CN117247869A - Paenibacillus with plant growth regulating activity and preparation and application thereof - Google Patents
Paenibacillus with plant growth regulating activity and preparation and application thereof Download PDFInfo
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- 241000179039 Paenibacillus Species 0.000 title claims abstract description 62
- 230000008635 plant growth Effects 0.000 title claims abstract description 16
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 238000000855 fermentation Methods 0.000 claims abstract description 48
- 230000004151 fermentation Effects 0.000 claims abstract description 48
- 239000002024 ethyl acetate extract Substances 0.000 claims abstract description 30
- 244000025254 Cannabis sativa Species 0.000 claims abstract description 10
- 238000004321 preservation Methods 0.000 claims abstract description 10
- 241000193397 Paenibacillus pabuli Species 0.000 claims abstract description 8
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 7
- 244000005700 microbiome Species 0.000 claims abstract description 6
- 230000001737 promoting effect Effects 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims description 23
- 239000007787 solid Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 230000012010 growth Effects 0.000 claims description 10
- 238000009630 liquid culture Methods 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 244000068988 Glycine max Species 0.000 claims description 6
- 235000010469 Glycine max Nutrition 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000005648 plant growth regulator Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000012137 tryptone Substances 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 241000234642 Festuca Species 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 230000002906 microbiologic effect Effects 0.000 claims description 3
- 238000007598 dipping method Methods 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 15
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- 241001465754 Metazoa Species 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 3
- 241000193830 Bacillus <bacterium> Species 0.000 abstract description 2
- 231100000989 no adverse effect Toxicity 0.000 abstract description 2
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- 235000010633 broth Nutrition 0.000 description 25
- 230000000694 effects Effects 0.000 description 12
- 241000501953 Aconitum pendulum Species 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 235000003826 Artemisia Nutrition 0.000 description 5
- 235000003261 Artemisia vulgaris Nutrition 0.000 description 5
- 244000030166 artemisia Species 0.000 description 5
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- 238000011282 treatment Methods 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 230000000249 desinfective effect Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
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- 241001361205 Dactylicapnos Species 0.000 description 2
- 244000117040 Dactylicapnos scandens Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000234643 Festuca arundinacea Species 0.000 description 2
- 241000825107 Hierochloe Species 0.000 description 2
- 235000014676 Phragmites communis Nutrition 0.000 description 2
- 244000273256 Phragmites communis Species 0.000 description 2
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- 230000018109 developmental process Effects 0.000 description 2
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- 230000035784 germination Effects 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
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- 241000227129 Aconitum Species 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- 101000993347 Gallus gallus Ciliary neurotrophic factor Proteins 0.000 description 1
- 235000015466 Hierochloe odorata Nutrition 0.000 description 1
- 241001646834 Mesona Species 0.000 description 1
- 240000008916 Oenothera biennis Species 0.000 description 1
- 235000004496 Oenothera biennis Nutrition 0.000 description 1
- 241001617298 Pedicularis kansuensis Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 240000003428 Tinospora crispa Species 0.000 description 1
- 244000137773 Viola philippica Species 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004790 biotic stress Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 229940045761 evening primrose extract Drugs 0.000 description 1
- 235000008524 evening primrose extract Nutrition 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 101150052159 maeA gene Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 230000005082 stem growth Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/25—Paenibacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P13/00—Herbicides; Algicides
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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Abstract
The invention relates to a paenibacillus with plant growth regulating activity, which is feed paenibacillus B03%Paenibacillus pabuli) It is prepared from herba ViolaeStipa purpurea) Is obtained by leaf separation; the feed paenibacillus B03%Paenibacillus pabuli) Tube for preserving Chinese microorganism strainThe preservation number of the common microorganism center of the Committee is CGMCC No.21386. Meanwhile, the invention also discloses a preparation method and application of the bacillus. The paenibacillus is derived from plant leaves, and has no adverse effect on the environment, plants, people, animals and other organisms; the fermentation liquor of the obtained strain and the ethyl acetate extract thereof act on pasture and toxic grass, can be used for recovering and improving the grassland productivity, increasing the yield of the pasture, improving the quality and inhibiting the spread of toxic weeds, thereby promoting the development of animal husbandry.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to paenibacillus with plant growth regulating activity and preparation and application thereof.
Background
Plant growth-promoting bacteria (PGPB) are a class of beneficial bacteria that mostly survive in rhizosphere soil or Plant tissue, and are capable of promoting Plant growth, and which protect plants from biotic and abiotic stresses. In recent years, based on the important value of PGPB in the fields of agriculture, environmental protection and the like, PGPB has become a hot spot for research of students at home and abroad. We have found that Pseudomonas speciesPseudomonas) BacillusBacillus) PaenibacillusPaenibacillus) Agrobacterium(s) aAgrobacterium) Flavobacterium (Fr.) KummerFlavobacterium) And the endophytic bacteria of more than 20 have the potential of preventing diseases and promoting growth.
In recent years, research shows that the grassland ecosystem of China is degenerated to different degrees due to various factors such as natural environment, artificial overdependence and the like. With the aggravation of the degradation degree of the grasslands, the soil nutrients are seriously lost, the dominant population structure of the plants is changed, the high-quality grasses are reduced, the proportion of the toxic grasses is greatly increased, the ecological balance of the grasslands is seriously threatened, and meanwhile, the development of animal husbandry is restricted. Thus, the management of degraded grasslands has become an important point of current research. The microorganism is used as an important biological resource, can promote the material circulation between plants and soil through the self life activities, further regulate and control the composition of a grassland ecological system and the stability of a structure, and plays an important role in recovering grassland ecology. In addition, it is widely used as a biotechnology tool to replace traditional fertilizers, and has been paid attention to by researchers because of its great potential for regulating plant growth.
Disclosure of Invention
The invention aims to provide paenibacillus with plant growth regulating activity.
Another technical problem to be solved by the present invention is to provide the preparation of Paenibacillus having plant growth regulating activity.
The third technical problem to be solved by the invention is to provide the application of the paenibacillus with plant growth regulating activity.
In order to solve the above problems, the invention provides a paenibacillus with plant growth regulating activity, which is characterized in that: the strain is feed paenibacillus B03%Paenibacilluspabuli) It is prepared from herba ViolaeStipapurpurea) Is obtained by leaf separation; the feed paenibacillus B03%Paenibacilluspabuli) The preservation number of the general microbiological center of China Committee for culture Collection of microorganisms is CGMCC No.21386 (preservation Unit address: beijing, chaoyang area, north Chen West Lu No. 1, 3 national academy of sciences of China, microbiology institute; preservation date: 12 months 17 days 2020).
Paenibacillus B03 fermentation broth prepared from Paenibacillus as described above.
The preparation method of the paenibacillus B03 fermentation broth is characterized by comprising the following steps: paenibacillus B03 stored on slant solid medium was dipped in inoculating loop and inoculated on TSAStreaking a solid culture medium, culturing at 28+/-2 ℃ in a dark place, and taking the culture medium as a first-stage strain after a macroscopic colony grows out; inoculating the activated primary strain to TSA liquid culture medium at 1% inoculum size, placing on shaking table at 35deg.C and rotation speed of 180 rpm for light-shielding culture, stopping shaking culture after the culture solution becomes turbid to obtain 1×10 strain 10 cfu·mL −1 Is prepared from the strain fermentation broth.
The TSA solid culture medium is prepared by adding 15 g tryptone, 5 g soybean peptone, 5 g sodium chloride and 18 g agar into 1000 mL deionized water, uniformly mixing, adjusting pH to be 7.1-7.5, and sterilizing at 121 ℃ for 20 min.
The TSA liquid culture medium is prepared by adding 15 g tryptone, 5 g soybean peptone and 5 g sodium chloride into 1000 mL deionized water, uniformly mixing, adjusting pH to be 7.1-7.5, and sterilizing at 121 ℃ for 20 min.
The macroscopic Colonies (CFU) were colonies with average diameter above 1 mm (containing 1 mm).
The application of the paenibacillus B03 fermentation broth is characterized in that: the Paenibacillus B03 is treatedPaenibacilluspabuli) The inactivated fermentation broth is diluted by 100 times by water to prepare 100 times of dilution liquid which is used as a plant growth regulator.
An ethyl acetate extract prepared from the Paenibacillus B03 fermentation broth as described above.
The preparation method of the ethyl acetate extract is characterized by comprising the following steps of: extracting Paenibacillus B03 fermentation broth with ethyl acetate 3 times of the volume of the fermentation broth, and distilling the obtained extract under reduced pressure to obtain an ethyl acetate extract.
The use of an ethyl acetate extract as described above, characterized in that: the ethyl acetate extract is used for inhibiting Artemisia malabarica of Phragmites communisPediculariskansuensis) Iron rod hammerAconitum pendulum) Or used as plant growth regulator to promote the growth of grass purple flower festuca arundinaceaStipapurpurea) Is a growth of (a).
Compared with the prior art, the invention has the following advantages:
1. the paenibacillus is derived from plant leaves, has no adverse effect on the environment and all organisms such as plants, people, animals and the like, has extremely important significance, and has the advantages of low production cost, simple production process, no environmental pollution and suitability for popularization and application.
2. The invention separates the strain from the plant leaf, and the fermentation liquor of the strain and the ethyl acetate extract thereof act on pasture and toxic grass, which can be used for recovering and improving the productivity of the pasture, increasing the yield of the pasture, improving the quality and inhibiting the spread of toxic weeds, thereby promoting the development of animal husbandry.
3. Experiments show that the feed paenibacillus is preparedPaenibacilluspabuli) The fermentation liquor and the ethyl acetate extract of the B03 obviously promote the growth of the grass purple flower festuca arundinacea, so that the root system of the grass purple flower festuca arundinacea is developed, the biomass of overground parts is increased, and the stress resistance activity of the grass is enhanced.
4. Experiments show that the feed paenibacillus is preparedPaenibacilluspabuli) B03 fermentation liquor and ethyl acetate extract obviously inhibit aconitum pendulum of poison grassAconitum pendulum) He Gansu Ma Hao (radix et rhizoma Hao)Pediculariskansuensis) Root, stem growth.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the drawings.
FIG. 1 shows the effect of Paenibacillus B03 fermentation broth of the present invention on fresh weight, root length and stem length of seedlings of Dactylicapnos, mr. malaiana and Oenothera biennis.
FIG. 2 shows the effect of ethyl acetate extract of Paenibacillus B03 broth of the present invention on dry weight, stem length and root length of Dactylicapnos scandens, hairyveromyces fragrans and Gansu Artemisia malaensis seedlings.
FIG. 3 shows the effect of ethyl acetate extract of Paenibacillus B03 broth of the present invention on Dactylicapnos, tinospora cordifolia and Gansu maea seedlings.
Detailed Description
Paenibacillus with plant growth regulating activity as feed bacillus Paenibacillus B03%Paenibacilluspabuli) It is prepared from herba ViolaeStipapurpurea) Is of the leaf part of (a)Separating to obtain the final product. Paenibacillus feed B03%Paenibacilluspabuli) The preservation number of the general microbiological center of China Committee for culture Collection of microorganisms is CGMCC No.21386 (preservation Unit address: beijing, chaoyang area, north Chen West Lu No. 1, 3 national academy of sciences of China, microbiology institute; preservation date: 12 months 17 days 2020).
The strain B03 provided by the invention belongs to Paenibacillus genus @Paenibacillus) The colony is yellow, transparent, smooth and moist, and has luster.
The bacterial strain preservation method comprises the following steps: short term storage can be achieved by streaking with TSA slant solid medium at 4deg.C. If the composition is preserved for a long time, the composition can be preserved in a mixed solution of TSA liquid culture medium and glycerol at the temperature of-70 ℃, and the volume ratio of the TSA liquid culture medium to the glycerol is 3:1.
A paenibacillus B03 fermentation broth prepared by paenibacillus comprises the following steps:
dipping a small amount of bacteria in paenibacillus B03 stored on a slant solid culture medium by using an inoculating loop, streaking on the TSA solid culture medium, culturing in a dark place at 28+/-2 ℃, and taking the bacteria as a first-stage strain after macroscopic colonies grow; inoculating the activated primary strain to TSA liquid culture medium at 1% inoculum size, culturing in dark on shaking table at 35deg.C and rotation speed of 180 rpm, periodically observing and recording, stopping shaking culture after the culture solution becomes turbid (about 72 h), and obtaining 1×10 concentration 10 cfu·mL −1 Is prepared from the strain fermentation broth.
Wherein: the TSA solid culture medium is prepared by adding 15 g tryptone, 5 g soybean peptone, 5 g sodium chloride and 18 g agar into 1000 mL deionized water, mixing uniformly, adjusting pH to 7.1-7.5, and sterilizing at 121deg.C for 20 min.
The TSA liquid culture medium is prepared by adding 15 g tryptone, 5 g soybean peptone and 5 g sodium chloride into 1000 mL deionized water, mixing uniformly, adjusting pH to 7.1-7.5, and sterilizing at 121deg.C for 20 min.
Macroscopic Colonies (CFU) were colonies with average diameters above 1 mm (containing 1 mm).
The application of the Paenibacillus B03 fermentation liquid comprises the following steps: the Paenibacillus B03 is treatedPaenibacilluspabuli) The inactivated fermentation broth is diluted by 100 times by water to prepare 100 times of dilution liquid which is used as a plant growth regulator.
An ethyl acetate extract prepared by using a paenibacillus B03 fermentation broth, which comprises the following steps: extracting Paenibacillus B03 fermentation broth with ethyl acetate 3 times of the volume of the fermentation broth, and distilling the obtained extract under reduced pressure to obtain an ethyl acetate extract.
The application of the ethyl acetate extract comprises the following steps: the ethyl acetate extract is used for inhibiting Artemisia malabarica of Phragmites communisPediculariskansuensis) Iron rod hammerAconitum pendulum) Or used as plant growth regulator to promote the growth of grass purple flower festuca arundinaceaStipapurpurea) Is a growth of (a).
The experimental methods used in the following examples are conventional methods unless otherwise specified.
EXAMPLE 1 isolation of plant leaf bacterial strains Using conventional isolation methods
Strain B03 was isolated from the purple needle Mao Shebu of Xiuxiu Longxiang (102℃51'10 "E, 37℃09' 07" N) from the city of Gansu, wu Weishi, tibetan, zhu nationality, county, and the like. The method comprises the following steps:
selecting healthy purple sweet grass plants with leaves, disinfecting She Buqie of purple sweet grass Mao Xinxian into small sections (0.2-0.5 cm), disinfecting the surfaces of 75% ethanol for 30 s, disinfecting the surfaces of 3% sodium hypochlorite for 5 min, washing the samples three times by using sterilized distilled water, placing the samples on sterile filter paper to absorb water, placing the samples on a TSA solid culture medium, culturing the samples at 28+/-2 ℃ in a dark place, picking a small number of bacterial colonies by using sterile toothpicks after macroscopic bacterial colonies grow out, transferring the bacterial colonies onto a new TSA solid culture medium for purification, and transferring the samples into the TSA inclined solid culture medium without pollution, and preserving the samples at 4 ℃ for a short period of time.
EXAMPLE 2 cultivation of the bacteria and preparation of the fermentation broth and ethyl acetate extract
1. Culturing a first-stage strain: the strain B03 stored on the inclined plane solid culture medium is dipped with a small amount of strain by an inoculating loop, streaked on the TSA solid culture medium, cultivated in a dark place at 28-30 ℃, and used as a first-stage strain after macroscopic colonies are grown.
2. Culturing a secondary strain: inoculating the activated primary strain to TSA liquid culture medium according to 1% of inoculum size, placing on a shaking table at 35 ℃ and rotating at 180 rpm for light-shielding culture, periodically observing and recording, stopping shaking culture after the culture solution becomes turbid (about 72 h), and transferring to a refrigerator at 4 ℃ for short-term preservation.
3. Preparation of fermentation broths with different concentrations: water is used as a diluent, the fermentation liquor of the strain B03 is prepared into 100-time and 200-time diluent, and the fermentation liquor of the strain B03 inactivated is prepared into 100-time and 200-time diluent.
4. Preparation of ethyl acetate extracts of different concentrations: using feed paenibacillusPaenibacilluspabuli) And B03 living bacterial liquid is taken as a raw material, ethyl acetate with the volume being 3 times that of the raw material is extracted, and the ethyl acetate extract is obtained through reduced pressure distillation. Prepared to 8 mg mL by adding DMSO -1 Is diluted to a concentration of 800, 400, 200, 100, 50 and 25 mug.mL -1 Is a solution of (a) and (b).
EXAMPLE 3 plant growth regulating Activity of Paenibacillus B03 fermentation broth and ethyl acetate extract
Seed germination is carried out by taking grass purple fescue and Mesona procumbens and aconitum pendulum as receptor plants and adopting a petri dish filter paper bed method.
The pasture and the seeds of the toxicant are placed in a culture dish (diameter 90 mm) of double-layer filter paper, soaked in distilled water, placed in an artificial climate incubator at 25 ℃ and 18 ℃ for germination accelerating, and seedlings with regular germination are selected and placed in a 6-hole plate for activity test.
Respectively diluting the fermentation liquor and the inactivated fermentation liquor according to the raw liquor, 100 times dilution and 200 times dilution, and the concentration of ethyl acetate extract according to 800, 400, 200, 100, 50 and 25 mug.mL -1 Adding into 6-well plate, placing 6 seedlings in each well, and setting 3 parallel treatments with water as control. After continuous cultivation in a climatic chamber (light 10 h/dark 14h, temperature: 25 ℃) for 7 d, the seedlings were taken out and measured for fresh weight, dry weight, root length and stem length. The experimental results were statistically analyzed using Sigmaplot 12.0 software.
Results
1. Plant growth regulating Activity of Paenibacillus B03 fermentation broth
The plant growth promoting activity of Paenibacillus subtilis B03 fermentation broth was examined using Paenibacillus purpureus as a receptor, and the results are shown in FIG. 1 and Table 1. Compared with the control group, the inactivated fermentation liquor diluted by 100 times has best effect, and the fresh weight, root length and stem length of the inactivated fermentation liquor are 102.58 percent, 163.11 percent and 73.72 percent of CK1 respectively, which reach extremely obvious difference levelp<0.01 Most preferably, the Paenibacillus B03 is diluted 100-fold to inactivate the pro-active effect of the fermentation broth.
The inhibitory activity of the Paenibacillus B03 fermentation liquor on the aconitum pendulum and the Artemisia kansui is shown in figure 1 and table 1, and the inactivated fermentation liquor diluted 100 times by B03 obviously inhibits the growth of the aconitum pendulum and the Artemisia kansui. The inhibition rate of the inactivated fermentation liquor diluted by 100 times of B03 on the root length of the aconitum pendulum is 46.52 percent, the inhibition rate on the root length of the pedicellus et pericarpium citri reticulatae is 67.05 percent, and the inactivated fermentation liquor shows extremely obvious difference levelsp<0.05). The Paenibacillus B03 has the best effect of inhibiting the toxic grass by diluting the 100-time inactivated fermentation broth.
TABLE 1 Effect of Paenibacillus B03 fermentation broth on fresh weight, root length and Stem length of seedlings of Dactylicapnos scandens, mr. malayan and Monkshood
2. Plant growth regulating Activity of Paenibacillus B03 ethyl acetate extract
Plant life-promoting properties of the B03 ethyl acetate extract were evaluated using Viola yedoensis seedlings as receptors, as shown in FIG. 2 and FIG. 3-a. The result shows that the Paenibacillus B03 ethyl acetate extract has obvious promotion effect on the purple needle Mao Jingchang and the dry weight, when the concentration of the ethyl acetate extract is 800 mug.mL -1 When the method is used, the promotion rate of the root length and the dry weight of the purple needle Mao Jingchang is 36.99 percent, 30.20 percent and 35.57 percent, and the difference reaches a significant level compared with the control groupp<0.05). When the concentration of the ethyl acetate extract is more than or equal to 100 mug.mL -1 When the purple needle Mao Jing is used, the difference between the long-treatment group and the control group reaches a significant levelp<0.05）。
From fig. 2 and fig. 3-b and 3-c, it can be seen that the root and stem elongation and seedling growth of aconitum pendulum and artemisia rupestris seedlings are significantly inhibited by the treatment of the extracts with different concentrations, and the inhibition effect has a significant dose dependency. When the treatment concentration is more than or equal to 100 mug.mL -1 When compared with the control group, the aconitum pendulum and the sweet artemisia rupestris treatment group reach the difference significance of the differencep<0.05 The inhibition rate of the radix aconiti szechenyiani and the root length of the pedicellus et pericarpium citri reticulatae is more than 40 percent.
Claims (10)
1. A paenibacillus having plant growth regulating activity, characterized in that: the strain is feed paenibacillus B03%Paenibacillus pabuli) It is obtained from the leaf part of the purple flower fescue; the feed paenibacillus B03%Paenibacillus pabuli) The preservation number of the general microbiological center of China Committee for culture Collection of microorganisms is CGMCC No.21386.
2. A Paenibacillus B03 fermentation broth prepared by using the Paenibacillus according to claim 1.
3. The method for preparing the Paenibacillus B03 fermentation broth according to claim 2, wherein the method comprises the following steps: dipping paenibacillus B03 stored on a slant solid culture medium by an inoculating loop, streaking on the TSA solid culture medium, culturing in dark at 28+/-2 ℃ and taking the bacteria as a first-stage strain after macroscopic colonies grow; inoculating the activated primary strain to TSA liquid culture medium at 1% inoculum size, placing on shaking table at 35deg.C and rotation speed of 180 rpm for light-shielding culture, stopping shaking culture after the culture solution becomes turbid to obtain 1×10 strain 10 cfu·mL −1 Is prepared from the strain fermentation broth.
4. The method for preparing the Paenibacillus B03 fermentation broth according to claim 3, wherein the method comprises the following steps: the TSA solid culture medium is prepared by adding 15 g tryptone, 5 g soybean peptone, 5 g sodium chloride and 18 g agar into 1000 mL deionized water, uniformly mixing, adjusting pH to be 7.1-7.5, and sterilizing at 121 ℃ for 20 min.
5. The method for preparing the Paenibacillus B03 fermentation broth according to claim 3, wherein the method comprises the following steps: the TSA liquid culture medium is prepared by adding 15 g tryptone, 5 g soybean peptone and 5 g sodium chloride into 1000 mL deionized water, uniformly mixing, adjusting pH to be 7.1-7.5, and sterilizing at 121 ℃ for 20 min.
6. The method for preparing the Paenibacillus B03 fermentation broth according to claim 3, wherein the method comprises the following steps: the macroscopic colonies were colonies with an average diameter above 1 mm.
7. Use of a paenibacillus B03 fermentation broth according to claim 2, wherein: the fermentation broth inactivated by the paenibacillus B03 is diluted by 100 times by water to prepare 100 times of dilution liquid which is used as a plant growth regulator.
8. An ethyl acetate extract prepared using the Paenibacillus B03 broth of claim 2.
9. The method for preparing ethyl acetate extract according to claim 8, wherein: extracting Paenibacillus B03 fermentation broth with ethyl acetate 3 times of the volume of the fermentation broth, and distilling the obtained extract under reduced pressure to obtain an ethyl acetate extract.
10. Use of an ethyl acetate extract according to claim 8, characterized in that: the ethyl acetate extract can be used for inhibiting growth of radix et rhizoma Hirudinae or radix Aconiti Szechenyiani, or used as plant growth regulator for promoting growth of grass and herba Violae.
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