Application of scenedesmus obliquus in improvement of saline-alkaline tolerance of suaeda glauca

Abstract

The invention discloses application of scenedesmus obliquus to improvement of saline-alkaline tolerance of suaeda glauca, and belongs to the technical field of saline-alkaline tolerance of suaeda glauca. The purpose of the invention is to improve the saline-alkali resistance of suaeda salsa and improve saline-alkali soil. The invention provides a method for improving saline-alkaline tolerance of suaeda glauca, which comprises the following steps: inoculating scenedesmus obliquus into a BG11 culture medium, culturing under the conditions of 30 DEG C and 200rpm oscillation, introducing 2.5% CO2 and giving 2000lux continuous illumination, after an algae solution reaches a logarithmic phase, centrifugally collecting algae under the condition of 5000rpm, washing the algae with distilled water for three times, and adding the algae into salt-containing wastewater until the final concentration is 0.05 g / L. The invention further provides a method for improving the saline-alkaline tolerance of suaeda glauca. The research reveals composition and functional differences of suaeda glauca rhizosphere bacterial communities under different irrigation treatments, and lays a foundation for promoting plant growth and improving saline-alkali soil by using microalgae.

Description

Technical Field

[0001] The invention belongs to the technical field of salt-alkali resistance of Suaeda salsa, and particularly relates to application of Scenedesmus obliquus in improving the salt-alkali resistance of Suaeda salsa. Background Art

[0002] Currently, commonly used saline-alkali land improvement technologies primarily include engineering measures, agronomic interventions, and chemical improvements. However, these methods are generally associated with high costs and the potential for secondary pollution. The limitations of these traditional methods urgently require the development of innovative technologies. To this end, the development of environmentally friendly bioremediation technologies has become a current research hotspot.

[0003] Suaeda salsa, a typical halophyte, is not only highly salt-tolerant but also serves as a high-quality feed resource. Improving the salt-alkali resistance of Suaeda salsa aims to provide more salt-alkali tolerance technologies and improve saline-alkali soils. Summary of the Invention

[0004] The invention aims to improve the salt-alkali resistance of Suaeda salsa and improve saline-alkali soil.

[0005] The invention provides an application of Scenedesmus obliquus in improving the salt-alkali tolerance of Suaeda salsa.

[0006] It is further defined that the saline-alkali land is soil treated with saline wastewater, and the saline wastewater quality parameters are as follows: TP 0.03±0.00mg / L, COD 70.56±3.46mg / L, EC 5.76±0.20mS / cm.

[0007] It is further defined that the salt-alkali tolerance ability is to promote the growth of Suaeda salsa.

[0008] It is further defined that the promoting the growth of Suaeda salsa refers to increasing plant height, rhizome and dry weight.

[0009] The present invention provides a method for improving the salt-alkali tolerance of Suaeda salsa. The method comprises inoculating Scenedesmus obliquus into a BG11 culture medium, culturing the culture medium at 30°C and 200 rpm with shaking, introducing 2.5% CO2, and providing continuous light of 2000 lux. After the algal liquid reaches a logarithmic growth phase, the algal bodies are collected by centrifugation at 5000 rpm, washed three times with distilled water, and added to saline wastewater to a final concentration of 0.05 g / L.

[0010] The present invention provides an application of Scenedesmus obliquus in improving saline-alkali soil.

[0011] Further defined, the improved saline-alkali land refers to reducing soil salinity, electrical conductivity, increasing soil nutrient content or increasing the diversity of rhizosphere soil bacterial communities; the saline-alkali land is soil treated with saline wastewater, and the saline wastewater quality parameters are as follows: TP0.03±0.00mg / L, COD 70.56±3.46mg / L, EC 5.76±0.20mS / cm.

[0012] To further define, improving soil nutrients means improving soil pH, total nitrogen, available nitrogen, available phosphorus and organic carbon content.

[0013] Further defined, improving the diversity and richness of rhizosphere soil bacterial communities specifically refers to increasing α-diversity indicators such as the number of observed species, Chao1 index, ACE index, Shannon index and Faith phylogenetic diversity index through irrigation treatment.

[0014] The present invention provides a method for improving saline-alkali soil. The method comprises inoculating Scenedesmus obliquus into a BG11 culture medium, culturing the medium at 30°C and 200 rpm with shaking, introducing 2.5% CO2, and providing continuous light of 2000 lux. After the algal liquid reaches a logarithmic growth phase, the algal bodies are collected by centrifugation at 5000 rpm, washed three times with distilled water, added to fresh water to a final concentration of 0.05 g / L, irrigated with Suaeda salsa, and then planted in saline-alkali soil.

[0015] Beneficial effects: The addition of microalgae reduced soil salinity, increased soil nutrient content, and altered the diversity and composition of rhizosphere soil bacterial communities. Microalgae treatment significantly increased the abundance of functional genes associated with salt stress tolerance and nitrogen metabolism in rhizosphere bacteria, particularly nitrogen metabolism, which effectively promoted the growth of Suaeda salsa. This study revealed differences in the composition and function of rhizosphere bacterial communities in Suaeda salsa under different irrigation treatments, laying the foundation for the use of microalgae to promote plant growth and improve saline-alkali soils. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The average growth of Suaeda salsa under different irrigation water treatments (± standard deviation). Different lowercase letters indicate significant differences between different treatments (LSD, p < 0.05);

[0017] Figure 2 The relative abundances of the top 10 bacterial phyla (a), the relative abundances of bacterial phyla with significant differences among different treatments (b), the relative abundances of the top 30 bacterial genera (c), and the relative abundances of bacterial genera with significant differences among different treatments (d);

[0018] Figure 3The taxonomic hierarchy of rhizospheric soil bacteria identified based on the linear discriminant analysis effect size (LEfSe) (LDA>4, p<0.05) (a) and the linear discriminant analysis results (b);

[0019] Figure 4 Mantel test between bacterial communities and environmental factors (a); * indicates that the correlation of environmental factors is significant at the p < 0.05 level. Collinear network diagram shows the interaction between environmental factors and phylum (b) and genus (c);

[0020] Figure 5 Functional profiles of bacteria at the primary level (a), secondary level (metabolism) (b), and tertiary level (glycan biosynthesis and metabolism) (c) based on PICRUSt analysis;

[0021] Figure 6 Circos diagram based on FAPROTAX analysis, showing the differences in carbon and nitrogen metabolism-related functions among the four treatments (a), and the predicted involvement of dominant bacterial communities in rhizosphere soil in nitrogen cycling in the saline wastewater + microalgae (SWM) treatment (b);

[0022] Figure 7 The effective number of sequences (a), rarefaction curve (b), and Shannon-Weil curve (c) of the four treatment groups;

[0023] Figure 8 Figure 3 Venn diagram of the relationship between amplicon sequence variant (ASV) richness (a) and principal coordinate analysis (PCoA) of the bacterial community structure in soil samples (b). DETAILED DESCRIPTION

[0024] The saline soil used in the experiment was collected from Huyanghe City, the Seventh Division of Xinjiang Production and Construction Corps (44°20′~47°04′N, 83°51′~85°51′E). The irrigation water used was the effluent from the Huyanghe City Wastewater Treatment Plant as saline wastewater.

[0025] The water quality parameters are as follows: (ammonia nitrogen) 1.18±0.02mg / L, (nitrate nitrogen) 0.26±0.02mg / L, (orthophosphate) 0.02±0.00mg / L, TP (total phosphorus) 0.03±0.00mg / L, COD (chemical oxygen demand) 70.56±3.46mg / L, EC (electrical conductivity) 5.76±0.20mS / cm.

[0026] Tetradesmus obliquus ZYY1 is described in the article Sustainable Treatment of Swine Wastewater: Optimizing the Culture Conditions of Tetradesmus cf. obliquus to Improve Treatment Efficiency.

[0027] BG11 medium: 1,500 mg / L NaNO3, 40 mg / L K2HPO4·3H2O, 75 mg / L MgSO4·7H2O, 36 mg / L CaCl2·2H2O, 6 mg / L citric acid, 6 mg / L ammonium ferric citrate, 1 mg / L Na2EDTA, 20 mg / L Na2CO3, 2.86 mg / L H3BO3, 1.81 mg / L MnCl2·H2O, 0.22 mg / L ZnSO4·7H2O, 0.079 mg / L CuSO4·5H2O, 0.39 mg / LNa2MoO4·2H2O, and 0.049 mg / L Co(NO3)2·6H2O.

[0028] Example 1. Method for preparing irrigation water

[0029] 1. 0.5 mg of Scenedesmus obliquus ZYY1 was inoculated into BG11 medium and cultured at 30°C, 200 rpm, and shaken for 6 days with 2.5% CO₂ and 2000 lux of continuous light to ensure the microalgae reached the logarithmic growth phase. After the microalgae reached the logarithmic growth phase, they were harvested by centrifugation at 5000 rpm (TGL-18000CR, Shanghai Anting Scientific Instrument Factory) for 5 minutes, washed three times with distilled water, and then added to saline wastewater to a concentration of 0.05 g / L.

[0030] Concentration control method: According to the established OD 680 Standard curve of dry weight (g / L). Operation steps: Take the logarithmic phase algae solution to measure OD 680 Calculate the biomass concentration using the standard curve and adjust to a target concentration of 0.05 g / L by centrifugation and resuspending. (For example, if the algae concentration is 0.5 g / L, centrifuge 10 mL and resuspend in 100 mL of saline wastewater.)

[0031] 2. In August 2023, a 100-day potted experiment was conducted in Shihezi City, Eighth Division of the Xinjiang Production and Construction Corps (44°15′–44°19′N, 85°59′–86°08′E). The outdoor temperature ranged from 10–35°C, the humidity from 30% to 45%, the photoperiod was 24 h, and the light intensity was 2000 lux. Each pot (bottom diameter 24 cm, top diameter 28 cm, height 30 cm) contained 20 kg of soil. Six seeds were sown in each pot and irrigated every five days, with the irrigation rate adjusted based on evaporation (determined by weighing). In the experimental setup, a separate set of control pots (with identical structure and soil volume to the experimental pots) without plants were placed under the same conditions. Every five days, the control pots were weighed using an electronic balance, and their mass loss compared to their initial water content (or the state after the last irrigation) was recorded as the evaporation loss mass ΔW (unit: g), which corresponds to the amount of evaporated water (1 g ≈ 1 mL). Each pot in the experimental treatment group was directly replenished with water based on this ΔW, i.e., each pot was replenished with deionized water equal to the water loss in the control pot each time, ensuring that the soil moisture in the treatment pots remained consistent with the set moisture baseline.

[0032] Three irrigation treatments were used: freshwater irrigation (FW), saline wastewater (SW), and saline wastewater plus microalgae (SWM). Three replicates were used for each treatment. At the end of the experiment, soil adhering to the roots was gently removed with a soft, sterile brush to obtain the rhizosphere soil. Plant height, root length, and total dry weight were recorded. Soil samples were passed through a 2 mm sieve to remove impurities, and the physicochemical properties and microbial community of the rhizosphere soil were analyzed.

[0033] 3. Determination of Suaeda salsa biomass and rhizosphere soil properties

[0034] Plant height and root diameter of Suaeda salsa were measured using a ruler. The roots of the plants were washed with distilled water and dried at 70°C for 48 hours. The plants were then weighed to obtain the total dry weight. Soil pH and EC were determined using a pH meter and conductivity meter at a dilution ratio of 1 (soil):5 (water) (w / v). Soil salinity was determined gravimetrically, total nitrogen (TN) by the Kjeldahl method, available nitrogen (AN) by the alkaline diffusion method, and available phosphorus (AP) by the sodium bicarbonate solution-molybdenum antimony counterspective method. Available potassium (AK) was determined by flame photometry, and soil organic carbon (SOC) was determined by potassium dichromate oxidation titration.

[0035] result:

[0036] 3.1. Growth of Suaeda salsa

[0037] There were significant differences in the effects of different irrigation treatments on the growth indicators of Suaeda salsa (p<0.05). Compared with freshwater irrigation (FW), saline wastewater (SW) and saline wastewater + microalgae (SWM) treatments significantly promoted the growth of plant height, root diameter and total dry weight of Suaeda salsa ( Figure 1 ), confirming that Suaeda salsa has good physiological adaptability to moderate salinity environments. Notably, the SWM treatment exhibited a more significant growth-promoting effect than the SW treatment, with plant height increasing by 14.6% (36.67±1.67cm), root diameter by 7.2% (3.03±0.30mm), and dry weight by 56.4% (3.71±0.17g).

[0038] 3.2. Soil physical and chemical properties

[0039] Analysis of variance results showed that irrigation water significantly affected all soil physical and chemical properties (Table 1). Compared with the original soil (OS), Suaeda salsa cultivation significantly reduced soil pH (by 0.9-3.8%) while increasing total nitrogen (37.04-48.15%), available potassium (52.23-58.69%), and organic carbon (95.04-142.98%). Under SW irrigation, soil pH, total nitrogen, available nitrogen, available phosphorus, and organic carbon contents increased by 5.41-8.11%, 32.82-39.26%, 13.81-39.20%, and 0.42-24.58%, respectively, compared with those under FW irrigation. The addition of microalgae further significantly improved these parameters. The electrical conductivity and salt content of the rhizosphere soil were significantly higher under freshwater and saline wastewater irrigation than under the original soil. The addition of microalgae significantly reduced the soil salinity (7.74 ± 0.08 g / kg) and electrical conductivity (2.03 ± 0.03 mS / cm) values ​​(p < 0.05), by 6.63-13.52% and 5.58-21.62%, respectively (Table 1).

[0040] Table 1. Soil physical and chemical properties

[0041]

[0042] The data are presented as mean ± standard deviation (n = 3). Different lowercase letters indicate significant differences among different treatments (LSD, p < 0.05).

[0043] 4. Analysis of rhizosphere soil bacterial communities

[0044] use Bacterial DNA was extracted from soil samples using a soil DNA extraction kit (OmegaBio-Tek, Norcross, Georgia, USA). Primers 341F and 806R were used to amplify the V3-V4 region of the bacterial 16S rRNA gene. Amplicon libraries were then sequenced by Shanghai Lingen Biotechnology Co., Ltd. (Shanghai, China) on an Illumina MiSeqPE250 platform (Illumina, San Diego, California, USA). Raw reads were deposited in the NCBI Sequence Read Archive database (accession number: PRJNA1199149). Raw DNA data were processed using Qiime2 (including quality control, filtering, and chimera removal), and amplicon sequence variants (ASVs) were identified using the DADA2 plugin in Qiime2. Alpha diversity, beta diversity, community composition, biomarker discovery, associations with environmental factors, and 16S rRNA function were analyzed.

[0045] Data processing and statistical analysis: Statistical analysis of the experimental data was performed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and SPSS version 20.0 (IBM Corporation, Armonk, NY, USA). The least significant difference (LSD) test (p < 0.05) was used to control for multiple comparisons of samples. Data are presented as mean ± standard deviation (SD).

[0046] result:

[0047] 4.1 Diversity of soil bacterial communities

[0048] In this study, 34,671, 39,938, 34,600, and 34,587 valid sequences were obtained from the OS, FW, SW, and SWM groups, respectively. The number of sequences differed significantly between treatments (p < 0.05, Table 2). Sequencing quality analysis showed that 99.08% of the sequences were within the 401-500 bp range. The average sequence lengths for the treatment groups were 417.91 bp for the OS group, 415.35 bp for the FW group, 415.51 bp for the SW group, and 416.82 bp for the SWM group. Figure 7 a). The convergence trend of the rarefaction curve and the Shannon-Weiner curve indicates that the sequencing depth has fully covered the bacterial diversity in the sample ( Figure 7b and 7c). Diversity index analysis (Table 2) showed significant differences among irrigation treatments in richness (number of observed species, Chao1 index, ACE index) and diversity (Shannon index, Simpson index, Pielou_J index, and Faith phylogenetic diversity index) (p < 0.05). The FW treatment exhibited the highest microbial diversity, with significantly higher numbers of observed species (1,244.00), Chao1 index (1,244.08), ACE index (1,244.88), Shannon index (6.50), and Pielou's evenness index (0.9115) than the other treatments (p < 0.05). In contrast, the number of observed species (955.00), Chao1 index (955.00), ACE index (955.19), Shannon index (6.12), Pilo evenness index (0.8918) and faith phylogenetic diversity index (59.83) under the SW treatment were the lowest, which decreased by 23.23%, 23.24%, 23.27%, 5.85%, 2.16% and 14.88% respectively compared with the FW irrigation treatment (p < 0.05), indicating that SW irrigation significantly reduced the diversity and evenness of the soil bacterial community. After the addition of microalgae, the observed species number, Chao1 index, ACE index, Shannon index, and Faith phylogenetic diversity index values ​​were 1,160.00, 1,160.00, 1,160.00, 6.29, and 74.93, respectively, representing increases of 21.47%, 21.47%, 21.44%, 2.78%, 0.01%, and 20.15%, respectively, compared to the SW irrigation treatment (p < 0.05). Notably, the Faith phylogenetic diversity index value was the highest in the SWM irrigation treatment, indicating that the addition of microalgae significantly increased phylogenetic diversity.

[0049] Table 2. Alpha diversity of soil bacterial communities

[0050]

[0051] Different lowercase letters indicate significant differences among different treatments (LSD, p < 0.05).

[0052] Beta diversity was assessed using Venn diagrams and principal coordinate analysis (PCoA). Figure 8), revealing the connections, commonalities and differences between the soil sample treatments studied. The Venn diagram shows the number of common and unique ASVs in multiple samples. A total of 2,995 ASVs were detected in the four groups, of which 161 ASVs (accounting for 16.5%, 12.7%, 16.7% and 13.6% of the total ASVs in each group, respectively) were shared by the four groups. The number of unique ASVs in the OS, FW, SW and SWM groups was 978, 1266, 962 and 1182 ASVs, respectively, indicating that different treatments significantly changed the bacterial community structure. The results of PCoA analysis showed that the first principal component explained 87% of the variation in the data matrix, and the contribution rates of the first and second axes were 61% and 26%, respectively. The four sample treatments were clustered separately. Therefore, the cultivation of Suaeda salsa, irrigation with saline wastewater and the addition of microalgae had a significant effect on the structure of the rhizospheric soil bacterial community.

[0053] 4.2 Composition of soil bacterial communities

[0054] A total of 33 bacterial phyla were identified in this study, including some unclassified bacteria ( Figure 2 The 10 dominant phyla with the highest relative abundance showed significant differences among the treatments. The bacterial communities in all treatment groups were dominated by Proteobacteria (25.14-44.51%), Actinobacteriota (14.90-33.61%), Chloroflexi (6.91-12.69%), Gemmatimonadota (6.95-14.02%), Bacteroidota (4.20-8.91%), and Acidobacteriota (3.75-6.34%). Figure 3 In a), the total relative abundance of these six phyla exceeded 85%. Figure 3 b further shows the response of soil bacterial communities to different irrigation treatments. Compared with the original soil (OS) group, the relative abundance of Chloroflexi in the rhizosphere soil of Suaeda salsa was significantly increased. SW irrigation significantly increased the relative abundance of Proteobacteria and Bacteroidetes, but reduced the relative abundance of Actinobacteria. In contrast, SWM irrigation increased the relative abundance of Chloroflexi (12.69%), Gemmatimonadetes (9.25%), Bacteroidetes (8.91%), and Acidobacteria (6.34%), while reducing the abundance of Proteobacteria and Actinobacteria. It is worth noting that among all four treatments, the relative abundance of Chloroflexi and Bacteroidetes was the highest under SWM irrigation ( Figure 2 b) in the above example.

[0055] The top 30 bacterial genera in relative abundance are Figure 2As shown in Figure c, Sphingomonas (2.78-5.89%) was the most common genus in all soil samples. Compared with other treatment groups, Sphingomonas (5.89%), Haliangium (2.64%) and Enterobacter (2.28%) were the dominant groups in the OS group ( Figure 2 Cultivation of Suaeda salsa (i.e., FW, SW, and SWM treatments) significantly increased the relative abundance of bacterial genera such as Cellvibrio (0.07-2.35%), Nocardioides (0.99-1.58%), Arthrobacter (0.74-1.92%), Bradyrhizobium (0.66-1.36%), Rheinheimera (0.09-0.13%), and Mesorhizobium (0.53-1.74%). Compared with FW irrigation, SW and SWM treatments significantly increased the relative abundance of Sphingomonas, Cellvibrio, Labrys, Enterobacter, Pseudomonas, and Mesorhizobium. Notably, SWM increased the abundance of Sphingomonas, Vibrionimonas, Haliangium, Nocardioides, Bradyrhizobium, and Pseudomonas compared with SW irrigation.

[0056] To further analyze and identify the differential characteristics of bacterial communities between different treatment groups, the species (i.e., biomarkers) with significant differences between the groups were analyzed using linear discriminant analysis effect size (LEfSe) ( Figure 3Fifteen bacterial species differed in abundance between the original soil samples and the rhizosphere soil samples of Suaeda salsa under the three irrigation treatments (linear discriminant analysis score (LDA) > 4, p < 0.05). Biomarkers in OS samples included f_Geminicoccaceae (o_Tistrellales, c_Alphaproteobacteria, p_Proteobacteria), c_Gammaproteobacteria (p_Proteobacteria), and p_Gemmatimonadota. Biomarkers for FW irrigation included c_MB-A2-108 (p_Actinobacteriota), c_Acidimicrobiia (p_Actinobacteriota), and c_Thermoleophilia (p_Actinobacteriota). Under SW irrigation, o_Rhizobiales (c_Alphaproteobacteria, p_Proteobacteria), o_Micrococcales (c_Actinobacteria, p_Actinobacteriota), and o_Pseudomonadales (c_Gammaproteobacteria, p_Proteobacteria) were more abundant. Under SWM irrigation, o_Burkholderiales (c_Gammaproteobacteria, p_Proteobacteria), c_Bacteroidia (p_Bacteroidota), and p_Chloroflexi were more abundant. Thus, significant differences in bacterial community composition were observed between soil, Suaeda salsa, and irrigation water.

[0057] 4.3 Correlation between soil bacterial communities and soil physicochemical properties

[0058] In this study, the Mantel test was used to evaluate the relationship between the bacterial community structure (including α diversity, dominant phyla and genera) and environmental factors in the four treatment groups ( Figure 4 (a in the figure). The results showed that SOC and TN were significantly positively correlated (Table 3), with both reaching their highest levels under the SWM treatment. Although soil environmental factors had no significant effect on bacterial α-diversity (Table 4), analysis revealed that pH significantly affected bacterial phylum-level community composition (p < 0.05), while total nitrogen and soil organic carbon significantly affected bacterial genus-level community composition.

[0059] Irrigation with saline wastewater and the application of microalgae altered the correlations between microbial abundance and rhizosphere soil physicochemical properties. These interactions were further analyzed by constructing a network diagram, where node size is proportional to the number of connections, and green and blue segments indicate positive and negative correlations, respectively ( Figure 4 The network diagram shows that the analysis of 10 dominant phyla and 30 dominant genera revealed 20 significant correlations, and the specific values ​​are shown in Tables 5 and 6. At the phylum level ( Figure 4 In b), the abundance of Actinobacteria and Bacteroidetes was negatively correlated with pH and positively correlated with available phosphorus, respectively. It is worth noting that Chloroflexi showed the most environmental factor connections, and its abundance was positively correlated with total nitrogen, available potassium, and soil organic carbon content. At the genus level ( Figure 4 In c), available potassium had the most connections, followed by available phosphorus and total nitrogen. Specifically, available potassium and total nitrogen were negatively correlated with the abundances of Haliangium, Enterobacter, and Rubellimicrobium. Furthermore, available potassium and available nitrogen were negatively correlated with the abundances of Arenimonas and Ellin6055. Conversely, available phosphorus was positively correlated with the abundances of Vibrionimonas and Pseudomonas, but negatively correlated with the abundance of Altererythrobacter.

[0060] 4.4 Functions of soil bacterial communities

[0061] (1) Metabolic pathway analysis based on PICRUSt2

[0062] Figure 5 The biological metabolic pathway analysis based on PICRUSt2 is presented, which helps to reveal the potential benefits of bacterial communities. Metabolic pathways are divided into three levels for display. In the first level classification ( Figure 5 In a), metabolic function (69.69-70.75%) was the main functional category, followed by genetic information processing (13.64-14.25%), environmental information processing (9.13-9.92%), cellular processes (3.28-3.80%), human diseases (1.35-1.67%), and organismal systems (1.13-1.19%). Since most genes were identified as participating in metabolic processes, genes specifically related to metabolism were further analyzed in the secondary classification ( Figure 5b). The relative abundance of carbohydrate metabolism (15.70-15.99%) and metabolism of cofactors and vitamins (6.51-6.58%) was higher in rhizosphere soil compared with OS. In SWM-irrigated soil, bacterial communities related to energy metabolism (8.88%) and metabolism of cofactors and vitamins (6.56%) were more abundant than those in SW-irrigated soil. Carbohydrate synthesis is a key functional trait for enhancing plant stress tolerance. Therefore, the relative abundance of glycan synthesis-related genes was analyzed in the three-level classification ( Figure 5 c). Unlike OS, soils associated with Suaeda salsa expressed numerous genes related to glycosphingolipid biosynthesis (globo and isoglobo series), N-glycan biosynthesis, other glycan degradation, peptidoglycan biosynthesis, and various types of N-glycan biosynthesis. The abundance of nearly all genes related to glycan biosynthesis was higher under SW and SWM irrigation than under FW irrigation. Among them, the abundance of genes involved in peptidoglycan biosynthesis was slightly lower under SW and SWM irrigation than under FW irrigation. Notably, the abundance of all genes involved in carbohydrate synthesis was higher under SWM irrigation than under SW irrigation. Furthermore, enrichment of two-component systems was observed in both OS and SW samples (Table 7). Nitrogen metabolism, which is associated with plant growth, was highest in abundance under SWM irrigation, followed by SW, FW, and OS groups. This suggests that Suaeda salsa cultivation, saline wastewater (SW and SWM) irrigation, and microalgae application enhanced metabolic processes in the bacterial community. Analysis of these functional traits is crucial for understanding the ecological role of bacterial communities in maintaining Suaeda salsa health and soil resilience.

[0063] (2) Bacterial community function prediction based on FAPROTAX

[0064] The results of the predicted bacterial community functions (top 30 by relative abundance) for the four treatment groups based on the FAPROTAX database are shown in Table 8. In the four groups of samples, chemoheterotrophy (28.82-29.89%), aerobic chemoheterotrophy (21.04-31.30%), fermentation, and processes related to nitrogen metabolism (including nitrate reduction, ureolysis, nitrogen fixation, nitrate respiration, and nitrogen respiration) dominated, with the relative abundance of each process exceeding 1%. Figure 6 Figure a shows the differences in carbon and nitrogen metabolism-related functions among the four treatments. Compared with the OS sample, the abundance of gene functions related to chemoheterotrophy, aerobic heterotrophy, aromatic compound degradation (aromatic_compound_degradation), and nitrogen fixation (nitrogen_fixation) increased by more than 1% in the rhizosphere bacterial community of Suaeda salsa. Furthermore, compared with FW, the SW and SWM treatments enhanced the abundance of gene functions related to nitrogen metabolism (nitrate reduction, nitrate respiration, nitrogen respiration, nitrite respiration (nitrite_respiration), and urea decomposition), fermentation, methylotrophy (methylotrophy, methanol oxidation (methanol_oxidation)), and phototrophy. Furthermore, the relative abundance of genes related to nitrogen metabolism (nitrate reduction, nitrogen fixation, nitrate respiration, nitrogen respiration, and nitrite respiration) significantly increased under SWM irrigation compared with SW irrigation. These results indicate that Suaeda salsa cultivation and saline wastewater irrigation (SW and SWM) enhance microbial functions related to carbon cycling and nitrogen metabolism to varying degrees, while the addition of microalgae further promotes soil nitrogen cycling processes.

[0065] Table 3. Intergroup correlations of soil physical and chemical properties

[0066]

[0067] *Indicates that the correlation between environmental factors is significant at the 0.05 level.

[0068] Table 4. Mantel test correlation

[0069]

[0070] *Indicates that the correlation between environmental factors is significant at the 0.05 level.

[0071] Table 5. Interactions between environmental factors and bacterial phyla

[0072]

[0073]

[0074] *Indicates that the correlation between environmental factors is significant at the 0.05 level.

[0075] Table 6 Interactions between environmental factors and bacterial genera

[0076]

[0077]

[0078] * and ** indicate that the correlations between environmental factors are significant at the 0.05 and 0.01 levels, respectively.

[0079] Table 7. The top 40 functional groups ranked by abundance based on the three-level taxonomic level of PICRUSt2.

[0080]

[0081]

[0082]

[0083] Table 8. Top 30 functional classes ranked by abundance based on FAPROTAX

[0084]

[0085]

[0086] Conclusion: Physicochemical properties of rhizosphere soil

[0087] Improvements in soil quality are often reflected in enhanced soil biochemical properties. Results showed that irrigation water significantly affected soil physicochemical properties (Table 1). Significant changes in pH were observed in all four samples. Specifically, cultivation with Suaeda salsa (FW) resulted in a decrease in soil pH, while SW irrigation resulted in significantly higher pH than the FW treatment, and the pH increased further after the addition of microalgae.

[0088] Regarding soil nutrient dynamics, Suaeda salsa cultivation alone significantly increased total nitrogen, available potassium, and soil organic carbon. Compared with freshwater irrigation, irrigation with saline wastewater resulted in significantly higher levels of total nitrogen, available nitrogen, available phosphorus, and soil organic carbon in the soil, which increased further after the addition of microalgae. These results suggest that wastewater has significant potential as a nutrient source. In summary, Suaeda salsa grown in saline wastewater combined with microalgae contributes to soil desalination and increases soil nitrogen, phosphorus, and carbon content, significantly improving soil quality.

[0089] Diversity and composition of bacterial communities: Soil microbial diversity is an important indicator of soil quality and can reflect the overall dynamic characteristics of the microbial community. The results showed that the diversity and richness of the bacterial community under FW treatment were significantly higher than those in other samples. Previous studies have shown that Suaeda salsa, a halophyte, can enhance the activity of microorganisms in saline soils and promote the healthy development of saline-alkali ecosystems. However, SW irrigation significantly reduced the diversity and uniformity of the soil bacterial community.

[0090] Principal coordinate analysis (PCoA) results showed that Suaeda salsa cultivation, saline wastewater irrigation, and microalgae addition all significantly affected the structure of rhizosphere soil bacterial communities. This may be because plant roots secrete hormones that influence rhizosphere soil bacterial communities. Furthermore, the bacterial communities in the microalgae addition treatment showed the greatest differences compared to the other treatments, indicating that this treatment plays an important role in ecosystem function and that microalgae addition can alter soil bacterial community structure.

[0091] At the phylum level, Proteobacteria and Actinobacteriota were the dominant bacterial phyla in the rhizosphere soil; these phyla are also common in other saline soils and halophytes. The abundance of Actinobacteria was significantly lower in alkaline soils than in neutral pH soils. Furthermore, both Actinobacteria and Proteobacteria exhibited adaptability to low-carbon environments. This may explain the increase in the relative abundance of Proteobacteria and the decrease in Actinobacteria under SW irrigation, as well as the further decrease in the abundance of these two phyla after microalgae addition. The relative abundance of Chloroflexi was significantly increased in the rhizosphere soil of Suaeda salsa (FW, SW, and SWM treatments) compared to the OS treatment, consistent with previous findings showing a positive correlation between the abundance of Chloroflexi and soil organic matter content. The relative abundance of Bacteroidetes increased under SW irrigation, possibly due to their positive response to nutrient levels. Under SWM irrigation treatment, the abundance of Gemmatimonadota increased, and Chloroflexi and Bacteroidetes were enriched, indicating that this treatment may create favorable conditions for the growth and reproduction of functional microbiota by improving the availability of carbon and nitrogen sources in the rhizosphere soil.

[0092] At the genus level, some salt-tolerant bacterial genera, such as Sphingomonas, Haliangium, and Enterobacter, were enriched in OS. After planting Suaeda salsa, the abundance of aerobic or strictly aerobic bacteria, including Cellvibrio, Nocardioides, Arthrobacter, Bradyrhizobium, Rheinheimera, and Mesorhizobium, increased significantly. This may be related to changes in available oxygen levels caused by changes in soil moisture. The abundance of Pseudomonas, which has denitrifying potential, increased significantly under irrigation with saline wastewater and reached its highest level after the addition of microalgae. Nitrogen addition promotes the proliferation of denitrifying bacteria. In addition to Pseudomonas, the SWM irrigation group enriched bacterial genera such as Sphingomonas, Vibrionimonas, Nocardioides, and Bradyrhizobium. Studies have shown that changes in nitrogen and phosphorus content in the soil can modify root exudates, thereby supporting the growth of different bacterial species. In addition, studies have shown that biochar can promote the proliferation of phosphate-solubilizing bacteria (such as Pseudomonas).

[0093] Effects of Soil Physicochemical Properties on Bacterial Community Composition: The rhizosphere soil bacterial community constitutes a complex network of interactions, whose structure is influenced by multiple driving factors, particularly interactions between soil physicochemical properties. Under SWM irrigation, soil organic carbon and total nitrogen reached their highest levels and exhibited a significant positive correlation. Previous studies have shown that soils with high total organic carbon content indicate ecosystem health. Notably, soil environmental factors did not significantly affect alpha diversity, suggesting that soil bacterial communities can maintain high stability despite environmental changes. The composition of the rhizosphere microbiome was significantly influenced by soil pH, total nitrogen, and soil organic carbon. Actinobacteria play a crucial ecological role in saline-alkali ecosystems. In this study, the abundance of Actinobacteria was significantly negatively correlated with soil pH (p < 0.05), consistent with previously discussed results showing a higher abundance of Actinobacteria in neutral soils than in alkaline soils. Furthermore, the abundance of Bacteroidetes was positively correlated with available phosphorus, while the abundance of Chloroflexi was positively correlated with total nitrogen, available potassium, and soil organic carbon.

[0094] In this study, nitrogen showed a strong correlation with specific bacterial genera. Haliangium, Enterobacter, and Rubellimicrobium were negatively correlated with total nitrogen, while Ellin6055 was negatively correlated with available nitrogen. Haliangium is a genus of bacteria with denitrification function. Enterobacter and Rubellimicrobium belong to the Proteobacteria phylum, and there is a significant negative correlation between the abundance of Proteobacteria and nitrogen content. Ellin6055 is related to nitrite oxidation. The increase in the abundance of Haliangium and Ellin6055 may promote NO in the soil. -2 and NO -3 Phosphorus is converted into N2, resulting in a decrease in soil nitrogen content. Furthermore, the abundance of Vibrionimonas and Pseudomonas was positively correlated with available phosphorus, while Rubellimicrobium was negatively correlated with available phosphorus. Vibrionimonas and Altererythrobacter participate in the biogeochemical cycle of phosphorus. Pseudomonas is a common phosphate-solubilizing microorganism that can increase available phosphorus content in soil. Furthermore, the abundance of Streptomyces was negatively correlated with pH, ​​indicating that this genus has the ability to regulate environmental pH.

[0095] Functional prediction of rhizosphere soil bacteria: Based on the functional prediction results of 16S rRNA gene sequences, carbohydrate metabolism functions dominated in all samples, and the carbohydrate metabolism level in the rhizosphere soil of Suaeda salsa was significantly higher than that in the OS group. Enhanced carbohydrate metabolism helps to improve plant stress tolerance. In the SWM-irrigated soil, bacterial communities related to energy metabolism, cofactor and vitamin metabolism were more abundant than those in the SW-irrigated soil, indicating that the addition of microalgae may enhance energy metabolism processes by promoting the synthesis of osmotic substances such as ester compounds and soluble sugars. In addition, the accumulation of soluble sugars may play a key role in plant salt tolerance. It is worth noting that the abundance of two-component systems was higher in OS and SW samples, which may provide bacteria with a selective advantage in coping with adverse environmental conditions.

[0096] Nitrogen cycle is a key link in soil nutrient cycling and showed significant differences among the treatment groups in this study. Functional prediction results based on PICRUSt2 and FAPROTAX showed that functional bacteria involved in nitrogen cycle were significantly enriched under SWM irrigation ( Figure 2c), which includes Bradyrhizobium, which is associated with nitrogen fixation, and Pseudomonas, which participates in denitrification and dissimilatory nitrate reduction to ammonium. Nitrospira, a nitrifying bacterium that mediates nitrite oxidation under aerobic conditions, is less abundant under SWM irrigation than under OS, but still contributes to nitrogen accumulation and Suaeda salsa growth in the soil. Based on the above analysis of bacterial community processes involved in nitrogen cycling under SWM irrigation, a remediation method ( Figure 6 (b) The increased nitrogen content in the rhizosphere soil after adding microalgae indicates a close connection between functional bacterial enrichment and nitrogen metabolism. This enrichment lays a key foundation for nitrogen accumulation in soil under SWM irrigation, providing a theoretical basis for saline-alkali land remediation and ecosystem restoration.

Claims

1. Application of Scenedesmus obliquus in improving the salt-alkali tolerance of Suaeda salsa.

2. The use according to claim 1, characterized in that The saline-alkali land is the soil treated with saline wastewater, and the saline wastewater quality parameters are as follows: TP 0.03±0.00mg / L, COD 70.56±3.46mg / L, EC 5.76±0.20mS / cm.

3. The use according to claim 1, characterized in that The salt-alkali tolerance is to promote the growth of Suaeda salsa.

4. The use according to claim 3, characterized in that The promoting the growth of Suaeda salsa refers to increasing plant height, rhizome and dry weight.

5. A method for improving the salt-alkali resistance of Suaeda salsa, characterized in that: Scenedesmus obliquus was inoculated into BG11 culture medium and cultured at 30°C and 200 rpm with shaking, 2.5% CO2 was introduced and 2000 lux of continuous light was given. After the algal liquid reached the logarithmic growth phase, the algal bodies were collected by centrifugation at 5000 rpm, washed three times with distilled water, and added to saline wastewater to a final concentration of 0.05 g / L.

6. Application of Scenedesmus obliquus in improving saline-alkali soil.

7. The use according to claim 6, characterized in that The improved saline-alkali land refers to reducing soil salinity, electrical conductivity, increasing soil nutrient content or increasing the diversity of rhizosphere soil bacterial communities; the saline-alkali land is soil treated with saline wastewater, and the saline wastewater quality parameters are as follows: TP 0.03±0.00mg / L, COD 70.56±3.46mg / L, EC 5.76±0.20mS / cm.

8. The use according to claim 7, characterized in that Improving soil nutrients means increasing soil pH, total nitrogen, available nitrogen, available phosphorus and organic carbon content.

9. The use according to claim 7, characterized in that Improve the diversity, richness and composition of rhizosphere soil bacterial communities, specifically by increasing α-diversity indicators such as the number of observed species, Chao1 index, ACE index, Shannon index and Faith phylogenetic diversity index through irrigation treatment.

10. A method for improving saline-alkali soil, characterized in that: Scenedesmus obliquus was inoculated into BG11 culture medium, cultured at 30°C and 200rpm shaking conditions, 2.5% CO2 was introduced and 2000lux of continuous light was given. After the algal liquid reached the logarithmic growth phase, the algal bodies were collected by centrifugation at 5000rpm, washed three times with distilled water, added to saline wastewater at a final concentration of 0.05g / L, irrigated with Suaeda salsa, and planted in saline-alkali land.

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