A method for promoting nodulation and growth of alfalfa

Abstract

The invention provides a method for promoting nodulation and growth of alfalfa, which relates to the field of biological breeding, and comprises the following steps: selection and preservation of test materials, selection of breeding, preparation of culture solution, seed treatment, seed germination test, seed germination test, data analysis, and single factor variance statistical analysis by SPSS23.0 to analyze the fresh and dry weight, length, root index, etc. of seeds, seedling growth index, chlorophyll content determination, relative conductivity, and physiological index of seeds. The invention uses alfalfa "Ju Neng 551" seeds as test materials to explore the effects of different concentrations (0.6 g / L ‑1 , 0.9gL ‑1 , 1.2gL ‑1 , 1.5gL ‑1 )The effects of combined inoculation of Cp2‑EPS and rhizobia (R) on seed germination and seedling growth of alfalfa were studied. It was found that combined inoculation of 0.6-1.5 g / L Cp2‑EPS and rhizobia could effectively promote the growth and root nodulation of alfalfa seedlings. Further comprehensive analysis by Topsis showed that combined inoculation of 1.2 g / L Cp2‑EPS and rhizobia had the best effect on promoting the growth of alfalfa seedlings.

Description

Technical Field

[0001] The invention relates to the field of biological breeding, and in particular to a method for promoting nodulation and growth of alfalfa. Background Art

[0002] Alfalfa (Medicago sativa L.) is a high-quality leguminous alfalfa with the characteristics of high yield, rich nutrition, strong adaptability and good ecological benefits. It is known as the "king of forage". In recent years, driven by policies and market demand, alfalfa has become the main crop of plant protein feed, and its related products such as straw bales and grass meal have become common alfalfa products in the agricultural market, and the quantity of products is in short supply. Therefore, how to increase the yield of forage such as alfalfa has become one of the research hotspots.

[0003] Microbial exopolysaccharides (EPS) are high molecular weight polymers associated with or secreted outside some special microbial cells. They are usually produced under harsh environmental or nutritional conditions and are positively correlated with abiotic stress. As a physiological stress response and a protective strategy for coping with the environment, EPS is of great significance to the growth, colonization and stress resistance of bacteria. EPS has a large yield, a short production cycle and a high extraction rate. It has broad development and application prospects. It has been widely studied for its excellent anti-inflammatory, antioxidant and anti-tumor effects. In recent years, a large number of scholars have begun to explore the effects of EPS on promoting plant growth. Gong Jian et al. found that cyanobacterial exopolysaccharides have an effect on the growth of sand. It has different degrees of promoting effect on the germination of desert herb seeds. Huang Shichen et al. found through soaking seeds with lactic acid bacteria extracellular polysaccharides that adding lactic acid bacteria extracellular polysaccharides to the soil has a significant promoting effect on the growth of rice seedlings and can significantly promote the synthesis of photosynthetic pigments. Ma Xiaoying et al. studied the effects of different microbial polysaccharides on plant growth and found that fungal fermentation broth polysaccharides and mycelium polysaccharides have a significant promoting effect on the growth of corn, especially on the growth of roots. At present, there are few studies on EPS in plant growth promotion, and its mechanism of action has not yet been clarified. Therefore, in-depth exploration of the potential of EPS in the field of plant growth promotion is of great significance to its development and improvement of agricultural production.

[0004] Erwinia persicina is a typical Gram-negative bacterium (G-). Its Cp2 strain can produce a large amount of slimy EPS on King's B medium and is widely present in the natural environment. However, there are few studies on the effect of Cp2-EPS on plant growth promotion, and there are even fewer reports on the combined inoculation of Cp2-EPS and rhizobia. Therefore, this experiment used the combined inoculation of Erwinia persicina Cp2-EPS and rhizobia to explore its effects on seed germination and seedling growth of alfalfa, in order to provide a theoretical basis for promoting the growth of alfalfa and optimizing rhizobia inoculation. Summary of the invention

[0005] The purpose of the present invention is to provide a method for promoting nodulation and growth of alfalfa to solve the technical problem of alfalfa growth.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for promoting nodulation and growth of alfalfa comprises the following steps:

[0008] S1. Selection and preservation of test materials

[0009] It was isolated from the mature nodules of alfalfa "Jueneng 551". The strain was stored at -80℃, while Cp2-EPS was stored at room temperature.

[0010] S2. Planting and breeding selection

[0011] Select alfalfa "Jueneng 551" seeds with sufficient full grains and no pests and diseases, and store the samples at a constant temperature of 4°C;

[0012] S3. Preparation of culture medium

[0013] Five Cp2-EPS solutions of different concentrations, 0, 0.6, 0.9, 1.2, and 1.5 gL-1, were prepared, 190 mL of each of the five Cp2-EPS solutions were added to growth bottles, and sterilized in an autoclave at 121°C and 0.11 MPa for 21 min before use, and then a bacterial suspension was prepared with sterile water, wherein OD600 = 0.12, and 10 mL of each was added to growth bottles, respectively, to be determined as the culture solution for seed germination and seedling growth;

[0014] S5. Seed treatment

[0015] The selected seeds were surface sterilized with 95% ethanol, then soaked in 5% NaClO for 5 min, and then washed with sterile water in turn;

[0016] S5. Seed germination test

[0017] Place three layers of filter paper in a culture dish and sterilize them, add culture solution to completely wet them, evenly place 50 seeds, repeat the process 4 times, seal the culture dish, place it in a light incubator, and culture it at 25±1℃, 60% humidity, and 4000lx light intensity. Record the number of seeds that germinate each day.

[0018] S6. Seed germination test

[0019] The treated seeds were placed equidistantly on the germination bed of the growth bottle, 25 seeds per bottle, and 8 replicates were made. The growth bottle was placed in a growth room with a temperature of 23°C, a humidity of 45%, a light (18Klux) of 18h, and a dark of 6h for cultivation. Samples were taken at 21 days to measure the indicators;

[0020] Determination index: Seed germination rate = number of seeds germinated on the 10th day / 25×100%; germination potential = (number of seeds germinated on the 5th day / 25×100%; germination index = ∑Gt / Dt;

[0021] Gt: number of germinations on day td;

[0022] Dt: corresponding germination days).

[0023] Preferably, the method further comprises the following steps:

[0024] Data analysis: SPSS 23.0 was used for one-way ANOVA statistical analysis to analyze seed fresh and dry weight, length, root indexes, seedling growth indexes, chlorophyll content determination, relative conductivity, physiological indexes, and calculate the mean and standard error of the mean, which was determined by the least significant difference (p < 0.05) test, and then data were sorted and plotted.

[0025] The beneficial effects of the present invention are:

[0026] The present invention uses alfalfa "Ju Neng 551" seeds as experimental materials to explore the effects of different concentrations (0.6 gL -1 , 0.9gL -1 , 1.2gL -1 , 1.5gL -1 )The effect of combined inoculation of Cp2-EPS and rhizobia (R) on seed germination and seedling growth of alfalfa was studied. It was found that combined inoculation of 0.6-1.5 g / L Cp2-EPS and rhizobia can effectively promote the growth and root nodulation of alfalfa seedlings. Further comprehensive analysis by Topsis showed that combined inoculation of 1.2 g / L Cp2-EPS and rhizobia had the best effect on promoting the growth of alfalfa seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the effect of combined inoculation on above-ground and underground fresh and dry weights of alfalfa seedlings;

[0028] Figure 2 Schematic diagram of the effect of combined inoculation on alfalfa seedling length, nodule number and root indexes;

[0029] Figure 3 This is a schematic diagram showing the effect of combined inoculation on photosynthetic indices of alfalfa seedlings;

[0030] Figure 4 This is a schematic diagram of the effects of combined inoculation on the physiological parameters of alfalfa seedlings;

[0031] Figure 5 It is a schematic diagram of correlation analysis and principal component analysis of the present invention;

[0032] Figure 6 It is a schematic diagram of correlation analysis of various indicators of the present invention; DETAILED DESCRIPTION

[0033] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments and drawings, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0034] The specific embodiments of the present invention are described below with reference to the accompanying drawings.

[0035] Example 1

[0036] A method for promoting nodulation and growth of alfalfa comprises the following steps:

[0037] S1. Selection and preservation of test materials

[0038] It was isolated from the mature nodules of alfalfa "Jueneng 551". The strain was stored at -80℃, while Cp2-EPS was stored at room temperature.

[0039] S2. Planting and breeding selection

[0040] Select alfalfa "Jueneng 551" seeds with sufficient full grains and no pests and diseases, and store the samples at a constant temperature of 4°C;

[0041] S3. Preparation of culture medium

[0042] Five Cp2-EPS solutions of different concentrations, 0, 0.6, 0.9, 1.2, and 1.5 gL-1, were prepared, 190 mL of each of the five Cp2-EPS solutions were added to growth bottles, and sterilized in an autoclave at 121°C and 0.11 MPa for 21 min before use, and then a bacterial suspension was prepared with sterile water, wherein OD600 = 0.12, and 10 mL of each was added to growth bottles, respectively, to be determined as the culture solution for seed germination and seedling growth;

[0043] S5. Seed treatment

[0044] The selected seeds were surface sterilized with 95% ethanol, then soaked in 5% NaClO for 5 min, and then washed with sterile water in turn;

[0045] S5. Seed germination test

[0046] Place three layers of filter paper in a culture dish and sterilize them, add culture solution to completely wet them, evenly place 50 seeds, repeat the process 4 times, seal the culture dish, place it in a light incubator, and culture it at 25±1℃, 60% humidity, and 4000lx light intensity. Record the number of seeds that germinate each day.

[0047] S6. Seed germination test

[0048] The treated seeds were placed equidistantly on the germination bed of the growth bottle, 25 seeds per bottle, and 8 replicates were made. The growth bottle was placed in a growth room with a temperature of 23°C, a humidity of 45%, a light (18Klux) of 18h, and a dark of 6h for cultivation. Samples were taken at 21 days to measure the indicators;

[0049] Determination index: Seed germination rate = number of seeds germinated on the 10th day / 25×100%; germination potential = (number of seeds germinated on the 5th day / 25×100%; germination index = ∑Gt / Dt;

[0050] Gt: number of germinations on day td;

[0051] Dt: corresponding germination days).

[0052] A method for promoting nodulation and growth of alfalfa, further comprising the following steps:

[0053] 1. Data analysis: SPSS 23.0 was used to perform one-way variance statistical analysis to analyze seed fresh and dry weight, length, root indexes and other seedling growth indicators, chlorophyll content, relative conductivity, physiological indexes, and calculate the mean and standard error of the mean. The least significant difference (p < 0.05) test was used to determine the data, and then data were sorted and plotted.

[0054] 2. Results and Analysis

[0055] 2.1 Effect of combined inoculation on seed germination

[0056] Please refer to Table 1. Compared with CK, the germination rate and germination potential of single inoculation (R / EPS) and combined inoculation were inhibited. The germination rate was significantly reduced under 1.5g EPS treatment (P<0.05), while other treatments were not significant. The germination potential of single inoculation R was not significantly different from that of CK, and other treatments were significantly lower than CK (P<0.05). This may be related to the initial concentration of the solution. In terms of relative germination index, the germination index of combined inoculation was not significantly different from that of CK (P>0.05), but was improved compared with single inoculation (R / EPS).

[0057] Table 1 Effect of combined inoculation on germination of alfalfa seeds

[0058] deal with Germination rate% Germination potential% Germination Index CK <![CDATA[97±0.66 a ]]> <![CDATA[95.5±0.50 a ]]> <![CDATA[63.97±1.12 a ]]> A <![CDATA[96.5±0.91 ab ]]> <![CDATA[94.5±1.05 ab ]]> <![CDATA[61.90±0.46 ab ]]> B <![CDATA[96±1.07 ab ]]> <![CDATA[93±0.66 bc ]]> <![CDATA[62.17±0.92 ab ]]> C <![CDATA[95.5±0.91 ab ]]> <![CDATA[93±0.66 bc ]]> <![CDATA[62.70±0.91 ab ]]> D <![CDATA[95.5±0.91 ab ]]> <![CDATA[92.5±0.91 bc ]]> <![CDATA[60.71±0.67 bc ]]> E <![CDATA[94±0.76 b ]]> <![CDATA[91±0.66 c ]]> <![CDATA[59.01±0.76 c ]]> F <![CDATA[95.5±0.50 ab ]]> <![CDATA[93±0.66 bc ]]> <![CDATA[63.29±0.38 a ]]> G <![CDATA[95±1.00 ab ]]> <![CDATA[93±0.66 bc ]]> <![CDATA[63.85±0.45 a ]]> H <![CDATA[96.5±0.91 ab ]]> <![CDATA[93±1.00 bc ]]> <![CDATA[63.78±0.46 a ]]> I <![CDATA[96±1.07 ab ]]> <![CDATA[92±0.76 bc ]]> <![CDATA[62.55±0.38 bc ]]>

[0059] *Note: CK, A, B, C, D, E, F, G, H, and I represent sterile water, R + sterile water, 0.6g EPS + sterile water, 0.9g EPS + sterile water, 1.2g EPS + sterile water, 1.5g EPS + sterile water, 0.6g EPS + R + sterile water, 0.9g EPS + R + sterile water, 1.2g EPS + R + sterile water, and 1.5g EPS + R + sterile water (R represents rhizobium); different lowercase letters indicate significant differences among treatments (P<0.05), the same below.

[0060] 2.2 Effects of combined inoculation on fresh and dry weight of seedlings

[0061] See also Figure 1 The minimum values ​​of aboveground and underground fresh-dry weights were observed in CK. Compared with CK, the aboveground and underground fresh-dry weights of single inoculation (R / EPS) and combined inoculation increased significantly (P<0.05), and the change trends were similar. Compared with single inoculation (R / EPS), the aboveground and underground fresh-dry weights of combined inoculation increased significantly (P<0.05). Among them, combined inoculation H showed the maximum value, which increased by 386%, 297%, 441% and 511% respectively compared with CK. The results showed that single inoculation (R / EPS) and combined inoculation significantly promoted the aboveground and underground fresh-dry weights of alfalfa seedlings. Compared with single inoculation (R / EPS), combined inoculation expressed significant interaction.

[0062] 2.3 Effects of combined inoculation on seedling length, root system parameters and nodule number

[0063] See also Figure 2 -A, single inoculation (R / EPS) and combined inoculation had different effects on the aboveground and underground lengths of seedlings. Compared with CK, except for single inoculation of R, the aboveground lengths of other treatments increased significantly (P<0.05). Among them, combined inoculation of H expressed the maximum value, which was significantly increased by 155% compared with CK (P<0.05). Relative to the underground length, with the increase of EPS concentration, the underground length was significantly inhibited when the addition amounts of EPS were 1.2g and 1.5g (P<0.05). This inhibition was alleviated by combined inoculation. There was no significant difference compared with CK. Combined inoculation had a significant interactive effect on the aboveground length of seedlings compared with single inoculation (R / EPS), and also alleviated the inhibitory effect of high concentration EPS on the underground length.

[0064] See also Figure 2-B, single inoculation (R / EPS) and combined inoculation had different promoting effects on seedling root nodulation. No nodules appeared in CK, but nodules were produced in single inoculation (R / EPS), and the difference between the two was not significant (P>0.05). Among them, the number of nodules with single addition of EPS showed a trend of first increasing and then decreasing with the increase of EPS concentration. Compared with single inoculation (R / EPS), combined inoculation had a significant promoting effect on seedling nodules (P<0.05). Among them, combined inoculation H expressed the maximum value. Compared with the single addition of 1.2g EPS, the whole bottle and single plant increased significantly by 275% and 100% respectively (P<0.05). This shows that compared with single inoculation (R / EPS), combined inoculation has a significant interactive effect on the growth of root nodules of alfalfa seedlings.

[0065] See also Figure 2 -D / E / F / G, compared with CK, the average root diameter, total root surface area, total root volume and number of root tips of seedlings in each treatment were significantly increased (P < 0.05), and the effect pattern was similar. Compared with single inoculation (R / EPS), the total root surface area, total root volume and number of root tips of combined inoculation were significantly increased (P < 0.05), among which, the combined inoculation H showed the maximum value, which increased by 135%, 152% and 178% ( Figure 2 -D / E / F / G).

[0066] 2.4 Effects of combined inoculation on photosynthetic indices of seedlings

[0067] See also Figure 3 -A, compared with CK, single inoculation (R / EPS) and combined inoculation had different degrees of effect on the chlorophyll content of alfalfa leaves. Among them, the chlorophyll a, chlorophyll b and total chlorophyll contents of single inoculation R were not significantly different from those of CK (P>0.05). With the increase of single EPS concentration, the chlorophyll a, chlorophyll b and total chlorophyll contents showed a gradual downward trend. Except for the chlorophyll b content of 0.6g EPS, the chlorophyll a, chlorophyll b and total chlorophyll contents of other single EPS additions were significantly reduced (P<0.05). This inhibitory effect was alleviated by combined inoculation. Compared with CK, the chlorophyll a content was not significantly different (P>0.05). The chlorophyll b content of combined inoculation F, H, I and the total chlorophyll content of H and I were significantly increased (P<0.05). Among them, the chlorophyll a, chlorophyll b and total chlorophyll contents of combined inoculation H all showed the maximum value, which were increased by 7.8%, 22% and 13% respectively compared with CK.

[0068] See also Figure 3-B / C / D / E, compared with CK, the transpiration rate, stomatal conductance and intercellular CO2 concentration of leaves of single inoculated (R / EPS) and combined inoculated seedlings were significantly increased (P < 0.05), among which the combined inoculation H showed the maximum value, which was significantly different from the single inoculation (R / EPS), and increased by 84%, 102% and 34% respectively compared with CK (P < 0.05). With respect to the net photosynthetic rate, the combined inoculation was significantly higher than that of CK (P < 0.05), among which the combined inoculation G showed the maximum increase of 520% ​​compared with CK.

[0069] 2.5 Effects of combined inoculation on physiological parameters of seedlings

[0070] See also Figure 4 Compared with CK, single inoculation (R / EPS) and combined inoculation significantly increased O2 - content, H2O2 content and MDA content (P<0.05), among which, when EPS was added alone, O2 - The content, H2O2 content and MDA content showed an upward trend with the increase of EPS concentration, which indicated that high concentration of EPS had an inhibitory effect on the growth and physiology of alfalfa seedlings, while in the combined inoculation, O2 - Compared with the single addition of EPS, the content, H2O2 content and MDA content showed different degrees of decrease. Compared with the SOD, CAT and DHA activities, they all showed the minimum values ​​in CK. Both single inoculation (R / EPS) and combined inoculation significantly increased the SOD, CAT and DHA activities (P < 0.05). There was a significant difference between the combined inoculation and the single inoculation (R / EPS) (P < 0.05), and there were different degrees of increase. Among them, the combined inoculation H showed the maximum value, which was significantly increased by 135%, 281% and 291% compared with CK, respectively.

[0071] 2.6 Correlation Analysis and Principal Component Analysis

[0072] See also Figure 5 -A, correlation analysis showed that SOD, CAT, DHA activities and photosynthetic indexes E, A, Ci and GH2O were positively correlated with growth indexes; MDA, H2O2 and O2 - There was a negative correlation between the content and growth indexes.

[0073] See also Figure 5 -B, Table 2, Table 3, principal component analysis shows that the first three principal components cumulatively account for more than 91%, so they can better reflect the changes in the main growth-promoting physiological and biochemical indicators of seedlings after combined inoculation. The contribution rate of principal component 1 is more than 75%, and the indicators that contribute most to principal component 1 are DHA, CAT activity and Ci.

[0074] Table 2 Principal component analysis of physiological indicators

[0075]

[0076] Table 3 Composition matrix

[0077]

[0078] See also Figure 6 Through comprehensive analysis of various indicators, it was concluded that compared with the control CK, single inoculation (R / EPS) and combined inoculation promoted the germination of alfalfa seeds and the growth of seedlings to varying degrees, and the combined inoculation expressed a significant interaction, among which the combined inoculation H inoculation had the best effect.

[0079] 3 Discussions

[0080] 3.1 Effects of combined inoculation on seed germination and seedling growth of alfalfa seedlings

[0081] The symbiotic nitrogen-fixing relationship between rhizobia and legumes has been fully verified to promote the growth of host plants. At present, a large number of research results have shown that exogenous application of EPS can effectively improve the growth performance of crops and has great potential in improving plant yield and quality. This study explored the effects of EPS and rhizobia on seed germination and seedling growth of alfalfa seedlings by co-inoculation. Seed germination, as a prerequisite for plant seedling growth, can directly affect the yield of alfalfa. The results of this study showed that the combined inoculation had no significant effect on seed germination, while the addition of EPS alone had a certain inhibitory effect on seed germination as the concentration increased. The combined inoculation method had a certain alleviating effect on its inhibitory effect, which may be related to the fact that the germination rate of alfalfa seeds itself is already very high (>90%). Huang Shichen et al. found that lactic acid bacteria extracellular polysaccharides had no significant effect on rice seed germination in their study on the effects of lactic acid bacteria polysaccharides on rice seed germination and seedling growth, which is consistent with the results of this study.

[0082] 3.2 Effect of EPS concentration on seed nodulation of alfalfa seedlings

[0083] The mutual support between the above-ground and underground parts of plants is the key to the overall development of plants. The root system is an important organ for plants to absorb water and nutrients. Its growth directly affects the growth of the above-ground plants. In this study, each treatment promoted the root structure of seedlings, significantly increased the average root diameter, total root surface area, total root volume and number of root tips, and there was a more significant interaction between the combined inoculations. Single inoculation of rhizobia and single addition of EPS both significantly promoted root nodulation of seedlings. The combined inoculation further improved root nodulation. The number of root nodules in single addition of EPS and combined inoculation showed a trend of first increasing and then decreasing with the increase of EPS concentration, indicating that EPS It has the effect of low promotion and high inhibition on the nodulation of roots of alfalfa seedlings. The combined inoculation improves the root structure of seedlings, promotes root nodulation and provides some nutrients, thereby maximizing the root's acquisition of water and nutrients, indirectly promoting the growth of seedlings, and significantly promoting the above-ground and underground fresh-dry weight and above-ground length, while the underground length of seedlings is inhibited with the increase of EPS concentration. The inhibition is effectively alleviated by combined inoculation. Photosynthesis is another important way for plants to obtain nutrients. The combined inoculation increases the chlorophyll content, significantly promotes the transpiration rate, stomatal conductance, net photosynthetic rate and intercellular CO2 concentration, increases the photosynthesis intensity, and indirectly promotes the growth of seedlings.

[0084] 3.3 Effects of combined inoculation on the growth of alfalfa seedlings

[0085] The increase in free radical content can cause damage to plant cell tissues, while the increase in antioxidant enzyme activity can effectively remove excessive free radicals and catalyze the decomposition of hydrogen peroxide. It is a key enzyme in the biological defense system and is essential for protecting the integrity of cell membrane structure. The results of this study showed that the addition of EPS alone increased the concentration of MDA, H2O2 and O2 in seedlings. - The content increased significantly, which had a certain inhibitory effect on the elongation growth of the seedling roots. The combined inoculation significantly increased the activities of the antioxidant enzymes SOD, CAT and DHA, effectively reduced their content, alleviated the inhibitory effect, and further promoted the growth of the seedlings above and below the ground.

[0086] In summary, the combined inoculation of Cp2-EPS and rhizobia has a significant promoting effect on the growth of alfalfa seedlings, revealing the multiple mechanisms of the promoting effect and providing a theoretical basis for subsequent field trials and promotion and application.

[0087] 4 Conclusion

[0088] The experiment showed that the combined inoculation of 0.6-1.5 g / L Cp2-EPS and rhizobia can effectively promote the growth and root nodulation of alfalfa seedlings. Further comprehensive analysis through Topsis showed that the combined inoculation of 1.2 g / L Cp2-EPS and rhizobia had the best effect on promoting the growth of alfalfa seedlings.

[0089] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for promoting nodulation and growth of alfalfa, characterized in that: The following steps are involved: S1. Selection and preservation of test materials It was isolated from the mature nodules of alfalfa "Ju Neng 551", and the strain was stored at -80℃. The Erwinia persica Cp2-EPS was stored at room temperature. S2. Planting and breeding selection Select alfalfa "Jueneng 551" seeds with sufficient full grains and no pests and diseases, and store the samples at a constant temperature of 4℃; S3. Preparation of culture medium Prepare 0.6, 0.9, 1.2, 1.5gL -1 Five different concentrations of Erwinia cerevisiae Cp2-EPS solutions were added to the growth bottles at 190 mL, sterilized in an autoclave at 121°C and 0.11 MPa for 21 min, and then prepared with sterile water to prepare a bacterial suspension with OD600=0.

12. 10 mL was added to the growth bottles to determine the culture solution for seed germination and seedling growth. S5. Seed treatment The selected seeds were surface sterilized with 95% ethanol, then soaked in 5% NaClO for 5 min, and then washed with sterile water in turn; S5. Seed germination test Place three layers of filter paper in a culture dish and sterilize it, add culture solution to completely wet it, evenly place 50 seeds, repeat the process 4 times, seal the culture dish, place it in a light incubator, and culture it at 25±1℃, 60% humidity, and 4000lx light intensity. Record the number of seeds that germinate every day. S6. Seed germination test The treated seeds were placed equidistantly on the germination bed of the growth bottle, 25 seeds per bottle, and 8 replicates were made. The growth bottle was placed in a growth room with a temperature of 23°C, a humidity of 45%, a light of 18Klux for 18 hours, and a dark of 6 hours for cultivation. Samples were taken at 21 days to measure the indicators. Determination index: Seed germination rate = number of seeds germinated on the 10th day / 25×100%; germination potential = number of seeds germinated on the 5th day / 25×100%; germination index = ∑Gt / Dt; Gt: number of germinations on day td; Dt: corresponding germination days.

2. A method for promoting nodulation and growth of alfalfa according to claim 1, characterized in that The following steps are also included: Data analysis: SPSS 23.0 was used for one-way ANOVA statistical analysis to analyze seed fresh and dry weight, length, root index seedling growth index, chlorophyll content, relative conductivity, physiological indexes, and calculate the mean and standard error of the mean, which was determined by the least significant difference p < 0.05 test, and then data were sorted and plotted.

Images