CN118460385B - George strain capable of improving salt and alkali tolerance of corn and application thereof - Google Patents

Abstract

The invention provides a Grignard strain capable of improving the salt and alkali tolerance of corn and application thereof, wherein the strain is named as FJ0037, the preservation number is CGMCC No.41320, the strain belongs to the common microorganism center of China general microbiological culture Collection center preserved in the 5 month 27 of 2024, and the preservation address is North Star Xie Jiu No. 1 and No. 3 in the Korean region of Beijing city. The strain was isolated from healthy leaves of goosegrass. Strain FJ0037 has an ultra-short propagation cycle, propagation coefficient and effective yield, and it is easy to survive in an industrial dish, thus providing a rich experimental material. The strain FJ0037 can promote growth, enhance stress resistance of crops, promote germination of corn seeds, has good salt and alkali resistance, and effectively slows down the inhibition of salt and alkali on plants.

Description

A strain of the genus Lachnosporium capable of improving the salt-alkali tolerance of corn and its application

Technical Field

The invention belongs to the field of salt-alkali resistant bacterial strains, and particularly relates to a strain of the genus Lycosporium capable of improving the salt-alkali resistance of corn and an application thereof.

Background Art

The western part of Jilin Province is one of the concentrated distribution areas of soda saline-alkali land in my country. The western part of Jilin Province is flat and belongs to an arid and semi-arid area. The salt and alkali brought by the river are precipitated here. A large amount of evaporation causes the groundwater in the soil to evaporate continuously, resulting in salt remaining in the surface soil. In recent years, improving the saline-alkali land in western Jilin, deeply exploring the ecological and economic benefits of the saline-alkali land in western Jilin, and awakening the "sleeping" soda saline-alkali land resources have become the primary issues.

Soil salinization has posed a huge threat to agricultural production, and researchers around the world have paid close attention to this issue. For a long time, both domestic and foreign researchers have conducted extensive and in-depth research on the improvement and utilization of saline-alkali soils, and have invested a lot of manpower and material resources. In the 1980s, foreign countries mostly focused on the application of chemical improvers and deep plowing to destroy the alkaline layer. my country has adopted methods such as planting rice to improve alkali, applying organic fertilizers, planting green manure, and leveling the land. According to the nature of the improvement measures, the treatment measures for salinized land can be roughly divided into three categories: (1) physical remediation methods; (2) chemical remediation methods; (3) biological remediation methods.

Physical restoration methods are non-biological measures, mainly including drainage, flushing, loosening the soil and fertilizing, spreading sand and pressing alkali, etc.

Chemical remediation methods are also non-biological measures, mainly including the addition of improvers such as polymers, gypsum, zeolite, furfural residue, etc. Liu Yingxian and others invented a saline-alkali soil improver, which is made of polymaleic acid, sodium alkylbenzene sulfonate, zinc sulfate or copper sulfate and water.

Bioremediation of saline-alkali soil mainly includes phytoremediation and microbial remediation.

Phytoremediation mainly involves planting salt-tolerant plants. Microbial remediation mainly involves using salt-resistant microorganisms as agents to promote plant growth in the soil, thereby achieving the purpose of improving saline-alkali soil.

For a long time, people have used physical or chemical methods to desalinate the soil to control saline-alkali land. This requires a lot of money and fresh water, and it is difficult to promote it comprehensively. Only biological improvement can change the structure of the soil and essentially improve the physical and chemical properties of the soil. Therefore, in recent years, the research on the improvement and utilization of saline-alkali land has gradually shifted from engineering measures to biological measures. The microbial method breaks the traditional method of improving saline-alkali land. With very little human input, it improves the vegetation and physical and chemical properties of the soil in saline-alkali land in a short period of time, thus providing new ideas for exploring saline-alkali land restoration methods. It is of great significance to restore the local ecosystem and has become the current research focus.

Eleusine indica is an annual weed of the genus Eleusine in the Poaceae family. It has a very developed root system, tufted culms, flattened and ridged leaf sheaths on both sides, loose leaves, and flat leaves. It is one of the world's "top ten" malignant weeds. Eleusine indica has strong adaptability and reproductive capacity. It grows mostly in wasteland, farmland, orchards and roadsides. It is distributed in temperate and tropical regions around the world. In my country, it is mainly distributed in the Yellow River Basin and a wide area in the south. Most weed seeds have a certain dormancy period to resist adverse environmental conditions. During the dormancy period, they still have vitality in the soil for many years. The whole plant of Eleusine indica can be used as feed, and it is also an excellent soil conservation plant. It has the effects of dispelling wind and dampness, clearing heat and detoxifying, and dispersing blood stasis and stopping bleeding. The whole plant can be boiled in water and taken to prevent and treat Japanese encephalitis.

Endophytic fungi have a response relationship to saline-alkali stress, improve the host plant's ability to tolerate saline-alkali, and effectively reduce the damage to plants caused by saline-alkali stress. At the same time, endophytic fungi also improve saline-alkali soil and effectively improve and repair polluted soil. Therefore, cultivating more beneficial endophytes is of great value to the research on improving saline-alkali land.

Soil salinization is an important factor affecting agricultural production and the ecological environment. How to improve the salt-alkali tolerance of crops and promote the biological treatment and comprehensive development of saline soils is a major issue in future agriculture.

At present, the improvement and utilization of saline-alkali soil is mainly based on ecological restoration methods. Among them, salt-alkali tolerant microorganisms have broken the shortcomings of traditional methods of improving saline-alkali land and improved the vegetation and physical and chemical properties of soil in saline-alkali land in a short period of time. Research on salt-alkali tolerant microorganisms will become a development trend of saline-alkali land improvement in the future. However, the screening and research of endophytic fungi that have salt-dissolving and growth-promoting effects and can survive in saline-alkali environments are still relatively rare.

By investigating the effects of salt-alkali tolerant fungi on plants, it was found that salt-alkali tolerant strains promoted plant germination and enhanced the salt-alkali tolerance of plants. Therefore, the study of salt-alkali tolerant strains to improve the salt-alkali tolerance of plants has important agricultural, environmental and ecological significance. By studying salt-alkali tolerant endophytic fungi, the salt-alkali tolerance of plants can be improved, saline-alkali land improvement can be promoted, and agricultural yield and quality can be increased. It is expected to become a breakthrough in the salt-alkali resistance technology of crops and contribute to the sustainable development of green agriculture.

Summary of the invention

The purpose of the present invention is to solve the shortcomings of the prior art, aiming to screen a strain of the genus Pseudomonas that can improve the salt-alkali tolerance of corn, adopt plant and microbial improvement in biological improvement, screen endophytic fungi in saline-alkali land in western Jilin, discover endophytic fungi with salt-alkali resistance, explore the process of screening salt-alkali resistant endophytic fungi, and lay an experimental foundation for the development of endophytic fungi in agriculture.

The technical solution adopted by the present invention is: a strain of the genus Pseudomonas that can improve the salt-alkali tolerance of corn, the strain is named FJ0037, the preservation number is CGMCC No.41320, and it was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 27, 2024, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

Preferably, the strain is isolated from healthy leaves of Glechoma longituba.

Application of a strain of the genus Glehnia in promoting the growth of crops in saline-alkali environments.

Preferably, the germination of corn seeds is promoted.

Preferably, the stress resistance of crops is enhanced.

The beneficial effects obtained by the present invention are: the strain FJ0037 has an ultra-short reproduction cycle, reproduction coefficient and effective yield, and is easy to survive in an industrial culture dish, thus providing rich experimental materials.

Strain FJ0037 can not only promote growth, but also enhance the stress resistance of crops, promote the germination of corn seeds, has good salt-alkali tolerance, and can effectively reduce the inhibitory effects of salt and alkali on plants.

As a highly salt-alkali tolerant strain, strain FJ0037 has broad prospects for use in the agricultural field. In the practical application of solving saline-alkali land problems, strain FJ0037 can be cultivated in large quantities and made into microbial agents or fertilizers, which can be truly applied to the agricultural market or the improvement of saline-alkali land, contributing to increasing crop yields and improving saline-alkali land. It is expected to become a breakthrough in crop salt-alkali resistance technology, and also contribute to the green development of agriculture and provide new ways and possibilities for sustainable economic development.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the number of strains isolated from Glechoma longituba in 6 culture media in the embodiment;

Figure 2 is the growth diameter change curve of FJ0037 under 4% saline-alkali conditions;

Figure 3 shows the number of corn germinations per day under saline-alkali stress conditions;

Figure 4 shows the number of germinations per day of corn under the influence of strain FJ0037;

Figure 5 shows the cumulative germination number of corn seeds.

DETAILED DESCRIPTION

The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

Plant sample: Gnaphalium truncatum, collected from western Jilin Province (N44°27′13.82″ E123°23′20.49″) in September 2020.

A salt- and alkali-tolerant strain was isolated from the healthy leaves of Glechoma longituba. The strain was named FJ0037 and stored at low temperature in the biochemistry laboratory.

1. Screening and identification of salt-alkali tolerant strains

A native plant sample, Glechoma longituba, was collected from saline-alkali land in western Jilin Province, and salt-alkali tolerant strains were isolated and screened. The screened salt-alkali tolerant strains were identified through morphological observation and ITS sequence analysis to clarify their taxonomic status.

1. Test materials

1.1 Culture medium

Six common culture media were selected, including potato dextrose agar medium (PDA medium), Sabouraud agar medium (SAB), Czapek medium (sucrose sodium nitrate medium), Martin medium, malt extract medium (malt extract 30 g, tryptone 5 g, agar 20 g, nalidixic acid 0.02 g, water 1000 mL), fungus medium No. 2 (glucose 10 g, glycol dew 20 g, yeast extract 3 g, MgSO ₄ •H ₂ O 0.3 g, monosodium glutamate 1 g, KH ₂ PO ₄ 0.5 g, maltose 20 g, agar 20 g, chloramphenicol 0.1 g, corn steep liquor 1 g). All the above six culture media need to be sterilized at 121 ℃ for 30 min and cooled for use.

2. Research content

2.1 Isolation and purification of strains

Thoroughly rinse the collected plant samples with clean tap water to remove soil and other attachments on the surface. After drying at room temperature, place them in a clean bench. Perform surface disinfection according to the following steps: Spray the plant samples with 75% ethanol for 30 seconds. After disinfection, wash the samples with distilled water for 3 to 5 times.

Use sterilized scissors to cut the sample tissue material into small pieces of about 1 cm in size, add them into a mortar and grind them, while adding 10 mL of sterile water to make sure that they are fully ground. Use a pipette to draw 100 μL of the mixed liquid in the mortar onto culture medium No. 1 to 6, and use a sterilized coating rod to evenly spread the bacterial solution on the culture medium. After the bacterial solution has fully penetrated into the culture medium, place it in a biological incubator and culture it at 28°C.

Under the sterile clean bench operating environment, these strains were preliminarily classified by observing the colony boundaries, color, and height of the endophytic fungi growing around the tissue blocks. Then, a small amount of hyphae was selected from different hyphae tips using a sterilized inoculation stick and transferred to a new PDA medium. Under the same culture conditions, the culture was repeated to obtain separate purified colonies, and this purification process needed to be repeated many times.

2.2 Screening of salt-alkali tolerant strains

Determination of fungal growth under saline-alkali stress conditions: _NaHCO3 and _{Na2CO3 were} used to simulate the soda saline-alkali land in western Jilin for saline-alkali stress. A mixture of _NaHCO3 and _Na2CO3 with salinity concentrations of 1.6%, 2.4%, 3.2%, and 4.0%, respectively, was set and added to PDA medium under sterile conditions. All strains isolated previously were inoculated on the PDA medium simulating saline-alkali stress. The culture medium was placed in a constant temperature incubator at 28° _C for 24 to 48 hours, with light shielding throughout the process. In the later stage, as the salinity increased, the culture time was appropriately increased to observe the growth of the strains.

2.3 Identification of strain species

2.3.1 Morphological identification of strains

The purified fungal strain was inoculated into PDA medium and cultured under appropriate conditions for 7 days. After the culture, the colony size, hyphae growth rate, color, surface characteristics, etc. were observed. The growth was further observed by the glass slide wet chamber culture method and stained and fixed. Finally, the hyphae and spore morphological characteristics were observed under an optical microscope. Combined with relevant literature such as "Fungal Taxonomy", the isolated fungi were preliminarily identified.

2.3.2 Molecular biological identification of strains

In this experiment, the genomic DNA of the isolated strain was extracted based on the instructions of the Ezup column fungal genomic DNA extraction kit, and PCR amplification was performed using this DNA as a template. The primers used were ITSI (5′-TCCGTAGGTGAACCTGCGG-3′0) and ITS4 (5′-TCCTCCGCTTATTG ATATGC-3′).

Subsequent sequencing work was completed by Sangon Biotechnology Co., Ltd. The obtained sequences were submitted to the NCBI database for BLAST homology comparison analysis to find the most similar sequence information, construct a phylogenetic tree, and combine morphological identification with ITS sequence identification results to determine the taxonomic status of the strain (order, family or genus level).

3. Research Results

3.1 Isolation results of strains

A total of 25 strains were isolated from Glechoma longituba (including 16 strains isolated from leaves and 9 strains isolated from roots), as shown in FIG1 .

3.2 Results of initial screening of salt-alkali tolerant strains

A strain with obvious salt-alkali resistance was selected and named FJ0037. This strain can grow normally on PDA with a mass concentration of 4% and a Na ₂ CO ₃ :NaHCO ₃ = 1:3. Figure 2 shows the curve of the growth diameter of FJ0037 strain under 4% salt-alkali stress.

3.3 Morphological identification results of endophytic fungi FJ0037

The FJ0037 strain with salt-alkali tolerance was selected as the research object of this experiment to identify the morphology of the FJ0037 strain. In this study, the NCBI software was used to perform BLAST sequence alignment on the ITS full sequence of the strain FJ0037, and similar sequences were further searched in the GenBank database. Then, the MEGA-X-10.1.7 software was combined with the Neighbor-Joining method to construct the phylogenetic tree of the FJ0037 strain.

The strain FJ0037 was preliminarily identified as Pleosporaceae sp. The characteristics of this strain are as follows: the colonies are gray-black, it grows fast on PDA plates, the hyphae are gray-white with diaphragms, and the conidia are nearly spherical with smooth walls.

2. Salt-alkali tolerant strain FJ0037 improves the salt-alkali tolerance of corn plants

1. Test materials

1.1 Test strain: FJ0037 strain isolated from saline-alkali land in western Jilin

1.2 Plant species: corn

1.3 Culture medium: Potato dextrose agar (PDA)

2. Cultivation of salt-alkali tolerant strains

Under sterile conditions, the FJ0037 mycelium block was placed on a new PDA culture medium plate for activation. After sealing with a sealing film, it was placed in a constant temperature biochemical incubator and cultured at 25°C in the dark.

3. Corn seed germination experiment

Select corn seeds that are uniform in size, plump and free of insect pests. Rinse them with sterile water 2 to 3 times, disinfect the surface of the corn seeds with 70% alcohol for 5 minutes, then soak and disinfect them with 0.1% potassium permanganate for 30 minutes, and finally rinse them repeatedly with sterile water until no color is washed out.

Four different concentrations of Na ₂ CO ₃ and NaHCO ₃ mixed solutions of 1.6%, 2.4%, 3.2% and 4.0% were prepared with sterile water for later use.

Set up the following three sets of experiments:

Blank control group: Take 25 corn seeds of the same size and shape and place them evenly on a 90 mm culture dish with two layers of filter paper on the bottom. Add 3 mL of sterile water to the culture medium every day for the growth of corn seeds. Perform three groups of experiments in parallel. Observe the growth every day and record the physiological indicators of corn seedlings. Pay attention to whether the corn seeds are contaminated and deal with them in time.

Salt-alkali stress group: Take 25 corn seeds of the same size and shape and place them evenly on a 90 mm culture dish with two layers of filter paper on the bottom. Add 3 mL of Na ₂ CO ₃ and NaHCO ₃ mixed solution of different concentrations to the culture medium every day to stress the growth of corn seeds. Three groups of experiments were carried out in parallel. The growth was observed regularly every day, and various physiological indicators of corn seedlings were recorded.

Salt-alkali stress + FJ0037 strain: Take 25 corn seeds of the same size and shape and place them evenly on a 90 mm culture dish full of FJ0037 strains. Add 3 mL of Na ₂ CO ₃ and NaHCO ₃ mixed solution of different concentrations to the culture medium at regular intervals every day, and conduct three groups of experiments in parallel. Observe the growth at regular intervals every day, and measure and record various physiological indicators of corn seedlings with a ruler.

Each dish was cultured at a constant temperature of about 25°C and away from light. When the length of the bud was greater than 2 mm, the seed was considered to have germinated. The filter paper was changed every 24 hours. After the first seed germinated, the seed was observed for 7 days. The germination status of the seed was observed and recorded every day. When the seed did not germinate for three consecutive days, the observation was terminated.

Seed germination rate GP (%) = (GN / SN) × 100;

Germination potential GE (%) = (number of germinated seeds on the second day / SN) × 100;

Germination index GI = Σ (Gt / Dt);

Relative salt damage rate Rd (%) = [(control germination rate - saline-alkali solution treatment germination rate) / control germination rate] × 100;

GN-total number of germinating seeds;

SN-total number of seeds tested;

Gt-number of seeds germinated on the day;

Dt-corresponding germination days;

CK-control group.

4. Research Results

4.1 Results of the number of corn seeds germinated per day

Figures 3 and 4 show that with the continuous increase of experimental days, the daily germination curve of corn seeds grown under saline-alkali stress conditions showed a single-peak change trend. On the second day of the control group, the number of corn seeds germinated was 12, which was the maximum value of single-day germination in 7 days. Compared with the data of the control group, the maximum single-day germination days under saline-alkali stress conditions were delayed by 1 to 2 days. Therefore, in this research experiment, the second day was selected as the standard for evaluating seed germination potential.

Figure 5 shows the cumulative germination number of corn seeds in 7 days. It can be seen from Figure 5 that the cumulative germination curves of corn seeds under different conditions have similar trends, and the cumulative germination number of corn seeds increases most significantly in the first three days. As the number of experimental days increases, the cumulative germination curve of seeds presents a gentle state. At the same time, as the salinity concentration continues to rise, the cumulative germination amount of seeds decreases significantly; the cumulative germination number of corn seedlings grown on the FJ0037 strain is significantly higher than the cumulative germination number of corn seedlings under the same salt concentration stress, and the CK control group has the largest cumulative number of germinated grains. The experiment shows that under the same concentration of salinity stress, the FJ0037 strain can significantly promote the germination of corn seeds.

4.3 Results of corn seedling indicators

Table 1 Corn index information

deal with Seed germination rate% Germination Index Germination potential% Relative salt damage rate% Water (control) 98.67 16.83 86.67 0.0 1.6% salt 76 10.24 54.67 22.98 2.4% salt 57.33 6.31 33.33 41.90 3.2% Salt 30.67 2.83 9.33 68.92 4% salt 21.33 2.25 5.33 78.38 1.6% salt + bacteria 89.33 11.90 70.67 9.47 2.4% salt + bacteria 89.33 13.07 72.33 9.47 3.2% salt + bacteria 86.67 13.25 76 12.16 4% salt + bacteria 53.33 8.22 44 45.95

As shown in Table 1, after 7 days of treatment, the germination rate of the control group of corn seeds was 98.67%. With the increase of salt-alkali stress concentration, the germination rate of the treated group seeds gradually decreased compared with the control group, and the lowest seed germination rate under 4% salt concentration treatment was 21.33%; the germination rate of corn seeds after adding FJ0037 strain was higher than that under salt-alkali stress, increasing by 13.33%, 32.00%, 56.00%, and 32.00%, respectively. At the same time, the germination index and germination potential of corn seeds showed a downward trend with the gradual increase of salt-alkali stress concentration, and the relative salt damage rate gradually increased. In the control group, the germination potential and germination index of corn seeds reached 86.67% and 16.83%, respectively, which was significantly higher than the performance of seeds under salt-alkali stress. When FJ0037 was added, the relative germination index increased by 1.66%, 6.76%, 10.42%, and 5.97%, respectively, the germination potential increased by 16.00%, 39.00%, 66.67%, and 38.67%, respectively, and the relative salt damage rate decreased by 13.51%, 32.43%, 56.76%, and 32.43%, respectively. This indicates that FJ0037 can improve the salt-alkali tolerance of plants.

4.4 Effects of the tested salt-alkali tolerant strains on the growth of corn seedlings

Table 2 Information on morphological indexes of corn seeds

Treatment Control Root length (cm) Seedling height (cm) Root weight (g) Seedling weight (g) Water (control) 5.09 1.49 0.03 0.038 1.6% salt 1.02 0.6 0.007 0.010 2.4% salt 0.63 0.36 0.003 0.005 3.2% salt 0.24 0.05 0.001 0.0005 4% salt 0.15 0.03 0.0006 0.0002 1.6% salt + bacteria 1.08 1.97 0.007 0.060 2.4% salt + bacteria 0.84 1.42 0.009 0.050 3.2% salt + bacteria 0.61 0.76 0.005 0.015 4% salt + bacteria 0.45 0.54 0.004 0.014

Compared with the control group, the growth and development of corn seeds under saline-alkali stress were significantly affected, and the quality of root length and seedling height were reduced to varying degrees. After inoculation with the salt-alkali tolerant endophytic fungus FJ0037, the plant biomass changed significantly. Specifically, the taproot length increased by 0.06, 0.21, 0.37, and 0.30 cm respectively compared with the saline-alkali stress group; the seedling height increased by 1.37, 1.06, 0.40, and 0.31 cm respectively, which were significantly higher than the saline-alkali stress group at the same concentration. The FJ0037 strain has the ability to promote growth and can improve various ecological indicators of corn.

Conclusion

The native plant Goosegrass was collected in the saline-alkali land in western Jilin, and the endophytic fungi were isolated and purified from it. The salt-alkali tolerant strains were screened by simulating salt-alkali tolerance, and it was found that the endophytic fungus FJ0037 from the healthy leaves of Goosegrass could grow rapidly on the medium with 4% salt concentration, that is, FJ0037 was selected as the research strain of this experiment. The strain was identified by morphological and molecular biological methods, combined with the determination of ITS sequences, BLAST comparative analysis was performed, phylogenetic trees were constructed, and the species classification of the target strain was determined. The strain FJ0037 was identified as Pleosporaceae sp.

By measuring the effects of the salt-alkali tolerant strain FJ0037 on corn seed germination, seedling growth, root development and other indicators, the results showed that the tested salt-alkali tolerant strain can effectively alleviate the inhibitory effect of salt-alkali stress on corn seed germination, and has the effect of promoting seed germination.

Under the same concentration of saline-alkali stress, after inoculation with strain FJ0037, the biomass of corn seeds increased significantly, and the root length and seedling height increased to varying degrees, proving that strain FJ0037 improves the plant's resistance to saline-alkali environments.

The salt-alkali tolerant FJ0037 strain can be actually applied to saline-alkali land management, which has important agricultural, environmental and ecological significance. The salt-alkali tolerant FJ0037 strain can improve the salt-alkali tolerance of plants, promote the improvement of saline-alkali land, and improve agricultural output and quality. It is a breakthrough in the salt-alkali resistance technology of crops and provides important value for the sustainable development of green agriculture.

The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not deviate from the spirit and scope of the present invention, and should be within the scope of protection of the claims attached to the present invention.

Claims (3)

1. A strain of Pleosporaceae sp. FJ0037 that can improve the salt-alkali tolerance of corn, characterized by: the preservation number is CGMCC No.41320, and it was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on May 27, 2024, and the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. 2. Use of the Pleosporaceae sp. FJ0037 described in claim 1 in promoting the growth of corn in a saline-alkali environment. 3. The use of Pleosporaceae sp. FJ0037 according to claim 2 in promoting the growth of corn in a saline-alkali environment, characterized in that it promotes corn seed germination.