Apple scar skin viroid (ASSVd), belongs to Apscaviroid, usually contains 330 nucleotides with the central conserved region but no enzyme activity, forming an asymmetrical rolling loop replication (Li and Sano, 2000). ASSVd was firstly reported to infect pome fruit trees (Malus, Pyrus and Cydonia spp.) (Hashimoto and Koganezawa, 1982). Subsequently, Chen et al (1986) also detected the viroid from the shoots of disease trees, cloned and analyzed its nucleic acid sequence and demonstrated its pathogenicity of this viroid, and then people have confirmed that apple scar skin disease is a disease cause of viroid (Zhou et al., 2002). Guo et al (2005) have cloned and analyzed the sequence of ASSVd inLiaoning province, China.
 

ASSVd mainly infected apple trees and pear trees (Hadidi et al., 1991; Yang et al., 1992; Koganezawa et al., 2003; Kyriakopoulou et al., 2003). Because the viroid was replicated and accumulated in nucleus of it’s hosts, it can be detected in the leaf, stem, epidermis and rootstock of the hosts disease-susceptibility, as well as the epidermis, pulp and seed of the fruits (Hadidi et al., 1991). Consequently, the fruit tree which was infected with the viroid would carry it all its life, and it can be spread via grafting and prune tools.
 

In this study, we detected and amplified 42 ASSVd isolates, and then the full length sequences of the 42 ASSVd were sequenced from apple, pear, apricot and peach trees in Xinjiang of China. The distribution of ASSVd was confirmed by in situ RT-PCR method. Thus, the results of our study also showed that ASSVd was a pathogen of peach and apricot trees. Meanwhile, we established the optimized detection methods of RT-PCR and in situ RT-PCR, which would provide a basis for the rapid identification of ASSVd.
 

1 Results and Analysis

1.1 Total RNA extraction

The conventional method of fast detection of RNA is high voltage electrophoresis, which can reduce the degradation of the RNA in a relatively short time. Gel electrophoresis analysis of the total RNA samples showed that the total RNA we obtained was in a good integrity and the value of OD₂₆₀/OD₂₈₀ was more than 1.80, which displayed that it was suitable for RT-PCR experiment (data was not shown here) (Figure 1).
 

Figure 1 Total RNA for apple, pear, apricot and peach trees

1.2 RT-PCR amplification products

Using specific primers for ASSVd, RT-PCR was carried out, and the expected fragments about 330 nt were obtained in these four kinds of trees.
 

1.2.1 RT-PCR detection for apple ASSVd

The specific product can be amplified in 15 strains by RT-PCR (Figure 2). The results showed as follows, ASSVd were detected in cultivars of Ralls (Aa3, Aa7 and Aa15), in cultivars of Red Fuji (Ad17, Ad23), and in cultivars of Starkrimson (Ae5, Ae23) in Yanqi county; similarly, in cultivars of Ralls (Aa98, Aa117) and Starkrimson (Ae35 ) in Korla region; in cultivars of Ralls (Aa123, Aa149), Red Fuji (Ad79) and Starkrimson (Ae86) in Heshuo regeion; as well as Aa176 of Ralls in Aksu. However, ASSVd were not detected in all sampling sites in Red delicious and Golden delicious cultivars.
 

Figure 2 RT-PCR detection results for apple ASSVd

1.2.2 RT-PCR detection for pear ASSVd.

The specific products were amplified in 13 trains by RT-PCR (Figure 3). The results showed that the ASSVd were detected in cultivars of Korla pear, Pa2, Pa14 and Pa27 and in cultivars of Apple pear, Pe3, Pe17, in Yanqi county; similarly, in cultivars of Korla pear, Pa39, Pa46, Pa52 and in cultivars of Apple pear, Pe93, Pe97, Pe116, in Korla region; as well as Pa63 of Korla pear and Pe136 of Apple pear in Heshuo regeion. However, ASSVd were not detected in all sampling sites in Jinfeng pear, Ya pear and Dangshan pear cultivars.
 

Figure 3 RT-PCR detection results for pear ASSVd

1.2.3 RT-PCR detection for peach ASSVd

With the same approach, 11 strains were obtained (Figure 4). The results demonstrated that the ASSVd were detected in cultivar of P. persica Sieb. et Zucc (Ta1, Ta4, Ta5, Ta9, Ta10, Ta12, Ta17, Ta21, Ta24, Ta25 and Ta29) only in Yanqi county. And the ASSVd were not detected in others sampling sites.
 

Figure 4 RT-PCR detection results for peach ASSVd

1.2.4 RT-PCR detection for apricot ASSVd

Similarly, we obtained 12 strains with specific product from 300 apricot samples (Figure 5). The ASSVd were detected in cultivar of apricot trees (X3, X7, X12, X14, X19, X22, X25, X27, X31, X34, X42 and X47) only in Yanqi County. And other sampling sites were not detected ASSVd at all.

Figure 5 RT-PCR detection results for apricot ASSVd

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

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).

Figure 6 Homology comparison of 42 cloned squences with NC_001340 (Puchta et al., 1990)

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 heavily in Yanqi County.

Table 2 Geographical distribution of viroids detected in Xinjiang province, China for plants assayed by RT-PCR for ASSVd

1.5 The ASSVd distribution of leaf tissues in four kind fruit trees

Blue-purple positive signals were detected in the nucleus of palisade tissues while it was not detected in control experiments (Figure 7). This data further showed that ASSVd was in the nulceus of leaf tissue cells.
 

Figure 7 Location of ASSVd in leaf tissues paraffin slice detected by in situ RT-PCR

2 Discussion

ASSVd can be spread via grafting and prune tools, it would cause a loss to fruit tree production if it widely spread. Although there is no effective method to prevent occurrence of the disease, we could discover the source of infection and handle it in time though suitable molecular detection methods.
 

In our study, the RT-PCR results showed that apple scar skin disease have occurred in Xinjiang of China. It has been shown that ASSVd can infect apple and pear tree (Zhu et al., 1995; Puchta et al., 1990; Guo et al., 2005). But there was no report that ASSVd was discovered in drupe trees. Our results showed that ASSVd was detectable in peach and apricot.
 

The results of cloning and sequence analysis showed a certain degree of sequence variation for these four kinds of fruit trees isolates of ASSVd in Xinjiang province, China. The 42 sequences of ASSVd had 85%~100% nucleotide sequence homology with NC_001340 and X17696 (Puchta et al., 1990) by the software DNAMAN. The analysis showed that the variation was small varieties of apple, peach and apricot trees and no significant differences in different regions, but showed that relatively significant difference in pear. In situ RT-PCR further confirmed the existence of ASSVd-specific nucleotide sequences in fruit trees. In addition, in situ RT-PCR showed that ASSVd was mainly distributed in the nucleus of leaf tissue cells.
 

Conclusionally, in this study, we established a good foundation for further study of the origin, recombination, evolution, geographical distribution, host specificity, structure periodicity and strain differentiation of viroids in these four kinds of fruit trees in the future.
 

3 Materials and Methods

3.1 Plant material and growth places

Tender leaves and shoots of apple, pear, apricot and peach samples were collected from Yanqi, Hejing, Bohu, Heshuo, Xinhe, Korla and Aksu in Xinjiang province, China. Biological characteristics of ASSVd samples were showed in table 3. 
 

Table 3 Sample information of apple, pear, apricot and peach trees for this experiment

3.2 RNA extraction and RT-PCR detection

Total RNA of ASSVd isolated from each sample was obtained as described previously (Zhao and Niu, 2006). Then the first-strand cDNA was synthesized by using reverse transcriptase (RT). Primers were designed according to the reported sequences by The National Center for Biotechnology Information of USA (NCBI, GenBank accession number X17696) (Puchta et al.,1990): ASSVd₁: 5′-CAGCACCACAGGAACCTCACGG-3′ (antisense, 10~32 sites); ASSVd₂: 5′-CTCGTCGTCGACGAAGG-3′ (sense, 80~97 sites); ASSVd₃:5′-CCTTCGTCGACGACGA-3′ (antisense, 82~97 sites), ASSVd₄: 5′-CCGGTGAGAAAGGAGCT GCCAGCA-3′ (sense, 98~121 sites) and were synthesized by Shanghai Biological Engineering Technology & Service Corp. The antisense primer ASSVd₁/ASSVd₃ was used as a primer for RT by Moloney Murine Leukemiavirus Reverse Transcriptase (Shanghai Biological Engineering Technology & Service Corp.) at 42℃ for 1 h in 50 mmol/L Tris-Cl pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl₂, and 10 mmol/L DDT. PCR was performed using a reaction mixture containing 10 mmol/L dNTPs, 2 mmol/L Mg²⁺, antisense and sense (ASSVd₁/ASSVd₃ and ASSVd₂/ASSVd₄ 20 µmol/L), cDNA (3 µg) and 2.5 U Taq DNA polymerase (Shanghai Biological Engineering Technology & Service Corp.). Samples were amplified for 32 cycles as follows: denaturation at 95℃ for 3 min, 62℃ for 45 s and 72℃ for 60 s, with the final extensions at 72℃ for 7 min. The products were denatured at 95℃ (or above) for 4 min after removal from the thermal cycler. The amplified products were separated by electrophoresis on 2% (w/v) non-denaturing agarose gel in 1×TAE buffer at 120 V for 40 min, and visualized by staining with Gold Viewer Dye (Beijing Liuyi Factory, China). PCR markers (Hua Mei, China) were used to determine the sizes of the amplified fragments. Expected fragments of about 330 nt were obtained through RT-PCR using specific primers for ASSVd.
 

3.3 Cloning and sequencing

The amplification fragments were separated by electrophoresis on a 2% agarose gel in 1×TAE buffer, excised from the gel under ultraviolet light and recovered using UNIQ-10 Gel Extraction Kit (Sangon) according to manufacturer’s protocol. The recovered fragments were cloned into pUCm-T vector, which is 2 773 nt in length with Amp resistance and enables blue/white color screening (Shanghai Biological Engineering Technology & Service Corp.), followed by transformation into competent E.coli cells. The desired clones were identified by restriction digestion and PCR after plasmid DNA isolated from the white colonies. Selected clones were then submitted to Shanghai Biological Engineering Technology & Service Corp. for sequencing. Sequences were analyzed by Basic Local Alignment Search Tool (BLAST) on www.ncbi.nlm.nih.gov and the DNAMAN software.
 

3.4 In situ RT-PCR

The distribution of ASSVd within leaf tissues of these four kinds of fruit trees was investigated by in situ RT-PCR. Paraffin sections were prepared according to the method of Niu et al(2007). Complementary DNA synthesis was performed according to the method of Zhao and Niu (2006) and in situ RT-PCR amplification and immunological detection used the methods of Niu et al(2007).
 

Authors’ Contributions

YTW and YZ designed this experiment and carried out the experiment; YTW also took part in the sequence analysis and wrote manuscript; JXN directed the experimental and modified the manuscript. All authors have read and approved the final manuscript.
 

Acknowledgments

This work was supported by the National Natural Science Foundation of China program (30360066) and the National Key Technologies R&D Program (2003BA546C).

Zhu S.F., Hadidi A., and Hammond R.W., 1995, Nucleotide sequence and secondary structure of pome fruit viroids from apple disease apples, pear rusty skin diseased pears and apple scar skin symptomless pear, Acta Horticulturae, 386: 554-559