Canadian Journal of Microbiology

Salt-tolerant and plant growth-promoting bacteria isolated from high-yield paddy soil

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2017-0571.R4

Manuscript Type: Article

Date Submitted by the 10-Apr-2018 Author:

Complete List of Authors: Shi-Ying, Zhang; Yunnan Institute of Microbiology; Yunnan Agricultural University Cong, Fan; Yunnan Institute of Microbiology; Yunnan Agricultural University Yong-xia, Wang; Yunnan Institute of Microbiology Yun-sheng, Xia; Yunnan Agricultural University Wei, Xiao; Yunnan Institute of Microbiology Xiao-Long,Draft Cui; Yunnan Institute of Microbiology

Rice, plant-growth promoting bacteria, diversity, salinity tolerance, 1- Keyword: aminocyclopropane-1-carboxycarboxylate deaminase

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

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1 Salt-tolerant and plant growth-promoting bacteria isolated from high-yield paddy soil

2

3 Shiying Zhang 1, 2  , Cong Fan 1, 2 , Yongxia Wang 1, Yunsheng Xia 2,

4 Wei Xiao 1, Xiaolong Cui 1

5 1 Yunnan Institute of Microbiology, Yunnan University, Kunming, China

6 2 Yunnan Engineering Laboratory of Soil Fertility and Pollution Remediation, Yunnan Agricultural

7 University, Kunming, China

8  These authors contributed equally to this work.

Draft

 Correspondence Xiaolong Cui, Yunnan Institute of Microbiology, Yunnan University, Kunming, 650091, PR China. Tel:86-871-65033543, E-mail: [email protected]. Wei Xiao, Yunnan Institute of Microbiology, Yunnan University, Kunming, 650091, PR China. Tel:86-871-65033543, E-mail: [email protected]. 1

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9 Abstract: Growth and productivity of rice is negatively affected by soil salinity. However, some

10 salt-tolerant bacteria improve plant health in saline stress. In this study, 305 of bacteria were isolated

11 from paddy soil in Taoyuan, China. Among these, 162 strains were tested its salt-tolerance, 67.3%,

12 28.4%, and 9.3% of the strains could grow in media with NaCl concentrations of 50, 100, and 150 g/L,

13 respectively. The phylogenic analysis to 74 of 162 strains indicates that these bacteria belong to

14 Bacillales (72%), Actinomycetales (22%), Rhizobiales (1%), and Oceanospirillales (4%). Among 162

15 strains,30 salt-tolerant strains were screened for their plant-promoting activities under axenic

16 conditions at 3, 6, 9 and 12 g/L NaCl, 43-97% of the strains could improve rice germination energy or

17 germination capacity, while 63-87% of the strains could increase shoot and root lengths. Among

18 various PGPB, TY0307 was the most effective strain for promoting the growth of rice, even at high salt

19 stress. This was associated with its production of 1-aminocyclopropane-1-carboxycarboxylate

20 deaminase, IAA and siderophore, and inducing accumulation of proline, while reducing the salt 21 induced malondialdehyde content. These Draftresults suggest that several strains isolated from paddy soil 22 could improve rice salt tolerance and may be used in the development of biofertilizer.

23 Keywords: Rice, plant-growth promoting bacteria, diversity, salinity tolerance,

24 1-aminocyclopropane-1-carboxycarboxylate deaminase

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Introduction Salinity is a major factor that detrimentally affects crop productivity worldwide. According to Food and Agricultural Organization (FAO) report, more than 800 million hectares of land and 20% of irrigated agricultural land are affected by salinity around the globe in 2008 (Singh and Jha, 2016). In addition, anthropogenic global warming is exacerbating the problem, causing secondary salinization (García-Cristobal et al., 2015). Because of its growth conditions, rice (Oryza sativa) is particularly susceptible to salt stress (Kohler et al., 2009; Hong et al., 2009; Lucas et al., 2014). In many rice production areas, salt stress limits yield (García-Cristobal et al., 2015). A number of approaches are used to address the negative impacts of salinity, including gypsum applications, organic matter amendments, and irrigation optimization to limit the quantities of salts applied and to effectively leach salts from the root zone, and planting salt-tolerant crop varieties (Nadeem et al., 2016). The use of beneficial bacteria, known as plant-growth promoting bacteria (PGPB), to increase the productivity of agriculturalDraft crops under stress conditions and decrease the use of chemical fertilizers and pesticides that have a strong negative impact on the environment is becoming an increasingly intriguing biotechnological alternative (Saharan and Nehra, 2011). A number of reports demonstrate the efficacy of PGPB in promoting plant growth under normal conditions as well as in saline soils and other stressed environments (Saharan and Nehra, 2011; Egamberdieva, 2009; Zahir et al., 2003, 2009; Bernardr et al., 2007; Nadeem et al., 2014). These bacteria promote plant growth either by directly providing nitrogen, phosphorous, and iron nutrition, stimulating plant growth by production of phytohormones, dissolved phosphorus, or indirectly by inhibiting the growth of pathogenic microorganisms (Nadeem et al., 2016; Dimkpa et al., 2009). The effectiveness of PGPB in mitigating the adverse effects of salinity stress has been reported for several vegetable and other crop plants (Zahir et al., 2009; Mayak et al., 2004). Among these strains, only a small number can improve the salt resistance of rice (García-Cristobal et al., 2015; Yuan et al., 2016; Jha and Subramanian, 2014; Nautiyal et al., 2013; Forni et al., 2016). Since the existence of rich microorganisms in hypersaline environments was discovered in the early 20th century, a large number of bacteria and archaea have been isolated from hypersaline environments, e.g., salt mines, salt fields, ancient salt crystals, and pickled food (Xiao et al., 2013).

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According to FAO standards (Brouwer et al., 1985), soil with a salt concentration of 0-3 g/L in the water extracted from a saturated soil is considered non-saline soil. However a small number of studies have found salt-tolerant bacteria in non-saline soil (Chen et al., 2010; Echigo et al., 2005). Surprisingly, researcher discovered a large number of halophiles and salt-tolerant bacteria in non-saline soils, Bacillaceae was the most frequently occurring family. Yet, where these salt-tolerant bacteria come from is unclear. With respect salt-tolerant bacteria widely distributed in other non-saline soil, it is hypothesized that salt-tolerant bacteria can be isolated from paddy soil, and these salt-tolerant bacteria can be correlated with improved rice growth under salt stress. Taoyuan village, Yunnan province of China, which was reported in several papers with the high rice yields above 13 t/ha, has been famous as a special eco-site for rice high yield due to its superior light and temperature conditions (Katsura et al., 2008; Li et al., 2009). The aim of the present study was to provide the first detailed characterization of salt-tolerant bacteria in high-yield paddy soil in Taoyuan village, and examine their effectDraft on plant growth, osmolyte content of rice plants growing under salt stress.

Materials and Methods Sample collection: Paddy soil samples were collected from Taoyuan village（35° 56' N, 104° 08' W） on December, 2010. Paddy soil approximately 5-10 cm beneath the ground surface was collected. Sampling was performed at five locations, and the samples were mixed together. All samples were collected into sterilized sample bags using a small sterilized shovel and transported to the laboratory at room temperature. Microbial isolation was performed within 24 h. Soil salt content was determined to be 1.01 g/kg using the NY/T1121.16-2006 method (Chinese Agricultural Standard). According to FAO standards, the collected samples were non-saline soils. Culture medium: R2A (1 L): yeast extract 0.5 g, peptone 0.5 g, soluble starch 0.5 g, casein acid hydrolysate 0.5 g, glucose 0.5 g, K2HPO4 0.3 g, MgSO4 0.024 g, sodium pyruvate 0.3 g. Modified LB (MLB, 1 L): tryptone 1 g, yeast extract 0.5 g, NaCl 0.5 g, sodium pyruvate 2 g. Marine broth agar (MBA, 1 L): peptone 5.0 g, yeast extract 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 8.8 g, sodium sulphate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34.0 mg, boric acid 22.0 mg, 4

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sodium silicate 4.0 mg, sodium fluoride 2.4 mg, ammonium nitrate 1.6 mg, disodium phosphate 8.0 mg, agar 15.0 g. Strain isolation: Three types of media were employed as isolation media. To each medium, 16 mg/L of nystatin was added. A 10 g soil sample was placed in an Erlenmeyer flask that contained 90 ml sterilized 0.85% (w/v) NaCl solution and glass beads. The flask was placed on a shaker for 2 h at 120 r/min at room temperature to yield a soil suspension. Next, a 10× gradient dilution was performed using 0.85% (w/v) NaCl solution. The 0.2 ml soil suspension with dilution at 10-5-10-7 was plated on medium and cultured at 28℃. After 7-30 days, single colonies were cultured in a corresponding medium for the streaking isolation of tetrads. The obtained pure cultures were freeze-dried in milk, subsequently inoculated onto a slant culture medium, and stored at 4℃ for future use. Evaluation of NaCl tolerance of the isolates: To test NaCl tolerance, isolates growth at various NaCl concentrations (0, 50, 100, 150, and 200 g/L) was investigated on R2A. Survival rate were calculated as: Number of viable strains/tested total numberDraft strains×100%. The experiment was repeated three times. To provide clearer descriptions, strains that grew in media with NaCl concentration higher than 50 g/L are referred to as salt-tolerant bacteria.

16S rRNA gene-based phylogenic analysis: Bacterial DNA was extracted and the 16S rRNA gene fragments were amplified using the method described in Xiao et al. (2012). The amplified products were sent to Sangon Biotech (Shanghai, China) for sequencing. Sequence alignment was performed online using EzBioCloud (http://eztaxon-e.ezbiocloud.net) to confirm the relative species of each strain. The taxonomic hierarchy above the genus level was analysed online on RDP (http://rap.cme.msu.edu/). Analysis of rice seed salt tolerance improvement by salt-tolerant bacteria: Salt-tolerant strains were separately inoculated into Erlenmeyer flasks that contained 150 ml medium and cultured on a shaker at 180 r/min at 28℃ for 24 h. Strains were collected by centrifuge at 5000 r/min for 5 min.

Pellets were re-suspended in 0, 3, 6, 9, and 12 g/L NaCl solutions. The OD600 values of the strain suspensions were determined by comparison with sterile water. The bacteria suspensions were adjusted

to OD600 at 0.3±0.05 for further use. Rice seeds were rinsed with sterile water to remove surface dust.

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Subsequently, the seeds were soaked in 75% ethanol for 2 min and then in 1% sodium hypochlorite for 5 min. Finally, they were rinsed three times with sterile water. Ten millilitres of strain suspensions were placed in culture bottles lined with filter paper. In each bottle, 10 surface-sterilized rice seeds were added and cultivated for 10 days at 28℃. The germination energy, germination capacity, root length, and shoot length were measured. Germination energy reflects the rate at which this process occurs and is given as the proportion of seeds which germinate half-way into the expected germination period. Germination capacity is the proportion of planted seeds which form normal sprouts at the maximum time needed for all viable seeds to germinate (Aniszewska and Słowiński, 2016) The experiment was repeated three times.. The measured parameters included the following: Germination energy = (the number of germinated seeds on the 5 th day / total number of seeds) × 100% Germination capacity = (the number of germinated seeds on the 10 th day / total number of seeds) × 100% Draft Screening for plant growth promoting traits: For quantifying 1-aminocyclopropane-1- carboxycarboxylate (ACC) deaminase activity, isolate TY0307 (Genbank accession number KF477150) was grown in R2A up to late log phase. ACC deaminase activity was determined by measuring the production of α-ketobutyrate, as described by Honma and Shimomura (1978). ACC deaminase activity was expressed as μmol α-ketobutyrate/mg protein/h. For quantification, a standard curve was plotted from OD540 of different concentrations of α-ketobutyrate ranging between 0.1 and 1.0 μmol. The protein concentration in the sample was determined by Lowry method (Lowry et al., 1951). Siderophore production by TY0307 was determined following the universal assay of Schwyn and Neilands (1987). In this assay, one can identify the siderophore producing bacteria through color change of the blue media. IAA production was estimated according to a standard method described by Gordon and Weber (1951) in which the hormone present in the culture reacts with Salkowski reagent, quantitatively measured on a spectrophotometer at 530 nm. Biochemical analysis of plants: Proline content in leaves of experimental plants was determined by spectrophotometric method (Bates et al., 1973) using standard curve of pure L-proline. Lipid

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peroxidation (reflected by malondialdehyde [MDA] content) was measured by grinding leaf tissue (200 mg) into a fine powder in liquid nitrogen and further process based on the method of Hodges et al. (1999). The protein concentration in the sample was determined by Lowry method (Lowry et al. 1951). All tests were carried out in triplicate. Statistical analysis: Analysis of variance (ANOVA) and standard deviation (SD) was calculated using IBM SPSS program version 19.

Results Screening of salt-tolerant bacteria A total of 305 strains of bacteria were isolated from paddy soil samples using three culture media. Based on the bacterial colony morphologies and bacteria growth rates, a salt-tolerance test was performed on 162 strains. The test results indicate that all tested strains could not grow in a medium with a 200 g/L NaCl. When the NaCl concentrationDraft was 50, 100, or 150 g/L, the strain survival rates were 67.3%, 28.4%, and 9.3%, respectively. As the concentration of NaCl increased, the number of surviving strains decreased. Diversity of salt-tolerant bacteria 16S rRNA gene sequencing was performed on 74 strains of salt-tolerant bacteria isolated from paddy soil. The results showed that the 74 strains belong to three phyla (Proteobacteria, Firmicutes, Actinobacteria), four orders (Bacillales, Actinomycetales, Rhizobiales, Oceanospirillales), nine families, 15 genera, and 35 species (Figure 1; Table 1). The 15 genera were as follows (number of strains in parentheses): Bacillus (39), Streptomyces (9), Fictibacillus (5), Halomonas (3), Nocardia (3), Microbacterium (2), Nesterenkonia (2), Oceanobacillus (1), Arthrobacter (1), Rhizobium (1), Jeotgalibacillus (1), Bhargavaea (1), Chryseomicrobium (1), Paenisporosarcina (1), Rhodococcus (1), Brevibacterium (1), Arsenicicoccus (1), and Solibacillus (1). Bacillus exhibited the highest abundance (52.7% of the strains), followed by Streptomyces and Fictibacillus (12.2% and 6.8% of the strains, respectively). A total of 50 strains could tolerate NaCl concentrations of 10% and 15%. Of these strains, 88% belonged to Bacillales, Rhizobiales, and Oceanospirillales, with most of the strains belonging to Bacillales (80%).

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Influence of salt-tolerant bacteria on rice seed germination under salt stress Based on characteristics such as salt tolerance and growth rate, 30 salt-tolerant strains were selected to test their effect on the salt tolerance of rice seeds. The effects of the 30 tested strains on the seed germination capacity and germination energy were not significant when under the stress of 3 g/L NaCl. Under the stress of 6 g/L NaCl, 67% of the salt-tolerant strains improved the germination energy but had no significant effect on the germination capacity.. Under the stress of 9 g/L and 12 g/L NaCl, the germination energy and germination capacity of the control group suddenly decreased to 0, while 97% and 43%, respectively, of the salt-tolerant bacteria-treated seeds exhibited germination energy higher than that of the control group. Additionally, 93% and 83% of the salt-tolerant bacteria-treated seeds, respectively, displayed a germination capacity higher than that of the control group. As the level of NaCl stress increased, the germination energy and germination capacity of the un-treated and the inoculation-treated group both decreased. DraftHowever, under the stress of a high salt concentration, the growth-promoting effects of the bacteria strains were superior. Among the tested strains, TY0121, TY0407, TY0509, and TY0239 significantly improved rice seed germination energy and germination capacity at different salt concentrations (Figure 2). However, without NaCl stress, the effects of these four strains on germination energy and the germination capacity did not significantly differ from that of control. These results suggest that these four strains of bacteria significantly relieved the toxic effects of high NaCl concentration on rice seed germination.

Effects of salt-tolerant bacteria on rice seedling shoot length Among the 30 tested strains, 73%, 90%, 77%, 90%, and 83% of the strains displayed better shoot lengths than the control group when under stress of 0, 3, 6, 9, and 12 g/L NaCl, respectively (Table 2). When under no NaCl stress, TY0130, TY0111, TY0406, TY0121, and TY0307 promoted a 30% increase in the shoot length of rice seedlings. TY0130 promoted a 45.1% increase compared to the control group. Under the stress of 3 g/L NaCl, TY0256, TY0119, TY0231, and TY0130 exhibited over 110% shoot length promotion in seedlings. TY0256 promoted the most significant growth increase (162.5%). Under the stress of 6 g/L NaCl, TY0256, TY0130-1, TY0545, TY0111, and TY0132

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displayed growth promotions over 176%, TY0256 promoted the most significant increase (278.6%) compared to the control group. Under 6 g/L NaCl stress, salt-tolerant bacteria exhibited the most significant growth promotion in the shoot length of rice seedlings. Under the stress of 9 g/L NaCl, no germination was found in the seeds of the control group. With inoculation of TY0307, TY0545, TY0407, TY0463, and TY0340, the shoot lengths of rice seedlings were over 2.5 cm. TY0307 and TY0545 promoted shoot lengths of 4.67 and 3.14 cm, respectively. Under the stress of 12 g/L NaCl, there was again no germination in the seeds of the control group. Seeds inoculated with TY0029 and TY0105 exhibited shoot lengths over 1 cm. Salt stress from high concentration of salt significantly inhibited the germination of rice seeds. However, with inoculation of salt-tolerant bacteria, the salt tolerance of rice seeds was significantly improved. As the salt stress increased, the promotive effects were more apparent. However, we did not find a strain that performed well under all salt concentrations. Draft Effects of salt-tolerant bacteria on rice seedling root length Under the stress of the different NaCl concentrations, 13%, 83%, 77%, 93%, and 77% of the strains, respectively, exhibited growth promotion for root lengths of rice (Table 2). The effects were similar to those of salt-tolerant bacteria on shoot length, where the promotive effects were more apparent under high NaCl concentrations. Without the NaCl stress, only TY0130-1 promoted root length growth 21.4%. The remaining strains exhibited less than 20% or no promotion. Under the stress of 3 g/L NaCl, TY0123, TY0119, TY0231, TY0114, and TY0239 promoted a growth rate over 179% for seedling roots; TY0123 promoted a growth rate of 200.7%. Under the stress of 6 g/L NaCl, five strains promoted a root length growth rate over 200%. TY0545 promoted a 244.2% increase compared to the control group. Under the stress of 9 g/L NaCl, no germination was found in seeds of the control group. Strains TY0307, TY0509, TY0222, TY0340, and TY0545 resulted in root lengths of 5.09, 4.38, 4.06, 4.03, and 3.45 cm, respectively. Under the stress of 12 g/L NaCl, again no germination was found in seeds of the control group. Seeds inoculated with TY0406, TY0407, TY0114, TY0132, and TY0119 produced root lengths of 3.84, 2.71, 2.53, 2.43, and 2.03 cm, respectively.

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Strains that promoted growth in both root and shoot length Without NaCl stress, four strains, i.e., TY0110, TY0111, TY0130-1, and TY0210, promoted growth in root and shoot lengths (Figure 3). Under the stress of 3 g/l NaCl, 80% of the strains had promotive effects on both shoot and root length. The five strains with the most apparent effects were TY0111, TY0114, TY0119, TY0231, and TY0239. Of these strains, TY0119 exhibited promotions of 196.4% and 128.3% in root and shoot length, respectively. The increase in root length promoted by these five strains was more than the increase in shoot length (Figure 3). A total of 63% of the strains promoted growth in shoot and root length under the stress of 6 g/l NaCl. TY0545 displayed the most significant growth promotion: 244.2% for root length and 190.7% for shoot length (Figure 3). Under the stress of 9 and 12 g/l NaCl, no germination occurred in seeds of the control group without bacteria inoculation. The high salt content severely inhibited the growth of rice seeds. A total of 87% and 77% of the strains could preserve rice seeds from growth inhibition under the stress of 9 and 12 g/l NaCl, respectively, and promote the growth of Draft roots and shoots. Under the stress of 9 g/l NaCl, TY0307 exhibited the most significant growth promotion: root length is 5.09 cm; shoot length is 4.67 cm (Figure 3).

Plant growth promoting features of TY0307 TY0307 was one of the most effective strains for promoting the growth of rice, even at high salt stress. It was screened for its various plant growth promoting features. ACC deaminase activity was found to be 280 ± 17 nmol α-ketobutyrate/mg protein/h. Similarly, it was also positive for the production of siderophore. Production of IAA was quantified 0.45 ± 0.05 μg/ml.

Effect of TY0307 on plant biochemical parameters under salt stress Proline content in uninoculated rice increased in the range of 0.9-5.7 times under salt stress, while it increased in the range of 0.3-4.8 times in bacterium-treated plants (Figure 4A). Osmotic stress inhibits plant growth and increases reactive oxygen species (ROS). Excessive ROS levels result in oxidative stress, for which lipid peroxidation (reflected by MDA content) is one of the biochemical markers. The amount of MDA content resulting from oxidative damages increased between 20.4 to 108.0% at

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different concentrations of NaCl. However, TY0307 significantly decreased the MDA content from 14.8 to 29.0 % in treated plants. The highest reduction in MDA content of 29 % was observed at 12 g/L NaCl (Figure 4B).

Discussion Since the discovery of halophiles and salt-tolerant microorganisms in high-salt environments, continuous research on the origins of these halophiles and salt-tolerant bacteria has been performed. With the expansion of the research field, researchers surprisingly also discovered a large number of halophiles and salt-tolerant bacteria in non-saline soils. Chen et al. (2010) screened 114 strains of halophiles and salt-tolerant bacteria from orchards, paddy, sandy soil, and forest soils using five media and different NaCl concentrations (5%, 10%, 15%, 20%). Of the 114 strains, 20.2% could tolerate NaCl concentrations in the range of 0-20%, and two strains were halophiles. Bacillaceae (33 strains; 54.1%) was the most frequently occurringDraft family. Echigo et al. (2005) isolated 176 strains of salt-tolerant bacteria from orchards, lawn, pasture, and woodlands around Tokyo, Japan, using one medium (20% NaCl concentration) and three pH gradients. Of the 176 strains, the majority were Halobacillus litoralis (66 strains), Halobacillus trueperi (28 strains), and Filobacillus milosensis (17 strains). In this study, 305 strains of bacteria were isolated from paddy soil using three culture media, and 162 of the strains were tested for salt tolerance. Among the tested strains, 67.3%, 28.4%, and 9.3% could grow in NaCl concentrations of 50, 100, and 150 g/L, respectively. When the NaCl concentration was 200 g/L, there was no growth of any strain. Phylogeneic analysis was performed on 74 strains and found that Bacillus was the most frequently occurring strain. Our result is consistent with that of Chen et al. (2010). The majority strains found by Echigo et al. (2005), i.e., Halobacillus and Filobacillus, were not found in this study. However, multiple strains of Bacillus megaterium, a major group in the study by Chen et al.(2010), were isolated, which could be a result of factors such as sample sources and isolation strategies. Similar to the findings by Chen et al. (2010) and Echigo et al. (2005), strains of the Bacillaceae were of absolute majority. Additionally, the Bacillus genus represented the most numerous group in the Yunnan Kunming salt mine (Xiao et al., 2006) as well as in brine and soil of the Yipinglang Salt Mine

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(Chen et al., 2007), composing 43.2% and 15.8%, respectively, of the isolated strains. However, Bacillus is rarely found in ancient salt crystals. To date, only one strain of Bacillus has been isolated from ancient salt crystals that are hundreds of millions years old. All other strains are archaea and non-spore forming (StanLotter et al., 2002). We speculate that the archaea were the first to appear in high-salt environments, followed by bacteria. The halophiles and salt-tolerant bacteria in non-saline environments originated in high-salt environments (spread by methods such as winds). Salt-tolerant bacteria further evolved in the non-saline environment. A portion of the halophiles was weeded out, while several enter dormancy or survive in high-salt microenvironment. Therefore, “salt-tolerant” instead of “salt-liking” may be the more favourable survival strategy. If spore formation is a widespread strategy to resist stress, why are spores rarely found in ancient halophiles in salt crystals? Our understanding of the stress resistance physiology of microorganisms is substantially less developed than desirable. In Chen et al. (2010), one strain of Pseudomonas was isolated, and no Pseudomonas was found in our study or thatDraft of Echigo et al. (2005). Rangarajan et al. (2002) isolated 59 strains of Pseudomonas from paddy soil (i.e., non-saline soil) of Southern India using a Pseudomonas-selective culture medium. Among the isolated strains, 18 were tolerant to 0.5 M (2.92%) NaCl, seven were tolerant to 1.0 M (5.85%) NaCl, and four were tolerant to 1.5 M (8.27%) NaCl. An eco-physiological approach suggests that the plant-associated microbial community may be the key factor for understanding the adaptation of plants to their habitat (Redman et al., 2002). Many investigations of the interaction of PGPB with other microbes and their effect on the physiological response of crop plants under different soil salinity regimes remain at an incipient stage (Singh et al., 2011). During the last decade, a number of reports have appeared on the beneficial effects of microorganisms, such as Pseudomonas, Bacillus, Pantoea, Burkholderia, and Rhizobium, that help enhance the tolerance of crops (e.g., rice, wheat, maize, cotton, lettuce, tomato, and pepper) to drought, salinity, heat stress, and chilling injury under controlled conditions (Jha and Subramanian, 2014； Natarajan et al., 2016). A large number of halophiles or salt-tolerant bacteria occur in nature. The screening of crop-beneficial microorganisms could substantially expand the applications of salt-tolerant bacteria and develop the resources of such bacteria. Researchers have isolated strains capable of promoting the salt

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tolerance of rice, corn, peanuts, and wheat from saline soils of beaches and other locations (Singh and Jha, 2016; Yuan et al., 2016; Nautiyal et al., 2013; Principe et al., 2007; Raheem and Ali, 2015; Shukla et al., 2012). Our results indicate that 80% of the growth-promoting salt-tolerant bacteria isolated from non-saline paddy soil belong to Bacillus spp. This finding is consistent with that of other research. The majority of the strains that are known to be salt-tolerant and capable of growth promotion are Bacillus, including Bacillus pumilus (Jha, 2013), Bacillus amyloliquefaciens (Nautiyal et al., 2013), and Bacillus thuringiensis (Raheem and Ali, 2015). Pseudomonas is another commonly observed salt-tolerant bacterium that promotes growth (Yuan et al., 2016; Jha and Subramanian, 2014; Forni et al., 2016; Raheem and Ali, 2015). However, no Pseudomonas was isolated in our study. It is possible that the isolation media we used were unsuited for the growth of Pseudomonas or that the number of Pseudomonas in the sampling environment was low. Because of the different culture media that were used, we isolated more growth-promoting actinomycetes, such as Streptomyces sp., which is rarely observed in other studies. Developing actinomycetesDraft into bio-fertilizer could provide advantages that other bacteria do not possess. That is, it is easy to culture, store, ship, and colonize, and it offers stronger resistance. Thus far, salt-tolerant bacteria capable of promoting rice salt tolerance are less frequently observed (García-Cristobal et al., 2015; Yuan et al., 2016; Jha and Subramanian, 2014; Nautiyal et al., 2013; Forni et al., 2016). Under the stress of different salt concentrations, this study found that 63%-87% of the strains could promote growth in both the shoot length and the root length of rice (fig 3). Numerous bacterial traits have been suggested to be involved in conferring salt tolerance to treated plants. These traits include the production of cytokinin, indoleacetic acid, ACC deaminase, abscisic acid, trehalose, volatile organic compounds, and exopolysaccharides. Several strains could even regulate gene transcription in rice to improve salt tolerance (Nautiyal et al., 2013). Thus, TY0307 capable of producing IAA, ACC deaminase and siderophore could enhance growth and alleviate salt stress in rice. The effect of promoting rice salt tolerance is found not only in single strains of bacteria but also in strain mixtures. Yuan et al. (2016) found that transplanting salt-tolerant bacteria from the plant rhizosphere to the roots of rice could also increase rice salt tolerance. These striking findings imply that microbial-mediated plant traits rely not only on individual members in a community but also on the cooperation and the functions of the entire microbiome.

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Certainly, rice salt tolerance involves a complicated biochemical and physiological process, such as protein synthesis, lipid metabolism, photosynthesis, ionic homeostasis, and nitrogen fixation (Parida and Das, 2005). PGPB improve plant growth under salt stress by maintaining favourable K+/Na+ ratio, or by enhancing the production of certain osmolytes such as proline, total soluble sugar, total protein content etc., or by both these methods (Singh and Jha, 2016). We observed a decrease in proline levels as a consequence of salinity in TY0307 inoculated rice and conclude this is a reflection of a lower severity of salt-induced stress in these plants. This was further corroborated by results of the MDA level which represents the degree of damage induced as a consequence of increased salinity and, is often used to evaluate plant salt tolerance (Han et al., 2014). Inoculation with TY0307 resulted in decreased MDA content in treated plants (Figure 4). In this study, the promotive effect of bacterial strains on the germination energy, germination capacity, shoot length, and root length of rice was found to be stronger under the stress of higher concentrations of salt than under the stressDraft of lower concentrations (Figure 3). This finding is consistent with the results of research by Wen et al. (2009). A possible explanation is that the high salt concentration resulted in physiological damage to rice that can be repaired by the bacteria. Additionally, the bacterial promotion of root growth under salt stress was more apparent than that of shoot growth (Figure 3). A possible explanation is that the leaves of rice are more sensitive to damage by osmotic pressure (Forni et al., 2016).

Conclusion Admittedly, our understanding of the mechanism of how microorganisms increase plant salt tolerance is limited. However, this limited understanding should not be an obstacle to developing the microorganisms into bio-fertilizer. Our results suggest the presence of rich and diverse salt-tolerant bacteria in the paddy soil. The significance of TY0307 treatment to rice under hydroponic conditions in ameliorating the salt stress possibly through increased producing of ACC deaminase, IAA and siderophore, induced reduction in MDA content, enhanced proline accumulation, and indicates its utility to be used as potential biofertilizer strain.

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This work was funded by grants from the National Natural Science Foundation of China (31200138, 31660042, 31660001, 31660089 and 41201321), the National Infrastructure of Natural Resources for Science and Technology Program of China (NIMR-2016-8) and the Yunnan Provincial Sciences and Technology Department (2014FB104, 2009CD012, 2015IC022, 2015HC018, 2018IA100).

References Ahmad M, Zahir ZA, Asghar HN, Asghar M. 2011. Inducing salt tolerance in mung bean through coinoculation with rhizobia and plant-growth-promoting rhizobacteria containing 1-aminocyclopropane-1-carboxylate deaminase. Can. J. Microbiol. 57(7): 578-89. Aniszewska M, Słowiński K. 2016. Effects of microwave irradiation by means of a horn antenna in the process of seed extraction on Scots pine ( Pinus sylvestris L.) cone moisture content and seed germination energy and capacity Eur JDraft Forest Rese, 135 (4) :633-642. Bates L, Waldren RP, Teare ID. 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39: 205-207 Bernardr G, Cheng Z, Jennifer C, Jin D. 2007. Promotion of plant growth by acc deaminase-producing soil bacteria. Eur. J. Plant. Pathol. 119(3): 329-339. Brouwer C, Goffeau A, Heibloem M. 1985. Irrigation Water Management. Training manual No. 1, In Introduction to irrigation; a manual. Prov. ed. FAO. Chen Q, Liu Z, Peng Q, Huang K, He J. 2010. Diversity of halophilic and halotolerant bacteria isolated from non-saline soil collected from Xiaoxi national natural reserve, Hunan province. Acta. Microbiol. Sin. 50(11): 1452-1459. Chen YG, Li HM, Li QY, Chen W, Cui XL. 2007. Phylogenetic diversity of culturable bacteria in the ancient salt deposits of the Yipinglang salt mine, P. R. China. Acta. Microbiol. Sin. 47(4): 571-577. Dimkpa C, Weinand T, Asch F. 2009. Plant-rhizobacteria interactions alleviate abiotic stress conditions. Plant Cell Environ. 32(12): 1682-1694. Echigo A, Hino M, Fukushima T, Mizuki T, Kamekura M. 2005. Endospores of halophilic bacteria of

15

https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 16 of 33

the family Bacillaceae isolated from non-saline Japanese soil may be transported by kosa event (Asian dust storm). Aquat. Biosyst. 1(1): 1-13. Egamberdieva D. 2009. Alleviation of salt stress by plant growth regulators and IAA producing bacteria in wheat. Acta. Physiol. Plant. 31(4): 861-864. Forni C, Duca D, Glick BR. 2016. Mechanisms of plant response to salt and drought stress and their alteration by rhizobacteria. Plant Soil 410(1): 335-356. García-Cristobal J, García-Villaraco A, Ramos B, Gutierrez-Mañero J, Lucas JA. 2015. Priming of pathogenesis related-proteins and enzymes related to oxidative stress by plant growth promoting rhizobacteria on rice plants upon abiotic and biotic stress challenge. J. Plant. Physiol. 188: 72-79. Gordon SA, Weber RP. 1951. Colorimetric estimation of indole acetic acid. Plant Physiol. 26: 192-195. Han QQ, Lü XP, Bai JP, Qiao Y, Paré PW. 2014. Beneficial soil bacterium Bacillus subtilis (GB03) augments salt tolerance of white clover. Front Plant Sci. 5: 525. Hodges DM. Delong JM, Forney CF, PrangeDraft RK. 1999. Improving the thiobarbituric acid reactive substances assay for estimating lipid peroxidation in plant tissue containing anthocyanin and other interfering compounds. Planta. 207: 604-611. Hong CY, Chao YY, Yang MY, Cheng SY, Cho SC. 2009. NaCl-induced expression of glutathione reductase in roots of rice (Oryza sativa L.) seedlings is mediated through hydrogen peroxide but not abscisic acid. Plant Soil. 320(1): 103-115. Honma M, Shimomura T. 1978. Metabolism of 1-aminocyclopropane-1-carboxylic acid. Agric. Biol. Chem. 42: 1825-1831. Jha Y. 2013. Paddy inoculated with PGPR show better growth physiology and nutrient content under salinity. Chil. J. Agr. Res. 73(1): 213-219. Jha Y, Subramanian RB. 2014. PGPR regulate caspase-like activity, programmed cell death, and antioxidant enzyme activity in paddy under salinity. Physiol. Mol. Bio. Plants 20(2): 201-207. Katsura K, Maeda S, Lubis I, Horie T, Cao W, Shiraiwa T. 2008. The high yield of irrigated rice in Yunnan China: a cross-location analysis. Field Crop Res. 107: 1-11. Kohler J, Hernández JA, Caravaca F, Roldán A. 2009. Induction of antioxidant enzymes is involved in the greater effectiveness of a PGPR versus am fungi with respect to increasing the tolerance of

16

https://mc06.manuscriptcentral.com/cjm-pubs Page 17 of 33 Canadian Journal of Microbiology

lettuce to severe salt stress. Environ. Exp. Bot. 65(3): 245-252. Li GH, Xue LH, Gu W, Yang CD, Wang SH. 2009. Comparison of yield components and plant type characteristics of high-yield rice between Taoyuan, a ‘special eco-site’ and Nanjing, China. Field Crop Res. 112: 214–221. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. Lucas JA, García-Cristobal J, Bonilla A, Ramos B, Gutierrez-Mañero J. 2014. Beneficial rhizobacteria from rice rhizosphere confers high protection against biotic and abiotic stress inducing systemic resistance in rice seedlings. Plant Physiol. Bioch. 82(3): 44-53. Mayak S, Tirosh T, Glick BR. 2004. Plant growth-promoting bacteria confer resistance in tomato plants to salt stress. Plant Physiol. Bioch. 42(6): 565-572. Ministry of agriculture of People’s Republic of China. 2006. Method for determination of total water-soluble salt (NY/T1121.16-2006).Draft Chinese Agricultural Standard. Nadeem SM, Ahmad M, Naveed M, Imran M, Zahir ZA. 2016. Relationship between in vitro characterization and comparative efficacy of plant growth-promoting rhizobacteria for improving cucumber salt tolerance. Arch. Microbiol. 198(4): 379-387. Nadeem SM, Ahmad M, Zahir ZA, Javaid A, Ashraf M. 2014. The role of mycorrhizae and plant growth promoting rhizobacteria (PGPR) in improving crop productivity under stressful environments. Biotechnol. Adv. 32(2): 429-448. Natarajan A, Kumar K, Madhuri K, Usharani GK. 2016. Isolation and characterization of salt tolerant plant growth promoting rhizobacteria from plants grown in affected regions of Andaman and Nicobar Islands. Geomicrobiol. J. 33 (10): 942-947. Nautiyal CS, Srivastava S, Chauhan PS, Seem K, Mishra A. 2013. Plant growth-promoting bacteria Bacillus amyloliquefaciens, NBRISN13 modulates gene expression profile of leaf and rhizosphere community in rice during salt stress. Plant Physiol. Bioch. 66(5): 1-9. Parida AK, Das AB. 2005. Salt tolerance and salinity effects on plants: a review. Ecotox. Environ. Safe 60(3): 324-349. Principe A, Alvarez F, Castro MG, Zachi L, Fischer SE. 2007. Biocontrol and PGPR features in native

17

https://mc06.manuscriptcentral.com/cjm-pubs Canadian Journal of Microbiology Page 18 of 33

strains isolated from saline soils of Argentina. Curr. Microbiol. 69(4): 314-322. Raheem A, Ali B. 2015. Halotolerant rhizobacteria: beneficial plant metabolites and growth enhancement of Triticum aestivum L. in salt-amended soils. Arch. Agron. Soil Sci. 61(12): 1691-1705. Rangarajan S, Saleena LM, Nair S. 2002. Diversity of Pseudomonas spp. isolated from rice rhizosphere populations grown along a salinity gradient. Microbial. Ecol. 43(2): 280-290. Redman RS, Sheehan KB, Stout RG, Rodriguez RJ, Henson JM. 2002. Thermotolerance generated by plant/fungal symbiosis. Science 298(5598): 1581. Saharan BS, Nehra V. 2011. Plant growth promoting rhizobacteria: a criticalre view. Life Sci. Med. Res. 21: 1-30. Schwyn B, Neilands JB. 1987. Universal chemical assay for the detection and determination of siderophores. Anal Biochem. 160: 47-56. Shukla PS, Agarwal PK, Jha B. 2012. ImprovedDraft salinity tolerance of Arachis hypogaea (L.) by the interaction of halotolerant plant-growth-promoting rhizobacteria. J. Plant Growth Regul. 31: 195-206. Singh JS, Pandey VC, Singh DP. 2011. Efficient soil microorganisms: a new dimension for sustainable agriculture and environmental development. Agr. Ecosyst. Environ. 140 (3-4): 339-353. Singh RP, Jha PN. 2016. Alleviation of salinity-induced damage on wheat plant by an ACC deaminase-producing halophilic bacterium serratia sp. SL-12 isolated from a salt lake. Symbiosis. 69(2): 101-111. StanLotter H, Radax C, Gruber C, Legat A, Pfaffenhuemer M, Wieland H, Leuko S, Weidler G, Kömle N, Kargl G. 2002. Astrobiology with haloarchaea from permo-triassic rock salt. Int. J. Astrobiol. 1(4): 271-284. Wen FP, Zhang T, Zhang ZH, Pan, YH. 2009. Proteome analysis of relieving effect of gibberellin on the inhibition of rice seed germination by salt stress. Acta. Agronomica. Sin. 35(3): 483-489. Xiao W, Wang ZG, Wang YX, Schneegurt MA, Li ZY, Cui XL. 2013. Comparative molecular analysis of the prokaryotic diversity of two salt mine soils in southwest China. J. Basic. Microb. 53(11): 942-952.

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Xiao W, Wang YX, Liu JH, Wang ZG, Zhang XX, Ji KY, Lai YH, Wen ML, Cui XL. 2012. Roseivivax sediminis sp. nov., a moderately halophilic bacterium isolated from salt mine sediment. Int. J. Syst. Evol. Microbiol. 62: 1890-1895. Yuan Z, Druzhinina IS, Labbé J., Redman R, Yuan Q. 2016. Specialized microbiome of a halophyte and its role in helping non-host plants to withstand salinity. Sci. Rep. 6: 32467. Zahir ZA, Arshad M, Wtjr F. 2003. Plant growth promoting rhizobacteria: applications and perspectives in agriculture. Adv. Agron. 81(03): 97-168. Zahir ZA, Ghani U, Naveed M, Nadeem SM, Asghar HN. 2009. Comparative effectiveness of Pseudomonas and Serratia sp. containing ACC-deaminase for improving growth and yield of wheat (Triticum aestivum L.) under salt-stressed conditions. Arch. Microbiol. 191(5): 415-424.

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26 Fig. 1 Distribution of salt-tolerant bacteria in paddy soil at Order level.

27 Fig. 2 Strains that significantly improved the germination energy (A) and germination capacity

28 (B) of rice seeds under salt stress.

29 Fig. 3 Strains that exhibit apparent growth promotion under the stress of different

30 concentrations of salt. Values are mean of three replicates ± SD. *means different significantly.

31 Fig. 4 Effect of TY0307 inoculation on proline (A) and MDA (B) content under different

32 concentrations of NaCl. Values are mean of three replicates ± SD. *means different significantly.

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1 Table 1 BLAST results and NaCl tolerance of the bacteria isolated from paddy soil.

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Strains Nearest phylogenetic neighbour in GenBank Similarity NaCl tolerance Phylum Order (accession number) (accession number) (%) (g/L)

Rhodococcus TY0433(KF477161) Rhizobium grahamii (AEYE01000061) 98.78 0-150 TY0207(KF477133) Halomonas nitrilicus (EU447162) 98.32 0-100 Proteobacteria Oceanospirillales TY0222(KF477137) Halomonas venusta (AJ306894) 99.25 0-150 TY0539(KF477169) Halomonas hydrothermalis (AF212218) 98.20 0-100

TY0004(KF477097) Bacillus horikoshii (X76443) 97.84 0-100 TY0010(KF477100) Bacillus megaterium (JJMH01000057) 98.27 0-100 TY0012(KF477101) Fictibacillus nanhaiensis (GU477780) 100 0-100 TY0015(KF477103) DraftFictibacillus nanhaiensis (GU477780) 97.80 0-100 TY0017(KF477104) Fictibacillus nanhaiensis (GU477780) 99.09 0-100 TY0018(KF477105) Bacillus vietnamensis (AB099708) 97.86 0-100 TY0028(KF477108) Brevibacterium frigoritolerans (AM747813) 99.50 0-50 Firmicutes Bacillales TY0029(KF477109) Bacillus marisflavi (AF483624) 99.36 0-150 TY0030(KF477110) Bacillus megaterium (JJMH01000057) 98.62 0-50 TY0033(KF477111) Bacillus oceanisediminis (GQ292772) 98.40 0-100 TY0036(KF477112) Bacillus megaterium (JJMH01000057) 98.57 0-150 TY0038(KF477113) Bacillus horikoshii (X76443) 98.52 0-50 TY0045(KF477114) Bacillus oceanisediminis (GQ292772) 98.19 0-150 TY0105(KF477117) Bacillus megaterium (JJMH01000057) 98.12 0-100 TY0108(KF477118) Bacillus simplex (AB363738) 98.98 0-100

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TY0111(KF477120) Bhargavaea cecembensis (AM286423) 97.72 0-100 TY0114(KF477121) Bacillus megaterium (JJMH01000057) 97.46 0-100 TY0115(KF477122) Bacillus selenatarsenatis (AB262082) 99.17 0-50 TY0119(KF477123) Fictibacillus nanhaiensis (GU477780) 100 0-100 TY0123(KF477125) Bacillus megaterium (JJMH01000057) 97.58 0-100 TY0124(KF477126) Bacillus aerophilus (AJ831844) 98.75 0-150 TY0125(KF477127) Bacillus oceanisediminis (GQ292772) 98.55 0-150 TY0127(KF477128) Jeotgalibacillus salarius (EU874389) 99.76 0-100 TY0128(KF477129) Bacillus marisflavi (AF483624) 98.87 0-100 TY0130(KF477130) DraftBacillus aerophilus (AJ831844) 98.43 0-100 TY0130-1(KF477131) Bacillus aerophilus (AJ831844) 97.70 0-100 TY0210(KF477134) Bacillus idriensis (AY904033) 98.28 0-100 TY0211(KF477135) Bacillus horikoshii (X76443) 99.43 0-100 TY0215(KF477136) Bacillus jeotgali (FR733689) 99.32 0-50 TY0223(KF477138) Bacillus subterraneus (FR733689) 98.86 0-150 TY0224(KF477139) Bacillus horikoshii (X76443) 99.50 0-100 TY0226(KF477140) Paenisporosarcina quisquiliarum (DQ333897) 99.57 0-50 TY0230(KF477141) Fictibacillus nanhaiensis (GU477780) 98.64 0-50 TY0236(KF477142) Bacillus barbaricus (AJ422145) 99.42 0-150 TY0239(KF477143) Oceanobacillus kimchii (AOCX01000002) 100 0-100

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TY0253(KF477145) Chryseomicrobium imtechense (GQ927308) 100 0-50 TY0256(KF477147) Bacillus horikoshii (X76443) 99.43 0-100 TY0301(KF477148) Bacillus safensis (ASJD01000027) 98.82 0-100 TY0307(KF477150) Bacillus nanhaiensis (GU477780) 98.94 0-100 TY0312(KF477151) Bacillus aryabhattai (EF114313) 99.01 0-50 TY0312A(KF477152) Bacillus aryabhattai (EF114313) 100 0-100 TY0334(KF477153) Bacillus simplex (AB363738) 98.63 0-50 TY0338(KF477154) Bacillus aerophilus (AJ831844) 99.30 0-150 TY0340(KF477155) Bacillus aerophilus (AJ831844) 97.72 0-150 TY0406(KF477157) DraftBacillus simplex (AB363738) 99.48 0-150 TY0407(KF477158) Bacillus megaterium (JJMH01000057) 99.09 0-100 TY0407-1(KF477159) Bacillus megaterium (JJMH01000057) 98.47 0-100 TY0439(KF477162) Solibacillus isronensis (AMCK01000046) 99.78 0-50 TY0463(KF477166) Bacillus megaterium (JJMH01000057) 98.56 0-150 TY0509(KF477167) Bacillus aerophilus (AJ831844) 98.29 0-150 TY0525(KF477168) Bacillus safensis (ASJD01000027) 98.89 0-100

TY0003(KF477096) Streptomyces tunisiensis (KF697135) 98.70 0-50 TY0007(KF477098) Streptomyces pratensis (JQ806215) 98.57 0-100 Actinobacteria Actinomycetales TY0009(KF477099) Streptomyces tunisiensis (KF697135) 98.93 0-50 TY0014(KF477102) Streptomyces luteogriseus (AB184379) 98.61 0-50

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TY0019(KF477106) Microbacterium oleivorans (AJ698725) 100 0-50 TY0027(KF477107) Microbacterium oxydans (Y17227) 97.77 0-50 TY0101(KF477115) Streptomyces xiamenensis (EF012099) 98.05 0-50 TY0103(KF477116) Streptomyces luteogriseus (AB184379) 99.01 0-50 TY0110(KF477119) Streptomyces longispororuber (AB184440) 99.42 0-100 TY0121(KF477124) Streptomyces cellulosae (AB184265) 100 0-100 TY0134(KF477132) Arthrobacter oxydans (X83408) 97.36 0-50 TY0240(KF477144) Nesterenkonia sandarakina (AY588277) 97.23 0-100 TY0255(KF477146) Nesterenkonia halotolerans (AY226508) 99.86 0-50 TY0304(KF477149) DraftArsenicicoccus bolidensis (AJ558133) 100 0-100 TY0400(KF477156) Streptomyces longispororuber (AB184440) 96.00 0-50 TY0429(KF477160) Rhodococcus ruber (X80625) 99.88 0-150 TY0450(KF477163) Nocardia abscessus (BAFP01000036) 98.18 0-50 TY0457(KF477164) Nocardia abscessus (BAFP01000036) 99.63 0-50 TY0459(KF477165) Nocardia abscessus (BAFP01000036) 99.88 0-50

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3 Table 2 Effects of salt-tolerant bacteria on rice seedling root and shoot length increase

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Rate of root length increase (%) Root length increase (cm) Rate of shoot length increase (%) Shoot length increase (cm)

NaCl(g/l) 0 3 6 9 12 0 3 6 9 12 TY0029 -65.3±7.1 89.2±2.2 -84.2±2.8 2.18±0.9 0.77±0.2 -71.3±1.9 87.2±5.2 -17.6±3.5 2.39±1.0 1.47±0.5 TY0033 -52.8±2.8 -0.6±1 -45.9±1.8 -0.34±0.1 1.4±0.5 -48.2±4.5 72.4±2.3 54.7±2.1 0 0.8±0.4 TY0036 -49.1±1.3 -10.4±1.0 -10.3±0.8 0.25±0.2 0.6±0.1 -45.1±3.9 105.3±8.7 88.2±10.1 0.05±0.01 0.8±0.1 TY0045 -36.8±1.6 -1.04±0.5 -78.6±5.2 -0.46±0.1 0.63±0.2 -30.5±2.3 -37.9±7.1 5.9±1.1 0.2±0.05 1.0±0.5 TY0105 -82.5±14.5 -62.3±11.2 -54.9±10.1 0.29±0.01 0.46±0.02 -79.4±15.4 108.4±12.3 -10.1±1.2 1.6±0.5 1.2±0.6 TY0110 7.9±5.1 -100.0±5.9 203.9±31.9 2.60±0.9 1.0±0.3 23.3±5.2 -100±5.8 160.7±30.1 2.4±0.8 0.5±0.1 TY0111 1.5±1.0 178.9±21.5 120.9±18.7 1.97±0.8 0.68±0.07 40.9±1.2 97.6±20.9 183.2±20.9 0.23±0.05 0.1±0.05 TY0114 -6.8±0.9 179.9±20.2 107.2±9.4 2.4±0.8 2.53±0.4 24.7±1.5 105.6±20.2 100.7±10.2 0.40±0.04 0.2±0.06 TY0119 -14.4±2.5 196.4±20.3 149.7±10.9 2.1±0.8 2.03±0.5 10.6±1.5 128.3±20.8 33.2±6.8 0.08±0.05 0.18±0.03 TY0121 -14.3±2.8 114.9±5.7 98.3±10.1 2.2±1.1 1.6±0.8 37.8±9.1 101.1±23.1 -87.5±12.9 0.12±0.01 0.07±0.05 TY0123 -40.3±9.8 200.7±34.1 42.0±21.1 0.9±0.04 0 -18.7±6.4 67.0±2.9 130.4±38.1 0.16±0.07 0 TY0127 -11.5±5.0 130.4±24.1 233.7±23.5 2.6±0.6Draft 1.97±0.2 4.6±0.8 103.9±12.6 133.6±21.3 0.20±0.02 0.22±0.05 TY0130 -15.0±4.7 103.3±13.9 -89.0±21.3 3.11±0.9 0 45.1±6.5 112.6±2.6 100±10.3 1.72±0.03 0.20±0.02 TY0130-1 21.4±10.2 124.8±21.4 135.9±19.4 3.22±1.2 0.92±0.09 27.3±10.2 103.4±10.6 191.4±28.4 1.39±0.6 0.13±0.4 TY0132 -4.72±1.1 4.0±0.9 211.1±40.5 1.38±0.5 2.43±0.6 15.3±1.0 -56.6±2.1 176.4±20.1 0.14±0.04 0.24±0.02 TY0210 3.47±1.2 65.0±3.1 164.1±5.2 1.79±0.8 1.74±0.5 29.4±1.2 104.1±5.1 154.3±10.1 0.39±0.08 0.21±0.06 TY0222 -7.08±1.0 125.4±5.3 164.1±7.1 4.06±0.6 1.63±0.4 24.1±2.0 77.3±3.1 139.3±4.3 0.32±0.04 0.05±0.05 TY0231 -12.8±1.9 192.4±20.1 75.1±5.1 2.38±0.5 2.0±0.4 16.0±0.8 124.8±21.2 38.6±4.3 0.40±0.01 0.28±0.06 TY0238 -15.1±1.5 83.5±5.2 148.6±2.4 2.65±0.8 1.20±0.6 -31.2±12.4 89.3±2.8 112.9±1.8 0.52±0.1 0.12±0.03 TY0239 -14.0±2.1 179.5±30.1 42.5±4.1 1.4±0.4 1.00±0.3 17.4±2.1 98.1±30.2 -34.6±2.1 0 0.30±0.04 TY0256 -19.6±1.8 90.1±2.4 164.6±4.3 1.55±0.8 0 25.9±2.1 162.5±5.3 278.6±2.5 0 0 TY0301 -2.22±0.8 69.3±4.3 4.9±0.9 1.20±0.5 0 27.6±0.8 96.8±2.8 -82.1±31.4 0.13±0.1 0 TY0307 -0.28±0.2 117.2±5.1 219.9±20.1 5.09±0.5 0 30.1±2.4 96.2±5.4 140.4±10.5 4.67±0.8 0.50±0.2 TY0338 0 56.1±2.8 0 0.85±0.4 0 -100.0±10.2 16.3±4.1 -83.9±5.6 0.10±0.02 0 TY0340 -7.08±2.1 121.5±2.6 95.1±5.4 4.03±0.8 0.90±0.5 29.0±5.1 82.8±7.6 94.6±8.1 2.6±0.2 0.27±0.1 TY0406 -2.22±0.8 45.8±10.9 54.7±8.9 1.93±0.8 3.84±0.9 37.5±8.3 16.1±4.1 -86.8±10.1 0.29±0.05 0.60±0.1 TY0407 -15.7±5.1 147.9±4.9 145.9±7.9 3.0±0.5 2.71±0.5 17.2±2.1 85.7±5.8 137.1±4.9 2.80±0.3 0.17±0.05 TY0463 -2.08±1.0 144.9±5.9 151.9±10.7 3.2±0.1 0 4.2±1.9 98.5±2.9 148.6±5.1 2.56±0.5 0

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TY0509 -12.8±5.2 87.5±1.3 180.7±40.2 4.38±1.5 0.73±0.5 9.8±2.1 59.7±5.2 160.7±40.1 0.60±0.5 0.20±0.5 TY0545 -6.81±1.1 106.6±2.8 244.2±31.1 3.45±0.2 0.83±0.2 11.0±2.1 66.8±5.8 190.7±10.1 3.14±0.6 0.18±0.1 The data are the averages ± standard deviations of three bottles replicates, with ten seedlings per bottles for each treatment.

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Distribution of salt-tolerant bacterial orders in paddy soils.

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Strains that significantly improved the germination energy (A) and germination capacity (B) of rice seeds under salt stress.

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