Sodium-resistant plant growth-promoting rhizobacteria isolated from a halophyte , Salsola grandis , in saline-alkaline soils of Turkey

Ryota Kataoka a, Esra Güneri b, Oguz Can Turgay b,*, Ahmet Emre Yaprak c, Bahar Sevilir b, Isa Başköse c a University of Yamanashi, Faculty of Life & Environmental Sciences, Department of Environmental Sciences, Takeda, Kofu, Yamanashi, Japan b Ankara University, Faculty of Agriculture Department of Soil Science and Plant Nutrition, Ankara, Turkey c Ankara University, Faculty of Science Department of Biology, Ankara, Turkey


Introduction
The introduction of irrigation systems has raised agricultural productivity in dry and semi-dry regions.Although only 18% of the cultivated area is irrigated globally, 40% of global food production comes from irrigated agriculture (Siebert et al., 2005).In these areas soil salinization is a major problem and leads to low agricultural productivity.Soil salinity is a worldwide problem affecting about 20% of the world's cultivated land and nearly half of all irrigated land (Zhu, 2001).According to the FAO/UNESCO soil map of the world, approximately 1.5 million hectares of land in Turkey are suffering from salinity and sodicity problems (Kendirli et al., 2005).
Phytoremediation has attracted rising attention as a suitable technology that can be applied not only in polluted but also in saline soil remediation because of its low economic cost and environmentally friendly process (Qadir et al., 2002(Qadir et al., , 2003)).Some reports suggest that the harvested plant, if not plowed back to the same soil, can contribute significantly to the removal of salts such as sodium (Na) from salt-affected soils (Ravindrana et al., 2007;Qadir et al., 2007).The genus Salsola (Chenopodiaceae) comprises about 120 species; they are widespread herbaceous or shrubby halophytic plants, especially in the brackish grounds of the moderate and subtropical regions of Europe, Asia, Africa and North America (Tundis et al., 2008).Salsola soda, typically grows in coastal regions and saline conditions, was used as a companion plant because of its high accumulation of Na (Colla et al., 2006;Zuccarini, 2008).In 1999, Salsola grandis, a species closely related to S. soda, was reported as a new species of genus Salsola (Freitag et al., 1999).This plant is an annual halophytic plant growing in open habitats with sparse vegetation where local semi-desert climate creates an extreme saline soil habitat (eg.pH over 9 and Na over 3500 mg•kg -1 ) that forms on highly clayey salt-containing marly substrate, in Nallıhan Region, Ankara Province, Turkey.To the best of our knowledge, there is no information on how this plant manages abiotic stress factors and survives in such a harsh environment and also no previous report about its Na uptake from soil.
Various soil microorganisms were shown to tolerate unwanted elements in soil using various mechanisms including exclusion, active removal, biosorption, precipitation, and extra or intracellular bioaccumulation (Silver, 1996;Whiting et al., 2001;Lasat, 2002;Gadd, 2004).These processes are known to effect the solubility and the bioavailability of toxic elements, such as heavy metals, to plants and therefore interactions between hyperaccumulator plants, metals and plant growth-promoting rhizobacteria (PGPR) have gained increasing attention for the last two decades (Turgay and Bilen, 2012).PGPR are free-living soil bacteria that can either directly or indirectly facilitate rooting and growth of plants.These bacteria serve the functions of N-fixation, production of siderophores, solubilization of minerals like phosphorus and synthesis of phytohormones such as indole-3-acetic acid (IAA).To date, some bacteria such as Klebsiella sp.D5A showed the high plant growth promoting activity on the glycophytic crop in saline-alkaline soils (Liu et al., 2014;Liu et al., 2016).Likewise, many studies have been published on beneficial effects of bacterial application on growth of wheat under salt stress (Ashraf et al., 2004;Sadeghi et al., 2012;Upadhyay et al., 2012;Chakraborty et al., 2013;Paul and Lade, 2014).
According to Ruppel et al. (2013), "micro-organisms use similar approaches to cope with oxidative stress and it is likely that microorganisms use the same mechanisms to alleviate salt stress effects in halophytes as in other tolerant plants.However, little is known about the effect of PGPR on the salt accumulation in halophytic plants.The application of PGPR may prove useful when developing strategies to facilitate plant growth in saline soils.Therefore, in the present study, we studied Na-resistant rhizobacteria isolated from the rhizosphere of a halophytic plant, S. grandis, and a halotolerant plant, wheat (Triticum aestivum).The aims of this study were to explore Na-resistant indigenous PGPR on S. grandis, and determine the effect of these isolates on the salt accumulation in S. grandis.

Sampling sites and soil properties
The seeds and five seedlings (2-3 month old) of Salsola grandis Freitag, Vural and N. Adıgüzel (Amaranthaceae) were collected from harsh ecological conditions which is including raw gypsum and saltcontaining marly substrate located at 28km east-southeast of Nallıhan region (40°05ʹ29.57ʺN,031°36ʹ10.26ʺE) in Ankara Province in November 2015 and in April 2016, respectively.The seeds collected were stored in paper bags at 4°C until use and the seedlings were used to isolate bacteria from rhizosphere soil on the next day after sampling.For the greenhouse experiment, soil was collected from a depth of 0-20 cm in a non-agricultural area (38°20ʹ08.6ʺN,033°59ʹ06.5ʺE)located at the campus of Aksaray University, Aksaray Province, Turkey.This soil was air-dried and stored in room conditions prior to soil analysis and the pot experiment.The soil characteristics are outlined in Table 1.

Pot experiment
We conducted a pot experiment, setting up S. grandis and Triticum spp., in triplicate for 82 days on a soil collected from Aksaray district to examine Na uptake of S. grandis.Triticum spp.(wheat) was used as control to compare the Na uptake with S. grandis.Five seedlings (82 days old) of S. grandis per pot and three seedlings (82 days old) of wheat per pot were grown at a greenhouse (Ankara University, Ankara, Turkey).The analysis of the Na concentration in S. grandis and wheat was performed as follows.Harvested plants were washed carefully using tap water and then distilled water, and then stem-leaves and roots were separated.After the measurement of the fresh weight, all plant samples were dried at 60°C, and their dry weight was measured.Then, the plants were ground and dry ashing was conducted in a muffle furnace at temperatures of 500 ± 50°C for approximately 20 h.The ash was dissolved in 10 M of nitric acid (HNO3) and the extract solution was analyzed using ICP-OES (Optima 2100 DV; Perkin Elmer, Inc., Shelton, CT, USA) (Kacar and Inal, 2010).

Isolation of rhizobacteria from Salsola grandis and wheat
Bacteria were isolated from 0.5 g of rhizosphere soils of S. grandis and wheat taken from the pot experiment mentioned above and that of naturally growing S. grandis taken from Nallıhan.The soil samples were mixed with 5 ml of sterile distilled water with a sufficient vortex and then diluted to 10 − 3 , 10 − 4 , and 10 − 5 .We then placed 50 µl of the resulting soil solutions onto plates of nutrient agar and of potato dextrose agar adjusted to pH 7.0.The plates were then incubated at 25°C for 3 days.

Screening of Na-resistant bacteria
Potato dextrose broth was adjusted to pH 7.0 using 1M potassium hydroxide (KOH) and the media with Na (750 mM) and without Na (0 mM) were prepared we then divided 5 ml of the media into 15-ml centrifugation tubes.Na concentration was set referring to Hotchkiss (1923).Individual bacteria were inoculated to each tube and incubated at 25°C for two days.After incubation, the optimal density for bacterial growth was measured at 600 nm.The strains which grew to more than half the number of those growing in the control (0 mM Na) tubes were selected as Na-resistant bacteria.

Screening of PGPR
In order to handle high number of strains in experimental condition, a sterile conical tube was set to test 50 strains with 4 replications at the same time in terms of microbes-Na uptake association.Saline soil collected from Aksaray was sieved (<2 mm) and autoclaved at 121 0 C for 120 min, and then 20 g of the sieved soil was put into a 50-ml conical centrifuge tube treated with 0.5 ml mineral nutrient solution (N, 100 mg kg -1 ; P, 100 mg kg -1 ; K, 125 mg kg -1 ).The bacterial suspension was adjusted to an optimal density of 1.0 at 600 nm.It was then inoculated with 1 ml bacterial suspension into each tube.The seeds were soaked in 0.5% of sodium hypochlorite (NaOCl) for 2 min for brief surface-sterilization and then rinsed using distilled water.Seeds were then individually transplanted into the tube and we prepared four replications for each bacterial isolate.The seedlings were grown in the plant growth chamber (15°C / 25°C night/day temperature, 55% relative humidity and 12 hours photo period) and harvested at 35 days of the plant development.The yield of shoots and roots was determined based on fresh and dry weight.Moreover, the Na concentration in the harvested plant was also measured using ICP-OES.The data obtained were statistically analyzed using Student's t-test to compare the plant growth between seedlings with and without each bacterial species (P < 0.05).

Determination of IAA
The cultures were grown for 4 days at 25°C in IAA production media (30 g of glucose, 2g of beef extract, 3g of CaCO3, 1mM (final concentration) of tryptophan, pH 7, 1 L of distilled water) and centrifuged at 10000g for 10 min.The IAA was determined in 300 µl of supernatant using 1.2 ml of Salkowski's reagent at 535 nm (Acuña et al., 2011).

Determination of phosphate solubilization
The media developed by Pikovskaya (1948) was used for quantitative estimation of tri-calcium phosphate solubilization by the isolates.Strains selected as PGPR were inoculated to the media and incubated at 25°C for 7days.As evaluation of P solubilization, we used following index; -No clear zone, ± detectable of the clear zone but very weak activity, + detectable of clear zone.

Siderophore production
Chrome azurol S (CAS) medium was prepared using a slightly modified method of Schwyn and Neilands (1987) and Pérez-Miranda et al. (2007).The medium for 100 ml of overlay was prepared as follows: CAS 6.04 mg, hexadecyltrimetyl ammonium bromide (HDTMA) 7.3 mg, Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) 3.04 g, and 1 ml of 1 mM FeCl3•6H2O.Siderophore detection was achieved after 10 mL overlays of this medium were applied over the agar plates containing cultivated microorganisms to be tested for siderophore production.The CAS-blue agar changed to light orange or yellow if bacteria produce the siderophore.− No color change, + change the color, ++ color change detected in all over the medium.

16S rRNA analysis of selected bacteria
To identify the isolated microorganisms, the nucleotide sequences of their 16S rDNA were investigated using molecular techniques.DNA was extracted using 100 μl of 2 × CTAB buffer [2.0% (w/v) CTAB; 1.4 M NaCl; 100 mM Tris-HCl (pH 8.0); 20 mM EDTA (pH 8.0)].After vortexing, the sample solution was incubated for 30 min at 60°C.An equal volume of chloroform was added to the sample solution and mixed to emulsify the mixture.The mixture was centrifuged at 15300g for 20 min at room temperature, and the supernatant was transferred to a new tube.A 2.5 times volume of ethanol was added and the mixture was incubated at room temperature for 20 min, then centrifuged at 15300g for 20 min at 4°C.The resulting DNA pellet was rinsed with 70% ethanol and dried.Each dried DNA pellet was dissolved in 50 µl sterilized distilled water.The 16S rRNA gene (700 bp) of each isolate was amplified by a T100™ Thermal Cycler (Bio-rad, CA, USA), with 1 cycle of 95°C for 5 min, 30 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 1 min and a final extension at 72°C for 7 min.The 40-µl polymerase chain reaction (PCR) mixtures contained 1.5 µl DNA extract, 1.5 µl of 10 mM each primer; 800F (GGATTAGATACCCTGGTA) and 1500R (TACCTTGTTACGACTT), 10 µl of 5×FIREPol ® MasterMix (SolisBioDyne, Tartu, Estonia).The nucleotide sequences of PCR-amplified fragments were determined by PCR-direct sequencing.The nucleotide sequences determined were compared with those in GenBank.All sequence data, including newly obtained and retrieved sequences, were aligned using the computer program BioEdit (available at http://www.mbio.ncsu.edu/BioEdit/bioedit.html).Distancebased phylogenetic trees were generated using the model of Jukes and Cantor (1969) and a neighbor-joining algorithm (Saito and Nei, 1987).The topology of phylogenetic trees was evaluated by bootstrap resampling (1000 replicates).Clustal W, provided by the DNA Data Bank of Japan (available at http://www.ddbj.nig.ac.jp/Welcome-j.html), was used for the analyses.

Pot experiment
The pot experiment was conducted to confirm the Na uptake and allocation by S. grandis.The Na uptake by S. grandis was compared with wheat in this study.The mean dried weight after the growth period of 82 days was 12.4 mg and 6.36 mg for S. grandis and wheat, respectively.The growth of stems and leaves in S. grandis were higher than those of wheat, but root biomass showed the opposite result (Table 2).Under Na uptake, S. grandis transported high amounts of Na to the stem and leaves from the roots (Table 2), whereas wheat retained Na in the roots.
A total of 131 strains-50, 50 and 31 strains from rhizosphere of wheat, S. grandis (Aksaray soil) and S. grandis (Nallıhan soil), respectively-were found during the screening of Na-resistant bacteria.In the screening of Na-resistant bacteria, a total of 36 strains were able to grow more than 80% above the growth levels of the control in the medium with 750 mM NaCl. 14 strains were grown in the medium with 750 mM of NaCl than the control (Figure 1).

Screening of PGPR
The plant growth promotion effect of the strains was shown in Figure 2 and Table 3.Total eight strains of bacteria isolated from the rhizosphere of S. grandis (strains AP20, B14, A22, B2 and A3) and wheat (strains WN4, WP18 and WP5) enhanced the growth of S. grandis, but little variation was observed in bacterial effectiveness among parameters (Table 3).Differences between treatments in the dry weight of stem and leaves were statistically greater than the control in all cases except for strain AP20.The strains A3 and A22 showed significant increases in the fresh weight and dry weight, and means of dried stem and leaves of S. grandis in tubes inoculated with these strains increased 15.5% and 17.4%, respectively, above the nonbacterial control.Moreover, the mean dried root weight increased 53.9% and 59.2% in strains A3 and A22, respectively, relative to the non-bacterial control.There was no significant difference between the control and other bacterial treatments (Table 3).For PGPR traits, all strains showed a negative activity on the phosphate solubility, but there was a positive activity on the siderophore and IAA production in most strains.Strains A22, WP5, B14 and AP20 produced more than 19 mg L −1 of IAA after the 4-day incubation (Table 4).All strains were either siderophore producers or IAA producers.Four strains out of the eight strains (A22, WP5, B14, AP20) showed both traits of siderophore and IAA producers.16SrRNA sequencing analysis

Discussion
In the present study, we revealed that Salsola grandis was able to uptake Na from soil and located it in aboveground plant tissues in higher amounts.Moreover, eight new PGPR, seven strains of Arthrobacter spp.and one strain of Bacillus sp., were isolated from rhizosphere soils.
S. grandis was reported as a new species of genus Salsola, and it is an annual halophytic plant which is distributed in Nallıhan region as the region has semi-arid climate conditions and clayey soil environments (Freitag et al., 1999).However, there has been no previous report about the Na uptake from soil by this plant.It is said that the halophytes are associated with a correlation between cation accumulation and plant succulence (Maimaiti et al., 2012), and S. grandis is also succulent.Moreover, Salsola soda, a relatively closely related species, is also able to uptake Na at the value of around 45,980 mg kg −1 (Hasanuzzaman et al., 2014).Thus S. grandis also has a high potential to uptake Na.In this study, we revealed that S. grandis is able to uptake Na, as shown in Table 2.We tried to show the sodium accumulation of S. grandis in comparison with that of wheat.Wheat (Triticum aestivum L.) is moderately salt-tolerant and one of the most important staple crops in the world (Maas and Hoffman, 1977).Moreover, it occupies 32.8% (7860,000 ha in 2015) in the cultivated land in Turkey.The comprehensive surveys conducted on salt tolerant crops indicated that wheat can tolerate and grow with no yield loss up to salinity level of 6-8 dS m −1 EC corresponding 60-80 mM NaCl.
All bacteria isolated and screened as PGPR were able to grow under the medium with 750 mM of NaCl.In the sequencing analysis, seven out of eight strains belonged to genus Arthrobacter.Strains of Arthrobacter species are among the most frequently isolated indigenous aerobic bacterial genera found in soils.Moreover, there are some reports that indicate Arthrobacter spp.has high levels of resistance to not only a variety of toxic metals (Hanbo et al., 2004;Megharaj et al., 2003;Henn et al., 2009) but also NaCl (Upadhyay et al., 2009(Upadhyay et al., , 2012)).In this study, thus, we suggest that the high isolation of Arthrobacter spp. was caused by the high levels of Na stress.In the process of screening, there were many more resistant bacteria in the rhizosphere soil of S, grandis than in that of wheat as the frequency was 42.6%, 77.4%, 18% in Aksaray (rhizosphere soil of S. grandis), Nallıhan (rhizosphere soil of S. grandis), Aksaray (rhizosphere soil of wheat), respectively.The salt concentration might become higher in both soils surrounding of S. grandis because of the Na uptake from soil and the non-Na-resistant bacteria might be eliminated from the rhizosphere soil.Therefore, Na-resistant bacteria may be more easily isolated from S. grandis than from wheat.
IAA production is an important trait of PGPR (Goswamia et al., 2014), as this phytohormone enables plants to develop their root systems.There are only few reports about IAA production by Arthrobacter species (Forni et al., 1992;Siddikee et al., 2010).Our results are in line with these previous findings and indicated that Arthrobacter spp.had various types of species with production of IAA even in the genus Arthrobacter.Therefore, Arthrobacter species in the present study can be considered as potential PGPR.Isolated strains A22, WP5, B14, WP18 and AP20 produced IAA in the presence of L-Tryptophan.Moreover, strains A22, WP5, B14 and AP20 also produced siderophore.The production of siderophores is important and possible to bind with the available form of iron Fe 3+ in the rhizosphere (Ahmad et al., 2008).This suggests that these strains enhance the growth of S. grandis as a result of their enhanced IAA and siderophore production.
We previously expected that Na-resistant PGPR might increase Na uptake of S. grandis from soil which may facilitate phytoremediation of saline soils.In this study, however, inoculation with Na-resistant PGPR (AP20, B14, WN4 and A22) did not change Na uptake from soil significantly but they showed higher Na uptake compared to un-inoculated control within 35 days laboratory experiment (Figure 2 and Table 3).This may be attributed to short experimental growth period in the present study.Therefore, experimental studies with longer period should be conducted for a better understanding of the association between Na-resistant PGPR and S. grandis.
In conclusion, eight strains appear to be suitable candidates for PGPR of S. grandis.These strains belong to genus of Arthrobacter and Bacillus and this is the first report on the Na-resistant PGPR of S. grandis.These PGPR candidates on S. grandis have possibility of achievement to remove salt from saline soils using microbe assisted phytoremediation.

Figure 1 .
Figure 1.Relative frequency of Na-resistant bacteria from rhizosphere soil

Table 1 .
Soil properties collected from Aksaray and Nallıhan.The soil from Aksaray was used at both of pot experiment and conical tube one.

Table 2 .
Na uptake by Salsola grandis over the short-term cultivation (82 days)

Table 3 .
Growth and Na uptake of Salsola grandis by inoculating bacterial strains.These values showed means ± standard deviation.Asterisks indicate significant differences from the control values (t-test, P < 0.05)