Beauveria bassiana YJ-YC1 and application thereof in prevention and treatment of lepidoptera pests

Abstract

The invention discloses a strain of Beauveria bassiana YJ-YC1 and an application of the Beauveria bassiana YJ-YC1 in prevention and treatment of lepidoptera pests, and particularly discloses a strain of Beauveria bassiana YJ-YC1. The beauveria bassiana YJ-YC1 provided by the invention is obtained by separating from the female ozostoma canarium bombyx batryticatus collected from a mangrove forest in Guangdong province, the preservation number of the beauveria bassiana YJ-YC1 is GDMCC NO: 66725, the beauveria bassiana YJ-YC1 has very strong pathogenicity to larvae and pupae of a plurality of outbreak lepidoptera pests (female ozostoma canarium, teak camel moth and heortia vitessoides), and the beauveria bassiana YJ-YC1 has the advantages of high bombyx batryticatus rate, no toxic or side effect and no toxic or side effect. The strain is a biological control bacterium with potential value in the control of lepidoptera pests, and has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial pest control technology, specifically involving a strain of Beauveria bassiana YJ-YC1 and its application in controlling lepidopteran pests. Background Technology

[0002] Lepidoptera insects are the most diverse insect group currently known, accounting for nearly 70% of important agricultural and forestry pests. Mangrove forests, as woody biological communities in tidal wetlands, are one of the world's most biodiverse ecosystems, playing vital ecological roles such as resisting wind and waves, protecting coastlines, degrading pollution, and regulating climate. However, the fragility and unique characteristics of mangrove ecosystems present significant challenges to pest control. The sea olive borer (*Hemiberlesia lataniae*) is one such example. Ptyomaxia syntaractis ) and teak camel moth ( Hyblaea puera The mangrove leafminer (Spodoptera exigua) is a major lepidopteran pest of mangroves. Its larvae can devour the leaves, fruits, and tender branches of female mangroves in a short period, causing widespread leaf drop and necrosis, severely impacting normal photosynthesis and interfering with the plant's growth and function. In addition, the yellow leafminer (Spodoptera exigua) is another major pest. Heortia vitessoides ) is an economic tree species, agarwood ( Aquilaria sinensis This is an important lepidopteran pest. The larvae feed on the leaves of agarwood, causing the leaves to become incomplete. In a large outbreak, they can eat all the leaves of agarwood in the affected area, causing the agarwood tree to weaken or even die.

[0003] Currently, the main methods for controlling lepidopteran pests include chemical control, physical control, and biological control. Chemical control has advantages such as rapid effectiveness and high efficacy, but long-term improper use can easily lead to the "3Rs" problem (resistance, resurgence, and pesticide residues) in pests, harm beneficial forest organisms, and cause environmental pollution. Biological control is a method that uses organisms or their metabolites to control pests, offering higher safety and longer-lasting effects. Entomopathogenic fungi originate from nature, exhibit high selectivity towards pests, and are therefore safer for the environment.

[0004] Currently, Beauveria bassiana ( ) is being used Beauveria bassiana No reports have been found on techniques for controlling various lepidopteran pests in mangroves, such as the sea olive female tussock moth, the teak camel moth, and the yellow field moth. Summary of the Invention

[0005] Based on the shortcomings and defects of existing technologies, this invention aims to provide a strain of Beauveria bassiana (Beauveria bassiana). Beauveria bassiana YJ-YC1 (the preservation number of this fungus is GDMCC NO: 66725, 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 lepidopteran pests.

[0006] The first objective of this invention is to provide a strain of Beauveria bassiana (Beauveria bassiana). Beauveria bassiana YJ-YC1, with accession number GDMCC NO: 66725.

[0007] A second objective of this invention is to provide a biological agent containing live cells of Beauveria bassiana YJ-YC1 or a culture of live cells of Beauveria bassiana YJ-YC1 as an active ingredient.

[0008] Preferably, the culture of live Beauveria bassiana YJ-YC1 cells in the formulation is a suspension of Beauveria bassiana YJ-YC1 conidia.

[0009] Preferably, the concentration of the Beauveria bassiana YJ-YC1 conidial suspension in the formulation is 1×10⁻⁶. 6 cfu / mL or higher.

[0010] Preferably, the concentration of the Beauveria bassiana YJ-YC1 conidial suspension in the formulation is 1×10⁻⁶. 6 ~1×10 8 cfu / mL.

[0011] A third objective of this invention is to provide the application of the aforementioned Beauveria bassiana YJ-YC1 or the aforementioned formulation in the control of lepidopteran pests.

[0012] Preferably, the application includes the step of applying live Beauveria bassiana YJ-YC1 cells or the preparation thereof to lepidopteran pests.

[0013] Preferably, the lepidopteran pest is *Hemiberle spp.*, *Hemiberle spp.*, or *Hemiberle spp.*

[0014] Preferably, the lepidopteran pests are the 2nd or 3rd instar larvae or pupae of the sea olive female tussock moth, the teak camel moth, or the yellow field moth.

[0015] Preferably, the method of application is impregnation.

[0016] The beneficial effects of this invention are: The *Beauveria bassiana* YJ-YC1 strain provided by this invention exhibits strong pathogenicity against the larvae and pupae of various explosive lepidopteran pests (*Ceratophorus spp.*, *Ceratophorus spp.*, and *Ceratophorus spp.*), with a pathogenicity of 1×10⁻⁶. 8 After treatment with a cfu / mL conidial suspension for 7 days, the mortality rates of 2nd and 3rd instar larvae were 100%, 87.34%, and 100%, respectively, while the mortality rates of pupae were 93.02%, 79.49%, and 90.91%, respectively. The high rate of molted insects indicates that this is a biological control fungus with potential value in the control of lepidopteran pests and has good application prospects.

[0017] Preservation Instructions The Beauveria bassiana YJ-YC1 provided by this invention ( Beauveria bassiana YJ-YC1 was deposited on July 22, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC NO: 66725. The depository 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 Beauveria bassiana YJ-YC1 on PDA medium on day 7. In the image, A is the front view of a Beauveria bassiana YJ-YC1 colony, and B is the back view of a Beauveria bassiana YJ-YC1 colony.

[0019] Figure 2 The images show the morphology of conidia, conidiophores, and hyphae of Beauveria bassiana YJ-YC1; where A is a conidia of Beauveria bassiana YJ-YC1, B is a conidiophore of Beauveria bassiana YJ-YC1, and C is a hyphae of Beauveria bassiana YJ-YC1.

[0020] Figure 3 This is a phylogenetic tree constructed for Beauveria bassiana YJ-YC1 based on ITS site sequences.

[0021] Figure 4 A suspension of conidia of Beauveria bassiana YJ-YC1 (1×10⁻⁶) 8 The pathogenicity of (cfu / mL) to the pupae of the sea olive female tussock moth, the teak camel moth, and the yellow field moth.

[0022] Figure 5 This study determined the toxicity of Beauveria bassiana YJ-YC1 to the yellow-spotted moth. A represents healthy yellow-spotted moth larvae from the control group; B represents yellow-spotted moth larvae treated with Beauveria bassiana YJ-YC1 for 7 days; C represents healthy yellow-spotted moth pupae from the control group; D represents yellow-spotted moth pupae infected with Beauveria bassiana YJ-YC1 and dead for 3 days; and E represents yellow-spotted moth pupae infected with Beauveria bassiana YJ-YC1 and dead for 7 days. Detailed Implementation

[0023] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0024] Example 1 1. Isolation and Identification of YJ-YC1 1.1 Strain Isolation In June 2024, *Bombyx mori* was collected from female *Bombyx mori* in a mangrove forest in Guangdong Province. A small number of conidia were picked from the *Bombyx mori* 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 edge of the colony were picked and isolated on fresh PDA medium. The above operation was repeated to obtain pure strain YJ-YC1.

[0025] 1.2 Morphological identification The isolated and purified strain YJ-YC1 was inoculated onto PDA medium plates (9 cm in diameter) and cultured at 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.

[0026] from Figure 1 It can be seen that on PDA medium, the YJ-YC1 strain is white and fluffy in the early stage of culture, with a dense texture, slightly raised on the front, thick in the middle and thin at the edge, and the edge is regularly radial; when sporulation occurs, the front of the colony is raised in the middle and is a light yellow powder, while the back of the colony is light yellow.

[0027] from Figure 2 It can be seen that the vegetative hyphae of strain YJ-YC1 are slender and colorless, and the conidiophores are opposite or scattered to form spike-like conidiophore structures. The conidia are colorless, elliptical or nearly spherical, with a few being oval. The spore outer wall is smooth, and the spore size is (2.74±0.07)μm × (1.81±0.05)μm (N=50).

[0028] 1.3 Molecular identification Using genomic DNA of strain YJ-YC1 as a template, the rDNA-ITS sequence of the strain was amplified by PCR using universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO.2) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO.3). The PCR reaction mixture (50 μL) consisted of 25 μL 2×Es TaqMaster Mix (Dye), 1 μL DNA template, 0.2 μL of 10 μmol / L upstream primer, 0.2 μL of 10 μmol / L downstream primer, 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. PCR products were analyzed by 1.0% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing results were compared against the GenBank database, and similar sequences were selected, trimmed at both ends, and a phylogenetic tree was constructed using the neighbor-joining method. Repeatability was tested 1000 times using the bootstrap method.

[0029] Based on the ITS sequencing results, poor-quality sequences at both ends were removed, resulting in the nucleotide sequence shown in SEQ ID NO.1. A BLAST comparison of the obtained nucleotide sequence with the NCBI database revealed that this strain was similar to those in the NCBI database. Beauveria bassiana The coverage of isolate SA1I1F6 was 97%, and the similarity was 100%.

[0030] Phylogenetic analysis results ( Figure 3 The results showed that strain YJ-YC1 was clearly separated from *Metarhizium anisopliae* strains, but clustered with *Beauveria bassiana* strains, indicating that this strain has a high similarity to *Beauveria bassiana* strains in the database. Therefore, based on morphological and molecular identification, this strain was identified as *Beauveria bassiana* and named *Beauveria bassiana* (also known as *Beauveria bassiana*). Beauveria bassiana )YJ-YC1.

[0031] 2. Pathogenicity determination of Beauveria bassiana YJ-YC1 against several forest pests. The pathogenicity of Beauveria bassiana YJ-YC1 against the larvae and pupae of *Beauveria bassiana*, *Beauveria bassiana*, and *Beauveria bassiana* was determined using the immersion method. First, conidia of Beauveria bassiana YJ-YC1 were prepared into 1×10⁻⁶ concentrations using a 0.5% (v / v) Tween-80 aqueous solution. 8 1×10 7 and 1×10 6A conidial suspension of cfu / mL was prepared. Second- to third-instar larvae or new pupae were immersed in the conidial suspension for 10 seconds, then immediately transferred to disposable plastic containers for rearing (temperature 25±1℃, relative humidity 70±5%, photoperiod 14 L:10 D). Larvae or pupae treated with a 0.5% (v / v) Tween-80 aqueous solution served as controls. Each treatment was repeated three times, with 15–20 larvae or pupae per replicate. The larvae of *Gnaphalium affine* and *Gnaphalium affine* were fed fresh *Gnaphalium affine* leaves daily, while the larvae of *Gnaphalium affine* were fed fresh *Aquilaria sinensis* leaves daily. Dead insects were removed and transferred to new plastic containers for humidified culture, and mycelial growth was observed.

[0032] Daily counts of larval or pupa mortality are recorded, and mortality rate, corrected mortality rate, and stunted larvae rate are 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%.

[0033] Table 1. Pathogenicity of Beauveria bassiana YJ-YC1 against 2nd-3rd instar larvae of the sea snub-nosed moth, the teak camel moth, and the yellow field moth. Table 1 shows that the conidial suspension of Beauveria bassiana YJ-YC1 exhibited strong pathogenicity against all three lepidopteran pests, and the mortality rate was positively correlated with conidial concentration and treatment time. At lower conidial concentrations (1×10⁻⁶), the mortality rate was significantly lower than that of other pests. 6 Under treatment with cfu / mL, the cumulative corrected mortality rate of 2nd-3rd instar larvae of the sea moth (CFU / mL) reached 73.32% after 7 days; while at a concentration of 1×10⁻⁶ CFU / mL, the mortality rate of larvae of the sea moth (CFU / mL) reached 73.32%; 8 At cfu / mL, the corrected mortality rate of 2nd to 3rd instar larvae of the sea olive female twig borer and the yellow field borer reached over 80% on day 5 and 100% on day 7. The cumulative corrected mortality rate of the larvae of the teak camel moth also reached 87.34% on day 7.

[0034] Beauveria bassiana YJ-YC1 can also effectively induce mushy curvature in insects, forming stiff worms. In summary, the larvae of the sea moth (Beauveria bassiana) are most sensitive to Beauveria bassiana YJ-YC1, even at 1×10⁻⁶ ppm. 6 At a concentration of CFU / mL, the silicosis rate was 81.99%; while at a concentration of 1×10⁻⁶, the silicosis rate was 81.99%. 8 At cfu / mL, the silicosis rate of *Hemiberlesia lataniae* reached 96.67%, followed by *Hemiberlesia lataniae* larvae at 92.16%; while the silicosis rate of *Hemiberlesia lataniae* was 74.17% (Table 1).

[0035] Table 2. Lethal median time (LT) of Beauveria bassiana YJ-YC1 against 2nd-3rd instar larvae of the sea snub-nosed moth, the teak camel moth, and the yellow field moth. 50 ) Table 2 shows the median lethal time (LT) of Beauveria bassiana YJ-YC1 against three pests. 50 The LT (transmission time) of all three pests decreased significantly with increasing conidial concentration. At the same treatment concentration, the susceptibility of the three pests differed. The LT of *Trichoderma harzianum* was... 50 The minimum value is 1×10. 8 At a CFU / mL concentration, the survival rate was 3.05 days; followed by *Pterocarya stenoptera*, and then LT. 50 The value was 3.44 d; while the LT of the teak moth was... 50 The value was 4.28 days. In the linear regression equation, the correlation coefficient (R2) was greater than 0.95, indicating that the cumulative number of larval deaths was highly positively correlated with the number of days of infection.

[0036] Beauveria bassiana YJ-YC1 also showed extremely strong pathogenicity to the pupae of three pests, at a concentration of 1×10⁻⁶. 8 Under conidial concentrations of CFU / mL, the corrected mortality rates of pupae of *Spodoptera litura*, *Pterocarya stenoptera*, and *Pterocarya stenoptera* were 93.02%, 79.49%, and 90.91%, respectively, with cumulative stunted larvae rates of 88.02%, 78.42%, and 92.99%, respectively. Figure 4 ).

[0037] Among them, the pathogenic phenotype of Beauveria bassiana YJ-YC1 against the larvae and pupae of the yellow field borer is as follows: Figure 5 As shown, in 1×10 8 At a concentration of CFU / mL, Beauveria bassiana YJ-YC1 showed significant lethality against larvae and pupae of the yellow field borer. Compared with the control group larvae ( Figure 5 Compared to A), white mycelium gradually grew on the surface of larvae that died from infection with Beauveria bassiana YJ-YC1, eventually becoming covered by a large amount of mycelium and beginning to produce sporulation. Figure 5 (B in the text). The infection process in pupae was also significantly different from that in the control group pupae ( Figure 5 Compared to C), mycelial growth was observed on the surface of pupae that had died for 3 days. Figure 5 (D in the text), by day 7, the hyphae proliferate extensively and begin to produce conidia ( Figure 5 (E in the text). These results show that Beauveria bassiana YJ-YC1 has a strong infectivity for both larvae and pupae of the yellow field borer.

[0038] 3. Comparison of virulence of different entomopathogenic fungi to third instar larvae of the teak moth. Teak moths are prone to rapid outbreaks and severe damage, and are highly resistant to various insecticidal fungi. This experiment further selected *Beauveria bassiana* SW-2 (… Beauveria bassiana The toxicity of SW-2 and Beauveria bassiana YJ-YC1 against the third instar larvae of the teak camel moth was compared.

[0039] The pathogenicity of the pathogenic fungus against the larvae of the teak camel moth was determined using the immersion method. First, the pathogenic fungus was prepared into solutions with a concentration of 1.5 × 10⁻⁶ using a 0.5% (v / v) Tween-80 aqueous solution. 8 1.5×10 7 1.5×10 6 Teak moth larvae of uniform growth were selected and immersed in a conidial suspension of CFU / mL for 10 s, then transferred to disposable plastic containers for rearing (temperature 25±1℃, relative humidity 70±5%, photoperiod 14 L:10 D). Teak moth larvae treated with a 0.5% (v / v) Tween-80 aqueous solution served as controls. Each treatment was repeated three times, with 10–20 larvae per replicate. Fresh female leaves of *Rhizophora stylosa* were introduced daily, and dead larvae were removed and transferred to new plastic containers for humidified culture. Mycelial growth was observed. The number of dead larvae was counted daily, and the mortality rate and corrected mortality rate were calculated.

[0040] Table 3. Toxicity of different entomopathogenic fungi to 3rd instar larvae of the teak moth Table 4. Lethal time (LT) of different entomopathogenic fungi on 3rd instar larvae of the teak moth 50 ) Table 3 shows that when the conidial concentration is 1×10⁻⁶, 8 At a concentration of 1×10⁻⁶ CFU / mL, the corrected mortality rate of third-instar larvae of the teak moth treated with *Beauveria bassiana* YJ-YC1 for 3 days was higher than that treated with *Beauveria bassiana* SW-2. After 7 days of treatment, the corrected mortality rate of third-instar larvae of the teak moth treated with *Beauveria bassiana* YJ-YC1 was 87.34%, which was superior to that of *Beauveria bassiana* SW-2 (corrected mortality rate of 84.62%). Table 4 shows that at a concentration of 1×10⁻⁶ CFU / mL, the corrected mortality rate of third-instar larvae of the teak moth was significantly higher than that of SW-2. 6 ~1×10 8 LT of Beauveria bassiana YJ-YC1 conidial suspension at cfu / mL 50 Both were smaller than those of Beauveria bassiana SW-2. Therefore, Beauveria bassiana YJ-YC1 is more effective than Beauveria bassiana SW-2 in controlling the teak moth.

[0041] In summary, Beauveria bassiana YJ-YC1 exhibits highly effective pathogenicity against pests, effectively infecting and killing the larvae and pupae of lepidopteran pests such as the sea olive female tussock moth, teak camel moth, and yellow field moth, with a high silicosis rate. It is a biological control fungus with potential value in the control of lepidopteran pests.

Claims

1. A strain of Beauveria bassiana Beauveria bassiana YJ-YC1, characterized in that, Its accession number is GDMCCNO:66725.

2. A biological agent, characterized in that, The active ingredient is a live bacterial cell of Beauveria bassiana YJ-YC1 as described in claim 1 or a culture of live bacterial cells of Beauveria bassiana YJ-YC1 as described in claim 1.

3. The formulation according to claim 2, characterized in that, The culture of live Beauveria bassiana YJ-YC1 cells in the formulation is a suspension of conidia of Beauveria bassiana YJ-YC1.

4. The formulation according to claim 3, characterized in that, The concentration of the Beauveria bassiana YJ-YC1 conidial suspension in the formulation is 1×10⁻⁶. 6 cfu / mL or higher.

5. The formulation according to claim 4, characterized in that, The concentration of the Beauveria bassiana YJ-YC1 conidial suspension in the formulation is 1×10⁻⁶. 6 ~1×10 8 cfu / mL.

6. The use of Beauveria bassiana YJ-YC1 as described in claim 1 or the preparation as described in claim 2 in the control of lepidopteran pests.

7. The application according to claim 6, characterized in that, The method includes the step of applying live Beauveria bassiana YJ-YC1 cells or the preparation thereof to lepidopteran pests.

8. The application according to claim 6, characterized in that, The lepidopteran pests mentioned are the sea olive female tussock moth, the teak camel moth, or the yellow field moth.

9. The application according to claim 8, characterized in that, The lepidopteran pests mentioned are the 2nd to 3rd instar larvae or pupae of the sea olive female tussock moth, the teak camel moth, or the yellow field moth.

10. The application according to claim 7, characterized in that, The method of application is impregnation.

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