CN114836350B - Strain for producing auxin IAA and application thereof - Google Patents
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Abstract
A strain for producing auxin IAA and application thereof relate to a strain and application thereof. The strain for producing the auxin IAA is Pantoea ananatis XBH-10 which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.24197. The application of the strain for producing the auxin IAA in rice planting is disclosed. The application of the strain for producing the auxin IAA in the crab cultivation in the paddy field is disclosed. The Pantoeaannatis XBH-10 bacterial colony is round, milky white and opaque, has a small single bacterial colony and a neat edge, and is gram-negative bacteria after gram staining; has IAA secretion ability and plant growth promoting effect.
Description
Technical Field
The invention relates to a strain and application thereof.
Background
The bacteria not only are decomposers, but also can be used as indirect or direct baits for aquatic organisms, and meanwhile, the mineralization of the putrefactive carbon by the bacteria in the water body plays an important role in the carbon circulation of the water body. A large number of experiments prove that the soil microbial community of the rice field plays an important role in promoting the accumulation, conversion, mineralization and nitrogen release processes of soil organic matters. In view of the important role of microorganisms in the ecological system, the deep research on the quantity and functions of microorganisms in the environment under different culture modes has very important significance for further understanding the role and mechanism of microorganisms in the artificial ecological system.
Disclosure of Invention
The invention provides a strain for producing auxin IAA and application thereof.
The strain for producing the auxin IAA is Pantoea ananatis XBH-10 which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.24197.
The application of the strain for producing the auxin IAA in rice planting is disclosed.
The application of the strain for producing the auxin IAA in the crab cultivation in the paddy field is disclosed.
The Pantoea ananatis XBH-10 bacterial colony is circular, milky white and opaque, has a small single bacterial colony and neat edge, and is gram-negative bacteria after gram staining; has IAA secretion ability and plant growth promoting effect. The diameter D of a phosphate solubilizing ring of Pantoea ananatis XBH-10 on a Monkina organophosphorus solid culture medium is 10.90mm, the diameter D of a bacterial colony is 2.43mm, and the D/D is 4.49; has strong organic phosphorus decomposing capacity.
The survival rate of the Pantoea ananatis XBH-10 in the environment with 0.6 percent of the concentration of bile salt is still kept to be 75 percent at 37 ℃; and Pantoea ananatis XBH-10 has excellent acid resistance.
The Pantoea ananatis XBH-10 is obtained by separating and purifying intestinal contents of Eriocheir sinensis, has negative hemolysis experiment result and high safety, can promote plant growth and increase crop yield, and is suitable for rice planting, especially for use in rice field crab culture.
Pantoea ananatis XBH-10 is Pantoea ananatis belonging to Pantoea (Pantoea); is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of Xilu No. 1 of Beijing republic of China, the microbial research institute of China academy of sciences, the preservation number is CGMCC No.24197, and the preservation date is 2021 year, 12 months and 27 days.
Drawings
FIG. 1 is a graph showing the result of measurement of IAA-producing ability of Pantoea ananatis XBH-10 by Salkowski colorimetry;
FIG. 2 is a graph showing the results of a phosphorus release test of Pantoea ananatis XBH-10 on Monkina organophosphorus solid medium;
FIG. 3 is a phylogenetic tree constructed from Pantoea ananatis XBH-10;
FIG. 4 is a graph showing a comparison of the growth of root buds of rice seeds of each treatment group after culturing at 28 ℃ for 7 days in example 5.
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 of the present invention may be combined with each other without conflict.
The first embodiment is as follows: the strain for producing the auxin IAA is Pantoea ananatis XBH-10 which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.24197.
Selecting Eriocheir sinensis cultured on water surface of Panjin photosynthetic crab Co Ltd in Panjin region of Panjin City of Liaoning province in 8 months 2021, placing intestinal content of Eriocheir sinensis in a conical flask containing glass beads and 50ml PBS, oscillating at room temperature for 30min at a rotation speed of 180r/min, performing gradient dilution, and subjecting 10 to dilution -3 、10 -4 、10 -5 And coating 100 mu L of the gradient on a Monkina organophosphorus culture medium plate, repeating each gradient for 3 times, and culturing at 28 ℃ for 24-48 h. And (5) observing whether a phosphorus-dissolving ring appears, and selecting the strain which generates the transparent ring for separation and purification. Then, the purified strain was inoculated to R containing L-tryptophan 2 Placing the mixture in a 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; and then, 50 mu L of the bacterial suspension is sucked and dropped on a white ceramic plate, 50 mu L of Salkowski colorimetric solution is added, the white ceramic plate is placed at room temperature and in a dark place and is stored for 30min, and color change is observed (if the color changes red, the strain has the function of generating IAA), wherein after the Salkowski colorimetric solution is dropped on the bacterial suspension of the strain XBH-10, the color in the white ceramic plate changes into reddish color at room temperature and in the dark place, as shown in figure 1, the strain XBH-10 has the capability of generating auxin and has strong growth promoting effect on plants.
10mg of IAA is accurately weighed, firstly, a small amount of absolute ethyl alcohol is used for dissolving, then distilled water is added for fixing the volume to 100mL, an IAA solution with the concentration of 100 mu g/mL is prepared to be used as a stock solution, and then the stock solution is diluted to be prepared into a series of standard solutions with the concentrations of 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 and 25.0 mu g/mL respectively to be used as working solution. Respectively adding 2mL of the working solution into the mixture in sequence2 times of Salkowski colorimetric solution is added into 8 test tubes, the test tubes are placed at 40 ℃ in a dark condition for heat preservation for 30min, and then the light absorption value at the wavelength of 530nm is measured. By OD 530 An IAA standard curve is drawn by taking the abscissa and the IAA concentration as the ordinate.
Inoculating the purified strain XBH-10 to R containing L-tryptophan 2 Placing the mixture in a 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; and then absorbing the XBH-10 bacterial suspension, measuring the OD value of the XBH-10 bacterial suspension at the wavelength of 600nm by using a spectrophotometry, then taking the XBH-10 bacterial suspension, centrifuging for 10min at the rotating speed of 10000r/min, taking the supernatant, adding an isovolumetric Salkowski colorimetric solution, standing at 40 ℃ in a dark place for 30min to develop color, and measuring the OD value at the wavelength of 530 nm. Calculating OD 600 The results are given in Table 1 for the IAA concentration per volume of fermentation broth at a value of 1. The quantitative analysis result shows that each milliliter of strain XBH-10 fermentation liquor contains 86.53 mu g of IAA, and the strain XBH-10 has strong IAA secretion capacity.
TABLE 1
The diameter D of a phosphate solubilizing ring of the strain XBH-10 on the Monkina organophosphorus solid medium is 10.90mm, the diameter D of a bacterial colony is 2.43mm, and the D/D is 4.49 (shown in figure 2).
16S rRNA identification of Strain XBH-10: the bacterial genome DNA extraction kit of Beijing Solebao biotechnology company is adopted to extract the 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, 2. Mu.L of dNTPs, 0.5. Mu.L of primer 27F, 0.5. Mu.L of primer 1492R0.5. Mu.L of DNA template, and ddH 2 O18. Mu.L. The PCR amplification reaction program was set as: pre-denaturation at 95 ℃ for 5min; denaturation at 94 ℃ for 50s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1.5min, wherein the cycle times are 30 times; further extension at 72 ℃ for 10min and storage at 4 ℃. The PCR amplification product was sent to RuiBiotech for sequencing, and the 16S rRNA of strain XBH-10 had 99% similarity to the gene sequence of Pantoea ananatis (KY 458567.1). Comparing sequencing results of 16S rRNA of the strain through NCBI database, and constructing system evolutionA tree (as shown in fig. 3). It can be seen from the phylogenetic tree of strain XBH-10 in FIG. 3 that strain XBH-10 has a smaller distance to evolve than the smallest branch of Pantoea ananatis (KY 458567.1).
Physiological and biochemical identification of the strain XBH-10: the strain XBH-10 is streaked on a three-region of a solid LB culture medium plate, a single colony is separated, the morphology of the single colony is described, and gram staining and physiological and biochemical identification are carried out on the strain according to a manual for identifying a common bacteria system. Bacterial colonies of the strain XBH-10 are circular, milky white and opaque, single bacterial colonies are small and have tidy edges, and the bacterial colonies are gram-negative bacteria after gram staining. The physiological and biochemical indexes of the strain XBH-10 are shown in Table 2.
The colony characteristics of the XBH-10 on the LB solid medium are as follows: the bacterial colony is circular, is milk white, and is opaque, and single bacterial colony is little and the edge is comparatively neat, becomes gram-negative bacterium through gram-stain. Part of the physiological and biochemical indicators of XBH-10 are shown in the following table. According to the description of physiological characteristics of Pantoea in Bergey's Manual of identification of bacteria, XBH-10 has the same physiological and biochemical characteristics as Pantoea ananatis model species, and the XCR-1 strain is inferred to be Pantoea ananatis from various physiological and biochemical characteristics.
TABLE 2
The strain XBH-10 has the same characteristics with the physiological and biochemical strain of Pantoea ananatis model species, and the strain XBH-10 is determined to be Pantoea ananatis according to the physiological and biochemical strain and the identification result of 16S rRNA, and is named as Pantoea ananatis XBH-10.
The second embodiment is as follows: in this embodiment, a bacterial solution of Pantoea ananatis XBH-10 was prepared.
Adding 100mL of water into 500g of soybean sprouts, boiling for 1h, filtering, supplementing water, performing moist heat sterilization at 121 ℃, and storing for later use, namely the soybean sprout juice with the mass fraction of 50%.
The Pantoea ananatis XBH-10 strain liquid culture medium consists of 1000mL of 10 mass percent bean sprout juice, 18.87 g of glucose, 2.23g of ammonium chloride and 0.47g of monopotassium phosphate.
Inoculating the seed solution of Pantoea ananatis XBH-10 into the culture medium of Pantoea ananatis XBH-10 with the inoculation amount of 0.2%, culturing at 37 ℃ and 180r/min for 24h, wherein the viable count of the Pantoea ananatis XBH-10 bacterial solution reaches 2.13 multiplied by 10 9 cfu/mL, (and the viable count of the XBH-10 seed liquid is 9.82 multiplied by 10) 8 cfu/mL; the XBH-10 seed liquid is prepared by inoculating Pantoea ananatis XBH-10 into LB liquid culture medium).
Example 1
Acid resistance test of Pantoea ananatis XBH-10:
preparing LB liquid culture medium, adjusting pH to 2.0, 3.0, 4.0 with 0.1mol/L hydrochloric acid, and inoculating 2% of Pantoea ananatis XBH-10 bacterial liquid (viable count of bacterial liquid is 2.13 × 10) 9 cfu/mL) were inoculated in the above liquid medium, respectively, and a medium without hydrochloric acid was set as a control. After incubation at 37 ℃ for 3h, viable bacteria were counted. The appropriate dilutions were selected, 100 μ L of liquid was pipetted to coat the plates, two plates per dilution, and blown dry in a clean bench.
Survival = viable count of acid conditioned medium/viable count of non-hydrochloric acid medium × 100%.
The results of the acid resistance test of Pantoea ananatis XBH-10 are shown in Table 3. It is shown that Pantoea ananatis XBH-10 has excellent acid resistance.
TABLE 3
Example 2
Pantoea ananatis XBH-10 bile salt resistance experiment:
the bacterial liquid of Pantoea ananatis XBH-10 (viable count of the bacterial liquid is 2.13X 10) 9 cfu/mL) were inoculated in liquid media of different concentrations of bile salts 0.2%, 0.4% and 0.6% at an inoculum size of 2%, and a medium without bile salts was set as a control, and cultured at 37 ℃ for 4 hours, after which viable cells were counted. Two plates per dilution were blown dry in a clean bench.
Survival = viable count of cholate medium/viable count of cholate-free medium × 100%.
The results of the Pantoea ananatis XBH-10 bile salt resistance experiment are shown in Table 4. The Pantoea ananatis XBH-10 has excellent bile salt resistance.
TABLE 4
Example 3
Activated Pantoea ananatis XBH-10 was spotted on a TSA plate, 4 inoculum spots were spotted on one plate, and the pathogenic fungus F.moniliforme was spotted at the center of the 4 inoculum spots, cultured at 30 ℃ for 1 week and the results were observed. If the antifungal agent has antagonism on pathogenic fungi, the pathogenic fungi can not cover the growth of bacteria, namely, an obvious inhibition zone can be formed.
Pantoea ananatis XBH-10 does not form a zone of inhibition and has no antibacterial activity.
Example 4
Pantoea ananatis XBH-10 was streaked on each fresh blood plate and observed after incubation at 30 ℃ for 48h. Hemolysis is classified into alpha hemolysis (incomplete hemolysis, producing a grass green hemolytic cycle), beta hemolysis (complete hemolysis, a well-defined, colorless and transparent hemolytic cycle) and non-hemolysis.
The hemolytic experiment result of Pantoea ananatis XBH-10 is negative, which can preliminarily show that Pantoea ananatis XBH-10 has no risk of human and animal diseases.
Example 5
The Pantoea ananatis XBH-10 is inoculated into 50mL LB liquid culture medium, shaking culture is carried out at 28 ℃ and 180r/min overnight, and the culture solution is diluted by sterile water until the concentration of the bacterial body is 10 8 CFU/mL, as bacterial suspension, for use.
And (3) selecting complete, full and consistent Longjing 31 rice seeds, placing the seeds in a small beaker, adding pure water to immerse the seeds, and keeping the seeds for 30min. Then removing blighted grains, sterilizing the surface of the blighted grains by using 75 percent ethanol for 5min, then soaking the blighted grains in 1 percent NaClO for 5min, and cleaning the blighted grains for 3 to 5 times by using sterile water; then placing the rice seeds into a beaker, adding sterile water to immerse the rice seeds, and accelerating germination for 1d at the temperature of 28 ℃.
Pregermination of 1dRice seeds are soaked in Pantoea ananatis XBH-10 bacterial suspension for 1h and then placed in sterile culture dishes with wet filter paper, and 20 seeds are placed in each culture dish. Culturing in an artificial climate incubator at 30 ℃, relative humidity of 70% and light-dark ratio of 1698 h, and keeping the filter paper moist by adding water. The treatment settings were divided into 2 groups, wherein 10mL of distilled water was injected into the CK group; the treatment group was injected with 10mL of two different concentrations of Pantoea ananatis XBH-10 bacterial suspensions (diluted to 10% concentration) -4 And 10 -5 ) Each treatment was repeated 3 times. Culturing at 28 deg.C for 7 days in a constant temperature incubator, and measuring root length and bud length.
As shown in Table 5, the concentration of the Pantoea ananatis XBH-10 bacterial suspension is increased, the bud length, the root length and the number of divided roots of the rice seeds are increased to different degrees after the rice seeds germinate for 7 days, wherein the concentration of the Pantoea ananatis XBH-10 bacterial suspension is 10 -4 The group had significantly increased shoot and root length (P) compared to the control group>0.05 Thereby proving that Pantoea ananatis XBH-10 has stronger growth promotion effect on the germination of rice seeds.
TABLE 5
Claims (2)
1. A bacterial strain for producing auxin IAA, which is Pantoea ananatis (A)Pantoea ananatis) XBH-10 preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.24197.
2. Use of the auxin IAA-producing strain of claim 1 in rice planting.
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