CN113684158A

CN113684158A - Siamese bacillus JY-1 and preparation and application thereof

Info

Publication number: CN113684158A
Application number: CN202111178812.1A
Authority: CN
Inventors: 蒋军喜; 欧阳慧; 虞凡霜; 刘冰; 熊桂红; 彭文文; 李帮明; 涂贵庆; 黄英金; 周庆红; 肖遥
Original assignee: Jiangxi Agricultural University
Current assignee: Jiangxi Agricultural University
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2021-11-23

Abstract

The invention discloses a Siamese bacillus JY-1 and a preparation and application thereof, which are preserved in China general microbiological culture Collection center in 2021, 9 and 6 months, and the preservation number is CGMCC NO. 23363. The Siamese bacillus JY-1 provided by the invention not only has a remarkable control effect on a target bacterium, namely kiwi fruit rot, but also has remarkable bacteriostatic activity on various plant pathogenic fungi and bacteria, such as rice blast, yam penicillium, rice leaf blight and the like, and has an important biocontrol application prospect.

Description

Siamese bacillus JY-1 and preparation and application thereof

Technical Field

The invention belongs to the field of application of microbial technology, and particularly relates to a Siamese bacillus JY-1, a preparation and application thereof.

Background

The kiwi fruit has rich nutrition, moderate sour and sweet taste and unique flavor, has the efficacies of cancer prevention, cancer resistance, aging resistance and the like, and is deeply favored by consumers; meanwhile, because of the characteristics of high planting benefit, relatively easy cultivation and the like, the kiwi fruits also get the relative favor of the mass producers. Therefore, although the cultivation history is not long, the kiwi fruit develops rapidly at home and abroad. At present, more than 30 countries such as China, New Zealand, Italy, Chilean and the like are planted in the world, and the total area reaches 28 kilohm²The total yield is nearly 452 ten thousand t; china is the first major country for producing kiwi fruits, and the cultivation area and the yield respectively reach 19.3 kilohm²And 229 million (2020). In recent years, the mature rot of kiwi fruit (fruit rot for short) occurs in large area, the disease mainly causes infection in young fruit stage, and gradually shows symptoms in the near-mature stage of the fruit, which often causes a large amount of fruit drop in the later stage of fruit bearing and explosive rot during the after-mature period of storage, and the fruit rot seriously affects the yield and quality of kiwi fruit, and has become an important limiting factor for the development of kiwi fruit in China.

Chemical control, namely pesticide control, has the advantages of quick response, remarkable effect, convenient use, no limitation of regions and seasons and the like, is suitable for large-area control, is an important means for controlling plant diseases, and has attracted social extensive attention due to the following negative effects such as environmental pollution, pesticide residue of agricultural products exceeding standards, formation of drug resistance of pathogenic bacteria and the like along with the use of a large amount of chemical agents in production. The plant pathogens are affected by the host plants and external biological and non-biological factors in the growth and development and pathogenic process. The method of controlling the occurrence and development of diseases by using beneficial microorganisms is called as plant disease biological control, and the beneficial microorganisms are called as plant disease biocontrol bacteria. The biological control can replace chemical pesticides to a certain extent, is safe to people and livestock, has no residual toxicity, and has no pollution to the environment, so that natural enemies can be effectively protected, ecological balance is protected, and the continuous pest control effect is exerted.

Disclosure of Invention

The invention obtains a biocontrol Siamese Bacillus strain JY-1 through screening, the name of the biocontrol Siamensis strain JY-1 is Bacillus siamensis J Y-1, the biocontrol Siamesis strain is stored in the common microorganism center of the China general microbiological culture Collection center at 9 and 6 months in 2021, and the storage number is CGMCC NO. 23363. The 16S rDNA sequence is shown in SEQ ID No: 1 is shown.

SEQ ID No：1：

GGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAGG

The bacterial strain not only has obvious control effect on the target bacteria kiwi fruit rot, but also has obvious bacteriostatic activity on various plant pathogenic fungi and bacteria such as rice blast, yam penicillium, rice bacterial blight and the like.

The invention also aims to provide a microbial preparation, which comprises the fermentation liquor of the Siamese bacillus JY-1. The preparation method comprises the following steps: connecting the Siamese bacillus JY-1 to an NA flat plate, performing activation culture at 30 ℃ for 24h, then transferring to 50mL NB optimized culture solution, performing culture for 72h under the conditions of 30 ℃ and 220r/min, and adjusting the concentration to be 1 multiplied by 10⁸CFU/mL to obtain the microbial preparation; the NB optimization culture solution comprises the following components: 2% of sucrose, 0.3% of yeast extract, 3% of peptone, 0.5% of NaCl, 0.09% of MgSO₄·7H₂O。

The invention has the beneficial effects that:

(1) the screened Bacillus siamensis JY-1 has strong bacteriostatic effects on grape white rot pathogen (Coniella castanaceae), grape bitter rot pathogen (Greeneria uvicola), Chinese yam colletotrichum gloeosporioides, Chinese gooseberry brown spot pathogen (Alternaria alternata), rice blast pathogen (Magnaporthe oryzae), lotus rot pathogen (Fusarium oxysporum), sesame stem spot pathogen (Macrophoma phaseoli), pear ring rot pathogen (Botryosphaeria bergeriana. sp.piricola), Phytophthora capsici (Phytophorta capsici), Chinese gooseberry fruit rot pathogen (Botryosphaeria dothidea), Penicillium sclerotium (Peillum candidum), citrus canker (Xanthomonas campestris), and yellow rice yellow pepper (Xanthomonas oryzae) and black sesame leaf blight (Xanthomonas oryzae).

(2) The microbial preparation has remarkable control effects on the fruit rot of kiwi fruits and the penicilliosis of Chinese yams; the active metabolite of the fermentation liquor of the Siamese bacillus JY-1 is high temperature resistant, and the metabolite has the highest bacteriostatic effect when placed at 40 ℃; the active substance with antagonistic effect of the strain is insensitive to the irradiation duration of ultraviolet rays.

Drawings

FIG. 1 shows a morphology diagram of a colony of Bacillus siamese JY-1 in different periods;

FIG. 2 is a diagram showing the morphology of gram-stained and spore-stained mycelia of Bacillus siameses JY-1;

FIG. 3 is a diagram of a phylogenetic tree of Bacillus constructed based on the 16S rDNA sequence;

FIG. 4 is a graph showing the effect of different pH treatments on the stability of active metabolites of strain JY-1;

FIG. 5 is a graph showing the effect of different temperature treatments on the stability of active metabolites of strain JY-1;

FIG. 6 is a graph showing the effect of different ultraviolet ray treatments on the stability of active metabolites of strain JY-1;

FIG. 7 shows a bacteriostasis spectrum test of a Bacillus siamensis JY-1 strain on plant pathogenic fungi, wherein A is grape white rot pathogen; b, bitter rot of grape; c, yam colletotrichum gloeosporioides; d, phytophthora capsici; e, rice blast germ; f, lotus rot pathogen; g, brown spot pathogen of kiwi fruit; h, pear ring rot bacteria; i, sesame rhizoctonia solani; j, the fruit rot of the kiwi fruit; k, yam penicillium, wherein in each picture, the left dish is treated, and the right dish is compared;

FIG. 8 shows a bacteriostasis spectrum test of a Bacillus siamensis JY-1 strain on plant pathogenic bacteria, wherein A is citrus canker pathogen; b, sesame bacterial wilt; c, rice bacterial leaf blight; d, rice bacterial streak disease germ; where in each photograph, the left dish is treated and the right dish is control.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and the accompanying drawings to fully understand the objects, aspects and effects of the present invention.

The strain culture media used in the following examples are as follows:

beef extract peptone medium NA (g/L): 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar strips and 1000mL of distilled water, wherein the pH value is 7.0-7.2, and the beef extract is sterilized at 121 ℃ for 20 min.

Nutrient broth NB (g/L): the NA medium without agar strips was sterilized at 121 ℃ for 20 min.

PDA solid medium (g/L): 200g of potato, 20g of glucose, 20g of agar, 1000mL of distilled water, pH 7.0-7.2, and sterilizing at 121 ℃ for 20 min.

Example 1:

obtaining of Siam bacillus JY-1:

1. source of soil

Rhizosphere soil of healthy kiwi plants in Yichun city of Jiangxi province of China Feng Xin county Shankou kiwi fruit base.

2. Screening of strains

(1) Soil collection

And (4) carrying out soil sample collection by a random sampling method. And randomly and respectively taking 50g of soil samples from different collection points 5-10 cm away from the ground, filling the soil samples into self-sealing bags, and storing the self-sealing bags in a refrigerator at 4 ℃ for bacterial separation.

(2) Isolation of bacteria

Plate gradient dilution separation method: weighing 5g of a soil sample by using an electronic balance, adding the soil sample into a sterilized triangular flask (containing a plurality of ground glass beads with the diameter of 3 mm) containing 45mL of physiological saline, oscillating, uniformly mixing, and standing for 20-30 min. Aseptically adding 5mL of the suspension to an empty sterilized tube, performing a water bath at 90 ℃ for 10min, subsequently adding 1mL of the liquid to a tube containing 9mL of seed culture solution, and performing enrichment culture at 37 ℃ for 36 h. According to the gradient dilution method, respectively taking the enriched bacterial liquid to dilute into 5 gradients, namely adding 0.5mL of bacterial liquid into a test tube (containing 4.5mL of seed culture medium), and lightly dilutingShaking up, repeating the operation in sequence, diluting to 10⁵And (4) doubling. Then each draw a gradient of 10^-3、10^-4And 10^-5The mixture was added to 200. mu.L of the selective medium plate, and uniformly spread using a spreading bar. Each experiment was repeated 3 times and incubated at 37 ℃ for 1 day.

(3) Bacterial purification

Selecting partial thallus in single colony with different characteristics such as shape, color and size from separated plate with sterilized toothpick, streaking and purifying on NA culture medium, and culturing at 37 deg.C for 1-2 days.

(4) Preservation of bacteria

Selecting pure strains belonging to the genus Bacillus from the purified plates according to the characteristics of color, shape, transparency, texture, etc. of the colonies, sequentially inoculating to NA slant culture medium with inoculating loop, numbering sequentially, culturing at constant temperature of 37 deg.C for 1-2 days, and storing in refrigerator at 4 deg.C for use.

(5) Screening of antagonistic bacteria

Screening strains with antagonistic effect on indicator bacteria by adopting a punching method and a plate opposing method, preparing a PDA culture medium plate, and punching holes at equal intervals around the periphery of 3cm away from the center of the plate by using a 5mm puncher. Inoculating the bacterial cake of the pathogenic bacteria of the fruit rot of the kiwi fruit to the center of a flat plate by using an inoculating needle, respectively sucking 100 mu L of fermentation liquor by using a liquid transferring gun, adding the fermentation liquor into 4 holes, taking NB culture solution as a control, carrying out 3 times of treatment, placing the inoculated fermentation liquor at the constant temperature of 28 ℃ for 3 days, measuring the diameter of a bacterial colony (the linear distance of the bacterial colony between two inhibition zones on a diagonal line) by using a cross method, and calculating the inhibition rate.

The strain JY-1 is obtained through the screening process, the colony morphology of the strain is shown in figure 1 on a beef extract peptone (NA) plate, a single colony is circular and slightly raised in the initial stage, the surface is glossy, and the surface is milky opaque; the bacterial colony is milk white, opaque and irregular round at the later culture period, the edge is serrated, the periphery is raised, and the surface is provided with wrinkles. The thallus is rod-shaped, has a length of about 1.0-2.5 μm and a width of about 0.5-1.0 μm, and produces spores. Gram staining was positive (the result is shown in FIG. 2, where A is gram staining and B is spore staining). Can grow at the temperature of 30-37 ℃; the growth pH range is 5-7; the salinity tolerance range is 2% -7% NaCl; anaerobic growth; the starch hydrolysis test, the methyl red reaction test, the catalase test, the V.P. test, the gelatin test, the citrate test and the nitrate reduction test are positive; the oxidase test, indole test, tryptophan deaminase test, phenylalanine deaminase test and urease test are negative. Can utilize glucose, maltose, sucrose, D-xylose, starch, arabinose; alpha-lactose cannot be utilized. Inositol, mannitol, sorbitol can be used; sweet alcohol cannot be utilized.

Extracting genomic DNA of the strain JY-1, and using primers: 27F (5 '-AGAGTTTGATCMTGGCTCA-3') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3') were used to amplify the 16S rDNA sequence of JY-1 strain and primers were synthesized from Shanghai. The total reaction (50. mu.L) was: DNA template 2. mu.L, 2 XTaq PCR Master Mix 25. mu. L, ddH₂O21. mu.L, and 1. mu.L of each primer. PCR amplification reaction procedure: pre-denaturation at 95 deg.C for 5 min; denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 2min, for 35 cycles; fill in 72 deg.C for 7 min. Subsequently, the obtained PCR amplification product was subjected to 1% agarose gel electrophoresis to determine the result. Adopting the method of the Beijing Tiangen agarose gel DNA recovery box to further cut and recover the amplification product. The purified product meeting the requirements is sent to the company of biological engineering (Shanghai) to complete the sequence determination. The strain JY-1 is subjected to physiological and biochemical experiments according to the standards, species classification characteristics and determination methods introduced in a common bacteria system identification manual and a concise Bergey judgment manual.

The results show that: the characteristics of the strain JY-1, such as morphology, 16S rDNA sequence analysis, physiological and biochemical characteristics (shown in Table 1) and the like are combined to be compared with similar strains of the strain, and the sequence is submitted to NCBI to obtain an accession number (KY 807041). The homology comparison of the sequence and the sequence results submitted in a GenBank database finds that the homology of the strain JY-1 and Siamese Bacillus (Bacillus siamensis) KCTC 13613 (the accession number is KY643639) is the highest and is up to 100 percent. Finally, the bacillus is identified as Siamese bacillus. In order to better distinguish the classification status of the strain, related gene sequences of the bacillus are selected from a database, and a phylogenetic tree is constructed (as shown in figure 3).

TABLE 1 physiological and biochemical characteristics of strain JY-1

Note: "+" indicates a positive reaction, "-" indicates a negative reaction, and "w" indicates a weak reaction.

Example 2:

the Bacillus siamensis JY-1 culture solution (the culture solution comprises 2 percent of cane sugar, 0.3 percent of yeast extract, 3 percent of peptone, 0.5 percent of NaCl and 0.09 percent of MgSO) obtained by screening in example 1 in NB culture solution₄·7H₂O), the temperature of the culture solution is 30 ℃, the pH is 7.0, the liquid loading amount is 50m L, the inoculation amount is 1 percent, the rotating speed is 220r/min, the time is 72h, and the culture solution is adjusted to the concentration of 1 multiplied by 10⁸And (3) CFU/mL to obtain the Siamese bacillus JY-1 microbial agent (fermentation stock solution) of the invention.

Example 3:

1) antagonistic activity determination of Siam bacillus JY-1 on 11 pathogenic fungi

Inoculating 11 pathogenic fungi including grape white rot, grape bitter rot, yam anthracnose, pepper phytophthora capsici, rice blast, lotus rot, kiwi fruit brown spot, pear ring rot, sesame stem blight, kiwi fruit rot and yam penicillium onto a PDA plate, and at a constant temperature of 25 ℃, until the colony grows over the plate 2/3 for later use. The JY-1 strain is streaked and activated to the NA culture medium, and is cultured in an oven at 30 ℃ for 2d for standby. Preparing a PDA plate on a sterile operating platform by adopting a plate confronting method, inoculating 9 different pathogenic fungus cakes in the center of a culture medium, carrying out point grafting on JY-1 thalli by using a sterile toothpick at a distance of 2.5cm from the middle, culturing in a constant temperature box at 28 ℃, and respectively taking the non-antagonistic strain as a control, and repeating the test for 3 times. When the hyphae of the control group overgrow the 2/3 position of the whole dish, the diameter of the hyphae of the pathogenic bacteria is measured by adopting a cross method, and the data is recorded by photographing. The results are shown in table 2 and fig. 7.

TABLE 2 inhibition of 11 pathogenic fungi by antagonistic JY-1 strains

Note: the data in the table are the bacteriostasis rate +/-standard deviation, and upper and lower case letters respectively represent the significant level of difference of p 0.01(p 0.05).

2) Antagonistic activity determination of Siam bacillus JY-1 on 4 pathogenic bacteria

Activating pathogenic bacteria to be tested, streaking on an NA plate, and culturing in an incubator at 30 ℃ for 1-2 days for later use.

Selecting a small amount of activated pathogenic bacteria by adopting a filter paper method, respectively diluting to 106cfu/mL by using sterile water on a sterile workbench, uniformly coating 100 mu L of the pathogenic bacteria on an NA culture medium plate, placing 4 sterilized 6mm filter paper sheets at a position 2.5cm away from the middle, air-drying, dropwise adding 10 mu L of 108cfu/mL antagonistic JY-1 strain diluent, placing the plate in an incubator at 30 ℃ for culturing for 1-2d, and repeating each test for 3 times. And measuring the width of the bacteriostatic zone according to the cross principle, photographing and calculating the bacteriostatic rate. The results are shown in table 3 and fig. 8.

TABLE 3 inhibitory Effect of antagonistic JY-1 strains on 4 pathogenic bacteria

3) Stability determination of active metabolite of Siam bacillus JY-1

Acid-base stability: centrifuging fermentation stock solution of a Siamese bacillus JY-1 strain for 30min at 4 ℃ and 8000r/min to obtain supernatant, respectively using 0.1mol/L HCl and 0.1mol/L NaOH to adjust the sterilization supernatant into different pH values (2, 4, 6, 8 and 10), placing the sterilization supernatant at room temperature for 24h, continuously oscillating the sterilization supernatant up and down for a plurality of times, adjusting the sterilization supernatant back to pH7, setting unadjusted supernatant as a control, and measuring the diameter of pathogenic bacteria according to cross. The result is shown in fig. 4, and the acid-base stability measurement result of the fermentation filtrate of the strain JY-1 shows that the bacteriostasis effect is optimal when the fermentation filtrate is not subjected to acid-base treatment (the pH value is 7.0), the diameter of a bacterial colony is only 24.00mm, when the pH value is 6.0 and 8.0, the active metabolite is gradually reduced, and the diameters of the bacterial colony are respectively 26.33mm and 26.00 mm. The bacteriostatic activity of the bacterial strain treated by strong acid and strong alkali is obviously reduced, namely the diameter of a colony is as high as 33.67mm when the pH value is 2.0, and the diameter of the colony is 30.33mm when the pH value is 10.0. The strain active metabolite is not resistant to strong acid and strong alkali, and the suitable pH value is 6.0-8.

Thermal stability: taking 2mL of the degerming supernatant fluid into a sterile centrifuge tube, respectively setting the degerming supernatant fluid in a constant-temperature water bath kettle at 40 ℃, 60 ℃, 80 ℃ and 100 ℃, carrying out water bath treatment for 30min and 60min respectively, taking the room-temperature treatment as a reference, and measuring the diameter of the pathogenic bacteria by adopting a cross method. The heat stability test result of the antibacterial activity product generated by the JY-1 strain shows that different temperatures and treatment times have different effects on the antibacterial action of the strain. As shown in FIG. 5, the bacteriostatic effect was the best when the temperature was 40 ℃, and the bacterial colony diameters were only 24.33mm and 23.00mm at 0.5h and 1h treatment, which indicates that the bacteriostatic effect was higher than that of the room temperature treatment. When the temperature is 60 ℃ and 80 ℃, the bacteriostatic activity is gradually reduced along with the increase of the treatment time, but the difference is not large compared with the room temperature treatment, and when the temperature reaches 100 ℃, the bacteriostatic activity is reduced compared with the room temperature treatment for 0.5 h. The strain is proved to be high temperature resistant in active metabolite, and the most suitable temperature is 40 ℃.

Ultraviolet stability: taking 2mL of sterilized supernatant liquid into a culture dish, opening a cover of the culture dish, placing the culture dish on a sterile workbench, respectively irradiating for 5min, 15 min, 25 min, 35min, 45min and 60min at a distance of 30cm from an ultraviolet lamp, setting the supernatant liquid without ultraviolet treatment as a control, and measuring the diameter of pathogenic bacteria according to a cross method. As shown in FIG. 6, the results show that the metabolic product of the strain JY-1 has almost unchanged antibacterial activity after being treated by different ultraviolet irradiation time, and the difference of the antibacterial effect among the treatments is not obvious. When the ultraviolet treatment time is 5-35 min, the bacteriostatic activity of the metabolite is in a descending trend, and when the irradiation time is 45min, the bacteriostatic activity of the metabolite is mild and then tends to be stable, and compared with the treatment without ultraviolet irradiation, the variation difference of the growth diameter of hyphae among the treatments is only about 1 mm. The results show that the active substances with antagonistic effect of the strain are insensitive to the irradiation duration of ultraviolet rays.

Example 4:

indoor fruit rot control effect of different treatment liquids of Siamese bacillus JY-1 on in-vitro kiwi fruit:

selecting healthy fruits with consistent ripeness and uniform size of kiwi fruits, washing the fruits clean under tap water, respectively soaking the fruits in 2% NaClO solution for 2min, taking out the fruits, washing off residual NaClO solution with tap water, and airing the fruits for later use. JY-1 strain was inoculated with pre-optimized NB medium (0.3% beef extract, 1% peptone, 0.5% NaCl) and post-optimized NB medium (2% sucrose, 0.3% yeast extract, 3% peptone, 0.5% NaCl, 0.09% MgSO₄·7H₂O) performing fermentation culture, and preparing fermentation liquor into fermentation stock solution (with the concentration of 1 × 10)⁸CFU/mL), bacterial suspension (centrifugation of culture stock solution and discarding supernatant, washing with sterile water 2 times, concentration of 1 × 10⁸CFU/mL) and sterile filtrate (supernatant was sterile filtered through 0.22 μm microporous membrane) were added to the 3 treatment solutions, and positive controls (sterile water + pathogenic bacteria cake) were set. The treated fruits were punched with a hole (5 mm. times.5 mm) in the middle of the fruit using a sterilized razor blade, air-dried, and 50. mu.L each of the above 3 treatment solutions and sterile water was added by using a pipette. After drying, respectively inoculating Chinese gooseberry fruit rot pathogen fungus cakes (the diameter is 5mm) by using a sterilized inoculating needle, covering sterilized cotton wetted by sterile water, putting the fruits in a magnetic disk, sealing by using a preservative film, placing in a constant temperature box with the humidity of 95% and the temperature of 28 ℃, measuring the diameters of disease spots when the number 3 and the number 6 are measured, calculating the prevention and treatment effects, treating 10 fruits each, and repeating the test for 3 times. The results are shown in Table 4. The control effects of the optimized 3 different treatment solutions are obviously improved, wherein the filtrate has the best control effect on the fruit rot of the kiwi fruit, the control effect of the inoculation 3d is as high as 92.21%, and the control effect is increased by 24.34% compared with the control effect before optimization. The control effect of the bacterial suspension is the worst and excellentNo effect is generated before the treatment, and the control effect of 3d and 6d after the optimization is 47.54 percent and 32.99 percent respectively. Shows that the active metabolite of the JY-1 strain has the best disease prevention effect on the pathogenic bacteria, and the fermentation stock solution is inferior.

TABLE 4 prevention and control effect of different treatment liquids of the strain JY-1 on in-vitro kiwi fruit rot

Note: s represents the area of attack; c represents a control effect.

Example 5:

inhibition of spore germination of penicillium dioscoreae germs by different treatment liquids of Siam bacillus JY-1

Scraping off pathogenic fungi cultured on PDA for 7 days with sterile water, transferring into conical flask containing glass beads, shaking to disperse uniformly, filtering with 4 layers of sterile gauze, removing mycelium, counting with blood counting chamber, and diluting to 5 × 10⁶spores/mL for standby. Preparing 7 treatment liquids, and preparing fermentation stock solution (with the concentration of 1 × 10) from fermentation liquid of the strain JY-1⁸CFU/mL), three concentrations of bacterial suspension (concentration 1X 10 separately)⁶、1×10⁷、1×10⁸CFU/mL), sterile filtrate, heat kill (stock solution autoclaved at 121 ℃ for 20min), and sterile water (distilled water autoclaved at 121 ℃ for 20 min). By adopting a slide germination method, 20 mu L of mixed liquid drops (14 mu LPDB, 4 mu L of spore suspension and 2 mu L of treatment liquid) are dripped on the surface of a sterile slide glass, the mixed liquid drops are placed in a culture dish for moisturizing, the culture dish is placed in a constant-temperature incubator at 25 ℃, the treatment is repeated three times every time, the germination condition of spores is observed every four hours, when the length of a germ tube is greater than or equal to the diameter of the spores, the germination of the spores is considered, and the germination rate of the pathogenic bacteria spores and the length of the germ tube are measured and calculated. The results are shown in Table 5, which shows that the bacterial suspension (1X 10)⁸) And the strain stock solution has the best effect of inhibiting the germination of the spores of the dry rot of the Chinese yam and has the germination rate0.64% and 2.02%, respectively. The second was sterile filtrate, with a germination rate of 4.05%. Bacterial suspension (1X 10)⁶) The control effect is the worst, and the germination rate is 38.55 percent. Bacterial suspension of JY-1 Strain (1X 10)⁸) The bacteria stock solution and the sterile filtrate have good effects on preventing diseases of the pathogenic bacteria.

TABLE 5 spore germination inhibiting effect of biocontrol bacteria on dry rot of Chinese yam

The above description is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiment, and the present invention shall fall within the protection scope of the present invention as long as the technical effects of the present invention are achieved by the same means. The invention is capable of other modifications and variations in its technical solution and/or its implementation, within the scope of protection of the invention.

SEQUENCE LISTING

<110> university of agriculture in Jiangxi

<120> Bacillus calsius JY-1 and preparation and application thereof

<130> 2021

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1492

<212> DNA

<213> Bacillus caldovelox strain JY-1

<400> 1

ggctcaggac gaacgctggc ggcgtgccta atacatgcaa gtcgagcgga cagatgggag 60

cttgctccct gatgttagcg gcggacgggt gagtaacacg tgggtaacct gcctgtaaga 120

ctgggataac tccgggaaac cggggctaat accggatggt tgtttgaacc gcatggttca 180

gacataaaag gtggcttcgg ctaccactta cagatggacc cgcggcgcat tagctagttg 240

gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt gatcggccac 300

actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa 360

tggacgaaag tctgacggag caacgccgcg tgagtgatga aggttttcgg atcgtaaagc 420

tctgttgtta gggaagaaca agtgccgttc aaatagggcg gcaccttgac ggtacctaac 480

cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg 540

tccggaatta ttgggcgtaa agggctcgca ggcggtttct taagtctgat gtgaaagccc 600

ccggctcaac cggggagggt cattggaaac tggggaactt gagtgcagaa gaggagagtg 660

gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg 720

actctctggt ctgtaactga cgctgaggag cgaaagcgtg gggagcgaac aggattagat 780

accctggtag tccacgccgt aaacgatgag tgctaagtgt tagggggttt ccgcccctta 840

gtgctgcagc taacgcatta agcactccgc ctggggagta cggtcgcaag actgaaactc 900

aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc 960

gaagaacctt accaggtctt gacatcctct gacaatccta gagataggac gtccccttcg 1020

ggggcagagt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta 1080

agtcccgcaa cgagcgcaac ccttgatctt agttgccagc attcagttgg gcactctaag 1140

gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta 1200

tgacctgggc tacacacgtg ctacaatgga cagaacaaag ggcagcgaaa ccgcgaggtt 1260

aagccaatcc cacaaatctg ttctcagttc ggatcgcagt ctgcaactcg actgcgtgaa 1320

gctggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt 1380

acacaccgcc cgtcacacca cgagagtttg taacacccga agtcggtgag gtaaccttta 1440

tggagccagc cgccgaaggt gggacagatg attggggtga agtcgtaaca gg 1492

Claims

1. A Siamese Bacillus JY-1 is characterized in that the Siamese Bacillus JY-1 is named as Bacillus siamenn sis JY-1, is stored in the common microorganism center of the China general microorganism culture preservation management committee at 9 and 6 months in 2021, and the storage number is CGMCC NO. 23363.

2. The Siamese bacillus JY-1 of claim 1, wherein a 16S rDNA sequence thereof is shown as SEQ ID No: 1 is shown.

3. The use of bacillus siamensis JY-1 of claim 1 for inhibiting grape white rot pathogen, grape bitter rot pathogen, yam anthracnose pathogen, kiwi fruit brown spot pathogen, rice blast pathogen, lotus rot pathogen, sesame stem blight pathogen, pear ring rot pathogen, pepper phytophthora capsici, kiwi fruit rot pathogen, yam penicillium pathogen, citrus canker pathogen, rice bacterial streak pathogen, rice white leaf blight pathogen and sesame ralstonia solanacearum.

4. A microbial preparation comprising the fermentation broth of bacillus siamensis JY-1 of any one of claims 1 to 2.

5. A process for the preparation of a microbial preparation according to claim 4, comprising the steps of: connecting the Siamese bacillus JY-1 to an NA flat plate, performing activation culture at 30 ℃ for 24h, then transferring to 50mL NB optimized culture solution, performing culture for 72h under the conditions of 30 ℃ and 220r/min, and adjusting the concentration to be 1 multiplied by 10⁸CFU/mL to obtain the microbial preparation; the NB optimization culture solution comprises the following components: 2% of sucrose, 0.3% of yeast extract, 3% of peptone, 0.5% of NaCl, 0.09% of MgSO₄·7H₂O。