Sources of Resistance to Powdery Mildew in Wild Barley (Hordeum vulgare subsp. spontaneum) Collected in Jordan, Lebanon, and Libya

1. Introduction

Among cereals, barley (Hordeum vulgare L.) is the fourth most important in the world. However, the importance of the crop in many areas of the world lies in the fact that it is often the only crop possible to grow in semi-arid areas and at elevations higher than other cereals. In the Near East and North Africa, barley is a typical crop in hostile environments. The importance of this crop is growing because of climate change and more unpredictable weather conditions in many regions of the world [1,2,3]. Barley is considered as one of the oldest domesticated crops. It is used for feed, malt, and food. In many areas, especially with arid and semi-arid climates, barley straw yield is the most important for farmers. Recently, more and more popular is the use of barley as food due to its health properties [3,4,5,6].

Barley's primary gene pool includes two subspecies: domesticated barley (Hordeum vulgare subsp. vulgare) and wild barley (Hordeum vulgare subsp. spontaneum) (Hvs). Wild barley differs from cultivated barley in several traits including a brittle rachis and it is considered a progenitor of cultivated barley [3,6]. Hvs occurs in Southwest Asia and, most probably due to human activities, populations of Hvs are present in Morocco, Ethiopia, and Tibet [3,6,7].

In the last century because of increasing crop erosion, many gene banks were established. The main goal of gene banks is to preserve key plant genetic resources in order to meet current and future needs concerning food production. It is achieved by introducing them into breeding programs to achieve biological progress and for use in direct production. To do this effectively there is a need for phenotyping and genotyping data for major gene bank collections [8,9,10]. There are relatively large collections of the genus Hordeum stored in many gene banks worldwide. It is estimated that about 485,000 accessions of this genus are stored at more than 200 institutions worldwide. These collections include H. vulgare ssp. vulgare (299 165 accessions), wild barley Hvs (32 385 accessions), and wild species of Hordeum (4 681 accessions). [3,6,11].

In West Asia and North Africa (WANA) including Jordan, Lebanon, and Libya are present diverse agroecological zones and different types and intensity of agriculture [12,13]. Wild barley is a widespread species in this region and genetically diverse populations of Hvs are reported to be collected [12,13,14,15]. Many studies showed that landraces and Hvs are very diverse and represent great value for breeding barley as the source of resistance to both abiotic and biotic stresses [16,17,18]. Recently there is also increasing interest in the study of both landraces and Hvs as a potential source of economically important characteristics to breed cultivars well adapted to changing climate conditions and more frequent weather anomalies [16,18].

Blumeria graminis (DC.) Golovin ex Speer f.sp. hordei Em. Marchal (Bgh) is a fungus that causes barley powdery mildew (BPM). It is considered as one of the most economically important pathogens on barley which can cause significant yield losses. Many studies have shown that Bgh is relatively rapidly developing many new races and that its spores are dispersed by wind over long distances in Europe [19,20,21,22,23,24,25,26]. It occurs in many barley growing regions of the world but it is especially important in Europe. This is due to the maritime climate in most of the Europe areas suitable for the development of BPM. In addition, barley is grown in Europe on relatively large areas and more than 60% of barley world production originates from this continent [21,25,26]. The average annual losses caused by this disease in barley production in Central Europe are estimated at about 10%. However, in many experiments, barley yield losses due to the occurrence of heavy infestation by BPM usually exceed 25%. The grain yield obtained from barley fields where BPM was present very often is characterized by lower quality characteristics important in malt production such as higher grain protein content and lack of proper grain size uniformity [27,30].

Chemical control and agronomic practices are used to reduce BPM incidence. However, these methods are often not effective, and in addition, there is a growing emergence of BPM resistance to fungicides [31,32]. A commonly used way to control BPM was the incorporation of new effective genes for powdery mildew resistance into barley cultivars [19,21,25]. It is the most effective and environmentally safe method to control this disease. Effective resistance not only protects the cultivated varieties but also reduces the production of inoculum and the spread of the pathogen to larger areas, leading to epiphytosis [24,25,33,34,35]. In most cases of growing cereals, including barley, in agricultural practice, the control against fungal pathogens is based on Integrated Pest Management (IPM) principles [36,37]. This approach to managing pests is based on combining biological, cultural, physical, and chemical methods to minimize economic, health, and environmental risks. Recently the use of genetic resistance as a component of IPM become more important due to the implementation of more environmentally friendly agricultural policies in many countries of the world [37,38].

From the very beginning of genetics studies of the resistance of barley to BPM, the Hvs was used. The very first study was conducted by Biffen in 1907 who analyzed the mode of inheritance of BPM resistance in progenies from crossing H. vulgare with Hvs [39]. Since that time race-specific resistance genes have been identified mainly in cultivated barley landraces [40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55] and wild barley [56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71] mostly originating from the WANA region [19,21,25]. Based on genetic studies many specific resistant genes were described in wild barley: Mla16-Mla21, Mla25-Mla29, Mla32, MlaLv, Mlf, Mlj, mlt, Ml(Ro), Ml(Ve) [25,58,59,65,66,68]. Barley breeders used many BPM resistance genes, especially in the Mla locus and Mlra, Mlk, MlLa, Mlg, Mlh [19,21,25]. However, many of these genes have lost their effectiveness as a result of pathogen adaptation and the emergence of virulent races to these genes [19,20,21,22,23,24,25,26]. In the last 40 years, only barley cultivars with Mlo resistance have been characterized as those with durable resistance to BPM because no known virulence for mlo genes was identified. This type of resistance to BPM was identified in barley mutants and in landraces, but not in Hvs [19,21,25,72,73,74].

Many studies proved that studies on the genetics of resistance to BPM using a differential set of BPM isolates can be successfully used for investigations to determine the presence of specific resistance genes in barley genetic resources [19,21,25,26]. The new efficient sources of resistance to BPM for proper crosses in breeding programs are crucial to conducting resistance breeding [25,33,34,35]. The use of seedlings in studies conducted to postulate specific BPM resistance genes using a differential set of Bgh isolates was proven to be an effective and sufficient method. This method is commonly used for the characterization of barley germplasm concerning its BPM resistance [26,40,43,50,51,52,54,55].

The presented investigation goal was to detect new sources of BPM resistance in accessions of Hvs collected in Jordan, Lebanon, and Libya.

1.1. Plant material

Eighty-one accessions of wild barley (H. vulgare subsp. spontaneum) (Hvs) collected in Jordan, Lebanon, and Libya were obtained from the ICARDA gene bank. These accessions were collected in 10 expeditions (LBY81, LBY82, LBY90, LBN92-2, LBN93, LBN94-1, JOR81-2, JOR85, JOR88-1, JOR95) during period 1981-1995. (Table 1).

1.2. Pathogen

Twenty B. graminis f. sp. hordei Em Marschal (Bgh) isolates were used to determine the resistance genes present in the tested accessions. These isolates possessed virulence genes corresponding to the most known resistance genes used in barley resistance programs (Table 2). Isolates originated from the collections in Risø National Laboratory, Roskilde, Denmark; Danish Institute for Plant and Soil Science, Lyngby, Denmark; Edigenossische Technische Hochschule – ETH, Zurich, Switzerland and Plant Breeding and Acclimatization Institute – National Research Institute (PBAI-NRI) IHAR-PIB Radzików, Poland. The isolates were chosen according to differences in virulence spectra that were observed on the Pallas isolines differential set [75] and on additional cultivars with resistance genes not present in Pallas isolines. Each of them represented a different pathotype, determined using the selected set of 20 Pallas isolines differential. Isolate Bgh 33 was the most avirulent isolate in the collection.

They were purified by single pustule isolation and were maintained and propagated on young seedlings of the powdery mildew susceptible cultivar Manchuria (CI 2330). Frequent virulence checks were made to ensure the purity of isolates throughout the experiment.

2.3. Populations and single plant lines resistance tests

In the preliminary study thirty plants per accession were evaluated with the Bgh33 isolate. Next, the selected single plant lines were tested with 20 differential isolates of Bgh.

All these tests were conducted under controlled conditions with a 16/8 h day/night photoperiod and a 22/16°C temperature regime. In all tests, the cultivar Manchuria CI 2330 was used as a susceptible control.

Seedlings with a fully expanded first leaf were inoculated with Bgh by shaking conidia from the susceptible cv. Manchuria CI 2330. After 8-10 days, the reaction type (RT) of plants to infection by Bgh was scored. A five-point RT scale was used: 0, no visible symptoms; 1, minute necrotic flecks, no mycelial growth, and no sporulation; 2, frequent chlorosis, reduced mycelial growth and no or very scarce sporulation; 3, moderate mycelial growth, moderate sporulation, and occasional chlorosis; 4, profuse sporulation of well-developed colonies, 0(4) sparse small colonies originating from the stomatal subsidiary cells [42,76]. Plants with RT of 0, 0(4), and 1 were classified as highly resistant (R), plants that scored 2 as moderately resistant (M), and ratings of 3 and 4 as susceptible and very susceptible.

The postulation of the presence of resistant genes was based on a comparison of reaction spectra observed on tested accessions and the barley differential set (Table 2). This was done based on the gene-for-gene hypothesis [77]. The RT observed on each accession was compared with the Bgh virulence spectrum on the set of barley differential set.

3. Results

In the preliminary study, among 81 tested accessions of H. vulgare subsp. spontaneum collected in Jordan (47), Lebanon (23), and Libya (11) 15 expressed resistance to isolate Bgh33 of B. graminis f. sp. hordei (Table 3). Eleven of them originated from Jordan and 4 from Libya. None of the plants of accessions from Lebanon showed powdery mildew resistance in preliminary testing with isolate Bgh33. Twelve of the tested accessions in which plants were resistant to isolate Bgh33 showed heterogenous RT to powdery mildew: 3 of them showed only one type of reaction, 11 showed two different types and 1 showed 3 types (0, 2, 4).

Among scored resistance RT in tested lines with 20 differential isolates the most common reaction was 0 (immunity) (Table 4). It was observed in all tested lines with 65.5%. The rest of RT occurred with frequencies: 1 – 0.96%, 2 – 31.1%, and 4 – 2.4%. Reactions type 3 and 0(4) were not observed. In total 97.6 observed reactions represented resistance RT (0, 1, and 2).

The spectrum of RT of 31 tested lines to infection by 20 differential isolates was compared with results observed on a differential set of barley. Based on this analysis it was concluded that all of the tested lines have unknown gene or genes for resistance which are not represented in the differential set. Twenty-six of these lines (83,9%) showed resistance RT to all isolates used. After analysis of the obtained results, it was concluded that all selected 31 single plant lines of wild barley have unknown genes for resistance which are not represented in the Pallas isolines differential set.

4. Discussion

In Jordan, Lebanon, and Libya are many mountainous regions and different climate zones from relatively humid, with moderate temperature maritime to very dry and hot desert climates. Such conditions are favorable for the evolution of very diverse genotypes of plants including wild barley [12,13,14,15]. Powdery mildew occurs commonly in this area on barley and wild barley. Because the area of the Fertile Crescent is considered the center of origin and diversification of barley it is also the center of the presence of very diverse resistance genes to BPM [3,6,21,22]. This was confirmed in the present study in which were identified new sources of resistance to BPM in selections from accessions of Hvs from Jordan and Libya.

The Fertile Crescent area is considered the center of origin and diversification of barley Taking this fact into account, due to host-pathogen coevolution it is also the area of the very diverse Bgh population. Many studies have shown that this particular area is a very rich source od resistance to BPM [3,6,21,22]. This was confirmed in the present study in which were identified new sources of resistance to BPM in selections from accessions of Hvs from Jordan and Libya.

In the preliminary study, among 81 tested accessions of Hvs collected in Jordan (47), Lebanon (23), and Libya (11) in 15 was observed BPM resistance to isolate Bgh33. Eleven of them originated from Jordan, 4 from Libya, and none from Lebanon. Twelve tested accessions showed heterogenous resistance reactions to powdery mildew: 3 of them showed only one type of reaction, 11 showed two types and 1 showed 3 types (0,2,4). Heterogenous reactions of Hvs accessions to powdery mildew were also reported in other studies [70,71]. In populations of Hvs, BPM is not developing to levels that significantly damage plants. This is the result of both the stabilizing effect of the genetic heterogeneity within the populations of Hvs and the presence of resistance sufficient to control the limited disease development [69,70,71,76].

In tested lines with 20 differential BPM isolates the most common RT was 0 (immunity). This kind of RT was observed in all tested lines and with 65.5 % of all observed RT. The rest of RT occurred with frequencies: 1 – 0.96 %, 2 – 31.1%, and 4 – 2.4%. Reactions type 3 and 0(4) were not observed. In total 97.6 observed reactions represented resistance RT (0, 1, and 2). Such a relatively high percentage of resistance RT showed that Hvs collected in Jordan and Libya are valuable sources of resistance for European barley breeding which is in agreement with other studies [56,57,63,64,69,70].

Fungus Bgh is characterized by a high level of genetic variability. It can develop during a relatively short time a new races that can spread to long distances [19,20,21,22,23,24,25,26]. This resulted in a rapidly reduced number of resistance genes effectively controlling the occurrence and spread of Bgh to be available for barley breeders [25,26]. At the same time, modern barley cultivars which were grown in large areas across Europe often had no partial type of host resistance due to breeding for a race-specific type of BPM resistance in most modern breeding programs [21,25,33,34,85]. This fact was recognized a long time ago by plant pathologists and plant breeders and several ways to increase the durability of resistance genes were proposed. Major strategies were proposed and implemented: the use of multiline cultivars, the combining (‘pyramiding’) different resistance genes into one variety, and the deployment of many cultivars with different resistance genes in space (e.g. cultivar mixtures) or time (winter versus spring barley) [19,25,33,34,35]. However, for such BPM strategies of genetic control very useful is to introduce into breeding materials new effective sources of resistance. Such newly identified sources of BPM resistance are still being found in barley landraces and wild relatives [25,50,51,52,53,54,55,69,70,71]. There are many examples of successful use by barley breeders the new sources of resistance to BPM originating from Hvs populations to develop new resistant cultivars. In most cases, these new resistance genes were deployed in new cultivars under different strategies to prolong the time of their effectiveness against BPM [19,25,33,34,35].

The additional advantage of using germplasm from Hvs by barley breeders is the possibility to introduce other desirable agronomic traits e.g. tolerance to drought conditions and other biotic and abiotic stresses [16,18]. However, for many barley breeders, a heterogeneity of Hvs accessions is a problem because it complicates and prolongs the breeding process. Often pre-breeding activities resulting in well-characterized single plant lines of Hvs with many important economically traits including resistance to BPM are needed. Pre-breeding activities presented here provide breeders with new BPM sources of resistance. To be used in different breeding strategies [19,21,33,34,35]. The big advantage of the use of Hvs genetic resources in barley breeding is a lack of problems with sterility. Such problems are often present if H. bulbosum or mutants are used [19,21,25].

Two major strategies for BPM control are available. The first is to grow resistant cultivars and the second one is the application of fungicides [36,37,38]. However, in many countries, Bgh races resistant to commonly used fungicides have been described [31,32]. In addition, the cost of fungicides and concerns about the environment led many countries to restrict their use in disease control [36,38]. Taking this into account, the BPM control using effective resistance genes is increasingly important in IPM strategies. The understanding of BPM genetic control and how to properly use resistance genes resulted from many genetic studies of barley resistance to BPM and the relatively good characterization of the genetics of powdery mildew/barley interactions [19,21,25]. This is one of the best-described host-pathogen genetic interactions and more than 100 mildew resistance genes have been identified [21,25]. This kind of knowledge about well-characterized sources of BPM resistance resulted in many successful BPM resistance breeding programs using new sources of resistance to BPM [19,21,25,33,34,35]. Breeding for resistance as a strategy to control BPM is increasingly understood and acceptable by societies as ecologically safe.

In the presented study to identify new sources of resistance in wild barley accessions the test with a set of differential BPM isolates was used and the selection of single plant lines was conducted. This method was described in many studies to identify specific resistance genes in barley accessions and breeding lines [19,21,25,26]. In addition, it was successfully used in many studies to screen both landraces and wild barleys for new effective resistance genes [26,40,43,50,51,52,54,55,69,70,71]. However, for the description of the partial type of resistance to BPM, this kind of test is not sufficient. For the detection of this kind of resistance, there is a need to get, in addition to the RT, the measurements of resistance parameters in different stages of plant development (e.g. at the adult plant stage) [79,80,81,82,83]. Adult plant resistance of tested accessions should be investigated in additional specific tests because almost all wild barleys contain major specific resistance genes which very often mask minor resistance genes determining the partial resistance or the presence of adult resistance [19,21,25,79,80,81,82,83,84,85].

A very interesting genetic resource for the breeding of resistant barley is 31 single plant lines of wild barley which have genes for resistance not represented in the BPM differential set. These identified new sources of highly effective resistance to BPM in single plant lines of Hvs from Jordan and Libya will be used in the barley pre-breeding program.

Further studies are needed to determine the mode of action of resistance genes in identified new sources of BPM resistance described in the presented study based on results of testing of hybrids resulting from crosses among appropriate genotypes [40,45,52]. In the future, some other available methods for the characterization of resistant lines have to be used, especially those for the study of partial and adult resistance [79,80,81,82,83,84,85].

In addition, modern molecular methods have to be used for further characterization of identified resistance genes to be efficiently used in barley breeding [86,87,88].

5. Conclusions

The Hvs populations from Jordan and Libya are valuable sources of BPM resistance.

Selected single plant lines of Hvs may be used in pre-breeding programs to provide barley breeders with new well-characterised sources of BPM resistance.

Future studies will concentrate on determining the genetic basis of resistance occurring in 31 Hvs selections. They will include the crosses of investigated selections with well-chosen parents and the development of molecular markers.

To successfully introduce described new sources of BPM resistance into barley elite cultivars, pre-breeding work is needed in the creation of initial well-characterized plant materials. This a needed step to use barley germplasm from gene banks: first, to use it in breeding programs, and second, in agricultural practice as elite cultivars.