Characteristics of Pseudomonas syringae pv. actinidifoliorum causing bacterial leaf spot of Actinidia spp. in Japan

To investigate whether Pseudomonas syringae pv. actinidifoliorum (Psaf) exists in Japan, bacterial strains isolated from Actinidia spp. were screened using a PCR targeting the hopO1 gene. As a result, three Hokkaido isolates derived from leaf spots of Actinidia arguta and A. kolomikta, two Shizuoka isolates derived from leaf spots of A. arguta, and two Ehime isolates derived from a symptomless branch of A. deliciosa gave positive reactions. The Hokkaido and Shizuoka isolates were obtained by Ushiyama et al. (Ann. Phytopath. Soc. Japan 58: 426-430) in 1988. All 7 isolates were demonstrated to be pathogenic on leaves of A. deliciosa after inoculation of leaves and reisolation of the isolates, but they were not pathogenic on the stems. They were gram-negative, aerobic rods with one to three polar flagella, and formed entire colonies that were pale, yellowish, and circular. On the basis of biochemical and physiological characterization, MALDI-TOF MS analysis using the Bruker MALDI Biotyper system, PCR assays targeting pathogenicity-related genes, and a multilocus sequence analysis (MLSA) using concatenated sequences of four housekeeping genes (cts [= gltA], gapA, gyrB and rpoD), we identified these isolates as Psaf. However, the results of the MLSA revealed that these isolates clustered into three independent groups (the Hokkaido isolates, Shizuoka isolates, and Ehime isolates), each of which formed a monophyletic group that was separate from the four recognized lineages (lineages 1, 2, 3 and 4) of Psaf. Also, phenotypic characterization and PCR assays showed that the Hokkaido isolates utilized D-fucose, the Shizuoka isolates had relatively poor capacity for levan formation and did not possess hopF1, and the Ehime isolates gave a positive reaction in gelatin hydrolysis and did not have either hopE1 or hopF1, indicating that these three groups differed from the four recognized lineages. The combined results of genotypic and phenotypic analyses therefore support the classification of the Hokkaido isolates, Shizuoka isolates, and Ehime isolates as novel lineages of Psaf, for which the respective names 'lineage 5', 'lineage 6' and 'lineage 7' are proposed. We also propose the name 'bacterial leaf spot' for the symptoms caused by the isolates in lineages 5 and 6 on A. arguta and A. kolomikta. To our knowledge, this report is the first in Japan of leaf spot of Actinidia spp. caused by Psaf. Our results also show that Psaf strains are heterogeneous and that each lineage has its own specific characteristics, which can be utilized as markers to detect Psaf strains and to differentiate Psaf lineages.