CN114908025A - Paenibacillus mucilaginosus HB-02 strain and application thereof in promoting crop growth - Google Patents
Paenibacillus mucilaginosus HB-02 strain and application thereof in promoting crop growth Download PDFInfo
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- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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Abstract
The invention relates to a paenibacillus mucilaginosus HB-02 strain and application thereof in promoting crop growth, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC) in 04/06/2022 with the preservation number of CGMCC No. 24636; the preservation address is No. 3 of Xilu No.1 of Beijing, Chaoyang, Beijing. The paenibacillus mucilaginosus HB-02 strain has strong phosphorus dissolving, potassium dissolving and nitrogen fixing capacities and strong capability of generating IAA, can be prepared into a microbial fertilizer, is beneficial to reducing the use of the fertilizer, further lightens the adverse effect of the fertilizer on the soil nutrient structure, the quality of agricultural products and the ecological environment, and can promote the crop production and improve the crop yield.
Description
Technical Field
The invention relates to the technical field of microorganisms and microbial fertilizers, in particular to a paenibacillus mucilaginosus HB-02 strain and application thereof in promoting crop growth.
Background
The large amount of fertilizer used in the agricultural production process causes the imbalance of soil nutrition structure, the quality reduction of agricultural products and the serious ecological environment pollution, the side effects are highly regarded by people, and the microbial fertilizer has the advantages of improving the crop nutrition level, promoting the transformation of substances in soil, promoting the crop yield and the crop quality, preventing and controlling harmful microorganisms and the like, makes up the defects of chemical fertilizers, is widely applied to the production of various crops, and makes the research and application of the microbial fertilizer become a hotspot. The specific microorganism in the microbial fertilizer decomposes substances which are difficult to utilize in soil through the self life activity, promotes the absorption of nutrient elements by plants, secretes various growth hormones or synthesizes substances which are beneficial to the growth and development of plants, inhibits the growth of pathogenic bacteria, and indirectly or directly improves the quality and the yield of the plants.
Plant growth-promoting rhizobacteria (PGPR) are a group of beneficial strains that live in the plant rhizosphere or colonize the surface of the root system to promote plant growth. Some PGPR live in the plant body and attached to the root system or soil around the root, and release organic acid to the outside of the cell to dissolve insoluble inorganic phosphorus, which is called as the acidolysis of inorganic phosphorus; while organophosphorus is converted to soluble phosphorus mainly by the hydrolysis of phosphate catalyzed by some phosphatases synthesized by PGPR, which is called mineralization of organophosphorus. Some PGPR can perform acidolysis and mineralization simultaneously so as to improve the utilization rate of the plant to phosphorus. Meanwhile, some PGPR can surround rock mineral particles by self capsules, and decompose the nutrient elements of feldspar, mica and phosphorus and potassium in apatite in soil into a form which can be directly absorbed and utilized by plants. The nitrogen-fixing microorganisms colonize the surface of the plant root system and sometimes invade the interior of the plant root system, and the plant is promoted to obtain more minerals and nutrients by promoting the growth of the plant root system, so that the plant growth is finally promoted. Some of the IAAs secreted by PGPR can interfere with various physiological processes of plants by changing the endogenous IAA pool of the plants, such as promoting division, extension and differentiation of plant cells; controlling the vegetative growth process; promoting seed and tuber germination; the growth speed of xylem and root is improved; stimulating the formation of lateral and adventitious roots, etc. However, not all PGPR have ideal functions of dissolving phosphorus, dissolving potassium, fixing nitrogen, promoting IAA secretion, and the like.
Disclosure of Invention
Aiming at the technical problems, the invention provides a paenibacillus mucilaginosus HB-02 strain and application thereof in promoting crop growth. The paenibacillus mucilaginosus HB-02 strain has strong phosphorus dissolving, potassium dissolving and nitrogen fixing capacities and strong capacity of generating 3-indoleacetic acid (IAA), can reduce the use of chemical fertilizers so as to reduce the adverse effect of the chemical fertilizers on the environment and promote the growth of crops.
In order to solve the technical problem, the embodiment of the invention adopts the following technical scheme:
in one aspect, the embodiment of the invention provides a Paenibacillus mucilaginosus HB-02 strain, and the classification name of the strain is Paenibacillus mucilaginosus (B.mucilaginosus) ((B.mucilaginosus))Paenibacillus mucilaginosus) The strain is preserved in China general microbiological culture Collection center (CGMCC) at 04/06/2022, with the preservation number of CGMCC 24636; the preservation address is No. 3 Xilu Beijing province of Chaozhou, the Beijing city, Chaoyang district。
The paenibacillus mucilaginosus HB-02 strain provided by the invention is separated from rhizosphere soil of robust wheat seedlings in wheat planting areas, belongs to the bacillus, has strong phosphorus dissolving, potassium dissolving and nitrogen fixing capabilities and strong capability of generating IAA, can be prepared into microbial fertilizer, is beneficial to crops to absorb phosphorus, potassium and nitrogen from the soil, thereby reducing the use of chemical fertilizer and further lightening the adverse effects of the chemical fertilizer on soil nutritional structure, agricultural product quality and ecological environment, and can promote crop production and improve crop yield. The 16S rDNA sequence of the strain is shown in SEQ ID NO. 1.
The method for screening the paenibacillus mucilaginosus HB-02 strain specifically comprises the following steps:
separating single colony from strong wheat seedling rhizosphere soil in a wheat planting area, and then screening a strain with obvious phosphorus and potassium dissolving capacity;
quantitatively measuring the phosphorus dissolving capacity, the potassium dissolving capacity and the nitrogen fixing capacity of the strains obtained in the step I respectively, and screening out strains with the best comprehensive phosphorus dissolving capacity, potassium dissolving capacity and nitrogen fixing capacity;
thirdly, detecting the capability of synthesizing IAA of the strains obtained in the second step;
fourthly, the bacterial strain obtained in the second step is applied to the field, and the influence of the bacterial strain on the growth of crops is inspected.
Through the experimental steps, the paenibacillus mucilaginosus HB-02 strain is obtained through screening.
In a second aspect, the embodiment of the invention also provides application of the paenibacillus mucilaginosus HB-02 strain in promoting crop growth.
The paenibacillus mucilaginosus HB-02 strain provided by the invention has strong phosphorus dissolving, potassium dissolving and nitrogen fixing capabilities and IAA synthesis capabilities, and can effectively promote the growth of crops. The field test data prove that the paenibacillus mucilaginosus HB-02 strain is used for seed dressing and irrigation of wheat, the color of leaves is dark green when seedlings emerge, the wheat seedlings are strong and robust, the growth vigor is better after green turning in winter, the leaves are green, the plants in the jointing stage are strong and dense, the leaves are dark green and thick, and the plant height and the yield of the wheat can be obviously increased.
In a third aspect, the embodiment of the invention also provides a method for promoting crop growth, which is to seed crop seeds after dressing the seeds with the zymocyte liquid of the paenibacillus mucilaginosus HB-02 strain.
Preferably, the crop is wheat.
Preferably, the content of the paenibacillus mucilaginosus HB-02 strain in the zymocyte liquid is 0.5 multiplied by 10 10 ~1.5×10 10 cfu/mL. At the concentration, the inoculation amount of the strain during seed dressing is preferably 15-25 mL/kg of seeds.
Preferably, the method further comprises applying the zymogen fluid of the paenibacillus mucilaginosus HB-02 strain in a pouring mode in the green turning period.
Preferably, the content of the paenibacillus mucilaginosus HB-02 strain in the zymocyte liquid for irrigation is 0.5 multiplied by 10 8 ~1.5×10 8 cfu/mL. At this concentration, the inoculum dose for irrigation was 1000 mL/m 2 It is preferable.
Preferably, the preparation method of the fermentation broth of the paenibacillus mucilaginosus HB-02 strain comprises the following steps: inoculating the paenibacillus mucilaginosus HB-02 strain to a colloid solid culture medium, culturing at the constant temperature of 36 ℃ for 12-24 h for activation, selecting a single colony to be inoculated into a colloid liquid culture medium, performing constant temperature shaking culture at the constant temperature of 36 ℃ for 12-24 h, inoculating the culture to the colloid liquid culture medium by 10 percent of inoculum size, performing constant temperature shaking culture at the constant temperature of 36 ℃ for 48-60 h, centrifugally recovering thalli, and adjusting the spore concentration to 0.5 multiplied by 10 by using sterile water 8 ~1.5×10 10 cfu/mL to obtain the zymogen liquid.
In a fourth aspect, the embodiment of the invention also provides a microbial inoculum, which comprises a fermentation product of the paenibacillus mucilaginosus HB-02 strain.
The microbial inoculum can be used for treating seed dressing and irrigation of crops to achieve the effects of improving the growth vigor of the crops, promoting the growth of the crops and increasing the yield.
Preferably, the preparation method of the fermentation product of the paenibacillus mucilaginosus HB-02 strain comprises the following steps: inoculating the paenibacillus mucilaginosus HB-02 strain to a colloid solid culture medium, culturing at the constant temperature of 36 ℃ for 12-24 h for activation, selecting a single colony to be inoculated into a colloid liquid culture medium, performing constant temperature shaking culture at the constant temperature of 36 ℃ for 12-24 h, inoculating the culture to the colloid liquid culture medium by 10% of inoculation amount, and performing constant temperature shaking culture at the constant temperature of 36 ℃ for 48-60 h.
Drawings
FIG. 1 shows the results of measuring the IAA-producing ability of HB-02 strain in example 1;
FIG. 2 is the colony morphology of HB-02 strain in example 1;
FIG. 3 shows the cell morphology of HB-02 strain in example 1;
FIG. 4 is a phylogenetic tree of HB-02 strain in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The experimental procedures used in the following examples are all conventional in the art unless otherwise specified.
The raw materials, reagents and the like used in the following examples were obtained commercially unless otherwise specified.
The experimental data in the following examples were analyzed using SPSS13.0 software, ANOVA with one-way ANOVA, mean. + -. standard deviation, and multiple comparisons using the Duncan method.
Example 1
The embodiment of the invention provides a paenibacillus mucilaginosus HB-02 strain, which is obtained by screening through the following steps:
test method 1
1.1 preparation of culture Medium and reagent
PKO inorganic phosphorus medium: glucose 10.0g, Ca 3 (PO 4 ) 2 5 g，(NH 4 ) 2 SO 4 0.5 g，NaCl 0.2 g，KCl 0.2 g，MgSO 4 ·7H 2 O 0.3 g，MnSO 4 ·H 2 O 0.03 g，FeSO 4 ·7H 2 0.03 g of O, 0.5g of yeast extract powder, adding water to supplement the mixture to 1000 mL, adjusting the pH value to 6.8-7.0, and sterilizing the mixture for 20min at 115 ℃.
Monkina organophosphorus culture medium: glucose 10.0g, (NH) 4 ) 2 SO 4 0.5 g，NaCl 0.3 g，KCl 0.3 g，FeSO 4 ·7H 2 O 0.03 g，MnSO 4 ·H 2 O 0.03 g，CaCO 3 5.0g, 0.4 g of yeast extract powder, 0.2 g of egg yolk lecithin, 1000 mL of deionized water, pH7.0 and sterilization at 115 ℃ for 20 min.
Potassium-dissolving culture medium: 10.0g of sucrose, 0.5g of magnesium sulfate heptahydrate, 1.0 g of calcium carbonate, 1.0 g of ammonium sulfate, 0.l g g of sodium chloride, 0.5g of yeast extract, 2.0 g of disodium hydrogen phosphate, 10.0g of potassium feldspar powder, 1000 mL of deionized water, pH of 7.2-7.4, and sterilization at 115 ℃ for 20 min.
YMA medium: weighing 0.5g of monopotassium phosphate, 1.0 g of yeast powder, 10.0g of mannitol, 0.2 g of magnesium sulfate, 0.1 g of sodium chloride, 100 mg of tryptophan and 1000 mL of distilled water, carrying out sterilization at the temperature of 121 ℃ for 15min, wherein the pH value is 6.8-7.2.
The nitrogen-free liquid culture medium of the aberra babylonica: glucose 10.0g, K 2 HPO 4 0.2g，NaCl 0.2g，MgSO 4 ·H 2 O0.2g，K 2 SO 4 0.2g，CaCO 3 5.0g, yeast extract powder 0.5g, pH7.0, 1000 mL water, 115 ℃ sterilization for 20 min.
The Artobia pipiens nitrogen-free solid culture medium: adding agar into nitrogen-free liquid culture medium of Artobia sibirica, adding agar 20 g per 1000 mL liquid culture medium, and sterilizing at 115 deg.C for 20 min.
Colloid liquid culture medium: 5g of white sugar and Na 2 HPO 4 2 g，MgSO 4 0.5 g，CaCO 3 0.1 g，FeCl 3 0.1 g, 1000 mL of water, pH 7.2-7.5, and sterilizing at 115 ℃ for 20 min.
Colloid solid medium: adding agar into colloid liquid culture medium, adding agar 20 g per 1000 mL liquid culture medium, and sterilizing at 115 deg.C for 20 min.
Molybdenum antimony stock solution: 126 mL of concentrated sulfuric acid was slowly added to 400 mL of water, and after stirring and cooling, 10 g of ammonium molybdate was weighed and added to 300 mL of water at about 60 ℃ and stirred until completely dissolved and cooled. And slowly adding the diluted sulfuric acid solution into the ammonium molybdate solution while stirring. Adding 100 mL of 0.5% antimony potassium tartrate solution, diluting with distilled water to a constant volume of 1L, and storing in dark place.
Molybdenum antimony color-developing resisting agent: 1.50 g ascorbic acid was completely dissolved in 100 mL molybdenum antimony stock solution.
2, 4-dinitrophenol solution: 0.25 g of 2, 4-dinitrophenol is weighed out and dissolved in 100 mL of distilled water.
Phosphorus standard solution: weighing KH dried at 45 deg.C for 6 hr 2 PO 4 0.4394 g, dissolving in 400 mL of distilled water, adding 5mL of concentrated sulfuric acid, diluting with distilled water to 1000 mL, wherein the concentration of the solution is 100 mg/L, and further diluting to obtain 5.0 mg/L of phosphorus standard solution.
Potassium standard solution: accurately weighing 1.5830 g of analytically pure KCl dried at 105 ℃ for 4-6 hours, dissolving in distilled water, and diluting to 1000 mL to obtain the product containing K 2 O1000 mg/L potassium standard solution. This solution was further diluted to give 100 mg/L of a potassium standard solution.
Preparing a PC colorimetric solution: weighing 12 g FeCl 3 Dissolving the mixture in 300 mL of distilled water, slowly adding 429.7 mL of concentrated sulfuric acid, and cooling to reach the volume of 1000 mL.
1.2 screening method
1.2.1 soil sample Collection
Selecting strong wheat seedling rhizosphere soil in a wheat growing area of an agricultural garden of Hebei agricultural university, weighing 10 g of strong wheat seedling rhizosphere soil, putting the strong wheat seedling rhizosphere soil into a sterilized triangular flask containing 90 mL of sterile water and glass beads, oscillating the triangular flask by using a shaking table for 1 h, performing 10-time series gradient dilution on suspension by using sterile water, selecting 100 mu L of diluent with proper gradient, coating the diluent on a colloid culture medium plate, and performing inversion culture at the constant temperature of 36 ℃ for 40 h to separate a single bacterial colony. And carrying out co-separation to obtain 863 strains of the bacterial strain.
1.2.2 isolation culture of phosphorus-solubilizing Potassium-solubilizing bacteria
And (3) inoculating the strain separated by 1.2.1 onto a Monkina organic phosphorus culture medium, a PKO inorganic phosphorus culture medium and a potassium-dissolving culture medium flat plate, culturing for 5-7 d at 36 ℃, observing the growth condition of the strain in the flat plate, judging whether a decomposition ring appears or not, preliminarily judging the phosphorus-dissolving and potassium-dissolving capacity of the strain according to the diameter of the decomposition ring, and co-screening to obtain 18 strains of bacteria with remarkable action effect.
And (3) further purifying the 18 strains with the phosphorus and potassium dissolving capacity, storing the purified strains, and quantitatively detecting the phosphorus, potassium and nitrogen dissolving capacity.
1.2.3 quantitative determination of the phosphorus solubilizing ability of bacteria
The detection of the inorganic phosphorus dissolved by bacteria is carried out by adopting a molybdenum-antimony colorimetric resistance method. The strain screened out at 1.2.2 was activated by shake cultivation overnight in a colloid liquid medium at 36 ℃ in a shaker, inoculated at 1% inoculum size in 50 mL of PKO liquid medium, and shake-cultured at 36 ℃ for 7 d. Centrifuging the fermentation liquor at 10000 r/min for 10 min, sucking 5mL of supernatant into a 50 mL volumetric flask, dropwise adding two drops of 2, 6-dinitrophenol solution, dropwise adding dilute hydrochloric acid to adjust the solution to be colorless, adding 5mL of molybdenum-antimony anti-color development liquid, fixing the volume, reacting at room temperature for 30 min by taking a blank culture medium as a reference, and then measuring the absorbance at 720 nm. The detection of bacteria dissolving organophosphorus adopts Monkina organophosphorus culture medium, and the detection method is the same as the above. Respectively sucking 0 mL, 0.5 mL, 1.0 mL, 1.5 mL, 3 mL, 5mL, 10 mL and 15 mL of 5 mg/L phosphorus standard solution into a 50 mL volumetric flask, adding double distilled water to scale, uniformly mixing, and drawing a standard curve by using the light absorption value as a vertical coordinate and the standard phosphorus concentration as a horizontal coordinate in the same detection method.
1.2.4 quantitative determination of the Potassium-resolving Capacity of bacteria
The strains screened out at 1.2.2 are activated by shaking culture overnight in a shaking table at 36 ℃ in a colloid liquid culture medium, and are inoculated in 50 mL of potassium-dissolving liquid culture medium according to the inoculum size of 1 percent, and are cultured for 7 d in a shaking table at 36 ℃. Centrifuging the fermentation liquid at 10000 r/min for 15min, recovering supernatant, and detecting potassium ion content with an atomic absorption spectrophotometer by using a sterile culture medium as a blank control. Adding a proper amount of potassium standard solution into a 50 mL volumetric flask respectively, performing constant volume by using distilled water to obtain potassium ion concentration gradient solutions of 0, 10, 20, 30, 40, 50, 60 and 70 mg/L, detecting by using an atomic absorption spectrophotometer, and drawing a potassium standard curve by taking the potassium ion concentration as an abscissa and the detection reading as an ordinate.
1.2.5 detection of Nitrogen-fixing ability of bacteria
Inoculating the strain screened out by 1.2.2 into a colloid solid culture medium slant, and culturing at the constant temperature of 36 ℃ overnight for activation. The culture was carried out in an incubator at 36 ℃ by streaking using an inoculating loop on an Abutilus solid medium. And observing the growth condition of the strain on an Abelmoschus manihot solid plate.
The method adopts a bacterial azotase kit (ELISA) to detect the azotase activity of azotobacter, and comprises the following specific steps: the panel was left at room temperature for 20min before being removed from the aluminum foil bag and 50. mu.L of sample was added to each well and 50. mu.L of standard series solution was added as above, with 3 replicates of each treatment. 100 μ L of detection antibody labeled with horseradish peroxidase (HRP) was added to each well, sealed with a sealing plate, incubated at 37 ℃ for 60min, and the water was removed. Adding washing solution 350 μ L per well, standing for 1min, removing water as much as possible, and repeating for 5 times. Substrate A, B in the kit was added in an amount of 50. mu.L each in each well in turn, and incubated at 37 ℃ for 15min in the absence of light. mu.L of stop solution was added to each well, and the OD value of each well was immediately measured at a wavelength of 450 nm. And (3) taking the light absorption value of the series of concentrations as a vertical coordinate and the azotase activity as a horizontal coordinate to obtain a regression equation, and calculating the azotase activity of the sample.
The results of the above-described experiments 1.2.3 to 1.2.5 are shown in Table 1.
TABLE 1 detection of phosphorus-dissolving, potassium-dissolving and nitrogen-fixing abilities of the screened bacteria
| Strain numbering | Organophosphorus content (μ g/mL) | Inorganic phosphorus content (μ g/mL) | Potassium content (μ g/mL) | Nitrogenase activity (U/L) |
| HB-01 | 12.17 | 16.53 | 7.49 | 15.67 |
| HB-02 | 79.54 | 112.13 | 37.42 | 26.53 |
| HB-35 | 68.43 | 77.42 | 18.37 | 21.48 |
| HB-68 | 16.79 | 13.58 | 23.62 | 15.69 |
| HB-84 | 8.22 | 36.16 | 26.33 | 19.56 |
| HB-125 | 35.27 | 123.58 | 10.11 | 23.49 |
| HB-168 | 79.89 | 37.63 | 19.58 | 24.21 |
| HB-224 | 62.36 | 95.21 | 37.54 | 20.16 |
| HB-276 | 81.36 | 58.71 | 23.74 | 34.29 |
| HB-297 | 63.17 | 36.89 | 15.43 | 8.54 |
| HB-351 | 39.22 | 89.58 | 13.58 | 27.59 |
| HB-465 | 32.16 | 25.97 | 26.52 | 31.85 |
| HB-477 | 68.73 | 59.22 | 25.79 | 20.01 |
| HB-534 | 47.28 | 35.42 | 29.16 | 21.33 |
| HB-673 | 44.29 | 76.28 | 36.21 | 18.76 |
| HB-725 | 59.37 | 43.25 | 21.69 | 23.58 |
| HB-798 | 62.41 | 59.72 | 35.57 | 27.00 |
| HB-829 | 13.19 | 28.93 | 40.71 | 23.69 |
As can be seen from the comprehensive analysis of 4 detection indexes, the strain with the number of HB-02 has the strongest comprehensive capability of dissolving phosphorus, potassium and nitrogen, the capability of dissolving inorganic phosphorus reaches 112.13 mu g/mL, the capability of dissolving organic phosphorus reaches 79.54 mu g/mL, the release amount of potassium is 37.42 mu g/mL, and the activity of the azotobacter is 26.53U/L, so that the strain is selected for subsequent test detection.
1.2.6 HB-02 Strain Synthesis 3-Indolylacetic acid (IAA) detection
Preparing IAA standard solutions with the concentrations of 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5 and 20.0 mg/L in sequence, adding 4 mL of each 3-indoleacetic acid standard solution into 4 mL of PC colorimetric solution, standing in the dark for 0.5 h, taking out, and immediately determining OD (optical density) of the solution 530 And adding 4 mL of distilled water into 4 mL of PC colorimetric solution for zero setting, repeating the measurement for 3 times, and respectively averaging the obtained data to draw a standard curve.
Transferring the HB-02 strain into a colloid liquid culture medium, performing shake culture at 36 ℃ for 24h by using a shaking table, transferring the strain into a YMA culture medium according to the inoculation amount of 1%, and performing shake culture at 36 ℃ for 4 d by using a shaking table. Centrifuging the culture solution at 10000 r/min for 15min, collecting supernatant 4 mL, adding equivalent PC colorimetric solution, standing in dark for 0.5 h, taking out, and immediately measuring OD with ultraviolet spectrophotometer 530 The OD of each treated sample was measured by zeroing with YMA medium which was centrifuged and added with PC colorimetric solution as a control 530 And (5) recording the value, and calculating the amount of the 3-indoleacetic acid produced by the strain according to a standard curve.
The results showed that the concentration of IAA synthesized by HB-02 strain was 69.87. mu.g/mL (as shown in FIG. 1).
1.2.7 HB-02 Strain growth promoting Effect in the field test
The HB-02 strain is streaked and inoculated in a colloidal solid medium, and is cultured overnight at a constant temperature of 36 ℃ for activation, a single colony is picked up and is cultured overnight in a colloidal liquid medium under shaking at a temperature of 36 ℃, and is inoculated in a fresh colloidal liquid medium according to the inoculum size of 10%, and is cultured for 48 hours under shaking at a temperature of 36 ℃. Centrifuging the fermentation liquid 10000 Xg for 10 min to recover thallus, suspending and diluting with sterile water to obtain HB-02 strain with viable bacteria concentration of about 1X 10 8 cfu/mL or 1X 10 10 cfu/mL of bacterial suspension, and bacterial counting was performed using a bacterial counting plate.
Selecting a wheat planting plot on an experimental farm of Hebei agricultural university, wherein the area of the plot is about 666.7 m 2 Divided into 3 small blocks, each block is about 222.2 m 2 The control group, treatment 1 group and treatment 2 group were used. The control group was not inoculated with the inoculum; the viable bacteria concentration of HB-02 strain adopted in both treatment 1 and treatment 2 was about 1.0X 10 10 Bacterial suspension of cfu/mLSeed dressing, wherein the inoculation amount is 20 mL/kg of seeds; in the green-turning period, the viable bacteria concentration of HB-02 strain is about 1.0X 10 8 Inoculating growth-promoting bacteria by pouring cfu/mL bacterial suspension, wherein the inoculation dose is 1000 mL/m 2 . The control group and the treatment group do not fertilize, mechanical seeding is adopted, and the management mode is the same as that of a common field.
In the wheat jointing stage, a 5-point sampling method is adopted, 10 plants are continuously selected at each point, 50 plants are selected for each treatment, the same position of the middle part of the fully-extended blade at the uppermost part is selected, a portable chlorophyll content measuring instrument is used for measuring the relative content of chlorophyll (SPAD value), and the average value is calculated. Plant height was measured and the average was calculated. And (4) taking the overground part of the wheat plant back to a laboratory, drying and weighing, and measuring the dry weight of the overground part. Sampling by adopting a 5-point sampling method in the milk stage of the wheat, taking 3 continuous lines at each point, taking 0.4 m in each line, investigating and counting the plant height and the average grain number per spike (30 plants at each random sampling point and 150 plants sampled at each group), weighing the thousand seed weight after the seeds are dried in the air, and measuring the yield per square meter by single threshing and single harvesting.
The investigation on the growth effect of the wheat shows that the wheat seedlings emerge 7 days after sowing, and basically all emerge 10 days after sowing. The results of investigation on the emergence conditions of wheat fields in test plots show that the emergence conditions of the microbial inoculum treatment groups are good, the leaves are dark green, and the wheat seedlings are strong and strong. After the wheat is turned green in winter, the treated wheat plants have better growth vigor and green leaves; the control group showed weak growth and yellow leaf tips. The growth conditions of wheat plants are investigated in the jointing stage, and the wheat seedlings of the treatment group are good in growth vigor, strong and dense in plants, dark green and thick in leaves, and obviously higher in plant height than those of the control group. The plant height of the treatment group 1 is 8.65 percent higher than that of the control group, the plant height of the treatment group 2 is 9.21 percent higher than that of the control group, and the difference between the two treatment groups and the control group is obvious. The chlorophyll relative content of the group 1 is 8.25% higher than that of the control group, the chlorophyll relative content of the group 2 is 11.14% higher than that of the control group, and the difference between the two groups is obvious compared with the control group. The dry weight of the treatment group 1 was 7.03% higher than that of the control group, and the dry weight of the treatment group 2 was 10.46% higher than that of the control group, and the difference between the two treatment groups was significant compared with the control group. In the detection of indexes such as plant height, chlorophyll relative content and dry weight, the treatment 2 groups are higher than the treatment 1 group, but the difference is not significant, which shows that the HB-02 strain inoculated twice is favorable for promoting the growth of wheat (as shown in Table 2).
TABLE 2 detection of growth promoting effect of Paenibacillus mucilaginosus HB-02 strain in wheat jointing stage
| Group of | Plant height/cm | Relative content of chlorophyll | Dry weight/g |
| Control group | 28.57±1.27b | 40.75±1.23b | 21.04±1.12a |
| Treatment of group 1 | 31.04±1.31a | 44.11±2.13a | 22.52±1.01b |
| Treatment of 2 groups | 31.69±1.49a | 45.29±2.89a | 23.24±2.35b |
Note: the data in the table are mean ± standard deviation. The data of different groups and different letters after the same column of data represent the same detection index are detected by the LSD methodP<The difference at the 0.05 level was significant.
In the wheat milk stage, the growth promoting effect of the growth promoting bacteria on wheat is analyzed by investigating indexes such as plant height, spike number, spike grain number, thousand grain weight, yield and the like. Compared with the control group, the indexes of the wheat of the treatment 1 group and the treatment 2 group are increased, wherein the increase of the number of ears, the number of grains per ear and the thousand grain weight is not obvious, the plant height and the yield are obviously increased, and the yield of the wheat is 709.86 g/m 2 Increased to 830.68 g/m 2 The growth promoting effect is obvious. In the detection of indexes such as plant height, grain number per ear, thousand grain weight and yield, the treatment 2 groups are superior to the treatment 1 group, but the difference between the two groups is not significant (as shown in table 3). The HB-02 inoculated strain can obviously improve the wheat yield, and the effect of two inoculations is better.
TABLE 3 detection of growth promoting effect of Paenibacillus mucilaginosus HB-02 strain in wheat milk stage
| Group of | Plant height/cm | Ear number/m 2 | Grain number per ear | Thousand grain weight/g | Yield/(g/m 2) |
| Control | 80.14±2.03b | 768.24±48.75a | 31.28±2.72a | 29.54±2.35a | 709.86±33.27b |
| Treatment of group 1 | 84.38±2.11a | 813.54±55.71a | 32.17±2.96a | 30.82±3.43a | 806.61±40.15a |
| Treatment of 2 groups | 85.76±3.54a | 815.36±49.72a | 32.79±3.03a | 31.07±2.94a | 830.68±36.27a |
Note: the data in the table are mean ± standard deviation. The data of different groups and different letters after the same column of data represent the same detection index are detected by the LSD methodP<The difference at the 0.05 level was significant.
2. Genus identification of HB-02 Strain
The HB-02 strain with better performances in all aspects is subjected to species identification.
2.1 colony and cell morphology Observation
The HB-02 strain is streaked and inoculated in a colloid solid medium, and is cultured overnight at a constant temperature of 36 ℃, and the colony morphology is observed. Cell morphology observation the cell morphology was observed under a 100 × objective microscope using crystal violet staining.
On a colloid solid culture medium, the HB-02 strain colony is circular or elliptical, is milk white or light yellow, is transparent or semitransparent, has regular edges, is raised into a steamed bun shape, is wet and sticky, and is purulent (as shown in figure 2). The thallus is rod-shaped, is uniformly dyed, and can form endogenic spores, spore mesogenesis and oval (as shown in figure 3).
2.216S rDNA gene sequence phylogenetic analysis
Extracting bacterial genome DNA as a template, and amplifying a 16S rDNA sequence by adopting a universal primer 27 f: AGAGTTTGATCCTGGCTCAG and 1495 r: CTACGGCTACCTT GTTACGA are provided. The primers were synthesized by Biotechnology engineering (Shanghai) GmbH. And designing a PCR reaction program according to the annealing temperature of the designed primer and the size of the amplified fragment. The reaction system was 5. mu.L of 10 XPCR Buffer, 4. mu.L of dNTP, 2. mu.L of each primer, 0.25. mu.L of DNA polymerase, 1. mu.L of DNA template, and ddH 2 O to 50. mu.L. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 45 s, annealing at 50 ℃ for 30 s, extension at 72 ℃ for 1min for 30 s, and 35 cycles; saturation extension at 72 ℃ for 10 min. After the reaction, the PCR result was detected by 1% agarose gel electrophoresis. The PCR products were sequenced by Beijing Hua Dagong Co., Ltd. Performing Blast comparison and homology comparison on the sequenced 16S rDNA sequence in a GenBank nucleic acid database, sequencing the sequence by using a Clustal algorithm in MegAlign software, calculating an evolution distance by using a Kimura two-parameter model, and constructing a phylogenetic tree by using a Neighbor-Joining method (as shown in figure 4).
Determining HB-02 strain as paenibacillus mucilaginosus according to the sequence similarity analysis of 16S rDNA (Paenibacillus mucilaginosus）。
The paenibacillus mucilaginosus HB-02 strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 2022, 04 and 06 days, and the preservation number is CGMCC number 24636; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang.
Example 2
The embodiment provides a microbial inoculum, which is a fermentation product of paenibacillus mucilaginosus HB-02 strain, and a preparation method of the fermentation product comprises the following steps: inoculating the strain HB-02 of Paenibacillus mucilaginosus obtained in example 1 into a solid medium containing pectin, performing constant temperature culture at 36 ℃ overnight (16 h) for activation, selecting a single colony, inoculating the single colony into a liquid medium containing pectin, performing constant temperature shaking culture at 36 ℃ overnight (16 h), inoculating the bacterial liquid into the liquid medium containing 10% of the inoculum size, performing constant temperature shaking culture at 36 ℃ for 48h, centrifuging the fermentation broth at 10000 Xg for 10 min to recover the bacterial body, and adjusting the spore concentration to a desired concentration, such as 0.5X 10, by using sterile water 8 ~1.5×10 8 cfu/mL or 0.5X 10 10 ~1.5×10 10 cfu/mL, and encapsulating to obtain the product.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
SEQUENCE LISTING
<110> Hebei Ji microbial technology Co., Ltd, Hebei agricultural university
<120> Paenibacillus mucilaginosus HB-02 strain and application thereof in promoting crop growth
<130> 20220518
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1492
<212> DNA
<213> 16SrDNA
<400> 1
aagagtttga tcatggctca ggacgaacgc tggcggcgtg cctaatacat gcaagtcgag 60
cggagcactt cggtgcttag cggcggacgg gtgagtaaca cgtaggcaac ctgcctgtaa 120
gatcgggata actaccggaa acggtagcta agaccggata gctggtttcg gtgcatgccg 180
gaatcatgaa acacggggca acctgtggct tacggatggg cctgcggcgc attagctagt 240
tggcggggta atggcccacc aaggcgacga tgcgtagccg acctgagagg gtgatcggcc 300
acactgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg aatcttccgc 360
aatgggcgca agcctgacgg agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa 420
gctctgttgc cagggaagaa tgtcgtggag agtaactgct ctgcgaatga cggtacctga 480
gaagaaagcc ccggctaact acgtgccagc agccgcggta atacgtaggg ggcaagcgtt 540
gtccggaatt attgggcgta aagcgcgcgc aggcggtctt ttaagtctgg tgtttaagcc 600
cggggctcaa ccccggttcg caccggaaac tggaagactt gagtgcagga gaggaaagcg 660
gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg 720
gctttctgga ctgtaactga cgctgaggcg cgaaagcgtg gggagcaaac aggattagat 780
accctggtag tccacgccgt aaacgatgag tgctaggtgt taggggtttc gatacccttg 840
gtgccgaagt aaacacaata agcactccgc ctggggagta cgctcgcaag agtgaaactc 900
aaaggaattg acggggaccc gcacaagcag tggagtatgt ggtttaattc gaagcaacgc 960
gaagaacctt accaggtctt gacatccctc tgaaagccct agagataggg ccctccttcg 1020
ggacagaggt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta 1080
agtcccgcaa cgagcgcaac ccttgacttt agttgccagc attgagttgg gcactctaga 1140
gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta 1200
tgacctgggc tacacacgta ctacaatggc cggtacaacg ggaagcgaag tcgcgagatg 1260
gagcgaatcc ttagaagccg gtctcagttc ggattgcagg ctgcaactcg cctgcatgaa 1320
gtcggaattg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggtcttgt 1380
acacaccgcc cgtcacacca cgagagttta caacacccga gtaacccgta agggagccag 1440
ccgtcgaagg tggggtagat gattggggtg aagtcgtaac aaggtagccg ta 1492
Claims (10)
1. A Paenibacillus mucilaginosus HB-02 strain is characterized in that the strain is classified as Paenibacillus mucilaginosus (HB-02)Paenibacillus mucilaginosus) The strain is preserved in China general microbiological culture Collection center (CGMCC) at 04/06/2022, with the preservation number of CGMCC 24636; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, the district of rising Yang.
2. The use of the Paenibacillus mucilaginosus HB-02 strain of claim 1 for promoting crop growth.
3. A method for promoting the growth of crops, which is characterized in that seeds of the crops are sown after being dressed with the zymocyte liquid of the paenibacillus mucilaginosus HB-02 strain of claim 1.
4. The method of claim 3, wherein the crop is wheat.
5. The method according to claim 3, wherein the content of the Paenibacillus mucilaginosus HB-02 strain in the fermentation broth is 0.5X 10 10 ~1.5×10 10 cfu/mL。
6. The method of claim 3, further comprising applying the zymogen fluid of the paenibacillus mucilaginosus strain HB-02 by pouring in a green-turning period.
7. The method according to claim 6, wherein the content of said Paenibacillus mucilaginosus HB-02 strain in said fermented liquid used for irrigation is 0.5X 10 8 ~1.5×10 8 cfu/mL。
8. The method according to claim 3, 4 or 6, wherein the preparation method of the fermentation broth of the Paenibacillus mucilaginosus HB-02 strain comprises: inoculating the paenibacillus mucilaginosus HB-02 strain to a colloid solid culture medium, culturing at the constant temperature of 36 ℃ for 12-24 h for activation, selecting a single colony to be inoculated into a colloid liquid culture medium, performing constant temperature shaking culture at the constant temperature of 36 ℃ for 12-24 h, inoculating the culture to the colloid liquid culture medium by 10 percent of inoculum size, performing constant temperature shaking culture at the constant temperature of 36 ℃ for 48-60 h, centrifugally recovering thalli, and adjusting the spore concentration to 0.5 multiplied by 10 by using sterile water 8 ~1.5×10 10 cfu/mL to obtain the zymogen liquid.
9. A microbial preparation comprising a fermentation product of the strain of Paenibacillus mucilaginosus HB-02 according to claim 1.
10. The microbial inoculum according to claim 9, wherein the fermentation product of the paenibacillus mucilaginosus HB-02 strain is prepared by a method comprising:
inoculating the paenibacillus mucilaginosus HB-02 strain to a colloid solid culture medium, culturing at the constant temperature of 36 ℃ for 12-24 h for activation, selecting a single colony to be inoculated into a colloid liquid culture medium, performing constant temperature shaking culture at the constant temperature of 36 ℃ for 12-24 h, inoculating the culture to the colloid liquid culture medium by 10% of inoculation amount, and performing constant temperature shaking culture at the constant temperature of 36 ℃ for 48-60 h.
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