CN110982746B

CN110982746B - Spore strain and application

Info

Publication number: CN110982746B
Application number: CN201911352661.XA
Authority: CN
Inventors: 王琼; 王浩宇; 于潇洋; 曾书林
Original assignee: Shaanxi Zirui Biotechnology Co ltd
Current assignee: Shaanxi Zirui Biotechnology Co ltd
Priority date: 2019-12-25
Filing date: 2019-12-25
Publication date: 2022-03-18
Anticipated expiration: 2039-12-25
Also published as: CN110982746A

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses a spore strain and application thereof. The invention firstly obtains a spore strain, and the preservation number of the spore strain is CGMCC No. 18773. The bacillus strain can utilize kitchen waste as a carbon source to produce a biosurfactant. The culture prepared from the spore strain can remarkably promote the growth of tomatoes and corns, and shows double biological regulation functions of both drug effect and fertilizer effect. And the culture prepared from the spore strain can be applied to biological control of plant pathogenic bacteria, can show good bacteriostatic activity to various plant pathogenic bacteria, and further expands the application of the biosurfactant in the field of agricultural production. In addition, the invention also provides a novel process for preparing the biosurfactant, which adopts the kitchen waste as the carbon source, reduces the production cost and realizes the cyclic utilization of resources.

Description

Spore strain and application

Technical Field

The invention relates to the technical field of microorganisms, in particular to a spore strain and application thereof.

Background

Biosurfactants are natural surfactants produced by microorganisms or animals and plants that have not only solubilizing and emulsifying properties comparable to chemical surfactants, but also many advantages not possessed by chemical surfactants: such as better stability, biodegradability, and environmental safety. Therefore, biosurfactants are also widely used in various fields such as petrochemical industry, medicine and food. More and more researches concern about the excavation of surfactant producing bacteria, and Bacillus spp (Bacillus spp.) is taken as an important source bacteria of biosurfactant producing bacteria and is also highly valued by researchers at present.

However, the production of biosurfactants requires that the strain be obtained by fermenting glucose with high concentration, which increases the production cost of the biosurfactants and limits the application of the biosurfactants in agriculture. Therefore, the utilization of cheap substrates such as kitchen waste and other cheap fermentation substrates to replace glucose as a carbon source and the further reduction of the production cost of the biosurfactant are key to promote the application of the biosurfactant, but no relevant report is found at present. In addition, researchers currently focus on the field of industry and environmental remediation for biosurfactant-producing bacteria, and little attention is paid to their application in the field of agricultural production.

Disclosure of Invention

The invention mainly solves the technical problem of providing a bacillus strain and also provides application of the bacillus.

Firstly, the invention provides a spore strain, and the preservation number of the spore strain is CGMCC No. 18773.

The spore strain with the preservation number of CGMCC No.18773 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the No. 3 of the Xilu-1 of the Beijing area facing the sun, the institute of microbiology, China academy of sciences, zip code 100101, the preservation number of CGMCC No.18773, the preservation date 2019, 10 months and 31 days, and the classification and the nomenclature are as follows: bacillus (Bacillus sp.).

The spore strain provided by the invention has the 16S rRNA gene sequence shown in SEQ ID NO. 1.

The bacillus strain provided by the invention can be used for producing a biosurfactant by using kitchen waste as a carbon source.

The invention also provides a culture prepared from the bacillus strain, wherein the culture is prepared from the bacillus strain and kitchen waste serving as a carbon source.

The invention provides the application of the culture as a plant growth promoter; preferably, as tomato and/or corn growth promoting agent.

The invention also provides the application of the culture as a biosurfactant.

The invention also provides application of the culture in preventing and treating plant diseases, preferably, the plant diseases are diseases caused by round spot pathogenic bacteria, small spot pathogenic bacteria, fusarium oxysporum, fusarium equiseti, fusarium graminearum, fusarium verticillioides and/or fusarium laminarinum.

The culture provided by the invention is prepared from a fermentation culture solution prepared from the spore strain, wherein the fermentation culture medium adopted by the fermentation culture solution takes kitchen waste as a carbon source.

Further, the culture provided by the invention is prepared from a fermentation culture solution prepared from the spore strain, wherein the fermentation culture solution adopts a fermentation culture medium which comprises the following components in percentage by mass: k₂HPO₄ 0.08～0.12％，KH₂PO₄0.08～0.12％，MgSO₄﹒7H₂O 0.01～0.03％，NaNO₃ 0.04～0.06％，(NH₄)₂SO₄ 0.04～0.06％，CaCl₂ 0.0008～0.0012％，FeSO₄0.0004-0.0006% of yeast powder, 0.01-0.03% of kitchen waste and 6.0-7.5% of pH.

More preferably, the fermentation medium adopted by the fermentation culture solution comprises the following components in percentage by mass: k₂HPO₄ 0.1％，KH₂PO₄ 0.1％，MgSO₄﹒7H₂O 0.02％，NaNO₃ 0.05％，(NH₄)₂SO₄ 0.05％，CaCl₂0.001％，FeSO₄0.0005%, 0.02% of yeast powder and 6% of kitchen waste, and the pH value is 7.0.

Further preferably, the culture is prepared by:

(1) inoculating the spore strain to a liquid seed culture medium, culturing to obtain a seed culture solution, centrifuging the seed culture solution, and suspending the collected thalli by using sterile normal saline to prepare a seed suspension;

(2) inoculating the seed suspension into a fermentation culture medium, and controlling the initial OD of a bacterial liquid in the fermentation culture medium after inoculation₆₀₀Culturing to obtain 1-1 fermentation culture solution;

(3) and centrifuging the fermentation culture solution, taking supernatant, then adjusting the pH value of the supernatant to 1.5-3, then centrifuging and collecting precipitates, suspending the obtained precipitates with deionized water to obtain precipitate suspension, adjusting the pH value of the precipitate suspension to 7.5-8.5 to obtain weak alkaline liquid, and freeze-drying the weak alkaline liquid to obtain the culture.

The prepared culture can be used as a biosurfactant; the culture can be used as a plant growth promoting agent and has remarkable effect on promoting the growth of tomatoes and corns; meanwhile, the culture can be applied to preventing and treating plant diseases, and has good effect on preventing and treating diseases caused by round spot disease pathogenic bacteria, small spot disease pathogenic bacteria, fusarium oxysporum, fusarium equiseti, fusarium graminearum, fusarium verticillioides and fusarium exserotinate.

The invention has the beneficial effects that:

(1) the spore strain provided by the invention is a high-yield biosurfactant strain, can be used for producing biosurfactants, and has an obvious effect on promoting the growth of plants, especially on promoting the growth of tomato seedlings and corn seedlings, as proved by experiments;

(2) when the spore strain is used for preparing the biosurfactant, the used fermentation medium can use kitchen waste as a carbon source, the kitchen waste can be leftovers generated in canteens, restaurants and daily family life, and the kitchen waste is used for replacing high-concentration glucose, so that the resource recycling can be realized, and the production cost of the biosurfactant is obviously reduced;

(3) the biosurfactant prepared by the spore strain has good surfactant performance, has good inhibition effect on various plant pathogenic bacteria such as round spot pathogenic bacteria, small spot pathogenic bacteria, fusarium oxysporum, fusarium equiseti, fusarium graminearum, fusarium verticillioides and fusarium laminarinum, and is the first discovery that the biosurfactant can be used as an active substance to antagonize various pathogenic fungi.

The invention provides a new spore strain, which can remarkably promote the growth of tomatoes and corns and has double biological regulation functions of both pesticide effect and fertilizer effect. The surfactant produced by the spore strain can be applied to biological control of plant pathogenic bacteria, can show good bacteriostatic activity to various plant pathogenic bacteria, and further expands the application of the biosurfactant in the field of agricultural production. In addition, the invention also provides a novel process for preparing the biosurfactant, which adopts the kitchen waste as the carbon source, reduces the production cost and realizes the cyclic utilization of resources.

The kitchen waste is used as a carbon source for fermentation, and is commonly called swill, namely swill and hogwash. Kitchen waste is mainly classified into food waste and kitchen waste according to different sources. The former is produced from leftovers in restaurants, dining halls and other catering industries, and has the characteristics of large production amount, multiple sources and wide distribution, and the latter mainly refers to leftovers discarded in daily cooking of residents, and the quantity of the leftovers is not as large as that of food wastes. The kitchen waste has high moisture content, is easy to decay and deteriorate, emits foul smell, is easy to grow harmful substances such as pathogenic microorganisms and mycotoxin, and has bad influence on environmental sanitation; but the kitchen waste is rich in organic matter, the main components of the kitchen waste comprise rice and flour food residues, vegetables, animal and vegetable oil, meat and bones and the like, and the kitchen waste rich in nutrition is a valuable renewable resource due to the chemical composition of starch, cellulose, protein, lipid and inorganic salt. According to the invention, the novel spore strain is provided, the spore strain can utilize the kitchen waste as a carbon source to produce the biosurfactant, and the biosurfactant has obvious effects in plant growth promotion and plant disease control and has wide application prospects in agriculture, so that the novel spore strain provides a novel way for resource utilization of the kitchen waste and has obvious economic benefits and environmental protection benefits.

Drawings

FIG. 1 is a graph showing emulsification values of surfactants against various hydrophobic substances, which were measured in Experimental example 1 of the present invention;

FIG. 2 is a graph showing a comparison of the root lengths of tomato seedlings measured after 3 days, 4 days and 5 days of culture in Experimental example 2 of the present invention, respectively;

FIG. 3 is a graph showing the growth of the aerial and underground parts of young maize plants after 20 days of continuous growth in Experimental example 2 of the present invention;

FIG. 4 is a graph of the dry weight of the aerial parts of maize seedlings after 20 days of continued growth in Experimental example 2 of the present invention (in the graph,. indicates. < 0.05,. indicates. < 0.01,. indicates. < 0.001);

FIG. 5 is a graph of the dry weight of the underground portion of maize seedlings after 20 days of continued growth in Experimental example 2 of the present invention (in the graph,. indicates. < 0.05,. indicates. < 0.01,. indicates. < 0.001).

Detailed Description

The technical solution of the present invention will be explained in detail below.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

EXAMPLE 1 preparation of Bacillus strains

The spore strain is obtained by carrying out gradient dilution, coating, purification and screening on a plant rhizosphere soil sample collected near a Liaohe oil field, and comprises the following specific steps:

(1) adding 10g of plant rhizosphere soil sample collected from near Liaohe oil field into 90ml of sterile water, and performing shake culture at 37 deg.C and 200rpm for 30min to obtain 10^-1Diluting the soil dilution solution to 10^-4～10^-8Concentration to obtain soil suspension with concentration gradients at all levels; each 100. mu.l of the soil suspension was applied to LB solid medium (composition of LB solid medium: yeast)5g of powder, 10g of tryptone, 10g of NaCl, 20g of agar and 1000ml of deionized water, wherein the pH value is 7.0); 3 times of each gradient, and starting to pick a single colony for purification after culturing at 30 ℃ for 24 hours;

(2) carrying out amplification culture on the purified strain in 25ml of LB liquid culture medium, and culturing for 12h at 30 ℃ and 200 rpm; collecting strain fermentation liquor to carry out surfactant production determination, wherein the determination content comprises the following steps: hemolysis experiment, liquid drop collapse experiment, oil discharge ring experiment and surface tension measurement;

(3) after the strains producing the surfactant are continuously passaged for a plurality of times, the capability of the strains producing the surfactant is measured again according to the method in the step (2) to determine the character stability; finally obtaining a bacterial strain with high yield of the biosurfactant, and the serial number of the bacterial strain is ZR 19.

And performing strain species identification on the strain by adopting 16S rRNA gene sequence analysis, wherein the strain is identified as Bacillus (Bacillus sp), and the 16S rRNA gene sequence of the Bacillus strain is shown as SEQ ID NO: 1:

GCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCG(SEQ ID NO:1)。

the strain obtained by screening is subjected to strain preservation and is preserved in the China General Microbiological Culture Collection Center (CGMCC), No. 3 of the Xilu No.1 of the Beijing area of the sunny ward, the institute of microbiology of the Chinese academy of sciences, zip code 100101, the preservation number is CGMCC No.18773, the preservation date is 2019, 10 months and 31 days, and the classification and the nomenclature are as follows: bacillus (Bacillus sp.).

EXAMPLE 2 preparation of biosurfactant

The invention also provides a biosurfactant which is prepared from a fermentation culture solution prepared from the spore strain, adopts kitchen waste as a carbon source and is prepared by the following steps:

(1) inoculating the spore strain into a liquid seed culture medium, and culturing at 37 ℃ and 200rpm for 10h to obtain a seed culture solution, wherein the liquid seed culture medium comprises the following components in percentage by mass (w/v): tryptone 1%, yeast powder 0.5%, NaCl 1%, pH 7.0; centrifuging the obtained seed culture solution at 12000rpm for 3min, collecting thallus, and suspending the collected thallus with sterile physiological saline to obtain seed suspension;

(2) inoculating the seed suspension into a fermentation culture medium, and controlling the initial OD of a bacterial liquid in the fermentation culture medium after inoculation ₆₀₀1 at 37 ℃ and 200rpm for 3 days to prepare a fermentation culture medium, wherein the fermentation culture medium consists of (w/v, mass-to-volume ratio): k₂HPO₄ 0.1％，KH₂PO₄ 0.1％，MgSO₄﹒7H₂O 0.02％，NaNO₃ 0.05％，(NH₄)₂SO₄ 0.05％，CaCl₂ 0.001％，FeSO₄0.0005%, 0.02% of yeast powder, 6% of kitchen waste and 7.0% of pH; wherein the kitchen waste is leftovers generated in canteens, restaurants and daily family life;

(3) centrifuging the fermentation culture solution at 12000rpm for 10min, collecting supernatant, adding 6M HCl into the supernatant to adjust pH to 2.0, and standing at 4 deg.C overnight; centrifuging at 12000rpm at 4 deg.C for 30min, collecting precipitate, and suspending the precipitate with small amount of deionized water to obtain precipitate suspension; the pH of the precipitation suspension is adjusted to 8.0 by 1M NaOH, and then the precipitation suspension is lyophilized, so that the biosurfactant is obtained, and the yield can reach 8.11 +/-0.18 g/L.

Comparative preparation of biosurfactant

The invention also provides another biosurfactant, which is prepared from a fermentation culture solution prepared from the spore strain, adopts glucose as a carbon source and is prepared by the following steps:

(2) inoculating the seed suspension into a fermentation culture medium, and controlling the initial OD of a bacterial liquid in the fermentation culture medium after inoculation ₆₀₀1 at 37 ℃ and 200rpm for 3 days to prepare a fermentation culture medium, wherein the fermentation culture medium consists of (w/v, mass-to-volume ratio): glucose 4%, yeast powder 0.1%, NaNO₃ 0.3％，KH₂PO₄ 0.025％，NaCl 0.03％，K₂HPO₄·3H₂O 0.001％，MgCl₂·6H₂O 0.001％，NH₄Cl 0.0003％，KCl 0.0003％，pH 7.0；

(3) Centrifuging the fermentation culture solution at 12000rpm for 10min, collecting supernatant, adding 6M HCl into the supernatant to adjust pH to 2.0, and standing at 4 deg.C overnight; centrifuging at 12000rpm at 4 deg.C for 30min, collecting precipitate, and suspending the precipitate with small amount of deionized water to obtain precipitate suspension; the precipitate suspension was adjusted to pH 8.0 with 1M NaOH and then lyophilized to obtain biosurfactant in a yield of 4.03. + -. 0.21 g/L.

Through the experiment of preparing the biosurfactant by the embodiment 2 and the comparative example, the spore strain provided by the invention can be used for preparing the biosurfactant by using glucose as a carbon source, can also be used for preparing the biosurfactant by using kitchen waste as a carbon source, and has higher yield in preparing the biosurfactant by using the kitchen waste as a carbon source.

Experimental example 1 measurement of the Properties of biosurfactants obtained in example 2 and comparative example

The performance of the surfactant was determined by its emulsifying ability for hydrophobic materials.

Dissolving the biosurfactant prepared in the example 2 in deionized water, ensuring that the final concentration of the biosurfactant is 200mg/L, adding 3ml of surfactant solution into a 10ml centrifuge tube, and respectively adding different kinds of hydrophobic substances with the same volume, wherein the hydrophobic substances are specifically as follows: n-hexadecane, peanut oil, olive oil, soybean oil, paraffin and n-hexane. Placing the mixture obtained after adding the hydrophobic substance on a vortex oscillator, shaking for 2min, standing for 24h, and determining the proportion of the height of the emulsified layer in the total height of the mixture. The emulsifying ability (i.e., the emulsifying coefficient E) of the surfactant prepared in example 2 of the present invention to the above-mentioned various hydrophobic substances₂₄Values) of 69%, 62%, 68%, 62%, 66%, 60%, respectively, the emulsification values of the surfactant for various hydrophobic materials are shown in fig. 1.

Dissolving the biosurfactant prepared in the comparative example in deionized water to ensure that the final concentration of the biosurfactant is 200mg/L, adding 3ml of surfactant solution into a 10ml centrifugal tube, and respectively adding different kinds of hydrophobic substances with the same volume, wherein the hydrophobic substances are specifically as follows: n-hexadecane, peanut oil, olive oil, soybean oil, paraffin and n-hexane. Placing the mixture obtained after adding hydrophobic substance on vortex oscillator, shaking for 2min, standing for 24 hr, and measuring the height of emulsified layerThe ratio of the total height of the mixture. The emulsifying ability (i.e., the emulsifying coefficient E) of the surfactant prepared according to the comparative example of the present invention to the above-mentioned various hydrophobic substances₂₄Values) of 65%, 61%, 63%, 58%, 65%, 58%, respectively, the emulsification values of the surfactant for various hydrophobic materials are shown in fig. 1.

Experimental example 2 growth promotion experiment of spore strain ZR19

The spore strain ZR19 was inoculated into LB liquid medium and cultured at 30 ℃ and 200rpm for 12 hours. Then, the cells were centrifuged at 12000rpm for 5min to collect ZR19 cells, the collected cells were resuspended in sterile water, and the OD of the resuspended cells was controlled₆₀₀＝0.5。

Growth promotion experiment on tomatoes

Soaking tomato seeds in the resuspended bacterial liquid for 2h, spreading the tomato seeds on a water agar culture medium (agar 0.9% (w/v)), germinating in dark at 28 ℃ for 48h, and then transferring the tomato seeds to 1/2MS solid culture medium, wherein the MS culture medium consists of (w/v, mass-to-volume ratio): KNO₃ 0.19％，NH₄NO₃ 0.16％，KH₂PO₄ 0.017％，MgSO₄﹒7H₂O 0.037％，CaCl₂﹒2H₂O 0.044％，KI 0.000083％，H₃BO₃ 0.00062％，MnSO₄﹒4H₂O 0.00223％，ZnSO₄﹒7H₂O 0.00086％，NaMoO₄﹒2H₂O 0.000025％，CuSO₄﹒5H₂O 0.0000025％，CoCl₂0.0000025％，EDTA-2Na 0.00373％，FeSO₄﹒7H₂O0.00278%, Inositol (Inositol) 0.01%, Glycine (Glycine) 0.0002%, VB 10.00001%, VB 60.00005%, VB 50.00005%, and agar 0.9%; root length was determined after 3, 4 and 5 days of culture on 1/2MS medium at 28 ℃.

FIG. 2 is a graph comparing the root length of tomato seedlings measured after 3 days, 4 days and 5 days of culture.

The results in fig. 2 show that the tomato seeds treated with the resuspended suspension (treated group) had a 41.6% and 12.2% increase in root length after 3 and 4 days, respectively, compared to the untreated tomato seeds (control group). The bacillus strain ZR19 can obviously promote the root growth of tomato seedlings and has the growth promoting effect on the tomato seedlings.

Second, growth promotion experiment for corn

The corn seeds are disinfected (the disinfection method is that the corn seeds are soaked in 1% NaClO for 10min and then washed with sterile water for 3 times), the disinfected corn seeds are laid on a water agar culture medium (agar is 0.9% (w/v)), germinate in a dark place at 28 ℃ for 48h and then are transferred to soil for planting (vermiculite: soil is 1: 9), 50ml of bacterial liquid after heavy suspension is added into each corn seedling after 20 days of planting, and the dry weight of the overground and underground parts of the corn is measured after the corn seedlings continue to grow for 20 days.

FIG. 3 is a graph of the growth vigor of the aerial and underground parts of maize seedlings after 20 days of continued growth. As can be seen from the figure, the corn seedlings treated with the resuspended inoculum solution (treated group) grew more vigorously in both root system and seedling vigor than the untreated corn seedlings (control group).

FIG. 4 is a dry weight of the aerial parts of maize seedlings after 20 days of continued growth, and FIG. 5 is a dry weight of the underground parts of maize seedlings after 20 days of continued growth. As can be seen from fig. 4 and 5, the above-ground and under-ground dry weights of the maize seedlings were increased by 40.7% and 42.4%, respectively, in the treated group compared to the control group.

The spore strain ZR19 can obviously promote the growth of the root system and the seedling body of the corn seedling and has the growth promoting effect on the corn seedling.

Experimental example 3 bacteriostatic experiment of biosurfactant

The biological surfactant prepared by the spore strain ZR19 is measured by a hyphal growth rate method to 7 plant pathogenic fungi: the inhibition of cercospora, fusarium equiseti, fusarium graminearum, fusarium verticillioides and fusarium exserotina.

Adding the biosurfactant prepared in the example 2 into a PDA solid culture medium, wherein the addition amount is 1 percent, and preparing a PDA drug sensitive plate; inoculating a pathogen bacteria mycelium block with the diameter of 5mm in the center of the plate, and taking a PDA plate without adding the biosurfactant as a control; culturing at 28 deg.C for 5 days, measuring the diameter of pathogenic fungi, and calculating the antibacterial rate according to the following formula:

the bacteriostatic ratio = (colony diameter control group-colony diameter experimental group)/colony diameter control group × 100%.

The results are shown in Table 1.

TABLE 1

As can be seen from Table 1, the biosurfactant produced by the spore strain ZR19 has good inhibition effect on 7 pathogenic fungi, the inhibition rate reaches 60.9-77.8%, the bacteriostasis spectrum is wide, and the bacteriostasis capability is strong.

SEQUENCE LISTING

<110> Shanxi purple Biotech limited

<120> spore strain and application

<130> RYP1910511.9

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1419

<212> DNA

<213> Bacillus spp.

<400> 1

gcaagtcgag 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 ttttaggagc cagccgccg 1419

Claims

1. A Bacillus strain (Bacillus sp.) is characterized in that the preservation number of the strain is CGMCC No. 18773.

2. The use of the spore strain of claim 1 for the production of a biosurfactant using kitchen waste as a carbon source.

3. A culture, wherein said culture is produced by the steps of:

(1) inoculating the spore strain of claim 1 into a liquid seed culture medium, culturing to obtain a seed culture solution, centrifuging the seed culture solution, and suspending the collected cells with sterile physiological saline to obtain a seed suspension;

(2) inoculating the seed suspension into a fermentation culture medium, and controlling the initial OD of a bacterial liquid in the fermentation culture medium after inoculation₆₀₀=1, then culturing to prepare a fermentation culture solution; the fermentation medium comprises the following components in percentage by mass: k₂HPO₄0.08～0.12%，KH₂PO₄ 0.08～0.12%，MgSO₄﹒7H₂O 0.01～0.03%，NaNO₃ 0.04～0.06%，(NH₄)₂SO₄0.04～0.06%，CaCl₂ 0.0008～0.0012%，FeSO₄0.0004-0.0006% of yeast powder, 0.01-0.03% of kitchen waste and 6.0-7.5% of pH;

4. The culture of claim 3, wherein the fermentation medium comprises the following components by mass: k₂HPO₄ 0.1%，KH₂PO₄ 0.1%，MgSO₄﹒7H₂O 0.02%，NaNO₃ 0.05%，(NH₄)₂SO₄ 0.05%，CaCl₂ 0.001%，FeSO₄0.0005%, 0.02% of yeast powder and 6% of kitchen waste, and the pH value is 7.0.

5. Use of a culture according to claim 3 or 4 for the preparation of a plant growth promoting agent, wherein the plant is selected from tomato and/or maize.

6. Use of the culture of claim 3 or 4 as a biosurfactant.

7. Use of the culture according to claim 3 or 4 for controlling plant diseases, wherein the plant diseases are diseases caused by cercospora, microsporum, fusarium oxysporum, fusarium equiseti, fusarium graminearum, fusarium verticillium and/or fusarium laminarinum.