The pangenome of the wheat pathogen Pyrenophora tritici-repentis reveals novel transposons associated with necrotrophic effectors ToxA and ToxB.

BACKGROUND: In fungal plant pathogens, genome rearrangements followed by selection pressure for adaptive traits have facilitated the co-evolutionary arms race between hosts and their pathogens. Pyrenophora tritici-repentis (Ptr) has emerged recently as a foliar pathogen of wheat worldwide and its populations consist of isolates that vary in their ability to produce combinations of different necrotrophic effectors. These effectors play vital roles in disease development. Here, we sequenced the genomes of a global collection (40 isolates) of Ptr to gain insights into its gene content and genome rearrangements. RESULTS: A comparative genome analysis revealed an open pangenome, with an abundance of accessory genes (~ 57%) reflecting Ptr's adaptability. A clear distinction between pathogenic and non-pathogenic genomes was observed in size, gene content, and phylogenetic relatedness. Chromosomal rearrangements and structural organization, specifically around effector coding genes, were detailed using long-read assemblies (PacBio RS II) generated in this work in addition to previously assembled genomes. We also discovered the involvement of large mobile elements associated with Ptr's effectors: ToxA, the gene encoding for the necrosis effector, was found as a single copy within a 143-kb 'Starship' transposon (dubbed 'Horizon') with a clearly defined target site and target site duplications. 'Horizon' was located on different chromosomes in different isolates, indicating mobility, and the previously described ToxhAT transposon (responsible for horizontal transfer of ToxA) was nested within this newly identified Starship. Additionally, ToxB, the gene encoding the chlorosis effector, was clustered as three copies on a 294-kb element, which is likely a different putative 'Starship' (dubbed 'Icarus') in a ToxB-producing isolate. ToxB and its putative transposon were missing from the ToxB non-coding reference isolate, but the homolog toxb and 'Icarus' were both present in a different non-coding isolate. This suggests that ToxB may have been mobile at some point during the evolution of the Ptr genome which is contradictory to the current assumption of ToxB vertical inheritance. Finally, the genome architecture of Ptr was defined as 'one-compartment' based on calculated gene distances and evolutionary rates. CONCLUSIONS: These findings together reflect on the highly plastic nature of the Ptr genome which has likely helped to drive its worldwide adaptation and has illuminated the involvement of giant transposons in facilitating the evolution of virulence in Ptr.

Fusarium Root Rot Complex in Soybean: Molecular Characterization, Trichothecene Formation, and Cross-Pathogenicity.

Soybean is threatened by many pathogens that negatively affect this crop's yield and quality, such as various Fusarium species that cause wilting and root rot diseases. Fusarium root rot (FRR) in soybean can be caused by F. graminearum and other Fusarium spp. that are associated with Fusarium head blight (FHB) in cereals. Therefore, it was important to inquire whether Fusarium pathogens from soybean can cause disease in wheat and vice versa. Here, we investigated the FRR complex in Manitoba (Canada) from symptomatic plants, using both culture- and molecular-based methods. We developed a molecular diagnostic toolkit to detect and differentiate between several Fusarium spp. involved in FHB and FRR, then we evaluated cross-pathogenicity of selected Fusarium isolates collected from soybean and wheat, and the results indicate that isolates recovered from one host can infect the other host. Trichothecene production by selected Fusarium spp. was also analyzed chemically via liquid chromatography mass spectrometry in both soybean (root) and wheat (spike) tissues. Trichothecenes were also analyzed in soybean seeds from plants with FRR to check the potentiality of trichothecene translocation from infected roots to the seeds. All of the tested Fusarium isolates were capable of producing trichothecenes in wheat spikes and soybean roots, but no trichothecenes were detected in soybean seeds. This study provided evidence, for the first time, that trichothecenes were produced by several Fusarium spp. (F. cerealis, F. culmorum, and F. sporotrichioides) during FRR development in soybean.

Overexpression of Solanum tuberosum Respiratory Burst Oxidase Homolog A (StRbohA) Promotes Potato Tolerance to Phytophthora infestans.

Reactive oxygen species (ROSs) represent one of the first lines of plants' biochemical defense against pathogens. Plants' respiratory burst oxidase homologs (RBOHs) produce ROSs as byproducts in several cellular compartments. In potato tubers, Solanum tuberosum respiratory burst oxidase homolog (StRBOHs) are involved in suberization and healing of wounded tissues. StRbohA has been tested in the model plant Arabidopsis thaliana, which led to enhanced plant defense against the soilborne pathogen Verticillium dahliae. Here, we showed that overexpressing StRbohA in potato plants increases plant tolerance to the oomycete Phytophthora infestans, the causal agent of late blight disease. Transgenic potato plants expressing StRbohA showed reduced disease symptoms (necrosis) compared with the wild type. In parallel, the expression of pathogenesis-related genes (PRs); RBOHs; antioxidation-related genes CPRX1, PRX2, APRX1, CAT1, and CAT2; and genes involved in the biosynthesis pathways of jasmonic and salicylic acids (ICS, PAL1, PAL2, LOX1, LOX2, and LOX3) exhibited significant increases in transgenic plants in response to infection. After higher expression of RBOHs, ROSs accumulated more in inoculation sites of the transgenic plants. ROSs act as signals that activate gene expression in the salicylic acid (SA) biosynthesis pathway, leading to the accumulation of SA and triggering SA-based defense mechanisms. SA-responsive PRs showed higher expression in the transgenic plants, which resulted in the restriction of pathogen growth in plant tissues. These results demonstrate the effective role of StRbohA in increasing potato defense against P. infestans.

Specific Detection and Identification of Fusarium graminearum Sensu Stricto Using a PCR-RFLP Tool and Specific Primers Targeting the Translational Elongation Factor 1α Gene.

Fusarium graminearum is a toxigenic plant pathogen that causes Fusarium head blight (FHB) disease on cereal crops. It has recently shown to have cross-pathogenicity on noncereals (i.e., Fusarium root rot [FRR] on soybean) in Canada and elsewhere. Specific detection and differentiation of this potent toxigenic, trichothecene-producing pathogen among other closely related species is extremely important for disease control and mycotoxin monitoring. Here, we designed a PCR restriction fragment length polymorphism protocol based on the DNA sequence of the translational elongation factor 1α (TEF1α) gene. A unique restriction site to the enzyme HpaII is only found in F. graminearum sensu stricto strains among different Fusarium strains in the F. graminearum species complex (FGSC) and other Fusarium spp. associated with FHB in cereals and FRR in soybean. Partial amplification of the TEF1α gene with newly designed primers mh1/mh2 generated a 459-bp PCR fragment. Restriction digestion of the generated fragments with the HpaII enzyme generated a unique restriction pattern that can rapidly and accurately differentiate F. graminearum sensu stricto among all other Fusarium spp. A primer pair (FgssF/FgssR) specific to F. graminearum sensu stricto also was designed and can distinguish F. graminearum sensu stricto from all other Fusarium spp. in the FGSC and other closely related Fusarium spp. involved in FHB and FRR. This finding will be very useful for the specific detection of F. graminearum sensu stricto for diagnostic purposes as well as for the accurate detection of this pathogen in breeding and other research purposes.

Transcriptome Analysis of Rlm2-Mediated Host Immunity in the Brassica napus-Leptosphaeria maculans Pathosystem.

Our study investigated disease resistance in the Brassica napus-Leptosphaeria maculans pathosystem using a combination of laser microdissection, dual RNA sequencing, and physiological validations of large-scale gene sets. The use of laser microdissection improved pathogen detection and identified putative L. maculans effectors and lytic enzymes operative during host colonization. Within 24 h of inoculation, we detected large shifts in gene activity in resistant cotyledons associated with jasmonic acid and calcium signaling pathways that accelerated the plant defense response. Sequencing data were validated through the direct quantification of endogenous jasmonic acid levels. Additionally, resistance against L. maculans was abolished when the calcium chelator EGTA was applied to the inoculation site, providing physiological evidence of the role of calcium in B. napus immunity against L. maculans. We integrated gene expression data with all available information on cis-regulatory elements and transcription factor binding affinities to better understand the gene regulatory networks underpinning plant resistance to hemibiotrophic pathogens. These in silico analyses point to early cellular reprogramming during host immunity that are coordinated by CAMTA, BZIP, and bHLH transcription factors. Together, we provide compelling genetic and physiological evidence into the programming of plant resistance against fungal pathogens.

Genome Analysis and Development of a Multiplex TaqMan Real-Time PCR for Specific Identification and Detection of Clavibacter michiganensis subsp. nebraskensis.

The reemergence of the Goss's bacterial wilt and blight disease in corn in the United States and Canada has prompted investigative research to better understand the genome organization. In this study, we generated a draft genome sequence of Clavibacter michiganensis subsp. nebraskensis strain DOAB 395 and performed genome and proteome analysis of C. michiganensis subsp. nebraskensis strains isolated in 2014 (DOAB 397 and DOAB 395) compared with the type strain, NCPPB 2581 (isolated over 40 years ago). The proteomes of strains DOAB 395 and DOAB 397 exhibited a 99.2% homology but had 92.1 and 91.8% homology, respectively, with strain NCPPB 2581. The majority (99.9%) of the protein sequences had a 99.6 to 100% homology between C. michiganensis subsp. nebraskensis strains DOAB 395 and DOAB 397, with only four protein sequences (0.1%) exhibiting a similarity <70%. In contrast, 3.0% of the protein sequences of strain DOAB 395 or DOAB 397 showed low homologies (<70%) with the type strain NCPPB 2581. The genome data were exploited for the development of a multiplex TaqMan real-time polymerase chain reaction (PCR) tool for rapid detection of C. michiganensis subsp. nebraskensis. The specificity of the assay was validated using 122 strains of Clavibacter and non-Clavibacter spp. A blind test and naturally infected leaf samples were used to confirm specificity. The sensitivity (0.1 to 1.0 pg) compared favorably with previously reported real-time PCR assays. This tool should fill the current gap for a reliable diagnostic technique.

Extreme resistance as a host counter-counter defense against viral suppression of RNA silencing.

RNA silencing mediated by small RNAs (sRNAs) is a conserved regulatory process with key antiviral and antimicrobial roles in eukaryotes. A widespread counter-defensive strategy of viruses against RNA silencing is to deploy viral suppressors of RNA silencing (VSRs), epitomized by the P19 protein of tombusviruses, which sequesters sRNAs and compromises their downstream action. Here, we provide evidence that specific Nicotiana species are able to sense and, in turn, antagonize the effects of P19 by activating a highly potent immune response that protects tissues against Tomato bushy stunt virus infection. This immunity is salicylate- and ethylene-dependent, and occurs without microscopic cell death, providing an example of "extreme resistance" (ER). We show that the capacity of P19 to bind sRNA, which is mandatory for its VSR function, is also necessary to induce ER, and that effects downstream of P19-sRNA complex formation are the likely determinants of the induced resistance. Accordingly, VSRs unrelated to P19 that also bind sRNA compromise the onset of P19-elicited defense, but do not alter a resistance phenotype conferred by a viral protein without VSR activity. These results show that plants have evolved specific responses against the damages incurred by VSRs to the cellular silencing machinery, a likely necessary step in the never-ending molecular arms race opposing pathogens to their hosts.

Botrytis cinerea manipulates the antagonistic effects between immune pathways to promote disease development in tomato.

Plants have evolved sophisticated mechanisms to sense and respond to pathogen attacks. Resistance against necrotrophic pathogens generally requires the activation of the jasmonic acid (JA) signaling pathway, whereas the salicylic acid (SA) signaling pathway is mainly activated against biotrophic pathogens. SA can antagonize JA signaling and vice versa. Here, we report that the necrotrophic pathogen Botrytis cinerea exploits this antagonism as a strategy to cause disease development. We show that B. cinerea produces an exopolysaccharide, which acts as an elicitor of the SA pathway. In turn, the SA pathway antagonizes the JA signaling pathway, thereby allowing the fungus to develop its disease in tomato (Solanum lycopersicum). SA-promoted disease development occurs through Nonexpressed Pathogen Related1. We also show that the JA signaling pathway required for tomato resistance against B. cinerea is mediated by the systemin elicitor. These data highlight a new strategy used by B. cinerea to overcome the plant's defense system and to spread within the host.

Goss's Wilt Resistance in Corn Is Mediated via Salicylic Acid and Programmed Cell Death but Not Jasmonic Acid Pathways.

A highly aggressive strain (CMN14-5-1) of Clavibacter nebraskensis bacteria, which causes Goss's wilt in corn, induced severe symptoms in a susceptible corn line (CO447), resulting in water-soaked lesions followed by necrosis within a few days. A tolerant line (CO450) inoculated with the same strain exhibited only mild symptoms such as chlorosis, freckling, and necrosis that did not progress after the first six days following infection. Both lesion length and disease severity were measured using the area under the disease progression curve (AUDPC), and significant differences were found between treatments. We analyzed the expression of key genes related to plant defense in both corn lines challenged with the CMN14-5-1 strain. Allene oxide synthase (ZmAOS), a gene responsible for the production of jasmonic acid (JA), was induced in the CO447 line in response to CMN14-5-1. Following inoculation with CMN14-5-1, the CO450 line demonstrated a higher expression of salicylic acid (SA)-related genes, ZmPAL and ZmPR-1, compared to the CO447 line. In the CO450 line, four genes related to programmed cell death (PCD) were upregulated: respiratory burst oxidase homolog protein D (ZmrbohD), polyphenol oxidase (ZmPPO1), ras-related protein 7 (ZmRab7), and peptidyl-prolyl cis-trans isomerase (ZmPPI). The differential gene expression in response to CMN14-5-1 between the two corn lines provided an indication that SA and PCD are involved in the regulation of corn defense responses against Goss's wilt disease, whereas JA may be contributing to disease susceptibility.

Reducing progoitrin and enriching glucoraphanin in Brassica napus seeds through silencing of the GSL-ALK gene family.

The hydrolytic products of glucosinolates in brassica crops are bioactive compounds. Some glucosinolate derivatives such as oxazolidine-2-thione from progoitrin in brassica oilseed meal are toxic and detrimental to animals, but some isothiocyanates such as sulforaphane are potent anti-carcinogens that have preventive effects on several human cancers. In most B. rapa, B. napus and B. juncea vegetables and oilseeds, there is no or only trace amount of glucoraphanin that is the precursor to sulforaphane. In this paper, RNA interference (RNAi) of the GSL-ALK gene family was used to down-regulate the expression of GSL-ALK genes in B. napus. The detrimental glucosinolate progoitrin was reduced by 65 %, and the beneficial glucosinolate glucoraphanin was increased to a relatively high concentration (42.6 µmol g(-1) seed) in seeds of B. napus transgenic plants through silencing of the GSL-ALK gene family. Therefore, there is potential application of the new germplasm with reduced detrimental glucosinolates and increased beneficial glucosinolates for producing improved brassica vegetables.

Plants versus fungi and oomycetes: pathogenesis, defense and counter-defense in the proteomics era.

Plant-fungi and plant-oomycete interactions have been studied at the proteomic level for many decades. However, it is only in the last few years, with the development of new approaches, combined with bioinformatics data mining tools, gel staining, and analytical instruments, such as 2D-PAGE/nanoflow-LC-MS/MS, that proteomic approaches thrived. They allow screening and analysis, at the sub-cellular level, of peptides and proteins resulting from plants, pathogens, and their interactions. They also highlight post-translational modifications to proteins, e.g., glycosylation, phosphorylation or cleavage. However, many challenges are encountered during in planta studies aimed at stressing details of host defenses and fungal and oomycete pathogenicity determinants during interactions. Dissecting the mechanisms of such host-pathogen systems, including pathogen counter-defenses, will ensure a step ahead towards understanding current outcomes of interactions from a co-evolutionary point of view, and eventually move a step forward in building more durable strategies for management of diseases caused by fungi and oomycetes. Unraveling intricacies of more complex proteomic interactions that involve additional microbes, i.e., PGPRs and symbiotic fungi, which strengthen plant defenses will generate valuable information on how pathosystems actually function in nature, and thereby provide clues to solving disease problems that engender major losses in crops every year.

Genome-Based Analysis of Verticillium Polyketide Synthase Gene Clusters.

Polyketides are structurally diverse and physiologically active secondary metabolites produced by many organisms, including fungi. The biosynthesis of polyketides from acyl-CoA thioesters is catalyzed by polyketide synthases, PKSs. Polyketides play roles including in cell protection against oxidative stress, non-constitutive (toxic) roles in cell membranes, and promoting the survival of the host organisms. The genus Verticillium comprises many species that affect a wide range of organisms including plants, insects, and other fungi. Many are known as causal agents of Verticillium wilt diseases in plants. In this study, a comparative genomics approach involving several Verticillium species led us to evaluate the potential of Verticillium species for producing polyketides and to identify putative polyketide biosynthesis gene clusters. The next step was to characterize them and predict the types of polyketide compounds they might produce. We used publicly available sequences from ten species of Verticillium including V. dahliae, V. longisporum, V. nonalfalfae, V. alfalfae, V. nubilum, V. zaregamsianum, V. klebahnii, V. tricorpus, V. isaacii, and V. albo-atrum to identify and characterize PKS gene clusters by utilizing a range of bioinformatic and phylogenetic approaches. We found 32 putative PKS genes and possible clusters in the genomes of Verticillium species. All the clusters appear to be complete and functional. In addition, at least five clusters including putative DHN-melanin-, cytochalasin-, fusarielien-, fujikurin-, and lijiquinone-like compounds may belong to the active PKS repertoire of Verticillium. These results will pave the way for further functional studies to understand the role of these clusters.

Role of Exopolygalacturonase-Related Genes in Potato-Verticillium dahliae Interaction.

Verticillium dahliae is a hemibiotrophic pathogen responsible for great losses in dicot crop production. An ExoPG gene (VDAG_03463,) identified using subtractive hybridization/cDNA-AFLP, showed higher expression levels in highly aggressive than in weakly aggressive V. dahliae isolates. We used a vector-free split-marker recombination method with PEG-mediated protoplast to delete the ExoPG gene in V. dahliae. This is the first instance of using this method for V. dahliae transformation. Only two PCR steps and one transformation step were required, markedly reducing the necessary time for gene deletion. Six mutants were identified. ExoPG expressed more in the highly aggressive than in the weakly aggressive isolate in response to potato leaf and stem extracts. Its expression increased in both isolates during infection, with higher levels in the highly aggressive isolate at early infection stages. The disruption of ExoPG did not influence virulence, nor did it affect total exopolygalacturonase activity in V. dahliae. Full genome analysis showed 8 more genes related to polygalacturonase/pectinase activity in V. dahliae. Transcripts of PGA increased in the △exopg mutant in response to potato leaf extracts, compared to the wild type. The expression pattern of those eight genes showed similar trends in the △exopg mutant and in the weakly aggressive isolate in response to potato extracts, but without the increase of PGA in the weakly aggressive isolate to leaf extracts. This indicated that the △exopg mutant of V. dahliae compensated by the suppression of ExoPG by activating other related gene.

NOXA Is Important for Verticillium dahliae's Penetration Ability and Virulence.

NADPH oxidase (Nox) genes are responsible for Reactive Oxygen Species (ROS) production in living organisms such as plants, animals, and fungi, where ROS exert different functions. ROS are critical for sexual development and cellular differentiation in fungi. In previous publications, two genes encoding thioredoxin and NADH-ubiquinone oxidoreductase involved in maintaining ROS balance were shown to be remarkably induced in a highly versus a weakly aggressive Verticillium dahliae isolate. This suggested a role of these genes in the virulence of this pathogen. NoxA (NADPH oxidase A) was identified in the V. dahliae genome. We compared in vitro expression of NoxA in highly and weakly aggressive isolates of V. dahliae after elicitation with extracts from different potato tissues. NoxA expression was induced more in the weakly than highly aggressive isolate in response to leaf and stem extracts. After inoculation of potato detached leaves with these two V. dahliae isolates, NoxA was drastically up-regulated in the highly versus the weakly aggressive isolate. We generated single gene disruption mutants for NoxA genes. noxa mutants had significantly reduced virulence, indicating important roles in V. dahliae pathogenesis on the potato. This is consistent with a significant reduction of cellophane penetration ability of the mutants compared to the wild type. However, the cell wall integrity was not impaired in the noxa mutants when compared with the wild type. The resistance of noxa mutants to oxidative stress were also similar to the wild type. Complementation of noxa mutants with a full length NoxA clones restored penetration and pathogenic ability of the fungus. Our data showed that NoxA is essential for both penetration peg formation and virulence in V. dahliae.

Gene Expression of Putative Pathogenicity-Related Genes in Verticillium dahliae in Response to Elicitation with Potato Extracts and during Infection Using Quantitative Real-Time PCR.

Quantitative real-time PCR was used to monitor the expression of 15 Verticillium dahliae's genes, putatively involved in pathogenicity, highly (HAV) and weakly aggressive (WAV) V. dahliae isolates after either (i) elicitation with potato leaf, stem, or root extracts, or (ii) inoculation of potato detached petioles. These genes, i.e., coding for Ras-GAP-like protein, serine/threonine protein kinase, Ubiquitin-conjugating enzyme variant-MMS2, NADH-ubiquinone oxidoreductase, Thioredoxin, Pyruvate dehydrogenase E1 VdPDHB, myo-inositol 2-dehydrogenase, and HAD-superfamily hydrolase, showed differential upregulation in the HAV versus WAV isolate in response to plant extracts or after inoculation of potato leaf petioles. This suggests their potential involvement in the observed differential aggressiveness between isolates. However, other genes like glucan endo-1,3-alpha-glucosidase and nuc-1 negative regulatory protein VdPREG showed higher activity in the WAV than in the HAV in response to potato extracts and/or during infection. This, in contrast, may suggest a role in their lower aggressiveness. These findings, along with future functional analysis of selected genes, will contribute to improving our understanding of V. dahliae's pathogenesis. For example, expression of VdPREG negatively regulates phosphorus-acquisition enzymes, which may indicate a lower phosphorus acquisition activity in the WAV. Therefore, integrating the knowledge about the activity of both genes enhancing pathogenicity and those restraining it will provide a guild line for further functional characterization of the most critical genes, thus driving new ideas towards better Verticillium wilt management.

Secretome Analysis of Clavibacter nebraskensis Strains Treated with Natural Xylem Sap In Vitro Predicts Involvement of Glycosyl Hydrolases and Proteases in Bacterial Aggressiveness.

The Gram-positive bacterium Clavibacter nebraskensis (Cn) causes Goss's wilt and leaf blight on corn in the North American Central Plains with yield losses as high as 30%. Cn strains vary in aggressiveness on corn, with highly aggressive strains causing much more serious symptoms and damage to crops. Since Cn inhabits the host xylem, we investigated differences in the secreted proteomes of Cn strains to determine whether these could account for phenotypic differences in aggressiveness. Highly and a weakly aggressive Cn strains (Cn14-15-1 and DOAB232, respectively) were cultured, in vitro, in the xylem sap of corn (CXS; host) and tomato (TXS; non-host). The secretome of the Cn strains were extracted and processed, and a comparative bottom-up proteomics approach with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine their identities and concentration. Relative quantitation of peptides was based on precursor ion intensities to measure protein abundances. In total, 745 proteins were identified in xylem sap media. In CXS, a total of 658 and 396 proteins were identified in strains Cn14-5-1 and DOAB232, respectively. The unique and the differentially abundant proteins in the secretome of strain Cn14-5-1 were higher in either sap medium compared to DOAB232. These proteins were sorted using BLAST2GO and assigned to 12 cellular functional processes. Virulence factors, e.g., cellulase, ß-glucosidase, ß-galactosidase, chitinase, ß-1,4-xylanase, and proteases were generally higher in abundance in the aggressive Cn isolate. This was corroborated by enzymatic activity assays of cellulase and protease in CXS. These proteins were either not detected or detected at significantly lower abundance levels in Cn strains grown in non-host xylem sap (tomato), suggesting potential factors involved in Cn-host (corn) interactions.

MinION Nanopore-based detection of Clavibacter nebraskensis, the corn Goss's wilt pathogen, and bacteriomic profiling of necrotic lesions of naturally-infected leaf samples.

The Goss's bacterial wilt pathogen, Clavibacter nebraskensis, of corn is a candidate A1 quarantine organism; and its recent re-emergence and spread in the USA and Canada is a potential biothreat to the crop. We developed and tested an amplicon-based Nanopore detection system for C. nebraskensis (Cn), targeting a purine permease gene. The sensitivity (1 pg) of this system in mock bacterial communities (MBCs) spiked with serially diluted DNA of C. nebraskensis NCPPB 2581T is comparable to that of real-time PCR. Average Nanopore reads increased exponentially from 125 (1pg) to about 6000 reads (1000 pg) after a 3-hr run-time, with 99.0% of the reads accurately assigned to C. nebraskensis. Three run-times were used to process control MBCs, Cn-spiked MBCs, diseased and healthy leaf samples. The mean Nanopore reads doubled as the run-time is increased from 3 to 6 hrs while from 6 to 12 hrs, a 20% increment was recorded in all treatments. Cn-spiked MBCs and diseased corn leaf samples averaged read counts of 5,100, 11,000 and 14,000 for the respective run-times, with 99.8% of the reads taxonomically identified as C. nebraskensis. The control MBCs and healthy leaf samples had 47 and 14 Nanopore reads, respectively. 16S rRNA bacteriomic profiles showed that Sphingomonas (22.7%) and Clavibacter (21.2%) were dominant in diseased samples while Pseudomonas had only 3.5% relative abundance. In non-symptomatic leaf samples, however, Pseudomonas (20.0%) was dominant with Clavibacter at 0.08% relative abundance. This discrepancy in Pseudomonas abundance in the samples was corroborated by qPCR using EvaGreen chemistry. Our work outlines a new useful tool for diagnosis of the Goss's bacterial wilt disease; and provides the first insight on Pseudomonas community dynamics in necrotic leaf lesions.

Naturally Occurring Fusarium Species and Mycotoxins in Oat Grains from Manitoba, Canada.

Fusarium head blight (FHB) can lead to dramatic yield losses and mycotoxin contamination in small grain cereals in Canada. To assess the extent and severity of FHB in oat, samples collected from 168 commercial oat fields in the province of Manitoba, Canada, during 2016-2018 were analyzed for the occurrence of Fusarium head blight and associated mycotoxins. Through morphological and molecular analysis, F. poae was found to be the predominant Fusarium species affecting oat, followed by F. graminearum, F. sporotrichioides, F. avenaceum, and F. culmorum. Deoxynivalenol (DON) and nivalenol (NIV), type B trichothecenes, were the two most abundant Fusarium mycotoxins detected in oat. Beauvericin (BEA) was also frequently detected, though at lower concentrations. Close clustering of F. poae and NIV/BEA, F. graminearum and DON, and F. sporotrichioides and HT2/T2 (type A trichothecenes) was detected in the principal component analysis. Sampling location and crop rotation significantly impacted the concentrations of Fusarium mycotoxins in oat. A phylogenetic analysis of 95 F. poae strains from Manitoba was conducted using the concatenated nucleotide sequences of Tef-1α, Tri1, and Tri8 genes. The results indicated that all F. poae strains belong to a monophyletic lineage. Four subgroups of F. poae strains were identified; however, no correlations were observed between the grouping of F. poae strains and sample locations/crop rotations.

Proteomic analysis of the phytopathogenic soilborne fungus Verticillium dahliae reveals differential protein expression in isolates that differ in aggressiveness.

Verticillium dahliae is a soilborne fungus that causes a vascular wilt disease of plants and losses in a broad range of economically important crops worldwide. In this study, we compared the proteomes of highly (Vd1396-9) and weakly (Vs06-14) aggressive isolates of V. dahliae to identify protein factors that may contribute to pathogenicity. Twenty-five protein spots were consistently observed as differential in the proteome profiles of the two isolates. The protein sequences in the spots were identified by LC-ESI-MS/MS and MASCOT database searches. Some of the identified sequences shared homology with fungal proteins that have roles in stress response, colonization, melanin biosynthesis, microsclerotia formation, antibiotic resistance, and fungal penetration. These are important functions for infection of the host and survival of the pathogen in soil. One protein found only in the highly aggressive isolate was identified as isochorismatase hydrolase, a potential plant-defense suppressor. This enzyme may inhibit the production of salicylic acid, which is important for plant defense response signaling. Other sequences corresponding to potential pathogenicity factors were identified in the highly aggressive isolate. This work indicates that, in combination with functional genomics, proteomics-based analyses can provide additional insights into pathogenesis and potential management strategies for this disease.