Method for improving combined nitrogen fixation of nitrogen-fixing bacteria and sugarcane

Abstract

The invention discloses a method for improving combined nitrogen fixation of nitrogen-fixing bacteria and sugarcane, and relates to the technical field of sugarcane cultivation, according to the method, sugarcane seed stems are inoculated with rhizobium sp.FSGRY3, Enterobacter sp.GG49E and Klebsilasp.GG160E, compared with uninoculated sugarcane, the total nitrogen content of sugarcane inoculated with the three nitrogen-fixing bacteria in a mixed mode is increased by 20.90%, and the total nitrogen content of sugarcane inoculated with the three nitrogen-fixing bacteria in a mixed mode is increased by 20.90%. The nitrogen fixation efficiency of the sugarcane inoculated with the three nitrogen-fixing bacteria in a mixed manner is improved by 67.67% compared with that of plants inoculated with single nitrogen-fixing bacteria. The three nitrogen-fixing bacteria have a synergistic effect on the sugarcane, so that the nitrogen fixation efficiency of the sugarcane is improved. The dependence of sugarcane on chemical nitrogen fertilizer is reduced, the agricultural production cost is reduced, and the pollution of chemical fertilizer to the environment is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of sugarcane cultivation, and in particular to a method for improving nitrogen fixation by combining nitrogen-fixing bacteria with sugarcane. Background Art

[0002] Sugarcane is a plant of the Gramineae family and a common cultivated plant in southern my country. It has the effects of clearing away heat and promoting body fluid, moistening dryness and harmonizing the stomach, and detoxifying. Sugarcane is an important sugar crop and energy crop. China is a major sugarcane producer, but the high cost of planting, especially the high nitrogen fertilizer usage and low utilization rate, also leads to the deterioration of farmland ecology, which has become an important limiting factor for the sustainable development of the sugarcane industry.

[0003] Rhizobia settle in the roots, and plants provide them with mineral nutrients and energy. Rhizobia fix free nitrogen in the atmosphere and provide nitrogen nutrients for plants. The two are in a state of equilibrium in the antagonistic parasitic relationship and show symbiosis. Rhizobia are a type of Gram-negative bacteria widely distributed in the soil. They coexist with legumes and form nodules by infecting the roots of legumes and fixing nitrogen in the air to provide nutrition for plants. The symbiosis between rhizobia and legumes is the most powerful system in the biological nitrogen fixation system, and the nitrogen fixed accounts for about 65% of the total biological nitrogen fixation. It plays an extremely important role in agricultural production and nitrogen fixation ecosystems.

[0004] In the prior art, sugarcane can obtain nitrogen by fixing nitrogen with nitrogen-fixing bacteria. The nitrogen-fixing bacteria are inoculated in the sugarcane seed stems, and an appropriate amount of fertilizer is applied during the sugarcane seedling stage to support the growth and photosynthesis of the sugarcane seedlings, provide a carbon source for the functional bacterial community, establish a stable combination, and then achieve combined nitrogen fixation. However, there are few studies on the high-level combined nitrogen fixation of sugarcane. The high-level combined nitrogen fixation of sugarcane may be achieved by a variety of nitrogen-fixing bacteria. Different bacteria can synergistically promote the growth of sugarcane, promote the elongation and development of roots by producing plant growth regulators, and improve the root absorption of nutrients such as water and nitrogen, and then combine with sugarcane to fix nitrogen. Research has not yet confirmed which bacteria are the main contributors to the biological nitrogen fixation of Chinese sugarcane. It is urgent to further study how to cultivate different nitrogen-fixing bacteria so that the nitrogen-fixing bacteria can achieve a stable high-level nitrogen fixation efficiency for sugarcane. In view of this, the present invention seeks a method that can improve the efficiency of combined nitrogen fixation of nitrogen-fixing bacteria and sugarcane, and provide basic information for achieving efficient combined nitrogen fixation of sugarcane and rhizobia in the field. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides a method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane, which can improve the total N content and nitrogen fixation efficiency of sugarcane. The specific technical scheme is as follows:

[0006] A method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane, inoculating sugarcane seed stems with nitrogen-fixing bacteria, planting the inoculated sugarcane seed stems, and then determining the nitrogen content of the sugarcane and 15N isotope abundance.

[0007] The nitrogen-fixing bacteria is one or more of Rhizobium sp.FSGRY3, Enterobacter sp.GG49E, and Klebsiella sp.GG160E.

[0008] Furthermore, the nitrogen-fixing bacteria are Rhizobium sp.FSGRY3, Enterobacter sp.GG49E, and Klebsiella sp.GG160E;

[0009] The rhizobium Rhizobium sp.FSGRY3 is derived from the root system of Scutellaria baicalensis, and its 16S rDNA accession number is KJ185035; the Enterobacter sp.GG49E is derived from the root system of sugarcane, and its 16S rDNA accession number is HQ204300; the Klebsiella sp.GG160E is derived from the root system of sugarcane, and its 16S rDNA accession number is HQ204303; the above-mentioned strains are preserved in the Guangxi Key Laboratory of Sugarcane Genetic Improvement.

[0010] Furthermore, the method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane comprises:

[0011] (1) A single bud section of sugarcane seed stem is taken, soaked in hot water at 50-55° C. for 25-35 minutes, then soaked in a 50% carbendazim wettable powder diluted with 500-600 times water for 5-6 minutes, taken out and dried, and then soaked in a nitrogen-fixing bacteria agent for 30 minutes to obtain a sugarcane seed stem inoculated with the nitrogen-fixing bacteria agent; the preparation method of the nitrogen-fixing bacteria liquid is as follows: activating on a YMA solid culture medium plate for 40-50 hours, taking a single colony and culturing it in a YMA liquid culture medium for 24-36 hours, washing the bacteria with sterile water by centrifugation for 2-3 times, and adjusting the concentration of the nitrogen-fixing bacteria liquid to 1-2×10 8 CFU / ml. YMA medium: mannitol 10g, potassium dihydrogen phosphate 0.5g, yeast powder 1g, magnesium sulfate 0.2g, sodium chloride 0.1g, calcium chloride 0.1g, 1000ml distilled water.

[0012] (2) According to 10mg ( 15 NH 4 ) 2 SO 4 (10.11atom%) / kg soil standard for soil pretreatment. 15 NH 4 ) 2 SO 4After dissolving with sterile water, spray it into the soil, stir twice a day, and after continuous stirring for 10 days, sprinkle 0.03-0.05g / kg of soil with 10% clothianidin· insecticide granules to control sugarcane pests to obtain pre-treated soil, plant sugarcane seed stems inoculated with nitrogen-fixing bacteria in the pre-treated soil, and pour 0.03-0.1L / kg of distilled water into the soil every 2 days after planting to keep the soil moisture at 70% of the maximum water holding capacity;

[0013] (3) After the sugarcane seed stems have sprouted and grown 2-3 leaves, pour 0.07-0.1 L / kg of soil 1 / 10 MS nutrient solution into the soil;

[0014] (4) When the sugarcane has grown 7-8 true leaves and begins to tiller, loosen the soil and apply 18-22g / kg of nitrogen-fixing bacteria agent to the soil. Loosen the soil once every 20 days to increase the root zone temperature, reduce weeds, and increase the number of nitrogen-fixing bacteria in the soil. Spray 0.01mg / L molybdate solution once on the 40th, 50th and 60th days after sowing.

[0015] (5) During the peak growth period of sugarcane (when the plant is 50 cm tall and grows an average of more than 15 cm every 15 days), the sugarcane plants were washed, the roots, stems and leaves were dried, ground into powder, and the nitrogen content and 15 N isotope abundance.

[0016] Furthermore, the preparation method of the nitrogen-fixing bacteria agent is as follows: the nitrogen-fixing bacteria are activated and then inoculated into a nitrogen-free culture medium for cultivation at 30°C and 150rpm / min for 24h until the logarithmic growth phase, to obtain a rhizobium solution, an enterobacterial solution, and a Klebsiella solution, and the concentration of each solution is adjusted to 1×10 7 CFU / mL, the adjusted concentrations of rhizobium, enterobacter and Klebsiella were inoculated at 1% on the sterilized bagasse-vermiculite solid fermentation medium, the strains were fully mixed with the bagasse-vermiculite solid medium, the temperature was adjusted to 30°C and the humidity was adjusted to 65%-75%, and the fermentation was carried out for 3 days. The concentration of the strains was not less than 1×10 8 CFU / g, and evenly mix the fermented rhizobium solution, enterobacter solution and Klebsiella solution to obtain the nitrogen-fixing bacteria agent.

[0017] Furthermore, the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage: 58-62% bagasse, 32-36% vermiculite, 2.9-3.2% sugarcane leaf biochar, 2.6-3.0% stone powder, 0.003-0.005% manganese sulfate, 0.06-0.14% dipotassium hydrogen phosphate, and 0.037-0.055% magnesium sulfate.

[0018] Furthermore, the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage: 60% bagasse, 33.846% vermiculite, 3% sugarcane leaf biochar, 3% stone powder, 0.004% manganese sulfate, 0.1% dipotassium hydrogen phosphate, and 0.05% magnesium sulfate.

[0019] Furthermore, the mixing weight ratio of the fermented rhizobium solution, enterobacter solution and Klebsiella solution in the nitrogen-fixing bacteria agent is 1:1:1.

[0020] Furthermore, the activation method of the nitrogen-fixing bacteria is as follows: taking nitrogen-fixing bacteria stored at -80°C, activating them on a YMA solid culture medium plate for 40-50 hours, taking a single colony in a nitrogen-fixing bacteria activation medium, culturing them at 28-32°C, 200-220rpm for 24-36 hours, and then washing the bacteria by centrifugation with sterile water for 2-3 times to complete the activation;

[0021] Furthermore, the nitrogen-fixing bacteria activation medium consists of 10 g sucrose, 1 g soybean extract, 2 g potassium dihydrogen phosphate, 0.05 g ferric chloride, 0.1 g magnesium sulfate, 1 g mannitol, 0.05 g alginate, 0.01 g boric acid, 0.05 g guar gum, and 1000 ml distilled water.

[0022] Furthermore, the bagasse-vermiculite solid fermentation medium also includes 0.05 g / ml cobalt chloride hexahydrate.

[0023] Furthermore, the water content of the bagasse-vermiculite solid fermentation medium is 48-53%.

[0024] Furthermore, the water content of the bagasse-vermiculite solid fermentation medium is 50%.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention inoculates sugarcane seed stems with a combination of nitrogen-fixing bacteria including rhizobium sp. FSGRY3, Enterobacter sp. GG49E, and Klebsiella sp. GG160E. The inventors found that Enterobacter sp. GG49E has strong adaptability and metabolic diversity, can play a nitrogen-fixing role under different environmental conditions, and increase the stability and reliability of nitrogen fixation. Klebsiella sp. GG160E has efficient nitrogenase activity, and can quickly convert nitrogen in the air into a nitrogen form that can be used by sugarcane. The three bacteria synergize to improve the nitrogen fixation efficiency of sugarcane. The nitrogen fixation efficiency is increased by 67.67% compared with plants inoculated with a single nitrogen-fixing bacteria, and the total nitrogen content is increased by 20.90% compared with plants not inoculated. The dependence of sugarcane on chemical nitrogen fertilizers is reduced, the agricultural production cost is reduced, and the pollution of chemical fertilizers to the environment is reduced.

[0027] 2. The present invention activates the nitrogen-fixing bacteria separately and then cultures them, which can ensure the activity and growth ability of the strains. Alginic acid and boric acid in the nitrogen-fixing bacteria activation culture medium can passivate a variety of heavy metal elements, reduce their toxic side effects, and have a certain inhibitory effect on nitrates, which can improve the tolerance of crops to drought, high temperature, salt stress, oxidation, etc., and have a strong effect on repairing crop diseases; mannitol and guar gum compound can play a buffering and protective role on nitrogen-fixing bacteria, and improve the survival rate of nitrogen-fixing bacteria in the soil; soybean extract contains some substances that can chelate metal ions, such as organic acids, polypeptides, etc., and nitrogenase is an enzyme containing metal ions (such as iron, molybdenum, etc.). The presence of metal ions is crucial to the activity of nitrogenase. Organic acids, polypeptides, etc. in soybean extracts can combine with metal ions to form stable complexes to prevent metal ions from precipitating or being adsorbed by other substances, thereby ensuring that nitrogenase can obtain sufficient metal ions and maintain its activity. Antioxidant effect: Nitrogenase is sensitive to oxygen and easily inactivated by oxidation. Soybean extract contains some antioxidants, such as phenolic compounds, flavonoids, etc. These antioxidants can scavenge free radicals and reduce the damage of oxidative stress to nitrogenase, thereby protecting the activity of nitrogen-fixing bacteria.

[0028] 3. Bagasse-vermiculite solid fermentation medium recycles bagasse and sugarcane leaf biochar resources. Bagasse contains rich organic substances such as cellulose, hemicellulose and lignin, which can be used as the carbon source for the growth of nitrogen-fixing bacteria. During the growth process, nitrogen-fixing bacteria use these carbon sources for metabolic activities and synthesize the substances they need to achieve growth and reproduction; sugarcane leaf biochar has a large specific surface area and rich pore structure, which can adsorb nutrients and water in the culture medium. This adsorption can prevent the loss of nutrients, maintain the stability of nutrients in the culture medium, and provide a continuous supply of nutrients for nitrogen-fixing bacteria. At the same time, sugarcane leaf biochar can also improve the physical structure of the soil, increase the water retention and air permeability of the soil, and improve the fertility and sustainability of the soil; at the same time, the introduction of trace elements such as cobalt can promote the growth and reproduction of nitrogen-fixing bacteria after separation and accelerate the reproduction rate of nitrogen-fixing bacteria. DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0030] The sugarcanes in the examples of the present application are all RB928064 sugarcanes.

[0031] Example 1

[0032] The nitrogen-fixing bacteria in this embodiment are Rhizobium sp. FSGRY3, Enterobacter sp. GG49E, and Klebsiella sp. GG160E.

[0033] A method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane in this embodiment specifically comprises the following steps:

[0034] 1. Preparation of nitrogen-fixing bacteria agent

[0035] Take nitrogen-fixing bacteria stored at -80°C, activate them on a YMA solid culture medium plate for 45 hours, take a single colony in the nitrogen-fixing bacteria activation medium, culture it at 30°C, 200rpm for 28 hours, and then wash the bacteria twice by centrifugation with sterile water to complete the activation; the nitrogen-fixing bacteria activation medium consists of 10g sucrose, 1g soybean extract, 2g potassium dihydrogen phosphate, 0.05g ferric chloride, 0.1g magnesium sulfate, 1g mannitol, 0.05g alginate, 0.01g boric acid, 0.05g guar gum, and 1000ml distilled water.

[0036] The activated nitrogen-fixing bacteria were inoculated into 100 ml of liquid nitrogen-free culture medium and cultured at 30 °C and 150 rpm / min for 24 h until the logarithmic growth phase to obtain rhizobium, Enterobacter and Klebsiella bacterial solutions. The concentration of each bacterial solution was adjusted to 1 × 10 7CFU / mL, the adjusted concentrations of rhizobium, enterobacter and Klebsiella were inoculated at 1% inoculation amount on the sugarcane bagasse-vermiculite solid fermentation medium sterilized at 121℃ for 30min, the nitrogen-fixing bacteria and the sugarcane bagasse-vermiculite solid medium were fully mixed, the temperature was adjusted to 30℃ and the humidity was adjusted to 70%, and the fermentation was carried out for 3 days. The concentration of the detected bacteria was not less than 1×10 8 CFU / g, and evenly mixing the fermented rhizobium solution, enterobacter solution and Klebsiella solution in a weight ratio of 1:1:1 to obtain a nitrogen-fixing bacteria agent; the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage, 60% bagasse, 33.846% vermiculite, 3% sugarcane leaf biochar, 3% stone powder, 0.004% manganese sulfate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate and 0.05g / ml cobalt chloride hexahydrate; the water content of the bagasse-vermiculite solid fermentation medium is 50%.

[0037] 2. Take a single bud section of sugarcane seed stem, soak it in 52°C hot water for 30 minutes, then soak the seed stem in 500 times the amount of 50% carbendazim wettable powder diluted with water for 6 minutes, take it out and dry it, then soak it in nitrogen-fixing bacteria solution for 30 minutes to obtain the sugarcane seed stem inoculated with nitrogen-fixing bacteria agent.

[0038] 3. According to 10mg ( 15 NH4) 2 SO 4 (10.11atom%) / kg soil standard for soil pretreatment. 15 NH 4 ) 2 SO 4 Dissolve it in sterile water and spray it on the soil. Stir it twice a day. After continuous stirring for 10 days, sprinkle 0.04g / kg of soil with 10% clothianidin· insecticide granules to control sugarcane pests. Get pre-treated soil, divide the pre-treated soil into several buckets, and plant the sugarcane seed stems inoculated with nitrogen-fixing bacteria in 25kg of stable isotope-containing 15 In the barrels pretreated with N ammonium sulfate, 2 single bud seed stems were planted in each barrel, and 10 barrels were planted. After planting, 2L of distilled water was poured into each barrel every 2 days to maintain the soil moisture at 70% of the maximum water holding capacity.

[0039] 4. After the sugarcane seed stems have grown two leaves, pour 2L of 1 / 10MS nutrient solution into each bucket, and then pour 2L of nitrogen-free 1 / 10MS nutrient solution into each bucket every 15 days; when the sugarcane grows to 7 true leaves and begins to tiller, loosen the soil, apply 500g of nitrogen-fixing bacteria agent per bucket, loosen the soil once every 20 days, and spray 0.01mg / L molybdate solution once on the 40th, 50th and 60th days after sowing.

[0040] 5. During the peak growth period of sugarcane, when the sugarcane plant is up to 50 cm and the average growth is more than 15 cm every 15 days, take the sugarcane plant, wash it, dry the roots, stems and leaves, grind them into powder, and test the nitrogen content and 15 N isotope abundance.

[0041] Example 2

[0042] The nitrogen-fixing bacteria in this embodiment are Rhizobium sp. FSGRY3, Enterobacter sp. GG49E, and Klebsiella sp. GG160E.

[0043] A method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane in this embodiment specifically comprises the following steps:

[0044] 1. Preparation of nitrogen-fixing bacteria agent

[0045] Take nitrogen-fixing bacteria stored at -80℃, activate them on YMA solid culture medium plates for 45h, take a single colony in nitrogen-fixing bacteria activation medium, culture it at 30℃, 200rpm for 28h, then wash the bacteria twice by centrifugation with sterile water to complete the activation;

[0046] The activated nitrogen-fixing bacteria were inoculated into 100 ml of liquid nitrogen-free culture medium and cultured at 30 °C and 150 rpm / min for 24 h until the logarithmic growth phase to obtain rhizobium, Enterobacter and Klebsiella bacterial solutions. The concentration of each bacterial solution was adjusted to 1 × 10 7 CFU / mL, the adjusted concentrations of rhizobium, enterobacter and Klebsiella were inoculated at 1% inoculation amount on the sugarcane bagasse-vermiculite solid fermentation medium sterilized at 121℃ for 30min, the nitrogen-fixing bacteria and the sugarcane bagasse-vermiculite solid medium were fully mixed, the temperature was adjusted to 30℃ and the humidity was adjusted to 70%, and the fermentation was carried out for 3 days. The concentration of the detected bacteria was not less than 1×10 8 CFU / g, and evenly mixing the fermented rhizobium solution, enterobacter solution and Klebsiella solution in a weight ratio of 1:1:1 to obtain a nitrogen-fixing bacteria agent; the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage, 60% bagasse, 33.846% vermiculite, 3% sugarcane leaf biochar, 3% stone powder, 0.004% manganese sulfate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate and 0.05g / ml cobalt chloride hexahydrate; the water content of the bagasse-vermiculite solid fermentation medium is 50%.

[0047] 2. Take a single bud section of sugarcane seed stem, soak it in 52°C hot water for 30 minutes, then soak the seed stem in 500 times the amount of 50% carbendazim wettable powder diluted with water for 6 minutes, take it out and dry it, then soak it in nitrogen-fixing bacteria solution for 30 minutes to obtain the sugarcane seed stem inoculated with nitrogen-fixing bacteria agent.

[0048] 3. According to 10mg ( 15 NH4) 2 SO 4 (10.11atom%) / kg soil standard for soil pretreatment. 15 NH 4 ) 2 SO 4 Dissolve it in sterile water and spray it on the soil. Stir it twice a day. After continuous stirring for 10 days, sprinkle 0.04g / kg of soil with 10% clothianidin· insecticide granules to control sugarcane pests. Get pre-treated soil, divide the pre-treated soil into several buckets, and plant the sugarcane seed stems inoculated with nitrogen-fixing bacteria in 25kg of stable isotope-containing 15 In the barrels pretreated with N ammonium sulfate, 2 single bud seed stems were planted in each barrel, and 10 barrels were planted. After planting, 2L of distilled water was poured into each barrel every 2 days to maintain the soil moisture at 70% of the maximum water holding capacity.

[0049] 4. After the sugarcane seed stems have grown two leaves, pour 2L of 1 / 10MS nutrient solution into each bucket, and then pour 2L of nitrogen-free 1 / 10MS nutrient solution into each bucket every 15 days; when the sugarcane grows to 7 true leaves and begins to tiller, loosen the soil, apply 500g of nitrogen-fixing bacteria agent per bucket, loosen the soil once every 20 days, and spray 0.01mg / L molybdate solution once on the 40th, 50th and 60th days after sowing.

[0050] 5. During the peak growth period of sugarcane, when the sugarcane plant is up to 50 cm and the average growth is more than 15 cm every 15 days, take the sugarcane plant, wash it, dry the roots, stems and leaves, grind them into powder, and test the nitrogen content and 15 N isotope abundance.

[0051] Example 3

[0052] The nitrogen-fixing bacteria in this embodiment is Rhizobium sp. FSGRY3.

[0053] A method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane in this embodiment specifically comprises the following steps:

[0054] 1. Preparation of nitrogen-fixing bacteria agent

[0055] Take nitrogen-fixing bacteria stored at -80°C, activate them on a YMA solid culture medium plate for 45 hours, take a single colony in the nitrogen-fixing bacteria activation medium, culture it at 30°C, 200rpm for 28 hours, and then wash the bacteria twice by centrifugation with sterile water to complete the activation; the nitrogen-fixing bacteria activation medium consists of 10g sucrose, 1g soybean extract, 2g potassium dihydrogen phosphate, 0.05g ferric chloride, 0.1g magnesium sulfate, 1g mannitol, 0.05g alginate, 0.01g boric acid, 0.05g guar gum, and 1000ml distilled water.

[0056] The activated nitrogen-fixing bacteria were inoculated into 100 ml of liquid nitrogen-free culture medium and cultured at 30 °C and 150 rpm / min for 24 h until the logarithmic growth phase to obtain rhizobium bacterial solution, and the bacterial solution concentration was adjusted to 1 × 10 7 CFU / mL, the rhizobium solution after concentration adjustment was inoculated with 1% inoculation amount on the sugarcane bagasse-vermiculite solid fermentation medium sterilized at 121℃ for 30min, the nitrogen-fixing bacteria and the sugarcane bagasse-vermiculite solid medium were fully mixed, the temperature was adjusted to 30℃ and the humidity was 70%, and the fermentation was carried out for 3 days. The concentration of the detected bacteria was not less than 1×10 8 CFU / g, the nitrogen-fixing bacteria agent is obtained; the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage: 60% bagasse, 33.846% vermiculite, 3% sugarcane leaf biochar, 3% stone powder, 0.004% manganese sulfate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, and 0.05g / ml cobalt chloride hexahydrate; the water content of the bagasse-vermiculite solid fermentation medium is 50%.

[0057] 2. Take a single bud segment of sugarcane seed stem, soak it in 52°C hot water for 30 minutes, then soak the seed stem in 500 times the amount of 50% carbendazim wettable powder diluted with water for 6 minutes, take it out and dry it, then soak it in nitrogen-fixing bacteria agent for 30 minutes to obtain the sugarcane seed stem inoculated with nitrogen-fixing bacteria agent.

[0058] 3. According to 10mg ( 15 NH4) 2 SO 4 (10.11atom%) / kg soil standard for soil pretreatment. 15 NH 4 ) 2 SO 4 Dissolve it in sterile water and spray it on the soil. Stir it twice a day. After continuous stirring for 10 days, sprinkle 0.04g / kg of soil with 10% clothianidin· insecticide granules to control sugarcane pests. Get pre-treated soil, divide the pre-treated soil into several buckets, and plant the sugarcane seed stems inoculated with nitrogen-fixing bacteria in 25kg of stable isotope-containing 15In the barrels pretreated with N ammonium sulfate, 2 single bud seed stems were planted in each barrel, and 10 barrels were planted. After planting, 2L of distilled water was poured into each barrel every 2 days to maintain the soil moisture at 70% of the maximum water holding capacity.

[0059] 4. After the sugarcane seed stems have grown two leaves, pour 2L of 1 / 10MS nutrient solution into each bucket, and then pour 2L of nitrogen-free 1 / 10MS nutrient solution into each bucket every 15 days; when the sugarcane grows to 7 true leaves and begins to tiller, loosen the soil, apply 500g of nitrogen-fixing bacteria agent per bucket, loosen the soil once every 20 days, and spray 0.01mg / L molybdate solution once on the 40th, 50th and 60th days after sowing.

[0060] 5. During the peak growth period of sugarcane, when the sugarcane plant is up to 50 cm and the average growth is more than 15 cm every 15 days, take the sugarcane plant, wash it, dry the roots, stems and leaves, grind them into powder, and test the nitrogen content and 15 N isotope abundance.

[0061] Example 4

[0062] The nitrogen-fixing bacteria in this embodiment is Enterobacter sp. GG49E.

[0063] A method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane in this embodiment specifically comprises the following steps:

[0064] 1. Preparation of nitrogen-fixing bacteria agent

[0065] Take nitrogen-fixing bacteria stored at -80°C, activate them on a YMA solid culture medium plate for 45 hours, take a single colony in the nitrogen-fixing bacteria activation medium, culture it at 30°C, 200rpm for 28 hours, and then wash the bacteria twice by centrifugation with sterile water to complete the activation; the nitrogen-fixing bacteria activation medium consists of 10g sucrose, 1g soybean extract, 2g potassium dihydrogen phosphate, 0.05g ferric chloride, 0.1g magnesium sulfate, 1g mannitol, 0.05g alginate, 0.01g boric acid, 0.05g guar gum, and 1000ml distilled water.

[0066] The activated nitrogen-fixing bacteria were inoculated into 100 ml of liquid nitrogen-free culture medium and cultured at 30 °C and 150 rpm / min for 24 h until the logarithmic growth phase to obtain Enterobacter bacterial solution, and the bacterial solution concentration was adjusted to 1 × 10 7 CFU / mL, the Enterobacterium liquid after concentration adjustment was inoculated with 1% inoculation amount on the sugarcane bagasse-vermiculite solid fermentation medium sterilized at 121℃ for 30min, the nitrogen-fixing bacteria and the sugarcane bagasse-vermiculite solid medium were fully mixed, the temperature was adjusted to 30℃ and the humidity was 70%, and the fermentation was carried out for 3 days. The concentration of the detected bacteria was not less than 1×10 8CFU / g, the nitrogen-fixing bacteria agent is obtained; the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage: 60% bagasse, 33.846% vermiculite, 3% sugarcane leaf biochar, 3% stone powder, 0.004% manganese sulfate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, and 0.05g / ml cobalt chloride hexahydrate; the water content of the bagasse-vermiculite solid fermentation medium is 50%.

[0067] 2. Take a single bud segment of sugarcane seed stem, soak it in 52°C hot water for 30 minutes, then soak the seed stem in 500 times the amount of 50% carbendazim wettable powder diluted with water for 6 minutes, take it out and dry it, then soak it in nitrogen-fixing bacteria agent for 30 minutes to obtain the sugarcane seed stem inoculated with nitrogen-fixing bacteria agent.

[0068] 3. According to 10mg ( 15 NH4) 2 SO 4 (10.11atom%) / kg soil standard for soil pretreatment. 15 NH 4 ) 2 SO 4 Dissolve it in sterile water and spray it on the soil. Stir it twice a day. After continuous stirring for 10 days, sprinkle 0.04g / kg of soil with 10% clothianidin· insecticide granules to control sugarcane pests. Get pre-treated soil, divide the pre-treated soil into several buckets, and plant the sugarcane seed stems inoculated with nitrogen-fixing bacteria in 25kg of stable isotope-containing 15 In the barrels pretreated with N ammonium sulfate, 2 single bud seed stems were planted in each barrel, and 10 barrels were planted. After planting, 2L of distilled water was poured into each barrel every 2 days to maintain the soil moisture at 70% of the maximum water holding capacity.

[0069] 4. After the sugarcane seed stems have grown two leaves, pour 2L of 1 / 10MS nutrient solution into each bucket, and then pour 2L of nitrogen-free 1 / 10MS nutrient solution into each bucket every 15 days; when the sugarcane grows to 7 true leaves and begins to tiller, loosen the soil, apply 500g of nitrogen-fixing bacteria agent per bucket, loosen the soil once every 20 days, and spray 0.01mg / L molybdate solution once on the 40th, 50th and 60th days after sowing.

[0070] 5. During the peak growth period of sugarcane, when the sugarcane plant is up to 50 cm and the average growth is more than 15 cm every 15 days, take the sugarcane plant, wash it, dry the roots, stems and leaves, grind them into powder, and test the nitrogen content and 15 N isotope abundance.

[0071] Example 5

[0072] The nitrogen-fixing bacteria in this embodiment is Klebsiella sp. GG160E.

[0073] A method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane in this embodiment specifically comprises the following steps:

[0074] 1. Preparation of nitrogen-fixing bacteria agent

[0075] Take nitrogen-fixing bacteria stored at -80°C, activate them on a YMA solid culture medium plate for 45 hours, take a single colony in the nitrogen-fixing bacteria activation medium, culture it at 30°C, 200rpm for 28 hours, and then wash the bacteria twice by centrifugation with sterile water to complete the activation; the nitrogen-fixing bacteria activation medium consists of 10g sucrose, 1g soybean extract, 2g potassium dihydrogen phosphate, 0.05g ferric chloride, 0.1g magnesium sulfate, 1g mannitol, 0.05g alginate, 0.01g boric acid, 0.05g guar gum, and 1000ml distilled water.

[0076] The activated nitrogen-fixing bacteria were inoculated into 100 ml of liquid nitrogen-free culture medium and cultured at 30 °C and 150 rpm / min for 24 h until the logarithmic growth phase to obtain Klebsiella bacterial solution, which was adjusted to a concentration of 1 × 10 7 CFU / mL, the Klebsiella liquid after adjusting the concentration was inoculated with 1% inoculation amount on the sugarcane bagasse-vermiculite solid fermentation medium sterilized at 121℃ for 30min, the nitrogen-fixing bacteria and the sugarcane bagasse-vermiculite solid medium were fully mixed, the temperature was adjusted to 30℃ and the humidity was 70%, and the fermentation was carried out for 3 days. The concentration of the detected bacteria was not less than 1×10 8 CFU / g, the nitrogen-fixing bacteria agent is obtained; the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage: 60% bagasse, 33.846% vermiculite, 3% sugarcane leaf biochar, 3% stone powder, 0.004% manganese sulfate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, and 0.05g / ml cobalt chloride hexahydrate; the water content of the bagasse-vermiculite solid fermentation medium is 50%.

[0077] 2. Take a single bud segment of sugarcane seed stem, soak it in 52°C hot water for 30 minutes, then soak the seed stem in 500 times the amount of 50% carbendazim wettable powder diluted with water for 6 minutes, take it out and dry it, then soak it in nitrogen-fixing bacteria agent for 30 minutes to obtain the sugarcane seed stem inoculated with nitrogen-fixing bacteria agent.

[0078] 3. According to 10mg ( 15 NH4) 2 SO 4 (10.11atom%) / kg soil standard for soil pretreatment. 15 NH 4 ) 2 SO 4Dissolve it in sterile water and spray it on the soil. Stir it twice a day. After continuous stirring for 10 days, sprinkle 0.04g / kg of soil with 10% clothianidin· insecticide granules to control sugarcane pests. Get pre-treated soil, divide the pre-treated soil into several buckets, and plant the sugarcane seed stems inoculated with nitrogen-fixing bacteria in 25kg of stable isotope-containing 15 In the barrels pretreated with N ammonium sulfate, 2 single bud seed stems were planted in each barrel, and 10 barrels were planted. After planting, 2L of distilled water was poured into each barrel every 2 days to maintain the soil moisture at 70% of the maximum water holding capacity.

[0079] 4. After the sugarcane seed stems have grown two leaves, pour 2L of 1 / 10MS nutrient solution into each bucket, and then pour 2L of nitrogen-free 1 / 10MS nutrient solution into each bucket every 15 days; when the sugarcane grows to 7 true leaves and begins to tiller, loosen the soil, apply 500g of nitrogen-fixing bacteria agent per bucket, loosen the soil once every 20 days, and spray 0.01mg / L molybdate solution once on the 40th, 50th and 60th days after sowing.

[0080] 5. During the peak growth period of sugarcane, when the sugarcane plant is up to 50 cm and the average growth is more than 15 cm every 15 days, take the sugarcane plant, wash it, dry the roots, stems and leaves, grind them into powder, and test the nitrogen content and 15 N isotope abundance.

[0081] Example 6

[0082] The nitrogen-fixing bacteria in this embodiment include Rhizobium sp. FSGRY3 and Enterobacter sp. GG49E.

[0083] A method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane in this embodiment specifically comprises the following steps:

[0084] 1. Preparation of nitrogen-fixing bacteria agent

[0085] Take nitrogen-fixing bacteria stored at -80°C, activate them on a YMA solid culture medium plate for 45 hours, take a single colony in the nitrogen-fixing bacteria activation medium, culture it at 30°C, 200rpm for 28 hours, and then wash the bacteria twice by centrifugation with sterile water to complete the activation; the nitrogen-fixing bacteria activation medium consists of 10g sucrose, 1g soybean extract, 2g potassium dihydrogen phosphate, 0.05g ferric chloride, 0.1g magnesium sulfate, 1g mannitol, 0.05g alginate, 0.01g boric acid, 0.05g guar gum, and 1000ml distilled water.

[0086] The activated nitrogen-fixing bacteria were inoculated into 100 ml of liquid nitrogen-free culture medium and cultured at 30 °C and 150 rpm / min for 24 h until the logarithmic growth phase to obtain rhizobium and enterobacterial cultures. The concentration of each culture was adjusted to 1 × 10 7CFU / mL, the adjusted concentrations of rhizobium and enterobacterium were inoculated at 1% on the sugarcane bagasse-vermiculite solid fermentation medium sterilized at 121°C for 30 min, the nitrogen-fixing bacteria were fully mixed with the sugarcane bagasse-vermiculite solid medium, the temperature was adjusted to 30°C and the humidity was adjusted to 70%, and the fermentation was carried out for 3 days. The concentration of the detected bacteria was not less than 1×10 8 CFU / g, and evenly mixing the fermented rhizobium bacterial solution and the enterobacterial bacterial solution in a weight ratio of 1:1 to obtain a nitrogen-fixing bacteria agent; the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage, 60% bagasse, 33.846% vermiculite, 3% sugarcane leaf biochar, 3% stone powder, 0.004% manganese sulfate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, and 0.05g / ml cobalt chloride hexahydrate; the water content of the bagasse-vermiculite solid fermentation medium is 50%.

[0087] 2. Take a single bud segment of sugarcane seed stem, soak it in 52°C hot water for 30 minutes, then soak the seed stem in 500 times the amount of 50% carbendazim wettable powder diluted with water for 6 minutes, take it out and dry it, then soak it in nitrogen-fixing bacteria agent for 30 minutes to obtain the sugarcane seed stem inoculated with nitrogen-fixing bacteria agent.

[0088] 3. According to 10mg ( 15 NH4) 2 SO 4 (10.11atom%) / kg soil standard for soil pretreatment. 15 NH 4 ) 2 SO 4 Dissolve it in sterile water and spray it on the soil. Stir it twice a day. After continuous stirring for 10 days, sprinkle 0.04g / kg of soil with 10% clothianidin· insecticide granules to control sugarcane pests. Get pre-treated soil, divide the pre-treated soil into several buckets, and plant the sugarcane seed stems inoculated with nitrogen-fixing bacteria in 25kg of stable isotope-containing 15 In the barrels pretreated with N ammonium sulfate, 2 single bud seed stems were planted in each barrel, and 10 barrels were planted. After planting, 2L of distilled water was poured into each barrel every 2 days to maintain the soil moisture at 70% of the maximum water holding capacity.

[0089] 4. After the sugarcane seed stems have grown two leaves, pour 2L of 1 / 10MS nutrient solution into each bucket, and then pour 2L of nitrogen-free 1 / 10MS nutrient solution into each bucket every 15 days; when the sugarcane grows to 7 true leaves and begins to tiller, loosen the soil, apply 500g of nitrogen-fixing bacteria agent per bucket, loosen the soil once every 20 days, and spray 0.01mg / L molybdate solution once on the 40th, 50th and 60th days after sowing.

[0090] 5. During the peak growth period of sugarcane, when the sugarcane plant is up to 50 cm and the average growth is more than 15 cm every 15 days, take the sugarcane plant, wash it, dry the roots, stems and leaves, grind them into powder, and test the nitrogen content and 15 N isotope abundance.

[0091] Example 7

[0092] The nitrogen-fixing bacteria in this embodiment include Rhizobium sp. FSGRY3 and Klebsiella sp. GG160E.

[0093] A method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane in this embodiment specifically comprises the following steps:

[0094] 1. Preparation of nitrogen-fixing bacteria agent

[0095] Take nitrogen-fixing bacteria stored at -80°C, activate them on a YMA solid culture medium plate for 45 hours, take a single colony in the nitrogen-fixing bacteria activation medium, culture it at 30°C, 200rpm for 28 hours, and then wash the bacteria twice by centrifugation with sterile water to complete the activation; the nitrogen-fixing bacteria activation medium consists of 10g sucrose, 1g soybean extract, 2g potassium dihydrogen phosphate, 0.05g ferric chloride, 0.1g magnesium sulfate, 1g mannitol, 0.05g alginate, 0.01g boric acid, 0.05g guar gum, and 1000ml distilled water.

[0096] The activated nitrogen-fixing bacteria were inoculated into 100 ml of liquid nitrogen-free culture medium and cultured at 30 °C and 150 rpm / min for 24 h until the logarithmic growth phase to obtain rhizobium and Klebsiella bacterial solutions. The concentration of each bacterial solution was adjusted to 1 × 10 7 CFU / mL, the Rhizobium and Klebsiella solutions after concentration adjustment were inoculated at 1% inoculation amount on the sugarcane bagasse-vermiculite solid fermentation medium sterilized at 121℃ for 30min, the nitrogen-fixing bacteria and the sugarcane bagasse-vermiculite solid medium were fully mixed, the temperature was adjusted to 30℃ and the humidity was adjusted to 70%, and the fermentation was carried out for 3 days. The concentration of the detected bacteria was not less than 1×10 8 CFU / g, and evenly mixing the fermented rhizobium solution and the Klebsiella solution in a weight ratio of 1:1 to obtain a nitrogen-fixing bacteria agent; the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage, 60% bagasse, 33.846% vermiculite, 3% sugarcane leaf biochar, 3% stone powder, 0.004% manganese sulfate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, and 0.05g / ml cobalt chloride hexahydrate; the water content of the bagasse-vermiculite solid fermentation medium is 50%.

[0097] 2. Take a single bud segment of sugarcane seed stem, soak it in 52°C hot water for 30 minutes, then soak the seed stem in 500 times the amount of 50% carbendazim wettable powder diluted with water for 6 minutes, take it out and dry it, then soak it in nitrogen-fixing bacteria agent for 30 minutes to obtain the sugarcane seed stem inoculated with nitrogen-fixing bacteria agent.

[0098] 3. According to 10mg ( 15 NH4) 2 SO 4 (10.11atom%) / kg soil standard for soil pretreatment. 15 NH 4 ) 2 SO 4 Dissolve it in sterile water and spray it on the soil. Stir it twice a day. After continuous stirring for 10 days, sprinkle 0.04g / kg of soil with 10% clothianidin· insecticide granules to control sugarcane pests. Get pre-treated soil, divide the pre-treated soil into several buckets, and plant the sugarcane seed stems inoculated with nitrogen-fixing bacteria in 25kg of stable isotope-containing 15 In the barrels pretreated with N ammonium sulfate, 2 single bud seed stems were planted in each barrel, and 10 barrels were planted. After planting, 2L of distilled water was poured into each barrel every 2 days to maintain the soil moisture at 70% of the maximum water holding capacity.

[0099] 4. After the sugarcane seed stems have grown two leaves, pour 2L of 1 / 10MS nutrient solution into each bucket, and then pour 2L of nitrogen-free 1 / 10MS nutrient solution into each bucket every 15 days; when the sugarcane grows to 7 true leaves and begins to tiller, loosen the soil, apply 500g of nitrogen-fixing bacteria agent per bucket, loosen the soil once every 20 days, and spray 0.01mg / L molybdate solution once on the 40th, 50th and 60th days after sowing.

[0100] 5. During the peak growth period of sugarcane, when the sugarcane plant is up to 50 cm and the average growth is more than 15 cm every 15 days, take the sugarcane plant, wash it, dry the roots, stems and leaves, grind them into powder, and test the nitrogen content and 15 N isotope abundance.

[0101] Example 8

[0102] The nitrogen-fixing bacteria in this embodiment include Enterobacter sp. GG49E and Klebsiella sp. GG160E.

[0103] A method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane in this embodiment specifically comprises the following steps:

[0104] 1. Preparation of nitrogen-fixing bacteria agent

[0105] Take nitrogen-fixing bacteria stored at -80°C, activate them on a YMA solid culture medium plate for 45 hours, take a single colony in the nitrogen-fixing bacteria activation medium, culture it at 30°C, 200rpm for 28 hours, and then wash the bacteria twice by centrifugation with sterile water to complete the activation; the nitrogen-fixing bacteria activation medium consists of 10g sucrose, 1g soybean extract, 2g potassium dihydrogen phosphate, 0.05g ferric chloride, 0.1g magnesium sulfate, 1g mannitol, 0.05g alginate, 0.01g boric acid, 0.05g guar gum, and 1000ml distilled water.

[0106] The activated nitrogen-fixing bacteria were inoculated into 100 ml of liquid nitrogen-free culture medium and cultured at 30 °C and 150 rpm / min for 24 h until the logarithmic growth phase to obtain Enterobacter and Klebsiella bacterial liquids. The concentration of each bacterial liquid was adjusted to 1 × 10 7 CFU / mL, the Enterobacter and Klebsiella solutions after concentration adjustment were inoculated at 1% inoculation amount on the sugarcane bagasse-vermiculite solid fermentation medium sterilized at 121℃ for 30min, the nitrogen-fixing bacteria and the sugarcane bagasse-vermiculite solid medium were fully mixed, the temperature was adjusted to 30℃ and the humidity was adjusted to 70%, and the fermentation was carried out for 3 days. The concentration of the detected bacteria was not less than 1×10 8 CFU / g, and evenly mixing the fermented Enterobacter bacterial solution and Klebsiella bacterial solution in a weight ratio of 1:1 to obtain a nitrogen-fixing bacteria agent; the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage, 60% bagasse, 33.846% vermiculite, 3% sugarcane leaf biochar, 3% stone powder, 0.004% manganese sulfate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, and 0.05g / ml cobalt chloride hexahydrate; the water content of the bagasse-vermiculite solid fermentation medium is 50%.

[0107] 2. Take a single bud segment of sugarcane seed stem, soak it in 52°C hot water for 30 minutes, then soak the seed stem in 500 times the amount of 50% carbendazim wettable powder diluted with water for 6 minutes, take it out and dry it, then soak it in nitrogen-fixing bacteria agent for 30 minutes to obtain the sugarcane seed stem inoculated with nitrogen-fixing bacteria agent.

[0108] 3. According to 10mg ( 15 NH4) 2 SO 4 (10.11atom%) / kg soil standard for soil pretreatment. 15 NH 4 ) 2 SO 4 Dissolve it in sterile water and spray it on the soil. Stir it twice a day. After continuous stirring for 10 days, sprinkle 0.04g / kg of soil with 10% clothianidin· insecticide granules to control sugarcane pests. Get pre-treated soil, divide the pre-treated soil into several buckets, and plant the sugarcane seed stems inoculated with nitrogen-fixing bacteria in 25kg of stable isotope-containing15 In the barrels pretreated with N ammonium sulfate, 2 single bud seed stems were planted in each barrel, and 10 barrels were planted. After planting, 2L of distilled water was poured into each barrel every 2 days to maintain the soil moisture at 70% of the maximum water holding capacity.

[0109] 4. After the sugarcane seed stems have grown two leaves, pour 2L of 1 / 10MS nutrient solution into each bucket, and then pour 2L of nitrogen-free 1 / 10MS nutrient solution into each bucket every 15 days; when the sugarcane grows to 7 true leaves and begins to tiller, loosen the soil, apply 500g of nitrogen-fixing bacteria agent per bucket, loosen the soil once every 20 days, and spray 0.01mg / L molybdate solution once on the 40th, 50th and 60th days after sowing.

[0110] 5. During the peak growth period of sugarcane, when the sugarcane plant is up to 50 cm and the average growth is more than 15 cm every 15 days, take the sugarcane plant, wash it, dry the roots, stems and leaves, grind them into powder, and test the nitrogen content and 15 N isotope abundance.

[0111] Comparative Example 1

[0112] The difference from Example 1 is that the activation method of nitrogen-fixing bacteria is: take nitrogen-fixing bacteria stored at -80°C, culture them in YMA liquid culture medium for 45 hours, and then wash the bacteria twice by centrifugation with sterile water to complete the activation. The other conditions are the same as in Example 1.

[0113] Comparative Example 2

[0114] The difference from Example 1 is that the nitrogen-fixing bacteria activation medium consists of 10 g sucrose, 1 g soybean extract, 2 g potassium dihydrogen phosphate, 0.05 g ferric chloride, 0.1 g magnesium sulfate, 0.05 g alginate, 0.01 g boric acid, and 1000 ml distilled water, and the other conditions are the same as in Example 1.

[0115] Comparative Example 3

[0116] The difference from Example 1 is that the nitrogen-fixing bacteria activation medium consists of 10 g sucrose, 1 g soybean extract, 2 g potassium dihydrogen phosphate, 0.05 g ferric chloride, 0.1 g magnesium sulfate, 1 g mannitol, 0.05 g guar gum, and 1000 ml distilled water, and the other conditions are the same as those in Example 1.

[0117] Comparative Example 4

[0118] The difference from Example 1 is that the nitrogen-fixing bacteria activation medium consists of 10 g sucrose, 1 g soybean extract, 2 g potassium dihydrogen phosphate, 0.05 g ferric chloride, 0.1 g magnesium sulfate, 1 g mannitol, 0.06 g humic acid, 0.05 g guar gum, and 1000 ml distilled water, and the other conditions are the same as those in Example 1.

[0119] Comparative Example 5

[0120] The difference from Example 1 is that the nitrogen-fixing bacteria activation medium consists of 10 g sucrose, 1 g yeast powder, 2 g potassium dihydrogen phosphate, 0.05 g ferric chloride, 0.1 g magnesium sulfate, 1 g mannitol, 0.05 g alginate, 0.01 g boric acid, 0.05 g guar gum, and 1000 ml distilled water, and the other conditions are the same as those in Example 1.

[0121] Comparative Example 6

[0122] The difference from Example 1 is that the nitrogen-fixing bacteria activation medium consists of 10 g sucrose, 2 g potassium dihydrogen phosphate, 0.05 g ferric chloride, 0.1 g magnesium sulfate, 1 g mannitol, 0.05 g alginate, 0.01 g boric acid, 0.05 g guar gum, and 1000 ml distilled water, and the other conditions are the same as those in Example 1.

[0123] Comparative Example 7

[0124] The difference from Example 1 is that the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage, 45% glucose, 34% vermiculite, 15% agar, 3% calcium sulfate, 2.85% stone powder, 0.005% manganese sulfate, 0.1% dipotassium hydrogen phosphate, 0.045% magnesium sulfate, and 0.05 g / ml cobalt chloride hexahydrate, and the other conditions are the same as those in Example 1.

[0125] Comparative Example 8

[0126] The difference from Example 1 is that the formula of the bagasse-vermiculite solid fermentation medium includes, by weight percentage, 60% bagasse, 34% vermiculite, 3% sugarcane leaf biochar, 2.85% stone powder, 0.005% manganese sulfate, 0.1% dipotassium hydrogen phosphate, and 0.045% magnesium sulfate, and the other conditions are the same as in Example 1.

[0127] Comparison Example

[0128] 1. Take a single bud section of sugarcane seed stem, soak it in 52℃ hot water for 30min, then soak the stem in 500 times diluted 50% carbendazim wettable powder for 6min, take it out, dry it, and soak it in sterile water for 30min to obtain the sterilized sugarcane seed stem.

[0129] 2. According to 10mg ( 15 NH4) 2 SO 4 (10.11atom%) / kg soil standard for soil pretreatment. 15 NH 4 ) 2 SO 4Dissolve it in sterile water and spray it on the soil. Stir it twice a day. After continuous stirring for 10 days, sprinkle 0.04g / kg of soil with 10% clothianidin· insecticide granules to control sugarcane pests to obtain pre-treated soil. Dispense the pre-treated soil into several barrels and plant the sterilized sugarcane seed stems in 25kg of barrels containing stable isotopes. 15 In the barrels pretreated with N ammonium sulfate, 2 single bud seed stems were planted in each barrel, and 10 barrels were planted. After planting, 2L of distilled water was poured into each barrel every 2 days to maintain the soil moisture at 70% of the maximum water holding capacity.

[0130] 3. After the sugarcane seed stems emerge and grow two leaves, pour 2L of 1 / 10MS nutrient solution into each bucket, and then pour 2L of nitrogen-free 1 / 10MS nutrient solution into each bucket every 15 days; when the sugarcane grows 7 true leaves and begins to tiller, loosen the soil, and then loosen the soil every 20 days, and spray 0.01mg / L molybdate solution once on the 40th, 50th and 60th days after sowing.

[0131] 4. During the peak growth period of sugarcane, when the sugarcane plant is up to 50cm and the average growth is more than 15cm every 15 days, take the sugarcane plant, wash it, dry the roots, stems and leaves, grind them into powder, and test the nitrogen content and 15 N isotope abundance. Nitrogen fixation efficiency is based on the formula Ndfa = 100 × [1-(inoculated plants 15 N isotope abundance / control plants 15 N isotope abundance)] calculation.

[0132] Detection of nitrogen content in sugarcane and 15 The N isotope abundance is shown in the table below.

[0133]

[0134]

[0135] As can be seen from the table above, compared with the uninoculated control, the single inoculation of three nitrogen-fixing bacteria and the mixed inoculation of RB928064 sugarcane with nitrogen-fixing bacteria can increase the nitrogen content of sugarcane plants, among which the nitrogen content of the mixed inoculation of three nitrogen-fixing bacteria increased by 20.90%. Compared with the uninoculated control, the single inoculation of Rhizobium sp.FSGRY3, the mixed inoculation of Rhizobium sp.FSGRY3+Enterobactersp.GG49E, the mixed inoculation of Rhizobium sp.FSGRY3+Klebsiella sp.GG160E, and the mixed inoculation of three nitrogen-fixing bacteria can all improve the nitrogen fixation efficiency of sugarcane, and the difference is significant at the 5% confidence level. Among them, the nitrogen fixation efficiency of the mixed inoculation of three nitrogen-fixing bacteria is the highest, which is 67.67% higher than that of the plants inoculated with a single nitrogen-fixing bacteria, and the total nitrogen content is 20.90% higher than that of the uninoculated plants. The three nitrogen-fixing strains co-inoculated in sugarcane produced a synergistic effect and improved the nitrogen fixation efficiency of sugarcane.

[0136] The present invention inoculates sugarcane seed stems with a nitrogen-fixing bacteria combination of rhizobium sp. FSGRY3, Enterobacter sp. GG49E and Klebsiella sp. GG160E. The inventor finds that Enterobacter sp. GG49E has strong adaptability and metabolic diversity, can play a nitrogen-fixing role under different environmental conditions, and increases the stability and reliability of nitrogen fixation. Klebsiella sp. GG160E has efficient nitrogenase activity and can quickly convert nitrogen in the air into a nitrogen form that can be used by sugarcane. The three bacteria synergize to improve the nitrogen fixation efficiency of sugarcane, reduce the dependence of sugarcane on chemical nitrogen fertilizers, reduce agricultural production costs, and reduce the pollution of chemical fertilizers to the environment.

[0137] Activating nitrogen-fixing bacteria separately before culturing can ensure the activity and growth ability of the bacteria. Alginate and boric acid in the nitrogen-fixing bacteria activation medium can passivate a variety of heavy metal elements, reduce their toxic side effects, and have a certain inhibitory effect on nitrates. They can improve the tolerance of crops to drought, high temperature, salt stress, oxidation, etc., and have a strong effect on repairing crop diseases; mannitol and guar gum compound can buffer and protect nitrogen-fixing bacteria and improve the survival rate of nitrogen-fixing bacteria in the soil; soybean extract contains some substances that can chelate metal ions, such as organic acids and polypeptides. Nitrogenase is an enzyme containing metal ions (such as iron, molybdenum, etc.). The presence of metal ions is essential for the activity of nitrogenase. Organic acids, polypeptides, etc. in soybean extracts can combine with metal ions to form stable complexes to prevent metal ions from precipitating or being adsorbed by other substances, thereby ensuring that nitrogenase can obtain sufficient metal ions to maintain its activity. Antioxidant effect: Nitrogenase is sensitive to oxygen and easily inactivated by oxidation. Soybean extract contains some antioxidants, such as phenolic compounds, flavonoids, etc. These antioxidants can scavenge free radicals and reduce the damage of oxidative stress to nitrogenase, thereby protecting the activity of nitrogen-fixing bacteria.

[0138] Bagasse-vermiculite solid fermentation medium recycles bagasse and sugarcane leaf biochar resources. Bagasse contains rich organic substances such as cellulose, hemicellulose and lignin, which can be used as carbon sources for the growth of nitrogen-fixing bacteria. During the growth process, nitrogen-fixing bacteria use these carbon sources for metabolic activities and synthesize the substances they need to achieve growth and reproduction; sugarcane leaf biochar has a large specific surface area and rich pore structure, which can adsorb nutrients and water in the culture medium. This adsorption can prevent the loss of nutrients, maintain the stability of nutrients in the culture medium, and provide a continuous supply of nutrients for nitrogen-fixing bacteria. At the same time, sugarcane leaf biochar can also improve the physical structure of the soil, increase the water retention and air permeability of the soil, and improve the fertility and sustainability of the soil; at the same time, the introduction of trace elements such as cobalt can promote the growth and reproduction of nitrogen-fixing bacteria after separation and accelerate the reproduction rate of nitrogen-fixing bacteria.

[0139] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane, characterized in that: include: The sugarcane seed stems are inoculated with nitrogen-fixing bacteria, and the inoculated sugarcane seed stems are planted; the nitrogen-fixing bacteria are one or more of Rhizobium sp. FSGRY3, Enterobacter sp. GG49E, and Klebsiella sp. GG160E.

2. The method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane according to claim 1, characterized in that: The nitrogen-fixing bacteria are Rhizobium sp. FSGRY3, Enterobacter sp. GG49E, and Klebsiella sp. GG160E.

3. The method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane according to claim 1, characterized in that: The method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane comprises: (1) A single bud section of sugarcane seed stem is taken, soaked in hot water at 50-55° C. for 25-35 minutes, then soaked in a 50% carbendazim wettable powder diluted with 500-600 times water for 5-6 minutes, taken out and dried, and then soaked in a nitrogen-fixing bacteria solution for 30 minutes to obtain a sugarcane seed stem; the preparation method of the nitrogen-fixing bacteria solution is as follows: activate on a YMA solid culture medium plate for 40-50 hours, take a single colony and culture it in a YMA liquid culture medium for 24-36 hours, wash the bacteria with sterile water by centrifugation for 2-3 times, and adjust the concentration of the nitrogen-fixing bacteria solution to 1-2×10 8 CFU / ml. (2) According to 10mg ( 15 The soil was pretreated with the standard of NH4)2SO4 (10.11atom%) / kg soil, and sugarcane seed stems were planted in the pretreated soil; (3) After the sugarcane seed stems have sprouted and grown 2-3 leaves, pour 1 / 10MS nutrient solution into the soil; (4) When the sugarcane has grown 7-8 true leaves and begins to tiller, loosen the soil and apply nitrogen-fixing bacteria. Loosen the soil once every 20 days and spray 0.01 mg / L molybdate solution once on the 40th, 50th and 60th days after sowing.

4. The method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane according to claim 1, characterized in that: The preparation method of the nitrogen-fixing bacteria agent is as follows: the nitrogen-fixing bacteria are activated and then inoculated into a nitrogen-free culture medium for cultivation to obtain a rhizobium bacterial solution, an enterobacterial bacterial solution, and a Klebsiella bacterial solution, and the concentration of each bacterial solution is adjusted to 1×10 7 CFU / mL, the Rhizobium solution, Enterobacter solution and Klebsiella solution after adjusting the concentration were inoculated into the sugarcane bagasse-vermiculite solid fermentation medium respectively, the strains were mixed evenly with the sugarcane bagasse-vermiculite solid medium and then fermented, and the strain concentration was detected to be no less than 1×10 8 CFU / g, and the fermented rhizobium solution, enterobacterium solution and Klebsiella solution are mixed evenly to obtain the nitrogen-fixing bacteria agent.

5. The method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane according to claim 4, characterized in that: The formula of the bagasse-vermiculite solid fermentation culture medium comprises, by weight percentage, 58-62% bagasse, 32-36% vermiculite, 2.9-3.2% sugarcane leaf biochar, 2.6-3.0% stone powder, 0.003-0.005% manganese sulfate, 0.06-0.14% dipotassium hydrogen phosphate, and 0.037-0.055% magnesium sulfate. The water content of the bagasse-vermiculite solid fermentation culture medium is 48-53%.

6. The method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane according to claim 5, characterized in that: The formula of the bagasse-vermiculite solid fermentation medium comprises, by weight percentage, 60% bagasse, 33.846% vermiculite, 3% sugarcane leaf biochar, 3% stone powder, 0.004% manganese sulfate, 0.1% dipotassium hydrogen phosphate, and 0.05% magnesium sulfate, and the water content of the bagasse-vermiculite solid fermentation medium is 50%.

7. The method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane according to claim 4, characterized in that: The mixing weight ratio of the fermented rhizobium solution, enterobacter solution and Klebsiella solution in the nitrogen-fixing bacteria agent is 0.8-1.2:0.8-1.2:0.8-1.

2.

8. The method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane according to claim 4, characterized in that: The mixing weight ratio of the fermented rhizobium solution, the enterobacterium solution and the Klebsiella solution in the nitrogen-fixing bacteria agent is 1:1:

1.

9. The method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane according to claim 4, characterized in that: The activation method of the nitrogen-fixing bacteria is as follows: taking nitrogen-fixing bacteria stored at -80°C, activating them on a YMA solid culture medium plate for 40-50 hours, taking a single colony in a nitrogen-fixing bacteria activation medium, culturing at 28-32°C, 200-220rpm for 24-36 hours, and then washing the bacteria with sterile water by centrifugation for 2-3 times to complete the activation; The nitrogen-fixing bacteria activation culture medium consists of 10g sucrose, 1g soybean extract, 2g potassium dihydrogen phosphate, 0.05g ferric chloride, 0.1g magnesium sulfate, 1g mannitol, 0.05g alginic acid, 0.01g boric acid, 0.05g guar gum and 1000ml distilled water.

10. The method for improving nitrogen fixation by nitrogen-fixing bacteria and sugarcane according to claim 4, characterized in that: The bagasse-vermiculite solid fermentation medium also includes 0.05 g / ml of cobalt chloride hexahydrate.

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