CN111748485A - Screening method of silicate bacteria with high potassium-decomposing capacity suitable for low-temperature slightly-acidic environment and potassium-decomposing bacterial fertilizer - Google Patents
Screening method of silicate bacteria with high potassium-decomposing capacity suitable for low-temperature slightly-acidic environment and potassium-decomposing bacterial fertilizer Download PDFInfo
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Abstract
The invention discloses a method for screening silicate bacteria with high potassium-decomposing capacity, which is suitable for a low-temperature slightly acidic environment, and mainly comprises the following steps: separating silicate bacteria with potassium-decomposing capacity by using a selective culture medium through a dilution inverted plate method and a marking method, measuring the potassium-decomposing capacity of the separated silicate bacteria through a shake flask fermentation test, selecting a strain with strong potassium-decomposing capacity, preparing a potassium bacterial fertilizer by using mushroom bran as a carrier and adopting a composting decomposition method, and evaluating the toxicity and the fertilizer efficiency of the potassium bacterial fertilizer by using water spinach as a material. The potassium-dissolving capacity test of the silicate bacteria obtained by separation shows that partial strains still have strong potassium-dissolving capacity at low temperature, the potassium-dissolving bacterial fertilizer prepared by taking mushroom bran as a carrier shows that the potassium-dissolving bacterial fertilizer has no toxicity to seeds through a seed germination index test, and indexes of plant height, leaf area and the like of the water spinach of the bacterial fertilizer applying group are obviously higher than those of a control group. The bacterial strain screened by the method has high potassium-dissolving capacity and strong stress resistance, and is suitable for preparing microbial fertilizer.
Description
Technical Field
The invention relates to the technical field of microbial fertilizers, in particular to a screening method of silicate bacteria with high potassium-decomposing capability in a low-temperature slightly acidic environment and a potassium-decomposing bacterial fertilizer.
Background
The soil itself is a natural potassium reservoir, but more than 95% of potassium exists in the form of silicate minerals such as potassium feldspar, mica and the like, so that the solubility is low, the potassium is difficult to dissolve, and plants are difficult to utilize. This creates a conflict between potassium-rich and potassium-deficient soils.
The potassium-decomposing bacteria can decompose the fixed potassium in the soil and release quick-acting potassium which can be absorbed and utilized by crops. The potassium-solubilizing bacteria include Bacillus extorquens (Bacillus extorquens), Bacillus mucilaginosus (Bacillus mucilaginosus), Bacillus circulans (Bacillus circulans) and the like.
Bacillus mucilaginosus is also called silicate bacteria, can decompose minerals such as potassium feldspar, mica and the like, release potassium and silicon, decompose apatite to release phosphorus from the apatite, and enhance the disease resistance of crops by secreting plant growth stimulin and a plurality of enzymes[8]。
The silicate bacteria can be made into biological potassium fertilizer, and can promote the growth of tobacco, soybean, eggplant, etc., increase yield and improve quality. The biological potassium fertilizer can increase the plant height and leaf area of hot pepper and eggplant, thereby improving the photosynthetic capacity of plants, promoting the absorption and utilization of nitrogen and phosphorus, and improving the fresh weight of fruits and the fruiting rate. The silicate bacteria are used in the microbial fertilizer, can decompose insoluble aluminosilicate inorganic mineral substances such as potassium feldspar, apatite and the like, promote nutrient elements such as insoluble potassium, phosphorus, silicon, magnesium and the like to be converted into soluble nutrients, and increase the content of the quick-acting nutrients in soil. However, researches show that the silicate bacteria agent can not completely replace quick-acting potash fertilizer, and the potassium supply effect can be enhanced by matching the silicate bacteria agent with chemical fertilizer.
The production and development of the microbial fertilizer require a carrier, and the carrier has the function of providing a buffer environment for the functional bacteria, so that the functional bacteria are not easily interfered by the change of the external environment. Common carriers of microbial fertilizers in the current market comprise vermiculite, perlite, peat, decomposed organic fertilizers and the like. Peat is used as the most commonly used carrier of microbial fertilizer, has the disadvantages of long regeneration period, large environmental destruction during mining, high price and the like, and the finding of a cheap and good carrier is a problem to be considered in the production of the microbial fertilizer. The mushroom bran is the leftover of edible mushroom production, and is usually directly discarded or combusted in the past, which not only causes resource waste, but also causes breeding of mould and pests, increases the number of harmful spores and pests in the air and causes environmental pollution. As the total yield and the total export of edible fungi in China leap the world first, the fungus chaff produced every year is at least 400 ten thousand. The traditional method for treating the fungus chaff is to explore the feasibility of replacing other materials with the fungus chaff as a microbial fertilizer carrier, and has important significance for environmental protection and resource saving.
Disclosure of Invention
The invention aims to provide a screening method of silicate bacteria with high potassium decomposing capacity suitable for a low-temperature slightly acidic environment, a potassium decomposing bacterial fertilizer and a potassium decomposing bacterial fertilizer containing the silicate bacteria.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for screening silicate bacteria with high potassium-decomposing capacity suitable for a low-temperature slightly acidic environment comprises the following steps:
(1) collecting soil from the rhizosphere of the crops in the vegetable field or the banana field;
(2) fully and uniformly mixing the collected soil, putting 5g of soil into a sterile test tube, adding 45mL of sterile water, shaking for 15min, standing for 5min, taking 1mL of mixed solution from the upper-layer liquid into a test tube containing 9mL of sterile water, and preparing the soil-water-based biological membraneInto 10-1Then diluted to 10-2，10-3，10-4，10-510 will be-1，10-2，10-3，10-4，10-5And 5 dilutions of soil diluent are coated on a silicate bacteria separation culture medium, and the silicate bacteria separation culture medium is inverted in a 30 ℃ culture box, and colonies which are large, round, neat in edge, high in transparency, obvious in bulge, quite adhesive and have a wire drawing phenomenon are selected for streak separation and purification. Repeating the steps for 3-4 times, and performing microscopic examination on the purity until a pure single bacterial colony is obtained, namely the silicate bacteria is obtained, and preserving the strain slope at 4 ℃ for later use.
(3) Selecting a plurality of strains of single colonies according to different colony forms of the single colonies of silicate bacteria;
(4) inoculating a plurality of selected strains of single colonies to a silicate bacterial plate and culturing for 48 hours at the temperature of 30 ℃;
(5) and (4) respectively measuring the potassium-dissolving capacity of the strains of the single bacterial colonies in the step (4), and selecting the strains with relatively small change of the potassium-dissolving capacity and strong potassium-dissolving capacity under different temperature conditions as strains for preparing the microbial fertilizer.
Further, after the step (4), further separating, purifying and preserving the selected multiple single colony strains.
Further, the step (4) also comprises gram staining, spore staining and capsule staining tests on the strain.
Further, the different temperatures described in step (5) include 30 ℃, 25 ℃, 20 ℃, 15 ℃.
Further, the step (5) is followed by inoculating the selected strain according to the proportion of inoculating 5ml into 95ml of liquid culture medium, and performing shake culture at 28 ℃ for 48h to obtain a bacterial liquid for preparing the microbial fertilizer.
The potassium-decomposing bacterial fertilizer comprises bacterial liquid, bacterial bran and sterile water, wherein the bacterial liquid is prepared by the method.
Further, the ratio of the bacterial liquid to the bacterial bran to the sterile water is 1:10: 10.
further, the potassium-solubilizing bacterial manure is prepared by the following method: and (3) bacterial liquid: fungus chaff: mixing sterile water at a certain proportion, placing into a plastic box, covering with preservative film, pricking several air holes, covering with preservative film for 2 times, culturing at 28 deg.C for 4 days, and storing at normal temperature in shade.
Further, the pH value of the potassium-decomposing bacterial manure is 6.0-3.0.
Further, the water content of the potassium-solubilizing bacterial manure is 30-45%.
The method comprises the steps of separating bacteria in soil, screening silicate bacteria with potassium-dissolving capacity, and selecting a silicate bacteria strain with strong potassium-dissolving capacity suitable for low-temperature slightly acidic environment as a strain for preparing microbial fertilizer through potassium-dissolving capacity test; the bacterial bran is used as a carrier to prepare the potassium-dissolving bacterial fertilizer by adopting a composting decomposition method, the bacterial strain obtained by the method still has strong potassium-dissolving capacity under a low-temperature slightly acidic environment through testing, the potassium-dissolving bacterial fertilizer prepared by using the bacterial bran as the carrier shows that the potassium-dissolving bacterial fertilizer has no toxicity to seeds through seed germination index testing, and the indexes of plant height, leaf area and the like of the swamp cabbage of the bacterial fertilizer applying group are obviously higher than those of a control group. In conclusion, the strain obtained by the method has the advantages of suitability for low-temperature slightly acidic environment, high potassium-dissolving capacity and strong stress resistance, and is suitable for preparing microbial fertilizers.
Drawings
FIG. 1 is a characteristic diagram of the colony shape of a silicate bacterium of the present invention on a silicate bacterium medium;
FIG. 2 is a morphogram of silicate bacterium strain K2 according to example 1 of the present invention;
FIG. 3 is an electrophoretogram of a PCR product of strain K2 of example 1 of the present invention;
FIG. 4 is a phylogenetic tree of the K2 strain of example 1 according to the present invention;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, but the technical solutions do not limit the scope of the present invention.
Example 1
1. Culture medium
1) Silicate bacteria isolation medium:
2) silicate bacteria fermentation medium:
2. separation, purification and preliminary identification of silicate bacteria
Mixing the collected soil sample, adding 5g of the soil sample into a triangular conical flask filled with 45mL of sterile water, shaking for 15min, standing for 5min, and making into 10-1Then taking 10-1Adding 1ml of the bacterial suspension into 9ml of sterile water to prepare 10-2The bacterial suspension is sequentially diluted to 10-3，10-4，10-5And coating the soil sample suspension with the first 5 dilutions on a silicate bacteria culture medium plate, performing inverted culture at 30 ℃ for 2d, and selecting a colony which is in a water-drop shape, is transparent, protrudes, is adhered and has a wiredrawing effect, namely the silicate bacteria. Streaking on solid plates and picking single colonies for gram, spore and capsule staining. Inoculating the pure culture to silicate slant culture medium, culturing for 48 hr, and preserving at 4 deg.C.
The silicate bacteria are cultured for 2-3d to form transparent raised circular colony with diameter of 3-13mm, and the colony has neat edge, wet, viscous, smooth, glossy, transparent or translucent surface and wire drawing phenomenon. According to the different colony forms of silicate bacteria, 4 strains with single colony are selected and respectively numbered as K1, K2, K3 and K4.
FIG. 1 is a colony morphology feature diagram of silicate bacteria.
The purified strains K1, K2, K3, K4 were subjected to gram stain, capsule stain and spore stain, and the results are shown in table 1 and fig. 2. The strain K2 is a gram-positive bacterium, has elliptical spores and thick large capsule. The other 3 strains are gram-negative bacteria, all of which have capsules but no spores. In view of the strong stress resistance of spore bacteria, which is an important consideration index for screening microbial fertilizer functional bacteria, the strain K2 is intensively researched.
TABLE 1.4 gram stain, spore stain and capsular stain of silicate bacteria
FIG. 2 is an individual morphological diagram of silicate bacterium strain K2, wherein gram stain (A) is G +, (B) capsular stain has large capsular, and (C) spore stain has spore, in order from left to right.
3. API physiological and biochemical identification of K2 strain
Referring to Berger's Manual of bacteria identification (eighth edition) and Manual of identification of common bacteria systems, 30 physiological and biochemical indicators such as sugar alcohol utilization of K2 strain can be measured, the strain can ferment and utilize saccharides such as glucose, sucrose, fructose, maltose, etc., can utilize alcohols such as glycerol, mannitol, inositol, etc., but cannot utilize sugar alcohol substances such as mannose, rhamnose, lactose, arabinose, erythritol, etc., has gelatinase activity but does not have enzyme activities such as β -galactosidase, urease, tryptophan deaminase, etc., and does not produce H2S, no indole substance is produced, citric acid cannot be utilized, but 3-hydroxy butanone can be produced. Specific results are shown in the API50CH sugar alcohol fermentation test and the major physiological properties of API20E in table 1, "+" indicates positive and "-" indicates negative.
TABLE 2 sugar alcohol fermentation test and other physiological characteristics of Strain K2
4. 16S rDNA sequencing analysis for strain identification of strain K2
DNA of the strain K2 was extracted by the CTAB method, the universal amplification primers (F: 5'-AGA GTT TGA TCC TGG CTCAG-3', R: 5'-AAG GAG GTG ATC CAG CC-3') for the bacteria were synthesized by Yiwei Weiji (Shanghai) trade company Limited, a specific target band of about 1.6kb in size was observed in the PCR amplification product by agarose gel electrophoresis, and FIG. 3 is an electrophoretogram of the PCR product of the strain K2.
The determined sequences were submitted to GenBank database and subjected to homology comparison using Blast program, and the results showed that the PCR-amplified sequence of K2 strain had 99% similarity to the gene sequence of Paenibacillus mucoginosus (GenBank accession No. JF 499917.1). The sequence was compared with the sequence between different genera of silicate bacteria downloaded in GenBank using Clustal X software and a phylogenetic tree was established using MEGA software, see FIG. 4. The K2 strain and P.mucoginoses belong to the same great branch in evolution, BootStrap is 100, which indicates that the homologous relationship between the K2 strain and P.mucoginoses is credible and belongs to Bacillus mucilaginosus. FIG. 4 is a phylogenetic tree of strain K2.
5. Test for Potassium resolving ability
Adding the purified strain into silicate fermentation medium (bacterial liquid: medium: 5:250), and shake culturing at 28 deg.C and 150r/min for 3 d. Preparing a potassium decomposing culture medium, subpackaging 95ml of the potassium decomposing culture medium, adding 0.5g of accurately weighed potassium mineral powder into a 250ml triangular flask, sterilizing for 20 minutes at 121 ℃, adding 5ml of inoculation liquid into the potassium decomposing culture medium, setting a blank control without inoculation, adjusting the rotation speed of a shaking table incubator to 150r/min, respectively culturing for 3 days at different temperatures of 30 ℃, 25 ℃, 20 ℃ and 15 ℃, centrifuging the culture liquid for 5 minutes at 2000r/min, reserving a supernatant, and measuring the content of quick-acting potassium by using flame spectrophotometry.
The comprehensive potassium-decomposing capacity of the 4 strains is measured, and it can be seen from table 2 that the potassium-decomposing capacity of the silicate bacteria changes with the change of temperature, the potassium-decomposing capacity of the strain K2 under different temperature conditions changes relatively little, and the potassium-decomposing capacity is also strong, so that the strain K2 has economic value only when being used as a fertilizer in a region with a large range, and therefore, the strain III is selected as a strain for preparing the microbial bacterial fertilizer.
TABLE 3 Potassium-solubilizing ability of the strains at different temperatures
5. Analysis of stress resistance results of silicate bacteria
The acid and alkali resistance test results show that the strain K2 can grow in the range of pH 5-pH 10, does not grow well in the environment of pH3 and pH4, and grows well in the slightly alkaline environment.
The temperature tolerance test result shows that the strain can grow at 20-40 ℃, does not grow at 0 ℃ and 10 ℃, and grows best at 30 ℃. But the K2 strain still has the potassium-dissolving capability at 15 ℃.
The salinity tolerance test result shows that the strain can grow in a NaCl silicate bacteria culture medium with the concentration of 0.1-0.6 percent, and a layer of thin gelatinous lawn is paved on the surface of the culture medium
Example 2
1. Preparation of bacterial manure for decomposing potassium
Culturing a bacterial liquid: the strain k2 with the highest potassium-solubilizing ability was shake-cultured at 30 ℃ for 48 hours, and when the OD value was about 0.5, the number of viable cells was considered to be 108.
And (3) treatment of mushroom bran: naturally drying, pulverizing, oven drying at 160 deg.C for 1h, and air drying for use.
And (3) bacterial liquid: fungus chaff: uniformly mixing sterile water according to the ratio of 1:10:10, placing into a plastic box, performing film-covering culture at room temperature for 4d, turning over for 2 times, and storing at normal temperature in a cool and dry place for later use.
2. Bacterial manure index detection
Effective viable count detection
Plate counting was performed with silicate bacteria selection medium, according to the industry standard for complex microbial fertilizers (NY/T798-2004). And (3) taking sterile water as a blank control, culturing at 28 ℃ for 48h, counting colonies, calculating the number of effective viable bacteria, and tracking and detecting for 1 time every 2 weeks to evaluate the stability of the bacterial manure. The results are shown in table 3, and the effective viable count of the potassium-decomposing bacterial manure is stable after continuous tracking monitoring for 8 weeks, which indicates that the bacterial manure has good stability.
TABLE 4 effective viable count tracking monitoring of potassium-solubilizing bacterial manure
pH value detection
The detection is carried out according to the method of the compound microbial fertilizer (NY/T398-2004). The silicate bacterial manure was detected to have a pH of 6.5.
Moisture detection
According to the agricultural industry standard of the people's republic of China, namely a method for preparing a compound microbial fertilizer (NY/T798-.
Determination of germination index
The leaching liquor of the potassium-decomposing bacterial manure is diluted into 100 percent, 50 percent, 25 percent and 10 percent by volume fraction (volume of the leaching liquor/volume after dilution). Spreading 25 Chinese cabbage seeds in each portion on wet filter paper of a culture dish, and adding 5mL of leaching liquor with different concentrations. The germination test was carried out by culturing in an incubator at 20 ℃ in the dark with distilled water as a control. The germination rate and root length of the seeds were measured at 36h and 60h, respectively.
The germination index GI (%) (treated seed germination rate · seed root length)/(control seed germination rate · seed root length) × 100 the germination index of the pakchoi seeds was calculated as shown in table 5. The bacterial manure leaching liquor with low concentration is shown to promote seed germination, and the fertilizer is safe and nontoxic.
TABLE 5 germination index of Chinese cabbage seeds with leaching solution of potassium-solubilizing bacterial manure
Fertilizer efficiency test of potassium bacterial fertilizer
Effect on stem height: selecting water spinach seedlings with similar plant heights to perform potting test, wherein 12 seedlings are potted in each pot, 8 pots (6 pots are regularly and quantitatively applied with bacterial manure, 2 pots are blank control without applying potassium bacterial manure), and measuring indexes such as stem length, leaf number and the like after 15 days. From tables 6, 7 and 8, it can be seen that the growth of the seedlings of the water spinach in the flowerpot added with the silicate bacterial manure is better.
TABLE 6 influence of Potassium bacterial manure on Stem height of swamp cabbage
TABLE 3 influence of Potassium bacterial manure on leaf length of swamp cabbage
TABLE 8 leaf number of Potassium bacterial manure to swamp cabbage
The silicate bacteria can be primarily determined by the classical identification technology of the microorganisms. In a potassium-dissolving experiment, at normal temperature, the potassium-dissolving capacity of each strain is similar, and at low temperature, the potassium-dissolving capacity of the strain K2 is stronger, so that the strain K2 has better stress resistance and is suitable for serving as a strain of a microbial fertilizer. After the bacterial manure is manufactured, the effective viable count is periodically measured, and the bacterial manure has certain stability through statistics. In the toxicity test of seed germination, the germination indexes of bacterial manure leaching solutions with different concentrations are all larger than 75%, the lower the concentration is, the larger the germination index of the seeds is, when the germination index of the seeds is larger than 70%, the bacterial manure leaching solution is considered to be non-toxic to the seeds, and when the germination index of the seeds is larger than 100%, the bacterial manure leaching solution has a promotion effect on seed germination. The fertilizer efficiency test shows that the water spinach plant applied with the silicate bacterial fertilizer has better growth vigor than the water spinach plant only applied with the bacterial bran, and has obvious advantages of high stem height and leaf area.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. A method for screening silicate bacteria with high potassium-decomposing capacity suitable for a low-temperature slightly acidic environment is characterized by comprising the following steps of:
(1) collecting soil from the rhizosphere of the crops in the vegetable field or the banana field;
(2) fully and uniformly mixing the collected soil, putting 5g of soil into a sterile test tube, adding 45mL of sterile water, shaking for 15min, standing for 5min, taking 1mL of mixed solution from the upper-layer liquid into a test tube containing 9mL of sterile water, and preparing into 10-1Then sequentially diluting to 10-2，10-3，10-4，10-510 will be-1，10-2，10-3，10-4，10-5Coating the 5-dilution soil dilution on a silicate bacteria separation culture medium, inverting the silicate bacteria separation culture medium in an incubator at 30 ℃, culturing for 2d, and selecting a water-drop-shaped colony which is transparent, convex, adhesive and has wiredrawing, namely silicate bacteria;
(3) carrying out streak culture on the silicate bacteria obtained in the step (2) on a silicate bacteria separation culture medium to obtain a silicate bacteria single colony;
(4) selecting a plurality of strains of single colonies according to different colony forms of the single colonies of silicate bacteria;
(5) inoculating a plurality of selected strains of the single colonies onto a silicate bacterial plate and culturing at 33 ℃ for 48 hours;
(6) and (5) respectively measuring the potassium-dissolving capacity of the strains of the single colonies in the step (5), and selecting the strains with relatively small change of the potassium-dissolving capacity and strong potassium-dissolving capacity under different temperature conditions as strains for preparing the microbial fertilizer.
2. The method for screening silicate bacteria having high potassium-solubilizing ability suitable for use in a low-temperature slightly acidic environment according to claim 1, wherein: and (4) further separating, purifying and preserving the selected multiple single colony strains after the step (4).
3. The method for screening silicate bacteria having high potassium-solubilizing ability suitable for use in a low-temperature slightly acidic environment according to claim 1, wherein: and (5) performing gram staining, spore staining and capsule staining tests on the strain.
4. The method for screening silicate bacteria having high potassium-solubilizing ability suitable for use in a low-temperature slightly acidic environment according to claim 1, wherein: the different temperatures in step (6) include 30 ℃, 25 ℃, 20 ℃ and 15 ℃.
5. The method for screening the silicate bacteria having a high potassium-solubilizing ability suitable for a low-temperature slightly acidic environment according to any one of claims 1 to 4, wherein: and (6) inoculating the selected strain according to the proportion that 5ml of the strain is inoculated into 95ml of liquid culture medium, and performing shake culture at 28 ℃ for 2 days to obtain a bacterial liquid for preparing the microbial fertilizer.
6. The potassium-decomposing bacterial fertilizer comprises bacterial liquid, bacterial bran and sterile water, and is characterized in that: the bacterial suspension produced by the method according to claim 5.
7. The potassium solubilizing bacterial fertilizer as claimed in claim 6, wherein: the ratio of the bacterial liquid to the fungus bran to the sterile water is 1:10: 10.
8. the potassium solubilizing bacterial fertilizer as claimed in claim 3, wherein: the potassium-solubilizing bacterial fertilizer is prepared by the following method: and (3) bacterial liquid: fungus chaff: mixing sterile water at a certain proportion, placing into a plastic box, covering with preservative film, pricking several air holes, covering with preservative film for 2 times, culturing at 28 deg.C for 4 days, and storing at normal temperature in shade.
9. The potassium solubilizing bacterial fertilizer as claimed in claim 8, wherein: the pH value of the potassium-decomposing bacterial fertilizer is 6.0-3.0.
10. The potassium solubilizing bacterial fertilizer as claimed in claim 8, wherein: the water content of the potassium-decomposing bacterial fertilizer is 30-45%.
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| CN108102962A (en) * | 2017-12-26 | 2018-06-01 | 中国热带农业科学院热带生物技术研究所 | A kind of Xiamen bacillus and its application |
| CN108330085A (en) * | 2018-01-30 | 2018-07-27 | 湖南农业大学 | A kind of fermentation process preparing probiotics suitable for the potassium decomposing bacterial strain of low temperature environment and based on the bacterial strain |
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Application publication date: 20201009 |