viruses Review Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review Gajanan T. Mehetre 1, Vincent Vineeth Leo 1 , Garima Singh 2 , Antonina Sorokan 3, Igor Maksimov 3, Mukesh Kumar Yadav 4, Kalidas Upadhyaya 5,*, Abeer Hashem 6,7, Asma N. Alsaleh 6 , Turki M. Dawoud 6, Khalid S. Almaary 6 and Bhim Pratap Singh 8,* 1 Department of Biotechnology, Mizoram University, Aizawl, Mizoram 796004, India; [email protected] (G.T.M.); [email protected] (V.V.L.) 2 Department of Botany, Pachhunga University College, Aizawl, Mizoram 796001, India; [email protected] 3 Institute of Biochemistry and Genetics, Ufa Federal Research Center of the Russian Academy of Sciences, pr. Oktyabrya 71, 450054 Ufa, Russia; [email protected] (A.S.); [email protected] (I.M.) 4 Department of Biotechnology, Pachhunga University College, Aizawl, Mizoram 796001, India; [email protected] 5 Department of Forestry, Mizoram University, Aizawl, Mizoram 796004, India 6 Botany and Microbiology Department, College of Science, King Saud University, P.O. Box. 2460, Riyadh 11451, Saudi Arabia; [email protected] (A.H.); [email protected] (A.N.A.); [email protected] (T.M.D.); [email protected] (K.S.A.) 7 Mycology and Plant Disease Survey Department, Plant Pathology Research Institute, ARC, Giza 12511, Egypt 8 Department of Agriculture and Environmental Sciences, National Institute of Food Technology Entrepreneurship & Management (NIFTEM), Industrial Estate, Kundli 131028, India * Correspondence: [email protected] (K.U.); [email protected] (B.P.S.); Tel.: +91-9436374242 (K.U.); Citation: Mehetre, G.T.; Leo, V.V.; +91-9436353807 (B.P.S.) Singh, G.; Sorokan, A.; Maksimov, I.; Yadav, M.K.; Upadhyaya, K.; Hashem, Abstract: Plant viral diseases are the foremost threat to sustainable agriculture, leading to several A.; Alsaleh, A.N.; Dawoud, T.M.; et al. billion dollars in losses every year. Many viruses infecting several crops have been described in Current Developments and the literature; however, new infectious viruses are emerging frequently through outbreaks. For the Challenges in Plant Viral Diagnostics: effective treatment and prevention of viral diseases, there is great demand for new techniques that can A Systematic Review. Viruses 2021, 13, provide accurate identification on the causative agents. With the advancements in biochemical and 412. https://doi.org/10.3390/v13030 molecular biology techniques, several diagnostic methods with improved sensitivity and specificity 412 for the detection of prevalent and/or unknown plant viruses are being continuously developed. Currently, serological and nucleic acid methods are the most widely used for plant viral diagnosis. Academic Editor: Nuredin Habili Nucleic acid-based techniques that amplify target DNA/RNA have been evolved with many variants. However, there is growing interest in developing techniques that can be based in real-time and Received: 7 January 2021 Accepted: 18 February 2021 thus facilitate in-field diagnosis. Next-generation sequencing (NGS)-based innovative methods have Published: 5 March 2021 shown great potential to detect multiple viruses simultaneously; however, such techniques are in the preliminary stages in plant viral disease diagnostics. This review discusses the recent progress in the Publisher’s Note: MDPI stays neutral use of NGS-based techniques for the detection, diagnosis, and identification of plant viral diseases. with regard to jurisdictional claims in New portable devices and technologies that could provide real-time analyses in a relatively short published maps and institutional affil- period of time are prime important for in-field diagnostics. Current development and application iations. of such tools and techniques along with their potential limitations in plant virology are likewise discussed in detail. Keywords: plant viruses; diagnostics; next-generation sequencing; omics technologies; nanopore Copyright: © 2021 by the authors. sequencing; quasi-species; in-field analysis Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons 1. Introduction Attribution (CC BY) license (https:// Viruses are the most genetically diverse organisms that cause infections in plants, creativecommons.org/licenses/by/ animals, and humans. In general, viruses consist of a small genome that encodes only 4.0/). Viruses 2021, 13, 412. https://doi.org/10.3390/v13030412 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 412 2 of 31 a few proteins, which makes it difficult to control viruses with a variety of methods [1]. In plants, among all the disease-causing pathogens, viruses present the substantial risk for agricultural production and have been reported to cause around half of the emergent infectious diseases among a variety of crop plants that cause about 40% of total crop losses upon viral infection [2]. Therefore, viruses represent one of the major constraints in agricultural production worldwide by decreasing both the quality and quantity of food crops [3]. Globally, more than twenty-five families of plant viruses are known to infect a variety of crop species, leading to higher economic losses [4]. Plant viral species are predominantly transmitted and spread by vertical and horizontal modes of transmission. In the vertical mode, the passage of the infectious agent occurs through the parent plants, either through vegetative propagation or by sexual reproduction via infected seeds. In the horizontal mode, spreading typically occurs through insect vectors, agricultural tools, as well as other direct or external forms of contamination [5]. Upon infection by a viral species, various symptoms, such as mosaic damage, yellowing, chlorosis, stunting, and necrosis are observed in infected plants, leading to loss of proper growth and reproductive functions of the plants [6]. Presently, very few treatments are available to manage or cure plant viral infectious diseases under field conditions. Therefore, early detection of the relevant causative agents represents the most crucial step to prevent the possible spread of the infectious disease [7]. The application of specific and sensitive methods for accurate detection is, therefore, re- quired for efficient and effective plant disease management. In this context, innovative techniques and methods are continuously being developed and applied in viral diagnostics. However, most of the diagnostics methods used for viral detection and identification are expensive, time-consuming, and remain unsuitable for capturing emerging viruses. Never- theless, there is a call to update these diagnostic methods for capturing new and emerging diverse viral species and their infections [8]. The most important difficulty with plant-based viral disease diagnostics is that plants are often infected simultaneously with different virus species or different variants of the same viral species [8,9]. In this context, the simultaneous detection of multiple viruses is the primary bottleneck in viral diagnostics. Moreover, due to the extraordinary genetic diversity among plant viruses, the use of one particular method for accurate detection becomes more complicated. Typically, viruses consist of genetic material, either RNA or DNA, with high mutation rates [10]. Currently, the preferred methods and techniques, such as electron microscopy (EM), immuno-serological methods (ELISA), molecular methods, and biosensor-based methods, are widely used to achieve rapid and reliable detection [11]. To increase the sensitivity and specificity, combinations of several techniques are predominantly employed in disease diagnosis. There is growing interest in assessing plant viral dynamics; however, effective strategies for the reliable detection of viruses in the infected plants were reported to be limited compared to those for human and animal viruses [12]. The recent developments in molecular biology and genomics techniques have enabled us to develop methods for plant viral diagnostics by obviating the need to culture viruses. The advent of next-generation sequencing (NGS) technologies has invariably led to a revolution in plant virus diagnosis [13,14]. These methods do not require any previ- ous knowledge of viral sequences and can sequence millions or billions of nucleotides in parallel, enabling the detection of all viruses, including those previously unreported [15]. Moreover, the use of such modern techniques to understand the ecology of the disease risk, emergence, and dynamics of viral diseases, alongside prior information about the virus and its diagnostics, is an emerging field of research [16,17]. In NGS-based methods, metage- nomics is gaining more considerable attention for viral diagnostics. Various sequencing platforms, like those developed for second-generation and third-generation sequencing are mostly used for diagnostics. Second-generation sequencing platforms, however, need capital investments, and their high sample turnover rates are not very cost-effective [18]. Therefore, third-generation sequencing technologies such as the nanopore sequencing plat- form could provide valuable and affordable diagnostic tools for the accurate diagnostics of Viruses 2021, 13, x FOR PEER REVIEW 3 of 31 Viruses 2021, 13, 412 3 of 31 cost-effective [18]. Therefore, third-generation sequencing technologies such as the na- nopore sequencing platform could provide valuable and affordable diagnostic tools for phyto-pathogensthe accurate diagnostics in crop of health.