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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 chip-
bud 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
16 samples anaylsed
nucleotide divergence between 15 master sequences was 0.098% (one sample being negative). from single tree.
From six seasonal shoots
in different parts of the
Analysis of the nucleotide polymorphism in the different samples
collected from single plum tree, F= fruit, LA= apical leaf, LB= basal Since sequencing potentially leads to an underestimation of the tree, 6 basal leaves (LB)
leaf extent and structure of PPV intra-host genetic diversity, the 15 and 6 apical leaves (LA)
were collected. In
Sample Number of Haplotype Nucleotide Average number
PCR products were cloned and 105 individual cDNA clones addition, the skin of 4
sequences/Number of
haplotypes
diversity (h) diversity (Pi) of nucleotide
differences (k)
were sequenced (7 clones per sample). developed fruits (F) from
different parts of tree was
F1 7/6 0.952 0.00312 2.190 The average pairwise genetic distance within this dataset was also analysed.
F2 7/2 0.286 0.00081 0.571
F3 7/3 0.667 0.00149 1.048
0.304%, over three fold higher than the estimate obtained from
F4 7/3 0.524 0.00122 0.857 directly sequenced PCR products.
LA1 7/3 0.524 0.00163 1.143
LA2 negative In total, the 105 sequences yielded 51 variants, two of which
LA3
LA4
7/5
7/7
0.857
1
0.00326
0.00393
2.286
2.762
were highly predominant, representing respectively 29.5% and
LA5 7/4 0.714 0.00190 1.333 19% of the sequences. The other 49 sequence variants were
LA6 7/5 0.857 0.00244 1.714
LB1 7/4 0.714 0.00244 1.714 observed at significantly lower frequencies (0.9-3.8%).
LB2 7/5 0.857 0.00285 2.000
LB3 7/3 0.524 0.00081 0.571 Analysis of the nucleotide polymorphism in the different samples depending of plant organ
LB4 7/5 0.857 0.00204 1.429
organ Number of Haplotype diversity Nucleotide diversity Average number of
LB5 7/5 0.857 0.00204 1.429
sequences/Number of (h) (Pi) nucleotide differences
LB6 7/4 0.714 0.00163 1.143
haplotypes (k)

whole dataset 105/51 0.879 0.00304 2.132

Fruits (F) 28/12 0.836 0.00259 1.815

Apical leaves (LA) 35/21 0.864 0.00301 2.114

whole dataset 15/4 0.600 0.00098 0.685
direct PCR Basal leaves (LB) 42/22 0.892 0.00307 2.152
sequencing

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.

References:
This work was supported by the grant 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.
HUSK/0901/1.2.1/0126 and APVV-0042-10. 3. Domingo E. 2002. J. Virol. 76: 463-465

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