Aleš Eichmeier

Comprehensive Virus Detection Using Next Generation Sequencing in Grapevine Vascular Tissues of Plants Obtained from the Wine Regions of Bohemia and Moravia (Czech Republic)

Comprehensive next generation sequencing virus detection was used to detect the whole spectrum of viruses and viroids in selected grapevines from the Czech Republic. The novel NGS approach was based on sequencing libraries of small RNA isolated from grapevine vascular tissues. Eight previously partially-characterized grapevines of diverse varieties were selected and subjected to analysis: Chardonnay, Laurot, Guzal Kara, and rootstock Kober 125AA from the Moravia wine-producing region; plus Müller-Thurgau and Pinot Noir from the Bohemia wine-producing region, both in the Czech Republic. Using next generation sequencing of small RNA, the presence of 8 viruses and 2 viroids were detected in a set of eight grapevines; therefore, confirming the high effectiveness of the technique in plant virology and producing results supporting previous data on multiple infected grapevines in Czech vineyards. Among the pathogens detected, the Grapevine rupestris vein feathering virus and Grapevine yellow speckle viroid 1 were recorded in the Czech Republic for the first time.

DETECTION OF GRAPEVINE PINOT GRIS VIRUS IN CERTIFIED GRAPEVINE STOCKS IN MORAVIA, CZECH REPUBLIC

Twenty one grapevine mother plants used by nurseries for propagation in South Moravia, Czech Republic, were tested for the presence of Grapevine Pinot gris virus (GPGV) and other viruses by simplex and multiplex RT-PCR. GPGV was found in all vines tested and Grapevine virus A, Grapevine fanleaf virus and Grapevine fleck virus were detected only in some of them. Part of the movement and coat protein coding regions of 21 GPGV isolates was sequenced. Phylogenetic analysis revealed that south Moravian GPGV isolates grouped with isolates from other regions and countries. This study provides the first comprehensive survey of the GPGV occurrence in South Moravia.

Diaporthe diversity and pathogenicity revealed from a broad survey of grapevine diseases in europe

Species of Diaporthe are considered important plant pathogens, saprobes, and endophytes on a wide range of plant hosts. Several species are well-known on grapevines, either as agents of pre- or post-harvest infections, including Phomopsis cane and leaf spot, cane bleaching, swelling arm and trunk cankers. In this study we explore the occurrence, diversity and pathogenicity of Diaporthe spp. associated with Vitis vinifera in major grape production areas of Europe and Israel, focusing on nurseries and vineyards. Surveys were conducted in Croatia, Czech Republic, France, Hungary, Israel, Italy, Spain and the UK. A total of 175 Diaporthe strains were isolated from asymptomatic and symptomatic shoots, branches and trunks. A multi-locus phylogeny was established based on five genomic loci (ITS, tef1, cal, his3 and tub2), and the morphological characters of the isolates were determined. Preliminary pathogenicity tests were performed on green grapevine shoots with representative isolates. The most commonly isolated species were D. eres and D. ampelina. Four new Diaporthe species described here as D. bohemiae, D. celeris, D. hispaniae and D. hungariae were found associated with affected vines. Pathogenicity tests revealed D. baccae, D. celeris, D. hispaniae and D. hungariae as pathogens of grapevines. No symptoms were caused by D. bohemiae. This study represents the first report of D. ambigua and D. baccae on grapevines in Europe. The present study improves our understanding of the species associated with several disease symptoms on V. vinifera plants, and provides useful information for effective disease management.

Use of Combined MSAP and NGS Techniques to Identify Differentially Methylated Regions in Somaclones: A Case Study of Two Stable Somatic Wheat Mutants.

The appearance of somaclonal variability induced by in vitro cultivation is relatively frequent and can, in some cases, provide a valuable source of new genetic variation for crop improvement. The cause of this phenomenon remains unknown; however, there are a number of reports suggesting that epigenetics, including DNA methylations, are an important factor. In addition to the non-heritable DNA methylation changes caused by transient and reversible stress-responsive gene regulation, recent evidence supports the existence of mitotically and meiotically inherited changes. The induction of phenotypes via stable DNA methylation changes has occasionally great economical value; however, very little is known about the genetic or molecular basis of these phenotypes. We used a novel approach consisting of a standard MSAP analysis followed by deep amplicon sequencing to better understand this phenomenon. Our models included two wheat genotypes, and their somaclones induced using in vitro cultivation with a changed heritable phenotype (shortened stem height and silenced high molecular weight glutenin). Using this novel procedure, we obtained information on the dissimilarity of DNA methylation landscapes between the standard cultivar and its respective somaclones, and we extracted the sequences and genome regions that were differentially methylated between subjects. Transposable elements were identified as the most likely factor for producing changes in somaclone properties. In summary, the novel approach of combining MSAP and NGS is relatively easy and widely applicable, which is a rather unique feature compared with the currently available techniques in the epigenetics field.

Identification of 17 ‘Candidatus Phytoplasma pyri’ genotypes based on the diversity of the imp gene sequence

M. Bohunick a*, L. Valentov a, J. Such a, T. Ne cas, A. Eichmeier, T. Kiss and R. Cmejla Research and Breeding Institute of Pomology Holovousy Ltd., Holovousy 129, 508 01 Ho r ice; Department of Biology, Faculty of Science, University of Hradec Kr alov e, Rokitansk eho 62, 500 03 Hradec Kr alov e; Department of Pomology, Faculty of Horticulture, Mendel University in Brno, Valtick a 337, 691 44 Lednice; and Mendeleum – Institute of Genetics, Mendel University in Brno, Valtick a 334, 691 44 Lednice, Czech Republic

High-Throughput Sequencing Analysis of the Bacterial Community in Stone Fruit Phloem Tissues Infected by “Candidatus Phytoplasma prunorum”

Abstract“Candidatus Phytoplasma prunorum” (CPp) is a highly destructive phytopathogenic agent in many stone fruit-growing regions in Europe and the surrounding countries. In this work, we focused on documenting entire bacterial community in the phloem tissues of 60 stone fruit trees. Nested PCR and two real-time PCR assays were used to select CPp-positive (group A) and CPp-negative samples (group B). Afterwards, high-throughput amplicon sequencing was performed to assess bacterial community compositions in phloem tissues. The bacterial composition in phloem tissue consisted of 118 distinct genera, represented mainly by Pseudomonas, Acinetobacter, Methylobacterium, Sphingomonas, and Rhizobium. Statistics showed that CPp influenced the bacterial composition of infected plants (group A) and that the bacterial community depended on the geographical origin of the sample. This is the first work focusing on an analysis of the influence of CPp on the bacteria coexisting in the phloem tissues of stone fruit trees.

Evaluation of Three Approaches For The Measurement of Rna Integrity, Concentration and Purity in Tissues of Apricot Flower Buds

The RNA quantity and quality are basic factors for successful studies of gene expression. On absorbance and fluorescence based measurements are routinely used techniques for RNA measurement. A new technique for measurement of RNA is available with Agilent 2100 bioanalyzer in combination with the RNA 6000 Nano LabChip® kit. This method offers in addition the advantage of assessing RNA integrity. Agilent 2100 Bioanalyzer is compared with UV spectrophotometry and fluorometry quantitation systems assessing the quantity, purity and RNA integrity. This study was focused on comparison of RNA isolated from apricot flower buds which were evaluated by mentioned methods. Obtained results show important differences in RNA which was isolated in 2008 (stored at -80 oC) and RNA which was newly isolated from the same stored tissues in 2013. Study also compares techniques of mRNA quantity, quality and integrity evaluation. Evaluation of Three Approaches For The Measurement of Rna Integrity, Concentration and Purity in Tissues of Apricot Flower Buds