Sample collection
In May 2009, China ixeris fasciation was observed in Guanzhong Area, Shaanxi Province, China. To verify disease aetiology, 30 symptomatic and three asymptomatic plants were collected in field. Some China ixeris plants were also transplanted into an insect-proof greenhouse for observation of symptom development.
Transmission electron microscopy
Fasciated stems of ca. 2 × 2 mm2 from symptomatic samples were processed for transmission electron microscope examination. The tissues were fixed in a buffer (pH 7.2) containing 3% (v/v) glutaraldehyde and 4% (v/v) paraformaldehyde, incubated at 4°C for 4 h, and subsequently in 1% (v/v) osmium tetroxide at room temperature for 2 h. Then the fixed samples were dehydrated in concentration gradients of ethanol (10-70%) and acetone (0-100%), and finally were embedded in Epon 812 Kamińska et al., (2001). The ultra-thin sections were stained with uranyl acetate and led citrate, and then examined.
PCR amplification
Total DNA of each sample was extracted following cetyltrimethyl ammonium bromide (CTAB) method (Kollar et al., 1990), and used as template in PCR assays. Total DNA of samples infected with a phytoplasma related to wheat blue dwarf phytoplasma (WBDp) (16SrI-C subgroup) (Wu et al., 2010) and chinaberry witches’ broom phytoplasma (CWBp) (16SrI-B subgroup) (Wu et al., 2010) were as positive controls.
Primers P1 (Deng and Hiruki, 1991) and P7 (Schneider et al., 1995) were used in amplification of phytoplasma 16S rRNA gene, spacer region between 16S and 23S rRNA genes and the start of 23S rRNA gene. The PCR products were diluted 1: 29 with sterile double-distilled water prior to the nested amplification using the general primer pair R16F2n/R2 (Gundersen and Lee, 1996). Each PCR mixture (25 μL) contained: 2 μL DNA template (10 ng/uL), 1 μL (10 pM) of each primer, 2 μL dNTP (2.5 mM), 2 μL MgCl2 (25 mM), 2.5 μL 10 × Taq buffer and 1 U Taq DNA polymerase (Thermo Fisher Scientific Inc.), and sterile double-distilled water to the final volume. The PCR amplification program was as follows: preheating at 94°C for 3 min, and then subjected them to 35 amplification cycles, of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min, with a final elongation of 72°C for 10 min.
Amplification of phytoplasma tuf gene was primed by primer pair fTufu/rTufu (Schneider and Gibb, 1997). The reaction mixture was set as above. After 3 minutes’ preheating at 94°C, 30 amplification cycles were carried out: denaturation at 94°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 1 min, with a final elongation of 72°C for 10 min.
PCR products were separated in 1% agarose gel by electrophoresis, stained with ethidium bromide and visualized using UV transilluminator.
Cloning, sequencing and sequence analysis
PCR products of 1.8 kb (phytoplasma 16S rRNA gene) and 0.8 kb (tuf gene) were purified using a commercial PCR Purification Kit (Bio Teke Corporation, Beijing, China) and cloned. The clones contained recombinant plasmid were selected by blue-white screen and for each sample, three clones were selected and sequenced by TaKaRa Biotechnology (Dalian) Co., Ltd. The primers used for sequencing of 16S rRNA gene were M13F(-47): CGCCAGGGTTTTCCCAGTCACGAC/M13R(-48): AGCGGATAACAATTTCACACAGGA and of tuf gene were M13F(-77): GATGTGCTGCAAGGCGATTA/M13R(-48), which were designed based on the sequence of pMD18-T simple vector and offered by the TaKaRa Biotehnology Co. Ltd.
Sequences were aligned using the Lasergene software (version 7.0; DNASTAR, Madison, USA) and used for searching against the database of National Center for Biotechnology Information (NCBI) by BLASTn.
Published phytoplasma sequences were retrived from GenBank; 33 sequences of 16S rRNA gene and 17 sequences of tuf genes from groups 16SrI, -III, -V, -X and -XII were selected. Phylogenetic trees were built by neighbor-joining (16S rRNA) or maximum parsimony (tuf gene) methods with a 1000-replicate bootstrap search using MEGA4 (Saitou and Nei, 1987; Tamura et al., 2007).
The nested PCR products of 16S rRNA gene (1.2 kb) from symptomatic samples were concentrated, and digested with eight restriction enzymes Alu I, Bfa I, Hae III, Hha I, Hpa II, Kpn I, Mse I and Rsa I (Lee et al., 1998). The digested PCR products were separated in 8% polyacrylamide gel by electrophoresis and visualized using UV tranilluminator after ethidium bromide staining.