Microbial agent capable of increasing yield of rehmannia and relieving continuous cropping obstacles and application of microbial agent

Abstract

The invention provides a microbial agent capable of increasing the yield of rehmannia and relieving successive cropping obstacles and application of the microbial agent, and belongs to the technical field of microorganisms. The microbial agent provided by the invention comprises bacillus amyloliquefaciens S15, the preservation number of the bacillus amyloliquefaciens S15 is CGMCC (China General Microbiological Culture Collection Center) NO.30976, and / or fusarium oxysporum S25, the preservation number of the fusarium oxysporum S25 is CGMCC NO.41373, and the microbial agent provided by the invention can be used for preparing a microbial agent. The method has a remarkable yield increasing effect on the rehmannia, can relieve continuous cropping obstacles of the rehmannia and increase the yield of the rehmannia during continuous cropping, is free of pollution to soil and can effectively promote sustainable development of the rehmannia planting industry.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a microbial agent capable of increasing the yield of Rehmannia glutinosa and alleviating continuous cropping obstacles, and application thereof. Background Art

[0002] Rehmannia glutinosa, the fresh or dried root of Rehmannia glutinosa Libosch., a plant of the Scrophulariaceae family, is one of the Chinese medicinal materials with the longest cultivation history in my country. As early as the Southern and Northern Dynasties, Jia Sixie recorded the "method of planting Rehmannia" in "Qimin Yaoshu". Lu Zhiyi of the Ming Dynasty recorded: "After planting Rehmannia, the soil becomes bitter... After ten years, the soil tastes sweet, and then Rehmannia can be planted again. Otherwise, it tastes bitter and has a thin shape, and it is not suitable for use as medicine." It can be seen that there was an obvious continuous cropping obstacle in the cultivation of Rehmannia at that time. However, the cause of the continuous cropping obstacle of Rehmannia glutinosa is not clear. Many researchers believe that allelopathic autotoxicity is one of the main reasons for the continuous cropping obstacle of Rehmannia glutinosa. The continuous cropping of Rehmannia glutinosa is usually manifested as: the crown width and leaves of the aboveground part of the plant are small, and the number of leaves is reduced; the underground root tubers cannot expand normally, the fibrous roots increase, and it cannot form a commercial medicinal material that can be used as medicine. Some experimental results show that the growth of Rehmannia glutinosa begins to show a continuous cropping effect about 50 days after planting. As Rehmannia grows, the difference between the first-crop Rehmannia and the second-crop Rehmannia becomes more and more obvious.

[0003] In the traditional Chinese medicine system, Rehmannia root undoubtedly plays a pivotal role. This precious Chinese herbal medicine, known as the "Chinese medicine", is one of the core raw materials of many Chinese patent medicines. However, the continuous cropping obstacle of Rehmannia root cultivation has seriously restricted the development of the Rehmannia root industry. Therefore, it is urgent to provide a method that can alleviate the continuous cropping obstacle of Rehmannia root and increase the yield of Rehmannia root. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a microbial agent in increasing the yield of Rehmannia root and alleviating the obstacle of continuous cropping, so as to alleviate the problem that the yield of Rehmannia root is significantly reduced during continuous cropping, and can effectively increase the yield of Rehmannia root under continuous cropping conditions.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of a microbial agent in improving the yield of Rehmannia glutinosa and alleviating the continuous cropping obstacle. The microbial agent comprises Bacillus amyloliquefaciens S15 and / or Fusarium oxysporum S25;

[0007] The Bacillus amyloliquefaciens S15 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of June 17, 2024 and a deposit number of CGMCCNO.30976;

[0008] The Fusarium oxysporum S25 is deposited in the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 17, 2024, and the deposit number is CGMCCNO.41373.

[0009] Preferably, when the microbial agent is Bacillus amyloliquefaciens S15, the concentration of the fermentation broth of Bacillus amyloliquefaciens S15 is 10 5 ~10 7 cfu / ml.

[0010] Preferably, when the microbial agent is Fusarium oxysporum S25, the concentration of the spore suspension of Fusarium oxysporum S25 is 10 6 ~10 8 cfu / ml.

[0011] Preferably, when the microbial agent is composed of a mixture of Bacillus amyloliquefaciens S15 and Fusarium oxysporum S25, the fermentation broth concentration of Bacillus amyloliquefaciens S15 is 10 5 ~10 7 cfu / ml, the spore suspension concentration of Fusarium oxysporum S15 is 10 6 ~10 8 cfu / ml, and the weight ratio of the fermentation broth to the spore suspension is 1-2:1-2.

[0012] Preferably, the method for applying the microbial agent is: inoculating the microbial agent onto Rehmannia glutinosa seedlings.

[0013] Preferably, the inoculation amount of the microbial agent is 8-12 ml / strain.

[0014] The present invention also provides a Bacillus amyloliquefaciens S15 in the above-mentioned application, wherein the Bacillus amyloliquefaciens S15 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of June 17, 2024 and a deposit number of CGMCC NO.30976.

[0015] The present invention also provides a Fusarium oxysporum S25 for the above-mentioned application, wherein the Fusarium oxysporum S25 is deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms, with the address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the preservation date being June 17, 2024, and the preservation number being CGMCC NO.41373.

[0016] The present invention also provides a microbial agent for use in the above application, comprising Bacillus amyloliquefaciens S15 and Fusarium oxysporum S25.

[0017] Preferably, the fermentation broth concentration of the Bacillus amyloliquefaciens S15 is 10 5 ~10 7 cfu / ml, the spore suspension concentration of Fusarium oxysporum S15 is 10 6 ~10 8 cfu / ml, and the weight ratio of the fermentation broth to the spore suspension is 1-2:1-2.

[0018] The Bacillus amyloliquefaciens S15 and Fusarium oxysporum S25 provided by the present invention both have the effects of alleviating the continuous cropping obstacle of Rehmannia glutinosa and increasing the yield of Rehmannia glutinosa, and the two bacteria have a synergistic effect, and when applied together, the alleviating effect of the continuous cropping obstacle of Rehmannia glutinosa can be further improved, and the yield-increasing effect of Rehmannia glutinosa is more significant. The microbial agent provided by the present invention is safe and environmentally friendly, does not cause harm to the soil, can effectively alleviate the reduction in Rehmannia glutinosa production caused by continuous cropping obstacles, and has a significant promoting effect on the development of the Rehmannia glutinosa industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The Bacillus amyloliquefaciens S15 in the screening culture medium in Example 1 (the left picture is a top view of the culture medium, and the right picture is a bottom view of the culture medium);

[0020] Figure 2 This is the screening of Fusarium oxysporum S25 in the culture medium in Example 1 (the left picture is a top view of the culture medium, and the right picture is a bottom view of the culture medium). DETAILED DESCRIPTION

[0021] The 16S sequence of Bacillus amyloliquefaciens S15 is shown in SEQ ID No. 1:

[0022] GTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGC

[0023] The ITS sequence of Fusarium oxysporum S25 is shown in SEQ ID No. 2:

[0024] TGCGGAGGGATCATTACCGAGTTTACAACTCCCAAACCCCTGTGAACATACCACTTGTTGCCTCGGCGGATCAGCCCGCTCCCGGTAAAACGGGACGGCCCGCCAGAGGACCCTAAACTCTGTTTCTATATGTAACTTCTGAGTAAAACCATAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCAAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCG AATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCACAGCTTGGTGTTGGGACTCGCGTTAATTCGCGTTCCCCAAATTGATTGGCGGT CACGTCGAGCTTCCATAGCGTAGTAGTAAAACCCTCGTTACTGGTAATCGTCGCGGCCACGCCGTTAAACCCCAACTTCTGAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATC

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Example 1 Bacterial strain screening

[0027] 1. Culture medium

[0028] Screening medium: 10 g stachyose, 4 g peptone, 1 g (NH4)2SO4, 0.1 g K2HPO4, 0.8 g KHPO4, 0.5 g MgSO4·7H2O, 0.1 g CaCl2·2H2O, 0.1 g NaCl, 18 g agar, add 15 mL 0.5% (95% ethanol dissolved) bromocresol purple as an indicator, and dilute to 1000 mL with distilled water.

[0029] Expansion medium: Potato dextrose agar (PDA) (OXOID, UK): 4.0 g potato powder, 20 g glucose, 15 g agar, 1000 mL distilled water, pH adjusted to 5.6±0.2.

[0030] 2. Preparation of Soil Suspension

[0031] The soil was from the first crop of Rehmannia glutinosa planted in Jiaozuo, Henan. Weigh 10 g of the first crop of soil and add it to 90 ml of cooled sterile distilled water sterilized with high pressure steam at 121°C for 20 min. Shake it on a shaker at 200 r / min for 30 min to obtain a concentration of 10 -1 g / ml soil suspension.

[0032] 3. Screening methods

[0033] Sterilize the screening medium with high pressure steam at 121°C for 20 min, then pour the medium into a petri dish and cool it down in a clean room. Dilute the soil bacteria suspension prepared above to 10 -5 After that, it was evenly spread on the screening culture medium, sealed and cultured in a constant temperature biological incubator at 30°C for 7 days, and the strain that caused the culture medium to turn yellow was selected for purification as the candidate strains. Two strains, Bacillus amyloliquefaciens S15 and Fusarium oxysporum S25, were screened (such as Figure 1 and Figure 2 shown).

[0034] Example 2 The first potted experiment

[0035] The two strains screened above were expanded and cultured in liquid potato dextrose agar (PDB) medium. The cell concentration of Bacillus amyloliquefaciens S15 was 10 8 CFU, the spore concentration of Fusarium oxysporum S25 is 10 8 When the number of CFU is reached, the culture is stopped and the inoculated fermentation liquid and spore liquid are used as spare.

[0036] The seedlings were planted in pots of 19 cm × 18 cm × 15 cm filled with peat soil (peat soil: vermiculite = 2:1) sterilized at 121 ° C for 60 min. The germplasm was derived from the cultivated Rehmannia glutinosa in Jiaozuo, Henan Province. They were divided into 4 groups, namely the control group, the Bacillus amyloliquefaciens S15 group, the Fusarium oxysporum S25 group and the mixed bacterial agent group, with 5 replicates in each group. After growing for 10 days in a greenhouse with a light intensity of 3000 lx and a photoperiod of white light / darkness of 16 h / 8 h, 10 ml of sterilized PDB solution was applied to the control group (FPCK), and 10 ml of 10% saturated fatty acid solution was inoculated to the Bacillus amyloliquefaciens S15 group (FP-S15). 6 cfu / ml Bacillus amyloliquefaciens S15 fermentation broth (diluted from the above-prepared Bacillus amyloliquefaciens S15 fermentation broth) and Fusarium oxysporum S25 group (FP-S25) were inoculated with 10 ml of the concentration of 10 7cfu / ml of Fusarium oxysporum S25 spore solution (obtained by diluting the Fusarium oxysporum S25 spore solution prepared above), and a mixed bacterial agent group (FPMM) inoculated with 10 ml of a concentration of 10 6 cfu / ml Bacillus amyloliquefaciens S15 fermentation broth and concentration of 10 7 cfu / ml of Fusarium oxysporum S25 spore solution mixed with bacterial agent (mixing weight ratio is 1:1). After culturing the Rehmannia glutinosa seedlings in each group in a greenhouse (conditions: temperature 23°C, humidity 50%) for 100 days, samples were taken and the underground fresh weight of Rehmannia glutinosa was weighed as the yield index. The results are shown in Table 1 below:

[0037] Table 1 Underground fresh weight of Rehmannia glutinosa in each group

[0038] Grouping Underground fresh weight / g Catalpol% Rehmannia glutinosin D% FPCK 23.79c 1.98a 0.26a FP-S15 35.44b 1.88a 0.23a FP-S25 37.00b 1.95a 0.26a FPMM 48.62a 2.00a 0.25a

[0039] As shown in Table 1, both Bacillus amyloliquefaciens S15 and Fusarium oxysporum S25 can significantly increase the yield of Rehmannia glutinosa, and the effect of increasing the yield of Rehmannia glutinosa is more obvious after the two bacteria are mixed, indicating that there is a synergistic effect between the two bacterial agents.

[0040] Example 3 Second potted experiment

[0041] The peat soil used for the first planting of Rehmannia was mixed according to the groups, and then put back into the original pots and used as the continuous soil for planting Rehmannia. Then, the tissue culture seedlings of Rehmannia (the tissue culture seedlings of Rehmannia that had been planted and cultured for 30 days, the germplasm came from the cultivated Rehmannia in Jiaozuo, Henan) were planted in the continuous soil and divided into 4 groups, namely the continuous control group, the continuous Bacillus amyloliquefaciens S15 group, the continuous Fusarium oxysporum S25 group and the continuous mixed bacterial agent group. A group of peat soil that had not been planted with Rehmannia (peat soil: vermiculite = 2:1) was added as the control group, with 5 replicates in each group. After growing for 10 days in a greenhouse with a light intensity of 3000lx and a photoperiod of white light / darkness of 16h / 8h, the continuous control group (SPAP) and the control group (SPCK) were applied with 10ml of sterilized PDB solution, and the continuous Bacillus amyloliquefaciens S15 group (SP-S15) was inoculated with 10ml of 10 6 cfu / ml Bacillus amyloliquefaciens S15 fermentation broth (diluted from the above-prepared Bacillus amyloliquefaciens S15 fermentation broth) and the continuous crop Fusarium oxysporum S25 group (SP-S25) were inoculated with 10 ml of the concentration of 10 7 cfu / ml of Fusarium oxysporum S25 spore solution (obtained by diluting the Fusarium oxysporum S25 spore solution prepared above), and a continuous mixed microbial agent group (SPMM) were inoculated with 10 ml of a concentration of 10 6 cfu / ml Bacillus amyloliquefaciens S15 fermentation broth and concentration of 10 7cfu / ml of Fusarium oxysporum S25 spore solution mixed with bacterial agent (mixing weight ratio is 1:1). After culturing the Rehmannia glutinosa seedlings in each group in a greenhouse (conditions: temperature 23°C, humidity 50%) for 100 days, samples were taken and the underground fresh weight of Rehmannia glutinosa was weighed as the yield index. The results are shown in Table 2 below:

[0042] Table 2 Underground fresh weight of Rehmannia glutinosa in each group

[0043]

[0044]

[0045] As shown in Table 2, the grouping of Bacillus amyloliquefaciens S15 and Fusarium oxysporum S25 can significantly increase the yield of Rehmannia glutinosa compared with the continuous cropping control group (SPAP), and the yield exceeds the control group (SPCK). It can be seen that Bacillus amyloliquefaciens S15 and Fusarium oxysporum S25 have excellent anti-repeat effect, can effectively alleviate the continuous cropping obstacles of Rehmannia glutinosa, and increase the yield of Rehmannia glutinosa, and the two bacteria have a synergistic effect, and after mixing, they have a better effect of alleviating the continuous cropping obstacles of Rehmannia glutinosa, further increasing the yield of Rehmannia glutinosa.

[0046] The contents of catalpol and rehmannia glycoside D in all samples are much higher than the pharmacopoeia requirements that raw rehmannia glutinosa should contain no less than 0.20% catalpol and no less than 0.10% rehmannia glutinosa glycoside D. Moreover, single strain bacterial agent or mixed bacterial agent will not reduce the contents of the two components, indicating that the bacterial agent of the present application will not reduce the quality of rehmannia glutinosa while increasing the yield.

[0047] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of a microbial agent in increasing the yield of Rehmannia glutinosa and alleviating the obstacle of continuous cropping, characterized in that: The microbial agent includes Bacillus amyloliquefaciens S15 and / or Fusarium oxysporum S25; The Bacillus amyloliquefaciens S15 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of June 17, 2024 and a deposit number of CGMCCNO.30976; The Fusarium oxysporum S25 is deposited in the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 17, 2024, and the deposit number is CGMCCNO.41373.

2. The use according to claim 1, characterized in that When the microbial agent is Bacillus amyloliquefaciens S15, the concentration of the fermentation liquid of Bacillus amyloliquefaciens S15 is 10 5 ~10 7 cfu / ml.

3. The use according to claim 1, characterized in that When the microbial agent is Fusarium oxysporum S25, the concentration of the spore suspension of Fusarium oxysporum S25 is 10 6 ~10 8 cfu / ml.

4. The use according to claim 1, characterized in that When the microbial agent is composed of a mixture of Bacillus amyloliquefaciens S15 and Fusarium oxysporum S25, the fermentation liquid concentration of Bacillus amyloliquefaciens S15 is 10 5 ~10 7 cfu / ml, the spore suspension concentration of Fusarium oxysporum S15 is 10 6 ~10 8 cfu / ml, and the weight ratio of the fermentation broth to the spore suspension is 1-2:1-2.

5. The use according to claim 4, characterized in that The application method of the microbial agent is: inoculating the microbial agent onto the Rehmannia glutinosa seedlings.

6. The use according to claim 5, characterized in that The inoculation amount of the microbial agent is 8-12 ml / strain.

7. The Bacillus amyloliquefaciens S15 for use according to any one of claims 1 to 6, characterized in that The Bacillus amyloliquefaciens S15 is deposited in the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 17, 2024, and the deposit number is CGMCCNO.30976.

8. The Fusarium oxysporum S25 for use according to any one of claims 1 to 6, characterized in that The Fusarium oxysporum S25 strain is deposited in the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 17, 2024, and the deposit number is CGMCCNO.41373.

9. The microbial agent for use according to any one of claims 1 to 6, characterized in that: These include Bacillus amyloliquefaciens S15 and Fusarium oxysporum S25.

10. The microbial agent according to claim 9, characterized in that The fermentation broth concentration of the Bacillus amyloliquefaciens S15 is 10 5 ~10 7 cfu / ml, the spore suspension concentration of Fusarium oxysporum S15 is 10 6 ~10 8 cfu / ml, and the weight ratio of the fermentation broth to the spore suspension is 1-2:1-2.

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