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Summary 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, H2O2 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.

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.

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.

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.

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, β-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.

Wheat leaf rust, caused by Puccinia triticina Eriks., is of worldwide concern for wheat producers. The disease has been an annual problem for Canadian wheat producers since the early days of wheat cultivation in the 1800s, and research focused on combating this disease began in the early 1900s. Significant progress was made towards understanding the epidemiology of wheat leaf rust and developing genetic resistance in many countries worldwide. This review paper focuses exclusively on the research and development done in whole, or in part, in Canada. An integrated approach to controlling wheat leaf rust consisted of research in the following areas: the early research on wheat leaf rust in Canada, breeding and commercialization of high quality rust resistant wheat cultivars, discovery and genetic analysis of leaf rust resistance genes, the population biology and genetics of the P. triticina/wheat interaction. This review summarizes the research in each of these areas and the connections between the d...

Thatcher near-isogenic lines (NILs) of wheat carrying resistance gene Lr2a, Lr3, LrB or Lr9 were inoculated with Puccinia triticina races of virulence phenotype BBBD, MBDS, SBDG and FBDJ. Puccinia triticina infection structures were analysed under the fluorescence microscope over a course of 14 days after inoculation (dai). The relative proportion of P. triticina and wheat genomic DNA in infected leaves was estimated with a semiquantitative multiplex PCR analysis using P. triticinaand wheat-specific primers. The occurrence of a hypersensitive response (HR), cellular lignification and callose deposition in inoculated plants was investigated microscopically. In interactions producing highly resistant infection type (IT) ‘0;’, a maximum of two haustorial mother cells per infection site were produced, and there was no increase in the proportion of P. triticina genomic DNA in infected leaves, indicating the absence of P. triticina growth. In comparison, sizes of P. triticina colonies increased gradually in interactions producing moderately resistant IT ‘1’ and ‘2’, with the highest proportion of P. triticina genomic DNA found in leaves sampled at 14 dai. In interactions producing susceptible IT ‘3‐4’, the highest proportion of P. triticina genomic DNA was found in leaves sampled at 10 dai (45AE5‐51AE5%). HR and cellular lignification were induced in interactions producing IT ‘0;’ and ‘1’ at 1 dai but they were not observed in interactions producing IT ‘2’ until 2 dai. No HR or cellular lignification were induced in interactions producing susceptible IT ‘3‐4’. Furthermore, a strong deposition of callose was induced in Lr9 +B BBD andLr9 + FBDJ (IT ‘0;’), whereas this defence response was not induced in resistant or susceptible interactions involving Lr2a, Lr3 or LrB, indicating that Lr9 mediated resistance was different from that conditioned by Lr2a, Lr3 or LrB.

Cereal rust fungi are pathogens of major importance to agriculture, threatening cereal production worldwide. Targeted breeding for resistance, based on information from fungal surveys and population structure analyses of virulence, has been effective. Nevertheless, breakdown of resistance occurs frequently and continued efforts are needed to understand how these fungi overcome resistance and to determine the range of available resistance genes. The development of genomic resources for these fungi and their comparison has released a torrent of new ideas and approaches to use this information to assist pathologists and agriculture in general. The sequencing of gene transcripts and the analysis of proteins from haustoria has yielded candidate virulence factors among which could be defence-triggering avirulence genes. Genome-wide computational analyses, including genetic mapping and transcript analyses by RNA sequencing of many fungal isolates, will predict many more candidates. Functional assays, such as leaf infiltration using Agrobacterium for delivery of cloned fungal effectors, are being developed. This will allow the screening of wheat germplasm for novel resistance genes for breeding. Comparative analyses have also revealed fungal virulence genes, providing fungal targets for disease control in host-produced RNAi approaches.

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.

Gene-associated simple sequence repeat (SSR) markers were developed for Puccinia triticina through the data mining of existing EST libraries. Analysis of 7134 expressed sequence tags (ESTs) from cDNA libraries of P. triticina detected 204 EST-SSRs with a minimum of 12 repeating nucleotides. The majority of EST-SSRs contained short di- or tri-nucleotide repeats. These EST-SSRs were evaluated on 35 P. triticina isolates collected in Canada and 21 EST-SSRs were polymorphic and informative in determining intraspecific genetic diversity. A comparison of virulence and EST-SSR genotypes showed a strong correlation between virulence to Lr2a, Lr2c and Lr17a and EST-SSRs genotypes. The differentiation of the P. triticina population based on EST-SSR genotypes was comparable to that obtained with genomic SSRs, despite differences between two types of SSR markers. Eight of the 21 EST-SSRs produced the cross amplification in Puccinia coronata and Puccinia graminis, suggesting that EST-SSRs are more applicable ...

Defence responses were investigated in two potato cultivars with different levels of resistance to late blight, Russet Burbank (susceptible) and Kennebec (moderately resistant), after inoculation with single isolates representing Phytophthora infestans genotypes US-1 (previously predominant, mildly aggressive) and US-8 (currently predominant, highly aggressive). The accumulation of brown lignin-like materials and an increase in the cell wall affinity to trypan blue 24 h after inoculation were observed in cv. Kennebec inoculated with US-1, but not in Kennebec inoculated with US-8, or cv. Russet Burbank inoculated with either US-1 or US-8. The expression of PAL-1, HMG-2, PR-1 and PR-5 was investigated in three leaf strata (local, proximal and distal) and at different times after inoculation, using SYBR real-time RT-PCR. The activation of these defence-related genes was affected not only by P. infestans genotype, but also by the potato cultivar and the proximity to the inoculation site. These genes were up-regulated earlier in Kennebec than in Russet Burbank and in response to US-1 than to US-8. Over all, the earliest and strongest up-regulation of these genes occurred in Kennebec inoculated with US-1. Furthermore, PAL-1 and HMG-2 were down-regulated at the site of infection while such down-regulation was not observed for PR-1 or PR-5. In parallel, the accumulation level and location of phenolics and rishitin matched those of PAL-1 and HMG-2 transcripts, respectively. These results strongly suggest that changes in either the activation or suppression of defence responses by the pathogen shape the level of susceptibility of potato cultivars to late blight.

Race-specific resistance of wheat to Puccinia graminis f. sp. tritici is primarily posthaustorial and often involves the induction of a hypersensitive response (HR). The aim of this study was to investigate host defense responses induced in interactions between P. graminis f. sp. tritici races and wheat lines carrying different race-specific stem rust resistance (Sr) genes. In incompatible interactions between wheat lines carrying Sr36 in three genetic backgrounds (LMPG, Prelude, or W2691) and avirulent P. graminis f. sp. tritici races MCCFC or RCCDM, callose accumulated within 24 h in wheat guard cells contacted by a P. graminis f. sp. tritici appressorium, and P. graminis f. sp. tritici ingress was inhibited following appressorium formation. Accordingly, the expression of transcripts encoding a callose synthase increased in the incompatible interaction between LMPG-Sr36 and avirulent P. graminis f. sp. tritici race MCCFC. Furthermore, the inhibition of callose synthesis through the infiltration of 2-deoxy-D-glucose (DDG) increased the ability of P. graminis f. sp. tritici race MCCFC to infect LMPG-Sr36. A similar induction of callose deposition in wheat guard cells was also observed within 24 h after inoculation (hai) with avirulent P. graminis f. sp. tritici race HKCJC on LMPG-Sr5 plants. In contrast, this defense response was not induced in incompatible interactions involving Sr6, Sr24, or Sr30. Instead, the induction of an HR and cellular lignification were noted. The manifestation of the HR and cellular lignification was induced earlier (24 hai) and was more extensive in the resistance response mediated by Sr6 compared with those mediated by Sr24 or Sr30. These results indicate that the resistance mediated by Sr36 is similar to that mediated by Sr5 but different from those triggered by Sr6, Sr24, or Sr30. Resistance responses mediated by Sr5 and Sr36 are prehaustorial, and are a result of very rapid recognition of molecules derived from avirulent isolates of P. graminis f. sp. tritici, in contrast to the responses triggered in lines with Sr6, Sr24, and Sr30.

The time-course and the spatial accumulation of PR-proteins pr-1 and pr-5 gene transcripts were investigated in two potato cultivars differing in their levels of susceptibility to late blight, caused by Phytophthora infestans (Mont.) de Bary. Cultivars Russet Burbank (RB, susceptible) and Kennebec (KB, moderately tolerant) were inoculated with either P. infestans genotype US-1 (old lineage) or US-8 (new lineage). A strong induction of both genes was detected in both cultivars inoculated with either P. infestans genotype, as compared to the healthy-controls. The accumulation of transcripts from both genes occurred earlier in KB than in RB leaflets. By comparing the two P. infestans isolates tested, a stronger and earlier induction of both PR genes was recorded in response to US-1 as compared to US-8. The spatio-temporal profiling of pr-1 and pr-5 genes expression showed a strong and early accumulation of transcripts at the local infection site, a late and intermediate level of induction at the proximal site, and no or very weak induction at a distal site remote from the infection site. These results show that pr-1 and pr-5 genes both are related to the defense mechanisms of potato to late blight, and that the higher infection success of P. infestans US-8 as compared to US-1 might be due to the late and/or the weak induction of these defense genes.

Late blight (LB) caused by Phytophthora infestans (Mont.) de Bary is a major constraint to potato production worldwide. In North America, this disease has re-emerged as a major concern to potato producers due to an important shift in its population structure. To design sustainable control strategies, it is necessary to have a better understanding of differential interactions that potato has developed in response to the currently pre-dominant versus the old populations of the pathogen. In this study, isolates from two P. infestans genotypes, US-1 (old, mildly aggressive) and US-8 (currently predominant, highly aggressive), were used to inoculate two potato cultivars, Russet Burbank (susceptible) and Kennebec (moderately resistant). Induction patterns of genes encoding three PR proteins (pr-2, pr-3 and pr-9) in response to isolates from those two genotypes were studied using northern blot analysis in three leaf strata (Local: inoculated leaflets, Proximal: un-inoculated leaflets in the same leaf inoculated with P. infestans, and Distal: un-inoculated leaflets from the leaf adjacent to the inoculated leaf) and different times after inoculation. Our results indicated that these pr genes were activated earlier in both cultivars when inoculated with P. infestans US-1 as compared to US-8 genotype. In addition, the induction of these genes occurred earlier in Kennebec than in Russet Burbank in response to both genotypes. Furthermore, pr-2 and pr-3 were found to be induced first locally, then systemically while pr-9 transcripts could be detected only locally at the site of inoculation and only in Kennebec inoculated with P. infestans US-1.

Differential expression of pal1 and hmgr2 was investigated using northern blot analysis in two potato cultivars (Russet Burbank (RB), susceptible and Kennebec (KB), moderately tolerant) after inoculation with two Phytophthora infestans isolates from the formerly (US-1) and currently predominant genotypes (US-8). The accumulation of pal1 transcripts was weaker in response to US-8 as compared to US-1 and occurred earlier in KB than in RB. The stronger expression of pal1 in response to US-1, as compared to US-8, is suggested to be due to defense gene suppression by the latter. No apparent strong accumulation of hmgr2 transcripts was recorded in RB as compared to KB inoculated with either US-1 or US-8. The induction of pal1 and hmgr2 was first observed in un-inoculated (proximal) close to the inoculated leaflets, then in un-inoculated (distal) leaflets of leaves adjacent to the inoculated leaf, and finally in local inoculated leaflets. The stronger expression of the two genes in proximal and distal leaflets, as compared to the local site of inoculation suggests the translocation of signal(s) from this site to healthy parts of the plant.

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 × 106 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.

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.