Aspergillus for preventing and treating monochamus alternatus and application thereof
By isolating and identifying the Aspergillus austwickii HUZU6 strain, a spore suspension was prepared for the control of pine sawyer beetle, which solved the problem of insufficient pathogenic fungi in the existing technology and achieved effective control of pine sawyer beetle and pine wilt disease.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HUZHOU UNIVERSITY
- Filing Date
- 2022-12-12
- Publication Date
- 2026-07-07
AI Technical Summary
The existing technologies for controlling the pine sawyer beetle have limited types of pathogenic fungi, and there is a lack of investigation and development of other pathogenic fungal resources, resulting in single control methods for the pine sawyer beetle with limited effectiveness.
A strain of Aspergillus austwickii HUZU6, which controls the pine sawyer beetle, was provided. It causes the beetle to die by infecting its body wall. The spore suspension was prepared for the control of the pine sawyer beetle and has low requirements for culture conditions.
Aspergillus austwickii HUZU6 exhibits strong lethal activity against the pine sawyer beetle, effectively controlling the spread of pine wilt disease caused by the pine sawyer beetle. It is environmentally friendly, easy to cultivate, and has good application prospects.
Smart Images
Figure CN117736875B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbiology, and more particularly to an Aspergillus strain for controlling the pine sawyer beetle and its application. Background Technology
[0002] Pine trees are among the most important tree species in the global ecosystem. They are not only a major component of parks, nature reserves, and urban ornamental plants, but also a valuable source of timber and various wood products, supplying half of the world's timber. In China, Masson pine forests account for approximately 10% of forest resources and are a primary source of construction timber, generating significant economic benefits for the country. Pine wilt disease, caused by the pine wilt nematode, is a major disease affecting pine forests worldwide, infecting many pine species and causing widespread damage. Originating in North America, pine wilt disease has now spread to countries such as Spain, Portugal, South Korea, and Japan. The first case of pine wilt infection was reported in Nanjing, China in 1982. To date, pine wilt disease has invaded 731 counties (districts) in 17 provinces, 1 autonomous region, and 1 municipality in my country, causing economic losses exceeding hundreds of billions of US dollars. In my country, the pine sawyer beetle is the main vector insect for pine wilt disease, capable of carrying large numbers of pine wilt nematodes into pine trees, causing disease and ultimately death. Therefore, effectively controlling the occurrence of pine sawyer beetle and interrupting the infection cycle of pine wilt nematode are key to the integrated prevention and control of pine wilt disease in pine trees.
[0003] Currently, the main control methods for the pine sawyer beetle are biological control, chemical control, and integrated management. Among these, biological control has become a key measure, playing a crucial role in regulating and controlling the beetle's population. This includes releasing natural enemy insects, pathogenic microorganisms, and predatory birds. Insectivorous fungi, as an important means of microbial control, are widely developed to replace chemical pesticides due to their diverse species, wide distribution in nature, strong pathogenicity against various pests, and environmental friendliness. However, due to the host specialization of pathogenic fungi and their co-evolution in habitats, further exploration of pathogenic fungal resources in local ecosystems is necessary for effective control of local pests.
[0004] Studies have shown that the pathogenic fungi of the pine sawyer beetle include Beauveria bassiana, Metarhizium anisopliae, Verticillium, and Paecilomyces. However, current research on the pathogenic fungi of the pine sawyer beetle is mostly focused on Beauveria bassiana and Metarhizium anisopliae, and commercial preparations of related pathogenic fungi are also concentrated on Beauveria bassiana and Metarhizium anisopliae, lacking investigation and development of other pathogenic fungal resources of the pine sawyer beetle. Summary of the Invention
[0005] To address the limited number of pathogenic fungi available for controlling the pine wilt disease under existing technologies, this invention provides an Aspergillus strain for controlling the pine wilt disease. This strain exhibits strong lethal activity against the pine wilt disease, capable of infecting the body wall of the pine wilt disease and causing its death, thereby effectively controlling the spread of pine wilt disease by the pine wilt disease. Furthermore, this strain has low requirements for culture conditions and shows great promise for development and application.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A strain of Aspergillus austwickii, strain number HUZU6, which controls the pine sawyer beetle, is deposited at the China Center for Type Culture Collection (CCTCC), Hubei Province, Room 211, Wuhan University, Wuchang District, Wuhan City, Hubei Province, China. The accession number is CCTCC M 20221601, and the deposit date is October 19, 2022.
[0008] The Aspergillus austwickii HUZU6 described in this invention was isolated by the inventors from adult pine sawyer beetles covered with fungal hyphae, collected from an area inhabited by the pine sawyer beetle in Anji County, Huzhou City, Zhejiang Province. Morphological and gene sequence analysis confirmed the strain to be Aspergillus austwickii. After culturing on PDA medium at 25°C for 7 days, the colony front changed from white to dark green, while the back became brownish-green. The conidiophores were smooth and contained pear-shaped vesicles. The conidia were spherical with a rough surface, measuring 3.5–4.4 × 3.1–3.6 μm in diameter, and grew on ampoule-shaped pedicels.
[0009] A culture containing the aforementioned Aspergillus, wherein the culture is a bacterial solution or a bacterial agent.
[0010] Preferably, the fungal agent is a spore suspension.
[0011] Preferably, the spore concentration in the spore suspension is not less than 1×10⁻⁶. 8 per mL.
[0012] Preferably, the spore concentration in the spore suspension is 1×10⁻⁶. 8 per mL.
[0013] The above-mentioned Aspergillus fungi were used in the control of pine sawyer beetle.
[0014] The application of the above-mentioned Aspergillus cell culture in the control of pine sawyer beetle.
[0015] Compared with the prior art, the beneficial effects of the present invention are specifically described as follows: (1) Aspergillus austwickii HUZU6 can infect the body wall of the pine sawyer beetle and cause its death, and has strong lethal activity against the pine sawyer beetle, thereby effectively controlling the spread of pine wilt disease by the pine sawyer beetle; (2) The biological agent source of Aspergillus austwickii HUZU6 is environmentally friendly and non-toxic, and has little impact on the ecological environment; (3) Aspergillus austwickii HUZU6 has low requirements for culture conditions and has a good prospect for development and application. Attached Figure Description
[0016] Figure 1 The images show the appearance of a single colony of the strain of the present invention on PDA medium, where A is the front side of the colony and B is the back side of the colony.
[0017] Figure 2 These are images of the conidiophores and conidia of the strain of this invention under an optical microscope, where A is a conidiophore and B is a conidia.
[0018] Figure 3 Images of the conidiophores and conidia of the strain of this invention under a scanning electron microscope, where A is a conidiophore and B is a conidia.
[0019] Figure 4 This is a phylogenetic tree composed of the strain of this invention and other Aspergillus fungi based on multiple gene sequences (ITS, tef-1α, LSU, SSU, rpb2, TUB).
[0020] Figure 5 This is a survival curve diagram for determining the pathogenicity of the strain of this invention against adult *Spodoptera litura*.
[0021] Figure 6 Images showing the phenotypic pattern of adult pine sawyer beetles infected by the strain of this invention.
[0022] Figure 7 Images of the conidiophores and conidia of the strain of this invention infecting the body surface of an adult pine sawyer beetle under a scanning electron microscope, where A is a conidiophore and B is a conidia. Detailed Implementation
[0023] The present invention will be further described below with reference to the accompanying drawings and specific implementation methods.
[0024] Example 1
[0025] A strain of Aspergillus austwickii, used to control the pine sawyer beetle, with strain number HUZU6, is deposited at the China Center for Type Culture Collection (CCTCC), Hubei Province, Room 211, Wuhan University, Wuchang District, Wuhan City, Hubei Province, China. The accession number is CCTCC M 20221601, and the deposit date is October 19, 2022.
[0026] Isolation of Aspergillus austwickii HUZU6:
[0027] Samples of the pine sawyer beetle (Acer truncatum) used for isolating pathogenic fungi were collected from the beetle's habitat in Anji County, Huzhou City, Zhejiang Province. Under aseptic conditions, the collected beetles were dissected using sterile scissors into seven parts: head, antennae, thorax, abdomen, wings, legs, and eggs (female). The tissues were inoculated onto PDA agar plates containing 0.05 g / L streptomycin sulfate, penicillin G, and tetracycline, and incubated at 25°C until the tissues were covered by hyphae. Colonies with significant differences in size, shape, color, texture, and surface structure were selected and purified 2-3 times on antibiotic-containing PDA plates to obtain pure colonies. Finally, the purified strains were cultured on standard PDA plates and stored at the Zhejiang Provincial Key Laboratory of Vector Biology and Pathogen Control, Huzhou University, for future use.
[0028] Morphological identification of Aspergillus austwickii HUZU6:
[0029] Aspergillus austwickii HUZU6 was activated using PDA medium and cultured for 7 days. Morphological characteristics of the colonies were recorded by photographing both sides. Under aseptic conditions, bacterial blocks were picked up with an inoculation needle and placed on a glass slide with water droplets. A coverslip was then placed on the slide, and the morphological characteristics of spores and conidiophores were observed under an optical microscope. Under aseptic conditions, bacterial blocks were picked up with an inoculation needle and placed in pre-cooled 2.5% glutaraldehyde fixative at 4°C for 2 days. The fixed bacterial blocks were washed three times with 0.1% phosphate buffer (pH 7.2-7.4), 5 min each time. After washing, the bacterial blocks were dehydrated for 10 min each time with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol, respectively. The treated bacterial blocks were freeze-dried, coated with platinum using a sputtering coating device, and observed using a scanning electron microscope.
[0030] The single colony morphological characteristics of Aspergillus austwickii HUZU6 are as follows: Figure 1 As shown, the colonies were initially white on the front, turning dark green later due to spore production, while the back of the colonies was brownish-green. The spores appear as follows... Figure 2 and Figure 3As shown, the conidiophores are smooth and have pear-shaped vesicles, while the conidia are spherical with a rough surface, measuring 3.5–4.4 × 3.1–3.6 μm in diameter, and grow on ampoule-shaped pedicels.
[0031] Molecular genetic classification and identification of Aspergillus austwickii HUZU6:
[0032] Aspergillus austwickii HUZU6 was activated using PDB medium. Hyphae were collected in sterile centrifuge tubes and thoroughly ground in a pre-chilled mortar with liquid nitrogen. Genomic DNA of Aspergillus austwickii HUZU6 was extracted using a rapid fungal genomic DNA extraction kit and used as a template for PCR. The rDNA-ITS region was amplified using universal fungal primers ITS1 and ITS4 (forward primer ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', reverse primer ITS4: 5'-TCCTCCGCTTATTGATATGC-3'). The 50 μL PCR reaction mixture included 1 μL template DNA, 2 μL forward primer ITS1, 2 μL reverse primer ITS4, 25 μL 2×Taq PCR PreMix, and 20 μL ddH2O. The PCR reaction conditions were: 95℃ pre-denaturation for 4 min, 94℃ denaturation for 60 s, 55℃ annealing for 60 s, 72℃ extension for 2 min, for a total of 35 cycles of amplification, and finally 72℃ extension for 10 min, and stored at 4℃.The genomic DNA extracted from *Aspergillus austwickii* HUZU6 was used as a template for PCR reactions. The rDNA-SSU region was amplified using NS1 and NS4 primers (NS1: 5'-GTAGTCATATGCTTGTCTC-3', NS4: 5'-CTTCCGTCAATTCCTTTAAG-3'); the rDNA-LSU region was amplified using LR7 and LROR primers (LR7: 5'-ACCCGCTGAACTTAAGC-3', LROR: 5'-TACTACCACCAAGATCT-3'); and the rDNA-rpb2 region was amplified using RPB2-5'F and RPB2-5'R primers (RPB2-5'F: 5'-CCCATRGCTTGTYYRCCC). The rDNA-tef-1α region was amplified using primers EF-983F and EF-2218R (forward primer EF-983: 5'-GCYCCYGGHCAYGGTGAYTTYAT-3', reverse primer EF-2218: 5'-GACTTGACTTCRGTVGTGAC-3'); the rDNA-TUB region was amplified using primers TUB1 and TUB22 (forward primer TUB1: 5'-AACATGCGTGAGATTGTAAGT-3', reverse primer TUB22: 5'-TCTGGATGTTGTTGGGAATCC-3'), further clarifying the species information of Aspergillus austwickii HUZU6. The PCR amplification products of multiple gene sequences from Aspergillus austwickii HUZU6 strain were subjected to 1.5% agarose gel electrophoresis. After sequencing, the sequences were compared online with known related sequences in the database using the BLAST tool, and alignment was performed using Clustal X2.0 and MEGA6 software. A phylogenetic tree was constructed using the maximum likelihood method.
[0033] The ITS-rDNA sequence of Aspergillus austwickii strain HUZU6 is shown in SEQ ID No. 1, with a nucleotide sequence length of 568 bp. After comparison, the strain was identified as Aspergillus austwickii. Figure 4As shown, based on the polygenic sequence of Aspergillus austwickii HUZU6, a phylogenetic tree was constructed using the maximum likelihood method. Aspergillus austwickii HUZU6 and Aspergillus austwickii DTO 228-F7 clustered in one branch with a sequence similarity of 99.42%. The polygenic phylogenetic analysis results are consistent with the morphological characteristics of insect pathogenic fungi.
[0034] Example 2
[0035] Pathogenicity assay of Aspergillus austwickii HUZU6 against the pine sawyer beetle:
[0036] The tested pine sawyer beetles were collected from their habitat in Anji County, Huzhou City, Zhejiang Province. Each beetle was individually transferred to a clean 50mL centrifuge tube and fed with fresh pine branches. The day before the experiment, healthy adult pine sawyer beetles were selected and feeding was stopped, keeping them in a state of starvation.
[0037] Aspergillus austwickii HUZU6 was inoculated onto PDA medium and cultured at 25°C for 14 days. After the HUZU6 strain had fully conjugated, a piece of mycelium was picked up with an inoculation needle and placed in a 0.01% Tween-80 solution containing glass beads. The mixture was shaken thoroughly and filtered through two layers of sterile gauze to remove mycelia. The concentration of the spore suspension was adjusted with 0.01% Tween-80 solution to obtain 1×10⁻⁶ spores. 8 A spore suspension of strain HUZU6 per mL.
[0038] Sixty healthy adult pine sawyer beetles were immersed in a spore suspension for 10 seconds using an immersion method. After immersion, the beetles were transferred to sterile 50 mL centrifuge tubes (one treated beetle per tube) and fed fresh pine branches (also immersed in the spore suspension for 10 seconds). A control group of 60 beetles treated with 0.01% Tween-80 solution served as the control group. The pine branches fed to the control group were immersed in 0.01% Tween-80 solution for 10 seconds and then incubated at 25°C. The activity of the beetles was assessed by observing the amount of debris produced after feeding, and mortality was recorded over 15 days. Dead beetles were transferred to new 50 mL sterile centrifuge tubes (containing sterile moist cotton balls) to observe fungal infection on their body surface. After the surface of the tested *Pinus sylvestris* was covered with mycelium, Koch's postulates were used for verification.
[0039] Under aseptic conditions, body tissues of *Acer truncatum* covered with distinct green aerial hyphae were dissected using sterile scissors and fixed in pre-cooled 2.5% glutaraldehyde fixative at 4°C for 2 days. The fixed tissues were washed three times with 0.1% phosphate buffer (pH 7.2-7.4), 5 min each time. After washing, the tissues were dehydrated for 10 min each time with 30%, 50%, 70%, 80%, 90%, 95%, and 100% ethanol, respectively. The treated tissues were freeze-dried, coated with platinum using a sputtering coating apparatus, and observed using a scanning electron microscope.
[0040] like Figure 5 As shown, the survival rate of adult pine sawyer beetles treated with Aspergillus austwickii HUZU6 spore suspension for 15 days decreased to 5%, significantly lower than the control group. The appearance of adult pine sawyer beetles that died after treatment with Aspergillus austwickii HUZU6 spore suspension is as follows. Figure 6 As shown, its surface is covered with green aerial hyphae, and the morphological characteristics of the fungus isolated from the surface are consistent with those of strain HUZU6, satisfying Koch's postulates. Figure 7 As shown, the surface of the adult pine sawyer beetle was observed to be infected by Aspergillus austwickii HUZU6 using a scanning electron microscope.
[0041] The above experimental results show that the Aspergillus austwickii HUZU6 strain exhibits strong lethal activity against adult pine sawyer beetles and can be used for the control of pine sawyer beetles.
Claims
1. A strain of Aspergillus that controls the pine sawyer beetle, characterized by: The name of the Aspergillus is Aspergillus austwickii The strain number is HUZU6, and it is deposited at the China Center for Type Culture Collection (CCTCC), Hubei Province, Room 211, Wuhan University, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC M 20221601 and deposit date of October 19, 2022.
2. A culture containing Aspergillus as described in claim 1, characterized in that, The bacterial culture is a bacterial solution or bacterial agent.
3. The Aspergillus cell culture according to claim 2, characterized in that, The bacterial agent is a spore suspension.
4. The Aspergillus cell culture according to claim 3, characterized in that, The spore concentration in the spore suspension is not less than 1×10⁻⁶. 8 per mL.
5. A culture of Aspergillus according to claim 3 or 4, characterized in that, The spore concentration in the spore suspension was 1×10⁻⁶. 8 per mL.
6. The application of Aspergillus as described in claim 1 in the control of pine sawyer beetle.
7. The application of Aspergillus cell culture as described in any one of claims 2-5 in the control of pine sawyer beetle.