Application of metal-phenol network coating on purpureocillium lilacinum nano coating

Abstract

The invention discloses an application of a metal-phenol network coating on a Purpureocillium lilacinum nano-coating, the nano-coating is prepared on the surface of Purpureocillium lilacinum conidia by taking tannic acid and iron ions as materials, the operation is simple, the Purpureocillium lilacinum conidia has biocompatibility, the method can effectively protect Purpureocillium lilacinum from being influenced by high temperature, and the service life of the Purpureocillium lilacinum is prolonged. The room-temperature storage activity is prolonged, the prevention and control potential of the Purpureocillium lilacinum on root-knot nematode is not influenced, and the Purpureocillium lilacinum has a relatively high application value in the aspect of prolonging the shelf life of

Description

Technical Field

[0001] The invention relates to the technical field of plant protection, and in particular to application of a metal-phenol network coating on a nano coating of Pseudomonas lilacinus. Background Art

[0002] Purpureocillium lilacinum, formerly known as Paecilomyces lilacinus, is one of the biocontrol fungi with good application prospects for controlling plant parasitic nematodes. Like most fungal biocontrol agents, Purpureocillium lilacinum is used in the form of living fungal spores, but the low activity of fungal spores leads to a short shelf life of Purpureocillium lilacinum products, which seriously affects its commercial value.

[0003] Metal-phenolic networks (MPNs) are a class of self-assembled nanomaterials composed of polyphenols and metal ions. Since they were first reported by Ejima et al. in 2013, they have been widely used in various fields, but most of the current work focuses on the application of tannic acid (TA) and iron as MPN components. Studies have shown that microbial and mammalian cells are coated with MPN coatings to protect the internal cells from harmful, often lethal enzymes, nanoparticles, heat or ultraviolet attacks, but there are relatively few reports on the application of MPN coatings in agriculture. Burke et al. (2023) demonstrated that the use of self-assembled MPN coatings can protect Pseudomonas chlororaphis from freeze-drying, high temperature (50°C) and high humidity. Therefore, the use of MPNs has the potential to extend the shelf life of microorganisms. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an application of a metal-phenolic network coating on a nano-coating of P. lilacinus. The coating as a nano-coating can effectively protect P. lilacinus from the influence of high temperature, prolong the storage vitality at room temperature, and does not affect its control potential for root-knot nematodes.

[0005] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0006] The invention also provides an application of a metal-phenol network coating on the coating of Pseudomonas lilacinus.

[0007] Furthermore, the application is to form a nano coating on the surface of the conidia of Vibrio lilacinus using tannic acid and iron ions as materials.

[0008] The present invention also provides a method for preparing spores of Psoralea corylifolia having a nano-coating, comprising the following steps:

[0009] 1) activating and culturing Purpureocillium lilacinum, collecting conidia of Purpureocillium lilacinum, and obtaining a conidia suspension; wherein the Purpureocillium lilacinum is Purpureocillium lilacinum strain 36-1;

[0010] 2) adding tannic acid solution and ferric chloride solution to the conidia suspension in sequence and mixing; then adding PBS (for stabilizing the pH value of the solution), centrifuging after the reaction is completed, removing the supernatant, and resuspending; the spore suspension obtained by resuspending is the spores of Psoralea lilacinus with the nano-coating.

[0011] The nano-coating of the Pseudomonas lilacinus spores having the nano-coating is determined by the number of repetitions of the entire step 2).

[0012] The nano-coated purpurogenous spores have the following characteristics:

[0013] 1. Maintain vitality under high temperature conditions;

[0014] 2. Maintain activity stability for a long time at room temperature.

[0015] Furthermore, the conidia suspension concentration is 10 7 CFU / mL.

[0016] Furthermore, the concentration of the tannic acid solution is 0.2 mg / mL, the concentration of the ferric chloride solution is 0.05 mg / mL, and the volume ratio of the tannic acid solution to the ferric chloride solution is 1:1.

[0017] Furthermore, the volume ratio of the total volume of the tannic acid solution and the ferric chloride solution to the conidia suspension is 1:49.

[0018] The present invention also provides an application of the spores of Pseudomonas lilacinus with a nano-coating prepared by the above method in preventing and controlling plant infection with root-knot nematodes.

[0019] The present invention also provides a biocontrol agent for preventing and treating plant root-knot nematode disease, wherein the biocontrol agent contains the spores of Pseudomonas lilacinus with a nano-coating prepared by the above method.

[0020] The present invention also provides an application of the above-mentioned biocontrol agent in preventing and controlling plant infection by root-knot nematodes while promoting plant growth.

[0021] The present invention also provides a method for preventing and controlling plant root-knot nematode disease, which comprises irrigating the roots of plants with the above-mentioned biocontrol agent.

[0022] Preferably, the plant is tomato.

[0023] Beneficial effects of the present invention:

[0024] The present invention provides a metal-phenolic networks (MPNs) coating as a spore coating, which is composed of tannic acid (TA) and iron ions (Fe III ) is used as the material to prepare the MPN coating on the conidia surface of Pseudomonas lilacinus. The operation is simple and biocompatible. This method can effectively protect Pseudomonas lilacinus from the influence of high temperature, prolong its storage vitality at room temperature, and does not affect its potential for controlling root-knot nematodes. It has high application value in extending the shelf life of Pseudomonas lilacinus products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 For TA-Fe III Coated conidia of Psoralea lilacinus Pl@[TA-Fe III ] Schematic diagram of preparation and characterization results

[0026] Figure 2 For TA-Fe III Coated conidia of Psoralea lilacinus Pl@[TA-Fe III ] Vitality protection under high temperature

[0027] Figure 3 For TA-Fe III Coated conidia of Psoralea lilacinus Pl@[TA-Fe III ] Long-term stability at room temperature storage

[0028] Figure 4 Effects of biocontrol agents on the growth of tomato plants DETAILED DESCRIPTION

[0029] For ease of understanding, the present invention is further described in detail with reference to specific embodiments.

[0030] Example 1: Spores of Psoralea purpurogena with a nano-coating Pl@[TA-Fe III Preparation of 1

[0031] like Figure 1 The spores of Psoralea lilacinus with a nano-coating are shown in Fig. III Preparation of 1:

[0032] 1) activating Purpureocillium lilacinum (the Purpureocillium lilacinum is Purpureocillium lilacinum strain 36-1, the strain 36-1 has a preservation number of CCTCC NO: M 20231462, which is disclosed in an application number of 2024101667478, and the invention name is an invention patent of Purpureocillium lilacinum strain 36-1 and its application) and culturing, collecting Purpureocillium lilacinum conidia and adjusting the concentration to 1×10 7 CFU / mL, and a conidia suspension was obtained;

[0033] 2) Take 490 μL of conidia suspension in a 2 mL centrifuge tube, add 5 μL of TA (20 mg / mL) and 5 μL of FeCl3·6H2O (5 mg / mL) in sequence, mix for 10 seconds, then add 500 μL of PBS (10 mM, pH 7.2-7.4) to stabilize the pH value of the solution. After the reaction is completed, centrifuge the mixture at 4000 g for 3 minutes, remove the supernatant, add deionized water to resuspend, and obtain a TA-Fe layer. III Nano-coated spores of Pseudomonas lilacinus, namely Pl@[TA-Fe III ]1.

[0034] Example 2

[0035] Repeat step 2) multiple times using the method of Example 1 to obtain two layers of TA-Fe III Nano-coated conidia of Psoralea lilacinus Pl@[TA-Fe III ]2. With three layers of TA-Fe III Nano-coated conidia of Psoralea lilacinus Pl@[TA-Fe III ]3 and has four layers of TA-Fe III Nano-coated conidia of Psoralea lilacinus Pl@[TA-Fe III ]4.

[0036] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the Pl@[TA-Fe III ]4 was characterized, and the results were as follows Figure 1 As shown in B: Both SEM and TEM images show that TA-Fe III Coating formation.

[0037] Example 3Pl@[TA-Fe III ] Vitality protection under high temperature

[0038] The uncoated conidia of Psoralea lilacinus (P1) and the conidia with two layers of TA-Fe III Nano-coated conidia of Psoralea lilacinus (Pl@[TA-FeIII ]2) Heat treatment at 54°C for 1 hour in a water bath, and count the number of live spores using the dilution plate method. After each treatment is fully mixed, 100 μL of spore suspension is drawn into 900 μL of pure water to obtain a 10-fold diluted spore suspension. Similarly, serial dilutions are prepared to a final concentration of 10 4 CFU / mL. Then, 100 μL of the above solution was applied to the surface of the PDA plate. Each treatment was repeated three times, and two plates were applied for each repeat. The plate was cultured at 28°C for 3 days until colonies were visible, and the number of colonies was counted and photographed for record.

[0039] The results are as follows Figure 2 As shown in the figure: After being treated at 54℃ for 1 hour, the uncoated spores at the same dilution multiple almost did not survive, while the spores coated twice still survived to a certain extent, and the number of colonies generated on the plate was nearly 10 times that of the uncoated spores. This significant difference in survival rate under high temperature indicates that the nano-coating (TA-Fe III The coating) is effective in protecting the survival of Psoralea corylifolia spores.

[0040] Example 4Pl@[TA-Fe III ] Long-term stability at room temperature storage

[0041] The uncoated conidia of Psoralea lilacinus and the TA-Fe III Coated conidia of Psoralea lilacinus (Pl@[TA-Fe III ]1. Pl@[TA-Fe III ]2. Pl@[TA-Fe III ]3) Store in pure water without any nutrients at room temperature. Use the dilution plate method to count the number of viable spores. After each treatment is fully mixed, take 100 μL of spore suspension and place it in 900 μL of pure water to obtain a spore suspension diluted 10 times. Similarly, prepare serial dilutions to a final concentration of 10 3 CFU / mL. Then 100 μL of the above solution was applied to the surface of the PDA plate, and each treatment was repeated three times, and each repetition was applied to 3 plates. The plate was cultured at 28°C for 3 days until colonies were visible. The initial activity of P. lilacinus in each treatment was evaluated. During the whole process, samples were taken and applied to the plate every month to count the number of live spores of P. lilacinus for a period of 6 months.

[0042] The results are as follows Figure 3 As shown in the figure, under room temperature, the activity of uncoated Psoralea purpurogena spores (Pl) continued to decrease with the extension of storage time. By the sixth month, the activity had dropped below 20%. III Coated spores (Pl@[TA-Fe III ]1. Pl@[TA-FeIII ]2. Pl@[TA-Fe III ]3) Although the spore activity continued to decline in the first four months, the overall activity was higher than that of uncoated spores, and the spore activity did not change much after four months, and was maintained at about 50% overall. As shown in Table 1, after one month of storage at room temperature, the uncoated spores were basically able to survive, and there was no significant difference between the treatments; from the second month, the uncoated spores and the coated spores began to show significant differences, and TA-Fe III The viability of coated spores was significantly higher than that of uncoated spores, and the difference gradually increased with time.

[0043] Table 1 Pl and Pl @ [TA-Fe III ]Comparison of activity after 6 months storage at room temperature

[0044]

[0045] Example 5

[0046] Biocontrol agent 1 for controlling plant root-knot nematode disease contains Pl@[TA-Fe III ]1, where Pl@[TA-Fe III The concentration of ]1 is 10 7 CFU / mL.

[0047] Example 6

[0048] Biocontrol agent 2 for controlling plant root-knot nematode disease contains Pl@[TA-Fe III ]2, where Pl@[TA-Fe III The concentration of ]2 is 10 7 CFU / mL.

[0049] Example 7

[0050] Biocontrol agents for controlling plant root-knot nematode diseases contain Pl@[TA-Fe III ]3, where Pl@[TA-Fe III The concentration of ]3 is 10 7 CFU / mL.

[0051] Comparative Example 1

[0052] Uncoated conidia of Psoralea lilacinus P1, the concentration of which was 10 7 CFU / mL.

[0053] Comparative Example 2

[0054] TA-Fe III The solution was prepared by mixing TA (20 mg / mL) and FeCl3·6H2O (5 mg / mL) in a volume ratio of 1:1.III The solution is diluted 100 times to obtain 1 / 100TA-Fe III .

[0055] Example 8 Evaluation of the efficacy of biological control agents against southern root-knot nematodes in indoor potted plants 1. Treatment of tomato seedlings:

[0056] Select plump tomato seeds of the same size, add 75% ethanol to disinfect the surface for 2 minutes, rinse with sterile water 3 times, and place in a petri dish covered with germination paper for germination. Then sow the tomato seeds in sterilized nutrient soil for seedling cultivation, cultivate under 28℃ light until the tomato grows 4 true leaves, and then transplant it into a 10×10cm pot. After the transplanted tomatoes are planted, different treatments are carried out, and 12 replicates are planted for each treatment; among them, the different treatment schemes are as follows:

[0057] Biocontrol agent 1 treatment group: 20 mL of biocontrol agent 1 and 1,500 nematodes were inoculated simultaneously;

[0058] Biocontrol agent 2 treatment group: 20 mL of biocontrol agent 2 and 1,500 nematodes were inoculated simultaneously;

[0059] Biocontrol agent 3 treatment group: 20 mL of biocontrol agent 3 and 1500 nematodes were inoculated simultaneously;

[0060] Pl treatment group: inoculated with 20 mL of uncoated P. lilacinus conidia Pl and 1500 nematodes simultaneously;

[0061] 1 / 100TA-Fe III Treatment group: 20 mL of 1 / 100 TA-Fe was inoculated at the same time III and 1,500 nematodes;

[0062] H2O treatment group: 20 mL of water and 1500 nematodes were inoculated at the same time.

[0063] 2. Investigation of tomato root-knot nematode disease

[0064] 60 days after the nematode inoculation, the control effect was calculated according to the tomato root knot index grading standard. The disease grading standard of root knot nematodes is as follows:

[0065] Level 0: No root knot;

[0066] Level 1: There are a small number of root knots, accounting for 1%-25% of the total root system;

[0067] Level 2: The number of root nodes is moderate, accounting for 26%-50% of the total root system;

[0068] Level 3: There are a large number of root nodes, accounting for 51%-75% of the entire root system;

[0069] Level 4: There are a large number of root nodes, accounting for 76%-100% of the entire root system (Benjamn et al 1987).

[0070] The disease index and relative prevention efficacy survey results are shown in Table 1.

[0071]

[0072]

[0073] Table 2 Effects of different treatments on the control of southern root-knot nematodes

[0074]

[0075] From the analysis in Table 2, it is found that 60 days after the nematode inoculation, the application of a certain concentration of TA-Fe III The solution had no control effect on root-knot nematodes, but the conidia suspension of P. lilacinus with or without coating had a good control effect on root-knot nematodes, and the above coating times had no significant effect on the biocontrol effect of P. lilacinus.

[0076] 3. Tomato biomass survey

[0077] 60 days after nematode inoculation, the biomass of tomatoes, including plant height, fresh weight of aboveground parts, root length, and root fresh weight, was measured. The number of root-knot nematodes in the soil and the number of eggs in tomato roots were also counted.

[0078] The results of the potted plant survey are shown in Figure 4. It can be seen that the biocontrol agents can not only protect against root-knot nematode diseases but also promote tomato growth, as follows:

[0079] a. Effects of root-knot nematode disease

[0080] Compared with the control (H2O treatment group), the Pl treatment group and the treatment groups of biocontrol agents 1 to 3 can effectively reduce the number of tomato root eggs and soil nematodes. The number of root eggs in the Pl treatment group and the treatment groups of biocontrol agents 1 to 3 decreased by 41.44%, 50.65%, 50.29%, and 55.38% respectively; the number of soil nematodes decreased by 51.06%, 38.30%, 44.68%, and 44.68% respectively. 1 / 100TA-Fe III There was no significant difference in the number of tomato root eggs and soil nematodes between the treatment group and the control group.

[0081] b. Promote tomato growth

[0082] Different treatments can promote the growth of tomato plants compared with the control, which is reflected in the increase of plant height, seedling fresh weight, root length and root fresh weight compared with the blank control (H2O treatment group). IIIThe solution significantly promoted the aboveground part of tomatoes, and significantly increased the plant height and seedling fresh weight of tomatoes. The plant height increased from 21.26cm in the control to 23.97cm, and the seedling fresh weight increased from 10.43g in the control to 12.44g. The Pl treatment group and the biocontrol agents 1 to 3 treatment groups had significant effects on the underground part of tomatoes, and the root growth was significantly better than that of the H2O treatment group and the 1 / 100TA-Fe III The root length and fresh weight of the treatment group were significantly higher than those of the H2O treatment group and the 1 / 100TA-Fe III Treatment group. The root lengths of the Pl treatment group and the biocontrol agents 1 to 3 treatment groups increased by 1.52 cm, 2.13 cm, 2.59 cm, and 2.59 cm, respectively, and the root fresh weights increased by 0.80 g, 0.73 g, 0.85 g, and 0.92 g, respectively, compared with the control (H2O treatment group).

[0083] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Application of a metal-phenolic network coating on the coating of Pseudomonas lilacinus.

2. The use according to claim 1, characterized in that: The application is to use tannic acid and iron ions as materials to form a nano coating on the surface of the conidia of Vibrio lilacinus.

3. A method for preparing spores of Psoralea corylifolia having a nano-coating, characterized in that: The following steps are involved: 1) activating and culturing Psoralea lilacinus, collecting conidia of Psoralea lilacinus, and obtaining a conidia suspension; 2) adding tannic acid solution and ferric chloride solution to the conidia suspension in sequence and mixing; then adding PBS, centrifuging after the reaction is completed, removing the supernatant, and resuspending; the obtained spore suspension is the spores of Pseudomonas lilacinus with the nano-coating.

4. The preparation method according to claim 3, characterized in that: The concentration of the conidia suspension is 10 7 CFU / mL.

5. The preparation method according to claim 3 or 4, characterized in that: The concentration of the tannic acid solution is 0.2 mg / mL, the concentration of the ferric chloride solution is 0.05 mg / mL, and the volume ratio of the tannic acid solution to the ferric chloride solution is 1:

1.

6. The preparation method according to claim 3 or 4, characterized in that: The volume ratio of the total volume of the tannic acid solution and the ferric chloride solution to the conidia suspension is 1:

49.

7. Use of the nano-coated spores of Pseudomonas lilacinus prepared by the method according to claim 3 in preventing and controlling plant infection with root-knot nematodes.

8. A biological control agent for preventing and controlling plant root-knot nematode disease, characterized in that: The biocontrol agent contains the spores of Pseudomonas lilacinus with a nano-coating prepared by the method according to claim 3.

9. Use of the biocontrol agent according to claim 8 in preventing and controlling plant infection by root-knot nematodes while promoting plant growth.

10. A method for preventing and controlling plant root-knot nematode disease, characterized in that: The method comprises irrigating the roots of plants with the biocontrol agent according to claim 8.

Images