Beauveria bassiana GIZ-0fBb1 for preventing and controlling two invading ants and application thereof

Abstract

The invention discloses beauveria bassiana GIZ-0fBb1 for preventing and treating two types of invading ants and application of the beauveria bassiana GIZ-0fBb1. The beauveria bassiana is named as Beauveria bassiana GIZ-0fBb1, and the preservation number of the beauveria bassiana GIZ-0fBb1 is GDMCC No: 67403. The GIZ-0fBb1 is high in growth speed, high in sporulation speed, large in sporulation quantity and good in insecticidal effect, is a biocontrol strain with a good application prospect in sustainable prevention and control of the solenopsis invicta and the small imported fire ants, and can be widely applied to biological prevention and control of the solenopsis invicta and the small imported fire ants.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a Beauveria bassiana GIZ-0fBb1 for preventing and treating two invasive ants and application thereof. BACKGROUND

[0002] Solenopsis invicta Solenopsis invicta Buren belongs to the order of Hymenoptera, the family of Formicidae and the genus of Solenopsis Solenopsis As of June 2025, the red imported fire ant has invaded 703 counties (cities, districts) in 13 provinces (municipalities) of China. Solenopsis molesta Wasmannia auropunctata belongs to the order of Hymenoptera, the family of Formicidae and the genus of Solenopsis Wasmannia is an important invasive species newly discovered in China, and the potential suitable area of Solenopsis molesta is south of 30°N in China. Both the red imported fire ant and Solenopsis molesta can seriously threaten the local public health, ecological system, agricultural production and power equipment in the invaded area, and cause huge economic losses, and are both recognized as one of the top 100 most dangerous invasive species in the world.

[0003] Chemical control is the most common and effective means for controlling the red imported fire ant and Solenopsis molesta. The nest of Solenopsis molesta is mainly distributed in shaded and humid habitats such as litter layer, trunk crevices of trees and rock crevices, and it particularly prefers stable microenvironments such as large tree trunks. The red imported fire ant also has a wide distribution and great harm in forest and grass wetlands and urban green spaces. Therefore, there are special difficulties and higher requirements for chemical control of the red imported fire ant and Solenopsis molesta in special areas such as nature reserves and wetlands with rich biodiversity. Due to the ecological sensitivity of these areas, conventional chemical insecticides may be washed into natural water bodies and soil by rainwater, causing pollution of the ecological environment and posing a threat to non-target organisms, while entomopathogenic fungi biological control can become one of the most promising core means for preventing and controlling the red imported fire ant and Solenopsis molesta in these sensitive areas due to its high targeting, environmental safety and ecological compatibility. SUMMARY

[0004] The first object of the present application is to provide a Beauveria bassiana (Bb) GIZ-0fBb1 capable of preventing and treating the red imported fire ant and / or Solenopsis molesta. Beauveria bassiana The Bb GIZ-0fBb1 was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on December 4, 2025, and the address is No. 59, Building 5, 100, Xianlie Middle Road, Guangzhou, Guangdong, China, with a postal code of 510070, and the deposit number is GDMCC No: 67403.

[0005] The second object of the present application is to provide the application of the above-mentioned Bb GIZ-0fBb1 in preventing and treating the red imported fire ant and / or Solenopsis molesta.

[0006] Preferably, the red imported fire ant and / or Solenopsis molesta comprises workers.

[0007] A third objective of this invention is to provide the application of the aforementioned Beauveria bassiana GIZ-0fBb1 in the preparation of products for the control of red imported fire ants and / or small imported fire ants.

[0008] A fourth objective of this invention is to provide a biocontrol agent containing the aforementioned Beauveria bassiana GIZ-0fBb1.

[0009] Preferably, the biocontrol agent is a bacterial suspension of Beauveria bassiana GIZ-0fBb1 with a concentration of 1.0 × 10⁻⁶. 5 -1.0×10 9 spores / mL.

[0010] A fifth objective of the present invention is to provide a method for controlling red imported fire ants and / or small imported fire ants, comprising the step of contacting Beauveria bassiana GIZ-0fBb1 or a culture thereof with the bodies of red imported fire ants and / or small imported fire ants.

[0011] The beneficial effects of this invention are as follows:

[0012] The Beauveria bassiana GIZ-0fBb1 strain of this invention has strong pathogenicity against both red imported fire ants and small imported fire ants. It grows rapidly, produces spores quickly and in large quantities, and has good insecticidal effects. It is a biocontrol strain with good application prospects in the sustainable control of red imported fire ants and small imported fire ants, and can be widely used for the biological control of red imported fire ants and small imported fire ants.

[0013] Beauveria bassiana ( Beauveria bassiana GIZ-0fBb1 was deposited on December 4, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 67403. Attached Figure Description

[0014] Figure 1 This is the morphology of strain GIZ-0fBb1 from Example 1 grown on PDA medium (d=9cm).

[0015] Figure 2 These are symptoms of red imported fire ant worker ants infected with Beauveria bassiana GIZ-0fBb1.

[0016] Figure 3 These are symptoms of worker fire ants infected with Beauveria bassiana GIZ-0fBb1.

[0017] Figure 4 This is the result of the toxicity test of Beauveria bassiana GIZ-0fBb1 against red imported fire ant worker ants in Example 2.

[0018] Figure 5This is the result of the toxicity test of Beauveria bassiana GIZ-0fBb1 on worker fire ants in Example 2. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0020] Example 1: Isolation and Identification of Strain GIZ-0fBb1

[0021] I. Experimental Methods

[0022] 1. Isolation of strains

[0023] Using sterilized tweezers, the mycelia on the black-winged termite bodies were inoculated onto PDA medium (potato dextrose agar medium formula: potato (peeled) 200.0 g, glucose 20.0 g, agar 14.0 g, water 1000 mL, final pH 5.6). The medium was then incubated at a constant temperature of (25±1)℃ in an artificial climate chamber. After the mycelia grew on the medium, they were promptly transferred to new PDA medium for purification and culture for 7 days.

[0024] 2. Morphological identification of the strain

[0025] Morphological identification was performed on the strains grown in PDA medium.

[0026] 3. Molecular identification of the strain

[0027] Mycelia were scraped into 1.5 cm centrifuge tubes, and DNA was extracted according to the Omega Fugal DNA Kit instructions. PCR amplification was performed using primer pairs ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The PCR program was: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 50℃ annealing for 15 s, 72℃ extension for 50 s, 32 cycles; 72℃ extension for 5 min. The PCR products were analyzed by 1% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained ITS sequences were compared with known fungal ITS sequences in the NCBI database using BLAST. Sequences from strains with high homology in GenBank were selected for further analysis. Metarhizium eburneumFor outgroups, multiple sequence alignment analysis was performed using MEGAX software. A phylogenetic tree was constructed using the maximum likelihood method, with 1000 bootstrap replicates to test its reliability. A bootstrap value of 75% or higher is generally considered reliable.

[0028] II. Experimental Results

[0029] 1. Morphological characteristics

[0030] A fungus, GIZ-0fBb1, was isolated from a diseased black-winged subterranean termite queen. On PDA medium, the initial colonies appeared as white, fluffy colonies with radially radiating, hairy hyphae at the edges. Upon sporulation, the colonies became slightly convex in the center, the sporophyte layer became powdery, and the growth was uniform, resulting in a cottony colony appearance. Figure 1 ).

[0031] 2. Sequence alignment

[0032] The rDNA-ITS of strain GIZ-0fBb1 was amplified by PCR, and the sequenced gene fragment was 574 bp in length. The sequenced ITS fragment was then compared with the NCBI database using BLAST, and the results showed that this strain is similar to *Beauveria bassiana*. Beauveria bassiana ATMSB01 (GenBank accession number: MZ855506.1) shows a 99.82% similarity. Its taxonomic position is: Deuteromycotina, Hyphomycetes, Moniliales, Moniliaceae, Beauveria bassiana. Beauveria ).

[0033] The ITS sequence of GIZ-0fBb1 is shown in SEQ ID NO.1 (574 bp), specifically: .

[0034] The strain GIZ-0fBb1 was named Beauveria bassiana GIZ-0fBb1 was deposited on December 4, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 67403.

[0035] Example 2: Virulence determination of strain GIZ-0fBb1

[0036] I. Experimental Methods

[0037] 1. Preparation of spore suspension

[0038] Collect Beauveria bassiana GIZ-0fBb1 mycelial powder cultured on PDA plates for 10 days using a sterile scraper. Wash with sterile 0.05% Tween-80 aqueous solution, and stir with a magnetic stirrer to ensure thorough mixing of the spore suspension. Measure the number of spores per unit volume (mL) using a hemocytometer, and then prepare a 1.0 × 10⁻⁶ spore suspension using 0.05% Tween-80 aqueous solution. 9 10 8 10 7 10 6 and 10 5 Five spore suspensions of different concentrations were prepared at 4°C and stored for later use.

[0039] 2. Pathogenicity test of the strain against red imported fire ants and small imported fire ants

[0040] Worker ants of roughly the same size (either red imported fire ants or small imported fire ants) were immersed in a prepared spore suspension for 5 seconds. They were then removed and placed in disposable petri dishes lined with moistened filter paper. The rim of each dish was coated with Teflon to prevent escape. Inside each dish were one sterile water tube plugged with cotton and one 5 mL tube containing 10 g / mL sugar water. Thirty ants were inoculated into each dish, with three replicates per nest, and each nest was replicated five times. A sterile 0.05% Tween-80 aqueous solution was used as a control. The incubator temperature was 27℃ and the humidity was 60%.

[0041] Observation and recording: The number of dead test insects was observed and recorded daily, and they were transferred to petri dishes lined with moist filter paper for moist culture. The growth of mycelium was observed. The growth of mycelium and spores on the dead insects was counted as infection (mute insects). The experiment was observed continuously for 10 days.

[0042] II. Experimental Results

[0043] The toxicity test results showed that Beauveria bassiana GIZ-0fBb1 had high toxicity to both red imported fire ant workers and small imported fire ant workers. With the increase of spore concentration and the extension of treatment time, the mortality rate of both red imported fire ant workers and small imported fire ant workers gradually increased. Figure 4 and Figure 5 On day 10 post-treatment, spore suspensions of different concentrations (1×10⁻⁶) were compared. 5 ~1×10 9 The mortality rates of red imported fire ant workers treated with concentrations of 1×10⁻⁶ spores / mL were 12.00%, 30.44%, 55.33%, 83.56%, and 97.11%, respectively, especially at high concentrations (1×10⁻⁶ spores / mL). 9 A spore suspension of 1×10⁶ spores / mL showed strong pathogenicity against red imported fire ants, with a cumulative mortality rate of 93.33%–100% among worker ants on day 10 post-treatment. On day 10 post-treatment, different concentrations of spore suspension (1×10⁶ spores / mL) showed varying degrees of pathogenicity.5 ~1×10 9 The mortality rates of fire ant workers treated with spores / mL were 13.33%, 28.67%, 53.11%, 87.56%, and 97.11%, respectively, especially for high-concentration spore suspensions (1×10⁻⁶ spores / mL). 9 The treatment (1 spore / mL) showed strong pathogenicity to red imported fire ants, with a cumulative mortality rate of 93.33% to 100% among worker ants on day 10 after treatment.

[0044] Using the Probit model, the regression equation for the pathogenicity of Beauveria bassiana GIZ-0fBb1 on red imported fire ant workers on day 10 was obtained as y = -5.803 + 0.843x (χ² = 81.88, df = 72, P = 0.199), with an LC50 of 7.65 × 10⁻⁶. 7 Spores / mL, 95% confidence interval is 4.83 × 10⁻⁶. 7 ~1.11 × 10 8 Spores / mL; the pathogenicity regression equation for worker fire ants is y = -6.622 + 0.949x (χ² = 95.50, df = 72, P = 0.052), and the LC50 is 9.41 × 10⁻⁶. 7 Spores / mL, 95% confidence interval is 6.11 × 10⁻⁶. 7 ~1.35 × 10⁶ spores / mL. This indicates that Beauveria bassiana GIZ-0fBb1 has strong pathogenicity against both red imported fire ant workers and small imported fire ant workers.

[0045] in conclusion

[0046] Beauveria bassiana GIZ-0fBb1 is a strain isolated from naturally infected black-winged subterranean termites that exhibits strong pathogenicity against both red imported fire ant (Periplaneta americana) and small imported fire ant (Periplaneta americana) workers. This strain grows well on PDA medium at 26±1℃, exhibiting rapid growth, high sporulation rate, and high sporulation yield, demonstrating good insecticidal efficacy. It is a promising biocontrol strain for the sustainable control of red imported fire ants and small imported fire ants and can be widely used for their biological control.

Claims

1. Beauveria bassiana ( Beauveria bassiana )GIZ-0fBb1, its accession number is: GDMCC No: 67403.

2. The application of Beauveria bassiana GIZ-0fBb1 as described in claim 1 in the control of red imported fire ants and / or small imported fire ants.

3. The application according to claim 2, characterized in that, The red imported fire ants and / or small fire ants include worker ants.

4. The use of Beauveria bassiana GIZ-0fBb1 as described in claim 1 in the preparation of red imported fire ant and / or small imported fire ant control products.

5. A biocontrol agent, characterized in that, Contains Beauveria bassiana GIZ-0fBb1 as described in claim 1.

6. The biocontrol agent according to claim 5, characterized in that, The biocontrol agent is a bacterial suspension of Beauveria bassiana GIZ-0fBb1 with a concentration of 1.0 × 10⁻⁶. 5 -1.0×10 9 spores / mL.

7. A method for controlling red imported fire ants and / or small imported fire ants, characterized in that, The method includes the step of contacting the Beauveria bassiana GIZ-0fBb1 or its culture as described in claim 1 with the bodies of red imported fire ants and / or small imported fire ants.

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