A Pseudomonas nitroreducens and its application in drought resistance and growth promotion of rice

Abstract

The present invention discloses a Pseudomonas nitroreducens and its application in drought resistance and growth promotion of rice. The strain is classified and named as Pseudomonas nitroreducens LSF-6, which was deposited at the China Center for Type Culture Collection on May 12, 2022, with the deposit number CCTCC NO: M 2022607. The Pseudomonas nitroreducens LSF-6 provided by the present invention can regulate the formation of rice root sheaths under drought conditions, promote the growth of rice and improve its water use efficiency, thereby enhancing the drought stress tolerance of rice, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and in particular, to a Pseudomonas nitroreducens LSF-6 and its application in drought resistance and growth promotion of rice. Background Art

[0002] Drought and water shortage are one of the main limiting factors affecting the sustainable development of global agriculture. Root sheaths, also known as sand sheaths, were first discovered in water-scarce areas such as deserts and are adaptive structures produced by plants in response to water stress. Some gramineous plants such as barley, wheat, corn, and sorghum under drought stress can form root sheaths. A root sheath is a sheath-like structure with root hairs as the skeleton and rhizosphere soil particles tightly adhered to the plant root surface by root surface mucus, which is the result of the interaction between plant roots, soil, and microorganisms. The formation of the root sheath structure promotes the information exchange and the exchange of water and nutrients at the root-soil interface. Microorganisms are one of the main influencing factors in the construction of plant root sheaths. Therefore, isolating and applying microorganisms in the soil that can promote the construction of rice root sheaths is an effective way to improve the drought tolerance of rice. Summary of the Invention

[0003] To this end, the present invention provides a Pseudomonas nitroreducens LSF-6 and its application in drought resistance and growth promotion of rice.

[0004] To achieve the above object, the technical solutions provided by the present invention are as follows:

[0005] A Pseudomonas nitroreducens LSF-6, the classification and naming of the Pseudomonas nitroreducens LSF-6 is Pseudomonas nitroreducens ( Pseudomonas nitroreducens ), which was deposited at the China Center for Type Culture Collection on May 12, 2022. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M2022607.

[0006] Further, the above-mentioned Pseudomonas nitroreducens LSF-6 was isolated from rice root sheaths.

[0007] Further, the 16S rDNA sequence of the above-mentioned Pseudomonas nitroreducens LSF-6 is as shown in SEQ ID NO.1.

[0008] Further, the colonies of the above-mentioned Pseudomonas nitroreducens LSF-6 on the LB solid medium are light yellow, semi-transparent, round, and the boundaries are not clear; the cell morphology is spherical.

[0009] A drought-resistant and growth-promoting microbial agent, the microbial agent includes Pseudomonas nitroreducens LSF-6 with the deposit number of CCTCC NO: M 2022607.

[0010] Use of the above-mentioned Pseudomonas nitroreducens LSF-6 or the above-mentioned microbial agent in promoting the growth of rice.

[0011] Use of the above-mentioned Pseudomonas nitroreducens LSF-6 or the above-mentioned microbial agent in improving the drought resistance of rice.

[0012] Use of the above-mentioned Pseudomonas nitroreducens LSF-6 or the above-mentioned microbial agent in promoting the growth of rice while improving the drought resistance of rice.

[0013] The remarkable advantages of the present invention are as follows:

[0014] The Pseudomonas nitroreducens LSF-6 of the present invention has the characteristic of improving the root sheath formation of rice. The re-inoculation test found that under drought treatment, compared with the control treatment, inoculation with Pseudomonas nitroreducens LSF-6 can significantly increase the root hair length, total root length, unit root sheath and water use efficiency of rice. At the same time, the chlorophyll content, net photosynthetic rate, stomatal conductance and transpiration rate of rice plants inoculated with Pseudomonas nitroreducens LSF-6 also increased significantly. In short, the Pseudomonas nitroreducens LSF-6 of the present invention is a new growth-promoting bacterium, which can significantly improve the drought tolerance of rice and thus promote the green production of rice. Description of the Drawings

[0015] Figure 1 : Colony morphology of Pseudomonas nitroreducens LSF-6.

[0016] Figure 2 : Cell morphology of Pseudomonas nitroreducens LSF-6.

[0017] Figure 3 : Effect of Pseudomonas nitroreducens LSF-6 on the biomass of rice.

[0018] Figure 4 : Effect of Pseudomonas nitroreducens LSF-6 on the photosynthetic parameters of rice.

[0019] Figure 5 : Effect of Pseudomonas nitroreducens LSF-6 on the root development of rice.

[0020] Figure 6 : Effect of Pseudomonas nitroreducens LSF-6 on the root sheath formation and water use efficiency of rice. Detailed Embodiments

[0021] The following describes the preferred embodiments of the present invention. It should be understood that the preferred embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not used to limit the present invention.

[0022] Unless otherwise specified, the raw materials and reagents used in the following examples are all obtained commercially.

[0023] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0024] In the following examples, the preparation method of the Pseudomonas CFC selective solid medium used is as follows: 16 g of gelatin peptone, 10 g of acid-hydrolyzed casein, 10 g of K2SO4, 1.4 g of MgCl2, 15 g of agar, and 10 g of glycerol are dissolved in 1 L of deionized water, sterilized at 121 °C for 15 min, and when cooled to about 50 °C, 2 mg of cetyltrimethylammonium bromide, 2 mg of fusidic acid sodium, and 10 mg of cephalothin II are added to every 200 mL of the medium, mixed well, and poured into plates for standby.

[0025] Example 1 Isolation and Screening of Strain RS63

[0026] The dry soil is screened through a 4 mm sieve, then water is added to the specified water content (220 mL of water is added to every 1 kg of dry soil). After the moist soil is fully mixed, it is screened a second time through a 4 mm sieve to remove any coarse substances, and then it is filled into plastic cups with a capacity of about 600 mL (bottom diameter: 6 cm, top diameter: 9 cm, height: 15 cm) (about 480 g of soil is filled in each plastic cup). Select rice seedlings with consistent growth and transplant them into the soil, and place them in a greenhouse for cultivation. The greenhouse is set with a 24 h cycle, the light intensity is 300 μmol m -2 s -1 , the relative humidity is 70%, and the light is 14 h (26 °C) / dark 10 h (22 °C). After the seedlings are transplanted into the soil, a one-week acclimation treatment is first carried out. During the acclimation period, 20 mL of water is watered every day to ensure the normal growth of rice. After the acclimation is over, the samples are adjusted to a unified initial water content by weighing, and a two-week drought treatment is started. During the drought treatment, the soil water content is maintained at 80% of the maximum field water holding capacity.

[0027] Take 1 g of the rice root sheath after drought treatment and place it in 10 mL of sterile water, and mix well. Take 1 mL of the soil suspension and dilute it stepwise with sterile water. Since the bacterial abundance in the soil is relatively high, when screening for bacteria in the soil, 10 -3 and 10 -4 100 μL of each concentration gradient bacterial suspension are respectively spread on the Pseudomonas CFC selective solid medium, and then inverted and incubated in the dark at 28 °C for 3 days. During this period, pay attention to observing the growth state of the colonies. After 3 days, pick single colonies and perform streak purification to obtain pure cultures.

[0028] The pure culture of the strain isolated from the rice root sheath was tested for its ability to build root sheaths: The strain stored at -80 °C was streaked on an LB solid medium and cultured in an incubator at 28 °C. After monoclonal colonies grew, a monoclonal colony was picked and transferred to 5 mL of LB liquid medium for culture, and cultured on a shaker at 28 °C and 200 rpm until the OD 600 of the bacterial solution reached 1.0. 500 μL of the cultured bacterial solution was transferred to 50 mL of LB liquid medium and continuously cultured for 2 - 3 days at 28 °C and 200 rpm until the OD 600 reached 1.5. Subsequently, the cultured bacterial solution was centrifuged at 5000 rpm for 10 min, the cell pellet was collected and suspended with sterile water, and the OD 600 value was adjusted to 0.2. 20 rice seeds were planted per pot in a pot filled with paddy soil. Rice seedlings with consistent growth were selected, and one surface-sterilized rice seedling was transplanted into each pot. The experiment was set up with an inoculation treatment and a non-inoculation treatment. In the experimental group, 10 mL of the bacterial solution with an OD 600 of 0.2 was applied to the substrate in each pot, and the control group was not inoculated with the strain. Five pots were planted in both the experimental group and the control group. During the growth period of the rice, the soil water content was adjusted to 80% of the maximum field water holding capacity every two days. After a total of 3 weeks of culture, the root sheath formation of the rice was statistically analyzed, and a Pseudomonas strain that could promote the formation of rice root sheaths was screened and named LSF-6.

[0029] Example 2 Identification of Strain LSF-6

[0030] Morphological identification:

[0031] The colonies of strain LSF-6 on the LB solid medium were light yellow, semi-transparent, round, and the boundaries were not clear ( Figure 1 ); the cell morphology was spherical ( Figure 2 ).

[0032] Molecular identification:

[0033] PCR was used to amplify the 16s rDNA of strain LSF-6. The upstream primer was 16s-rRNA-F, and the downstream primer was 16s-rRNA-R. The 16s rDNA sequence of strain LSF-6 is shown in SEQ ID NO.1. This sequence was BLAST aligned in the Genbank database. According to the results of homology analysis, strain LSF-6 was identified as Pseudomonas nitroreducens ( Pseudomonas nitroreducens ).

[0034] The sequences of primers 16s-rRNA-F / 16s-rRNA-R are as follows:

[0035] 16s-rRNA-F: 5’-AGAGTTTGATCCTGGCTCAG-3’,

[0036] 16s-rRNA-R: 5’-TACGGCTACCTTGTTACGACTT-3’.

[0037] Example 3 Application of Strain LSF-6 in Drought Resistance and Growth Promotion of Rice

[0038] Inoculate a single colony of strain LSF-6 into 5 mL of LB liquid medium and culture it on a shaker at 28°C and 200 rpm until the OD of the bacterial solution 600 = 1.0. Take 500 μL of the cultured bacterial solution and transfer it to 50 mL of LB liquid medium, and continue to culture it at 28°C and 200 rpm for 2 - 3 days until the OD 600 = 1.5. Subsequently, centrifuge the cultured bacterial solution at 5000 rpm for 10 min, collect the cell pellet and suspend it with sterile water, and adjust the OD 600 value to 0.2. Plant 20 rice seeds per pot in pots filled with paddy soil, select rice seedlings with consistent growth, transplant one surface-sterilized rice seedling per pot, set the experiment as inoculation treatment and non-inoculation treatment, apply 10 mL of bacterial solution with OD 600 = 0.2 to the substrate of each pot in the experimental group, and do not inoculate the strain in the control group. Plant 5 pots each in the experiment and the control group. During the growth period of rice, adjust the soil water content to 80% of the maximum field water holding capacity every two days. After culturing for 3 weeks, measure the dry weights of the above-ground parts and roots, photosynthetic parameters, root sheath weight and water use efficiency of the plants.

[0039] The measurement results are as follows:

[0040] 1) Under drought treatment, compared with the control treatment, the dry weights of the above-ground parts and roots of rice inoculated with strain LSF-6 increased by 62.5% and 64.9% respectively ( Figure 3 ).

[0041] 2) Compared with the control treatment, the SPAD value, stomatal conductance, transpiration rate and net photosynthetic rate of rice inoculated with strain LSF-6 were all significantly increased ( Figure 4 ). At the same time, the total root length and root hair length of rice inoculated with strain LSF-6 increased by 45.8% and 38.1% respectively compared with the control ( Figure 5 ).

[0042] 3) Compared with the control treatment, the water use efficiency of rice inoculated with strain LSF-6 increased by 34.5% ( Figure 6). The above results indicate that under drought stress, strain LSF-6 can enhance the drought tolerance of rice by regulating the formation of rice root sheaths, improving rice growth and water use efficiency, and thus has good application prospects.

[0043] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A Pseudomonas nitroreducens Pseudomonas nitroreducens strain LSF-6, characterized in that: It was deposited at the China Center for Type Culture Collection on May 12, 2022. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2022607.

2. The Pseudomonas nitroreducens LSF-6 according to claim 1, characterized in that: The colonies of Pseudomonas nitroreducens LSF-6 on LB solid medium are light yellow, semi-transparent, round, and have unclear boundaries; the cell morphology is spherical.

3. A drought-resistant growth-promoting bactericide, characterized in that: It includes Pseudomonas nitroreducens LSF-6 with the deposit number CCTCC NO: M 2022607.

4. Use of Pseudomonas nitroreducens LSF-6 according to claim 1 or the bacterial agent according to claim 3 in improving the drought resistance of rice, characterized in that: The improvement of the drought resistance of rice is to increase the dry weight of the unit root sheath of rice under drought growth conditions and to improve the water use efficiency of rice under drought growth conditions; the drought growth conditions refer to maintaining the soil water content at 80% of the maximum water holding capacity during the growth period of rice.

5. Use of Pseudomonas nitroreducens LSF-6 according to claim 1 or the bacterial agent according to claim 3 in promoting the growth of rice while improving the drought resistance of rice, characterized in that: The promotion of rice growth is to increase the above-ground dry weight of rice, increase the root dry weight of rice, increase the SPAD value of rice, increase the stomatal conductance of rice, increase the transpiration rate of rice, increase the net photosynthetic rate of rice, increase the total root length of rice, and increase the root hair length of rice; the improvement of the drought resistance of rice is to increase the dry weight of the unit root sheath of rice under drought growth conditions and to improve the water use efficiency of rice under drought growth conditions; the drought growth conditions refer to maintaining the soil water content at 80% of the maximum water holding capacity during the growth period of rice.

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