Resistance to leaf rust (Puccinia hordei) in Greek barley cultivars and breeding lines

Abstract

Thirty barley (Hordeum vulgare L.) cultivars and breeding lines bred in Cereal Institute - NAGREF, Thermi-Thesaloniki, Greece, were tested for resistance to leaf rust (Puccinia hordei) at the seedling stage with 8 different P. hordei isolates. Nineteen of the cultivars/lines (63%) showed resistance reaction for at least one isolate. The presence of specific resistance alleles was postulated for 13 cultivars/lines (43%), while 11 entries showed susceptible reaction to all isolates. Based on the virulence profile of the isolates, the latter cultivars had either no resistance gene to P. hordei or one or more of resistance genes Rph1, Rph10, and Rph11, respectively. Two cultivars and breeding lines showed segregation for P. hordei resistance. Five different resistance alleles, Rph2, Rph3, Rph4, Rph6, and Rph8, were detected singly or in combinations. The most common resistance allele was Rph3, which occurred in 6 cultivars/lines. Presence of alleles Rph4 and Rph8 was postulated in 5 cultivars, whereas, Rph2 and Rph6 were postulated to be present in two cultivars. Different strategies for control of leaf rust using resistance genes are discussed.

Introduction

Barley leaf rust, caused by the fungal pathogen Puccinia hordei Otth, is an important foliar disease in most barley growing regions throughout the world including Europe (Clifford, 1985; Mazaraki & Grabowska, 1998), North America (Reinhold & Sharp, 1982; Griffey et al,. 1994), Near East (Anikster et al., 1992), New Zealand (Lim & Gaunt, 1986), Australia (Park et al., 1992; Park, 2003) and North Africa (Parlevliet et al., 1981; Yahyaoui & Sharp, 1987). In Central Europe leaf rust ranks second after powdery mildew among the most common diseases of barley (Dreiseitl & Jurecka 1996, 1997; Czembor et al., 2006). In recent years the economic importance of barley leaf rust has increased in Europe especially in central and northwestern Europe (Mazaraki & Grabowska, 1998; Dreiseitl & Steffenson, 2000; Niks et al., 2000; Czembor et al., 2006). Most probably this it is caused mainly by observed increases in fitness of leaf rust populations to cultivar Vada (Rients E. Niks & Henryk J. Czembor, personal communications).

The use of disease-resistant barley cultivars has been an efficient means for controlling the disease and preventing yield losses. Barley yield losses may reach 30% in susceptible cultivars due to infection by P. hordei (Griffey et al., 1994; Whelan et al., 1997). However, the average yield losses of barley due to leaf rust reach usually 10-25% (Dreiseitl & Jurecka 1996, 1997; Niks et al., 2000). At present, 19 loci with major genes for resistance to leaf rust are described: Rph1, Rph2bj, Rph2k, Rph2l, Rph2m, Rph2n, Rph2q, Rph2r, Rph2s, Rph2t, Rph2u, Rph2y, Rph3c, Rph3w, Rph3aa, Rph4, Rph5, Rph6, Rph7g, Rph7ac, Rph8, Rph9, Rph10, Rph11, Rph12, Rph13, Rph14, Rph15, Rph16, Rph17, Rph18, Rph19 (Franckowiak et al., 1997; Park & Karakousis, 2002; Steffenson, 2002; Chelkowski et al., 2003; Park et al., 2003).

According to the most accepted theory, barley was derived from its wild ancestor Hordeum spontaneum (Bothmer et al., 1995; Willcox, 1995). It is assumed that the original area of cultivation and the centre of origin of H. vulgare L. the Fertile Crescent (Rasmusson, 1985; Bothmer et al., 1995; Willcox, 1995). From this area, cultivation of barley most probably first spread westward, reaching Greece (Knossos, Crete) around 6000 BC and was grown commonly on Balkan Peninsula around 3000 BC (Evans, 1968; Rasmusson, 1985). In Greece the wild barley species H. spontaneum, H. bulbosum, and H. murinum grow frequently on the edge of cultivated fields with H. vulgare (Bothmer et al., 1995; Penelope Bebeli, personal communication). The abundance of wild barley species in Greece greatly supports as "green bridge" outbreaks of barley diseases including leaf rust (Eyal et al., 1973; Czembor, 1996; Penelope Bebeli, personal communication). The abundance of wild Hordeum species most probably contributes to genetic diversity of leaf rust in agreement with the concept of correlated host-pathogen evolution (Wolfe, 1988; Czembor, 2000; Brown & Hovmøller, 2002; McDonald & Linde, 2002). Taking this into account resistance of barley to major diseases including leaf rust plays major role in barley production in Greece (Czembor & Bladenopoulos, 2001). The resistance alleles present in cultivars and breeding lines used in agriculture have to be known in order to interpret interactions between populations of the P. hordei and barley. Therefore, tests of the cultivars and breeding lines had to be carried out for identifying alleles for leaf rust resistance (Dreiseitl & Steffenson, 2000; Czembor & Czembor, 2007a,b). This identification is conducted on the basis of the gene-for-gene hypothesis to postulate such genes after inoculation of plants with pathogen isolates that have a defined, well-known virulence spectrum and the subsequent reading of infection types. This method is commonly used in breeding programmes of barley for resistance to infection by obligate pathogens such as rusts and powdery mildews (Dreiseitl & Steffenson, 2000).

Materials and Methods

Plant material A total of 30 barley cultivars and breeding lines bred in Cereal Institute - NAGREF, Thermi-Thesaloniki, Greece were used (Table 1).

Pathogen Eight differential isolates of P. hordei were used (Table 2). These isolates originated from IHAR Radzikow collection and were chosen according to differences in virulence spectra observed on 12 differential cultivars. None of the isolates used was able to differentiate genes Rph4 from Rph8 and Rph1 from Rph10 and Rph11.

Testing procedure This study was carried out in the IHAR Radzikow greenhouse. Cultivar L94, which does not carry any known genes for resistance to P. hordei, was used as a susceptible control. The plants were grown with 16 h light and temperature range of 18-22C. Urediniospores of P. hordei were suspended in deionized water with couple drops of "Tween 20" and inoculated onto one-week old seedling plants (primary leaf fully expanded) using a rate 3 mg urediniospores and 10 ml of water- for 100 plants. Inoculated plants were incubated for 24 hours in a chamber in which the humidity was maintained near saturation by mist from ultrasonic humidifiers n complete darkness and with a temperature range of 12-15C. Then plants were transferred to a greenhouse bench.

Disease assessment Reactions of each accession were evaluated after an incubation period of 12-14 days in a greenhouse at 20-24C. Disease symptoms were assessed on the primary leaf of the seedlings according to 0-4 scale adapted from Levine & Cherewick (1952) (Table 3). Infection types 0, 0;, 1 and 2 were considered indicative of incompatibility whereas infection types 3 and 4 of compatibility.

Postulation of leaf-rust resistance genes was done by comparisons of patterns of reaction of differential lines with known resistance genes and the cultivars/lines investigated.

Results

Of the 30 cultivars/lines investigated, 19 (63%) showed resistance reaction to at least one isolate. The presence of specific resistance alleles was postulated for 13 cultivars/lines (43%), while 11 entries showed susceptible reaction to all isolates. Based on the virulence profile of the isolates, the latter cultivars had either no resistance gene to P. hordei or one or more of resistance genes Rph1, Rph10, and Rph11, respectively. Two cultivars and breeding lines Athinaida and GD-11, showed segregation for P. hordei resistance. Five different resistance alleles, Rph2, Rph3, Rph4, Rph6, and Rph8, were detected singly or in combinations. The most common resistance allele was Rph3, which occurred in 6 cultivars/lines: Niki, GD-21, GD-32, GD-42, GD-59, GD-84. Presence of one of these alleles was postulated in 5 cultivars: Erato, Kronos, Paros, Kos, Thermi. Alleles Rph2 and Rph6 were postulated to be present in cultivars Ellasson and Kamiros. Among infection types observed only 19.9% were classified as leaf rust resistance (scores 0, 0;, 1 and 2) (Table 4). Considering infection types classified as leaf rust resistance the 0 (14.9%) was the most frequent and the 1 (0.8%) was the least frequent.

Discussion

Farmers in Europe frequently apply fungicide treatments on barley to protect it against fungal leaf pathogens, including P. hordei. However, there is increasing opposition to the application of large amounts of pesticides in agriculture, because of environmental and health risks (Gullino & Kuijpers, 1994; Nieróbca et al., 2003; Czembor, 2005). The obvious alternative to fungicide treatment against plant diseases is the use of resistant cultivars (Alemayehu, 1995; Czembor, 2005; Czembor & Czembor, 2007a, b). The obtained results indicated lack of resistance or very low number of resistance genes to P. hordei in barley cultivars and breeding lines bred in Greece. Taking this into account, it is recommended to use integrated control against this pathogen. This method includes the use of fungicides and various strategies for major and minor resistance gene deployment (Czembor, 1996, 2005; Finckh et al., 2000). In addition, there is need to identify and use new sources of resistance to this pathogen in barley breeding programmes including those in Greece (Jin et al., 1995; Manisterski & Anikster,1995; Alemayehu & Parlevliet, 1996; Backes et al., 2003).

P. hordei is characterised by large genetic variability (Park, 2003). Interesting fact is that races of P. hordei in Europe, North Africa and the Middle East have virulences to genes that have not been widely deployed in these regions (Parlevliet, 1976; 1983, Reinhold & Sharp, 1982). Use of specific resistance genes in barley may result in selection of virulent races of P. hordei. Example for this situation is deployment in barley cultivars of gene Rph7 from Cebada Capa. This gene was the most effective leaf rust resistance genes in barley and cultivars with this gene were widely grown in the southeastern USA beginning in the late 1960s. However, this gene remained effective until the early 1990s, when virulence was detected in collections from the South-East of USA and California (Steffenson et al., 1993). The origin of this virulence in the US was considered most likely to be due to mutation and selection (Steffenson et al., 1993; Alemayehu, 1995; Niks et al., 2000).

Until 1970, most leaf rust resistance breeding programs utilized specific Rph genes as sources of resistance, but few of the genes have been widely used commercially (Alemayehu, 1995; Niks et al., 2000). The resistance genes that were used usually occurred singly in released cultivars. Because of this fact, the resistance conferred by these genes was not durable (Lindhout, 2002). Parlevliet (1983) concluded that, excepted for Rph7, individual Rph genes were not worth deploying commercially. Interest in more durable resistance to leaf rust began in Europe in about 1970 by the observation of non-hypersensitive resistance called also as slow rusting (Alemayehu, 1995; Niks et al., 2000). Generally, during 1970s most European barley cultivars carried no Rph genes, but partial resistance was widespread and relatively easy to introduce to breeding programs. During 1970s and 1980s the partial resistance to leaf rust in barley was effective and it has shown little erosion. However, there were observations of increases in fitness of leaf rust populations to cultivar Vada (Parlevliet, 1983; Alemayehu, 1995; Niks et al., 2000).

In recent years, it is observed in Central Europe that leaf rust is causing more and more yield losses in most commonly grown barley cultivars. Most probably it is caused by dilution or loss of partial resistance to this pathogen in the breeding process (Rients E. Niks, personal communication). Because of this fact it is recommended to conduct tests of breeding material for leaf rust resistance. In addition, it seems that the pathogen is able to overcome any R-gene rapidly. Based on this fact, the best strategy for barley breeders to control this pathogen is to try to increase the level of partial resistance, preferably based on different sources of resistance (Alemayehu, 1995; Niks et al., 2000; Hovmøller personal communication). In the past, one of the most commonly used gene in barley breeding in Europe was Rph3 (Walther, 1996; Brooks et al. 2000). This was confirmed in our study because the most common resistance allele in tested cultivars and breeding lines was Rph3 (6 cultivars and breeding lines).

Studies were carried out on seedlings. However, partial resistance is generally better expressed at the adult plant stage (Parlevliet & van Ommeren, 1975; Smit & Parlevliet, 1990).It will be interesting to extend studies to adult plants as well. Determination of the number of genes and the type of their action in lines may be established by crosses and backcrosses among appropriate genotypes (Jin & Steffenson, 1994; Alemayehu, 1995; Martinez et al., 2001).

The present investigation resulted in information about resistance alleles of leaf rust in cultivars and breeding lines used in Greece. This kind of information will help breeders to use proper breeding initial material and to use the most effective breeding techniques to breed barley resistant to this pathogen (Finckh et al., 2000; Vallavieille-Pope et al., 2000). This kind of information is valuable in interpret interactions between populations of P. hordei and barley in Greece and to recommend the most effective method for deployment of available resistance genes in cultivars.

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