Transmission studies of the newly described apple chlorotic fruit spot viroid using a combined RT-qPCR and droplet digital PCR approach

Abstract

The transmission of the apscaviroid tentatively named apple chlorotic fruit spot viroid (ACFSVd) was investigated using a one-step reverse-transcription (RT) droplet digital PCR assay for absolute quantification of the viroid, followed by quantification of relative standard curves by RT-qPCR. Our results indicate that ACFSVd is effectively transmitted by grafting, budding and seeds. No transmission has yet been observed to the viroid-inoculated pome fruit species Pyrus sp. and Cydonia sp. ACFSVd was detected in viruliferous aphids (Myzus persicae, Dysaphis plantaginea) and in codling moths (Cydia pomonella). The viroid was also detected systemically in the infected hemiparasitic plant Viscum album subsp. album (mistletoe).

Modes of Viroid Transmission

“…ASSVd is seed-borne in apple [ 46 ], and seedlings germinated from ASSVd-positive apple seeds demonstrated a 7.7% infection rate [ 29 ]. ACFSVd is seed-transmitted from symptomatic apple fruit; seedlings that germinated from infected apple seeds showed an infection rate of 2.8% [ 10 ]. Transmission of GYSVd-1 via infected seeds in eight grapevine varieties has been demonstrated [ 56 , 57 ].…”

“…ACFSVd was transmitted by two species of aphids and by the larvae of a codling moth that had been feeding directly on symptomatic, viroid-infected apple trees [ 10 ]. Desvignes [ 72 ] reported that Myzus persicae (the green peach aphid) experimentally transmitted PLMVd, but at a low rate, suggesting minor relevance under natural peach-orchard conditions [ 82 ].…”

“…The hemi-parasitic plant mistletoe is frequently found in apple orchards. Mistletoe comes directly into contact with apple trees through haustoria, during which it may become systemically infected with ACFSVd and transmit the viroid from infected to uninfected trees [ 10 ]. Domesticated goats have been reported to transmit viroids when their horns were rubbed against viroid-infected trees [ 80 , 81 ].…”

“…Thus, the unassigned viroids in the genus Apscaviroid are apple-fruit-crinkle viroid (AFCVd) and grapevine-latent viroid (GLVd), in the genus Coleviroid are coleus-blumei viroid 5 (CbVd-5) and coleus-blumei viroid 6 (CbVd-6), and in the genus Pospiviroid is portulaca-latent viroid (PoLVd). Recently, two novel viroids have been described, apple-chlorotic-fruit-spot viroid (ACFSVd), a tentative new viroid in the genus Apscaviroid [ 9 , 10 ], and coleus-blumei viroid 7 (CbVd-7), a tentative new member of the genus Coleviroid [ 11 ].…”

Digital PCR: What Relevance to Plant Studies?

“…Moreover, this means that dPCR is even suitable for the preparation of in-house reference materials, which is particularly important in the field of plant pathogen diagnostics, where no reference materials are commercially available. Reverse-transcription (RT) digital PCR has been applied for the absolute quantification of viruses and viroids [60,64], with the aim of identifying the main transmission routes, main transmission vectors, and the plants carrying latent infections-a step of great relevance in the control and certification of nurseries' plants.…”