3.7.33
16S RDNAS OF PAULOWNIA WITCHES' BROOM PHYTOPLASMA TRANSMITTED BY HALYOMORPHA MISTA

S OKUDA, Y NAKANO, T GOTO and T NATSUAKI

1Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan

Background and objectives
Paulownia witches' broom (PaWB) caused by a phytoplasma is the most serious problem in the production of paulownia (Paulownia tomentosa) timber in Japan. Experiments were performed (i) to reconfirm the transmission of PaWB by Halyomorpha mista, a species of the stink bug family Pentatomidae (Hemiptera:Heteroptera) [1], and (ii) to attain PaWB-infected periwinkle (Catharanthus roseus) plants for further studies.

Materials and methods
Non-viruliferous 2-4th instar larvae and adult bugs were fed for 3-9 days on PaWB-infected paulownia leaves for aquisition, and removed to healthy periwinkle seedlings or soybean seeds. After 28-40-day incubation on the healthy plants, the bugs were transferred to healthy periwinkle plants for inoculation. Proof of infection was given by phytoplasma-specific PCR, sequencing of DNA clones of PCR products and electron microscopy.

Results and conclusions
Faint symptoms of vein clearing and yellows appeared in leaves of twelve of 16 periwinkle plants 2-3 months after inoculation. Virescence was observed in 6 of 12 plants later. Electron microscopy revealed the presence of phytoplasma bodies in preparations from leaves and flowers which showed clear symptoms of phytoplasma infection. 16S rDNAs were amplified by PCR using universal and phytoplasma-specific primers from 12 plants with symptoms. DNA products by PCR from PaWB-infected paulownia plants, PaWB-inoculated periwinkle plant and viruliferous bugs were cloned into 11,4 and 4,clones, respectively, and sequenced. Two variants were involved in any of the three DNA samples. The results strongly suggest that these variants were transmitted from paulownia to periwinkle by the vector bug.

References
1. Shiozawa H 1986. Bulletin of the Research Institute of Japan Plant Protection Society 4,45-50.