Saline-alkali soil microorganism combination, complex microbial inoculant and application of complex microbial inoculant

Abstract

The invention provides a saline-alkali soil microbial composition, a complex microbial inoculant and application thereof, and belongs to the technical field of microorganisms. The microbial composition comprises microbacterium NO1 and Zobellassp. NO15, the preservation number of the microbacterium NO1 is CGMCC (China General Microbiological Culture Collection Center) No.32676, the preservation number of the Zobellassp. NO15 is CGMCC No.32677, the preservation number of the microbacterium NO1 is CGMCC No.32676, and the preservation number of the Zobellassp. NO15 is CGMCC No.32677. The microbial composition is high in saline-alkali tolerance, can efficiently utilize organic matters in saline-alkali soil, and has a growth promoting effect on plants. The microbial composition disclosed by the invention can be used for saline-alkali soil treatment, and has important significance in improving soil quality, guaranteeing human health and improving ecological environment.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a saline-alkali land microbial combination, a composite bacterial agent and applications thereof. Background Art

[0002] Soil salinization is a global ecological resource problem and an important environmental disaster or risk caused by natural or human activities. At present, key technologies such as well irrigation and drainage, rice planting and salt washing, large water pressure salt and sub-film drip irrigation have been used to achieve large-scale utilization of regional mild and moderate saline-alkali land that is easy to transform. A large number of mild and moderate saline-alkali lands have been successfully transformed, but some cultivated land is prone to repeated salinization, and moderate and severe stubborn saline-alkali wasteland is difficult to transform, which is still a historical problem.

[0003] Soil microorganisms can potentially adapt to saline-alkali environments and bring benefits to the improvement of saline-alkali soils by regulating organic matter circulation and promoting plant growth. At present, most of the microbial technologies for improving saline-alkali land are focused on the identification of microbial communities and changes in abundance. The exploration of functional microorganisms generally remains at the salt-alkali tolerance research of a single strain, which has limitations such as single function, weak tolerance, and weak organic matter regulation level. Summary of the invention

[0004] The purpose of the present invention is to provide a saline-alkali land microbial combination, a composite bacterial agent and their application. The microbial combination of the present invention has a strong salt-alkali tolerance, can efficiently utilize organic matter in saline-alkali land soil and has a growth-promoting effect on plants.

[0005] The present invention provides a microbial combination, comprising Microbacterium ( Microbacterium sp .) NO1 and Chabelella ( Zobellellasp .) NO15; the preservation number of the Microbacterium NO1 is CGMCC No.32676, and the preservation number of the C. truncatula NO15 is CGMCC No.32677.

[0006] Preferably, the ratio of the effective viable counts of Microbacterium NO1 and Chapelaeella NO15 is 1:1.

[0007] The present invention also provides a fermentation product of the microbial combination described in the above scheme.

[0008] The present invention also provides a composite bacterial agent, comprising the microbial combination or the fermentation product described in the above scheme.

[0009] Preferably, the OD of the microbial combination in the composite bacterial agent is 600 Value ≥ 1.

[0010] The present invention also provides a method for preparing the composite bacterial agent described in the above scheme, comprising the following steps: inoculating Microbacterium NO1 and Cleaverella NO15 into liquid culture medium respectively, culturing to obtain Microbacterium NO1 bacterial solution and Cleaverella NO15 bacterial solution respectively; mixing the Microbacterium NO1 bacterial solution and Cleaverella NO15 bacterial solution to obtain the composite bacterial agent.

[0011] Preferably, the liquid culture medium comprises 1 / 10 LB liquid culture medium.

[0012] The present invention also provides the use of the microbial combination, the fermentation product, the composite bacterial agent or the composite bacterial agent prepared by the preparation method described in the above scheme in the treatment of saline-alkali land.

[0013] Preferably, the treatment of saline-alkali land includes one or more of the following 1) to 4): 1) Improve the enzyme activity level of BG in the governance system; 2) Promote and activate carbon metabolism levels within the governance system; 3) Improve the level of organic matter transformation within the governance system; 4) Promote crop growth.

[0014] Preferably, the crop comprises corn.

[0015] The present invention provides a microbial combination, comprising Microbacterium ( Microbacterium sp.) NO1 and Chabelella ( Zobellella sp.) NO15; the preservation number of the Microbacterium NO1 is CGMCC No.32676, and the preservation number of the Chabelella NO15 is CGMCC No.32677. The microbial combination of the present invention has a strong salt-alkali tolerance, can efficiently utilize organic matter in saline-alkali soil and has a growth-promoting effect on plants. Among them, the plants to which the microbial combination is applied under the conditions of severe saline-alkali land can increase root length by 52%, root weight by 49%, fresh weight by 44%, and surface length by 39.5%. Therefore, the microbial combination of the present invention can be used for saline-alkali land management, which is of great significance to improving soil quality, protecting human health and improving the ecological environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0017] Figure 1 is a nucleotide sequence alignment diagram of Microbacterium NO1; Figure 2 This is a nucleotide sequence alignment of Zybelsella NO15; Figure 3 It is the accumulation curve of BG enzyme activity level (carbon utilization efficiency) in the microbial combination system; Figure 4 This is a real picture of the potted plant; Figure 5 This is a bar chart of the potted corn phenotype; Figure 6 A metabolic schematic diagram of carbon metabolism-related pathways in the microbial combination system; red indicates significant upregulation (p-value<0.05).

[0018] Biological Deposit Description Microbacterium Microbacterium sp .) NO1 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the deposit number CGMCC No.32676, the deposit date is November 18, 2024, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the status is alive; Chabelella Zobellellasp .) NO15 is deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), with the deposit number CGMCC No.32677, the deposit date is November 18, 2024, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the status is alive. DETAILED DESCRIPTION

[0019] The present invention provides a microbial combination (microbial combination S "NO1+NO15"), comprising Microbacterium ( Microbacterium sp .) NO1 and Chabelella ( Zobellellasp .) NO15; the Microbacterium NO1 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with a deposit number of CGMCC No.32676, and the Chabelella NO15 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, with a deposit number of CGMCC No.32677.

[0020] The Microbacterium NO1 and the Chabelella NO15 of the present invention are collected from the coastal saline-alkali area of ​​Jiangsu Province and are screened and separated by using a plate condition screening method; the Microbacterium NO1 and the Chabelella NO15 have strong salt-alkali resistance, can efficiently utilize soil organic matter in soil with a high salinization level and a low organic matter content, and greatly promote the organic matter utilization capacity in the system in a combined state, and the soil microorganisms can potentially adapt to the saline-alkali environment, regulate the organic matter cycle, promote plant growth, and bring benefits to the improvement of saline-alkali land.

[0021] The microbial combination of the present invention has an interaction mechanism in the organic matter transformation process and has a high level of transformation effect on organic matter.

[0022] In the specific implementation process of the present invention, the ratio of the effective live bacteria count of Microbacterium NO1 and that of Chabelella NO15 is 1:1.

[0023] The present invention also provides a fermentation product of the microbial combination described in the above scheme.

[0024] The present invention also provides a composite bacterial agent, comprising the microbial combination or the fermentation product described in the above scheme.

[0025] In the specific implementation process of the present invention, the OD of the microbial combination in the composite bacterial agent is 600 Value ≥ 1.

[0026] The present invention also provides a method for preparing the composite bacterial agent described in the above scheme, comprising the following steps: Microbacterium NO1 and Czabel's bacteria NO15 are inoculated into liquid culture medium respectively, and cultured to obtain Microbacterium NO1 bacterial liquid and Czabel's bacteria NO15 bacterial liquid respectively; the Microbacterium NO1 bacterial liquid and Czabel's bacteria NO15 bacterial liquid are mixed to obtain a composite bacterial agent.

[0027] In the specific implementation of the present invention, the liquid culture medium includes 1 / 10 LB liquid culture medium; the volume ratio of the Microbacterium NO1 bacterial solution to the Chabelella NO15 bacterial solution is 1:1.

[0028] In one embodiment of the present invention, the composite bacterial agent is prepared by mixing the seed liquid of Microbacterium NO1 and Chabelella NO15 in equal proportions.

[0029] In the present invention, the Microbacterium NO1 and the Chabelella NO15 can be cultured alone or in combination.

[0030] The present invention also provides the use of the microbial combination, the fermentation product, the composite bacterial agent or the composite bacterial agent prepared by the preparation method described in the above scheme in the treatment of saline-alkali land.

[0031] In the specific implementation of the present invention, the saline-alkali land includes severe saline-alkali land; the alkalinity of the saline-alkali land is ≥9, preferably 9-11. In an embodiment of the present invention, the alkalinity of the saline-alkali land is 9, 10 or 11; the salinity of the saline-alkali land is ≥100 g / L, preferably 100-200 g / L. In an embodiment of the present invention, the salinity of the saline-alkali land is 100, 140, 200 g / L; the saline-alkali land includes coastal saline-alkali land; the soil of the coastal saline-alkali land is soil with a high salinization level and a low organic matter content; the coastal saline-alkali land is the coastal saline-alkali land in Jiangsu Province.

[0032] In the specific implementation process of the present invention, the treatment of saline-alkali land includes one or more of the following 1) to 4): 1) improving the enzyme activity level of BG in the treatment system; 2) promoting and activating the carbon metabolism level in the treatment system; 3) improving the organic matter conversion level in the treatment system; 4) promoting crop growth.

[0033] The present invention also provides the above scheme, wherein the crop comprises corn.

[0034] In order to further illustrate the present invention, a saline-alkali land microbial combination, a composite bacterial agent and applications thereof provided by the present invention are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 Isolation of potential functional strains from saline-alkali soil in Jiangsu coastal area using conditional screening (1) Sample collection: In 2024, soil samples will be collected along different latitudinal gradients in the coastal saline-alkali land of Jiangsu Province (Lianyungang-Yancheng-Nantong).

[0036] (2) Sample purification: Accurately weigh 10 g of saline-alkali soil, add 90 mL of deionized water and shake at 150 rpm for 3 h. Then let it stand for 1 h, take the supernatant and dilute it in a gradient manner and spread it on a Gibbson modified medium plate with a salt concentration of 10% and pH 9.0, and culture it at 37°C for 5 d.

[0037] (3) Strain isolation and purification: Single colonies were picked from the culture medium and streaked onto new culture medium. The bacteria were repeatedly isolated and purified by the plate streak method. Fifteen culturable strains were initially screened out and the purified strains were inoculated onto slant culture medium and cultured at 37 °C for 4 days. They were then stored in a 4 °C refrigerator for later use.

[0038] (4) Strain screening: The salinity and alkalinity of Gibbons modified medium were adjusted to achieve the purpose of screening salt-alkali tolerant strains. Three groups of experiments were set up. In the first group of experiments, NaCl was added to the medium by exogenous means to adjust the salinity of the medium while ensuring the pH of the medium was 9.0. The NaCl concentrations in each medium were 100, 120, 140, 160, 180 and 200 g / L, respectively. In the second group of experiments, 1 mol / L NaOH solution was added to the medium by exogenous means to adjust the alkalinity of the medium while ensuring the NaCl concentration of the medium was 100 g / L. The pH values ​​of the medium were adjusted to 9.0, 10.0, 11.0 and 12.0, respectively. The third group of experiments was designed to simultaneously increase the alkalinity and salinity of the medium to 9.0 and 100 g / L, 11.0 and 140 g / L, and 12.0 and 200 g / L, respectively. Finally, the isolated and purified strains were inoculated on culture media with different salinity and alkalinity, and the colony growth was observed after static culture at 37°C for 4 days to screen out strains with stronger salt-alkali tolerance.

[0039] Under the premise that the pH value of Gibbson's modified medium was 9.0, strains 8, 9, and 13 had the lowest salt tolerance and could tolerate medium with a salt content of up to 100 g / L; strains 1, 5, 6, 10, 11, and 15 had the best salt tolerance and could tolerate medium with a salt content of more than 200 g / L.

[0040] Table 1 Salt tolerance test of strains

[0041] Note: +: strain grew, −: strain did not grow.

[0042] When the NaCl concentration of Gibbson's modified medium was maintained at 100 g / L, strains 3, 8, 9, and 13 had the worst alkali resistance and could only grow in an alkaline environment of pH 9.0 at most; strains 1, 5, 11, and 15 had the best alkali resistance and could grow in an alkaline environment above pH 12.0 at most.

[0043] Table 2 Alkali resistance test of strains

[0044] Note: +: strain grew, −: strain did not grow.

[0045] All 21 strains could grow at 100 g / L NaCl and pH 9.0. By continuously increasing the salinity and alkalinity of the culture medium, strains 1, 5, and 15 could grow well at 200 g / L NaCl and pH 12.0, showing strong salt-alkali tolerance.

[0046] Table 3 Detection of salt and alkali tolerance of strains

[0047] (5) Classification and identification of strains: The strains obtained in (4) were compared with the signature species detected in soil samples and bioinformatics analysis, and were identified using 16S rDNA, i.e., PCR amplification and sequencing were performed using the prokaryotic 16S rDNA universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′, SEQ ID NO. 1) and 1492R (5′-GGYTACCTTGTTACGACTT-3′, SEQ ID NO. 2), and then compared with the international NCBI GenBank (www.ncbi.nlm.nih.gov) nucleotide database, with a nucleotide homology of 99%, see Figure 1 and Figure 2 ; The strain numbered NO1 was identified as Microbacterium ( Microbacterium sp .), the strain numbered NO15 is Zombieella ( Zobellellasp .); The identified Microbacterium NO1 and C. chuberella NO15 were deposited in the China General Microbiological Culture Collection Center, the deposit number of Microbacterium NO1 is CGMCC No.32676, and the deposit number of C. chuberella NO15 is CGMCC No.32677.

[0048] Example 2 Detection and screening of organic matter conversion ability of functional strains The monoclonal strains of Microbacterium NO1 and Chabelella NO15 obtained by screening in Example 1 were selected and inoculated in the basal medium (BM): 0.47 g / L ammonium sulfate, 5.49 g / L sodium succinate, 0.2 g / L magnesium sulfate, 0.5 g / L disodium hydrogen phosphate, 0.1 g / L calcium chloride, 0.5 g / L potassium hydrogen phosphate, pH value 7.2. The organic matter conversion ability of the strain was initially screened: 1% to 3% glucose and acetic acid were added to the culture medium as the starting carbon source, and the organic matter conversion ability of the strain was characterized by β-glucosidase BG, cellulase CBH, and soil microbial carbon utilization rate CUE indicators.

[0049] The strains identified in Example 1 were combined into bacterial communities based on the principles of gene level differences, evolutionary relationships, and metabolic complementarity. After subculture of the seed solution stored at 4 °C for 24 h, it was inoculated in BM culture medium in equal proportions (effective viable count 1:1), and glucose and acetic acid were used as the only carbon sources in the BM culture medium to preliminarily detect the potential organic matter conversion ability of the bacterial community, which was characterized by the BG enzyme activity level in the system. Afterwards, samples were taken to detect the enzyme activity and organic matter conversion ability levels in the system, and the organic matter conversion ability of the microbial combination was compared with that of a single bacterium.

[0050] Results Figure 3 The results showed that when they existed alone, there was a low level of BG enzyme activity accumulation in the system. When the bacterial communities were combined, the microbial combinations S "NO1 + NO15" and S "NO5 + NO15" could significantly increase the enzyme activity level of BG in the system, and the promotion efficiency of S "NO1 + NO15" was more significant. The above results preliminarily revealed the interaction mechanism of microbial combinations in the process of organic matter transformation and their high-level transformation effect on organic matter.

[0051] Example 3 Detection of the growth-promoting ability of microbial combinations on corn in saline-alkali soil In-situ soil was collected from the experimental area of ​​Yandong Town, Tinghu District, Yancheng City, Jiangsu Province (120.36 °N, 33.47 °E), a typical coastal saline-alkali land, to set up a corn pot experiment. The microbial combination NO1+NO15 was mixed in equal proportions with an inoculation amount of 107 cfu and applied to the rhizosphere soil. Two experimental groups, moderate salinity (salt = 5 g / L) and severe salinity (salt = 8 g / L), were set up. Samples were taken continuously every 3 days, and the plant growth characteristics and soil physical and chemical properties were tested after sieving through a 2 mm sieve. The results are shown in Figure 4 and Figure 5 The results showed that under the condition of severe saline-alkali land, the root length of corn treated with the microbial combination increased by 52%, the root weight increased by 49%, the fresh weight increased by 44%, and the surface length increased by 39.5%. It can be seen that in the severe saline-alkali experimental group, the root length, root weight, surface length, fresh weight and other growth characteristics of corn potted plants were better than those in the moderate saline-alkali experimental group, which means that the microbial combination has a good growth-promoting ability for corn in saline-alkali land.

[0052] Metabolome analysis was performed using the screened microbial combination S "NO1+NO15", and the carbon metabolism-related pathways that were significantly upregulated compared with single bacteria were analyzed. Figure 6 The results show that the microbial combination can significantly promote and activate the carbon metabolism level in the system, thereby improving the level of organic matter conversion in the system. The efficient organic matter conversion ability of the microbial combination has been preliminarily confirmed at the molecular level.

[0053] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A microbial combination, characterized in that: Including Microbacterium ( Microbacterium sp .) NO1 and Chabelella ( Zobellellasp .)NO15; The deposit number of the Microbacterium NO1 is CGMCC No.32676, and the deposit number of the Chabelella NO15 is CGMCC No.32677.

2. The microbial combination according to claim 1, characterized in that The ratio of the effective live bacteria count of Microbacterium NO1 to that of Chapelaria NO15 is 1:

1.

3. A fermentation product of the microbial combination according to claim 1 or 2.

4. A composite bacterial agent, characterized in that: Comprising the microbial combination according to claim 1 or 2 or the fermentation product according to claim 3.

5. The composite bacterial agent according to claim 4, characterized in that: The OD of the microbial combination in the composite bacterial agent 600 Value ≥ 1.

6. The method for preparing the composite bacterial agent according to claim 4 or 5, characterized in that: The following steps are involved: Inoculating Microbacterium NO1 and Chabelella NO15 into liquid culture medium respectively, culturing, and obtaining Microbacterium NO1 bacterial liquid and Chabelella NO15 bacterial liquid respectively; The Microbacterium NO1 bacterial solution and the Chabelella NO15 bacterial solution are mixed to obtain a composite bacterial agent.

7. The preparation method according to claim 6, characterized in that: The liquid culture medium includes 1 / 10 LB liquid culture medium.

8. Use of the microbial combination according to claim 1 or 2, the fermentation product according to claim 3, the composite bacterial agent according to claim 4 or 5, or the composite bacterial agent prepared by the preparation method according to claim 6 or 7 in the treatment of saline-alkali land.

9. The use according to claim 8, characterized in that: The saline-alkali land treatment includes one or more of the following 1) to 4): 1) Improve the enzyme activity level of BG in the governance system; 2) Promote and activate carbon metabolism levels within the governance system; 3) Improve the level of organic matter transformation within the governance system; 4) Promote crop growth.

10. The use according to claim 9, characterized in that: The crops include corn.

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