Institut für Sonderkulturen und Produktionsphysiologie

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Untersuchungen zur physikalischen Kartierung des Genoms der Weinrebe
(2000) Böhm, Andreas; Blaich, Rolf
In this study research on physical mapping of the grapevine genome was done and a method for preparation of high molecular weight DNA from Vitis-protoplasts was established. In the first part of the project the usefulness of megarestriction-fragments for physical mapping in different grapevine-varieties was analyzed. Hybridization experiments with a probe for repetitive DNA showed that most of the restriction fragments were only about 200 Kb in length with a maximum of 400 Kb. Due to the distance of 300 Kb/cM in the genome of Vitis most fragments only cover a distance of less than 1 cM. For this reason physical mapping with megarestriction-fragments would be less successful. As an alternative strategy for physical mapping a genomic BAC-library was constructed for the variety 'Vidal blanc' which is less sensitive to fungal pathogens. 800 BAC-clones with an average insert size of about 49 Kb have been randomly picked and analyzed. After transforming the complete ligation-mixture 30000 single BACs are expected. They represent more than 3-fold genomesize of Vitis and each single-copy sequence would be contained in the library with a probability of 95 For a first screening of the partial library specific probes for genes from the metabolism of plants or for genes with correlation to pathogen resistance have been developed. The amplification of partial sequences from these Vitis-genes was done with specific oligonucleotides. The PCR-products have been cloned and sequenced. A special probe specific for chloroplast-DNA was used to detect BACs with inserts from the genome of chloroplasts. As a first result of colony-hybridization experiments eight clones with inserts of chloroplast-DNA could be identified. One BAC-clone contains a gene similar to an osmotin-like protein.
Untersuchungen zur Abundanz der Reblaus (Dactylosphaera vitifolii Shimer) und zur Nodositätenbildung in Abhängigkeit von Umweltfaktoren
(2000) Kopf, Andreas; Blaich, Rolf
The aim of the examinations was to investigate the abundance of Phylloxera (Dactylosphaera vitifolii Shimer), the occurrence of different biotypes of Phylloxera, the reaction of rootstocks to the infestation by Phylloxera and the influence of abiotic environmental conditions on the interaction between insect and plant. To investigate this interaction galls on rootlets (nodosities) and leaf galls were examined. The abundance of Phylloxera and the issue of the holocyclical reproduction in the wine region palatinate were evaluated in a field monitoring. In a special field trial the occurrence of different stages of Phylloxera and their damages on the rootstock were registered. With a dual aseptical in vitro system Phylloxera of different origins were examined on their aggressiveness to different varieties of rootstocks. In pot trials the influence of the type of soil and the effect of N-fertilization on the development of nodosities were investigated. The results of the examination show that Phylloxera can be found in nearly every part of the palatinate and that the improper cultivation of grafted rootstocks promotes the spreading of Phylloxera. Through shoots of rootstocks ? as they can be found in vineyards run wild - a holocyclical development of Phylloxera is made possible under appropriate climatical circumstances. Fitness, population dynamics of Phylloxera and the number of nodosities caused by the insects are correlating with their adaptation to a host rootstock. Pot studies have demonstrated that Phylloxera populations develop better in clay soil than sandy soil. High densities of Phylloxera in combination with a lack of N-supply increase a growth depression on grafted roots. It could also be proved that N-fertilization reduces the Phylloxera populations and the development of nodosities up to 98 %.
Mechanisms of frost adaptation and freeze damage in grapevine buds
(2002) Badulescu Valle, Radu Virgil; Blaich, Rolf
Mechanisms of frost hardening in compound (latent) buds of the grapevine cultivar ?Bacchus? were tested with different methods during three winters. The investigated parameters were LTE/HTE (low temperature exotherm/high temperature exotherm), water content, starch, sugar- and anions combination and bud histology. Water content from wood and buds was determined regularly every 2 weeks from March 1998 until Mai 2000. The lowest water content in wood and buds (about 40 %) was found between November and February. In general shoot sections and buds from the apical shoot area contained less water than in the basal area. Sugars and anions were analyzed with HPLC. The highest concentrations of soluble sugars were found in basal buds of the shoot, the lowest concentration in buds of the apical shoot area. Sucrose was the predominant soluble sugar, it was accompanied by glucose, fructose, sucrose, raffinose, and also stacchyose which was hitherto not described for grapevine buds. The concentration of soluble sugars increased during autumn and reached its maximum (around 150 mg/g dry matter) in November/December until the beginning of January then it decreased again to around 30 mg/g at bud burst. The predominant anion was sulphate while chloride could be detected only in traces. The anions reached their maximum at the beginning of January and in mid April. To evaluate the exotherm measuring method, model experiments were carried out with water drops (1µl) on filter paper and with small plant parts (leaf, stems, flower parts). Both the plant parts and the destilled water on the cellulose fiber freeze mainly between ?8 and ?15°C (an influence of the low osmotic value of the plant sap could not be found). After the first freezing the specimen were thawed and freezing repeated. The freezing points of the first and the second freezing cycle were significantly correlated. This shows that freezing does not occur at random, but is determined by ice nucleation sites characteristic for each sample. These sites even survive the physical destruction of the cells by the ice cristals. Further model experiments were carried out to get indications on possible barriers to ice cristal growth in plant tissue. Exotherm analysis was used to determine the freezing point of grapevine buds which is accompanied by a transient temperature rise called exotherm. The grapevine buds show 2 or more exotherms, one or two HTEs (high temperature exotherms) between ? 5 °C and ?10°C and the LTE (low temperature exotherm, sometimes more than one ) between ?10°C and ?25°C depending on the frost adaption of the buds. The HTEs are assumed to indicate the freezing of surface water or apoplastic water in the subtending tissue (bud pad), whereas the LTE (or LTEs) seem to be caused by freezing of the primary (and secondary) buds (shoot primordiy of the compound bud). The temperature minimum of the LTEs (down to ? 25 °C) is reached in January/February and is not influenced by humidity which, however, changes the THE values occuring usually around ? 10 ° and ? 4 °C, which are influenced by water in the bud scales. The LTEs of the buds in the lower area of the shoot were higher as compared to the buds in the middle and upper area of the shoot. The LTE analysis clearly shows the frost adaptation of the latent buds which usually reaches a maximum by the end of January but a clear relation to the changing air temperatures could not be established. Histological and cytological analyses were used to test for frost damage in bud parts and for changes during the cold adaptation. A modified staining method was developed to differentiate the cells. During automn and winter the buds contained a lot of starch grains which dissolved at bud burst. A permeability barrier between bud pad and shoot primordia could not be found, however it could be directly shown, that a HTE causes no cell damage in the buds, while after the appearence of the LTE(s) a disintegration of protoplasts in primary and secondary buds could be found. This is a direct evidence that LTEs indicates the death of the eyes in the complex grapevine bud. If after the appearance of the HTE the buds were held one day at this temperature before further cooling, no LTEs would appear. This and similar observations during the frost storage of grapevine cuttings is discussed in terms of the (harmless) ice formation in the bud base at moderate minus temperatures which would result in a freeze drying effect due to the lower water potential of the bud pad (in comparison to the non frozen eyes) and a further increase of the frost resistance of the growing points. If frost adapted grapevine shoots from the field were kept at 20°C deacclimation occurred after about 10 days. Accidentally wetted buds showed exotherms above ?4°C. In these buds and the watering water ice nucleating bacteria (Pseudomonas fluorescens) could be found.
Molekulare Analyse des Himbeerringflecken Nepovirus (RpRSV) und Herstellung eines Konstrukts zur Induktion von RpRSV?Resistenz in Reben
(2003) Ebel, Rainer; Reustle, Götz
The Raspberry Ringspot Nepovirus (RpRsV) is one of the viruses responsible for the fanleaf disease of grapevine. Two different serological strains of RpRSV exist: the grapevine strain (RpRSV-g) and the cherry strain (RpRSV-ch), which occurs in Germany and Switzerland. RpRSV has two RNAs (RNA1 and RNA2). Both have a genome-linked protein at the 5' ends and are polyadenylated at the 3' ends. RNA1 and RNA2 have one open reading frame flanked by 5' and 3' non-coding regions. The ORFs encode for one large polyprotein which is proteolytically cleaved in smaller functional proteins. The aims of the work were to produce RpRSV grapevine plants and the closer characterisation of RpRSV. The strategy was to create a gene construct, which should induce a gene silencing against the viruses in the grapevine rootstocks. Nicotiana benthamiana was chosen as a test system for the constructs. Because there was no sequence data available both strains were sequenced. The RpRSV strains were propagated in Chenopodium quinoa. The viral RNAs were purified, cDNA synthesized, cloned and sequenced. The sequences were compared and multiple alignments were performed. A sequence from the RNA2 3' non-coding region of RpRSV-ch was selected for the gene construct. An inverted repeat of this sequence was generateted, separated by a plant intron sequence. This construct under the control of a 35S promotor was cloned in the transformation vectors pPZPnptII (antibiotic resistance) and pPZPbar (herbicide resistance). Agrobacterium mediated transformation of Nicotiana benthamiana has been carried out. The T2 generation of the regenerated transgenic tobacco lines were tested for RpRSV resistance. Furthermore full-length clones of the RNA1 and RNA2 of RpRSV-g were produced. Therefore the full-length ds cDNA of both RNA strains were cloned under the control of a 35S promotor. Infection experiments in Chenopodium quinoa with the full clones have successfully been carried out.
Die Hypersensibilität der Europäischen Pflaume (Prunus domestica L.) gegenüber dem Scharkavirus (Plum pox virus)
(2005) Neumüller, Michael; Wünsche, Jens Norbert
In terms of economic damage, Sharka is the most important virus disease in stone fruit crops. The causative agent of this disease is the Plum pox virus (PPV). Some genotypes of European plum (Prunus domstica L.) show a hypersensitive response when infected with PPV. Results about the inheritance of this type of resistance mediated by hypersensitivity and different types of symptoms characteristic for the hypersensitive defence response in the interavtion PPV/Prunus domestica are presented. From the biochemichal point of view, it is shown that the defence response is indeed a hypersensitive response. A strategy for the development of DNA markers for hypersensitivity to PPV in European plum is developed. The potential use of hypersensitivity for solving the Sharka problem is discussed.
Entwicklung von „screening“-Methoden zur Analyse von PTGS-basierter Resistenz gegen Nepoviren in Pflanzen
(2006) Winterhagen, Patrick; Reustle, Götz
Nepoviruses are the causal agent of the fanleaf disease which leads to severe loss in viticulture (Raski et al., 1983). To induce virus resistance by post transcriptional gene silencing (PTGS) against Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV) and Raspberry ringspot virus (RpRSV), grapevine rootstocks were transformed with inverted repeat constructs or constructs containing sequences of the target virus and the defective interfering (DI) sequence of Tomato bushy stunt virus (Reustle et al., 2005). The induction und efficiency of PTGS by different constructs were investigated on the model plant Nicotiana benthamiana. Transgene-induced PTGS was demonstrated by the detection of small interfering (si)RNA in N. benthamiana. Using Agrobacterium for infiltration of a GFP-sensor construct, consisting of the GFP expression cassette and the sequence of the target virus, the efficiency of established transgene-induced PTGS was investigated. GFP-expressing plants accumulated mRNA of the sensor construct after the first day post infiltration in infiltrated leaves. After the second day the accumulation of siRNA with GFP- und virus-specific sequences was detected. In plants, which did not show any GFP-fluorescence after infiltration, GFP or viral sequence specific siRNA were not detectable. Generally, in virus resistant plants GFP-fluorescence was absent after infiltration. A correlation of virus resistance und accumulation of virus- or transgene-specific siRNA was not found. Several systems to evaluate PTGS and virus resistance in transgenic grapevine were tested. Transgenic grapevine did not accumulate transgene specific siRNA. An elevated resistance of transgenic grapevine was discovered by grafting experiments onto virus infected rootstocks. Whereas virus infected grapevine accumulated virus- and transgene-specific RNA and siRNA, in the non-infected grafts viral RNA was not detectable. Obviously, degradation of viral RNA in resistant grapevine und N. benthamiana was rapid und highly efficient without leading to accumulation of siRNA. However, due to the high inoculum pressure, grafting experiments are difficult to interprete and a possible field resistance against natural infection by the vector nematodes is probably not detectable. For investigation of PTGS in transgenic grapevine in vivo a system for vacuum infiltration to transfer the GFP-sensor construct into leaf tissue was established. For inoculation of grapevine using the natural GFLV vector nematode Xiphinema index an in vitro dual culture was developed. This space saving system allows analysis of resistance of grapevine under controlled conditions within a short time. An incubation time of about only six weeks was sufficient for the inoculation of control plants.
Development of a strategy to induce RNA-silencing in squash against virus diseases by genetic transformation
(2007) Khidr, Yehia; Reustle, Götz
Viral diseases of Cucurbits are an important limitation in the production of the crop. Zucchini yellow mosaic virus (ZYMV) and Watermelon mosaic virus-2 (WMV-2) are the most important squash (Cucurbita pepo L.) infecting viruses. Mixed infections with these viruses are deleterious for cucurbitaceous plants leading annually to significant yield losses world wide. All varieties of economical importance are susceptible for these viruses and classical breeding did not yield resistance. Therefore, a transgenic approach was chosen to induce resistance against both viruses by post-transcriptional gene silencing (PTGS). Highly conserved regions in the coat protein genes of ZYMV and WMV-2 were chosen to establish an inverted repeat construct. This construct was cloned into binary vector under control of the 35S promoter. Embryogenic callus was induced from different organs of three squash cultivars as target tissues for Agrobacterium transformation. The embryogenic callus was developed within 13-20 weeks incubation on MS medium containing different plant growth regulator combinations of auxin and cytokinin. Induction of embryogenesis in different explants ranged from 5 to 100 % depending on the organ and genotype used. Efficiency of embryo maturation, conversion and germination into entire plants from squash embryogenic callus was found to be callus age depended. Regeneration with young (2 months old) material was efficient, whereas regeneration of material maintained under in vitro conditions for more than 2 years was not possible. Agrobacterium-mediated transformation of squash embryogenic callus was established using transient GUS-gene expression. The highest transformation efficiency was obtained with the supervirulent ATHV strain, bacterial density of 0.85, washing procedure of the embryogenic material prior to Agrobacterium co-culture, application of 1 mM Acetosyringone in induction medium and sub-culturing of embryogenic callus on fresh MS medium 5-9 days prior co-culturing with the Agrobacterium. Selection strategy was optimized using GFP as reporter gene. For the genotype CX3006 300 mg/l Kanamycin showed the highest number of green areas but most efficient selection agent was Paromomycin 150 mg/l. For the genotype Dundoo 200 mg/l Paromomycin was the effective selection agent and showed the highest number of green areas. Selection of transformed calli could be efficient with the used selection agents but regeneration of transgenic plant was not possible because the old material was only one to be used for transformation experiments. It seems that these old materials may have lost their competency when they were maintained for long term in tissue culture. Therefore, the functionality of the inverted repeat construct was evaluated in Nicotiana benthamiana as a model plant. Transgenic lines were analyzed by PCR, Southern blot analysis and segregation analysis of T1 offspring. The transgene-induced PTGS in transgenic lines was confirmed by infiltration of GFP-sensor constructs containing viral derived sequences as silencing target and /or a construct containing the p19 silencing suppressor. In all transgenic lines tested, GFP fluorescence in infiltrated leaves was extinguished three days post-infection with GFP-sensor constructs. In contrast, all transgenic lines showed GFP fluorescence in infiltrated leaves when GFP-sensor constructs were co-infiltrated with a binary vector containing the viral silencing suppressor p19. With this work, tools have been developed to engineer virus resistance in squash. Using the optimized Agrobacterium-mediated transformation procedure together with the efficient RNA-silencing of the inverted inverted repeat construct and freshely induced embryogenic material it is quite possible to establish virus resistance in squash.
Molecular evidence of intraclonal variation and implications for adaptational traits of grape phylloxera populations (Daktulosphaira vitifoliae, Fitch)
(2007) Vorwerk, Sonja; Blaich, Rolf
Grape phylloxera (Daktulosphaira vitifoliae Fitch; Homoptera: Phylloxeridae) is an economical important insect pest of grapevine (Vitis spp.) worldwide. The insect was introduced with contaminated plant material from North America in the 1850s and spread rapidly across all European viticultural regions. In the 19th century, nearly three-forths of the ungrafted and highly susceptible European grape species were destroyed by the insect pest. European viticulture did not recover until the development of grafting, combining European Vitis vinifera varieties with resistant rootstocks, bred from American Vitis species. Grape phylloxera is still present in viticulture. Today, grape phylloxera populations mainly persist in abandoned vineyards and rootstock nurseries. Grape phylloxera populations seem to be variable in terms of genotypic composition and host adaptability. The lifecycle described by Fitch (1854) and others in the 19th century does not seem to match actual conditions anymore. This thesis aimed at redefining the genetic structure of European grape phylloxera populations by employing genetic markers. It was shown, that the insect has turned away from its classical holocycle and now mainly reproduces asexually, as already demonstrated for Australian grape phylloxera populations. Despite asexual reproduction, all examined populations revealed a high grade of genotypic diversity. The reports on the emergence of new and more aggressive strains raised the question, how a population composed of asexually reproducing organisms would change and adapt to such an extent. Using a multilocus marker system, eight single founder lineages were genetically monitored over at least 15 generations. All lineages revealed a high grade of intraclonal variation. Sequencing of polymorphic fragments showed, that the genetic variation was not due to contaminating plant or bacterial DNA, but was due to variation within the insect genome. Furthermore, mutations occured already in early generations and were not observed to accumulate in later generations. Mutations were rather generated constantly and only few mutation specific markers were identified to be stable over all following generations. The here documentated genetic variation reveals the great adaptational potential of this insect pest. The adaptability of single founder lineages was further assessed by measuring physiological parameters in single isolation chambers in the greenhouse. Parameters as the number of surviving individuals per generation, the number of eggs or the number of ovarioles per generation exposed differences in performance among the lineages and also within the lineages a high grade of intraclonal variation. A direct correlation of specific multilocus markers and particularly adapted individuals or lineages was not possible in this assay. Two markers, though, were observed to occure in several lineages which performed well on the new host plant. These markers may be a first step to the development of adaptation-related markers and need to be tested on further populations and host plants. When analysing intraclonal variation, the question of putative contaminating factors within the system arises. Symbiotic bacteria occuring in nearly all aphid species certainly are the first to be suspected as a source of genetic variation among single individuals tested. Endosymbiotic bacteria, as Buchnera aphidicola in other aphid species, influencing nutritional condition and fitness of the insect population, were not identified in D. vitifoliae. A bacterium, closely related to Pantoea agglomerans, however, was identified in several grape phylloxera populations, using universal 16S rDNA primers and later specifically developed markers, which were also employed for in situ hybridization. The bacterium was localized in the salivary pump of D. vitifoliae. PCR analysis of in vitro reared populations revealed that the bacterium is present in root- and leaf-feeding parthenogenetic populations of grape phylloxera and, moreover, seems to be transmitted from generation to generation. In other insect species, this bacterium has been demonstrated to produce antifungal and antibacterial substances, which were also found in first in vitro tests with grape phylloxera associated bacteria. The insect may benefit from the antagonistic potential of these bacteria. P. agglomerans may be a further participant in the certainly complex interaction of grape phylloxera and grapevine. This thesis represents a broad approach to elucidate the development of grape phylloxera populations in Europe. Using new molecular marker systems, it has become possible to gain more information on the genetic structure of the insect and its adaptational potential. The predominant clonal reproduction mode of the insect confronts grapevine breeders and pest management with the task to continously develop new resistant rootstocks and to keep up with new pest management systems.
Physiological responses of 'Jonagold' apple (Malus domestica Borkh.) following postharvest 1-Methylcyclopropene (1-MCP) application
(2009) Heyn, Claudia Susanne; Wünsche, Jens Norbert
Storage technologies such as controlled atmosphere (CA) storage and recently 1-Methylcyclopropene (1-MCP) treatments have led to an all-year-round global supply of high qualitative apple fruit. As a consequence, pressure of competition between several apple growing areas is increasing and in the same way consumers demands and expectations for apple fruit quality. However, throughout storage fruit quality is generally preserved at a high level whereas conditions at several points throughout the distribution chain are often not adequate for fresh commodities. It is critically important to maintain consistently high fruit quality throughout the marketing period to the final consumer and that fruit quality at the point of sale meets consumer requirements. Although decision for purchasing apple fruit is mainly due to appearance and firmness, consumer are increasingly concerned about nutritional quality and health-protecting compounds in foods. The plant hormone ethylene influences many of the ripening processes in climacteric fruit such as apple. Several storage conditions, such as reduced storage temperatures, controlled storage atmospheres with low O2- and elevated CO2-concentrations and recently 1-MCP treatments are known methods to minimize ethylene biosynthesis, ethylene sensitivity and responses of harvested climacteric fruit and by that to slow metabolic changes during ripening. 1-MCP is an effective tool for maintaining fruit quality during storage and post-storage handling. 1-MCP, a synthetic unsaturated cyclic olefin, is thought to act as a competitive substance to ethylene, occupying the ethylene receptor site so that ethylene cannot bind. In general, 1-MCP is able to counteract ripening effects triggered by ethylene during and after storage by blocking its action in fruit rather than inhibiting its production. The present research project consists of three studies. The aim of the first study was to determine the effect of 1-MCP treatment, storage condition and ?duration on apple fruit quality and consumer acceptability. The second part of the study focused on the effect of 1-MCP treatment, storage condition and ?duration on climacteric characteristics of apple fruit. The effect of 1-MCP treatment, storage condition and ?duration on antioxidant capacity of apple fruit was studied in the third part of the research. ?Jonagold? apple fruit were picked at commercial maturity in 2004, 2005 and 2006. Fruit were treated with 1-MCP on the day of harvest (0 days after harvest, 0 DAH) in 2004 and 7 DAH in 2005 and 2006 and stored the following day either in cold storage, CA- or ultra low oxygen- (ULO) storage. Fruit was held in cold storage prior to commencement of storage in 2005 and 2006. After 2, 4 and 6 months in 2004/05, 3, 6 and 9 months in 2005/06 and 3 and 5 months in 2006/07 fruit samples from each storage atmosphere ± 1-MCP were removed. Fruit quality parameters were assessed after harvest, commencement of storage and after each sample removal in 2004/05, 2005/06 and 2006/07 following 10 days shelf-life at 20°C. Consumer preference mapping was performed after 3 and 5 months of cold- and ULO-storage in 2006/07. Shelf-life respiration rate and fruit ethylene production was measured after harvest, commencement of storage and after each sample removal in 2004/05 and 2005/06, respectively. In 2005/06 ATP and ADP concentration was additionally determined. Nutritional quality and health-protecting compounds were examined by means of ascorbic acid concentration (L-AA), phenolic compounds and total non-enzymatic antioxidant capacity in 2005/06 following 10 days shelf-life after harvest, commencement of storage and after each sample removal. The results of the first part of the study showed that fruit quality generally decreased during storage and shelf-life depending on 1-MCP treatments, storage condition and ?duration. However, 1-MCP delayed ripening more and maintained fruit quality better than CA- or even ULO-storage alone. In consumer preference mapping most consumers, regardless of age or gender, preferred the 1-MCP treated fruit from ULO-storage. This effect was particularly seen when fruit were stored longer. Though sensory evaluation studies are time-consuming and there might be some flaws and difficulties to generate representative results from consumer taste panels, they are a useful tool to assess food quality and consumer preference. The results of the second part of the study proved that 1-MCP is a potent antagonist in terms of reducing and delaying ethylene production and respiratory rise. Although CA- and ULO-storage reduced ethylene production significantly in ?Jonagold? apples, 1-MCP treatment inhibited ethylene biosynthesis and accompanied respiration rate more than CA- and ULO-storage alone. The present study clearly shows that apple fruit shall be exposed as soon as possible to 1-MCP treatment and appropriate storage conditions after harvest for achieving a maximum effect on reduction of climacteric characteristics and maintenance of postharvest and post-storage apple fruit quality. L-AA concentration significantly decreased during storage, irrespective of storage condition and 1-MCP treatment. At commencement of storage L-AA concentration in 1-MCP treated fruit was higher than in untreated control fruit. However, following 9 months of storage L-AA concentration was lower in all 1-MCP treated fruit when compared with untreated fruit. Vitamin C equivalent phenolic concentration and vitamin C equivalent antioxidant capacity (VCEAC) decreased after 6 months of storage and gradually increased again after 9 months of storage. 1-MCP treatment had no effect on phenolics and VCEAC, respectively. In general, the results of the third part of the study showed that the nutritional value of apple fruit was not influenced by 1-MCP and storage condition.

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