The combined Cp2-EPS and rhizobium inoculation enhance the growth of alfalfa under salt stress

doi:10.21203/rs.3.rs-4969705/v1

Download PDF

Article

The combined Cp2-EPS and rhizobium inoculation enhance the growth of alfalfa under salt stress

https://doi.org/10.21203/rs.3.rs-4969705/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

The interaction of plant and soil microbial communities can promote plant growth and increase stress tolerance. Exopolysaccharide (EPS) is a secondary metabolite produced by certain bacteria and important signal molecules between plants and microorganisms, which has the potential to alleviate salt stress in plant. The study explored the effects of combined Erwinia persicina Cp2-EPS and rhizobium strain (Gz5) inoculation on seed germination and seedling growth of alfalfa under salt stress. The results showed that under 100 mmol·L^-1 NaCl stress, each treatment had a very significant positive effect on seed germination and seedling growth compared with the control. The germination index aboveground and underground fresh and dry weight, aboveground and underground length, root index, chlorophyll content, SOD and CAT activities and SS content of combined Cp2-EPS and Gz5 inoculation both achieved maximum value, MDA and H₂O₂ content reached a minimum value. And relative conductivity and preserved amino acid content were significantly improved compared with the control. Therefore, combined inoculation had positive effects on seed germination and seedling growth of alfalfa under salt stress, and had a more remarkable effects than single inoculation (R/EPS). Our findings provided valuable insights for enhancing the salt tolerance of alfalfa and saline-alkali land improvement.

Biological sciences/Microbiology

Biological sciences/Physiology

Combined inoculation

Exopolysaccharide

Rhizobium

Alfalfa

Erwinia persicina

Salt stress

Salinity is one of the most significant environmental factors limiting the productivity of plants (Abhinandan et al. 2018). Currently, over 10% of arable land and 25–30% of irrigated land in the world are salt-affected (Eliasson et al. 2003; Zaman et al. 2018), and the area of salinization is increasing (Shrivastava and Kumar, 2015). Soil salinization has been shown to reduce the diversity and relative abundance of soil microorganisms, and have adverse effects on the interaction between microorganisms and plants (Banerjee et al. 2019). This is thought to be an important reason for indirectly reducing salt tolerance and yield reduction of plants. Alfalfa (Medicago sativa) is a perennial leguminous forage, which is one of the most widely cultivated forage forages in the world, and it is also well-known as the “king of forages”, due to rich nutrition and good ecological value. It is cultivated in over 80 countries across various continents, with an area of more than 35 million hectares, making it the most widely cultivated in the world (Radović et al. 2009; Moot et al. 2012). Compared with other forages, alfalfa has superior stress resistance and is widely cultivated in mining areas, degraded grasslands and saline-alkali lands in Northwest China (Peng et al. 2020; Wang et al. 2016). Therefore, enhancing the salt tolerance of alfalfa is crucial for the improvement of saline-alkali land in China. Currently, it has been found that symbiotic microorganisms have an important impact on their adaptation to adversity stress and can serve as an alternative source of stress relief to improve plant yield and plant growth conditions (Vimal et al. 2017). For example, Rhizobia and legumes have formed a stable symbiotic nitrogen fixation system during the long-term interaction, such as providing fixed nitrogen to plants to increase biomass (Kudoyarova et al. 2015), stimulating plants to produce antioxidant enzymes (Kong et al. 2015; Duan et al. 2018; Rajkumar et al. 2010; Lee et al. 2009), and directly or indirectly stimulating plant growth (Khan and Bano, 2019; He et al. 2020; Abdelkrim et al. 2018; Saadani et al. 2016). However, excessive NaCl also affects rhizobial colonization and interaction in legume roots (Jofré et al. 1998; Sandhya et al. 2009).

In recent years, studies have found that polysaccharides have practical utility value in inducing plant stress resistance and are a promising agricultural biomaterial (Upadhyay et al. 2011). EPS are natural higher molecular weight polymers secreted during the growth and metabolism of microorganisms (Laspidou and Rittmann, 2002), which are usually produced under harsh environmental or nutritional conditions and have a positive correlation with abiotic stress (Sengupta and Dey, 2019). As a physiological stress response and environmental protection strategy, they play a crucial important role in the growth, colonization and stress resistance of bacteria (Pulsawat et al. 2003; Nandal et al. 2005). EPS have rich functional groups, which enable acidic EPS to precipitate metal cations and produce mediated acid resistance and aluminum resistance (Corzo et al. 1994; Avelar et al. 2012). They can also provide antioxidants, store carbon and promote plant growth nutrients (Upadhyay et al. 2011), significantly promoting seed germination, growth and stress resistance (Ashraf et al. 2004). In addition, heavy metal-resistant strains producing EPS have protective effects on heavy metal-sensitive strains that do not produce EPS in co-culture experiments, which is called “rescue” non-resistant strains (Nocelli et al. 2016). This also means a stronger ability to symbiosis with other soil microorganisms and host plants, which has an impact on microbial ecology and alfalfa production (Primo et al. 2020; Primo et al. 2020). Erwinia persicina Cp2 strain can produce large amounts of mucus-like Cp2-EPS on King’s B medium, and they widely exist in the environment (Zhang, 2013). Some findings have shown that the combined EPS and rhizobium inoculation exhibited a significantly higher the growth and nitrogen fixation of alfalfa compared to single rhizobacterial treatments (Jin et al. 2021). However, whether the combined EPS and rhizobium inoculation can alleviate under salt stress in alfalfa have rarely been reported. Plant salt tolerance mainly includes morphological, physiological and biochemical changes (Koyro, 2006). Among them, seed germination and seedling growth are the basis of plant growth and development, and they are also the most vulnerable links. Therefore, this experiment used combined different concentrations of Cp2-EPS and rhizobium inoculation to investigate its effect on the growth of alfalfa seedlings under 100 mmol·L^− 1 NaCl stress. This study provides a new approach for enhancing the salt tolerance of alfalfa and improving saline-alkali soil.

2.1 Experimental materials

Seeds of alfalfa (Medicago sativa) variety used in this study was ‘Juneng551’. The rhizobium strain is Gz5, which was isolated from the mature root nodules of ‘Juneng551’ alfalfa and stored at -80℃. Cp2-EPS were extracted from E. persicina Cp2 strain. Bacterial culture was grown in yeast extract mannitol agar media (YMA: Mannitol 10.0 g, Yeast powder 1.0 g, K₂HPO₄·3H₂O 0.5 g, MgSO₄·7H₂O 0.2 g, NaCl 0.1 g, CaCO₃ 3.0 g, pH 7.0-7.2 and 18 g agar) at 25℃. All test materials were provided by the Forage Pathology Laboratory, Pratacultural College, Gansu Agricultural University.

2.2 Experimental treatments

2.2.1 Salt tolerance evaluation of Rhizobium Gz5

YMB medium was used with 0‰, 5‰, 10‰, 15‰, 20‰, 25‰, 30‰, 35‰, 40‰ and 50‰ NaCl treatments. An equal amount of sterile water of YMB medium inoculated with bacterial suspension was used as a control to access rhizobia, and after 48 h of shaking cultivation (28℃, 180 rpm), the absorbance value was determined at OD600 and the growth curve of salt tolerance was made (Din et al. 2019).

2.2.2 Test Method

Using an electronic balance, 0, 0.6, 0.9, 1.2, and 1.5 g of Cp2-EPS were weighed, respectively, and dissolved by adding 100 mmol·L^-1 NaCl solution, and the solution was fixed to 1 L with 100 mmol·L^-1 NaCl solution, and then transferred to the growth vials, and 190 mL was added to each vial, and then sterilized by autoclaving (121℃, 0.11 Mpa, 20 min). 0, 0.6, 0.9, 1.2, and 1.5 g·L^-1 of sterile mixed solution of Cp2-EPS and NaCl were obtained, i.e., treatments CK, B, C, D, and E.

Rhizobium Gz5 stored in the refrigerator at -80°C was activated, inoculated into YMB medium, and incubated in a constant temperature oscillating incubator at 28°C, 180 r·min^-1 for 3 d. After centrifugation of the bacterial body, Gz5 suspensions were prepared with the above five different concentrations of Cp2-EPS and NaCl, and the cell densities of the rhizobium were not less than 10⁸ CFU·mL^-1 (OD600 = 0.1), and the OD600 = 0.1 was calculated from the standard curve of Rhizobium Gz5. The cell density of Gz5 was not less than 10⁸ CFU·mL^-1 with OD600 = 0.1. The bacterial suspension prepared at each Cp2-EPS concentration was added in 10 mL to the above growth vials corresponding to them containing different Cp2-EPS concentrations (0, 0.6, 0.9, 1.2, and 1.5 g·L^-1), i.e., treatments A, F, G, H, and I. Ten (CK, A, B, C, D, E, F, G, H, and I) treatments were finally identified (Table 1).

Table 1

Processing settings
Treatment	CK	A	B	C	D	E	F	G	H	I
	Sterile water	Gz5	0.6 g·L^− 1Cp2-EPS	0.9 g·L^− 1Cp2-EPS	1.2 g·L^− 1Cp2-EPS	1.5 g·L^− 1Cp2-EPS	0.6 g·L^− 1Cp2-EPS +Gz5	0.9 g·L^− 1Cp2-EPS +Gz5	1.2 g·L^− 1Cp2-EPS +Gz5	1.5 g·L^− 1Cp2-EPS +Gz5

Table 2

Effects of combined Cp2-EPS and Gz5 inoculation on seed germination of alfalfa under salt stress
Treatments	Germination rate (%)	Germination potential (%)
CK	91.00 ± 2.52^c	88.00 ± 1.63^c	57.00 ± 0.53^d
A	96.00 ± 1.63^ab	94.00 ± 1.15^ab	60.00 ± 0.36^c
B	94.00 ± 1.15^bc	93.00 ± 1.00^b	61.00 ± 0.46^bc
C	97.00 ± 1.00^ab	94.00 ± 1.15^ab	62.00 ± 0.53^abc
D	98.00 ± 1.15^ab	96.00 ± 0.00^ab	63.00 ± 0.38^a
E	97.00 ± 1.00^ab	95.00 ± 1.00^ab	61.00 ± 1.01^bc
F	99.00 ± 1.00^a	97.00 ± 1.00^a	63.00 ± 0.93^a
G	98.00 ± 1.15^ab	96.00 ± 0.00^ab	62.00 ± 0.42^abc
H	98.00 ± 1.15^ab	97.00 ± 1.00^a	62.00 ± 0.29^ab
I	97.00 ± 1.00^ab	94.00 ± 1.15^ab	61.00 ± 0.31^bc
Different letters indicate significant differences among different treatments, P < 0.05, and error bars are expressed in the form of standard error (SE). The abscissa CK, A, B, C, D, E, F, G, H, I represent NaCl, NaCl + R, 0.6 g Cp2-EPS + NaCl, 0.9 g Cp2-EPS + NaCl, 1.2 g Cp2-EPS + NaCl, 1.5 g Cp2-EPS + NaCl, 0.6 g Cp2-EPS + R + NaCl, 0.9 g Cp2-EPS + R + NaCl, 1.2 g Cp2-EPS + R + NaCl, 1.5 g Cp2-EPS + R + NaCl respectively (R stands for Rhizobium strain Gz5).

2.3 Index determination

2.3.1 Growth indicators

The germination rate (GR), Germination potential (GP) and Germination index (GI) were measured according to the equations reported by (Hashem et al. 2016). After 14 d, the dry and fresh weights of alfalfa were determined separately. Root morphological indicators: the roots of the seedlings were rinsed with distilled water, and 16 plants were taken for each treatment. Scanned by the root scanner, the output pictures are analyzed by Win-RHIZO root analysis system software, and the total root surface area, total root volume, average root diameter and root tip number are respectively obtained.

2.3.2 Physiological and biochemical indicators

Chlorophyll content is measured with reference to the method of Wassie et al (2020). Conductivity was determined according to the method of Dacosta et al (2004). Soluble sugar (SS) content, Proline (Pro) content, Malondialdehyde (MDA), Hydrogen peroxide (H₂O₂), Superoxide dismutase (SOD) and Catalase (CAT) were determined according to the commercial kits (Suzhou Michy Biomedical Technology Co., Ltd).

2.4 Statistical analysis

One-way ANOVA statistical analysis was carried out by SPSS23.0, and the mean value and standard error of the mean value were calculated, determined by the least significant difference test (P < 0.05). Data collation and drawing are performed through Microsoft Excel and Origin2021.

2.5 Ethical approval

We confirm that the use of plants in the present study complies with international, national and/or institutional guidelines.

3.1 Determination of salt tolerance of rhizobium strain Gz5

24 h growth status of rhizobium strain Gz5 under different NaCl levels. The results showed that when the concentration of NaCl in the culture solution was > 30‰, the absorbance OD600 values were all < 0.2, and almost no growth occurred. When the concentration of NaCl in the culture medium is less than 30‰, the rhizobia grow well (Figure 1).

3.2 Effects of combined inoculation on seed germination

Salinity can inhibit the germination of all plant seeds (Table2). Under salt stress, the lowest germination potential, germination rate and germination index were observed for the CK. Compared with CK, single inoculation of Gz5 or Cp2-EPS (R/EPS) and combined inoculation all significantly promoted germination potential, germination rate and germination index (P<0.05). Among them, the combined inoculation F treatment showed the maximum value, which was 9 %, 11.4 % and 11.9 % higher than CK, respectively. Overall, both single inoculation (R/EPS) and combined inoculation alleviated the adverse effects of salinity on seed germination. Among them, F treatment had the best promoting effect in combined inoculation (Table2).

3.3 Effects of combined inoculation on fresh weight and dry weight of seedlings

3.3.1 Effects of combined inoculation on dry weight of seedlings aboveground and underground

The combined inoculation on seedling growth is shown in Figure 2A. The effect of salt stress on plants will eventually lead to a decrease in biomass. Under salt stress, the shoot and root dry weight of CK was the lowest. Compared with CK, the aboveground and underground dry weights of single inoculation (R/EPS) and combined inoculation were significantly increased (P<0.05), and the trend of change was similar. Compared with single inoculation (R/EPS), combined inoculation G, H, I aboveground dry weight and F, G, H, I below ground dry weight were significantly increased (P<0.05). Among them, the underground dry weight of H and the aboveground dry weight of G show the maximum, which are respectively increased by 375% and 383% compared with CK (Figure 2B).

3.3.2 Effects of combined inoculation on fresh weight of seedlings aboveground and underground

Salt stress negatively affected seedling fresh weight in a similar pattern to dry weight. Compared with CK, the aboveground and belowground fresh weights of single inoculation (R/EPS) and combined inoculation were significantly increased (P<0.05). Compared with single inoculation (R/EPS), the combined inoculation G, H, I aboveground fresh weight and F, G, H, I underground fresh weight increased significantly (P<0.05). Among them, the aboveground and underground fresh weights of combined inoculation H showed the maximum value, which increased by 290% and 563% respectively compared with CK (Figure 2B, C).

3.4 Effects of combined inoculation on the aboveground and underground length of seedlings

The root system is the primary site for sensing salt stress, and salinity will inhibit the growth of the root system of seedlings, thereby affecting the development of aboveground parts. Under salt stress, the minimum lengths above and below ground were observed in the CK. Compared with CK, the aboveground and underground lengths of single inoculation (R/EPS) and combined inoculation were significantly increased (P<0.05). Compared with single inoculation (R/EPS), the aboveground lengths of G and H and the underground lengths of F, G, H and I were significantly increased (P<0.05) in combined inoculation. Among them, the underground length of G and the aboveground length of H show the largest increase, which are respectively 209% and 404% higher than CK. The trend of aboveground growth of seedlings was similar to that of belowground growth, and these results indicated that combined inoculation (R/EPS) had a more positive interaction on aboveground and belowground growth of seedlings under salt stress than single inoculation (R/EPS) (Figure 2E).

3.5 Effects of combined inoculation on root structure of seedlings

Compared with CK, the root diameter, total root surface area and total root volume of other treatments were significantly increased (P<0.05), except that the root diameter of A was significantly lower than that of CK, and the number of root tips of each treatment increased compared with CK. Among them, the root diameter, total root surface area and root tip number of combined inoculation H all showed the maximum value, which increased by 45.9%, 623% and 650% respectively compared with CK. The combined inoculation of G showed the maximum total root volume, which increased by 650% compared with CK (Figure 2D).

3.6 Effect of combined inoculation on Chlorophyll content of seedlings

The effect of salt stress on chlorophyll content was as drastic as other morphological and physiological parameters of plant seedlings. Compared with the CK, both single inoculation (R/EPS) and combined inoculation significantly increased the chlorophyll a content of seedlings (P<0.05), indicating that either Cp2-EPS or rhizobium had a positive effect on chlorophyll a content. Among them, combined inoculation F showed the maximum value, which increased by 172% compared with CK. Similarly, the effects of single inoculation (R/EPS) and combined inoculation on the chlorophyll b content of seedlings were similar to those of chlorophyll a, and each treatment was significantly higher than CK (P<0.05). Among them, combined inoculation F showed the maximum value, which increased by 144% compared with CK. In general, combined inoculation can effectively reduce the effect of salinity on the chlorophyll content of seedlings and promote photosynthesis (Figure 3A, B, C).

3.7 Effects of combined inoculation on electrical conductivity of seedling leaves

Electrical conductivity is the response of plants to environmental stress, and salinity will cause an increase in electrical conductivity in plants. Compared with CK, the relative conductivity of single inoculation (R/EPS) and combined inoculation were significantly lower (P<0.05), both of which could help plants alleviate the damage caused by salt stress. And the effect patterns of single Cp2-EPS and combined inoculation were similar (Figure 3D).

3.8 Effects of combined inoculation on physiological indexes of seedlings

Under salt stress, the content of proline and soluble sugar in the CK both expressed the minimum value, compared with CK, the content of single inoculation (R/EPS) and combined inoculation increased significantly (P<0.05). Among them, the soluble sugar content of combined inoculation H expressed the maximum value, which increased by 350% compared with CK (Figure 4A, 4B). As far as MDA and H₂O₂ were concerned, the contents of MDA and H₂O₂ in single inoculation (R/EPS) and combined inoculation were significantly lower than those in CK (P<0.05). Among them, compared with CK, combined inoculation with G showed the largest reduction of MDA content of 35% and H₂O₂ content of 39.3%, indicating that there was a significant interaction between the two (Figure 4C, 4D). Compared with control, both single inoculation (R/EPS) and combined inoculation SOD and CAT activities were significantly increased (P<0.05). Among them, the SOD and CAT activities of combined inoculated H both expressed the maximum, which increased by 71.9% and 97.6% compared with CK, and also expressed a significant interaction (Figure 4E, 4F).

3.9 Principal component analysis and correlation analysis

3.9.1 Correlation of physiological traits and growth traits

The correlation between physiological characteristics and growth traits of seedlings was analyzed, and the results showed that soluble sugar content, proline content, SOD activity and CAT activity were all positively correlated with growth indicators; MDA and H₂O₂ contents were negatively correlated with growth indicators. This indicates that changes in physiological characteristics have certain effects on seedling growth (Figure 5A).

3.9.2 Principal component analysis of physiological indexes

According to the results of principal component analysis, the first three principal components accounted for more than 94%, so it can better reflect the changes of the main physiological indicators of seedlings under salt stress by combined inoculation. The contribution rate of principal component 1 was more than 69, and the indicators that contributed more to principal component 1 were SOD activity and CAT activity. Therefore, when alfalfa is subjected to salt stress, SOD activity and CAT activity in seedlings can better reflect the physiological changes of alfalfa. Provide a basis for follow-up research (Figure 5B).

3.10 The comprehensive evaluation of various indexes of combined vaccination

According to the contribution rate value of each comprehensive index, the comprehensive evaluation index of different treatments was calculated, and the effects of different treatments were sorted according to the comprehensive evaluation. It was found that each treatment had a promoting effect. Among them, combined vaccination ranked the top 4, and combined vaccination H was the best. On the whole, the order of inoculation effect of each treatment is H, G, I, F, D, C, A, E, B, CK (Figure 6).

The response of plants to salt stress is a complex process, and its salt tolerance is a comprehensive expression of various metabolisms. Alfalfa can tolerate a certain salinity through its own mechanism, and the intervention of exogenous biotic or abiotic factors can help it break through the upper limit of salinity tolerance (Buysse and Merckx, 1993; Bhaskara et al. 2015). Salt stress will limit the water supply of seeds and seedlings, destroy various enzyme structures, interrupt protein metabolism, respiration and photosynthesis, and cause seed germination failure or immature seedling development (Bharathi et al. 2004). Whether seeds can quickly and efficiently absorb water from the surrounding environment under salt stress is an important indicator for testing the salt tolerance of seeds. In this study, each treatment significantly promoted alfalfa seed germination, and this induction mechanism provided the basis for better seedling establishment. It was also found through experiments that with the increase of Cp2-EPS concentration, the germination of seeds showed a trend of first increasing and then decreasing. This may be related to Cp2-EPS as a solute and its own hydrophilic properties. High concentration of Cp2-EPS will reduce the water potential, thereby inhibiting seed imbibition and germination (Upadhyay et al. 2011).

Salt stress can lead to ion toxicity. When plants cannot effectively excrete toxic ions, cells will be destroyed, cell division and expansion will be hindered, thereby reducing plant growth vigor, and ultimately leading to restricted growth of seedlings (Kronzucker et al. 2013; Gupta and Huang, 2014; Ilangumaran and Smith, 2017). In this study, inoculation of rhizobium strain Gz5 or Cp2-EPS improved root structure, helped plants maintain ion balance, and promoted root growth. The positive response of Cp2-EPS to the growth of lateral roots may be due to the fact that the Cp2-EPS polymer matrix contains 97% of water and compounds and the limitation of Na⁺ ions, which can directly protect the roots of seeds and seedlings from physiological desiccation (Lutts et al. 1996). However, combined inoculation produced a significant interaction compared with single inoculation (R/EPS), thereby helping seedlings to maximize water and nutrient uptake. In addition, EPS can provide cell adhesion, signal transduction and cell protection, and provide a better guarantee for the interaction between PGPR and plants, thereby promoting the growth of seedlings. This is why combined inoculation has a better promotion effect on seedling growth attributes than single inoculation (R/EPS). Studies have shown that plant height and biomass vary greatly among alfalfa varieties under salt stress, which can be used as indicators to indicate the response of alfalfa to salt stress at seedling stage (Bernstein, 2019). Compared with uninoculated and single inoculation (R/EPS), combined inoculation showed a significant increase in aboveground and belowground length and aboveground and belowground fresh dry weight.

Salt stress can damage the integrity of the plasma membrane of plants, increase its permeability, and change the ion content and electrolyte permeability of plants (Rahneshan et al. 2018; Yan et al. 2006). Chlorophyll content is considered to be a biochemical indicator of plant salt tolerance. Salt-tolerant plants exhibited unchanged or increased chlorophyll levels under salt stress, whereas a decrease in chlorophyll content was found in salt-sensitive plants (Afridi et al. 2019). In this study, the chlorophyll content and relative electrical conductivity of leaves were significantly improved by combined inoculation, which promoted photosynthesis and reduced electrolyte extravasation to some extent. Under adverse conditions, plant tissues perform osmotic adjustment through inorganic ions and organic osmotic substances, reducing the osmotic potential of plant cells, allowing plants to absorb water, and improving plant survival (Abd et al. 2015). Combined inoculation significantly increased the content of proline and soluble sugar in seedlings, thereby enhancing the tolerance of seedlings to salinity. Excessive reactive oxygen species (ROS) in plants can seriously damage the structure and integrity of the plasma membrane of the cell, leading to cell oxidative stress damage or increased membrane lipid peroxidation, and ultimately inhibiting its growth (Mahakham et al. 2017; Hu et al. 2018). In this study, combined inoculation enhanced the stress resistance of alfalfa seedlings by significantly enhancing SOD and CAT activities, significantly reducing MDA and H₂O₂ contents, and reducing cell membrane lipid peroxidation. Correlation analysis and principal component analysis showed that there was a significant correlation between the physiological characteristics of seedlings and growth traits, and the activities of SOD and CAT could better reflect the physiological changes of seedlings. Topsis comprehensive analysis showed that combined inoculation had a more significant promotion effect.

Combined Cp2-EPS and rhizobium Gz5 inoculation had the function of relieving salt stress. Specifically, Cp2-EPS of 0.6-1.5 g·L^-1 and rhizobium strain Gz5 could alleviate the toxic effect of 100 mmol·L^-1 NaCl stress, and promote alfalfa seed germination and seedling growth. And the combined 1.2 g·L^-1 Cp2-EPS and rhizobium Gz5 inoculation had the best mitigation effect on alfalfa under 100 mmol·L^-1 NaCl.

Credit authorship contributions statement

R.H. (Rong Huang) performed the experiments, analyzed the data. Z.J., J.J. and X.N. made helpful comments on our work and manuscript. Z.Z. (Zhenfen Zhang) conceived and supervised the project. All authors have read and agreed to the published version of the manuscript.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Natural Science Foundation of China (32060396), Key Laboratory of Grassland Ecosystem of the Ministry of Education (KLGE202203), Grassland monitoring and evaluation in Gansu Province (GSZYTC-ZCJC-21010) and the Natural Science Foundation of Gansu Province, China (20JR10RA562).

Abd_Allah E F, Hashem A, Alqarawi A A, et al. Enhancing growth performance and systemic acquired resistance of medicinal plant Sesbania sesban Merr using arbuscular mycorrhizal fungi under salt stress. Saudi Journal of Biological Sciences, 2015, 22(3): 274-283. http://doi.org/10.1016/j.sjbs.2015.03.004
Abdelkrim S, Jebara S H, Jebara M. Antioxidant systems responses and the compatible solutes as contributing factors to lead accumulation and tolerance in Lathyrus sativus inoculated by plant growth promoting rhizobacteria. Ecotoxicology and Environmental Safety, 2018, 166: 427-436. http://doi.org/10.1016/j.ecoenv.2018.09.115
Abhinandan K, Skori L, Stanic M, et al. Abiotic stress signaling in wheat-an inclusive overview of hormonal interactions during abiotic stress responses in wheat. Frontiers in plant science, 2018, 9: 734. http://doi.org/10.3389/fpls.2018.00734
Afridi M S, Mahmood T, Salam A, et al. Induction of tolerance to salinity in wheat genotypes by plant growth promoting endophytes: Involvement of ACC deaminase and antioxidant enzymes. Plant Physiology and Biochemistry, 2019, 139: 569-577. http://doi.org/10.1016/j.plaphy.2019.03.041
Al-Khatib M M, McNeilly T, Collins J C. Between and within cultivar variability in salt tolerance in lucerne (Medicago sativa L.). Genetic Resources and Crop Evolution, 1994, 41: 159-164. https://doi.org/10.1007/BF00051632
Ashraf M, Hasnain S, Berge O, et al. Inoculating wheat seedlings with exopolysaccharide-producing bacteria restricts sodium uptake and stimulates plant growth under salt stress. Biology and Fertility of Soils, 2004, 40: 157-162. http://doi.org/10.1007/s00374-004-0766-y
Avelar Ferreira P A, Bomfeti C A, Lima Soares B, et al. Efficient nitrogen-fixing Rhizobium strains isolated from amazonian soils are highly tolerant to acidity and aluminium. World Journal of Microbiology and Biotechnology, 2012, 28: 1947-1959. http://doi.org/10.1007/s11274-011-0997-7
Banerjee S, Walder F, Büchi L, et al. Agricultural intensification reduces microbial network complexity and the abundance of keystone taxa in roots. The ISME Journal, 2019, 13(7): 1722-1736. http://doi.org/10.1038/s41396-019-0383-2
Bernstein N. Plants and salt: Plant response and adaptations to salinity[M]//Model ecosystems in extreme environments. Academic Press, 2019: 101-112.
Bharathi R, Vivekananthan R, Harish S, et al. Rhizobacteria-based bio-formulations for the management of fruit rot infection in chillies. Crop Protection, 2004, 23(9): 835-843. http://doi.org/10.1016/j.cropro.2004.01.007
Bhaskara G B, Yang T H, Verslues P E. Dynamic proline metabolism: importance and regulation in water limited environments. Frontiers in Plant Science, 2015, 6: 484. http://doi.org/10.3389/fpls.2015.00484
Buysse J A N, Merckx R. An improved colorimetric method to quantify sugar content of plant tissue. Journal of Experimental Botany, 1993, 44(10): 1627-1629. https://doi.org/10.1093/jxb/44.10.1627
Corzo J, León-Barrios M, Hernando-Rico V, et al. Precipitation of metallic cations by the acidic exopolysaccharides from Bradyrhizobium japonicum and Bradyrhizobium (Chamaecytisus) strain BGA-1. Applied and Environmental Microbiology, 1994, 60(12): 4531-4536. http://doi.org/10.1128/aem.60.12.4531-4536.1994
DaCosta M, Wang Z, Huang B. Physiological adaptation of Kentucky bluegrass to localized soil drying. Crop Science, 2004, 44(4): 1307-1314. http://doi.org/10.2135/cropsci2004.1307
Din B U, Sarfraz S, Xia Y, et al. Mechanistic elucidation of germination potential and growth of wheat inoculated with exopolysaccharide and ACC-deaminase producing Bacillus strains under induced salinity stress. Ecotoxicology and Environmental Safety, 2019, 183: 109466. http://doi.org/10.1016/j.ecoenv.2019.109466
Duan C, Fang L, Yang C, et al. Reveal the response of enzyme activities to heavy metals through in situ zymography. Ecotoxicology and Environmental Safety, 2018, 156: 106-115. http://doi.org/10.1016/j.ecoenv.2018.03.015
Eliasson Å, Faurès J M, Frenken K, et al. AQUASTAT–Getting to grips with water information for agriculture. Land and Water Development Division FAO, Rome, Italy (http://www. fao. org/ag/aquastat. Accessed January 2011), 2003.
Gupta B, Huang B. Mechanism of salinity tolerance in plants: physiological, biochemical, and molecular characterization. International Journal of Genomics, 2014, 2014: 1-18. http://doi.org/10.1155/2014/701596
Hashem A, Abd_Allah E F, Alqarawi A A, et al. The interaction between arbuscular mycorrhizal fungi and endophytic bacteria enhances plant growth of Acacia gerrardii under salt stress. Frontiers in Microbiology, 2016, 7: 1089. http://doi.org/10.3389/fmicb.2016.01089
He H, Wu M, Guo L, et al. Release of tartrate as a major carboxylate by alfalfa (Medicago sativa L.) under phosphorus deficiency and the effect of soil nitrogen supply. Plant and Soil, 2020, 449: 169-178. http://doi.org/10.1007/s11104-020-04481-9
Hu T, Li H, Li J, et al. Absorption and bio-transformation of selenium nanoparticles by wheat seedlings (Triticum aestivum L.). Frontiers in Plant Science. 2018, 9:1-10. http://doi.org/10.3389/fpls.2018.00597
Ilangumaran G, Smith D L. Plant growth promoting rhizobacteria in amelioration of salinity stress: a systems biology perspective. Frontiers in Plant Science, 2017, 8: 1768. http://doi.org/10.3389/fpls.2017.01768
Jin J, Wang T, Cheng Y, et al. Current situation and prospect of forage breeding in China. Bulletin of Chinese Academy of Sciences (Chinese Version), 2021, 36(06): 660-665. https://doi.org/10.16418/j.issn.1000-3045.20210511003
Jofré E, Fischer S, Rivarola V, et al. Saline stress affects the attachment of Azospirillum brasilense Cd to maize and wheat roots. Canadian Journal of Microbiology, 1998, 44(5): 416-422. http://doi.org/10.1016/0300-483x(95)03196-m
Kasotia A, Varma A, Tuteja N, et al. Amelioration of soybean plant from saline-induced condition by exopolysaccharide producing Pseudomonas-mediated expression of high affinity K⁺-transporter (HKT1) gene. Current Science: A Fortnightly Journal of Research, 2016: 1961-1967. http://doi.org/10.18520/cs/v111/i12/1961-1967
Khan N, Bano A. Exopolysaccharide producing rhizobacteria and their impact on growth and drought tolerance of wheat grown under rainfed conditions. PLoS One, 2019, 14(9): e0222302. http://doi.org/10.1371/journal.pone.0222302
Kong Z, Glick B R, Duan J, et al. Effects of 1-aminocyclopropane-1-carboxylate (ACC) deaminase-overproducing Sinorhizobium meliloti on plant growth and copper tolerance of Medicago lupulina. Plant and Soil, 2015, 391: 383-398. http://doi.org/10.1007/s11104-015-2434-4
Koyro H W. Effect of salinity on growth, photosynthesis, water relations and solute composition of the potential cash crop halophyte Plantago coronopus L. Environmental and Experimental Botany, 2006, 56(2): 136-146. http://doi.org/10.1016/j.envexpbot.2005.02.001.
Kronzucker H J, Coskun D, Schulze L M, et al. Sodium as nutrient and toxicant. Plant and soil, 2013, 369: 1-23. http://doi.org/ 10.1007/s11104-013-1801-2
Kudoyarova G R, Arkhipova T N, Melent’ev A I. Role of bacterial phytohormones in plant growth regulation and their development. Bacterial Metabolites in Sustainable Agroecosystem, 2015: 69-86. http://doi.org/10.1007/978-3-319-24654-34
Laspidou C S, Rittmann B E. A unified theory for extracellular polymeric substances, soluble microbial products, and active and inert biomass. Water research, 2002, 36(11): 2711-2720. http://doi.org/ 10.1016/s0043-1354(01)00413-4.
Lee S H, Kim E Y, Hyun S, et al. Metal availability in heavy metal-contaminated open burning and open detonation soil: assessment using soil enzymes, earthworms, and chemical extractions. Journal of Hazardous Materials, 2009, 170(1): 382-388. http://doi.org/10.1016/j.jhazmat.2009.04.088
Lutts S, Kinet J M, Bouharmont J. Effects of salt stress on growth, mineral nutrition and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L.) cultivars differing in salinity resistance. Plant Growth Regulation, 1996, 19: 207-218. https://doi.org/10.1007/BF00037793
Mahakham W, Sarmah AK, Maensiri S, et al. Nanopriming technology for enhancing germination and starch metabolism of aged rice seeds using phytosynthesized silver nanoparticles. Scientific Reports, 2017, 7: 8263-9284. http://doi.org/10.1038/s41598-017-08669-5
Moot D J, Teixeira E, Brown H. Alfalfa. Food and Agriculture Organisation of the United Nations, 2012.
Nandal K, Sehrawat A R, Yadav A S, et al. High temperature-induced changes in exopolysaccharides, lipopolysaccharides and protein profile of heat-resistant mutants of Rhizobium sp. (Cajanus). Microbiological Research, 2005, 160(4): 367-373. http://doi.org/10.1016/j.micres.2005.02.011
Nocelli N, Bogino P C, Banchio E, et al. Roles of extracellular polysaccharides and biofilm formation in heavy metal resistance of rhizobia. Materials, 2016, 9(6): 418. http://doi.org/10.3390/ma9060418
Peng Q, Wu M, Zhang Z, et al. The interaction of arbuscular mycorrhizal fungi and phosphorus inputs on selenium uptake by alfalfa (Medicago sativa L.) and selenium fraction transformation in soil. Frontiers in Plant Science, 2020, 11: 966. http://doi.org/10.3389/fpls.2020.00966
Primo E D, Cossovich S, Nievas F, et al. Exopolysaccharide production in Ensifer meliloti laboratory and native strains and their effects on alfalfa inoculation. Archives of microbiology, 2020, 202: 391-398. http://doi.org/10.1007/s00203-019-01756-3
Primo E, Bogino P, Cossovich S, et al. Exopolysaccharide II is relevant for the survival of Sinorhizobium meliloti under water deficiency and salinity stress. Molecules, 2020, 25(21): 4876. http://doi.org/10.3390/molecules25214876
Pulsawat W, Leksawasdi N, Rogers P L, et al. Anions effects on biosorption of Mn (II) by extracellular polymeric substance (EPS) from Rhizobium etli. Biotechnology Letters, 2003, 25: 1267-1270. http://doi.org/10.1023/a:1025083116343
Radović J, Sokolović D, Marković J. Alfalfa-most important perennial forage legume in animal husbandry. Biotechnology in Animal Husbandry, 2009, 25(5-6-1): 465-475. http://doi.org/10.2298/BAH0906465R
Rahneshan Z, Nasibi F, Moghadam A A. Effects of salinity stress on some growth, physiological, biochemical parameters and nutrients in two pistachio (Pistacia vera L.) rootstocks. Journal of Plant Interactions, 2018, 13(1): 73-82. https://doi.org/10.1080/17429145.2018.1424355
Rajkumar M, Ae N, Prasad M N V, et al. Potential of siderophore-producing bacteria for improving heavy metal phytoextraction. Trends in Biotechnology, 2010, 28(3): 142-149. http://doi.org/10.1016/j.tibtech.2009.12.002
Saadani O, Fatnassi I C, Chiboub M, et al. In situ phytostabilisation capacity of three legumes and their associated plant growth promoting bacteria (PGPBs) in mine tailings of northern Tunisia. Ecotoxicology and Environmental Safety, 2016, 130: 263-269. http://doi.org/10.1016/j.ecoenv.2016.04.032
Sandhya V, SK. Z A, Grover M, et al. Alleviation of drought stress effects in sunflower seedlings by the exopolysaccharides producing Pseudomonas putida strain GAP-P45. Biology and Fertility of Soils, 2009, 46: 17-26. http://doi.org/10.1007/s00374-009-0401-z
Sengupta S, Dey S. Microbial exopolysaccharides (EPS): role in agriculture and environment. Agriculture and Food: e-Newsletter, 2019, 1(12): 4-8. https://www.researchgate.net/publication/337672441
Shrivastava P, Kumar R. Soil salinity: a serious environmental issue and plant growth promoting bacteria as one of the tools for its alleviation. Saudi Journal of Bioloical Science, 2015, 22: 123–131. http://doi.org/10.1016/j.sjbs.2014.12.001
Upadhyay S K, Singh J S, Singh D P, et al. Exopolysaccharide-producing plant growth-promoting Rhizobacteria under salinity condition. Pedosphere, 2011, 21(02): 214-222. http://doi.org/10.1016/S1002-0160(11)60120-3
Vimal S R, Singh J S, Arora N K, et al. Soil-plant-microbe interactions in stressed agriculture management: a review. Pedosphere, 2017, 27(2): 177-192. http://doi.org/10.1016/S1002-0160(17)60309-6
Wang Y, Zhang Z, Zhang P, et al. Rhizobium symbiosis contribution to short-term salt stress tolerance in alfalfa (Medicago sativa L.). Plant and Soil, 2016, 402: 247-261. http://doi.org/10.1007/s11104-016-2792-6
Wassie M, Zhang W, Zhang Q, et al. Exogenous salicylic acid ameliorates heat stress-induced damages and improves growth and photosynthetic efficiency in alfalfa (Medicago sativa L.). Ecotoxicology and Environmental Safety, 2020, 191: 110206. http://doi.org/10.1016/j.ecoenv.2020.110206
Yan S H, Ji J, Wang G. Effects of salt stress on plants and the mechanism of salt tolerance. World Science-Technology Research and Development, 2006, 28(4): 70-76. http://doi.org/10.11867/j.issn.1001-8166.2018.04.0361
Zaman M, Shahid S A, Heng L, et al. Soil salinity: Historical perspectives and a world overview of the problem. Guideline for salinity assessment, mitigation and adaptation using nuclear and related techniques, 2018: 43-53.
Zhang Z F. Seed- borne bacteria of lucerne (Medicago sativa) and their pathogenicity[D]. Lanzhou: Lanzhou University, 2013.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

The combined Cp2-EPS and rhizobium inoculation enhance the growth of alfalfa under salt stress

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Experimental materials

2.2 Experimental treatments

2.2.1 Salt tolerance evaluation of Rhizobium Gz5

2.2.2 Test Method

2.3 Index determination

2.3.1 Growth indicators

2.3.2 Physiological and biochemical indicators

2.4 Statistical analysis

3. Results

4. Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1