Fungus Zymoseptoria Tritici Research Articles

DMI fungicide resistance in Zymoseptoria tritici is unlinked to geographical origin and genetic background: a case study in Europe.

The hemibiotrophic fungus Zymoseptoria tritici causing Septoria tritici blotch (STB), is a devastating foliar pathogen of wheat worldwide. A common group of fungicides used to control STB are the demethylation inhibitors (DMIs). DMI fungicides restrict fungal growth by inhibiting the sterol 14-α-demethylase, a protein encoded by CYP51 gene and essential for maintaining fungal cell permeability. However, the adaptation of Z. tritici populations in response to intensive and prolonged DMI usage has resulted in a gradual shift towards reduced sensitivity to this group of fungicides. In this study, 311 isolates were collected pre-treatment from nine wheat-growing regions in Europe in 2019. These isolates were analysed by high-throughput amplicon-based sequencing of nine housekeeping genes and the CYP51 gene. Analyses based on housekeeping genes and the CYP51 gene revealed a lack of population structure in Z. tritici samples irrespective of geographical origin. Minimum spanning network (MSN) analysis showed clustering of multilocus genotypes (MLGs) based on CYP51 haplotypes, indicating an effect of selection due to DMI fungicide use. The majority of the haplotypes identified in this study have been reported previously. The diversity and frequencies of mutations varied across regions. Using a high-throughput amplicon-sequencing approach, we found several mutations in the CYP51 gene combined in different haplotypes that are likely to cause fungicide resistance. These mutations occurred irrespective of genetic background or geographical origin. Overall, these results contribute to the development of effective and sustainable risk monitoring for DMI fungicide resistance. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

ZymoSoups: A high-throughput forward genetics method for rapid identification of virulence genes in Zymoseptoria tritici.

Septoria tritici blotch is caused by the fungus Zymoseptoria tritici and poses a major threat to wheat productivity. There are over twenty mapped loci in wheat that confer strong (gene-for-gene) resistance against this pathogen, however the corresponding genes in Z. tritici that confer virulence against distinct R genes remain largely unknown. In this study, we developed a rapid forward genetics methodology to identify genes that enable Z. tritici to gain virulence on previously resistant wheat varieties. We used the known gene-for-gene interaction between Stb6 and AvrStb6 as a proof-of-concept that this method could quickly recover single candidate virulence genes. We subjected the avirulent Z. tritici strain IPO323, which carries the recognized AvrStb6 allele, to UV mutagenesis and generated a library of over 66,000 mutants. We screened these mutants on leaves of the resistant wheat variety Cadenza, in mixtures (soups) ranging from 100-500 mutants per soup. We identified five soups with a gain-of-virulence (GoV) phenotype relative to the IPO323 parental strain and re-sequenced 18 individual isolates, including four control isolates and two mutants lacking virulence, when screened individually. Of the 12 confirmed GoV mutants, one had a single nucleotide polymorphism (SNP) in the AvrStb6 coding region. The other 11 GoV mutants exhibited large (~70Kb) deletions at the end of chromosome 5, including the AvrStb6 locus. Our findings demonstrate the efficiency of this forward genetic approach in elucidating the genetic basis of qualitative resistance to Z. tritici and the potential to rapidly identify other, currently unknown, Avr genes in this pathogen.

Embracing Biological Control of Septoria Tritici Blotch for Sustainable Wheat Protection

ABSTRACTWheat, one of the top‐produced cereals worldwide, is affected by many abiotic and biotic stresses, such as the ascomycete fungus Zymoseptoria tritici, the causal agent of Septoria tritici blotch (STB). STB has historically been managed with fungicides, but the pathogen readily overcomes chemical control because of its rapid genetic evolution. In addition, many fungicides are now being banned or limited by governments aiming for more environment‐friendly methods for pest management. This scenario gave rise to thinking about alternative control means such as biological control agents (BCAs) and organism‐derived biomolecules (ODBs). In this work, we review microbial BCA candidates and ODBs currently studied for the control of STB. Key studies have identified successful candidates including bacterial strains of the genera Pseudomonas and Bacillus, and fungal strains such as Trichoderma harzianum, Penicillium olsonii and Acremonium alternatum. In addition, lesser‐studied fungi, bacteria and compounds have been tested. Despite promising research, no BCA or ODB has been registered or commercially used against STB, and field trials are notably lacking, with existing studies being limited in scale. Further understanding of the interactions between Z. tritici and the wheat microbiome may uncover new potential candidates for STB biocontrol.

Multiple routes to fungicide resistance: Interaction of Cyp51 gene sequences, copy number and expression.

We examined the molecular basis of triazole resistance in Blumeria graminis f. sp. tritici (wheat mildew, Bgt), a model organism among powdery mildews. Four genetic models for responses to triazole fungicides were identified among US and UK isolates, involving multiple genetic mechanisms. Firstly, only two amino acid substitutions in CYP51B lanosterol demethylase, the target of triazoles, were associated with resistance, Y136F and S509T (homologous to Y137F and S524T in the reference fungus Zymoseptoria tritici). As sequence variation did not explain the wide range of resistance, we also investigated Cyp51B copy number and expression, the latter using both reverse transcription-quantitative PCR and RNA-seq. The second model for resistance involved higher copy number and expression in isolates with a resistance allele; thirdly, however, moderate resistance was associated with higher copy number of wild-type Cyp51B in some US isolates. A fourth mechanism was heteroallelism with multiple alleles of Cyp51B. UK isolates, with significantly higher mean resistance than their US counterparts, had higher mean copy number, a high frequency of the S509T substitution, which was absent from the United States, and in the most resistant isolates, heteroallelism involving both sensitivity residues Y136+S509 and resistance residues F136+T509. Some US isolates were heteroallelic for Y136+S509 and F136+S509, but this was not associated with higher resistance. The obligate biotrophy of Bgt may constrain the tertiary structure and thus the sequence of CYP51B, so other variation that increases resistance may have a selective advantage. We describe a process by which heteroallelism may be adaptive when Bgt is intermittently exposed to triazoles.

Stomatal penetration: the cornerstone of plant resistance to the fungal pathogen Zymoseptoria tritici

BackgroundSeptoria tritici blotch (STB), caused by the foliar fungus Zymoseptoria tritici, is one of the most damaging disease of wheat in Europe. Genetic resistance against this fungus relies on different types of resistance from non-host resistance (NHR) and host species specific resistance (HSSR) to host resistance mediated by quantitative trait loci (QTLs) or major resistance genes (Stb). Characterizing the diversity of theses resistances is of great importance for breeding wheat cultivars with efficient and durable resistance. While the functional mechanisms underlying these resistance types are not well understood, increasing piece of evidence suggest that fungus stomatal penetration and early establishment in the apoplast are both crucial for the outcome of some interactions between Z. tritici and plants. To validate and extend these previous observations, we conducted quantitative comparative phenotypical and cytological analyses of the infection process corresponding to 22 different interactions between plant species and Z. tritici isolates. These interactions included four major bread wheat Stb genes, four bread wheat accessions with contrasting quantitative resistance, two species resistant to Z. tritici isolates from bread wheat (HSSR) and four plant species resistant to all Z. tritici isolates (NHR).ResultsInfiltration of Z. tritici spores into plant leaves allowed the partial bypass of all bread wheat resistances and durum wheat resistance, but not resistances from other plants species. Quantitative comparative cytological analysis showed that in the non-grass plant Nicotiana benthamiana, Z. tritici was stopped before stomatal penetration. By contrast, in all resistant grass plants, Z. tritici was stopped, at least partly, during stomatal penetration. The intensity of this early plant control process varied depending on resistance types, quantitative resistances being the least effective. These analyses also demonstrated that Stb-mediated resistances, HSSR and NHR, but not quantitative resistances, relied on the strong growth inhibition of the few Z. tritici penetrating hyphae at their entry point in the sub-stomatal cavity.ConclusionsIn addition to furnishing a robust quantitative cytological assessment system, our study uncovered three stopping patterns of Z. tritici by plant resistances. Stomatal resistance was found important for most resistances to Z. tritici, independently of its type (Stb, HSSR, NHR). These results provided a basis for the functional analysis of wheat resistance to Z. tritici and its improvement.

The Egyptian wheat cultivar Gemmeiza-12 is a source of resistance against the fungus Zymoseptoria tritici

BackgroundWheat is one of the world’s most important cereal crops. However, the fungal pathogen Zymoseptoria tritici can cause disease epidemics, leading to reduced yields. With climate change and development of new agricultural areas with suitable environments, Z. tritici may advance into geographical areas previously unaffected by this pathogen. It is currently unknown how Egyptian wheat will perform in the face of this incoming threat. This project aimed to assess the resistance of Egyptian wheat germplasm to Z. tritici, to identify cultivars with high levels of resistance and characterise the mechanism(s) of resistance present in these cultivars.ResultsEighteen Egyptian wheat cultivars were screened against two Z. tritici model isolates and exhibited a wide spectrum of responses. This ranged from resistance to complete susceptibility to one or both isolates tested. The most highly resistant cultivars from the initial screen were then tested under two environmental conditions against modern UK field isolates. Disease levels under UK-like conditions were higher, however, symptom development on the cultivar Gemmeiza-12 was noticeably slower than on other Egyptian wheats. The robustness of the resistance shown by Gemmeiza-12 was confirmed in experiments mimicking Egyptian environmental conditions, where degree of Z. tritici infection was lower. The Kompetitive allele-specific PCR (KASP) diagnostic assay suggested the presence of an Stb6 resistant allele in several Egyptian wheats including Gemmeiza-12. Infection assays using the IPO323 WT and IPO323ΔAvrStb6 mutant confirmed the presence of Stb6 in several Egyptian cultivars including Gemmeiza-12. Confocal fluorescence microscopy demonstrated that growth of the IPO323 strain is blocked at the point of stomatal penetration on Gemmeiza-12, consistent with previous reports of Stb gene mediated resistance. In addition to this R-gene mediated resistance, IPO323 spores showed lower adherence to leaves of Gemmeiza-12 compared to UK wheat varieties, suggesting other aspects of leaf physiology may also contribute to the resistance phenotype of this cultivar.ConclusionThese results indicate that Gemmeiza-12 will be useful in future breeding programs where improved resistance to Z. tritici is a priority.

The collembolan Heteromurus nitidus grazes the wheat fungal pathogen Zymoseptoria tritici on infected tissues: opportunities and limitations for bioregulation.

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is a foliar disease affecting wheat crops against which conventional control methods are not totally effective. During inter-epidemic periods the fungus survives in wheat residues left on the ground. In this study, we tested the potential of the collembolan Heteromurus nitidus - a springtail species present in field soils and known to interact with different fungal species - as a potential bioregulation agent of Z. tritici on wheat residues through a choice and consumption experiment. Springtails preferred inoculated fresh residues but did not have a preference between inoculated and uninoculated old residues. Springtails grazed on Z. tritici fruiting bodies and reduced pycnidiospore numbers by ten-fold compared to control inoculated fresh residues. Attraction toward fresh inoculated residues and pycnidiospore reduction support the hypothesis that Z. tritici is a food source for springtails. Heteromurus nitidus showed no preference between inoculated and uninoculated 18-month-old residues, probably because they no longer produced ascospores. Attraction towards fresh residues and spore reduction support our hypothesis that H. nitidus may contribute to the bioregulation of Z. tritici. Perspectives for field application would be determined by the ability of H. nitidus and Z. tritici to interact at key epidemiological stages. The impact of H. nitidus on the quantity of pathogen primary inoculum over time should be estimated using residues of intermediate age. This would help to identify the optimal period for enhancing the effectiveness of springtails as consumers of Z. tritici. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

H4K20me3 is important for Ash1-mediated H3K36me3 and transcriptional silencing in facultative heterochromatin in a fungal pathogen.

Facultative heterochromatin controls development and differentiation in many eukaryotes. In metazoans, plants, and many filamentous fungi, facultative heterochromatin is characterized by transcriptional repression and enrichment with nucleosomes that are trimethylated at histone H3 lysine 27 (H3K27me3). While loss of H3K27me3 results in derepression of transcriptional gene silencing in many species, additional up- and downstream layers of regulation are necessary to mediate control of transcription in chromosome regions enriched with H3K27me3. Here, we investigated the effects of one histone mark on histone H4, namely H4K20me3, in the fungus Zymoseptoria tritici, a globally important pathogen of wheat. Deletion of kmt5, the gene encoding the sole methyltransferase responsible for H4K20 methylation, resulted in global derepression of transcription, especially in regions of facultative heterochromatin. Derepression in the absence of H4K20me3 not only affected known genes but also a large number of novel, previously undetected transcripts generated from regions of facultative heterochromatin on accessory chromosomes. Transcriptional activation in kmt5 deletion strains was accompanied by a complete loss of Ash1-mediated H3K36me3 and chromatin reorganization affecting H3K27me3 and H3K4me2 distribution in regions of facultative heterochromatin. Strains with H4K20L, M or Q mutations in the single histone H4 gene of Z. tritici recapitulated these chromatin changes, suggesting that H4K20me3 is important for Ash1-mediated H3K36me3. The ∆kmt5 mutants we obtained were more sensitive to genotoxic stressors than wild type and both, ∆kmt5 and ∆ash1, showed greatly increased rates of accessory chromosome loss. Taken together, our results provide insights into an unsuspected mechanism involved in the assembly and maintenance of facultative heterochromatin.

Fungal plant pathogen "mutagenomics" reveals tagged and untagged mutations in Zymoseptoria tritici and identifies SSK2 as key morphogenesis and stress-responsive virulence factor.

"Mutagenomics" is the combination of random mutagenesis, phenotypic screening, and whole-genome re-sequencing to uncover all tagged and untagged mutations linked with phenotypic changes in an organism. In this study, we performed a mutagenomics screen on the wheat pathogenic fungus Zymoseptoria tritici for altered morphogenetic switching and stress sensitivity phenotypes using Agrobacterium-mediated "random" T-DNA mutagenesis (ATMT). Biological screening identified four mutants which were strongly reduced in virulence on wheat. Whole genome re-sequencing defined the positions of the T-DNA insertion events and revealed several unlinked mutations potentially affecting gene functions. Remarkably, two independent reduced virulence mutant strains, with similarly altered stress sensitivities and aberrant hyphal growth phenotypes, were found to have a distinct loss of function mutations in the ZtSSK2 MAPKKK gene. One mutant strain had a direct T-DNA insertion affecting the predicted protein's N-terminus, while the other possessed an unlinked frameshift mutation towards the C-terminus. We used genetic complementation to restore both strains' wild-type (WT) function (virulence, morphogenesis, and stress response). We demonstrated that ZtSSK2 has a non-redundant function with ZtSTE11 in virulence through the biochemical activation of the stress-activated HOG1 MAPK pathway. Moreover, we present data suggesting that SSK2 has a unique role in activating this pathway in response to specific stresses. Finally, dual RNAseq-based transcriptome profiling of WT and SSK2 mutant strains revealed many HOG1-dependent transcriptional changes in the fungus during early infection and suggested that the host response does not discriminate between WT and mutant strains during this early phase. Together these data define new genes implicated in the virulence of the pathogen and emphasise the importance of a whole genome sequencing step in mutagenomic discovery pipelines.

Will Climate Change Affect the Disease Progression of Septoria Tritici Blotch in Northern Europe?

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici Desm., is the most important disease affecting wheat in Northern Europe. There is a strong correlation between STB and weather variables; therefore, research on climate change and epidemiology is essential. In a long-term survey across 25 years, we evaluated the epidemiological development of STB at a representative location under maritime climatic conditions. The surveys conducted between 1996 and 2021 showed an increase in disease severity of STB with respect to time. At the survey location, the plants were also evaluated for other diseases, but other foliar diseases were only observed with negligible severities. However, a continuous increase in the severity of STB was observed throughout the survey. During the survey period, there was no significant relationship between disease severity and single weather parameters (e.g., temperature and precipitation). However, seasonal changes in the progression of conducive STB conditions within the season were observed during the survey. Therefore, STB infections occurred at increased temperatures due to infections later during the growth season. In general, the distribution of conducive weather conditions, which supports an infection, determines the epidemiological behaviour of STB during the growing season. Due to these enhanced STB epidemics, a decline in wheat production has been observed, especially in agronomic practices of maritime climates. This is particularly the case if temperature and precipitation during the growing season are affected by climate change.

Combined pangenomics and transcriptomics reveals core and redundant virulence processes in a rapidly evolving fungal plant pathogen

BackgroundStudying genomic variation in rapidly evolving pathogens potentially enables identification of genes supporting their “core biology”, being present, functional and expressed by all strains or “flexible biology”, varying between strains. Genes supporting flexible biology may be considered to be “accessory”, whilst the “core” gene set is likely to be important for common features of a pathogen species biology, including virulence on all host genotypes. The wheat-pathogenic fungus Zymoseptoria tritici represents one of the most rapidly evolving threats to global food security and was the focus of this study.ResultsWe constructed a pangenome of 18 European field isolates, with 12 also subjected to RNAseq transcription profiling during infection. Combining this data, we predicted a “core” gene set comprising 9807 sequences which were (1) present in all isolates, (2) lacking inactivating polymorphisms and (3) expressed by all isolates. A large accessory genome, consisting of 45% of the total genes, was also defined. We classified genetic and genomic polymorphism at both chromosomal and individual gene scales. Proteins required for essential functions including virulence had lower-than average sequence variability amongst core genes. Both core and accessory genomes encoded many small, secreted candidate effector proteins that likely interact with plant immunity. Viral vector-mediated transient in planta overexpression of 88 candidates failed to identify any which induced leaf necrosis characteristic of disease. However, functional complementation of a non-pathogenic deletion mutant lacking five core genes demonstrated that full virulence was restored by re-introduction of the single gene exhibiting least sequence polymorphism and highest expression.ConclusionsThese data support the combined use of pangenomics and transcriptomics for defining genes which represent core, and potentially exploitable, weaknesses in rapidly evolving pathogens.

Induction of distinct plant cell death programs by secreted proteins from the wheat pathogen Zymoseptoria tritici

Cell death processes in eukaryotes shape normal development and responses to the environment. For plant–microbe interactions, initiation of host cell death plays an important role in determining disease outcomes. Cell death pathways are frequently initiated following detection of pathogen-derived molecules which can lead to resistance or susceptibility to disease depending on pathogen lifestyle. We previously identified several small secreted proteins (SSPs) from the wheat-infecting fungus Zymoseptoria tritici that induce rapid cell death in Nicotiana benthamiana following Agrobacterium-mediated delivery and expression (agroinfiltration). Here we investigated whether the execution of host cells was mechanistically similar in response to different Z. tritici SSPs. Using RNA sequencing, we found that transient expression of four Z. tritici SSPs led to massive transcriptional reprogramming within 48 h of agroinfiltration. We observed that distinct host gene expression profiles were induced dependent on whether cell death occurs in a cell surface immune receptor-dependent or -independent manner. These gene expression profiles involved differential transcriptional networks mediated by WRKY, NAC and MYB transcription factors. In addition, differential expression of genes belonging to different classes of receptor-like proteins and receptor-like kinases was observed. These data suggest that different Z. tritici SSPs trigger differential transcriptional reprogramming in plant cells.

Zymoseptoria tritici white-collar complex integrates light, temperature and plant cues to initiate dimorphism and pathogenesis.

Transitioning from spores to hyphae is pivotal to host invasion by the plant pathogenic fungus Zymoseptoria tritici. This dimorphic switch can be initiated by high temperature in vitro (~27 °C); however, such a condition may induce cellular heat stress, questioning its relevance to field infections. Here, we study the regulation of the dimorphic switch by temperature and other factors. Climate data from wheat-growing areas indicate that the pathogen sporadically experiences high temperatures such as 27 °C during summer months. However, using a fluorescent dimorphic switch reporter (FDR1) in four wild-type strains, we show that dimorphic switching already initiates at 15–18 °C, and is enhanced by wheat leaf surface compounds. Transcriptomics reveals 1261 genes that are up- or down-regulated in hyphae of all strains. These pan-strain core dimorphism genes (PCDGs) encode known effectors, dimorphism and transcription factors, and light-responsive proteins (velvet factors, opsins, putative blue light receptors). An FDR1-based genetic screen reveals a crucial role for the white-collar complex (WCC) in dimorphism and virulence, mediated by control of PCDG expression. Thus, WCC integrates light with biotic and abiotic cues to orchestrate Z. tritici infection.

LC-MS/MS-Based Fungicide Accumulation Assay to Demonstrate Efflux Activity in the Wheat Pathogen Zymoseptoria tritici.

Increased drug efflux compromises the efficacy of a large panel of treatments in the clinic against cancer or bacterial, fungal, and viral diseases, and in agriculture due to the emergence of multidrug-resistant pathogenic fungi. Until recently, to demonstrate increased drug efflux, the use of labeled drugs or fluorescent dyes was necessary. With the increasing sensitivity of detection devices, direct assessment of drug efflux has become realistic. Here, we describe a medium-throughput method to assess the intracellular drug concentration in the plant pathogenic fungus Zymoseptoria tritici cultivated in the presence of a sublethal fungicide concentration. As a model fungicide, we used the succinate-dehydrogenase inhibitor boscalid. The boscalid concentration was assessed in the different culture fractions using mass spectrometry linked to liquid chromatography (LC-MS/MS). The ratio between the intracellular and total boscalid amount was used as an inversed proxy for the efflux activity. Using isogenic mutant strains known for their differential efflux capacities, we validated the negative correlation between the intracellular boscalid concentration and efflux activity. In addition, intra-cellular fungicide accumulation explains the susceptibility of the tested strains to boscalid. This assay may be useful in lead development when a new molecule displays good inhibitory activity against its isolated target protein but fails to control the target organism.

Blocked at the Stomatal Gate, a Key Step of Wheat Stb16q-Mediated Resistance to Zymoseptoria tritici.

Septoria tritici blotch (STB), caused by the fungus Zymoseptoria tritici, is among the most threatening wheat diseases in Europe. Genetic resistance remains one of the main environmentally sustainable strategies to efficiently control STB. However, the molecular and physiological mechanisms underlying resistance are still unknown, limiting the implementation of knowledge-driven management strategies. Among the 22 known major resistance genes (Stb), the recently cloned Stb16q gene encodes a cysteine-rich receptor-like kinase conferring a full broad-spectrum resistance against Z. tritici. Here, we showed that an avirulent Z. tritici inoculated on Stb16q quasi near isogenic lines (NILs) either by infiltration into leaf tissues or by brush inoculation of wounded tissues partially bypasses Stb16q-mediated resistance. To understand this bypass, we monitored the infection of GFP-labeled avirulent and virulent isolates on Stb16q NILs, from germination to pycnidia formation. This quantitative cytological analysis revealed that 95% of the penetration attempts were unsuccessful in the Stb16q incompatible interaction, while almost all succeeded in compatible interactions. Infectious hyphae resulting from the few successful penetration events in the Stb16q incompatible interaction were arrested in the sub-stomatal cavity of the primary-infected stomata. These results indicate that Stb16q-mediated resistance mainly blocks the avirulent isolate during its stomatal penetration into wheat tissue. Analyses of stomatal aperture of the Stb16q NILs during infection revealed that Stb16q triggers a temporary stomatal closure in response to an avirulent isolate. Finally, we showed that infiltrating avirulent isolates into leaves of the Stb6 and Stb9 NILs also partially bypasses resistances, suggesting that arrest during stomatal penetration might be a common major mechanism for Stb-mediated resistances.

The Zymoseptoria tritici white collar-1 gene, ZtWco-1, is required for development and virulence on wheat

The fungus Zymoseptoria tritici causes Septoria Tritici Blotch (STB), which is one of the most devastating diseases of wheat in Europe. There are currently no fully durable methods of control against Z. tritici, so novel strategies are urgently required. One of the ways in which fungi are able to respond to their surrounding environment is through the use of photoreceptor proteins which detect light signals. Although previous evidence suggests that Z. tritici can detect light, no photoreceptor genes have been characterised in this pathogen. This study characterises ZtWco-1, a predicted photoreceptor gene in Z. tritici. The ZtWco-1 gene is a putative homolog to the blue light photoreceptor from Neurospora crassa, wc-1. Z. tritici mutants with deletions in ZtWco-1 have defects in hyphal branching, melanisation and virulence on wheat. In addition, we identify the putative circadian clock gene ZtFrq in Z. tritici. This study provides evidence for the genetic regulation of light detection in Z. tritici and it open avenues for future research into whether this pathogen has a circadian clock.

Bioinspired Rhamnolipid Protects Wheat Against Zymoseptoria tritici Through Mainly Direct Antifungal Activity and Without Major Impact on Leaf Physiology.

Rhamnolipids (RLs), glycolipids biosynthesized by the Pseudomonas and Burkholderia genera, are known to display various activities against a wide range of pathogens. Most previous studies on RLs focused on their direct antimicrobial activity, while only a few reports described the mechanisms by which RLs induce resistance against phytopathogens and the related fitness cost on plant physiology. Here, we combined transcriptomic and metabolomic approaches to unravel the mechanisms underlying RL-induced resistance in wheat against the hemibiotrophic fungus Zymoseptoria tritici, a major pathogen of this crop. Investigations were carried out by treating wheat plants with a bioinspired synthetic mono-RL with a 12-carbon fatty acid tail, dodecanoyl α/β-L-rhamnopyranoside (Rh-Est-C12), under both infectious and non-infectious conditions to examine its potential wheat defense-eliciting and priming bioactivities. Whereas, Rh-Est-C12 conferred to wheat a significant protection against Z. tritici (41% disease severity reduction), only a slight effect of this RL on wheat leaf gene expression and metabolite accumulation was observed. A subset of 24 differentially expressed genes (DEGs) and 11 differentially accumulated metabolites (DAMs) was scored in elicitation modalities 2, 5, and 15 days post-treatment (dpt), and 25 DEGs and 17 DAMs were recorded in priming modalities 5 and 15 dpt. Most changes were down-regulations, and only a few DEGs and DAMs associated with resistance to pathogens were identified. Nevertheless, a transient early regulation in gene expression was highlighted at 2 dpt (e.g., genes involved in signaling, transcription, translation, cell-wall structure, and function), suggesting a perception of the RL by the plant upon treatment. Further in vitro and in planta bioassays showed that Rh-Est-C12 displays a significant direct antimicrobial activity toward Z. tritici. Taken together, our results suggest that Rh-Est-C12 confers protection to wheat against Z. tritici through direct antifungal activity and, to a lesser extent, by induction of plant defenses without causing major alterations in plant metabolism. This study provides new insights into the modes of action of RLs on the wheat-Z. tritici pathosystem and highlights the potential interest in Rh-Est-C12, a low-fitness cost molecule, to control this pathogen.

Phenotyping Mediterranean Durum Wheat Landraces for Resistance to Zymoseptoria tritici in Tunisia.

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.

Differential Regulation and Production of Secondary Metabolites among Isolates of the Fungal Wheat Pathogen Zymoseptoria tritici.

ABSTRACTThe genome of the wheat-pathogenic fungus Zymoseptoria tritici represents extensive presence-absence variation in gene content. Here, we addressed variation in biosynthetic gene cluster (BGC) content and biochemical profiles among three isolates. We analyzed secondary metabolite properties based on genome, transcriptome, and metabolome data. The isolates represent highly distinct genome architecture but harbor similar repertoires of BGCs. Expression profiles for most BGCs show comparable patterns of regulation among the isolates, suggesting a conserved biochemical infection program. For all three isolates, we observed a strong upregulation of a putative abscisic acid (ABA) gene cluster during biotrophic host colonization, indicating that Z. tritici interferes with host defenses by the biosynthesis of this phytohormone. Further, during in vitro growth, the isolates show similar metabolomes congruent with the predicted BGC content. We assessed if secondary metabolite production is regulated by histone methylation using a mutant impaired in formation of facultative heterochromatin (H3K27me3). In contrast to other ascomycete fungi, chromatin modifications play a less prominent role in regulation of secondary metabolites. In summary, we show that Z. tritici has a conserved program of secondary metabolite production, contrasting with the immense variation in effector expression, and some of these metabolites might play a key role during host colonization.IMPORTANCE Zymoseptoria tritici is one of the most devastating pathogens of wheat. So far the molecular determinants of virulence and their regulation are poorly understood. Previous studies have focused on proteinaceous virulence factors and their extensive diversity. In this study, we focus on secondary metabolites produced by Z. tritici. Using a comparative framework, we characterize core and noncore metabolites produced by Z. tritici by combining genome, transcriptome, and metabolome data sets. Our findings indicate highly conserved biochemical profiles with contrasting genetic and phenotypic diversity of the field isolates investigated here. This discovery has relevance for future crop protection strategies.

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici.

Acquired resistance is a threat to antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms and highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the resistance risk to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complex III of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely used strobilurins which inhibit the same complex at the ubiquinol oxidation site (Qo site). We identified the G37V change within the cytochrome b Qi site as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. We identified negative cross-resistance between isolates harbouring G37V or G143A, a Qo site change previously selected by strobilurins. Double mutants were less resistant to both QiIs and quinone outside inhibitors compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides and provides new perspectives for the management of QiIs.