A microbial combination for saline-alkali land, a compound microbial agent and its application

Abstract

The present invention provides a saline-alkali soil microbial combination, a compound microbial agent and their applications, belonging to the field of microbial technology. The microbial combination of the present invention includes Microbacterium ( Microbacterium sp .) NO1 and ZoBellia ( Zobellella sp .) NO15. The preservation number of Microbacterium NO1 is CGMCC No. 32676, and the preservation number of ZoBellia NO15 is CGMCC No. 32677; the microbial combination has strong saline-alkali tolerance, can efficiently utilize the organic matter in saline-alkali soil and has a growth-promoting effect on plants. The microbial combination of the present invention can be used for saline-alkali soil treatment, which is of great significance for improving soil quality, ensuring human health and improving the ecological environment.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a microbial combination for saline-alkali land, a compound microbial agent and their applications. Background Art

[0002] Soil salinization, as a worldwide ecological resource problem, is an important environmental disaster or risk caused by natural or human activities. At present, key technologies such as well irrigation and drainage, rice planting for salt washing, large water pressure for salt leaching, and drip irrigation under plastic film have been used to achieve large-scale utilization of lightly and moderately salinized and easily transformable saline-alkali land in the region. A large number of lightly and moderately saline-alkali lands have been successfully transformed. However, the salinization of some cultivated lands is prone to recurrence, and the difficult transformation of moderately and severely stubborn saline-alkali waste lands remains a long-standing problem left over from history.

[0003] Soil microorganisms can potentially adapt to saline-alkali environments and bring benefits to the improvement of saline-alkali soils by regulating organic matter cycling and promoting plant growth. At present, most of the microbial technologies for saline-alkali land improvement focus on the identification of microbial communities and changes in abundance, and the excavation of functional microorganisms generally stays at the study of the salt and alkali tolerance of single strains, with 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 microbial combination for saline-alkali land, a compound microbial agent and their applications. The microbial combination of the present invention has strong salt and 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, including Microbacterium sp. Microbacterium sp . NO1 and Zobellia sp. Zobellella sp . NO15; the preservation number of Microbacterium sp. NO1 is CGMCC No. 32676, and the preservation number of Zobellia sp. NO15 is CGMCC No. 32677.

[0006] Preferably, the ratio of the effective viable count of Microbacterium sp. NO1 to Zobellia sp. NO15 is 1:1.

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

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

[0009] Preferably, the OD 600 value of the microbial combination in the compound microbial agent is ≥1.

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

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

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

[0013] Preferably, the treatment of saline-alkali land includes one or more of the following 1) to 4):

[0014] 1) Improving the enzyme activity level of BG in the treatment system;

[0015] 2) Promoting and activating the carbon metabolism level in the treatment system;

[0016] 3) Improving the organic matter conversion level in the treatment system;

[0017] 4) Promoting crop growth.

[0018] Preferably, the crop includes corn.

[0019] The present invention provides a microbial combination, which includes Microbacterium ( Microbacterium sp.) NO1 and ZoBellia ( Zobellella sp.) NO15; the preservation number of Microbacterium NO1 is CGMCC No. 32676, and the preservation number of ZoBellia NO15 is CGMCC No. 32677. The microbial combination of the present invention has strong saline-alkali tolerance, can efficiently utilize the organic matter in saline-alkali land soil and has a growth-promoting effect on plants. Among them, for plants to which the microbial combination is applied under the conditions of heavy saline-alkali land, the root length can increase by 52%, the root weight can increase by 49%, the fresh weight can increase by 44%, and the surface length can increase by 39.5%. Therefore, the microbial combination of the present invention can be used for treating saline-alkali land, which is of great significance for improving soil quality, ensuring human health and improving the ecological environment. Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 It is the nucleotide sequence alignment diagram of Microbacterium sp. NO1;

[0022] Figure 2 It is the nucleotide sequence alignment diagram of ZoBellia sp. NO15;

[0023] Figure 3 It is the curve graph of the accumulation of BG enzyme activity level (carbon utilization efficiency) in the microbial consortium;

[0024] Figure 4 It is the actual picture of the potted plant;

[0025] Figure 5 It is the column graph of the phenotype of potted corn;

[0026] Figure 6 It is the metabolic schematic diagram of the carbon metabolism related pathways in the microbial consortium; among them, red indicates significant up-regulation (p-value < 0.05).

[0027] Biological deposit description

[0028] Microbacterium sp. ( Microbacterium sp .) NO1 was deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit number CGMCC No. 32676, the deposit date being November 18, 2024, the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the status being viable;

[0029] ZoBellia sp. ( Zobellella sp .) NO15 was deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit number CGMCC No. 32677, the deposit date being November 18, 2024, the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the status being viable. Detailed implementation manners

[0030] The present invention provides a microbial consortium (microbial consortium S "NO1 + NO15"), comprising Microbacterium sp. ( Microbacterium sp .) NO1 and ZoBellia sp. ( Zobellella sp .) NO15; the Microbacterium sp. NO1 was deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 32676, and the ZoBellia sp. NO15 was deposited in the China General Microbiological Culture Collection Center, with the deposit number CGMCC No. 32677.

[0031] Microbacterium sp. NO1 and ZoBellia sp. NO15 of the present invention were collected from the coastal saline-alkali area of Binhai, Jiangsu Province, and were screened and isolated by the method of screening under plate conditions; the Microbacterium sp. NO1 and ZoBellia sp. NO15 have strong saline-alkali tolerance, can efficiently utilize soil organic matter in soil with high salinization level and low organic matter content, and greatly promote the organic matter utilization ability in the combined state. Soil microorganisms can potentially adapt to the saline-alkali environment, regulate the organic matter cycle, and promote plant growth, bringing benefits to the improvement of saline-alkali land.

[0032] There is an interaction mechanism in the process of organic matter transformation by the microbial combination of the present invention and it has a high-level transformation effect on organic matter.

[0033] In the specific implementation process of the present invention, the ratio of the effective viable count of Microbacterium sp. NO1 to ZoBellia sp. NO15 is 1:1.

[0034] The present invention also provides a fermentation product of the microbial combination described in the above scheme.

[0035] The present invention also provides a composite microbial agent, comprising the microbial combination or the fermentation product described in the above scheme.

[0036] In the specific implementation process of the present invention, the OD 600 value of the microbial combination in the composite microbial agent ≥ 1.

[0037] The present invention also provides a preparation method of the composite microbial agent described in the above scheme, comprising the following steps:

[0038] Inoculate Microbacterium sp. NO1 and ZoBellia sp. NO15 into a liquid medium respectively and culture them to obtain Microbacterium sp. NO1 bacterial liquid and ZoBellia sp. NO15 bacterial liquid respectively; mix the Microbacterium sp. NO1 bacterial liquid and ZoBellia sp. NO15 bacterial liquid to obtain a composite microbial agent.

[0039] In the specific implementation process of the present invention, the liquid medium comprises 1 / 10 LB liquid medium; the volume ratio of the Microbacterium sp. NO1 bacterial liquid to the ZoBellia sp. NO15 bacterial liquid is 1:1.

[0040] In an embodiment of the present invention, the composite microbial agent is prepared by mixing the seed liquids of the single strains of Microbacterium sp. NO1 and ZoBellia sp. NO15 in equal proportions.

[0041] In the present invention, the Microbacterium sp. NO1 and ZoBellia sp. NO15 can be cultured separately or in combination.

[0042] The present invention also provides the application of the microbial combination, the fermentation product, the composite microbial agent or the composite microbial agent prepared by the preparation method described in the above scheme in the treatment of saline-alkali land.

[0043] 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.

[0044] 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.

[0045] The present invention also provides the above scheme, wherein the crop comprises corn.

[0046] 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.

[0047] Example 1 Isolation of potential functional strains from saline-alkali soil in Jiangsu coastal area using conditional screening

[0048] (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).

[0049] (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.

[0050] (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.

[0051] (4)Strain screening: Adjust the salinity and alkalinity of the Gibbons modified medium to screen for salt- and alkali-tolerant strains. Three groups of experiments were set up. In the first group of experiments, under the condition of ensuring that the pH of the medium was 9.0, NaCl was added externally to the medium to adjust the salinity of the medium, so that the NaCl concentrations in each medium were 100, 120, 140, 160, 180, and 200 g / L respectively. In the second group of experiments, under the condition of ensuring that the NaCl concentration of the medium was 100 g / L, 1 mol / L NaOH solution was added externally to the medium to adjust the alkalinity of the medium, and 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 increase the alkalinity and salinity of the medium simultaneously to 9.0 and 100 g / L, 11.0 and 140 g / L, 12.0 and 200 g / L respectively. Finally, the isolated and purified strains were inoculated on the media with different salinities and alkalinities, and the colony growth status was observed after static culture at 37 °C for 4 d to screen out the strains with stronger salt- and alkali-tolerant abilities.

[0052] On the premise that the pH value of the Gibbson modified medium was 9.0, the salt tolerance of strains 8, 9, and 13 was the lowest, and they could tolerate the medium with a maximum salt content of 100 g / L; the salt tolerance of strains 1, 5, 6, 10, 11, and 15 was the best, and they could tolerate the medium with a salt content of more than 200 g / L.

[0053] Table 1 Detection of Strain Salt Tolerance

[0054]

[0055] Note: +: Strain growth, −: Strain non-growth.

[0056] When the NaCl concentration of the Gibbson modified medium was maintained at 100 g / L, the alkali tolerance of strains 3, 8, 9, and 13 was the worst, and they could only grow in the alkaline environment with a pH of 9.0 at most; the alkali tolerance of strains 1, 5, 11, and 15 was the best, and they could grow in the alkaline environment with a pH of more than 12.0 at most.

[0057] Table 2 Detection of Strain Alkali Tolerance

[0058]

[0059] Note: +: Strain growth, −: Strain non-growth.

[0060] Under the conditions of an NaCl concentration of 100 g / L and a pH of 9.0, all 21 strains were able to grow. By continuously increasing the salinity and alkalinity of the culture medium, when the NaCl concentration was 200 g / L and the pH was 12.0, three strains, namely 1, 5, and 15, could grow well, showing strong salt and alkali tolerance.

[0061] Table 3 Detection of the salt and alkali tolerance of strains

[0062]

[0063] (5) Classification and identification of strains:

[0064] The strains obtained in (4) were compared with the landmark identification species in the detection and bioinformatics analysis of soil samples, and were identified using 16S rDNA. That is, the universal primers for prokaryotic 16S rDNA, 27F (5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGYTACCTTGTTACGACTT-3', SEQ ID NO.2), were used for PCR amplification and sequencing, and then compared with the nucleotide database of the international NCBI GenBank (www.ncbi.nlm.nih.gov). The nucleotide homology was 99%. See Figure 1 and Figure 2 ; the strain numbered NO1 was identified as Microbacterium ( Microbacterium sp .), and the strain numbered NO15 was identified as Zobellia ( Zobellella sp .).

[0065] The identified Microbacterium NO1 and Zobellia NO15 were respectively deposited in the China General Microbiological Culture Collection Center. The deposit number of Microbacterium NO1 is CGMCC No. 32676, and the deposit number of Zobellia NO15 is CGMCC No. 32677.

[0066] Example 2 Detection and screening of the organic matter conversion ability of functional strains

[0067] The monoclonal strains of Microbacterium NO1 and ZoBellia NO15 screened in Example 1 were picked and inoculated in a basal medium (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, with a pH value of 7.2. The primary screening of the organic matter conversion ability of the strains was carried out: 1% - 3% glucose and acetic acid were respectively added as the starting carbon sources in the medium, and the organic matter conversion ability of the strains was characterized by the β-glucosidase BG, cellulase CBH, and soil microbial carbon utilization efficiency CUE indicators.

[0068] The strains identified in Example 1 were combined into microbial communities based on the principles of genetic level differences, evolutionary relationships, and metabolic complementarity. After the seed liquid stored at 4 °C was subcultured for 24 h, it was inoculated into the BM medium in equal proportions (effective viable cell count 1:1), and glucose and acetic acid were used as the starting sole carbon sources in the BM medium respectively to preliminarily detect the potential organic matter conversion ability of the microbial communities, which was characterized by the BG enzyme activity level in the system. Then, 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 single bacteria.

[0069] The results are shown in Figure 3 , and the results show that when they exist alone, there is a low level of BG enzyme activity accumulation in the system. When the microbial communities are combined, both the microbial combinations S “NO1 + NO15” and S “NO5 + NO15” can significantly increase the BG enzyme activity level in the system, and the promotion efficiency of S “NO1 + NO15” is more significant. The above results preliminarily reveal the interaction mechanism and the high-level conversion effect on organic matter during the organic matter conversion process of the microbial combination.

[0070] Example 3 Detection of the growth promotion ability of microbial combinations on corn in saline-alkali soil

[0071] In-situ soil 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, was collected to set up a corn pot experiment. The microbial combination NO1 + NO15 was evenly mixed at an inoculation amount of 107 cfu and applied to the rhizosphere soil. Two experimental groups of moderate salinity (salt content = 5 g / L) and severe salinity (salt content = 8 g / L) were set up. Samples were continuously taken every 3 days, and after passing through a 2 mm sieve, the plant growth characteristics and soil physical and chemical property indicators were detected. The results are shown in Figure 4 and Figure 5, The results show that for corn with the microbial combination applied under the conditions of severely saline-alkali land, the root length can increase by 52%, the root weight can increase by 49%, the fresh weight can increase by 44%, and the surface length can increase by 39.5%. It can be seen that in the severely saline-alkali experimental group, the growth characteristics of corn potted plants such as root length, root weight, surface length, and fresh weight are all better than those in the moderately saline-alkali experimental group, which indicates that this microbial combination has good growth-promoting ability for corn in saline-alkali land.

[0072] Metabolome analysis was carried out using the screened microbial combination S “NO1 + NO15”, and the carbon metabolism-related pathways that were significantly up-regulated compared with single bacteria were analyzed. See the omics results in 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. It is preliminarily confirmed at the molecular level that the microbial combination has high-efficiency organic matter conversion ability.

[0073] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all belong to 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 composite bacterial agent, characterized in that: Comprising the microbial combination according to claim 1 or 2.

4. The composite bacterial agent according to claim 3, characterized in that: The OD of the microbial combination in the composite bacterial agent 600 Value ≥ 1.

5. The method for preparing the composite bacterial agent according to claim 3 or 4, 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.

6. The preparation method according to claim 5, characterized in that: The liquid culture medium includes 1 / 10 LB liquid culture medium.

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

8. The use according to claim 7, 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.

9. The use according to claim 8, characterized in that: The crops include corn.

Images