Pathogenicity assay
In both, scale and bulb pathogenicity assays, the bacterial strain 20TX0166T caused a pathogenic response, inducing necrosis of the fleshy scales (Fig. S3) and whole bulbs (Fig. 1). Two isolates (20TX0166b from fleshy scale and 20TX0166c from bulb) with similar morphology to the original isolate were re-isolated from each of the two pathogenicity tests. When these re-isolated daughter strains were tested further using the scale and bulb assays, the symptoms they produced were identical to that of the original strain. After sequencing the 16S rRNA gene, the daughter strains (GenBank OM863553 and OM863552) were confirmed as identical to the original strain.
Phylogenetic analysis based on 16S rRNA
Upon searching on BLAST, the 16S rRNA gene sequence (GenBank accession number OK275102) of the strain 20TX0166T shared 100% similarity with Curtobacterium flaccumfaciens LMG 3645T (Table 1). The novel strain sequence also had > 99% similarity with the sequences of type strains of six other species of Curtobacterium. Such a high level of similarity makes it difficult to assign 20TX0166T as one of the previously documented species based on 16S rRNA gene similarity. In the phylogenetic tree created using 16s rRNA sequences, the novel strain did not produce separate branching from the C. flaccumfaciens LMG 3645T, indicating the strain was most closely related to this species (Fig. 2). However, previous studies on Brevundimonas and Streptomyces have reported that multiple bacterial species with distinct ecological niches can share identical 16S rRNA gene sequences (Antony-Babu et al. 2017; Jaspers and Overmann 2004). Also, in a taxonomical classification study conducted using 1142 actinobacterial type strain genomes (Nouioui et al. 2018), it was suggested that similarity based on a single gene sequence cannot be fully relied for species identification. Thus, the genome of the novel strain isolated from rotting onion was analysed further to confirm the species identity.
Table 1
Comparison of 16s rRNA gene similarity, digital DNA-DNA hybridization (dDDH), and average nucleotide identity (ANI) of Curtobacterium allii 20TX0166T, obtained from a rotting onion bulb in Texas, USA with the most similar species of Curtobacterium.
Strain
|
16S rRNA similarity (%)†
|
dDDH (d4, %)‡
|
OrthoANIu (%)§
|
ANIb (%)¶
|
Curtobacterium flaccumfaciens LMG 3645T
|
100.0
|
63.3
|
95.7
|
95.4
|
Curtobacterium pusillum DSM 20527T
|
99.5
|
28.3
|
84.9
|
84.4
|
Curtobacterium oceanosedimentum ATCC 31317T
|
99.5
|
26.4††
|
83.3††
|
83.0††
|
Curtobacterium ammoniigenes NBRC 101786T
|
99.3
|
20.1
|
75.6
|
76.4
|
Curtobacterium luteum DSM 20542T
|
99.3
|
25.9
|
83.3
|
82.8
|
Curtobacterium citreum DSM 20528T
|
99.3
|
26.3
|
83.4
|
83.1
|
Curtobacterium albidum DSM 20512T
|
99.1
|
26.4
|
83.6
|
83.2
|
Curtobacterium herbarum DSM 14013T
|
98.3
|
25.3
|
82.4
|
82.2
|
†, ‡, §, ¶ Values were computed using BLAST, TYGS webserver, OAT software v0.93.1, and pyANI, respectively. |
†† The dDDH and ANI values were calculated using the genome of C. oceanosedimentum strain NS263, as genome for the type strain was not available. |
Phylogenetic analysis based on Whole genome
The genome (GenBank accession number JAIWKR000000000) of the novel strain was estimated to be 3,986,426 nt (35 contigs, N50 = 502,963 nt, and the longest contig = 865,032 nt) with a G + C content of 70.8%. The draft genome consisted of 3,762 coding DNA sequences (CDS), 55 tRNA genes, 3 rRNA genes, and 1 tmRNA gene. The othoANIu, ANIb and dDDH values (Table 1) between the novel strain and C. flaccumfaciens LMG 3645T were 95.7%, 95.4%, and 63.3%, respectively, which were lower than the minimum threshold values of 96% (ANI) and 70% (dDDH) for species determination (Auch et al. 2010; Ciufo et al. 2018; Wayne et al. 1987). Although the ANI values between the novel strain and C. flaccumfaciens LMG 3645T were closer to the species delineation threshold, the dDDH value between them was well below the threshold, which was a scenario similar to Sawada et al. (Sawada et al. 2021). Also, the novel species formed a separate branching away from C. flaccumfaciens LMG 3645T on the phylogenetic tree, supported with a 100% bootstrap value, indicating 20TX0166T is a novel species (Fig. 3).
Upon comparing the 35 strains deposited as C. flaccumfaciens in the NCBI Genome database (Table 2), the 15 strains (CFBP 3418, BRIP 70607, DSM 20129, BRIP 70606, BRIP 70601, BRIP 70614, BRIP 70615, VKM Ac-1386, CFBP 8820, P990, VKM Ac-1795, BRIP 70624, CFBP 8825, CFBP 8821, and 208) had dDDH and ANI values greater than the species cut-off threshold of 70% and 96%, respectively, compared with C. flaccumfaciens LMG 3645T, suggesting the 15 strains are all C. flaccumfaciens strains. However, the dDDH and ANI values of 6 strains (Cff1037, CFBP 1384, CFBP 8822, CFBP 2402, CFBP 3401, and CFBP 3423), compared with C. allii 20TX0166T were greater than the species cut-off threshold of 70% and 96%, respectively, suggesting the species assignations should be moved from C. flaccumfaciens and reclassified as C. allii. While the remaining 14 strains (BRIP 70610, CFBP 3417, CFBP 2403, MEB126, CFBP 3422, UCD-AKU, CFBP 3415, S5.26, CFBP 8818, CFBP 8819, CFBP 8823, CFBP 8824, CFBP 3400, and Jub65) had dDDH and ANI values lesser than the species cut-off threshold compared to both C. flaccumfaciens LMG 3645T and C. allii 20TX0166T, indicating these 14 strains may potentially be new species. A phylogenetic tree was created using the TYGS web server to show the relationships among these strains (Fig. S4). The 14 strains branched into 5 species clusters, which indicated these could be classified potentially as five new species.
Table 2
Genomic assessment of bacterial strains designated as Curtobacterium flaccumfaciens in NCBI, compared with the novel strain 20TX0166T obtained from a rotting onion bulb in Texas, USA, and C. flaccumfaciens LMG 3645T.
|
dDDH (d4, %)†
|
OrthoANIu (%)‡
|
|
Strain
|
20TX0166T
|
LMG 3645T
|
20TX0166T
|
LMG 3645T
|
Curtobacterium species status
|
Cff1037ѱ
|
83.7
|
64.5
|
98.3
|
95.9
|
C. allii
|
CFBP 1384
|
82.2
|
63.5
|
98.1
|
95.7
|
C. allii
|
CFBP 8822
|
80.5
|
63.9
|
97.9
|
95.8
|
C. allii
|
CFBP 2402
|
80.4
|
63.9
|
97.9
|
95.7
|
C. allii
|
CFBP 3401
|
80.4
|
63.8
|
97.8
|
95.7
|
C. allii
|
CFBP 3423
|
77.0
|
65.0
|
97.5
|
95.9
|
C. allii
|
CFBP 3418
|
63.2
|
100.0
|
95.6
|
99.9
|
C. flaccumfaciens
|
BRIP 70607
|
62.9
|
77.2
|
95.3
|
97.3
|
C. flaccumfaciens
|
DSM 20129
|
62.8
|
77.0
|
95.4
|
97.4
|
C. flaccumfaciens
|
BRIP 70606
|
62.9
|
76.3
|
95.6
|
97.5
|
C. flaccumfaciens
|
BRIP 70601
|
63.0
|
76.2
|
95.6
|
97.4
|
C. flaccumfaciens
|
BRIP 70614
|
62.9
|
76.2
|
95.6
|
97.4
|
C. flaccumfaciens
|
BRIP 70615
|
62.8
|
76.1
|
95.6
|
97.5
|
C. flaccumfaciens
|
VKM Ac-1386
|
61.8
|
75.6
|
95.3
|
97.3
|
C. flaccumfaciens
|
CFBP 8820
|
63.2
|
75.5
|
95.6
|
97.3
|
C. flaccumfaciens
|
P990ф
|
63.0
|
75.4
|
95.6
|
97.3
|
C. flaccumfaciens
|
VKM Ac-1795
|
62.6
|
75.3
|
95.6
|
97.2
|
C. flaccumfaciens
|
BRIP 70624
|
63.4
|
75.1
|
95.7
|
97.2
|
C. flaccumfaciens
|
CFBP 8825
|
63.4
|
75.0
|
95.6
|
97.3
|
C. flaccumfaciens
|
CFBP 8821
|
63.4
|
73.9
|
95.6
|
97.1
|
C. flaccumfaciens
|
208
|
63.5
|
73.2
|
95.8
|
97.0
|
C. flaccumfaciens
|
BRIP 70610
|
58.3
|
61.6
|
94.9
|
95.3
|
Potential new species
|
CFBP 3417
|
50.7
|
54.5
|
93.3
|
94.1
|
Potential new species
|
CFBP 2403
|
50.5
|
53.1
|
93.3
|
93.9
|
Potential new species
|
MEB126
|
50.5
|
53.1
|
93.4
|
93.9
|
Potential new species
|
CFBP 3422
|
50.8
|
53.0
|
93.3
|
93.9
|
Potential new species
|
UCD-AKUδ
|
50.7
|
53.0
|
93.3
|
93.8
|
Potential new species
|
CFBP 3415
|
50.3
|
53.0
|
93.3
|
93.8
|
Potential new species
|
S5.26
|
50.8
|
52.9
|
93.4
|
93.8
|
Potential new species
|
CFBP 8818
|
50.4
|
52.5
|
93.3
|
93.8
|
Potential new species
|
CFBP 8819
|
50.4
|
52.5
|
93.3
|
93.7
|
Potential new species
|
CFBP 8823
|
50.1
|
52.4
|
93.2
|
93.7
|
Potential new species
|
CFBP 8824
|
50.4
|
51.0
|
93.4
|
93.4
|
Potential new species
|
CFBP 3400
|
32.7
|
32.9
|
87.4
|
87.4
|
Potential new species
|
JUb65
|
28.8
|
28.8
|
85.4
|
85.5
|
Potential new species
|
†, ‡ Values were computed using TYGS web server and OAT software v0.93.1. dDDH and OrthoANIu values > 70% and > 96%, respectively are highlighted in bold font. |
ѱ (O’Leary and Gilbertson 2020). ф (Chen et al. 2021). δ (Flanagan et al. 2013). |
Physiological and chemotaxonomic analyses
The cells of 20TX0166T were Gram positive (Fig. S5), non-spore-forming rods (Fig. S6), obligately aerobic, non-motile, catalase positive, and oxidase negative. The TEM images showed that bacterial cells were rod-shaped with an absence of flagella, 1.5-2.0 µm long x 0.6-1.0 µm wide (Fig. 4). No soft rotting of potato slices was observed after 24 h at 22°C, indicating no pectolytic activity by strain 20TX0166T. The tobacco hypersensitivity test was negative, in which the strain did not elicit a hypersensitive response, indicating the bacterium is non-pathogenic to tobacco.
Growth was observed at 4 to 39°C but not at 41°C on all media tested. The optimum temperature for growth was 25°C and, although growth was observed at 4°C, the rate of growth was slow as it took 5–7 d to form visible colonies at that temperature. After 24 h of incubation at 25°C on YPGA and NBYA media, the bacterium produced 0.5-1.0 mm wide, smooth, creamy, round, and entire colonies. Colony size increased to 2–3 mm in 3 d, and became somewhat mucoid with light yellow pigmentation (Fig. S7). Bacterial growth was observed at a pH of 5–10 with an optimal pH of 7. Growth was observed on YPGA medium with up to 9% NaCl, while optimum growth was observed at 0 to 1% NaCl.
Significant differences were noted in the Biolog, API Coryne, and API 20NE test reactions between the novel strain and C. flaccumfaciens LMG 3645T, as well as the type strains of other related species (Table 3). These results further support that the onion strain is a novel species of Curtobacterium. The comprehensive results of the Biolog, API Coryne, and API 20NE of the novel strain and the closely related species are provided in Table S1. The major fatty acids profile from FAME analysis were C15:0 anteiso and C17:0 anteiso (Table 4). Other fatty acids detected in lower amounts were C15:0 iso and C16:0 iso. The cellular fatty acid content of the novel strain was different from that of C. flaccumfaciens JCM 9670T and the type strains of other related species, supporting the evidence that strain 20TX0166T is a novel species of Curtobacterium.
Table 3
Comparison of selected physiological and phenotypic characteristics of strain 20TX0166T of Curtobacterium obtained from a rotting onion bulb in Texas, USA, and of closely related Curtobacterium type strains.
Reactions
|
Strains of Curtobacterium†
|
1‡
|
2‡
|
3§
|
4§
|
5§
|
6§
|
7§
|
Catalase
|
+¶
|
+
|
+
|
+
|
+
|
+
|
+
|
Oxidase
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Motility
|
-
|
-
|
+
|
+
|
+
|
-
|
-
|
Optimum pH
|
7
|
7
|
7
|
7
|
7
|
7
|
4
|
Biolog
|
|
|
|
|
|
|
|
N-Acetyl-DGalactosamine
|
+
|
+
|
-
|
-
|
-
|
-
|
-
|
D-Arabitol
|
+
|
+
|
w
|
-
|
-
|
-
|
-
|
myo-Inositol
|
+
|
+
|
-
|
-
|
-
|
-
|
-
|
L-Alanine
|
+
|
+
|
-
|
-
|
-
|
w
|
-
|
D-Glucuronic Acid
|
+
|
+
|
-
|
-
|
-
|
-
|
-
|
L-Malic Acid
|
-
|
+
|
ND
|
ND
|
ND
|
ND
|
ND
|
Bromo-Succinic Acid
|
-
|
+
|
-
|
w
|
-
|
-
|
-
|
API Coryne
|
|
|
|
|
|
|
|
Pyrazinamidase
|
+
|
+
|
ND
|
-
|
-
|
ND
|
ND
|
Alkaline Phosphatase
|
+
|
-
|
ND
|
ND
|
ND
|
ND
|
ND
|
Gelatin hydrolysis/protease
|
-
|
-
|
+
|
-
|
-
|
+
|
-
|
L-ribose (Fermentation)
|
+
|
-
|
+
|
+
|
+
|
-
|
+
|
D-mannitol (Fermentation)
|
+
|
-
|
ND
|
ND
|
ND
|
ND
|
ND
|
D-maltose (Fermentation)
|
-
|
-
|
+
|
+
|
+
|
+
|
ND
|
D-lactose (Fermentation)
|
+
|
-
|
ND
|
ND
|
ND
|
ND
|
ND
|
glycogen (Fermentation)
|
+
|
-
|
-
|
+
|
+
|
-
|
-
|
API 20NE
|
|
|
|
|
|
|
|
Gelatin hydrolysis/protease
|
-
|
-
|
+
|
-
|
-
|
+
|
-
|
D-mannitol (Assimilation)
|
-
|
+
|
ND
|
+
|
-
|
ND
|
ND
|
N-acetyl-glucosamine (Assimilation)
|
-
|
+
|
+
|
+
|
-
|
-
|
-
|
Potassium gluconate (Assimilation)
|
+
|
+
|
-
|
-
|
-
|
-
|
+
|
Malic acid/Malate (Assimilation)
|
-
|
+
|
ND
|
+
|
+
|
ND
|
ND
|
† Strains: 1, 20TX0166T; 2, C. flaccumfaciens LMG 3645T; 3, C. pusillum JCM 1350T; 4, C. citreum JCM 1345T; 5, C. luteum JCM 1480T; 6, C. albidum NBRC 15078T; 7, C. ammoniigenes B55T. |
‡ Data are from this study. |
§ Data are from (Aizawa et al. 2007). |
¶ +, Positive reaction; –, negative reaction; w, weakly positive reaction; ND, No data available. |
Table 4
Comparison of fatty acid composition (%) of strain 20TX0166T of Curtobacterium obtained from a rotting onion bulb in Texas, USA, and of closely related Curtobacterium type strains.
Fatty acid %
|
Strains of Curtobacterium†
|
1‡
|
2§
|
3§
|
4§
|
5§
|
6§
|
C15:0 iso
|
3.96
|
2.8
|
2.8
|
1.6
|
7.9
|
0.1
|
C15:0 anteiso
|
48.14
|
31.3
|
19.6
|
37.2
|
39.5
|
1.3
|
C16:0 iso
|
2.63
|
12.3
|
3.9
|
11.8
|
24
|
0.4
|
C17:0 anteiso
|
39.44
|
29.3
|
18.6
|
36.6
|
18.4
|
2.4
|
† Strains: 1, 20TX0166T; 2, C. flaccumfaciens JCM 9670T; 3, C. pusillum JCM 1350T; 4, C. citreum JCM 1345T; 5, C. luteum JCM 1480T; 6, C. ammoniigenes B55T. |
‡ Data are from this study |
§ Data are from (Aizawa et al. 2007). |