CN114317371A - Biocontrol growth-promoting bacterium for rice blast and application thereof - Google Patents
Biocontrol growth-promoting bacterium for rice blast and application thereof Download PDFInfo
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Abstract
A rice blast biocontrol growth-promoting bacterium and application thereof relate to biocontrol growth-promoting microorganisms and application thereof. The rice blast biocontrol growth-promoting bacteria are Bacillus belgii (Bacillus velezensis)499G3, and are preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 23719. The application of the rice blast biocontrol growth-promoting bacterium Bacillus velezensis 499G3 in rice blast biocontrol. After 30 days of spraying, the yield of overground part of rice can be increased by 39.61 percent, the chlorophyll content of the rice is increased by 20.24 percent, the IAA content of the rice is increased by 6.82 percent, and the total nitrogen phosphorus potassium content of the rice is increased by 10.96 percent, 17.01 percent and 8.17 percent respectively by Bacillus velezensis 499G 3.
Description
Technical Field
The invention relates to a biocontrol growth-promoting microorganism and application thereof.
Background
Rice is the main grain crop in China and also the most important economic crop. Rice, especially rice flower-fragrant varieties in northeast China, is easily damaged by rice blast in the growth process, and the crop yield is seriously influenced.
At present, the prevention and the treatment of rice blast mainly adopt pesticides as main materials, and the growth promotion of rice mainly adopts the application of chemical fertilizers, but the utilization of microbial agents is slightly deficient, and the development of corresponding biocontrol growth-promoting microbial agents can cater to the green, environment-friendly and sustainable agroecological concept.
Disclosure of Invention
In order to practice a green, environment-friendly and sustainable agricultural ecological concept, the invention provides a rice blast biocontrol growth-promoting bacterium and application thereof.
The rice blast biocontrol growth-promoting bacteria are Bacillus belgii (Bacillus velezensis)499G3, and are preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 23719.
The application of the rice blast biocontrol growth-promoting bacterium Bacillus velezensis 499G3 in rice blast biocontrol.
The application of the rice blast biocontrol growth-promoting bacterium Bacillus velezensis 499G3 in promoting the growth of rice.
Further, the application of Bacillus velezensis 499G3 in promoting the production of chlorophyll and IAA in rice and increasing the total nitrogen phosphorus potassium content in rice is provided.
The colony color of Bacillus velezensis 499G3 is light yellow, the surface of the colony has folds (as shown in figure 1), the growth is rapid, the colony can grow after being inoculated into LB culture medium for 12 hours, and the antagonistic effect is generated on rice blast.
By adopting a bacterial liquid spraying mode, the overground part of the rice can be promoted to gain 39.61% 30 days after the Bacillus subtilis 499G3 is sprayed, the chlorophyll content in the rice is increased by 20.24%, the IAA content in the rice is increased by 6.82%, and the total nitrogen phosphorus potassium content in the rice is increased by 10.96%, 17.01% and 8.17% respectively.
Bacillus belgii (Bacillus velezensis)499G3 is Bacillus belgii, belongs to Bacillus (Bacillus), is preserved in China general microbiological culture Collection center, has a preservation address of No. 3 of Sai Luo No. 1 of Beijing Korean area, and a preservation number of CGMCC No.23719, and has a preservation date of 2021, 11 months and 04 days.
Drawings
FIG. 1 is a morphological diagram of Bacillus subtilis 499G3 on LB plate;
FIG. 2 is a phylogenetic tree of Bacillus velezensis 499G3 molecular characterization;
FIG. 3 is a graph showing antagonistic effects against rice blast by Bacillus velezensis 499G3 (left panel is an experimental group of Bacillus velezensis; right panel is a control group);
FIG. 4 is a graph showing a comparison of rice growth after spraying with Bacillus velezensis 499G 3;
FIG. 5 is a graph showing a comparison of chlorophyll and IAA contents of rice after spraying with Bacillus velezensis 499G 3;
FIG. 6 is a graph showing the comparison of total nitrogen, phosphorus and potassium contents of rice after spraying with Bacillus velezensis 499G 3.
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.
Example 1
Selecting roots and leaves of wild rice potted in the northeast geographical and agricultural ecological research institute of Chinese academy of sciences of Changchun, Jilin province in 2021 month, washing with distilled water, placing into a sterilization culture dish, adding alcohol, soaking for 30s, continuously overturning with tweezers during the period to sterilize the surface, then rinsing with distilled water to remove the alcohol on the surface, placing into a mortar for grinding, adding distilled water with the same equal amount into grinding fluid for dilution, and uniformly coating the water diluted grinding fluid on a solid LB culture medium. The growth of colonies was observed after 1 day of culture. Respectively selecting colonies with different shapes such as rapid growth, morphology, color and the like on the solid LB culture medium for pure culture until the colonies are single, and screening out the strains. The strains and rice blast pathogenic bacteria are subjected to antagonism experiments one by one, bacteria with remarkable antagonism effects are screened out to be subjected to pot growth promotion experiments, and then corresponding strains (499G3) with the highest rice growth amount are selected from the strains.
Wherein, LB culture medium: 10g of NaCl, 10g of tryptone, 5g of yeast powder, 18g of agar and 1L of distilled water, and sterilizing for 30min by high-pressure steam at 121 ℃. About 20ml of medium was poured per 9cm X9 cm plate.
The strain 499G3 was subjected to molecular characterization, the genome of the strain 499G3 was extracted using a genome extraction Kit (FastDNA Spin Kit for Soil), and the V3-V4 region of the 16S rRNA gene was selected for detection (sequences of primer pairs were 341F: 5'-ACTCCTACGGGAGGCAGCA-3' and 785R: 5 '-GGACTACHVGGGTWTCTAAT-3'). The reaction system was 25. mu.L, Premix version 2.012.5. mu.L, 27F (10nM) 1. mu.L, 1492R (10nM) 1. mu. L, DNA template 1. mu.L, and made up to 25. mu.L with sterile water. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 50s, and extension at 72 ℃ for 90s for 25 cycles; final extension at 72 ℃ for 5 min. After PCR amplification, the product was checked by 1.5% agarose gel electrophoresis and had a distinct characteristic band. The PCR amplification product was sent to Biotechnology engineering (Shanghai) Inc. for sequencing, and the resulting sequence was uploaded to NCBI. Homology alignment with sequences in the gene bank was performed by Blast analysis and phylogenetic evolutionary trees were constructed using Neihbor-joining in MEGA5.1 software (as shown in fig. 2).
The strain 499G3 has the highest homology with Bacillus subtilis (Bacillus velezensis) up to 99%, and is determined by identification combined with morphological characteristics, growth conditions and the like of the thallus, and the strain 499G3 is the Bacillus subtilis (Bacillus velezensis) and is named as Bacillus subtilis (Bacillus velezensis)499G 3.
The rice blast biocontrol growth-promoting bacteria is Bacillus licheniformis (Bacillus velezensis)499G3, and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 23719.
Bacillus belgii (Bacillus velezensis)499G3 can be grown on solid LB medium.
Example 2
Placing the rice blast pathogenic bacteria in the form of fungus cakes in the center of a culture dish filled with a PDA culture medium, inoculating Bacillus velezensis 499G3 at intervals of 20mm around the rice blast pathogenic bacteria for the confrontation experiment; and (4) selecting a culture dish which is not inoculated with bacteria and only inoculated with rice blast pathogenic bacteria as a control, and observing the bacteriostatic effect after culturing for 7 days. Bacillus licheniformis (Bacillus velezensis)499G3 experimental group Magnaporthe grisea does not grow, as shown in the left panel of FIG. 3; the control group of Magnaporthe grisea colonies grew and covered the surface of the medium as shown in the right panel of FIG. 3. It was confirmed that Bacillus subtilis 499G3 has an excellent rice blast-inhibiting effect. Wherein, PDA culture medium: 200g of potatoes, 20g of glucose, 1L of distilled water and 18g of agar powder.
Example 3
Transplanting rice seedlings with consistent growth and development for 15 days into a flowerpot, spraying Bacillus subtilis 499G3 bacterial liquid on 3 plants per pot, spraying secondary bacterial liquid on 20 th day of transplantation, and identifying growth indexes and physiological and biochemical indexes of the plants on 30 th day of transplantation.
The preparation method and the spraying mode of the microbial inoculum are as follows: culturing Bacillus subtilis 499G3 in LB liquid shake flask for 1 day, centrifuging to remove supernatant, and diluting with distilled water to give final concentrateDegree of 106cfu/ml, the bacterial liquid is uniformly sprayed on the rice leaves by a spray can, and 20ml of the bacterial liquid is sprayed on each pot of rice. The control group was not sprayed with Bacillus subtilis 499G 3.
The rice was taken out of the pot and the length of the above-ground portion was measured with a ruler.
The total nitrogen and the total phosphorus of the plants are measured by a continuous flow analyzer, and the total potassium is measured by inductively coupled plasma emission spectrum.
The chlorophyll is determined by ethanol extraction, removing coarse vein of each leaf, cutting into pieces, weighing 0.2g of the leaves respectively, extracting chlorophyll by 95% ethanol grinding method and soaking method, determining absorbances at 649nm and 665nm respectively with 95% ethanol as reference, calculating chlorophyll concentration, and converting into fresh weight chlorophyll content (mg/g FW).
Ca＝13.95D665－6.88D649；
Cb＝24.96D649－7.32D665；
CT＝Ca+Cb
In the formula, Ca: the content of chlorophyll a; cb: the content of chlorophyll b; CT: the total chlorophyll content.
The plant IAA is determined by a chelation method:
(1) IAA standard solution: accurately weighing 10mg of IAA, dissolving with a small amount of ethanol, and diluting to 100mL (the concentration is 100 mu g/mL) with distilled water as stock solution; then, the stock solution was used to prepare 0 (blank), 0.5. mu.g/mL, 1.0. mu.g/mL, 5.0. mu.g/mL, 10.0. mu.g/mL, 15.0. mu.g/mL, 20.0. mu.g/mL, 25.0. mu.g/mL series of standard solutions as working solutions (ready-to-use).
(2) Reagent A: 15mL of FeCl solution containing 0.5mol/L, concentrated H2300mL of SO (density 1.84g/mL) and 500mL of distilled water were mixed and shaken before use, and stored in the dark, and 4mL of this reagent was added to 1mL of the sample solution.
(3) And (3) reagent B: contains 0.5mol/L FeCl310mL of the solution and 500mL of 35% perchloric acid were mixed and shaken before use, and the mixture was stored in the dark, and 2mL of the reagent was added to 1mL of the sample solution. Reagent A and reagent B may be either one of them (reagent B is more sensitive than reagent A).
(4)0.1mol/L NaOH solution.
(5) Methanol.
Taking 8 clean large test tubes (No. 0-7), sequentially adding 2mL of IAA series concentration working solution, respectively adding 4mL of reagent B (or 8mL of reagent A), and carrying out dark heat preservation in an incubator at 40 ℃ for 30min (to accelerate color reaction); a standard curve was drawn by colorimetry at 530nm with absorbance values on the ordinate and IAA concentrations (μ g/mL) on the abscissa.
Air-drying experimental rice leaves and grinding into fine powder; weighing 0.5g of powder, placing into a beaker, adding 40mL of 0.1mol/L NaOH solution, boiling in a water bath at 100 deg.C for 15min (covering to prevent water evaporation); taking out the beaker, adding 25mL of 0.1mol/L NaOH solution into the beaker, fully shaking the beaker, fixing the volume to 100mL by using methanol, standing the beaker for 30min, taking the supernatant, and centrifuging the supernatant for 30min to obtain the IAA extract of the experimental sample.
Taking 2 clean large test tubes, sequentially adding 2mL of the IAA extract solution of the experimental sample and 4mL of the reagent B (or 8mL of the reagent A), developing in an incubator at 40 ℃ for 30min, carrying out color comparison at 530nm (zero adjustment is carried out by using '0' of a standard curve), and recording the absorbance value.
Sample indole acetic acid content (. mu.g/g) ═ CVT/W
C: the IAA concentration, mu g/mL, found on the standard curve;
VT: the total volume of the extracting solution is mL;
w: sample weight, g.
The experimental results are as follows: compared with a control group, the overground part of the rice in the experimental group is increased by 39.61%, the chlorophyll content in the rice is increased by 20.24%, the IAA content in the rice is increased by 6.82%, and the total nitrogen, phosphorus and potassium content in the rice is increased by 10.96%, 17.01% and 8.17% respectively.
Claims (3)
1. A rice blast biocontrol growth-promoting bacterium is Bacillus belgii (Bacillus velezensis)499G3, which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 23719.
2. Use of the rice blast biocontrol growth-promoting bacterium of claim 1 in biocontrol of rice blast.
3. Use of the rice blast biocontrol growth-promoting bacterium of claim 1 for promoting the growth of rice.
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