Puccinia graminis f. sp. tritici (Pgt) race TKTTF was reported as the dominant race in the wheat stem rust epidemics in Ethiopia during 2014–15 (Olivera et al. 2015). The race and variants hereof have also been recorded elsewhere in Africa, the Middle East, and Europe (www.wheatrust.org/stem-rust-tools-maps-and-charts/race-frequency-map). Here, we report the presence of additional virulence to Sr25 in the TKTTF population, a resistance gene transferred to several Australian and CIMMYT wheat genotypes. At the seedling stage, Sr25 confers infection type (IT) 2 or lower for isolates in the Ug99 race group and up to IT 2+ toward race TKTTF (Newcomb et al. 2016; Olivera et al. 2015). Our results are based on Pgt isolates of the TKTTF race from Ethiopia (2012, 2013, 2015), Egypt (2014), Azerbaijan (2014), Iran (2009, 2011, 2014), Iraq (2014), Lebanon, Sudan, and Turkey (2012), Denmark and Germany (2013), and Sweden (2014). Race typing was carried out at the Global Rust Reference Center according to Jin et al. (2008), except that we scored IT on both leaf 1 and 2; additional single pustule isolates of each sample were raised and stored in liquid nitrogen (–196°C). Sr25 response was assayed using seedling leaves and stems of adult plants of Misr1 (Oasis/Skauz//4*BCN/3/2*Pastor) and Agatha/9*LMPG (Sr25 carriers) along with two reference lines, Triumph 64 (SrTmp) and NA101/MqSr7a (Sr7a), and Morocco as a control. Seedling ITs were scored 17 days post-inoculation at 18 ± 2°C using a 0 to 4 scale (McIntosh et al. 1995). Isolates showing ITs of 33+ to 4 on Misr1, Agatha/9*LMPG, and susceptible check were considered Sr25 virulent, and clearly different from ITs conferred by Sr25 avirulent isolates. Results were confirmed for each isolate by race typing additional single-pustule isolates derived from cultivars Misr1 and/or Agatha, along with avirulent reference isolates. Virulence for Sr25 was observed in race TKTTF isolates from Azerbaijan, Egypt, Ethiopia, Iran, Iraq, and Sweden, collected in 2014 or 2015, but not in any sample collected earlier than 2014. The results were confirmed on adult plants of Misr1 and Agatha/9*LMPG by Sr25 virulent and avirulent isolates of TKTTF, TTKSK, and TTKST, respectively. Spore suspensions of ∼0.5 ml at concentration of ∼3 × 105 spores/ml were injected into the stem internodes at Zadoks 45. The adult plant and seedling tests were carried out concurrently using the environmental conditions described above. The plants containing Sr25 were susceptible to the Sr25 virulent isolate and moderately resistant to moderately susceptible to the Sr25-avirulent isolates of TKTTF, TTKSK, and TTKST. The experiments were repeated two times with three replicates, using cv. Morocco as a susceptible check. Emergence of virulence to Sr25 in the race TKTTF is considered significant due to its spread into new areas and the potential loss of a significant source of resistance against Ug99.
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Stem rust is a potentially destructive fungal disease of wheat worldwide. In 1998 Pgt pathotype TTKSK virulent to Sr31 was detected in Uganda. The same pathotype was confirmed in Lorestan and Hamedan provinces of Iran in 2007. We used a derivative of race TTKSK to phenotype 62 Iranian wheat landraces (resistant to stripe rust in a previous study) at the seedling stage to this new pathotype (TTSSK). Twenty eight accessions were evaluated for the presence of resistance genes Sr2, Sr22, Sr24, Sr25, Sr26, Sr35, Sr36 and Srweb using SSR markers. None carried Sr2, Sr24 or Sr26, but the presence of Sr22, Sr25, Sr35 and Sr36 was indicated. Some susceptible landraces predicted to carry Sr2 by marker analysis require further investigation. To evaluate defense gene expression in compatible and incompatible stem rust interactions we sampled resistant and susceptible cultivars at 0, 12, 18, 24, 72 hours post-inoculation (hpi). ?-1,3 glucanase expression was studied using qGLU-S and qGLUU-AS primers and a real-time PCR step-one ABI machine, with ?-tubulin and EF1-? genes used as internal controls. In incompatible interactions defense gene expression was increased at 24 hpi, but in compatible interactions the highest level of expression occurred at 12 hpi and was significantly decreased at 18 hpi. The results revealed that expression of defense genes such as ?-1,3 glucanase was earlier in compatible than in incompatible interactions but the expression level was less in incompatible interactions. On the other hand, in susceptible genotypes the expression of defense genes increased immediately after inoculation and declined sharply after infection. In contrast defense gene expression in resistant genotypes began to increase after establishment of the pathogen.
Stem rust (SR) resistance is required for CIMMYT durum germplasm to keep relevance in Ethiopia, where Ug99 and other Pgt races are a major yield-limiting constraint, and in countries along the possible dissemination paths of these races. Resistance to Ug99 is widespread in most durum germplasm groups when tested in Kenya, but resistance is lost when exposed to Ethiopian races; hence selection at the Debre Zeit site in Ethiopia is essential for durum wheat. Due to difficulties with shuttling segregating populations between Mexico and Ethiopia, we have adopted a strategy involving the identification of resistant/moderately resistant lines at Debre- Zeit, and inter-crossing in Mexico followed by selection for resistance to leaf rust and agronomic type and finally screening for SR reaction in the resulting F6 lines at Debre-Zeit at the same time as they are tested for yield and quality in preliminary yield trials in Mexico. This has generated a significant increase in the proportion of resistant and moderately resistant genotypes within outgoing CIMMYT germplasm, from less than 3% at the onset of the initiative in 2008 to 16% in 2011, and 38% in 2013. SR-resistant germplasm was characterized by similar frequency distributions to other traits in the overall germplasm such as yield potential, drought tolerance and industrial quality parameters. Advances have also been realized using marker-assisted selection (MAS) to introgress Sr22 from bread wheat and to combine it with Sr25, producing advanced lines with 2-gene stacks with confirmed outstanding resistance and superior quality attributes. Since the two genes are closely linked but from different sources bringing them together required a very rare recombination event finally detected via MAS among thousands of plants. They are now essentially inherited together with a very low likelihood of generating recombinant individuals with either gene. The yield potential and stability of these lines are under evaluation in Ethiopia and the best lines are being used in a second round of breeding.
Wheat stem rust (Puccinia graminis f. sp. tritici Eriks. and E. Henn.) is one of the most destructive diseases world-wide. Races belonging to Ug99 (or TTKSK) continue to cause crop losses in East Africa and threaten global wheat production. Developing and deploying wheat varieties with multiple race-specific genes or complex adult plant resistance is necessary to achieve durability. In the present study, we applied genome-wide association studies (GWAS) for identifying loci associated with the Ug99 stem rust resistance (SR) in a panel of wheat lines developed at the International Maize and Wheat Improvement Center (CIMMYT). Genotyping was carried out using the wheat 9K iSelect single nucleotide polymorphism (SNP) chip. Phenotyping was done in the field in Kenya by infection of Puccinia graminis f. sp. tritici race TTKST, the Sr24-virulent variant of Ug99. Marker-trait association identified 12 SNP markers significantly associated with resistance. Among them, 7 were mapped on five chromosomes. Markers located on chromosomes 4A and 4B overlapped with the location of the Ug99 resistance genes SrND643 and Sr37, respectively. Markers identified on 7DL were collocated with Sr25. Additional significant markers were located in the regions where no Sr gene has been reported. The chromosome location for five of the SNP markers was unknown. A BLASTN search of the NCBI database using the flanking sequences of the SNPs associated with Ug99 resistance revealed that several markers were linked to plant disease resistance analogues, while others were linked to regulatory factors or metabolic enzymes. A KASP (Kompetitive Allele Specific PCR) assay was used for validating six marker loci linked to genes with resistance to Ug99. Of those, four co-segregated with the Sr25-pathotypes while the rest identified unknown resistance genes. With further investigation, these markers can be used for marker-assisted selection in breeding for Ug99 stem rust resistance in wheat.
Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is one of the most destructive diseases of wheat. A new race of the pathogen named TTKSK (syn. Ug99) and its derivatives detected in East Africa are virulent to many designated and undesignated stem rust resistance genes. The emergence and spread of those races pose an imminent threat to wheat production worldwide. Genes Sr25 and Sr26 transferred into wheat from Thinopyrum ponticum are effective against these new races. DNA markers for Sr25 and Sr26 are needed to pyramid both genes into adapted germplasm. The previously published dominant markers Gb for Sr25 and Sr26#43 for Sr26 were validated with eight wheat lines with or without Sr25 or Sr26. We tested six published STS (sequence tagged site) markers amplifying diagnostic bands of Th. ponticum. Marker BF145935 consistently amplified well and can be used as a co-dominant marker for Sr25. Among 16 STS markers developed from wheat ESTs mapped to deletion bin 6AL8-0.90-1.00, none was co-dominant for tagging Sr26. However, five 6A-specific markers were identified. Multiplex PCR with marker Sr26#43 and 6A-specific marker BE518379 can be used as a co-dominant marker for Sr26. The co-dominant markers for Sr25 and Sr26 were validated with 37 lines with known stem rust resistance genes. A diverse set of germplasm consisting 170 lines from CIMMYT, China, USA and other counties were screened with the co-dominant markers for Sr25 and Sr26. Five lines with the diagnostic fragment for Sr25 were identified, and they all have Ã¢â‚¬ËœWheatearÃ¢â‚¬â„¢ in their pedigrees, which is known to carry Sr25. None of the 170 lines tested had Sr26, as expected.