Paecilomyces lilacinus as well as fungicide and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to paecilomyces lilacinus as well as a fungicide and application thereof. The invention discloses a novel paecilomyces lilacinum Pli-25, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.41932, and the 16S rDNA (Reverse Deoxyribose Nucleic Acid) sequence of the paecilomyces lilacinum Pli-25 is as shown in SEQ ID NO: 1. A liquid-state fermentation process and a solid-state fermentation process of the strain are studied, a liquid-state fungicide and a powdery fungicide of the paecilomyces lilacinus strain Purpureocillium lilacinus Pli-25 are developed and obtained, a field test result shows that the prevention and treatment effect on the plant field nematode disease by applying the fungicide is remarkable, a new microbial resource and fungicide are provided for biological prevention and treatment of the plant nematode disease, and the application of the fungicide to the field nematode disease is facilitated. The application prospect is wide.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Paecilomyces lilacinus and a bacterial agent and application thereof. Background Art

[0002] Nematode diseases are highly destructive agricultural diseases, causing significant economic losses to global agriculture. Root-knot nematodes, cyst nematodes, and other nematode species parasitize the roots of a wide range of crops, severely disrupting their growth and development.

[0003] When nematodes invade plant roots, they damage the root's cellular structure, impairing root function. The roots' ability to absorb water and nutrients is significantly reduced, leading to slow plant growth, yellowing leaves, wilting, and even death. In severe cases, nematode disease can cause widespread crop yield reductions or even complete crop failure. For example, in vegetable cultivation, root-knot nematode disease can reduce the yield of vegetables like cucumbers and tomatoes by 30% to 70%, severely impacting their quality. Fruits become smaller, deformed, and taste worse, significantly reducing their market value.

[0004] Currently, chemical control remains one of the primary means of combating nematode diseases in agricultural production. However, the use of chemical pesticides presents numerous challenges. First, chemical pesticides remain in the soil for long periods of time, potentially contaminating soil, water sources, and agricultural products, posing a potential threat to human health. Long-term consumption of agricultural products containing pesticide residues can lead to various diseases, such as cancer and neurological disorders. Second, nematodes can easily develop resistance to chemical pesticides, leading to a gradual decline in their effectiveness. To achieve effective control, the dosage and frequency of pesticide application must be continuously increased, which not only increases production costs but also further exacerbates environmental pollution and ecological damage.

[0005] Biological control, as a green and environmentally friendly pest control method, has garnered increasing attention. However, some biocontrol products currently on the market suffer from issues such as inconsistent effectiveness and a narrow range of action. Some microbial agents have poor survival and colonization in soil and are easily affected by environmental factors, resulting in suboptimal control effectiveness. Furthermore, most existing biocontrol products are limited in their functionality, effectively controlling nematodes but failing to simultaneously promote plant growth and development.

[0006] Paecilomyces lilacinus is an endoparasitic fungus with significant application value, demonstrating tremendous potential in the field of biological control. Paecilomyces lilacinus exhibits strong environmental adaptability, capable of surviving and reproducing in diverse soil types, temperatures, and humidity conditions. It can colonize and form stable communities in the soil, exerting a sustained effect against nematodes. Compared to chemical pesticides, Paecilomyces lilacinus is environmentally friendly, meeting the requirements of green agriculture.

[0007] Obtaining new Paecilomyces lilacinus through screening is of great significance for the biological control of nematode diseases. Developing the screened Paecilomyces lilacinus into a bacterial agent has broad application prospects. Summary of the Invention

[0008] To address the above technical problems, the present invention obtained a strain of Paecilomyces lilacinus Pli-25 by sampling soil around severely diseased tomato plants. This strain has excellent protective effects against nematode diseases in fruits and vegetables and can be used in the prevention and control of plant nematode diseases, providing a new technical solution for the biological control of plant nematode diseases.

[0009] In one aspect, the present invention provides a strain of Paecilomyces lilacinus, the biological deposit information of which is as follows:

[0010] Strain name: Pli-25;

[0011] Classification name: Paecilomyces lilacinus;

[0012] Date of receipt by the depository: May 9, 2025;

[0013] Time of issuance of the preservation certificate: May 22, 2025;

[0014] Depository: China General Microbiology Center (CGMCC);

[0015] Deposit number: CGMCC No.41932.

[0016] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0017] Furthermore, the 16S rDNA sequence of Paecilomyces lilacinus Pli-25 is shown in SEQ ID NO: 1.

[0018] In a second aspect, the present invention further provides a microbial agent, comprising the Paecilomyces lilacinus Pli-25 strain and an agriculturally acceptable carrier.

[0019] Furthermore, the microbial agent is a liquid fermentation product or a solid fermentation product.

[0020] Furthermore, in the microbial agent, the method for preparing the liquid fermentation product comprises the following steps:

[0021] Step 1, preparing a liquid fermentation medium, wherein the medium comprises algae, Artemisia annua, cornmeal, cottonseed meal and calcium phosphate;

[0022] Step 2, inoculating the Paecilomyces lilacinus strain according to claim 1 or 2 into the liquid fermentation medium, and fermenting at 25-30° C. for 3-5 days;

[0023] Step 3: Collect the fermentation broth and obtain the liquid fermentation product after centrifugation.

[0024] Furthermore, in the microbial agent, the method for preparing the solid-state fermentation product comprises the following steps:

[0025] S1. preparing a solid fermentation substrate, wherein the substrate comprises algae, cornmeal, Artemisia annua, magnesium sulfate and calcium phosphate;

[0026] S2. inoculating the fermentation liquid containing the Paecilomyces lilacinus strain according to claim 1 or 2 into the solid fermentation substrate, and fermenting at 25-30° C. for 4-5 days;

[0027] S3. Collect the solid fermentation product, dry and crush it to obtain powder.

[0028] In a third aspect, the invention also provides a use of the Paecilomyces lilacinusPli-25 strain or the microbial agent described in the present invention in preventing and controlling plant nematode diseases.

[0029] Furthermore, in the application, the plant nematode disease is root knot nematode disease or cyst nematode disease.

[0030] Furthermore, in the application, the plant is tomato, peanut or cucumber.

[0031] Finally, the present invention also provides a method for preventing and controlling plant nematode diseases, comprising applying the Paecilomyces lilacinus Pli-25 strain of the present invention or the microbial agent of the present invention to plants or a plant growth environment.

[0032] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0033] The present invention isolated a new Paecilomyces lilacinus Pli-25 strain from plant root soil, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with a deposit number of CGMCC No. 41932. Its 16S rDNA sequence is shown in SEQ ID NO: 1.

[0034] The present invention develops a liquid inoculum of the Paecilomyces lilacinus Pli-25 strain and a powdered inoculum of the Paecilomyces lilacinus Pli-25 strain through research on liquid fermentation technology and solid-state fermentation technology. Field test results show that the application of the inoculum has a nematode control effect of 89% to 93% on plants in the field, with significant control effects. The invention provides an effective technical solution for the biological control of plant nematodes and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Colony morphology of Paecilomyces lilacinus Pli-25. DETAILED DESCRIPTION

[0036] The technical solutions of the present invention are described below with reference to the following embodiments; however, the present invention is not limited to the following embodiments.

[0037] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below with reference to specific embodiments and drawings, but the embodiments are not intended to limit the present invention.

[0038] The experimental methods and detection methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0039] Example 1

[0040] This example is to screen and obtain the Paecilomyces lilacinus strain.

[0041] Select a plot with severe tomato disease and collect soil from relatively disease-free plants. Remove the surface soil and sample 10 grams of soil from the rhizosphere, approximately 3 to 7 centimeters below the ground. Place the collected soil in a sterile Erlenmeyer flask, add 100 mL of sterile water, shake thoroughly, and let it sit for 10 minutes. Dilutions are then aspirated and graded. 100 μL of each dilution is evenly spread on polydimethylsiloxane (PDA) medium and incubated at 28°C for 3 to 5 days until colonies emerge. Colonies are then selected for expansion and screened for Septoria lycopersici using the plate standoff method. The leaf spot pathogen is inoculated on one quarter of one side of a solid PDA medium, while the strain to be screened is inoculated on the other side using a sterile toothpick. After 7 days, the standoff culture is observed and the diameter of the inhibition zone is measured. The most effective control strain, designated Pli-25, is selected based on its antibacterial activity and stored at -80°C.

[0042] Example 2

[0043] This example is to identify the strain Paecilomyces lilacinus Pli-25.

[0044] The strain Pli-25 obtained by screening was inoculated into PDA medium and cultured at 28°C for 5 days. The morphology was observed. Figure 1 As shown, the strain was white in the initial culture, and the colonies turned light purple after 5 days of culture, and the colony morphology was oval.

[0045] The strain Pli-25 obtained above was streaked and cultured, and the cells were collected and genomic DNA was extracted. The 16S rDNA gene of Pli-25 was amplified using universal primers, and the amplified product was sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequencing result is shown in SEQ ID NO: 1, and the specific sequence is as follows:

[0046] The nucleotide sequence shown in SEQ ID NO: 1 was compared by Blast in NCBI, and combined with bacterial morphology observation, the strain was determined to be Paecilomyces lilacinus, which was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) in 2025 with the deposit number GCMCC No. 41932.

[0047] Example 3

[0048] This example is about preparing the fermentation product of Paecilomyces lilacinus Pli-25.

[0049] Resuscitate the strain: Remove Paecilomyces lilacinus Pli-25 from the refrigerator and streak it onto potato dextrose agar. Incubate at 28°C, 75%-80% relative humidity, in the dark for 3-5 days. Observe the growth of white to purple colonies with a diameter of at least 0.5 cm on the surface of the culture medium. Set aside.

[0050] Preparation of fermentation substrate: remove impurities from seaweed such as Zostera marina, dry them, and crush them into 60-80 mesh; remove impurities from Artemisia annua, dry them and set aside; set aside fresh, mold-free, food-grade raw materials such as corn flour and cottonseed cake powder; and set aside calcium phosphate.

[0051] 3.1 Liquid fermentation

[0052] The above raw materials were weighed in a ratio of 20 kg of crushed Sargassum, 42 kg of crushed Artemisia annua, 12.5 kg of cornmeal, 2.5 kg of cottonseed meal, and 0.2 kg of calcium phosphate per 1000 kg of water, mixed thoroughly, adjusted to pH 7.2, and sterilized under high pressure at 121° C. for 30 minutes to prepare a liquid fermentation medium for later use.

[0053] The revived Paecilomyces lilacinus Pli-25 was inoculated into the liquid fermentation medium prepared above, and cultured at 28°C, a stirring speed of 160, and a relative humidity of 60%. After 3 days, the fermentation broth was collected and centrifuged at 3000 rpm for 10 minutes. The concentrated solution was collected to obtain the liquid fermentation product of Paecilomyces lilacinus Pli-25.

[0054] 3.2 Liquid fermentation

[0055] The above raw materials were weighed in a ratio of 25 kg of crushed green algae, 40 kg of crushed Artemisia annua, 8.5 kg of cornmeal, 2 kg of cottonseed meal, and 0.1 kg of calcium phosphate per 1000 kg of water, mixed thoroughly, adjusted to pH 7.0, and sterilized by autoclaving at 121°C for 30 minutes to prepare a liquid fermentation medium for later use.

[0056] The revived Paecilomyces lilacinus Pli-25 was inoculated into the liquid fermentation medium prepared above, and cultured at 28°C, a stirring speed of 120, and a relative humidity of 80%. After 5 days, the fermentation broth was collected and centrifuged at 3000 rpm for 10 minutes. The concentrated solution was collected to obtain the liquid fermentation product of Paecilomyces lilacinus Pli-25.

[0057] 3.3 Solid-state fermentation

[0058] The invention relates to a method for preparing a solid-state fermentation product of Paecilomyces lilacinus Pli-25. The solid-state fermentation product is prepared by mixing 70% of Zostera marina, 15% of cornmeal, 14.95% of Artemisia annua, 0.03% of magnesium sulfate, and 0.02% of calcium phosphate, and sterilizing the mixture at 80°C for 40 min. The solid-state fermentation product of Paecilomyces lilacinus Pli-25 is obtained by inoculating the recovered Paecilomyces lilacinus Pli-25 immediately after the mixture is cooled. The solid-state fermentation product is fermented at 26°C, 75% of relative humidity, and protected from light for 5 days. The solid-state fermentation product is collected after 5 days, dried at <50°C, crushed into 60 mesh, and packaged to obtain the solid-state fermentation product of Paecilomyces lilacinus Pli-25.

[0059] 3.4 Solid-state fermentation

[0060] The invention relates to the invention wherein 70% of sargassum thunbergii, 15% of cornmeal, 14.95% of artemisia annua, 0.03% of magnesium sulfate and 0.02% of calcium phosphate are weighed in percentage by mass, the raw materials are mixed and sterilized at 80°C for 40 minutes; after cooling, the recovered Paecilomyces lilacinus Pli-25 is inoculated immediately, and the mixture is fermented at 28°C, 80% relative humidity and in the dark for 4 days; after 4 days, the solid fermentation product is collected, dried at <50°C, crushed into 80 mesh size and packaged to obtain the solid-state fermentation product of Paecilomyces lilacinus Pli-25.

[0061] Example 4

[0062] This example is a field test of the efficacy of the Paecilomyces lilacinus Pli-25 agent applied to tomatoes.

[0063] 4.1 Preparation of Paecilomyces lilacinus Pli-25 inoculum

[0064] Prepare the fermentation broth: Weigh 68 kg of Zostera marina, crushed to 60 mesh, weigh 15 kg of cornmeal, 10.5 kg of cottonseed meal, 0.5 kg of magnesium sulfate, 0.1 kg of calcium phosphate, and 1000 kg of first-grade RO-purified water. Mix these ingredients thoroughly, sterilize at 121°C for 25 minutes, and cool to below 30°C to prepare the fermentation broth.

[0065] Take out the preserved Paecilomyces lilacinus Pli-25 strain, streak it on PDA culture medium, culture it at 28℃ for 3-5 days, and observe the growth of white to purple colonies with a diameter of more than 0.5 cm on the surface of the culture medium.

[0066] In a clean bench, wash the revived Paecilomyces lilacinus Pli-25 colonies from the surface of the PDA medium with sterile water and inoculate them into the fermentation broth using the flame method. Incubate at 28°C, natural pH, and 220-260 rpm for 3 days. When the fermentation broth becomes viscous, sample it and examine it microscopically for the absence of foreign bacteria. Fermentation is complete when more than 80% of the hyphae have transformed into spores. Collect the fermentation broth and centrifuge it at 3000 rpm for 10 minutes. Collect the concentrated fermentation broth to obtain the Paecilomyces lilacinus Pli-25 inoculum.

[0067] 4.2 Tomato field efficacy test

[0068] A greenhouse with years of continuous tomato cultivation and repeated cropping was selected as the test site. The experimental group used 28-day-old tomato seedlings for transplanting and planting, with a plant spacing of 8 inches and a row spacing of 8 inches. Paecilomyces lilacinus Pli-25 was applied to the tomato root soil at a rate of 2 kg per mu (approximately 2 kg) per acre (approximately 1 acre) via root irrigation. Applications of Paecilomyces lilacinus Pli-25 began half a month before the start of the planting season and were applied every two weeks. A control group, where no Paecilomyces lilacinus Pli-25 was used, maintained the same planting parameters (plant spacing, row spacing, and seedling age) as the experimental group.

[0069] The incidence of tomato nematodes in the experimental group and the control group was recorded during the planting process. Thirty days after transplantation, the number of tomato plants infected with nematodes in the experimental group and the control group was counted. The results are shown in Table 1. The incidence rate was calculated as follows:

[0070] Incidence rate (%) = (number of diseased plants / number of surveyed plants) × 100

[0071] Control effect (%) = (1-incidence rate of control group / incidence rate of experimental group) × 100

[0072] Table 1 Statistical results of the control effect of different treatments on tomato nematode disease

[0073] Trial Groups Incidence Control effect control group 71.3% - experimental group 4.9% 93.13%

[0074] The results showed that the Paecilomyces lilacinus Pli-25 inoculant in the experimental group demonstrated significant efficacy in controlling tomato root-knot nematode disease compared to the control group. Statistical analysis showed that the inoculant achieved a 93.13% efficacy against tomato root-knot nematodes, providing an effective biological control method for tomato root-knot nematode disease.

[0075] Example 5

[0076] This example is a field test of the efficacy of the fungicide Paecilomyces lilacinus Pli-25 applied to peanuts.

[0077] 5.1 Preparation of Paecilomyces lilacinus Pli-25 inoculum

[0078] Prepare the fermentation broth: Weigh 30 kg of Zostera marina (60-mesh kelp), 12.5 kg of cornmeal, 8 kg of cottonseed meal, 0.5 kg of calcium phosphate, and 1000 kg of first-grade RO-purified water. Mix these ingredients thoroughly, sterilize at 121°C for 25 minutes, and cool to below 30°C to prepare the fermentation broth.

[0079] Take out the preserved Paecilomyces lilacinus Pli-25 strain, streak it on PDA culture medium, culture it at 28℃ for 3-5 days, and observe the growth of white to purple colonies with a diameter of more than 0.5 cm on the surface of the culture medium.

[0080] In a clean bench, wash the revived Paecilomyces lilacinus Pli-25 colonies from the surface of the PDA medium with sterile water and inoculate them into the fermentation broth using the flame method. Incubate at 28°C, natural pH, and 220-260 rpm for 3 days. When the fermentation broth becomes viscous, sample it and examine it microscopically for the absence of foreign bacteria. Fermentation is complete when more than 80% of the hyphae have transformed into spores. Collect the fermentation broth and centrifuge it at 3000 rpm for 10 minutes. Collect the concentrated fermentation broth to obtain the Paecilomyces lilacinus Pli-25 inoculum.

[0081] 5.2 Peanut field efficacy test

[0082] A greenhouse with a continuous peanut crop, where disease outbreaks were more frequent in previous years, was selected as the experimental site. The experimental group used drip irrigation, applying the Paecilomyces lilacinus Pli-25 inoculant prepared in 5.1 once before sowing, during the seedling stage, and during the growing season, at a rate of 2 kg per mu. A control group, which did not receive the Paecilomyces lilacinus Pli-25 inoculant, served as the control group. All other field management procedures were routine. At harvest, the incidence of peanut nematode disease in the experimental and control groups was statistically analyzed. The incidence and control efficacy were calculated using the same formulas as in 4.2. The statistical results are shown in Table 2.

[0083] Table 2 Statistical results of peanut nematode control efficacy under different treatments

[0084] Trial Groups Incidence Control effect control group 61.30% - experimental group 6.10% 90.05%

[0085] The results showed that compared with the control group, the experimental group treated with Paecilomyces lilacinus Pli-25 had a significant effect on the prevention and control of peanut nematode disease. The application of Paecilomyces lilacinus Pli-25 significantly reduced the incidence of peanut nematode disease.

[0086] Example 6

[0087] This example is a field test of the efficacy of the fungicide Paecilomyces lilacinus Pli-25 applied to cucumbers.

[0088] 6.1 Preparation of Paecilomyces lilacinus Pli-25 Powder

[0089] The fermentation broth prepared in 4.1 of Example 4 was inoculated into the solid fermentation substrate at an inoculum size of 15% by weight of the solid fermentation substrate, mixed with the solid fermentation substrate, and aerobically fermented in a koji pan at room temperature of 29°C, a relative humidity of 80%, protected from light, and a humidity of 75% to 90% for 5 days. The fermentation was completed. The solid fermentation substrate consisted, by weight, of 70% sargassum, 15% cornmeal, 5% cottonseed meal, 0.03% magnesium sulfate, 0.02% calcium phosphate, and the balance Artemisia annua.

[0090] After the fermentation is completed, the culture medium is dried and crushed at <50°C to make a 60-80 mesh powder, which is the Paecilomyces lilacinus Pli-25 agent powder, and stored in a dark, cool place.

[0091] 6.2 Cucumber field control efficacy test

[0092] A cucumber greenhouse with a high incidence of disease in previous years was selected as the experimental plot. During transplanting, the experimental group applied a powdered inoculant of Paecilomyces lilacinus Pli-25 to the roots of the cucumber seedlings using a hole-drying method. A control group, which did not receive the powdered inoculant, followed conventional practices. At harvest, the incidence of cucumber nematodes in the experimental and control groups was statistically analyzed. The incidence and control efficacy were calculated using the same formulas as in 4.2. The statistical results are shown in Table 3.

[0093] Table 3 Statistical results of the control effect of cucumber nematode disease under different treatments

[0094] Trial Groups Incidence Control effect control group 79.80% - experimental group 8.10% 89.85%

[0095] The results showed that compared with the control group, the experimental group treated with the powdered inoculant of Paecilomyces lilacinus Pli-25 was significantly more effective in preventing and controlling cucumber nematode disease. The application of the powdered inoculant of Paecilomyces lilacinus Pli-25 significantly reduced the incidence of cucumber nematode disease, providing a practical and operational technical means for the biological control of nematode disease in fruits and vegetables, and is of great significance in promoting the development of green agricultural prevention and control technologies.

[0096] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. A strain of Paecilomyces lilacinus, characterized in that The Paecilomyces lilacinum is named Purpureocillium lilacinumPli-25 and is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No.41932.

2. The Paecilomyces lilacinus according to claim 1, characterized in that The 16S rDNA sequence of Cossackia coli is shown in SEQ ID NO:

1.

3. A microbial agent, characterized in that: The invention comprises the Purpureocillium lilacinum Pli-25 strain according to claim 1 or 2 and an agriculturally acceptable carrier.

4. The microbial agent according to claim 3, characterized in that The bacterial agent is a liquid fermentation product or a solid fermentation product.

5. The microbial agent according to claim 4, characterized in that The method for preparing the liquid fermentation product comprises the following steps: Step 1, preparing a liquid fermentation medium, wherein the medium comprises algae, Artemisia annua, cornmeal, cottonseed meal and calcium phosphate; Step 2, inoculating the Paecilomyces lilacinus strain according to claim 1 or 2 into the liquid fermentation medium, and fermenting at 25-30° C. for 3-5 days; Step 3: Collect the fermentation broth and obtain the liquid fermentation product after centrifugation.

6. The microbial agent according to claim 4, characterized in that The method for preparing the solid-state fermentation product comprises the following steps: S1. preparing a solid fermentation substrate, wherein the substrate comprises algae, cornmeal, Artemisia annua, magnesium sulfate and calcium phosphate; S2. inoculating the fermentation liquid containing the Paecilomyces lilacinus strain according to claim 1 or 2 into the solid fermentation substrate, and fermenting at 25-30° C. for 4-5 days; S3. Collect the solid fermentation product, dry and crush it to obtain powder.

7. Use of the Purpureocillium lilacinum Pli-25 strain according to claim 1 or 2 or the microbial agent according to any one of claims 3 to 6 in preventing and controlling plant nematode diseases.

8. The use according to claim 7, characterized in that The plant nematode disease is root knot nematode disease or cyst nematode disease.

9. The use according to claim 7 or 8, characterized in that The plant is tomato, peanut or cucumber.

10. A method for preventing and controlling plant nematode diseases, characterized in that: The method comprises applying the Purpureocillium lilacinum Pli-25 strain according to claim 1 or 2 or the microbial agent according to any one of claims 3 to 6 to plants or a plant growth environment.

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