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.

Pseudomonas allii sp. nov., a pathogen causing soft rot of onion in Japan.

Six phytopathogenic bacterial strains, MAFF 301512, MAFF 301513, MAFF 301514T, MAFF 301515, MAFF 301516 and MAFF 301517, were isolated from soft rot lesions of onion (Allium cepa L.) in Japan. The cells were Gram-reaction-negative, aerobic, non-spore-forming, motile with one or two polar flagella and rod-shaped. Analysis of their 16S rRNA gene sequences showed that they belong to the genus Pseudomonas, with the highest similarities to Pseudomonas poae DSM 14936T (99.86 %), Pseudomonas simiae OLiT (99.85 %), Pseudomonas trivialis DSM 14937T (99.79 %) and Pseudomonas extremorientalis KMM 3447T (99.79 %). Their genomic DNA G+C content was 60.9 mol% and the major fatty acids (>5 % of the total fatty acids) present were C16 : 0, summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), summed feature 8 (C18 : 1 ω7c /C18 : 1 ω6c) and C17 : 0 cyclo. Phylogenetic and phylogenomic analyses based on the rpoD gene and whole genome sequences, respectively, demonstrated that the strains belong to the Pseudomonas fluorescens subgroup, but form a monophyletic and robust clade, with Pseudomonas azotoformans as their neighbour. Between the strains and P. azotoformans, the average nucleotide identity scores were 95.63-95.70 %, whereas the digital DNA-DNA hybridization scores of the strains against their closest relatives, including P. azotoformans, were 65.4 % or less, which are lower than the 70 % cut-off for prokaryotic species delineation. The strains were differentiated from their closest relatives by phenotypic characteristics, pathogenicity in onion and cellular fatty acid composition. The phenotypic, chemotaxonomic and genotypic data showed that the strains represent a novel Pseudomonas species, proposed to be named Pseudomonas allii sp. nov., with MAFF 301514T (=ICMP 23680T) being the type strain.