Multiplex PCR Assay for Simultaneous Detection of Korean Quarantine Phytoplasmas

Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang 430-757, Korea(Received on August 23, 2011; Revised on October 16, 2011; Accepted on October 17, 2011)Multiplex PCR assays were developed for the simul-taneous detection of ten important Korean quarantinephytoplasmas. The species-specific primers were design-ed based on ribosomal protein, putative preproteintranslocase Y, immunodominant protein, elongationfactor TU, chaperonin protein and the 16S rRNA genesof ‘Candidatus (Ca.) Phytoplasma’ species. Three mainprimer sets were prepared from ten designed primerpairs to limit nonspecific amplification as much aspossible. The multiplex PCR assay using the threeprimer sets successfully amplified the correct conservedgenes for each ‘Ca. Phytoplasma’ species. In addition,ten important ‘Ca. Phytoplasma’ species could be easilydetermined by recognizing band patterns specific foreach phytoplasma species from three primer sets.Moreover, a high sensitivity of multiplex PCR for eachprimer set was observed for samples containing a lowDNA concentration (10 ng/µl). This study provides theuseful multiplex PCR assay as a convenient method todetect the presence of ten important quarantinephytoplasmas in Korea.Keywords : ‘Candidatus Phytoplasma’ species, Detection,Multiplex PCR, QuarantinePhytoplasma associated plant diseases occur in many cropsworldwide and more than 300 distinct plant diseases havebeen attributed to phytoplasmas, affecting hundreds of plantgenera (Hoshi et al., 2007). Phytoplasmas severely affectherbaceous and woody plants and are the primary limitingfactors for many important crops all over the world. Manyof the most important diseases from an economic stand-point are those affecting woody plants, including coconutlethal yellowing, peach X-disease, grapevine yellows andapple proliferation, and those affecting herbaceous plants,including vegetable crops, ornamental plants and legumes(Bertaccini and Duduk, 2009). Because of these diseases,the movement of the affected plant species should beinternationally restricted through quarantine regulations(Lee et al., 2000). In Korea, phytoplasma related diseases have been report-ed in about 50 different plant species and the phytoplasmaswere found to be associated with 6 ‘Candidatus (Ca.)Phytoplasma (P.)’ species including ‘Ca. P. asteris’, ‘Ca. P.pruni’, ‘Ca. P. trifolii’, ‘Ca. P. solani’, ‘Ca. P. castaneae’and ‘Ca. P. ziziphi’. Among them, ‘Candidatus Phyto-plasma asteris’ associated plant diseases were found toaffect the widest range of plants (Lee et al., 2004). TheKorea National Plant Quarantine Services (NPQS) listed 20phytoplasma diseases that should be prohibited and re-gulated including apple proliferation, strawberry witches’broom, walnut witches’ broom, etc. Those diseases wereassociated with 10 important ‘Ca. Phytoplasma’ speciesand among them; ‘Ca. P. asteris’ associated plant diseaseswere reported to be present in 54 plant families of 350 plantspecies (http://www.nqps.go.kr). Nowadays, molecularbased identification has been widely used to quickly detectpathogens. Nested PCR following PCR primed by phyto-plasma-universal primers are commonly used in phyto-plasma detection in Korea. Generally, identification of anunknown phytoplasma species from one disease requires asignificant amount of time. After PCR analyses, sequencingor restriction fragment length polymorphism analyses areneeded to further differentiate pathogen at the phytoplasmaspecies level. Thus, quick and efficient detection methodsfor ten important phytoplasma species are needed inregards to quarantine services. Simultaneous detection oftwo or more DNA targets can be achieved by duplex ormultiplex PCR in a single reaction by adding severalspecific primers into the PCR cocktail. Lopez et al. (2006)demonstrated that the multiplex PCR is a valuable tool fordetection and identification purposes in plant pathologybecause more than one pathogen frequently infect a singlecrop or host. Therefore, in this study, we developed amultiplex PCR assay by designing phytoplasma species-specific primer pairs based on various conserved genesincluding 16S rRNA, ribosomal protein gene operon (rp),