Effect of Encapsulated Pseudomonas Putida Rs-198 ...

Journal of Plant Nutrition

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Effect of Encapsulated Pseudomonas Putida Rs-198 Strain on Alleviating Salt Stress of Cotton Yanhui He, Wu Zhansheng, Liang Tu & Chunhui Shan To cite this article: Yanhui He, Wu Zhansheng, Liang Tu & Chunhui Shan (2017): Effect of Encapsulated Pseudomonas Putida Rs-198 Strain on Alleviating Salt Stress of Cotton, Journal of Plant Nutrition, DOI: 10.1080/01904167.2016.1264595 To link to this article: http://dx.doi.org/10.1080/01904167.2016.1264595

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Date: 18 January 2017, At: 00:17

ACCEPTED MANUSCRIPT Effect of Encapsulated Pseudomonas Putida Rs-198 Strain on Alleviating Salt Stress of Cotton Yanhui He, Zhansheng Wu, Liang Tu, Chunhui Shan School of Chemistry and Chemical Engineering, Shihezi University, Shihezi 832003, PR China Address Correspondence to Zhansheng Wu. Email: [email protected]

ABSTRACT The present investigation was carried out to determine the inter-relationship between some physiological attributes of cotton and encapsulated Rs-198 strain. The pot experiment had seven treatments (with or without encapsulated bacteria under different salt stress, 10 replicates) and carried out in the greenhouse during 2014, April to June. Pot results showed that the inoculated encapsulated Rs-198 significantly increased the plant biomass under 0.5% salt stress. Besides, an approximately 19.47% increased in the soluble protein content of cotton with encapsulated Rs-198 inoculated in salt condition and relatively higher chlorophylls a, b and carotenoid concentrations were maintained at 0.626, 0.304, and 0.564 mg/g. Moreover, 42.30% proline content and 24.98% peroxidase activity were reduce when inoculated Rs-198 under 0.5% salt stress. In conclusion, application of encapsulated Rs-198 strain was effective in relieving salt stress under saline conditions. The microcapsules bioinoculants are potential alternatives for sustainable agriculture due to their low cost of production. Keywords P. putida Rs-198; cotton; Microencapsulation; alleviate salt stress; plant growth promoting

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ACCEPTED MANUSCRIPT INTRODUCTION Salinity is a major abiotic stress that not only reduce the average yield but also lead to poor-quality production of crops. Unfortunately, the excessive and continued use of chemical fertilizers to alleviate salt stress and improve plant yields involves various environmental concerns, such as low soil fertility, low soil biodiversity, eutrophication of water bodies, etc. Thus, novel fertilization strategies are needed to reduce fertilizer inputs and their environmental consequences in crops from agro-ecosystems. Soluble proteins are the important osmolytes that help in the osmotic adjustment in plants under stress. It may be due to upregulation of biosynthesis pathway of osmosis regulated compound, that helped in maintaining the water status of cell and protect the membranes under drought stress (Naseem and Bano, 2014). Chlorophyll content is often measured in plants in order to assess the impact of environmental stress, as changes in pigment content are linked to visual symptoms of plant illness and photosynthetic productivity (Hamdia et al., 2004). Free proline provides protection against stress by acting as an N-storage compound, osmolite, hydrophilic protectant for enzymes and cellular structures, and as a free radical scavenger (López-Gómez et al., 2014). It is reported that peroxidase (POD) has been the most popular indicator enzyme could be related to the depressive effect of salinity on plant growth because of its high concentration in most plant tissues (Liu et al 2013). In recent years, plant growth promoting bacteria (PGPB) has been found can grow around the plant root tissues, stimulating plant growth directly or indirectly by a wide variety of mechanisms like phosphate solubilization, fixing atmospheric nitrogen

and phytohormone

production (Paul and Lade, 2014). Besides, bacteria assist the associated plants in the uptake of

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ACCEPTED MANUSCRIPT mineral nutrients and water and also they increase tolerance to environmental stresses (Peng et al., 2013; Armada et al., 2014). PGPB inoculation increased aerial biomass production, harvest index, and grain yield of the Supremo 13 cultivar by 4.7%, 16%, and 20.2%, respectively (de Salamone et al., 2012). Liu et al. reported that the plant growth-promoting bacteria (PGPR)+ fertilizer treated seedlings had a higher dry matter accumulation than the fertilizer-only-treated after 3 months of incubation (Liu et al., 2013). In other reports, root and shoot fresh, dry weight of all treated plants inoculated with exopolysaccharides-producing bacteria (Proteus penneri, Pseudomonas aeruginosa, and Alcaligenes faecalis) was significantly higher in comparison with uninoculated control in both stress and nonstress conditions which show the resistance to drought effect (Naseem et al., 2014). Similarly, Egamberdieva reported that cucumber and tomato plant height and fruit yield (from 8 to 16%) was also significantly increased after inoculation with P. clororaphis TSAU13 strain in greenhouse experiments (Egamberdieva, 2012). Biofertilization or inoculation with plant growth-promoting bacteria is a sustainable alternative for agro-ecosystems. Inoculation of plant seedlings with Psedomonas spp. increased protein concentration under stress (Islam et al., 2014). Chlorophyll (Chl) a and Chl b values decreased rapidly from 2.73 to 1.71 mg g-1 dry weight (DW) (Chl a) and 0.84 to 0.46 mg g-1 DW. (Chl b) after 15-35 days of water deficit (Ge et al., 2014). However, inoculation of cells into soil may be difficult because of competition from indigenous microflora, unfavorable physicochemical conditions, and fluctuation in pH and temperature (Wu et al., 2011). Therefore, numerous scholars have focused on improve the survival of the bacterial strain via microencapsulation techniques (Bashan and Gonzales, 1999; Young et al., 2006). So far, it appears that alginate is the most promising of the encapsulating

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ACCEPTED MANUSCRIPT materials tested (Schoebitz et al., 2013). However, because of the limited published research on alginate beads related to agriculture because of their higher price. Some authors attempted to provide solution by blending of sodium alginate (NaAlg) with cost-effective bentonite and starch to develop a biodegradable and controlled-release bacterial fertilizer (Wu et al., 2014). Using alginate and starch by internal gelation technology can be a suitable procedure for protecting the probiotic strain and can be more efficient than using alginate alone (Martin et al., 2013). The slower release with increasing clay contents is due to the lower swelling behavior of bentonite (Singh et al., 2009). The basic industrial concept underlying encapsulated microbial cells is to entrap live microorganisms into a polymeric matrix and maintain their viability (Bashan et al., 2014). Extensive research resulted in the development of different kinds of free microbial inoculants, but application of microbead alginate formulations to inoculate plants in the soil was done a few times (Bashan et al., 2009a; 2009b; 2011; 2014). In our previous works, Pseudomonas putida Rs-198 was isolated and certified relieving salt stress and promoting cotton growth (Yao et al., 2010). Moreover, it is quite interesting and informative to investigate the promoting growth effect of encapsulated Rs-198 on cotton under salt stress. Furthermore, the objectives of this study are to evaluate the inter-relationship between some physiological attributes of cotton and encapsulated Rs-198 strain under different salt stress. The effect of salt stress on the physiological attributes such as plant height, fresh weight, dry weight, and germination of seeds, soluble protein, chlorophylls a, b, carotenoid, proline and peroxidase (POD) activity of cotton leaves when inoculated with encapsulated Rs-198 strain were evaluated in a pot experiment with vermiculite. The microencapsulated rhizobacteria may

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ACCEPTED MANUSCRIPT have broad application prospects to meet the needs of agricultural and help farmers improve production. MATERIALS AND METHODS Microcapsules Preparation The strain Rs-198 used in this study was previously isolated from salinization soil in Xinjiang (Yao et al., 2010). The Rs-198 was cultured in nutrient broth liquid medium (5 g beef extract, 10 g peptone, 5 g sodium chloride (NaCl), 1000 ml (H2O), pH 7.0-7.2) with shaking at 200 rpm and at 30 °C for 48 h. NaAlg, bentonite and starch were mixed at a ratios of 1.5%: 4%: 3% and then sterilized at 121°C for 20 min. Pseudomonas putida Rs-198 microcapsules were prepared according to the methods described by Wu et al., (2011) via extrusion. Briefly, the cell population of fermentation broth was set to 1013 cfu/ml and the cell suspension was suspended in the NaAlg-bentonite-starch (NaAlg-B-S) matrix solution and thoroughly mixed to make a homogenous solution. These mixtures were extruded through a sterile injection needle (needle size 0.9 mm) drop wise into a pre-cooled sterile 2% (w/v) crosslinking agent solution of calcium chloride (CaCl2). The water-soluble sodium alginate was converted into water-insoluble calcium alginate beads. Thus instantaneously formed beads were allowed to film-formation about 2 hours at room temperature. Wet microcapsules were collected by sieving and washed several times and then dried in Intelligent Ovens (Equipment Co., Ltd. Shanghai Jing Mai) at 40°C until constant weight was obtained. The dry beads were stored in sterile glass bottles for further analysis.

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ACCEPTED MANUSCRIPT Pot Experiment Seeds Sterilization The cotton seeds were sterilized by immersion in 70% ethanol for 5 min and subsequently in 0.1% mercury chloride (HgCl2) for 10 min, washed several times with sterile water, and allowed to germinate. Greenhouse Pot Experiment Plants were placed in plastic pots (9 cm diameter; 12 cm deep) containing 250 g of sterilized vermiculite (diameter 0.5 mm). The pot experiment had seven treatments: CK-0%, non-bacteria and non-saline stress; CK-0.5%, non-bacteria and 0.5% saline stress; C-0%, encapsulated P. putida Rs-198 and non-saline stress; C-0.5%, encapsulated P. putida Rs-198 and 0.5% saline stress; C-1.0%, encapsulated P. putida Rs-198 and 1.0% saline stress; C-2.0%, encapsulated P. putida Rs-198 and 2.0% saline stress; C-5.0% encapsulated P. putida Rs-198 and 5.0% saline stress (Table 1). Ten cottonseeds were sown in each pot, and each treatment included 10 replicates. Subsequently, approximately 0.35 g of encapsulated P. putida Rs-198 strain was added to the cottonseeds. The plants were grown in greenhouse conditions with day/night temperatures of 30°C and 25°C, respectively, and 400 μmol photons (m2 s)−1 of light supplied for 14 h during the day. Humidity was maintained at 60% by irrigated sterile water via injection four to five times a week. Scanning Electron Microscope (SEM) The microcapsules lie in the cotton rhizosphere were found for investigating the micrographs of the bacterial on the surface of the microcapsules. Samples of microcapsules were mounted on the stub and then coated with gold particles and observed with an SEM (JSM-6700F, Jeol, Tokyo,

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ACCEPTED MANUSCRIPT Japan). Growth Parameters The number of germination, germination rate was calculated at 10 d after planting. 50 days after sowing, five plants from each replicate were randomly harvested, and data on plant growth variables, such as shoot fresh weight, root fresh weight, shoot dry weight, and root dry weights were collected. The plant material for dry weight was dried at 105°C for 10 min and then 75°C for constant weight. Soluble Protein Contents Soluble protein content in green leaves was estimated following the method of Bradford (1976) and Daud et al., (2015). Freshly cut leaf disks were homogenized in 5 ml of distilled water. The homogenate was centrifuged in a refrigerated centrifuge at 10000 g for 10 min, and the supernatant obtained was used for protein determination. A reaction mixture consisting of 1 mL leaf extract and 2 mL of Bradford reagent was incubated at room temperature for 2 min. Absorbance was measured at 595 nm and total soluble protein concentration (μg mL-1) was calculated using bovine serum albumin standard curve. Chlorophyll Determination For chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids (Car) determination, fully expanded young leaves were detached from different plants and two cleaned leaf from each leaf were homogenized in 5 ml of 80% acetone at 4°C. Three milliliter of the supernatant was taken to measure absorption at 663, 646 and 470 nm. The pigments were calculated following the formula proposed by Lichtenthaler and Wellburn (1955) and expressed as mg g-1 DW. Chlorophy ll a (mg/g) = (12.72OD663-2.59OD646) ×V/M；

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ACCEPTED MANUSCRIPT Chlorophy ll b (mg/g)= (22.88OD646-4.67OD661) ×V/M； Total leaf chlorophy ll = (20.29OD646+8.05OD661) ×V/M； Carotenoid (mg/g) ＝ (1000OD470-3.27C (a) - 104C (b) –V)/M Note: V in the above formula to extract volume (L), M for the leaf fresh weight (g). Estimation of Proline Content Free proline (Pro) content was estimated following the method of Bates et al. (1973) and Ge et al. (2014). Freshly cut leaf disks were extracted with 4 ml of 3% sulphosalicylic acid for 10 min in boiling water. 2 ml of the filtrate was mixed with 2 ml acid-ninhydrin and 2 ml of glacial acetic acid in a test tube for 30 min at 100 °C in water bath. The reaction was terminated by placing the test tubes on ice followed by proline extraction with 4 ml toluene. Proline content was measured at the absorbance at 520 nm and calculated as lg g-1 DW against the standard proline curve after chromophore for 15 min. Peroxidase Activity Determination Peroxidase activity was assayed by the methods of Liu et al. with some modifications (Liu et al., 2013). About 5.0 g samples was homogenized in ice-water bath with 25 ml phosphate buffer (pH 6.0, 4°C), then centrifuged at 4°C, 10000×g for 30 min, the supernatant was collected and then quickly cooled in liquid nitrogen for analysis of POD enzyme activity. The reaction mixture contained 28 μl of guaiacol, 19 μl of 30% (v/v) H2O2, 50 mL of 0.1 mol/L phosphate buffer (pH 6.0). 1 ml of enzyme extract were added by 3 ml of reaction mixture and the blank sample contained the same mixture solution with 1 ml phosphate buffer. The maximal initial reaction velocity was calculated over 20 to 140 s linear increase in absorbance at 470 nm. One unit of

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ACCEPTED MANUSCRIPT enzymatic activity was defined as amount of enzyme that oxidizes 1 mM of guaiacol per minute at 25°C and pH 6.0 under assay conditions. Data Analysis Data were tested for statistical significance using the analysis of variance (ANOVA) package included in Microsoft Excel 2007 and comparison was done using a least significant difference (LSD) t-test. Mean comparisons were conducted using a least significant difference (LSD) test (P = 0.05). Standard error and LSD result were calculated. RESULTS SEM The use of SEM provided information on the release degree of bacteria from the microcapsules. As shown in Figure 1, the Strains can intensity distributed on the surface of the capsules and the diameter of the bacteria released from microcapsules in this study almost approached to 2 μl. NJN-6 strain colonization on banana roots was also studied in which the root systems were examined by SEM for the presence of the bacterial cells one day after inoculation with NJN-6 (Figure 1b). The results clearly showed that NJN-6 could colonize on the surface of banana roots successfully even after 7 days (Yuan et al., 2013). Salt Resistance on Physiological and Biochemical Indexes of Cotton Effect of Encapsulated Rs-198 on Cotton Growth To assess plant growth-promoting effects on cotton, the germination rates of seeds treated with P. putida Rs-198 microcapsules with and without the addition of the different concentrations of NaCl were monitored and compared with the control treatments. The results are summarized in Table 1. When cotton seedlings were grown in the vermiculite system under growth cabinet

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ACCEPTED MANUSCRIPT conditions for 7 d, germination reduced from 40.6% to 35.1% under 0.5% salt (Table 1). Salt stress also reduced plant growth; plant length, fresh weight, and dry weight decreased by 0.41%, 17.68%, and 13.07% under 0.5% NaCl, respectively, presumably because of the reduced availability of nutrients required for growth. Inhibition of plant growth by salinity was also explained as a result of the toxic effects of NaCl and the ability of the root system to control the entry of ions to the shoot and to slow down the water uptake of plants (Egamberdieva, 2011). Bacterial treatments used in the study increased germination, fresh weight, dry weight, and plant length compared with the non-treated plants under 0% and 0.5% NaCl (in low concentration salinated soil) (Table 1). Germination, plant length, fresh weigh, and dry weight of cotton significantly increased at low NaCl concentration, but decreased with increasing salt concentration when inoculated with encapsulated Rs-198 on cotton. The effects of salt stress were noticeably alleviated at a high salt concentration, which may be due to the fact that the promoting effect of bacteria on cotton should be greater than that of salt stress at low salt concentration until high salt stress surpasses the promoting effect of bacteria. Plant hormones (i.e., IAA and GA) produced by bacteria could improve the fitness of the plant–bacterium interaction and stimulate the development of the root system of the host plant (Egamberdieva, 2009). Diby et al. also observed that the population of Pseudomonas pseudoalcaligenes MSP-538 does not considerably change with increasing salinity in soil (Diby et al., 2005). Seed bacterization of maize with EPS-producing bacterial strains improved plant biomass, root and shoot length, and leaf area (Naseem et al., 2014). Other authors also reported that pre-sowing wheat seeds with plant growth regulators, such as IAA and gibberellins, alleviated the growth-inhibiting effect of salt stress (Egamberdieva, 2009).

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ACCEPTED MANUSCRIPT Soluble Proteins Soluble protein is the main osmotic adjustment of halophytes and the carbon source and energy source of organic material synthesis. It has a stabilizing effect on the cell membrane and protoplasm colloid. The soluble protein content in plants increases under adversity to improve the osmotic regulation ability of plant cells and reduce the degree of damage in plants. Moreover, it is important in resistance to adversity stress (Ge et al., 2014). Salinity stress induced a significant decrease in the soluble proteins in cotton plants (Figure 2). At low doses of NaCl (0.5%), the soluble protein content increase approximately 19.47% in cotton when inoculated with encapsulated Rs-198 compared with that of the control plants. At high doses of NaCl (1%, 2%, 5%), the opposite was observed, and the levels of soluble protein of the cotton seedlings were higher than that of cotton under 0.5% NaCl stress and zero inoculation. Rs-198 not only alleviated the inhibitory effect of salinity on soluble protein production but also induced a progressive increase in protein concentration compared with the control plants at higher salinity. A similar trend was found by Hamdia et al. in cotton under salinity stress when inoculated with Azospirillum brasilense (Hamdia et al., 2004). In addition, Pseudomonas mirabilis enhanced the total protein contents in the shoot and root parts of maize under Zn stress, showing that P. mirabilis may delay protein degradation and maintain steady protein metabolism, thereby reducing the stress of ammonia-like substances and increasing the ability of plants to withstand stressful conditions (Islam et al., 2014). Photosynthetic Pigments Chlorophyll content is often measured in plants to assess the impact of environmental stress, as changes in pigment content are linked to visual symptoms of plant illness and photosynthetic

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ACCEPTED MANUSCRIPT productivity. The chlorophyll and carotenoid contents of cotton were affected by the different salinity levels (Figure 3). The chlorophyll content significantly decreased under salt stress in comparison with the control. Plant growth-promoting properties of Rs-198 showed that cotton with strain inoculation maintained relatively higher chlorophylls a and b and carotenoid concentrations at 0.626, 0.304, and 0.564 mg/g, respectively, under salt stress compared with cotton without strain inoculation. Hassan et al. also showed that inoculation with either 1-aminocyclopropane-1-carboxylate (ACC)-deaminase or nitrogen-fixing activity-containing rhizobacteria significantly increased the contents of chlorophylls a and b and carotenoid under lead (Pb)-contaminated soils (p < 0.05) (Hamdia et al., 2004). However, the photosynthetic pigment content sharply declined from 1% to 5% (Figure 3). High salt inhibits metabolic processes by inhibiting the action of enzymes, which may be the most important cause of inhibition. Salt inhibits chlorophyll synthesis by causing an impaired uptake of essential elements, such as magnesium (Mg) and iron (Fe), by plants (Hassan et al., 2014). The chlorophyll a and chlorophyll b contents increased until the salt concentration was more than 2.0% in the inoculation treatment (Figure 4). We derived the increments of the photosynthetic pigments and obtained the rate of increase. The rate of increase in chlorophyll a was determined using the following equation: y = −0.058x + 0.1081. The rate of increase in chlorophyll b was determined using the following equation: y = −0.0234x + 0.0356. The depletion rate was considerably greater with increasing stress. In the comparison of chlorophylls a and b, chlorophyll a was found to be more susceptible to the effects of salt stress, and the effect of salinity on the chlorophyll a/b ratio did not follow any certain trend/pattern.

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ACCEPTED MANUSCRIPT Proline Content Regarding proline production by plants as a compatible solute able to help cells in the osmoregulation processes and facilitate water uptake in response to stress, we determined that the reference cotton exhibited low proline production under stressful conditions (Armada et al., 2014). Proline is one of the most effective osmotic regulatory substances that can reflect the resistance of plants to a certain extent when subjected to stress. When confronted with stress, plants accumulate proline to improve cell sap concentration, reduce the osmotic potential, and maintain cell expansion pressure for regulating the balance of original metabolism (Naseem et al., 2014). Proline concentration in cotton significantly increased with increasing salinity higher than 1% NaCl, and the accumulation rate was considerably greater with the increase in salt concentration (Figure 5). To cope with drought, higher salt stress required greater proline accumulation than lower salt stress. Besides, the proline content of cotton seedlings in the encapsulated Rs-198 inoculation treatment was significantly reduced by 23.88%, 42.30% compared with those that were not inoculated under 0%, 0.5% salt stress. Therefore, the relieving salt stress can increase under salt stress condition. The results of free proline content in Rs-198 inoculation and non-inoculation under NaCl stress conditions supported the hypothesis of a positive correlation between proline accumulation and salt stress tolerance of plants. The results of proline accumulation in cotton leaves reported in this study were consistent with those of Hamdia et al. on maize with A. brasilense inoculation (Hamdia et al., 2004). POD Activity One important mechanism related to stress tolerance is the alteration in oxidative stress

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ACCEPTED MANUSCRIPT necessary for plant survival. The accumulation of reactive oxygen species (ROS) in plant cells under stress is removed by enzymatic systems, and the increase in antioxidant enzymatic activities is correlated with the severity of stress (Koussevitzky et al., 2008). POD is a major protective enzyme of the defense system, which maintains low levels of free radicals in the body to prevent damage. In the present study of Ge et al. activity of the antioxidant enzymes, POD increased under drought conditions (Ge et al., 2014). The effects of salt stress and Rs-198 on POD activity under different salt conditions are shown in Figure 5. In cotton, POD activity highly decreased by 5.75% and 24.98% when inoculated with Rs-198 under 0% and 0.5% salt stress. This result showed that the strains could alleviate salt stress more effectively under the salinization condition. However, POD enzyme activity also slowly increased with the increase of salt concentration. This may be due to the higher salt concentration leading to higher salt stress effect of cotton and surpassed the alleviate effect. Thus, POD is considered the key antioxidant enzyme in the defense system, and it plays an important role in scavenging reactive oxygen species (ROS). Notably, drought stress and antioxidant enzyme activity demonstrated a significant interaction, but inoculation with PGPR lessened the adverse effect of stress on antioxidant enzyme activity (Naseem et al., 2014). However, studies with different plant species showed different increases in POD activity; higher POD activity was observed in soybean plants inoculated with pathogens (Choudhary, 2011) and oat leaves inoculated with Acinetobacter sp. (Xun et al., 2015). CONCLUSIONS Salinity stress induced a significant decrease in the soluble proteins, chlorophyll, carotenoid content and increased in the proline content, peroxidase (POD) enzyme levels in cotton plants at

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ACCEPTED MANUSCRIPT low doses of NaCl. However, the soluble protein, chlorophyll, carotenoid content increased and proline content, peroxidase (POD) enzyme decreased when inoculated with encapsulated Pseudomonas putida Rs-198 strain. We conclude that the strains could alleviate salt stress more effectively under the salinization condition. Soluble protein, chlorophyll and carotenoid continued to decrease, whereas the proline content and POD enzyme exhibited a sustained increase with salt stress increasing. Thus, the encapsulated Rs-198 strain could significantly alleviate salt stress and promote the growth of cotton at low salt concentrations until the alleviating efficiency was surpassed by salt stress. So, microencapsulation of NaAlg-B-S Pseudomonas putida Rs-198 strain is a novel way to improving plant salt alleviate efficiency. These new advancements importantly contribute towards provided insights into the promising application of using encapsulated bacterial fertilizers in farmlands. ACKNOWLEDGEMENTS This study was financially supported by National Natural Science Foundation of China (31260022, 21566035), Science and Technology Fund Projects of Shihezi University (2013ZRKXJQ01).

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ACCEPTED MANUSCRIPT from starch-alginate-clay based formulation. Applied Clay Science 45: 76-82. Wu. Z. S., Y. F. Zhao, I. Kaleem, C. Li. 2011. Preparation of calcium alginate microcapsuled microbial fertilizer coating Klebsiella oxytoca Rs-5 and its performance under salinity stress. European Journal of Soil Biology 47: 152-159. Wu. Z. S., Y. H. He, L. J. Chen, Y. J. Han, C. Li. 2014 Characterization of Raoultella planticola Rs-2 microcapsule prepared with a blend of alginate and starch and its release behavior. Carbohydrate Polymers 110: 259-267. Xun, F. F., B. M. Xie, S. S. Liu, C. H. Guo. 2015. Effect of plant growth-promoting bacteria (PGPR) and arbuscular mycorrhizal fungi (AMF) inoculation on oats in saline-alkali soil contaminated by petroleum to enhance phytoremediation. Environmental Science and Pollution Research 22: 598-608. Yao. L. X., Z. S. Wu, Y. Y. Zheng, I. Kaleem, C. Li. 2010. Growth promotion and protection against salt stress by Pseudomonas putida Rs-198 on cotton. European Journal of Soil Biology 46: 49-54. Young, C. C., P. Rekha, W. A. Lai, A. B. Arun. 2006. Encapsulation of plant growth-promoting bacteria in alginate beads enriched with humic acid. Biotechnology and Bioengineering 95: 76-83. Yuan, J., Y. Z. Ruan, B. B. Wang, J. Zhang, R. Waseem, Q. W. Huang, Q. R. Shen. 2013. Plant growth-promoting rhizobacteria strain Bacillus amyloliquefaciens NJN-6-Enriched bio-organic fertilizer suppressed fusarium wilt and promoted the growth of banana plants. Journal of Agricultural and Food Chemistry 61: 3774-3780.

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ACCEPTED MANUSCRIPT TABLE 1 Cotton seedling biomass and germination rate of inoculated and non-inoculated encapsulated Rs-198 under non-salt and salt stress conditions. Samples

Plant height (cm)

Fresh weight (g)

Dry weight (g)

Germination (%)

CK-0%

18.500±0.525d

1.595±0.082b

0.153±0.024a

0.406b

CK-0.5%

18.425±0.480d

1.313±0.080bc

0.133±0.006b

0.351b

C-0%

21.850±0.409bc

1.885±0.063a

0.150±0.005a

0.510a

C-0.5%

22.225±0.662ab

1.793±0.089a

0.160±0.006a

0.550a

C-1.0%

23.350±0.528a

1.743±0.066ab

0.175±0.006a

0.555a

C-2.0%

20.600±0.460c

1.645±0.085ab

0.143±0.005ab

0.560a

C-5.0%

14.500±0.444e

1.090±0.0480c

0.100±0.003b

0.420b

Note: Means ± SE (n=3) at p < 0.05.

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a

b

FIGURE 1 SEM of P. putida Rs-198 strains on the surface of microcapsules (a: ×3000, b: ×8000).

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Soluble protein content (μg/g)

390 370

Soluble protein content

350 330 310 290 270

250 CK-0% CK-0.5% C-0%

C-0.5% C-1.0% C-2.0% C-5.0%

FIGURE 2 Effects of encapsulated strain Rs-198 on the soluble protein content of cotton seedlings under salt stress (Mean ± SE (n = 3) at p < 0.05).

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1 Chlorophyll b

Chlorophyll content (mg/g)

0.9

Chlorophyll a

0.8

Carotenoids

0.7 0.6 0.5 0.4 0.3

0.2 0.1 0 CK-0% CK-0.5%

C-0%

C-0.5%

C-1.0%

C-2.0%

C-5.0%

FIGURE 3 Effect of salt stress and encapsulated Rs-198 on photosynthetic pigment content of the cotton seedlings (Mean ± SE (n = 3) at p < 0.05).

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0.15 y = -0.029x2 + 0.1081x R² = 0.9959

0.10

y = -0.058x + 0.1081

Rate (%)

0.05 0.00

-0.05 chlorophyll a -0.10

y = -0.0117x2 + 0.0356x R² = 0.9994

chlorophyll b chl a IR

-0.15

chl b IR

y = -0.0234x + 0.0356

-0.20

0.0

1.0

2.0

3.0

4.0

5.0

6.0

NaCl (%)

FIGURE 4 Effect of encapsulated Rs-198 on relieving salt stress and promoting photosynthetic pigments in cotton seedlings (chl a IR: chlorophyll a increase rate, chl b IR: and chlorophyll b increase rate).

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4000

Proline content

3.5

3500

Proline content (µg/g)

POD activety 3.0

3000

2.5

2500

2.0

2000

1.5

1500

1.0

1000

0.5

500

0.0

POD activity (U/gFw·min)

4.0

0

CK-0% CK-0.5% C-0%

C-0.5% C-1.0% C-2.0% C-5.0%

FIGURE 5 Proline and POD activity in cotton leaves treated with encapsulated Rs-198 under salt stress (Mean ± SE (n = 3) at p < 0.05).

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