Browsing by Subject "Sonnenblume"

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Der Einfluss von Ölgehalt und Fettsäuremuster auf die Lagerfähigkeit von Saatgut
(2007) Ghiasvand Ghiasi, Kambiz; Kruse, Michael
Seed storage with the objective of maintaining the quality for the later sowing is a constant challenge, not only in gene banks but also in agriculture, forestry and horticulture. In 1980 ELLIS and ROBERTS established ?The improved seed viability equation? for the prediction of the storability of seeds. With this equation the loss-of-germination-curve can be computed as a function of the initial seed germination, the storage temperature and the seed moisture content for each species. However, with oil-rich seeds the equation very often misses its aim. With this seeds, the variety and lot specific oil content considerably determines water activity and aging rate. Therefore the objective of the present work was to describe the influence of oil content on the aging behavior of seeds of the oil crops during storage quantitatively, to integrate this influence in the most reasonable way into the viability equation and to improve accuracy of its prediction. The investigation was carried out with storage experiments under controlled conditions in the laboratory at higher temperatures and running times between a few days and six months. First it was examined, whether the usually determined oil content of the entire seed is an informative parameter for water activity in the embryo axis. Experiments with sunflowers with an oil content between 28 and 48 % showed that oil-rich seeds need an about 1 % higher seed moisture content than seeds with lower oil content to have the same water activity in the embryonic axis. The storage of these seeds as well as the seeds of rape with oil contents between 39 and 50 % and flax with oil contents between 36 and 43 % showed that the loss of germination is more consistent with uniform water activity than with uniform seed moisture content. This has not been taken into account in the previous viability equation, so that its prediction contains systematic errors. Therefore extensive storage experiments were carried out with altogether 28 seed lots of rape, sunflower, flax and corn with different moisture contents and a uniform temperature of 45°C. Only for few of the stored lots the prediction of the seed viability equation was found to be correct. To include the oil content into the seed viability equation eight different suggestions were compiled. These were applied in three nonlinear regression models with different restrictions to the results of the storage experiments. The first model permitted the species specific determination of the weighing factors (constants) for the seed viability equation. The second model only allowed to determine the weighing factors for the absolute term and the seed moisture content specifically. Oil content was provided however with a species-nonspecific weighing factor. In the third model all weighing factors were species-nonspecific determined. All eight suggestions achieved better estimations for the aging rate in the species-specific models than the previous viability equation. Where this could be examined statistically, the improvements were significant. The same was found for the models with species-nonspecific weighting factors for the oil content. However, not all suggestions led to a converging result of the regression analysis. All examined species-nonspecific models did not improve the adjustment compared to the previous viability equation. The suggestions were then validated with a new and independent dataset with a storage temperature of seeds of 32°C. It was shown that the change of the temperature reduced the accuracy of the estimations of the nonlinear regression models. The new suggestions nevertheless corresponded better to the observed results than the previous viability equation. Based on these results a suggestion was then selected for the extension of the viability equation by ELLIS and ROBERT, which does not introduce a new weighing factor to the equation as the weighing factor might potentially contribute to a decrease of the precision of a prediction due to its standard error. Finally it was examined whether the fatty acid composition before storage in addition to the oil content affects the aging rate and whether the change of the fatty acid composition is directly connected to the loss of germination during storage. Only with rape, significant relations between the proportion of the fatty acid 14:0, 18:0, 20:0, 22:0 and the aging rate of the seed lots were determined. A uniform change of the fatty acid composition of all examined species could not be observed. Therefore this characteristic could not contribute to the further improvement of the prediction accuracy of the seed viability equation. To summarize, a suggestion to include the oil content into the viability equation was designed that clearly improves the accuracy of the prediction of the viability equation for oil-rich seeds and that contributes to a more appropriate and efficient storage of seeds.
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.
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.
QTL mapping of resistance to Sclerotinia sclerotiorum (Lib.) De Bary in sunflower (Helianthus annuus L.)
(2005) Micic, Zeljko; Melchinger, Albrecht E.
Sclerotinia sclerotiorum (Lib.) de Bary is one of the most important pathogens of sunflower. Three different disease symptoms can be caused by S. sclerotiorum: Sclerotinia wilt, midstalk- and head rot. An improvement of the resistance against S. sclerotiorum would contribute to yield security and thus increase the profitability of sunflower cultivation. We investigated resistance to midstalk rot with respect to the prospects of marker-assisted selection (MAS). The bjectives were to (1) identify quantitative trait loci (QTL) involved in resistance against Sclerotinia sclerotiorum, (2) map their position in the genome, (3) characterize their gene effects, and (4) estimate their consistency across generations of the cross NDBLOSsel x CM625. Two sunflower lines with high resistance level to S. sclerotiorum and different genetic origins (NDBLOSsel and TUB-5-3234) were used as parents. They were crossed with a highly susceptible line CM625 to develop two mapping populations. A modified leaf test was used for the evaluation of midstalk-rot resistance. Three resistance traits and two morphological traits were measured. Disease resistance of 354 F3 families of the population NDBLOSsel x CM625 was screened in field trials with two different sowing times in 1999. A total 317 recombinant inbred lines (RIL) derived from F3 families were tested in 2002/2003. The 434 F3 families of cross CM625 x TUB-5-3234 were screened in 2000/2001. The field trials were conducted by using generalized lattice designs with three replications and five infected plants per replication. Highly significant genetic variation between F3 families and RIL was observed for the resistance traits in all field trials. Heritabilities ( ) were highest for stem lesion and lowest for leaf lesion for all three experiments. The resistance traits were moderately correlated with each other. For the construction of the genetic map of population NDBLOSsel x CM625, 352 F2 individuals were analyzed with 117 SSR marker loci. On the basis of results from the QTL mapping in F3 families, 41 markers were selected and genotyped in 248 RIL. A "selective genotyping" (SG) approach was used for population CM625 x TUB-5-3234. Based on the results measured in F3 families for stem lesion, the SSR genotype at 72 marker loci was determined for the 60 most resistant and 60 most susceptible F2 individuals. For QTL mapping and estimation, the method of the "composite interval of mapping" was used. For stem lesion in the population NDBLOSsel x CM625, eight QTL were detected explaining 33.7% of the genetic variance ( ). The QTL on LG8 explained 36.7% of the phenotypic variance (R2adj). All other QTL for this trait explained between 3.3 and 6.0% of R2adj. Nine QTL were detected for leaf lesion. The proportion of the phenotypic variance explained by individual QTL ranged from 3.4 to 11.3%. All detected QTL for leaf lesion explained 25.3% of the genetic variance in cross validation. For speed of fungal growth, 6 QTL were detected, which explained from 4.6 to 10.2% R2adj. Cross validation explained 24.4% of. Most QTL showed additive gene action. QTL occurring consistently across generations can be recommended for MAS and therefore, the QTL results between RIL and F3 families of population NDBLOSsel x CM625 were compared. One common QTL was identified for leaf lesion, two for stem lesion and three for speed of fungal growth. In population CM625 x TUB-5-3234, four QTL for stem lesion, three QTL for leaf lesion and three QTL for speed of fungal growth were identified. Owing to the SG approach we conjecture that not all QTL were found. The comparison of QTL results between two F3 populations showed two common QTL for stem lesion on LG4 and LG8. The QTL on LG4 originated from the susceptible parent CM625. The QTL on LG8 probably corresponds to the QTL with the largest effect determined in the population NDBLOSsel x CM625. Regarding MAS, our results indicate that two QTL detected for stem lesion and speed of fungal growth in population NDBLOSsel x CM625 are promising. They were consistent across environments, and showed no adverse correlation to leaf morphology in trials with the RIL. In mapping population CM625  TUB-5-3234, it remained unclear whether TUB-5-3234 can contribute new alleles with sufficiently large effects for resistance that were not identified in line NDBLOSsel and would be useful in MAS. The genomic region on LG10 should be analyzed in more detail with respect to its importance for resistance in multiple plant parts (head and stalk) and to verify its association with leaf morphology. Resistance breeding of sunflower against S. sclerotiorum is difficult due to the complex inheritance of the trait. This study showed that both the resistance source NDBLOSsel and the identified markers are promising in improving resistance by MAS. For a broader resistance against S. sclerotiorum, it is necessary to detect new resistance genes from different sources to pyramide them in elite lines.
Socio-economic evaluation of sunflower agri-food chains in Brazil in view of the potential implementation of innovative plant protein ingredients for human consumption
(2018) Sousa, Lucas Oliveira de; Berger, Thomas
This study aimed at performing a socio-economic analysis of an agri-food chain focused on a non-established crop in view of the potential implementation of food innovations, using sunflower agri-food chains in Brazil and upcoming sunflower high-quality food protein ingredients as a case study. Thus, fieldwork was carried out in the main sunflower-producing areas between April and August 2016 for data collection among sunflower chain agents from the input, farming, and processing segments, besides representatives from the research sector. Section 1 applied a multiple case study embedded design to describe and analyze the dynamics of operation of sunflower agri-food chains in Brazil. The analysis followed a theory-driven approach based on concepts from transaction costs economics and the social network approach. The findings indicated an environment of high transaction costs, in which the economic transactions are ruled by formal and relational governance structures, and made possible through knowledge diffusion, under the coordination of a processing company. Nevertheless, the sustainable long-term operation of the sunflower chains is constrained by typical limitations of non-established crops, such as restricted market structure, land use competition with well-established crops, and technological limitations regarding plant breeding, and control of pests and diseases. Moreover, Section 1 revealed that a farmer-led sunflower chain in the state of Mato Grosso (MT) stood out regarding the operational stability, suggesting a closer analysis of this farmers’ collective endeavor, which was performed in the subsequent section. Thus, Section 2 adopted a single case study embedded design to describe and analyze the establishment process of the leading Brazilian sunflower agri-food chain located in MT under the regime of farmers. The analysis followed a framework that regarded the agri-food chain establishment as an entrepreneurial process. The findings indicated that the process of establishment of this sunflower chain has been a complex social-economic endeavor stemming from a set of interconnected driving forces composed of entrepreneurial skills, social network, resource availability, and crop suitability. Furthermore, Section 2 suggested the existence of a supportive institutional environment for the establishment of new sunflower agri-food chains in MT among soybean farmers, besides indicating the need of examining the potential for sunflower production expansion in MT, which was the focus of the next section. Thus, Section 3 applied an integrated assessment approach that combines an agent-based model (ABM) with a crop growth model to investigate the potential for sunflower land use expansion in double-cropping systems prevailing in MT. The ABM was implemented using the software package Mathematical Programming-based Multi-Agent Systems (MPMAS), and the crop yields simulations were implemented using the process-based model for nitrogen and carbon in agro-ecosystems (MONICA). The findings indicated the existence of a potential for the expansion of the sunflower production in MT. Nevertheless, this potential is constrained by the distance between the producing areas and the processing facilities. Moreover, the simulations confirmed the land use competition between sunflower and maize, showing that sunflower land use is strongly associated with agents’ expectations regarding prices and yields of sunflower and maize. However, the results also revealed a complementary effect between these two crops due to the different water deficit tolerance of these crops. Section 3 also highlighted that the simulated potential production of sunflower would require further increases in the current processing capacity installed in MT. To conclude, the analyses performed in Sections 1, 2, and 3 indicated relevant aspects to be considered by innovators interested in implementing food innovations related to non-established crops. The scarcity of feedstock suppliers requires the adoption of contractual and relational governance structures coupled with the provision of technical assistance at the farming level. Moreover, farmers with a recognized professional and social reputation as well as leadership abilities play an important role in influencing other farmers to adopt a non-established crop. Finally, the suitability of the crop for the agricultural system prevailing in the region is essential for ensuring a minimum level of farmers’ willingness to adopt a non-established crop. In this regard, particular attention should be given to the land use competition with well-established crops.
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.