Isolation, identification, and growth-promotion function of Streptomyces sp. in wetland rhizosphere

doi:10.21203/rs.3.rs-2112434/v1

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Isolation, identification, and growth-promotion function of Streptomyces sp. in wetland rhizosphere

https://doi.org/10.21203/rs.3.rs-2112434/v1

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Wetlands are the most biologically diverse ecosystems on Earth. The isolation of Streptomyces strains from wetlands is helpful to study their diversity and functions in such habitats. In this study, six strains of Streptomyces were isolated from the rhizosphere soil of three plant species in Huaxi Wetland of Guiyang and were identified as Streptomyces galilaeus, S. avidinii, S. albogriseolus, S. hydrogenans, S. spororaveus, and S. cellulosae, respectively. The six strains all solubilized phosphate, fixed nitrogen, and produced ACC deaminase and siderophores, and four strains also secreted indole-3-acetic acid. The six strains grew well in the ranges of 0%–3% NaCl, 0%–25% PEG, and pH 5.0–10.0, which showed that the screened strains were tolerant to certain degrees of salinity, drought, and acidic/alkaline pH stress. In addition, the S. avidinii WL3 and S. cellulosae WL9 strains (especially the former) significantly promoted seed germination of mung bean, pepper, and cucumber. A pot experiment further showed that WL3 significantly promoted the growth of cucumber seedlings compared with that of the control, with plant height, root length, lateral root number, and fresh weight increased by 104.99%, 49.99%, 94.12%, and 82.86%, respectively. Thus, strains of six species of Streptomyces with multiple plant growth-promoting characteristics were isolated from the wetland. These results lay a foundation for their potential use as microbial agents and seed-coating treatments.

Wetland

Streptomyces

Isolation and identification

Growth-promoting effect

Stress resistance

In recent years, plant-growth promoting rhizobacteria (PGPR) have become an important research focus owing to their many growth-promoting characteristics, such as the production of organic acids, siderophores, plant hormones, and extracellular polysaccharides, and induction of enhanced plant stress tolerance (Grover et al. 2011). Species of Acetobacter, Actinomyces, Azospirillum, Bacillus, Burkholderia, Lactobacillus, Paenibacillus, Pseudomonas, Rhizobium, Serratia, and other PGPR genera improve crop yields, and several strains have been commercialized (Backer et al. 2018). Streptomyces is an actinomycete genus with unique mycelial morphology, and the abundant secondary metabolites synthesized are a rich source of natural bioactive products, medicinal chemicals, and new drugs (Lacey & Rutledge 2022). Members of the genus produce an abundance of antibiotics and plant growth hormones, inhibit pathogens, promote plant growth, and improve plant tolerance to biotic and abiotic stresses (Kim et al. 2011). Streptomyces sp. GMKU3100, isolated from rice by Rungin et al. (2012), increases the length and biomass of roots and shoots. Streptomyces sp. RP1A-12, isolated from peanut rhizosphere soil, can promote peanut seed germination, and increases stem and shoot length (Jacob et al. 2018). Greenhouse and field experiments have shown that S. rimosus C-2012 and S. monomycini C801 isolated from wheat fields increase the fresh and dry aboveground biomass of mint (Esmaeil et al. 2019). Under waterlogging stress, inoculation with Streptomyces sp. MM10 isolated from the wheat rhizosphere effectively increases the content of peppermint oil (Aly et al. 2012). However, most of these strains were isolated from well-ventilated soil environments, sediments, seawater, and freshwater systems (Seipke et al. 2012; van der Meij et al. 2017).

A wetland is a transitional zone between land and water, which contains both animal and plant resources. Therefore, wetlands are the most biologically diverse ecosystem on Earth and are often referred to as the “kidneys of the Earth.” Wetland plant roots contain unique physiological structures that can utilize light, water, and nutrients, thus attracting countless heterotrophic biota, and their interaction with the surrounding environment determines the ecological function of vegetation wetlands. In addition, the biota in the rhizosphere of wetland plants is unique and more abundant compared with that in most flooded soils (Neori & Agami 2017). However, current research on wetlands often focuses on microbe-mediated treatment of environmental pollution or characterization of microbial populations and communities, whereas few studies investigate the function of plant rhizosphere microorganisms (Jessica et al. 2006). The climate and natural resources of Guizhou province are especially suitable for the growth of actinomycetes. Therefore, in this study, we isolated and screened multiple species of Streptomyces from the rhizosphere soil of three plant species in Huaxi Wetland, Guiyang. We measured their plant growth-promoting and growth characteristics, and analyzed their growth-promoting effects by means of seed germination and pot experiments. The results will enrich available Streptomyces resources with growth-promoting functions, and lay a foundation for an improved understanding of the ecological functions and growth-promoting mechanisms of Streptomyces species.

Isolation, screening, and identification of Streptomyces strains

Rhizosphere soils of canna (Canna indica), reed (Phragmites australis), and rice (Oryza sativa) were collected from Huaxi Wetland Park in Guiyang (26°26′34.29″N, 106°40′43.22″E), Guizhou, China. The soils were shaken in sterile saline containing glass beads and soil suspensions of different concentrations were generated using a double dilution method. The appropriate dilution was spread on solid Gao’s No. 1 medium and cultured at 28 °C for 10 days. Different forms of single colonies were selected and purified using a streaking method and were preserved (Li et al. 2020). The isolated strains were inoculated on multiple types of agar medium (including glucose yeast extract medium, yeast medium solution (YMS) medium, Kirschner synthesis No. 1 medium, Czapek’s medium, glucose asparagine medium, starch medium, and Gao’s No. 1 medium), and cultured at 28 °C for 5 days. The colony morphological characteristics and soluble pigment production of each strain were observed, and mycelial morphology was observed using the coverslip insertion method. Physiological and biochemical tests for bacterial identification were conducted in accordance with Bergey’s Manual of Determinative Bacteriology (Buchanan & Gibbons 1984), Classification Technology of Actinomycetes (Guan et al., 2016), and other relevant literature (Guo et al. 2014; Khanna & Solanki 2012; Rani et al. 2018; Qin et al. 2009; Sheng et al. 2015). Molecular identification was performed by sequencing the 16S rRNA gene. Total genomic DNA was extracted from each strain using the E.Z.N.A. Bacterial DNA Kit (Omega Bio-Tek, Norcross, GA, USA). PCR amplification of the 16S rRNA gene was performed using the bacterial universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CTACGGCTACCTTGTTACGA-3′). Reactions were performed in a 25 μL volume containing 1 μL genomic DNA, 12.5 μL of 2´ Taq PCR Mastermix, 1 μL each primer, and 9.5 μL ddH₂O. The amplification protocol was as follows: pre-denaturation at 94 °C for 5 min, then 30 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 45 s, and extension at 72 °C for 90 s, and final extension at 72 °C for 10 min (Lane et al. 1985). The PCR products were sent to Shanghai Lifei Biotechnology Co., Ltd. (Shanghai, China) for sequencing. The sequences were compared to those deposited in GenBank using the BLASTn search tool and in the EzBioCloud database. The type strains with high homology to the sequences were selected, and a dendrogram was constructed using the neighbor-joining method with MEGA 7.0 software.

Determination of plant growth-promoting and growth characteristics of Streptomyces strains

Preparation of spore suspension and determination of spore number

The isolated strains were inoculated into liquid Gao’s No. 1 medium and activated at 28 °C and 150 rpm for 3 days, then transferred to an inclined plane of Gao’s No. 1 medium and cultured at 28 °C for 5 days. A small volume of sterile water was applied to allow the spores to be scraped off. The spore suspension was placed in a sterile 50 mL triangular flask containing glass beads and filtered with sterilized absorbent cotton after sufficient oscillation. The filter residue was washed with sterile water two or three times to obtain the spore suspension. A sample (10 μL) of the suspension was added to the counting chamber of a hemocytometer, covered with a glass slide, and allowed to stand for 5 min before the spores were counted under a microscope.

Determination of 1-aminocyclopropane-1-carboxylic acid deaminase activity

The spore suspension (1.0 × 10⁷/mL) of each isolated strain was inoculated into a test tube containing 15 mL tryptic soy broth culture medium and cultured with shaking for 24 h under identical conditions with three replicates. The culture was centrifuged at 3,500 ´ g for 20 min. The bacterial cells were collected and washed twice with DF medium. The pellet was suspended in ADF medium for 24 h until the strain was induced to produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. After centrifugation, the pellet was washed twice with 5 mL of 0.1 mol/L Tris-HCl (pH 7.6) buffer, and frozen at −20 °C for 2 h. The frozen bacterial cells were suspended in 1200 μL of 0.1 mol/L Tris-HCl (pH 8.5) buffer and 108 μL of 20 mg/mL lysozyme was added. After incubation in a water bath at 37 °C for 1 h, 30 μL toluene was added and the mixture was shaken quickly to obtain a crude enzyme solution. The concentration of bacterial protein was determined using the Bradford method. The α-ketobutyric acid content in the crude enzyme solution was determined using the 2,4-dinitrophenylhydrazine method. The ACC deaminase activity was expressed as the number of micromoles per milligram bacterial protein that catalyzes ACC to form α-ketobutyric acid per hour (Jaemsaeng et al. 2018).

Determination of indole-3-acetic acid secretion

Following the method of Han et al. (2018), the spore suspension of each activated strain was inoculated into 100 mL liquid Gao’s No. 1 medium containing 0.05 g tryptophan and cultured with shaking for 5 days under identical conditions. The culture was centrifuged at 3,500 ´ g for 25 min. The supernatant was mixed with the same volume of Salkowski reagent and incubated at room temperature for 30 min in the dark. When indole-3-acetic acid (IAA) was produced and the solution assumed a pink color, the absorbance was immediately measured at 530 nm with a spectrophotometer. The supernatant of non-inoculated medium was used as a control for calibration. The content of IAA per unit volume of culture was calculated based on a standard curve.

Determination of phosphorus solubilization capability

Molybdenum blue colorimetry (Elizabeth et al. 2007) was used to determine the phosphorus solubilization ability of each strain. The spore suspension of the activated strain was inoculated into 100 mL National Botanical Research Institute’s phosphate growth medium (NBRIP) and cultured under identical conditions for 7 days. The culture was centrifuged at 3,500 ´ g for 25 min. After mixing the supernatant with the colorimetric solution, the absorbance was measured at 660 nm. The supernatant of the non-inoculated medium was mixed with the colorimetric solution as a blank control. The soluble phosphate content was calculated based on a standard curve.

Determination of siderophore production capability

For qualitative determination of the siderophore production ability of each strain, the method of Han et al. (2018) was used. Four sterile filter paper discs of diameter 6 mm were placed on a chrome azurol S (CAS) blue detection plate, the spore suspension was inoculated onto its surface, and then cultured at 28 °C for 5 days. Development of an orange transparent ring around the colony indicated that the strain produced siderophores. For quantitative determination, single colonies of the selected strains were cultured in liquid MKB medium for 48 h and the culture was then centrifuged at 3,500 ´ g for 15 min. The supernatant was mixed with the same volume of CAS detection solution and the OD₆₃₀ absorbance (A_s) was measured at room temperature for 1 h. The supernatant of the non-inoculated medium was used as a reference (A_r) to calculate the relative content of siderophore units (SU; %) as (A_r − A_s)/A_r × 100 (Machuca & Milagres 2003).

Determination of potential nitrogen fixation ability

The activated strain was inoculated on nitrogen-free solid medium and cultured at 28 °C for 7 days. The strains that were capable of growth were transferred to fresh medium, and if the strain was capable of growth after five generations of serial cultivation, it was determined that the strain had potential nitrogen fixation activity (Zhang et al. 2021).

Determination of stress tolerance

The salt tolerance, acidic and alkaline pH tolerance, and drought tolerance of the strains were assessed. The activated strain was streaked on the surface of Gao’s No. 1 plates containing different concentrations of NaCl (0%–5%) and cultured in an incubator at 28 °C for 7 days for observation of growth. In addition, the strains were inoculated on Gao’s No.1 plates of different pH (4.0–10.0) to observe their acidic and alkaline pH tolerance. The drought tolerance of the strains was evaluated by inoculating the strains on solid plates containing different concentrations (0%–35%) of polyethylene glycol 6000 (PEG-6000) (Wang et al. 2019).

Effects of Streptomyces strains on seed germination and seedling growth of vegetables

Uniform seeds of mung bean (Vigna radiata) and pepper (Capsicum annuum) were surface sterilized by soaking in 2% sodium hypochlorite solution for 10 min, and cucumber (Cucumis sativus) seeds were soaked in 10% sodium hypochlorite solution for 10 min. The seeds were rinsed with a large volume of sterile water and then soaked in the spore suspension (1.0 × 10⁷/mL) of the six Streptomyces strains for 2 h. Sterile water was used as the control. Sterile filter paper was used to absorb the bacterial liquid on the seed surface and the seeds were then placed in a sterile petri dish with two layers of moist sterile filter paper. Three replicates were performed for each treatment, with 20 mung bean seeds, 50 pepper seeds, and 10 cucumber seeds per replicate. Germination was conducted in a light incubator at 28 °C and the filter paper was kept moist. The number of germinated seeds was recorded every 24 h, with a radicle length of 2 mm deemed to characterize germination. The germination rate (GR; %) was calculated with the formula GR = (number of all normal germinated seeds at the end of the germination period/number of seeds for inspection) × 100. The vigor index (VI) was calculated as VI = S × ∑(G_t/D_t), where G_t is the number of germinated seeds per day corresponding to D_t, D_t is the germination time, and S is the seedling root length in a certain period (mm). Germinated seeds were randomly selected from each replicate and the root length, stem length, and fresh weight were measured. Combined with the seed germination data, the strain with the strongest effect on seed germination was selected for the seedling growth experiment. In this pot experiment, the vegetable seeds were sterilized and germinated, and then transplanted into pots containing sterile soil for cultivation, with one seedling per pot. At the two-leaf stage, seedlings of similar growth were selected for irrigation treatment; the experimental group was irrigated with 30 mL spore suspension (1.0 × 10⁷/mL) per pot, whereas the control group was irrigated with 30 mL Gao’s No. 1 liquid solution per pot. All seedlings were irrigated once every 3 days. After 15 days, physiological indicators, such as plant height, root length, lateral root number, and fresh weight, were measured to assess the growth-promoting effect of each Streptomyces strain (Horstmann et al. 2020).

Isolation, purification, and identification of Streptomyces strains

Six Streptomyces strains were isolated, of which the WL1 strain was isolated from the rice rhizosphere, WL3 and WL8 were obtained from the reed rhizosphere, and WL4, WL5, and WL9 were isolated from the canna rhizosphere. Their morphology and colony characteristics showed obvious diversity (Fig. 1). On Gao’s No. 1 medium, the aerial mycelium of the WL1 strain was white-gray and the basal mycelium was red, and the colony was small and compact, smooth, and dry with pink-brown pigment. The aerial mycelium of the WL3 strain was grayish white, the substrate mycelium was white, the colony was dry, and the edge was white villiform. The aerial mycelium and substrate mycelium of strain WL4 were white, the colony was dry, the middle depression was green, fluffy, and the edge was radial. The mycelium of the WL5 strain was yellow-white, and the colony was dry and with concentric ring powder. The aerial mycelium of the WL8 strain was gray-white, the basal mycelium was white, the colony was dry, gray, convex, small and compact, and the edge was white. The aerial mycelium of strain WL9 was yellow-white, the substrate mycelium and the soluble pigment were yellow-green, and the colony was dry, fluffy, and the edge was translucent. The spore filaments of the strains were observed by the coverslip insertion method. The spore filaments of the six strains were all clustered, curved, or wavy (Fig. 1). The culture characteristics of the strains in different media are summarized in Table 1 and Fig. 2. The WL1, WL3, and WL8 strains produced brownish-black pigment when cultured on glucose yeast extract and YMS agar medium. The WL9 strain produced yellow-green soluble pigments on Kirschner synthesis No. 1 and glucose asparagine medium, and pink soluble pigments on starch agar medium. The other strains did not produce pigments. Thus, the strains each exhibited unique morphological characteristics on different media.

Analysis of the physiological and biochemical characteristics (Table 2) revealed that the WL1 strain could not utilize L-arabinose and D-mannitol. The WL3 strain could not utilize L-rhamnose, D-fructose, and raffinose, and showed weak utilization of D-glucose, L-arabinose, and D-mannitol, and the other reactions of these two strains were positive. The WL4 strain showed weak utilization of L-arabinose, the WL4, WL5, and WL9 strains were negative for hydrogen sulfide and melanin utilization, whereas the other reactions were positive. The WL8 strain utilized raffinose and D-mannitol, produced hydrogen sulfide and melanin, and promoted nitrate reduction and starch hydrolysis, but other reactions were negative.

The total genomic DNA of each strain was extracted, and the 16S rRNA gene was amplified by PCR and sequenced. The amplified sequence for each strain was compared against the EzBioCloud and GenBank databases, and a neighbor-joining dendrogram was constructed (Fig. 3). The placement of the six actinomycete strains in the tree was consistent with the species that showed the highest sequence similarity (Table 3). Thus, strains WL1, WL3, WL4, WL5, WL8, and WL9 were identified as S. galilaeus, S. avidinii, S. albogriseolus, S. hydrogenans, S. spororaveus, and S. cellulosae, respectively, based on morphological, physiological, and biochemical characteristics and the molecular analyses.

Growth-promoting characteristics and stress tolerance of the six Streptomyces strains

The growth-promoting characteristics of the six strains of Streptomyces were assessed (Table 4). All six strains produced ACC deaminase and solubilized phosphorus. Among the strains, the WL1 strain had the highest capacity for these two properties. The WL5 strain secrete IAA of up to 164.548 μg/mL, whereas WL4 and WL8 did not secrete IAA. After the strains were inoculated on the CAS blue detection plate, orange transparent circles were observed around the colonies of the six strains, thus all had the ability to produce siderophores (Fig. 4). Quantitative determination showed that the WL4 and WL5 strains had the strongest abilities to produce siderophores. The six strains grew well on nitrogen-free solid medium (Fig. 5), showing that all strains had strong nitrogen fixation ability.

The stress tolerance characteristics of each strain are summarized in Table 5. With increase in salt concentration and PEG-6000 concentration, the stress tolerance of each strain gradually weakened and the colony morphology became smaller, whereas the WL3, WL4, WL5, and WL8 strains showed stronger alkali tolerance (Table 5). The WL5 strain had the strongest stress tolerance overall and was able to grow well under the conditions of 0%–5% NaCl, pH 5–10, and 0%–15% PEG-6000, followed by the WL4 and WL8 strains. The WL1 strain showed the weakest stress tolerance.

Effects of Streptomyces strains on seed germination and seedling growth of vegetables

Considering the growth-promoting and stress-tolerant properties, the WL1, WL3, WL5, and WL9 strains with multiple growth-promoting properties were selected for seed germination experiments. With the exception of the WL1 strain, the other three strains stimulated increase in the VI, root length, and fresh weight of mung bean seedlings to varying degrees. The WL3 strain had the most distinct effect on promoting germination of mung bean seeds, and the root length of seedlings increased by 36.70% compared with that of the control (P < 0.05) (Table 6). The WL3 and WL9 strains had favorable effects on pepper seeds and seedlings; the WL3 strain increased the germination frequency (6.87%), VI (18.29%), root length (15.38%), and fresh weight (11.76%). With regard to the effect on cucumber seeds and seedlings, the four strains increased the VI, root length, and fresh weight to different degrees. The WL3 strain significantly increased the VI and root length of cucumber seedlings (P < 0.05), which increased by 71.00% and 27.10% compared with those of the control. These results indicated that the WL3 strain had the strongest growth-promoting effects on seed germination and seedling growth.

The pot experiment of cucumber seedlings inoculated with the WL3 strain showed that this strain could significantly promote the growth of cucumber seedlings (Fig. 6). Specifically, compared with the control group, the WL3 strain increased the plant height, root length, lateral root number, and fresh weight of seedlings by 104.99%, 49.99%, 94.12%, and 82.86%, respectively (Table 7).

Actinomycetes are widely distributed with more than 40 species known. Metabolites of actinomycetes have the effect of inhibiting pathogens and promoting plant growth, which has become an important research focus. Of the metabolites that exhibit these activities produced by microorganisms, actinomycetes account for 45%, fungi for 38%, and unicellular bacteria for 17% (Mahyarudin et al. 2015). Streptomyces spp. are dominant because they are widely distributed and are adaptable to diverse environments, which contributes to their ability to compete with other microorganisms (Sreevidya et al. 2016). Streptomyces is known to be a potential biocontrol agent that produces a variety of bioactive metabolites (Nimaichand et al. 2013). Members of this genus have also been reported to play a role in promoting plant growth in recent years. For example, S. griseoluteus WT can produce relatively high levels of polyamines (putrescine, spermidine, and spermine), which can promote increases in the root and shoot fresh weight, dry weight, and length of legumes grown in sandy soil (Nassar et al. 2003). Streptomyces caviscabies, S. globisporus subsp. caucasicus, and S. griseorubens can produce cellulase, hydrocyanic acid, and IAA, which significantly increase the tiller number, panicle number, filled grain number, and grain weight of rice (Gopalakrishnan et al. 2014). Streptomyces filipinensis can produce ACC deaminase and IAA, and promotes increase in tomato root length, fresh weight, and plant height (El-Tarabily 2008).

The microbial community in wetland soils is rich. In the present experiment, six strains of actinomycetes were isolated and screened, which were identified as S. galilaeus WL1, S. avidinii WL3, S. albogriseolus WL4, S. hydrogenans WL5, S. spororaveus WL8, and S. cellulosae WL9. At present, in addition to S. hydrogenans, previous reports on Streptomyces spp. have focused on antibiotics production and bactericidal effects. It has been reported that S. galilaeus can produce anthracycline antibiotics of medicinal value and is a causative pathogen of potato common scab (Bartel et al. 1990; Zhang et al. 2020). Streptavidin secreted by S. avidinii can bind to the coenzyme factor biotin as a molecular marker, protect its environmental niche, and inhibit the invasion of plant pathogens (Laitinen et al. 2021). Streptomyces albogriseolus can use polyethylene as the sole carbon source, degrade the toxic herbicide sulfosulfuron, act as an antibacterial agent, and produce an active ingredient with strong nematicidal activity (Samundeeswari et al. 2012; Zeng et al. 2013; Arya et al., 2016; Shao et al. 2019). The high-molecular-weight antibiotic AF1 produced by S. spororaveus strain has antibacterial activity against certain fungi and G⁺ bacteria (Chang et al. 2022). Streptomyces cellulosae can produce potent compounds with antioxidant activity and inhibit the growth of Sclerotium rolfsii mycelia (Rani et al. 2018; Abo-Zaid et al. 2021). However, little information is available on the growth-promoting effects of these strains. This is the first report that strains of S. galilaeus, S. avidinii, S. albogriseolus, S. spororaveus, and S. cellulosae have multiple growth-promoting characteristics. The present results indicate that the Huaxi Wetland contained abundant Streptomyces resources with growth-promoting characteristics.

Streptomyces hydrogenans has been used as a plant growth promoter, photosynthetic pigment enhancer, and biocontrol agent. For example, S. hydrogenans DH16 produces IAA (36 μg/mL), secretes ACC deaminase (0.363 μmol/(mg·h)) and biofungicides, promotes the germination and seedling growth of tomato, mung bean, and pea, and reduces invasion of the plant-parasitic Meloidogyne incognita (Sharma et al. 2020; Kaur & Manhas 2022). Compared with the DH16 strain, the S. hydrogenans WL5 strain isolated in the present research had stronger growth-promoting ability and also promoted the germination of mung bean and cucumber seedlings, but did not show growth-promoting effects on pepper seedlings, which may be associated with the interaction between rhizosphere microorganisms and plants. IAA has been identified as an effector molecule in plant–microbe interaction, and both interacting parties regulate their ability to produce IAA in accordance with the respective content of IAA produced (Ahemad & Kibret 2014). Streptomyces sp. KLBMP 5084 secretes 8.24 μg/mL IAA, which promotes seed germination and seedling growth of Limonium sinense (Qin et al. 2014), whereas Streptomyces sp. CLV45 secretes as much as 398.53 μg/mL IAA, which promotes the growth of soybean plants (Horstmann et al. 2020). In the present study, S. hydrogenans WL5 produced the highest IAA quantity (164.548 μg/mL), followed by S. cellulosae WL9, which could secrete 49.55 μg/mL of IAA. However, the promotion of germination of three vegetable seeds was weaker than that of S. avidinii WL3 (23.119 μg/mL of IAA). Therefore, for different plant species, inoculation with a PGPR strain that produces an appropriate IAA concentration may achieve the optimal stimulation effect.

Similar to most rhizosphere growth-promoting bacteria, the mechanism by which Streptomyces promotes plant growth predominantly involves phosphorus solubilization, nitrogen fixation, siderophore secretion, and plant hormone production. The siderophore is a low-molecular-weight organic compound with more than 500 types. Based on the characteristics of the chelating group, siderophores can be divided into catechol esters, hydroxamates, and α-hydroxycarboxylate esters, which combine with Fe³⁺ to form a soluble iron–siderophore complex for the plant or self-absorption (Hider & Kong 2010). The strain Streptomyces sp. GMKU3100 produces abundant siderophores, which chelate Fe³⁺ and competitively inhibit pathogen growth, and are able to improve the growth of rice and mung bean seedlings (Rungin et al. 2012). In the current study, the strains WL4, WL5, and WL9 had strong ability to secrete siderophores, and WL5 and WL9 had certain germination-promoting effects on mung bean and cucumber seeds. Subsequent determination of the anti-pathogenic activity of these three strains may expand their potential utilization.

Ethylene is an important regulator of plant developmental processes, such as seed germination, senescence, stress signal transduction, and root elongation. Under abiotic stress, high concentrations of ethylene inhibit root and shoot proliferation and promote leaf senescence, thereby hindering plant growth and development (Nazar et al. 2014). ACC deaminase decomposes the ethylene precursor ACC into α-ketobutyric acid and ammonia, which reduces the damage of ethylene to plants and promotes plant growth. In addition, PGPR, which produce ACC deaminase, can further use ACC as a carbon and nitrogen source, which lays the foundation for the regulation of healthy growth of plants under stress (Orozco-Mosqueda et al. 2020). Although PGPR containing ACC deaminase also secrete auxin, gibberellin, and other substances, the existence of ACC deaminase activity is the main mechanism to improve plant tolerance to stress, and it has been reported that PGPR containing ACC deaminase can improve the salt tolerance of tomato, peanut, rape, wheat, and other crops (Wang et al. 2021). For example, Pseudomonas syringae S1 and P. fluorescens S2 harboring ACC deaminase activity significantly increase maize yield and nutrient uptake under drought and soil salinization stress (Zafar-ul-Hye et al. 2014). The acdS gene encoding ACC deaminase has been gradually mined, and rice inoculated with an acdS deletion mutant of P. stutzeri A1501 shows lower tolerance to salt or heavy metals than the wild type (Han et al. 2015). The acdS gene cloned from a PGPR strain shows potential as a stress-resistant alternative to transgenic plants (Naing et al. 2021). The six strains of Streptomyces isolated in the present study all had the ability to secrete ACC deaminase, were capable of growth in the ranges 0%–3% NaCl, 0%–25% PEG-6000, and pH 5.0–10.0, and each showed distinctive tolerance properties. Among the strains, WL4, WL5, and WL8 showed superior growth under the tested salt concentrations, pH range, and PEG concentrations, and thus show potential for their utilization as microbial agents for application under stress conditions.

Seeds are vital for efficient growth and development of plants. To improve the seed germination percentage and survival of seedlings, seed priming technology has gradually emerged. Bio-priming is a new technology by which seeds are soaked in a PGPR culture solution to initiate physiological processes within the seeds and enhance bacterial abundance in the seed body, thereby improving seed germination, vigor, root growth, and stress tolerance (Miljaković et al. 2022). Kthiri et al. (2021) reported that wheat seeds treated with Meyerozyma guilliermondii INAT showed enhanced development and photosynthesis, and decreased flavonol and anthocyanin contents, whereas Bacillus subtilis 10-4 alleviated the negative effects of drought stress on wheat seed germination and seedling growth (Lastochkina et al. 2020). Inoculation with S. cyaneus ZEA17I, S. anulatus CMJ58I, and S. albidoflavus VT111I significantly promoted radicle development of roquette and tomato seeds, and all inoculated strains were able to colonize and grow on the seed surface after 24 h (Kunova et al. 2016). Streptomyces has a positive effect on seed germination owing to its mycelium morphology, which enables it to quickly colonize plants and produce a large number of beneficial secondary metabolites (Bonaldi et al. 2015; Boruta 2021). The six strains of Streptomyces sp. isolated in the present study, except the WL4 and WL8 strains, did not produce IAA, but had multiple growth-promoting characteristics. The S. avidinii WL3 strain significantly improved the germination frequency, VI, root length, stem length, and fresh weight of mung bean, pepper, and cucumber seedlings, and significantly promoted the growth of cucumber seedlings. Thus, the WL3 strain showed considerable potential as a candidate for utilization as a biological fertilizer and seed coating.

In this study, six strains were isolated from the rhizosphere soil of the wetland plants reed, canna, and rice. The strains were identified as S. galilaeus WL1, S. avidinii WL3, S. albogriseolus WL4, S. hydrogenans WL5, S. spororaveus WL8, and S. cellulosae WL9, which revealed the diversity of Streptomyces in wetlands. All six strains shared the characteristics of secreting ACC deaminase, phosphorus solubilization, nitrogen fixation, and siderophore production. With the exception of WL4 and WL8, the remaining four strains secreted IAA. The isolated strains were able to grow under 3% NaCl, 25% PEG, and pH 10.0, and showed certain stress tolerances. Thus, the wetland-isolated Streptomyces strains had multiple growth-promoting characteristics and strong environmental adaptability. The WL3 and WL9 strains significantly promoted the germination of mung bean, pepper, and cucumber seeds, especially WL3. Moreover, WL3 significantly increased the root length of mung bean and cucumber seedlings after germination, and an inoculation experiment showed that the WL3 strain significantly promoted the increase in plant height, root length, lateral root number, and fresh weight of cucumber seedlings. Therefore, S. avidinii WL3 strain shows superior growth-promoting effects and has potential for utilization as a microbial agent.

Acknowledgment

This study was funded by the National Natural Science Foundation of China (32060028). We thank Robert McKenzie, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript.

Funding: National Natural Science Foundation of China (32060028).

Confilcts of interest: The authors declare no conflict of interest.

Availability of data and material: All data generated or analysed during this study are included in this published article.

Code availability: Not applicable

Authors’ contributions: Lizhen Han is the corresponding author, Hong Zhang, Xue Bai and Yujie Han are Lizhen Han’s graduate students. Lizhen Han designed the experiments, Hong Zhang wrote the manuscript, these three students performed the experiments, especially Hong Zhang. All authors read and approved the final manuscript.

Ethics approval: Not applicable

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Table 1 Culture characteristics of six strains of Streptomyces grown on different media

Medium

Substrate mycelium

Aerial mycelium (Soluble pigment)

WL1

WL3

WL4

WL5

WL8

WL9

WL1

WL3

WL4

WL5

WL8

WL9

Glucose yeast extract

beige

brown

beige (brown black)

offwhite (brown black)

)

offwhite

white

white (brown black)

white

YMS

beige

brown

beige (brown black)

white (brown black)

offwhite

white

white (brown black)

white

Glucose asparagine

light green

white

yellow white

white

ivory

light green

pale yellow

white

yellow white

white

ivory (yellow-green)

Kirschner synthesis No.1

light green

white

yellow white

white

ivory

light green

pale yellow

white

pale yellow

white

ivory (yellow-green)

Starch

light green

white

yellow white

white

beige

light green

white

pale yellow

beige (pink)

Czapek

red

white

yellow white

white

pale yellow

Table 2 Physiological and biochemical characteristics of six Streptomyces strains

Strain number	Physiology and biochemistry
Strain number	D- glucose	L- arabinose	L- rhamnose	D- fructose	Raffinose	D- mannitol	Nitrate reduction	hydrogen sulfide	Melanin	Gelatin liquefaction	Starch hydrolysis
WL1	+	-	+	+	+	-	+	+	+	+	+
WL3	+(-)	+(-)	-	-	-	+(-)	+	+	+	+	+
WL4	+	+(-)	+	+	+	+	+	-	-	+	+
WL5	+	+	+	+	+	+	+	-	-	+	+
WL8	+	-	-	-	+	+	+	+	+	-	+
WL9	+	+	+	+	+	+	+	-	-	+	+

Note: +, positive reaction; -, negative reaction

Table 3 Homology comparison of the six Streptomyces strains isolated from wetland rhizosphere soils in Guizhou

Strain number	Species	Type strain	Similarity	Genbank accession
WL1	*Streptomyces galilaeus*	JCM 4757^T	100%	AB045878
WL3	*Streptomyces avidinii*	NBRC 13429^T	100%	AB184385
WL4	*Streptomyces albogriseolus* *Streptomyces viridodiastaticus*	NRRL B-1305^T NBRC 13106^T	99.93% 99.93%	AJ494865 AB184317
WL5	*Streptomyces hydrogenans* *Streptomyces albidoflavus* *Streptomyces violascens*	NBRC 13475^T DSM 40455^T ISP 5183^T	100% 100% 100%	AB184868 Z76676 AY999737
WL8	*Streptomyces nojiriensis* *Streptomyces spororaveus*	LMG 20094^T LMG 20313^T	100% 100%	AJ781370 AJ81370
WL9	*Streptomyces cellulosae*	NBRC 13027^T	100%	AB184265

Table 4 Plant growth-promoting characteristics of six strains of Streptomyces

Strain number	ACC deaminase (μmol/(mg·h))	IAA (μg/mL)	Phosphorus solubilization (μg/mL)	Siderophore (%)	Nitrogen fixation
WL1	2.190±0.421^a	5.619±0.119^d	44.143±1.640^a	9.299±0.704^f	+
WL3	0.162±0.074^c	23.119±0.630^c	9.361±0.900^d	20.458±1.039^e	+
WL4	1.257±0.229^b	0	27.941±1.300^b	89.127±0.559^a	+
WL5	1.130±0.060^b	164.548±1.237^a	12.894±1.790^cd	85.408±0.372^b	+
WL8	1.090±0.319^b	0	2.928±0.870^e	37.84±0.398^d	+
WL9	1.192±0.227^b	49.548±1.326^b	16.280±2.360^c	53.29±1.929^c	+

Note: +, positive nitrogen fixation capacity

Table 5 Stress tolerance of six Streptomyces strains

Strain number

NaCl concentration

PEG-6000 concentration

15%

25%

35%

WL1

+(-)

WL3

+(-)

WL4

+++

WL5

+++

WL8

+++

WL9

+++

Note: +++, ++, + indicate the degree of growth; -, no growth

Table 6 Effects of four Streptomyces strains on seed germination

Treatment	Strain	Germination rate (%)	Vigor index	Root length (cm)	Fresh weight (mg)
Mung bean	CK	100.00±0.00a	724.51±130.82ab	22.57±2.87b	1.62±0.08a
	WL1	100.00±0.00a	583.31±98.60b	23.07±1.79ab	1.23±0.12b
	WL3	100.00±0.00a	997.89±10.09a	30.83±2.33a	1.78±0.05a
	WL5	100.00±0.00a	982.33±130.50a	29.00±3.12ab	1.74±0.05a
	WL9	98.33±1.67a	810.52±80.28ab	27.60±1.10ab	1.65±0.03a
Capsicum	CK	87.33±3.53a	508.86±47.91ab	16.90±1.61ab	0.17±0.00a
	WL1	86.67±5.33a	460.39±27.33ab	15.27±0.27ab	0.17±0.01a
	WL3	93.33±0.67a	601.95±89.42a	19.50±2.15a	0.19±0.01a
	WL5	89.33±2.91a	372.46±82.82b	13.27±2.32b	0.19±0.00a
	WL9	88.67±2.91a	520.51±40.56ab	17.401.03ab	0.18±0.01a
Cucumber	CK	86.67±8.82a	294.28±17.43b	41.07±2.32b	0.56±0.06a
	WL1	80.00±5.77a	350.96±13.93b	44.53±2.03ab	0.65±0.07a
	WL3	93.33±3.33a	503.23±51.33a	52.20±3.21a	0.70±0.08a
	WL5	86.67±3.33a	339.93±9.51b	43.60±1.78ab	0.72±0.07a
	WL9	76.67±13.33a	361.14±84.34ab	48.20±3.58ab	0.67±0.04a

Note: Lowercase letters indicate a significant difference between groups (p < 0.05)

Table 7 Effects of the WL3 strain of Streptomyces on cucumber seedling growth

Treatment	Fresh weight (g)	Root length (mm)	Plant height (mm)	Lateral root number
CK	0.35±0.02	42.29±2.98	34.29±3.56	9.86±0.59
WL3	0.64±0.04**	63.43±2.40**	70.29±2.21**	19.14±1.10**

Note: * p < 0.05, ** p < 0.01

No competing interests reported.

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Isolation, identification, and growth-promotion function of Streptomyces sp. in wetland rhizosphere

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Abstract

Figures

Introduction

Materials And Methods

Isolation, screening, and identification of Streptomyces strains

Determination of plant growth-promoting and growth characteristics of Streptomyces strains

Preparation of spore suspension and determination of spore number

Determination of 1-aminocyclopropane-1-carboxylic acid deaminase activity

Determination of indole-3-acetic acid secretion

Determination of phosphorus solubilization capability

Determination of siderophore production capability

Determination of potential nitrogen fixation ability

Determination of stress tolerance

Effects of Streptomyces strains on seed germination and seedling growth of vegetables

Results

Isolation, purification, and identification of Streptomyces strains

Growth-promoting characteristics and stress tolerance of the six Streptomyces strains

Effects of Streptomyces strains on seed germination and seedling growth of vegetables

Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

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