Resistance against barley leaf rust (Puccinia hordei) in West-European spring barley germplasm

HAL Id: hal-00886084 https://hal.archives-ouvertes.fr/hal-00886084 Submitted on 1 Jan 2000 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Resistance against barley leaf rust (Puccinia hordei) in West-European spring barley germplasm Rients Niks, Ursula Walther, Heidi Jaiser, Fernando Martinez, Diego Rubiales To cite this version: Rients Niks, Ursula Walther, Heidi Jaiser, Fernando Martinez, Diego Rubiales. Resistance against barley leaf rust (Puccinia hordei) in West-European spring barley germplasm. Agronomie, EDP Sciences, 2000, 20 (7), pp.769-782. <10.1051/agro:2000174>. <hal-00886084> Plant Genetics and Breeding Original article Resistance against barley leaf rust (Puccinia hordei) in West-European spring barley germplasm Rients E. NIKSa*, Ursula WALTHERb, Heidi JAISERc, Fernando MART Í NEZd, Diego RUBIALESd, Ole ANDERSEN**, Kerstin FLATH**, Paul GYMER**, Fritz HEINRICHS**, Rickard JONSSON**, Lissy KUNTZE**, Morten RASMUSSEN**, Edeltraut RICHTER** a Laboratorium voor Plantenveredeling, Wageningen University, Postbus 386, 6700 AJ Wageningen, The Netherlands b Bundesanstalt für Züchtungsforschung an Kulturpflanzen, Theodor Roemer-Weg 4, 4320 Aschersleben, Germany c Pajbjergfonden, Gersdorffslundvej 1, Hou, 8300 Odder, Denmark d Instituto Agricultura Sostenible, CSIC, Apdo 4084, 14080 Córdoba, Spain (Received 28 April 2000; revised 9 July 2000; accepted 15 August 2000) Abstract – The level and type of resistance against leaf rust (Puccinia hordei) was determined in modern spring barley germplasm. In field trials all over Europe most accessions were in some locations and years significantly less infected than the moderately resistant reference ‘Grit’. Differentiating P. hordei isolates indicated that most accessions carried hypersensitivity (Rph) genes. A virulence survey indicated that among the known resistance genes, only Rph7 is still fully effective in Europe. Some accessions carried undetermined hypersensitivity resistance gene(s) that were effective to all isolates tested. The level of non-hypersensitivity or partial resistance was assessed from the latency period of the fungus and the percentage of early aborted infection units not associated with plant cell necrosis. These parameters indicated that several accessions had a level of partial resistance higher than that of the highly partially resistant ‘Vada’. We concluded that barley breeders have achieved very high levels of partial resistance against P. hordei in spring barley germplasm. barley / leaf rust / partial resistance / virulence / hypersensitivity Résumé – Résistance à la rouille brune (Puccinia hordei) du germplasme d’orge de printemps d’Europe de l’ouest. Le niveau et le type de résistance contre la rouille brune (Puccinia hordei) ont été déterminé chez le germplasme d’orge de printemps. Dans des champs expérimentaux présents dans l’Europe entière, la plupart des accessions ont été pour certains sites et années, moins infectées comparé à la référence ‘Grit’ qui est modérément résistante. Des isolats de P. hordei différenciés ont indiqué que la plupart des accessions contenaient les gènes d’hypersensitivité Rph. Un test de virulence a indiqué que des gènes Rph connus, seul Rph7 est encore complètement efficace en Europe. Certaines Agronomie 20 (2000) 769–782 769 © INRA, EDP Sciences 2000 Communicated by Hanne Østergård (Roskilde, Denmark) * Correspondence and reprints [email protected] ** Affiliations are in Appendix R.E. Niks et al. 770 1. Introduction Barley leaf rust, caused by Puccinia hordei Otth, occurs wherever barley (Hordeum vulgare L.) is cultivated. The economic importance of the rust depends on the region in the world, and varies from year to year. In Europe, the disease is generally more important in the temperate and warmer regions, like the United Kingdom and France, and less so in the more northern parts of Europe. In 1998 and 1999, for example, leaf rust was among the most important barley pathogens in the UK (Clarkson, NIAB, Cambridge, UK, pers. comm.). Losses of about 10 to 25% have been cited for the Czech Republic [3]. It appears that the economic importance of barley leaf rust has increased in recent years [2, 4]. In Europe, farmers apply repeated fungicide treatments on barley to protect against fungal leaf pathogens, including barley leaf rust. There is increasing opposition to the application of pesticides in agriculture, because of the environmental and health risks. The most obvious alternative to fungicide treatment is the use of resistant cultivars. Resistance in barley to P. hordei is widely available. There are two types of resistance to this pathogen: hypersensitivity resistance and non-hypersensitivity resistance [1]. The hypersensitivity resistance is governed by major genes (Rph), that are race-specific [5]. This resistance is often complete, and associated with necrosis of plant cells that are attacked by the pathogen sporelings. Some of the Rph genes have a delayed or rather weak effect on the pathogen. Such Rph genes confer incomplete resistance, in which the fungus forms small uredinia that are surrounded by chlorotic or necrotic plant tissue (i.e. low to intermediate infection types). The non-hypersensitivity resistance, also called “partial resistance” [18], is not associated with plant cell necrosis. It has a typically quantitative inheritance [13, 20]. In field trials this resistance is characterised by low levels of infection despite a compatible (high) type of infection. In monocyclic tests in the greenhouse, partially resistant barley genotypes are identified by the long latency period of the fungus and the low infection density as compared to the susceptible reference cultivars [15]. Breeding for resistance to leaf rust in barley has not received top priority in European barley programmes. However, selection against very susceptible phenotypes occurs. In several barley breeding programmes, artificial inoculations are made on breeding lines to facilitate this selection. Such procedures might result in the gradual accumulation of quantitative genes for resistance [14, 17]. Here we describe the resistance of a set of modern West-European spring barley germplasm against P. hordei. The accessions were tested at several locations with the objective of assessing the level and the type of their resistance. This research was complemented by a virulence survey to determine which Rph genes are still effective to the pathogen in Europe, and the frequency of possible virulence factors in the P. hordei population. Histological and macroscopic observations were carried out in a monocyclic greenhouse test to determine the level of non-hypersensitivity resistance in the tested lines. accessions contenaient des gènes d’hypersensitivité indéterminés qui se sont avérés efficaces contre tous les isolats testés. Le niveau de non-hypersensitivité ou résistance partielle a été évalué en fonction de la période latente du champignon et du pourcentage des parties infectées après avortement hâtif non associés à la nécrose des cellules végétales. Ces paramètres ont indiqué que plusieurs accessions présentent un niveau de résistance partielle plus élevé que celui de la référence avec résistance partielle, ‘Vada’. Nous pouvons donc conclure que les sélectionneurs d’orge ont atteint des très hauts niveaux de résistance partielle contre P. hordei chez le germplasme de l’orge de printemps. orge / rouille brune / résistance partielle / virulence / hypersensitivité Resistance to leaf rust in spring barley 771 2. Materials and methods 2.1. Multi-location field test for resistance 2.1.1. Plant material, locations and experimental design Twenty-nine cultivars and breeding lines of spring barley were obtained from seven breeding companies or research institutes in Germany, Denmark and the Netherlands. Almost all those accessions were cultivars that had been released since 1990, or breeding lines just about to be released. They had displayed consistently low levels of leaf rust infection in the breeders’ field trials, and hence were presumed to be partially resistant. ‘Alexis’ was added as a susceptible reference (three times), and ‘Grit’ as a moderately resistant reference (four times). ‘Grit’ was used as reference, since it had shown a moderate and stable level of resistance during 12 years of experiments between 1981 and 1995 [29]. The set also contained the lines L94 (extremely susceptible), 116-5 and ‘Vada’ (high level of partial resistance) and 175-16 (extreme level of partial resistance, [19]). In 1998, the set was tested at eight locations in four countries: Aschersleben, Hadmersleben and Langenstein (Germany); Abed, Sejet and Pajbjerg (Denmark); Sandon (UK); and Wageningen (the Netherlands). In 1999 the set was planted at the same locations, but instead of Wageningen, Landskrona (Sweden) and Radzikow (Poland) were included. Four of the lines tested in 1998 were replaced by new accessions in the 1999 set. This paper reports only on the accessions that were included in both years’ trials. The planting and leaf rust evaluations were carried out according to the RESI procedure [7]. At each testing site, the set was sown in three or four complete randomised blocks. The accessions were planted as microplots (double rows), or as single rows. Each plant row was 90–120 cm long with an inter-row spacing of about 25 cm. For each accession about 50 seeds were available per replication. A spreader row, consisting of a mixture of very susceptible barley lines or cultivars, was sown in the alleyways, perpendicular to the test entries, or alternating with them as microplots. At some locations, the leaf rust epidemics were initiated by artificial inoculation of the spreader rows, whereas in other locations, the epidemic occurred spontaneously. 2.1.2. Evaluation of infection levels Where possible, the trials were evaluated three times during the growing season starting at the tillering stage (growth stages 21–23, [31]). The assessed parameter was the average percentage of leaf area covered by the rust uredinia. A disease severity scale was provided to the evaluators at each location in order to reduce bias between locations and evaluators. Data analysis was performed with the SASapplication RESI [7]. The mean disease severity per accession per location per year was calculated as a mean of scores and replications. 2.2. Evaluation for race-specific hypersensitivity resistance At the Bundesanstalt für Züchtungsforschung und Kulturpflanzen, Aschersleben, Germany, seedlings of the accessions were inoculated with six barley leaf rust isolates, representing different virulence patterns (Tab. I). In the 1999 test the isolate 16-3 was replaced by 23. At the Laboratory of Plant Breeding in Wageningen, the seedlings were inoculated at the seedling stage with isolate 24 (Tab. I). Infection types were recorded to aid in the postulation of Rph genes in the accessions. 2.3. Virulence surveys Samples of the P. hordei population were collected in 1998 in seven European countries: Germany (5 locations), France (4), the United Kingdom (3), Switzerland (1), Austria (1), Denmark (1) and Belgium (1). The sampling was carried out in 1998 by Felsenstein, Munich, Germany, by use of mobile spore traps [25]. Most of the German isolates had been provided by various breeding stations. Spores from single uredinia Plant Genetics and Breeding R.E. Niks et al. 772 Table I. Infection types (on 0–4 scale)1of isolates of Puccinia hordei that differentiate between the various Rph resistance genes in barley. Cultivar Rph-gene Isolates 54-3 16-3 23 I 80 8-2 8-1 30-1+4280 24 ‘Sudan’ Rph1 0-2 2-4 4 4 4 4 4 ‘Peruvian’ Rph2 4 3 4 4 224 4 Hor679-3 Rph3 0 0 0 3 3 0 0 0 ‘Gold’ Rph4 4 4 4 4 4 4 4 4 ‘Quinn’ Rph2+Rph5 4 2-4 0 0 0 0 0 ‘Bolivia’ Rph2+Rph6 4 4 4 3 0-2 0-2 3 4 Hor4279 Rph7 0 0 0 0 0 0 0 0 Egypt 4 Rph8 4 3 4 4 4 4 4 4 Hor2596 Rph9 0 0 0 3 0 0 4 0 Hor500-1 0 0 0 3 4 4 4 n.t. Hor1132 sel. 3 0 0 0 0 0 0 n.t. ‘Trumpf’ Rph12 0 0 0 3 0 0 4 4 ‘Lada’ Rph12 0 0 0 3 0 0 4 4 1infection types 3 and 4 indicate virulence; 0 to 2 indicate avirulence. n.t.: not tested. Table II. Percentage of mono-uredinia-derived isolates of the barley leaf rust fungus (Puccinia hordei), collected in 1998, that carry virulence to resistance factors in 15 differential barley lines. Barley line Resistance Country of origin and number of isolates tested gene(s) Germany1France2UK3Switzerland Austria Danmark Belgium 98 82 72 17 33 17 22 ‘Sudan’ Rph1 100 100 100 100 100 100 100 ‘Peruvian’ Rph2 100 100 100 100 100 100 100 RikaxF1 Rph3 58 59 21 35 30 71 55 ‘Estate’ Rph3 69 58 28 65 70 71 68 Hor679-3 Rph3 75 84 40 71 85 88 77 ‘Gold’ Rph4 100 100 100 100 100 100 100 ‘Quinn’ Rph2+Rph5 20 50 83 71 21 35 50 ‘Bolivia’ Rph2+Rph6 100 100 100 100 100 100 100 ‘Cebada Capa’ Rph7 0000000 Egypt 4 Rph8 98 95 97 100 100 100 86 Hor2596 Rph9 100 100 100 100 100 100 100 Hor500-1 90 51 34 53 91 71 86 Hor1132 sel. 3 8 3 12 9 0 5 ‘Trumpf’ Rph12 97 100 100 100 100 100 100 ‘Lada’ Rph12 98 100 100 100 100 100 100 1Collected around Hamburg, Magdeburg, Hannover, Leipzig and Karlsruhe. 2Collected around Lille, Paris, Toulouse, Bourge. 3Collected around Harrogate, Cambridge, Edinburgh. Resistance to leaf rust in spring barley 773 were applied to seedling leaves of a susceptible cultivar to produce mono-uredinia-derived isolates. A total of 341 of such isolates were applied to seedlings of 15 barley lines and cultivars to determine the virulence frequencies in the P. hordei populations. Most of these 15 barley accessions (Tab. II) belong to the regular differential series for barley leaf rust [1, 2, 26, 28]. Three accessions with Rph3 were included. Hor679-3 probably only carries Rph3. ‘Estate’ has been reported to differ from Hor679-3 by one and Rika ×F1 by two additional resistance genes [28]. Similar samplings and virulence surveys had been carried out in other years and other locations in the framework of the present study. The results of those surveys served as back-up for the 1998 results. 2.4. Evaluation of the level of non-hypersensitivity resistance The set of barley lines was grown in greenhouse compartments at the Laboratory of Plant Breeding, Wageningen, to quantify the level of non-hypersensitivity resistance in each accession. The set was sown in two series for seedling assessments and in four series for adult plant assessments. About 11 days after sowing, the first leaves were fixed in a horizontal position and inoculated in a settling tower. About four seedlings per accession were available for inoculation per series. Each box received 4 mg inoculum, which amounts to about 200 spores per cm2. The procedure and conditions were as described by Niks and Rubiales [10]. For the adult plant evaluation, three plants were raised per accession per series. When the plants had developed about six or seven leaves, the upper surface of the uppermost fully expanded leaf (one per plant) was inoculated by dusting with inoculum. Isolate 24 of P. hordei was used for both the seedling and the adult plant tests. After five days, three inoculated leaves of each accession were sampled for microscopic observations. A central segment of each leaf was cut, fixed in lactophenol-ethanol, and stained with Uvitex for fluorescence microscopy [9, 24]. For each sample, 100 infection units were inspected for their stage of development and whether they were associated with autofluorescent plant cells, indicating plant cell necrosis. The percentage of early aborted infection units not associated with plant cell necrosis was determined. Early aborted infection units have no more than six haustorial mother cells [8]. For each accession, the infection type, on a 0–9 scale, was recorded [6]. These observations were carried out on inoculated leaves that had not been sampled and/or on the leaf stubs that remained after having sampled the leaf for microscopy. On the seedling leaves, the latency period of the rust also was determined [11]. 3. Results 3.1. Resistance levels in spring barley accessions At three locations in 1998 and five locations in 1999, the level of infection was too low to allow for a rust severity rating. Moreover, in some instances, only one or two of the three proposed evaluations were made before crop maturation. At those locations, the number of uredinia per three tillers per microplot were counted (Wageningen, 1998), or a semi-quantitative rating on a 1–9 or 0–10 scale was performed (Langenstein, Abed, both years; Landskrona, 1999). At Sejet (1999) and Radzikow (1999), the level of infection was negligible, and no data were collected. At five (1998) and four (1999) locations, the level of infection was sufficient to collect data that could be considered reliable and discriminatory among the accessions (Fig. 1). In both years Hadm.18091-96 was the most resistant accession, and L94 the most susceptible. The mean disease severity values differed substantially across the locations, but the ranking of the accessions was similar, especially among the most resistant and most susceptible accessions. Between the years there were only a few striking differences. ‘Cork’ was less severely infected in 1999 than in 1998. This effect seems mainly due to the lower infection at Hadmersleben. ‘Ria’, ‘Henni’ and Hadm.3500-96 were relatively more Plant Genetics and Breeding R.E. Niks et al. 774 Figure 1. Mean disease severities in 27 spring barley accessions infected by Puccinia hordei in field trials in two years and for five and four locations, respectively. For each accession the left-hand bar presents1998 data, the right-hand bar 1999 data. LA: Landskrona, Sweden; PF: Pajbjerg, Denmark; SJ: Sejet, Denmark; SAN: Sandon, UK; HAD: Hadmersleben, Germany; AS: Aschersleben, Germany. Resistance to leaf rust in spring barley 775 severely infected in 1999 than in 1998. With ‘Ria’ and ‘Henni’, this was likely due to higher infection at Hadmersleben. In contrast, Hadm.3500-96 had higer infection at all locations. The test sites differed in the discrimination between levels of infection on the various accessions. In 1998, 15 accessions at Pajbjerg and 19 accessions at Hadmersleben were significantly more resistant than the reference ‘Grit’. This was likely due to the relatively poor expression of resistance in ‘Grit’ at those locations. At Hadmersleben, where infection levels were high, ‘Grit’ was not significantly more resistant than the susceptible reference ‘Alexis’. In the other 1998 locations, some accessions, including ‘Alexis’, were significantly more susceptible than ‘Grit’, and none significantly more resistant than ‘Grit’. Also in 1999 at Hadmersleben many accessions (21) were significantly more resistant than ‘Grit’. Again, ‘Grit’ seemed to express its resistance poorly at this location. Also in that year, ‘Grit’ was not significantly different in leaf rust severity from the susceptible reference ‘Alexis’ at Hadmersleben. In 1999, several of the accessions were at the other locations more resistant than ‘Grit’ and a few were more susceptible than ‘Grit’. The number of locations in which the accessions had a mean disease severity significantly different from reference ‘Grit’ is presented in Table III. Also for this criterium line Hadm.18091-96 was in both years the most resistant accession, and L94 the most susceptible. ‘Henni’ appeared rather variable in its performance, since in both years, it had at some locations a significantly higher and in other locations a significantly lower level of infection than ‘Grit’. Seven accessions (Hadm. 18091-96, 17-5-6, ‘Libelle’, ‘Meltan’, ‘Jacinta’, ‘Polygena’ and ‘Mentor’) were among the ten most resistant accessions in both years. The mean disease severity (averaged over the locations) for all accessions in 1998 correlated very well with those in 1999 (r = 0.95, significant at 0.01 level, Tab. IV). The results from the locations where low infection occurred (see above) still resulted in a similar ranking of the accessions. We conclude that the large majority of the spring barley lines were at least as resistant as ‘Grit’. With the exception of some cvs (e.g. ‘Henni’ and ‘Cork’), the ranking of the accessions between locations and years were, in general, not very different. 3.2. Genes for race-specific hypersensitivity resistance Conclusions on the presence of race specific resistance genes were based on the seedling tests with the differentiating isolates listed in Table I. The postulated resistance genes present in the accessions are presented in Table III. Nine accessions were resistant to all isolates except I 80, 301+4280 and 24, suggesting the presence of Rph12. ‘Barke’, susceptible only to I 80 and 30-1+4280, probably carries Rph9. Six accessions were only susceptible to I 80, which may be explained by assuming the presence of both Rph3 and Rph9/Rph12. Isolate 24, the only isolate that distinguishes Rph9 from Rph12, is avirulent to Rph3 (Tab. I). This makes it impossible to determine whether the six accessions carry Rph9 or Rph12. The reaction pattern of ‘Henni’ (only resistant to 54-3, not tested against 16-3) suggested the presence of Rph1, and the reaction of Libelle (only susceptible to 8-2 and I 80) suggested the presence of Rph3. One accession, Hadm. 3500-96, was only susceptible to isolate 24, which cannot be explained by any of the Rph gene (combinations) for which the isolates differentiated (Tab. I). That line may therefore carry an unknown Rph gene. Four accessions were resistant to all isolates. One of these, ‘Hanka’, was derived from a parent known to carry Rph7, a gene effective to all isolates used in this study (Tab. I). In the other four accessions this or other Rph gene(s) may occur. Four accessions gave a susceptible infection type to all isolates, and therefore may not carry any of the Rph-genes to which at least one of the isolates carries avirulence. Since all isolates possess virulence for Rph4 and Rph8, either of those genes Plant Genetics and Breeding R.E. Niks et al. 776 Table III. Resistance of 27 spring barley accessions to barley leaf rust, Puccinia hordei: the number of locations in which each accession was significantly less (<) or significantly more (>) infected than the reference ‘Grit’ in field tests in 1998 and 1999 (number of locations), the putative Rph-genes present in each accession, and greenhouse experiments with isolate 24 to determine the level of non-hypersensitivity resistance: the latency period relative to line L94 (relative LP), and the percentage of early aborted infection units not associated with necrosis (% EA–N) determined in seedlings and in adult plants. The infection type (IT on a 0–9 scale) to isolate 24 is also presented. Isolate 24 Accession Number of locations Putative Rph gene(s) present4IT10 Relative LP7 % EA – N6 1998119992Seedlings Seedlings8 Adult plants9 Hadm18091-96 2< 4< Rph? 5 - 13 21 17-5-16 2< 2< -7 144 35 49 ‘Meltan’ 2< 3< Rph3+Rph9/12 3 - 23 44 ‘Libelle’ 2< 3< Rph3 1 - 17 36 ‘Jacinta’ 2< 1< Rph? 5 - 44 56 ‘Ria’ 2< 1< Rph12 7 144 45 53 ‘Polygena’ 2< 2< Rph3+Rph9/12 2 - 13 26 ‘Mentor’ 2< 2< Rph3+Rph9/12 1 - 26 23* ‘Hanka’ 2< 1< Rph751 - 12 24 ‘Vada’ 1< 2< -7 126 27 43 ‘Barke’ 2< 3< Rph9 2 - 6* 3 GS1568 1< 2< Rph3+Rph9/12 1 - 17 24 ‘Cooper’ 1< 2< Rph12 7 125 42 45 Hadm.3500-96 2< 0 Rphx 7 114 10 33 ‘Corniche’ 2< 1< Rph12 8 110 11 36* 116-5 1<1> 2< -8 124 16 21 ‘Henni’ 2<1> 1<1> Rhp1 8 111 28 29 ‘Fergie’ 2< 1< Rph12 8 114 9 15 ‘Cork’ 1< 2< Rph3+Rph9/12 1 - 36 54 ‘Lisbet’ 1<1> 0Rph12 7 111 9 7 ‘Optic’ 0 1< Rph12 7 121 23 28 ‘Bartok’ 1> 1> Rph12 7 127 22 44 ‘Grit’300Rph12 8 124 9 13 ‘Alliot’ 2> 0Rph? 2 - 18 12 ‘Madeira’ 2> 2> Rph12 7 105 2* 7* ‘Alexis’ 4> 3> Rph3+Rph9/12 3-916 L94 5> 4> - 9 100 3 2 1Total number of locations: 5. 2Total number of locations: 4. 3‘Grit’ was included as accession, and tested against three plots per replication in which ‘Grit’ served as reference. 4-: no evidence for any Rph-gene; Rph?: unknown Rph gene(s) effective to all differentiating isolates; Rphx: unknown Rph gene(s) effective to all differentiating isolates except isolate 24. 5as Rph?, but Rph7 gene presumed because of ancestry. 6Figures marked by * are based on only one replication. 7-: not measured because of low infection type. 8Based on two replications, three leaf segments per replication, 100 infection units per leaf segment. 9Based on four replications, three leaf segments per replication, 100 infection units per leaf segment. 10 Based on the 0–9 scale of McNeal et al. [6]. For virulence spectrum of this isolate, see Table I.