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

Thank you for your attention!!!