Tetrahydropyrimidine fermentation liquor and application thereof in growth promotion and salt resistance of rice

Abstract

The invention discloses ectoine fermentation liquor and application thereof in growth promotion and salt resistance of rice. The ectoine fermentation liquor is efficiently synthesized by using a low-quality carbon source, the yield and fermentation efficiency of ectoine are improved while the cost is saved, the yield of ectoine can reach 134 g / L, and the production rate of ectoine reaches 3.73 g / L / h. Furthermore, an application technology of unpurified fermentation liquor in rice seedling growth promotion and salt stress resistance is developed. The ectoine fermentation liquor can effectively promote the growth of the rice and the tolerance of the rice to saline-alkali soil. The invention provides an economical and feasible technical scheme for a low-inferior carbon source fermentation process of ectoine and the field of agricultural application of ectoine.

Description

Technical Field

[0001] The invention relates to a low-carbon source fermentation process for efficiently synthesizing ectoine and an application method of ectoine fermentation liquid in promoting rice growth and salt resistance and improving the tolerance of rice seedlings in saline-alkali soil environment, belonging to the fields of fermentation engineering and agriculture. Background Art

[0002] With the continuous change of global climate and the continuous emergence of extreme climate, the growth and yield of rice have been greatly reduced due to various stresses, among which the saline-alkali stress caused by soil salinization is more serious. Rice is sensitive to saline-alkali environment. At present, the main way to improve rice growth and yield is to use industrial fertilizers and pesticides, but they have many disadvantages, such as greater harm and unsustainable effect, which can be compensated by pollution-free, long-acting, low-cost biofertilizers or other yield-increasing agents. Ectoine, as a cyclic amino acid derivative of L-aspartic acid, is an important natural secondary metabolite in halophilic microorganisms. As a protective agent and stabilizer, it plays an important role in various external pressures (such as high temperature, high osmotic pressure, drying and radiation) in cells. Ectoine can prevent water loss under drought or saline-alkali conditions, and has good application prospects in rice salt resistance and other aspects. At present, some progress has been made in the biosynthesis of ectoine, which has enabled the fermentation yield of ectoine to reach about 100g / L. However, due to the price of the fermentation raw material glucose and the complexity of the subsequent fermentation and separation process, ectoine biofermentation is difficult to be used in agricultural production.

[0003] The present invention develops a more economical and efficient strategy for fermenting and using ectoine. Using low-quality carbon resources to replace glucose as the carbon source for ectoine fermentation not only improves the fermentation efficiency of ectoine, but also significantly increases the yield of ectoine. And by using the technology of directly using the fermentation liquid, the effects of promoting the growth of rice and alleviating salt stress are achieved. In general, the present invention proposes a strategy for efficiently fermenting ectoine with low-quality carbon sources and directly using the fermentation liquid without separation, which provides a feasibility for the application of biological fermentation ectoine. Summary of the invention

[0004] The purpose of the present invention is to provide a method for efficiently synthesizing ectoine by fermenting a low-quality carbon source and applying the fermentation liquid to improve the growth of rice seedlings and the tolerance in a saline-alkali environment.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] The invention discloses an ectoine fermentation broth, which is obtained by inoculating ectoine-producing bacteria into a low-quality carbon source fermentation medium for fermentation.

[0007] The fermentation medium comprises: 10-30 g / L of low-quality carbon source, 3-10 g / L of nitrogen source and 1-10 g / L of inorganic salt.

[0008] The low-quality carbon source is at least one of straw hydrolyzate, inulin, molasses or sugarcane molasses.

[0009] The nitrogen source was ammonium chloride;

[0010] The inorganic salt is at least one of sodium citrate dihydrate, potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, and manganese sulfate.

[0011] Further preferred are: 10-30 g / L of low-quality carbon source, 3-10 g / L of ammonium chloride, 0.5-2 g / L of sodium citrate dihydrate, 0.5-3 g / L of potassium dihydrogen phosphate, 0.5-2 g / L of magnesium sulfate, 0.5-2 g / L of iron sulfate, and 0-0.5 g / L of manganese sulfate.

[0012] More preferably: 20 g / L straw hydrolyzate, 6 g / L ammonium chloride, 1 g / L sodium citrate dihydrate, 2 g / L potassium dihydrogen phosphate, 1.2 g / L magnesium sulfate, 0.1 g / L iron sulfate, and 0.1 g / L manganese sulfate.

[0013] It is further preferred that the carbon source is straw hydrolyzate, which is obtained by processing straw, and the straw hydrolyzate contains glucose, xylose, arabinose, galactose and a small amount of fructose.

[0014] The fermentation was further carried out by feeding and the carbon source of the feeding was straw hydrolyzate. During the fermentation process, the glucose concentration was detected and the feeding was carried out by feeding and the carbon source concentration in the fermentation liquid was kept at 1 g / L.

[0015] The strain used for fermentation is a genetically engineered bacterium obtained by knocking out the crr gene and overexpressing the ectABC gene cluster and the lysC gene in Escherichia coli MG1655.

[0016] The fermentation is carried out at 35-39° C. for 36-100 hours to obtain tetrahydropyrimidine fermentation liquid.

[0017] The method for preparing the tetrahydropyrimidine fermentation broth comprises the following steps:

[0018] 1) Streak the ectoine-producing bacteria onto solid LB medium and culture at 35-39°C for 8-16 hours;

[0019] 2) Pick the cultured bacteria, inoculate into liquid LB medium, and culture at 35-39°C for 8-16 hours to obtain seed liquid;

[0020] 3) Inoculate the seed liquid into the fermentation medium, add ampicillin, streptomycin and IPTG inducer, and ferment and culture at 35-39° C. for 36-100 hours.

[0021] The addition amount of ampicillin is 50-100 mg / L, the addition amount of streptomycin is 30-50 mg / L, and the addition amount of IPTG inducer is 0.05-0.2 mol / L.

[0022] The fermented tetrahydropyrimidine fermentation liquid can be directly used to enhance rice stress resistance and promote growth without purification.

[0023] Furthermore, ectoine is applied to rice by spraying or root irrigation with ectoine fermentation liquid.

[0024] Further, spraying or root irrigation treatment is performed starting from the bud stage after transplanting the rice seedlings.

[0025] Further spraying treatment: the ectoine fermentation liquid is configured to a 0.1mM-10mM concentration of ectoine fermentation liquid solution, and evenly sprayed on the plants. From the bud stage, the spraying amount is controlled at 10mL, once every 2 days. Root irrigation treatment: the ectoine fermentation liquid is configured to a 0.1mM-10mM concentration of ectoine fermentation liquid solution, and the root irrigation treatment is carried out to the roots of the plants from the bud stage after transplanting. The addition amount is controlled at 10mL, once every 2 days.

[0026] The ectoine fermentation liquid was applied to the roots of the plants starting from the bud stage after transplanting. The optimum ectoine concentration for root irrigation was 5mM.

[0027] In rice growth promotion, spraying 5mM tetrahydropyrimidine fermentation liquid can effectively promote the growth of rice seedlings;

[0028] In rice salt resistance, ectoine fermentation liquid can alleviate the damage of rice in severe saline-alkali environment. Different application methods and different application concentrations of ectoine affect the resistance of rice to salt stress. Among them, spraying ectoine fermentation liquid has a better effect on alleviating rice salt stress damage.

[0029] In the case of soils with a salt content of 0.4-0.6%, which is called severe saline-alkali soil, spraying ectoine fermentation liquid has a significant effect on the stress resistance of rice seedlings during growth. Spraying treatment with 0.5-3mM ectoine fermentation liquid is the best, followed by root irrigation treatment with 0.5-3mM ectoine fermentation liquid. The damage caused by salt stress can be basically alleviated.

[0030] Spraying begins at the bud stage after transplanting rice seedlings. The optimum concentration of spraying ectoine fermentation liquid is 3mM.

[0031] When spraying or root irrigation is further performed on hydroponic rice, the optimum concentration of the ectoine fermentation solution is 5 mM. A special plant hydroponic box is used for the growth of rice seedlings, with a specification of 96 holes, a hole diameter of 6.3 mm, a length of 127 mm, a width of 87 mm, and a height of 114 mm.

[0032] When spraying or root irrigation is performed on hydroponic rice containing 100 mM sodium chloride, the optimum concentration of the ectoine fermentation solution is 0.5 mM. A special plant hydroponic box is used for the growth of rice seedlings, with a specification of 96 holes, a hole diameter of 6.3 mm, a length of 127 mm, a width of 87 mm, and a height of 114 mm.

[0033] When spraying or root irrigation is performed on rice in moderate saline-alkali soil, the optimum concentration of tetrahydropyrimidine is 3 mM. The soil loading amount of the rice pot experiment is 500 g / pot, and the pot specifications are 13 cm high, 12 cm top diameter, and 9.5 cm bottom diameter.

[0034] Beneficial effects:

[0035] 1. The ectoine fermentation liquid of the present invention is fermented from straw hydrolyzate, which saves costs and improves yield and efficiency. The yield of ectoine can reach 134 g / L, and the production rate of ectoine can reach 3.73 g / L / h.

[0036] 2. The use of ectoine fermentation liquid of straw hydrolyzate is more conducive to the realization of stress resistance and growth promotion function of rice seedlings than the use of commercial ectoine. Compared with the application of commercial ectoine, the rice plant height, root length, plant fresh weight, plant dry weight, root fresh weight, root dry weight and total chlorophyll concentration in this experiment were significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Ectoelastine was produced by fermentation in a 5L fermenter using straw hydrolysate as carbon source. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be described in detail below. However, the described embodiments are only a part of the embodiments of the present invention rather than all the embodiments. The present invention can be better understood according to the following embodiments.

[0039] The strain used for fermentation in the following examples is engineered Escherichia coli MG1655, and the construction process of the strain is shown in patent CN118028204A.

[0040] Ectoin (98%) and inulin (90%) were purchased from Shanghai Titan Technology Co., Ltd., sugarcane molasses was from China National Bluestar Group Co., Ltd., and straw hydrolyzate was from Suzhou Juwei Yuanchuang Biotechnology Co., Ltd., with the main components being glucose (426 g / L), xylose (194 g / L), arabinose (8.8 g / L), cellobiose (63 g / L) and a small amount of fructose (3.1 g / L). Other experimental materials were purchased from commercial sources.

[0041] The preparation of tetrahydropyrimidine fermentation broth comprises the following steps:

[0042] (1) Plate culture: streak the engineered E. coli onto LB solid medium and culture at 37°C for 12-18 hours;

[0043] (2) Seed culture: Pick a spot from the plate containing the bacterial strain or take the bacterial strain from the seed tube, inoculate it into a test tube containing 5 mL of LB medium, place it in a shaking incubator at 37°C and culture it at 180 rpm for 12 h as the seed solution;

[0044] (3) Fermentation tank culture: The activated seed liquid is inoculated into the fermentation tank with an inoculation amount of 1-10% (v / v), a stirring speed of 600-800rpm, and the dissolved oxygen is controlled at 40%. The fermentation culture is carried out at 37°C, and the pH is controlled to 7.0 with ammonia water. The fermentation medium is 6g / L ammonium chloride, 1g / L sodium citrate dihydrate, 2g / L potassium dihydrogen phosphate, 1.2g / L magnesium sulfate, 0.1g / L iron sulfate, and 0.1g / L manganese sulfate. At the beginning of fermentation, 0.1mg / L ampicillin and 0.05mg / L streptomycin are added. The initial hydrolyzate concentration is 20g / L. The glucose content is detected during the fermentation process. When the glucose is consumed, the hydrolyzate is replenished to maintain the glucose concentration in the fermentation system at 1g / L, OD 600 When the pH reaches 8, add 0.1 mmol / L IPTG.

[0045] Examples 1-4

[0046] (1) Plate culture: streak the engineered E. coli Fect3 onto LB solid medium and culture at 37°C for 12-18 h;

[0047] (2) Seed culture: Pick a spot from the plate containing the bacterial strain or take the bacterial strain from the seed tube, inoculate it into a test tube containing 5 mL of LB medium, place it in a shaking incubator at 37°C and culture it at 180 rpm for 12 h as the seed solution;

[0048] (3) Shake flask development: The activated seed liquid was inoculated into a shake flask with an inoculation amount of 1% (v / v). The fermentation medium was 6 g / L ammonium chloride, 1 g / L sodium citrate dihydrate, 2 g / L potassium dihydrogen phosphate, 1.2 g / L magnesium sulfate, 0.1 g / L ferrous sulfate, 0.1 g / L manganese sulfate, 100 mg / L ampicillin, 50 mg / L streptomycin, and an additional 20 g / L of a low-grade carbon source of reducing sugar from different sources was added as a carbon source. Place in a 37°C shaker and ferment at 180 rpm for 48 hours. After culturing for 5 hours, add 0.1 mmol / L IPTG. Add ammonia water every 12 hours to adjust the pH to 7. The fermentation results are shown in Table 1:

[0049] Table 1 Shake flask fermentation results of Examples 1-4

[0050]

[0051] As can be seen from Table 1, most cheap carbon sources are not conducive to the production of ectoine, while straw hydrolysate can effectively promote the synthesis of ectoine. In the process of Escherichia coli fermentation to synthesize ectoine, glucose as a carbon source is more conducive to the synthesis of ectoine than oligofructose, sucrose and other sugars. The main reducing sugar in inulin is oligofructose, and the main component of sugarcane molasses is sucrose. The yield of ectoine using inulin and sugarcane molasses as carbon sources is relatively low. Straw hydrolysate uses glucose as the main sugar, as well as reducing sugars such as xylose, arabinose, cellobiose, and galactose. It is speculated that these sugars have a synergistic metabolic effect and promote the synthesis of ectoine.

[0052] Example 5

[0053] The straw hydrolysate was used to produce ectoine by fermentation in a 5L fermenter.

[0054] (1) Plate culture: streak the engineered E. coli onto LB solid medium and culture at 37°C for 12-18 hours;

[0055] (2) Seed culture: Pick a spot from the plate containing the bacterial strain or take the bacterial strain from the seed tube, inoculate it into a test tube containing 5 mL of LB medium, place it in a shaking incubator at 37°C and culture it at 180 rpm for 12 h as the seed solution;

[0056] (3) Fermentation tank culture: The activated seed liquid is inoculated into the fermentation tank with an inoculation amount of 10% (v / v), a stirring speed of 600-800rpm, and the dissolved oxygen is controlled at 40%. The fermentation culture is carried out at 37°C, and the pH is controlled to 7.0 with ammonia water. The fermentation medium is 6g / L ammonium chloride, 1g / L sodium citrate dihydrate, 2g / L potassium dihydrogen phosphate, 1.2g / L magnesium sulfate, 0.1g / L iron sulfate, and 0.1g / L manganese sulfate. At the beginning of fermentation, 0.1mg / L ampicillin and 0.05mg / L streptomycin are added. The initial hydrolyzate concentration is 20g / L. The glucose content is detected during the fermentation process. When the glucose is consumed, the hydrolyzate is replenished to maintain the glucose concentration in the fermentation system at 1g / L, OD 600 When the concentration reaches 8, add IPTG 0.1mmol / L. After the fermentation is completed, the upper fermentation liquid is filtered and sterilized through a microfiltration membrane to obtain a sterilized fermentation liquid for subsequent rice growth promotion and stress resistance verification.

[0057] The results are as follows Figure 1 As shown, E. coli OD 600 The maximum yield of ectoine can reach 134.1 g / L at 36 h. The production rate can reach 3.73 g / L / h.

[0058] This result shows that the use of straw hydrolysate can significantly shorten the fermentation time while increasing the yield. Compared with the production rate of other carbon sources, which is generally lower than 2g / L / h, the straw hydrolysate significantly increases the production rate to 3.73g / L / h. This method further improves the production efficiency of tetrahydropyrimidine.

[0059] The detection method of ectoine in this embodiment is as follows:

[0060] Take 2 mL of sample every 4-12 hours, take a portion and dilute to an appropriate multiple, and use a spectrophotometer to detect the absorbance at 600 nm to reflect the amount of bacteria. The remaining sample is centrifuged at 12000 rpm for 2 minutes, and the supernatant is aspirated. A portion is used to detect the remaining sugar concentration using a biosensor analyzer; a portion is diluted to an appropriate multiple and filtered with a 0.22 micron pore size filter membrane, and added to a liquid sample bottle, and detected using high performance liquid chromatography (HPLC): Liquid chromatography uses C 18 The chromatographic column and the mobile phase were 20% acetonitrile and 80% water. The concentration of ectoine was calculated by comparing the peak area with that of the standard sample.

[0061] Example 6

[0062] Growth-promoting effect of tetrahydropyrimidine fermentation liquid on hydroponic rice seedlings.

[0063] In this example, commercial pure ectoine and the ectoine fermentation liquid obtained in Example 5 were prepared into an aqueous solution of a certain concentration for use in subsequent experiments.

[0064] (1) Seed disinfection: Wash the rice seeds with running tap water to remove surface impurities, then stir and disinfect with 2.5% sodium hypochlorite for 15 min, then stir and disinfect with 75% ethanol twice, each time for 5 min, and finally rinse with pure water several times;

[0065] (2) Seed germination: Spread the sterilized rice seeds on a double-layer sterilized gauze in a germination box, add tap water or pure water to immerse half of the seeds, place in a greenhouse at about 30°C, change the water every 24 hours, and germinate for 2-3 days until the buds are about 5 mm long;

[0066] (3) Transplantation: Transplant seedlings with uniform growth into rice culture boxes. To prevent the rice roots from intertwining and breaking during sampling, the seeds can be placed at intervals.

[0067] A special plant hydroponic box is used for growing rice seedlings. The box has 96 holes, a hole diameter of 6.3 mm, a length of 127 mm, a width of 87 mm, and a height of 114 mm.

[0068] After 4-5 days of cultivation with Kimura B nutrient solution, spraying treatment began. 9 groups of experiments were set up, all of which were cultured with Kimura B nutrient solution, and the spraying treatments were different. Group A, spraying water, that is, 0mM concentration of ectoine; Group B, spraying 0.5mM concentration of ectoine; Group C, spraying 1mM concentration of ectoine; Group D, spraying 3mM concentration of ectoine; Group E, spraying 5mM concentration of ectoine; Group F, spraying 10mM concentration of ectoine; Group EC1, root irrigation with 5mM concentration of ectoine; Group EC2, spraying 5mM ectoine fermentation liquid; Group EC3, root irrigation with 5mM ectoine fermentation liquid. The application amount is 10mL, placed in a light incubator, light for 12h, 30℃, dark for 12h, 28℃. Treat once every 2 days, and change the nutrient solution every 4 days. Data were collected two weeks after treatment, and three groups were taken for parallel testing of plant height, root length, plant fresh weight, plant dry weight, root fresh weight, root dry weight and total chlorophyll concentration, and the mean was calculated.

[0069] The chlorophyll concentration detection method is: take about 5cm of the latest grown fresh rice leaves from the top, cut them into 1mm thin filaments or thin segments and mix them evenly, take 25mg-50mg of leaves, put them into a 10mL stoppered test tube, add 1mL DMSO to soak the plant tissue in DMSO, extract at 65℃ in the dark until the leaves turn white or transparent. After cooling, add 4mL 80% acetone to dilute, and measure the absorbance outside 663.6 and 646.6nm. DMSO: 80% acetone = 1:4 when the control is zeroed, 80% acetone is diluted with weak alkaline water (pH = 7.8), and the colorimetric dish is washed with 80% acetone. The absorbance is more accurate within 0.2-0.8, otherwise it is necessary to add 80% acetone to dilute to the appropriate absorbance, and then calculate the absorbance before dilution.

[0070] The calculation formula for chlorophyll concentration is:

[0071] C a (mg·L -1 )=12.27A 663.6 -2.52A 646.6

[0072] C b (mg·L -1 )=20.10A 646.6 -4.92A 663.6

[0073] C T (mg·L -1 )=C a +C b =7.35A 663.6 +17.58A 646.6

[0074] Three parallel experiments were performed in each treatment group.

[0075] The average growth of rice is shown in Table 2.

[0076] Table 2

[0077]

[0078] As can be seen from the above table, compared with the spraying treatment group E and the root irrigation treatment group EC1, the spraying of tetrahydropyrimidine has a significant growth-promoting effect on rice seedlings during hydroponic growth. In the spraying treatment, the EC2 group sprayed with 5mM tetrahydropyrimidine fermentation liquid was significantly better than the E group sprayed with 5mM tetrahydropyrimidine, and was the treatment group with the most significant growth-promoting effect. Compared with the control group A, the EC2 group sprayed with 5mM tetrahydropyrimidine fermentation liquid showed an increase of 19.1% in plant height, 57% in root length, 36.3% in plant fresh weight, 49.2% in plant dry weight, 48.7% in root fresh weight, 50.1% in root dry weight, and 25.3% in total chlorophyll concentration. The above results show that spraying 5mM tetrahydropyrimidine fermentation liquid can effectively promote the growth of rice seedlings.

[0079] Example 7

[0080] Effect of ectoine fermentation broth on salt stress resistance of rice seedlings grown in hydroponics at 100 mM sodium chloride concentration.

[0081] In this embodiment, commercial ectoine and ectoine fermentation liquid are used in the same manner as in Example 6. Germination is accelerated until the buds are about 5 mm long, and the seeds are moved to a rice culture box. After 4-5 days of culture in Kimura B nutrient solution, ectoine spraying treatment is started. Eight groups of experiments were set up, all of which were cultured with Kimura B nutrient solution plus 100mM sodium chloride, and the application treatments were different. Group A, spraying water, that is, 0mM concentration of ectoine; Group B, spraying 0.1mM concentration of ectoine; Group C, spraying 0.25mM concentration of ectoine; Group D, spraying 0.5mM concentration of ectoine; Group E, spraying 1mM concentration of ectoine; Group DC1, root irrigation with 0.5mM concentration of ectoine; Group DC2, spraying fermentation liquid containing 0.5mM ectoine; Group DC3, root irrigation with fermentation liquid containing 0.5mM ectoine. The spraying amount was 10 mL, and the plants were placed in a light incubator with 12 h of light at 30 ° C and 12 h of darkness at 28 ° C. The plants were treated once every 2 days and the nutrient solution was changed every 4 days. Data was collected after two weeks of treatment, and three groups were taken for parallel testing of plant height, root length, plant fresh weight, plant dry weight, root fresh weight, root dry weight and total chlorophyll concentration, and the mean was calculated. The chlorophyll detection method was the same as in Example 6.

[0082] The average growth of rice is shown in Table 3.

[0083] Table 3

[0084]

[0085] As can be seen from the above table, compared with the spraying treatment group D and the root irrigation treatment group DC1, the spraying of tetrahydropyrimidine has a significant effect on the stress resistance of rice seedlings during hydroponic growth under salt stress. In the spraying treatment, the DC2 group sprayed with 0.5mM tetrahydropyrimidine fermentation liquid had the best effect, followed by the DC3 group with 0.5mM tetrahydropyrimidine fermentation liquid for root irrigation. Compared with the control group A, the DC2 group showed an increase of 21.9% in plant height, 16.6% in root length, 55.3% in plant fresh weight, 37.2% in plant dry weight, 115% in root fresh weight, 31.5% in root dry weight, and 30.6% in total chlorophyll concentration. In summary, the above results show that spraying 0.5mM tetrahydropyrimidine fermentation liquid can effectively promote the growth of rice under salt stress. Compared with the control group without stress in Example 1-A, the total height was 1.05 times, the fresh weight was 1.007 times, the dry weight was 1.54 times, and the chlorophyll was 0.9 times, which basically alleviated the damage of salt stress.

[0086] At present, saline-alkali soil is divided into 0.1%-0.2% salt content is called light saline-alkali soil, 0.2%-0.4% salt content is called moderate saline-alkali soil, and 0.4%-0.6% salt content is called severe saline-alkali soil. This embodiment uses 100mM (0.58%) sodium chloride, which belongs to the severe saline-alkali environment range. The above results show that different application methods and different application concentrations of tetrahydropyrimidine affect the resistance of rice to salt stress, among which spraying tetrahydropyrimidine fermentation liquid has a better effect on alleviating rice salt stress damage.

[0087] Example 8

[0088] Tetrahydropyrimidine fermentation liquid improves the tolerance of rice seedlings in saline-alkali soil environment.

[0089] In this example, commercial ectoine and ectoine fermentation liquid were used in the same manner as in Example 6. After germination, the seeds were transplanted into soil pots until the buds were about 3 cm long. Six rice seedlings were transplanted into each soil pot. The amount of soil in the rice pot experiment was 500 g / pot. The pot specifications were 13 cm high, 12 cm in top diameter, and 9.5 cm in bottom diameter.

[0090] Eight groups of experiments were set up with different spraying treatments. Group A, spraying water, i.e. 0mM ectoine; Group B, spraying 1mM ectoine; Group C, spraying 3mM ectoine; Group D, spraying 5mM ectoine; Group E, spraying 10mM ectoine; Group CC1, root irrigation with 3mM ectoine; Group CC2, spraying with 3mM ectoine fermentation liquid; Group CC3, root irrigation with 3mM ectoine fermentation liquid. The spraying volume was 10mL, and the plants were placed in a light incubator with 12h of light, 30℃, and 12h of darkness, 28℃. The plants were treated once every 2 days, and watered according to the soil moisture to keep it moist. Data were collected three weeks after the treatment, and the rice seedlings were carefully removed from the soil to avoid breaking the rice roots. Three groups were selected for parallel testing of plant height, root length, plant fresh weight, plant dry weight, root fresh weight, root dry weight and total chlorophyll concentration, and the mean was calculated. The chlorophyll detection method was the same as in Example 6.

[0091] The indicators of saline-alkali soil in this experiment are: pH=8.78, soil salt content is 3.436g / kg, which is moderate saline soil and strong alkaline soil.

[0092] The soil testing results and methods are shown in Tables 4 and 5.

[0093] Table 4

[0094]

[0095] Table 5

[0096]

[0097] The average growth of rice is shown in Table 6.

[0098] Table 6

[0099]

[0100] As can be seen from the table above, compared with the spraying treatment group C and the root irrigation treatment group CC1, the spraying of tetrahydropyrimidine has a significant effect on improving the tolerance of rice seedlings in saline-alkali soil environment. In the spraying treatment, the CC2 group spraying with 3mM tetrahydropyrimidine fermentation liquid had the best effect. Compared with the control group A, the CC2 group showed an increase of 55.6% in plant height, 90.5% in root length, 89.6% in plant fresh weight, 99.5% in plant dry weight, 173.4% in root fresh weight, 136% in root dry weight, and 64.6% in total chlorophyll concentration. The above results show that when rice seedlings grow in a severe saline-alkali soil environment, tetrahydropyrimidine fermentation liquid can effectively improve the ability of rice to resist salt and alkali and promote the growth of rice.

[0101] In summary, adding ectoine fermentation liquid by spraying can significantly promote the growth of rice seedlings and improve the salt resistance of rice. It can alleviate the damage of salt-alkali stress to rice during growth while preventing water loss and promoting rice growth.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An ectoine fermentation broth, characterized in that: The fermentation liquid is obtained by inoculating ectoine-producing bacteria into a low-quality carbon source fermentation medium and fermenting.

2. The tetrahydropyrimidine fermentation broth according to claim 1, characterized in that: The low-quality carbon source is straw hydrolyzate.

3. The tetrahydropyrimidine fermentation broth according to claim 1, characterized in that: The low-quality carbon source fermentation medium comprises: 10-30 g / L of low-quality carbon source, 3-10 g / L of ammonium chloride, 0.5-2 g / L of sodium citrate dihydrate, 0.5-3 g / L of potassium dihydrogen phosphate, 0.5-2 g / L of magnesium sulfate, 0-0.5 g / L of iron sulfate, and 0-0.5 g / L of manganese sulfate.

4. The tetrahydropyrimidine fermentation broth according to claim 1, characterized in that: Fed-batch fermentation was adopted, and during the fermentation process, the glucose concentration was detected and fed-batch was performed to maintain the glucose concentration in the fermentation liquid at 1 g / L.

5. The tetrahydropyrimidine fermentation broth according to claim 1, characterized in that: The fermentation is carried out at 35-39° C. for 36-100 h to obtain tetrahydropyrimidine fermentation liquid.

6. Use of the tetrahydropyrimidine fermentation liquid according to any one of claims 1 to 5 in promoting rice growth and resisting salt.

7. The use according to claim 6, characterized in that: Tetrahydropyrimidine is applied to hydroponic rice by spraying or root irrigation.

8. The use according to claim 7, characterized in that After transplanting rice seedlings, spray or irrigate the roots at the bud stage, and the concentration of the ectoine fermentation liquid is 0.1 mM-10 mM.

9. The use according to claim 8, characterized in that: The ectoine fermentation liquid was applied to the roots of the plants starting from the bud stage after transplanting. The optimum ectoine concentration for root irrigation was 5mM.

10. The use according to claim 8, characterized in that: Spraying begins at the bud stage after transplanting rice seedlings. The optimum concentration of spraying ectoine fermentation liquid is 3mM.

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