Stress-resistant growth-promoting microbial agent as well as preparation method and application thereof

Abstract

The invention discloses a stress-resistant growth-promoting microbial agent as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. The stress-resistant and growth-promoting microbial agent comprises bacillus siamensis, Burkholderia bidirectionally and sphingomonas yunnanensis which are mixed in equal proportion and cooperate with one another, so that the stress-resistant and growth-promoting microbial agent is remarkable in stress-resistant and growth-promoting effect, and particularly, the stress resistance index is improved in the growth process of wheat plants; the activity of antioxidant enzyme SOD in the plants is improved, the content of osmotic regulation substance proline is increased, and the content of MDA is reduced. The microbial agent disclosed by the invention is matched with conventional fertilization, so that the growth of wheat seedlings under salt stress is remarkably promoted, and the wheat yield is increased.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a stress-resistant and growth-promoting microbial inoculant, a preparation method thereof, and an application thereof. Background Art

[0002] Wheat ( Triticum aestivum L. ) is an annual or biennial herbaceous plant of the genus Triticum in the family Poaceae. The caryopsis of wheat can be eaten and is one of the most important food crops at present.

[0003] Soil salinization seriously affects plant growth and crop yields. Many agricultural irrigation lands are troubled by salt stress. And with the unreasonable use of cultivated land by humans and environmental pollution, the problem of land salinization is becoming increasingly serious. The physical and chemical properties of saline-alkali soil are relatively poor and it is easy to harden. In the soil, not only the activity of enzymes is inhibited, but also the activity of microorganisms is affected. Eventually, plant growth is inhibited and soil fertility decreases. With the intensification of global climate change, agricultural production faces increasingly serious abiotic stresses such as drought and salinization. Traditional chemical fertilizers and plant growth regulators have limited effects under adversity conditions and may cause environmental pollution. As an environmentally friendly biological fertilizer, microbial inoculants can promote plant growth and improve plant stress resistance through various mechanisms.

[0004] However, the microbial inoculants currently on the market generally have problems such as insufficient stress resistance, single function, and unstable field effects, and there is no microbial inoculant specifically for wheat to resist salt stress. Therefore, there is an urgent need to develop a microbial inoculant that can improve the stress resistance and growth promotion of wheat. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a stress-resistant and growth-promoting microbial inoculant for wheat growing in saline-alkali land, and through the synergistic effect of a specific functional strain combination and other synergistic components, the growth performance of wheat under the adversity conditions of saline-alkali land is significantly improved.

[0006] To achieve the above technical objectives, the technical solution adopted by the present invention is as follows: A stress-resistant and growth-promoting microbial inoculant, wherein the microbial inoculant comprises the following raw materials in parts by weight: 15 - 25 parts of functional microbial inoculant, 3 - 5 parts of trehalose-proline composite nanoparticles, 35 - 45 parts of composite carrier, and 2 - 3 parts of trace element-containing substance.

[0007] Further, the functional microbial inoculum includes *Bacillus siamensis*, *Burkholderia ambifaria*, and *Sphingomonas yunnanensis*. The *Bacillus siamensis* is purchased from the China Center for Type Culture Collection, with the preservation number CCTCC AB2022048 and the original preservation date being January 7, 2022. The *Burkholderia ambifaria* is purchased from the China General Microbiological Culture Collection Center, with the preservation number CGMCC No. 1.10511 and the original preservation date being March 29, 2010. The *Sphingomonas yunnanensis* is purchased from the China General Microbiological Culture Collection Center, with the preservation number CGMCC No. 1.15275 and the original preservation date being June 20, 2015.

[0008] Further, the preparation method of the functional microbial inoculum is as follows: (1) Thaw the *Bacillus siamensis* and *Burkholderia ambifaria* and activate them in LB solid medium. Separate single colonies are picked and inoculated into LB liquid medium, and cultured at 27 °C and 180 r / min until OD 600 = 0.6 to obtain seed solutions. The seed solutions are inoculated into 250 mL LB liquid medium at a ratio of 1% respectively and cultured at 27 °C and 180 r / min. The viable cell count of the bacterial liquid is detected. After reaching 1×10 8 cfu / mL, the culture is terminated, and the *Bacillus siamensis* bacterial liquid and *Burkholderia ambifaria* bacterial liquid are obtained respectively. (2) Place the *Sphingomonas yunnanensis* in nutrient broth agar medium for activation. Pick a single colony and inoculate it into nutrient broth liquid medium, and culture at 30 °C and 200 r / min until OD 600 = 0.6 to obtain a seed solution. The seed solution is inoculated into 250 mL LB liquid medium at a ratio of 1% and cultured at 30 °C and 200 r / min. The viable cell count is detected. After reaching 1×10 8 cfu / mL, the culture is terminated, and the *Sphingomonas yunnanensis* bacterial liquid is obtained. (3) Mix the *Bacillus siamensis* bacterial liquid and *Burkholderia ambifaria* bacterial liquid in step (1) and the *Sphingomonas yunnanensis* bacterial liquid in step (2) at a volume ratio of 1:1:1 to obtain the functional microbial inoculum.

[0009] Further, the preparation method of the trehalose-proline composite nanoparticles is as follows: Dissolve trehalose and L-proline in deionized water at a mass ratio of 2:1 to prepare a 10% solution. Then add 0.1% chitosan as a stabilizer. Treat it 5 times under the condition of 100 MPa by high-pressure homogenization to obtain composite nanoparticles with a particle size of 50 - 100 nm. Spray drying is used to obtain powdery trehalose-proline composite nanoparticles.

[0010] Further, the composite carrier includes humic acid, bentonite, and biochar, and the mass ratio of the three is 3:2:1.

[0011] Further, the trace element-containing substance includes chelated zinc, chelated iron, and chelated manganese, and the mass ratio of the three is 4:3:2.

[0012] A preparation method of an anti-stress and growth-promoting microbial inoculant includes the following steps: Expand and culture three strains respectively to obtain the bacterial liquids of the three strains. After mixing them in equal volume to obtain a functional microbial inoculant, first mix the functional microbial inoculant with the composite carrier, then add trehalose-proline composite nanoparticles and the trace element-containing substance, mix well, adjust the water content to 25-30%, ferment at 30 °C for 48 h, dry at low temperature until the water content is less than 10%, and pulverize through 80 meshes to obtain the anti-stress and growth-promoting microbial inoculant.

[0013] The present invention also provides the application of the described anti-stress and growth-promoting microbial inoculant in promoting the growth of wheat and effectively improving the salt and alkali tolerance of wheat.

[0014] All raw materials used in the present invention are commercially available.

[0015] Beneficial effects (1) The microbial inoculant of the present invention is prepared by mixing three functional strains, namely Bacillus siamensis, Burkholderia ambifaria, and Sphingomonas yunnanensis, in equal proportions and cooperating synergistically, effectively improving the activity of antioxidant enzyme SOD in plants, increasing osmotic adjustment substances such as proline, and reducing membrane lipid peroxidation - the content of MDA is reduced, significantly promoting the growth of wheat seedlings under salt stress and increasing the wheat yield.

[0016] (2) The present invention first combines trehalose and proline through nanotechnology to form stable particles, which are used as microbial protectants and plant anti-stress inducers and slowly release under saline-alkali conditions. In addition, the nanoscale particles are more easily absorbed and utilized by microorganisms and plant roots. In addition, trace elements iron, manganese, and zinc are added to the inoculant in chelated form, which can be effectively absorbed and utilized by plants, and comprehensively improve the stress resistance and growth performance of wheat plants through the synergistic effect with the composite microbial community.

[0017] (3) The microbial inoculant of the present invention, when applied in combination with conventional fertilization, significantly promotes the growth and stress resistance of wheat seedlings under salt stress and increases the wheat yield. Description of the drawings

[0018] Figure 1 It is the growth state diagram of the selected strains of the present invention on the LB medium; note: 1 represents Bacillus siamensis, 2 represents Burkholderia ambifaria, and 3 represents Sphingomonas yunnanensis. Specific embodiments

[0019] The technical solution of the present invention will be further described below in conjunction with specific embodiments, but not limited thereto.

[0020] Example 1 An adversity-resistant and growth-promoting microbial inoculant, the microbial inoculant comprising the following raw materials in parts by weight: 15 parts of a functional microbial inoculant, 3 parts of trehalose-proline composite nanoparticles, 35 parts of a composite carrier, and 2 parts of a trace element-containing substance.

[0021] The functional microbial inoculant includes Bacillus siamensis, Burkholderia ambifaria, and Sphingomonas yunnanensis; the preservation number of Bacillus siamensis is CCTCC AB 2022048; the preservation number of Burkholderia ambifaria is CGMCC No. 1.10511; the preservation number of Sphingomonas yunnanensis is CGMCC No. 1.15275.

[0022] The preparation method of the functional microbial inoculant is as follows: (1) After thawing Bacillus siamensis and Burkholderia ambifaria, they are activated in an LB solid medium. Single colonies are respectively picked and inoculated into an LB liquid medium, and cultured at 27°C and 180 r / min until OD 600 = 0.6 to obtain seed solutions. The seed solutions are respectively inoculated into 250 mL of LB liquid medium at 1% and cultured at 27°C and 180 r / min. The viable count of the bacterial solution is detected. After reaching 1×10 8 cfu / mL, the culture is terminated, and Bacillus siamensis bacterial solution and Burkholderia ambifaria bacterial solution are respectively obtained; (2) Sphingomonas yunnanensis is placed in a nutrient broth agar medium for activation. Single colonies are picked and inoculated into a nutrient broth liquid medium, and cultured at 30°C and 200 r / min until OD 600 = 0.6 to obtain a seed solution. The seed solution is inoculated into 250 mL of LB liquid medium at 1% and cultured at 30°C and 200 r / min. The viable count is detected. After reaching 1×10 8 cfu / mL, the culture is terminated to obtain Sphingomonas yunnanensis bacterial solution; (3) The Bacillus siamensis bacterial solution and Burkholderia ambifaria bacterial solution in step (1) and the Sphingomonas yunnanensis bacterial solution in step (2) are mixed according to a volume ratio of 1:1:1 to obtain a functional microbial inoculant.

[0023] The preparation method of the trehalose-proline composite nanoparticles is as follows: Dissolve trehalose and L-proline in deionized water at a mass ratio of 2:1 to prepare a 10% solution; then add 0.1% chitosan as a stabilizer; treat it 5 times under the condition of 100 MPa by high-pressure homogenization to obtain composite nanoparticles with a particle size of 50-100 nm; spray-dry to obtain powdery trehalose-proline composite nanoparticles.

[0024] The composite carrier includes humic acid, bentonite, and biochar, and the mass ratio of the three is 3:2:1.

[0025] The trace element-containing substance includes chelated zinc, chelated iron, and chelated manganese, and the mass ratio of the three is 4:3:2.

[0026] A preparation method of a stress-resistant and growth-promoting microbial inoculant includes the following steps: Expand and culture three strains respectively to obtain the bacterial liquids of the three strains, mix them in equal volume to obtain a functional microbial inoculant, first mix the functional microbial inoculant with the composite carrier, then add the trehalose-proline composite nanoparticles and the trace element-containing substance, mix well, adjust the water content to 25-30%, ferment at 30 °C for 48 h, dry at low temperature until the water content is less than 10%, and pulverize through 80 meshes to obtain the stress-resistant and growth-promoting microbial inoculant.

[0027] Example 2 A stress-resistant and growth-promoting microbial inoculant, the microbial inoculant includes the following raw materials in parts by weight: 20 parts of functional microbial inoculant, 4 parts of trehalose-proline composite nanoparticles, 40 parts of composite carrier, and 2 parts of trace element-containing substance.

[0028] The functional microbial inoculant includes Bacillus siamensis, Burkholderia ambifaria, and Sphingomonas yunnanensis; the preservation number of Bacillus siamensis is CCTCC AB 2022048; the preservation number of Burkholderia ambifaria is CGMCC No. 1.10511; the preservation number of Sphingomonas yunnanensis is CGMCC No.1.15275.

[0029] The preparation method of the functional microbial inoculant is: (1) Thaw Bacillus siamensis and Burkholderia ambifaria and activate them in LB solid medium, pick single colonies and inoculate them into LB liquid medium respectively, culture at 27 °C and 180 r / min until OD 600 =0.6 to obtain seed liquid, inoculate the seed liquid into 250 mL LB liquid medium at a ratio of 1% respectively and culture at 27 °C and 180 r / min, detect the viable count of the bacterial liquid, and reach 1×10 8After the culture ended at cfu / mL, Bacillus siamensis liquid and Burkholderia ambifaria liquid were obtained respectively; (2) Sphingomonas yunnanensis was activated in nutrient broth agar medium, and single colonies were picked and inoculated into nutrient broth liquid medium. The culture was carried out at 30 °C and 200 r / min until OD 600 = 0.6 to obtain a seed solution. The seed solution was inoculated into 250 mL LB liquid medium at a ratio of 1% and cultured at 30 °C and 200 r / min. The viable cell count was detected. When it reached 1×10 8 cfu / mL, the culture ended, and Sphingomonas yunnanensis liquid was obtained; (3) The Bacillus siamensis liquid and Burkholderia ambifaria liquid in step (1) and the Sphingomonas yunnanensis liquid in step (2) were mixed at a volume ratio of 1:1:1 to obtain a functional microbial inoculant.

[0030] The preparation method of the trehalose-proline composite nanoparticles is as follows: Trehalose and L-proline were dissolved in deionized water at a mass ratio of 2:1 to prepare a 10% solution; then 0.1% chitosan was added as a stabilizer; the mixture was treated 5 times under the condition of 100 MPa by high-pressure homogenization to obtain composite nanoparticles with a particle size of 50 - 100 nm; spray drying was used to obtain powdery trehalose-proline composite nanoparticles.

[0031] The composite carrier includes humic acid, bentonite, and biochar, and the mass ratio of the three is 3:2:1.

[0032] The trace element-containing substance includes chelated zinc, chelated iron, and chelated manganese, and the mass ratio of the three is 4:3:2.

[0033] A preparation method of an anti-stress and growth-promoting microbial inoculant includes the following steps: Three strains were respectively enlarged in culture to obtain the liquid of the three strains. After mixing in equal volume to obtain a functional microbial inoculant, the functional microbial inoculant was first mixed with the composite carrier, then the trehalose-proline composite nanoparticles and the trace element-containing substance were added, and they were fully mixed evenly. The water content was adjusted to 25 - 30%, fermented at 30 °C for 48 h, dried at low temperature until the water content was less than 10%, and pulverized through 80 meshes to obtain the anti-stress and growth-promoting microbial inoculant.

[0034] Example 3 An anti-stress and growth-promoting microbial inoculant, the microbial inoculant includes the following raw materials in parts by weight: 25 parts of functional microbial inoculant, 5 parts of trehalose-proline composite nanoparticles, 45 parts of composite carrier, and 3 parts of trace element-containing substance.

[0035] The functional microbial inoculum includes *Bacillus siamensis*, *Burkholderia ambifaria*, and *Sphingomonas yunnanensis*; the preservation number of *Bacillus siamensis* is CCTCC AB 2022048; the preservation number of *Burkholderia ambifaria* is CGMCC No. 1.10511; the preservation number of *Sphingomonas yunnanensis* is CGMCC No.1.15275.

[0036] The preparation method of the functional microbial inoculum is as follows: (1) Thaw *Bacillus siamensis* and *Burkholderia ambifaria* and activate them in LB solid medium. Pick single colonies and inoculate them into LB liquid medium, and culture at 27 °C and 180 r / min until OD 600 = 0.6 to obtain seed solutions. The seed solutions are inoculated into 250 mL LB liquid medium at a ratio of 1% respectively and cultured at 27 °C and 180 r / min. Detect the viable count of the bacterial solution. After reaching 1×10 8 cfu / mL, the culture is terminated to obtain *Bacillus siamensis* bacterial solution and *Burkholderia ambifaria* bacterial solution respectively; (2) Place *Sphingomonas yunnanensis* in nutrient broth agar medium for activation. Pick a single colony and inoculate it into nutrient broth liquid medium, and culture at 30 °C and 200 r / min until OD 600 = 0.6 to obtain a seed solution. The seed solution is inoculated into 250 mL LB liquid medium at a ratio of 1% and cultured at 30 °C and 200 r / min. Detect the viable count. After reaching 1×10 8 cfu / mL, the culture is terminated to obtain *Sphingomonas yunnanensis* bacterial solution; (3) Mix the *Bacillus siamensis* bacterial solution and *Burkholderia ambifaria* bacterial solution in step (1) and the *Sphingomonas yunnanensis* bacterial solution in step (2) at a volume ratio of 1:1:1 to obtain the functional microbial inoculum.

[0037] The preparation method of the trehalose-proline composite nanoparticles is as follows: Dissolve trehalose and L-proline in deionized water at a mass ratio of 2:1 to prepare a 10% solution; then add 0.1% chitosan as a stabilizer; use high-pressure homogenization to treat it 5 times under the condition of 100 MPa to obtain composite nanoparticles with a particle size of 50 - 100 nm; spray dry to obtain powdery trehalose-proline composite nanoparticles.

[0038] The composite carrier includes humic acid, bentonite, and biochar, and the mass ratio of the three is 3:2:1.

[0039] The trace element-containing substance includes chelated zinc, chelated iron, and chelated manganese, and the mass ratio of the three is 4:3:2.

[0040] A preparation method of a stress-resistant and growth-promoting microbial inoculant, comprising the following steps: Expand and culture three strains respectively to obtain the bacterial liquids of the three strains, mix them in equal volumes to obtain a functional microbial inoculant, first mix the functional microbial inoculant with a composite carrier, then add trehalose-proline composite nanoparticles and trace element-containing substances, mix well, adjust the water content to 25-30%, ferment at 30 °C for 48 h, dry at low temperature until the water content is less than 10%, and pulverize through 80 meshes to obtain the stress-resistant and growth-promoting microbial inoculant.

[0041] Comparative Example 1 Compared with Example 3, in this comparative example, except that the volume ratio of Bacillus siamensis bacterial liquid, Burkholderia ambifaria bacterial liquid, and Sphingomonas yunnanensis bacterial liquid is 1:2:1, the other raw materials and steps are the same as those in Example 3.

[0042] Comparative Example 2 Compared with Example 3, in this comparative example, except that the volume ratio of Bacillus siamensis bacterial liquid, Burkholderia ambifaria bacterial liquid, and Sphingomonas yunnanensis bacterial liquid is 2:1:1, the other raw materials and steps are the same as those in Example 3.

[0043] Comparative Example 3 Compared with Example 3, in this comparative example, except that the volume ratio of Bacillus siamensis bacterial liquid, Burkholderia ambifaria bacterial liquid, and Sphingomonas yunnanensis bacterial liquid is 1:1:2, the other raw materials and steps are the same as those in Example 3.

[0044] Comparative Example 4 Compared with Example 3, in this comparative example, except that only Bacillus siamensis bacterial liquid and Burkholderia ambifaria bacterial liquid with a volume ratio of 1:1 are used, the other raw materials and steps are the same as those in Example 3.

[0045] Comparative Example 5 Compared with Example 3, in this comparative example, except that only Bacillus siamensis bacterial liquid and Sphingomonas yunnanensis bacterial liquid with a volume ratio of 1:1 are used, the other raw materials and steps are the same as those in Example 3.

[0046] Comparative Example 6 Compared with Example 3, in this comparative example, except that only Burkholderia ambifaria bacterial liquid and Sphingomonas yunnanensis bacterial liquid with a volume ratio of 1:1 are used, the other raw materials and steps are the same as those in Example 3.

[0047] Comparative Example 7 Compared with Example 3, in this comparative example, except that only Bacillus siamensis bacterial liquid is used, the other raw materials and steps are the same as those in Example 3.

[0048] Comparative Example 8 Compared with Example 3, in this comparative example, except that only Burkholderia ambifaria bacterial liquid is used, the other raw materials and steps are the same as those in Example 3.

[0049] Comparative Example 9 Compared with Example 3, in this comparative example, except that only Sphingomonas yunnanensis bacterial liquid was used, the other raw materials and steps were the same as those in Example 3.

[0050] Performance test Determination of compatibility between strains The filter paper method was used to determine the compatibility between strains: The three strains were respectively inoculated into LB liquid medium and shaken in a shaker for 24 h. The cultured bacterial liquid was adjusted to OD 600 = 1.0 with sterile water. 100 μL was taken and spread on LB medium respectively. After drying, sterile filter paper with a diameter of 5 mm was placed on the plate, and 3 μL of bacterial liquid of other strains was added dropwise. The plate was incubated at 30 °C in a constant temperature incubator and repeated 3 times. After 2 d, observe whether there is an inhibition zone to determine the compatibility between the three strains used. Note: 1 represents Bacillus siamensis, 2 represents Burkholderia ambifaria, and 3 represents Sphingomonas yunnanensis.

[0051] As shown by Figure 1 , there was no antagonistic zone among the three strains, indicating that Bacillus siamensis, Burkholderia ambifaria, and Sphingomonas yunnanensis were not antagonistic to each other and were compatible with each other.

[0052] Planting experiment The experiment was carried out in the moderately saline-alkali land experimental area of the Yellow River Delta National Agricultural Science and Technology Park. The tested wheat variety was Yannong 1212.

[0053] Experimental design: A total of 14 treatment groups were set up in the experiment, including a blank control group (CK0), a conventional fertilization group (CK1), and the microbial inoculants T1-T12 prepared by using Examples 1-3 and Comparative Examples 1-9 of the present invention. Each treatment group was repeated 3 times, and the area of each plot was 10 m 2 . The treatment groups were randomly arranged, and a protection row was set around. The specific fertilization conditions of each treatment group were as follows: CK0: No fertilization treatment; CK1: Conventional fertilization treatment (urea 340 kg / hm 2 , diammonium phosphate 500 kg / hm 2 , potassium sulfate 150 kg / hm 2 ); T1: 50% conventional fertilization + microbial inoculant of Example 1; T2: 50% conventional fertilization + microbial inoculant of Example 2; T3: 50% conventional fertilization + microbial inoculant of Example 3; T4: 50% conventional fertilization + microbial inoculant of Comparative Example 1; T5: 50% conventional fertilization + microbial inoculant of Comparative Example 2; T6: 50% conventional fertilization + microbial inoculant of Comparative Example 3; T7: 50% conventional fertilization + microbial inoculant of Comparative Example 4; T8: 50% conventional fertilization + microbial inoculant of Comparative Example 5; T9: 50% conventional fertilization + microbial inoculant of Comparative Example 6; T10: 50% conventional fertilization + microbial inoculant of Comparative Example 7; T11: 50% conventional fertilization + microbial inoculant of Comparative Example 8; T12: 50% conventional fertilization + microbial inoculant of Comparative Example 9; Wheat was planted with equal row spacing, and the seeding rate per mu was 15 kg. The application rate of the microbial inoculant in the examples and comparative examples was 3 kg / mu. The same as the conventional fertilizer, a one-time basal application fertilization method was used, and the mentioned conventional fertilization treatment also adopted a one-time basal application fertilization method. The management measures of each treatment group were the same as those of conventional field management measures.

[0054] Determination indexes and methods: (1) Determination of wheat growth indexes: One week before wheat harvest, five-point sampling method was used to sample wheat plants in each treatment group, five plants were measured in each treatment group, the height above the wheat roots was measured with a ruler, and the average value was finally taken.

[0055] (2) Determination of wheat stress resistance indexes: When wheat grew for 30 d, five-point sampling method was used to collect wheat leaves in each treatment group and then mixed. The proline content of wheat plants was measured by sulfosalicylic acid method, the malondialdehyde (MDA) content was measured by thiobarbituric acid (TBA) reaction method, and the superoxide dismutase (SOD) content measured by nitroblue tetrazolium (NBT) photoreduction method.

[0056] (3) Determination of wheat yield and its component factors indexes: At wheat harvest, three 1 m×1 m quadrats were randomly selected in each treatment group, the effective spike number, grains per spike, and 1000-grain weight were counted, and each treatment group was repeated three times, and the yield was converted.

[0057] The specific test results are shown in the following table: Table 1 Results of wheat plant growth and stress resistance indexes under different treatments

[0058] As can be seen from the data in Table 1, compared with CK0 and CK1, the height of wheat plants increased significantly after applying the microbial inoculant of the present invention, indicating that the microbial inoculant of the present invention can significantly promote the growth of wheat plants on saline-alkali land. At 30 d, the proline content of wheat plants treated with Example 3 of the present invention reached 133.4 μg g-1 , which was significantly increased by 113.8% and 77.4% compared with CK0 and CK1. Under salt stress conditions, plants improve their ability to resist stress by increasing the amount of osmoregulatory substances such as proline in their bodies, thereby maintaining water balance. This indicates that the microbial inoculant of the present invention can change the defense mechanism of wheat to cope with free radicals, the microbial inoculant of the present invention increases the proline content of wheat plants, and improves the stress resistance of wheat plants.

[0059] From the MDA data in Table 1, it can be seen that compared with the blank control group and the conventional fertilization group, after applying the microbial inoculant of the embodiment of the present invention, the MDA content in wheat plants is much lower than that in CK0 and CK1. The MDA content reflects the level of lipid peroxidation of plant membranes and the degree of damage to cell membranes. The higher the content, the higher the degree of membrane damage. The results of this study indicate that applying the microbial inoculant of the present invention can reduce the MDA content of wheat plants, improve the stress resistance of wheat plants, and reduce the degree of membrane damage.

[0060] Under soil salt stress, plants produce harmful substances such as reactive oxygen species (ROS) due to dehydration, resulting in an imbalance of reactive oxygen species in the plant body. Superoxide dismutase (SOD) is an important component of the antioxidant enzyme system, protecting cell membranes from damage caused by ROS and inhibiting membrane lipid peroxidation, thereby reducing the stress damage to plant cells under adversity. From the SOD data in Table 1, it can be seen that compared with the blank control group and the conventional fertilization group, after applying the microbial inoculant of the embodiment of the present invention, the SOD activity in wheat plants is significantly increased, indicating that the microbial inoculant of the present invention is beneficial to improving the stress resistance of wheat plants.

[0061] Table 2 Wheat yield and its component factors under different treatments

[0062] From the data in Table 2, it can be seen that there are significant differences in the number of spikes per plant, grains per spike, 1000-grain weight and yield at the wheat harvest stage under different treatments. Compared with the blank control group and the conventional fertilization group, after applying the microbial inoculant of the embodiment of the present invention, the number of spikes per plant, grains per spike, 1000-grain weight and theoretical yield at the wheat harvest stage are all significantly increased, indicating that after applying the microbial inoculant of the present invention, the stress resistance of wheat plants is improved, and it can significantly promote the absorption and utilization of nutrients by wheat plants in saline-alkali land and the formation of yield.

[0063] It should be noted that the above embodiments are only some embodiments of the preferred ways to implement the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Claims

1. A stress-resistant and growth-promoting microbial inoculant, characterized in that, The microbial inoculant comprises the following raw materials in parts by weight: 15-25 parts of functional microbial inoculant, 3-5 parts of trehalose-proline composite nanoparticles, 35-45 parts of composite carrier, and 2-3 parts of trace element-containing substance.

2. The stress-resistant growth-promoting microbial inoculant according to claim 1, characterized in that, The functional microbial inoculant includes Bacillus siamensis, Burkholderia ambifaria, and Sphingomonas yunnanensis; the preservation number of Bacillus siamensis is CCTCC AB 2022048; the preservation number of Burkholderia ambifaria is CGMCC No. 1.10511; the preservation number of Sphingomonas yunnanensis is CGMCC No. 1.15275.

3. The stress-resistant growth-promoting microbial inoculant according to claim 1, characterized in that, The preparation method of the functional microbial inoculant is as follows: (1) After thawing Bacillus siamensis and Burkholderia ambifaria, activate them in LB solid medium. Pick single colonies and inoculate them into LB liquid medium respectively, and culture at 27°C and 180 r / min until OD 600 = 0.6 to obtain seed solutions. The seed solutions are inoculated into 250 mL LB liquid medium at a ratio of 1% respectively and cultured at 27°C and 180 r / min. Detect the viable count of the bacterial solution. After reaching 1×10 8 cfu / mL, end the culture to obtain Bacillus siamensis bacterial solution and Burkholderia ambifaria bacterial solution respectively; (2) Activate Sphingomonas yunnanensis in nutrient broth agar medium, pick a single colony and inoculate it into nutrient broth liquid medium, and culture it at 30 °C and 200 r / min until OD 600 = 0.6 to obtain a seed solution. The seed solution is inoculated into 250 mL LB liquid medium at a ratio of 1% and cultured at 30 °C and 200 r / min. Detect the viable cell count, and end the culture after reaching 1×10 8 cfu / mL to obtain Sphingomonas yunnanensis bacterial liquid; (3) Mix the Bacillus siamensis bacterial liquid in step (1), the Burkholderia ambifaria bacterial liquid, and the Sphingomonas yunnanensis bacterial liquid in step (2) according to a volume ratio of 1:1:1 to obtain the functional microbial inoculant.

4. The stress-resistant growth-promoting microbial inoculant according to claim 1, characterized in that, The preparation method of the trehalose-proline composite nanoparticles is as follows: Dissolve trehalose and L-proline in deionized water according to a mass ratio of 2:1 to prepare a 10% solution; then add 0.1% chitosan as a stabilizer; treat it 5 times under the condition of 100 MPa by high-pressure homogenization to obtain composite nanoparticles with a particle size of 50-100 nm; spray-dry to obtain powdery trehalose-proline composite nanoparticles.

5. The stress-resistant growth-promoting microbial inoculant according to claim 1, characterized in that, The composite carrier includes humic acid, bentonite, and biochar, and the mass ratio of the three is 3:2:

1.

6. The stress-resistant and growth-promoting microbial inoculant according to claim 1, characterized in that, The trace element-containing substance includes chelated zinc, chelated iron, and chelated manganese, and the mass ratio of the three is 4:3:

2.

7. A preparation method of the stress-resistant and growth-promoting microbial inoculant according to any one of claims 1-6, characterized in that, It is prepared by including the following steps: Expand the culture of the three strains respectively to obtain the bacterial liquids of the three strains, mix them in equal volume to obtain the functional microbial inoculant, first mix the functional microbial inoculant with the composite carrier, then add the trehalose-proline composite nanoparticles and the trace element-containing substance, mix well, adjust the water content to 25-30%, ferment at 30 °C for 48 h, dry at low temperature until the water content is less than 10%, and pulverize through 80 meshes to obtain the stress-resistant and growth-promoting microbial inoculant.

8. Use of the stress-resistant growth-promoting microbial inoculant according to claim 1, characterized in that, The microbial inoculant is used to promote the growth of wheat and effectively improve the stress resistance of wheat growing in saline-alkali land.

Images