CN113929525A - Microbial carbon-based fertilizer, preparation method and application thereof - Google Patents
Microbial carbon-based fertilizer, preparation method and application thereof Download PDFInfo
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- CN113929525A CN113929525A CN202111255000.2A CN202111255000A CN113929525A CN 113929525 A CN113929525 A CN 113929525A CN 202111255000 A CN202111255000 A CN 202111255000A CN 113929525 A CN113929525 A CN 113929525A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
- C05D9/00—Other inorganic fertilisers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Fertilizers (AREA)
Abstract
The invention discloses a microbial carbon-based fertilizer, a preparation method and application thereof. The influence of the biochar on the growth of the bacillus amyloliquefaciens is taken as an index, the optimal biochar is selected to be the biochar of the wheat straw and the biochar of the rice hull, and the physicochemical properties of the biochar are measured. Further condition optimization finds that the optimal particle size of the two biochar for adsorbing the bacillus amyloliquefaciens B1408(CGMCC No.15110) is 180-200 meshes, the optimal adsorption time is 48h, the optimal oscillation speed is 170rpm/min, and the optimal mixture ratio is bacterial suspension: biochar is 20: 1. Then, the method is explored by adopting two process flows of adsorption and blending to prepare four bacillus amyloliquefaciens B1408 microbial carbon base fertilizers of wheat biochar adsorption B1408(WSBX), wheat biochar blending (WSBC), rice biochar adsorption (RSBX) and rice biochar blending (RSBC). Finally, the four microbial carbon-based fertilizers have good effects on cucumber growth promotion and cucumber fusarium wilt disease control, and have good development and application prospects.
Description
Technical Field
The invention belongs to the technical field of fertilizers, and particularly relates to a microbial carbon-based fertilizer, a preparation method and application thereof.
Background
Chemical fertilizers and pesticides play an important role in guaranteeing national food safety as important agricultural production data, but the problems of agricultural product quality safety, environmental pollution and the like caused by large-scale unreasonable use of the chemical fertilizers and the pesticides are increasingly serious, and the reduction and the improvement of the application of the chemical fertilizers and the pesticides become a national strategy for promoting the high-quality development of agriculture. The microbial pesticide (fertilizer) has better environmental compatibility, has unique advantages in promoting the reduction of the application and the efficiency of the fertilizer and the pesticide and promoting the plant health, and the research and the development of products are increasingly paid attention to. Bacillus amyloliquefaciens (Bacillus sp.) B1408(CGMCC No.15110) is a biocontrol bacterium separated from the rhizosphere soil of healthy cucumbers in the subject group, has the functions of preventing diseases and promoting growth, has good prevention and treatment effects on wheat take-all and cucumber fusarium wilt, and shows good growth promoting effect.
As a sustainable and low cost carbon-based material, biochar is obtained from a series of renewable biological wastes by thermochemical treatment. Biochar properties vary depending on the raw materials, thermochemical methods, processes and conditions. The abundant microporous structure, aromatic functional groups and surface area can adjust the physical characteristics of the soil, including porosity, density and particle size distribution, thereby changing the oxygen concentration, water storage capacity and nutrient conditions. The unique adsorption capacity of biochar enables carbon fixation and soil health and productivity by enrichment of nutrients and microorganisms. Biochar is also considered a high potential environmental protection technology for new effective carriers or nutrient storage and aids in environmental cleanliness. Research reports that the biochar enriches soil microorganisms and improves soil mineralization and biological oxidation. At the same time, it provides an additional place for the metabolism of the bacteria, thereby influencing the activity of the microorganisms through the change of nutrient utilization. Although biochar can benefit soil health by enhancing permeability, retaining water and nutrients, improving fertility, biochar alone cannot form a complete fertilizer to provide abundant nutrients for crops, and it is a better choice to combine biochar as a carrier with a strain of functional microorganisms to establish multiple associations or interactions to improve the efficiency of functional microorganisms.
In view of this, the research and development of the microbial carbon-based fertilizer of the bacillus amyloliquefaciens B140, the growth promotion effect of the bacillus amyloliquefaciens B140 on cucumbers and the evaluation of the cucumber fusarium wilt prevention effect are carried out, and the disease prevention and growth promotion capability of the bacillus amyloliquefaciens B1408 strain is further optimized, so that the problems of low colonization rate and poor activity of the existing disease prevention and growth promotion strain and the microbial strain after the existing microbial fertilizer is applied can be solved.
Disclosure of Invention
The invention aims to provide a microbial carbon-based fertilizer, a preparation method and an application thereof, wherein the microbial carbon-based fertilizer has good effects on cucumber growth promotion and cucumber fusarium wilt disease prevention and treatment, and has good development and application prospects.
The method specifically comprises the following steps:
bacillus amyloliquefaciens B1408(Bacillus sp.), the preservation number is CGMCC No.15110, and the preservation date is: 21/12/2017, the preservation unit is: the China general microbiological culture Collection center has the following preservation addresses: the western No.1 Hotel zip code of Beijing, Chaoyang, China, Shih & Xilu, 100101 telephone number 64807355; the bacillus amyloliquefaciens B1408 is applied to preparing a microbial carbon-based fertilizer or a medicament for preventing and treating plant soil-borne diseases.
A microbial carbon-based fertilizer comprises biological carbon and microorganisms attached to the biological carbon; the microorganism is Bacillus amyloliquefaciens B1408, and the preservation number is CGMCC No. 15110.
Optionally, the biochar is alkaline, the content of C is 65-66%, and the ash content is 70%71 percent of the total surface area of the alloy, and the specific surface area of the alloy is 60 to 62m2/g。
Optionally, the biochar is wheat straw biochar, corn straw biochar or rice hull biochar;
the particle size of the biochar is 180-200 meshes.
Optionally, the bacillus amyloliquefaciens B1408 is attached to the biochar by adsorbing, soaking or blending the bacterial suspension or fermentation liquor of the bacillus amyloliquefaciens B1408;
the adsorption, immersion or blending time is 48h, the oscillation speed is 170rpm/min, and the ratio of bacterial suspension or fermentation broth V: biochar W is 20: 1;
the microbial cell content of the bacterial suspension or fermentation broth is at least 108CFU/mL。
Optionally, the preparation of the bacterial suspension of bacillus amyloliquefaciens B1408 comprises:
inoculating the activated strain into an LB culture medium, and culturing at 28 ℃ and 170r/min for 12h to prepare a seed solution; inoculating into LB culture medium with 2% inoculum size, and culturing at 28 deg.C and 170r/min for 48 h; resuspending the harvested cells in sterile water, and adjusting the concentration of the bacterial solution to 108CFU/mL prepared bacterial suspension.
A preparation method of a microbial carbon-based fertilizer comprises adsorbing, impregnating or mixing with bacterial suspension or fermentation liquor of Bacillus amyloliquefaciens B1408 by using biological carbon;
the particle size of the biochar is 180-200 meshes; the adsorption, immersion or blending time is 48h, the oscillation speed is 170rpm/min, the ratio of bacterial suspension or fermentation broth V: biochar W is 20: 1;
the microbial cell content of the bacterial suspension or fermentation broth is at least 108CFU/mL。
Optionally, the preparation of the bacterial suspension of bacillus amyloliquefaciens B1408 comprises:
inoculating the activated strain into an LB culture medium, and culturing at 28 ℃ and 170r/min for 12h to prepare a seed solution; inoculating into LB culture medium with 2% inoculum size, and culturing at 28 deg.C and 170r/min for 48 h; resuspending the harvested cells in sterile water, and adjusting the concentration of the bacterial solution to 108CFU/mL prepared bacterial suspension.
A medicine for preventing and treating plant soil-borne diseases contains Bacillus amyloliquefaciens B1408.
Optionally, the medicament also contains biochar, and the bacillus amyloliquefaciens B1408 is attached to the biochar;
attaching the bacillus amyloliquefaciens B1408 to the biochar by adsorbing, dipping or blending a bacterial suspension or fermentation liquor of the bacillus amyloliquefaciens B1408;
the adsorption, immersion or blending time is 48h, the oscillation speed is 170rpm/min, and the ratio of bacterial suspension or fermentation broth V: biochar W is 20: 1;
the microbial cell content of the bacterial suspension or fermentation broth is at least 108CFU/mL。
The invention takes the influence of the biochar on the growth of the bacillus amyloliquefaciens as an index, selects the optimal biochar as the biochar of the wheat straws and the biochar of the rice hulls, and measures the physicochemical properties of the biochar. Further condition optimization finds that the optimal particle size of the two biochar to B1408 adsorption is 180-200, the optimal adsorption time is 48h, the optimal oscillation speed is 170rpm/min, and the optimal mixture ratio is bacterial suspension: biochar is 20: 1. Then, the method is explored by adopting two process flows of adsorption and blending to prepare four bacillus amyloliquefaciens B1408 microbial carbon base fertilizers of wheat biochar adsorption B1408(WSBX), wheat biochar blending (WSBC), rice biochar adsorption (RSBX) and rice biochar blending (RSBC). Finally, the four microbial carbon-based fertilizers have good effects on cucumber growth promotion and cucumber fusarium wilt disease control, and have good development and application prospects.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:
FIG. 1 is a graph of three biochar-neutral treatments and their effect on the growth of Bacillus amyloliquefaciens B1408 as provided in example 2 of the present invention; (A) natural biochar; (B) biochar after neutral treatment; (C) the bacterial count of B1408 after 48h of natural biochar and neutral treatment biochar;
FIGS. 2-6 are graphs showing the effect of different biochar types, biochar particle sizes, adsorption times, oscillation rates, and biochar usage on the adsorption of Bacillus amyloliquefaciens B1408 provided in example 3 of the present invention; concentration of original bacterial suspension: log10CFU/mL ═ 9.32; FIG. 3 effect of biochar of different particle size on the adsorption capacity of B1408, (A) wheat straw biochar; (B) rice straw biochar (original bacteria suspension concentration: log10 CFU/mL-9.85); FIG. 4 effect of different adsorption times of biochar on B1408 adsorption capacity, (A) wheat straw biochar; (B) b1408 rice straw biochar (original bacteria suspension concentration: log10 CFU/mL-10.27); FIG. 5 Effect of shaker rotation speed on charcoal adsorption B1408, (A) wheat straw biochar; (B) rice straw biochar (original bacteria suspension concentration: log10 CFU/mL-9.81); FIG. 6 influence of the ratio of bacterial suspension to biochar on biochar adsorption B1408; (B) rice straw biochar (original bacteria suspension concentration: log10 CFU/mL-10.47);
FIGS. 7 and 8 are scanning electron microscope and infrared spectra of two biochar of wheat and rice provided in example 4 of the present invention; (A) rice straw biochar; (B) wheat straw biochar;
FIGS. 9 and 10 show two processes for preparing microbial charcoal-based fertilizer by adsorption and blending provided in example 5 of the present invention;
FIGS. 11 to 14 are the evaluation of national standard indexes such as appearance, microcosmic effect, slow release effect and shelf life of four microbial carbon-based fertilizers provided in example 6 of the present invention; FIG. 11 is an appearance diagram of a carbon-based microbial fertilizer prepared by different processes, (A) a wheat straw carbon-based microbial fertilizer (WSBC) prepared by a blending method; (B) the rice straw carbon-based microbial fertilizer (RSBC) prepared by the blending method; (C) wheat straw carbon-based microbial fertilizer (WSBX) prepared by an adsorption method; (D) a rice straw carbon-based microbial fertilizer (RSBX) prepared by an adsorption method; FIG. 12 scanning electron micrographs of carbon-based microbial fertilizers prepared by different processes, (A) wheat straw carbon-based microbial fertilizer (WSBC) prepared by blending method; (B) the rice straw carbon-based microbial fertilizer (RSBC) prepared by the blending method; (C) wheat straw carbon-based microbial fertilizer (WSBX) prepared by an adsorption method; (D) a rice straw carbon-based microbial fertilizer (RSBX) prepared by an adsorption method; FIG. 13 the slow release effect of the carbon-based microbial fertilizer; FIG. 14 shows the effective viable count of the carbon-based microbial fertilizer under different storage conditions, (A) at 4 ℃; (B) the effective viable count of the carbon-based microbial fertilizer under the normal temperature condition;
FIGS. 15 to 17 are the statistics of the disease index of cucumber fusarium wilt and the results of disease prevention and growth promotion potting after the use of four microbial carbon-based fertilizers according to example 7 of the present invention; FIG. 15 the effect of carbon-based microbial fertilizer on the incidence of cucumber seedling blight, CK, clear water control; FOC, cucumber fusarium wilt; car + FOC, carbendazim + fusarium oxysporum; WSB + FOC, wheat straw biochar + cucumber fusarium wilt; WSBC + FOC, wheat straw carbon-based microbial fertilizer prepared by a blending method and cucumber fusarium wilt; WSBX + FOC, wheat straw carbon-based microbial fertilizer prepared by an adsorption method and cucumber fusarium wilt; RSB + FOC, rice straw charcoal + cucumber fusarium wilt; RSBC + FOC, rice straw carbon-based microbial fertilizer prepared by a blending method and cucumber fusarium wilt; RSBX + FOC, rice straw carbon-based microbial fertilizer prepared by an adsorption method and cucumber fusarium wilt; FIG. 16 shows the effect of Bacillus amyloliquefaciens microbial carbon-based fertilizer on the growth of cucumber plants inoculated with blight pathogenic bacteria, CK, and clear water control; FOC, cucumber fusarium wilt; car + FOC, carbendazim + fusarium oxysporum; WSB + FOC, wheat straw biochar + cucumber fusarium wilt; WSBC + FOC, wheat straw carbon-based microbial fertilizer prepared by a blending method and cucumber fusarium wilt; WSBX + FOC, wheat straw carbon-based microbial fertilizer prepared by an adsorption method and cucumber fusarium wilt; RSB + FOC, rice straw charcoal + cucumber fusarium wilt; RSBC + FOC, rice straw carbon-based microbial fertilizer prepared by a blending method and cucumber fusarium wilt; RSBX + FOC, rice straw carbon-based microbial fertilizer prepared by an adsorption method and cucumber fusarium wilt; FIG. 17 growth promoting effect of Bacillus amyloliquefaciens microorganism carbon-based fertilizer on cucumber plants, CK, clear water control; WSBX, a wheat straw carbon-based microbial fertilizer prepared by an adsorption method; WSBC (Wireless sensor and controller), namely a wheat straw carbon-based microbial fertilizer prepared by a blending method; WSB, wheat straw biochar; RSBX, a rice straw carbon-based microbial fertilizer prepared by an adsorption method; RSBC, a rice straw carbon-based microbial fertilizer prepared by a blending method; RSB, rice straw biochar.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail with reference to embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Bacillus amyloliquefaciens B1408(Bacillus sp.), the preservation number is CGMCC No.15110, and the preservation date is: 21/12/2017, the preservation unit is: the China general microbiological culture Collection center has the following preservation addresses: the western No.1 Hotel zip code of Beijing, Chaoyang, China, Shih & Xilu, 100101 telephone number 64807355; the bacillus amyloliquefaciens B1408 is applied to preparing a microbial carbon-based fertilizer or a medicament for preventing and treating plant soil-borne diseases.
The bacterial suspension or fermentation liquid or fermentation product of the bacillus amyloliquefaciens B1408 in the bacillus amyloliquefaciens B1408 microbial carbon-based fertilizer is also within the protection scope of the invention.
The optimal particle size of the wheat straw biochar and the rice husk biochar in the four bacillus amyloliquefaciens B1408 microbial carbon-based fertilizers for adsorbing the bacillus amyloliquefaciens B1408 is 180-fold 200, the optimal adsorption time is 48h, the optimal oscillation speed is 170rpm/min, and the optimal proportion is bacterial suspension: the optimal process parameters obtained by optimizing the biochar are 20:1, and the protection scope of the invention is also provided.
The biochar of wheat straws and the biochar of rice husks in the four bacillus amyloliquefaciens B1408 microbial carbon-based fertilizers are alkaline (pH is 11), the content of C is about 65%, the ash content is about 70%, and the specific surface area is 61m2The basic properties of the catalyst such as the concentration of the catalyst and the adsorption capacity are also within the protection scope of the invention.
The invention also provides an adsorption and blending process flow and a method used in the four bacillus amyloliquefaciens B1408 microbial carbon-based fertilizers.
The carbon-based fertilizers of the four bacillus amyloliquefaciens B1408 microorganisms have obvious adsorption effects; releasing bacillus amyloliquefaciens in 0-60d periodB1408 has strong slow release effect and viable count of 10 after being stored for 180 days at room temperature and 4 DEG C7-109Longer shelf life, etc. are also within the scope of the invention.
The application of the carbon-based fertilizer of the four bacillus amyloliquefaciens B1408 microorganisms in the control of cucumber wilt disease of the cucumber growth promoter is also within the protection scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. In particular to a method for obtaining the biochar, which adopts a conventional method in the field.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of seed solutions and suspensions of Bacillus amyloliquefaciens B1408
The activated strain B1408 was inoculated into LB medium, cultured at 28 ℃ at 170r/min for 12 hours on a constant temperature shaker to prepare a seed solution, inoculated into 100mL of LB medium at 2%, and cultured at 28 ℃ at 170r/min for 48 hours. Then, centrifuging at 8000r/min for 10min, collecting the precipitated cells, suspending the harvested cells in sterile water, and adjusting the concentration of the bacterial liquid to 108CFU/mL prepared bacterial suspension.
Example 2 three biochar neutral treatments and their effect on the growth of Bacillus amyloliquefaciens B1408
Soaking 3 kinds of biomass charcoal in 0.1mol/L HCl overnight, repeatedly washing with deionized water until pH is neutral, vacuum filtering, oven drying, and making into neutral wheat straw biochar, rice straw biochar, and corn straw biochar, which are respectively marked as WSB (neutral), RSB (neutral), and CSB (neutral).
Weighing three biochar with natural pH and 0.1g of each biochar subjected to neutral treatment, and performing moist heat sterilization at 121 ℃ for 25 min.
100mL of LB medium was placed in a 250mL Erlenmeyer flask, sterilized by moist heat at 121 ℃ for 25min, cooled and inoculated with the prepared B1408 seed solution in an amount of 2% of the medium. Meanwhile, the sterilized charcoal is inoculated into the culture medium and cultured for 60h at the temperature of 28 ℃ and the rpm/min of 170. Samples were taken at different time points of 0h, 6h, 24h, 36h, 48h, 60h and counted using a dilution-coated plate method with no biochar added as a control, and each treatment was repeated three times.
It can be seen from FIG. 1(A) that the growth rate of Bacillus amyloliquefaciens B1408 is higher than that of the medium without biochar addition. As can be seen from FIG. 1(B), B1408 showed a substantially uniform growth profile in the medium supplemented with 3 biochar species and the biochar species subjected to the neutral treatment. After 48h of culture, the number of thalli in the culture medium added with the biochar subjected to neutral treatment and the culture medium not subjected to neutral treatment is reduced, but the reduction speed of the biochar added is obviously lower than that of a control. The biochar has no effect on the growth of the bacillus amyloliquefaciens. And the biochar with and without neutral treatment had no significant difference in the maximum growth of bacillus amyloliquefaciens B1408 as shown in fig. 1 (C). In view of the subsequent adsorption test on B1408 and simplification of the formulation processing procedure, non-neutralized biochar, i.e., natural biochar, was used in the adsorption test and fertilizer processing.
Example 3 Condition optimization of biochar adsorption of Bacillus amyloliquefaciens B1408
(1) Influence of different types of biochar on B1408 thallus adsorption
Selecting 100-mesh and 120-mesh wheat straw biochar, rice straw biochar and corn straw biochar for screening the types of biochar.
(2) Influence of different particle sizes of biochar on biochar adsorption
And (2) screening three different particle size ranges of 40-60 meshes, 100-120 meshes and 180-200 meshes on the basis of the biochar with the best adsorption capacity obtained in the step (1).
(3) Effect of adsorption time on charcoal adsorption
The biochar is screened for different adsorption times of 6, 12, 24, 48, 60 and 72 hours on the basis of the best adsorption capacity in (1) and the best adsorption particle size in (2).
(4) Influence of oscillation speed on charcoal adsorption
On the basis of (1) the best adsorption capacity, (2) the best particle size and (3) the best time, the biochar is screened at different oscillation speeds of 80, 110, 140, 170 and 200 r/min.
(5) Influence of charcoal amount on charcoal adsorption
On the basis of the above four optimum conditions, the influence of the addition of biochar 2.5, 5, 10 and 25g on the adsorption of the cells was further screened.
In the above-described screening under the conditions, (1) to (4) used were each 100mL in volume of bacterial suspension, 5g in amount of biochar, and (5) used was 50mL in volume of bacterial suspension. The vessel used in all experiments was a 250mL Erlenmeyer flask and the incubation temperature was 28 ℃. In all experiments, the coating counting method is uniformly adopted to measure the quantity of the biochar adsorbed bacteria, and specifically comprises the following steps: respectively taking 1mL of the adsorbed bacterial suspension for gradient dilution, sucking 100 mu L of the dilution solution, coating the dilution solution on a flat plate of an NA culture medium, culturing in a biochemical incubator at 28 ℃, and counting after 24-48 h. Each treatment was repeated three times. The number of adsorbed cells of charcoal is equal to the number of cells before adsorption of the cell suspension — the number of cells after adsorption of the cell suspension.
The results of the screening of the biochar types show that (figure 2) the adsorption capacity of the three biochar types to the B1408 can reach 1011CFU/mL and no significant difference exist, but the adsorption capacity of the wheat straw biochar and the rice straw biochar to the B1408 is greater than that of the corn straw biochar to the B1408, and the wheat straw biochar and the rice straw biochar are used as adsorption carriers in subsequent adsorption experiments.
The biochar particle size range screening results (fig. 3) show that the amount of B1408 adsorbed on wheat straw biochar (fig. 3A) and rice straw biochar (fig. 3B) increases with decreasing particle size. This is probably due to the smaller particles increasing the contact area of the biochar with the bacterial cells, further increasing the adsorption capacity of the biochar B1408. In the subsequent adsorption test, charcoal with 180-200 meshes is selected for the adsorption test.
The results of biochar adsorption time screening (fig. 4) show that the adsorption amounts of wheat straw biochar (fig. 4A) and rice straw biochar (fig. 4B) to B1408 fluctuate up and down along with the change of time, the adsorption amounts decrease within 36h, but then the adsorption amounts reach the maximum value within 48h, and decrease after 48h and tend to be stable. Therefore, 48h was selected as the optimum adsorption time for the two biochar types.
The results of the screening of the oscillation speed required by the adsorption of the biochar show that (figure 5), the oscillation speed is increased from 80rpm/min to 200rpm/min to cause the adsorption amount of the two biochars to fluctuate, and the adsorption amount of the wheat straw biochar (figure 5A) is increased from 140rpm/min to 200rpm/min to cause the adsorption amount to increase continuously, which is probably because the biochar and B1408 are not mixed enough to achieve the optimal adsorption effect at low speed (namely 80rpm/min), and B1408 can be effectively attached to the biochar at high speed; the rice straw biochar (figure 5B) has no significant difference on the adsorption amount under different rotating speeds, but the adsorption amount under 170rpm/min is the largest, so the optimal adsorption rotating speed of comprehensively selecting the two biochars is 170 rpm/min.
The screening result of the usage amount of the biochar shows that (figure 6), when 2.5g of biochar is added into the B1408 bacterial suspension, the adsorption amount of the biochar to the B1408 is the highest per gram. The reason is that after a small amount of biochar is added, the dispersion effect of the biochar in the bacterial suspension is better. The adsorption amount of the biochar in the bacterial suspension is increased along with the reduction of the consumption of the biochar, and the adsorption amounts per gram of the biochar from the wheat straws (figure 6A) and the biochar from the rice straws (figure 6B) are consistent. There was a significant difference in absorption between the ratios. Therefore, the ratio of the bacteria suspension to the biochar (v/w) is 20:1, and the optimal condition for adsorbing the bacteria by the biochar is obtained.
Example 4 determination of physicochemical Properties of biochar
(1) And (3) pH measurement: 1.0g of each of the 3 biochar types was weighed and dissolved in 20mL of distilled water, and the solution was filtered by shaking for 10min, and the pH of the filtrate was measured using a pH meter.
(2) Elemental analysis: the C, H, N, S content of the biochar was measured using an elemental analyzer at an oven temperature of 1150 ℃ and the O content was calculated using a differential subtraction method.
(3) Specific surface area: the specific surface area of the biochar was measured by a BET method using a specific surface area meter. The samples were sent to Qingdao Standd detection, Inc. for measurement.
(4) Ash content: the samples were sent to Qingdao Standd detection, Inc. for measurement. Ash was calculated using a muffle furnace 800 ℃ open calcination for 2 h.
(5) Surface morphology: the surface morphology of the biochar is determined by a scanning electron microscope.
(6) The structure of the functional group is as follows: and (3) determining the structure of the functional group of the biochar by using a Fourier infrared spectrometer.
The basic property characterization result of the biochar shows that (table 1), the biochar is alkaline, the element content of the biochar is C > O > H > N, the C content of the biochar from the wheat straws and the biochar from the rice straws reaches 65.43% and 65.57%, and the difference between the aromaticity and the water absorbability of the biochar from the wheat straws and the biochar from the rice straws is small. The ash content of the wheat straw biochar and the ash content of the rice straw biochar reach 70.8% and 70.3% respectively, and the ash content is high, so that the wheat straw biochar and the rice straw biochar contain rich mineral substances. The specific surface areas of the wheat straw biochar and the rice straw biochar are 60.460m respectively2G and 61.609m2The specific surface area is larger, which shows that the two biochar have strong adsorption capacity.
TABLE 1 characterization of basic properties of biochar
WSB represents wheat straw biochar; RSB represents a rice straw organism
The results of the surface morphology of the biochar (fig. 7) show that the tubular structures in the rice straw biochar (fig. 7A) and the wheat straw biochar (fig. 7B) are densely distributed and regularly arranged, and the side surfaces of the biochar have various folds. The tubular diameter of the biochar is much greater than the length of the bacteria. Therefore, the tubular structure and the surface structure of the biochar can adsorb the bacillus amyloliquefaciens B1408 thallus, and space is provided for the growth and the propagation of the thallus. Thalli can be gathered in a charcoal tubular structure and lateral folds, and the thalli are favorably released into the soil environment. Most of thalli are attached to a tubular structure of the biochar, and the tubular structure and transverse folds of the biochar are suitable for adsorbing the thalli, so that the rice straw biochar and the wheat straw biochar are suitable carriers for PGPR in agricultural application.
Fourier infrared spectroscopyThe analysis results show (figure 8) that the main absorption peaks of the wheat straw biochar (figure 8A) and the rice straw biochar (figure 8B) appear at 3422, 1619, 1420.69 and 792.25cm-1Nearby. 3422.44 thereincm-1The nearby broad absorption peak is the stretching vibration peak of intermolecular hydrogen bond-OH, and the hydroxyl groups can be derived from carbohydrates in organic matters; 1619.80cm-1Nearby stretching vibration of C-C, C-O and antisymmetric stretching vibration of-COO-in the aromatic ring; 1420.69cm-1Nearby is-CH-in-plane bending vibration, is an alkane or alkene functional group, 792.25cm-1The C-H out-of-plane bending vibration peak is arranged nearby, so that the biochar has an obvious aromatic structure. Most of the carbon in the two biochar exists in a double-bond and aromatic ring structure, the structure is relatively stable, and the biochar is endowed with strong adsorption performance by the abundant chemical functional groups such as hydroxyl, carboxyl and the like on the surface, so that the biochar can be widely applied to the fertilizer industry and the environmental pollution prevention and control as an adsorption material.
Example 5 preparation of Bacillus amyloliquefaciens B1408 carbon-based microbial Fertilizer
Based on the operation feasibility, 2 methods of a blending method and an adsorption method are selected for the research of the carbon-based microbial fertilizer in the experiment, and the preparation method comprises the following steps:
(1) adsorption process
And (3) screening the biochar with the best adsorption performance as a carrier according to the experimental result of the example 3, carrying out B1408 adsorption according to the optimal carrier particle size, oscillation speed, proportion of the biochar to bacterial suspension and reaction time, carrying out suction filtration after adsorption, drying at low temperature of 30 ℃ to obtain a finished product, wherein the carbon-based microbial fertilizer prepared from the biochar of wheat straws is marked as WSBX, and the carbon-based microbial fertilizer prepared from the biochar of rice straws is marked as RSBX. The adsorption test process flow diagram is shown in figure 9.
(2) Blending method
Weighing the required wheat straw biochar and rice straw biochar, filling the wheat straw biochar and the rice straw biochar into a big beaker, carrying out damp-heat sterilization at high temperature of 121 ℃ for 25min, cooling, moistening with deionized water, stirring until the water content reaches 'hand-held conglobation, touch and dispersion', adding B1408 bacterial suspension with the same amount as the biocharThe bacteria content is about 1011And CFU/g, fully stirring and mixing, drying at a low temperature of 30 ℃ to obtain a finished product, wherein the carbon-based microbial fertilizer prepared from the wheat straw biochar is marked as WSBC, and the carbon-based microbial fertilizer prepared from the rice straw biochar is marked as RSBC. The process flow diagram is shown in FIG. 10.
Example 6 determination of microbial charcoal-based Fertilizer Properties
(1) Surface structure analysis
The surface structure of the four microbial carbon-based fertilizers is analyzed, the adsorption condition of the biological carbon prepared by different processes to the bacillus amyloliquefaciens B1408 can be visually seen, the microbial carbon-based fertilizers processed by an adsorption method and a blending method are selected in the experiment, the surface structure of the microbial carbon-based fertilizers is observed by a scanning electron microscope, and images are recorded.
(2) Determination of pH
And (3) measuring the pH value by adopting a composite electrode method, weighing 1g of the microbial carbon-based fertilizer, mixing the fertilizer and the water in a mass ratio of 1:20, and measuring the pH value by using a pH meter.
(3) Determination of shelf life of microbial carbon-based fertilizer
Weighing 30g of each microbial carbon-based fertilizer, putting the microbial carbon-based fertilizers into self-sealing bags in three parts, and respectively storing at room temperature and 4 ℃. 0.1g of each microbial charcoal-based fertilizer is weighed at different time points (0, 20, 40, 60, 80, 100, 120, 150d and 180d), 0.9mL of sterilized distilled water is added and mixed sufficiently to ensure that all the bacteria are separated, and counting is carried out by a dilution coating plate method. Each treatment was repeated three times.
(4) Slow release effect of microbial carbon-based fertilizer
10g of the microbial carbon-based fertilizer of the four processing technologies are respectively weighed and added into a 250mL triangular flask containing 100mL of sterile water, then the mixed solution is shaken for 60d under the conditions of 28 ℃ and 170rpm/min, and the solution is taken at different periods (0, 10, 20, 30, 40, 50 and 60d) for dilution and coating on a flat plate method for counting. Each treatment was repeated three times.
Appearance diagrams and scanning electron microscope image results of microbial carbon-based fertilizers prepared by different processes show that both the adsorption-type microbial carbon-based fertilizer and the blend-type microbial carbon-based fertilizer are fine powder (fig. 11), and A, B, C and D in fig. 11 respectively show (A) wheat straw carbon-based microbial fertilizer (WSBC) prepared by the blend method, (B) rice straw carbon-based microbial fertilizer (RSBC) prepared by the blend method, (C) wheat straw carbon-based microbial fertilizer (WSBX) prepared by the adsorption method, and (D) rice straw carbon-based microbial fertilizer (RSBX) prepared by the adsorption method). Due to the fact that B1408 thalli are adsorbed on the surface and the pores of the fertilizer, spots and bulges appear on the surfaces of the adsorption type microbial carbon-based fertilizer and the mixed type microbial carbon-based fertilizer. The surface and pores of the biochar can still be clearly seen after blending, the surface and pores of the biochar after adsorption are fully adsorbed by thalli, the adsorbed microbial charcoal base fertilizer has more bacterial load (figure 12), and the A, B, C and D in figure 12 respectively show ((A) wheat straw carbon base microbial fertilizer (WSBC) prepared by a blending method, (B) rice straw carbon base microbial fertilizer (RSBC) prepared by a blending method, (C) wheat straw carbon base microbial fertilizer (WSBX) prepared by an adsorption method, and (D) rice straw carbon base microbial fertilizer (RSBX) prepared by an adsorption method). The preparation processes of the microbial carbon-based fertilizer are different, so that the quantity of bacteria adsorbed by the biological carbon is greatly different. Generally, the microbial carbon-based fertilizer prepared by the adsorption method is more loaded with thalli than the microbial carbon-based fertilizer prepared by the blending method.
The slow release results of the microbial carbon-based fertilizer prepared by different processes show that (figure 13) the release curves of the fertilizer prepared by different processing processes are different, but the overall release trend is increased firstly and then reduced. After the release test is started, B1408 thalli attached to the surface and pores of the biochar quickly enter sterile water, so that the concentration of the thalli in the sterile water is quickly increased in an early stage. The carbon-based fertilizers WSBC and RSBC prepared by the blending method have the maximum release rate before 5d, and the thalli have a steady descending trend after 5d, possibly the death rate of the thalli is greater than the release rate of the thalli. The release trends of the carbon-based fertilizers WSBX and RSBX prepared by the adsorption method at the first 5d are similar to those of WSBC and RSBC, but the WSBX has an increasing trend at 10-20d after 5-10d is obviously reduced, and the RSBX has an increasing trend at 20-30d, which is probably because thalli on the surface of the biochar at the first stage are released and thalli in pores of the biochar at the later stage are released, so that the concentration of the thalli is increased. After 60d WSBX, WSBC, RSBX and RSBC released into sterile waterThe cell concentration can reach 5.00X 104、2.50×103、5.00×104And 5.00X 103CFU/mL. Therefore, the carbon-based microbial fertilizer processed by different processes has good slow release effect.
According to the regulation of the national standard GB 20287-2006 for the microbial fertilizer, the shelf life of the microbial fertilizer is 6 months, so 180d is taken as the end point of the test. The results show (FIG. 14) that the effective viable counts of the carbon-based microbial fertilizers were greatly influenced under different storage conditions (A, 4 ℃; B, room temperature), the viable counts of the four microbial carbon-based fertilizers all declined at 4 ℃, and at 180 days, the effective viable counts of WSBX, WSBC, RSBX and RSBC were 1.20 × 109、2.75×108、6.00×109、3.00×107CFU/g, the microbial carbon-based fertilizer prepared by the adsorption method still has high viable count, and the viable count of RSBC does not reach the national standard and is more than or equal to 2 multiplied by 108CFU/g and WSBC viable count reach the national standard. Under the condition of normal temperature, the effective viable count is 7.00 multiplied by 10 respectively after 6 months8、5.50×108、2.50×108、4.00×108CFU/g, all reach the effective viable count of the national standard. In a word, under the storage conditions of 4 ℃ and normal temperature, except that RSBC does not reach the effective viable count of the national standard, other viable counts reach the standard, but the effective viable count stored at 4 ℃ is better than that stored at normal temperature in general.
Example 7 evaluation of disease prevention and growth promotion effects of Bacillus amyloliquefaciens B1408 microorganism carbon-based fertilizer
Preparing a FOC bacterial liquid of cucumber fusarium wilt pathogenic bacteria: inoculating cucumber wilt bacterium FOC into potato glucose broth liquid culture medium, culturing at 28 deg.C and 170rpm/min for 7d to obtain pathogenic bacteria liquid, and regulating bacteria concentration to 10 with sterile water8CFU/mL is ready for use.
The cucumber seeds (Jinyan No. 4) are sterilized by 75 percent alcohol for 30s, washed by sterile water for three times, sterilized by 1.5 percent NaClO for 5min, washed by sterile water for six times, dried, put on moist gauze and germinated in a constant temperature incubator at 28 ℃ in the dark for 24h, and then the seeds after germination acceleration are sown in a seedling tray for greenhouse cultivation in the biological source pesticide engineering technology research center of Shanxi province of northwest agriculture and forestry science and technology university in northwest China. After the cotyledon grows out completely, the seedlings with consistent growth vigor are selected and transplanted into a plastic flowerpot containing 100g of matrix (10 multiplied by 9cm), and one seedling is planted in each pot. The matrix is soil mixed with nutrient soil (1:1) sieved by 2mm, when cucumber grows to two true leaves, a disease prevention and growth promotion test of cucumber fusarium wilt is carried out, and the setting treatment is as follows: (1) CK, do not do any treatment, only irrigate the root with 20mL sterile distilled water; (2) FOC, pouring 20mL of prepared FOC bacterial suspension into roots for treatment; (3) FOC + WSBX; (4) FOC + WSBC; (5) FOC + RSBX; (6) FOC + RSBC; (7) FOC + WSB; (8) FOC + RSB; (9) FOC + Car, (3) - (6) are treated by mixing 3% of each microbial carbon-based fertilizer with soil, and after one week, 20mL of prepared FOC bacterial suspension is irrigated to roots; (7) - (8) mixing 3% of wheat straw biochar and rice straw biochar with soil, and irrigating 20mL of prepared FOC bacterial suspension into roots after one week; (9) irrigating roots of 20mL medicament contrast carbendazim, and irrigating roots of 20mL FOC bacterial suspension after one week; simultaneously, carrying out a growth promotion test of the microbial carbon-based fertilizer, (10) WSBX; (11) WSBC; (12) RSBX; (13) RSBC; (14) WSB; (15) RSB, (10) - (13) are treated by mixing 3% of each microbial carbon-based fertilizer with soil; (14) - (15) treatment was two biochar soil treatments at 3%, each treatment of 5 cucumber plants, repeated three times. After the root irrigation treatment of FOC pathogenic bacteria, the disease index, the plant height, the stem thickness, the fresh weight and the dry weight are measured for four weeks.
The disease prevention results of the microbial carbon-based fertilizer show that (figure 15), the disease indexes of the biological carbon and the bacillus amyloliquefaciens biological carbon-based fertilizer are obviously lower than those of a positive control (only FOC is inoculated) and a carbendazim drug control, which shows that the biological carbon and the bacillus amyloliquefaciens biological carbon-based fertilizer have better prevention effect on cucumber fusarium wilt, and the prevention effect is from strong to weak:
WSBX>RSB>RSBX>WSBC>RSBC>WSB。
the results of the influence of the bacillus amyloliquefaciens microbial carbon-based fertilizer on the growth of plants after the cucumber plants are inoculated with the blight pathogenic bacteria show that (table 2 and figure 16), the inoculation of pathogenic bacteria FOC remarkably inhibits the growth of cucumbers, and the WSBX and RSBX microbial carbon-based fertilizers can obviously relieve the inhibition of the cucumber blight pathogenic bacteria on the growth of the cucumbers, and the effect has obvious advantage compared with a medicament contrast carbendazim. And the two microbial carbon-based fertilizers WSBC and RSBC have the relieving effect of inhibiting the growth of cucumbers by FOC pathogenic bacteria, but the relieving effect is not as obvious as that of WSBX and RSBX.
TABLE 2 relieving effect of carbon-based fertilizer for cucumber growth inhibition by cucumber fusarium wilt pathogens
CK: clear water control; FOC: root irrigation treatment of cucumber fusarium wilt pathogenic bacteria; mixing carbon-based microbial fertilizers with 3% of FOC + WSBX, FOC + WSBC, FOC + RSBX and FOC + RSBC in soil, and irrigating 20mL of prepared FOC bacterial suspension to roots after one week; mixing two biochar with 3% of FOC + WSB and FOC + RSB with soil, and irrigating 20mL of prepared FOC bacterial suspension to form roots after one week; FOC + Car: and (3) irrigating roots with 20mL of medicament contrast carbendazim, and irrigating roots with 20mL of FOC bacterial suspension after one week, wherein the concentration of the carbendazim is 500 mg/L. Data are mean ± standard values and experiments were repeated 3 times. Different letters in the same column represent significant differences (p < 0.05).
The results of the growth promotion effect of the bacillus amyloliquefaciens microbial carbon-based fertilizer on cucumber plants show that under the condition that cucumber fusarium wilt pathogenic bacteria FOC are not added, carbon-based microbial fertilizers WSBX, WSBC, RSBX and RSBC prepared by wheat straw biochar and rice straw biochar through a blending method and an adsorption method have growth promotion effects on cucumbers in different degrees. Compared with CK, the RSBX carbon-based fertilizer respectively increases the height, stem thickness, fresh weight and dry weight of the cucumber by 14.10%, 9.36%, 7.46% and 9.62%, and the WSBX carbon-based fertilizer respectively increases the height, stem thickness, fresh weight and dry weight of the cucumber by 5.77%, 13.95%, 8.87% and 11.65%, but the WSBC and RSBC carbon-based fertilizer has no obvious growth promoting effect on the height, fresh weight and dry weight of the cucumber and has a certain promotion effect on the stem thickness of the cucumber. In addition, the rice straw biochar also has a relatively obvious growth promoting effect on cucumber plants.
TABLE 3 growth promoting effect of microbial carbon-based fertilizer on cucumber plants
CK: clear water control; mixing the WSBX, the WSBC, the RSBX and the RSBC which are 3 percent of each microbial carbon-based fertilizer with soil; and the WSB and the RSB are treated by mixing two biochar with soil, wherein the content of the biochar is 3%. Data are mean ± standard values and experiments were repeated 3 times. Different letters in the same column represent significant differences (p < 0.05).
The preferred embodiments of the present disclosure are described in detail with reference to the accompanying drawings, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Claims (10)
1. The Bacillus amyloliquefaciens B1408(Bacillus sp.) with the preservation number of CGMCC No.15110 is used for preparing a microbial carbon-based fertilizer or a medicament for preventing and treating plant soil-borne diseases.
2. The microbial carbon-based fertilizer is characterized by comprising biological carbon and microorganisms attached to the biological carbon;
the microorganism is Bacillus amyloliquefaciens B1408, and the preservation number is CGMCC No. 15110.
3. The microbial carbon-based fertilizer according to claim 2, wherein the biochar is alkaline, the content of C is 65-66%, the ash content is 70-71%, and the specific surface area is 60-62 m2/g。
4. The microbial carbon-based fertilizer according to claim 1 or 2, wherein the biochar is wheat straw biochar, corn straw biochar or rice hull biochar;
the particle size of the biochar is 180-200 meshes.
5. The microbial carbon-based fertilizer according to claim 1 or 2, wherein the bacillus amyloliquefaciens B1408 is attached to the biochar by adsorbing, impregnating or blending a bacterial suspension or fermentation broth of the bacillus amyloliquefaciens B1408;
the adsorption, immersion or blending time is 48h, the oscillation speed is 170rpm/min, and the ratio of bacterial suspension or fermentation broth V: biochar W is 20: 1;
the microbial cell content of the bacterial suspension or fermentation broth is at least 108CFU/mL。
6. The microbial carbon-based fertilizer according to claim 1 or 2, wherein the preparation of the bacterial suspension of bacillus amyloliquefaciens B1408 comprises:
inoculating the activated strain into an LB culture medium, and culturing at 28 ℃ and 170r/min for 12h to prepare a seed solution; inoculating into LB culture medium with 2% inoculum size, and culturing at 28 deg.C and 170r/min for 48 h; resuspending the harvested cells in sterile water, and adjusting the concentration of the bacterial solution to 108CFU/mL prepared bacterial suspension.
7. A preparation method of a microbial carbon-based fertilizer is characterized in that the biological carbon adsorbs, impregnates or is mixed with bacterial suspension or fermentation liquor of bacillus amyloliquefaciens B1408;
the particle size of the biochar is 180-200 meshes; the adsorption, immersion or blending time is 48h, the oscillation speed is 170rpm/min, the ratio of bacterial suspension or fermentation broth V: biochar W is 20: 1;
the microbial cell content of the bacterial suspension or fermentation broth is at least 108CFU/mL。
8. The method for preparing a microbial carbon-based fertilizer according to claim 7, wherein the preparation of the bacterial suspension of the bacillus amyloliquefaciens B1408 comprises the following steps:
inoculating the activated strain into an LB culture medium, and culturing at 28 ℃ and 170r/min for 12h to prepare a seed solution; inoculating into LB culture medium with 2% inoculum size, and culturing at 28 deg.C and 170r/min for 48 h; resuspending the harvested cells in sterile water, and adjusting the concentration of the bacterial solution to 108CFU/mL prepared bacterial suspension.
9. The medicine for preventing and treating plant soil-borne diseases is characterized by comprising bacillus amyloliquefaciens B1408.
10. The pesticide for controlling plant soil-borne diseases according to claim 9, wherein the pesticide further comprises biochar to which bacillus amyloliquefaciens B1408 is attached;
attaching the bacillus amyloliquefaciens B1408 to the biochar by adsorbing, dipping or blending a bacterial suspension or fermentation liquor of the bacillus amyloliquefaciens B1408;
the adsorption, immersion or blending time is 48h, the oscillation speed is 170rpm/min, and the ratio of bacterial suspension or fermentation broth V: biochar W is 20: 1;
the microbial cell content of the bacterial suspension or fermentation broth is at least 108CFU/mL。
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