Molecular Pathogens 2012, Vol.3, No.3, 12
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Figure 4 RT-PCR detection results for peach ASSVd
Note: Lane 1: Products amplified with primers ASSVd1 and
ASSVd2 primers from RNA of healthy peach leaf tissues; Lane 2:
Products amplified from RNA isolates from sample Ta1 using the
antisense primer ASSVd1 only; Lanes 3~13: fragments amplified
using both ASSVd1 and ASSVd2 primers from RNA isolates from
peach samples Ta1, Ta4, Ta5, Ta9, Ta10, Ta12, Ta17, Ta21, Ta24,
Ta25 and Ta29, respectively; Lane 14: DNA Marker
Figure 5 RT-PCR detection results for apricot ASSVd
Note: Lane 1: Products amplified with primers ASSVd1 and
ASSVd2 primers from RNA of healthy apricot leaf tissues; Lane 2:
Products amplified from RNA isolates from sample X3 using the
antisense primer ASSVd1 only; Lanes 3~14: Fragments amplified
using both ASSVd1 and ASSVd2 primers from RNA isolates from
apricot samples X3, X7, X12, X14, X19, X22, X25, X27, X31,
X34, X42 and X47 respectively; Lane 15: DNA Marker
X14, X19, X22, X25, X27, X31, X34, X42 and X47)
only in Yanqi County. And other sampling sites were
not detected ASSVd at all.
1.3
Cloning and sequence analysis of RT-PCR
products of ASSVd
Sequence analysis of the selected clones revealed
that the full-lengths of viroid in the four kinds of
fruit trees were 330 nt and exhibited the highest
homology with the ASSVd (accession number
X17696) using BLAST. The 42 sequences obtained in
this study were submitted to GenBank (accession
numbers EU031455~EU031496). The accession
numbers of the 12 sequences on apple trees were
EU031455~EU031466, thereinto, nine of the twelve
sequences were 330 nt, the other three sequences were
315
nt, 317 nt and 328 nt, respectively. The accession
numbers of the 10 sequences on pear trees were
EU031467~EU031476, thereinto, four of the ten
sequences were 327 nt, three of the 10 sequences
were 320 nt, two of the 10 sequences were 321 nt and
the other one was 250 nt. Similarly, there were
EU031477~EU031486 in the 10 sequences on peach
trees with five of the ten sequences 330 nt, four of the
10
sequences 331 nt and the other one 332 nt. As well
as there were EU031487~EU031496 in the 10
sequences on apricot trees and all of them were 330 nt.
The detection rates were 2.1%, 2.1%, 2.8% and 3.3%
on apple, pear, peach and apricot trees, respectively
(
Table 1).
Table 1 Four kinds of fruit trees in Xinjiang of China tested by
RT-PCR for ASSVd
Sort of
fruit trees
No. of the
tested
plants
No. of the
tested positive
samples
No. of sequences
that submitted to
GenBank
Apple
570
15
12
Pear
480
13
10
Peach
360
11
10
Apricot
300
12
10
Total
1710
51
42
The ASSVd sequences obtained in this study were
97.97%, 88.64%, 98.14%, 98.35%
identical to
NC_001340 (Puchta et al.,
1990)
from apple, pear,
peach, apricot trees, respectively. The alignments of
ASSVd in these four kinds of fruit trees showed
variability in positions mainly at the left-terminal
region (TL), the pathopoiesis region (P), the variable
region (V) and the right-terminal region (TR). The 42
isolates had 85%~100% nucleotide sequence
homology with NC_001340 (Puchta et al., 1990) by
the software DNAMAN (Figure 6).
1.4
Analysis of geographical distribution of ASSVd
detected in Xinjiang province, China
All the samples were collected from Yanqi, Hejing,
Bohu, Heshuo, Xinhe, Korla and Aksu in Xinjiang,
China. It can be learned that the distribution of
ASSVd was mainly in Yanqi County and a little in
Korla, Heshuo and Aksu by using RT-PCR method,
and ASSVd were not detected in other places (Table 2).
The detection rates were 8.5%, 2.2%, 1.9% and 0.4%
in Yanqi, Korla, Heshuo and Aksu, respectively.
Therefore, we informed that the fruit trees in Xinjiang
province were infected with ASSVd, especially
