The sanitary status of peach fruit trees was assessed in central and coastal regions of Montenegro during a survey in September and October of 2011 and 2012. Leaf samples were collected from 58 (2011) and 47 (2012) trees showing chlorotic rings and spots, mosaic, necrosis, leaf distortion, and stunting. Total RNAs was extracted from each sample by RNeasy Plant Mini kit (Qiagen, Germany) and used as a template in PDO (polyvalent degenerate oligonucleotides) nested reverse transcription (RT)-PCR for the detection of fruit tree viruses belonging to the genera Trichovirus, Capillovirus, and Foveavirus (family Betaflexiviridae). PDO primer sets PDO-F1i/PDO-R3i/PDO-R4i and PDO-F2i/PDO-R1i (2) were used in the first RT-PCR and nested PCR, respectively. Total RNAs obtained from Italian Apple chlorotic leaf spot virus (ACLSV)-infected isolate and healthy peach leaves were used as positive and negative controls, respectively. A nested set of primers amplified a 362-bp product from 6 samples collected in 2011 (10.3%) and 13 samples collected in 2012 (27.7%). Sequence analysis included three isolates (367/11, 133/12, and 168/12) chosen from different peach cultivars (Ritastar, Spring Belle, and Redhaven, respectively). Amplified products of expected size of the partial RNA-dependent RNA polymerase from three positive samples were cloned into p-GEM-T Easy Vector (Promega, Madison, WI) and sequenced (MWG-Biotech AG, Germany). Sequences were deposited in GenBank under accession nos. KF534757, KF534769, and KF534766, respectively. BLAST analysis showed that the sequence of isolate 367/11 (KF534757) shared high nucleotide similarity (78.9 to 87.2%) with ACLSV isolates from GenBank, showing highest identity with isolate PBM1 (AJ243438) from Germany. Sequence analysis of isolate 133/12 (KF534769) proved that it is 90.5 to 93.3% identical to Cherry green ring mottle virus (CGRMV) isolates reported from other parts of the world. In particular, the highest nucleotide similarity was showed with isolate P1C124 (AJ291761) from France. Finally, analysis of sequence from the isolate 168/12 (KF534766) revealed high degree of identity (86.1 to 96.1%) with the corresponding nucleotide sequences of the Cherry necrotic rusty mottle virus (CNRMV) isolates, showing highest similarity with isolate 120/86 (AF237816) from Switzerland. To confirm virus infectivity, according to the FAO/IPGRI Technical Guidelines (1), budwood from 367/11, 133/12, and 168/12 samples were grafted into seedlings of peach (GF305), Prunus serrulata (cv. Shirofugen) and P. avium (cv. Sam) then maintained in a greenhouse with controlled conditions. Six months post inoculation, GF305 indexed with 367/11 sample reacts with a green depressed mottle on leaves typical of ACLSV infection. Cherry tree of cv. Shirofugen indexed with sample 133/12 showed symptoms attributable to CGRMV such as epinasty, twisting and curling of leaves while a tree of cv. Sam indexed with 168/12 sample exhibited classical necrotic shot holes in leaves induced by CNRMV infection (1). Sequence analysis of PCR products obtained from indicator plants by RT-PCR as described above showed full nucleotide identity with KF534757, KF534769, and KF534766 sequences and confirmed the presence of previous described viral agents. To our knowledge, this is the first report of ACLSV, CGRMV, and CNRMV occurrence on peach in Montenegro. Due to the economic importance of this crop, sanitation measures should be adopted to improve the control of imported plants and the use of virus-tested propagation material in order to prevent spreading of these viruses. References: (1) M. Diekmann and C. A. J. Putter. FAO/IPGRI Technical Guidelines for the Safe Movement of Germplasm. No. 16. Stone Fruits, 1996. (2) X. Foissac et al. Phytopathology 95:617, 2005.
The RT-PCR identification and sequence analysis of Apple chlorotic leaf spot virus from apple cultivars in Jiaodong Peninsula, China