicaceae family, with predominant weed hosts being shepherd's purse (Capsella bursa-pastoris), wild mustard (Sinapis arvensis), yellow rocket (Barbarea vulgaris), and pennycress (Thlaspi arvense). Identifying pathogenic X. campestris weed isolates was rare, with only eight isolates causing brown necrotic leaf spots or typical V-shaped lesions on cabbage. There was no evidence of cabbage-infecting weed isolates persisting in an infected field by overwintering in weed hosts; however, similar cabbage and weed X. campestris haplotypes were identified in the same field during an active black rot outbreak. X. campestris weed isolates are genetically diverse both within and between fields, but our findings indicate that X. campestris weed isolates do not appear to act as primary sources of inoculum for B. oleracea fields in New York.

Comparative Secretome Analyses of Toxigenic and Atoxigenic Rathayibacter Species.

Phytopathogenic Rathayibacter species are unique bacterial plant pathogens because they are obligately vectored by plant parasitic anguinid nematodes to the developing seedheads of forage grasses and cereals. This understudied group of plant-associated Actinomycetes includes the neurotoxigenic plant pathogen R. toxicus, which causes annual ryegrass toxicity in grazing livestock. R. toxicus is currently endemic to Australia and is listed as a plant pathogen select agent by the U.S. Department of Agriculture-Animal and Plant Health Inspection Service. The complex Rathayibacter disease cycle requires intimate interactions with the nematode vector and plant hosts, which warrants an increased understanding of the secretory and surface-associated proteins that mediate these diverse eukaryotic interactions. Here we present the first comparative secretome analysis for this complex, nematode-vectored Rathayibacter genus that compares the three agronomically damaging toxigenic and atoxigenic Rathayibacter species, R. toxicus, R. iranicus, and R. tritici. The exoproteomic comparison identified 1,423 unique proteins between the three species via liquid chromatography-tandem mass spectrometry, leading to the identification of putative pathogenicity-related proteins and proteins that may mediate nematode attachment. Of the uniquely identified proteins, 94 homologous proteins were conserved between the three Rathayibacter exoproteomes and comprised between 43.4 and 58.6% of total protein abundance. Comparative analyses revealed both conserved and uniquely expressed extracellular proteins, which, interestingly, had more similarities to extracellular proteins commonly associated with bacterial animal pathogens than classic plant pathogens. This comparative exoproteome analysis will facilitate the characterization of proteins essential for vector attachment and host colonization and assist in the development of serological diagnostic assays.

Bacterial Canker of Tomato: Revisiting a Global and Economically Damaging Seedborne Pathogen.

The gram-positive actinobacterium Clavibacter michiganensis is the causal agent of bacterial canker of tomato, an economically impactful disease with a worldwide distribution. This seedborne pathogen systemically colonizes tomato xylem leading to unilateral leaflet wilt, marginal leaf necrosis, stem and petiole cankers, and plant death. Additionally, splash dispersal of the bacterium onto fruit exteriors causes bird's-eye lesions, which are characterized as necrotic centers surrounded by white halos. The pathogen can colonize developing seeds systemically through xylem and through penetration of fruit tissues from the exterior. There are currently no commercially available resistant cultivars, and bactericidal sprays have limited efficacy for managing the disease once the pathogen is in the vascular system. In this review, we summarize research on epidemiology, host colonization, the bacterial genetics underlying virulence, and management of bacterial canker. Finally, we highlight important areas of research into this pathosystem that have the potential to generate new strategies for prevention and mitigation of bacterial canker.

Colletotrichum fioriniae infecting invasive Japanese hop (Humulus scandens) in the United States.

Japanese hop (Humulus scandens) is a non-native, invasive plant that colonizes disturbed riparian areas throughout the eastern United States and Canada, forming dense, monocultural stands that displace native plant communities due to a high reproductive rate, rapid growth, climbing bines, and dense shading (Balogh and Dancza 2008). It is capable of serving as a reservoir for agronomically important plant pathogens, such as the Tomato spotted wilt virus and powdery mildew species that infect commercial hemp and hop fields (Yoon et al. 2018; Weldon et al. 2020). In the spring of 2016, diseased populations of H. scandens were observed along the Monocacy River in Frederick County, Maryland with severe chlorotic and necrotic leaf lesions. Symptomatic leaves were surface sterilized and placed in moist chambers at 25°C for sporulation. Sporulating acervuli, lacking setae, developed on irregular, tan necrotic leaf lesions following 7 to 12 days in a moist chamber (Figure 1). Conidia were hyaline, aseptate, smooth-walled, fusiform to cylindrical with both ends acute (Figure 1B). Conidia measured (n = 100) [L x W; Average (+ Std. Err), range]: 12.42 µm (± 0.10), 8.41 - 14.48 µm; x 3.91 µm (±0.03), 3.03 - 4.91 µm. Monoconidial fungal cultures were obtained by transferring conidia with a sterile glass needle to acidified potato dextrose agar and incubated at 25°C for 2 to 3 days. Based on phenotypic characteristics and conidial morphology and size, the pathogen appeared to belong to the Colletotrichum acutatum complex (Damm et al. 2012). Therefore, six loci (ITS, GADPH, CHS1, HIS3, ACT, and TUB2) were amplified and sequenced from a representative isolate, 16-008, for species characterization (GenBank accessions MW023070 to MW023075) (Damm et al. 2012). For the ITS region and ACT, GADPH, and CHS1 loci, isolate 16-008 was 100% identical to C. fioriniae and shared 99% similarity to TUB2 and HIS3 for multiple accessions of C. fioriniae in GenBank. Gene sequences were aligned, trimmed, concatenated, and analyzed against 32 reference strains, within the C. acutatum complex (Damm et al. 2012). Concatenated loci were used to generate a maximum likelihood phylogeny using W-IQ-TREE (Trifinopoulos et al. 2016). Results from the phylogenetic analysis demonstrated that isolate 16-008 was most genetically similar to C. fioriniae with a bootstrap support of 100% (Figure 2). Based on phenotypic and sequence analyses, isolate 16-008 was identified as C. fioriniae. Humulus scandens seedlings from Maryland (n = 3) were inoculated with a conidia suspension (107 conidia mL-1) with 0.125% Tween 20® and applied with an atomizer until runoff. Inoculated plants were placed in a dew chamber at 25°C for 2 days. Experimental plants were distributed in a mist tent at 25°C with 14 h of light and monitored for 2 weeks. Negative control plants (n = 2) were sprayed with a sterile 0.125% Tween 20® water solution. All inoculated plants were symptomatic by 12 days post inoculation. No symptoms were observed on the mock-inoculated plants. Symptoms were identical to disease field samples. Inoculations were repeated with the same results. Colletotrichum fioriniae was reisolated and confirmed from excised leaf lesions via ITS and ACT sequencing. To our knowledge, this is the first report of C. fioriniae naturally infecting H. scandens within the United States (Farr and Rossman 2020). Future studies will evaluate the host range of this isolate due to the species broad host range and the weed's extensive distribution.

Comparative Evaluation of LAMP, qPCR, Conventional PCR, and ELISA to Detect Ralstonia solanacearum in Kenyan Potato Fields.

Bacterial wilt caused by Ralstonia solanacearum is considered among the most damaging diseases of potato in Sub-Saharan Africa and the most significant biotic constraint of potato production alongside late blight. Unlike late blight, which can be managed by chemical means, R. solanacearum can only be managed through cultural methods and clean seed. Laboratory testing to certify seed before planting is required to confirm the absence of the pathogen in Kenya. A loop-mediated isothermal amplification (LAMP) assay was developed using the UDP-(3-O-acyl)-N-acetylglucosamine deacetylase gene (IpxC) to screen seed potato for R. solanacearum strains. The assay was assessed using DNA extracted from R. solanacearum and other soil and potato pathogens to demonstrate specificity and sensitivity. The LAMP assay was validated using field samples from different potato growing regions of Kenya collected over two growing seasons and compared with established nucleic acid and protein-based assays. The IpxC LAMP assay was found to be specific and sensitive to R. solanacearum, detecting as low as 2.5 pg/µl of R. solanacearum DNA. Of the 47 potentially infected field samples collected, both IpxC LAMP and quantitative polymerase chain reaction (PCR) detected R. solanacearum DNA in 90% of the samples, followed by conventional PCR (86%) and ELISA (75%). This IpxC LAMP assay is a promising diagnostic tool to rapidly screen for R. solanacearum in seed potato with high sensitivity in Kenya. Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Partial Proteome of the Corynetoxin-Producing Gram-Positive Bacterium, Rathayibacter toxicus.

Rathayibacter toxicus is a Gram-positive bacterium that is the causative agent of annual ryegrass toxicity (ARGT), a disease that causes devastating losses in the Australian livestock industry. R. toxicus exhibits a complex life cycle, using the nematode Anguina funesta as a physical vector to carry it up to the seed head of the host plant. ARGT is caused by a tunicamycin-like corynetoxin that is produced in R. toxicus-infected seed galls. We analyzed protein expression in R. toxicus under stationary growth phase conditions to obtain a more complete understanding of the biology of this organism and identify potential targets for immunoassay development. A total of 323 unique proteins were identified, including those with putative roles in secondary metabolism and pathogenicity. The proteome analysis for this complex phytopathogenic Gram-positive bacterium will facilitate in the characterization of proteins necessary for host colonization and toxin production, and assist in the development of diagnostic assays. Data are available via ProteomeXchange with identifier PXD004238.

Characterizing the Genetic Diversity of the Clavibacter michiganensis subsp. michiganensis Population in New York.

New York Clavibacter michiganensis subsp. michiganensis isolates, collected from disparate bacterial canker of tomato outbreaks over the past 11 years, were characterized with a multilocus sequence analysis (MLSA) scheme that differentiated the 51 isolates into 21 haplotypes with a discriminatory power of 0.944. The MLSA scheme consisted of five housekeeping genes (kdpA, sdhA, dnaA, ligA, and gyrB) and three putative pathogenicity genes (celA, tomA, and nagA). Repetitive polymerase chain reaction (PCR), with the BOX-A1R primer, confirmed the high diversity of C. michiganensis subsp. michiganensis isolates in New York by demonstrating that all six PCR patterns (A, B, 13C, 65C, 81C, and D) were present, with PCR patterns C and A being the most common. The MLSA scheme provided higher resolving power than the current repetitive-PCR approach. The plasmid profiles of New York isolates were diverse and differed from reference strain NCPPB382. PCR analysis indicated that the presence of putative pathogenicity genes varied between isolates and highlighted the ephemeral nature of pathogenicity genes in field populations of C. michiganensis subsp. michiganensis. Analysis of molecular variance between Serbian and New York C. michiganensis subsp. michiganensis isolates demonstrated that the two populations were not significantly different, with 98% genetic variation within each population and only 2% genetic variation between populations.

Tomato fruit and seed colonization by Clavibacter michiganensis subsp. michiganensis through external and internal routes.

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial wilt and canker of tomato, is an economically devastating pathogen that inflicts considerable damage throughout all major tomato-producing regions. Annual outbreaks continue to occur in New York, where C. michiganensis subsp. michiganensis spreads via infected transplants, trellising stakes, tools, and/or soil. Globally, new outbreaks can be accompanied by the introduction of contaminated seed stock; however, the route of seed infection, especially the role of fruit lesions, remains undefined. In order to investigate the modes of seed infection, New York C. michiganensis subsp. michiganensis field strains were stably transformed with a gene encoding enhanced green fluorescent protein (eGFP). A constitutively eGFP-expressing virulent C. michiganensis subsp. michiganensis isolate, GCMM-22, was used to demonstrate that C. michiganensis subsp. michiganensis could not only access seeds systemically through the xylem but also externally through tomato fruit lesions, which harbored high intra- and intercellular populations. Active movement and expansion of bacteria into the fruit mesocarp and nearby xylem vessels followed, once the fruits began to ripen. These results highlight the ability of C. michiganensis subsp. michiganensis to invade tomato fruits and seeds through multiple entry routes.

Genome Sequence of Xanthomonas campestris Strain FDWSRU 18048, an Emerging Pathogen of Nonnative, Invasive Garlic Mustard (Alliaria petiolata).

Xanthomonas campestris infections of nonnative, invasive garlic mustard populations have been recently reported in the eastern United States. Here, we report the genome sequence of the pathogenic X. campestris strain FDWSRU 18048. The genome is 4,978,509 bp and closely related to the genome of X. campestris pv. incanae strain CFBP2527.

Uromyces rebeccae, sp. nov., a newly described rust on the federally endangered plant, California sea-blite (Suaeda californica).

Rust disease was observed on populations of Suaeda californica near Morro Bay, California. The pathogen was identified as a species of Uromyces based on teliospore and urediniospore morphology and nuc 28S rDNA sequence analysis. The isolate was compared with previously described species of Uromyces that infect members of Chenopodiaceae, prompting a taxonomic reevaluation of Uromyces species on Suaeda. Herein, Uromyces rebeccae is described. It can be differentiated from the closely related U. chenopodii (syn.: Aecidium chenopodii-fruticosi; U. giganteus) based on host range, teliospore morphology, and 28S sequence data. The new combination, Uromyces chenopodii-fruticosi, is made for Aecidium chenopodii-fruticosi, the oldest name for Eurasian Suaeda rust. Finally, it was determined that U. giganteus likely does not occur in the United States and that the rust of S. taxifolia in the United States likely comprises a third, yet unnamed taxon, different from both U. rebeccae and U. chenopodii-fruticosi. This is the first record of a rust fungus on S. californica. An identification key for Uromyces species reported on Chenopodiaceae is provided.

The Identification and Conservation of Tunicaminyluracil-Related Biosynthetic Gene Clusters in Several Rathayibacter Species Collected From Australia, Africa, Eurasia, and North America.

Tunicaminyluracil antibiotics are a novel class of toxigenic glycolipids that are synthesized by several soil-associated Actinomycetes. The acquisition of a tunicaminyluracil biosynthetic gene cluster (TGC) in Rathayibacter toxicus has led to the emergence of the only described, naturally occurring tunicaminyluracil-associated mammalian disease, annual ryegrass toxicity of livestock. Endemic to Australia, R. toxicus is obligately vectored by Anguinid seed gall nematodes to the developing seedheads of forage grasses, in which the bacteria synthesize tunicaminyluracils that may subsequently be consumed by livestock and result in high rates of mortality and morbidity. The potential impact of R. toxicus on U.S. agriculture has led the U.S. Department of Agriculture - Animal and Plant Health Inspection Service to list R. toxicus as a Plant Pathogen Select Agent. R. toxicus is the only characterized phytopathogenic bacterium to produce tunicaminyluracils, but numerous R. toxicus-like livestock poisonings outside Australia suggest additional bacterial sources of tunicaminyluracils may exist. To investigate the conservation of the TGC in R. toxicus and whether the TGC is present in other Rathayibacter species, we analyzed genome sequences of members of the Rathayibacter genus. Putative TGCs were identified in genome sequences of R. toxicus, R. iranicus, R. agropyri, and an undescribed South African Rathayibacter species. In the latter three species, the putative TGCs have homologs of tunicaminyluracil-related genes essential for toxin production, but the TGCs differ in gene number and order. The TGCs appear at least partially functional because in contrast to atoxigenic species, TGC-containing Rathayibacter species were each able to tolerate exogenous applications of tunicamycin from Streptomyces chartreusis. The North American R. agropyri TGC shows extensive diversity among the sequenced isolates, with presense/absense polymorphisms in multiple genes or even the whole TGC. R. agropyri TGC structure does not appear to correlate with date or location of isolate collection. The conservation and identification of tunicaminyluracil-related gene clusters in three additional Rathayibacter species isolated from South Africa, the Middle East, and the United States, suggests a wider global distribution of potentially neurotoxigenic plant-associated bacteria. This potential for additional endemic and exotic toxigenic Rathayibacter species could have widespread and severe implications for agriculture.

Plant-like bacterial expansins play contrasting roles in two tomato vascular pathogens.

Expansin proteins, which loosen plant cell walls, play critical roles in normal plant growth and development. The horizontal acquisition of functional plant-like expansin genes in numerous xylem-colonizing phytopathogenic bacteria suggests that bacterial expansins may also contribute to virulence. To investigate the role of bacterial expansins in plant diseases, we mutated the non-chimeric expansin genes (CmEXLX2 and RsEXLX) of two xylem-inhabiting bacterial pathogens, the Actinobacterium Clavibacter michiganensis ssp. michiganensis (Cmm) and the ß-proteobacterium Ralstonia solanacearum (Rs), respectively. The Cmm ΔCmEXLX2 mutant caused increased symptom development on tomato, which was characterized by more rapid wilting, greater vascular necrosis and abundant atypical lesions on distant petioles. This increased disease severity correlated with larger in planta populations of the ΔCmEXLX2 mutant, even though the strains grew as well as the wild-type in vitro. Similarly, when inoculated onto tomato fruit, ΔCmEXLX2 caused significantly larger lesions with larger necrotic centres. In contrast, the Rs ΔRsEXLX mutant showed reduced virulence on tomato following root inoculation, but not following direct petiole inoculation, suggesting that the RsEXLX expansin contributes to early virulence at the root infection stage. Consistent with this finding, ΔRsEXLX attached to tomato seedling roots better than the wild-type Rs, which may prevent mutants from invading the plant's vasculature. These contrasting results demonstrate the diverse roles of non-chimeric bacterial expansins and highlight their importance in plant-bacterial interactions.

Whole genome sequence of two Rathayibacter toxicus strains reveals a tunicamycin biosynthetic cluster similar to Streptomyces chartreusis.

Rathayibacter toxicus is a forage grass associated Gram-positive bacterium of major concern to food safety and agriculture. This species is listed by USDA-APHIS as a plant pathogen select agent because it produces a tunicamycin-like toxin that is lethal to livestock and may be vectored by nematode species native to the U.S. The complete genomes of two strains of R. toxicus, including the type strain FH-79, were sequenced and analyzed in comparison with all available, complete R. toxicus genomes. Genome sizes ranged from 2,343,780 to 2,394,755 nucleotides, with 2079 to 2137 predicted open reading frames; all four strains showed remarkable synteny over nearly the entire genome, with only a small transposed region. A cluster of genes with similarity to the tunicamycin biosynthetic cluster from Streptomyces chartreusis was identified. The tunicamycin gene cluster (TGC) in R. toxicus contained 14 genes in two transcriptional units, with all of the functional elements for tunicamycin biosynthesis present. The TGC had a significantly lower GC content (52%) than the rest of the genome (61.5%), suggesting that the TGC may have originated from a horizontal transfer event. Further analysis indicated numerous remnants of other potential horizontal transfer events are present in the genome. In addition to the TGC, genes potentially associated with carotenoid and exopolysaccharide production, bacteriocins and secondary metabolites were identified. A CRISPR array is evident. There were relatively few plant-associated cell-wall hydrolyzing enzymes, but there were numerous secreted serine proteases that share sequence homology to the pathogenicity-associated protein Pat-1 of Clavibacter michiganensis. Overall, the genome provides clear insight into the possible mechanisms for toxin production in R. toxicus, providing a basis for future genetic approaches.

Microbe-ID: an open source toolbox for microbial genotyping and species identification.

Development of tools to identify species, genotypes, or novel strains of invasive organisms is critical for monitoring emergence and implementing rapid response measures. Molecular markers, although critical to identifying species or genotypes, require bioinformatic tools for analysis. However, user-friendly analytical tools for fast identification are not readily available. To address this need, we created a web-based set of applications called Microbe-ID that allow for customizing a toolbox for rapid species identification and strain genotyping using any genetic markers of choice. Two components of Microbe-ID, named Sequence-ID and Genotype-ID, implement species and genotype identification, respectively. Sequence-ID allows identification of species by using BLAST to query sequences for any locus of interest against a custom reference sequence database. Genotype-ID allows placement of an unknown multilocus marker in either a minimum spanning network or dendrogram with bootstrap support from a user-created reference database. Microbe-ID can be used for identification of any organism based on nucleotide sequences or any molecular marker type and several examples are provided. We created a public website for demonstration purposes called Microbe-ID (microbe-id.org) and provided a working implementation for the genus Phytophthora (phytophthora-id.org). In Phytophthora-ID, the Sequence-ID application allows identification based on ITS or cox spacer sequences. Genotype-ID groups individuals into clonal lineages based on simple sequence repeat (SSR) markers for the two invasive plant pathogen species P. infestans and P. ramorum. All code is open source and available on github and CRAN. Instructions for installation and use are provided at https://github.com/grunwaldlab/Microbe-ID.