IntroductionTop

Durum wheat (Triticum turgidum subsp. durum) is an important crop in the Mediterranean basin. One of the most important biotic constraints affecting this crop is leaf rust, caused by the fungus Puccinia triticina.

For more than three decades, the leaf rust resistance gene Lr14a has been effective in protecting durum wheat against leaf rust in Spain The presence of this commonly occurring gene in durum wheat germplasm was first reported by Herrera-Foessel et al. (2008), who also found two closely linked SSR (Single Sequence Repeats) markers. The Lr14a gene is located on chromosome arm 7BL and was originally transferred from the emmer wheat ‘Yaroslav’ to common wheat, and from there to durum wheat. Many cultivars resistant to leaf rust are known to carry Lr14a, such as ‘Creso’, an Italian cultivar first released in 1974, its descendant ‘Colosseo’ (Maccaferri et al., 2010), and some CIMMYT derived cultivars like ‘Don Jaime’ (Soleiman et al., 2014a). All have been resistant to the prevailing leaf rust races in Spain until 2011 (Soleiman, 2013).

Lr72, previously known as Lr_Altar (Herrera-Foessel et al., 2014), was first described in the Mexican cultivar ‘Altar C84’, released in Spain as ‘Gallareta’. This gene effectively protected the majority of durum wheat cultivars against the Mexican leaf rust races for nearly two decades, but the resistance it provided was overcome by the race BBG/BN in 2001 in Mexico, and later in other parts of the world (Singh et al., 2004). Currently, Lr72 gene is ineffective against most leaf rust races from durum worldwide, but it is still capable of protecting durum wheat from the races that are adapted to common hexaploid wheat (Herrera-Foessel et al., 2014). In a survey on durum leaf rust in Spain in 2009-11 all races were avirulent to durum cultivars containing Lr14a and avirulent on ‘Altar’ (Soleiman, 2013). However, in France Goyeau et al. (2012) reported the existence of ‘durum’ races virulent on Lr14a but avirulent on ‘Altar’ (Lr72).

During the spring of 2013, uredinia with susceptible infection type were observed in field trials on durum wheat ‘Colosseo’ and ‘Don Jaime’ in the north and the south of Spain, confirming the virulence to Lr14a. The aim of this work was to characterize the virulence pattern of four Spanish isolates with virulence to Lr14a, and provide critical information to breeders to guide them in their gene deployment and use strategies.

Material and methodsTop

Leaves with uredinia of high infection type were collected in the field on ‘Don Jaime’ and ‘Gallareta’ durum wheat cultivars in April 2013 at two Spanish sites, Conil, near the city of Cadiz (36.27690° N, -6.08718°W) in Southern Spain, and La Tallada d’Empordà, near the city of Girona (42.08051° N, 3.05563° W) in Catalonia (northeast of Spain). Overall, four samples (one per cultivar and location) were analyzed. Spores from single uredinia from moderately infected leaves were inoculated on seedlings of the widely susceptible durum ‘Don Rafael’ in order to multiply each isolate separately. Inoculated plants were placed in a dark dew chamber for 14 h at 20 °C and 100% relative humidity.

A set of 19 near isogenic lines of the obsolete cultivar ‘Thatcher’ that have single gene for leaf rust resistance was used to determine the virulence spectrum of each isolate. ‘Thatcher’ was included in all tests as susceptible check. Nomenclature of races follows Soleiman et al. (2014b). The five sets of differential lines were: (1) Lr1, Lr2a, Lr2c, Lr3; (2) Lr9, Lr16, Lr24, Lr26; (3) Lr3ka, Lr11, Lr17, Lr30; (4) Lr3bg, Lr13, Lr15, Lr18; and (5) Lr10, Lr14a, Lr23 and‘Jupare’ (Lr27+Lr31, Lr72). Durum genotypes with known resistance genes were also included in the experiment: breeding line Somateria (Lr14a+), ‘Colosseo’ (Lr14a+), ‘Don Jaime’ (Lr14a+), ‘Gallareta’ (Lr72), ‘Guayacán INIA’ (Lr61), and breeding lines Storlom (Lr3+Lr72) and Camayo (Lr_Cam) (Herrera-Foessel et al., 2007). SSR markers Xgwm344 and Xgwm146 were used to detect the presence of the Lr14a gene. These markers are dominant and linked in coupling with that gene (Herrera-Foessel et al., 2008).

Plants were grown in soil trays (60×40×10 cm), each one containing ten genotypes with four plants per genotype. When plants were at DC 16 (Zadoks et al., 1974), the unfolded 5^th leaf of each plant was inoculated with 6 mg of urediniospores mixed with talcum powder (1:50, v/v) per tray. Plants were incubated as described above and transferred the next day to a greenhouse compartment at 18-25 ºC. Twelve days after inoculation the infection type (IT) was assessed according to a 0-4 scale (Stakman et al., 1962). Virulence spectrum of the four new isolates was compared to that of the two races that were predominant in Spain from 2009 to 2011 (Soleiman, 2013).

Results and discussionTop

The four isolates collected in 2013 had the same virulence spectrum, virulent on both Lr14a and Lr72, and the pathotype was designated as DBB/BS (Table 1). In a Spanish virulence survey in the period 2009-2011 on durum wheat leaf rust, seven races were identified among 75 isolates. The prevailing races were very closely related, with virulence on Lr72 and avirulence on Lr14a (Soleiman, 2013). The four isolates examined in the present study were very similar to the races identified during the 2009-2011 period, but with added virulence on Lr14a, and all durum cultivars carrying this gene (Table 1). One plausible origin of the new race is that it evolved from the commonly found race DBB/BN through stepwise mutation acquiring virulence for the Lr14a resistance. The simultaneous detection of DBB/BS in both south and north of Spain supports this hypothesis. Mutations of avirulence loci in urediniospores, which are produced in massive quantities in wheat fields, are an important mechanism to generate new races (Huerta-Espino et al., 2011).

Table 1. Infection types (IT)[1] at 5^th leaf stage on 20 near-isogenic Thatcher lines and eight genotypes with known R-genes inoculated with two leaf rust races collected on durum wheat in 2009-11 (DBB/BN and DBB/CN), and the new race collected in 2013 in Spain (DBB/BS).

The new race was also virulent on the wheat lines with Lr72 as observed in field trials in Conil (I. Solis, unpublished data) and Gerona (D. Villegas, unpublished data) and in our study. This indicates that this pathotype is different from the French races, many of them with virulence on lines with Lr14a and avirulent on Altar C84 (Lr72) as described by Goyeau et al. (2012). This report of races with virulence on both Lr14a and Lr72 is of critical relevance for durum wheat breeders as breeding programs and gene deployment strategies will need to be modified. For this reason, an additional test on flag leaves would clearly confirm the virulence on both genes. While Lr14a gene still provides effective protection in many areas worldwide, including the Americas, Central India, East Africa and the Middle East (K. Ammar, unpublished data), its use in new crossing programs should be avoided, even in the presence of Lr72, based on the appearance of races with Lr14a virulence in France, Spain and Tunisia (Gharbi et al., 2013), and likely in other areas of the Mediterranean Basin. Sources of resistance in addition to Lr14a gene (e.g. Lr3, LrCam, Lr27+Lr31, Lr61) will need to be used in order to maintain effective leaf rust resistance in durum wheat.