CN113717890B - Bacillus salis strain and application thereof - Google Patents
Bacillus salis strain and application thereof Download PDFInfo
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Abstract
The invention relates to a bacillus salis strain and application thereof. The bacillus salis is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2020558. The bacillus salis Y10312 can effectively antagonize pathogenic bacteria of ginger root rot, especially the antagonism rate of Pythium gracile and Fusarium solani reaches more than 79%, and the morbidity of rot of ginger root can be obviously reduced, so that the crop yield is greatly improved, and the invention has wide application prospect in the aspect of treating plant diseases.
Description
Technical Field
The invention relates to the technical field of plant disease control and microorganisms, in particular to a bacillus salis strain and application thereof.
Background
The plant root rot is a disease caused by fungi, the plant with early disease does not show obvious symptoms, the function of absorbing moisture and nutrient of the plant is gradually weakened along with the aggravation of the rot degree of the root, the overground part is first yellow due to the supply and the shortage of the nutrient, and when the illumination is strong before and after the midnoon and the evaporation capacity is large, the leaf on the upper part of the plant is wilted, but can recover at night. When the disease is serious, the wilting condition can not be recovered at night, and the whole plant leaves yellow and wither. At this time, the root bark turns brown and separates from the medulla, and finally the whole plant dies. The root rot can be caused by infection of various pathogens such as Pythium, fusarium, phytophthora, and the like, the pathogens overwintering in soil or on disease residues, become a primary infection source in the next year, and the pathogens invade from root wounds or rhizome parts and spread through rainwater or irrigation water.
Ginger belongs to a perennial monocotyledonous herbaceous plant, has extremely high economic value, and has wide application in the fields of food, medicines, health care products and the like. In the process of planting ginger, a planter often neglects the problem that ginger is easy to generate infection and disease, and the problem of ginger disease becomes a key factor for restricting the development of the ginger industry. In ginger planting production, more common ginger diseases are mainly ginger blast diseases caused by pathogenic bacteria, root rot diseases caused by pathogenic fungi, wilt diseases caused by pathogenic bacteria and fungi, and the like. Pathogenic bacteria firstly infects the root and stem parts of ginger and are often easily ignored by growers, and if timely measures are not taken, the root and even the whole plant of the plant are caused to attack, so that the yield of ginger is reduced, the quality is reduced, and huge economic losses are brought to the vast ginger growers.
Currently common disease control methods include physical control, chemical control and biological control methods. The physical control mainly changes the planting land block every year, avoids continuous cropping planting to prevent and treat the occurrence of ginger root diseases, and has the defect of long treatment period. The chemical control mainly uses various pesticides to control pathogenic bacteria, the method is easy to cause pollution, the symptoms and the root causes are not cured, and the method is easy to cause the defects of vicious circle of disease occurrence and deterioration of physical and chemical properties of soil. The biological control mainly uses biocontrol bacteria to prevent diseases, and the method is green and environment-friendly, but can achieve good prevention effect only by screening biocontrol strains with high efficiency and high pertinence. Therefore, screening of the high-efficiency antagonistic strain against ginger pathogenic bacteria is a key to effectively solving the problem of ginger diseases.
The mangrove wetland system has rich microbial resources and various functions, and is a valuable resource library for discovering microbial strains for medicine, industry and agriculture, so that the efficient antagonistic strain of plant pathogenic bacteria is excavated from the mangrove wetland system, the problem of serious plant soil-borne disease transmission can be effectively solved, and the bottleneck for restricting plant development is solved.
Disclosure of Invention
Based on the above, the invention aims to provide a novel bacillus salis Y10312 (Halobacillus sp.) which can effectively prevent and treat ginger root rot, and particularly has good antagonism effect on pathogenic fungi Pythium gracile (Pythium myriotylum) and Fusarium solani (Fusarium solani) so as to achieve the purpose of preventing and treating diseases.
The specific technical scheme is as follows:
a bacillus salis Y10312, the bacillus salis Y10312 is preserved in China center for type culture collection, and the preservation number is CCTCC NO: M2020558.
The invention also provides application of the bacillus salis Y10312 in preventing and treating ginger root rot.
The invention also provides application of the bacillus salis Y10312 in preparing a biological agent for preventing and treating ginger root rot.
The invention also provides application of the bacillus salis Y10312 in preparation of antagonistic Pythium gracile and/or Fusarium solani biological preparations.
The invention also provides a biological agent, and the active ingredient of the biological agent comprises the bacillus salis Y10312.
The invention also provides a method for preventing and treating plant root rot, which comprises the following steps: soaking ginger seeds in the bacterial liquid containing the bacillus salis Y10312, then planting, and drip irrigation the roots by using the bacterial liquid containing the bacillus salis Y10312 after the planted ginger grows out.
Further, salt spores in the bacterial liquidThe concentration of the Bacillus Y10312 was 1.0X10 8 cfu/ml～1.0×10 10 cfu/ml。
Further, the soaking time is 30-60 s.
Further, the drip irrigation method comprises the following steps: drip irrigation is carried out once every 12-18 days, at least 3 times.
In some of these embodiments, the control method comprises the steps of: planting rhizoma Zingiberis recens at a concentration of 1.0X10 of Bacillus salicus Y10312 8 cfu/ml～1.0×10 10 Soaking cfu/ml of bacterial liquid for 30-60 s, planting, and using the bacillus salis Y10312 with the concentration of 1.0X10 after ginger buds 8 cfu/ml～1.0×10 10 The root is drip-irrigated by cfu/ml bacterial liquid, 1 time every 12-18 days, at least 3 times.
Further, the control method comprises the following steps: planting rhizoma Zingiberis recens at a concentration of 1.0X10 of Bacillus salicus Y10312 8 cfu/ml～1.0×10 10 Soaking cfu/ml of the bacterial liquid for 30s, then planting, and using the bacillus salis Y10312 with the concentration of 1.0X10 after ginger buds 8 cfu/ml～1.0×10 10 The root is drip-irrigated by cfu/ml bacterial liquid, 1 time is carried out every 15 days, the bacterial liquid amount is 3-5L/mu in each drip-irrigation, and the total drip-irrigation is 3 times.
The invention also provides a culture method of the bacillus salis Y10312, which comprises the following steps: inoculating the bacillus salis Y10312 into a culture medium, and culturing at 28-37 ℃.
In some of these embodiments, the medium is 2216E medium or LB medium.
Further, the culture medium is 2216E liquid culture medium, wherein the 2216E liquid culture medium comprises the following raw materials in parts by weight: 5 parts of peptone, 1 part of yeast extract powder, 0.1 part of ferric citrate, 19.45 parts of sodium chloride, 5.98 parts of magnesium chloride, 3.24 parts of sodium sulfate, 1.8 parts of calcium chloride, 0.55 part of potassium chloride, 0.16 part of sodium carbonate, 0.08 part of potassium bromide, 0.034 part of strontium chloride, 0.022 part of boric acid, 0.004 part of sodium silicate, 0.0024 part of sodium fluoride, 0.0016 part of sodium nitrate, 0.008 part of disodium hydrogen phosphate and 1000 parts of sterile water.
When the culture medium is 2216E liquid culture medium, the culture method of the bacillus salis Y10312 comprises the following steps:
s1 inoculation: the bacillus salis Y10312 is added at the ratio of 1.0X10 5 cfu/ml～5.0×10 6 Inoculating the cfu/ml inoculating concentration and inoculating amount of bacterial liquid to culture medium volume ratio of 1:20-1:100 into 2216E liquid culture medium;
s2, culturing: 2216E liquid culture inoculated with the Bacillus salidus Y10312 was based on shaking culture at 28 ℃.
Further, the inoculation concentration in step S1 is 2.5X10 6 cfu/ml。
Further, the inoculation amount in the step S1 is that the volume ratio of the bacterial liquid to the culture medium is 1:100.
Further, the culture time in the step S2 is 24-48 hours; further, the incubation time was 28 hours.
Further, the oscillating speed in the step S2 is 180 rpm-220 rpm; still further, the oscillating speed was 180rpm.
In some embodiments, the culture medium is an LB liquid culture medium comprising the following raw materials in parts by weight: 10 parts of tryptone, 5 parts of yeast extract powder, 10 parts of sodium chloride and 1000 parts of sterile water.
When the culture medium is LB liquid culture medium, the culture method of the bacillus salidus Y10312 comprises the following steps:
s1 inoculation: the bacillus salis Y10312 is added at the ratio of 1.0X10 5 cfu/ml～5.0×10 6 Inoculating the cfu/ml inoculating concentration and inoculating amount of bacterial liquid to culture medium volume ratio of 1:20-1:100 into LB liquid culture medium;
s2, culturing: LB liquid culture inoculated with the salt spore rod Y10312 was shake-cultured at 37 ℃.
Further, the inoculation concentration in step S1 is 2.5X10 6 cfu/ml。
Further, the inoculation amount in the step S1 is that the volume ratio of the bacterial liquid to the culture medium is 1:100.
Further, the culture time in the step S2 is 24-48 hours; further, the incubation time was 24 hours.
Further, the oscillating speed in the step S2 is 180 rpm-220 rpm; still further, the oscillating speed was 180rpm.
The bacillus halosporidium Y10312 (Halobacillus sp.) of the invention is preserved in China center for type culture collection (CCTCC for short; address: china center for type culture collection in Wuhan university of Wuhan City, hubei province, china) on 9 months 29 days in 2020, and the preservation date is as follows: 9/29/2020, accession number: cctccc No. M2020558.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a novel bacillus salis Y10312 which can effectively antagonize pathogenic bacteria of ginger root rot, and particularly has an antagonism rate of more than 79% on Pythium gracile and Fusarium solani. When the method is used for planting ginger, the incidence rate of ginger root rot can be effectively reduced (from 36.62% to 3.30%), so that the ginger yield is greatly improved. The bacillus salis Y10312 has wide application prospect in the aspect of preventing and controlling ginger diseases.
Drawings
FIG. 1 is a colony morphology diagram of the Bacillus saliensis Y10312 of the present invention after 48h culture on 2216E agar solid medium;
FIG. 2 is a colony morphology diagram of the Bacillus salis Y10312 of the invention after 48h culture on LB solid medium;
FIG. 3 is a colony morphology of Pythium gracile after 48h culture on PDA solid medium;
FIG. 4 is a colony morphology of Fusarium solani after 48h culture on PDA solid medium;
FIG. 5 is a graph showing the results of an experiment for antagonizing Pythium insidiosum by Bacillus salis Y10312 according to the present invention; wherein A is an antagonism experiment graph of the bacillus salis Y10312 on Pythium insidiosum, and B is a negative experiment sterile water comparison graph;
FIG. 6 is a graph showing the results of an experiment for antagonizing Fusarium solani by the Bacillus salis Y10312; wherein A is an antagonism experiment graph of bacillus salis Y10312 on Fusarium solani, and B is a negative experiment sterile water comparison graph;
FIG. 7 is a nucleic acid electrophoresis chart of PCR amplification of the 16SrRNA sequence of Bacillus salidus Y10312 according to the present invention;
FIG. 8 is a tree of evolution constructed by the Bacillus salis Y10312 of the present invention and the 16S rRNA sequence bar codes of similar species.
Detailed Description
The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
In the present invention, the term "plurality" means two or more. "and/or", describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.
The present invention will be described in further detail with reference to specific examples.
The 2216E agar solid culture medium comprises the following components in parts by weight: 5 parts of peptone, 1 part of yeast extract powder, 0.1 part of ferric citrate, 19.45 parts of sodium chloride, 5.98 parts of magnesium chloride, 3.24 parts of sodium sulfate, 1.8 parts of calcium chloride, 0.55 part of potassium chloride, 0.16 part of sodium carbonate, 0.08 part of potassium bromide, 0.034 part of strontium chloride, 0.022 part of boric acid, 0.004 part of sodium silicate, 0.0024 part of sodium fluoride, 0.0016 part of sodium nitrate, 0.008 part of disodium hydrogen phosphate, 15 parts of agar and 1000 parts of sterile water. The preparation method comprises the following steps: weighing and measuring the components, stirring and dissolving the components in sterile water, subpackaging the sterile water into conical flasks, sealing the conical flasks with sterilizing paper, sterilizing the conical flasks in a sterilizing pot at 121 ℃ under 101KPa for 20min under high pressure steam, taking out the sterilizing pot when the temperature of the sterilizing pot is reduced to below 70 ℃ and the pressure is restored to 0KPa, pouring the sterilizing pot into culture dishes on an ultra clean bench, pouring about 20ml of culture medium into each culture dish, cooling and solidifying, and preserving the solid culture plates at 4 ℃.
The formula of 2216E liquid culture medium comprises the following components in parts by weight: 5 parts of peptone, 1 part of yeast extract powder, 0.1 part of ferric citrate, 19.45 parts of sodium chloride, 5.98 parts of magnesium chloride, 3.24 parts of sodium sulfate, 1.8 parts of calcium chloride, 0.55 part of potassium chloride, 0.16 part of sodium carbonate, 0.08 part of potassium bromide, 0.034 part of strontium chloride, 0.022 part of boric acid, 0.004 part of sodium silicate, 0.0024 part of sodium fluoride, 0.0016 part of sodium nitrate, 0.008 part of disodium hydrogen phosphate and 1000 parts of sterile water. Weighing and measuring the components, stirring and dissolving the components in sterile water, subpackaging the sterile water into conical flasks, sealing the conical flasks by using sterilizing paper, sterilizing the conical flasks by using high-pressure steam at 121 ℃ and 101KPa for 20min in a sterilizing pot, taking out the conical flasks when the temperature of the sterilizing pot is reduced to below 70 ℃ and the pressure is restored to 0KPa, and preserving the conical flasks for later use.
The formula of the PDA solid culture medium comprises the following components in parts by weight: 200 parts of potato, 20 parts of glucose, 3 parts of NaCl and KH 2 PO 4 1.5 parts, 15 parts of agar powder and 1000 parts of sterile water. Weighing and measuring the above componentsAnd then, stirring and dissolving the components in sterile water, sub-packaging the sterile water into conical flasks, sealing the conical flasks by using sterilizing paper, placing the conical flasks in a sterilizing pot for sterilizing for 20min by using high-pressure steam at 121 ℃ and 101KPa, taking out the conical flasks when the temperature of the sterilizing pot is reduced to below 70 ℃ and the pressure is restored to 0KPa, and preserving the conical flasks for later use.
The formula of the LB liquid medium comprises the following components in parts by weight: 10 parts of tryptone, 5 parts of yeast extract powder, 10 parts of sodium chloride and 1000 parts of sterile water. The manufacturing method comprises the following steps: accurately adding the components, metering volume, packaging into conical bottle, sterilizing in a sterilizing pot at 121deg.C under 101KPa for 20min, and taking out when the sterilizing pot temperature is reduced to below 70deg.C and pressure is restored to 0KPa, and storing.
Embodiment one: separation and purification method of bacillus salis Y10312
The separation and purification method of the salt spore rod Y10312 strain comprises the following steps:
s1: weighing 10g of a soil sample from mangrove soil of a wetland park in a mangrove national land of the sea-land island in the Guangdong province; then, adding 90ml of sterile water into the soil sample in an ultra-clean workbench, and placing the mixture on an oscillator for 60min to enable the soil sample to be uniformly dispersed in the diluent to form soil suspension; after the soil is dispersed, 100ul of soil suspension is absorbed into 900ul of sterile water to obtain 10 times of diluent, and then 10 times of diluent is sequentially diluted to obtain 10 2 Multiple dilutions, 10 3 Multiple dilutions, 10 4 Multiple dilutions, 10 5 Multiple dilutions and 10 6 The whole process is carried out in an ultra-clean bench.
S2: taking 100ul of 10 times of diluent and 10 2 Multiple dilutions, 10 3 Multiple dilutions, 10 4 Multiple dilutions, 10 5 Multiple dilutions and 10 6 The multiple dilutions were spread on 2216E agar solid plates, respectively, and then the plates were placed in an incubator at 28℃for 2-3 days, and bacterial plaques were grown on the plates.
S3: after the culture is finished, single bacterial plaques with different shapes, colors, sizes and the like are picked one by one from a proper dilution gradient flat plate according to the growth condition of bacterial colonies, and the flat plate streak is carried out, so that the salt spore rod Y10312 strain is obtained through separation.
S4: the purified and cultured salt spore rod Y10312 strain is selected from 2216E, inoculated into a 500ml conical flask containing 200ml 2216E liquid culture medium, and cultured for 28 hours under the conditions of 28 ℃ and 180rpm constant temperature shaking culture to prepare seed liquid. The seed liquid and glycerin are filled in tubes according to the volume ratio of 1:4 and uniformly mixed to obtain the concentration of 1.0x10 5 ～5.0×10 6 The glycerol seed solution per ml is stored at-20deg.C or-80deg.C for a long time after marking on the tube wall.
The morphology of the isolated salt spore rod Y10312 strain in this example cultured on 2216E agar solid culture plate for 48h is shown in FIG. 1, and the colony is gray, irregular, soft in texture, rough in surface and spread to the periphery.
The salt spore rod Y10312 obtained by separation in the embodiment is coated on LB solid medium, then the culture plate is placed in an incubator for constant temperature culture at 37 ℃ for 48 hours, the morphological diagram of the culture is shown in figure 2, the bacterial colony is in an off-white irregular shape, the texture is softer, the surface is rough, and the bacterial colony diffuses to the periphery.
Embodiment two: antagonism of Bacillus salis Y10312 against Pythium insidiosum and Fusarium solani
S1 inoculation: taking glycerol seed liquid preserved at the temperature of minus 20 ℃ in the first embodiment, wherein the volume ratio of the fungus liquid to the culture medium is 1: inoculating 100 inoculum size into 2216E liquid culture medium, placing into a constant temperature shaking table, and culturing at 28deg.C for 28 hr to obtain activated bacterial liquid;
s2, identifying a culture medium: a flat plate facing method is adopted. Taking pathogenic fungus mass Pythium gracile (figure 3) and Fusarium solani (figure 4) fungus block (diameter 0.8 cm) after culturing on PDA solid medium for 48 hr, placing in the center of PDA solid medium, and sucking 20ul activated fungus liquid at 2cm distance from the fungus block for spot grafting, wherein three parallel groups are total; meanwhile, the control group flat plate is only connected with pathogenic bacteria at the center of the flat plate; after inoculation, placing the strain in a biochemical incubator at a constant temperature of 28 ℃ for 3-5d, observing whether a bacteriostasis ring is generated, and measuring test data such as the size of the bacteriostasis ring.
The results are shown in fig. 5 and 6: in the embodiment of the invention, the isolated salt bacillus strain Y10312 grows well on a PDA culture medium and generates a bacteriostasis zone, and the growth of pathogenic bacteria Pythium gracile and Fusarium solani is inhibited. The results show that the bacillus salis strain Y10312 has the function of antagonizing pathogenic bacterial group Pythium gracile and Fusarium solani and can be used for preventing and treating plant diseases caused by pathogenic bacterial group Pythium gracile and Fusarium solani.
Embodiment III: bacillus salis Y10312 culture method
This example provides a method of culturing two B.salina strains Y10312.
The first culture method, using 2216E liquid medium, was as follows:
activation before culture: taking glycerol seed liquid preserved at the temperature of minus 20 ℃ in the first embodiment, wherein the volume ratio of the fungus liquid to the culture medium is 1:100 inoculum size was inoculated into 2216E liquid medium, and placed in a constant temperature shaking table for 28h of shaking culture at a constant temperature of 28℃to obtain an activated bacterial liquid.
S1 inoculation: taking activated bacterial liquid, wherein the volume ratio of the bacterial liquid to the culture medium is 1:100 in 500ml 2216E liquid culture medium;
s2, culturing: the culture flask inoculated with the Bacillus salis strain Y10312 is placed in a constant temperature shaking table, and is subjected to constant temperature shaking culture at a speed of 180 rpm-220 rpm (180 rpm in this example is preferred) for 24 h-48 h (28 h in this example) at 28 ℃ to obtain a high-density bacterial suspension.
The second culture method uses LB liquid medium, and the process is as follows:
activation before culture: taking glycerol seed liquid preserved at the temperature of minus 20 ℃ in the first embodiment, wherein the volume ratio of the fungus liquid to the culture medium is 1:100 inoculum size was inoculated into LB liquid medium, and the culture was carried out in a shaking table at 37℃for 28 hours under shaking to obtain an activated bacterial liquid.
S1 inoculation: taking activated bacterial liquid, wherein the volume ratio of the bacterial liquid to the culture medium is 1:100 is inoculated in 500ml LB liquid medium;
s2, culturing: the culture flask inoculated with the Bacillus salis Y10312 is placed in a constant temperature shaking table, and is subjected to constant temperature shaking culture at a speed of 180 rpm-220 rpm (180 rpm in the embodiment is preferred) for 24-48 h (24 h in the embodiment) at 37 ℃ to obtain a high-density bacterial suspension.
Embodiment four: PCR amplification of 16S rRNA sequence of Bacillus salidus Y10312 and sequencing
S1: extraction of genomic DNA
Genomic DNA was extracted using the Omega Bacterial DNA Kit (D3350-01) kit. Firstly, taking 2ml of seed liquid in the first embodiment in a sterile 2ml centrifuge tube, centrifuging at 12000rpm for 2min, and discarding the supernatant to keep sediment; then 100ul of 1×TE Buffer is added into the precipitate, vortex mixing is carried out, 10ul of lysozyme is added and mixing is carried out, and the temperature bath is carried out for 10min at 37 ℃; adding 100ul BTL Buffer and 20ul proteinase K, mixing, warm-bathing at 55deg.C for 1 hr, and mixing with shaking for three times; adding 5ul RNaseA enzyme, mixing, standing at room temperature for 5min, centrifuging at 10000rpm for 2min, collecting 200ul supernatant, and transferring to a new sterile 1.5ml centrifuge tube; adding 200ul BTL Buffer, mixing, and standing at 65deg.C for 10min; adding 200ul of absolute ethyl alcohol, mixing by vortex, transferring all samples into an adsorption column, centrifuging at 10000rpm for 2min, discarding supernatant and the adsorption column, and placing the adsorption column into a new collecting pipe; adding 500ul HBC Buffer,10000rpm to the adsorption column, centrifuging for 2min, and discarding supernatant; adding 700ul DNA Wash Buffer,10000rpm to the adsorption column, centrifuging for 2min, discarding supernatant, and repeating twice; putting the empty adsorption column into a collecting pipe again, and centrifuging at 10000rpm for 2min; adding 30 ul-50 ul Elution Buffer (preheated at 65 ℃) to dissolve DNA precipitate into an adsorption column to obtain genome DNA, and preserving at-20 ℃ for later use.
S2: PCR amplification of 16SrRNA sequence
PCR amplification was performed using the genomic DNA obtained in step S1 as a template and Eubac27F and Eubac1492R as primers, and the PCR reaction system (50. Mu.l) was as follows:
the PCR reaction procedure was: pre-denaturation at 94℃for 5min; denaturation at 94 ℃ for 30s, renaturation at 55 ℃ for 30s, extension at 72 ℃ for 1min, and repeated circulation for 30 times; the PCR amplification product was stored at 72℃for another 10min and at 4 ℃.
The sequence of the upstream primer Eubac27F is as follows: 5'-agagtttgat cctggctcag-3', SEQ ID NO:2.
the sequence of the downstream primer Eubac1492R is as follows: 5'-ggttaccttg ttacgactt-3', SEQ ID NO:3.
s3: nucleic acid electrophoresis of PCR products
After spotting 5ul of the PCR product obtained in step S2, nucleic acid electrophoresis was performed at 120V for 25min. As shown in FIG. 7, the 16S rRNA fragment amplified by using the genomic DNA of Bacillus saliicus Y10312 isolated in the first embodiment of the present invention as a template has single band and high brightness, and the length of the 16S rRNA sequence is about 1500bp.
S4: sequencing of the 16S rRNA sequence
After purifying and recovering the PCR product obtained in the step S2, taking 30ul of the purified product, and sending the purified product to a Guangzhou department biotechnology limited company for sequence two-way sequencing, wherein the sequencing result shows that the 16S rRNA sequence length of the bacillus halobacillus strain is 1480bp, and the specific sequence is SEQ ID NO:1 is shown as follows:
SEQ ID NO：1：
fifth embodiment: construction of phylogenetic tree
The 16S rRNA sequence of the Bacillus salis strain obtained in the fourth example was input into NCBI, BLAST alignment was performed, and an NJ phylogenetic tree was constructed based on the barcode 16S rRNA segment sequence, and as shown in FIG. 8, according to the phylogenetic tree, the isolated Bacillus salis Y10312 strain of the first example of the present invention had a sequence similarity of 99% with Bacillus salis Halobacillus salinus strain HSL-3 (NR 025244.1), and was highly similar to most of the sequences in the 16S rRNA barcode database of Bacillus salis but did not have complete overlap. Thus, it is understood that the strain obtained in the present invention belongs to the genus Bacillus salis in classification and is a novel species, and the present invention designates it as Halobacillus sp.Y10312.
Example six: control effect of bacillus salis Y10312 on ginger diseases
S1, activating strains: taking glycerol seed liquid preserved at the temperature of minus 20 ℃ in the first embodiment, wherein the volume ratio of the fungus liquid to the culture medium is 1: inoculating 100 inoculum size into 2216E liquid culture medium, placing into a constant temperature shaking table, and culturing at 28deg.C for 28 hr to obtain activated bacterial liquid;
fermentation of S2 Bacillus salis Y10312: inoculating the strain activated by the S1 into an LB liquid culture medium, and stopping culturing when the fermentation culture is carried out until the OD600 is about 1.5;
s3, ginger planting application: before ginger planting, diluting the fermentation broth obtained in the step S2 by 400 times, soaking ginger seeds in the diluted fermentation broth for about 30 seconds, and then planting. The planting test is carried out on an experiment base of the agricultural university of West Hua in the Qingchun district, the local small yellow ginger is planted, the experiment base is a continuous cropping planting base, the experiment group and the control group are 3 mu, and each of the three is repeated for 1 mu. About 2200 plants per acre.
S4, drip irrigation: after the ginger bud, the experiment group dilutes the fermentation broth obtained in the step S2 by 200-400 times (400 times is preferred in the embodiment), the ginger root is subjected to drip irrigation, 3-5L (4L is preferred in the embodiment) of the diluted fermentation broth is used for each mu of land, and the diluted fermentation broth is used for 3 times continuously every 15 days. The control group was directly irrigated with the same amount of water drops in the same way without adding bacterial liquid.
S5, statistics: and digging out roots 3.5 months after budding, counting the rotting percentage of the roots, and counting the acre yield when harvesting. The results are shown in Table 1:
TABLE 1 control effect of Bacillus salis Y10312 on ginger diseases
As is clear from Table 1, the root rot incidence of ginger is reduced from 36.62% to 3.30% and the yield per mu is increased from 2593kg to 4016kg after the treatment of the Bacillus salis Y10312 bacterial liquid obtained by the first separation in the embodiment of the invention. The results show that the bacillus salis Y10312 obtained by separation in the embodiment of the invention can effectively reduce the incidence of ginger root rot, improve the yield of ginger and has wide application prospect in ginger disease control.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention.
SEQUENCE LISTING
<110> agricultural university of south China; guangdong New technology Co., ltd
<120> a Bacillus salis strain and use thereof
<130> 2021-08-26
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1480
<212> DNA
<213> Artificial Sequence
<400> 1
tgggggcaca aggacctggc acttcggcgg ctggctccaa aaggttacct caccgacttc 60
gggtgttgcc aactctcgtg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca 120
ccgcggcatg ctgatccgcg attactagcg attccggctt catgcaggcg agttgcagcc 180
tgcaatccga actgagaatg gttttatggg atttgctaca cctcgcggct tcgctgccct 240
ttgttccatc cattgtagca cgtgtgtagc ccaggtcata aggggcatga tgatttgacg 300
tcatccccgc cttcctccgg tttgtcaccg gcagtcacct tagagtgccc aactaaatgc 360
tggcaactaa gattaggggt tgcgctcgtt gcgggactta acccaacatc tcacgacacg 420
agctgacgac aaccatgcac cacctgtcat ttggtccccg aagggaagac cctatctcta 480
gggtggtcca aagatgtcaa gacctggtaa ggttcttcgc gttgcttcga attaaaccac 540
atgctccacc gcttgtgcgg gcccccgtca attcctttga agtttcagcc ttgcggccgt 600
actccccagg cggagtgctt aatgcgttaa cttcagcact aaggggtgga agccccctaa 660
cacctagcac tcatcgttta cggcgtggac taccagggta tctaatcctg tttgctaccc 720
acgctttcgc acctcagcgt cagaaacaga ccagagagtc gccttcgcca ctggtgttcc 780
tccacatatc tacgcatttc accgctacac gtggaattcc actctcctct tctgtcctca 840
agttccccag tttccaatgg ccctccacgg ttgagccgtg gcctttcaca tcagacttaa 900
ggaaccgcct gcgcgcgctt tacgcccaat aattccggac aacgcttgcc ccctacgtat 960
taccgcggct gctggcacgt agttagccgg ggcttcctcg ttaggtaccg tcaaggtacc 1020
gctctattcg cacggtactt gttcttccct aacaacagaa ctttacgatc cgaagacctt 1080
catcgttcac gcggcgttgc tccgtcagac tttcgtccat tgcggaagat tccctactgc 1140
tgcctcccgt aggagtctgg gccgtgtctc agtcccagtg tggccgatca ccctctcagg 1200
tcggctacgc atcgtcgcct tggtgagccg ttacctcacc aactagctaa tgcgccgcgg 1260
gcccatctgt aagtgatagc gaaaagccat cttttaatgt tccttcatgc gaaggaacat 1320
gttacccggc attagctccg gtttcccgga gttattccga tcttacaggc aggttgccca 1380
cgtgttactc acccgtccgc cgctcgttcc acgagcgtca cccccgaagg ggatcagctc 1440
gcttcccgcg ctcgactgca ttatagtacc cgccctccaa 1480
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 2
agagtttgat cctggctcag 20
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 3
ggttaccttg ttacgactt 19
Claims (8)
1. The bacillus salis Y10312 is characterized in that the bacillus salis Y10312 is preserved in China center for type culture collection, and the preservation number is CCTCC NO: M2020558.
2. Use of bacillus salis Y10312 as claimed in claim 1 for controlling ginger root rot.
3. Use of bacillus salis Y10312 as claimed in claim 1 for the preparation of a biological agent for controlling ginger root rot.
4. Use of bacillus salis Y10312 as claimed in claim 1 for the preparation of antagonistic humicola and/or fusarium solani biologicals.
5. A biological agent, characterized in that the active ingredient of the biological agent comprises bacillus salis Y10312 according to claim 1.
6. The control method of plant root rot is characterized by comprising the following steps: soaking ginger seeds in the bacterial liquid containing the bacillus salis Y10312 in claim 1, then planting, and drip irrigation the roots after the planted ginger grows out and buds by using the bacterial liquid containing the bacillus salis Y10312 in claim 1.
7. The control method according to claim 6, wherein the concentration of Bacillus salis Y10312 in the bacterial liquid is 1.0X10 8 cfu/ml～1.0×10 10 cfu/ml；
And/or the soaking time is 30-60 s;
and/or, the drip irrigation method comprises the following steps: drip irrigation is carried out once every 12-18 days, at least 3 times.
8. The method for culturing bacillus salis Y10312 according to claim 1, comprising the steps of:
inoculating the bacillus salis Y10312 into a culture medium, and culturing at 28-37 ℃.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877943A (en) * | 2015-06-11 | 2015-09-02 | 福建农林大学 | Antagonistic bacterium for controlling radix rehmannia root rot and application of antagonistic bacterium |
| CN108070539A (en) * | 2017-08-28 | 2018-05-25 | 福建省农业科学院农业生物资源研究所 | The bacillus of one plant of biological control for root rot and bacterial wilt |
| CN110591963A (en) * | 2019-09-29 | 2019-12-20 | 山东佐田氏生物科技有限公司 | Saline-alkali-tolerant bacillus malassezia and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877943A (en) * | 2015-06-11 | 2015-09-02 | 福建农林大学 | Antagonistic bacterium for controlling radix rehmannia root rot and application of antagonistic bacterium |
| CN108070539A (en) * | 2017-08-28 | 2018-05-25 | 福建省农业科学院农业生物资源研究所 | The bacillus of one plant of biological control for root rot and bacterial wilt |
| CN110591963A (en) * | 2019-09-29 | 2019-12-20 | 山东佐田氏生物科技有限公司 | Saline-alkali-tolerant bacillus malassezia and application thereof |
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