CN111057665B - Cellulose degrading bacterium n3 for producing IAA and application thereof - Google Patents

Abstract

The invention provides an IAA-producing cellulose degrading bacterium n3 and application thereof, and relates to the technical field of agricultural microorganisms, wherein the cellulose degrading bacterium n3 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 18613. The cellulose degrading bacteria n3 have strong capability of producing CMC enzyme, and can produce IAA, the CMC enzyme has the highest activity reaching 24.96U/mL, and the highest secretion of IAA can reach 19.07 mg/L. Therefore, the cellulose degrading bacteria n3 can be used for preparing a straw decomposition promoting microbial inoculum for producing IAA and/or CMC enzyme or having a growth promoting function, so that the cellulose degrading bacteria n3 can be applied to straw decomposition promoting and crop growth promoting, and straw returning efficiency and crop yield improvement are realized.

Description

A cellulose-degrading bacteria n3 producing IAA and its application

technical field

The invention belongs to the technical field of agricultural microorganisms, in particular to an IAA-producing cellulose degrading bacteria n3 and its application.

Background technique

Straw returning to the field is a measure that is widely valued in the world today to increase the yield of crops that improve soil fertility. While eliminating the air pollution caused by the straw burning process, it can also improve soil physical properties, improve soil organic matter levels, and increase soil biological properties. It plays an important role in improving the level of soil nutrient supply and so on. Straw is rich in nutrients such as organic matter and nitrogen, phosphorus and potassium, which can be converted into nutrient forms that are easily absorbed and utilized by crops. Making full use of the positive effect of returning straw to the field can effectively improve resource utilization, increase crop yield, and promote nutrient cycling.

Straw is mainly composed of lignin, cellulose, hemicellulose and other refractory substances, of which cellulose content is the highest, and the decomposition rate of straw is slow in natural state. At present, scholars at home and abroad generally believe that when straw is returned to the field, stalk-decomposing fungi that produce cellulose-degrading enzymes (such as carboxymethyl cellulose degrading enzyme, CMC) are used to accelerate the decomposing of straw and other wastes. However, whether the functional microorganisms in the stalk agent can be successfully colonized will be affected by the abundance of soil resources and the competition intensity of indigenous microorganisms, so the regional matching of stalk rot agents is required. There should also be differences, so it is necessary to screen strains in the application area to prepare stalk rot inoculants to improve the degree and rate of stalk rot in this area.

Sand ginger black soil has the bad characters of dry shrinkage and wet expansion, easy drought and waterlogging, poor soil tillage and low fertility level, which has adverse effects on crop growth. It is a typical low-yield soil in my country. The sand ginger black soil is sticky and heavy, and the soil structure is poor. It is prone to problems such as "drought, waterlogging, stiffness and thinness" in production. Therefore, the general application of stalk rot agent on sand ginger black soil is less effective. Sand ginger black soil is mostly a wheat and jade rotation area, and the rotation interval is short. If the straw is not completely decomposed after the application of the stalk agent, on the one hand, the previous crop will not have time to decompose and will accumulate and stay in the soil for a long time, thus affecting the next season. Sowing of crops; on the other hand, the incompletely decomposed straw in the soil will cause the wheat and jade seeds to be unable to combine with the soil, making it difficult for the wheat and jade seeds to germinate, and will compete with the crops for nutrients, resulting in a decrease in the emergence rate and yield of the crops. .

Indoleacetic acid (IAA) is a kind of plant hormone, which is a signal substance that produces and regulates auxin. It is ubiquitous in plants and is an endogenous auxin that is ubiquitous in plants. The growth-promoting effect of auxin is mainly to promote the growth of cells, especially the elongation of cells, which has positive significance for crop growth and yield improvement. Therefore, the use of high-efficiency rot-promoting and growth-promoting strains is conducive to the growth and development of crops in the sand ginger black soil wheat-jade rotation system and maintains excellent soil properties.

At present, most scholars at home and abroad only study the cellulose-degrading ability or growth-promoting ability of a certain strain, and are committed to studying a certain function of a certain strain, but strains with multiple functions are rarely seen.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a high-efficiency CMC-producing cellulose-degrading bacteria n3 screened from sand ginger black soil, which can improve the cellulose degradation rate and promote the decomposition of straw, thereby promoting the decomposition of returning straw, and improving the return of straw to the field. In addition, the bacteria can also produce high IAA, promote the germination and growth of crop seeds, and increase crop yield.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides an IAA-producing cellulose-degrading bacteria n3, which is preserved in the China General Microorganism Culture Collection and Management Center, and the preservation number is CGMCC No.18613.

The present invention also provides the application of the cellulose-degrading bacteria n3 in the preparation of IAA and/or CMC enzymes.

Preferably, when using the cellulose-degrading bacteria n3 to prepare IAA, the following steps are included: adjusting the pH of the LB medium containing 100 mg/LL-tryptophan to 6.0-9.0, and inoculating the cellulose-degrading bacteria n3 The bacterial suspension is shaken and cultured; the volume of the bacterial suspension is 1% of the volume of the LB medium; the OD ₆₀₀ value of the bacterial suspension is 0.8-1.2.

Preferably, the temperature of the shaking culture is 28-30°C, and the shaking speed is 160-180 rpm.

Preferably, when using the cellulose-degrading bacteria n3 to prepare the CMC enzyme, the following steps are included: adjusting the pH of the liquid fermentation medium to 4.0-6.0, inoculating the cellulose-degrading bacteria n3, and shaking culture; the cellulose The inoculation volume of degrading bacteria n3 is 1% of the volume of the liquid fermentation medium; the liquid fermentation medium includes raw materials with the following concentrations: sodium chloride 6g/L, magnesium sulfate heptahydrate 0.1g/L, calcium chloride 0.1 g/L, potassium dihydrogen phosphate 0.5g/L, yeast extract 10g/L and straw 20g/L.

Preferably, the LB medium or the liquid fermentation medium further includes the following components by mass percentage: 0.1% carbon source and 1% nitrogen source.

Preferably, the carbon source includes one or more of glucose, mannitol, sucrose, maltose, xylose, lactose and fructose;

The nitrogen source includes one or more of potassium nitrate, ammonium sulfate, ammonium nitrate, yeast powder, glutamic acid, urea and peptone.

The present invention also provides the application of the cellulose-degrading bacteria n3 in the preparation of a decay-promoting and growth-promoting bacterial agent.

Preferably, the type of the decay-promoting bacteria agent is bacteria water agent; when the bacteria water agent is applied, the inoculation amount is 1-9×10 ⁶ CFU/ g straw or 1～9×10 ⁷ CFU/g soil.

The invention also provides the application of the cellulose-degrading bacteria n3 in promoting straw returning efficiency and increasing crop yield.

The invention provides an IAA-producing cellulose-degrading bacteria n3, which is preserved in the China General Microorganism Culture Collection and Management Center, and the preservation number is CGMCC No.18613. The cellulose-degrading bacteria n3 of the invention has a smooth surface, small colonies, neat edges, opaque, slightly yellow; the cellulose-degrading bacteria n3 are Gram-negative bacteria, aerobic, chemoheterotrophic, contact enzyme positive, M.R. The test was negative, the VP test was negative, the starch hydrolysis was negative, the gelatin hydrolysis was negative, and the citrate utilization was negative.

The cellulose-degrading bacteria n3 of the invention can produce high indole acetic acid, and has strong CMC enzyme production ability, and has higher crop growth-promoting and straw rot-promoting abilities. The highest activity can reach 24.96U/mL. Therefore, the cellulose-degrading bacteria n3 can be used to prepare a stalk rot inoculant with both a growth-promoting function, so as to be used in promoting straw rot, promoting straw returning efficiency and increasing crop yield.

The cellulose-degrading bacteria n3 of the invention can be used for multiple purposes, and exert the maximum efficiency of the strain. On the one hand, it can promote the growth of crops and increase the yield of crops; It has the potential as a functional microbial fertilizer and contributes to promoting the green development of agriculture.

biological deposit information

Cellulose degrading bacteria n3, classified as Azospirillum zeae, was deposited in the China General Microorganism Collection and Management Center on September 23, 2019, and the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing Institute of Microbiology, Chinese Academy of Sciences, the deposit number is CGMCC No.18613.

Description of drawings

Fig. 1 is the colony diagram of cellulose degrading bacteria n3 provided by the present invention;

Fig. 2 is the ability of producing CMC enzyme of different strains;

Fig. 3 is the IAA production ability of different strains;

Fig. 4 phylogenetic tree of n3 strain constructed according to 16S rDNA sequence;

Figure 5 is a graph showing the effect of different pH on the activity of CMC enzyme produced by cellulose-degrading bacteria n3;

Figure 6 is a graph showing the effect of different liquid loadings on the activity of CMC enzyme produced by cellulose-degrading bacteria n3;

Figure 7 is a graph showing the effect of different nitrogen sources on the activity of CMC enzyme produced by cellulose-degrading bacteria n3;

Figure 8 is a graph showing the effect of different incubation times on the production of IAA by cellulose-degrading bacteria n3;

Figure 9 is a graph showing the effect of different incubation times on the growth of cellulose-degrading bacteria n3;

Figure 10 is a graph showing the effect of different liquid loadings on the production of IAA by cellulose-degrading bacteria n3;

Figure 11 is a graph showing the effect of different liquid loadings on the growth of cellulose-degrading bacteria n3;

Figure 12 is a graph showing the effect of different initial pH on the production of IAA by cellulose-degrading bacteria n3;

Figure 13 is a graph showing the effect of different initial pH on the growth of cellulose-degrading bacteria n3;

Figure 14 is a graph showing the effect of different carbon sources on the production of IAA by cellulose-degrading bacteria n3;

Figure 15 is a graph showing the effect of different carbon sources on the growth of cellulose-degrading bacteria n3;

Figure 16 is a graph showing the effect of different nitrogen sources on the production of IAA by cellulose-degrading bacteria n3;

Figure 17 is a graph showing the effect of different nitrogen sources on the growth of cellulose-degrading bacteria n3;

Figure 18 is a graph showing the effect of different strains on the rot-promoting ability of straw.

Detailed ways

The invention provides an IAA-producing cellulose-degrading bacteria n3, which is preserved in the China General Microorganism Culture Collection and Management Center, and the preservation number is CGMCC No.18613.

The cellulose-degrading bacteria n3 of the present invention is screened from the black soil collected in the agricultural demonstration science and technology park in Mengcheng County, Anhui Province. After verification, the cellulose-degrading bacteria n3 are Gram-negative bacteria, aerobic, Chemoheterotrophic, contact enzyme positive, M.R test negative, VP test negative, starch hydrolysis negative, gelatin hydrolysis negative, citrate utilization negative; the colony structure is shown in Figure 1, the surface is smooth, the colonies are small, the edges are neat and opaque , slightly yellowish.

The present invention also provides the application of the cellulose-degrading bacteria n3 in the preparation of IAA and/or CMC enzymes.

When using the cellulose-degrading bacteria n3 to prepare IAA, the present invention preferably includes the following steps: adjusting the pH of the LB medium containing 100 mg/L L-tryptophan to 6.0-9.0, inoculating the cellulose-degrading bacteria n3 The bacterial suspension was shaken and cultivated; the bacterial suspension was to use an inoculation loop to pick a ring of thalli from the solid medium, and insert it into a 25ml test tube containing 6ml of LB medium, and then the test tube was stoppered and placed at 30 Obtained by overnight culture in a shaker at 180 rpm; the OD ₆₀₀ value of the bacterial suspension is 0.8-1.2, and the inoculation volume is 1% of the volume of the LB medium. In the present invention, when carrying out the shaking culture, the liquid filling amount of the medium is preferably 25mL/250mL, and the culture time is preferably 15h. At this time, the cultivated cellulose-degrading bacteria n3 produces the highest amount of IAA and the best strain growth ability. good. The temperature of the shaking culture in the present invention is preferably 28-30° C., and the shaking speed is preferably 160-180 rpm.

When using the cellulose-degrading bacteria n3 to prepare the CMC enzyme, the present invention preferably includes the following steps: adjusting the pH of the liquid fermentation medium to 4.0-6.0, inoculating the cellulose-degrading bacteria n3, and shaking culture; the cellulose The inoculation volume of degrading bacteria n3 is 1% of the volume of the LB medium; the liquid fermentation includes raw materials with the following concentrations: sodium chloride 6g/L, magnesium sulfate heptahydrate 0.1g/L, calcium chloride 0.1 g/L , potassium dihydrogen phosphate 0.5g/L, yeast extract 10g/L and straw 20g/L.

In the present invention, the LB medium or the liquid fermentation medium preferably further includes the following components by mass: 0.1% carbon source, 1% nitrogen source. The carbon source in the LB medium of the present invention preferably includes one or more of glucose, mannitol, sucrose, maltose, xylose, lactose and fructose, more preferably mannitol; the nitrogen source preferably includes potassium nitrate, One or more of ammonium sulfate, ammonium nitrate, yeast powder, glutamic acid, urea and peptone, more preferably yeast powder. The nitrogen source in the liquid medium of the present invention preferably includes one or more of potassium nitrate, ammonium sulfate, ammonium nitrate, yeast powder, glutamic acid, urea and peptone, more preferably yeast powder or peptone.

The present invention also provides the application of the cellulose-degrading bacteria n3 in the preparation of a decay-promoting and growth-promoting bacterial agent.

The type of the decay-promoting bacteria agent of the present invention is preferably bacteria water agent; when the bacteria water agent is applied, the inoculation amount is preferably 1-9×10 ⁶ based on the amount of the cellulose-degrading bacteria n3 CFU/g straw or 1-9×10 ⁷ CFU/g soil, more preferably 5×10 ⁶ CFU/g straw or 5×10 ⁷ CFU/g soil.

The invention also provides the application of the cellulose-degrading bacteria n3 in promoting straw returning efficiency and increasing crop yield. The use method and application amount of the cellulose-degrading bacteria n3 in the application of the present invention are the same as above, and will not be repeated here.

A cellulose-degrading bacterium n3 producing IAA and its application will be described in detail below with reference to the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

1. Reagent preparation:

LB medium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 1000 mL of distilled water, adjusted to pH 7.0-7.2, and sterilized at 121°C for 20 minutes (add 20 g of agar to this recipe for solid medium).

Inorganic salt medium: ammonium sulfate 2.0g, sodium dihydrogen phosphate 0.5g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate heptahydrate 0.2g, calcium dichloride 0.1g, distilled water 1000mL, adjusted to pH 7.0～7.2, sterilized at 121°C bacteria for 20 minutes.

Liquid fermentation medium: 6 g of sodium chloride, 0.1 g of magnesium sulfate heptahydrate, 0.1 g of calcium chloride, 0.5 g of potassium dihydrogen phosphate, 10 g of yeast extract, 20 g of straw, 1000 mL of distilled water, sterilized at 121°C for 20 minutes.

Enrichment medium: sodium carboxymethyl cellulose 20g, microcrystalline cellulose 5g, cellulose powder 5g, dipotassium hydrogen phosphate 1g, nitric acid 1g, magnesium sulfate heptahydrate 0.2g, copper chloride dihydrate 0.1g, trichloride Iron 0.02g, distilled water 1000mL. Sterilize at 121°C for 20 minutes.

Carboxymethyl cellulose medium: 15 g of sodium carboxymethyl cellulose, 1 g of ammonium nitrate, 1 g of yeast extract, 0.5 g of magnesium sulfate heptahydrate, 1 g of potassium dihydrogen phosphate, 15 g of agar, and 1000 mL of distilled water. Sterilize at 121°C for 20 minutes.

2. Strain screening

Weigh 10g soil samples from the sand ginger black soil collected in the Agricultural Demonstration Science and Technology Park in Mengcheng County, Anhui Province, inoculate 10g soil samples in 90mL sterile water, shake at 28°C, 150rpm shaker for 30min, and take 1mL soil suspension in the sterile operation table. , add 9 mL of sterile water to make a stock solution with a concentration of 10 ^-1 . The strains were isolated and purified by the dilution plate method, and the pure cultured strains were stored in a refrigerator at 4°C.

The basic properties of the tested soils are shown in Table 1:

Table 1 Basic properties of the tested soils

The strains obtained by separation and purification were then inoculated on the sodium carboxymethyl cellulose selective medium, and the colony diameter (D) and the diameter of the transparent circle (H) were measured after the Congo red staining method was placed for 20 minutes. According to the H/D value, Preliminary judgment on the strength of the strain's ability to degrade cellulose.

All strains with cellulose-degrading ability were screened by Congo red staining. According to the H/D results, the n3 strain had the highest cellulose-degrading ability.

Preparation of crude enzyme solution: inoculate the strains in a liquid medium with wheat straw powder as the sole carbon source and cultivate in a liquid ^shake flask at 37°C for 60 hours. For the crude enzyme solution. Take 0.2 mL of supernatant into a 25 mL dry scale test tube, add 1.8 mL of 1% CMC-Na solution (pH 4.8, prepared with 0.1 mol/L citric acid-sodium citrate buffer) in a water bath at 50°C for 30 min and then add DNS reagent 3.0mL, boiling water bath for 5min, stop the reaction and develop color. Cool in a cold shower, dilute to 25 mL, shake well, and measure the absorbance at a wavelength of 520 nm. For the blank control, the enzyme solution was inactivated in a boiling water bath for 15 min, and other conditions remained unchanged.

The enzyme activity in this test is X=1000×G×25/0.2×30×180.

In the formula: X: the enzyme activity of the sample (U flail); 1000: conversion multiple; G: the number of milligrams of glucose corresponding to the light absorption value on the standard curve; 25: volume of constant volume (mL); 0.2: amount of enzyme added (mL) ); 30 is the action time (min); 180: the molecular weight of glucose (g/moL). Under the above conditions, the enzyme activity is defined according to the international unit regulations as: the amount of enzyme that catalyzes the hydrolysis of the substrate (sodium carboxymethyl cellulose) to generate 1 pmol of glucose per minute is 1 enzyme activity unit U.

Among the 8 strains screened out, n3 had the strongest ability to degrade cellulose, which was significantly higher than that of other strains, and the CMC enzyme activity could reach 20.60U·mL ^-1 .

Qualitative determination: Inoculate bacteria with cellulose degradation function in LB liquid medium containing L-tryptophan (100 mg·L ^-1 ) at 30°C, 180 rpm, culture for 1 d, take 100 μL of bacterial suspension and drop it on a white ceramic plate At the same time, 100μL Salkowski colorimetric solution (50mL 35% HClO ₄ , 1 mL 0.5mol·L ^-1 FeCl ₃ , stored in the dark) was added at the same time, and the white ceramic plate was placed under light for 30 minutes at room temperature to observe it, and the color turned red It can secrete indole acetic acid, the depth of color represents the level of its ability to produce indole acetic acid, and no color change means it does not produce indole acetic acid. The positive control is 100 μL of LB medium without inoculation, adding 100 μL of Salkowski colorimetric liquid.

Quantitative determination: quantitatively determine the strains with the ability to secrete IAA screened out by qualitative analysis, the culture conditions are qualitatively determined, first measure the OD ₆₀₀ value of the bacterial suspension by spectrophotometry, then draw 5mL of the bacterial suspension and centrifuge it at 10000rpm for 10 minutes. minutes, take 2 mL of supernatant and add an equal volume of Salkowski colorimetric solution, let stand for 30 minutes in the shade at room temperature, measure its OD value at a wavelength of 530 nm, and calculate the IAA content in the bacterial suspension by IAA standard curve.

Through the qualitative analysis of IAA, 5 strains have the ability to produce IAA, namely n1, n3, n4, n5, n8, and through quantitative determination, the results show that the n3 strain has the strongest ability to produce IAA, and the concentration can reach 19.07 mg·L ^{- 1} , significantly higher than other strains.

The cellulose-degrading bacteria n3, which has the strongest ability to produce CMC enzymes and IAA, and has a higher ability to promote the decay of wheat straw, can be screened through the above determination.

The strains screened and isolated by the above method were sequenced by Nanjing Company, and compared in the GenBank database according to the obtained 16S rDNA (SEQID NO.1) sequence results, Blast searched for homologous sequences, using MEGA5.0 software, Phylogenetic tree was constructed by Neighbour-Joining method. Combined with morphological analysis and physiological and biochemical characteristics of the strain, it was identified as azospirillumzeae. The phylogenetic tree of the n3 strain constructed according to the 16S rDNA sequence is shown in Figure 4.

Physiological and biochemical properties of the bacteria were counted, as shown in Table 2:

Table 2 Physiological and biochemical characteristics of cellulose-degrading bacteria n3

Note: + means positive reaction, - means negative reaction

Example 2

Effects of different pH, ventilation and nitrogen sources on the ability of strains to produce CMC enzyme

1. The effect of initial pH of medium on the ability to produce CMC enzyme

The strains were inoculated into the liquid fermentation medium with wheat straw powder as the sole carbon source, cultured in liquid shake flasks at 37°C for 60h, and their OD ₅₂₀ values were measured with a spectrophotometer. The initial pH was set at 4, 5, 6, 7, 8, 9, and 10, respectively. After culturing for 60 h, the content of CMC enzyme produced was measured with a spectrophotometer.

The results are shown in Figure 2: when the pH value is 5.0, the CMC enzyme activity is the highest, reaching 24.96U·mL ^-1 , and when the pH value is 4.0, the enzyme activity is the second, which is 24.85U·mL ^-1 , indicating that the n3 strain has more Strong acid resistance, CMC enzyme activity at pH 4.0 and 5.0 is significantly higher than that under other pH conditions.

2. The effect of ventilation volume on the ability to produce CMC enzyme

The strains were inoculated into the liquid fermentation medium with wheat straw powder as the sole carbon source, cultured in liquid shake flasks at 37°C for 60h, and their OD ₅₂₀ values were measured with a spectrophotometer. Set 25, 50, 75, 100, 150 mL of culture solution in a 250 mL Erlenmeyer flask. After culturing for 60 h, the content of CMC enzyme produced was measured with a spectrophotometer.

The results are shown in Figure 3: when the liquid volume of the 250mL conical flask is 25mL and the ventilation volume is the largest, the n3 strain produces the highest CMC enzyme activity, which is 15.33U·mL ^-1 .

3. The effect of nitrogen sources on the ability to produce CMC enzymes

0.1% (m/V) nitrogen source was added to the liquid fermentation medium with wheat straw powder as the sole carbon source. The nitrogen sources included potassium nitrate, ammonium sulfate, ammonium nitrate, yeast powder, glutamic acid, urea, and peptone. , 37 ℃ liquid shake flask cultured for 60h, using a spectrophotometer to determine the content of strain CMC enzyme.

The results are shown in Figure 4: Different nitrogen sources have different effects on the ability of n3 strain to produce CMC enzyme. When yeast powder and peptone are used as nitrogen sources, the ability to produce CMC enzyme is the most significant, up to 18.30 and 16.02 U·mL ^-1 , respectively.

Example 3

Test the effects of different pH, aeration, different time, different carbon sources, and different nitrogen sources on the yield of strain IAA and the growth of strains

1. The effect of fermentation time on strain IAA yield and strain growth

Put 50 mL of LB liquid medium (IAA detection medium) containing 100 mg·L ^-1 L-tryptophan in a 250 mL conical flask, and inoculate bacteria with an OD value of about 1 at an inoculum of 1% (V/V). The suspension was cultured at 30°C with a shaker at 180 rpm, and samples were dynamically sampled at 10, 15, 20, 32, 44, and 56 h, respectively, to determine the growth of the strain (OD ₆₀₀ ) and the ability to produce IAA (OD ₅₃₀ ). repeat.

The results are shown in Figure 5: OD ₆₀₀ reached the maximum value at 20h, and the growth of the strain showed a declining trend after 20h. The content of IAA produced was basically consistent with the growth status of the strain. At 10-15h, the content of IAA produced by the strain increased logarithmically, and at 15h The IAA content reached the highest at 18.66 mg·L ^-1 . After 15 h, the ability of the strain to produce IAA gradually decreased.

2. The effect of pH value on the yield of strain IAA and the growth of strain

The LB medium containing 100 mg·L ^-1 L-tryptophan was adjusted to different pH (4, 5, 6, 7, 8, 9, 10) respectively, and 50 mL of LB liquid medium was placed in a 250 mL conical flask. , according to the inoculum of 1% (V/V), inoculate a bacterial suspension with an OD value of about 1, cultivate at 30 ° C, 180 rpm shaker for 24 hours, and dynamically sample at 10, 15, 20, 32, 44, and 56 hours, respectively, and determine the strain. Growth ( _OD600 ) and IAA-producing capacity ( _OD530 ) were performed in triplicate for each treatment.

The results are shown in Figure 6: when the pH was 6.0, the OD ₆₀₀ and OD ₅₃₀ values of the strain both reached the maximum, the OD ₆₀₀ was 0.70, and the IAA concentration was 19.03 mg·L ^-1 . The pH of the sand ginger black soil itself is acidic, indicating that the living habits of the strains are consistent with the sand ginger black soil. Between pH 7.0 and 9.0, the growth and IAA content of the strain were relatively stable.

3. The effect of ventilation on the yield of strain IAA and the growth of strain

Pack the LB liquid medium containing 100mg·L ^-1 L-tryptophan into 250mL conical flasks in 25mL, 50mL, 75mL, 100mL, 150mL, and inoculate the OD value of about 1% (V/V) according to the inoculation amount. The bacterial suspension was cultured at 30°C with a shaker at 180 rpm, and samples were dynamically sampled at 10, 15, 20, 32, 44, and 56 h, respectively, to determine the growth of the strain (OD ₆₀₀ ) and the ability to produce IAA (OD ₅₃₀ ). Three repetitions.

The results are shown in Figure 7: Since strain n3 is aerobic bacteria, when the liquid volume of the 250mL conical flask is 25 mL, its growth status and IAA production conditions are optimal. The IAA content generally showed a downward trend.

4. The effect of nitrogen source on the yield and growth of strain IAA

0.1% (W/V) nitrogen sources were added to the inorganic salt medium (containing 100 mg·L ^-1 L-tryptophan) excluding ammonium sulfate, including potassium nitrate, ammonium sulfate, ammonium nitrate, yeast Powder, glutamic acid, urea, peptone, take 50 mL and put it in a 250 mL conical flask, inoculate a bacterial suspension with an OD value of about 1 according to the inoculum of 1% (V/V), and cultivate at 30 ° C, 180 rpm shaker for 24 h, Dynamic sampling was performed at 10, 15, 20, 32, 44, and 56 h, respectively, and the growth (OD ₆₀₀ ) and IAA-producing ability (OD ₅₃₀ ) of the strain were determined.

The results are shown in Figure 8: when the yeast powder was used as the nitrogen source, the growth amount (OD ₆₀₀ ) of the n3 strain was the largest, which was 0.64. At the same time, when the yeast powder was used as the nitrogen source, the strain produced the highest IAA content (OD ₅₃₀ ), which was as high as 36.18 mg·L ^-1 .

5. The effect of carbon source on the yield and growth of strain IAA

Add 1% (W/V) carbon source to the inorganic salt medium (containing 100 mg·L ^-1 L-tryptophan), respectively, the carbon sources are glucose, mannitol, sucrose, maltose, xylose, lactose, Fructose, take 50mL and put it in a 250mL conical flask, inoculate a bacterial suspension with an OD value of about 1 according to the inoculum of 1% (V/V), and culture it at 30 ° C and 180 rpm for 24 hours. Dynamic sampling was performed at 32, 44, and 56 h, and the growth status (OD ₆₀₀ ) and IAA-producing ability (OD ₅₃₀ ) of the strain were determined.

The results are shown in Figure 9: when mannitol was used as the carbon source, the ability to produce IAA was the highest, reaching 7.36 mg·L ^-1 , and the growth of the strain also reached the maximum.

Example 4:

Experiment of straw rot-promoting test of inoculum

Test process: Weigh 5g of wheat straw powder that has passed through a 20-mesh sieve after being crushed into a 250mL conical flask, add 30mL of water, add 2g of sodium nitrate, and add 10mL of bacterial liquid that has been centrifuged and resuspended in sterile water, so that the concentration of the bacterial liquid reaches 10 ⁸ About cfu·mL ^-1 , after culturing for 15 days, the straw was taken out, the side wall was repeatedly washed with distilled water, dried at 80 °C to constant weight, and sterile water was used to replace the bacterial liquid. The other steps were the same. As a control treatment, each treatment three repetitions.

The degradation test is shown in Figure 15: after 15 days of degradation, the degradation rate of the treated wheat straw with the inoculum reached 15.1%, which was 9.8% higher than that of the control.

Example 5:

Growth-promoting test of inoculum in corn

Test soil: Taken from the Agricultural Demonstration Park of Mengcheng County, Anhui Province

Test method: Spread corn seeds evenly in a petri dish with clean filter paper, soak them in distilled water at 28°C for 2 days, and select well-sprouted seeds for sowing. Take the flowerpot (diameter of upper mouth 5cm, diameter of lower bottom 3cm, height 5cm) as a culture container, each pot is filled with soil 5.0kg, and 2 corn plants are planted. The inoculum amount of ⁸ CFU/g was inoculated into the soil, and no inoculum was used as the control, with 5 replicates in each group. Before sowing, soak the germinated corn seeds with the above bacterial solution respectively. Put the soil into each pot at a distance of 1 cm from the upper edge, sow the soaked seeds into the pot, and then cover with a layer of floating soil. Sow one corn seed per pot, and plant 5 pots for each treatment. After sowing, the pots were placed in an artificial climate box (30° C. during the day and 25° C. at night. Lighting time was 16 hours). All treatments were randomly arranged under the same conditions, and immersion pot watering was performed at the same time. The dosages of N and K fertilizers were 2.0 g of urea and 1.4 g of potassium chloride per pot, respectively.

Sample harvesting: The corn was sampled after 49 days of growth, and the plant height, dry weight and fresh weight of the corn were measured.

Table 3 Effects of inoculated strain n3 on maize plants

Note: * means there is a significant difference between the two treatments (P<0.05), ** means there is a very significant difference between the two treatments (P<0.01)

The results are shown in Table 3: strain n3 has the effect of promoting plant growth. In this experiment, after inoculation treatment, the plant height and SPAD value of maize plants increased compared with the control treatment, and the difference was significant (P<0.5); Potassium was significantly different from the control treatment (P<0.01). Among them, the plant height and SPAD value of maize increased by 18.3% and 5.24% respectively compared with the control treatment, and the total potassium increased by 15.6% compared with the control. In conclusion: the inoculated plants were significantly better than the control treatment in terms of morphology and nutrient absorption.

The invention provides an IAA-producing cellulose-degrading bacterium n3 and its application, which has the ability to produce IAA and CMC enzymes, can promote the rot of straw and improve the cellulose degradation rate, thereby promoting the efficiency of straw returning to the field, reducing environmental pollution, and realizing Crop yields increased.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Anhui Agricultural University

<120> A cellulose-degrading bacterium n3 producing IAA and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 560

<212> DNA

<213> Azospirillum zeae

<400> 1

cctacgggag gcagcagtgg ggaatattgg acaatgggcg caagcctgat ccagcaatgc 60

cgcgtgagtg atgaaggcct tagggttgta aagctctttc gcacgcgacg atgatgacgg 120

cagcgtgaga agaagccccg gctaacttcg tgccagcagc cgcggtaata cgaagggggc 180

tagcgttgtt cggaattact gggcgtaaag ggcgcgtagg cggcctgttt agtcagaagt 240

gaaagctccg ggctcaacct gggaatagct tttgatactg gcaggcttga gttccggaga 300

ggatggtgga attcccagtg tagaggtgaa attcgtagat attgggaaga acaccggtgg 360

cgaaggcggc catctggacg gacactgacg ctgaggcgcg aaagcgtggg gagcaaacag 420

gattagatac cctggtagtc cacgccgtaa acgatgaatg ctagacgtcg gggtgcatgc 480

acttcggtgt cgccgctaac gcattaagca ttccgcctgg ggagtacggc cgcaaggtta 540

aaactcaaag gaattgacgg 560

Claims (10)

1. A cellulose-degrading bacteria Azospirillum zeae ( Azospirillum zeae ) n3 that produces IAA, is characterized in that, it is preserved in China General Microorganism Culture Collection and Management Center, and the deposit number is CGMCC No.18613. 2. The application of the cellulose-degrading bacteria Azospirillum zeae n3 according to claim 1 in the preparation of IAA and/or CMC enzymes. 3. application according to claim 2 is characterized in that, when utilizing described cellulose degrading bacteria Azospirillum zeae n3 to prepare IAA, comprising the following steps: regulate the LB medium containing 100mg/L L-tryptophan. The pH value is 6.0-9.0, inoculate the bacterial suspension of the cellulose-degrading bacteria Azospirillum zeae n3, and shake culture; the volume of the bacterial suspension is 1% of the volume of the LB medium; the bacterial suspension The OD ₆₀₀ value is 0.8 to 1.2. 4 . The application according to claim 3 , wherein the temperature of the shaking culture is 28-30° C., and the shaking speed is 160-180 rpm. 5 . 5. application according to claim 2, is characterized in that, when utilizing described cellulose degrading bacteria Azospirillum zeae n3 to prepare CMC enzyme, comprises the following steps: adjusting the pH value of liquid fermentation medium to 4.0～6.0, inoculating The cellulose-degrading bacterium Azospirillum zeae n3 was cultured with shaking; the inoculation volume of the cellulose-degrading bacterium Azospirillum zeae n3 was 1% of the volume of the liquid fermentation medium; the liquid fermentation medium included the following concentrations Raw materials: sodium chloride 6g/L, magnesium sulfate heptahydrate 0.1g/L, calcium chloride 0.1g/L, potassium dihydrogen phosphate 0.5g/L, yeast extract 10g/L and straw 20g/L. 6 . The application according to claim 5 , wherein the LB medium or the liquid fermentation medium further comprises the following components by mass: 0.1% carbon source and 1% nitrogen source. 7 . 7. The application according to claim 6, wherein the carbon source comprises one or more of glucose, mannitol, sucrose, maltose, xylose, lactose and fructose; The nitrogen source includes one or more of potassium nitrate, ammonium sulfate, ammonium nitrate, yeast powder, glutamic acid, urea and peptone. 8. The application of the cellulose-degrading bacteria Azospirillum zeae n3 according to claim 1 in the preparation of a decay-promoting and growth-promoting microbial agent. 9. application according to claim 8, it is characterized in that, the type of described decay-promoting bacteria agent is bacteria water agent; When described bacteria water agent is in application, with described cellulose degrading bacteria Azospirillum zeae n3 The inoculum amount is 1-9×10 ⁶ CFU/g straw or 1-9×10 ⁷ CFU/g soil. 10 . The application of the cellulose-degrading bacteria Azospirillum maize n3 according to claim 1 in promoting straw returning efficiency and increasing crop yield. 11 .

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