Fungicide tolerance of Peronospora tabacina in German tabacco cultures of 2003

In einem Biomonitoring wurden 19 Infektionsereignisse des Tabakblauschimmels Peronospora tabacina während der Anbausaison 2003 dokumentiert. Die Verbreitung des Pathogens konnte zeitlich und geografisch erfasst werden, und der Ausgangpunkt ließ sich auf einen gemeinsamen Ursprung in Südbaden eingrenzen. Alle Isolate erwiesen sich in Testkonzentrationen bis 100 ppm als gleichermaßen Metalaxyl-resistent und waren genetisch von Feldproben aus dem Vorjahr nicht zu unterscheiden, so dass von einer Übertragung aus dem Befall von 2002 ausgegangen werden muss. Acht alternative Fungizide und Pflanzenstärkungsmittel wurden auf ihre Wirksamkeit gegenüber den Metalaxyl-toleranten Feldisolaten getestet.In a biomonitoring during the tobacco season 2003, 19 infection events of bluemould caused by Peronospora tabacina were documentated. Spreading of the pathogen was monitored temporary and geographically and allowed tracing the origin to a source in Südbaden. All isolates showed similar tolerance to Metalaxyl at doses up to 100 ppm and were genetically indistinguishable from samples of the previous year. This supports the assumption that the pathogen survived from previous years infection. Eight alternative fungicides and bio-activators were tested on their efficacy against the resistant isolates

Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora.

BACKGROUND: Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily. RESULTS: Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic. CONCLUSIONS: The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors

Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes

<p>Abstract</p> <p>Background</p> <p>Sesquiterpene lactones are characteristic metabolites of Asteraceae (or Compositae) which often display potent bioactivities and are sequestered in specialized organs such as laticifers, resin ducts, and trichomes. For characterization of sunflower sesquiterpene synthases we employed a simple method to isolate pure trichomes from anther appendages which facilitated the identification of these genes and investigation of their enzymatic functions and expression patterns during trichome development.</p> <p>Results</p> <p>Glandular trichomes of sunflower (<it>Helianthus annuus </it>L.) were isolated, and their RNA was extracted to investigate the initial steps of sesquiterpene lactone biosynthesis. Reverse transcription-PCR experiments led to the identification of three sesquiterpene synthases. By combination of <it>in vitro </it>and <it>in vivo </it>characterization of sesquiterpene synthase gene products in <it>Escherichia coli </it>and <it>Saccharomyces cerevisiae</it>, respectively, two enzymes were identified as germacrene A synthases, the key enzymes of sesquiterpene lactone biosynthesis. Due to the very low <it>in vitro </it>activity, the third enzyme was expressed <it>in vivo </it>in yeast as a thioredoxin-fusion protein for functional characterization. In <it>in vivo </it>assays, it was identified as a multiproduct enzyme with the volatile sesquiterpene hydrocarbon δ-cadinene as one of the two main products with α-muuorlene, β-caryophyllene, α-humulene and α-copaene as minor products. The second main compound remained unidentified. For expression studies, glandular trichomes from the anther appendages of sunflower florets were isolated in particular developmental stages from the pre- to the post-secretory phase. All three sesquiterpene synthases were solely upregulated during the biosynthetically active stages of the trichomes. Expression in different aerial plant parts coincided with occurrence and maturity of trichomes. Young roots with root hairs showed expression of the sesquiterpene synthase genes as well.</p> <p>Conclusion</p> <p>This study functionally identified sesquiterpene synthase genes predominantly expressed in sunflower trichomes. Evidence for the transcriptional regulation of sesquiterpene synthase genes in trichome cells suggest a potential use for these specialized cells for the identification of further genes involved in the biosynthesis, transport, and regulation of sesquiterpene lactones.</p

Identification of a novel Plasmopara halstedii elicitor protein combining de novo peptide sequencing algorithms and RACE-PCR

<p>Abstract</p> <p>Background</p> <p>Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires <it>de novo </it>peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete <it>Plasmopara halstedii</it>, we first evaluated the performance of three different <it>de novo </it>peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i).</p> <p>Results</p> <p>The performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set.</p> <p>All three <it>de </it>novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of <it>P. halstedii</it>. We found ten <it>de novo </it>sequenced peptides that showed homology to a <it>Phytophthora infestans </it>protein, a closely related organism of <it>P. halstedii</it>. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase.</p> <p>Conclusions</p> <p>Our study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different <it>de novo </it>peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in <it>P. halstedii</it>.</p

"Jumping Jack": Genomic Microsatellites Underscore the Distinctiveness of Closely Related Pseudoperonospora cubensis and Pseudoperonospora humuli and Provide New Insights Into Their Evolutionary Past

Downy mildews caused by obligate biotrophic oomycetes result in severe crop losses worldwide. Among these pathogens, Pseudoperonospora cubensis and P. humuli, two closely related oomycetes, adversely affect cucurbits and hop, respectively. Discordant hypotheses concerning their taxonomic relationships have been proposed based on host-pathogen interactions and specificity evidence and gene sequences of a few individuals, but population genetics evidence supporting these scenarios is missing. Furthermore, nuclear and mitochondrial regions of both pathogens have been analyzed using microsatellites and phylogenetically informative molecular markers, but extensive comparative population genetics research has not been done. Here, we genotyped 138 current and historical herbarium specimens of those two taxa using microsatellites (SSRs). Our goals were to assess genetic diversity and spatial distribution, to infer the evolutionary history of P. cubensis and P. humuli, and to visualize genome-scale organizational relationship between both pathogens. High genetic diversity, modest gene flow, and presence of population structure, particularly in P. cubensis, were observed. When tested for cross-amplification, 20 out of 27 P. cubensis-derived gSSRs cross-amplified DNA of P. humuli individuals, but few amplified DNA of downy mildew pathogens from related genera. Collectively, our analyses provided a definite argument for the hypothesis that both pathogens are distinct species, and suggested further speciation in the P. cubensis complex