A Salt-Tolerant Streptomyces paradoxus D2-8 from Rhizosphere Soil of Phragmites communis Augments Soybean Tolerance to Soda Saline-Alkali Stress

Salinization-alkalization of soil is a serious threat worldwide, and the total area of saline soils exceeds 8.3 × 10⁸ hm² worldwide, including 53% alkaline soils and 47% saline soils. In China, it reaches 9.9 × 10⁷ hm². The Songnen Plain, an important plantation base of soybean production, is one of China's five largest salt-affected soil regions and has 3.42 × 10⁶ hm² salt soil (Huang et al. 2016). Salinity stress has detrimental effects on soybean growth and agronomy traits, such as nodulation, seed quality and quantity, and yield (Phang et al. 2008). Except for plant breeding and agricultural practices, applying a salt-tolerant bioagent may be an effective and sustainable means to enhance crop health and augment plant tolerance to salinity stress without environmental damage. To date, only several commercial bioagent products in the market have been applied in agriculture (Zeng et al. 2012; Panda et al. 2014). Moreover, the effect of the biological agent was influenced by variable conditions in the field (Chatterton and Punja 2010). Additionally, it is estimated that 50% of annual yield losses in major crops were caused by abiotic stress worldwide (Zörb et al. 2019). So, it is urgent to isolate more microorganisms with tolerance to various abiotic stress in the field, such as salt, alkaline, drought, etc., that could be applied as bioagents. Nowadays, some salt-tolerant plant growth-promoting rhizobacteria (PGPR) have shown great potential for alleviating salinity stress of many crops, such as Bacillus, Rhizobium, Pseudomonas, and others (Han et al. 2021). For soybean, Bacillus, Pseudomonas, and Rhizobacteria were reported to alleviate the harmful effects of salinity stress (Khan et al. 2019; Costa-Gutierrez et al. 2020; Abulfarajand Jalal 2021). To our best of knowledge, most PGPR were investigated under salt (NaCl) stress, and few actinobacteria were studied its potential to prime the alkaline or saline-alkali tolerance of soybean in soda alkaline soils. Alkaline or soda saline-alkali soils are composed primarily of NaHCO₃/Na₂CO₃ with excess Na⁺, HCO₃⁻/CO₃²⁻, high-pH (> 8.5), and poor soil structure imposes more damages on plant growth than saline soils. Hence, more microorganism resources with saline-alkali tolerance are essential to increase crop yields under soda saline-alkali stress.

Actinomycetes produce many bioactive compounds and have a great capacity to promote plant growth (Bhatti et al. 2017; Rehan et al. 2021). Nowadays, several commercial biocontrol agents are developed from actinobacteria, such as Actinovate based on Streptomyces lydicus, Mycostop based on Streptomyces griseoviridis, and Rhizovit based on Streptomyces sp. DSMZ 12424 (Lahdenperä et al. 1991; Berg et al. 2001; Minuto et al. 2006; Zeng et al. 2012). Actinobacteria can survive in various environments, such as saline or alkaline soils (Vasavada et al. 2006; Sadeghi et al. 2013). They are promising microorganisms to apply in abiotic stress environments for sustainable agriculture.

Phragmites communis (common reed), a tall perennial grass, is widely distributed in alkaline and saline soils of Songnen plain. The survival threshold of salt tolerance of the reed is 1%. It can grow normally in soil with a pH value of 6.5 ~ 9 and has strong salt and alkali resistance. Actinomycetes from rhizosphere soil of Phragmites were supposed to be salt and alkali tolerant and beneficial plant growth-promoting rhizobacteria. To test the above hypothesis and obtain soda saline-alkali tolerant PGPR to improve crop health in alkaline soils, the actinomyces were isolated from the rhizosphere soil of Phragmites, and antifungal activity of salt-tolerant strain D2-8 was screened. Soda saline-alkali tolerance was focused, and the survival ability of D2-8 in 0 ~ 120 mM mixed saline-alkali solution (NaCl, Na₂CO₃, NaHCO_3, and Na₂SO₄) was confirmed. Meanwhile, plant growth promotion characteristic of the strain was measured. Then, the polyphasic taxonomy analysis was carried out, and the genome was sequenced to reveal the antifungal, plant growth promotion, and saline-alkali tolerant mechanisms. Furthermore, pot and field experiments evaluated its soda saline-alkali tolerance improvement and growth promotion. This study provided the multifarious PGPR Streptomyces strain with soda saline-alkali tolerance, which has great potential to improve soybean productivity, and it is also an important source of new secondary metabolite substances.

Isolation of salt and saline-alkali tolerant actinomyces strain D2-8 with antagonistic activity. To obtain the biocontrol actinomyces resources with soda saline-alkali tolerance, we isolated 13 strains from the rhizosphere soil of P. communis using the dilution plate method. The antagonistic activity of the 13 strains (D2-1 ~ D2-13) against P. sojae displayed significant variation (0 ~ 28 ± 0.77 mm). Specifically, strain D2-8 significantly inhibited the mycelia growth of P. sojae by 28 ± 0.77 mm (62%) compared to the control and other strains. In addition, it also had the antagonistic activity against Fusarium and Gibberella (Table SI). So, strain D2-8 was further identified and studied in detail. We measured the resistance of D2-8 to abiotic stresses. It could grow in the presence of 0 ~ 10% NaCl (w/v) with an optimal level of 4% (w/v) (Fig. 1a). Furthermore, it could grow well in 10 ~ 80 mmol soda saline-alkali solution (NaCl, Na₂CO₃, NaHCO_3, and Na₂SO₄, molar ratio 1 : 1 : 9 : 9) except for the weak growth in 120 mmol saline-alkali solutions (Fig. 1b).

Fig. 1

Identification of strain D2-8. The morphological characteristics of strain D2-8 showed the typical characteristics of the genus Streptomyces. Strain D2-8 exhibited a white aerial spore mass and a grey substrate mycelium and formed chains of smooth spore. The spore (0.6 ~ 0.8 × 0.9 ~ 1.1 μm) was rhabditiform (Fig. 2a and 2b). The strain grew well and developed more abundant aerial mycelia on ISP 3 (oatmeal agar) and Czapek's agar (Table SII). The growth of strain D2-8 was observed at 15 ~ 40°C (optimum temperature, 28°C), and pH 5 ~ 9 (optimum pH 7). Detailed physiological characteristics are presented in Table I. Strain D2-8 could liquefy gelatin and produce H₂S and protease.

Fig. 2

The 16S rRNA gene sequence (1,487 bp) of strain D2-8 was closely related to members of the genus Streptomyces. The strain shared a high 16S rRNA gene sequence similarity of 99.4% to Streptomyces paradoxus NBRC 14887^T and Streptomyces hawaiiensis NBRC 12784^T. The neighbor-joining tree showed that strain D2-8 formed a distinct phyletic line and occupied a subclade with S. paradoxus NBRC 14887^T (Fig. 3). Based on morphological, physiological, and phylogenetic analysis, strain D2-8 was considered a species of the genus Streptomyces. The name of Streptomyces paradoxus D2-8 was proposed. It was deposited in the China Center for Type Culture Collection (Wuhan) CCTCC M2019949.

Fig. 3

Multiple plant growth promotion properties of strain D2-8. Stain D2-8 showed prominent properties for plant growth promotion. On the brilliant blue CAS plate, D2-8 could form an orange loop, which indicated the production of siderophore (Table II). It could produce major phytohormone indole acetic acid (IAA) in Gause No1 liquid medium with tryptophan (Table II), and the yield was 29.1 μg/ml. When testing for changes due to organic acids, the supernatant turned to yellow upon adding methyl red, which suggested D2-8 could produce organic acid (Table II). The ability of D2-8 to produce ACC deaminase was proved by the fact that it grew on the minimal salt medium supplemented with ACC (Table II). These results indicated that D2-8 produced IAA, organic acids, ACC deaminase, and siderophore, which likely contributed to the plant growth promotion by D2-8.

Identification of genes associated with plant growth promotion in the D2-8 genome. To reveal the molecular mechanism of antagonism and plant growth promotion, the D2-8 genome was sequenced. The assembled genome sequence of strain D2-8 was 8,732,707 bp long and composed of 72 contigs with N50 of 280,139 bp. The G+C content of the D2-8 genome was 71.15 mol %, and its coverage was 336×. The gene prediction resulted in 7,703 gene models. A total of 4,818 proteins were predicted (Table SIII). Annotation obtained via RAST assigned 18% sequences (1,504/8,377) to 24 subsystem categories and 82% sequences, not in subsystem. The high-ranking numbers in the subsystem categories were amino acids and derivatives, carbohydrates, protein metabolism, fatty acids, lipids, isoprenoids, respiration, nucleosides, and nucleotides (Fig. 4a). These subsystems belonged to the basic biological processes. Besides, the stress response and metabolism of aromatic compounds were relatively prominent.

Fig. 4

According to the functional annotation, various genes related to plant growth-promoting traits were found in the genome. Strain D2-8 can produce IAA with a maximal yield of 29.1 μg/ml. Several genes related to IAA biosynthesis were found in the genome of D2-8, such as the genes encoding for indole-3-glycerol phosphate synthase (contig1_962), phosphoribosyl anthranilate isomerase (contig1_953), and anthranilate phosphoribosyl transferase (contig1_852). Tryptophan 2-monooxygenase (contig19_5733) and amidase (contig20_5854, contig3_1725, contig3_1776, contig13_4655, contig17_5213) in IAA biosynthesis IAM pathway were found in the genome. Aldehyde dehydrogenase (NAD⁺) (contig7_3087, contig11_4153) in the IAA biosynthesis TAM pathway was also identified in the genome.

Additionally, genes of ammonia assimilation via both the GDH pathway using glutamate dehydrogenase (contig3_1507) and glutamine synthetase (GS)-glutamate synthase (GOGAT) pathway using glutamine synthetase (contig1_757, contig1_760, contig1_782, contig1_793, contig3_1528, contig3_1878) and glutamate synthase (contig1_209, contig1_975, contig1_976, contig1_1058) were identified. Furthermore, two genes encoding for 1-aminocyclopropane-1-carbo xy-late (ACC) deaminase (contig2_1144, contig8_3396) were found. These results were consistent with the potential ability of D2-8 to produce ACC deaminase.

Identification of genes responsible for the anti-fungal property in the D2-8 genome. Strain D2-8 had the potential to inhibit several plant fungi; therefore, the antiSMASH program and annotated genes sorting in the subsystem categories of RAST were performed to reveal the biologically active natural products. The anti-SMASH analysis predicted that strain D2-8 contains 23 individual clusters which share homology to known gene clusters whose metabolic products are known and seven clusters that show no similarities to known clusters (Table III). These different secondary metabolite gene clusters included four siderophores, two melanins, eight terpenes, one NRPS, two LAP, one ectoine, three bacteriocins, four T1PKS, two T2PKS, one T3PKS, one NRPS-like, and one PKS-like. Six clusters encoded the metabolites with antibacterial or antifungal activity, including collinomycin (72%) with antibacterial (enterococci), and no antifungal or antiviral activity, mediomiycin A (36%) with antifungal activity, calcimycin (10%) with inhibition of Gram-positive bacteria and some fungi, rustmicin (10%) with inhibition of elongation of germ tube of Puccinia graminis, ulleungmycin (8%) with antibacterial activity against Staphylococcus aureus, diazepinomicin (7%) with antibacterial, anticancer, and anti-inflammatory activity. Most such clusters displayed a low level of similarity to the known clusters. For antifungal metabolism, cluster 25 (Region 9.4) showed 36% similarity to the biosynthetic cluster of mediomiycin A in Kitasatospora mediocidica (Table III). It is a PKS gene cluster containing three PKS (contig 9_3944, contig 9_3945, contig 9_3946).

Cluster 15 (Region 3.2) and cluster 1 (Region 1) were predicted to produce siderophores (Table III). Cluster 15 showed 100% similarity to the biosynthetic cluster of coelichelin in Streptomyces coelicolor A3(2). Cluster 1 showed 83% similarity to the biosynthetic cluster of desferrioxamin B/desferrioxamine E in S. coelicolor A3(2). These genes further verified the ability of producing siderophores by strain D2-8.

Identification of genes responsible for salt stress adaptation of strain D2-8. For salt tolerance, several genes were found related to osmotic stress. Genes involved in production and uptake of choline and glycine betaine were found in D2-8, such as high-affinity choline uptake protein BetT, glycine betaine transporter OpuD, glycine betaine ABC transport system OpuAA, OpuAB, OpuAC, transcription regulator IclR family, choline dehydrogenase (BetA), and betaine aldehyde dehydrogenase (BetB) (Fig. 4b). These compounds could maintain membrane fluidity and enhance tolerance to salt (Guillot et al. 2000). D2-8 also contained an outer membrane protein A precursor gene. These genes were specific to D2-8, and absent from the genome of Streptomyces avermitilis MA-4680, S. coelicolor A3(2), and Streptomyces griseus subsp. griseus NBRC 13350.

Ectoine is a kind of compatible solute with protein-stabilizing properties. In addition, ectoine also stabilizes a higher-order nucleoprotein complex at the regulatory region of bacterial rRNA promoters (Pul et al. 2007). A group of microorganisms can synthesize the compatible solutes upon exposure to high salinity, including some Streptomyces species (Killham and Firestone 1984). In the strain D2-8 genome, there was an ectoine encoding gene cluster. Its ectoine biosynthetic gene cluster was highly like that of Streptomyces genus (Fig. S1). The compatible solutes ectoine and glycine betaine could influence (either as destabilizers or stabilizers) the melting temperature of the DNA helix (Rajendrakumar et al. 1997). These two substances both contributed to the salt tolerance of strain D2-8.

Plant growth-promoting activity of strain D2-8. D2-8 showed the potential to promote plant growth in the pot experiment, and the growth stage in the 10⁷ CFU/ml D2-8-treatment was earlier than that of control. The height of soybean seedlings increased by 17.6% at 12 d in the 10⁷ CFU-ml D2-8 treatment group compared to the control group (Fig. 5a). The treatment of strain D2-8 also affected the structure of the root. More lateral roots and longer main roots were observed. The root length increased significantly (21.3%) when the soybean seeds were soaked with 10⁷ CFU/ml D2-8 spore's suspension (Fig. 5b). Fresh weight of root and aboveground were significantly improved by 65.6% and 27.4% in 10⁷ CFU/ml D2-8 treatment (p < 0.05), respectively (Fig. 5c). These results implied that the concentration of 10⁷ CFU/ml of D2-8 spores had the best effect on plant growth promotion. So, 10⁷ CFU/ml of D2-8 spores were used further to investigate the plant growth under saline-alkali conditions. Compared with saline-alkali treatment (SA group), the D2-8 treatment increased the seedling's height of soybean under saline-alkali stress conditions (Fig. 5d and 5e). Additionally, the root length and fresh weight were improved by D2-8 treatment under saline-alkali stress conditions (Fig. 5f). The root length and fresh weight of aboveground increased by 44.1% and 37.5%, respectively. These results indicated D2-8 had the potential of alleviating the saline-alkali stress to promote soybean growth.

Fig. 5

In the field experiment, seeds coating of D2-8 also showed significant plant promotion. Soybean yield increased by 5.88% (Table IV). Above all, D2-8 has not only the potential to promote soybean growth and increase its production, but also this strain can augment the soybean tolerance to saline-alkali stress.

A diverse range of microorganism is dispersed in the rhizosphere of plant, and rhizosphere microbial communities play a crucial role in ensuring the stability and productivity of the agricultural ecosystem, including nutrition, disease suppression, and resistance to both biotic and abiotic stresses (Newitt et al. 2019). Lots of plants growth-promoting rhizobacteria have been isolated, and some were used as microbial agents and biofertilizers in agricultural production. Many actinomyces, especially the genus Streptomyces, were isolated from the plant rhizosphere and were known to enhance plant growth and protect plant health (Liu et al. 2019). Three Streptomyces strains have been developed into commercial biocontrol agents called Actinovate, Rhizovit, and Mycostop. P. australis is one of the most extensively distributed plant species used for phytoremediation of different types of wastewater, soil, and sediments. As a halophyte, the rhizosphere of P. australis harbored some new and vital microbial groups, which are of great importance in the vegetation restoration and ecological reconstruction of salinized soil. Although the bacterial community of rhizosphere of P. australis was revealed and some rhizobacteria were isolated (He et al. 2019), few Streptomyces with alkaline tolerance from the rhizosphere of P. australis were isolated (Pereira et al. 2015). In this study, Streptomyces strain D2-8 was isolated from the rhizosphere of Phragmites, which grows in the saline and alkaline land. It exhibited a broad antifungal activity against the pathogen of soybean rot diseases, such as P. sojae and Fusarium. Moreover, strain D2-8 showed moderate saline and alkaline stress tolerance. It can grow in the 10% NaCl and 120 mM soda saline and alkaline solution. Some Streptomyces strains also showed salt tolerance, such as Streptomyces sparsus sp. nov. (0–15% NaCl, pH 6–10.0) (Jiang et al. 2011), and Streptomyces clavuligerus strain Mit-1 (up to15% NaCl, pH 11.0) (Thumar and Singh 2009). However, their tolerance to soda alkaline solution was not measured. Although D2-8 was not a halophilic bacterium, its tolerance to saline and alkaline and antifungal activity make it a promising microorganism to apply in alkaline land to protect crops against disease and improve plant production.

The beneficial microorganism can promote plant growth through nitrogen fixation, potassium solubilization, and phosphorus solubilization; meanwhile, it produces siderophores, IAA, and ACC deaminase to enhance plant stress tolerance. In genus Streptomyces, two antagonistic Streptomyces WZS1-1 and WZS2-1 produced IAA, siderophores, solubilized phosphorus and potassium, harbored nitrogenase activity, and can promote wheat growth (Han et al. 2018). Streptomyces sp. NEAU-S7GS2 also produces IAA, siderophores, ACC deaminase, and inoculation of NEAU-S7GS2 showed the potential of growth promotion in soybean (Liu et al. 2019). In the current study, D2-8 was confirmed to produce IAA, siderophore, and organic acid. Its ability of growth promotion was further measured in soybean. D2-8 spores can promote the root length under normal conditions and the saline and alkaline condition in pod experiments. Moreover, seeds coated with D2-8 can increase soybean production in field experiments. The plant growth promotion of strain D2-8 under saline and alkaline condition would be valuable for agricultural production.

The genome analysis of D2-8 gave insight into its plant growth promotion mechanism. Multiple siderophores biosynthetic and uptake systems identified in this strain suggested functional duplication conferring an advantage for the bacterium as it colonizes different ecological niches. In this study, IAA biosynthesis genes were also found in D2-8, and IAA production of D2-8 consisted with the analysis results in silico. The plant intake of IAA released by Streptomyces was one of the reasons for soybean growth promotion. Streptomyces’ saprotrophic and spore-forming lifestyle is beneficial to survive well under unfavorable conditions. The detailed mechanism of plant growth promotion of Streptomyces D2-8 under saline and alkaline stress needs further research.

Streptomyces spp. have a high performance in producing various bioactive secondary metabolism and enzymes (Zhang et al. 2017). The genomic analysis of D2-8 identified many PKS and NRPS gene clusters. Six clusters encoded the metabolites with antibacterial or antifungal activity, but the similarity of gene cluster was low, which indicate that the strain may produce some new metabolisms. Although three PKS enzymes were found, whether D2-8 produced the mediomycin or its similar antibiotics still need more experiments. Moreover, RAST analysis results revealed that production and uptake of choline and glycine betaine genes in the strain genome is uniquely present in D2-8 but absent from the related Streptomyces complete genome of S. avermitilis MA-4680, S. coelicolor A3(2), and S. griseus subsp. griseus NBRC 13350. We proposed that ectoine, choline, and glycine betaine are particularly important for adaptation to saline and alkaline stress. Altogether, the presented data demonstrate that strain D2-8 is a moderate saline and alkaline stress tolerance streptomyces with the potential of plant growth promotion and antifungal activity and streptomyces to find novel bioactive compounds.

Saline and alkaline tolerant actinomyces, designated as S. paradoxus D2-8, was isolated from rhizosphere soil of Phragmites, which grown in the saline and alkaline land. Except for the antifungal activity, the strain has significant saline-alkali tolerance and plant growth promotion. Moreover, genome analysis showed that IAA and siderophore biosynthesis pathway, genes encoding ACC deaminase, and secondary metabolite gene clusters were identified in the D2-8 genome. Soybean growth promotion experiments further confirmed that the strain improved plant tolerance to saline and alkaline stress and promoted host health. The yield increase of soybean after D2-8 seed's coating treatment in the field suggested that strain D2-8 could be a potential candidate for the development of bioagent with abiotic stress tolerance used in sustainable agriculture in the future.