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Evaluation of genetic diversity of Plum pox

virus in a single plum tree.
Conference Paper August 2011

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EVALUATION OF THE GENETIC DIVERSITY OF PLUM POX VIRUS

IN A SINGLE PLUM TREE.
Luk PREDAJA Albeta NAGYOV Zdeno UBR Miroslav GLASA

Institute of Virology, Slovak Academy of Sciences, Dbravsk cesta 9, 84505 Bratislava, Slovakia
Plum pox virus (PPV, genus Potyvirus, family Potyviridae) is an aphid-borne RNA virus infecting perennial stone fruits of Prunus spp. crops worldwide [1].
Despite substantial progress in the ability to analyze and describe PPV variability, less attention is paid to understand the PPV diversity and evolution within
single hosts. Dynamic genetic structure of virus populations has a significant role in the epidemiology of the virus, going up to the selection of variants with
increased pathogenicity [2]. The fast mutation and large population size of RNA viruses produce populations of viral genomes known as quasispecies [3]. The
quasispecies nature of PPV may imply a high adaptive potential, allowing for the rapid selection of biologically distinct variants with the highest fitness in new
environments.
A PPV-free two years old Prunus domestica cv. Oullins Gage tree (grafted on St. Julien rootstock) grown in a pot under controlled conditions was triple inoculated in May 2003 by chip-budding with PPV-M
(isolate VAR-2), PPV-D (isolate BOR-1) and PPV-Rec (isolate BOR-3) isolates. In spring 2004 the tree was replanted in an open field and let to develop untreated. In September 2010, seven years after chipbud inoculation, the tree was sampled for analysis of PPV diversity. A 746-bp fragment spanning the C-ter NIb/N-ter CP region was amplified using the TaKaRa LA Taq polymerase (TaKaRa, Bio Inc.) and
primer pair NCuniFor 5-GAGGCAATTTGTGCTTCAATGG-3 (sense) and NCuniRev 5-CGCTTAACTCCTTCATACCAAG-3(antisense). The RT-PCR products were directly sequenced and
simultaneously, an aliquot of the same PCR products were cloned into the pGEM-T Easy cloning vector (Promega) and 7 randomly chosen cDNA clones were sequenced for each PCR product (in total, 105
individual clones were sequenced). Sequence analyses were performed using the Molecular Evolutionary Genetics Analysis (MEGA v.4.1) and DNA Sequence Polymorphism (DnaSP v.5) software.

Successful inoculation of all 3 PPV isolates was documented by strain-specific RT-PCR one year post inoculation. From 2nd year on, only the PPV-M and PPV-Rec isolates could be
detected, PPV-D no longer being detected. 5th year post inoculation only presence of PPV-M could be detected.

The alignment of the sequences of 15 PCR products showed that 9 of them were identical. The other 6 sequences each
differed by a single point mutation. Only one substitution resulted in an amino acid change. The average pairwise
nucleotide divergence between 15 master sequences was 0.098% (one sample being negative).
Analysis of the nucleotide polymorphism in the different samples
collected from single plum tree, F= fruit, LA= apical leaf, LB= basal
leaf

Sample

Number of
sequences/Number of
haplotypes

Haplotype
diversity (h)

Nucleotide
diversity (Pi)

Average number
of nucleotide
differences (k)

F1

7/6

0.952

0.00312

2.190

F2

7/2

0.286

0.00081

0.571

F3

7/3

0.667

0.00149

1.048

F4

7/3

0.524

0.00122

0.857

LA1

7/3

0.524

0.00163

1.143

LA2

negative

LA3

7/5

0.857

0.00326

2.286

LA4

7/7

0.00393

2.762

LA5

7/4

0.714

0.00190

1.333

LA6

7/5

0.857

0.00244

1.714

LB1

7/4

0.714

0.00244

1.714

LB2

7/5

0.857

0.00285

2.000

LB3

7/3

0.524

0.00081

0.571

LB4

7/5

0.857

0.00204

1.429

LB5

7/5

0.857

0.00204

1.429

LB6

7/4

0.714

0.00163

1.143

0.879

0.00304

2.132

whole dataset 105/51

whole dataset
direct PCR
sequencing

15/4

0.600

0.00098

Since sequencing potentially leads to an underestimation of the

extent and structure of PPV intra-host genetic diversity, the 15
PCR products were cloned and 105 individual cDNA clones
were sequenced (7 clones per sample).
The average pairwise genetic distance within this dataset was
0.304%, over three fold higher than the estimate obtained from
directly sequenced PCR products.
In total, the 105 sequences yielded 51 variants, two of which
were highly predominant, representing respectively 29.5% and
19% of the sequences. The other 49 sequence variants were
observed at significantly lower frequencies (0.9-3.8%).

16 samples anaylsed
from single tree.
From six seasonal shoots
in different parts of the
tree, 6 basal leaves (LB)
and 6 apical leaves (LA)
were collected. In
addition, the skin of 4
developed fruits (F) from
different parts of tree was
also analysed.

Analysis of the nucleotide polymorphism in the different samples depending of plant organ
organ

Number of
sequences/Number of
haplotypes

Haplotype diversity
(h)

Nucleotide diversity
(Pi)

Average number of
nucleotide differences
(k)

Fruits (F)

28/12

0.836

0.00259

1.815

Apical leaves (LA)

35/21

0.864

0.00301

2.114

Basal leaves (LB)

42/22

0.892

0.00307

2.152

0.685

Conclusions:
Sequence analysis revealed that after 7 years of infection, only PPV-M was still detectable in the tree and that the two other isolates (PPV-Rec and
PPV-D) have been displaced.
Existence of PPV genetic variability within single tree was demonstrated.
Within PPV population, a total of 51 different haplotypes could be identified from the 105 individual sequences, two of which were largely
dominant. However, no clear-cut pattern of the viral population by the tree architecture could be highlighted.
The intra-isolate variability observed for the PPV-M isolate was consistent with the quasi-species theory of RNA virus populations.

This work was supported by the grant

HUSK/0901/1.2.1/0126 and APVV-0042-10.
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References:
1. Garcia, J.A., and Cambra, M. 2007. Plant Viruses 1:69-79
2. Garcia-Arenal, F et al. 2001. Annu. Rev. Phytopathol. 39:157-186.
3. Domingo E. 2002. J. Virol. 76: 463-465