Method for producing indoleacetic acid by using aroma-removing alcaligenes YB082 and plant growth regulator

Abstract

The invention discloses an application of aroma-removing alcaligenes sp. YB082 in production of indoleacetic acid (IAA). According to the regulator, a YB082 strain is inoculated into an LB-lt liquid culture medium, fermentation liquor containing IAA is obtained after aerobic culture is conducted for 24-72 h, the strain density OD600 in the fermentation liquor is larger than 1.2, and the IAA content is larger than 35 mg / L. Besides, the strain is used as a growth regulator to be applied to a seedling raising substrate to promote germination and growth of Chinese cabbage seeds, results show that all plant seeds in a YB082 strain treatment group germinate and break soil in the 48th hour, and the seed germination promotion and Chinese cabbage seed growth promotion are obviously superior to those in a blank group.

Description

TECHNICAL FIELD

[0001] The present application relates to a bacterial strain Alcaligenes odorans YB082 (YB082) for producing indole acetic acid (IAA) and a method for producing IAA and a plant growth regulator, belonging to the technical field of environmental microorganisms. Alcaligenes defragrans The present application relates to a bacterial strain Alcaligenes odorans YB082 (YB082) for producing indole acetic acid (IAA) and a method for producing IAA and a plant growth regulator, belonging to the technical field of environmental microorganisms. BACKGROUND

[0002] IAA and GA are the core components of plant growth regulators, and their biosynthetic mechanisms and regulation are crucial for plant growth and development. IAA has the functions of cell elongation, root development, and maintenance of apical dominance, mainly acting on shoot apical meristem, root cap, and other parts. GA has the functions of stem elongation, seed germination, and flowering induction, mainly acting on stem tips and young embryos. Microorganisms are important producers of IAA and GA, and their synthesis pathways are diverse. Numerous experiments have shown that IAA-producing bacteria have a positive effect on the growth regulation of various crops, such as promoting seed germination, stimulating root development, and increasing yield. In practical applications, bacteria are more widely used due to their easy cultivation and rapid expansion. However, most of the IAA and GA-producing bacteria found so far belong to the genera Bacillus, Pseudomonas, Enterobacter, and Actinomyces. Strains that can produce both IAA and GA are rare and have low yields. Therefore, discovering more high-yield IAA and GA-producing bacteria is an important part of the application of plant growth regulators.

[0003] The present application reports for the first time a strain of Alcaligenes odorans that can produce IAA, with good IAA yield and effective promotion of seed germination, and has good prospects in the field of plant growth regulators. SUMMARY

[0004] The present application provides a bacterial strain, Alcaligenes odorans YB082 (YB082), for the fermentation production of IAA. Alcaligenes defragrans The present application provides a bacterial strain, Alcaligenes odorans YB082 (YB082), for the fermentation production of IAA.

[0005] Alcaligenes odorans YB082, classification and naming Alcaligenes defragrans YB082, deposited at the China Center for Type Culture Collection, located in Wuhan, Wuhan University, with the accession number CCTCC M 20231957, and the deposit date of October 20, 2023.

[0006] Method for YB082 to ferment IAA: (1) Activation method of preserved strain The YB082 strain preserved in glycerol was activated using LB medium (tryptone 1 g / L, yeast extract powder 0.5 g / L, NaCl 1 g / L, agar powder 15 g / L). The YB082 strain preserved in glycerol at -80℃ was streaked on solid LB medium with a sterile inoculation loop and cultured in a biochemical incubator at 30℃ until colonies appeared, and the strain was activated.

[0007] (2) Fermentation broth preparation LB-lt broth (ingredients: tryptone 8.0-10.0 g / L, yeast powder 3.0-5.0 g / L, sodium chloride 8.0-10.0 g / L, L-tryptophan 0.5-1.0 g / L) was prepared in 200 ml, sterilized at 121 °C for 10-30 min, and cooled for standby.

[0008] In some preferred cases, the liquid culture is LB-lt liquid medium, and the ingredients of the LB-lt liquid medium are tryptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, and L-tryptophan 1.0 g / L.

[0009] (3) YB082 fermentation culture inoculation YB082 strain was inoculated in LB liquid culture medium (without agar powder) to expand the culture. The 10 OD (Optical Density λ=600 nm optical density) strain culture liquid was centrifuged at 10,000 r / min for 5 min, and the precipitate was collected, suspended with sterile water, and inoculated in the fermentation broth in step (2) above. The initial OD of fermentation was 0.05.

[0010] (4) YB082 fermentation process conditions for IAA production The inoculated culture liquid was incubated at 28-32 °C under aerobic conditions. A biochemical incubator can be used for culture, and the shaking speed is 180-200 r / min. The culture time is 24-48 hours. The culture liquid OD exceeds 1.2, and the culture is terminated.

[0011] (5) Detection of IAA content in fermentation products 10 mL of fermentation broth was centrifuged at 10,000 r / min for 10 min. 5 mL of supernatant was mixed with an equal volume of Salkowcki color developing solution, and the reaction was carried out in the dark for 30 min. The absorbance value was detected at 530 nm by spectrophotometer. The content of IAA in the fermentation product was determined. When the content of IAA in the fermentation product is greater than 35 mg / L, the fermentation requirement is met.

[0012] (6) IAA separation and purification To obtain the purified IAA product, the pH of the fermentation broth can be adjusted to 2-3, and the product can be obtained by ethyl acetate extraction and concentration, and crystallization.

[0013] The present application also provides a plant growth regulator, which comprises the bacterial strain, Pseudomonas denitrificans YB082 ( Alcaligenes defragrans YB082). The preparation method is as follows: (1) The fermentation broth containing IAA is obtained by the method of producing IAA by fermentation of YB082, and the strain density OD600 in the fermentation broth is greater than 1.2, and the content of IAA is greater than 35 mg / L.

[0014] (2) The fermentation broth obtained above is added with trace Ca(NO3)2 (final concentration 0.6-1 mM) and MgSO4 (final concentration 0.2-05 mM), and the pH is adjusted to 6.8-7.2. Thus, a plant growth regulator is obtained.

[0015] The present application has the following beneficial effects: The present application discloses a new bacterial strain capable of producing IAA, Alcaligenes odorans YB082 (YB082), and a method for preparing a fermentation product containing IAA. In addition, a plant growth regulator mainly comprising the bacterial strain is disclosed. Alcaligenes defragrans

[0016] The present application has the advantages of simple operation, high efficiency, harmless product, and the like, and has the potential for popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 IAA production capacity of Alcaligenes odorans YB082 strain. A, color development pictures after reaction of sterile culture solution (left) and Ylb culture solution (right) with Salkowcki color reagent; B, IAA production of YB082 strain at different culture times.

[0018] Figure 2 Effect of different tryptophan substrate concentrations on IAA production of YB082 strain.

[0019] Figure 3 GA production capacity of YB082.

[0020] Figure 4 YB082 strain as a growth regulator promotes germination of Chinese cabbage seeds in planting substrate.

[0021] Figure 5 Pictures of growth of Chinese cabbage seeds treated in different ways. DETAILED DESCRIPTION

[0022] The present application will be described in detail below with reference to specific examples, but those skilled in the art will understand that the following examples are only for illustration of the present application, and should not be regarded as limiting the scope of the present application.

[0023] Example 1 IAA production characteristics of strain YB082 (1) Activation method of preserved strain ​The YB082 strain, preserved in frozen glycerol, was activated using LB medium (1 g / L tryptone, 0.5 g / L yeast extract (purchased from Angel Yeast Co., Ltd.), 1 g / L NaCl, and 15 g / L agar powder). YB082 strains preserved in frozen glycerol at -80℃ were picked, streaked onto solid LB medium using a sterile inoculation loop, and incubated in a 30℃ biochemical incubator until colonies appeared, indicating activation of the strain.

[0024] (2) IAA production method A small number of colonies of the activated YB082 strain were inoculated into LB liquid culture medium (without agar powder) for expansion. A culture medium with an OD of 100 (Optical Density λ = 600 nm) was harvested, centrifuged at 10000 rpm for 5 min, and the precipitate was collected, resuspended in sterile water, and inoculated into 1 L of LB-lt culture medium. The LB-lt culture medium consisted of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 1.0 g / L L-tryptophan. The inoculated culture was then incubated aerobically at 30-36℃. A biochemical shaker could be used for cultivation at 200 rpm. The incubation times were set to 24 hours, 48 ​​hours, and 72 hours to obtain fermentation broths (i.e., fermentation broths were obtained at 24, 48, and 72 hours for IAA detection).

[0025] (3) Detection of IAA content in fermentation products Centrifuge 10 mL of fermentation broth at 10000 rpm for 10 min. Take 5 mL of the supernatant and mix it with an equal volume of Salkowcki colorimetric solution. Incubate in the dark for 30 min, and detect the absorbance at 530 nm using a spectrophotometer. Determine the IAA content in the fermentation product. Fermentation is considered complete when the IAA content in the fermentation product is greater than 35 mg / L.

[0026] (4) IAA separation and purification To obtain purified IAA, the pH of the fermentation broth was adjusted to 2.8, and the mixture was extracted and concentrated with ethyl acetate. After crystallization, the purified IAA product was obtained.

[0027] The ability of strain YB082 to produce IAA is as follows: Figure 1 As shown. YB082 was cultured using the IAA-producing culture method of this invention. From Figure 1 As shown in Figure A, the culture medium of strain YB082 exhibits a distinct color change after reacting with Salkowcki chromogenic reagent. A pink color indicates a positive result, signifying the presence of IAA in the culture medium; the deeper the color, the higher the concentration of IAA. No color change indicates a negative result, signifying the absence of IAA. Figure 1The right side of the middle A is YB082 culture solution treatment, and the color reaction is all changed into pink, indicating that YB082 has the ability to produce IAA Figure 1 The left side of the middle A is a sterile blank culture solution as a control.

[0028] Further, the strains are respectively cultured for 24, 48 and 72 hours, and the content of IAA in the culture solution is detected, as shown in Figure 1 Middle B, YB082 can obtain a higher IAA yield at 48h, and the IAA concentration reaches 41.4 mg / L Figure 1 Middle B). It can be known from Figure 1 Middle B that YB082 has a good ability to produce IAA.

[0029] Example 2 In order to investigate the effect of L-tryptophan on the production of IAA by YB082, the method of Example 1 is used for culture, and the culture time is set to 48 hours, and different concentration gradients of L-tryptophan are added to the LB-lt culture solution as a substrate (such as 0 g / L, 0.2 g / L, 0.5 g / L, 1 g / L, 1.5 g / L), and the content of IAA in the fermentation solution is detected. The results show that with the increase of the concentration of L-tryptophan in the culture solution, the yield of IAA increases. When the concentration of L-tryptophan in the culture solution is 0.5 g / L, the yield of IAA after 24h culture is 30.5 mg / L. When the concentration of L-tryptophan in the culture solution reaches 1 g / L, the yield of IAA reaches 41.4 mg / L.

[0030] Example 3 In addition, the present application also finds that YB082 also has the activity of producing gibberellin (GA).

[0031] The specific method is: (1) The activated YB082 strain (activated by the method of Example 1) is inoculated into the LB liquid culture solution, and the initial concentration of the strain culture is 0.5 OD. It is placed in the condition of 28-32℃ for aerobic culture. Biochemical shaker culture can be used, and the shaking speed is 150-200 r / min. The culture time is 24-72 hours. The fermentation product containing GA can be obtained. The results are shown in Figure 3 , the culture solution is obtained at 24, 48 and 72 h of strain culture, 10000 r / min centrifugation for 10 min, and the supernatant is detected for GA concentration. The results show that the GA content of YB082 can reach 29.1 mg / L at 24h of fermentation.

[0032] Example 4 YB082 has the ability to produce plant growth regulators such as IAA and GA, thus the strain has the effect of promoting seed germination and growth. In order to verify the effect of the strain, the present application carries out Chinese cabbage seed germination and growth experiment. The specific method is as follows: Planting substrate preparation: Collecting pruning trees, dry branches, fallen leaves and other materials floating in the Yangtze River in Yichang Sanxia Dam area, crushing them into wood chips with a diameter of 1-3 mm, weighing the corresponding mass of crushed wood chips, and uniformly stacking them into the substrate groove of the culture plate, compacting, as the planting substrate.

[0033] YB082 bacterial liquid preparation: after centrifugation of the cultured YB082 bacterial liquid at 8 000 r / min for 10 min, the supernatant was discarded, the bacterial body was collected, resuspended with sterile water, and the strain concentration was adjusted to 0.8-1.0 OD / mL.

[0034] Experimental scheme: 3 Chinese cabbage seeds were put into each substrate groove (containing planting substrate). The experiment was divided into two groups, namely treatment group and control group. The treatment group added 3 mL of prepared YB082 bacterial liquid into each substrate groove; the control group replaced the bacterial liquid with sterile water. Each group was repeated 3 times. The results are shown in Figure 4

[0035] Figure 4 A is the germination rate of Chinese cabbage seeds, the germination rate of the treatment group reaches 89%, which is significantly higher than that of the blank group of 77.7%, and has good seed germination promoting effect. Figure 4 B is the average root length of Chinese cabbage seedlings after 7 days of cultivation, the average root length of the treatment group is 3.738 cm, which is significantly higher than that of the blank group of 1.443 cm, and has excellent root elongation promoting effect. Figure 4 C is the average stem thickness of Chinese cabbage seedlings after 7 days of cultivation, the average stem thickness of the treatment group is 0.955 mm, which is significantly higher than that of the blank group of 0.883 mm, and has good stem thickness promoting effect. Figure 4 D is the average stem length of Chinese cabbage seedlings after 7 days of cultivation, the average stem length of the treatment group is 5.664 cm, which is significantly higher than that of the blank group of 4.518 cm, and has obvious stem elongation promoting effect. Figure 4 E is the fresh weight of Chinese cabbage seedlings after 7 days of cultivation, the fresh weight of the treatment group is 44 mg, which is higher than that of the blank group of 41 mg, and can significantly increase the fresh weight of Chinese cabbage seedlings. Figure 4 ​F is the Chinese cabbage seed cultivation 7 days, collect each Chinese cabbage seedlings, place them in 75 °C oven drying 3 h, then take out the detection of its dry weight, the blank group dry weight is 0.026 mg, the treatment group is 0.025 mg, the difference between the two is not big.

[0036] By Figure 5 It can be seen that the treatment group in the 48th hour all the Chinese cabbage seeds have germinated and broken out, while the blank group has 4 Chinese cabbage seeds growing in the 96th hour. The growth of the treatment group is far superior to the blank group. It can be seen that YB082 strain as a growth regulator, applied to Chinese cabbage seedling raising substrate, has obvious effect on Chinese cabbage seed germination, and also has obvious gain effect on seedling growth.

Claims

1. Use of Alcaligenes defragrans YB082 in the production of indole acetic acid (IAA).

2. Use according to claim 1, characterized in that, Alcaligenes odorans YB082, taxonomic designation Alcaligenes defragrans YB082, deposited with the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, under accession number CCTCC M 20231957 on October 20, 2023.

3. Use according to claim 1, characterized in that, YB082 is cultured in a liquid medium to produce IAA.

4. Use according to claim 3, characterized in that, The liquid medium is LB-lt liquid medium, and the components of the LB-lt liquid medium are as follows: 8.0-10.0 g / L of tryptone, 3.0-5.0 g / L of yeast powder, 8.0-10.0 g / L of sodium chloride, and 0.5-1.0 g / L of L-tryptophan.

5. Use according to claim 4, characterized in that, The liquid medium is LB-lt liquid medium, and the components of the LB-lt liquid medium are as follows: 10.0 g / L of tryptone, 5.0 g / L of yeast powder, 10.0 g / L of sodium chloride, and 1.0 g / L of L-tryptophan.

6. A method for producing indole acetic acid (IAA), characterized by, The method comprises the following steps: inoculating YB082 into LB-lt liquid medium, and culturing the YB082 in an aerobic manner for 24-72 h to obtain IAA in the fermentation broth of the strain, wherein the aerobic culture is carried out in a shaking incubator at a rotation speed of 180-200 r / min and at a temperature of 30-36℃.

7. Plant growth regulator, characterized in that, The regulator is obtained by inoculating YB082 into LB-lt liquid medium, and culturing the YB082 in an aerobic manner for 24-72 h to obtain a fermentation broth containing IAA, wherein the density of the strain in the fermentation broth is greater than 1.2 at OD600, and the content of IAA is greater than 35 mg / L.

8. The plant growth regulator according to claim 7, characterized in that, In the plant growth regulator, the obtained fermentation broth is added with trace amounts of Ca(NO3)2 and MgSO4, so that the final concentration of Ca(NO3)2 is 0.6-1 mM, the final concentration of MgSO4 is 0.2-0.5 mM, the pH is adjusted to 6.8-7.2, and the plant growth regulator is obtained.

9. Use of the plant growth regulator according to claim 7 or 8 in promoting rapid germination of seeds.

10. Use according to claim 9, characterized in that, The plant seeds include any one of Chinese cabbage, radish, pepper, eggplant, and rice. The plant seeds include any one of Chinese cabbage, radish, pepper, eggplant, and rice.

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