The Complete Genome Sequence of Xanthomonas theicola, the Causal Agent of Canker on Tea Plants, Reveals Novel Secretion Systems in Clade-1 Xanthomonads.

Xanthomonas theicola is the causal agent of bacterial canker on tea plants. There is no complete genome sequence available for X. theicola, a close relative of the species X. translucens and X. hyacinthi, thus limiting basic research for this group of pathogens. Here, we release a high-quality complete genome sequence for the X. theicola type strain, CFBP 4691T. Single-molecule real-time sequencing with a mean coverage of 264× revealed two contigs of 4,744,641 bp (chromosome) and 40,955 bp (plasmid) in size. Genome mining revealed the presence of nonribosomal peptide synthases, two CRISPR systems, the Xps type 2 secretion system, and the Hrp type 3 secretion system. Surprisingly, this strain encodes an additional type 2 secretion system and a novel type 3 secretion system with enigmatic function, hitherto undescribed for xanthomonads. Four type 3 effector genes were found on complete or partial transposons, suggesting a role of transposons in effector gene evolution and spread. This genome sequence fills an important gap to better understand the biology and evolution of the early-branching xanthomonads, also known as clade-1 xanthomonads.

Current Understanding of the History, Global Spread, Ecology, Evolution, and Management of the Corn Bacterial Leaf Streak Pathogen, Xanthomonas vasicola pv. vasculorum.

Bacterial leaf streak of corn, caused by Xanthomonas vasicola pv. vasculorum, has been present in South Africa for over 70 years, but is an emerging disease of corn in North and South America. The only scientific information pertaining to this disease on corn came from work done in South Africa, which primarily investigated host range on other African crops, such as sugarcane and banana. As a result, when the disease was first reported in the United States in 2016, there was very limited information on where this pathogen came from, how it infects its host, what plant tissue(s) it is capable of infecting, where initial inoculum comes from at the beginning of each crop season, how the bacterium spreads from plant to plant and long distance, what meteorological variables and agronomic practices favor disease development and spread, how many other plant species X. vasicola pv. vasculorum is capable of infecting or using as alternate hosts, and if the bacterium will be able to persist in all corn growing regions of the United States. There were also no rapid diagnostic assays available which initially hindered prompt identification prior to the development of molecular diagnostic tools. The goal of this synthesis is to review the history of X. vasicola pv. vasculorum and bacterial leaf streak in South Africa and its movement to North and South America, and highlight the recent research that has been done in response to the emergence of this bacterial disease.

Genomic Acquisitions in Emerging Populations of Xanthomonas vasicola pv. vasculorum Infecting Corn in the United States and Argentina.

Xanthomonas vasicola pv. vasculorum is an emerging bacterial plant pathogen that causes bacterial leaf streak on corn. First described in South Africa in 1949, reports of this pathogen have greatly increased in the past years in South America and in the United States. The rapid spread of this disease in North and South America may be due to more favorable environmental conditions, susceptible hosts and/or genomic changes that favored the spread. To understand whether genetic mechanisms exist behind the recent spread of X. vasicola pv. vasculorum, we used comparative genomics to identify gene acquisitions in X. vasicola pv. vasculorum genomes from the United States and Argentina. We sequenced 41 genomes of X. vasicola pv. vasculorum and the related sorghum-infecting X. vasicola pv. holcicola and performed comparative analyses against all available X. vasicola genomes. Time-measured phylogenetic analyses showed that X. vasicola pv. vasculorum strains from the United States and Argentina are closely related and arose from two introductions to North and South America. Gene content comparisons identified clusters of genes enriched in corn X. vasicola pv. vasculorum that showed evidence of horizontal transfer including one cluster corresponding to a prophage found in all X. vasicola pv. vasculorum strains from the United States and Argentina as well as in X. vasicola pv. holcicola strains. In this work, we explore the genomes of an emerging phytopathogen population as a first step toward identifying genetic changes associated with the emergence. The acquisitions identified may contain virulence determinants or other factors associated with the spread of X. vasicola pv. vasculorum in North and South America and will be the subject of future work.

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

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

Genome Resource of Barley Bacterial Blight and Leaf Streak Pathogen Xanthomonas translucens pv. translucens strain UPB886.

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.

Rice phenylalanine ammonia-lyase gene OsPAL4 is associated with broad spectrum disease resistance.

Most agronomically important traits, including resistance against pathogens, are governed by quantitative trait loci (QTL). QTL-mediated resistance shows promise of being effective and long-lasting against diverse pathogens. Identification of genes controlling QTL-based disease resistance contributes to breeding for cultivars that exhibit high and stable resistance. Several defense response genes have been successfully used as good predictors and contributors to QTL-based resistance against several devastating rice diseases. In this study, we identified and characterized a rice (Oryza sativa) mutant line containing a 750 bp deletion in the second exon of OsPAL4, a member of the phenylalanine ammonia-lyase gene family. OsPAL4 clusters with three additional OsPAL genes that co-localize with QTL for bacterial blight and sheath blight disease resistance on rice chromosome 2. Self-pollination of heterozygous ospal4 mutant lines produced no homozygous progeny, suggesting that homozygosity for the mutation is lethal. The heterozygous ospal4 mutant line exhibited increased susceptibility to three distinct rice diseases, bacterial blight, sheath blight, and rice blast. Mutation of OsPAL4 increased expression of the OsPAL2 gene and decreased the expression of the unlinked OsPAL6 gene. OsPAL2 function is not redundant because the changes in expression did not compensate for loss of disease resistance. OsPAL6 co-localizes with a QTL for rice blast resistance, and is down-regulated in the ospal4 mutant line; this may explain enhanced susceptibility to Magnoporthe oryzae. Overall, these results suggest that OsPAL4 and possibly OsPAL6 are key contributors to resistance governed by QTL and are potential breeding targets for improved broad-spectrum disease resistance in rice.

Sensitive detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by loop-mediated isothermal amplification.

Molecular diagnostics for crop diseases can enhance food security by enabling the rapid identification of threatening pathogens and providing critical information for the deployment of disease management strategies. Loop-mediated isothermal amplification (LAMP) is a PCR-based tool that allows the rapid, highly specific amplification of target DNA sequences at a single temperature and is thus ideal for field-level diagnosis of plant diseases. We developed primers highly specific for two globally important rice pathogens, Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) disease, and X. oryzae pv. oryzicola, the causal agent of bacterial leaf streak disease (BLS), for use in reliable, sensitive LAMP assays. In addition to pathovar distinction, two assays that differentiate X. oryzae pv. oryzae by African or Asian lineage were developed. Using these LAMP primer sets, the presence of each pathogen was detected from DNA and bacterial cells, as well as leaf and seed samples. Thresholds of detection for all assays were consistently 10(4) to 10(5) CFU ml(-1), while genomic DNA thresholds were between 1 pg and 10 fg. Use of the unique sequences combined with the LAMP assay provides a sensitive, accurate, rapid, simple, and inexpensive protocol to detect both BB and BLS pathogens.

Development of a Genomics-Based LAMP (Loop-Mediated Isothermal Amplification) Assay for Detection of Pseudomonas fuscovaginae from Rice.

The vast amount of data available through next-generation sequencing technology is facilitating the design of diagnostic marker systems. This study reports the use of draft genome sequences from the bacterial plant pathogen Pseudomonas fuscovaginae, the cause of sheath brown rot of rice, to describe the genetic diversity within a worldwide collection of strains representing the species. Based on a comparative analysis with the draft sequences, primers for a loop-mediated isothermal amplification (LAMP) assay were developed to identify P. fuscovaginae. The assay reported here reliably differentiated strains of P. fuscovaginae isolated from rice from a range of other bacteria that are commonly isolated from rice and other plants using a primer combination designated Pf8. The LAMP assay identified P. fuscovaginae purified DNA, live or heat-killed cells from pure cultures, and detected the bacterium in extracts or exudates from infected host plant material. The P. fuscovaginae LAMP assay is a suitable diagnostic tool for the glasshouse and laboratory and could be further developed for in-field surveys.

Understanding the complexity of disease-climate interactions for rice bacterial panicle blight under tropical conditions.

Bacterial panicle blight (BPB) caused by Burkholderia glumae is one of the main concerns for rice production in the Americas since bacterial infection can interfere with the grain-filling process and under severe conditions can result in high sterility. B. glumae has been detected in several rice-growing areas of Colombia and other countries of Central and Andean regions in Latin America, although evidence of its involvement in decreasing yield under these conditions is lacking. Analysis of different parameters in trials established in three rice-growing areas showed that, despite BPB presence, severity did not explain the sterility observed in fields. PCR tests for B. glumae confirmed low infection in all sites and genotypes, only 21.4% of the analyzed samples were positive for B. glumae. Climate parameters showed that Montería and Saldaña registered maximum temperature above 34°C, minimum temperature above 23°C, and Relative Humidity above 80%, conditions that favor the invasion model described for this pathogen in Asia. Our study found that in Colombia, minimum temperature above 23°C during 10 days after flowering is the condition that correlates with disease incidence. Therefore, this correlation, and the fact that Montería and Saldaña had a higher level of infected samples according to PCR tests, high minimum temperature, but not maximum temperature, seems to be determinant for B. glumae colonization under studied field conditions. This knowledge is a solid base line to design strategies for disease control, and is also a key element for breeders to develop strategies aimed to decrease the effect of B. glumae and high night-temperature on rice yield under tropical conditions.

Genomics-Based Diagnostic Marker Development for Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola.

A computational genomics pipeline was used to compare sequenced genomes of Xanthomonas spp. and to rapidly identify unique regions for development of highly specific diagnostic markers. A suite of diagnostic primers was selected to monitor diverse loci and to distinguish the rice bacterial blight and bacterial leaf streak pathogens, Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola, respectively. A subset of these primers was combined into a multiplex polymerase chain reaction set that accurately distinguished the two rice pathogens in a survey of a geographically diverse collection of X. oryzae pv. oryzae, X. oryzae pv. oryzicola, other xanthomonads, and several genera of plant-pathogenic and plant- or seed-associated bacteria. This computational approach for identification of unique loci through whole-genome comparisons is a powerful tool that can be applied to other plant pathogens to expedite development of diagnostic primers.

Repeated gain and loss of a single gene modulates the evolution of vascular plant pathogen lifestyles.

Vascular plant pathogens travel long distances through host veins, leading to life-threatening, systemic infections. In contrast, nonvascular pathogens remain restricted to infection sites, triggering localized symptom development. The contrasting features of vascular and nonvascular diseases suggest distinct etiologies, but the basis for each remains unclear. Here, we show that the hydrolase CbsA acts as a phenotypic switch between vascular and nonvascular plant pathogenesis. cbsA was enriched in genomes of vascular phytopathogenic bacteria in the family Xanthomonadaceae and absent in most nonvascular species. CbsA expression allowed nonvascular Xanthomonas to cause vascular blight, while cbsA mutagenesis resulted in reduction of vascular or enhanced nonvascular symptom development. Phylogenetic hypothesis testing further revealed that cbsA was lost in multiple nonvascular lineages and more recently gained by some vascular subgroups, suggesting that vascular pathogenesis is ancestral. Our results overall demonstrate how the gain and loss of single loci can facilitate the evolution of complex ecological traits.

A Pathovar of Xanthomonas oryzae Infecting Wild Grasses Provides Insight Into the Evolution of Pathogenicity in Rice Agroecosystems.

Xanthomonas oryzae (Xo) are globally important rice pathogens. Virulent lineages from Africa and Asia and less virulent strains from the United States have been well characterized. Xanthomonas campestris pv. leersiae (Xcl), first described in 1957, causes bacterial streak on the perennial grass, Leersia hexandra, and is a close relative of Xo. L. hexandra, a member of the Poaceae, is highly similar to rice phylogenetically, is globally ubiquitous around rice paddies, and is a reservoir of pathogenic Xo. We used long read, single molecule real time (SMRT) genome sequences of five strains of Xcl from Burkina Faso, China, Mali, and Uganda to determine the genetic relatedness of this organism with Xo. Novel transcription activator-like effectors (TALEs) were discovered in all five strains of Xcl. Predicted TALE target sequences were identified in the Leersia perrieri genome and compared to rice susceptibility gene homologs. Pathogenicity screening on L. hexandra and diverse rice cultivars confirmed that Xcl are able to colonize rice and produce weak but not progressive symptoms. Overall, based on average nucleotide identity (ANI), type III (T3) effector repertoires, and disease phenotype, we propose to rename Xcl to X. oryzae pv. leersiae (Xol) and use this parallel system to improve understanding of the evolution of bacterial pathogenicity in rice agroecosystems.

Analysis of the Pythium ultimum transcriptome using Sanger and Pyrosequencing approaches.

BACKGROUND: Pythium species are an agriculturally important genus of plant pathogens, yet are not understood well at the molecular, genetic, or genomic level. They are closely related to other oomycete plant pathogens such as Phytophthora species and are ubiquitous in their geographic distribution and host rage. To gain a better understanding of its gene complement, we generated Expressed Sequence Tags (ESTs) from the transcriptome of Pythium ultimum DAOM BR144 (= ATCC 200006 = CBS 805.95) using two high throughput sequencing methods, Sanger-based chain termination sequencing and pyrosequencing-based sequencing-by-synthesis. RESULTS: A single half-plate pyrosequencing (454 FLX) run on adapter-ligated cDNA from a normalized cDNA population generated 90,664 reads with an average read length of 190 nucleotides following cleaning and removal of sequences shorter than 100 base pairs. After clustering and assembly, a total of 35,507 unique sequences were generated. In parallel, 9,578 reads were generated from a library constructed from the same normalized cDNA population using dideoxy chain termination Sanger sequencing, which upon clustering and assembly generated 4,689 unique sequences. A hybrid assembly of both Sanger- and pyrosequencing-derived ESTs resulted in 34,495 unique sequences with 1,110 sequences (3.2%) that were solely derived from Sanger sequencing alone. A high degree of similarity was seen between P. ultimum sequences and other sequenced plant pathogenic oomycetes with 91% of the hybrid assembly derived sequences > 500 bp having similarity to sequences from plant pathogenic Phytophthora species. An analysis of Gene Ontology assignments revealed a similar representation of molecular function ontologies in the hybrid assembly in comparison to the predicted proteomes of three Phytophthora species, suggesting a broad representation of the P. ultimum transcriptome was present in the normalized cDNA population. P. ultimum sequences with similarity to oomycete RXLR and Crinkler effectors, Kazal-like and cystatin-like protease inhibitors, and elicitins were identified. Sequences with similarity to thiamine biosynthesis enzymes that are lacking in the genome sequences of three Phytophthora species and one downy mildew were identified and could serve as useful phylogenetic markers. Furthermore, we identified 179 candidate simple sequence repeats that can be used for genotyping strains of P. ultimum. CONCLUSION: Through these two technologies, we were able to generate a robust set (approximately 10 Mb) of transcribed sequences for P. ultimum. We were able to identify known sequences present in oomycetes as well as identify novel sequences. An ample number of candidate polymorphic markers were identified in the dataset providing resources for phylogenetic and diagnostic marker development for this species. On a technical level, in spite of the depth possible with 454 FLX platform, the Sanger and pyro-based sequencing methodologies were complementary as each method generated sequences unique to each platform.

Management of Xanthomonas Leaf Blight of Onion with Bacteriophages and a Plant Activator.

Xanthomonas leaf blight of onion (Allium cepa), caused by Xanthomonas axonopodis pv. allii, continues to be a challenging and yield-threatening disease in Colorado and other regions of onion production worldwide. Studies were conducted to develop management strategies for this disease that are equally effective and more sustainable than the current practices of making multiple applications of copper bactericides. Mixtures of bacteriophages and the plant defense activator, acibenzolar-S-methyl, were evaluated under field and greenhouse conditions for their abilities to reduce Xanthomonas leaf blight severity. Bacteriophage populations in the phyllosphere of onion were monitored over time. Bacteriophage populations persisted on onion leaves for at least 72 to 96 h under field and greenhouse conditions, respectively. Under field conditions at one location, biweekly or weekly applications of bacteriophages reduced disease severity by 26 to 50%, which was equal to or better than weekly applications of copper hydroxide plus mancozeb. Acibenzolar-S-methyl also successfully reduced disease severity by up to 50% when used alone preventatively or followed by biweekly bacteriophage applications. Reductions in disease severity generally were not associated with improvements in onion bulb size or yield. Integration of bacteriophage mixtures with acibenzolar-S-methyl appears to be a promising strategy for managing Xanthomonas leaf blight of onion, and could reduce grower reliance on conventional copper bactericide applied with ethylenebisdithiocarbamate fungicides.

Epiphytic Survival of Xanthomonas axonopodis pv. allii and X. axonopodis pv. phaseoli on Leguminous Hosts and Onion.

Xanthomonas leaf blight of onion (Allium cepa), caused by Xanthomonas axonopodis pv. allii, and common bacterial blight of dry bean (Phaseolus vulgaris), caused by Xanthomonas axonopodis pv. phaseoli, are perennial problems in the Central High Plains of the United States. Onion and dry bean are commonly grown in rotation in Colorado, but it is unknown if X. axonopodis pv. allii and X. axonopodis pv. phaseoli survive epiphytically or pathogenically on dry bean and onion, respectively. Under high humidity growth chamber conditions, epiphytic X. axonopodis pv. allii populations increased on alfalfa, chickpea, dry bean, lentil, and soybean, but the epiphytic populations were at least 10-fold greater on onion. When artificially inoculated under field conditions, epiphytic populations of X. axonopodis pv. allii were recovered from dry bean, lentil, and onion, but the bacterium did not persist on chickpea or soybean. Epiphytic X. axonopodis pv. phaseoli was recovered from symptomless onion plants in fields cropped to dry bean the prior year, but not from fields cropped to a host other than dry bean. Close rotation of onion and dry bean may allow X. axonopodis pv. allii and X. axonopodis pv. phaseoli to persist epiphytically, and crop rotation schemes may need to be altered to reduce survival of these pathogens in onion and dry bean cropping systems.

Inoculum Sources and Survival of Xanthomonas axonopodis pv. allii in Colorado.

Xanthomonas leaf blight, caused by the bacterium Xanthomonas axonopodis pv. allii, is an emerging disease of onion in the western United States and worldwide, but few management strategies have been developed because little is known about disease epidemiology and pathogen survival. Therefore, we sought to identify and quantify primary inoculum sources of the pathogen in Colorado. Growth chamber and field studies evaluated survival and dissemination of X. axonopodis pv. allii in association with weed, alternate host, and volunteer onion plants, irrigation water, and crop debris. Epiphytic X. axonopodis pv. allii was recovered from the foliage of nine asymptomatic weed species and Medicago sativa, but the bacterium was not recovered from plants in locations where an epidemic of Xanthomonas leaf blight did not occur the prior year. The bacterium also was isolated from volunteer onion with characteristic Xanthomonas leaf blight symptoms. A rifampicin mutant of X. axonopodis pv. allii strain O177 was recovered consistently from the irrigation tail water of onion fields inoculated with the bacterium; populations as large as 3.02 × 104 CFU/ml were recovered. X. axonopodis pv. allii was recovered from infested onion leaves 9 months after they were placed on the soil surface or buried to a depth of 25 cm, but culturable populations of the pathogen decreased 104 to 106 more in buried leaves. Cultural practices that avoid or eliminate X. axonopodis pv. allii inoculum sources should reduce Xanthomonas leaf blight losses to onion.