CN116622589B

CN116622589B - Bacillus bailii and application thereof in preventing and controlling fruit tree diseases

Info

Publication number: CN116622589B
Application number: CN202310793634.6A
Authority: CN
Inventors: 孙伟波; 刘凤权; 赵延存; 李朝辉; 明亮
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2023-06-30
Filing date: 2023-06-30
Publication date: 2024-05-24
Anticipated expiration: 2043-06-30
Also published as: CN116622589A

Abstract

The invention belongs to the field of biological control of plant diseases, and particularly discloses a bacillus beijerinus (Bacillus velezensis) strain LS21 and application thereof in the aspect of controlling fruit tree diseases; the strain is preserved in China general microbiological culture collection center (CGMCC) with the number of CGMCC No.24447; the strain and the fermentation liquor thereof have better indoor antagonistic activity on pathogenic fungi of apples and pear trees, and the biocontrol microbial inoculum prepared by the strain fermentation liquor can prevent and treat fungal diseases such as apple anthracnose, pear black spot and the like in the field; the strain and the product thereof are safe to human and livestock, are environment-friendly, can reduce the use of chemical pesticides in the field, ensure the ecological environment safety and the fruit safety, and are excellent biocontrol resources.

Description

Bacillus bailii and application thereof in preventing and controlling fruit tree diseases

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus beijerinckii and application thereof in fruit tree disease control.

Background

The fruit growing area and the yield of the fruits always stably occupy the 1 st world in the first large country of the world fruit industry, and the fruit growing area and the yield of the fruits always occupy the 1 st world in the first large country of the world fruit retail market with hundreds of billions. Citrus, apples and pears are the top three fruit types in the planting scale of China, wherein the area and the yield of apples occupy about half of the world, and the area and the yield of pears occupy more than two thirds of the world, so that the healthy development of fruit tree industry is significant for the fruit tree industry in China and the world. The occurrence of various diseases seriously affects the development of fruit tree industry and the improvement of fruit quality, and is a problem which is always concerned in production. The disease control in China mainly takes chemical control, and the long-term use of chemical pesticides brings problems of enhanced drug resistance, environmental pollution, high risk of food safety and the like, so that the development of environment-friendly pollution-free green prevention and control technology is urgently needed.

Biological control utilizes antagonism of microorganisms themselves or their metabolites to control diseases, and is an important component for green control. The biological control technology using bacillus as the main material is widely applied in China, and the currently registered biological pesticide strains mainly include bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and the like. Bacillus bailii (Bacillus velezensis) is a homonymous bacillus amyloliquefaciens, has the functions of promoting plant growth and resisting pathogenic microorganisms in agricultural production, shows broader antibacterial activity, and is a microorganism with huge potential for developing biological pesticides. The use of bacillus berryis in the presently disclosed patent or article for controlling fruit tree diseases has been reported for controlling diseases （CN 111100820 B、CN 112553129 B、CN 112980721 B、CN 109880764 B、CN 112493253 B、CN 108330089 B、CN 112322560 B、, such as apple ring rot, apple mildew heart disease, apple southern blight, apple rot, pear ring rot, pear rot, anthracnose of mango, strawberry root rot, banana wilt, etc., 36 (2) 220-230, 37 (1) 172-177, 37 (1) 165-171.

Prepared from fruit-borne sclerotinia fraseri

Colletotrichum fructicola

) The caused anthracnose of apples and pear is an important disease of the two big fruit trees, and the anthracnose and early leaf-falling phenomena are caused. Since 2008, anthracnose is almost spread over yellow river hometown apple and pear producing areas in China, and spread to Shandong and south producing areas, so that the most serious disease occurs in Jiangsu Feng county, anhui Dangshan and other areas, and the disease incidence rate of some orchards is almost 100%. Pear black spot is one of the main diseases of pears, and serious damage is caused in Yangtze river basin and other places in China. At present, the bacillus belicus is used for preventing and controlling diseases such as anthracnose of apples and pears, and the like, and has been reported freshly.

Disclosure of Invention

Aiming at the problem of lack of green prevention and control products in fruit tree disease control, the invention provides bacillus beljalis with broad-spectrum antibacterial effect, which has better antagonism effect on various pathogenic bacteria on apples and pear trees, can make up the defect of disease control in the currently disclosed patent, and has wide application prospect in the technical field of biological pesticides.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

The biocontrol strain LS21 is obtained by separating and screening from rhizosphere soil of an orchard by adopting a dilution plating method, and the biocontrol strain LS21 has a strong antagonism effect on pathogenic fungi such as apple anthracnose pathogen, pear black spot pathogen and the like on a solid flat plate. LS21 was identified as Bacillus bailii (Bacillus velezensis) based on colony and fungus morphology observations, physiological and biochemical indicators, 16S and gyrA gene sequence measurements, and the like. The strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 24447 (address: north Star Xiyu No. 1,3 of the Korean area of Beijing, china academy of sciences microbiological study, post code 100101) at 25 days of 2022.

Colony characteristics of the strain on LB solid medium: round or irregular, white opaque, with folds. When observed under a microscope, the thallus is rod-shaped and can produce spores.

The invention provides a bacillus beijerinckii fermentation broth and a preparation method thereof; the preparation method of the fermentation broth comprises the following steps: and (3) selecting an activated LS21 strain on a solid culture medium, inoculating the strain to an LB liquid culture medium, culturing the strain at 28 ℃ and 180 rpm rotating speed for 24h, adjusting OD ₆₀₀ = 1.0, inoculating the strain to a soybean meal culture medium for fermentation culture according to the proportion of 1% by volume, and fermenting for 48-72 hours to obtain a fermentation broth. The yield of the spores (bacillus bailii Bacillus velezensisLS) in the fermentation broth is more than 5 multiplied by 10 ⁸ cfu/mL.

In order to prolong the survival time of thalli in the microbial inoculum and make the dosage form more stable, the storage time of the product is longer; the invention also provides a biocontrol microbial inoculum prepared from the bacillus beijerinckii fermentation broth and a preparation method thereof, wherein the preparation method of the biocontrol microbial inoculum comprises the following steps: and (3) regulating the pH value of the obtained fermentation liquor to 3.5-4, and adding preservative potassium sorbate, thickener xanthan gum, wetting agent glycerol and auxiliary alcohol ether glucoside AEG050, wherein the optimized contents are respectively 0.05%, 0.1%, 10% and 10%, so as to prepare a suspending agent product, namely the biocontrol microbial agent. The microbial inoculum product is stable at room temperature, and has no obvious sedimentation layering phenomenon.

The invention also provides application of the fermentation liquor of bacillus belicus in fruit tree disease control, and application of the biocontrol microbial inoculum prepared from the fermentation liquor of bacillus belicus in fruit tree disease control.

The application of the invention uses the strain as a basic material to prepare fermentation liquor and a biocontrol microbial inoculum, and the strain is used for preventing and controlling fungal diseases such as apple anthracnose, pear black spot and the like.

Further, the application specific method comprises the following steps: 7-10 days after flower falling of the fruit tree, adding water into the fermentation liquor to dilute the fermentation liquor by 50 times, spraying the fermentation liquor, and carrying out spraying on the fermentation liquor in time after raining according to weather and 15-20 days in the growing period until the fermentation liquor is finished before bagging.

Further, the application specific method comprises the following steps: 7-10 days after flower falling of the fruit tree, adding water into the biocontrol microbial inoculum to dilute the biocontrol microbial inoculum by 50 times, spraying the biocontrol microbial inoculum, and carrying out one-time supplementary spraying in time after raining according to weather and 15-20 days in the growing period until the bagging is finished.

The beneficial effects of the invention are as follows:

the bacillus belicus LS21 provided by the invention is a biocontrol microorganism resource with broad-spectrum antagonistic activity, has a good control effect on fruit tree diseases, can inhibit the occurrence of diseases such as anthracnose and black spot of an orchard, improves the microenvironment of the orchard, reduces the dosage of chemical pesticides, protects the ecological environment, improves the fruit quality, and has good social, economic and ecological benefits.

Drawings

FIG. 1 is a colony morphology of Bacillus belicus LS21 single cell grown on LB plates;

FIG. 2 is a graph showing the effect of Bacillus belicus LS21 on PDA plates against pathogenic fungi of fruit trees: wherein, the diagram A is an inoculation schematic diagram, pathogenic fungus blocks with the diameter of 5 mm are inoculated in the center of a PDA flat-plate culture dish with the diameter of 9 cm, 2 mu L LS21 fermentation liquid is connected on the upper side, the lower side and the left side of the dish, and fungus liquid is not connected on the right side of the dish, and is left as a CK blank control; FIG. B, C, D is a graph showing antagonistic effects after inoculation of apple anthracnose (Colletotrichum fructicola), pear anthracnose (Colletotrichum fructicola) and pear black spot (ALTERNARIA ALTERNATA) respectively and cultivation for 5 days;

FIG. 3 shows the inhibitory effect of Bacillus belicus LS21 fermentation broth on apple anthracnose silk growth in a room; wherein, the graph A is a CK control without fermentation broth, and the graph B, C, D is a hypha growth graph on a 100-fold, 50-fold and 10-fold fermentation broth dilution plate respectively;

FIG. 4 shows the inhibitory effect of Bacillus belicus LS21 inoculant product on pear anthracnose spot in a room; wherein CK is the disease spot treatment without spraying bacterial agent, 10 times diluted bacterial agent is sprayed before and after inoculation of bacteria, 10 times prevention and 10 times treatment are respectively carried out, and the disease spot growth result and the disease spot length are shown in the graph and the table.

Description of the embodiments

The present invention is further illustrated in the following drawings and detailed description, which are to be understood as being merely illustrative of the invention and not limiting the scope of the invention.

Example 1

The invention provides bacillus belicus with a fruit tree disease prevention and control effect, the strain code is LS21, the strain is separated from rhizosphere soil of a pear garden in Li water area of Nanjing city of Jiangsu province, biocontrol bacteria are separated and purified from water dilution of soil samples by adopting a dilution plate coating method, a plate antagonism experiment is used for screening high-activity antagonistic strain, and LS21 shows better antagonistic activity. LS21 was identified as Bacillus bailii (Bacillus velezensis) based on colony and fungus morphology observations, physiological and biochemical indicators, 16S and gyrA gene sequence measurements, and the like. The 16S gene sequence of bacillus belicus (Bacillus velezensis) strain LS21 is shown as SEQ ID No.01, and the gyrA gene sequence is shown as SEQ ID No. 02. The strain is preserved in China general microbiological culture Collection center with a preservation number of CGMCC NO. 24447. The form, the characteristics and the application effect of the strain are described by combining relevant experimental data.

SEQ ID NO.01：

CGGACGCGTGCTATAATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGCTCACAGA

SEQ ID NO.02：

CTGCTGCTGAGCGTTATCGTATCCCGGGCGCTTCCGGATGTGCGTGACGGTCTGAAGCCGGTTCACAGACGGATTTTGTACGCAATGAATGATTTAGGCATGACCAGTGACAAACCATATAAAAAATCTGCCCGTATCGTCGGTGAAGTTATCGGTAAGTACCACCCGCACGGTGACTCAGCGGTTTACGAATCAATGGTCAGAATGGCGCAGGATTTTAACTACCGCTACATGCTTGTTGACGGACACGGCAACTTCGGTTCGGTTGACGGCGACTCAGCGGCCGCGATGCGTTACACAGAAGCGAGAATGTCAAAAATCGCAATGGAAATTCTGCGTGACATTACGAAAGACACGATTGACTATCAAGATAACTATGACGGTTCAGAAAGAGAGCCTGCCGTCATGCCTTCGAGATTTCCGAATCTGCTCGTAAACGGGGCTGCCGGTATTGCGGTCGGAATGGCGACAAACATTCCCCCGCATCAGCTTGGGGAAGTCATTGAAGGCGTGCTTGCCGTAAGTGAGAATCCTGAGATTACAAACCAGGAGCTGATGGAATACATCCCGGGCCCGGATTTTCCGACTGCAGGTCAGATTTTGGGCCGGAGCGGCATCCGCAAGGCATATGAATCCGGACGGGGATCAATCACGATCCGGGCTAAGGCTGAAATCGAAGAGACTTCATCGGGAAAAGAAAGAATTATTGTCACGGAACTTCCTTATCAGGTGAACAAAGCGAGATTAATTGAAAAAATCGCGGATCTTGTCCGGGACAAAAAAATCGAAGGAATTACCGATCTGCGAGACGAATCCGACCGTAACGGAATGAGAATCGTCATTGAGATCCGCCGTGACGCCAATGCTCACGTCATTTTGAATAACCTGTACAAACAAACGGCCCTGCAGACGTCTTTCGGAATCAACCTGCTGGCGCTCGTTGACGAACAGCCAAAGGATAGCTGACACG

1. The solid culture conditions of the strain were as follows: LB solid medium composition: tryptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L, agar powder 15 g/L, adding water to constant volume, adjusting pH to 7.0, sterilizing with high pressure steam at 121deg.C for 20 min, cooling, and culturing strain.

2. The colony characteristics of LS21 on LB solid medium are shown in figure 1, after 2-3 days of growth, the colony is round or irregular, white and opaque, has folds and bulges in the center. The cells were observed under a microscope to have a short rod shape. The strain can produce spores by using spore staining solution for staining and observation.

3. Molecular characterization: PCR amplification and sequencing are carried out on 16S genes and gyrA genes of the strain by adopting a primer pair 27F/1492R and gyrA-F/gyrA-R, the obtained sequence is placed in NCBI for Blast comparison, bacillus benralis (Bacillus velezensis) with the highest sequence similarity is found, the similarity is more than 99%, and bacillus benralis is identified. The strain has been identified and confirmed by the China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms.

4. LS21 fermentation liquid preparation method comprises the following steps: the LS21 strain activated on the LB solid medium is selected and inoculated on the LB liquid medium, and is cultured on a constant temperature shaking table (28 ℃ C., 180 rpm rotating speed) for 24 h, and the OD ₆₀₀ =1.0 is regulated to obtain seed liquid. Inoculating soybean meal culture medium in a volume ratio of 1% for fermentation culture, and fermenting for 48-72 hours to obtain fermentation liquor. The bean pulp culture medium comprises 16 g/L, 6 g/L, 1.5 g/L, 0.75 g/L, 0.75 g/L and 5 mg/L of bean pulp, corn starch, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate and microelements; the microelements are mixed solution of zinc sulfate, ferrous sulfate, copper sulfate and manganese sulfate with the same concentration, and the total concentration is 5 mg/L. Under the substrate, the spore yield after LS21 fermentation is above 5X 10 ⁸ cfu/mL.

5. The fermentation broth is used for preparing the birth control microbial inoculum. The preparation process comprises the following steps: and (3) regulating the pH value of the LS21 fermentation liquid to 3.5-4 by using hydrochloric acid, adding preservative potassium sorbate, thickener xanthan gum, wetting agent glycerol and auxiliary agent alcohol ether glycoside AEG050, wherein the optimized contents are respectively 0.05%, 0.1%, 10% and 10%, and the prepared product is in the form of a suspending agent. The microbial inoculum product is stable at room temperature, and has no obvious sedimentation layering phenomenon.

The test and verification of the antagonistic pathogenic fungi effect of the strain are carried out by taking the prepared fermentation liquor sample or the biocontrol microbial inoculum as a material, so as to further illustrate the applicability of the strain material.

Example 2

Plate antagonistic activity assay of LS21 strain against pathogenic fungi.

The test site was in laboratory at plant protection institute of academy of agriculture, jiangsu province. The LS21 strain activated on the LB solid medium is selected and inoculated on the LB liquid medium, and is cultured on a constant temperature shaking table (28 ℃ C., 180 rpm rotating speed) for 24h, and the LS21 culture solution is obtained by adjusting OD ₆₀₀ = 1.0. Activating apple anthracnose, pear anthracnose and pear black spot germs stored in a laboratory on a PDA flat plate, growing round uniform colonies after 4-5 days, and punching holes from the edges by using a puncher of 5mm to obtain fungus blocks. Taking a PDA plate culture dish with the diameter of 9 cm, connecting pathogenic fungi blocks with the diameter of 5mm in the center of the dish, connecting 2 mu L LS21 culture solution on the upper side, the lower side and the left side of the dish, and leaving the culture solution on the right side as a CK blank. The inoculation scheme is shown as a in figure 2.

After inoculation, the petri dishes are placed in a constant temperature incubator at 28 ℃ for 5 days, photographed, and the growth radiuses of fungus blocks in the up, down, left and right directions are recorded to evaluate the antagonistic effect. Antagonistic effect (%) = (control group colony radius-treatment group colony radius)/control group colony radius×100%.

The antagonistic effect of LS21 strain on apple anthracnose (Colletotrichum fructicola), pear anthracnose (Colletotrichum fructicola) and pear black spot (ALTERNARIA ALTERNATA) is shown as B, C, D in figure 2. The statistical results of the antagonistic effect are shown in table 1. As can be seen from FIG. 2 and Table 1, LS21 strain shows remarkable antagonistic effect on the growth of pathogenic fungus hyphae on PDA plate medium, and according to the statistical result of colony radius, the average antagonistic effect of LS21 strain is 67.6% -70.4%, namely the hyphae growth inhibition rate is about two thirds.

TABLE 1 Flat plate antagonistic Activity of LS21 Strain against pathogenic fungi

Example 3

An indoor antagonism test of pathogenic fungi was performed using the fermentation broth prepared in example 1.

The test site was in laboratory at plant protection institute of academy of agriculture, jiangsu province. And activating the apple anthracnose pathogen, pear anthracnose pathogen and pear black spot pathogen stored indoors on a PDA flat plate, and growing a colony with uniform circles after 4-5 days. Taking LS21 strain fermentation liquor, centrifuging (5000 rpm,3 min), taking supernatant, filtering with a sterile filter, removing thallus and spore in the supernatant, respectively adding into PDA culture medium in molten and non-coagulated state at 10 times, 50 times and 100 times of dilution concentration, mixing uniformly, and pouring into a flat plate for coagulation. And (3) inoculating fungus blocks to the center of the PDA culture medium by using a 5mm puncher, and culturing in a constant temperature incubator at 28 ℃ for 4-5 days in a dark place. The treatment without fermentation broth was repeated 3 times as a control. The lesion diameter size was measured for each treatment group. The calculation formula is as follows: inhibition = (control colony diameter-treatment colony diameter)/control colony diameter x 100%, data statistics are shown in table 2, and inhibition effect of fermentation broth on apple anthracnose hypha growth indoors is shown in fig. 3.

The result shows that the sterile fermentation broth of LS21 strain has better antagonistic activity to the tested pathogenic fungi, the inhibition rate of 10-time diluent is more than 85%, and the inhibition rate of 50-time diluent is more than 75%. Indicating that metabolite components exhibiting antagonistic activity may be present in the sterile fermentation broth. There is potential for further investigation.

TABLE 2 indoor antagonistic Activity of sterile filtrate of LS21 fermentation broth against pathogenic fungi

Example 4

Indoor control experiments of pear anthracnose were performed using the biocontrol microbial inoculum product prepared in example 1.

The test site was in laboratory at plant protection institute of academy of agriculture, jiangsu province. And activating the pear anthracnose stored in a laboratory on a PDA (personal digital assistant) plate, growing a round uniform colony after 4-5 days, and punching holes from the edge by using a puncher of 5 mm to obtain fungus blocks. In the growing period of pear fruits, young pear leaves are collected in a pear garden in the academy of agricultural sciences of Jiangsu province, the variety is green crown, the tree age is 8 years, and the pear leaves are cleaned again for later use indoors with sterile water. Taking a plastic storage box, adding a small amount of sterile water, placing a pallet, placing blades on the pallet at a height of about 5 cm a away from the bottom of the box, needling both sides of the blades to create wounds, and moisturizing the stems with absorbent cotton. Inoculating fungus block to wound, and culturing in a 28 deg.C constant temperature incubator in dark place. The microbial inoculum products are divided into two treatments of prevention and treatment, namely spraying microbial inoculum before 24 h and spraying microbial inoculum after 24 h after fungus inoculation. The microbial inoculum is diluted 10 times and sprayed on the leaves. After culturing for 10 days, photographing, measuring leaf spot length, wherein CK is spot treatment without spraying a microbial inoculum.

The leaf spot control effect and data are shown in figure 4. The result shows that the LS21 biocontrol microbial inoculum product has better control effect on the anthracnose of pear leaves. The average length of the lesion without spraying the microbial inoculum is 12.8 mm, the average length of the lesion with the 10-time diluent for preventing treatment is 9.0 mm, and the average length of the lesion with the 10-time diluent for treating treatment is 7.7 mm. The bacterial agent treatment reduced the anthrax plaque length by 39.8% relative to the control.

Example 5

The biocontrol microbial agent product prepared in example 1 is used for apple anthracnose prevention and control experiments.

The test site is located in the Xuzhou city, fengxian and Shake river orchard. The apple variety is Fuji red, and the tree age is 11 years. 3 non-adjacent 5 multiplied by 5 apple tree blocks are selected in the orchard as test treatment groups for spraying LS21 biocontrol microbial inoculum products, and 3 blocks are selected as clear water control treatment. The spraying agent is the biocontrol microbial inoculum prepared in the example 1 diluted by 50 times of water, and the spraying is started from 15 days after flower falling and is continued until the spraying is carried out for 4 times before bagging. The clear water control zone did not spray the agent. At near maturity, apple tree leaves in the clear water control area have anthracnose, part of branches start to have leaf blight symptoms, and a small amount of fallen leaves are displayed. And randomly selecting 100 leaves from the apple tree in the pesticide test treatment area to count the size of anthracnose spots. The disease grade number and the disease index are counted according to the grading standards of 0,1, 3, 5, 7 and 9. Disease index = Σ (number of diseased leaves at each level×number of levels)/(total number of leaves investigated×9) ×100. Control effect= (control disease index-treatment disease index)/control disease index x 100%.

The test results are shown in Table 3. The results show that the prepared LS21 suspending agent can effectively reduce the incidence rate of anthracnose of apple leaves, and compared with the incidence rate of 81% of a control group, the incidence rate of a microbial inoculum treatment group is 36%. The data obtained by calculating the disease index show that the control effect of the microbial inoculum treatment group is 72.0%, so that the microbial inoculum treatment group has a certain control effect on apple anthracnose.

TABLE 3 prevention and treatment effects of LS21 suspension microbial inoculant products on apple anthracnose

Example 6

The biocontrol microbial agent product prepared in example 1 is used for a control test of pear anthracnose.

The test site is located in Dangshan garden in the city of Anhui province. The pear variety is Dangshan pear, and the tree age is 15 years. 3 non-adjacent 5 multiplied by 5 pear blocks are selected in the orchard as test treatment groups for spraying LS21 biocontrol microbial inoculum products, and 3 blocks are selected as clear water control treatment. The spraying agent is the biocontrol microbial inoculum prepared in the example 1 diluted by 50 times of water, and the spraying is started from 15 days after flower falling and is continued until the spraying is carried out for 4 times before bagging. The clear water control zone did not spray the agent. In the near maturity stage, the leaves of the pear tree in the clear water control area have anthracnose, and part of branches start to have fruits which show anthracnose round lesions and a small amount of fallen leaves. And randomly selecting 100 leaves from the pear tree in the pesticide test treatment area to count the size of anthracnose spots. The disease grade number and the disease index are counted according to the grading standards of 0, 1, 3, 5, 7 and 9. Disease index = Σ (number of diseased leaves at each level×number of levels)/(total number of leaves investigated×9) ×100. Control effect= (control disease index-treatment disease index)/control disease index x 100%.

The test results are shown in Table 4. The results show that the prepared LS21 suspending agent can effectively reduce the incidence rate of anthracnose of pear leaves, and compared with the incidence rate of 83% of a control group, the incidence rate of the bacterial agent treatment group is 29%. The data obtained by calculating the disease index show that the control effect of the microbial inoculum treatment group is 85.0%, so that the microbial inoculum product has a good control effect on pear anthracnose.

TABLE 4 prevention and treatment effects of LS21 suspension inoculant products on pear anthracnose

Example 7

The biocontrol microbial agent product prepared in example 1 was used for the control test of pear black spot.

The test site is located in Fengyuan fruit Cooperation in Jurong city of Jiangsu province. The pear cultivars are rich water and water. And (5) a trellis type cultivation mode and conventional management. 5 pear trees are selected as test treatment groups in each row in the pear garden, 3 rows of treatment groups are selected to spray LS21 biocontrol microbial inoculum, and 3 rows of spraying Shi Qingshui rows are selected as control treatment in each row. The spraying time of the agent is 15 days after flower falling, and the total spraying is 4 times before bagging, and the spraying agent is the spraying of the biocontrol microbial inoculum prepared in the example 1 diluted by 50 times of water. And (5) spraying again in time after rain. Two weeks before fruit harvest, 100 leaves were randomly selected for black spot disease spot size in each of the agent treatment group and the clear water control group. The disease grade number and the disease index are counted according to the grading standards of 0, 1,3, 5, 7 and 9. Disease index = Σ (number of diseased leaves at each level×number of levels)/(total number of leaves investigated×9) ×100. Control effect= (control disease index-treatment disease index)/control disease index x 100%.

The test results are shown in Table 5. The results show that the prepared LS21 suspending agent can effectively reduce the incidence rate of pear leaf black spot, and compared with the incidence rate of 73% of the control group, the incidence rate of the microbial inoculum treatment group is 14%. The data obtained by calculating the disease index show that the control effect of the microbial inoculum treatment group is 83.3%, so that the microbial inoculum product has a good control effect on pear black spot.

TABLE 5 prevention and treatment Effect of LS21 suspension inoculant product on pear black spot

The experiment shows that the bacillus belicus LS21, the fermentation liquid and the biocontrol microbial inoculum can effectively control main fungal diseases of fruit trees, have good control effects on leaf diseases such as apple anthracnose, pear black spot and the like, and have good application prospects in biological control of fruit tree diseases.

It should be noted that the foregoing merely illustrates the technical idea of the present invention and is not intended to limit the scope of the present invention, and that a person skilled in the art may make several improvements and modifications without departing from the principles of the present invention, which fall within the scope of the claims of the present invention.

Claims

1. The bacillus beleiensis strain is characterized in that the preservation name of the bacillus beleiensis strain is bacillus beleiensis Bacillus velezensis LS21, and the bacillus beleiensis strain is preserved in China general microbiological culture collection center (CGMCC) No. 24447, and the preservation date is 2022, 2 and 25; the preservation address is North Star Xili No. 1, 3 of the Korean area of Beijing, china academy of sciences of microorganisms.

2. Bacillus beleimeris according to claim 1, characterized in that the colony characteristics of the strain on LB solid medium: round or irregular, white and opaque, with folds, the thallus is rod-shaped, and can produce spores.

3. The fermentation broth of bacillus beljavensis according to claim 1 or 2.

4. A method for preparing a fermentation broth of bacillus belgium according to claim 3, comprising the steps of:

And (3) inoculating the bacillus belicus Bacillus velezensis LS strain Bacillus velezensis LS activated on the solid culture medium to an LB liquid culture medium, culturing 24 h at 28 ℃ and 180: 180 rpm, adjusting OD ₆₀₀ =1.0, inoculating the bacillus belicus Bacillus velezensis LS strain into a soybean meal culture medium in a volume ratio of 1% for fermentation culture, and fermenting for 48-72 hours to obtain a fermentation broth.

5. The method for preparing the fermentation broth of bacillus beljalis according to claim 4, wherein the bean pulp culture medium comprises bean pulp 16 g/L, corn starch 6 g/L, sodium chloride 1.5 g/L, magnesium sulfate 0.75 g/L, potassium dihydrogen phosphate 0.75 g/L and trace elements; the microelements are mixed solution of zinc sulfate, ferrous sulfate, copper sulfate and manganese sulfate with the same concentration, and the total concentration is 5 mg/L.

6. The method for producing a fermentation broth of Bacillus bailii according to claim 4, wherein the yield of Bacillus bailii Bacillus velezensis LS/mL is 5X 10/⁸ cfu/mL or more.

7. A biocontrol agent prepared from the fermentation broth of bacillus belgium according to claim 3.

8. The method for preparing a biocontrol agent prepared from a fermentation broth of bacillus beljalis according to claim 7, comprising the steps of: taking the fermentation liquor to adjust the pH value to 3.5-4, and adding preservative potassium sorbate, thickener xanthan gum, wetting agent glycerol and auxiliary alcohol ether glycoside AEG050 to prepare a biocontrol microbial agent; the preservative comprises 0.05% of potassium sorbate, 0.1% of thickening agent xanthan gum, 10% of wetting agent glycerin and 10% of auxiliary agent.

9. Use of a fermentation broth of bacillus beljalis according to claim 3 for the control of fruit tree diseases, including apple anthracnose, pear black spot; the application specifically comprises the following steps: 7-10 days after the flowers of the fruit trees fall, adding water into the fermentation liquor to dilute the fermentation liquor for 50 times, then spraying the fermentation liquor on the leaf surfaces, spraying the fermentation liquor once in 15-20 days according to weather conditions, and then spraying the fermentation liquor in time after raining until the fermentation liquor is finished before bagging.

10. The application of the biocontrol microbial inoculum prepared by the fermentation broth of bacillus belicus according to claim 7 in the prevention and treatment of fruit tree diseases, wherein the fruit tree diseases comprise apple anthracnose, pear anthracnose and pear black spot; the application specifically comprises the following steps: 7-10 days after flower falling of the fruit tree, adding water into the biocontrol microbial inoculum to dilute the biocontrol microbial inoculum by 50 times, then spraying the biocontrol microbial inoculum on leaf surfaces, spraying the biocontrol microbial inoculum once in 15-20 days according to weather conditions, and then spraying the biocontrol microbial inoculum in time after raining until the biocontrol microbial inoculum is finished before bagging.