Fruit trees host a wide range of viral pathogens, mainly as a result of their vegetative mode of propagation and perennial nature . Notably, among fruit trees, 44 viruses have been identiﬁed in the major cultivated Prunus species . Some of these pathogens are causing diseases adversely affecting yield and/or decreasing the fruit quality . The identiﬁcation, detection, and characterization of the causal agents of such diseases are challenging, due to the low titer and irregular within-plant distribution of viruses in perennial plants, the occurrence of both inter- and intra-species mixed-infections in single trees, the frequent symptomless infections or ﬂuctuation of symptom intensity during the season, and the complex and heterogeneous nature of viral populations [3–9]. Viruses 2018, 10, 436; doi:10.3390/v10080436 www.mdpi.com/journal/viruses Viruses 2018, 10, 436 2 of 23 Several biological, serological, and molecular assays have been applied, either alone or in combination, for the detection of fruit tree viruses. Biological assays, also known as biological indexing, were, in the past, an essential tool for the evaluation of the health status of individual plants and for the detection and/or characterization of a particular virus. In the case of fruit tree viruses, bioassays took advantage of the increased susceptibility of some fruit tree genotypes to viral infection, resulting in pronounced and easily identiﬁable symptoms expression. A number of fruit tree genotypes have been used for this purpose, e.g., Malus platycarpa, Malus micromalus, Malus pumila “Virginia Crab” or “Spy227”, for apple and pear virus pathogens [10,11]; and Prunus persica GF305, Prunus tomentosa, Prunus serrulata “Kwanzan” or “Shirofugen” [12–14], for stone fruit viral pathogens. Besides woody indicators, several herbaceous plant species, showing either local or systemic symptoms, have also been used [3,10,15], although no universal indicators for all fruit viruses are available. Effectiveness of bioassays vary considerably and can be hampered by several factors. Firstly, there is a need for controlled greenhouse facilities for plant maintenance, due to the vector-mediated transmission of several viruses, thus limiting a large scale and easy-to-use evaluation. Although symptoms on woody indicators could appear a few weeks post-inoculation, they may take from several months to years in the case of some viruses (e.g., Plum pox virus, PPV). In addition, a high intra-species genetic diversity of fruit tree viruses may result, not only in variable symptomatology [16,17], but also in symptomless reactions (e.g., PPV-Rec isolates in GF305 peach seedlings) . However, a question arises concerning the health status of used indicators, if not issued from in vitro meristem culture or thermotherapy sanitation, due to the possibility of hidden infections possibly inﬂuencing the interpretation of results. Nowadays, although still necessary in fruit tree virus research (assessment of biological properties, pathotyping etc.) and certiﬁcation schemes, the labor- and time-consuming biological indexing, with problematic reliability and subjective evaluation, has less practical application in virus diagnosis. Serological methods, ELISA (enzyme-linked immunosorbent assay) and its modiﬁcations,although limited in sensitivity, enable cheap and fast parallel diagnosis of many samples, using either polyclonal, monoclonal, or recombinant antibodies [19–21]. However, immunogenicity of some fruit tree viruses is scarce, producing antibodies which are not reliable enough for mass-scale diagnosis. Therefore, speciﬁc and sensitive detection of most fruit tree viruses and virus-like pathogens is mainly based on molecular diagnosis. Generally, detection of viral pathogens by genome ampliﬁcation techniques (Polymerase chain reaction: PCR, Reverse transcription polymerase chain reaction: RT-PCR and Loop-mediated isothermal ampliﬁcation: LAMP) is more sensitive than ELISA, while offering the possibility of multiple detection [22–25]. However, the development of speciﬁc molecular tests is strongly dependent on the knowledge of the pathogen genome and its molecular diversity, making primer design highly dependent on (or limited by) available sequence datasets. Especially in the case of recently discovered or poorly studied viruses, the available sequences might not represent the real pathogen diversity present in the ﬁeld, resulting in the development of insufﬁciently polyvalent tests, which might provide false negative results. Similar situations also occurred with well-known and widely spread viruses, such as Prune dwarf virus (PDV)  and Apple chlorotic leaf spot virus (ACLSV) , highlighting the need for continuous assessment of the viral molecular variability as a prerequisite to develop polyvalent and efﬁcient detection tools. Such problems can be overcome by the use of modern next generation sequencing (NGS) or high-throughput sequencing (HTS) technologies, which, theoretically, can produce sequence data of every putative viral agent present in a sample without the need of any former knowledge about their genome . This fact, in addition to the relatively little time needed for the completion of a sequence run and the analysis of the resulting data, make HTS the undisputed leading technology in diagnostics, nowadays.
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