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Received: 24 December 2024
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Published: 27 January 2025
Citation: Xu, W.; Liu, Y.; Cheng, Y.;
Zhang, J. Plant Growth-Promoting
Effect and Complete Genomic
Sequence Analysis of the Beneficial
Rhizosphere Streptomyces sp. GD-4
Isolated from Leymus secalinus.
Microorganisms 2025,13, 286.
https://doi.org/10.3390/
microorganisms13020286
Copyright: © 2025 by the authors.
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This article is an open access article
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licenses/by/4.0/).
Article
Plant Growth-Promoting Effect and Complete Genomic
Sequence Analysis of the Beneficial Rhizosphere
Streptomyces sp. GD-4 Isolated from Leymus secalinus
Wanru Xu, Yimeng Liu, Yiping Cheng and Jie Zhang *
Key Laboratory of Biological Resources and Ecological Environment of Ministry of Education, College of Life
Sciences, Sichuan University, Chengdu 610064, China; xuwanru@stu.scu.edu.cn (W.X.);
liuyimeng@stu.scu.edu.cn (Y.L.); chenyiping@stu.scu.edu.cn (Y.C.)
*Correspondence: zhangjfy@scu.edu.cn
Abstract: Plant growth-promoting rhizobacteria (PGPR) are beneficial bacteria residing in
the rhizosphere and are capable of enhancing plant growth through various mechanisms.
Streptomyces sp. GD-4 is a plant growth-promoting bacterium isolated from the rhizosphere
soil of Leymus secalinus. To further elucidate the molecular mechanisms underlying the
beneficial effects of the strain on plant growth, we evaluated the growth-promoting effects
of Streptomyces sp. GD-4 on forage grasses and conducted comprehensive genome mining
and comparative genomic analysis of the strain. Strain GD-4 effectively colonized the
rhizosphere of three forages and significantly promoted the growth of both plant roots
and leaves. Genome sequence functional annotation of GD-4 revealed lots of genes associ-
ated with nitrogen, phosphorus, and sulfur metabolism. Additionally, genes potentially
involved in plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, tre-
halose production, siderophore production, and phosphate solubilization were annotated.
Whole-genome analysis revealed that GD-4 may possess molecular mechanisms involved
in soil nutrient cycling in rhizosphere soil and plant growth promotion. The bacteria also
possess genes associated with adaptability to abiotic stress conditions, further supporting
the ability of Streptomyces sp. GD-4 to colonize nutrient-poor soils. These findings provide
a foundation for further research into soil remediation technologies in plateau regions.
Keywords: plant growth promotion; Streptomyces; genome analysis; dissimilatory nitrate
reduction to ammonium; alpine grassland restoration
1. Introduction
The ecological quality of the ecological environment in the Qinghai–Tibet Plateau
region has an indicative effect on the global climate. Zoige is located in the hinterland
of the Qinghai–Tibet Plateau and is an important water conservation area for the Yellow
River and Yangtze River [
1
]. Due to nearly 40 years of overgrazing, some grasslands in
the Zoige area have become desertified and soil fertility has been severely depleted [
2
].
Numerous studies have shown that the soil fertility of Zoige degraded sandy land is far
lower than that of normal grassland; in particular, the nitrogen content is dozens of times
different than that of normal grassland soil [
3
]. It is difficult for most replanted grasses to
colonize and grow in the sandy soil. The pioneer plant Leymus secalinus is a native perennial
herbaceous plant in alpine grasslands [
4
]. As an important plant to resist desertification, it
can grow in severely desertified soil, thanks to its rapid growth and well-developed root
system. Through previous rhizosphere metagenomic studies [
4
,
5
], we speculated that in
Microorganisms 2025,13, 286 https://doi.org/10.3390/microorganisms13020286
Microorganisms 2025,13, 286 2 of 31
hotspots of microbial activity, rhizosphere microorganisms play an indispensable role in
the colonization process of pioneer plants. Plant growth-promoting rhizobacteria (PGPR)
can promote plant growth through a variety of mechanisms, including increasing the
absorption of nutrient uptake [
5
], the production of plant hormones [
6
], resistance to biotic
and abiotic stress, and the promotion of improvement of the rhizosphere environment [
7
].
However, for plants in plateau and poor-soil areas, limited nutrients, extreme climate,
and high altitude will limit the effects of these mechanisms. Therefore, isolating plant
growth-promoting rhizobacteria from native plants can effectively harness adaptation to
harsh soil conditions.
In areas experiencing grassland desertification, nitrogen typically originates from
atmospheric deposition [
8
], animal urine [
6
], and the microbial nitrogen cycle [
9
]. The
nitrogen required for restoring degraded grasslands typically takes a prolonged accumula-
tion period to support sufficient plant growth. To enhance plant growth and colonization
efficiency, improve soil fertility, and leverage the growth of pioneer plants to combat
desertification, it is essential to explore strategies for increasing nitrogen content in the
soil [
10
]. From the perspective of plant growth, the absorption and conversion of existing
inorganic nitrogen sources are critical. Therefore, utilizing the interactions between plants
and rhizosphere microorganisms is key to improving nitrogen use efficiency [11–13].
Grassland degradation is often accompanied by nitrogen loss, which may become
an important condition limiting the growth of pioneer plants [
14
]. For instance, nitrogen
sources available to plants include both direct and indirect forms, such as nitrate and
ammonium. Ammonium can be directly absorbed and assimilated by plants, while nitrate
typically enters the plant and is subsequently converted into ammonium [
15
]. However,
studies have shown that nitrate in sandy soils is prone to loss through leaching or denitrifi-
cation, which converts it into gaseous nitrogen, whereas ammonium nitrogen tends to be
more readily retained in the soil [
16
]. As a result, the challenge in Zoige’s restored sandy
lands, which are deficient in nitrogen sources, is to find effective strategies for nitrogen
retention that can promote plant growth in these nitrogen-limited environments.
The nitrogen cycle of soil microorganisms typically encompasses six distinct nitrogen
transformation processes [
17
]: nitrogen fixation, assimilation, ammonification, nitrification,
denitrification, and anaerobic ammonium oxidation. Dissimilatory nitrate reduction to am-
monium (DNRA) refers to the ability of certain microorganisms to utilize electron donors
in the process of reducing nitrate to ammonium [
18
]. Numerous potentials remain to be
explored in the current research on DNRA bacteria. Previous studies highlighted that the
pathway of the dissimilatory reduction of nitrate to ammonium can preserve bioavailable
nitrogen in soils deficient in organic matter [
13
,
19
]. Related reports from metagenomic
research indicate that Miscanthus condensatus, a perennial grass species, serves as the pri-
mary pioneer plant in the acidic volcanic sediments of Miyake Island, Japan. In conditions
characterized by soil nitrogen deficiency, a significant number of nitrogen cycle-related
genes associated with DNRA (nirB, nirD) are present in its rhizosphere [
12
]. To restore the
nitrogen balance affected by production imbalances, utilizing microorganisms for nitrogen
conversion presents a sustainable solution [
20
]. Many articles currently emphasize the
abundance of nitrogen cycle-related genes from microbial communities. For instance, there
are reports utilizing metagenomic sequencing technology to investigate how the rhizo-
sphere of alpine coniferous forests enhances plants’ acquisition of ammonium ions through
the activities of rhizosphere microorganisms [
11
]. However, there are few relevant literature
reports on the DNRA bacteria that promote growth from the rhizosphere of pioneer plants.
To improve the practical application of DNRA bacteria in production, it is essential to
identify and utilize superior bacterial strain resources.
Microorganisms 2025,13, 286 3 of 31
In this study, we isolated growth-promoting bacteria from the roots of Lycium chinensis
and obtained an Actinomycete strain. Following screening, culture, and whole-genome
sequencing, we discovered that this strain has functional genes related to plant growth
promotion. The annotation results of functional genes related to the nitrogen cycle indicated
that the strain had the potential to dissimilate nitric acid to ammonium. Through pot
experiments, it was observed that the subject exhibits a growth-promoting effect on various
dominant grassland plants, including Lolium perenne L., Elymus dahuricus Turcz., and Elymus
sibiricus L.
2. Materials and Methods
2.1. Sample Collection
Samples were collected from Axi Township (33
◦
41
′
0
′′
N, 102
◦
56
′
7
′′
E), Zoige County,
Sichuan Province, where the average annual temperature ranges from 0.6
◦
C to 1.2
◦
C, and
the average annual precipitation is between 600 mm and 800 mm [
21
]. For each
5 m ×5 m
plot, 10 plants from similar growth patterns were randomly selected. The top 10 cm of
soil was removed with sterilized shovels, and plants were carefully dug out. The roots
were shaken to remove loosely attached soil, and the firmly adhered soil was collected as
rhizosphere soil using a sterile brush. After cleaning and collecting the sandy soil attached
to the plant root system, the soil was transplanted into a pot for storage.
2.2. Isolation and Subculture of Bacteria
We weighed 10 g of Leymus secalinus root soil into a 250 mL Erlenmeyer flask and
added 90 mL of sterile water. We then placed the flask in a constant-temperature shaking
incubator set to 28
◦
C and to shake it at 120 rpm for 20 min. We allowed the mixture to settle
and carefully collected the supernatant, representing the 10
−1
dilution. Serial dilutions
were then performed to obtain 10
−2
, 10
−3
, and 10
−4
dilutions. From each dilution, 50
µ
L
was spread onto nitrogen-free solid medium (Ashby) plates. Each concentration gradient
was plated in triplicate. The plates were incubated at 28
◦
C for 3 to 5 days, during which
strains exhibiting rapid growth were selected and subjected to streak purification. The
purification process was repeated 2 to 3 times to isolate pure single colonies.
2.3. Plant Growth-Promoting Assay of Streptomyces sp. GD-4
We selected three grass species native to the local grassland (Lolium perenne L., Elymus
dahuricus Turcz., and Elymus sibiricus L.) for a growth promotion experiment using potted
plants. Before plant inoculation, the collected repaired grassland sandy soil was sieved and
sterilized by autoclaving at 121
◦
C for 30 min. The sterilized plant seeds were germinated
in the dark and subsequently transplanted into pots in a greenhouse for 2 days after one
week. A bacterial suspension of Streptomyces sp. GD-4 was prepared. Once the plants
had established stable growth in the pots, 5 mL of the actinomycete bacterial suspension
(OD = 0.1) was injected via root irrigation, followed by appropriate watering for continued
cultivation. After 30 days, measurements were taken for root length, leaf length, above-
ground and under-ground biomass, root absorption area, and plant chlorophyll content.
2.4. Root Colonization of Streptomyces sp. GD-4
Strain GD-4 was cultured for 14 days at 28
◦
C using the slide culture method. The
coverslips with attached hyphae were fixed in 5% glutaraldehyde, followed by dehydration
through a graded series of ethanol solutions (30%, 50%, 70%, 90%, 95%, and absolute
ethanol) [
21
]. After the initial sample preparation, the specimens were sputter-coated
with a thin layer of gold–palladium, and their morphological characteristics were then
observed using a scanning electron microscope (SEM). The observation method for the
Microorganisms 2025,13, 286 4 of 31
experimental group followed the procedure described above. Plant roots inoculated with
bacteria were carefully excised from the pots and thoroughly rinsed with sterile water until
no soil remained on the root surfaces. The roots were then fixed in 5% glutaraldehyde
for 8 h, after which 1–2 cm segments of the main root were cut. After dehydration and
drying in a graded ethanol series, the samples were examined using a scanning electron
microscope (SEM) to assess microbial colonization on the root surfaces. The control group
of plants was processed and imaged using the same procedure.
2.5. Ammonia Production
Ammonia production of the test strains was tested in peptone water (g/L: peptone
10.0; sodium chloride 5.0). Fresh culture (48 h age) was inoculated into 50 mL of peptone
water and cultivated at 28
◦
C, 150 rpm, for 7 days. Nessler’s reagent (0.5 mL) (Macklin,
Shanghai, China) was added to each bacterial suspension. Development of golden yellow
color was noted as a positive result for ammonia production.
2.6. Comparative Genome Analysis
The average nucleotide identity (ANI) values among 11 genome sequences, including
Streptomyces sp. GD-4 and other 10 Streptomyces strains, were calculated using the Majorbio
online service [
22
]. ANI results were used for hierarchical cluster analysis using MUMmer
v3.23 software.
2.7. Library Construction and Genome Sequencing
Genomic DNA was sequenced using a combination of PacBio Sequel IIe and Illu-
mina sequencing platforms. For Illumina sequencing, genomic DNA was used for each
strain in sequencing library construction. DNA samples were sheared into 400–500 bp
fragments using a Covaris M220 Focused Acoustic Shearer following the manufacturer’s
protocol. Illumina sequencing libraries were prepared from the sheared fragments using
the NEXTFLEX Rapid DNA-Seq Kit (Illumina, San Diego, CA, USA). Briefly, 5
′
prime ends
were first end-repaired and phosphorylated. Next, the 3
′
ends were A-tailed and ligated to
sequencing adapters. The third step was to enrich the adapters-ligated products using PCR.
The prepared libraries were then used for paired-end Illumina sequencing (2
×
150 bp) on
an Illumina Novaseq 6000 (Illumina Inc., San Diego, CA, USA).
For PacBio sequencing, genomic DNA was fragmented at ~10 kb. The fragmented
DNA was then purified, end-repaired, and ligated with SMRT bell sequencing adapters
following the manufacturer’s recommendations (Pacific Biosciences, Menlo Park, CA, USA).
Next, the PacBio library was prepared and sequenced on one SMRT cell using standard
methods.
2.8. Genome Assembly and Annotation
The data generated from the PacBio Sequel IIe and Illumina platforms were used for
bioinformatics analysis. The detailed procedures are as follows.
The raw Illumina sequencing reads generated from the paired-end library were sub-
jected to quality filtering using fastp v0.23.0. HiFi reads were generated from the PacBio
platform for analysis. Then, the clean short reads and HiFi reads were assembled to con-
struct complete genomes using Unicycle v0.4.8 [
23
] and Pilon v1.22 to polish the assembly
using short-read alignments, reducing the rate of small errors. The final assembled genome
was submitted to the NCBI database (accession number: PRJNA1200240). The coding
sequences (CDs) of chromosomes and plasmids were predicted using Glimmer or Prodigal
v2.6.3 and GeneMarkS [
24
], respectively. tRNA-scan-SE (v2.0) [
25
] was used for tRNA
prediction, and Barrnap v0.9 (https://github.com/tseemann/barrnap (accessed on 1 De-
cember 2024)) was used for rRNA prediction. The predicted CDs were annotated from NR,
Microorganisms 2025,13, 286 5 of 31
Swiss-Prot, Pfam, GO, COG, KEGG, and CAZY databases using sequence alignment tools
such as BLAST, Diamond, and HMMER. Briefly, each set of query proteins was aligned with
the databases, and annotations of best-matched subjects (e-value < 10
−5
) were obtained
for gene annotation. Biosynthetic gene clusters (BGCs) of secondary metabolites were
identified by antiSMASH v5.1.2 software.
3. Results
3.1. SEM Observation of the Morphology and Colonization of Strain GD-4
Streptomyces sp. GD-4 was isolated from the rhizosphere of the pioneer plant Leymus
secalinus and has demonstrated the ability to grow in nitrogen-deficient media (Ashby).
Scanning electron microscopy (Figure 1) revealed that strain GD-4 exhibits the typical
morphological characteristics of Streptomyces. SEM images taken after culturing on medium
for 14 days show that GD-4 has highly branched hyphae. The hyphae in the culture
medium formed curved spiral spore chains, and the free spores were smooth and rod-
shaped (Figure 1a). The shrinkage of the bacterial cells observed in the electron microscope
may be related to the dehydration treatment used in the experiment. Scanning electron
microscopy observations of Streptomyces colonization in the roots of three grass species
revealed clear colonization traces of Streptomyces sp. GD-4 in the rhizosphere of Elymus
dahuricus Turcz. (Figure 1b), Lolium perenne L. (Figure 1c), and Elymus sibiricus L. (Figure 1d).
Colonization was primarily concentrated in the taproot region. SEM observations revealed
that Streptomyces sp. GD-4 formed a dense colonization structure on the main root of grass
plants, closely interacting with the root surface through hyphae and extracellular secretions.
Microorganisms 2025, 13, x FOR PEER REVIEW 5 of 35
NR, Swiss-Prot, Pfam, GO, COG, KEGG, and CAZY databases using sequence alignment
tools such as BLAST, Diamond, and HMMER. Briefly, each set of query proteins was
aligned with the databases, and annotations of best-matched subjects (e-value < 10−5) were
obtained for gene annotation. Biosynthetic gene clusters (BGCs) of secondary metabolites
were identified by antiSMASH v5.1.2 software.
3. Results
3.1. SEM Observation of the Morphology and Colonization of Strain GD-4
Streptomyces sp. GD-4 was isolated from the rhizosphere of the pioneer plant Leymus
secalinus and has demonstrated the ability to grow in nitrogen-deficient media (Ashby).
Scanning electron microscopy (Figure 1) revealed that strain GD-4 exhibits the typical
morphological characteristics of Streptomyces. SEM images taken after culturing on me-
dium for 14 days show that GD-4 has highly branched hyphae. The hyphae in the culture
medium formed curved spiral spore chains, and the free spores were smooth and rod-
shaped (Figure 1a). The shrinkage of the bacterial cells observed in the electron micro-
scope may be related to the dehydration treatment used in the experiment. Scanning elec-
tron microscopy observations of Streptomyces colonization in the roots of three grass spe-
cies revealed clear colonization traces of Streptomyces sp. GD-4 in the rhizosphere of Ely-
mus dahuricus Turcz. (Figure 1b), Lolium perenne L. (Figure 1c), and Elymus sibiricus L. (Fig-
ure 1d). Colonization was primarily concentrated in the taproot region. SEM observations
revealed that Streptomyces sp. GD-4 formed a dense colonization structure on the main
root of grass plants, closely interacting with the root surface through hyphae and extra-
cellular secretions.
Figure 1. The observation of bacterial morphology and colonization by scanning electron micros-
copy (SEM), The red arrow indicates Streptomyces sp. GD-4. (a) Scanning electron micrograph of
Streptomyces sp. GD-4 cells. (b) The root colonization of Elymus dahuricus Turcz. after inoculation of
GD-4 by SEM. (c) The root colonization of Lolium perenne L. after inoculation of GD-4 by SEM. (d)
The root colonization of Elymus sibiricus L. after inoculation of GD-4 by SEM.
Figure 1. The observation of bacterial morphology and colonization by scanning electron microscopy
(SEM), The red arrow indicates Streptomyces sp. GD-4. (a) Scanning electron micrograph of Strepto-
myces sp. GD-4 cells. (b) The root colonization of Elymus dahuricus Turcz. after inoculation of GD-4 by
SEM. (c) The root colonization of Lolium perenne L. after inoculation of GD-4 by SEM. (d) The root
colonization of Elymus sibiricus L. after inoculation of GD-4 by SEM.
3.2. Effect of Strain GD-4 Inoculation on the Physiological Index of Plants
Greenhouse pot experiments were conducted using three grass species in degraded
sandy soil. The results indicated that Streptomyces sp. GD-4 had varying degrees of growth-
promoting effects on the development of gramineous plants under oligotrophic conditions
(Figure 2).
Microorganisms 2025,13, 286 6 of 31
Microorganisms 2025, 13, x FOR PEER REVIEW 6 of 35
3.2. Effect of Strain GD-4 Inoculation on the Physiological Index of Plants
Greenhouse pot experiments were conducted using three grass species in degraded
sandy soil. The results indicated that Streptomyces sp. GD-4 had varying degrees of
growth-promoting effects on the development of gramineous plants under oligotrophic
conditions (Figure 2).
Figure 2. Plant growth promotion assay of Streptomyces sp. GD-4 on pasture after 30 days of inocu-
lation. (a) From left to right: the Lolium perenne L. control group and the GD-4 inoculation group. (b)
From left to right: the Elymus sibiricus L. control group and the GD-4 inoculation group. (c) From
left to right: the Elymus dahuricus Turcz control group and the GD-4 inoculation group.
Both the above-ground and below-ground biomass of the plants showed significant
increases (Figure 3), particularly in the below-ground biomass of Elymus sibiricus L. and
Elymus dahuricus Turcz (Figure 3b). Inoculation with strain GD-4 significantly increased
the root length of Elymus dahuricus Turcz., and the root length and leaf length of Elymus
sibiricus L. showed an increasing trend, but it was not statistically significant (Figure A1).
In addition, inoculation with GD-4 significantly increased the chlorophyll content of the
leaves of three plants (Figure 3c), and the active root absorption areas of two plants were
increased (Figure 3d).
Figure 2. Plant growth promotion assay of Streptomyces sp. GD-4 on pasture after 30 days of
inoculation. (a) From left to right: the Lolium perenne L. control group and the GD-4 inoculation group.
(b) From left to right: the Elymus sibiricus L. control group and the GD-4 inoculation group. (c) From
left to right: the Elymus dahuricus Turcz control group and the GD-4 inoculation group.
Both the above-ground and below-ground biomass of the plants showed significant
increases (Figure 3), particularly in the below-ground biomass of Elymus sibiricus L. and
Elymus dahuricus Turcz (Figure 3b). Inoculation with strain GD-4 significantly increased
the root length of Elymus dahuricus Turcz., and the root length and leaf length of Elymus
sibiricus L. showed an increasing trend, but it was not statistically significant (Figure A1).
In addition, inoculation with GD-4 significantly increased the chlorophyll content of the
leaves of three plants (Figure 3c), and the active root absorption areas of two plants were
increased (Figure 3d).
Microorganisms 2025, 13, x FOR PEER REVIEW 7 of 35
Figure 3. Effects of Streptomyces sp. GD-4 strain on growth parameters of different pasture species
cultured in sandy soil for 30 Days: (a) leaf dry weight; (b) root dry weight; (c) chlorophyll content
index. (d) Active root absorption area. The values represent the means of repl icates (n = 3) ± standard
deviations. Asterisks in superscript indicate a significant difference from the control at 95% between
treatments. Each data point is the average of three replicates, and error bars represent ±SD. * Signif-
icance at p < 0.05; ** significance p < 0.01.
3.3. Comparative Genomics and Phylogenetic Analysis
Phylogenetic analysis based on 31 housekeeping gene sequences (Figure 4) indicated
that strain GD-4 is classified within the genus Streptomyces. It shares the highest sequence
similarity with Streptomyces canus (95.9%) and exhibits the closest genetic relationship
with Streptomyces fulvoviolaceus (96.2%). The strain forms a distinct clade with a bootstrap
support value of 93.8%. However, since strain GD-4 could not be identified at the species
level, it was designated as Streptomyces sp. GD-4. Although GD-4 shares many similarities
with several Streptomyces species, there have been no reports on the growth-promoting
mechanisms of this bacterium in the roots of pioneer plants in alpine sandy soils. The
successfully assembled GD-4 genome was analyzed by average nucleotide identity (ANI)
with 10 Streptomyces species (Table A1). The results showed that the ANI values for all 11
Streptomyces strains, including GD-4, were less than 95%; Streptomyces sp. HP-A2021 has
the highest ANI value of 86.48%, followed by Streptomyces rochei S32 with 85.96%.
Figure 3. Effects of Streptomyces sp. GD-4 strain on growth parameters of different pasture species
cultured in sandy soil for 30 Days: (a) leaf dry weight; (b) root dry weight; (c) chlorophyll con-
tent index. (d) Active root absorption area. The values represent the means of replicates (n= 3)
±standard
deviations. Asterisks in superscript indicate a significant difference from the control at
95% between treatments. Each data point is the average of three replicates, and error bars represent
±SD. * Significance at p< 0.05; ** significance p< 0.01.
Microorganisms 2025,13, 286 7 of 31
3.3. Comparative Genomics and Phylogenetic Analysis
Phylogenetic analysis based on 31 housekeeping gene sequences (Figure 4) indicated
that strain GD-4 is classified within the genus Streptomyces. It shares the highest sequence
similarity with Streptomyces canus (95.9%) and exhibits the closest genetic relationship
with Streptomyces fulvoviolaceus (96.2%). The strain forms a distinct clade with a bootstrap
support value of 93.8%. However, since strain GD-4 could not be identified at the species
level, it was designated as Streptomyces sp. GD-4. Although GD-4 shares many similarities
with several Streptomyces species, there have been no reports on the growth-promoting
mechanisms of this bacterium in the roots of pioneer plants in alpine sandy soils. The
successfully assembled GD-4 genome was analyzed by average nucleotide identity (ANI)
with 10 Streptomyces species (Table A1). The results showed that the ANI values for all 11
Streptomyces strains, including GD-4, were less than 95%; Streptomyces sp. HP-A2021 has
the highest ANI value of 86.48%, followed by Streptomyces rochei S32 with 85.96%.
Microorganisms 2025, 13, x FOR PEER REVIEW 8 of 35
Figure 4. (a) Phylogenetic tree constructed based on 31 housekeeping genes using the Neighbor-Join-
ing (NJ) method in MEGA 6.0 software. The red line represents Streptomyces sp. GD-4. (b) The heat
maps of ANI (average nucleotide identity) between strain GD-4 and other 10 Streptomyces genus.
3.4. Genome Characteristics of Strain GD-4
To further elucidate the mechanism by which bacterial GD-4 promotes the growth of
pioneer plants under oligotrophic stress conditions, we analyzed its entire genome. We
deposited the sequence information in NCBI GenBank. The analysis revealed that the
chromosome length of strain GD-4 is 9,994,786 bp, with an average G + C content of 70.46%
(Table 1). The complete GD-4 genome is estimated to contain 9352 coding sequences
(CDSs), 18 rRNA genes, 71 tRNA genes, 62 sRNA genes (Table 1), a phage region, and a
plasmid. A total of 9352 coding sequences (CDSs) were predicted. The Gene Ontology
(GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Ge-
nomes (KEGG) databases were used to predict and annotate the GD-4 genes, resulting in
5435 (63.66%), 7009 (74.9%), and 6107 (65.3%) annotations, respectively (Table 2). GO an-
notations (Figure A2) revealed involvement in Biological Processes (2727), Cellular Com-
ponents (2187), and Molecular Functions (4879). The major categories include integral
components of membranes (1534), DNA binding (769), and ATP binding (539).
Table 1. General features and genomic assembly of Streptomyces sp. GD-4.
Features Chromosome
Total size of the contigs (In Megabases) 9,994,786 bp
Number of protein-coding genes 9352
Number of rRNA genes 18
Number of tRNA genes 71
Number of sRNA genes 62
G + C% 70.46%
Signal transduction 138
Prophage regions 1
Figure 4. (a) Phylogenetic tree constructed based on 31 housekeeping genes using the Neighbor-
Joining (NJ) method in MEGA 6.0 software. The red line represents Streptomyces sp. GD-4. (b) The
heat maps of ANI (average nucleotide identity) between strain GD-4 and other 10 Streptomyces genus.
3.4. Genome Characteristics of Strain GD-4
To further elucidate the mechanism by which bacterial GD-4 promotes the growth
of pioneer plants under oligotrophic stress conditions, we analyzed its entire genome.
We deposited the sequence information in NCBI GenBank. The analysis revealed that
the chromosome length of strain GD-4 is 9,994,786 bp, with an average G + C content
of 70.46% (Table 1). The complete GD-4 genome is estimated to contain 9352 coding
sequences (CDSs), 18 rRNA genes, 71 tRNA genes, 62 sRNA genes (Table 1), a phage
region, and a plasmid. A total of 9352 coding sequences (CDSs) were predicted. The
Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia
of Genes and Genomes (KEGG) databases were used to predict and annotate the GD-4
genes, resulting in 5435 (63.66%), 7009 (74.9%), and 6107 (65.3%) annotations, respectively
(Table 2). GO annotations (Figure A2) revealed involvement in Biological Processes (2727),
Cellular Components (2187), and Molecular Functions (4879). The major categories include
integral components of membranes (1534), DNA binding (769), and ATP binding (539).
A total of 7009 COG-annotated genes were classified into 4 categories (Figure A3)
and 24 distinct COG types. The most abundant functional clusters identified include
Carbohydrate Transport and Metabolism (830 genes), Transcription (1071 genes), General
Function Prediction Only (830 genes), Signal Transduction Mechanisms (610 genes), and
Amino Acid Transport and Metabolism (581 genes). The KEGG database annotation
identified six major pathway categories (Figure 5a): Cellular Processes (217 pathways),
Microorganisms 2025,13, 286 8 of 31
Metabolism (5495 pathways), Genetic Information Processing (275 pathways), Human
Diseases (210 pathways), and Environmental Information Processing (412 pathways).
Table 1. General features and genomic assembly of Streptomyces sp. GD-4.
Features Chromosome
Total size of the contigs (In Megabases) 9,994,786 bp
Number of protein-coding genes 9352
Number of rRNA genes 18
Number of tRNA genes 71
Number of sRNA genes 62
G + C% 70.46%
Signal transduction 138
Prophage regions 1
Table 2. Summary of functional annotations.
Functional Annotations Number of Protein-Coding
Genes (CDs) Percentage (%)
Total 9352 100
COG 7009 74.9
KEGG 6107 65.3
GO 5435 63.66
NR 9258 58.11
Swiss-port 5902 63.1
Pfam 7426 79.4
Microorganisms 2025, 13, x FOR PEER REVIEW 9 of 35
Table 2. Summary of functional annotations.
Functional Annotations Number of Protein-Coding Genes (CDs) Percentage (%)
Total 9352 100
COG 7009 74.9
KEGG 6107 65.3
GO 5435 63.66
NR 9258 58.11
Swiss-port 5902 63.1
Pfam 7426 79.4
A total of 7009 COG-annotated genes were classified into 4 categories (Figure A3)
and 24 distinct COG types. The most abundant functional clusters identified include Car-
bohydrate Transport and Metabolism (830 genes), Transcription (1071 genes), General
Function Prediction Only (830 genes), Signal Transduction Mechanisms (610 genes), and
Amino Acid Transport and Metabolism (581 genes). The KEGG database annotation iden-
tified six major pathway categories (Figure 5a): Cellular Processes (217 pathways), Metab-
olism (5495 pathways), Genetic Information Processing (275 pathways), Human Diseases
(210 pathways), and Environmental Information Processing (412 pathways).
Figure 5. Classification of Streptomyces sp. GD-4 based on KEGG database annotation. (a) The ordi-
nate indicates the level2 classification of the KEGG pathway, and the ordinate indicates the number
of genes annotated under that classification. The column colors represent the level1 classification of
the KEGG pathway. The right-most column shows the number of genes in different level1 catego-
ries. (b) Circular genome map of strain Streptomyces sp. GD-4. From the outer circle to the inner
circle: The first and fourth rings represent the coding sequences (CDSs) on the forward and reverse
strands. The second and third rings show the distribution of CDSs, tRNA, and rRNA on the positive
and negative strands, respectively. The fifth ring depicts the GC content, while the sixth ring dis-
plays the GC-skew values.
3.5. Genes Associated with Plant Growth Promotion in GD-4 Genome
Functional analysis of the GD-4 genome revealed the presence of multiple genes in-
volved in various plant growth-promoting (PGP) functions, including nitrogen metabo-
lism, sulfur metabolism, auxin and siderophore biosynthesis, phosphate solubilization,
root colonization, and abiotic stress tolerance (Tables 3 and 4).
Figure 5. Classification of Streptomyces sp. GD-4 based on KEGG database annotation. (a) The ordinate
indicates the level2 classification of the KEGG pathway, and the ordinate indicates the number of
genes annotated under that classification. The column colors represent the level1 classification of the
KEGG pathway. The right-most column shows the number of genes in different level1 categories.
(b) Circular genome map of strain Streptomyces sp. GD-4. From the outer circle to the inner circle: The
first and fourth rings represent the coding sequences (CDSs) on the forward and reverse strands. The
second and third rings show the distribution of CDSs, tRNA, and rRNA on the positive and negative
strands, respectively. The fifth ring depicts the GC content, while the sixth ring displays the GC-skew
values.
3.5. Genes Associated with Plant Growth Promotion in GD-4 Genome
Functional analysis of the GD-4 genome revealed the presence of multiple genes in-
volved in various plant growth-promoting (PGP) functions, including nitrogen metabolism,
sulfur metabolism, auxin and siderophore biosynthesis, phosphate solubilization, root
colonization, and abiotic stress tolerance (Tables 3and 4).
Microorganisms 2025,13, 286 9 of 31
Table 3. Genes related to nitrogen, phosphorus, and sulfur metabolism.
Function Gene ID KO Name KO ID KO Description Enzyme
Nitrogen
metabolism
gene2544 gltB K00265 glutamate synthase (NADPH), large
chain [EC:1.4.1.13]
gene5942 nirD K00363 nitrite reductase (NADH), small
subunit [EC:1.7.1.15]
gene5943 nirB K00362 nitrite reductase (NADH), large
subunit [EC:1.7.1.15]
gene4307 narK K02575 MFS transporter, NNP family,
nitrate/nitrite transporter -
gene0326 narL K07684
nitrate/nitrite response regulator
NarL, two-component system, NarL
family
-
gene2048 narH K00371 nitrate reductase/nitrite
oxidoreductase, beta subunit [EC:1.7.5.11.7.99.-]
gene2049 narJ K00373
nitrate reductase molybdenum
cofactor assembly chaperone
NarJ/NarW
-
gene2544 gltB K00265 glutamate synthase (NADPH), large
chain [EC:1.4.1.13]
gene6510 gltD K00266 glutamate synthase (NADPH), small
chain [EC:1.4.1.13]
gene1739 glnA K01915 glutamine synthetase [EC:6.3.1.2]
gene2808 glnB K04751 nitrogen regulatory protein P-II 1 -
gene2807 glnD K00990 [protein-PII] uridylyltransferase [EC:2.7.7.59]
gene6281 glnE K00982
[glutamine synthetase]
adenylyltransferase/[glutamine
synthetase]-adenylyl-L-tyrosine
phosphorylase
[EC:2.7.7.42
2.7.7.89]
gene6662 iscU K04488 nitrogen fixation protein NifU and
related proteins -
gene2809 amt K03320 ammonium transporter, Amt family -
gene0085 nasT K07183
two-component system, response
regulator/RNA-binding
antiterminator
-
gene0517 moaC K03637 cyclic pyranopterin monophosphate
synthase [EC:4.6.1.17]
gene3219 moaE K03635 molybdopterin synthase catalytic
subunit [EC:2.8.1.12]
gene3830 moaA K03639 GTP 3′,8-cyclase [EC:4.1.99.22]
gene4835 moaD K03636 sulfur-carrier protein -
gene8041 urtE K11963 urea transport system ATP-binding
protein -
gene8042 urtD K11962 urea transport system ATP-binding
protein -
gene8043 urtC K11961 urea transport system permease
protein -
gene8044 urtB K11960 urea transport system permease
protein -
gene8045 urtA K11959 urea transport system
substrate-binding protein -
sulfur metabolism
gene1472 cysJ K00380 sulfite reductase (NADPH)
flavoprotein alpha-component [EC:1.8.1.2]
gene2334 cysH K00390 phosphoadenosine phosphosulfate
reductase
[EC:1.8.4.8 1.8.4.10]
gene2335 cysC K00860 adenylylsulfate kinase [EC:2.7.1.25]
gene2336 cysD K00957 sulfate adenylyltransferase subunit 2 [EC:2.7.7.4]
gene2337 cysN K00956 sulfate adenylyltransferase subunit 1 [EC:2.7.7.4]
Microorganisms 2025,13, 286 10 of 31
Table 3. Cont.
Function Gene ID KO Name KO ID KO Description Enzyme
sulfur metabolism
gene2339 ssuA K15553 sulfonate transport system
substrate-binding protein -
gene2340 ssuB K15555 sulfonate transport system
ATP-binding protein [EC:7.6.2.14]
gene2341 ssuC K15554 sulfonate transport system permease
protein -
gene5061 ssuD K04091 alkanesulfonate monooxygenase [EC:1.14.14.5
1.14.14.34]
gene2574 sseA K01011 thiosulfate/3-mercaptopyruvate
sulfurtransferase [EC:2.8.1.1 2.8.1.2]
gene4389 tauA K15551 taurine transport system
substrate-binding protein -
gene4390 tauB K10831 taurine transport system
ATP-binding protein [EC:7.6.2.7]
gene4388 tauC K15552 taurine transport system permease
protein -
gene4391 tauD K03119 taurine dioxygenase [EC:1.14.11.17]
gene6420 thiS K03154 sulfur carrier protein -
Phosphorus
metabolism
gene4333 pstC K02037 phosphate transport system
permease protein -
gene4334 pstA K02038 phosphate transport system
permease protein -
gene4335 pstB K02036 phosphate transport system
ATP-binding protein [EC:7.3.2.1]
gene1629 phoD K01113 alkaline phosphatase D [EC:3.1.3.1]
gene6104 phoA K01077 alkaline phosphatase [EC:3.1.3.1]
gene4080 ppx-gppA K01524
exopolyphosphatase/guanosine-5′-
triphosphate,3′-diphosphate
pyrophosphatase [EC:3.6.1.11
3.6.1.40]
-
gene4131 ppa K01507 inorganic pyrophosphatase [EC:3.6.1.1]
gene3124 phoP K07658
two-component system, OmpR
family, alkaline phosphatase
synthesis response regulator PhoP
-
gene3254 phoH K06217 phosphate starvation-inducible
protein PhoH and related proteins -
gene4251 phoU K02039 phosphate transport system protein -
gene5771 phoH2 K07175 PhoH-like ATPase -
gene0729 gdh K00034 glucose 1-dehydrogenase [EC:1.1.1.47]
Table 4. Genes and protein products present in the genome of Streptomyces sp. GD-4.
Function Gene ID KO Name KO ID KO Description Enzyme
Auxin
biosynthesis
gene6498 trpA K01695 tryptophan synthase alpha chain [EC:4.2.1.20]
gene6497 trpB K01696 tryptophan synthase beta chain [EC:4.2.1.20]
gene6496 trpC K01609 indole-3-glycerol phosphate
synthase [EC:4.1.1.48]
gene6381 trpD K00766 anthranilate
phosphoribosyltransferase [EC:2.4.2.18]
gene6492 trpE K01657 anthranilate synthase component I [EC:4.1.3.27]
gene5439 nthB K20807 nitrile hydratase subunit beta [EC:4.2.1.84]
gene5441 nthA K01721 nitrile hydratase subunit alpha [EC:4.2.1.84]
gene0620 - K00128 aldehyde dehydrogenase (NAD+) [EC:1.2.1.3]
gene1290 - K01593 aromatic-L-amino-acid/L-
tryptophan decarboxylase
[EC:4.1.1.28 4.1.1.105]
gene9104 - K01501 nitrilase [EC:3.5.5.1]
gene4607 hisC K00817 histidinol-phosphate
aminotransferase [EC:2.6.1.9]
Microorganisms 2025,13, 286 11 of 31
Table 4. Cont.
Function Gene ID KO Name KO ID KO Description Enzyme
Abiotic stress
tolerance
gene2632 ectB K00836 diaminobutyrate-2-oxoglutarate
transaminase [EC:2.6.1.76]
gene6739 ectD K10674 ectoine hydroxylase [EC:1.14.11.55]
gene6740 ectC K06720 L-ectoine synthase [EC:4.2.1.108]
gene6742 ectA K06718 L-2,4-diaminobutyric acid
acetyltransferase [EC:2.3.1.178]
gene3683 groEL K04077 chaperonin GroEL [EC:5.6.1.7]
gene3684 groES K04078 chaperonin GroES -
gene3608 betB K00130 betaine-aldehyde dehydrogenase
[EC:1.2.1.8] -
gene1092 betA K00108 choline dehydrogenase
[EC:1.1.99.1] -
gene1761 betI K02167
TetR/AcrR family transcriptional
regulator, transcriptional repressor
of bet genes
gene5096 cspA K03704 cold-shock protein -
gene4328 hspR K13640 MerR family transcriptional
regulator, heat-shock protein HspR -
gene4731 hslJ K03668 heat-shock protein HslJ -
gene3770 htpX K03799 heat-shock protein HtpX [EC:3.4.24.-]
gene6571 hslR K04762 ribosome-associated heat-shock
protein Hsp15 -
gene5866 hrcA K03705 heat-inducible transcriptional
repressor -
gene1087 proW K02001 glycine betaine/proline transport
system permease protein -
gene1088 proV K02000 glycine betaine/proline transport
system ATP-binding protein [EC:7.6.2.9]
gene1866 proP K03762 MFS transporter, MHS family,
proline/betaine transporter -
gene2716 proS K01881 prolyl-tRNA synthetase [EC:6.1.1.15]
gene4311 proA K00147 glutamate-5-semialdehyde
dehydrogenase [EC:1.2.1.41]
gene5828 proB K00931 glutamate 5-kinase [EC:2.7.2.11]
gene2163 ggt K00681
gamma-
glutamyltranspeptidase/glutathione
hydrolase
[EC:2.3.2.2 3.4.19.13]
gene0962 gst K00799 glutathione S-transferase [EC:2.5.1.18]
gene7747 trxA K03671 thioredoxin 1 -
gene4564 trxB K00384 thioredoxin reductase (NADPH) [EC:1.8.1.9]
gene2356 treX K01214 isoamylase [EC:3.2.1.68]
gene2358 treY K06044 (1->4)-alpha-D-glucan
1-alpha-D-glucosylmutase [EC:5.4.99.15]
gene2985 treS K05343
maltose alpha-D-
glucosyltransferase/alpha-
amylase
[EC:5.4.99.16 3.2.1.1]
gene4925 otsB K01087 trehalose 6-phosphate phosphatase [EC:3.1.3.12]
gene4926 otsA K00697 trehalose 6-phosphate synthase
[EC:2.4.1.15 2.4.1.347]
Iron uptake and
transport
gene8395 fhuB K23228 ferric hydroxamate transport
system permease protein -
gene8396 fhuD K23227 ferric hydroxamate transport
system substrate-binding protein -
gene8397 fhuC K10829 ferric hydroxamate transport
system ATP-binding protein [EC:7.2.2.16]
gene3465 fepD K23186 iron-siderophore transport system
permease protein -
gene3466 fepG K23187 iron-siderophore transport system
permease protein -
gene6856 fepC K23188 iron-siderophore transport system
ATP-binding protein [EC:7.2.2.177.2.2.-]
gene5573 desE K25287 iron-desferrioxamine transport
system substrate-binding protein -
gene5604 entS K08225 MFS transporter, ENTS family,
enterobactin (siderophore) exporter
-
Microorganisms 2025,13, 286 12 of 31
3.5.1. Nitrogen Metabolism
The ammonium production assay proved that GD-4 could produce ammonium nitro-
gen. In the prediction of nitrogen cycle genes in GD-4, genes related to the dissimilatory
nitrate reduction to ammonium (DNRA) pathway were identified. Key annotations in-
cluded genes encoding nitrate reductase (narH), nitrite reductase large subunit (nirB),
nitrite reductase small subunit (nirD), and genes for related regulatory factors and molyb-
denum cofactors (mobA, moeB, and moaACDE) (Table 3). In addition, some genes related
to nitrogen metabolism were identified. For instance, genes involved in the regulation of
the nitrogen cycle include gltD (glutamate synthase small-subunit protein), gltB (gluta-
mate synthase large-subunit protein), glnA (glutamine synthetase), and glnD (bifunctional
nitrogen sensor protein) [26].
Among the genes related to nitrogen metabolism, we also identified genes involved in
nitrogen transport, including nitrate/nitrite transporter (nark) and ammonium transporter
(amt) [
27
]. Additionally, core genes involved in the regulation of nitrogen metabolism
in prokaryotes were annotated, including those encoding nitrogen regulatory protein P-
II 1 (glnB) and the related genes glnAED (Table A2). P-II proteins are multifunctional
regulators in bacteria, archaea, and plastids, controlling nitrogen and carbon metabolism,
transporters, and signaling molecules [
28
]. In addition, the gene NarL (nitrate/nitrite
response regulator) is also present, typically involved in transcriptional activation as a
nitrate response regulator in E. coli [
29
]. At the same time, genes related to nitric oxide
reductase-activating proteins (NorD, NorQ) have not been identified [30].
Additionally, among the genes implicated in nitrogen metabolism in the GD-4 genome,
the gene encoding the urea transport system permease protein (urtC) and its associated
urtABDE gene cluster were identified (Table 3). These genes play a crucial role in the
efficient transport of extracellular urea into the cell, thereby facilitating the acquisition of a
stable nitrogen source from animal urine under nitrogen-limited conditions.
3.5.2. Sulfur Metabolism
The GD-4 genome contains the gene clusters cysJHCDN and ssuABC, which are in-
volved in sulfur metabolism and sulfate transport. These clusters encode key enzymes
such as adenylylsulfate kinase (cysC), sulfite reductase (cysJ), and phosphoadenosine
phosphosulfate reductase (cysH). Genes related to transport include those encoding 3-
mercaptopyruvate sulfurtransferase (sseA). Through the action of the ssuABC gene cluster,
GD-4 is capable of converting organic sulfides and sulfates into H
2
S. Studies have demon-
strated that exogenous H
2
S, acting as a signaling molecule, responds to environmental
stresses such as heavy metals, drought, and salinity, thereby regulating plant growth
[31–33].
The tau family genes (tauA, tauB, tauC, tauD) are involved in the thiophenylalanine (tau-
rine) metabolic pathway, assisting GD-4 in utilizing sulfur from taurine to meet its sulfur
requirements. The genome annotation revealed various sulfur ABC transporters, membrane
proteins, and the cysteine desulfurase subfamily, which includes sulfur carrier proteins
(thiQ, thiP, thiB, thiE, and thiS). Additionally, cysteine desulfurase (sufS) and the Fe-S
cluster assembly protein gene cluster (sufBCD) were also identified.
3.5.3. Phosphate Solubilization
The GD-4 genome contains genes involved in phosphate transport and assimilation,
including the phosphate transport system substrate-binding protein (pstS), ATP-binding
protein (pstB), permease protein (pstAC), and alkaline phosphatase (phoAD). Additionally,
it harbors genes encoding inorganic pyrophosphatase (PPA) and the phosphate metabolism
regulatory gene exopolyphosphatase/guanosine-5
′
-triphosphate,3
′
-diphosphate pyrophos-
phatase (ppx-gppA). The GD-4 genome also includes the glucose 1-dehydrogenase (gdh)
Microorganisms 2025,13, 286 13 of 31
gene, which aids in the oxidation of glucose to synthesize GA and is involved in the regula-
tion of phosphate (Table A3). However, the gene encoding the cofactor pyrroloquinoline
quinone (PQQ) for GA production has not been annotated. Additionally, the genome
contains genes for phosphonoacetate (phnA), phosphate transport, binding proteins, and
sensing and signal transduction genes (phoUHP).
3.5.4. Auxin Biosynthesis
The GD-4 genome has three annotated tryptophan-dependent synthesis pathway com-
plete or partial genes, IAM, IAN, and TAM Pathway, in which indole-3-acetaldehyde serves
as an intermediate from tryptophan through L-tryptophan decarboxylase [EC:4.1.1.105]
and monoamine oxidase (aofH) and is catalyzed by aldehyde dehydrogenase [EC:1.2.1.3]
to finally generate IAA (Figure 6). In the other two pathways, IAN is formed by indole-3-
acetonitrile catalyzed by nitrilase [EC:3.5.5.1], and TAM is formed by indole-3-acetamide
catalyzed by the enzyme encoded by gene amiE (Table A4). At the same time, indole-3-
acetonitrile can also form indole-3-acetamide through the enzyme encoded by the gene
nthAB (nitrile hydratases) (Table 4) and finally generate IAA [
34
]. Tryptophan synthesis
genes are annotated in the GD-4 genome, and tryptophan (Trp) is a general precursor for
the synthesis of IAA by most bacteria. Among them, the tryptophan operon (trpABCDE)
generates L-tryptophan through the catalysis of a series of enzymes and enters the IAA
synthesis pathway.
Microorganisms 2025, 13, x FOR PEER REVIEW 14 of 35
Figure 6. Schematic overview of metabolic pathways and transpo rt systems in Streptomyces sp. GD-4. Individual pathways are denoted by single-headed arrows, while
reversible pathways are denoted by double-headed arrows. Dashed arrows represent genes missing in genomes. The figure was created with BioRender.com.
Figure 6. Schematic overview of metabolic pathways and transport systems in Streptomyces sp. GD-4.
Individual pathways are denoted by single-headed arrows, while reversible pathways are denoted by
double-headed arrows. Dashed arrows represent genes missing in genomes. The figure was created
with BioRender.com.
Microorganisms 2025,13, 286 14 of 31
3.5.5. Identification of Genes Responsible for Bacterial Biocontrol
Streptomyces serve as a rich source of natural antibiotics and related compounds, and
they are extensively studied in both agricultural and medical fields [
35
]. The GD-4 genome
contains genes involved in the synthesis of phenazine, enediyne antibiotics, ansamycins,
vancomycin, and tetracycline (Table A5). For example, the phzEFS genes that are reported
to regulate phenazine biosynthesis are annotated in GD-4 [36].
3.6. Abiotic Stress Tolerance
The ectB gene (betaine-aldehyde dehydrogenase) annotated in GD-4 plays a role
in responding to environmental stress conditions, such as high salinity or drought, by
promoting the accumulation of ectoine. Simultaneously, the betIBA operon encodes a
group of enzymes regulated by the betI gene. This operon facilitates the conversion of
choline into glycine betaine, enabling bacteria to adapt to osmotic stress [
37
]. The groEL
and groES genes encode molecular chaperones that assist cells in managing heat stress.
Additionally, the cspA gene, encoding a cold-shock protein (CspA), was identified in
GD-4, while the hspR gene, a heat-shock protein transcriptional regulator, plays a role in
modulating stress responses. The hslJ, hslR, dnaJ, and dnaK genes are involved in protein
folding and repair processes [38].
Additionally, genes involved in oxidative stress response, including gamma-glutamylt-
ranspeptidase (ggt), glutathione S-transferase (gst), and thioredoxin reductase (trxAB),
were identified. Genes related to salt stress, such as proABPSVWX, which encodes
the proline transport system substrate-binding protein, were also predicted. Further-
more, genes associated with trehalose synthesis, including isoamylase (TreZ), trehalose
6-phosphate synthase (OtsA), trehalose 6-phosphate phosphatase (OtsB), and maltose
alpha-D-glucosyltransferase (TreS), were annotated.
3.7. Specific Gene Clusters in Streptomyces sp. GD-4
AntiSMASH analysis of the GD-4 genome identified 25 gene clusters associated with
secondary metabolite biosynthesis on the chromosome and 2 additional clusters on the
plasmid. These include four clusters encoding non-ribosomal peptide synthetases (NRPSs),
three NRPS-like clusters, four siderophore clusters, six terpene clusters, two type I polyke-
tide synthase (T1PKS) clusters, two melanin clusters, and seven clusters associated with
other secondary metabolites (Table 5).
The secondary metabolites predicted from the chromosomal gene clusters are pri-
marily antibiotics, siderophores, and terpenes. Salinomycin and chlorinated polyketides,
encoded by plasmid gene clusters, along with istamycin, synthesized by chromosomal
gene clusters, are all associated with antibacterial activity [
39
]. Naphthomycin A [
40
] and
albaflavenone [
41
] are antibiotics or antimicrobial compounds produced by Streptomyces
GD-4, exhibiting strong bacteriostatic and antifungal properties. Hopene, a terpene com-
pound widely found in bacterial membranes, has a similar secondary metabolite synthesis
gene cluster annotated in Streptomyces collinus Tü 365 [
42
]. In addition, ectoine is a natural
amino acid derivative that plays a vital role in stress resistance [
43
]. It provides osmotic
protection, exhibits antioxidant properties, and stabilizes protein structures.
Siderophores play a crucial role in chelating iron ions. The synthetic gene cluster re-
sponsible for the secondary metabolite amychelin belongs to the type I polyketide synthase
(T1PKS) pathway (Table 5). Amychelin is a non-peptide siderophore containing multiple
functional groups, such as phenolic hydroxyl and carboxyl groups, that chelate iron to form
high-affinity complexes [
44
]. As a vital secondary metabolite of plant growth-promoting
microorganisms, it enhances iron absorption by plant root systems.
Microorganisms 2025,13, 286 15 of 31
Table 5. Predicted secondary metabolite biosynthetic gene clusters in Streptomyces sp. GD-4.
Cluster ID Type Similar Cluster Similarity (%) Gene No.
cluster1 butyrolactone salinomycin 4 11
cluster2 butyrolactone merochlorin A/merochlorin B 78 118
cluster1 NRPS griseochelin 100 85
cluster2 RiPP-like informatipeptin 37 6
cluster3 terpene hopene 92 22
cluster4 siderophore grincamycin 8 13
cluster5 NRPS-like s56-p1 11 35
cluster6 terpene geosmin 100 18
cluster7 RiPP-like - - 12
cluster8 siderophore - - 7
cluster9 terpene albaflavenone 100 21
cluster10 terpene naphthomycin A 9 22
cluster11 NRPS-like A40926 7 41
cluster12 siderophore desferrioxamin B/desferrioxamine
E83 9
cluster13 melanin istamycin 5 11
cluster14 NRPS-like cyphomycin 2 35
cluster15 ectoine ectoine 100 10
cluster16 NAPAA - - 32
cluster17 NRPS herboxidiene 4 36
cluster18 T1PKS amychelin 81 74
cluster19 melanin melanin 71 9
cluster20 T1PKS spore pigment 83 130
cluster21 terpene 2-methylisoborneol 100 17
cluster22 NRPS lasalocid 9 75
cluster23 NRPS tylactone 6 21
cluster24 RRE-containing mycotrienin I 7 30
cluster25 terpene - - 21
4. Discussion
Plant growth-promoting rhizobacteria (PGPR) are extensively applied in agricultural
production and soil ecological restoration projects [
45
–
47
]. Streptomyces, a prevalent Gram-
positive bacterium found in soil, establishes a symbiotic relationship with plants in the
rhizosphere [
48
]. In pot experiments, strain GD-4 significantly enhanced chlorophyll
content and root biomass, indicating its potential to promote plant growth in low-fertility
soils [
49
]. Studies have shown that Streptomyces species can promote the growth of Pinus
massoniana seedlings and increase root biomass. Also, in field experiments, the grain yield
of wheat supplemented with Streptomyces is equal to or even higher than that achieved
with nitrogen fertilizer application [
50
]. Therefore, it is speculated that Streptomyces may
enhance the ability of plants to absorb and utilize nitrogen from the soil in nitrogen-limited
environments, including both nitrate and ammonium forms of nitrogen. In this study, a
strain of Streptomyces with nitrogen-retention capabilities was isolated from the rhizosphere
of a pioneer plant in restored grassland. Inoculation experiments confirmed its ability
to colonize the rhizosphere of plants and promote plant growth under oligotrophic soil
conditions. To evaluate its potential as a PGPR, whole-genome sequencing using third-
generation technology was performed. Genome mining revealed the presence of functional
genes associated with both plant growth promotion and abiotic stress alleviation.
For the strain GD-4 genome, primary involvements in nitrogen metabolism and trans-
port, phosphorus metabolism, sulfur metabolism, siderophore production, and auxin
synthesis were annotated. Similar genes have also been reported in other plant growth-
promoting rhizobacteria (PGPR). Effective colonization of microorganisms in the rhizo-
sphere is fundamental to the functional realization of PGPR [
49
,
51
–
54
]. Scanning electron
microscopy results showed that GD-4 successfully colonized the root systems of three
different grasses, and the hyphae and spore structures were visible (Figure 1). Bacterial
colonization in plants relies on the recognition of chemical messages and chemotaxis. The
GD-4 genome encodes methyl-accepting chemotaxis protein (MCP) and chemotaxis protein
Microorganisms 2025,13, 286 16 of 31
methyltransferase (CheR) (Table A6). Methyltransferase (CheR) catalyzes the methylation
of the cytoplasmic signaling domain of chemoreceptors and is a core component of the
chemosensory cascade [
55
,
56
]. Studies have shown that mutations or deletions of CheR
can disrupt the chemotactic behavior in various species [
56
]. Additionally, some research
suggests that CheR may play a role in biofilm formation [
57
]. Meanwhile, according to the
gene annotation results, it can use a series of plant-derived compounds as carbon sources
for growth (Table A6), transport nutrients such as cellobiose and glucose through ABC
transports, and achieve a good symbiotic relationship with plants [58].
It is well established that the most effective method for biological nitrogen fixation is
the symbiotic relationship between legumes and rhizobia [
59
–
61
]. For plants that cannot
rely on microbial nitrogen fixation to establish a stable nitrogen source, other nitrogen
cycle pathways may exist to provide nitrogen for plant growth [
62
]. A key characteristic
of strain GD-4 is its ability to produce ammonia (Figure A4), a nitrogen source readily
available for plant uptake. Ammonia is typically generated through processes such as
amino acid degradation, urease-mediated hydrolysis, deamination, and other biological
activities. Nitrate, nitrite, and ammonia serve as the primary nitrogen sources in the
environment, undergoing transformation and regulation by various environmental factors.
Notably, the GD-4 genome predominantly features annotations for the dissimilatory nitrate
reduction to ammonium (DNRA) pathway (Figure 6). Current studies on metagenomic and
plant growth-promoting bacteria have shown that this pathway plays a role in nitrogen
retention in the soil or plant rhizosphere [
13
,
63
–
65
]. Several articles have reported that
Pseudomonas spp. possessing the genes nirB and genes nirD can also perform dissimilatory
nitrate reduction to ammonium (DNRA) and convert nitrate nitrogen into ammonium
under aerobic conditions [
66
]. This study predicts that dissimilatory nitrate reduction to
ammonium (DNRA) may serve as an effective mechanism for GD-4 to retain nitrogen in
this environment [12]. Moreover, studies have shown that the direct assimilation of urine-
derived nitrogen into microbial organic nitrogen pools is a crucial process for nitrogen
retention in urine patches. This process subsequently supports plant nitrogen supply
during microbial turnover [67].
Phosphorus is a crucial nutrient for plants, and its deficiency in available form restricts
plant growth and development. The related genes pstA, pstB, and pstC related to phospho-
rus metabolism were found in the gene annotation of GD-4 and are phosphate transporters,
participating in bacterial phosphate transport as phosphate-specific transport (pst) operons.
The alkaline phosphatase encoded by the gene phoA can decompose organic phosphorus
and release inorganic phosphorus to provide nutritional support for plants [
68
]. The GD-4
genome encodes multiple genes related to sulfur metabolism and transport (Table A7).
Sulfur, as a key element for plant growth, is related to plant stress resistance to a certain
extent [
69
]. For example, the annotated H
2
S synthesis genes can affect plant hormone
regulation and participate in plant responses to abiotic stress [
32
]. Auxin is an essential
substance for plant growth, and IAA secretion is one of the important characteristics of
some PGPRs [
70
]. In tryptophan operon, tryptophan synthase alpha subunit (trpA) and
beta subunit (trpB) catalyze the conversion of indole to tryptophan, while trpC catalyzes
the cyclization reaction to produce indole-3-glycerol phosphate. Additionally, trpD cat-
alyzes the formation of phosphoribosylanthranilate (PRA), and trpE catalyzes the reaction
between chorismate and glutamine to generate anthranilate, which serves as a precursor
for IAA biosynthesis. Streptomyces sp. AC40 has been reported to contain annotated genes
encoding nitrile hydratases and to produce IAA via the IAN pathway [
71
]. There are
five tryptophan-dependent IAA biosynthesis pathways in organisms, including the IAM,
IAN, indole-3-pyruvic acid (IPyA), tryptamine (TAM), and tryptophan side chain oxidase
Microorganisms 2025,13, 286 17 of 31
(TSO) pathways [
70
,
72
]. In GD-4, the IAA synthesis pathway integrates multiple pathways,
revealing the flexibility of IAA biosynthesis in bacteria (Figure 6).
Another key role of plant growth-promoting bacteria is the production of
siderophores
[73–75]
. The GD-4 genome encodes genes involved in siderophore production
and transport, including coproporphyrin ferrochelatase, methylglutaconyl-CoA hydratase,
shikimate kinase, and ABC transporter permeases. AntiSMASH analysis identified BGCs
of the siderophore desferrioxamin B, aerobactin, in the GD-4 genome. Desferrioxamine
B and desferrioxamine E are siderophores predicted to be produced by Streptomyces sp.
GD-4. These compounds primarily support microbial survival and growth in iron-deficient
environments by chelating ferric iron (Fe
3+
) and facilitating its transport into cells [
76
].
Siderophore production by actinomycetes is an important factor in the antagonism of plant
pathogens and can produce indirect PGP effects on plants [
77
,
78
]. In addition, we have
also found many genes responsible for the synthesis of antibacterial compounds. The pos-
sible synthesized products include enediyne antibiotics, ansamycins, vancomycin-group
antibiotics, and tetracycline. Some secondary metabolites can chelate iron ions, destroy the
formation of biofilm, and have good antibacterial effects (Table A8).
Many studies have demonstrated that Streptomyces can regulate the structure of soil
microbial communities [
79
]. For instance, Zhang et al.’s study showed that inoculating
Streptomyces Act12 and D74 into cucumber root soil increased bacterial diversity and
recruited more nitrogen-fixing bacteria [
80
]. Similarly, Hu et al.’s research found that
inoculating Streptomyces TOR3209 into tomato plants not only enhanced the abundance
of microorganisms critical to the nitrogen cycle but also facilitated the recruitment of the
endophytic growth-promoting bacterium Bacillus velezensis WSW007 [
81
]. Preliminary
research in our lab indicates that as grassland degradation deepens, the abundance of Strep-
tomyces increases. This suggests that Streptomyces species may be particularly well adapted
to nutrient-poor soils and have the potential to enhance plant growth by influencing the
root microbial community.
To adapt to the abiotic stressors of plateau soils, the genome of Streptomyces sp.
GD-4 contains genes encoding osmotic regulators, including transporters for trehalose,
polyamines, and proline (Table A9). The identification of these genes in the alpine sandy
environment provides insight into how GD-4 maintains stable cell morphology under stress.
Additionally, the gene cluster responsible for the biosynthesis of glycine betaine further elu-
cidates the bacterium’s capacity to withstand harsh environmental conditions. For example,
ectoine, a common compatible solute, enables bacteria to adapt to high-osmotic-pressure
environments. Its biosynthesis begins with diaminobutyrate-2-oxoglutarate transaminase
(ectB) [
82
]. This gene cluster (ectABCD), encoding the enzymes for ectoine biosynthesis,
was previously identified in the genome of Streptomyces coelicolor A3(2) [
43
]. Moreover, the
annotated protein secretion systems in Streptomyces sp. GD-4 are primarily the Sec system
(post-translational translocation) and the Tat system (twin-arginine translocation), with the
core coding genes being secYEG/yajC/yidC and tatABC, respectively [
69
,
83
] (Table A10).
These secretion systems facilitate the export of adhesion proteins and factors involved in
the synthesis of extracellular polysaccharides (EPSs) and auxins, all of which contribute
to bacterial colonization and symbiosis with plants [
84
]. The predicted stress-resistant
genes, matching the environmental conditions of isolation, confirm GD-4
′
s adaptive sur-
vival abilities and its potential for promoting plant growth in sandy soil for ecological
restoration.
5. Conclusions
Our research identified a plant rhizosphere growth-promoting bacterium, GD-4, which
promotes the growth of plants in alpine desert grasslands. Additionally, it was confirmed
Microorganisms 2025,13, 286 18 of 31
that Streptomyces sp. GD-4 exhibits strong colonization capabilities and growth-promoting
effects across three species of grasses. This study further elucidates the potential growth-
promoting mechanisms of GD-4 through whole-genome and comparative genomics anal-
yses. It is hypothesized that the key mechanism involves its capacity to assist plants in
retaining nitrogen in the soil by reducing dissimilatory nitrate reduction to ammonium
(DNRA). Nitrate nitrogen is converted into ammonium nitrogen, which is more readily
absorbed by plants, thereby indirectly facilitating the initial colonization of plants in des-
olate grasslands. Additionally, it was found that the bacterium possesses specific stress
resistance genes, including those responsible for the production of siderophores, trehalose,
and cold- and heat-shock proteins, enabling it to survive in environments characterized
by abiotic stress. This suggests that GD-4 is particularly well adapted to the oligotrophic
soils of the plateau, thereby promoting plant health and facilitating adaptive growth. These
findings indicate that GD-4 has the potential for the development of microbial agents aimed
at ecological restoration engineering.
Author Contributions: Data curation, W.X.; formal analysis, Y.C.; funding acquisition, J.Z.; methodol-
ogy, W.X.; validation, Y.L.; writing—original draft, W.X.; writing—review and editing, J.Z. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Second Tibetan Plateau Scientific Expedition and Research
Program grant number [2019QZKK0404] And The APC was funded by the Second Tibetan Plateau
Scientific Expedition and Research Program.
Data Availability Statement: Dataset available on request from the authors.
Acknowledgments: This work was financially supported by the Second Tibetan Plateau Scientific
Expedition and Research Program (STEP) (2019QZKK0404).
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A
Table A1. Features of the GD-4 genome in comparison with other Streptomyces strains from different
ecological niches.
Name Source of
Isolation Genome Size CD’s G + C (%) Potential Function Accession No. Reference
Streptomyces
rochei S32 soil 8.0 Mb 7486 72.5
Plant growth promotion,
nitrogen fixation,
production of bioactive
substances
CP133098 [85]
Streptomyces
griseoincarna-
tus HNS054
Marine sponge 7.5 Mb 6678 72.3
Heterologous expression
host for secondary
metabolites, salinity
tolerance
CP139576 [86]
Streptomyces sp.
AFD10 desert soil 7.9 Mb 7246 72.6
Antibiotic production,
bioactive secondary
metabolite production
JASSVZ000000000
[87]
Streptomyces sp.
Z38 root tissues 7.3 Mb 6446 72.4
Plant growth promotion,
heavy metal resistance,
bio-nanoparticle
synthesis
WPIR00000000 [88]
Streptomyces
luteireticuli
ASG80
Sisal roots 8.7 Mb 7867 71.77 Biocontrol agent against
Phytophthora diseases CP167927 [89]
Streptomyces sp.
UYFA156 seeds 7.1 Mb - 73.4 Plant growth promotion CP040466 [90]
Microorganisms 2025,13, 286 19 of 31
Table A1. Cont.
Name Source of
Isolation Genome Size CD’s G + C (%) Potential Function Accession No. Reference
Streptomyces sp.
C8S0 sediment 6.9 Mb 6,841 73.5 Bioactive metabolite
production CP045031 [91]
Streptomyces sp.
JL1001
Rhizosphere
soil 7.9 Mb 7315 71.71
Production of novel
bioactive natural
products,
leinamycin-type gene
cluster
CP136798 [92]
Streptomyces
sp.CH9-7TRhizosphere
soil 8.8 Mb 7664 71.7
Nocardamine production,
bioactive secondary
metabolite production
JAERRI000000000
[93]
Streptomyces_sp_HP-
A2021
Rhizosphere
soil 9.6 Mb 8534 71.07
Bioactive secondary
metabolite production,
antimicrobial activity
CP094344.1 [94]
Table A2. Genes involved in nitrogen metabolism predicted for Streptomyces sp. GD-4.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Nitrogen metabolism
gene0092 cynT K01673 carbonic anhydrase [EC:4.2.1.1]
gene1739 glnA K01915 glutamine synthetase [EC:6.3.1.2]
gene1760 gdhA K00261 glutamate dehydrogenase (NAD(P)+) [EC:6.3.1.2]
gene2048 narH K00371
nitrate reductase/nitrite oxidoreductase,
beta subunit [EC:1.7.5.1 1.7.99.-]
gene2544 gltB K00265 glutamate synthase (NADPH) large
chain [EC:1.4.1.13]
gene4307 narK K02575 MFS transporter, NNP family,
nitrate/nitrite transporter -
gene4308 nasC K00372 assimilatory nitrate reductase catalytic
subunit [EC:1.7.99.-]
gene4309 nasB K00360 assimilatory nitrate reductase electron
transfer subunit [EC:1.7.99.-]
gene5295 nirK K00368 nitrite reductase (NO-forming) [EC:1.7.2.1]
gene5356 - K15371 glutamate dehydrogenase [EC:1.4.1.2]
gene5868 npd K00459 nitronate monooxygenase [EC:1.13.12.16]
gene5942 nirD K00363
nitrite reductase (NADH), small subunit
[EC:1.7.1.15]
gene5943 nirB K00362
nitrite reductase (NADH), large subunit
[EC:1.7.1.15]
gene6510 gltD K00266 glutamate synthase (NADPH), small
chain [EC:1.4.1.13]
gene9104 - K01501 nitrilase [EC:3.5.5.1]
gene6662 iscU K04488 nitrogen fixation protein NifU and
related proteins -
gene0326 narL K07684 two-component system, NarL family,
nitrate/nitrite response regulator NarL -
gene2048 narH K00371
nitrate reductase/nitrite oxidoreductase,
beta subunit [EC:1.7.5.1/1.7.99.-]
gene2049 narJ K00373 nitrate reductase molybdenum cofactor
assembly chaperone NarJ/NarW -
gene4268 narA K25150 polyether ionophore transport system
ATP-binding protein -
gene4269 narB K25149 polyether ionophore transport system
permease protein -
Molybdenum
cofactor
gene4499 mobA K03752 molybdenum cofactor
guanylyltransferase [EC:2.7.7.77]
gene4379 moeB K21029 molybdopterin-synthase
adenylyltransferase [EC:2.7.7.80]
gene0517 moaC K03637 cyclic pyranopterin monophosphate
synthase [EC:4.6.1.17]
gene3219 moaE K03635 molybdopterin synthase catalytic
subunit [EC:2.8.1.12]
gene3830 moaA K03639 GTP 3′,8-cyclase [EC:4.1.99.22]
gene4835 moaD K03636 sulfur-carrier protein -
Microorganisms 2025,13, 286 20 of 31
Table A2. Cont.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Nitrogen transporters
gene0085 nasT K07183 two-component system, response
regulator/RNA-binding antiterminator -
gene2809 amt K03320 ammonium transporter, Amt family -
gene1739 glnA K01915 glutamine synthetase [EC:6.3.1.2]
gene2807 glnD K00990 [protein-PII] uridylyltransferase [EC:2.7.7.59]
gene2808 glnB K04751 nitrogen regulatory protein P-II 1 -
gene6281 glnE K00982
[glutamine synthetase]
adenylyltransferase/[glutamine
synthetase]-adenylyl-L-tyrosine
phosphorylase
[EC:2.7.7.42/2.7.7.89]
gene4307 narK K02575 MFS transporter, NNP family,
nitrate/nitrite transporter -
Table A3. Genes involved in phosphate solubilization predicted for Streptomyces sp. GD-4.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Phosphate
solubilization
gene1931 zwf K00036 glucose-6-phosphate 1-dehydrogenase
[EC:1.1.1.49/1.1.1.363]
gene4131 ppa K01507 inorganic pyrophosphatase [EC:3.6.1.1]
gene0144 pqqE K06139 PqqA peptide cyclase [EC:1.21.98.4]
gene2585 pqqL K07263 zinc protease [EC:3.4.24.-]
gene0729 gdh K00034 glucose 1-dehydrogenase [EC:1.1.1.47]
gene1760 gdhA K00261 glutamate dehydrogenase(NAD(P)+) [EC:1.4.1.3]
gene0424 phoD K01113 alkaline phosphatase D [EC:3.1.3.1]
gene8518 phoP K07658
two-component system, OmpR family,
alkaline phosphatase synthesis response
regulator PhoP
-
gene3368 phoH2 K07175 PhoH-like ATPase -
gene4887 phoU K02039 phosphate transport system protein -
gene5884 phoH K06217 phosphate starvation-inducible protein
PhoH and related proteins -
gene6104 phoA K01077 alkaline phosphatase [EC:3.1.3.1]
Phosphate transport
gene4252 pstS K02040 phosphate transport
systemsubstrate-binding protein -
gene4803 pstB K02036 phosphate transport
systemATP-binding protein [EC:7.3.2.1]
gene4804 pstA K02038 phosphate transport systempermease
protein -
gene4805 pstC K02037 phosphate transport systempermease
protein -
gene6940 pstI K20754 aqualysin 1 [EC:3.4.21.111]
gene1103 phnB K04750 PhnB protein -
gene1789 phnW K03430 2-aminoethylphosphonate-pyruvate
transaminase [EC:2.6.1.37]
gene4587 phnO K09994 (aminoalkyl)phosphonate
N-acetyltransferase [EC:2.3.1.280]
gene7793 phnA K19670 phosphonoacetate hydrolase [EC:3.11.1.2]
Table A4. Genes involved in auxin metabolism predicted for Streptomyces sp. GD-4.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
IAA biosynthesis
gene0221 paaF K01692 enoyl-CoA hydratase [EC:4.2.1.17]
gene0256 amiE K01426 amidase [EC:3.5.1.4]
gene0482 gcdH K00252 glutaryl-CoA dehydrogenase [EC:1.3.8.6]
gene0485 atoB K00626 acetyl-CoA C-acetyltransferase [EC:2.3.1.9]
gene0620 - K00128 aldehyde dehydrogenase (NAD+) [EC:1.2.1.3]
gene1290 - K01593 aromatic-L-amino-acid/L-
tryptophan decarboxylase [EC:4.1.1.28/4.1.1.105]
gene1432 cypD_E K14338
cytochrome
P450/NADPH-cytochrome P450
reductase
[EC:1.14.14.1/1.6.2.4]
gene1474 pdhD K00382 dihydrolipoyl dehydrogenase [EC:1.8.1.4]
gene1502 - K03392 aminocarboxymuconate-
semialdehyde decarboxylase [EC:4.1.1.45]
Microorganisms 2025,13, 286 21 of 31
Table A4. Cont.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
IAA biosynthesis
gene1818 fadJ K01782
3-hydroxyacyl-CoA
dehydrogenase/enoyl-CoA
hydratase/3-hydroxybutyryl-CoA
epimerase
[EC:1.1.1.35/4.2.1.17/5.1.2.3]
gene2069 katE K03781 catalase [EC:1.11.1.6]
gene2370 phsA K20219 o-aminophenol oxidase [EC:1.10.3.4]
gene2538 katG K03782 catalase-peroxidase [EC:1.11.1.21]
gene4279 kynU K01556 kynureninase [EC:3.7.1.3]
gene4282 kynA K00453 tryptophan 2,3-dioxygenase [EC:1.13.11.11]
gene4301 aofH K00274 monoamine oxidase [EC:1.4.3.4]
gene5439 nthB K20807 nitrile hydratase subunit beta [EC:4.2.1.84]
gene5441 nthA K01721 nitrile hydratase subunit alpha [EC:4.2.1.84]
gene7158 - K22450 aralkylamine N-acetyltransferase [EC:2.3.1.87]
gene9104 - K01501 nitrilase [EC:3.5.5.1]
gene6498 trpA K01695 tryptophan synthase alpha chain [EC:4.2.1.20]
gene6497 trpB K01696 tryptophan synthase beta chain [EC:4.2.1.20]
gene6496 trpC K01609 indole-3-glycerol phosphate
synthase [EC:4.1.1.48]
gene6381 trpD K00766 anthranilate
phosphoribosyltransferase [EC:2.4.2.18]
gene6492 trpE K01657 anthranilate synthase component I [EC:4.1.3.27]
gene4607 hisC K00817 histidinol-phosphate
aminotransferase [EC:2.6.1.9]
ACC Deaminase
gene2178 map K01265 methionyl aminopeptidase [EC:3.4.11.18]
gene1750 kbl K00639 glycine C-acetyltransferase [EC:2.3.1.29]
gene1790 pgsA K00995
CDP-diacylglycerol---glycerol-3-
phosphate
3-phosphatidyltransferase
[EC:2.7.8.5]
gene2753 glpQ K01126 glycerophosphoryl diester
phosphodiesterase [EC:3.1.4.46]
Table A5. Genes involved in antibiotics predicted for Streptomyces sp. GD-4.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Enediyne antibiotics
gene1384 ncsB3 K20420 2-hydroxy-5-methyl-1-naphthoate
7-hydroxylase [EC:1.14.15.31]
gene2301 mdpB3 K21193 acetyltransferase/esterase -
gene2929 ncsB4 K21209 acyltransferase -
gene3244 ncsC4 K21214 NDP-hexose 4-ketoreductase -
gene4917 sgcE6 K21185 flavin reductase -
gene4990 sgcE11 K21167 enediyne biosynthesis protein E11 -
gene6898 sgcF K21159 epoxide hydrolase -
gene7800 cepH K16431 FAD-dependent halogenase [EC:1.14.19.-]
gene8258 sgcD3 K21177 cytochrome P450 hydroxylase [EC:1.14.-.-]
gene8273 calE5 K21172 enediyne biosynthesis protein CalE5 -
Ansamycins gene1929 tktA K00615 transketolase [EC:2.2.1.1]
gene8433 asm10 K16038 N-methyltransferase [EC:2.1.1.-]
Vancomycin-group
antibiotics
gene0345 evaC K16437 methylation protein EvaC -
gene1329 rfbB K01710 dTDP-glucose 4,6-dehydratase [EC:4.2.1.46]
gene7800 cepH K16431 FAD-dependent halogenase [EC:1.14.19.-]
gene7828 cepJ K16434 thioesterase CepJ -
gene8312 nocN K16422 4-hydroxymandelate oxidase [EC:1.1.3.46]
pA_gene0134 cepA K16428 nonribosomal peptide synthetase CepA -
Tetracycline
gene1025 tetX K18221 tetracycline 11a-monooxygenase,
tetracycline resistance protein [EC:1.14.13.231]
gene3288 oxyQ K14254 aminotransferase -
gene5733 oxyS K14256
anhydrotetracycline
6-monooxygenase/5a,11a-
dehydrotetracycline 5-monooxygenase
[EC:1.14.13.38/1.14.13.234]
gene8337 oxyF K14251 C-methyltransferase [EC:2.1.1.-]
pA_gene0186 oxyA K05551 minimal PKS ketosynthase (KS/KS
alpha)
[EC:2.3.1.-
/2.3.1.260/2.3.1.235]
pA_gene0187 oxyB K05552 minimal PKS chain-length factor
(CLF/KS beta)
[EC:2.3.1.-2.3.1.260
2.3.1.235]
Microorganisms 2025,13, 286 22 of 31
Table A5. Cont.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Tetracycline
pA_gene0188 ctcP K14257 tetracycline 7-halogenase/FADH2
O2-dependent halogenase [EC:1.14.19.49/1.14.19.-]
pA_gene0199 oxyC K05553 minimal PKS acyl carrier protein -
pA_gene0200 oxyJ K12420 ketoreductase [EC:1.1.1.-]
Phenazine
gene2251 phzS K20940 5-methylphenazine-1-carboxylate
1-monooxygenase [EC:1.14.13.218]
gene6250 phzF K06998 trans-2,3-dihydro-3-
hydroxyanthranilate isomerase [EC:5.3.3.17]
gene7406 phzF K06998 trans-2,3-dihydro-3-
hydroxyanthranilate isomerase [EC:5.3.3.17]
gene6410 phzE K13063 2-amino-4-deoxychorismate synthase [EC:2.6.1.86]
Table A6. Genes involved in plant–bacteria interactions predicted for Streptomyces sp. GD-4.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Root colonization
Motility
gene8106 flgS K02482
two-component system, NtrC family, sensor
kinase [EC:2.7.13.3]
gene2789 fliA K02405 RNA polymerase sigma factor FliA -
gene2607 rpoD K03086 RNA polymerase primary sigma factor -
Root colonization
and interactions
gene3612 mdh K00024 malate dehydrogenase [EC:1.1.1.37]
gene0159 xerD K04763 integrase/recombinase XerD -
Chemotaxis
gene0567 - K11354 two-component system, chemotaxis family,
sensor kinase Cph1 [EC:2.7.13.3]
gene0888 rbsB K10439 ribose transport system substrate-binding
protein -
gene8627 mcp K03406 methyl-accepting chemotaxis protein -
gene3596 cheR K00575 chemotaxis protein methyltransferase CheR [EC:2.1.1.80]
Cellulose
degradation
gene0446 bglX K05349 beta-glucosidase [EC:3.2.1.21]
gene0445 - K01179 endoglucanase [EC:3.2.1.4]
gene0447 cbhA K19668 cellulose 1,4-beta-cellobiosidase [EC:3.2.1.91]
gene1904 celF K01222 6-phospho-beta-glucosidase [EC:3.2.1.86]
Exopolysaccharide
biosynthesis
gene1005 exoZ K16568
exopolysaccharide production protein ExoZ
-
gene1536 exoY K16566
exopolysaccharide production protein ExoY
-
gene3072 wecA K02851
UDP-GlcNAc:undecaprenyl-
phosphate/decaprenyl-phosphate
GlcNAc-1-phosphate transferase
[EC:2.7.8.33/2.7.8.35]
gene5303 exoA K16557 succinoglycan biosynthesis protein ExoA [EC:2.4.-.-]
gene5659 gumD K13656 undecaprenyl-phosphate glucose
phosphotransferase [EC:2.7.8.31]
gene5724 cysE K00640 serine O-acetyltransferase [EC:2.3.1.30]
Biofilm formation
gene1169 oxyR K04761 LysR family transcriptional regulator,
hydrogen peroxide-inducible gene activator
-
gene1609 glgC K00975 glucose-1-phosphate adenylyltransferase [EC:2.7.7.27]
gene2789 fliA K02405 RNA polymerase sigma factor FliA -
gene2982 glgP K00688 glycogen phosphorylase [EC:2.4.1.1]
gene3721 bcsA K00694 cellulose synthase (UDP-forming) [EC:2.4.1.12]
gene3765 crp K10914 CRP/FNR family transcriptional regulator,
cyclic AMP receptor protein -
gene4594 gcvA K03566
LysR family transcriptional regulator,
glycine cleavage system transcriptional
activator
-
gene5656 pgaB K11931 poly-beta-1,6-N-acetyl-D-glucosamine
N-deacetylase [EC:3.5.1.-]
gene6457 dksA K06204 RNA polymerase-binding transcription
factor
gene7372 crr K02777 sugar PTS system EIIA component [EC:2.7.1.-]
gene8276 rcdA K23778
TetR/AcrR family transcriptional regulator,
regulator of biofilm formation and stress
response
-
gene4092 icaR K21453 TetR/AcrR family transcriptional regulator,
biofilm operon repressor -
Microorganisms 2025,13, 286 23 of 31
Table A7. Genes involved in sulfur metabolism predicted for Streptomyces sp. GD-4.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Sulfur metabolism
gene0895 sfnG K17228 dimethylsulfone monooxygenase [EC:1.14.14.35]
gene0932 - K00387 sulfite oxidase [EC:1.8.3.1]
gene1472 cysJ K00380
sulfite reductase (NADPH) flavoprotein
alpha-component [EC:1.8.1.2]
gene2332 sir K00392 sulfite reductase (ferredoxin) [EC:1.8.7.1]
gene2334 cysH K00390 phosphoadenosine phosphosulfate
reductase [EC:1.8.4.8/1.8.4.10]
gene2335 cysC K00860 adenylylsulfate kinase [EC:2.7.1.25]
gene2336 cysD K00957 sulfate adenylyltransferase subunit 2 [EC:2.7.7.4]
gene2337 cysN K00956 sulfate adenylyltransferase subunit 1 [EC:2.7.7.4]
gene2574 sseA K01011 thiosulfate/3-mercaptopyruvate
sulfurtransferase [EC:2.8.1.1/2.8.1.2]
gene3444 metB K01739 cystathionine gamma-synthase [EC:2.5.1.48]
gene3652 doxD K16937 thiosulfate dehydrogenase (quinone)
large subunit [EC:1.8.5.2]
gene4896 doxD K16937 thiosulfate dehydrogenase (quinone)
large subunit [EC:1.8.5.2]
gene3878 aprA K00394 adenylylsulfate reductase, subunit A [EC:1.8.99.2]
gene4304 metX K00641 homoserine O-acetyltransferase/O-
succinyltransferase [EC:2.3.1.31/2.3.1.46]
gene4469 dmdC K20035 3-(methylsulfanyl)propanoyl-CoA
dehydrogenase [EC:1.3.99.41]
gene4657 dmdB K20034 3-(methylthio)propionyl---CoA ligase [EC:6.2.1.44]
gene5061 ssuD K04091 alkanesulfonate monooxygenase [EC:1.14.14.5/
1.14.14.34]
gene5724 cysE K00640 serine O-acetyltransferase [EC:2.3.1.30]
gene5725 cysK K01738 cysteine synthase [EC:2.5.1.47]
gene6420 thiS K03154 sulfur carrier protein -
gene6037 iscR K13643
Rrf2 family transcriptional regulator,
iron–sulfur cluster assembly
transcription factor
-
gene6382 sufS K11717 cysteine desulfurase/selenocysteine
lyase [EC:2.8.1.7/4.4.1.16]
gene6657 sufB K09014 Fe-S cluster assembly protein SufB -
gene6658 sufD K09015 Fe-S cluster assembly protein SufD -
gene6660 sufC K09013 Fe-S cluster assembly ATP-binding
protein -
Sulfur transport
gene2339 ssuA K15553 sulfonate transport system
substrate-binding protein -
gene2340 ssuB K15555
sulfonate transport system ATP-binding
protein [EC:7.6.2.14]
gene2341 ssuC K15554 sulfonate transport system permease
protein -
gene4388 tauC K15552 taurine transport system permease
protein -
gene4389 tauA K15551 taurine transport system
substrate-binding protein -
gene4390 tauB K10831 taurine transport system ATP-binding
protein [EC:7.6.2.7]
gene4391 tauD K03119 taurine dioxygenase [EC:1.14.11.17]
Table A8. Genes involved in siderophore biosynthesis predicted for Streptomyces sp. GD-4.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Siderophore
biosynthesis
gene7262 aroC K01736 chorismate synthase [EC:4.2.3.5]
gene6884 aroH K06208 chorismate mutase [EC:5.4.99.5]
gene2570 hemH K01772 protoporphyrin/coproporphyrin
ferrochelatase [EC:4.98.1.14.99.1.9]
gene7265 aroK K00891 shikimate kinase [EC:2.7.1.71]
gene6413 bfr K03594 bacterioferritin [EC:1.16.3.1]
gene6412 bfd K02192 bacterioferritin-associated ferredoxin -
gene3085 lysA K01586 diaminopimelate decarboxylase [EC:4.1.1.20]
gene6037 iscR K13643
Rrf2 family transcriptional regulator,
iron–sulfur cluster assembly transcription
factor
-
Microorganisms 2025,13, 286 24 of 31
Table A8. Cont.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Siderophore
biosynthesis
gene2932 iscS K04487 cysteine desulfurase [EC:2.8.1.7]
gene6243 efeU K07243 high-affinity iron transporter -
gene7987 pvdQ K07116 acyl-homoserine-lactone acylase [EC:3.5.1.97]
gene0300 mbtH K05375 MbtH protein -
gene4994 mbtN K00257 acyl-ACP dehydrogenase [EC:1.3.99.-]
gene7826 mbtI K04781 salicylate synthetase [EC:5.4.4.2/4.2.99.21]
gene7834 mbtB K04788 mycobactin phenyloxazoline synthetase -
gene2630 iucB K03896 acetyl CoA:N6-hydroxylysine acetyl
transferase [EC:2.3.1.102]
gene5568 iucC K03895 aerobactin synthase [EC:6.3.2.39]
gene5570 iucD K03897 lysine N6-hydroxylase [EC:1.14.13.59]
gene0303 dhbF K04780 glyine—[glycyl-carrierprotein]ligase [EC:6.2.1.66]
gene2631 asbA K24108 spermidine-citrateligase [EC:6.3.2.-]
gene5422 entF K02364 L-serine—[L-seryl-carrierprotein]ligase [EC:6.3.2.1/46.2.1.72]
gene7825 entE K02363 2,3-dihydroxybenzoate—[aryl-
carrierprotein]ligase [EC:6.3.2.1/46.2.1.71]
gene8325 menF K02552 menaquinone-
specificisochorismatesynthase [EC:5.4.4.2]
Iron uptake and
transport
gene8395 fhuB K23228 ferric hydroxamate transport system
permease protein -
gene8396 fhuD K23227 ferric hydroxamate transport system
substrate-binding protein -
gene8397 fhuC K10829 ferric hydroxamate transport system
ATP-binding protein [EC:7.2.2.16]
gene3465 fepD K23186 iron-siderophore transport system
permease protein -
gene3466 fepG K23187 iron-siderophore transport system
permease protein -
gene6856 fepC K23188 iron-siderophore transport system
ATP-binding protein [EC:7.2.2.1/77.2.2.-]
gene5573 desE K25287 iron-desferrioxamine transport system
substrate-binding protein -
gene5604 entS K08225 MFS transporter, ENTS family,
enterobactin (siderophore) exporter -
gene6244 efeB K16301 deferrochelatase/peroxidase EfeB [EC:1.11.1.-]
gene6245 efeO K07224 iron uptake system component EfeO -
gene7837 pvdA K10531 L-ornithine N5-monooxygenase
[EC:1.14.13.1951.14.13.196]
Table A9. Genes involved in abiotic stress response predicted for Streptomyces sp. GD-4.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Heat tolerance
gene4731 hslJ K03668 heat-shock protein HslJ -
gene3770 htpX K03799 heat-shock protein HtpX [EC:3.4.24.-]
gene6571 hslR K04762 ribosome-associated heat-shock protein
Hsp15 -
gene5866 hrcA K03705 heat-inducible transcriptional repressor -
Cold-shock protein
gene4329 dnaJ K03686 molecular chaperone DnaJ -
gene0076 dnaK K04043 molecular chaperone DnaK -
gene0819 cspA K03704 cold-shock protein -
gene3683 groEL K04077 chaperonin GroEL [EC:5.6.1.7]
gene3684 groES K04078 chaperonin GroES -
Salinity tolerance
gene1089 betB K00130 betaine-aldehyde dehydrogenase [EC:1.2.1.8]
gene1092 betA K00108 choline dehydrogenase [EC:1.1.99.1]
gene1761 betI K02167
TetR/AcrR family transcriptional
regulator, transcriptional repressor of
bet genes
-
gene1086 proX K02002 glycine betaine/proline transport
system substrate-binding protein -
gene1087 proW K02001 glycine betaine/proline transport
system permease protein -
gene1088 proV K02000 glycine betaine/proline transport
system ATP-binding protein [EC:7.6.2.9]
gene1866 proP K03762 MFS transporter, MHS family,
proline/betaine transporter -
gene2716 proS K01881 prolyl-tRNA synthetase [EC:6.1.1.15]
gene4068 proC K00286 pyrroline-5-carboxylate reductase [EC:1.5.1.2]
Microorganisms 2025,13, 286 25 of 31
Table A9. Cont.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Salinity tolerance
gene4311 proA K00147 glutamate-5-semialdehyde
dehydrogenase [EC:1.2.1.41]
gene5828 proB K00931 glutamate 5-kinase [EC:2.7.2.11]
gene2892 putR K23253
PucR family transcriptional regulator,
proline-responsive transcriptional
activator
-
Oxidative stress
tolerance
gene2069 katE K03781 catalase [EC:1.11.1.6]
gene2538 katG K03782 catalase-peroxidase [EC:1.11.1.21]
gene2163 ggt K00681
gamma-
glutamyltranspeptidase/glutathione
hydrolase
[EC:2.3.2.2]
[EC:3.4.19.13]
gene0778 trxA K03671 thioredoxin 1 -
gene4564 trxB K00384 thioredoxin reductase (NADPH) [EC:1.8.1.9]
Polyamine
biosynthesis
gene4295 speE K00797 spermidine synthase [EC:2.5.1.16]
gene4902 speG K00657 diamine N-acetyltransferase [EC:2.3.1.57]
gene8127 speA K01585 arginine decarboxylase [EC:4.1.1.19]
gene0359 argS K01887 arginyl-tRNA synthetase [EC:6.1.1.19]
gene7176 argH K01755 argininosuccinate lyase [EC:4.3.2.1]
gene7525 argO K22477 N-acetylglutamate synthase [EC:2.3.1.1]
gene2745 potC K11070
spermidine/putrescine transport system
permease protein -
gene2746 potB K11071
spermidine/putrescine transport system
permease protein -
gene2747 potA K11072
spermidine/putrescine transport system
ATP-binding protein [EC:7.6.2.11]
gene2748 potD K11069
spermidine/putrescine transport system
substrate-binding protein -
Trehalose
gene2358 treY K06044 (1->4)-alpha-D-glucan
1-alpha-D-glucosylmutase [EC:5.4.99.15]
gene2363 treZ K01236
maltooligosyltrehalose trehalohydrolase
[EC:3.2.1.141]
gene2985 treS K05343 maltose alpha-D-
glucosyltransferase/alpha-amylase
[EC:5.4.99.16]
[EC:3.2.1.1]
gene1426 otsB K01087 trehalose 6-phosphate phosphatase [EC:3.1.3.12]
gene4926 otsA K00697 trehalose 6-phosphate synthase
[EC:2.4.1.15/2.4.1.347]
Tolerance against
metal toxicity
gene1479 chrR K19784 chromate reductase, NAD(P)H
dehydrogenase (quinone) -
gene8824 chrA K07240 chromate transporter -
gene0716 cusR K07665
two-component system, OmpR family,
copper resistance phosphate regulon
response regulator CusR
-
gene5624 copA K17686 P-type Cu+ transporter [EC:7.2.2.8]
gene5625 copZ K07213 copper chaperone -
gene0790 arsR K03892
ArsR family transcriptional regulator,
arsenate/arsenite/antimonite-
responsive transcriptional repressor
-
gene1363 arsB K03893 arsenical pump membrane protein -
gene4206 arsA K01551 arsenite/tail-anchored
protein-transporting ATPase [EC:7.3.2.7/7.3.-.-]
gene6310 arsC K00537 arsenate reductase (glutaredoxin) [EC:1.20.4.1]
gene8604 arsB K03325 arsenite transporter -
gene5916 znuB K09816 zinc transport system permease protein -
gene8791 znuB K09816 zinc transport system permease protein -
gene5917 znuC K09817 zinc transport system ATP-binding
protein [EC:7.2.2.20]
gene5918 znuA K09815
zinc transport system substrate-binding
protein -
gene0517 moaC K03637 cyclic pyranopterin monophosphate
synthase [EC:4.6.1.17]
gene5137 moaC K03637 cyclic pyranopterin monophosphate
synthase [EC:4.6.1.17] -
gene3219 moaE K03635 molybdopterin synthase catalytic
subunit [EC:2.8.1.12]
gene3830 moaA K03639 GTP 3′,8-cyclase [EC:4.1.99.22]
gene6814 moaA K03639 GTP 3′,8-cyclase [EC:4.1.99.22] -
gene8622 moaA K03639 GTP 3′,8-cyclase [EC:4.1.99.22] -
gene4835 moaD K03636 sulfur-carrier protein -
Microorganisms 2025,13, 286 26 of 31
Table A9. Cont.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
Carotenoid
biosynthesis
gene0366 crtQ K00514 zeta-carotene desaturase [EC:1.3.5.6]
gene0713 crtO K02292 beta-carotene ketolase (CrtO type) -
gene1782 crtB K02291 15-cis-phytoene synthase [EC:2.5.1.32]
gene2161 crtI K10027 phytoene desaturase
[EC:1.3.99.26]
[EC:1.3.99.28 ]
[EC:1.3.99.29 ]
[EC:1.3.99.31]
gene4907 crtU K09879 carotenoid phi-ring
synthase/carotenoid chi-ring synthase
[EC:1.3.99.39]
[EC:1.3.99.40]
gene6428 crtP K10210 diapolycopene oxygenase [EC:1.14.99.44]
gene7872 cruC K14597 chlorobactene glucosyltransferase -
Table A10. Genes involved in secretory systems predicted for Streptomyces sp. GD-4.
Activity Description Gene ID KO Name KO ID KO Description Enzyme
General secretory
(Sec)
gene3731 secY K03076 preprotein translocase subunit SecY -
gene3781 secE K03073 preprotein translocase subunit SecE -
gene5351 secA K03070 preprotein translocase subunit SecA [EC:7.4.2.8]
gene6638 secG K03075 preprotein translocase subunit SecG -
gene7245 secD K03072 preprotein translocase subunit SecD -
gene7246 secF K03074 preprotein translocase subunit SecF -
gene8418 secDF K12257 SecD/SecF fusion protein -
Twin-arginine
translocation system
gene2382 tatA K03116 sec-independent protein translocase
protein TatA -
gene7067 tatA K03116 sec-independent protein translocase
protein TatA -
gene8221 tatA K03116 sec-independent protein translocase
protein TatA -
gene3274 tatB K03117 sec-independent protein translocase
protein TatB -
gene5174 tatD K03424 TatD DNase family protein [EC:3.1.21.-]
gene7068 tatC K03118 sec-independent protein translocase
protein TatC -
Microorganisms 2025, 13, x FOR PEER REVIEW 29 of 35
Figure A1. Effects of Streptomyces sp. GD-4 strain on growth parameters of different pasture species
cultured in sandy soil for 30 days: (a) root length; (b) leaf fresh weight; (c) leaf length; (d) root fresh
weight. The values represent the means of replicates (n = 3) ± standard deviations. Asterisks in
superscript indicate a significant difference from the control at 95% between treatments. Each data
point is the average of three replicates, and error bars represent ±SD. * Significance at p < 0.05; **
significance at p < 0.01.
Figure A2. Classification of Streptomyces sp. GD-4 based on GO database annotation.
Figure A1. Effects of Streptomyces sp. GD-4 strain on growth parameters of different pasture species
cultured in sandy soil for 30 days: (a) root length; (b) leaf fresh weight; (c) leaf length; (d) root
fresh weight. The values represent the means of replicates (n= 3)
±
standard deviations. Asterisks
in superscript indicate a significant difference from the control at 95% between treatments. Each
data point is the average of three replicates, and error bars represent
±
SD. * Significance at p< 0.05;
** significance at p< 0.01.
Microorganisms 2025,13, 286 27 of 31
Microorganisms 2025, 13, x FOR PEER REVIEW 29 of 35
Figure A1. Effects of Streptomyces sp. GD-4 strain on growth parameters of different pasture species
cultured in sandy soil for 30 days: (a) root length; (b) leaf fresh weight; (c) leaf length; (d) root fresh
weight. The values represent the means of replicates (n = 3) ± standard deviations. Asterisks in
superscript indicate a significant difference from the control at 95% between treatments. Each data
point is the average of three replicates, and error bars represent ±SD. * Significance at p < 0.05; **
significance at p < 0.01.
Figure A2. Classification of Streptomyces sp. GD-4 based on GO database annotation.
Figure A2. Classification of Streptomyces sp. GD-4 based on GO database annotation.
Figure A3. Classification of Streptomyces sp. GD-4 based on COG database annotation.
Microorganisms 2025, 13, x FOR PEER REVIEW 30 of 35
Figure A3. Classification of Streptomyces sp. GD-4 based on COG database annotation.
Figure A4. Results of ammonium production experiment: (a) before adding Nessler’s reagent
(K
2
HgI
4
); (b) after adding Nessler’s reagent (K
2
HgI
4
).
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Microorganisms 2025,13, 286 28 of 31
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