Reliable RT‐PCR detection of Apple stem pitting virus in pome fruits and its association with quince fruit deformation disease

Abstract

In this study a spot nested RT-PCR assay was developed for the detection of Apple stem pitting virus (ASPV). A one step RT-PCR for the generic detection of foveaviruses using degenerate primers that target a conserved region of the RNA-dependent RNA polymerase (RdRp) gene was followed by a nested PCR that amplifies a 312 bp ASPV specific product. The method is rapid, simple and displays high sensitivity and broad detection range, overcoming the virus molecular variability. The optimum sampling conditions for reliable virus detection were also investigated. ASPV was detected throughout the year in different plant tissues of affected trees, thus the method could be used for routine screening and in certification schemes of pome fruits. ASPV was detected in quince orchards in Greece in all trees that were tested, showing a fruit deformation disorder. Sequencing and phylogenetic analysis of amplicons generated by RT-PCR from plant tissue affected with the deformation disease indicated that the agent responsible was a variant of ASPV.

Cydonia japonica, Pyrus calleryana and P. amygdaliformis: three new ornamental or wild hosts of Apple stem pitting virus

“…Analyses of the small region of the RdRp gene studied here preliminarily indicate that ASPV isolates from these new hosts are generally not closely related together and that they also show significant divergence with ASPV isolates from cultivated/rootstock species. It is noticeable that the ornamental pear isolate (PCT88) showed 87% aa identity with two cultivated apple and pear isolates (83 and 76% at nt level, respectively) (GT11, AFT5; [7]) grown in the same area.…”

“…The virus has also been detected in widely used apple-(M9, EM9, MM106) and pear-rootstocks (Pyrus ussuriensis) [3][4][5]. ASPV has also been isolated from commercial used Pyrus species such as the Japanese pear (P. serotina-pyrifolia) [6,7] and P. sinkiangensis cv. Korla [8].…”

“…The tested samples consisted of mixed plant tissues of bark, leaf and petioles. For template preparation spotting of plant sap on nylon membranes or total RNAs extraction was used as described previously [7].…”

“…For ASPV detection, a well-established spot nested RT-PCR assay was applied at point throughout the growing season and dormancy, using degenerate primers (SP nest1, SP nest2) with a broad detection range amplifying a 312 bp fragment of the RNA-dependent RNA polymerase (RdRp) gene [7]. In a second assay to confirm the presence of ASPV in positive samples, the specific primers SPcp1 and SPcp2, targeting a 789 bp fragment of the capsid protein (CP) gene were used in a separate RT-PCR assay [15].…”

“…The 312 bp RdRp PCR products were excised from the agarose gels, purified using a matrix gel extraction system (Marligen Bioscience, USA) and directly sequenced [7]. BLASTX nucleotide query comparison of the obtained sequences with the NCBI database was performed, confirming that the amplified fragments correspond to ASPV isolates.…”