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.     

Differential expression of putative cell death regulator genes in near-isogenic,resistant and susceptible barley lines during interaction with the powdery mildew fungus

We analysed pathogenesis-related expression of genes, that are assumed to be involved in ubiquitous plant defence mechanisms like the oxidative burst, the hypersensitive cell death reaction (HR) and formation of localized cell wall appositions (papillae). We carried out comparative northern blot and RT-PCR studies with near-isogenic barley (Hordeum vulgareL. cv. Pallas) lines (NILs) resistant or susceptible to the powdery mildew fungus race A6 (Blumeria graminis f.sp. hordei, BghA6). The NILs carrying one of the R-genes Mla12, Mlg or the mlo mutant allele mlo5 arrest fungal development by cell wall appositions (mlo5) or a HR (Mla12) or both (Mlg). Expression of an aspartate protease gene, an ascorbate peroxidase gene and a newly identified cysteine protease gene was up-regulated after inoculation with BghA6, whereas the constitutive expression-level of a BAS gene, that encodes an alkyl hydroperoxide reductase, was reduced. Expression of a newly identified barley homologue of a mammalian cell death regulator, Bax inhibitor 1, was enhanced after powdery mildew inoculation. An oxalate oxidase-like protein was stronger expressed in NILS expressing penetration resistance. A so far unknown gene that putatively encodes the large subunit of a superoxide generating NADPH oxidases was constitutively expressed in barley leaves and its expression pattern did not change after inoculation. A newly identified barley Rac1 homologue was expressed constitutively, such as the functionally linked NADPH oxidase gene. Gene expression patterns are discussed with regard to defence mechanisms and signal transduction.     

Stomatal lock-open, a consequence of epidermal cell death, follows transient suppression of stomatal opening in barley attacked by Blumeria graminis

Blumeria graminis f.sp. hordei (Bgh) attack disrupted stomatal behaviour, and hence leaf water conductance (g(l)), in barley genotypes Pallas and Ris?-S (susceptible), P01 (with Mla1 conditioning a hypersensitive response; HR), and P22 and Ris?-R (with mlo5 conditioning papilla-based penetration resistance). Inoculation caused some stomatal closure well before the fungus attempted infection. Coinciding with epidermal cell penetration, stomatal opening in light was also impeded, although stomata of susceptible and mlo5 lines remained largely able to close in darkness. Following infection, in susceptible lines stomata closed in darkness but opening in light was persistently impeded. In Ris?-R, stomata recovered nearly complete function by approximately 30 h after inoculation, i.e. after penetration resistance was accomplished. In P01, stomata became locked open and unable to close in darkness shortly after epidermal cells died due to HR. In the P22 background, mlo5 penetration resistance was often followed by consequential death of attacked cells, and here too stomata became locked open, but not until approximately 24 h after pathogen attack had ceased. The influence of epidermal cell death was localized, and only affected stomata within one or two cells distance. These stomata were unable to close not only in darkness but also after application of abscisic acid and in wilted leaves suffering drought. Thus, resistance to Bgh based on HR or associated with cell death may have previously unsuspected negative consequences for the physiological health of apparently 'disease-free' plants. The results are discussed in relation to the control of stomatal aperture in barley by epidermal cells.     

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.