Morphology Of The Pathogen Of Citrus Huanglongbing In Japan And It's Detection By PCR

Citrus Huanglongbing caused by Candidatus Liberibacter is one of the most severe citrus diseases, which has been reported in many countries and areas. In Japan, citrus Huanglongbing was observed in the island of Iriomote in Okinawa and in the main island of Okinawa in 1988 and in 1994 respectively. The disease was also observed in kagoshima island recently. It is intend to spread to the north area.The prokaryote associated with citrus Huanglongbing was first observed in 1970 by Lafleche and Bove in the phloem of infected sweet orange leaves. The electron microscope is one of the most important direction methods to study the morphology and to detect the pathogen associated with citrus Huanglongbing. According to the sequence of 16S RNA, a fast and accurate method based on PCR has been developed to detect the pathogen and now it is the most frequently used method.Because the pathogen of citrus huanglongbing is restricted in the sieve tubes in phloem which is surrounded by xylem tissue, it is difficult to fix the vascular tissue. In order to get good fixation, we wiped off the xylem tissue and cut the slices into 1mm cubes. Application of a few drops of Fixative No.1(2% glutaraldehyde in 0.1M cacodylate buffer at pH7.2) to the tissue before cutting into slices can prevent sticking and drying and initiate the fixing process. The air in the plant tissues prevent the resin to reach the organelles, which makes the fixation difficult. During the pre-embedding process, application of vacuum will make a rather good embedding. The pathogen of citrus huanglongbing found in Japan was detected in ultrathin section of infected citrus leaves and a comparation with the pathogen found in other areas was also carried out. It has show that the pathogen associated with Japanese citrus huanglongbing differ from others in some characters as the pathogen is mostly in round or oval forms, rarely in filamentous forms, diameter in 100-500nm with a 10-50nm envelope. A rapid and accurate detection method, with an improved DNA extraction process and Ready-To-GoTM PCR procedure was also developed.