Powdery Mildew Resistance Genes in Barley Varieties Bred for Human Consumption

Abstract

Barley has properties that can improve and maintain human health, but to upgrade the positive characteristics of grain, specific breeding programs are required. Consumption of chemically protected food is unpopular and, therefore, genetic sources of disease resistance to the most frequent diseases are essential. The aim of this contribution is to postulate genes for powdery mildew resistance in spring barley varieties bred for human consumption. One hundred and twenty-seven breeding strains selected from thirty-three crosses, commercial varieties AF Lucius and AF Cesar developed in the program, and eight other check varieties were tested with a set of numerous pathogen isolates. Fifteen known resistance genes were found including a nonspecific resistance Mlo detected in breeding lines selected from 21 crosses. For spring barley, the utilization of Mlo is generally recommended, but its importance in varieties earmarked for human consumption should be highlighted because alternative sources of genetically more complicated resistance, derived from distant relatives or based on the accumulation of minor genes, could be economically ineffective. The presented findings enable varieties for human consumption with fully effective and durable resistance to powdery mildew to be selected.

1. Introduction

Barley (Hordeum vulgare L.) was one of the first domesticated crops and ranks among the most important cultivated cereals. There is evidence that wild barley (H. v. subsp. spontaneum = Hvs) seeds were collected by humans from around 17–19 thousand years ago and its domestication started about 7–9 thousand years later [1]. Barley grain was first used for human consumption and later for feeding domesticated animals and other uses. It has continued as a staple food in the mountainous regions of Central Asia, in southwest Asia, and northern Africa, where it is still used mainly for bread and porridge [2].

At present, barley is an important crop grown on a total area of approximately 52 Mha with a production of ca. 157 Mt in 2020 [3]. It is bred mainly for providing malt in the brewing and distilling industries; although, the greatest part of its production is used as livestock feed. Nowadays, people seek safe food to obtain essential nutrients and also for potential health benefits [4]. Foods containing barley are high in plant sterols/stanols as well as soluble fiber (e.g., beta-glucans) that has LDL cholesterol-lowering properties, resulting in the prevention of cardiovascular diseases [5]. Barley grain is also a possible tool for controlling type II diabetes, digestive system diseases, or compromised immune systems [6]; although, its positive effects are sometimes not easy to prove conclusively [7,8]. It is a good source of other bioactive constituents, such as vitamin E, B-complex vitamins, enzymes, minerals, and phenolic compounds [9], and has potential for protein production [10]. There are some possibilities of changing the content of these substances in barley grain through breeding. However, this task is rather new, and there are only a few programs aimed at raising the content of these constituents, increasing the beneficial effect on human health.

Powdery mildew is one of the most common diseases of barley and is caused by the airborne ascomycete fungus Blumeria graminis f. sp. hordei (Bgh). This, as well as many other plant pathogens, can be efficiently controlled by inexpensive and environmentally friendly genetic resistance. However, Bgh is extraordinarily adaptable and some commonly recommended strategies of using genetic resistance may be ineffective.

The research presented in this contribution is a part of a genome-wide association study (GWAS) with a large-scale analysis of almost 500 spring barley accessions. The project aims to identify genes associated with powdery mildew resistance, drought tolerance, qualitative parameters of grain for different uses, and resistance to ear fusariosis. The goal of the powdery mildew study within the project was to identify major resistance genes in the set of barleys, including the postulation of these genes in a subset designated for human consumption in which the detection of fully effective resistance was prioritized.

2. Materials and Methods

2.1. Plant Material

A total of 137 barley accessions were studied, including 10 checks, of which 2 commercial varieties (AF Lucius and AF Caesar) were bred during the course of the program. The core of the group was represented by 33 crosses resulting here in 61 breeding strains and 66 SSP grown from 11 of them.

2.2. Pathogen Isolates

For resistance tests, 51 selected reference isolates of Bgh were used, which had been collected in 12 countries, all in nonpolar continents, over a period of 66 years (1953–2018), which comprised the global virulence/avirulence diversity of the pathogen. The responses of 35 standard barley genotypes carrying different specific resistance genes to these isolates have been described previously [11]. Before inoculation, all isolates were checked for their purity, and their correct pathogenicity phenotypes were verified on standard barley lines [12]. The isolates were multiplied on leaf segments of the susceptible cv. Stirling.

2.3. Testing Procedure

About 50 seeds of each accession were sown in two pots (80 mm diameter) filled with a gardening peat substrate and placed in a mildew-proof greenhouse under natural daylight. The primary leaves were excised when the second leaves were emerging, and leaf segments 15 mm long were cut from the middle part of healthy fully expanded leaves. Three segments of each accession were placed on the surface of the media (0.8% water agar containing 40 mg^−L of benzimidazole—a leaf senescence inhibitor) in a 150 mm Petri dish. Leaf segments were placed adjacent to each other along with four segments of Stirling oriented diagonally with their adaxial surfaces facing upward.

For inoculation, a cylindrical metal settling tower of 150 mm diameter and 415 mm in height closed at the top was used, and a dish with segments was placed at the bottom of the tower. Conidia of each isolate taken from a leaf segment of the susceptible variety with fully developed pathogen colonies were shaken onto a square piece of 40 × 40 mm of black paper to visually control the amount of inoculum deposited. Then, the paper was rolled to form a blowpipe, and conidia of the isolate were blown through a side hole of a 13 mm diameter with its center 50 mm from the upper end into the settling tower over the Petri dish at a concentration of ca. 10 conidia mm⁻². The dishes with inoculated leaf segments were incubated at 20 ± 1 °C under cool-white fluorescent lamps providing 12 h light at 30 ± 5 μmol m⁻² s⁻¹.

2.4. Evaluation

Seven days after inoculation, infection responses (IR = phenotype of accession x isolate interaction) were scored on a scale of 0–4 [13], where 0 = no mycelium and sporulation, and 4 = strong mycelial growth and sporulation. IRs 3, 3–4, and 4 were considered susceptible (Figure 1). Each accession was tested with a minimum of two replications. If there were significant differences in IRs between replicates, additional tests were performed. A set of 51 IRs provided an infection response array (IRA) for each accession. Based on the gene-for-gene hypothesis [14], the resistance genes in accessions were postulated by comparing their IRAs with previously determined IRAs of standard barley genotypes possessing known resistance genes. During phenotyping, special attention was paid to boundary IRs 2–3 and 3 separating resistance and susceptibility, which pose the greatest risk of error in distinguishing between resistance and susceptibility [15]. Other details of Materials and Methods have been recently described [16].

3. Results

Resistance tests were carried out on a set of 137 barley accessions including 10 check varieties and 127 accessions selected from 33 crosses represented by 61 breeding strains and 66 single-seed progenies (SSP) derived from 11 of them. All accessions were inoculated with 51 powdery mildew isolates resulting in 20 infection response arrays (IRAs) reflecting phenotypes of all Ml genes and their combinations, including a fully susceptible IRA demonstrating the absence of any major resistance gene. Eight isolates were sufficient to characterize the IRAs (

Table 1. Infection response arrays produced by eight Blumeria graminis f. sp. hordei isolates on 137 barley genotypes and the corresponding powdery mildew resistance genes.

Ml Gene(s)	Blumeria graminis f. sp. hordei Isolates, Their Country of Origin, and Year of Collection
	Race I	J-462	EA30	PF512	I-20	M-3	GH	X-30
	JAP	ISR	SWE	CZE	CZE	CZE	AUS	CZE
	1953	1979	1976	2001	2011	2014	2005	2012
none	4	4	4	4	4	4	4	4
a3	0	4	0	0	0	0	0	4
a8	0	4	4	4	4	4	4	4
a8, k1	0	4	2	4	4	2	4	2
a12	1	4	4	4	4	4	1	4
a12, g	0	4	0	4	0	4	1	4
a12, La	0	4	2–3	4	4	4	0	2–3
a12, VIR	0	4	4	1	4	4	0	4
a13	0	0	0	0	4	4	0	4
a13, g	0	0	0	0	0	4	0	4
a13, La	0	0	0	0	4	4	0	2–3
Ab	2	4	2	4	4	4	4	4
aLo	0	0	4	4	4	4	4	4
aLo, g	0	0	0	4	0	4	4	4
Ch	2	4	4	4	4	4	4	4
g	0	4	0	4	0	4	4	4
g, Dr2	0	4	0	4	0	4	2	4
IM9	0	0	0	4	4	0	0	0
IM9, g	0	0	0	4	0	0	0	0
mlo	0(3) 1	0(3)	0(3)	0(3)	0(3)	0(3)	0(3)	0(3)
Ve	0	0	0	0	0	4	0	4

¹ Wild phenotype typical for mlo gene reflecting IR0 with occurrence of a small number of less developed colonies of the pathogen.

). A complete list of 137 tested accessions with their detailed designation used in the GWAS project and their postulated powdery mildew resistance genes is presented in Table S1.

3.1. Homogeneous Strains

Breeding strains selected from 23 crosses were homogeneous; in crosses 2161 and 2624 (in this section the prefix “KM” is omitted), first resistance genes were not postulated. Therefore, six SSPs were tested from each and an identical known resistance gene of all SSPs within a cross was revealed.

3.2. Heterogeneous Strains

In strains selected from the remaining 10 crosses, heterogeneity of resistances was found. In crosses 2454, 2693, 2881, and 3488, different genes of individual strains were postulated. From the other six crosses, nine breeding strains were selected (three from 2986, two from 3387, and one from 1057–1924, 2910, 3372, and 3375) and from each, six SSP were tested. In SSPs, of the seven strains selected from five crosses (2910, 3372, 3375, one strain of the cross 3387, and all three strains of the cross 2986) lines with different genes were discovered, whereas in strains 1057–1924 and in one of two strains derived from 3387, only lines with identical resistances were found and their heterogeneity was, therefore, not confirmed.

A total of 15 resistance genes against powdery mildew were found; 10 were present at least once alone (a3, a8, a12, a13, Ab, aLo, Ch, g, mlo, and Ve) and 5 (Dr2, IM9, k1, La, and VIR) only in combination with other genes. Altogether nine combinations of known resistance genes were found. In seven breeding strains of four crosses (2283, 2696, 2881, and 3238) unknown resistances were detected. Twenty-four crosses were represented only by homogeneous breeding strains. In 21 out of 33 crosses, the mlo gene was found, in three of which (2693, 3372, and 3488), there were lines containing also other resistances. Check varieties and all 49 breeding strains having different resistance genes are listed in

Table 2. Postulated Ml powdery mildew resistance genes in 10 check barley varieties and 49 breeding strains selected from 33 crosses having different Ml genes.

Check Variety	Ml Resistance	Designation of Cross	Ml Resistance
or Designation of Cross	Gene(s)		Gene(s)
AF Cesar	mlo	KM2986	Dr2, g
AF Lucius	a13	KM2986	g
CDC Fibar	a8	KM2986	mlo
CDC Rattan	a8, k1	KM3083	mlo
Clearwater	none	KM3189	mlo
Harriman	a3	KM3191	mlo
Jersey	mlo	KM3222	mlo
LP3	aLo, g + g	KM3227	mlo
Nudimelanocrithon	mlo	KM3238	a13, g, u
Tadmor	aLo	KM3255	mlo
KM1057	a13, La	KM3313	mlo
KM2161	g, IM9	KM3322	mlo
KM2283	a13, g, u	KM3339	mlo
KM2454	a8 + Ch	KM3340	mlo
KM2454	Ch	KM3341	mlo
KM2460	mlo	KM3342	mlo
KM2551	mlo	KM3372	a12
KM2624	Ab	KM3372	a12, g
KM2693	a13, g	KM3372	a12, VIR
KM2693	a13, g + mlo	KM3372	mlo
KM2696	a12, g, u	KM3375	a12
KM2881	a8 + a8, u	KM3375	a12, La
KM2881	a8, u	KM3375	Ve
KM2881	a12, g, u	KM3387	a12
KM2910	a8	KM3387	a12, g
KM2910	Dr2, g	KM3488	mlo
KM2910	g, IM9	KM3488	Ve
KM2942	a13	Je_x_Ta	mlo
KM2975	mlo	Ta_x_Je	mlo
KM2986	a13, g

+ Heterogeneous variety compared with two or more genotypes.

.

4. Discussion

4.1. Check Varieties

Prior to this study, the resistance of only five out of ten check varieties was known. AF Lucius, released in the Czech Republic in 2009, was one of the first European barley varieties bred for human consumption. It carries Mla13 [17], which predominated in the 1980s in that country, and the breakdown of this gene resulted in a rapid increase and the wide spread of virulent pathotypes [18] causing a disease epidemic in much of Europe [19]. Five years later another Czech variety, AF Cesar, also bred for the same use was registered, but with improved powdery mildew resistance based on mlo, a recessive gene conditioning nonspecific durable resistance [20]. This gene was found in a Dutch malting variety, Jersey [20], and in Melanocrithon, and had arisen naturally in the latter [21,22]. The Syrian variety, Tadmor, was one of the first barleys in which a new gene MlaLo was detected [23]. This gene was located in or close to the Mla locus [24] and can be distinguished from Mla8 with just two known isolates collected from Hvs in Israel. Tadmor was the only known spring barley carrying MlaLo compared with winter barley varieties where this resistance gene is the most frequent [25]. Thus, the present tests confirmed the authenticity [26] of these five check varieties.

In the remaining check varieties, their resistance was not known before the present tests. Mla8 was present in CDC Fibar and also CDC Rattan, which also contained Mlk1. LP3 was the only heterogeneous check variety with two lines; one carried Mlg and the other Mlg and MlaLo. Hence, LP3 is the second spring barley variety known to contain MlaLo. In Harriman, Mla3 was found, whereas Clearwater is the only variety from 137 accessions tested that has no major powdery mildew resistance gene.

Genotypic heterogeneity frequently occurs in segregating populations in breeding programmes and also in accessions of wild barley [27], in gene bank accessions [28], and in some commercial cultivars [20] including hybrids (F1 generation) [24]. Such heterogeneity, even in check varieties is, therefore, not unusual.

4.2. Breeding Strains

Twelve known resistance genes were postulated in breeding strains but in contrast to the set of check varieties, Mla3, MlaLo, and Mlk1 were not found in any of them. The importance of these specific genes for the resistance of the respective varieties in the field is generally low. This statement is valid for all the resistances found here, and for some of them (Mla8, MlaLo, or MlCh), the frequency of avirulent genes in the population is zero and, therefore, these genes cannot eliminate any virulent pathotypes from populations of the pathogen. Moreover, even if the frequency of avirulent pathotypes in a population is high and approaching 100%, the contribution of specific genes to varietal resistance in the field can be negligible as a result of rapid directional selection [29]. In four crosses, unknown resistances were found in all respective genotypes together with some identified genes. Because many isolates were virulent to each of them, none of these unknown resistances can positively affect the resistance of the given strains in the field.

Resistance gene postulation in barley varieties is based on comparing their IRAs with previously determined IRAs of standard genotypes possessing known genes. For that, two characteristics of resistance genes are important: (1) IR of the host genotype carrying a resistance gene and (2) specified sets of avirulent and virulent isolates to individual genes. This can be demonstrated on combinations of Mla13 and other genes. IR0 is a typical phenotype of both Mla13 and Mlg; three from eight Bgh isolates shown in Table 2 were virulent to the first, and five to the second, gene. One host genotype was susceptible only to two of the three isolates virulent to Mla13, and the resistance of this genotype to isolate I-20 was based on the joint presence of both these genes (Mla13 and Mlg). In the case of the combination of Mla13 and MlLa, one of three isolates virulent to Mla13 was avirulent to a genotype, but its IR was 2–3, which is a typical phenotype of MlLa. If a genotype with Mla13, Mlg, and MlLa was present (such a genotype was not found here), then only the isolate M-3 is virulent to this gene combination. If an isolate is avirulent to both Mla13 and MlLa, then the phenotype of this gene combination is IR0 because lower IR (in the given case IR0) is epistatic with regard to higher IR (here, IR2–3).

Virulence frequencies in Central European populations have been frequently studied. For example, in 2015–2017, six out of 11 specific resistance genes found in breeding strains studied here confirmed that there was a low protective effect because of the high virulence frequency to these genes [30]. However, even a low virulence frequency to some resistance genes does not necessarily mean that such varieties are less infected. If the virulence frequency to a resistance gene is, e.g., 2%, and a variety carrying this gene is grown on 1% of the crop area, then the infection incidence can be more than twice the average. Specific resistances are short-lived, and may be overcome in one growing season or over a period of only a few years and, thus, inappropriate for breeding varieties with efficient and durable resistance.

Bread wheat is one of the most important sources of human and animal food, and powdery mildew is a serious disease of this crop. Breeding and growing resistant cultivars are an effective and environmentally friendly way of reducing the adverse impact of this disease. Therefore, a similar GWAS project has been previously established for bread wheat. The project has several aims including the accumulation of minor resistance genes to improve the resistance durability of this widely grown crop [31].

In contrast to wheat, barley cultivated for human consumption occupies small areas and consequently, varietal disease resistance of this specific part of the crop has little importance in the epidemiology of the pathogen. However, the consumption of chemically protected food is unpopular and, therefore, genetic sources of disease resistance to one of the most frequent diseases [32] are essential. The adoption of Mlo resistance is recommended for spring barley, but in varieties earmarked for human consumption, the importance of this gene should be highlighted because alternative sources of genetically more complicated resistance, derived from distant relatives [33,34] or based on the accumulation of minor genes [18,35], could be economically ineffective for breeding companies. The set of breeding strains studied herein enables barley varieties for human consumption with the recessive gene mlo to be selected, providing fully effective and durable resistance to powdery mildew.