CN112195142A

CN112195142A - Biocontrol bacillus beleisi ZHX-7 and application thereof

Info

Publication number: CN112195142A
Application number: CN202011286285.1A
Authority: CN
Inventors: 张霞; 郭志青; 迟玉成; 许曼琳; 于静; 李莹; 宋新颖; 何康
Original assignee: Shandong Peanut Research Institute
Current assignee: Shandong Peanut Research Institute
Priority date: 2020-11-17
Filing date: 2020-11-17
Publication date: 2021-01-08
Anticipated expiration: 2040-11-17
Also published as: CN112195142B

Abstract

The invention belongs to the field of bacterial strains and application thereof, and particularly relates to biocontrol bacillus beilesiensis ZHX-7 with the preservation number of CGMCC No. 20374. The Bacillus beleisi ZHX-7 has obvious inhibition effect on peanut pathogenic bacteria, can be used as a potential biocontrol strain, applies the strain ZHX-7 to peanut disease control, improves the resistance of peanuts to crown rot, southern blight, net blotch, ring spot, root rot and peanut sheath blight, and can remarkably promote the growth and yield of the peanuts. Tests prove that the strain ZHX-7 can also inhibit the contents of peanut aspergillus flavus and toxins thereof. The application method of the strain ZHX-7 belongs to the field of biological control, is safe, quick and effective, is beneficial to green and healthy production of peanuts, and provides technical support for comprehensive control of peanut diseases.

Description

Biocontrol bacillus beleisi ZHX-7 and application thereof

Technical Field

The invention belongs to the technical field of biology, particularly relates to a strain and an application field thereof, and particularly relates to separation and identification of Bacillus belgii with broad-spectrum resistance to various plant pathogenic bacteria and an application of the Bacillus belgii in promotion of peanut growth and prevention and control of peanut pathogenic fungi.

Background

Peanuts are important oil crops and economic crops in China and are one of crops commonly planted in China. However, with the annual increase of peanut diseases, the disease becomes a main obstacle for further development of peanuts in China. Because of the wide variety of peanut soil-borne disease pathogenic bacteria and the difference of pathogenic bacteria in different areas, diseases are always the difficult point of prevention and control in peanut production, especially the peanut crown rot, the peanut southern blight, the peanut root rot and the like which are more serious in recent years occur in most peanut producing areas in China. In the last decade, due to the change of cultivation system, the large-area popularization of high-yield new varieties and the change of climatic conditions, the peanut diseases are aggravated year by year, the healthy production of peanuts in China is seriously influenced, and the peanut diseases become important factors for restricting the yield and the quality of the peanuts.

In recent years, in China, due to the lack of continuous cropping and disease-resistant varieties, pathogenic bacteria are accumulated year by year, so that the occurrence of diseases is more serious, the peanut cultivation method is limited by the current peanut industry structure and cultivation system, the chemical prevention is still mainly used for controlling underground peanut diseases and insect pests, however, the chemical pesticide is excessively depended on, so that soil, agricultural products, air, water quality and the like are polluted to different degrees, the healthy development of the peanut industry is seriously influenced, the ecological environment health and the food safety in China are threatened, and the method is also against the great promotion of pesticide reduction and synergy measures in China. Based on the consideration of human health and environmental problems, people are concerned about finding alternatives to chemical pesticides, and the use of microorganisms for controlling plant diseases is a good choice.

Disclosure of Invention

The invention aims to solve various problems in the aspect of peanut disease control by using chemical pesticides in the prior art, and provides biocontrol Bacillus belgii ZHX-7 and application thereof. The method belongs to the field of biological control, is safe, quick and effective, is beneficial to green and healthy production of peanuts, and provides technical support for comprehensive control of peanut diseases.

The technical scheme of the invention is as follows:

the biocontrol Bacillus beleisi ZHX-7 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 20374.

Further, the 16S rDNA sequence of the strain ZHX-7 is shown in SEQ ID NO: 1 is shown in the specification; the gyrB gene sequence is shown as SEQ ID NO: 2, respectively.

The biocontrol Bacillus belezii ZHX-7 is applied to prevention and control of peanut crown rot, peanut reticulum, peanut verticillium, peanut root rot, peanut southern blight and peanut sheath blight. Further, the application form is any one of bacterial suspension, sterile fermentation liquor, volatile gas and bacteria-containing fermentation liquor of the strain ZHX-7.

The biocontrol Bacillus beleisi ZHX-7 is applied to promoting the growth of peanuts.

The biocontrol Bacillus beleisi ZHX-7 can be used for inhibiting peanut aspergillus flavus and the toxin content thereof. Further, the application form is any one of bacterial suspension, sterile fermentation liquor and volatile gas of the strain ZHX-7.

Biological material sample preservation information:

the strain ZHX-7 is Bacillus velezensis (Bacillus velezensis), is preserved in China general microbiological culture Collection center (CGMCC), and has a preservation address as follows: china, West Lu No. 1 of Beijing Chaoyang district, No. 3, institute of microbiology, national academy of sciences, date of deposit: 16 days 07 and 16 in 2020, the preservation number is CGMCC No. 20374.

The invention has the beneficial effects that:

the Bacillus beleisi ZHX-7 provided by the invention has an obvious inhibiting effect on peanut pathogenic bacteria, can be used as a potential biocontrol strain, applies the strain ZHX-7 to peanut disease control, improves the resistance of peanuts to crown rot, southern blight, net blotch, ring spot, root rot and peanut sheath blight, and can remarkably promote the growth and yield of peanuts. Tests prove that the strain ZHX-7 can also inhibit the contents of peanut aspergillus flavus and toxins thereof. The application method of the strain ZHX-7 belongs to the field of biological control, is safe, quick and effective, is beneficial to green and healthy production of peanuts, and provides technical support for comprehensive control of peanut diseases.

Drawings

FIG. 1 is a colony morphology of strain ZHX-7 on LB medium;

FIG. 2 is a phylogenetic tree constructed by the strain ZHX-7 based on 16S rDNA and gyrB genes;

FIG. 3 shows antagonistic action of strain ZHX-7 on target pathogenic bacteria;

FIG. 4 is the determination of the bacteriostatic ability of the strain ZHX-7 fermentation liquid on 3 pathogenic bacteria;

FIG. 5 is the measurement of the bacteriostatic ability of the strain ZHX-7 volatile gas on 3 pathogenic bacteria;

FIG. 6 shows the inhibitory effect of the ZHX-7 strain on other peanut pathogens tested;

FIG. 7 shows the control effect of the strain ZHX-7 fermented liquid on peanut crown rot;

FIG. 8 is the effect of strain ZHX-7 on peanut growth;

FIG. 9 is the effect of strain ZHX-7 on the growth of Aspergillus flavus;

FIG. 10 shows the effect of suspension of strain ZHX-7 on the content of peanut aflatoxins.

Detailed Description

The technical solutions of the present invention will be described in detail and fully with reference to the following specific embodiments, and it should be understood 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.

For a further understanding of the present invention, reference will now be made in detail to the following examples.

Unless otherwise specified, experimental materials, reagents, instruments and the like used in the examples of the present invention are commercially available, and unless otherwise specified, technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

Separation and identification of Bacillus beleisi ZHX-7

1. Screening and isolation of the strain ZHX-7:

the sample is soil from the disease of peanut continuous cropping field, near the rhizosphere of healthy plant and at the depth of 6cm from the ground surface, and is stored at 4 ℃.

The method comprises the following steps: (1) weighing 10g of the soil sample, adding the soil sample into 90mL of sterile water, shaking at 28 ℃ and 140rpm for 15min, standing for 5min, taking supernatant, and sequentially diluting to 10 times by adopting a 10-time series dilution method^-2、10^-3、10^-4And 10^-5The suspension was applied to LB solid medium at 100. mu.L/concentration, and the suspension was incubated at 28 ℃ in the dark, 3 times for each concentration. (2) When bacterial colonies appear on the culture medium, a suspension of spores of coronaria arachidicola (Aspergillus niger) is sprayed. And after culturing for 2d, selecting bacterial colonies with obvious bacteriostatic zones around, picking single bacterial colonies by using a bacterium transferring ring, and performing purification culture on the LA solid culture medium. (3) Finally, selecting a strain with an inhibition zone of over 2cm, wherein the thallus is light yellow, the bacterial colony is opaque and irregular, the strain is named as ZHX-7, and the strain morphology chart is shown in figure 1. (4) After single bacterial colony of the strain ZHX-7 obtained by screening is shaken by an LB liquid culture medium, glycerol is added, and the mixture is preserved at the temperature of minus 80 ℃.

2. Strain identification of strain ZHX-7:

the method comprises the following steps: (1) according to the kit "TRANSGEN" for extracting bacterial genomes

The Bacteria Genomic DNA Kit "extracted the Genomic DNA of ZHX-7.

(2) The genome of the strain ZHX-7 was PCR-amplified using 16S rDNA universal primers 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') (SEQ ID NO: 3) and 1492R (5 '-GGYTACCTTGTTACGACTT-3') (SEQ ID NO: 4), gyrB gene primers F (5'-GAAGTCATCATGACCGTTCTGC-3') (SEQ ID NO: 5) and R (5'-AGCAGGGTACGGATGTGCGAGCC-3') (SEQ ID NO: 6), respectively, and the PCR products were sent to Producer organism (Shanghai) Co., Ltd for sequencing. The full length of the 16S rDNA sequence is 1394bp, and the sequence is shown as SEQ ID NO: 1 is shown in the specification; the total length of the gyrB gene sequence is 1079bp, and the sequence is shown as SEQ ID NO: 2, respectively.

(3) The obtained sequences were submitted to GenBank database for BLAST analysis alignment and phylogenetic trees were constructed using MEGA6.06 software in combination with the 16S rDNA sequence and the gyrB gene sequence (see fig. 2). The result shows that the similarity of the strain ZHX-7 and the Bacillus velezensis CP023320 reaches 95 percent, which indicates that the strain ZHX-7 is the Bacillus velezensis.

Example 2

Inhibition of growth of mycelium of peanut pathogenic bacteria by using strain ZHX-7

1. Inhibition effect of strain ZHX-7 bacterial suspension on growth of 3 peanut pathogenic bacteria hypha

(1) Peanut crown rot bacteria: picking single colony of strain ZHX-7 with toothpick, placing into a triangular flask containing 20mLLB culture medium, performing shake culture at 28 deg.C for 14 hr to obtain product with OD concentration₄₂₀Bacterial suspension equal to 0.3. Sucking 10 mu L of bacterial suspension, inoculating the bacterial suspension to the center of a PDA solid culture medium with the diameter of 9cm, and after the bacterial suspension is fully absorbed, culturing at the constant temperature of 28 ℃ for 24 hours. Uniformly spraying OD into PDA culture medium₄₂₀A spore suspension of coronafia arachidii No. 0.3, as a control without spore spray. Culturing at 25 deg.C for 4 days, counting the size of zone of inhibition, and taking pictures (see FIG. 3A, B). FIG. 3A is the control group of crown rot fungus of peanut, and FIG. 3B is the pair of crown rot fungus of peanut by strain ZHX-7The confrontation effect is achieved; the result shows that the strain ZHX-7 has obvious antagonistic activity on the peanut crown rot bacteria, and the size of a bacteriostatic zone is 24.17 +/-0.98 mm.

(2) Peanut latticed bacteria: preparation of the Strain ZHX-7 at OD concentration₄₂₀Bacterial suspension 0.3, ready for use. A bacterial cake of pathogenic bacteria with a diameter of 5mm is inoculated into the center of the plate, 10 mu L of bacterial suspension of the strain ZHX-7 is inoculated at the position of about 22mm on both sides of the bacterial cake, and the non-inoculated bacterial suspension is used as a control. Carrying out constant-temperature dark culture at 25 ℃ for about 5 days, counting the bacteriostasis condition, and calculating the bacteriostasis rate (%) (the growth diameter of a control colony-the growth diameter of a treated colony)/the growth diameter of a control colony multiplied by 100. FIG. 3C shows the control group of peanut blotch germs, and FIG. 3D shows the antagonistic effect of the strain ZHX-7 on peanut blotch germs; as a result, the strain ZHX-7 has a remarkable bacteriostatic effect on the peanut reticulospora (see figure 3C, D), and the hypha growth inhibition rate is 52.13%.

(3) Alternaria alternata: preparation of the Strain ZHX-7 at OD concentration₄₂₀The bacterial suspension was prepared in a concentration OD of alternaria arachidicola (Beechia arachidicola)₄₂₀Spore suspension of 0.3 for use. 10. mu.L of spore suspension was aspirated and inoculated into the center of a 9 cm-diameter PDA solid medium at a distance of about 22mm from both sides of the center of the plate with 10. mu.L of each of the bacterial suspensions of strain ZHX-7, and the uninoculated bacterial suspension was used as a control. Carrying out constant-temperature dark culture at 25 ℃ for about 4d, counting the bacteriostasis, and calculating the bacteriostasis rate (%) (the diameter of a control colony-the diameter of a treated colony)/the diameter of the control colony multiplied by 100. FIG. 3E shows the control group of peanut verticillium dahliae, and FIG. 3F shows the effect of ZHX-7 on peanut verticillium dahliae; as a result, the strain ZHX-7 has a remarkable bacteriostatic effect on alternaria arachidicola (see figure 3E, F), and the hypha growth inhibition rate is 58.90%.

2. Inhibition effect of strain ZHX-7 sterile fermentation liquid on growth of 3 peanut pathogenic bacteria hypha

Picking out single colony of the strain ZHX-7 by using toothpick, putting the single colony into a triangular flask containing 100mL of LB culture medium, carrying out shake culture at 28 ℃ for 36h, carrying out high-speed centrifugation, collecting supernatant, and filtering by using a 0.22 mu m filter membrane to obtain sterile fermentation liquor. Mixing sterile fermentation broth with PDA culture medium at ratio of 1:10, cooling to about 50 deg.C, pouring into flat plate, and inoculating into straight plateThe diameter of the seed cake is 5mm, or 10 μ L spore suspension (crown rot or brown rot, OD)₄₂₀0.3), 6 replicates per treatment were used as controls, and the colony diameters of the peanut netspot pathogen, the peanut verticillium wilt pathogen, and the peanut crown rot pathogen were measured after 5d, and 7d, respectively, for each treatment, and the culture was performed at 25 ℃.

As a result, compared with the corresponding control, the sterile fermentation liquid of the strain ZHX-7 remarkably inhibits the growth of hypha of peanut crown rot, peanut netspot and peanut verticillium (see figure 4), and the hypha growth inhibition rates are 14.87%, 27.13% and 32.21% respectively. In FIG. 4, A represents the bacteriostatic ability of the fermentation broth of the strain ZHX-7 against peanut crown rot pathogen, B represents the inhibition against peanut net blotch pathogen, and C represents the inhibition against peanut verticillium; indicates that at p <0.01 levels, the difference was very significant.

3. Inhibition effect of strain ZHX-7 volatile gas on growth of 3 peanut pathogenic bacteria hypha

Picking single colony of strain ZHX-7 with toothpick, placing into a triangular flask containing 20mLLB culture medium, performing shake culture at 28 deg.C for 14 hr to obtain product with OD concentration₄₂₀Bacterial suspension equal to 0.3. And pouring LB culture medium into one side of the two-partition culture dish, and pouring PDA culture medium with the same volume into the other side of the two-partition culture dish. Uniformly coating 50uL of bacterial suspension on one side of LB culture medium, inoculating bacterial cake of peanut netospora bacteria with diameter of 5mm or 10 uL of spore suspension (peanut crown rot or peanut ring spot, OD)₄₂₀0.3), 50uL of sterile water was applied as a control group, 6 replicates were set for each treatment, and colony diameters of coronafora arachidicola, verticillium arachidicola and porphyridium arachidicola were measured after 3d, 3d and 5d, respectively. As a result, as shown in FIG. 5, the growth of hyphae of crown rot, ring rot and reticulospora of peanut was significantly reduced by the volatile gas of the strain ZHX-7, and the hypha growth inhibition rates were 11.53%, 14.24% and 11.11%, respectively.

FIG. 5A shows the bacteriostatic ability of the strain ZHX-7 volatile gas against peanut crown rot, FIG. 5B shows the inhibition against peanut net blotch, and FIG. 5C shows the inhibition against peanut verticillium; indicates that at p <0.01 levels, the difference was very significant.

4. Bacteriostatic action of strain ZHX-7 on other peanut pathogenic fungi

(1) Peanut root rot (Fusarium solani and Fusarium oxysporum): preparation of the Strain ZHX-7 at OD concentration₄₂₀Sucking 10. mu.L of the bacterial suspension, inoculating the bacterial suspension in the center of a PDA solid culture medium with the diameter of 9cm, and culturing at the constant temperature of 28 ℃ for 24 hours after the bacterial suspension is fully absorbed. Uniformly spraying OD into PDA culture medium₄₂₀Spore suspensions of fusarium solani and fusarium oxysporum of 0.3 were compared with no spore spray suspension. Culturing at constant temperature of 25 deg.C for about 3D, counting and photographing the size of inhibition zone, as shown in FIGS. 6A-D, wherein FIGS. 6A and 6C are control groups of Fusarium solani and Fusarium oxysporum, respectively, and FIGS. 6B and 6D are inhibition effects of strain ZHX-7 on Fusarium solani and Fusarium oxysporum, respectively. The result shows that the strain ZHX-7 has obvious antagonistic activity on the fusarium solani and fusarium oxysporum, and the sizes of inhibition zones are respectively 24.67 +/-0.75 mm and 31.33 +/-0.94 mm.

(2) Southern blight and rhizoctonia solani of peanuts: preparation of the Strain ZHX-7 at OD concentration₄₂₀Bacterial suspension 0.3, ready for use. A bacterial cake of pathogenic bacteria with a diameter of 5mm is inoculated into the center of the plate, 10 mu L of bacterial suspension of the strain ZHX-7 is inoculated at the position of about 22mm on both sides of the bacterial cake, and the non-inoculated bacterial suspension is used as a control. Carrying out constant-temperature dark culture at 25 ℃ for about 5 days, counting the bacteriostasis conditions, and obtaining a chart shown in fig. 6E-H, wherein the chart shown in fig. 6E and 6G are respectively a control group of the peanut sclerotinia sclerotiorum and the peanut sheath blight germ, the chart shown in fig. 6F and 6H are respectively the bacteriostasis effects of the strain ZHX-7 on the peanut sclerotinia sclerotiorum and the peanut sheath blight germ, and calculating the bacteriostasis rate (%) (the growth diameter of a control bacterial colony-the growth diameter of a treated bacterial colony)/the growth diameter of the control bacterial colony multiplied by 100. The result shows that the strain ZHX-7 has obvious bacteriostatic effect on the southern blight and the peanut sheath blight, and the hypha growth inhibition rates are 65.57 percent and 55.98 percent respectively.

Example 3

Potted plant test for preventing and treating peanut crown rot by bacterial strain ZHX-7 bacteria fermentation liquid

Bacterial liquid (OD) of strain ZHX-7 for culturing 956 seed of peanut₄₂₀0.3) soaking for 4h, and sowing in single seedA disposable plastic cup of 320mL was simultaneously inoculated with 2mL of fermentation broth. Soaking LB culture medium, inoculating LB culture medium with the same amount as the blank control, and treating with 800 times of 50% carbendazim water solution as the treatment control. Meanwhile, peanut crown rot fungi are cultured on a PDA plate, after 6 days, spore suspension of the peanut crown rot fungi is prepared by using sterilized water, 5mL of the spore suspension is added into 150mL of PDB, and the mixture is subjected to shaking culture at 25 ℃ for 24 hours and is used for inoculating peanuts. Each peanut was inoculated with 2mL of a peanut crown rot spore suspension, and an equal amount of PDB was used as a control. And (3) contrasting and treating each 30 pots, repeating for 3 times, counting the mortality of each group after 30 days, and calculating the prevention and treatment effect. Control effect (%) - (control mortality-treatment mortality)/control mortality × 100%. According to statistics, the control effect of carbendazim and the strain ZHX-7 on the peanut crown rot is respectively 63.94% and 81.97% compared with a control, the death rate of peanuts treated by the strain ZHX-7 is lowest, and the control effect is best (see figure 7, wherein different capital letters in the figure are indicated as p in<At the 0.01 level, the difference was very significant).

Example 4

Influence of strain ZHX-7 strain fermentation liquid on peanut growth

The strain ZHX-7 bacteria-containing fermentation liquid is diluted by 500 times (the final volume is 750mL) by using sterilized water, added into a peanut water culture box, and the diluent is changed every 5 days by using LB culture medium with 500 times dilution as a control. And (3) selecting 956 seeds of peanut seeds with consistent germination tendency (soaking in sterile water for 3d in advance), uniformly placing in peanut water culture boxes, and placing 30 seeds in each water culture box. Contrasting and processing each 3 water culture boxes, repeating for 3 times, carrying out post-treatment on peanuts for 15 days, weighing the total fresh weight of the plants, the fresh weight of the overground part and the fresh weight of the roots, and weighing the total dry weight of the plants, the dry weight of the overground part and the dry weight of the roots after drying. Statistically, the bacterial fermentation broth of strain ZHX-7 was found to significantly or very significantly increase the fresh and dry weight of the flower-cultivated 956 plants compared to the control, total fresh weight of treatment, fresh weight of stems, fresh weight of roots, total dry weight of treatment, dry weight of stems, dry weight of roots by 48.27%, 55.13%, 126.14%, 11.65%, 5.69% and 96.12%, respectively (see fig. 8). In FIG. 8, A is a comparison of the overall growth of the peanuts and B is the effect of the strain ZHX-7 on the fresh and dry weight of the peanuts (note: the difference is very significant at p <0.01 level; the difference is significant at p <0.05 level).

Example 5

Influence of strain ZHX-7 on contents of Aspergillus flavus and toxin thereof

1. Effect of strain ZHX-7 on growth of aspergillus flavus of peanut:

according to the method for controlling the growth of the aspergillus flavus by the strain ZHX-7 by referring to the peanut crown rot fungi, the influence of the bacterial suspension, the sterile fermentation liquid and the volatile gas of the strain ZHX-7 on the growth of the aspergillus flavus is carried out, and the result shows that the diameter of a inhibition zone of the bacterial suspension is 2.07 +/-0.07 cm, the hypha growth inhibition rates of the sterile fermentation liquid and the volatile gas are respectively 3.81% and 3.38%, wherein the volatile gas lightens the yellow color of the aspergillus flavus (see figure 9, in figure 9, A, C, E is a control group, and B, D, F is the influence of the bacterial suspension, the sterile fermentation liquid and the volatile gas of the strain ZHX-7 on the growth of the aspergillus flavus).

2. Effect of strain ZHX-7 on peanut aflatoxin content:

(1) soaking rice in sterilized water for 18h, draining, weighing 20g, packaging into sterilized conical flask, adding 6mL sterilized water into conical flask, and sterilizing at high temperature (121 deg.C, 15 min). Cooling the sterilized rice to room temperature, and treating: inoculating 1mL of Aspergillus flavus spore suspension and 50 μ L of ZHX-7 bacterial suspension onto sterilized rice; and (5) processing: inoculating 1ml of Aspergillus flavus spore suspension and 10. mu.L of ZHX-7 bacterial suspension to sterilized rice; the negative control group rice is not added with the bacterial suspension, and sterilized water is used for replacing the spore suspension; the positive control rice was supplemented with only spore suspension. After culturing in the dark at 25 ℃ for 7d, freezing and drying the sample, crushing the sample by a crusher, sieving the crushed sample by a 20-mesh sieve, and detecting the content of the aflatoxin in the sample by using an aflatoxin total amount ELISA kit (Beijing Hua an Mai Co., Ltd.).

(2) Crushing No. 6 peanut seeds in peanut varieties by using a crusher, weighing 20g of the crushed seeds, subpackaging the crushed seeds into sterilized conical flasks, adding 6mL of sterilized water into the conical flasks, and sterilizing at high temperature (121 ℃, 15 min). Cooling the sterilized peanut pieces to room temperature, treating: inoculating 1mL of Aspergillus flavus spore suspension and 50uL of ZHX-7 bacterial suspension to the sterilized chopped peanuts; and (5) processing: inoculating 1mL of Aspergillus flavus spore suspension and 10 uL of ZHX-7 bacterial suspension to the sterilized chopped peanuts; the negative control group of crushed peanuts are not added with the bacterial suspension, and sterilized water is used for replacing the spore suspension; the positive control group was ground peanuts with only spore suspension added. After dark culture at 25 ℃ for 7 days, the sample is frozen and dried, and is sieved by a 20-mesh sieve, and the content of aflatoxin in the sample is detected by an aflatoxin total amount ELISA kit (Beijing Hua Anmai Co., Ltd.).

(3) Using enzyme-linked immunosorbent assay (iMark)^TMBIO-RAD, USA) measuring wavelength 450nm and reference wavelength 630nm to determine the OD value of each well of the microplate. Net software was used to analyze the data and calculate the aflatoxin concentration in each sample based on the standard curve. The increase of the aflatoxin content in the sample is equal to the aflatoxin content in the sample-the aflatoxin content of the negative control group, and the aflatoxin degradation rate (%) is equal to (the aflatoxin increase of the positive control group-the aflatoxin increase of the treatment group)/the aflatoxin increase of the positive control group x 100.

As a result, the 10. mu.L and 50. mu.L of ZHX-7 bacterial suspensions were found to have the aflatoxin degradation rates of 64.43% and 94.86% in rice and 46.99% and 87.70% in peanut, respectively, and it can be seen that the ZHX-7 bacterial suspensions can significantly reduce the aflatoxin content, and at the same time, the effect of more thalli than less thalli is better (see FIG. 10, different capital letters in the figure indicate that the difference is significant under the level of p < 0.01).

The above description is only for the preferred embodiment of the present invention and should not be taken as limiting the invention, and any modifications, equivalents, improvements and the like made within the scope of the present invention should be included in the patent protection scope of the present invention.

Sequence listing

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agctatacaa gcaatattta ttctttcaca aataatatca acacatacga aggcggcacg 480

cacgaggccg gatttaaaac cggtctgacc cgtgtcataa acgactatgc aagaagaaaa 540

gggattttca aagaaaatga tccgaattta agcggggatg atgtgagaga agggctgact 600

gccattattt caattaagca ccctgatccg caattcgaag ggcagacgaa aaccaagctc 660

ggcaactccg aagcgagaac gatcactgat acgctgtttt cttctgcgct ggaaacattc 720

cttcttgaaa atccggactc agcccgcaaa atcgttgaaa aaggtttaat ggccgcaaga 780

gcgcggatgg cggcgaaaaa agcccgggaa ttgacccggc gcaaaagtgc gcttgagatt 840

tccaatctgc cgggcaaact ggcggactgt tcttctaaag atccgagcat ttccgagctg 900

tatatcgtag agggtgactc tgcgggcgga tcagcgaaac agggacggga ccgtcatttc 960

caagccattc tgccgctgcg cggtaagatt ctgaacgttg agaaagccag acttgataag 1020

attctctcaa acaatgaggt cagatcaatg atcacggccc tcggaacagg gatcggaga 1079

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agagtttgat cmtggctcag 20

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggytaccttg ttacgactt 19

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaagtcatca tgaccgttct gc 22

<210> 6

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agcagggtac ggatgtgcga gcc 23

Claims

1. The biocontrol Bacillus beleisi ZHX-7 is characterized in that the strain ZHX-7 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 20374.

2. The biocontrol Bacillus beleisi ZHX-7 of claim 1, wherein the 16S rDNA sequence of said strain ZHX-7 is as set forth in SEQ ID NO: 1 is shown in the specification; the gyrB gene sequence is shown as SEQ ID NO: 2, respectively.

3. Use of the biocontrol Bacillus belezii ZHX-7 of claim 1 or 2 for controlling crown rot, blotch, verticillium, rhizoctonia, southern blight and rhizoctonia solani.

4. Use according to claim 3, in the form of any one of a bacterial suspension, a sterile fermentation broth, a volatile gas, and a bacterial fermentation broth of strain ZHX-7.

5. Use of the biocontrol Bacillus belgii ZHX-7 of claim 1 or 2 to promote peanut growth.

6. The use of the biocontrol bacillus beilaiensis ZHX-7 of claim 1 or 2 for inhibiting peanut aspergillus flavus and its toxin content.

7. Use according to claim 6, in the form of any one of a suspension of the strain ZHX-7, a sterile fermentation broth, a volatile gas.