Implications of Crop Rotation and Fungicide on Fusarium and Mycotoxin Spectra in Manitoba Barley, 2017-2019.

Fusarium head blight (FHB) is one of the most important diseases of barley in Manitoba province (western Canada), and other major barley producing regions of the world. Little is known about the Fusarium species and mycotoxin spectra associated with FHB of barley in Manitoba. Hence, barley grain samples were collected from 149 commercial fields from 2017 to 2019, along with information on respective cropping history, and analyzed with respect to Fusarium species spectra, abundance, chemotype composition, and mycotoxin profiles. Fusarium poae was the predominant Fusarium species associated with FHB of barley in Manitoba, followed by F. graminearum, and F. sporotrichioides; F. equiseti and F. avenaceum were also detected but at low levels. F. poae strains with the nivalenol (NIV) chemotype and F. graminearum strains with 3-acetyl deoxynivalenol (3-ADON) and 15-acetyl deoxynivalenol (15-ADON) chemotypes were commonly detected in the barley grain samples. Nivalenol (597.7, 219.1, and 412.4 µg kg-1) and deoxynivalenol (DON) (264.7, 56.7, and 65.3 µg kg-1) were the two most prevalent mycotoxins contaminating Manitoba barley in 2017, 2018 and 2019, respectively. A substantially higher DON content was detected in grain samples from barley fields with cereals as a preceding crop compared to canola and flax. Furthermore, F. poae proved less sensitive to four triazole fungicides (metconazole, prothioconazole+tebuconazole, tebuconazole, and prothioconazole) than F. graminearum. Findings from this research will assist barley producers with improved understanding of FHB threat levels and optimizing practices for the best management of FHB in barley.

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.

Population Genetic Structure and Chemotype Diversity of Fusarium graminearum Populations from Wheat in Canada and North Eastern United States.

Fusarium head blight (FHB) is a major disease in wheat causing severe economic losses globally by reducing yield and contaminating grain with mycotoxins. In Canada, Fusarium graminearum is the principal etiological agent of FHB in wheat, producing mainly the trichothecene mycotoxin, deoxynivalenol (DON) and its acetyl derivatives (15-acetyl deoxynivalenol (15ADON) and 3-acetyl deoxynivalenol (3ADON)). Understanding the population biology of F. graminearum such as the genetic variability, as well as mycotoxin chemotype diversity among isolates is important in developing sustainable disease management tools. In this study, 570 F. graminearum isolates collected from commercial wheat crops in five geographic regions in three provinces in Canada in 2018 and 2019 were analyzed for population diversity and structure using 10 variable number of tandem repeats (VNTR) markers. A subset of isolates collected from the north-eastern United States was also included for comparative analysis. About 75% of the isolates collected in the Canadian provinces of Saskatchewan and Manitoba were 3ADON indicating a 6-fold increase in Saskatchewan and a 2.5-fold increase in Manitoba within the past 15 years. All isolates from Ontario and those collected from the United States were 15ADON and isolates had a similar population structure. There was high gene diversity (H = 0.803-0.893) in the F. graminearum populations in all regions. Gene flow was high between Saskatchewan and Manitoba (Nm = 4.971-21.750), indicating no genetic differentiation between these regions. In contrast, less gene flow was observed among the western provinces and Ontario (Nm = 3.829-9.756) and USA isolates ((Nm = 2.803-6.150). However, Bayesian clustering model analyses of trichothecene chemotype subpopulations divided the populations into two clusters, which was correlated with trichothecene types. Additionally, population cluster analysis revealed there was more admixture of isolates among isolates of the 3ADON chemotypes than among the 15ADON chemotype, an observation that could play a role in the increased virulence of F. graminearum. Understanding the population genetic structure and mycotoxin chemotype variations of the pathogen will assist in developing FHB resistant wheat cultivars and in mycotoxin risk assessment in Canada.

The role of reactive oxygen species in the virulence of wheat leaf rust fungus Puccinia triticina.

Reactive oxygen species (ROS) play an important role during host-pathogen interactions and are often an indication of induced host defence responses. In this study, we demonstrate for the first time that Puccinia triticina (Pt) generates ROS, including superoxide, H2 O2 and hydroxyl radicals, during wheat infection. Through pharmacological inhibition, we found that ROS are critical for both Pt urediniospore germination and pathogenic development on wheat. A comparative RNA-Seq analysis of different stages of Pt infection process revealed 291 putative Pt genes associated with the oxidation-reduction process. Thirty-seven of these genes encode known proteins. The expressions of five Pt genes, including PtNoxA, PtNoxB, PtNoxR, PtCat and PtSod, were subsequently verified using RT-qPCR analysis. The results show that the expressions of PtNoxA, PtNoxB, PtNoxR, PtCat and PtSod are up-regulated during urediniospore germination. In comparison, the expressions of PtNoxA, PtNoxB, PtNoxR and PtCat are down-regulated during wheat infection from 12 to 120 h after inoculation (HAI), whereas the expression of PtSod is up-regulated with a peak of expression at 120 HAI. We conclude that ROS are critical for the full virulence of Pt and a coordinate down-regulation of PtNox genes may be important for successful infection in wheat.

Temporal Quantitative Changes in the Resistant and Susceptible Wheat Leaf Apoplastic Proteome During Infection by Wheat Leaf Rust (Puccinia triticina).

Wheat leaf rust caused by the pathogenic fungus, Puccinia triticina, is a serious threat to bread wheat and durum production in many areas of the world. This plant-pathogen interaction has been studied extensively at the molecular genetics level however, proteomics data are still relatively scarce. The present study investigated temporal changes in the abundance of the apoplastic fluid proteome of resistant and susceptible wheat leaves infected with P. triticina race-1, using a label-free LC-MS-based approach. In general, there was very little difference between inoculated and control apoplastic proteomes in either host, until haustoria had become well established in the susceptible host, although the resistant host responds to pathogen challenge sooner. In the earlier samplings (up to 72 h after inoculation) there were just 46 host proteins with significantly changing abundance, and pathogen proteins were detected only rarely and not reproducibly. This is consistent with the biotrophic lifestyle of P. triticina, where the invading pathogen initially causes little tissue damage or host cell death, which occur only later during the infection cycle. The majority of the host proteins with altered abundance up to 72 h post-inoculation were pathogen-response-related, including peroxidases, chitinases, ß-1-3-endo-glucanases, and other PR proteins. Five days after inoculation with the susceptible apoplasm it was possible to detect 150 P. triticina proteins and 117 host proteins which had significantly increased in abundance as well as 33 host proteins which had significantly decreased in abundance. The latter represents potential targets of pathogen effectors and included enzymes which could damage the invader. The pathogen-expressed proteins-seen most abundantly in the incompatible interaction-were mostly uncharacterized proteins however, many of their functions could be inferred through homology-matching with pBLAST. Pathogen proteins also included several candidate effector proteins, some novel, and some which have been reported previously. All MS data have been deposited in the PRIDE archive (www.ebi.ac.uk/pride/archive/) under Project PXD012586.

Transcriptome analysis of Hpa1Xoo transformed cotton revealed constitutive expression of genes in multiple signalling pathways related to disease resistance.

The transcriptome profile in leaves and roots of the transgenic cotton line T-34 expressing hpa1(Xoo) from Xanthomonas oryzae pv. oryzae was analysed using a customized 12k cotton cDNA microarray. A total of 530 cDNA transcripts involved in 34 pathways were differentially expressed in the transgenic line T-34, in which 123 differentially expressed genes were related to the cotton defence responses including the hypersensitive reaction, defence responses associated with the recognition of pathogen-derived elicitors, and defence signalling pathways mediated by salicylic acid, jasmonic acid, ethylene, auxin, abscicic acid, and Ca(2+). Furthermore, transcripts encoding various leucine-rich protein kinases and mitogen-activated protein kinases were up-regulated in the transgenic line T-34 and expression of transcripts related to the energy producing and consuming pathway was also increased, which suggested that the enhanced metabolism related to the host defence response in the transgenic line T-34 imposed an increased energy demand on the transgenic plant.

Genetic transformation of cotton with a harpin-encoding gene hpaXoo confers an enhanced defense response against different pathogens through a priming mechanism.

BACKGROUND: The soil-borne fungal pathogen Verticillium dahliae Kleb causes Verticillium wilt in a wide range of crops including cotton (Gossypium hirsutum). To date, most upland cotton varieties are susceptible to V. dahliae and the breeding for cotton varieties with the resistance to Verticillium wilt has not been successful. RESULTS: Hpa1Xoo is a harpin protein from Xanthomonas oryzae pv. oryzae which induces the hypersensitive cell death in plants. When hpa1Xoo was transformed into the susceptible cotton line Z35 through Agrobacterium-mediated transformation, the transgenic cotton line (T-34) with an improved resistance to Verticillium dahliae was obtained. Cells of the transgenic T-34, when mixed with the conidia suspension of V. dahliae, had a higher tolerance to V. dahliae compared to cells of untransformed Z35. Cells of T-34 were more viable 12 h after mixing with V. dahliae conidia suspension. Immunocytological analysis showed that Hpa1Xoo, expressed in T-34, accumulated as clustered particles along the cell walls of T-34. In response to the infection caused by V. dahliae, the microscopic cell death and the generation of reactive oxygen intermediates were observed in leaves of T-34 and these responses were absent in leaves of Z35 inoculated with V. dahliae. Quantitative RT-PCR analysis indicated that five defense-related genes, ghAOX1, hin1, npr1, ghdhg-OMT, and hsr203J, were up-regulated in T-34 inoculated with V. dahliae. The up-regulations of these defense-relate genes were not observed or in a less extent in leaves of Z-35 after the inoculation. CONCLUSIONS: Hpa1Xoo accumulates along the cell walls of the transgenic T-34, where it triggers the generation of H2O2 as an endogenous elicitor. T-34 is thus in a primed state, ready to protect the host from the pathogen. The results of this study suggest that the transformation of cotton with hpa1Xoo could be an effective approach for the development of cotton varieties with the improved resistance against soil-borne pathogens.

Fusion body formation, germ tube anastomosis, and nuclear migration during the germination of urediniospores of the wheat leaf rust fungus, Puccinia triticina.

ABSTRACT Vegetative or parasexual recombination is thought to be a key mechanism for the genetic diversity of cereal rust fungi. The process of germ tube fusion leading to hyphal anastomosis and nuclear recombination was analyzed in wheat leaf rust fungus, Puccinia triticina. Germ tube anastomosis was observed in 27 P. triticina isolates, each representing a different virulence phenotype. Germ tube fusion bodies (GFBs), which appeared as viscid globules formed at tips of germ tubes, were essential for germ tube anastomosis. The formation of GFBs was affected by the urediniospore density and the length of illumination during germination. GFBs were formed at the highest frequency when urediniospores were spread to a concentration of 1 x 10(6) urediniospores/ml and incubated in dark for 12 to 24 h during germination. GFB attached to either the side of another germ tube ("tip to side") or to another GFB formed at the tip of a second germ tube ("tip to tip"). In "tip to side" anastomosis, two nuclei in the germ tube bearing the GFB migrated into the second germ tube through the GFB which resulted in four nuclei within this germ tube. In "tip to tip" anastomosis, nuclei in both germ tubes migrated into the fused GFB and all four nuclei came into close proximity. Urediniospores of isolates MBDS-3-115 and TBBJ-5-11 were stained with DAPI (4',6'diamine-2-phenylindole) and Nuclear Yellow (Hoechst S769121), respectively, and then mixed and germinated on water agar. Some fused GFBs contained nuclei stained with DAPI and nuclei stained with Nuclear Yellow in close proximity, demonstrating the fusion between genetically different P. triticina isolates. In some fused GFBs, "bridge-like" structures connecting different nuclei were observed.

Mutations in the N-terminal coding region of the harpin protein Hpa1 from Xanthomonas oryzae cause loss of hypersensitive reaction induction in tobacco.

Harpins encoded by many gram-negative phytopathogenic bacterial hrp genes induce hypersensitive response (HR) and associated defense responses on nonhost plants. Hpa1(Xoo) and Hpa1(Xoc), two harpin proteins from Xanthomonas oryzae pathovars, induce HR when infiltrated into tobacco leaves. N- and C-terminal mutations of Hpa1(Xoo) and Hpa1(Xoc), respectively, were tested for their ability to elicit HR on tobacco. Deletion of codons for 12 highly hydrophilic amino acids (H(2)N-QGISEKQLDQLL-COOH) that partially overlap the N-terminal alpha-helical regions of respective proteins was found to be critical for the elicitation of HR in tobacco. Furthermore, two single missense mutants Hpa1(Xoo) (L51P) and Hpa1(Xoc) (L53P) that are predicted to destroy the coiled-coil integrity and inhibit the dimer formation eliminated HR elicitation activity in tobacco. However, both wild-type proteins and derivative mutants retained the ability to induce systemic acquired resistance in tobacco against tobacco mosaic virus. Accumulations of npr1 (nonexpressor of pathogenesis-related protein 1), hsr515 (hypersensitivity-related protein 515), and pr2 (pathogenesis-related protein 2) transcripts were found in tobacco plants infiltrated with wild-type or mutated proteins.