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Browsing by Person "Spring, Otmar"

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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.
Die linearen Drüsenhaare der Sonnenblume : Morphologie, Verbreitung, Metabolitprofil und Sesquiterpenbiosynthese
(2014) Aschenbrenner, Anna-Katharina; Spring, Otmar
The sunflower H. annuus and related species of Heliantinae possess, in addition to the very intensively studied capitate glandular trichomes, a second type of multicellular and biosynthetically active trichome which was so far underinvestigated. In the present work, these linear glandular trichomes (LGT) were studied in detail. Microscopic investigations revealed a wide distribution of LGT within sunflower and species of the subtribe Heliantinae. LGT occurred on leaves and stems of sunflower, but the highest accumulation was found at the involucral bracts of the flower head. This suggests that they may play a chemoecological role in plant-insect or plant-herbivore interaction. The fast development of LGT within only 32h was found to take place during the emergence of leaf primordia on seedlings between the second and third day of germination. The biosynthetic activity got visible with the accumulation of substances successively in the tip cell and progressed towards the trichome base. The deposition of metabolites was accompanied by a loss of cellular vitality and was indicated by the degradation of the nucleus. Through examining the metabolite profile of LGT, it was revealed that they contain oxidized flavones. These compounds were subsequently isolated and structurally characterized. In addition, a wide variety of bisabolene-type sesquiterpenes was found. Among the identified structures were several Glandulones and Heliannuoles already known from previous metabolic studies of sunflower, but also several new compounds. Since the biosynthesis of these compounds from sunflower is fairly unknown, the focus of the present study was on the discovery and characterization of key enzymes that catalyze the first step of bisabolene biosynthesis. From the transcriptome of LGT, candidate genes for the formation of bisabolenes were identified by homology-based experiments. The complete sequences of the transcribed genes from cDNA was isolated by RACE experiments and confirmed by comparison with genomic sequences from the sunflower genome project. The functional identity of two candidate genes was achieved by means of heterologous expression in a yeast system specialized for the characterization of sesquiterpenes. The product of the transformed yeast cells was isolated and spectroscopically identified as cis-γ-bisabolene. Based on these experiments, these two enzymes could be identified as isoforms of a cis-gamma-bisabolene synthase, and represents the first known enzymes of this type characterized in sunflower.
Molekulare und entwicklungsbiologische Charakterisierung von Schlüsselenzymen der Naturstoffbiosynthese in Drüsenhaaren der Sonnenblume
(2008) Göpfert, Jens C.; Spring, Otmar
Glandular trichomes from anther appendages of sunflower were collected and their RNA was isolated. Sequence comparison with known plant sesquiterpene synthases was used to identify sunflower synthases in RT-PCR reactions. Three enzymes, HaGAS1, HaGAS2 and HaCS with high similarities to already characterized sesquiterpene synthases were identified. Their nucleotide sequences were completely established on the genomic level and as RNA transcripts. The nucleotide sequences as well as the deduced amino acid sequences showed typical characteristics of terpene synthases. In order to characterize the enzymes, the sesquiterpene synthase genes were cloned and expressed in E. coli. In vitro assays with the recombinant enzymes were carried out using the native substrate farnesyldiphosphate. The resulting products were extracted and analysed by GC-MS. They were identified by comparison of data base MS-data and using reference samples under identical analytical conditions. Two expressed enzymes, HaGAS1 and HaGAS2, synthesized germacrene A as a single product. Heterologous in vivo expression of both germacrene A-synthases in S. cerevisiae confirmed the in vitro result, since the analysis of the synthesized product showed a single germacrene A peak. Due to a very low in vitro activity of HaCS, the products of the third synthase could not be directly determined by MS-analysis. Therefore, the enzyme was expressed as a thioredoxin-fusion protein in vivo in transgenic yeast. This attempt resulted in a much higher rate of product yield. Two main and at least six minor products were traced in GC-analysis. They were confirmed as sesquiterpene hydrocarbons by GC-MS analysis. One of the two main products was identified as gamma-cadinene, whereas the second main peak could not be determined conclusively. Among the minor compounds alpha-copaene, alpha-muurolene und beta-caryophyllene were identified. Screening of a H. annuus EST library (established at the Berkeley Center for Synthetic Biology, University of California, Berkeley, USA) from mRNA of trichomes revealed the presence of a cytochrome P450 protein which showed high similarity to an Artemisia annua enzyme involved in artemisinic acid biosynthesis. This enzyme and another similar protein from Lactuca sativa were cloned and coexpressed with the germacrene A-synthase HaGAS2 in yeast. The resulting product was indirectly determined as germacrene A carboxylic acid using GC-MS analysis. These novel cytochrome P450 enzymes from sunflower and lettuce can be characterized as multifunctional germacrene A-monooxygenases. They catalyse a three-step oxidation leading from germacrene A to germacrene A carboxylic acid. This oxidation process represents an essential step towards the biosynthesis of sesquiterpene lactones. Semiquantitative RT-PCR analysis demonstrated that the expression of all three sesquiterpene synthases and the sunflower P450 monooxygenase occurred directly within trichome cells. The expression was highly upregulated during the secretory stage of the capitate glandular trichomes. This developmentally regulated expression was shown for the first time in trichomes. Additionally to sesquiterpene synthase activity in trichomes of anthers and leaves, it also was detected in sunflower roots. In addition, 5-deoxynevadensin was identified as a new constituent of the glandular trichomes of sunflower. This 5-deoxy-flavone is responsible for the bright blue fluorescence of sunflower trichomes detected by fluorescence microscopy. The newly identified component may act as protectant for the STL against UV-degradation.
Neue Cytochrom P450 Enzyme des Sesquiterpenlacton Stoffwechsels der Sonnenblume (Helianthus annuus L.)
(2016) Frey, Maximilian; Spring, Otmar
In the present work additional steps towards the elucidation of the biosynthetic pathway of H. annuus sesquiterpene lactones (STL) were achieved. Firstly candidate sequences were retrieved from a transcriptome database by filtering according to expression pattern and similarity to P450 enzymes known to participate in STL biosynthetic pathways. Open reading frames (ORFs) were obtained using 3´-and 5´-RACE-PCR. Previously described and newly identified candidate genes were then transformed in yeast vectors and expressed in combination with different substrate vectors. A high throughput micro approach was developed that allowed the expression and analysis of many yeast strains at the same time. For the transient expression in N. benthamiana the genes of known and putative enzymes were introduced via Agrobacterium mediated transformation. Using the in planta expression system the complete STL pathway of sunflower to costunolide was reconstructed de novo in a step-by-step approach. Previously described Michael-addition reactions of α-methylene-γ-lactone type STL to the thiol group of cysteine or glutathione in tobacco expression systems could be observed for all STL investigated. Chemically synthesized STL adducts were used as reference for the identification of in planta produced STL adducts. Enzyme characterization was conducted in two different in vivo expression systems, in yeast (Saccharomyces cervisiae) and tobacco (Nicotiana benthamiana). For the investigated biosynthetic pathway, differences between these two expression systems were discussed. Candidate gene M4 showed an unexpected product in yeast (farnesyl-δ-lactone) and led in combination with HaG8H to the production of costunolide. In the plant expression system, germacrene A acid was converted to costunolide by M4 in the absence of HaG8H. In both cases, M4 was involved in the synthesis of costunolide and should therefore be assigned Helianthus annuus costunolide synthase the underlying reaction mechanism should however be investigated more thoroughly. Helianthus annuus costunolide 14-hydroxylase HaC14H (candidate M33) was characterized in yeast and tobacco. A classification into subfamily CYP71CB, together with Tp8878 the Tanacetum parthenium costunolide/parthenolide 3β-hydroxylase is proposed. It was shown that in planta the main product of HaG8H exists most likely as inunolide, which would be the entry point for the biosynthesis of 8-epixanthatine and tomentosine. Candidate S2 from Ikezawa et al. (2011) was found to convert 8β-hydroxy-germacrene A acid to 8β-hydroxy-costunolide (eupatolide) in tobacco, but not in yeast, producing several byproducts. The name Helianthus annuus eupatolide synthase HaES is proposed accordingly. HaES has 47 % amino acid identity to the parthenolide synthase from T. parthenium (TpPTS). A classification into a new CYP71 subfamily is proposed. Two alternative metabolic routes led to 8β-hydroxy-costunolide in the expression studies in tobacco, the underlying mechanisms are discussed. Enzymes involved in STL biosynthesis were expressed in inner tissues of young Helianthus annuus plants; the induction of expression of STL biosynthesis enzymes in leaf primordia correlates with the development of capitate glandular trichomes (CGT) and STL synthesis. HaC14H was found in a chromosomal region in proximity to several P450 enzyme candidates, that share the same subfamily and the expression in capitate glandular trichomes (CGT). Therefore involvement of these enzymes in later steps of the biosynthesis of the elaborate STL structures found in CGT is likely.
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.
Strategies and mechanisms of cellular interaction between the parasitic weed Orobanche cumana WALLR. and its host Helianthus annuus L.
(2020) Krupp, Anna Clarissa; Spring, Otmar
Sunflower broomrape, Orobanche cumana WALLR., is a root parasitic plant causing considerable yield losses in sunflower cultivation in Europe, North Africa and Asia. Comprehensive knowledge about early interaction stages between host and parasite is necessary to find new ways of controlling this weed. In this thesis, three aspects regarding the biology of O. cumana were studied: 1) the chemotropism of O. cumana germtubes which bend towards the host root, 2) the development of O. cumana on resistant and susceptible sunflower lines and 3) the development of the phloem connection between the O. cumana haustorium and the sunflower host root. Sesquiterpene lactones in sunflower root exudates act as germination stimulants for O. cumana. As sesquiterpene lactones are known inhibitors of plant elongation growth and seem to play a role in the phototropic curvature of sunflower hypocotyls, a chemotropism bioassay on water agar was established to test if they also serve as chemotropic signals for the host-finding of O. cumana germtubes. When sesquiterpene lactone containing sunflower root exudate, sunflower seed oil extract or the sesquiterpene lactone reference costunolide were applied on filter discs, 70 % of the germtubes showed orientation towards them. The artificial strigolactone GR24, however, did not induce chemotropism. A concentration gradient of sesquiterpene lactones exudated from the host root is likely to be responsible for a stronger inhibition of elongation growth on the host-facing flank of the germtube. This would confer a double role of sesquiterpene lactones from root exudates in the sunflower-broomrape-interaction, namely as germination stimulants and as chemotropic signals. One way of controlling O. cumana is the cultivation of resistant sunflower lines. However, this resistance is rapidly overcome by more aggressive pathotypes of the parasite. Therefore, the resistance or tolerance reaction of the sunflower genotype T35001 was investigated in comparison to six other sunflower genotypes with different resistance characteristics. The development of O. cumana was monitored in a root chamber system which allowed permanent assessment of germination, attachment and tubercle formation in the different host-parasite-combinations. All seven tested sunflower lines induced germination and attachment of O. cumana, independent of the expected resistance or susceptibility of the host. A difference between compatibility or incompatibility of the interactions was only observed at the tubercle stage. On T35001, tubercles never occurred, neither in root chamber nor in pot experiments. To find out why the development stopped before the tubercle stage, samples of sunflower roots with attached O. cumana seedlings were analysed by bright field-, fluorescence- and transmission electron microscopy. Histological studies revealed that O. cumana penetrated the host root, but never reached the host’s vascular bundle. The root cortex cells surrounding the Orobanche haustorium showed no ultrastructural changes such as cell wall thickening. Fluorescence microscopy revealed no callose depositions or signs of phytoalexin release. However, ultrastructural examination of the host-parasite-interface showed degeneration processes in both cortex and haustorial cells. Cortex cells were flooded with bacteria, haustorium cells showed degeneration of cytoplasm and nuclei. The resistance mechanism that prevented further development of the O. cumana haustorium did not express itself in a histologically visible way. As holoparasite, O. cumana acquires its entire demand for water, minerals and organic nutrients from the host’s vascular system. The development of the xylem connection between O. cumana and sunflower had previously been reported, but the phloem connection is far more relevant for the parasite in terms of organic nutrients. Accordingly, the ultrastructure of the phloem connection between the haustorium of young O. cumana tubercles and the sunflower root was examined. Parasite and host tissues were intermingled at the contact site and difficult to distinguish, but sieve-tube elements of O. cumana and sunflower could be differentiated according to their plastid ultrastructure. While sieve-element plastids of O. cumana were larger, often irregular in shape and contained few, small starch inclusions, sieve-element plastids of the host were significantly smaller, always round with more and larger starch inclusions. This made it possible to trace the exact contact site of host and parasite sieve elements to show a direct symplastic phloem connection between the two species. The interspecific sieve plate showed more callose on the host side. This allowed detection of newly formed plasmodesmata between host sieve-tube elements and parenchymatic parasite cells, thus showing that undifferentiated cells of the parasite can connect to fully differentiated sieve elements of sunflower.
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.

Browsing by Person "Spring, Otmar"

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