CN115895960A - Strain for comprehensive planting and breeding of rice and fish and application thereof - Google Patents
Strain for comprehensive planting and breeding of rice and fish and application thereof Download PDFInfo
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Abstract
The invention discloses a strain for comprehensive planting and breeding of rice and fishery and application thereof, and relates to the strain and application thereof in the field of agriculture. The strain for the comprehensive breeding of rice and fishing is Bacillus marinus HL-44, is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC:24990. the bacillus marinus HL-44 disclosed by the invention has the capability of producing siderophores and IAA and can promote the growth of crops, the bacillus marinus HL-44 also has a strong capability of producing DDP-IV inhibitors, the bacillus marinus HL-44 can effectively reduce the blood sugar of fishes, is beneficial to the absorption of sugar in the living environment of the fishes and improves the yield, and the bacillus marinus HL-44 disclosed by the invention is particularly suitable for the application of comprehensive cultivation of rice and fisheries.
Description
Technical Field
The invention relates to a strain and application thereof in the field of agriculture.
Background
The fish culture in the rice field has the advantages of saving a large amount of water resources and land resources, promoting the sustainable development of the aquaculture industry and reducing the use of chemical fertilizers, and is popularized by the Chinese government on a large scale. By 2020, the culture area of fish culture in the rice field is as high as 1439.41 ten thousand mu, the yield of aquatic products is as high as 85.69 ten thousand tons, and the total yield of the aquatic products accounts for 41.41 percent of the total culture area of rice and fish culture in China and 29.41 percent of the total yield of the aquatic products, and occupies an important position. Researches prove that in a fish culture ecosystem in a rice field, proper fish culture does not reduce the yield of rice, but can reduce plant diseases and insect pests and control weeds, so that the use of herbicides and pesticides is reduced, and the yield and the quality of cultivated plants and animals can be improved. In addition, compared with conventional rice planting or fish culture, the rice in the comprehensive rice and fish culture can reduce the input of exogenous nitrogen and accelerate the recycling of nitrogen by utilizing the fixation effect of nitrogen-containing elements and microorganisms in the excretion of cultured animals, thereby reducing the use of fertilizers. The microorganism is used as an important medium of nutrient circulation in the comprehensive planting and breeding of rice and fishery, and plays an important role in nutrient element circulation, formation and maintenance of soil fertility and improvement of ecological environment on one hand; on the other hand, the fish colonizes in the intestinal tract to become intestinal indigenous flora, so that a microecosystem in which intestinal microorganisms are mutually restricted and dependent with the host and the aquatic environment is formed.
Disclosure of Invention
The invention provides a strain for comprehensive planting and breeding of rice and fishery and application thereof.
The bacterial strain for the comprehensive planting and breeding of rice and fishing is Bacillus marinus (Bacillus oceaniedinis) HL-44, is preserved in the China general microbiological culture Collection center, and has the preservation number of CGMCC:24990.
the application of the strain Bacillus marinus (HL-44) for the comprehensive planting and breeding of rice and fisheries in the comprehensive planting and breeding of the rice and fisheries.
The bacterial colony of the Bacillus marinus Bacillus oceanediminis HL-44 on the LB solid culture medium is characterized in that: the bacterial colony is milky white, round, opaque, moist and smooth in surface, glossy, irregular in edge and gram-positive through gram staining.
The bacillus marinus HL-44 is cultured on a CAS culture medium for a period of time, and then an obvious color change ring is formed around a bacterial colony, which indicates that the bacillus marinus HL-44 can generate an iron carrier with high iron chelating capacity, and the Su value of the iron carrier generated by the bacillus marinus HL-44 strain at 37 ℃ is 62%, which indicates that the strain has strong iron carrier generating capacity.
After Salkowski colorimetric solution is dripped into the bacterial liquid of the bacillus marinus HL-44, the color of the bacillus marinus HL-44 is changed into reddish color in a 1mL glass bottle under the condition of room temperature and light, and the result shows that the bacillus marinus HL-44 has the capability of producing auxin and has strong growth promotion effect on plants. The IAA production capacity of the bacillus marinus HL-44 is quantitatively detected, 86.53 microgram of IAA is contained in each milliliter of bacillus marinus HL-44 bacterial liquid, and the strain IAA has strong secretion capacity.
The bacillus marinus HL-44 is inoculated on an SKM culture medium, an obvious hydrolysis ring is formed around a bacterial colony after the bacillus marinus HL-44 is cultured for a period of time, the diameter D of the hydrolysis ring of the bacillus marinus HL-44 is 18.72mm, the diameter D of the bacterial colony is 4.31mm, and the D/D is 4.34; the yield of the DDP-IV inhibitor of the Bacillus marinus HL-44 strain at 37 ℃ is as high as 58.39 percent, which shows that the Bacillus marinus HL-44 has stronger capability of producing the DDP-IV inhibitor.
The bacillus marinus HL-44 disclosed by the invention has the capability of producing siderophores and indole-3-acetic acid (IAA) at the same time. The iron-producing carrier can chelate iron ions in the environment, maintain the stability of chloroplast and be beneficial to the growth and development of crops, and the IAA is used as a plant growth regulating substance, can promote the growth of plant roots, increase the length of the roots and the growth quantity of lateral roots, accelerate the absorption and conversion efficiency of the crops to soil nutrients in the growth process, and can stimulate the plants to secrete ACC deaminase. The bacillus marinus HL-44 disclosed by the invention can promote the growth of crops and increase the yield of the crops, and is particularly suitable for application in rice planting. The bacillus marinus HL-44 also has strong ability of producing DDP-IV inhibitor, and the bacillus marinus HL-44 can effectively reduce the blood sugar of fish, is beneficial to the absorption of sugar in the living environment of the fish and improves the yield. Therefore, the bacillus marinus HL-44 is particularly suitable for the application of comprehensive planting and breeding of rice and fishery, and the bacillus marinus HL-44 can promote the growth of rice and is beneficial to the growth of fish.
The Bacillus marinus HL-44 is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of West Lu No. 1 of the Korean-Yang district in Beijing, and the preservation number is CGMCC:24990 and the preservation date is 2022, 5 months and 30 days.
Drawings
FIG. 1 shows the result of screening bacterial siderophore of Bacillus oceanidianis HL-44;
FIG. 2 is a diagram showing the results of the Salkowski colorimetric determination of IAA production ability of Bacillus oceanic diagnosis HL-44;
FIG. 3 is a milk protein hydrolysis loop on Bacillus oceanic deminis HL-44% skimmed milk powder culture medium;
FIG. 4 is a phylogenetic tree constructed by Bacillus oceanisediminis HL-44;
FIG. 5 shows the result of the water culture box test of Bacillus oceanidianis HL-44.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and 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.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The first embodiment is as follows: the strain for the comprehensive planting of rice and fishing in the embodiment is Bacillus marinus HL-44, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC:24990.
1. method for obtaining the strains of the present embodiment
1. Selecting carp colony cultured in paddy field in large depression area of Panjin City in Liaoning 9 months in 2021 years, randomly selecting 3 healthy 1-year-old carps (150 +/-10 g), anesthetizing with MS-222 anesthetic (250 mg/L), wiping the surface of the carps with absolute ethyl alcohol, dissecting with sterilized scissors and tweezers in a super clean bench, taking out the whole intestinal tract of the carps, slightly extruding the content, placing in a conical flask containing glass beads and 50mL of sterile water, oscillating at room temperature for 30min at the rotating speed of 180r/min, performing gradient dilution, and diluting 10 th day -3 、10 -4 、10 -5 Coating 100 mu L of the gradient on an LB solid medium plate, repeating each gradient for 3 times, culturing at 28 ℃ for 48h, and selecting strains with different shapes for separation.
2. Identification of siderophore
And transferring the strain obtained by reactivating the separated and purified strain to an LB plate for culturing for 24h, picking a single colony by using a sterilized toothpick, inoculating the single colony to a chromium azure S (Chromeazurol S, CAS) solid detection culture medium, carrying out inverted culture at 37 ℃ for 2-3d, and observing the size of a color-changing ring around the colony. The isolated and purified strain HL-44 is inoculated on a CAS culture medium, and after a period of culture, a remarkable color-changing ring is formed around a colony (shown in figure 1), which indicates that the strain HL-44 can generate an iron carrier with high iron chelating capacity.
The strain HL-44 with obvious color change circle is further tested:
(1) Inoculating the activated strain HL-44 lawn in an iron-limiting SA liquid culture medium, and performing shake culture at 37 ℃ for 48h to obtain HL-44 bacterial liquid;
(2) Transferring the HL-44 bacterial liquid to be tested which grows for 48 hours into a sterilized 10mL centrifuge tube, and centrifuging for 15min at 13000 rpm;
(3) Transferring the supernatant into a test tube treated by concentrated hydrochloric acid, adding a certain amount of the CAS detection solution prepared in situ to ensure that the volume ratio of the supernatant to the detection solution is 1:1, fully and uniformly mixing, and standing at room temperature for 1h;
(4) Measuring absorbance value (A) at the wavelength of 630nm, taking double distilled water as a control for zero adjustment, taking the absorbance value at the wavelength of 630nm after mixing the uninoculated SA iron-limiting culture medium and the detection solution which are measured by the same method as a reference value (Ar), and expressing the activity unit of the siderophore by the following formula:
Su≈(Ar-As)/Ar×100；
in the formula: su is the siderophore content; ar is the OD value of the SA iron-limiting culture medium which is not inoculated and the supernatant of the detection liquid; as is the OD of the supernatant of the medium tested.
The activity unit of the siderophore is less than 10, which is generally considered as negative, and the mixture of the siderophore and the detection liquid has no color change.
The Su value of the strain HL-44 for producing the siderophore at 37 ℃ is 62%, which indicates that the strain has stronger siderophore production capability.
3. Determination of indole-3-acetic acid (IAA) produced by strain HL-44
3.1 inoculation of the isolated Strain HL-44 from the intestinal content of Cyprinus Carpio to the L-tryptophan-containing R 2 Placing the mixture in the liquid culture medium A, and placing the mixture in a constant-temperature shaking table at 28 ℃ for shake culture for 4d at 180 r/min. Sucking 500. Mu.L of the bacterial liquid into a 2mL glass bottle, and adding 500. Mu.L of the bacterial liquid into the glass bottleSalkowski colorimetric solution of L. Meanwhile, 500mg/L IAA is added into Salkowski colorimetric solution to be used as a positive control. The 2mL glass bottle was stored at room temperature in the dark for 30min, and the color change was observed. If the color turns red, the strain has the function of producing IAA.
As shown in figure 2, after Salkowski colorimetric solution is dripped into the bacterial liquid of the strain HL-44, the color of the bacterial liquid turns into reddish color in a 1mL glass bottle under the condition of room temperature and light shielding, which shows that the strain HL-44 has the ability of producing auxin.
3.2 accurately weighing 10mg of IAA, dissolving with a small amount of absolute ethyl alcohol, adding distilled water to a constant volume of 100mL, preparing an IAA solution with the concentration of 100 mu g/mL as a stock solution, and diluting the stock solution to prepare a series of standard solutions with the concentrations of 0 (blank), 0.5, 1.0, 5.0, 10.0, 15.0, 20.0 and 25.0 mu g/mL as working solutions. Respectively taking 2mL of the working solution, sequentially adding into 8 test tubes, adding 2 times of Salkowski colorimetric solution, placing at 40 ℃ in a dark condition, keeping the temperature for 30min, and measuring the light absorption value at the wavelength of 530 nm. By OD 530 An IAA standard curve is drawn by taking the abscissa and the IAA concentration as the ordinate. The IAA-producing ability of the strain HL-44 is quantitatively determined under the same culture condition as that of the primary screening. Firstly, measuring the OD value of a bacterial liquid at the wavelength of 600nm by a spectrophotometry, then centrifuging the bacterial liquid at the rotating speed of 10000r/min for 10min, taking the supernatant, adding an equal volume of Salkowski colorimetric solution, standing at 40 ℃ in a dark place for 30min for developing color, and measuring the OD value at the wavelength of 530 nm. Calculating OD 600 The IAA concentration per unit volume of the bacterial suspension of the strain HL-44 was 1. (bacteria liquid concentration higher class appropriate dilution)
The IAA production capacity of the strain HL-44 is quantitatively detected, the strain HL-44 is inoculated by using an LB liquid culture medium containing L tryptophan, after the culture at 30 ℃ and 180rpm is carried out for 24 hours, the result is shown in Table 1, 86.53 mu. G IAA is produced in bacterial liquid of the strain HL-44 per ml, and the IAA secretion capacity of the strain is strong.
TABLE 1 test results of the strain HL-44IAA
4. Determination of DDP-IV inhibitor produced by strain HL-44
4.1 the bacterial strain HL-44 is inoculated on an SKM culture medium, after 48H culture at 28 ℃, an obvious hydrolysis ring is formed around a bacterial colony, the diameter D of the hydrolysis ring of the bacterial strain HL-44 is 18.72mm, the diameter D of the bacterial colony is 4.31mm, the D/D is 4.34 (shown in figure 3), and the bacterial strain HL-44 has the capability of producing a DDP-IV inhibitor.
4.2 inoculating the strain HL-44 to a 96-pore plate, and accurately dropwise adding 25 mu L of glycyl-prolyl-p-nitroaniline and 25 mu L of CFS or CFE into each pore; reacting at 37 ℃ for 15min, adding 50 mu L of 0.01U/mL DDP-IV (dipeptidyl peptidase IV), continuing the reaction at 37 ℃ for 1h, adding 100 mu L of 1mol/L sodium acetate buffer solution (pH = 4.0) to stop the reaction, and detecting the absorbance of the reaction solution at 405nm by using a microplate reader to obtain the DDP-IV inhibition rate of the strain.
Wherein, the DDP-IV inhibition rate calculation formula is as follows:
A sample to be tested :25 μ L of sample +25 μ L LGly-pro-phy +50 μ L LDDP-IV +100 μ L sodium acetate;
A sample blank :25 μ L of sample +50 μ L of LTris-HCl +25 μ L of LGly-pro-phy +100 μ L of sodium acetate;
A negative control :25 μ L of Tris-HCl +25 μ L of LGly-pro-phy +50 μ L of LDDP-IV +100 μ L of sodium acetate;
A negative blank :75 μ L of Tris-HCl +25 μ L of LGly-pro-phy +100 μ L of sodium acetate.
The yield of the DDP-IV inhibitor of the strain HL-44 at 37 ℃ is as high as 58.39 percent, which indicates that the HL-44 strain has stronger capability of producing the DDP-IV inhibitor.
5. Identification of Strain HL-44
5.1 physiological and biochemical identification: the three regions of the preserved strain are streaked on a solid LB culture medium plate, a single colony is separated, the morphology of the single colony is described, and the gram staining and physiological and biochemical identification are carried out on the strain according to a manual for identifying a common bacteria system.
The colony characteristics of the strain HL-44 on the LB solid medium are as follows: the bacterial colony is milky white, round, opaque, moist and smooth in surface, glossy, irregular in edge and gram-positive through gram staining. Some physiological and biochemical indicators of HL-44 are shown in the following table. According to the description of Bacillus physiological characteristics in Bergey's Manual of identification of bacteria, HL-44 has the same characteristics with the physiological and biochemical characteristics of Bacillus oceanicus (Bacillus oceanic diagnosis) model species, and it is concluded from various physiological and biochemical characteristics (shown in Table 2) that the strain HL-44 is probably Bacillus oceanic diagnosis.
TABLE 2 physiological and biochemical results of the strain HL-44
5.2 16S rRNA identification: a bacterial genome DNA extraction kit of Beijing Soilebao Biotechnology company is selected to extract separated and purified bacterial strain DNA. Carrying out PCR amplification by using a bacterial universal primer 27F/1492R, wherein the PCR amplification system is a 25 mu L system: 2.5. Mu.L of 10 Xbuffer, 0.5. Mu.L of Taq enzyme, 0.5. Mu.L of primer 27F, 0.5. Mu.L of primer 1492R, 1. Mu.L of DNA template, ddH 2 O20. Mu.L. The reaction program is set to be pre-denatured at 95 ℃ for 5min; denaturation at 94 ℃ for 50s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1.5min, cycle times of 30 times, re-extension at 72 ℃ for 10min, and storage at 4 ℃. The PCR amplification product was sent to RuiBiotech for sequencing. And comparing sequencing results of the 16S rRNA of the strain through an NCBI database, and constructing a phylogenetic tree.
After sequencing the 16S rRNA sequence, BLAST alignment in NCBI found that the similarity of the 16S rRNA gene sequence of the strain HL-44 and Bacillus marinus (Bacillus oceanisediminis) is 99%. As shown in FIG. 4, the phylogenetic tree of HL-44 shows that the strain HL-44 has a minimum branch with Bacillus oceanisediminis (MT 457444.1) and has a shorter evolutionary distance, and the HL-44 strain is identified as Bacillus oceanisediminis through comprehensive physiological and biochemical indexes.
The second embodiment is as follows: the embodiment prepares the Bacillus oceanisediminis HL-44 bacterial liquid.
Adding 1000mL of water into 100g of soybean sprout, boiling for 1h, filtering, adding water to 1L, performing moist heat sterilization at 121 ℃, and storing for later use, namely the soybean sprout juice with the mass fraction of 10%.
The sea mud Bacillus oceanidianisis HL-44 liquid culture medium consists of 1000mL of bean sprout juice with the mass fraction of 10%, 10.87g of soluble starch, 2.48g of yeast extract, 1.83g of ammonium nitrate and 0.82g of ferric sulfate.
Inoculating Bacillus oceanic diminis HL-44 into a Bacillus oceanic diminis HL-44 liquid culture medium with the inoculation amount of 0.2 percent, culturing at 37 ℃ for 180r/min for 24h to obtain the Bacillus oceanic diminis HL-44 bacterial liquid, wherein the viable count of the Bacillus oceanic diminis HL-44 reaches 1.98 multiplied by 10 9 cfu/mL。
Example 1 Rice Germination experiment
Preparing Bacillus oceanisediminis HL-44 bacterial liquid according to the method of the second embodiment, diluting the bacterial liquid until the bacterial concentration is 1.0 multiplied by 10 5 cfu/mL of bacterial liquid.
Rice LJ31 was selected as the test rice. And (3) selecting rice seeds with full shapes and consistent sizes in each treatment group, immersing the seeds in 70% ethanol for sterilization for 15min, and washing with sterile water for three times to remove ethanol residues. Placing the disinfected rice seeds into a 100mL conical flask, adding 50mL of corresponding seed soaking solution, placing the rice seeds into an incubator at 28 +/-0.5 ℃ for soaking for 2 days, accelerating germination for 2 days, placing the seeds with consistent germination into a water culture box, placing the rice seeds into a plant illumination incubator for 28 ℃ normal temperature culture for 14 days, wherein the day/night illumination time is 12h/12h, the light intensity is 12000lx, and the humidity in the plant illumination incubator is set to be 60%.
Experimental setup 2 groups of treatments:
control group: the seed soaking solution is sterile water;
HL-44 treatment group: the seed soaking solution has a thallus concentration of 1.0 × 10 5 cfu/mL Bacillus oceaniensis HL-44And (5) bacterial liquid.
Influence of Bacillus oceanicus HL-44 strain on the growth of rice seedlings:
the planting mode of the rice mainly adopts seedling raising and transplanting, so the overground part length of the rice seedling and the morphological structure of the root system have very important functions on the colonization of the rice after the seedling is transplanted and the growth of the rice plant in the later period. Under hydroponic box culture conditions, rice growth is shown in FIG. 5. Experimental statistics as shown in table 3, the overground part of the experimental group rice was significantly increased (P < 0.01), and the overground part of the treatment group was increased by 40.59% compared with the control group. The root length, fresh weight and dry weight of the rice in the experimental group are improved by 15.59 percent, 18.69 percent and 20.90 percent respectively (P is less than 0.05) compared with those in the control group to different degrees.
TABLE 3 Effect of HL-44 Strain on Rice seedling growth, n =20
The bacillus marinus HL-44 disclosed by the invention has the capability of producing siderophores and IAA and can promote the growth of rice.
Claims (2)
1. The strain for the comprehensive planting and breeding of rice and fisheries is characterized in that the strain for the comprehensive planting and breeding of rice and fisheries is Bacillus marinus (Bacillus oceaniedinis) HL-44 which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC:24990.
2. the use of the strain of claim 1 in the integrated cultivation of rice and fishery.
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| CN117187130A (en) * | 2023-09-05 | 2023-12-08 | 北京市水产技术推广站(北京市鱼病防治站) | Pantoea capable of producing DDP-IV inhibitor and application thereof |
| CN117229966A (en) * | 2023-09-25 | 2023-12-15 | 东北农业大学 | Pseudomonas glycine capable of producing DDP-IV inhibitor and strong phosphate solubilizing and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117187130A (en) * | 2023-09-05 | 2023-12-08 | 北京市水产技术推广站(北京市鱼病防治站) | Pantoea capable of producing DDP-IV inhibitor and application thereof |
| CN117187130B (en) * | 2023-09-05 | 2024-02-20 | 北京市水产技术推广站(北京市鱼病防治站) | Pantoea capable of producing DDP-IV inhibitor and application thereof |
| CN117229966A (en) * | 2023-09-25 | 2023-12-15 | 东北农业大学 | Pseudomonas glycine capable of producing DDP-IV inhibitor and strong phosphate solubilizing and application thereof |
| CN117229966B (en) * | 2023-09-25 | 2024-03-26 | 东北农业大学 | Pseudomonas glycine capable of producing DDP-IV inhibitor and strong phosphate solubilizing and application thereof |
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