CN114774300A

CN114774300A - Korean pseudomonas and application thereof

Info

Publication number: CN114774300A
Application number: CN202111678875.3A
Authority: CN
Inventors: 郭俏; 景悦曦; 杨珊珊; 孙晨瑜; 李海洋; 来航线; 薛泉宏
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-07-22
Anticipated expiration: 2041-12-31
Also published as: CN114774300B

Abstract

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

Description

Korean pseudomonas and application thereof

Technical Field

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

Background

The growth of crops is influenced by a series of environmental factors, wherein drought, salinity, low temperature and the like are main factors causing the yield of crops to be reduced. Drought stress severely affects plant growth, development and reproduction, resulting in substantial crop losses. It is one of the most serious natural disasters that drought can cause up to 90% of agricultural economic loss. China is one of the countries with serious water shortage in the world, the drought soil area accounts for over 1/2 of the national soil area, the soil water resource is very short, and the crop cultivation often faces the risk of drought stress. Drought stress directly affects various stages of plant growth and development, and causes a series of negative effects on the physiological and biochemical levels of plants. For example, drought stress causes increased cell membrane permeability of plants, hormone imbalance, large accumulation of active oxygen, reduction of plant photosynthetic efficiency, and disturbance of nutrient metabolism, and finally, plant growth is inhibited and yield is reduced. The changes of indexes such as plant biomass, water utilization efficiency, photosynthetic capacity, osmotic adjustment capacity, stability of cell membranes, defense capacity of an antioxidant system, hormone level and the like are often used for judging the standard of the drought stress resistance of plants. In addition to drought stress, low temperature stress is a common disaster encountered in crop cultivation, and it not only causes a reduction in plant yield, but also, in severe cases, causes plant death. Statistically, the world loses every year for low temperatures amounting to $ 2000 billion. Therefore, improving the resistance of crops to low temperature stress and reducing the adverse effects of low temperature on crops are of great importance in agricultural production. According to the different degrees of low temperature, the low temperature damage of plants can be divided into two categories, namely cold damage (damage to plants by zero-above low temperature) and freezing damage (damage to plants by zero-below low temperature). The cold injury in low-temperature stress is more common in the occurrence area, the cold injury damages the normal functions and structures of cells, particularly the cytoplasmic membranes and the organelle membrane systems are damaged or interfere with normal activities, so that the metabolism is disordered, the functions are interfered, the structures and the tissues are also damaged frequently, and then the scars appear rapidly in the form. Cold damage caused by low temperature stress can cause adverse effects throughout the entire growth process of the plant, such as seed germination, plant growth, photosynthesis, fruit set, yield and quality development, etc. The cold injury results in weak seedling, slow growth, wilting, yellowing, local necrosis, low fruit bearing rate, low yield, low quality, etc. Researchers have attempted to improve the cold resistance of crop plants by different means based on the study of the physiological and biochemical effects of cold damage on plants. Tomato (Lycopersicon esculentum Mill.) is a vegetable commonly planted in China and has important economic value. Tomatoes are annual or perennial herbaceous plants, and fruits are rich in various mineral elements, carotene, B and C vitamins and the like. Tomatoes are very sensitive to drought stress and water shortage can severely affect tomato yield. Research shows that water stress can inhibit the germination of tomato seedlings and the growth of radicles and hypocotyls, and reduce the biomass of the tomato seedlings. In addition, under drought stress, the tomatoes have corresponding negative characteristics at all levels of cells, organs, individuals and groups, the growth and development of the tomatoes are inhibited, and the fruit yield is reduced.

At present, the harm of cold damage to crops is generally reduced by improving agricultural measures, for example, greenhouse planting, film mulching planting and other modes are adopted in winter to improve the soil temperature and the crop growth environment; the cold-resistant variety is cultivated by adopting cross breeding or genetic engineering, and the adverse effect of low temperature is reduced. However, the improvement of the agronomic measures is time-consuming and labor-consuming, and in addition, because the cold resistance of the plant is a comprehensive expression rather than caused by the expression of a single gene, and the cold resistance includes changes of other characteristics of the plant cell, such as cell membrane fluidity, synthesis and accumulation of low molecular weight or high molecular weight cryoprotective substances, and the like, no breakthrough progress is made in obtaining the low temperature resistant plant variety by the traditional breeding means or the transgenic technology. A simple and easy way to improve the cold resistance of crops is to use some exogenous substances, including plant growth regulating substances, such as hormone substances, such as abscisic acid, brassinolide, salicylic acid and the like;osmolytes such as soluble sugars, betaines, etc.; inorganic salt ions such as Ca⁺，K⁺And the like to improve the cold resistance of crops. The beneficial microorganisms can also improve the cold resistance of plants, can be stably planted at the roots of the plants, and have the functions of improving the soil structure, preserving water, promoting the absorption of minerals by the plants and the like to promote the growth of the plants, so the application of the beneficial microorganisms has an important effect on improving the cold resistance of crops.

In addition to drought resistance, in recent years, it has been discovered that beneficial microorganisms can promote plant growth under drought conditions. The rhizosphere growth-promoting bacteria (PGPR) can generate hormone substances such as indoleacetic acid, cytokinin and the like to promote the growth of Plant roots, increase the number of secondary roots and root hairs, facilitate the absorption of water and nutrients by plants, promote the growth of plants and enhance the tolerance of the plants to adverse conditions such as drought, pathogenic bacteria infection and the like. The beneficial microorganism can also induce and enhance the drought tolerance of the plant and adjust the physiological and biochemical processes related to the drought resistance of the plant. For example, rhizosphere bacterium Bacillus amyloliquefaciens can obviously improve the activity of antioxidant related enzyme in plants, reduce the content of active oxygen and malonaldehyde and relieve the oxidative damage caused by drought stress; the streptomyces clausii Act12 can increase the biomass of wheat, improve the content of water-soluble total sugar, regulate the content of proline and glutathione, and enhance the drought tolerance of wheat through the conduction of ABA dependent signals; and B, the subtilis GB03 can induce plants to accumulate more osmoregulation substances such as proline, betaine and the like, and relieve the damage of drought stress on plant cell membranes and biomacromolecules.

Pseudomonas is a gram-negative bacterium belonging to the family pseudomonas, which is widely distributed in soil, fresh water, seawater, and organisms. The pseudomonas has wide applicable temperature range, can grow at 4-43 ℃, and has the optimal growth temperature of about 30 ℃. The pH value range suitable for the growth of the bacteria is 7.0-8.5, and most of the bacteria cannot grow in an environment with the pH value of 6 or below 6. Non-pathogenic pseudomonas is active in the plant rhizosphere and is one of the most prominent PGPR. The pseudomonas fluorescens belongs to pseudomonas of the pseudomonadaceae of the thin-walled bacterium family, is widely distributed on plant rhizosphere soil and surfaces of fruits and vegetables, and can promote the growth and yield increase of the fruits and vegetables while effectively inhibiting pathogens by a plurality of strains, so that the pseudomonas fluorescens is concerned by researchers in various countries and is a type of biocontrol bacteria and rhizosphere growth-promoting bacteria which are researched and reported most at the earliest 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 the chlorophyll content of the plants, producing glutathione, ACC deaminase and the like. In addition, Pseudomonas fluorescens can also improve the ability of plant bodies to resist the invasion of pests and diseases. Researches find that pseudomonas fluorescens has an inhibiting effect on root-knot nematodes in and out of plants, and can inhibit the incidence of root-knot nematode diseases of mung beans so as to improve the yield of seeds of mung beans; the nitropyrrolidin and the pyrrolomycin generated by the pseudomonas fluorescens strains Ps170 and Ps117 have great potential in the aspect of preventing and controlling Erwinia amylovora. Korean Pseudomonas korea (Pseudomonas koreensis) is a subspecies of Pseudomonas fluorescens, however, the application of Korean Pseudomonas to the research of the mechanism for improving the stress resistance of plants is rarely reported.

Disclosure of Invention

In order to solve the above technical problems in the background art, the present invention provides a korean pseudomonas that can improve the growth status of tomato plants under drought stress conditions, increase the tomato resistance enzyme activity, reduce the cell MDA content, and improve the tomato drought tolerance, and applications thereof.

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

a korean pseudomonas, 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 months, and the preservation number is NO. 23459.

The Korean Pseudomonas as described above, characterized in that: the Korean pseudomonas was isolated from southern foot of lesser Khingan in Heilongjiang province with geographical coordinates of 46 ° 57 'N and 128 ° 16' E.

Use of the Korean Pseudomonas korea Koreaensis GS for plant growth promotion as described above.

The use of Pseudomonas korea GS in tomato growth promotion as described above.

Use of the Korean Pseudomonas korea Koreaensis GS for promoting tomato growth under stress conditions as described above.

Use of the Korean Pseudomonas korea Koreaensis GS for promoting tomato growth under drought stress or cold injury stress as described above.

Use of the Korean Pseudomonas korea Koreaensis GS for promoting tomato seed germination under drought stress or cold damage stress as described above.

Use of the Korean Pseudomonas korea Koreaensis GS for promoting the growth of tomato plants under drought stress or cold damage stress as described above.

The use of Pseudomonas korea Koreaensis GS for improving drought tolerance of tomato or enhancing cold tolerance of tomato as described above.

The invention has the advantages that:

the invention takes tomatoes and P.koreensis GS as the objects of the invention, drought stress is carried out by respectively adopting methods of adding polyethylene glycol and controlling watering amount through in-dish and pot experiments, and the effect of the P.koreensis GS cell-free fermentation filtrate on the growth promotion and drought tolerance of tomato plants is revealed from the measurement of the biological form and physiological and biochemical indexes of the tomatoes. In a dish germination test, 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 increase rates are respectively 9.5%, 29.08%, 7.74% and 28.96%; under drought stress, the treatment of the P.koreensis GS fermentation filtrate 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 a pot experiment, the plant height, fresh weight, dry weight, root length, dry weight and relative chlorophyll content (SPAD) of a potted tomato (30d) treated by P.koreensis GS fermentation filtrate are respectively and obviously improved by 4.75%, 4.15%, 1.81%, 23.25%, 9.83% and 9.89%; under drought stress treatment, the plant height, root length, fresh weight, dry weight, SPAD value and root dry weight of potted tomato (30d) are respectively and obviously improved by 1.56%, 9.82%, 11.05%, 3.77%, 13.60% and 1.69%. Therefore, the p.koreensis GS fermentation filtrate improves the growth status of tomatoes under drought stress treatment, and improves the drought tolerance of tomatoes. In addition, a germination test in a dish shows that after the P.koreensis GS fermentation filtrate is treated, activities of Peroxidase (POD) and catalase (CATALase) of seedlings in the dish are respectively and remarkably improved by 11.85% and 97.07% under the non-drought condition; the CAT enzyme activity of the tomato seedlings in the dish is obviously improved by 50.16 percent under drought stress; in a pot experiment, the CAT enzyme activity of a leaf blade of a tomato plant (30d) treated by the P.koreensis GS fermentation filtrate is remarkably increased by 27.30 percent under a non-drought condition, and the CAT enzyme activities of the leaf blade POD and the CAT enzyme activities are respectively and remarkably increased by 64.51 percent and 358.25 percent under drought stress; the contents of the anti-osmotic adjusting substances of proline and Malondialdehyde (MDA) in leaves of a tomato plant (30d) are not obviously changed under the non-drought condition, while the content of the MDA (representing the degree of membrane lipid peroxidation) is obviously reduced by 38.30 percent and the content of proline is obviously improved by 2.59 percent under the drought stress. The invention shows that the P.koreensis GS can improve the growth condition of tomato plants under drought stress conditions, improve the crop resistance enzyme activity, reduce the MDA content of cells and further improve the drought tolerance of tomatoes. In addition, the p.koreensis GS cell-free fermentation liquor can promote the growth of the plant height of the tomato seedlings, has obvious growth promotion effect under the cold damage condition, can obviously improve the content 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 Koreensis GS cell-free fermentation filtrate can improve the activities of defense enzymes SOD and POD of tomato seedlings and enhance the cold damage resistance of the 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 difference (P < 0.05));

fig. 2 is a comparative plot of the effect of p. koreensis GS cell-free fermentation filtrate on tomato plant biological indicators under drought and non-drought conditions;

fig. 3 is a bar graph of the effect of p.koreensis GS cell-free fermentation filtrate treatment on the defense enzyme activity of tomato seedlings under drought and non-drought conditions (different letters indicate significant differences (P < 0.05));

fig. 4 is a comparative plot of the effect of p. koreensis GS cell-free fermentation filtrate treatment on biological indicators of tomato plants under drought and non-drought conditions;

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

fig. 6 is a graph of the effect of P. koreensis GS cell-free fermentation filtrate treatment on the 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 plant height of tomato seedlings under cold damage and moderate temperature conditions;

fig. 8 is the effect of p. koreensis GS cell-free fermentation filtrate treatment on tomato seedling growth under cold damage and moderate temperature conditions.

Detailed Description

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

1. The invention will be explained in detail by the following tests

(1) Influence of Pseudomonas koreensis GS (hereinafter referred to as P.koreensis GS) fermentation filtrate on tomato seed germination condition under drought stress

The influence of the p.koreensis GS cell-free fermentation filtrate after seed soaking on the response of tomatoes to drought stress is revealed through a polyethylene glycol dish in-situ simulated drought stress test.

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

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

The influence of the P.koreensis GS cell-free fermentation filtrate on the biological properties (plant height, root length, fresh weight, dry weight, stem thickness and the like) and the physiological and biochemical properties (malondialdehyde, proline, antioxidant enzyme and the like) of the tomatoes is revealed by taking the tomatoes as test plants, controlling the watering amount as a stress means through a nutrition pot drought stress test.

The invention researches the influence effect of P.koreensis GS cell-free fermentation filtrate on the growth, drought resistance and cold resistance of tomatoes under the drought stress condition through in-dish germination and pot culture tests.

2. Detailed experiments

2.1 materials and methods

2.1.1 test strains

Korean Pseudomonas koreana koreensis GS (NCBI accession No. PRJNA517377) was isolated from the ginseng base of Tiande ginseng, Inc. of peach Shanzhen peach Shandong province, Heilongjiang province, south foot of lesser Xinganling (46 ° 57 'N, 128 ° 16' E), and brown soil, and was collected by the laboratory of microbiological resources of the resource environmental institute of university of agriculture and forestry, northwest. Meanwhile, the strain has been submitted to China general microbiological culture Collection center (CGMCC) for preservation 22/9/2021, and the preservation number is NO. 23459.

2.1.2 test tomato varieties

Tomato seeds (golden greenhouse number one), purchased from west andringpeng germchit limited.

2.1.3 test Medium

Beef extract-peptone broth (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 raising substrate comprises high-quality turf, substrate raw materials, perlite, vermiculite and the like, the content of organic matters is more than 35%, the content of water is more than 20%, the pH value is neutral, and the soilless culture seedling raising substrate is purchased from Shen Lu Yuan seedling raising substrate company.

2.1.4 drought stress test

A) Germination in dish and growth-promoting drought-enduring test

Indoor culture dish bioassay was used. After culturing the culture at 28 ℃ for 72h with shaking (180rpm) in a beef extract peptone liquid medium, centrifuging the culture at 4 ℃ for 15min at 10,000 rpm, filtering the culture through a sterile microfiltration membrane (0.22 mu m pore size), collecting a P.koreensis GS sterile fermentation filtrate, and diluting the P.koreensis GS sterile fermentation filtrate into a 100-fold diluent with sterile water for later use. The method comprises sterilizing 600 tomato seeds with 75% ethanol for 30s, washing with sterile water for at least 5 times, and uniformly placing sterilized tomato seeds in culture dishes (20 seeds per dish) with sterilized double-layer filter paper.

The sterilized tomato seeds are divided into two groups, one group is soaked in sterile water, the other group is soaked in 100-fold diluted P.koreensis GS sterile fermentation filtrate (the invention is explained in detail by taking 100-fold dilution as an example, the specific dilution and screening are detailed in table 1), and after treatment, the culture dish is placed in a constant-temperature illumination incubator at 25 ℃ for germination in the dark. Each dish was replaced with 10mL of the corresponding treated solution every 1 day from the 2 nd day, and the number of seeds germinated from each dish was observed and recorded every day. The number of days that the white germs of the 1 st seed are exposed is used as the starting time of the germination of the treated seeds, and the number of days that no germs of the seed are exposed for 3 consecutive days is used as the ending time of the treatment test.

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

Table 1 effect of p. koreensis GS cell-free fermentation filtrate treatment on biological indicators of tomato seedlings under drought and non-drought conditions

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

After tomato seedlings grow for 10 days, polyethylene glycol solution (hydrophilic macromolecules, strong water absorption, which can cause difficulty in plant water absorption and simulate drought stress) with the concentration of 12% is prepared by sterile water and cell-free fermentation filtrate diluted by 100 times respectively for drought stress treatment, so that four treatments are set, namely: the method comprises the following steps of adding 7mL of treatment fluid into a control group (CK) only with sterile water, a control group (PK) only with P.koreensis GS sterile fermentation filtrate treatment (PK), sterile water + polyethylene glycol (DK), and a control group (DPK) only with sterile water, sterile fermentation filtrate treatment (PK) only with P.koreensis GS sterile fermentation filtrate treatment (DPK), wherein each dish is provided with 20 tomato seeds, each dish is a repetition, each treatment is provided with 5 repetitions, and each group treats 100 tomato seeds. And harvesting tomato seedlings 0h and 96h after drought stress treatment for measuring the biological characters and physiological and biochemical indexes of the plants.

B) Growth promotion in dishes and cold resistance test

Germination design of tomato seeds: after culturing the culture medium of the koreensis GS in a beef extract peptone liquid medium at 28 ℃ for 72 hours with shaking at 180rpm, centrifuging the culture at 4 ℃ at 10000rpm for 15 minutes, filtering the culture through a sterile microfiltration membrane (with the pore diameter of 0.22 mu m), collecting the cell-free fermentation liquor of the p.koreensis GS, and diluting the filtrate into 50-fold and 100-fold diluted solutions by sterile water.

Taking a proper amount of tomato seeds, disinfecting the tomato seeds for 30s by using 75% alcohol, washing the tomato seeds for at least more than 5 times by using sterile water, and uniformly placing the disinfected tomato seeds into culture dishes paved with sterilized double-layer filter paper (30 seeds per dish) for 30 culture dishes. The culture dish is divided into three groups, each group comprises 10 culture dishes, the seeds are soaked in sterile water and cell-free fermentation liquor diluted by 50 times and 100 times respectively, the required treatment liquor mainly soaks the filter paper and the seeds and records the initial mass, and after treatment, the culture dish is placed in a constant-temperature illumination incubator at 25 ℃ and germinates in the dark. And (3) adding the correspondingly treated solution to each culture dish from the 2 nd day at regular time, ensuring the quality to be the same as the initial quality, and observing and recording the germination number of the seeds in each culture dish at regular time every day. The germination was completed by taking the germ of 2mm exposed from the seed as an indication of the germination of the seed and 3 days after which no seed was germinated. After the tomato germination is finished, respectively using sterile water and cell-free fermentation liquor diluted by 50 times and 100 times to carry out cold damage stress and temperature-adapted treatment. A total of six treatments were set, each of which was repeated 5 times, in the manner shown in table 2.

TABLE 2 different temperature stress treatment conditions

Placing the culture dish subjected to cold damage stress treatment in an illumination constant-temperature incubator for culture, wherein the day/night time is set to be 14h/10h, the temperature is 17 ℃/10 ℃, the air relative humidity is 65%, and the illumination intensity is 8000 lx; the culture dish with proper temperature treatment is placed in another illumination constant temperature incubator for culture, the day/night time is set to be 14h/10h, the temperature is 26 ℃/20 ℃, the air relative humidity is 65%, the illumination intensity is 8000lx, and 15ml of corresponding treatment liquid is added every day for each treatment. And (5) measuring the main plant height of the tomato seedlings at the 10 th treated stage, and harvesting the tomato seedlings at the 14 th treated stage for measuring the physiological and biochemical indexes of the plants.

2.1.5 potting test

The culture medium is soilless culture medium, and is sterilized by moist heat at 121 ℃ for 2h and placed in a nutrition pot. Sterilizing about 800 tomato seeds with 75% alcohol for 30s, washing with sterile water for 5 times, dividing the sterilized seeds into two parts, soaking one part with sterile P.koreensis GS fermentation filtrate diluted by 100 times, and soaking one part with equal volume of sterile water. Soaking for 72h, after the seeds are exposed to white, respectively sowing the two treated tomato seeds in a nutrition pot (3 seeds in each pot), and reserving 2 seeds in each pot after seedling establishment. The experiment design was a randomized complete block design with 3 blocks each containing 80 pots. Potting of each block was randomly divided into Control (CK) and treatment (PK) groups of 40 pots each for a total of 240 pots.

Culturing in a light constant temperature incubator with light irradiation time of 12h/12h, temperature of 25 deg.C, relative humidity of 65%, and light irradiation of 1000 μmol/m^-2s^-1. And (3) irrigating 20mL of 1/2Hoagland nutrient solution to the experimental group and the control group every 1-25 days, and supplementing the nutrient solution prepared from 100-time diluted P.koreensis GS sterile fermentation filtrate to the experimental group every 3 days. And thinning at 6d, dividing the treatment components into normal water groups and drought stress groups at 25d, continuously watering 1/2Hoagland nutrient solution in the normal water groups, and performing drought treatment without watering until tomato seedlings wither. Four treatments were thus set, namely a control group (CK) with nutrient solution only, a treatment (PK) with p.koreensis GS sterile fermentation filtrate, a nutrient solution + drought stress treatment (DK), and a treatment (DPK) with p.koreensis GS sterile fermentation filtrate + drought stress treatment, each consisting of 20 pots (2 plants per pot) for a total of 480 plants.

2.2 determination of biological indicators

2.2.1 in-dish test

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

2.2.2 potting test

Potted tomato

25d, 20 tomato plants were harvested from each of the experimental and control groups, and the biological indicators such as plant height, root length, fresh weight, stem thickness and the like were measured to analyze the growth promoting effect of the strains. Potted tomato 30 days, and quickly measuring the SPAD value of tomato leaves by using a chlorophyll meter at 11:00 noon; measuring the plant height, root length, fresh weight and dry weight of the plant, and the fresh weight and dry weight of the root; 3 samples are respectively mixed and collected from the four treatments, and each biological sample consists of specific leaves (5 th leaves from a growth point downwards) of 3 random tomato plants for enzyme activity measurement; from each treatment 6 samples were taken in a mix, each biological sample consisting of a specific leaf (leaf 4 down from the growing point) of 3 random tomato plants for the determination of proline and malondialdehyde. After sampling, the leaf samples are directly placed into an ice box and taken back to a laboratory to be placed into a refrigerator at the temperature of minus 80 ℃ for storage for later use.

2.2.3 enzyme Activity assay

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

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

Catalase (CAT): the reaction system is measured to contain 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 preheating for 3min at 25 ℃, 0.2mL of 200mmol/L hydrogen peroxide is added tube by tube, and the absorbance changes by 0.01 per minute at 240nm as 1 enzyme activity unit U, and the enzyme activity is expressed as U/min.

2.2.4 determination of proline content

Determination of proline content: taking 0.5g of fresh sample, adding 10mL of 3% sulfosalicylic acid, leaching for 15min in a boiling water bath, centrifuging and taking supernatant for measuring the proline content. 6mL of the reaction system was: 2mL of leaching liquor, 2mL of glacial acetic acid and 2mL of acidic ninhydrin, reacting for 30min at 100 ℃, and then putting into ice water to terminate the reaction. The reaction mixture was extracted with 5mL of toluene, and the supernatant was taken to measure the absorbance at 520nm, and the proline content was calculated by a standard curve.

2.2.5 determination of the Malondialdehyde (MDA) content

The content of the malondialdehyde can reflect the lipid peroxidation level of the membrane, and the content of the malondialdehyde is measured by a 2-thiobarbituric acid (TBA) method. Grinding and extracting malondialdehyde from tomato leaf with 5% trichloroacetic acid (TCA), adding 2mL extractive solution and 2mL thiobarbituric acid into test tube, mixing, boiling in water bath for 12min, rapidly cooling to stop reaction, and centrifuging at 4500rpm for 10min if precipitate is formed. Absorbance of the supernatant at 532nm, 600nm and 450nm was measured using thiobarbituric acid solution as a blank. The calculation formula is as follows:

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

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

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. Plotting was performed using Excel 2007 software. Data in the graph are mean ± sd.

2.4 germination and growth promotion drought tolerance test results in dish

Effect of Korensis GS fermentation filtrate on growth of tomato seedlings in dishes

Effect of Koreensis GS fermentation filtrate on germination percentage in tomato dishes

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

2.4.1.2 P.Koreaensis GS fermentation filtrate effects on biological Properties of tomato in dish

Table 3 influence of p. koreensis GS cell-free fermentation filtrate on biological indicators of tomato seedlings under drought and non-drought conditions

Note: different capital letters in the same column indicate significant difference between groups (P <0.01) and different lowercase letters in the same column indicate significant difference between groups (P < 0.05).

The same goes for

As shown in table 3, the p.koreensis GS fermented filtrate under non-stress treatment has a promoting effect on the growth of tomato plants, and the p.koreensis GS fermented filtrate significantly increases the plant height, root length, fresh weight and dry weight of tomatoes by 2.72%, 14.80%, 10.60% and 26.47%, respectively. Under drought stress, the P.koreensis GS fermentation filtrate has a remarkable growth promotion effect on tomato plants, the height of the tomato plants in the DPK group is remarkably improved by 1.06 percent compared with the DK group, the root length is remarkably improved by 14.86 percent, the fresh weight is remarkably improved by 18.18 percent, and the dry weight is remarkably improved by 3.33 percent. The results show that the P.koreensis GS fermentation filtrate can promote the growth of tomato seedlings under the drought condition.

2.4.1.3P.Koreensis GS fermentation filtrate treatment on the Effect of on the Pepper tomato defensive enzyme Activity

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

Effect of Korensis GS fermentation filtrate on the biological characteristics of potted tomato plants

Effect of Koreensis GS fermentation filtrate on growth of potted tomato plants

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

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

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

As shown in table 5, when the plants in PK group were treated at 30d, the plant height, fresh weight, dry weight, root length and SPAD value of tomato were significantly increased by 4.75%, 4.15%, 1.81%, 23.25%, 9.89%, and the dry root weight of tomato plant was significantly increased by 9.83%, respectively. Under drought stress, the p.koreensis GS fermentation filtrate has a promoting effect on the plant height, root length, fresh weight, dry weight, and SPAD value of tomatoes. Compared with the DK group, the plant height, root length, fresh weight, dry weight and SPAD value of the DPK group are respectively and obviously improved by 1.56%, 9.82%, 11.05%, 3.77% and 13.60%, and the dry weight of tomato roots is obviously improved by 1.69%. The Koreaensis GS fermentation filtrate has a remarkable growth promotion effect on tomato plants, and the drought tolerance of the tomato plants is effectively improved.

Influence of koreensis GS fermentation filtrate on potted tomato defensive enzyme activity

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

2.4.2.3 P.Korensis GS fermentation filtrate influence on proline and malondialdehyde content of potted tomato

As shown in fig. 6, the treatment of p. koreensis GS fermentation filtrate did not significantly affect the proline and MDA content of tomato plants under non-drought conditions, the proline content of DPK group was significantly increased by 2.59% compared to DK group (fig. 6a), and the MDA content was significantly decreased by 38.30% compared to DK group (fig. 6 b). Under the non-stress condition, the contents of proline and MDA in the plant of the tomato are not obviously changed; under drought stress, the change of the MDA content in tomato plants is obvious and the tomato plants are sensitive to the treatment of P.koreensis GS fermentation filtrate.

Drought stress causes damage to plant growth and development from many aspects: excessive accumulation of Reactive Oxygen Species (ROS), damage to cell membranes and nuclear structures, inhibition of leaf and root growth, decreased photosynthetic rates, and the like. The leaves and root system of the plant are damaged, the activity of the root system is reduced, and the absorption of water and mineral elements by the plant can be inhibited. Under drought stress, indexes such as tomato plant height, fresh weight, root length, SAPD and the like after treatment of the P.koreensis GS fermentation filtrate are obviously improved, 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 primary site, and high concentrations of ROS have a toxic effect on plant cell membrane systems and the like. A complete set of ROS scavenging system is arranged in the plant cell, and can regulate and improve the activity of antioxidant enzymes or antioxidant substances in adverse circumstances, so that the ROS level in the cell is maintained within a certain range, and further the oxidative stress caused by stress is relieved. Osmotic regulatory systems in plants promote the accumulation of large amounts of solutes (sugar alcohol compounds, amino acids, secondary metabolites, organic acids, etc.) in plants 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 proline content is obviously increased, and the MDA content is obviously reduced, which indicates that the P.koreensis GS fermentation filtrate participates in the osmotic regulation of tomatoes, reduces the accumulation of ROS in the leaves, and relieves the lipid peroxidation degree (MDA) of tomato leaf cell membranes, thereby relieving the damage of ROS to cell membranes.

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

2.5 germination and growth promoting Cold resistance test results in dishes

2.5.1 height of tomato seedlings

As shown in fig. 7 and 8, the plant height (distance between cotyledon and plant growth point) of tomato seedlings was measured with a ruler after 10d of in-dish test treatment. 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. Compared with the sterile water treatment, the cell-free fermentation filtrate diluted by 50 times and 100 times increases the plant height of the tomato seedlings by 20.72 percent and 13.81 percent at low temperature and increases the plant height of the tomato seedlings by 10.70 percent and 26.49 percent under the moderate temperature treatment.

2.5.2 chlorophyll content determination

The method comprises the following steps: the chlorophyll content is determined by 96% ethanol extraction method. Adding appropriate amount of 96% ethanol and quartz sand into 0.2g fresh sample, grinding until plant tissue turns white, filtering with single-layer filter paper to 25ml brown volumetric flask, repeatedly washing the filter paper with 96% ethanol until it turns white, and fixing volume. Absorbance values at 665nm, 449nm and 470nm wavelengths were determined (96% ethanol in zero adjustment cups) and chlorophyll content was calculated with reference to the plant physiology (zhangong 28557) textbook and repeated 3 times for each treatment.

Results and analysis:

as shown in table 6 (in table 6, a ═ Δ LCK%; B ═ Δ SCK%), the total chlorophyll content of the tomato leaves increased with the increase of dilution factor under the low-temperature and moderate-temperature treatment, and the chlorophyll content of each type significantly increased under the low-temperature treatment as compared with the moderate-temperature treatment. Based on the total chlorophyll content, the low-temperature treatment of the cell-free fermentation filtrate diluted by 50 times is increased by 32.87 percent compared with the proper-temperature treatment, and the low-temperature treatment of the cell-free fermentation filtrate diluted by 100 times is increased by 28.78 percent compared with the proper-temperature treatment, and the difference is obvious.

Table 6 content of chlorophyll in tomato leaves treated with koreensis GS cell-free fermentation filtrate under cold damage and moderate temperature conditions p

2.5.3 determination of antioxidant enzyme Activity of tomato seedlings

The method comprises the following steps: preparing a crude enzyme solution: weighing 0.5g fresh sample, adding 5ml pre-cooled 0.1mol/L sodium phosphate buffer solution (pH 7.0), rapidly mixing, centrifuging the grinding solution at 4 deg.C at 10000 × g for 10min, collecting supernatant as crude enzyme solution, and standing in refrigerator at 4 deg.C. The superoxide dismutase (SOD) activity was measured by nitro blue tetrazolium reduction method, and the Peroxidase (POD) activity was measured by guaiacol method, each treatment being repeated 3 times.

Results and analysis:

as shown in table 7 (in table 7, a ═ Δ LCK%; B ═ Δ SCK%), the activities of SOD and POD enzymes of tomato seedlings treated with p.koreensis GS cell-free fermentation broth at different dilution times showed a significantly increased tendency compared to sterile water under low-temperature and moderate-temperature treatment. Under low-temperature treatment, the SOD enzyme activity is respectively increased by 26.55 percent and 35.62 percent, and the POD enzyme activity is respectively increased by 18.82 percent and 9.09 percent. Under the same treatment liquid, the enzyme activity of low-temperature treatment is obviously improved compared with that of moderate-temperature treatment, and the SOD activity is respectively increased by 43.95%, 48.20% and 37.07%; POD activity increased by 12.48%, 24.37% and 17.84%, respectively.

TABLE 7 antioxidant enzyme activity of tomato seedlings treated with cold damage and thermophilic conditions P

Summary of cold resistance:

(1) the koreensis GS cell-free fermentation liquor can promote the growth of the plant height of tomato seedlings and has obvious growth promotion effect under the cold damage condition.

(2) The content of chlorophyll in leaves of tomato seedlings can be obviously improved by using a koreensis GS cell-free fermentation filtrate. Under the stress of cold damage, the fertilizer is not degraded, and can improve the photosynthesis of the tomato seedlings.

(3) The Koreensis GS cell-free fermentation filtrate can improve the activities of defense enzymes SOD and POD of tomato seedlings and enhance the cold resistance of the tomato seedlings.

Claims

1. A korean pseudomonas, characterized in that: the Korean Pseudomonas is Pseudomonas koreensis GS, which has been 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. The Korean Pseudomonas as claimed in claim 1, wherein: the Korean pseudomonas was isolated from southern foot of lesser Khingan in Heilongjiang province with geographical coordinates of 46 ° 57 'N and 128 ° 16' E.

3. The use of the Korean Pseudomonas koreana Koreensis GS as defined in claim 1 for plant growth promotion.

4. The use of the Korean Pseudomonas korea Koreaensis GS as claimed in claim 1 for promoting tomato growth.

5. Use of the Korean Pseudomonas korea Koreaensis GS as defined in claim 1 for tomato growth promotion under stress conditions.

6. Use of the Korean Pseudomonas korea Koreaensis GS as defined in claim 1 for tomato growth promotion under drought stress or cold damage stress.

7. Use of the Korean Pseudomonas korea Koreaensis GS as defined in claim 1 for promoting germination of tomato seeds under drought stress or cold damage stress.

8. Use of the Korean Pseudomonas korea Koreaensis GS as defined in claim 1 for promoting the growth of tomato plants under drought stress or cold damage stress.

9. The use of the Korean Pseudomonas korea Koreaensis GS as claimed in claim 1 for improving drought tolerance or enhancing cold tolerance of tomato.