Ambihai Shayanthan
Anna Motnenko
Justin P. Hawkins
Ivan J. Oresnik- 1Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada
- 2Department of Agricultural Chemistry, University of Jaffna, Kilinochchi, Sri Lanka
Phosphorus is a key nutrient needed for plant growth and is often found in soils in an insoluble form. While phosphate fertilizers promote quick plant growth, they can be easily converted to insoluble forms through soil processes or lost via runoff. This results in poor phosphate use efficiency, which is economically and environmentally costly. A possible way to remediate these problems is to introduce phosphate-solubilizing bacteria as a biological fertilizer. In this work, we report the isolation of eight phosphate-solubilizing bacteria from agricultural soils in Manitoba. Their ability to solubilize Ca3(PO4)2 ranged from 95 to 144 mg/dL. Based on whole-genome sequencing, the isolates consisted of six Paraburkholderia strydomiana isolates, comprising at least three distinct strains, a Paraburkholderia graminis, and a Burkholderia ambifaria isolate. In addition to solubilizing phosphate, the P. strydomiana strains visibly influenced soybean seedling growth. Utilizing the closed genomes from the isolates in this study, we were able to scaffold the type strain and show that P. strydomiana genomes appear to consist of two large replicons as well as a larger plasmid. Further genomic analysis also demonstrated that P. strydomiana appears to contain RuBisCO and a complete Calvin-Benson-Bassham pathway. Unlike the type strain, the isolates in this study did not carry genes associated with nitrogen fixation or the ability to form symbiotic associations.
Graphical Abstract.
1 Introduction
Phosphorus is the second most limiting nutrient in plant growth and development, playing a vital role in plant metabolic processes (Rawat et al., 2021). The amount of inorganic phosphate (Pi) determines the success of plants in both natural and agricultural ecosystems (Cheng et al., 2023). However, the amount of total phosphate in soil is low relative to other nutrients, and of the total phosphorus, only 0.1% is available as Pi, which is directly usable by the plant (Zhu et al., 2016; Zou et al., 1992). Consequently, the demand for phosphate fertilizers has increased to maintain plant productivity.
However, Pi can be easily unavailable due to immobilization, either by combining with aluminum or iron in acid soils or binding to calcium in alkaline-calcareous soils. The accumulation of immobilized phosphate in soils and potential transfer to water bodies ultimately leads to environmental eutrophication concerns (Gatiboni et al., 2020). Therefore, it is beneficial to explore ways to enhance the bioavailability of immobilized phosphorus in agricultural soils to mitigate the overuse of phosphorus fertilizers (Yu et al., 2019).
Soil microorganisms, such as bacteria, can recover nutrients from natural reservoirs, enriching soil with essential nutrients (Kamble et al., 2024; Philippot et al., 2024). Bacteria are abundant, especially in the plant rhizosphere (Banerjee and Van Der Heijden, 2023; Shayanthan et al., 2022). Applying phosphate-solubilizing bacteria is a promising strategy to enhance phosphorus use efficiency, as it converts insoluble phosphorus into soluble forms (Li et al., 2020; Yu et al., 2022). Bacterial isolates capable of solubilizing phosphate can be obtained from various diverse environments, including the rhizosphere and agricultural soils (Song et al., 2021).
Several mechanisms are involved in bacterial phosphate solubilization, including the production of organic acids, protons, siderophores, extracellular enzymes, and substrate mineralization (Cheng et al., 2023; Gatiboni et al., 2020; Rawat et al., 2021). Many bacterial genera can do this, such as Pseudomonas, Enterobacter, Bacillus, Serratia, Pantoea, Rhizobium, Arthrobacter, and Burkholderia (Janati et al., 2023; Rawat et al., 2021; Song et al., 2021). The genus Paraburkholderia, recognized as distinct from the genus Burkholderia in 2014, includes many plant-beneficial and environmental taxa (Paulitsch et al., 2021; Sawana et al., 2014). Bacteria in this genus are also able to solubilize phosphate (Kaur et al., 2016; Paulitsch et al., 2021).
Bacterial isolates selected for phosphate solubilization often exhibit additional plant-growth-promoting traits, including seedling growth promotion, nitrogen fixation, pathogen suppression through metabolite secretion, indole acetic acid production, ACC deaminase activity to modify hormones, and stress resistance to heavy metals (Bashan and de-Bashan, 2010; Cheng et al., 2023). Phosphate-solubilizing bacteria with plant-growth-promoting traits are widely reported, yet expression of these traits can vary substantially across different soils. In addition, edaphic factors can significantly influence phosphate-solubilizing efficiency and directly impact the composition of bacterial communities (Janati et al., 2023). We hypothesize that screening local agricultural soils may lead to the identification of bacterial isolates that can withstand the indigenous microbiota and contribute to sustainable agricultural production. This study aims to isolate phosphate-solubilizing bacteria from Manitoba soils and to characterize their potential for plant growth promotion.
2 Materials and methods
2.1 Culture conditions and isolation of phosphate-solubilizing bacteria
Bacterial strains were grown at 28 °C using a Tryptone Yeast Extract (TY) medium containing 5 g Tryptone and 3 g yeast extract per liter of water (Beringer, 1974). Pikovskaya (PVK) medium was used to visualize phosphate solubilization. This contained (per liter): 10 g glucose, 5 g Ca3(PO4)2–5 g (NH4)2SO4, 0.5 g NaCl, 0.2 g MgSO4, 0.2 g KCl, 0.2 g yeast extract, 0.5 g MnSO4, and 0.002 g Fe SO4 (Nautiyal, 1999). To isolate phosphate-solubilizing bacteria from soil samples, 5 g of soil was mixed with 50 ml of sterile water, diluted, and plated onto PVK agar (Nautiyal, 1999). Colonies forming larger zones of clearing were picked and single colony purified three times.
For long-term storage, purified cultures were grown overnight in TY to late log phase and mixed with a freezing solution (TY containing 24% vol/vol DMSO) such that the final concentration of DMSO was 8%. These were stored at -80 °C.
2.2 Soil collection
Soil samples were collected from the University of Manitoba Ian N. Morrison Research Farm near Carman, Manitoba (49.492261 N, 98.042497 W) in 2018. The plots sampled were part of a rotation study focused on incorporating soybean into a rotation with canola, corn, and wheat. In 2018, all four crops were grown. Soils were collected as 2 x 9 cm cores taken immediately adjacent to plant roots at crop maturity. Samples were stored at 4 °C, with subsamples frozen at -80 °C the same day.
2.3 DNA extraction and amplification of the V4 rRNA
To extract bacterial genomic DNA from isolated strains, colonies were picked with a sterile onoculating stick from an agar plate and resuspended in 0.1 ml ddH2O. From this, 50 μL was extracted using Qiagen’s DNeasy PowerSoil kit using the manufacturer’s protocol. Initial identification of bacterial isolates was done by sequencing the V4 region of the 16S rRNA. Briefly, the V4 region was amplified using the 515F; 5’-GTGCCAGCMGCCGCGG-3’ (Parada et al., 2016) – 806R; 5’-GGACTACNVGGGTWTCTAAT-3’ (Apprill et al., 2015) primer pair as previously described (Yudistira et al., 2021). The isolated PCR products sent to Centre d’expertise et de services Génome Québec for sequencing. Initial taxonomy was assigned to sequences using BLAST (2.11.0) against the NCBI nucleotide database.
2.4 Quantification of phosphate solubilization
To quantify phosphate solubilization by bacterial cultures, a QuantiChrom™ Phosphate Assay Kit was utilized. Briefly, three independent colonies were grown overnight in 5 mL TY broth. The optical densities (OD600) of the cultures were normalized to 1.0 and 1 mL was added to 5 mL of PVK broth and incubated at 28 °C for three days. The cultures were then pelleted for one minute at top speed in a microfuge, and the supernatants were assayed. The phosphate solubilization assays were carried out in a 150 µL final volume in a 96-well clear bottom plate that included 50 µL of sample or standard and 100 µL of the provided reagent which contained Maclchite Green and molybdate for color development. The contents were mixed and incubated in the dark for 30 minutes at room temperature. Color development was assayed at 620 nm using a plate reader. The phosphate concentration was calculated as follows:
ODBlank, ODStandard, and ODSample are the OD620nm values of the 0 Pi Blank, the 0.28 mg dL-1 Pi Standard, and the sample, respectively. Conversions: 1 mg/dL Pi equals 105.3 µM, 0.001%, or 10 ppm.
2.5 Assessment of plant growth-promoting capacity
Plant growth-promoting activity was determined using a vigor index (Abdul‐Baki and Anderson, 1973). Briefly, this index measures growth promotion using the mean root length, mean shoot length, and seed germination.
To carry out these assays, seeds were first surface sterilized for 20 minutes using a 1% hypochlorite solution. Subsequently, they were washed with approximately 10 volumes of sterile ddH2O over a period of approximately 30 minutes. Bacteria being tested were first grown in 5 mL broth culture of TY and grown overnight. The overnight culture was then used to inoculate a 100 mL broth culture of TY. The OD600 of these cultures was adjusted to an optical density of 1. A volume of 20 ml of the adjusted cultures was used to soak the surface sterilized seeds. Seeds were treated with 20 mL of the bacterial culture for 30 minutes. Control seeds were soaked in 20 mL of sterilized water ddH2O. Finally, the seeds were individually planted using sterile forceps into seed-starting trays that contained a sterile sand/vermiculite (1:1) mixture. These were subsequently covered with plastic cling film to reduce the drying of the seedlings. A total of 24 seeds were planted for each treatment. Seedlings were harvested 15 days after planting, and root and shoot lengths were measured. Significance was determined using a two-way ANOVA using Dunnett’s test for post hoc analysis.
2.6 Whole genome sequencing
Whole genome sequencing was carried out essentially as previously described using a MinION sequencer (Hawkins et al., 2022). To extract genomic DNA, bacteria were grown overnight in TY broth, and genomic DNA was extracted using the PureLink Genomic DNA mini kit (Invitrogen). Library preparation was performed using SQK-LSK-109 and EXP-NBD-104 kits following the manufacturer’s instructions (Oxford Nanopore Technologies, Oxford, UK). Sequencing was carried out using a Nanopore MinION Mk1B system with R10.3 flow cells. Sequencing was stopped once sufficient data were obtained to achieve approximately 100x coverage across all genomes. Sequencing reads were then base-called using Guppy-GPU (Wick et al., 2019). The generated FASTQ files were subjected to quality control and adaptor trimming. Adaptor trimming was performed using BBduk (Bushnell et al., 2017). de novo genome assembly was achieved using Flye, followed by three rounds of polishing using minimap2 (Kolmogorov et al., 2019; Li, 2018). Genome completion was estimated using CheckM (Parks et al., 2015) and found to be above 99%, with an estimated 1% contamination in all assemblies. Default parameters were used for all software in the analysis.
2.7 Analysis of genomic sequences
Phylogenetic analysis was carried out using programs and packages available on the Type Strain Genome Server (TYGS), found at https://tygs.dsmz.de/background/show. Assembled genomes were submitted to the Type Strain Genome Server (TYGS) to obtain DNA-DNA Hybridization (dDDH) values as well as G+C content (Meier-Kolthoff et al., 2022). Phylogenetic trees were also generated on the TYGS server using the Genome BLAST Distance Phylogeny (GBDP) approach, with the ‘coverage’ algorithm and distance formula d5.n (Meier-Kolthoff et al., 2013). Average Nucleotide Identity (ANI) values were calculated using fastANI (Jain et al., 2018). ANIclustermap (Shimoyama, 2022), found at https://github.com/moshi4/ANIclustermap) was used to visualize ANI data.
The WK1.1f assembly was scaffolded using RagTag (v2.1.0). The scaffold module was utilized with SI02 designated as the reference genome. The default minimap2 module was used to align the contigs from WK1.1f. Unplaced contigs were combined into a single scaffold labeled “ch0” with the -C option. All other parameters were left at their default settings. To compare the genomes, BRIG (v0.95) was employed. The concatenated WK1.1f fasta sequence formed the backbone for the alignment. GC content and GC skew rings were added, followed by a BLASTn alignment of the SI02 and SI08 genomes. The BLASTn thresholds were set at 50% and 70% identity. Symbiosis genes were annotated separately, following the layout of the WK1.1f backbone.
Genome synteny was assessed using SYNY (Julian and Pombert, 2024). Assembled genomes were annotated with Prokka (v1.14.6) using default settings, and the resulting GenBank files were used as input for the analysis. All isolates were subjected to pairwise comparisons using default settings for modification parameters. Synteny plots were generated based on collinearity inference using DIAMOND (Buchfink et al., 2021).
Pangenome analysis was conducted using Roary (v3.13). Genome assemblies were annotated with Prokka (v1.14.6). The GFF3 output files served as input for Roary. The pangenome was calculated with default settings, using the -e and -n options. The resulting presence-absence file was used to visualize the pangenome with an UpSet plot generated by the R package Complexupset version 1.33.
The genomes of SI01, SI02, SI04, SI08, and SI09 were aligned using Mauve within Geneious. Each aligned block was manually inspected for significant gaps in homology. Protein-coding regions unique to a cluster and containing more than 10 genes were extracted and concatenated. Each replicon was then subjected to cluster of orthologous groups (COGs) classification using eggNOG-mapper (v2.1.12), with DIAMOND selected for annotation. The total counts for each COG category per replicon were used to generate a heatmap in R.
Pathway completeness was assessed using eggNOG-mapper (v2.1.12). Protein-coding FASTA files generated by Prokka (v1.14.6) were used as input. The DIAMOND BLASTx option was selected to annotate coding sequences. To group and visualize metabolic pathways, KEGGaNOG (v0.5.6) was used. The annotation files produced by eggnog-mapper served as input to KEGGaNOG, using all default options.
3 Results
3.1 Isolation of phosphate−solubilizing bacteria
Bacteria that have phosphate-solubilizing capabilities often exhibit plant growth-promoting traits (Cheng et al., 2023; Shen et al., 2021). Many of the phosphate-solubilizing bacteria previously isolated have been isolated from the rhizosphere of several crop plants. These tended to be more metabolically active than those isolated from non-rhizosphere sources (Gyaneshwar et al., 2002). We collected soil samples adjacent to the roots from a rotation study focused on incorporating soybeans into rotations with canola, corn, and wheat.
After soils from each crop were initially plated onto PVK agar, 20 putative phosphate-solubilizing bacteria were found. Following single-colony purification and retesting for phosphate-solubilizing activity, nine strains consistently produced larger zones of clearing and were selected for further analysis. These were labeled as SI01-SI09. Based on the sequencing results of the V4 regions of the isolates, as well as the sample/plate from which each of the strains was initially isolated, and the growth characteristics, it was deemed likely that SI02 and SI03 were likely siblings, so SI03 was not further characterized. This left 8 isolates capable of solubilizing phosphate on PVK agar (Figures 1A, B). SI01 and SI09 were isolated from the corn plot, SI02-SI05 and SI07 from the soybean plot, and SI06 and SI08 from the canola and wheat plots, respectively. Based on the V4 sequences, except for SI05 and SI06, the strains were tentatively identified as Paraburkholderia strydomiana. SI05 and SI06 were tentatively identified as Burkholderia ambifaria and Paraburkholderia graminis, respectively.
Figure 1. Qualitative and quantitative analysis of phosphate solubilization. Isolated strains were either spotted onto PVK agar (A) and (B) to demonstrate phosphate solubilization or directly quantified (C), as described in materials and methods. (A) Strains are listed clockwise from the top: SI09, SI06, SI07, and SI08. (B) Strains are listed clockwise from the top: SI05, SI01, SI02, and SI04. (C) Data are presented as the average and standard deviation of three biological replicates (n = 3). All isolates solubilize significantly more phosphate than the uninoculated control (p=0.0001). Significance was determined using a one-way ANOVA and Dunnett’s test for post hoc analysis.
To quantify their ability, these strains were grown overnight in TY, subcultured into PVK broth for three days, and the supernatants were assayed for soluble Pi (Figure 1C). The results show that in all cases, the isolates could solubilize between 95–144 mg dL-1 phosphate, in contrast to the 10 mg dL-1 normally found in PVK medium. The quantification also corroborated the visual observation that each of the strains looked similar on PVK agar.
3.2 Plant growth enhancement through PSB inoculation
Seed germination and seedling emergence are crucial in plant development and linked to better later health (Gardarin et al., 2016). To assess if the strains promoted growth, they were tested on soybean seeds.
When soybean seeds were inoculated with the phosphate-solubilizing strains, it appeared that seeds treated with SI02, SI04, SI07, and SI09 looked larger and greener than the control plants that were treated with only water (Figure 2). Seeds treated with SI05 and SI06 looked smaller and were negatively impacted compared to the control plants (Figure 2). When these observations were used to calculate a vigor index, it was found that treating soybean seeds with these strains resulted in significant differences (Figure 3). Seeds treated with SI02, SI04, SI07, and SI09 increased the vigor index, whereas SI05 and SI06 decreased the vigor index (Figure 3C). Looking through the individual measures that were used to determine the vigor index, it was noted that changes that were seen in the soybean seedlings were related to the percentage of seeds that germinated as well as the differences in the root and shoot lengths (Figures 2, 3). We note that all the strains that affected the germination rate of soybean were P. strydomiana (Figure 3A). Due to inconsistent growth, strains SI01 and SI08 were excluded from these assays.
Figure 2. Effect of phosphate-solubilizing isolates on soybean seedling growth. Surface-sterilized seeds were treated with phosphate-solubilizing isolates and planted aseptically. Seedlings were harvested 15 days after planting. Representative plants were selected for visualization. Treatments are labeled as shown.
Figure 3. Quantification of the effects of phosphate-solubilizing isolates on soybean seedlings. Phosphate-solubilizing isolates were applied to surface-sterilized seeds, which were then planted in sand-vermiculite as described in the materials and methods. The data come from two independent trials, each with at least 24 seeds. (A) Seed germination. The germination rate of treated seeds was expressed relative to the uninoculated control, which was set at 100%. (B) Root and shoot measurements. (C) Vigor Index was calculated as (mean shoot length + mean root length) * (% germination). Significance, where shown, was determined using a two-way ANOVA followed by Dunnett’s post hoc test. *p < 0.05.
3.3 Genome sequencing
P. strydomiana strains have previously been isolated from nitrogen-fixing nodules taken from Hypocalyptus sophoroides in South Africa (Beukes et al., 2019). Additionally, Paraburkholderia is strongly associated with plant growth-promoting traits. Since the isolates in this study were not derived from nodules but were selected based on their ability to solubilize phosphate, sequencing these isolates was of interest to see if their genome sequences might provide insights into their capabilities.
Whole genome sequencing using the MinION yielded greater than 35x sequencing depth per sample (Table 1). As expected, based on preliminary sequencing of the V4 region, six of the eight isolates belonged to the species P. strydomiana (SI01, SI02, SI04, SI07, SI08, and SI09), and the other two were P. graminis (SI06) and B. ambifaria (SI05).
Table 1. Genomic features of isolated PSB strains.
The genome assembly of B. ambifaria showed 5 contigs with a total size of 8.076 Mbp, an N50 of 3.02 Mbp, a GC content of 66.3%, and coverage of 75x. Using the Rapid Annotation Subsystem Technology (RAST), this genome was predicted to contain 7130 protein-coding genes.
The genome of P. graminis is 7.243 Mbp with 3 contigs, an N50 of 4.256 Mbp, 60.4% GC content, and 50x coverage, comprising 6385 protein-coding and 69 tRNA genes. P. strydomiana assemblies mostly had 3 contigs, except SI07 with 9. (Table 1). If other assemblies were used as scaffolds, SI07 could also be resolved to have 3 contigs. Their sizes ranged 7.2–7.791 Mbp, N50 3.9–4.33 Mbp, GC 58.2–61.8%, coverage 35x–118x, with 6915–7226 protein-coding and 65–68 tRNA genes.
3.4 Relatedness of P. strydomiana isolates
Using assembled genomes, we calculated pairwise ANI and dDDH values for each isolate and its closest relatives with the Type Strain Genome Server (TYGS) (Table 2). The nearest related species for each query is shown in Table 2. Each strain’s ANI compared to its match was over 95%, and its dDDH exceeded 70%, which are the currently accepted cut-offs for species identity, when compared to the type strain.
Table 2. Genome comparison with type strains.
Using the genome sequences from the three identified subject strains (Table 2), an all-against-all ANI analysis was conducted. As expected, isolates SI05 and SI06, which were identified as P. ambifaria and B. graminis, respectively, had no relation to the any of the other sequences in the comparison (Figure 4). The isolates identified as P. strydomiana were divided into three groups with 100% identity (Figure 4). These groups consisted of SI01, SI108, and SI109, SI02 and SI04, as well as SI07. We note that while all of these had greater than 95% identity with Wk1.1fT, these groups appeared to be distinct based on the analysis (Figure 4).
Figure 4. Average nucleotide identity of phosphate-solubilizing isolates. A heat map of whole genome average nucleotide identity among phosphate-solubilizing isolates, including P. stryodmiana WK1.1fT. The average nucleotide identity was calculated using fastANI and visualized with ANIclustermap.
To corroborate the ANI analysis, a phylogenetic analysis was performed using the Genome Blast Distance Phylogeny method on the TYGS server. The results show that the resultant tree supported the ANI analysis (Figure 5). Collectively, the data support the hypothesis that these isolates appear to represent three distinct strains, which are in turn distinct from the type strain Wk1.1f (Figure 5).
Figure 5. TYGS whole genome phylogenetic tree. The tree was inferred with FastME 2.1.6.1 (Lefort et al., 2015) from Genome BLAST Distance Phylogeny (GBDP) distances calculated from genome sequences. Branch lengths are scaled by GBDP distance d5. Numbers above branches are GBDP bootstrap support values over 60% from 100 replications, with an average of 77.3%. The tree was rooted at the midpoint (Shimoyama, 2022). User-submitted strains are marked on the figure with blue crosses. Other strains used were selected by the server for comparison.
3.5 Genomic architecture of P. strydomiana
The genome of strain WK1.1fT, with over 120X coverage, was assembled into 259 contigs (Beukes et al., 2019). Other P. strydomiana genomes in NCBI (https://www.ncbi.nlm.nih.gov/datasets/genome/?taxon=1245417) also vary from 38 to 259 contigs, showing an incomplete understanding of their genome architecture. We aimed to see if WK1.1fT shared the same genome structure as our isolates.
Since WK1.1fT shared the greatest similarity with SI02, the WK1.1fT contigs were aligned to SI02 using the minimapp2 option in RagTAG (Alonge et al., 2022), and the existing assembly was scaffolded. Contigs that were not placed were concatenated into a single scaffold. Utilizing BRIG (Alikhan et al., 2011), the resultant contigs were then subsequently compared with SI02, SI07, and SI08 (Figure 6). From the results, it is clear that the genomes show high similarity (Figure 6). Aside from some minor differences, the WK1.f1 genome was similar to our sequenced isolates across the two largest contigs.
Figure 6. BRIG visualization of the 3 isolate subgroups to a scaffolded WK1.1fT. Ring content in order from the inside out is the GC content, GC skew, contig size, BLASTn alignment with SI02, BLASTn alignment with SI07, BLASTn alignment with SI08, and lastly the location of symbiotic genes on the WK1.1fT genome. Note that the symbiotic genes were not found in either SI02, SI07, or SI08. The red contig is a concatenation of the elements that could not be scaffolded to SI02 and consists of 127 individual sequences.
Additionally, 127 sequences that did not align well from WK1.f1 were placed in the concatenated scaffold showed identity with the smallest replicon from SI02 and SI08. This region exhibits multiple regions of low or no similarity to the other isolates and contains numerous insertion elements and recombinases, suggesting it may be highly dynamic. Notably, SI07, which we also could not close, aligned well with WK1.1f and exhibited similar gaps to the other isolates. Generally, based on our analysis P. strydomiana genomes appear to consist of 3 replicons, Contig 1, which is between 3.8 - 4.3 Mb, Contig 2 which is between 3.2 - 3.3 Mb, and Contig 3 which shows the greatest variability with strains S102 and S104 having a replicon of approximately 0.24 Mb, whereas S101, S108, and S109 had a replicon of approximately 0.5 Mb.
Using SYNY to visualize genome collinearity (Julian and Pombert, 2024), the analysis revealed high concordance within P. strydomiana groups (SI02 vs. SI04, and SI08 vs SI09) across all three replicons (Supplementary Figures 1A, B, respectively). When comparing SI02 to SI08, there is substantial synteny between the two largest replicons, and very limited synteny with the smallest replicon (Supplementary Figure 1C). When SI02 and SI08 are compared to SI07, there is significant collinearity across the two largest replicons; however, qualitatively, each has unique regions (Supplementary Figures 1D, E). Similar results were seen when comparing these strains with WK1.1fT (Supplementary Figures 1F–H). Collectively, these analyses corroborate the average nucleotide identity as well as the phylogenetic analysis (Figures 4, 5) and suggest the major differences in the genome make-up are due to the variability of the smallest replicon.
Although the two largest replicons were largely syntenic, it was of interest to determine if these regions of variability affected the organism’s overall physiology. To determine the functionality in these variable regions, the genomes of SI01, SI02, SI04, SI08, and SI09 were first aligned with Mauve. Since SI07 was not a closed genome, it was excluded from the analysis. Each aligned block from the larger replicons was manually checked for gaps in homology. Protein-coding regions unique to each cluster (SI01, SI08, SI09, or SI02 and SI04), with more than 10 genes, were extracted and concatenated. Due to the dissimilarity of the smallest replicons, the entire replicon was considered. The concatenated variable regions were then classified using eggNOG-mapper (Cantalapiedra et al., 2021).
Comparison of the gaps in the largest replicon revealed that both strain groups mainly contain genes in COG groups S, P, M, and V. Group M covers cell wall/membrane/envelope biogenesis, Group P involves inorganic ion transport and metabolism, Group V includes defense mechanisms, and Group S has genes for unknown proteins (Figure 7).
Figure 7. COG analysis of P. strydomiana isolates’ variable regions. Regions >10 genes on each replicon were identified and concatenated. Due to the high variability of the smallest replicon (labelled plasmid), the entire replicon was used in full. Protein coding regions were classified with eggNOG-mapper, and category abundances shown in a heat map. COG categories are classified as follows: A, RNA processing and modification; B, Chromatin structure and dynamics; D, Cell cycle control, cell division, chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; G, Carbohydrate transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism; J, Translation, ribosomal structure, and biogenesis; K, Transcription; L, Replication, recombination, and repair; M, Cell wall/membrane/envelope biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport, and metabolism; R, General function prediction only; S, Function unknown; T, signal transduction mechanisms; U, Intracellular trafficking, secretion, and vesicular transport; V, Defense mechanisms; W, Extracellular structures; X, mobilome; Y, Nuclear structure; Z, Cytoskeleton.
Comparing the variable regions of replicon two shows that both strain groups contain COG groups C, G, K, and L. Group L includes genes for replication, recombination, and repair, while Group K involves proteins related to transcription. Group G encodes carbohydrate transport and metabolism, and group C includes genes for energy production and conversion (Figure 7).
Comparing the plasmids, the highest proportion of shared genes are from Group S, which encodes proteins of unknown function. Plasmids from SI01, SI08, and SI09 have more recognized COG groups than SI02 and SI04. Notably, these plasmids are nearly twice as large as those in other strains (Figure 7).
3.6 Pan-genome analysis of P. strydomiana
Genome structure analysis indicated some variability among strains. To evaluate this, a pangenome analysis was performed (Figure 8). The pangenome includes 12,123 genes, with the core genome consisting of 5,195 genes. The type strain, WK1.1f, has 2551 additional genes not present in the other isolates. The number of unique shared genes aligns with hierarchical clustering from ANI analysis and phylogeny (Figures 4 and 5). Strains SI01, SI08, and SI09, which are closely related phylogenetically, share 920 unique genes; SI02 and SI04 share 723; SI07 has an additional 598 unique genes. Aside from SI01, each strain also contains unique genes: SI02 and SI04 have 14 and 12, respectively, while SI08 and SI09 have 69 and 73, respectively. Notably, the isolates share 267 unique genes not found in the type strain.
Figure 8. Pan-genome analysis of P. strydomiana. Pan-genome analysis was conducted using Roary (v3.13) with Prokka (v1.14.6) annotated genome assemblies. Results are visualized with an UpSet plot created by the R package ComplexUpSet version 1.33. The set size shows the total gene count in each genome, while the intersection size indicates the shared sets between genomes, highlighted by the black lines.
3.7 Comparative genomic analysis of P. strydomiana
Using annotated genomes, P. strydomiana was analyzed with KEGGaNOG to visualize pathway completeness (Popov et al., 2025). Core metabolic pathways, including glycolysis, pentose phosphate pathway, Entner-Doudoroff pathway, TCA cycle, glyoxylate shunt, and gluconeogenesis, are present in all strains (Figure 9). All strains contain RuBisCO and genes for the Calvin-Benson-Bassham cycle (Figure 9). In addition, all amino acid biosynthetic pathways are present (Figure 9). The presence of complete cytochrome c, cytochrome bd, and electron transport oxidoreductases suggests an aerobic lifestyle. All strains also have many genes for Type II, III, and VI secretion systems (Figure 9). Regarding phosphate use, all strains have phosphate transport and bidirectional polyphosphate pathways (Figure 9).
Figure 9. Presence or absence of common metabolic pathways. Annotated genomes of the P. strydomiana isolates, as well as the type strain WK1.1fT, were analyzed using KEGGaNOG. Pathway completeness is scored with a value between 0 (absent) and 1 (complete) and is visualized on a blue scale.
There are notable differences between the type strain and the strains isolated in this study. The type strain WK1.1f contains a phosphonate transporter and genes for breaking carbon-phosphate bonds. It also possesses genes necessary for nitrogen fixation and has been demonstrated to be capable of symbiotic nitrogen fixation (Beukes et al., 2019). We note that the majority of the genes associated with nitrogen fixation are located on the third replicon (Figure 6). Additionally, the annotated genome predicts that strains SI01, SI02, SI04, SI07, SI08, and SI09 are capable of performing mixed acid fermentation to produce lactate.
3.8 Presence of genes associated with plant growth promotion
Although many plant growth-promoting bacteria have been reported (Abou Jaoudé et al., 2024; Khoso et al., 2024), very little is known about the molecular basis for their ability to promote plant growth. Several genes have been correlated with plant growth promotion, including pyrroloquinoline quinone encoding genes (pqq), nifHDK, which encodes nitrogenase, acdS, which encodes 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, hcnABC, which encode enzymes that produce hydrogen cyanide; phlABCD, which encode genes necessary for the biosynthesis of 2,4-diacetylphloroglucinol, and budABC, which has been shown to affect stomatal activity (Bruto et al., 2014). To determine if any of these might be present, the genomes of each strain were queried for their presence. The results show that all the strains contained pqqBCDE, hcnABC, and acdS. The strains appeared to contain two genes necessary for the synthesis of acetoin/2,3-butanediol but were missing the initial step of the pathway. The strains did not contain genes indicative of nitrogenase or 2,4-diacetylphloroglucinol (Table 3).
Table 3. Presence of genes associated with plant growth promotion.
4 Discussion
This study isolated phosphate-solubilizing strains from Manitoba agricultural fields and tested their plant growth-promoting traits. The strains belonged to three Gram-negative Betaproteobacteria species: P. graminis, B. ambifaria, and P. strydomiana, all of which are associated with promoting plant growth. The predominant mechanism of phosphate solubilization in many Gram-negative bacteria is the direct oxidation of an aldo-sugar (Rawat et al., 2021). This oxidation is carried out by a membrane-bound glucose dehydrogenase that requires pyrroloquinoline quinone (PQQ) as a cofactor. PQQ is a small, active redox cofactor encoded by the pqq operon, consisting of the core genes pqqABCDEF responsible for dehydrogenase activity and phosphate solubilization. Although the pqq operon can contain up to six genes, it has been shown that pqqA, pqqC, pqqD, and pqqE are essential for phosphate solubilizing activity (Shen et al., 2012). All analyzed strains contained pqqBCDE within a single operon but lacked pqqF or pqqA. The gene pqqA encodes a small peptide of about twenty amino acids and is often overlooked or misannotated within genome assemblies, so it has not been systematically studied. Notably, in Burkholderia ambifaria (SI05), the pqqA gene is located in an operon with the other pqq genes. Since PqqA is essential for function, we assume that the other strains also carry this component. However, although BLASTP analysis using PqqA from Pseudomonas fluorescens could detect pqqA-like genes, these were often not found within a genetic context that clearly indicated they were genuine hits, and further functional characterization is needed to confirm or refute these candidates.
Several studies have reported that B. ambifaria enhances plant growth in a variety of crops, including wheat, maize, Anoectochilus roxburghii, Amaranthus cruentus, A. hypochondriacus, and soybean. In contrast, inoculating soybean seeds with of B. ambifaria, SI05, resulted in significantly lower seedling growth in our study (Figures 2, 3). While we did not explore the presence or absence of genes that might be correlated with this phenotype, it is worth noting that the ability of bacteria to be plant growth-promoting is not a phenotype that is delineated by species.
The previous isolation of P. strydomiana was from the nodules of the Hypocalyptus sophoroides. Its nodulation ability has been proven on either cowpea (Vigna unguiculata) or siratro (Macroptilium atropurpureum) (Beukes et al., 2019). P. strydomiana is closely related to other nodulating species, such as P. kirstenboschensis, P. dilworthii, and P. rhynchosiae. Our P. strydomiana strains were isolated as free-living phosphate-solubilizing bacteria. The previous genome sequence for this species was deposited as a draft genome consisting of 259 contigs with an estimated genome size of 8,397,958 bp (Beukes et al., 2019). Our data indicate that we have isolated three distinct strains with genomes consisting of 3 replicons, with genome sizes ranging from 7,767,803 to 7,849,785 bp (Table 1). We found that by using contigs from the draft genome of the type strain WK1.1f and utilizing our P. strydomiana genomes as scaffolds, we could reduce the type strain to three replicons. This genome configuration is not atypical of what has been found within the family Burkholderiaceae, in that P. strydomiana has a multipartite genome consisting of a chromosome, a chromid, and a large plasmid (diCenzo et al., 2019). Additionally, the contig labelled as the chromosome contains greater than 44% of the genetic material in concordance with what has been previously reported (diCenzo et al., 2019).
The original P. strydomiana strains were isolated from nodules and shown to interact symbiotically, while the strains isolated in this study were free-living bacteria capable of solubilizing phosphate. Analysis indicates that most nodulation and nitrogen fixation genes are located on the large plasmid of WK1.1fT, and the isolated strains lack these genes (Figures 6, 9). The plasmids in these strains are highly variable (Figure 6), supporting the idea that they may aid environmental adaptation (diCenzo et al., 2019).
The goal of this study was to isolate phosphate-solubilizing bacteria that could serve as biological amendments to improve plant nutrient uptake. A limitation of the current study is that the isolates were tested on only one crop related to the original rotation study. Furthermore, our isolation process was not exhaustive, so many phosphate-solubilizing bacteria were likely missed due to limited sampling or the isolates’ inability to grow on PVK medium. Some of these candidates are now being tested under less controlled conditions and across different crops to assess their potential in agricultural settings. Using our closed genomes to scaffold the type strain, along with comparative genomic analysis, provides an opportunity to use P. strydomiana as a model organism for more detailed genetic and physiological studies, which could enhance our understanding of plant growth promotion.
Data availability statement
Genomes have been deposited under NCBI BioProject PRJNA1353154. BioSample Assessions described in this paper are as follows: SI01, SAMN52930736; SI02, SAMN52930737; SI04, SAMN52930738; SI05, SAMN52930739; SI06, SAMN52930740; SI07, SAMN52930741; SI08, SAMN52930742; and SI09, SAMN52930743.
Author contributions
AS: Writing – original draft, Investigation, Writing – review & editing, Methodology, Formal Analysis, Visualization, Data curation, Conceptualization, Validation. AM: Data curation, Methodology, Visualization, Writing – original draft, Writing – review & editing, Software, Formal Analysis, Investigation. JH: Data curation, Investigation, Writing – review & editing, Methodology, Software. IO: Supervision, Writing – original draft, Funding acquisition, Writing – review & editing, Resources, Project administration, Methodology.
Funding
The author(s) declared financial support was received for this work and/or its publication. Funding for this work was provided by the Manitoba Pulse and Soybean Growers and the Canadian Agricultural Partnership (Task No. 1000227246), and a Natural Science and Engineering Research Council Discovery Grant (RGPIN-2018-04966) to IO. AS gratefully acknowledges support from the University of Manitoba Faculty of Science Enhancement of Grant Stipends (SEGS) program.
Acknowledgments
The authors thank Patricia A. Ordoñez for facilitating soil collection.
Conflict of interest
The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The author IO declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbrio.2025.1735305/full#supplementary-material
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Keywords: Burkholderia, comparative genomics, Paraburkholderia, phosphate solubilization, plant-growth-promoting
Citation: Shayanthan A, Motnenko A, Hawkins JP and Oresnik IJ (2025) Isolation of non-symbiotic phosphate-solubilizing plant growth-promoting Paraburkholderia strydomiana. Front. Bacteriol. 4:1735305. doi: 10.3389/fbrio.2025.1735305
Received: 29 October 2025; Accepted: 28 November 2025; Revised: 27 November 2025;
Published: 19 December 2025.
Edited by:
Anil K. H. Raghavendra, New South Wales Department of Primary Industries, AustraliaReviewed by:
Nitin Kamble, University of Cincinnati Medical Center, United StatesKattie Weigh, NSW Government, Australia
Copyright © 2025 Shayanthan, Motnenko, Hawkins and Oresnik. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Ivan J. Oresnik, aXZhbi5vcmVzbmlrQHVtYW5pdG9iYS5jYQ==
†These authors have contributed equally to this work