CN110042075B - Water chestnut endophytic bacillus subtilis and application thereof - Google Patents

Abstract

The invention provides a water chestnut endophytic Bacillus subtilis HUB0219 which is preserved in Guangdong province microbial strain preservation center (GDMCC), wherein the preservation number is GDMCC No. 60639, and the preservation date is 2019, 5 months and 6 days. The bacillus subtilis HUB0219 has good antagonistic action on various common plant pathogenic fungi, has an obvious effect on inhibiting the water chestnut rot, and has the potential of being developed into agricultural biocontrol microbial agents and biological preservatives.

Description

Water chestnut endophytic bacillus subtilis and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, relates to bacillus subtilis and application thereof, and particularly relates to water chestnut endophytic bacillus subtilis and application thereof.

Background

With the improvement of living standard and the change of consumption concept, people pay more attention to food safety and ecological safety, and the search for a novel bacteriostatic agent which is safe, effective, low in toxicity and low in residue becomes a hotspot of researches on plant protection and postharvest preservation of fruits and vegetables. The biological control and biological preservation by using beneficial microorganisms through bacteria inhibition are one of important strategies and trends of sustainable development of modern agriculture, and particularly, the biological control of soil-borne diseases or the storage and preservation of underground organs of plants are effective measures and are important components in plant protection and biological preservation and preservation.

Among the biocontrol bacteria, bacillus is representative, and nearly 50 commercial preparations have been successfully developed, mainly including Bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, bacillus mycoides, and Bacillus pumilus. They can colonize plant rhizosphere, body surface or in vivo, and have biocontrol effects on rice sheath blight disease, bean root rot, apple moldy core disease, cotton blight, cotton wilt and the like caused by filamentous fungi by competing with pathogenic bacteria for peripheral nutrition, secreting antibacterial substances such as peptides, phospholipids and polyenes, or inducing a plant defense system to resist the invasion of the pathogenic bacteria. In the past, environmental microorganisms are mostly discovered from plant roots for the research and development of biocontrol bacteria, and the colonization ability and efficiency of the environmental microorganisms are often one of important factors influencing the application effect.

Water chestnuts, also known as water chestnuts, are sweet, crisp, tasty and refreshing, have much juice and no residues, and are important characteristic fruits in south of the Ridge in winter and spring. The total yield of the water chestnuts is not high, the fresh water chestnuts are mainly sold and are on the market due to the short harvesting period, the water chestnuts are thin in peel and much in juice, the water chestnuts are easy to dehydrate, wither, rot and deteriorate, the storage period is short, the market demand cannot be met, and the income of farmers and the development of the water chestnut industry are also severely restricted. Therefore, the research on the water chestnut storage and preservation technology and products is very key for solving the contradiction between seasonal production and perennial consumption of the water chestnuts. The storage and preservation with mud are not only low in cost and simple in treatment, but also easy to implement in large scale, and are the most important preservation way.

Pathogenic fungal infestation is the most important cause of rottenness of water chestnut stores. The most important fungi causing the corm rot of chufa in the storage period include Trichoderma asperellum, fusarium oxysporum, rhizoctonia solani (Thielaviopsis paradoxa), and the like. The traditional water chestnut storage and preservation method comprises a stacking method, a ceramic jar storage method, a fine sand storage method, a soil storage method, a cellaring method and the like, but the storage effect is unstable, even large-area rotting and storage failure can be caused due to the complexity of the influence of factors such as different collection time, different storage temperature, environmental microorganism distribution and the like on the water chestnut physiology. Therefore, the proper bacteriostasis pretreatment is carried out before the water chestnuts are stored in a warehouse, so that the fungal density in a microenvironment in the storage period can be greatly reduced, and the storage and preservation effects can be improved.

CN103907674A discloses a compound preservative for storing water chestnuts and a preparation method thereof, wherein the compound preservative comprises 1-2% of pullulan, 0.2-0.4% of lauric acid monoglyceride, 0.01-0.03% of ascorbic acid and the balance of water by mass percent.

CN106359568A discloses a fresh-keeping method for fresh-cut water chestnuts, which comprises the following steps: (1) Removing soil from corm Eleocharitis peel, soaking in ozone water, and cleaning with clear water; (2) soaking the whole water chestnut in hot water; (3) naturally drying the water chestnut fruits after heat treatment; (4) removing the water chestnut peels, and cleaning with clear water; (5) Soaking the peeled corm Eleocharitis in a mixed solution containing citric acid, malic acid and corm Eleocharitis peel extract; (6) Coating the fresh-cut water chestnuts with chitosan, and drying the water chestnuts by cold air; (7) And packaging the fresh-cut water chestnuts dried by cold air in a vacuum packaging machine within 20 minutes to obtain finished products, but the taste of the water chestnuts is influenced by the method, and the residues are serious.

Therefore, the novel bacteriostatic agent which is safe, effective, low in toxicity and low in residue is provided for storing and preserving the water chestnuts, and has important significance in the field of modern agriculture.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the water chestnut endophytic bacillus subtilis and the application thereof, wherein the bacillus subtilis is a plant endophyte from edible healthy water chestnuts and has the advantages of high fungus antagonistic activity, wide antibacterial spectrum, strong colonization capability, environmental friendliness, high safety and the like.

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

in a first aspect, the invention provides Bacillus subtilis named as Bacillus subtilis HUB0219, which is deposited in Guangdong province microbial culture collection center (GDMCC) and is addressed to Guangdong province microbial research institute of No. 59 great institute of Michelia Tokor, michelia, guangzhou, 510070 with the deposition number of GDMCC No. 60639 and the deposition date of 2019, 5 months and 6 days.

According to the invention, the bacillus subtilis HUB0219 is separated from healthy water chestnut bulbs, the bacterial colony is light white and opaque, the bacterial colony is a gram-positive bacterium and is multi-single-grown, the bacterial strain forms an oval spore, the bacterial strain has a good antagonistic effect on various common plant pathogenic fungi, has an obvious effect on inhibiting water chestnut rot, and has the potential of being developed into agricultural biocontrol microbial agents and biological preservatives.

In a second aspect, the invention provides an application of the bacillus subtilis in the first aspect in preparing a biocontrol microbial inoculum.

Preferably, the concentration of Bacillus subtilis is (1.0-2.0). Times.10 ⁸ CFU/mL, for example, may be 1.0X 10 ⁸ CFU/mL、1.1×10 ⁸ CFU/mL、1.2×10 ⁸ CFU/mL、1.3×10 ⁸ CFU/mL、1.4×10 ⁸ CFU/mL、1.5×10 ⁸ CFU/mL、1.6×10 ⁸ CFU/mL、1.7×10 ⁸ CFU/mL、1.8×10 ⁸ CFU/mL、1.9×10 ⁸ CFU/mL or 2.0X 10 ⁸ CFU/mL。

In a third aspect, the present invention provides a biocontrol agent comprising a bacillus subtilis according to the first aspect.

Preferably, said subtillis areThe concentration of bacillus is (1.0-2.0) x 10 ⁸ CFU/mL, for example, may be 1.0X 10 ⁸ CFU/mL、1.1×10 ⁸ CFU/mL、1.2×10 ⁸ CFU/mL、1.3×10 ⁸ CFU/mL、1.4×10 ⁸ CFU/mL、1.5×10 ⁸ CFU/mL、1.6×10 ⁸ CFU/mL、1.7×10 ⁸ CFU/mL、1.8×10 ⁸ CFU/mL、1.9×10 ⁸ CFU/mL or 2.0X 10 ⁸ CFU/mL。

Preferably, the biocontrol bacterial agent further comprises any one or a combination of at least two of pharmaceutically acceptable carriers, excipients or diluents.

In a fourth aspect, the present invention provides a method for preparing the biocontrol microbial inoculum as described in the third aspect, said method comprising the steps of:

(1) Inoculating bacillus subtilis into an LB culture medium, and performing shaking culture to obtain a seed solution;

(2) Absorbing the seed liquid, inoculating the seed liquid into a fermentation culture medium, and performing fermentation culture;

(3) And filtering the fermentation liquor to obtain the biocontrol microbial inoculum.

Preferably, the temperature of the shaking culture in the step (1) is 25 to 35 ℃, for example, 25 ℃, 26 ℃, 27 ℃,28 ℃, 29 ℃,30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃ or 35 ℃, preferably 25 to 28 ℃.

Preferably, the shaking culture speed in step (1) is 120-200 r/min, such as 120r/min, 130r/min, 140r/min, 150r/min, 160r/min, 170r/min, 180r/min, 190r/min or 200r/min, preferably 150-200 r/min.

Preferably, the shaking culture time in the step (1) is 20-25 h, for example, 20h, 21h, 22h, 23h, 24h or 25h, preferably 24h.

Preferably, the pH of the fermentation medium in step (2) is 6.5 to 7.5, and may be, for example, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.

Preferably, the fermentation culture temperature in step (2) is 25 to 35 ℃, for example, 25 ℃, 26 ℃, 27 ℃,28 ℃, 29 ℃,30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃ or 35 ℃, preferably 25 to 28 ℃.

Preferably, the time for fermentation culture in step (2) is 20-25 h, for example, 20h, 21h, 22h, 23h, 24h or 25h, preferably 24h.

Preferably, the filtration of step (3) is performed using a 0.22 μm bacterial filter.

As a preferable technical solution, the present invention provides a method for preparing the biocontrol microbial inoculum according to the third aspect, the method comprising the steps of:

(1) Inoculating bacillus subtilis into an LB culture medium, and carrying out shaking culture at the temperature of 25-35 ℃ and the speed of 120-200 r/min for 20-25 h to obtain seed liquid;

(2) Absorbing the seed liquid, inoculating the seed liquid into a fermentation culture medium with the pH value of 6.5-7.5, and fermenting and culturing for 20-25 h at the temperature of 25-35 ℃;

(3) And filtering the fermentation liquor by using a 0.22-micron bacteria filter to obtain the biocontrol microbial inoculum.

In a fifth aspect, the present invention provides the use of a bacillus subtilis according to the first aspect and/or a biocontrol agent according to the third aspect for inhibiting phytopathogens.

Preferably, the phytopathogen comprises any one of, or a combination of at least two of, moniliforme (Thielaviopsis paradoxa), trichoderma asperellum (Trichoderma asperellum), viridae (Penicillium digitorum), penicillium citrinum (p. Italicum), mango anthrax (Colletotrichum gloeosporioides) or maize microsporum maydis.

In a sixth aspect, the invention provides an application of the bacillus subtilis in the first aspect and/or the biocontrol microbial inoculum in the third aspect in fruit fresh-keeping.

Preferably, the plant comprises any one or a combination of at least two of water chestnut, citrus, mango or corn, preferably water chestnut.

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

(1) The bacillus subtilis HUB0219 has good antagonistic effect on a plurality of common plant pathogenic fungi such as moniliforme (Thielavisosis paradoxa), trichoderma asperellum (Trichoderma asperellum), viride (Penicillium digitatum), penicillium citrinum (P.italicum), mango anthrax (Colletotrichum gloeosporioides) and maize microsporum maydis;

(2) The bacillus subtilis HUB0219 has an obvious effect of inhibiting the generation of rottenness of the water chestnuts in the storage process, and the perfectness rate of the water chestnuts is as high as 97.5 percent and 95 percent when the water chestnuts are stored for 30 days and 60 days;

(3) The bacillus subtilis HUB0219 is expected to be developed into agricultural biocontrol bacteria and biological antistaling agent.

Drawings

FIG. 1 shows the colony morphology of Bacillus subtilis HUB 0219;

FIG. 2 shows the gram stain result of Bacillus subtilis HUB 0219;

FIG. 3 shows the spore staining result of Bacillus subtilis HUB 0219;

FIG. 4 (A) is the result of verifying the pathogenicity of Bacillus subtilis HUB0219 to water chestnut, and FIG. 4 (B) is the enlarged result of FIG. 4 (A);

FIG. 5 (A) is a result of culturing Bacillus subtilis HUB0219 in opposition to Rhinoconosis (Thielavia paradoxa), FIG. 5 (B) is a result of culturing Bacillus subtilis HUB0219 in opposition to Trichoderma asperellum, FIG. 5 (C) is a result of culturing Bacillus subtilis HUB0219 in opposition to Trichoderma citricola, FIG. 5 (D) shows the result of culturing Bacillus subtilis HUB0219 in the presence of Penicillium citrinum in opposition, FIG. 5 (E) shows the result of culturing Bacillus subtilis HUB0219 in the presence of mango anthrax (Colletotrichum gloeosporioides), and FIG. 5 (F) shows the result of culturing Bacillus subtilis HUB0219 in the presence of corn microsporum maydis in the presence of Bacillus subtilis HUB0219 in the absence of Bacillus subtilis in opposition;

FIG. 6 (A) is a graph showing the inhibitory effect of Bacillus subtilis HUB0219 on Rhinocytosis (Thielavirosis paradoxa) on water chestnut, and FIG. 6 (B) is a blank control;

FIG. 7 (A) is the inhibitory effect of Bacillus subtilis HUB0219 on Trichoderma asperellum (Trichoderma asperellum) on water chestnut, and FIG. 7 (B) is a blank control.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and that no limitation of the invention is intended.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 acquisition and morphological characterization of Bacillus subtilis HUB0219

(1) Strain acquisition

The strain HUB0219 was isolated from healthy water chestnuts in the Ridge Tochu village of northern village of Lechang, guangdong province by the inventor Xu Liangxiong at 2016, 01, 14.

(2) Morphological identification of strains

Inoculating the separated strain to an NA solid culture medium, and culturing at 30 ℃ for 48h, wherein the single colony is in a form shown in figure 1, and the colony is light white and opaque, has a dry, flat and unsmooth surface, irregular and lusterless edge, uniform texture, easy picking and no wire drawing phenomenon; the colonies are inoculated in an LB liquid culture medium and are subjected to constant temperature shaking culture at 30 ℃ for 18-24 hours, the culture medium becomes turbid, and no mycoderm is generated on the surface.

The microscopic examination result after gram staining is shown in FIG. 2, the thallus is purple, is gram-positive bacteria, bacillus and polymorphous, and has the size of (0.5-1) × (1.5-2.5) μm; as shown in fig. 3, the strain formed oval spores.

Example 2 physiological and Biochemical characterization of Bacillus subtilis HUB0219

Refer to Berger's Manual of identification of bacteria (eighth edition) and Manual of identification of common bacteria System, for utilization of sugar alcohol, nitrate-reduced macromolecules, and various enzymes of HUB0219 strainAnd 30 physiological and biochemical indexes such as growth temperature and salt tolerance. The strain can ferment and utilize saccharides such as glucose, sucrose, fructose, mannose, maltose, and alcohols such as glycerol, mannitol, inositol, but cannot utilize sugar alcohol substances such as arabitol, rhamnose, lactose; has gelatinase activity, but does not have beta-galactosidase, urease, tryptophan deaminase and other enzyme activities; failure to produce H ₂ S, no indole substance is produced, citrate cannot be utilized, but 3-hydroxy butanone can be produced. Specific results are shown in Table 1 for the API50CH sugar alcohol fermentation test and API20E main physiological characteristics of Bacillus subtilis HUB0219, "+" indicates positive and "-" indicates negative.

TABLE 1

Example 3 molecular characterization of Bacillus subtilis HUB0219

Obtaining a single colony by streaking the strain through a plate, picking the single colony in a bacteria shaking tube containing 2mL of TSB, and placing the bacteria shaking tube at 28 ℃ for shaking culture until the bacteria is turbid; taking 1mL of bacterial liquid in a centrifuge tube, centrifuging at 10000rpm for 3min, removing supernatant, adding 50 mu L of sterile water, vibrating uniformly, performing heat preservation treatment at 95 ℃ for 2min, and placing in a 0 ℃ refrigerator for later use;

the primers 27F and 1492R were selected according to the literature for PCR amplification of the genome of the strain under conditions of 94 ℃ pre-denaturation for 3min,35 cycles (94 ℃ denaturation 30s,50 ℃ annealing for 45s,72 ℃ extension for 100 s), 72 ℃ extension for 7min, and the PCR amplification was introduced as follows:

27F(SEQ ID NO:1)：5’-TACGGYTACCTTGTTACGACTT-3’；

1492R(SEQ ID NO:2)：5’-TACGGYTACCTTGTTACGACTT-3’.

TABLE 2 PCR reaction System

Adding 1 mul sample buffer solution into 5 mul amplification product, mixing, performing agarose gel electrophoresis for 45min, and detecting the amplification result under an ultraviolet lamp; recovering the DNA fragment by using a TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 Kit;

the recovered fragments are sent to Guangzhou Ai Ji biotechnology limited company for sequencing, the sequencing result shows that the 16S rRNA sequence fragment SEQ ID NO:3 (1396 bp in total) of the strain is compared with a Bacillus subtilis sequence in GenBank through BLAST, the result shows that the similarity (maxidiversity) of the strain and the Bacillus subtilis sequence is 100%, the coverage range (query cover) of the comparison sequence is 100%, and the E value is 0, so that the strain is identified as Bacillus subtilis HUB0219, and the identification result is reliable.

SEQ ID NO:3：

GAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTT.

Example 4 strain preservation

Based on the morphological observation, physiological and biochemical analysis and molecular identification results of the embodiments 1-3, the strain HUB0219 belongs to Bacillus subtilis, and the Bacillus subtilis HUB0219 is preserved in Guangdong province microbial cultures collection center (GDMCC) in 2019, 5 and 6 days, and the address is Michelia Tokyo 100 # Okayao 59-Guangdong province microbial research institute in Guangzhou city, zip code 510070, and the preservation number is GDMCC No. 60639.

Example 5 pathogenicity verification of Bacillus subtilis HUB0219 on Water chestnut

Cleaning fresh harvested healthy water chestnuts, soaking the water chestnuts in 70% ethanol for 1min for surface disinfection, and cutting two cuts with the diameter of about 10mm and the depth of about 3mm at the position above the equator of the corm of the water chestnuts by using a sterile knife after air drying; dipping with sterile cotton swab to obtain the product with concentration of 1.0 × 10 ⁸ Cutting with CFU/mL bacterial suspension, processing 5 corm Eleocharitis per strain with sterile water as blank control, placing in sterile transparent lunch box, and culturing in dark room at 25 deg.C.

The results are shown in fig. 4 (A) and fig. 4 (B), the water chestnut morbidity is observed on the 4 th day after inoculation, and the bacillus subtilis HUB0219 is found not to infect the water chestnuts, the water chestnut epidermis is intact, the meat quality is hard, and the cross-cut meat quality is white; no obvious rottenness phenomena such as sinking, liquid seepage and the like of the water chestnuts near the wound still appear at 10 days after inoculation.

Example 6 in vitro bacteriostatic assay for Bacillus subtilis HUB0219

The fungal antagonistic activity of the bacillus subtilis HUB0219 is tested by adopting a plate confronting method, and the method comprises the following steps: use of a sterile punch with an internal diameter of 6mm in pathogenic fungiCutting small bacterial cake at the edge of bacterial colony, inoculating small pathogenic bacterial cake 20mm away from the edge of the plate on PDA culture medium by using sterile inoculating needle, and inoculating 2 μ L of small bacterial cake with concentration of 1.0 × 10 at 20mm away from the edge of the plate ⁸ CFU/mL HUB0219 bacterial suspension;

the preparation method of the HUB0219 bacterial suspension comprises the following steps: inoculating single colony strain into 3mL LB culture medium, performing shaking culture at 28 deg.C for 24h at 150r/min to obtain first-stage seed solution, sucking 1mL of first-stage seed solution, inoculating into a triangular flask containing 100mL LB culture medium, and culturing for 24h.

Culturing at 28 deg.C for 4 days, and observing antagonistic effect of Bacillus subtilis HUB0219 on pathogenic fungi. As a result, it was found that Bacillus subtilis HUB0219 exhibited remarkable antagonistic effects against 6 pathogenic fungi, among which the antagonistic effect against Rhinocosoma (Thielavissis paradoxa) (FIG. 5 (A)), against Citrus reticulata (Penicillium digitatum) (FIG. 5 (C)), against Penicillium citrinum (FIG. 5 (D)), against Rhinochloropsis mango (Colletrichoides) (FIG. 5 (E)), and against Pseudocerus zeae (Helminthosporum maydis) (FIG. 5 (F)) was superior to that against Trichoderma asperellum (FIG. 5 (B)).

Example 7 bacteriostatic experiment of controlling infection in whole fruit of water chestnut

Treating corm Eleocharitis by wound-cutting inoculation method as in example 5, and picking with sterile cotton swab to obtain corm Eleocharitis with concentration of 2.0 × 10 ⁸ Coating CFU/mL HUB0219 bacterial solution on the incision, inoculating a small pathogenic bacterium cake on the surface of the incision, treating 10 water chestnuts by each bacterium by using sterile water and a pathogenic bacterium agar block as a blank control, loading into a sterile transparent lunch box, and preserving in a dark room at normal temperature. And observing the disease condition of the water chestnuts after 4 days, and counting the perfectness rate of the water chestnut cuts and the inhibition rate of the bacillus subtilis HUB0219 on the cut rotting.

The calculation formula of the completeness rate of the water chestnut cuts is as follows:

G＝(N ₀ -N)/N ₀ ×100％

wherein G is the cut perfectness rate of water chestnut and N ₀ The number of the cuts is the original number of the chufa, and N is the number of the cuts and rot of the chufa;

the calculation formula of the inhibition rate of bacillus subtilis HUB0219 on the canker is as follows:

inhibition ratio (%) = (100-G)%

As a result, as shown in FIGS. 6 (A) and 6 (B), the inhibition rate of Bacillus subtilis HUB0219 against Rhinoceros (Thielavirosis paradoxa) was 75%, and as shown in FIGS. 7 (A) and 7 (B), the inhibition rate of Bacillus subtilis HUB0219 against Trichoderma asperellum (Trichoderma asperellum) was 30%.

Example 8 preservation experiment of whole water chestnut with mud

Collecting soil of water chestnut field, air drying, pulverizing, sieving with 60 mesh sieve, dividing into 6 equal parts, each 500g, respectively spraying 125mL Bacillus subtilis HUB0219 suspension to 3 parts, and spraying equal amount of sterile water to three parts as blank control;

selecting 60 healthy water chestnuts with proper sizes and intact pericarps, dividing into 6 groups, placing in a carton, respectively and uniformly mixing the soil and the water chestnuts, sealing the carton, placing in a dark room at normal temperature for storage, observing and recording the completeness rate of the water chestnuts after 1 month, wherein the calculation formula of the completeness rate is as follows:

G _n ＝(N ₀ -N)/N ₀ ×100％

in the formula, G _n The perfectness rate of the water chestnuts in N days is N ₀ The number of the water chestnuts is the original number of the water chestnuts, and N is the number of the water chestnuts rotted.

Experimental results show that the completeness rates of the water chestnuts stored for 30 days and 60 days are 97.5% and 95% respectively in the treatment group added with the bacillus subtilis HUB0219 bacterial suspension, and are superior to the completeness rates (65% and 60%) of the control group, so that the bacillus subtilis HUB0219 has an obvious effect of inhibiting the rottenness caused by fungal infection of the water chestnuts in the storage process.

Example 9

Using the same method as in example 7, cut-inoculated citrus, mango and corn were examined for the inhibitory effect of HUB0219 on Rhinoceros (Thielavirosis paradoxa).

Comparative example

Selecting commercially available bacillus subtilis, treating water chestnut, orange, mango and corn by a cutting inoculation method, and detecting the inhibition effect of the commercially available bacillus subtilis on rhizopus moniliforme (Thielavirosis paradoxa).

As a result, as shown in Table 3, HUB0219 exhibited a better inhibitory effect against Rhinoceros (Thielavirosis paradoxa) than commercial Bacillus subtilis.

TABLE 3

Variety of fruit	HUB0219	Commercially available Bacillus subtilis
			Water chestnut	75％	33％
Citrus fruit	71％	42％
			Mango (mango)	66％	45％
Corn (corn)	69％	26％

In conclusion, the bacillus subtilis HUB0219 has good antagonistic action on a plurality of common plant pathogenic fungi such as moniliforme (Thielaviopsis paradoxa), trichoderma asperellum (Trichoderma asperellum), viridis (Penicillium digitatum), penicillium citrinum (P.italicum), mango anthrax (Colletotrichum gloeosporioides) and maize small spot pathogen (Helminthosporium maydis); the water chestnut preservative has an obvious effect of inhibiting the generation of rottenness in the water chestnut storage process, the perfectness rate of the water chestnut is as high as 97.5 percent and 95 percent when the water chestnut is stored for 30 days and 60 days, and the water chestnut preservative is expected to be developed into agricultural biocontrol bacteria and biological preservative.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Sequence listing

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tgcagaagag gagagtggaa ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa 660

caccagtggc gaaggcgact ctctggtctg taactgacgc tgaggagcga aagcgtgggg 720

agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag 780

ggggtttccg ccccttagtg ctgcagctaa cgcattaagc actccgcctg gggagtacgg 840

tcgcaagact gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt 900

ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac atcctctgac aatcctagag 960

ataggacgtc cccttcgggg gcagagtgac aggtggtgca tggttgtcgt cagctcgtgt 1020

cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgatcttagt tgccagcatt 1080

cagttgggca ctctaaggtg actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1140

aatcatcatg ccccttatga cctgggctac acacgtgcta caatggacag aacaaagggc 1200

agcgaaaccg cgaggttaag ccaatcccac aaatctgttc tcagttcgga tcgcagtctg 1260

caactcgact gcgtgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat 1320

acgttcccgg gccttgtaca caccgcccgt cacaccacga gagtttgtaa cacccgaagt 1380

cggtgaggta accttt 1396

Claims (17)

1. The Bacillus subtilis is named as Bacillus subtilis HUB0219 and is preserved in Guangdong province microbial culture collection center (GDMCC), the preservation number is GDMCC No. 60639, and the preservation date is 2019, 5 months and 6 days.

2. Use of the bacillus subtilis according to claim 1 for preparing a biocontrol microbial inoculum.

3. The use according to claim 2, wherein the Bacillus subtilis is present at a concentration of (1.0 to 2.0) x 10 ⁸ CFU/mL。

4. A biocontrol microbial inoculum comprising the bacillus subtilis of claim 1.

5. The biocontrol microbial inoculum of claim 4 wherein the concentration of Bacillus subtilis is (1.0-2.0) x 10 ⁸ CFU/mL。

6. The biocontrol agent of claim 4, further comprising any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

7. A method of preparing the biocontrol bacterial agent as claimed in claim 4, wherein said method comprises the steps of:

(1) Inoculating bacillus subtilis into an LB culture medium, and performing shaking culture to obtain a seed solution;

(2) Absorbing the seed liquid, inoculating the seed liquid into a fermentation culture medium, and performing fermentation culture;

(3) And filtering the fermentation liquor to obtain the biocontrol microbial inoculum.

8. The method according to claim 7, wherein the temperature of the shaking culture in the step (1) is 25 to 35 ℃.

9. The method according to claim 7, wherein the shaking culture in the step (1) is performed at a speed of 120 to 200r/min.

10. The method according to claim 7, wherein the shaking culture in the step (1) is carried out for 20 to 25 hours.

11. The method according to claim 7, wherein the pH of the fermentation medium in the step (2) is 6.5 to 7.5.

12. The method according to claim 7, wherein the temperature of the fermentation culture in the step (2) is 25 to 35 ℃.

13. The method according to claim 7, wherein the fermentation culture time in the step (2) is 20 to 25 hours.

14. The method according to claim 7, wherein the filtration in the step (3) is performed using a 0.22 μm bacterial filter.

15. The method for preparing a peptide according to any one of claims 7 to 14, characterized in that it comprises the following steps:

(1) Inoculating the bacillus subtilis into an LB culture medium, and carrying out shaking culture at the temperature of 25-35 ℃ and the speed of 120-200 r/min for 20-25 h to obtain a seed solution;

(2) Absorbing the seed liquid, inoculating the seed liquid into a fermentation culture medium with the pH of 6.5-7.5, and fermenting and culturing for 20-25 h at the temperature of 25-35 ℃;

(3) And filtering the fermentation liquor by adopting a 0.22-micron bacterial filter to obtain the biocontrol microbial inoculum.

16. Use of the bacillus subtilis of claim 1 and/or the biocontrol agent of claim 4 for inhibiting phytopathogens;

the plant pathogenic bacteria are any one or a combination of at least two of moniliforme (Thielavissis paradoxa), trichoderma asperellum (Trichoderma asperellum), viridae (Penicillium digitorum), penicillium citrinum (P. Italicum), mango anthrax (Colletotrichum gloeosporioides) or maize macula maydai.

17. Use of the bacillus subtilis of claim 1 and/or the biocontrol agent of claim 4 for fruit preservation;

the fruit is water chestnut.

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