CN117844705A - Endophytic listeria, microbial agent prepared from endophytic listeria and application of microbial agent - Google Patents
Endophytic listeria, microbial agent prepared from endophytic listeria and application of microbial agent Download PDFInfo
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- CN117844705A CN117844705A CN202410076122.2A CN202410076122A CN117844705A CN 117844705 A CN117844705 A CN 117844705A CN 202410076122 A CN202410076122 A CN 202410076122A CN 117844705 A CN117844705 A CN 117844705A
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- listeria
- plant
- priestia
- endophytica
- cgmcc
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Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to a microbial strain and application thereof, in particular to a plant endophytic listeria, a microbial agent prepared by using the same and application thereof. The invention provides a plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 with the functions of promoting the effect of tomato and improving the cold resistance, which can synthesize siderophores to produce auxin (IAA) and 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) and has the capability of fixing nitrogen. The microbial agent or fermentation liquor prepared by the strain can be applied to tomato production to improve the plant height and leaf area of tomato plants, and has better promotion effect; the cold resistance of tomatoes can be obviously improved in the aspects of tomato plant height, biomass, accumulation of protective substances, antioxidase activity, chlorophyll content, photosynthetic parameters and the like.
Description
Technical Field
The invention relates to a microbial strain and application thereof, in particular to a plant endophytic listeria, a microbial agent prepared by using the same and application thereof.
Background
Tomato is one of the main categories of greenhouse cultivated vegetables in northern China, is a warm-loving vegetable crop, and low-temperature stress is a main obstacle factor affecting the yield and quality of the warm-loving vegetable crop. At present, three methods for improving plant cold resistance at home and abroad are mainly adopted: the low-temperature resistant varieties are cultivated, the low-temperature exercise and the chemical reagent treatment are carried out, the traditional breeding approach can cultivate excellent low-temperature resistant varieties, such as tomato varieties 'Fuyou 109' which can resist low temperature of 2-3 ℃ are cultivated by Dutch specialists, the low-temperature resistant varieties 'strawberry tomatoes', 'Provence' and the like which are bred in China are cultivated, but the time is longer; the low-temperature exercise is targeted, the physiological and biochemical changes of the plants are required to be treated, the required time is long, and the effect is unstable; although the chemical reagent takes effect quickly, the chemical molecule residues pose a potential threat to vegetables and the environment, and a green and environment-friendly prevention and control technology is developed to relieve the damage of low temperature to the facility tomatoes, so that the method is extremely important for the production of high-yield and high-quality tomatoes. In recent years, with the increase of the requirements of people on resource environment and food quality, beneficial microorganisms play an increasingly important role in ecological green agriculture, and the improvement of plant cold resistance by the microorganisms has become a new research hot spot.
The invention patent application with the publication number of CN116463242A discloses a strain of Paenibacillus polymyxa (Paenibacillus polymyxa) X-11, the preservation number is CGMCC No.20476, the strain can promote germination of tomato seeds and elongation of radicle, the optimal growth promotion concentration of the strain is different according to different tomato varieties, the too high concentration of Paenibacillus polymyxa X-11 can inhibit seed germination and elongation of radicle, and the Paenibacillus polymyxa X-11 can simultaneously induce PAL activity of tomato leaves to be improved, and the induction effect is better than that of carbendazim. The cold damage investigation result of the cold resistance of tomatoes shows that the disease index of paenibacillus polymyxa X-11 treatment is obviously lower than that of a control, and the applicant does not find out that endophytic Listeria in plants can promote tomato growth and resist cold.
Disclosure of Invention
One of the purposes of the invention is to provide a plant endophytic listeria monocytogenes (Priestia endophytica) CGMCC No.29318 which has a pro-active effect on tomatoes and can improve the cold resistance function of tomatoes.
The strain in the invention is separated from tomato root systems with cold resistance. The strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) in 12 months and 15 days of 2023, and is classified as plant endophytic Listeria (Priestia endophytica), the preservation number is CGMCC No.29318, and the address of the preservation institution is in China general microbiological institute (CGMCC) of national institute No.3 of sciences of North Chen West road No.1 in the Korean region of Beijing city.
The colony morphology and the physiological and biochemical characteristics of the strain are as follows:
the bacterial colony of the strain is round, has unsmooth edges, milky white and opaque, can synthesize siderophores, generates auxin (IAA) and 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD), and has nitrogen fixation capability. Similarity to Priestia endophytica strain S2-11 is 100%.
The second object of the present invention is to provide a microbial agent comprising plant endophytic listeria (Priestia endo phytica) CGMCC No.29318.
The invention further aims to provide a plant endophytic listeria monocytogenes (Priestia endo phytica) CGMCC No.29318, fermentation liquor containing the plant endophytic listeria monocytogenes CGMCC No.29318 and application of a microbial agent in promoting plant growth.
The invention aims at providing a fermentation broth containing plant endophytic listeria monocytogenes (Priestia endo phytica) CGMCC No.29318 and application of a microbial agent to improvement of cold resistance of plants, wherein the plants are tomatoes.
Specifically, the plant endophytic listeria is obtained by inoculating Latin name Priestia endophytica and the preservation number is CGMCC No.29318 in a sterilized culture medium containing a carbon source, a nitrogen source and inorganic salt for aeration culture.
Specifically, the culture medium is selected from sterilized LB culture medium.
The culture conditions of the strain are as follows:
1. culturing slant strain: the strain is inoculated into the inclined surface of a test tube, the culture temperature is 25-32 ℃, the pH=7.0-7.2, and the culture time is 1-3 days.
2. Shake flask culture: inoculating the slant strain into triangular flask with LB culture medium, culturing at 25-32deg.C, pH=7.0-7.2 and rotation speed 180-220 rpm for 24-60 hr to make the bacterial count 2×10 8 cfu/ml。
The essential characteristics and remarkable technical progress of the invention are as follows:
the screening method of the invention obtains a plant endophytic Listeria with Latin name of Priestia endophytica and preservation number of CGMCC No.29318, and experiments of the applicant prove that the strain can synthesize siderophores to produce auxin (IAA) and 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD) and has nitrogen fixation capability. The method is applied to tomato production, can improve the plant height and leaf area of tomato plants, and has better promotion effect; the cold resistance of tomatoes can be obviously improved from the aspects of tomato plant height, biomass, accumulation of protective substances, antioxidase activity, chlorophyll content, photosynthetic parameters and the like.
The strain of the invention is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) in the 12 th month 15 of 2023, and is classified as plant endophytic Listeria (Priestia endoph ytica), the preservation number is CGMCC No.29318, and the address of the preservation mechanism is in China institute of sciences (CGMCC) No.3 of the national academy of sciences 1 in the North China, the Korean yang area of Beijing city.
Drawings
The drawings of the invention are as follows:
FIG. 1 is a schematic representation of the colony of the plant endophytic Listeria (Pri estia endophytica) CGMCC No.29318 of the present invention cultured using a planar culture dish.
FIG. 2 is a schematic representation of the effect of plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 on tomato growth comparison with a blank control plant in the present invention.
In the figure, the pot plants on the left side are tomato seedlings with the application of plant endophytic listeria (Priestia endophytica) CGMCC No.29318, the pot plants on the right side are tomato seedlings without the application of plant endophytic listeria (Priestia endophytica) CGMCC No.29318, the difference between the tomato seedlings is large, and the tomato seedlings with the application of plant endophytic listeria (Priestia endophytica) CGMCC No.29318 have strong stems, more leaves and luxuriant branches and leaves; tomato seedlings without application of plant endophytic listeria monocytogenes (Priestia endophytica) CGMCC No.29318 have sparse leaves.
FIG. 3A is a graph showing the effect of plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 on leaf growth comparison with a blank control after tomato inoculation in accordance with the present invention.
FIG. 3B is a bar graph comparing leaf area of tomato inoculated with CGMCC No.29318 of Listeria endophyte (Priestia endophytica) of the present invention with that of a control leaf.
FIG. 3C is a bar graph comparing leaf length of plant-grown Listeria (Priestia endophytica) CGMCC No.29318 post-inoculation tomato with control leaves of the present invention.
FIG. 3D is a bar graph comparing leaf widths of plant-grown Listeria (Priestia endophytica) CGMCC No.29318 post-inoculation tomato with control leaves of the present invention.
The left leaf in FIG. 3A is tomato leaf with CGMCC No.29318 applied to plant endophytic Listeria (Priestia endophyti ca), the right leaf is tomato leaf without CGMCC No.29318 applied to plant endophytic Listeria (Priestia endophytica), and the leaf length, leaf width and leaf area of tomato leaf with CGMCC No.29318 applied to plant endophytic Listeria (Priestia endophytica) are all greater than those of control leaf. Fig. 3B, fig. 3C, and fig. 3D show specific comparison data of leaf length, leaf width, and leaf area of two groups of treated tomatoes, and TC3 in fig. 3B, fig. 3C, and fig. 3D is plant endophytic listeria (Priestia endophytica) CGMCC No.29318, showing that plant endophytic listeria (Priestia endophytica) CGMCC No.29318 can significantly promote increase in area of tomato leaves.
FIG. 4A is a graph showing the qualitative assay plate test effect of plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 on ferrite synthesis in the present invention.
The strain in FIG. 4A produces an orange-yellow aperture on CAS medium, indicating its function to synthesize siderophores.
FIG. 4B is a graph showing the effect of qualitative measurement of hemolysis of Listeria endophyte (Priestia endophytica) CGMCC No.29318 in the present invention.
Since the function of the blood plate is to observe the hemolytic properties of the bacteria, the plant endophytic listeria monocytogenes (Priestia endophytica) CGMCC No.29318 in fig. 4B does not produce a transparent ring on the blood plate, indicating that it is not hemolytic.
FIG. 4C is a plot of the qualitative determination of nitrogen fixation capacity of plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 of the present invention.
The strain in FIG. 4C can be grown on Ashby medium, indicating that it has nitrogen fixation properties.
FIG. 4D is a graph showing the effect of a dish test for detecting the ability of plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 to secrete ACC deaminase by using DF medium.
FIG. 4E is a graph showing the effect of a plate assay for detecting the ability of plant-derived listeria (Pries tia endophytica) CGMCC No.29318 to secrete ACC deaminase using ADF media.
The strain in FIG. 4E was grown on ADF plates, indicating that it was able to produce ACC deaminase.
FIG. 4F is a graph showing the effect of a qualitative assay of the ability of plant Endophytene Listeria (Priestia endophytica) CGMCC No.29318 to secrete auxin (IAA) in the present invention.
The left side of FIG. 4F is a control, the right side is a strain of the invention, and the right side is pink in color, indicating that it has the ability to secrete auxin.
FIG. 5 is a schematic representation of the effect of plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 on tomato growth with blank control seedlings in the present invention.
In FIG. 5, tomato seedlings of the strain of the present invention were applied from left to right in order from the low temperature (5 ℃) treatment, tomato seedlings of the strain of the present invention were not inoculated at the low temperature (5 ℃) treatment, tomato seedlings of the strain of the present invention were applied at the normal temperature (25 ℃) treatment, and tomato seedlings of the strain of the present invention were not inoculated at the normal temperature (25 ℃) treatment. Figure 5 illustrates that the strain of the present invention maintains normal growth of tomato seedlings at low temperature, with plant height and root length significantly higher than the low temperature control, and maintains the level of the normal temperature control.
Fig. 6A is a graph comparing MDA content of tomato leaves after treatment with low temperature stress.
In fig. 6A, it can be seen that after stress treatment at 5 ℃, the Malondialdehyde (MDA) content of tomato leaves is significantly increased, which indicates that the cell membrane is damaged and membrane lipid is peroxidized, resulting in MDA accumulation, and the MDA content of contrast + low temperature is highest, and the MDA content of plants inoculated with endophytic listeria (Priestia endophytica) cgmccno.29318 is always lower than that of contrast treatment, which indicates that the cold damage of tomato leaves treated by the strain of the invention is lighter.
FIG. 6B is H of tomato leaf after treatment with low temperature stress 2 O 2 Content versus bar graph.
In FIG. 6B it can be seen that stress treatment at 5℃induces hydrogen peroxide (H 2 O 2 ) Significant increase in content, causing oxidative stress damage to leaf, control+low temperature H 2 O 2 The content is highest, and the plant H of the CGMCC No.29318 is inoculated with the endophytic Listeria pulvis (Priestia endophytica) 2 O 2 The content is always lower than that of the control treatment, which shows that the strain treatment can effectively relieve the membrane lipid peroxidation damage to the tomato leaves caused by low-temperature stress.
Fig. 6C is a graph comparing POD activity of tomato leaves after treatment with low temperature stress.
FIG. 6C illustrates that tomato plants treated with plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 up-regulates Peroxidase (POD) activity, resulting in improved ability of scavenging active oxygen free radicals in plant cells, maintaining balance between intracellular free radical generation and scavenging, reducing membrane lipid peroxidation, and alleviating cell membrane damage caused by low temperature.
Figure 6D is a graph comparing SOD activity of tomato leaves after treatment with low temperature stress.
FIG. 6D illustrates that tomato plants treated with endophytic Listeria (Priestia endophytica) CGMCC No.29318 have increased superoxide dismutase (SOD) activity and scavenge H caused by low temperature stress 2 0 2 Maintains oxidation-reduction balance in vivo, and improves the low temperature resistance of tomato plants.
Fig. 6E is a graph of PRO content versus bar graph of tomato leaf after treatment with low temperature stress.
FIG. 6E illustrates the effect of Proline (PRO) in regulating cell penetration, storing nitrogen, scavenging hydroxyl radicals and protecting intracellular enzymatic activity. The proline content of the plant endophytic listeria monocytogenes (Priestia endophytica) CGMCC No.29318 treated leaf is obviously improved compared with a control, which shows that the strain treatment of the invention increases the proline content of tomato plant cells, lowers the freezing point and enhances the cold injury resistance.
FIG. 7A is a bar graph comparing photosynthetic rates of a plant of the present invention, listeria endophyte (Priestia endophytica) CGMCC No.29318, after tomato inoculation, with a control plant.
FIG. 7B is a bar graph comparing transpiration rates of a plant of the invention, i.e., listeria endophyte (Priestia endophytica) CGMCC No.29318, after grafting tomato to a control plant.
FIG. 7C is a bar graph comparing the water use efficiency of the plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 of the present invention after tomato inoculation with a control plant.
FIG. 7D is a bar graph comparing stomatal conductance of a plant of the invention, i.e., listeria endophyte (Priestia endophytica) CGMCC No.29318, to that of a control plant after tomato inoculation.
As can be seen from fig. 7A, 7B, 7C and 7D, the plant endophytic listeria pulvis (Priestia endophytica) CGMCC No.29318 can increase the photosynthetic rate of tomato, slow down the transpiration rate, and alleviate the limitation of low temperature stress on the photosynthetic capacity of plants to a certain extent. The Water Utilization Efficiency (WUE) and the stomatal conductance (Gs) of the plants connected with the strain are always higher than those of the plants not connected with the strain under the conditions of normal temperature or low temperature.
Detailed Description
The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the invention, but are instead provided with equivalents in accordance with the description of the invention without departing from the scope of the invention.
Example 1
Isolation and identification of plant endophytic listeria (Priestia endophytica) CGMCC No.29318
1. Cleaning the surfaces of root systems and stems of tomatoes under running water, wiping the surfaces with sterile filter paper, taking 1g of main roots and lateral roots from the roots, transferring the roots into an ultra-clean bench for surface disinfection, firstly, carrying out shaking and soaking in 75% alcohol for 1 minute, then carrying out shaking and soaking in 1% sodium hypochlorite solution for 5-10 minutes, and then rinsing with sterile water for 3 times. And (3) respectively coating the final rinsing liquid on an R2A plate and an NR plate, and observing whether a colony grows out after 72 hours, and sterilizing the surface thoroughly after the colony grows out in a sterile manner. Placing 1g of the surface sterilized material into a sterile mortar, adding 9 ml of sterile physiological saline, and grinding into homogenate to obtain 10 -1 Diluent and then draw 10 ml of 10 -1 Performing gradient dilution on the soil diluent to 10 -3 Take 10 -1 、10 -2 、10 -3 Concentration dilutions were plated on NR and R2A media with 3 replicates per gradient. Incubation was performed at 30℃for 48 hours, combined with colony morphology observation and 16SrDNA gene sequence amplification and sequencing verification as shown in SEQ No. 3.
The oligotrophic culture medium R2A consists of the following raw materials: beef extract 3.0 g, peptone 10.0 g, yeast powder 1.0 g, glucose 3.0 g, sodium pyruvate 3.0 g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1.0 g, sodium chloride 5.0 g, agar 20 g and water 1 liter.
The nutrient rich medium NR consists of the following raw materials: yeast powder 0.50 g, tryptone 0.50 g, glucose 0.50 g, soluble starch 0.50 g, dipotassium hydrogen phosphate 0.30 g, magnesium sulfate 0.02 g, sodium pyruvate 0.30 g, agar 15.00 g and water 1 liter.
2. Morphological characterization of strains
The colony of plant endophytic listeria (Priestia endophytica) CGMCC No.29318 is circular, has a non-smooth edge, is milky white and is not transparent, and the specific result is shown in figure 1.
3. Physiological and biochemical characteristics of strain
The applicant believes that the growth promoting capacity of plant endophyte listeria (Priestia endophytica) cgmccno.29318 on tomatoes may be related to its own biochemical characteristics, so that the identification of biochemical characteristics of plant endophyte listeria (Priestia endophy tica) CGMCC No.29318 can be performed from whether it produces auxin (IAA), 1-aminocyclopropane-1-carboxylic Acid (ACC) deaminase, whether it has nitrogen fixing capacity, and whether it is possible to synthesize ferrites.
The experimental method comprises the following steps:
(1) Culture medium for strain culture
LB liquid medium: distilled water was added to 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride, the pH was adjusted to=7.0, the volume was adjusted to 1 liter, and then the mixture was sterilized at 121℃for 15 minutes, and the mixture was cooled and used.
LB solid medium: agar was added to the LB liquid medium to a concentration of 15 g/l; sterilizing at 121deg.C for 15 min, pouring LB liquid medium cooled to 55deg.C into a culture dish, and naturally cooling.
And (3) strain preservation: inoculating the single colony to LB liquid medium, and culturing at 30 ℃ for 16 hours to obtain a culture bacterial liquid. 1 part by volume of the culture broth and 1 part by volume of a 50% (v/v) glycerol aqueous solution were mixed and stored at-80 ℃.
(2) Qualitative determination of siderophore synthesis
(1) The CAS medium includes the following ingredients: chrome azure 0.060 g, cetyl trimethyl ammonium bromide 0.073 g, ferric trichloride hexahydrate 2.645 mg, sodium dihydrogen phosphate dihydrate 0.295 g, sodium dihydrogen phosphate dodecahydrate 1.213 g, ammonium chloride 0.125 g, potassium dihydrogen phosphate 0.0375 g, sodium chloride 0.0625 g, magnesium sulfate heptahydrate 0.1 g, glucose 10 g, peptone 10 g, acid hydrolyzed casein 10 g, agar 15-20 g.
(2) The test strains were inoculated onto the plates, respectively, each treatment was repeated 3 times, the incubation was reversed at 30℃for 3 days, the color change of each plate was observed, and the results were recorded, and the specific results are shown in FIG. 4A.
(3) The ferrite produced by the microorganism activates indissolvable iron in soil, improves the solubility and mobility of the iron and improves the effectiveness of the iron in the soil. The strain in FIG. 4A produces an orange-yellow aperture on CAS medium, indicating its function to synthesize siderophores.
(3) Detection of bacterial strain hemolysis
(1) Blood agar plates were purchased from Beijing Liqiao Corp (product number: PB 001), activated for 24 hours, and plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 were inoculated onto blood agar plates, the plates were placed in an incubator at 30℃for inverted culture, and whether the strain produced transparent circles was observed, if the transparent circles were produced, the presence of hemolysis was indicated. The specific results are shown in FIG. 4B.
(2) Since no transparent circles were produced on the blood plates, this strain was not hemolyzed.
(4) Analysis of Nitrogen fixation Capacity of Strain
(1) Each liter of Ashby medium consists of the following raw materials: potassium dihydrogen phosphate 0.2 g, sodium chloride 0.2 g, magnesium sulfate heptahydrate 0.2 g, potassium sulfate dihydrate 0.2 g, calcium carbonate 5 g, glucose 5 g, mannitol 5 g, agar 15.0 g, balance water, ph=7.0.
(2) The strain selected was inoculated on Ashby solid medium, set up and repeated 3 times, placed in an incubator at 28℃for inverted culture, and observed for growth of the strain, and the specific results are shown in FIG. 4C.
(3) The strain can be seen to grow on Ashby medium, indicating that it has nitrogen fixing properties.
(5) Analysis of the ability of the Strain to secrete 1-aminocyclopropane-1-carboxylic acid deaminase (ACCD)
(1) Each liter of DF culture medium consists of the following raw materials: component a was used in an amount of 0.1 ml, component b was used in an amount of 0.1 ml, potassium dihydrogen phosphate was 4.0 g, disodium hydrogen phosphate was 6.0 g, magnesium sulfate heptahydrate was 0.2 g, glucose was 2.0 g, 50% d-gluconic acid solution (shanghai source leaf company, product number s 11155) was 4 ml, citric acid was 2.0 g, ammonium sulfate was 2.0 g, agar was 15.0 g, and the balance was water, ph=7.0 to 7.2.
Wherein:
every 100 ml of component a consists of the following raw materials: 10 mg of boric acid, 11.19 mg of manganese sulfate monohydrate, 124.6 mg of zinc sulfate heptahydrate, 78.22 mg of copper sulfate pentahydrate, 10 mg of molybdenum trioxide and the balance of water.
Every 10 ml of component b consists of the following raw materials: ferrous sulfate heptahydrate 100 mg, and water in balance.
Per liter ADF medium: ammonium sulfate in DF medium was replaced with 1-aminocyclopropane-1-carboxylic Acid (ACC) at a concentration of 0.5M.
(2) The screened strain is activated, inoculated on DF solid culture medium, cultured for 3-4 days, transferred to ADF solid culture medium, placed in a 30 ℃ incubator for inverted culture, and observed whether the strain grows or not, and the specific results are shown in FIG. 4D and FIG. 4E.
(3) ACC deaminase is one of the indicators for determining the ability of a rhizosphere growth-promoting bacterium to promote plant growth. ACC deaminase can degrade the ethylene precursor ACC, thereby reducing ethylene levels during plant growth and contributing to plant growth. If the strain is able to grow on ADF medium it is indicated that the bacteria are able to produce ACCase. The strain in FIG. 4E was grown on ADF plates, indicating that it was able to produce ACC deaminase.
(6) Analysis of the ability of strains to secrete auxin (IAA)
(1) Preparation of Salkouski Reagent colorimetric solution
30 ml of concentrated sulfuric acid is slowly added into 20 ml of H 2 O, 0.15 g FeCl is added 3 ·6H 2 O. And (5) storing in a dark place.
(2) Preparation of 100 mg/ml tryptophan mother liquor
A20 ml mother liquor was prepared and 0.5 g tryptophan (L-Trp) was weighed, and filtered for sterilization. (1 ml of LB medium was used at a concentration of 100 mg/l, and thus 4. Mu.l of tryptophan mother liquor at a concentration of 100 mg/ml was added).
The amplified culture test bacteria solution was centrifuged at 12000 rpm for 2 minutes to obtain a supernatant, and after mixing 0.5 ml supernatant and 0.5 ml Salkouski Reagent colorimetric solution, 500. Mu.l of non-inoculated LB liquid medium was added as a control, and the mixture was subjected to a dark reaction for 15 minutes. The color turns pink to positive, which indicates that IAA can be secreted, and the darker the color, the greater the secretion intensity, and the specific result is shown in FIG. 4F.
(3) The left side of FIG. 4F is a control, the right side is a strain of the invention, and the right side is pink in color, indicating that it has the ability to secrete auxin.
4. 16S rDNA sequence of plant endophytic Listeria (Priestia endophytica) CGMCC No.29318
Bacterial DNA is extracted by adopting a CTAB/NaCl method, and primer 27F (AGA GTT TGA TCC TGG CTC AG) shown as SEQ No.1 is synthesized according to the conserved sequence of bacterial 16S rDNA, and 1492R (TAC GGC TAC CTT GTT ACG ACT T) shown as SEQ No. 2. Then carrying out PCR reaction, using rTaq enzyme, wherein the reaction system is 40 microliters, and the reaction program is 98 ℃ pre-denaturation for 3 minutes; denaturation at 98℃for 20 sec, annealing at 56℃for 20 sec, extension at 72℃for 20 sec, 29 cycles; extension was carried out at 72℃for 5 minutes. And (3) performing 0.8% agarose nucleic acid electrophoresis, sequencing positive amplification products by Beijing qing department biotechnology Co., ltd, obtaining a 16SrDNA gene sequence shown as SEQ No.3, and comparing the sequencing results.
5. Identification and preservation of strains
Priestia endophytica strain S2-11 similarity is 100%, and specific results are shown in Table 1; the bacterial species in this application is plant endophytic listeria monocytogenes (Priestia endophytica) by combining the bacterial morphology, physiological and biochemical characteristics and the 16S rDNA gene sequence. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 15 days of 2023, with the preservation number of CGMCC No.29318, and the classification name of the strain is plant endophytic Listeria (Priestia endophytica).
TABLE 1 comparison of isolated strains with typical strains of database
Example 2
Preparation of microbial agent containing plant endophytic listeria (Priestia endophytica) CGMCC No.29318
Inoculating plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 into bacterial culture medium, and culturing to obtain OD 600 The bacterial liquid with the value of 0.5-1.0 is the bacterial agent.
The culture conditions of the strain are as follows:
1. culturing slant strain: the strain is inoculated into the inclined surface of a test tube, the culture temperature is 25-32 ℃, the pH=7.0-7.2, and the culture time is 1-3 days.
2. Shake flask culture: inoculating the slant strain into triangular flask with LB culture medium, culturing at 25-32deg.C, pH=7.0-7.2 and rotation speed 180-220 rpm for 24-60 hr to make the bacterial count 2×10 8 cfu/ml。
Example 3
Plant endophytic listeria monocytogenes (Priestia endophytica) CGMCC No.29318 growth-promoting capability test
Selecting sterilized tomato seeds with consistent size after germination acceleration, sowing the tomato seeds in a plastic nutrition pot, and culturing the tomato seeds by adopting a special seedling culture matrix. Culturing tomato seedlings to 4-leaf stage in a sunlight greenhouse with natural illumination and temperature of 20-25 ℃, transplanting when the tomato seedlings grow to 4 true leaves, and setting 2 groups of treatments when transplanting: inoculating plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 and non-inoculated plant (CK), irrigating root of tomato root system during transplanting, adding 10 ml OD 600 Bacterial liquid (2×10) with a value of about 1.0 8 CFU/ml), 15 days after transplanting, 10 ml OD was added again 600 Bacterial liquid (2×10) with a value of about 1.0 8 CFU/ml), with equal amount of clear water as control, phenotype was observed 30 days after transplantingFor leaf area measurements for 2 sets of treatments at 25℃see FIGS. 2, 3A, 3B, 3C, and 3D.
Experimental results: the detection finds that the plant endophytic listeria pulvis (Priestia endophytica) CGMCC No.29318 can promote the growth of tomato leaves, and compared with a control, the tomato leaves have thick stems and obviously increased leaf areas.
Example 4
Plant endophytic listeria monocytogenes (Priestia endophytica) CGMCC No.29318 for improving low temperature resistance of tomato
The experimental method comprises the following steps: selecting sterilized tomato seeds with consistent size after germination acceleration, sowing the tomato seeds in a plastic nutrition pot, and culturing the tomato seeds by adopting a special seedling culture matrix. Culturing tomato seedlings to 4-leaf stage in a sunlight greenhouse with natural illumination and temperature of 20-25 ℃, transplanting when the tomato seedlings grow to 4 true leaves, and setting 2 groups of treatments when transplanting: inoculating plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 (TC 3) and non-inoculated plant (CK), irrigating root of tomato root system during transplanting, adding 10 ml OD 600 Bacterial liquid (2×10) with a value of about 1.0 8 CFU/ml), 15 days after transplanting, 10 ml OD was added again 600 Bacterial liquid (2×10) with a value of about 1.0 8 CFU/ml), with equal amount of fresh water as a control, at 30 days after transplanting, with 10 pots each, all treatments transferred to a climatic chamber for 10 days, divided into two temperature conditions: normal temperature 25 ℃ and low temperature 5 ℃, photoperiod 16L:8D. After 10 days of treatment at 5 ℃, phenotype observation is carried out on the plants treated at low temperature, measurement of plant height and root length is carried out on 4 groups of treatment, fresh weight is measured on the overground part and the underground part respectively, fixation is carried out for 15 minutes at 105 ℃, and dry weight of the overground part and the underground part is measured after constant-temperature drying at 70 ℃. The cold tolerance function of plant endophytic listeria monocytogenes (Priestia endophytica) CGMCC No.29318 is detected, and the specific results are shown in Table 2 and FIG. 5.
TABLE 2 Effect of plant Endophytic Listeria (Priestia endophytica) CGMCC No.29318 on tomato seedling growth under Low temperature stress
Experimental results: the endophytic listeria (Priestia endoph ytica) CGMCC No.29318 is inoculated in the tomato seedling stage, so that the low temperature resistance of the tomato seedlings can be obviously improved. At normal temperature, the plant height, fresh weight and dry weight of the tomato seedlings treated by inoculation are all obviously higher than those of a control (P is less than 0.05), and are respectively improved by 8.93%, 13.09% and 19.03% compared with the normal temperature control; the low-temperature stress obviously reduces the plant height, fresh weight and dry weight of tomato plants, and compared with normal-temperature control, the low-temperature stress obviously reduces the plant height, fresh weight and dry weight by 12.05 percent, 29.70 percent and 13.98 percent respectively, and has an inhibition effect on the growth of tomato seedlings; and inoculating plant endophytic listeria (Priestia endoph ytica) CGMCC No.29318, increasing the fresh weight and dry weight of the tomato plant by increasing the plant height, and respectively increasing 19.19%, 41.65% and 28.83% by the lower temperature control, so that the growth index returns to the normal temperature control level, thereby relieving the inhibition of low temperature on the growth of tomato seedlings.
Example 5
Plant endophytic listeria (Priestia endophytica) CGMCC No.29318 for promoting accumulation of protective substances and activity of antioxidant enzyme
The experimental method comprises the following steps: the third fully developed leaf of each treated tomato plant was taken and the fresh sample was about 0.5 g. The Malondialdehyde (MDA) content was measured using a kit (cat# MDA-1-Y, micro method) from Suzhou Kogyo Ming Biotechnology Co., ltd, and the difference in absorbance at 532 nm and 600 nm was used to calculate the MDA content. The measurement of Proline (PRO) was performed at 520 nm using a kit (product number: PRO-1-Y, micro method) from Suzhou Ming Biotechnology Co., ltd; hydrogen peroxide (H) 2 O 2 ) The content was determined using a kit (cat No.: h 2 O 2 -1-Y, microfabrication), measured at 415 nm; the detection of Peroxidase (POD) was carried out using a kit (cat# POD-1-Y, micro method) from Suzhou Ministry Biotechnology Co., ltd, and the change of A470 per minute per gram of tissue per ml of the reaction system was 0.01 as an enzyme activity unit. The superoxide dismutase (SOD) activity was measured using a kit (product number: SOD-1-Y, micro method) from Suzhou Ming Biotechnology Co., ltd. Each processing arrangement 3The specific results are shown in fig. 6A, 6B, 6C, 6D, and 6E.
Experimental results: the cold damage of tomato leaves treated by the strain of the invention is light; the MDA content in the inoculated plants is lower than that of the non-inoculated plants, and meanwhile, the activities of the antioxidant enzymes POD and SOD are improved by 49.64 percent and 16.01 percent respectively compared with those of the non-inoculated plants, so that the cell membrane damage caused by low temperature is relieved; the proline content of cells is obviously improved, the accumulation of cell protective substances is promoted, and the cold injury resistance is enhanced.
Example 6
Plant endophytic listeria monocytogenes (Priestia endophytica) CGMCC No.29318 for improving chlorophyll content of tomato leaves
The experimental method comprises the following steps: the control is tomato leaf which is not treated by plant endophytic Listeria (Priestia endophyti ca) CGMCC No.29318, the third leaf of tomato plant which is treated by four treatments of fungus inoculation, fungus non-inoculation, normal temperature and low temperature is taken to be fully unfolded, the sample leaf is cut into fragments, 0.2 g of leaf is weighed, 3 ml of 95% ethanol is added into the leaf, the leaf is ground into slurry, 25 ml of 95% ethanol is added into the slurry, and the slurry is soaked in the dark for 48 hours, and the slurry is shaken for 3 times. After the leaves were completely whitened, the absorbance of the extract was measured at 649 nm, 665 nm and 470 nm. Chlorophyll a (chla) =13.95×a 665 -6.88×A 649 The method comprises the steps of carrying out a first treatment on the surface of the Chlorophyll b (Chl b) =24.96×a 649 -7.32×A 665 The method comprises the steps of carrying out a first treatment on the surface of the Total chlorophyll (chlt) =6.63×a 665 +18.8×A 649 . The specific results are shown in Table 3.
Experimental results: under low temperature stress, the content of chlorophyll a, b and a+b in leaves treated by the plant endophytic listeria monocytogenes (Priestia endophytica) CGMCC No.29318 is higher than that of the control (P is less than 0.05).
TABLE 3 Effect of plant endophytic Listeria (Priestia endophytica) CGMCC No.29318 on chlorophyll content of tomato leaves under Low temperature stress
Example 7
Plant endophytic listeria pulmonarius (Priestia endophytica) CGMCC No.29318 for improving photosynthetic parameters of tomato leaves
The experimental method comprises the following steps: the net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and Water Utilization (WUE) of leaves 2-3 were measured by a photosynthetic measurement system (Ciras-3, PP-systems, UK) under good lighting conditions between 9:00 and 11:00 am. The illumination intensity of She Shina source is 600 mu mol m -2 ·s -1 ，O 2 Content 21%, CO 2 The concentration is 360 mu L.L -1 Specific results are shown in fig. 7A, 7B, 7C, and 7D.
Experimental results: the photosynthetic rate of the grafted plants is obviously increased, and it can be seen that the endophytic bacillus can help the plants to relieve the basic metabolism of photosynthesis under low-temperature stress. The photosynthetic rate (Pn) and the transpiration rate (Tr) of a tomato plant are remarkably reduced by low-temperature stress, the photosynthetic rate of the tomato can be improved by endophytic bacillus, 40.45% compared with a control, the transpiration rate is slowed down, 44.53% compared with the control, and the limitation of the low-temperature stress on the photosynthetic capacity of the plant is relieved to a certain extent. The Water Utilization Efficiency (WUE) and the air pore conductivity (Gs) of the inoculated plants are always higher than those of the non-inoculated plants under normal temperature or low temperature conditions, the difference does not reach a significant level although the difference is improved under normal temperature conditions, and the improvement range is larger under low temperature conditions and is respectively 27.77 percent and 78.81 percent higher than that of the control.
Claims (7)
1. The plant endophytic listeria is named Priestia endophytica in Latin, and the preservation number is CGMCC No.29318.
2. A microbial inoculant comprising the latin designation Priestia endophytica, accession number CGMCC No.29318, of a plant endophytic listeria.
3. Use of a plant endophytic listeria of CGMCC No.29318 or a fermentation broth containing same or a microbial inoculant according to claim 2 for promoting plant growth comprising latin designation Priestia endophytica according to claim 1.
4. Use of a plant endophytic listeria of CGMCC No.29318 or a fermentation broth containing same or a microbial agent of claim 2 for improving cold resistance of a plant, said plant being tomato, comprising latin name Priestia endophytica of claim 1.
5. The use according to claim 3 or 4, wherein the plant-borne listeria is obtained by inoculating a plant-borne listeria with the accession number cgmccno.29318, which is sterilized and aerated in a medium comprising a carbon source, a nitrogen source and an inorganic salt, and culturing the plant-borne listeria under the name Priestia endophytica.
6. The method according to claim 5, wherein the medium is LB medium.
7. The use according to claim 5, wherein the culture conditions of the bacterial species comprise the steps of:
A. culturing slant strain: inoculating the strain into a test tube inclined plane, wherein the culture temperature is 25-32 ℃, the pH=7.0-7.2, and the culture time is 1-3 days;
B. shake flask culture: inoculating the slant strain into triangular flask with LB culture medium, culturing at 25-32deg.C, pH=7.0-7.2 and rotation speed 180-220 rpm for 24-60 hr to make the bacterial count 2×10 8 cfu/ml。
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