0051-01-0021-0003-2.pdf2.12MB

Introduction

 Viroids are plant pathogens that are non-encapsulated, single-stranded, circular RNA molecules consisting of 246-475 nucleotides (Flores et al. 2012). They do not contain any coding regions for protein synthesis but instead replicate autonomously in susceptible cells (Flores et al. 2000). In chrysanthemum (Dendrnthema grandiflorum), two different viroids, Chrysanthemum stunt viroid (CSVd) (Pospiviroidae) and Chrysanthemum chlorotic mottle viroid (CChMVd) (Avsunviroidae), have been reported (Bouwen and Zaayen 2004; Hadidi and Candresse 2003; Matsushita et al. 2007).

 CSVd was firstly recognized in United States in 1945 and rapidly spread into all over the world. Currently it is one of the most serious pathogens affecting chrysanthemum cultivation worldwide (Flores et al. 1997; Lawson 1987). Since CSVd was firstly identified from chrysanthemum ‘Chunkwang’ varieties in 1997 in Korea, it has spread into many chrysanthemum cultivars throughout the country (Chung et al. 2001; 2005). Its major symptoms include reduced plant height and flower size, leaf blemishes and specks, yellowish pigmentations, early flowering, and photoperiod flowering response disturbances (Chung et al. 2005; Flores et al. 2005; Horst et al. 1977; Hosokawa et al. 2004). Disease incidence can vary and is highly dependent on both the cultivar and environmental conditions, especially temperature and light (Handley and Horst 1988). Further, the viroid can be transmitted by the seeds and pollens as well as plant sap on knives and pruning tools by grafting (Chung et al. 2001; Chung and Pak 2008; Matsushita et al. 2007). 

 As mass production of chrysanthemum is achieved by implantation of seedlings, it is necessary to diagnose CSVd infection in seedlings in order to prevent the introduction and propagation of CSVd disease into the field. However, early detection of CSVd infection is difficult due to the lack of disease symptoms during the breeding stage of chrysanthemum seedlings (Chung et al. 2005). It has been reported previously that RT-PCR and nested PCR are highly sensitive for the diagnosis of CSVd infection in seedlings, even at a very low level (Hadidi and Candresse 2003; Monger et al. 2010). In the present study, we conducted molecular diagnosis, using RTPCR and nested PCR, of CSVd infection rates in seedlings from various domestic or imported chrysanthemum varieties. In addition, we determined possible route of infection in the seedlings by comparing nucleotide sequences of identified CSVd from various cultivars.

Materials and Methods

Collection of Chrysanthemum samples

 Seedlings of spray-type chrysanthemum varieties were collected from greenhouse cultivars in Chilgok County in Gyeongbuk province in South Korea. All samples for RT-PCR were collected from 30 varieties, including 5 domestic, 6 Japanese, and 19 European varieties (Table 2).

Table 2. Chrysanthemum variants examined in this study

Extraction of nucleic acids

 Total RNA was extracted from young leaves (0.1 g) of various seedlings using either Trizol reagent (Invitogen, USA) or an RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. To eliminate DNA contamination, RNase-free DNase I was used according to the manufacturer's protocols. RNA concentrations of purified samples were determined using a Nanophotometer (Implen, Germany).

RT-PCR and nested-PCR

 RT-PCR and nested PCR were carried out according to Hosokawa et al. (2004), with modifications. Reverse transcriptase (RT) reactions were performed with 1 μg of RNA as a template for cDNA synthesis using High Capacity cDNA Reverse Transcription (Applied Biosystems) according to the manufacturer's instructions. PCR reaction was performed using 20 μl of Smart Taq Pre-Mix (Solgent, Korea) containing 1 μl of cDNA as a template and 10 pmoles of each primer set of either RT-PCR 1 or RT-PCR 2 (Table 1). The mixtures using RT-PCR 1 primer set were amplified for 40 cycles (94℃ for 30 sec, 53℃ for 60 sec, and 72℃ for 60 sec). The mixtures using RT-PCR 2 primer set were amplified for 35 cycles (94℃ for 30 sec, 62℃ for 30 sec, and 72℃ for 30 sec) using a PTC-200 thermal cycler (MJ Research, USA). The resulting RT-PCR products were separated on a 1% agarose gel and visualized by ethidium bromide staining.

Table 1. Oligonucleotide primer information of RT-PCR and nested PCR for the detection of CSVd in Chrysanthemum

 The nested PCR reaction was performed using 20 μl of Smart Taq Pre-Mix (Solgent, Korea) containing 1 μl of PCR product mixture as a template and 10 pmole of each primer. The mixtures were amplified for 35 cycles (94℃ for 30 sec, 62℃ for 30 sec, and 72℃ for 30 sec) using a PTC-200 thermal cycler (MJ Research, USA). CSVd primers for RT-PCR and nested PCR are listed in Table 1.

Nucleotide sequence analysis

 The nested PCR products were separated on a 1% LMP agarose gel, excised, purified using the Wizard PCR preps DNA purification system (Promega, USA), and subcloned into pGEM-T Easy vector (Promega, USA). Cloned cDNAs were sequenced using an Applied Biosystems 3100 Capillary DNA Sequence using BigDye Terminator Cycle Sequencing kit (Applied Biosystems, USA) at Solgent Sequencing Facility (Solgent, Korea). Computer analysis of DNA sequences was performed using DNASIS version 2.1 (Hitachi). Databases were searched using the BLASTN algorithm (Schaffer et al. 2001), and nucleotide sequences were aligned using CLUSTAL W (Thompson et al. 1994) online (http://www.ebi.ac.uk/Tools/ msa/clustalw2/). Prediction of the secondary structures of viroids was carried out using the WEB-based Mfold program Version 3.1 (Zuker 2003). Structures were generated based on the minimum energy folding principle of an RNA sequence (Zuker and Stiegler 1981).

Results and Discussion

 Here we demonstrated whether the combination of RT-PCR and nested PCR is successful to detect CSVd infection in various chrysanthemum seedlings. Seedling samples were collected from 30 varieties, including 5 domestic, 6 Japanese, and 19 European varieties (Table 2). Diagnosis was conducted in three seedlings of each variant, which were planted for 1 month and 2 months (Fig. 1 and Fig. 3). RTPCR analysis using two different primer sets did not show any band (either 354 bp or 253 bp) for CSVd in any of the tested samples, whereas nested PCR analysis detected CSVd infection (221 bp) in 10 varieties: one domestic, two Japanese, and seven European varieties (Fig. 1 and Table 2). The total CSVd infection rate was 33.3%. In addition, we assume that CSVd infection in seedlings is very low level because it is detected at the second nested PCR analysis but not at the first analysis of RT-PCR. Our results suggest that the combined analysis of RT-PCR and nested PCR is successful to screen CSVd detection in chrysanthemum seedlings. 

Fig. 1. Diagnosis of CSVd from selected seedlings of chrysanthemum varieties by RT-PCR using RT-PCR primer set 2 (A) and nested PCR (B). Leaves were collected from seedlings of various variants which are implanted for 1 month in the field. Positive control (+), Negative control (–), Confetti (1), Confetti dark (2), Froggy (3), Harley (4), Woodstar (5), Annecy lemon (6), Motown (7), Sound (8), Caitlynn (9), Cinzia (10), Fortune (11), Neva (12), Vatican (13), Mild ND (14), Orange ND (15), Power ND (16), Bride ND (17). Expected size of RT-PCR and nested PCR products (arrow heads) was 253 bp and 221 bp, respectively.

Fig. 3. The second diagnosis of CSVd from seedlings of 10 chrysanthemum varieties which are detected CSVd in the first diagnosis by RTPCR using RT-PCR primer set 2 (A) and nested PCR (B). Leaves were collected from seedlings implanted for 2 months in the field. Positive control (+), Negative control (–), Mild ND (1), Kohama (2), Shiju (3), Argus (4), Caitlynn (5), Cinzia (6), Ford (7), Fortune (8), Harley (9), Neva (10). Expected size of RT-PCR and nested PCR products (arrow heads) was 253 bp and 221 bp, respectively.

 Detection efficiency of plant viroids can be affected by various steps during the procedures of analysis such as sampling, grinding methods of tissues, nucleic acid extraction and assay specificity. CF11 cellulose column is used to improve efficiency of RNA extraction and purity of seed and woody plant tissues (Chung et al. 2009; Chung and Pak 2008; Shiwaku et al. 1996). Recently, real-time RT-PCR is used as a more powerful diagnostic technique and has been applied to diagnose various plant pathogens including viroids (Monger et al. 2010). Further researches are required to improve the detection efficiency of CSVd using real-time RTPCR protocols.

 To determine molecular variation among the identified CSVd samples, we conducted a comparison of CSVd nucleotide sequences from infected samples. We were unable to obtain RT-PCR products from seedlings of infected varieties using the primer set of Chung et al. (2001) (Table 1) even though PCR conditions are adjusted in various ways to improve detection efficiency such as annealing temperature and thermal cycles. Nucleotide sequence comparison of nested PCR products showed that the identified sequences consisted of two regions (from 1 to 145 and from 296 to 354 nucleotides) of the circular form of CSVd RNA molecules, and the sequences were highly conserved (98-100%) among the identified isolates (Table 3). However, there were nucleotide differences at three positions in several of the CSVd isolates. The major difference was at position 49-50 and could be divided into two groups. The first change was AC and appeared in eight varieties (Caitlynn, Cinzia, Ford, Harley, Kohama, Mild ND, Neva, and Shijue), whereas the other was GA and appeared in two varieties (Argus and Fortune). Another nucleotide substation appeared at position 321 (G A) in an isolate identified from the Caitlynn variant. A nucleotide deletion appeared at position 347 (U -) in an isolate identified from the Fortune variant. These results indicate that there are some nucleotide variations of identified CSVd in seedlings of the investigated chrysanthemum varieties. However, it is still uncertain whether these nucleotide differences are the characteristics of genetically different CSVd varieties which can express different pathogenesis. Since CSVd is firstly recognized in the United State in 1945, numerous CSVd varieties have been reported in Chrysanthemum and various other host plants including the families of Solanaceae, Asteraceae and Cucurbitaceae (Bouwen and van Zaayen 2004). In Korea, CSVd is firstly identified from Chunkwang variant (K1) in 1997 and currently at least 6 varieties, which have less than 98% nucleotide homology, have been identified in various chrysanthemum cultivars (Chung et al. 2001; Chung et al. unpublished observation). 

Table 3. Comparison of nucleotide sequences of nested PCR products of CSVd isolated from 10 chrysanthemum varieties in this study.

 Screening of the CSVd infection rate has been reported in various cultivated chrysanthemum varieties and wild species in Japan, and the presence of five sequence varieties has been identified based on differences in nucleotide sequences (Matsushita et al. 2007). Our predominant CSVd isolates containing AC at position 49-50 were identical in nucleotide sequence with variant 6 (AB006737), which was identified from C. morifolium in Hokkaido prefecture in Japan (Table 3). Further investigation of 31 CSVd sequences identified in the NCBI database showed that AC at position 49-50 only appeared in three more isolates (D88895, AB279771, and AB279769) reported in Japan. For the others, GA appeared in 24 isolates and CA in three isolates (V01107, NC_002015, and E13156) reported in Australia and Japan. It is believed that the sequence GA at position 49-50 is common in many identified CSVd sequences, including those of Korean strain K1 (AF394452), China strain (HQ891018), and Japan strain (X16408) (Shiwaku et al. 1996; Chung et al. 2001). This suggests that our CSVd isolate originated uniquely from Hokkaido in Japan. Among our eight chrysanthemum varieties infected by CSVd (AC), both Kohama and Siju were Japanese varieties; the other five varieties (Harley, Caitlynn, Cinzia, Ford, and Neva) were imported from European companies while one variant, Mild ND, was a domestic variant. This result suggests that seedlings were contaminated by CSVd during implantation process into the field. To confirm this, further diagnosis of CSVd infection was conducted on imported seedlings not implanted into the field (Fig. 2). Seedlings of two European varieties, Harley and Caitlynn, which showed CSVd infection in the first diagnosis, were subjected to RNA extraction before implantation into the field. Our results show that CSVd was not detected in those samples by either RT-PCR or nested PCR analysis. Thus, this result suggests that seedlings could be infected during implantation process rather than the seedlings themselves. CSVd can be easily transmitted by sap during handling and implantation process but not directly by soil (Hollings and Stone 1973). Thus, further research is required to investigate the possibility of CSVd contamination during implantation process such as hands, cutting knives.

Fig. 2. The diagnosis of CSVd from seedlings of two imported chrysanthemum varieties by RT-PCR using RT-PCR primer set 1 (A) and nested PCR (B). Leaves were collected directly from seedlings before implanting into the field. Positive control (+), Negative control (–), Caitlynn (C), Harley (H). Expected size of RT-PCR and nested PCR products (arrow heads) was 354 bp and 221 bp, respectively.

 According to our results, nucleotide position 126 was U in all identified isolates (Table 3). This was similar with variant 6 (Hokkaido prefecture), but different from the other five isolates from Japan (Matsushita et al. 2007). However, position 126 (C) was present in many CSVd isolates, including both China and Korea varieties. Korean isolate K1 had a substitution at position 126 (C) as well as at position 49-50 (GA). Thus, CSVd isolates of this study could be different from previous Korean isolate K1. This suggests that it could be often in an invasion of new types of CSVd in field. 

 Plant viroids share five structural and functional domains; P domain is associated with pathogenicity, V domain shows high sequence variability, and C domain has a highly conserved central region with two terminal domains (T1 and T2) that are interchangeable between viroids (Keese and Symons 1985) (Fig. 4). The nucleotide sequence 49-52 is located in the P domain, but it is unknown whether or not this nucleotide substitution (ACGA) is related with pathogenicity (Keese and Symons 1985). 

Fig. 4. Positions of nucleotide substitution and deletion of CSVd isolates identified in this study. Mutated nucleotides were positioned in the secondary structure of completed sequence of CSVd (AB006737). Regions of five domains are positioned as like left and right terminal domains (TL and TR), pathogenic domain (P), Central conserved domain (C) and variable domain (V).

 In summary, we screened CSVd infection in seedlings from 30 chrysanthemum varieties using both RT-PCR and nested PCR analysis. Molecular diagnosis indicated that 10 varieties were infected by CSVd. The nucleotide sequences were the most similar with an isolate (AB006737) identified in Hokkaido, Japan. However, there were a few mutations in the nucleotide sequences of some isolates. Our study suggests that viroid diagnosis of seedlings before cultivation is necessary to prevent the introduction and propagation of viroid infection into the field. 

Acknowledgements

 We thank Jung-Teck Park for providing various chrysanthemum varieties. The authors are grateful to Drs Jae-Dong Jung, Moo-Ung Jang for their scientific comments on an earlier draft. This study was supported by a grant of the Rural Development Administration in Korea.