Development and characterisation of novel durum wheat-H. chilense 4Hch chromosome lines as a source for resistance to Septoria tritici blotch.

The use of wild species as a source of genetic variability is a valued tool in the framework of crop breeding. Hordeum chilense Roem. et Schult is a wild barley species that can be a useful genetic donor for sustainable wheat breeding which carries genes conferring resistance to some diseases or increasing grain quality, among others. Septoria tritici blotch (STB), caused by the Zymoseptoria tritici fungus, is one of the most important wheat diseases worldwide, affecting both bread and durum wheat and having a high economic impact. Resistance to STB has been previously described in H. chilense chromosome 4Hch. In this study, we have developed introgression lines for H. chilense chromosome 4Hch in durum wheat using interspecific crosses, advanced backcrosses, and consecutive selfing strategies. Alien H. chilense chromosome segments have been reduced in size by genetic crosses between H. chilense disomic substitution lines in durum wheat and durum wheat lines carrying the Ph1 deletion. Hordeum chilense genetic introgressions were identified in the wheat background through several plant generations by fluorescence in situ hybridisation (FISH) and simple sequence repeat (SSR) markers. An STB infection analysis has also been developed to assess STB resistance to a specific H. chilense chromosome region. The development of these H. chilense introgression lines with moderate to high resistance to STB represents an important advance in the framework of durum breeding and can be a valuable tool for plant breeders.

Resistance of wheat genotypes to Mycosphaerella graminicola isolates at seedling stage under greenhouse conditions.

One of the most devastating foliar diseases of wheat worldwide is Septoria leaf blotch (STB), caused by Mycosphaerella graminicola (asexual stage/Anamorph: Septoria tritici) which has been recently intensified in some regions in Iran. In this study, 49 wheat genotypes and 20 wheat differential genotypes were evaluated for their reaction to infection by six isolates of M. graminicola collected from infected fields during 2016-2017 at seedling stage under greenhouse conditions. According to the analysis of variance (ANOVA) of leaf pycnidia coverage percentage, a significant difference (p < .01) was observed between M. graminicola isolates and wheat cultivars. The interaction between genotypes and isolates was also significant (p < .01) and the results indicated a specific interaction between genotypes and isolates. The results presented Dezful and West Azerbaijan isolates that were the most virulent with more pathogenesis on differential genotypes. Although 47 of the wheat genotypes were susceptible to all isolates, some genotypes, including Wc-46,224 (Austria), Wc-45,425 (Portugal), Wc-45,565 (Turkey), P.S.No4 (Italy), Dehdasht, M3 Synthetic, KavKaz-k4500, Arina, Flame, and Riband were resistant to all isolates. In addition, the isolates exhibited different virulence patterns on wheat genotypes. The results of this study revealed high virulence of M. graminicola isolates, and Iranian and foreign wheat genotypes, commonly used in the region, presented high susceptibility, and the resistance sources had been identified among genotypes that can be applied in the wheat breeding programs.

Large-scale study validates that regional fungicide applications are major determinants of resistance evolution in the wheat pathogen Zymoseptoria tritici in France.

In modern cropping systems, the near-universal use of plant protection products selects for resistance in pest populations. The emergence and evolution of this adaptive trait threaten treatment efficacy. We identified determinants of fungicide resistance evolution and quantified their effects at a large spatiotemporal scale. We focused on Zymoseptoria tritici, which causes leaf blotch in wheat. Phenotypes of qualitative or quantitative resistance to various fungicides were monitored annually, from 2004 to 2017, at about 70 sites throughout 22 regions of France (territorial units of 25 000 km2 on average). We modelled changes in resistance frequency with regional anti-Septoria fungicide use, yield losses due to the disease and the regional area under organic wheat. The major driver of resistance dynamics was fungicide use at the regional scale. We estimated its effect on the increase in resistance and relative apparent fitness of each resistance phenotype. The predictions of the model replicated the spatiotemporal patterns of resistance observed in field populations (R2 from 0.56 to 0.82). The evolution of fungicide resistance is mainly determined at the regional scale. This study therefore showed that collective management at the regional scale could effectively complete local actions.

Septoria Leaf Blotch and Reduced Nitrogen Availability Alter WRKY Transcription Factor Expression in a Codependent Manner.

A major cause of yield loss in wheat worldwide is the fungal pathogen Zymoseptoria tritici, a hemibiotrophic fungus which causes Septoria leaf blotch, the most destructive wheat disease in Europe. Resistance in commercial wheat varieties is poor, however, a link between reduced nitrogen availability and increased Septoria tolerance has been observed. We have shown that Septoria load is not affected by nitrogen, whilst the fungus is in its first, symptomless stage of growth. This suggests that a link between nitrogen and Septoria is only present during the necrotrophic phase of Septoria infection. Quantitative real-time PCR data demonstrated that WRKYs, a superfamily of plant-specific transcription factors, are differentially expressed in response to both reduced nitrogen and Septoria. WRKY39 was downregulated over 30-fold in response to necrotrophic stage Septoria, whilst changes in the expression of WRKY68a during the late biotrophic phase were dependent on the concentration of nitrogen under which wheat is grown. WRKY68a may therefore mediate a link between nitrogen and Septoria. The potential remains to identify key regulators in the link between nitrogen and Septoria, and as such, elucidate molecular markers for wheat breeding, or targets for molecular-based breeding approaches.

The transcription factor Zt107320 affects the dimorphic switch, growth and virulence of the fungal wheat pathogen Zymoseptoria tritici.

Zymoseptoria tritici is a filamentous fungus causing Septoria tritici blotch in wheat. The pathogen has a narrow host range and infections of grasses other than susceptible wheat are blocked early after stomatal penetration. During these abortive infections, the fungus shows a markedly different gene expression pattern. However, the underlying mechanisms causing differential gene expression during host and non-host interactions are largely unknown, but likely include transcriptional regulators responsible for the onset of an infection programme in compatible hosts. MoCOD1, a member of the fungal Zn(II)2 Cys6 transcription factor family, has been shown to directly affect pathogenicity in the rice blast pathogen Magnaporthe oryzae. Here, we analyse the role of the putative transcription factor Zt107320, a homologue of MoCOD1, during infection of compatible and incompatible hosts by Z. tritici. We show for the first time that Zt107320 is differentially expressed in host versus non-host infections and that lower expression corresponds to an incompatible infection of non-hosts. Applying reverse genetics approaches, we further show that Zt107320 regulates the dimorphic switch as well as the growth rate of Z. tritici and affects fungal cell wall composition in vitro. Moreover, ∆Zt107320 mutants showed reduced virulence during compatible infections of wheat. We conclude that Zt107320 directly influences pathogen fitness and propose that Zt107320 is involved in the regulation of growth processes and pathogenicity during infection.

Rapid loss of virulence during submergence of Z. tritici asexual spores.

Zymoseptoria tritici, the causal agent of Septoria tritici blotch, is a notable pathogen of temperate-grown wheat. To better understand the mechanisms underpinning pathogenicity, leaf infection assays are commonly used to compare either the virulence of Z. tritici wildtype or mutant strains, or the susceptibility of wheat cultivars. These assays, which control for many biotic, abiotic and experimental variables, involve the application of known spore numbers to leaves. To achieve this, spore numbers are quantified during a period of aqueous suspension. Published methods rarely state the period in which spores are held in suspension, suggesting that this variable may be uncontrolled. Using simple, agar-based plating experiments, this work firstly demonstrates that blastospore culturability (the ability to form a colony when plated on appropriate agar) decreases rapidly over time during maintenance in aqueous suspension. It is subsequently shown that this reduction in culturability correlates to a reduction in the virulence of the blastospore population. This is shown in three wild type Z. tritici strains. From this, it is concluded that suspension time is a variable of major importance in experimental design and one which, if not controlled, may lead to erroneous conclusions from inter-strain comparisons. The conidia of the unrelated fungus Magnaporthe oryzae also rapidly lose culturability when stored in aqueous suspension, whereas the microspores of Fusarium oxysporum f. sp. cubense do not, suggesting that this phenomenon occurs in some but not all other fungi. Finally, a droplet method of inoculations is proposed to decrease the variability in the numbers of spores applied, within and between experiments.

Spatiotemporal dynamics of fungicide resistance in the wheat pathogen Zymoseptoria tritici in France.

BACKGROUND: Management of pesticide resistance is a major issue in modern agricultural systems, particularly in the context of the broader challenge of reducing pesticide use. However, such management must be adapted to resistance dynamics, which remains challenging to predict due to its dependence on many biological traits of pests, interactions with the environment and pesticide use. We retrospectively studied the evolution of reported resistances to four modes of action (benzimidazoles, quinone outside inhibitors, sterol demethylation inhibitors and succinate dehydrogenase inhibitors), in French populations of the wheat pathogen Zymoseptoria tritici. RESULTS: We used statistical models to analyse the Performance trial network data set (2004-2017; ∼ 70 locations in France yearly). They highlighted contrasting behaviours between phenotypes, for example: (i) stable spatial distributions and colonization front structures over time, and (ii) different frequency growth rates at the national scale and between regions. CONCLUSION: We provide a quantitative description of the spatiotemporal patterns of resistance evolution for fungicides with several modes of action. Moreover, we highlight some unexpected resistance dynamics in France, with major differences between the north and south. This complex pattern of resistance evolution in French populations is consistent with previous descriptions of dynamics at the European scale. These results should make it easier to anticipate evolution locally and to improve the management of resistance. © 2019 Society of Chemical Industry.

Non-target site SDHI resistance is present as standing genetic variation in field populations of Zymoseptoria tritici.

BACKGROUND: A new generation of more active succinate dehydrogenase (Sdh) inhibitors (SDHIs) is currently widely used to control Septoria leaf blotch in northwest Europe. Detailed studies were conducted on Zymoseptoria tritici field isolates with reduced sensitivity to fluopyram and isofetamid; SDHIs which have only just or not been introduced for cereal disease control, respectively. RESULTS: Strong cross-resistance between fluopyram and isofetamid, but not with other SDHIs, was confirmed through sensitivity tests using laboratory mutants and field isolates with and without Sdh mutations. The sensitivity profiles of most field isolates resistant to fluopyram and isofetamid were very similar to a lab mutant carrying SdhC-A84V, but no alterations were found in SdhB, C and D. Inhibition of mitochondrial Sdh enzyme activity and control efficacy in planta for those isolates was severely impaired by fluopyram and isofetamid, but not by bixafen. Isolates with similar phenotypes were not only detected in northwest Europe but also in New Zealand before the widely use of SDHIs. CONCLUSION: This is the first report of SDHI-specific non-target site resistance in Z. tritici. Monitoring studies show that this resistance mechanism is present and can be selected from standing genetic variation in field populations. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A role for random, humidity-dependent epiphytic growth prior to invasion of wheat by Zymoseptoria tritici.

Zymoseptoria tritici causes Septoria leaf blotch of wheat. The prevailing paradigm of the Z. tritici-wheat interaction assumes fungal ingress through stomata within 24-48h, followed by days of symptomless infection. This is extrapolated from studies testing the mode of fungal ingress under optimal infection conditions. Here, we explicitly assess the timing of entry, using GFP-tagged Z. tritici. We show that early entry is comparatively rare, and extended epiphytic growth possible. We test the hypotheses that our data diverge from earlier studies due to: i. random ingress of Z. tritici into the leaf, with some early entry events; ii. previous reliance upon fungal stains, combined with poor attachment of Z. tritici to the leaf, leading to increased likelihood of observing internal versus external growth, compared to using GFP; iii. use of exceptionally high humidity to promote entry in previous studies. We combine computer simulation of leaf-surface growth with thousands of in planta observations to demonstrate that while spores germinate rapidly on the leaf, over 95% of fungi remain epiphytic, growing randomly over the leaf for ten days or more. We show that epiphytic fungi are easily detached from leaves by rinsing and that humidity promotes epiphytic growth, increasing infection rates. Together, these results explain why epiphytic growth has been dismissed and early ingress assumed. The prolonged epiphytic phase should inform studies of pathogenicity and virulence mutants, disease control strategies, and interpretation of the observed low in planta growth, metabolic quiescence and evasion of plant defences by Zymoseptoria during symptomless infection.

Selection and characterization of Argentine isolates of Trichoderma harzianum for effective biocontrol of Septoria leaf blotch of wheat.

Species of the genus Trichoderma are economically important as biocontrol agents, serving as a potential alternative to chemical control. The applicability of Trichoderma isolates to different ecozones will depend on the behavior of the strains selected from each zone. The present study was undertaken to isolate biocontrol populations of Trichoderma spp. from the Argentine wheat regions and to select and characterize the best strains of Trichoderma harzianum by means of molecular techniques. A total of 84 out of the 240 strains of Trichoderma were able to reduce the disease severity of the leaf blotch of wheat. Thirty-seven strains were selected for the reduction equal to or greater than 50% of the severity, compared with the control. The percentage values of reduction of the pycnidial coverage ranged between 45 and 80%. The same last strains were confirmed as T. harzianum by polymerase chain reaction amplification of internal transcribed spacers, followed by sequencing. Inter-simple sequence repeat was used to examine the genetic variability among isolates. This resulted in a total of 132 bands. Further numerical analysis revealed 19 haplotypes, grouped in three clusters (I, II, III). Shared strains, with different geographical origins and isolated in different years, were observed within each cluster. The origin of the isolates and the genetic group were partially related. All isolates from Paraná were in cluster I, all isolates from Lobería were in cluster II, and all isolates from Pergamino and Santa Fe were in cluster III. Our results suggest that the 37 native strains of T. harzianum are important in biocontrol programs and could be advantageous for the preparation of biopesticides adapted to the agroecological conditions of wheat culture.

Comparative transcriptomic analyses of Zymoseptoria tritici strains show complex lifestyle transitions and intraspecific variability in transcription profiles.

Zymoseptoria tritici causes Septoria tritici blotch (STB) on wheat. The disease interaction is characterized by clearly defined temporal phases of infection, ultimately resulting in the death of host tissue. Zymoseptoria tritici is a highly polymorphic species with significant intraspecific variation in virulence profiles. We generated a deep transcriptomic sequencing dataset spanning the entire time course of an infection using a previously uncharacterized, highly virulent Z. tritici strain isolated from a Swiss wheat field. We found that seven clusters of gene transcription profiles explained the progression of the infection. The earliest highly up-regulated genes included chloroperoxidases, which may help the fungus cope with plant defences. The onset of necrotrophy was characterized by a concerted up-regulation of proteases, plant cell wall-degrading enzymes and lipases. Functions related to nutrition and growth characterized late necrotrophy and the transition to saprotrophic growth on dead plant tissue. We found that the peak up-regulation of genes essential for mating coincided with the necrotrophic phase. We performed an intraspecies comparative transcriptomics analysis using a comparable time course infection experiment of the genome reference isolate IPO323. Major components of the fungal infection transcriptome were conserved between the two strains. However, individual small, secreted proteins, proteases and cell wall-degrading enzymes showed strongly differentiated transcriptional profiles between isolates. Our analyses illustrate that successful STB infections involve complex transcriptomic remodelling to up-regulate distinct gene functions. Heterogeneity in transcriptomes among isolates may explain some of the considerable variation in virulence and host specialization found within the species.

Accurate Assessment of Wheat and Triticale Cultivar Resistance to Septoria tritici and Stagonospora nodorum Infection by Biotin/Avidin ELISA.

Specific and quantitative biotin/avidin-enzyme-linked immunosorbent assays (BA-ELISA) were evaluated for their ability to assess resistance of wheat and triticale cultivars to Septoria tritici (leaf blotch) and Stagonospora nodorum (leaf and glume blotch) in field trials. Using BA-ELISAs, the antigen amounts of S. tritici and of Stagonospora nodorum were measured in the flag leaf (F) and the first leaf below it (F-1) of five cultivars of triticale at Zadok's growth stage (GS) 75-80 and in 11 cultivars of wheat at GS 73-75 in 2001 and 2002. The presence of the pathogens was found to be specific to parts of the plants, cultivar, and plant species. Stagonospora nodorum was the dominant leaf blotch pathogen in triticale, while both Septoria tritici and Stagonospora nodorum occurred commonly in wheat. Close correlations were obtained between the pathogen amount measured by BA-ELISA and the percentage of necrotic leaf area in the tested cultivars. The BA-ELISA values for the tested triticale and wheat cultivars were ranked, and they correlated well with the susceptibility ratings given in the cultivar list recommended by Bundessortenamt (German Federal Office of Plant Variety), which is based on visual assessment of the leaf blotch complex caused by S. tritici and Stagonospora nodorum. The relative susceptibilities of individual wheat cultivars to both pathogens were similar. In conclusion, BA-ELISA provided for an accurate diagnosis and quantification of S. tritici and Stagonospora nodorum in infected plant tissue, and therefore can be used to assess resistance to these fungi in a disease complex in both early-stage breeding lines and field trials.