Abstract
Background and aims
Soil salinity is a worldwide environmental problem that can hinder plant development and therefore negatively impact crop production. Inoculation of halophytic plants with plant growth-promoting (PGP) actinobacteria has been suggested as one strategy to improve salt tolerance. Here we performed a glasshouse experiment to test the effect of a PGP halotolerant endophytic actinomycete strain, KLBMP 5084 on the performance of the halophyte Limonium sinense under conditions of salt stress.
Methods
Strain KLBMP 5084 was identified and screened for multiple PGP traits. The complete genome of strain KLBMP 5084 was sequenced and analyzed. L. sinense control seedlings (no inoculation) and seedlings inoculated with KLBMP 5084 were given different NaCl (0, 100 and 250 mM) salt-stress treatments. Growth parameters and physiological responses of L. sinense were determined after harvest.
Results
Compared with the control, plants inoculated with strain KLBMP 5084 had greater in fresh weight, root length, leaf length and total chlorophyll and proline contents under both normal and high salinity conditions. Compared with control, inoculated plants had significantly lower leaf malondialdehyde (MDA) content and significantly more antioxidant enzymes. Moreover, inoculated plants had significantly lower accumulation of Na+ in both leaves and roots under high salt-stress conditions. Genomic analysis of strain KLBMP 5084 revealed many PGP related genes, including some genes putatively involved in salt tolerance and harsh environment adaptation.
Conclusion
Strain KLBMP 5084 seems to confer salt tolerance to host plant L. sinense through more than one mechanism, suggesting KLBMP 5084 could be a strong PGP agent to improve plant yields and tolerance to salinity stress.
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Introduction
Soil salinity is one of the major stresses that limit global agricultural productivity, affecting more than 45 million ha of irrigated land worldwide (Munns and Tester 2008). It is predicted that increased salinization of soil will result in the loss of up to 50% of arable land by the year 2050 (Wang et al. 2003; Singh et al. 2015). Presence of high concentrations of salts, especially the Na+ in soil, induces both osmotic and ionic stress to plant cells (Kohler et al. 2009; Chavez-Aviles et al. 2013). Some plants, termed halophytes, have evolved complex mechanisms to adapt to saline stress, such as synthesizing antioxidant enzymes and non-enzymatic antioxidants to scavenge the excess reactive oxygen species (ROS) (Ahmad et al. 2010), and producing secondary metabolites and phytohormones (Ramakrishna and Ravishankar 2011). However, most crops used for human/animal nutrition are relatively susceptible to high salt stress (Ondrasek et al. 2011). Thus, it is still important and necessary to develop effective strategies to enhance the salt tolerance of plants in agricultural production.
An attractive and environmentally friendly strategy to mitigate stress effects on plants is the application of microorganisms with plant growth-promoting (PGP) traits. These microorganisms (which include both endophytic and rhizospheric bacteria) can exhibit beneficial effects on plant growth and development, because they can mediate a large number of biotic and abiotic stresses in plants through various mechanisms, such as lowering the level of ethylene by production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, biosynthesizing phytohormones, and raising the soil phosphate solubilization and increasing its absorption for plants (Yang et al. 2009; Qin et al. 2011; Glick 2014). PGP bacteria have been applied to many important crops, and have resulted in enhanced plant development and protection against different stressors (Tiwari et al. 2011; Chen et al. 2016; Akram et al. 2016). For example, the use of microorganisms that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD) has become a promising strategy to promote plant growth and alleviate salinity stress (Yaish et al. 2015; Singh et al. 2015; Nadeem et al. 2016). However, most of these studies used growth-promoting rhizobacteria. Actinobacteria, especially endophytic actinobacteria, have not been tested as thoroughly as a tool for improving plant growth under stress conditions (Barnawal et al. 2014). Species of the genus Streptomyces have been obtained from different habitats and may become important PGP organisms for agriculture (Viaene et al. 2016). It has been reported that some species of Streptomyces promoted plant growth via the producing of ACC deaminase, indole-3-acetic acid (IAA) and siderophore under saline soil conditions (Sadeghi et al. 2012; Palaniyandi et al. 2014).
Limonium sinense (Girard) Kuntze is a halophytic dicot species endemic to China, mainly distributed along seashores and salt marshes in southern China, and used for salinity soil improvement. This traditional Chinese medicine herb has been used to treat hemorrhage and recently it was also demonstrated to have anti-tumor and hepatitis C virus properties (Tang et al. 2014; Hsu et al. 2015). Recently, due to human exploitation and habitat deterioration, L. sinense has become one of the endangered species in China, needing urgent protection (Dong 2005). It has been demonstrated that the endophytic bacteria inhabiting halophytes are adapted to survive under saline conditions and they exhibit both direct and indirect plant growth promoting effects by affecting the fertility of the soil and the salinity tolerance of host plants (Ruppel et al. 2013; Niu et al. 2016). However, few studies have investigated the capacity of native actinobacteria associated with halophyte to enhance plant growth under salinity stress. Thus, the potential PGP mechanisms of actinobacteria are not well understood.
In this study, we tested the effect of an endophytic actinomycete strain on the growth and physiological responses of its host, L. sinense, under higher salt stress. The genome of the strain was sequenced and analyzed for PGP traits, including mechanisms that confer environmental adaptation and increased metabolic potential. This work aimed to improve our understanding of how this beneficial endophyte may help regulate salt tolerance in L. sinense and the results may provide a path to develop actinomycete strains as biofertilizers to improve growth and salt tolerance of this endangered halophyte and important agricultural crops.
Materials and methods
Isolation of endophytic actinobacteria
Endophytic actinobacteria were isolated according to the previously described surface-sterilization procedure (Qin et al. 2009) from healthy halophyte L. sinense sampled from the coastal area of Jiangsu Province, eastern China. An endophyte, designated as strain KLBMP 5084, obtained from the leaf of L. sinense was selected for further studies.
Phenotypic characterization and molecular identification of strain KLBMP 5084
Strain KLBMP 5084 was cultured for 21 days at 28 °C and its morphological characteristics were examined using a light microscopy and scanning electron microscopy (SEM) (Hitachi, S-3400 N). Growth on different carbon source was tested using the method described by Gordon et al. (1974). Determination of NaCl tolerance was performed using ISP 2 agar medium (Shirling and Gottlieb 1966) after incubation at 28 °C for two weeks. Molecular identification of strain KLBMP 5084 via 16S rRNA gene was done using a previously described method (Qin et al. 2012). Sequencing of the 16S rRNA gene was performed and the gene sequences obtained were compared with type strains in the EzTaxon-e database (Kim et al. 2012). The phylogenetic tree was constructed using the neighbor-joining algorithm with MEGA 5.0 software (Tamura et al. 2011), and tree topology was evaluated by 1000 replicates of bootstrap analysis.
PGP traits detection and heavy metal resistances of strain KLBMP 5084
The production of ACC-deaminase by strain KLBMP 5084 was detected according to a previously described method (Penrose and Glick 2003). The putative nitrogen-fixing ability was tested using N-free semi-solid JNFb medium (Döbereiner et al. 1995). The production of IAA was examined when the strain was grown in a minimal salts medium supplemented with 0.5 mg ml−1 tryptophan (Qin et al. 2015), and was detected by the Salkowski’s reagent reaction. The isolate was screened on Ca3(PO4)2 agar medium to determine phosphate solubilization ability. The ability of the strain to produce siderophores was checked according to the chrome azurol-S (CAS) assay (Schwyn and Neilands 1987). Heavy metal resistance in KLBMP 5084 was evaluated by measuring the minimal inhibitory concentration of different heavy metal ions by ISP 2 agar plates incubated at 28 °C for five days.
Plant materials and pot experiment
Strain KLBMP 5084 was first cultured in ISP 2 broth for five days at 28 °C. Then, the cells were collected by centrifugation, washing, and re-suspending in sterilized water to a density of about 106 cfu mL−1 for inoculation. The seeds of L. sinense were surfaced sterilized in 75% ethanol for 30 s, followed by 0.1% HgCl2 for 7 min, and rinsed with sterilized water five times. Then, 10 seeds were incubated on NA agar for 5 days at 28 °C to confirm the sterilization efficacy. Seeds were then sown in plastic pots filled with sterilized vermiculite and soil mix, and moistened with sterilized half strength Hoagland’s nutrient solution. After germination, uniform seedlings were thinned to one plant per pot (diameter 7 cm, depth 10 cm) filled with 160 g sterilized soil. Three days later, each pot was inoculated with a 10 mL strain-suspension culture directly into the soil. Treatment plants were inoculated every 5 d for a total of four inoculations; control seedlings were watered with sterile water. Pots in the salt-stress treatments were watered with 0, 100 and 250 mM NaCl. All plants were grown in a greenhouse (25 °C/20 °C, 16/8 h light/dark). Each treatment consisted of five biological replicates. Plants were harvested after 30 days for growth parameters determination.
Verification of inoculation
To verify that strain KLBMP 5084 had successfully colonized each treatment plant, after harvest, all treatment plants were surface sterilized according to the protocol described above. Roots tissues were cut into small sections (about 0.3 cm × 0.3 cm), plated onto ISP 2 agar medium, and incubated for 5–7 days at 28 °C. For each plant, the colonized strain was identified by morphological characteristics and comparing the sequence of the 16S rRNA gene to the sequence of the strain used in the original inoculation.
Plant biomass and physiological measurements
Plants growth attributes (root length, leaf length and total fresh weight) were recorded after 30 days. Frozen plant samples were used for the physiological index measurements. Total chlorophyll content of leaves of different treatments was analyzed following a previously described protocol (Ali et al. 2014). Proline concentrations of the leaves were measured according to Bates et al. (1973). Leaf malondialdehyde (MDA) content was measured via the thiobarbituric (TBA) reaction using the kit (A003, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). About 0.2 g of the leaf samples was homogenized in 1.8 mL of 0.1 M phosphate buffer (pH 7.0) on ice, followed by centrifugation at 10000 rpm for 10 min. The supernatant was collected to analyze antioxidant enzymatic activities. Peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) of leaf tissues were analyzed using the detection kits (A001 for SOD, A084 for POD and A007 for CAT, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instruction.
Analysis of ion contents
Na+ and K+ contents of leaves and roots were determined followed the method of Niu et al. (2016). Ion analysis was performed by inductively coupled plasma atomic emission spectrometry (ICP-AES, Opmina 8000; PerkinElmer Co., USA).
Genome sequencing, assembly and annotation
Strain KLBMP 5084 was inoculated into 250 mL ISP 2 broth medium and shaken at 150 rpm at 28 °C for 5 days. Collected cells by centrifugation were used for genomic DNA extraction according to a previously described method (Li et al. 2007). The DNA concentration and quality were analyzed using a NanoDrop spectrophotometer (Thermo Scientific, Inc., USA). Then the extracted DNA was used to construct a 20 kb genomic library and sequenced on the single molecule real time (SMRT) sequencing platform by Pacific Bioscience (PacBio) RS II system (Biomarker Technologies Corporation, Beijing, China), which produced a 204 fold coverage genome. The filtered subreads were then assembled de nove using the PacBio hierarchical genome assembly process (HGAP) algorithm 2.3 (Chin et al. 2013). Gene prediction was performed using Prodigal v. 2.6 (Hyatt et al. 2010), whereas tRNA and rRNA were analyzed using tRNAscan-SE v1.31 and Barrnap 0.4.2 software. Their functions were predicted according to the databases of GO, COG and KEGG. The secondary metabolites gene clusters were analyzed using antiSMASH 3.0 (Medema et al. 2011). The complete genome sequence and nearly full length 16S rRNA gene sequence of Streptomyces sp. KLBMP 5084 were deposited in NCBI with the accession numbers CP016795 and JX993791, respectively.
Statistical analysis
The obtained data were analyzed using Sigma Plot Software (10.0). Results were represented as mean values ± standard deviation, and analyzed with one-way ANOVA. P < 0.05 was considered as significant.
Results
Characterization of strain KLBMP 5084
Strain KLBMP 5084 was isolated from a healthy leaf of the halophyte L. sinense and tested for the presence of a number of PGP traits. The results of the characterization analysis showed that KLBMP 5084 had the ability to produce ACC deaminase. Moreover, KLBMP 5084 was found to be positive for phosphate solubilization, siderophore production and growth in N-deficient media (Table 1). KLBMP 5084 tolerated NaCl concentrations of up to 7% and was capable of utilizing different carbon sources for growth. It also exhibited tolerance to multiple heavy metals, including Cu2+, Pb2+, Zn2+ and Hg2+ (Table 1).
SEM observations revealed that strain KLBMP 5084 morphology was typical of the genus Streptomyces. It displayed highly branched aerial hyphae that differentiated into flexuous to spiral spore chains with smooth spores. Phylogenetic analysis using the 16S rRNA gene sequences showed that strain KLBMP 5084 was affiliated with the genus Streptomyces, and was most closely related to Streptomyces pactum NBRC 13433T and Streptomyces olivaceus NRRL B-3009T (99.9% sequence similarity). Strain KLBMP 5084 clustered with S. pactum NBRC 13433T in the phylogenetic tree (Fig. 1), and they formed a distinct subclade with a high bootstrap value of 99%. However, it was not possible to identify strain KLBMP 5084 at the species level in this analysis, so we designated the strain as Streptomyces sp. KLBMP 5084.
Strain KLBMP 5084 improves host growth under NaCl stress
Strain KLBMP 5084 enhanced both root and leaf lengths of L. sinense under both non-salt conditions and NaCl stress (Fig. 2a, b). At 100 mM NaCl stress, the KLBMP 5084 inoculation resulted in enhanced root length by 14.8%. In the 250 mM NaCl treatment, root length of inoculated plants was significantly increased by 27.3% (Fig. 2c). Leaf length was 24.6% longer than control treatment under non salt condition, and KLBMP 5084 significantly improved leaf length by 38.1% and 13.0% under 100 and 250 mM NaCl treatments, respectively (Fig. 2d). Total seedling fresh weight was increased by 77.6% without salt stress compared with un-inoculated plants. Application of KLBMP 5084 restored the host fresh weight and increase of 150% and 66.9% was observed at 100 and 250 mM NaCl treatments, respectively over control plants (Fig. 2e). The results clearly suggest that the inoculation of strain KLBMP 5084 significantly enhances the growth and salt tolerance of L. sinense.
Establishment of strain KLBMP 5084 in plant tissue
Strain KLBMP 5084 was re-isolated from roots to examine the colonization in the inner tissues of L. sinense after 30 days. The re-isolated strains from roots that had the same colony characteristics with strain KLBMP 5084 were analyzed by 16S rRNA gene sequencing, and the results showed they were 100% identical to the original strain. This demonstrated that the inoculated strain KLBMP 5084 had colonized the host root.
Effect of strain KLBMP 5084 inoculation on leaf chlorophyll and proline contents
The results showed that NaCl stress decreased the total chlorophyll content of leaf (Table 2). However, application of strain KLBMP 5084 not only promoted the leaf growth, but also restored the chlorophyll content. In the 0 mM NaCl treatment, leaf total chlorophyll content was 1.7% higher in inoculated plants compared to the controls. Strain KLBMP 5084 also significantly increased leaf chlorophyll content; inoculated plants had 4.2% greater chlorophyll content in the 100 mM NaCl treatment and 7.0% greater content in the 250 mM NaCl treatments compared with the controls. The greatest leaf proline content (28.6 μg g−1 FW) was observed in the control plants treated with 250 mM NaCl. In the 0 mM NaCl treatment, no significant differences were observed in leaf proline content of plants with (15.2 μg g−1 FW) or without (14.5 μg g−1 FW) KLBMP 5084 inoculation. However, in the salt-stress treatments, plants inoculated with KLBMP 5084 had higher proline content than controls by 9.3% in the 100 mM NaCl treatment and by 20.8% in the 250 mM NaCl treatment (Fig. 3). Increased contents of leaf total chlorophyll and proline by strain KLBMP 5084 demonstrated its potential to be protective against NaCl stress for L. sinense seedlings.
Effect of strain KLBMP 5084 inoculation on host antioxidants
In the salt-stress treatments, the leaf MDA content of inoculated plants was 33.7% higher than controls in the 100 mM NaCl treatment and 253.4% higher than controls in the 250 mM, NaCl treatment. However, strain KLBMP 5084 inoculation significantly decreased the leaf MDA accumulation under salt stress. The highest decrease in MDA content (61.4%) was observed in the 100 mM NaCl stress treatment compared with the control (Table 2). A similar trend was observed under the 250 mM NaCl stress, where inoculated plants exhibited a 27.4% decrease in MDA content. The results indicated that inoculation with KLBMP 5084 reduced lipid peroxidation and the degree of cell damage, therefore increasing the tolerance of the host plant to salt stress.
To explore how endophyte inoculation of L. sinense influences the ability of the plant to counter oxidative stress induced by salinity, we analyzed the activity of three antioxidant enzymes: POD, SOD and CAT. The inoculation of strain KLBMP 5084 was found to significantly enhance the antioxidant capacities in L. sinense leaves (Table 2). For example, with KLBMP 5084 inoculation, SOD activity significantly increased by 19.4% (0 mM), 25.3% (100 mM) and 29.8% at 250 mM NaCl compared with the control. POD activity was significantly higher in treated compared to control plants: 49.9% higher in the 100 mM treatment and 13.6% in the 250 mM NaCl treatment. CAT activity in inoculated plants was 6.9% higher in the 0 mM treatment and 8.3% higher in the 100 mM NaCl treatment compared to controls. At the higher 250 mM NaCl salt level, the KLBMP 5084 treated plants had 120.3% greater CAT activity compared with the control plants.
Effect of strain KLBMP 5084 inoculation on host ion accumulation
To test whether the inoculation of strain KLBMP 5084 influenced ion accumulation in L. sinense seedlings, endogenous Na+, K+ content and Na+/ K+ ratio were determined under 0, 100 and 250 mM NaCl treatments. Compared with control plants, inoculated plants had significantly reduced Na+ accumulation in their leaves (Fig. 4a). Na+ accumulation in leaves was 10.1% (0 mM NaCl), 6.0% (100 mM NaCl) and 10.3% (250 mM NaCl) lower in inoculated leaves compared with controls. Similarly, root Na+ content (Fig. 4b) was lower in treatment plants by 46.2% (0 mM NaCl), 63.2% (100 mM NaCl) and 35.3% (250 mM NaCl) compared with control plants. The results showed that soil inoculation of KLBMP 5084 was more effective in reducing Na+ content in roots than leaves. The seedlings K+ content decreased with the increase in the salt stress level. Leaf K+ accumulation was unaffected by KLBMP 5084 inoculation (Fig. 4c). However, an increase in K+ content was observed in endophyte-treated roots, by 16.5% in the 100 mM NaCl treatment and by 20.2% in the 250 mM NaCl treatment (Fig. 4d). Therefore, the Na+/ K+ ratio was reduced significantly in L. sinense seedlings inoculated with endophyte KLBMP 5084 under salt stress. Compared to the control, strain KLBMP 5084 reduced leaf Na+/ K+ ratio (Fig. 4e) by 16.7% and 8.9%, root Na+/ K+ ratio (Fig. 4f) by 33.4% and 37%, respectively, in the 100 and 250 mM NaCl treatments.
Genome characteristics of strain KLBMP 5084
The complete genome (one scaffold) of Streptomyces sp. KLBMP 5084 consists of a single circular chromosome of 8,180,260 bp (Fig. 5). The genome G + C content was 72.41%. No plasmid was found in the genome. The genome contains 7034 genes, including 6433 predicted coding genes. A total of 87 RNA genes consisting of 3 miRNA, 18 rRNA and 66 tRNA genes were identified in the strain KLBMP 5084 genome (Table 3). The number of genes associated with the COG functional categories is present in Fig. 6.
Genes related to PGP and salt environment adaption
In the genome of strain KLBMP 5084, genes related to PGP were observed, including N2-fixation, phosphate solubilization, production of IAA, sideropheres, pyridoxal and hydrogen cyanide (HCN). Strain KLBMP 5084 contains a nifU gene, which is involved in the nitrogen fixation. The genome also contains a gene (iaaM) potentially involved in the biosynthesis of IAA via the indole-3-acetamide pathway. It carries genes gdh [encoding glucose dehydrogenase (GDH)] and pqqD (involved in biosynthesis of the pyrroloquinoline quinone cofactor of GDH). The two genes are responsible for the synthesis of gluconic acid, which is considered as one of the major organic acids for the solubilization of mineral phosphates by bacteria (Farhat et al. 2015). Strain KLBMP 5084 also has a set of genes coding for the synthesis of siderophores (rhbCDEF) and for the transport of the siderophores (iucA) (Table 4). The gene coding for ACC deaminase was also detected, which could enhance plant growth under stress condition (Ali et al. 2014). In addition, genes coding for hydrolytic enzymes, including chitinase, β-glucosidase, lipase, cellulose, protease and amylase were also found in the genome. The anti-pathogen compounds γ-aminobutyric acid (GABA) and phenazine synthesis genes were also detected. The presence of these genes indicate that strain KLBMP 5084 can act as a plant growth promoting bacteria as the detected genes are involved in direct and indirect plant growth promotion (Table 4). In total, 36 putative secondary metabolites biosynthesis gene clusters were found according to antiSMASH 3.0.4 analysis, including 9 PKS (5 type I PKS, 1 II PKS and 3 III PKS), 7 NRPS, 3 hybrid NRPS-PKS, and other antibiotics.
Genome analysis also revealed that KLBMP 5084 has many genes related to salinity tolerance. For example, genes encoding Na+/H+ antiporters for pumping out Na+ and a K+ transporter for K+ accumulation (Epstein 2003) were found, all of which may serve to resist hyperosmotic stress. In addition, the KLBMP 5084 genome has a set of genes to encode proteins to protect the strain from oxidative stress, including superoxide dismutases, peroxidases, and catalases. We also found proteins that help with heavy-metal resistance, cold-shock proteins and heat-shock proteins, all of which could potentially benefit endophyte when it colonizes plants in coastal salt marshes under harsh conditions.
Discussion
The use of PGPB is gaining increasing interest in agriculture as a promising alternative for enhancing seed germination, plant development and crop yields under stress conditions (Nadeem et al. 2014; Chowdhury et al. 2015; Qu et al. 2016). Some studies have reported the isolation and characterization of salt tolerant bacteria to improve crops growth under salinity stress (Ramadoss et al. 2013; Fan et al. 2016). In the current study, we reported the detailed classification status and phenotypic characterization of one plant growth promoting endophyte KLBMP 5084 obtained from the leaf of the halophytic medicinal plant L. sinense. Our previous study demonstrated that strain KLBMP 5084 promoted host seed germination under salinity stress when grown on Murashige and Skoog media (Qin et al. 2014), meriting further exploration of its potential for PGP effects in conditions of soil salinity. The present pot experiment showed the beneficial endophyte significantly improved host plant growth under salt-stress conditions (Fig. 2). The 16S rRNA gene phylogenetic analysis indicated that strain KLBMP 5084 is most closely related to Streptomyces pactum NBRC 13433T (Fig. 1). Strains of S. pactum have been reported to produce antitumor antibiotic pactamycin (Bhuyan 1962). Recently, a soil actinobacterium S. pactum Act12, obtained from the Qinghai-Tibet Plateau in China, was found to improve plant growth and exhibited strong drought tolerance and phytoremediation potential (Yan et al. 2014; Cao et al. 2016). Many Streptomyces strains have been found to act as PGP bacteria by synthesizing phytohormones, phosphate solubilization, and producing ACCD and antibiotics (Schrey and Tarkka 2008; Jog et al. 2012). Possession of ACC deaminase is considered as an important factor for a PGP endophyte because this compound induces stress tolerance in plants by decreasing ethylene concentrations (Santoyo et al. 2016). Palaniyandi et al. (2014) reported that a halotolerant ACC deaminase-producing Streptomyces strain PGPA39 promoted tomato growth under salt stress. Although soil-borne ACC deaminase-producing Streptomyces have been reported to promote tomato growth (El-Tarabily 2008), there is scarce information on endophytic actinobacteria production of ACC deaminase and how this might result in PGP effects when plants are under salinity stress.
Apart from ACC deaminase production, strain KLBMP 5084 possessed a series of other traits that are important for plant growth promoting activities, including phosphate solubilization and siderophore production (Nadeem et al. 2014). Endophytic bacteria with the capacity for nitrogen fixation have been isolated from many plants, and they can provide this limiting nutrient to their host plants. N2-fixation is one of the best characterized traits of many PGP rhizobacteria (Dastager et al. 2011). Phosphate solubilization by plant-associated growth promoting microorganisms has also been well documented. Application of these strains can promote the phosphate mobilization and improve P supply for plants (Jha and Kumar 2009). Actinomycete siderophore production is an important factor for phytopathogen antagonism and may have indirect PGP effect on plants (Franco-Correaa et al. 2010). Luo et al. (2012) found that endophytes that produced ACC deaminase, siderophore and IAA are critical to plant growth. From the bacterial point of view, the present results are in consistent with early reports that the PGP effect by KLBMP 5084 is attributed to its multiple PGP traits (El-Tarabily 2008; Palaniyandi et al. 2014; Qin et al. 2015), which was also demonstrated by its genome analysis.
Strain KLBMP 5084 was also found to be salt tolerant and exhibit characteristics related to heavy-metal resistance (Table 1), which suggests it could potentially be used as a PGP agent for phytoremediation of coastal heavy metal polluted soils. It utilized a series of plant derived compounds as carbon sources for growth, such as cellulose, xylose, and glucose. Previous studies have demonstrated that plant associated endophytes can utilize the carbon sources of plants to thrive within their hosts (Taghavi et al. 2009; Luo et al. 2012). The effective re-isolation of KLBMP 5084 in our experimental plants suggests that this strain successfully colonized the roots of host plants. However, because of the absence of unequivocal microscopic or GFP-tagged molecular proof, we refer to this strain as a putative endophyte.
Plant leaf chlorophyll concentration is an indicator of salt tolerance and responds to increasing salinity (Habib et al. 2016). Studies have demonstrated that plants produce less chlorophyll when exposed to salinity stress due to chlorophyll peroxidation (Han et al. 2014; Akram et al. 2016). In this study, increasing the salt concentration from 0 to 250 mM had the effect of progressively decreasing the leaf total chlorophyll contents in both control and inoculated plants. However, the leaves of inoculated seedlings contained higher chlorophyll content under both 100 and 250 mM NaCl salt conditions, which indirectly indicated improved photosynthetic activity. Our findings are congruent with the results of Singh et al. (2015) who reported an increase in total biomass and chlorophyll content of wheat after treated with the halotolerant PGPB strain Klebsiella sp. SBP-8.
Proline is a known osmoprotectant that also plays antioxidant role under NaCl stress. Hyperaccumulation of free proline in plants is known in conferring salinity tolerance (Kaur et al. 2016). Proline accumulation has been reported as one of the mechanisms of stress alleviation by PGP bacteria (Kim et al. 2014; Akram et al. 2016). In our study, leaf proline content was higher under NaCl stress and KLBMP 5084-inoculated L. sinense leaves showed the highest accumulation of proline (Fig. 3), which probably served to protect plant cells from oxidative damage.
Lipid peroxidation is estimated through the accumulation of MDA, which has been used as the criterion to determine the sensitivity of plants to stress conditions. The significantly reductions in leaf MDA content by strain KLBMP 5084 inoculation (Table 2) compared with non-treated plants suggest its capability to prevent soil salt induced lipid peroxidation of leaf cell membranes under salt stress. Our results are in agreement with findings from other studies (Xun et al. 2015; Pereira et al. 2016) that plants treated with PGP rhizobacteria show less accumulation of MDA.
The salinity-induced increase in ROS-scavenging enzymes, such as SOD, POD, CAT and ascorbate peroxidase (APX), is a common mechanism for detoxifying the plant of ROS (Kumar et al. 2015). Inoculated seedling leaves under both NaCl treatments (100 and 250 mM) showed significant increases in SOD, POD and CAT in comparison with control plants, indicating the induction of oxidative damage repair mechanisms by endophyte KLBMP 5084 (Table 2).
Salt tolerance is associated with a plant’s ability to avoid Na+ accumulation and promote K+ influx (Shabala and Cuin 2008; Pereira et al. 2016). Our data showed that both leaf and root Na+ increased with rise in NaCl concentrations. However, the endophyte-inoculated plants (both leaves and roots) showed significant decrease in Na+ content, and increase in K+ uptake compare to control, therefore increasing the K+/ Na+ ratios in both leaves and roots of L. sinense under salinity stress, which is shown for some species to be as more important as maintaining a low level of Na+ in plant tissues (Maathuis and Amtmann 1999). Our results are in accordance with the findings of Niu et al. (2016), who reported a significant increase in K+ and decrease in Na+ accumulation when the halophyte grass Puccinellia tenuiflora was inoculated by beneficial bacteria GB03. However, whether strain KLBMP 5084 improved salinity tolerance of L. sinense by host specific regulation of related ion transportation genes expression merits further exploration.
The genome sequencing and annotation of strain KLBMP 5084 provided useful information for interpreting its PGP mechanisms. Nitrogen-fixing bacteria have been isolated for many years using different formulations of N-free semi-solid media (Baldani et al. 2014). However, this method can provide false-positive results. Although KLBMP 5084 contains a gene (nifU) involved in the nitrogen fixation, the real ability of nitrogen-fixing of strain KLBMP 5084 still needs to be confirmed using acetylene reduction assays or other methods. Strain KLBMP 5084 also contains genes attribute to ACC deaminase and IAA production, HCN, siderophore synthesis and phosphate solubilization. These genes are all considered to have PGP effects (Nadeem et al. 2014). Besides the direct PGP effects, PGP rhizobacteria can enhance plant development indirectly by suppressing pathogens (Santoyo et al. 2016). The genus Streptomyces is well known for its capacity of producing a large number of biological compounds. In the genome of strain KLBMP 5084, we identified many genes to be responsible for the synthesis of antimicrobial compounds, including chitinase, phenazine, γ-aminobutyric acid, and many secondary metabolites biosynthesis gene clusters for antibiotics synthesis. The presence of these genes could better explain the antifungal pathogens activities of endophyte KLBMP 5084 (unpublished). Strain KLBMP 5084 was isolated from the salt marsh plant and can tolerate up to 7% NaCl. The genes involved in Na+/H+ antiporter, K+ transporter, glycine-betaine transport protein, trehalose and antioxidant enzymes biosynthesis were detected in the genome, which may help strain KLBMP 5084 adapt to salt environmental conditions. The genome data support the phenotypic characteristics of strain KLBMP 5084 and host plant physiological response after endophyte inoculation under salt stress as we observed in this study.
Conclusion
Plants growing on coastal salt marsh are exposed to extreme conditions such as high salt concentration and low organic matter content. Thus, application of PGP organisms may benefit the growth and environmental tolerance of halophytes in these harsh environments. The halotolerant endophyte Streptomyces sp. KLBMP 5084 has multiple potential PGP traits and genomic analysis showed evidence that it may be resistant to multiple heavy metals. Inoculation with Streptomyces sp. KLBMP 5084 effectively promoted L. sinense growth, increased photosynthetic activity and cellular antioxidant activities (SOD, POD, CAT and proline), and decreased the lipid peroxidation, reactive oxygen species and Na+ accumulation under salt stress. Genomic analysis of KLBMP 5084 further supports its potential as a PGP strain. In conclusion, this study provides physiological and genomic evidence that Streptomyces sp. KLBMP 5084 contributes to the salt tolerance of the halophyte L. sinense. The results from our study help elucidate the functional roles of endophytic actinobacteria associated with coastal halophytes, and may be useful for developing biofertilizers from beneficial endophytes to improve plant growth in areas with high soil salinity. Future research should focus on understanding the molecular mechanisms of the PGP traits of strain KLBMP 5084 under salt stress.
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Acknowledgements
This research was supported by grant from the National Natural Science Foundation of China (No.31370062), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Sheng Qin was supported by the Qing Lan Project of Jiangsu Province (2014). The authors are grateful to the reviewers for their valuable comments on the manuscript.
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Sheng Qin and Wei-Wei Feng contributed equally to this work.
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Qin, S., Feng, WW., Wang, TT. et al. Plant growth-promoting effect and genomic analysis of the beneficial endophyte Streptomyces sp. KLBMP 5084 isolated from halophyte Limonium sinense . Plant Soil 416, 117–132 (2017). https://doi.org/10.1007/s11104-017-3192-2
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DOI: https://doi.org/10.1007/s11104-017-3192-2