Introduction
Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV), and Apple stem pitting virus (ASPV) are major viruses of apple which lead to economicdamage and top working disease (Campbell, 1963; Posnette et al., 1963; Schmidt, 1972; Yanase, 1983; Zahn, 1996; Wang et al., 2011). All three viruses belong to genera within the Betaflexiviridae, with ASGV belonging to genus Capillovirus, ACLSV to the genome Trichovirus, and ASPV to the genome Foveavirus.
These three viruses are transmitted by grafting in the field or mechanically to diagnostic species (e.g. Chenopodium quinoa or Chenopodium amaranticolor etc.) (Yanase, 1983; Llacer et al., 1985; Kinard and Scott, 1996). ASGV (under the synonym Citrus tatter leaf virus) has been reported to be transmitted through seed of lily and C. quinoa (Inouye et al., 1979), and at a low rate in seed of citrus (Tanner et al., 2011). Viruses or viroids that are able to transmit through apple seed include Apple latent spherical virus (ALSV), Tomato bushy stunt virus (TBSV), Tobacco mosaic virus (TMV), Apple scar skin viroid (ASSVd), and Dapple apple viroid (DAV) (Allen, 1969; Hadidi et al., 1991; Nakamura et al., 2011). However, there were no reports of seed transmission in apple of any of the three viruses examined here.
We tested apple seedlings to check the possibility of seed transmission of the above three viruses. Apple seedlings were germinated from seeds of three commercially sold apple cultivars, and RT-PCR was used for virus detection. ASGV was detected in two seedlings but ACLSV and ASPV were not detected.
The ASGV genome is composed of a positive sense single stranded RNA with a length about 6,495-6,497 nucleotides (Yoshikawa et al., 1992). Open reading frame 1 (ORF1) encodes the replication-associated protein (Rep) and coat protein (CP) as a continuous polyprotein separated by a variable region. Rep contains several conserved domains (methyltransferase, papain-like protease, RNA helicase, and RNA-dependent RNA polymerase) and is translated directly from genomic RNA. Although the 27 kDa CP sequence is present in the ORF1 polyprotein, CP is expressed from a 3’-co-terminal subgenomic RNA (Magome et al., 1997; Tatineni et al., 2009). ORF2 encodes the 37 kDa movement protein (MP) which is located between Rep and CP in a different reading frame, overlapping the variable region of ORF1.
To carry out further analysis of the detected ASGV, seven coat protein clones (of isolate S) were isolated and sequenced. There was a conserved nucleotide at CP position 299 and amino acid at CP residue 100, which differ from other reported ASGV CP sequences. Phylogenetic tree analysis suggests that the detected ASGV-S CP is grouped with those of other Korean ASGV isolates reported in 2015 (Han et al., 2015).
Materials and Methods
Apple seeds
All apple seeds were obtained from commercially sold apple fruits during September to December in 2014. Cultivars were Busa, Hongok, and Aori, and mixed randomly. Obtained seeds were washed and kept at 4℃ until used. In order to break seed dormancy, seeds were soaked in water and kept at 4℃ for 4-7 days. Subsequently, seeds were placed on wet tissue in petri dishes covered with aluminum foil and kept at 24℃ until seeds germinated. Germinated seeds were planted in soil and kept at 24℃ under light for 16 hours and darkness for 8 hours.
Diagnosis
Apple leaves were frozen in liquid nitrogen and ground with beads. 50-100 mg of ground tissue were mixed with 450 µl RLT buffer from the RNeasy Plant Mini Kit (Qiagen) containing 4.5 µl of 2-mercaptoethanol. Total RNA extraction steps were performed according to the manufacturer’s protocol. Total RNAs were then reverse transcribed into cDNA using SuPrimeScript RT Premix (2x) (GENET BIO CO., Ltd) as recommended by the manufacturer’s protocol. PCR was performed in a 20 µl total volume containing 1 µl cDNA, 10 pmol of virus-specific primers (Table 1), 2 µl of 10X Reaction Buffer, 2 µl of 10 mM dNTPs mixture, and 1 unit of Prime Taq DNA polymerase (GENET BIO CO., Ltd). The PCR conditions were as follows: 5 min at 94 °C for pre-denaturing, 37 cycles of 30 sec at 94 °C for denaturing, 30 sec at 56 °C for annealing, and 30 sec at 72 °C for extension, followed by 5 minutes at 72 °C for final extension. Plants were recorded as positive if PCR products of the expected sizes (Table 1) were obtained, and no products were obtained from negative controls.
|
Table 1. Primers used in this study.
|
|
|
zPrimers designed based on alignment of multiple ASGV sequences (Han et al., 2015) |
|
Cloning and Sequencing
The ASGV CP gene was amplified from cDNAs of ASGV positive samples, with PCR performed as described above with ASGV CP specific primers (Han et al., 2015; Table 1), except that 30 cycles were carried out, with a 1-minute extension time. The PCR products were cloned into pGEM®-T Easy vector (Promega CO., LTD.). Plasmids with PCR product inserts were confirmed using restriction enzyme digestion with SalI and BamHI (New England BioLabs® Inc.). Positive plasmids were sequenced by Macrogen Inc.
Sequence analysis and phylogenetic tree construction
The nucleotide sequences and amino acid sequences were compared using DNAMAN software (Version 5. 2.10, Lynnon BioSoft). Phylogenetic trees were constructed using neighbor-joining method with 1,000 bootstrap replicates in the MEGA version 6 (Tamura et al., 2013). 48 ASGV CP nucleotide sequences were obtained from NCBI GenBank. Cherry virus A (CVA; genus: Capillovirus) CP nucleotide sequence was used as an outgroup to root the ASGV CP phylogenetic tree.
Results and Discussion
Diagnosis of apple viruses
Seeds of three commercially obtained apple cultivars (Busa, Hongok, and Aori) were germinated and tested to detect apple viruses. Total RNAs from 3-6 true leaves were subjected to RT-PCR using three sets of virus-specific primers for ASGV, ACLSV, and ASPV (Table 1). Two of nineteen seedling plants were positive for ASGV. Neither ACLSV or ASPV were detected in any plants (Table 2).
Comparison of ASGV CP sequences
Complete ASGV CP genes were cloned and sequenced for analysis of sequence variability. A total of seven ASGV CP sequences were determined from the seedlings (isolate S: S8, S20, S22, S27, S31, S33, and S38, respectively). All of them showed slightly different nucleotide sequences. However, S20, S22, and S31 had an identical sequence at the amino acid level. Overall sequence identities were 99.84% at the nucleotide level and 99.76% at the amino acid level.
ASGV CP isolate S sequences were compared with reported ASGV CP sequences from multiple countries and hosts (Table 2). Nucleotide and amino acid sequence results showed that a single amino acid sequence variation was observed only in the seven isolate S clones. All isolate S clones showed thymine at nt 299, whereas previously reported ASGV CP sequences showed cytosine at that position (Fig. 1). Amino acid sequence corresponding to nt 299 showed Isoleucine for all clones of isolate S, compared to Threonine for all previously reported ASGV CP sequences.
Phylogenetic tree analysis
An ASGV CP phylogenetic tree was constructed using nucleotide sequences of seven isolate S clones with other sequences from multiple countries and hosts (Fig. 2). All isolate S clones were grouped with seven Korean isolates, three Chinese isolates from apple, two isolates from kiwi, and an Indian isolate from apple. Average nucleotide sequence identity among all isolates used for constructing the phylogenetic tree was 95.92%. Average nucleotide identity of the group of all isolate S sequences and their most closely related isolates was 99.81%.
