A Survey of Xylella fastidiosa in the U.S. State of Virginia Reveals Wide Distribution of Both Subspecies fastidiosa and multiplex in Grapevine.

Global travel and trade in combination with climate change are expanding the geographic distribution of plant pathogens. The bacterium Xylella fastidiosa is a prime example. Native to the Americas, it has spread to Europe, Asia, and the Middle East. To assess the risk that pathogen introductions pose to crops in newly invaded areas, it is key to survey their diversity, host range, and disease incidence in relation to climatic conditions where they are already present. We performed a survey of X. fastidiosa in grapevine in Virginia using a combination of quantitative PCR, multilocus sequencing, and metagenomics. We also analyzed samples from deciduous trees with leaf scorch symptoms. X. fastidiosa subspecies fastidiosa was identified in grapevines in all regions of the state, even in Northern Virginia, where the temperature was below -9°C for 10 days per year on average in the years preceding sampling. Unexpectedly, we also found for the first time grapevine samples infected with X. fastidiosa subspecies multiplex (Xfm). The Xfm lineage found in grapevines had been previously isolated from blueberries in the Southeastern United States and was distinct from that found in deciduous trees in Virginia. The obtained results will be important for risk assessment of X. fastidiosa introductions in other parts of the world.

Genotypic and Phenotypic Analyses Show Ralstonia solanacearum Cool Virulence is a Quantitative Trait Not Restricted to "Race 3 biovar 2".

Most Ralstonia solanacearum species complex strains cause bacterial wilts in tropical or subtropical zones, but the group known as Race 3 biovar 2 (R3bv2) is cool virulent and causes potato brown rot at lower temperatures. R3bv2 has invaded potato-growing regions around the world but is not established in the United States. Phylogenetically, R3bv2 corresponds to a subset of the R. solanacearum phylotype IIB clade, but little is known about the distribution of the cool virulence phenotype within phylotype IIB. Therefore, genomes of 76 potentially cool virulent phylotype IIB strains and 30 public genomes were phylogenetically analyzed. A single clonal lineage within the sequevar 1 subclade of phylotype IIB that originated in South America has caused nearly all brown rot outbreaks worldwide. To correlate genotypes with relevant phenotypes, we quantified virulence of ten Ralstonia strains on tomato and potato at both 22°C and 28°C. Cool virulence on tomato did not predict cool virulence on potato. We found that cool virulence is a quantitative trait. Strains in the sequevar 1 pandemic clonal lineage caused the most disease, while other R3bv2 strains were only moderately cool virulent. However, some non-R3bv2 strains were highly cool virulent and aggressively colonized potato tubers. Thus, cool virulence is not consistently correlated with strains historically classified as R3bv2 group. To aid detection of sequevar 1 strains, this group was genomically delimited in the LINbase web server and a sequevar 1 diagnostic primer pair was developed and validated. We discuss implications of these results for the R3bv2 definition.

Open Access and Reproducibility in Plant Pathology Research: Guidelines and Best Practices.

The landscape of scientific publishing is experiencing a transformative shift toward open access, a paradigm that mandates the availability of research outputs such as data, code, materials, and publications. Open access provides increased reproducibility and allows for reuse of these resources. This article provides guidance for best publishing practices of scientific research, data, and associated resources, including code, in The American Phytopathological Society journals. Key areas such as diagnostic assays, experimental design, data sharing, and code deposition are explored in detail. This guidance aligns with that observed by other leading journals. We hope the information assembled in this paper will raise awareness of best practices and enable greater appraisal of the true effects of biological phenomena in plant pathology.

Reference-Free Plant Disease Detection Using Machine Learning and Long-Read Metagenomic Sequencing.

Surveillance for early disease detection is crucial to reduce the threat of plant diseases to food security. Metagenomic sequencing and taxonomic classification have recently been used to detect and identify plant pathogens. However, for an emerging pathogen, its genome may not be similar enough to any public genome to permit reference-based tools to identify infected samples. Also, in the case of point-of care diagnosis in the field, database access may be limited. Therefore, here we explore reference-free detection of plant pathogens using metagenomic sequencing and machine learning (ML). We used long-read metagenomes from healthy and infected plants as our model system and constructed k-mer frequency tables to test eight different ML models. The accuracy in classifying individual reads as coming from a healthy or infected metagenome were compared. Of all models, random forest (RF) had the best combination of short run-time and high accuracy (over 0.90) using tomato metagenomes. We further evaluated the RF model with a different tomato sample infected with the same pathogen or a different pathogen and a grapevine sample infected with a grapevine pathogen and achieved similar performances. ML models can thus learn features to successfully perform reference-free detection of plant diseases whereby a model trained with one pathogen-host system can also be used to detect different pathogens on different hosts. Potential and challenges of applying ML to metagenomics in plant disease detection are discussed. IMPORTANCE Climate change may lead to the emergence of novel plant diseases caused by yet unknown pathogens. Surveillance for emerging plant diseases is crucial to reduce their threat to food security. However, conventional genomic based methods require knowledge of existing plant pathogens and cannot be applied to detecting newly emerged pathogens. In this work, we explored reference-free, meta-genomic sequencing-based disease detection using machine learning. By sequencing the genomes of all microbial species extracted from an infected plant sample, we were able to train machine learning models to accurately classify individual sequencing reads as coming from a healthy or an infected plant sample. This method has the potential to be integrated into a generic pipeline for a meta-genomic based plant disease surveillance approach but also has limitations that still need to be overcome.

LINbase: a web server for genome-based identification of prokaryotes as members of crowdsourced taxa.

High throughput DNA sequencing in combination with efficient algorithms could provide the basis for a highly resolved, genome phylogeny-based and digital prokaryotic taxonomy. However, current taxonomic practice continues to rely on cumbersome journal publications for the description of new species, which still constitute the smallest taxonomic units. In response, we introduce LINbase, a web server that allows users to genomically circumscribe any group of prokaryotes with measurable DNA similarity and that uses the individual isolate as smallest unit. Since LINbase leverages the concept of Life Identification Numbers (LINs), which are codes assigned to individual genomes based on reciprocal average nucleotide identity, we refer to groups circumscribed in LINbase as LINgroups. Users can associate with each LINgroup a name, a short description, and a URL to a peer-reviewed publication. As soon as a LINgroup is circumscribed, any user can immediately identify query genomes as members and submit comments about the LINgroup. Most genomes currently in LINbase were imported from GenBank, but users can upload their own genome sequences as well. In conclusion, LINbase combines the resolution of LINs with the power of crowdsourcing in support of a highly resolved, genome phylogeny-based digital taxonomy. LINbase is available at http://www.LINbase.org.

Genome Resource: Draft Genome of Fusarium avenaceum, Strain F156N33, Isolated from the Atmosphere Above Virginia and Annotated Based on RNA Sequencing Data.

Fusarium avenaceum is a filamentous fungus commonly associated with plants and soil. It is a causal agent of Fusarium head blight (FHB) on maize and small-grain cereals and blights on other plant species, and is one of the very few fungal species known to have ice nucleation activity (i.e., it catalyzes ice formation). Here, we report the draft genome of the ice-nucleation-active F. avenaceum strain F156N33 isolated from the atmosphere above Virginia. The genome assembly is 41,175,306 bp long, consists of 214 contigs, and is predicted to encode 11,233 proteins, which were annotated using RNA-sequencing data obtained from the same strain.

Population Genomic- and Phylogenomic-Enabled Advances to Increase Insight Into Pathogen Biology and Epidemiology.

Population genetics has been a key discipline in phytopathology for many years. The recent rise in cost-effective, high-throughput DNA sequencing technologies, allows sequencing of dozens, if not hundreds of specimens, turning population genetics into population genomics and opening up new, exciting opportunities as described in this Focus Issue. Without the limitations of genetic markers and the availability of whole or near whole-genome data, population genomics can give new insights into the biology, evolution and adaptation, and dissemination patterns of plant-associated microbes.

Genome Resource: Ralstonia solanacearum Phylotype II Sequevar 1 (Race 3 Biovar 2) Strain UW848 From the 2020 U.S. Geranium Introduction.

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.

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.

An AlgU-Regulated Antisense Transcript Encoded within the Pseudomonas syringae fleQ Gene Has a Positive Effect on Motility.

Production of bacterial flagella is controlled by a multitiered regulatory system that coordinates the expression of 40 to 50 subunits and ordered assembly of these elaborate structures. Flagellar expression is environmentally controlled, presumably to optimize the benefits and liabilities of having these organelles on cell growth and survival. We recently reported a global survey of AlgU-dependent regulation and binding in Pseudomonas syringae pv. tomato DC3000 that included evidence for strong downregulation of many flagellar and chemotaxis motility genes. Here, we returned to those data to look for other AlgU-dependent influences on the flagellar regulatory network. We identified an AlgU-dependent antisense transcript expressed from within the fleQ gene, the master regulator of flagellar biosynthesis in Pseudomonas We tested whether expression of this antisense RNA influenced bacterial behavior and found that it reduces AlgU-dependent downregulation of motility. Importantly, this antisense expression influenced motility only under conditions in which AlgU was expressed. Comparative sequence analysis of the locus containing the antisense transcript's AlgU-dependent promoter in over 300 Pseudomonas genomes revealed sequence conservation in most strains that encode AlgU. This suggests that the antisense transcript plays an important role that is conserved across most of the genus PseudomonasIMPORTANCEPseudomonas syringae is a globally distributed host-specific bacterial pathogen that causes disease in a wide-range of plants. An elaborate gene expression regulation network controls flagellum production, which is important for proper flagellum assembly and a key aspect of certain lifestyle transitions. P. syringae pv. tomato DC3000 uses flagellum-powered motility in the early stages of host colonization and adopts a sessile lifestyle after entering plant tissues, but the regulation of this transition is not understood. Our work demonstrates a link between regulation of motility and global transcriptional control that facilitates bacterial growth and disease in plants. Additionally, sequence comparisons suggest that this regulation mechanism is conserved in most members of the genus Pseudomonas.

Genetic Diversity and Virulence of Wheat and Barley Strains of Xanthomonas translucens from the Upper Midwestern United States.

Bacterial leaf streak (BLS) of wheat and barley, caused by Xanthomonas translucens pv. undulosa and X. translucens pv. translucens, has been of growing concern in small grains production in the Upper Midwestern United States. To optimize disease resistance breeding, a greater awareness is needed of the pathovars and genetic diversity within the pathogens causing BLS in the region. Multilocus sequencing typing (MLST) and analysis (MLSA) of four common housekeeping genes (rpoD, dnaK, fyuA, and gyrB) was used to evaluate the genetic diversity of 82 strains of X. translucens isolated between 2006 and 2013 from wheat, barley, rye, and intermediate wheatgrass. In addition, in planta disease assays were conducted on 75 strains to measure relative virulence in wheat and barley. All strains were determined by MLSA to be related to X. translucens pv. undulosa and X. translucens pv. translucens. Clustering of strains based on Bayesian, network, and minimum spanning trees correlated with relative virulence levels in inoculated wheat and barley. Thus, phylogeny based on rpoD, dnaK, fyuA, and gyrB correlated with host of isolation and was an effective means for predicting virulence of strains belonging to X. translucens pv. translucens and X. translucens pv. undulosa.

A proposal for a portal to make earth's microbial diversity easily accessible and searchable.

Estimates of the number of bacterial species range from 107 to 1012. At the pace at which descriptions of new species are currently being published, the description of all bacterial species on earth will only be completed in thousands of years. However, even if one day all species were named and described, these names and descriptions would still be of little practical value unless they could be easily searched and accessed, so that novel strains could be easily identified as members of any of these species. To complicate the situation further, many of the currently known species contain significant genotypic and phenotypic diversity that would still be missed if description of microbial diversity were limited to species. The solution to this problem could be a database in which every bacterial species and every intra-specific group is anchored to a genome-similarity framework. This ideal database should be searchable using complete or partial genome sequences as well as phenotypes. Moreover, the database should include functions to easily add newly sequenced novel strains, automatically place them into the genome-similarity framework, identify them as members of an already named species, or tag them as members of yet to be described species or new intra-specific groups. Here, we propose the means to develop such a database by taking advantage of the concept of genome sequence similarity-based codes, called Life Identification Numbers or LINs.

A Proposal for a Genome Similarity-Based Taxonomy for Plant-Pathogenic Bacteria that Is Sufficiently Precise to Reflect Phylogeny, Host Range, and Outbreak Affiliation Applied to Pseudomonas syringae sensu lato as a Proof of Concept.

Taxonomy of plant pathogenic bacteria is challenging because pathogens of different crops often belong to the same named species but current taxonomy does not provide names for bacteria below the subspecies level. The introduction of the host range-based pathovar system in the 1980s provided a temporary solution to this problem but has many limitations. The affordability of genome sequencing now provides the opportunity for developing a new genome-based taxonomic framework. We already proposed to name individual bacterial isolates based on pairwise genome similarity. Here, we expand on this idea and propose to use genome similarity-based codes, which we now call life identification numbers (LINs), to describe and name bacterial taxa. Using 93 genomes of Pseudomonas syringae sensu lato, LINs were compared with a P. syringae genome tree whereby the assigned LINs were found to be informative of a majority of phylogenetic relationships. LINs also reflected host range and outbreak association for strains of P. syringae pathovar actinidiae, a pathovar for which many genome sequences are available. We conclude that LINs could provide the basis for a new taxonomic framework to address the shortcomings of the current pathovar system and to complement the current taxonomic system of bacteria in general.

Genome-Enabled Phylogeographic Investigation of the Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 and Screening for Sources of Resistance Against Its Core Effectors.

Phylogeographic studies inform about routes of pathogen dissemination and are instrumental for improving import/export controls. Genomes of 17 isolates of the bacterial wilt and potato brown rot pathogen Ralstonia solanacearum race 3 biovar 2 (R3bv2), a Select Agent in the United States, were thus analyzed to get insight into the phylogeography of this pathogen. Thirteen of fourteen isolates from Europe, Africa, and Asia were found to belong to a single clonal lineage while isolates from South America were genetically diverse and tended to carry ancestral alleles at the analyzed genomic loci consistent with a South American origin of R3bv2. The R3bv2 isolates share a core repertoire of 31 type III-secreted effector genes representing excellent candidates to be targeted with resistance genes in breeding programs to develop durable disease resistance. Toward this goal, 27 R3bv2 effectors were tested in eggplant, tomato, pepper, tobacco, and lettuce for induction of a hypersensitive-like response indicative of recognition by cognate resistance receptors. Fifteen effectors, eight of them core effectors, triggered a response in one or more plant species. These genotypes may harbor resistance genes that could be identified and mapped, cloned, and expressed in tomato or potato, for which sources of genetic resistance to R3bv2 are extremely limited.

LINgroups as a Robust Principled Approach to Compare and Integrate Multiple Bacterial Taxonomies.

As a central organizing principle of biology, bacteria and archaea are classified into a hierarchical structure across taxonomic ranks from kingdom to subspecies. Traditionally, this organization was based on observable characteristics of form and chemistry but recently, bacterial taxonomy has been robustly quantified using comparisons of sequenced genomes, as exemplified in the Genome Taxonomy Database (GTDB). Such genome-based taxonomies resolve genomes down to genera and species and are useful in many contexts yet lack the flexibility and resolution of a fine-grained approach. The Life Identification Number (LIN) approach is a common, quantitative framework to tie existing (and future) bacterial taxonomies together, increase the resolution of genome-based discrimination of taxa, and extend taxonomic identification below the species level in a principled way. Utilizing LINgroup as an organizational concept helps resolve some of the confusion and unforeseen negative effects resulting from nomenclature changes of microorganisms that are closely related by overall genomic similarity (often due to genome-based reclassification). Our experimental results demonstrate the value of LINs and LINgroups in mapping between taxonomies, translating between different nomenclatures, and integrating them into a single taxonomic framework. They also reveal the robustness of LIN assignment to hyper-parameter changes when considering within-species taxonomic groups.

Genetic and Functional Diversity Help Explain Pathogenic, Weakly Pathogenic, and Commensal Lifestyles in the Genus Xanthomonas.

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

The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity.

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.

Nonagricultural reservoirs contribute to emergence and evolution of Pseudomonas syringae crop pathogens.

While the existence of environmental reservoirs of human pathogens is well established, less is known about the role of nonagricultural environments in emergence, evolution, and spread of crop pathogens. Here, we analyzed phylogeny, virulence genes, host range, and aggressiveness of Pseudomonas syringae strains closely related to the tomato pathogen P. syringae pv. tomato (Pto), including strains isolated from snowpack and streams. The population of Pto relatives in nonagricultural environments was estimated to be large and its diversity to be higher than that of the population of Pto and its relatives on crops. Ancestors of environmental strains, Pto, and other genetically monomorphic crop pathogens were inferred to have frequently recombined, suggesting an epidemic population structure for P. syringae. Some environmental strains have repertoires of type III-secreted effectors very similar to Pto, are almost as aggressive on tomato as Pto, but have a wider host range than typical Pto strains. We conclude that crop pathogens may have evolved through a small number of evolutionary events from a population of less aggressive ancestors with a wider host range present in nonagricultural environments.

Allelic variation in two distinct Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses but not bacterial motility.

The bacterial flagellin (FliC) epitopes flg22 and flgII-28 are microbe-associated molecular patterns (MAMPs). Although flg22 is recognized by many plant species via the pattern recognition receptor FLS2, neither the flgII-28 receptor nor the extent of flgII-28 recognition by different plant families is known. Here, we tested the significance of flgII-28 as a MAMP and the importance of allelic diversity in flg22 and flgII-28 in plant-pathogen interactions using purified peptides and a Pseudomonas syringae ∆fliC mutant complemented with different fliC alleles. The plant genotype and allelic diversity in flg22 and flgII-28 were found to significantly affect the plant immune response, but not bacterial motility. The recognition of flgII-28 is restricted to a number of solanaceous species. Although the flgII-28 peptide does not trigger any immune response in Arabidopsis, mutations in both flg22 and flgII-28 have FLS2-dependent effects on virulence. However, the expression of a tomato allele of FLS2 does not confer to Nicotiana benthamiana the ability to detect flgII-28, and tomato plants silenced for FLS2 are not altered in flgII-28 recognition. Therefore, MAMP diversification is an effective pathogen virulence strategy, and flgII-28 appears to be perceived by an as yet unidentified receptor in the Solanaceae, although it has an FLS2-dependent virulence effect in Arabidopsis.