Stripe rust resistance in Triticum durum

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and T. durum – T. urartu amphiploids. Sania Ahmed & Hadi Bux & Awais Rasheed & Alvina Gul Kazi & Abdul Rauf &. Tariq Mahmood & Abdul Mujeeb-Kazi.

Australasian Plant Pathol. DOI 10.1007/s13313-013-0237-8

Stripe rust resistance in Triticum durum – T. monococcum and T. durum – T. urartu amphiploids Sania Ahmed & Hadi Bux & Awais Rasheed & Alvina Gul Kazi & Abdul Rauf & Tariq Mahmood & Abdul Mujeeb-Kazi

Received: 12 October 2012 / Accepted: 5 June 2013 # Australasian Plant Pathology Society Inc. 2013

Abstract Stripe rust is recognized as a significant constraint to wheat production worldwide. Sustainable control against this destructive disease is achieved preferably by deploying stripe rust resistance genes (Yr) in wheat cultivars. To identify new sources of effective Yr genes against stripe rust, the response of 194 wheat amphiploids (AABBAmAm and AABBAuAu) to stripe rust at seedling and adult plant stage was evaluated. Among the amphiploids tested at seedling stage, 26 (13.40 %) were classified as resistant (IT 0–3), 9 (4.63 %) intermediate (IT 4–6) and 159 (81.95 %) as susceptible (IT 7–9). Out of 26 seedling-resistant amphiploids 17 (8.76 %) showed resistance at the adult-plant stage. Comparative analysis of amphiploids and their durum parents identified the putative source of Yr resistance. The role

Electronic supplementary material The online version of this article (doi:10.1007/s13313-013-0237-8) contains supplementary material, which is available to authorized users. S. Ahmed : A. Rauf PMAS Arid Agriculture University Rawalpindi, Rawalpindi, Pakistan H. Bux Institute of Plant Sciences, University of Sindh, Jamshoro 76080, Pakistan A. Rasheed (*) : T. Mahmood Department of Plant Sciences, Quaid-i-Azam University, Islamabad 44000, Pakistan e-mail: [email protected] A. G. Kazi Atta-ur-Rehman School of Applied Biosciences (ASAB), National University of Science and Technology (NUST), Islamabad, Pakistan A. Mujeeb-Kazi National Institute of Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan

of durum cultivar in resistance suppression was observed in many cases. These novel amphiploid (2n=6x=42) stocks are derivatives of 20 durum cultivars and accessions of the Am and Au genome effectively assembled AB genome diversity that is user friendly for maximizing their exploitation for applied agricultural targets. This initial screening facilitates the characterization of these amphiploids for stripe rust resistance breeding goals. Keywords Yellow rust . Amphiploids . Durable resistance . A-genome . Interspecific hybridization

Introduction Stripe rust (yellow rust), caused by Puccinia striiformis f. sp. tritici, is an important foliar disease of wheat. It occurs in wheat growing areas of temperate, moist and cool regions on all continents except Antarctica (Chen 2005). Its wider prevalence is a global threat to wheat production, inflicting potential losses of 30 to 100 % and affects the quality of grain and forage (Chen 2005). China, India and Pakistan are the top wheat producers in Asia where 59.3 million hectares are under wheat cultivation. Stripe rust is prevalent in 24.8 million hectares of this area i.e., ~ 40 % of wheat grown in the region (Singh et al. 2004). The identification of new stripe rust resistant genes and their deployment in new cultivars is the most effective method to protect wheat productivity. So far 84 Yr genes have been designated in wheat out of which 36 genes have temporary designations (McIntosh et al. 2010). Additionally, 52 QTLs have also been identified conferring resistance to stripe rust in bread and durum wheats (McIntosh et al. 2010). Utilization of genetic resistance to stripe rust and its incorporation in wheat demands characterization of genetic resources with potentially new or unidentified genes for resistance to the disease. These genetic resources include

S. Ahmed et al.

wild relatives, elite cultivars and landraces (Bux et al. 2012). There are numerous examples of successful transfers of genes carrying resistance to stripe rust (Kilian et al. 2011) from wild relatives. At the diploid level, there are two species of einkorn wheat: Triticum monococcum L. and T. urartu Thum. Biosystematic treatments and sterility of their hybrids (Johnson and Dhaliwal 1976) indicated that they are valid biological species. T. monococcum comprises both wild and cultivated forms and has important wild subspecies, T. boeoticum, that are wide spread in Western Asia and southern Balkans (Harlan and Zohary 1966). Triticum urartu was identified in 1937 but its existence remained fairly obscure until Johnson (1975) began investigating this species as the possible B genome donor to the polyploid wheats. Johnson (1975) argued that T. urartu could be the source of the B genome, but the T. urartu chromosome pairing with the A genome chromosomes of hexaploid wheat (T. aestivum) indicated that T. urartu must have an A genome akin to T. monococcum (Dvorak 1976; Zohary and Hopf 2000); an interpretation currently well accepted. As resistance genes become ineffective due to increased virulence in pathogen populations, breeders introduce new genes. These initially came from common wheat, however, there has been an increased use of resistance genes introduced from wheats with a lower ploidy level or from related species. The resistance genes introgressed from species of lower ploidy often confer lower levels of resistance than in the original source genotypes or there is complete suppression of resistance. The possibility of genetic suppression of phenotypic effects in wide crosses of wheat across both interspecific and intergeneric categories (Ma et al. 1997; Mujeeb-Kazi et al. 1987, 1989) has been a recurrent problem for many years, but there is no plausible explanation of how suppression actually occurs, assuming the presumed genes are present. This study was conducted to explore the stripe rust resistance in a wide array of wheat amphiploids, derived from durum cultivar crossed with Am and Au genome diploid species.

Yr3, Yr5, Yr10, Yr15, YrSP and YrCV. The isolate carries broader virulence and represents the important virulence spectrum of the current PST populations in Pakistan. Evaluation for disease resistance The germplasm was planted in a completely randomized design with three replications in disposable pots (14×14×15 cm) under glass house conditions at the Crop Disease research Program (CDRP) Murree, Pakistan. Seedlings at the two-leaf stage were inoculated with urediospore suspension of the isolate suspended in a mixture of 30:70 mineral oil: petroleum ether. Inoculated seedlings were placed in open air for 2 h and then transferred to a dew chamber maintained at 10 °C with 16 h/8 h light/ dark photoperiod for 48 h (Rizwan et al. 2007). Seedlings were transferred to a glass house with a temperature range of 18–20 °C. Infection types (IT) were recorded 3 weeks post-inoculation following the 0–9 scale (Line and Qayyum 1992). Plants having infection types 0–3 were considered resistant to stripe rust; those having ITs 4–6 and 7–9 were classified as intermediate and susceptible, respectively. Field evaluation for stripe rust resistance was done at the National Agriculture Research Centre (NARC), Islamabad (33°43′N 73°04′E, 518 m a.s.l). This location is a stripe rust intensive location in Pakistan with ideal conditions conducive for infection and spread. The entries were planted in triplicate following randomized block design in single 1-meter rows at a distance of 60 cm between rows. Stripe rust epidemic was initiated approximately 6 weeks after planting by inoculating spreader rows (Morocco) and lines to be screened with urediospores suspension (mineral oil: petroleumether 30:70). The disease severity and infection types were recorded when the cultivar Morocco reached maximum stripe rust severity level. Data were recorded twice at 10 day intervals according to the modified Cobb scale (Petersen et al. 1948). For analysis of data, severity value of all replicates were multiplied to 0.1, 0.2, 0.4, 0.8 and 1 if the infection type recorded was resistant, moderately resistant, moderately susceptible and susceptible, respectively.

Materials and methods Results Plant and pathogen materials Evaluation for disease resistance A collection of 194 A-genome amphiploids (AABBAuAu and AABBAmAm) was studied for stripe rust resistance. The production protocol of these amphiploids was reported earlier (Mujeeb-Kazi 2006). Twenty durum wheat parental lines were also studied for genomic comparison. Urediniospores of Puccinia striiformis f. sp. tritici (Pst) isolate PK2009 were used for testing at seedling and adult stage. This isolate of PST has the virulence/avirulence Yr1, Yr2, Yr6, Yr7, Yr8, Yr9, Yr17, Yr24, Yr26, Yr28, Yr29, Yr31 /

Analysis of variance revealed that highly significant variations existed among the genotypes at the seedling and adult plant stages (Table 1). A similar trend was observed for durum parents of these amphiploids (Table 1). A variety of infection types (ITs) was recorded in the germplasm. Frequencies of resistant, intermediate and susceptible genotypes are given in Table 2. Of 194Am/Au genome amphiploids tested at seedling stage, 26 (13.40 %) were classified as resistant (IT 0–3), 9

Stripe rust resistance in Triticum durum – T. monococcum Table 1 Analysis of variance (ANOVA) for severity response to stripe rust (Yr) of A-genome amphiploids (2n=6x=42) and durum parents (2n=4x=28)

Table 3 Association between seedling and adult plant evaluation to stripe rust severity in A-genome amphiploids (2n=6x=42) and their durum (2n=4x=28) parents



A-genome amphiploids Seedling-Adult

Durums (AABB)

t-test χ2-test

11.21*** 38.33***

1.79* 7.61*

Replicates Genotypes (G) Stages (S) G×S Error

A-genome amphiploids








2 193 1 193 774

5.14 2754.4 364500 2897.6 2.14

NS *** *** *** NS

2 23 1 23 94

1.15 480.2 2025 678.6 0.541

NS *** *** *** NS

NS Non-significant at P=0.05 ***Significant difference at P=0.001

(4.63 %) as intermediate (IT 4–6) and 159 (81.95 %) as susceptible (IT 7–9). Out of 26 amphiploids resistant at seedling stage, 17 (8.76 %) genotypes (accessions 6, 8, 9, 11, 12, 14, 16, 19, 28, 29, 30, 31, 34, 43, 44, 49 and 57) were also found resistant at the adult-plant stage. This indicated the presence of seedling resistance (or All-stage resistance) for stripe rust in these genotypes whereas 62 (31.95 %) genotypes were susceptible at the seedling stage and showed resistance at adult-plant stage. The genotypes in latter group may possess adult-plant resistance (APR) against stripe rust pathogen. In durum parents, 19 were found resistant and five durums (Decoy1, Altar84, SHAG-22, Yarmuk and Araus) were found susceptible. However, at the adult plant stage, SHAG-22 was found resistant which indicated the presence of adult plant resistance (APR) gene(s). In all such cases, where amphiploids are derived from susceptible durum, the derived amphiploids are equivocally getting resistance from wild diploid parent. Some statistical analyses were also carried out to analyze the association between seedling and adult plant data (Table 3). Among amphiploids, t-test revealed that differences existed between results of the seedling and adult plant stages. The same was observed for the durum parents of the amphiploids. Further validation was carried out by the chi-square test which indicated that resistant, intermediate and susceptible frequencies were

Table 2 Frequency distribution for response to stripe rust in 94 Agenome amphiploids (2n=6x=42) and their 24 durum (2n=4x=28) parents at seedling and adult plant stage Category

A-genome amphiploids

Durums (AABB)

Seedling test Field testing Seedling test Field testing Resistant 26 Intermediate 9 Susceptible 159

73 19 102

23 0 1

18 6 –

*, **, *** Significant differences at P≤0.05, 0.01, 0.001 respectively

different at seedling and adult plant stages in both durums and amphiploids. Response of durum wheat genotypes to stripe rust at seedling and adult plant stage is given in Table 4. Number of amphiploids derived from each durum and their frequency to stripe rust at seedling and adult plant stage are also depicted in Table 4. Because resistance suppression is an important phenomenon while transferring resistance from a lower ploidy level (2n=2x=14, Am/Au) to a higher, therefore higher frequency of resistance suppression was observed both at seedling and adult plant stages (Table 4). However, due to analysis of a large collection of amphiploids, we were able to identify a significantly large number of amphiploids resistant at both seedling and adult plant stage. This resistant amphiploid collection is also important because of carrying resistance from both sources i.e., durum and A genome diploid accessions, and the isolate used to screen against Stripe rust carries latest gene virulence information.

Discussion Wild progenitors of wheat possess abundant unutilized genetic diversity. There are several stripe rust resistance genes derived from wild relatives like Yr5 from T. spelta (Kema 1992), Yr8 from Ae.comosa (Riley et al. 1968), Yr9 from Secale cereale (Zeller 1973), Yr28 from Ae. tauschii (Singh et al. 1998), Yr37 from Ae. kotschyi (Marais et al. 2005), Yr38 from Ae. sharonensis (Marais et al. 2006), Yr40 from Ae. geniculata (Kuraparthy et al. 2007) and Yr42 from Ae. neglecta (Marais et al. 2009). Unfortunately, no resistance gene to stripe rust has been identified and transferred to wheat from the A-genome diploid progenitors T. monococcum and T. urartu. We have explored diversity for stripe rust resistance in 194 A-genome amphiploids where the resistance sources from different subspecies of A-genome related species will enrich the existing gene pool with the potential to improve both durum and bread wheats. Production of amphiploids involving A-genome related species and T. turgidum has been successful (Gill et al. 1988; Ma et al. 1997) and their potential in wheat breeding has been discussed earlier (Gill et al. 1988). Resistant amphiploids are being used at CIMMYT to introduce the resistance gene(s)

S. Ahmed et al. Table 4 Durum wheats and Yr response in their derived A-genome amphiploids (2n=6x=42; AABBAA) Durum

Yr (S)


Yr (A)

No. of amphiploids derived

Frequency to Yr(S)

Frequency to Yr(A)

Durum resistance suppression in amphiploids









At seedling stage (n)

At adult stage (n)

Common genotypes (n)




5 29 1 6 7

– – – – 2

– 1 – – 1

5 28 1 6 4

1 9 – – 1

4 5 1 2 –

– 15 – 4 6

5 29 – 6 5

4 14 – 4 5

4 14 – 4 4



31 9 10 2 13 17 2 13 4 3 2 16

5 – – – 4 2 – 4 1 2 – 4

1 – – – 1 – – 1 1 – 1 –

25 9 10 2 8 15 2 8 2 1 1 12

12 4 3 2 5 7 1 6 2 2 – 10

2 1 1 – 2 1 – 1 – – – 2

17 4 6 – 6 9 1 6 2 1 2 4

26 9 10 2 8 15 2 – 3 1 2 12

17 5 5 – 5 9 1 – 2 – 2 4

15 5 5 – 3 8 1 – 2 – 2 3




















– 2

2 –

– 4

2 5

– 4

– 4



4 1 2 7

– 1 – 1

– – – 1

4 – 2 5

2 1 1 1

– – – 1

2 – 1 5

4 – 2 –

1 – 1 5

1 – 1 –

Yr (S) Response to Yr at seedling stage, Yr (A) Response to Yr at adult plant stage, S Susceptible, I Intermediate, R Resistant

from T. monococcum or T. urartu to T. turgidum. Although some degree of genetic divergence is known to exist between the A genomes of the diploid and tetraploid species of wheat (Gill et al. 1988), high bivalent chromosome pairing and presence of multivalents are common in meiosis of the AAAABB amphiploids. The F1 (2n=3x=21, AAB) hybrids show bivalent associations up to a maximum of six bivalents in a majority of the meiocytes (Gul-Kazi 2011). This should allow the transfer of resistance gene(s) from the diploid to the tetraploid T. turgidum. Nevertheless, the lack of resistance expression or the existence of suppressors of resistance is a constraint that limits the potential use of some of these alien genetic resources. It may be offset by producing a wider range of amphiploid combinations as observed in this study. Finding genotypes with non-suppressing alleles or artificially mutating the suppressors may overcome this barrier. In this study we did not find any resistant durum which inhibited suppression except for LCK59.1.

However only one amphiploid was produced from this durum and it has to be analyzed further if it could be a candidate with nonsuppressing allele. One important point of this study is the testing at a single location with an isolate having limited virulence information. Testing of material at additional locations with additional isolates may lead to the information with more clarity facilitating the selection of the material for further exploitation and research. Most importantly a large number of amphiploids (62 accessions) were susceptible at seedling stage and resistant to moderately resistant at adult plant stage. These accessions may possess adult-plant resistance (APR) against stripe rust. This type of resistance unlike seedling resistance is race-non specific and durable (Chen and Moore 2002) and is more attractive for breeders. These genotypes prove to be an important genetic resource for the improvement of wheat against stripe rust and this initial screening has provided basic information about their resistance status acting as the foundation to extensive in depth

Stripe rust resistance in Triticum durum – T. monococcum

studies on the molecular characterization of the novel gene(s) and will provide allelic richness for the breeders having enthusiasm to transfer resistance along with new diversity. Moreover authentication of durable resistance in these genotypes can be carried out through genetic studies and application of molecular markers linked to the QTLs conferring this type of resistance. The significance of this initial study with novel genomic diversity is that stable hexaploid stocks have been produced to allow global exploitation for Yr resistance targeted programs through free sharing of valuable germplasm. The user friendly genetic stocks shall permit rapid exploitation and studying gene penetration and suppression aspects. Further of greater importance these stocks are an excellent conduit of exploiting major and minor genes for developing wheat cultivars with durable resistance that have a better chance to provide sustainable outputs and assist food security targets of the future that in the light of the global increase figures of 8.2 billion by 2025 and 9.2 billion by 2050 are a huge challenge to combat. True that durums (2n=4x=28, AABB) are rich source via the intraspecific hybridization route the added integration of the Am and Au diploid diversity offers unique means of pyramiding resistance factors across the durum and the diploid progenitor accessions that have been scarcely utilized in wheat breeding. The present study opens up doors to genetically diversify the wheat gene pools across the greater A genome holdings (T. boeoticum, T. monococcum and T. urartu) and the exotic tetraploids (T. dicoccum, T. dicoccoides and T. carthlicum) accessions. Acknowledgments We acknowledge Dr Kent Evans (USDA-ARS, Plant Pathology Dept. Washington State University, Pullman WA 99) for technical and English language editing of manuscript.

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