1. Introduction
The total area of saline soils worldwide is estimated to reach 9.5 × 10
8 ha
−1, accounting for 20% of the total global arable land area [
1]. High soil salinity can expose plants to ionic toxicity and osmotic stress, leading to nutrient imbalance, hormone disturbance, and oxidative damage, all of which severely limit crop yields [
2]. Current methods for alleviating soil salinity include physical (foreign soil improvement technology, sand paving, and open/blind ditch salt drainage) [
3] and chemical (calcium, alkali reduction, organic, and mineral resource modifiers) approaches [
4]. However, issues such as high cost, large engineering volume, and environmental unfriendliness restrict the use of the abovementioned methods.
In recent years, improving saline soil and mitigating the salt stress of crops using microbial agents have become key issues in agricultural research. Plant growth-promoting bacteria (PGPB) are beneficial bacteria that colonize plant roots, either on the root surface or within the root interior, and promote crop growth directly or indirectly [[
5], [
6], [
7]]. However, only microorganisms that are inherently resistant to different stress conditions can promote plant growth through various mechanisms [
8]. Halotolerant PGPB can survive and grow under salt stress conditions and are more effective than non-salt-tolerant bacteria in improving the salt tolerance of plants [
9]. In general, halotolerant PGPB can promote crop development by improving plant water transport and hormone status [
10], inducing plant systemic resistance [
11], and increasing nutrient availability in the soil. However, the ability of PGPB to perform their intended role depends largely on the survival and proliferation of the PGPB in the plant root zone [
12], which, in turn, depends on the application methods of the PGPB. The application methods available include seed inoculation [
13], vegetative infestation [
14], and soil basal inoculation [
15], which are usually applied only once during the crop cycle. PGPB colonization is influenced not only by biological factors such as competition with the autochthonous soil microbial community [
16], chemotaxis, cell surface properties, and interroot secretions [
17] but also by the soil environment, including the soil texture, temperature, oxygen content, moisture, and nutrient contents [
18], as well as agronomic measures such as irrigation, fertilization, intercropping, and crop rotation [
19].
Drip irrigation provides the required water and nutrients to the crop evenly and slowly into the soil near the roots, thus maximizing plant growth and production [
20]. We hypothesize that the application of halotolerant PGPB to the crop root zone along with a water-fertilizer solution using a drip irrigation system is expected to increase the survival of the applied bacteria due to greater moisture and nutrient contents in the drip zone and to promote plant benefits. In this study, the potential effectiveness of drip irrigation using halotolerant PGPB to improve crop growth and soil fertility in saline soils was evaluated. In addition, the effects of drip irrigation with halotolerant PGPB on the composition, diversity and potential functions of rhizosphere bacterial communities were investigated. Our study also included co-occurrence networks of bacterial communities because the functioning of soil bacterial communities depends not only on community composition but also on interactions among populations, which can be studied through such network analyses [
21,
22]. This study provides a basis for improving saline soil and alleviating salt stress in plants via the use of halotolerant PGPB.
2. Materials and methods
2.1. Description of the experimental area
Halotolerant PGPB experiments were conducted from 2020 to 2022 at the Water Resources Management and Irrigation Experimental Station in Xinjiang Uygur autonomous region, China (40°6′N, 81°2′E). The region is characterized by a typical continental arid climate, with an average annual sunshine duration of 2865 h, a temperature of 10.7 °C, a precipitation of 67 mm, and an evaporation of 2110 mm. The soil texture is sandy, and at a depth of 0-40 cm, it has a pH of 8.9, an electrical conductivity (EC) of 873 μs·cm−1, a total soil salinity (percentages of chloride, sulfate, and carbonate in the soil) of 4.3 g·kg−1, an available nitrogen (AN) of 15.1 mg·kg−1, an available phosphorus (AP) of 9.4 mg·kg−1, an available potassium (AK) of 68.4 mg·kg−1, and an organic matter (OM) content of 3.81 mg·kg−1. The soil bulk density and field capacity were 1.41 g·cm−3 and 23.6%, respectively.
2.2. Experimental design
Red jujube is one of the main and characteristic agricultural products in Xinjiang Uygur autonomous region, accounting for more than 34% of the planting area and 50% of the yield in China [
23]. Red jujube trees (variety of
Ziziphus jujuba Mill.) were planted in 2008 and used for experiments, with flood irrigation used throughout the year. The current experiment started on April 15, 2020, and was conducted for two consecutive years. Halotolerant PGPB reported previously were selected from field trials [
24]; they include
Bacillus subtilis (BS),
Pseudomonas fluorescens (PF),
Bacillus thuringiensis (BT),
Bacillus megaterium (BMe),
Bacillus licheniformis (BL),
Bacillus cereus (BC), and
Bacillus mucilaginous (BM). The PGPB used in the study were produced by Beihai Qiangxing Biotechnology Co., Ltd. (China). The bacterial agents were in powdered form, and the standard was GB20287-2006. The living bacteria counts were greater than 5.0 × 10
8 colony forming units (CFU)·g
−1. Jujube plantations without added microbes were used as a control (CK). Each treatment included six replications, and each plot (2.8 m × 5.0 m) consisted of 30 jujubes. The entire growth stage of jujube is divided into five periods, namely, the twig growth period (Tw, April 3rd to June 22nd), the flowering period (FL, June 23rd to July 23rd), the fruit enlargement period (FE, July 24th to August 23rd), the white ripening stage (WR, August 24th to September 22nd), and the full ripening period (FR, September 23rd to November 10th). There were 16 irrigations during the whole growth stage. During late Tw, fertilization and PGPB application were carried out alternately, with seven applications of fertilization and seven applications of PGPB during the jujube growing season (from April to November) (Table S1 in Appendix A). Fifty milliliters of halotolerant PGPB at a concentration of 10
8 CFU·mL
−1 was dissolved in water and transported to the jujube root zone through a drip irrigation system. A venturi injector installed at the head of each plot was used for halotolerant PGPB application (
Fig. 1).
The jujube trees were planted in narrow (80 cm) and wide (200 cm) rows, with 100 cm spacing. Drip irrigation was arranged in parallel on both sides of the jujube trees, approximately 20 cm from the tree roots. The drip irrigation system had a flow rate of 3.0 L·h−1. The irrigation amount was 8210 m3·ha−1, including 318 kg·ha−1 of N, 180 kg·ha−1 of P2O5, and 359 kg·ha−1 of K2O (Table S2 in Appendix A). The CK treatment received the same amounts of water and nutrients as the other treatments but without bacterial agents.
2.3. Soil sampling and determination of soil chemical properties
Jujube rhizosphere soils were sampled on the fifth day after halotolerant PGPB application. We conducted four soil samplings during the experimental period, corresponding to the jujube FL, FE, WR, and FR. The soil samples were sieved (1 mm). For these samples, the soil pH was determined using a compound electrode (InPro2000; Mettler-Toledo, Switzerland) with a soil-to-water ratio of 1:2.5. The soil EC was determined with a conductivity meter (DDS-11A; Shanghai Dapu Instruments Co., Ltd., China).
We collected rhizosphere soil samples during the harvest of jujube trees at the end of the FR in 2021. Six replicates were established for each treatment (
n = 6). Within each plot (2.8 m × 5.0 m = 14 m
2), a composite sample made from five subsamples was obtained after sampling following an “S” sampling pattern. The collected soils were then split into two subsamples after being transported to the laboratory on ice. One portion was air-dried and then passed through a 2.0-mm sieve to determine the soil chemical properties, and the other portion was used for soil enzyme activity measurements (4 °C) and for DNA extraction and subsequent 16S ribosomal RNA (rRNA) gene amplicon sequencing (−80 °C). The soil AN, AP, and AK contents were determined by the Kjeldahl digestion method, molybdenum blue method and flame spectrophotometry methods, respectively, and the soil OM content was measured by loss-on-ignition [
25].
2.4. Polymerase chain reaction (PCR) amplification and amplicon sequencing
Among all the PGPB, the top 3 PGPB treatments that resulted in greater jujube yield and superior fruit quality were selected for sampling for genomic DNA extraction. In addition, plants not treated with PGPB were used as the CK group. Each treatment had six replicates. The genomic DNA of the 24 soil samples was extracted using the MP FastDNA Spin Kit (MP Biomedicals, USA) according to the manufacturer’s instructions. The bacterial community structure of the jujube rhizosphere was investigated by sequencing the V4 region of the 16S rRNA gene, which was amplified using the primer pair 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). After electrophoresis on a 2% agarose gel, PCR products with bright main bands between 400 and 450 bp were mixed in equidensity ratios and purified with a Qiagen Gel Extraction Kit (Qiagen, Germany). The sequencing libraries were generated using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) following the manufacturer’s protocol, and index codes were added. The libraries were sequenced using the Illumina HiSeq 2500 platform (Majorbio, China). A total of 1 010 634 valid 16S rRNA gene sequences were assembled, ranging from 19 504-72 708 per sample after quality control. The number of sequences per sample was rarefied to 19 504 via random sampling. In total, 3795 operational taxonomic units (OTUs) were clustered from sequences at 97% similarity using UPARSE software. Potential functions of the soil bacterial community were calculated using FAPROTAX, which extrapolates taxonomic bacterial community profiles into putative functional profiles based on a database of cultured microorganisms [
26].
2.5. Determination of plant antioxidative enzymes, yield, and quality
Fresh leaves (0.15 g) were homogenized and precooled to 4 °C in normal saline (1.35 mL). The homogenate was subsequently centrifuged at 3500 r·min−1 for 10 min at 4 °C. The supernatants were used for the determination of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, which were determined by the hydroxylamine method, ultraviolet-visible spectroscopy (UV-VIS) spectrophotometry, and the ammonium molybdate method, respectively.
After the jujube trees had entered the FR stage, five jujube fruits were picked from the eastern, southern, western, and northern aspects of each tree to calculate the plant yield. Each treatment was replicated five times. Twenty waxy fruits with no spots were randomly selected for quality measurements. The fruits were mixed after their pits were removed. The soluble sugar (SS) content was determined by the anthrone colorimetric method, the organic acid (OA) content was determined by the acid-alkali neutralization titrimetric method, and the vitamin C (VC) content was determined by the 2,6-dichloro-indophenol titration method.
2.6. Statistical analyses
The Richness and Shannon indices were used to assess the bacterial community diversity of the jujube rhizosphere soils, which were obtained by counting the OTU numbers and calculated according to species abundance, respectively. Principal component analysis (PCA) based on the UniFrac matrix was used to evaluate the changes in the bacterial community structure. Network analysis (
n = 6) was conducted to determine the bacterial co-occurrence patterns under PGPB treatments. Only OTUs with average relative abundances > 0.05% were retained to improve the network’s reliability. Spearman correlations between two OTUs were considered valid if the coefficient (
ρ) was > 0.6 and the false discovery rate (FDR)-corrected
p value was < 0.01 [
27]. All the robust correlations identified from the pairwise comparisons of OTU abundance formed a correlation network. Each node represents an OTU, and each edge represents a solid and significant correlation between the nodes. The topological parameters were estimated using the igraph package to describe the topology of the networks. The networks were visualized in Gephi [
28]. In addition, the OTUs with the highest betweenness centrality values were considered putative keystone taxa, which indicated that the nodes could hold together communicating nodes [
29].
A structural equation model (SEM) was used to elucidate the pathway by which drip irrigation halotolerant PGPB improve soil salinity and jujube agronomic performance (AMOS v22.0; IBM, USA). Pearson correlation was performed to assess the relationships among soil salinity (pH and EC), soil fertility (AN, AP, AK, and OM), jujube antioxidative enzymes (SOD, POD, and CAT), bacterial composition (the first component of PCA (PC1) of the community), diversity (Shannon index), potential function (PC1 of FAPROTAX), and co-occurrence patterns (keystones). The fitted model satisfied the significance criteria when χ2/df < 3 (χ2: Chi-square statistic, df: degree of freedom), p > 0.05 and comparative fit index (CFI) > 0.95. Analysis of variance (ANOVA) was performed to evaluate the differences in soil salinity, soil nutrients, plant antioxidant enzymes, yield, and quality among the four treatments in the field trials (Tukey honestly significant difference (HSD), *p < 0.05).
3. Results
3.1. Jujube yield and quality
Jujube irrigated with PGPB presented a significant increase in yield, approximately 3.3%-22.8% greater than that of the CK without PGPB (
Fig. 2). Compared with those in the CK treatment, jujube growth levels under drip irrigation with BL, BC, and BM increased by 18.7%, 22.8%, and 13.8%, respectively (
p < 0.05). Compared with that of the CK treatment, the SS contents of the plants in the PGPRB drip irrigation treatment significantly increased by 2.5%-24.1%, with significant differences (
p < 0.05) among the BL, BC, and BM treatments and the CK treatment, increasing by 24.1%, 23.8%, and 24.0%, respectively. In addition, the VC content increased by 0.6%-21.5% in plants with PGPB drip irrigation, indicating significant differences among the BL, BC, BM, and CK treatments (
p < 0.05), with 12.2%, 19.1%, and 21.5% increases in VC content, respectively. However, there was no significant difference in the titratable acid content among the treatments.
3.2. Plant antioxidative enzyme activity
The effects of drip irrigation with halotolerant PGPB on the antioxidant enzymes of jujube in saline soil are shown in
Fig. 3. Compared with the CK treatment, drip irrigation with halotolerant PGPB significantly improved the antioxidant properties of jujube, with the SOD, POD, and CAT contents increasing by 0.7%-12.5%, 1.1%-16.4%, and 2.5%-15.4%, respectively. The SOD contents of jujube significantly differed among the BC, BM, and CK treatments (
p < 0.01), increasing by 12.5% and 10.5%, respectively. There was also a significant difference (
p < 0.01) in the CAT content of jujube among the BS, BC, BM, and CK treatments, which increased by 10.8%, 15.4%, and 13.4%, respectively.
3.3. Soil fertility and salinity
The effects of drip irrigation with halotolerant PGPB on soil salinity indices, i.e., soil pH and EC, in jujubes at each reproductive stage are shown in Figs. 4(a)-(d). Drip irrigation with halotolerant PGPB had significant impacts on soil pH and EC, reducing the soil pH and EC content by 0.5%-3.4% and 0.9%-21.3% at the FL stage, 1.8%-6.7% and 0.8%-22.9% at the expansion stage, and 1.8%-6.7% and 0.8%-22.9% at the maturity stage, respectively, compared with those of the CK treatment. The values in the WR stage were 1.3%-8.8% and 5.3%-26.3% lower than those in the CK stage, and the values in the FR stage were 0.8%-9.2% and 0.2%-15.1% lower than those in the CK stage. Among the microbial agent treatments, the BL, BC, and BM treatments had more prominent effects on reducing the soil pH, and the ranking of effects was BM > BL > BC. These treatments also significantly reduced the soil EC throughout the entire jujube growth period, which was ranked as BM > BS > BL.
The effects of drip irrigation with halotolerant PGPB on soil available nutrients are shown in
Fig. 4(e). Compared with the CK treatment, drip irrigation with PGPB significantly influenced soil fertility and altered the AN, AP, AK, and OM contents of the soil by −3.1%-37.8%, 0-24.3%, −8.3%-15.2%, and −1.2%-41.4%, respectively. Compared with those in the CK, the soil AN contents increased by 36.8%, 37.8%, 35.3%, and 19.8% in BS, BL, BC, and BM, respectively. The soil AP content increased by 27.4%, 30.8%, 19.3%, and 24.3%, respectively. Compared with the CK treatment, the BC and BM treatments increased the soil AK content by 9.8% and 15.2%, respectively. Compared with the CK treatment, the BS, BMe, BL, and BC treatments increased the soil OM content by 31.8%, 36.3%, 41.4%, and 37.2%, respectively.
3.4. Bacterial community diversity and composition
16S rRNA amplicon sequencing of the bacterial community was performed for the BL, BC, BM, and CK treatments based on their positive impacts on saline soil and crop performance. The results revealed that drip irrigation with PGPB significantly altered the jujube rhizosphere bacterial community (
Fig. 5). Compared with the control, drip irrigation with PGPB significantly increased bacterial community diversity (
p < 0.01). For example, the Richness index increased by 23.6%, 22.8%, and 25.0% compared with that of the control (
Fig. 5(a)), and the Shannon index increased by 14.6%, 16.7%, and 8.0% (
Fig. 5(b)) for the BL, BC, and BM treatments, respectively. The number of OTUs for these treatments was significantly greater than that for the CK treatment (Fig. S1 in Appendix A). The relative abundance of
Bacillus spp. associated with the three PGPB strains significantly increased (
Fig. 5(c)).
In terms of microbial community composition, Proteobacteria, Actinobacteriota, Bacteroidota, and Firmicutes were the dominant phyla (
Fig. 5(d)), accounting for more than 82.6% of the total sequences. Furthermore, the relative abundances of Proteobacteria, Cyanobacteria, and Nitrospirota significantly differed among the treatments (
p < 0.05, Fig. S2 in Appendix A). Halotolerant PGPB increased the relative abundances of
Psychrobacter,
Flavobacterium,
Steroidobacter,
unclassified f_Sphingomonadaceae, and other genera (
p < 0.05, Fig. S3 in Appendix A). Individually, BL increased the relative abundances of
Ensifer and
Devosia; BM increased the relative abundances of
f_Xanthobacteraceae and
o__Enterobacterales; and BC increased the relative abundances of
Cellulosimicrobium and
Hyphomicrobium (
p < 0.05, Fig. S3).
Using the FAPROTAX database, we found that the BL, BC, and BM treatments all reduced the functions of nitrate and nitrogen (N) respiration, plant pathogens, and aromatic compound degradation, whereas the functions of each PGPB treatment in enhancing the bacterial community differed: the BL treatment enhanced nitrogen fixation and photoheterotrophy; the BC treatment caused nitrate reduction, ligninolysis, and hydrocarbon degradation; and the BM treatment promoted fermentation, chitinolysis, and aerobic chemoheterotrophy (
Fig. 5(e)). The co-occurrence networks of the bacterial communities were constructed using a correlation matrix and visualized for OTUs with relative abundances > 0.5% (
Fig. 5(f)). There were 150 nodes and 932 links in the CK treatment network and 178, 185, and 170 nodes in the BL, BC, and BM treatment networks, respectively. The halotolerant PGPB treatment significantly increased the average degree (AD) and average path length (APL) of the network and reduced the network modularity. Moreover, there was a significant change in keystones in the networks, which were
c_S0134_terrestrial_group,
f_D05-2, and
f_Kineosporiaceae in the CK network;
Bauldia,
Subgroup_10, and
f_Rhizobiales in the BL network;
Gaiella,
Terrimonas, and
g_unclassified in the BC network; and
f_Devoslaceae,
c_KD4-96, and
Microvirga in the BM network.
3.5. Relationships among soil chemical properties, jujube yield and quality, and the bacterial community
Pearson correlations were studied among pH and EC, soil available nutrients and organic matter (AN, AP, AK, and OM), plant antioxidant enzymes (SOD, POD, and CAT), and bacterial community characteristics (composition, diversity, interactions, and potential functions) (
Fig. 6(a)). We found significant correlations among the above indicators in the PGPB treatment and significant negative correlations between the soil salinity index and most bacterial indicators. However, the correlations among these different parameters in the BL, BC, and BM treatments were different. For example, there were significant negative correlations between the soil pH and the relative abundance of
Steroidobacter in the BL and BC treatments but not in the BM treatment. There were significant positive correlations between the soil nutrient OM and the Shannon and Richness diversity indices of the bacterial communities in the BL and BC treatments but not in the BM treatment. We subsequently constructed a structural equation model to elucidate the pathways through which drip irrigation with PGPB enhances crop yield and quality (
Fig. 6(b)). The SEM results indicated that drip irrigation with PGPB restrains soil salinity and improves crop yield and quality by positively influencing the composition and potential function of the bacterial community in the rhizosphere and simultaneously increases the content of soil nutrients to improve crop yield and quality. The fitted model (
χ2/df = 2.74,
p = 0.055, CFI = 0.963, RMSEA = 0.179) met the significance criteria. Additionally, community function positively influenced the plant antioxidant enzyme content, enhancing crop quality. The variations in soil salinity, soil nutrient contents and plant antioxidant enzymes under the BL, BC, and BM treatments had different contributions to the increase in crop yield/quality (
Fig. 6(c)). For example, soil salinity contributed 0.8%, 0.1%, and 1.2% of the nutrients in the BL, BC and BM treatments, respectively; soil nutrients contributed 49.6%, 11.9%, and 25.8%; and plant antioxidant enzymes contributed 0.3%, 0.1%, and 1.1%, respectively, to the increases in jujube yield and quality.
4. Discussion
4.1. Drip irrigation with halotolerant PGPB alters the rhizosphere bacterial community
The rhizosphere, where soil microorganisms intensely interact with plant roots, provides a unique environment for bacterial community development [
30]. The observed increase in bacterial diversity in soils irrigated with halotolerant bacteria aligns with previous studies in which BM, BC, and BL were inoculated to promote soil nutrient availability and plant growth under drought or salinity conditions [
31,
32]. Notably, the soil bacterial diversity in the BM treatment was relatively low compared with the levels in the other bacterial treatments. BC can produce lipopeptides, polyketides, cyclic polypeptides and volatile antimicrobial substances [
33], providing antagonistic action against some bacterial species in the rhizosphere. This mechanism may explain the reduced soil bacterial diversity observed in soils irrigated with BC compared with the other treatments. Furthermore, drip irrigation with halotolerant PGPB increased the relative abundances of Cyanobacteria and Nitrospirota at the phylum level (
Fig. 5(e) and Fig. S2).
Psychrobacter,
Flavobacterium and
Steroidobacter, which are enhanced by drip irrigation with haloPGPB (
Fig. 5(e) and Fig. S3), are functional microorganisms involved in soil nutrient cycling and transformation [[
34], [
35], [
36]]. In addition to the large amounts of water and nutrients delivered by drip irrigation, halotolerant PGPB provide an additional nutrient source by releasing nutrients from soil OM and minerals. For example, BL is able to decompose OM through the production of hemicellulases and cellulases [
37], and BM can solubilize the otherwise insoluble K in mica ores and provide more available K in soils [
38]. The increased availability of nutrients may increase the relative abundances of functional groups following the application of halotolerant PGPB via drip irrigation. Additionally, the
Bacillus used in the present study was proven to supply sufficient substrate through OAs, amino acids, and hormones produced by metabolism [
37,
38], which also provided suitable conditions for the proliferation of indigenous microorganisms. The individual effects of halotolerant PGPB on the bacterial community may be associated with their unique characteristics. For example, BC is reported to produce amylase, cellulase, protease, and lipase extracellular hydrolytic enzymes [
39], leading to increased soil nitrogen (
Fig. 4(e)) and carbohydrate contents and increasing the relative abundances of
Hyphomicrobium (a type of denitrifying bacteria [
40]) and soil N cycling
.Bacterial keystone species, identified by network hubs, significantly contribute to the formation of bacterial communities [
29]. Drip irrigation with halotolerant PGPB increased the number of network nodes, links, and AD, fostering a more complex bacterial network with intensive interactions among species [
41]. Regardless of their relative abundance in a bacterial community, keystones exert significant influences on bacterial community composition and function through intermediate and effective groups [
42]. In this study, significant changes in keystone species were observed under halotolerant PGPB conditions.
F. kineosporiaceae was identified as a keystone in the CK network, whereas
Bauldia,
Terrimonas and
Microvirga were identified as keystones in the BL, BC and BM networks, respectively, and closely interacted with
Bacillus (
Fig. 5(c)).
Bauldia is considered a N-fixing bacterium [
43], and
Terrimonas plays a crucial role in the biogeochemical cycling of sulfur [
44]. Conversely,
F. kineosporiaceae, the keystone of CK, is reported to be enriched in salty environments such as seawater [
45].
4.2. Drip irrigation with halotolerant PGPB enhances plant adaptation to salt stress
Soil salinity is considered one of the most adverse environmental factors limiting crop productivity and is projected to be exacerbated in the coming decades [
46]. Soil salinity is typically negatively correlated with crop yield and quality [
31]. Our results indicate that treatments with halotolerant PGPB reduced soil salinity. Several mechanisms can explain the positive effects of
Bacillus species within the soil-plant system.
Bacilli enter and colonize the crop root zone via the drip irrigation system (
Fig. 5(c)), releasing acidic metabolites such as low-molecular-weight OAs [
47]. Halotolerant PGPB facilitate the movement of salt ions such as Na
+ and Cl
− from the external environment into specific areas of the plant cytoplasm [
48], thereby reducing the Na
+ and Cl
− contents in saline soils [
49].
Plants exposed to adverse environmental factors, such as salt stress, require more nutrients to adapt and resist [
50]. Drip irrigation with halotolerant PGPB significantly increased the available soil nutrient contents of AN, AP, and AK (
Fig. 4(e)). For example, other
Bacillus species, such as BM, can solubilize potassium (K) by various mechanisms, including the production of OAs, as mentioned above, which improves the chelation of cations bound to K and helps microbes dissolve K [
48,
51]. Consequently, the AK content was relatively high in response to BM treatment (
Fig. 4(e)). The altered bacterial community composition, indicated by PC1, was significantly positively correlated with the soil nutrient indicators (
Fig. 6(b)). This is primarily because bacterial species involved in nutrient cycling, which have relatively high relative abundances under halotolerant PGPB conditions, are positively related to the available soil nutrient content in the plant rhizosphere [
52]; for example,
Flavobacterium participates in the decomposition of soil OM [
53], and
f_Sphingomonadaceae, affiliated with Sphingomonadales, harbors the pyrroloquinoline quinone gene, which is involved in P solubilization [
54]. Additionally, changes in bacterial community keystones may further contribute to improvements in soil fertility. For example,
Bauldia, a N-fixing bacterium [
43], was a keystone in the BL network.
Higher soil salt levels lead to hyperosmotic conditions in the plant root zone, disrupting the dynamic balance between plant reactive oxygen species (ROS) production and scavenging [
55]. ROS production in plants is associated with damage, such as growth inhibition, oxidative stress, and cell structure injury [
56]. The application of halotolerant PGPB significantly increased the activities of antioxidant enzymes such as SOD, POD, and CAT in jujube, thereby reducing the oxidative damage caused by excessive ROS in the plant [
57]. Halotolerant PGPB can activate plant antioxidant defense mechanisms by increasing the expression levels of genes encoding antioxidant enzymes in plants. For example, BC can stimulate the upregulation of mRNAs encoding SOD, POD, and CAT in potato [
58]. Additionally, halotolerant PGPB indirectly promote antioxidant enzyme activities by providing sufficient substrates for the synthesis of metalloenzymes such as SOD [
59].
4.3. Great potential of halotolerant PGPB drip irrigation in engineering applications
In this study, small amounts of halotolerant PGPB were supplied to the jujube root zone through a drip irrigation system at high frequency. The simultaneous supply of water and nutrients creates a favorable environment for bacterial growth, facilitating bacterial colonization and proliferation, resulting in better effects than soil basal application and seed dressing [
60]. Although all the halotolerant PGPB treatments had positive impacts on the soil and crop, significant differences in their promoting effects were observed among the different bacterial treatments. For example, the fruit VC and SS contents following the BM treatment were significantly greater than those following the other treatments. K is an important source of osmotic fluid and positive charge for plant electrostability and enzyme activation, managing carbohydrate synthesis, the rate of mineral uptake, and the transport of assimilation products [
61]. An increase in the soil AK content promotes the synthesis of proteins, sugars, and starch in plants [
62]. Additionally, we evaluated the performance of the drip irrigation system after two years of trials and reported that drip irrigation with halotolerant PGPB did not aggravate emitter clogging; in contrast, it largely alleviated it (Table S4 in Appendix A). A fundamental cost analysis was conducted to assess the feasibility of PGPB. The PGPB used in the study were acquired through commercial means, and the prices of the PGPB in 500 g packages varied from 2.6 to 3.0 USD. The application dose of PGPB for each jujube tree was 70 g. For example, the input for using BL was 0.38 USD·tree
−1, whereas the income was 0.58 USD·tree
−1. The additional income from PGPB is approximately 0.2 USD·tree
−1. Importantly, halotolerant PGPB can be applied using existing fertilizer devices in drip irrigation systems without additional equipment, effectively reducing the cost of adding halotolerant PGPB. In summary, drip irrigation is an inexpensive, convenient, and effective method for applying halotolerant PGPB to improve saline soil. Future research should focus on the effects and underlying mechanisms of water-fertilizer coupling at different bacterial agent concentrations.
5. Conclusions
This study demonstrated the effectiveness of using halotolerant PGPB administered through a drip irrigation system to increase the growth of jujube trees in saline soils. This approach notably decreased the soil pH and EC while significantly improving the jujube yield, VC content, and SS content. Among the seven halotolerant PGPB strains tested, BL and BM were identified as the most effective at reducing soil salinity and increasing plant yield and quality. The application of halotolerant PGPB not only mitigated soil salinity but also increased soil nutrient availability and the activity of plant antioxidant enzymes. These benefits were accompanied by significant shifts in the composition, diversity, and interaction patterns of the soil bacterial community, contributing to alleviating plant stress and culminating in increased crop yield and quality. This study underscores the potential of halotolerant PGPB as a sustainable strategy to combat the adverse effects of soil salinity on agricultural productivity. Halotolerant PGPB reduced soil salinity and increased the availability of soil nutrients and plant antioxidant enzyme activities, and these changes occurred in parallel with changes in bacterial community composition, diversity, and co-occurrence patterns, alleviating plant stress and increasing crop yield and quality.
CRediT authorship c ontributionstatement
Yunpeng Zhou: Writing - original draft, Investigation, Visualization, Writing - review & editing, Formal analysis, Data curation. Bernard R. Glick: Writing - review & editing. Hassan Etesami: Writing - review & editing. Hongbang Liang: Formal analysis, Investigation. Felipe Bastida: Writing - review & editing. Xin Wu: Writing - review & editing. Naikun Kuang: Data curation, Formal analysis. Yunkai Li: Supervision, Investigation, Funding acquisition, Writing - review & editing, Project administration, Resources.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The research is funded by the National Natural Science Foundation of China (52339004), the Pinduoduo-China Agricultural University Research Fund (PC2023A02002), and the Bintuan Science and Technology Program (2023AB071).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.eng.2025.03.040.
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