Isozyme Variation and Genetic Diversity of the European Barley Powdery Mildew Population

Isozyme and virulence analyses of Erysiphe graminis bordei were performed with samples collected from different sites from nearly all over Europe. Isozymes and unspecific proteins extracted from conidia were separated by starch gel electrophoresis and isoelectric focusing, respectively, and the resulting isozyme banding patterns were compared with the corresponding virulence data. One isozyme phenotype prevailed in all samples. Only 7.9% of 280 isolates showed divergent banding patterns. Expected frequencies of isolates with divergent banding patterns were calculated for each subsample. In the Italian subsample, isolates with divergent banding patterns were significantly more frequent than expected. At the same time, isolates from Italy had significantly fewer virulence factors than those from N.W. Europe, indicating weaker selection by host resistance genes. It is suggested that isozyme uniformity in the homogeneous north-western European barley powdery mildew population has arisen from strong selection pressures for specific virulence genes. The direction of this selection, acting upon a mainly asexually reproducing population, has changed over space and time due to the introduction of new resistance genes, forcing local populations through bottlenecks. This may have led to random loss of genetic variation (genetic drift) in the barley powdery mildew gene pool.     

Sources of powdery mildew resistance in barley landraces from morocco

A total of forty eight accessions of barley landraces from Morocco were screened for resistance to powdery mildew. Twenty two (46%) of tested landraces showed resistance reactions and thirty four single plant lines were selected. Eleven of these lines were tested in seedling stage with seventeen and another twenty three lines with twenty three isolates of powdery mildew respectively. The isolates were chosen according to the virulence spectra observed on the ‘Pallas’ isolines differential set. Line 229–2–2 was identified with resistance to all prevalent in Europe powdery mildew virulence genes. Lines 230–1–1, 248–1–3 showed susceptible reaction for only one and lines 221–3–2, 227–1–1, 244–3–4 for only two isolates respectively. Three different resistance alleles (Mlat, Mla6, and MLA14) were postulated to be present in tested lines alone or in combination. In thirty (88%) tested lines it was impossible to determine which specific gene or genes for resistance were present. Most probably these lines possessed alleles not represented in the ‘Pallas’ isolines differential set. The distribution of reaction type indicated that about 71% of all reaction types observed were classified as powdery mildew resistance (scores 0, 1 and 2). Majority (79%) of resistance reaction types observed in tested lines was intermediate resistance reaction type two and twenty three lines (68%) showed this reaction for inoculation with more than 50% isolates used. The use of new effective sources of resistance from Moroccan barley landraces for diversification of resistance genes for powdery mildew in barley cultivars was discussed.     

Virulence Gene Frequency Change in Erysiphe graminis f. sp. hordei Due to Selection by Non-Corresponding Barley Mildew Resistance Genes and Hitchhiking

Individual isolates (in total 4040) of Erysiphe graminis f. sp. hordei were collected between 1989 and 1991 in fields of spring barley varieties with resistance genes Mla9, Mla12 and Ml(La) in the local air spora, i.e. remote from barley fields, in Hesse, Germany. Their virulence pattern was determined on a 13-partite differential set. In the air spora. virulence complexity (the number of virulence genes per isolate) increased by 30% (4.2 vs. 5.5) between 1989 and 1991. This was mainly due to an increase of pathotypes with virulence genes Va7, Val3, Vk and V(La). In each mildew field population, frequencies of several non-corresponding virulence genes, e.g. V(La) on Mla9 variety, greatly increased during the period. Such change can be caused by hitchhiking selection, i.e. indirect selection resulting from asexual reproduction, and direct selection by non-corresponding resistance genes. To separate the two effects, the aerial barley mildew population of 1991 was sampled on cv. Pallas and near-isogenic lines of Pallas. Frequencies of non-corresponding virulence genes as observed in samples from the near-isogenic lines were compared to expected non-corresponding virulence frequencies which were obtained by subdividing the sample on Pallas into 12 subsamples, comprising all isolates virulent to line 1 through 12, respectively. Among 90 resistance/virulence gene combinations selection for non-cor-responding virulence was significant in five cases (e.g. Va9 on Mla7 host) and against non-corresponding virulence in nine cases (e.g. Vk on Ml(La) host). Hitchhiking selection was significant in eight cases. In 11 cases the two types of selection were significant in combination but not individually. It is suggested to monitor the stability of selection against non-corresponding virulence genes in race surveys and utilize it in diversified barley crops such as variety mixtures in order to retard the evolution of complex races.     

AB-QTL analysis in spring barley. I. Detection of resistance genes against powdery mildew, leaf rust and scald introgressed from wild barley

The objective of this study was to map new resistance genes against powdery mildew (Blumeria graminis f. sp. hordei L.), leaf rust (Puccinia hordei L.) and scald [Rhynchosporium secalis (Oud.) J. Davis] in the advanced backcross doubled haploid (BC₂DH) population S42 derived from a cross between the spring barley cultivar Scarlett and the wild barley accession ISR42-8 (Hordeum vulgare ssp. spontaneum). Using field data of disease severity recorded in eight environments under natural infestation and genotype data of 98 SSR loci, we detected nine QTL for powdery mildew, six QTL for leaf rust resistance and three QTL for scald resistance. The presence of the exotic QTL alleles reduced disease symptoms by a maximum of 51.5, 37.6 and 16.5% for powdery mildew, leaf rust and scald, respectively. Some of the detected QTL may correspond to previously identified qualitative (i.e. Mla) and to quantitative resistance genes. Others may be newly identified resistance genes. For the majority of resistance QTL (61.0%) the wild barley contributed the favourable allele demonstrating the usefulness of wild barley in the quest for resistant cultivars.     

Characterization of a Single-Spore Isolate Population of Plasmodiophora brassicae Resulting from a Single Club

The single‐spore isolates ‘e₃’ and ‘e₆’ of Plasmodiophora brassicae with different virulence patterns were distinguished by restriction fragment length polymorphisms in fingerprint‐like patterns and by electrophoretic karyotypes using repetitive fragments as hybridization probes. These molecular tools were used to characterize a set of isolates originated from an infected root, which was inoculated with a mixture of the two single‐spore isolates (e₃ and e₆). Spores harvested from mixed‐infected roots were used to establish 53 new single‐spore isolates. All these single‐spore isolates revealed parental patterns according to their molecular fingerprints and their virulence pattern. No sexual recombination could be detected with repetitive molecular probes. However, one isolate (M₃₆ES49) showed the same fingerprint pattern and virulence pattern but different sizes of small chromosomes than the parental type ‘e₆’, which is taken as an indication of chromosome rearrangement during the infection cycle.     

RFLP markers to identify the alleles on the Mla locus conferring powdery mildew resistance in barley

Summary To identify the mildew resistance locus Mla in barley with molecular markers, closely linked genomic RFLP clones were selected with the help of near-isogenic lines having the Pallas and Siri background. Out of 22 polymorphic clones 3 were located around the Mla locus on chromosome 5 with a distance of 5.1 + 2.9 cM (MWG 1H068), 4.2±1.7 cM (MWG 1H060) and 0.7 ± 0.7 cM (MWG 1H036), respectively. The polymorphic clone MWG 1H036 displayed the same RFLP pattern in both Pallas and Siri near-isogenic lines and in different varieties digested with six restriction enzymes possessing the same mildew resistance gene. The alleles of the Mla locus were grouped in 11 classes according to their specific RFLP patterns; 3 of these groups contain the majority of Mla alleles already used in barley breeding programs in Europe.     

Chromosome landing at the Mla locus in barley (Hordeum vulgare L.) by means of high-resolution mapping with AFLP markers

 The complex Mla locus of barley determines resistance to the powdery mildew pathogen Erysiphe graminis f. sp. hordei. With a view towards gene isolation, a population consisting of 950 F₂ individuals derived from a cross between the near-isogenic lines ‘P01’ (Mla1) and ‘P10’ (Mla12) was used to construct a high-resolution map of the Mla region. A fluorescence-based AFLP technique and bulked segregant analysis were applied to screen for polymorphic, tightly linked AFLP markers. Three AFLP markers were selected as suitable for a chromosome-landing strategy. One of these AFLP markers and a closely linked RFLP marker were converted into sequence-specific PCR markers. PCR-based screening of approximately 70 000 yeast artificial chromosome (YAC) clones revealed three identical YACs harbouring the Mla locus. Terminal insert sequences were obtained using inverse PCR. The derived STS marker from the right YAC end-clone was mapped distal to the Mla locus. Received: 17 July 1998 / Accepted: 9 August 1998     

Virulence analysis of wheat powdery mildew population (Erysiphe graminis f.sp.tritici) from the region of Slovakia and Hungary

In the year 1992 a total of 163 isolates of wheat powdery mildew were tested. The samples of mildew isolates were obtained by means of a mobile spore catching apparatus. The populations from 4 regions of Slovakia and 3 regions of Hungary were analyzed. The resistance due toPm5, Pm8 andMl-i genes at the observed locations has already been overcome. The resistance genesPm1, Pm2 and a gene combinationPm2+Pm6 ensure the protection only against a part of the patho-types of powdery mildew population. Virulence corresponding to thePm4b gene has been low so far. The regional patterns of pathogen virulence are in good agreement with the gene resistance spectrum by the cultivars grown regionally. Little differences in virulence among the populations from the regions of Slovakia and Hungary indicate that this part of Eastern Europe should be considered as an epidemiologic unit.     

Mapping of powdery mildew resistance genes in a newly determined accession of Hordeum vulgare ssp. spontaneum

The accession PI466197 of wild barley (Hordeum vulgare ssp. spontaneum) with a newly identified resistance to powdery mildew caused by Blumeria graminis f.sp. hordei was studied with the aim to localise the genes determining resistance on a barley genetic map using DNA markers. Molecular analysis was performed in the F₂ population of the cross between the winter variety ‘Tiffany’ and the resistant accession PI466197, consisting of 113 plants. DNA markers, 17 simple sequence repeats (SSRs), four sequence-tagged sites (STSs) and one cleaved amplified polymorphic sequence (CAPS) marker developed from the Mla locus sequence were used for genetic mapping and a two-locus model of resistance was shown. One of the resistance genes originating from H. vulgare ssp. spontaneum PI466197 was localised between the markers RGH1aE1 and Bmac0213 on the short arm of chromosome 1H, which is the position consistent with the Mla locus. The other gene was proven to be highly significantly linked with GBMS247, Bmac0134 and MWG878 on the short arm of chromosome 2H. The flanking markers were Bmac0134 and MWG878, assigned 4 and 8 cM from the resistance gene, respectively. Until now, no gene conferring powdery mildew resistance originating from H. vulgare has been located on the short arm of barley chromosome 2H.     

Genetical and RFLP studies at the Mla locus conferring powdery mildew resistance in barley

Summary The complex structure of the multigene family at the Mla locus conferring powdery mildew resistance in barley was studied by making diallel crosses between several near-isogenic lines carrying different Mla alleles. The mode of inheritance of the Mla alleles investigated was determined to be dominant for Mla1, Mla6, Mla7 and Mla13 and semidominant for Mla3, Mla12 and Mla20. F₁ plants were backcrossed to the susceptible recurrent parent in order to identify susceptible and double-resistant recombinants in the BC₁F₁ generation. Out of 17605 progenies tested in the BC₁F₁ generation, two susceptible recombinants, one between Mla1 and Mla12 and one between Mla13 and Mla20 were confirmed. The former was also verified by RFLP analysis.     

Comparative virulence of Pyrenophora teres f. teres from Syria and Tunisia and screening for resistance sources in barley: implications for breeding

Aims: The aim of this study is to investigate the pathogenic diversity and virulence groups among Pyrenophora teres f. teres isolates, sampled from Syria and Tunisia, and to identify the most effective source of resistance in barley that could be used in breeding programmes to control net blotch in both countries. Methods and Results: One hundred and four isolates of P. teres f. teres were collected from barley in different agroecological zones of Tunisia and Syria. Their virulence was evaluated using 14 barley genotypes as differential hosts. The upgma clustering identified high pathogenic variability; the isolates were clustered onto 20 pathotypes that were sheltered under three virulence groups, with high, intermediate and low disease scores. According to susceptibility/resistance frequencies and mean disease ratings, CI05401 cultivar ranked as the best differential when inoculated with the Syrian isolates. However, CI09214 cultivar was classified as the best effective source of resistance in Tunisia. Conclusions: All P. teres f. teres isolates were differentially pathogenic. CI09214 and CI05401 cultivars were released as the most effective sources of resistance in Syria and Tunisia. Significance and Impact of the Study: National and international barley breeding programmes that seek to develop resistance against P. teres f. teres in barley should strongly benefit from this study. This resistance cannot be achieved without the proper knowledge of the pathogen virulence spectrum and the sources of host resistance.     

Mapping multiple disease resistance genes using a barley mapping population evaluated in Peru, Mexico, and the USA

We used a well-characterized barley mapping population (BCD 47 × Baronesse) to determine if barley stripe rust (BSR) resistance quantitative trait loci (QTL) mapped in Mexico and the USA were effective against a reported new race in Peru. Essentially the same resistance QTL were detected using data from each of the three environments, indicating that these resistance alleles are effective against the spectrum of naturally occurring races at these sites. In addition to the mapping population, we evaluated a germplasm array consisting of lines with different numbers of mapped BSR resistance alleles. A higher BSR disease severity on CI10587, which has a single qualitative resistance gene, in Peru versus Mexico suggests there are differences in pathogen virulence between the two locations. Confirmation of a new race in Peru will require characterization using a standard set of differentials, an experiment that is underway. The highest levels of resistance in Peru were observed when the qualitative resistance gene was pyramided with quantitative resistance alleles. We also used the mapping population to locate QTL conferring resistance to barley leaf rust and barley powdery mildew. For mildew, we identified resistance QTL under field conditions in Peru that are distinct from the Mla resistance that we mapped using specific isolates under controlled conditions. These results demonstrate the long-term utility of a reference mapping population and a well-characterized germplasm array for locating and validating genes conferring quantitative and qualitative resistance to multiple pathogens.     

CAPS and DHPLC Analysis of a Single Nucleotide Polymorphism in the Cytochrome b Gene Conferring Resistance to Strobilurins in Field Isolates of Blumeria graminis f. sp. hordei

A single nucleotide polymorphism in the wheat powdery mildew (Blumeria graminis f. sp. tritici) cytochrome b gene is responsible for resistance to inhibitors of the quinol outer binding site of the cytochrome bc₁ complex (QoI) fungicides. Analysis of a partial sequence of the cytochrome b gene from field isolates resistant and sensitive to QoI fungicides revealed the same point mutation in barley powdery mildew (B. graminis f. sp. hordei). Analysis of 118 and 40 barley powdery mildew isolates using a cleaved amplified polymorphic sequence assay and denaturing high performance liquid chromatography, respectively, confirmed that this single nucleotide polymorphism also confers resistance to QoI fungicides in barley powdery mildew.     

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F₁ hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F₁s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.     

Characterisation of pathogenic and molecular diversity in Sclerospora graminicola,the causal agent of pearl millet downy mildew

Genotypic diversity among 46 isolates of Sclerospora graminicola collected from seven states in India during 1992–2005 was determined through pathotyping and AFLP analysis. A high level of variation was observed among the isolates for downy mildew incidence, latent period and virulence index. Based on the reaction on a set of nine pearl millet lines, 46 isolates were classified in 21 pathotypes. Quantitative differences in virulence levels of the test isolates were assessed by calculating the virulence index (disease incidence × latent period ^? 1). A dendrogram generated by the average linkage cluster analysis of virulence index clustered the 46 isolates into eight groups. Region-specific grouping of five isolates from Gujarat and six from Rajasthan was observed within two distinct groups. Temporal variation was also observed among the isolates collected from the same location and same host over the years. A total of 297 bands were scored following selective amplification with three primer combinations E-TT/M-CAG, E-AT/M-CAG and E-TG/M-CAT and all of them were polymorphic. Cluster analysis of AFLP data clustered the test isolates into seven groups. Analysis of molecular variance indicated that variation in the S. graminicola populations was largely due to differences among the isolates within the states.     

Pathogenic variation and sexual reproduction in Swedish populations of Bremia lactucae

Summary The host-pathogen interaction between lettuce (Lactuca sativa) and downy mildew (Bremia lactucae) is mainly differential and the resistance so far utilized in the host is vertical. As in many other obligate parasites, the introduction of cultivars with new vertical resistance has exerted a strong selection pressure on the pathogen resulting in significant changes in virulence frequencies and in the establishment of races with new combinations of virulence. Genetic diversity in pathogen populations may arise through mutation and gene flow, and new virulence genotypes may then be established through parasexuality and sexual recombination. In Swedish populations of Bremia lactucae, the pattern of variation in the parasite agrees well with that which might be expected in a diploid, outcrossing organism with frequent sexual reproduction. This is supported by: two or more isolates, different in virulence and mating type, may occur together on the same lettuce leaf; zygotes (oospores) are formed in all populations investigated and the frequency varies from 22% to 98%; oospores germinate rather frequently under suitable conditions. To breed for resistance in dynamic host-pathogen systems such as this one is difficult and the program should preferably be based on race-non-specific resistance.     

Molecular mapping of powdery mildew resistance gene <Emphasis Type="Italic">Eg-3</Emphasis> in cultivated oat (<Emphasis Type="Italic">Avena sativa</Emphasis> L. cv. Rollo)

Powdery mildew is a prevalent fungal disease affecting oat (Avena sativa L.) production in Europe. Common oat cultivar Rollo was previously shown to carry the powdery mildew resistance gene Eg-3 in common with cultivar Mostyn. The resistance gene was mapped with restriction fragment length polymorphism (RFLP) markers from Triticeae group-1 chromosomes using a population of F₃ lines from a cross between A. byzantina cv. Kanota and A. sativa cv. Rollo. This comparative mapping approach positioned Eg-3 between cDNA-RFLP marker loci cmwg706 and cmwg733. Since both marker loci were derived from the long arm of barley chromosome 1H, the subchromosomal location of Eg-3 was assumed to be on the long arm of oat chromosome 17. Amplified fragment length polymorphism (AFLP) marker technology featured as an efficient means for obtaining markers closely linked to Eg-3.     

Virulence Pattern of Venturia inaequalis Field Isolates and Corresponding Differential Resistance in Malus x domestica

All commercially important apple cultivars are susceptible in the field to scab caused by Venturia inaequalis. The scope of this study was to investigate variation in virulence in Venturia inaequalis populations towards commercial apple cultivars. For this purpose, primary lesions were sampled in orchards with different varietal compositions and diversities. The virulence pattern of monosporic isolates, obtained by isolation of single conidia, was assessed by cross inoculations of the cv. Ananas Reinette, Boskoop, Glockenapfel, Golden Delicious, Gravenstein, James Grieve, Jonathan, Maigold, Reinette de Champagne, Spartan and Yellow Transparent. All cultivars were susceptible to some isolates and resistant to others. No cultivar behaved the same way, which suggests the presence of differential resistance in each cultivar and corresponding virulence in some isolates. Isolates from a monoculture of Golden Delicious consisted mainly of a pathotype that was virulent to Golden Delicious but not to other cultivars. In the samples from cultivar mixtures, virulence pattern variation was considerable. The results give further evidence of the existence of a large pool of differential and ephemeral resistances in Malus, which were overcome by the local scab populations during co‐evolution.     

Genetic diversity of <Emphasis Type="Italic">Rhynchosporium secalis</Emphasis> in Tunisia as revealed by pathotype,AFLP, and microsatellite analyses

Genetic variability among 122 Rhynchosporium secalis isolates collected from barley in three regions of Tunisia was investigated using host differentials, amplified fragment length polymorphism (AFLP), and microsatellite markers. The isolates were collected from a widely grown scald-susceptible barley cultivar Rihane and a range of local landrace cultivars in geographically distinct regions with different agroclimatic conditions. Pathotypic diversity (the proportion of unique pathotypes) was high in R. secalis populations from the high (100% diversity), moderate (95%), and low (100%) rainfall areas of Tunisia, and from both Rihane (which is the sole variety grown in the high rainfall region) and local landraces (which predominate in the low rainfall area). This may reflect a general adaptability for aggressiveness and suggests that the widely grown cultivar Rihane has exerted little or no selection pressure on the pathogen population since its release in 1983. Genotypic diversity (GD), defined as the probability that two individuals taken at random had different genotypes, was high for populations from Rihane, local landraces, and different agro-ecological zones (GD = 0.96–0.99). There was low genetic differentiation among pathogen populations from different host populations (G _ST ≤ 0.08, θ ≤ 0.12) and agro-ecological zones (G _ST ≤ 0.05, θ ≤ 0.04), which may be partly explained by gene flow due to the movement of infected stubble around the country. There was no correlation (r = 0.06, P = 0.39) between virulence phenotype and AFLP haplotype. A phenetic tree revealed groups with low bootstrap values that did not reflect the grouping of isolates based on host, pathotype, or agro-ecological region. The implications of these findings for R. secalis evolutionary potential and scald-resistance breeding in Tunisia are discussed.     

Polymorphism at the Hor 1 locus of barley (Hordeum vulgare L.)

The Hor 1 locus of barley encodes a group of seed storage polypeptides called C hordein. Two-dimensional electrophoretic analysis of C-hordein fractions from six cultivars with different alleles at the Hor 1 locus showed extensive polymorphism. A total of 34 major polypeptides was mapped, with between 4 and 18 present in each cultivar. There was less variation among the same cultivars in the numbers (6 to 10) of restriction fragments of genomic DNA which hybridized to a cDNA clone related to C hordein. The total number of restriction fragments was also lower (22), and most pairs of cultivars had more restriction fragments than polypeptides in common. A total number of about 20–30 C-hordein genes per haploid genome was estimated. The results indicate that cultivars differ mainly in the extent of gene and polypeptide divergence, rather than in the degree of gene reiteration. They are consistent with the proposed origin of the multiple structural genes at the Hor 1 locus by the duplication and divergence of a single ancestral gene.NACB was supported by a grant from the Home Grown Cereals Authority.