Variability of the wheat powdery mildew pathogen Blumeria graminis f. sp. tritici in the 2003 crop season

Wheat (Triticum aestivum) powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici, is one of the most severe foliar diseases attacking this crop, reducing grain yields by 10% to 62% in Brazil. The disease can be controlled by genetic resistance of the host, but the pathogen has physiological specialization, which enables it to infect wheat cultivars that have remained resistant for years. The objective of this work was to evaluate the variability of pathogenic strains of B. graminis f. sp. tritici collected in Brazil and the effectiveness of wheat resistance genes to powdery mildew in the 2003 crop season. Plants of a differential series were inoculated with each monopustular isolate. Thirty-one combinations of effective and ineffective resistance genes were identified. Only the gene Pm4a+... remained totally effective to all isolates, and gene Pm6 was highly effective (below 10% of susceptibility), whereas genes Pm3a and Pm8 were totally ineffective (susceptible to all isolates). Genes Pm3c, D1, and D2 showed low effectiveness (above 50% of susceptibility), and genes Pm1, 2, 4a, 1+?, and 2+Mld had mean effective results to most strains (susceptibility between 10% and 49%). The virulence formula Pm1, 3c, 4a, 6, 1+?, 2+Mld, 4a+..., D2 (effective genes) / 2, 3a, 8, D1 (ineffective genes) was most frequently found, accounting for 15% of the occurrences. The most frequent number of ineffective genes was seven, ranging from three to ten.

Sensibilidade de Blumeria graminis f.sp. tritici a alguns fungicidas

Em experimentos conduzidos no campo, na safra 2000, avaliou-se a sensibilidade do oídio do trigo nas cultivares de trigo (Triticum aestivum) BR 23 e OR 1 a alguns fungicidas. As avaliações da intensidade da doença foram feitas com base na incidência, severidade e área abaixo da curva de progresso da doença, realizadas aos sete, 14 e 21 dias após a pulverização dos fungicidas. O fungo agente causal do oídio, Blumeria graminis f.sp. tritici, mostrou-se sensível aos fungicidas sistêmicos em ambas as cultivares. A maior sensibilidade foi ao fungicida triadimenol, considerando-se os valores absolutos na porcentagem de controle da doença. O enxofre, pelo curto período de proteção, não tem potencial de uso em trigo. Os danos no rendimento de grãos foram de 32% e 79% respectivamente para 'BR 23' e 'OR 1'. O controle do oídio, mesmo em cultivares altamente suscetíveis, como 'OR 1', pode ser eficientemente realizado com fungicidas sistêmicos recomendados pela pesquisa, não sendo detectada insensibilidade do fungo aos mesmos. No entanto, deve-se evitar o uso de sub-dose e controlar a doença com a incidência recomendada pela pesquisa com base no limiar de dano econômico.

Efetividade de genes de resistência de trigo a oídio

Levantamentos periódicos da freqüência de virulência do patógeno causador de oídio (Blumeria graminis f. sp. tritici) em trigo (Triticum aestivum)auxiliam na escolha de fontes de resistência para uso em cruzamentos. Este trabalho relata resultados de cinco anos de verificação de efetividade de genes de resistência para populações patogênicas de B. graminis f. sp. tritici oriundas do Brasil e do Chile. Amostras de oídio foram recebidas pela Embrapa Trigo, Passo Fundo, RS, em 1995, 1996, 1997, 1999 e 2000. Inoculou-se cada isolado monopustular em plantas de trigo da série diferencial para raças. Foram identificadas 90 combinações diferentes de genes efetivos e inefetivos para resistência. Em 1995 e 1997, a maioria dos isolados foram virulentos com relação aos genes Pm3a, Pm3c, Pm8, Pm1+... e PmD1; em 1999, para Pm3a, Pm3c, Pm4b, Pm8, PmD1 e PmD2; e, em 2000, para Pm3a, Pm3c, Pm4b, Pm6, Pm8, PmD1 e PmD2. Em todos os anos analisados, permaneceram efetivos a todos os isolados os genes Pm2 e Pm4a+.... O número de genes inefetivos por isolado variou entre um e nove, no total de 13 genes ou combinações de genes testados. Isolados apresentando virulência a cinco genes ou combinações foram mais freqüentes em 1995 e em 1997, a seis em 1999 e a sete em 2000, o que pode significar aumento na complexidade da composição racial do patógeno a cada ano.

Extratos de Lentinula edodes e Agaricus blazei sobre Bipolaris sorokiniana e Puccinia recondita f. sp. tritici, in vitro

Com o objetivo de buscar medidas alternativas para o controle de Bipolaris sorokiniana e Puccinia recondita f. sp. tritici testou-se o efeito fungitóxico in vitro dos cogumelos Lentinula edodes e Agaricus blazei sobre esses fungos. Os extratos brutos aquosos de ambos os cogumelos não tiveram efeito significativo tanto no crescimento micelial quanto na germinação de esporos de B. sorokiniana. Por outro lado, os extratos dos cogumelos inibiram a germinação de esporos de P. recondita f. sp. tritici, com destaque para o isolado LE 96/17 de L. edodes que apresentou inibição da ordem de 52,4%.

Sensitivity reduction in Blumeria graminis f. sp. hordei to triadimenol fungicide applied as barley seed treatment

Experiments were carried out in a growth chamber with controlled temperature and photoperiod to test two populations of Blumeria graminis f. sp. hordei from Guarapuava, Paraná State, and Passo Fundo, Rio Grande do Sul State, Brazil. Treatments consisted in application of the fungicide triadimenol (Baytan 150 SC®) at three rates of its commercial formulation: 150, 250, 350 mL/100 Kg barley seeds. The experiments were conducted separately in a growth chamber for each population, adopting the same temperature and photoperiod. For inoculation, pots containing barley seedlings colonized by the fungus were placed among the plots. After emergence of the first symptoms, the disease severity was assessed at two-day intervals. The experiments were repeated twice for each fungus population. Data were expressed as area under the disease progress curve and as powdery mildew control by comparing the severity after the fungicide treatments to that of control. Data were subjected to analysis of variance and regression analysis; the area under the disease progress curve was also calculated. Comparing the data obtained in the present study with those reported in the literature and the control, the maximum value of 26.1% is considered insufficient to prevent the damages caused by the disease. The control response to the fungicide rate was significant. We can conclude that there was a reduction in the sensitivity of both B. graminis f.sp. hordei populations to the fungicide triadimenol, which explains the control failure observed in barley farms.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen blumeria graminis f. sp hordei

To further our understanding of powdery mildew biology during infection, we undertook a systematic shotgun proteomics analysis of the obligate biotroph Blumeria graminis f. sp. hordei at different stages of development in the host. Moreover we used a proteogenomics approach to feed information into the annotation of the newly sequenced genome. We analyzed and compared the proteomes from three stages of development representing different functions during the plant-dependent vegetative life cycle of this fungus. We identified 441 proteins in ungerminated spores, 775 proteins in epiphytic sporulating hyphae, and 47 proteins from haustoria inside barley leaf epidermal cells and used the data to aid annotation of the B. graminis f. sp. hordei genome. We also compared the differences in the protein complement of these key stages. Although confirming some of the previously reported findings and models derived from the analysis of transcriptome dynamics, our results also suggest that the intracellular haustoria are subject to stress possibly as a result of the plant defense strategy, including the production of reactive oxygen species. In addition, a number of small haustorial proteins with a predicted N-terminal signal peptide for secretion were identified in infected tissues: these represent candidate effector proteins that may play a role in controlling host metabolism and immunity. Molecular & Cellular Proteomics 8: 2368-2381, 2009.

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.

Physiologic races of Puccinia recondita f. sp. tritici in the United States from 1926 through 1960

Bibliography: p. 18.

Identifying races of Puccinia graminis f. sp. avenae; a modified international system

Bibliography: p. 19-23.

Pathogenic specialization in cereal rust fungi, especially Puccinia recondita f. sp. tritici: concepts, methods of study, and application

Issued in cooperation with Kansas Agricultural Experiment Station.

Analyse transcriptomique de l'interaction tripartite "Pseudozyma flocculosa-Blumeria graminis f.sp. hordei-Hordeum vulgare"

Afin d’améliorer nos pratiques agricoles dans le contexte d’une agriculture durable, plusieurs agents de lutte biologique (ALB) ont été développés, testés et sont maintenant utilisés dans le monde pour combattre les pertes de rendements causées par les maladies. Blumeria graminis f. sp. hordei ( Bgh) est l’agent pathogène responsable du blanc de l’orge et peut réduire les rendements de cette culture jusqu’à 40%. Un champignon épiphyte, Pseudozyma flocculosa, a été découvert et identifié en 1987 en association étroite avec le blanc du trèfle. Les chercheurs ont alors remarqué que ce champignon exhibait une forte activité antagoniste contre le blanc en détruisant les structures de l’agent pathogène. Suite à d’autres travaux, il est apparu que ce comportement antagoniste était dirigé contre tous les membres des Erysiphales et semblait lié à la synthèse d’un glycolipide antifongique soit la flocculosine. Toutefois, on n’est toujours pas parvenus à associer l’efficacité de l’ALB avec la production de ce glycolipide. Ces observations suggèrent que d’autres facteurs seraient impliqués lorsque les deux protagonistes, l’ALB et le blanc, sont en contact. L’objectif principal de ce projet était donc de chercher d’autres mécanismes moléculaires pouvant expliquer l’interaction P. flocculosa-blanc et orge, en faisant une analyse transcriptomique complète des trois protagonistes en même temps. L’interaction tripartite a été échantillonnée à différents temps suivant l’inoculation de P. flocculosa sur des feuilles d’orge présentant déjà une intensité de blanc d’environ 50%. Les échantillons de feuilles prélevés ont ensuite été utilisés pour l’extraction de l’ARN qui ont été ensuite transformés en ADNc pour la préparation des librairies. Cinq répliquats ont été effectués pour chaque temps et le tout a été séquencé à l’aide de séquençage par synthèse Illumina HiSeq. Les séquences obtenues (reads) ont ensuite été analysées à l’aide du logiciel CLC Genomics Workbench. Brièvement, les séquences obtenues ont été cartographiées sur les trois génomes de réf��rence. Suite à la cartographie, les analyses d’expression ont été conduites et les gènes exprimés de façon différentielle ont été recherchés. Cette étape a été conduite en portant une attention particulière aux gènes codant pour un groupe de protéines appelées CSEP pour “candidate secreted effector proteins” qui seraient possiblement impliquées dans l’interaction tripartite. Parmi les protéines exprimées de façon différentielle en présence du blanc ou en absence de ce dernier, nous avons pu constater que certaines CSEP étaient fortement exprimées en présence du blanc. Ces résultats sont prometteurs et nous offrent une piste certaine pour l’élucidation des mécanismes impliqués dans cette interaction tripartite.

Analysis of epidermis- and mesophyll-specific transcript accumulation in powdery mildew-inoculated wheat leaves.

Powdery mildew is an important disease of wheat caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici. This pathogen invades exclusively epidermal cells after penetrating directly through the cell wall. Because powdery mildew colonizes exclusively epidermal cells, it is of importance not only to identify genes which are activated, but also to monitor tissue specificity of gene activation. Acquired resistance of wheat to powdery mildew can be induced by a previous inoculation with the non-host pathogen B. graminis f. sp. hordei, the causal agent of barley powdery mildew. The establishment of the resistant state is accompanied by the activation of genes. Here we report the tissue-specific cDNA-AFLP analysis and cloning of transcripts accumulating 6 and 24 h after the resistance-inducing inoculation with B. graminis f. sp. hordei. A total of 25,000 fragments estimated to represent about 17,000 transcripts were displayed. Out of these, 141 transcripts, were found to accumulate after Bgh inoculation using microarray hybridization analysis. Forty-four accumulated predominantly in the epidermis whereas 76 transcripts accumulated mostly in mesophyll tissue.

Efeito do tratamento de sementes com fungicida sobre o controle de doenças na parte aérea do trigo

O controle das doenças foliares do trigo (Triticum aestivum) através do tratamento de sementes foi avaliado em Passo Fundo, RS em 1999. A cultivar usada no experimento foi OR 1, conhecida por sua suscetibilidade ao oídio e à ferrugem da folha. Todos os fungicidas diferiram (p > 0,05) da testemunha, não tratada em rendimento de grãos (REND) e peso do hectolitro (PH). Os maiores rendimentos foram obtidos nos tratamentos fluquinconazole + procloraz nas doses de 250,0 g + 51,0 g e 125,0 g + 25,0 g (2.943 kg/ha e 2.864 kg/ha), 132 e 125 %, respectivamente, superiores ao rendimento da testemunha (1.270 kg/ha). Os fungicidas triazóis indicados pela pesquisa (triticonazole, difenoconazole e triadimenol) produziram 1.908 kg/ha, 1.821 kg/ha e 1.815 kg/ha, respectivamente e não diferiram entre si. Não foram observados efeitos fitotóxicos dos tratamentos de sementes com os diferentes fungicidas e doses em teste. O controle do oídio (Blumeria graminis f. sp. tritici) e da ferrugem da folha (Puccinia triticina) foi efetivo até aos 98 DAE (dias após a emergência), a partir do qual atingiu-se o limiar indicado para pulverização na parte aérea recomendado. Aos 108 DAE os fungicidas começaram a perder a persistência e aos 118 DAE, as severidades médias de oídio e ferrugem, dos diferentes tratamentos, foram de 6,52% e 33,90% e o da testemunha de 16,5% e 78,0%, respectivamente. O tratamento de sementes com fungicidas como demonstrado nesse trabalho, quando associado com rotação cultural e cultivares mais resistentes, poderá ter importante papel no controle das doenças foliares do trigo reduzindo o custo e a poluição ambiental.

Eficiência e persistência de fungicidas no controle do oídio do trigo via tratamento de sementes

O oídio do trigo, causado por Blumeria graminis f.sp. tritici, ocorre com alta freqüência e intensidade em cultivares suscetíveis, na região Sul do Brasil. Em experimento conduzido em casa-de-vegetação, avaliou-se a eficiência e o período de proteção dos fungicidas difenoconazole (15 SC 100, 200 e 300 mL), flutriafol (12,5 SC 200, 300 e 400 mL) triadimenol (15 SC 200, 300 e 400 mL) e triticonazole (20 SC 135, 270 e 400 mL/ 100 kg de sementes), em três doses da formulação comercial, aplicados via tratamento de semente para o controle do agente causal do oídio. Sementes de trigo, cultivar BR 23, suscetível à doença, foram tratadas com os fungicidas e semeadas em vasos plásticos, contendo como substrato uma mistura de solo, areia e vermiculita. Vasos contendo plantas de trigo, contendo sinais do fungo, foram colocados entre os tratamentos para servir como fonte de inóculo primário. As avaliações foram realizadas diariamente, no afilho principal, desde o surgimento dos primeiros sinais. Utilizou-se como critério de quantificação da doença a incidência de folhas infectadas, até que a planta estivesse com a quarta folha totalmente expandida. Curvas ajustadas segundo o modelo logístico foram obtidas para cada fungicida em suas três doses. O período de proteção conferido pelos fungicidas foi menor para o produto difenoconazole, aumentando o período com o flutriafol e, posteriormente, com o triticonazole. O maior período de proteção foi obtido com o fungicida triadimenol.