Fungicide composition capable of promoting soybean growth and increasing yield in saline soil and application thereof

Abstract

The invention discloses a microbial agent composition capable of promoting growth and yield increase of soybeans in saline soil and application of the microbial agent composition. A microbial agent is an enterobacter strain THD10 or a complex microbial agent formed by the enterobacter strain THD10. The microbial agent provided by the invention can significantly improve the plant height, fresh weight, dry weight, nodulation number and nitrogenase activity of soybeans. In saline soil, compared with treatment without inoculation of the microbial inoculum, inoculation treatment can significantly improve the soybean biomass, and the nitrogen fixation efficiency of a single soybean plant is improved; the SOD, CAT and POD contents of soybean leaves are increased, and the malondialdehyde content of the leaves is reduced; the method can improve the soybean yield and promote the improvement of soybean quality indexes, has very important significance for enlarging the soybean cultivation area and improving the per unit yield in China, and has important guidance and reference significance for cultivation of other crops in saline soil.

Description

Technical Field

[0001] The invention belongs to the field of agricultural microorganisms and relates to a bacterial agent combination capable of promoting soybean growth and yield in saline soil and application thereof in soybean production. Background Art

[0002] Soil salinity is one of the abiotic factors that affect crop growth and yield. Higher soil salinity can cause plant ion and osmotic stress, which in turn leads to oxidative stress, nutrient imbalance, and tissue senescence. Soybean is a typical legume that can form a symbiotic relationship with rhizobia, thereby achieving efficient symbiotic nitrogen fixation. Previous investigations by our research team found that soybean roots grown in saline soil had significantly fewer nodules than those grown under conventional conditions. This may be due to the inhibitory effect of soil salinity on rhizobia growth. Plant salt tolerance is related to many factors, which are not only related to the genetic characteristics of the plant itself, but also to its rhizosphere microbiome. Under salt stress, salt-tolerant microorganisms in the plant rhizosphere regulate plant growth metabolism and improve the host's salt tolerance. The main mechanisms include the production of some plant hormones (auxin, gibberellins, cytokinins), the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, the production of exopolysaccharides and osmotic regulatory substances (proline, glycine betaine, etc.), and the regulation of plant defense systems and antioxidant enzyme activity. Research on salt-tolerant bacterial agents is of great significance for the growth and yield improvement of soybeans in saline soils. It will also be beneficial to expand the soybean planting area and provide technical reference for saline soil agriculture. Summary of the Invention

[0003] The purpose of the present invention is to provide a bacterial agent combination that promotes soybean nitrogen fixation efficiency and yield improvement and its application in saline soil, targeting the special environment of saline soil, to solve the problem of soybean growth inhibition and low yield in saline soil, and to achieve sustainable development of the soybean industry.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] The present invention provides an Enterobacter strain THD10, which is classified and named Enterobacter ludwigii. The strain was deposited in the General Microbiology Center of the China Culture Collection Administration on December 12, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the culture collection number is CGMCC NO.29193.

[0006] The present invention also provides a bacterial agent containing the strain THD10. The bacterial agent of the present invention can be obtained by conventional methods in the art, such as by culturing the strain THD10, or by preparing the strain THD10 according to conventional methods, such as by resuspending it in water. The total bacterial content of the bacterial agent can be a conventional content, such as 10 7~10 9 CFU / mL.

[0007] The present invention also provides the use of the strain THD10 or a microbial agent containing it to promote soybean growth and / or soybean yield in saline soil. In some embodiments, the use in promoting soybean growth in saline soil specifically refers to increasing soybean plant height, fresh weight, and / or dry weight. In some embodiments, the use in promoting soybean yield in saline soil specifically refers to increasing soybean grain number and / or total yield.

[0008] The present invention also provides a composite bacterial agent, which includes the Enterobacter strain THD10 and the Sinorhizobium freundii CCBAU45436, and the bacterial amount of the Enterobacter strain THD10 and the Sinorhizobium freundii CCBAU45436 is 1: (0.8-1.2); in some embodiments, the mixed bacterial amount ratio is 1:1.

[0009] Sinorhizobium fredii CCBAU45436 involved in the present invention is a broad-host rhizobium commonly used both at home and abroad, and has been reported in many documents (e.g., Global transcriptional repression of diguanylate cyclases by MucR1 is essential for Sinorhizobium-soybean symbiosis).

[0010] In some embodiments, the total bacterial count of the composite bacterial agent described in this application is 10 7 ~10 9 CFU / mL.

[0011] The present invention also provides application of the composite bacterial agent of the present invention in promoting soybean growth in saline soil.

[0012] The inventors found that the composite bacterial agent formed by the two can significantly increase the plant height, fresh weight and dry weight of soybeans in saline soil, and increase the number of nodules and nitrogenase activity.

[0013] The present invention also provides use of the composite bacterial agent of the present invention in increasing the number of soybean nodules and nitrogen fixation activity in saline soil.

[0014] The present invention also provides the use of the composite bacterial agent of the present invention in improving the net photosynthetic rate and efficiency of soybeans in saline soil and improving the chlorophyll content.

[0015] The present invention also provides the use of the composite bacterial agent described in the present invention in improving the yield and / or quality of soybeans in saline soil, preferably, in increasing the number of soybean pods, the number of grains and / or the total yield; or preferably, in increasing the protein content and / or linoleic acid content in soybeans.

[0016] The inventors found that the composite microbial agent formed by the two can significantly improve the yield and quality of soybeans in saline soil.

[0017] The strain THD10 or composite bacterial agent described in the present invention can be applied to the roots according to conventional methods. In one embodiment, the present invention also provides a method for the above application, comprising the following steps:

[0018] (1) Soybean seeds, soil, microbial agent, and water were thoroughly mixed in a ratio of 15-25 g soybean seeds: 15-25 g soil: 1 mL microbial agent: 3-5 mL water, so that the soil containing the microbial solution was evenly wrapped around the soybean seeds to form soil-wrapped seeds;

[0019] (2) Tilling the soil appropriately and evenly sowing the soybean seeds covered with the soil formed in step (1) so that the seeds are not exposed on the soil surface, and watering the soil appropriately.

[0020] In some embodiments, soybean seeds are sown in mid-June of each year and harvested in October of the same year.

[0021] The appropriate tillage and watering described in the present invention can be performed according to conventional operations in the art.

[0022] The strain Enterobacter sp. THD10 of the present invention can be cultured according to conventional methods in the art, for example, by inoculating it into LB medium and shaking it at 150 rpm and 30°C. The preparation of the inoculum is to shake the culture for 20 hours, collect the bacterial solution, centrifuge it at 6000 rpm for 10 minutes, remove the supernatant, and resuspend it in sterile water. Repeat this process five times to obtain a concentration of approximately 1.0×10 8 CFU / mL.

[0023] The strain CCBAU45436 described herein can be cultured according to conventional methods in the art. For example, CCBAU45436 is inoculated in TY medium (formula: mannitol, 10 g; K2HPO4, 0.5 g; NaCl, 0.1 g; yeast extract, 0.5 g; CaCO3, 3.0 g; MgSO4·7H2O, 0.2 g; 1000 mL water; pH 7.2) and cultured with shaking at 180 rpm and 28°C. The inoculum is prepared by shaking the culture for 24 hours, collecting the bacterial solution, centrifuging it at 6000 rpm for 10 minutes, removing the supernatant, and resuspending it in sterile water. This is repeated five times until the bacterial concentration is adjusted to 1.0 × 10 8CFU / mL.

[0024] Beneficial effects of the present invention:

[0025] (1) The Enterobacter sp. THD10 described in the present invention can further improve soybean growth and symbiotic nitrogen fixation efficiency based on soybean inoculation with Sinorhizobium fredii CCBAU45436. Compared with inoculation with CCBAU45436 alone, co-inoculation of THD10 and CCBAU45436 significantly improved soybean growth and symbiotic nitrogen fixation, demonstrating a significant synergistic effect.

[0026] (2) The bio-infectant combination provided by the present invention can significantly increase soybean plant height, biomass, symbiotic nitrogen fixation capacity, and photosynthetic characteristics during the flowering period, enhance SOD, CAT, and POD enzyme activities, increase leaf proline content, and reduce malondialdehyde content. This system can promote multiple soybean growth indicators in saline soil, thereby alleviating salt stress on soybeans.

[0027] (3) The bio-microbial agent provided by the present invention can increase the number of soybean pods and grains during the harvest period, significantly increase the yield, and achieve increased soybean income in saline soil.

[0028] (4) The biological agent provided by the present invention can increase the protein or oil content of soybeans during the harvest period, thereby improving the quality of soybeans. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the colony morphology of strain THD10;

[0030] Figure 2 The effects of different treatments on soybean plant height, fresh weight and dry weight at flowering stage;

[0031] Figure 3 The effects of different treatments on soybean nodule number and nitrogenase activity at flowering stage;

[0032] Figure 4 The effects of different treatments on the net photosynthetic rate and chlorophyll content of soybean at flowering stage;

[0033] Figure 5 The effects of different treatments on soybean reductase at flowering stage;

[0034] Figure 6 The effect of different treatments on the malondialdehyde content in soybean at the flowering stage;

[0035] Figure 7 The results show the effects of different treatments on soybean yield indicators at harvest time. DETAILED DESCRIPTION

[0036] The present invention is described below with reference to the following examples, but the present invention is not limited to the following examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

[0037] The materials in the present invention include: LB culture medium, sterile water, sulfuric acid, ferrous sulfate, nitric acid, acetylene, ruler, etc., which can be purchased through public channels. The equipment and instruments used in the process are common equipment in the field.

[0038] All materials, reagents and instruments used in the present invention are well known in the art, but do not limit the implementation of the present invention. Other reagents and equipment well known in the art can be applied to the implementation of the following embodiments of the present invention.

[0039] The following examples further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0040] The strain THD10 of the present invention was isolated from the soybean nodules cultivated in a soybean plantation in Huai'an City, Jiangsu Province in 2020. The colony morphology of the strain THD10 in LB medium is as follows: Figure 1 As shown. Based on the alignment of the 16S rRNA gene sequence of strain TGB1 (sequence shown in SEQ ID NO. 1) and combined with the strain's colony morphology, THD10 was determined to belong to Enterobacter ludwigii. This strain has been deposited with the General Microbiology Center of the China Culture Collection Administration, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, under the culture collection number CGMCC No. 29193.

[0041] The present invention also relates to Sinorhizobium fredii CCBAU45436, which is a widely-hosted rhizobium commonly used at home and abroad and has been reported in many documents (e.g., Global transcriptional repression of diguanylate cyclases by MucR1 is essential for Sinorhizobium-soybean symbiosis).

[0042] Unless otherwise specified, the soybean varieties used in the following examples are NJAU-C101 and NJAU-C105, which are available to the public during the patent period.

[0043] Example 1: Test microbial strains and preparation methods thereof

[0044] Enterobacter sp. THD10 was inoculated into LB medium and cultured with shaking at 150 rpm and 30°C for 20 h. The culture was collected and centrifuged at 6000 rpm for 10 min. The supernatant was removed and resuspended in sterile water. This step was repeated five times. The final concentration of the culture was adjusted to 1.0 × 10 8 CFU / mL, reserve for future use.

[0045] Rhizobium CCBAU45436 was inoculated into TY medium (formula: mannitol, 10 g; K2HPO4, 0.5 g; NaCl, 0.1 g; yeast extract, 0.5 g; CaCO3, 3.0 g; MgSO4·7H2O, 0.2 g; 1000 mL water; pH 7.2) and cultured with shaking at 180 rpm and 28°C. The inoculum was prepared by shaking the culture for 24 h, collecting the culture medium, centrifuging it at 6000 rpm for 10 min, removing the supernatant, and resuspending it in sterile water. This was repeated five times until the concentration of the culture medium was adjusted to 1.0 × 10 8 CFU / mL, reserve for future use.

[0046] Example 2: Effects of different strains inoculated in saline soil on soybean growth

[0047] A soybean cultivation experiment was conducted on a farm in Tinghu District, Yancheng City, where the soil salinity was approximately 0.1%-0.15%. Soybean planting was conducted in mid-June 2023. Two soybean varieties (NJAU-C101 and NJAU-C105) were selected, with each variety divided into four treatments: (1) no inoculation (CK); (2) inoculation with the growth-promoting bacterium THD10 (THD10); (3) inoculation with the rhizobium CCBAU45436 alone; and (4) double inoculation (THD10 + CCBAU45436). Each treatment had 15 replicates. The preparation of the bioinoculation agent was similar to that in Example 1. For the CK treatment, seeds were directly sown in holes, with 5 seeds per hole. For the single- and double-inoculation treatments, seed dressing was performed as follows: 200g of soybean seeds, 200g of soil, 10mL of Rhizobium THD10 or CCBAU45436 solution (for the double-inoculation treatment, 5mL of Rhizobium CCBAU45436 solution and 5mL of the growth-promoting strain THD10 solution) and 40mL of sterile water were thoroughly mixed. The soil containing the solution was evenly coated around the soybean seeds, forming soil-encased seeds. When the soybeans reached the flowering stage, parameters such as plant height, biomass, photosynthetic characteristics, nitrogen fixation characteristics, and stress resistance were measured.

[0048] like Figure 2 As shown, except for THD10 which did not significantly increase the plant height of soybean C105, all inoculation treatments significantly increased the plant height of soybean ( Figure 2A), fresh weight ( Figure 2 B) and dry weight ( Figure 2 C). Overall, compared to the uninoculated CK treatment, the dual-inoculation treatment exhibited the greatest growth-promoting effect, with increases in plant height, fresh weight, and dry weight by 44.0% and 22.8%, 128.9% and 64.3%, and 188.6% and 102.5%, respectively. These results demonstrate that THD10 of the present invention promotes soybean growth during the flowering stage, with the THD10 + CCBAU45436 combination being even more effective.

[0049] Example 3: Effects of different inoculation treatments on soybean nodule number and nitrogen fixation activity

[0050] Based on the treatment settings in Example 2, the number of soybean root nodules and the nitrogenase activity of the nodules were determined. The results showed that no nodules were found in the treatments without rhizobia inoculation (CK and THD10), regardless of the soybean variety, indicating that the abundance of indigenous rhizobia in the test saline soil was low. After inoculation with CCBAU45436, the number of soybean root nodules increased significantly. For soybean variety C105, the number of nodules in the double inoculation treatment was significantly greater than that in the treatment with only CCBAU45436 inoculation (p < 0.05) ( Figure 3 A). The nitrogenase activity of soybean nodules was determined using the acetylene reduction method. The results showed that rhizobium CCBAU45436 significantly improved the nitrogen fixation ability of rhizobia (p<0.05). These results indicate that inoculation with rhizobium CCBAU45436 can significantly increase the number of nodules and nitrogen fixation efficiency of soybeans grown in saline soil. Adding the growth-promoting bacteria THD10 can further improve the nitrogen fixation efficiency of soybeans and maintain the nitrogen supply of soybeans. The two have a synergistic effect ( Figure 3 B).

[0051] Example 4: Effects of different inoculation treatments on soybean net photosynthetic rate and chlorophyll content

[0052] Based on the treatment settings in Example 2, the net photosynthetic rate and chlorophyll content of soybean leaves were measured. Figure 4 It can be seen that inoculation with growth-promoting bacteria THD10 could not increase the net photosynthetic rate of soybean (p>0.05), but after compounding with CCBAU45436, it could significantly enhance the net photosynthetic rate of soybean leaves ( Figure 4A; p < 0.05). A portable chlorophyll meter was used to analyze the chlorophyll content of soybean leaves. The results showed that inoculation with rhizobium CCBAU45436 did not significantly increase the chlorophyll content of the leaves (p > 0.05), while inoculation with the growth-promoting bacteria THD10 promoted the chlorophyll content of soybean C101 (p < 0.05). The double inoculation treatment significantly increased the chlorophyll content of soybean leaves regardless of the soybean variety, indicating that the combined bacterial agent exhibited an unexpected synergistic effect (p < 0.05).

[0053] Example 5: Effects of different inoculation treatments on soybean antioxidant enzyme substances

[0054] When plants are subjected to salt stress, they tend to accumulate more reactive oxygen species (ROS) in their bodies, causing oxidative stress. Based on the treatment settings in Example 2, three enzymes related to the elimination of oxidative stress in soybean plants were analyzed. Figure 5 As shown, for soybean variety C101, double inoculation can significantly increase CAT activity. For soybean variety C105, all inoculation treatments can increase SOD activity; rhizobium inoculation treatment and double inoculation treatment can significantly increase CAT activity; double inoculation treatment can significantly increase POD activity. In addition, the malondialdehyde content in leaves ( Figure 6 The results showed that both inoculation with rhizobium CCBAU45436 and double inoculation significantly reduced malondialdehyde (MDA) levels in leaves. The double inoculation treatment had the lowest MDA levels, significantly different from the other treatments (p<0.05). These results suggest that, compared to single-agent inoculation, combined bacterial inoculants can alleviate oxidative stress, improve antioxidant capacity, and mitigate salt stress in soybeans to a certain extent.

[0055] Example 6: Effects of different inoculation treatments on soybean yield-related indicators

[0056] Based on the treatment settings in Example 2, the number of soybean pods, number of grains, 100-grain weight and yield were measured at the soybean maturity stage. Figure 7 As shown, for variety C101, all inoculation treatments significantly promoted soybean pod number, grain number and yield ( Figure 7 A, B and D; p < 0.05); For variety C105, the inoculation treatment significantly increased the number of soybean grains, 100-grain weight and total yield ( Figure 7 B, C, and D; p < 0.05). The double-inoculation treatment also significantly increased the number of pods in soybean C105 (p < 0.05). Total yield remained high in both double-inoculation treatments. This suggests that the tested microbial agents or combinations can increase soybean yield in saline soils.

[0057] At the same time, near-infrared spectroscopy was used to measure the quality indicators of C101 soybeans (Table 1). The results showed that the dual inoculation treatment increased the protein content and linoleic acid content of soybean variety C101 by 1.2% and 4.3%, respectively (p < 0.05). These results indicate that the composite microbial inoculant can improve soybean quality to a certain extent and is beneficial for improving the overall quality of soybeans.

[0058] Table 1 Effects of different treatments on soybean quality indicators

[0059]

[0060] The above examples fully demonstrate that the combined inoculum of the inoculated strains Enterobacter sp.THD10 and Sinorhizobium frediiCCBAU45436 can promote soybean plant height, fresh weight, and dry weight in saline soil, improve the efficiency of symbiotic nitrogen fixation, alleviate salt stress on soybeans, increase the content of stress-resistant substances, and improve soybean yield and quality indicators.

[0061] The above embodiments are merely examples for the purpose of illustrating the present invention and are not intended to limit the embodiments. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions may be made. It is not necessary and impossible to enumerate all embodiments here, and obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An Enterobacter sp. THD10, classified as Enterobacter sp. THD10, with a deposit number of CGMCCNO: 29193.

2. A bacterial agent containing the Enterobacter strain THD10 according to claim 1; preferably, the bacterial amount of the bacterial agent is 10 7 ~10 9 CFU / mL.

3. Use of the strain THD10 according to claim 1 or the microbial agent according to claim 2 in promoting soybean growth and / or soybean yield in saline soil; preferably, in increasing soybean plant height, fresh weight and / or dry weight; more preferably, in increasing soybean fresh weight and / or dry weight; or preferably, in increasing soybean grain number and / or total yield.

4. A composite bacterial agent, characterized in that: The composite bacterial agent comprises the Enterobacter strain THD10 and Sinorhizobium freundii CCBAU45436 according to claim 1. Preferably, the bacterial amount of the Enterobacter strain THD10 and Sinorhizobium freundii CCBAU45436 is 1:(0.8-1.2); more preferably, the mixed bacterial amount ratio is 1:

1.

5. The composite bacterial agent according to claim 4, characterized in that The total bacterial count of the composite bacterial agent is 10 7 ~10 9 CFU / mL.

6. Use of the composite bacterial agent according to claim 4 or 5 in promoting soybean growth in saline soil; preferably, in increasing soybean plant height, fresh weight and / or dry weight; or in increasing soybean nodule number and / or nitrogenase activity.

7. Use of the composite bacterial agent according to claim 4 or 5 in improving the net photosynthetic rate and efficiency of soybean and increasing the chlorophyll content in saline soil, or in improving the antioxidant properties of soybean.

8. Use of the composite bacterial agent according to claim 4 or 5 for increasing soybean yield and / or soybean quality in saline soil; preferably, for increasing the number of soybean pods, the number of grains and / or the total yield; or preferably, for increasing the protein content and / or linoleic acid content in soybeans.

9. The use according to any one of claims 3, 6 to 8, characterized in that: Specifically, the strain THD10 according to claim 1, the bacterial agent according to claim 2, or the composite bacterial agent according to claim 4 or 5 is applied to the roots of plants.

10. The use according to any one of claims 3, 6 to 8, characterized in that: The steps include: (1) Soybean seeds, soil, a microbial agent, and water are thoroughly mixed in a ratio of 15-25 g soybean seeds: 15-25 g soil: 1 mL microbial agent: 3-5 ml water, so that the soil containing the microbial solution is evenly wrapped around the soybean seeds to form soil-wrapped seeds; the microbial agent is the strain THD10 described in claim 1, the microbial agent described in claim 2, or the composite microbial agent described in claim 4 or 5; (2) tilling the soil appropriately and sowing the soybean seeds covered with the soil formed in step (1) evenly using the hole-drilling method so that the seeds are not exposed on the soil surface, and watering appropriately; preferably, the soybean seeds are sown in mid-June each year.

Images