Viroids of the Mediterranean Basin.

There has been substantial progress in the Mediterranean countries regarding research on viroids. Twenty-nine viroid species, all belonging to Pospiviroidae and Avsunviroidae genera, have been detected in the Mediterranean Basin. Not only have detection methods, such as reverse transcription-quantitative polymerase chain reaction and next-generation sequencing, been used for viroid detection, along with molecular hybridization techniques allowing for rapid detection, identification, and characterization of known and novel viroids in these countries, but eradication measures have also been taken that allowed for the efficient elimination of certain viroids in a number of Mediterranean countries. The eradication measures were followed as recommended by the European and Mediterranean Plant Protection Organization, which is known by its abbreviation, EPPO. The Mediterranean Region has been a niche for viroids since ancient times due to the warm climate and the socio-cultural conditions that facilitate viroid transmission among different host plant species.

Tissue Print Hybridization for Detection and Characterization of Viroids.

Simplifying sample preparation by transferring plant sap (from plant sections) directly onto a membrane is advantageous for any routine viroid detection technique, such as tissue printing. After fixing the samples on the membrane, hybridization steps similar to Northern or dot blot can be successfully implemented as long as factors like stringency and low viroid titer are properly adjusted to enable enhancement of the detection limit. The protocol described allows for indexing hundreds of field samples as a phytosanitary control measure.

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) at the Service of Viroid Detection.

A wide range of nucleic acid-based detection methods is available for viroid diagnosis. In this chapter, we describe a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP). The technique involves the use of nucleic acids, plant sap, or even traces of plant tissue. Adding a set of four to six primers, Bst DNA polymerase, AMV, or another thermostable reverse transcriptase, allows the fast detection of RNA targets like viroids after a short (20-40 min) incubation at 63 °C either in an open or close-tube system.