日 植 病 報 62: 177-180 (1996) 短 報 Ann. Phytopathol. Soc. Jpn. 62: 177-180 (1996) Phytopathological Note PCR (Polymerase Chain Reaction) Detection of Phytoplasma in Phloem Sap Collected by Laser Stylectomy Mamoru SATO*, Shigetou NAMBA**, Maki KATSUHARA*,***, Hiromu KAWAKITA*, Wataru MITSUHASHI* and Susumu KAWABE*,•õ Key words: phytoplasma, rice yellow dwarf disease, PCR, laser stylectomy. Phytoplasmas are nonculturable Mollicutes associated the PCR method using the primer sets constructed by with diseases of several hundred plant species and are Namba et al.11) present in the sap of phloem sieve tubes. A phloem sap PCR detection of phytoplasma in phloem sap. can be collected from the inflorescens of certain groups Rice plants (Oryzae sativa cv. Kinuhikari) with brown of woody monocotyledons in Agavaceae and Palmae17) planthoppers (Nilaparvata lugens) were set in front of or from severed stylets of insects by the •gaphid tech- the condensing lens of a YAG laser system. The stylet nique•h, which is applicable to various kinds of plants bundle was cut by a pulse of the beam focused on the including woody or herbaceous species7). The phloem proboscis. The phloem sap exuding through the severed sap of rice plants can be obtained by a modified aphid stylet bundle inserted into the sieve element of the plant technique called •glaser stylectomy•h in which the insect was collected in a micro-capillary tube. A total of 0.5 to stylets are severed by YAG (yttrium-aluminum garnet) 1.0ƒÊl of phloem sap was collected from diseased plants, laser6). while 5 to 10ƒÊl was obtained from healthy plants. The On the other hand, polymerase chain reaction (PCR) collected phloem sap was stored at -20•Ž until PCR has been developed for the in vitro amplification of assay. nucleic acid sequences. Recent studies suggest the utility One ƒÊl of phloem sap collected from diseased or of PCR as a diagnostic tool for phytoplasma diseases. healthy rice plants were diluted in 20ƒÊl of distilled Deng & Hiruki (1991) reported that 16S rRNA genes of water and heated at 95•Ž for 5min. A total of 2.5ƒÊl of phytoplasmas could be amplified by PCR using specific the heated sample was used as the DNA templates for primers2). Then, specific oligonucleotide primer pairs PCR assay. In another method, about one ,ƒÊl of non- designed on the basis of well-characterized cloned DNA heated phloem sap was directly put into the PCR reac- sequences or from 16S rRNA sequences of animal tion mixture. The amplification was performed in a 50 mycoplasma have been employed in PCR detection of ƒÊl PCR reaction mixture containing 250ƒÊM each of phytoplasmas1,3,4,8-10,14,15) Moreover, Namba et al.11,12) dATP, dGTP, dCTP and dTTP, 1ƒÊM of each upstream described the detection, differentiation and phylogeny of and downstream primer, 5ƒÊl of 10•~PCR reaction buffer phytoplasmas by PCR analysis using specific primers (Promega Corp., Madison WI), 0.5U Taq DNA polymer- originating from 16S rRNA genes. However, there has ase (Promega Corp) and 100ƒÊl paraffin liquid. The not been any reports of direct PCR detection of phyto- universal primer set (SN910601, SN910502) and phyto- plasmas isolated in vitro. plasma specific primer set (SN910601, SN920204) were Recently, our research group reported that phyto- used in this experiment. They had been designed for plasmas were detectable in the phloem sap of rice plants detection of Mollicutes or phytoplasma, respectively by collected by laser stylectomy using a fluorescence Namba et al.11) The sequence of each primer is as technique with DNA fluorochrome 4•Œ-6•Œ-diamidino-2- follows: SN910601; GTTTGATCCTGGCTCAGGATT, phenylindole (DAPI)5). It strongly suggests that a new SN910502; AACCCCGAGAACGTATTCACC, detection system of phytoplasmas may be developed by SN920204; CCTCAGCGTCAGGTAA. Using the univer- combining the laser stylectomy with PCR assay. In the sal primer set, PCR was carried out for 50 cycles under present study, we investigated whether or not phyto- the following conditions: the first cycle, denaturation 90 plasmas in the phloem sap collected from rice plants sec at 94•Ž; ramping over 40sec to 60•Ž; annealing 2 with yellow dwarf disease can be detected directly by min at 60•Ž, ramping 30sec to 72•Ž, extension for 3min * National Institute of Sericultural and Entomological Science , Tsukuba 305, Japan 蚕 糸 ・昆 虫 農 業 技 術 研 究 所 ** Division of Agriculture and Agricultural Life Science , Graduate School, The University of Tokyo, Tanashi 188, Japan 東京大学大学院農学生命科学研究科 *** Present address: Research Institute of Bioresources , Okayama University, Kurashiki 710, Japan 岡 山 大 学 資 源 生 物 科 学 研 究 所 † Present address: Tohoku Agricultural Experiment Station , Morioka 020-01, Japan 東 北 農 業 試 験 場 178 日本植物病理学会報 第62巻 第2号 平成8年4月 Table 1. Phytoplasma detection in phloem sap col- 1 2 3 4 5 6 7 8 9 lected from yellow dwarf-diseased or healthy rice plants treated by PCR assay using univer- sal or phytoplasma specific primer sets a) The phloem sap was heated at 95•Ž for 5min. b) Figures show PCR-positive sample numbers against tested sample numbers. 1 2 3 4 5 6 7 8 9 10 11 12 13 Fig. 2. PCR amplification of 16S rRNA fragment of phytoplasmas in phloem sap collected from rice plants without any DNA extraction procedures. 1: Lambda phage DNA digested with HindIII (molecular weight marker). 2-3: Samples col- lected from rice plants with yellow dwarf dis- ease using the universal primer set. 4-5: Sam- ples collected from healthy rice plants using the universal primer set. 6-7: Samples collected from rice plants with yellow dwarf disease using the specific primer set. 8-9: Samples col- lected from healthy rice plants using the specific primer set. Arrows: 1.37kb and 0.75kb band amplified by PCR using the universal or specific Fig. 1. PCR amplification of 16S rRNA fragment of primer set, respectively. phytoplasmas from heating-treated phloem sap collected from rice plants using universal or phytoplasma-specific primer sets. 1: Lambda observed in 9 of 10 samples collected from diseased phage DNA digested with HindIII (molecular plants. In some samples, phytoplasmas were detected weight marker). 2-4: Samples collected from even when diluted 100-fold or 10-fold with distilled water rice plants with yellow dwarf disease using the universal primer. 5-7: Samples collected from (data not shown). However, no band was observed for all ten samples collected from healthy rice plants (Table 1 healthy rice plants using the universal primer. 8-10: Samples collected from rice plants with and Fig. 1). Similar results were obtained by the PCR assay using another phytoplasma specific primer set. All yellow dwarf disease using the specific primer. 11-13: Samples collected from healthy rice tested samples collected from diseased plants produced plants using the specific primer. Arrows: 1.37 0.75kb target DNA amplified by the primers, whereas kb and 0.75kb band amplified by PCR using the no bands were produced by samples collected from universal or specific primer set, respectively. healthy plants (Table 1, Fig. 1). Similar results were obtained even when non-heated samples were used as DNA templates for PCR assay at 72℃; the continuing cycles, denaturation 2 min at using two sets of primer pair (Table 1, Fig. 2). 94℃; the final cycle, extension for 7min at 72℃. Unidentified bacteria were sometimes isolated on LB Denaturation and annealing conditions were the same as agar plates from phloem sap of rice plants (unpublished for the first cycle. As for the phytoplasma specific data). To confirm the efficacy of the universal primer primer, thermocycling was done for 20 cycles. The sets for detecting phytoplasmas in the phloem sap, PCR annealing temperature was 40℃ and other conditions assay using template DNA of these bacteria prepared by were as mentioned above. heating (95•Ž, 5min) was attempted. No DNA band was Samples of phloem sap collected from diseased or detectable in any of the samples of bacterial strains healthy rice plants were heated and tested for PCR (data not shown). detection using the universal primer set. he 1.37kb Molecular analyses of PCR products. To DNA fragment of the 16S rRNA gene was predicted to confirm that the DNA fragments in phloem sap be amplified by PCR if the samples contained phytoplas- amplified by PCR had originated from 16S rRNA genes ma particles. A clear single DNA band (1.37kb) was of RYD-phytoplasma, both PCR products from •gphloem Ann. Phytopathol. Soc. Jpn. 62 (2). April, 1996 179 1 2 3 4 5 6 7 signature oligonucleotides of RYD-phytoplasma report- ed by Namba et al.12) No difference was found between the nucleotide sequences (data not shown). Our results revealed that RYD-phytoplasmas in the phloem sap collected from diseased rice plants by the laser stylectomy were detectable by PCR assay. This is the first report of PCR detection of phytoplasma parti- cles suspended in phloem sap isolated outside of plants. The detection method using DAPI stain described previously5) was very excellent, but our new detection method is superior in the reliability of phytoplasma- identification to that. Because, some species of bacteria were detectable sometimes in the phloem sap collected by the laser stylectomy (our unpublished data), which were easily stained by DAPI. The PCR using two sets of primers was clearly distinguishable phytoplasmas from these bacteria. Fig. 3. Restriction analysis of PCR products from Our newly developed method is unique in a respect of DNA in phloem sap amplified using the univer- not requiring any special phytoplasma-DNA extraction sal primer set. 1: Molecular weight markers (ƒÉ procedures to prepare template DNA. It required, but HindIII), 2: Non-digested, 3: EcoRV-digested, not essentially, the heating (95•Ž, 5min) of diluted 4: HincII-digested, 5: HhaI-digested, 6: HaeIII- phloem sap including phytoplasma-DNA.