Browsing by Subject "Falscher Mehltau"

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Epidemiologische Aspekte der Falschen Mehltauinfektion durch Plasmopara viticola an Vitis
(2007) Keil, Sven; Spring, Otmar
The obligate biotrophic oomycete Plasmopara viticola (Berk. & Curt.) Berl. & de Toni causes downy mildew on grapevine. Plasmopara viticola is one of the economically most important pathogens in viticulture, with severe losses in yield of up to 70%. Existing prognosis models for plant protection in viticulture only allow yes/no statements on possible infection events in the vineyard. On the basis of these models, the severity of infections remain uncertain, although this represents a crucial point for an efficient application of fungicides. At low infestation severity, the application of protective fungicides at the end of the incubation period usually is sufficient. The low level of infestation can be tolerated and only further propagation of the pathogen must be prevented. In contrast, at high infestation severity curative fungicides have to be applied as soon as possible, because otherwise too much host tissue would be destroyed. Based on epidemiological studies and field experiments a prognostic concept has been designed, which enables the user to evaluate the relevance of infection events of grapevine downy mildew. This work has been carried out in the context of the research project ?Optimierung der Peronospora-Bekämpfung im Rebschutz auf der Basis eines erweiterten Prognosemodells (Forschungsvorhaben des Bundesministeriums für Ernährung, Landwirtschaft und Verbraucherschutz Nr. 514-33.54/01HS048)?. The developed concept was then integrated into the existing prognosis model and should support both consultants and winegrowers in using plant protection agents only in case of an expected increase of infestation frequency and severity. In this way, an effective and more economical use of fungicides is possible, which contributes to economic savings and reduces pesticide contamination of the environment. In the present study, aspects of sporangiogenesis, infestation of host tissue and hibernation, spreading of sporangia, interaction between vine leaves and sporangia as well as the climatic conditions during infection were analysed and evaluated with respect to the impact on epidemiology. The results improve existing literature data and deliver new insight to the epidemiology and biology of Plasmopara viticola.
Molekulare und biochemische Charakterisierung von NEP1 - ähnlichen Proteinen (NLPs) aus Plasmopara viticola
(2017) Schumacher, Stefan; Vögele, Ralf
Plasmopara viticola, the causal agent of grapevine downy mildew is one of the most important diseases in viticulture and leads to significant losses in crop in years with beneficial weather conditions. The molecular processes during the interaction between this pathogen and vine are yet poorly understood. Adopted pathogens achieve an infection by avoidance or suppression of plant innate immunity. This suppression takes place through pathogen secreted effector molecules, which can modulate plant defense mechanisms in all kinds of ways. One of these effector families are the necrosis and ethylene inducing peptide 1 – like proteins (NLP). These proteins occur in a vast variety of microorganisms and can on the one hand act as virulence factors and on the other hand induce a broad spectrum of defense responses in plants. In necrotrophic or hemibiotrophic pathogens these proteins are formed when the organism starts to feed from dead plant material. Beside these cytotoxic proteins many non-cytotoxic NLPs are known from hemibiotrophic or biotrophic microorganisms. However, the particular function of these NLPs is so far unknown. To date NLPs from Hyaloperonospora arabidopsidis, causal agent of downy mildew on the model plant Arabidopsis thaliana, are the only known example for these proteins from an obligate biotroph plant pathogen. These NLPs are not able to induce necrosis and their roll during infection by the pathogen is so far unknown. During this work two NLPs of complete size as well as one truncated version were identified in the genome of the obligate biotrophic oomycete Plasmopara viticola. During further experiments these proteins had been characterized by the use of molecular biological and biochemical techniques. The studies revealed a high degree of conservation of the corresponding genes isolated from several resistant and susceptible grapevine cultivars. Gene expression analysis showed high PvNLP expression during early time points of infection and even before first contact with host plant material, respectively. Necrosis-inducing activity of PvNLPs was neither observed in the model plant Nicotiana benthamiana nor in different susceptible and resistant Vitis species. To further investigate the reasons for the non-cytotoxic character of these proteins several experiments were conducted to clarify the relevance of different structural regions, their affinity to form homo- and hetero oligomers, as well as their subcellular localization. The crucial component for the lack of cytotoxicity was not identified. Neither the presence of a signal peptide nor a Nterminal region from another NLP with cytotoxic characteristics were able to form a necrosis inducing fusion protein with one of the identified NLPs from P. viticola. Formation of homodimers was observed for PvNLPs in vitro, but apparently does not occur during expression in planta. Furthermore PvNLPs are localized in the cytoplasm of N. benthamiana cells and show a possible association with the plant cell nucleus. This pattern of subcellular localization was also observed for a NLP with necrosis-inducing activity. The ability to induce plant innate immunity in Vitis could not be attested, suggesting a possible lack of the corresponding receptor in this plant genus. The results of this work further suggest a different role of non-cytotoxic NLPs which in P. viticola may fulfill a function during early infection stages ranging from zoospore release until the successful penetration of the host plant Vitis vinifera.
Plasmopara viticola, the downy mildew of grapevine : phenotypic and molecular characterization of single sporangium strains infecting hosts with different resistance levels
(2015) Gómez Zeledón, José Javier; Spring, Otmar
The downy mildew of grapevine, Plasmopara viticola, is one of the most important pathogens in viticulture. Its genetic diversity had been assessed in some previous studies using molecular markers, but the diversity of the infection behavior has not yet been addressed adequately. Therefore, the development of a fast, reliable and uncomplicated assay to screen for pathogen phenotypes on host with different resistance levels was a major task of this work. A leaf disc test was proposed, evaluating sporulation and necrosis produced by the pathogen on Vitis plants with different susceptibility. Using this bioassay, interesting strains were assessed and kept for future studies. The urgent need to work with genetic homogeneous inoculum was shown, because the assays revealed a high phenotypic diversity in isolates collected from the field as a bulk sample. Hence, a cloning technique to obtain single sporangium strains was found useful to avoid working with mixed genotypes. The leaf disc bioassay also allowed screening for fungicide resistance in P. viticola populations. Isolates resistant to dimethomorph and metalaxyl, two important fungicides for oomycetes control, were detected. Higher resistance was associated with fields were the fungicide application was high as well. Some strains were even resistant to doses where the fungicide exhibits phytotoxic activity to grapevine. The approach of characterizing P. viticola pathotypes on different host plants of Vitis vinifera cultivars and Vitis species from North America and Asia revealed a broad spectrum of fully susceptible to completely resistant reactions. This information is of direct practical value in future plant breeding programs, but also provides the chance to select specific host-pathogen combinations to study the mechanisms of resistance or susceptibility. Fluorescence microscopy revealed how the infection progress of highly and lowly virulent strains advance in tolerant and susceptible hosts, and which points of the infection are interesting for future studies. On the molecular level, effectors were investigated to trace their possible involvement in the infection process. It was found that RXLR 1, NLP 1, Elicitin like 2, Glucanase inhibitor 2 and 4 , and 1,3-ß Glucanase 2 are candidates which are upregulated in the earliest infection stages. Following the here established methodology and suggested strategy it should be possible in the future to get a better insight in the mechanisms of infection and resistance of grapevine downy mildew.
Das Potenzial von Falschem Mehltau als Quelle von Omega-3-Fettsäuren für die menschliche Ernährung
(2009) Anderle, Ann-Marie; Spring, Otmar
The absolute EPA ((5Z, 8Z, 11Z, 14Z, 17Z)-eicosapentaenoic acid) contents of the downy mildews of sunflowers (Plasmopara halstedii) and lettuce (Bremia lactucae) were quantified by means of GC-FID. The EPA content in sporangia of Bremia lactucae varied between 8 to 13 mg per dry weight. As at the institute of botany a collection of genetically identical sporangia strains of Plasmopara halstedii was established, the natural variation of EPA in four genetically different sporangia strains of Plasmopara halstedii on 10 different sunflower cultivars or -lines (15 days old seedlings) was investigated. The variance in EPA between the sporangia strains (LS-13.12.05-C6, BL-11.06.02-A4z, GG-16.10.97-A25, HE-10.01.06-A8) was only low (18-25 mg EPA per g dry weight). In contrast, the specific variation of EPA in infected sunflower seedlings (Giganteus, HA 821, HA 304, RHA 265, RHA 274, PM 13, 799-2, PM 17, 803-1, DM-2) was relatively high (0,28 to 1,10 mg EPA per g dry weight). Additionally three stages (minimum, optimum, maximum) of nitrogen fertilization were tested for their influence on the EPA content in infected sunflower seedlings. Statistical analysis was carried out by the program SAS. Analysis of variance based on F-tests and multiple t-tests. The influence of hydroponic fertilization (0.1 mM, 1 mM, 5 mM) on EPA in infected sunflower seedlings was high. The nitrogen dose of 5 mM almost doubled the EPA content in infected sunflower seedlings from 1 to almost 2 mg EPA per g dry weight. However, this content is not enough (by factor 10) to serve directly for human nutrition. Therefore at last a food chain trial with Bremia lactucae- infected lettuce was carried out in a cooperation project by several institutes of the Universtiy of Hohenheim. Infected lettuce was fed to hens. The omega-3-fatty acid content per egg was almost doubled (80 to 136 mg) if 10% lettuce was given to hen´s food. The infection with EPA containing downy mildew showed no effects.
Untersuchungen zum asexuellen Gentransfer bei biotrophen Oomyceten anhand der Fallbeispiele Plasmopara halstedii und Peronospora tabacina
(2009) Hammer, Timo; Spring, Otmar
Evidence for gene transfer during the asexual life cycle of certain biotrophic oomycetes was searched for in this study in order to evaluate the potential impact of such parasexual recombination on the variability of these important plant pathogens. Therefore, two case studies with Plasmopara halstedii, the causal agent of sunflower downy mildew, and with the tobacco blue mould pathogen, Peronospora tabacina, were conducted. Although the life cycles of both pathogens lack the possibility of genetic recombination, the organisms differ significantly in their variability. Using molecular methods, several indications for interspecific parasexual recombination between the near relatives Plasmopara halstedii and Plasmopara angustiterminalis were found, giving a possible explanation for the unexpected variability of these pathogens. Asexually formed offspring from dual infection experiments with the two Plasmopara species showed pheno- and genotypic parental traits in new combination and could be cultivated under double selective pressure. The recombinant strain ?R? was studied over 30 generations. Up to the 9th generation and after single sporangium infection, nuclear and mitochondrial traits of both parents were detected in ?R?, indicating a heterokaryon with nuclei and mitochondria of both parental strains. Starting with the 10th generation only fungicide-resistance remained as Pl. halstedii-specific trait, whereas all other detected signals were Pl. angustiterminalis-specific, which indicated true genetic recombination. As one possible mechanism for the genetic exchange it was shown that nuclei can be exchanged between neighboured hyphae via anastomoses and that more than one nucleus can be distributed into one developing sporangium. To prove anastomoses between the different Plasmopara species, specific optical labelling of the participating hyphae is a prerequisit. However, only transient expression of an optical marker gene was achieved, yet. Additional experiments for establishing a stable transformation system were conducted, but were not yet successful in selecting transgenic strains. In contrast to the study on Plasmopara, there was no evidence for recombination in Peronospora tabacina. Neither anastomoses nor heterokaryotic sporangia were found throughout the study. The results are concordant with the findings that tobacco blue mould shows very low variability and that only two phenotypes are known so far from studies in Europe. As the two types of the pathogen, which differ in fungicide sensitivity, did not interact during parallel infection of the same host tissue, they were characterized in detail. Several new pheno- and genotypic differences could be revealed, showing the genetic distance between the two types of the pathogen. A simple PCR detection system to differentiate the two genotypes was created and more than 50 European isolates of tobacco blue mould were monitored.
Verbreitung, Diversität und Übertragung des Mykovirus PhV und seine Auswirkung auf Plasmopara halstedii, den Falschen Mehltauerreger der Sonnenblume
(2015) Grasse, Wolfgang; Spring, Otmar
The Plasmopara halstedii Virus (PhV) is a ss(+)RNA virus with two segments. It occurs only in its host Plasmopara halstedii, the downy mildew pathogen of the sunflower. The two RNA strands encode for an RNA depending RNA Polymerase (RdRp) and a coat protein (CP), respectively. So far the phylogenetic analysis has shown similarities of the RdRp with the family of Nodaviruses while the CP seems to belong to the family of Tombusviruses. Phylogentic comparison based on both sequences now suggest a new clade, containing PhV and the Sclerophthora macrospora Virus A, which is basal to both mentioned virus families. Studies about diversity and the occurrence of PhV have shown that the virus existed in samples from 17 countries from five continents which were collected over the past 40 years. Its presence in more than 90% of these samples was documented. No correlation was found between the geographic origin and age of the samples, and presence or absence of PhV sequences. The calculated genetic diversity among all samples was surprisingly low. For 22 fully sequenced samples from 13 countries, only 18 SNP positions were reported. Genetic distances were extremely low with means of 0.001 for the RdRp and 0.002 for the CP. Investigations of the influence of PhV on the aggressiveness and pathogenicity of P. halstedii have shown a hypovirulent effect of the virus. In this study, isogenic strains of the Oomycete were infected with PhV and used for a series of bioassays on sunflowers. The production of sporangia was lowered by ca. 30% in case of virus presence and the latent period, i.e. the day of the first observed sporulation, was delayed by one day. The potential for systemic infections of the sunflower was also lowered by one third when PhV was present. Experiments to generate PhV by means of active cDNA clones in P. halstedii were performed with two different vectors and three transformation methods. It was shown that elctroporation techniques were useful to transport plasmids into the zoospores of P. halstedii and that the T7 promotor was able to start the transcription. The following generation of sporangia, however, lacked these sequences. This indicates that there was a transient transformation which produced PhV RNA sequences, but these sequences were unable to rebuild the virus itself.