Genome-wide association analysis of tan spot disease resistance in durum wheat accessions from Tunisia.

Background: Tunisia harbors a rich collection of unexploited durum wheat landraces (Triticum durum ssp. durum) that have been gradually replaced by elite cultivars since the 1970s. These landraces represent an important potential source for broadening the genetic background of elite durum wheat cultivars and for the introgression of novel genes for key traits, including disease resistance, into these cultivars. Methods: In this study, single nucleotide polymorphism (SNP) markers were used to investigate the genetic diversity and population structure of a core collection of 235 durum wheat accessions consisting mainly of landraces. The high phenotypic and genetic diversity of the fungal pathogen Pyrenophora tritici-repentis (cause of tan spot disease of wheat) in Tunisia allowed the assessment of the accessions for tan spot resistance at the adult plant stage under field conditions over three cropping seasons. A genome-wide association study (GWAS) was performed using a 90k SNP array. Results: Bayesian population structure analysis with 9191 polymorphic SNP markers classified the accessions into two groups, where groups 1 and 2 included 49.79% and 31.49% of the accessions, respectively, while the remaining 18.72% were admixtures. Principal coordinate analysis, the unweighted pair group method with arithmetic mean and the neighbor-joining method clustered the accessions into three to five groups. Analysis of molecular variance indicated that 76% of the genetic variation was among individuals and 23% was between individuals. Genome-wide association analyses identified 26 SNPs associated with tan spot resistance and explained between 8.1% to 20.2% of the phenotypic variation. The SNPs were located on chromosomes 1B (1 SNP), 2B (4 SNPs), 3A (2 SNPs), 3B (2 SNPs), 4A (2 SNPs), 4B (1 SNP), 5A (2 SNPs), 5B (4 SNPs), 6A (5 SNPs), 6B (2 SNPs), and 7B (1 SNP). Four markers, one on each of chromosomes 1B, and 5A, and two on 5B, coincided with previously reported SNPs for tan spot resistance, while the remaining SNPs were either novel markers or closely related to previously reported SNPs. Eight durum wheat accessions were identified as possible novel sources of tan spot resistance that could be introgressed into elite cultivars. Conclusion: The results highlighted the significance of chromosomes 2B, 5B, and 6A as genomic regions associated with tan spot resistance.

Wheat Stem Rust Detection and Race Characterization in Tunisia.

Climate changes over the past 25 years have led to conducive conditions for invasive and transboundary fungal disease occurrence, including the re-emergence of wheat stem rust disease, caused by Puccinia graminis f.sp. tritici (Pgt) in East Africa, Europe, and the Mediterranean basin. Since 2018, sporadic infections have been observed in Tunisia. In this study, we investigated Pgt occurrence at major Tunisian wheat growing areas. Pgt monitoring, assessment, and sampling from planted trap nurseries at five different locations over two years (2021 and 2022) revealed the predominance of three races, namely TTRTF (Clade III-B), TKKTF (Clade IV-F), and TKTTF (Clade IV-B). Clade III-B was the most prevalent in 2021 as it was detected at all locations, while in 2022 Pgt was only reported at Beja and Jendouba, with the prevalence of Clade IV-B. The low levels of disease incidence during these two years and Pgt population diversity suggest that this fungus most likely originated from exotic incursions and that climate factors could have caused disease establishment in Tunisia. Further evaluation under the artificial disease pressure of Tunisian wheat varieties and weather-based modeling for early disease detection in the Mediterranean area could be helpful in monitoring and predicting wheat stem rust emergence and epidemics.

Deciphering resistance to Zymoseptoria tritici in the Tunisian durum wheat landrace accession 'Agili39'.

BACKGROUND: Septoria tritici blotch (STB), caused by Zymoseptoria tritici (Z. tritici), is an important biotic threat to durum wheat in the entire Mediterranean Basin. Although most durum wheat cultivars are susceptible to Z. tritici, research in STB resistance in durum wheat has been limited. RESULTS: In our study, we have identified resistance to a wide array of Z. tritici isolates in the Tunisian durum wheat landrace accession 'Agili39'. Subsequently, a recombinant inbred population was developed and tested under greenhouse conditions at the seedling stage with eight Z. tritici isolates and for five years under field conditions with three Z. tritici isolates. Mapping of quantitative trait loci (QTL) resulted in the identification of two major QTL on chromosome 2B designated as Qstb2B_1 and Qstb2B_2. The Qstb2B_1 QTL was mapped at the seedling and the adult plant stage (highest LOD 33.9, explained variance 61.6%), conferring an effective resistance against five Z. tritici isolates. The Qstb2B_2 conferred adult plant resistance (highest LOD 32.9, explained variance 42%) and has been effective at the field trials against two Z. tritici isolates. The physical positions of the flanking markers linked to Qstb2B_1 and Qstb2B_2 indicate that these two QTL are 5 Mb apart. In addition, we identified two minor QTL on chromosomes 1A (Qstb1A) and chromosome 7A (Qstb7A) (highest LODs 4.6 and 4.0, and explained variances of 16% and 9%, respectively) that were specific to three and one Z. tritici isolates, respectively. All identified QTL were derived from the landrace accession Agili39 that represents a valuable source for STB resistance in durum wheat. CONCLUSION: This study demonstrates that Z. tritici resistance in the 'Agili39' landrace accession is controlled by two minor and two major QTL acting in an additive mode. We also provide evidence that the broad efficacy of the resistance to STB in 'Agili 39' is due to a natural pyramiding of these QTL. A sustainable use of this Z. tritici resistance source and a positive selection of the linked markers to the identified QTL will greatly support effective breeding for Z. tritici resistance in durum wheat.

Characterization of Mediterranean Durum Wheat for Resistance to Pyrenophora tritici-repentis.

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.

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.

Characterization of Pyrenophora tritici-repentis in Tunisia and Comparison with a Global Pathogen Population.

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.

Stress and sexual reproduction affect the dynamics of the wheat pathogen effector AvrStb6 and strobilurin resistance.

Host resistance and fungicide treatments are cornerstones of plant-disease control. Here, we show that these treatments allow sex and modulate parenthood in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that the Z. tritici-wheat interaction complies with the gene-for-gene model by identifying the effector AvrStb6, which is recognized by the wheat resistance protein Stb6. Recognition triggers host resistance, thus implying removal of avirulent strains from pathogen populations. However, Z. tritici crosses on wheat show that sex occurs even with an avirulent parent, and avirulence alleles are thereby retained in subsequent populations. Crossing fungicide-sensitive and fungicide-resistant isolates under fungicide pressure results in a rapid increase in resistance-allele frequency. Isolates under selection always act as male donors, and thus disease control modulates parenthood. Modeling these observations for agricultural and natural environments reveals extended durability of host resistance and rapid emergence of fungicide resistance. Therefore, fungal sex has major implications for disease control.

Proteome catalog of Zymoseptoria tritici captured during pathogenesis in wheat.

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/MS(E)) 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 MS(E) 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.

FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici.

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.

Effector discovery in the fungal wheat pathogen Zymoseptoria tritici.

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.

Horizontal gene and chromosome transfer in plant pathogenic fungi affecting host range.

Plant pathogenic fungi adapt quickly to changing environments including overcoming plant disease resistance genes. This is usually achieved by mutations in single effector genes of the pathogens, enabling them to avoid recognition by the host plant. In addition, horizontal gene transfer (HGT) and horizontal chromosome transfer (HCT) provide a means for pathogens to broaden their host range. Recently, several reports have appeared in the literature on HGT, HCT and hybridization between plant pathogenic fungi that affect their host range, including species of Stagonospora/Pyrenophora, Fusarium and Alternaria. Evidence is given that HGT of the ToxA gene from Stagonospora nodorum to Pyrenophora tritici-repentis enabled the latter fungus to cause a serious disease in wheat. A nonpathogenic Fusarium species can become pathogenic on tomato by HCT of a pathogenicity chromosome from Fusarium oxysporum f.sp lycopersici, a well-known pathogen of tomato. Similarly, Alternaria species can broaden their host range by HCT of a single chromosome carrying a cluster of genes encoding host-specific toxins that enabled them to become pathogenic on new hosts such as apple, Japanese pear, strawberry and tomato, respectively. The mechanisms HGT and HCT and their impact on potential emergence of fungal plant pathogens adapted to new host plants will be discussed.

Meiosis drives extraordinary genome plasticity in the haploid fungal plant pathogen Mycosphaerella graminicola.

Meiosis in the haploid plant-pathogenic fungus Mycosphaerella graminicola results in eight ascospores due to a mitotic division following the two meiotic divisions. The transient diploid phase allows for recombination among homologous chromosomes. However, some chromosomes of M. graminicola lack homologs and do not pair during meiosis. Because these chromosomes are not present universally in the genome of the organism they can be considered to be dispensable. To analyze the meiotic transmission of unequal chromosome numbers, two segregating populations were generated by crossing genetically unrelated parent isolates originating from Algeria and The Netherlands that had pathogenicity towards durum or bread wheat, respectively. Detailed genetic analyses of these progenies using high-density mapping (1793 DArT, 258 AFLP and 25 SSR markers) and graphical genotyping revealed that M. graminicola has up to eight dispensable chromosomes, the highest number reported in filamentous fungi. These chromosomes vary from 0.39 to 0.77 Mb in size, and represent up to 38% of the chromosomal complement. Chromosome numbers among progeny isolates varied widely, with some progeny missing up to three chromosomes, while other strains were disomic for one or more chromosomes. Between 15-20% of the progeny isolates lacked one or more chromosomes that were present in both parents. The two high-density maps showed no recombination of dispensable chromosomes and hence, their meiotic processing may require distributive disjunction, a phenomenon that is rarely observed in fungi. The maps also enabled the identification of individual twin isolates from a single ascus that shared the same missing or doubled chromosomes indicating that the chromosomal polymorphisms were mitotically stable and originated from nondisjunction during the second division and, less frequently, during the first division of fungal meiosis. High genome plasticity could be among the strategies enabling this versatile pathogen to quickly overcome adverse biotic and abiotic conditions in wheat fields.

G(alpha) and Gbeta proteins regulate the cyclic AMP pathway that is required for development and pathogenicity of the phytopathogen Mycosphaerella graminicola.

We identified and functionally characterized genes encoding three Galpha proteins and one Gbeta protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian Galpha(i) and Galpha(s) families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB), MgGpa1 mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation. MgGpa3 mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of MgGpb1 mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of MgGpb1 and MgGpa3 mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in MgGpa1 mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of MgGpa1, MgGpa3, and MgGpb1 are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by MgGpa3 and MgGpb1 regulate the cAMP pathway that is required for development and pathogenicity in M. graminicola.

Transposon-tagging identifies novel pathogenicity genes in Fusarium graminearum.

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.

Transposition of a fungal miniature inverted-repeat transposable element through the action of a Tc1-like transposase.

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.