RFLP mapping of three new loci for resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) in barley

Three new major, race-specific, resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) were identified in three barley lines, RS42-6*O, RS137-28*E, and HSY-78*A, derived from crosses with wild barley (Hordeum vulgare ssp. spontaneum). The resistance gene origining from wild barley in line RS42-6*O, showed a recessive mode of inheritance, whereas the other wild barley genes were (semi)-dominant. RFLP mapping of these three genes was performed in segregating F₂ populations. The recessive gene in line RS42-6*O, was localized on barley chromosome 1S (7HS), while the (semi)-dominant genes in lines RS137-28*E, and HSY-78*A, were localized on chromosomes 1L (7HL) and 7L (5HL), respectively. Closely linked RFLP clones mapped at distances between 2.6cM and 5.3 cM. Hitherto, specific loci for powdery mildew resistance in barley had not been located on these chromosomes. Furthermore, tests for linkage to the unlocalized resistance gene Mlp revealed free segregation. Therefore, these genes represent new loci and new designations are suggested: mlt (RS42-6*O), Mlf (RS137-28*E), and Mlj (HSY-78*A). Comparisons with mapped QTLs for mildew resistance were made and are discussed in the context of homoeology among the genomes of barley (H-vulgare), wheat (Triticum aestivum), and rye (Secale cereale). Duplications of RFLP bands detected in the neighbourhood of Mlf and mlt might indicate an evolutionary interrelationship to the Mla locus for mildew resistance.     

Half sib selection for resistance to powdery mildew (Erysiphe graminis DC. f.sp. secalis Marchal) in rye populations

Summary Three rye populations originating from different cultivars were subjected to half sib progeny selection for three cycles to improve their resistance to powdery mildew (Erysiphe graminis DC. f.sp. secalis Marchal). Selection was carried out in the greenhouse (measurement of spore production at the three-leaf stage), in the field (scoring of mildew infections at ear emergence) or at both locations. All selections resulted in significant responses, the level of which varied depending on the genetic base of mildew resistance in the populations. Greenhouse selection was most effective when the frequency of dominant genes could be increased, whereas field selection was less effective. Estimates for heritability in the narrow sense ranged from 0.42–0.94 indicating that mildew resistance can be increased effectively. Inbred lines should be selected in base populations with a high level of quantitative resistance and/or a high frequency of dominant genes. For an effective resistance strategy in hybrid breeding such dominant genes are most valuable, and can be selected in young growth stages. In Strain CG the increase of dominant resistance genes by early selection resulted in a significant improvement of kernel yield.     

Polyamines in discrete regions of barley leaves infected with the powdery mildew fungus, Erysiphe graminis

The concentrations of putrescine, spermidine and spermine and the activities of arginine decarboxylase (ADC; EC 4.1.1.19) and ornithine decarboxylase (ODC: EC 4.1.1.17) were determined in discrete regions of barley leaves ( Hordeum vulgare L. cv. Golden Promise) infected with the powdery mildew fungus ( Erysiphe graminis f.sp. hordei Marchal). Polyamine concentrations and the activities of both enzymes were always greatest within the region surrounding the fungal pustule, with the lowest values always being found in the region furthest away from the pustule. Although the concentrations of the three amines and ADC and ODC activities within the fungal pustule were always less than values from the zone surrounding the pustule, these differences were never significant. Polyamine concentrations and ODC activity were not significantly reduced, and ADC activity remained unchanged in mildewed leaves with all surface fungal growth removed. It would appear therefore that not only does most of the increase in amines and ODC activity reside in the leaf itself, but that very little of this increase is due to fungal growth and sporulation. Furthermore, it seems possible that the increase in polyamines in mildewed barley could be involved in 'green-island' formation, where regions around mildew pustules remain green and physiologically active while the rest of the leaf senesces.     

Seedling resistance of wheat to the powdery mildew fungus,Erysiphe graminis f. sp. tritici. I. Resistance of first leaves

During primary infection by conidia ofErysiphe graminis f. sp.tritici, three mechanisms of resistance operate in first leaves of 8-day-old seedlings of both resistant and susceptible wheats. The first mechanism, operating at the penetration site, is responsible for the failure of penetrations attempted by primary germ tubes (PGT). The second mechanism is concerned with the abortion of haustoria in normal-appearing host cells. The third mechanism relates to the abortion of haustoria and the hypersensitivity of the penetrated host cells.With the inoculum-level of 19–24 conidia/mm², the three mechanisms together prevented 89.3 % of the attempted penetrations by PGT from producing normal haustoria in resistant wheat Purdue 5752C1-7-5-1 and 37.4 % in the susceptible wheat Vermillion. The first mechanism accounted for the prevention of 73.3 % of the attempted PGT penetrations on Purdue 5752C1-7-5-1 and 36 % on Vermillion. The second mechanism was responsible for stopping 19 % of all the successful penetrations in Purdue 5752C1-7-5-1 and 0.8 % in Vermillion. The third mechanism accounted for the failure of 41 % of all the successful penetrations in Purdue 5752C1-7-5-1 and 1.4% in Vermillion. Thirty-six hours after inoculation, 10.7% of all the attempted PGT penetrations appeared to be developing normally in first leaves of 8-day-old seedlings of resistant wheat Purdue 5752C1-7-5-1 as compared to 62.6 % in the susceptible wheat Vermillion.This appears to be the first report showing the relative effectiveness of various mechanisms of resistance concerning any powdery mildew fungus.     

Polyamine metabolism in barley reacting hypersensitively to the powdery mildew fungus Blumeria graminis f. sp. hordei

Polyamine levels and activities of enzymes of polyamine biosynthesis and catabolism were examined in the barley cultivar Delibes (Ml1al + Ml(Ab)) reacting hypersensitively to the powdery mildew fungus, Blumeria graminis f. sp. hordei (race CC220). Levels of free putrescine and spermine and of conjugated forms of putrescine, spermidine and spermine were greatly increased 1–4 d following inoculation of barley with the powdery mildew. These changes in polyamine levels were accompanied by elevated activities of the polyamine biosynthetic enzymes ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and S‐adenosylmethionine decarboxylase (AdoMetDC) and the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO). Activities of two enzymes involved in conjugating polyamines to hydroxycinnamic acids, putrescine hydroxycinnamoyl transferase (PHT) and tyramine feruloyl‐CoA transferase (TFT) were also examined and were found to increase significantly 1–4 d after inoculation. The possibility that the increased levels of free spermine, increased polyamine conjugates, and increased DAO and PAO activities are involved in development of the hypersensitive response of Delibes to powdery mildew infection is discussed.     

Components of adult plant resistance to powdery mildew (Erysiphe graminis f. sp. avenae) in oats

The adult plant reaction to mildew (Erysiphe graminis f. sp. avenae) of four oat genotypes was studied. A negative relationship existed in these genotypes between length of latent period and the percentage leaf area with mildew. The very susceptible CI 4877 had the shortest latent period and the largest area of leaf infected, while the highly resistant Cc 4761 had the longest latent period and the least leaf area covered with mildew. The same association also occurred between leaves within a genotype, particularly those genotypes with adult plant resistance, i.e., Maldwyn and Cc 4761. The more recently developed upper leaves showed the longest incubation period and least percentage area affected compared with lower leaves. A visual score of pustule sporulation indicated that CI 4877, Milford and Maldwyn had similar well-sporulating foci, but in Cc 4761 the pustules showed significantly less sporulation. Estimates of the relative number of spores produced per day over a 15 day period reflected the level of resistance. Thus the most susceptible genotype CI 4877 released over 50 times more spores than Cc 4761, while Milford and Maldwyn respectively produced 9 and 4.5 times more spores than the highly resistant Cc 4761. The longer latent period of Cc 4761 was reflected in the delay this genotype showed in reaching its relatively low peak production of spores. The possible use of the latent period as a factor when selecting for adult plant resistance in early growth stages is discussed.     

A proteomic analysis of powdery mildew (Blumeria graminis f.sp. hordei) conidiospores

Conidiospores are the asexual propagation units of many plant-pathogenic fungi. In this article, we report an annotated proteome map of ungerminated conidiospores of the ascomycete barley powdery mildew pathogen, Blumeria graminis f.sp. hordei . Using a combination of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, we have identified the proteins in 180 spots, which probably represent at least 123 distinct fungal gene products. Most of the identified proteins have a predicted function in carbohydrate, lipid or protein metabolism, indicating that the spore is equipped for the catabolism of storage compounds as well as for protein biosynthesis and folding on germination.     

DNA polymorphism among barley NILs of cv. Pallas, carrying genes for resistance to powdery mildew (Blumeria graminis f. sp. hordei)

Barley powdery mildew, caused by the pathogen Blumeria graminis f. sp. hordei is an important disease of barley (Hordeum vulgare L.). The random amplified polymorphic DNA (RAPD) method was used to detect DNA polymorphism among 7 Pallas near-isogenic lines (NILs) carrying Mla3, Mla12, Mlk, Mlp, Mlat, Mlg and MlLa genes for resistance to B. graminis f. sp. hordei. From among 500 random 10-mer primers tested, 3 were specific for NIL P2 (Mla3), 1 for P10 (Mla12), 6 for P17 (Mlk), 46 for P19 (Mlp), 4 for P20 (Mlat), 6 for P21 (Mlg), and 4 for P23 (MlLa). The results of this study demonstrated that the RAPD technique is a useful tool for detecting DNA polymorphism among Pallas NILs.     

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.     

Proteogenomics and in silico structural and functional annotation of the barley powdery mildew Blumeria graminis f. sp. hordei

Blumeria graminis is an economically important obligate plant-pathogenic fungus, whose entire genome was recently sequenced and manually annotated using ab initio in silico predictions (Spanu et al. 2010, Science 330, 1543-1546). Employing large scale proteogenomic analysis we are now able to verify independently the existence of proteins predicted by ∼24% of open reading frame models. We compared the haustoria and sporulating hyphae proteomes and identified 71 proteins exclusively in haustoria, the feeding and effector-delivery organs of the pathogen. These proteins are significantly smaller than the rest of the protein pool and predicted to be secreted. Most do not share any similarities with Swiss–Prot or Trembl entries nor possess any identifiable Pfam domains. We used a novel automated prediction pipeline to model the 3D structures of the proteins, identify putative ligand binding sites and predict regions of intrinsic disorder. This revealed that the protein set found exclusively in haustoria is significantly less disordered than the rest of the identified Blumeria proteins or random (and representative) protein sets generated from the yeast proteome. For most of the haustorial proteins with unknown functions no good templates could be found, from which to generate high quality models. Thus, these unknown proteins present potentially new protein folds that can be specific to the interaction of the pathogen with its host.     

Effects of sowing date and volunteers on the infectivity of soil infested with Gaeumannomyces graminis var. tritici and on take-all disease in successive crops of winter wheat

In a field experiment on winter wheat, take‐all on plants and the infectivity of the soil were studied in crop sequences with different combinations of sowing dates. Take‐all was negligible in the first wheat crop, but thereafter the mean disease intensity (measured using a take‐all rating, TAR, with a maximum of 300) was 108, 190, 118 and 251 in the second to fifth successive crops. In each growing season, the disease differed amongst sequences and built up more rapidly and was more intense on plants sown in mid‐September than on plants sown in mid‐October. In late‐sown plots, where volunteers had been present during the mid‐September to mid‐October period, take‐all reached an intensity intermediate between that in early‐sown plots and that in late‐sown plots that had been kept free of volunteers. Volunteers, therefore, partially offset the expected beneficial effect of decreased disease with later sowing. Differences in take‐all amongst sequences were most pronounced in the second wheat crop and early sowing of the previous wheat increased intensity of disease. In the following (third) crop, differences in disease intensity amongst sequences were smaller. Soil infectivity (measured by seedling bioassay after harvest) built up progressively from a low level after the first crop to peak after the third crop. In this build‐up phase, soil infectivity estimates were always numerically greater after harvest of early‐sown treatments than after later‐sown treatments, although never significant at P= 0.05. The greatest difference (P= 0.06) was recorded in October before sowing of the third crop, where the comparison was between soil after two previous early sowings and soil after two previous later sowings and control of volunteers. In the same autumn, presence of green cover (i.e. volunteers) was associated with a smaller loss of soil infectivity between harvest and later sowing than occurred in an absence of green cover. In 2^nd–4^th crops, where comparisons were available and mean TARs indicated moderate levels of take‐all, sowing later had no yield benefit, despite more take‐all and greater soil infectivity associated with early sowing. Important considerations for the management of crops at risk of take‐all are 1) choosing appropriate sowing dates to minimize take‐all or to encourage take‐all decline and 2) controlling volunteers and weed hosts where crops are sown late to minimise take‐all.     

Apoplastic pH signaling in barley leaves attacked by the powdery mildew fungus Blumeria graminis f. sp. hordei

To investigate apoplastic responses of barley (Hordeum vulgare L.) to the barley powdery mildew fungus Blumeria graminis f. sp. hordei, noninvasive microprobe techniques were employed. H(+)- and Ca(2+)-selective microprobes were inserted into open stomata of barley leaves inoculated with Blumeria graminis f. sp. hordei race A6 conidia. Resistance gene-mediated responses of barley genotype Ingrid (susceptible parent line) and the near-isogenic resistant Ingrid backcross lines (I-mlo5, I-Mla12, and I-Mlg) were continuously monitored from 20 min to 4 days after inoculation. The main events were categorized as short-term responses around 2 h after inoculation (hai), intermediate responses around 8 and 12 hai, and long-term responses starting between 21 and 24 hai. Short-term responses were rapid transient decreases of apoplastic H(+)- and Ca2+ activities that lasted minutes only. Kinetics were similar for all genotypes tested, and thus, these short-term responses were attributed as nonspecific first encounters of fungal surface material with the host plasma membrane. This is supported by the observation that a microinjected chitin oligomer (GlcNAc)8 yielded similar apoplastic alkalinization. Intermediate responses are trains of H+ (increase) spikes that, being different in susceptible Ingrid and penetration-resistant I-mlo5 (or I-Mlg), were interpreted as accompanying specific events of papillae formation. Long-term events were massive slow and long-lasting alkalinizations up to two pH units above control. Since these latter changes were only observed with near-isogenic hypersensitive reaction (HR)-mounting genotypes I-Mla12 and I-Mlg but not with I-mlo5 or, to a smaller extent, with susceptible Ingrid, both lacking significant rates of HR, they were rated as cell death specific. It is concluded that apoplastic pH changes are important indicators of host-pathogen interactions that correlate with both the different stages of fungal development and the different types of host defense response.     

The effect of sowing date on adult plant resistance to Erysiphe graminis f.sp. avenae in oats

A technique developed for detecting small differences in mildew infection under glasshouse/laboratory conditions is described. A satisfactory correlation was found between results obtained by this method and infections in the field. Detailed examination of the levels of infection in cultivars Maldwyn and Milford showed that the upper and more recently developed leaves of Maldwyn had a significantly smaller area infected than those of Milford, although seedling leaves of both genotypes were very susceptible. This was most marked when plants were in the 6- to 7-leaf stage. Comparison of host plants sown on successive dates during the spring and early summer showed that with the acceleration of the rate of growth occurring in later-sown plants, resistance was expressed progressively earlier in the life of the plant. Newly expanded leaves which show very low level of infection at the 6- to 7-leaf stage become susceptible as they age, resistance being then expressed in the younger leaves.     

Respiratory burst oxidase homologue A of barley contributes to penetration by the powdery mildew fungus Blumeria graminis f. sp. hordei

Reactive oxygen intermediates (ROI) are closely related to defence reactions of plants against pathogens. A prominent role in the production of ROI has been attributed to the plant respiratory burst oxidase homologues (RBOH) of the human phagocyte GP91(phox). A barley RBOH, which encodes a putative superoxide (O2*-)) producing NADPH oxidase, is described here. Histochemical analysis of the barley-Blumeria graminis f. sp. hordei (Bgh) interaction showed that O(2*-) is produced locally at the site of penetration. In contrast, hydrogen peroxide (H2O2) is produced in non-penetrated cell wall appositions. A barley RBOHA cDNA was isolated and a minor induction of expression of RBOHA was observed during the interactions of barley with Bgh. Transient RNA interference-mediated gene silencing of HvRBOHA during the penetration process of Bgh led to an increase of basal penetration resistance. The results support a potential role of HvRBOHA in cellular accessibility to Blumeria graminis.