Abstract

Twenty barley (Hordeum vulgare L.) cultivars bred in Cereal Institute - NAGREF, Thermi-Thesaloniki, Greece were tested for resistance to powdery mildew (Blumeria graminis f. sp. hordei). These cultivars were tested at the seedling stage with 21 differential isolates of powdery mildew. The isolates were chosen according to their virulence spectra as observed on the 'Pallas' isoline differential set and on 7 additional differential cultivars. In 11 cultivars it was not possible to postulate presence of specific resistance genes and in another 9 cultivars presence of Mlg, Ml(CP), Mla7 and Mla12 were detected. Among tested cultivars with detected resistance genes 5 (55%) cultivars had only one gene for resistance. Resistance genes at Mla locus (Mla7 and Mla12) were present in 6 (67%) cultivars with specific detected resistance genes. The most common resistance gene was Mla12. This gene was present in 5 (25%) cultivars (Paros, Cypros, Rodos, Dimitra, and Persefoni). Combination of genes Mlg and Ml(CP) was postulated to be present in 3 cultivars (Kamiros, Kadmos and Kos). Gene Mla7 was postulated to be present in one cultivar (Erato). No resistance for powdery mildew was observed for 4 (20%) cultivars (Athinaida, Grammos, Kithira and Triptolemos). The majority (61.7%) of the observed infection types was classified as susceptibility (score 4) to infection by B. graminis f.sp. hordei. About 38.3% of all infection types observed among tested lines were classified as powdery mildew resistance (scores 0, 1 and 2). The most frequent resistant infection type was 0 (27.4%) and the least frequent (1.4%) was infection type 1. Resistant infection type 2 occurred with frequency of 9.5%. Major strategies for control of powdery mildew using resistance genes are discussed.

Introduction

Powdery mildew, caused by the pathogen Blumeria graminis (DC.) Golovin ex Speer f. sp. hordei Em. Marchal (synamorph Erysiphe graminis DC. f. sp. hordei Em. Marchal), is one of the most destructive foliar diseases of barley in regions with a maritime climate including Greece (Rasmusson, 1985; Czembor, 1996; Atzema, 1998). In countries where mildew is a problem, yield losses in experimental tests usually exceed 25%, although average losses are smaller and about 10% (Zwatz 1987; Czembor & Czembor, 2001).

In the most accepted theory, barley was derived from its wild ancestor Hordeum spontaneum and the original area of cultivation and the centre of origin of H. vulgare L. is assumed to be the area of 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). However, wild progenitors of barley were exploited in Greece (Franchthi cave on Peloponnisos Peninsula ~ 12,000 BC) for at least several thousand years before the appearance of domesticated barley (Willcox, 1995). The wild barley species H. spontaneum, H. bulbosum, and H. murinum grow abundantly in Greece often on the edge of a cultivated fields with H. vulgare (Bothmer et al., 1995; Hawkes, 1995). These species are represented by ubiquitous populations greatly varying in morphology and growth habits. H. bulbosum and H. murinum introgress occasionally and H. spontaneum and H. spontaneum may cross with domesticated barley (Eyal et al., 1973; Bothmer et al., 1995; Hadjichristodoulou, 1995). The mentioned Hordeum species in Mediterranean region (including Greece) annually support outbreaks of powdery mildew of barley (Eyal et al., 1973; Czembor, 1996). The concept of correlated host-pathogen evolution implies that genetic diversity in the populations of the indigenous Hordeum species in Greece is matched by diversity in populations of E. graminis f.sp. hordei (Eyal et al., 1973; Wolfe, 1988). Taking this into account resistance of barley plays major role in barley production in Greece.

In Europe, the use of specific resistance genes to control barley powdery mildew began in the 1930s with the work of Honecker. Since that period, barley breeders commonly used resistance alleles such as Mla6, Mla7, Mla9, Mla12 and Mla13 at the Mla locus and the resistance genes Mlk, Mlg, Ml(La), Mlh and Mlra (Czembor & Czembor, 1998; 1999; 2001). However, resistance conferred by most resistance genes used on a large acreage has not been effective for more than a few years with the exception of mlo. Despite the fact that since 1979 the mlo resistance gene has been deployed in many barley cultivars throughout Europe there is no known virulence towards it (Jorgensen, 1994). This lack of durability of the other resistance genes was caused by a high level of pathogenic variability encountered in natural populations of E. graminis f. sp. hordei. It has been demonstrated many times that E. graminis f. sp. hordei is able to develop many new races and that its spores are spread in vast numbers by wind over large distances across Europe (Brändle, 1994; Limpert et al., 1999; Hovmøller et al., 2000).

In order to predict performance of specific powdery mildew resistance and to use them properly in different strategies of control it is essential to know the resistances of already registered cultivars and cultivars to be registered (breeding lines and cultivars included in registration trials) to infection by this fungus (Czembor & Gacek, 1990; Czembor & Czembor, 1998; 1999; 2001). Therefore, frequent tests of cultivars have to be carried out for identifying alleles for powdery mildew resistance. This is done on the basis of the gene-for-gene hypothesis by inoculation of plants with pathogen isolates that have a defined virulence spectrum. The subsequent reading of infection types determines a reaction spectrum for each entry and then the possible resistance phenotype of tested plant material can be derived (Czembor & Czembor, 1998; 1999; 2001; Dreiseitl & Jørgensen, 2000).

The objective of this study was to determine the identity of powdery mildew resistance genes in Greek cultivars of barley.

Materials and Methods

Twenty barley cultivars bred in Cereal Institute - NAGREF, Thermi-Thesaloniki, Greece were used (Table 1). These cultivars were tested in greenhouse with 21 isolates of B. graminis f. sp. hordei (Table 2). The isolates originated from collections of the Risø National Laboratory, Roskilde, Denmark; Danish Institute for Plant and Soil Science, Lyngby, Denmark, ETH, Zurich, Switzerland and Plant Breeding and Acclimatization Institute - IHAR, Radzików, Poland. The isolates were chosen according to differences in virulence spectra that were observed on 'Pallas' isoline differential set and additional 7 cultivars with known powdery mildew resistance genes (Kølster et al., 1986). Isolates were purified by single pustule isolation. Young seedlings of the cultivar 'Manchuria' (CI 2330) were used to maintain and propagate all isolates used. Isolates were tested frequently on host differentials to assure their purity throughout the experiment. During tests the plants were grown in plastic pots (5 cm upper diameter) filled with a mixture of Radzików sandy soil and peat in a 3 : 1 ratio with 16 h light and 8 h dark at 16 - 22ºC. At least ten plants from each selected line were tested together with seedlings of the cultivar 'Manchuria' CI 2330 (used as susceptible control) and differential set (to assure the purity of the isolates). The inoculation was carried out when plants were 10 - 12 days old (two leaf stage) by shaking or brushing conidia from diseased plants. After 8 - 10 days of incubation the disease reaction types were scored on the primary leaf of the seedlings. This scoring was done according to a 0 - 4 scale described in the previous study (Czembor, 2000). Seedlings were classified into susceptible or resistant groups. Plants with infection types 0 - 2 were classified as resistant, while plants that scored 3 and 4 were classified as susceptible.

Hypotheses about the specific resistance genes present were made from the comparison of the reaction spectra of the tested lines with those of differential lines. Identification of resistance genes was made by eliminating resistance genes not present in tested lines. The next step was determination of postulated and possible resistance genes present and was done on the basis of the gene for gene hypothesis. In the case when a compatible reaction (scores 3 and 4) was observed with one given isolate, it meant that the cultivar did not possess the resistance alleles for which the isolate was avirulent. Incompatible reactions (scores 0-2) with isolates possessing only one avirulence allele among the remaining possible resistance alleles made it possible to postulate that the matching resistance allele was present (Czembor & Czembor, 2001; Dreiseitl & Jørgensen, 2000).

Results

In 11 cultivars it was not possible to postulate presence of specific resistance genes (Table 3). In another 9 cultivars presence of Mlg, Ml(CP), Mla7 and Mla12 were detected. Among tested cultivars with detected resistance genes 5 (55%) cultivars had only one gene for resistance. Resistance alleles at Mla locus ( Mla7 and Mla12) were present in 6 (67%) cultivars with specific detected resistance genes. The most common resistance allele was Mla12. This was present in 5 (25%) of all the cultivars (Paros, Cypros, Rodos, Dimitra, and Persefoni). Combination of genes Mlg and Ml(CP) was postulated to be present in 3 cultivars (Kamiros, Kadmos and Kos). The Mla7 allele was postulated to be present in one cultivar (Erato). No resistance for powdery mildew was observed for 4 (20%) cultivars (Athinaida, Grammos, Kithira and Triptolemos).

The majority (61.7%) of all the observed infection types was classified as susceptibility (score 4) to infection by B. graminis f.sp. hordei (Table 4). About 38.3% of the remainder were classified as powdery mildew resistance (scores 0, 1 and 2). The most frequent (27.4%) resistant infection type was 0 (immunity) and the least frequent (1.4%) was infection type 1 (hypersensivity). Resistant infection type 2 occurred with frequency of 9.5%.

Discussion

Currently powdery mildew is one of the most common and most widespread disease of barley. However it was, for a long time, not an important factor in barley production. The first devastating epidemic of barley powdery mildew was observed in Europe on winter barley in 1901 and on spring barley in 1903 (Wolfe & Schwarzbach, 1978). It happened at the advent of modern agricultural methods such as the large scale cultivation of uniform varieties, the use high crop densities and the application of nitrogen fertilisers (Wolfe & Schwarzbach, 1978; Wolfe, 1984). The main means of control of powdery mildew are using of fungicides and growing of resistant varieties. However, future strategies for the control of powdery mildew will have to focus increasingly on ecologically acceptable methods because any usage of chemicals (pesticides, fungicides, herbicides, and mineral fertilisers) in agriculture is increasingly criticised in many countries. This kind of method is breeding for resistance. Application of resistance is considered also as relatively inexpensive and convenient for the farmer because use of fungicides require investments in machinery, labour and special training (Czembor & Gacek, 1990; 1995; Gullino & Kuijpers, 1994; Jacobsen, 1997).

A number of genes for specific resistance have been used in commercial barley cultivars. However, 28 of the 33 the most common alleles are closely linked or allelic (Jørgensen 1994). This limits the possible number of combination in breeding of new cultivars and all these genes were successively overcome by the appearance of pathotypes with matching virulence (Czembor & Gacek, 1990; Czembor & Czembor, 1998; 1999; 2001). This was confirmed in this study by the presence of alleles at the Mla locus in 67% Greek barley cultivars with detected specific resistances. Because of this situation the durability of resistance genes may be increased by use of multiline cultivars or by combining ('pyramiding') different resistance genes into one cultivar (Czembor & Gacek, 1990). Also deploying many cultivars with different resistance genes in space (e.g. cultivar mixtures, growing different cultivars in different regions) or time (spring versus winter) may be used (Czembor & Gacek, 1990; 1996; Czembor & Czembor, 1998; 1999; 2001; Finckh et al., 2000). However we have to admit that there is lack of new sources of powdery mildew resistance, especially Mlo, in tested Greek cultivars. The Mlo resistance has become a very important source of powdery mildew resistance in barley because there is no known virulence towards it (Hovmøller et al., 2000). The Mlo resistance is unique kind of resistance because it is monogenic and non-race-specific. Negative pleiotropic effects that were common when mlo was used in earlier crosses have been overcome by recent breeding and this type of resistance is at present utilised with increasing intensity in spring barley production. In the last few years 20-30% spring barley cultivars grown in EU and Central Europe carry Mlo resistance (Jørgensen, 1994; Atzema, 1998; Czembor & Czembor, 2001). Also relatively small number of resistance genes are present in tested cultivars and most of these cultivars (55%) had only one gene for resistance. Future work of Greek barley breeders should focus on pyramiding newly described genes for resistance, especially the mlo gene.

The results presented here come from the tests performed on seedlings, which does not necessary predict adult plant resistance and field performance of the selected resistant lines. However, determination of powdery mildew resistance genes based on tests performed on seedlings is effective and sufficient for breeders and pathologist needs (Czembor & Czembor, 1998; 1999; 2001; Dreiseitl & Jørgensen, 2000). Also different levels of partial resistance in tested lines may have influence on conclusions concerning postulation of presence of specific resistance genes (Jørgensen, 1994; Czembor, 1996).

Based on this study it may be concluded that Greek cultivars of barley posses several genes for resistance and may be used in different gene deployment strategies for efficient control of powdery mildew.

Acknowledgements

The authors thank Dr. H. J. Schaerer (ETH Zürich, Switzerland) for the powdery mildew isolates and Dr. L. Munk (Royal Agricultural and Veterinary University, Copenhagen, Denmark) for the Pallas near-isogenic lines.

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