CN111172060A - Bacillus with banana vascular wilt prevention and treatment function and preparation method and application thereof - Google Patents
Bacillus with banana vascular wilt prevention and treatment function and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus with a function of preventing and treating banana vascular wilt, and a preparation method and application thereof, wherein the bacillus is bacillus subtilis YX-11. The bacillus subtilis is obtained by the steps of culture material preparation, separation culture, screening of antagonistic endophytic bacteria and identification of antagonistic endophytic bacteria YX-11. The microbial inoculum of the bacillus subtilis YX-11 is obtained by performing primary preparation of seed liquid, secondary preparation of the seed liquid and fermentation on the bacillus subtilis YX-11. The preparation method of the bacillus subtilis YX-11 microbial inoculum comprises the steps of primary seed liquid preparation, secondary seed liquid preparation and fermentation liquid preparation. The preparation with the bacillus subtilis YX-11 as the active ingredient is green, environment-friendly and residue-free, has a remarkable prevention and treatment effect on banana wilt and has a promotion effect on plant growth.
Description
Technical Field
The invention belongs to the technical field of bacillus microorganism separation culture, and particularly relates to bacillus with a function of preventing and treating banana vascular wilt, and a preparation method and application thereof.
Background
The banana wilt is a vascular fungal soil-borne disease caused by fusarium oxysporum cubense, which can cause the blockage of the absorption and conduction of nutrients by roots and stems, poor growth, wilting and dwarfing, and the loss of yield and quality, so that the yield is reduced, and the failure is caused seriously, and the disease causes serious harm and economic loss to the worldwide banana industry. At present, the disease area of banana vascular wilt is gradually enlarged, and because the prevention and treatment effect of the conventional medicament on banana vascular wilt is not ideal and is difficult to prevent and treat, no specific medicament for preventing and treating banana vascular wilt exists at present, the specific medicament becomes the bottleneck of banana industry development, and the banana production and development are severely restricted. Because chemical agents and physical prevention and control have poor prevention and control effects on diseases, and agricultural microbial agents are adopted to prevent and control the blight based on the rapid development of the biological industry, so that the crops can be prevented from generating drug resistance, and the environment-friendly effect without residues can be realized, so that the development of a functional strain for efficiently preventing the banana wilt and a prevention and control product prepared by the functional strain are particularly important for the healthy and continuous development of the banana industry.
Disclosure of Invention
The first purpose of the invention is to provide a safe, environment-friendly and remarkable-control-effect Bacillus subtilis YX-11; the second purpose is to provide a preparation method of the bacillus subtilis YX-11; the third purpose is to provide the microbial inoculum of the bacillus subtilis YX-11; the fourth purpose is to provide a preparation method of the Bacillus subtilis YX-11 microbial inoculum; the fifth purpose is to provide the application of the Bacillus subtilis YX-11 microbial inoculum in the aspect of preventing and treating the banana wilt.
The first object of the present invention is achieved by: the Bacillus with the function of controlling banana wilt is Bacillus subtilis YX-11, wherein the preservation unit of the Bacillus subtilis YX-11 is as follows: the China general microbiological culture Collection center has the following preservation addresses: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on the day of deposit: 09 and 02 in 2019, with the preservation number: CGMCC No. 18456.
The second object of the present invention is achieved by: a preparation method of bacillus YX-11 with a function of preventing and treating banana vascular wilt comprises the following steps
1) Preparing a culture material:
selecting banana leaves for later use, wherein the banana leaves are used for picking healthy and disease-free banana plants;
melting LB solid culture medium and PDA culture medium, respectively pouring into a sterilized culture dish, and cooling to obtain LB culture medium flat plate and PDA culture medium flat plate; wherein, the LB culture medium plate is used for separating and preserving endophytic bacteria, and the PDA culture medium plate is used for culturing banana wilt bacteria and oppositional culture;
2) and (3) separation culture:
after the banana leaves are disinfected by alcohol, washing the banana leaves for 3-5 times by using sterilized water, placing the banana leaves in a sterilized mortar to be ground into juice, and coating the juice obtained by grinding on an LB culture medium plate; randomly picking colonies with different forms, carrying out purification culture and preservation, and naming one strain obtained by purification culture as YX-11;
3) screening antagonistic endophytic bacteria:
using banana fusarium oxysporum as an indicator bacterium, culturing the selected strain YX-11 by adopting a PDA (personal digital Assistant) plate confronting culture method, observing the bacteriostatic action of the strain YX-11 on the banana fusarium oxysporum in the culture process, and measuring the width of a bacteriostatic band;
4) identification of antagonistic endophytic bacteria YX-11:
according to the analysis result of the sequence similarity of the 16SrDNA and a phylogenetic tree, antagonistic endophytic bacteria YX-11 has the closest genetic relationship with strains Bacillus methylotrophicus, Bacillus amyloliquefaciens and Bacillus subtilis, the homology is the highest, the similarity reaches 99 percent, according to the similarity and homology analysis of the phylogenetic tree, the strains YX-11 and Bacillus subtilis are positioned in the same branch, the evolutionary relationship and the distance are the closest, the similarity reaches 100 percent, and the strain is identified to be the Bacillus subtilis by combining morphological characteristics, culture characteristics and physiological and biochemical characteristics.
The third object of the present invention is achieved by: the microbial inoculum of the Bacillus subtilis YX-11 is obtained by performing primary preparation of seed liquid, secondary preparation of the seed liquid and fermentation on the Bacillus subtilis YX-11.
The fourth object of the present invention is achieved by: a preparation method of a bacillus subtilis YX-11 microbial inoculum comprises the following specific steps
A, preparing a first-stage seed solution, namely selecting activated Bacillus subtilis YX-11 to inoculate the activated Bacillus subtilis YX-11 to a seed culture medium in an inoculation amount of 5-10%, and culturing for 10-15 hours to obtain a first-stage seed solution of a strain;
b, preparing a secondary seed solution, namely transferring the primary seed solution to a secondary seed culture medium in a seed tank according to the inoculation amount of 1-3%, and culturing for 10-15 h to obtain a secondary seed solution;
c, preparing fermentation liquor, namely inoculating the secondary seed liquid onto a fermentation medium in a fermentation tank according to the inoculation amount of 1-3%, and culturing for 46-48 h to obtain the effective viable bacteria number not less than 1 × 1010cfu/mL of fermentation liquor of Bacillus subtilis YX-11, namely the Bacillus subtilis microbial inoculum with the function of preventing and treating the banana wilt.
The fifth object of the present invention is achieved by: the bacillus subtilis YX-11 microbial inoculum is applied to the prevention and treatment of banana vascular wilt.
The invention has the beneficial effects that: the Bacillus subtilis YX-11 is a beneficial microorganism, and a preparation taking the Bacillus subtilis YX-11 as an active ingredient can be applied to controlling banana wilt, can be used as a microbial bactericide and has the following advantages: the coating is non-toxic, pollution-free, residue-free, safe to people and livestock, and has strong environmental adaptability and environmental friendliness; the banana vascular wilt prevention agent has strong inhibition effect on fungi and bacteria, has good effect on preventing banana vascular wilt and has promotion effect on plant growth; not only can avoid the generation of pesticide resistance of crops, but also can realize green environmental protection without residue; low production cost, simple and easy application method, and is beneficial to industrial production and application popularization of the preparation.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to be limiting in any way, and any modifications or alterations based on the teachings of the present invention are intended to fall within the scope of the present invention.
The Bacillus with the function of preventing and treating the banana wilt is Bacillus subtilis YX-11, and the preservation unit of the Bacillus subtilis YX-11 is as follows: the China general microbiological culture Collection center has the following preservation addresses: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on the day of deposit: 09 and 02 in 2019, with the preservation number: CGMCC No. 18456.
A preparation method of bacillus YX-11 with a function of preventing and treating banana vascular wilt comprises the following steps
1) Preparing a culture material:
selecting banana leaves for later use, wherein the banana leaves are used for picking healthy and disease-free banana plants;
melting LB solid culture medium and PDA culture medium, respectively pouring into a sterilized culture dish, and cooling to obtain LB culture medium flat plate and PDA culture medium flat plate; wherein, the LB culture medium plate is used for separating and preserving endophytic bacteria, and the PDA culture medium plate is used for culturing banana wilt bacteria and oppositional culture;
2) and (3) separation culture:
after the banana leaves are disinfected by alcohol, washing the banana leaves for 3-5 times by using sterilized water, placing the banana leaves in a sterilized mortar to be ground into juice, and coating the juice obtained by grinding on an LB culture medium plate; randomly picking colonies with different forms, carrying out purification culture and preservation, and naming one strain obtained by purification culture as YX-11;
3) screening antagonistic endophytic bacteria:
using banana fusarium oxysporum as an indicator bacterium, culturing the selected strain YX-11 by adopting a conventional PDA (personal digital assistant) plate confrontation culture method, observing the bacteriostatic action of the strain YX-11 on the banana fusarium oxysporum in the culture process, and measuring the width of a bacteriostatic band;
4) identification of antagonistic endophytic bacteria YX-11.
The microbial agent of the bacillus subtilis YX-11 is obtained by performing primary preparation of seed liquid, secondary preparation of the seed liquid and fermentation on the bacillus subtilis YX-11.
A preparation method of a bacillus subtilis YX-11 microbial inoculum comprises the steps of A first-stage seed liquid preparation, B second-stage seed liquid preparation and C fermentation liquid preparation, and comprises the specific steps of
A, preparing a first-stage seed solution, namely selecting activated Bacillus subtilis YX-11 to inoculate the activated Bacillus subtilis YX-11 to a seed culture medium in an inoculation amount of 5-10%, and culturing for 10-15 hours to obtain a first-stage seed solution of a strain;
b, preparing a secondary seed solution, namely transferring the primary seed solution to a secondary seed culture medium in a seed tank according to the inoculation amount of 1-3%, and culturing for 10-15 h to obtain a secondary seed solution;
and C, preparing fermentation liquor, namely inoculating the secondary seed liquid to a fermentation medium in a fermentation tank according to the inoculation amount of 1-3%, and culturing for 46-48 h to obtain the fermentation liquor of Bacillus subtilis YX-11, namely the Bacillus subtilis microbial inoculum with the function of preventing and treating the banana wilt.
In the step A, the strain is subjected to shake cultivation for 10-15 hours at the cultivation temperature of 35-37 ℃ and the rotation speed of 155-165 rpm/min, the seed culture medium is a conventional liquid LB culture medium, and the pH value is 7.0-8.0.
In the step B, the primary seed liquid is at the temperature of 35-38 ℃, the rotating speed is 100rpm/min, and the ventilation volume is 1.5 multiplied by 106mL/h, and culturing for 10-15 h under the condition that the tank pressure is kept at 0.04-0.06 MPa.
In the step C, the secondary seed liquid is at 35-38 ℃, the rotating speed is 100rpm/min, and the ventilation volume is 1.5 multiplied by 106mL/h, and culturing for 46-48 h under the condition that the tank pressure is kept at 0.04-0.06 MPa.
The secondary seed culture medium in the step B and the fermentation culture medium in the step C both comprise the following components in percentage by mass: 1.4% of soybean meal, 3% of cane sugar, 3% of peptone, 3% of corn flour, 2% of fish meal, 0.03% of sodium chloride, 0.05% of magnesium sulfate, 6% of calcium carbonate, 0.03% of monopotassium phosphate, 0.03% of dipotassium phosphate, 0.02% of manganese sulfate and the balance of water, wherein the pH value is 7.0-8.0.
The effective viable count of the bacillus subtilis preparation prepared in the step C is not less than 1 multiplied by 1010cfu/mL。
The application of the bacillus subtilis YX-11 microbial inoculum in the aspect of preventing and treating banana wilt is to dilute 10 times, 20 times or 30 times of the bacillus subtilis microbial inoculum with the function of preventing and treating banana wilt to irrigate roots (6000 mL/plant) of banana plants in a greenhouse and spray the diluted solution to prevent and treat the banana wilt.
The invention is further illustrated by the following specific examples:
example 1
This example provides a method for preparing Bacillus subtilis YX-11.
1) Preparing a culture material:
selecting banana leaves for later use, wherein the banana leaves are used for picking healthy and disease-free banana plants;
melting conventional LB solid culture medium and PDA culture medium, respectively pouring into a sterilized culture dish, cooling, and making into LB culture medium flat plate and PDA culture medium flat plate; wherein, the LB culture medium plate is used for separating and preserving endophytic bacteria, and the PDA culture medium plate is used for culturing banana wilt bacteria and oppositional culture;
2) and (3) separation culture:
after the banana leaves are disinfected by alcohol, washing the banana leaves for 3-5 times by using sterilized water, placing the banana leaves in a sterilized mortar to be ground into juice, and coating the juice obtained by grinding on an LB culture medium plate; randomly selecting colonies with different forms, carrying out conventional purification culture and storing, and naming one strain obtained by purification culture as YX-11;
3) screening antagonistic endophytic bacteria:
using banana fusarium oxysporum as an indicator bacterium, culturing the selected strain YX-11 by adopting a conventional PDA (personal digital assistant) plate confrontation culture method, observing the bacteriostatic action of the strain YX-11 on the banana fusarium oxysporum in the culture process, and measuring the width of a bacteriostatic band;
4) identification of antagonistic endophytic bacteria YX-11:
through molecular identification, the antagonistic endophytic bacterium YX-11 is closest to the genetic relationship of strains Bacillus methylotrophicus, Bacillus amyloliquefaciens and Bacillus subtilis, the homology is highest, the similarity reaches 99%, according to the similarity and homology analysis of phylogenetic trees, the strains YX-11 and Bacillus subtilis are positioned in the same branch, the evolutionary relationship and the distance are closest, the similarity reaches 100%, and the strain is identified as the Bacillus subtilis by combining morphological characteristics, culture characteristics and physiological and biochemical characteristics.
Example 2
The embodiment provides a preparation method of a Bacillus subtilis YX-11 microbial inoculum.
A preparation method of a bacillus subtilis YX-11 microbial inoculum comprises the following specific steps:
a, preparing a first-stage seed solution, namely selecting activated Bacillus subtilis YX-11 to inoculate a seed culture medium with a pH value of 7.0 by 5% of inoculum size, wherein the seed culture medium is a conventional liquid LB culture medium, and performing shake culture on the strain for 10-15 hours at a culture temperature of 35-37 ℃ and a rotation speed of 155-165 rpm/min to obtain the first-stage seed solution of the strain;
b, preparing a second-stage seed solution, namely transferring the first-stage seed solution to a second-stage seed culture medium with the pH value of 7.0 in a seed tank according to the inoculation amount of 1 percent, and enabling the first-stage seed solution to be transferred to the second-stage seed culture medium in the seed tankThe seed liquid is cultured at 35-38 deg.C, rotation speed of 100rpm/min, and ventilation amount of 1.5 × 106mL/h, and maintaining the tank pressure at 0.04-0.06 MPa, and culturing for 10-15 h to obtain a secondary seed solution;
c, preparing fermentation liquor, namely inoculating the secondary seed liquid to a fermentation culture medium with the pH value of 7.0 in a fermentation tank according to the inoculation amount of 1%, and enabling the secondary seed liquid to have the culture temperature of 35-38 ℃, the rotating speed of 100rpm/min and the ventilation volume of 1.5 multiplied by 106The effective viable count is not less than 1 multiplied by 10 after the culture for 46 to 48 hours under the conditions that the mL/h and the tank pressure are kept between 0.04 and 0.06MPa10cfu/mL of fermentation liquor of Bacillus subtilis YX-11, namely the Bacillus subtilis microbial inoculum with the function of preventing and treating the banana wilt.
Wherein the secondary seed culture medium in the step B and the medium fermentation culture medium in the step C both comprise the following components in percentage by mass: 1.4 percent of soybean meal, 3 percent of cane sugar, 3 percent of peptone, 3 percent of corn flour, 2 percent of fish meal, 0.03 percent of sodium chloride, 0.05 percent of magnesium sulfate, 6 percent of calcium carbonate, 0.03 percent of monopotassium phosphate, 0.03 percent of dipotassium phosphate, 0.02 percent of manganese sulfate and the balance of water, wherein the pH value is 7.0.
Example 3
The embodiment provides a preparation method of a Bacillus subtilis YX-11 microbial inoculum.
A preparation method of a bacillus subtilis YX-11 microbial inoculum comprises the following specific steps:
a, preparing a first-stage seed solution, namely selecting activated Bacillus subtilis YX-11 to inoculate a seed culture medium with a pH value of 7.5 in an inoculation amount of 7.5%, wherein the seed culture medium is a conventional liquid LB culture medium, and performing shake culture on the strain for 10-15 hours at a culture temperature of 35-37 ℃ and a rotation speed of 155-165 rpm/min to obtain the first-stage seed solution of the strain;
b, preparing a secondary seed solution, namely transferring the primary seed solution to a secondary seed culture medium with the pH value of 7.5 in a seed tank according to the inoculation amount of 2%, and enabling the primary seed solution to have the culture temperature of 35-38 ℃, the rotating speed of 100rpm/min and the ventilation volume of 1.5 multiplied by 106mL/h, and the tank pressure is kept at 0.04-0.06 MPa, and the culture is carried out for 10-15 h to obtain the productTo secondary seed liquid;
c, preparing fermentation liquor, namely inoculating the secondary seed liquid to a fermentation culture medium with the pH value of 7.5 in a fermentation tank according to the inoculation amount of 2%, and enabling the secondary seed liquid to have the culture temperature of 35-38 ℃, the rotating speed of 100rpm/min and the ventilation volume of 1.5 multiplied by 106The effective viable count is not less than 1 multiplied by 10 after the culture for 46 to 48 hours under the conditions that the mL/h and the tank pressure are kept between 0.04 and 0.06MPa10cfu/mL of fermentation liquor of Bacillus subtilis YX-11, namely the Bacillus subtilis microbial inoculum with the function of preventing and treating the banana wilt.
Wherein the secondary seed culture medium in the step B and the medium fermentation culture medium in the step C both comprise the following components in percentage by mass: 1.4 percent of soybean meal, 3 percent of cane sugar, 3 percent of peptone, 3 percent of corn flour, 2 percent of fish meal, 0.03 percent of sodium chloride, 0.05 percent of magnesium sulfate, 6 percent of calcium carbonate, 0.03 percent of monopotassium phosphate, 0.03 percent of dipotassium phosphate, 0.02 percent of manganese sulfate and the balance of water, wherein the pH value is 7.5.
Example 4
The embodiment provides a preparation method of a Bacillus subtilis YX-11 microbial inoculum.
A preparation method of a bacillus subtilis YX-11 microbial inoculum comprises the following specific steps:
a, preparing a first-stage seed solution, namely selecting activated Bacillus subtilis YX-11 to inoculate a seed culture medium with the pH value of 8.0 in an inoculation amount of 10%, wherein the seed culture medium is a conventional liquid LB culture medium, and performing shake culture on the strain for 10-15 hours at a culture temperature of 35-37 ℃ and a rotation speed of 155-165 rpm/min to obtain the first-stage seed solution of the strain;
b, preparing a second-stage seed solution, namely transferring the first-stage seed solution to a second-stage seed culture medium with the pH value of 8.0 in a seed tank according to the inoculation amount of 3 percent, and enabling the first-stage seed solution to have the culture temperature of 35-38 ℃, the rotating speed of 100rpm/min and the ventilation volume of 1.5 multiplied by 106mL/h, and maintaining the tank pressure at 0.04-0.06 MPa, and culturing for 10-15 h to obtain a secondary seed solution;
c, preparing fermentation liquor, namely inoculating the secondary seed liquid into the fermentation tank according to the inoculation amount of 3 percent, wherein the pH value of the secondary seed liquid is 8.0The fermentation medium of (1) and the secondary seed liquid is cultured at a temperature of 35-38 deg.C, a rotation speed of 100rpm/min, and an aeration rate of 1.5 × 106The effective viable count is not less than 1 multiplied by 10 after the culture for 46 to 48 hours under the conditions that the mL/h and the tank pressure are kept between 0.04 and 0.06MPa10cfu/mL of fermentation liquor of bacillus subtilis YX-11, namely the bacillus subtilis microbial inoculum with the function of preventing and treating the banana wilt.
Wherein the secondary seed culture medium in the step B and the medium fermentation culture medium in the step C both comprise the following components in percentage by mass: 1.4 percent of soybean meal, 3 percent of cane sugar, 3 percent of peptone, 3 percent of corn flour, 2 percent of fish meal, 0.03 percent of sodium chloride, 0.05 percent of magnesium sulfate, 6 percent of calcium carbonate, 0.03 percent of monopotassium phosphate, 0.03 percent of dipotassium phosphate, 0.02 percent of manganese sulfate and the balance of water, wherein the pH value is 8.0.
Example 5
The embodiment provides application of a Bacillus subtilis YX-11 microbial inoculum in preventing and treating banana wilt.
The concrete mode is as follows:
the field test is carried out on microbial strains in Anning City of Kunming, Yunnan province and a test greenhouse applying national local combined engineering research center, and the pathogenic bacteria of banana vascular wilt are inoculated by adopting a root-damaging inoculation method with the inoculation concentration of 107CFU/mL, the inoculation amount is 50 mL/strain, and clear water is used as a control.
The test was divided into treatment group and control group. Respectively diluting YX-11 biological bactericide by 10 times, 20 times and 30 times of liquid to carry out root irrigation (6000 mL/plant) and spraying treatment on banana plants in a greenhouse, taking 1000 times of liquid of pesticide carbendazim (50 percent of effective component) as a positive control and clear water as a negative control, carrying out spraying treatment for 1 time every 7 days, and continuously spraying for 5 times. Each treatment consisted of 3 replicate groups, each replicate group planting 12 banana plants (i.e. each treatment consisted of 3 banana plant groups set under the same planting conditions, each replicate group planting 12 banana plants). The disease incidence was investigated and the control effect was calculated 6 months after each treatment was sprayed, and the control effect (%) was [ (control disease incidence-treatment disease incidence) control disease incidence ] × 100, and the test results are shown in table 1.
Grading standard of leaf diseases:
level 1: no stripe or yellow leaf appears, and the plant is healthy;
and 2, stage: slight streaking or yellowing of the bottom leaves;
and 3, level: streaking or substantial yellowing of the bottom leaves;
4, level: a large number of streaks or almost all leaf yellowing occurs;
and 5, stage: the whole plant died.
From the experimental statistics data in table 1, we can see that the banana plants treated by the stock solution of the bacillus subtilis YX-11 fermentation broth and the fermentation broth diluted by 10 times thereof have better control effect; the 40-time diluted fermentation liquor of the bacillus subtilis YX-11 has similar control effect with the plant treated by the carbendazim; the control effect of 60 times diluted fermentation liquor of the bacillus subtilis YX-11 is only 16.17 percent, and the control effect is poor, so the application is not recommended.
Claims (10)
1. The bacillus with the function of preventing and treating banana vascular wilt is characterized in that: the Bacillus is Bacillus subtilis YX-11, and the preservation unit of the Bacillus subtilis YX-11 is as follows: the China general microbiological culture Collection center has the following preservation addresses: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on the day of deposit: 09 and 02 in 2019, with the preservation number: CGMCC No. 18456.
2. The preparation method of the bacillus YX-11 with the function of preventing and treating banana vascular wilt, which is disclosed by claim 1, is characterized in that: comprises the following steps
1) Preparing a culture material:
selecting banana leaves for later use, wherein the banana leaves are used for picking healthy and disease-free banana plants;
melting LB solid culture medium and PDA culture medium, respectively pouring into a sterilized culture dish, and cooling to obtain LB culture medium flat plate and PDA culture medium flat plate; wherein, the LB culture medium plate is used for separating and preserving endophytic bacteria, and the PDA culture medium plate is used for culturing banana wilt bacteria and oppositional culture;
2) and (3) separation culture:
after the banana leaves are disinfected by alcohol, washing the banana leaves for 3-5 times by using sterilized water, placing the banana leaves in a sterilized mortar to be ground into juice, and coating the juice obtained by grinding on an LB culture medium plate; randomly picking colonies with different forms, carrying out purification culture and preservation, and naming one strain obtained by purification culture as YX-11;
3) screening antagonistic endophytic bacteria:
using banana fusarium oxysporum as an indicator bacterium, culturing the selected strain YX-11 by adopting a PDA (personal digital Assistant) plate confronting culture method, observing the bacteriostatic action of the strain YX-11 on the banana fusarium oxysporum in the culture process, and measuring the width of a bacteriostatic band;
4) identification of antagonistic endophytic bacteria YX-11.
3. The bacillus subtilis YX-11 microbial inoculum of claim 1, which is characterized in that: the Bacillus subtilis YX-11 is obtained by primary preparation of seed liquid, secondary preparation of seed liquid and fermentation.
4. The preparation method of the bacillus subtilis YX-11 microbial inoculum according to claim 3, which comprises the steps of A primary seed liquid preparation, B secondary seed liquid preparation and C fermentation liquid preparation, and is characterized in that: comprises the following specific steps
A, preparing a first-stage seed solution, namely selecting activated Bacillus subtilis YX-11 to inoculate the activated Bacillus subtilis YX-11 to a seed culture medium in an inoculation amount of 5-10%, and culturing for 10-15 hours to obtain a first-stage seed solution of a strain;
b, preparing a secondary seed solution, namely transferring the primary seed solution to a secondary seed culture medium in a seed tank according to the inoculation amount of 1-3%, and culturing for 10-15 h to obtain a secondary seed solution;
and C, preparing fermentation liquor, namely inoculating the secondary seed liquid to a fermentation medium in a fermentation tank according to the inoculation amount of 1-3%, and culturing for 46-48 h to obtain the fermentation liquor of Bacillus subtilis YX-11, namely the Bacillus subtilis microbial inoculum with the function of preventing and treating the banana wilt.
5. The method for preparing Bacillus subtilis YX-11 microbial inoculum according to claim 4, which is characterized in that: in the step A, the strain is subjected to shake cultivation for 10-15 hours at the cultivation temperature of 35-37 ℃ and the rotation speed of 155-165 rpm/min, the seed culture medium is a conventional liquid LB culture medium, and the pH value is 7.0-8.0.
6. The method for preparing Bacillus subtilis YX-11 microbial inoculum according to claim 4, which is characterized in that: in the step B, the primary seed liquid is at the temperature of 35-38 ℃, the rotating speed is 100rpm/min, and the ventilation volume is 1.5 multiplied by 106mL/h, and culturing for 10-15 h under the condition that the tank pressure is kept at 0.04-0.06 MPa.
7. The method for preparing Bacillus subtilis YX-11 microbial inoculum according to claim 4, which is characterized in that: in the step C, the secondary seed liquid is at 35-38 ℃, the rotating speed is 100rpm/min, and the ventilation volume is 1.5 multiplied by 106mL/h, and culturing for 46-48 h under the condition that the tank pressure is kept at 0.04-0.06 MPa.
8. The method for preparing Bacillus subtilis YX-11 microbial inoculum according to claim 4, which is characterized in that: the secondary seed culture medium in the step B and the fermentation culture medium in the step C both comprise the following components in percentage by mass: 1.4% of soybean meal, 3% of cane sugar, 3% of peptone, 3% of corn flour, 2% of fish meal, 0.03% of sodium chloride, 0.05% of magnesium sulfate, 6% of calcium carbonate, 0.03% of monopotassium phosphate, 0.03% of dipotassium phosphate, 0.02% of manganese sulfate and the balance of water, wherein the pH value is 7.0-8.0.
9. The method for preparing Bacillus subtilis YX-11 microbial inoculum according to claim 4, which is characterized in that: the effective viable count of the bacillus subtilis preparation prepared in the step C is not less than 1 multiplied by 1010cfu/mL。
10. The application of the bacillus subtilis YX-11 microbial inoculum of claim 4 in the aspect of preventing and treating banana vascular wilt.
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| CN112006036A (en) * | 2020-09-08 | 2020-12-01 | 仲恺农业工程学院 | Microbial suppository for disease resistance and yield increase of bananas as well as preparation method and application of microbial suppository |
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| CN113502246A (en) * | 2021-07-08 | 2021-10-15 | 广西科学院 | Compound microbial agent and preparation method and application thereof |
| CN116064275A (en) * | 2022-07-06 | 2023-05-05 | 海南大学 | Spore bacteria for preventing and treating banana vascular wilt and application thereof |
| CN117327599A (en) * | 2023-04-17 | 2024-01-02 | 广州澄华农业科技有限公司 | Bacillus subtilis subspecies desert strain and application thereof |
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| CN112006036A (en) * | 2020-09-08 | 2020-12-01 | 仲恺农业工程学院 | Microbial suppository for disease resistance and yield increase of bananas as well as preparation method and application of microbial suppository |
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| CN116064275A (en) * | 2022-07-06 | 2023-05-05 | 海南大学 | Spore bacteria for preventing and treating banana vascular wilt and application thereof |
| CN116064275B (en) * | 2022-07-06 | 2024-01-30 | 海南大学 | Spore bacteria for preventing and treating banana vascular wilt and application thereof |
| CN117327599A (en) * | 2023-04-17 | 2024-01-02 | 广州澄华农业科技有限公司 | Bacillus subtilis subspecies desert strain and application thereof |
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