Jillian M. Lang

Analysis of the Pythium ultimum transcriptome using Sanger and Pyrosequencing approaches

BackgroundPythium 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.ResultsA 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.ConclusionThrough these two technologies, we were able to generate a robust set (~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.

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.

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.

Sensitive Detection of Xanthomonas oryzae Pathovars oryzae and oryzicola by Loop-Mediated Isothermal Amplification

ABSTRACT 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 104 to 105 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.

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.

Rice Galaxy: an open resource for plant science

Background Rice molecular genetics, breeding, genetic diversity, and allied research (such as rice-pathogen interaction) have adopted sequencing technologies and high density genotyping platforms for genome variation analysis and gene discovery. Germplasm collections representing rice diversity, improved varieties and elite breeding materials are accessible through rice gene banks for use in research and breeding, with many having genome sequences and high density genotype data available. Combining phenotypic and genotypic information on these accessions enables genome-wide association analysis, which is driving quantitative trait loci (QTL) discovery and molecular marker development. Comparative sequence analyses across QTL regions facilitate the discovery of novel alleles. Analyses involving DNA sequences and large genotyping matrices for thousands of samples, however, pose a challenge to non-computer savvy rice researchers. Findings We adopted the Galaxy framework to build the federated Rice Galaxy resource, with shared datasets, tools, and analysis workflows relevant to rice research. The shared datasets include high density genotypes from the 3,000 Rice Genomes project and sequences with corresponding annotations from nine published rice genomes. Rice Galaxy includes tools for designing single nucleotide polymorphism (SNP) assays, analyzing genome-wide association studies, population diversity, rice-bacterial pathogen diagnostics, and a suite of published genomic prediction methods. A prototype Rice Galaxy compliant to Open Access, Open Data, and Findable, Accessible, Interoperable, and Reproducible principles is also presented. Conclusions Rice Galaxy is a freely available resource that empowers the plant research community to perform state-of-the-art analyses and utilize publicly available big datasets for both fundamental and applied science.