CN113373092B - Composite microbial preparation and preparation method thereof - Google Patents
Composite microbial preparation and preparation method thereof Download PDFInfo
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Abstract
The invention provides a compound microbial preparation and a preparation method thereof, wherein bacillus subtilis and streptomyces roseoflash are compounded into the compound microbial preparation, and the prevention and control effects are realized through various disease prevention mechanisms such as secretion of resistant substances, re-mailing effect, induction of host cells to generate defending structures, competition effect and the like, and the two strains have synergistic effect, so that soil-borne disease pathogenic bacteria such as gray mold, powdery mildew, banana wilt and soybean root rot can be effectively and stably prevented and treated, and the compound microbial preparation can be also used for soil improvement of crop fields such as vegetables, flowers, melons and fruits, forest trees, chinese herbal medicines and the like after continuous cropping.
Description
Technical Field
The invention relates to the technical field of biological agents, in particular to a compound microbial agent and a preparation method thereof.
Background
At present, chemical control is mainly used for controlling plant diseases, however, long-term use of chemical control agents not only can generate pollution, but also can cause various diseases to have larger and larger resistance, and probiotics in plants and soil are killed by mistake while harmful bacteria are killed, so that the immune system of the plants is destroyed, and the plant diseases are more difficult to control.
In recent years, as the pollution problem of chemical pesticides is becoming serious, biological control is becoming more and more important because of the advantages of no pollution, no residue, difficult drug resistance, safety to ecological environment and human health and the like, and the application of biological control technology in plant disease control is attracting attention. The biological control technology refers to the use of biological control microorganisms existing in nature, and the use of living bodies or metabolites thereof to inhibit the number and activity of pathogenic microorganisms, thereby reducing the occurrence rate of diseases or slowing down the occurrence rate of the diseases and reducing the severity of the diseases.
At present, researches on biological control agents are mostly single strains, CN103131657A (bacillus subtilis and biological control preparations and applications thereof) discloses application of the bacillus subtilis in the biological control preparations, but the disease prevention mechanism of the single strain is single, the control effect is low and unstable. The composite bacterial preparation has a plurality of strains, so that the preservation and the application of the composite bacterial preparation are limited by a plurality of conditions.
Disclosure of Invention
The invention provides a compound microorganism preparation and a preparation method thereof.
The invention relates to a compound microorganism preparation, which comprises the following components: bacillus subtilis (Bacillus subtilis) and streptomyces roseoflash (Streptomyces roseoflavus).
Further, the viable bacteria content in the composite microbial preparation is 10 6 -10 9 cfu/g。
Further, the CFU ratio of bacillus subtilis and streptomyces roseoflash in the compound microbial preparation is (4-7): (1-2).
Further, the composite microbial preparation further comprises: and (3) a solid agent.
Further, the solid agent is at least one of diatomite, kaolin, bentonite and mica powder.
The invention also provides a preparation method of the compound microbial preparation, which comprises the following steps:
(1) Inoculating bacillus subtilis and streptomyces roseoflash respectively to respective activation culture mediums for activation culture, and inoculating the bacillus subtilis and the streptomyces roseoflash respectively to respective fermentation culture mediums for fermentation culture to obtain respective fermentation bacterial solutions;
(2) Filtering fermentation bacteria liquid respectively, collecting mycoplasm, drying, pulverizing to obtain bacterial powder, mixing bacterial powder of each strain to obtain composite bacterial powder;
(3) Mixing the residual bacterial liquid filtered in the step (2), adding 75-95% v/v ethanol solution with the bacterial liquid volume of 4-7 times, and performing ultrasonic extraction for 5-10min with the ultrasonic power of 200-230W to obtain composite bacterial liquid;
(4) Mixing the compound bacterial liquid with a solid agent, drying, and adding compound bacterial powder to obtain the compound microbial preparation.
Further, the activation medium may be a conventional activation medium capable of culturing bacillus subtilis and streptomyces roseoflash.
Further, each liter of the bacillus subtilis fermentation medium comprises the following components: 2-5g/L ammonium sulfate, 1-4g/L calcium carbonate, 0.2-0.5g/L magnesium sulfate, 0.1-0.5 g/L sodium chloride, 3-6g/L peptone, 10-15g/L corn starch, 10-15g/L malt flour and the balance of water.
Further, each liter of the Streptomyces roseoflash fermentation medium comprises the following components: 0.2-0.5g/L dipotassium hydrogen phosphate, 0.3-0.8g/L ammonium nitrate, 2-4g/L calcium carbonate, 5-10g/L potassium sodium tartrate, 0.2-0.8g/L sodium citrate, 10-20g/L starch, 8-12g/L peanut cake powder, 14-22g/L corn steep liquor and the balance water.
Furthermore, the invention also provides application of the compound microbial preparation in preventing and treating pathogenic bacteria of soil-borne diseases and/or preparing soil conditioner.
Further, the soil-borne disease pathogens include, but are not limited to, botrytis cinerea (Botrytis cinerea), chaetomium monofil (Sphaerotheca fuliginea), fusarium oxysporum (Fusarium oxysporum), verticillium dahliae (Verticiullim dahliae Kleb), and rhizoctonia solani (Rhizoctonia solani).
Compared with the prior art, the invention has the beneficial effects that:
the invention compounds bacillus subtilis and streptomyces roseofluosus into a compound microbial preparation, realizes the control effect by secreting resistant substances, re-mailing, inducing host cells to generate a plurality of disease prevention mechanisms such as defending structures, competing effects and the like, has a synergistic effect between two strains, can effectively and stably control soil-borne disease pathogenic bacteria such as gray mold, powdery mildew, banana wilt and soybean root rot, and can also be used for soil improvement of crop fields such as vegetables, flowers, melons and fruits, trees, chinese herbal medicines and the like after continuous cropping.
The invention carries out fermentation culture after activating strains, separates the bacterial and the fermentation bacterial liquid after fermentation is finished, prepares the bacterial into dry powder, mixes and extracts active substances in the fermentation bacterial liquid, then prepares the extracted composite bacterial liquid and a solid agent into a powdery preparation, and finally mixes the powdery preparation with the bacterial powder to effectively preserve the bacterial powder.
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
The bacillus subtilis used in the invention has the commercial number of ACCC 03221 and the streptomyces roseoflavone has the commercial number of ACCC 40415.
Streptomyces lilacinus used in the invention has a commercial number ACCC40024.
The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
Example 1
A complex microbial formulation comprising: bacillus subtilis, streptomyces roseoflavone and kieselguhr, wherein the CFU ratio of the bacillus subtilis to the streptomyces roseoflavone is 5:2.
the viable cell amount contained in the composite microbial preparation was 10 8 cfu/g。
The preparation method of the compound microbial preparation comprises the following steps:
(1) Inoculating bacillus subtilis and streptomyces roseofluosus into a PDA culture medium respectively, performing activation culture at 28 ℃ for 6 hours, performing activation culture, inoculating bacillus subtilis into a bacillus subtilis fermentation culture medium, and culturing for 72 hours at 32-35 ℃ under the condition of pH7 to obtain bacillus subtilis fermentation broth; inoculating streptomyces roseoflavae into a streptomyces roseoflavae fermentation medium, and culturing for 144 hours at 30-32 ℃ to obtain streptomyces roseoflavae fermentation broth;
each liter of the bacillus subtilis fermentation medium comprises the following components: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5g/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L malt flour and the balance water;
each liter of the Streptomyces roseoflash fermentation medium comprises the following components: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium nitrate, 3g/L calcium carbonate, 6g/L potassium sodium tartrate, 0.5g/L sodium citrate, 15g/L starch 10g/L peanut cake powder, 18g/L corn steep liquor and the balance water;
(2) Filtering fermentation bacteria liquid respectively, collecting mycoplasm, drying, pulverizing to obtain bacterial powder, mixing bacterial powder of each strain to obtain composite bacterial powder;
(3) Mixing the residual bacterial liquid filtered in the step (2), adding an 80% v/v ethanol solution with the bacterial liquid volume being 6 times, and carrying out ultrasonic extraction for 6min, wherein the ultrasonic power is 230W, so as to obtain a composite bacterial liquid;
(4) Mixing the composite bacterial liquid with diatomite, drying, and adding the composite bacterial powder to obtain the composite microbial preparation.
Example 2
A complex microbial formulation comprising: bacillus subtilis, streptomyces roseoflash and mica powder, wherein the CFU ratio of the bacillus subtilis to the streptomyces roseoflash is 4:1.
the viable cell amount contained in the composite microbial preparation was 10 6 cfu/g。
The preparation method of the compound microbial preparation comprises the following steps:
(1) Inoculating bacillus subtilis and streptomyces roseofluosus into a PDA culture medium respectively, performing activation culture at 28 ℃ for 6 hours, performing activation culture, inoculating bacillus subtilis into a bacillus subtilis fermentation culture medium, and culturing for 72 hours at 32-35 ℃ under the condition of pH7 to obtain bacillus subtilis fermentation broth; inoculating streptomyces roseoflavae into a streptomyces roseoflavae fermentation medium, and culturing for 144 hours at 30-32 ℃ to obtain streptomyces roseoflavae fermentation broth;
each liter of the bacillus subtilis fermentation medium comprises the following components: 2g/L ammonium sulfate, 1g/L calcium carbonate, 0.2g/L magnesium sulfate, 0.4 g/L sodium chloride, 6g/L peptone, 15g/L corn starch, 10g/L malt flour and the balance water;
each liter of the Streptomyces roseoflash fermentation medium comprises the following components: 0.5g/L dipotassium hydrogen phosphate, 0.3-0.8g/L ammonium nitrate, 2g/L calcium carbonate, 5g/L sodium potassium tartrate, 0.2g/L sodium citrate, 10g/L starch, 12g/L peanut cake powder, 22g/L corn steep liquor and the balance water;
(2) Filtering fermentation bacteria liquid respectively, collecting mycoplasm, drying, pulverizing to obtain bacterial powder, mixing bacterial powder of each strain to obtain composite bacterial powder;
(3) Mixing the residual bacterial liquid filtered in the step (2), adding a 75% v/v ethanol solution with the bacterial liquid volume being 7 times, and carrying out ultrasonic extraction for 5min, wherein the ultrasonic power is 210W, so as to obtain a composite bacterial liquid;
(4) Mixing the composite bacterial liquid with mica powder, drying, and adding the composite bacterial powder to obtain the composite microbial preparation.
Example 3
A complex microbial formulation comprising: bacillus subtilis, streptomyces roseoflavone and kieselguhr, wherein the CFU ratio of the bacillus subtilis to the streptomyces roseoflavone is 5:2.
the viable cell amount contained in the composite microbial preparation was 10 8 cfu/g。
The preparation method of the compound microbial preparation comprises the following steps:
(1) Inoculating bacillus subtilis and streptomyces roseofluosus into a PDA culture medium respectively, performing activation culture at 28 ℃ for 6 hours, performing activation culture, inoculating bacillus subtilis into a bacillus subtilis fermentation culture medium, and culturing for 72 hours at 32-35 ℃ under the condition of pH7 to obtain bacillus subtilis fermentation broth; inoculating streptomyces roseoflavae into a streptomyces roseoflavae fermentation medium, and culturing for 144 hours at 30-32 ℃ to obtain streptomyces roseoflavae fermentation broth;
each liter of the bacillus subtilis fermentation medium comprises the following components: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5g/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L malt flour and the balance water;
each liter of the Streptomyces roseoflash fermentation medium comprises the following components: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium nitrate, 3g/L calcium carbonate, 6g/L potassium sodium tartrate, 0.5g/L sodium citrate, 15g/L starch 10g/L peanut cake powder, 18g/L corn steep liquor and the balance water;
(2) Filtering fermentation bacteria liquid respectively, collecting mycoplasm, drying, pulverizing to obtain bacterial powder, mixing bacterial powder of each strain to obtain composite bacterial powder;
(3) Mixing the residual bacterial liquid obtained in the step (2) by filtering to obtain a composite bacterial liquid;
(4) Mixing the composite bacterial liquid with diatomite, drying, and adding the composite bacterial powder to obtain the composite microbial preparation.
Example 4
A complex microbial formulation comprising: bacillus subtilis, streptomyces roseoflavone and kieselguhr, wherein the CFU ratio of the bacillus subtilis to the streptomyces roseoflavone is 5:2.
the viable cell amount contained in the composite microbial preparation was 10 8 cfu/g。
The preparation method of the compound microbial preparation comprises the following steps:
(1) Inoculating bacillus subtilis and streptomyces roseofluosus into a PDA culture medium respectively, performing activation culture at 28 ℃ for 6 hours, performing activation culture, inoculating bacillus subtilis into a bacillus subtilis fermentation culture medium, and culturing for 72 hours at 32-35 ℃ under the condition of pH7 to obtain bacillus subtilis fermentation broth; inoculating streptomyces roseoflavae into a streptomyces roseoflavae fermentation medium, and culturing for 144 hours at 30-32 ℃ to obtain streptomyces roseoflavae fermentation broth;
each liter of the bacillus subtilis fermentation medium comprises the following components: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5g/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L fish meal and the balance water;
each liter of the Streptomyces roseoflash fermentation medium comprises the following components: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium nitrate, 3g/L calcium carbonate, 6g/L potassium sodium tartrate, 0.5g/L sodium citrate, 15g/L starch 10g/L peanut cake powder, 18g/L corn steep liquor and the balance water;
(2) Filtering fermentation bacteria liquid respectively, collecting mycoplasm, drying, pulverizing to obtain bacterial powder, mixing bacterial powder of each strain to obtain composite bacterial powder;
(3) Mixing the residual bacterial liquid filtered in the step (2), adding an 80% v/v ethanol solution with the bacterial liquid volume being 6 times, and carrying out ultrasonic extraction for 6min, wherein the ultrasonic power is 230W, so as to obtain a composite bacterial liquid;
(4) Mixing the composite bacterial liquid with diatomite, drying, and adding the composite bacterial powder to obtain the composite microbial preparation.
Example 5
A complex microbial formulation comprising: bacillus subtilis, streptomyces roseoflavone and kieselguhr, wherein the CFU ratio of the bacillus subtilis to the streptomyces roseoflavone is 5:2.
the viable cell amount contained in the composite microbial preparation was 10 8 cfu/g。
The preparation method of the compound microbial preparation comprises the following steps:
(1) Inoculating bacillus subtilis and streptomyces roseofluosus into a PDA culture medium respectively, performing activation culture at 28 ℃ for 6 hours, performing activation culture, inoculating bacillus subtilis into a bacillus subtilis fermentation culture medium, and culturing for 72 hours at 32-35 ℃ under the condition of pH7 to obtain bacillus subtilis fermentation broth; inoculating streptomyces roseoflavae into a streptomyces roseoflavae fermentation medium, and culturing for 144 hours at 30-32 ℃ to obtain streptomyces roseoflavae fermentation broth;
each liter of the bacillus subtilis fermentation medium comprises the following components: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5g/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L malt flour and the balance water;
each liter of the Streptomyces roseoflash fermentation medium comprises the following components: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium nitrate, 3g/L calcium carbonate, 6g/L potassium sodium tartrate, 15g/L starch 10g/L peanut cake powder, 18g/L corn steep liquor and the balance water;
(2) Filtering fermentation bacteria liquid respectively, collecting mycoplasm, drying, pulverizing to obtain bacterial powder, mixing bacterial powder of each strain to obtain composite bacterial powder;
(3) Mixing the residual bacterial liquid filtered in the step (2), adding an 80% v/v ethanol solution with the bacterial liquid volume being 6 times, and carrying out ultrasonic extraction for 6min, wherein the ultrasonic power is 230W, so as to obtain a composite bacterial liquid;
(4) Mixing the composite bacterial liquid with diatomite, drying, and adding the composite bacterial powder to obtain the composite microbial preparation.
Comparative example 1
A microbial formulation comprising: bacillus subtilis and diatomite, wherein the living bacteria content in the microbial preparation is 10 8 cfu/g。
A method for preparing a microbial preparation, comprising the steps of:
(1) Inoculating bacillus subtilis into a PDA culture medium, performing activation culture at 28 ℃ for 6 hours, performing activation culture, inoculating into a bacillus subtilis fermentation culture medium, and culturing for 72 hours at 32-35 ℃ under the condition of pH7 to obtain bacillus subtilis fermentation broth;
each liter of the bacillus subtilis fermentation medium comprises the following components: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5g/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L malt flour and the balance water;
(2) Filtering fermentation broth, collecting mycoplasm, oven drying, and pulverizing to obtain fungus powder;
(3) Filtering the residual bacterial liquid in the step (2), adding an 80% v/v ethanol solution with the bacterial liquid volume being 6 times, and carrying out ultrasonic extraction for 6min, wherein the ultrasonic power is 230W to obtain bacterial liquid;
(4) Mixing the bacterial liquid extracted in the step (3) with diatomite, drying, and adding bacterial powder to obtain the composite microbial preparation.
Comparative example 2
A microbial formulation comprising: streptomyces roseoflavatus and diatomite, wherein the viable bacteria content in the microbial preparation is 10 8 cfu/g。
A method for preparing a microbial preparation, comprising the steps of:
(1) Inoculating Streptomyces roseoflavatus into PDA culture medium, activating and culturing at 28deg.C for 6h, activating and culturing, inoculating into Streptomyces roseoflavatus fermentation culture medium, and culturing at 30-32deg.C for 144h to obtain Streptomyces roseoflavatus fermentation broth;
each liter of the Streptomyces roseoflash fermentation medium comprises the following components: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium nitrate, 3g/L calcium carbonate, 6g/L potassium sodium tartrate, 0.5g/L sodium citrate, 15g/L starch 10g/L peanut cake powder, 18g/L corn steep liquor and the balance water;
(2) Filtering fermentation broth, collecting mycoplasm, oven drying, and pulverizing to obtain fungus powder;
(3) Filtering the residual bacterial liquid in the step (2), adding an 80% v/v ethanol solution with the bacterial liquid volume being 6 times, and carrying out ultrasonic extraction for 6min, wherein the ultrasonic power is 230W to obtain bacterial liquid;
(4) Mixing the bacterial liquid extracted in the step (3) with diatomite, drying, and adding bacterial powder to obtain the composite microbial preparation.
Comparative example 3
A complex microbial formulation comprising: bacillus subtilis and streptomyces lavendulae (Streptomyces lavendulae) and kieselguhr, wherein the CFU ratio of the bacillus subtilis to the streptomyces lavendulae is 5:2.
the viable cell amount contained in the composite microbial preparation was 10 8 cfu/g。
The preparation method of the compound microbial preparation comprises the following steps:
(1) Inoculating bacillus subtilis and streptomyces lilacinus into a PDA culture medium respectively, performing activation culture for 6 hours at 28 ℃, performing activation culture, inoculating bacillus subtilis into a bacillus subtilis fermentation culture medium, and culturing for 72 hours at 32-35 ℃ under the condition of pH7 to obtain bacillus subtilis fermentation bacterial liquid; inoculating Streptomyces lilacinus into Streptomyces lilacinus fermentation medium, and culturing at 30-32 ℃ for 144h to obtain Streptomyces lilacinus fermentation broth;
each liter of the bacillus subtilis fermentation medium comprises the following components: 3g/L ammonium sulfate, 2g/L calcium carbonate, 0.3g/L magnesium sulfate, 0.5g/L sodium chloride, 35g/L peptone, 12g/L corn starch, 12g/L malt flour and the balance water;
each liter of the Streptomyces lilacinus fermentation medium comprises the following components: 0.3g/L dipotassium hydrogen phosphate, 0.5g/L ammonium carbonate, 3g/L calcium carbonate, 0.5g/L magnesium sulfate, 15g/L starch 10g/L soybean meal, 18g/L corn steep liquor and the balance water;
(2) Filtering fermentation bacteria liquid respectively, collecting mycoplasm, drying, pulverizing to obtain bacterial powder, mixing bacterial powder of each strain to obtain composite bacterial powder;
(3) Mixing the residual bacterial liquid filtered in the step (2), adding an 80% v/v ethanol solution with the bacterial liquid volume being 6 times, and carrying out ultrasonic extraction for 6min, wherein the ultrasonic power is 230W, so as to obtain a composite bacterial liquid;
(4) Mixing the composite bacterial liquid with diatomite, drying, and adding the composite bacterial powder to obtain the composite microbial preparation.
Test example 1 effect of use
Test strain: botrytis cinerea), sphaerotheca (Sphaerotheca fuliginea (Schlect. Ex Fr. Poll.), fusarium oxysporum (Fusarium oxysporum), verticillium dahliae (Verticiullim dahliae Kleb), rhizoctonia solani (Rhizoctonia solani);
the measuring method comprises the following steps: diluting a dissolved bacterial preparation with a proper amount of double distilled water to prepare a bacterial preparation solution, uniformly mixing the bacterial preparation solution with a PDA culture medium according to a volume ratio of 1:3, preparing a PDA drug-containing culture medium, adding an equal amount of double distilled water into the culture medium as a blank control, setting three repetitions in each group, cutting bacterial cakes along the outer edge of hypha by using a puncher with a diameter of 5mm, inoculating the bacterial cakes into the PDA drug-containing culture medium, culturing the bacterial cakes in a constant temperature incubator with a temperature of 27+/-1 ℃ for 5d, measuring the diameter of bacterial colonies, taking an average value of the results, comparing the blank control, calculating the antibacterial rate, and calculating the formula as follows:
antibacterial ratio (%) = [ (colony diameter of blank control group-colony diameter of treated group)/(colony diameter of blank control group-cake diameter) ]100%
TABLE 1
Experimental data shows that the compound microbial preparation prepared by the invention can inhibit Botrytis cinerea, sphaerotheca fuliginea, fusarium oxysporum, verticillium dahliae and Rhizoctonia solani, and has better antibacterial effect than single bacteria.
Test example 2 control Effect on cucumber gray mold
Selecting a seedling of cucumber with 'jin you No. 1' growing to three leaves and one heart, and evaluating the control effect of the microbial preparation on cucumber gray mold by adopting a potting test method;
the test method comprises the following steps: after the cucumber seedlings are inoculated with Botrytis cinerea for 24 hours, the bacterial preparation solution (same as in test example 1) is uniformly sprayed on the front and the back of the leaf blade, the spraying amount is 10 mL/basin, the leaf blade is sprayed once every 7 days, the leaf blade is placed in an environment with the temperature of 27+/-1 ℃ and the relative humidity of 90% for culture, the blank group is sprayed with the same amount of clear water of the bacterial preparation solution, each treatment is repeated for 5 basins, and the disease is observed and classified after 7 days and 14 days of bacterial preparation application. The grading criteria are as follows:
level 0: no disease spots;
stage 1:3 disease spots are arranged on a single leaf;
3 stages: 4-6 disease spots are arranged on a single blade;
5 stages: 7-10 disease spots are arranged on a single blade;
7 stages: the single leaf has 11-20 disease spots, and part of the single leaf is densely packed into a sheet;
stage 9: the single leaf has dense disease spots and occupies more than 1/4 of the leaf area;
disease index= [ Σ (number of leaves at each stage×representative value at that stage) ]/(total number of leaves examined×9) ×100;
control effect= (blank disease index-treatment disease index)/blank disease index x 100%;
TABLE 2
From the above table, it can be seen that the compound microorganism preparation of the invention has better control effect on cucumber gray mold, wherein the compound microorganism preparation prepared in example 2 has the best control effect.
Test example 3 control Effect on banana vascular wilt
Selecting a 'Nandina yellow' banana seedling with 5-7 leaves, and evaluating the control effect of the microbial preparation on banana vascular wilt by adopting a potting test method;
the test method comprises the following steps: after banana seedlings are inoculated with fusarium oxysporum for 24 hours, bacterial preparation solutions (same as test example 1) are uniformly poured into soil of the banana seedlings, the pouring amount is 30 mL/basin, the banana seedlings are sprayed once every 7d, the banana seedlings are placed in an environment with the temperature of 25+/-2 ℃ and the relative humidity of 60% for culture, a blank group is poured with clear water with the same amount as the bacterial preparation solutions, each treatment is repeated for 5 basins, and the disease is observed and classified after 7 days and 14 days of bacterial preparation application, wherein the classification standard is as follows:
level 0: the plants have no symptoms of withered and yellow;
stage 1: the plant leaves have slight withered and yellow symptoms, tender leaves are intact, a small part of root systems are slightly brown, and water stain brown stains appear on the stem parts;
2 stages: the plant leaves have obvious withered and yellow symptoms, tender leaves are intact, the root system is browned, and the stem and the pseudo-stem are water-stain-like browned;
3 stages: the whole plant has the symptoms of withered and yellow, the root system is browned, the rotten stem and the pseudo stem are browned and connected, and a few of the petioles are reddish and browned;
4 stages: plant withers and dies, and root systems are severely browned and rotten;
disease index= [ Σ (number of leaves at each stage×representative value at that stage) ]/(total number of leaves examined×4) ×100;
control effect= (blank disease index-treatment disease index)/blank disease index x 100%;
TABLE 3 Table 3
As can be seen from the above table, the compound microbial preparation of the invention has a better control effect on rubber wilt, wherein the compound microbial preparation prepared in example 2 has the best control effect.
Test example 4 field test
The test is carried out in Xiuying area of the sea city in 3 months in 2020, a test field after 1 year of continuous cropping is selected, the test is carried out by adopting cucumber seedlings grown to 'jin you No. 1' with three leaves and one heart, the field planting density is 1000 plants/mu, each group of test is 0.5 mu, a bacterial preparation is applied before the cucumber seedlings are planted, the application amount is 1 kg/mu, 1 time is applied every 10d, the bacterial preparation is diluted 1000 times and used, meanwhile, a blank control group without the bacterial preparation is arranged, the incidence rate of the cucumber is observed and recorded in the planting process, the organic matter content of the soil is measured after the harvest is completed in 5 months in the same year, the cucumber yield is recorded, and the test result is shown in table 4;
TABLE 4 Table 4
As can be seen from the field test results, the cucumber yield is increased by 14.7-16.9% after the compound microbial preparation is applied, the incidence rate is low, compared with a blank control group, the soil organic matter content after continuous cropping is obviously improved, and the effect of the compound microbial preparation is obviously better than that of a single microbial preparation and other compound microbial preparations.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (5)
1. Composite microThe biological agent is characterized by comprising bacillus subtilisBacillus subtilis) ACCC 03221 and Streptomyces roseoflashStreptomyces roseoflavus) ACCC 40415 and a solidifying agent; the CFU ratio of Bacillus subtilis and Streptomyces roseoflash is (4-7): (1-2) the solid agent is at least one of diatomite, kaolin, bentonite and mica powder;
the preparation method of the compound microbial preparation comprises the following steps:
(1) Inoculating bacillus subtilis and streptomyces roseoflash respectively to respective activation culture mediums for activation culture, and inoculating the bacillus subtilis and the streptomyces roseoflash respectively to respective fermentation culture mediums for fermentation culture to obtain respective fermentation bacterial solutions;
(2) Filtering fermentation bacteria liquid respectively, collecting mycoplasm, drying, pulverizing to obtain bacterial powder, mixing bacterial powder of each strain to obtain composite bacterial powder;
(3) Mixing the residual bacterial liquid filtered in the step (2), adding an ethanol solution with the bacterial liquid volume of 4-7 times, and performing ultrasonic extraction for 5-10min with the ultrasonic power of 200-230W to obtain a composite bacterial liquid;
(4) Mixing the compound bacterial liquid with a solid agent, drying, and adding compound bacterial powder to obtain the compound microbial preparation.
2. The composite microbial preparation according to claim 1, wherein the viable bacteria content in the composite microbial preparation is 10 6 -10 9 cfu/g。
3. The composite microbial preparation of claim 1, comprising the following ingredients per liter of bacillus subtilis fermentation medium: 2-5g/L ammonium sulfate, 1-4g/L calcium carbonate, 0.2-0.5g/L magnesium sulfate, 0.1-0.5 g/L sodium chloride, 3-6g/L peptone, 10-15g/L corn starch and 10-15g/L malt flour.
4. The composite microbial preparation of claim 1, comprising per liter of streptomyces roseoflavus fermentation medium: 0.2-0.5g/L dipotassium hydrogen phosphate, 0.3-0.8g/L ammonium nitrate, 2-4g/L calcium carbonate, 5-10g/L potassium sodium tartrate, 0.2-0.8g/L sodium citrate, 10-20g/L starch, 8-12g/L peanut cake powder and 14-22g/L corn steep liquor.
5. The use of a compound microbial preparation according to any one of claims 1 to 4, wherein said compound microbial preparation is used for controlling botrytis cinerea @Botrytis cinerea) Sphaerotheca fuliginea (L.) KuntzeSphaerotheca fuliginea) Fusarium oxysporum (F.oxysporum)Fusarium oxysporum) Verticillium dahliae (Fr.) KummerVerticiullim dahliae Kleb) And rhizoctonia solani @Rhizoctonia solani) And/or in the preparation of soil amendments.
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