CN116144531A - Phosphate-dissolving bacteria for promoting crop growth and application thereof - Google Patents
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Abstract
The invention discloses phosphate solubilizing bacteria for promoting crop growth and application thereof, and relates to phosphate solubilizing bacteria and application thereof. The invention solves the problem. The phosphate-dissolving bacteria for promoting the growth of crops are Klebsiella migrata Klebsiella michiganensis HLM-8, and are preserved in the China general microbiological culture Collection center (CGMCC) No:25358. the Michigan klebsiella HLM-8 has good capability of dissolving organic phosphorus and inorganic phosphorus at the same time, and promotes the growth of crops. The Michigan klebsiella HLM-8 also has good capacity of producing siderophores, chelates iron ions in the environment, is absorbed and utilized by crops, and promotes the growth of the crops. The Klebsiella mil HLM-8 is particularly suitable for planting rice.
Description
Technical Field
The invention relates to a phosphate solubilizing bacterium and application thereof.
Background
Phosphorus is one of the essential nutrient elements for plant growth and plays a particularly critical role in the growth and development process of plants. Most of the soil in China has the condition of phosphorus deficiency, and phosphorus elements in the soil mainly exist in a indissolvable mineral state. If the phosphorus element in the soil can be activated and fully utilized, the current situation of phosphorus deficiency of the soil can be solved to a certain extent. The chemical fertilizer used at present can well solve the problem, but can bring about the problem of environmental pollution. In order to increase crop yield in actual agricultural planting processes, the yield exceeds 90kg/hm 2 The phosphate fertilizer is applied to the soil, part of the phosphate fertilizer is absorbed and utilized by crops, and most of the phosphate fertilizer is converted into insoluble phosphorus to be applied back to the soil. The application of large amounts of phosphate fertilizers is increasingly serious with soil hardening, soil acidification, and soil impoverishment. The indissolvable phosphorus in the soil is released by a microorganism activation method, so that the growth of plants is promoted, and the preparation of the bacterial strain into the biological fertilizer is a green, efficient and environment-friendly method. Studies show that phosphate-solubilizing bacteria can solubilize phosphorus elements in the environment through two ways of secreting organic acid, protons, siderophores and other substances or extracellular enzymes. Therefore, reasonable utilization of phosphorus-dissolving microorganisms and development of microbial fertilizers have great prospect for improving crop yield, reducing chemical fertilizer use and reducing environmental soil pollution.
At present, a certain research is also carried out on the separation and application of phosphorus-dissolving microorganisms, but a plurality of bacillus and fungi are concentrated, and reports on the phosphorus-dissolving organic phosphorus, inorganic phosphorus and siderophores of the Michigan klebsiella are still the first time.
Disclosure of Invention
The invention provides phosphate solubilizing bacteria for promoting crop growth and application thereof.
The phosphate-dissolving bacteria for promoting the growth of crops are Klebsiella migrata Klebsiella michiganensis HLM-8, and are preserved in the China general microbiological culture Collection center (CGMCC) No:25358.
the application of the Michigan klebsiella Klebsiella michiganensis HLM-8 in promoting the growth of crops.
The application of the Michigan Klebsiella Klebsiella michiganensis HLM-8 in promoting rice growth.
The colony characteristics of the klebsiella michigan HLM-8 on the LB solid medium are as follows: the bacterial colony is milky white and opaque, has no flagella and motility, is a facultative anaerobe, has negative gram staining result, and has short rod-shaped cells, smooth surface and regular edges.
The Michigan klebsiella HLM-8 strain is inoculated on a Meng Jinna organophosphorus medium, obvious organophosphorus dissolving rings are formed around colonies after the bacterial strains are cultured for a period of time, the diameter D of the organophosphorus dissolving rings is 16.68mm, the diameter D of the colonies is 3.93mm, and the D/D is 4.24, which indicates that the Michigan klebsiella HLM-8 has a strong organophosphorus dissolving function; the Michigan klebsiella HLM-8 is inoculated on a Meng Jinna inorganic phosphorus culture medium, obvious inorganic phosphorus dissolving rings are formed around colonies after the colonies are cultured for a period of time, the diameter D of each inorganic phosphorus dissolving ring is 7.24mm, the diameter D of each colony is 3.84mm, and the D/D is 1.89, so that the Michigan klebsiella HLM-8 has a certain function of dissolving inorganic phosphorus.
The Michigan klebsiella HLM-8 strain is inoculated on a CAS culture medium, obvious color-changing rings are formed around colonies after the culture for a period of time, the diameter D of the color-changing rings is 8.38mm, the diameter D of the colonies is 2.26mm, the D/D of the colonies is 3.71, and the Su value of siderophores produced by the Michigan klebsiella HLM-8 at 37 ℃ is calculated to be 51%, which indicates that the Michigan klebsiella HLM-8 can produce siderophores with high iron chelating capacity.
The Michigan klebsiella HLM-8 has good capability of degrading organic phosphorus and inorganic phosphorus, and can convert the phosphorus which is difficult to be absorbed by plants into phosphorus which is easy to be absorbed, thereby achieving the purpose of promoting the growth of crops. The Michigan klebsiella HLM-8 also has the function of forming iron elements by iron ions in the chelate-forming environment of the siderophore, participates in the synthesis of chloroplast protein and chlorophyll, maintains the stability of chloroplasts, and is beneficial to the growth and development of crops. The Klebsiella mil HLM-8 is particularly suitable for planting rice.
The invention discloses michigan klebsiella Klebsiella michiganensis HLM-8 which is michigan klebsiella and is preserved in China general microbiological culture Collection center (CGMCC) with a preservation address of Beichen Xielu No. 1 and No. 3 in the Chaiyang area of Beijing city. The collection number is CGMCC No.25358, and the collection date is 2022, 7 months and 20 days.
Drawings
FIG. 1 shows the results of Klebsiella michiganensis HLM-8 organophosphorus identification;
FIG. 2 shows the results of Klebsiella michiganensis HLM-8 inorganic phosphorus identification;
FIG. 3 shows the identification result of Klebsiella michiganensis HLM-8 siderophores;
FIG. 4 is a phylogenetic tree constructed by Klebsiella michiganensis HLM-8;
FIG. 5 is a graph of Klebsiella michiganensis HLM-8 promoting seed germination results.
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 phosphate-solubilizing bacteria for promoting the growth of crops in the embodiment are Klebsiella migratory Klebsiella michiganensis HLM-8, and are preserved in the China general microbiological culture Collection center (CGMCC) No:25358.
the application of the Klebsiella midwifery Klebsiella michiganensis HLM-8 in the embodiment for promoting the growth of crops, in particular to the application in promoting the growth of rice.
1. Method for obtaining Klebsiella mil Klebsiella michiganensis HLM-8 of the present embodiment
1. 2021, selecting carp population cultivated in Panshan county Hu Guzhen Zhang Gucun of Panjin, liaoning, 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 forceps in an ultra-clean workbench, taking out the whole intestinal tract of the carps, slightly extruding the content, placing into a conical flask containing glass beads and 50mL of sterile water, oscillating at 180r/min at room temperature for 30min, performing gradient dilution, and concentrating 10 -3 、10 -4 、10 -5 100 μl of each gradient was plated on LB solid medium plates, and each gradient was repeated 3 times and incubated at 28deg.C for 24-48h. After 48h of culture, strains with different shapes are selected for separation.
2. Identification of strains with the ability to resolve organophosphorus
The isolated and purified strains were inoculated with sterile toothpicks onto plates of Meng Jinna organophosphorus bacterial medium, respectively. Each gradient was repeated 3 times and incubated at 28℃for 24-48h. And selecting colonies with organophosphorus-dissolving rings, measuring the diameter D of the colonies and the diameter D of the organophosphorus-dissolving rings by a crisscross method, calculating D/D, selecting strains with large ratio for purification, and repeatedly verifying the organophosphorus-dissolving effect.
After the separated and purified strain is cultured on Meng Jinna organophosphorus medium for a period of time, obvious organophosphorus dissolving ring is formed around bacterial colony, as shown in figure 1, the diameter D of the organophosphorus dissolving ring of the strain HLM-8 is 16.68mm, the diameter D of the bacterial colony is 3.93mm, and the D/D is 4.24, which indicates that the strain HLM-8 has a stronger organophosphorus dissolving function.
3. Identification of the ability of the Strain HLM-8 to resolve inorganic phosphorus
Bacterial strain HLM-8 was inoculated with sterile toothpicks onto plates of Meng Jinna inorganic phosphorus bacteria medium, respectively. Each gradient was repeated 3 times and incubated at 28℃for 24-48h. After the bacterial strain HLM-8 is inoculated on a Meng Jinna inorganic phosphorus culture medium for a period of time and cultured, obvious inorganic phosphorus dissolving rings are formed around bacterial colonies, as shown in figure 2, the diameter D of the inorganic phosphorus dissolving rings of the bacterial strain HLM-8 is 7.24mm, the diameter D of the bacterial colony is 3.84mm and the D/D is 1.89, which indicates that the bacterial strain HLM-8 has a certain inorganic phosphorus dissolving function.
4. Identification of Strain HLM-8 siderophores
The isolated and purified strain HLM-8 is re-activated and transferred to an LB plate for culture 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 2-3 days, so that the size of a color-changing ring around the colony is observed.
Bacterial strain HLM-8 was inoculated on CAS medium and after a period of incubation, a distinct color circle formed around the colony, as shown in FIG. 3, with a color circle diameter D of 8.38mm, a colony diameter D of 2.26mm, and a D/D of 3.71, indicating that bacterial strain HLM-8 is capable of producing siderophores with high iron chelating capacity.
Further experiments were performed on strain HLM-8, which exhibited a distinct color circle:
(1) Inoculating activated strain HLM-8 lawn in iron-limiting SA liquid culture medium, and shake culturing at 37deg.C for 48 hr;
(2) Transferring the bacterial liquid to be detected which grows for 48 hours into a sterilized 10mL centrifuge tube, and centrifuging at 13000rpm for 15 minutes;
(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 experimental results show that: the strain HLM-8 had a Su value of 51% for siderophores at 37℃indicating that the strain had a stronger siderophore-producing capacity.
5. Identification of Strain HLM-8
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.
Colony characteristics of strain HLM-8 on LB solid medium were: the bacterial colony is milky white and opaque, has no flagella and motility, is a facultative anaerobe, has negative gram staining result, and has short rod-shaped cells, smooth surface and regular edges. The physiological and biochemical indexes of part of the strain HLM-8 are shown in the following Table 1. According to the description of the physiological characteristics of Bacillus in the Bojv's Manual of bacteria identification, strain HLM-8 has the same characteristics as the physiological biochemistry of the species of Morganella morganii (Klebsiella michiganensis), from which it was deduced that strain HLM-8 might be Morganella morganii (Klebsiella michiganensis).
TABLE 1 Klebsiella michiganensis HLM-8 physiological and Biochemical results
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, primer27F 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 HLM-8 was 99% similar to Klebsiella Michigan (Klebsiella michiganensis). As shown in FIG. 4, the phylogenetic tree of strain HLM-8 shows the smallest branch of strain HLM-8 Klebsiella Mitsubii (Klebsiella michiganensis) (MH 985186.1) and has a relatively short evolutionary distance, and the strain HLM-8 is identified as Klebsiella Mitsubii (Klebsiella michiganensis) by comprehensive physiological and biochemical indexes.
6. Detection of stress resistance of Klebsiella Michigan (Klebsiella michiganensis) HLM-8
Acid resistance detection: inoculating Michigan klebsiella HLM-8 into LB liquid medium with pH of 2.0, 3.0 and 4.0, and starting spore number of 2×10 8 cfu/mL, the bacterial liquid is sucked for 3 hours and coated on a plate, after culturing for 24 hours at 37 ℃, the spore number is measured, and the survival rate is calculated.
The acid resistance of the Michigan klebsiella HLM-8 is shown in Table 2, and the Michigan klebsiella HLM-8 has better acid resistance.
And (3) detecting the bile salt resistance: 1mL of the bacterial liquid (initial spore number: 2X 10) was aspirated by Klebsiella Michigan HLM-8 8 cfu/mL) was placed in a sterilized dish, and then the dish was poured with LB solid medium containing 0.5%, 1.0% and 1.5% sodium taurocholate while culturing for 48 hours at 37 ℃ with MRS solid medium containing no sodium taurocholate as a control group, colony counting was performed, and survival rate of the strain was calculated.
The bile salt resistance of the Michigan klebsiella HLM-8 is shown in Table 2, and the Michigan klebsiella HLM-8 has better bile salt resistance.
TABLE 2 Klebsiella michiganensis HLM-8 acid and bile salt resistance test
The second embodiment is as follows: in this embodiment, a Klebsiella Michigan Klebsiella michiganensis HLM-8 strain solution was 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 Klebsiella midwifery Klebsiella michiganensis HLM-8 liquid medium consists of 1000mL of 10% bean sprout juice, 9.45g of arabinose, 3.21g of soybean peptone, 1.77g of ammonium dihydrogen phosphate and 0.94g of magnesium sulfate.
Inoculating 0.2% of seed solution of Klebsiella Mitsui bacteria HLM-8 into Klebsiella michiganensis HLM-8 bacterial liquid culture medium, culturing at 37deg.C and 180r/min to obtain 24h,Klebsiella michiganensis HLM-8 bacterial liquid with viable count of 1.24X10 9 cfu/mL (while the viable count of the Klebsiella michigan HLM-8 seed solution is 7.18X10) 8 cfu/mL, the Klebsiella Michigan HLM-8 seed solution is inoculated in LB liquid culture medium.
Example 1 Rice germination experiment
According to the method of the second embodiment, the Klebsiella Mitsui Klebsiella michiganensis HLM-8 bacterial solutions are respectively diluted to 2.0X10 of bacterial concentration 4 cfu/mL and 2.0X10 5 cfu/mL is used as bacterial liquid for standby.
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. The sterilized rice seeds are placed in a 100mL conical flask, 50mL of corresponding seed soaking solution is added, the rice seeds are placed in a 28+/-0.5 ℃ incubator for seed soaking for 2 days, after germination is accelerated for 2 days, the seeds with consistent germination are placed in a hydroponic box, and the seeds are placed in a plant illumination incubator for culturing at the normal temperature of 28 ℃ for 14 days.
Setting 3 groups of treatments:
the seed soaking solution of the control CK group is sterile water;
10 4 the seed soaking solution has a thallus concentration of 2.0X10% 4 cfu/mL of Klebsiella Michigan Klebsiella michiganensis HLM-8 bacteria solution;
10 5 the seed soaking solution has a thallus concentration of 2.0X10% 5 cfu/mL of Klebsiella Michigan Klebsiella michiganensis HLM-8 bacterial solution.
Wherein the day/night illumination time length in the plant illumination incubator is 12h/12h, the light intensity is 12000lx, and the humidity in the plant illumination incubator is set to be 60%.
Under the culture condition of the water planting box, the growth condition of each treatment is shown in FIG. 5 and 10 4 Group sum 10 5 The rice growth condition of the group is obviously better than that of the control CK group, and the growth promotion effect is in positive correlation with the concentration of Klebsiella mil Klebsiella michiganensis HLM-8 in the seed soaking solution. As can be seen from Table 3, 10 5 The root length is longer than 10 4 Group and control CK increased by 12.83% and 27.59%, respectively; 10 5 The upper part of the group is 10 4 Group and control CK increased 11.76% and 36.52%, respectively; 10 5 The fresh weight of the product is 10 4 Group and control CK increased by 12.70% and 20.71%, respectively; 10 5 Dry weight of group is 10 4 The group and control CK increased 11.21% and 31.57%, respectively. Further, the method has the advantages that the overground part, root length, fresh weight and dry weight of rice seedlings can be greatly improved by immersing the rice seedlings in the Klebsiella mil Klebsiella michiganensis HLM-8 bacterial liquid, and germination of rice plants is facilitated.
Table 3 effect of klebsiella michigan HLM-8 on rice seedling growth, n=20
The Michigan klebsiella HLM-8 has good capability of degrading organic phosphorus and inorganic phosphorus, can convert the phosphorus which is difficult to be absorbed by plants into phosphorus which is easy to be absorbed, and can further promote the growth of crops in the field practical application. Iron ions in the iron carrier producing environment of the klebsiella michigan HLM-8 can be chelated to form iron elements, participate in synthesis of chloroplast protein and chlorophyll, maintain stability of chloroplasts, and are beneficial to growth and development of crops.
Claims (2)
1. The phosphate solubilizing bacterium for promoting the growth of crops is Klebsiella Mitsui Klebsiella michiganensis HLM-8, and is preserved in China general microbiological culture Collection center (CGMCC) No:25358.
2. use of a strain according to claim 1 for promoting crop growth.
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