Race Ug99 Research Articles

First report of Ug99 Wheat Stem Rust caused by Puccinia graminis f. sp. tritici in South Asia.

Since the emergence of Ug99 wheat stem rust in Uganda in 1998 (Pretorius et al. 2000), the threat of movement into South Asia has been a concern due to long-distance dispersal capacity of airborne spores (Brown and Hovmøller 2002; Singh et al. 2008; Meyer et al. 2017). Increased preparedness by comprehensive rust surveillance efforts and development and deployment of resistant cultivars in advance of an incursion into South Asia has been one of the success stories of the Borlaug Global Rust Initiative (Sharma et al. 2013). In November 2023, an off-season rust survey was conducted in Marpha, Gandaki and Bagmati provinces in Nepal. Rust was only observed at two sites, Dangdunge of Dolakha district and Mude of Sindhupalchok district, where spring wheat was grown as fodder crop outside the main cropping season. Rust infected wheat leaves (10-15 leaves per site) were air dried and sealed in envelopes that were shipped under permit to the Global Rust Reference Center, Denmark. Bulk samples of stem rust, Puccinia graminis f.sp. tritici (Pgt), were recovered from both envelopes, and single pustule isolates were raised and multiplied on Morocco and McNair. Meanwhile, specimens of dry leaves were subjected to SSR genotyping according to standard procedures (Patpour et al. 2022). One distinct multi-locus Pgt genotype was observed, identical to and representing 99% of Ug99 isolates within Clade I collected in East Africa between 2012-2022. A Pgt single pustule isolate from each of the sampling sites were inoculated onto 20 internationally agreed stem rust differential lines using standard procedures, and 14 supplementary lines providing additional resolution of pathogen virulence (Patpour et al. 2022). The pathotyping was repeated in two independent experiments, which resulted in the infection type pattern of Pgt race TTKTT (Supplementary Table 1). Additional independent SSR genotype assays of recovered isolates confirmed the prevalent genotype of Clade I (Patpour et al. 2022; Szabo et al. 2022). This first detection of Ug99 race TTKTT in South Asia emphasizes the need for continued coordinated international surveillance efforts and utilization of diverse sources of resistance to control stem rust in wheat. New surveillance efforts in Nepal during February-March 2024 did not reveal additional cases of wheat stem rust. However, more detailed and sustained rust surveillance efforts, assessment of the vulnerability of current wheat crops to Ug99 and other races of stem-, stripe/yellow- and leaf rust, as well as intensified breeding for rust resistance throughout the region is strongly recommended to meet current and future plant health risks.

Molecular cytogenetic characteristics of new spring bread wheat introgressive lines resistant to stem rust.

Anticipatory wheat breeding for pathogen resistance is key to preventing economically significant crop losses caused by diseases. Recently, the harmfulness of a dangerous wheat disease, stem rust, caused by Puccinia graminis f. sp. tritici, was increased in the main grain-producing regions of the Russian Federation. At the same time, importation of the Ug99 race (TTKSK) is still a possibility. In this regard, the transfer of effective resistance genes from related species to the bread wheat breeding material followed by the chromosomal localization of the introgressions and a marker analysis to identify known resistance genes is of great importance. In this work, a comprehensive analysis of ten spring bread wheat introgressive lines of the Federal Center of Agricultural Research of the South-East Region (L657, L664, L758, L935, L960, L968, L971, L995/1, L997 and L1110) was carried out. These lines were obtained with the participation of Triticum dicoccum, T. timopheevii, T. kiharae, Aegilops speltoides, Agropyron elongatum and Secale cereale. In this study, the lines were evaluated for resistance to the Ug99 race (TTKSK) in the Njoro, Kenya. Evaluation of introgression lines in the field for resistance to the Ug99 race (TTKSK) showed that four lines were immune, two were resistant, three were moderately resistant, and one had an intermediate type of response to infection. By cytogenetic analysis of these lines using fluorescent (FISH) and genomic (GISH) in situ hybridization, introgressions from Ae. speltoides (line L664), T. timopheevii (lines L758, L971, L995/1, L997 and L1110), Thinopyrum ponticum = Ag. elongatum (2n = 70) (L664, L758, L960, L971, L997 and L1110), as well as introgressions from T. dicoccum (L657 and L664), T. kiharae (L960) and S. cereale (L935 and L968) were detected. Molecular markers recommended for marker-oriented breeding were used to identify known resistance genes (Sr2, Sr25, Sr32, Sr1A.1R, Sr36, Sr38, Sr39 and Sr47). The Sr36 and Sr25 genes were observed in lines L997 and L1110, while line L664 had the Sr39+Sr47+Sr25 gene combination. In lines L935 and L968 with 3R(3D) substitution from S. cereale, gene resistance was presumably identified as SrSatu. Thus, highly resistant to both local populations of P. graminis and the Ug99 race, bread wheat lines are promising donors for the production of new varieties resistant to stem rust.

Phylogenesis of virulent races of Puccinia graminis f. sp. tritici based on phenotyping and genotyping using newly developed SNP markers for genetic groups in clades

Ug99 (TTKSK) and Digalu (TKTTF) race groups of Puccinia graminis f. sp. tritici (Pgt), are thought to be the most virulent, rendering many wheat stem rust resistance genes (Sr) ineffective. A total of 600 single pustule Pgt isolates were collected from eight Egyptian provinces during 2016–2022, undergoing both phenotyping and genotyping assays. Among these, 129 viable samples (v-samples) underwent phenotyping using the North American differentials delineated to Pgt races in the TTKS (Ug99) and TKTTF race groups, identifying 24 distinct physiologic races of Pgt. Notably, seven virulent races, TKTTF (Digalu), TKKTF, TKPTF, TKKTP, TTRTF, TTTTF, and TKFTF, were discovered in Egypt. The host reaction of wheat germplasm to these seven races indicated a high level of susceptibility. A set of 437 dead samples (d-samples) of Pgt isolates from Egypt were genotyped using newly created 17 core SNP markers. Genotypic analysis revealed that these seven races could be classified into two major clades: III and IV. Within these clades, six subclades were identified, namely III-B, IV-A.2, IV-B, IV-E.1, IV-E.2, and IV-F. These SNP genotypes exhibited a broad geographic distribution, highlighting the global movement of Pgt populations across Europe, Asia, and Africa. Notably, none of these genetic groups were present in Egypt before 2016. The most prevalent genotypes were subclades IV-A.2 (TKTTF; Digalu), IV-E.2 (TKKTF, TKPTF, TKFTF), and III-B (TTRTF). The prevalence of these unique genotypes/races suggests clonal reproduction and evolution within them. Race TKKTP (subclade IV-E.1) is particularly concerning because of its ability to overcome the resistance conferred by gene Sr24 against race TTKSK (Ug99). Phylogenetic analysis of Pgt genetic groups revealed that the majority of identified races belong to TKTTF (Digalu) race group (subclade IV-A.2). Notably, no races were found to belong to the TTKS (Ug99) race group (clade I). This information should help breeders select a broader range of resistance genes that are both diverse and effective to enhance the durability of stem rust resistance.

Identification and introgression of a novel leaf rust resistance gene from Thinopyrum intermedium chromosome 7Js into wheat.

A novel leaf rust resistance locus located on a terminal segment (0-69.29 Mb) of Thinopyrum intermedium chromosome arm 7JsS has been introduced into wheat genome for disease resistance breeding. Xiaoyan 78829, a wheat-Thinopyrum intermedium partial amphiploid, exhibits excellent resistance to fungal diseases in wheat. To transfer its disease resistance to common wheat (Triticum aestivum), we previously developed a translocation line WTT26 using chromosome engineering. Disease evaluation showed that WTT26 was nearly immune to 14 common races of leaf rust pathogen (Puccinia triticina) and highly resistant to Ug99 race PTKST of stem rust pathogen (P. graminis f. sp. tritici) at the seedling stage. It also displayed high adult plant resistance to powdery mildew (caused by Blumeria graminis f. sp. tritici). Cytogenetic and molecular marker analysis revealed that WTT26 carried a T4BS·7JsS chromosome translocation. Once transferred into the susceptible wheat genetic background, chromosome 7JsS exhibited its resistance to leaf rust, indicating that the resistance locus was located on this alien chromosome. To enhance the usefulness of this locus in wheat breeding, we further developed several new translocation lines with small Th. intermedium segments using irradiation and developed 124 specific markers using specific-locus amplified fragment sequencing, which increased the marker density of chromosome 7JsS. Furthermore, a refined physical map of chromosome 7JsS was constructed with 74 specific markers, and six bins were thus arranged according to the co-occurrence of markers and alien chromosome segments. Combining data from specific marker amplification and resistance evaluation, we mapped a new leaf rust resistance locus in the 0-69.29Mb region on chromosome 7JsS. The translocation lines carrying the new leaf rust resistance locus and its linked markers will contribute to wheat disease-resistance breeding.

Analysis of resistance to stem rust and identification of <i>Sr</i> genes in introgressive lines of spring bread wheat

Background. Due to the increase in the harmfulness of wheat stem rust (Puccinia graminis f. sp. tritici Erikss. & E. Henn.) in the Volga region, and the likelihood of the spread of the aggressive Ug99 race, an assessment of the genetic diversity of wheat breeding lines and identification of effective Sr genes are of fundamental importance.Materials and methods. Ninety spring bread wheat introgressive lines with stem rust resistance, developed at the Federal Center of Agriculture Research of the South-East Region, were analyzed. Molecular markers were used to identify resistance genes: Sr24/Lr24, Sr25/Lr19, Sr26, Sr28, Sr31/Lr26, Sr32, Sr36, Sr38/Lr37, Sr39 and Sr57/Lr34. The analysis of plants for resistance was carried out according to standard methods; the Stakman scale was applied to determine the type of reaction.Results. The genes Sr31/Lr26, Sr25/Lr19, Sr57/Lr34, Sr38/Lr37 and Sr39/Lr35 were identified in the analyzed breeding lines. Sr25 was found in 51 lines (56,7% of samples), Sr31 in 41 lines (45.6%), Sr57/Lr34 in 5 lines, Sr38 in 10 lines and Sr39 in one line. Combinations of resistance genes were identified: Sr31+Sr25 in 28 lines (31.1%), Sr25+Sr38 in 5 lines, and Sr25+Sr39 in one line. The Sr24/Lr24, Sr26, Sr28, Sr32 and Sr36 genes were not identified.Conclusion. As a result, promising highly resistant introgressive wheat lines with promising combinations of Sr31+Sr25, Sr25+Sr38 and Sr25+Sr39 genes were identified. They can be used in Russian breeding programs for immunity.

The Screening of Pakistani Wheat Landraces to Stem Rust (Puccinia gramminis f. sp. tritici) resistance under field conditions.

The stem rust diseases of wheat have historically been the major biotic stress responsible for destabilizing production in Asia and other parts of the world .Stem rust has continuously been in surge of destroying wheat genome especially after its race Ug99 that surfaced in Uganda in year 1999 that suppressed vital stem rust resistance gene Sr31.It was the pivotal gene that had immunity to wheat against lethal stem rust fungus.In order to trace genetic resources inside the wheat genome; a set of 112 landraces was evaluated for stem rust resistance under field condition at Jhuddo, a hot spot of stem rust in southern Pakistan. The results indicated that 80% landraces were susceptible to intensity of stem rust at Jhuddo,4% moderately suscesptible,11% moderately resistant while 17% shown moderetly resistance moderetly susceptible types of reactions according to modified Cobbs Scale. .

What Was the Reason for the Durable Effect of Sr31 against Wheat Stem Rust?

Common wheat cultivars have been protected from stem rust for several decades worldwide by the Sr31 resistance gene transferred from Secale cereale L. (cv. Petkus). In 1998, Sr31 was overcome in Uganda by the Ug99 race of Puccinia graminis f. sp. tritici Eriks. & Henn. (Pgt). The Ug99 race and its derivatives have spread widely in Africa, neighboring regions and Europe. However, Sr31 remains effective in other areas of the world, including Russia. To breed wheat with durable resistance, it is promising to research the resistance mechanisms of nonhost species and introgressive cultivars. The aim of the research was to estimate the resistance of S. cereale and Triticum aestivum cultivars with Sr31 to stem rust and to study the mechanisms of incompatibility of Pgt with plants at the cellular level. The research was carried out in Western Siberia (Russia, Omsk region) in 2018–2022. Rye and wheat with Sr31 (cvs. Kavkaz, Seri 82, Bacanora (=Kauz’s’), NIL Thatcher TcLr26/Sr31) were resistant at the stages of seedling and adult plant, and cv. PWB343 was more susceptible to disease. Cytological studies have shown that Pgt died on the rye plants on the surface, and cv. Petkus intensively suppressed the development of the appressoria necessary to penetrate into tissues. Wheat cultivars inhibited the Pgt development mainly on the surface and while it attempted to penetrate into the stomata (pre-haustorial resistance). It has been demonstrated that Pgt has to adapt step-by-step to the surface and tissue properties for compatible interaction, which may be the reason for the durable effectiveness of Sr31.

Puccinia graminis f. sp. tritici Population Causing Recent Wheat Stem Rust Epidemics in Kazakhstan Is Highly Diverse and Includes Novel Virulence Pathotypes

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a reemerging disease that caused severe epidemics in northern Kazakhstan and western Siberia in the period of 2015 to 2019. We analyzed 51 stem rust samples collected between 2015 and 2017 in five provinces in Kazakhstan. A total of 112 Pgt races were identified from 208 single-pustule isolates. These races are phenotypically and genotypically diverse, and most of them are likely of sexual origin. No differentiation of phenotypes and single-nucleotide polymorphism genotypes was observed between isolates from Akmola and North Kazakhstan provinces, supporting the idea of a wide dispersal of inoculum in the northern regions of the country. Similarities in virulence profiles with Pgt races previously reported in Siberia, Russia, suggest that northern Kazakhstan and western Siberia constitute a single stem rust epidemiological region. In addition to the races of sexual origin, six races reported in Europe, the Caucasus, and East Africa were detected in Kazakhstan, indicating that this epidemiological region is not isolated, and spore inflow from the west occurs. Virulence alone or in combination to several genes effective against the Ug99 race group was detected, including novel virulence on Sr32 + Sr40 and Sr47. The occurrence of a highly diverse Pgt population with virulence to an important group of Sr genes demonstrated the importance of the pathogen's sexual cycle in generating new and potentially damaging virulence combinations.

Virulence Survey of Puccinia striiformis in Israel Revealed Considerable Changes in the Pathogen Population During the Period 2001 to 2019.

A total of 353 urediniospore isolates of Puccinia striiformis f. sp. tritici (Pst) collected in Israel during 2001 to 2019 were analyzed. Pst pathogenicity was studied with a set of 20 differentials (17 Avocet and 3 other lines). Three periods were compared: 2001 to 2007, 2009 to 2016, and 2017 to 2019. No virulence to Yr5 or Yr15 was detected. Virulence frequencies on Yr4, Yr10, Yr24, and YrSp genes rose to the moderate level (0.28 to 0.44) in 2017 to 2019. Virulence frequencies to Yr2 and Yr9 decreased. One Pst phenotype was identified in all three periods, but its frequency drastically decreased from 0.74 in 2001 to 2016 to 0.21 in 2017 to 2019. The most probable scenario of emergence of wheat yellow rust in Israel is wind dissemination of Pst urediniospores from the Horn of Africa. Variability of the Pst population increased amid considerable evolution with two major transformations in 2009 and 2017. The first modification can be attributed to changes in wheat genetic background in Israel due to deployment of new cultivars resistant to yellow rust since 2004. The second shift in 2017 can be primarily explained by intensive deployment of wheat cultivars resistant to the stem rust race Ug99 in the 2010s in the Horn of Africa. This led to changing genetic backgrounds of the cultivated wheats in the donor region and development and long-distance spread of new Pst phenotypes to Israel. Two singular multivirulent Pst phenotypes were identified in 2019, one of them being closely related to the aggressive Warrior race. Such phenotypes may potentially defeat existing resistances.

Geographical Correlation and Genetic Diversity of Newly Emerged Races within the Ug99 Lineage of Stem Rust Pathogen, Puccinia graminis f. sp. tritici, in Different Wheat-Producing Areas.

Wheat stem rust caused by Puccinia graminis f. sp. tritici is one of the most destructive wheat diseases worldwide. Identifying stem rust races in general, Ug99 lineage particularly, and determining resistance genes are critical goals for disease assessment. Thirty wheat varieties and monogenic lines with major stem rust resistance genes (Sr) were examined here over the course of three succeeding seasons from 2020 to 2022. Fourteen stem rust races have been identified in ten African countries, as well as Central and West Asia and North Africa (CWANA) and ten European countries. The Ug99 group (Clade I) included four races (TTKSK, TTKST, TTKTK, and TTKTT) and was reported in five African countries (Egypt, Kenya, Rwanda, Tanzania, and Uganda) and Iran, but none of the European countries. On the other hand, none of the races in Clade III-B (TTRTF) and Clade IV-B (TKTTF and TTTTF) were found in Egypt. Furthermore, Egyptian races were clustered separately from races identified from other countries, and six races were found only in Egypt, including PKSTC, RKTTH, TKTTC, TTTSK, TCKTC, and TKTTH. Races from Kenya, Tanzania, Uganda, Rwanda, and Iran were all closely associated with one another, according to correlation analysis. However, most races identified from other investigated regions, including Eritrea, Spain, Ethiopia, Morocco, Italy, Poland, Kenya, Tanzania, and Uganda, were adversely linked with Egyptian races. The diagnostic 350 bp long PCR fragment linked with virulence to Sr31, Clement (Sr31), and Brigardier (Sr31) was used to identify the TTKSK (Ug99) race. The identification of the regional associations and genetic diversity of newly emerged races within the Ug99 lineage of P. graminis tritici in Africa, Asia, and Europe is one of the key goals of this study. It will help plant breeders to develop new resistant lines against the virulent races, especially TTKSK (Ug99) and TTTSK. This helps in ensuring global food security in the context of climate change.

Development of a Diagnostic Assay for Differentiation Between Genetic Groups in Clades I, II, III, and IV of Puccinia graminis f. sp. tritici.

Wheat stem rust has reemerged as a serious disease caused by new variants of Puccinia graminis f. sp. tritici. Variants with significant virulence and broad geographic distribution (Africa, Central Asia, and Europe) include the Ug99 race group, race TTRTF, and TKTTF race group. Genetic analysis has placed isolates representing these critical new virulent races into 12 genetic groups that make up clades I to IV. Development of molecular diagnostic assays for these 12 genetic groups will be an important component of global surveillance efforts. A single-nucleotide polymorphism database was mined for candidate markers that would differentiate between these 12 genetic groups. Thirty-five candidate markers were screened, and a core set of 17 markers was tested against a set of 94 isolates representing a broad range of genotypes and race phenotypes. These core markers were 100% accurate in identifying the 12 genetic groups for 52 isolates in clades I to IV, and no false positives were observed with nontarget isolates. The assay has built-in redundancy so that minor genetic changes or errors in genotyping calling will not affect the accuracy of the results. This assay is also effective in identifying the genetic groups in clade V from Germany and Georgia, the three main subgroups in North American clade VI, and clade VII consisting of race TTTTF found in North and South America. This assay provides a rapid diagnostic tool for both living and nonliving samples to detect these critical new races or race groups of P. graminis f. sp. tritici.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Study of new spring bread wheat cultivar quartet in multi location test according to the KASIB system

The results of the study of a new spring bread wheat cultivar Quartet, production by introgressive breeding for economically useful traits are presented. This cultivar have a combination genes Lr39(=Lr41) (from Aegilops tauschii), Lr19/Sr25 (7DS-7DL-7Ae#1L translocation from Agr. elonga-tum) and Lr26/Sr31/Yr9/Pm8 (1BL·1R#1S translocation from S. cereale) It has been established that this cultivar has good adaptability and plasticity both in the conditions of the Saratov region Russia and when tested according to the KASIB system (. This cultivar was not inferior to local cultivar standards, but in many regions significantly exceeded them in grain productivity. At the same time, the cultivar Quartet have the good bread making quality, even though it contains the 1BL·1R#1S translocation from S. cereale. The resistance of the cultivar Quartet to leaf rust populations has been established, both in the Lower Volga region and in Western Siberia and Kazakhstan, as well as to most local stem rust populations, including the dangerous Ug99 race of this pathogen. Cultivar Quartet was slightly affected by powdery mildew. When evaluating this cultivar according to the KASIB system, it gave a differentiated degree of damage by local powdery mildew populations and had a reaction from 0 to 3 points and a degree of damage from 0% to 40%, but it should be noted that Quartet was resistant to most pathogen populations. Evaluation of a new spring bread wheat cultivar Quartet for a number of agronomic valuable traits and adaptability, revealed the prospects of using a combination of translocations 7DS-7DL-7Ae#1L + 1BL•1R#1S, in combination with chromatin from Aegilops tauschii. At the same time, a compensatory effect of the cultivar L505 was observed in the Quartet, which reduces the negative impact on the quality of flour and dough of 1BL•1R#1S translocation.

A unique race of the wheat stem rust pathogen with virulence on Sr31 identified in Spain and reaction of wheat and durum cultivars to this race

AbstractWheat stem rust, caused by Puccinia graminis f. sp. tritici, is a re‐emerging disease, posing a threat to wheat production. In Spain, stem rust has been rarely detected since the 1970s, but infection was observed in wheat fields in 2018. We analysed six stem rust samples collected in Rota, Cádiz province and one from Monteagudo del Castillo, Teruel province. All the samples from Rota were typed as race TKTTF, whereas the sample from Monteagudo del Castillo, collected in a wheat field adjacent to barberry bushes, was typed as race TKHBK. This race has a unique and significant virulence combination that includes virulence to Sr31, Sr33, Sr53 and Sr59, and is avirulent to Rusty, a durum line developed for universal susceptibility to the wheat stem rust pathogen. TKHBK is the first race outside the Ug99 race group with virulence to Sr31 and the first known race with virulence to Sr59. Genotyping studies indicate that race TKHBK does not belong to the Ug99 or TKTTF race groups and constitutes a previously unknown lineage. Two hundred bread and durum wheat cultivars and breeding lines from Spain were evaluated against TKHBK, TKTTF, and six additional races. Resistance was observed to all the races evaluated. Molecular markers confirmed the presence of Sr7a, Sr24, Sr31, Sr38 and Sr57 in bread wheat, and Sr13 in durum wheat. The re‐emergence of wheat stem rust in Spain and the occurrence of unique virulences underscore the need to continue surveying and monitoring this disease.

Molecular Characterization of Genomic Regions for Adult Plant Resistance to Stem Rust in a Spring Wheat Mapping Population.

Adult plant resistance (APR) to wheat stem rust has been one of the approaches for resistance breeding since the evolution of the Ug99 race group and other races. This study was conducted to dissect and understand the genetic basis of APR to stem rust in spring wheat line 'Copio'. A total of 176 recombinant inbred lines (RILs) from the cross of susceptible parent 'Apav' with Copio were phenotyped for stem rust resistance in six environments. Composite interval mapping using 762 genotyping-by-sequencing markers identified 16 genomic regions conferring stem rust resistance. Assays with gene-linked molecular markers revealed that Copio carried known APR genes Sr2 and Lr46/Yr29/Sr58 in addition to the 2NS/2AS translocation that harbors race-specific genes Sr38, Lr37, and Yr17. Three quantitative trait loci (QTLs) were mapped on chromosomes 2B, two QTLs on chromosomes 3A, 3B, and 6A each, and one QTL on each of chromosomes 2A, 1B, 2D, 4B, 5D, 6D, and 7A. The QTL QSr.umn.5D is potentially a new resistance gene and contributed to quantitative resistance in Copio. The RILs with allelic combinations of Sr2, Sr38, and Sr58 had 27 to 39% less stem rust coefficient of infection in all field environments compared with RILs with none of these genes, and this gene combination was most effective in the U.S. environments. We conclude that Copio carries several genes that provide both race-specific and non-race-specific resistance to diverse races of stem rust fungus and can be used by breeding programs in pyramiding other effective genes to develop durable resistance in wheat.

First Report of Races TKTTP and TKKTP of Puccinia graminis f. sp. tritici with Virulence to Wheat Stem Rust Resistance Gene Sr24 in Turkey and Tunisia

Severe wheat stem rust caused by Puccinia graminis Pers.:Pers. f. sp. tritici Erikss. (Pgt) can result in complete crop failure. In recent years, the increasing frequency and the early onset of stem rust in Central West Asia and North Africa (CWANA) has become a big concern. The Sr24 resistance gene, one of the most effective stem rust resistance genes effective against most P. graminis f. sp. tritici races worldwide, has been widely deployed. Until the recent establishment of virulence to Sr24 within the Ug99 lineage of the pathogen in Africa (Hei et al. 2020; Jin et al. 2008; Patpour et al. 2015), Iraq (Nazari et al., 2021), occasional detections of races virulent to Sr24 were reported in South Africa (Le Roux and Rijkenberg 1987), India (Bhardwaj et al. 1990), Germany (Olivera Firpo et al. 2017), Georgia (Olivera, et al. 2019), and Western Siberia (Skolotneva et al., 2020). During the rust surveys conducted in Sinops, Samsun, and Kastomonu in the Black Sea region in northern Turkey in 2018, 19 isolates were collected. Single pustule (SP) isolates were developed and used in race analysis in the Biosafety Rust Laboratory, Regional Cereal Rust Research Center (RCRRC), Izmir, Turkey. Sample recovery, experimental procedures for pre-inoculation, inoculation, incubation, and race typing were conducted as previously described (Nazari et al. 2021). Among the tested SP isolates, two isolates showed a high infection type (IT) of 33+ on the Sr24 tester line (Little Club/Agent) and a low infection type of 11+ for the source of Sr31 (Benno/6*LMPG-6). Eight SP isolates were further developed from the high IT 33+ pustules collected from the Sr24 tester line. After spore multiplications, they were used in inoculation of the 20 North American stem rust single-gene lines used to differentiate races of P. graminis f. sp. tritici, plus Trident (Sr38+), Siouxland (Sr24+Sr31), and Sisson (Sr31+Sr36). Five SP-derived isolates with IT 33+ on the Sr24 single-gene line collected from Samsun (Alacam - Etyemez; Location: N 41.61889 E 35.55722) and Sinop (Merkez-Sanlıoglu; Location: N 41.85556 E 35.04889) were identified as race TKKTP and the remaining three SP isolates as race TKTTP. In 2020, we detected two isolates of TKKTP among the stem rust samples from Tunisia submitted to RCRRC. These two isolates were collected from bread wheat cultivars Heydna and Tahmet at a trial site near Bou Salem in Western Tunisia (Location: N 36.5351 E 8.95486). Based on the negative results of the Stage 1 test using a suite of four real-time polymerase chain reaction assays diagnostic for the Ug99 race group developed by Szabo (2012), these two races should not belong to the Ug99 race group when compared to the reference Ug99 race TTKTT from Kenya. These races were virulent to Sr5, Sr21, Sr9e, Sr7b, Sr6, Sr8a, Sr9g, Sr9b, Sr30, Sr17, Sr9a, Sr9d, Sr10, SrTmp, Sr24, Sr38, and SrMcN. In addition to these genes, race TKTTP was virulent to Sr36. Both races were avirulent to Sr11 and Sr31. To our knowledge, this is the first report of P. graminis f. sp. tritici races with the Sr24 virulence in Turkey and Tunisia. The results reflect an increasing trend of virulence to Sr24 in the pathogen populations, and raise a great concern given the deployment of the Sr24 resistance gene in widely grown wheat cultivars worldwide.

Screening of Some Ethiopian Wheat Lines Against Stem Rust Races at Seedling Stage

In Ethiopia, stem rust caused by Puccinia graminis f. sp. tritici (Pgt) is one of the most serious wheat diseases. On vulnerable cultivars, it has the ability to cause yield losses of up to 100%. The infection responses of forty-two wheat lines at the seedling stage against eleven isolates of stem rust race TTKSK (Ug99 race), TTRTF, TKTTF (Digalu race), JRCQC, TKKTF, TRTTF, TTTTF, TTKTF, TTKTT, RRTTF, and TKPTF were evaluated in a study. Wheat lines were experimentally infected with urediniospores of the races under controlled conditions at Ambo Plant Protection Research Center in 2021. Against the Pgt races that were examined, different seedling resistance responses were seen. Thirteen wheat lines, WL-1, WL-4, WL-21, WL-35, WL-37, WL-38, WL-41, WL-43, WL-45, WL-48, WL-49, WL-61, WL-64, had low infection types against all races, whereas six wheat lines, WL-26, WL-31, WL-33, WL-47, WL-51, and WL-66, had the resistant wheat lines discovered in this study can be exploited as a source of resistant genes for breeding program.

Identification and characterization of Sr22b, a new allele of the wheat stem rust resistance gene Sr22 effective against the Ug99 race group.

SummaryWheat stem (or black) rust, caused by Puccinia graminis f. sp. tritici (Pgt), has been historically among the most devastating global fungal diseases of wheat. The recent occurrence and spread of new virulent races such as Ug99 have prompted global efforts to identify and isolate more effective stem rust resistance (Sr) genes. Here, we report the map‐based cloning of the Ug99‐effective SrTm5 gene from diploid wheat Triticum monococcum accession PI 306540 that encodes a typical coiled‐coil nucleotide‐binding leucine‐rich repeat protein. This gene, designated as Sr22b, is a new allele of Sr22 with a rare insertion of a large (13.8‐kb) retrotransposon into its second intron. Biolistic transformation of an ~112‐kb circular bacterial artificial chromosome plasmid carrying Sr22b into the susceptible wheat variety Fielder was sufficient to confer resistance to stem rust. In a survey of 168 wheat genotypes, Sr22b was present only in cultivated T. monococcum subsp. monococcum accessions but absent in all tested tetraploid and hexaploid wheat lines. We developed a diagnostic molecular marker for Sr22b and successfully introgressed a T. monococcum chromosome segment containing this gene into hexaploid wheat to accelerate its deployment and pyramiding with other Sr genes in wheat breeding programmes. Sr22b can be a valuable component of gene pyramids or transgenic cassettes combining different resistance genes to control this devastating disease.

A prebreeding study of introgression spring bread wheat lines carrying combinations of stem rust resistance genes, Sr22+Sr25 and Sr35+Sr25.

The Sr22, Sr35, and Sr25 genes attract the attention of bread wheat breeders with their effectiveness against Puccinia graminis f. sp. tritici race Ug99 and its biotypes. The effectiveness and impact of Sr22+Sr25 and Sr35+Sr25 gene combinations on agronomic traits have not yet been studied. In the present article, these traits were studied using the spring bread wheat lines L503/W3534//L503, L503/Sr35//L503/3/L503 carrying the Sr22+Sr25 and Sr35+Sr25 genes during 2016–2020. These lines were assessed for resistance to P. graminis f. sp. tritici under natural epiphytotics and to the Saratov, Lysogorsk and Omsk populations of the pathogen and to the PgtZ1 (TKSTF) and PgtF18.6 fungus isolates in laboratory conditions (TKSTF + Sr33). The presence of the studied Sr-genes was conf irmed by using molecular markers. Prebreeding studies were conducted during 2018–2020 vegetation periods. Under the natural epiphytotics of the pathogen and in the laboratory conditions, the Sr22+Sr25 combination was highly effective, while Sr35+Sr25 was ineffective. For grain yield, the lines with the Sr22+Sr25 and Sr35+Sr25 genes were superior to the recipient cultivar L503 in one year (Sr22+Sr25 in 2019; Sr35+Sr25 in 2018), with a decrease in 2020, but in general there were no differences. For the period 2018–2020, both combinations showed a decrease in 1000 grains weight and an increase in the germination-earing period. The line with Sr22+Sr25 genes showed insignif icant effects on gluten and dough tenacity, but the ratio of dough tenacity to extensibility was higher, and f lour strength, porosity and bread volume were lower; in the line with Sr35+Sr25 genes, the gluten content was lower, but the strength, tenacity of the dough and the ratio of dough tenacity to extensibility were higher, f lour strength and the porosity of the bread were at the recipient level, but the volume of bread was lower.

Detection of Lr19/Sr25 in segregating populations of wheat (Triticum aestivum L.) using robust molecular markers

Marker assisted breeding (MAB) technology has been proved effective to transfer the genes of interest and also increased the accuracy of selection in wheat improvement programmes. The Agropyron elongatum derived 7D.7Ag translocation carrying Lr19/Sr25 is not only effective against leaf rust pathotypes but also effective against stem rust race Ug99 and its variants. Therefore, wheat breeding lines carrying Lr19/ Sr25 translocation segment have been developed worldwide. The present study was aimed for developing wheat genotypes with Lr19/ Sr25 using Xwmc221, PSY1-E1 and Gb DNA markers in segregating populations of wheat. We could infer that Xwmc221, PSY1-E1 and Gb are effective molecular markers to tag Lr19/Sr25 in segregating generations of wheat. The data also revealed consistency between host pathogen interaction (HPI) test and Xwmc221, PSY1-E1 and Gb molecular markers for selecting lines with Lr19/Sr25 in segregating generations. This work not only confirmed the robustness of the three Lr19/Sr25 markers but also demonstrated the application of both genotyping and phenotyping in making full-proof selection of superior progenies with gene of interest in wheat.

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.