Rapid and precise identification, this is the goal when responding to threats that are affecting (plant) health. High-throughput sequencing (HTS) is a key technology to guide decision making and is taking the lead in the field of diagnostics. Dr. Paolo Margaria from the department Plant Viruses describes how HTS and bioinformatics are routinely employed to rapidly find answers to the origin of diseases and are essential to the “international character” of the Plant Virus Department.
We have recently become very sensitive to health threats from alien viruses and how important it is to understand the origin and direction of outbreaks affecting our health. Let´s put this on a plant perspective. Viruses can cause serious plant diseases, and early detection and identification is essential for acting and implementing adequate countermeasures: we need to be fast, we need to be accurate and, we need to have the capacity to check for novel, unknown, even unexpected pathogens. Fantastic tools have been developed to address this task, and these powerful and exciting techniques are more and more dominating the field of diagnostics. High-throughput sequencing (HTS), i.e. the parallel sequencing of multiple molecules to generate hundreds of millions of sequences, supports the work of the Plant Virus Department to rapidly find answers to the origin of diseases. HTS is the basis for virus discovery, studies on virus diversity [1], surveys and surveillance [2], the safe exchange of plant materials over intercontinental borders, and ensures the highest quality standards of the DSMZ reference Plant Virus Collection materials.
As a brief overview on diagnostics, investigations using a transmission electron microscope (TEM) allow for morphological observation of virus particles in leaf sap derived from infected plants. This verifies presence of virus-like particles and points into the right direction for identification, for example by visualization of small-round versus long-flexuous particles. That is why we value that we have access to the TEM facility at the Julius Kühn-Institute in Braunschweig as a first look into virus presence. The standard serological and molecular diagnostic tests for virus detection, ELISA assays, polymerase chain reaction etc can reach high sensitivity and specificity, however they require specific antisera or oligonucleotides as primers for the amplification reaction, and thus knowledge about the pathogen or at least its group or family is necessary to guide the detection. Nowadays, HTS is taking the lead in the field of diagnostics, with the main advantage that no a priori knowledge on the target is required, thus enabling a global assessment of the spectrum of organisms present in a sample. Wonderful news! In combination with the “high-throughput” sequencing power of the chemistry and equipment, high numbers of sequences and multiple samples can be assessed in a single sequencing run. In a very short time, we can detect “all” organisms, known pathogens or new ones, and we are able to reconstruct complete genomes! Knowledge on the virus presence in crops and ornamental plants allows further investigations of a disease, can clarify its etiology and virus/plant interactions, and support appropriate reactions towards plant protection and prevention of occurrence of epidemic events.
The high processivity combined with immediate sequence information at genomic level make HTS ideal for being at the core of outbreak events, and rapidly associate a disease with organisms. Biological assays remain fundamental to accurately interpret what we are finding with modern technologies, especially when we want to understand a disease rather than describing the presence of sequences in the analyzed data. However, the global information provided by HTS allows unprecedented ways for rapid identification. That is why HTS has become a core methodology and fundamental competence of the Plant Virus Department, with all steps of the workflow conducted in house, from plant sample preparation to library synthesis and bioinformatic analyses. The sequencing infrastructure present at the DSMZ allows us to get information on viruses and other organisms in a plant sample in a matter of days. The whole workflow is continuously subject to improvements, by exploring and establishing novel methods and technologies (Oxford Nanopore Sequencing is in the forefront as platform for rapid diagnostics [3]), and implementing bioinformatic pipelines for detection of viruses and beyond, of plant pathogens on global scale.
HTS activities are significant parts of our services, and allow us to “travel” all over the world, by investigating the pathogen ensembles in a wide spectrum of crops, ornamentals and wild plants, with a diversity that can be envied, from maize samples from Ecuador, to yam in Brazil, tomatoes from Turkey, citrus from Montenegro, papaya from Jordan, pineapple from Mauritius, pomegranate from Saudi Arabia, groundnut from Burkina Faso, soybean from Congo, cassava from Sri Lanka, passiflora from Cambodia, apple plantlets on the way to the USA… or mycoviruses in Fusarium! Our discoveries are then only the beginning of new “stories” about new viruses, diseases and host plants and an enormous contribution for us, our partners and customers.
With our HTS activities, we also support the international movement of germplasm to foster crop improvement; the exchange of genetic resources is indeed necessary, however it introduces the risk of spreading (new) viruses and other pathogens [4]. Verification of the sanitary status of the materials is key to prevent these scenarios. The biggest effort in this context concerns the exchange of cassava genetic material between South America and Africa, in the program of breeding and genetic improvement against virus diseases: here it is essential to avoid introductions across continents! HTS allows us to verify pathogen freedom in materials which are subjected to growing-on tests conducted in the glasshouse for a few months, or even in plantlets from tissue culture propagation, thus reducing the time necessary for plant health verification and further actions.
In the frame of authentication and verification of purity of the DSMZ reference Plant Virus Collection, all our PV accessions are currently subjected to HTS, virus genomes are reconstructed and made available in public databases to customers and scientific community. More than 600 virus isolates have been sequenced so far, and sequences have been openly released via GenBank to provide the complete genomic information of the isolates offered in our catalogue.
Overall, we can not renounce to HTS anymore! All these activities and scenarios bring the Department into an “international environment”, which is a true character of our daily modus operandi, and contributes to our excitement to look for next and for more.
Author: Dr. Paolo Margaria
References
- Sareli K, Winter S, Chatzivassiliou EK, Knierim D, Margaria P. High molecular diversity of full-length genome sequences of zucchini yellow fleck virus from Europe. Archives of Virology, 2022, 167:2305-2310.
- Mendoza AR, Margaria P, Nagata T, Winter S, Blawid R. Characterization of yam mosaic viruses from Brazil reveals a new phylogenetic group and possible incursion from the African continent. Virus Genes, 2022, 58:294-307.
- Margaria P, Menzel, W, Winter S. Automated and real-time profiling of plant virus infections to support diagnostics and quick response in outbreak events. International Conference of Plant Pathology, Lyon, 2023
- Margaria P, Winter S. High-Throughput Sequencing for pathogen indexing to safeguard the international movement of clonal crops. International Conference of Plant Pathology, Lyon, 2023