Plant growth-promoting rhizobacterium Pseudomonas PS01 induces salt tolerance in Arabidopsis thaliana

Objectives Plant growth-promoting rhizobacteria (PGPR) may contribute to sustainable crop production by improving plant growth and/or plant tolerance to abiotic stresses. Soil salinity, which limits the productivity of crop plants, is one of the major concerns of modern agriculture, especially in countries heavily affected by climate change as Vietnam. Currently, only a few reports have studied local PGPR isolated in Vietnam, particular Pseudomonas. Therefore, our study aimed to isolate and identify a region-specific Pseudomonas strain and evaluate the effects of this strain on germination, growth promotion and gene expression of Arabidopsis thaliana under salt stress. Results The Pseudomonas named PS01 was isolated from maize rhizosphere collected in Ben Tre province, Vietnam. This strain was identified as a member of the Pseudomonas putida subclade. Pseudomonas PS01 could improve the germination rate of Arabidopsis seeds in 150 mM NaCl. A. thaliana plants inoculated with Pseudomonas PS01 survived under salt stress conditions up to 225 mM NaCl, while all non-inoculated plants were dead above 200 mM NaCl. The transcriptional levels of genes related to stress tolerance showed that only LOX2 was up-regulated, while APX2 and GLYI7 were down-regulated in inoculated plants in comparison to the non-inoculated controls. In turn, RD29A and RD29B did not show any significant changes in their expression profiles. Electronic supplementary material The online version of this article (10.1186/s13104-019-4046-1) contains supplementary material, which is available to authorized users.


Introduction
Soil salinity is a widespread problem that limits crop yield and cultivation throughout the world, including the Mekong Delta, Vietnam [1]. Salinity creates ion imbalance and generates highly reactive oxygen species (ROS) in plants, which causes ion toxicity and oxidative stress [2,3]. This, in turn, leads to plant growth inhibition, slower development, senescence and death. To improve plant salinity tolerance, several strategies, such as the use of fertilizers, traditional breeding and genetic engineering, have been extensively studied for decades [4]. The application of plant growth-promoting rhizobacteria (PGPR) is one of the most promising alternative approaches to improve crop production in saline soils [2,4,5]. Various salt-tolerant PGPR including Azospirillum, Burkholderia, Rhizobium, Pseudomonas, Acetobacter and Bacillus have been successfully applied or tested for plant growth promotion under salt stress [6,7]. The fluorescent Pseudomonas is considered an important model to assess beneficial plant-bacteria interactions, including plant growth promotion under abiotic stress [8,9]. Inoculation of plants with Pseudomonas was found to alleviate salinity effects on plant development by reducing the uptake of toxic ions, inducing systemic resistance, producing phytohormones, increasing nutrient uptake and establishing root colonization [10][11][12][13][14]. Pseudomonas-induced salt tolerance has been mainly studied at the physiological and biochemical levels in plants. However, little is known about the transcriptional changes of plant saltresponsive genes in an interactive process [14].
To cope with salt stress, early plant responses include synthesis of ROS scavengers, detoxification of ROS and abscisic acid (ABA) signaling [15][16][17]. The glyoxalase pathway, which degrades methylglyoxal, is one of the main detoxification pathways [18]. In the ABA response, the expression of RD29 (Responsive to Desiccation) genes including RD29A and RD29B is induced by salt stress [19]. PGPR may significantly enhance plant antioxidant activities, and thus protect plants from salt toxicity, by increasing the expression of enzymes such as ascorbate peroxidase (APX), superoxide dismutase (SOD) and catalase [16,19,20]. Interestingly, A. thaliana inoculated with PGPR such as Burkholderia phytofirmans PsJN and Enterobacter spp. EJ01 showed an enhanced tolerance to salt stress that involved transcriptional changes of genes related to early stress responses [16,19].
It is necessary to identify native microbial strains which can be used in regional crops as potential plant growth promoters to achieve desirable yields [21]. The application of indigenous PGPR will provide more advantages for regional crops since PGPR can easily acclimatize to the local environmental conditions and enhance the plant-microbe interactions [22]. In previous study, we successfully isolated and identified some Pseudomonas spp. capable of enhancing plant growth in Vietnam [23]. Therefore, in this study, we aimed to isolate a Pseudomonas strain that can increase salt stress tolerance and to investigate the underlying molecular mechanisms.

Bacterial isolation
Rhizobacteria were isolated from maize (Zea mays L.) rhizosphere collected in Ben Tre province, Vietnam. A soil suspension was obtained by shaking roots with adhered soil in phosphate-buffered saline for 10 min at 180 rpm. The suspension was serially diluted, spread onto King'B medium (KB) [24] and incubated at 30 °C for 24 h. A single colony was picked up and re-cultured a few times on solid KB to obtain a pure culture. The presence of fluorescent Pseudomonas was examined under UV light. Total isolates were grown on different concentration NaCl (0-10%) to evaluate salt tolerance property.

Bacterial identification
For phenotyping, the bacterial strain was identified according to morphological and chemotaxonomic characters based on the Bergey's Manual of Determinative Bacteriology. For genotyping, bacterial genomic DNA was extracted and purified using the Wizard Genomic DNA purification kit (Promega, USA). The complete 16S rDNA was amplified by using PCR with the universal bacterial primers 27F and 1492R (Additional file 1: Table S1) [25]. The PCR product was sequenced and analyzed with BLASTn to identify the strain genus. Simultaneously, a fragment of rpoD gene was amplified by using the primers rpoD 70F and rpoD 70R (Additional file 1: Table S1) [26,27]. The sequences of related species and genera were obtained from GenBank. Multiple sequence alignments were performed by ClustalX; the phylogenetic analysis was determined by employing the neighbor-joining method. The phylogenetic tree was constructed with MEGA version 6 Software [28].

Plant growth conditions and treatments
To define whether the bacteria had an effect on the germination of Arabidopsis in normal and saline condition, sterilized and synchronized seeds were inoculated with bacterial suspension (10 6 CFU/ml), or MgCl 2 solution as a control or nongrowth-promoting strain Escherichia coli DH5α as a negative control, and germinated on solid, half-strength Murashige and Skoog medium (MS ½) plates with or without 150 mM NaCl. Plates were placed in a growth chamber at 22 °C with a photoperiod of 16/8 h (light/dark). Four days after sowing (DAS), the seed germination percentage was determined.
To investigate the effect of Pseudomonas PS01 on Arabidopsis salt tolerance under different NaCl concentrations, 4-day-old seedlings were transferred to solid MS ½ supplemented with different concentrations of this salt. Plates were placed vertically in a growth chamber at 22 °C with a photoperiod of 16/8 h (light/dark). After 7 days, the plant survival rate was determined.

RNA extraction and real-time PCR (RT-PCR) analyses
RNA extraction was performed on plantlets after being transplanted to MS ½ alone or supplemented with 150 mM NaCl for 24 h. RNA was obtained by using the Trizol (Invitrogen ™ , USA) method. RT-PCR was performed by using the Luna Universal One-Step RT-PCR Kit (New England Biolabs, USA). PCR primers are shown in Additional file 2: Table S2. The relative transcript level (RTL) was calculated by normalizing to ACT2 as follows: RTL = 2 ∆∆Ct , where ∆∆Ct = ∆Ct(gene) − ∆Ct(ACT2). All experiments were performed with three biological and two technical replicates.

Statistical analysis
For comparison between treatments, ANOVA was performed with Graphpad Prism 7.0.

Identification of bacteria
Seventeen rhizobacterial strains were isolated from maize rhizosphere. The salt tolerant properties results revealed that out of 17 bacterial isolates tested; only isolate named PS01 was able to grow in the presence of 8% NaCl (Additional file 3: Fig. S1). Based on its growth curve, PS01 strain has an optimal growth temperature of 30 °C. Morphological and chemotaxonomic analyses revealed that PS01 is rod-shaped, Gram-negative, aerobic, non-sporeforming, catalase-positive and oxidase-positive, and fluoresces under UV light at 365 nm (Additional file 4: Fig.  S2). The BLAST search of 16S rDNA against the Gen-Bank indicated that PS01 is most similar to Pseudomonas spp. In this genus, the rpoD gene has been identified as one of the best biomarkers for gene phylogeny, which correlates well with that of the 16S rRNA gene [26,27,29,30]. Therefore, rpoD was used to identify PS01 in our study. The phylogenetic tree of rpoD gene indicated that PS01 belongs to the Pseudomonas putida subclade (Fig. 1).

Pseudomonas PS01 enhances seed germination rate in salt stress conditions
To test whether PS01 could enhance the germination rate of A. thaliana, its effect was examined in MS ½ media with or without 150 mM NaCl. We observed that the germination rate in the 150 mM NaCl treatment was significantly increased in PS01-inoculated seeds when compared to the control. PS01-treated A. thaliana seeds showed 30.7% germination rate, while this value was only 9.5% in the control (Fig. 2a). However, no significant difference in the seed germination rate could be observed between the inoculated and non-inoculated seeds in MS ½, suggesting that the PS01 treatment has no effect on A. thaliana germination in normal conditions.

Pseudomonas PS01 enhances salt stress tolerance
To address the effects of NaCl and PS01 on A. thaliana salt tolerance in vitro, the survival rates (%) of PS01inoculated and non-inoculated plants grown on media supplemented with NaCl concentrations ranging from 75 mM to 225 mM were evaluated. On the 7th day after being transferred to the salt stress media, Arabidopsis seedlings were observed and photographed to identify the number of surviving plants.
PS01 root colonization was shown to increase the survival of plants exposed to saline concentrations ranging from 75 to 225 mM NaCl (Additional file 5: Fig. S3). For instance, all plants inoculated with PS01 could survive the 175 mM NaCl treatment as opposed to only 30-40% of the controls (Fig. 2b). These results suggest that Pseudomonas PS01 may enhance A. thaliana survival under salt stress.

Pseudomonas PS01 induces transcriptional changes in salt-stressed A. thaliana plants
To investigate the molecular mechanisms of PS01induced salt stress tolerance in Arabidopsis, some genes related to early salt stress responses such as ROS scavenging (APX2), detoxification (GLYI7), ABA signaling (RD29A and RD29B) and JA synthesis (LOX2) were After 24 h salt stress, the transcriptional expression of all five genes was remarkably up-regulated compared to the control (seedlings grown on MS ½ alone). The analyses showed no differences in RD29A and RD29B expression in PS01-inoculated and non-inoculated seedlings under the NaCl treatment. By contrast, LOX2 expression was up-regulated in PS01-inoculated, salt-stressed plants, while APX2 and GLYI7 were significantly downregulated (Fig. 3).

Discussion
PS01 is the first Pseudomonas strain isolated in Vietnam which alleviates the effect of salinity on plant growth. PS01 belongs to the Pseudomonas putida subclade based on the rpoD gene tree (Fig. 1), being closely related to P. taiwanensis (GenBank accession number HE577796.1). Interestingly, P. putida was already known to reduce the detrimental effect of salinity on germination and plant growth [7,31]. In this study, Pseudomonas PS01 improved the germination rate of A. thaliana on salt stress medium (MS ½ supplemented with 150 mM NaCl). Compared to previous studies focusing on the effect of bacteria on plant growth, the germination process was accelerated and its period was also extended [32]. For example, P. putida R4 and P. chlororaphis R5 improved cotton seed germination in response to NaCl stress up to 64 and 73%, respectively [7]. In contrast, Pseudomonas spp. PDMZnCd2003 negatively affected rice germination, and reduced seedling shoot and root length [33]. We have shown that PS01 helps improve salt stress tolerance in A. thaliana seedlings. Under salt stress conditions, the transcriptional level increase of GLYI7 and APX2 in seedlings inoculated with PS01 was lower than that of non-inoculated seedlings. Glyoxalase can be considered as an alarm in the abiotic stress response in plants [34]. In turn, ascorbate peroxidases catalyze the conversion of H 2 O 2 into H 2 O. The down-regulation of GLYI7 and APX2 suggests that inoculation with PS01 may reduce stress level in plants. Therefore, the mechanisms contributing to the survival of inoculated plants during salt stress can be related to other molecular pathways. Alternatively, the production of biofilms by the bacteria could be beneficial to plant survival under stress conditions as shown in recent reports, in which bacterial exopolysaccharide (EPS) and biofilm formation stimulated plant growth under salt stress by reducing Na + uptake by the plant [20,35]. However, further evidences for EPS production or other putative mechanisms participating in the PS01-mediated salt tolerance of plants need to be verified. Jasmonate (JA) is a positive regulator of the salt stress response that accumulates rapidly when plants are submitted to stress [20,36]. LOX2 encodes a lipoxygenase that constitutes an essential component of the JA synthesis pathway [37]. Compared to non-inoculated seedlings in response to salt stress, the increase of LOX2 expression in PS01 inoculated seedlings in our study is in agreement with Cho et al. [14] and Poupin et al. [38], who also reported that PGPR such as Pseudomonas chlororaphis O6 and Burkholderia phytofirmans PsJN mediated systemic resistance against abiotic stress by increasing the expression of defense genes regulated by the JA pathway [14,38]. The phenotypic changes, transcriptional profile and spatiotemporal responses under salt stress of plants inoculated with PS01 will be analyzed at different plant growth stages to clarify this pathway. Fig. 3 Gene expression analysis of abiotic stress-responsive genes of Arabidopsis seedlings inoculated with Pseudomonas PS01 after 24 h of being transferred to salt stress conditions. The transcriptional levels of RD29A, RD29B, LOX2, APX2 and GLYI7 of PS01-inoculated and non-inoculated A. thaliana seedlings grown in MS ½ alone or transferred to MS ½ supplemented with 150 mM NaCl are represented. Experiment was performed with three biological and two technical replicates (*) and (**) indicate the significant difference between both treatments (150 mM NaCl vs 150 mM NaCl + PS01 inoculated) at p value < 0.05 and p < 0.01, respectively

Conclusion
Our study has shown that Pseudomonas PS01 can improve Arabidopsis thaliana germination and survival rate under salt stress.

Limitations
Although, mechanism underlying PS01-Arabidopsis interaction to enhance plant salt tolerance has not been fully discovered, further studies have been conducting in our group to provide new insights into this interaction including whole genome analysis and transposon mutant library screening of PS01, as well as long-term transcriptomic analysis of plants inoculated with this isolate. PS01 will be inoculated with maize under salt stress and be developed as a biofertilizers in the future prospect.

Additional files
Additional file 1: Table S1. Primers used to amplify 16S DNA and rpoD gene.
Additional file 2: Table S2. List of RT-PCR primers used in this study.
Additional file 3: Figure S1. Growth of Pseudomonas PS01 on TSB medium at different NaCl concentrations: 0% (A), 2% (B), 4% (C), 6% (D), 8% (E) and 10% (F). Authors' contributions TNC, BTHT and MTTH designed this research, performed the experiments, analyzed the data and wrote the manuscript. LVB reviewed of the literature and provided technical assistance to TNC. All authors read and approved the final manuscript.