CN117187138B - High IAA secretion capacity Hulman atlantic bacillus and application thereof - Google Patents
High IAA secretion capacity Hulman atlantic bacillus and application thereof Download PDFInfo
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 15
- 230000028327 secretion Effects 0.000 title claims abstract description 15
- 241000588732 Atlantibacter hermannii Species 0.000 claims abstract description 33
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 26
- 235000009566 rice Nutrition 0.000 claims abstract description 26
- 241000588724 Escherichia coli Species 0.000 claims abstract description 25
- 230000001488 breeding effect Effects 0.000 claims abstract description 6
- 238000009395 breeding Methods 0.000 claims abstract description 5
- 238000009629 microbiological culture Methods 0.000 claims abstract description 5
- 240000007594 Oryza sativa Species 0.000 claims abstract 2
- 241000238557 Decapoda Species 0.000 claims description 11
- 239000000589 Siderophore Substances 0.000 abstract description 12
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 abstract description 7
- 239000011574 phosphorus Substances 0.000 abstract description 7
- 229910052698 phosphorus Inorganic materials 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 6
- 238000000855 fermentation Methods 0.000 abstract description 4
- 230000004151 fermentation Effects 0.000 abstract description 4
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- 238000001514 detection method Methods 0.000 description 8
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 4
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- 229910052742 iron Inorganic materials 0.000 description 4
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- 238000000034 method Methods 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
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- 244000046052 Phaseolus vulgaris Species 0.000 description 3
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
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- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 244000153158 Ammi visnaga Species 0.000 description 2
- 235000010585 Ammi visnaga Nutrition 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229940093761 bile salts Drugs 0.000 description 2
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- JAJWGJBVLPIOOH-IZYKLYLVSA-M sodium taurocholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 JAJWGJBVLPIOOH-IZYKLYLVSA-M 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 229960004799 tryptophan Drugs 0.000 description 2
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- -1 (NH) 4 ) 2 SO 4 2g Chemical compound 0.000 description 1
- 241000593729 Atlantibacter Species 0.000 description 1
- 229930192334 Auxin Natural products 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
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- 241000049865 Ecdyonurus helveticus Species 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010022971 Iron Deficiencies Diseases 0.000 description 1
- 240000002605 Lactobacillus helveticus Species 0.000 description 1
- 235000013967 Lactobacillus helveticus Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
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- 239000002363 auxin Substances 0.000 description 1
- NGPGDYLVALNKEG-UHFFFAOYSA-N azanium;azane;2,3,4-trihydroxy-4-oxobutanoate Chemical compound [NH4+].[NH4+].[O-]C(=O)C(O)C(O)C([O-])=O NGPGDYLVALNKEG-UHFFFAOYSA-N 0.000 description 1
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- 239000003337 fertilizer Substances 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 229940054346 lactobacillus helveticus Drugs 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 235000010958 polyglycerol polyricinoleate Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
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Classifications
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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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A strain of Escherichia coli with high IAA secretion and application thereof relate to a strain and application thereof. The invention relates to a high IAA secretion-amount Bacillus helveticus, which is Bacillus helveticus (Atlantibacter hermannii) JF33 and is preserved in China general microbiological culture Collection center (CGMCC) No.28083. The invention relates to application of the high IAA secretion quantity of the Huffman atlantic bacillus in comprehensive breeding of rice and crab. The fermentation broth of the Escherichia coli (Atlantibacter hermannii) JF33 contains 27.28 mu g IAA, has strong IAA secretion capacity, and has the effect of decomposing inorganic phosphorus and producing siderophores.
Description
Technical Field
The invention relates to a strain and application thereof.
Background
The paddy field is one of the largest constructed wetland systems in China, the paddy field crab cultivation belongs to a novel planting and cultivating mode, and the paddy field crab cultivation and the crab cultivation are implemented, so that the planting industry and the cultivation industry are combined with each other. According to the actual situation analysis of breeding, the eriocheir sinensis can play a role of a protector, so that weeding and deinsectization of a paddy field are facilitated, and the pesticide usage amount for paddy field planting can be reduced; meanwhile, the excrement of the eriocheir sinensis can be used as a fertilizer for rice, and the growth of the rice is promoted.
Besides various organic matters beneficial to rice growth, the eriocheir sinensis excrement can be used for planting the root system of the rice by utilizing the growth promoting strain in the eriocheir sinensis excrement to act on the root system of the rice through PGPR (the beneficial bacteria which survive in the range of plant root circles and have the effect of promoting plant growth or antagonizing pathogenic bacteria are collectively called as the bacterial strain), so that the growth of the rice is beneficial.
Disclosure of Invention
The invention aims to provide a strain of Escherichia coli with high IAA secretion and application thereof.
The invention relates to a high IAA secretion-amount Bacillus helveticus, which is Bacillus helveticus (Atlantibacter hermannii) JF33 and is preserved in China general microbiological culture Collection center (CGMCC) No.28083.
The invention relates to application of the high IAA secretion quantity of the Huffman atlantic bacillus in comprehensive breeding of rice and crab.
The invention adds the high IAA secretion amount of the Bacillus helveticus (Atlantibacter hermannii) JF33 bacterial liquid into the rice field of the rice-crab comprehensive planting and breeding mode to promote the growth of rice and rice-field crabs.
IAA secretion ability is considered to be characteristic of rice growth-promoting bacteria having high colonisation efficiency, and is closely related to whether or not the growth-promoting strain can exert a growth-promoting effect stably in the plant body. The invention provides a high IAA secretion an amount of a bacterium of the genus escherichia coli, will help to improve the comprehensive breeding effect of rice and crab. The fermentation broth of the Escherichia coli (Atlantibacter hermannii) JF33 contains 27.28 mu g IAA, and the IAA secretion capacity is high.
The bacterial colony of the Escherichia coli (Atlantibacter hermannii) JF33 on the LB solid medium is yellow, round, smooth and moist, and has neat edges, obvious bulges at the center, opacity and gram-negative bacteria after gram staining.
The JF33 of escherichia coli (Atlantibacter hermannii) in helamann produced siderophores with high iron chelating ability, and the Su value of siderophores in 37 ℃ environment was 59.2%. Under the iron deficiency condition, the Escherichia coli (Atlantibacter hermannii) JF33 can achieve the antibacterial effect by generating a siderophore with higher affinity and competing pathogenic bacteria for iron elements, so that the immune defensive capability of the rice crabs is improved, and the proportion of pathogenic bacteria in intestinal flora of the rice crabs is reduced.
The Escherichia coli (Atlantibacter hermannii) JF33 can form obvious inorganic phosphorus-dissolved rings on a Meng Jinna inorganic phosphorus culture medium, the diameter D of the inorganic phosphorus-dissolved rings is 13.93mm, the colony diameter D is 9.61mm, and the D/D is 1.45; has a certain function of dissolving inorganic phosphorus and can promote the growth of rice.
The survival rate of the escherichia coli (Atlantibacter hermannii) JF33 of the invention under the condition of 37 ℃ in the environment with the bile salt concentration of 1.5% is still kept to be 87.5%; and strain spore survival rate of the strain of the Escherichia coli (Atlantibacter hermannii) JF33 at the pH of 2.0 can reach 84.0%, and the strain has extremely strong tolerance to the growth environment with lower acidity and higher bile salt concentration. Thus, the Escherichia coli (Atlantibacter hermannii) JF33 can survive and act in the intestinal tract of rice crabs.
The strain height and fresh weight of rice seedlings can be greatly improved by the aid of the Escherichia coli (Atlantibacter hermannii) JF33, root length and stem thickness of the rice seedlings can be slightly improved, and growth of rice plants is facilitated.
The Bacillus helveticus (Atlantibacter hermannii) JF33 is Bacillus helveticus belonging to the genus Bacillus helveticus (Atlantibacter); the culture medium is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is 1 # 3 of North West Lu of the Korean area of Beijing, the preservation number is 28083 of CGMCC, and the preservation date is 2023, 08 and 02.
Drawings
FIG. 1 is a graph of the results of a siderophore screening assay of E.helamabilis (Atlantibacter hermannii) JF33 on CAS solid detection media;
FIG. 2 is a graph of experimental results of the lysis of inorganic phosphorus by E.helamabilis (Atlantibacter hermannii) JF33 on Meng Jinna inorganic phosphorus bacteria medium;
FIG. 3 is a graph showing the results of measuring IAA production capacity of Escherichia coli (Atlantibacter hermannii) JF33 by Salkowski colorimetry;
FIG. 4 is a phylogenetic tree constructed from E.helveticus (Atlantibacter hermannii) JF 33;
FIG. 5 is a graph showing the comparative effect of the strain of E.helman (Pseudomonas glycinae) JF33 on the growth of seedlings of rice in example 1.
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 high IAA secretion amount of the embodiment is provided by the Bacillus helveticus (Atlantibacter hermannii) JF33 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 28083.
Selecting a eriocheir sinensis population cultivated in paddy fields in the south China sea village of the ocean city of the Anshan of Liaoning in 9 months 2022, randomly selecting 30 healthy paddy fields to cultivate eriocheir sinensis, wherein the weight of male crabs is 75+/-5 g; the weight of the female crabs is (60+/-5) g, the collected eriocheir sinensis (hereinafter referred to as paddy crabs) cultivated in the paddy field is brought back to the university of northeast agricultural science and technology academy of animal science by adopting a cold chain transportation mode, 20 healthy paddy crabs are selected, and the healthy paddy crabs are anesthetized by adopting an MS-222 anesthetic (250 mg/L). The surface of the rice field crabs is wiped by absolute ethyl alcohol, and the ultra-clean work is performedDissecting with sterilized scissors and forceps, taking out whole intestinal tract of rice field crab, gently squeezing out the content, placing into conical flask containing glass beads and 50mL sterile water, oscillating at 180r/min for 30min at room temperature, performing gradient dilution, and collecting extract 10 -3 、10 -4 、10 -5 100 mu L of the gradient is coated on an LB solid culture medium plate, each gradient is repeated for 3 times, and the mixture is placed at 28 ℃ for culturing for 24-48 hours. After 48h of culture, strains with different shapes are selected for separation and purification, wherein the strain JF33 is obtained.
Then, the purified strain JF33 is re-activated and 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 (Chromeazrol S, CAS) solid detection medium, the culture is inverted at 37 ℃ for 2-3 days, obvious color-changing rings (shown in figure 1) are observed to form around the colony of the strain JF33, the colony diameter D and the color-changing ring diameter D are measured by adopting a crisscross method, the color-changing ring diameter D of the strain JF33 is 16.36mm, the colony diameter D is 7.95mm, and the D/D is 2.06, so that the strain JF33 has a siderophore with strong Gao Tieao synthesis capacity.
Strain JF33 was further tested:
(1) Inoculating the activated strain JF33 lawn in an iron-limiting SA liquid culture medium, and performing shake culture at 37 ℃ for 48 hours;
(2) Transferring the bacterial suspension to be tested, 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. (when the unit of siderophore activity is less than 10, it is generally considered negative, and the mixture of siderophore and detection liquid has no color change.)
As proved by experiments, the Su value of the siderophore of the strain JF33 at 37 ℃ is 59.2%, which shows that the strain has extremely strong siderophore production capacity.
After purification of strain JF33, the bacterial strain JF33 was inoculated with a sterile toothpick onto a plate of Meng Jinna inorganic phosphorus bacteria medium. Each gradient was repeated 3 times and incubated at 28℃for 24-48 h. The colony diameter D and the diameter D of the inorganic phosphate ring were measured by the crisscross method, and D/D was calculated by forming an apparent inorganic phosphate ring around the colony of the strain JF33 (as shown in FIG. 2). The diameter D of the inorganic phosphate ring of the strain JF33 is 13.93mm, the colony diameter D is 9.61mm, and the D/D is 1.45. The strain JF33 has a certain function of decomposing inorganic phosphorus.
Bacterial strain JF33 colony isolate inoculated 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 suspension 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. After the Salkowski colorimetric solution is dripped into the bacterial suspension of the strain JF33, the color in a 1mL glass bottle is changed into red at room temperature under the dark condition (shown in figure 3); the strain JF33 has the capability of producing auxin and has a certain growth promoting effect on plants.
The IAA10mg is precisely weighed, firstly, a small amount of absolute ethyl alcohol is used for dissolution, then distilled water is added for volume fixation to 100mL, IAA solution with the concentration of 100 mug/mL is prepared as stock solution, and then the stock solution is diluted to prepare serial standard solutions with the 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. Bacterial strain JF33 colony isolate was inoculated into a strain containingR of L-tryptophan 2 And (3) in the liquid culture medium A, placing the liquid culture medium A in a shaking table at a constant temperature of 28 ℃ for 180r/min for shake culture for 4d, then measuring the OD value of the bacterial suspension at the wavelength of 600nm by using a spectrophotometry, taking the bacterial suspension, centrifuging the bacterial suspension at a rotating speed of 10000r/min for 10min, taking the supernatant, adding an equal volume of Salkowski colorimetric solution, placing the supernatant in a dark place at 40 ℃ for 30min for color development, and measuring the OD value at the wavelength of 530 nm. Calculation of OD 600 When the value is 1, the IAA concentration per unit volume of the fermentation liquid (when the concentration of the bacterial liquid is too high, dilution is required). The measurement result shows that the strain JF33 contains 27.28 mug IAA in per milliliter of fermentation broth, and the secretion capacity of the strain JF33IAA is strong.
Strain JF33 stress resistance assay:
acid resistance detection: the strain JF33 was inoculated into LB liquid medium with pH of 2.0, 2.5 and 3.0 according to an inoculum size of 2% (v/v), and the bacterial liquid was sucked and plated for 0, 1, 2 and 3 hours, respectively, and cultured at 37℃for 24 hours, and then the viable count was measured.
And (3) detecting the bile salt resistance: the activated strain JF33 is diluted by a sterile physiological saline solution in a multiple ratio, a proper dilution gradient is selected, and 1mL of the dilution is sucked and placed in a sterilized plate. Plates were then poured with LB solid medium containing 0.3% and 1.0% sodium taurocholate, while MRS solid medium without sodium taurocholate was used as a control group, cultured at 37℃for 48 hours, colony counts were performed, and the survival rate of the strain was calculated.
The results of the test are shown in Table 1, in which the decrease in pH had a smaller effect on the number of spores of the strain JF33, and the survival rate of spores of the strain JF33 was 84.0% when the pH was 2.0. The concentration of bile salts has little influence on the spore number of the strain JF33, and when the concentration of bile salts is increased to 1.5%, the spore survival rate of the strain JF33 is still as high as 87.5%. The strain JF33 has extremely strong tolerance to the growth environment with lower acidity and higher bile salt concentration.
TABLE 1
Physiological and biochemical identification of strain JF 33: the deposited strain JF33 is marked on a solid LB culture medium plate in three areas, single colonies are separated, the morphology of the single colonies is described, and gram staining and physiological and biochemical identification are carried out on the strain according to the general bacterial System identification handbook.
Bacterial colony of the strain JF33 is yellow, round, smooth and moist, has regular edges, has obvious bulges in the center, is opaque and is gram-negative bacteria after gram staining. The physiological and biochemical indexes of the strain JF33 are shown in Table 2. According to the description of physiological characteristics of Enterobacter from the Bojie's Manual of bacteria identification, it is inferred from various physiological and biochemical strains that JF33 may be Escherichia coli (Atlantibacter hermannii).
TABLE 2
16S rRNA identification of Strain JF 33: 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. Sequencing results of strain 16S rRNA were aligned by NCBI database and phylogenetic tree was constructed (as shown in FIG. 4). A BLAST alignment in NCBI revealed that the 16S rRNA gene sequence of strain JF33 was 99% similar to that of E.helamabilis (Atlantibacter hermannii). From phylogenetic tree of strain JF33, it can be seen that the smallest branch of strain JF33 and Healman atlanta (Atlantibacter hermannii) (NR 104940.1) is close in evolutionary distance, and the comprehensive physiological and biochemical index identifies JF33 strain as Healman atlantaBacillus (Atlantibacter hermannii).
Example 1
Inoculating strain of Escherichia coli (Atlantibacter hermannii) JF33 into 50mL LB liquid medium, shake culturing at 28deg.C and 180r/min overnight, diluting the culture solution with sterile water to thallus concentration of 1×10 5 CFU/mL was used as bacterial suspension for further use. 150 seeds of Longjing 31 rice with plump and consistent grains are selected, soaked in warm water at 55 ℃ for 18 hours, and then are grouped and respectively placed in 1 multiplied by 10 5 CFU·mL -1 Soaking in suspension or clear water of Escherichia coli (Atlantibacter hermannii) JF33 for 5 hr; the seeds after soaking are washed 3 times by distilled water, then are evenly placed in a culture dish (diameter is 10 cm) filled with 3 layers of filter paper, and then are respectively sprayed with 5mL of the bacterial suspension (the seeds are soaked by adopting the suspension of the Lactobacillus helveticus JF33 strain before) or clear water (the seeds are soaked by adopting the clear water before) on the surfaces of the seeds. After accelerating germination at 28 ℃, sowing the seedlings in 15kg paddy field soil transplanting pots, taking 30 seedlings from each pot after 28 days, and measuring root length, plant height, stem thickness and fresh weight of the seedlings, wherein the seedlings are repeated for 3 times.
The experimental results are shown in Table 4 and FIG. 5, and the Humulan atlantic E.coli (Atlantibacter hermannii) JF33 has obvious growth promoting effect on rice under potting conditions. As can be seen from Table 4, the root length, plant height, stem thickness and fresh weight of the rice plants subjected to seed soaking treatment by the Escherichia coli (Atlantibacter hermannii) JF33 are respectively increased by 12.34%, 16.37%, 12.34% and 21.67%, so that the Escherichia coli (Atlantibacter hermannii) JF33 can greatly improve the plant height and fresh weight of the rice seedlings, and the root length and stem thickness of the rice seedlings are slightly improved, thereby being very beneficial to the growth of the rice plants.
TABLE 4 Table 4
1000ml of the culture medium of the strain of Escherichia coli (Atlantibacter hermannii) JF33 in this example consists of 40g of sucrose, 7.8g of ammonium tartrate, 4g of monopotassium phosphate, 2.7g of potato starch and the balance of distilled water.
Inoculating the seed solution of the Escherichia coli (Atlantibacter hermannii) JF33 into the culture medium of the Escherichia coli (Atlantibacter hermannii) JF33 bacterial liquid with the inoculum size of 6%, and culturing for 24 hours under the conditions of 70mL/250mL, 32 ℃, 200r/min and pH 7.5 until the viable count of the Escherichia coli (Atlantibacter hermannii) JF33 bacterial liquid reaches 6.72 multiplied by 10 8 CFU/mL, but the number of viable bacteria before the optimization is 1.88×10 8 CFU/mL (non-optimized medium is bean sprout juice medium, bean sprout juice medium: 100mL of bean sprout juice, 10g of sucrose, (NH) 4 ) 2 SO 4 2g，NaCl 0.4g，ZnSO 4 0.08g, distilled water to 1000mL, pH 7, and autoclaved at 115℃for 30 min).
Claims (2)
1. A strain of high IAA secretion of Bacillus helveticus is Bacillus helveticus (Atlantibacter hermannii) JF33, and is preserved in China general microbiological culture Collection center (CGMCC) No.28083.
2. Use of a high IAA secretion amount of escherichia coli as defined in claim 1 for comprehensive breeding of rice crabs.
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