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. 2026 Feb 17;16:9424. doi: 10.1038/s41598-025-33275-1

Pseudomonas rhizozeae sp. nov., originated from rhizosphere soil of corn field

Sándor Kocsubé 1,3, Dóra Anna Papp 1, Attila Farkas 2, Thu Huynh 1, Henrietta Allaga 1, András Szekeres 1, Csaba Vágvölgyi 1, Judit Ámon 1,4, Zsuzsanna Hamari 1, Mónika Varga 1,✉
PMCID: PMC13003124  PMID: 41702933

Abstract

A novel Pseudomonas species was isolated from the rhizosphere of Zea mays in Hungary. Phylogenetic analysis based on concatenated 16S rRNA, rpoD, and rpoB sequences placed the isolate within the Fluorescens superclade, while a proteome-based phylogenetic analysis elucidated the accurate phylogenetic position, a distinct lineage between the Corrugata and Jessenii clades. Whole-genome sequencing of the type strain SZMC 28357T (GenBank accession: JAVLSB000000000) revealed a 5.9 Mb genome with a G + C content of 61%. Comparative genomic analyses (ANI and dDDH) further confirmed that SZMC 28357T represents a novel species. In silico screening identified 12 secondary metabolite biosynthetic clusters, including non-ribosomal peptide synthetases (NRPSs), polyketide synthases (PKS), and ribosomally synthesized peptides (RiPPs). The production of hydrogen cyanide, ectoine and NAGGN metabolites was confirmed through analytical methodologies. Based on genomic, phylogenetic, phenotypic and biochemic evidence, we propose the name Pseudomonas rhizozeae sp. nov. for this newly characterized species.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-33275-1.

Subject terms: Biotechnology, Microbiology, Molecular biology, Plant sciences

Introduction

The genus Pseudomonas was introduced by Migula in 18941. According to the List of Prokaryotic Names with Standing in Nomenclature, the genus Pseudomonas (family of Pseudomonadaceae, class of Gammaproteobacteria) comprises over 300 validly described species2. These bacteria are Gram-stain-negative, aerobic, rod-shaped and polar flagellated. Pseudomonas species are found in diverse environments, including soil, water, plants, animals, clinical settings, and air3,4. Their ability to scavenge iron, exhibit swarming motility, and form biofilms may contribute to their pathogenic potential5–7. Meanwhile, their capacity to produce bioactive secondary metabolites, such as siderophores8 and antibiotics9, allows them to engage in competitive and cooperative interactions within microbial communities, particularly in the rhizosphere. Although many species are utilized as biocontrol agents against soil-borne plant pathogens10, rich taxonomical and metabolomical diversity within Pseudomonas keeps the genus an attractive source of biocontrol agents or bioactive compounds. Our recent screen for potent Pseudomonas isolates capable of inhibiting aflatoxin production by Aspergillus flavus resulted in the identification of 50 Pseudomonas strains isolated from corn rhizosphere and characterization of their antagonistic properties11. A phylogenetic analysis based on the 16S rRNA and rpoD sequences revealed five identical isolates that were clearly distinct from known Pseudomonas species11. Although these five strains did not perform any antagonistic activity against A. flavus in solid-phase confrontation assays, they strongly reduced fungal biomass production and completely inhibited aflatoxin biosynthesis in liquid-phase experiments11. When the previously performed phylogenetic analysis was repeated using the concatenated sequences of the 16S rRNA-rpoD-rpoB genes and including one additional isolate, the five strains together with the new isolate formed a distinct clade within the Pseudomonas fluorescens superclade.

This study aims to describe and formally name a novel Pseudomonas species from the maize rhizosphere, integrating genomic, phenotypic, and chemotaxonomic evidence. One isolate, Ps_91 (subsequently deposited as SZMC 28357), was selected for whole-genome sequencing. Comparative genome analysis including average nucleotide identity (ANI), digital DNA-DNA hybridization (dDDH), and phylogenomic analysis based on orthologs of 1343 SZMC 28357 proteins across 85 species confirmed that SZMC 28357 represents a novel species within Pseudomonas between the Corrugata and Jessenii clades, for which we propose the name of Pseudomonas rhizozeae sp. nov. Alongside the comparative physiological and biochemical characterisation, analytical techniques were used to confirm the production of metabolites encoded by specific secondary metabolite gene clusters.

Results and discussion

Phylogenetic analysis and genome characterization of SZMC 28357

Five Pseudomonas isolates (Ps_89, 90, 91, 96 and 100) from our previous study11, along with a newly isolated one from corn rhizosphere in Madaras, Hungary (for details see Supplementary Table S1), were subjected to phylogenetic analysis based on concatenated sequences of 16S rRNA-rpoD-rpoB genes. Maximum likelihood analysis confirmed that all six isolates form a well separated clade with high statistical support, sister to Pseudomonas cucumis, a member of the Mandelii clade within the Fluorescens superclade of Pseudomonas (Fig. 1, Supplementary Fig. S1). The isolates were deposited to the Szeged Microbiological Collection (SZMC) under accession numbers SZMC 28355 to SZMC 28360. One isolate, SZMC 28357 (formerly Ps_91 11), was selected for further investigations and designated as the type strain (SZMC 28357T). SZMC 28357T was additionally deposited in the NCCB, NCAIM and DSMZ collections as NCCB 100969T, NCAIM B.02685T and DSM 117247T, respectively.

Fig. 1.

Fig. 1

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Collapsed maximum likelihood phylogram based on concatenated alignments of 16S rRNA, rpoB and rpoD sequences extracted from reference genomes. Clade designations were adopted following the classification proposed by Rudra and Gupta et al.12 and Girard et al.4. Statistical support of the best tree was calculated with UFBoot approximation. Only values higher than 95% are shown. Members of the genus Perlucidibaca were used as outgroup. Type strains are marked with superscript T.

The whole genome of SZMC 28357T was sequenced (NCBI GenBank accession: JAVLSB000000000) and annotated. The genome was assembled into 76 contigs with an estimated genome size of 5.9 Mb with a G + C content of 61%, which falls within the 58–69% range typical of Pseudomonas. Genome statistics were determined by the PGAP pieline with an N50 and L50 value of 171.1 kb and 12, respectively. Quality analysis performed by CheckM v1.2.313 determined 99.36% completeness and 0.08% contamination of the assembly. ANI comparisons with 36 Pseudomonas species (including members of the Corrugata, Mandelii and Kielensis and Protegens clades) yielded values below the species delineation threshold of 95%, with the highest being 85.7% and 85.5% to P. nunensis and P. hormoni, respectively (Fig. 2).

Fig. 2.

Fig. 2

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Heatmap of ANI values between SZMC 28357 and the most closely related genomes determined by phylogenomic analysis. The ANI values were obtained using FastANI. ANI values lower than 95% indicate distinct species. Heatmap was generated by using ANIclustermap version 1.3.0.

dDDH values (using Formula 2) were also well below the 70% species cutoff. The closest related species (P. nunensis, P. migulae, P. hormoni, and P. frederiksbergensis) all exhibited dDDH values of 28.3–28.4 (Supplementary Table S2).

Both the phylogenetic analysis based on three genes and the whole-genome comparisons placed SZMC 28357T within the Mandelii clade of the Fluorescens superclade. However, the closest relatives identified by ANI and dDDH analyses were slightly different as based on ANI values P. mandelii was more closely related to SZMC 28357T than P. frederiksbergensis. To elucidate the exact phylogenetic position of SZMC 28357T, we conducted a phylogenomic analysis based on 1343 conserved proteins across 85 Pseudomonas species (114,155 total proteins), including representatives from the Corrugata, Mandelii, Kielensis and Jessenii and Protegens clades. While the phylogram topology generally matched that of the 16S rRNA-rpoD-rpoB phylogenetic tree (Fig. 1), SZMC 28357T resolved as a separate lineage between the Corrugata and Jessenii clades (Fig. 3). Based on our results obtained from all six isolates, we concluded that they represent a novel species, with strain SZMC 28357T designated as the type strain. In accordance with species nomenclature rules, we propose the new species to be called Pseudomonas rhizozeae, referring to the rhizosphere of Zea mays, where from the type strain was isolated.

Fig. 3.

Fig. 3

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Maximum likelihood phylogenetic tree based on 1343 concatenated single copy orthologous proteins. Statistical support of the best tree was calculated with UFBoot support. Only values higher than 95% are shown. Pseudomonas saponiphila and Pseudomonas protegens were used as outgroup. Type strains are marked with superscript T.

In silico identification and analytical confirmation of secondary metabolite gene clusters of Pseudomonas rhizozeae SZMC 28357T sp. nov.

We identified twelve putative secondary metabolite gene clusters by in silico analysis, including 4 NRPS (non-ribosomal peptide synthetase), 3 RiPP (ribosomally synthesized and post-translationally modified peptide), 1 PKS (polyketide synthase) and 4 miscellaneous clusters (Supplementary Fig. S2, panel A-L).

Three NRPS clusters showed no significant similarity to any known clusters, while one was 97% similar to an NRPS of unknown function found in P. mandelii.

Among RiPPs, a redox-cofactor and a linear azole-containing peptide (LAP) cluster was identified that showed 72% and 62% similarity to clusters in P. viridiflava and P. izuensis, respectively. LAP production suggests potential biocontrol properties14. A third RiPP cluster had 50% similarity to an uncharacterized RiPP-like cluster of P. fluorescens.

The predicted PKS cluster showed 97% similarity to the arylpolyene pigment biosynthesis cluster of P. vancouverensis, which is associated with oxidative stress resistance15.

Two clusters encoded hydrogen cyanide biosynthesis genes, with 75% and 83% similarity to those in P. gingeri and P. arsenicoxydans, respectively. Through the execution of HCN-detection plate assay we confirmed that SZMC 28357T produces HCN, which is a useful trait of biocontrol agents due to its nematocidal and aphicidal effect16,17.

The remaining two clusters were predicted to be involved in N-γ-acetylglutaminyl glutamine 1-amide (NAGGN) production (100% similarity to NAGGN cluster from P. nunensis) and ectoine formation (60% similarity to the cluster of Rhizobium bangladeshense). Both ectoine and NAGGN are known to be synthetized in response to osmotic stress18,19. Presence of these compounds was detected by HPLC-HRMS in aqueous extracts from salt-stressed SZMC 28357T cultures (Supplementary Fig. S3), suggesting that the strain exhibits osmotic stress tolerance18,19.

Phenotypic and biochemical characterization of Pseudomonas rhizozeae SZMC 28357T sp. nov.

Colonies of SZMC 28357T on LB agar (28 °C for 24 h) are white-yellowish, mucoid, smooth, round, opaque with entire edges and 0.5–1.0 mm in diameter. Cells are 1.1–1.9 μm long and 0.2–0.5 μm wide. Gram-stain-negative, aerobic, non-spore forming and rod-shaped (Fig. 4). According to motility tests (swimming, swarming and twitching tests) the strain is non-motile, which is in line with the scanning electron microscopy image of the bacterial cells (Fig. 4).

Fig. 4.

Fig. 4

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Scanning electron microscopy image of rod-shaped non-flagellated cells of Pseudomonas rhizozeae sp. nov SZMC 28357T. Scale bar represents 200 nm.

The strain grows between 10 and 45 °C (optimum: 24–30 °C), in 0–5% NaCl (optimum: 3–4%), and at pH 6.0–10.0 (optimum: pH 6.0) (Table 1). Without any change in colony colour on King’s B agar, the strain shows weak fluorescence under UV light (see Supplementary Fig. S4), indicating the production of fluorescent compounds. It shows mucoid phenotype on sucrose-supplemented medium as an indication of levan production (Table 1). The strain shows β-haemolytic activity on blood agar and positive for catalase and cytochrome oxidase activity (Table 1). In API 20 NE tests, SZMC 28357T was positive for aesculin hydrolysis and assimilation of N-acetylglucoseamine, potassium gluconate, capric acid, trisodium citrate and adipic acid. It was negative for d-glucose fermentation, nitrate reduction, indole production, arginine dihydrolase activity, urease activity, gelatine hydrolysis, β-galactosidase activity and assimilation of maltose, l-arabinose, d-mannitol, mannose, malic acid and phenylacetic acid (Table 2).

Table 1.

Physiological characteristics of Pseudomonas rhizozeae sp. nov.

Characteristics P. rhizozeae sp.nov,. P. mandelii20–23 P. migulae24–26 P. lini21,22,25–28 P.arsenicoxydans21,22,25,26,28 P. koreensis29–33 P. jessenii20,29–34 P. laurylsulfativorans34 P. glycinae30 P. kribbensis30,31 P. vancouverensis30–34 P. atacamensis33 P. frederiksbergensis21–23,28,35 P. mucoides21 P. prosekii26 P. cucumis28 P. silesiensis22,28 P. nunensis23
Temperature tolerance (°C) 10–45 4–35 4–35 4–36 4–37 4–30 4–35 8–42 4–36 4–30 ND 4–37 4–30 4–37 5–30 4–37 4–35 4–28
Temperature optimum (°C) 24–30 30 30 ND 30 ND 30 30 28–30 30 30 28 ND 28 ND 28 15–30 20–25
NaCl conc. tolerance (%) 0–5 ND 0–5 0–4 0–4 1 < 7 ND 0–6 5–6 1–3 ND ND 0–4 0-4.5 ND 0–3 0–4 0–4
Optimal NaCl conc. (%) 3–4 ND ND ND 0 ND ND 0–2 ND 1 ND 0–4 ND ND ND ND ND ND
pH tolerance 6–10 ND ND ND 6.5–10 ND ND 5–10 4–10 5–10 ND ND ND 7–9 ND 6–9 6–9 5–9
pH optimum 6 ND ND ND 7.5-8 ND ND 7 6–7 7 ND ND ND ND ND 7 7–8 6–7
Fluorescent pigments on King’s B agar (+) + + + – + + + + - + + – + + + – ND
Levan from sucrose + + + VC ND – VC ND ND ND - ND – ND ND ND ND ND
Cytochrome-oxidase + + + + + + + + + + + + + + + + + ND
Catalase + + + ND + + + + + + + + + + + ND ND ND
Hemolysis β γ γ ND ND ND γ ND ND ND ND ND ND γ ND ND ND ND
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In comparison to the closely related species of the genus Pseudomonas. Characteristics are scored as: +, positive reaction; −, negative reaction; ND, no data; VC, variable characteristics among strains and/or experiments. (+) on King’s B agar: without changing of the colony colour, a weak fluorescence was observed under UV light (see supplementary Fig. S4).

Table 2.

Results of the API 20 NE analysis of Pseudomonas rhizozeae sp. nov.

Characteristics P. rhizozeae sp.nov,. P. mandelii20–23 P. migulae24–26 P. lini21,22,25–28 P.arsenicoxydans21,22,25,26,28 P. koreensis29–33 P. jessenii20,29–34 P. laurylsulfativorans34 P. glycinae30 P. kribbensis30,31 P. vancouverensis30–34 P. atacamensis33 P. frederiksbergensis21–23,28,35 P. mucoides21 P. prosekii26 P. cucumis28 P. silesiensis22,28 P. nunensis23
Reduction of NO3 to NO2 ï- + + + – – + + – – + – + + – + + –
Indole production – – – ND – – – – – – ND - - - ND -– – ND
Glucose fermentation - ND ND ND ND – ND – – – ND - - ND ND – – ND
Arginine hydrolysis - VC + ND + + VC – + + - + – - ND – – +
Urea hydrolysis - VC - ND + VC - – – – - - – – – – – ND
Aesculin hydrolysis + - - - - ND VC - - - ND - - ND – + ND +
Gelatin hydrolysis - - - + - VC - - + - - - VC + + + - +
Assimilation
 Glucose + + + + + + + + + +  ND + + + + + + +
 Arabinose - + + + + + + + + +  - + + + + + + +
 Mannose - + + + + + + + + +  - + + + - + + +
 Mannitol - + + + + + + - + +  + + + + - + + +
 N-acetyl-glucosamine + + + VC + + + + + +  + + + + - + - +
 Maltose - VC - - VC - VC - - -  ND - - - - - + ND
 Gluconate + + + + + + + + + +  + + + + + + + +
 Caprate + + + + + + + + + +  ND + + + ND ND + ND
 Adipic acid + ND ND ND ND - ND - - -  ND ND - - ND - - ND
 Malic acid - + + + ND + + + + +  + + + + ND + + +
 Citrate + + + + - + + + + +  + + + + + + - +
 Phenyl-acetate - VC + - + - + + - -  + - - - ND - - ND
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In comparison to the closely related species of the genus Pseudomonas. Characteristics are scored as: +, positive reaction; −, negative reaction; ND, no data; VC, variable characteristics among strains and/or experiments.

The overall phenotype of SZMC 28357T was distinct from that of the closely related Pseudomonas species (Tables 1 and 2).

Chemotaxonomic characteristics of Pseudomonas rhizozeae SZMC 28357T sp. nov.

Since cellular fatty acid profiles have diagnostic values for distinguishing and identifying plant pathogenic species in genus Pseudomonas, cellular fatty acid methyl ester profiling was carried out on SZMC 28357T by GC analysis. The profile shows quantitatively and/or qualitatively important typical saturated fatty acids C12:0, C14:0, C16:0, C18:0, C10:0 3-OH, C12:0 2-OH, C12:0 3-OH, C17:0 cyclo, with C16:0 as the dominant fatty acid component (Table 3). The fatty acid profile of SZMC 28357T was quantitatively similar to that of P. jessenii and P. vancouverensis, except for C10:0 3-OH and C17:0 cyclo (Table 3). Based on the diagnostic value of the presence of fatty acids C10:0 3-OH and C17:0cyclo at 0.7% and 5.5%, respectively, these fatty acids can be used for the identification of Pseudomonas rhizozeae sp. nov. Major polar lipids in SZMC 28357T included phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, lysophosphatidylethanolamine, and lysophosphatidylglycerol (Supplementary Table S3), consistent with other Pseudomonas species36,37. Isoprenoid quinone analysis showed ubiquinone 9 as the major component, alongside ubiquinone 8 (Supplementary Table S3), in agreement with known Pseudomonas profiles37–39. Additionally, ubiquinone 4 and 2 were also detected as minor quinones (Supplementary Table S3).

Table 3.

Cellular fatty acid composition (%) of Pseudomonas rhizozeae sp. nov.

Fatty acid P. rhizozeae P. mandelii P. migulae P. lini P. arsenicoxydans P. koreensis P. jessenii P. laurylsulfativorans P. glycinae P. kribbensis P. vancouverensis P. atacamensis P. frederiksbergensis P. mucoides P. cucumis P. silasiensis
C12:0 2.7 ± 0.2 2.5 5.5 6.3 5 1.6 3.6 4.1 2.9 n.d. 3.4 n.d. 2.5 4.8 1.4 6.5
C14:0 0.4 ± 0.02 0.6 0.7 0.6 4.6 0.7 0.5 n.d. 0.6 1.2 0.7 n.d. n.d. n.d. n.d. n.d.
C16:0 36.4 ± 2.8 28 28.3 25.4 27.9 33.3 34.9 31.6 22.9 33.4 35.7 25.0 30.4 36.4 14.9 27.9
C18:0

1±

0.1

 0.3 0.9 1 0.8 0.7 0.7 n.d. 0.3 0.8 0.8 n.d. n.d. n.d. n.d. n.d.

C10:0

3-OH

 0.7 ± 0.1 3.9 3 3.1 3 2.2 3.9 3.9 6.6 5.4 3.4 n.d. 5.7 4.5 13.6 3.7

C12:0

2-OH

 3.7 ± 0.1 5.4 3.3 2 3.8 5.0 3.4 3.2 5.5 6.8 3.4 10.3 4.6 3.3 n.d. 1

C12:0

3-OH

 3.5 ± 0.1 4.3 4.2 3 4.6 4.0 3.9 4.2 6.7 7.5 3.9 n.d. 6.5 4.5 12 3.1

C17:0

cyclo

 5.5 ± 0.3 31.4 7.2 2 12.2 2.1 1.3 1.3 10.3 15.1 13.9 n.d. 1.1 1.5 n.d. 1
Summed features 3 and 8*
 3 a 30.8 ± 0.8 15.5 28.3 32.4 21.2 36.7 30.5 38.8 23.6 16.8 22.1 26.9 35.5 36 19.2 36.9
 8 b 14.3 ± 0.5 7.1 15.3 21.3 17.1 13.0 12.5 11.2 13.4 8.9 9.8 9.5 11.6 6.4 7.4 n.d.
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Type strain SZMC 28357T and the type strains of the closely related Pseudomonas species. * groups of two or three fatty acids that are not separated by GC-MS with the Sherlock microbial identification System; n.d., not detected.

Conclusions

Phylogenetic analysis based on the concatenated sequences of the 16S rRNA-rpoD-rpoB genes placed strain SZMC 28357T, isolated from the rhizosphere of Zea mays, in a distinct clade within the genus Pseudomonas. Comparative ANI and dDDH genome analyses confirmed that SZMC 28357T is clearly differentiated from all closely related Pseudomonas species. Phylogenomic analysis further supported this result and positioned SZMC 28357T on a separate lineage between the Corrugata and Jessenii clades of the Fluorescens superclade. Biochemical and chemotaxonomic characteristics of SZMC 28357T also showed unique features compared with those of closely related species. Therefore, strain SZMC 28357T should be classified as a novel species with the name Pseudomonas rhizozeae sp. nov. Furthermore, SZMC 28357T inhibited aflatoxin production by Aspergillus flavus during liquid phase confrontation11.

Description of Pseudomonas rhizozeae sp. nov.

Pseudomonas rhizozeae (rhi.zo.ze’ae. Gr. fem. n. rhiza, a root; L. fem. n. zea, Zea mays, maize; N.L. gen. fem. n. rhizozeae, of maize root), referring to the rhizosphere of Zea mays, where from the type strain was isolated.

Colonies are white-yellowish, mucoid, smooth, round, opaque, and have entire margins, 0.5–1.0 mm in diameter. Cells are 1.1–1.9 μm long and 0.2–0.5 μm wide, non-motile, Gram-stain-negative, aerobic, non-spore forming and rod-shaped. The strain grows between 10 and 45 °C (optimum 24–30 °C), in 0–5% NaCl (optimum 3–4%), and at pH 6.0–10.0 (optimum pH 6.0). It weakly produces fluorescent pigment on King’s B agar and produces levan. It shows β-haemolytic activity and is positive for catalase and cytochrome oxidase activity. SZMC 28357T is positive for aesculin hydrolysis and for the assimilation of N-acetylglucoseamine, potassium gluconate, capric acid, trisodium citrate and adipic acid and negative for d-glucose fermentation, nitrate reduction, indole production, arginine dihydrolase activity, urease activity, gelatine hydrolysis, β-galactosidase activity and for the assimilation of maltose, l-arabinose, d-mannitol, mannose, malic acid and phenylacetic acid. The cellular fatty acid methyl ester profile is typical of Pseudomonas species, however, the presence of 0.7% and 5.5% of fatty acids C10:0 3-OH and C17:0cyclo, respectively, has diagnostic value and can be used for the identification of the novel species represented by strain SZMC 28357T.

The type of Pseudomonas rhizozeae sp. nov. is SZMC 28357T (= NCCB 100969T = NCAIM B.02685T = DSM 117247T) isolated from corn rhizosphere in Madaras, Hungary. The genome size of the type strain is estimated to be 5.9 Mb with G + C content of 61 mol%. Genbank accession number of the 16S rRNA and whole genom annotation of SZMC 28357T are OR563981.1 and JAVLSB000000000, respectively.

In silico analysis revealed 12 putative secondary metabolite gene clusters (four non-ribosomal peptide synthetase, three ribosomally synthesized and post-translationally modified peptide, one polyketide synthase and four miscellaneous clusters). These include a redox-cofactor, a linear azole-containing peptide cluster, an arylpolyene pigment biosynthesis cluster, two hydrogen cyanide biosynthesis clusters, an N-γ-acetylglutaminyl glutamine 1-amide biosynthesis cluster, and an ectoine biosynthesis cluster. Production of arylpolyene, N-γ-acetylglutaminyl glutamine 1-amide, ectoin and HCN was experimentally confirmed. The biosynthesis of linear azole-containing peptides and hydrogen cyanide confers fungicidal and nematocidal-aphicidal properties, respectively. Production of arylpolyene pigment, ectoine and N-γ-acetylglutaminyl glutamine 1-amide contributes to resistance against oxidative and osmotic stress. Collectively, these properties indicate that SZMC 28357T represents a novel Pseudomonas species with biocontrol potential.

Methods

Strains

Strains Ps_91, Ps_89, Ps_90, Ps_96, and Ps_100 originated from our previous work11 and were deposited in the Szeged Microbiological Collection (SZMC) of the Department of Biotechnology and Microbiology, University of Szeged, Hungary under accession numbers SZMC 28357, SZMC 28355, SZMC 28356, SZMC 28358 and SZMC 28359, respectively. An additional strain was isolated from a rhizosphere soil sample of maize as described earlier11, collected from a corn field in Madaras, Hungary and deposited as SZMC 28360.

DNA work

DNA was extracted from the isolates using the Zymo Research Quick DNA Fungal/Bacterial Miniprep Kit according to the manufacturer’s instructions. For species identification, partial sequences of the 16S rRNA, rpoB and rpoD genes40–42 were amplified by using 5’-cctacgggaggcagcag-3’ forward with 5’-attaccgcggctgctggca-3’ reverse, 5′-tggccgagaaccagttccgcgt-3′ forward with 5′-cggcttcgtccagcttgttcag-3′ reverse and 5’-gcgcaatcgcgcacttccc-3’ forward with 5’-gacatgcgacggttgatgtc-3’ reverse primer pairs, respectively. Amplicons were sequenced, and resulted sequences were deposited in NCBI GenBank under accession numbers OR571460-OR571469 and OR563981.

Whole-genome was sequenced by using Illumina NextSeq platform with paired-end reads. Library preparation was carried out by using the NEBNext Ultra II Library Prep Kit. The genome sequencing was performed by Illumina with NextSeq/NovaSeq chemistry (2 × 150 nt PE) (NextSeq 500/550 High Output Kit v2.5/NovaSeq 6000 SP Reagent Kit v1.5). Quality of the 150 bp paired-end raw reads was assessed using FastQC version 0.12.1, and adapter trimming and filtering of low-quality bases were performed with Trimmomatic version 0.3943. The assembly of the filtered reads was carried out by using SKESA version 2.5.044 (with 240x coverage) and submitted for annotation to the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).

Comparative genome analysis

ANI values were calculated using FastANI version 1.3345 and visualized with ANIclustermap version 1.3.0. Additionally ANIb and ANIm (Supplementary Fig. S5) values were also determined using pyANI-plus v1.046. dDDH values were obtained from the Genome-to-Genome Distance Calculator (GGDC 3.0) web server (https://ggdc.dsmz.de/ggdc.php)47.

Secondary metabolite biosynthetic gene clusters were identified by using antiSMASH 7.1.048 with relaxed detection parameters.

Phylogenetic analysis

In order to obtain a three-genes-based (16S rRNA, rpoB and rpoD) phylogenetic tree, sequences of 16S rRNA, rpoB and rpoD genes from the six Pseudomonas isolates were determined, while those of the reference strains belonging to the family Pseudomonadaceae were retrieved from NCBI Datasets. The dataset included synonymized and reclassified species and comprised 495 species (see Supplementary excel). Reference sequences were aligned with partial sequences of the six strains using MAFFT version 7.4949, with the G-INS-i iterative refinement alignment strategy. The alignments were concatenated by using FASconCAT-G50, and the partitioned dataset was submitted for evolutionary model selection using the inbuilt model selection tool51 of IQ-TREE version 2.0.752. The model selection included all DNA models with gamma or free rate heterogeneity and tested with a maximum of 6 categories. Maximum likelihood analysis with the best-fit evolutionary models for each partition was performed using IQ-TREE 2 with statistical support determined by UFBoot53 in 5000 replicates.

In order to elucidate the plylogenetic position of SZMC 28357T, a proteom-based phylogenetic analysis was carried out by identifying single-copy orthologous genes (using OrthoFinder version 2.5554 with default parameters) across the available Pseudomonas genomes. After alignment with MAFFT v 7.49 (with G-INS-I), proteins shorter than 100 amino acids were excluded. The final dataset consisted of 1343 protein sequences (438,532 residues) from 85 species (with total protein number 114,155). Model testing and maximum likelihood analysis were carried out as described above.

Scanning electron microscopy

The detailed surface topography and three-dimensional cellular architecture of SZMC 28357T cells were examined using scanning electron microscopy. Cells were fixed overnight at 4 °C in 2.5% (v/v) glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2), then spotted onto silicon disc coated with 0.01% (w/v) poly-L-lysine. Samples were washed twice with PBS, dehydrated in graded ethanol (30–100% v/v), and dried using a critical point dryer. Gold coating (12 nm) was applied prior to imaging on a Zeiss Sigma 300 Field-Emission scanning electron microscope. Cell size was measured from 100 cells using ImageJ, and average values were calculated.

Phenotypic and biochemical characterization

Growth of SZMC 28357T was tested on Tryptic Soy Agar (TSA; Difco) at 4, 8, 22, 24, 30, 37 and 45 °C to determine the optimal temperature. For pH tolerance, growth was assessed in TSB (Difco) adjusted to pH 6.0–10.0 in 0.5 unit increments using buffer systems of 0.1 M KH2PO4/0.1 M citric acid/0.1 M sodium citrate (for pH 6.0–8.0) and 0.1 M NaHCO3/0.1 M Na2CO3 (for pH 9.0–10.0) for 4 days at 28 °C with vigorous shaking55. NaCl tolerance was evaluated in TSB supplemented with 0–5% NaCl (w/v) at 1% intervals. Gram-staining was performed using standard protocols56. Swimming, swarming and twitching motility test was performed according to Telma de Sousa57. Fluorescent pigment production was assessed on King’s B agar58, where yellowish pigmentation indicated the production of fluorescent pigments. Catalase activity was confirmed by observing O2 production upon treatment with 3% (w/v) H2O2, and cytochrome oxidase activity was tested by colony color change upon application of 1% (w/v) tetramethyl-p-phenylenediamine59. β-haemolytic activity was evaluated on 5% sheep blood agar by observing clearance zone. Levan production was tested on nutrient agar (NA) medium supplemented with 5% sucrose60. Mucoid colonies after 2 days of incubation at 28 °C indicated a positive results. Additional biochemical and physiological characteristics were assessed using API 20 NE kit (bioMérieux) according to the manufacturer’s instructions.

Detection of HCN production

Qualitative determination of HCN production was conducted according to Castric and Castric61 with minor modifications. The bacterium was inoculated onto 3 ml of LB agar within a 24-well plate. A previously prepared dried Whatman 3MM chromatography paper strip, which contained the HCN detection reagent of Freigl and Anger62 (5 mg of copper(II) ethyl acetoacetate and 5 mg of 4,4′-methylenebis-(N, N-dimethylaniline) 1 ml of chloroform) was positioned over the well. Subsequently, the 24-well was sealed and incubated at 28 °C for 48 h. Dark blue colorization of Whatman paper indicated HCN production. P. aeruginosa PaO1 strain (DSMZ 22644) served as a positive control.

Metabolite analysis

Fatty acid methyl ester profiling

Extraction and analysis of fatty acid methyl esters were carried out on TSBA (Becton, Dickinson and Company, Sparks, NV, USA) cultures (28 °C, 24 h) following the standard protocol of the MIDI (Sherlock® Microbial Identification System version 6.0) utilizing a Nexera GC-2030 gas chromatograph equipped with an AOC-20i Plus autoinjector (Shimadzu, Kyoto, Japan) and Sherlock CAS Software version 6.4 (Microbial ID Inc., Newark, DE, USA) operating through the LabSolution version 5.97 software for the data analysis63.

Analysis of isoprenoid quinones and polar lipides

Isoprenoid quinones and polar lipids were extracted from freeze-dried cells (grown in LB broth, 28 °C, 24 h) according to the method detailed by Minnikin et al.64. Both types of compounds were analyzed utilizing a DionexUltimate 3000 UHPLC system (Dionex) coupled to a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer equipped with a heated electrospray interface. The analytical conditions are described in the Supplementary methods.

Analysis of ectoine and NAGGN

The bacteria was grown in 10 ml MM63 broth containing 8.9% at 37 °C for 18 h, then transferred to 20 ml MM63 broth containing 17.5% NaCl and incubated at 28 °C, 150 rpm, for 24 h. Cells were harvested (5,000 g for 20 min), freeze-dried, and 20 mg samples were disrupted by using a bead-beater (25 Hz, 80 s) in 1 ml water with silica beads. After centrifugation (18, 000 g, 10 min) the supernatant was analyzed utilizing a DionexUltimate 3000 UHPLC system (Dionex) coupled to an Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer that operated with a heated electrospray interface.

Ectoine and NAGGN were separated using an ACQUITY BEH amide (1.7 μm, 100 × 2.1 mm) column equipped with an ACQUITY BEH amide guard column (1.7 μm, 5 × 2.1 mm) at 25 °C. Separation was achieved with a mobile phase consisting of water (A) and acetonitrile (B) both supplemented with 0.1% formic acid. The linear gradient program started after an isocratic step (0.5 min, 97% B), and decreased to 60% B in 8 min, followed by another isocratic step (1 min, 60% B). After reaching the initial solvent composition in 0.5 min, it was held for 5 min for re-equilibration.

All samples were analyzed in positive ionization mode. The ion source had the following settings: the temperature of the probe heater and ion transfer capillary, spray voltage, sheath gas flow rate, auxiliary gas flow rate and S-lens RF level were set to 300 °C, 350 °C, 3.5 kV, 40 arbitrary unit, 10 arbitrary unit and 50 arbitrary unit, respectively.

To detect ectoine and NAGGN the mass spectrometer acquired data using a parallel reaction monitoring method (PRM). The PRM data were acquired at a resolution of 17,500 with a maximum injection time of 100 ms and automatic gain control target of 2.00 × 105. The precursor isolation window was 0.4 m/z. The precursor ions were [M + H]+ m/z 143.0821 for ectoine and [M + H]+ m/z 316.1621 for NAGGN.

LC-HRMS data were acquired and processed using Trace Finder 4.0 software.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (37.9KB, xlsx)
Supplementary Material 2 (6MB, pdf)

Acknowledgements

We would like to thank Zita Szalai for her help in the latin naming of the species.

Author contributions

Conceptualization, data curation, D.P., S.K., M.V.; investigation, D.P., S.K., A.F., T.H.,H.A., J.Á., M.V.; methodology, S.K. Zs.H.; funding acquisition, M.V.; writing-original draft, D.P.; visualization, S.K.; writing-review and editing, Zs.H.; formal analysis, A.Sz.

Funding

Work was supported by the Hungarian National Research, Development and Innovation Office (NKFIH K139312 and 2023 − 1.1.1-PIACI_FÓKUSZ-2024-00044). A.F. was supported by the European Union’s Horizon 2020 research and innovation program grant number 739593.

Data availability

The genome sequence of SZMC 28357T was deposited at the NCBI GenBank under accession number JAVLSB000000000. Partial sequences of the 16S rRNA, *rpoB* and *rpoD* loci of strains SZMC 28355, SZMC 28356, SZMC 28357T*, SZMC 28358, SZMC 28360 and SZMC 28359 have been deposited at NCBI GenBank under the accession numbers OR571460-OR571469 and OR563981.*.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (37.9KB, xlsx)
Supplementary Material 2 (6MB, pdf)

Data Availability Statement

The genome sequence of SZMC 28357T was deposited at the NCBI GenBank under accession number JAVLSB000000000. Partial sequences of the 16S rRNA, *rpoB* and *rpoD* loci of strains SZMC 28355, SZMC 28356, SZMC 28357T*, SZMC 28358, SZMC 28360 and SZMC 28359 have been deposited at NCBI GenBank under the accession numbers OR571460-OR571469 and OR563981.*.

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