Anna Kicherer

An automated field phenotyping pipeline for application in grapevine research.

Due to its perennial nature and size, the acquisition of phenotypic data in grapevine research is almost exclusively restricted to the field and done by visual estimation. This kind of evaluation procedure is limited by time, cost and the subjectivity of records. As a consequence, objectivity, automation and more precision of phenotypic data evaluation are needed to increase the number of samples, manage grapevine repositories, enable genetic research of new phenotypic traits and, therefore, increase the efficiency in plant research. In the present study, an automated field phenotyping pipeline was setup and applied in a plot of genetic resources. The application of the PHENObot allows image acquisition from at least 250 individual grapevines per hour directly in the field without user interaction. Data management is handled by a database (IMAGEdata). The automatic image analysis tool BIVcolor (Berries in Vineyards-color) permitted the collection of precise phenotypic data of two important fruit traits, berry size and color, within a large set of plants. The application of the PHENObot represents an automated tool for high-throughput sampling of image data in the field. The automated analysis of these images facilitates the generation of objective and precise phenotypic data on a larger scale.

Postponing First Shoot Topping Reduces Grape Cluster Compactness and Delays Bunch Rot Epidemic

Field trials investigating the impact of the timing of first shoot topping in a vertical shoot positioning (VSP) trellis system on (i) the cluster morphology, (ii) the canopy structure in the cluster-zone, and (iii) the progress of the bunch rot disease severity, were conducted in two white Vitis vinifera (Pinot gris, Riesling) cultivars in the years 2012 and 2013 in Luxembourg. The first shoot topping was carried out at seven different times from one week prior to beginning of flowering (BBCH 57) to four weeks after the end of flowering (BBCH 75–79) at approximately weekly intervals. Late first shoot topping reduced cluster compactness, delayed bunch rot epidemics, and thereby prolonged the potential ripening period compared to the standard treatment, in which the first shoot topping was performed one week postbloom. First shoot topping four weeks postbloom delayed the moment when 5% disease severity was reached up to eleven days and increased total soluble solids by 0.77 to 2.24 Brix compared to the standard treatment, with no significant impact on yield. Postponing the first shoot topping to the latest technically possible moment is recommended to improve grape architecture and health as well as to optimize grape maturity and potential wine quality under cool climate conditions. Late first shoot topping can be implemented in bunch rot control strategies without imposing additional costs and might support the reduction of pesticide use.

Towards Automated Large-Scale 3D Phenotyping of Vineyards under Field Conditions

In viticulture, phenotypic data are traditionally collected directly in the field via visual and manual means by an experienced person. This approach is time consuming, subjective and prone to human errors. In recent years, research therefore has focused strongly on developing automated and non-invasive sensor-based methods to increase data acquisition speed, enhance measurement accuracy and objectivity and to reduce labor costs. While many 2D methods based on image processing have been proposed for field phenotyping, only a few 3D solutions are found in the literature. A track-driven vehicle consisting of a camera system, a real-time-kinematic GPS system for positioning, as well as hardware for vehicle control, image storage and acquisition is used to visually capture a whole vine row canopy with georeferenced RGB images. In the first post-processing step, these images were used within a multi-view-stereo software to reconstruct a textured 3D point cloud of the whole grapevine row. A classification algorithm is then used in the second step to automatically classify the raw point cloud data into the semantic plant components, grape bunches and canopy. In the third step, phenotypic data for the semantic objects is gathered using the classification results obtaining the quantity of grape bunches, berries and the berry diameter.

Phenoliner: A New Field Phenotyping Platform for Grapevine Research

In grapevine research the acquisition of phenotypic data is largely restricted to the field due to its perennial nature and size. The methodologies used to assess morphological traits and phenology are mainly limited to visual scoring. Some measurements for biotic and abiotic stress, as well as for quality assessments, are done by invasive measures. The new evolving sensor technologies provide the opportunity to perform non-destructive evaluations of phenotypic traits using different field phenotyping platforms. One of the biggest technical challenges for field phenotyping of grapevines are the varying light conditions and the background. In the present study the Phenoliner is presented, which represents a novel type of a robust field phenotyping platform. The vehicle is based on a grape harvester following the concept of a moveable tunnel. The tunnel it is equipped with different sensor systems (RGB and NIR camera system, hyperspectral camera, RTK-GPS, orientation sensor) and an artificial broadband light source. It is independent from external light conditions and in combination with artificial background, the Phenoliner enables standardised acquisition of high-quality, geo-referenced sensor data.

Automatic image-based determination of pruning mass as a determinant for yield potential in grapevine management and breeding

Background and Aims Vine balance is defined as a relation between vegetative (mass of dormant pruning wood) and generative (yield) growth. For grapevine breeding, emphasis is usually placed on the evaluation of individual seedlings. In this study, we calculated the mass of dormant pruning wood with the assistance of an automated image-based method for estimating the pixel area of dormant pruning wood. The evaluation of digital images in combination with depth map calculation and image segmentation is a new and non-invasive tool for objective data acquisition. Methods and Results The proposed method was tested on a set of seedlings planted at the Institute for Grapevine Breeding Geilweilerhof, Germany. All images taken in the field were geo-referenced, and the automated method was validated by manual segmentation. Together with additional yield parameters, the vine balance indices can be used to classify seedlings for breeding purposes. Conclusion The computed pruning mass obtained using image-based methods is an accurate, inexpensive and easy method to estimate pruning mass compared with the manual time-consuming measurements. Together with the yield parameters, it is a suitable method for seedling evaluation and can also be used in precision viticulture. Significance of the Study This study demonstrates an image-based evaluation of the pruning mass to be a highly valuable tool for grapevine research and grapevine breeding. Moreover, the tool might be used by industry to monitor vine balance. The key findings reported have the potential to increase grapevine breeding efficiency by using an accurate and objective phenotyping method.

Development of methods for pre-symptomatic detection of grapevine diseases like esca, phytoplasmoses and viruses using hyperspectral sensors

Ryegrass (Lolium spec.) is the most important cool-season forage crop in temperate regions. Though, the seed production is considerably affected by several fungal and bacterial obligate biotrophic pathogens. The overall purpose of this study is directed to developing ryegrass cultivars with multiple pathogen resistance and agronomic adaption to Germany’s agricultural conditions. This aim shall be achieved by combining genes for resistances to stem rust, crown rust and bacterial wilt. The pyramidisation shall be accomplished by the use of specific molecular markers which will be derived by bulked segregant analysis combined with next generation sequencing based massive analysis of cDNA ends (MACE) transcriptome profiling. RNA was isolated from bulks of infected and noninfected leaf segments from susceptible and resistant genotypes of various fullsibling mapping populations (n ≥ 200) and their respective parental lines for every investigated pathogen. After MACE was performed, bioinformatic analysis detects SNPs and transcripts that were exclusively expressed in the resistant bulk. Thus, 30 molecular markers were genetically mapped to a 50.8 cM spanning region surrounding the stem rust resistance locus LpPg1. The development of this high efficient molecular selection tool marks MACE as a fast and reliable method that detects polymorphisms for genetic mapping of candidate genes and obtains to be the method of choice for investigating the molecular and genetic base of resistances to stem rust, crown rust and bacterial wilt.The current production systems in arable farming have reached their limits. Sizes of machinery are continuously increasing. Compaction and limits on the road are the consequences. Productionrelated restrictions like nitrogen pressure and development of resistances against plant protection products are further problems. Last but not least, the sociopolitical acceptance of crop production is questioned in public opinion. Due to these circumstances the question arises if the system of crop farming which has been adapted to the machinery available on the market is the right strategy for the future. Why not going the other way round and decide what a plant production system has to look like to be at an optimum and then decide what kind of machinery is needed to cultivate? Following this idea the plants must be in the focus.

Non-invasive phenotyping methods for abiotic and biotic stress in grapevine breeding

Ryegrass (Lolium spec.) is the most important cool-season forage crop in temperate regions. Though, the seed production is considerably affected by several fungal and bacterial obligate biotrophic pathogens. The overall purpose of this study is directed to developing ryegrass cultivars with multiple pathogen resistance and agronomic adaption to Germany’s agricultural conditions. This aim shall be achieved by combining genes for resistances to stem rust, crown rust and bacterial wilt. The pyramidisation shall be accomplished by the use of specific molecular markers which will be derived by bulked segregant analysis combined with next generation sequencing based massive analysis of cDNA ends (MACE) transcriptome profiling. RNA was isolated from bulks of infected and noninfected leaf segments from susceptible and resistant genotypes of various fullsibling mapping populations (n ≥ 200) and their respective parental lines for every investigated pathogen. After MACE was performed, bioinformatic analysis detects SNPs and transcripts that were exclusively expressed in the resistant bulk. Thus, 30 molecular markers were genetically mapped to a 50.8 cM spanning region surrounding the stem rust resistance locus LpPg1. The development of this high efficient molecular selection tool marks MACE as a fast and reliable method that detects polymorphisms for genetic mapping of candidate genes and obtains to be the method of choice for investigating the molecular and genetic base of resistances to stem rust, crown rust and bacterial wilt.The current production systems in arable farming have reached their limits. Sizes of machinery are continuously increasing. Compaction and limits on the road are the consequences. Productionrelated restrictions like nitrogen pressure and development of resistances against plant protection products are further problems. Last but not least, the sociopolitical acceptance of crop production is questioned in public opinion. Due to these circumstances the question arises if the system of crop farming which has been adapted to the machinery available on the market is the right strategy for the future. Why not going the other way round and decide what a plant production system has to look like to be at an optimum and then decide what kind of machinery is needed to cultivate? Following this idea the plants must be in the focus.

NoViSys: Novel Viticulture Systems for sustainable production and products

The project is dealing with the improvement of a universal pneumatic seeder which is suitable for sowing a wide range of different seeds in order to decrease particulate emissions of seed dressings. Aim of the project is to identify leakages in the pneumatic system in order to develop opportunities for the subsequent improvement of existing equipment to fulfill the high demands of user and environmental protection. Pneumatic seeders are characterized by a central, funnelshaped hopper. The batch feeder being an airlock and dosing feeder is located in the outlet of the seeder. The metered seed is fed into the air stream and transported to the sowing distributor by a conveying air stream.Due to climatic change, phenology traits are becoming increasingly important in grapevine breeding, since a premature flowering and ripening time could be observed for grapevine in the last decades. However, knowledge about these traits is still limited as they are genetically very complex and highly influenced by environmental factors. The analysis of the genetic basis of flowering time therefore will enable the development of tightly linked molecular markers useful for markerassisted selection of especially late flowering breeding lines.One recently occurred invasive insect pest has caught the attention for investigating biological control mechanisms and systems: The spotted wing drosophila (SWD, Drosophila suzukii Matsumura) is endemic in East China and Japan but has been introduced to the western hemisphere about 10 years ago and has been found in Europe since 2009. Nowadays, it has emerged to one of the most harmful pests to commercially grown fruit plants like stone fruits and nearly all kind of berries while it prefers ripe and overripe fruits. Our intention is to investigate the possible usage of natural antagonists for biological control. Therefore, we examine the natural load of parasites and pathogens (i.e. fungi, bacteria, viruses, microsporidia and protista) in fruit flies, isolate them and re-infect lab populations of D. suzukii for investigating the antagonistic potential. Furthermore, we will integrate the fruit pest codling moth (Cydia pomonella), which is an ongoing problem in apple orchards also because the pest develops resistance against commercially available insecticides. The long-term aim is to establish a stable system for pathogen detection that can be used for rapid identification of microbial antagonists in natural populations.Two main problems have to be considered in the development of control agents against the Fire Blight pathogen Erwinia amylovora: First, the ability of exponential growth leads to high cell densities in a short amount of time. Second, the most critical phase of Fire Blight infection occurs during blossoming, when the pathogen is transported to open flowers by various insects. To prevent infection, it is important to avoid invasion inside the plants tissue by interfering with growth of E. amylovora cells.The entomopathogenic fungus Isaria fumosorosea, formerly known as Paecilomyces fumosoroseus, has got a relatively wide host range. Within the scope of the EU project BIOCOMES investigations were done to validate the use of I. fumosorosea as a BCA against several pest insects. Under laboratory conditions it could be shown that I. fumosorosea seems to be a suitable BCA against Bemisia tabaci (silverleaf whitefly) and Spodoptera exigua (beet armyworm).Since the last two decades a number of fingerprinting methods have been developed to analyze microbial communities and their dynamics, including Terminal Restriction Fragment Length Polymorphism (T-RFLP), Length Heterogeneity-Polymerase Chain Reaction (LHPCR) and Automated Ribosomal Intergenic Spacer Analysis (ARISA). Because the latter provides a quick and cheap way together with high accuracy, we have chosen this method to investigate the fungal communities on grapevine, wood, leaves and berries.Saoussen Ben Tiba, Andreas Larem, Eva Fritsch, Karin Undorf-Spahn, Asma Laarif, Sami Fattouch, Johannes A. Jehle 1 Julius Kuhn-Institut, Institute for Biological Control, Darmstadt, Germany 2 Regional Center for Agriculture and Biological Agriculture, Chott Meriem, Tunisia 3 National Institute for applied technological Science Tunis (INSAT), Carthage University, Tunisia Email of corresponding author: saoussen.ben.tiba@jki.bund.deOne promising approach to a more environmentally friendly viticulture is growing fungus resistant cultivars with the novel cultivation method of the minimal pruning of trellis trained grapevines (MPTS). This practice reduces the tremendous amounts of fungicides needed to protect traditional cultivars and is expected to increase biodiversity compared to vineyards with traditional trellis trained grapevines (TS).Meiosis as the specialized cell division of sexual reproduction plays a crucial role in the exchange and reorganization of genetic material between two individuals by dividing the chromosome set in half and forming gametes. Even though in the last years major findings in the field of meiosis have been achieved, especially in plants, some key questions remain concealed. For a proper meiosis the initiation of double strand breaks (DSBs) during early prophase I is essential. Without DSBs no physical connection can occur between homologous chromosomes and recombination, pairing, and crossing over are excluded. So far in all analyzed eukaryotes SPO11, a meiosis specific transesterase, is the key enzyme inducing DSBs. But other than in animals and fungi where a single SPO11 is sufficient, plants need at least two different SPO11, referred to as SPO11-1 and SPO11-2, for proper meiosis. In Arabidopsis thaliana both have crucial functions and are essential in a functional form for the induction of meiotic DSBs as single knock out mutants are leading to near sterility by random chromosome distribution. Despite the same function of the homologs SPO111 and -2, the identity between both proteins is quite low. Homology of the orthologous SPO11 from different organisms is much higher. By exchanging SPO11-1 and -2 in Arabidopsis by their orthologs from various organisms we could demonstrate a species specific function of each SPO11, as a functional complementation of sterility could only be achieved with SPO11 from closely related species from the Brassicaceae. By exchanging non conserved regions between SPO11-1 and -2 of Arabidopsis we additionally could show a sequence specific function for each SPO11, as a functional rescue could not be achieved with all chosen regions. Interestingly, we could reveal a specific pattern of aberrant spliced isoforms for each SPO11 which are also sequence as well as species specific. By producing antibodies against AthSPO11-1 and -2 we were able to analyze for the first time the binding of SPO11-2 onto the DNA and perform co-immunolocalization studies with SPO11-1 and -2.European Stone Fruit Yellows (ESFY) is one of the most serious diseases in European fruit production. Infected Prunus cultivars yield poorly and lead to high economic losses. ESFY is caused by a specialized bacterium located in the phloem tissue of Prunus ssp., the Phytoplasma ‘Candidatus Phytoplasma prunorum’. It is spread by the phloemfeeding plum psyllid (Cacopsylla pruni) which acquires the bacterium by feeding on infected plants and is able to transmit it to healthy plants.Leaf rust caused by Puccinia triticina can cause yield losses up to 60 % and is the most common rust disease of wheat in the world. Vertical leaf rust resistance genes (Lr-genes) have been introduced in cultivars. Many of these resistances are broken down by virulent pathotypes. Horizontal resistances which are independent from races of a pathogen are known but show a quantitative characteristic which is carried by a few cultivars.

HT-Phenotyping methods for yield parameters in grapevine

The main role of MADS-box transcription factors in plant developmental processes has been well described in the model plant Arabidopsis thaliana. However, little is known about their function in crops of important agricultural and commercial value. Our study aims to investigate their role in two agronomical relevant Rosaceae crops: apple (Malus x domestica Borkh.) and strawberry (Fragaria vesca). Expression studies using qPCR and RNA seq have identified two apple Dormancy Associated MADS-box (DAM) genes. They group with the StMADS11 clade, and were named MdDAM1 and MdDAM2, the last one discovered ex novo. Real time expression studies in dormant buds collected during the chilling period and chromatin immunoprecipitation (ChIP) analyses confirmed that the genes are downregulated by exposure to cold and MdDAM1 is epigenetically repressed, as it has been demonstrated for Arabidopsis FLC and peach DAM genes. In parallel we worked on strawberry MADS-box genes of known function involved in flower development. We chose three MADS-box genes that are homologs of Arabidopsis PISTILLATA and AGAMOUS to perform gene expression and functional analysis using a RNA interference approach to obtain post-transcriptional gene silencing. The positive transgenic lines of each transformation were evaluated at the molecular and phenotypic level. Single gene mutants does not show altered flower phenotype, suggesting a different mechanism of flower development in strawberry, probably due to the peculiar flower structure.The quarantine pest pine wood nematode (PWN) Bursaphelenchus xylophilus, native in North America, can be found in wood and root parts of infected conifer trees with focus on Pinus species. At the beginning of the 20 century this species was introduced into Japan supposedly with timber imports where it induced pine wilt expression on susceptible native pine species. Further outbreaks outside Europe are reported from China, Korea, Taiwan and Mexico. In 1999 PWN was found in Portugal, which today is declared as complete infested, since followed by first single tree outbreaks in Spain. The European Union (EU) member states have to undertake measures to prevent further spread of this nematode according to the emergency measures of the EU Commission.The box tree pyralid Cydalima perspectalis (Walker 1859) is an invasive alien moth from East Asia which occurs in Central Europe since 2007. It is an insect pest on plants of the genus Buxus, causing serious damage. Because of the rapid spread in Germany and nearby countries like Switzerland, one part of this study was testing different ways of eco-friendly regulation with commercially available beneficials and biological control agents.1 Julius Kühn-Institute, Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany. 2 Geisenheim Research Center, Section Phytomedicine, Geisenheim, Germany. 3 Área de Genética, Facultad de Ciencias, Universidad de Málaga, Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM-UMA-CSIC), Málaga (Spain). 4 Suez Canal University, Faculty of Agriculture, Ismailia, Egypt. Email of corresponding author: namis.eltlbany@jki.bund.deExcessive use of mineral nitrogen (N) fertilizer is a common practice in rice-wheat rotations in southeastern China. At the same time N use efficiencies (NUEs) in these rice-based cropping system are very low (Peng et al., 2006). The consequences are high N losses from arable land to water bodies (surfaceand groundwater) and to the atmosphere (Richter and Roelcke, 2000). To investigate the scope and scale of reductions in mineral N fertilizer inputs, demonstration field experiments on farmers’ field sites were conducted for three consecutive winter wheat-summer rice double crop rotations in two counties of Jiangsu Province from 2008 to 2011. Results from the first two years are presented here. NUEsPolycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants which often persist in soil. The biochemistry of microbe-driven degradation of PAHs is well-studied though less is known about abiotic soil environmental factors influencing the microbe-phenanthrene interaction. Since minerals and charcoal are major soil components we matured different microbial communities in artificial soils based only on variation in the mineral content (montmorillonite, illite, ferrihydrite) and presence of charcoal for 2 years after addition of Luvisol microorganisms and sterile manure. Established microbial communities in soils were exposed to PAHs’ model compound phenanthrene (2 mg/g) to study microbial functionality depending on soil composition. Furthermore, litter which was previously shown to enhance microbial activity was added to the soils (1 wt%). Both treatments with corresponding controls were further incubated for 63 days with sampling on day 0, 7, 21 and 63. A natural Luvisol soil was treated similarly for control. Fingerprints by denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA or ITS gene fragments, respectively, amplified from extracted total community DNA were generated. The presence of phenanthrene-degradative genes was screened by PCR-Southern Blot detection and soil treatments were subjected to a chemical phenanthrene analysis. DGGE fingerprints revealed that the addition of phenanthrene and litter caused a shift in microbial community composition. Differences in bacterial response to phenanthrene were seen depending on litter addition and soil composition. The incubation time was determined as additional influencing factor. Furthermore, a dominance of a specific genotype for phenanthrene degradation is assumed based on PCR-Southern Blot analysis.1 Julius Kühn-Institute Federal Research Centre for Cultivated Plants (JKI), Institute for Epidemiology and Pathogen Diagnostics, Messeweg 11-12, D-38104 Braunschweig 2 Institute of Crop Science and Resource Conservation, Soil Science and Ecology, University of Bonn, Nussallee 13, D-53115 Bonn 3 Institute of Bioand Geosciences 3, Agrosphere, Forschungszentrum Jülich GmbH, D-52425 Jülich Email of corresponding author: sven.jechalke@jki.bund.de