First report of expansion of virulence in Puccinia striiformis f. sp. tritici to wheat resistance genes Yr10 and Yr24 in the Middle East.

Over the last decade, field assessments of the yellow rust differential lines for resistance genes Yr10 and Yr24 and race analysis in the Middle East have demonstrated efficient yellow rust control by Yr10 and Yr24 (=Yr26). Yellow rust samples collected during 2018-21 in Central West Asia & North and sub-Saharan Africa underwent race analysis at the Regional Cereal Rust Research Center in Izmir, Türkiye. The infected leaf segments were subjected to rehydration at 20°C for three hours. Subsequently, the leaf segments were rubbed on the first leaves of seedlings of susceptible cultivar Morocco. Inoculated seedlings were placed at 10°C in dark conditions with 95% humidity for 24 hrs, then moved to a growth chamber with a 16-hr light (220 µmolm-2s-1) cycle at 15°C and an eight-hour dark period at 12°C. Urediniospores were collected 15 days post-inoculation. A set of yellow rust differential lines including Morocco, Avocet 'S', Avocet 'R', Yr1/6* Avocet 'S', Kalyansona (Yr2), Vilmorin 23 (Yr3), Hybrid 46 (Yr4), Yr6/6* Avocet 'S', Yr7/6* Avocet 'S', Yr8/6* Avocet 'S', Yr9/6* Avocet 'S', Yr10/6* Avocet 'S', Moro (Yr10+), Yr17/6*Avocet 'S', Yr24/6* Avocet 'S', TP1295 (Yr25), Yr27/6* Avocet 'S', YrSp/6* Avocet 'S', Spalding Prolific (YrSP), Strubes Dickkopf (YrSD), Tres/6*Avocet'S', Cham 1, and Ambition was used in race analysis. A mixture of 2 mL Soltrol® and 0.5 mg fresh urediniospores was used to inoculate 10-day-old seedlings of the 23 differential varieties. Pre-inoculation, incubation, and post-inoculation conditions were the same as above. Seedling infection types (ITs) were recorded 15 days post-inoculation on a scale of 0 to 9 (McNeal et al. 1971), where ITs 0 to 6 are classified as low infection types (LITs= avirulent) and ITs 7 to 9 categorized as high infection types (HITs= virulent). HITs of 7 to 9 were observed for the first time on Yr10/6* Avocet 'S', Yr24/6* Avocet 'S', as well as on Moro (Yr10+) for 25 sample of the total 50 isolates from Lebanon and Türkiye in 2018. During the race analysis in 2019 to 2021, virulence for Yr10 and Yr24 was identified among tested samples from Egypt, Lebanon, Jordan, Syria, and Türkiye, indicating the expansion of virulence for Yr10 and Yr24 into new regions. HITs were observed for the durum wheat cultivar Cham 1 and wheat cultivar Ambition in all races. Virulence for YrA, Yr2, Yr6, Yr7, Yr8, Yr17, and 32 was common within the Yr10 and Yr24 virulent races, and virulence for YrSp and Yr27 were observed in low frequency. Molecular genotyping of 209 isolates, including the Yr10 virulent races, was performed using 19 microsatellite markers (Ali et al. 2017; Rodriguez-Algaba et al. 2017) and aligned with the Puccinia striiformis nomenclature system of the Global Rust Reference Center (GRRC). The results showed that 66 isolates were identical to the genotyping lineage "ME2018" identified in Egypt in 2018 by GRRC. This genetic lineage has now been designated as PstS17 (Hovmøller et al. 2023). The durum wheat cultivars have always been resistant to yellow rust in the Middle East. Seedling tests of 50 durum advanced lines from CIMMYT's International Durum Wheat Yield Nursery showed LITs in 45 accessions (90%) against an avirulent race for Yr10 and Yr24 (PstS2), but only 12% remained resistant while tested with a PstS17 (virulent for Yr10 and Yr24). This observation provides compelling evidence of the Yr10 and/or Yr24 presence within tested durum wheat germplasm. Continued monitoring of virulence and resistance of wheat germplasm to yellow rust is critical for successful breeding for rust resistance.

Exploring the Genetic Diversity and Population Structure of Wheat Landrace Population Conserved at ICARDA Genebank.

Landraces are considered a valuable source of potential genetic diversity that could be used in the selection process in any plant breeding program. Here, we assembled a population of 600 bread wheat landraces collected from eight different countries, conserved at the ICARDA's genebank, and evaluated the genetic diversity and the population structure of the landraces using single nucleotide polymorphism (SNP) markers. A total of 11,830 high-quality SNPs distributed across the genomes A (40.5%), B (45.9%), and D (13.6%) were used for the final analysis. The population structure analysis was evaluated using the model-based method (STRUCTURE) and distance-based methods [discriminant analysis of principal components (DAPC) and principal component analysis (PCA)]. The STRUCTURE method grouped the landraces into two major clusters, with the landraces from Syria and Turkey forming two clusters with high proportions of admixture, whereas the DAPC and PCA analysis grouped the population into three subpopulations mostly according to the geographical information of the landraces, i.e., Syria, Iran, and Turkey with admixture. The analysis of molecular variance revealed that the majority of the variation was due to genetic differences within the populations as compared with between subpopulations, and it was the same for both the cluster-based and distance-based methods. Genetic distance analysis was also studied to estimate the differences between the landraces from different countries, and it was observed that the maximum genetic distance (0.389) was between the landraces from Spain and Palestine, whereas the minimum genetic distance (0.013) was observed between the landraces from Syria and Turkey. It was concluded from the study that the model-based methods (DAPC and PCA) could dissect the population structure more precisely when compared with the STRUCTURE method. The population structure and genetic diversity analysis of the bread wheat landraces presented here highlight the complex genetic architecture of the landraces native to the Fertile Crescent region. The results of this study provide useful information for the genetic improvement of hexaploid wheat and facilitate the use of landraces in wheat breeding programs.

QTL Mapping of Adult Plant Resistance to Stripe Rust in a Doubled Haploid Wheat Population.

Stripe rust caused by Puccinia striiformis Westend. f. sp. tritici. is a major bread wheat disease worldwide with yield losses of up to 100% under severe disease pressure. The deployment of resistant cultivars with adult plant resistance to the disease provides a long-term solution to stripe rust of wheat. An advanced line from the International Winter Wheat Improvement Program (IWWIP) 130675 (Avd/Vee#1//1-27-6275/Cf 1770/3/MV171-C-17466) showed a high level of adult plant resistance to stripe rust in the field. To identify the adult plant resistance genes in this elite line, a mapping population of 190 doubled haploid (DH) lines was developed from a cross between line 130675 and the universal stripe rust-susceptible variety Avocet S. The DH population was evaluated at precision wheat stripe rust phenotyping platform, in Izmir during 2019, 2020, and 2021 cropping seasons under artificial inoculations. Composite interval mapping (CIM) identified two stable QTLs QYr.rcrrc-3B.1, and QYr.rcrrc-3B.2, which were detected in multiple years. In addition to these two QTLs, five more QTLs, QYr.rcrrc-1B, QYr.rcrrc-2A, QYr.rcrrc-3A, QYr.rcrrc-5A, and QYr.rcrrc-7D, were identified, which were specific to the cropping year (environment). All QTLs were derived from the resistant parent, except QYr.rcrrc-3A. The significant QTLs explained 3.4-20.6% of the phenotypic variance. SNP markers flanking the QTL regions can be amenable to marker-assisted selection. The best DH lines with high yield, end-use quality, and stripe rust resistance can be used for further selection for improved germplasm. SNP markers flanking the QTL regions can aid in identifying such lines.

Comparison of Genomic Prediction Methods for Yellow, Stem, and Leaf Rust Resistance in Wheat Landraces from Afghanistan.

Wheat rust diseases, including yellow rust (Yr; also known as stripe rust) caused by Puccinia striiformis Westend. f. sp. tritici, leaf rust (Lr) caused by Puccinia triticina Eriks. and stem rust (Sr) caused by Puccinia graminis Pres f. sp. tritici are major threats to wheat production all around the globe. Durable resistance to wheat rust diseases can be achieved through genomic-assisted prediction of resistant accessions to increase genetic gain per unit time. Genomic prediction (GP) is a promising technology that uses genomic markers to estimate genomic-assisted breeding values (GBEVs) for selecting resistant plant genotypes and accumulating favorable alleles for adult plant resistance (APR) to wheat rust diseases. To evaluate GP we compared the predictive ability of nine different parametric, semi-parametric and Bayesian models including Genomic Unbiased Linear Prediction (GBLUP), Ridge Regression (RR), Least Absolute Shrinkage and Selection Operator (LASSO), Elastic Net (EN), Bayesian Ridge Regression (BRR), Bayesian A (BA), Bayesian B (BB), Bayesian C (BC) and Reproducing Kernel Hilbert Spacing model (RKHS) to estimate GEBV's for APR to yellow, leaf and stem rust of wheat in a panel of 363 bread wheat landraces of Afghanistan origin. Based on five-fold cross validation the mean predictive abilities were 0.33, 0.30, 0.38, and 0.33 for Yr (2016), Yr (2017), Lr, and Sr, respectively. No single model outperformed the rest of the models for all traits. LASSO and EN showed the lowest predictive ability in four of the five traits. GBLUP and RR gave similar predictive abilities, whereas Bayesian models were not significantly different from each other as well. We also investigated the effect of the number of genotypes and the markers used in the analysis on the predictive ability of the GP model. The predictive ability was highest with 1000 markers and there was a linear trend in the predictive ability and the size of the training population. The results of the study are encouraging, confirming the feasibility of GP to be effectively applied in breeding programs for resistance to all three wheat rust diseases.

Genetic Diversity and Population Structure Analysis of Triticum aestivum L. Landrace Panel from Afghanistan.

Landraces are a potential source of genetic diversity and provide useful genetic resources to cope with the current and future challenges in crop breeding. Afghanistan is located close to the centre of origin of hexaploid wheat. Therefore, understanding the population structure and genetic diversity of Afghan wheat landraces is of enormous importance in breeding programmes for the development of high-yielding cultivars as well as broadening the genetic base of bread wheat. Here, a panel of 363 bread wheat landraces collected from seven north and north-eastern provinces of Afghanistan were evaluated for population structure and genetic diversity using single nucleotide polymorphic markers (SNPs). The genotyping-by-sequencing of studied landraces after quality control provided 4897 high-quality SNPs distributed across the genomes A (33.75%), B (38.73%), and D (27.50%). The population structure analysis was carried out by two methods using model-based STRUCTURE analysis and cluster-based discriminant analysis of principal components (DAPC). The analysis of molecular variance showed a higher proportion of variation within the sub-populations compared with the variation observed as a whole between sub-populations. STRUCTURE and DAPC analysis grouped the majority of the landraces from Badakhshan and Takhar together in one cluster and the landraces from Baghlan and Kunduz in a second cluster, which is in accordance with the micro-climatic conditions prevalent within the north-eastern agro-ecological zone. Genetic distance analysis was also studied to identify differences among the Afghan regions; the strongest correlation was observed for the Badakhshan and Takhar (0.003), whereas Samangan and Konarha (0.399) showed the highest genetic distance. The population structure and genetic diversity analysis highlighted the complex genetic variation present in the landraces which were highly correlated to the geographic origin and micro-climatic conditions within the agro-climatic zones of the landraces. The higher proportions of admixture could be attributed to historical unsupervised exchanges of seeds between the farmers of the central and north-eastern provinces of Afghanistan. The results of this study will provide useful information for genetic improvement in wheat and is essential for association mapping and genomic prediction studies to identify novel sources for resistance to abiotic and biotic stresses.

First Report of Ug99 Race TTKTT of Wheat Stem Rust (Puccinia graminis f. sp. tritici) in Iraq.

A wheat rust survey was conducted in Iraq in 2019 and collected 27 stem rust (caused by Puccinia graminis Pers.:Pers. f. sp. tritici Erikks. & E. Henn.) samples. Seven samples were viable, and they were tested for races of P. graminis f. sp. tritici at the Regional Cereal Rust Research Center (RCRRC) in Izmir, Turkey under strict quarantine procedures. Two 0.5 cm segments of each infected stem sheath were incubated in a petri dish at 20°C for three hours for re-hydration of urediniospores, which were multiplied on 10-day old seedlings of susceptible cultivar Morocco grown in a spore free growth chamber at 18°C and 16 hours light. Inoculated seedlings underwent a dew period at 18°C for 16 hours dark and 8 hours fluorescent light and 95% relative humidity. Three days after moving the pots to a growth chamber with eight hours dark at 18°C and 16 hours light (300 µmol m-2s-1), each pot was covered using a cellophane bag. Bulk urediniospores of each collection were collected 14 days post-inoculation from a cellophane bag using a mini cyclone spore collector connected to a gelatin capsule. One ml of 3M Novec™ oil was added to each capsule, and spores were inoculated onto 20 North American stem rust differential lines using the standard procedures (Jin et al. 2008). Pre-inoculation, inoculation, incubation, and post-inoculation conditions were the same as above. Seedling infection types (ITs) were recorded 14 days post-inoculation using 0 to 4 scale (Stakman et al. 1962). Race designation followed the five- letter code nomenclature described by Jin et al. (2008). Out of the seven samples, four were typed as TKKTF, two as TKTTF, and one collected from an advanced breeding bread wheat line "Shahoo 2" (Inqalab 91*2/Tukuru) in a trial site at Halabja governorate showed mixed ITs of 11+ and 3+ for lines carrying Sr11, Sr24, Sr36, and Sr31. Three single pustule (SP) isolates were developed from the IT of 3+ pustules collected from the Sr31 tester line, and one SP isolate was developed from the IT 11+ pustule collected from the Sr11 source. After spore multiplication, the SP-derived isolates were inoculated on the 20 North American differential lines. To confirm virulence/avirulence on Sr24, Sr31, and Sr36, cultivars Siouxland (PI 483469, Sr24+Sr31) and Sisson (PI 617053, Sr36+Sr31) were also inoculated. All seedling assays were repeated three times. The three SP isolates virulent on Sr31 were designated as race TTKTT, and the SP isolate avirulent on Sr11 was designated as TKTTF. Seedling ITs of 3+ and 0; were recorded for Siouxland and Sisson against TTKTT, respectively, and both cultivars showed IT of 1+ against TKTTF. Race TKTTF was similar to TKKTF except for additional virulence on Sr36, and TTKTT differed from the other two races being virulent on Sr24 and Sr31. DNA analysis of three TTKTT isolates from Kenya and the TTKTT isolate from Iraq using a diagnostic qPCR assay developed by the USDA-ARS Cereals Disease Laboratory (Ug99 RG stage 1, Szabo unpublished) confirmed that all tested isolates belonged to the Ug99 lineage. Race TTKTT was first reported from Kenya in 2014 (Patpour et al. 2016), and in 2018 from Ethiopia (Hei et al. 2020). We report the first detection of TTKTT in Iraq and the Middle East region. This represents only the third instance of a member of the Ug99 race group outside of Africa since first detection of race TTKSK in Yemen in 2006, and Iran in 2007 (Nazari et al. 2009). The continued spread of stem rust races with complex virulence and the increasing frequency and early onset of stem rust infections in the Middle East is a cause for concern. Continuous support for rust surveillance and race typing in this region remains crucial. References: Hei, N. B., et al. 2020. Plant Dis. 104:982. Jin, Y., et al. 2008. Plant Dis. 92:923-926. Nazari, K., et al. 2009. Plant Dis. 93:317. Patpour, M., et al. 2016. Plant Dis. 100:522. Stakman, E. C., et al. 1962. Identification of physiological races of Puccinia graminis var. tritici. U. S. Dep. Agric. ARS E-617.

Genome-wide association study of resistance to PstS2 and Warrior races of Puccinia striiformis f. sp. tritici (stripe rust) in bread wheat landraces.

Stripe or yellow rust, caused by Puccinia striiformis Westend. f. sp. tritici is a major threat to bread wheat production worldwide. The breakdown in resistance of certain major genes and newly emerging aggressive races of stripe rusts pose serious concerns in all main wheat growing areas of the world. To identify new sources of resistance and associated QTL for effective utilization in future breeding programs an association mapping (AM) panel comprising of 600 bread wheat landraces collected from eight different countries conserved at ICARDA gene bank were evaluated for seedling and adult plant resistance against the PstS2 and Warrior races of stripe rust at the Regional Cereal Rust Research Center (RCRRC), Izmir, Turkey during 2016, 2018 and 2019. A set of 25,169 informative SNP markers covering the whole genome were used to examine the population structure, linkage disequilibrium and marker-trait associations in the AM panel. The genome-wide association study (GWAS) was carried out using a Mixed Linear Model (MLM). We identified 47 SNP markers across 19 chromosomes with significant SNP-trait associations for both seedling stage and adult plant resistance. The threshold of significance for all SNP-trait associations was determined by the false discovery rate (q) ≤ 0.05. Three genomic regions (QYr.1D_APR, QYr.3A_seedling and QYr.7D_seedling) identified in this study do not correspond to previously reported Yr genes or QTL, suggesting new genomic regions for stripe rust resistance.

Yellow Rust Epidemics Worldwide Were Caused by Pathogen Races from Divergent Genetic Lineages.

We investigated whether the recent worldwide epidemics of wheat yellow rust were driven by races of few clonal lineage(s) or populations of divergent races. Race phenotyping of 887 genetically diverse Puccinia striiformis isolates sampled in 35 countries during 2009-2015 revealed that these epidemics were often driven by races from few but highly divergent genetic lineages. PstS1 was predominant in North America; PstS2 in West Asia and North Africa; and both PstS1 and PstS2 in East Africa. PstS4 was prevalent in Northern Europe on triticale; PstS5 and PstS9 were prevalent in Central Asia; whereas PstS6 was prevalent in epidemics in East Africa. PstS7, PstS8 and PstS10 represented three genetic lineages prevalent in Europe. Races from other lineages were in low frequencies. Virulence to Yr9 and Yr27 was common in epidemics in Africa and Asia, while virulence to Yr17 and Yr32 were prevalent in Europe, corresponding to widely deployed resistance genes. The highest diversity was observed in South Asian populations, where frequent recombination has been reported, and no particular race was predominant in this area. The results are discussed in light of the role of invasions in shaping pathogen population across geographical regions. The results emphasized the lack of predictability of emergence of new races with high epidemic potential, which stresses the need for additional investments in population biology and surveillance activities of pathogens on global food crops, and assessments of disease vulnerability of host varieties prior to their deployment at larger scales.

Molecular markers for tracking the origin and worldwide distribution of invasive strains of Puccinia striiformis.

Investigating the origin and dispersal pathways is instrumental to mitigate threats and economic and environmental consequences of invasive crop pathogens. In the case of Puccinia striiformis causing yellow rust on wheat, a number of economically important invasions have been reported, e.g., the spreading of two aggressive and high temperature adapted strains to three continents since 2000. The combination of sequence-characterized amplified region (SCAR) markers, which were developed from two specific AFLP fragments, differentiated the two invasive strains, PstS1 and PstS2 from all other P. striiformis strains investigated at a worldwide level. The application of the SCAR markers on 566 isolates showed that PstS1 was present in East Africa in the early 1980s and then detected in the Americas in 2000 and in Australia in 2002. PstS2 which evolved from PstS1 became widespread in the Middle East and Central Asia. In 2000, PstS2 was detected in Europe, where it never became prevalent. Additional SSR genotyping and virulence phenotyping revealed 10 and six variants, respectively, within PstS1 and PstS2, demonstrating the evolutionary potential of the pathogen. Overall, the results suggested East Africa as the most plausible origin of the two invasive strains. The SCAR markers developed in the present study provide a rapid, inexpensive, and efficient tool to track the distribution of P. striiformis invasive strains, PstS1 and PstS2.

Genome-wide DArT and SNP scan for QTL associated with resistance to stripe rust (Puccinia striiformis f. sp. tritici) in elite ICARDA wheat (Triticum aestivum L.) germplasm.

KEY MESSAGE: Identified DArT and SNP markers including a first reported QTL on 3AS, validated large effect APR on 3BS. The different genes can be used to incorporate stripe resistance in cultivated varieties. Stripe rust [yellow rust, caused by Puccinia striiformis f. sp. tritici (Pst)] is a serious disease in wheat (Triticum aestivum). This study employed genome-wide association mapping (GWAM) to identify markers linked to stripe rust resistance genes using Diversity Arrays Technology (DArT(®)) and single-nucleotide polymorphism (SNP) Infinium 9K assays in 200 ICARDA wheat genotypes, phenotyped for seedling and adult plant resistance in two sites over two growing seasons in Syria. Only 25.8 % of the genotypes showed resistance at seedling stage while about 33 and 44 % showed moderate resistance and resistance response, respectively. Mixed-linear model adjusted for false discovery rate at p < 0.05 identified 12 DArT and 29 SNP markers on chromosome arms 3AS, 3AL, 1AL, 2AL, 2BS, 2BL, 3BS, 3BL, 5BL, 6AL, and 7DS significantly linked to Pst resistance genes. Of these, the locus on 3AS has not been previously reported to confer resistance to stripe rust in wheat. The QTL on 3AS, 3AL, 1AL, 2AL, and 2BS were effective at seedling and adult plant growth stages while those on 3BS, 3BL, 5BL, 6AL and 7DS were effective at adult plant stage. The 3BS QTL was validated in Cham-6 × Cham-8 recombinant inbred line population; composite interval analysis identified a stripe resistance QTL flanked by the DArT marker, wPt-798970, contributed by Cham-6 parent which accounted for 31.2 % of the phenotypic variation. The DArT marker "wPt-798970" lies 1.6 cM away from the 3BS QTL detected within GWAM. Epistatic interactions were also investigated; only the QTL on 1AL, 3AS and 6AL exhibited interactions with other loci. These results suggest that GWAM can be an effective approach for identifying and improving resistance to stripe rust in wheat.

Pathogenicity Spectra and Screening for Resistance in Barley Against Tunisian Pyrenophora teres f. teres.

This work aimed to determine patterns of pathogenicity in Pyrenophora teres f. teres and to identify potentially effective resistance sources that could be used as breeding material to control net blotch in Tunisia. Extensive pathogenic variability was detected in 85 isolates of P. teres causing net blotch of barley in Tunisia. Based on unweighted pair-group method with arithmetic averaging clustering and mean disease rating scores, three distinct virulence groups were identified. The isolates were classified into 23 pathotypes. Pathogenic variability within the groups was higher than that between the groups, a finding that can guide a rational choice of isolates for screening lines as part of a breeding program. Conversely, studying the relationship between geographic and pathotypic structure allowed us to detect a significant isolation by distance pattern, suggesting a regular and gradual dispersal of the pathogen over this spatial scale. Using specific resistance properties of individual barley genotypes as virulence markers, all the differential barley genotypes were shown to be distinct, and no single source of resistance was totally effective against all isolates.