A biocontrol fungus, Cordyceps militaris CJ1, and its application
By using the biocontrol strain Cordyceps militaris CJ1 to prepare spore suspensions for spraying to treat termites, the environmental and health hazards of chemical control have been solved, achieving efficient and green control of termites.
Patent Information
- Application Number
- CN202610499081.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-15
- Publication Date
- 2026-05-26
AI Technical Summary
Existing chemical methods for termite control are harmful to the environment and human health, and there is a lack of effective eco-friendly microbial insecticides.
We provide a biocontrol fungus, Cordyceps militaris CJ1, and through cultivation, prepare a spore suspension and use it to treat termites, achieving highly effective lethality against black-winged subterranean termites and Formosan subterranean termites.
Java Cordyceps CJ1 exhibits excellent insecticidal activity against termites, achieving green control without chemical residues and reducing environmental and health risks.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biological control technology, specifically to a biocontrol fungus, Cordyceps militaris CJ1, and its applications. Background Technology
[0002] Termites are ancient social insects with highly complex social structures. Colonies typically consist of individuals of different castes, including queens, kings, soldiers, and workers. Workers comprise over 80% of the colony and are the primary cause of termite infestations (Xu et al., 2016; Ji et al., 2021). Termites are global social insects, and their covert and gradual destructive characteristics lead to enormous economic losses. According to the Food and Agriculture Organization of the United Nations (FAO), direct economic losses due to termite damage worldwide reach as high as US$40 billion annually. More than 3,000 termite species are known globally, with over 470 species recorded in my country. Among them, more than 10 species, such as the black-winged subterranean termite *Odontotermes formosanus* (Shiraki) and the Formosan subterranean termite *Coptotermes formosanus* (Shiraki), pose significant threats to human activities (Huang et al., 2004; Yang et al., 2015). Black-winged subterranean termites are among the most damaging termite species in my country, affecting over 100 plant species with a broad and non-selective diet (Zhong et al., 1997; Xiang Jianping, 2012). In forestry, they bore into roots, causing trees to collapse; in construction, they erode wooden structures, building nests inside earthen embankments, disrupting the original integrity of buildings and leading to embankment collapse (Xu Zhide et al., 2007). Formosan subterranean termites are the most damaging termite species to buildings, with large colonies, rapid reproduction, and high destructiveness; besides buildings, they also cause significant damage to garden trees (Li Guangsheng et al., 2025).
[0003] Currently, termite control measures include physical, chemical, and biological methods, with chemical control being the most widely used. Chemical control offers advantages such as ease of operation, significant effectiveness, and rapid action; however, while effective, chemical pesticides can also harm the environment and human health, and damage ecosystems. In contrast, microbial insecticides are highly efficient and environmentally friendly. Currently, commonly used insect pathogens for pest control include Bacillus thuringiensis, Beauveria bassiana, and Metarhizium anisopliae, but the variety is limited, and there are no reports on the use of Cordyceps militaris for termite control, either domestically or internationally. Therefore, providing a biocontrol strain that is both highly toxic to termites and environmentally friendly is of great significance for achieving green termite control. Summary of the Invention
[0004] The purpose of this invention is to provide a biocontrol fungus, *Cordyceps militaris* CJ1, and its application in termite control. *Cordyceps militaris* CJ1 was first discovered by the applicant in *Subterranean subterranean termite*, and biological testing results show that *Cordyceps militaris* CJ1 has a high mortality rate against both *Odontotermes acutissima* and *Subterranean subterranean termite*.
[0005] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:
[0006] A biocontrol fungus, Cordyceps javanica CJ1, isolated from the Formosan subterranean termite, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is January 26, 2026, and the accession number is CGMCC No. 42564.
[0007] The morphological characteristics of Cordyceps militaris CJ1 are as follows: After 7 days of culture in PPDA medium at 25±1 ℃, the colonies are round on the front with neat edges, dark gray to grayish-brown in the center, gradually turning white, with a velvety or cottony surface and concentric rings; the back is light yellow with radial grooves on the edge; the conidiophores have flask-shaped phialides, solitary or whorled on the hyphae, broad at the base and narrow at the apex, with conidia falling off from the apex; the conidia are elliptical or nearly round, about 3.1~5.8μm×1.7~2.5μm in size.
[0008] The present invention also provides the application of Cordyceps militaris in the control of termites, wherein the Cordyceps militaris is any of the Cordyceps militaris CJ1 described above.
[0009] The present invention also provides a method for applying the above-described Cordyceps militaris in the control of termites, wherein the method involves culturing the described Cordyceps militaris CJ1 strain and preparing a spore suspension for treating termites.
[0010] Furthermore, the concentration of the Cordyceps militaris CJ1 strain spore suspension is 1×10⁻⁶. 7 ~1×10 9 spores / mL.
[0011] Furthermore, the Javan Cordyceps CJ1 was first cultured in potato dextrose peptone (PPDA) medium for 7 days, and then mycelia and spores were picked and inoculated into saccharomyces salsa var. salsa (SDY) medium and cultured with shaking for 4 days.
[0012] Furthermore, the termites mentioned for termite control are black-winged subterranean termites and Formosan subterranean termites.
[0013] Furthermore, the aforementioned prevention and control method is the spraying method.
[0014] The beneficial effects of this invention are:
[0015] The Cordyceps javanica CJ1 strain of this invention was isolated and purified from dead Formosan subterranean termites. Tests on the effects of Cordyceps javanica CJ1 on both *Odontotermes acutissima* and *Formosan subterranean termites* revealed that it exhibits good insecticidal activity against both species. This strain's mechanism of action is natural parasitic lethality, leaving no chemical residue. It can be developed into a microbial insecticide, achieving reduced pesticide use with increased efficacy and ecological safety, thus promoting green termite control. Attached Figure Description
[0016] Figure 1 Image showing a Formosan subterranean termite that died from fungal infection, covered by fungal hyphae.
[0017] Figure 2 Morphological characteristics and culture traits of Cordyceps militaris CJ1;
[0018] Note: AB: Front and back views of Cordyceps militaris CJ1 colonies on PPDA medium; CD: Sporulation structure and conidia of Cordyceps militaris CJ1;
[0019] Figure 3 Electrophoresis diagram of PCR amplification products in the molecular identification of Cordyceps militaris CJ1
[0020] Figure 4 This is a schematic diagram of a phylogenetic tree;
[0021] Figure 5 Survival rate of black-winged subterranean termites after treatment with different concentrations of Cordyceps militaris CJ1 spore suspension;
[0022] Figure 6 Survival rate of Formosan subterranean termites after treatment with different concentrations of Cordyceps militaris CJ1 spore suspension. Detailed Implementation
[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.
[0024] Experimental materials and methods involved in the embodiments:
[0025] (1) Test insects: Black-winged subterranean termites were collected from Nanjing, Jiangsu Province. After the termite nests were collected, they were kept in an insect incubator at 25±1℃ and 90±5% relative humidity in the dark. Formosan subterranean termites were collected from Guangzhou, Guangdong Province. After the nests were collected, they were kept in an insect incubator at 25±1℃ and 90±5% relative humidity in the dark.
[0026] (2) Culture medium composition: PPDA solid culture medium consists of 200 g potato extract, 10 g peptone, 20 g glucose, 15 g agarose and 1 L distilled water; SDY liquid culture medium consists of 10 g tryptone, 40 g glucose, 10 g yeast extract and 1 L distilled water.
[0027] (3) The method for determining the toxicity of Java Cordyceps CJ1 spore suspension to black-winged subterranean termites and Formosan subterranean termites was based on Part 9 "Spraying Method" of the "Guidelines for Indoor Biological Testing of Pesticides" and modified accordingly.
[0028] The present invention will be further described in detail below with reference to specific embodiments. The embodiments will help to understand the present invention, but the scope of protection of the present invention is not limited to the following embodiments.
[0029] Example 1: Isolation and Identification of Cordyceps Javanae CJ1
[0030] Isolation of the strain: The strain was derived from Formosan subterranean termites whose bodies were covered with mycelium after death during normal rearing. Figure 1 In a clean bench, the Formosan subterranean termite bodies coated with mycelium were immersed in 75% ethanol for 3 seconds, followed by rinsing the bodies three times with sterilized ultrapure water. The rinsed Formosan subterranean termite bodies were then placed on PPDA medium. After mycelium and conidia grew, single colonies were picked and transferred to fresh PPDA medium for further isolation and purification until purified colonies were obtained.
[0031] Morphological identification of the strain: After inoculating *Cordyceps militaris* strain CJ1 on PPDA medium and culturing at 25±1 ℃ for 7 days, the colonies were generally circular with neat edges and a distinct color gradient. The central area was dark gray to grayish-brown, gradually transitioning to white towards the edge; the surface texture was fluffy or cottony, with clearly visible concentric rings; the reverse side of the colony was light yellow, with radial grooves at the edge. The conidiophores of *Cordyceps militaris* CJ1 have flask-shaped phialides, solitary or whorled on the hyphae. The phialides are wider at the base and abruptly narrow at the top, with conidia falling off from the top; the conidiophores are elliptical or nearly circular, approximately 3.1–5.8 μm × 1.7–2.5 μm in size. Figure 2 ).
[0032] Molecular identification of the strain: CJ1 hyphae that had grown well on PPDA medium were picked up using an inoculation needle in a clean bench, placed in a mortar, flash-frozen in liquid nitrogen, and then ground into powder. Total fungal DNA was extracted using the SteadyPure Universal Genomic DNA Extraction Kit (Hunan Aike Rui Biotechnology Co., Ltd.). The primers for PCR amplification of fungal DNA are shown below:
[0033] ITS1 F:TCCGTAGGTGAACCTGCGG
[0034] ITS4 R:TCCTCCGCTTATTGATAGC
[0035] The PCR amplification system is shown in Table 1. The PCR amplification reaction conditions are: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 54 ℃ annealing for 30 s, 72 ℃ extension for 30 s, for a total of 35 cycles; 72 ℃ final extension for 5 min.
[0036] Table 1
[0037]
[0038] PCR products were detected by agarose gel electrophoresis. Products meeting the expected amplification length were sent to Qingke Biotechnology Co., Ltd. for sequencing (Figure 3). After sequencing, the obtained sequences were BLAST-aligned in the GenBank database to obtain sequences highly homologous to the target sequence. A phylogenetic tree was constructed using MEGA-X software via Neighbor-Joining (NJ). The phylogenetic tree constructed based on the ITS sequence of Cordyceps militaris CJ1 is shown below. Figure 4 As shown.
[0039] Example 2: Toxicity determination of Cordyceps militaris CJ1 spore suspension against black-winged subterranean termites.
[0040] (1) Cultivation of Cordyceps militaris CJ1:
[0041] First, *Cordyceps militaris* CJ1 was inoculated onto PPDA plates and cultured at 25±1 ℃ for 7 days. Then, in a clean bench, 2 mL of sterile ddH2O was added to the cultured PPDA plates, and mycelia and spores were scraped from the PPDA plates into the ddH2O using a spreader. Finally, the ddH2O containing mycelia and spores was transferred to SDY medium using a pipette and cultured at 25±1 ℃ with shaking at 180 rpm / min for 4 days.
[0042] (2) Preparation of spore suspension:
[0043] The culture was centrifuged at 7500 rpm for 5 min, and the supernatant was discarded. The precipitate was resuspended in 0.01% Tween-80 sterile water and stirred with a magnetic stirrer for 30 min to ensure that the conidia were fully detached from the hyphae. The hyphae were then filtered through four layers of 50-mesh gauze, and the filtrate became the spore suspension stock solution. Finally, the spore concentration in the stock solution was measured using a hemocytometer, and the stock solution was diluted with 0.01% Tween-80 sterile water to a concentration of 1×10⁻⁶. 9 1×10 8 1×10 7 1×10 6 and 1×10 5 A spore suspension of spores / mL.
[0044] (3) Toxicity test of Java Cordyceps CJ1 against black-winged subterranean termites:
[0045] The lethality of *Cordyceps militaris* CJ1 spore suspension against *Odontotermes acutissima* was tested using the Potter spray method. The specific method is as follows: A culture dish (75 mm in diameter) containing 30 viable *Odontotermes acutissima* worker ants was placed on the platform of a Potter spray tower. The operating pressure of the spray tower was set to 0.6 bar, the spray volume was 2 mL, and the droplet deposition was approximately 5 mg / cm³. 2 The treatment group used different concentrations of Cordyceps militaris CJ1 spore suspensions, while the control group used 0.01% Tween-80 sterilized water instead of the spore suspension. The treated black-winged subterranean termites were then placed in a dark environment at 25±1 ℃ and 90±5% relative humidity, and the number of dead termites was recorded every 12 hours.
[0046] Toxicity assays showed that Cordyceps militaris CJ1 exhibited good toxicity against black-winged subterranean termites. At 96 h, the LC50 of Cordyceps militaris CJ1 against black-winged subterranean termites was [not specified]. 20 It is 1.0456×10 6 spores / mL, LC 50 1.4909×10 7 spores / mL (Table 2). At 96 h, after 1×10 8 and 1×10 9 The mortality rates of black-winged subterranean termites after treatment with a suspension of Cordyceps militaris CJ1 spores per spore (Figure 5) were 75.33% and 99.33%, respectively.
[0047] Table 2
[0048]
[0049] Example 3: Toxicity determination of Java Cordyceps CJ1 spore suspension against Formosan subterranean termites
[0050] (1) Cultivation of Cordyceps militaris CJ1:
[0051] First, *Cordyceps militaris* CJ1 was inoculated onto PPDA plates and cultured at 25±1 ℃ for 7 days. Then, in a clean bench, 2 mL of sterile ddH2O was added to the cultured PPDA plates, and mycelia and spores were scraped from the PPDA plates into the ddH2O using a spreader. Finally, the ddH2O containing mycelia and spores was transferred to SDY medium using a pipette and cultured at 25±1 ℃ with shaking at 180 rpm / min for 4 days.
[0052] (2) Preparation of spore suspension:
[0053] The culture was centrifuged at 7500 rpm for 5 min, and the supernatant was discarded. The precipitate was resuspended in 0.01% Tween-80 sterile water and stirred with a magnetic stirrer for 30 min to ensure that the conidia were fully detached from the hyphae. The hyphae were then filtered through four layers of 50-mesh gauze, and the filtrate became the spore suspension stock solution. Finally, the spore concentration in the stock solution was measured using a hemocytometer, and the stock solution was diluted with 0.01% Tween-80 sterile water to a concentration of 1×10⁻⁶. 9 2×10 8 4×10 7 8×10 6 and 1.6×10 6 A spore suspension of spores / mL.
[0054] (3) Toxicity test of Java Cordyceps CJ1 against Formosan subterranean termites:
[0055] The lethality of *Cordyceps militaris* CJ1 spore suspension against *Termitomyces formosanus* was tested using the Potter spray method. The specific method is as follows: A culture dish (75 mm in diameter) containing 20 viable *Termitomyces formosanus* worker ants was placed on the platform of a Potter spray tower. The operating pressure of the spray tower was set to 0.6 bar, the spray volume was 2 mL, and the droplet deposition was approximately 5 mg / cm³. 2 The treatment group used different concentrations of Cordyceps militaris CJ1 spore suspensions, while the control group used 0.01% Tween-80 sterilized water instead of the spore suspension. The treated Formosan subterranean termites were then placed in a dark environment at 25±1 ℃ and 90±5% relative humidity, and the number of dead Formosan subterranean termites was recorded every 1 day.
[0056] Bioassay results showed that Cordyceps militaris CJ1 exhibits high toxicity to Formosan subterranean termites. At 5 days, the LC50 of Cordyceps militaris CJ1 against Formosan subterranean termites was... 20 1.5024×10 7 spores / mL, LC 50 It is 5.3559×10 7 spores / mL (Table 3). 1×10 9 2×10 8 4×10 7 and 8×10 6 The lethality of *Cordyceps militaris* CJ1 spore suspension at spores / mL against *Termitomyces formosanus* reached 100.00% at 5, 7, 8, and 9 days, and was 1.6 × 10⁻⁶. 6 A suspension of Cordyceps militaris CJ1 spores at spores / mL could also cause 97.00% mortality in Formosan subterranean termites after 9 days (Figure 6).
[0057] Table 3
[0058]
[0059] The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. All components not explicitly stated in this embodiment can be implemented using existing technology.
Claims
1. A biocontrol strain of Cordyceps javanica (CJ1), characterized in that, Cordyceps javanica ) CJ1, characterized in that, It is preserved in China General Microbiological Culture Collection Center, the preservation address is No. 1 Xibei Road, Beichen, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, the preservation date is January 26, 2026, and the preservation number is CGMCC No. 42564.
2. The Java ginseng CJ1 of claim 1, wherein, The morphological characteristics of the Cordyceps javanica CJ1 are as follows: after being cultured in a PPDA medium at 25±1 DEG C for 7 days, the colony is round in front view, with neat edges, a dark gray-brown to gray-brown center gradually changing to white, a surface of villus or cotton-like, and a concentric ring belt; the back is light yellow, with radial grooves on the edge; the sporiferous structure has a flask-shaped phialide, which is single or whorled in mycelium, with a wide base and a narrow top, and the conidia are shed from the top; the conidia are oval or nearly round, with a size of about 3.1-5.8 mu m x 1.7-2.5 mu m.
3. Use of J. javanica in the control of termites, characterized in that, The Cordyceps javanica is the Cordyceps javanica CJ1 of claim 1 or 2.
4. The method of using the Java fungus of claim 3 for controlling termites, characterized in that, After the Cordyceps javanica CJ1 strain is cultured, a spore suspension is prepared to treat termites.
5. The method of use as claimed in claim 4, wherein, The culture is that the Cordyceps javanica CJ1 is inoculated on a solid culture medium for 7 days, and then mycelium and spores are picked and inoculated in a liquid culture medium for further culture for 4 days.
6. The method of use as claimed in claim 5, wherein, The solid culture medium is a potato glucose proteose peptone medium, and the liquid culture medium is a Sabouraud glucose yeast extract medium.
7. The use according to claim 3, wherein the compound is ###00003### 3 The termites are Odontotermes formosanus or Coptotermes formosanus.
8. Use of the Java fungus in the preparation of a medicine for controlling termites, characterized in that, The Cordyceps javanica is the Cordyceps javanica CJ1 of claim 1 or 2.
9. A medicine for controlling termites, characterized by comprising the compound according to claim 1. The Cordyceps javanica comprises the Cordyceps javanica CJ1 of claim 1 or 2.
10. The use according to claim 4, wherein The concentration of the spore suspension of the strain of Cordyceps javanica CJ1 was 1 x 10 7 ~ 1 x 10 9 spores / mL.