Diagnostic techniques for viroids

Abstract

The fast growth of the human population forces us to produce more food, but higher crop production also leads to the fast spread of diseases. Plant pathology deploys a wide range of methods that do not provide an adequate solution to all disease losses. In the case of viroids, therapeutic means of control are not available; therefore control strategies are more focused on the development of reliable detection methods to quickly exclude the infected plant material. Although viroids are the smallest and simplest plant pathogens, their identification and detection is not straightforward. Each viroid–host combination is specific, and for reliable identification, all steps from sampling to final detection must be performed accurately. In this review, several methods for viroid detection in various host plants are discussed, including their advantages and disadvantages. Even though relatively new molecular methods enable fast and sensitive detection of viroids, a combination of different methods gives the most reliable identification. Techniques based on nucleic acids may be the future for viroid detection but they still cannot replace biological indexing, which is usually essential in epidemiological and aetiological studies.

Rasta Disease of Tomato in Ghana is Caused by the PospiviroidsPotato spindle tuber viroidandTomato apical stunt viroid

“…Thus, additional diagnostic tests are necessary to confirm viroid infection. However, RT-PCR tests can also be unreliable for viroid detection and identification due to differences in primer or viroid sequences, primer annealing conditions, RNA extraction methods, and viroid titer (Cohen et al 2006;Gucek et al 2017). Indeed, our initial attempts to detect viroids in RNA extracts prepared from rasta disease samples applied to FTA cards in RT-PCR tests with degenerate viroid primer pairs were inconclusive due to nonspecific amplification of presumably host-derived nucleic acids.…”

“…Viroids replicate in the nucleus (members of the family Pospiviroidae) or in plastids (members of the family Avsunviroidae) of infected cells (Branch and Robertson 1984;Daros et al 1994;Di Serio et al 2014;Ding 2009;Flores et al 2000Flores et al , 2009. A number of comprehensive reviews on viroids have been published (Diener 2003;Ding 2009;Flores et al 2005Flores et al , 2014Gucek et al 2017;Hull 2013;Navarro et al 2012;Tabler and Tsagris 2004).…”

Impact of Nucleic Acid Sequencing on Viroid Biology

“…Due to the non-coding nature of viroid RNA, the standard serological based virus diagnostic techniques are not possible. Hence, the conventional detection of viroids was accomplished by a combination of biological indexing (i.e., bioassays) and molecular biology techniques as well as by plant certification and quarantine programs [168][169][170]. In the 1980s, different types of polyacrylamide gel electrophoreses (e.g., 2D-PAGE and reverse PAGE) and RNA gel blot assays were used for routine diagnosis [170].…”

“…In the 1980s, different types of polyacrylamide gel electrophoreses (e.g., 2D-PAGE and reverse PAGE) and RNA gel blot assays were used for routine diagnosis [170]. These techniques were later replaced by the introduction of RT-PCR combined with the cloning and sequencing of the amplified viroid cDNAs using first-generation sequencing machines due to their precision, sensitivity, reliability, and speed [168]. Since the first RT-PCR based detection of viroids in both apple and pear trees in the 1990s [171], RT-PCR, combined with the Sanger sequencing method, has played a crucial role in the detection of a previously known viroids, or of their variants, in a new host plant or geographical area.…”

The occurrence of viruses and viroids in ornamental citrus mother plants in Tuscany (Central Italy)

“…Commonly, their control is based on preventive measures as availability of viroid-free budwood as a source of propagation material follow by adequate indexing procedures (Eiras et al, 2009). Even if studies are more oriented to fruit trees, these alteration may also play a significant role in ornamental citrus and many molecular diagnostic techniques for viroids are available (Luigi and Faggioli, 2013;Gucek et al, 2017).…”