First Report of Potato Blackleg Caused by Pectobacterium carotovorum subsp brasiliense in Japan

Potato (Solanum tuberosum) is one of the important main crops in Hokkaido, the northernmost island in Japan. In 2013 and 2015, potato plants (cv. Toyoshiro) with blackleg symptoms were found in seed potato fields in Tokachi subprefecture of Hokkaido. Blackleg and stem rot are caused by various bacterial species belonging to the genera Pectobacterium and Dickeya (Czajkowski et al. 2011). In Japan, P. carotovorum subsp. carotovorum (former scientific name: Erwinia carotovora subsp. carotovora), P. atrosepticum (E. atroseptica), and Dickeya sp. (E. chrysanthemi, species not specified), are the main causal agents of blackleg disease (Tanii 1984). Bacterial strains kbs-1 and kbs-2 were isolated in 2013, and strains pcbm-1, pcbm-2, and pcbm-3 were isolated in 2015 from symptomatic stems of potato plants in the fields. These strains were negative in PCR analyses using specific primers EXPCCF/EXPCCR for the detection of P. carotovorum subsp. carotovorum, Y45/Y46 for P. atrosepticum and ADE1/ADE2 for Dickeya (Czajkowski et al. 2011). In contrast, all strains yielded a specific amplicon (322 bp) in a PCR with primers Br1f and L1r for P. carotovorum subsp. brasiliense (Duarte et al. 2004). The 16S rDNA sequences of kbs-1 (DDBJ/GenBank accession LC146474), kbs-2 (LC146475), pcbm-1 (LC146476), pcbm-2 (LC146477), and pcbm-3 (LC146478) had 99% identities to P. carotovorum subsp. brasiliense strains (e.g., JF926720 in Canada, JF926723 in Brazil, and JF926718 in Syria) by BLAST analysis. In addition, a multilocus sequence analysis (Ma et al. 2007) was performed by MEGA6.06 using concatenated DNA sequences of seven housekeeping genes: acnA, gapA, icdA, mdh, mtlD, pgi, and proA from kbs-1 (LC145701 to 07), kbs-2 (LC145708 to 14), pcbm-1 (LC145715 to 21), pcbm-2 (LC145722 to 28), and pcbm-3 (LC145729 to 35). As a result, these five strains shared high identity with each other (>99%). The clade containing these five strains was consistently placed in a larger clade with the other P. carotovorum subsp. brasiliense and 100% bootstrap support for its separation from other Pectobacterium species available in GenBank in the consensus tree constructed using a maximum likelihood method. Pathogenicity of these strains against 60 potato plants (cv. Konahubuki) was confirmed in field experiments with seed tubers placed for 5 s in a bacterial suspension (1 × 107 cfu/ml). Four plants showed severe blackleg or tuber rot a month after planting; water-inoculated plants remained symptomless. Strains reisolated from the artificially diseased stems were confirmed as P. carotovorum subsp. brasiliense using the methods described. These strains produced indole and grew at 37°C, but did not produce acid from a-methyl glucoside or reduce substances from sucrose. On the basis of the disease symptoms, the cultural, molecular, and pathological features of the strains, we concluded that the blackleg symptoms of potato plants in Hokkaido in 2013 and 2015 were caused by P. carotovorum subsp. brasiliense. To our knowledge, this is the first report of P. carotovorum subsp. brasiliense as the blackleg disease agent of potato in Japan. It will be necessary to examine the epidemiology of P. carotovorum subsp. brasiliense because the occurrence of potato blackleg disease has increased recently in Japan. © The American Phytopathological Society.