1. A biocontrol strain is Dm97 with the preservation number of GDMCC No. 61459.

2. A microbial inoculant comprising the biocontrol strain of claim 1; or prepared by fermentation culture of the biocontrol strain of claim 1.

3. The microbial agent according to claim 2, wherein the microbial agent is prepared by mixing wet bacteria and a bacteria preservation solution according to the ratio of 1: 20-60 g/mL.

4. The microbial agent according to claim 3, wherein the total concentration of viable bacteria in the microbial agent is 5 x 10⁹～1×10¹⁰CFU/mL。

5. A method for culturing biocontrol strains is characterized by comprising the following steps: inoculating the biocontrol strain Dm97 as defined in claim 1 into a culture medium for culture, after single colony grows out, selecting the single colony to be inoculated into a culture solution, and performing shake culture to prepare a seed solution; inoculating the seed liquid into the culture liquid for shake cultivation to obtain a culture bacterial liquid of the biocontrol strain Dm 97.

6. The method for culturing biocontrol strain as claimed in claim 5, wherein the formulation of said culture medium or culture solution comprises: 10g/L of sodium chloride, 10g/L of tryptone and 5g/L of yeast extract powder, and the pH value is adjusted to 7.2.

7. The biocontrol strain as defined in claim 1 or the microbial agent as defined in any one of claims 2-4 for preventing and treating diseases of and/or promoting growth of Fritillaria thunbergii.

8. The use of claim 7, wherein the disease of Fritillaria thunbergii comprises soft rot of Fritillaria thunbergii.

9. A method for preventing and treating diseases of Fritillaria thunbergii and/or promoting growth of Fritillaria thunbergii, which is characterized in that the biocontrol strain as claimed in claim 1 or the microbial agent as claimed in any one of claims 2-4 is applied by irrigation when mother seedlings of Fritillaria thunbergii are transplanted.

10. The method for preventing and treating diseases and/or promoting growth of fritillaria thunbergii according to claim 9, wherein the step of applying the microbial agent comprises: diluting the microbial agent, adopting soil treatment in the early stage, and adopting root treatment in the later stage.

Background

The fritillary bulb is a traditional Chinese medicine of fritillary, is a bulk common traditional Chinese medicine used as both medicine and food, has the effects of reducing phlegm and relieving cough, clearing heat and resolving masses, and takes alkaloid as a main component for reducing phlegm and relieving cough. Zhejiang fritillaria is suitable for growing in hilly shadow or under bamboo forest at lower altitude, and mainly produced in south Jiangsu, north Zhejiang and Hunan provinces.

Main diseases such as gray mold, black spot, root rot, soft rot and the like easily occur to thunberg fritillary bulb in the growth process, and are increasingly serious along with continuous cropping. Soft rot of thunberg fritillary bulb is a soil-borne disease, and is caused by Erwinia carotovora, damaged parts of diseased bulbs begin to be brown water stain-shaped and spread quickly, and the damaged bulbs become bean curd residue shaped like vinasse or become sticky and smooth nasal discharge; sometimes the hazard ceases and the surface loses water and becomes a cavity that is insect-bite-like. The boundaries of rotten and healthy parts are evident. The epidermis is not damaged, the inner part is soft, rotten and dried, and empty shells are left, and the rotten bulbs have special wine sour taste. At present, various bactericides and mite-killing bang are mainly used for preventing and treating seeds before seeding. The method comprises the following steps of soaking seeds for 10-15 minutes by using a mixed solution of 800 times of 20% wettable trichlorfon, 2000 times of 80% dichlorvos emulsion and 1000 times of 40% kewensan emulsion before planting. However, chemical control causes pollution to soil and water resources, and pesticide residues also cause harm to the health of people and livestock.

Based on the above-mentioned drawbacks of chemical control, there is an urgent need for an effective, safe and reliable biological control method for controlling these diseases. In agricultural measures, rotation crop and stubble rotation are effective, particularly paddy-upland rotation crop, the bacterial load in soil can be effectively reduced, and the occurrence of diseases is reduced, but certain difficulties exist in practical application. Compared with other methods, the biological control method has the characteristics of safety, effectiveness and durability, and particularly avoids a series of problems caused by chemical control. The biocontrol agent has good disease control effect, is nontoxic to human and livestock, does not pollute the environment and has no residue; the killing specificity to plant diseases and insect pests is strong, natural enemies are not damaged, beneficial organisms are obtained, and ecological balance can be kept; the production raw materials and the active ingredients are natural products which are easy to degrade and can return to the nature, thereby ensuring sustainable development; the microorganism can be modified by biotechnology and genetic engineering; various factors and components play roles, and pathogenic bacteria are difficult to generate drug resistance. Therefore, the biocontrol microbial inoculum for preventing and treating soft rot of thunberg fritillary bulb is a promising method for preventing and treating the soft rot of thunberg fritillary bulb, but at present, the biocontrol microbial inoculum capable of effectively preventing and treating the soft rot of thunberg fritillary bulb is still lacking.

Disclosure of Invention

In order to overcome the defects in the prior art, the biocontrol strain Dm97 is separated and screened, the microbial agent prepared by the strain can effectively prevent soft rot of Fritillaria thunbergii and promote the growth of Fritillaria thunbergii, and the microbial agent and agricultural cultivation measures are combined for use, so that the biocontrol strain Dm97 can exert the prevention and control potential of Fritillaria thunbergii as much as possible.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a biocontrol strain, which is Dm97 (Bacillus) and is classified and named as Bacillus sp, wherein the preservation number is GDMCC No. 61459, the preservation date is 2021, 01 and 20 days, the preservation unit is Guangdong province microorganism culture preservation center (GDMCC), and the preservation unit address is Guangzhou city Mr. 100, large institute No. 59, building 5, Guangdong province microorganism research institute.

The second aspect of the invention provides a microbial agent, which comprises the biocontrol strain Dm97 or is prepared by fermenting and culturing the biocontrol strain Dm 97.

Further, the microbial agent adopts wet bacteria and a bacteria preservation solution according to the ratio of 1: 20-60 g/mL. Wherein the wet bacteria are culture bacteria liquid of the prepared biocontrol strain Dm 97; the thallus preserving liquid comprises one or more of corn meal, bean cake powder and bean cake powder, one or more of glucose, sucrose and maltose, fish meal and mineral nutrient elements. Specifically, the formula of the bacteria preservation solution comprises 5-10 g of one or more of corn flour, bean cake powder and soybean meal powder, 3-6 g of one or more of glucose, sucrose and maltose, 2-8 g of fish meal, 0.1-0.5 g of monopotassium phosphate, 0.1-0.5 g of ferrous sulfate, 1-5 g of magnesium sulfate, 0.1-0.5 g of manganese sulfate and 0.1-0.5 g of calcium carbonate, and the balance is 1L. Furthermore, the preparation concentration of the wet bacteria and the bacteria preservation solution is 1: 40 g/mL.

Furthermore, the total concentration of viable bacteria in the microbial agent is 5 multiplied by 10⁹～1×10¹⁰CFU/mL。

Further, the microbial agent can be stored at room temperature for 2-3 years.

Further, the microbial agent also comprises an agriculturally and pharmaceutically acceptable auxiliary agent, including but not limited to: dispersants, stabilizers, carriers, and the like. The formulation of the microbial agents include, but are not limited to: liquid preparations, solid preparations (such as powders), and the like.

Further, the microbial agent is applied after dilution, the dilution multiple is 50-1000 times, and the concentration after dilution is 1 multiplied by 10⁷～9×10⁷CFU/mL。

The third aspect of the present invention provides a method for culturing a biocontrol strain, which comprises the steps of: inoculating the biocontrol strain Dm97 into a culture medium for culture, after single bacteria grow out, selecting the single bacteria colony to be inoculated into a culture solution, and performing shake culture to prepare a seed solution; inoculating the seed liquid into the culture liquid for shake cultivation to obtain a culture bacterial liquid of the biocontrol strain Dm 97.

Further, the formula of the culture medium or the culture solution comprises: 10g/L of sodium chloride, 10g/L of tryptone and 5g/L of yeast extract powder, and the pH value is adjusted to 7.2. Specifically, the formula is an LB formula.

Further, the culture method comprises the following specific steps: inoculating a biocontrol strain Dm97 into an LB culture medium, streaking on a flat plate, culturing for 12-18h in a biochemical incubator at 25-32 ℃, picking a single colony to be inoculated into an LB culture solution after the single colony grows out, and culturing for 20-30 h in a shaking table at 25-32 ℃ and 150-250 r/min to prepare a seed solution; and inoculating the seed solution into an LB culture solution, and culturing for 36-60 h in a shaking table at the temperature of 25-32 ℃ and at the speed of 150-250 r/min to obtain a culture bacterial solution of the biocontrol strain Dm 97. Further, inoculating the single colony cultured for 14-16h into a test tube containing 5mL of LB culture solution, and culturing in a shaking table at 28 ℃ and 200r/min for 25h to prepare a seed solution; inoculating the seed solution into a triangular flask filled with 500mL of LB culture solution at the inoculation amount of 1 wt%, and culturing for 48h in a shaking table at 28 ℃ and 200r/min to obtain a culture bacterial solution of a biocontrol strain Dm 97.

Further, the culture bacterial liquid and the thallus preservation liquid are adopted according to the ratio of 1: 20-60 g/mL of microbial agent with the total viable bacteria concentration of 5 multiplied by 10⁹～1×10¹⁰CFU/mL. Further, the microbial agent is diluted to 1 × 10⁷～9×10⁷CFU/mL for application.

The fourth aspect of the invention provides an application of biocontrol strain Dm97, the microbial agent prepared by fermenting and culturing the biocontrol strain Dm97 or the biocontrol strain Dm97 in controlling the disease of Fritillaria thunbergii and/or promoting the growth of Fritillaria thunbergii.

Further, the disease of the thunberg fritillary bulb comprises soft rot of the thunberg fritillary bulb.

The fifth aspect of the invention provides a method for preventing and treating diseases of fritillaria thunbergii and/or promoting the growth of fritillaria thunbergii, which adopts the biocontrol strain Dm97, comprises the biocontrol strain Dm97 or a microbial agent prepared by fermentation culture of the biocontrol strain Dm97 and is applied by irrigation when the mother seedlings of fritillaria thunbergii are transplanted.

Further, the step of administering the microbial agent comprises: diluting the microbial agent, and adopting soil treatment in the early stage and root treatment in the later stage.

Further, the microbial agent is diluted by the following steps: the adopted microbial agent comprises a culture bacterial liquid and a thallus preservation liquid of an anti-microbial strain Dm97, and can also comprise an auxiliary agent acceptable in the agricultural pharmacy, including but not limited to: dispersants, stabilizers, carriers, and the like; diluting the microbial agent with clear water, mixing, and diluting to 1 × 10⁷～9×10⁷CFU/mL (preferably 5X 10)⁷CFU/mL)。

Further, the frequency and amount of administration are: diluting the microbial inoculum by 100 times to prepare applied bacterial liquid, spraying 2L of the applied bacterial liquid per mu of soil before transplanting on a seedbed, treating roots on the same day as transplanting, and treating 15d, 30d and 75d in a root irrigation manner, wherein 1L of the applied bacterial liquid per mu is applied each time.

The sixth aspect of the invention provides a method for separating and screening a biocontrol strain Dm97, which comprises the following steps: the rhizosphere soil bacteria of the diseased plants are separated according to a dilution plate coating method. And sequentially coating 100 mu L of diluent on an NA culture medium, repeating 3 gradients each, culturing at 30 ℃ for 24-48 h, observing and selecting single colonies with different morphological characteristics, purifying and storing in a refrigerator at 4 ℃ for later use. And screening the separated and purified strains on an LB culture medium by adopting a point grafting method, and selecting the strains with obvious inhibition zones through primary screening and secondary screening.

Compared with the prior art, the invention has the following beneficial effects by adopting the technical scheme:

the biocontrol strain Dm97 screened by the invention is a biological preparation developed for the disease of the thunberg fritillary bulb, in particular to the soft rot of the thunberg fritillary bulb, and belongs to a biological preparation, so that a series of problems caused by the use of chemical pesticides are completely avoided, the pollution-free production of the thunberg fritillary bulb is facilitated, the use amount of other chemical pesticides is not needed or reduced, the planting cost is reduced, and the development of the breeding industry of the thunberg fritillary bulb is facilitated. According to field experiments, the microbial agent prepared from the biocontrol strain Dm97 can prevent soft rot through soil treatment in the early stage, can better control the soft rot through root treatment in the later stage, can effectively control infection of the soft rot on Fritillaria thunbergii to achieve the effect of controlling the occurrence of the illness state, and particularly, the Dm97 microbial agent has the effects of preventing and controlling the soft rot of Fritillaria thunbergii by more than 90 percent, promoting the growth of disease-free plants by more than 70 percent and increasing the yield of Fritillaria thunbergii by more than 50 percent, which shows that the Dm97 microbial agent has good effects of preventing diseases and increasing the yield, and has great potential to be developed into a commercial viable bacteria biocontrol agent.

Drawings

FIG. 1 shows the control effect of the microbial inoculum Dm97 on soft rot of Fritillaria thunbergii in a field test (330 days after sowing) in one embodiment of the invention; wherein, A is blank control, and B is treated by microbial inoculum Dm 97.

FIG. 2 shows the growth promoting effect of the microbial inoculum Dm97 on Fritillaria thunbergii in a field test (145 days after sowing); wherein, A is blank control, and B is treated by microbial inoculum Dm 97.

The bio-control strain-Bacillus Dm97 is preserved and named as Bacillus sp, and has a preservation number of GDMCC No. 61459, a preservation date of 2021, 01-20 days, a preservation unit of Guangdong province microbial culture Collection (GDMCC), and a preservation unit address of Guangzhou city Mr. 100 college No. 59 building, 5 building, Guangdong province microbial research institute.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental methods of the following examples, which are not specified under specific conditions, are generally determined according to national standards. The experimental materials not shown in the following examples are all commercially available materials. The equipment used in the steps in the following examples is conventional. If there is no corresponding national standard, it is carried out according to the usual international standards, to the conventional conditions or to the conditions recommended by the manufacturer. Unless otherwise indicated, all parts are parts by weight and all percentages are percentages by mass. Unless defined or indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention.

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.

Example 1 isolation screening and identification of biocontrol Strain Dm97

The embodiment relates to a screening and identifying process of a biological control strain Dm97 (Bacillus sp), which specifically comprises the following steps:

(1) screening of biocontrol strain Dm97

The strain source is as follows: dm97 is separated from the soil surrounding the root of Nantong Zhejiang fritillaria;

the strain separation method comprises the following steps: adopting a plate dilution method: 6 tubes each containing 9mL of water were sterilized and pressed from 10 to 10⁶The order of (1) is numbered, 1mL of the cultured bacteria solution is sucked from a liquid transfer test tube, the bacteria solution is injected into a test tube diluted by 10 times, the rubber on the liquid transfer tube is lightly pressed by fingers, the air suction is carried out for three times, the bacteria solution is fully and uniformly mixed with the water, 1mL of the diluted solution is sucked from the test tube diluted by 10 times, and the diluted solution is injected into the test tube diluted by 10 times²In the double diluted tubes, the second mixing step was repeated. And so on until the dilution of the last test tube is completed.

Immersing the spreader in a beaker containing alcohol, taking a small amount of diluted bacteria liquid (not more than 0.1mL, specifically 100 muL) and dripping the diluted bacteria liquid on the surface of the NA culture medium, igniting the spreader stained with a small amount of alcohol on flame, and cooling for 8-10 s after the alcohol is burnt out. The bacterial liquid is evenly coated on the surface of the NA culture medium by using a coater, and the culture dish can be rotated during coating, so that the bacterial liquid is evenly distributed. Repeating each gradient for 3 times, culturing at 30 ℃ for 24-48 h, observing and selecting single colonies with different morphological characteristics, purifying and storing in a refrigerator at 4 ℃ for later use; and screening the separated and purified strains on an LB culture medium by adopting a point grafting method, and selecting the strains with obvious inhibition zones through primary screening and secondary screening.

(2) Identification of biocontrol strain Dm 97-molecular biology identification

The sequence of the 16S rRNA coding gene fragment of the prokaryote is identified by using a sequencing method, and the amplification and the sequence analysis of the 16S rRNA coding gene are specifically as follows: culturing the strain in LB culture medium at 28 ℃ to logarithmic phase, centrifuging at 12000r/min for 5min to collect thallus, extracting bacterial genome DNA by using a genome DNA rapid extraction kit of Shanghai Saibaosheng Gene technology Limited, taking the extracted DNA product as a template, and adopting the following bacterial 16S rRNA coding gene amplification universal primers:

Farword：U8-27(5’-AGAGTTTGATC(AC)TGGCTCAG-3’)

Reserve：L1494-1514(5’-CTACGG(AG)TACCTTGTTACGAC-3’)

the 16S rRNA-encoding gene fragment was amplified using the genomic DNA as a template. After purifying the PCR product, the PCR product is sent to Shanghai Bioengineering Co., Ltd for sequencing. And (3) carrying out homology comparison on the sequencing result through NCBI BLAST software, and finally identifying the strain Dm97 as Bacillus (Bacillus sp.) and preserving the strain, wherein the preservation numbers are as follows: GDMCC No. 61459.

Example 2 cultivation of biocontrol Strain Dm97 and preparation of its inoculum

This embodiment is a preferable cultivation method of the biocontrol strain Dm97 obtained by screening in example 1 and a method for preparing the microbial inoculum thereof, and the method includes the following steps:

(1) culture of biocontrol strain Dm97

Inoculating biocontrol strain Dm97 to LB culture medium (LB formula: sodium chloride 10g/L, tryptone 10g/L, yeast extract 5g/L, adjusting pH to 7.2). Streaking on a plate, culturing in a biochemical incubator at 28 ℃ for 14-16h, after single colony grows out, selecting the single colony, inoculating into a test tube containing 5mL of LB culture solution, and culturing in a shaking table at 28 ℃ and 200r/min for 25h to prepare seed solution; inoculating the seed solution into a triangular flask filled with 500mL of LB culture solution with the inoculation amount of 1%, and culturing for 48h in a shaking table at 28 ℃ and 200r/min to obtain the culture bacterial liquid of the prepared biocontrol strain Dm 97.

(2) Preparation of microbial agent

The microbial agent adopts the culture bacterial liquid and the thallus preservation liquid of the biocontrol strain Dm97 prepared in the step (1) according to the ratio of 1: 40(g/mL), and can be stored at room temperature for 2-3 years. The formula of the thallus preservation solution comprises 5-10 g of one or more of corn meal, bean cake powder and bean pulp powder, 3-6 g of one or more of glucose, sucrose and maltose, 2-8 g of fish meal, 0.1-0.5 g of monopotassium phosphate, 0.1-0.5 g of ferrous sulfate, 1-5 g of magnesium sulfate, 0.1-0.5 g of manganese sulfate and 0.1-0.5 g of calcium carbonate, and the balance is 1L. The total viable bacteria concentration of the above microbial preparation is 5 × 10⁹～1×10¹⁰CFU/m. The microbial inoculum can be diluted by 50-1000 times (the dilution concentration is 1 multiplied by 10)⁷～9×10⁷CFU/mL) before transplanting of mother seedling of Bulbus Fritillariae ThunbergiiAnd (3) irrigating and applying (namely irrigating roots after diluting the strain preparation while transplanting crops).

Example 3 use of microbial Agents prepared from biocontrol Strain Dm97

Example 3 the microbial inoculum prepared from biocontrol strain Dm97 prepared in example 2 was diluted to 5X 10 with clear water⁷Shaking uniformly after CFU/mL, and verifying the aspect of controlling the diseases of the thunberg fritillary bulb, wherein the method comprises the following specific steps:

(1) disease prevention test in field (Zhejiang fritillaria soft rot)

The field experiment is carried out in the professional cooperative society for planting traditional Chinese medicinal materials in east city of Hanyan county, Hainan city, south China, Jiangsu province, and the plant to be tested is thunberg fritillary bulb (Fritillaria thunbergii Miq.).

The experimental treatment comprises the following steps: and (4) preparing a diluent of the microbial agent by the biocontrol strain Dm97 and comparing with clear water. The treatment method comprises the following steps: the transplanting day is treated, 15d, 30d and 75d are treated in a root irrigation mode, and each time is 1L/mu. Each processing 3 repetitions, each cell area 24m²(6m 4m), the row spacing is 20cm, and the plant spacing is 10 cm. 1200 thunberg fritillary bulb in each cell.

The disease grade standard of soft rot disease:

level 0: no lesion spots are found on the whole bulb surface;

level 1: the lesion area of the whole bulb is less than or equal to 5%;

and 2, stage: the area of the whole bulb lesion spots is 5 to 20 percent;

and 3, level: the area of the whole bulb lesion spot is 20 to 50 percent;

4, level: the whole lesion area of the bulb is 50 to 70 percent

And 5, stage: the lesion area of the whole bulb is more than 70 percent.

Disease severity (%). 100 x Σ (diseased plant number x disease progression)/(total plant number x highest disease progression)

Disease prevention effect (%) < 100% × (control disease severity-treatment disease severity)/control disease severity.

The results of the field disease prevention test are shown in the following table 1 and figure 1:

TABLE 1 prevention and treatment of soft rot of Fritillaria thunbergii by Middling fungicide Dm97 in the field (330 days after sowing)

Note: values are mean ± sem, with different letters indicating significant variability between treatments at a significant level of P ═ 0.05.

The results shown in the table show that the control effect of the Dm97 microbial inoculum on soft rot of Fritillaria thunbergii reaches 92.58%, and the Dm97 microbial inoculum can well control the soft rot of Fritillaria thunbergii by combining the graph 1.

(2) Growth promoting and yield increasing effect for field

The field experiment is carried out in the professional cooperative society for planting traditional Chinese medicinal materials in east city of Hanyan county, Hainan city, south China, Jiangsu province, and the plant to be tested is thunberg fritillary bulb (Fritillaria thunbergii Miq.).

The experimental treatment comprises the following steps: and (4) preparing a diluent of the microbial agent by the biocontrol strain Dm97 and comparing with clear water. The treatment method comprises the following steps: the transplanting day is treated, 15d, 30d and 75d are treated in a root irrigation mode, and each time is 1L/mu. Each processing 3 repetitions, each cell area 24m²(6m 4m), the row spacing is 20cm, and the plant spacing is 10 cm. 1200 thunberg fritillary bulb in each cell. The growth indexes such as leaf number, plant height and the like are counted in the growth period respectively, the yield is counted after harvesting, and specific growth promoting results are shown in the following table 2, table 3 and figure 2.

TABLE 2 root growth promoting effect of Nantong Zhejiang fritillaria bulb (145 days after sowing)

TABLE 3 growth promoting effect of the fungicide Dm97 in the field (145 days after sowing)

Note: values are mean ± sem, with different letters indicating significant variability between treatments at a significant level of P ═ 0.05.

As can be seen from the above table, the Dm97 microbial inoculum has 75.78% of root system growth promoting effect, the overground part growth promoting effect is 10% -20%, the conventional yield is 530 kg/mu, the field treatment yield is 800 kg/mu, the yield increasing rate is 50.94%, and the Dm97 microbial inoculum has excellent yield increasing effect and great potential to be developed into a commercial viable bacteria biocontrol agent by combining figure 2.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.