Evolution of the Toxb Gene in Pyrenophora tritici-repentis and Related Species.

Pyrenophora tritici-repentis (tan spot) is a destructive foliar pathogen of wheat with global impact. This ascomycete fungus possesses a highly plastic open pangenome shaped by the gain and loss of effector genes. This study investigated the allelic variations in the chlorosis-encoding gene ToxB across 422 isolates representing all identified pathotypes and worldwide origins. To gain better insights into ToxB evolution, we examined its presence and variability in other Pyrenophora spp. A ToxB haplotype network was constructed, revealing the evolutionary relationships of this gene (20 haplotypes) across four Pyrenophora species. Notably, toxb, the homolog of ToxB, was detected for the first time in the barley pathogen Pyrenophora teres. The ToxB/toxb genes display evidence of selection that is characterized by loss of function, duplication, and diverse mutations. Within the ToxB/toxb open reading frame, 72 mutations were identified, including 14 synonymous, 55 nonsynonymous, and 3 indel mutations. Remarkably, a, ∼5.6-kb Copia-like retrotransposon, named Copia-1_Ptr, was found inserted in the toxb gene of a race 3 isolate. This insert disrupted the ToxB gene's function, a first case of effector gene disruption by a transposable element in P. tritici-repentis. Additionally, a microsatellite with 25 nucleotide repeats (0 to 10) in the upstream region of ToxB suggested a potential mechanism influencing ToxB expression and regulation. Exploring ToxB-like protein distribution in other ascomycetes revealed the presence of ToxB-like proteins in 19 additional species, including the Leotiomycetes class for the first time. The presence/absence pattern of ToxB-like proteins defied species relatedness compared with a phylogenetic tree, suggesting a past horizontal gene transfer event during the evolution of the ToxB gene. [Formula: see text] Copyright © 2024 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food. This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A New ToxA Haplotype in the Wheat Fungal Pathogen Bipolaris sorokiniana.

The necrotrophic effector ToxA is a well-studied virulence factor produced by several fungal necrotrophs. Initially cloned from the wheat tan spot pathogen Pyrenophora tritici-repentis in 1996, ToxA was found almost a decade later in another fungal pathogen, Parastagonospora nodorum, and its sister species, Parastagonospora pseudonodorum. In 2018, ToxA was detected in a third wheat fungal pathogenic species, Bipolaris sorokiniana, which causes spot blotch disease. However, unlike the case with P. tritici-repentis and P. nodorum, the ToxA in B. sorokiniana has only been investigated in recent years. In this report, five Australian B. sorokiniana isolates were assessed for the presence of ToxA. Four isolates were found to contain ToxA. While one isolate harbored the previously reported ToxA haplotype sequence (ToxA19), three isolates contain a different haplotype, designated herein as ToxA25, which has a nonsynonymous mutation resulting in an amino acid change of glycine to arginine at position 168. Both B. sorokiniana ToxA isoforms, when heterologously expressed in Escherichia coli, exhibited the classic ToxA necrosis-inducing activity on ToxA-sensitive Tsn1 cultivars. Preliminary analysis of the B. sorokiniana isolates in Australian wheat cultivars showed that isolates with ToxA19, ToxA25, or ToxA-deficient displayed various degrees of virulence, with the most aggressive isolates observed for those producing ToxA. Differences in spot blotch disease severity between Tsn1 and tsn1 cultivars were observed; however, this was not limited to the ToxA-producing isolates. The overall results suggest that the virulence of the Australian B. sorokiniana isolates is diverse, with the significance of ToxA-Tsn1 interactions depending on individual isolates.

A Revised Nomenclature for ToxA Haplotypes Across Multiple Fungal Species.

ToxA is one of the most studied proteinaceous necrotrophic effectors produced by plant pathogens. It has been identified in four pathogens (Pyrenophora tritici-repentis, Parastagonospora nodorum, Parastagonospora pseudonodorum [formerly Parastagonospora avenaria f. sp. tritici], and Bipolaris sorokiniana) causing leaf spot diseases on cereals worldwide. To date, 24 different ToxA haplotypes have been identified. Some P. tritici-repentis and related species also express ToxB, another small protein necrotrophic effector. We present here a revised and standardized nomenclature for these effectors, which could be extended to other poly-haplotypic genes found across multiple species.

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.

Comparison of single-trait and multi-trait genomic predictions on agronomic and disease resistance traits in spring wheat.

KEY MESSAGE: This study performed comprehensive analyses on the predictive abilities of single-trait and two multi-trait models in three populations. Our results demonstrated the superiority of multi-traits over single-trait models across seven agronomic and four to seven disease resistance traits of different genetic architecture. The predictive ability of multi-trait and single-trait prediction models has not been investigated on diverse traits evaluated under organic and conventional management systems. Here, we compared the predictive abilities of 25% of a testing set that has not been evaluated for a single trait (ST), not evaluated for multi-traits (MT1), and evaluated for some traits but not others (MT2) in three spring wheat populations genotyped either with the wheat 90K single nucleotide polymorphisms array or DArTseq. Analyses were performed on seven agronomic traits evaluated under conventional and organic management systems, four to seven disease resistance traits, and all agronomic and disease resistance traits simultaneously. The average prediction accuracies of the ST, MT1, and MT2 models varied from 0.03 to 0.78 (mean 0.41), from 0.05 to 0.82 (mean 0.47), and from 0.05 to 0.92 (mean 0.67), respectively. The predictive ability of the MT2 model was significantly greater than the ST model in all traits and populations except common bunt with the MT1 model being intermediate between them. The MT2 model increased prediction accuracies over the ST and MT1 models in all traits by 9.0-82.4% (mean 37.3%) and 2.9-82.5% (mean 25.7%), respectively, except common bunt that showed up to 7.7% smaller accuracies in two populations. A joint analysis of all agronomic and disease resistance traits further improved accuracies within the MT1 and MT2 models on average by 21.4% and 17.4%, respectively, as compared to either the agronomic or disease resistance traits, demonstrating the high potential of the multi-traits models in improving prediction accuracies.

Identification of a Novel ToxA Haplotype of Pyrenophora tritici-repentis from Japan.

Pyrenophora tritici-repentis was described first as a pathogen of wheat (tan spot) in Japan in the 1920s, but, since then, no reports on P. tritici-repentis race structure or its effectors in Japan have been published. In this study, 10 single-spore isolates of P. tritici-repentis were collected from bread wheat in Japan. These isolates were evaluated for virulence on four differential wheat genotypes and tested for the presence/absence of the effector-encoding genes, ToxA and ToxB, in multiplex PCR assays. These isolates were identified as ToxA producers, of which eight were designated as race 2 (ToxA producers) and two were classified as race 1 (ToxA and ToxC producers) based on their virulence patterns. Sequence analysis of the ToxA amplicons from these 10 isolates indicated the presence of a novel ToxA haplotype (denoted PtrA2). A comparative sequence analysis and resequencing of ToxA from reference P. tritici-repentis isolates showed that all previously published ToxA haplotypes in P. tritici-repentis were identical, and are hence denoted PtrA1 in this study. A total of 163 PtrToxA sequences from global origins were already deposited in GenBank and were confirmed identical to PtrA1. Sequence variation in PtrA1 and PtrA2 open reading frames were found at three positions: one synonymous mutation at position 412 (C/G) and two nonsynonymous mutations at positions 342 and 362 that alter amino acid sequence. These mutations did not seem to affect the necrosis development on a ToxA-sensitive wheat genotype when rated for symptoms 5 to 7 days after inoculation. This is the first report correctly confirming the presence of an additional novel ToxA haplotype in P. tritici-repentis for which we have predicted its isoform and updated the ToxA haplotype evolutionary network.

Diversity of Fusarium spp. Associated with Wheat Node and Grain in Representative Sites Across the Western Canadian Prairies.

Fusarium head blight (FHB) and Fusarium crown and root rot (FCRR) are major wheat diseases. Populations of FHB and FCRR pathogens are highly dynamic, and shifts in these populations in different regions is reported. Analyzing fungal populations associated with wheat node and grain tissues collected from different regions can provide useful information and predict diseases that might affect subsequent crops and effective disease management practices. In this study, wheat node and grain samples were collected from four representative sites across the western Canadian prairies in the 2018 growing season to characterize the major Fusarium spp. and other mycobiota associated with wheat in these regions. In total, 994 fungal isolates were recovered, and based on culture and molecular diagnostic methods, three genera constituted over 90% of all fungal isolates, namely Alternaria (39.6%), Fusarium (27.8%), and Parastagonospora (23.9%). A quantitative PCR (qPCR) diagnostic toolkit was developed to quantify the most frequently isolated Fusarium spp. in infected wheat tissues: Fusarium avenaceum, F. culmorum, F. graminearum, and F. poae. This qPCR specificity was validated in silico, in vitro, and in planta and proved specific to the target species. The qPCR results showed that F. graminearum was not detected frequently from wheat node and grain samples collected from four locations in this study. F. poae was the most abundant Fusarium species in grain samples in all tested locations. However, in node samples, F. culmorum (Beaverlodge and Scott) and F. avenaceum (Lacombe and Lethbridge) were the most abundant species. Trichothecene genotyping showed that the 3ADON is the most dominant trichothecene genotype (68%), followed by type-A trichothecenes (29.5%), whereas the 15ADON trichothecene genotype was least dominant (2.5%) and the NIV genotype was not detected. Moreover, a total of 129 translation elongation factor 1-alpha (TEF1α) sequences from nine Fusarium spp. were compared at the haplotype level to evaluate genetic variability and distribution. F. avenaceum and F. poae exhibited higher diversity as reflected by higher number of haplotypes present in these two species compared with the rest.

The Changing Virulence of Stripe Rust in Canada from 1984 to 2017.

Stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici, is an important wheat disease worldwide. In this study, the P. striiformis f. sp. tritici population in Canada, representing a time period from 1984 to 2017, was analyzed for virulence diversity and geographical distribution. Virulence of 140 P. striiformis f. sp. tritici isolates was evaluated on 17 near-isogenic wheat lines in the 'Avocet S' background, each containing a single resistance gene along with an 18th line 'Tyee'. Seedlings were inoculated with a urediniospore/talc mixture and infection types were evaluated on a scale of 0 to 9. In total, 89 races were identified with various combinations of defeated Yr genes. Clear changes in pathogen virulence have been observed through time that are confirmed by clustering algorithms. The results showed that the tested P. striiformis f. sp. tritici isolates remained avirulent on Yr1, Yr5, and Yr15, and have very low frequency of virulence on Yr76, but had high frequencies of virulence on Yr6, Yr7, Yr8, Yr9, Yr17, Yr43, Yr44, YrTr1, and YrExp2. P. striiformis f. sp. tritici virulence spiked on Yr7, Yr8, and Yr9 for the first time in 2000, and on Yr10 and Yr27 in 2010. Overall, the predominant races in Canada were very similar to those reported in the United States (PSTv-37, PSTv-41, and PSTv-52), which indicates long-distance migration of P. striiformis f. sp. tritici from the United States to Canada. Sixty-four races had unique virulence combinations that had not been previously reported in the United States, which suggested that evolution of virulence/avirulence for host resistance by mutation at local scale, is possible. Analysis of diversity between Canadian isolates and races from the United States since 2010 showed that the P. striiformis f. sp. tritici population in western Canada is similar to that in the western states of the United States, and that the population in eastern Canada is similar to the eastern and/or central regions of the United States, supporting the hypothesis that specific P. striiformis f. sp. tritici populations in North America travel through different wind trajectories.

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.

Parastagonospora nodorum and Related Species in Western Canada: Genetic Variability and Effector Genes.

Parastagonospora nodorum is an important fungal pathogen that causes Septoria nodorum blotch (SNB) in wheat. This pathogen produces several necrotrophic effectors that act as virulence factors; three have been cloned, SnToxA, SnTox1, and SnTox3. In this study, P. nodorum and its sister species P. avenaria f. tritici (Pat1) were isolated from wheat node and grain samples collected from distanced sites in western Canada during 2018. The presence of effector genes and associated haplotypes were determined by PCR and sequence analysis. An internal transcribed spacer-restriction fragment length polymorphism test was developed to distinguish between leaf spotting pathogens (P. nodorum, Pat1, Pyrenophora tritici-repentis, and Bipolaris sorokiniana). P. nodorum was mainly recovered from wheat nodes and to a lesser extent from the grains, while Pat1 was exclusively isolated from grain samples. The effector genes were present in almost all P. nodorum isolates, with the ToxA haplotype 5 (H5) being most prevalent, while a novel ToxA haplotype (denoted here H21) is reported for the first time. In Pat1, only combinations of SnTox1 and SnTox3 genes were present. A ToxA haplotype network was also constructed to assess the evolutionary relationship among globally found haplotypes to date. Finally, cultivars representing wheat development in Canada for the last century were tested for sensitivity to Sn-effectors and to the presence of Tsn1, the ToxA sensitivity gene. Of tested cultivars, 32.9 and 56.9% were sensitive to SnTox1 and SnTox3, respectively, and Tsn1 was present in 59% of the cultivars. In conclusion, P. nodorum and Pat1 were prevalent wheat pathogens in Canada with a potential tissue-specific colonization capacity, while producing necrotrophic effectors to which wheat is sensitive.

Specific Detection and Quantification of Major Fusarium spp. Associated with Cereal and Pulse Crops.

Plant pathogenic Fusarium spp. are widespread and cause important diseases on a wide host range, including economically important cereal and pulse crops. A number of molecular methods have been used to detect, identify, and quantify a long list of plant pathogenic Fusarium spp. In general, these methods are much faster, highly specific, more sensitive, and more accurate than culture-based methods and can be performed and interpreted by personnel with no specialized taxonomical expertise. The accurate isolation and identification of these pathogens is required to effectively manage diseases caused by pathogenic Fusarium spp. In this chapter, we present detailed molecular methods for detection, quantification, and differentiation between many of the Fusarium spp. associated with cereal and pulse crops.

Genomic Predictions for Common Bunt, FHB, Stripe Rust, Leaf Rust, and Leaf Spotting Resistance in Spring Wheat.

Some studies have investigated the potential of genomic selection (GS) on stripe rust, leaf rust, Fusarium head blight (FHB), and leaf spot in wheat, but none of them have assessed the effect of the reaction norm model that incorporated GE interactions. In addition, the prediction accuracy on common bunt has not previously been studied. Here, we investigated within-population prediction accuracies using the baseline M1 model and two reaction norm models (M2 and M3) with three random cross-validation (CV1, CV2, and CV0) schemes. Three Canadian spring wheat populations were evaluated in up to eight field environments and genotyped with 3158, 5732, and 23,795 polymorphic markers. The M3 model that incorporated GE interactions reduced residual variance by an average of 10.2% as compared with the main effect M2 model and increased prediction accuracies on average by 2-6%. In some traits, the M3 model increased prediction accuracies up to 54% as compared with the M2 model. The average prediction accuracies of the M3 model with CV1, CV2, and CV0 schemes varied from 0.02 to 0.48, from 0.25 to 0.84, and from 0.14 to 0.87, respectively. In both CV2 and CV0 schemes, stripe rust in all three populations, common bunt and leaf rust in two populations, as well as FHB severity, FHB index, and leaf spot in one population had high to very high (0.54-0.87) prediction accuracies. This is the first comprehensive genomic selection study on five major diseases in spring wheat.

Genomic Prediction Accuracy of Stripe Rust in Six Spring Wheat Populations by Modeling Genotype by Environment Interaction.

Some previous studies have assessed the predictive ability of genome-wide selection on stripe (yellow) rust resistance in wheat, but the effect of genotype by environment interaction (GEI) in prediction accuracies has not been well studied in diverse genetic backgrounds. Here, we compared the predictive ability of a model based on phenotypic data only (M1), the main effect of phenotype and molecular markers (M2), and a model that incorporated GEI (M3) using three cross-validations (CV1, CV2, and CV0) scenarios of interest to breeders in six spring wheat populations. Each population was evaluated at three to eight field nurseries and genotyped with either the DArTseq technology or the wheat 90K single nucleotide polymorphism arrays, of which a subset of 1,058- 23,795 polymorphic markers were used for the analyses. In the CV1 scenario, the mean prediction accuracies of the M1, M2, and M3 models across the six populations varied from -0.11 to -0.07, from 0.22 to 0.49, and from 0.19 to 0.48, respectively. Mean accuracies obtained using the M3 model in the CV1 scenario were significantly greater than the M2 model in two populations, the same in three populations, and smaller in one population. In both the CV2 and CV0 scenarios, the mean prediction accuracies of the three models varied from 0.53 to 0.84 and were not significantly different in all populations, except the Attila/CDC Go in the CV2, where the M3 model gave greater accuracy than both the M1 and M2 models. Overall, the M3 model increased prediction accuracies in some populations by up to 12.4% and decreased accuracy in others by up to 17.4%, demonstrating inconsistent results among genetic backgrounds that require considering each population separately. This is the first comprehensive genome-wide prediction study that investigated details of the effect of GEI on stripe rust resistance across diverse spring wheat populations.

Identification of Disease Resistance Parents and Genome-Wide Association Mapping of Resistance in Spring Wheat.

The likelihood of success in developing modern cultivars depend on multiple factors, including the identification of suitable parents to initiate new crosses, and characterizations of genomic regions associated with target traits. The objectives of the present study were to (a) determine the best economic weights of four major wheat diseases (leaf spot, common bunt, leaf rust, and stripe rust) and grain yield for multi-trait restrictive linear phenotypic selection index (RLPSI), (b) select the top 10% cultivars and lines (hereafter referred as genotypes) with better resistance to combinations of the four diseases and acceptable grain yield as potential parents, and (c) map genomic regions associated with resistance to each disease using genome-wide association study (GWAS). A diversity panel of 196 spring wheat genotypes was evaluated for their reaction to stripe rust at eight environments, leaf rust at four environments, leaf spot at three environments, common bunt at two environments, and grain yield at five environments. The panel was genotyped with the Wheat 90K SNP array and a few KASP SNPs of which we used 23,342 markers for statistical analyses. The RLPSI analysis performed by restricting the expected genetic gain for yield displayed significant (p < 0.05) differences among the 3125 economic weights. Using the best four economic weights, a subset of 22 of the 196 genotypes were selected as potential parents with resistance to the four diseases and acceptable grain yield. GWAS identified 37 genomic regions, which included 12 for common bunt, 13 for leaf rust, 5 for stripe rust, and 7 for leaf spot. Each genomic region explained from 6.6 to 16.9% and together accounted for 39.4% of the stripe rust, 49.1% of the leaf spot, 94.0% of the leaf rust, and 97.9% of the common bunt phenotypic variance combined across all environments. Results from this study provide valuable information for wheat breeders selecting parental combinations for new crosses to develop improved germplasm with enhanced resistance to the four diseases as well as the physical positions of genomic regions that confer resistance, which facilitates direct comparisons for independent mapping studies in the future.

Identification of a Locus Conferring Dominant Susceptibility to Pyrenophora tritici-repentis in Barley.

The fungus Pyrenophora tritici-repentis (Ptr) causes tan spot, a destructive foliar disease of wheat worldwide. The pathogen produces several necrotrophic effectors, which induce necrosis or chlorosis on susceptible wheat lines. Multiple races of Ptr have been identified, based on their ability to produce one or more of these effectors. Ptr has a wide host range of cereal and non-cereal grasses, but is known to cause damage only on wheat. Previously, we showed that Ptr can interact specifically with cultivated barley (Hordeum vulgare ssp. vulgare), and that the necrotrophic effector Ptr ToxB induces mild chlorosis in a highly selective manner when infiltrated into certain barley genotypes. In the present study, a barley doubled-haploid (DH) population was evaluated for reaction to Ptr race 5, a Ptr ToxB-producer. Then a comprehensive genetic map composed of 381 single nucleotide polymorphism (SNP) markers was used to map the locus conditioning this chlorosis. The F1 seedlings, and 92 DH lines derived from a cross between the resistant Japanese malting barley cultivar Haruna Nijo and the susceptible wild barley (H. vulgare ssp. spontaneum) OUH602 were inoculated with a conidial suspension of Ptr race 5 isolate at the two-leaf stage. The seedlings were monitored daily for symptoms and assessed for chlorosis development on the second leaf, 6 days after inoculation. All tested F1 seedlings exhibited chlorosis symptoms similar to the susceptible parent, and the DH lines segregated 1:1 for susceptible:resistant phenotypes, indicating the involvement of a single locus. Marker-trait linkage analysis based on interval mapping identified a single locus on the distal region of the short arm of chromosome 2H. We designate this locus Susceptibility to P. tritici-repentis1 (Spr1). The region encompassing this locus has 99 high confidence gene models, including membrane receptor-like kinases (RLKs), intracellular nucleotide-binding, leucine-rich repeat receptors (NLRs), and ankyrin-repeat proteins (ANKs). This shows the involvement of a dominant locus conferring susceptibility to Ptr in barley. Further work using high-resolution mapping and transgenic complementation will be required to identify the underlying gene.

Pyrenophora tritici-repentis in Tunisia: Race Structure and Effector Genes.

Tan spot is a destructive foliar wheat disease worldwide and caused by the ascomycete fungus Pyrenophora tritici-repentis (Ptr); it has become more frequent in Tunisia over the last decade. In this study, the virulence of 73 single-spore isolates, collected from durum and bread wheat fields during 2017-2018 growing season, was evaluated on four differential wheat genotypes. This was followed by polymerase chain reaction tests with specific primers for the effector genes ToxA, ToxB, and toxb (ToxB-homolog). Sequence analysis to validate the identity of the amplified genes was followed, and ToxA amplicons from a subset of 22 isolates were analyzed to determine its haplotype identity. Ptr isolates from Tunisia were grouped in races 2, 4, 5, and 7, and 44% of the tested isolates did not fit under any known race, and were denoted here as atypical. These atypical isolates induced the same symptoms as race 7 isolates, extensive necrosis, and chlorosis on susceptible genotypes, but lacked the ToxA gene. ToxA is the only identified necrosis-inducing effector in Ptr, and was amplified in 51% of tested isolates, and shared identical sequence to previously identified haplotype (H15). ToxB and its homolog toxb were present in 97% and 93% of tested isolates, respectively. Ptr in Tunisia lacked Ptr ToxC activity, and none of the tested isolates induced the specific symptoms of that effector. Race 7 and the atypical isolates dominated the Tunisian Ptr population, while races 2, 4, and 5 were found at low percentages. In conclusion, ToxB and its homolog were the most dominant genes in Ptr from Tunisia, and the majority of the isolates induced necrosis and chlorosis on Ptr ToxA and Ptr ToxB susceptible wheat genotypes. However, only about half of that necrosis can be attributed to ToxA presence, this result necessitates further research to investigate the prevalence of additional necrotic effector(s). Terminology: in this paper, Pyrenophora tritici-repentis abbreviated as Ptr, the effectors are referred to by Ptr ToxA, Ptr ToxB and Ptr ToxC, and the genes coding for them are written in italic as ToxA, ToxB, and ToxC, respectively.

An exo-1,3-ß-glucanase GLU1 contributes to the virulence of the wheat tan spot pathogen Pyrenophora tritici-repentis.

Tan spot, caused by Pyrenophora tritici-repentis, is an important foliar disease of wheat. In the present study, a gene named glucanase gene (GLU1) encoding a putative exo-1,3-ß-glucanase was cloned from a race five isolate of P. tritici-repentis. Transcription profile analysis of the GLU1 gene showed a carbon source control of the accumulation of transcript, which is strongly induced in minimal medium but depressed in rich medium. A time-course study of the infection process of the wild-type isolate on a susceptible wheat genotype revealed that the transcript level of GLU1 increased more than 8000-fold 8 h after inoculation. To study its potential function in pathogenicity, GLU1 was silenced via a sense and antisense mediated silencing mechanism. One transformant named C1 showed significantly reduced growth and sporulation relative to the wild-type. Cytological analysis of the infection revealed that C1 produced significantly lower numbers of germ tubes and appressoria than the wild-type strain on susceptible wheat leaves. This strain, as well as another two transformants, caused significantly less disease symptoms relative to the wild-type after inoculation onto a susceptible wheat genotype. These results indicate that GLU1 contributes to the development and virulence of P. tritici-repentis.

RNA-mediated gene silencing of ToxB in Pyrenophora tritici-repentis.

The fungus Pyrenophora tritici-repentis causes tan spot, a wheat leaf disease of worldwide importance. The pathogen produces three host-selective toxins, including Ptr ToxB, which causes chlorophyll degradation and foliar chlorosis on toxin-sensitive wheat genotypes. The ToxB gene, which codes for Ptr ToxB, was silenced in a wild-type race 5 isolate of the fungus thorough a sense- and antisense-mediated silencing mechanism. Toxin production by the silenced strains was evaluated in culture filtrates of the fungus via Western blotting analysis, and plant bioassays were conducted to test the virulence of the transformants in planta. The chlorosis-inducing ability of the silenced strains was correlated with the quantity of Ptr ToxB, and transformants in which toxin production was strongly decreased also caused very little disease on toxin-sensitive wheat genotypes. Cytological analysis of the infection process revealed that, in addition to a reduced capacity to induce chlorosis, the silenced strains with the greatest decrease in the levels of Ptr ToxB produced significantly fewer appressoria than the wild-type isolate, 12 and 24 h after inoculation onto wheat leaves. The results provide strong support for the suggestion that the amount of Ptr ToxB protein produced by fungal isolates plays a significant role in the quantitative variation in the virulence of P. tritici-repentis.