Composition for alleviating the obstacle of continuous cropping of Panax notoginseng and its application method

Abstract

The present invention relates to a composition for alleviating the continuous cropping obstacle of Panax notoginseng and an application method thereof, belonging to the field of Chinese medicine planting, comprising cinnamic acid and Burkholderia. The present invention also provides a method for using the composition for alleviating the continuous cropping obstacle of Panax notoginseng and related uses thereof.

Description

Technical Field

[0001] The invention relates to a composition for alleviating the obstacle of continuous cropping of Panax notoginseng and an application method thereof, and belongs to the field of traditional Chinese medicine planting. Background Art

[0002] Yunnan is the main production area of ​​the traditional Chinese medicine Panax notoginseng. Panax notoginseng prefers a warm, humid growing environment. Continuous cropping is a key factor restricting the healthy development of the Panax notoginseng industry, which seriously affects the yield and quality of Panax notoginseng. At present, the main means to alleviate the continuous cropping of Panax notoginseng include soil fumigation, crop rotation and biological control. Soil fumigation often uses some highly toxic chemical fumigants, and its harm to the environment limits its application in actual production; while the crop rotation cycle is long, and suitable crops for alleviating the continuous cropping of Panax notoginseng have not yet been used in production. Using beneficial microorganisms to alleviate the continuous cropping of Panax notoginseng has the advantages of low toxicity, no pollution, and environmental friendliness, and is an effective way to prevent and control the green ecology of Panax notoginseng.

[0003] Burkholderia B36 was isolated from the rhizosphere soil of Panax notoginseng in the early stage. It can not only degrade the autotoxic saponins secreted into the rhizosphere soil during the growth of Panax notoginseng, but also significantly antagonize the beneficial microorganisms of root rot pathogens, and can significantly alleviate the continuous cropping obstacles of Panax notoginseng. For example, Chinese patent CN 202110087107 discloses a Burkholderia and its application in preventing and controlling the main pathogens of Panax notoginseng root rot, which has a good effect on alleviating the soil obstacles of Panax notoginseng continuous cropping for about 3 years.

[0004] Cinnamic acid is an organic acid isolated from cinnamon bark or benzoin. It is a colorless needle-shaped crystal or white crystalline powder, slightly soluble in water, soluble in ethanol, methanol, petroleum ether, chloroform, and easily soluble in benzene, ether, acetone, glacial acetic acid, carbon disulfide and oils. It is mainly used to prepare raw materials for esters, spices and medicines. However, there are no reports on the combined application of Burkholderia and cinnamic acid in Panax notoginseng cultivation. Summary of the invention

[0005] A first aspect of the present invention is to provide a composition for alleviating the disorder of continuous cropping of Panax notoginseng, which comprises cinnamic acid and Burkholderia.

[0006] In one embodiment, the cinnamic acid is a 10 μg / mL cinnamic acid aqueous solution; the Burkholderia is 10 6 CFU / mL of a water suspension of Burkholderia spp.

[0007] In another embodiment, the volume ratio of the cinnamic acid aqueous solution to the Burkholderia aqueous suspension is 1:0.1-10. In a preferred embodiment, the volume ratio of the cinnamic acid aqueous solution to the Burkholderia aqueous suspension is 1:0.5-2; in a further embodiment, the volume ratio of the cinnamic acid aqueous solution to the Burkholderia aqueous suspension is 1:1.

[0008] In another embodiment, the Burkholderia is Burkholderia B36. The Burkholderia B36 has been disclosed in a Chinese patent application numbered 2021100871074, a Burkholderia and its application in preventing and controlling the main pathogens of Panax notoginseng root rot.

[0009] The second aspect of the present invention is to provide a method for using the composition to alleviate the disorder of continuous cropping of Panax notoginseng, which specifically comprises:

[0010] 1) preparing a cinnamic acid aqueous solution and a Burkholderia suspension of corresponding concentrations, and mixing them in proportion;

[0011] 2) Before planting Panax notoginseng, press 0.1ml / cm 3 Water the soil for continuous cropping of Panax notoginseng and transplant the Panax notoginseng plants after 3 days.

[0012] In one embodiment, the Panax notoginseng continuous cropping soil refers to Panax notoginseng continuous cropping soil for 3 years or more.

[0013] The third aspect of the present invention is to provide the application of the composition in Panax notoginseng planting.

[0014] In one embodiment, the Panax notoginseng planting refers to alleviating the obstacles of continuous cropping of Panax notoginseng in the soil.

[0015] The present invention effectively alleviates the soil continuous cropping disorder of Panax notoginseng by combining Burkholderia and cinnamic acid, especially has a significant alleviating effect on the soil continuous cropping disorder of Panax notoginseng for 3 years or more, and it is found that the two have a significant effect on alleviating the soil continuous cropping disorder of Panax notoginseng.

[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 :Effects of cinnamic acid and Burkholderia B36 synergistic treatment on the survival rate of Panax notoginseng seedlings and the occurrence of root rot in continuous cropping soil;

[0019] Figure 2 :Determination of the chemotactic effect of Burkholderia B36 on cinnamic acid, where a, b, c, and d represent significant differences at the P<0.05 level tested by Duncan's new multiple range method.

[0020] Figure 3:The effect of cinnamic acid on the colonization ability of Burkholderia B36 in soil, where a and b represent significant differences at the P<0.05 level tested by Duncan's new multiple range method. ;

[0021] Figure 4 :Determination of the effect of cinnamic acid on the biofilm formation of Burkholderia B36, where a, b, c, and d represent significant differences at the P<0.05 level tested by Duncan's new multiple range method. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0023] Example 1 Cinnamic acid cooperates with Burkholderia B36 to alleviate the obstacle of continuous cropping of Panax notoginseng

[0024] A single colony of Burkholderia B36 was added to LB liquid culture medium and shaken for 2 days to increase the bacterial concentration to OD 590 The value reached 0.6. The following watering was carried out in the flower pots filled with soil from the continuous cropping of Panax notoginseng planted for five years: ① the concentration was 10 6 CFU / mL of Burkholderia B36 water suspension; ② 10μg / mL of cinnamic acid; ③ A mixture of Burkholderia B36 and cinnamic acid in a 1:1 ratio, at 0.1ml / cm 3 The soil of continuous cropping of Panax notoginseng was watered, and the same volume of 0.1% methanol was added as a control. One-year-old healthy Panax notoginseng plants were transplanted 3 days after treatment. Each treatment had 6 pots, and 8 plants were planted in each pot. After 25 days, the number of surviving Panax notoginseng seedlings was investigated, and the survival rate was calculated according to the following formula; the plants with obvious root rot symptoms were investigated, and the incidence of root rot was calculated according to the following formula.

[0025] Survival rate (%) = number of surviving seedlings / total number of seedlings × 100

[0026] Root rot incidence (%) = number of root rot plants / total number of plants × 100

[0027] Test results:

[0028] 1) Seedling survival rate and root rot incidence rate Figure 1 As shown in the figure, compared with the control, the synergistic treatment of cinnamic acid and Burkholderia B36 can significantly increase the survival of Panax notoginseng plants and significantly reduce the occurrence of root rot. This shows that the synergistic treatment of cinnamic acid and Burkholderia is beneficial to reduce the occurrence of root rot and alleviate the obstacles of continuous cropping of Panax notoginseng.

[0029] 2) Calculation of synergistic index between cinnamic acid and Burkholderia B36: The Burgi method was used. For the specific design principle and calculation method, please refer to the non-patent literature: Dai Tijun. Quantitative analysis of combined medication [J]. Chinese Pharmacological Bulletin, 1998, 14(5): 2. The calculation formula is:

[0030] q=E(A / 2+B / 2) / EA(or EB)

[0031] E(A / 2+B / 2) refers to the biological effect of a half-dose combination of two drugs

[0032] EA or EB is the biological effect of a single drug at a single dose.

[0033] When q>1, it means that the two have a synergistic effect, q=1 means that the two are superimposed, and q<1 means that the two are antagonistic.

[0034] The results are as follows:

[0035]

[0036]

[0037] The results in the above table show that in terms of seedling survival rate, the q value of the combination compared with the single drug is much greater than 1, indicating that the two drugs have achieved a very obvious synergistic effect in terms of seedling survival rate. In terms of resistance to root rot, the combination has a significant synergistic effect with cinnamic acid, but only an additive effect with Burkholderia.

[0038] Example 2 Investigation of the synergistic mechanism of the composition of cinnamic acid and Burkholderia B36

[0039] 1) Chemotaxis of Burkholderia B36 to cinnamic acid

[0040] Take 100 μl of bacterial solution and add it to 900 μl LB liquid medium to culture the strain for 2 days until the bacterial concentration OD 590 The value reached 0.6, and a sterile yellow gun tip, 1ml syringe, and LB plate culture medium were prepared. Cinnamic acid was prepared into solutions of different concentrations (1μg / mL, 10μg / mL, 50μg / mL, 100μg / mL, 1000μg / mL). Under the sterile conditions of the ultra-clean workbench, 100μl of bacterial solution was transferred to the yellow gun tip with a pipette, 100μl of solution was drawn into the syringe, and then the syringe needle was inserted into the yellow gun tip. After being placed flat on the workbench and allowed to stand for 40 minutes, the liquid in the syringe was transferred to a sterile and clean 2ml tube, and diluted 10 times, 100 times, and 1000 times with sterile water, and then 100μl was taken for plate coating. After culturing at 28℃ for 1 day, the number of colonies was observed and counted.

[0041] 2) Cinnamic acid promotes the colonization of Burkholderia B36 in rhizosphere soil

[0042] The Burkholderia B36 marked with chloramphenicol and rifampicin resistance was selected, and the bacterial concentration OD590 value reached 0.6 after 2 days of culture. Each tissue culture bottle was filled with rhizosphere soil of Panax notoginseng, and 17mL of solution (5mL B36 bacterial solution + 7mL cinnamic acid + 5mL water / antibiotics) was added. After stirring evenly, the lid was covered and cultured for 10 days. On the 5th day of the culture period, 5g of soil was added to 50mL of sterilized water, and cultured on a shaker for 30 minutes. After diluting 10 times, 100 times, and 1000 times, 100μL was taken with a pipette and plated on TSA medium with antibiotics added. After overnight culture at 28°C, the number of colonies formed was recorded to evaluate the colonization effect of Burkholderia B36 in the soil.

[0043] 3) Cinnamic acid promotes biofilm formation of Burkholderia B36

[0044] Take 100 μl of bacterial solution and add it to 900 μl LB liquid medium. Incubate overnight at 28°C until the bacterial concentration reaches OD 590 When the value reached 0.4, 20 μl of bacterial solution was added to 180 μl of 1 / 10 LB solution of cinnamic acid with different concentrations (1 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 1000 μg / mL) in a 96-well plate and cultured at 28°C for 52 h. After 52 h of culture, the crystal violet method was used to determine the biofilm formation of the strain under different concentrations of metabolites.

[0045] Test results:

[0046] 1) Chemotaxis of Burkholderia B36 to cinnamic acid: Results Figure 2 As shown, strain B36 has a significant chemotactic effect on cinnamic acid at concentrations of 1μg / mL, 10μg / mL, and 50μg / mL, but high concentrations (100μg / mL, 1000μg / mL) significantly reduce the chemotactic activity of the strain. This indicates that the chemotactic effect of strain B36 on cinnamic acid is a phenomenon of low promotion and high inhibition, that is, low concentrations promote and high concentrations inhibit.

[0047] 2) Cinnamic acid promotes the colonization of Burkholderia B36 in rhizosphere soil: The results are as follows Figure 3 As shown, compared with the blank control, the number of colonies of Burkholderia B36 in the soil increased significantly in the presence of cinnamic acid, indicating that the synergistic addition of 1-10 μg / mL cinnamic acid is beneficial to promote the colonization of Burkholderia in the rhizosphere of Panax notoginseng.

[0048] 3) Cinnamic acid promotes biofilm formation of Burkholderia B36: Results Figure 4As shown, after 52 hours of culture, 1-1000 μg / mL cinnamic acid can promote the formation of biofilm of the strain, among which 10 and 100 μg / mL concentrations have significant promoting effects.

[0049] The above results indicate that the synergistic effect of cinnamic acid and Burkholderia in alleviating the continuous cropping problem of Panax notoginseng soil may be related to the chemotactic effect of Burkholderia on cinnamic acid and the promotion of Burkholderia colonization and biofilm formation in the rhizosphere soil of Panax notoginseng by cinnamic acid. The specific synergistic mechanism needs further study.

Claims

1. A composition for use in alleviating the disorder of continuous cropping of Panax notoginseng, the composition comprising cinnamic acid and Burkholderia; the cinnamic acid is a 10 μg / mL cinnamic acid aqueous solution; the Burkholderia is 10 6 CFU / mL of a water suspension of Burkholderia; the volume ratio of the cinnamic acid aqueous solution to the Burkholderia water suspension is 1:0.5-2, and the Burkholderia is Burkholderia B36.

2. The use according to claim 1, characterized in that The volume ratio of the cinnamic acid aqueous solution to the Burkholderia aqueous suspension is 1:

1.

3. A method for alleviating the continuous cropping obstacle of Panax notoginseng, the composition comprising cinnamic acid and Burkholderia; the cinnamic acid is a 10 μg / mL cinnamic acid aqueous solution; the Burkholderia is 10 6 CFU / mL of a water suspension of Burkholderia; the volume ratio of the cinnamic acid aqueous solution to the Burkholderia water suspension is 1:0.5-2, and the Burkholderia is Burkholderia B36; Specifically include: 1) Prepare cinnamic acid aqueous solution and Burkholderia suspension of corresponding concentrations and mix them in proportion; 2) Before planting Panax notoginseng, press 0.1 ml / cm 3 Water the soil for continuous cropping of Panax notoginseng and transplant the Panax notoginseng plants after 3 days.

4. The method for alleviating the obstacle of continuous cropping of Panax notoginseng according to claim 3, characterized in that: The Panax notoginseng continuous cropping soil refers to the Panax notoginseng continuous cropping soil for 3 years or more.

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