Paecilomyces lilacinus and application thereof

Abstract

The invention discloses paecilomyces lilacinus and application thereof, and relates to the technical field of microorganisms, and the technical key points are as follows: the classification name of the paecilomyces lilacinus is Purpureobacterium lilacinus, the paecilomyces lilacinus is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation date is January 16, 2025, the preservation number is CGMCC No.41756, and the preservation number is CGMCC No.41756. The paecilomyces lilacinus can be applied to prevention and control of pine wood nematodes. According to the method, the pine wood nematodes are controlled through microorganisms, the death rate of the pine wood nematodes is high, the insect killing time is short, and compared with a traditional pine wood nematode killing method, the method is high in insect killing efficiency and low in pollution.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a Purpureocillium lilacinum and application thereof. BACKGROUND

[0002] The pine wood nematode is a plant pathogenic nematode that mainly parasitizes in Pinus plants, causing rapid death of pine trees. The Purpureocillium lilacinum is an endophytic fungus that can parasitize eggs, larvae and female insects of plant parasitic nematodes, and can invade and destroy cell components by secreting chitinase and serine protease to degrade chitin and protein components of the nematode cuticle.

[0003] Therefore, the Purpureocillium lilacinum can be applied in agricultural prevention and control, but the existing research has not studied the specific application of the Purpureocillium lilacinum in preventing and controlling the pine wood nematode. SUMMARY

[0004] The present application aims to solve the above problems, and provides a Purpureocillium lilacinum and application thereof.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0006] The present application provides a Purpureocillium lilacinum, the classification name of which is Purpureocillium lilacinum, which is preserved in the China General Microbiological Culture Collection Center on January 16, 2025, and the preservation number is CGMCC No.41756.

[0007] The present application is further provided as follows: the application of the Purpureocillium lilacinum in preventing and controlling the pine wood nematode, the Purpureocillium lilacinum traps the pine wood nematode by forming a ring, thereby killing the pine wood nematode and absorbing nutrients in the pine wood nematode for its own growth and reproduction, and further decomposing the pine wood nematode.

[0008] The present application is further provided as follows: the Purpureocillium lilacinum extract is obtained by ethyl acetate extraction.

[0009] The present application is further provided as follows: preferably, the use amount of the Purpureocillium lilacinum extract is 15%.

[0010] Compared with the prior art, the present application has the beneficial effects: the present application can prevent and control the pine wood nematode by microorganisms, the mortality rate of the pine wood nematode is high, and the insecticidal time is fast, compared with the traditional pine wood nematode killing method, the present application has high insecticidal efficiency and low pollution. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a Purpureocillium lilacinum spore bottle picture in the embodiment of the present application;

[0012] Figure 2 is a picture of spores of Purpureocillium lilacinum in an embodiment of the present application;

[0013] Figure 3 is a picture of Purpureocillium lilacinum conidial chain in an embodiment of the present application;

[0014] Figure 4 is a picture of Purpureocillium lilacinum mycelium binding Bursaphelenchus xylophilus in an embodiment of the present application;

[0015] Figure 5 is a survival curve of Bursaphelenchus xylophilus in a dichloromethane extract virulence determination in an embodiment of the present application;

[0016] Figure 6 is a survival curve of Bursaphelenchus xylophilus in an ethyl acetate extract virulence determination in an embodiment of the present application. DETAILED DESCRIPTION

[0017] In order to enable persons skilled in the art to better understand the present application scheme, the technical solutions of the present application will be further described in detail below in combination with the embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should belong to the scope of protection of the present application.

[0018] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below in combination with the embodiments.

[0019] Embodiment:

[0020] The classification and naming of the Purpureocillium lilacinum fungus is Purpureocillium lilacinum, which is preserved in the China General Microbiological Culture Collection Center, the preservation date is January 16, 2025, the preservation number is CGMCC No. 41756, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing.

[0021] Potato dextrose agar medium (PDA): 200 g of peeled potato, 20 g of glucose, 18 g of agar, 1000 mL of water, 121 ℃ high pressure steam sterilization for 20 min. The strain is inoculated on PDA medium and cultured at 25 ℃ for 7 days.

[0022] Method for extracting Purpureocillium lilacinum metabolites by dichloromethane:

[0023] (1) Preparation and sterilization of PD. Potato dextrose medium (PD): 200 g of peeled potato, 20 g of glucose, 1000 mL of water.

[0024] (2) Inoculate the PD with the fungus Paecilomyces lilacinus.

[0025] (3) Place the PD inoculated with the bacteria in a shaker and culture at 26°C for 72 hours.

[0026] (4) Separate the Paecilomyces lilacinus and the fermentation liquid through a filtration device, and a total of 500 ml of filtrate needs to be separated.

[0027] (5) Check for leaks in the separatory funnel.

[0028] (6) Add liquid: first add 250 mL of dichloromethane and then add 250 mL of filtrate.

[0029] (7) Shake and mix.

[0030] (8) Standing and stratifying: Place the mixed solution after shaking on an iron stand and let it stand for 30 minutes. The upper layer is the filtrate and the lower layer is the organic solution.

[0031] (9) Liquid separation: Separate the lower organic solution into a conical flask, and add an equal amount of dichloromethane to the upper solution for secondary extraction, and extract continuously for 3 times.

[0032] (10) The lower organic solution extracted three times was collected for distillation.

[0033] (11) When the liquid is distilled to about 2 mL, a portion is taken out for identification. The extract is then distilled separately until only solid remains. The solid is then dissolved in 2 mL of dimethyl sulfoxide as the original drug and stored in a refrigerator for toxicity determination against pine wood nematodes.

[0034] Method for extracting metabolites of Paecilomyces lilacinus with ethyl acetate:

[0035] Steps (1) to (7) and (11) are the same as above.

[0036] (8) Standing and stratifying: Place the mixed solution after shaking on an iron stand and let it stand for 30 minutes. The upper layer is the organic solution and the lower layer is the filtrate.

[0037] (9) Separation: First, separate the lower filtrate into a conical flask, then pour the upper organic solution from the top of the separatory funnel and save it. Then add 250 mL of ethyl acetate to the separatory funnel and then add the lower filtrate into it. Extract continuously for a total of 3 times.

[0038] (10) The upper organic solution after three extractions was collected for distillation.

[0039] After evaporating to a solid by rotary evaporation, 1 mL of DMSO was used to dissolve the original drug.

[0040] Results of toxicity test of the extract against pine wood nematode

[0041] Table 1 shows the average mortality rates of pine nematodes at different time points in the dichloromethane extract toxicity assay. The mortality rate of pine nematodes gradually increased over time, reaching a peak of 98.22% at 72 hours, with a corrected mortality rate of 98.98%. A higher dosage of the technical pesticide resulted in faster pine nematode death and a higher mortality rate.

[0042] Table 1 Toxicity determination of dichloromethane extract Average mortality of pine wood nematodes

[0043]

[0044] Table 2 shows the average mortality rate of pine nematodes at different time periods, as determined by the ethyl acetate extract. The mortality rate gradually increased over time, reaching a peak of 99.71% at 72 hours, with a corrected mortality rate of 100%. A higher dosage of the technical pesticide resulted in faster pine nematode death and a higher mortality rate.

[0045] Table 2 Toxicity determination of ethyl acetate extract Average mortality of pine wood nematodes

[0046]

[0047] By comparing the toxicity of the original drug extracted with dichloromethane and ethyl acetate to pine wood nematodes and measuring the average mortality rate, it was found that the original drug extracted with ethyl acetate was more effective than the original drug extracted with dichloromethane.

[0048] Survival curve of toxicity assay

[0049] Figure 5 It can be seen that the survival rate of pine wood nematodes was lowest when the dichloromethane-extracted dosage was 15% compared to 5%. 15% had the lowest survival rate, followed by 10%, and 5% had the highest survival rate. Over time, the 15% dosage of the original drug had the highest nematode kill rate at 12 hours, followed by 10% and 5%. This shows that for the same concentration of original drug used within the same time period, the greater the number of pine wood nematodes killed. When the dichloromethane-extracted dosage of the original drug was 15%, the highest number of nematode deaths occurred between 0 and 12 hours. When the dosage of the original drug was 10%, the highest number of nematode deaths occurred between 24 and 36 hours. However, when the dosage of the original drug was 5%, the number of nematode deaths remained similar across all time periods. At 48 and 60 hours, the nematode kill rates of the 10% and 15% dosages were similar.

[0050] Figure 6It can be seen that the survival rate of pine wood nematodes was lowest when the ethyl acetate-extracted technical drug was used at a dosage of 15% compared to 5%. 15% had the lowest survival rate, followed by 10%, and 5% had the highest survival rate. Over time, the 15% technical drug had the highest nematode kill rate at 12 hours, followed by 10% and 5%. This indicates that at the same concentration, a higher dosage of the technical drug within the same timeframe resulted in a greater number of pine wood nematodes dying. When the 15% technical drug was used at a dosage of ethyl acetate, the highest number of nematodes died between 0 and 12 hours; when the 10% dosage was used at a dosage of 10%, the highest number of nematodes died between 24 and 36 hours; and when the 5% dosage was used at a dosage of 48 to 60 hours. At 72 hours, the 10% and 15% technical drug dosages showed similar nematode-killing efficacy against pine wood nematodes.

[0051] By comparison Figure 5 and Figure 6 The survival curve showed that the original drug extracted with ethyl acetate had a better insecticidal effect on pine wood nematodes when the original drug was used at the same dosage and within the same time.

[0052] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A fungus Paecilomyces lilacinus, characterized by: The classification name of the described Paecilomyces lilacinum is Purpureocillium lilacinum, and it is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms. The deposit date is January 16, 2025, and the deposit number is CGMCC No.41756.

2. Use of the Paecilomyces lilacinus according to claim 1 in preventing and controlling pine wood nematodes.

3. The use of Paecilomyces lilacinus for controlling pine wood nematodes according to claim 2, characterized in that: The extract of Paecilomyces lilacinus was obtained by extraction with ethyl acetate.

4. The use of Paecilomyces lilacinus for controlling pine wood nematodes according to claim 3, characterized in that: Preferably, the dosage of the Paecilomyces lilacinus extract is 15%.

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