Cordyceps javanica strain yj-y1 and its application in preventing and treating mangrove leaf-eating pests

Abstract

The application discloses a strain of Cordyceps javanicus (L. Cordyceps javanica ) YJ-Y1 and application of the strain in prevention and treatment of mangrove leaf-eating pests. The Cordyceps javanicus YJ-Y1 is separated from the sea holly female borer pupae in mangroves in Yangjiang City of Guangdong Province, has a preservation number of GDMCC NO: 66724, has strong pathogenicity to various mangrove outburst lepidoptera pests (sea holly female borer, xylotrupes dichotomus and acigona actyx), has good salt tolerance and high temperature tolerance, can help growth of the strain under the condition of 0.5% to 2% NaCl or 30 DEG C, has fast mycelium growth and large spore yield, is a biological control strain with application value in prevention and treatment of mangrove leaf-eating pests, and can be used for pest prevention and treatment in high-temperature and high-salt environments.

Description

Technical Field

[0001] This invention belongs to the technical field of biological pest control, specifically relating to a strain of Cordyceps militaris YJ-Y1 and its application in controlling leaf-eating pests in mangroves. Background Technology

[0002] Mangroves, as unique woody biological communities in coastal tidal wetlands, are among the world's most biodiverse ecosystems. Guangdong Province accounts for approximately 40% of China's mangrove area, with *Rhizophora stylosa*, *Symplocos buergeriana*, and *Kandelia candel* covering relatively large areas. Due to their relatively simple community structure and harsh living environment, they are prone to large-scale pest and disease outbreaks. *Rhizophora stylosa* (also known as the sea olive borer) is a significant pest. Ptyomaxia syntaractis Teak camel moth ( Hyblaea puera ) and the tung tree hairy-mandibleed leafroller ( Lasiognatha cellifera The mangrove larvae are the primary and voracious leaf-eating pests of the mangrove species *Symplocos rubrum* and *Symplocos lucida*. Their larvae can devour the leaves, fruits, and tender branches of these trees in a short period, causing widespread leaf drop and necrosis. This severely impacts normal photosynthesis, interfering with the plant's growth and function. Furthermore, affected mangrove plants are susceptible to other minor pests and diseases, increasing the risk of death.

[0003] The fragility and unique characteristics of mangrove ecosystems make pest control far more challenging than in terrestrial forest systems. Mangroves are periodically submerged by seawater, and traditional chemical pesticides are rapidly diluted and washed away by the tides after application, resulting in significantly reduced efficacy and difficulty in effectively controlling target pests. Furthermore, the long-term and irrational use of chemical agents can easily cause environmental pollution and lead to pesticide resistance in pests. Biological control, a nature-based method, utilizes entomopathogenic fungi, which are widely distributed and diverse in nature, playing a crucial role in regulating insect populations within ecosystems. Biological control also offers greater safety and persistence, making it a promising alternative to chemical pesticides.

[0004] Patent application CN119530023A discloses a strain of Metarhizium anisopliae YEC isolated from soil in Guangdong Province. Metarhizium flavoviride YEC), which can infect the teak moth, a mangrove pest, and can cause high mortality and stunted growth of the teak moth in a short period of time. Patent application CN121369421A discloses the Javan Cordyceps fungus Bd01 (… Cordyceps javanica Bd01 has an infectious and lethal effect on fall armyworm larvae, significantly reducing larval pupation and emergence rates. Currently, there is a lack of entomopathogenic fungi suitable for the high-salt environment of mangroves, and no technology has been reported for isolating Cordyceps militaris from the mummified remains of mangrove pests to control them. Summary of the Invention

[0005] Based on existing defects and shortcomings, the present invention aims to provide a strain of Java Cordyceps fungus ( Cordyceps javanica YJ-Y1 (the preservation number of this fungus is GDMCC NO: 66724, the preservation date is July 22, 2025, the preservation unit is GDMCC-Guangdong Provincial Microbial Culture Collection Center) and its application in the control of leaf-eating pests in mangroves.

[0006] The first objective of this invention is to provide a strain of Cordyceps militaris (Java Cordyceps fungus) Cordyceps javanica YJ-Y1, with accession number GDMCC NO: 66724.

[0007] A second objective of this invention is to provide a biological agent containing live cells of *Cordyceps militaris* YJ-Y1 or a culture of live cells of *Cordyceps militaris* YJ-Y1 as an active ingredient.

[0008] Preferably, the culture of live Cordyceps militaris YJ-Y1 in the preparation is a spore suspension of Cordyceps militaris YJ-Y1.

[0009] Preferably, the concentration of the spore suspension of *Cordyceps militaris* YJ-Y1 is 1×10⁻⁶. 4 cfu / mL or higher.

[0010] Preferably, the concentration of the spore suspension of *Cordyceps militaris* YJ-Y1 is 1×10⁻⁶. 6 cfu / mL or higher.

[0011] A third objective of this invention is to provide the application of the aforementioned Cordyceps militaris YJ-Y1 or any of the formulations described herein in the control of leaf-eating pests in mangroves.

[0012] Preferably, the method includes the step of applying the *Cordyceps militaris* YJ-Y1 or any of the preparations described herein to the mangrove leaf-eating pests.

[0013] Preferably, the mangrove leaf-eating pests are the sea olive female twig borer, the teak camel moth, and / or the tung tree hairy-jawed leafroller.

[0014] Preferably, the mangrove leaf-eating pests are the third instar larvae of the sea olive female twig borer, the teak camel moth, and / or the tung tree hairy-jawed leafroller.

[0015] Preferably, the application is by spraying.

[0016] The beneficial effects of this invention are: The *Cordyceps militaris* YJ-Y1 strain provided by this invention exhibits strong pathogenicity against various outbreaks of lepidopteran pests in mangroves (*Hemiberlesia lataniae*, *Hemiberlesia lataniae*, and *Hemiberlesia lataniae*). It also demonstrates strong salt tolerance, with 0.5%–2% NaCl promoting its growth. Furthermore, it exhibits rapid mycelial growth and high sporulation at high temperatures (30°C), making it a biological control strain with excellent control effects against mangrove leaf-eating pests in high-salt, high-temperature environments, demonstrating significant application potential and value.

[0017] Preservation Instructions The present invention Cordyceps javanica YJ-Y1 (Cordyceps militaris YJ-Y1) was deposited on July 22, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC NO: 66724. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0018] Figure 1 The image shows the colony morphology of Cordyceps militaris YJ-Y1 on PDA medium on day 10; where A is the front view of the colony of Cordyceps militaris YJ-Y1 and B is the back view of the colony of Cordyceps militaris YJ-Y1.

[0019] Figure 2 The images show the mycelium, conidiophores, conidia, and morphology of the mummified insect of Cordyceps militaris YJ-Y1. A represents the mycelium of Cordyceps militaris YJ-Y1, B represents the conidiophore of Cordyceps militaris YJ-Y1, C represents the conidia of Cordyceps militaris YJ-Y1, and D shows the grayish-white mycelium and pale purple sporosphere that grew after the conidia of Cordyceps militaris YJ-Y1 were inoculated back into the larvae (left) and pupae (right) of the sea moth (Cordyceps militaris) and died.

[0020] Figure 3 This is a phylogenetic tree constructed based on ITS site sequences for Cordyceps militaris YJ-Y1. Detailed Implementation

[0021] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0022] Example 1: Isolation and Identification of Strains YJ-Y1 1. Strain Isolation In June 2024, *Bombyx mori*, a female *Bombyx mori* larvae, were collected from mangrove forests along the coast of Yangjiang City, Guangdong Province. A small number of conidia were picked from the larvae in a clean bench and inoculated onto PDA medium (200 g fresh potato, 15 g agar, 20 g glucose, and water to 1000 mL) using the spot inoculation method. After 5-7 days, mycelia from the colony edges were picked and isolated onto fresh PDA medium. The above operations were repeated to obtain a pure strain YJ-Y1. This strain was deposited on July 22, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number GDMCC NO: 66724, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology.

[0023] 2. Morphological identification The isolated and purified strain YJ-Y1 was inoculated onto PDA medium plates (6 cm in diameter) and cultured at a constant temperature of 25°C. The colony morphology was observed and recorded daily. After 7 days, mycelia were picked and the morphology of mycelia and conidiophores of the strain was observed under an optical microscope. Mature conidia were picked and their morphology and size were measured.

[0024] like Figure 1 As shown, on PDA medium, strain YJ-Y1 initially appeared as grayish-white fluffy colonies. After 7-10 days of continued culture, the colonies became round, 20-30 mm in diameter, with a pale purple spore layer at the center. Figure 1 (A) The reverse side of the colony is pale yellow ( Figure 1 (B in the middle).

[0025] The hyphae of strain YJ-Y1 are slender, smooth, and transparent. Figure 2 (A in the text); the phialpinioidea is attached to the conidiophore, its base is elliptical and swollen, gradually tapering upwards ( Figure 2 (B in the text); conidia are transparent, elongated and elliptical, measuring (4.06±0.07) μm × (2.35±0.03) μm (N=55) Figure 2 (C in the text); Conidia were re-inoculated onto the larvae and pupae of the sea olive female twig borer. After the larvae and pupae died, white mycelium grew, which later covered a pale purple spore layer (C in the text). Figure 2 (D in the middle).

[0026] 3. Molecular identification Using the genomic DNA of strain YJ-Y1 as a template, the rDNA-ITS sequence of strain YJ-Y1 was amplified by PCR using universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO.1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO.2). The PCR reaction mixture (50 μL) consisted of 25 μL 2×Es TaqMaster Mix (Dye), 1 μL DNA template, 0.2 μL each of 10 μmol / L forward and reverse primers, and ddH2O to a final volume of 50 μL. The amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; 72℃ extension for 5 min. The PCR products were detected by 1.0% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing results were compared with the GenBank gene database, and similar sequences were selected and pruned at both ends for alignment. A phylogenetic tree was constructed using the neighbor-joining method, and repeatability was tested 1000 times using the bootstrap method.

[0027] Based on the sequencing results of the ITS sequence, the nucleotide sequence of the ITS of strain YJ-Y1 was obtained as shown in SEQ ID NO.3. After BLAST comparison with the NCBI database, it was found that strain YJ-Y1 was consistent with the ITS sequence in the NCBI database. Cordyceps javanica The ITS nucleotide sequence of strain SC53B05 (NCBI accession number: MW113581) had the highest alignment score, with a coverage rate of 99% and a similarity of 99.83%.

[0028] Phylogenetic analysis ( Figure 3 The results showed that strain YJ-Y1 was related to Beauveria bassiana ( ). Beauveria bassiana ) and Metarhizium anisopliae ( Metarhizium anisopliae The strain was clearly isolated from the genus Cordyceps ( ). Cordyceps The strains are closer together and are associated with Cordyceps militaris ( ). Cordyceps javanica The clustering of these cells indicates that strain YJ-Y1 has a high similarity to *Cordyceps militaris* in the database. Therefore, based on morphological and molecular identification, strain YJ-Y1 was identified as *Cordyceps militaris* and named *Cordyceps militaris* YJ-Y1 (…). Cordyceps javanica YJ-Y1).

[0029] Example 2: Pathogenicity test of Cordyceps militaris YJ-Y1 against mangrove leaf-eating pests The pathogenicity of *Cordyceps militaris* YJ-Y1 against the larvae of *Gnaphalium affine*, *Pterocarya stenoptera*, and *Sclerotium truncatella* was determined using a spray method. First, conidia of *Cordyceps militaris* YJ-Y1 were prepared into 1×10⁻⁶ concentrations using a 0.5% (v / v) Tween-80 aqueous solution. 8 1×10 7 1×10 6 1×10 5 and 1×10 4 A spore suspension of CFU / mL was used to evenly spray 1 mL of the spore suspension onto every 10 larvae of the tested 3rd instar larvae, which were then transferred to disposable plastic containers for rearing (rearing conditions: temperature 25±1℃, relative humidity 70±5%, photoperiod 14 L:10 D). 3rd instar larvae treated with a 0.5% (v / v) Tween-80 aqueous solution served as a blank control. Each treatment was repeated three times, with 15–20 larvae per replicate. Fresh leaves from female *Rhizoctonia solani* were fed daily to the larvae of *Rhizoctonia solani* and *Rhizoctonia solani*, while fresh leaves from *Paulownia tomentosa* were fed to the larvae of *Paulownia tomentosa*. Dead larvae were removed and transferred to new plastic containers for humidified culture, and mycelial growth was observed. The number of larval deaths was counted daily, and the mortality rate, corrected mortality rate, and stunted larvae rate were calculated using the following formulas: Mortality rate (%) = (Number of deaths in treatment / Control mortality rate) × 100%; Corrected mortality rate (%) = (treatment mortality rate) (Control mortality rate) / (100) (Control mortality rate) × 100%; Stunted worm rate = number of stunted worms / number of dead worms treated × 100%.

[0030] Table 1. Pathogenicity of Cordyceps militaris YJ-Y1 against the third instar larvae of *Cordyceps militaris*, *Cordyceps javanica*, and *Cordyceps tuldosum*. As shown in Table 1, the spore suspension of *Cordyceps militaris* YJ-Y1 exhibited strong pathogenicity against three mangrove leaf-eating pests, and the mortality rate was positively correlated with spore concentration and treatment time. At lower spore concentrations (1×10⁻⁶), the mortality rate was significantly higher than that of other mangrove pests. 5 Under treatment with cfu / mL, the cumulative corrected mortality rates of 3rd instar larvae of *Gnaphalium affineum*, *Gnaphalium affineum*, and *Gnaphalium affineum* were 55.56%, 55.00%, and 59.52% after 7 days, respectively; spore concentrations of 1×10⁻⁶ CFU / mL were also observed. 6 At cfu / mL, the cumulative corrected mortality rate of the three pests all reached over 80%; at a spore concentration of 1×10⁻⁶, the cumulative corrected mortality rate of the three pests all reached over 80%; 7 At cfu / mL, the cumulative corrected mortality rate of 3rd instar larvae of the sea olive female twig borer and the tung tree hairy-jawed leafroller was 100% after 7 days, while that of the teak camel moth was 97.50%.

[0031] Cordyceps militaris YJ-Y1 can also effectively cause insect stiffening, forming mummified worms. At lower spore concentrations (1×10⁻⁶), this effect is also observed. 4 Even under treatment with cfu / mL, the rate of stunted insect death remained above 80%, while the spore concentration was 1×10⁻⁶. 6 At cfu / mL, the rate of stunted death of worms reached 100%.

[0032] Table 2. Median lethal concentration of Cordyceps militaris YJ-Y1 against 7-day-old larvae of the following species: *Cordyceps militaris*, *Cordyceps javanica*, and *Cordyceps tungus*. Table 2 shows that the three mangrove leaf-eating pests exhibit different susceptibility to Cordyceps militaris YJ-Y1. After 7 days of treatment, the LC50 of the *Symplocos cuspidata* moth was... 50 The smallest value is 3.47 × 10. 4 cfu / mL; followed by *Spodoptera litura*, LC... 50 The value is 4.04 × 10 4 cfu / mL; while the LC50 of the teak moth 50 The value is 5.22 × 10 4 cfu / mL.

[0033] Table 3. Cordyceps militaris YJ-Y1 (1×10⁻⁶) 8 The median time to death (cfu / mL) of the third instar larvae of the sea olive female twig borer, the teak camel moth, and the tung tree hairy roller moth. Table 3 shows the median time to death (LT) of Cordyceps militaris YJ-Y1 against three mangrove leaf-eating pests. 50 The LT (long-term susceptibility) of all three mangrove leaf-eating pests decreased significantly with increasing spore concentration, and their susceptibility differed. The LT of *Triplophysa hainanensis* was significantly shortened. 50 The minimum value is 1×10. 8 At a concentration of CFU / mL, the d was 2.88; the second highest was the small leafroller moth of the tung tree, LT. 50 The value was 2.95 d; while the LT of the teak moth was... 50 The value is 3.13 days. In the linear regression equation, the correlation coefficient (R²) is... 2 The values ​​were all greater than 0.95, indicating a strong positive correlation between the cumulative number of larval deaths and the number of days of infection.

[0034] Example 3: Salt tolerance test of Cordyceps militaris YJ-Y1 First, SMAY medium plates (40 g maltose, 10 g yeast extract, 10 g peptone, 15 g agar, and water to a final volume of 1000 mL) with NaCl concentrations of 0.5%, 1%, 2%, and 3% (w / w) were prepared, with the untreated plate serving as a control. A 0.5 mm diameter mycelial disc of *Cordyceps militaris* YJ-Y1 was inoculated into the center of each medium plate at different salt concentrations. After incubation at 25°C for 15 days, the diameter of the mycelial rings under different salt concentrations was measured. The spores were washed with a 0.5% (v / v) Tween-80 aqueous solution, diluted, and the number of conidia was counted using a hemocytometer. The sporulation yield of the strain under different salt concentrations was calculated. The experiment was repeated three times.

[0035] Table 4. Effects of different salt concentrations on the growth and sporulation of Cordyceps militaris YJ-Y1. Note: Different letters in the same column indicate significant differences in the average diameter of the mycelial rings or the average sporulation rate under different salt concentrations (P<0.05).

[0036] Table 4 shows that adding a certain concentration of NaCl promotes mycelial growth and increases sporulation of *Cordyceps militaris* YJ-Y1. By day 15 of cultivation, compared with the control, treatment with 0.5%–2% NaCl significantly promoted mycelial growth and significantly increased sporulation of *Cordyceps militaris* YJ-Y1, while the results of treatment with 3% NaCl were similar to the control. These results indicate that *Cordyceps militaris* YJ-Y1 has good salt tolerance, promoting mycelial growth and sporulation at certain salt concentrations, making it suitable for use in areas with high salinity.

[0037] Example 4: Determination of the thermocompatibility of Cordyceps militaris YJ-Y1 A 0.5 mm diameter mycelial disc of *Cordyceps militaris* YJ-Y1 was inoculated into the center of a SMAY agar plate and incubated at 15, 20, 25, and 30 °C. After 15 days, the size of the mycelial rings under different temperature treatments was measured, and the spores were washed with a 0.5% (v / v) Tween-80 aqueous solution. After dilution, the number of conidia was counted using a hemocytometer, and the sporulation yield under different temperature treatments was calculated. The experiment was repeated three times.

[0038] Table 5. Effects of different culture temperatures on the growth and sporulation of Cordyceps militaris YJ-Y1. Note: Different letters in the same column indicate significant differences in the average diameter of the mycelial ring or the average sporulation at different temperatures (P<0.05).

[0039] Table 5 shows that the mycelial growth and sporulation of *Cordyceps militaris* YJ-Y1 vary significantly under different temperature conditions. At 15℃, mycelial growth is slow, but increases rapidly at 20℃, and peaks between 25-30℃, significantly higher than at 15℃ and 20℃. Regarding sporulation, it is low between 15-20℃, increases rapidly at 25℃, and peaks at 30℃, significantly higher than at other temperatures. Considering both mycelial growth rate and sporulation, *Cordyceps militaris* YJ-Y1 grows best at 25-30℃, with high sporulation at 30℃, making it suitable for applications in areas with higher temperatures.

[0040] In summary, *Cordyceps militaris* YJ-Y1, an entomopathogenic fungus isolated from the mummified pupae of *Cordyceps militaris* in mangroves, exhibits highly effective pathogenicity against outbreaks of mangrove leaf-eating pests such as *Cordyceps militaris*, *Camelus chinensis*, and *Sclerotium tungatum*, effectively infecting and killing them with a high rate of mummification. Furthermore, *Cordyceps militaris* YJ-Y1 demonstrates strong salt tolerance and rapid mycelial growth and high sporulation at high temperatures (30℃), making it highly valuable for the biological control of mangrove leaf-eating pests.

Claims

1. A strain of Javanese Cordyceps militaris Cordyceps javanica YJ-Y1, characterized in that, Its accession number is GDMCCNO:66724.

2. A biological agent, characterized in that, The active ingredient is a live bacterial cell of *Cordyceps militaris* YJ-Y1 as described in claim 1 or a culture of live bacterial cells of *Cordyceps militaris* YJ-Y1 as described in claim 1.

3. The formulation according to claim 2, characterized in that, The culture of live Cordyceps militaris YJ-Y1 in the preparation is a spore suspension of Cordyceps militaris YJ-Y1.

4. The formulation according to claim 3, characterized in that, The concentration of the spore suspension of Cordyceps militaris YJ-Y1 is 1×10⁻⁶. 4 cfu / mL or higher.

5. The formulation according to claim 4, characterized in that, The concentration of the spore suspension of Cordyceps militaris YJ-Y1 is 1×10⁻⁶. 6 cfu / mL or higher.

6. The application of the Java Cordyceps militaris YJ-Y1 as described in claim 1 or the preparation as described in any one of claims 2 to 5 in the control of leaf-eating pests in mangroves.

7. The application according to claim 6, characterized in that, The method includes the step of applying the Cordyceps militaris YJ-Y1 of claim 1 or the preparation of any one of claims 2 to 5 to the body of mangrove leaf-eating pests.

8. The application according to claim 6, characterized in that, The mangrove leaf-eating pests mentioned are the sea olive female tumor moth, the teak camel moth, and / or the tung tree hairy-jawed leafroller.

9. The application according to claim 8, characterized in that, The mangrove leaf-eating pests mentioned are the third instar larvae of the sea olive female tussock moth, the teak camel moth, and / or the tung tree hairy-jawed leafroller.

10. The application according to claim 7, characterized in that, The application is described as a spray.

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