CN111944710B - Shewanella and application thereof in improving stress resistance of plants - Google Patents
Shewanella and application thereof in improving stress resistance of plants Download PDFInfo
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Abstract
The invention discloses Shewanella and application thereof in improving stress resistance of plants. The Shewanella is Shewanella (Shewanella sp.) YJD-CS2, the preservation number of which in China center for type culture Collection is CCTCC NO: and M2019330. Experiments demonstrated that the application of Shewanella (Shewanella sp.) YJD-CS2CCTCC NO: m2019330 can improve the saline-alkali stress resistance of the plant, and is specifically characterized in that the dry weight, the fresh weight, the total root length and the root surface area of the plant are obviously increased. The invention has important application value.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to Shewanella and application thereof in improving stress resistance of plants.
Background
The salinization of soil has become a serious problem affecting agricultural production, and crops are often stressed by abiotic stress factors such as high alkali and high salt in the growth and development process of the crops, and the stress factors cause a large amount of yield reduction of the crops in the world. In recent years, adverse factors such as abnormal climate, scarcity of water resources, reduction of available cultivated land and increase of saline-alkali soil seriously threaten national food safety of China. At present, people carry out a great deal of research on the mechanism of the plant responding to the adverse stress such as saline-alkali stress and the like from the aspects of physiology, biochemistry, metabolism, ecology, heredity, evolution and the like, accumulate abundant data, and particularly along with the development of molecular biology, people can know the adverse resistance mechanism of the plant to the saline-alkali stress on the molecular level of gene composition, expression regulation, signal conduction and the like, thereby opening up a new way for improving the adverse resistance of the plant by utilizing a genetic engineering means. Because of the complexity of the plant stress resistance character, the traditional breeding method is very difficult to improve the stress resistance of the plant, and although the genetic engineering means opens up a new way for the plant stress resistance breeding, the separation of the high-efficiency stress resistance gene becomes a main factor for limiting the plant stress resistance genetic engineering.
Disclosure of Invention
The invention aims to improve the stress resistance of plants.
The invention firstly protects Shewanella (Shewanella sp.) YJD-CS2, the strain is preserved in China center for type culture Collection (CCTCC for short, the address is Wuhan university in Wuhan, china) in 2019 and 05 and 07 days, and the preservation number is CCTCC NO: and M2019330. Shewanella (Shewanella sp.) YJD-CS2CCTCC NO: m2019330 is abbreviated as Shewanella (Shewanella sp.) YJD-CS2 or Shewanella YJD-CS2.
The invention also protects a microbial inoculum which contains the Shewanella YJD-CS2.
The microbial inoculum can be used for improving the saline-alkali stress resistance of plants.
The preparation method of the microbial inoculum comprises the following steps: shewanella YJD-CS2 is inoculated to a bacterial culture medium and cultured to obtain OD 600nm The bacteria solution with the value of 0.5-1.0 is the microbial inoculum.
The bacterial culture medium can be LB liquid culture medium.
In the preparation method of the microbial inoculum, the culture conditions can be as follows: culturing at 25-35 deg.C (such as 25-30 deg.C, 30-35 deg.C, 25 deg.C, 30 deg.C or 35 deg.C), 100-150r/min (such as 100-130r/min, 130-150r/min, 100r/min, 130r/min or 150 r/min) for 30-60 h (such as 30-48 h, 48-60 h, 30h, 48h or 60 h).
The microbial inoculum may include a carrier in addition to the active ingredient. The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymeric compound. The mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth. The plant material may be at least one of corn flour, bean flour and starch. The high molecular compound may be polyvinyl alcohol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water. The organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and a filtrate. The composition can be prepared into various dosage forms, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.
According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.
The Shewanella sp (Shewanella sp.) YJD-CS2CCTCC NO: the application of M2019330 or any of the above bactericides in regulating and controlling the stress resistance of plants also belongs to the protection range of the invention.
In the application, the regulation and control of the plant stress resistance can be the improvement of the saline-alkali resistance of the plant (namely the improvement of the saline-alkali stress resistance of the plant).
The Shewanella sp (Shewanella sp.) YJD-CS2CCTCC NO: the application of M2019330 or any one of the above bactericides in preparation of products for improving stress resistance of plants.
In the above application, the product may be a microbial fertilizer.
In any of the above applications, the stress resistance may be three, any two or any one of salt resistance, alkali resistance and salt and alkali resistance.
In any of the above applications, the plant may be any of the following c 1) to c 11): c1 A dicotyledonous plant; c2 A monocot plant; c3 Gramineous plants; c4 Solanaceae plants; c5 Cruciferous plants; c6 Corn); c7 Maize inbred line B73; c8 ) tomatoes; c9 Tomato variety M82; c10 Arabidopsis thaliana; c11 A Columbia-0 subtype of wild type Arabidopsis thaliana.
The invention also provides a microbial fertilizer which contains the Shewanella sp YJD-CS2CCTCC NO: m2019330 or any of the microbial agents.
The invention also discloses a method for improving the stress resistance of plants, which can be used for treating the plants by using the Shewanella, thereby improving the stress resistance of the plants.
In the above method, the "treating a plant with Shewanella" may be performed by applying Shewanella to a root system of the plant.
In the above method, the "treating the plant with Shewanella" may be carried out by spraying Shewanella onto the aerial parts (e.g., leaves) of the plant.
In the above method, the step of treating the plant with Shewanella may be specifically a step of treating the plant with any of the above-mentioned microbial agents.
In the method, the step of treating the plant by using any of the microbial inoculum can be realized by applying any of the microbial inoculum to a root system of the plant.
In the method, the step of treating the plant by using any of the microbial inoculum can be realized by spraying any of the microbial inoculum to the overground part (such as leaves) of the plant.
In any of the above methods, the Shewanella may be the Shewanella sp YJD-CS2CCTCC NO: and M2019330.
In the above method, the step of "using said Shewanella (Shewanella sp.) YJD-CS2CCTCC NO: the M2019330 or the microbial inoculum for treating the plant can be applied to the root system of the plant or sprayed to the overground part (such as leaves) of the plant.
In the method, the stress resistance can be three, any two or any one of salt resistance, alkali resistance and saline-alkali resistance.
In the above method, the plant may be any one of the following c 1) to c 11): c1 A dicot plant; c2 A monocot plant; c3 Gramineous plants; c4 ) plants of the Solanaceae family; c5 Cruciferous plants; c6 Corn); c7 Maize inbred line B73; c8 ) tomatoes; c9 Tomato variety M82; c10 Arabidopsis thaliana; c11 A Columbia-0 subtype of wild type Arabidopsis thaliana.
Any one of the microbial fertilizers can be a compound microbial fertilizer and/or a biological organic fertilizer. The compound microbial fertilizer can be a fertilizer compounded by microbial inoculum, nutrient substances and organic matters. The compound microbial fertilizer has the functions of both microbes and chemical fertilizers. The biological organic fertilizer can be a fertilizer formed by compounding a microbial inoculum and a decomposed organic fertilizer. The dosage form of the compound microbial fertilizer and/or the biological organic fertilizer can be granules.
Any of the above mentioned improvements in plant stress resistance may in particular be manifested in an increase in the dry weight and/or fresh weight and/or total root length and/or root surface area of the plant. Any of the above-described improvements in plant stress resistance may also be manifested by an increase in the height of the aerial parts (i.e., an increase in plant height).
Experiments prove that the Shewanella sp YJD-CS2CCTCC NO provided by the invention is as follows: the M2019330 can improve the resistance of tomatoes, corns and arabidopsis thaliana to saline-alkali stress, and is specifically characterized in that the dry weight, the fresh weight, the total root length and the root surface area are obviously increased, and the height of the overground part is increased (namely the plant height is increased). The invention has important application value.
Drawings
FIG. 1 shows the morphology of the bacterium YJD-CS2 observed by high-resolution transmission electron microscopy.
FIG. 2 shows the effect of YJD-CS2 microbial inoculum on the growth of root system of Arabidopsis seedlings under saline-alkali stress conditions.
FIG. 3 is a statistical result of root length of Arabidopsis seedlings treated by YJD-CS2 microbial inoculum under the saline-alkali treatment condition.
FIG. 4 shows the effect of YJD-CS2 inoculum on whole seedlings and aerial parts of maize seedlings under alkali stress conditions.
FIG. 5 is the effect of YJD-CS2 inoculum on root morphology of maize seedlings under alkaline stress conditions.
FIG. 6 is a statistical result of the average total root length, the average root surface area, the average fresh weight and the average dry weight of corn seedlings treated by YJD-CS2 microbial inoculum under the alkali stress condition.
FIG. 7 shows the effect of YJD-CS2 inoculum on whole seedlings and aerial parts of tomato seedlings under alkaline stress conditions.
FIG. 8 is the effect of YJD-CS2 inoculum on root morphology of tomato seedlings under alkaline stress conditions.
FIG. 9 is a statistical result of the average total root length, average root surface area, average fresh weight and average dry weight of tomato seedlings treated with YJD-CS2 microbial inoculum under alkali stress conditions.
Deposit description
The strain name: shewanella genus
Latin name: shewanella sp.
The strain number is as follows: YJD-CS2
The preservation organization: china center for type culture Collection
The preservation organization is abbreviated as: CCTCC (China center for cell communication)
Address: wuhan university in China
The preservation date is as follows: 5/7/2019
The registration number of the collection center: CCTCC NO: m2019330
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental procedures in the following examples are all conventional ones unless otherwise specified.
The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
In the quantitative experiments in the following examples, three replicates were set up and the results averaged.
LB liquid medium: adding distilled water into 10g tryptone, 5g yeast extract and 10g sodium chloride, adjusting pH to 7.0, adjusting volume to 1L, sterilizing at 121 deg.C for 20min, and cooling.
LB solid medium: adding agar into LB liquid culture medium to make the concentration of agar 15g/L; then sterilized at 121 ℃ for 20min. The medium cooled to about 55 ℃ was poured into a petri dish and allowed to cool naturally.
The Columbia-0 subtype of wild type Arabidopsis thaliana (Arabidopsis thaliana) is described in the following references: kim H, hyun Y, park J, park M, kim M, kim H, lee M, moon J, lee I, kim J.A genetic link between colored responses and flowing time through FVE in Arabidopsis thaliana. Nature genetics.2004, 36. The Columbia-0 subtype of wild type Arabidopsis (Arabidopsis thaliana) is hereinafter referred to as Arabidopsis thaliana for short.
Example 1, shewanella sp YJD-CS2CCTCC NO: isolation, identification and preservation of M2019330
1. Isolation of bacterium YJD-CS2
1. Adding 5g of soil sample (rhizosphere soil of plant Artemisia salina in West beach saline-alkali soil in suburb of Yinchuan city of Hui nationality of Ningxia, china) into 45mL of sterile distilled water, stirring for 15min, standing for 10min, collecting 1mL of supernatant, adding into a sterile test tube containing 9mL of sterile water, and mixing completely (the dilution is 10) -1 ) Then 1mL of the mixture is sucked from the test tube and added into another sterile test tube containing 9mL of sterile water to be uniformly mixed, and the like is repeated to prepare 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 Bacterial suspensions of various dilutions. 0.1mL of each dilution was uniformly applied to LB solid medium and incubated at 30 ℃ for 2-3 days.
2. After completing the step 1, picking a single colony on an LB solid culture medium, and repeatedly purifying for more than 3 times. The selected bacterium is named as the bacterium YJD-CS2.
2. Identification of bacteria YJD-CS2
1. Morphological identification
(1) The bacterium YJD-CS2 was inoculated into LB solid medium, and the morphology of a single colony was observed after 3 days.
The result shows that the bacterial colony of the bacteria YJD-CS2 is circular, has the diameter of 2.0-4.0 mm, has neat edges, smooth and wet surface, light yellow color and opaque bacterial colony.
(2) The bacterium YJD-CS2 was stained and identified as a gram-negative bacterium.
(3) And observing the morphology of the bacteria YJD-CS2 by adopting a high-resolution transmission electron microscope.
The results are shown in FIG. 1 (10000 times for the left image and 20000 times for the right image). The size of the bacterium YJD-CS2 was 1.07. Mu. M.times.3.084. Mu.m, short rod shape.
2. 16S rDNA sequence homology analysis
The 16S rDNA of the bacterium YJD-CS2 is shown as sequence 1 in the sequence table.
The double-stranded DNA molecule shown in the sequence 1 in the sequence table is compared with the sequence in GenBank by utilizing Clustal X software. The results showed that the homology of the bacterium YJD-CS2 with Shewanella strain FDAARGOS _354 was the highest, reaching 99.38%.
3. Preservation of
According to the above morphology and the results of 16S rDNA sequence homology analysis, the bacterium YJD-CS2 separated and purified in the step is identified as a bacterium belonging to the genus Shewanella. The bacterium YJD-CS2 is preserved in China center for type culture Collection (CCTCC for short, the address is Wuhan university, china) in 2019, 5 months and 7 days, and the preservation number is CCTCC NO: and M2019330. The bacterium YJD-CS2 is known as Shewanella sp YJD-CS2CCTCC NO: m2019330, YJD-CS2 or Shewanella YJD-CS2 for short, shewanella sp.
And (3) strain preservation: inoculating a single colony of Shewanella YJD-CS2 to an LB liquid culture medium, and culturing at 30 ℃ for 16h to obtain a culture solution. 1 part by volume of the culture broth and 1 part by volume of 50% (v/v) aqueous glycerol solution were mixed and stored at-80 ℃.
Example 2 preparation of YJD-CS2 microbial inoculum
1. The strain stored at-80 ℃ in example 1 was activated on LB solid medium.
2. Selecting LB solid medium single colony, inoculating into conical flask (500 mL) containing 100mL LB liquid medium, culturing at 30 deg.C and 130r/min for 48 hr to obtain OD 600nm A bacterial solution with a value of about 1.0. The bacterial liquid is the prepared YJD-CS2 bacterial agent.
Example 3 application of Shewanella YJD-CS2 in improving saline-alkali resistance of Arabidopsis thaliana
The specifications of the culture dish are 10cm multiplied by 10cm.
1. Preparation of the culture Medium
MS solid medium 1: dissolving 2.37g MS basic culture medium powder and 13g agar in distilled water, then adding distilled water to a constant volume of 1L, adjusting pH to 5.8, sterilizing at 121 deg.C for 20min, and cooling for use.
MS solid medium 2: dissolving 2.37g MS basic culture medium powder and 13g agar in distilled water, adding distilled water to a constant volume of 1L, adjusting pH to 8.5, sterilizing at 121 deg.C for 20min, and cooling.
Salt solid medium: dissolving 2.37g of MS basic culture medium powder, 13g of agar and 5.844g of NaCl in distilled water, then using the distilled water to fix the volume to 1L, and adjusting the pH value to 8.0; the concentration of NaCl in the system is 100mM; then, the mixture was sterilized at 121 ℃ for 20min, and the medium at about 55 ℃ was poured into petri dishes (25 mL each), and allowed to cool.
Alkali solid medium: 2.37g MS minimal medium powder, 13g agar and 0.168g NaHCO 3 Dissolving in distilled water, then using distilled water to fix the volume to 1L, and adjusting the pH value to 8.0; naHCO 2 3 The concentration in the system was 2mM; then, the mixture was sterilized at 121 ℃ for 20min, and the medium at about 55 ℃ was poured into petri dishes (25 mL each), and allowed to cool.
Saline-alkali solid culture medium: 2.37g MS minimal medium powder, 13g agar, 5.844g NaCl and 0.168g NaHCO 3 Dissolving in distilled water, then using distilled water to fix the volume to 1L, and adjusting the pH value to 8.0; naHCO 3 The concentration of NaCl in the system was 2mM and the concentration of NaCl in the system was 100mM; then, the mixture was sterilized at 121 ℃ for 20min, and the medium at about 55 ℃ was poured into petri dishes (25 mL each), and allowed to cool.
MS1+ microbial inoculum solid medium: a petri dish containing the MS solid culture medium 1 is taken, 0.1mL of YJD-CS2 microbial inoculum is coated on the surface of the culture medium below one fourth of the lower part of the petri dish, and the petri dish is naturally air-dried.
MS2+ microbial inoculum solid medium: a petri dish containing the MS solid culture medium 2 is taken, 0.1mL of YJD-CS2 microbial inoculum is coated on the surface of the culture medium below one fourth of the lower part of the petri dish, and the petri dish is naturally air-dried.
Salt + microbial inoculum solid medium: taking a culture dish filled with a salt solid culture medium, coating 0.1mL of YJD-CS2 microbial inoculum on the surface of the culture medium below one fourth of the culture dish, and naturally drying.
Alkali + microbial inoculum solid medium: taking a culture dish filled with an alkali solid culture medium, coating 0.1mL of YJD-CS2 microbial inoculum on the surface of the culture medium below one fourth of the culture dish, and naturally drying.
Saline-alkali + microbial inoculum solid culture medium: taking a culture dish filled with a saline-alkali solid culture medium, coating 0.1mL of YJD-CS2 microbial inoculum on the surface of the culture medium below one fourth of the lower part of the culture dish, and naturally drying in the air.
2. Influence of YJD-CS2 microbial inoculum on saline-alkali stress resistance of arabidopsis thaliana
The culture conditions were: 22 ℃;12h light/12 h dark; the illumination intensity was 12000Lx.
1. Taking Arabidopsis seeds, treating with 70% (v/v) ethanol water for 5min, sterilizing with 2.6% (v/v) sodium hypochlorite water solution for 10min, and washing with sterilized warm water for 5 times.
2. After the step 1 is completed, sowing the arabidopsis seeds in a solid culture medium (salt solid culture medium, alkali solid culture medium, saline-alkali solid culture medium, salt + microbial inoculum solid culture medium, alkali + microbial inoculum solid culture medium, saline-alkali + microbial inoculum solid culture medium, MS solid culture medium 1, MS solid culture medium 2, MS1+ microbial inoculum solid culture medium or MS2+ microbial inoculum solid culture medium), and vernalizing at 4 ℃ for three days. It should be noted that the seeds are not in direct contact with the microbial inoculum during sowing, namely the seeds are sown in more than one fourth of the culture dish filled with the solid culture medium, and after subsequent vertical culture, the root of the arabidopsis thaliana does not grow to the culture medium coated with the microbial inoculum (the experiment researches the influence of the secretion of the microbial inoculum on the root growth of the arabidopsis thaliana).
3. The solid medium which completed step 2 was vertically cultured for 6 days, and the growth state of Arabidopsis thaliana was observed.
The growth state of arabidopsis thaliana is shown in fig. 2 (1 is an MS solid culture medium 1,2 is an MS1+ microbial inoculum solid culture medium, 3 is an MS solid culture medium 2,4 is an MS2+ microbial inoculum solid culture medium, 5 is an alkali solid culture medium, 6 is an alkali + microbial inoculum solid culture medium, 7 is a salt solid culture medium, 8 is a salt + microbial inoculum solid culture medium, 9 is a saline-alkali solid culture medium, and 10 is a saline-alkali + microbial inoculum solid culture medium).
4. The length of the main root of the arabidopsis seedlings in the step 3 is counted (the average value is obtained by repeating the experiment for three times, and 80 arabidopsis seeds are used each time).
The statistical results are shown in table 1 and fig. 3 (1 is that MS solid culture medium 1,2 is MS1+ microbial inoculum solid culture medium, 3 is MS solid culture medium 2,4 is MS2+ microbial inoculum solid culture medium, 5 is alkali solid culture medium, 6 is alkali + microbial inoculum solid culture medium, 7 is salt solid culture medium, 8 is salt + microbial inoculum solid culture medium, 9 is saline alkali solid culture medium, 10 is saline alkali + microbial inoculum solid culture medium, blank is culture medium, S2-12 is culture medium containing YJD-CS2 microbial inoculum).
TABLE 1
| Type of solid Medium | Average main root length (cm) |
| MS |
28.54 |
| MS1+ microbial inoculum solid medium | 34.67 |
| MS |
8.70 |
| MS2+ microbial inoculum solid medium | 31.96 |
| Alkali solid culture medium | 1.18 |
| Solid culture medium of alkali and microbial inoculum | 25.32 |
| Salt solid culture medium | 7.87 |
| Solid culture medium of salt and microbial inoculum | 12.64 |
| Saline-alkali solid culture medium | 1.01 |
| Saline-alkali + microbial inoculum solid culture medium | 6.74 |
The results show that the average main root length of the arabidopsis seedlings on the MS solid culture medium 1 and the MS1+ microbial inoculum solid culture medium is obviously different under the condition of no salt and alkali stress, the average main root length of the arabidopsis seedlings on the MS solid culture medium 2 and the MS2+ microbial inoculum solid culture medium is obviously different, and the arabidopsis seedlings coated with the YJD-CS2 microbial inoculum grow better; under the condition of saline-alkali stress, the average main root length of the arabidopsis seedlings on the solid culture medium coated with the YJD-CS2 microbial inoculum is obviously longer than that of the corresponding solid culture medium not coated with the YJD-CS2 microbial inoculum. Therefore, the Shewanella YJD-CS2 can improve the saline-alkali resistance of Arabidopsis.
Example 4 application of Shewanella YJD-CS2 in improving alkali stress resistance of corn
Agar solid medium: 5g of agar powder was dissolved in 500mL of distilled water and sterilized at 121 ℃ for 20min. Pouring the agar culture medium with the temperature of about 55 ℃ into a culture dish, and naturally cooling.
Alkali liquor: 1 part by volume of 500 Xalkali mother liquor and 499 parts by volume of water. 500 Xthe mother liquor of alkali: 10.599g of Na 2 CO 3 And 8.4g NaHCO 3 Dissolved in 500mL of deionized water, and the pH value is adjusted to 10.0 for later use.
The culture conditions are as follows: 28 ℃;12h light/12 h dark; the illumination intensity was 12000Lx.
1. Taking 80 maize inbred line B73 seeds, sterilizing for 5min by using 70% (v/v) ethanol water solution, and washing for 1 time by using sterilized water; then sterilizing with 2.6% (v/v) sodium hypochlorite aqueous solution for 15min, and washing with sterilized water for 5 times.
2. Sowing corn seeds in an agar solid culture medium, and vertically culturing for 7 days to obtain seedlings.
3. Transplanting the seedlings into pots filled with nutrient soil (2 seedlings per pot) to obtain 80 seedlings of 40 pots of corn.
4. 20 pots of corn seedlings are watered with 200mL of alkali liquor every week. And (3) taking another 20 pots of corn seedlings, and irrigating the corn seedlings with deionized water once a week, wherein 200mL of the deionized water is irrigated each time. The mixture was irrigated for 5 weeks.
5. And randomly dividing 20 pots of corn seedlings poured with alkali liquor into an alkali liquor control group and an alkali liquor fungicide group, wherein each group comprises 10 pots. The 20 pots of corn seedlings watered with deionized water are randomly divided into two groups, namely a water control group and a water microbial inoculum group, and each group comprises 10 pots. The experiments were as follows:
dripping 200 mu L of YJD-CS2 microbial inoculum to the root of each corn seedling every 3 days for the alkaline microbial inoculum group, and culturing for 35 days; the alkaline solution control group was cultured under the same conditions for 35 days without applying a microbial inoculum.
Dripping 200 mu L of YJD-CS2 microbial inoculum to the root of each corn seedling every 3 days for the aquatic microbial inoculum group, and culturing for 42 days; the water control group was cultured under the same conditions for 35 days without applying a microbial inoculum.
6. And observing and analyzing the phenotype of the growth and development of each group of corn, and recording the root form of each group of corn seedlings by using a WiRHIO root scanning analyzer.
The phenotypes of the overground part and the whole seedling of the corn seedling are shown in a figure 4 (1 is the overground part of a water treatment group, the left side is a water control group, and the right side is a water microbial inoculum group, 2 is the overground part of an alkali liquor treatment group, the left side is an alkali liquor control group and the right side is an alkali liquor microbial inoculum group, 3 is the whole seedling of the water treatment group, the left side is a water control group, and the right side is a water microbial inoculum group, and 4 is the whole seedling of the alkali liquor treatment group, and the left side is an alkali liquor control group and the right alkali liquor microbial inoculum group).
The shape of corn root is shown in figure 5 (1 is water control group, 2 is water bacterial agent group, 3 is alkali solution control group, and 4 is alkali solution bacterial agent group).
7. And (3) carrying out statistics on the total root length, root surface area, fresh weight and dry weight of each corn seedling by treating the corn seedlings with YJD-CS2 microbial inoculum for 35 days under the alkali stress condition, and averaging.
The results are shown in Table 2 and FIG. 6. Under the condition of water treatment, the dry weight, fresh weight, total root length and root surface area of the maize seedlings applied with the YJD-CS2 microbial inoculum are all obviously higher than those of the maize seedlings not applied with the YJD-CS2 microbial inoculum; under the condition of alkali stress, the dry weight, the fresh weight, the total root length and the root surface area of the maize seedlings applied with the YJD-CS2 microbial inoculum are all obviously higher than those of the maize seedlings not applied with the YJD-CS2 microbial inoculum. Therefore, the Shewanella YJD-CS2 can improve the alkali stress resistance of the corn.
TABLE 2
Example 5 application of Shewanella YJD-CS2 to improvement of tomato alkali stress resistance
The culture conditions are as follows: 28 ℃;12h light/12 h dark; the light intensity was 5000Lx.
Alkali liquor: 1 part by volume of 500 Xalkali mother liquor and 499 parts by volume of water. 500 Xthe mother liquor of alkali: 10.599g of Na 2 CO 3 And 8.4g NaHCO 3 Dissolved in 500mL of deionized water, and the pH value is adjusted to 10.0 for later use.
1. Placing 80 tomato variety M82 seeds into a culture dish, adding a small amount of water, and placing the culture dish in a constant-temperature incubator for culturing until the seeds germinate and have short roots.
2. Transplanting the germinated tomato seedlings into pots (2 in each pot) filled with nutrient soil to obtain 80 tomato seedlings in 40 pots.
3. And (3) pouring alkali liquor once a week in 20 pots of tomato seedlings, wherein 200mL of alkali liquor is poured once each time. And irrigating 20 pots of tomato seedlings with deionized water once a week, wherein 200mL of deionized water is irrigated every time. The mixture was irrigated for 6 weeks.
4. The 20 pots of tomato seedlings watered with the alkali liquor are randomly divided into two groups, namely an alkali liquor control group and an alkali liquor fungicide group, and each group comprises 10 pots. Randomly dividing 20 pots of tomato seedlings watered with deionized water into two groups, namely a water control group and a water microbial inoculum group, and 10 pots of tomato seedlings are respectively arranged in each group. The experiments were as follows:
dripping 60 mu L of YJD-CS2 microbial inoculum to the root of each tomato seedling every 3 days for the alkali microbial inoculum group, and culturing for 42 days; the control group of alkali liquor was cultured for 42 days under the same conditions without applying any microbial inoculum.
Dripping 60 mu L of YJD-CS2 microbial inoculum to the root of each tomato seedling every 3 days for the aquatic microbial inoculum group, and culturing for 42 days; the water control group was cultured for 42 days under the same conditions without applying the microbial inoculum.
5. And observing and analyzing the phenotype of the growth and development of each group of tomatoes, and recording the root system morphology of each group of tomato seedlings by using a WiRHIO root system scanning analyzer.
The phenotypes of the overground part and the whole seedling of the tomato seedling are shown in figure 7 (1 is the overground part of a water treatment group, the left side is a water control group, and the right side is a water fungicide group, 2 is the overground part of an alkali liquor treatment group, the left side is an alkali liquor control group and the right side is an alkali liquor fungicide group, 3 is the whole seedling of the water treatment group, the left side is a water control group, and the right side is an alkali liquor control group, and 4 is the whole seedling of the alkali liquor treatment group, and the left side is an alkali liquor control group and the right side is an alkali liquor fungicide group).
The tomato root morphology is shown in FIG. 8 (1 is water control group, 2 is water microbial inoculum group, 3 is alkali liquor control group, and 4 is alkali liquor microbial inoculum group).
7. And (3) carrying out statistics on the total root length, the root surface area, the fresh weight and the dry weight of each tomato seedling after the tomato seedlings are treated by the YJD-CS2 microbial inoculum for 42 days under the alkali stress condition, and taking an average value.
The results are shown in Table 3 and FIG. 9. Under the condition of water treatment, the dry weight, fresh weight, total root length and root surface area of the tomato seedlings applied with the YJD-CS2 microbial inoculum are all obviously higher than those of the tomato seedlings not applied with the YJD-CS2 microbial inoculum; under the alkali stress condition, the dry weight, the fresh weight, the total root length and the root surface area of the tomato seedlings applied with the YJD-CS2 microbial inoculum are obviously higher than those of the tomato seedlings not applied with the YJD-CS2 microbial inoculum. Therefore, the Shewanella YJD-CS2 can improve the saline alkali stress resistance of tomatoes.
TABLE 3
<110> northeast university of forestry
Shewanella and application thereof in improving stress resistance of plants
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1459
<212> DNA
<213> Shewanella sp.
<400> 1
ggggctgttg ggggcaggct aacacatgca agtcgagcgg cagcacaagg gagtttactc 60
ctgaggtggc gagcggcgga cgggtgagta atgcctaggg atctgcccag tcgaggggga 120
taacagttgg aaacgactgc taataccgca tacgccctac gggggaaaga aggggacctt 180
cgggccttcc gcgattggat gaacctaggt gggattagct agttggtgag gtaatggctc 240
accaaggcga cgatccctag ctgttctgag aggatgatca gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagtg gggaatattg cacaatgggg gaaaccctga 360
tgcagccatg ccgcgtgtgt gaagaaggcc ttcgggttgt aaagcacttt cagtagggag 420
gaaagggtga gtcttaatac ggctcatctg tgacgttacc tacagaagaa ggaccggcta 480
actccgtgcc agcagccgcg gtaatacgga gggtccgagc gttaatcgga attactgggc 540
gtaaagcgtg cgcaggcggt ttgttaagcg agatgtgaaa gccctgggct caacctagga 600
atagcatttc gaactggcga actagagtct tgtagagggg ggtagaattc caggtgtagc 660
ggtgaaatgc gtagagatct ggaggaatac cggtggcgaa ggcggccccc tggacaaaga 720
ctgacgctca tgcacgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgtctactcg gagtttggtg tcttgaacac tgggctctca agctaacgca 840
ttaagtagac cgcctgggga gtacggccgc aaggttaaaa ctcaaatgaa ttgacggggg 900
cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa cgcgaagaac cttacctact 960
cttgacatcc acagaagagt gcagagatgc ggttgtgcct tcgggaaccg tgagacaggt 1020
gctgcatggc tgtcgtcagc tcgtgttgtg aaatgttggg ttaagtcccg caacgagcgc 1080
aacccctatc cttatttgcc agcacgtaat ggtgggaact ctagggagac tgccggtgat 1140
aaaccggagg aaggtgggga cgacgtcaag tcatcatggc ccttacgagt agggctacac 1200
acgtgctaca atggcgagta cagagggttg caaagccgcg aggtggagct aatctcacaa 1260
agctcgtcgt agtccggatt ggagtctgca actcgactcc atgaagtcgg aatcgctagt 1320
aatcgtggat cagaatgcca cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380
caccatggga gtgggctgca aaagaagtgg gtagcttaac cttcgggggg gcgctcacca 1440
actttggttt cattggtgc 1459
Claims (7)
1. Shewanella (Shewanella sp.) YJD-CS2, the preservation number of which in China center for type culture Collection is CCTCC NO: and M2019330.
2. A microbial inoculum, which is characterized in that: the microbial agent contains the Shewanella sp (Shewanella sp.) YJD-CS2CCTCC NO: and M2019330.
3. The Shewanella (Shewanella sp.) YJD-CS2CCTCC NO of claim 1: the application of the M2019330 or the microbial inoculum of claim 2, which is at least one of A1) to A3):
a1 Improving the saline-alkali resistance of arabidopsis;
a2 Improving the alkali resistance of corn;
a3 ) increase the alkali resistance of tomatoes.
4. The Shewanella sp (Shewanella sp.) YJD-CS2CCTCC NO: use of M2019330 or the microbial inoculum of claim 2 in the preparation of a product; the product has the functions of at least one of A1) to A3):
a1 Improving the saline-alkali resistance of arabidopsis thaliana;
a2 Improving the alkali resistance of corn;
a3 ) increase the alkali resistance of tomatoes.
5. A method for improving the saline-alkali resistance of Arabidopsis thaliana, which comprises using the Shewanella sp (Shewanella sp.) YJD-CS2CCTCC NO: m2019330 or the microbial inoculum disclosed by claim 2 is used for treating arabidopsis thaliana so as to improve the saline-alkali resistance of arabidopsis thaliana.
6. A method for improving alkali resistance of corn by using the Shewanella sp (Shewanella sp.) YJD-CS2CCTCC NO: m2019330 or the microbial inoculum of claim 2 is used for treating corn so as to improve the alkali resistance of the corn.
7. A method for improving alkali resistance of tomato, which comprises using the Shewanella sp YJD-CS2CCTCC NO: m2019330 or the microbial inoculum of claim 2, so as to improve the alkali resistance of the tomatoes.
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| CN103173383A (en) * | 2012-12-26 | 2013-06-26 | 清华大学深圳研究生院 | Shewanella piezotolerans 34# and application thereof to algae inhibition |
| CN104962494A (en) * | 2015-06-25 | 2015-10-07 | 东北农业大学 | Shewanella having good degradation effect on herbicide atrazine |
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| CN109593686A (en) * | 2019-01-18 | 2019-04-09 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | A kind of Shewanella and its application in control vegetable field soil nitrogen pollution |
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| CN104962494A (en) * | 2015-06-25 | 2015-10-07 | 东北农业大学 | Shewanella having good degradation effect on herbicide atrazine |
| JP2017176128A (en) * | 2016-03-31 | 2017-10-05 | 株式会社前川製作所 | Improvement in salt resistance and/or osmotic pressure resistance of plant using comamonas bacteria |
| CN109593686A (en) * | 2019-01-18 | 2019-04-09 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | A kind of Shewanella and its application in control vegetable field soil nitrogen pollution |
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