Susceptibility-inducing factor (suppressor) from Blumeria graminis f. sp. hordei has no effect on the primary infection of the fungus

When fungal germlings, after forming haustoria of Blumeria graminis f. sp. hordei (B. graminis), were removed from the surfaces of barley coleoptiles by cellulose acetate, followed by challenge inoculation with the non-pathogen Erysiphe pisi, they infected the nonhost barley coleoptile cells. This phenomenon was not observed on the coleoptile surface when the fungal germlings of B. graminis were removed before the formation of haustoria. Also, when the surface was inoculated with the pathogen of barley B. graminis as a challenger, after removing the fungal germlings of inducer post haustorial formation, the penetration efficiency of the fungi increased significantly compared with that of the control. Furthermore, when we extracted the crude-susceptibility inducing factor (suppressor) from coleoptiles before and after the formation of haustoria of B. graminis, suppressor activity against infection with E. pisi was observed only in the extract of barley coleoptiles that included haustoria of B. graminis about 18 h or later after inoculation. Surprisingly, however, the extract did not increase the penetration efficiency of B. graminis significantly. Thus, we hypothesize that the suppressor extracted from barley coleoptiles in which B. graminis had formed haustoria has no effect on increasing the penetration efficiency of the primary infection from the appressorium of B. graminis but has an effect on the infection with non-pathogen E. pisi.     

Cytological events in tomato leaves inoculated with conidia of <Emphasis Type="Italic">Blumeria graminis</Emphasis> f. sp. <Emphasis Type="Italic">hordei</Emphasis> and <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> KTP-01

Leaves of tomato and barley were inoculated with conidia of Blumeria graminis f. sp. hordei race 1 (R1) or Oidium neolycopersici (KTP-01) to observe cytological responses in search of resistance to powdery mildew. Both conidia formed appressoria at similar rates on tomato or barley leaves, indicating that no resistance was expressed during the prepenetration stage of these fungi. On R1-inoculated tomato leaves, appressoria penetrated the papillae, but subsequent haustorium formation was inhibited by hypersensitive necrosis in the invaded epidermal cells. On the other hand, KTP-01 (pathogenic to tomato leaves) successfully developed functional haustoria in epidermal cells to elongate secondary hyphae, although the hyphal elongation from some conidia was later suppressed by delayed hypersensitive necrosis in some haustorium-harboring epidermal cells. Thus, the present study indicated that the resistance of tomato to powdery mildew fungi was associated with a hypersensitive response in invaded epidermal cells but not the prevention of fungal penetration through host papilla.     

A microscopic study of powdery mildew on barley after application of the systemic compound wepsyn

5-Amino, 3-phenyl, 1-bis (dimethylamido) phosphoryltriazole 1, 2, 4 (wepsyn) was applied to the roots of barley seedlings, var. ‘Balder’ in concentrations of 10, 25, 62 and 156 ppm, and the infection process ofErysiphe graminis f.sp.hordei on these plants studied microscopically. During the first few hours after inoculation a retardation of the germination occurred at the two highest concentrations, thus before the fungus had established contact with the host protoplasm. Penetration and formation of a lignituber was retarded at all wepsyn concentrations. At 10 and 25 ppm the formation of primary haustoria was reduced about thirty fold in relation to that on control plants. Mycelium that developed was often stunted. At higher wepsyn concentrations almost no haustoria were formed. Some leaf tip necrosis occurred, especially at the higher concentrations of wepsyn.     

Differential effects of -mannose and 2-deoxy- -glucose on attempted powdery mildew fungal infection of inappropriate and appropriate ramineae

Barley, oat and wheat were used as both inappropriate hosts (IH) and appropriate hosts (AH) for three formae speciales of the fungus Blumeria graminis, the causal agent of powdery mildew disease. Treatment with either the glucose analog 2-deoxy- -glucose (DDG) or with -mannose dramatically suppressed penetration resistance in IH and to a much lesser extent in AH combinations. Other effects of DDG and -mannose were strikingly dissimilar. DDG greatly reduced localized autofluorescence at fungal attack sites on epidermal cells, and prevented hypersensitive epidermal cell death (HR). -mannose had little effect on autofluorescence or HR. DDG arrested the development of fungal haustoria and apparently prohibited biotrophy leading to secondary hyphae. -mannose allowed haustorial development and functional biotrophy leading to the production of elongating secondary hyphae. This suggests that B. graminis is in some way capable of utilizing -mannose as a carbon substrate. Results with IH combinations paralleled those of known mlo -barley responses to DDG and -mannose. Results are discussed in relation to specific physiological processes known to be influenced by either DDG or by -mannose, or by both compounds.     

Fungal suppression of resistance against inappropriate Blumeria graminis formae speciales in barley,oat and wheat

When barley, wheat or oat leaf epidermal cells were attacked by their appropriate forma specialis (f.sp.) of Blumeria graminis DC. Speer (f.sp. hordei, tritici and avenae, respectively), many attempted penetrations succeeded, functional haustoria were formed and very few plant cells died. When attacked by either of the two possible inappropriate ff.spp., penetration attempts failed in association with papilla deposition by epidermal cells, attacked cells died, or if visible haustoria were formed the plant cell died very soon afterwards. Double inoculation experiments were performed where each cereal species was first attacked by its appropriate f.sp., as inducer, and later by the different ff.spp. as challenger. Infection by the appropriate inducer profoundly affected cellular responses to challenger attack. Suppression of defensive responses was dramatic within epidermal cells containing the inducer haustorium, evident to some extent in adjacent cells, but undetectable at two cells distance. Suppression of penetration resistance allowed most challenger attacks, even by inappropriate ff.spp., to form a haustorium. Furthermore, death of penetrated epidermal cells was also suppressed so that haustoria of the inappropriate ff.spp. functioned to support colony development. In oat, delayed epidermal cell death prevented full colony development by inappropriate ff.spp., but in barley and wheat, no cell death was apparent by four days after inoculation and colonies of the inappropriate ff.spp. produced extensive hyphae, secondary haustoria and conidial chains.     

Two class III chitin synthases specifically localized in appressoria and haustoria of Puccinia graminis f. sp. tritici

Rust fungi like Puccinia graminis f. sp. tritici are known to change their cell wall properties upon entering the plant tissue. Immunohistochemistry revealed the cellular localization of two class III chitin synthase isoforms in rust mycelia developing on and in the host plant. Isoform IIIa is restricted to fungal infection structures growing on the surface of the plant, such as germ tubes and, predominantly, appressoria. Isoform IIIb is found exclusively in haustoria developed inside the plant. Thus, the rust fungus uses at least two chitin synthase isoforms with specialized functions in the differentiation of infection structures during the biotrophic plant-pathogen-interaction.     

Isolation of genes expressed during compatible interactions between powdery mildew (Blumeria graminis) and wheat

Powdery mildew of wheat is an important disease caused by the obligately biotrophic fungus Blumeria graminis. In compatible interactions, the powdery mildew fungus undergoes a series of developmental stages to form haustoria within host cells through which nutrients are obtained. In this study, we utilized the cDNA-AFLP technique to isolate wheat genes expressed at 5 days' post-inoculation. The expression patterns of several sequences identified in the cDNA-AFLP profiling were further investigated by Northern hybridization and RT-PCR analyses. Genes with sequence similarity to GenBank accessions AAR91119, P20076, BT009372, ABA99697, BAD81963 or AAO72574 showed greater expression in susceptible rather than resistant or mock-inoculated leaves. In addition, several sequences with similarity to CAD27894, XM_466672, AAT79487 or AAM64566 were expressed only in the compatible interactions. Possible involvement of these genes in susceptibility of host wheat upon powdery mildew infection is further discussed.     

Hydroxypyrimidine fungicides inhibit adenosine deaminase in barley powdery mildew

Adenine and adenosine are metabolized by the adenine salvage pathway during primary infection of barley powdery mildew, Erysiphe graminis f.sp. hordei. Operation of this pathway was affected by the hydroxypyrimidine fungicide, ethirimol. Adenosine deaminase, ADAase, which was detected in mildew conidia and infected plants, but not in healthy barley, was the only enzyme in this pathway inhibited by the fungicide in in vitro assays. This feature of the mildew enzyme was unusual, and correlates with the specificity of hydroxypyrimidines which act against powdery mildews only. Other properties of this enzyme were similar to ADAase from other sources. In structure/activity studies with dimethirimol analogs, poor fungicidal activity was often associated with failure to inhibit ADAase, especially when assayed during appressoria formation. Purine derivatives were much less specific, and their mode of action against powdery mildew is probably different. Ethirimol resistance was not related to changes in ADAase, nor was the fungicide altered to an inactive metabolite. It is concluded that ADAase is one site of hydroxypyrimidine action.     

Identification of eight effector candidate genes involved in early aggressiveness of the barley powdery mildew fungus

The barley powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), expresses 491 candidate for secreted effector proteins (CSEPs), identified based on presence of a predicted signal peptide and relative sequence uniqueness. Virtually nothing is known about the mechanism of action of CSEPs. The present study used host‐induced gene silencing (HIGS) to identify genes involved in early fungal aggressiveness. Screening of 22 selected CSEP‐encoding genes suggested that CSEP0007, CSEP0025, CSEP0128, CSEP0211, CSEP0247, CSEP0345, CSEP0420 and CSEP0422 are individually required for normal levels of aggressiveness of Bgh. Expression data showed that these genes were up‐regulated at initial stages of infection, suggesting their involvement in early fungal aggressiveness. Altogether, the findings expand the current knowledge of genes contributing to Bgh pathogenesis and provide the basis for future functional characterization.     

Infection-inhibition activity of avenacin saponins against the fungal pathogens Blumeria graminis f. sp. hordei, Bipolaris oryzae, and Magnaporthe oryzae

Triterpenoid saponins are sugar-modified triterpene derivatives. Cereals and other grasses are generally deficient in these secondary metabolites with the exception of oat. Oat accumulates antimicrobial triterpenoid saponins in its roots. These oat-root-derived compounds, called avenacins, confer broad-spectrum resistance to soil-borne pathogens. Here, we tested the effect of avenacins on the development of infection structures of fungal pathogens Blumeria graminis f. sp. hordei and Bipolaris oryzae and Magnaporthe oryzae. We show that avenacins are able to inhibit the infection process of these phytopathogens on plant hosts.     

Antifungal activity of silver nanoparticles synthesized using turnip leaf extract (Brassica rapa L.) against wood rotting pathogens

The synthesis of metal nanoparticles using greener methodology and its application in biological fields is a burgeoning field of research. We demonstrated the synthesis of silver nanoparticles using turnip leaf extract and its interaction with wood-degrading fungal pathogens such as Gloeophyllum abietinum, G. trabeum, Chaetomium globosum, and Phanerochaete sordida. TEM images revealed that the silver nanoparticles were predominantly spherical and loosely agglomerated with an average size of 16.14 nm. DLS measurement of silver nanoparticles revealed an average hydrodynamic diameter of ca. 39.5 nm with a zeta potential of ?14.8 mV at pH 7.5. FTIR spectra revealed the presence of amine and aliphatic esters, which were involved in the reduction and stabilization of silver nanoparticles. The as-synthesized silver nanoparticles showed broad spectrum antifungal activity against wood-degrading fungi by inhibiting growth. Thus, the greener-synthesized silver nanoparticles can be used as an antifungal agent against wood-degrading fungal pathogens.     

A transient expression system to assay putative antifungal genes on powdery mildew infected barley leaves

A transient expression system has been established to assay the regulation and impact of putative antifungal proteins at the single cell level in barley (Hordeum vulgareL.) epidermal leaf cells infected with powdery mildew (Erysiphe graminisf.sp.hordei).The system combines biolistic transformation with direct comparison of the fungal attack in transfected and wild type cells. Many epidermal cells showed detectable expression of marker genes including a novel improved version of green fluorescent protein (GFP). The marker was detectable for at least 10 days during which individual host cells supported an unaffected asexual life-cycle of the pathogen.     

Evidence that ethirimol may interfere with adenine metabolism during primary infection of barley powdery mildew

Ethirimol, a hydroxypyrimidine fungicide active against powdery mildews only, inhibited the formation of appressoria during primary infection of barley powdery mildew, Erysiphe graminis f.sp hordei. It also affected other stages of mildew development. Several adenine analogs had similar effects and ethirimol-resistant mildew strains were generally cross-resistant to these. Adenine and adenosine reduced the fungitoxicity of ethirimol. During the formation of appressoria [³H]adenine was incorporated into RNA but [¹⁴C]glycine was not, suggesting that purine biosynthesis did not occur. Ethirimol inhibited this RNA synthesis and it is concluded that the fungicide may interfere with adenine metabolism at some site subsequent to its synthesis.     

Virulence phenotypes of Blumeria graminis f. sp. hordei in South Africa

Virulence analysis of 224 isolates of Blumeria graminis f. sp. hordei (barley powdery mildew) from South Africa was performed. The isolates were collected from eight fields and a greenhouse in 2004 and 2007. The isolates were tested for virulence on a set of 20 differential varieties. All isolates were virulent on the resistance genes Mla8 and Ml(Ch) and avirulent for the resistance genes Mla3, Mla6, Mla7, Mla9, Mla13, Mla23, Mlp1 and MlaN81. Virulence frequencies of field isolates for the resistance genes Mla12 + MlaEm2, Mlat, Mla22, Mlk1 and Mlh were 52.9–99.5 % and for Mla1 + MlaAl2, MlLa, Mlra, Mlg + MlCP and Ml(Ru2) were 0.5–23.5 %. In total, 46 pathotypes were detected in the field and seven other pathotypes in the greenhouse. Only nine pathotypes were found in both years, but they included 61.8 % of the isolates. The predominant pathotype represented 15.9 % of the isolates, and was the only one common to all three field populations. The average relative virulence complexity per field isolate increased from 0.405 in 2004 to 0.486 in 2007. Two powdery mildew metapopulations in geographically distant and separated areas (North West and Western Cape) were deduced. The South African population of Blumeria graminis f. sp. hordei had unique virulence frequencies and virulence associations when compared to populations from other parts of the world.     

Macroscopic and Histological Characterisation of Genes er1 and er2 for Powdery Mildew Resistance in Pea

In pea, two single recessive genes, er1 and er2, have been identified for resistance to powdery mildew caused by Erysiphe pisi, but little is known about their mode of action. Pea accessions carrying the genes er1 or er2 and other accessions displaying resistance to powdery mildew in the field were studied. In accessions carrying gene er1, epidermal cell penetration was prevented and very few haustoria or colonies were formed. Under controlled conditions, er1 conferred complete or almost complete resistance to the fungal isolates used and this resistance was not associated with macroscopically visible necrosis. Under field conditions these accessions developed a low level of disease. Resistance in line JI2480 (carrying er2) increased with temperature and leaf age, and complete resistance was expressed only at high temperature (25 °C) or in mature leaves. This resistance was based mainly on post-penetration cell death, complemented by a reduction of percentage penetration success in mature leaves. Combining the resistance provided by gene er1 and by line JI2480 into new cultivars is likely to increase their level of resistance and enhance durability of the protection.     

Cytology of infection of apple leaves by Diplocarpon mali

Diplocarpon mali, the causal agent of Marssonina leaf blotch of apple, causes severe defoliation during the growing season. Little information is available on the mode of infection and the infection process. In this study, the infection strategies of D. mali in apple leaves were investigated using fluorescence and electron microscopy. Conidia attached to leaf surface apparently by mucilage and germinated on both sides of leaves 6 h post-inoculation (hpi). The pathogen penetrated the cuticle by infection pegs formed either in germ tubes or appressoria in 6 hpi, and then formed haustoria in host epidermal and mesophyll cells accompanied by extension of subcuticular and intercellular hyphae. Five days post-inoculation (dpi), the intracellular hyphae were observed. At the same time, the subcuticular hyphal strands (SHS) were produced as a means for fast expansion and reproduction. About 7 dpi, acervuli formed on inoculated leaves. This was the first observation that D. mali formed haustoria and SHS as infection strategies. Our results suggest that D. mali may behave like a hemibiotroph, which can use both biotrophic and necrotrophic strategies to establish infections on apple leaves.     

Encasement of Erysiphe graminis haustoria after treatment with bromuconazole

Preventive application of bromuconazole caused reduction in size and increased encasement rate of haustoria of Erysiphe graminis DC. For example, seven days after inoculation, 60 and 70% of haustoria had been encased in leaves treated with 8 mg litre⁻¹ and 16 mg litre⁻¹ respectively; the average length of the digitations was 8–10 μm in treated cells compared to 24 μm in untreated cells. The encasement process extended from the neck region to the whole haustorium. Haustorial bodies from treated plants had electron-dense cytoplasm and their organelles were more difficult to identify than in control plants. Extrahaustorial matrix was reduced to an unusually thin, osmiophilic pellicle, surrounded by abundant heterogeneous encasement material. Curative treatment induced similar changes, especially in the margin of the colonies. In the centre of the colony, haustoria were less affected by the fungicide; deposition of collar-like material, modification of extrahaustorial matrix and membrane and accumulation of plant cytoplasm around the digitations resulted in an intermediate, ‘swollen’ state of digitate haustoria. The possible pathway of encasement events is discussed.