CN114774300B - Pseudomonas koraiensis and application thereof - Google Patents

Abstract

The invention belongs to the field of microorganisms, and relates to Korean pseudomonas which is Pseudomonas koreensis GS and is submitted to China general microbiological culture collection center (CGMCC) for preservation in 2021, 9 and 22 days, wherein the preservation number is NO.23459. The invention provides Korean pseudomonas capable of improving the growth condition of tomato plants under drought stress conditions, improving the resistance enzyme activity of tomatoes, reducing the MDA content of cells and improving the drought resistance of tomatoes and application thereof.

Description

Pseudomonas koraiensis and application thereof

Technical Field

The invention belongs to the field of microorganisms, and relates to Korean pseudomonas and application thereof.

Background

The growth of crops is affected by a series of environmental factors, wherein drought, salinity, low temperature and the like are main factors which lead to the reduction of the yield of crops. Drought stress severely affects plant growth, development and propagation, resulting in substantial crop yield loss. It is counted that drought can cause up to 90% of agricultural economic losses, which is one of the most serious natural disasters. Drought stress directly affects various stages of plant growth and development, causing a series of negative effects on the physiological and biochemical level of plants. For example, drought stress causes an increase in plant cell membrane permeability, hormone imbalance, accumulation of active oxygen in large amounts, decrease in photosynthetic efficiency of plants, and disturbance of nutrient metabolism, and eventually plant growth is suppressed, yield is reduced. The plant biomass, water utilization efficiency, photosynthetic capacity, osmotic regulation capacity, cell membrane stability, antioxidant system defense capacity, hormone level and other index changes are often used as standards for judging the drought stress resistance of plants. In addition to drought stress, low temperature stress is also a disaster frequently encountered in crop cultivation, which not only results in reduced plant yield, but also causes plant death when severe. The world is statistically costing $ 2000 billion annually for low temperature losses. Therefore, improving the low temperature stress resistance of crops and reducing the adverse effect of low temperature on crops is of great importance in agricultural production. According to the degree of low temperature, the low temperature damage of plants can be divided into two major categories, namely cold damage (damage to plants at low temperature) and freeze damage (damage to plants at low temperature). The cold injury in the low temperature stress is more common in the occurrence area, and damages the normal functions and structures of cells, especially the cytoplasmic membrane and the organelle membrane system, or interferes with normal activities, so that metabolism is disturbed, not only is the functions disturbed, but also the structures and tissues are often damaged, and further flaws appear rapidly in morphology. The low temperature stress causing cold damage can cause adverse effects in the whole growth process of plants, such as seed germination, plant growth, photosynthesis, fruit setting, yield, quality formation and the like. The result of cold injury is weak seedling, slow plant growth, wilting, yellowing, local necrosis, low fruit setting rate, reduced yield, reduced quality, etc. Researchers have attempted to improve the cold resistance of crop plants by different means based on studies of the physiological and biochemical effects of cold injury on plants. Tomato (Lycopersicon esculentum Mill.) is a commonly planted vegetable in China and has important economic value. Tomato is a annual or perennial herb plant, and the fruits are rich in various mineral elements, carotene, B and C vitamins and the like. Tomatoes are very sensitive to drought stress and water deficiency can severely affect tomato yield. It was found that water stress inhibited tomato seedling germination and radicle and hypocotyl growth, reducing tomato seedling biomass. In addition, under drought stress, tomatoes can show corresponding negative characterization at various levels of cells, organs, individuals and populations, tomato growth and development are inhibited, and fruit yield is reduced.

At present, the harm of cold injury to crops is generally reduced by improving agricultural measures, such as improving the soil temperature by adopting greenhouse planting, film-covered planting and other modes in winter, and improving the crop growth environment; adopts cross breeding or genetic engineering to cultivate cold-resistant varieties, and reduces the adverse effect of low temperature. However, improvement of agronomic measures is time-consuming and labor-consuming, and further, since cold resistance of plants is a comprehensive expression not caused by expression of a single gene, which includes changes in other characteristics of plant cells such as cell membrane fluidity, synthesis and accumulation of low-molecular or high-molecular weight cryoprotectants, etc., no breakthrough progress has been made in obtaining a variety of plants resistant to low temperatures by conventional breeding means or transgenic techniques. A simple and easy way to improve the cold resistance of crops is through some exogenous substances, including plant growth regulating substances such as hormone substances such as abscisic acid, brassinolide, salicylic acid and the like; osmoregulating substances such as soluble sugars, betaines, and the like; inorganic salt ions such as Ca ⁺ ，K ⁺ And the like, improves the cold resistance of crops. The beneficial microorganism can also improve the cold resistance of plants, can be stably planted in the rhizosphere of the plants, and has the functions of improving soil structure, preserving water, promoting the absorption of mineral nutrition by the plants and the like to promote the growth of the plants, so that the application of the beneficial microorganism has an important role in improving the cold resistance of crops.

In addition to drought resistance, research has recently found that beneficial microorganisms can promote plant growth under drought conditions. The growth promoting rhizobacteria (PGPR) can produce hormone substances such as indoleacetic acid, cytokinin and the like to promote the growth of Plant root systems, increase secondary roots and root hair quantity, facilitate the absorption of water and nutrients by plants, promote the growth of plants and enhance the tolerance of the plants to drought, pathogenic bacteria infection and other stress. The beneficial microorganism can also induce and enhance drought tolerance of plants and regulate physiological and biochemical processes related to drought resistance of the plants. For example, rhizosphere bacteria Bacillus amyloliquefaciens can significantly improve the activity of antioxidant related enzymes in plants, reduce the content of active oxygen and malondialdehyde, and alleviate oxidative damage caused by drought stress; streptomyces melarsonii Act12 can increase wheat biomass, improve water-soluble total sugar content, regulate proline and glutathione content, and can enhance drought tolerance of wheat through ABA dependent signal transmission; the subilis GB03 can induce the plants to accumulate more permeation regulating substances such as proline, betaine and the like, and relieve damage to plant cell membranes and biological macromolecules caused by drought stress.

Pseudomonas belongs to the Pseudomonas family of gram-negative bacteria, which are widely distributed in soil, fresh water, sea water and organisms. The pseudomonas has a wider adaptation temperature range, can grow at 4-43 ℃ and has an optimal growth temperature of about 30 ℃. The proper pH value range for the growth of the bacteria is between pH 7.0 and 8.5, and most bacteria cannot grow in an environment with pH of 6 or below. Non-pathogenic pseudomonas is active in plant rhizosphere and is one of the most predominant PGPR. The fluorescent pseudomonas belongs to the Pseudomonas genus of the Pseudomonas family of the Phanerochaete phylum, is widely distributed on the surfaces of plant rhizosphere soil and fruits and vegetables, and a plurality of strains can effectively inhibit pathogens and promote the growth and yield increase of fruits and vegetables, so that the fluorescent pseudomonas is concerned by researchers in various countries, and is also a type of biocontrol bacteria and rhizosphere growth-promoting bacteria which are the earliest in research and most reported at home and abroad. Pseudomonas fluorescens can promote the growth of plants by producing auxin, enhancing the effectiveness of phosphorus and potassium in soil, promoting the increase of chlorophyll content of plants, producing glutathione, ACC deaminase and the like. In addition, the pseudomonas fluorescens can also improve the invasion resistance of plant bodies against insect pests. The research shows that the pseudomonas fluorescens has an inhibition effect on the root knot nematode in and out of the plant body, and can inhibit the incidence rate of mung bean root knot nematode disease so as to improve the seed yield of mung beans; nitropyrrolin and pyrrolomycin produced by Pseudomonas fluorescens strains Ps170 and Ps117 have great potential in controlling Pyricularia pyriformis (Erwinia amylovora). Pseudomonas korea (Pseudomonas koreensis) is a subspecies of Pseudomonas fluorescens, however, the use of Pseudomonas korea for the study of mechanisms for improving stress resistance in plants has been recently reported.

Disclosure of Invention

In order to solve the technical problems in the background art, the invention provides Korean pseudomonas capable of improving the growth condition of tomato plants under drought stress, improving the resistant enzyme activity of tomatoes, reducing the MDA content of cells and improving the drought resistance of tomatoes and application thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a pseudomonas korea, characterized in that: the Korean pseudomonas is Pseudomonas koreensis GS, which is submitted to China general microbiological culture collection center (CGMCC) for preservation in 2021, 9 and 22 days, and the preservation number is No.23459.

A korean pseudomonas as described above, characterized in that: the Korean Pseudomonas was isolated from the south foot of the Khingan mountain of Heilongjiang province with geographical coordinates of 46 DEG 57'N,128 DEG 16' E.

Use of pseudomonas koraiensis Pseudomonas koreensis GS as described above for plant growth promotion.

Use of pseudomonas koraiensis Pseudomonas koreensis GS as described above for tomato growth promotion.

Use of pseudomonas koraiensis Pseudomonas koreensis GS as described above for tomato growth promotion under stress conditions.

The use of pseudomonas koraiensis Pseudomonas koreensis GS as described above for tomato growth promotion under drought stress or under cold stress.

The use of pseudomonas koraiensis Pseudomonas koreensis GS to promote germination of tomato seeds under drought stress or under cold stress as described above.

The use of pseudomonas koraiensis Pseudomonas koreensis GS as described above to promote the growth of tomato plants under drought stress or under cold stress.

The use of pseudomonas koraiensis Pseudomonas koreensis GS as described above for improving drought tolerance or for enhancing cold tolerance of tomatoes.

The invention has the advantages that:

according to the invention, tomato and P.koreensis GS are taken as objects of the invention, and through dish and pot experiments, drought stress is carried out by respectively adopting methods of adding polyethylene glycol and controlling watering amount, and the effect of P.koreensis GS cell-free fermentation filtrate on growth promotion and drought tolerance of tomato plants is revealed from the biological form and physiological and biochemical index measurement of the tomato. In the germination test in a dish, the P.koreensis GS fermentation filtrate can improve the germination rate of tomato seeds, and simultaneously obviously improve the plant height, root length, fresh weight and stem thickness of tomato seedlings, wherein the amplifications are 9.5%, 29.08%, 7.74% and 28.96% respectively; under drought stress, the P.koreensis GS fermentation filtrate treatment has obvious growth promoting effect on tomato seedlings, and the plant height, root length, fresh weight and dry weight of the tomato seedlings are obviously improved by 1.05%, 14.86%, 16.99% and 6.10%. In the potting test, the plant height, fresh weight, dry weight, root length, root dry weight and chlorophyll relative content (SPAD) of the potted tomato (30 d) after the treatment of the p.koreensis GS fermentation filtrate are obviously improved by 4.75%, 4.15%, 1.81%, 23.25%, 9.83% and 9.89%, respectively; under drought stress treatment, the plant height, root length, fresh weight, dry weight, SPAD value and root dry weight of the potted tomato (30 d) are obviously improved by 1.56%, 9.82%, 11.05%, 3.77%, 13.60% and 1.69%, respectively. Therefore, the P.koreensis GS fermentation filtrate improves the growth condition of tomatoes under drought stress treatment and improves the drought tolerance of tomatoes. In addition, the germination test in the dish shows that after the P.koreensis GS fermentation filtrate is treated, the activities of Peroxidase (POD) and Catalase (CAT) in seedlings in the dish are obviously improved by 11.85 percent and 97.07 percent respectively under the non-drought condition; the CAT enzyme activity of tomato seedlings in the dish is obviously improved by 50.16% under drought stress; in a potting test, the leaf CAT enzyme activity of a tomato plant (30 d) treated by the P.koreensis GS fermentation filtrate is obviously increased by 27.30% under a non-drought condition, and the leaf POD and CAT enzyme activities are respectively obviously increased by 64.51% and 358.25% under drought stress; the content of permeation-resistant regulating substances proline and Malondialdehyde (MDA) in the leaves of the tomato plant (30 d) is not obviously changed under the non-drought condition, and the MDA content (representing the peroxidation degree of membrane lipid) is obviously reduced by 38.30% and the proline content is obviously improved by 2.59% under drought stress. The invention shows that the P.koreensis GS can improve the growth condition of tomato plants under drought stress, improve the resistant enzyme activity of crops, reduce the MDA content of cells and further improve the drought tolerance of tomatoes. In addition, the P.koreensis GS cell-free fermentation liquid can promote the growth of the plant height of the tomato seedlings, has obvious growth promoting effect under the condition of cold damage, can obviously improve the contents of various chlorophyll in the leaves of the tomato seedlings, and can improve the photosynthesis of the tomato seedlings under the stress of cold damage; the korensis GS cell-free fermentation filtrate can improve the activity of tomato seedling defense enzyme SOD and POD, and enhance the cold damage resistance of tomato seedlings.

Drawings

FIG. 1 is a bar graph of the effect of P.koreensis GS cell-free fermentation filtrate on germination rate in tomato dishes under drought and non-drought conditions (different letters indicate significant differences (P < 0.05));

FIG. 2 is a graph showing the potted plant comparison of the effect of P.koreensis GS cell-free fermentation filtrate on tomato plant biological index under drought and non-drought conditions;

FIG. 3 is a bar graph of the effect on tomato seedling defensive enzyme activity after treatment of P.koreensis GS cell-free fermentation filtrate under drought and non-drought conditions (the different letters represent significant differences (P < 0.05));

FIG. 4 is a graph showing potting contrast of the effect of P.koreensis GS cell-free fermentation filtrate treatment on tomato plant biological indicators under drought and non-drought conditions;

FIG. 5 is the effect on tomato plant defensive enzyme activity after treatment of P.koreensis GS cell-free fermentation filtrate under drought and non-drought conditions (the different letters indicate significant differences (P < 0.05));

FIG. 6 is the effect of P.koreensis GS cell-free fermentation filtrate treatment on proline and MDA content of tomato plants under drought and non-drought conditions (different letters indicate significant differences (P < 0.05));

FIG. 7 is the effect of P.koreensis GS cell-free fermentation filtrate treatment on tomato seedling height under cold damage and warm conditions;

FIG. 8 is the effect of P.koreensis GS cell-free fermentation filtrate treatment on tomato seedling growth under cold damage and warm conditions.

Detailed Description

The invention provides Korean pseudomonas and application thereof, in particular application of Korean pseudomonas to promotion of tomato growth and improvement of drought resistance of tomatoes under drought stress.

1. The invention is described in detail by the following test

(1) Influence of fermentation filtrate of Pseudomonas koreensis GS under drought stress (hereinafter abbreviated as P.koreensis GS) on germination status of tomato seeds

The effect of P.koreensis GS cell-free fermentation filtrate seed soaking on tomato response to drought stress is revealed by a polyethylene glycol dish simulated drought stress test.

(2) Effect of P.koreensis GS fermentation filtrate on tomato seedling growth morphology and drought tolerance index under drought stress

(3) Effect of p.koreensis GS fermentation filtrate on tomato seedling growth morphology and cold tolerance under cold stress.

The tomato is taken as a plant to be tested, the watering quantity is controlled to be a stress means through a nutrition pot drought stress test, and the influence of P.koreensis GS cell-free fermentation filtrate on biological properties (plant height, root length, fresh weight, dry weight, stem thickness and the like) and physiological and biochemical properties (malondialdehyde, proline, antioxidant enzyme and the like) of the tomato is revealed.

According to the invention, through in-dish germination and potting experiments, the effect of the P.koreensis GS cell-free fermentation filtrate on tomato growth, drought tolerance and cold tolerance under drought stress conditions is explored.

2. Detailed description of the invention

2.1 materials and methods

2.1.1 test strains

Korean Pseudomonas Pseudomonas koreensis GS (NCBI accession number: PRJNA 517377) isolated from the Protopanax persicae, mountain Tiande Ginseng radix Limited company Ginseng radix base, heilongjiang, federation, sorption mountain, inc., south America, odonia, 46 DEG 57'N,128 DEG 16' E, brown loam, deposited by the resource environment, resource laboratory, university of North agriculture and forestry, north, and technology. Meanwhile, the strain is submitted to China general microbiological culture collection center (CGMCC) for preservation in 2021, 9 and 22 days, and the preservation number is No.23459.

2.1.2 test tomato variety

Tomato seeds (golden shed number one) were purchased from the western amp gold peng seedling limited company.

2.1.3 test Medium

Beef extract-peptone liquid medium (NB) was used for liquid fermentation culture of pseudomonas korea;

beef extract-peptone agar medium (NA) was used for culture and preservation of pseudomonas korea.

The soilless culture seedling substrate consists of high-quality turf, substrate raw materials, perlite, vermiculite and the like, wherein the organic matter content is more than 35%, the water content is more than 20%, the pH value is neutral, and the soilless culture seedling substrate is purchased from the county Lu Yuan seedling substrate limited company.

2.1.4 drought stress test

A) In-dish germination and growth-promoting drought-enduring test

An in-house dish bioassay was used. After shaking (180 rpm) the p.koreensis GS in beef extract peptone liquid medium at 28 ℃ for 72 hours, the culture was centrifuged at 10,000 rpm for 15 minutes at 4 ℃ and filtered through a sterile microfiltration membrane (0.22 μm pore size), p.koreensis GS sterile fermentation filtrate was collected and diluted with sterile water to 100-fold dilution for use. 600 tomato seeds were sterilized with 75% alcohol for 30s, washed with sterile water for at least 5 times, and the sterilized tomato seeds were uniformly placed in culture dishes (20 seeds per dish) with sterilized double-layered filter paper.

The sterilized tomato seeds were divided into two groups, one group was soaked with sterile water and the other group was soaked with 100-fold diluted p.koreensis GS sterile fermentation filtrate (the invention is described in detail by taking 100-fold dilution as an example, specific dilution screening is shown in table 1), and after treatment the dishes were placed in a constant temperature light incubator at 25 ℃ for dark germination. A new 10mL of the corresponding treated solution was replaced to each dish at 1d intervals from 2d, and the number of seed germination per dish was observed and recorded at regular intervals each day. The start time of germination of the seeds under this treatment was indicated by the exposure of the embryo to the seeds, the day 1 of exposure of the white embryo to the seeds, and the end time of the treatment test was indicated by the absence of the exposure of the embryo to the seeds for 3 consecutive days.

As can be seen in table 1, first, the growth of tomato seedlings under drought stress conditions was significantly inhibited compared to normal growth conditions. Secondly, under the condition of non-drought and drought stress, the dry weight, root length and plant height of the ground fresh weight of tomato seedlings are all increased compared with those of a control under the treatment of P.koreensis GS cell-free fermentation filtrate with different dilution factors. Under the non-stress condition, the fresh weight, dry weight, root length and plant height increase rate of the ground under the treatment of the P.koreensis GS cell-free fermentation filtrate with different dilution factors are respectively 0.55 to 14.78 percent, 7.76 to 17.24 percent, 28.72 to 45.61 percent and 0.086 to 6.02 percent; under drought stress, the increase rate is 17.78% -25.40%, 27.50% -45.83%, 12.00% -36.27% and 0.97% -4.87% respectively. Under drought conditions, the fresh weight, dry weight and root length of tomato seedlings on the ground are obviously improved under the condition that the maximum dilution is 1000 times, and the increase rates are 25.40%, 27.50% and 12% respectively. From the results, the P.koreensis GS fermentation filtrate has obvious growth promoting effect on the growth of tomato plants under the dilution gradient of 100-1000 times, can effectively improve the drought tolerance of the tomato plants, and has stronger effect on improving the dry weight of tomato seedlings under drought stress conditions. Based on Table 1, the invention was described in detail with 100-fold dilution.

TABLE 1 influence of P.koreensis GS cell-free fermentation filtrate treatment on biological index of tomato seedlings under drought and non-drought conditions

Note that: different letters in the same column indicate significant differences between groups (P < 0.05).

After tomato seedlings grow for 10d, preparing polyethylene glycol solution (hydrophilic macromolecules, strong water absorption, which can cause water absorption difficulty of plants and simulate drought stress) with the concentration of 12% by using sterile water and 100 times of diluted acellular fermentation filtrate respectively, and performing drought stress treatment, thereby setting four treatments respectively as follows: control group (CK) with only sterile water, treatment (PK) with only P.koreensis GS sterile fermentation filtrate, sterile water+polyethylene glycol (DK), P.koreensis GS sterile fermentation filtrate+polyethylene glycol (DPK), and 7mL of treatment solution were added, 20 tomato seeds in each dish, one repetition per dish, 5 repetitions per treatment were set, and 100 tomato seeds per group were treated. And (3) after drought stress treatment, harvesting tomato seedlings for 0h and 96h, and measuring biological traits and physiological and biochemical indexes of plants.

B) In-dish growth promotion and cold resistance test

Tomato seed germination design: after shaking culture of P.koreensis GS in beef extract peptone liquid medium at 180rpm for 72 hours at 28℃the culture was centrifuged at 10000rpm for 15min at 4℃and filtered through a sterile microfiltration membrane (0.22 μm pore size) to collect P.koreensis GS cell-free fermentation broth, and the filtrate was diluted 50-fold and 100-fold with sterile water.

Taking a proper amount of tomato seeds, sterilizing for 30s by using 75% alcohol, washing for at least 5 times by using sterile water, and uniformly placing the sterilized tomato seeds into culture dishes (30 seeds per dish) paved with sterilized double-layer filter paper, wherein the total number of the culture dishes is 30. The culture dishes are divided into three groups of 10, the seeds are soaked with sterile water and cell-free fermentation liquid diluted 50 times and 100 times respectively, the required treatment liquid mainly wets filter paper and seeds, the initial mass is recorded, and the culture dishes are placed in a constant-temperature illumination incubator at 25 ℃ for dark germination after treatment. Each dish was periodically replenished with the corresponding treated solution daily starting from day 2d, ensuring the same initial mass, and the number of seed germination per dish was periodically observed and recorded daily. The germination mark is 2mm of the exposed embryo of the seed, and the germination of no seed is continued for 3 days as the end of germination. After the tomato sprouting is finished, respectively carrying out cold injury stress and temperature adaptation treatment by using sterile water and cell-free fermentation liquid diluted by 50 times and 100 times. Six treatments were set up, each 5 replicates, in the manner shown in table 2.

TABLE 2 different temperature stress treatment conditions

Placing the cold damage stress treated culture dish in an illumination constant temperature incubator for culturing, and setting day/night time for 14h/10h, wherein the temperature is 17 ℃/10 ℃, the air relative humidity is 65%, and the illumination intensity is 8000lx; the culture dish suitable for the temperature treatment is placed in another illumination constant temperature incubator for culture, day/night time is arranged for 14h/10h, the temperature is 26 ℃/20 ℃, the relative air humidity is 65%, the illumination intensity is 8000lx, and 15ml of corresponding treatment liquid is added every day for each treatment. And measuring the main plant height of the tomato seedlings at 10d after treatment, and harvesting the tomato seedlings at 14d after treatment for measuring physiological and biochemical indexes of plants.

2.1.5 potted plant test

The culture medium is soilless culture medium, and the culture medium is sterilized by damp heat at 121 ℃ for 2 hours and is placed in a nutrition pot. About 800 tomato seeds were sterilized with 75% alcohol for 30s, washed with sterile water for 5 times, and the sterilized seeds were aliquoted into two portions, one portion was soaked with the p.koreensis GS sterile fermentation filtrate having a dilution factor of 100 times, and one portion was soaked with an equal volume of sterile water. Soaking seeds for 72 hours, respectively sowing two treated tomato seeds into a nutrition pot (3 seeds per pot), and reserving 2 plants per pot after seedling establishment. The experimental design was a random complete block design with 3 blocks, each containing 80 potting. The potting of each granule was randomly aliquoted into Control (CK) and treatment (PK) groups, 40 pots per group, 240 pots total.

The culture pot is according to the followingThe complete machine block is cultured in a constant temperature illumination incubator under the conditions of illumination time of 12h/12h, temperature of 25 ℃ and relative humidity of 65 percent and illumination of 1000 mu mol.m ^-2 s ^-1 . The experimental group and the control group were irrigated with 20mL of 1/2Hoagland nutrient solution every day at 1-25 d, and the experimental group was supplemented with nutrient solution prepared from 100-fold diluted P.koreensis GS sterile fermentation filtrate every 3 d. All treatments were thinning at 6d and at 25d the treatment groups were divided into normal water groups and drought stress groups, the normal water groups continued to be watered with 1/2Hoagland nutrient solution, without being watered for drought treatment, and drought stress was followed until wilting of tomato seedlings occurred. Four treatments were thus set, namely a control group (CK) with only nutrient solution, a treatment (PK) with P.koreensis GS sterile fermentation filtrate, a treatment (DK) with nutrient solution+drought stress, and a treatment (DPK) with P.koreensis GS sterile fermentation filtrate+drought stress, 20 pots per component (2 plants per pot), total 480 plants.

2.2 determination of biological indicators

2.2.1 in-dish test

Immediately measuring plant height, root length (main root length) and biological fresh weight of the tomato seedlings after harvesting, absorbing the surface moisture of the material by using absorbent paper, deactivating enzymes of the material in a blast oven at 105 ℃ for 10min, continuously drying the material at 75 ℃ to constant weight, and weighing the dry weight respectively.

2.2.2 potted plant experiments

And (3) collecting 20 tomato plants from each of the experimental group and the control group, and measuring biological indexes such as overground plant height, root length, fresh weight, stem thickness and the like of the tomato plants to analyze the growth promotion effect of the strains. Potted tomatoes 30d, wherein the SPAD value of tomato leaves is respectively and rapidly determined by a chlorophyll meter at the midday of 11:00; measuring plant height, root length, fresh weight, dry weight and fresh weight of the root; mixing 3 samples from four treatments, each biological sample consisting of specific leaves (5 th leaf from growing point) of random 3 tomato plants for enzyme activity measurement; from each treatment 6 samples were taken in a mix, each biological sample consisting of specific leaves (4 th leaf from the growing point down) of random 3 tomato plants for the determination of proline and malondialdehyde. The leaf samples are directly put into an ice box after being sampled, brought back to a laboratory and put into a refrigerator at the temperature of minus 80 ℃ for preservation for later use.

2.2.3 enzyme Activity assay

Preparing a crude enzyme solution: adding liquid nitrogen into the sample, rapidly grinding, adding precooled 0.1M sodium phosphate buffer solution (pH 7.0), rapidly mixing, centrifuging at 4deg.C for 10min at 000Xg, taking supernatant as crude enzyme solution, and standing at 4deg.C in refrigerator for use.

Peroxidase (POD): the reaction system measured comprises 0.2mL of crude enzyme solution (blank control plus 0.2mL of pre-deactivated crude enzyme solution), 1.0mL of 0.1% guaiacol, 1mL of 0.18% hydrogen peroxide, and 7.6mL of distilled water; the absorbance at 470nm was varied by 0.01 per minute as 1 enzyme activity unit U, the enzyme activity being expressed as U/min.

Catalase (CAT): the assay reaction system contained 0.1mL of crude enzyme solution (blank plus 0.1mL of pre-inactivated crude enzyme solution), 1.0mL of Tris-HCl (pH 7.0), 1.7mL of distilled water, and after 3min of pre-heating at 25℃0.2mL of 200mmol/L hydrogen peroxide was added tube by tube, the absorbance varied by 0.01 per minute at 240nm as 1 enzyme activity unit U, and the enzyme activity was expressed as U/min.

2.2.4 determination of proline content

Determination of proline content: 0.5g of fresh sample is taken, 10mL of 3% sulfosalicylic acid is added, the mixture is leached in a boiling water bath for 15min, and the supernatant is centrifugally taken for measuring the proline content. The 6mL reaction system is: 2mL of the leaching solution, 2mL of glacial acetic acid, 2mL of acidic ninhydrin, and after reaction at 100 ℃ for 30min, the reaction was terminated in ice water. The reaction mixture was extracted with 5mL of toluene, the absorbance at 520nm was measured on the supernatant, and the proline content was calculated by a standard curve.

2.2.5 determination of Malondialdehyde (MDA) content

The content of malondialdehyde reflects the peroxidation level of membrane lipid, and the content of malondialdehyde is determined by using a 2-thiobarbituric acid (TBA) method. Malondialdehyde in tomato leaves was extracted by grinding with 5% trichloroacetic acid (TCA), adding 2mL of the extract and 2mL of thiobarbituric acid to the tube, mixing well, boiling the water bath for 12min, rapidly cooling to terminate the reaction, if any, precipitating, and centrifuging at 4500rpm for 10min. The absorbance of the supernatant was measured at 532nm, 600nm and 450nm with thiobarbituric acid solution as a blank. The calculation formula is as follows:

MDA(μmol·g ^-1 FW)＝[6.452×(A652－A600)－0.559×A450]×Vt/Vs/FW

wherein Vt is total volume of the extract (mL); vs is the volume (mL) of the extract for measurement; FW is the fresh weight of the sample (g).

2.3 data analysis

Statistical analysis of the data was performed using Excel 2007 and SPSS 21.0 software. Analysis of variance and multiple comparisons (α=0.05) were performed using one-way ANOVA and LSD methods. Mapping was performed using Excel 2007 software. Data in the graph are mean ± standard deviation.

2.4 results of germination and growth-promoting drought-enduring test in dish

2.4.1 Effect of the fermentation filtrate of the koreesis GS on the growth of tomato seedlings in dishes

2.4.1.1 Effect of the fermentation filtrate of the koreesis GS on the germination rate in the tomato dish

As can be seen from FIG. 1, the germination rate of tomatoes seeded with P.koreensis GS cell-free fermentation filtrate (referred to as fermentation filtrate hereinafter) was higher than that of the control group. The germination rate of PK group is obviously improved by 26.88% in 1d compared with CK group, 31.25% in 2d and 25.0% in 3 d. The germination rate of the DPK group is also obviously improved compared with that of the DK group, which shows that the P.koreensis GS fermentation filtrate can improve the germination rate of tomato seeds under the drought stress condition.

2.4.1.2 Effect of the fermentation filtrate of the koreensis GS on the biological properties of tomatoes in dishes

TABLE 3 influence of P.koreensis GS cell-free fermentation filtrate on biological index of tomato seedlings under drought and non-drought conditions

Note that: the same column of different uppercase letters indicates significant differences between groups (P < 0.01), and the same column of different lowercase letters indicates significant differences between groups (P < 0.05). The following is the same as

As shown in Table 3, the P.koreensis GS fermentation filtrate has a promoting effect on tomato plant growth under non-stress treatment, and the P.koreensis GS fermentation filtrate remarkably improves the plant height, root length, fresh weight and dry weight of tomatoes, which are remarkably improved by 2.72%, 14.80%, 10.60% and 26.47%, respectively. Under drought stress, the P.koreensis GS fermentation filtrate has obvious growth promoting effect on tomato plants, the tomato plants in the DPK group are obviously improved by 1.06% compared with DK group plants, the root length is obviously improved by 14.86%, the fresh weight is obviously improved by 18.18%, and the dry weight is obviously increased by 3.33%. The results show that P.koreensis GS fermentation filtrate can promote the growth of tomato seedlings under drought conditions.

2.4.1.3 Effect of treatment of fermentation filtrate of koreensis GS on tomato defensive enzyme activity in dishes

As shown in FIG. 3, POD and CAT enzyme activities in tomato seedlings of PK group were significantly improved by 11.85% and 97.07% compared with those of CK group (FIG. 3a and FIG. 3 b). The POD enzyme activity of DPK group was not significantly changed compared with DK group (FIG. 3 a), and CAT enzyme activity was significantly increased by 50.16% compared with DK group (FIG. 3 b). It is demonstrated that the application of P.koreensis GS fermentation filtrate can increase the activities of tomato plant defense enzymes POD and CAT, and that under drought stress, tomato plant CAT enzyme activity is more sensitive to P.koreensis GS fermentation filtrate treatment.

2.4.2 Effect of the fermentation filtrate of the p.koreensis GS on the biological properties of potted tomato plants

2.4.2.1 Effect of the fermentation filtrate of the p.koreensis GS on the growth of potted tomato plants

As shown in Table 4, at 25d of treatment, the P.koreensis GS fermentation filtrate significantly increased the plant height, root length, and stem thickness of tomato plants by 13.35%, 27.76% and 12.50%, respectively, and the fresh weight of tomato plants was also significantly increased by 16.05% compared with the control group. The result shows that the P.koreensis GS fermentation filtrate has growth promoting effect on potted tomato plants.

TABLE 4 influence of P.koreensis GS cell-free fermentation filtrate treatment on biological indicators of tomato plants under drought conditions (25 d)

TABLE 5 influence of P.koreensis GS cell-free fermentation filtrate treatment on biological indicators of tomato plants under drought and non-drought conditions (30 d)

As shown in table 5, at 30d treatment, the plant height, fresh weight, dry weight, root length and SPAD values of tomato were significantly increased by 4.75%, 4.15%, 1.81%, 23.25%, 9.89% and root dry weight of tomato plants were significantly increased by 9.83% respectively. Under drought stress, the P.koreensis GS fermentation filtrate has promoting effects on tomato plant height, root length, fresh weight, dry weight and SPAD value. Compared with DK group, DPK group has plant height, root length, fresh weight, dry weight and SPAD value increased by 1.56%, 9.82%, 11.05%, 3.77% and 13.60%, and tomato root dry weight increased by 1.69%. The fermentation filtrate of the KORESSIS has remarkable growth promoting effect on tomato plants, and effectively improves drought tolerance of the tomato plants.

2.4.2.2 Effect of the fermentation filtrate of the koreesis GS on the defensive enzymatic activity of potted tomatoes

As shown in FIG. 4 and FIG. 5, CAT enzyme activity of tomato plants treated with P.koreensis GS fermentation filtrate was significantly improved by 27.30% as compared with CK group (FIG. 5 b), and POD enzyme activity was not significantly changed (FIG. 5 a). The POD and CAT enzyme activities of the tomato plants in the DPK group are obviously improved by 64.51 percent and 358.25 percent respectively compared with those in the DK group (figures 5a and b). The result shows that the P.koreensis GS fermentation filtrate treatment under drought stress has an activating effect on the activity of POD and CAT enzyme in tomato plants.

2.4.2.3 Effect of korensis GS fermentation filtrate on proline and malondialdehyde content of potted tomatoes

As shown in fig. 6, the treatment of the p.koreensis GS fermentation filtrate had no significant effect on the proline and MDA contents of tomato plants under non-drought conditions, the proline content of the DPK group was significantly increased by 2.59% compared with the DK group (fig. 6 a), and the MDA content was significantly decreased by 38.30% compared with the DK group (fig. 6 b). Under the non-stress condition, the proline and MDA content in the tomato plant is not obviously changed; under drought stress, MDA content in tomato plants is obviously changed and is sensitive to treatment of P.koreensis GS fermentation filtrate.

Drought stress causes damage to plant growth and development from many aspects: reactive Oxygen Species (ROS) accumulate excessively, cell membrane and nuclear structure are damaged, leaf and root growth is inhibited, photosynthetic rate is lowered, etc. The plant leaves and root systems are damaged, the activity of the root system is reduced, and the absorption of moisture and mineral elements by plants can be inhibited. Under drought stress, the indexes such as tomato plant height, fresh weight, root length, SAPD and the like are obviously improved after the P.koreensis GS fermentation filtrate is treated, which indicates that the P.koreensis GS fermentation filtrate can improve the drought tolerance of tomato plants. When plants are subjected to drought stress, the biofilm system is a sensitive and initial part, and the high concentration of ROS has toxic effects on plant cell membrane systems and the like. The plant cells have a complete ROS scavenging system, and can regulate and improve the activities of antioxidase or antioxidant substances in adverse circumstances, so that the ROS level in the cells is maintained within a certain range, and further, the oxidative stress caused by the stress is relieved. Osmotic regulation systems in plants promote plants to accumulate large amounts of solutes (sugar alcohol compounds, amino acids, secondary metabolites, organic acids, etc.) to adapt to stress. Under drought stress, the activities of POD and CAT enzymes in tomato leaves treated by the P.koreensis GS fermentation filtrate are obviously improved, the content of proline is obviously increased, and the content of MDA is obviously reduced, which shows that the P.koreensis GS fermentation filtrate participates in the osmotic adjustment of tomatoes, reduces the accumulation of ROS in the leaves, and eases the lipid peroxidation degree (MDA) of the cell membranes of the tomato leaves, thereby reducing the damage of the ROS to the cell membranes.

According to the technical scheme provided by the invention, the P.koreensis GS fermentation filtrate treatment has remarkable growth promotion and drought tolerance regulation effects on tomatoes under drought stress, and shows that the P.koreensis GS fermentation filtrate has important application value for improving drought tolerance of crops.

2.5 results of germination and growth-promoting cold resistance test in a dish

2.5.1 tomato seedling height

After 10d of the in-dish test treatment, the plant height (distance from cotyledon to plant growth point) of the tomato seedling was measured with a ruler, as shown in fig. 7 and 8. The result shows that the P.koreensis GS has a promoting effect on the growth of tomato seedlings and can obviously improve the plant height of the tomato seedlings. The cell-free fermentation filtrate diluted 50 times and 100 times increases the tomato seedling height by 20.72% and 13.81% at low temperature and increases the tomato seedling height by 10.70% and 26.49% at moderate temperature compared with the aseptic water treatment.

2.5.2 chlorophyll content determination

The method comprises the following steps: the chlorophyll content is determined by 96% ethanol extraction method. 0.2g of fresh sample, adding a proper amount of 96% ethanol and quartz sand, grinding until plant tissues turn white, filtering a single-layer filter paper to a 25ml brown volumetric flask, repeatedly flushing the filter paper with 96% ethanol until the filter paper turns white, and fixing the volume. The absorbance at wavelengths 665nm, 449nm and 470nm (zeroing cup 96% ethanol) was measured, chlorophyll content was calculated with reference to a plant physiology (code Zhang Ji) textbook, and each treatment was repeated 3 times.

Results and analysis:

as shown in table 6 (a=Δlck;b=Δsck;) in table 6, the total chlorophyll content of the tomato leaf tended to increase with increasing dilution factor at low temperature and at moderate temperature, with low temperature treatment significantly increasing chlorophyll content versus moderate temperature treatment. The total chlorophyll content of the cell-free fermentation filtrate diluted by 50 times is increased by 32.87% compared with the cell-free fermentation filtrate diluted by 100 times by 28.78% compared with the cell-free fermentation filtrate diluted by 100 times at low temperature, and the difference is obvious.

TABLE 6 chlorophyll content of tomato leaves under cold injury and temperature adapted conditions P.koreensis GS cell-free fermentation filtrate treatment

2.5.3 determination of antioxidant enzyme Activity of tomato seedlings

The method comprises the following steps: preparing a crude enzyme solution: 0.5g of fresh sample is weighed, 5ml of precooled 0.1mol/L sodium phosphate buffer solution (pH 7.0) is added for rapid and uniform mixing, the grinding fluid is centrifuged for 10min at 10000 Xg at 4 ℃, the supernatant is taken as crude enzyme solution, and the mixture is placed in a refrigerator at 4 ℃. Superoxide dismutase (SOD) activity was measured using the azotetrazolium reduction method, and Peroxidase (POD) activity was measured using the guaiacol method, with each treatment repeated 3 times.

Results and analysis:

as shown in Table 7 (A=ΔLCK; B=ΔSCK% in Table 7), the activity of SOD and POD enzyme of tomato seedlings treated by different dilution factors of P.koreensis GS cell-free fermentation broth at low temperature and proper temperature showed a remarkable trend of increasing compared with that of sterile water. Under the low-temperature treatment, the SOD enzyme activities are respectively increased by 26.55 percent and 35.62 percent, and the POD enzyme activities are respectively increased by 18.82 percent and 9.09 percent. Under the treatment of the same treatment fluid, the enzyme activity of the low-temperature treatment is obviously improved compared with that of the moderate-temperature treatment, and the SOD activity is respectively increased by 43.95%, 48.20% and 37.07%; POD activity was increased by 12.48%, 24.37% and 17.84%, respectively.

TABLE 7 antioxidant Activity of tomato seedlings under Cold injury and temperature-adapted conditions P.koreensis GS cell-free fermentation filtrate treatment

Summary of cold resistance:

(1) The koreesis GS cell-free fermentation liquid can promote the growth of tomato seedling plant height, and has obvious growth promoting effect under the condition of cold injury.

(2) The korensis GS cell-free fermentation filtrate can obviously improve the content of various chlorophyll in tomato seedling leaves. Under the stress of cold damage, the tomato seedlings are not degraded, and the photosynthesis of the tomato seedlings can be improved.

(3) The cell-free fermentation filtrate of the KORENSIS GS can improve the activity of SOD and POD of tomato seedlings and enhance the cold resistance of tomato seedlings.

Claims (7)

1. A pseudomonas korea, characterized in that: the Korean pseudomonas is Pseudomonas koreensis GS, and the Korean pseudomonas is submitted to China general microbiological culture collection center (CGMCC) for preservation in 2021, 9 and 22 days, and the preservation number is No.23459.

2. Use of pseudomonas Pseudomonas koreensis GS according to claim 1 for tomato growth promotion.

3. The use of pseudomonas koraiensis Pseudomonas koreensis GS as claimed in claim 1 for promoting tomato growth under stress conditions.

4. The use of pseudomonas koraiensis Pseudomonas koreensis GS as claimed in claim 1 for promoting tomato growth under drought stress or under cold damage stress.

5. The use of pseudomonas koraiensis Pseudomonas koreensis GS as claimed in claim 1 for promoting germination of tomato seeds under drought stress or cold damage stress.

6. The use of pseudomonas koraiensis Pseudomonas koreensis GS as claimed in claim 1 for promoting the growth of tomato plants under drought stress or under cold damage stress.

7. The use of pseudomonas koraiensis Pseudomonas koreensis GS as claimed in claim 1 for improving drought tolerance or for enhancing cold tolerance of tomatoes.

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