INCIDENCE OF VIRUS INFECTIONS ON DIFFERENT PEACH CULTIVARS IN MONTENEGRO

INCIDENCE OF VIRUS INFECTIONS ON DIFFERENT PEACH CULTIVARS IN MONTENEGRO Zindović J., 1 Božović V., 1 Miladinović Z., 2 Rubies Autonell C. 3, Ratti C. 3 1 2 3

Peach production in Montenegro Stone fruit production Total surface (ha) Enterprises and collective farms Private farms Plum ca. 1210 ha ca. 5 ha ca. 1205 ha Peach ca. 185 ha ca. 85 ha ca. 100 ha Cherries ca. 125 ha ca. 3 ha ca. 122 ha Peach ranking second in planted surface in stone fruit production in MNE Podgorica s region is the main peach-growing area 45% of peach production posses enterprises (AD Plantaze 13. jul) AD Plantaze 13. jul (locality Ćemovsko field): 85 ha with 19 different cultivars

Investigation on peach viruses Previous surveys This survey Presence of PPV on plum (PPV-D and PPV-Rec) and peach (PPV-M) was confirmed in 2007 Zindovic et al., 2008 Viršček-Marn et al., 2008 PPV was confirmed in 17 out of 18 tested samples Presence of PLMVd was confirmed in 2011 Mavrič-Pleško et al. 2011 Survey is conducted in Podgorica region (ca. 80 ha) Locality: Ćemovsko field 9 peach cultivars were examined for the presence of 9 peach viruses

Aim of investigation Presence of different economically important peach viruses in Montenegro Incidence of 9 peach viruses in different peach cultivars Molecular characterization of the most prevalent peach virus Molecular variability of selected PPV isolates Reconstruction of phylogenetic trees based on sequences of investigated virus isolates

Examined cultivars and viruses Cultivars (out of 19): Adriana Caldesi Gloria Maria Marta May Crest Morsiani Rita Star Spring Belle Spring Crest Viruses (9): Plum pox virus (PPV) Prunus necrotic ringspot virus (PNRSV) Prune dwarf virus (PDV) Apple chlorotic leaf spot virus (ACLSV) Peach mosaic virus (PMV) Cherry mottle leaf virus (CMLV) Strawberry latent ringspot virus (SLRSV) Tobacco ringspot virus (TRSV) Tomato ringspot virus (ToRSV)

Field survey In September and October 2011, 58 symptomatic leaves samples were collected Symtoms: chlorotic ringspots, veinal chlorosis, netting, leaf distortion, stunting, rosette formation blotches and deformations

Field survey cv. Ritastar cv. Ritastar Symptoms on Prunus persica indicative of PNRSV infection: hlorotic rings and spots, mosaic

cv. Ritastar 370/11 cv. Ritastar 372/11 Symptoms on Prunus persica indicative of PPV infections: vein chlorosis and netting

Molecular methods used for the detection of stone fruit viruses Virus Primers Primers sequences (5-3 ) ACLSV CMLV PMV PNRSV PDV PPV TRSV ToRSV SLRSV ACLSRV F ACLSV R PM16AFF PM16AFR PM16AFF CML-26R Ilar 2F Ilar 1R PDV 2F PDV 1R PPV 8511-8532 F PPV 9763-9784 R P241 P316D P316M PPV-DM probe TRSVf1 TRSVr1 ToRSVf1 ToRSVr1 3DR F1 2R2 5DF GGCAACCCTGGAACAGA CAGACCCTTATTGAAGTCGAA CAAACATGGCTTTCACCTTCTGCA TCTTGCCCCACCCTTCAACAAATG CAAACATGGCTTTCACCTTCTGCA AGATCCTCTTTCCCTTCTAAAATG CCACCGAGAGGTTGGCA TTCTAGCAGGTCTTCATCGA ATGGATGGGATGGATAAAATAGT TAGTGCAGGTTAACCAAAAGGAT CGATATCTTGAAGCTTTTTACG CTCTTGCACAAGAACTATAACC CGTTTATTTGGCTTGGATGGAA GATTAACATCACCAGCGGTGTG GATTCACGTCACCAGCGGTGTG FAM-CGTCGGAACACAAGAAGAGGACACAGA- TAMRA GGAAGCTGTATAAACTCAGC GTGTTGGACAAACACGACAC GGAAGCTGTATAAACTCAGC GTCCTCATGGAACCTTTCTC AGGCTCAAGAAAACACAC GGCTGATCCTCGTGAGAC GCGTGTGGCGTCGCTAAT CCCTTGGTTACTTTTACCTCCT Size of amplicon 358 bp Reference Nemichov et al., 1995 Method RT-PCR 419 bp Delano and RT-PCR 705 bp Upton, 1999 RT-PCR 206 bp 173 bp Candresse et al., 1997 Parakh et al., 1995 RT-PCR RT-PCR 1274 bp This work RT-PCR 243 bp Olmos et al., 2005 Real Time PCR 338 bp Martin et al., RT-PCR 512 bp 2009 RT-PCR 739 bp RT-PCR 203 bp Ratti et al., 2008 Nested PCR

Incidence of different stone fruit s viruses in Montenegrin peach 70.7% of samples were infected by at least one of ascertained viruses Single and double infections were confirmed, respectively, in 56.9% and 13.8% of the assayed plants Incidence of single and double infections not infected 29.3% duble infections 13.8% single infections 56.9% Molecular analysis revealed: Presence of PPV, PNRSV and PDV Double infection were found between PPV+PNRSV and PPV+PDV Absence of ACLSV, PMV, CMLV, SLRSV, TRSV and ToRSV from all tested samples

Incidence of PPV, PNRSV and PDV in examined peach cultivars Cultivar PPV PNRSV PDV PPV was detected in 60,3% of assayed samples PNRSV was detected in 18,9% of assayed samples PDV was detected in 1,7% of assayed samples Maria Marta Spring Belle Spring Crest Adriana May Crest Gloria Morsiani Rita Star Caldesi 83.3% 0% 0% 83.3% 0% 0% 75.0% 25.0% 0% 66.7% 0% 16.7% 66.7% 66.7% 0% 62.5% 0% 0% 62.5% 0% 0% 55.5% 55.5% 0% 0% 0% 0%

Detection of PPV by Real-Time RT-PCR 39 out of 58 samples showed positive results in Real-Time PCR 8 highly infected PPV isolates (from each infected cultivar) were chosen for further analysis Molecular variability is determined using RT-PCR and primers targeting CP region (1276 bp) 1276 bp negative control

Purification of PCR products Cloning in pgem-t Easy vector 8 PPV isolates were purified, cloned and sequenced Screening of colonies was performed by PCR using M13-for/M13-rev primers Purification of plasmid-dna ~1500 bp Sequencing Screening of colonies

Molecular characterization Nucleotide sequences of complete CP gene derived from Montenegrin isolates were compared with previously described PPV sequences deposited in GenBank (92,2 99,1%) BLAST analysis showed: MNE isolates from Gloria, May Crest, Adriana and Rita Star were the most closely related (97,1 99,1%) with isolate SK68 (M92280.1) MNE isolates from Morsiani, Maria Marta and Spring Crest shared most identity (97,6 98,4%) with previously reported Montenegrin isolate Godinje 1 (HQ452396) from region of Bar MNE isolate from cv. Spring Belle was most similar with sequence of Greek isolate N1 (FJ361234) from peach.

Phylogenetic analysis Phylogenetic tree was constructed using Neighborn-Joining method with bootstrap analyses of 1000 replicates within MEGA 4.1 software (Tamura et al., 2007) Phylogenetic tree was generated from complete nucleotide sequence of CP gene of 44 PPV isolates: 8 from this study 36 from NCBI database

To date 7 PPV strains were defined (PPV-D, PPV-M, PPV-Rec, PPV-EA, PPV-W, PPV-T and PPV-C) All isolates clustered within 5 strain groups (PPV-D, PPV-M, PPV-Rec, PPV-T and PPV-C) Phylogenetic analysis using sequences of complete CP gene showed that all Montenegrin isolates from peach belong to PPV-M strain

Molecular analysis for PNRSV Detection of PNRSV by RT- PCR using Ilar2F/Ilar1R primers 206 bp 11 out of 58 assayed peach samples were positive on PNRSV PNRSV was confirmed in 3 cultivars: Rita Star, May Crest and Spring Crest Highest infection rate (66,7%) was confirmed in cv. May Crest RT-PCR using Ilar2F/Ilar 1R primers

Molecular characterization 4 PNRSV isolates derived from cv.s Rita Star (2), May Crest (1) and Spring Crest (1) were chosen for further analysis Complete CP gene was amplified by RT-PCR using MG1/MG2 primers (Glasa et al., 2008) Isolates were cloned and sequenced Screening of PNRSV isolates by PCR using M13-for/M13-rev

Sequence analyses of CP gene from PNRSV isolates proved to be 89.3 100% identical with corresponding sequences of isolates previously described 2 PNRSV isolates from cv. Rita Star were most closely related to Chilean NctCl.augl isolate from nectarine (EF565253) PNRSV isolates from cv.s May Crest and Spring Crest were most closely related to Italian isolate PchIt.may1 from peach cv. May Crest

Sequences of 4 PNRSV isolates were deposited in GenBank (JX569825 - JX569828) Isolates clustered within three groups: PE-5, PV-32 and PV- 96 2 MNE isolates from cv. Rita Star clustered in PE-5 group Isolates from cv. May Crest and Spring Crest clustered in PV-96 Phylogenetic tree was constructed using minimum-evolution method with Tamura-Nei corrected nt distance and bootstrap analyses of 10000 replicates within MEGA 4.1 software

Molecular analysis for PDV RT-PCR method using PDV-2F/PDV-1R primers (173 bp) Presence of PDV was confirmed in only 1 out of 58 samples BLAST analysis revealed high similarity, ranging from 94.2 to 96%, with isolates reported from other parts of the world Highest value MNE isolates showed with Ch 137 isolate (L28145) 173 bp RT-PCR for PDV (CP)

Conclusions RT-PCR were used to identify infection by PPV, PDV, PNRSV, ACLSV, PMV, CMLV, TRSV, ToRSV and SLRSV in stone fruits. Nested-PCR (n-pcr) method was also used to detect SLRSV and Real-time RT-PCR to detect PPV The study reported presence of one quarantine virus (PPV) and two quality viruses (PDV and PNRSV) The study reported very high incidence of PPV (60.3%). PNRSV was detected in 18.9% and PDV in 1.7% assayed samples All other assayed peach viruses were not detected in the collected samples

Real Time RT-PCR identified more PPV infected samples than RT-PCR indicating higher sensitivity Amplification of different amplicons from the detected viruses by RT-PCR, followed by cloning of complete and partial sequences of CP gene allowed phylogenetic analysis of isolates of different detected viruses: PPV (8), PNRSV (4) and PDV (1) Sequence analyses revealed that all Montenegrin PPV isolates shared from 92.2 to 99.1% nucleotide identity with corresponding PPV-M strain sequences deposited in GenBank

Results indicate the most probable introduction of PPV in Montenegro through peach propagation material which is mainly imported from Greece where this strain is the most prevailing one Phylogenetic analysis suggesting two possible introductions of PNRSV in Montenegro Molecular grouping of PNRSV isolates wasn t in correlation with geographical region and host Urgent sanitation measures should be taken: eradication of infected trees severe control related to importation of plant material

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