Fermentation product of bacillus amyloliquefaciens from chrysanthemum straw and its application in preventing and controlling soil-borne bacterial wilt

Abstract

The application discloses a chrysanthemum straw fermentation product of bacillus amyloliquefaciens T-5, which is prepared by solid fermentation and leaching with bacillus amyloliquefaciens T-5 as a functional microorganism and chrysanthemum straw as a substrate; the strain preservation number of the bacillus amyloliquefaciens T-5 is CGMCC No.8547. The chrysanthemum straw is fermented by T-5 to obtain the fermentation product, and a small amount of leaching liquor can extremely significantly inhibit the growth of the bacterial wilt bacteria, and the order of magnitude of the T-5 after the fermentation is between 10 9 ‑10 10 The metabolomics and verification experiment results show that part of phenolic acid and other substances after the T-5 fermentation are the key to the bacteriostatic effect, and the secondary metabolites of p-hydroxyphenylpropionic acid and 2-hydroxy-3-phenylpropionic acid have a significant inhibitory effect on the growth of the bacterial wilt bacteria. The application also discloses application of the chrysanthemum straw fermentation product of the bacillus amyloliquefaciens T-5 in the prevention and control of the soil-borne bacterial wilt.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and relates to a chrysanthemum straw fermentation product of bacillus amyloliquefaciens and application thereof in prevention and control of soil-borne bacterial wilt. BACKGROUND

[0002] Microbial fermentation technology is one of important means for functional microorganisms to synthesize certain or certain types of compounds, and has a wide application in many fields such as medicine, food and agriculture. In the past, most compounds were dependent on chemical synthesis and plant extraction, which can meet the demand to a great extent, but inevitably cause serious damage to the environment and ecology. With the progress of society and the urgent demand of people for high quality of life, microbial fermentation technology gradually replaces the above two means of obtaining substances and becomes a research hotspot.

[0003] Soil-borne bacterial wilt is caused by Ralstonia solanacearum, which is a soil-borne bacterial disease that causes irreversible damage to solanaceous crops such as tomatoes, potatoes and peppers. Although the conventional chemical control method (such as chemical pesticide spraying) can alleviate the spread of the disease and the damage to crops to a certain extent, with the passage of time, the pathogenic bacteria also gradually evolve into pesticide resistance. Moreover, the large amount of pesticide application also brings food safety problems, soil environmental problems and pesticide residue problems. SUMMARY

[0004] The inventors take various different straws as substrates, bacillus amyloliquefaciens T-5 as functional microorganisms, explore the inhibitory effect of different straw fermentation products on bacterial wilt, and analyze the functional substances that play a key inhibitory effect. The inventors find that only the chrysanthemum straw fermented by bacillus amyloliquefaciens T-5 can significantly inhibit the growth of bacterial wilt; further combined with metabolomics, it is found that after the chrysanthemum straw is fermented by bacillus amyloliquefaciens T-5, alkaloids, flavonoids and phenolic acids and other secondary metabolites are enriched.

[0005] The purpose of the present application is achieved by the following technical solutions:

[0006] A chrysanthemum straw fermentation product of bacillus amyloliquefaciens T-5, which is prepared by solid fermentation and extraction with bacillus amyloliquefaciens T-5 as functional microorganisms and chrysanthemum straw as substrate.

[0007] The chrysanthemum straw fermentation product contains p-hydroxyphenylpropionic acid and 2-hydroxy-3-phenylpropionic acid.

[0008] Bacillus amyloliquefaciens T-5, the taxonomic name of which is Bacillus amyloliquefaciens, was preserved in China General Microbiological Culture Collection Center on December 6, 2013, at No. 1, Beichen West Road, Haidian District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the strain preservation number is CGMCC No. 8547. Bacillus amyloliquefaciens T-5 is a known microorganism, and the separation and identification and physiological and biochemical characteristics of Bacillus amyloliquefaciens T-5 are described in Chinese Patent Application CN 108835127 A, Example 2.

[0009] Another object of the present application is to provide a preparation method of the chrysanthemum straw fermentation product of Bacillus amyloliquefaciens T-5, comprising: drying, grinding, sieving and sterilizing chrysanthemum straw, adjusting the moisture content of the chrysanthemum straw powder to 65-75% with sterile water, inoculating Bacillus amyloliquefaciens T-5 liquid at an inoculation amount of 1-2%, performing solid fermentation, and then adding sterile water for extraction at a solid-liquid ratio (mass ratio) of 1:5-1:10 to obtain the fermentation product.

[0010] Preferably, the chrysanthemum straw powder is sieved through a 20-mesh sieve.

[0011] Preferably, the sterilization is sterilization at 115°C for 30 min.

[0012] Preferably, the concentration of the Bacillus amyloliquefaciens T-5 liquid is OD 600 = 0.5-0.6.

[0013] The Bacillus amyloliquefaciens T-5 liquid is prepared by the following method: picking a single colony of T-5 and inoculating it in NA liquid medium, culturing overnight at a rotation speed of 160-200 rpm and a temperature of 25-35°C, aspirating the liquid into a sterile centrifuge tube, centrifuging, discarding the supernatant, washing away the medium with sterile water, and adjusting the OD 600 of the Bacillus amyloliquefaciens T-5 liquid to 0.5-0.6 with sterile water.

[0014] Preferably, the solid fermentation is performed at an ambient temperature of 26-35°C, a relative air humidity of 70-80%, and for a time of 7-10 days.

[0015] Preferably, the extraction is performed at a rotation speed of 160-200 rpm at room temperature for 0.5-2 h.

[0016] Another object of the present application is to provide the application of the chrysanthemum straw fermentation product of Bacillus amyloliquefaciens T-5 in the prevention and control of soil-borne bacterial wilt.

[0017] A method for preventing and controlling soil-borne bacterial wilt, comprising: after 2 weeks of transplanting tomato seedlings at the three-leaf stage, diluting the chrysanthemum straw fermentation product by 10-15 times, and applying 20-50 mL of the diluted chrysanthemum straw fermentation product to each tomato.

[0018] Preferably, the chrysanthemum straw fermentation product is diluted with water by 10-15 times.

[0019] Another object of the present application is to provide the application of the chrysanthemum straw fermentation product of Bacillus amyloliquefaciens T-5 in the preparation of a pesticide preparation or fertilizer for preventing and controlling soil-borne bacterial wilt.

[0020] Another object of the present application is to provide the application of p-hydroxyphenylpropionic acid or 2-hydroxy-3-phenylpropanoic acid in the preparation of a pesticide preparation or fertilizer for preventing and controlling soil-borne bacterial wilt.

[0021] The present application has the following beneficial effects:

[0022] The chrysanthemum straw is fermented by T-5 to obtain a fermentation product, and a small amount of extract can significantly inhibit the growth of bacterial wilt bacteria. 9 -10 10 The results of metabolomics and verification experiments show that part of the phenolic acid and other substances after T-5 fermentation are the key to the bacteriostatic effect, and the secondary metabolites p-hydroxyphenylpropionic acid and 2-hydroxy-3-phenylpropanoic acid have a significant inhibitory effect on the growth of bacterial wilt bacteria. When the concentration of p-hydroxyphenylpropionic acid or 2-hydroxy-3-phenylpropanoic acid is 0.2 mM, the growth of bacterial wilt bacteria can be significantly inhibited. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The influence of chrysanthemum straw extract on the biomass of bacterial wilt bacteria.

[0024] Figure 2 The influence of chrysanthemum straw on the biomass of T-5.

[0025] Figure 3 The abundance comparison of different secondary metabolites in the fermented straw.

[0026] Figure 4 The influence of different concentrations of secondary metabolite 3-(4-Hydroxyphenyl)-propionic acid (p-hydroxyphenylpropionic acid) on the growth of bacterial wilt bacteria.

[0027] Figure 5 The influence of different concentrations of secondary metabolite 2-Hydroxy-3-phenylpropanoic acid (2-hydroxy-3-phenylpropanoic acid) on the growth of bacterial wilt bacteria.

[0028] Figure 6The effects of the leaching solution of different treatments on the disease index of tomato bacterial wilt.

[0029] Biological material preservation information

[0030] T-5, the classified name of which is Bacillus amyloliquefaciens, was preserved in China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on December 6, 2013, and the strain preservation number is CGMCC No. 8547. DETAILED DESCRIPTION

[0031] The technical solutions of the present application will be further described in combination with the specific embodiments.

[0032] Preparation of T-5 bacterial solution:

[0033] The T-5 bacterial solution glycerol tube preserved under the condition of-80℃ was streaked on NA solid culture medium and placed under the condition of 30℃ until single colonies were grown. Single bacteria were picked and inoculated in NA liquid medium, and cultured overnight under the condition of 170 rpm and 30℃. Then 2 mL of bacterial solution was taken in a sterile centrifuge tube, centrifuged at 6000 rpm for 4 minutes, and the supernatant was discarded. Then sterile water was used for resuspension, and the above operation was repeated for 2-3 times to sufficiently wash away the culture medium. The absorbance was measured by using an enzyme label instrument, and the absorbance of the bacterial solution was 600. According to the absorbance, the bacterial solution was diluted or concentrated to OD 600 0.5.

[0034] Preparation of Ralstonia solanacearum (RS) bacterial solution:

[0035] The Ralstonia solanacearum (RS) bacterial solution was prepared as follows: QL-Rs1115, a Ralstonia solanacearum with strong pathogenicity, was isolated from diseased tomato plants in Qilin Town, Nanjing, and was named RS (reference: Wei, Z., Yang, X. M., Yin, S. X., Shen, Q. R., Ran, W. & Xu, Y. C.. Efficacy of Bacillus-fortified organic fertiliser in controlling bacterial wilt of tomato in the field. Appl. Soil Ecol., 2011, 48, 152-159.). The RS bacterial solution glycerol tube preserved under the condition of-80℃ was streaked on NA solid culture medium and placed under the condition of 30℃ until single colonies were grown. Single bacteria were picked and inoculated in NA liquid medium, and cultured overnight under the condition of 170 rpm and 30℃. Then 2 mL of bacterial solution was taken in a sterile centrifuge tube, centrifuged at 6000 rpm for 4 minutes, and the supernatant was discarded. Then sterile water was used for resuspension, and the above operation was repeated for 2-3 times to sufficiently wash away the culture medium. The absorbance was measured by using an enzyme label instrument, and the absorbance of the bacterial solution was 600. According to the absorbance, the bacterial solution was diluted or concentrated to OD600 RS bacterial solution with RS of 0.5.

[0036] Example 1

[0037] Inhibitory effect of different straw extracts on Ralstonia solanacearum

[0038] Straw: chrysanthemum straw, corn straw, wheat straw, soybean straw, pepper straw

[0039] Take the straw, remove the roots and leaves, dry, grind and pass through a 20 mesh sieve. Weigh 0.80 g of straw powder into a 50 mL centrifuge tube, seal with a sealing film, sterilize at a temperature of 115°C for 30 min; set up straw-T-5 fermentation group and straw-unfermented group (not inoculated); straw-T-5 fermentation group: adjust the moisture content (mass fraction) to 70% with sterile water in a clean bench, inoculate T-5 bacterial solution (OD 600 = 0.5) at a proportion of 1%, place in a condition of air relative humidity of 80% and temperature of 30°C, and stand for fermentation culture for 7 d; after the fermentation is completed, take out the centrifuge tube, add sterile water at a solid-liquid ratio (i.e. mass ratio) of 1:6, and extract at room temperature at a rotation speed of 170 rpm for 1.5 h. Chrysanthemum straw-unfermented group: do not inoculate T-5 bacterial solution, and control other conditions to be consistent with those of chrysanthemum straw-T-5 fermentation group.

[0040] After the extraction is completed, stand for half an hour, take the supernatant, filter to remove bacteria (0.22 μm filter head) to obtain sterile extract. In a 96-well plate, mix 10% NA medium (i.e. 10 times dilution of NA medium), extract of straw-T-5 fermentation group and RS bacterial solution (OD 600 = 0.5) at a volume ratio of 178:20:2 to a total volume of 200 μL, and at the same time, use an equal volume of sterile water (CK) and extract of chrysanthemum straw-unfermented group as controls, cultivate at 30°C and 170 rpm for 48 h, measure the OD 600 of Ralstonia solanacearum under different treatments, and analyze the effect of extract of T-5 fermented straw on the growth of Ralstonia solanacearum, and the results are shown in Table 1. It can be seen that the extract of chrysanthemum straw after fermentation by Bacillus amyloliquefaciens T-5 can significantly inhibit the growth of Ralstonia solanacearum. Figure 1

[0041] Table 1. Effect of different treatments on the biomass of Ralstonia solanacearum

[0042]

[0043] Example 2

[0044] Determination of T-5 biomass

[0045] Straw: chrysanthemum straw, corn straw, wheat straw, soybean straw, pepper straw ​

[0046] Straw was taken, roots and leaves were removed, and the straw was dried, ground, and sieved to 20 mesh. 0.80 g of straw powder was weighed into a 50 mL centrifuge tube, which was sealed with a sealing film, sterilized at a temperature of 115°C for 30 min, and then adjusted to a moisture content of 70% with sterile water in a clean bench. T-5 bacterial liquid (OD 600 = 0.5) was inoculated at a proportion of 1%, and the mixture was placed in a condition of an air relative humidity of 80% and a temperature of 30°C for static fermentation culture for 7 days. After the fermentation was completed, the centrifuge tube was taken out, sterile water was added at a solid-liquid mass ratio of 1:6, and the mixture was extracted at room temperature at a rotation speed of 170 rpm for 1 h. After the extraction was completed, 1 mL of the extract was immediately subjected to density gradient dilution, and plate counting was performed on V8 selective medium (V8 medium: 340 mL V8 fruit and vegetable juice, 33 g NaCl, 0.8 g glucose, deionized water to 1 L, pH 5.2). The results are shown in Table 2. Figure 2

[0047] As can be seen from the results, when the chrysanthemum straw was used as a substrate for fermentation, the effective viable cell count of T-5 could reach 3.58 ± 0.95 (10 9 CFU / g of straw).

[0048] Table 2. Effect of different straws on the biomass of T-5

[0049] Straw type [CD AT T-5 viable cell count (10 9 CFU / g straw)] Soybean straw 3.75±0.66 Corn straw 7.37±2.34 Wheat straw 2.19±0.32 Pepper straw 2.51±0.49 Chrysanthemum straw 3.58±0.95

[0050] Example 3

[0051] Metabolomics analysis of different straws before and after fermentation

[0052] Different straws were taken, roots and leaves were removed, and the straws were dried, ground, and sieved to 20 mesh. 0.80 g of straw powder was weighed into a 50 mL centrifuge tube, which was sealed with a sealing film, sterilized at a temperature of 115°C for 30 min, and then adjusted to a moisture content of 70% with sterile water in a clean bench. T-5 bacterial liquid (OD 600 = 0.5) was inoculated at a proportion of 1%, and the mixture was placed in a condition of an air relative humidity of 80% and a temperature of 30°C for static fermentation culture for 7 days to obtain different straw fermentation samples for determination of metabolomics, with unfermented straw as a control.

[0053] The sample metabolomics analysis process is as follows:

[0054] Sample extraction process:

[0055] (1) The biological sample was placed in a freeze dryer (Scientz-100F) for vacuum freeze drying;

[0056] (2) The sample was ground to a powder state by using a grinder (MM 400, Retsch) at a frequency of 30 Hz for 1.5 min; ​

[0057] (3) Weigh 100 mg of powder and dissolve it in 1.2 mL of 70% methanol extract solution;

[0058] (4) Vortex once every 30 minutes for 30 seconds, a total of 6 times, and the sample is placed in a 4°C refrigerator overnight;

[0059] (5) After centrifugation (12000 rpm for 10 minutes), the supernatant is aspirated, the sample is filtered with a microporous filter membrane (0.22 μm pore size), and stored in an injection bottle for UPLC-MS / MS analysis.

[0060] Chromatography-mass spectrometry collection conditions:

[0061] The data collection instrument system mainly includes ultra-high performance liquid chromatography (Ultra Performance Liquid Chromatography, UPLC, SHIMADZU Nexera X2, https: / / www.shimadzu.com.cn / ) and tandem mass spectrometry (Tandem mass spectrometry, MS / MS, Applied Biosystems 4500QTRAP, http: / / www.appliedbiosystems.com.cn / ).

[0062] In combination with metabolomics, first, the secondary metabolites significantly enriched after fermentation of chrysanthemum straw are analyzed, and compared with the metabolomics information of unfermented chrysanthemum straw, and the secondary metabolites with the highest relative abundance in the sample after fermentation of chrysanthemum straw are screened out; then, horizontal comparison is made with other fermented straw metabolomics, to further narrow down the screening range of key bacteriostatic substances.

[0063] Results: After fermentation of chrysanthemum straw, 45 kinds of secondary metabolites were significantly increased, and the total relative abundance of Figure 4 Among them, the total relative abundance of 8 substances is the highest, accounting for more than 10%, and mainly including phenolic acid substances.

[0064] The inventors use indoor bacteriostatic experiments for verification: DMSO is used as a solvent to prepare a standard mother liquor with a concentration of 100 mM, DMSO is used for gradient dilution to obtain standard solutions with final concentrations of 20, 40, 60, 80 and 100 mM, and 0.22 μm organic phase filter membrane is used for filtration and sterilization. In a 96-well plate, 178 μL of 10% NA culture medium, 18 μL of sterile water, 2 μL of each concentration of standard solution and 2 μL of Qingguibacteria liquid (OD 600 = 0.5) are added, 2 μL of DMSO is used as a control instead of the standard solution, and the culture is incubated at 30°C, 170 rpm for 24 h, and the biomass of Qinggui bacteria is measured

[0065] The results of the indoor antibacterial experiment are shown in Table 1. Figure 4 and Figure 5 It is shown that both p-hydroxyphenylpropionic acid and 2-hydroxy-3-phenylpropionic acid have significant inhibitory effect on the growth of P. solanacearum, and the growth of P. solanacearum can be significantly inhibited when the concentration of the above two substances is 0.2 mM.

[0066] Example 4

[0067] Prevention and control effect of chrysanthemum straw fermentation extract on bacterial wilt

[0068] Tomato seedlings growing to the three-leaf stage and having consistent growth were transplanted into 6-hole seedling trays with 100 g of substrate per hole, and were placed in a greenhouse for cultivation, with the environmental temperature controlled at 30-35°C and the air humidity controlled at 60-80%; after two weeks, P. solanacearum was inoculated, with the concentration of P. solanacearum controlled at 10 6 CFU / g of substrate; after P. solanacearum was inoculated for 3 days and stable colonization, three treatment groups were set: RSF1 group: the extract prepared by the chrysanthemum straw-T-5 fermentation group of Example 1 was diluted 10 times with water, and 20 mL of the diluted chrysanthemum straw fermentation extract was applied to each seedling by root irrigation; RSNF1 group: the extract prepared by the chrysanthemum straw-unfermented group of Example 1 was diluted 10 times with water, and 20 mL of the diluted chrysanthemum straw-unfermented group extract was applied to each seedling by root irrigation; CK: 20 mL of water was added to each seedling by root irrigation. Then the disease prevention of tomato was recorded every day, and the disease index at different times was calculated according to the disease grade, with the plant disease grade divided into 5 levels: 0 = healthy plant, 1 = 1%-25% of the plant part showing disease symptoms, 2 = 26%-50% of the plant part showing disease symptoms, 3 = 51%-75% of the plant part showing disease symptoms, and 4 = the plant is completely diseased or the plant has died (reference: Biological control of bacterial wilt of potatoes caused by Pseudomonas solanacearum).

[0069] Plant disease index (%) = [∑(number of plants at each level x corresponding level) / (total number of plants surveyed x highest level value)] x 100 (reference: Biological control of bacterial wilt of potatoes: Attempts to induce resistance by treating tubes with bacteria).

[0070] The results are shown in Table 2. Figure 6As shown in Figure 2, the addition of the extract of the Chrysanthemum straw-T-5 fermentation group significantly reduced the incidence of tomato bacterial wilt.

Claims

1. A fermented product of chrysanthemum straw of Bacillus amyloliquefaciens T-5, characterized by: It is with Bacillus amyloliquefaciens T-5 as functional microorganism, and chrysanthemum straw as substrate, prepared by solid fermentation and extraction; Bacillus amyloliquefaciens T-5, the taxonomic name of which is Bacillus amyloliquefaciens Bacillus amyloliquefaciens , and was preserved in China General Microbiological Culture Collection Center on December 6, 2013, with a preservation number of CGMCC No. 8547.

2. The fermented product of chrysanthemum straw of Bacillus amyloliquefaciens T-5 according to claim 1, characterized in that: The chrysanthemum straw fermentation product contains p-hydroxyphenylpropionic acid and 2-hydroxy-3-phenylpropionic acid.

3. A method of preparing a fermented product of chrysanthemum straw of Bacillus amyloliquefaciens T-5 according to claim 1, characterized by: The application relates to a chrysanthemum straw fermentation product and a method for preparing the chrysanthemum straw fermentation product. The chrysanthemum straw is dried, ground, sieved and sterilized, sterile water is used to adjust the water content of the chrysanthemum straw powder to 65-75%, Bacillus amyloliquefaciens T-5 bacterial liquid is inoculated into the chrysanthemum straw powder according to an inoculation amount of 1-2%, solid fermentation is carried out, sterile water is added according to a solid-liquid ratio of 1:5-1:10 for extraction, and the fermentation product is obtained.

4. The method for preparing the fermentation product of Bacillus amyloliquefaciens T-5 from chrysanthemum straw according to claim 3, characterized in that: The chrysanthemum straw is ground and sieved through a 20-mesh screen; the sterilization is sterilization at 115 DEG C for 30 min.

5. The method for preparing the fermentation product of Bacillus amyloliquefaciens T-5 from chrysanthemum straw according to claim 3, characterized in that: The concentration of Bacillus amyloliquefaciens T-5 bacterial solution was OD 600 = 0.5-0.

6.

6. The method for preparing the fermentation product of Bacillus amyloliquefaciens T-5 from chrysanthemum straw according to claim 3, characterized in that: The solid fermentation is carried out at an ambient temperature of 26-35 DEG C and an air relative humidity of 70-80%, and the solid fermentation time is 7-10 days.

7. The method for preparing the fermentation product of Bacillus amyloliquefaciens T-5 from chrysanthemum straw according to claim 3, characterized in that: The extraction is carried out at a rotating speed of 160-200 rpm at room temperature for 0.5-2 h.

8. Use of the chrysanthemum straw fermentation product of Bacillus amyloliquefaciens T-5 of claim 1 in the prevention and control of soil-borne bacterial wilt, which is a soil-borne bacterial disease caused by Ralstonia solanacearum (Eelli) Yabuuchi et al. Ralstonia solanacearum .

9. A method for preventing and controlling soil-borne bacterial wilt caused by Ralstonia solanacearum (formerly known as Pseudomonas solanacearum) which is a soil-borne bacterial disease, characterized by: Ralstonia solanacearum ) which is a soil-borne bacterial disease, characterized by: The application relates to a chrysanthemum straw fermentation product and a method for preparing the chrysanthemum straw fermentation product. After the tomato seedlings at the three-leaf stage are transplanted for 2 weeks, the chrysanthemum straw fermentation product in claim 1 is diluted by 10-15 times, 20-50 mL of the diluted chrysanthemum straw fermentation product is applied to each tomato in a root irrigation mode.

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