Phylogenetic analysis
A 1444 bp fragment of the 16S rRNA gene was amplified from the P. eucalypticola strain NP-1 T, sequenced and the sequence deposited in GenBank under accession number MN 238,862. A similarity search with this sequence was performed using EzBioCloud. Thirty valid species belonging to P. fluorescens intrageneric group (IG) proposed by Mulet et al.15 exhibited at least 97% similarity with NP-1 T, and these include P. vancouverensis ATCC 700688 T (98.8% similarity), P. moorei DSM12647T (98.8% similarity), P. koreensis Ps9-14 T (98.8% similarity), P. parafulva NBRC16636T (98.5% similarity) and P. reinekei Mt-1 T (98.5% similarity). The similarities with the other 25 species are provided in Supplementary Table S1. A phylogenetic tree based on the 16S rRNA sequence was constructed and is shown in Fig. 1. Strain NP-1 T forms a weakly supported cluster with P. kuykendallii NRRL B-59562 T, but both strains are situated on separate branches. Strain NP-1 T grouped in none known group or subgroup within P. fluorescens lineage, and it clusters of the outer edge of a much larger group containing several Pseudomonas groups/subgroups. However, Pseudomonas species cannot be identified based only on 16S rRNA analysis.
Neighbor-joining phylogenetic tree based on the 16S rRNA gene of Pseudomonas eucalypticola NP-1T and phylogenetically close members of Pseudomonas. The evolutionary distances were computed using the Jukes-Cantor method. The optimal tree with a sum of branch length = 0.23535266 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Cellvibrio japonicus Ueda107T was used as outgroup.
The MLSA approach based on the concatenated sequences of the partial 16S rRNA, gyrB, rpoB and rpoD genes, has been demonstrated to greatly facilitate the identification of new Pseudomonas strains16. According to the 16S rRNA alignment, 33 species from P. fluorescens IG and one species from P. pertucinogena IG were selected for MLSA. The concatenated sequences of the type strains of each selected species comprised a total of 3813 bp (Supplementary Table S2) and were used for phylogenetic tree construction. The analysis of concatenated gene sequences indicated that strain NP-1 T belongs to the P. fluorescens lineage, and this finding was supported by a bootstrap value of 91% (Fig. 2).However, NP-1 T still cannot be determined which group belongs to17.
Neighbor-joining phylogenetic tree based on concatenated 16S rRNA, gyrB, rpoB and rpoD gene partial of Pseudomonas eucalypticola NP-1T and the type strains of other Pseudomonas species. The evolutionary distances were computed using the Jukes-Cantor method. The evolutionary distances were computed using the Jukes-Cantor method e optimal tree with the sum of branch length = 1.37677586 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.
For further identification of NP-1 T, a phylogenomic tree inferred with GBDP was constructed by using Type (Strain) Genome Server (TYGS)18, and all reference type strains and their genome sources are listed in Supplementary Table S3. The result showed the presence of an independent branch supported by a bootstrap value of 88% that can be differentiated from the other Pseudomonas species type strains (Fig. 3) and revealed that NP-1 T clustered with P. coleopterorum LMG 28558 T and P. rhizosphaerae LMG 21640 T which affiliated with P. fluorescens IG, but does not belong to any group. Strain NP-1 T was not be affiliated with any previously described Pseudomonas species and can thus be considered to represent a novel species. Based on above-described the results, P. coleopterorum, P. rhizosphaerae, P. graminis and P. lutea were selected for further analysis with NP-1 T.
Phylogenomic tree of strain NP-1T and related type strains of the genus Pseudomonas available on the TYGS database. The tree inferred with FastME 2.1.6.1 based on GBDP distances calculated from the genome sequences. The branch lengths are scaled in terms of the GBDP distance formula d5. The numbers above the branches show the GBDP pseudo-bootstrap support values > 60% from 100 replications, and the average branch support is 94.6%.
General taxonomic genome feature
The draft genome assembly of strain NP-1 T contains 6,401,699 bp. The genome of NP-1 T, which consists of one chromosome and one plasmid, has been deposited in GenBank under the accession numbers CP056030 and CP056031, respectively. The genome has a G + C content of 63.96 mol%, as determined from the complete genome sequence, and 83.45% of the genome is coding and consists of 5,788 genes. The similarity of the genome of P. eucalypticola NP-1 T to other publicly available genomes of closely related Pseudomonas species was determined using ANI, digital DDH and G + C mol %5,6,7,8,9. Each of these comparisons yielded different ANIm and ANIb values, but the highest ANIb and ANIm values of 78.7 and 86.5 were obtained for NP-1 T and P. rhizosphaerae LMG 21640 T. The similarity between P. coleopterorum LMG 28558 T and NP-1 T was higher than that between P. graminis DSM 11363 T and P. lutea LMG 21974 T (Table 1). All ANIb and ANIm values obtained from the comparisons of NP-1 T with the other tested species were below 95%, which confirmed that strain NP-1 T belongs to an independent species. The TETRA frequencies between NP-1 T and the other tested type strains were lower than 0.99, which is the recommended cutoff value for species (Table 2). The digital DNA-DNA hybridization (dDDH) comparison with the draft genome of the type strain NP-1 T yielded low percentages (< 30%) with all tested species (Table 2, the same species share at least70% in silico DDH). The G + C mol % differences between NP-1Tand related species were higher than 1 (Table 2). These results, together with the ANI, and DDH values, confirm that the NP-1 T strain represents a novel species in the genus Pseudomonas.
Morphology and phenotypic characteristics
The colonies were round and beige with smooth surfaces and edges after incubation on LA medium for 48 h at 25 °C (Fig. 4A). The cells of NP-1 T were Gram-negative (Supplementary Fig. S1), rod-shaped (1.0 μm wide, 2.0 μm longth averages), and motile due to the presence of a single polar flagellum, as observed by transmission electron microscopy (TEM, Fig. 4B and C). NP-1 T grow at temperatures between 4 and 37 °C, although 25 °C was found to be the optimal temperature for growth, and no growth was detected at 42 °C. In addition, growth was observed on LB medium in the presence of 0–2% NaCl (optimum 0.5%), and at pH values of 3.0–7.0 (optimal at pH 6) (Table 3). Strain NP-1 T, as well as P. graminis DSM11363T, P. lutea LMG21974T, and P. rhizosphaerae LMG21640T, failed to produce fluorescent pigments after growth for 24–48 h at 25 °C on King B medium. The tested type strains with the exception of P. coleopterorum LMG 28558 T, exhibited positive oxidase activity. Similar to P. coleopterorum LMG28558T, P. graminis DSM11363T and P. lutea LMG21974T, the NP-1 T nitrate reduction is negative. In the Biolog GN2 plates, NP-1 T utilized dextrin, glycogen, L-arabinose, D-fructose, D-galactose, gentiobiose, α-D-glucose, D-mannose, D-psicose, L-rhamnose, D,L-lactic acid, quinic acid, succinic acid, bromo, succinic acid, succinamic acid, glucuronamide, L-aspartic acid, D-trehalose, formic acid, D-galacturonic acid, D-gluconic acid, D-glucuronic acid. And α-keto glutaric acid, D-saccharic acid, L-alaninamide, L-asparagine, hydroxy-L-proline, turanose, methyl pyruvate, cis-aconitic acid, D-galactonic acid lactone tests were variable. Other tests were negative in the Biolog GN2 plate. The differential phenotypic characteristics in the Biolog GN2 test are indicated in Table 3; starch hydrolysis reaction was positive in NP-1 T, but negative in P. gramis and P. rhizosphaerae; NP-1 T can use D-sorbitol, which is different from P. gramis, P. lutea and P. rhizosphaerae; NP-1 T could not utilize propionate, but P. coleopterorum, P. gramis and P. rhizosphaerae could.
Colonies of Pseudomonas eucalypticola NP-1T grown on LA medium at 25 °C for 48 h (A). The bacterial morphology was obtained by scanning electron microscopy (B), and the transmission electron microscopy showed the presence of a single polar flagellum (C, black arrows). Bar = 1 μm.
Chemotaxonomic analysis
A cellular fatty acid analysis of NP-1 T and various reference type strains, namely, P. graminis DSM 11363 T, P. lutea LMG 21974 T, P. coleopterorum LMG 28558 T and P. rhizosphaerae LMG 21640 T was performed. The results from the chemotaxonomic analyses are shown in Table 4. The major cellular fatty acids of strain NP-1 T were 3-hydroxydodecanoic acid (C12:0 3-OH), dodecanoic acid (C12:0), 2-hydroxydodecanoic acid (C12:0 2-OH), 3-hydroxydecanoic acid (C10:0 3-OH), hexadecanoic acid (C16: 0), 17-carbon cyclopropane fatty acid (C17:0 cyclo), C16:1 w6c/C16:1 w7c (Summed Feature 3) and summed Feature 8 (C18:1 w6c/C16:1 w7c). Hexadecanoic acid (C16: 0) was the most abundant fatty acid in all tested samples. This fatty acid profile is characteristic of strains from group I which have C10:3 3-OH and C12:0 3-OH19. The cellular fatty acid profile of strain NP-1 T matched that of P. lutea. The main difference between strain NP-1 T and the reference strains is related to the presence of C19:0 cyclo w8c, which was was only detected in NP-1 T and P. lutea.
Antifungal activity
P. eucalypticola NP-1 T exhibited antifungal activity against five tested fungal species (Fig. 5). Specifically, NP-1 T exhibited strong antifungal activity against C. pseudoreteaudii, M. oryzae, and S. sclerotiorum, as demonstrated by the formation of an inhibition zone with a width greater than 30 mm. The contrast, the inhibition zones between NP-1 T and the two Fusarium species had a width less than 30 mm.
Antagonistic interaction between Pseudomonas eucalypticola NP-1T and selected phytopathogenic fungi.
Source: Ecology - nature.com
