Whole-genome sequencing uncovers the plant growth-promoting potential of Bacillus licheniformis G41, isolated from the rhizosphere soil of Gannan navel orange

doi:10.21203/rs.3.rs-5302746/v1

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Research Article

Whole-genome sequencing uncovers the plant growth-promoting potential of Bacillus licheniformis G41, isolated from the rhizosphere soil of Gannan navel orange

https://doi.org/10.21203/rs.3.rs-5302746/v1

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Journal Publication

published 11 Apr, 2025

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Background

Gannan navel orange orchards currently rely heavily on inorganic fertilizers, which have significantly degraded soil quality. Therefore, developing sustainable methods aligned with modern green agriculture is crucial. Plant growth-promoting rhizobacteria (PGPR) are beneficial microorganisms that can promote plant growth and contribute to soil ecological balance. In-depth research and application of PGPR can support both agricultural productivity and environmental sustainability.

Results

In this study, Bacillus licheniformis G41 was isolated from the rhizosphere soil of navel orange in southern Jiangxi Province. It was found that this strain possesses the ability to produce indole-3-acetic acid (IAA), synthesize siderophores, and solubilize phosphate. To further validate its PGP effects, strain G41 was inoculated into navel orange seedlings. After the inoculation experiment, plant height, biomass accumulation, chlorophyll content, and antioxidant enzyme activities increased in the inoculated group compared to the control group. Whole-genome sequencing revealed a genome size of 4610067 bp, with a total of 21 scaffolds, an average GC content of 46.17%, 4,700 predicted genes, 82 tRNAs, and 3 rRNAs. By comparing the predicted genes with the KEGG database, key functional genes related to IAA biosynthesis, siderophore biosynthesis and transport, and phosphorus cycle were identified.

Conclusion

Overall, genomic analyses and PGP experiments suggest that B. licheniformis G41, which possesses multiple plant growth-promoting traits, can effectively promote the growth of navel orange seedlings and exhibits potential as an efficient and environmentally friendly microbial fertilizer.

PGPR

Bacillus licheniformis

Navel orange

Whole-genome sequencing

Navel orange is a citrus fruit belonging to the Rutaceae family (Xiang et al. 2020). The Gannan navel variety is widely grown in Ganzhou City, Jiangxi Province, China, and has been awarded as a national geographic indication fruit because of its appealing appearance, delicious taste, and high nutritional value (Zhang et al. 2022). However, in recent years, the soil quality of navel orange orchards has suffered an unprecedented deterioration due to irrational fertilization strategies and negligence of agricultural management, which have drastically reduced the soil health and the yield of navel orange (Zhou et al. 2021). Therefore, there is an urgent need to explore and develop an environmentally friendly and efficient method to improve the soil quality of navel orange orchards and to promote the growth of this crop.

Plant growth-promoting rhizobacteria (PGPR) are beneficial to agriculture due to their cost-effectiveness, practicality, and sustainability(Bhat et al. 2023; Perveen et al. 2023; Nagrale et al. 2023), making them increasingly attractive for both researchers and farmers. PGPR live freely in the soil or associate with plant roots, promoting plant growth by secreting specific compounds while inhibiting the growth of other harmful bacteria. The growth-promotion effect of these microorganisms on plants can be classified into two categories. The first is direct growth-promotion, in which microorganisms can increase nutrient availability (phosphate solubilization, potassium solubilization, and nitrogen fixation), secrete growth-regulating molecules (indole-3-acetic acid, 1-aminocyclopropane-1-carboxylate deaminase, gibberellins, abscisic acid), and produce siderophores. The second is indirect promotion, in which PGPR recruit other beneficial microorganisms, inhibit pathogen proliferation, improve soil conditions, and mitigate abiotic stresses (Zhou et al. 2023). A variety of PGPR have been identified, including Azospirillum, Azotobacter, Gluconacetobacter, Pseudomonas, Bacillus, Burkholderia, Serratia, Streptomyces, Coniothyrium, Erwinia, Flavobacterium, Agrobacterium, and Trichoderma, etc. (Gray and Smith 2005; Tabassum et al. 2017; Fadiji et al. 2022). The abundance of these microbial resources offers a wide range of applications and innovation possibilities for agricultural production.

Bacillus spp. is regarded as one of the most promising PGPR due to its ability to promote plant growth (Woo et al. 2007; Berg et al. 2014; Qessaoui et al. 2019). These bacteria are now widely applied in the fields of food (Kim et al. 2024), medicine (Irshad et al. 2018), and agriculture (Khan et al. 2022). In this study, Bacillus licheniformis G41 was isolated from the rhizosphere soil of Gannan navel orange. The strain was found to possess the ability to produce IAA, secrete siderophores, and to solubilize phosphate. In order to validate the effectiveness of the strain in practical application, it was inoculated into the roots of navel orange seedlings, and its impact on plant growth was monitored. This approach not only verifies the growth-promoting properties observed under laboratory conditions but also aims to provide evidence for future field applications. Although PGPR have shown potential in the agricultural field, the molecular mechanisms underlying their growth-promoting traits remain to be fully elucidated. To address this, whole-genome sequencing technology was used to conduct an in-depth analysis of functional genes involved in the regulation of plant growth, thus laying a solid genetic foundation for future agricultural biotechnology development.

2.1 Collection of soil samples

The soil samples were collected from a navel orange orchard in Ganzhou City, Jiangxi Province, China (25°48′N, 115°09′E). The orchard was divided into six different areas, with five high-yield navel orange trees selected as sampling objects in each area. Rhizosphere soil was collected near the drip line of the selected navel orange trees, placed in sterile sealable bags and immediately brought back to the laboratory, where they were stored in a refrigerator at 4°C for future use.

2.2 Isolation and purification of isolates

Soil samples collected from the same area were mixed. After mixing uniformly, 5 g of the soil sample were poured into a conical flask containing 95 mL of sterile water. The conical flask with the soil suspension was placed in a constant temperature oscillating water bath and heat for 15 minutes (80°C, 180 r/min). After the treatment, the flask was left to rest for 20 minutes. The soil suspension was then diluted using a serial dilution method. Then, 100 µL from each dilution was plated on LB solid medium plates, in triplicate. The inoculated plates were incubated at 37°C. Finally, single colonies with distinct morphologies were selected and purified three times on LB solid medium. The purified strains were then inoculated onto LB slant medium and stored at a low temperature for subsequent experiments.

2.3 Characterization of PGP properties

The ability of the strain to secrete IAA was determined by the Salkowski assay (Glickmann and Dessaux 1995). The isolated strain was inoculated into LB liquid medium with 100 mg/L L-tryptophan and incubated for 24 hours at 37°C, shaking at 150 r/min. The bacterial suspension (10 mL) was then centrifuged at 8000 r/min for 10 minutes. The supernatant was combined with Salkowski reagent at the ratio of 1:1 by volume, incubated in the dark for 30 minutes, and the absorbance of the mixture was measured at 530 nm. Finally, the IAA content was calculated based on a standard curve.

Siderophore production was quantified as in Payne's method (Payne 1994), in MSA medium (Meng et al. 2022). The strain was cultured in MSA medium at 37°C under agitation at 180 r/min for 48 hours. After that, 10 mL of the culture was centrifuged at 8000 r/min for 10 minutes, and 2 mL of the supernatant were mixed with 2 mL of the CAS assay solution. After 1 hour, OD630 nm (denoted as As) was determined. As a negative control/baseline (denoted as Ar), 2 mL of CAS assay solution was mixed with 2 mL of supernatant from uninoculated MSA medium and measured as described above. The siderophores yield was quantified in siderophore units (SU) according to the formula: SU (%) = [(Ar - As) / Ar] × 100%.

To determine phosphate solubilization ability, inorganic phosphorus medium was prepared as in (Liu et al. 2014). The strain was inoculated in 50 ml liquid medium in a conical flask and incubated at 37°C with shaking at 180 r/min for 7 days. At the end of the experiment, the liquid medium was centrifuged at 8000 r/min for 10 minutes, and finally the available phosphorus content of the supernatant was determined according to the Mo-Sb anti-spetrophotometry method (Huang et al. 2010).

2.4 In-soil PGP experiment design

Navel orange seedlings of uniform size, robust growth, and free from disease or injury were purchased from a local nursery in Ganzhou City. They were cultivated in red soil, one of the four major soil types in southern Jiangxi Province. One of the isolated strains was cultured in 50 ml of liquid LB medium at 37°C under agitation at 180 r/min until reaching the logarithmic stage. Then the preinoculum was inoculated into fresh liquid LB medium and incubated under the same conditions (37°C, 180 r/min) for 24 hours. The bacterial culture was diluted to the required OD600 of 1 by adding an appropriate amount of sterile water. The strain-inoculated experimental group and the uninoculated control group, each counted six replicates, totaling 12 pots. The experiment was carried out on the rooftop canopy of Yifu Building, Gold Campus, Jiangxi University of Science and Technology, over a period of nine weeks. Seedlings in the experimental group were inoculated weekly with 200 mL of the bacterial solution, while the control group received sterile liquid LB medium in the same manner. Daily watering was conducted, with the frequency adjusted based on actual weather conditions.

2.5 Evaluation of in-soil PGP effect

Plant height, above-ground biomass, and below-ground biomass of navel orange seedlings were measured as in (Zong and Wang 2011): (1) Plant height was measured from the graft union to the highest point of the navel orange seedlings; (2) The fresh weight of above-ground and below-ground biomass was recorded after washing the soil from the roots with water. The content of chlorophyll in the leaves was determined using the ethanol extraction method as described by Lichtenthaler and Wellburn (Lichtenthaler and Wellburn 1983). Superoxide dismutase (SOD) was determined by the SOD kit of Nanjing Jiancheng Bioengineering Institute. Peroxidase (POD) and catalase (CAT) activity were measured using POD activity detection kit and CAT activity detection kit of Beijing Solarbio Science & Technology Co., Ltd.

2.6 Whole-genome sequencing and analysis

In this study, de novo sequencing technology was employed for the whole-genome sequencing of the isolated strain. This sequencing approach allows for the analysis of the bacterial genome without relying on any reference sequences, using bioinformatics tools to reconstruct the genomic sequence from scratch. The sequencing was performed using the second-generation sequencing platform, Illumina, where DNA samples were used to construct fragments with insert sizes of approximately 400 bp, followed by paired-end (PE150) and single-end sequencing with a read length of 150 bp. Each sample was sequenced to achieve a genome coverage depth of 100x, which was subsequently used to assemble multiple genomic scaffolds. Predicted genes and biological functions were annotated by comparison with reference databases. The KEGG database was used to identify the KEGG names, KO IDs, KO descriptions, and other functional annotations, providing insights into gene functions at a systemic level. The whole-genome sequencing was performed by Shanghai Majorbio Bio-Pharm Technology Co., Ltd.

According to sequencing data, 31 housekeeping genes (including dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB and tsf) were selected for sequence alignment in the NCBI database (URL: http://blast.ncbi.nlm.nih.gov/) using the BLAST + software (version 2.3.0) (URL: http://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/2.3.0/). Through this analysis, 19 strains were identified as the closest to the experimental strains at the genus level. Finally, the phylogenetic tree of the isolate was constructed using the NJ (Neighbor-Joining) method in the software MEGA (Version 10.1.7) (URL: https://www.megasoftware.net/).

2.7 Statistical analysis

All experimental data were recorded using Microsoft Excel 2013. Statistical t-test analysis of the pot experiment data was conducted using GraphPad Prism 10, and the results were graphically represented. All results are presented as mean ± standard deviation (SD).

3.1 Plant growth promoting activities

The screened strain exhibited a variety of growth-promoting properties (Table 1). After one day of incubation in LB liquid medium containing L-tryptophan (100 mg/L), the IAA yield of strain G41 was 23.22 ± 0.03 mg/L. The quantitative determination of siderophores was measured in siderophore units, resulting in 62.17 ± 0.37% after two days on incubation in MSA medium. The phosphate solubilization capacity of the strain was determined by the Mo-Sb anti-spetrophotometry method as 100.18 ± 5.87 mg/L.

Table 1

Plant growth-promoting properties of strain G41
PGP trait	IAA production (mg/L)	Siderophores (%)	Phosphate solubilization (mg/L)
Activity	23.22 ± 0.03	62.17 ± 0.37	100.18 ± 5.87

3.2 Effect of strain inoculation on the growth of navel orange seedlings

Inoculated navel orange seedlings exhibited a significantly enhanced growth trend compared to the control group (Figs. 1, 2), as evidenced by metrics such as plant height (Fig. 2A), above-ground biomass (Fig. 2B), and below-ground biomass (Fig. 2C). The average height of navel orange seedlings in the uninoculated control group was 55.7 cm, the above-ground and below-ground biomass weighed 49.76 g and 20.23 g, respectively. In contrast, the navel orange seedlings inoculated with the strain grew to a height of 65.8 cm, representing an increase of 18.13%; the above-ground biomass showed a significant increase of 60.14 g, corresponding to an increase of 20.86%; the below-ground biomass also showed a significant increase of 17.8%. In summary, the inoculation with the strain G41 promoted the overall growth of navel orange seedlings, with effects on biomass accumulation. The average content of chlorophyll a in the control group reached 0.97 mg/g, while chlorophyll b was 0.34 mg/g, resulting in a total average chlorophyll content of 1.31 mg/g (Figs. 2D, 2E and 2F). In contrast, the inoculated plants showed an average content of chlorophyll a of 1.33 mg/g and chlorophyll b of 0.49 mg/g, leading to a total average chlorophyll content of 1.82 mg/g and an increase of 38.93% in total chlorophyll content. The average activity levels of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the control group were 819.95 U/g, 7180.55 U/g, and 323.18 U/g, respectively. In the inoculated plants, the average activity levels of SOD, POD, and CAT were 1054.77 U/g, 13552.7 U/g, and 759.36 U/g, respectively, representing an increase of 28.64%, 88.74%, and 134.97% compared to the control group. These results indicate that there were significant differences in the activities of SOD, POD, and CAT between the inoculated treatment group and the control group.

3.3 Whole-genome overview and phylogenetic analysis

To further investigate the PGP capabilities of the strain G41 at the genetic level, whole-genome sequencing was performed. The sequencing indicated that the genome size of this strain is 4,610,067 bp, with a total number of 21 scaffolds. The average GC content is 46.17%, and the total number of genes is 4,700, including 82 tRNAs and 3 rRNAs (Table 2).

Table 2

Overview of the *B. licheniformis* G41 genome
Genome size (bp)	Scaffold no.	GC content (%)	CDS no.	tRNA no.	rRNA no.
4610067	21	46.17	4700	82	3

The characteristics of strain G41 genome are shown in the Fig. 3. The genome is circular, with different COG functional classifications distinguished by various colors. Specifically, the CGView genomic circle map is divided into seven concentric rings, arranged from the outermost to the innermost as follows: the first ring represents coding sequences (CDS) on the positive strand; the second and third rings display positive and negative strand sequence features, respectively; the fourth ring shows the CDS on the negative strand; the fifth circle displays the GC content, where outward and inward peaks indicate regions with higher and lower GC content than the genome-wide average, respectively; the sixth circle represents the GC-Skew value which is used to determine the leading and lagging strands, as well as the start and end of replication. The GC-skew is calculated using the formula: GC-skew = (G – C) / (G + C); the innermost circle represents the genome size of the strain.

For the identification of the taxonomic affiliation of strain G41, its house-keeping gene sequences were analyzed against the corresponding sequences in the NCBI database using the software BlAST+. The results revealed a sequence similarity of up to 99.1% between strain G41 and Bacillus licheniformis (GCF_000011645.1). Based on these alignment results, strain G41 was classified as Bacillus licheniformis and accordingly named Bacillus licheniformis G41. A phylogenetic tree was constructed by the software MEGA as shown in Fig. 4.

3.4 Functional annotation of the strain genome

The 4,700 predicted genes were compared with the COG, GO, and KEGG databases to obtain corresponding functional annotations. According to the COG database annotations, 3,779 genes from strain G41 were classified into 23 specific categories. Among these, 402 genes related to carbohydrate transport and metabolism represented the most abundant functional category, followed by transcription, which included 394 genes, and amino acid transport and metabolism, involving 367 genes. The extracellular structures category showed the lowest number of annotated genes, with a total of five (Fig. 5). A total of 3,117 genes were annotated in the GO database and were assigned functional tags, covering three major types: biological process, cellular component, and molecular function, corresponding to 1,591, 1,545, and 2,471 gene annotations, respectively. Within their respective classifications, proteolysis, membrane, and ATP binding genes occupied dominant positions (Fig. 6). In the KEGG database, 3,447 genes were annotated, which were divided into 6 primary and 43 secondary categories. In the pathways of environmental information processing and metabolism, 404 and 2,616 genes were annotated, respectively. In the environmental information processing pathway, 212 and 189 genes associated with signal transduction and membrane transport accounted for 52.48% and 46.78% of the total number of genes, respectively. In the metabolism pathway, genes were predominantly found in global and overview maps, carbohydrate metabolism, and amino acid metabolism, with the relevant gene numbers being 1,019, 381, and 283, accounting for 38.95%, 14.56%, and 10.82% (Fig. 7).

3.5 Genomic Mining of Plant Growth-Promoting Genes in B. licheniformis G41

The genome sequencing results indicate that strain G41 harbors numerous genes associated with growth-promoting properties. As shown in Table 3, a total of 39 functional genes were identified, including 9 genes involved in IAA biosynthesis, 11 genes related to siderophore biosynthesis and transport, and 19 genes involved in the phosphorus cycle. Among these functional genes, those related to IAA biosynthesis include aofH, amiE, a gene encoding aldehyde dehydrogenase, trpA, trpB, trpC, trpD, trpE and trpF. Monoamine oxidase plays a role in the conversion of tryptamine to indole-3-acetaldehyde in the tryptamine (TAM) pathway, amidase is able to convert indole-3-acetamide to indole-3-acetic acid in the indole-3-acetamide (IAM) pathway, and aldehyde dehydrogenase is a key enzyme in the indole-3-pyruvate (IPyA) pathway catalyzing the conversion of indole-3-acetaldehyde to indole-3-acetic acid (Tang et al. 2023). The tryptophan-linked gene trpABCDEF is associated with the biosynthesis of tryptophan, a precursor substance of indole-3-acetic acid (Singh et al. 2021). The genes entABCE (Pakarian and Pawelek 2016), dhbF (Abe et al. 2019), asbA (Lyngwi et al. 2016), and menF (Dahm et al. 1998)are involved in the biosynthesis of nonribosomal peptide siderophores. Three genes related to siderophores were found in the cellular ABC transporters pathway, namely fecB, fecC, and fecD (Braun and Herrmann 2007). The genes hemH is associated with the synthesis of protoporphyrin/coproporphyrin ferrochelatase in the porphyrin metabolism pathway, which is also related to siderophore production (Zhou et al. 2024). Among the phosphorus cycle genes, 6 genes related to phosphate ester mineralization were identified, specifically glpQ (Schwan et al. 2003), glpA (Schryvers and Weiner 1982), glpK (Rawls et al. 2011), phoA (Liu et al. 2018), phoD (Liu et al. 2018), and a 3-phytase-encoding gene (Liu et al. 2018). A total of 7 genes involved in inorganic phosphate solubilization, all related to organic acid production were also found, including gdh, ackA, gltA, pyk, pyc, buk, and aroK (Guo 2024). Additionally, genes encoding proteins of the high-affinity inorganic phosphate transport system, namely pstA, pstB, pstC, and pstS (Liu et al. 2018) were found, along with phosphate starvation-induced response regulatory genes phoR (Liu et al. 2018) and phoP (Choi et al. 2009).

Table 3

PGP trait-associated genes identified in the genome of strain G41
Function	KO ID	KO name	KO description
IAA biosynthesis	K00274	aofH	monoamine oxidase [EC:1.4.3.4]
	K01426	amiE	amidase [EC:3.5.1.4]
	K00128	-	aldehyde dehydrogenase (NAD+) [EC:1.2.1.3]
	K01695	trpA	tryptophan synthase alpha chain [EC:4.2.1.20]
	K01696	trpB	tryptophan synthase beta chain [EC:4.2.1.20]
	K01609	trpC	indole-3-glycerol phosphate synthase [EC:4.1.1.48]
	K00766	trpD	anthranilate phosphoribosyltransferase [EC:2.4.2.18]
	K01657	trpE	anthranilate synthase component I [EC:4.1.3.27]
	K01817	trpF	phosphoribosylanthranilate isomerase [EC:5.3.1.24]
Siderophore biosynthesis and transport	K00216	entA	2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase [EC:1.3.1.28]
	K01252	entB	bifunctional isochorismate lyase/aryl carrier protein [EC:3.3.2.1 6.3.2.14]
	K02361	entC	isochorismate synthase [EC:5.4.4.2]
	K02363	entE	2,3-dihydroxybenzoate—[aryl-carrier protein] ligase [EC:6.3.2.14 6.2.1.71]
	K04780	dhbF	glyine—[glycyl-carrier protein] ligase [EC:6.2.1.66]
	K24108	asbA	spermidine-citrate ligase [EC:6.3.2.-]
	K02552	menF	menaquinone-specific isochorismate synthase [EC:5.4.4.2]
	K23181	fecB	ferric citrate transport system substrate-binding protein
	K23183	fecC	ferric citrate transport system permease protein
	K23182	fecD	ferric citrate transport system permease protein
	K01772	hemH	protoporphyrin/coproporphyrin ferrochelatase [EC:4.98.1.1 4.99.1.9]
Phosphorus cycle	K01126	glpQ	glycerophosphoryl diester phosphodiesterase [EC:3.1.4.46]
	K00111	glpA	glycerol-3-phosphate dehydrogenase [EC:1.1.5.3]
	K00864	glpK	glycerol kinase [EC:2.7.1.30]
	K01077	phoA	alkaline phosphatase [EC:3.1.3.1]
	K01113	phoD	alkaline phosphatase D [EC:3.1.3.1]
	K01083	-	3-phytase [EC:3.1.3.8]
	K00034	gdh	glucose 1-dehydrogenase [EC:1.1.1.47]
	K00925	ackA	acetate kinase [EC:2.7.2.1]
	K01647	gltA	citrate synthase [EC:2.3.3.1]
	K00873	pyk	pyruvate kinase [EC:2.7.1.40]
	K01958	pyc	pyruvate carboxylase [EC:6.4.1.1]
	K00929	buk	butyrate kinase [EC:2.7.2.7]
	K00891	aroK	shikimate kinase [EC:2.7.1.71]
	K02038	pstA	phosphate transport system permease protein
	K02036	pstB	phosphate transport system ATP-binding protein [EC:7.3.2.1]
	K02037	pstC	phosphate transport system permease protein
	K02040	pstS	phosphate transport system substrate-binding protein
	K07636	phoR	two-component system, OmpR family, phosphate regulon sensor histidine kinase PhoR [EC:2.7.13.3]
	K07658	phoP	two-component system, OmpR family, alkaline phosphatase synthesis response regulator PhoP

In this work, a strain capable of producing IAA, siderophores, and solubilizing phosphate was isolated from a navel orange orchard in the Gannan region. The strain was inoculated into the roots of navel orange seedlings in a in-soil experiment, and the results suggested its effectiveness in promoting various plant traits. This finding is consistent with numerous literature reports, about the important role of Bacillus spp. in promoting plant growth. Specifically, Shin et al. found that B. velezensis BS1, capable of producing hydrolytic enzymes (cellulase and protease) and siderophores, could promote the growth of pepper seedlings (Shin et al. 2021). Similarly, Sharma et al. isolated the strain B. subtilis KU21 from the roots of Rosmarinus officinalis, which exhibited phosphate solubilization, nitrogen fixation, IAA and siderophores production, hydrogen cyanide (HCN) production, lytic enzyme activity, and ACC deaminase activity; furthermore, growth-promoting experiments on tomato showed enhanced seed germination, nutrient acquisition, and soil quality parameters (NPK) compared to the control group (Sharma et al. 2024). Devi et al. recovered two plant growth-promoting Bacillus strains (B. licheniformis MNNITSR2 and B. velezensis MNNITSR18) with multiple growth-promoting traits, which were found to significantly increase rice plant height, root length, root number, tiller number, leaf number, dry weight, and yield in both single inoculation and mixed inoculation treatment groups as compared to the control in a subsequent pot experiment.(Devi et al. 2023). A recent study proved that inoculating B. subtilis CG-6 not only significantly increased the plant height, root length, fresh weight, and dry weight of alfalfa but also significantly increased the levels of antioxidant enzymes in alfalfa leaves, with increases ranging from 15.52–34.03% (Chen et al. 2024).

As the earliest discovered plant hormones, indole-3-acetic acid (IAA, also known as indoleacetic acid or auxin) is an indispensable key factor in the regulation of plant growth and development (Brown 1974; Teale et al. 2006; Spaepen and Vanderleyden 2011). It is worth noting that the existence of this biologically active substance is not limited to plants, and a variety of microorganisms, including bacteria (Kang et al. 2023), yeasts (Soponputtaporn et al. 2024), and fungi (Abdelhamid et al. 2024), have also been found to possess the ability to synthesize IAA. Delving deeper into the mechanism of microbial synthesis of IAA, five major L-tryptophan-dependent pathways have been revealed in the existing literature: indole-3-acetamide (IAM), indole-3-acetonitrile (IAN), tryptophan side-chain oxidase (TSO), tryptamine (TAM), and indole-3-pyruvic acid (IPyA) (Etesami and Glick 2024). In this study, strain G41 had key genes on the three pathways of IAM, TAM, and IPyA, as well as tryptophan-linked gene. Similar to our results, Ji et al. observed a complete IPyA pathway in the whole-genome sequencing analysis of B. amyloliquefaciens Ba13 (Ji et al. 2021). In addition, Batista et al. found the presence of two synthesis pathways (IPyA and TAM) in B. thuringiensis RZ2MS9 (Batista et al. 2021). In the research field of Lysinibacillus, the genes amiE and aldH related to the IAM and IPyA pathways (Hilário et al. 2024), and found the key genes of the IPyA and TAM pathways (Pantoja-Guerra et al. 2023). The genome annotation of Bacillus subtilis confirmed the presence of the tryptophan-linked gene trpABCDEFS (Fang et al. 2023).

Siderophores are low-molecular-weight compounds synthesized by bacteria, actinomycetes, fungi, certain algae, and plants under iron-limiting conditions and are unique in their ability to specifically chelate \(\:{\text{Fe}}^{\text{3+}}\), thereby effectively alleviating the pressure of iron deficiency (Kumar et al. 2018). To date, over 500 compounds have been identified as belonging to the siderophores family, which can be primarily classified into three major types based on the chemical groups that chelate trivalent iron ions: catecholate siderophores, hydroxamate siderophores, and carboxylate siderophores (Boukhalfa and Crumbliss 2002). In the process of exploring the mechanisms of siderophores synthesis, Sheng et al. firstly analyzed the siderophores synthesis genes of Brevibacillus brevis GZDF3 using genome mining technology and constructed the phylogenetic tree of each synthesis gene, respectively, and ultimately confirmed the strain's ability to produce catechol-type siderophores using CAS liquid phase detection and the Arnow method (Sheng et al. 2018). Moreover, the whole-genome sequencing results of B. subtilis TY-1 also contained a cluster of genes related to the synthesis of bacillibactin, a catechol-type siderophore (Tian et al. 2023). Chandwani et al. sequenced the whole-genome of a strain of B. subtilis (CWTS 5) and identified a number of genes involved in the biosynthesis and transport of siderophores (Chandwani et al. 2023). In the present work, we systematically categorized and organized the genes related to siderophore synthesis and transport from three aspects, namely, the biosynthesis of siderophore group nonribosomal peptides, the ABC transporters pathway, and the porphyrin metabolism pathway.

Phosphate solubilizing microorganisms (PSMs) can not only effectively promote the dissolution of inorganic phosphorus (Pi) by secreting protons, organic acids, inorganic acids, and other substances but also mineralize organic phosphorus (Po) through the secretion of various phosphatases, thereby increasing the content of soluble phosphorus in the soil (Tian et al. 2021; Liu et al. 2024). This process enriches the soil's phosphorus nutrient pool and enhances the efficiency of plant uptake and utilization of available phosphorus. Xu et al. analyzed the putative mechanisms by which B. subtilis YB-04 promotes cucumber seedlings growth from the perspective of genome sequencing and discovered that the genome of this strain contains genes encoding proteins related to the phosphate-specific transport system (pstA, pstB, pstC, pstS), response regulatory genes under phosphate starvation conditions (phoR, phoP), and alkaline phosphatase genes (phoA, phoD), which collectively constituted the strategy for the strain to adapt to a low-phosphorus environment (Xu et al. 2022). Another article not only identified genes encoding multiple phosphatases as well as proteins related to the phosphate-specific transport system but also found several genes involved in the secretion of organic acids (Zhao et al. 2022). The high affinity phosphate transport system pstABCS can be utilized under environmental conditions in which phosphate is limiting (Martín and Liras 2021). The phosphate starvation response regulation in strain G41 is putatively regulated by the two-component regulatory system phoR-phoP. PhoR responds to phosphate deficiency by phosphorylating phoP, and the phosphorylated phoP then binds to specific sequences on DNA, precisely activating or repressing the transcription of genes (Santos-Beneit 2015). The regulation and expression of this key genes provide valuable insights into the molecular mechanisms of the phosphorus cycle in the microbe-soil-plant system.

This study focused on the microbial resources in the rhizosphere soil of Gannan navel orange. A bacterial strain named G41 was isolated and was found to possess the capability to produce IAA, siderophores, and solubilize phosphate. Further validation through pot experiments showed that inoculation with strain G41 increased the plant height, biomass, chlorophyll content, and antioxidant enzyme activities of navel orange seedlings, suggesting it to possess PGP capabilities in vivo. Subsequently, whole-genome sequencing of strain G41 was performed. Based on the analysis of housekeeping genes, the strain was identified as Bacillus licheniformis and officially designated as Bacillus licheniformis G41. Notably, this strain harbors a rich repository of functional genes, and this work found a total of 39 genes associated with PGP, which are broadly distributed across key processes of IAA biosynthesis, siderophore biosynthesis and transport, and the phosphorus cycle. In summary, B. licheniformis G41 holds great potential for application as a biofertilizer in sustainable agriculture. The whole-genome sequencing provides a solid theoretical foundation for further investigation into the genetic mechanisms underlying its plant growth-promoting activities.

Ethics approval and consent to participate: Not applicable.

Consent for publication: Not applicable.

Availability of data and materials: The research data supporting this publication are available through the data platform of Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (https://www.majorbio.com/). All other relevant data supporting the findings of this study are available from the corresponding author upon reasonable request.

Competing interests: The authors declare that they have no competing interests.

Funding: This study was funded by the Key Research and Development Project of Ganzhou Science and Technology Bureau (2022B-NY9223).

Authors' contributions: H.C. designed and implemented experiments, conducted data analysis, and drafted the initial manuscript. T.P. assisted with experiments execution and participated in data analysis. W.Z. assisted in the conduct of the experiments and provided technical support. H.H. contributed to data interpretation and offered critical feedback on the manuscript. S.Y. and Y.Z. as the corresponding authors, supervised the entire project, designed the experiments, and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Acknowledgments: Not applicable.

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Whole-genome sequencing uncovers the plant growth-promoting potential of Bacillus licheniformis G41, isolated from the rhizosphere soil of Gannan navel orange

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Journal Publication

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Abstract

Background

Results

Conclusion

Figures

1. Introduction

2. Materials and Methods

2.1 Collection of soil samples

2.2 Isolation and purification of isolates

2.3 Characterization of PGP properties

2.4 In-soil PGP experiment design

2.5 Evaluation of in-soil PGP effect

2.6 Whole-genome sequencing and analysis

2.7 Statistical analysis

3. Results

3.1 Plant growth promoting activities

3.2 Effect of strain inoculation on the growth of navel orange seedlings

3.3 Whole-genome overview and phylogenetic analysis

3.4 Functional annotation of the strain genome

3.5 Genomic Mining of Plant Growth-Promoting Genes in B. licheniformis G41

4. Discussion

5. Conclusion

Declarations

References

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