CN107988108B

CN107988108B - Mold-resistant bacillus and antibacterial application thereof

Info

Publication number: CN107988108B
Application number: CN201711385074.1A
Authority: CN
Inventors: 杨水云; 吴丹; 苏婷婷; 李少锋; 赵浩淼
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2017-12-20
Filing date: 2017-12-20
Publication date: 2020-10-27
Anticipated expiration: 2037-12-20
Also published as: CN107988108A

Abstract

The invention discloses a bacillus for antagonizing mould and antibacterial application thereof, belonging to the technical field of microbial control. The bacillus for antagonizing the mould is a bacillus with broad-spectrum antibacterial capability. The strain is preserved in the China center for type culture collection, and the sequence alignment analysis finds that the strain is 100 percent of the known Bacillus amyloliquefaciens Holycan 1(Bacillus amyloliquefaciens Holycan 1, KM54172), and the strain is identified to belong to the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). The bacillus for antagonizing the mold has very short time for reaching the stationary phase, is favorable for fast colonization and occupying ecological niche, and competes with other molds for nutrients and living space. Therefore, the penicillium antagonistic bacillus amyloliquefaciens provided by the invention has a good inhibition effect on common crop disease fungi, shows a wide antibacterial spectrum, has a large biocontrol potential, and has a good application prospect.

Description

Mold-resistant bacillus and antibacterial application thereof

Technical Field

The invention belongs to the technical field of microbial control, relates to bacillus, and particularly relates to mould-antagonistic bacillus and antibacterial application thereof.

Background

The mold is ubiquitous and has strong stress resistance due to sporulation. Since the mold belongs to chemoheterotrophic bacteria and can live by organic matters, mold pollution or infection can be caused in places with organic matters if the temperature and the humidity are proper, and a lot of troubles are brought to the human society. For example, fruits, vegetables and grain feeds are easily polluted by mould in storage, so that economic loss and even poisoning of people and livestock are caused; in the livestock house, the mold is bred in the livestock manure, so that diseases are brought to the livestock; the mold also causes many plant diseases, affects the growth of plants, causes a reduction in yield, and the like. Prevention and management of mold contamination is a persistent battle that we have long and may go on for a long time.

The bacillus is an aerobic and spore-producing gram-positive bacterium widely existing in nature, has strong stress resistance and is easy to culture. Bacillus amyloliquefaciens is a kind of bacillus, is widely distributed in the natural world and belongs to probiotics, and some strains have bacteriostatic activity on bacteria or fungi and have good application potential (Zhang Juan and the like, the Bacillus amyloliquefaciens and application thereof as probiotics [ J ]. animal nutrition report, 2014,26(4):863 and 867), so the bacillus amyloliquefaciens can be an ideal screening object of biological control strains (biocontrol bacteria). In recent years, many researchers have focused on screening of biocontrol strains, but not many of the wide-spectrum antagonistic strains screened up to now.

Fruits and vegetables, as fresh food, are damaged to different degrees during picking, transportation and storage, and are further polluted by mold and rotten, and even if the fruits and vegetables are stored in a refrigerator, the fruits and vegetables are infected by the mold due to high humidity. Pathogenic bacteria of fruits and vegetables invade the fruits and vegetables through wounds to decompose nutritional ingredients in the fruits and vegetables, so that the quality of the fruits and vegetables is deteriorated, huge economic loss is caused, potential food safety hazards are brought, and the pathogenic bacteria become one of important factors influencing the fruit and vegetable planting industry. Biocontrol bacteria harmless to human and livestock can be used for bacteria prevention, so that rot of fruits and vegetables in the storage period after picking is reduced (Bishihui et al. application research of microorganisms in storage and preservation of fruits and vegetables is advanced [ J ]. food industry science and technology, 2017, (20): 347-. Some researches have focused on the screening and application of mold antagonistic bacteria, such as the antagonism and application of paenibacillus strains not belonging to the genus bacillus to penicillium (screening and application of penicillium antagonistic bacteria after harvest of gujuan. Jiangxi agriculture university, 2013) and preservation of apples by using bacillus cereus fermentation broth (preliminary research on apple preservation by bacillus cereus fermentation broth [ J ] food industry science and technology, 2006, (11): 516-.

In addition to affecting fruit and vegetable storage, mold contamination can also cause deterioration of environmental hygiene in poultry houses, leading to deterioration of animal feed mildew and deterioration, and further to the health of animals, leading to reduced meat quality (Zhang 38387, Yuan et al. Aphelenchomyces and mycotoxins for the hazard and prevention measures of poultry production [ J ]. Jilin zootechnical veterinarian, 2013, (06): 55; Zhang ren Kun et al. common mycotoxins for the hazard and prevention measures of swine [ J ]. modern agriculture technology, 2017, (17): 24-25). In addition to the sanitary management of poultry and livestock houses and the prevention of mold, the inhibition of mold contamination during feed storage is currently a focus of research in the feed industry (auspicious army et al. research on the damage of mycotoxins in feeds and their degradation methods [ J ]. China poultry, 2016,38(5): 34-37).

Similarly, the mold is also the main cause of heating and mildew of the grains. After grains are polluted by the mold, not only the grains are deteriorated, but also the food safety is brought to people (Borui et al. research progress of the mold and the toxin thereof in the paddy [ J ]. Hunan agricultural science, 2009, (11): 85-87).

Fungal diseases of plants are an important problem causing agricultural production and food safety, and invasion of pathogenic microorganisms can cause slow growth, stagnation, influence on appearance and quality of fruits, and even cause destructive death of crops and no grain harvest, thereby causing huge economic loss (thank you for research on screening, identification and antagonistic mechanism of antagonistic microorganisms against phytopathogens [ D ]: Sichuan university, 2004). Conventionally, the prevention and control of plant diseases mainly depend on the use of chemical pesticides and fertilizers, and although the effects are remarkable, the pollution of soil and underground water is caused, which is a more serious ecological problem. The biological pesticide is a new type of green and environment-friendly pesticide, and has great superiority and application potential. It has been theoretically demonstrated that the application of bacillus in the control of plant fungi is feasible (chrysus et al. application of bacillus in the control of plant fungal diseases [ J ]. freshness preservation and processing, 2012,12(5):39-43, 47).

There are several mechanisms by which bacillus antagonises moulds, one of which is known as the "competitive mechanism". The meaning is that in the same environment, ecological site competition exists between bacillus and mould so that nutrient substances (including oxygen) are competed, if the growth rate of the bacillus is greater than that of the mould, the bacillus can occupy the wind and occupy the invasive ecological site early and form protection for animals and plants, so that the mould cannot invade (treet al. biological control research of the bacillus advances [ J ]. Shandong chemical industry, 2015, (12): 55-57). It has been found that during the re-growth of Bacillus, a biofilm is formed on the host surface to prevent the invasion of other pathogenic bacteria (Niu DD et al. the Plant growth-promoting rhizobacterium Bacillus cereus AR156 induced system resistance in Arabidopsis thaliana substrate activating polysaccharide-and japonate/ethylene-dependent amplifying vaccine [ J ]. Mol Plant Microbe interaction, 2011,24(5): 533-.

Bacillus has been shown to be a safe and beneficial microorganism. Toxicity test research on aquaculture animals shows that the bacillus amyloliquefaciens microcapsule is non-toxic and harmless (Anjia et al. safety analysis of bacillus amyloliquefaciens [ J ]. animal medicine progress, 2013, (01): 16-19). The bacillus subtilis viable bacteria agent can be used as oral liquid for treating human intestinal diseases, and can be used as a health product for improving animal intestinal functions (Huiming et al. application research of bacillus subtilis J. Anhui agricultural science, 2008, (27): 11623-. The live Bacillus licheniformis preparation is used as the common medicine for infant's acute and chronic enteritis and diarrhea.

Disclosure of Invention

The invention aims to provide the bacillus for antagonizing the mould and the antibacterial application thereof, the bacillus is safe and non-toxic, can antagonize the mould in a broad spectrum, and can be effectively applied to the fresh keeping of fruits and vegetables.

The invention is realized by the following technical scheme:

the invention discloses a Bacillus for antagonizing mould, belonging to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and preserved in China center for type culture collection with a preservation number of CCTCC No. M2017455.

Preferably, the nucleotide sequence of the bacillus antagonistic to mould is as shown in SEQ ID NO: 1 is shown.

Preferably, the mold-antagonistic bacillus has a rapid growth rate, and the mold-antagonistic bacillus grows for 1h to the logarithmic phase and for 6h to the stationary phase.

Preferably, the mould-antagonistic bacillus has broad spectrum antagonistic properties.

Preferably, the mold-antagonistic bacillus is capable of resisting the growth of penicillium, banana anthracnose, rubber anthracnose, botrytis cinerea, cornus officinalis, aspergillus niger, rhizopus, fusarium oxysporum, and chrysosporium megalophyllum.

The invention also discloses application of the bacillus for antagonizing mould in preparation of a fruit and vegetable antibacterial agent.

The invention also discloses application of the bacillus for antagonizing mould in preparation of biological pesticides.

The invention also discloses application of the bacillus for antagonizing mould in preparation of feed and grain preservation mildewproof agent.

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

1. the bacillus for antagonizing the mould disclosed by the invention is bacillus amyloliquefaciens, belongs to different species and has different application ranges compared with the bacillus subtilis LPL-NM45 disclosed by the prior art with the patent number of 2013.10263918.0.

2. The bacillus for antagonizing the mould disclosed by the invention has higher growth speed, occupies ecological niche and utilizes nutrient substances more quickly, so that the bacillus has competitive advantage and better antagonistic effect. As compared with Bacillus amyloliquefaciens disclosed in application No. 201710100745.9, the time for the strain to enter logarithmic phase is 4h, and the stationary phase is 12h, while the strain provided by the invention reaches the logarithmic phase for 1h and reaches the stationary phase for 6 h. Meanwhile, in order to verify the growth space occupation competitive advantage, the growth curve of the fungus-resistant bacillus disclosed by the invention is determined and compared with the growth curve of the fungus, penicillium is selected as a representative of the fungus in an experiment, and the result shows that the delay period of the penicillium reaches 15-20 h. It is expected that if the mold-antagonistic bacillus of the present invention and the penicillium coexist, the penicillium is not ready for growth when the mold-antagonistic bacillus of the present invention reaches stationary phase at 6 hours. At that time, the bacillus for antagonizing the mold of the invention is already used up locally possible nutrients and forms a protective mycoderm, and the penicillium has no nutrients available and has no chance to grow again. Therefore, the bacillus for antagonizing the mould has great biological site competition capability and can protect relevant objects from being invaded by the mould.

3. The bacillus liquid for antagonizing mildew is combined with a biochemical mildew-proof preparation, and has a positive synergistic effect on the mildew-proof effect. Experiments show that the good fruit rate and the green-keeping efficiency of the winter jujubes after being stored for 90 days are higher than those of the control, and no mildew occurs. The positive synergistic effect of the strain on the biochemical mildew-proof preparation is not reported.

In conclusion, the bacillus for antagonizing the mold, provided by the invention, is safe and non-toxic, can antagonize the mold in a broad spectrum, and can be used for fruit and vegetable preservation, poultry and livestock house environment treatment, refrigeration house and warehouse mold prevention and plant pathogenic bacteria prevention and treatment.

The following collections were made of the present invention for said mould-antagonistic bacillus:

preservation time: 28/8/2017, storage location: wuhan, Hubei, Wuchang Lojia mountain, China Center for Type Culture Collection (CCTCC); the preservation number is CCTCC No. M2017455.

Drawings

FIG. 1 is a lawn morphology of a mold-antagonistic Bacillus species disclosed herein;

FIG. 2 is a graphical representation of the morphological characterization of antagonistic molds disclosed herein, wherein a is gram positive; b is spore staining;

FIG. 3 is a starch hydrolysis loop for mold antagonism as disclosed herein;

FIG. 4 shows PCR amplification products of antagonistic 16S rDNA of molds disclosed in the present invention (lane M is molecular weight standard, lane 1 is PCR product of strain 16S rDNA);

FIG. 5 is a cluster analysis of the 16S rDNA sequence of the antagonistic mold disclosed herein. The KM454172 which is the minimum aggregation class of the antagonistic mold disclosed in the present invention is a known Bacillus amyloliquefaciens (Bacillus amyloliquefaciens Holycan 1);

FIG. 6 is a comparison of the growth rate measurements for antagonistic molds and penicillium disclosed herein;

FIG. 7 shows that the antagonistic mold disclosed in the present invention prevents the colonization and diffusion of Penicillium on the surface of the Ludian navel orange;

FIG. 8 shows that antagonistic mold disclosed in the present invention prevents colonization and spread of Penicillium on the surface of Daxian navel orange;

FIG. 9 shows that the antagonistic mold disclosed in the present invention prevents the colonization and growth of Penicillium on the surface of Citrus unshiu in Hubei;

FIG. 10 is a graph showing the antagonistic mold disclosed herein preventing the colonization of Penicillium on the surface of Citrus sinensis in Han Dynasty;

FIG. 11 shows the inhibitory effect of antagonistic mold disclosed herein on banana anthracnose;

FIG. 12 is a graph showing the inhibitory effect of antagonistic mold disclosed herein on rubber anthrax;

FIG. 13 shows the inhibitory effect of antagonistic molds disclosed herein on Botrytis cinerea;

FIG. 14 shows the inhibitory effect of antagonistic mold disclosed herein on Cornus officinalis Glossium;

FIG. 15 is a graph showing the inhibitory effect of antagonistic mold disclosed herein on Aspergillus niger;

FIG. 16 shows the inhibitory effect of antagonistic mold disclosed herein on Rhizopus;

FIG. 17 shows the inhibitory effect of antagonistic mold disclosed in the present invention on Fusarium oxysporum;

FIG. 18 shows the inhibitory effect of antagonistic mold disclosed herein on Buxus major;

FIG. 19 shows the preservation effect of the method for soaking winter jujubes with antagonistic mold bacteria liquid disclosed by the present invention; the control is tap water immersion;

FIG. 20 shows the synergistic preservative effect of the antagonistic mold bacterial liquid and sodium diacetate disclosed in the present invention; control was sodium diacetate dip.

Detailed Description

The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention.

The screening and identification process of the CCTCC M2017455 strain of the present invention is further described in detail as follows:

1. the culture medium used

(1) LB culture medium: prepared according to a conventional formula and used for culturing bacteria.

(2) Potato culture medium: prepared according to a conventional formula and used for culturing moulds.

(3) Starch hydrolysis culture medium: prepared according to a conventional formula and used for testing the starch hydrolysis capability.

(4) Common medium (m/v):

boiling 6.6% of potato for 20 min, filtering with gauze to obtain juice. Adding the following substances into the potato juice according to the following proportion: glucose 0.66%, peptone 1.25%, beef extract 1.25%, sodium chloride 1.5%, natural pH. The method is used for measuring the antibacterial spectrum of the invention bacteria by a plate confrontation method and measuring and comparing the growth curves of the invention bacteria and the mould.

2. Screening of antagonistic Bacillus

2.1 Source soil sample Collection

The method randomly collects soil samples with humus at 68 positions for later use.

2.2 indicator suspension preparation

The penicillium is inoculated on a slant culture medium of a plurality of potatoes by a slant streak method, and the potatoes are cultured for 7 days at a constant temperature of 28 ℃. Sterile normal saline is added into each tube for 3 times to be 2mL, and the washed spore suspension is collected and combined to be used as indicator bacterial suspension of antagonistic bacteria for spraying.

2.3 Strain selection

Dissolving the humus soil sample in water, and boiling for 5min to kill microorganism trophosome. Then, ten times of dilution is adopted for dilution. Absorbing 100 mu L of soil suspension with different dilutions, coating the soil suspension on an LB culture medium, uniformly spraying indicator bacterium suspension on a flat plate, culturing at 28 ℃ for 48h after surface moisture is basically absorbed, observing antagonistic effect, selecting bacteria with the largest bacterial colony or inhibition zone, performing microscopic examination, and storing strains if the bacteria are bacillus. 6 strains of bacillus which grow rapidly are screened out, and finally, the bacillus belongs to the same strain through identification.

Determination of antimicrobial spectra of 3 antagonistic strains

Inoculating pathogenic bacteria in the center of a common culture medium by adopting a plate confronting method, inoculating the screened antagonistic bacteria at two symmetrical points 2.5cm away from the center when the diameter of the lawn of the pathogenic bacteria is 5mm, observing the growth characteristics of the colony of pathogenic fungi, and judging whether the antagonistic effect is achieved. If the mould colony is round or can cover the screened antagonistic bacteria, no antagonism is shown; if the mold colony is deformed, such as square, diamond, petal shape, etc., it is considered as antagonistic.

Identification of 4 antagonistic strains

4.1 Moss morphology Observation

The bacillus strain is spotted on an LB culture medium, the bacillus strain is cultured for 12-24h at 37 ℃, and when bacterial lawn appears, the characteristics are observed as follows in the figure 1: the color of the lawn is milky white; the central bulge is thickened, and the outer edge is slightly thinner; surface drying; irregular edges or radial growth; the surface wrinkles were severe. The growth characteristics of the bacillus are met.

4.2 gram and spore staining

According to the routine experimental method, see fig. 2, wherein, a, gram-positive staining; and b, spore staining, wherein the staining result accords with the characteristics of the bacillus.

4.3 starch hydrolysis experiments

According to a conventional starch hydrolysis identification method, the bacillus strain is spotted on a starch culture medium, and the bacillus strain is proved to have the capability of starch hydrolysis. Referring to FIG. 3, it is illustrated that the mold-antagonistic Bacillus of the present invention has the characteristics of Bacillus amyloliquefaciens.

4.4 physiological and biochemical assays

And carrying out physiological and biochemical identification on the bacillus with the assistance of a third party. Through identification, the bacillus for antagonizing the mould meets the characteristics of the bacillus amyloliquefaciens. The specific results are shown in table 1 below:

TABLE 1 physiological and biochemical identification of fungi antagonistic Bacillus

Note: positive; -, negative

4.516S rDNA sequence similarity analysis

The bacillus genome DNA is used as a template for PCR amplification, as shown in figure 4, and the sequencing result of the bacillus is shown in SEQ ID NO: 1 (1415bp), and the clustering dendrogram constructed according to the method is shown in FIG. 5. The sequencing results were compared with the sequence of the known Bacillus amyloliquefaciens (KM 454172)16S rDNA sequence in NCBIGeneBank to find that the similarity was 99%, and the Bacillus amyloliquefaciens was highly likely to be Bacillus amyloliquefaciens.

The bacillus is preserved in China center for type culture Collection of microorganisms at 8 months and 28 days in 2017, and the preservation number is CCTCCM 2017455.

The bacillus of the invention has the following characteristics:

1. the color of the bacterial lawn is milky white; the central bulge is thickened, and the outer edge is slightly thinner; surface drying; irregular edges or radial growth; surface wrinkles are severe;

2. the invented bacterium is in the form of short rod, gram-positive, mesogenic spore;

3. the bacterium can secrete amylase to form a starch ring, and has the capability of starch decomposition;

4. the invention bacteria accord with the characteristics of the bacillus amyloliquefaciens by the aid of a third party to carry out physiological and biochemical identification;

5. the 16S rDNA sequence of the invention strain is amplified and compared to find that the similarity of the strain and the corresponding sequence of the known Bacillus amyloliquefaciens KM454172 is 99.0 percent, and the strain is judged to be most likely to belong to the Bacillus amyloliquefaciens;

6. the growth speed of the invented bacteria is very fast (the fastest of the available data). The time for entering the logarithmic phase and the stationary phase is very short, which is beneficial to occupying ecological sites early. The speed of utilizing nutrient substances is faster, and the growth points are protected to prevent the invasion of the mould;

7. the antibacterial spectrum of the bacteria is measured by adopting a plate confronting method, and the inhibition effect on pathogenic bacteria of banana anthracnose, rubber anthracnose mould, botrytis cinerea, cornus officinalis leaf blight mould, aspergillus niger, rhizopus, mountain leaf blight mould and buxus buergeriana is obvious;

8. the invention bacterium is combined with a biochemical mildew-proof preparation, and has positive synergistic effect on the mildew-proof effect.

The effects of the present invention will be further described in detail with reference to the following specific experimental contents:

example 1 comparison of growth rates of mold-antagonistic Bacillus and Penicillium bacteria of the present invention

The mold-antagonistic bacillus and penicillium of the present invention were inoculated into a common culture medium, and the growth curves of the two were measured.

The growth curve of the mold-antagonistic bacillus of the present invention measured spectrophotometrically is shown as line a in fig. 6. It can be seen that the mould-antagonistic bacillus of the present invention has a short time to reach the logarithmic phase and stationary phase, 1h and 6h respectively.

The growth curve of penicillium measured by a dry weight method of the bacteria is shown as a line B in figure 6, and the time for reaching the logarithmic phase and the stationary phase is very long, namely 20h and 60h respectively, which is 10 times as long as the bacillus for antagonizing the mold.

The comparison shows that the bacillus for antagonizing the mould has fast growth speed, can fast colonize and occupy ecological niches, fast consume nutrients, protect animals and plants and prevent pathogenic bacteria from invading.

Example 2 inhibition of colonization growth of Penicillium Rodianum by mold-antagonistic Bacillus bacteria of the present invention

The preparation of the bacillus suspension for antagonizing the mould comprises the following steps: inoculating the mould-resistant bacillus into an LB culture solution, and performing shaking culture at 37 ℃ for 8-15h to obtain the mould-resistant bacillus suspension.

Preparation of penicillium spore suspension: the penicillium is inoculated on a slant culture medium of a plurality of potatoes by a slant streak method, and the potatoes are cultured for 7 days at a constant temperature of 28 ℃. Adding sterile normal saline into each tube for 3 times to obtain 2mL, collecting and combining washed spore suspensions, and obtaining penicillium spore suspension.

Stabbing a square wound with the side length of about 4mm and the depth of 3mm on the waist surface of a commercial Ludian navel orange by using sterile scissors, immersing the wound in the bacterial suspension of the invention for preventive inoculation, and immersing the wound in the penicillium spore suspension after the bacterial liquid is dried for infection inoculation. In the control group, the wound was first immersed in sterile water, dried, and then immersed in a penicillium spore suspension for infection inoculation. Keeping the relative humidity at about 95%, storing at 28 ℃ for two weeks, and observing the infection condition of the mold.

The observation results are shown in FIG. 7. The left part is that fruits are rotten and soft in a large area when the spore bacteria liquid for antagonizing the mold is not sprayed, and the right part is that the wounds of the fruits are intact when the spore bacteria liquid for antagonizing the mold is sprayed on the wounds in advance, which shows that the spore bacteria liquid for antagonizing the mold has good antagonistic effect on the penicillium of the navel orange of the Ludian.

Example 3 inhibition of colonization growth of Penicillium funiculosum Daxian with mold-antagonistic Bacillus bacteria of the present invention

Stabbing a square wound with the side length of about 4mm and the depth of 3mm on the waist surface of a commercially available Daxian navel orange, immersing the wound in the bacterial suspension of the invention for preventive inoculation, drying the bacterial liquid, immersing the wound in the bacterial suspension of penicillium, and performing infection inoculation. In the control group, the wound was first immersed in sterile water, dried, and then immersed in a penicillium spore suspension for infection inoculation. Keeping the relative humidity at about 95%, storing at 28 ℃ for two weeks, and observing the infection condition of the mold.

The observation results are shown in FIG. 8. The left part is the fruit peel which is rotten and soft and crimpled when the bacillus liquid for antagonizing the mould is not sprayed, and the right part is sprayed on the wound in advance, so that the wound of the fruit is intact, which shows that the bacillus liquid for antagonizing the mould has good antagonistic effect on the penicillium funiculosum Daxian orange.

Example 4 inhibition of colonization by Penicillium hupehensis with the mold-antagonistic Bacillus of the present invention

Stabbing a square wound with the side length of about 4mm and the depth of 3mm on the waist surface of the commercially available Hubei emperor orange by using sterile scissors, immersing the wound in the bacterial suspension of the invention for preventive inoculation, and immersing the wound in the penicillium spore suspension after the bacterial liquid is dried for infection inoculation. In the control group, the wound was first immersed in sterile water, dried, and then immersed in a penicillium spore suspension for infection inoculation. Keeping the relative humidity at about 95%, storing at 28 ℃ for two weeks, and observing the infection condition of the mold.

The observation results are shown in fig. 9. The fact that the penicillium spore suspension is sprayed under the condition that the mould-antagonistic bacillus liquid is not sprayed in the upper row leads fruits to be soft and have a large number of mould spores, the fact that the fungi-antagonistic bacillus liquid is sprayed on wounds in advance in the lower row leads the fruits to be soft and have a large number of mould spores, the fact that the fruits are intact and have no mould even if the fungi-antagonistic bacillus liquid is sprayed on the wounds in advance shows that the fungi-antagonistic bacillus liquid has good antagonistic effect on the penicillium of citrus unshiu in Hubei.

Example 5 inhibition of colonization by Penicillium citrosum in Hanzhong by mold-antagonistic Bacillus bacteria of the present invention

Stabbing a square wound with the side length of about 4mm and the depth of 3mm on the waist surface of sweet orange in Hanzhong sold in the market by using sterile scissors, immersing the wound in the bacterial suspension of the invention for preventive inoculation, and immersing the wound in the penicillium spore suspension after the bacterial liquid is dried for infection inoculation. In the control group, the wound was first immersed in sterile water, dried, and then immersed in a penicillium spore suspension for infection inoculation. Keeping the relative humidity at about 95%, storing at 28 ℃ for two weeks, and observing the infection condition of the mold.

The observation results are shown in fig. 10. The titer of penicillium spores sprayed on the wound is sequentially improved from left to right; the upper row of bacillus liquid which is not sprayed with the antagonistic mould of the invention causes the fruits to be soft and have a large amount of mould spores, and the lower row of bacillus liquid which is sprayed with the antagonistic mould of the invention in advance on the wounds leads the fruits to be intact and have no mould; the bacillus liquid for antagonizing the mold has good antagonistic effect on penicillium citrosum in Hanzhong orange.

Example 6 Experimental testing of Bacillus antagonising mold of the invention for antagonism of Musa paradisiaca

The banana anthracnose mould is selected, and the bacteriostatic ability of the mould-antagonistic bacillus is measured by adopting a plate confrontation method. The results are shown in FIG. 11, where the mold lawn did not exhibit circular growth or the growth of Bacillus species covering the mold-antagonistic bacteria of the present invention, indicating that the mold-antagonistic Bacillus species of the present invention have antagonistic activity against banana anthracnose.

Example 7 Experimental testing of mold-antagonizing Bacillus of the invention for antagonizing rubber anthracnose mold

The rubber anthrax is selected, and the bacteriostatic ability of the bacillus for antagonizing the mould is determined by adopting a plate confrontation method. The results are shown in FIG. 12, where the mold lawn did not exhibit circular growth or the growth of Bacillus species covering the mold-antagonistic bacteria of the present invention, indicating that the mold-antagonistic Bacillus species of the present invention have antagonistic activity against B.rubus.

Example 8 Experimental testing of Bacillus antagonistic to mold the invention antagonizes Botrytis cinerea

The botrytis cinerea is selected, and the plate confrontation method is adopted to measure the bacteriostatic ability of the bacillus for antagonizing the mould. The results are shown in FIG. 13, where the mold lawn did not exhibit circular growth or the growth of Bacillus species covering the antagonistic mold of the present invention, indicating that the mold antagonistic Bacillus species of the present invention have antagonistic activity against Botrytis cinerea.

Example 9 Experimental testing of Bacillus antagonising mold and antagonising Douglas Cornus officinalis of the present invention

Selecting the cornus officinalis subtillis, and determining the bacteriostatic ability of the mould-antagonistic bacillus by adopting a plate confrontation method. The results are shown in FIG. 14, where the mold lawn did not exhibit circular growth or the growth of Bacillus covered with the mold-antagonistic bacterium of the present invention, indicating that the mold-antagonistic Bacillus of the present invention has antagonistic ability against Paraquat B.

Example 10 Experimental testing of Bacillus antagonistic to mold and Aspergillus niger antagonistic to mold of the invention

Aspergillus niger is selected, and the bacteriostatic ability of the mould-antagonistic bacillus is measured by adopting a flat plate confronting method. The results are shown in FIG. 15, where the mold lawn did not exhibit circular growth or the growth of Bacillus species covering the mold-antagonistic bacteria of the present invention, indicating that the mold-antagonistic Bacillus species of the present invention are antagonistic to Aspergillus niger.

Example 11 Experimental testing of Bacillus antagonistic to mold and Rhizopus of the invention

Selecting rhizopus, and determining the bacteriostatic ability of the bacillus for antagonizing the mould by adopting a plate confronting method. The results are shown in FIG. 16, where the mold lawn did not exhibit circular growth or the growth of Bacillus covered the mold-antagonistic bacteria of the present invention, indicating that the mold-antagonistic Bacillus of the present invention has antagonistic activity against Rhizopus.

Example 12 Experimental testing of Bacillus antagonistic to mold antagonistic to Fusarium oxysporum mold of the present invention

The mountain leaf blight mould is selected, and the bacteriostatic ability of the mould-antagonistic bacillus is measured by adopting a plate confronting method. The results are shown in FIG. 17, where the mold lawn did not exhibit circular growth or the growth of Bacillus covered with the antagonistic mold of the present invention, indicating that the Bacillus antagonistic to mold of the present invention has antagonistic ability against Fusarium oxysporum.

Example 13 Experimental testing of mold-antagonizing Bacillus of the invention for antagonizing Buxus megalophylla

The chrysosporium megalophyllum mould is selected, and the bacteriostatic ability of the mould-antagonistic bacillus is determined by adopting a plate confronting method. The results are shown in FIG. 18, where the mold lawn did not exhibit circular growth or the growth of Bacillus covered the mold-antagonistic bacteria of the present invention, indicating that the mold-antagonistic Bacillus of the present invention has antagonistic activity against Buxus megatherium.

Example 14 use of mold-antagonistic Bacillus bacteria of the present invention for fresh fruit preservation of winter jujubes

The preparation of the bacillus suspension for antagonizing the mould comprises the following steps: inoculating the mould-resistant bacillus into an LB culture medium, and performing shaking culture at 37 ℃ for 8-15h to obtain the mould-resistant bacillus suspension.

The Chinese dates are soaked in the bacillus suspension for antagonizing the mold for fresh-keeping inoculation, and the Chinese dates are soaked in sterile water for comparison.

Each group is treated with 10 winter jujubes, the winter jujubes are put into perforated plastic bags after being naturally aired, stored in a freezer with the relative humidity of about 95 percent and the temperature of 4 ℃, and the states of the winter jujubes are observed in 45 days and 90 days respectively. The good fruit rate, the green-keeping efficiency and the mildew-proof efficiency of the winter jujubes are calculated by the following formulas.

The results are shown in fig. 19, after the control group (sterile water group) is stored for 90 days, the good fruit rate of the winter jujubes is only 20%, the green-keeping efficiency is 20%, and the mildew-proof efficiency is 50%; if the bacillus suspension for antagonizing the mould is adhered to the surface of the winter jujube, the good fruit rate of the winter jujube is 70 percent after the winter jujube is stored for 90 days, and the good fruit rate is improved by 40 percent compared with the control; the green-keeping efficiency is 50%, which is improved by 30% compared with the control; the mildew-proof efficiency is 100 percent, which is improved by 50 percent compared with the control group. The fungus-resistant bacillus liquid has the mildew-proof effect on winter jujubes and also has the effects of keeping green and preserving freshness.

Example 15 synergistic freshness test of mold-antagonistic bacillus solution and sodium diacetate of the present invention

Preparing a chemical mildew-proof fresh-keeping solution: 0.04g/ml sodium diacetate solution is prepared to be used as chemical fresh-keeping solution.

Preparation of biochemical compound mildew-proof fresh-keeping liquid: sodium diacetate was added to the bacillus suspension of antagonistic mould of the invention to make the concentration 0.04g/ml, as a compound preservative solution.

The experimental group soaks winter jujubes in biochemical compound mildew-proof fresh-keeping liquid, and the control group soaks winter jujubes in chemical mildew-proof fresh-keeping liquid. Processing 10 fruits in each group, air drying winter jujube, placing into perforated plastic bag, storing in refrigerator at 4 deg.C with relative humidity of 95%, and observing winter jujube state in 45 days and 90 days respectively. The good fruit rate, the green-keeping efficiency and the mildew-proof efficiency of the winter jujubes are calculated by the following formulas.

The results are shown in fig. 20, after the winter jujubes soaked by the chemical mildew-proof preservative solution are stored for 90 days, the good fruit rate of the winter jujubes is 50%, the green-keeping efficiency is 20%, and the mildew-proof efficiency is 80%; after the winter jujubes soaked by the compound mildew-proof fresh-keeping liquid are stored for 90 days, the good fruit rate of the winter jujubes is 80 percent, which is improved by 30 percent compared with the single chemical group protection and 10 percent compared with the single bacteria liquid protection; the green-keeping efficiency is 70%, which is improved by 50% compared with single chemical group protection and 20% compared with single bacteria liquid protection; the mildew-proof efficiency is 100 percent, is improved by 20 percent compared with the single chemical group protection, and has the same mildew-proof effect with the single bacteria liquid. The combination of the bacillus liquid for antagonizing the mold and the chemical mildew preventive (sodium diacetate) has the positive synergistic green-keeping, hard-keeping and fresh-keeping effects.

In conclusion, the invention discloses a bacillus capable of antagonizing mould and application thereof. The Bacillus strain for antagonizing the mould belongs to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is as follows: CCTCC M2017455, and the preservation date is 8 months and 28 days in 2017.

The bacillus with mold antagonism can resist the growth of penicillium, banana anthracnose, rubber anthracnose, botrytis cinerea, cornus officinalis leaf blight, aspergillus niger, rhizopus, mountain leaf blight mold and buxus chrysosplenium, and has broad-spectrum antagonistic activity on plant pathogenic mold.

The bacillus which antagonizes mould belongs to probiotics and is safe to human and animals.

The bacillus with antagonistic mould can be used for keeping fruits and vegetables fresh and preventing mould of a refrigeration house, so that the fruits and vegetables are prevented from mildewing; can be used for the sanitary management of livestock houses and preventing the occurrence of diseases of the livestock; can prevent feed and grain mould and ensure the safety of feed and grain; can be used as biological pesticide for preventing plant mould diseases. Therefore, the bacillus with mould antagonism has larger biological control potential and good application prospect.

Sequence listing

<110> university of west ampere traffic

<120> bacillus for antagonizing mould and antibacterial application thereof

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>1415

<212>DNA

<400>1

tgcagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa 60

cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120

tggttgtctg aaccgcatgg ttcagacata aaaggtggct tcggctacca cttacagatg 180

gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcgac gatgcgtagc 240

cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact cctacgggag 300

gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc cgcgtgagtg 360

atgaaggttt tcggatcgta aagctctgtt gttagggaag aacaagtgcc gttcaaatag 420

ggcggcacct tgacggtacc taaccagaaa gccacggcta actacgtgcc agcagccgcg 480

gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagggct cgcaggcggt 540

ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcattgg aaactgggga 600

acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc gtagagatgt 660

ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa ctgacgctga ggagcgaaag 720

cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctaa 780

gtgttagggg gtttccgccc cttagtgctg cagctaacgc attaagcact ccgcctgggg 840

agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 900

atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctctgacaat 960

cctagagata ggacgtcccc ttcgggggca gagtgacagg tggtgcatgg ttgtcgtcag 1020

ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga tcttagttgc 1080

cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga aggtggggat 1140

gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggacagaac 1200

aaagggcagc gaaaccgcga ggttaagcca atcccacaaa tctgttctca gttcggatcg 1260

cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc agcatgccgc 1320

ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag tttgtaacac 1380

ccgaagtcgg tgaggtaacc tttatggagc cagcc 1415

Claims

1. The bacillus for antagonizing the mold is characterized in that the bacillus is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) which is preserved in China center for type culture collection with the preservation number of CCTCC No. M2017455.

2. Use of a mould-antagonistic bacillus of claim 1 for the preparation of an antimicrobial agent for fruits and vegetables.

3. Use of a mould-antagonistic bacillus bacterium according to claim 1 for the preparation of a biopesticide.

4. The use of a mold-antagonistic bacillus of claim 1 in the preparation of a feed and grain preservation fungicide.