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Projects / Programmes source: ARRS

Combination of next generation sequencing and metagenomic analysis in the diagnostics of severe hop stunting

Research activity

Code Science Field Subfield
4.03.01  Biotechnical sciences  Plant production  Agricultural plants 

Code Science Field
B225  Biomedical sciences  Plant genetics 

Code Science Field
4.04  Agricultural and Veterinary Sciences  Agricultural biotechnology 
Keywords
metagenomics, transcriptome, RNA-seq, bioinformatics, next generation sequencing technologies, hop, Humulus lupulus L., plant diseases, diagnostics, epidemiology
Evaluation (rules)
source: COBISS
Researchers (17)
no. Code Name and surname Research area Role Period No. of publications
1.  16324  PhD Janez Demšar  Computer science and informatics  Researcher  2011 - 2014  339 
2.  24785  Alenka Ferlež Rus  Plant production  Researcher  2011 - 2014  113 
3.  31143  Nataša Hren    Technician  2011 - 2014 
4.  16379  PhD Jernej Jakše  Plant production  Principal Researcher  2011 - 2014  629 
5.  05994  PhD Branka Javornik  Plant production  Researcher  2011 - 2014  1,282 
6.  23044  Gregor Leskošek  Plant production  Researcher  2011 - 2014  174 
7.  31918  PhD Aljaž Majer  Plant production  Junior researcher  2011 - 2014  26 
8.  26537  PhD Stanislav Mandelc  Pharmacy  Researcher  2011 - 2014  73 
9.  34401  Mitar Milutinović  Computer science and informatics  Researcher  2012  16 
10.  20162  PhD Sebastjan Radišek  Plant production  Researcher  2011 - 2014  568 
11.  31175  Gregor Rot  Computer science and informatics  Researcher  2011 - 2014  45 
12.  32017  Tina Svetek  Plant production  Junior researcher  2011 - 2014 
13.  19184  PhD Nataša Štajner  Plant production  Researcher  2011 - 2014  290 
14.  30142  PhD Marko Toplak  Computer science and informatics  Researcher  2011 - 2012  27 
15.  15577  Nevenka Valič  Plant production  Researcher  2011 - 2014  89 
16.  12536  PhD Blaž Zupan  Computer science and informatics  Researcher  2011 - 2014  513 
17.  23195  Silvo Žveplan  Plant production  Researcher  2011 - 2014  188 
Organisations (3)
no. Code Research organisation City Registration number No. of publications
1.  0416  Slovenian Institute of Hop Research and Brewing  Žalec  5051762000  3,983 
2.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  63,768 
3.  1539  University of Ljubljana, Faculty of Computer and Information Science  Ljubljana  1627023  14,205 
Abstract
In 2006, hop growers in the Savinja valley reported the appearance of hop plants showing stunted vigor. Such symptoms were visible in various cultivars (notably ‘Celeia’, ‘Aurora’ and ‘Bobek’), showing the disease agent’s ability to affect different hop genotypes. Infected plants reached only up to 50% of normal height. Symptoms were even more severe the next year and affected plants eventually declined. It was confirmed that the disease was spreading around the first observed point of infection, suggesting agrotechnical practice as the most likely cause of transmission of the disease to healthy plants, although disease spread by means of insect vectors could not be ruled out. Infected planting material exchange between hop farmers has been confirmed as a source of new outbreaks over longer distances. Plant pathologists have conducted several analyses, which have ruled out fungi as possible disease agents. Symptom exhibiting plants also proved free of various viruses, but proved positive for the presence of hop latent viroid (HLVd), which does not cause the observed symptoms. The stunt disease symptoms resemble those of hop stunt disease caused by hop stunt viroid (HSVd) discovered in Japan, but it has not been widely described in other hop growing regions of the world. Affected plants from Slovenian fields have been repeatedly tested by several RT-PCR assays for the presence of HSVd and always exhibited unspecific or non-positive signals. The speed of spread and severity of this disease require quick action. In the proposed project, we aim to accomplish several tasks, which will subsequently allow prompt identification of the pathogen, by developing a a method for its routine detection, comparing the transcriptome response of diseased and healthy plants and establishing guidelines for proper disease management. In order to identify the pathogen, we propose to use a combination metagenomic approach of sequence comparison and next generation sequencing technology (NGS), which will allow us to identify as yet unknown pathogens present in stunted hop plants. There have been several successful studies utilizing metagenomics coupled with NGS for the diagnostics of human, animal and plant diseases, which leads us to believe that this approach will allow prompt novel pathogen detection or show a synergistic infection of several pathogens. On the basis of the retrieved sequence, we will develop a diagnostic method for routine detection of the pathogen. We will then study the epidemiological characteristics of the disease, aimed at clarifying whether weed plant species present in hop gardens are possible reservoirs of the pathogen or contribute its spread, and whether the pathogen can spread in a hop garden by means of mechanical injuries to plants. In addition to identifying the pathogen(s), we propose to study differential gene expression in transcriptomes of 3 experimental groups: 1) HLVd positive hop plants with evident new disease symptoms, 2) HLVd positive symptom-free hop plants and 3) HLVd/viruses negative symptom-free hop plants. To understand the molecular mechanisms underlying host-pathogen interactions, it is necessary to identify the gene transcripts differentially expressed in infected and healthy plants. This study will be carried out using an RNA-seq analysis of the 3 described experimental groups and will include de novo assembly of the hop transcriptome. Although RNA-seq had been developed fairly recently, it has already greatly contributed to the understanding of gene expression and regulation. We anticipate that the proposed combination of metagenomic analysis of NGS data and RNA-seq studies will result in successful identification of the novel hop pathogen or explain the synergistic infection of several pathogens, while providing a detailed insight into the transcriptome of infected hop plants.
Significance for science
Metagenomics analysis, applying next generation sequencing technology (NGS), was used for the first time in the study of an unknown hop disease. In the framework of these project activities, the sequence of total RNA, as well as a fraction of small RNAs, was determined. We gained specific bioinformatics data skills and practical experience by analysing the results with selected software packages. The analysis enabled us to identify citrus bark cracking viroid (CBCVd) in susceptible plants. This is the first known description of this viroid in hop and its first occurrence in areas not specific for citrus production. Analysis of small RNAs demonstrated the possibility of using such data and analytical approach for routine diagnostics, because it is very affordable. Approaches for identifying pathogens causing unknown diseases in use to date have been immensely improved, with our new detection analysis focusing on faster, more sensitive and comprehensive analysis. NGS technology enabled us to obtain a detailed transcriptome of hop with broad applicability for research into hop response to infection with the above mentioned pathogen. Analysis of the transcriptome confirmed the existence of several transcripts differentially expressed among susceptible plants. The plant immune defence system generates substantial amounts of viroid/virus small RNAs on interaction of the viroid/virus with the plant. These small RNAs can silence plant genes by RNA interference mechanisms. In our analysis, based on the bioinformatics approaches of target identification in the hop transcriptome and verification with RT-qPCR, we were able to demonstrate that genes are differentially expressed in infected plants compared to those with no viroid infection, but we did not observe a full silencing pattern. Epidemiological studies during the course of our project have supplemented knowledge of how the viroid spreads in hop and helped to form a recommendation on how to manage and prevent such infections. Nucleotide sequences obtained during the course of the project are available to other research groups in the publicly accessible NCBI SRA data base.
Significance for Slovenia
Hop growing represents a significant part of Slovenian agricultural activities and is one of the most export oriented activities of Slovenian agriculture, with more than 95% of hop cones being exported to foreign countries. In the Savinja valley and other parts of Slovenia, world renowned, highly aromatic domestic hop varieties are being grown (varieties commercially known as Super Styrian). Because of the specific microclimate conditions, these varieties only give an optimal yield in this area. The occurrence of a new, previously unidentified hop disease, which spreads rapidly and independently among hop varieties, is a serious threat to domestic hop growing and a potential cause of major economic losses. Within the framework of our project, we contributed to pathogen identification by implementing new research approaches and set strategies for preventing the spread of the infection by studying the epidemiological properties of the pathogen. We developed new diagnostic methods with applicative value in breeding new varieties, ensuring healthy planting material and controlling the spread of the pathogen. The research results, and the development and introduction of new identification techniques, have significant importance, since they can be applied for resolving similar issues of new disease occurrences, both in the field of plant growing and otherwise. Knowledge of the hop transcriptome response to viroid infection enables us, in addition to gaining basic knowledge, to use this knowledge in designing plant defence strategies. The results have an impact on obtaining new knowledge at the University of Ljubljana and the Slovenian Institute for Hop Research and Brewing and, consequently, on the introduction of the knowledge into new techniques in Slovenia and in the wider European and global environment. The new technologies can help reduce the environmental burden, contribute to food safety, reduce energy use and thus enable more sustainable development. The project was applied in the context of research of both a postgraduate and an undergraduate student, thus having a direct impact on the educational process at the University of Ljubljana. The acquired nucleotide sequences also enable direct use of the obtained knowledge and data in undergraduate teaching processes.
Most important scientific results Annual report 2012, 2013, final report, complete report on dLib.si
Most important socioeconomically and culturally relevant results Annual report 2011, 2012, 2013, final report, complete report on dLib.si
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