The first report of bacterial streak and rot of onion caused by Pseudomonas viridiflava in Japan.

In March 2020, a bacterial streak and rot symptom was observed on the onion (Allium cepa L.) leaves in Akita Prefecture of Japan. On the beginning, oval and dark-greenish water-soaked lesions with grayish-white necrotic center, 2-3 mm in diameter, appeared on the middle or the tip of upper leaves. Lesions, frequently surrounded by light yellow halo, expanded along veins and overlapped together. As lesions grew, the center of the lesions turned to light brown necrosis. The basal areas of diseased leaves often rotted, causing the withering of a whole leaf at last. From the water-soaked tissues of young lesions, a bacterium forming cream white colonies and producing fluorescent pigment on King's medium B was consistently isolated, and suggested to be a member of genus Pseudomonas. The isolates were positive for potato soft rot and tobacco hypersensitive reaction, and negative for levan production, oxidase and arginine dihydrolase activity, indicating that they belonged to LOPAT group II, Pseudomonas viridiflava, defined by Lelliott et al. (1966). P. viridiflava is known as a pathogen of bacterial streak and bulb rot of onion in United States and Uruguay, though it has not been reported in Japan. Four isolates were chosen for further examinations. Pathogenicity tests on onion leaves stubbed with selected isolates reproduced similar symptoms as observed in field's samples. After 2 days of inoculation, isolates produced water-soaking around the stabbed holes, developed into grayish-white necrosis on 4 days after inoculation. After 10 days of inoculation, lesions grew to necrotic streaks with light blown center surrounded by water-soaking and light yellow halo. Additionally, on onion scales, isolates caused water-soaked rot, yellow- to brown-colored, in 1-2 days after inoculation. From water-soaked areas of both of leaves and scales, inoculum was reisolated and fulfilled Koch´s postulates. In phenotypic properties, isolates showed consistent results as P. viridiflava strains identified in former studies. They were gram negative and aerobic, positive for hydrolysis of esculin, casein and gelatin, and negative for nitrate reduction and indole production. Regarding the utilization of carbohydrates, positive for 2-keto-gluconate, glucose, fructose, D-galactose, mannose, L(+)arabinose, glycerol, mannitol, sorbitol, myo-inositol, meso-erythritol, meso-tartrate, D(-)-tartrate, gluconate, n-caprate, dl-malate, citrate, L-arginine, L-aspartate, L-glutamate, and negative for D-arabinose, maltose, sucrose, raffinose, adonitol, trehalose, L-tartrate, L-rhamnose, acetate. The 16S rRNA gene sequences (Frank et al 2008) of four isolates (1,377 bp) showed 100% similarity as that of P. viridiflava type strain LMG 2352T (GenBank Accession No. Z76671) in BLASTN search. In phylogenetic analysis using gyrB (910 bp) and rpoD (801 bp) genes (Maeda et al 2006), isolates formed a cluster with P. viridiflava strains deposited in public databases, independent from other closely related Pseudomonas species. Sequences of 16S rRNA (GenBank Accession Nos. LC597475-LC597478), gyrB (LC597479-LC597482) and rpoD (LC597483-LC597486) genes were deposited in DNA Data Bank of Japan. According to these results, the isolates were identified as P. viridiflava (Burkholder 1930) Dowson 1939. This is the first report of the occurrence of bacterial streak and rot of onion caused by P. viridiflava in Japan, causing severe damage on onion growth.