Characterizing the pathotype structure of barley powdery mildew and effectiveness of resistance genes to this pathogen in Kazakhstan

Powdery mildew caused by the biotrophic fungus Bgh (syn. Erysiphe graminis f.sp. hordei), is one of the most destructive diseases of barley (Hordeum vulgare L.) worldwide [1–3]. The disease is especially prevalent in moderate to temperate growing regions where yield losses can reach 40% [4]. Powdery mildew has a number of characteristics that support rapid evolution, such as large numbers of asexual haploid spores, sexual recombination during the growing season, and airborne dispersal over large distances [5].

Barley is the second after wheat most important for Kazakhstan cereal crop with total annual grain yield of more than 2 mln tons per hectare. Spring two-rowed barley represents over 90% of all barleys in the country [6]. Such a low output can be explained by strong pressure from abiotic factors, such as drought, heat, and heavy rains in autumn, and by periodic invasions of devastating barley pathogens [7]. Powdery mildew is one of the most powerful factors affecting barley production in Kazakhstan. Annually the pathogen affects crops of winter and spring barley in Kazakhstan. In recent years, disease epidemics have often been observed in the southern and south-eastern regions [8, 9], indicating an expansive spread in the country. Most of the cultivated commercial varieties of barley lack sufficient resistance to powdery mildew and thus, the disease is of economic importance particularly in locations where conditions are conducive for disease development [9]. Breeding of resistant cultivars of barley is still impeded by high intrapopulation variability of Bgh and by the ability of the pathogen to overcome host resistance [1–3]. Consequently finding effective and durable control measures to constrain powdery mildew fungi represents an important challenge in crop protection research.

There are several ways of controlling the disease. The primary one is the use of genetically mildew-resistant cultivars. This is a cheap and environmentally safe method. There is a large number of mapped resistance genes that could provide protection against barley powdery mildew infection. More than 85 race-specific resistance genes for powdery mildew have been identified in barley [1]. Many near-isogenic lines have been developed by backcrossing different resistance gene donors into common genetic background, resulting in, e.g., the Pallas differential set [10]. Mla, one of the genetically most thoroughly characterized race-specific loci conferring resistance to powdery mildew, spans 32 known alleles and represents a true allelic series on chromosome 1H [11]. The Mlo resistance is a kind of resistance that is unique primarily because it is monogenic [12], although it does not conform to the gene-for-gene system [13], and also because its resistance mechanism differs substantially from that of the other kinds of resistance [1, 12].

Powdery mildew can also be controlled with fungicides but these are ecologically undesirable and their frequent use may speed up the evolution towards resistance to fungicides [14]. The efficacy of using genetically mildew-resistant cultivars depends on pathogen virulence [15]. Additionally, the continuous use of these genes often results in selection in favor of pathotypes with the matching virulence genes in the pathogen population, and therefore in the breakdown of the resistance [16].

Globally, populations of Bgh have changed and new and virulent pathotypes have emerged [17–23]. Individual genes of specific resistances differ substantially in their effectiveness against the pathogen population comprising both virulent and avirulent pathotypes [24]. New pathotypes not only cause severe epidemics but substantially limit production of commercial cultivars [17–23]. Despite the increasing importance of powdery mildew in Kazakhstan, the population structure of Bgh in this region has not been characterized and so far there were no attempts to determine effectiveness of powdery mildew resistance genes for future application in local breeding programs. This study was therefore undertaken to characterize the virulence structure of populations of Bgh in barley-growing regions and identify effective powdery mildew resistance genes in the Republic of Kazakhstan.

Prior to this study, pathogenic variability of Bgh populations has been investigated in a number of countries around the globe [17–23]. However, the virulence structure of Bgh populations in Kazakhstan and other countries in Central Asia (Uzbekistan, Turkmenistan, Kyrgyzstan, and Tajikistan) was still unknown. This study characterized the virulence structure of Bgh populations in barley-growing regions of Kazakhstan and identified pathotypes of Bgh and the frequency of their occurrence in two regions of Kazakhstan. To the best of our knowledge, this study represents the first documentation of the virulence structure of Bgh in Central Asia.

Based on 107 isolates collected during a 2 year period, 23 pathotypes with virulence complexity ranging from 1 to 6 were identified (Table 3). Two pathotypes, 24 and 64, were identified to be of importance in the barley production in Kazakhstan due to their occurrence across the entire study area. They differ only by one virulence gene, i.e. pathotype 24 is avirulent to gene Mlk, and pathotype 64 is virulent. In Kazakhstan all tested isolates were avirulent on lines with genes Mla9, Mla1 + MlaAl2, Mla6 + Mla14, Mla13 + MlRu3, Mla7 + MlNo3, Mla10 + MlDu2, Mla13 + MlRu3, Mlo-5 and virulent on the line with gene Mla8 (Table 1). Frequency of isolates virulent to genes, Mlg + MlCP and Mla12 + MlEm2 was very low. In some populations (Saryagash, Tulkubas, Ordabasy) isolates virulent to genes Mla22 and Mlat were detected at frequency of 11.1–33.3%. The isolates virulent to these two genes occurred frequently in European, Australian [19] and South African populations [21]. It has been recently reported that line P12 [10] with gene Mla22 is the most useful differential cultivar, i.e. possesses the maximal pathogen differentiating ability. However, none of commercial barley varieties includes this gene [20]. In our experiments isolates virulent to genes Mla3, Mlg + MlCP, Mla12 + MlEm2, Mla22 and Mlat were found only in some areas of South Kazakhstan region where winter varieties of barley are cultivated. In total virulence diversity of Bgh pathotypes was much higher in populations on winter barley varieties versus spring barley. It has been shown previously that the virulent pathotype (Va22) to gene Mla22 occurs only on winter barley varieties in France and fails to infect spring barley [25]. In central Europe, winter barley is infected by a broader spectrum of pathogens than spring barley and powdery mildew fungus is dominant among them on susceptible cultivars [26]. Moreover, Bgh pathotypes virulent to many resistance genes including genes Mla3, Mlg + MlCP, Mla12 + MlEm2 и Mlat were detected just on winter barley varieties [27]. According to Dreiseitl and Kosman [21] directional selection might be responsible for an increasing virulence frequency to resistance genes Mla12 + MlaEm2, Mlg + MlCP, and MlLa, which may be present in some varieties. Confinement of the most virulent pathotypes (72, 60, 57) to winter varieties of barley (Yuzhno-Kazakhstanskiy 43 and Bereke 54) could sufficiently effect the progress of an epiphytoty, particularly in the areas where these cultivars cover considerable acreages. There is also possibility of their future occurrence and spread on commercial varieties of spring barley. Therefore any strategy based on major resistance genes must incorporate two or more of the following genes Mla9, Mla1 + MlaAl2, Mla6 + Mla14, Mla13 + MlRu3, Mla7 + MlNo3, Mla10 + MlDu2, Mla13 + MlRu3. By present many interesting findings on sources of resistance genes to barley powdery mildew have been published worldwide. Postulation of resistance genes to powdery mildew of barley is a major method of their identification in barley varieties. For instance, some researchers [1, 2, 24, 26, 28] postulated Ml-genes in winter and spring barley varieties by this method. Among them the following barley varieties are of particular interest for breeders in Kazakhstan: Algerian (Mla1) [1], Agra, Arve, Thule (sources of gene Mla9) [2, 28], Shamu, Baronesse (sources of gene Mla3) [2], Atlas (source of gene Mlat) [1], Kristaps, Rasa (sources of genes Mla7, MlNo3, Mlg) [28]. They are carriers of effective resistance genes to Kazakhstanean Bgh isolates. Besides experience from Europe suggests the best ways of achieving durable resistance is to use either mlo [29] or combinations of minor genes. The recessive resistance gene mlo has remained effective for more than 50 years and is the mainstay of mildew control in European winter barley plantings [5].

Minimal intrapopulation diversity judging on all statistical indices (H _W , H _G , D) was displayed by the population from Zhambyl region, and maximal one by the population from South Kazakhstan population (Table 5). The results of the study show significant difference even within the limits of one host variety. Spring barley variety Baisheshek is a source of infection in some areas of South Kazakhstan and Zhambyl regions (Table 2). Analysis of pathotype structure of the fungus isolated from this cultivar showed higher virulence diversity of South Kazakhstan isolates versus Zhambyl isolates. Since there is no information on resistance genes in commercial barley varieties it is impossible to assess reliably which genes could influence the results of the virulence analysis. Powdery mildew in Kazakhstan is mainly an infection that is introduced from other countries, so one of the factors causing this difference could be different ways of the colony migration. Primary infection could be brought into South Kazakhstan region by airflows from adjacent Central Asian countries (Uzbekistan, Tajikistan), and into Zhambyl region by airflows from Kyrgyzstan and China. Virulence of the pathogens from Zhambyl population actually does not differ from that of the isolates from Chinese population [22], and the structure of Bgh populations in Uzbekistan and Tajikistan is still not known. The greatest virulence polymorphism of isolates was observed on lines with genes Mlra, Mlk, MlLa and Mlh (from 10.0 to 78.6%).

The data of many studies show that the virulence of the barley powdery mildew pathogen differs greatly in different eco-geographical regions. For instance, Czech population demonstrates high phenotypical diversity and the lowest mean complexity of virulence versus Israel population [18]. Virulence of Chinese Bgh populations differs substantially from the same of European populations; the only similarity consists in virulence to genes Mla8 and Ml(Ch) [22]. It has been recently shown that the structure of Bgh populations in South Africa is very variable, contains unique virulence frequencies and associations and differs from populations in other parts of the globe [21]. Comparison of our own results with published data displays low similarity of populations in Kazakhstan to European, African, Australian and South-West Asian populations. Particularly, the virulence rate of Bgh populations in Kazakhstan is much lower than in Europe [19, 20], South Africa [21] and South-West Asia [18]. The difference between Kazakhstanean and Chinese populations consists mainly in the fact that the pathogens isolated in South Kazakhstan are virulent to some effective resistance genes (Mla3, Mlat, Mlg, Mla22) in China [22]. The Australian population also differs from population in Kazakhstan, because the population of Bgh in our country contains the isolates virulent to gene Mlra that is effective in Australia [5].

Differentiation of Kazakhstani barley powdery mildew population resulted in isolation and propagation of 23 pathotypes with different virulence to isogenic Pallas lines. Indices of diversity showed that virulence was very diverse within the pathogen population and the Gleason, Simpson and Shannon indices were high in South Kazakhstan region (H _G  = 4.31, D = 0.89, Sh = 2.44) but low in populations from Zhambyl (H _G  = 2.16, D = 0.78, Sh = 2.03). The predominant pathotypes 24 and 64 comprised 19.13–21.37% of the isolates from South Kazakhstan region and 10.78–14.19% of the isolates from Zhambyl region. High virulence (virulence complexity 5–6) is characteristic for unique pathotypes 72, 60 and 57 isolated from winter barley varieties. No isolate was virulent on differential lines possessing the resistance genes Mla9, Mla1 + MlaAl2, Mla6 + Mla14, Mla13 + MlRu3, Mla7 + MlNo3, Mla10 + MlDu2, Mla13 + MlRu3 and Mlo-5, therefore these genes are effective in Kazakhstan. Besides high effectiveness to the most part of the agent pathotypes was demonstrated by Mla3, Mlg + MlCP and Mla12 + MlEm2. The use of sources of the above-mentioned genes ensures the successful breeding of barley for powdery mildew immunity. Since different selections of susceptible barley varieties are grown in Kazakhstan, micro-evolutionary changes of Bgh may result in emergence and propagation of new pathotypes within the pathogen population. The present study provides a foundation for future studies on the pathogenic variability within of Bgh populations in Kazakhstan and addresses the knowledge gap on the virulence structure of Bgh in Central Asia.