Evaluating barley landraces collected in North Africa and the Middle East for powdery mildew infection at seedling and adult plant stages

Barley (Hordeum vulgare L.) is one of the most widely grown cereal crops. Numerous pathogens impair the amount and quality of the grain yield. Blumeria graminis f. sp. hordei (Bgh) is a fungal pathogen causing powdery mildew, a widespread and economically important foliar disease. Since there is a limited number of known resistance genes, efforts of scientists and breeders are focused on searching for new sources of resistance. Barley landraces are a known, but still underexploited source of diversity. A set of 79 barley landraces collected in North Africa and the Middle East was tested against powdery mildew at seedling and adult plant stages. Under a controlled environment, 50% of accessions showed resistance conditioned by major genes. Among them, seven accessions showed broad resistance to Bgh isolates that were virulent to most of the known resistance genes. The field experiments carried out under natural infection over several years indicated all accessions as potential sources for resistance breeding. Twelve landraces were found to be medium resistant or resistant during all six seasons. This report relates to barley landraces as a promising source of potentially novel resistance to powdery mildew.


Introduction
Barley (Hordeum vulgare L.) is the fourth most important cereal crop according to harvesting area (http://www.fao. org/faost at). It is resistant to harsh environmental conditions, such as soil salinity, high altitude and low rainfall (von Bothmer et al. 2003a), and is used for feeding and malting purposes. The challenge is to control over 250 pathogens infecting the barley crop (Singh et al. 2019). Most of them are a significant economic problem through reduction of the crop quality and yield. Prevention of crop loss is one of the goals of food security (Savary et al. 2012). Powdery mildew caused by Blumeria graminis (D.C.) Golovin ex Speer f. sp. hordei Em. Marchal (Bgh) is one of the most important barley diseases. Bgh is an airborne fungus which causes yield loss of up to 50%, while average losses are about 10-20% (Tratwal and Weber 2006). In contrast to chemical agents, resistance breeding is an economically effective and environmentally friendly method of controlling the spread of the pathogen.
Several race-specific major resistance genes were already mapped on the barley genome: Mla, Mlat, MlGa, Mlk, Mlnn, Mlra on chromosome 1H; MlLa and MlMor on 2H; Mlg and MlBo on 4H; Mlj on 5H; Mlh on 6H; and mlt and Mlf on 7H (Jørgensen 1994;Schönfeld et al. 1996;Chełkowski et al. 2003;Piechota et al. 2019). Some of them, as well as other major resistance genes with unknown location on the barley genome, like Ml(Ab), Ml(Lv); Ml(Lo) and Ml (St), have been introduced to modern European cultivars (Dreiseitl 2017a). Resistance conditioned by major genes is non-durable. Newly appearing fungal pathotypes can overcome resistant genes in a few years. Also the effectiveness of pyramiding two or more major genes is limited in time (Dreiseitl 2003. The recessive allele mlo carries non-race-specific resistance to Bgh. It is widely used in elite cultivars due to its durability and lack of selection pressure to the pathogen population. In the face of the limited available gene pool, searching for new resistance sources is in the interests of scientists and breeders. The selection process has narrowed the gene pool of modern crops (Wulff and Dhugga 2018), so exploration should extend beyond cultivars (Pietrusińska et al. 2018). Barley landraces could be promising as they were cultivated according to traditional practices and not subjected to strong selection pressure. They are highly diverse and heterogeneous populations with a variable gene pool (Camacho Villa et al. 2005). Landraces belong to the primary gene pool and can be easily used in breeding programs. The lack of crossing barriers reduces breeding costs and efforts (von Bothmer et al. 2003b;Yun et al. 2006). The aim of this study was to select promising sources of powdery mildew resistance in North African and Middle East barley landraces.

Plant material
A set of 79 spring barley landraces from the collection of the Plant Breeding and Acclimatization Institute -National Research Institute (Radzików, Poland) was screened for resistance to Bgh. All accessions were obtained from The International Centre for Agricultural Research in the Dry Areas (ICARDA) collection (Table 1). Barley landraces originated from North Africa and the Middle East and included 27 accessions from Algeria, 14 from Jordan, six from Egypt, six from Morocco, six from Libya, one from Tunisia and 19 of unknown origin.

Seedling resistance screening
Seedling tests were performed for resistance against each of six Bgh isolates: Bgh27, Bgh133, Bgh111, Bgh131, Bgh4714, Bgh314. For each test ca. 25 seeds per barley accession were sown. They were grown under controlled chamber conditions with a 16/8 h day/night photoperiod and a 22/16 °C temperature regime. Seedlings with a fully expanded first leaf (DC: 12 (Zadoks et al. 1974)) were inoculated with Bgh by shaking conidia from the susceptible cv. Manchuria. After 8-10 days, infection types were scored according to a 5-level scale (Mains and Dietz 1930), where 0, 1 and 2 represented resistant plants and 3 and 4 represented susceptible plants. Postulation of resistant genes was based on a comparison of reaction spectra designated on landraces and the barley differential set (Table S1). In case of a mixed reaction against Bgh isolate, postulation was performed for the resistance score. Possibility of resistance to Avr genes was concluded on the basis of the gene-forgene hypothesis (Flor 1956). The infection response spectrum of each landrace was compared with the Bgh virulence spectrum previously found on the set of barley differential varieties.

Adult plant resistance screening
Barley landraces were screened for adult plant resistance under natural infection conditions. Plants were field grown in Radzików, central Poland (52° 13′ 38″ N, 20° 36′ 55″ E) during six seasons: 2010-2013, and 2016-2017. Plants were sown in a 2-m plot with 20 cm row spacing and 7 cm distance between seeds. Susceptible Manchuria was sown every 20 rows as a disease spreader. Disease symptoms were scored on adult plants at the flowering stage (DC: 60-69; Zadoks et al. 1974) according to a 9-level scale, were 1 indicates a very susceptible reaction and extreme infection of the entire plant and 9 indicates a totally resistant plant without visible symptoms of infection (Dreiseitl 2003 and the lowest infection score per accession and per season was recorded.

Adult stage screening
The set of 79 barley landraces was tested under field conditions during six seasons. The lowest infection scores noted during the trial were: 5 (medium resistance) recorded for 21 (26%) landraces and 3 (which is medium susceptible) observed for 58 (74%) accessions (Table 1). Average infection scores calculated for six seasons were: 7 (resistant) for 13 (16%) accessions, 5-6 (medium resistant) for 65 (82%) accessions, and 4 (medium susceptible) for one accession. A set of 12 (612,613,621,631,647,648,651,694,727,730,748,753) accessions showed an average score of 7 (resistant) with the lowest score of 5 (medium resistant). These accessions were medium resistant or resistant during all six seasons.
Since major genes are usually overcome in a few years, field resistance determined by minor quantitative genes is more promising. It has longer durability and is effective against various pathotypes. That kind of resistance increases during continuous cultivation and quantitative minor genes are still diversifying and changing (Jensen et al. 2012). The multiyear trial examined resistant landraces under different weather conditions and variable pathogen pressure. A set of 12 (612,613,621,631,647,648,651,694,727,730,748,753) accessions revealed a good level of resistance during six seasons. These accessions could be valuable material for a more detailed analysis and a good source for resistance breeding. Additionally, selected landraces were collected from the area of barley origin and domestication. Field resistance maintained by long-term coevolution with a pathogen is valuable and economically important for farmers and breeders (Jensen et al. 2012).
This multiyear study did not reveal consistent field resistance to natural pathogen pressure with seedling resistance against artificial inoculation in a climate chamber. Reaction patterns were variable among growth stages. Expression of quantitative resistance becomes more visible at the adult stage and against multipathotype infection. Previous data identified wild barley (H. vulgare ssp. spontaneum) genotypes resistant at seedling stage and highly susceptible at adult stage under field conditions (Dreiseitl and Bockelman 2003). Nevertheless, all landraces resistant to at least three Bgh isolates (701,720,695,719,737,740,698,694,729,794,730) at the seedling stage were at least medium resistant at the adult stage.
According to Dreiseitl (2017b) searching for and detecting new resistance is desirable for its utility in breeding programs, for easier identification of minor resistance genes frequently masked by major genes and for improving knowledge of resistance. New resistance genes can be useful by combining them with other known genes or partial Mlo resistance in barley cultivars. That approach makes resistance more durable and inhibits Mlo resistance erosion (Dreiseitl 2017b). Six-year field trials and artificial testing against highly virulent powdery mildew isolates, presented in this report, provided the list of barley landraces resistant to B. graminis f. sp. hordei. Acknowledgements The authors thank to Professor Henryk Czembor for significant and valuable advices provided in their work with barley-powdery mildew pathosystem. The research was conducted within program "Creation of scientific basis for biological improvement and plant genetic resources protection as source of innovation and support of sustainable agriculture and national food security", Project No. 3-2-00-0-02 (PW task 2.2): "Broadening of barley gene pool" supported by The Ministry of Agriculture and Rural Development Poland.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.