CN115895960B - 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a strain for comprehensively planting rice and fish and application thereof, and relates to a strain and application thereof in the field of agriculture. The strain for comprehensively planting and culturing rice and fish is bacillus marinus Bacillus oceanisediminis HL-44 and is preserved in the China general microbiological culture Collection center (CGMCC), and the preservation number is: 24990. the bacillus marinus HL-44 has the capability of producing siderophores and IAA at the same time, can promote the growth of crops, has stronger capability of producing DDP-IV inhibitor, can effectively reduce the blood sugar of fish, is beneficial to the absorption of sugar in the living environment of fish and improves the yield, and the bacillus marinus HL-44 is particularly suitable for the application of comprehensive planting and breeding of rice and fish.
Description
Technical Field
The invention relates to a strain and application thereof in the field of agriculture.
Background
The fish culture in the paddy 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 China government on a large scale. By 2020, the cultivation area of the fish in the paddy field is up to 1439.41 mu, the yield of the aquatic products is up to 85.69 mu ton, and the cultivation area is 41.41% of the total cultivation area of the Chinese rice and fish and the total yield of the aquatic products is 29.41%, so that the cultivation area is very important. In the ecological system for raising fish in paddy field, proper fish raising can not reduce the yield of paddy rice, but can reduce plant diseases and insect pests and control weeds, so that the use of herbicide and pesticide can be reduced, and the yield and quality of raising plants and animals can be even improved. In addition, compared with conventional rice planting or fish culture, the rice in the rice and fish comprehensive planting and culture can utilize the fixation effect of nitrogen element and microorganism in the excretion of cultured animals, reduce the input of exogenous nitrogen, accelerate the recycling of nitrogen element, and further reduce the use of fertilizer. Microorganisms are used as important media of nutrition circulation in comprehensive planting and breeding of rice and fish, and play an important role in nutrition element circulation, formation and maintenance of soil fertility and ecological environment improvement; on the other hand, the fish is colonized in the intestinal canal of the fish, so that the colonization becomes intestinal indigenous flora to form a microecological system which is mutually restricted and interdependent with host and aquatic environment.
Disclosure of Invention
The invention provides a strain for comprehensive planting and breeding of rice and fish and application thereof.
The strain for comprehensively planting and culturing rice and fish is bacillus marinus (Bacillus oceanisediminis) HL-44 and is preserved in the China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC:24990.
the strain bacillus marinus (Bacillus oceanisediminis) HL-44 for rice and fish comprehensive planting and breeding is applied to rice and fish comprehensive planting and breeding.
The bacterial colony characteristics of the bacillus marinus Bacillus oceanisediminisHL-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 after gram staining.
The bacillus marinus HL-44 forms an obvious color-changing ring around a colony after being cultured on a CAS culture medium for a period of time, which indicates that bacillus marinus HL-44 can produce siderophores with high iron chelating capacity, and the Su value of siderophores produced by bacillus marinus HL-44 strain at 37 ℃ is 62%, which indicates that the strain has stronger siderophore producing capacity.
After Salkowski colorimetric solution is added into the bacillus marinus HL-44 in a dropwise manner, the color in a 1mL glass bottle is changed into reddish color at room temperature under the condition of light shading, which indicates that the bacillus marinus HL-44 has the capability of producing auxin and has stronger growth promoting effect on plants. The IAA production capacity of the bacillus marinus HL-44 is quantitatively detected, 86.53 mug IAA is contained in each milliliter of bacillus marinus HL-44 bacterial liquid, and the IAA secretion capacity of the strain is strong.
The bacillus marinus HL-44 is inoculated on a SKM culture medium, after a period of culture, obvious hydrolysis rings are formed around bacterial colonies, the diameter D of the hydrolysis rings of the bacillus marinus HL-44 is 18.72mm, the diameter D of the bacterial colonies is 4.31mm, and the D/D is 4.34; the yield of the DDP-IV inhibitor of the bacillus marinus HL-44 strain reaches 58.39 percent at 37 ℃, which shows that the bacillus marinus HL-44 has stronger ability of producing the DDP-IV inhibitor.
The bacillus sea mud of the invention HL-44 has the capacity of producing siderophores and indole-3-acetic acid (IAA) at the same time. The siderophores can chelate iron ions in the environment, maintain the stability of chloroplasts, and are beneficial to the growth and development of crops, and IAA is used as a plant growth regulating substance, so that the growth of plant roots can be promoted, the length of the roots and the growth quantity of lateral roots can be increased, the absorption and conversion efficiency of soil nutrients in the growth process of crops can be accelerated, and the secretion of ACC deaminase by plants can be stimulated. The bacillus marinus HL-44 promotes the growth of crops and increases the yield of the crops, and is particularly suitable for the application of paddy rice planting. The bacillus marinus HL-44 also has stronger ability of producing DDP-IV inhibitor, and the bacillus marinus HL-44 can effectively reduce blood sugar of fish, is beneficial to absorption of sugar in the living environment of fish and improves yield. Therefore, the bacillus marinus HL-44 is particularly suitable for the comprehensive planting and breeding of rice and fish, and the bacillus marinus HL-44 can promote the growth of rice and fish.
The bacillus marinus Bacillus oceanisediminis HL-44 is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is the North Xili No. 1, 3 of the Korean area of Beijing, and the preservation number is CGMCC:24990, the date of preservation is 2022, 5, 30.
Drawings
FIG. 1 shows the screening result of bacillus marinus Bacillus oceanisediminis HL-44 bacterial siderophores;
FIG. 2 is a graph showing the results of Salkowski colorimetry of Bacillus marinus Bacillus oceanisediminis HL-44 for IAA production;
FIG. 3 shows the lactoproteolytic loop on B.seamud Bacillus oceanisediminis HL-44% nonfat milk powder medium;
FIG. 4 is a phylogenetic tree constructed by Bacillus seamud Bacillus oceanisediminis HL-44;
FIG. 5 shows the results of the test of the bacillus marinus Bacillus oceanisediminis HL-44 hydroponic cassette.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The first embodiment is as follows: the strain for comprehensive rice and fish planting and breeding in the embodiment is bacillus marinus Bacillus oceanisediminis HL-44 and is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC:24990.
1. method for obtaining the Strain of this embodiment
1. 2021, selecting carp population cultivated in paddy field in Dali area of Panjin city of Liaoning, randomly selecting 3 healthy 1 year old carps (150+ -10 g), anaesthetizing with MS-222 anaesthetic (250 mg/L), wiping the surface of the carps with absolute ethyl alcohol, dissecting with sterilized scissors and forceps in an ultra clean bench, taking out the whole intestinal tract of the carps, lightly extruding the content, placing into conical flask containing glass beads and 50mL sterile water, oscillating at 180r/min at room temperature for 30min, performing gradient dilution, and concentrating 10 -3 、10 -4 、10 -5 100 mu L of the culture medium is coated on an LB solid culture medium plate in a gradient way, each gradient is repeated for 3 times, and strains with different shapes are selected for separation after the culture is carried out for 48 hours at 28 ℃.
2. Identification of siderophores
The strain obtained by reactivating the separated and purified strain is transferred to an LB plate for culturing for 24 hours, then a sterilized toothpick is used for picking a single colony to be connected to a chrome azure S (Chromeazurol S, CAS) solid detection medium, and the culture medium is inverted at 37 ℃ for culturing for 2-3 days, so that the size of a color-changing ring around the colony is observed. The isolated and purified strain HL-44 was inoculated on CAS medium and after a period of incubation, a distinct color-changing ring formed around the colonies (as shown in FIG. 1), indicating that strain HL-44 was able to produce siderophores with high iron sequestration capacity.
Further experiments were carried out on the strain HL-44, which shows a distinct color shift circle:
(1) Inoculating activated bacterial strain HL-44 lawn in an iron-limiting SA liquid culture medium, and performing shake culture at 37 ℃ for 48 hours to obtain HL-44 bacterial liquid;
(2) Transferring the HL-44 bacterial liquid to be detected, which grows for 48 hours, into a sterilized 10mL centrifuge tube, and centrifuging at 13000rpm for 15min;
(3) Transferring the supernatant into a test tube treated by concentrated hydrochloric acid, adding a certain amount of freshly prepared CAS detection solution to make the volume ratio of the supernatant to the detection solution be 1:1, fully mixing uniformly, and standing at room temperature for 1h;
(4) Measuring an absorbance value (A) at a wavelength of 630nm, taking double distilled water as a control for zeroing, taking the absorbance value (Ar) at the wavelength of 630nm after the unvaccinated SA limited iron culture medium and the detection liquid which are measured by the same method are mixed as a reference value, and expressing the activity unit of the siderophore by the following formula:
Su≈(Ar-As)/Ar×100；
wherein: su is the siderophore content; ar is the OD value of the SA limited iron culture medium and the supernatant of the detection liquid which are not inoculated; as is the OD of the culture supernatant.
The siderophore activity unit was less than 10, and was generally considered negative, and the mixture of siderophores and the detection liquid had no color change.
The Su value of the siderophore produced by the strain HL-44 at 37 ℃ is 62%, which indicates that the strain has stronger siderophore producing capacity.
3. Determination of indole-3-acetic acid (IAA) production by Strain HL-44
3.1 inoculating the strain HL-44 isolated from the intestinal content of carp into R containing L-tryptophan 2 And (3) in the liquid culture medium A, placing the culture medium A in a shaking table at a constant temperature of 28 ℃ for 180r/min for shake culture for 4d. mu.L of the bacterial liquid was pipetted into a 2mL glass bottle and 500. Mu.L of Salkowski broth was added. A Salkowski colorimetric solution was added simultaneously with 500mg/L IAA as a positive control. The 2mL glass bottle was kept at room temperature in a dark place for 30min, and the color change was observed. If the color turns red, the strain has the function of producing IAA.
As shown in FIG. 2, after the Salkowski colorimetric solution is added dropwise to the strain HL-44, the color in a 1mL glass bottle becomes reddish at room temperature under the dark condition, which indicates that the strain HL-44 has the capability of producing auxin.
3.2 accurately weighing IAA 10mg, dissolving with a small amount of absolute ethyl alcohol, adding distilled water to a volume of 100mL, preparing IAA solution with a concentration of 100 mug/mL as a stock solution, and diluting the stock solution to prepare serial standard solutions with concentrations of 0 (blank), 0.5, 1.0, 5.0, 10.0, 15.0, 20.0 and 25.0 mug/mL respectively as working solutions. 2mL of the working solution are respectively taken and sequentially added into 8 test tubes, salkowski colorimetric solution with the volume of 2 times is added, the temperature is kept for 30min under 40 ℃ in dark condition, and then the absorbance at the wavelength of 530nm is measured. At OD 530 IAA concentration is plotted on the abscissa as the ordinate as the IAA standard curve. In the same culture strip as the primary screenThe IAA-producing ability of the strain HL-44 was quantitatively determined under the test piece. Firstly, measuring the OD value of a bacterial liquid at the wavelength of 600nm by using a spectrophotometry, then taking the bacterial liquid, centrifuging for 10min at the rotating speed of 10000r/min, taking supernatant, adding an equal volume of Salkowski colorimetric liquid, placing for 30min at 40 ℃ in a dark place to develop color, and measuring the OD value at the wavelength of 530 nm. Calculation of OD 600 At a value of 1, IAA was present in the bacterial liquid of strain HL-44 in a unit volume. (dilution of the bacterial liquid at a higher concentration)
The IAA production capacity of the strain HL-44 is quantitatively detected, the strain HL-44 is inoculated by using an LB liquid medium containing L tryptophan, and after the strain is cultured for 24 hours at 30 ℃ and 180rpm, the result is shown in a table 1, 86.53 mu g IAA is produced in each milliliter of bacterial liquid of the strain HL-44, and the IAA secretion capacity of the strain is strong.
TABLE 1 detection results of Strain HL-44IAA
4. Determination of DDP-IV inhibitor produced by Strain HL-44
4.1 bacterial strain HL-44 was inoculated on SKM medium and after 48H of cultivation at 28℃a distinct hydrolytic circle was formed around the colony, the hydrolytic circle diameter D of the bacterial strain HL-44 was 18.72mm, the colony diameter D was 4.31mm and D/D was 4.34 (as shown in FIG. 3), and the bacterial strain HL-44 had the ability to produce DDP-IV inhibitor.
4.2 inoculating the strain HL-44 into a 96-well plate, and accurately dripping 25 mu L, 1.6mmol/L glycyl-prolyl-p-nitroaniline and 25 mu L CFS or CFE into each well; the reaction was carried out at 37℃for 15 minutes, 50. Mu.L of DDP-IV (dipeptidyl peptidase IV) was further added thereto at 0.01U/mL, the reaction was continued at 37℃for 1 hour, and then 100. Mu.L of sodium acetate buffer solution (pH=4.0) was further added thereto to terminate the reaction, and the absorbance of the reaction solution was measured at 405nm using an enzyme-labeled instrument to obtain the inhibition ratio of the strain DDP-IV.
Wherein, the formula of the DDP-IV inhibition rate is:
A sample to be measured :25 mu L sampleProduct +25. Mu.LGly-pro-phy +50. Mu.LDDP-IV +100. Mu.L sodium acetate;
A sample blank : 25. Mu.L of sample +50. Mu.L of LTris-HCl +25. Mu.L of LGly-pro-phy +100. Mu.L of sodium acetate;
A negative control :25 mu LTris-HCl+25 mu LGly-pro-phy+50 mu LDDP-IV+100 mu L sodium acetate;
A negative blank :75 mu LTris-HCl+25 mu LGly-pro-phy+100 mu L sodium acetate.
The productivity of the DDP-IV inhibitor of the strain HL-44 reaches up to 58.39 percent at 37 ℃, which shows that the strain HL-44 has stronger ability of producing the DDP-IV inhibitor.
5. Identification of Strain HL-44
5.1 physiological and biochemical identification: the preserved strain is streaked on a solid LB culture medium plate in three areas, single colony is separated and the form of the strain is described, and gram staining and physiological and biochemical identification are carried out on the strain according to the conventional bacterial System identification handbook.
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 after gram staining. The physiological and biochemical indicators of HL-44 are shown in the following table. According to the description of the physiological characteristics of Bacillus in the Bojie's Manual of bacteria identification, HL-44 has the same characteristics as the physiological biochemistry of the model species Bacillus seamud (Bacillus oceanisediminis), and it is inferred from the physiological biochemistry (shown in Table 2) that the strain HL-44 may be Bacillus seamud (Bacillus oceanisediminis).
TABLE 2 physiological and biochemical results of strain HL-44
5.2 16S rRNA identification: and (3) selecting a bacterial genome DNA extraction kit of Beijing Soxhaust biological technology company, and extracting, separating and purifying the strain DNA.The bacterial universal primer 27F/1492R is adopted for PCR amplification, and the PCR amplification system is a 25 mu L system: 10 Xbuffer 2.5. Mu.L, taq enzyme 0.5. Mu.L, primer 27F 0.5. Mu.L, primer 142R 0.5. Mu.L, DNA template 1. Mu.L, ddH 2 O20. Mu.L. The reaction procedure is set to 95 ℃ for 5min of pre-denaturation; denaturation at 94℃for 50s, annealing at 56℃for 30s, extension at 72℃for 1.5min, cycle times for 30 times, extension at 72℃for 10min again, and preservation at 4 ℃. The PCR amplified product was sent to RuiBiotech company for sequencing. And comparing sequencing results of the strain 16S rRNA through NCBI database, and constructing a phylogenetic tree.
After sequencing the 16S rRNA sequence, BLAST alignment in NCBI found that the 16S rRNA gene sequence of strain HL-44 was 99% similar to Bacillus sea mud (Bacillus oceanisediminis). As shown in FIG. 4, the phylogenetic tree of HL-44 shows that the minimum branch of the strain HL-44 and the bacillus marinus (Bacillus oceanisediminis) (MT 457444.1) is close in evolutionary distance, and the comprehensive physiological and biochemical index identifies the strain HL-44 as the bacillus marinus (Bacillus oceanisediminis).
The second embodiment is as follows: in the embodiment, bacillus marinus Bacillus oceanisediminis HL-44 bacterial liquid is prepared.
100g of soybean sprouts is added with 1000mL of water, boiled for 1h, filtered and supplemented with water to 1L, and the soybean sprouts are subjected to damp-heat sterilization at 121 ℃ and stored for standby, namely 10% of soybean sprout juice by mass fraction.
The bacillus sea mud Bacillus oceanisediminis HL-44 liquid culture medium consists of 1000mL of 10% bean sprout juice, 10.87g of soluble starch, 2.48g of yeast extract, 1.83g of ammonium nitrate and 0.82g of ferric sulfate.
Inoculating Bacillus marinus Bacillus oceanisediminis HL-44 into Bacillus oceanisediminis HL-44 liquid culture medium at 37deg.C and 180r/min for 24 hr to obtain Bacillus oceanisediminis HL-44 bacterial liquid, wherein the viable count of Bacillus oceanisediminis HL-44 reaches 1.98X10 9 cfu/mL。
Example 1 Rice germination experiment
According to the method of the second embodiment, the bacillus marinus Bacillus oceanisediminis HL-44 bacterial liquid is diluted to the bacterial concentration1.0×10 5 cfu/mL of bacterial liquid.
Rice LJ31 was selected as the test rice. The rice seeds with full shapes and consistent sizes are selected from each treatment group respectively, the seeds are immersed in 70% ethanol for sterilization for 15min, and then the rice seeds are washed three times by sterile water to remove ethanol residues. Placing sterilized rice seeds into a 100mL conical flask, adding 50mL of corresponding seed soaking solution, soaking the seeds in a 28+/-0.5 ℃ incubator for 2 days, accelerating germination for 2 days, placing the seeds with consistent germination into a hydroponic box, placing the hydroponic box into a plant illumination incubator for normal-temperature cultivation at 28 ℃ for 14 days, wherein the day/night illumination time length is 12h/12h, the light intensity is 12000lx, and the humidity in the plant illumination incubator is set to 60%.
Experimental setup 2 sets of treatments:
control group: the seed soaking solution is sterile water;
HL-44 treatment group: the seed soaking solution has a cell concentration of 1.0X10 5 cfu/mL bacillus marinus Bacillus oceanisediminis HL-44 bacterial liquid.
Influence of Bacillus sea mud Bacillus oceanisediminis HL-44 strain on rice seedling growth:
the planting mode of the rice mainly comprises seedling raising and transplanting, so that the overground part length of the seedling and the morphological structure of the root system have very important effects on the colonization of the rice after transplanting and the growth of later-stage rice plants. Under the culture condition of the water planting box, the growth condition of the rice is shown in figure 5. Experimental statistics as shown in table 3, the aerial parts of the rice of the experimental group had extremely significant elevation (P < 0.01), and the length of the aerial parts of the treatment group was increased by 40.59% as compared with the control group. The root length, fresh weight and dry weight of the rice in the experimental group are also improved to different degrees compared with those in the control group, and are respectively improved by 15.59%,18.69% and 20.90% (P < 0.05).
Table 3 effect of HL-44 strain on rice seedling growth, n=20
The bacillus sea mud HL-44 has the capability of producing siderophores and IAA, and can promote the growth of rice.
Claims (2)
1. A bacterial strain for comprehensive planting and breeding of rice and fish is characterized in that the bacterial strain for comprehensive planting and breeding of rice and fish is bacillus marinusBacillus oceanisediminis) HL-44, deposited in China general microbiological culture Collection center (CGMCC) with the accession number of CGMCC No:24990.
2. the use of the strain according to claim 1 in rice and fish integrated farming, said use being in a method of non-disease treatment.
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