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 [7]. 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.

The influence of powdery mildew infection on photosynthesis, chlorophyll fluorescence, leaf chlorophyll and carotenoid content of three woody plant species

The effect of powdery mildew development on photosynthesis, chlorophyll fluorescence, leaf chlorophyll and carotenoid concentrations on three woody plants frequently planted in urban environments was studied. Rates of photosynthetic CO2 fixation were rapidly reduced in two of the three genotypes tested prior to visible signs of infection. Effects on chlorophyll fluorescence (Fo, Fv/Fo, Fv/Fm), leaf chlorophyll and carotenoid content were not manifest until >25 per cent of the leaf area was observed to be covered by mycelial growth indicating reduced photo-synthetic rates during the early stages of infection were not due to degradation of the leaf chloroplast structure. Observation of the fluorescence transient (OJIP curves) showed powdery mildew infection impairs photosynthetic electron transport system by reducing the size but not heterogeneity of the plastoquninone pool, effecting both the acceptor and donor side of photosystem II. Impairment of the photosynthetic electron transport system was reflected by reduced values of a performance index used in this investigation as a measure of photochemical events within photosystem II electron transport. In addition interpretation of the fluorescence data indicated powdery mildew infection may impair the photo-protective process that facilitates the dissipation of excess energy within leaf tissue.

Relationships between Oak powdery mildew incidence and severity and commensal fungi

Oak (Quercus robur) powdery mildew is a common and damaging fungal disease. In a local survey at Reading, UK, oak powdery mildew was common on trees of all height classes but was most common on trees of 3-9m. A variety of other fungal species were commonly found growing in association with oak powdery mildew colonies. The abundance of such fungi was estimated through stratified sample surveys for 2.5 years. The taxa most commonly associated with oak powdery mildew were Acremonium sp., Trichoderma sp., Ampelomyces/Phoma sp. and Leptosphaerulina australis. Nearly 90% of mildew colonies were associated with L. australis, which is not generally considered as a mycoparasite or antagonist, in contrast with the other three fungi. Abundance varied between June and October surveys. Acremonium sp. abundance was greater in summer samplings whereas L. australis and Trichoderma sp. abundances were greater in autumn samplings. Ampelomyces/Phoma sp. was never observed in the absence of powdery mildew. Relationships between the mildew-associated fungi and oak powdery mildew appeared curved and differed significantly between sampling years. L. australis was positively correlated with the other three associated fungi studied when powdery mildew was also present. The variety and high population densities of the mildew associated fungi suggest that they may be important in determining the final density of oak mildew and the damage caused by it.

Global transcriptome analysis and identification of differentially expressed genes after infection of Fragaria vesca with powdery mildew (Podosphaera aphanis)

Background: Podosphaera aphanis, the causal agent of strawberry powdery mildew causes significant economic loss worldwide. Methods: We used the diploid strawberry species Fragaria vesca as a model to study plant pathogen interactions. RNA-seq was employed to generate a transcriptome dataset from two accessions, F. vesca ssp. vesca Hawaii 4 (HW) and F. vesca f. semperflorens Yellow Wonder 5AF7 (YW) at 1 d (1 DAI) and 8 d (8 DAI) after infection. Results: Of the total reads identified about 999 million (92%) mapped to the F. vesca genome. These transcripts were derived from a total of 23,470 and 23,464 genes in HW and YW, respectively from the three time points (control, 1 and 8 DAI). Analysis identified 1,567, 1,846 and 1,145 up-regulated genes between control and 1 DAI, control and 8 DAI, and 1 and 8 DAI, respectively in HW. Similarly, 1,336, 1,619 and 968 genes were up-regulated in YW. Also 646, 1,098 and 624 down-regulated genes were identified in HW, while 571, 754 and 627 genes were down-regulated in YW between all three time points, respectively. Conclusion: Investigation of differentially expressed genes (log2 fold changes �5) between control and 1 DAI in both HW and YW identified a large number of genes related to secondary metabolism, signal transduction; transcriptional regulation and disease resistance were highly expressed. These included flavonoid 3´-monooxygenase, peroxidase 15, glucan endo-1,3-β-glucosidase 2, receptor-like kinases, transcription factors, germin-like proteins, F-box proteins, NB-ARC and NBS-LRR proteins. This is the first application of RNA-seq to any pathogen interaction in strawberry

Interactions between the powdery mildew effector BEC1054 and barley proteins identify candidate host targets

There are over 500 candidate secreted effector proteins (CSEPs) or Blumeria effector candidates (BECs) specific to the barley powdery mildew pathogen Blumeria graminis f.sp. hordei. The CSEP/BEC proteins are expressed and predicted to be secreted by biotrophic feeding structures called haustoria. Eight BECs are required for the formation of functional haustoria. These include the RNase-like effector BEC1054 (synonym CSEP0064). In order to identify host proteins targeted by BEC1054, recombinant BEC1054 was expressed in E. coli, solubilized, and used in pull-down assays from barley protein extracts. Many putative interactors were identified by LC-MS/MS after subtraction of unspecific binders in negative controls. Therefore, a directed yeast-2-hybrid assay, developed to measure the effectiveness of the interactions in yeast, was used to validate putative interactors. We conclude that BEC1054 may target several host proteins, including a glutathione-S-transferase, a malate dehydrogenase, and a pathogen-related-5 protein isoform, indicating a possible role for BEC1054 in compromising well-known key players of defense and response to pathogens. In addition, BEC1054 interacts with an elongation factor 1 gamma. This study already suggests that BEC1054 plays a central role in barley powdery mildew virulence by acting at several levels.

Genetics of resistance to powdery mildew (Microsphaera diffusa) in Brazilian soybean populations

Crosses between resistant and susceptible soybean cultivars were performed and the F2 populations were obtained to study the inheritance of soybean resistance to powdery mildew and to estimate the number and action of genes related to resistance. The reaction to powdery mildew was studied in a greenhouse and pots carrying plants with symptoms were distributed among the pots carrying the genotypes to be tested as a source of inoculum. Individual plants were scored according to the method of Yorinori (1997), with modifications, and classified as resistant or susceptible. The results showed that adult soybeans plants can present resistance to powdery mildew, which is controlled by one major gene with a dominant effect.

Validation of microsatellite markers for assisted selection of soybean genotypes resistant to powdery mildew

Powdery mildew is one of the most serious diseases of soybean and is found in all producing countries. The purpose of this study was to validate microsatellite markers previously identified as associated with resistance to powdery mildew in soybean. The study was conducted in two F, parent populations with contrasting resistance to powdery mildew, In the analysis 10 SSR primers were used for the populations. and tour polymorphic markers were identified for cross I (MGBR95-20937 x IAC-Foscarin 31) and three for cross 2 (MGBR-46 x Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) 48). The Chi-square analysis of the phenotypic evaluation confirmed the expected segregation (3: 1) of a dominant gene related to resistance. The polymorphic markers also segregated as expected (1:2:1). The markers Sat 366 and Sat 393 in the crosses 1 and 2. respectively, located at 9.41 and 12.45 cM from the gene. were considered promising for marker-assisted selection for resistance to powdery mildew in soybean. at a selection efficiency of 92.7% and 60.3% respectively.

Inheritance of resistance to powdery mildew in pea and pathogenesis-related aspects

A herança da resistência ao oídio na cultivar de ervilha MK-10 e alguns aspectos histológicos da infecção foram estudados. Para o estudo da herança, as gerações F1, F2, retrocuzamentos e geração F3 de MK-10 com duas populações suscetíveis foram avaliadas. Nas avaliações histológicas observou-se a porcentagem de conídios germinados, porcentagem de conídios que formaram apressório, porcentagem de conídios que estabeleceram colônia e número de haustórios por colônia. Para comparar as razões de segregação obtidas no estudo da herança da resistência, adotou-se o teste do Qui-quadrado (X²) e para os dados das análises histológicas, utilizou-se o teste Tukey a 5% de probabilidade. Concluiu-se que a resistência de MK-10 ao oídio é devida a um par de alelos recessivos e que a resistência é expressa na fase de pré-penetração, completada por uma morte celular localizada pós-penetração, característica da presença do par de alelos recessivos er1er1.

Molecular phylogenetic and morphological analyses of Oidium heveae, a powdery mildew of rubber tree

Powdery mildew of rubber tree caused by Oidium heveae is an important disease of rubber plantations worldwide. Identification and classification of this fungus is still uncertain because there is no authoritative report of its morphology and no record of its teleomorphic stage. In this study, we compared five specimens of the rubber powdery mildew fungus collected in Malaysia, Thailand, and Brazil based on morphological and molecular characteristics. Morphological results showed that the fungus on rubber tree belongs to Oidium subgen. Pseudoidium. Nucleotide sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region and the large subunit rRNA gene (28S rDNA) were conducted to determine the relationships of the rubber powdery mildew fungus and to link this anamorphic fungus with its allied teleomorph. The results showed that the rDNA sequences of the two specimens from Malaysia were identical to a specimen from Thailand, whereas they differed by three bases from the two Brazilian isolates: one nucleotide position in the ITS2 and two positions in the 28S sequences. The ITS sequences of the two Brazilian isolates were identical to sequences of Erysiphe sp. on Quercus phillyraeoides collected in Japan, although the 28S sequences differed at one base from sequences of this fungus. Phylogenetic trees of both rDNA regions constructed by the distance and parsimony methods showed that the rubber powdery mildew fungus grouped with Erysiphe sp. on Q. phillyraeoides with 100% bootstrap support. Comparisons of the anamorph of two isolates of Erysiphe sp. from Q. phillyraeoides with the rubber mildew did not reveal any obvious differences between the two powdery mildew taxa, which suggests that O. heveae may be an anamorph of Erysiphe sp. on Q. phillyraeoides. Cross-inoculation tests are required to substantiate this conclusion. © The Mycological Society of Japan and Springer-Verlag 2005.

Obtaining resistant lettuce progenies to downy mildew

A alface é a hortaliça folhosa mais consumida no Brasil. No entanto, a dificuldade em produzi-la vem aumentando, principalmente pela infestação das áreas de produção por Bremia lactucae, sendo o uso de cultivares com resistência horizontal, a alternativa mais viável no controle da doença. Diante do exposto, o objetivo do presente trabalho foi obter progênies de alface crespa resistentes às raças de míldio SPBl:01, SPBl:02, SPBl:03, SPBl:04, SPBl:05, SPBl:06 e SPBl:07. O trabalho de melhoramento consistiu de duas etapas: cruzamento dos parentais para obtenção das progênies de alface crespa resistentes e teste de resistência das progênies às raças de B. lactucae. Os parentais utilizados na obtenção das progênies resistentes foram Argeles e linhagem JAB 4-13-7, visando a obtenção de progênies de alface do tipo crespa, com os fatores de resistência R-18 e R-38. Para tanto, adotou-se o método genealógico, tendo como padrão, para as seleções, a cultivar Hortência e o genótipo JAB 4-13-7. Após a seleção e autofecundação das plantas no campo, efetuou-se o teste de resistência ou suscetibilidade, por meio da inoculação nas progênies oriundas dos cruzamentos, de uma mistura de água destilada + esporângios de B. lactucae das raças SPBl:01, SPBl:02, SPBl:03, SPBl:04, SPBl:05, SPBl:06 e SPBl:07 obtidas de isolados coletados nos anos de 2008 a 2010. Quinze dias após a inoculação, as plântulas foram selecionadas, descartando aquelas que possuíam esporulação e pontos necróticos causados por B. lactucae. Pelo método genealógico, selecionaram-se 69 progênies F3 com boas características agronômicas. No entanto, após o teste de resistência ou suscetibilidade, somente 19 apresentaram todas as plantas resistentes ao míldio.

Effect of potassium silicate on epidemic components of powdery mildew on melon

The effects of silicon (Si) supplied in the form of potassium silicate (PS) were evaluated on epidemic components of powdery mildew of melon under greenhouse conditions. The PS was applied to the roots or to leaves. In the first experiment, epidemic components were evaluated after inoculation with Podosphaera xanthii. In the second experiment, the disease progress rate was evaluated on plants subjected to natural infection. The area under the disease progress curve was reduced by 65% and 73% in the foliar and root treatments, respectively, compared to control plants, as a consequence of reductions in infection efficiency, colony expansion rate, colony area, conidial production and disease progress rate. However, root application of PS was more effective than foliar application in reducing most of the epidemic components, except for infection efficiency. This can be explained by the high Si concentration in leaf tissues with root application, in contrast to the foliar treatment where Si was only deposited on the external leaf surfaces. The effects of PS reported in this study demonstrated that powdery mildew of melon can be controlled, and that the best results can be achieved when PS is supplied to the roots.

Inheritance of resistance to powdery mildew in pea and pathogenesis-related aspects

The inheritance of resistance to powdery mildew in the pea cultivar MK-10 and some histological aspects of infection were assessed. For the inheritance study, F1, F2, backcrosses and F3 generations of MK-10 crossed with two susceptible populations were evaluated. Histological evaluations included percentage of germinated conidia, percentage of conidia that formed appresoria, percentage of conidia that established colonies, and number of haustoria per colony. Segregation ratios obtained in the resistance inheritance study were compared by Chi-square (ײ) test and the histological data were analyzed by Tukey's test at 5% probability. It was concluded that resistance of MK-10 to powdery mildew is due to a pair of recessive alleles since it is expressed in the pre-penetration stage and completed by post-penetration localized cellular death, characteristic of the presence of the pair of recessive alleles er1er1.