Application of fusarium equiseti in prevention and treatment of Japanese pine scale
By using Fusarium thirst spore suspension to infect Japanese pine thorn scales, the problems of environmental pollution and poor control effects of chemical control methods in the existing technology were solved, and efficient biological control effects were achieved and good applications were achieved. prospect.
Patent Information
- Application Number
- CN202411319189.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-21
- Publication Date
- 2024-12-31
AI Technical Summary
The chemical control methods used in the prior art for the prevention and control of pine pine squama in Japan have strong environmental pollution and ecological destructiveness, and there are existing pathogenic microorganisms that have poor control and control effects and are difficult to expand and proliferate, making it difficult to effectively apply to the prevention and control of pine squamama.
The spore suspension of Gibberella intricans Wollenw. was used to infect the second instar nymphs and female adults of the Japanese pine thorn scale. The mycelial infection caused the insect body to die, and the spore suspension was easy to expand, which increased the infection rate. and lethality rate.
Fusarium thief spore suspension can effectively infect Japanese pine thorn scales. The second-instar nymph showed infection symptoms 2-5 days after infection with bacteria, with a mortality rate of 64%. After infection with female adults, the activity is weakened and the mortality rate is 100%, which is high. Infection rate and infection effect are suitable for the biological control of pine pine scales.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological control of forest pests and diseases, and in particular to an application of Fusarium equisetifolia in controlling Japanese pine scale. Background Art
[0002] Currently, chemical control methods are mostly used for the prevention and control of Japanese pine scale, including scraping the bark at the base of the trunk and applying pesticides (monocrophate, omethoate) or drilling holes in the trunk to inject pesticides to control fixed-stage nymphs; when the nymphs emerge, spraying chemical agents such as omethoate emulsifiable concentrate and carbofuran emulsifiable concentrate to control the emerging nymphs; however, chemical control methods cause great environmental pollution and strong ecological destructiveness, and the control effect is not sustainable.
[0003] Microbial pathogens, as biological insecticides, have the advantages of strong targeting and no environmental pollution, and have good killing properties against scale insect pests. Currently, there are 6 types of pathogenic microorganisms recorded for Japanese pine scale, namely, Bacillus thuringiensis wax moth variant pine scale strain, Lecanus lecanii, Fusarium spp., Lecanus mycoides, Beauveria bassiana and Penicillium cairnsense However, the above-mentioned pathogenic microorganisms have a series of shortcomings such as poor infection effect, limited infection conditions, and difficulty in strain propagation, which make them unable to be used in the prevention and control of Japanese pine scale; therefore, it is particularly important to find a pathogenic microorganism that can be practically applied and can effectively prevent and control Japanese pine scale. Summary of the invention
[0004] In view of this, the present invention provides an application of Fusarium equiseti in preventing and controlling Japanese pine scale. When using the spore suspension prepared by the Fusarium equiseti can not only effectively infect the second-instar nymphs and female adults of the Japanese pine scale, but also is easier to propagate, so it has good application prospects in the biological control of the Japanese pine scale.
[0005] The technical solution of the present invention is as follows: Application of Fusarium equisetifolia in preventing and controlling Japanese pine scale, wherein the strain number of Fusarium equisetifolia is BNCC124121 and the Latin name is Gibberella intricans Wollenw.; specifically, the application of spore suspension of Fusarium equisetum in controlling Japanese pine scale.
[0006] Preferably, the number of spores in the spore suspension is 1-5×10 7 spores / mL.
[0007] Preferably, the spore suspension is prepared as follows: Step 1: Strain culture and expansion. The slant strain of Fusarium equiseti was transferred to a comprehensive PDA medium plate and expanded at 25°C until the plate was full of growth; Step 2: Preparation of spore suspension. Take a plate full of strains, wash the spores with 0.05% Tween-80 sterile water on a clean bench, vortex oscillator to fully oscillate to disperse the spores as much as possible, count them with a hemocytometer, and prepare 1-5×10 7 spores / mL of spore suspension.
[0008] Preferably, the use of Fusarium equisetum in preventing and controlling Japanese pine scale is specifically to use a spore suspension to infect Japanese pine scale insects.
[0009] Preferably, the Japanese pine scale insect body includes second-instar nymphs and female adults.
[0010] Compared with the prior art, the beneficial effects of the present invention are: In the present invention, it is found for the first time that Fusarium equisetifolia can be used in preventing and controlling Japanese pine scale; the spore suspension of Fusarium equisetifolia is applied to the second-instar nymphs of Japanese pine scale, and the second-instar nymphs show infection symptoms 2-5 days after being infected with the bacteria, white hyphae grow on the surface of the insect body, and the insect body becomes concave and black in the later stage, with a cumulative mortality rate of 64% in 10 days; the spore suspension of Fusarium equisetifolia is applied to the female adults of Japanese pine scale, and the activity of the female adults is weakened after being infected with the bacteria, the insect body becomes black and shriveled on the second day, and a small amount of hyphae grows on the insect body on the second to fourth days, with a mortality rate of 100%.
[0011] The Fusarium equiseti provided by the present invention has the advantage of being easy to propagate. A spore suspension prepared using the Fusarium equiseti can quickly take effect on Japanese pine scale, has the advantages of high infection rate and good infection effect, and has good application prospects in the biological control of Japanese pine scale. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0013] Figure 1 This is an informational photo of Fusarium equisetum of the present invention.
[0014] Figure 2 These are photos of the strain after culture and expansion.
[0015] Figure 3 Photo 1 is of the second instar nymph before infection.
[0016] Figure 4 This is a photograph of a second-instar nymph infected with the spore suspension of Example 1.
[0017] Figure 5 Photo 2 is of the second instar nymph before infection.
[0018] Figure 6 This is a photograph of a second-instar nymph infected with the spore suspension of Example 2.
[0019] Figure 7 These are photos of second-instar nymphs and female adults before infection.
[0020] Figure 8 These are photographs of second-instar nymphs and female adults after infection with the spore suspension of Example 3.
[0021] Fig. 9 These are photographs of second-instar nymphs and female adults after infection with the spore suspension of Example 4.
[0022] Fig.10 This is a photo of the colony morphology of common Fusarium.
[0023] Fig.11 This is a photo of the second-instar nymph after infection with common Fusarium.
[0024] Fig.12 This is a photo of the colony morphology of Paecilomyces lilacinus.
[0025] Fig.13 This is a photo of the second-instar nymph after infection with Paecilomyces lilacinus.
[0026] Fig.14 This is a photo of the colony morphology of Mucor circinelloides.
[0027] Fig.15 This is a photo of the second-instar nymph after infection with Mucor circinelloides. DETAILED DESCRIPTION
[0028] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.
[0029] In this application, the strain is sourced from the Henan Industrial Microbial Strain Engineering Technology Research Center, and the comprehensive PDA medium used for strain culture is composed of: 1L potato extract, 20g glucose, 3g KH2PO4, 1.5g MgSO4•7H2O, 0.008g thiamine, 15g agar, pH = 6. The specimen insect source used in the present invention was collected from Tai'an Culaishan Forest Farm on June 26, August 28, and September 5, 2024. The branches collected from the forest were observed and cut into 3-6cm pine wood sections with insects, wax-sealed at both ends, and placed in a sterilized culture dish for use as the insect source used in the experiment.
[0030] Example 1 Application of Fusarium equisetifolia in preventing and controlling Japanese pine scale, wherein the strain number of Fusarium equisetifolia is BNCC124121 and the Latin name is Gibberella intricans Wollenw.; The product for controlling Japanese pine scale is a spore suspension of Fusarium equisetum; The spore suspension was prepared as follows: Step 1: strain culture and expansion. The slant strain of Fusarium equiseti was transferred to a comprehensive PDA medium plate, which was placed upside down in an incubator and expanded for 5 days at 25°C until the strain covered the plate. Step 2: Preparation of spore suspension. Take a plate full of strains, wash the spores with 0.05% Tween-80 sterile water on a clean bench, use a pipette to draw the spores into a sterilized centrifuge tube with 3-4 glass beads, vortex the spores to disperse them as much as possible, filter them into a new sterilized centrifuge tube with 2 layers of gauze, rinse several times to 10 ml, count them with a hemocytometer, and prepare 1×10 7 spores / mL of spore suspension.
[0031] Example 2 Application of Fusarium equisetifolia in preventing and controlling Japanese pine scale, wherein the strain number of Fusarium equisetifolia is BNCC124121 and the Latin name is Gibberella intricans Wollenw.; The product for controlling Japanese pine scale is a spore suspension of Fusarium equisetum; The spore suspension was prepared as follows: Step 1: strain culture and expansion. The slant strain of Fusarium equiseti was transferred to a comprehensive PDA medium plate, which was placed upside down in an incubator and expanded for 5 days at 25°C until the strain covered the plate. Step 2: Preparation of spore suspension. Take a plate full of strains, wash the spores with 0.05% Tween-80 sterile water on a clean bench, use a pipette to draw the spores into a sterilized centrifuge tube with 3-4 glass beads, vortex the spores to disperse them as much as possible, filter them into a new sterilized centrifuge tube with 2 layers of gauze, rinse several times to 10 ml, count them with a hemocytometer, and prepare 2×10 7 spores / mL of spore suspension.
[0032] Example 3 Application of Fusarium equisetifolia in preventing and controlling Japanese pine scale, wherein the strain number of Fusarium equisetifolia is BNCC124121 and the Latin name is Gibberella intricans Wollenw.; The product for controlling Japanese pine scale is a spore suspension of Fusarium equisetum; The spore suspension was prepared as follows: Step 1: Strain culture and expansion. The slant strain of Fusarium equiseti was transferred to a comprehensive PDA medium plate, which was placed upside down in an incubator and expanded for 7 days at 25°C until the strain covered the plate. Step 2: Preparation of spore suspension. Take a plate full of strains, wash the spores with 0.05% Tween-80 sterile water on a clean bench, use a pipette to draw the spores into a sterilized centrifuge tube with 3-4 glass beads, vortex the spores to disperse them as much as possible, filter them into a new sterilized centrifuge tube with 2 layers of gauze, rinse several times to 10 ml, count them with a hemocytometer, and prepare 5×10 7 spores / mL of spore suspension.
[0033] Example 4 Application of Fusarium equisetifolia in preventing and controlling Japanese pine scale, wherein the strain number of Fusarium equisetifolia is BNCC124121 and the Latin name is Gibberella intricans Wollenw.; The product for controlling Japanese pine scale is a spore suspension of Fusarium equisetum; The spore suspension was prepared as follows: Step 1: Strain culture and expansion. The slant strain of Fusarium equiseti was transferred to a comprehensive PDA medium plate, which was placed upside down in an incubator and expanded for 7 days at 25°C until the strain covered the plate. Step 2: Preparation of spore suspension. Take a plate full of strains, wash the spores with 0.05% Tween-80 sterile water on a clean bench, use a pipette to draw the spores into a sterilized centrifuge tube with 3-4 glass beads, vortex the spores to disperse them as much as possible, filter them into a new sterilized centrifuge tube with 2 layers of gauze, rinse several times to 10 ml, count them with a hemocytometer, and prepare 4×10 7 spores / mL of spore suspension.
[0034] test Test 1: The spore suspension of Fusarium equisetifolia provided in Examples 1-4 was used to infect the second-instar nymphs and / or female adults of Japanese pine scale, and the process was as follows: Using the insect immersion method, 50 healthy second-instar nymphs and 20 healthy female adults of Japanese pine stem scale were screened out, and the insect-infested branches were immersed in the prepared spore suspension for infection for 1 min, then air-dried for 6 min, transferred to a sterilized culture dish with moistened filter paper at the bottom of the culture dish for moisture retention, and then placed in a constant temperature and light incubator at 25℃ for culture, and the insect body changes and mortality were observed and recorded daily.
[0035] The mortality calculation results are shown in Table 1, as follows: Table 1 Infection effect of spore suspension obtained in Example 1 on Japanese pine scale
[0036] The changes of the insect body are as follows: The spore suspension obtained in Example 1 was used to infect second-instar nymphs, and the results were as follows: Before infection, the second-instar nymphs live in the cracks of the bark. Figure 3 ; See photos after infection Figure 4 , Figure 4 Middle, the left picture shows a photo of a second-instar nymph infected for 5 days, and the right picture shows a photo of a second-instar nymph infected for 7 days; Combination Figure 3 It can be seen that before infection, the second-instar nymphs have plump and round bodies. Figure 4 It can be seen that after 5 days of infection, the bodies of the second-instar nymphs became sunken and a small amount of hyphae attached to the surface of the bodies; after 7 days of infection, the bodies of the second-instar nymphs became obviously sunken and the amount of hyphae on the surface of the bodies increased; The spore suspension obtained in Example 2 was used to infect second-instar nymphs, and the results were as follows: Before infection, the second-instar nymphs live in the cracks of the bark. When the bark is peeled off, the second-instar nymphs are exposed. Figure 5 ; See photos after infection Figure 6 , Figure 6Middle, the left picture shows a photo of a second-instar nymph infected for 3 days, and the right picture shows a photo of a second-instar nymph infected for 5 days; Combination Figure 5 It can be seen that before infection, the second-instar nymphs have plump and round bodies. Figure 6 It can be seen that after 3 days of infection, hyphae grew on the bodies of the second-instar nymphs. After 5 days of infection, more hyphae grew on the bodies of the second-instar nymphs, almost covering the surface of the bodies, and the colors turned black and died. The spore suspension obtained in Example 3 was used to infect second-instar nymphs and female adults, and the results were as follows: Before infection, the second-instar nymphs and female adults parasitize in the cracks of the bark. After the bark is peeled off, the exposed second-instar nymphs and female adults can be seen. Figure 7 ; See photos after infection Figure 8 , the left picture is a photo of the infection for 2 days, and the right picture is a photo of the infection for 4 days; Figure 7 and Figure 8 In the figure, A is a second-instar nymph and B is a female adult; Combination Figure 8 It can be seen that after 2 days of infection, hyphae grew on the body surface of the second-instar nymph (A); the body color of the female adult (B) darkened, a small amount of hyphae grew on the surface, and the insect died; after 4 days of infection, the hyphae on the body surface of the second-instar nymph (A) were obvious, the insect body darkened and died; the hyphae on the surface of the female adult (B) were obvious; The spore suspension obtained in Example 4 was used to infect second-instar nymphs and female adults, and the results were as follows: See photos after infection Fig. 9 , the left picture is a photo of the infection for 3 days, and the right picture is a photo of the infection for 5 days; Fig. 9 In the figure, A is a second-instar nymph and B is a female adult; Combination Fig. 9 It can be seen that after 3 days of infection, hyphae grew on the body surface of the second-instar nymph (A); the body color of the female adult (B) darkened, a small amount of hyphae grew on the surface, and the insect died; after 5 days of infection, the body of the second-instar nymph (A) was obviously sunken, hyphae grew on the body surface; the female adult (B) completely turned black and shrunken and was covered with hyphae. After observation, 5×10 7 When the spore suspension with spores / mL was used to infect Japanese pine scale, the second-instar nymphs showed infection symptoms as early as the second day after infection, and infection symptoms appeared gradually within 2-7 days. White hyphae grew on the surface of the insect body, and the insect body became concave and black in the later stage. The overall infection rate was 56%, and the cumulative mortality rate in 10 days was 64%; After being infected with the bacteria, the activity of female adults weakens. On the second day, the body turns black and shriveled. From the second to the fourth day, a small amount of hyphae grows on the body. The mortality rate is 100% within 2-4 days.
[0037] Test 2: 1. Common Fusarium 1. Strain culture (1) Source of strains: Culture Collection Center of Institute of Microbiology, Chinese Academy of Sciences; (2) Culture medium formula: The first type: malt extract medium: 12Brix. malt extract 1L, agar 15g, natural pH; The second type: glucose 1%, peptone 1%, yeast extract 0.5%, agar 2%, natural pH; (3) Transfer training: The slant strains were transferred to the above two culture medium plates and cultured at 25°C until sufficient spore production; (4) Strain growth: Growth in the first medium was slow, almost non-existent.
[0038] In the second medium, the growth was faster, the colonies were white, spread, and covered the entire plate in 5 days; (5) Colony morphology: see Fig.10 ; 2. Implementation of infection experiment (1) Experimental plan: Insect source: The second-instar fixed nymphs of Japanese pine scale (the insect body is swollen, parasitizing under the cracks of the bark or appearing in the cracks of the bark) were used as the infected objects. The specimens were collected from Culai Mountain in Tai'an on June 6. The branches collected from the forest were observed and cut into 3-6cm pine wood sections with insects, both ends were sealed with wax, and placed in a sterilized culture dish for later use; Preparation of spore suspension: Take a plate full of strains, wash the spores with 0.05% Tween-80 sterile water, vortex thoroughly to disperse the spores as much as possible, count them with a hemocytometer, and prepare 1.2×10 8 spores / mL of spore suspension; Infection with second-instar nymphs: Immerse the branches with insects in the prepared spore suspension for 1 minute, then air-dry for 5 minutes, transfer to a sterilized culture dish with moistened filter paper at the bottom for moisture retention, and then culture in a 25°C constant temperature and light incubator. Observe and record the infection and lethality of each strain to the insects regularly every day and take photos; 3. Experimental results See Fig.11 , Fig.11 The left picture is a photo taken 5 days after infection, and the right picture is a photo taken 8 days after infection. Fig.11 It can be seen that 5 days after infection, the second-instar nymphs had no symptoms of infection, and 8 days after infection, the second-instar nymphs were still plump, that is, common Fusarium could not infect the second-instar nymphs, so the second-instar nymphs had no obvious symptoms of infection.
[0039] 2. Paecilomyces lilacinus BNCC336533 1. Strain culture (1) Strain source: Henan Industrial Microbial Strain Engineering Technology Research Center; (2) Culture medium formula: Comprehensive PDA medium: potato extract 1L, glucose 20g, KH2PO43g, MgSO4▪7H2O 1.5g, thiamine 0.008g, agar 15g, pH 6.0±0.2; (3) Transfer training: The slant strain was transferred to a comprehensive PDA medium plate and cultured at 25°C until sufficient spore production; (4) Strain growth: The colony is white with pale yellow on the back; (5) Colony morphology: see Fig.12 ; 2. Implementation of infection experiment (1) Experimental plan: Insect source: The second-instar fixed nymphs of Japanese pine scale (the insect body is swollen, parasitizing under the cracks of the bark or appearing in the cracks of the bark) were used as the infected objects. The specimens were collected from the Haoshan Forest Farm in Yiyuan, Zibo on June 26. The branches collected from the forest were observed and cut into 3-6cm pine wood sections with insects, both ends were wax-sealed, and placed in a sterilized culture dish for later use; Preparation of spore suspension: Take a plate full of strains, wash the spores with 0.05% Tween-80 sterile water, vortex thoroughly to disperse the spores as much as possible, count them with a hemocytometer, and prepare 5×10 7 Spores / mL spore suspension; Infection with second-instar nymphs: Immerse the branches with insects in the prepared spore suspension for 1 minute, then air-dry for 5 minutes, transfer to a sterilized culture dish with moistened filter paper at the bottom for moisture retention, and then culture in a 25°C constant temperature and light incubator. Observe and record the infection and lethality of each strain to the insects regularly every day and take photos; 3. Experimental results See Fig.13 , Fig.13 The left picture shows a photo of a second-instar nymph infected for 4 days, and the right picture shows a photo of a second-instar nymph infected for 7 days; Fig.13 It can be seen that after infection with Pseudomonas lilacinus, the plumpness of the body of the second-instar nymphs did not change.
[0040] 3. Mucor circinelloides 1. Strain culture (1) Source of strains: Culture Collection Center of Institute of Microbiology, Chinese Academy of Sciences; (2) Culture medium formula: Glucose 1%, peptone 1%, yeast extract 0.5%, agar 2%, natural pH; (3) Transfer training: Transfer the slant strain to the above-mentioned medium plate and expand the culture at 25°C until sufficient spore production; (4) Strain growth: In the early stage, the colonies are white with yellow back, spreading and growing rapidly, covering the entire plate in 5 days; (5) Colony morphology: See Fig.14 ; 2. Implementation of infection experiment (1) Experimental plan: Insect source: The second-instar fixed nymphs of Japanese pine scale (the insect body is swollen, parasitizing under the cracks of the bark or appearing in the cracks of the bark) were used as the infected objects. The specimens were collected from Culai Mountain in Tai'an on June 6. The branches collected from the forest were observed and cut into 3-6cm pine wood sections with insects, both ends were sealed with wax, and placed in a sterilized culture dish for later use; Preparation of spore suspension: Take a plate full of strains, wash the spores with 0.05% Tween-80 sterile water, vortex thoroughly to disperse the spores as much as possible, count them with a hemocytometer, and prepare 1×10 8 spores / mL spore suspension; Infection with second-instar nymphs: Immerse the branches with insects in the prepared spore suspension for 1 minute, then air-dry for 5 minutes, transfer to a sterilized culture dish with moistened filter paper at the bottom for moisture retention, and then culture in a 25°C constant temperature and light incubator. Observe and record the infection and lethality of each strain to the insects regularly every day and take photos; 3. Experimental results See Fig.15 , Fig.15 The left picture shows a photo of a second-instar nymph infected for 4 days, and the right picture shows a photo of a second-instar nymph infected for 8 days. Fig.15 It can be seen that when the second-instar nymphs were infected with Mucor circinelloides, the bodies of the second-instar nymphs were fresh and plump, with no obvious symptoms of infection.
[0041] Combining the results of Experiment 1 and Experiment 2, it can be seen that the Fusarium equisetum provided by the present invention is pathogenic to the second-instar nymphs and female adults of Japanese pine scale, and has a high mortality rate, and can be widely used in the prevention and control of Japanese pine scale.
[0042] Although the present invention has been described in detail by reference to the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.
Claims
1. An application of Fusarium equisetum in preventing and controlling Japanese pine scale, characterized in that: The strain number of this Fusarium equisetum is BNCC124121, and its Latin name is Gibberella intricans Wollenw.; specifically, the application of spore suspension of Fusarium equisetum in controlling Japanese pine scale.
2. The use of Fusarium equisetifolia in preventing and controlling Japanese pine scale as claimed in claim 1, characterized in that: The number of spores in the spore suspension is 1-5×10 7 spores / mL.
3. The use of Fusarium equisetifolia in preventing and controlling Japanese pine scale as claimed in claim 1, characterized in that: The spore suspension was prepared as follows: Step 1: Strain culture and expansion. The slant strain of Fusarium equiseti was transferred to a comprehensive PDA medium plate and expanded at 25°C until the plate was full of growth; Step 2: Preparation of spore suspension. Take a plate full of strains, wash the spores with 0.05% Tween-80 sterile water, vortex thoroughly to disperse the spores as much as possible, count them with a hemocytometer, and prepare 1-5×10 7 spores / mL of spore suspension.
4. The use of Fusarium equisetifolia in preventing and controlling Japanese pine scale as claimed in claim 3, characterized in that: Specifically, a spore suspension is used to infect Japanese pine scale insects.
5. The use of Fusarium equisetifolia in preventing and controlling Japanese pine scale as claimed in claim 4, characterized in that: The Japanese pine scale insect body includes a second-instar nymph and a female adult.