CN110713956B

CN110713956B - Lysine bacillus S12 and application thereof

Info

Publication number: CN110713956B
Application number: CN201911131186.3A
Authority: CN
Inventors: 李冠喜; 于颖媛; 张璐; 焦玉茹; 李秋月; 亓超凡; 张雯倩; 郭帅
Original assignee: Qufu Normal University
Current assignee: Qingdao Rbt Biotechnology Co ltd
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2022-02-08
Anticipated expiration: 2039-11-19
Also published as: CN110713956A

Abstract

The invention discloses a lysine bacillus S12 strain and application thereof, and relates to the technical field of edible fungus biology. The bacillus Lysinibacillus sp S12 is classified and named, and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019713; the preservation date is as follows: 9 month 11 year 2019; and (4) storage address: wuhan university in Wuhan, China. The lysine bacillus S12 is applied to the growth of the agaricus bisporus, can effectively promote the growth of agaricus bisporus hyphae, shortens the production period of the agaricus bisporus, improves the yield of the agaricus bisporus, ensures that products are green, healthy and pollution-free, is nontoxic and harmless, and improves the economic and social benefits of edible mushroom production.

Description

Lysine bacillus S12 and application thereof

Technical Field

The invention relates to the technical field of edible fungus biology, and particularly relates to a lysine bacillus S12 and application thereof.

Background

Agaricus bisporus is one of the most common edible fungi, and has fleshy texture. The hypha is silvery white, the growth speed is relatively fast, the fungus quilt is not easy to form, the sporocarp is more single, round, white and has no scale, the pileus is thick and not easy to open the umbrella, the middle of the stipe is thicker and shorter, the flesh is white, the tissue is solid, the stipe is provided with semi-membranous fungus rings, and the spore is silvery brown. The agaricus bisporus is an edible fungus with wide cultivation, high production capacity and large consumption, is rich in protein, sugar, various nucleotides, vitamins and unsaturated fatty acid, and has rich nutrition, delicious taste and higher edible value. Has effects of resisting tumor, enhancing immunity, lowering blood pressure, etc., and has obvious medicinal and tonic effects. Since europe and north america, humans have been eating at least since ancient greek times, and artificial cultivation has been started in france of the 17 th century, and now has been widely cultivated all over the world. The traditional agaricus bisporus planting method has low yield and more chemical pollution, and cannot meet the requirements of consumers. The microorganism is vital to the growth of the plant, can decompose organic substances in soil to form humus and release nutrients, maintain the normal growth of plant cells, can convert soil carbon and fix inorganic nutrient elements, increases the utilization rate of the soil and achieves the effect of continuous culture. Therefore, the method is important for overcoming the defects of the traditional technology, reducing the use of chemical fertilizers and searching for suitable microorganisms, not only can promote the yield increase of the agaricus bisporus, but also can not cause harm to human health and environmental pollution, is nontoxic and harmless, and ensures that the agaricus bisporus is green, healthy and pollution-free.

Disclosure of Invention

Aiming at the defects of the prior art, the technical problem to be solved by the invention is to provide a bacterium for promoting the growth of agaricus bisporus; the invention aims to solve another technical problem of providing the application of the bacillus lysinate S12 in promoting the growth of agaricus bisporus so as to solve the problems of low yield and much chemical pollution of the traditional agaricus bisporus planting method.

In order to solve the problems, the invention adopts the technical scheme that:

the bacterium for promoting the growth of the agaricus bisporus is classified and named as bacillus Lysinibacillus sp S12 and is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019713; the preservation date is as follows: 9 month 11 year 2019; and (4) storage address: wuhan university in China.

Application of lysine bacillus S12 in promoting growth of Agaricus bisporus.

The application comprises the following steps: preparing culture materials, culturing bacterial liquid, cultivating in an earth covering mode and managing fruiting.

Further, the bacteria are sprayed in the casing soil in the form of fermentation broth.

Further, the preparation method of the covering soil comprises the steps of adding crushed dry cow dung, wheat hull or rice chaff, lime and calcium superphosphate into soil, uniformly stirring, then irrigating, taking soil after fermentation, and disinfecting by a physical method.

Further, the ratio of the thickness of the covering soil to the thickness of the culture material is 3.5-4.0: 18 to 22.

An Agaricus bisporus growth promoting microbial inoculum comprises lysine bacillus S12.

Has the advantages that: compared with the prior art, the invention screens, separates and identifies a bacterium lysine bacillus S12 for promoting the growth of the agaricus bisporus from the agaricus bisporus planting soil covering rhizosphere soil, and the agaricus bisporus added with the growth-promoting bacterium has large diameter of mushroom cap, large thickness of mushroom cap, long degree of stipe and high total quality of strain sporocarp through bacterium liquid culture, soil covering cultivation and fruiting management. The yield of the agaricus bisporus can be improved, the agaricus bisporus is non-toxic and harmless, the green, healthy and pollution-free products are guaranteed, meanwhile, the using amount of auxiliary materials is reduced, and the economic and social benefits of edible mushroom production are improved.

Drawings

FIG. 1 is a graph showing the results of co-culturing the selected bacteria with Agaricus bisporus strains on a plate for 7 days; the upper panel is a co-culture group; the lower panel is a control group;

FIG. 2 is a graph showing the effect of the selected strains on the hyphal growth radius of the Agaricus bisporus strain;

FIG. 3 is a phylogenetic tree diagram of Bacillus lysinate S12;

FIG. 4 is a graph showing the results of measurement of the growth ability of Bacillus lysinate S12 on Agaricus bisporus hyphae;

FIG. 5 is a graph showing the effect of Bacillus lysinate S12 on the yield of Agaricus bisporus strains;

FIG. 6 is a graph showing the effect of Bacillus lysinate S12 on the diameter of agaricus bisporus pileus;

FIG. 7 is a graph showing the effect of Bacillus lysinate S12 on the pileus thickness of Agaricus bisporus;

FIG. 8 is a graph showing the effect of Bacillus lysinate S12 on the diameter of the stipe of Agaricus bisporus.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to be limiting.

Example 1

1) Isolation screening and identification of bacteria

Randomly collecting agaricus bisporus planting earthing rhizosphere soil samples from a planting base of an edible fungus science and technology industry park of Shandong Taian Yang town, performing co-separation on the agaricus bisporus planting earthing rhizosphere soil samples by a gradient dilution method to obtain 78 bacteria isolates, respectively selecting the bacteria isolates to be cultured for 5-7 d by adding agar-rich culture media (5 g of beef extract, 10g of peptone, 5g of sodium chloride, 20g of agar and pH 7.2-7.4), and then selecting the bacteria single colony isolate around which a transparent ring is formed.

Inoculating the strains which are separated and purified by primary screening and stored into a conical flask of an NA culture medium, culturing for 48h at 28 ℃ at 180r/min in a shaking table to obtain a cultured saturated bacterial liquid, inoculating 0.2mL of the bacterial liquid into a PDA plate (20 g of glucose, 200g of potato, 10g of peptone, 3g of yeast extract, 15-20g of agar, 1000mL of water and pH7.0) according to the aseptic operation requirement by using a pipette, uniformly coating by using a coater, taking 1cm of agaricus bisporus fungi by using a puncher, and placing the agaricus bisporus fungi in the center of the plate coated with the bacteria. And adding 0.2mL of sterile water into the control group, inoculating, culturing in an incubator at 28 ℃, observing the growth condition of hyphae every day, and photographing and recording. The results of the culture for 7 days are shown in FIG. 1, and the effect on the radius of hyphal growth is shown in FIG. 2.

The screened lysine bacillus S12 is subjected to biological characteristic observation by methods such as morphological characteristics, thallus, gram stain, 3% KOH solubility test, spore stain and the like. The morphological characteristics are as follows: the short rod-shaped thallus has no spore and no capsule, the bacterial colony is circular, the surface is smooth and opaque, and the color is milky white. The biochemical characteristics of the screened lysine bacillus S12 are measured by citrate utilizing the methods of experiments, indole experiments, methyl red experiments, Vopu (VP) experiments and the like. The results are shown in table 1:

TABLE 1 physiological and biochemical characteristics of Bacillus lysinate S12

Item	Results	Item	Results
				Acid production from sucrose	-	Fructose acid production	+
v.P. reaction	-	Citric acid salt	-
				Indole formation	-	Oxidase enzyme	+
Gram stain	-	Hydrolyzed starch	+
				Nitrate reduction	-	Hydrolyzed gelatin	+

The 16S rDNA sequence of the strain is amplified, and the product after PCR amplification is sent to Shanghai biological engineering company to complete sequence determination, and the sequence is shown as SEQ ID NO. 1. Blast analysis was performed on the sequencing results at NCBI and a phylogenetic tree was constructed using MEGA7.0, as shown in figure 3. The sequencing result is subjected to Blast alignment, and the strain is determined to be the bacillus Lysinibacillus sp S12.

2) Measurement of hypha feeding and growth ability

The bacillus lysinate S12 was inoculated into the culture medium, and the amount of hypha sprouting and growth was observed every day from day 2, and the results are shown in fig. 4. The agaricus bisporus mycelium inoculated into the lysine bacillus S12 is found to grow vigorously, the daily growth amount of the mycelium is gradually increased from the 4 th day, and the growth length of the mycelium is obviously longer than that of a control group from the 6 th day to the 8 th day, and the advantages are obvious.

Example 2

Under natural conditions, soil is mixed with organic substances to effectively perform anaerobic fermentation, a small piece of land is taken to cultivate bisporous mushroom in the field of wheat or rape afterreap or in the place close to water source, the land is taken to dig 25-30cm deeply, the soil is crushed and beaten to be fine, no mud block is formed as far as possible, and then crushed dry cow dung, wheat hull or rice chaff, lime and calcium superphosphate are added. The materials are fully and evenly mixed with the soil, and then are irrigated to play a role in heat preservation. The soil is taken out after the soil is smashed and fermented evenly, the soil is taken out and then disinfected by a physical and chemical method, and the soil is put into a mushroom bed after the disinfection is finished, and the prepared compost is put into the mushroom bed. The thickness of the covering soil is determined according to the thickness of the culture material, the thickness of the culture material for high-yield cultivation is 18-22 cm, and the thickness of the covering soil is 3.5-4.0 cm. After the covering soil is prepared, 200mL of the bacillus lysinate S12 bacterial liquid is diluted to 1L by sterile water, the liquid is uniformly sprayed in the covering soil, the mixture is fully mixed, a proper temperature and moisture environment is kept, the fruiting speed and yield of the agaricus bisporus are observed, and the sterile water with the same total volume is added to serve as a control group. The yields of the first tide, the second tide and the third tide of strains are shown in figure 5, the influence of the edible fungus growth promoting bacteria on the properties of the sporocarp of the agaricus bisporus strains is shown in figures 6 to 8, the agaricus bisporus strains added with the growth promoting bacteria have large cap diameter, thick cap, long stipe and high total sporocarp quality.

Sequence listing

<110> university of Qufu Master

<120> lysine bacillus S12 and application thereof

<130> 1

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1459

<212> DNA

<213> Lysinibacillus sp.S12

<400> 1

aagggcggcg tgctatacat gcaagtcgag cgaacagaga aggagcttgc tccttcgacg 60

ttagcggcgg acgggtgagt aacacgtggg caacctacct tatagtttgg gataactccg 120

ggaaaccggg gctaataccg aataatctgt ttcacctcat ggtgaaacac tgaaagacgg 180

tttcggctgt cgctatagga tgggcccgcg gcgcattagc tagttggtga ggtaacggct 240

caccaaggcg acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300

acggcccaga ctcctacggg aggcagcagt agggaatctt ccacaatggg cgaaagcctg 360

atggagcaac gccgcgtgag tgaagaagga tttcggttcg taaaactctg ttgtaaggga 420

agaacaagta cagtagtaac tggctgtacc ttgacggtac cttattagaa agccacggct 480

aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg aattattggg 540

cgtaaagcgc gcgcaggtgg tttcttaagt ctgatgtgaa agcccacggc tcaaccgtgg 600

agggtcattg gaaactggga gacttgagtg cagaagagga tagtggaatt ccaagtgtag 660

cggtgaaatg cgtagagatt tggaggaaca ccagtggcga aggcgactat ctggtctgta 720

actgacactg aggcgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac 780

gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct gcagctaacg 840

cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg aattgacggg 900

ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 960

gtcttgacat cccgttgacc actgtagaga tatggtttcc ccttcggggg caacggtgac 1020

aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080

gcgcaaccct tgatcttagt tgccatcatt tagttgggca ctctaaggtg actgccggtg 1140

acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga cctgggctac 1200

acacgtgcta caatggacga tacaaacggt tgccaactcg cgagagggag ctaatccgat 1260

aaagtcgttc tcagttcgga ttgtaggctg caactcgcct acatgaagcc ggaatcgcta 1320

gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380

cacaccacga gagtttgtaa cacccgaagt cggtgaggta accttttgga gccagccgcc 1440

gaaaggttgg atttgaagt 1459

Claims

1. A bacterium promoting the growth of agaricus bisporus, which is classified and named as lysine bacillus (B) (Lysinibacillussp.) S12, deposited in China center for type culture Collection with the preservation number of CCTCC NO: m2019713; the preservation date is as follows: 9 month 11 year 2019; and (4) storage address: wuhan university in China.

2. The use of lysine bacillus S12 of claim 1 to promote the growth of agaricus bisporus.

3. The use of claim 2, comprising the steps of: preparing culture materials, culturing bacterial liquid, cultivating in an earth covering mode and managing fruiting.

4. Use according to claim 3, wherein the bacteria are sprayed in the casing as a fermentation broth.

5. The use as claimed in claim 3, wherein the casing soil is prepared by adding ground dry cow dung, wheat hull or rice chaff, lime and calcium superphosphate into soil, stirring, irrigating, fermenting, taking out soil, and sterilizing by physical method.

6. The application of claim 3, wherein the ratio of the thickness of the covering soil to the thickness of the culture material is (3.5-4.0): (18-22).

7. An Agaricus bisporus growth promoting agent comprising the Bacillus lysii S12 of claim 1.