CN109609419B - Zollinia bacteria and application thereof in improving stress resistance of plants - Google Patents

Abstract

The invention discloses a Zygophyllella bacterium and application thereof in improving plant stress resistance. The bacterium belonging to genus Zolbecco is specifically Zobellella sp (Zobellella sp.) DQSA1, and the preservation number of the bacterium in the China center for type culture Collection is CCTCCNO: m2018587. Experiments demonstrated that application of Zobellella sp DQSA1 CCTCCNO to corn or mung bean roots: m2018587, can promote growth and improve salt and alkali stress resistance, and is specifically represented by increase of plant height, fresh weight, dry weight, chlorophyll, soluble sugar content, soluble protein content and superoxide dismutase activity. The invention has important application value.

Description

Zollinia bacteria and application thereof in improving stress resistance of plants

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a Zygophyllum bacterium and application thereof in improving plant stress resistance.

Background

In recent years, with the increase in the world population and the reduction in cultivated land, some saline-alkali soil is developed and utilized, and the saline-alkali soil tends to expand. The salinization of soil seriously restricts the development of the agriculture and animal husbandry in the whole world, particularly in arid and semiarid regions, the salinization harm and the arid stress are mutually superposed, the growth of plants is seriously threatened, the salinization resistance of the plants is improved, the growth of the plants is promoted, and the problem to be solved in the salinization region is urgently needed.

The damage of saline-alkali stress to plants is mainly shown in the following three points: (1) physiological drought: the water potential of the root cells is higher than that of the soil, so that the root is difficult to absorb water, and even the water in the plant body has the risk of exosmosis; thus, the usual manifestations of salt damage are actually drought damage, which is more severe with increasing transpiration, especially in the case of low atmospheric relative humidity; the dehydration of plant cells leads to the destruction of the membrane structure, which leads to a decrease in the stability of the membrane structure and an increase in the permeability of the plasma membrane. (2) Poisoning of ions: the reason of poor plant growth in the soil with excessive salt is not physiological drought or difficult water absorption, but the absorption of certain salt is excessive, so that the absorption of other nutrient elements is excluded, and the effect similar to single salt poison is generated. (3) Disruption of normal metabolism: too much salt has a great influence on photosynthesis, respiration and protein metabolism. Excessive salt can inhibit chlorophyll biosynthesis and production of various enzymes, especially influence formation of chlorophyll-protein complex, reduce stomatal conductance and reduce plant photosynthetic capacity. Excessive salt also decreases PEP carboxylase and RuBP carboxylase activity, and enhances light respiration. Under saline-alkali stress, a large amount of active oxygen is generated in plants, which can damage the proton membrane, thus causing the membrane permeability to change and the structure and function to be damaged. At the same time, the hormone level in the plant body is changed, such as the accumulation of ethylene is increased, and the synthesis of other hormones (such as auxin and cytokinin) is reduced, so that the aging and death of the plant are accelerated.

Mung beans are annual cultivated species of vigna genus of Phaseolus of subfamily Papilionaceae of Leguminosae, and have more than 2000 years of cultivation history in China. Mung beans are important miscellaneous beans crops, are rich in nutrition, belong to low fat, medium starch and high protein, are main edible bean crops with homology of medicine and food, and are better export-earning crops. China is the largest export country of mung beans in the world, the planting area is about 600 million hectares throughout the year, the total yield is about 100 million tons, and the yield of mung beans in the world is increased at the speed of 2.5 percent each year. Meanwhile, the mung beans have shorter growth period, so that the mung beans are good crop rotation crops.

The corn belongs to the gramineae corn genus, is one of three main grain crops in China, is second to rice in planting area and total yield, and is the first to the grain crops in unit area yield. The corn is a high-quality feed for animal husbandry, and according to statistics, the corn is used as the feed for producing livestock products such as milk, meat, eggs and the like in the modern world and accounts for 75-80% of the total yield. The feeding value of the corn kernels is high, and the feeding value of 100kg of corn kernels is equivalent to 135kg of oat. Meanwhile, the fresh and tender branches and leaves of the corn are rich in nutrition, and the utilization value of the corn is high. In recent years, with the reform of the supply side of the large agriculture and the rearrangement of the animal husbandry in China, the role of the feeding corn in the agriculture and the animal husbandry is more and more important.

Disclosure of Invention

The invention aims to improve the stress resistance of plants.

The invention firstly protects the Zobellella sp (Zobellella sp.) DQSA1, the strain is preserved in China center for type culture Collection (CCTCC for short, address: Wuhan university in China) in 2018, 09 and 03 days, the preservation number is CCTCC NO: m2018587. Zobellella sp (Zobellella sp.) DQSA1CCTCC NO: m2018587 is abbreviated as zellella (Zobellella sp.) DQSA 1.

The invention also protects a microbial inoculum which contains the Zobellella sp DQSA1CCTCC NO: m2018587.

The microbial inoculum can be used for improving the stress resistance (such as saline-alkali stress resistance) of plants.

The preparation method of the microbial inoculum comprises the following steps: and (2) inoculating the DQSA1 of the Zobella (Zobellella sp.) to a bacterial culture medium and culturing to obtain a bacterial liquid, namely the microbial inoculum.

The bacterial culture medium may be an improved S-G liquid culture medium.

The preparation method of the improved S-G liquid culture medium specifically comprises the following steps: distilled water was added to 30g of sodium chloride, 10g of yeast extract, 5.3g of sodium carbonate, 8.4g of sodium bicarbonate, 3g of trisodium citrate, 2g of potassium chloride, 1g of magnesium sulfate heptahydrate, 0.05g of ferrous sulfate heptahydrate, and 0.0036g of manganese dichloride tetrahydrate, the pH was adjusted to 9.5, the volume was adjusted to 1L with distilled water, and then sterilization was carried out at 121 ℃ for 15 min.

In the preparation method of the microbial inoculum, the culture conditions can be as follows: culturing at 25-35 deg.C (such as 25-28 deg.C, 28-35 deg.C, 25 deg.C, 28 deg.C or 35 deg.C), 150-200rpm (such as 150-175rpm, 175-200rpm, 150rpm, 175rpm or 200rpm) for 20-30 h (such as 20-24 h, 24-30 h, 20h, 24h or 30 h).

The microbial inoculum may include a carrier in addition to the active ingredient. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymeric compound. The mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth. The plant material may be at least one of corn flour, bean flour and starch. The high molecular compound may be polyvinyl alcohol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water. The organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and a filtrate. The composition can be prepared into various dosage forms, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

The invention also protects the Zobellella sp DQSA1CCTCC NO: the application of M2018587 or any of the above bactericides can be at least one of A1) -A9):

A1) promoting the plant to take root;

A2) promoting the growth of plants;

A3) the stress resistance of the plants is improved;

A4) enhancing the scavenging capacity of the plant to active oxygen;

A5) improving the anti-aging capability of the plant;

A6) fixing nitrogen;

A7) synthesizing a siderophore;

A8) synthesizing ACC deaminase;

A9) synthesizing IAA.

The invention also protects the Zobellella sp DQSA1CCTCC NO: the application of M2018587 or any of the above bactericides in preparing products; the product may function as at least one of a1) -a 9):

A1) promoting the plant to take root;

A2) promoting the growth of plants;

A3) the stress resistance of the plants is improved;

A4) enhancing the scavenging capacity of the plant to active oxygen;

A5) improving the anti-aging capability of the plant;

A6) fixing nitrogen;

A7) synthesizing a siderophore;

A8) synthesizing ACC deaminase;

A9) synthesizing IAA.

In the above application, the product may be a microbial fertilizer.

In any of the above applications, the improvement of plant stress resistance may be an improvement of plant salt and alkali stress resistance. The salt and alkali stress resistance can be three, any two or any one of salt resistance, alkali resistance and salt and alkali resistance.

In the use of any of the above, the plant may be any of the following c1) to c 6): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) leguminous plants; c5) corn; c6) mung bean.

The invention also protects a microbial fertilizer which contains the Zobellella sp DQSA1CCTCC NO: m2018587 or any of the agents described above.

The invention also protects B1) or B2) or B3) or B4) or B5).

B1) The method for promoting the plant to take root can be to adopt the Zygolella bacteria to treat the plant, thereby promoting the plant to take root.

B2) The method for promoting plant growth comprises treating plant with bacteria of genus Zollinia to promote plant growth.

B3) The method for improving the stress resistance of the plants can be used for treating the plants by using the bacteria of the genus Zollinia, so that the stress resistance of the plants is improved.

B4) The method for enhancing the scavenging capacity of plants to active oxygen can be to adopt the bacteria of the genus Zollinia to treat the plants so as to enhance the scavenging capacity of the plants to active oxygen.

B5) A method for improving anti-aging ability of plant comprises treating plant with bacteria belonging to genus Zollinia, thereby enhancing scavenging ability of plant to active oxygen.

In any of the above methods, the "treating the plant with a bacterium of the genus Zygella" may be achieved by applying a bacterium of the genus Zygella to the root system of the plant.

In any of the above methods, the "treating the plant with the bacteria of the genus Zygophyllum" may be carried out by spraying the bacteria of the genus Zygophyllum onto the aerial parts (e.g., leaves) of the plant.

In any of the above methods, the "treating a plant with a bacterium belonging to the genus Zollinia" may be specifically treating a plant with any of the above microbial agents.

In any of the above methods, the "treating a plant with any of the above microbial agents" may be achieved by applying any of the above microbial agents to a plant root system.

In any of the above methods, the "treating a plant with any of the above microbial agents" may also be achieved by spraying any of the above microbial agents onto an above-ground part (e.g., a leaf) of a plant.

In any of the above processes, the bacterium of the genus zulbertzitia may be the bacterium of the genus zulbertella (Zobellella sp.) DQSA1CCTCC NO: m2018587.

In any of the above processes, the "using said Zobellella sp (Zobellella sp.) DQSA1CCTCC NO: m2018587 or any of the above inoculants "treating a plant may be applying the microbial fertilizer to the root system of the plant or spraying the microbial fertilizer to the aerial parts (e.g., leaves) of the plant.

In any of the methods described above, the plant may be any of the following c1) to c 6): c1) a dicotyledonous plant; c2) a monocot plant; c3) a gramineous plant; c4) leguminous plants; c5) corn; c6) mung bean.

Any one of the above promoting the rooting of the plant may be promoting the rooting of the cutting of the plant.

Any one of the above described methods for promoting plant growth and/or for improving plant stress resistance may specifically be characterized by an increase in at least one of plant height, fresh weight, dry weight, chlorophyll, soluble sugar content, soluble protein content and superoxide dismutase activity.

Any one of the above methods for improving stress resistance of a plant may be a method for improving salt and alkali resistance of a plant. The salt and alkali stress resistance can be three, any two or any one of salt resistance, alkali resistance and salt and alkali resistance.

Any of the above-described methods for enhancing the scavenging ability of plants to reactive oxygen species may be characterized by an increase in superoxide dismutase activity.

Any of the above described methods of increasing the anti-aging ability of a plant may be characterized by a reduction in malondialdehyde content.

Any one of the microbial fertilizers can be a compound microbial fertilizer and/or a biological organic fertilizer. The compound microbial fertilizer can be a fertilizer compounded by microbial inoculum, nutrient substances and organic matters. The compound microbial fertilizer has the functions of both microbes and chemical fertilizers. The biological organic fertilizer can be a fertilizer formed by compounding a microbial inoculum and a decomposed organic fertilizer. The dosage form of the compound microbial fertilizer and/or the biological organic fertilizer can be granules.

Experiments demonstrated that either maize or mung bean roots were subjected to Zobellella sp DQSA1CCTCC NO: m2018587, can promote growth and improve the resistance to saline-alkali stress, which is expressed by the increase of plant height, fresh weight, dry weight, chlorophyll, soluble sugar content, soluble protein content and superoxide dismutase activity; with Zobellella sp (Zobellella sp.) DQSA1CCTCC NO: m2018587 processing the mung bean cutting to promote the rooting of the mung bean cutting. The invention has important application value.

Drawings

FIG. 1 is a 16S rRNA phylogenetic tree of bacterial DQSA 1.

FIG. 2 is the result of statistics of the number of roots of mung bean cuttings under different treatment conditions.

FIG. 3 is a root phenotype of mung bean cuttings when soaked in water.

FIG. 4 shows the root phenotype of mung bean cutting when soaked in saline-alkali stress solution.

FIG. 5 phenotype of aerial parts of maize seedlings.

FIG. 6 phenotype of aerial parts of mung bean seedlings.

Deposit description

The strain name is as follows: genus Zuliella

Latin name: zobellella sp.

The strain number is as follows: DQSA1

The preservation organization: china center for type culture Collection

The preservation organization is abbreviated as: CCTCC (China center for cell communication)

Address: university of Wuhan, China

The preservation date is as follows: 09 month 03 of 2018

Registration number of the preservation center: CCTCC NO: m2018587

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The light-dark alternate culture in the following examples, namely 16h light culture and 8h dark culture were alternately performed, and the light intensity in the light culture was 60. mu. mol m^-2S^-1(ii) a The relative humidity was 70%.

Modified S-G liquid medium: distilled water was added to 30g of sodium chloride, 10g of yeast extract, 5.3g of sodium carbonate, 8.4g of sodium bicarbonate, 3g of trisodium citrate, 2g of potassium chloride, 1g of magnesium sulfate heptahydrate, 0.05g of ferrous sulfate heptahydrate, and 0.0036g of manganese dichloride tetrahydrate, the pH was adjusted to 9.5, the volume was adjusted to 1L with distilled water, and then sterilization was carried out at 121 ℃ for 15 min.

Modified S-G solid medium: adding agar into the improved S-G liquid culture medium to make the concentration of the agar to be 20G/L; then sterilized at 121 ℃ for 15 min.

Modifying the S-G solid plate: the modified S-G solid medium cooled to about 55 ℃ was poured into a petri dish and allowed to cool naturally.

The arbuscular mycorrhizal compound nitrogen-free culture medium: distilled water was added to 0.2g of monopotassium phosphate, 0.2g of sodium chloride, 0.5g of magnesium sulfate heptahydrate, 0.2g of calcium sulfate, 0.2g of potassium sulfate dihydrate, 5g of calcium carbonate, 10g of glucose and 20g of agar, the pH was adjusted to 7.0, the volume was adjusted to 1L with distilled water, and then sterilization was carried out at 121 ℃ for 15 min.

ADF culture medium: distilled water was added to 4.0g of potassium dihydrogen phosphate, 6.0g of disodium hydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, 0.1g of ferrous sulfate heptahydrate, 2.0g of glucose, 2.0g of gluconic acid, 2.0g of citric acid, 0.1mL of a trace element solution, and 3.0mmol of ACC, the pH was adjusted to 7.5, a constant volume of 1L was set with distilled water, and then sterilization was performed at 121 ℃ for 15 min. The microelement solution is an aqueous solution containing 100mg/L boric acid, 112mg/L magnesium sulfate heptahydrate, 1.246g/L zinc sulfate, 782mg/L copper sulfate pentahydrate and 100mg/L molybdenum trioxide.

King culture medium: to hydrolyzed peptone 20g, dipotassium hydrogen phosphate 1.5g, magnesium sulfate heptahydrate 1.5g and glycerol 10mL were added distilled water, the pH was adjusted to 7.2, a volume of 1L was made with distilled water, and then sterilization was carried out at 121 ℃ for 15 min.

And c, dye liquor c: and (4) slowly adding the solution a into the solution b along the bottle wall, and fully and uniformly mixing to obtain a dye solution c. Solution a: 0.024g of azure chrome is dissolved in 20mL of double distilled water, and then is mixed with 4mL of ferric trichloride solution (the solvent is water) (the concentration is 1mmol/L) uniformly to obtain solution a. Solution b: 0.030g of cetyltrimethylammonium bromide was dissolved in 16mL of double distilled water to obtain a solution.

And (3) a culture medium d: adding 40mL of 10 XMM 9 salt solution into a container filled with 300mL of double distilled water, and uniformly mixing; then adjusting the pH value to 6.8 by using a sodium hydroxide aqueous solution (the concentration is 1M); finally, 6.4g of agar powder is added to obtain a culture medium d. 10 × MM9 salt solution: 500mL of double distilled water is added into 30g of disodium hydrogen phosphate, 1.5g of monopotassium phosphate, 2.5g of sodium chloride and 5g of ammonium chloride, and the solution is fully dissolved to obtain 10 XMM 9 salt solution.

Sterilizing the culture medium d, the dye solution c, a calcium chloride solution (water as a solvent) (concentration of 1mmol/L), a magnesium sulfate solution (water as a solvent) (concentration of 1mmol/L), a glucose solution (water as a solvent) (concentration of 20g/100mL) and a casein amino acid solution (water as a solvent) (concentration of 10g/100mL) at 121 ℃ for 20min respectively, and cooling to about 50 ℃; adding 0.4mL of calcium chloride aqueous solution, 8mL of magnesium sulfate solution, 4mL of glucose aqueous solution and 12mL of casamino acid aqueous solution which are cooled to about 50 ℃ into the culture medium d cooled to about 50 ℃, slowly adding the dye solution c cooled to about 50 ℃ along the wall of the bottle, and fully shaking to obtain a blue qualitative detection culture medium, namely a CAS culture medium.

Example 1, Zobellella sp (Zobellella sp.) DQSA1CCTCC NO: isolation, identification and preservation of M2018587

Isolation of bacterial DQSA1

1. Adding 10g saline-alkali soil sample (salt alkali grassland collected from Dragon and phoenix region of Daqing city of Heilongjiang province, China) into 90mL of sterile water, oscillating at 28 deg.C for 20min, standing for 10min, collecting supernatant 1mL, adding into sterile test tube containing 9mL of sterile water, and mixing thoroughly (the dilution is 10 at this time)^-1) Then 1mL of the mixture is sucked from the test tube and added into another sterile test tube containing 9mL of sterile water to be uniformly mixed, and the like is repeated to prepare 10^-2、10^-3Bacterial suspensions of various dilutions.

2. After completion of step 1, 0.2mL of the dilution was diluted to 10^-3The bacterial suspension is evenly coated on an improved S-G solid plate and is statically cultured for 7 days at a constant temperature of 28 ℃.

3. After step 2 is completed, selecting single colonies with different colony morphologies on the improved S-G solid plate, repeatedly purifying for more than 3 times, and then preserving strains by using 50% (v/v) glycerol aqueous solution and preserving at-40 ℃ for later use.

4. The bacterial suspension of each strain purified in step 3 was spotted on the nitrogen-free Arbexate medium in an amount of 10. mu.L (each strain was repeated 3 times on the nitrogen-free Arbexate medium), cultured at 28 ℃ for 7 days, and then observed. If a certain strain can grow on the nitrogen-free culture medium of the arbuscular junction device, the strain is proved to have the nitrogen fixing function.

5. mu.L of the bacterial suspension of each strain purified in step 3 was spotted on ADF medium (each strain was repeated 3 times on ADF medium), cultured at 28 ℃ for 7 days, and then observed. If a strain can grow on ADF medium (i.e., can grow with ACC as the only nitrogen source), it indicates that the strain can synthesize ACC deaminase.

6. The bacterial suspension of each strain purified in step 3 was spotted on CAS medium in an amount of 10. mu.L (each strain was repeated 3 times on CAS medium), cultured at 28 ℃ for 7 days, and observed. If a certain strain can grow on the CAS culture medium and form an orange halo, the strain is proved to have the function of synthesizing the siderophore. The siderophore is a low molecular weight organic compound which is generated by microorganisms and partial plants under the condition of low iron stress and can complex ferric ions with high efficiency. The absorption of the ferric ions by plants can be influenced by the strong and weak capacity of the siderophore. The iron carrier can dissolve and combine iron elements in soil for plants to utilize, thereby increasing iron nutrition and promoting plant growth.

7. Respectively inoculating monoclonals of each strain purified in the step 3 (3 repeats for each strain) into a King culture medium containing 100mg/mL tryptophan, and carrying out shaking culture at 28 ℃ and 175rpm for 24h under the condition of keeping out of the sun to obtain a culture solution; centrifuging the culture solution at 12000rpm for 5min, and collecting supernatant; the supernatant was mixed with PC colorimetric solution (1.2 g FeCl₃Dissolving in 50mL of distilled water, slowly adding 42.97mL of concentrated sulfuric acid, cooling, diluting with distilled water to constant volume of 100mL), mixing in equal volume, placing on a white porcelain plate, placing in dark for 30min, and observing color development.

And (3) mixing the King culture medium containing 100mg/mL tryptophan and Salkowski colorimetric solution in equal volume, placing on a white porcelain plate, placing for 30min under a dark condition, and observing the color development condition (as a blank control).

If a certain strain can generate color reaction and the blank control cannot generate color reaction, the strain can synthesize IAA.

8. Through the steps 4-7, a strain of bacteria is screened and named as bacterial DQSA 1. The bacterium DQSA1 can be grown on Azurite medium and ADF medium, on CAS medium and form an orange halo, and on King medium with 100mg/mL tryptophan and develop a color reaction. Therefore, the bacterium DQSA1 has the functions of nitrogen fixation and siderophore synthesis, and can synthesize ACC deaminase and IAA.

II, identification of bacterial DQSA1

1. Morphological identification

(1) The bacterium DQSA1 was inoculated onto modified S-G solid plates and the morphology of single colonies was observed after 5 days.

The result shows that the bacterium DQSA1 is round, light pink, convex on the colony surface, moist, smooth, glossy and neat in edge.

(2) The bacterium DQSA1 was gram-stained and identified as a gram-negative bacterium.

(3) The morphology of the bacterium DQSA1 was observed using a high resolution transmission electron microscope.

As a result, the bacterial cells of the bacterium DQSA1 were rod-shaped.

2. Physiological and biochemical characteristic analysis

The physiological and biochemical characteristics of the bacterium DQSA1 were determined by methods in the literature of reference (Dongxu bead, Chuiziangying. Manual of systematic identification of common bacteria [ M ]. Beijing: published scientific and technological sciences, 2001: 365).

The results show that the bacterium DQSA1 is aerobic, the catalase test is positive, and the bacterium DQSA1 can utilize beta-methyl D-glucoside, D-xylose, D-mannitol, N-acetyl-D-glucosamine, D-cellobiose, D, L-alpha-glycerol, methyl pyruvate, alpha-hydroxybutyric acid, alpha-ketobutyric acid, D-malic acid, L-arginine, L-asparaginic acid, L-phenylalanine, L-serine, L-threonine, glycyl-L-glutamic acid, Tween 40, Tween 80, 4-hydroxybenzoic acid, glycogen and putrescine as the only carbon source or nitrogen source.

3. 16S rRNA sequence homology analysis

Through detection, the nucleotide sequence of 16S rRNA of the bacterium DQSA1 is shown as a sequence 1 in a sequence table.

The double-stranded DNA molecule shown in sequence 1 in the sequence table and the sequence in the EZ BioCloud database are compared and analyzed. A16S rRNA phylogenetic tree was prepared.

The 16S rRNA phylogenetic tree is shown in FIG. 1. The results showed that the bacterial DQSA1 resemblance to Zobenelladenitificans ZD1 was 96.59%.

Third, preservation of bacterial DQSA1

And identifying the bacterium DQSA1 separated and purified in the step one as the bacterium of the genus Zollinia according to the morphological identification result, the physiological and biochemical characteristic analysis result and the 16S rRNA sequence homology analysis result. The bacterium DQSA1 has been preserved in China center for type culture Collection (CCTCC for short, address: Wuhan university, China) in 2018, 09 month 03, with the preservation number of CCTCC NO: m2018587. The bacterial DQSA1 is collectively referred to as the genus geotrichum (Zobellella sp.) DQSA1CCTCC NO: m2018587, Callerobacter DQSA 1.

Example 2, Zollinia DQSA1 can promote rooting of mung bean cuttings

1. Obtaining of mung bean cutting

(1) And (4) selecting full mung bean seeds, and washing the seeds for 2 hours by using clear water.

(2) After the step (1) is finished, the mung bean seeds are taken, sterilized by 0.1% (v/v) sodium hypochlorite aqueous solution for 30min and then washed by sterile water for 10 min.

(3) And (3) after the step (2) is finished, placing the mung bean seeds in sterile water for soaking for 12 hours, then sowing the seeds in sterile vermiculite, carrying out dark culture for 48 hours at the temperature of 27 +/-2 ℃, and then carrying out light-dark alternate culture for 3 days at the temperature of 27 +/-2 ℃ to obtain mung bean seedlings.

(4) And (4) after the step (3) is finished, selecting mung bean seedlings with basically consistent growth vigor, cotyledon size and hypocotyl thickness, and cutting off the hypocotyls and root systems of the mung bean seedlings with the cotyledon thickness being less than 4cm to obtain the mung bean cutting slips.

2. Preparation of DQSA1 microbial inoculum

(1) A single colony of DQSA1 of Zygotella was inoculated into a conical flask (250 mL) containing 100mL of modified S-G liquid medium, and cultured at 28 ℃ for 24h with shaking at 175rpm to obtain bacterial suspension 1.

(2) After the step (1) is finished, taking bacterial liquid 1, centrifuging for 10min at 10000g, and collecting thalli.

(3) After the completion of the step (2), the cells were taken, washed three times with sterile physiological saline, and then resuspended in sterile physiological saline to obtain OD_600nmBacterial liquid 2 with a value of about 0.5.

The bacterial liquid 2 is the prepared DQSA1 bacterial agent.

3. Rooting of mung bean cuttings

(1) And (3) taking 4 mung bean cutting sticks prepared in the step (1), and soaking in DQSA1 microbial inoculum (aiming at inoculation) or water for 24 hours.

(2) After the step (1) is finished, the base part of the mung bean cutting is washed by sterile water, and then is inserted into a centrifugal tube filled with saline-alkali stress liquid (10 mM sodium carbonate aqueous solution) or water, and the mung bean cutting is cultured for 7 days in light and dark alternately at the temperature of 27 +/-2 ℃. During the incubation period, the solution in the centrifuge tubes was changed daily.

(3) And (3) after the step (2) is completed, counting the rooting number of the mung bean cutting strips, and then taking an average value according to groups.

The statistical result of the rooting number of the mung bean cutting is shown in figure 2(CK is water soaking and no inoculation, S is saline-alkali stress liquid soaking and no inoculation, B is water soaking and inoculation, and BS is saline-alkali stress liquid soaking and inoculation). When soaked in water, the root phenotype of the mung bean cutting is shown in figure 3 (the left figure shows that the bacteria are not inoculated, and the right figure shows that the bacteria are inoculated). When the mung bean cutting is soaked in the saline-alkali stress solution, the root phenotype of the mung bean cutting is shown in figure 4 (the left figure shows that the bacteria are not inoculated, and the right figure shows that the bacteria are inoculated). The results show that the Zollinia DQSA1 can promote the rooting of the mung bean cutting. Under normal growth conditions (i.e., water soak, no salt and alkali stress), the number of roots of mung bean cuttings inoculated with the tulip DQSA1 was increased by 60% compared to mung bean cuttings not inoculated with the tulip DQSA 1. Under the condition of saline-alkali stress (namely soaking in saline-alkali stress solution), compared with the mung bean cuttings which are not inoculated with the Zollinia DQSA1, the rooting number of the mung bean cuttings inoculated with the Zollinia DQSA1 is increased by 111.11%.

Example 3 Zollinger DQSA1 ability to improve saline and alkaline stress resistance of corn and mung bean

The saline-alkali soil is collected from saline-alkali grassland of Daqing city of Heilongjiang province in China, the pH value is 8.34, the alkalization degree is 34.28%, and the content of soluble salt is 0.77%. The non-saline-alkali soil is collected in the farmland in the same district of Daqing city of Heilongjiang province in China, the pH value is 6.56, the alkalization degree is 7.60 percent, and the content of soluble salt is 0.10 percent.

The method for detecting chlorophyll content of corn seedlings is referred to the following documents: horse jiamin, isolation of alfalfa growth-promoting rhizobacteria and growth-promoting action research [ D ]. university of harbin, 2013. The methods for detecting the soluble sugar content, the soluble protein content, the superoxide dismutase activity and the malonaldehyde content of the corn seedlings refer to the following documents: symplocos manshurica, dianthus superbus, plum cube, plant physiology experimental guidance [ M ]. beijing: higher education publishers, 2006: 100-227.

preparation of DQSA1 microbial inoculum

(1) A single colony of DQSA1 of Zygotella was inoculated into a conical flask (500 mL) containing 300mL of modified S-G liquid medium, and cultured at 28 ℃ for 24h with shaking at 175rpm to obtain bacterial suspension 1.

(2) After the step (1) is finished, taking bacterial liquid 1, centrifuging for 10min at 10000g, and collecting thalli.

(3) After the step (2) is finished, the thalli are taken, washed three times by using sterile normal saline and then resuspended by using sterile water to obtain the concentration of 10⁹cfu/mL of bacterial suspension 2.

The bacterial liquid 2 is the prepared DQSA1 bacterial agent.

DQSA1 of Zygotella and Zygolella can promote corn growth

1. Obtaining of maize seedlings

A. Obtaining saline-alkali soil + DQSA1 corn seedlings

(1) Taking a square groove filled with saline-alkali soil, inoculating bacterial liquid 2 (the inoculation dose is 10) into the saline-alkali soil⁷cfu/g soil).

(2) After 2 days of the step (1), sowing the sprouted corns, and alternately culturing the corns at the temperature of 27 +/-2 ℃ in light and dark.

(3) Inoculating bacterial liquid 2 (the inoculation dose is 0.5 multiplied by 10) into the saline-alkali soil after the 10 th day of corn seedling emergence in the step (2)⁶cfu/g soil), and then culturing alternately in light and dark at 27 +/-2 ℃ for 30 days to obtain saline-alkali soil + DQSA1 corn seedlings.

B. Obtaining saline-alkali soil corn seedlings

(1) And (3) taking a square groove filled with saline-alkali soil, sowing the corns after germination acceleration, and alternately culturing the corns in light and dark at the temperature of 27 +/-2 ℃.

(2) And (2) alternately culturing the corns in the step (1) in light and dark at 27 +/-2 ℃ for 30 days at the 10 th day of seedling emergence to obtain saline-alkali soil corn seedlings.

C. Obtaining non-saline-alkali soil corn seedlings

(1) And (3) sowing the maize after germination acceleration in a square groove filled with non-saline-alkali soil, and alternately culturing the maize at the temperature of 27 +/-2 ℃ in light and dark.

(2) And (2) alternately culturing the corns in the step (1) in light and dark at 27 +/-2 ℃ for 30 days at the 10 th day of seedling emergence to obtain non-saline-alkali soil corn seedlings.

2. The height, fresh weight, dry weight, chlorophyll content (expressed by SPAD value), soluble sugar content, soluble protein content, superoxide dismutase (SOD) activity and Malondialdehyde (MDA) content of corn seedlings (saline-alkali soil + DQSA1 corn seedlings, saline-alkali soil corn seedlings or non-saline-alkali soil corn seedlings) were tested.

The phenotype of the aerial parts of maize seedlings is shown in FIG. 5 (saline-alkali maize seedlings, saline-alkali + DQSA1 maize seedlings and non-saline-alkali maize seedlings in sequence from left to right). The results of the corn seedling tests are shown in tables 1 and 2: compared with the saline-alkali soil without the application of the tulobergia DQSA1, the plant height, fresh weight, dry weight, chlorophyll, soluble sugar content, soluble protein content and superoxide dismutase activity of the corn planted on the saline-alkali soil with the tulobergia DQSA1 are all increased remarkably, and the malondialdehyde content is reduced remarkably. The results show that the Zollinia DQSA1 can promote the growth of corn, enhance the active oxygen scavenging ability of corn plants and improve the anti-aging ability of corn plants.

TABLE 1

Treatment of	Plant height (cm)	Fresh weight (g)	Dry weight (g)	Chlorophyll (SPAD value)
					Corn seedling of non-saline-alkali soil	69.7±8.2	10.195±3.513	0.912±0.009	27.171±2.635
Saline-alkali soil corn seedling	63.0±5.1	7.320±1.523	0.460±0.005	26.286±2.608
					Saline-alkali soil + DQSA1 maize seedlings	66.0±4.6	8.238±1.963	0.765±0.006	32.971±2.111
Saline-alkali soil mung bean seedling	16.6±2.2	0.827±0.150	0.118±0.003	16.058±2.460
					Saline-alkali soil + DQSA1 mung bean seedlings	18.1±2.3	0.914±0.116	0.144±0.002	31.550±2.537

TABLE 2

DQSA1 of genus Trigonella and Turber can promote growth of semen Phaseoli Radiati

1. Obtaining of mung bean seedlings

A. Obtaining of seedling of mung bean with saline-alkali soil and DQSA1

(1) Taking a square groove filled with saline-alkali soil, inoculating bacterial liquid 2 (the inoculation dose is 10) into the saline-alkali soil⁷cfu/g soil).

(2) And (3) sowing the germinated mung beans 2 days after the step (1) is completed, and alternately culturing the mung beans in light and dark at the temperature of 27 +/-2 ℃.

(3) Inoculating the bacterial liquid 2 (the inoculation dose is 0.5 multiplied by 10) into the saline-alkali soil after the mung beans in the step (2) emerge at the 10 th day⁶cfu/g soil), and then culturing in light and dark alternately at 27 +/-2 ℃ for 30 days to obtain saline-alkali soil + DQSA1 mung bean seedlings.

B. Obtaining of mung bean seedlings in saline-alkali soil

(1) And (3) taking a square groove filled with saline-alkali soil, sowing the mung beans after germination acceleration, and alternately culturing the mung beans in light and dark at the temperature of 27 +/-2 ℃.

(2) And (2) alternately culturing the mung beans in the step (1) in light and dark at 27 +/-2 ℃ for 30 days at the 10 th day of seedling emergence of the mung beans to obtain the mung bean seedlings in the saline-alkali soil.

2. The plant height, fresh weight, dry weight, chlorophyll content (expressed by SPAD value), soluble sugar content, soluble protein content, superoxide dismutase (SOD) activity and Malondialdehyde (MDA) content of the mung bean seedlings (saline-alkali soil + DQSA1 mung bean seedlings or saline-alkali soil mung bean seedlings) are detected.

The phenotype of the aerial parts of mung bean seedlings is shown in FIG. 6 (saline-alkali soil + DQSA1 mung bean seedlings and saline-alkali soil mung bean seedlings in sequence from left to right). The detection results of the mung bean seedlings are shown in tables 1 and 2: compared with the saline-alkali soil without the application of the Zollinia DQSA1, the mung beans planted on the saline-alkali soil with the Zollinia DQSA1 have the advantages that the plant height, fresh weight, dry weight, chlorophyll, soluble sugar content, soluble protein content and superoxide dismutase activity are obviously increased, and the malondialdehyde content is obviously reduced. The results show that the Zollinia DQSA1 can promote the growth of mung beans, enhance the scavenging ability of mung bean plants on active oxygen and improve the anti-aging ability of mung bean plants.

Therefore, the Zollinia DQSA1 can improve the stress resistance of corn and mung bean, such as the capability of resisting saline-alkali stress.

<110> university of eight agricultural reclamation of Heilongjiang

<120> Zygophyllella bacteria and application thereof in improving plant stress resistance

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1439

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 1

tgcacatggc gggcaggcct aacacatgca agtcgagcgg cagcgggaaa gtagcttgct 60

acttttgccg gcgagcggcg gacgggtgag taaggcttgg gtatctgccc agtcgagggg 120

gataaccgtt ggaaacgacg gctaataccg catacgccct gcgggggaaa gcaggggatc 180

ttcggacctt gcgcgattgg atgagcccaa gtgagattag ctagttggtg aggtaatggc 240

tcaccaaggc gacgatctct agctggtctg agaggatgac cagccacact gggactgaga 300

cacggcccag actcctacgg gaggcagcag tggggaatat tgcacaatgg gggaaaccct 360

gatgcagcca tgccgcgtgt gtgaagaagg ccttcgggtt gtaaagcact ttcagtggtg 420

aggaaaggca agcggcgaat acccgcttgc tgtgacgtta accacagaag aagcaccggc 480

taactccgtg ccagcagccg cggtatacgg agggtgcaag cgttaatcgg aatgactggg 540

cgtaaagcgc acgcaggcgg tttgttaagc cagatgtgaa agccccgagc tcaacttggg 600

aactgcattt ggaactggca agctagagtc ttggagaggg gggcagaatt tccggtgtag 660

cggtgaaatg cgtagagatc ggaaggaata ccagtggcga aggcggcccc ctggccaaag 720

actgacgctc aggtgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac 780

gctgtaaacg atgtcaactt ggagtctgtg ccatttgagc gcgggttccg gagctaacgc 840

gttaagttga ccgcctgggg agtacggccg caaggttaaa actcaaatga attgacgggg 900

gcccgcacaa gcggtggagc atgtggttta attcgatgca acgcgaagaa ccttacctac 960

ccttgacata gtaagaactt ggcagagatg ccttggtgcc ttcgggaact tacatacagg 1020

tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080

caacccttat cctttgttgc cagcgattcg gtcgggaact caaaggagac tgccggtgat 1140

aaaccggagg aaggtgggga cgacgtcaag tcatcatggc ccttacgggt agggctacac 1200

acgtgctaca atggcgcgta cagagggcgg cgaaccagcg atggtaagcg aatcccaaaa 1260

agcgcgtcgt agtccggatc ggagtctgca actcgactcc gtgaagtcgg aatcgctagt 1320

aatcgtggat cagaatgcca cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380

caccatggga gtgggttgct ccagaagtag atagcttaac ctttcgggga ggggggccg 1439

Claims (11)

1. The Zobellella sp (Zobellella sp.) DQSA1 has a preservation number of CCTCC NO: m2018587.

2. A microbial inoculum, which is characterized in that: the microbial inoculum contains the genus Zobellella (Zobellella sp.) DQSA1 of claim 1.

3. Use of the agent of claim 1 of Zobellella sp (Zobellella sp.) DQSA1 or claim 2 as at least one of A1) -A3):

A1) promoting the rooting of the mung bean cutting;

A2) promoting the growth of mung beans or corns;

A3) the saline-alkali resistance of the mung bean or the corn is improved.

4. Use of the agent of the genus Zobellella (Zobellella sp.) DQSA1 of claim 1 or of the agent of claim 2 for the preparation of a product; the product has a function of at least one of A1) -A3):

A1) promoting the rooting of the mung bean cutting;

A2) promoting the growth of mung beans or corns;

A3) the saline-alkali resistance of the mung bean or the corn is improved.

5. The use of claim 4, wherein: the product is a microbial fertilizer.

6. A method for promoting rooting of mung bean cutting shoots, which is to treat mung bean cutting shoots by adopting Zobellella sp (Zobellella sp.) DQSA1 of claim 1 so as to promote rooting of mung bean cutting shoots.

7. The method of claim 6, wherein: the mung bean cutting treated by the Zobellella sp (Zobellella sp.) DQSA1 according to claim 1 is a mung bean cutting treated by the microbial inoculum according to claim 2.

8. A method for promoting plant growth, which comprises treating a plant with the Zobellella sp (Zobellella sp.) DQSA1 of claim 1 to promote plant growth;

the plant is mung bean or corn.

9. The method of claim 8, wherein: the plant treated with the Zobellella sp (Zobellella sp.) DQSA1 of claim 1 is a plant treated with the microbial inoculum of claim 2.

10. A method for improving the saline-alkali resistance of plants, which is to treat the plants with the Zobellella sp (Zobellella sp.) DQSA1 of claim 1, thereby improving the saline-alkali resistance of the plants;

the plant is mung bean or corn.

11. The method of claim 10, wherein: the plant treated with the Zobellella sp (Zobellella sp.) DQSA1 of claim 1 is a plant treated with the microbial inoculum of claim 2.

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