Disruption of barley immunity to powdery mildew by an in-frame Lys-Leu deletion in the essential protein SGT1

Barley (Hordeum vulgare L.) Mla (Mildew resistance locus a) and its nucleotide-binding, leucine-rich-repeat receptor (NLR) orthologs protect many cereal crops from diseases caused by fungal pathogens. However, large segments of the Mla pathway and its mechanisms remain unknown. To further characterize the molecular interactions required for NLR-based immunity, we used fast-neutron mutagenesis to screen for plants compromised in MLA-mediated response to the powdery mildew fungus, Blumeria graminis f. sp. hordei. One variant, m11526, contained a novel mutation, designated rar3 (required for Mla6 resistance3), that abolishes race-specific resistance conditioned by the Mla6, Mla7, and Mla12 alleles, but does not compromise immunity mediated by Mla1, Mla9, Mla10, and Mla13. This is analogous to, but unique from, the differential requirement of Mla alleles for the co-chaperone Rar1 (required for Mla12 resistance1). We used bulked-segregant-exome capture and fine mapping to delineate the causal mutation to an in-frame Lys-Leu deletion within the SGS domain of SGT1 (Suppressor of G-two allele of Skp1, Sgt1_{ΔKL308–309}), the structural region that interacts with MLA proteins. In nature, mutations to Sgt1 usually cause lethal phenotypes, but here we pinpoint a unique modification that delineates its requirement for some disease resistances, while unaffecting others as well as normal cell processes. Moreover, the data indicate that the requirement of SGT1 for resistance signaling by NLRs can be delimited to single sites on the protein. Further study could distinguish the regions by which pathogen effectors and host proteins interact with SGT1, facilitating precise editing of effector incompatible variants.     

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.     

Pathotypes of Blumeria graminis f. sp. tritici,the causal agent of wheat powdery mildew from some regions of Iran

Wheat powdery mildew is controlled mainly by race-specific resistance. To be effective, breeding wheat for resistance to powdery mildew requires knowledge of virulence diversity in local populations of the pathogen. Isolates of Blumeria graminis, collected in 2009 and 2010 from three areas of Iranian production, were analysed for virulence using a host differential series comprised of 16 known genes conferring resistance to powdery mildew. The results showed that high-virulence frequencies to genes Pm1, Pm2, Pm4a, Pm5, Pm6, Pm7, Pm8 and Pm9 were found over both years and across all three areas. Virulence frequencies for Pm3a and Pm3b were intermediate, while virulence frequencies for Pm3a, Pm3c, Pm4a and Pm2, 6 were low. Genes Pm1, 2, 9 and Pm2, 4b, 8 were highly resistant in all regions. Virulence to Pm8 increased to high levels, while virulence to Pm4a decreased across the area surveyed from 2009 to 2010.     

Susceptibility to take‐all of cereal and grass species,and their effects on pathogen inoculum

Two field trials were conducted to investigate different herbage grasses and cereals for their susceptibility to the disease take‐all, for their impact on concentrations of the pathogen, Gaeumannomyces graminis var. tritici (Ggt), in soil and for their effect on development of take‐all in a subsequent wheat crop. In the herbage grass trial, Bromus willdenowii was highly susceptible to Ggt, produced the greatest post‐senescence Ggt concentrations in soil and highest incidence of take‐all in following wheat crop. Lolium perenne, Lolium multiflorum and Festuca arundinacea supported low Ggt soil concentrations and fallow the least. The relationship between susceptibility to Ggt and post‐senescence concentrations in soil differed between pasture grasses and cereals. In a trial in which Ggt was added to half the plots and where wheat, barley, triticale, rye or fallow were compared, the susceptibility of the cereals to take‐all was not clearly linked to post‐harvest soil Ggt concentrations. In particular, triticale and rye had low and negligible take‐all infection respectively, but greater post‐harvest soil Ggt concentrations than barley or wheat. This indicates that low Ggt concentrations on roots may build up during crop senescence on some cereals. Soil Ggt concentrations were greater following harvest in inoculated plots sown to cereals, but in the second year there was more take‐all in the previously non‐inoculated than inoculated plots. Thus, the grass and cereal species differed in susceptibility to take‐all, in their impact on Ggt multiplication and in associated take‐all severity in following wheat crop.     

Protecting South Asian Wheat Production from Stem Rust (Ug99) Epidemic

The Ug99 group of stem rust races (Puccinia graminis Pers. f. sp. tritici Eriks. & E. Henn.) has evolved and migrated. While the original variant overcame the widely deployed gene Sr31, and Sr21 (in Chinese Spring background), but not Sr21 in Einkorn, a new strain of Ug99, virulent on Sr24, was detected in 2006 and caused a severe epidemic in 2007 in Kenya. Forms virulent on Sr36 and Sr21 were identified in 2007. Likewise, an Ug99 variant virulent to both Sr21 and Sr24 was identified in 2008 in Kenya. Simultaneously, the original strain spread to Yemen and Sudan in 2006. Fears of a spread into Asia were confirmed when this race was detected in Iran in 2007. This has raised serious concerns that Ug99 could follow the same migratory route from Africa to Asia as Yr9 and cause major epidemics across the epidemiological region of South Asia. In 2005–06, screening in Kenya and Ethiopia of wheat materials from Asian countries revealed a very low frequency of lines resistant to Ug99 and its variants. Under the umbrella of the Borlaug Global Rust Initiative (BGRI), significant efforts have been made to counter the challenges posed by Ug99 and its derivative races. Diverse sources of resistance to the pathogen have been identified and incorporated in high‐yielding wheat backgrounds. The most promising strategy has been to deploy spring wheat varieties possessing adult plant resistance (APR) in infested and bordering areas to decrease inoculum amounts and slow down the development of new virulence, for example four CIMMYT genotypes with Sr2+ have been released in Afghanistan and their seed is also distributed in region bordering Iran. For an immediate remedy, race‐specific resistance genes can be deployed in combinations using marker‐assisted selection. Several Ug99‐resistant varieties have already been released in South Asian countries (Afghanistan, India, Nepal, Bangladesh and Pakistan), and seed dissemination is underway. The Ug99 risk in the region can be reduced to minimum levels by identifying, releasing and providing seed of high‐yielding and resistant cultivars.     

Single residues in the LRR domain of the wheat PM3A immune receptor can control the strength and the spectrum of the immune response

The development of improved plant nucleotide‐binding, leucine‐rich repeat (LRR) immune receptors (NLRs) has mostly been based on random mutagenesis or on structural information available for specific receptors complexed with the recognized pathogen effector. Here, we use a targeted mutagenesis approach based on the natural diversity of the Pm3 powdery mildew resistance alleles present in different wheat (Triticum aestivum) genotypes. In order to understand the functional importance of the amino acid polymorphisms between the active immune receptor PM3A and the inactive ancestral variant PM3CS, we exchanged polymorphic regions and residues in the LRR domain of PM3A with the corresponding segments of PM3CS. These novel variants were functionally tested for recognition of the corresponding AVRPM3^A2/F2 avirulence protein in Nicotiana benthamiana. We identified polymorphic residues in four regions of PM3A that enhance the immune response, but also residues that reduce it or result in complete loss of function. We found that the identified critical residues in PM3A modify its activation threshold towards different protein variants of AVRPM3^A2/F2. PM3A variants with a lowered threshold gave a stronger overall response and gained an extended recognition spectrum. One of these variant proteins with a single amino acid change was stably transformed into wheat, where it conferred race‐specific resistance to mildew. This is a proof of concept that improved PM3A variants with an enlarged recognition spectrum can be engineered based on natural diversity by exchanging single or multiple residues that modulate resistance function.     

Barley mildew and its elicitor chitosan promote closed stomata by stimulating guard-cell S-type anion channels

Stomatal closure is known to be associated with early defence responses of plant cells triggered by microbe-associated molecular patterns (MAMPs). However, the molecular mechanisms underlying these guard-cell responses have not yet been elucidated. We therefore studied pathogen-induced changes in ion channel activity in Hordeum vulgare guard cells. Barley mildew (Blumeria graminis) hyphae growing on leaves inhibited light-induced stomatal opening, starting at 9 h after inoculation, when appressoria had developed. Alternatively, stomatal closure was induced by nano-infusion of chitosan via open stomata into the sub-stomatal cavity. Experiments using intracellular double-barreled micro-electrodes revealed that mildew stimulated S-type (slow) anion channels in guard cells. These channels enable the efflux of anions from guard cells and also promote K(+) extrusion by altering the plasma membrane potential. Stimulation of S-type anion channels was also provoked by nano-infusion of chitosan. These data suggest that MAMPs of mildew hyphae penetrating the cuticle provoke activation of S-type anion channels in guard cells. In response, guard cells extrude K(+) salts, resulting in stomatal closure. Plasma membrane anion channels probably represent general targets of MAMP signaling in plants, as these elicitors depolarize the plasma membrane of various cell types.     

The evaluation of rhizomania resistant sugar beet for the UK

Sugar beet rhizomania disease, caused by Beet necrotic yellow vein virus and transmitted by the soil‐borne parasite Polymyxa betae, was first recorded in the UK in 1987. Recently, breeding lines and cultivars with partial resistance to the virus derived from the ‘Holly’ source of resistance have been developed and their suitability for use under UK conditions is explored in this paper. Virus multiplication in the roots of resistant lines exposed to severe disease pressure in glasshouse tests, when quantified by ELISA, was less than one third of that in susceptible controls. More recently developed resistant lines had a lower virus content, on average, largely due to a reduced frequency of susceptible individuals. There was no evidence for resistance to the vector, P. betae, in virus resistant lines. However, the proportion of viruliferous P. betae resting spores in the roots, estimated using the most probable number (MPN) technique, was reduced by at least one third in resistant lines compared with the most susceptible control. A novel line, containing an additional gene to that in ‘Holly’, was the most effective, reducing the infection level to 3% of that in the susceptible control. In two field experiments on severely infested sites, the rate of infection of a resistant line, when assessed by ELISA, was reduced by half compared with a susceptible cultivar and sugar yields of resistant lines were consistently 2–3 times higher than those of susceptible cultivars. In 41 trials on rhizomania‐free sites, several recently introduced resistant lines exhibited sugar yields and agronomic performance comparable to that of three selected high yielding, susceptible cultivars. Results are discussed in relation to the specific UK requirements for rhizomania resistant cultivars. One resistant line, Beta 805 (cv. Concept), fulfilled the requirements for widespread use to control the disease.     

In vitro antifungal activity of the tea tree (Melaleuca alternifolia) essential oil and its major components against plant pathogens

AIMS: The aim of this study was to examine the effect of Melaleuca alternifolia essential oil (TTO) and its principal components on four cereal-pathogenic fungi. METHODS AND RESULTS: The antimycotic properties of TTO and of terpinen-4-ol, gamma-terpinen and 1,8-cineole (eucalyptol) were evaluated in vitro on Fusarium graminearum, Fusarium culmorum and Pyrenophora graminea. Moreover, barley leaves infected with Blumeria graminis were treated with whole TTO. All the tested fungi were susceptible to TTO and its components. CONCLUSIONS: TTO exerted a wide spectrum of antimycotic activity. Single TTO purified components were more active than the whole oil in reducing in vitro growth of fungal mycelium and, among the tested compounds, terpinen-4-ol was the most effective. SIGNIFICANCE AND IMPACT OF THE STUDY: TTO and its components can be considered potential alternative natural fungicides.