Synbiotics for enhancing insect resistance of Chinese cabbage and application of synbiotics

Abstract

The invention discloses application of synbiotics to prevention and control of plutella xylostella pests. Probiotics in the synbiotics are a pseudomonas strain PPF1, the pseudomonas strain PPF1 is classified and named as Pseudomonas putida, and the prebiotic composition is ornithine, alanine, valine, serine, leucine and cycloleucine. The invention further discloses a preparation method of the prebiotic composition. Wherein the pseudomonas can improve the resistance of Chinese cabbage to plutella xylostella. According to the prebiotic composition, the resistance of Chinese cabbage to plutella xylostella is improved, meanwhile, the growth of the pseudomonas strain PPF1 is remarkably promoted, and the colonization ability of the pseudomonas strain PPF1 is improved. The synbiotics are applied into soil by a root irrigation method, so that the feeding area of the cabbage by the plutella xylostella can be remarkably reduced, and the pest of the plutella xylostella in the cabbage can be effectively prevented and controlled.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and relates to the application of synbiotics in preventing and controlling insect pests. Background Art

[0002] The diamondback moth (Plutella xylostella) is one of the most devastating pests in the vegetable industry. It mainly harms common cruciferous crops such as cabbage, Shanghai greens, rapeseed, cauliflower, and kale, posing a huge threat to the overall yield and quality of vegetables. In agricultural production, farmers mainly control diamondback moth pests through chemical means such as pesticides. However, with the frequent use of a large number of chemical pesticides, the diamondback moth's resistance to pesticides has increased rapidly. The diamondback moth has the characteristics of not being dormant all year round, strong reproductive capacity, and a short reproductive cycle. Insects of all developmental stages can be seen everywhere, which will form a vicious cycle of repeated spraying, increased dosage, and increased pest resistance, leading to increasingly prominent agricultural product safety issues. Therefore, it is urgent to explore ecological and green means to prevent and control diamondback moth pests.

[0003] Rhizosphere Pseudomonas is an important class of beneficial microorganisms that can promote plant growth and improve plant tolerance to various stresses. The colonization of Pseudomonas in the roots of Arabidopsis thaliana will induce plant systemic resistance (ISR), thereby effectively responding to pest attacks. However, due to the limited number of nutrient resources in the soil, after the Pseudomonas agent is applied to the soil, there are problems such as low colonization efficiency and low survival rate, which seriously reduces the control efficiency of Pseudomonas. Therefore, it is necessary to explore resources that promote the colonization and growth of Pseudomonas in the rhizosphere and provide a suitable growth environment for Pseudomonas. Root exudates, as nutrient substrates or signal molecules for microorganisms, can serve as an important source of rhizosphere prebiotics. The combination of rhizosphere beneficial microorganisms and rhizosphere prebiotics is called rhizosphere synbiotics. The application of rhizosphere synbiotics will effectively improve the colonization and survival ability of beneficial microorganisms in the rhizosphere, which will provide a new way to prevent and control plant diseases and insect pests. More and more pesticides are being or may be banned. Therefore, there is an urgent need to explore prebiotics that can enhance the colonization of Pseudomonas in the rhizosphere of cabbage, construct synbiotic formulas in cabbage, and prevent and control the diamondback moth pest in cabbage. Summary of the invention

[0004] The present invention aims to provide a synbiotic, wherein the probiotics in the synbiotic are Pseudomonas, and the prebiotic substances are ornithine, alanine, valine, serine, leucine and cycloleucine, which can significantly promote the colonization and growth of Pseudomonas in the rhizosphere of cabbage and greatly improve the resistance of cabbage to diamondback moth.

[0005] The technical solution of the present invention is as follows:

[0006] The first object of the present invention is to provide a synbiotic for improving the resistance of cabbage to diamondback moth. The synbiotic comprises probiotics and prebiotic substances, wherein the probiotics are Pseudomonas, and the prebiotic substances are composed of ornithine, alanine, valine, serine, leucine, and cycloleucine.

[0007] Furthermore, the Pseudomonas strain PPF1, classified and named Pseudomonas putida, was deposited in the China Center for Type Culture Collection on January 17, 2024, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2024130.

[0008] Furthermore, the synbiotics include liquid A and liquid B:

[0009] The liquid A is a Pseudomonas suspension prepared with sterile water, and the concentration of the Pseudomonas suspension is 1×10 7 ~1×10 9 CFU / mL, preferably 1×10 8 CFU / mL;

[0010] In a specific embodiment, the Pseudomonas suspension is prepared by the following method: strain PPF1 is activated using CFC Pseudomonas selective solid culture medium; a single colony of PPF1 is picked up and placed in TSB liquid culture medium, cultured with shaking at 28°C for 24 hours, centrifuged, the upper layer of TSB liquid culture medium is discarded, the residual culture medium is washed with sterile water to remove the residual culture medium, the bacteria are collected, and the concentration of the bacterial solution is adjusted with sterile water to obtain a Pseudomonas suspension.

[0011] In a more specific embodiment, the Pseudomonas suspension is prepared by the following method: strain PPF1 is activated using CFC Pseudomonas selective solid culture medium; a single colony of PPF1 is picked up and placed in TSB liquid culture medium, cultured at 28°C with shaking for 24 hours, 180 rpm, centrifuged at 5000 rpm for 10 minutes, the upper layer of TSB liquid culture medium is discarded, and the bacteria are then resuspended with sterile water, and the supernatant is discarded after centrifugation. After repeating the sterile water resuspension and centrifugation three times in total, the concentration of the bacterial solution is adjusted to obtain a Pseudomonas suspension.

[0012] The B solution is an aqueous solution of prebiotic substances, in which the concentration of each amino acid is 0.05-5 mmol / L, preferably 0.5 mmol / L.

[0013] The second object of the present invention is to provide the use of the aforementioned synbiotics for improving the resistance of cabbage to diamondback moth in the preparation of a cabbage rhizosphere application preparation, wherein the preparation comprises at least one of the functions (1) and (2):

[0014] (1) Promote the growth and / or colonization of rhizospheric Pseudomonas;

[0015] (2) Improve the resistance of cabbage to Plutella xylostella.

[0016] Furthermore, the synbiotics are applied 14 days after the cabbage is sown; solution A and solution B are mixed and applied to the soil at the root of the cabbage, and each application of solution A should make the final concentration of Pseudomonas in the soil reach 10 10 CFU / kg, each application of solution B should make the final concentration of each amino acid in the soil reach 50μmol / kg; apply 1-2 times a week, and continue for no less than 3 weeks.

[0017] Compared with the prior art, the present invention has the following effects:

[0018] (1) Applying the Pseudomonas strain PPF1 to the rhizosphere of cabbage can significantly improve the resistance of cabbage to the diamondback moth.

[0019] (2) Prebiotics can significantly promote the growth of Pseudomonas strain PPF1 and improve its colonization ability.

[0020] (3) The combination of prebiotics and Pseudomonas PPF1 can significantly improve the effectiveness of Pseudomonas in controlling Plutella xylostella pests, significantly reduce the damaged area of ​​cabbage, and effectively control Plutella xylostella pests.

[0021] (4) The Pseudomonas strain and rhizosphere prebiotic substance of the present invention are easy to use, have good stability, low cost, significant benefits, and broad market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Effects of different combinations of substances on the defense of cabbage against Plutella xylostella

[0023] A is amino acid, S is sugar, O is other, N is positive control, W is negative control

[0024] Figure 2 Effect of Pseudomonas sp. PPF1 on the defense of cabbage against Plutella xylostella

[0025] Figure 3 Effects of amino acids on the growth of Pseudomonas sp. PPF1

[0026] Figure 4 Effects of amino acids on the colonization ability of Pseudomonas sp. PPF1

[0027] Figure 5 Effect of synbiotics on the defense of cabbage against Plutella xylostella

[0028] Pseudomonas putida PPF1; classification name: Pseudomonas putida (Latin name: Pseudomonas putida); deposited in China Center for Type Culture Collection, the deposit date is January 17, 2024, the deposit address: Wuhan University, China Center for Type Culture Collection, the deposit number is CCTCC NO: M 2024130. DETAILED DESCRIPTION

[0029] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0030] The experimental materials, sources and preparations in the following examples are as follows:

[0031] TSB liquid culture medium: 17 g tryptone, 3 g soy peptone, 5 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose, dilute to 1 L with deionized water, pH 7.0, sterilize at 115°C for 30 min.

[0032] 1 / 5TSB medium: Add 900 mL of deionized water to 100 mL of TSB medium and sterilize at 115°C for 30 min.

[0033] Pseudomonas CFC selective medium: 16g gelatin peptone, 10g acid hydrolyzed casein, 10g potassium sulfate, 1.4g magnesium chloride, 12g agar, pH 7.1, weigh 49.4g, take 10g glycerol, heat and dissolve in 1000mL deionized water, divide into 200mL bottles, sterilize at 121℃ high pressure for 15min, cool to about 50℃, add one bottle of Pseudomonas CFC selective medium additive to every 200mL medium and mix well.

[0034] Preparation of diamondback moth larvae: Diamondback moth eggs were purchased from Henan Keyun Biological Co., Ltd. (Jiyuan, China) and incubated in an incubator at 27 °C, 40%-50% relative humidity. After hatching, diamondback moth larvae were placed on cabbage plants for 4 days, and similarly developed larvae were harvested from the cabbage plants for subsequent experiments.

[0035] Cabbage varieties: Xiaoyi and autumn cabbage

[0036] Preparation of Pseudomonas: strain PPF1, classified and named Pseudomonas putida, was deposited in China Center for Type Culture Collection on January 17, 2024, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2024130.

[0037] Example 1 Obtaining a prebiotic formula

[0038] Diamondback moths were placed on cabbage leaves to feed, and the rhizosphere metabolome of the plants fed by the diamondback moths and the control plants (not fed by the diamondback moths) was measured one week later.

[0039] The results showed that after being fed by Plutella xylostella, there were differences in 32 components in the rhizosphere metabolites, including leucine, alanine, xylose, ribose, threonine, fucose, 2-6-diaminopimelic acid, 2-ketoisovaleric acid, maleic acid, N-acetyl-5-hydroxytryptamine, 4-hydroxycinnamic acid, serine, valine, maltotriose, sorbitol, salicin, naringenin, cycloleucine, 4-hydroxyphenylethanol, cis-eicosenoic acid, ornithine, glucose, fructose, threonic acid, 3-hydroxybutyric acid, glucoheptonic acid, 2-D-deoxyglucose, quinic acid, dihydroxyacetone, epigallocatechin-3,4-dihydroxymandelic acid, and N-acetylisoindolinone.

[0040] Further studies found that 7 substances (3,4-dihydroxymandelic acid, N-acetylisoindolinone, threonine, maleic acid, N-acetyl-5-hydroxytryptamine, 4-hydroxyphenylethanol, and cis-eicosenoic acid) were significantly enriched in the control plants, while leucine, alanine, xylose, ribose, fucose, 2-6-diaminopimelic acid, 2-ketoisovaleric acid, 4-hydroxycinnamic acid, serine, valine, maltotriose, sorbitol, salicin, naringenin, cycloleucine, ornithine, glucose, fructose, threonic acid, 3-hydroxybutyric acid, glucoheptonic acid, 2-D-deoxyglucose, quinic acid, dihydroxyacetone, and epigallocatechin, a total of 25 substances, were enriched in plants fed by the diamondback moth.

[0041] These 25 substances belong to three categories: amino acids (leucine, alanine, cycloleucine, ornithine, serine, valine); sugars (glucose, fructose, xylose, ribose, fucose, maltotriose, sorbitol, 2-D-deoxyglucose); others (threonic acid, 3-hydroxybutyric acid, glucoheptonic acid, 2-6-diaminopimelic acid, 2-ketoisovaleric acid, 4-hydroxycinnamic acid, quinic acid, dihydroxyacetone, epigallocatechin, salicin, naringenin). Therefore, these three types of substances were screened separately or in combination of two or three types.

[0042] The three types of substances were prepared into aqueous solutions separately, in combination of two types, or in combination of three types. The concentration of each substance in the aqueous solution was 0.5 mmol / L.

[0043] Cabbage seeds were sterilized with 75% ethanol for 1 min, then 2% sodium hypochlorite for 2 min, and then rinsed several times with sterile water. Cabbage seeds were then planted in seedling trays (50 holes per tray) and placed in a greenhouse at 23° C. After germination, one seedling was retained in each hole. Each hole contained 50 g of soil.

[0044] 14 days after sowing, each substance solution was added to the soil to a final concentration of 50 μmol / kg, twice a week for three consecutive weeks.

[0045] A mixed solution of 7 substances, 3,4-dihydroxymandelic acid, N-acetylisoindolinone, threonine, maleic acid, N-acetyl-5-hydroxytryptamine, 4-hydroxyphenylethanol, and cis-eicosenoic acid, was used as a positive control, and an equal amount of sterile water was added as a negative control. Six trays were used as six biological replicates. After the treatment, the fourth and fifth fully developed and similar true leaves in each treatment were harvested with sterilized scissors, and the petioles of the leaves were covered with cotton wool soaked in deionized water to prevent wilting. Five larvae of the diamondback moth (7 days old) were placed on one leaf, and the box was sealed with a sealing film. After 17 hours, the area of ​​each leaf eaten by the diamondback moth larvae was recorded. The area of ​​cabbage eaten by the diamondback moth was counted using Image software.

[0046] like Figure 1 As shown, the damaged area of ​​cabbage after amino acid application alone (A) was the lowest, significantly lower than all treatments. In addition, the damaged area of ​​cabbage after the combined treatment of amino acids and sugars (A+S), the combined treatment of amino acids and other substances (A+O), and the combined treatment of amino acids, sugars and other substances (A+S+O) was significantly lower than the positive control treatment (N) and the negative control (W). At the same time, it was found that sugars alone (S), other substances alone (O), or the combination of sugars and other substances (O+S) could not reduce the damaged area of ​​cabbage. Therefore, it was preliminarily determined that prebiotic products are composed of substances such as amino acids.

[0047] Example 2 Effect of Pseudomonas strains on inhibiting Plutella xylostella pests

[0048] Preparation of Pseudomonas suspension: strain PPF1 was activated with CFC solid medium; a single colony of PPF1 was picked up and placed in TSB liquid medium, cultured at 28°C and 180 rpm for 24 h, centrifuged at 4°C and 5000 rpm for 10 min, the upper layer of TSB medium was discarded, the bacterial precipitate was washed with sterile water, and centrifuged at 4°C and 5000 rpm for 10 min. After repeating this three times, the OD of the bacterial solution was adjusted with sterile water. 600 =0.4 (corresponding to 1×10 8 CFU / mL) to obtain Pseudomonas bacterial suspension.

[0049] The cabbage seeds were sterilized with 75% ethanol for 1 min, then 2% sodium hypochlorite for 2 min, and then rinsed several times with sterile water. The cabbage seeds were then planted in seedling trays (50 holes per tray) and placed in a greenhouse at 23°C. After germination, one seedling was retained in each hole. Each hole contained 50 grams of soil. Six trays were used as six biological replicates. The seedling trays were randomly replaced in the greenhouse every week. 2 mL of water was supplied to each hole every morning and evening. 14 days after sowing, the experimental setting treatment:

[0050] 1) Pseudomonas PPF1: Pseudomonas PPF1 bacterial solution was evenly poured into the soil near the roots of cabbage using the root irrigation method to reach a final concentration of 10 10 CFU / kg.

[0051] 2) Control group: Apply an equal amount of deionized water to the soil around the roots of the cabbage.

[0052] Add twice a week. After three weeks, the fourth and fifth fully developed and similar true leaves in each treatment were harvested with sterilized scissors, and the petioles of the leaves were covered with cotton wool soaked in deionized water to prevent wilting. Five diamondback moth (7-day-old) larvae were placed on one leaf, and the box was sealed with a sealing film. After 17 hours, the area of ​​each leaf eaten by diamondback moth larvae was recorded. The area of ​​cabbage eaten by diamondback moth was counted by Image software.

[0053] Results Figure 2 Compared with the control group, the Pseudomonas in the experimental group could significantly reduce the area of ​​cabbage eaten by the diamondback moth, indicating that Pseudomonas could significantly improve the resistance of cabbage to the diamondback moth (P<0.5).

[0054] Example 3 Effect of prebiotics on promoting the growth of Pseudomonas

[0055] The preparation of Pseudomonas PPF1 bacterial suspension is described in Example 2.

[0056] Test setup process:

[0057] Treatment 1 (Pseudomonas PPF1 + amino acid prebiotic substance group): Pseudomonas bacterial solution and amino acid (ornithine, alanine, valine, serine, leucine, cycloleucine) prebiotic substance solution were mixed and added;

[0058] Control group (Pseudomonas PPF1 bacterial solution): Pseudomonas bacterial solution was added, and the prebiotic substance was replaced with an equal volume of sterile water.

[0059] The preparation method of the prebiotic substance solution is as follows: dissolve the amino acid prebiotic substances composed of ornithine, alanine, valine, serine, leucine, and cycloleucine in deionized water, then draw them out with a sterile syringe, and then filter them through a 0.22 μm filter membrane to finally obtain a mixed solution in which the concentration of each amino acid substance is 0.5 mM.

[0060] Experimental group: 200 μL 1 / 5 TSB liquid medium and 2 μL Pseudomonas PPF1 bacterial solution (final concentration of 1×10 6 CFU / mL) and 2 μL of prebiotic substance solution.

[0061] Blank control group: 200 μL TSB liquid culture medium and 2 μL Pseudomonas PPF1 bacterial solution (final concentration of 1x10 6 CFU / mL) and 2 μL sterile deionized water.

[0062] Each treatment was repeated 6 times. After adding the sample, the 96-well plate was placed in a shaker at 28°C and 180 r / min for 24 h, and the absorbance (OD 600 ) to determine the effect of amino acid substances on the growth of Pseudomonas PPF1.

[0063] Results Figure 3 Compared with the control group, the preferred amino acid prebiotics can significantly increase the growth of Pseudomonas PPF1, indicating that the prebiotic substance can significantly promote the growth of Pseudomonas PPF1 (P<0.5).

[0064] Example 4 Effect of prebiotics on promoting colonization of Pseudomonas

[0065] The preparation of Pseudomonas PPF1 bacterial suspension is described in Example 2.

[0066] The configuration of the prebiotic substance solution is as in Example 3.

[0067] Fill 200 μl gun tip with 100 μl Pseudomonas PPF1 bacterial suspension (OD600=0.1) prepared from overnight TSB culture. Use 1 mL sterile syringe as chemotaxis capillary and inject 200 μl amino acid mixed solution or water (use sterile water as blank control). Connect the tip of the syringe to the bacterial suspension in the tip of the gun tip. After static incubation at 27°C for 4h, remove the syringe and count the number of Pseudomonas PPF1 in the syringe on CFC Pseudomonas selective medium by dilution coating plate method. Every 4 syringes are used as a replicate, and each treatment is repeated six times.

[0068] Results Figure 4Compared with the blank control group, the preferred amino acids (ornithine, alanine, valine, serine, leucine, and cycloleucine) can significantly increase the number of Pseudomonas PPF1, indicating that the preferred amino acid substances have a promoting effect on the chemotaxis of Pseudomonas PPF1 and can improve the colonization ability of Pseudomonas PPF1 (P<0.5).

[0069] Example 5 Application of synbiotic substances helps cabbage control diamondback moth pests

[0070] According to the preferred amino acid substances (ornithine, alanine, valine, serine, leucine, cycloleucine) that can enhance the growth and colonization ability of Pseudomonas strains in the rhizosphere, pot experiments were further conducted to investigate the effects of the amino acid prebiotic substances on Pseudomonas in enhancing the resistance of cabbage to Plutella xylostella.

[0071] The cabbage culture conditions refer to those in Example 2. 14 days after sowing, the experiment was set up and treated as follows:

[0072] 1) Pseudomonas PPF1 + amino acid: Pseudomonas PPF1 bacterial solution and amino acid mixture were evenly poured into the soil near the roots of cabbage by root irrigation method, and the bacterial solution was made to reach a final concentration of 10 10 CFU / kg (the preparation method is as described above), and the amount of each amino acid added to the amino acid mixture should make its final concentration in the soil reach 50 μmol / kg (the preparation method is as described above).

[0073] 2) Pseudomonas PPF1 group: Pseudomonas PPF1 bacterial solution was evenly poured into the soil near the roots of cabbage using the root irrigation method to make the final concentration reach 10 10 CFU / kg (preparation method as described above).

[0074] 3) Amino acid group: The amino acid mixture was evenly poured into the soil near the roots of the cabbage using the root irrigation method. The amount of each amino acid added to the amino acid mixture was such that its final concentration in the soil reached 50 μmol / kg (the preparation method was as described above).

[0075] 4) Control group: An equal amount of deionized water was applied to the soil around the roots of the cabbage.

[0076] Add twice a week. After three weeks, the fourth and fifth fully developed and similar true leaves in each treatment were harvested with sterilized scissors, and the petioles of the leaves were covered with cotton wool soaked in deionized water to prevent wilting. Five diamondback moth (7-day-old) larvae were placed on one leaf, and the box was sealed with a sealing film. After 17 hours, the area of ​​each leaf eaten by diamondback moth larvae was recorded. The area of ​​cabbage eaten by diamondback moth was counted by Image software.

[0077] The results are as follows Figure 5 As shown, compared with the single application of Pseudomonas PPF1 strain and the single application of amino acids, the combined use of amino acids (ornithine, alanine, valine, serine, leucine, cycloleucine) and Pseudomonas PPF1 strain can significantly reduce the feeding area of ​​cabbage by Plutella xylostella, indicating that the control effect of the combined application of amino acids and Pseudomonas PPF1 strain on Plutella xylostella pests is significantly higher than the control effect of the single application of Pseudomonas strains and the single application of amino acids, and the combined application of amino acids and Pseudomonas PPF1 strains has a synergistic effect.

[0078] 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. A synbiotic for improving the resistance of cabbage to diamondback moth, characterized in that: The synbiotics include probiotics and prebiotic substances, wherein the probiotics are Pseudomonas, and the prebiotic substances are composed of ornithine, alanine, valine, serine, leucine, and cycloleucine.

2. The synbiotic for improving the resistance of cabbage to diamondback moth according to claim 1, characterized in that: Pseudomonas strain PPF1, classified and named Pseudomonas putida, was deposited in China Center for Type Culture Collection on January 17, 2024, with the deposit address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2024130.

3. The synbiotic for improving the resistance of cabbage to diamondback moth according to claim 1, characterized in that: The synbiotics include liquid A and liquid B: The liquid A is a Pseudomonas suspension prepared with sterile water, and the concentration of the Pseudomonas suspension is 1×10 7 ~1×10 9 CFU / mL; The B solution is an aqueous solution of prebiotic substances, and the concentration of each amino acid in the aqueous solution of prebiotic substances is 0.05-5 mmol / L.

4. The synbiotic for improving the resistance of cabbage to diamondback moth according to claim 1, characterized in that: The concentration of the Pseudomonas suspension is 1×10 8 CFU / mL.

5. The synbiotic for improving the resistance of cabbage to diamondback moth according to claim 1, characterized in that: The concentration of each amino acid was 0.5 mmol / L.

6. Use of the synbiotic for improving the resistance of cabbage to diamondback moth according to any one of claims 1 to 5 in preparing a preparation for rhizosphere application of cabbage, characterized in that: The preparation comprises at least one of the functions of (1) and (2): (1) Promote the growth and / or colonization of rhizospheric Pseudomonas; (2) Improve the resistance of cabbage to Plutella xylostella.

7. The use according to claim 6, characterized in that: The synbiotics were applied 14 days after the cabbage was sown; solution A and solution B were mixed and applied to the soil at the root of the cabbage. Each application of solution A should make the final concentration of Pseudomonas in the soil reach 10 10 CFU / kg, each application of solution B should make the final concentration of each amino acid in the soil reach 50μmol / kg; apply 1-2 times a week, and continue for no less than 3 weeks.

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