Effects of salt stress levels on nutritional quality and microorganisms of alfalfa-influenced soil

Experimental site

This study was conducted in a field site in Baotou City, Inner Mongolia Autonomous Region, China (coordinates 40 ^∘17′N, 111 ^∘27′E). The experimental site was located on the north bank of the Yellow River, which is in the Hetao Plain. The study site was a typical saline area.

Alfalfa Preparation

ZhongMu No.3 alfalfa was planted on four kinds of land in the experimental base, and it was divided into CK, mild, moderate, and severe according to their salinity. The physical and chemical properties of the soil are shown in Table 1. ZhongMu No.3 alfalfa was a variety selected by the Beijing Institute of Animal Science and Veterinary Medicine of the Chinese Academy of Agricultural Sciences. Its characteristics are strong salt resistance, good palatability, high digestibility, nutrition, and rich in value (Ma et al., 2017). These seeds were also provided by the Beijing Institute of Animal Science and Veterinary Medicine of the Chinese Academy of Agricultural Sciences.

The alfalfa used in the experiment was cutting at the initial flowering stage, and the height of the stubble for cutting was five cm. The collected alfalfa were surface-disinfected with 70% ethanol, 2% sodium hypochlorite, followed by five times rinsing with sterile distilled water for 2 min. Alfalfa of 10 g that included leaves and stems sample was taken from each foil in each treatment group and immediately chopped into 1—2 cm lengths (without root). Then use a sterile grinder to ground into a powdery shape to better release endophytes. 500 g samples from each treatment group were put into kraft paper bags and dried for testing nutritional contents. There were three replicates for each treatment.

Analysis of soil characteristics and alfalfa chemical composition

Soil samples were air-dried indoors, soil EC (electrical conductivity) was determined by EC Meter (FieldScout EC 110 Meter), Na⁺ and K⁺cations were determined by atomic absorption spectrometry (AA-6800, Daojin, Japan) (Li et al., 2019), SO${}_4^{2-}$ and Cl⁻ were determined by ion chromatography (IC-2000; Dionex, Sunnyvale, CA, USA) (Li et al., 2019). The soil pH was determined with a PHS-3C type acidity meter.

The dry matter (DM) content of fresh plant samples was oven-dried (ULM 800; GmbH, Schwa Bach, Germany) at 65 ^∘C for 48 h. Dried samples were ground to one mm particles and crude protein (CP), soluble protein (SP), neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, and fatty acids (FA) were analyzed by near-infrared reflectance spectroscopy (NIRS). All results are reported as % dry mass (% DM). The spectra were analyzed using a large dataset of calibration samples from different kinds of grasslands available from the Institute VDLUFA Qualitätssicherung NIRS GmbH, Kassel, Germany.

High throughput sequencing of microbial population

Microbial DNA was extracted from fresh alfalfa endophytes microorganisms through the following steps. 10 g of plant samples that has been ground were mixed with 90 mL of sterile water and then treated with a table concentrator at 120r/m for 2 h. The sample was then filtered with carbasus and the liquid was centrifuged at 10,000 rpm for 10 min at 4 ^∘C. The supernatant was abandoned and the pellet was suspended in one mL of sterile water solution. The precipitate was used for DNA extraction.

Microbial DNA of alfalfa samples was extracted using the E.Z.N.A.®soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s protocols. The final DNA concentration and purification were determined by NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and DNA quality was checked by 1% agarose gel electrophoresis. The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) by thermocycler PCR system (GeneAmp 9700, ABI, USA). The PCR reactions were conducted using the following program: 3 min of denaturation at 95 ^∘C, 27 cycles of 30 s at 95 ^∘C, 30s for annealing at 55 ^∘C, and 45s for elongation at 72 ^∘C, and a final extension at 72 ^∘C for 10 min. PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase and 10 ng of template DNA. The resulted PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluo^TM-ST (Promega, USA) according to the manufacturer’s protocol.

Raw fastq files were demultiplexed, quality-filtered by Trimmomatic, and merged by FLASH with the following criteria: (a) the reads were truncated at any site receiving an average quality score <20 over a 50 bp sliding window; (b) primers were exactly matched allowing 2 nucleotide mismatching, and reads containing ambiguous bases were removed; (c) sequences whose overlap was longer than 10 bp were merged according to their overlap.

Operational taxonomic units (OTUs) were clustered with 97% similarity cutoff using UPARSE, and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier algorithm against the Silva (SSU123) 16S rRNA database using a confidence threshold of 70%. We uploaded the sequences data in the NCBI under the accession number PRJNA560790.

Statistical Analysis

SAS 9.3 software was used to analyze differences between treatment means using Tukey’s multiple comparison test, with a significance level of P < 0.05. The data from high throughput sequencing and Non-metric multidimensional scaling (NMDS) were analyzed on the Majorbio I-Sanger Cloud Platform (http://www.i-sanger.com).

Soil conditions of the study site

Data on field salinity is shown in Table 1. Soil pH, EC, Na⁺, and Cl⁻ were affected by soil salinization. The soil properties of the CK area were significantly different from other test areas (P < 0.05). The salt stress of three groups were higher than CK, and the differences between the mild area and the moderate area were small. The soil EC in the mild area was 0.59 mS/cm, whereas the EC in the severe area was 2.3 mS/cm. The content of Na⁺ in CK was 0.11 g/kg, which was significantly different from other treatment groups. The content of Na + in the severe group was more than twice that of the CK group, which was 0.25 g/kg. Overall, the cations and anions of the CK group were significantly different from those of the other groups, while the contents of Na⁺, Cl⁻, SO₄²⁻, pH of mild and moderate were not significant.

The chemical composition of alfalfa in fields under salt stress

As shown in Fig. 1, the abiotic stress of salt on alfalfa nutrition was obvious. There was no significant difference in the DM of each treatment, CP which is one of the most important indicators for judging the quality of alfalfa was significantly different between treatments. There was a significant difference between the three treatment groups and the CK, which was the salt stress alfalfa CP was higher than CK. The CP was up to 20.85% DM in moderate stress. At this time, the SP content under moderate salt stress treatment was 5.1% DM, which was also at a high level. However, when the soil EC reached severe salt stress (i.e., Na⁺ was 0.25 g/kg and Cl⁻ was 0.203 g/kg), the CP decreased, and SP content also decreased. FA did not show significant differences when faced with different salt stresses. There was no significant difference between NDF and ADF in salt stress (P > 0.01). When alfalfa was planted in moderate and severe site, it was difficult for the alfalfa to maintain it’s ion balance, resulting in a decrease in SP content, which in turn affected CP content. The severe group of salt stress affected the growth and development of alfalfa, as manifested by a decrease in the nutritional quality of alfalfa.

Microbial community composition under different salt stress

High-throughput assays were performed for variable regions 3 and 4 of 16srDNA, the rarefaction curve (Fig. 2) showed that the sequencing work was relatively comprehensive in covering the bacterial diversity, as the rarefaction curves tended to approach saturation. To identify the bacterial diversity of alfalfa treated with different salt stresses (Table 2), The coverage of all samples were greater than 99%, indicating that the sequencing breadth was relatively comprehensive, and the data was sufficient to represent the characteristics of bacterial microbial communities. According to the number of OTU and CHAO index, the abundance of endophytes bacterial communities under different salt stress treatments were different. The OTU number of CK was 154, this was less than mild group. But more than the moderate and severe groups. Similarly, the Shannon index was also the highest for CK. This also showed that the biological environment of the CK group was more diverse and more abundant.

Non-metric multidimensional scaling (NMDS) analysis was used to visualize the differences in the distribution and structure of bacterial microbial communities after different salt stress treatments. As shown in Fig. 3, there were significant differences in the endophytes microorganisms of alfalfa after treatments. The NMDS resulted have a stress value of 0.062, which meant that the NMDS analysis results indicate that there was no stress in 2-dimensional representation. Mild salt stress and moderate salt stress had similar microbial community structure. In the severe group, the microbial community structure changes from a structure with a higher similarity. There was no overlap with the original community.

Alfalfa microbial community composition is shown in Figs. 4 and 5, showing the distribution of at the genus level under different salt stress levels. Proteobacteria was the most abundant bacterial population. CK (92.9%) had 7.83% more Proteobacteria than the severe salt stress treatment (85.07%). As salt stress increases, the proportion of Proteobacteria decreased monotonically. Among the four groups of salt stress treatments, Pantoea had the highest abundance, followed by Pseudomonas. With the increasing of salt stress, the abundance of Methylobacterium and Sphingomonas decreased gradually. There was a big significant differences between CK and severe in Paenibacillaceae, Aurantimonadaceae, Caulobacteraceae, Nocardia, Kineosporiaceae, Comamonadaceae, Sphingobacteriaceae, Brucellaceae and Bacillaceae. Among them, the abundance of Orangeaceae, Petiaceae, Nocardaceae, Kineosporiaceae and Comamonadaceae were decreased compared with CK, and the others were increased. Planococcus can develop perchlorate-specific stress adaptations that were not (or only to a lower extent) used to counteract high Na⁺ concentrations. The response of plant microorganisms to different salt stresses was very interesting and beyond expectation.

To further understand the effects of different salt stress levels on the microbial genus, we performed a one-way analysis of variance for the top 15 genera in the abundance of different treatments. The results are shown in Fig. 6. With the gradual increase in salt stress, Pseudomonas, Sphingomonas, Methylobacterium, Sphingobacterium, and Aureimonas showed strong response patterns. Pseudomonas and Sphingobacterium increased with salt stress increased, which may be due to the fact that these two genera prefer to survive and reproduce in saline environments. In contrast, Sphingomonas, Methylobacterium, and Aureimonas decreased with increasing salt stress and showed salt-tolerant properties. It can be seen from Fig. 6 that although the microbes in the first 15 genus have a large difference in their responses to different salt stress levels, there was one genus with a significant difference, which was Exiguobacterium. The genus with extremely significant differences were Methylobacterium and Rhizobium. Methylobacterium had a content of 1.7% under severe salt stress. This indicated that Methylobacterium has better salt tolerance and also helps alfalfa to better cope with salt stress. Exiguobacterium was widely used for the industrial production of enzymes. In the CK, the content of Exiguobacterium was less at 0.57%.

As shown in Fig. 7, the microbial factor analysis (db-RDA) diagram shows the correlation between microbes, samples and major nutritional indicators at the genus level. Samples under the same salt stress were close, while samples under different salt stress were far. The microbial community structure of moderate and mild group was similar, and the microbial community structure of CK was different from that of the mild and the moderate, indicating that salt stress would change the structure of microbial community. The CP, SP and FA were positively correlated, and they were negatively correlated with lignin, ADF and NDF, respectively. The CP, SP, FA vector points to mild and moderate, indicating that CP, SP, FA were positively correlated with the mild and the moderate. The correlation between Sphingomycetes, Methylobacterium and CP, SP, and FA was high, while the correlation between Pseudomonas and Chryseobacterium and FA was high.

Alfalfa nutrition in fields under salt stress

Salt stress affects the normal growth and development of plants and affects the nutrients of plants. Differences between SP in different treatments can be considered as a detoxification effect of plants (Zhu, 2001) and there were also significant differences in SP between mild and moderate salt stress. SP was an important factor in relieving osmotic pressure in response to salt stress (Kanwal, Razzaq & Maqbool, 2019). The SP content in a low salt treatment increased significantly (Oprica et al., 2016), while the SP content in a high salinity growth environment was decreased (Agastian, Kingsley & Vivekanandan, 2000). FA was also important detoxifications for plants to respond to abiotic stresses (Constantino et al., 2013). But it has no significant differences among those treatments, or it may be that the FA represents the total fatty acid, not the finer molecular compound such as exogenous FA, which was protecting the tonoplast of the plants (Zhao & Qin, 2005).

Bacterial diversity under different salt stress

With an increasing in salt stress, the richness of bacterial communities decreased. The bacterial community shannon index in the moderate salinity treatment group was 2.1, lower than the other groups. In the mild and moderate salt stress group, richness was 158.27 and 159.24, respectively. This may be due to salt stress increasing the taxonomic groups in the bacterial family (Meng et al., 2019a; Wang et al., 2019b). This was very interesting, we need more research to confirm that.

When soil EC of 1.35 mS/cm, the structure of the flora changed greatly. This conclusion was consistent with the findngs of, (Wang et al., 2019b) who reported that the response of bacteria to salt stress was very active and sensitive. This may indicate that soil EC of 1.35 mS/cm was an inflection point, beyond which soil salt stress has a serious influence on plant microorganisms, and there was significant difference in the nutritional quality of alfalfa. This implied that in the face of such sensitive bacterial community structure, there were also different microbial community structures under different salt stresses. This phenomenon can also be compared to habitats in ecology. That was to say, there were many microbial resources and information that we do not understand under salt habitat (Shao et al., 2018). In the face of salt stress, Proteobacteria is still the main component of bacteria (Lucas et al., 2013). In addition, the same pattern was seen with Bacteroidetes in phylum, which dominates the microorganisms under salt stress (Vasim et al., 2018). In our study, when soil EC reached 1.35 mS/cm, the abundance of Bacteroidetes suddenly increased from less than 1% to 8.2%. However, when the soil EC was 0.59 mS/cm, it’s abundance dropped to 6.4%. It was very likely that Bacteroidetes were more suitable for living in environments with a certain level of salt content (Yadav & Saxeny, 2018), as was the case with halophilic and salt-tolerant bacteria, but when the soil EC reaches 1.35 mS/cm, Bacteroidetes content dropped by 21.9%. When the EC of the soil was too high, the tolerance of some bacteria is reached and inhibits the growth and reproduction of microorganisms.

These halophilic microorganisms significantly changed under salt stress, were likely to play a crucial role in plant growth and development (Yadav & Saxeny, 2018). This may also explain the reasons for the above-mentioned partial alfalfa protein enhancement. Therefore, in the face of abiotic salt stress, in addition to the inherent mechanism of the plant itself in response to salt stress and ionic stress, the endophytes microorganism with the plant (e.g., Bacteroidetes) may also help the plant to grow better.

Moreover, the presence of Rhizobium and Pseudomonas can also alleviate the effects of salt stress on plants (Dilfuza, Dilfuza & Stephan, 2013). However, it was also impossible to rule out the propagation of microorganisms from the roots to the plants. It can also be seen from Fig. 5. that Rhizobium was the most suitable for growth in the environment without salt stress, and it’s abundance was also the highest. This may be due to the fact that salt stress has an inhibitory effect on Rhizobium (Ahmad et al., 2013), which may also explain the decrease in Rhizobium as the increasing salt stress. Rhizobium symbiosis had a positive effect on alfalfa salt tolerance by improving the activity of antioxidant enzymes and osmotic adjustment capacity (Wang et al., 2016), at the same time Rhizobium symbiosis has positive effects on the salt tolerance of alfalfa by improving antioxidant enzyme activity and osmotic adjustment (Wang et al., 2016). The increase in enzyme activity and the increase in other active ingredients such as catalase (CAT) and peroxidase (POD) may also be factors in the increase in nutrients. Furthermore, the importance of Rhizobium to plants was self-evident. However, with the increase of salt stress, the differences between treatments were also significant. For Rhizobium, the most suitable part for growth was the root of the sputum. Methylobacterium was a genus of Proteobacteria that exhibits good colonization properties in a salt-stressed environment (Lee et al., 2015). Also, it contained some substances that can promote plants, such as IAA, cytokinin and so on (Madhaiyan et al., 2006).

In the severe salt stress treatment, the relative content of Exiguobacterium increased to 1.26%. Exiguobacterium, as a halophile, can grow in a high-salt environment and can produce protease, lipase, amylase, cellulase, mannanase, chitinase The presence of these enzymes can also help plants better adapt to halophytic environments (Johnson et al., 2017). It significantly increased the secondary metabolites in plants by inoculation of Exiguobacterium oxidotolerans on brahmi in a saline environment, and helped plants to effectively alleviate the ionic and osmotic stress caused by salt stress Bharti et al., 2013). Exiguobacterium has also been detected on corn under salt stress as a salt-tolerant bacteria. Its presence effectively helps alleviate the stress of corn in a salt stress environment (Aslam & Ali, (2018)). In this study, three strains of Methylobacterium, Rhizobium, and Exiguobacterium with significant differences were able to help the abiotic stress in the salt stress environment to a certain extent. The specific mechanism requires further research.