Streptomyces spp. enhance vegetative growth of maize plants under saline stress

Abstract

Saline stress is one of the abiotic stresses that most compromises the yield of crops and can be mitigated by plant growth-promoting rhizobacteria (PGPR). This work characterized rhizobacteria isolates from the genus Streptomyces as PGPR and evaluated their role on growth and alleviation of the effects caused by saline stress in maize (Zea mays L.). Production of indolic compounds (IC), siderophores, ACC deaminase, phenazines, and promotion of plant growth were determined to characterize bacterial isolates. Salinity tolerance was accessed by culturing the Streptomyces isolates under NaCl increasing concentrations (0–300 mM). Four Streptomyces isolates exhibiting PGPR traits and salinity tolerance were selected and their effect on tolerance of maize plants to saline stress was evaluated. Plants obtained from bacterized seeds and submitted to 100 and 300 mM NaCl were used. All Streptomyces spp. produced IC and siderophores, CLV178 being the best producer of these two compounds. ACC deaminase was detected in six of the 10 isolates (CLV95, CLV97, CLV127, CLV179, CLV193, and CLV205), while phenazines were found only in CLV186 and CLV194. All isolates were tolerant to salinity, growing at concentrations up to 300 mM NaCl, with exception of CLV188. Increased concentrations of IC were detected in most of the isolates exposed to salinity. CLV97 and CLV179 significantly promoted growth of roots and leaves of maize plants and attenuated the negative effects of salinity on plant growth. Root colonization by Streptomyces spp. was confirmed in plants cultivated 20 days under saline stress.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-021-00480-9.

Introduction

Plants are constantly exposed to biotic and abiotic stresses, which may negatively affect growth and limit crop productivity. Climate changes and inadequate agronomic practices such as improper irrigation and fertilization have led to the aggravation of soil salinization. Worldwide, salinization of the agricultural lands has compromise growth and caused major losses to crop production, especially in arid and semi-arid regions [1, 2].

Most crops are highly susceptible to saline soil [3]. Thus, plants that grow under high concentrations of NaCl have their development limited either by the osmotic effect of the salt present in the soil or by the toxic effect of salt absorption by the plant [4]. Intracellular accumulation of Na⁺ and Cl⁻ causes ionic imbalance and promotes nutritional damages, such as the reduction of Ca⁺² and K⁺ influx, metabolic damages to stomatal regulation and photosynthetic rate, as well as induced hyperosmotic stress [5]. Specifically, salinity stress generates reactive oxygen species (ROS) that disrupt normal metabolism through damaging the DNA, RNA, and proteins as well as cause lipid peroxidation [1, 6]. These ROS also cause chlorophyll destruction and damage the root meristem activity [1, 7]. Moreover, plants under saline stress show increased levels of ethylene, known as a stress hormone [8]. The negative effects of ethylene are root and shoot growth inhibition, leaf expansion suppression, and epinasty [9].

Toward a sustainable agricultural vision, crops need to present disease resistance; tolerance to salt, drought, and heavy metal stresses; as well as better nutritional value. One strategy to meet this challenge is to use soil microorganisms that facilitate plant growth while alleviate stresses [10]. The plant growth-promoting rhizobacteria (PGPR) are microbes able to colonize the roots and to enhance plant growth. These microorganisms act by direct mechanisms such as biofertilization (growth stimulus) and rhizoremediation (plant stress control), and by indirect means which are related to biological control, reducing the impact of diseases, including antibiosis and competition for nutrients and niches [11]. Several non-symbiotic rhizobacteria (Pseudomonas, Bacillus, Klebsiella, Azotobacter, Azospirillum) have been used as bio-inoculants to promote plant growth under various stresses [11, 12]. Indeed, there is clear evidence that a diverse group of root-associated microbes is essential for promoting plant adaptation to salinity [2, 13]. PGPR have been reported to induce tolerance to saline stress through the elicitation of the so-called induced systemic tolerance (IST), which involves modulation of hormone levels, antioxidant defense, osmotic adjustment, expression of stress response genes, as well as production of exopolysaccharides and volatile organic compounds [13, 14]. Auxin, mainly the indoleacetic acid (IAA), is a plant hormone related to regulatory responses of plants under both biotic and abiotic stress conditions [15]. Generally, when produced by the rhizobacteria, IAA interferes with many plant developmental processes since it is acquired by the plant and alters its endogenous pool [10]. By altering the root architecture, favoring its growth and assisting the water uptake, IAA secreted by PGPR may help the plant to overcome the negative effects of saline stress [11, 16]. In addition, some rhizobacteria can counteract the negative effects of increased levels of ethylene on plants under stress, as they have the enzyme aminocyclopropane-1-carboxylic acid (ACC) deaminase, which hydrolyzes ACC, the ethylene precursor, thereby reducing the excess ethylene and rescuing plants from its inhibitory effects on root growth under drought and salinity [17].

Although the PGPR traits mentioned above directly contribute to the tolerance of plants to abiotic stresses, several other rhizobacterial traits may affect the growth of plants under salt stress. For instance, PGPR produce siderophores, molecules of low molecular weight that interact with Fe⁺³, and transport it into bacterial cells, where it is reduced it to Fe⁺², which is biologically usable by plants. The production of siderophores by microorganisms such as rhizobacteria is important for plant development [18]. The production of molecules such as phenazines may have an indirect effect and promote plant’s overall health. Phenazines, for example, may aid plant development and mitigate stress, due to their ability to modify the redox potential of cells, regulate gene expression, and assist in biofilm formation, increasing bacterial survival [19].

Rhizobacteria from the genus Streptomyces have been increasingly studied in areas including health, agriculture, and other sectors. They are actinomycetes Gram-positive, filamentous (forming branching hypha and mycelium), and spore-producing bacteria, from which metabolites, including hormones, antibiotics, and extracellular enzymes, act directly and indirectly on plants, giving them the characteristics of PGPR [20]. Promotion of plant growth and increase of resistance to plant diseases mediated by Streptomyces spp. have been reported. Studies have shown that strains of Streptomyces promoted wheat growth (Triticum aestivum) and pigeon pea (Cajanus cajan) plants [21, 22]. The use of Streptomyces roseoflavus NKZ-259 reduced the incidence of gray mold in tomatoes, as well as promoted growth of tomato and pepper seedlings [23]. PGPR Streptomyces show a potential to survive in various conditions and are able to keep their PGPR characteristics active even under salt stress [2]. Sadeghi et al. [24] demonstrated that a Streptomyces isolate was tolerant to NaCl concentration up to 300 mM. Indeed, the production of IAA and siderophore increased in the presence of NaCl, as well as plant growth parameters in normal and salt stress conditions.

Maize (Zea mays L.) is one of the most important crops for both human and animal consumption, reaching a world production of approximately 1050 million metric tons of grain in 2018/2019 [25]. Considering that salinization is a worldwide problem and that maize is vulnerable to salinity [26], the identification of Streptomyces spp. that favor growth of maize plants and that are able to attenuate the deleterious effects of salinity may represent the possibility for formulation of a biofertilizer with application on environmentally sustainable agriculture systems.

Therefore, in the current study, we addressed the question whether halotolerant Streptomyces spp. could promote growth and salt tolerance of maize plants. For that, we initially characterized 10 Streptomyces isolates as PGP and investigated (1) whether they were halotolerant by evaluating bacterial growth and production of IAA under increased NaCl concentrations and (2) the effect of bacterization on growth of maize plants submitted to saline stress of 100 and 300 mM of NaCl, under greenhouse conditions. Our findings will help identify potential PGPR Streptomyces able to increase salinity stress tolerance of maize plants.

Material and methods

Selection and identification of rhizobacteria isolates

Rhizobacteria were isolated from soil samples collected from rhizosphere of Brachiaria sp. at Mato Grosso do Sul (22° 28′ 19.33′′ S, 54° 48′ 30.83′′ W), Triticum aestivum L. at Paraná (24° 57′ 20″ S, 54° 48′ 30.83′′ W), Phaseolus vulgaris L. at Mato Grosso (15° 33′ 32′′ S, 54° 17′ 46′′ W), and Cucumis melo L. at Ceará (04° 33′ 42′′ S, 37° 46′ 11′′ W), comprising different regions of Brazil. Soil samples were oven-dried at 30 °C for 7 days and stored at – 20 °C. Dissociation of the microorganisms from the roots was carried out by agitation in HCN liquid medium [27] at 100 rpm, 42 °C for 30 min. A volume of 100 μL of the dilution (1:10, v/v) was plated on ISP2 or ISP4 medium [28], supplemented with the antibiotics cycloheximide (100 μg mL⁻¹) and nalidixic acid (50 μg mL⁻¹), and the antifungal nystatin (100 μg mL⁻¹); plates were maintained at 28 °C and monitored for approximately 15 days. Actinobacteria were selected based on typical morphology of the genus Streptomyces, such as colony morphology, mycelia color, and microscopic traits [29]. The identified isolates were stored in 50% glycerol at – 80 °C as part of the Collection of Microorganisms of the Plant Biotechnology Laboratory (CLV)—PUCRS.

Isolates were, then, subjected to taxonomic identification using 16S rRNA gene sequencing. Isolates were cultured in ISP2/ISP4 agar medium, incubated at 28 °C for 3 days (exponential phase). Bacterial genomic DNA was extracted by the Wizard® Genomic DNA Purification Kit (Promega Biotecnologia de Brasil, Ltda) following manufacturer’s instructions. PCR amplification of the 16S rRNA gene was performed using the universal primers 9F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1542R (5′-AGAAAGGAGGTGATCCAGCC-3′), and Taq Platinum Enzyme Kit (Invitrogen™). The cycle parameters were as follows: an initial denaturation at 94 °C for 5 min, followed by 30 cycles at 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 60 s, and one last cycle at 72 °C for 6 min. The PCR-amplified fragments were subject to Sanger sequencing (Myleus Biotecnologia; Belo Horizonte, Brazil). The obtained nucleotide sequences were compared with sequences in the GenBank database from the National Center for Biotechnology information (NCBI). The phylogenetic analysis was carried out using the sequences and another 20 Streptomyces 16S rRNA sequences from different species retrieved from the NCBI. All sequences were aligned using CLUSTAL W [30]. To ensure the stability and reliability of the phylogenetic relationships among the strains used in this study, a phylogenetic tree was constructed through the maximum likelihood (ML) method and Tamura-Nei model, using MEGA X [31]. The topology of the phylogenetic trees was evaluated by bootstrap resampling (1000 replications). The sequences were submitted to NCBI GenBank for accession numbers.

Characterization of Streptomyces isolates as PGPR

The production of indolic compounds (IC), siderophores, ACC deaminase, and phenazines, and the promotion of plant growth by Streptomyces isolates were conducted using rhizobacterial suspensions. For all experiments, cultures were grown in 10 mL ISP2 or ISP4 liquid medium, depending on which the medium provided optimal growth for each isolate (data not shown), at 28 °C and 100 rpm from 4 to 5 days, depending on the experiment. Each bacterial suspension was centrifuged (2500g, 15 min) at room temperature; the pellet was resuspended in sterile distilled water and the final bacterial concentration was adjusted to 10⁸ cfu mL⁻¹.

The production of indolic compounds by the Streptomyces isolates was analyzed by Salkowski’s method, following Horstmann et al. [32]. The supernatant of the 4-day-old culture was combined with Salkowski’s reagent (1:1; v/v), incubated for 30 min at room temperature in the dark and absorbances were determined in a spectrophotometer at 530 nm. Culture medium was used as blank. Quantifications were carried out in at least 15 replicates. Fresh mass (g) of the pellet was determined. Concentration of IC was determined based on the calibration curve of IAA (from 5 to 200 μg of IAA mL⁻¹) and expressed in μg of IAA g⁻¹ cells.

Production of siderophores by Streptomyces spp. was determined by culturing the isolates into CAS agar plates (Chrome Azurol S) following [33]. Briefly, aliquots of 100 μL of the bacterial suspension were inoculated into wells (5 mm) made in the CAS agar. Sterile distilled water was used as control. Each isolate was inoculated into three wells, totaling nine samples per isolate. The plates were incubated at 28 ± 2 °C. The release of siderophores by the Streptomyces spp. was evaluated by the change of the color of medium from blue to yellowish orange. After 3 days, the halos formed around the wells were measured. It was considered positive for siderophore production the isolate that produced halos of at least 2 mm of border. Data were expressed as mean ± SE of the halo border.

The presence of ACC deaminase (E.C. 4.1.99.4) produced by Streptomyces isolates was determined following Horstmann et al. [32]. Briefly, 5-day-old cultures of Streptomyces isolates were centrifuged, and pellet was suspended in Dworkin and Foster (DF) minimal salt medium [34], without glucose or nitrogen salts and washed twice. In a 24-well plate, 5 μL of the bacterial suspension was inoculated on medium DF (positive control), on DF medium N-free (negative control), and DF medium + ACC (as the only source of nitrogen). The plates were incubated at 28 °C for 7 days. The presence of ACC deaminase was qualitatively confirmed by visually detecting the rhizobacteria capable of growing in DF + ACC medium.

The phenazines, 1-hydroxyphenazine (1-OH-PHZ), and phenazine 1-carboxylic acid (PCA) were evaluated in the supernatant of Streptomyces spp. cultures, grown in 10 mL ISP2 liquid medium, at 100 rpm and 26 ± 2 °C for 5 days (decline phase). The culture supernatant (1 mL) was dried under a stream of air to one-third of the initial volume. Analysis was performed by high performance liquid chromatography (HPLC) using a Sykam S600 Chromatography system with a detector UV/VIS Model 3345 DAD, set at 367 nm, 40 °C (Sykam, Germany). Phenazine separation was performed in a MetaSil ODS reverse phase column (5 μm, 250 × 4.6 mm), and the chromatographic data was obtained and processed by the Clarity Chromatography Software data system (Data Apex, The Czech Republic). The solvents used were (eluent A) water and (eluent B) acetonitrile, both acidified with 2.5% formic acid in water (v/v). The linear gradient of the mobile phase consisted of 0–15% of eluent B for 2 min, 15–83% B for 12 min, 83–0% B for 2 min, and 0% B for 4 min [32]. The flow was maintained in 1 mL min⁻¹ and injection volume was 20 μL. The chromatography was performed in duplicate. The areas obtained in the chromatograms were compared to the calibration curves established with phenazine standards (Sigma-Aldrich, Brazil). The concentration was expressed in μg of phenazine g⁻¹ cells.

Promotion of plant growth by Streptomyces isolates

The experiment of plant growth promotion was carried out under greenhouse conditions (19 to 28 °C and 800 μmol m⁻² s⁻¹ of photosynthetic photon flux density) using maize bacterized seeds. The isolates were grown in ISP2/ISP4 liquid medium at 28 °C for 5 days. After culturing, the bacterial suspension was centrifuged, rinsed, resuspended in water, and adjusted to approximately 10⁸ cfu mL⁻¹. Maize seeds (Zea mays L., SHS 5050; Santa Helena Sementes, Brazil) were aseptically bacterized for 20 min under gentle manual shaking. Afterwards, seeds were briefly blotted-dried and sown in plastic pots (1000 mm³) filled with commercial organic soil (13% clay, 7.7% organic matter and ground calcareous rock; pH 6.6 [measured in water 1:5 (w/v)]; EC: 0.26 mS cm⁻¹). The control was conducted with seeds treated with sterile water. The experiment was conducted in a greenhouse (photoperiod 18 h and 23–30 °C). The plants were irrigated when necessary. Thirty seeds were used per treatment, i.e., per Streptomyces isolate.

Plant growth analysis was performed 45 days after sowing, and growth parameters of length (cm) and dry biomass (g) of root and leaves were evaluated. Root length was recorded after uprooting the plant and washing the roots. The dry biomass was determined after oven-drying leaves and roots separately, at 70 °C, for 5 days. Data were expressed in increment (%) of growth related to the control treatment as average of 30 plants per treatment. Scanning electronic microscopy (SEM) was used to confirm the colonization of maize roots by rhizobacteria. Briefly, root samples (4 cm in length) were collected, gently rinsed in tap water in order to remove soil debris, and fixed in 0.2 M sodium phosphate buffer (pH 8) containing 2.5% glutaraldehyde. After 15 days, samples were divided in pieces of 1 cm, dehydrated through a graded acetone series, and critical point dried with carbon dioxide. Finally, samples were mounted on aluminum stubs and coated with a gold sputter coater (Quorum Q 150R ES Plus, England). Visualization was performed using a scanning electron microscope (FEI Inspect™ F50, Japan) under magnification of × 5000 and × 20,000.

Tolerance of Streptomyces spp. to salinity

The Streptomyces isolates were tested for tolerance to the saline stress. Isolates were cultured in ISP2 or ISP4 liquid medium supplemented with NaCl at the concentrations of 50, 100, 200, and 300 mM. As a control, the ISP2 or ISP4 media without NaCl supplementation was used. The cultures were maintained at 28 °C for 5 days. Bacterial growth was evaluated by the pellet mass (g) and expressed as mean of eight replicates. The isolates were also evaluated for their ability to produce indolic compounds under saline solutions, as described above.

Tolerance of maize plants to salt stress

Tolerance and survival of maize plants to salt were evaluated to determine sublethal concentrations of NaCl. In greenhouse, 45-day-old maize plants were treated with NaCl at concentrations of 50, 100, 200, and 300 mM (EC 1.64, 2.98, 3.07, and 5.66 mS cm⁻¹, respectively; determined at the end of the experiment). Plants submitted to the concentrations of 100, 200, and 300 mM NaCl were gradually adapted, starting the irrigation with 50 mM solution, and every 3 days increasing the NaCl concentration until reaching the concentration established for the treatment [35]. Control treatment (0 mM NaCl; original EC 0.26 mS cm⁻¹) consisted of irrigation with water. Water was supplied every 2 days at the base of the pots through the entire period of the experiment to avoid any drought effect. After 20 days, parameters of vegetative growth, such as root and leaf length (cm), and fresh and dry mass (g), were analyzed. Twenty plants were used per treatment (NaCl concentration), consisting of two seeds per pot.

Effect of Streptomyces isolates on tolerance to salt stress of maize plants

Four isolates were chosen to evaluate the effect of Streptomyces on growth of maize plants cultivated under salt stress. The criterion of choice was based on the isolate that presented at least two characteristics of PGPR, promoted plant growth and showed tolerance to salt stress. The selected isolates were cultured in ISP2/ISP4 liquid medium, for 5 days. Bacterization was performed as described above. Plants were cultivated for 45 days prior salinization to allow adequate plant growth and root colonization. Salt stress was established using the NaCl concentrations 0, 100, and 300 mM determined in previous experiment (EC 0.89; 1.84; 6.37 mS cm⁻¹, respectively). The control treatment consisted of non-bacterized (NB) seeds and plants irrigated with NaCl. Within one isolate, each parameter was also analyzed in comparison to the “stress control” which corresponded to no-salt-receiving plants (0 mM NaCl). Treatment with NaCl was performed only once in each pot. Every 2 days, plants were watered from the base of the pot. Plant growth was evaluated after 20 days after treatment. Growth parameters of root and leaf length (cm), fresh and dry mass (g), and stalk diameter (cm) were analyzed. Colonization of maize roots by rhizobacteria under saline stress was evidenced by SEM.

Statistical analysis

Data from PGPR characterization and from the experiments of salt stress were submitted to the homogeneity of variances test (Levene) and analyzed by ANOVA. Means were separated by the Tukey test (P = 0.05). Data from plant growth promotion by rhizobacteria were analyzed by Student’s T test, P = 0.05. All statistical analyses were performed using the software SPSS Statistics v. 22. Data from experiments were expressed as mean ± SE.

Results

Selection and identification of rhizobacteria isolates

Ten actinomycetes were isolated from the four rhizospheric soil samples. All of them showed typical morphology of Streptomyces and were able to grow in either ISP2 or ISP4 at 28 °C (Online Resource 1). BLAST analysis based on sequences of 16S rRNA gene from the 10 isolates showed that they belong to the genus Streptomyces. Sequences were deposited in GenBank under accession numbers CLV95-MN461005, CLV97-MN461006, CLV127-MN461007, CLV178- MN461008, CLV179-MN461009, CLV186-MN461010, CLV188-MN461011, CLV193-MN461012, CLV194-MN461013, and CLV205-MN461014.

Phylogenetic analysis revealed that the isolates were separated into two clades (Fig. 1). CLV178, CLV188, CLV193, and CLV194 are grouped close together and with Streptomyces rimosus (99%). CLV193 and CLV194 shared 100% homology, although they are morphologically different (Online Resource 1). It is evident that isolate CLV97 is closely related to the species Streptomyces cirratus and Streptomyces vinaceus. CLV95 and CLV127 were grouped with Streptomyces californicus with 85% homology. CLV179 showed 90% similarity to Streptomyces cellulosae and Streptomyces bellus. Comparison between CLV186 and CLV205 showed 100% homology. These two isolates showed similar morphology (Online Resource 1), however differed on the production of metabolites (Table 1).

Fig. 1.

Taxonomic identification of bacterial isolates of Streptomyces. Phylogenetic analyses were performed with reference sequences obtained from the NCBI GenBank database. Phylogenetic tree was constructed using the maximum likelihood method and Tamura-Nei model based on 16S rRNA partial gene sequences. Bootstrap percentages based on 1000 replications are shown at branch points

Table 1.

PGPR characteristics of Streptomyces isolates

Streptomyces isolates	Indolic compounds (μg g−1 cells)	Production of siderophore (cm)a	ACC deaminase activityb	Phenazines (μg g−1 cells)c
				PCA	1-OH-PHZ
CLV 95	168.19 ± 13.0 c*	1.11 ± 0.07 de	+	−	−
CLV 97	12.89 ± 3.2 e	1.72 ± 0.2 ab	+	−	−
CLV 127	8.62 ± 1.5 e	0.89 ± 0.04 e	+	−	−
CLV 178	414.63 ± 25.9 a	1.91 ± 0.2 a	−	−	−
CLV 179	18.58 ± 1.9 e	1.46 ± 0.06 bcd	+	−	−
CLV 186	301.60 ± 21.8 b	1.21 ± 0.08 cde	−	574.4	573.2
CLV 188	11.53 ± 0.7 e	1.07 ± 0.1 de	−	−	−
CLV 193	18.76 ± 2.4 e	1.52 ± 0.1 bc	+	−	−
CLV 194	100.10 ± 7.2 d	1.84 ± 0.1 ab	−	−	255.2
CLV 205	23.90 ± 2.0 e	1.05 ± 0.07 e	+	−	−

*Means (±SE) followed by the different letters in the columns indicate significant difference according Tukey test (P = 0.05)

^aValues are the average width of the halo border (cm) of at least three replicates

^bACC deaminase activity: (−) undetectable colony; (+) colony growth. Evaluated in duplicate

^cAverage of duplicates of phenazines: PCA, phenazine-1-carboxylic acid; 1-OH-PHZ, 1-hydroxyphenazine; (−): not detected

Characterization of Streptomyces isolates as PGPR

The rhizobacteria Streptomyces showed traits that are common to PGPR. All isolates produced indolic compounds and siderophores, although variations among the isolates were recorded. In addition, phenazines and ACC deaminase were produced by the isolates. Quantitative analysis of the supernatant from Streptomyces spp. cultures showed variable production of indolic compounds (IC) (Table 1). The isolate CLV178 produced the highest concentration of IC, followed by isolate CLV186. CLV95 and CLV194 also produced IC over 100 μg g⁻¹ cells. The other isolates produced less than 25 μg g⁻¹ cell. As observed in the production of IC, the 10 isolates were able to produce siderophores. CLV178, CLV194, and CLV97 showed a highlighted production of siderophores (1.72 to 1.91 cm of halo border), whereas CLV127 and CLV205 were not as efficient on producing these molecules (Table 1; Fig. 2). Production of the enzyme ACC deaminase by Streptomyces spp. was evaluated by culturing bacteria on DF semisolid medium containing ACC as the only source of nitrogen. Isolates CLV95, CLV97, CLV127, CLV179, CLV193, and CLV205 were able to grow on DF + ACC medium, indicating the presence of the enzyme ACC deaminase (Table 1). The remaining isolates did not grow in the presence of ACC as N source. Phenazines were only detected in the supernatant from CLV186 and CLV194 cultures. CLV186 produced PCA and 1-OH-PHZ, whereas only 1-OH-PHZ was detected in CLV186 suspension. From this isolate, production of 1-OH-PHZ was 2.2-fold compared to CLV194 (Table 1).

Fig. 2.

Siderophore production of the selected rhizospheric Streptomyces spp. a CLV178. b CLV186. c CLV127. Bar = 3 cm

Promotion of plant growth by Streptomyces spp.

Treating maize seeds with Streptomyces isolates significantly affected the vegetative growth, promoting leaf and root growth (Fig. 3). Compared to the control treatment, root length was increased by bacterization with several isolates (Fig. 3a). The most significant root growth was achieved by CLV127 and CLV97, with increase of 41% and 30.7%, respectively. CLV95 and CLV205 promoted root growth by an average of 26%. Growth of leaves was also recorded on plants cultivated in the presence of four Streptomyces isolates. The maximum increase in leaf length (49.8%) was observed when plants were associated to CLV179, even though other isolates positively affected this parameter. Similarly, significantly increment of dry mass of leaves and roots resulted from treatment with CLV179 (127.9% and 49% respectively). CLV95 and CLV194 significantly promoted leaf growth of maize plants as well (Fig. 3b). It is noteworthy that none of the isolates tested was detrimental to plant growth, with exception of CLV186 that caused a reduction of 23.5% on root dry mass (Fig. 3b). Colonization of the roots was evidenced by SEM (Fig. 4b, e; Online resource 2 a, d).

Fig. 3.

Growth of maize plants (root and leaf) obtained from seeds bacterized with Streptomyces isolates. a Increment of plant length. b Increment of dry mass. Values are the average of 30 plants. Bars with asterisk indicate significant difference of the treatment compared to the control plants (non-bacterized) according to Student’s T test (P = 0.05)

Fig. 4.

Colonization of maize roots by Streptomyces CLV97 (b–d) and CLV179 (e–g) in different concentrations of NaCl (100 and 300 mM). Non-bacterized (NB) root is shown in a. Insets show Streptomyces spp. attached to the root surface. Spores and hyphae are indicated by white and black arrows, respectively

Tolerance of Streptomyces isolates to salinity

All isolates were able to grow in a culture medium supplemented with various NaCl concentrations (0, 50, 100, 200, and 300 mM). Tolerance of Streptomyces to saline stress varied according to the isolate (Fig. 5a). Most of the isolates were tolerant to the NaCl concentrations tested. Three of the 10 isolates analyzed grew significantly better with 300 mM NaCl. At this concentration of NaCl, CLV127 showed 3-fold more cells than the control treatment, without salt, whereas CLV95 and CLV193 showed growth increment of approximately 65%. Only the isolates CLV188 showed deleterious response to NaCl at 300 mM. Results, then, showed that all isolates proved to be tolerant to this type of stress, with exception of CLV188.

Fig. 5.

Tolerance of Streptomyces spp. to saline stress (0 to 300 mM NaCl). a Bacterial growth. Data were evaluated as pellet mass (g) obtained after 5 days of culture. b Production of indolic compounds. Data are expressed in μg g⁻¹ cells. Values are the average of at least five bacterial suspensions. Means followed by different letters within the CLV indicate significant difference according to Tukey test (P = 0.05)

Once the tolerance to salinity was proven for the isolates, the ability to produce IC under stress was evaluated. All isolates continued to produce IC under stress conditions, although variation was observed (Fig. 5b). Overall, 200 mM NaCl significantly induced the production of IC by most of the isolates submitted to saline stress. At 300 mM, CLV95, CLV179, and CLV193 were stimulated to produce IC, and CLV179 was the most affected by the high salt concentration, resulting in a 10-fold IC production (Fig. 5b). CLV178 was the only isolate to show lower concentration of IC, compared to the control, regardless the concentration tested. Isolates CLV188 and CLV194 showed a markedly increase IC production at intermediate concentrations of salt (50 to 200 mM), although they did not maintain the production at 300 mM NaCl (Fig. 5b).

Tolerance of maize plants to salt stress

The tolerance of maize plants to salt stress was assessed by cultivating plants under different concentrations of NaCl (Fig. 6). Salt stress drastically affected the growth of maize as reflected by stunted growth and less vigor. Root growth was mostly affected by salt concentration from 200 and 300 mM, with the concentration of 300 mM leading to reduced root length and biomass (28.7% and 30.4%, respectively). More detrimental effect was noted on leaves regarding the effect of salt concentrations. There was a significant decrease in leaf growth from 50 mM NaCl and 37% of reduction in leaf biomass at 300 mM compared to control plants (Fig. 6). Considering that the root growth at 50 mM NaCl did not differ from the control plants and that there was no significant difference in the effect of the salt between 100 and 200 mM for all the growth parameters analyzed, the concentrations of 100 and 300 mM were chosen to test the effect of the Streptomyces isolates on the attenuation of salt stress in maize plants.

Fig. 6.

Growth of maize plants submitted to different concentrations of NaCl, cultivated for 20 days under saline stress. Values are the average of 20 plants. Means followed by different letters within each parameter indicate significant difference according to Tukey test (P = 0.05)

Effect of Streptomyces spp. on tolerance to salt stress of maize plants

Taking in account the results obtained with PGPR traits, promotion of growth of maize plants, and tolerance of Streptomyces spp. to salinity, four isolates (CLV95, CLV97, CLV178, and CLV179) were selected for evaluating whether they attenuate the effects of salt stress during growth of maize plants. Seeds were bacterized and then plants treated with 100 and 300 mM of NaCl were analyzed based on growth parameters.

All four Streptomyces isolates affected in some extent the growth of maize plants subjected to different salinity levels (Table 2). Plants inoculated with CLV95 and treated with 100 mM NaCl showed similar root and leaf growth to non-stressed plants (0 mM NaCl). When 300 mM NaCl was applied, the presence of CLV95 on the roots reduced stress effects and the leaf growth (length and fresh biomass) was comparable to control plants. Indeed, dry mass of leaves was increased 1.8 times compared to non-stressed plants. Isolate CLV97 was more efficient mitigating the salt stress effects, since length and biomass of leaves as well as stalk diameter were increased regardless the NaCl concentration. In fact, leaf length of salt-treated plants increased 24% and 16% under 100 and 300 mM NaCl, respectively, whereas an average of 40% increase of fresh biomass of leaves was observed. Moreover, root growth was promoted at 100 mM NaCl. Interestingly, leaf dry biomass was higher in plants treated with CLV97 and submitted to 300 mM NaCl than observed with the control plants (0 mM NaCl). Variation of responses to the salinity levels was recorded in plants treated with CLV178, although some mitigation of saline stress was detected at 300 mM treatment with respect to dry biomass of roots and leaves. No difference was detected on growth parameters when plants were treated with CLV179 and different salinity levels compared to non-salinized plants, with exception of leaf dry mass which was significant higher at 300 mM than the control plants.

Table 2.

Effect of Streptomyces spp. on growth of maize plants cultivated under saline stress (100 and 300 mM NaCl)

		NaCl	NB	CLV95	CLV97	CLV178	CLV179
Length	Root (cm)	0	47.2 ± 1.9aA	49.4 ± 2.4aA	41.0 ± 1.5bB	41.8 ± 1.7aB	39.81 ± .9aB
		100	38.2 ± 1.9bB	46.8 ± 2.0aA	45.2 ± 1.2aA	42.2 ± 1.2aAB	35.5 ± 1.2aB
		300	22.2 ± 1.8cA	34.5 ± 1.1bA	35.4 ± 0.9cA	37.3 ± 1.5bA	37.8 ± 1.4aA
	Leaves (cm)	0	55.7 ± 1.4aC	55.0 ± 2.6aC	45.4 ± 1.3bC	74.7 ± 4.2aB	95.6 ± 3.2aA
		100	54.8 ± 1.7aB	55.4 ± 2.4aB	56.5 ± 1.6aB	59.3 ± 3.3bB	92.1 ± 2.4abA
		300	33.3 ± 2.41bC	53.3 ± 1.4aC	53.0 ± 1.5aC	68.0 ± 4.0abB	85.3 ± 2.1bA
Fresh biomass	Root (g)	0	3.7 ± 0.2aC	3.9 ± 0.3abBC	3.3 ± 0.2aC	5.3 ± 0.4aB	8.2 ± 0.6aA
		100	3.6 ± 0.2aB	4.2 ± 0.2aB	3.4 ± 0.2aB	3.7 ± 0.3bB	6.3 ± 0.5bA
		300	2.9 ± 0.4bB	3.4 ± 0.2bB	3.4 ± 0.2aB	4.1 ± 0.4abB	7.6 ± 0.5abA
	Leaves (g)	0	4.2 ± 0.4aC	3.8 ± 0.3aC	3.0 ± 0.2bC	10.9 ± 1.2aB	14.6 ± 1.3aA
		100	4.0 ± 0.2aB	4.6 ± 0.4aB	4.1 ± 0.2aB	4.9 ± 0.5bB	14.1 ± 1.1aA
		300	2.8 ± 0.7bB	4.7 ± 0.3aB	4.2 ± 0.2aB	7.1 ± 1.1bB	13.7 ± 1.1aA
Dry biomass	Root (g)	0	0.4 ± 0.03aC	0.4 ± 0.04aC	0.4 ± 0.02aC	0.7 ± 0.06aB	0.9 ± 0.1aA
		100	0.4 ± 0.02aB	0.4 ± 0.02aB	0.4 ± 0.02aB	0.4 ± 0.03bB	0.8 ± 0.07aA
		300	0.2 ± 0.05bC	0.5 ± 0.03aBC	0.4 ± 0.02aC	0.6 ± 0.08aB	0.9 ± 0.05aA
	Leaves	0	0.6 ± 0.04aB	0.6 ± 0.05bB	0.4 ± 0.03cB	0.7 ± 0.3aB	2.1 ± 0.2bA
		100	0.6 ± 0.04aB	0.8 ± 0.07bB	0.7 ± 0.04bB	0.4 ± 0.1bB	2.5 ± 0.2bA
		300	0.7 ± 0.1aC	1.1 ± 0.08aB	0.9 ± 0.05aB	0.6 ± 0.4aC	4.7 ± 0.4aA
Stalk diameter (cm)		0	2.3 ± 0.1aC	2.3 ± 0.2aC	1.8 ± 0.1bC	3.6 ± 0.3aB	4.4 ± 0.2aA
		100	2.1 ± 0.08aB	2.1 ± 0.1aB	2.3 ± 0.1aB	2.5 ± 0.2bB	5.1 ± 0.2aA
		300	1.7 ± 0.2bB	2.4 ± 0.1aB	2.4 ± 0.1aB	3.0 ± 0.2abB	4.7 ± 0.2aA

NB, non-bacterized seeds

Means followed by different small letters within the columns (for each parameter) and capital letter within the lines indicate significant difference according to Duncan’s multiple range test (P = 0.05)

Comparing growth among Streptomyces-treated and non-bacterized (NB) maize plants, it was noticed that the reduction of root growth caused by 100 mM was attenuated by the presence of CLV95 and CLV97 (Table 2). Effect of salt stress caused by 300 mM was also reduced by these isolates regarding leaf dry mass, which increased 1.6- and 2-fold by CLV95 and CLV97, respectively. CLV97 was also efficient on attenuating salinity effect on leaf length and fresh biomass of plants treated with 300 mM NaCl, compared to NB plants. More significant results were obtained on the plants treated with CLV179, showing growth of all parameters superior to NB plants and those treated with the other isolates, with exception of root growth. Isolate CLV179 was able to maintain the promotion of plant growth even under salinity stress. Dry mass of roots and leaves were increased by 3.7- and 6.9-fold at 300 mM, respectively. Stalk diameter was also significantly larger with CLV179 than in the non-bacterized plants at 100 and 300 mM.

Colonization of the roots by Streptomyces was confirmed after 60 days from the beginning of the experiment. Spores and hyphae were observed on the root surface (Fig. 4c, d, f, g; Online resource 2, b, c; e, f).

Discussion

Plant growth-promoting rhizobacteria are microorganisms present in the rhizosphere that promote growth and help plants to cope with biotic and abiotic stresses [11, 36]. Identification of effective PGPR initiates with a screening of microorganisms followed by pure culture on solid medium to determine traits of plant growth promotion. These characteristics include production of phytohormones [21, 36] and siderophores [18], hydrolytic enzymes, and antibiotics [37, 38]. Among these, the production of indolic compounds seems to be the most common trait found among PGPR [21]. All Streptomyces spp. isolated in this work produced indolic compounds (IC), quantified based on the 3-indol acetic acid (IAA) standard. Although variation in concentration was observed, it was possible to select isolates with great capacity of IC production, such as CLV178 and CLV186. Production of indolic compounds by rhizobacteria is a desirable trait when these microorganisms are to be used to formulate biofertilizers due to its effects on root growth and hence water acquisition and nutrient uptake [11]. However, the most indolic-productive Streptomyces isolate did not promote either root or shoot growth of maize plants, likely due to the inhibitory effect on root growth resulting from high concentrations of auxin. Comparable to our results, an IAA-overproducing mutant of the bacterium Pseudomonas fluorescens BSP53a inhibited the development of roots in cherry cuttings [39]. Contrastingly, Streptomyces CLV97, CLV127, and CLV179 produced indolic compounds below 20 μg g⁻¹ of cells. It is well known that the profile of PGPR traits of each isolate may result in different effect on plant growth. Therefore, although IAA is an important PGPR trait, other characteristics may interfere on plant development. In addition to IAA, all Streptomyces spp. were able to produce siderophores, and among the isolates tested, several showed evidence of ACC deaminase in their cells. These traits are well studied on PGPR and have been associated to plant growth and defense against biotic and abiotic stress [18, 20] and have been demonstrated in some strains of Streptomyces [40–42]. Phenazines were detected on CLV186 and CLV194. These compounds affect bacterial survival and may induce plant tolerance to biotic stresses and inhibit pathogenic organisms [19, 43], which places them as an important characteristic of rhizobacteria.

As part of the characterization of the Streptomyces isolates as PGPR, growth of maize plants was analyzed, and our results showed that the isolates tested either promoted or showed neutral effect on maize vegetative growth. No isolate was significantly deleterious to plant growth. It is worth mentioning that CLV179 had a positive effect on plant development. The isolates CLV95, CLV97, and CLV127 also promoted root growth, varying from 25 to 40% increment compared to non-bacterized plants, which could be indirectly related to the IC production by these isolates. However, experimental evidence has suggested that plant growth stimulation is a net result of multiple mechanisms that may be activated simultaneously [40]. Therefore, we suggest that the combination of IAA with other PGPR traits as siderophores and ACC deaminase production may have been responsible for the results obtained with CLV97, CLV178, and CLV179 on the growth of maize plants. The PGPR characteristics identified in Streptomyces spp. have been pointed out as responsible for promoting growth of several species such as tomato [33], sunflower [44], chickpea [45], soybean [32], Araucaria angustifolia [46], and Eucalyptus [47].

Salinity may affect microorganisms in the soil. According to their salinity tolerance, bacteria can be classified as halotolerant when are able to survive in media containing a wide range of sodium chloride (up to 25%) or in the absence of sodium chloride [2]. Studies on salt-tolerant PGPR indicated that these microbes have developed complex physiological and biochemical mechanisms which maintain their survival and multiplication in saline conditions [5, 48]. Streptomyces are reported as salt-tolerant bacteria, able to survive in various conditions including saline soils [2]. Most isolates of Streptomyces tested in this study were tolerant to NaCl (50 to 300 mM), with exception of CLV188, in which growth was decreased at 300 mM. Indeed, salt stress promoted the growth of CLV95, CLV127, and CLV193. There was a stimulus in the production of IC by bacteria, even in those isolates where growth was impaired by salinity. Thereby, Streptomyces isolates evaluated in this study may be considered halotolerant.

Salinity causes severe reduction of growth in plants. This stress prevents plant from capturing nutrients by lowering the water potential and creating osmotic stress. The plant strategy to overcome this negative effect is to uptake Na⁺ and Cl⁻, leading to another level of stress, which is a substantial increase in cellular ion contents, negatively affecting cellular biochemistry [49]. It has been reported that halotolerant PGPR alleviate salt stress and help plants to maintain growth in several species, including maize [50–52]. In order to test the hypothesis that Streptomyces isolates attenuate the negative effects of salinity on growth of maize plants, the isolates CLV95, CLV97, CLV178, and CLV179 were chosen. When compared with non-bacterized plants, which suffered the effect of NaCl mainly at 300 mM, CLV97 and CLV179 stood out for alleviating the effect of salinity. Our results suggest that the bacteria acted in different ways. Plants bacterized with CLV179 grew regardless of salt concentration, adding a protective effect against salinity to the growth-promoting effect observed in non-saline conditions. On the other hand, CLV97 not only relieved stress but also promoted plant growth by increasing five of the seven parameters evaluated, regardless of salt concentration. Both isolates showed similar PGPR traits, such as high production of siderophores and presence of ACC deaminase. Salinity can reduce the availability of Fe in the soil and the presence of siderophore-producing PGPR in the rhizosphere can mitigate this detrimental effect [53]. However, the role of siderophores as a single plant growth-promoting trait on improving Fe acquisition by plants exposed to saline stress is still unclear, and an indirect contribution of bacterial siderophores to plant growth on saline soils has been suggested [53]. In addition to siderophores, both Streptomyces were able to grow in the presence of ACC as the only source of nitrogen, which suggests the presence of ACC deaminase. The ACC produced by the plants in response to saline stress is secreted and metabolized by soil microorganisms, by activation of ACC deaminase [9], which leads to decreased amount of ACC in the rhizosphere, lowering the ethylene [17, 52]. Although several works had related the mitigation of salt stress to the root colonization by PGPR-producing ACC deaminase (Pseudomonas, Enterobacter, and Streptomyces) [41, 52, 54], we propose that the increased tolerance to salinity in maize promoted by Streptomyces resulted from a combination of the PGPR traits, siderophores and ACC deaminase, shown by CLV97 and CLV179.

In conclusion, the Streptomyces spp. isolated in this study were characterized as salt-tolerant PGPR and showed potential to improve growth of maize plants under normal and saline conditions. The Streptomyces CLV97 and CLV179 helped plants to cope with the salinity stress up to 300 mM of NaCl. Our results highlight Streptomyces as a candidate for the formulation of a biofertilizer for plant management under salt stressful environments.

Author contribution

RM Nozari and ER Santarém designed the experiments. RM Nozari and F Ortolan performed the experiments. RM Nozari, ER Santarém, and LV Astarita contributed equally to data analyses and discussion of the results. RM Nozari and ER Santarém wrote the manuscript. LV Astarita reviewed the manuscript. Funding was obtained by E. Santarém and LV Astarita.

Funding

This work was supported by Ballagro Agro Tecnologia Ltda (Brazil) through fellowship of first author, by PUCRS—BPA Program by fellowship of the second author, and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 001. Financial support was provided by Ballagro Agro Tecnologia Ltda, São Paulo, Brazil (AGT/TA 01/2015-SIGPDI 194) and the National Council for Scientific and Technological Development (CNPq/Brazil; 403843/2013-8).

Declarations

Conflict of interest

The authors declare no competing interests.