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Pyrenophora tritici-repentis causes tan spot, an important foliar disease of wheat. A collection of P. tritici-repentis isolates from Tunisia, located in one of the main secondary centers of diversification of durum wheat, was tested for phenotypic race classification based on virulence on a host differential set and for the presence of the necrotrophic effector (NE) genes ToxA, ToxB, and toxb by PCR analysis. While races 2, 4, 5, 6, 7, and 8 were identified according to their virulence phenotypes, PCR testing indicated the presence of “atypical” isolates that induced necrosis on the wheat differential ‘Glenlea,’ but lacked the expected ToxA gene, suggesting the involvement of other NEs in the P. tritici-repentis/wheat interaction. Genetic diversity and the P. tritici-repentis population structure were explored further by examining 59 Tunisian isolates and 35 isolates from Algeria, Azerbaijan, Canada, Iran, and Syria using 24 simple sequence repeat markers. Average genetic diversity, overall gene flow, and percentage polymorphic loci were estimated as 0.58, 2.09, and 87%, respectively. Analysis of molecular variance showed that 81% of the genetic variance occurred within populations and 19% occurred between populations. Cluster analysis by the unweighted pair group method indicated that ToxB– isolates grouped together and were distantly related to ToxB+ isolates. Based on Nei’s analysis, the global collection clustered into two distinct groups according to their region of origin. The results suggest that geographic origin and the host specificity imposed by different NEs can lead to differentiation among P. tritici-repentis populations.

Septoria tritici blotch (STB) caused by Zymoseptoria tritici is the primary biotic stress for durum wheat production in Tunisia. The present study was aimed to decipher the genetic diversity and population structure of Z. tritici bread wheat isolates; in the northern regions of Tunisia this pathogen infected exclusively bread wheat. A total of 162 single-spore isolates were sampled during the 2015–2016 growing season from nine naturally infected bread wheat fields at four locations in the three main wheat-growing regions in Tunisia, previously reported as hot spots for Septoria disease (Cap Bon, Bizerte, and Beja). Collected isolates were fingerprinted using twelve polymorphic microsatellite markers to assess the genetic diversity and population structure of Z. tritici. All the microsatellite loci were polymorphic and a high genetic diversity was observed within the whole population. The highest Nei’s index value (0.42), Shannon Index (0.84) and private allele numbers (36) were found at the El Haouaria location (Cap Bon region). Furthermore, a moderate genetic differentiation within and among the surveyed locations (73% and 27%) was confirmed by analysis of molecular variance (AMOVA). On the other hand, the STRUCTURE program was shown to be less sensitive in revealing genetic structure especially at low levels of diversity. Thus, information on the genetic structure of the pathogen population collected from bread wheat is useful for designing and implementing durable and effective management strategies.

Septoria tritici blotch (STB) disease caused by Zymoseptoria tritici is the most predominant disease on durum wheat in Tunisia, while its occurrence on bread wheat is rare. In this study, we investigated the performance of 89 wheat cultivars from Morocco, Algeria and Tunisia, screened in Tunisia for their relative resistance to STB. Field experiments were carried out in an augmented design, during 2016–17 and 2017–18 cropping seasons at two locations in Northern Tunisia: Beja (Oued Beja station) and Cap Bon regions (Menzel Temim and El Haouaria). All trials were conducted under natural infection. Visual disease assessments were quantified using the percentage of leaf area covered by pycnidia (PC), necrotic area (N), area under disease progress curve of each genotype (AUDPC) and the relative area under disease progress curve (rAUDPC). Results indicated that the majority of Tunisian, Algerian and Moroccan durum wheat populations (TDM, ADW and MDW) were susceptible to STB at both locations in Tunisia ranging from susceptible to highly susceptible, with the rAUDPC, N and PC ranging from 0.5 to 0.8, 30 to 65% and 40 to 75% respectively. On the other hand, the Moroccan bread wheat genotypes (MBW) were susceptible in Cap Bon area and resistant at Beja. Tunisian bread wheat genotypes (TBW) were resistant at both locations; with levels varying from immune to resistant classes where rAUDPC, PC and N did not exceed 0.2 and 10% respectively, with the exception of the local bread wheat variety known as “Farina Arbi” which was susceptible at Cap Bon and resistant at Beja.

Durum wheat landraces have huge potential for the identification of genetic factors valuable for improving resistance to biotic stresses. Tunisia is known as a hot spot for Septoria tritici blotch disease (STB), caused by the fungus Zymoseptoria tritici (Z. tritici). In this context, a collection of 3166 Mediterranean durum wheat landraces were evaluated at the seedling and adult stages for STB resistance in the 2016–2017 cropping season under field conditions in Kodia (Tunisia). Unadapted/susceptible accessions were eliminated to reach the final set of 1059 accessions; this was termed the Med-collection, which comprised accessions from 13 countries and was also screened in the 2018–2019 cropping season. The Med-collection showed high frequency of resistance reactions, among which over 50% showed an immune reaction (HR) at both seedling and adult growth stages. Interestingly, 92% of HR and R accessions maintained their resistance levels across the two years, confirming the highly significant correlation found between seedling- and adult-stage reactions. Plant Height was found to have a negative significant effect on adult-stage resistance, suggesting that either this trait can influence disease severity, or that it can be due to environmental/epidemiological factors. Accessions from Italy showed the highest variability, while those from Portugal, Spain and Tunisia showed the highest levels of resistance at both growth stages, suggesting that the latter accessions may harbor novel QTLs effective for STB resistance.

This chapter reviews progress and challenges in breeding disease-resistant cereals in the West Asia and North Africa (WANA) region. It discusses challenges in ensuring durable resistance in the face of constantly-evolving pathogen threats. It includes case studies on progress in combatting rust diseases in Iran and combatting Septoria in North Africa.

Durum wheat (Triticum turgidumL. subsp.durum) landraces represent a prominent genetic resource for Mediterranean farming systems and breeding programs. Fourteen landraces sampled in Tunisia were genotyped with 9 microsatellite markers and characterized with 15 morphological descriptors, including resistance to the fungal diseaseSeptoria triticiblotch (STB). The genetic diversity, nearly was as important within landraces populations (45%) than between populations (54%). It was structured in seven genetic groups and was only partly explained by the variety name or the locality of origin. Populations were also greatly diversified phenotypically (Shannon-Weaver H’=0.54) with traits related to spike and awn colours being the most diversified. Resistance to STB was either qualitative in two populations or with varying degrees of quantitative resistance in the others. A Pst-Fstcomparison indicate a local adaptation of the populations. Overall, the genetic structure of Tunisian durum wheat landraces revealed a complex selection trajectory and seed exchanges between farmers.

Tan spot (TS), caused by the fugus Pyrenophora tritici-repentis (Ptr), has gained significant importance in the last few years, thereby representing a threat to wheat production in all major wheat-growing regions, including Tunisia. In this context, we evaluated a Mediterranean collection of 549 durum wheat accessions under field conditions for resistance to Ptr over two cropping seasons in Jendouba (Tunisia), a hot spot for Ptr. The relative disease severities showed significant phenotypic variation from resistance to susceptibility. The correlation between disease scores over the two trials was significant, as 50% of the accessions maintained good levels of resistance (resistant–moderately resistant). Seedling and adult-stage reactions were significantly correlated. The ANOVA analysis revealed that the genotype term is highly significant at the adult stage, thus emphasizing the high genetic variability of the tested accessions. Reaction-type comparison among and between countries revealed a high diversity of TS resistance. Plant height (PH) was negatively correlated to disease scores, indicating that PH might either have a significant effect on TS severity or that it can be a potential disease escape trait. The evaluation of this collection allowed for the identification of potential diverse resistance sources to Ptr that can be incorporated in breeding programs.

Mixtures of cultivars with contrasting levels of resistance can suppress infectious diseases in wheat, as demonstrated in numerous field experiments. Most studies focused on airborne pathogens in bread wheat, while splash-dispersed pathogens have received less attention, and no studies have been conducted in durum wheat. We conducted a two-year field experiment in Tunisia, to evaluate the performance of cultivar mixtures with varying proportions of resistance (0–100%) in controlling the polycyclic, splash-dispersed disease Septoria tritici blotch (STB) in durum wheat. To measure STB severity, we used a high-throughput method based on digital image analysis of 3074 infected leaves collected from 42 and 40 experimental plots during the first and second years, respectively. This allowed us to quantify pathogen reproduction on wheat leaves and to acquire a large dataset that exceeds previous studies with respect to accuracy and precision. Our analyses show that introducing only 25% of a disease-resistant cultivar into a pure stand of a susceptible cultivar provides a substantial reduction of almost 50% in disease severity compared to the susceptible pure stand. However, comprising the resistant component of two cultivars instead of one did not further improve disease control, contrary to predictions of epidemiological theory. Susceptible cultivars can be agronomically superior to resistant cultivars or be better accepted by growers for other reasons. Hence, if mixtures with only a moderate proportion of the resistant cultivar provide a similar degree of disease control as resistant pure stands, as our analysis indicates, such mixtures are more likely to be accepted by growers.

Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z.?tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In?planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z.?tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z.?tritici–wheat pathosystem

Zymoseptoria tritici is an economically important pathogen of wheat. However, the molecular basis of pathogenicity on wheat is still poorly understood. Here, we present a global survey of the proteins secreted by this fungus in the apoplast of resistant (cv. Shafir) and susceptible (cv. Obelisk) wheat cultivars after inoculation with reference Z. tritici strain IPO323. The fungal proteins present in apoplastic fluids were analyzed by gel electrophoresis and by data-independent acquisition liquid chromatography/mass spectrometry (LC/MSE) combined with data-dependent acquisition LC–MS/MS. Subsequent mapping mass spectrometry-derived peptide sequence data against the genome sequence of strain IPO323 identified 665 peptides in the MSE and 93 in the LC–MS/MS mode that matched to 85 proteins. The identified fungal proteins, including cell-wall degrading enzymes and proteases, might function in pathogenicity, but the functions of many remain unknown. Most fungal proteins accumulated in cv. Obelisk at the onset of necrotrophy. This inventory provides an excellent basis for future detailed studies on the role of these genes and their encoded proteins during pathogenesis in wheat.

International audience; With the increase of sequenced fungal genomes, high-throughput methods for functional analyses of genes are needed. We assessed the potential of a new transposon mutagenesis tool deploying a Fusarium oxysporum miniature inverted-repeat transposable element mimp1, mobilized by the transposase of impala, a Tc1-like transposon, to obtain knock-out mutants in Fusarium graminearum. We localized 91 mimp1 insertions which showed good distribution over the entire genome. The main exception was a major hotspot on chromosome 2 where independent insertions occurred at exactly the same nucleotide position. Furthermore insertions in promoter regions were over-represented. Screening 331 mutants for sexual development, radial growth and pathogenicity on wheat resulted in 19 mutants (5.7%) with altered phenotypes. Complementation with the original gene restored the wild-type phenotype in two selected mutants demonstrating the high tagging efficiency. This is the first report of a MITE transposon tagging system as an efficient mutagenesis tool in F. graminearum.

Transposon mutagenesis was applied to generate mutants in Fusarium graminearum. The mimp1/impala system originally identified in F. oxysporum proved very promising for mutagenesis as the transposon and reinserted at high frequency in (the vicinity) of genes. A collection of mutants was screened for growth, for pathogenicity and for perithecia production. Several mutants blocked in one or more functions were obtained. The wild-type phenotype of one such mutant could be restored by complementation with a non-disrupted copy of the gene. In addition reinsertions occurred on each of the four chromosomes of F. graminearum, making this system a powerful tool in the functional analyses of the > 10,000 genes predicted in the F. graminearum genome.

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed “mesosynteny” is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors., PLoS Genetics, 7 (6), ISSN:1553-7390, ISSN:1553-7404

Culture filtrates (CFs) of the fungal wheat pathogen Zymoseptoria tritici were assayed for necrosis-inducing activity after infiltration in leaves of various wheat cultivars. Active fractions were partially purified and characterized. The necrosis-inducing factors in CFs are proteinaceous, heat stable and their necrosis-inducing activity is temperature and light dependent. The in planta activity of CFs was tested by a time series of proteinase K (PK) co-infiltrations, which was unable to affect activity 30min after CF infiltrations. This suggests that the necrosis inducing proteins (NIPs) are either absent from the apoplast and likely actively transported into mesophyll cells or protected from the protease by association with a receptor. Alternatively, plant cell death signaling pathways might be fully engaged during the first 30min and cannot be reversed even after PK treatment. Further fractionation of the CFs with the highest necrosis-inducing activity involved fast performance liquid chromatography, SDS-PAGE and mass spectrometry. This revealed that most of the proteins present in the fractions have not been described before. The two most prominent ZtNIP encoding candidates were heterologously expressed in Pichia pastoris and subsequent infiltration assays showed their differential activity in a range of wheat cultivars.

The mimp1 element previously identified in the ascomycete fungus Fusarium oxysporum has hallmarks of miniature inverted-repeat transposable elements (MITEs): short size, terminal inverted repeats (TIRs), structural homogeneity, and a stable secondary structure. Since mimp1 has no coding capacity, its mobilization requires a transposase-encoding element. On the basis of the similarity of TIRs and target-site preference with the autonomous Tc1-like element impala, together with a correlated distribution of both elements among the Fusarium genus, we investigated the ability of mimp1 to jump upon expression of the impala transposase provided in trans. Under these conditions, we present evidence that mimp1 transposes by a cut-and-paste mechanism into TA dinucleotides, which are duplicated upon insertion. Our results also show that mimp1 reinserts very frequently in genic regions for at least one-third of the cases. We also show that the mimp1/impala double-component system is fully functional in the heterologous species F. graminearum, allowing the development of a highly efficient tool for gene tagging in filamentous fungi.