Migration and Distribution of Graft-inoculated Jujube Witches'-broom Phytoplasma within a Cantharanthus roseus Plant

Department of Plant Medicine, Chungbuk National University, Cheongju 361-763, Korea(Received on February 14, 2012; Revised on April 29, 2012; Accepted on May 2, 2012)Periwinkle seedlings (Cantharanthus roseus) were ino-culated with jujube witches’- broom (JWB) phytoplasmavia grafting to analyze the migration of JWB phyto-plasmas within the host plant. The phytoplasmas weredetected using nested polymerase chain reaction (PCR)and fluorescence microscopy. Fluorescence microscopywas a simple and easy method of detecting phytoplasmas;however, it was not sufficiently sensitive to detect verylow phytoplasma concentrations. Therefore, the migra-tion of JWB phytoplasma was investigated throughPCR. The first migration of JWB phytoplasma from aninfected tissue to healthy tissues occurred late. Aftergrafting, the phytoplasmas moved from the inoculatedtwig (or scion) to the main stem, which took 28 days.Afterward, the phytoplasma migrated faster and tookless than 4 days to spread into the roots from the mainstem. All twigs were then successively colonized by theJWB phytoplasmas from the bottom to the top. JWBphytoplasma was detected via nested PCR in all parts ofthe periwinkle seedling 82 days after inoculation. Basedon these results, the inoculated JWB phytoplasmaappeared to migrate downward to the roots along themain stem during the early stages, and then continuedto move upward, colonizing twigs along the way untilthey reached the apex.Keywords : grafting, migration, nested PCR, periwinkle,phytoplasmaAmong the diseases of jujube (Zizyphus jujube Mill.) trees,the jujube witches’-broom (JWB) disease is the mostserious and destructive. JWB is a systemic disease, with theinfected trees showing typical symptoms of phytoplasmassuch as witches’-broom, phyllody, and yellows. The patho-gen, Candidatus Phytoplasma ziziphi, is usually present inhigh density in plant tissues with or without visible externalsymptoms, even though it is limited to the sieve elements ofthe plant (Jung et al., 2003; Kuske and Kirkpatrick, 1992;Nakashima and Hayashi, 1995). However, phytoplasma issometimes difficult to detect even in tissues that displaydistinct external symptoms. For example, phytoplasmacells were observed via transmission electron microscopy(TEM) in young inflorescences of coconut palms, but not inmature inflorescences, leaves, or stems with symptoms ofthe lethal yellows disease (Parthasarathy, 1974). This obser-vation may indicate the involvement of phytoplasma-produced or phytoplasma-induced substances in symptomexpression. The metabolism of infected plants is known tobe disturbed by phytoplasmas, resulting in the degenerationof infected sieve elements (Credi, 1994; Douglas, 1993).Phytoplasmas are obligate endoparasites of plant cellsand cannot be mechanically transmitted, thereby the studyof their biological and phytopathological characteristicshave been hindered (Kirkpatrick, 1992; McCoy et al., 1989)for a long time. After the discovery of phytoplasma as aplant pathogen, most studies focused on new diseases,including the means of transmission and disease control.Due to the fastidious nature of phytoplasmas, their di-stribution in the organs of an individual plant has not yetbeen well elucidated, even though they can be detectedfrom the phloem sieve tubes of an infected plant. Fortu-nately, the progress of molecular biological techniques suchas polymerase chain reaction (PCR) has made the detectionof relatively low concentrations of phytoplasmas possibleeven in plants with extremely mild symptoms or asympto-matic (Ahrens and Seemuller, 1992; Christensen et al.,2004; Garcia-Chapa, 2003; Lee et al., 1995). Several studies on the localization and migration ofphytoplasmas within infected plant tissues have been con-ducted (Cha and Tattar, 1991; Seemuller et al., 1984; Yi etal., 2001). About a decade ago, Wei et al. (2000) verifiedthat mulberry dwarf phytoplasma is distributed to an entirevein and petiole of a mulberry leaf by using nested PCR. Adynamic analysis of onion yellows phytoplasmas in Chry-santhemum coronarium was also conducted (Wei et al.,2004). However, little is still known on the distribution andmigration of phytoplasmas in plant organs, including roots,buds, flowers, and fruits. Hence, findings on the within-plant distribution of phytoplasmas and their migration from