X-DISEASE CONFIRMATION AND DISTRIBUTION IN CHOKECHERRY IN NORTH-DAKOTA

A phytoplasma was observed in diseased chokecherry plants in North Dak ota by electron microscopy and identified with a phytoplasma-specific polyclonal antibody and with restriction fragment length polymorphism (RFLP) analysis of 16S ribosomal DNA (168 rDNA) sequence amplified wit h the nested polymerase chain reaction (PCR). The phytoplasma was part ially purified directly from infected chokecherry plants for use in ra ising a polyclonal antibody in mice. The specificity of the polyclonal antibody against phytoplasma was shown by its reaction with diseased, but not with healthy, chokecherries upon immunofluorescence staining. Immunofluorescence staining demonstrated the chokecherry phytoplasma in North Dakota to be serologically related to phytoplasmas associated with eastern X, western X, milkweed yellows, and pigeon pea witches'- broom disease. The antibody did not react with phytoplasmas associated with ash yellows, aster yellows, elm yellows, goldenrod yellows, spir ea stunt, and palm lethal yellowing. The identity as X-disease phytopl asma was supported by RFLP analysis of the amplified products obtained from the nested-PCR by using two phytoplasma-universal primer pairs. The RFLP patterns from MseI and HpaII digestion indicated that the X-d isease phytoplasma in chokecherry belongs to subgroup A in the 165 rRN A group III. The presence of X-disease symptoms, electron microscopic observation of phytoplasmas in symptomatic plants, detection with the polyclonal antibody, and detection and identification by PCR-RFLP anal ysis provided the first confirmation that chokecherry X-disease occurs in the Great Plains region. Chokecherry X-disease was found to be wid espread in North Dakota based on positive serological detection of X-d isease phytoplasma in chokecherry samples collected throughout North D akota. X-disease in chokecherry was also confirmed in South Dakota and in Saskatchewan, Canada.