CN112358995B

CN112358995B - Biocontrol bacillus beleisi ZHX-12 and application thereof

Info

Publication number: CN112358995B
Application number: CN202011286282.8A
Authority: CN
Inventors: 张霞; 迟玉成; 许曼琳; 郭志青; 于静; 何康; 李莹; 宋新颖
Original assignee: Shandong Peanut Research Institute
Current assignee: Shandong Peanut Research Institute
Priority date: 2020-11-17
Filing date: 2020-11-17
Publication date: 2022-05-27
Anticipated expiration: 2040-11-17
Also published as: CN112358995A

Abstract

The invention provides a biocontrol bacillus beleisi ZHX-12, the preservation number is CGMCC No.20375, and the 16S rDNA sequence is shown as SEQ ID NO: 1 is shown in the specification; the gyrB gene sequence is shown as SEQ ID NO: 2, respectively. The biocontrol Bacillus beialassius ZHX-12 is applied to preventing and controlling peanut crown rot, peanut netospora, peanut ring rot, peanut root rot, peanut southern blight and peanut sheath blight, and can promote peanut growth and inhibit peanut aspergillus flavus and toxin content thereof. The application method of the Bacillus beleisi ZHX-12 belongs to the field of biological control, is safe, quick and effective, is beneficial to green and healthy production of peanuts, provides technical support for comprehensive control of peanut diseases, and can promote growth of the peanuts and inhibit the contents of aspergillus flavus and toxins thereof in the peanuts.

Description

Biocontrol bacillus beleisi ZHX-12 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 ZHX-12 with broad-spectrum resistance to various plant pathogenic bacteria and an application thereof in promoting peanut growth and preventing and controlling peanut pathogenic fungi.

Background

Peanuts are one of important sources of food, feed and edible oil in the world, and are easy to infect diseases, particularly soil-borne diseases caused by fungi, including peanut crown rot, southern blight and the like. Peanut crown rot is caused by the infestation of Aspergillus niger damaging kernels, cotyledons and stem bases, mainly stem bases. The disease can survive in infected peanut seeds and spread through the seeds, wet kernels in a storage period are easy to be infected by germs, and the germ carrying rate of the seeds is as high as more than 90%. The soil-borne bacteria is another important initial infection source, and the bacteria can survive in the soil for 1-2 years. In recent years, due to continuous cropping and shortage of disease-resistant varieties, pathogenic bacteria accumulate in successive years, so that diseases become more serious, and the diseases generally cause seedling shortage by 10 percent and are more than 50 percent seriously. Southern blight is caused by Sclerotium rolfsii (Sclerotium rolfsii), the incidence rate of the plots is about 10% generally, the weight is more than 30%, and the yield of peanuts is reduced by 50-100%. Fungicides are the main route to control plant pathogens, but based on human health and environmental concerns, there is interest in finding alternatives to chemical pesticides, where the use of microorganisms to control plant diseases is a good choice.

Bacillus belgii is widely distributed in nature, has good characteristics of rapid growth and stability, is easy to separate and culture, is harmless to human beings and animals, does not pollute the environment, has rich metabolites, has broad-spectrum antibacterial activity and stronger anti-stress capability, and therefore plays an increasingly important role in many fields such as agriculture, environment, fermentation industry and the like. In recent years, scientists at home and abroad separate a large amount of Bacillus belgii from plant rhizosphere soil, culture ponds, deep sea and Daqu in succession, and researches show that a plurality of Bacillus belgii strains play a certain role in promoting plant growth, antagonizing pathogenic bacteria, brewing white spirit and the like, and have very important theoretical research and practical application values, but the Bacillus belgii is not applied to peanut disease control so far.

Disclosure of Invention

The invention aims to solve various defects of chemical pesticides in the aspect of peanut disease and pest control in the prior art, provides a biocontrol Bacillus belezii ZHX-12 and application thereof, wherein Bacillus belezii is applied to peanuts, and the resistance of the peanuts to peanut pathogenic bacteria such as crown rot, southern blight and the like is improved by virtue of the biocontrol strain. The method belongs to the field of biological control, is safe, quick and effective, is beneficial to green and healthy production of peanuts, provides technical support for comprehensive control of peanut diseases, and can promote growth of the peanuts and inhibit the contents of peanut aspergillus flavus and toxins thereof.

The technical scheme of the invention is as follows:

the biocontrol Bacillus beleisi ZHX-12 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 20375.

Further, the 16S rDNA sequence of the strain ZHX-12 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-12 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 biocontrol Bacillus belezii ZHX-12 can be applied in any one of a bacterial suspension, a sterile fermentation liquid, a volatile gas and a bacterial fermentation liquid of the strain ZHX-12.

The biocontrol Bacillus beleisi ZHX-12 is applied to promoting peanut growth.

Further, peanut seeds are soaked in the bacteria-containing fermentation liquor of the strain ZHX-12 for a period of time and then sowed, and a certain amount of fermentation liquor is inoculated while sowing.

The biocontrol Bacillus beleisi ZHX-12 is used for inhibiting peanut aspergillus flavus and toxin content thereof.

Furthermore, the application form of the biocontrol bacillus beilesensis ZHX-12 in inhibiting the contents of peanut aspergillus flavus and toxins thereof is any one of bacterial suspension, sterile fermentation liquor and volatile gas of the strain ZHX-12.

Biological material sample preservation information:

the strain ZHX-12 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 at 07 months in 2020, the preservation number is CGMCC No. 20375.

The invention has the beneficial effects that:

the Bacillus belgii ZHX-12 provided by the invention has an obvious inhibiting effect on peanut pathogenic bacteria, can be used as a potential biocontrol strain, applies the Bacillus belgii 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 Bacillus beilisi ZHX-12 can also inhibit the contents of peanut aspergillus flavus and toxin thereof. The application method of the Bacillus belgii ZHX-12 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-12;

FIG. 2 is a phylogenetic tree constructed by the strain ZHX-12 based on 16S rDNA and gyrB genes;

FIG. 3 shows antagonistic action of strain ZHX-12 on target pathogenic bacteria;

FIG. 4 is the determination of the bacteriostatic ability of the strain ZHX-12 fermentation liquid on 3 pathogenic bacteria;

FIG. 5 is the measurement of the bacteriostatic ability of the volatile gas of the strain ZHX-12 on 3 pathogenic bacteria;

FIG. 6 shows the inhibitory effect of the ZHX-12 strain on other peanut pathogens tested;

FIG. 7 shows the control effect of the strain ZHX-12 fermented liquid on peanut crown rot;

FIG. 8 shows the control effect of strain ZHX-12 strain fermentation liquid on southern blight;

FIG. 9 shows the effect of strain ZHX-12 on peanut growth;

FIG. 10 is the effect of strain ZHX-12 on the growth of Aspergillus flavus;

FIG. 11 shows the effect of suspension of strain ZHX-12 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 isolation and identification of Bacillus belgii ZHX-12

1. Screening and isolation of Strain ZHX-12:

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

The method comprises the following steps:

(1) weighing 10g of the soil sample, adding the soil sample into 90mL of sterile water, oscillating 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, spraying a peanut crown rot (Aspergillus niger) spore suspension, culturing for 2d, selecting the colonies with obvious bacteriostatic rings around, picking single colonies by using the bacteriostatic rings, and performing purification culture on an LA solid culture medium.

(3) Finally, selecting a strain with an inhibition zone of more than 2cm, wherein the thallus is light yellow, the colony is rough and opaque, and the strain is named as ZHX-12, and a strain morphology chart is shown in figure 1.

(4) After shaking the single colony of the strain ZHX-12 with LB liquid culture medium, adding glycerol and preserving at-80 ℃.

2. Strain identification of strain ZHX-12:

according to the kit "TRANSGEN" for extracting bacterial genomes

The Bacteria Genomic DNA Kit "extracted the Genomic DNA of ZHX-12. The genome of the strain ZHX-12 was PCR-amplified using 16S rDNA universal primers 9F (5'-GAGTTTGATCCTGGCTCAG-3') (SEQ ID NO: 3) and 1542R (5'-AGAAAGGAGGTGATCCAGCC-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) Ltd for sequencing.

The sequence determination result shows that the 16S rDNA sequence of the strain ZHX-12 has the full length of 1439bp, and the sequence is shown as SEQ ID NO: 1 is shown in the specification; the total length of the gyrB gene sequence is 1084bp, and the sequence is shown as SEQ ID NO: 2, respectively. The obtained sequence is submitted to a GenBank database for BLAST analysis and comparison, and a phylogenetic tree shown in figure 2 is constructed by MEGA6.06 software in combination with a 16S rDNA sequence and a gyrB gene sequence, and the result shows that the similarity of the strain ZHX-12 and the Bacillus subtilis velezensis CP047157 reaches 97 percent, which indicates that the strain ZHX-12 is the Bacillus subtilis.

Example 2 inhibition of growth of Coccomyza arachidicola, Neurospora reticulata and Phycomycetes verticillata by Bacillus belgii ZHX-12

Inhibition effect of strain ZHX-12 bacterial suspension on growth of 3 peanut pathogenic bacteria hypha

1. Peanut crown rot bacteria: picking single colony of strain ZHX-12 with toothpick, placing into a triangular flask containing 20mL LB culture medium, performing shake culture at 28 deg.C for 14h 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 about 8 days, and taking statistics of the size of zone of inhibition (see FIGS. 3A and 3B). FIG. 3A is a control group of crown rot germs of peanuts, and FIG. 3B is a counter effect of crown rot germs of peanuts; the result shows that the strain ZHX-12 has obvious antagonistic activity on the peanut crown rot bacteria, and the size of the inhibition zone is 28.67 +/-1.03 mm.

2. Peanut latticed bacteria: similarly, the concentration of the prepared strain ZHX-12 is OD₄₂₀Bacterial suspension 0.3, ready for use. Inoculating a bacterial cake of pathogenic bacteria with the diameter of 5mm into the center of the plate, inoculating 10 mu L of bacterial suspension of a bacterial strain ZHX-12 at the position of about 22mm on both sides of the bacterial cake respectively, taking the non-inoculated bacterial suspension as a control, carrying out constant-temperature dark culture at 25 ℃ for about 5d, counting the bacteriostasis condition, 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 x 100. FIG. 3C shows the control group of peanut leaf spot pathogens, and FIG. 3D shows the confronting effect of peanut leaf spot pathogens; the result shows that the strain ZHX-12 has obvious bacteriostatic effect on the peanut netspot pathogen, and the hypha growth inhibition rate is 67.26%.

3. Alternaria alternata: similarly, the concentration of the prepared strain ZHX-12 is OD₄₂₀The bacterial suspension was prepared in a concentration OD of alternaria arachidicola (Beechia arachidicola)₄₂₀Spore suspension of 0.3 for use. Sucking 10 mu L of spore suspension, inoculating the spore suspension into the center of a PDA solid culture medium with the diameter of 9cm, respectively inoculating 10 mu L of bacterial suspension of a strain ZHX-12 at positions which are about 22mm away from the two sides of the center of a plate, taking the non-inoculated bacterial suspension as a control, culturing at the constant temperature of 25 ℃ in dark for about 4 days, counting the bacteriostasis condition, 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, and FIG. 3F shows the antagonistic effect of peanut verticillium; the result shows that the strain ZHX-12 has a remarkable bacteriostatic effect on the alternaria arachidicola, and the hypha growth inhibition rate is 66.21%.

(II) inhibition effect of strain ZHX-12 sterile fermentation liquid on growth of 3 peanut pathogenic bacteria hypha

Picking single colony of strain ZHX-12 with toothpick, placing in a triangular flask containing 100mL LB medium, 28 deg.CShake culturing for 36h, high speed centrifuging, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain sterile fermentation liquid. Mixing sterile fermentation broth with PDA culture medium at ratio of 1:10, cooling to about 50 deg.C, pouring into flat plate, inoculating cake of peanut netospora bacteria with diameter of 5mm or 10 μ L spore suspension (peanut crown rot or peanut wheel spot, 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, as shown in FIG. 4, the growth of the hyphae of crown rot, netospora arachidis and verticillium arachidis of the strain ZHX-12 was significantly reduced compared to the corresponding control (wherein, FIG. 4A shows the bacteriostatic ability of the fermentation liquid of the strain ZHX-12 to the crown rot, FIG. 4B shows the inhibition to the netospora arachidis, FIG. 4C shows the inhibition to the physalospora arachidis, and the difference shows that the hyphae growth inhibition rates are 20.11%, 49.13% and 41.52% respectively at a p <0.01 level).

(III) inhibition effect of strain ZHX-12 volatile gas on growth of 3 peanut pathogenic bacteria hypha

Picking single colony of strain ZHX-12 with toothpick, placing into a triangular flask containing 20mL LB culture medium, performing shake culture at 28 deg.C for 14h 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 50 μ L of bacterial suspension on one side of LB culture medium, inoculating bacterial cake of peanut netospora bacteria with diameter of 5mm or 10 μ L of spore suspension (peanut crown rot or peanut wheel spot, OD)₄₂₀0.3), 50 μ L of sterile water was applied as a control group, 6 replicates were set for each treatment, and colony diameters of coronafora arachidis hypogaeae, netospora arachidis hypogaeae and verticillium arachnoideum were measured after 3d, 3d and 5d, respectively. As a result, as shown in FIG. 5, it was found that the growth of hyphae of crown rot, Neurospora arachidicola and Erythrophora arachidicola was extremely significantly reduced by the volatile gas of the strain ZHX-12 (FIG. 5A shows that the volatile gas of the strain ZHX-12 is responsible for the growth of peanutsThe bacteriostatic ability of crown rot pathogen, fig. 5B is the inhibition of peanut net spot pathogen, and fig. 5C is the inhibition of peanut wheel spot pathogen; is represented by p<At the 0.01 level, the difference was extremely significant), the inhibition rates of hyphal growth were 14.52%, 17.88%, and 16.76%, respectively.

Example 3 bacteriostatic action of Bacillus beilaisi ZHX-12 on peanut root rot, peanut southern blight and peanut sheath blight

1. Peanut root rot (Fusarium solani and Fusarium oxysporum): preparation of the Strain ZHX-12 at OD concentration₄₂₀Sucking 10 mu L of bacterial suspension liquid, inoculating the bacterial suspension liquid in the center of a PDA solid culture medium with the diameter of 9cm, and after the bacterial suspension liquid is fully absorbed, culturing for 24 hours at constant temperature of 28 ℃. Uniformly spraying OD into PDA culture medium₄₂₀Spore suspensions of 0.3 of Fusarium solani and Fusarium oxysporum, which are cultured at a constant temperature of 25 ℃ for about 3D without spraying the spore suspensions, and the sizes of inhibition zones are counted and photographed 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 the strain ZHX-12 on Fusarium solani and Fusarium oxysporum, respectively. As a result, the strain ZHX-12 has obvious antagonistic activity on fusarium solani and fusarium oxysporum, and the sizes of inhibition zones are respectively 28.50 +/-0.96 mm and 33.17 +/-1.77 mm.

2. Sclerotium rolfsii and rhizoctonia solani of peanuts: preparation of the Strain ZHX-12 at OD concentration₄₂₀Bacterial suspension 0.3, ready for use. Inoculating a bacterial cake of pathogenic bacteria with the diameter of 5mm into the center of a plate, inoculating 10 mu L of bacterial suspension of a strain ZHX-12 at the position of about 20mm of both sides of the bacterial cake respectively, taking the non-inoculated bacterial suspension as a control, carrying out constant-temperature dark culture at 25 ℃, carrying out statistics on the bacteriostasis conditions for about 5 days, and referring to fig. 6E-H, wherein fig. 6E and 6G respectively refer to control groups of peanut sclerotinia sclerotiorum and peanut sheath blight bacteria, fig. 6F and 6H respectively refer to the bacteriostasis effects of the strain ZHX-12 on the peanut sclerotinia sclerotiorum and peanut sheath blight bacteria, and calculating the bacteriostasis rate (%) (control colony diameter-treated colony growth diameter)/control colony growth diameter multiplied by 100. As a result, the strain ZHX-12 has obvious bacteriostatic effect on southern sclerotium blight and peanut sheath blight, and the hypha growth inhibition rates are 64.93% and 73.22% respectively。

Example 4 Bacillus belgii ZHX-12 control of peanut pathogens potting test

1. Potted plant test for preventing and treating peanut crown rot by using strain ZHX-12 bacteria fermentation liquid

Bacterial liquid (OD) of strain ZHX-12 for number 10 peanut seeds₄₂₀0.3) for 4h, and single-seed sowing in a disposable plastic cup of 320mL, while inoculating 2mL of fermentation broth in the cup. 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%. Statistically, compared with a control group, the control effects of the carbendazim and the strain ZHX-12 on the peanut crown rot are 64.41 percent and 81.36 percent respectively, the death rate of peanuts treated by the strain ZHX-12 is lowest, and the control effect is best (see figure 7, wherein different capital letters in the figure are represented as p<At the 0.01 level, the difference was very significant).

2. Potted plant test for preventing and treating peanut southern blight by using strain ZHX-12 bacteria fermentation liquid

Bacterial liquid (OD) of strain ZHX-12 for number 10 peanut seeds₄₂₀0.3) for 4h, and single-seed sowing in a disposable plastic cup of 320mL, while inoculating 2mL of fermentation broth in the cup. 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. After 7 days, inoculating 2 bacteria-carrying oat grains on the base of the peanut stem for each peanut. 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%. Statistics shows that compared with a control group, the control effects of the carbendazim and the strain ZHX-12 on the cotton blight are 59.09 percent and 84.85 percent respectively, and the strainThe ZHX-12 treated peanuts had the lowest mortality and the best control (see FIG. 8, in which the different capital letters are indicated at p<At the 0.01 level, the difference was very significant).

Example 5 Effect of Bacillus belgii ZHX-12 on peanut growth

1. Influence of strain ZHX-12 strain fermentation liquid on peanut growth

After the seeds of the high-oleic acid peanut variety Jinhua No. 10 are soaked in the bacteria-containing fermentation liquor of the strain ZHX-12 for 4 hours, the seeds are sowed in 320mL disposable plastic cups in single seeds, and 2mL fermentation liquor is inoculated at the same time. The LB medium was soaked and the same amount of LB medium was inoculated as a control. And (3) contrasting and treating 10 pots of peanuts, repeating for 3 times, treating the peanuts after 30 days, weighing the total fresh weight of the plants, and weighing the total dry weight of the plants after drying. Statistically, as shown in fig. 9 (×) showing that the difference is very significant at p <0.01 level, the fermented broth of the strain ZHX-12 can significantly increase the fresh weight and dry weight of the jin flower 10 plants compared to the control group by 26.03% and 22.54%, respectively.

Example 6 Effect of Bacillus belgii ZHX-12 on the content of Aspergillus flavus and its toxins

1. Effect of the Strain ZHX-12 on the growth of Aspergillus flavus of peanut:

referring to the method of peanut crown rot, an experiment of influence of bacterial suspension of the strain ZHX-12, sterile fermentation liquid and volatile gas on growth of the aspergillus flavus is carried out, as shown in fig. 10, the result shows that the diameter of a zone of inhibition of the bacterial suspension on the aspergillus flavus is 2.23 +/-0.05 cm, and the inhibition rates of hypha growth of the sterile fermentation liquid and the volatile gas are respectively 4.75% and 34.07%, wherein the volatile gas lightens the color of the aspergillus flavus (as shown in fig. 10, in the figure, 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-12 on the growth of the aspergillus flavus).

2. Effect of strain ZHX-12 on peanut aflatoxin content:

(1) soaking rice in sterilized water for 18h, draining, weighing 20g, packaging into sterilized conical flask, adding 6mL of sterilized water, 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 LZHX-12 bacterial suspension onto sterilized rice; and (5) processing: inoculating 1mL of Aspergillus flavus spore suspension and 10 μ LZHX-12 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 weighed seeds into a sterilized conical flask, adding 6mL of sterilized water into the conical flask, 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 50 uL of ZHX-12 bacterial suspension to the sterilized chopped peanuts; and (5) processing: inoculating 1mL of Aspergillus flavus spore suspension and 10 mu LZHX-12 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, as shown in FIG. 11 (different capital letters in the figure indicate that the difference is very significant under the p <0.01 level), the 10. mu.L and 50. mu.L of ZHX-12 bacterial suspensions have the aflatoxin degradation rates of 75.63% and 99.56% in rice, respectively, and the aflatoxin degradation rates of 55.03% and 90.94% in peanut respectively, and it can be seen that the ZHX-12 bacterial suspensions can reduce the aflatoxin content very significantly, and have better effect of reducing the aflatoxin content with more bacterial cells than with less bacterial cells.

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

<110> institute for peanut research in Shandong province

<120> biocontrol Bacillus belezii ZHX-12 and application thereof

<141> 2020-11-17

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1439

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

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gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360

aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtgccgtt caaatagggc 420

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atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc 540

ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact 600

tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga 660

ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt 720

ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg 780

ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt 840

acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900

tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacaatcct 960

agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg tcgtcagctc 1020

gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag 1080

cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac 1140

gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg acagaacaaa 1200

gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt cggatcgcag 1260

tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt 1320

gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg 1380

aagtcggtga ggtaaccttt tatggagcca gccgccgaag gtgggacaga tgattgggg 1439

<210> 2

<211> 1084

<212> DNA

<213> Bacillus belgii (Bacillus velezensis)

<400> 2

ccttgtcgac cactcttgac gttacggttc atcgtgacgg aaaaatccat tatcaggcgt 60

acgagcgcgg tgtacctgtg gccgatcttg aagtgatcgg cgaaactgat aagaccggaa 120

cgattacgca cttcgttccg gacccggaaa ttttcaaaga aacaactgta tatgactatg 180

atctgctttc aaaccgtgtc cgggaattgg ccttcctgac aaaaggcgta aacatcacga 240

ttgaagacaa acgtgaagga caagaacgga aaaacgagta ccactacgaa ggcggaatca 300

aaagctatgt tgagtactta aaccgttcca aagaagtcgt tcatgaagag ccgatttata 360

tcgaaggcga gaaagacggc ataacggttg aagttgcatt gcaatacaac gacagctata 420

caagcaatat ttattctttc acaaataata tcaacacata cgaaggcggc acgcacgagg 480

ccggatttaa aaccggtctg acccgtgtca taaacgacta tgcaagaaga aaagggattt 540

tcaaagaaaa tgatccgaat ttaagcgggg atgatgtgag agaagggctg actgccatta 600

tttcaattaa gcacccggat ccgcaatttg aagggcagac gaaaaccaag ctcggcaact 660

ccgaagcgag aacgatcact gatacgctgt tttcttctgc gctggaaaca ttccttcttg 720

aaaatccgga ctcagcccgc aaaattgttg aaaaaggttt aatggctgca agagcgcgga 780

tggcggcgaa aaaagcccgg gaattgaccc ggcgcaaaag tgcgcttgag atttccaatc 840

tgccgggcaa actggcggac tgttcttcta aagatccgag catttccgag ctgtatatcg 900

tagagggtga ctctgcgggc ggatcagcga aacagggacg ggaccgtcat ttccaagcca 960

ttctgccgct gcgcggtaag attctgaacg ttgagaaagc cagacttgat aagattctct 1020

caaacaatga ggtcagatca atgatcacgg ccctcggaac aggaatcgga gaagatttta 1080

atct 1084

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gagtttgatc ctggctcag 19

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

agaaaggagg tgatccagcc 20

<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-12 is used for inhibiting peanut aspergillus flavus and toxin content thereof, wherein the strain ZHX-12 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 20375.

2. The use according to claim 1, wherein the 16S rDNA sequence of strain ZHX-12 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. The use according to claim 1, wherein the biocontrol bacillus beilesensis ZHX-12 is used in the form of any one of a suspension of the strain ZHX-12, a sterile fermentation broth, a volatile gas.