Application of biopesticide in prevention and treatment of tea lesser leafhopper

Abstract

The invention belongs to the technical field of microbial fermentation and insect pest prevention and control, and particularly relates to application of a biopesticide to prevention and control of tea lesser leafhopper. The bacillus velezensis NM153 supernate is used for treating the tea lesser leafhopper, and the result shows that the tea lesser leafhopper can be observed to die in quantity after being treated for about 12 hours under the treatment of the bacillus velezensis NM153 supernate, and the death rate reaches 52.2%, so that the supernate has good quick-acting property, is suitable for practical application scenes in which insect pests need to be quickly controlled, and has wide application prospects. And the death rate of the tea lesser leafhopper at 24 hours reaches 91.1%. In addition, it is further verified that monensin, natamycin and dehydrohirsutine in metabolites of the bacillus velezensis NM153 have remarkable technical effects on prevention and treatment of tea lesser leafhopper.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation and pest control, and particularly relates to application of a biological pesticide in control of Empoasca (E.) Vitis Gothe. BACKGROUND

[0002] Empoasca (E.) Vitis Gothe, also known as tea aphid, is one of the main pests on tea trees in China, and its distribution area is very wide. The adult and nymph of the tea aphid pierce and suck the juice of tender stems and leaves of tea trees with needle-like mouthparts. In the initial stage of tea tree damage, the growth of tea buds is slow, the new leaves change from green to yellow, the leaf veins turn red, and as the density of the tea aphid increases, the buds and leaves shrink, the leaf quality becomes hard, and in severe cases, the buds and new leaves are scorched and shed. The tea aphid has a great impact on the yield and quality of summer and autumn tea. Tea gardens in all parts of the country have the tea aphid, and in the past, chemical control was mainly used for the control of the tea aphid. Although a certain control can be achieved, the effect is not significant, and at the same time, it also brings many side effects, such as causing high pesticide residues in tea, affecting the quality of tea, causing the pests to become rampant again due to the development of pesticide resistance, causing environmental pollution, and destroying the ecological balance. CN 104073441 A discloses the use of Metarhizium anisopliae for the control of the tea aphid, but there are few reports on the use of biological pesticides for the control of the tea aphid in the prior art. Therefore, it is of great significance to find more new species of microorganisms that can control the tea aphid. SUMMARY

[0003] To solve the above technical problems, the application provides the following technical solutions:

[0004] The application discloses a biological pesticide, which is obtained by fermentation of Bacillus velezensis NM153, and contains monensin, natamycin and dehydrohirsutidine.

[0005] Preferably, the biological pesticide does not contain Bacillus velezensis NM153 bacteria.

[0006] Preferably, the mass ratio of monensin, natamycin and dehydrohirsutidine is 1-6:4-8:1-5.

[0007] Preferably, the total mass of monensin, natamycin and dehydrohirsutidine is greater than or equal to 1% of the mass of the biological pesticide.

[0008] A method for preparing a biological pesticide, characterized in that the preparation method of the biological pesticide is as follows:

[0009] (1) inoculating Bacillus velezensis NM153 in LB solid culture medium to activate, to obtain activated Bacillus velezensis NM153;

[0010] (2) inoculating the activated Bacillus velezensis NM153 in LB liquid culture medium to ferment, filtering to obtain the supernatant to obtain the biological pesticide.

[0011] Preferably, in step (1), the preparation method of the LB solid culture medium is: weighing 8-12 g / L of tryptone, 4-7 g / L of yeast extract, 13-18 g / L of agar powder and 8-12 g / L of sodium chloride, and autoclaving at 121 DEG C for 15-25 min.

[0012] Preferably, in step (2), the inoculation amount of Bacillus velezensis NM153 is 100-500 single colonies of Bacillus velezensis NM153 per 100 ml of LB liquid culture medium. The preparation method of the LB liquid culture medium is: weighing 8-12 g / L of tryptone, 4-7 g / L of yeast extract and 8-12 g / L of sodium chloride, and autoclaving at 121 DEG C for 15-25 min.

[0013] Preferably, in step (2), the fermentation conditions are: the fermentation temperature is 28-32 DEG C, the rotation speed is 160-250 r / min, and the culture time is 48-72 h.

[0014] The biological pesticide is applied to the prevention and control of Empoasca vitis.

[0015] The biological pesticide is applied to the preparation of a product for preventing and controlling Empoasca vitis.

[0016] The beneficial effects of the present application are:

[0017] The present application treats Empoasca vitis with the supernatant of Bacillus velezensis NM153, and the results show that a large number of Empoasca vitis can be observed to die after about 12 hours of treatment under the supernatant of Bacillus velezensis NM153, and the mortality rate reaches 52.2%, which shows that the supernatant has good quick-acting property and is suitable for practical application scenarios requiring rapid control of pests, and the mortality rate of Empoasca vitis reaches 91.1% at 24 hours. In addition, it is further verified that monensin, natamycin and dehydroharringtonine in the metabolic products of Bacillus velezensis NM153 have significant technical effects on the prevention and control of Empoasca vitis. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Colony map of Bacillus velezensis NM153.

[0019] Figure 2 Phylogenetic tree of Bacillus velezensis NM153. DETAILED DESCRIPTION

[0020] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0021] Example 1 Classification and identification of microorganisms

[0022] The species identification of strain NM153 was performed by using 16S rDNA identification technology. The 16S rRNA gene amplification was performed by using universal primers, and after the PCR amplification was completed, the PCR product was detected by electrophoresis using 1% agarose gel. The gene product obtained by PCR was sequenced, and the 16S rRNA gene sequence determination result of strain NM153 is shown as SEQ ID NO. 1, and the picture of single colony is shown as Figure 1 .

[0023] SEQ ID NO. 1

[0024]

[0025] The 16S rRNA gene sequence of strain NM153 was obtained by PCR amplification. The alignment result shows that strain NM153 is most similar to Bacillus velezensis, and the phylogenetic tree is as follows Figure 2 Therefore, strain NM153 is identified as Bacillus velezensis, and the strain is named Bacillus velezensis NM153, which is preserved in China Center for Type Culture Collection, Wuhan, China, with the strain preservation number CCTCC M2025605 and the preservation time of March 27, 2025.

[0026] Example 2 Preparation of biopesticide

[0027] A method for preparing a biopesticide, characterized in that the biopesticide preparation method is:

[0028] (1) inoculate Bacillus velezensis NM153 in LB solid medium to activate, and obtain activated Bacillus velezensis NM153; the preparation method of the LB solid medium is: weigh 8-12 g / L of tryptone, 4-7 g / L of yeast extract, 13-18 g / L of agar powder, 13-18 g / L of agar powder and 8-12 g / L of sodium chloride, and autoclave at 121°C for 15-25 min.

[0029] (2) inoculate the activated Bacillus velezensis NM153 in LB liquid medium for fermentation, filter the supernatant to obtain the biopesticide. The preparation method of the LB liquid medium is: weigh 8-12 g / L of tryptone, 4-7 g / L of yeast extract, and 8-12 g / L of sodium chloride, and autoclave at 121°C for 15-25 min. In step (2), the inoculation amount of Bacillus velezensis NM153 is 100-500 single colonies of Bacillus velezensis NM153 per 100 ml of LB liquid medium. In step (2), the fermentation conditions are: fermentation temperature is 28-32°C, 160-250 r / min, and culture for 48-72 h.

[0030] The concentration of Bacillus velezensis NM153 in the fermentation broth is 2.5-3.9 x 10 9cfu / ml. Metabolomics analysis found that the supernatant of the fermentation broth contained monensin, natamycin and dehydroxy-uncariae alkaloids, and the relative mass ratio of monensin, natamycin and dehydroxy-uncariae alkaloids was 1-6:4-8:1-5, and the total mass of monensin, natamycin and dehydroxy-uncariae alkaloids was greater than or equal to 1% in the mass fraction of biological pesticide quality. The CAS registry numbers of monensin, natamycin and dehydroxy-uncariae alkaloids are 17090-79-8; 7681-93-8 and 35467-43-7.

[0031] Example 3 Application of Bacillus velezensis NM153 bacterial suspension and supernatant

[0032] The experiment set up two treatment substances, Bacillus velezensis NM153 bacterial suspension (bacterial body itself without metabolites, 3.2 x 10 9 cfu / ml) and supernatant (metabolites without bacterial body); for concentration, three concentration gradients of stock solution, 100-fold dilution and 300-fold dilution were set; and at 12 hours and 24 hours after treatment, the experimental effects of each treatment group were observed and recorded respectively, to comprehensively evaluate the time-effect relationship.

[0033] Healthy, flat and pesticide-free tea leaves were selected, and the tea leaf surface and back were treated with LB diluent of bacterial suspension (volume ratio of bacterial suspension and LB liquid medium was 1:1) and supernatant respectively, each tea leaf was treated with 5ml volume and placed in a culture dish, and then starved for 8h and fed with fresh pesticide-free tea leaves, 30 tea small green leafhopper low-age larvae per group, repeated 3 times, and the number of deaths and survival numbers were checked after 12h and 24h respectively, and the death standard was that the insect body and appendages were lightly touched with a small brush, and any reaction was considered as death

[0034] 2. Experimental results

[0035] Table 1-2 results show that the supernatant of Bacillus velezensis NM153 fermentation broth has significant insecticidal activity on tea small green leafhopper, and its effect is much better than that of bacterial suspension treatment group, indicating that the main insecticidal effect comes from the metabolites secreted by the bacterial body rather than the bacterial body itself. A large number of tea small green leafhoppers can be observed to die at about 12 hours after treatment, with a mortality rate of 52.2%, showing that the supernatant has good quick-acting property and is suitable for practical application scenarios requiring rapid control of pests, and the mortality rate of tea small green leafhoppers reaches 91.1% at 24 hours.

[0036] To further confirm that the active ingredients (monensin, natamycin and dehydro-uncarine) in the metabolites are the key factors affecting the insecticidal effect, monensin, natamycin and dehydro-uncarine were mixed in a mass ratio of 1:4:3, then mixed with the bacterial suspension to obtain a stock solution (the total mass fraction of monensin, natamycin and dehydro-uncarine in the stock solution is greater than or equal to 1%), and then diluted 100 times and 300 times with LB, respectively. The verification test was carried out according to the method described above. The results are shown in Table 3. The supernatant of the fermentation broth containing monensin, natamycin and dehydro-uncarine of Bacillus velezensis NM153 achieved a death rate of 45.6% for E. amurensis in 12 hours and a death rate of 86.7% for E. amurensis in 24 hours. It can effectively inhibit E. amurensis and has the potential to be developed into a high-efficiency biological pesticide.

[0037] Table 1 Death rate of E. amurensis treated by supernatant of Bacillus velezensis NM153

[0038]

[0039]

[0040] Table 2 Death rate of E. amurensis treated by bacterial suspension of Bacillus velezensis NM153

[0041]

[0042] Table 3 Death rate of E. amurensis treated by monensin, natamycin and dehydro-uncarine

[0043]

[0044]

[0045] Note: In Tables 1-3, the number before the slash is the number of dead E. amurensis, and the number after the slash is the total number of E. amurensis.

Claims

1. A biopesticide, characterized by, The biological pesticide is obtained by filtering the supernatant after fermentation of Bacillus velezensis NM153, and contains monensin, natamycin and dehydroxyuncarine; the preservation number of the Bacillus velezensis NM153 is CCTCC M2025605.

2. The biopesticide according to claim 1, characterized in that, The biological pesticide does not contain Bacillus velezensis NM153 bacteria.

3. The biopesticide according to claim 2, characterized in that, The mass ratio of the monensin, natamycin and dehydroxyuncarine is 1-6:4-8:1-5.

4. The biopesticide according to claim 3, characterized in that, The total mass of the monensin, natamycin and dehydroxyuncarine is greater than or equal to 1% of the mass of the biological pesticide.

5. A method of preparing the biopesticide according to any one of claims 1 to 4, characterized in that, The preparation method of the biological pesticide is as follows: (1) inoculating Bacillus velezensis NM153 in LB solid culture medium to activate, to obtain activated Bacillus velezensis NM153; (2) inoculating the activated Bacillus velezensis NM153 in LB liquid culture medium for fermentation, and filtering the supernatant to obtain the biological pesticide.

6. The production method according to claim 5, wherein In step (1), the preparation method of the LB solid culture medium is as follows: weighing 8-12 g / L of tryptone, 4-7 g / L of yeast extract, 13-18 g / L of agar powder and 8-12 g / L of sodium chloride, and autoclaving at 121 DEG C for 15-25 min.

7. The production method according to claim 6, wherein In step (2), the inoculation amount of Bacillus velezensis NM153 is 100-500 single colonies of Bacillus velezensis NM153 per 100 ml of LB liquid culture medium; and the preparation method of the LB liquid culture medium is as follows: weighing 8-12 g / L of tryptone, 4-7 g / L of yeast extract and 8-12 g / L of sodium chloride, and autoclaving at 121 DEG C for 15-25 min.

8. The production method according to claim 7, characterized by, In step (2), the fermentation conditions are as follows: the fermentation temperature is 28-32 DEG C, the rotation speed is 160-250 r / min, and the culture time is 48-72 h.

9. The biological pesticide of any one of claims 1-4 or the biological pesticide obtained by the preparation method of any one of claims 5-8 is applied in the prevention and control of Empoasca flavescens.

10. The biological pesticide of any one of claims 1-4 or the biological pesticide obtained by the preparation method of any one of claims 5-8 is applied in the preparation of a product for preventing and controlling Empoasca flavescens.

Images