NL2027089B1

NL2027089B1 - Iaa-producing and cellulose-degrading strain n3 and use thereof

Info

Publication number: NL2027089B1
Application number: NL2027089A
Authority: NL
Inventors: Zhang Ziyun; Nie Cai'e; Zhu Lin; Tian Da; Ma Chao; Gao Hongjian; Chai Rushan; Wu Jing; Wu Liangping; Wang Yubao
Original assignee: Univ Anhui Agricultural
Priority date: 2019-12-11
Filing date: 2020-12-10
Publication date: 2022-03-04
Also published as: CN111057665B; CN111057665A; NL2027089A

Abstract

The present disclosure belongs to the technical ﬁeld of agricultural microorganisms and provides an indole-3-acetic acid (IAA)-producing and cellulose-degrading strain n3 and use thereof. The cellulose-degrading strain n3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with a deposition number of CGMCC No. 18613. The cellulose- degrading strain n3 of the present disclosure can produce a high content of IAA which can reach 19.07 mg/L and carboxymethyl cellulose (CMC) enzyme whose activity can reach 24.96 U/mL. Therefore, the cellulose-degrading strain n3 can be used to prepare an IAA and/or CMC enzyme producing bacterial agent or a growth-promoting and straw-degrading bacterial agent for use in promoting straw degradation and crop growth to increase efficiency of the recycling and application of straw and crop yield.

Description

-1- IAA-PRODUCING AND CELLULOSE-DEGRADING STRAIN N3 AND

USE THEREOF

TECHNICAL FIELD The present disclosure belongs to the technical field of agricultural microorganisms, and specifically relates to an indole-3-acetic acid (IAA)- producing and cellulose-degrading strain n3 and use thereof.

BACKGROUND The recycling and application of straw is a measure taken to improve soil fertility to increase crop yields. It is generally well accepted in the world today since it eliminates air pollution caused by straw burning and improves physical properties and organic matter levels of soil. Moreover, it plays an important part in increasing soil biological activity and nutrient supply and other aspects. Straw is rich in organic matter and nutrient elements such as nitrogen, phosphorus and potassium, which can be converted into nutrient forms easily absorbed and utilized by crops. The recycling and application of straw can effectively improve resource utilization, increase crop yield, and promote nutrient recycling. Straw mainly includes lignin, cellulose, hemicellulose and other difficult-to-degrade substances, where the cellulose has a highest content. Naturally, the straw is decomposed at a relatively slow rate. At present, scholars at home and abroad generally believe that during the recycling and application of straw, straw-decomposing microorganisms which produce cellulose-degrading enzymes (such as carboxymethyl cellulose (CMC) degrading enzyme) can be added to accelerate decomposition of straw and other wastes. However, colonization of functional microorganisms in a straw-decomposing agent is affected by abundance of soil resources and competition with indigenous microorganisms and other factors. Therefore, the straw-decomposing agent is highly specific to regions, where different straw-decomposing agents shall be applied to different soils in different regions. As a result, it is necessary to screen strains in an application area to prepare a straw-

-2- decomposing microorganism agent to improve degree and rate of straw decomposition in the area.

Shajiang black soil has certain undesirable features.

It shrinks under dry conditions, swells under wet conditions, and is susceptible to drought and flooding.

Moreover, it is poorly arable with low fertility, which has an adverse effect on crop growth and is a typical low-yield soil in China.

Shajiang black soil has heavy stickiness and poor soil structure.

It can easily lead to problems such as drought, waterlogged, stiff and thinning during production.

It has low activities of soil microorganisms and low fertility in a cultivated layer, which is not conducive to the colonization of foreign strains.

Therefore, a general straw-decomposing agent has a relatively poor effect when applied to the Shajiang black soil.

The Shajiang black soil is mostly used for cultivating wheat and maize alternatively with a short rotation interval.

If straws cannot be completely decomposed by the straw-decomposing agent, straws of the previous crops will stay in soil for a long time, affecting sowing of later crops.

Moreover, the straws in the soil which are not completely decomposed will prevent contact of wheat or maize seeds with the soil, so that the seeds are difficult to germinate.

Furthermore, straws remaining in the soil will compete with the crops for nutrients, resulting in lower crop emergence rate and lower yield.

IAA is a plant hormone which produces a signal for regulation of plant growth.

It is present in a wide range of plants as an endogenous auxin.

The auxin promotes growth mainly by promoting cell growth, especially cell elongation, which has a positive effect on crop growth and yield increase.

Therefore, an efficient decomposition-promoting and growth- promoting strain is beneficial to growth and development of wheat and maize alternatively cultivated in the Shajiang black soil and helps maintain excellent soil properties.

At present, most scholars at home and abroad only conduct researches on cellulose-degrading capacity or growth-promoting capacity

-3- of a certain strain, and focus on a certain function of a certain strain. However, researches on strains with multiple functions are rarely seen.

SUMMARY In view of this, an objective of the present disclosure is to provide an efficient CMC enzyme-producing and cellulose-degrading strain n3 screened from Shajiang black soil. The strain can accelerate cellulose and straw degradation, and thereby promote the recycling and application of straw. Moreover, the strain can produce a high content of IAA to promote germination and growth of crop seeds to increase crop yields.

To achieve the above objective, the present disclosure provides the following technical solutions.

The present disclosure provides an IAA-producing and cellulose- degrading strain n3, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with a deposition number of CGMCC No. 18613.

The present disclosure also provides use of the cellulose-degrading strain n3 in preparation of IAA and/or CMC enzyme.

Preferably, the use of the cellulose-degrading strain n3 in preparation of IAA may include the following steps: adjusting pH of LB medium containing 100 mg/L L-tryptophan to 6.0-9.0, inoculating a suspension of the cellulose-degrading strain n3 with ODsoo of 0.8-1.2 in a volume of 1% of that of the LB medium, and culturing under shaking.

Preferably, the culturing under shaking may be carried out at 28-30°C at 160-180 rpm.

Preferably, the use of the cellulose-degrading strain n3 in preparation of CMC enzyme may include the following steps: adjusting pH of liquid fermentation medium to 4.0-6.0, inoculating the cellulose-degrading strain n3 in a volume of 1% of that of the liquid fermentation medium, and culturing under shaking, where the liquid fermentation medium includes the following raw materials: 6 g/L of sodium chloride, 0.1 g/L of magnesium sulphate heptahydrate, 0.1 g/L of calcium chloride, 0.5 g/L of

-4- potassium dihydrogen phosphate, 10 g/L of yeast extract, and 20 g/L of straw.

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

Preferably, the carbon source may include one or more of glucose, mannitol, sucrose, maltose, xylose, lactose and fructose; the nitrogen source may include one or more of potassium nitrate, ammonium sulphate, ammonium nitrate, yeast powder, glutamic acid, urea, and peptone.

The present disclosure also provides use of the cellulose-degrading strain n3 in preparation of a degradation-promoting and growth-promoting bacterial agent.

Preferably, the degradation-promoting and growth-promoting bacterial agent may be an aqueous bacterial agent. When in use, the aqueous bacterial agent may be inoculated to straw in an amount of (1- 9)x108 CFU/g or to soil in an amount of (1-9)x107 CFU/g based on the cellulose-degrading strain n3.

The present disclosure also provides use of the cellulose-degrading strain n3 in promoting the recycling and application of straw and increasing crop yield.

The present disclosure provides an IAA-producing and cellulose- degrading strain n3, which is deposited at the CGMCC with a deposition number of CGMCC No. 18613. The cellulose-degrading strain n3 of the present disclosure has a smooth surface, and opaque, slightly yellow and relatively small colonies with neat edges. It is an aerobic chemoheterotrophic gram-negative bacteria, which shows a positive result in contact enzyme assay and negative results in methyl red (M.R) test, Voges-Proskauer (VP) test, starch hydrolysis test, gelatine hydrolysis test, and citrate utilization test.

The cellulose-degrading strain n3 of the present disclosure can produce a high content of IAA which can reach 19.07 mg/L and CMC

-5. enzyme with a relatively high activity which can reach 24.96 U/mL, showing relatively strong capabilities in promoting crop growth and straw degradation. Therefore, the cellulose-degrading strain n3 can be used to prepare a growth-promoting and straw-degrading bacterial agent, so as to be used in promoting straw degradation and the recycling and application of straw and increasing crop yield.

The cellulose-degrading strain n3 of the present disclosure can be used for multiple purposes to achieve full use of the strain. It can be used to promote crop growth to increase crop yield, and accelerate cellulose and straw degradation to promote the recycling and application of straw. It has the potential as a functional microbial fertilizer to promote green agricultural development.

Information of Biological Deposit The cellulose-degrading strain n3, classified as Azospirillum zeae, was deposited at the CGMCC on September 23, 2019, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology of Chinese Academy of Sciences with a deposit number of CGMCC No.18613.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows colonies of the cellulose-degrading strain n3 provided by the present disclosure; FIG. 2 shows CMC enzyme production capacities of different strains; FIG. 3 shows IAA production capacities of different strains; FIG. 4 shows a phylogenetic tree of the strain n3 constructed based on 16S rDNA sequence; FIG. 5 shows effect of different pH on activity of CMC enzyme produced by the cellulose-degrading strain n3; FIG. 6 shows effect of different loaded liquid volumes on activity of CMC enzyme produced by the cellulose-degrading strain n3; FIG. 7 shows effect of different nitrogen sources on activity of CMC enzyme produced by the cellulose-degrading strain n3;

-6- FIG. 8 shows effect of different culturing time on IAA produced by the cellulose-degrading strain n3; FIG. 9 shows effect of different culturing time on growth of the cellulose-degrading strain n3; FIG. 10 shows effect of different loaded liquid volumes on IAA produced by the cellulose-degrading strain n3; FIG. 11 shows effect of different loaded liquid volumes on growth of the cellulose-degrading strain n3; FIG. 12 shows effect of different initial pH on IAA produced by the cellulose-degrading strain n3; FIG. 13 shows effect of different initial pH on growth of the cellulose- degrading strain n3; FIG. 14 shows effect of different carbon sources on IAA produced by the cellulose-degrading strain n3; FIG. 15 shows effect of different carbon sources on growth of the cellulose-degrading strain n3; FIG. 16 shows effect of different nitrogen sources on IAA produced by the cellulose-degrading strain n3; FIG. 17 shows effect of different nitrogen sources on growth of the cellulose-degrading strain n3; FIG. 18 shows straw degradation-promoting capacities of different strains.

DETAILED DESCRIPTION The present disclosure provides an IAA-producing and cellulose- degrading strain n3, which is deposited at the CGMCC with a deposition number of CGMCC No. 18813.

The cellulose-degrading strain n3 of the present disclosure is screened from Shajiang black soil collected in the Agricultural Science and Technology Demonstration Park in Mengcheng County, Anhui Province of China. The cellulose-degrading strain n3 is confirmed as an aerobic chemoheterotrophic gram-negative bacterium which shows a positive result in contact enzyme assay and negative results in M.R test,

-7- VP test, starch hydrolysis test, gelatine hydrolysis test and citrate utilization test. The strain is shown in FIG. 1 having a smooth surface, and opaque, slightly yellow and relatively small colonies with neat edges.

In the present disclosure, the use of the cellulose-degrading strain n3 in preparation of IAA may preferably include the following steps: adjusting pH of LB medium containing 100 mg/L L-tryptophan to 6.0-9.0, inoculating a suspension of the cellulose-degrading strain n3 with ODeoo of 0.8-1.2 in a volume of 1% of that of the LB medium, and culturing under shaking, where the suspension is obtained by picking a loop of bacteria from a solid medium with an inoculating loop, inoculating into a 25 ml test tube containing 6 ml of LB medium, stoppering the test tube, placing the test tube in a shaker, and culturing at 30°C and 180 rpm overnight. In the present disclosure, the culturing under shaking may be preferably carried out with a medium loaded liquid volume of 25 mL/250 mL, and culturing time of preferably 15 h. At this point, cultivated cellulose-degrading strain n3 produces the highest amount of IAA with optimal strain growth. Preferably, the culturing under shaking may be carried out at 28-30°C and 160-180 rpm.

In the present disclosure, the use of the cellulose-degrading strain n3 in preparation of CMC enzyme may preferably include the following steps: adjusting pH of liquid fermentation medium to 4.0-6.0, inoculating the cellulose-degrading strain n3 in a volume of 1% of that of the LB medium, and culturing under shaking, where the liquid fermentation medium includes the following raw materials: 6 g/L of sodium chloride,

0.1 g/L of magnesium sulphate heptahydrate, 0.1 g/L of calcium chloride,

0.5 g/L of potassium dihydrogen phosphate, 10 g/L of yeast extract, and 20 g/L of straw.

Preferably, the LB medium or the liquid fermentation medium may preferably further include the following components by mass percentage:

0.1% of carbon source and 1% of nitrogen source. In the present

-8- disclosure, the carbon source in the LB medium may preferably include one or more of glucose, mannitol, sucrose, maltose, xylose, lactose and fructose, more preferably mannitol, the nitrogen source may preferably include one or more of potassium nitrate, ammonium sulphate, ammonium nitrate, yeast powder, glutamic acid, urea, and peptone, more preferably yeast powder. In the present disclosure, the nitrogen source in the liquid medium may preferably include one or more of potassium nitrate, ammonium sulphate, ammonium nitrate, yeast powder, glutamic acid, urea, and peptone, more preferably yeast powder or peptone.

The present disclosure also provides use of the cellulose-degrading strain n3 in preparation of degradation-promoting and growth-promoting bacterial agent.

In the present disclosure, the degradation-promoting and growth- promoting bacterial agent may be preferably an aqueous bacterial agent.

When in use, the aqueous bacterial agent may be inoculated to straw in an amount of preferably (1-9)x108 CFU/g and more preferably 5x10° CFU/g, or to soil in an amount of preferably (1-9)x107 CFU/g and more preferably 5x107 CFU/g based on the cellulose-degrading strain n3.

The present disclosure also provides use of the cellulose-degrading strain n3 in promoting the recycling and application of straw and increasing crop yield. In the use of the present disclosure, the cellulose- degrading strain n3 is used with a method and an amount the same as those described above, and will not be repeated herein.

The IAA-producing and cellulose-degrading strain n3 and use thereof provided by the present disclosure will be described in detail in connection with the following embodiments, but they should not be construed as limiting the claimed scope of the present disclosure. Example 1

1. Preparation of reagents: LB medium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride and 1,000 mL of distilled water, adjusted to pH 7.0-7.2, sterilized

-9- at 121°C for 20 min (for a solid medium, 20 g of agar was added to this formula). Inorganic salt medium: 2.0 g of ammonium sulphate, 0.5 g of sodium dihydrogen phosphate, 0.5 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulphate heptahydrate, 0.1 g of calcium dichloride and 1,000 mL of distilled water, adjusted to pH 7.0-7.2, sterilized at 121°C for 20 min. Liquid fermentation medium: 6 g of sodium chloride, 0.1 g of magnesium sulphate heptahydrate, 0.1 g of calcium chloride, 0.5 g of potassium dihydrogen phosphate, 10 g of yeast extract, 20 g of straw and 1,000 mL of distilled water, sterilized at 121°C for 20 min. Enrichment medium: 20 g of sodium carboxymethyl cellulose (CMC- Na), 5 g of microcrystalline cellulose, 5 g of cellulose powder, 1 g of dipotassium hydrogen phosphate, 1 g of nitric acid, 0.2 g of magnesium sulphate heptahydrate, 0.1 g of copper chloride dihydrate, 0.02 g of ferric chloride and 1,000 mL of distilled water, sterilized at 121°C for 20 min. CMC medium: 15 g of CMC-Na, 1 g of ammonium nitrate, 1 g of yeast extract, 0.5 g of magnesium sulphate heptahydrate, 1 g of potassium dihydrogen phosphate, 15 g of agar, and 1,000 mL of distilled water, sterilized at 121°C for 20 min.

2. Screen of strain 10 g of Shajiang black soil was collected from the Agricultural Science and Technology Demonstration Park in Mengcheng County, Anhui Province of China, and added to 90 mL of sterile water, shaken on a shaker at 150 rpm and 28°C for 30 min. 1 mL of soil suspension was taken on a sterile operating table and added to 9 mL of sterile water to prepare a stock solution with a concentration of 101. Strains were isolated and purified by a dilution-plate method, and pure culture strains were stored in a refrigerator at 4°C. Test soil had basic properties as shown in Table 1:

-10 - Table 1 Basic properties of test soll Alkali- Organic Total Available Available hydrolyzable matter Nitrogen | phosphorus potassium nitrogen (g'kg™) (g'kg™) (mg-kg™) (mg-kg™) (mg-kg™)

12.46 0.137 80.2 15.4 100.3 Isolated and purified strains were respectively inoculated on CMC-Na selective media, and placed for 20 min based on a Congo red staining method. Then colony diameter (D) and transparent circle diameter (H) were measured. Based on H/D, cellulose degradation capacities of the strains were preliminary determined.

All the strains which can degrade cellulose were screened out with the Congo red staining method. H/D results showed that, the strain n3 had the strongest cellulose degradation capacity.

Preparation of crude enzyme solution: strains were inoculated to liquid media with wheat straw powder as the sole carbon source, and cultured in the liquid media in flasks under shaking at 37°C for 60 h. A fermentation broth was centrifuged at 4°C and 5,000 rpm for 10 min. A supernatant was obtained as a crude enzyme solution. 0.2 mL of the supernatant was taken and added into a 25 mL dry graduated test tube.

1.8 mL of 1% CMC-Na solution (pH 4.8, prepared with 0.1 mol/L citric acid-sodium citrate buffer) was added and incubated with a water bath at 50°C for 30 min. 3.0 mL of 3,5-dinitrosalicylic acid (DNS) reagent was added and incubated with a boiling water bath for 5 min. Then, a reaction was stopped and colour was developed. After cooling by flowing cold water, a solution was diluted to 25 mL, shaken uniformly, and measured for absorbance at 520 nm. A blank control was an enzyme solution treated under the same conditions except that it was inactivated in a boiling water bath for 15 min.

-11 - In this experiment, enzyme activity X=1,000xGx25/0.2x30x180. In the formula: X: enzyme activity of sample (U/g); 1,000: conversion factor; G: milligrams of glucose corresponding to light absorption value on a standard curve; 25: constant volume (mL); 0.2: amount of enzyme (mL); 30: action time (min); 180: molecular weight of glucose (g/mol). Under the above conditions, enzyme activity was defined based on international unit as follows: the amount of enzyme that catalysed hydrolysis of substrate (CMC-Na) to produce 1 pmol of glucose per minute was defined as 1 enzyme activity unit U.

Among the 8 selected strains, the strain n3 had the strongest cellulose-degrading capacity which was significantly higher than the other strains, where CMC enzyme activity thereof can reach 20.60 U-mL™".

Qualitative determination: bacteria with cellulose-degrading capacity were inoculated in LB liquid media containing L-tryptophan (100 mg-L™), cultured at 30°C and 180 rpm for 1 d. 100 pL of bacterial suspension was dripped on a white ceramic plate, and at the same time, 100 pL of Salkowski colorimetric solution (50 mL of 35% HCIO4, 1 mL of 0.5 mol-L-* FeCl, stored in the dark) was added. The white ceramic plate was then placed at room temperature for 30 min in the dark and observed. A colour changed to red indicated that the strain can secrete IAA. Darkness or lightness of the colour represented level of IAA-producing capacity. An unchanged colour indicated that the strain produced no IAA. A positive control was a mixture of 100 uL of LB medium without inoculation of bacteria and 100 pL of Salkowski colorimetric solution.

Quantitative determination: the strains which can secrete IAA as screened by the qualitative analysis were quantitatively determined. Culture conditions were the same as those in the qualitative determination. A bacterial suspension was determined for ODsoo value by spectrophotometry. Then 5 mL of bacterial suspension was centrifuged at 10,000 rpm for 10 min. 2 mL of supernatant was added to an equal volume of Salkowski colorimetric solution, allowed to stand still in the dark at room temperature for 30 min and measured at 530 nm for OD

-12- value. An IAA standard curve was used to calculate IAA content of the bacterial suspension.

After IAA qualitative analysis, 5 strains (namely n1, n3, n4, n5, and n8) were determined to have the capability to produce IAA. Subsequent quantitative determination showed that, the strain n3 had the strongest capability to produce IAA with a concentration of IAA up to 19.07 mg:L", which was significantly higher than those of other strains.

Through the above determinations, the cellulose-degrading strain n3 was screened with the strongest capabilities to produce CMC enzyme and IAA, showing relatively strong capability of promoting wheat straw decomposition.

The strain screened and isolated by the above method was sequenced by Nanjing Company. Based on obtained 16S rDNA (SEQ ID NO.1) sequence, comparison was made in the GenBank database with Blast searching for homologous sequences. The MEGAS.0 software was used to construct a phylogenetic tree with the Neighbour-Joining method. Based on morphological analysis and physiological and biochemical characteristics of the strain, the strain was identified as azospirillum zeae. The phylogenetic tree of the strain n3 constructed based on the 16S rDNA sequence was shown in FIG. 4.

Physiological and biochemical properties of the strain were sum up and shown in Table 2: Table 2 Physiological and biochemical characteristics of cellulose- degrading strain n3 Item result Gelatin hydrolysis - VP reaction - M.R reaction - Contact enzyme + Citrate utilization -

-13- Starch hydrolysis - Aerobic test Aerobic Gram staining - Note: + represented positive reaction, - represented negative reaction. Example 2 Effects of different pH, ventilation, and nitrogen source on CMC enzyme-producing capability of strain

1. Effect of initial pH of medium on CMC enzyme production The strain was inoculated into a liquid fermentation medium with wheat straw powder as the sole carbon source, cultured in liquid medium in a flask under shaking at 37°C for 60 h, and measured for ODs20 with a spectrophotometer. An initial pH was set to 4, 5, 6, 7, 8 9, and 10 respectively. After cultivation for 60 h, content of CMC enzyme produced was measured with a spectrophotometer. Results were shown in FIG. 2. CMC enzyme activity was the highest at pH 5.0, reaching 24.96 U-mL"", and lower at pH 4.0, being 24.85 U:mL: '. This indicated that the strain n3 had relatively strong acid resistance, and the CMC enzyme activity at pH 4.0 and 5.0 was significantly higher than that under other pH conditions.

2. Effect of ventilation on CMC enzyme production The strain was inoculated into a liquid fermentation medium with wheat straw powder as the sole carbon source, cultured in liquid medium in a flask under shaking at 37°C for 60 h, and measured for ODs20 with a spectrophotometer. 25, 50, 75, 100 and 150 mL of culture solution were loaded in a 250 mL Erlenmeyer flask respectively. After cultivation for 60 h, content of CMC enzyme produced was measured with a spectrophotometer. Results were shown in FIG. 3. When loaded liquid volume in the 250 mL Erlenmeyer flask was 25 mL with maximum ventilation, activity of

-14 - CMC enzyme produced by strain n3 was the highest, reaching 15.33 U:mL-.

3. Effect of nitrogen source on CMC enzyme production

0.1% (m/V) of nitrogen source was added to a liquid fermentation medium with wheat straw powder as the sole carbon source. The nitrogen source included potassium nitrate, ammonium sulphate, ammonium nitrate, yeast powder, glutamic acid, urea or peptone. The strain was cultured in liquid medium in a flask under shaking at 37°C for 60 h, and measured for CMC enzyme content with a spectrophotometer. Results were shown in FIG. 4. Different nitrogen sources had different effects on CMC enzyme-producing capacity of strain n3. Among the nitrogen sources, yeast powder and peptone resulted in the most significant CMC enzyme production, reaching 18.30 and 16.02 U:mL* respectively.

Example 3 Effects of different pH, ventilation, time, carbon source and nitrogen source on IAA production and growth of strain

1. Effect of fermentation time on IAA production and growth of strain 50 mL of LB liquid medium (IAA detection medium) containing 100 mg:Lt L-tryptophan was added in a 250 mL Erlenmeyer flask. A bacterial suspension with OD value of about 1 was inoculated in an amount of 1% (V/V) and cultivated on a shaker at 30°C and 180 rpm. Dynamic sampling was carried out at 10, 15, 20, 32, 44, and 56 h respectively to determine growth of the strain (ODsoo) and IAA produced by the strain (ODsao), with three repetitions for each treatment.

Results were shown in FIG. 5. ODeoo reached the maximum at 20 h and showed a decline trend after 20 h. The content of IAA produced was basically consistent with growth of the strain. At 10-15 h, the content of IAA produced by the strain increased logarithmically. The IAA content reached the highest 18.66 mg:L" at 15 h and gradually decreased after 15h.

-15-

2. Effect of pH on IAA production and growth of strain LB liquid medium containing 100 mg:L: L-tryptophan was adjusted to different pH (4, 5, 6, 7, 8, 9 and 10). 50 mL of LB liquid medium was taken and added to a 250 mL Erlenmeyer flask. A bacterial suspension with OD value of about 1 was inoculated in an amount of 1% (V/V) and cultivated on a shaker at 30°C and 180 rpm for 24 h. Dynamic sampling was carried out at 10, 15, 20, 32, 44, and 56 h respectively to determine growth of the strain (ODeoo) and IAA produced by the strain (ODs30), with three repetitions for each treatment.

Results were shown in FIG. 6. When the pH was 6.0, both the ODsoo and ODs3 values of the strain reached the maximum values with ODsoo of 0.70 and IAA concentration of 19.03 mg-L"'. The Shajiang black soil was acidic itself. The results indicated that living conditions of the strain matched Shajiang black soil. When pH was 7.0-9.0, growth of the strain and content of IAA produced by the strain were relatively stable.

3. Effect of ventilation on IAA production and growth of strain 25 mL, 50 mL, 75 mL, 100 mL and 150 mL of LB liquid medium containing 100 mg-L' L-tryptophan were added into a 250 mL Erlenmeyer flask respectively. A bacterial suspension with OD value of about 1 was inoculated in an amount of 1% (V/V) and cultivated on a shaker at 30°C and 180 rpm. Dynamic sampling was carried out at 10, 15, 20, 32, 44, and 56 h respectively to determine growth of the strain (ODsoo) and IAA produced by the strain (ODs39), with three repetitions for each treatment.

Results were shown in FIG. 7. Since the strain n3 was an aerobic bacterium, when the loaded liquid volume was 25 mL in a 250 mL Erlenmeyer flask, conditions were optimal for growth and IAA production. With increase of the loaded liquid volume, the strain showed an overall decreasing trend in growth and IAA production.

4. Effect of nitrogen source on IAA production and growth of strain

0.1% (W/V) nitrogen source was added to the inorganic salt medium (containing 100 mg-L-! L-tryptophan) without ammonium sulphate. The

-16 - nitrogen source included potassium nitrate, ammonium sulphate, ammonium nitrate, yeast powder, glutamic acid, urea or peptone. 50 mL of the medium was taken and added to a 250 mL Erlenmeyer flask. A bacterial suspension with OD value of about 1 was inoculated in an amount of 1% (V/V) and cultivated on a shaker at 30°C and 180 rpm for 24 h. Dynamic sampling was carried out at 10, 15, 20, 32, 44, and 56 h respectively to determine growth of the strain (ODesoo)} and IAA produced by the strain (ODsao). Results were shown in FIG. 8. When yeast powder was used as the nitrogen source, the strain n3 achieved maximum growth (ODeoo) of 0.64 and produced the highest content of IAA (OD:s30) which reached 36.18 mg-L™*.

5. Effect of carbon source on IAA production and growth of strain 1% (W/V) of carbon source was added to the inorganic salt medium (containing 100 mg:L’ L-tryptophan) respectively. The carbon source included glucose, mannitol, sucrose, maltose, xylose, lactose or fructose. 50 mL of the medium was taken and added to a 250 mL Erlenmeyer flask. A bacterial suspension with OD value of about 1 was inoculated in an amount of 1% (V/V) and cultivated on a shaker at 30°C and 180 rpm for 24 h. Dynamic sampling was carried out at 10, 15, 20, 32, 44, and 56 h respectively to determine growth of the strain (ODsoo) and IAA produced by the strain (ODsao). Results were shown in FIG. 9. When mannitol was used as the carbon source, IAA production was the highest, reaching 7.36 mg-L™, and the strain also achieved maximum growth. Example 4: Test of bacterial agent in promoting straw degradation Test process: 5 g of wheat straw powder which had passed through a 20 mesh sieve was added into a 250 mL Erlenmeyer flask. 30 mL of water and 2 g of sodium nitrate were added. 10 mL of bacterial solution obtained by centrifuging and resuspending in sterile water was added, so

-17 - that a bacterial concentration reached about 108 cfu-mL-'. After culturing for 15 d, the straw powder was taken out with side wall of the flask repeatedly washed by distilled water, and dried at 80°C to a constant weight. As a control, sterile water was used to replace the bacterial solution while other steps were the same. Each treatment was repeated three times. The degradation test results were shown in FIG. 15. After degradation for 15 d, wheat straw treated with the bacterial agent showed a degradation rate of 15.1%, which was higher than the control group by

9.8%. Example 5: Test of bacterial agent in promoting maize growth Test soil: collected from the Agricultural Science and Technology Demonstration Park in Mengcheng County, Anhui Province of China. Test method: maize seeds were spread uniformly in a petri dish laid with clean filter paper, and immersed in distilled water at 28°C for 2 d. Seeds with desired and neat germination were selected for sowing. Flower pots (with an upper mouth diameter of 5 cm, a lower bottom diameter of 3 cm, and a height of 5 cm) were used as culture containers. Each pot was filled with 5.0 kg of soil to cultivate 2 maize plants. The test strain n3 was cultivated, prepared into an aqueous bacterial agent, and inoculated into the soil in an amount of 5x10% CFU/g. A control had no bacterial agent. Each group had 5 repetitions. Before sowing, pre- germinated maize seeds were immersed with the above bacterial liquid respectively. The soil was filled in each pot with a surface of the soil lower than an upper edge of the pot by 1 cm. Immersed seeds were sowed into the pots, and covered with a layer of floating soil. One maize seed was sown to each pot, and 5 pots were used for each treatment. After sowing, the flower pots were placed in an artificial climate box (30°C during the day and 25°C at night, lighting time was 16 h). All treatments were arranged randomly under the same conditions, and watering was carried

-18- out at a same time point. Dosages of N and K fertilizers were 2.0 g of urea and 1.4 g of potassium chloride per pot. Sample harvest: maize plants were sampled after 49 d of growth to measure plant height, plant dry weight and fresh weight.

Table 3 Effect of inoculation of strain n3 on maize plants Fresh weight | | Dry weight of Total Plant height SPAD of | Treatment aboveground potassium (cm) value aboveground parts (9) (g/kg) parts (9)

3.91+0.4 5161£0.5 2.75+0.1 CK 23.50+2.29 40.23+1.62 2 9 4

4.12+0.2 5.4310.5 n3 27.80£5.02742.34x0.827 5 3.18+0.27** Note: * indicates that there was a significant difference between two treatments (P<0.05), and ** indicates that there was a very significant difference between two treatments (P<0.01).

Results in Table 3 showed that the strain n3 can promote plant growth. In this experiment, maize plants inoculated with the bacteria had increased plant height and SPAD value with a significant difference (P<0.5) and total potassium with a very significant difference (P<0.01) compared with the control treatment. The plant height and the SPAD value were increased by 18.3% and 5.24% respectively and the total potassium was increased by 15.6% compared with the control treatment. In summary, the plants inoculated with the bacteria were significantly better than the control treatment in morphology and nutrient absorption. The present disclosure provides an IAA-producing and cellulose- degrading strain n3 and use thereof. The strain n3 can produce IAA and CMC enzyme to promote degradation of straw and cellulose, thereby

-19- promoting the recycling and application of straw, reducing environmental pollution and increasing crop yield.

The above descriptions are merely preferred implementations of the present disclosure.

It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

-20-

SEQUENCE LISTING <110> Anhui agriculture university <120> |AA-producing and cellulose-degrading strain n3 and use thereof <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

-21- acttcggtgt cgccgctaac gcattaagca ttccgcctgg ggagtacggc cgcaaggtta 540 aaactcaaag gaattgacgg 560

2027089SEQ.TXTUSB Sequence Listing <110> Anhui agriculture university <120> IAA-producing and cellulose-degrading strain n3 and use thereof <140> N2027089 <141> 10/12/2020 <150> CN201911267554.7 <151> 11/12/2019 <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 Pagina 1

Claims

_22- Conclusions

An indole-3-acetic acid (IAA) producing a cellulose-degrading n3 strain, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with a deposit number of CGMCC No. 18613.

Use of the cellulose-degrading n3 strain according to claim 1 for the preparation of IAA and/or carboxymethylcellulose enzyme (CMC).

The use according to claim 2, wherein the use of the cellulose-degrading n3 strain in preparation of IAA comprises the steps of: adjusting pH of Lb medium with 100 mg/L L-tryptophan to 6.0-9.0 and a suspension of the cellulose-degrading n3 strain with OD 50 0.8 -1.2 in a volume of 1% that of the LB medium, and cultured by shaking.

The use according to claim 3, wherein the shaking culture at 28-30°C and 160-180 rpm is used.

The use according to claim 2, wherein the use of the cellulose-degrading n3 strain to prepare the CMC enzyme comprises the steps of: adjusting pH of a liquid fermentation medium to 4.0-6.0 and the cellulose -degrading n3 strain in a volume of 1% of that of the liquid fermentation medium and culturing with shaking, wherein the liquid fermentation medium contains the following raw materials: 6 g/l sodium chloride, 0.1 g/l magnesium sulphate heptahydrate, 0.1 g/l calcium chloride, 0.5 g/l potassium dihydrogen phosphate, 10 g/l yeast extract and 20 g/l straw.

The use according to claim 3 or claim 5, wherein the LB medium or liquid fermentation medium further contains the following components by mass percentage: 0.1% carbon source and 1% nitrogen source.

The use according to claim 6, wherein the carbon source contains one or more of glucose, mannitol, sucrose, maltose, xylose, lactose and fructose;

23.

the fuel source contains one or more of potassium nitrate, ammonium sulfate, ammonium nitrate, yeast powder, glumatic acid, urea and peptone.

Use of the cellulose-degrading n3 strain according to claim 1 for the preparation of a degradation-promoting and growth-promoting bacterial agent.

The use according to claim 8, wherein the degradation-promoting and growth-promoting bacterial agent is an aqueous bacterial agent and when used, the aqueous bacterial agent is inoculated to straw in an amount of (1-9)x10°® CFU/g or to soil at (1-9)x10 7 CFU/g based on the cellulose-degrading n3 strain.

Use of the cellulosic degrading n3 strain according to claim 1 in promoting straw recycling and utilization and increasing yield.