PCR (Polymerase Chain Reaction) Detection of Phytoplasma in Phloem Sap Collected by Laser Stylectomy Mamoru SATO*, Shigetou NAMB

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 phytoplasmas 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 universal 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 collected from rice plants with yellow dwarf disease 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 collected 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 reported 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. Whereas, con- digested. 7: SmaI-digested. Arrows: faint DNA ventional PCR detection methods of phytoplasmas bands. require complicated DNA-extraction procedures to pre-

pare template DNA. Our results also showed that the sap DNA•h and RYD-phytoplasma DNA extracted from plant components of phloem sap, at least from rice rice plants with yellow dwarf disease used as a positive plants, did not inhibit PCR amplification. Therefore, this control were analyzed by restriction digestion and direct system may be applicable to many kinds of plants if sequencing. Ten ƒÊl of reaction mixture including their phloem sap can be collected using laser stylectomy amplified DNA was purified using Takara Suprec-02 or other methods. We succeeded to detect phytoplasmas tubes and digested by restriction enzymes such as from the phloem sap of mulberry by the similar method EcoRV, HincII, HhaI, HaeIII, and SmaI. The molecular (unpublished data). weight was determined from fragments produced by the This new phytoplasma-detection system may be use- digestion of lambda phage DNA with HindIII (Toyobo ful for the study on in vitro cultivation of phytoplasma, Corp.). which is very important subject and has not yet been

The 1.37kb DNA from PCR using the universal possible. In the laser stylectomy, phytoplasmas as an primer set was clearly digested with HincII, HhaI and inoculum of primal culture are prepared in an ideal HaeIII, but not with SmaI and EcoRV. Two new frag- state, since phytoplasmas are suspended in pure phloem ments were observed with HincII and HhaI digestion, sap of •gthe natural medium•h of phytoplasma without and three with HaeIII digestion although the smallest any inhibitors from other plant organs except sieve DNA (77 bases) was missing from the gel (Fig. 3). The cells. Then, the growth of phytoplasma can be measured

0.75kb DNA from PCR using the phytoplasma-specific sensitively and specifically by PCR assay using two primer set was digested by HaeIII, but not by four other primer sets without any DNA extraction procedures. restriction enzymes. No differences were found in the We are presently conducting the in vitro cultivation of restriction digestion patterns between •gphloem sap phytoplasmas using this system. DNA•h and RYD-phytoplasma using the universal primer set and the specific primer set. Literature cited Moreover, part of the PCR-amplified sample DNA was purified by agarose gel electrophoresis as described 1. Ahrens, U. and Seemtiller, E. (1992). Detection of DNA of plant pathogenic mycoplasmalike organisms by a previously13) and sequenced with Sequenase Kit Version 2.0 (U.S. Biochemical Corp., Cleveland, OH) using syn- polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82: 828-832. thetic primers and 35S dATP. Manganese was used for 2. Deng, S. and Hiruki, C. (1991). Amplification of 16S the sequencing buffer to improve the extension accord- rRNA genes from culturable and nonculturable Mol- ing to the commercially supplied protocol16). The 1370- licutes. J. Microbiol. Methods 14: 53-61. nucleotide sequence of PCR product from •gphloem sap 3. Firrao, G., Gobbi, E. and Locci, R. (1993). Use of DNA•h was determined by direct sequencing of both polymerase chain reaction to produce oligonucleotide strands by using the 17 oligonucleotide primers12). This probes for mycoplasmalike organisms. Phytopathology nucleotide sequence was compared with the 16S rRNA 180 日本植物病理学会報第62巻第2号平成8年4月

83: 602-606. spring Harbor Laboratory Press, New York, pp. 164- 4. Gundersen, D.E., Lee, L.M., Rehner, S.A., Davis, RE. and 165. Kingsbury, D.T. (1994). Phylogeny of mycoplasma like 14. Schaff, D., Lee, I.M. and Davis, R.E. (1992). Sensitive organisms (phytoplasma): a basis for their classifica- detection and identification of mycoplasmalike organ- tion. J. Bacteriol. 176: 5244-5254. isms in plants by polymerase chain reactions. Biochem. 5. Kawabe, S., Kawakita, H., Nakashima, K. and Katsu- Biophys. Res. Commun. 186: 1503-1509. hara, M. (1991). Detection of mycoplasmalike organ- 15. Schneider, B., Ahrens, U., Kirkpatrick, C. and Seemul- isms in phloem sap collected from rice plants with ler, E. (1993). Classification of plant-pathogenic my- yellow dwarf disease. Ann. Phytopathol. Soc. Jpn. 57: coplasma-like organisms using restriction-site analysis 274-277. of PCR-amplified 16S rRNA. J. Gen. Microbiol. 139: 6. Kawabe, S., Fukumorita, T. and Chino, M. (1980). 519-527. Collection of rice phloem sap from stylets of homopter- 16. Tabor, S. and Richardson, C.C. (1989). Effect of manga- ous insects severed by YAG laser. Plant Cell Physiol. nese ions on the incorporation of dideoxynucleotides by 21: 1319-1327. bacteriophage T7 DNA polymease and Escherichia coli 7. Kennedy, J.S. and Mittler, T.E. (1953). A method for DNA polymerase. Proc. Natl. Acad. Sci. USA 86: 4076- obtaining phloem sap via the mouth-parts of aphids. 4080. Nature 171: 528. 17. Van Die J. (1975). In Phloem Transport (Aronoff, S. 8. Kirkpatrick, B.C., Stenger, D.C., Morris, T.J. and Pur- eds.), Plenum Press, New York and London, pp. 427-467. cell, A.H. (1987). Cloning and detection of DNA from a nonculturable plant pathogenic mycoplasma-like organ- 和文摘要 isms. Science 238: 197-200. 9. Lee, I.M., Davis, R.E., Scuclair, N., DeWitt, N.D. and 佐藤守・難波成任・且原真木・川北弘・三橋渡・河部 Conti, M. (1993). Genetic relatedness of mycoplasma- 遅:レーザ・スタイレクトミー法で採取した師管液中のphyto- like organisms detected in Ulmus spp. in the United plasmaのPCR法による検出 States and Italy by means of DNA probes and polymer- レーザ・スタイレクトミー法とPCR法を組み合わせた新しい ase chain reactions. Phytopathology 83: 829-833. 10. Lee, I.M., Hammond, R.W., Davis, R.E. and Gundersen, phytoplasma検出法の開発を行った。黄萎病(RYD)罹病イネを吸汁中のトビイロウンカの口針をレーザ光線で切断し,そこ D.E. (1993). Universal amplification and analysis of から流出する純粋な師管液をガラス細管に採取した。その師管 pathogen 165 rRNA for classification and identification 液中のphytoplasmaのPCR法による直接検出を試みた。師管 of mycoplasmalike organisms. Phytopathology 83: 834- 液をそのままで,あるいは95℃, 5分の熱処理を行った後,テン 842. プレートDNAとして用い,また,2組のプライマー・ペアーに 11. Namba, S., Kato, S., Iwanami, S., Oyaizu, H., Shiozawa, よりphytoplasmaの16S rRNA遺伝子領域(1.37kbと0.75 H. and Tsuchizaki, T. (1993). Detection and differentia- kb)をPCR増幅させた。その結果,罹病イネから採取したほと tion of plant-pathogenic mycoplasmalike organisms んどすべての標本から予想されたサイズのPCR産物が得られ using polymerase chain reaction. Phytopathology 83: たが,健全イネ由来のものからは全く得られなかった。PCR産 786-791. 物の制限酵素切断パターンおよび塩基配列の決定により,それ 12. Namba, S., Oyaizu, H., Kato, S., Iwanami, S. and らはRYD-phytoplasmaの16S rRNA遺伝子由来のものであ Tsuchizaki, T. (1993). Phylogenetic diversity of ることが証明された。以上により,レーザ・スタイレクトミー法 phytopathogenic mycoplasmalike organisms. Int. J. とPCR法を併用したこの新しい検出システムはphytoplasma Syst. Bacteriol. 43: 461-467. を鋭敏に,かつ特異的に検出出来ることが明らかになった。 13. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). (Received October 9, 1995; Accepted January 24, 1996) Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold