Emergence of a new race of leaf rust with combined virulence to Lr14a and Lr72 genes on durum wheat

Leaf rust is a foliar disease caused by the fungus Puccinia triticina that may severely reduce durum wheat yield. Resistance to this pathogen is common in modern durum germplasm but is frequently based on Lr72 and Lr14a. After accounts of races with virulence to Lr14a gene in France in 2000, the present study reports the detection in 2013 for the first time of a new race with virulence to Lr14a and Lr72. The aim of this work was to characterize the virulence pattern of four Spanish isolates with virulence to Lr14a, and to discuss the consequences of this presence. Rusted leaves from cultivars ‘Don Jaime’ (Lr14a) and ‘Gallareta’ (Lr72) were collected in 2013 in the field at two Spanish sites, one in the south (near Cadiz) and another in the north (near Girona). Spores from single pustule for each cultivar and site were multiplied on susceptible cultivar ‘Don Rafael’. Then, the four isolates were inoculated on a set of 19 isogenic lines Thatcher to characterize their virulence spectrum. All isolates presented the same virulence pattern. They were virulent on both Lr14a and Lr72 and the race was named DBB/BS. This race was very similar to those reported in 2009-11, but with added virulence to Lr14a. The resistance based on Lr14a has therefore been overcome in Spain, by a new race that has likely emerged via stepwise mutation from the local predominating races. This information is important to guide breeders in their breeding programmes and gene deployment strategies. additional key words: resistance; Puccinia triticina; SSR marker. authors’ contributions. Conceived and designed the experiments: IS and CR. Performed the experiments: NHS, DV, FA, JS. Analyzed the data: NHS and FMM. Contributed materials: IS, MHS and JCS. Wrote the paper: NHS, KA and FMM. citation: Soleiman, N. H.; Solis, I.; Soliman, M. H.; Sillero, J. C.; Villegas, D.; Alvaro, F.; Royo, C.; Serra, J.; Ammar, K.; Martinez, F. (2016). Short communication: Emergence of a new race of leaf rust with combined virulence to Lr14a and Lr72 genes on durum wheat. Spanish Journal of Agricultural Research, Volume 14, Issue 3, e10SC02. http://dx.doi.org/10.5424/sjar/2016143-9184. received: 22 Dec 2015. accepted: 17 Jun 2016. copyright © 2016 inia. This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial (by-nc) Spain 3.0 Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Funding: Agencia Española de Cooperación Internacional para el Desarrollo / Spanish Agency of International Cooperation for Development (AECID) funded the PhD fellowship of N. H. Soleiman. competing interests: The authors have declared that no competing interests exist. correspondence should be addressed to Fernando Martinez: fernan@us.es.


abstract
Leaf rust is a foliar disease caused by the fungus Puccinia triticina that may severely reduce durum wheat yield. Resistance to this pathogen is common in modern durum germplasm but is frequently based on Lr72 and Lr14a. After accounts of races with virulence to Lr14a gene in France in 2000, the present study reports the detection in 2013 for the first time of a new race with virulence to Lr14a and Lr72. The aim of this work was to characterize the virulence pattern of four Spanish isolates with virulence to Lr14a, and to discuss the consequences of this presence. Rusted leaves from cultivars 'Don Jaime' (Lr14a) and 'Gallareta' (Lr72) were collected in 2013 in the field at two Spanish sites, one in the south (near Cadiz) and another in the north (near Girona). Spores from single pustule for each cultivar and site were multiplied on susceptible cultivar 'Don Rafael'. Then, the four isolates were inoculated on a set of 19 isogenic lines Thatcher to characterize their virulence spectrum. All isolates presented the same virulence pattern. They were virulent on both Lr14a and Lr72 and the race was named DBB/BS. This race was very similar to those reported in 2009-11, but with added virulence to Lr14a. The resistance based on Lr14a has therefore been overcome in Spain, by a new race that has likely emerged via stepwise mutation from the local predominating races. This information is important to guide breeders in their breeding programmes and gene deployment strategies.
additional 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 Se-2 var '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 methods
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.
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 discussion
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).
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. ; [1] IT according to a 0-4 scale as described by Stakman et al. (1962) with some modifications. IT 0 to 2 (including ; that indicates the presence of only necrotic flecks on infected leaves) indicate an incompatible reaction (avirulent race), whereas IT 3 to 4 indicate a compatible response (virulent race). [2] Nomenclature of races follows Soleiman et al. (2014b). [3] Tested positive for the presence of Lr14a with the SSR markers Xgwm344 and Xgwm146.