CN114774290A - Beauveria bassiana microbial preparation and application thereof - Google Patents

Abstract

The application discloses a beauveria bassiana microbial preparation and application thereof. The beauveria bassiana microbial preparation comprises beauveria bassiana QJBb181 and/or beauveria bassiana QJBb181 active substances. The microbial preparation of beauveria bassiana has a killing effect on cutworms, grubs, spodoptera exigua, prodenia litura and wireworms, can effectively prevent and alleviate the occurrence of underground pests, has a promoting effect on the growth of crops such as tobacco seedlings and the like, and plays a role in preventing dead seedlings from dying.

Description

Beauveria bassiana microbial preparation and application thereof

Technical Field

The application relates to the technical field of microbial preparations, in particular to a beauveria bassiana microbial preparation and application thereof.

Background

The underground pests refer to pests living in soil and damaging roots, stems close to soil surface and other parts of plants, and mainly comprise mole cricket, grub, cutworm, wireworm, maggot and the like. The pests have various types and harm hosts. For example, eating the underground tissue of the seedling, such as the young root, the subterranean stem, etc., of the seedling, so that the seedling cannot come out of the soil or the whole seedling withers; underground pests such as cutworms and the like also mainly eat roots, stems, root tubers, seedlings, growing points and the like of crops, and often cause seedling shortage, ridge breaking or poor plant growth. Is a big problem in the crop planting industry.

At present, the control of underground pests is mainly based on chemical agents, such as chlorpyrifos, phoxim and the like, which can quickly and effectively kill grubs. Although the pesticide has the advantage of good quick-acting performance, the long-term use of the chemical pesticide has the following hazards or hidden dangers: firstly, the pesticide can kill and poison the pests, and simultaneously has a killing effect on beneficial insects and beneficial birds such as 'natural enemies' of the pests and pollinating insects, and the ecological balance of the natural world can be destroyed in the past. Secondly, the chemical pesticide is used in a large amount, so that the pesticide is gradually accumulated in the environment, particularly in the soil, and the problem of pesticide pollution to the environment is caused. Thirdly, the pesticide is absorbed by crops, enters the bodies of animals, and is subjected to biological enrichment and concentration to enable the pesticide to have higher toxicity, so that not only are natural enemies of pests more susceptible to toxic action, but also human health is threatened through a food chain; pesticide residues can also directly affect food safety and affect the production and trade of crops; fourthly, the chemical pesticide is used in large quantities for a long time, so that the pests easily generate extremely strong resistance, the pest control effect is influenced, and the vicious circle that the dosage of the chemical pesticide is increased continuously is involved. Currently, prevention of pesticide contamination has become a concern in many countries of the world today, and chemical pesticide abuse can create more serious and profound damage than insect damage.

In order to reduce the use of chemical pesticides, related studies have shown that microorganisms can be used for biological control of pests; however, the existing biocontrol agents capable of conveniently and effectively killing various underground pests are quite lacking. The vigorous development and development of microbial pesticides, particularly biocontrol agents based on filamentous fungi are imperative, and are an effective way for solving the problems (namely resistance, rampant re-growth and residue) of chemical pesticides 3R.

Disclosure of Invention

The application aims to provide a novel beauveria bassiana microbial preparation and application thereof.

The following technical scheme is adopted in the application:

one aspect of the present application discloses a beauveria bassiana microbial preparation, which includes beauveria bassiana QJBb181 and/or beauveria bassiana QJBb181 active substance.

The Beauveria bassiana (Beauveria bassiana) is obtained by separating from the cutworm and is marked as Beauveria bassiana QJBb 181; the method has the advantages of high production efficiency of liquid-solid two-phase fermentation and large spore yield, the content of conidia can reach 1000 hundred million/g, and the conidia has strong infection activity on various underground insects such as cutworms, grubs, spodoptera exigua, prodenia litura, wireworms and the like. Therefore, the beauveria bassiana microbial preparation for controlling the underground pests, which is creatively prepared by using the beauveria bassiana QJBb181 and/or the beauveria bassiana QJBb181 active substances, can effectively control the underground pests in the growth process of the roots of plants.

It should be noted that the inventors isolated three strains of fungus for controlling soil insects with different functions from the microbial flora of cutworm, identified the three strains of fungus by morphological and molecular biology respectively, and determined that the three strains of fungus are metarhizium anisopliae QJMa143, beauveria bassiana QJBb181, and metarhizium bleekii QJMr101, respectively. The application mainly protects beauveria bassiana QJBb181, and other two fungus strains are protected in other patent applications respectively.

In one implementation of the present application, the soil insects that the beauveria bassiana microbial preparation can control mainly include at least one of cutworms, grubs, spodoptera exigua, spodoptera litura and wireworms.

It can be understood that the beauveria bassiana QJBb181 is isolated from the cutworm and necessarily has a good killing effect on the cutworm; in an implementation mode of the application, experiments are carried out in a field with serious flammulina velutipes, and the beauveria bassiana QJBb181 has a good killing effect on the flammulina velutipes. In addition, the pesticide also has a killing effect on several soil insects with serious harm, such as grub, spodoptera litura and the like.

In one implementation of the present application, the beauveria bassiana QJBb181 active substance is a conidium and/or microsclerotia fermentation product of beauveria bassiana QJBb 181.

It is understood that the beauveria bassiana QJBb181 active substance means a substance having the same effect as beauveria bassiana QJBb181 after administration, such as conidia, microsclerotia fermentation product, and the like.

In one implementation of the present application, the beauveria bassiana microbial preparation further comprises a fermentation substrate.

The beauveria bassiana microbial preparation of the application has the main insecticidal active ingredient of beauveria bassiana QJBb181 and/or beauveria bassiana QJBb181 active substances; however, for better insecticidal effect, the microbial preparation of the present application contains not only the insecticidal active ingredient but also other nutritional ingredients, such as fermentation substrate; on one hand, the function of trapping and killing underground pests is achieved; on the other hand, the fungal hyphae can also be planted and symbiotic in roots of crops such as tobacco, so that the yield and quality of the crops are improved, and the economic benefit of the crops is improved. In an implementation of this application, adopt the beauveria bassiana microbial preparation of this application to carry out the insecticidal, can also promote the tobacco seedling to grow, increase of production.

In one implementation of the present application, the fermentation substrate is at least one of corn starch, wheat bran, and rice flour.

In one implementation manner of the application, the mass ratio of the beauveria bassiana QJBb181 and/or beauveria bassiana QJBb181 active substances to the fermentation substrate in the beauveria bassiana microbial preparation is 1: 100-200.

It should be noted that, although, the microbial preparation of beauveria bassiana of the present application can be used for various underground pest killing; however, it is not excluded that the microbial preparation of beauveria bassiana of the present application can also be used in combination with other biological or chemical drugs as long as the pesticidal activity of the microbial preparation itself is not affected. In addition, the microbial preparation of beauveria bassiana of the present application can also be made into various dosage forms according to product requirements, such as wettable powder, powder or granules, and the like, as long as corresponding auxiliary components are added, and the preparation is not specifically limited herein.

Another aspect of the application discloses the use of a microbial preparation of beauveria bassiana of the application in the preparation of a preparation for promoting the growth of tobacco seedlings.

In a further aspect, the application discloses the application of the microbial preparation of beauveria bassiana in the preparation of the preparation for preventing the death of the dead seedlings of the tobacco seedlings.

The research of the application shows that the microbial preparation of beauveria bassiana can kill various underground pests, promote the growth of tobacco seedlings and prevent dead seedlings from dying; therefore, the beauveria bassiana microbial preparation can be prepared into a preparation for promoting the growth of tobacco seedlings or a preparation for preventing the death of the tobacco seedlings.

In another aspect, the application discloses a method for controlling underground pests, which comprises the steps of mixing the beauveria bassiana microbial preparation with an organic fertilizer or fine soil to prepare a mixture, and applying the mixture to crops at the bottom of a upturned soil breaking period or a ridging period before transplanting to play a role in controlling the underground pests.

In one implementation of the present application, the weight ratio of the beauveria bassiana microbial preparation to the organic fertilizer or the fine soil of the mixture is 1: 10.

The beneficial effect of this application lies in:

the beauveria bassiana microbial preparation has a killing effect on cutworms, grubs, spodoptera exigua, prodenia litura and wireworms, can effectively prevent and alleviate underground pests, has a promoting effect on the growth of crops such as tobacco seedlings and the like, and plays a role in preventing dead seedlings from dying.

The beauveria bassiana QJBb181 is stored in China General Microbiological Culture Collection Center (CGMCC) at 2021, 05 and 17 months, with the preservation number: CGMCC No. 22425. The address of the preservation unit is Beijing of China.

Detailed Description

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Examples

First, strain isolation

The spore powder on the surface of the stiff insects was dipped with an inoculating needle under sterile conditions and streaked onto 1/4SDAY plate medium 90mm in diameter, or the stiff insects were gently and quickly shaken on the medium with sterilized forceps. The plate was placed in a 26 ℃ incubator for dark culture. After 3 days, colony edge hyphal tips on the picking medium were transferred to a new 90mm 1/4SDAY plate medium. Repeating the operation for 2-3 times to obtain the purified beauveria bassiana (B. The purified strain was cultured in dark in a constant temperature incubator at 26 ℃ for 2 weeks, and short-term storage and long-term storage with 20% glycerol freezing were performed, respectively.

II, identifying strains

1. Morphological characterization

After a fungus strain QJBb181 is cultured on an 1/4SDAY plate medium at a constant temperature of 25 ℃ for 14d, the morphological characteristics of colonies are observed, and the morphological characteristics of hyphae and spores of the strain are observed by a microscope, and the results are shown in Table 1.

TABLE 1 identifying results of the morphological characteristics of Beauveria bassiana

Species of Strain	Beauveria bassiana QJBb181
		Color of colony	White colour
Colony morphology	Cotton-like
		Matrix pigments	White with slight yellow color
Color and luster of spore	White colour
		Hyphal morphology	Branch division
Spore form	Most part is spherical
		Spore size	2～2.5×1.8～2.8μm

Molecular characterization of ITS sequences of QJBb181 Strain

Shaking and culturing QJBb181 strain in 1/4SDA liquid culture medium for 72h (250rpm, 26 ℃), collecting mycelium or thallus with four layers of sterilized filter cloth, grinding with liquid nitrogen into white powder, and extracting total DNA of strain with DNA kit. Using the total DNA as a template, PCR was performed using universal primers (TW81 and AB28) for amplifying the fungal ITS sequences, and the ITS sequences of the strains were amplified and sequenced by Shanghai Biometrics. The sequence was Blast-aligned with known sequences in the NCBI database to find out that homology was higher than 98% and E value of 0.0 was regarded as the same species.

Wherein, the universal primer TW81 is the sequence shown in Seq ID No.1, and AB28 is the sequence shown in Seq ID No. 2.

Seq ID No.1：5’-gtttccgtagctgaacctgc-3’

Seq ID No.2：5’-atatgcttaagttcagcgggt-3’

According to the biological culture characteristics of the QJBb181 strain and the comparison result of ITS sequences, the QJBb181 strain is determined to be Beauveria bassiana (Beauveria bassiana) and is marked as Beauveria bassiana QJBb 181.

Determination of insecticidal Activity of Beauveria bassiana QJBb181

In this example, the insecticidal activity of the obtained beauveria bassiana QJBb181 was measured as follows:

collecting Beauveria bassiana QJBb181 strain, culturing mature conidia at 26 deg.C in 1/4SDAY medium, and preparing into 1 × 10 with sterile water containing 0.5% Tween-80⁸Soaking 3-year-old golden insects in spore/mL spore suspension by using a micro dropper, wherein each end of the golden insects is 20 mu L, and the treatment time is 30-40 seconds; 3 treatments were set up, with 10 replicates. After inoculation, the strain is raised at the room temperature of 25 ℃ and the humidity of 50-60%, and a non-inoculated 0.5% Tween-80 solution is used as a blank control. The death was checked 1 time every 24h and 12d of continuous observation recorded the number of dead insects.

Indoor bioassay shows that: the beauveria bassiana QJBb181 strain has high toxicity to the larvae of the golden insects, the cumulative corrected mortality rate reaches 91.5%, and LT50 obtained by a probability value analysis method is 6.53 d.

Four, producing spore powder by Beauveria bassiana QJBb181 liquid-solid phase fermentation

Beauveria bassiana QJBb181 strain is inoculated on solid 1/4SDAY culture medium and cultured in dark in a constant temperature incubator at 26 ℃ for 2 weeks. Respectively scraping mature conidia under aseptic condition, and preparing into 1 × 10 in 0.1% Tween-80 sterile water⁸～1×10⁹20mL of spore/mL suspension is inoculated into 200mL of 1/4SDA liquid culture medium in a 1L triangular flask, the temperature is kept at 25-27 ℃, the rotation speed is 250rpm, and the suspension is subjected to shaking culture for 72 hours to serve as liquid seed liquid.

The solid phase fermentation material is rice, the rice is uniformly soaked according to the weight ratio of 2:1 of the rice to water, the soaked rice is filled into a 50 x 40 polyethylene fermentation bag according to the filling amount of 300g per bag after the rice is soaked for 1 hour, the opening of a ventilation bag is tightly tied by a thread rope, the bag is filled into a sterilization pot, and the sterilization is carried out for 30min at the temperature of 121 ℃. Naturally cooling on a clean bench, and inoculating liquid mother bacteria according to the inoculation ratio of 1:10 (V/W). And (3) after mixing, placing the mixed solution on a culture rack at 25-27 ℃, culturing for 12-15 days, collecting the culture, and drying the culture indoors. And collecting spore powder by using a vibrating screen with 200 meshes. And weighing the collected conidium powder, wherein the fermentation yield of the QJBb181 strain is 0.6-0.7%. And counting by a blood counting plate method to obtain that the number of live spores of each gram of spore powder of the QJBb181 strain is 1000 hundred million/g, and the heating decrement is 8-10%.

Production of microsclerotia by inducing fermentation of beauveria bassiana

The induced fermentation process parameters of the strain are determined by optimizing conditions such as a fermentation formula, the optimum pH, the temperature, the oxygen demand, the illumination and the like, 300L-3000L fermentation of the liquid fermentation of the microbial sclerotium of the strain is carried out, and the microbial sclerotium fermentation process flow of the beauveria bassiana is formulated:

cutting a 2 cm-sized fungus cake from a QJBb181 strain mature conidium plate, aseptically inoculating the fungus cake into 150mL of induction culture solution in a 1L triangular shake flask, keeping the temperature at 25-27 ℃, and culturing for 56h to obtain the strain liquid of the mother fungus in the triangular flask. Aseptically inoculating into a 30L seeding tank, adopting 1/4SDA liquid culture medium until the amount of seed liquid required by the culture of the fermentation tank and the canning coefficient: 80 percent.

Induction fermentation parameters: pot pressure 0.2cm²The ventilation volume is 1:1 according to v/v/min, the tank temperature is 27 +/-1 ℃, the culture time is 2-3d, the hypha quantity is large, the bacterial liquid becomes thick, a large number of spores are formed, and the detection is free from foreign bacteria pollution and can be used as a primary inoculum.

Transferring the qualified first-stage inoculum to a 300L fermentation tank according to the ratio of 1:10, adjusting the tank pressure and air inlet and outlet valves, starting stirring, and performing induced liquid culture.

Culture parameters: stirring speed of 200rpm, tank temperature of 27 +/-1 ℃ and tank pressure of 0.5kg/cm²The ventilation volume is 1: 0.5-0.7 in terms of v/v/min. Culturing for 48-72 h. Sampling inspection every 8h in the culture protocolOnce.

And (4) fermenting the liquid until the 6 th day, and stopping fermentation when 60-80% of mycelia are converted into microsclerotia. Discharging the fermentation liquor into a sterilized plastic barrel, and adding dried diatomite or other fillers according to the proportion of 1: 2-4. The filler is mixed by temperature-controlled vacuum drying, so that the water content of the microsclerotia and the rice flour is lower than 13%.

Preparation of bio-control fungicide of beauveria bassiana

The fermented beauveria bassiana strain dry spore powder and the fermentation substrate are crushed together and then added with auxiliary materials, or the microbore fermented in one step is added with diatomite according to the ratio of 1:3(V/W) to prepare beauveria bassiana powder with the effective content of more than or equal to 10 hundred million spores/g, fermented materials (grains) can be directly used as microbial preparations according to requirements, and various dosage forms such as powder, wettable powder or granules can be prepared according to product requirements as long as corresponding auxiliary components are added.

Seven, field experiment of beauveria bassiana biocontrol microbial inoculum for preventing and controlling cutworms in tobacco fields

1. Materials and methods

Test reagents: beauveria bassiana powder and local conventional treatment agent. The underground pests are spodoptera frugiperda.

Experiment design: 15 cells of 10 x 10m are randomly selected, the interval between every two cells is more than 2m, the mutual interference between the cells is prevented, and the cultivation management conditions between all the test cells are basically kept consistent. Selecting a tobacco curing field where tobacco flammulina velutipes occur all year round, setting 5 treatments for 3 times of repetition and 15 cells in total, uniformly mixing the beauveria bassiana powder and clear water in the tobacco field from a valve crushing period to a ridging period, then spraying the mixture with fertilizer, and synchronously applying local conventional treatment agents and rooting water in a transplanting link.

Processing one: 1 kg/mu of beauveria bassiana powder;

and (5) processing: beauveria bassiana powder, 2.5 kg/mu;

and (3) treatment III: beauveria bassiana powder, 5 kg/mu;

and (4) treatment: the drug control CK1 is adopted in the example, chlorpyrifos is adopted as the drug control, and the dosage is 2.7 kg/mu;

and (5) processing: blank control CK2, clear water control, i.e. no drug was administered.

Test investigation: investigating the tobacco seedlings once in 3 days, 5 days and 10 days after transplanting, investigating the number of the broken seedlings, the number of the damaged plants and the insect quantity in each cell for three times, and calculating the damaged plant rate and the control effect according to the formula (1) and the formula (2). Meanwhile, according to the pest damage symptoms of tobacco seedlings and the investigation condition of field pests, the number of the pest plants and the types of the pests are recorded.

Formula (1):

formula (2):

2. results and analysis

The statistical results of the investigation of the damage conditions of underground pests 3 days, 5 days and 10 days after the tobacco seedlings are transplanted in the beauveria bassiana powder treatment area, the conventional agent treatment area and the control area are shown in table 2.

TABLE 2 investigation statistical table of green prevention and control effect of beauveria bassiana underground pests with different treatment doses

In table 2, the values listed are mean ± sem (n: 172). The data were statistically analyzed by Student Newman Keuls (S-N-K) and the difference between the lower case letters and the upper case letters was significant at 0.05 and 0.01.

The investigation result shows that the underground pests in the test area are black cutworms. 3 days after the tobacco seedlings in the clear water control area are transplanted, a large number of seedling breakage and tobacco plant damage situations occur, 10 days after the transplanting, the seedling breakage rate of the blank control area is 10.72%, the damaged plant rate is 15.42%, the insect plant rate is 18.75%, and the quantity of the insect plants in the whole plant is 22.50.

The damage of underground pests after the conventional medicament treatment is obviously lighter than that of a control, and no obvious aggravation trend is generated over time. 10 days after transplanting, the pest rate of the treatment area is about 15.83 percent, the pest amount of the treatment area is about 19.58 heads, and the pest amount of the treatment area is not obviously different from that of the control area, but the seedling breakage rate of 2.08 percent and the pest rate of 3.33 percent are both obviously lower than that of the control (P is 0.01), and the final control effect reaches 78.38 percent.

After the beauveria bassiana powder is treated by different dosages, the damage degree of underground pests is obviously lower than that of a clear water control area, and the seedling breakage rates of beauveria bassiana preparations with different dosages of 1kg, 2.5kg and 5 kg/mu are respectively 3.33%, 2.08% and 1.67% 10 days after transplanting; the rates of damaged plants are respectively 7.5%, 3.33% and 2.92%; the pest rates are respectively 8.75%, 5.42% and 5.00%; the quantity of the hundred plants is respectively 10.42, 6.25 and 5.83, which are all obviously lower than that of a control (P is 0.01), wherein the dosage of 2.5 kg/mu and 5 kg/mu of beauveria bassiana powder has the best control effect on underground pests, the control effect after 10 days of transplantation is 78.38 percent and 81.08 percent respectively, and the control effect is equivalent to that of the conventional medicament treatment (P is 0.01).

Test results show that in areas with serious underground pests, the underground pests can be effectively prevented and controlled by conventional medicament treatment and beauveria bassiana treatment of 1 kg/mu, 2.5 kg/mu and 5 kg/mu, the seedling breakage rate is reduced, and the seedling protection rate is improved. The treatment during the upturned soil crushing period or ridging of the beauveria bassiana can effectively reduce seedling shortage and death in the field, and a good control effect is achieved on underground pests in the former period, wherein the prevention and control effects on the underground pests are optimal when 2.5 kg/mu and 5 kg/mu of the beauveria bassiana granules are treated, and the seedling breakage rate is 2.08-1.67% after 10 days; the final control effect reaches 81.08 percent. The control effect is similar to that of the conventional medicament, has no adverse effect on the growth of tobacco seedlings, and has obvious effect of promoting the growth of the tobacco seedlings. Therefore, the beauveria bassiana powder is recommended to be applied in a strip mode in a valve breaking period or a ridging period to carry out green prevention and control on underground pests, and the recommended dosage is 2.5 kg/mu.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

Claims (10)

1. A microbial preparation of beauveria bassiana is characterized in that: the beauveria bassiana microbial preparation comprises beauveria bassiana QJBb181 and/or beauveria bassiana QJBb181 active substances.

2. The microbial preparation of beauveria bassiana according to claim 1, characterized in that: the underground pests comprise at least one of cutworms, grubs, spodoptera exigua, spodoptera litura and wireworms;

preferably, the underground pests are wireworms and/or cutworms.

3. The microbial preparation of beauveria bassiana according to claim 1, wherein: the beauveria bassiana QJBb181 active substance is conidium and/or microsclerotia fermentation product of beauveria bassiana QJBb 181.

4. A microbial preparation of beauveria bassiana according to any one of claims 1 to 3, characterized in that: the beauveria bassiana microbial preparation further comprises a fermentation substrate.

5. The microbial preparation of beauveria bassiana according to claim 4, characterized in that: the fermentation substrate is at least one of corn starch, wheat bran and rice flour.

6. The microbial preparation of beauveria bassiana according to claim 4, characterized in that: the mass ratio of the beauveria bassiana QJBb181 and/or beauveria bassiana QJBb181 active substances to the fermentation substrate in the beauveria bassiana microbial preparation is 1: 100-200.

7. Use of a microbial preparation of beauveria bassiana according to any one of claims 1 to 6 in the manufacture of a preparation for promoting the growth of tobacco seedlings.

8. Use of a microbial preparation of beauveria bassiana according to any one of claims 1-6 in the preparation of a preparation for preventing death of tobacco seedlings.

9. A method for controlling underground pests, which is characterized by comprising the following steps: the beauveria bassiana microbial preparation of any one of claims 1 to 6 is mixed with an organic fertilizer or fine soil to prepare a mixture, and the mixture is applied to crops in a upturned furrow breaking period or ridging period before transplanting to play a role in controlling underground pests.

10. The control method according to claim 9, characterized in that: in the mixture, the weight ratio of the beauveria bassiana microbial preparation to the organic fertilizer or the fine soil is 1: 10.