CN111849815B - Plant growth promoting rhizosphere strain Gxun-20 and application thereof in plant growth promotion - Google Patents
Plant growth promoting rhizosphere strain Gxun-20 and application thereof in plant growth promotion Download PDFInfo
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Abstract
The invention provides a plant rhizosphere growth promoting strain Gxun-20 and application thereof in plant growth promotion, wherein a strain with efficient IAA secretion and phosphorus dissolving growth promoting capability is screened from mangrove rhizosphere soil through an ADF screening culture medium and an LB purification culture medium, 16S rRNA and physiological and biochemical characteristics of the strain are measured, the strain is determined to be brevibacillus brevis which is gram-positive bacterium, and the colony is round, smooth, convex and white. And 6, 13 days in 2019, and is preserved in Guangdong province microbial strain collection center. And optimizing the fermentation condition of Gxun-20, and performing a growth promotion test on the plant by using the bacterial liquid obtained by fermenting the Gxun-20. Compared with other strains, the strain Gxun-20 has higher IAA yield, and can be used for promoting the growth and development of plants, thereby effectively reducing the use of chemical fertilizers and the damage of the fertilizers to soil.
Description
Technical Field
The invention belongs to the technical field of plant growth promotion, and particularly relates to a plant rhizosphere growth promoting strain Gxun-20 and application thereof in plant growth promotion.
Background
According to research, the excessive use of the fertilizer causes heavy metal pollution of soil, reduces the biological activity of the soil, imbalances the pH value of the soil, soil hardening and the like, which are adverse to the healthy growth of crops. The heavy metal content of crops is seriously exceeded due to the influence, and the health of human beings is threatened when the human beings eat grains containing a large amount of heavy metals. In order to reduce the use of harmful fertilizers and to make human food healthy, it is necessary to introduce and research more healthful and effective fertilizers.
The plant growth promoting rhizobacteria is a beneficial bacteria capable of secreting surfactant, plant growth hormone such as 3-indoleacetic acid and the like. Part of the PGPR strains can not only synthesize the plant hormone IAA, but also promote the growth and development of plants. The soil components can be improved by utilizing the action mechanism of the plant rhizosphere growth-promoting bacteria, so that the soil fertility is more and more suitable for the growth of plants, and the efficiency of plant restoration is further improved.
The microbial fertilizer is prepared by utilizing PGPR (microbial growth promoting bacterium) bacteria, so that the use efficiency of soil is improved, and the problems of soil hardening and the like caused by chemical fertilizers are reduced. It has been found that such PGPR strains include Agrobacterium, bacillus, rhizobium, pseudomonas, and the like. Several studies have shown that PGPR has a promoting effect on plant growth. If plant growth-promoting bacteria are found in vegetarian plumBacillus.spYM-1 passivates soil for Pb andpromoting the growth of rape; wang Qianlong et al found that seven biocontrol bacteria can promote the growth of corn; tan Danyong et al found that the growth-promoting bacteria of ramie had the effect of promoting the growth of ramie. Researches also show that plant rhizosphere growth-promoting bacteria also have a repairing effect on heavy metal pollution of soil, for example, pan Fengshan researches find that rhodiola sachalinensis growth-promoting bacteria can improve cadmium extraction of plants, so that the effect of repairing soil is achieved; yang Yanan separates rhizosphere microorganisms from tomato rhizosphere, researches the growth promoting effect of the rhizosphere microorganisms on tomatoes, and improves the problems of soil fertility reduction, aggravation of soil-borne diseases, autotoxic substance accumulation and the like.
At present, the microbial fertilizer in China has the problems of low purity of bacterial strains, weak targeted growth promotion effect, unstable effect and the like. It is also necessary to purify the microorganisms around the plant rhizosphere soil in large quantities and to optimize the culture conditions.
Disclosure of Invention
Aiming at the problems of the microbial fertilizer in China at present, the invention provides a plant rhizosphere growth promoting strain Gxun-20 and application thereof in plant growth promotion, which can be used for promoting plant growth.
The invention is realized by the following technical scheme:
a plant growth-promoting rhizosphere strain Gxun-20, wherein the plant growth-promoting rhizosphere strain Gxun-20 is Bacillus brevis and is deposited in Guangdong province microorganism culture collection center, and the address is as follows: no. 59 building No. 5 of the first 100 college of the first furious Zhonglu, guangzhou, guangdong province, china, the name of classificationBrevibacillus parabreivsThe preservation number is GDMCC NO:60686 with a preservation time of 2019, 6 months and 13 days.
Furthermore, the plant growth-promoting rhizobacteria Gxun-20 is gram-positive, and the colony morphology is round, smooth, convex and white.
Meanwhile, the invention also provides application of the plant rhizosphere growth promoting strain Gxun-20 in plant growth promotion, which comprises the following steps:
(1) Selecting plump, undamaged and good-quality seeds, firstly soaking in warm water at 50 ℃ for 10min, pouring out, then soaking in sterile water for 8h, and finally using mass concentrationIs 10% of Na 3 PO 4 Soaking the seeds in the solution for 10-15 min; washing the soaked seeds with clear water, removing water, placing in a culture dish paved with paper towels, and spraying sterile water for soaking; finally, placing the seeds in a dark and ventilated constant-temperature incubator at 25 ℃, spraying sterile water every day to keep the seeds moist until most of the seeds are exposed to the white, and obtaining seeds accelerating germination;
(2) Mixing greenhouse surface soil, sawdust and organic fertilizer according to a ratio of 1; firstly, thoroughly watering the seedling culture nutrient soil with sterile water, then inoculating the seeds accelerating germination into the seedling culture nutrient soil, covering a layer of thin seedling culture nutrient soil, and finally paving a transparent mulching film, and timely tearing off the transparent mulching film when 75-80% of the seedlings break the soil to come out, so that the seedlings smoothly undergo photosynthesis;
(3) When 3-4 leaves grow out from the seedlings and the growth state is stable, 10mL of zymogen liquid diluted by 5-10 times is poured into each seedling in a liquid bacterial root pouring mode, the zymogen liquid is poured once every 7 days, and the treatment is carried out for 4-5 times.
The rhizobacteria screened by the invention have higher IAA yield than other strains, because R 2 Liquid A media may not be the optimal fermentation media for this strain, so media optimization is required. Optimization of IAA-producing media has been studied in many cases from the viewpoints of fermentation time, pH, carbon source, nitrogen source, inorganic salts, temperature, and the like. The invention firstly determines that the fermentation time of the strain is 26-40 h, and the IAA yield of the strain is 27.50mg/L. And when the strain selects corn steep liquor as a nitrogen source and the concentration is 0.05% (w/v), the strain produces IAA up to 65.16 mg/L. Thus, the strains screened by the invention have the potential to be developed into microbial fertilizers.
As a further improvement of the application of the plant rhizosphere growth promoting strain Gxun-20 in plant growth promotion, the fermentation method of the zymophyte liquid comprises the following steps,
(1) Inoculating plant growth promoting rhizosphere strain Gxun-20 into LB liquid culture medium, shaking culturing at constant temperature of 30 deg.C in 200r/min shaking table for activation, and measuring OD of culture solution every 2h 600 Value, plotPreparing a growth curve;
(2) R after sterilization 2 Adding 2 mg/L-tryptophan into the liquid culture medium A, adjusting the initial pH to 6.0-6.5, then inoculating the activated plant rhizosphere growth-promoting strain Gxun-20 in the logarithmic growth phase according to the inoculation amount of 5 mu L/mL culture medium, and carrying out oscillation fermentation for 26-40 h in a shaker at 25-30 ℃ at 200r/min to obtain zymogen liquid;
as a further improvement of the application of the plant rhizosphere growth promoting strain Gxun-20 in plant growth promotion, R is 2 The formula of the liquid culture medium A is as follows:
glucose 0.5g, yeast powder 0.5g, casamino acid 0.5g, K 2 HPO 4 0.1 to 0.4g of sodium pyruvate, 0.3g of sodium pyruvate, 0.5g of tryptone, 0.05 to 0.5g of soluble starch, 1.5 to 2.5mg of L-tryptophan and 0.05g of magnesium sulfate heptahydrate, dissolving the mixture in 1000mL of water, and dissolving the mixture in K 2 HPO 4 With KH 2 PO 4 Adjusting the pH value to 6.0-6.5, and sterilizing for 20min at 121 ℃.
Or, said R 2 The formula of the liquid culture medium A is as follows:
0.4g of sucrose, 0.5g of corn steep liquor, 0.5g of casamino acid, 0.1g of sodium chloride, 0.3g of sodium pyruvate, 0.5g of tryptone, 0.5g of soluble starch, 2mg of L-tryptophan and 0.05g of magnesium sulfate heptahydrate, dissolving the components in 1000mL of water, and dissolving the components in K 2 HPO 4 With KH 2 PO 4 Adjusting the pH value to 6.0-6.5, and sterilizing at 121 ℃ for 20 min.
Experiments show that the influence of the carbon source on the IAA production of the strain is that sucrose, fructose, maltose, honey, glucose and corn flour are sequentially reduced from large to small, and when the carbon source is sucrose, the IAA yield reaches the highest 22.76mg/L and is obviously higher than that of other carbon sources. When the carbon source is corn flour, the yield of the IAA is only 7.85mg/L, and when the carbon source is fructose, maltose and glucose, the yield of the IAA is respectively 16.99mg/L, 14.34mg/L and 14.13mg/L. The best carbon source selected is sucrose.
The nitrogen source generates IAA quantity from Gxun-20 in turn from more to less: yeast powder, peptone, corn steep liquor, potassium nitrate and ammonium sulfate. For the yield of the IAA, when the nitrogen source is yeast powder, the yield of the IAA reaches the highest 68.56mg/L and is obviously higher than that of other nitrogen sources, and when the nitrogen source is inorganic nitrogen sources such as potassium nitrate, ammonium sulfate and the like, the yield of the IAA is obviously less than that of organic nitrogen sources such as peptone, yeast powder and the like. The IAA yield of the corn steep liquor is 52.63mg/L which is slightly lower than organic nitrogen sources such as peptone and yeast powder, but the corn steep liquor is cheaper than the organic nitrogen sources such as peptone and yeast powder in terms of economic consumables, so the corn steep liquor is preferably used as the nitrogen source in the invention.
According to the invention, different inorganic salts are added in the fermentation process respectively to observe the influence of the inorganic salts on the production of the IAA by the Gxun-20 strain, and the inventor finds that after the sodium chloride is added under the same condition, the amount of the IAA produced by the Gxun-20 strain is up to 58.01mg/L, and the yield is higher compared with other inorganic salts (magnesium sulfate, sodium nitrate, calcium chloride, ammonium sulfate and dipotassium phosphate), so that the sodium chloride is most beneficial to the production of the IAA by the Gxun-20 strain.
As a further improvement of the application of the plant rhizosphere growth promoting strain Gxun-20 in plant growth promotion, the mass concentration of the sucrose is 0.4%; the mass concentration of the corn steep liquor is 0.05 percent; the mass concentration of the soluble starch is 0.05%.
The yield of the IAA of the Gxun-20 strain is gradually increased along with the increase of the sucrose concentration, and the yield of the IAA reaches 39.74 mg/L at the highest sucrose concentration of 0.4% (w/v). Thus, the optimal sucrose concentration was chosen to be 0.4% (w/v).
As the concentration of corn steep liquor increases, the IAA yield decreases sharply, significantly below the yield achieved at 0.05% (w/v), so the optimal concentration of corn steep liquor is selected to be 0.05% (w/v).
When the concentration of the soluble starch is 0.05% (w/v), the IAA yield of the Gxun-20 strain reaches 53.60mg/L, which is not much different from the highest IAA yield concentration, and 0.05% (w/v) is selected as the optimal concentration of the soluble starch in consideration of economic materials.
As a further improvement of the application of the plant rhizosphere growth promoting strain Gxun-20 in plant growth promotion, the content of organic fertilizer nitrogen is 0.7-0.8%, the content of phosphorus is 0.45-0.6%, and the content of potassium is 0.4-0.5%.
Meanwhile, the invention also provides a screening method of the plant rhizosphere growth promoting strain Gxun-20, which comprises the following steps:
(1) Taking 1g of mangrove root system soil sample, adding 20mL of sterile water, shaking in a shaking table at 200r/min at 30 ℃ for 30min, and standing for 5-10 min to obtain a soil suspension; the mangrove forest is from the north sea of Guangxi;
(2) Taking 5mL of soil suspension, carrying out enrichment culture in an LB liquid culture medium for 24h, carrying out gradient dilution on the enriched bacterial liquid, taking 100 mu L of bacterial liquid from each gradient, coating the bacterial liquid on an ADF solid culture medium, and then culturing the bacterial liquid in a constant-temperature incubator at 34 ℃ for 3d to obtain a single bacterial colony;
(3) Selecting single colonies with different shapes and sizes on the plate, and inoculating the single colonies on an LB solid culture medium for separation and purification;
(4) Inoculating the separated and purified strains into an LB liquid culture medium containing 50mg/mL L-tryptophan, culturing for 24h at 30 ℃ in a shaking table of 200r/min, then, in an aseptic environment, dropwise adding 2 mu L of each bacterial liquid onto a phosphorus-dissolving culture medium, inversely placing the bacterial liquid on a 34 ℃ constant-temperature incubator for culturing for 3d, observing the growth condition of bacteria on the phosphorus-dissolving culture medium and the size of a phosphorus-dissolving ring, wherein the occurrence of the phosphorus-dissolving ring indicates that the strains have phosphorus-dissolving capacity, and the larger the phosphorus-dissolving ring is, the stronger the phosphorus-dissolving capacity is;
(5) Centrifuging the centrifuge tube filled with the bacteria liquid for 2min at 12000r/min, taking 100 mu L of supernatant fluid on a white transparent 96 colorimetric empty plate, then dropwise adding 100 mu L of Salkowski colorimetric solution, taking 10mg/L IAA as a contrast, adding equivalent Salkowski colorimetric solution for reaction, placing the colorimetric plate in a 38 ℃ constant-temperature incubator in the dark for 40min, taking out for observation, wherein the color becomes red to indicate that the IAA can be generated, and the deeper the color is, the higher the IAA yield is;
(6) Taking a strain with strong phosphorus dissolving capacity and high IAA yield, namely a plant rhizosphere growth promoting strain, which is named as Gxun-20;
the Salkowski colorimetric solution is 0.1mL of 0.5mol/L FeCl 3 With 5mL of 35% HClO by mass concentration 4 Mixing and preparing.
As a further improvement of the screening method of the plant rhizosphere growth promoting strain Gxun-20, the ADF solid culture medium comprises the following specific components:
the component one: moO 3 10mg of manganese sulfate monohydrate 11.19mg of heptahydrateMagnesium sulfate 124.6mg; cuSO 4 ﹒5H 2 O 78.22 mg；H 3 BO 3 10mg; and (2) component two: ferrous sulfate heptahydrate 100 mg;
after weighing the formula, dissolving the component I in 100mL of distilled water, dissolving the component II in 10mL of distilled water, and refrigerating at low temperature for later use;
0.5mL of fraction one and 0.2mL of fraction two were added to the beaker, respectively, and the following reagents were added: mgSO (MgSO) 4 ﹒7H 2 0.2g of O, 2.0g of citric acid, 2.0g of 1-aminocyclopropane-1-carboxylic acid, 2.0g of gluconic acid, 2.0g of glucose, KH 2 PO 4 4.0g、Na 2 HPO 4 6.0g, then adding distilled water, adjusting the pH to 7.0, diluting to a constant volume of 1000mL, and finally sterilizing at 121 ℃ for 20min to obtain the ADF solid culture medium.
As a further improvement of the screening method of the plant growth promoting rhizosphere strain Gxun-20, the LB liquid culture formula is as follows: the yeast powder is 5g, the tryptone is 10g, the NaCl is 10g, the pH value is 7.0, and the LB solid culture medium is added with 20g of agar based on the LB liquid culture medium.
The phosphorus dissolving culture medium comprises: glucose 10g, (NH) 4 ) 2 SO 4 0.5g；MgSO 4 ﹒7H 2 O 0.3gz、MnSO 4 ﹒4H 2 O 0.03g、KCl 0.3g、FeSO 4 ﹒7H 2 O 0.03g、NaCl 0.3g、Ca 3 (PO 4 ) 2 20g of agar powder and 20g of agar powder, then adding distilled water, adjusting the pH to 7.0, fixing the volume to 1000mL, and sterilizing for 20min at 121 ℃.
The invention has the beneficial effects that:
1. the plant rhizosphere growth promoting strain Gxun-20 provided by the invention has higher IAA yield, corn steep liquor is selected as a nitrogen source, when the concentration is 0.05% (w/v), the IAA yield of the strain reaches 65.16 mg/L at most, and the plant rhizosphere growth promoting strain has a good application effect on plant growth promotion.
2. According to the invention, strains with IAA secretion and phosphorus dissolving growth promoting capability are screened out through the ADF screening culture medium and the LB purification culture medium, and the formula and the dosage of the fermentation culture medium are improved through the optimization of fermentation conditions, so that the IAA production efficiency and the IAA yield of the plant rhizosphere growth promoting strain Gxun-20 are improved, the fermentation cost is reduced, and the fermentation efficiency is improved; and finally, the fermentation liquor is utilized to irrigate the crops, so that the growth speed of the crops is effectively improved. The damage of using chemical fertilizer to the land is reduced.
Biological preservation description:
the plant rhizosphere growth promoting strain Gxun-20 is Bacillus brevis and is preserved in Guangdong province microorganism strain preservation center, and the address is as follows: no. 59 building 5 of the Jieli Zhonglu Dazhou No. 100 of Guangzhou, guangdong province, china, having a taxonomic nomenclature ofBrevibacillus parabreivsThe preservation number is GDMCC NO:60686 with a preservation time of 2019, 6 months and 13 days.
Drawings
FIG. 1 is a morphological feature diagram of a plant growth-promoting rhizosphere strain Gxun-20 of the present invention.
FIG. 2 is a table of results of identification of physiological and biochemical indicators of the plant growth-promoting rhizobacteria Gxun-20 of the present invention.
FIG. 3 is a graph showing the growth of plant growth promoting rhizosphere strain Gxun-20 of example 1 of the present invention.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
1. Screening of plant growth-promoting rhizosphere strain Gxun-20
(1) Taking 1g of mangrove root system soil sample, respectively adding the mangrove root system soil sample into five shaking bottles filled with 20mL of sterile water, sticking labels, placing the bottles into a shaking table with 200r/min, shaking for 30min at 30 ℃, taking out the bottles, and standing for 5-10 min to obtain a soil suspension;
(2) Respectively sucking 5mL of soil suspension from the five bottles of samples, carrying out enrichment culture in an LB liquid culture medium for 24h, carrying out gradient dilution on the enriched bacterial liquid, coating 100 mu L of bacterial liquid on an ADF solid culture medium in each gradient, wherein each coating is three in parallel, and then placing the coated flat plate in a constant-temperature incubator at 34 ℃ for culture for 3d to obtain single bacterial colonies;
(3) Selecting single colonies with different shapes and sizes on the plate, and inoculating the single colonies on an LB solid culture medium for separation and purification;
(4) Inoculating the separated and purified strains into an LB liquid culture medium containing 50mg/mL L-tryptophan, culturing for 24h at 30 ℃ in a shaking table of 200r/min, then in a sterile environment, dropwise adding 2 mu L of each bacterial liquid onto a phosphorus-dissolving culture medium, inversely placing the bacterial liquid on a 34 ℃ constant-temperature incubator for culturing for 3d, observing the growth condition of bacteria on a phosphorus-dissolving plate and the size of a phosphorus-dissolving ring, wherein the occurrence of the phosphorus-dissolving ring indicates that the strains have phosphorus-dissolving capacity, and the larger the phosphorus-dissolving ring is, the stronger the phosphorus-dissolving capacity is;
(5) Centrifuging the centrifuge tube containing the bacterial solution at 12000r/min for 2min, collecting 100. Mu.L of supernatant on a white transparent 96 colorimetric empty plate, and adding 100. Mu.L of Salkowski colorimetric solution (0.1mL of 0.5mol/L FeCl) dropwise 3 +5mL 35% HClO 4 ) Adding equivalent Salkowski colorimetric solution for reaction by taking 10mg/L IAA as a control, placing the colorimetric plate in a constant-temperature incubator at 38 ℃ in a dark place for 40min, taking out and observing, wherein the condition that the color turns red indicates that the IAA can be generated, and the darker the color indicates that the IAA yield is higher;
(6) Taking a strain corresponding to the bacteria liquid with high IAA yield and large phosphorus-dissolving circle, and taking a strain with strong phosphorus-dissolving capacity and high IAA yield, namely a plant rhizosphere growth promoting strain, which is named as Gxun-20;
the ADF solid medium is as follows:
the component one: moO 3 10mg, 11.19mg of manganese sulfate monohydrate and 124.6mg of magnesium sulfate heptahydrate; cuSO 4 ﹒5H 2 O 78.22 mg；H 3 BO 3 10mg; and (2) component two: ferrous sulfate heptahydrate 100 mg;
after weighing the formula, dissolving the component I in 100mL of distilled water, dissolving the component II in 10mL of distilled water, and refrigerating at low temperature for later use;
0.5mL of fraction one and 0.2mL of fraction two were added to the beaker, respectively, and the following reagents were added: mgSO (MgSO) 4 ﹒7H 2 0.2g of O, 2.0g of citric acid, 2.0g of 1-aminocyclopropane-1-carboxylic acid, 2.0g of gluconic acid, 2.0g of glucose and KH 2 PO 4 4.0g、Na 2 HPO 4 6.0g, then adding distilled water, adjusting the pH to 7.0, diluting to a constant volume of 1000mL, and finally sterilizing at 121 ℃ for 20min to obtain the ADF solid culture medium.
The LB liquid culture medium is as follows:
5g of yeast powder, 10g of tryptone, 10g of NaCl and pH7.0 in 1000mL, wherein the LB solid culture medium is formed by adding 20g of agar on the basis of an LB liquid culture medium;
the phosphorus dissolving culture medium comprises the following components:
glucose 10g, (NH) 4 ) 2 SO 4 0.5g；MgSO 4 ﹒7H 2 O 0.3gz、MnSO 4 ﹒4H 2 O 0.03g、KCl 0.3g、FeSO 4 ﹒7H 2 O 0.03g、NaCl 0.3g、Ca 3 (PO 4 ) 2 20g of agar powder and 20g of agar powder, then adding distilled water, adjusting the pH to 7.0, fixing the volume to 1000mL, and sterilizing for 20min at the temperature of 121 ℃.
In this example, after four generations of separation and purification in LB medium, a single colony was obtained and named Gxun-20. The Gxun-20 strain is characterized by morphological characteristics of Brevibacillus brevis, and the colony morphology is round, smooth, convex and white, as shown in figure 1, and is a gram-positive bacterium. According to some characteristic physiological and biochemical indexes, refer to the handbook of identifying common bacteria, and the results are shown in FIG. 2.
2. Preparation of plant rhizosphere growth promoting strain Gxun-20 zymocyte liquid
(1) Inoculating plant growth promoting rhizosphere strain Gxun-20 into LB liquid culture medium, shaking culturing at constant temperature of 30 deg.C in 200r/min shaking table for activation, and measuring OD of culture solution every 2h 600 Values, growth curves were plotted, as shown in fig. 3;
(2) R after sterilization 2 Adding 2 mg/L-tryptophan into the liquid culture medium A, adjusting the initial pH to 6.0, inoculating the activated target strain in logarithmic phase according to the inoculation amount of 5 mu L/mL culture medium, and performing oscillation fermentation for 26h in a shaking table of 200r/min at 25 ℃ to obtain zymogen liquid;
said R 2 Liquid medium A is as follows:
glucose 0.5g, yeast powder 0.5g, casamino acid 0.5g, K 2 HPO 4 0.1g, 0.3g sodium pyruvate, 0.5g tryptone, 0.05g soluble starch, 1.5mg L-tryptophan, 0.05g magnesium sulfate heptahydrate, dissolved in 1000mL water and applied with K 2 HPO 4 With KH 2 PO 4 Adjusting pH to 6.0, and sterilizing at 121 deg.C for 20min; the mass concentration of the corn steep liquor is 0.05 percent; the mass concentration of the soluble starch is 0.05%.
3. Plant growth promotion by using plant rhizosphere growth promoting strain Gxun-20
The crop treated in this example was corn.
(1) Selecting plump, undamaged and high-quality corn seeds, soaking in 50 deg.C warm water for 10min, pouring out, soaking in sterile water for 8 hr, and finally soaking in 10% Na 3 PO 4 Soaking the seeds in the solution for about 15min to kill various harmful bacteria on the surfaces of the seeds;
(2) Washing the soaked seeds with clear water, removing water, placing in a culture dish with paper towel, spraying sterile water for soaking, placing in a dark 25 deg.C ventilated constant temperature incubator, and spraying sterile water every day to keep moistening until most of seeds are white;
(3) Mixing greenhouse surface soil, sawdust and organic fertilizer according to a ratio of 1; firstly, thoroughly watering the seedling culture nutrient soil with sterile water, then inoculating the maize seeds accelerating germination into the seedling culture nutrient soil, covering a layer of thin seedling culture nutrient soil, and finally paving a transparent mulching film, and removing the transparent mulching film in time when 75% of seedlings break the soil and come out, so that the seedlings smoothly carry out photosynthesis; the nitrogen content of the organic fertilizer is 0.7%, the phosphorus content is 0.6%, and the potassium content is 0.5%.
(4) And after 3-4 leaves grow from the seedlings and the growth state is stable, irrigating 10mL of 5-time diluted zymogen liquid to each seedling in a way of irrigating roots with the bacteria liquid, irrigating the zymogen liquid once every 7 days, and treating for 4 times in total.
The height of the maize seedlings of this example was increased by an average of 12.68% compared to the control group (plants watered with sterile water).
Example 2
Compared with embodiment 1, the present embodiment is different in that:
said R 2 The preferable formula of the A liquid culture medium is as follows:
sucrose 0.4g, corn steep liquor 0.5g, and casein amino0.5g of acid, 0.1g of sodium chloride, 0.3g of sodium pyruvate, 0.5g of tryptone, 0.5g of soluble starch, 2mg of L-tryptophan and 0.05g of magnesium sulfate heptahydrate, dissolving in 1000mL of water, adding K 2 HPO 4 With KH 2 PO 4 Adjusting pH to 6.5, and sterilizing at 121 deg.C for 20 min. The mass concentration of the sucrose is 0.4%; the mass concentration of the corn steep liquor is 0.05 percent; the mass concentration of the soluble starch is 0.05%.
The fermentation condition is that the fermentation is carried out for 38 hours in a shaking table with 200r/min at the temperature of 28 ℃.
The crop treated in this example is wheat, and the specific method is as follows:
(1) Selecting plump, undamaged and high-quality wheat seeds, soaking in 50 deg.C warm water for 10min, pouring out, soaking in sterile water for 8 hr, and finally soaking in 10% Na 3 PO 4 Soaking the seeds in the solution for about 15min to kill various harmful bacteria on the surfaces of the seeds;
(2) Washing the soaked seeds with clear water, removing water, placing in a culture dish with paper towel, spraying sterile water for moistening, placing in a dark 25 deg.C ventilated constant temperature incubator, spraying sterile water every day for keeping moistening until most seeds are exposed;
(3) Mixing greenhouse surface soil with wood chips and organic fertilizers according to a ratio of 1; firstly, thoroughly watering the seedling culture nutrient soil with sterile water, then inoculating the maize seeds accelerating germination into the seedling culture nutrient soil, covering a layer of thin seedling culture nutrient soil, and finally paving a transparent mulching film, and removing the transparent mulching film in time when 75% of seedlings break the soil and come out, so that the seedlings smoothly carry out photosynthesis; the nitrogen content of the organic fertilizer is 0.8 percent, the phosphorus content is 0.5 percent, and the potassium content is 0.4 percent.
(4) And after 3-4 leaves grow from the seedlings and the growth state is stable, irrigating 10mL of 5-time diluted zymogen liquid to each seedling in a way of irrigating roots with the bacteria liquid, irrigating the zymogen liquid once every 7 days, and treating for 4 times in total.
The plant height of the wheat seedlings of this example was increased by 20.74% on average compared to the control group (plants watered with sterile water).
Example 3
Compared with embodiment 1, the present embodiment is different in that:
said R 2 The preferable formula of the A liquid culture medium is as follows:
0.4g of sucrose, 0.5g of corn steep liquor, 0.5g of casamino acid, 0.1g of sodium chloride, 0.3g of sodium pyruvate, 0.5g of tryptone, 0.5g of soluble starch, 2mg of L-tryptophan and 0.05g of magnesium sulfate heptahydrate, dissolving the components in 1000mL of water, and dissolving the components in K 2 HPO 4 With KH 2 PO 4 Adjusting pH to 6.2, and sterilizing at 121 deg.C for 20 min. The mass concentration of the sucrose is 0.4%; the mass concentration of the corn steep liquor is 0.05 percent; the mass concentration of the soluble starch is 0.05%.
The fermentation condition is that the fermentation is carried out for 38 hours in a shaking table with 200r/min at the temperature of 28 ℃.
The crop treated in this example is pepper, and the specific method is as follows:
(1) Selecting full, undamaged and high-quality pepper seeds, soaking in 50 deg.C warm water for 10min, pouring out, soaking in sterile water for 8 hr, and finally soaking in 10% Na 3 PO 4 Soaking the seeds in the solution for about 15min to kill various harmful bacteria on the surfaces of the seeds;
(2) Washing the soaked seeds with clear water, removing water, placing in a culture dish with paper towel, spraying sterile water for moistening, placing in a dark 25 deg.C ventilated constant temperature incubator, spraying sterile water every day for keeping moistening until most seeds are exposed;
(3) Mixing greenhouse surface soil with wood chips and organic fertilizers according to a ratio of 1; firstly, thoroughly watering the seedling culture nutrient soil with sterile water, then inoculating the maize seeds accelerating germination into the seedling culture nutrient soil, covering a layer of thin seedling culture nutrient soil, and finally paving a transparent mulching film, and removing the transparent mulching film in time when 75% of seedlings break the soil and come out, so that the seedlings smoothly carry out photosynthesis; the nitrogen content of the organic fertilizer is 0.7%, the phosphorus content is 0.45%, and the potassium content is 0.5%.
(4) And after 3-4 leaves grow from the seedlings and the growth state is stable, irrigating 10mL of 8-time diluted zymogen liquid to each seedling in a way of irrigating roots with the bacteria liquid, irrigating the zymogen liquid once every 7 days, and treating for 4 times in total.
The plant height of wheat seedlings of this example was increased by an average of 18.6% compared to the control group (plants watered with sterile water).
The above embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and the scope of the present invention is defined by the claims. Various modifications and equivalents may be made thereto by those skilled in the art within the spirit and scope of the present invention, and such modifications and equivalents should be considered as falling within the scope of the present invention.
Claims (5)
1. A plant rhizosphere growth promoting strain Gxun-20 is characterized in that: the plant rhizosphere growth promoting strain Gxun-20 is Bacillus brevis (B)Brevibacillus parabreivs) Deposited in the Guangdong province culture Collection of microorganisms with the following addresses: no. 59 building No. 5 of the first 100 college of the first furious Zhonglu, guangzhou, guangdong province, china, the name of classificationBrevibacillus parabreivsThe preservation number is GDMCC NO:60686 with a date of 6 months and 13 days 2019.
2. Use of the plant rhizosphere growth promoting strain Gxun-20 of claim 1 for plant growth promotion, wherein: the method comprises the following steps:
(1) Selecting plump, undamaged and good-quality seeds, firstly soaking in warm water at 50 ℃ for 10min, pouring out, then soaking in sterile water for 8h, and finally soaking in Na with the mass concentration of 10% 3 PO 4 Soaking the seeds in the solution for 10-15 min; washing the soaked seeds with clear water, removing water, placing in a culture dish paved with paper towels, and spraying sterile water for soaking; finally, placing the seeds in a dark and ventilated constant-temperature incubator at 25 ℃, spraying sterile water every day to keep moist until most of the seeds are exposed to be white, and obtaining seeds accelerating germination;
(2) Mixing greenhouse surface soil, sawdust and organic fertilizer according to a ratio of 1; firstly, thoroughly watering the seedling culture nutrient soil with sterile water, then inoculating the seeds accelerating germination into the seedling culture nutrient soil, covering a layer of thin seedling culture nutrient soil, and finally paving a transparent mulching film, and timely tearing off the transparent mulching film when 75-80% of the seedlings break the soil to come out, so that the seedlings smoothly undergo photosynthesis;
(3) When 3-4 leaves grow out from the seedlings and the growth state is stable, 10mL of zymogen liquid diluted by 5-10 times is poured into each seedling in a liquid bacterial root pouring mode, the zymogen liquid is poured once every 7 days, and the treatment is carried out for 4-5 times.
3. The application of the plant rhizosphere growth-promoting strain Gxun-20 in plant growth promotion according to claim 2, wherein the fermentation method of the zymophyte liquid comprises the following steps,
(1) Inoculating plant growth promoting rhizosphere strain Gxun-20 into LB liquid culture medium, performing constant temperature shaking culture at 30 deg.C in 200r/min shaker for activation, and measuring OD of culture solution every 2h 600 Value, drawing a growth curve;
(2) R after sterilization 2 Adding 2 mg/L-tryptophan into the liquid culture medium A, adjusting the initial pH to 6.0-6.5, then inoculating the activated plant rhizosphere growth promoting strain Gxun-20 in the logarithmic growth phase according to the inoculation amount of a culture medium of 5 mu L/mL, and performing oscillation fermentation for 26-40 h in a shaker of 200r/min at the temperature of 25-30 ℃ to obtain the zymophyte liquid.
4. The use of the plant rhizosphere growth promoting strain Gxun-20 according to claim 3 in plant growth promotion, wherein R is 2 The formula of the liquid culture medium A is as follows:
glucose 0.5g, yeast powder 0.5g, casamino acid 0.5g, K 2 HPO 4 0.1 to 0.4g of sodium pyruvate, 0.3g of sodium pyruvate, 0.5g of tryptone, 0.05 to 0.5g of soluble starch, 1.5 to 2.5mg of L-tryptophan and 0.05g of magnesium sulfate heptahydrate, dissolving the mixture in 1000mL of water, and dissolving the mixture in K 2 HPO 4 With KH 2 PO 4 Adjusting the pH value to 6.0-6.5, and sterilizing at 121 ℃ for 20min;
or, said R 2 The formula of the liquid culture medium A is as follows:
sucrose 0.4g, corn steep liquor 0.5g, and casein amino0.5g of acid, 0.1g of sodium chloride, 0.3g of sodium pyruvate, 0.5g of tryptone, 0.5g of soluble starch, 2mg of L-tryptophan and 0.05g of magnesium sulfate heptahydrate, dissolving in 1000mL of water, adding K 2 HPO 4 With KH 2 PO 4 Adjusting the pH value to 6.0-6.5, and sterilizing for 20min at 121 ℃.
5. The use of the plant growth-promoting rhizobacteria Gxun-20 according to claim 2 in plant growth promotion, wherein the organic fertilizer nitrogen content is 0.7-0.8%, the phosphorus content is 0.45-0.6%, and the potassium content is 0.4-0.5%.
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| CN112538443A (en) * | 2020-12-02 | 2021-03-23 | 无锡市三阳生态农业发展有限公司 | Fermentation method for increasing yield of rhizosphere growth-promoting bacteria |
| CN112899188A (en) * | 2021-01-29 | 2021-06-04 | 西南大学 | Microbial agent for promoting crop root development and preparation and application thereof |
| CN113278565A (en) * | 2021-07-09 | 2021-08-20 | 广西民族大学 | Brevibacillus parabrevis Gxun-20 and application thereof |
| CN114015629B (en) * | 2021-12-20 | 2022-04-29 | 南京林业大学 | Complex microbial inoculum for promoting growth of soybeans and improving abundance of probiotic microbial populations |
| CN115074268B (en) * | 2022-05-16 | 2023-10-13 | 云南农业大学 | Application of biological agent for promoting synthesis of truffle mycorrhiza |
| CN115011483A (en) * | 2022-06-10 | 2022-09-06 | 上海市农业科学院 | High-throughput screening method for IAA-producing endophytes of plant tissues |
| CN115011482A (en) * | 2022-06-10 | 2022-09-06 | 上海市农业科学院 | Method for separating plant rhizosphere microorganisms in high flux and culturing and screening IAA-producing strains |
| CN114990029B (en) * | 2022-07-15 | 2023-09-22 | 广西民族大学 | Microbial composite microbial agent and preparation method and application thereof |
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