Fine Structure and Instability of the Ml-a Locus in Barley

There are many naturally occurring variants at the Ml-a locus in barley that confer resistance to the powdery mildew fungus Erysiphe graminis f. sp. hordei. Since the Ml-a locus is bracketed by Hor-1 and Hor-2, genes that encode storage proteins in the endosperm, the Ml-a locus is amenable to fine structure analysis. Rare susceptible recombinants, as judged by exchange of flanking markers, were recovered in F3 families from the Ml-a10 X Ml-a1, Ml-a1 X Ml-a15 and Ml-a6 X Ml-a13 crosses. Some susceptible recombinants were recovered from the Ml-a6 X Ml-a13 cross that did not fit the expected F3 family segregation ratios. The Ml-a6/Ml-a13 recombinants often reverted to resistance in subsequent generations. No recombinants were recovered in the reciprocal cross, Ml-a13 X Ml-a6. The possibility of a transposable element and a possible linear order of six "alleles" at the Ml-a locus is discussed.     

Evidence for Resistance-inducing Compounds in Conidia of Erysiphe graminis f.sp. hordei

Preparations of Erysiphe graminis f.sp. hordei conidia were spray-applied to the first leaf of barley plants which were subsequently challenge inoculated with virulent powdery mildew. The powdery mildew reducing effect of the preparations was assessed by scoring the outcome of the challenge inoculation. Homogenates of ungerminated conidia, germinated conidia, and methanol-water extracts of germinated conidia reduced the number of powdery mildew colonies. Cell wall fragments from ungerminated conidia, germinated conidia, and conidial germination fluid obtained from conidia germinated in aqueous suspension did not reduce the number of powdery mildew colonies. Microsconical analysis of the infection course following challenge inoculation indicated that the powdery mildew reducing effect is due partly to induced resistance.     

Quantitative Studies of Resistance Induced By Avirulent Cultures of Erysiphe graminis f. sp. hordei in Barley

Quantification of resistance induced by avirulent cultures of Erysiphe graminis f. sp. hordei against virulent cultures in barley was attempted. Four mildew cultures and 4 barley varieties with known genes of virulence and resistance respectively were used. Pre, post and simultaneous inoculation of leaves was done with avirulent and virulent cultures. Pre-inoculation with avirulent cultures induced resistance in the host such that the pustule number and spore production by later inoculation of virulent cultures was reduced significantly. Once induced, such resistance was active up to 8 days. There was some indication of induced susceptibility if the inducing culture was characterized by medium virulence. Increase of inceulum density of the avirulent (inducer) culture increased the amount of induced resistance Further studies of the phenomenon of induced resistance are needed in relation to possible applications for disease control through inoculations. varietal mixtures and multilines.     

Quantitative Differences in Powdery Mildew Resistance Among Spring Barley Cultivars

Quantitative powdery mildew resistance in compatible host-pathogen-combinations was measured by the number of pastules/cm² leaf area. Spring barley cultivar ‘Proctor’ was significantly less infected than ‘Golden Promise”. Using these two cultivars (having no effective major resistance gene) as controls, MO- and AR-resistant cultivars were inoculated with virulent mildew isolates. ‘Mona”, ‘Grit’ and ‘Nudinka’ had a higher or, at least, the same level of quantitative resistance as ‘Proctor”. None of the remaining cultivars showed the high susceptibility expressed by ‘Golden Promise”. Ranking of host genotypes was nearly constant while that of mildew isolates varied considerably. Only a small portion of the observed variance was due to interaction between host cultivars and pathogen isolates. ‘Triesdorfer Diva’ gave a resistant infection type after inoculation with different AR-virulent isolates, indicating that this cultivar has major resistance other than that conditioned by gene Ml-a12.     

Germination of Erysiphe gratninis f.sp. hordei conidia on barley leaves

Germination of Erysiphe graminis f.sp. hordei conidia on leaves of several barley cultivars was studied in the laboratory. On both detached leaves and intact plants, within 48 h of inoculation a higher proportion of conidia had germinated on the basal and middle portions of the adaxial leaf surface than on the corresponding portions of the abaxial surface. Such differences between surfaces were not observed near the leaf tip. Similar results were obtained with all the cultivars and growth stages tested, and with five isolates of E. graminis, and are consistent with the observation that there is usually less powdery mildew on the abaxial than the adaxial surface of barley leaves. With most of the barley genotype/mildew isolate combinations tested, within 48 h of inoculation higher proportions of conidia germinated on seedlings and juvenile plants than on older plants. Inherited characteristics which affect spore germination on the leaf surface may be important factors in the development of adult-plant resistance of barley to powdery mildew, particularly in certain genotypes.     

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.     

Chance and selection in the evolution of barley mildew

Populations of the barley powdery mildew fungus are genetically very diverse. However, when a new resistance gene is introduced into barley to control mildew, the population of the pathogen may respond by rapid growth of a few virulent clones. These phases of rapid clonal evolution cause radical changes in the frequencies of mildew genotypes.     

Powdery Mildew Resistance in Barley Landraces from Morocco

Nineteen barley landraces collected from Morocco were screened for resistance to powdery mildew. The landraces originated from the collection at the Polish Gene Bank, IHAR Radzików, Poland. The fifteen landraces tested showed powdery mildew resistance reactions and 35 single plant lines were selected. Twenty-one of these lines were tested in the seedling stage with 30, four lines with 17 and another 10 lines with 23 differential isolates of powdery mildew, respectively. The isolates were chosen according to their virulence spectra observed on the Pallas isolines differential set. Nine lines (E 1029-1-1, E 1042-2-2, E 1050-1-1, E 1054-5-1, E 1056-2-5, E 1056-3-1, E 1061-1-1, E 1061-1-3 and E 1067-1-2) which originated from seven landraces showed resistance to all prevalent European powdery mildew virulence genes. The most frequent score was 2 and 16 lines showed this reaction for inoculation with most isolates used. The distribution of reaction type indicated that about 77% of all reaction types observed were classified as powdery mildew resistance (scores 0, 1 and 2). In all lines the presence of unknown genes alone or in combinations with specific ones was postulated. Four different resistance alleles ( Mlat , Mla6 , Mla14 and Mla12 ) were postulated to be present in 10 tested lines alone or in combination. Alleles Mlat , Mla6 and Mla14 were postulated to be present in four and Mla12 in two tested lines, respectively. The value of barley landraces for diversification of resistance genes for powdery mildew is discussed.     

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.     

Interaction of a Blumeria graminis f. sp. hordei effector candidate with a barley ARF‐GAP suggests that host vesicle trafficking is a fungal pathogenicity target

Filamentous phytopathogens, such as fungi and oomycetes, secrete effector proteins to establish successful interactions with their plant hosts. In contrast with oomycetes, little is known about effector functions in true fungi. We used a bioinformatics pipeline to identify Blumeria effector candidates (BECs) from the obligate biotrophic barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh). BEC1–BEC5 are expressed at different time points during barley infection. BEC1, BEC2 and BEC4 have orthologues in the Arabidopsis thaliana‐infecting powdery mildew fungus Golovinomyces orontii. Arabidopsis lines stably expressing the G. orontii BEC2 orthologue, GoEC2, are more susceptible to infection with the non‐adapted fungus Erysiphe pisi, suggesting that GoEC2 contributes to powdery mildew virulence. For BEC3 and BEC4, we identified thiopurine methyltransferase, a ubiquitin‐conjugating enzyme, and an ADP ribosylation factor‐GTPase‐activating protein (ARF‐GAP) as potential host targets. Arabidopsis knockout lines of the respective HvARF‐GAP orthologue (AtAGD5) allowed higher entry levels of E. pisi, but exhibited elevated resistance to the oomycete Hyaloperonospora arabidopsidis. We hypothesize that ARF‐GAP proteins are conserved targets of powdery and downy mildew effectors, and we speculate that BEC4 might interfere with defence‐associated host vesicle trafficking.     

Dual role of nitric oxide in Solanum spp.–Oidium neolycopersici interactions

The role of nitric oxide in the pathogenesis of Oidium neolycopersici was studied on leaf discs of three Solanum spp. genotypes differing in their susceptibility to powdery mildew infection. The germination of pathogen conidia, development of infection structures and reaction of host tissues were compared for S. lycopersicum (susceptible), S. chmielewskii (moderately resistant) and S. habrochaites f. glabratum (highly resistant genotype) in presence of compounds modulating NO levels. The effect of NO donor sodium nitroprusside varied among genotypes and studied time intervals whereas NO scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide accelerated fungal development in all three Solanum spp. genotypes. The exposure of leaf discs to NOS inhibitor N^G-nitro-l-arginine methyl ester decreased powdery mildew growth namely in S. chmielewskii. Confocal laser scanning microscopy using the fluorescent probe 4-amino-5-(N-methylamino)-2′,7′-difluorofluorescein diacetate localised NO accumulation both in pathogen germ tubes and appressoria and in penetrated cells of resistant genotypes of S. chmielewskii and S. habrochaites f. glabratum. Our results confirm an essential role for NO in powdery mildew pathogenesis including the penetration of biotrophic pathogen and the initiation of hypersensitive reaction, and suggest the contribution of NO to molecular mechanisms of diversity in interactions of Solanum spp. with O. neolycopersici.     

The effectivity of Tilletiopsis albescens in biocontrol of powdery mildew

Tilletiopsis albescens grows well on powdery mildew fungi inoculated on barley or cucumber leaves and causes collapse of the colonies. Application of ballistospores or cut mycelium was equally effective for biocontrol, and the effectiveness tended to increase exponentially with the concentration of germinating units (conidia and cut mycelium) applied. Seventy percent relative humidity or more is required for effective biocontrol. Two applications of T. albescens in the period from 3 days before to 3 days after inoculation with powdery mildew were more effective than one. Applications before inoculation or 7 days after inoculation with powdery mildew had little effect. T. albescens followed the powdery mildew as it was disseminated to uninoculated leaves, but this did not result in an effective biocontrol. The potential for using T. albescens for biocontrol of powdery mildews is discussed.     

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating (‘resting’) conidia were avirulent except when an inoculation density of 1 × 10⁷ conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 × 10⁶ and 1 × 10⁷ per insect. An inoculation density of 1 × 10⁵ conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 × 10⁷ or 1 × 10⁶ conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 μm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60–90 h if infection densities of 1 × 10⁶ or 1 × 10⁷ activated conidia (pre-incubated for 2–3 h) per insect are employed.     

Polymorphic change of appressoria by the tomato powdery mildew Oidium neolycopersici on host tomato leaves reflects multiple unsuccessful penetration attempts

The appressorial shapes of the powdery mildews are an important clue to the taxonomy of the powdery mildew fungi, but the conidia of the tomato powdery mildew Oidium neolycopersici KTP-01 develop non-lobed, nipple-shaped, and moderately lobed or multilobed appressoria on the same leaves. To remove this ambiguity, we performed consecutive observations of sequential appressorial development of KTP-01 conidia with a high-fidelity digital microscope. Highly germinative conidia of KTP-01, collected from conidial pseudochains formed on the tomato leaves, were inoculated into host tomato and nonhost barley leaves or an artificial hydrophobic membrane (Parafilm). Events from germination initiation to appressorium formation were synchronous in all conidia on all materials used for inoculation, but post-appressorial behaviors varied among the materials. Appressoria on the membrane-stuck glass slide formed several projections at different portions of the appressoria to repeat unsuccessful penetration attempts. Similar unsuccessful penetration behavior by KTP-01 conidia was observed in the inoculations into leaves of barley plants, wild tomato species Lycopersicon peruvianum LA2172 (carrying the Ol-4 gene for powdery mildew resistance), and a susceptible host tomato (Lycopersicon esculentum) that had been inoculated with the barley powdery mildew (Blumeria graminis f. sp. hordei, race 1) conidia. On the barley leaves, all penetrations of KTP-01 were impeded by the papillae formed beneath the sites of the appressorial projections. On both the wild tomato and the race 1-inoculated cultivated tomato plants, KTP-01 conidia were prevented from forming functional haustoria by hypersensitive epidermal cell death; this hypersensitive reaction involved the Ol-4 gene in the wild tomato plants or the 'induced resistance' acquired by the nonpathogenic conidia previously inoculated into the cultivated tomato plants. All these KTP-01 conidia produced several projections on the appressoria during the repeated unsuccessful penetration attempts and eventually exhibited multilobed appressoria. On the host tomato leaves inoculated singly with KTP-01 conidia, fewer than 20% of the conidia located appressoria on the central part of target epidermal cells and succeeded in forming functional haustoria at the first penetration attempt without forming an appressorial projection. These conidia exhibited non-lobed appressoria. The remaining conidia, however, whose appressoria were located on/near the border of the target epidermal cells, were more likely to fail to penetrate at the first penetration, and then to develop additional projections for subsequent penetrations. Most conidia succeeded in forming functional haustoria at the second to fourth penetration attempts, but a few conidia failed to produce haustoria at all attempted penetrations. Eventually, the conidia that succeeded at the second penetration possessed a single appressorial projection (exhibiting the nipple-shaped appressoria), whereas the remaining conidia exhibited moderately lobed appressoria with two to four appressorial projections and multilobed appressoria, with more projections. Thus, the present study revealed that the basic shape of appressoria of KTP-01 was the non-lobed type, and that polymorphic changes of the appressoria occurred as a result of successive production of projections during repeated unsuccessful penetration attempts.     

Development of powdery mildew on leaves of several barley varieties at different growth stages

On detached leaves and intact plants of several barley varieties at different growth stages, lower percentages of germinated conidia of Erysiphe graminis f.sp. hordei penetrated the host and initiated infection on the abaxial than adaxial surface. More and larger E. graminis colonies developed on the adaxial surface and these comprised more densely packed hyphae and produced more conidiophores than did colonies on the abaxial surface. These results are consistent with the observation that there is usually more powdery mildew on the adaxial than abaxial surface of barley leaves in the field. Smaller proportions of germinated E. graminis conidia penetrated and infected the host on leaves of adult or near-adult plants than on those of seedlings or juvenile plants. Older plants also supported fewer, smaller and less dense colonies with less sporulation than young plants. The effects of growth stage of the host plant on development of powdery mildew were much greater in some barley varieties, and with some E. graminis isolates, than others.     

ROPGAPs of Arabidopsis limit susceptibility to powdery mildew

The barley ROP GTPase HvRACB is a susceptibility factor of barley to powdery mildew caused by the biotrophic fungus Blumeria graminis f.sp. hordei (Bgh). In a recent publication, we reported about a MICROTUBULE-ASSOCIATED ROP GTPASE-ACTIVATING PROTEIN 1 (HvMAGAP1) of barley. Transient-induced gene silencing or overexpression of HvMAGAP1 resulted in enhanced or reduced susceptibility to Bgh, respectively, indicating a possible HvRACB-antagonistic function of HvMAGAP1 in interaction with Bgh. HvMAGAP1 also influences the polarity of cortical microtubules in interaction with Bgh. In AtROPGAP1 and AtROPGAP4, Arabidopsis homologs of HvMAGAP1, knock-out T-DNA insertions enhanced susceptibility of Arabidopsis to the virulent powdery mildew fungus Erysiphe cruciferarum, indicating functions of ROPGAPs in pathogen interaction of monocots and dicots. Here we discuss the role of AtROPGAP1 and AtROPGAP4 in Arabidopsis pathogenesis of powdery mildew in some more detail.     

Mango powdery mildew Oidium mangiferae an alternative food for the predatory mites Typhlodromus mangiferus and Typhlodromips swirskii (Phytoseiidae) in absence or presence increasing prey density of Oligonichus mangiferus (Tetranychidae) in Egypt

The predacious mites, Typhlodromus mangiferus Zaher and El-Borolossy and Typhlodromips swirskii (Athias-Henriot), reproduced successfully on mango powdery mildew Oidium mangiferae Berthet in absence or presence of spider mite prey Oligonichus mangiferus (Rahman and Sapra) under laboratory conditions of 25 ± 1°C and 60–65% R.H. Adult female of both predators consumed protonymphs of O. mangiferus at different experimental densities. The consumption rate increased with increasing prey densities up to 25 protonymphs/female/day and decreased significantly at 35 and 50 protonymphs/female/day for the two predatory mites. Addition of powdery mildew conidia to each prey density significantly reduced consumption of spider mites at 35 and 50 protonymphs/female/day. Mean eggs/female/day by T. swirskii and T. mangiferus was 0.30 and 0.72 when reared on powdery mildew conidia compared with 1.64 and 1.57 when fed on powdery mildew and tetranychid prey, respectively. This increase in reproduction would have compensated the reduction in protonymph prey consumption due to the presence of mildew conidia. Mite–mildew interactions are discussed.     

Isofemale Line Analysis of Meloidogyne incognita Virulence to Cowpea Resistance Gene Rk

Isofemale lines (IFL) from single egg masses were studied for genetic variation in Meloidogyne incognita isolates avirulent and virulent to the resistance gene Rk in cowpea (Vigna unguiculata). In parental isolates cultured on susceptible and resistant cowpea, the virulent isolate contained 100% and the avirulent isolate 7% virulent lineages. Virulence was selected from the avirulent isolate within eight generations on resistant cowpea (lineage selection). In addition, virulence was selected from avirulent females (individual selection). Virulence differed (P ≤ 0.05) both within and between cohorts of IFL cultured for up to 27 generations on susceptible or resistant cowpea. Distinct virulence profiles were observed among IFL. Some remained avirulent on susceptible plants and became extinct on resistant plants; some remained virulent on resistant and susceptible plants; some changed from avirulent to virulent on resistant plants; and others changed from virulent to avirulent on susceptible plants. Also, some IFL increased in virulence on susceptible plants. Single descent lines from IFL showed similar patterns of virulence for up to six generations. These results revealed considerable genetic variation in virulence in a mitotic parthenogenetic nematode population. The frequencies of lineages with stable or changeable virulence and avirulence phenotypes determined the overall virulence potential of the population.     

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.     

Effect of three suppressors on the expression of powdery mildew resistance genes in barley

Three recessive mutagen-induced alleles that partially suppress the phenotypic expression of the semidominant powdery mildew resistance gene Mla12 have been studied. When each suppressor is present in homozygous condition, the infection type 0, conferred by gene Mla12 when homozygous, is changed to intermediate infection types. The three suppressor lines were crossed with seven near-isogenic lines with different powdery mildew resistance genes and one, M100, was crossed with nine additional lines. Seedlings of parents and from the F1and F2 generations were tested with powdery mildew isolates that possessed the appropriate avirulence and virulence genes. The segregation of phenotypes in the F2 generation disclosed that the three suppressors affected the phenotypic expression of three resistance genes, whereas that of four resistance genes remained unaffected. The suppressor in mutant M100 affected the phenotypic expression of 9 of the 10 additional resistance genes present. It is suggested that the three suppressors are mutationally modified genes involved in host defence processes. This implies that different resistance genes employ different, but overlapping, spectra of defence processes, or signal transduction pathways. Key words : barley, Hordeum vulgare, powdery mildew, Erysiphe graminis hordei, mutation, resistance, suppressor.