Pseudomonas marginalis is one of the well-known pathogens of bacterial rot of onion (Allium cepa L.), Welsh onion (A. fistulosum L.), and rakkyo (A. chinense G.Don) (Iitomi and Umekawa 1983; Ohuchi et al 1983, Nishiwaki 2017; Tominaga and Tsuchiya 1958; Umekawa and Iitomi 1983). On onion, the disease symptoms start with dark green and water-soaking small spots on leaves in winter or spring. As the disease progress, water-soaking lesions coalesce into large ones accompanying light yellowish-green or light brown chlorotic area, then expand along veins with wave shaped margins. When the lesions reach to leaf sheathes and bulbs, it causes pink or brown colored soft rot and diseased plants die prematurely (Ohuchi et al 1983). On these reports, the bacterial isolates were identified as P. marginalis based on pathogenicity, bacteriological properties or 16S rRNA sequence. Later, several strains of onion isolates were reclassified as Pseudomonas allii (Sawada et al 2021), and Welsh onion isolates as Pseudomonas kitaguniensis (Sawada et al 2020). Therefore, P. kitaguniensis is considered as Welsh onion pathogen in Japan.
From February to June of 2020 to 2022, rot symptoms appeared on onion plants growing in fields in Iwate and Akita Prefecture. Onions were planted in fields in autumn of the previous year. The symptoms were water-soaking rot on leaves, and soft rot on leaf sheathes and bulbs (Fig. 1). On the beginning, dark green water-soaking rot was observed on the middle of leaves. The lesion gradually spread, and the color of the lesion turned to yellowish-green or light brown, producing necrotic area on the center of the lesion. The margins of water-soaking lesions were wavey. On the severe infection, water-soaking rot extended to leaf sheathes. Inside leaf sheathes and bulbs, pink or light brown colored soft rot was observed, though there was no odor unique to the soft rot caused by Pectobacterium carotovorum. As the disease progressed, leaves and sheathes dried and died, and then bulbs were disappeared with the outer brown scale left. These symptoms were corresponded to ‘spring rot’ of Ohuchi et al (1983), and similarly observed on samples from Iwate in February of 2020, Akita in March of 2021 and June of 2022. From diseased legions, bacterial ooze eructed under microscopic observation.
Total 34 isolates were obtained from independent plant samples of Iwate in 2020 (20 isolates), Akita in 2021 (11 isolates), Akita in 2022 (3 isolates). Isolation of bacterium was conducted using the section from the margin of water-soaking rot legion and healthy region, on yeast-peptone (YP) agar plates according to the method of Tsuji and Takikawa (2018). All isolates produced fluorescent pigment on King’s medium B (King et al 1954), therefore, preliminary examinations to discriminate Pseudomonas strains producing fluorescent pigment was conducted (Lelliott et al 1966). As the results of preliminary examinations, all isolates showed positive results on levan production, oxidase activity, potato soft rot and arginine dihydrolase; negative on tobacco hypersensitive reaction, indicating that isolates belong to LOPAT group IVa. Additionally, all isolates showed ability to cause water-soaking rot on onion bulb scales when tested following to the method of Tsuji and Kadota (2020). According to these results, isolates were considered to belong to P. marginalis sensu lato (Sawada et al 2023).
To clarify the phylogenetic positions of onion isolates in P. marginalis sensu lato, bacteriological characterization and genetic analyses were conducted using representative seven isolates (TS20-14, TS20-19, TS20-24, TS20-29, TS21-01, TS21-05, and KD2C) and reference strains (Table 1). In addition, ability to cause water-soaking rot symptoms on onion scales in different temperatures were investigated, because in the preliminary test isolates produced more severe water-soaking rot on onion scales than reference strains of identified P. marginalis (MAFF301377 and MAFF301674), in lower temperature than 28 °C (the temperature that bacterial strains are usually incubated). As the references, additional to two strains above, P. kitaguniensis MAFF212408T and MAFF301498, P. grimontii MAFF212084 and MAFF301378, P. allii MAFF301512, P. palleroniana MAFF301416, P. extremorientalis MAFF302031 and MAFF302398 were used. All the reference strains belong to P. marginalis sensu lato (Sawada et al 2023). Detail information of reference strains are shown in Genebank project, NARO database (https://www.gene.affrc.go.jp/index_en.php).
At first, bacteriological properties were evaluated as described by Suzuki et al. (2003), Takikawa et al. (1989) and Tsuji and Kadota (2020). Bacteriological properties of seven isolates did not vary among strains, showing corresponding results as reference strains of P. kitaguniensis (MAFF212408T and MAFF301498). Seven isolates and reference strains of P. kitaguniensis were distinguished from the reference of other species by utilization of D-arabinose as a sole carbon source (Table 2).
Genetic analyses were conducted using 16S rRNA, rpoD and gyrB gene sequences of seven isolates and reference strains as follows. Genomic DNA was extracted using a SimplePrepTM reagent (Takara Bio, Shiga, Japan) based on the manufacturer’s instructions. The 16S rRNA gene was amplified according to the method of Kusumoto et al. (2004), with primers fD1-sat (5’-AGAGTTTGATCCTGGTCAG-3’) and rP2-sat (5’-ACGGCTACCTTGTTACGACTT-3’) described by Takahashi et al. (2013). Housekeeping genes were amplified as described by Maeda et al. (2006) for rpoD gene with primers 70F2 (5’- ACGACTGACCCGGTACGCATGTAYATGMGNGARATGGG-3’) and 70R2 (5’-ATAGAAATAACCAGACGTAAGTTNGTRTAYTTYTTNGCDAT-3’), and Yamamoto et al. (1999) for gyrB gene with primers UP-1E (5’-GAGGAAACAGCTATGACCAYGSNGGNGGNAARTTYRA-3’) and AprU (5’-TGTAAAACGACGGCCAGTGCNGGRTCYTTYTCYTGRCA-3’). PCR conditions were followed to Tsuji and Takikawa (2018). Amplified products were sequenced using Applied Biosystems® 3500xL genetic analyzer (Thermo Fisher Scientific K.K., Tokyo, Japan) in Biotechnology center of Akita Prefectural University. Sequenced data were deposited in the DNA Data Bank of Japan (DDBJ) database (https://www.ddbj.nig.ac.jp) with the accessions indicated in Fig. 2 and 3 in parentheses. Accessions for reference sequences from public databases are also in Fig. 2 and 3. 16S rRNA gene sequences of reference strains were retrieved from Genebank project, NARO. Phylogenetic trees were constructed using both of the neighbor-joining method and the maximum likelihood method based on the Jukes-Canter model and the Tamura-Nei model in MEGA 7.0 (Kumar et al. 2016). In the phylogenetic tree constructed using 1376-bp of 16S rRNA gene (Fig. 3), seven isolates and reference strains of P. kitaguniensis formed an independent branch. In the tree based on concatenated sequence of rpoD (804-bp) and gyrB (910-bp) gene (Fig. 2), isolates and P. kitaguniensis reference strains formed a clearly discriminated cluster from other species of P. marginalis sensu lato. Although the topology among P. marginalis sensu lato varied depending on the methods or models used, the isolates consistently formed a branch only with the references of P. kitaguniensis.
Based on the results of bacteriological characterization and genetic analyses, these isolates from spring rot of onion were identified as P. kitaguniensis. This report will be the first report of P. kitaguniensis causing rot disease on onion, though P. kitaguniensis has been considered as the pathogen of Welsh onion in Japan.
As mentioned in the preliminary examinations, P. kitaguniensis isolates caused severe and large water-soaking rot symptoms on onion scale sections, when placed at room temperature (10–20 ºC) than in the incubator at 28 ºC. Therefore, ability of isolates to produce water-soaking rot on onion scales in different temperatures was tested. Onion bulbs were surface sterilized after outer brown scales and roots removed, then cut lengthwise to obtain appropriate sections. Two sections were placed in a plastic food cup with a lid (129 mm in diameter and 68 mm in depth), with the inner side to top. P. kitaguniensis isolates and the references were cultured on PPGA (Nishiyama and Ezuka 1977) medium for 24–48 h at 28 °C, then suspended in sterilized distilled water at approximately 2.0×107 cfu/ml. Two onion scales in each cup were stabbed with a sterilized tip of a micropipette, and 20 μl drops of bacterial suspension were put on the stabbed holes. Onion scales were incubated at 10, 15, 20, 25, 30 °C, respectively. Examination was repeated five times at each temperature. After inoculation of 24 h and 48 h, the size of water-soaking area was measured using the software Leafareacounter Plus (https://www.vector.co.jp/soft/win95/business/se488466.html).
As the result, 24 h after inoculation, P. kitaguniensis isolates and reference strains (MAFF212408T and MAFF301498) caused water-soaking rot on onion scales at every temperature that they were incubated. Among the references, P. grimontii MAFF301378 and P. extremorientalis MAFF302031 caused water-soaking rot symptoms in this method. Severity of water-soaking rot caused by P. kitaguniensis isolates was various among isolates. At 24 h after inoculation, water-soaking rot areas were larger at 15–25 °C than at 10 and 30 °C. At 48 h after inoculation, areas of water-soaking rot became much larger at 10–25 °C, especially in 10–20 °C, though that was relatively constant at 30 °C. Reference strains of P. kitaguniensis showed similar tendency (Fig. 4). According to these results, P. kitaguniensis strains cause strong water-soaking rot symptoms at comparatively lower temperature, 10–25 °C, than at the usual temperature of incubation, suggesting the relationship with the season that they cause ‘spring rot’ on onion plants. To support P. kitaguniensis ability to cause water-soaking rot at lower temperature, the production of pectinase at 15 °C and 30 °C was investigated. The examination was conducted using polygalacturonic acid, following to Tsuyumu et al (1989). However, P. kitaguniensis and the references did not show pectinase activity on the medium, though the reference strain (Pectobacterium carotovorum pv. carotovorum TS20-127) did (data not shown). This suggested that P. marginalis sensu lato may exhibit the ability to cause water-soaking rot on onion scales when they inhabit the host plant, or that other mechanisms are concerned to water-soaking rot symptoms on onion scales.
In Iwate and Akita, located in the northern part of Japan, it is difficult to use bacteriocides in winter and early spring season because fields are covered with snow. ‘Spring rot’ symptoms caused by P. kitaguniensis were already observed in February, near the end of winter in Japan, therefore, further study is needed to control this disease.