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.

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.

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.

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.

The Use of Pentaploid Crosses for the Introgression of Amblyopyrum muticum and D-Genome Chromosome Segments Into Durum Wheat.

The wild relatives of wheat provide an important source of genetic variation for wheat improvement. Much of the work in the past aimed at transferring genetic variation from wild relatives into wheat has relied on the exploitation of the ph1b mutant, located on the long arm of chromosome 5B. This mutation allows homologous recombination to occur between chromosomes from related but different genomes, e.g. between the chromosomes of wheat and related chromosomes from a wild relative resulting in the generation of interspecific recombinant chromosomes. However, the ph1b mutant also enables recombination to occur between the homologous genomes of wheat, e.g. A/B, A/D, B/D, resulting in the generation of wheat intergenomic recombinant chromosomes. In this work we report on the presence of wheat intergenomic recombinants in the genomic background of hexaploid wheat/Amblyopyrum muticum introgression lines. The transfer of genomic rearrangements involving the D-genome through pentaploid crosses provides a strategy by which the D-genome of wheat can be introgressed into durum wheat. Hence, a pentaploid crossing strategy was used to transfer D-genome segments, introgressed with either the A- and/or the B-genome, into the tetraploid background of two durum wheat genotypes Karim and Om Rabi 5 in either the presence or absence of different Am. muticum (2n = 2x = 14, TT) introgressions. Introgressions were monitored in backcross generations to the durum wheat parents via multi-color genomic in situ hybridization (mc-GISH). Tetraploid lines carrying homozygous D-genome introgressions, as well as simultaneous homozygous D- and T-genome introgressions, were developed. Introgression lines were characterized via Kompetitive Allele-Specific PCR (KASP) markers and multi-color fluorescence in situ hybridization (FISH). Results showed that new wheat sub-genomic translocations were generated at each generation in progeny that carried any Am. muticum chromosome introgression irrespective of the linkage group that the segment was derived from. The highest frequencies of homologous recombination were observed between the A- and the D-genomes. Results indicated that the genotype Karim had a higher tolerance to genomic rearrangements and T-genome introgressions compared to Om Rabi 5. This indicates the importance of the selection of the parental genotype when attempting to transfer/develop introgressions into durum wheat from pentaploid crosses.

Rapid global spread of two aggressive strains of a wheat rust fungus.

Rust fungi can overcome the effect of host resistance genes rapidly, and spores can disperse long distance by wind. Here we demonstrate a foreign incursion of similar strains of the wheat yellow rust fungus, Puccinia striiformis f. sp. tritici, in North America, Australia and Europe in less than 3 years. One strain defined by identity at 15 virulence loci and 130 amplified fragment length polymorphism (AFLP) fragments was exclusive to North America (present since 2000) and Australia (since 2002). Another strain of the same virulence phenotype, but differing in two AFLP fragments, was exclusive to Europe (present since 2000-2001) as well as Western and Central Asia and the Red Sea Area (first appearance unknown). This may be the most rapid spread of an important crop pathogen on the global scale. The limited divergence between the two strains and their derivatives, and the temporal-spatial occurrence pattern confirmed a recent spread. The data gave evidence for additional intercontinental dispersal events in the past, that is, many isolates sampled before 2000 in Europe, North America and Australia had similar AFLP fingerprints, and isolates from South Africa, which showed no divergence in AFLP, differed by only two fragments from particular isolates from Central Asia, West Asia and South Europe, respectively. Previous research has demonstrated that isolates of the two new strains produced up to two to three times more spores per day than strains found in USA and Europe before 2000, suggesting that increased aggressiveness at this level may accelerate global spread of crop pathogens.

Differential Selection on Rhynchosporium secalis During Parasitic and Saprophytic Phases in the Barley Scald Disease Cycle.

ABSTRACT Competition among eight Rhynchosporium secalis isolates was assessed during parasitic and saprophytic phases of the disease cycle in field experiments conducted at two locations and over two growing seasons. The eight isolates were inoculated onto six barley populations exhibiting varying degrees of resistance. Microsatellite analysis of 2,866 isolates recovered from the field experiments showed significant, and sometimes opposite, changes in the frequencies of R. secalis genotypes during the growing season (parasitic phase) and between growing seasons (saprophytic phase). Isolates that showed the most complex virulence in greenhouse seedling assays had the lowest fitness in the field experiment. Significant differences in isolate fitness were found on different host populations and in different environments. Selection coefficients were large, indicating that evolution can occur rapidly in field populations. Although inoculated isolates had the lowest overall fitness on the moderately resistant landrace cv. Arabi Aswad, some isolates were more virulent and consistently increased in frequency on this landrace, suggesting a risk of directional selection and possible erosion of the resistance following its widespread deployment in monoculture. These results provide the first direct evidence that R. secalis pathogen genotypes differ in their saprophytic ability and parasitic fitness under field conditions.

Pathotype and microsatellite analyses reveal new sources of resistance to barley scald in Tunisia.

We examined the variation and relationships between pathogenicity and a microsatellite-based haplotype in 79 Tunisian Rhynchosporium secalis isolates that were collected from the most commonly cultivated barley populations in Tunisia, Rihane cv. and local landraces, with the goal of finding genes that might be used to monitor resistance to scald. Isolates could be classified into three distinct virulence groups based on artificial inoculation of 19 differential cultivars with known scald resistance genes. The resistance gene BRR2 carried by the Astrix differential cultivar appeared to be the most effective in Tunisia. Pathotypes sampled from the Rihane host were more virulent than those sampled from local barley landraces. Because some differential cultivars that carried the same resistance genes showed different reaction patterns to 48 of the isolates, we postulated that other unknown resistance gene(s) specific to Tunisian isolates may be prevalent and could be used in Tunisian barley breeding programs. Microsatellite fingerprinting allowed the detection of 11 alleles linked to the virulence and pathogenic identification of 52% of the tested isolates. Thus, microsatellite analysis may provide a rapid tool for pathogen detection, without an inoculation step that requires long incubation periods before ultimate disease assessment.

The Horn of Africa as a centre of barley diversification and a potential domestication site.

According to a widely accepted theory on barley domestication, wild barley (Hordeum vulgare ssp. spontaneum) from the Fertile Crescent is the progenitor of all cultivated barley (H. vulgare ssp. vulgare). To determine whether barley has undergone one or more domestication events, barley accessions from three continents have been studied (a) using 38 nuclear SSR (nuSSRs) markers, (b) using five chloroplast SSR (cpSSR) markers yielding 5 polymorphic loci and (c) by detecting the differences in a 468 bp fragment from the non-coding region of chloroplast DNA. A clear separation was found between Eritrean/Ethiopian barley and barley from West Asia and North Africa (WANA) as well as from Europe. The data from chloroplast DNA clearly indicate that the wild barley (H. vulgare ssp. spontaneum) as it is found today in the "Fertile Crescent" might not be the progenitor of the barley cultivated in Eritrea (and Ethiopia). Consequently, an independent domestication might have taken place at the Horn of Africa.

Genetic diversity of Rhynchosporium secalis in Tunisia as revealed by pathotype, AFLP, and microsatellite analyses.

Genetic variability among 122 Rhynchosporium secalis isolates collected from barley in three regions of Tunisia was investigated using host differentials, amplified fragment length polymorphism (AFLP), and microsatellite markers. The isolates were collected from a widely grown scald-susceptible barley cultivar Rihane and a range of local landrace cultivars in geographically distinct regions with different agroclimatic conditions. Pathotypic diversity (the proportion of unique pathotypes) was high in R. secalis populations from the high (100% diversity), moderate (95%), and low (100%) rainfall areas of Tunisia, and from both Rihane (which is the sole variety grown in the high rainfall region) and local landraces (which predominate in the low rainfall area). This may reflect a general adaptability for aggressiveness and suggests that the widely grown cultivar Rihane has exerted little or no selection pressure on the pathogen population since its release in 1983. Genotypic diversity (GD), defined as the probability that two individuals taken at random had different genotypes, was high for populations from Rihane, local landraces, and different agro-ecological zones (GD = 0.96-0.99). There was low genetic differentiation among pathogen populations from different host populations (G(ST) < or = 0.08, theta < or = 0.12) and agro-ecological zones (G(ST) < or = 0.05, theta < or = 0.04), which may be partly explained by gene flow due to the movement of infected stubble around the country. There was no correlation (r = 0.06, P = 0.39) between virulence phenotype and AFLP haplotype. A phenetic tree revealed groups with low bootstrap values that did not reflect the grouping of isolates based on host, pathotype, or agro-ecological region. The implications of these findings for R. secalis evolutionary potential and scald-resistance breeding in Tunisia are discussed.