Complex microbial inoculant suitable for salt tolerance and growth promotion of corn and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant suitable for salt tolerance and growth promotion of corn and application of the complex microbial inoculant. The complex microbial inoculant is prepared from seven strains including gordonia lyophorae, microbacterium amyloliquefaciens, pseudomonas stutzeri, brevibacterium foodstuff, xanthomonas aeruginosa, South China sea bacillus and brevibacterium epidermidis, and has the remarkable characteristics of no antagonism among the strains, salt and alkali tolerance, high viable count and the like; the plant thickness, the ear thickness and the yield of the corn can be obviously promoted, and the empty stalk rate can be well reduced. The protein content, catalase activity, peroxidase activity, superoxide dismutase activity and total chlorophyll content of the plant under the salt stress condition are remarkably improved compared with those of a control group; meanwhile, the content of malonaldehyde and the content of proline are remarkably reduced, and it is indicated that the complex microbial inoculant plays a role in promoting and protecting corn in the process of resisting saline-alkali stress.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, specifically to the technical field of compound bacterial agents and their preparation methods, and more specifically to the technical field of a compound bacterial agent suitable for salt tolerance and growth promotion of corn and its application. Background Art

[0002] Corn is one of the cereal crops with the widest global planting range and the largest yield. Globally, the planting area of corn ranks third, after wheat and rice. Corn is of extremely important status in crops. It is not only an important food crop but also plays a key role in multiple fields such as feed, industry, and energy. Corn is a high-quality feed raw material and is widely used in animal husbandry. In developed animal husbandry countries, 70%-75% of corn consumption is used for feed. There are many types of corn deep-processing products, including corn starch, corn oil, alcohol, etc. Corn is also used to produce biofuels such as ethanol, which helps to relieve the energy pressure. The extensive planting area, high yield, rich nutritional value, and strong adaptability of corn make it an important crop for ensuring global food security and promoting agricultural economic development.

[0003] Saline-alkali stress can significantly inhibit the germination of corn seeds and the growth of seedlings. In a high saline-alkali environment, the osmotic pressure of the soil solution increases, resulting in difficult water absorption by seeds and a reduced germination rate. At the same time, the root growth of seedlings will also be inhibited, with a decrease in root length and root weight, affecting the early growth of seedlings. And it will also interfere with the photosynthetic performance of corn leaves, reducing the content of photosynthetic pigments (such as chlorophyll and carotenoids), resulting in a decline in photosynthetic efficiency. In addition, saline-alkali stress will also affect the dry matter accumulation and distribution of corn, reducing the accumulation of biomass. At the same time, it will cause a significant decrease in corn yield, mainly manifested as a reduction in the number of grains per ear and the 100-grain weight. In addition, saline-alkali stress may also affect the quality of corn, such as protein content, starch content, etc.

[0004] The use of compound bacterial agents is a green and sustainable agricultural practice, which can reduce the use of chemical fertilizers and lower agricultural production costs. At the same time, by improving the soil environment and enhancing plant stress resistance, compound bacterial agents contribute to improving the utilization efficiency of saline-alkali land and promoting the sustainable development of agriculture. Compound bacterial agents have significant potential in alleviating salt stress during corn growth and promoting growth, and are an agricultural technology means worthy of promotion. Therefore, developing a compound bacterial agent suitable for salt tolerance and growth promotion of corn is of great significance for the development of agriculture in arid areas. It not only has important significance for ensuring national food security and promoting regional economic growth but also plays an irreplaceable role in ecological protection, cultural inheritance, etc. Summary of the Invention

[0005] In view of the problem that there are few reports on compound microbial agents suitable for salt tolerance and growth promotion of maize in the prior art, the present invention provides a compound microbial agent suitable for salt tolerance and growth promotion of maize and its application. The present invention also provides the application of the compound microbial agent in promoting the growth of maize under salt stress conditions, which can significantly promote the plant height, ear diameter and yield of maize, and can well reduce the empty stalk rate. And under salt stress conditions, the protein content, catalase, peroxidase, superoxide dismutase activity and total chlorophyll content of the plants are significantly higher than those of the control group; at the same time, the contents of malondialdehyde and proline are significantly reduced, indicating that the compound microbial agent provided by the present application plays a promoting and protective role in the process of maize resisting saline-alkali stress.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions to achieve it: The present invention provides a compound microbial agent, which is obtained by mixing the resuspension of Gordonia hydrophobica ( Gordonia hydrophobica ), Microbacterium amylolyticum ( Microbacterium amylolyticum ), Pseudomonas stutzeri ( Pseudomonas stutzeri ), Brevibacterium alimentarium ( Brachybacterium alimentarium ), Xanthomonas huabeiensis ( Luteimonas huabeiensi s), Bacillus nanhaiensis ( Bacillus australimaris ), Brevibacterium epidermidis ( Brevibacterium epidermidis ).

[0007] Preferably, the microbial agent is obtained by mixing the resuspension of Gordonia hydrophobica ( Gordonia hydrophobica ), Microbacterium amylolyticum ( Microbacterium amylolyticum ), Pseudomonas stutzeri ( Pseudomonas stutzeri ), Brevibacterium alimentarium ( Brachybacterium alimentarium ), Xanthomonas huabeiensis ( Luteimonas huabeiensi s), Bacillus nanhaiensis ( Bacillus australimaris ), Brevibacterium epidermidis ( Brevibacterium epidermidis ) in a volume ratio of 1:1:1:1:1.

[0008] Preferably, the optical density OD 600 of the resuspension of each strain in the microbial agent is 0.2.

[0009] Preferably, the optical density OD 600 of the microbial agent is 0.02.

[0010] Furthermore, the present invention also provides a preparation method of the compound microbial agent, which is specifically prepared by the following steps: inoculating the strain in TSA containing 2% NaCl and culturing it until the exponential growth phase, centrifuging to collect cells, washing once with 1×PBS, and resuspending in 10 mM MgCl2 solution; adjusting the optical density OD 600Adjust to 0.2; mix the selected strains in the ratio of 1:1:1:1:1 to obtain a composite microbial agent, and then dilute the agent to OD 600 of 0.02 for standby.

[0011] Furthermore, the present application also provides an application of a composite microbial agent in promoting the growth of maize under salt stress conditions.

[0012] The application adopts the following steps: apply the synthetic functional microbial agent to maize by drip irrigation with water throughout the growth period, and the application rate is 2 L / mu. Preferably, for the application of a composite microbial agent in promoting the growth of maize under salt stress conditions, the applicable maize variety is the mid-late maturing forage maize variety A1589.

[0013] In the present application, in the following content, "Gordonia hydrophobica ( Gordonia hydrophobica )" is abbreviated as "Gordonia hydrophobica"; Microbacterium amylolyticum ( Microbacterium amylolyticum )" is abbreviated as "Microbacterium amylolyticum"; Pseudomonas stutzeri ( Pseudomonas stutzeri )" is abbreviated as "Pseudomonas stutzeri"; Brevibacillus alimentarius ( Brachybacterium alimentarium )" is abbreviated as "Brevibacillus alimentarius"; Xanthomonas luteola Huabei ( Luteimonas huabeiensi s)" is abbreviated as "Xanthomonas luteola Huabei"; Bacillus nanhaiensis ( Bacillus australimaris )" is abbreviated as "Bacillus nanhaiensis"; Brevibacterium epidermidis ( Brevibacterium epidermidis )" is abbreviated as "Brevibacterium epidermidis".

[0014] Compared with the prior art, the present invention has the following characteristics: (1) The composite microbial agent provided by the present invention uses seven strains of bacteria, namely Gordonia hydrophobica, Microbacterium amylolyticum, Pseudomonas stutzeri, Brevibacillus alimentarius, Xanthomonas luteola Huabei, Bacillus nanhaiensis, and Brevibacterium epidermidis. The microbial agent has the remarkable characteristics of no antagonism between strains, salt tolerance, and high viable bacteria count.

[0015] (2) For the composite microbial agent provided by the present invention, the synthetic functional microbial agent is applied to maize by drip irrigation with water throughout the growth period of saline-alkali stressed soil. The maize yield in the composite microbial agent treatment group is significantly increased, and after using the composite microbial agent, the empty stalk rate is significantly reduced and the ear diameter is increased. It can be seen that under salt stress conditions, the mixed microbial agent significantly promotes the growth of maize.

[0016] (3)The compound microbial agent provided by the present invention shows a significant increase in protein content, catalase, peroxidase, superoxide dismutase activity, and total chlorophyll content compared to the control group under salt stress conditions. At the same time, after treatment with the compound microbial agent, the contents of malondialdehyde and proline are significantly reduced, indicating that the compound microbial agent used in the compound microbial agent treatment group plays a promoting and protective role in the process of maize resisting saline-alkali stress. Description of the Drawings

[0017] Figure 1 The figure shows the comparison chart of maize yield results after applying the microbial agent.

[0018] Figure 2 The figure shows the comparison chart of maize protein content results after applying the microbial agent.

[0019] Figure 3 The figure shows the comparison chart of catalase content results after applying the microbial agent.

[0020] Figure 4 The figure shows the comparison chart of superoxide dismutase content results after applying the microbial agent.

[0021] Figure 5 The figure shows the comparison chart of peroxidase content results after applying the microbial agent.

[0022] Figure 6 The figure shows the comparison chart of chlorophyll content results after applying the microbial agent.

[0023] Figure 7 The figure shows the comparison chart of malondialdehyde content results after applying the microbial agent.

[0024] Figure 8 The figure shows the comparison chart of proline content results after applying the microbial agent.

[0025] Figure 9 The figure shows the comparison chart of the empty stalk rate results after applying the microbial agent.

[0026] Figure 10 The figure shows the comparison chart of maize ear diameter results after applying the microbial agent.

[0027] In the above drawings, CK is the control group, and MIX is the compound microbial agent application group. Detailed Embodiments

[0028] The following examples are used to further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps, or conditions of the present invention shall fall within the scope of the present invention.

[0029] The seven strains of bacteria used in the present invention, namely Gordonia amarae, Microbacterium amylolyticum, Pseudomonas stutzeri, Brevibacterium alimentarium, Xanthomonas huabeiensis, Bacillus nanhaiensis, and Brevibacterium epidermidis, are all harmless and safe strains. Among them, Gordonia amarae, Microbacterium amylolyticum, and Brevibacterium epidermidis were obtained by purchasing from the China General Microbiological Culture Collection Center (CGMCC), and the general public can also obtain them through other public channels such as the China General Microbiological Culture Collection Center (CGMCC); Pseudomonas stutzeri was obtained by purchasing from Ningbo Mingzhou Biotechnology Co., Ltd., and the general public can also obtain it through other public channels such as Ningbo Mingzhou Biotechnology Co., Ltd.; Brevibacterium alimentarium and Xanthomonas huabeiensis were obtained by purchasing from Beijing Bio-win Biotechnology Co., Ltd., and the general public can also obtain them through other public channels such as Beijing Bio-win Biotechnology Co., Ltd.; Bacillus nanhaiensis was obtained by purchasing from the Guangdong Provincial Culture Collection of Microorganisms (GDMCC), and the general public can also obtain it through other public channels such as the Guangdong Provincial Culture Collection of Microorganisms (GDMCC).

[0030] The "A1589 mid-late maturing forage maize" used in this application was purchased from China International Seed Co., Ltd., and the general public can obtain it by purchasing from China International Seed Co., Ltd. or other seed companies.

[0031] The tryptic soy agar medium (TSA), DF liquid medium, and ADF liquid medium used in this application are conventional media in the technical field.

[0032] In this application, a BCA method protein content assay kit, catalase (CAT) kit, superoxide dismutase (SOD) kit, peroxidase (POD) kit, malondialdehyde (MDA) content kit, proline (PRO) content assay kit, and plant chlorophyll content kit were used for plant enzyme activity detection. The above kits were all purchased from Suzhou Keming Biotechnology Co., Ltd.

[0033] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0034] Example 1: A compound microbial agent The present invention provides a compound microbial agent, which is obtained by mixing the resuspension liquids of Gordonia amarae, Microbacterium amylolyticum, Pseudomonas stutzeri, Brevibacterium alimentarium, Xanthomonas huabeiensis, Bacillus nanhaiensis, and Brevibacterium epidermidis.

[0035] The present invention also provides a method for preparing a composite bacterium agent, which is specifically obtained by the following steps: inoculating the strain in TSA containing 2% NaCl and culturing it until the exponential growth phase, centrifuging to collect cells, washing once with 1×PBS, and resuspending in 10 mM MgCl2 solution; adjusting the optical density OD 600 of each cell solution to 0.2; mixing the selected strains according to the condition of 1:1:1:1:1 to obtain a composite bacterium agent, and then diluting the bacterium agent to OD 600 of 0.02 for standby.

[0036] Furthermore, the present application also provides an application of the composite bacterium agent in promoting the growth of maize under salt stress conditions. Example 2: Hemolytic assay Spot inoculate Gordonia hydrophobica, Microbacterium amylolyticum, Pseudomonas stutzeri, Brevibacterium alimentarium, Xanthomonas huabeiensis, Bacillus nanhaiensis, Brevibacterium epidermidis strains onto the hemolytic medium, and culture at 30 °C for 3 - 7 days. A semi-transparent hemolytic ring of 1 - 2 mm can be observed around the colony, indicating that the strain has hemolytic property. The measurement results show that the selected Gordonia hydrophobica, Microbacterium amylolyticum, Pseudomonas stutzeri, Brevibacterium alimentarium, Xanthomonas huabeiensis, Bacillus nanhaiensis, Brevibacterium epidermidis do not have hemolytic reaction, indicating that the selected seven strains are biosafe strains and can be used for the construction of the composite bacterium agent in the future.

[0037] Example 3: Determination of the ability to produce 1-aminocyclopropane-1-carboxylic acid deaminase (ACC) Inoculate the strain into a liquid nitrogen-free medium and culture it at 30 °C with shaking at 200 rpm for 24 h. Pipette 0.1 ml of the bacterial liquid and inoculate it into 5 ml of DF liquid medium. After culturing with shaking for 24 h, pipette 0.1 ml of the above culture solution and transfer it to ADF liquid medium. After culturing with shaking for 48 h, repeat the transfer to ADF medium. The strain that can grow is determined as an ACC deaminase positive strain. Through detection, it is found that Gordonia hydrophobica, Microbacterium amylolyticum, Pseudomonas stutzeri, Brevibacterium alimentarium, Xanthomonas huabeiensis, Bacillus nanhaiensis, Brevibacterium epidermidis can all grow well, indicating that the seven selected strains all have good ability to produce 1-aminocyclopropane-1-carboxylic acid deaminase (ACC).

[0038] Example 4: Strain antagonism experiment For the obtained Gordonia phobias, Microbacterium amylolyticum, Pseudomonas stutzeri, Brevibacterium alimentarium, Xanthomonas huabeiensis, Bacillus nanhaiensis, Brevibacterium epidermidis, pairwise antagonistic experiments were conducted on the selected strains. One strain was selected for liquid culture, and the other strains were used for solid culture. The cultured bacterial liquid was evenly spread on the solid medium. A puncher was used to punch holes in the colonies of the solid-cultured strains, and the colony agar blocks were picked up with forceps and inverted on the plate coated with the indicator bacteria; the plate was covered and cultured in an incubator, and it was observed whether a clear antibacterial circle was formed around the agar block, and the diameter of the antibacterial circle was measured. The results showed that no clear antibacterial circles were formed between the selected strains, indicating that there was no antagonistic reaction between the selected strains, and they could be used for the construction of compound bacterial agents in the future.

[0039] Example 5: Construction of compound bacterial agent Based on the records in the above Examples 1 to 3, strains that tolerated 5% NaCl, had no hemolysis and no antagonistic effect on each other were selected from the above results. The strains were inoculated into TSA containing 2% NaCl and cultured until the exponential growth phase. The cells were collected by centrifugation, washed once with 1×PBS, and resuspended in 10 mM MgCl2 solution. The optical density (OD 600） of each cell solution was adjusted to 0.2. The selected strains were mixed under the condition of 1:1:1:1:1 to obtain a compound bacterial agent, and then the bacterial agent was diluted to OD 600 of 0.02 for standby.

[0040] Example 6: Promotion of maize growth by compound bacterial agent Based on the compound bacterial agent obtained from the records in the above Example 4 and Example 5, a plot experiment was carried out on the saline-alkali land of the Manas Comprehensive Experimental Station of the Xinjiang Academy of Agricultural Sciences. The total salt content of the selected saline-alkali land soil was between 1% and 1.5%, which was moderately salinized soil. A control group (CK) and a synthetic functional bacterial agent treatment group (MIX) were set up, and the experimental area of each treatment was 20 m 2 , with 3 replicates. The synthetic functional bacterial agent was applied to maize by drip irrigation with water throughout the growth period, and the application rate was 2 L / mu. At the end of the growth period, the yield, empty-stalk rate, and ear thickness of maize were measured. The yield measurement results of the control group (CK) and the synthetic functional bacterial agent treatment group (MIX) in the measurement results are shown in Appendix Figure 1 , Appendix Figure 9 , Appendix Figure 10 . The maize and maize yield in the compound bacterial agent treatment MIX group increased significantly. And by measuring the number of empty stalks in every 20 maize plants to determine the empty-stalk rate, the results showed that after using the compound bacterial agent, the empty-stalk rate decreased significantly. The ear thickness of maize was measured, and after using the compound bacterial agent, the ear thickness increased. Under salt stress conditions, the mixed bacterial agent significantly promoted the growth of maize.

[0041] Example 7: Detection of the Activity of Enzymes in Maize Plants Promoted by the Compound Bacterial Agent Based on the construction of the compound bacterial agent in Example 5 above and the treatment in Example 6, leaves were collected at the trumpet stage of maize growth and stored in a liquid nitrogen tank. A protein content assay kit, a catalase kit, a superoxide dismutase kit, a peroxidase kit, a malondialdehyde content kit, a proline content assay kit, and a plant chlorophyll content kit from Suzhou Keming Biotechnology Co., Ltd. were used to detect the activity of plant enzymes. The measurement results are shown in Appendix Figure 2 , Appendix Figure 3 , Appendix Figure 4 , Appendix Figure 5 , Appendix Figure 6 , Appendix Figure 7 , Appendix Figure 8 The results showed that the protein content, catalase, peroxidase, superoxide dismutase activity, and total chlorophyll content were all significantly higher than those of the control group; at the same time, after the MIX treatment, the contents of malondialdehyde and proline were significantly reduced, indicating that the compound bacterial agent used in the MIX treatment group played a promoting and protective role in the process of maize resisting saline-alkali stress.

[0042] Comparative Example 1: Comparison of the Growth-Promoting Effects of Different Strains and Combinations Based on the construction of the compound bacterial agent in Example 5 above and the treatment in Example 6, the seven selected strains were respectively compounded as single strains and in pairs at a ratio of 1:1, and the concentration of the bacterial agent was adjusted to OD 600 of 0.02 as the comparative example group. According to the bacterial agent constructed in Example 5, the concentration of the bacterial agent was adjusted to OD 600 of 0.01, OD 600 of 0.02, and OD 600 of 0.03 as Group 1 of Example 1, Group 2 of Example 2, and Group 3 of Example 3. And a maize planting experiment was carried out for comparison, and the results of the empty stalk rate and the ear thickness of maize were measured. The grouping treatment situation and the maize germination rate situation are shown in Table 1 below.

[0043] Table 1: Grouping Treatment Situation, Empty Stalk Rate of Maize, and Ear Thickness of Maize

[0044] By comparing the growth promotion effects of the above different strains and combinations, it can be seen that the growth promotion effect of individual strains on corn is lower than that obtained by combining the selected strains in pairs. The compound microbial agent prepared by mixing the seven selected strains in the present application has the best effect on promoting the growth of corn under saline-alkali stress. The blank stalk rate of corn is significantly lower than that of other treatment groups, and the ear diameter of corn is significantly higher than that of other comparative example treatment groups, which also confirms that the combination of the seven strains selected in the technical solution provided in the present application can exert excellent synergistic effects.

[0045] By comparing the growth promotion effects of the above different strains and combinations, it can be seen that the growth promotion effect of individual strains on corn and winter corn is lower than that obtained by combining the selected strains in pairs. The compound microbial agent prepared by mixing the seven selected strains in the present application has the best effect on promoting the growth of corn and winter corn under saline-alkali stress. The germination rates of corn and winter corn are significantly higher than those of other comparative example treatment groups, which also confirms that the combination of the seven strains selected in the technical solution provided in the present application can exert excellent synergistic effects and has a significant impact on agricultural development and sustainable development in arid regions.

[0046] The above embodiments are merely examples for clearly illustrating the present invention and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A composite bacterial agent, characterized in that: The bacterial agent is composed of hydrophobic Gordonia ( Gordonia hydrophobica )、Microbacterium amyloliquefaciens( Microbacterium amylolyticum )、Pseudomonas stutzeri( Pseudomonas stutzeri )、Brexibacterium parvum( Brachybacterium alimentarium )、Xanthomonas sphenanthera( Luteimonas huabeiensi s), South China Sea Bacillus ( Bacillus australimaris ) Brevibacterium epidermidis Brevibacterium epidermidis ) and mixed to obtain a resuspension.

2. A composite bacterial agent according to claim 1, characterized in that: The bacterial agent is composed of hydrophobic Gordonia ( Gordonia hydrophobica )、Microbacterium amyloliquefaciens( Microbacterium amylolyticum )、Pseudomonas stutzeri( Pseudomonas stutzeri )、Brexibacterium parvum( Brachybacterium alimentarium )、Xanthomonas sphenanthera( Luteimonas huabeiensi s), South China Sea Bacillus ( Bacillus australimaris ) Brevibacterium epidermidis Brevibacterium epidermidis ) The resuspension was obtained by mixing in a volume ratio of 1:1:1:1:

1.

3. A composite bacterial agent according to claim 1, characterized in that: The optical density OD of the resuspension of each strain in the bacterial agent 600 is 0.

2.

4. A composite bacterial agent according to claim 1, characterized in that: The optical density OD of the bacterial agent 600 is 0.

02.

5. The method for preparing a composite bacterial agent according to claim 1, characterized in that: The preparation was carried out in the following steps: the strain was inoculated in TSA containing 2% NaCl and cultured until the exponential growth phase, the cells were collected by centrifugation, washed once with 1× PBS, and resuspended in 10 mM MgCl2 solution; the optical density OD of each cell solution was calculated. 600 Adjust to 0.2; mix the selected strains at a ratio of 1:1:1:1:1 to obtain a composite bacterial agent, and then dilute the bacterial agent to OD 600 0.02 is reserved.

6. Use of a composite bacterial agent as described in any one of claims 1 to 5 in promoting corn growth under salt stress conditions.

7. The use of a composite bacterial agent as claimed in claim 6 in promoting corn growth under salt stress conditions, characterized in that: The application adopts the following steps: the synthetic functional bacterial agent is applied to corn by drip irrigation during the whole growth period, and the application amount is 2 L / mu.

8. The use of a composite bacterial agent as claimed in claim 6 in promoting corn growth under salt stress conditions, characterized in that: The applicable corn variety is A1589 medium-late maturing feed variety corn.

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