Pseudomonas chlororaphis, complex microbial inoculant and application of pseudomonas chlororaphis in restoration of degraded grassland

Abstract

The invention provides pseudomonas chlororaphis, a complex microbial inoculant and application of the complex microbial inoculant in restoration of degraded grassland, and relates to the technical field of microorganisms. The pseudomonas chlororaphis provided by the invention is pseudomonas chlororaphis PS08, and the preservation number of the pseudomonas chlororaphis PS08 is CGMCC (China General Microbiological Culture Collection Center) No.33616. The strain has the capacity of dissolving potassium and phosphorus, also has the capacity of producing siderophores, auxin and ACC deaminase, can improve the seed germination rate, promote plant growth, improve plant biomass and promote plant root development, and is further used for repairing degraded grassland. The complex microbial inoculant prepared from the strain is used for repairing degraded grassland, the soil fertility is improved by constructing a soil structure, nutrient and microorganism multi-dimensional microhabitat and enhancing the density and vitality of underground propagules, and on the basis, alfalfa and wild indigenous grass seeds are reseeded, so that synergistic repairing of vegetation and soil is achieved, and the soil fertility is improved. The above-ground biomass and the pasture quality are improved, and the grassland soil fertility is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Pseudomonas chlororaphis, a composite bacterial agent and application thereof in the restoration of degraded grasslands. Background Art

[0002] Grassland ecosystems have ecological functions such as conserving water resources, maintaining water and soil, preventing wind and sand, maintaining biodiversity, and fixing carbon. They also provide a variety of grass and livestock products, and are an important guarantee for the transformation of modern agriculture and animal husbandry. However, due to unreasonable grazing systems, blind reclamation, excessive logging and other human factors, as well as natural factors brought about by climate change, natural grasslands have been seriously degraded, mainly manifested in reduced area, poor nutrients, increased soil erosion, deterioration of soil structure, low grassland productivity, loss of biodiversity, poor quality, and slow restoration. These problems have seriously restricted the functionality and stability of grassland ecosystems and reduced the production level of grassland livestock products.

[0003] Due to the differences in the ecological niches of forage grasses and the different time and space utilization of light, heat, water, soil and nutrients, there are technical problems with the emergence and establishment of forage seeds after reseeding. Therefore, it is necessary to select appropriate reseeding methods and management measures to ensure the success of reseeding and the establishment of reseeding communities.

[0004] Plant rhizosphere microorganisms play an important role in nutrient efficiency, promoting plant growth, forming symbiotic relationships, and ecological restoration. However, the effects of rhizosphere microorganisms selected from nature on forage and land vary. Therefore, how to select and utilize rhizosphere microorganisms to better apply them to degraded grassland restoration is a technical problem that needs to be solved urgently. Summary of the invention

[0005] The object of the present invention is to provide a strain of Pseudomonas chlororaphis, a composite bacterial agent and the use thereof in the restoration of degraded grassland. The strain has the ability to dissolve potassium and phosphorus, produce siderophores, plant growth hormones and ACC deaminase, and also has a plant growth-promoting effect. Based on the function of the strain, the present invention also provides the use of the strain.

[0006] The present invention provides a strain of Pseudomonas chlororaphis, wherein the Pseudomonas chlororaphis is Pseudomonas chlororaphis PS08, and the preservation number is CGMCC No. 33616.

[0007] The present invention also provides a bacterial agent, which contains the Pseudomonas chlororaphis PS08 or is prepared from the Pseudomonas chlororaphis PS08.

[0008] The present invention also provides a plant growth promoter, which contains the Pseudomonas chlororaphis PS08 or is prepared from the Pseudomonas chlororaphis PS08.

[0009] The present invention also provides a biological fertilizer, which contains the Pseudomonas chlororaphis PS08 or is prepared from the Pseudomonas chlororaphis PS08.

[0010] The present invention also provides a seed dressing agent, which contains the Pseudomonas chlororaphis PS08 or is prepared from the Pseudomonas chlororaphis PS08.

[0011] The present invention also provides the use of the Pseudomonas chlororaphis or the bacterial agent or the plant growth promoter or the biological fertilizer or the seed dressing agent in at least one of the following: S1. Siderophore production; S2. Produces auxin; S3. Produces ACC deaminase; S4. Increase soil phosphorus content; S5. Promote plant growth; S6. Increase plant biomass; S7. Promote plant root development; S8. Improve seed germination rate.

[0012] The present invention also provides a composite bacterial agent, which comprises Bacillus brevisflower PS06 and Pseudomonas chlororaphis PS08.

[0013] The present invention also provides the use of the Pseudomonas chlororaphis or the bacterial agent or the plant growth promoter or the biological fertilizer or the seed dressing agent or the composite bacterial agent in repairing degraded grassland.

[0014] The present invention also provides a method for restoring degraded grassland, comprising the following steps: (1) On the 30th to 45th day after the grass turns green, cut off the rhizomes of the grass to be repaired and remove the roots by walking horizontally underground; (2) spraying the composite bacterial agent 2 to 3 times 15 to 60 days after the original pasture turns green; (3) From late May to late June, grass seeds are re-seeded in areas with insufficient grass coverage, wherein the re-seeded grass seeds are yellow alfalfa seeds and wild native grass seeds; the composite microbial agent is used for seed dressing before the re-seeding of the grass seeds; (4) After the reseeded grass seeds have grown, sprinkler irrigation 2 to 3 times.

[0015] Preferably, in step (2), the amount of the composite bacterial agent sprayed each time is 5-10 kg / hm 2 ; In step (3), when mixing seeds, the grass seeds and the composite bacterial agent are mixed at a mass ratio of 15-25:1; the mass ratio of the yellow alfalfa and the wild native grass seeds is 1:(1-4); the sowing rate of the grass seeds is 22.5-33 kg / hm2 .

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a strain of Pseudomonas chlororaphis PS08, with a deposit number of CGMCC No. 33616; the Pseudomonas chlororaphis PS08 is isolated from rhizosphere soil of Leymus chinensis. The strain has the ability to dissolve potassium and phosphorus, produce siderophores, auxins and ACC deaminase, and also has a plant growth-promoting effect, can increase soil phosphorus content, increase seed germination rate, promote plant growth, increase plant biomass, and promote plant root development.

[0017] The present invention provides a composite bacterial agent, comprising Bacillus brevisflower PS06 and Pseudomonas chlororaphis PS08. The composite bacterial agent is used for soaking grass seeds, which can significantly increase the germination rate of grass seeds, increase the aboveground biomass and forage quality, and improve the fertility of grassland soil.

[0018] The present invention also provides a method for restoring degraded grasslands, which uses the above-mentioned composite bacterial agent to reconstruct the soil structure, nutrients and multi-dimensional microbial microhabitats, enhance the density and vitality of underground reproductive bodies, and thus improve soil fertility. On this basis, yellow lucerne and wild native grass seeds are reseeded, and the above-ground biomass and forage quality are increased by spraying and mixing grass seeds with the above-mentioned composite bacterial agent, combined with reasonable management measures, to improve grassland soil fertility, achieve coordinated restoration of vegetation (above ground) and soil (underground); achieve natural grassland ecological restoration and sustainable animal husbandry.

[0019] Biodeposit Information: The Pseudomonas chlororaphis PS08 of the present invention is named as PS08 and is classified as Pseudomonas chlororaphis ( Pseudomonas chlororaphis ), deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC), the deposit date is February 24, 2025, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.33616. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the growth status of Pseudomonas chlororaphis PS08 in Example 1 on the Montana organophosphate medium.

[0021] Figure 2 This is a diagram showing the effects of phosphate solubilization, siderophore production and potassium solubilization of Pseudomonas chlororaphis PS08 in Example 1.

[0022] Figure 3 This is the growth-promoting effect of the composite bacterial agent in Example 2 on alfalfa seedlings.

[0023] Figure 4This is a diagram of the no-tillage overseeding process during the restoration of degraded grassland in Example 3.

[0024] Figure 5 This is a comparison chart of the effects of using the composite bacterial agent of Example 3 of the present invention in Experimental Example 2 compared with the treatment with only the bacterial solution of Stipa breviflora PS06 in Comparative Example 3. DETAILED DESCRIPTION

[0025] The present invention provides a strain of Pseudomonas chlororaphis, wherein the Pseudomonas chlororaphis is Pseudomonas chlororaphis PS08, and the preservation number is CGMCC No. 33616.

[0026] In the present invention, the chlororaphis PS08 is isolated from rhizosphere soil of Leymus chinensis. The colony morphology of the chlororaphis PS08 is rod-shaped, oval, slightly yellow, opaque, rough on the surface, aerobic, and with irregular edges. In the present invention, the 16S rDNA sequence length of the chlororaphis PS08 is 1432 bp, and the specific 16S rDNA sequence is shown in SEQ ID NO: 1.

[0027] In the present invention, the Pseudomonas chlororaphis PS08 has the function of dissolving potassium and phosphorus, and also has the function of producing iron carriers, plant growth hormones and ACC deaminase, and also has a plant growth-promoting effect, which can increase the soil phosphorus content, increase the seed germination rate, promote plant growth, increase plant biomass, and promote the development of plant root systems.

[0028] The present invention also provides a bacterial agent containing the chlororaphis PS08 or prepared from the chlororaphis PS08. In the present invention, the culture medium for preparing the chlororaphis PS08 includes at least one of LB culture medium, NB culture medium, and Montana organophosphate culture medium. As an implementation method, the fermentation conditions for preparing the bacterial agent are: 30°C, 150-200rpm. Ensure that the effective viable count of the chlororaphis PS08 is greater than 1×10 8 cfu / mL is sufficient.

[0029] The present invention also provides a plant growth promoter, which contains the Pseudomonas chlororaphis PS08 or is prepared from the Pseudomonas chlororaphis PS08. In the present invention, the plant growth promoter can promote plant growth, increase plant biomass, and promote plant root development; the plant is preferably forage grass.

[0030] The present invention also provides a biological fertilizer, which contains the Pseudomonas chlororaphis PS08 or is prepared from the Pseudomonas chlororaphis PS08. The Pseudomonas chlororaphis PS08 of the present invention has the characteristics of dissolving potassium and phosphorus, can promote the decomposition and transformation of nutrients (nitrogen, phosphorus, potassium and iron) in the soil, and is conducive to promoting the absorption of nutrients by plants.

[0031] In the present invention, the plant growth promoter or the biofertilizer mentioned above may further contain auxiliary materials permitted in the field of microbial preparations or biofertilizers.

[0032] The present invention also provides a seed dressing agent, which contains the Pseudomonas chlororaphis PS08 or is prepared from the Pseudomonas chlororaphis PS08. In the present invention, the seeds are treated with the Pseudomonas chlororaphis PS08, which can significantly reduce the average germination time and the number of hard seeds, and significantly improve the seed germination rate and germination potential. The seeds are preferably forage seeds, and more preferably alfalfa seeds.

[0033] The present invention also provides the use of the Pseudomonas chlororaphis or the bacterial agent or the plant growth promoter or the biological fertilizer or the seed dressing agent in at least one of the following: S1. Siderophore production; S2. Produces auxin; S3. Produces ACC deaminase; S4. Increase soil phosphorus content; S5. Promote plant growth; S6. Increase plant biomass; S7. Promote plant root development; S8. Improve seed germination rate.

[0034] In the present invention, the plant that produces auxin is preferably indoleacetic acid. The plant is preferably forage grass, and more preferably alfalfa.

[0035] The present invention also provides a composite bacterial agent, which includes Bacillus brevifolia PS06 and Pseudomonas chlororaphis PS08. In the present invention, as an implementable method, the composite bacterial agent is prepared by mixing Bacillus brevifolia PS06 and Pseudomonas chlororaphis PS08. Preferably, the volume ratio of Bacillus brevifolia PS06 and Pseudomonas chlororaphis PS08 is (1~3):(1~3). The composite bacterial agent is further preferably a composite bacterial agent mixed with the bacterial solution of Bacillus brevifolia PS06 and the bacterial solution of Pseudomonas chlororaphis PS08 in a volume ratio of 1:1; more preferably, the effective viable count of Bacillus brevifolia PS06 is 1×10 8 ~4×10 8 cfu / mL, the effective viable count of Pseudomonas chlororaphis PS08 is 1×10 8 ~4×10 8cfu / mL. In the present invention, the composite bacterial agent has a better effect than single Bacillus brevisflower PS06 or Pseudomonas chlororaphis PS08, reduces the average germination time and the number of hard seeds, and significantly improves the seed germination rate and germination potential; can promote seed germination, has a significant growth-promoting effect, and can also promote the plant height, root length, stem weight and root weight of seed seedlings; increases the aboveground biomass of pasture and pasture quality, and improves pasture soil fertility. The seeds or seedlings are preferably pasture seeds or seedlings, and are further preferably alfalfa.

[0036] The present invention also provides the use of the Pseudomonas chlororaphis or the bacterial agent or the plant growth promoter or the biological fertilizer or the seed dressing agent or the composite bacterial agent in repairing degraded grassland.

[0037] The present invention also provides a method for restoring degraded grassland, comprising the following steps: (1) On the 30th to 45th day after the grass turns green, cut off the rhizomes of the grass to be repaired and remove the roots by walking horizontally underground; (2) spraying the composite bacterial agent 2 to 3 times on the 15th to 60th day after the original pasture turns green; (3) From late May to late June, grass seeds are re-seeded in areas with insufficient grass coverage, wherein the re-seeded grass seeds are yellow alfalfa seeds and wild native grass seeds; the composite microbial agent is used for seed dressing before the re-seeding of the grass seeds; (4) After the reseeded grass seeds have grown, sprinkler irrigation 2 to 3 times.

[0038] In step (1) of the above-mentioned restoration method of the present invention, the root stems of rhizomatous grass grass to be repaired are cut and horizontally moved underground to promote the vegetative reproduction of the original grass and realize the colonization and renewal of the original grass population; the root cutting depth is preferably 10-15 cm, more preferably 12 cm, and the interval width is preferably 15-25 cm, more preferably 20 cm. After the root cutting is completed, 4000-5000 kg / hm 2 Organic fertilizer, preferably 4500kg / hm 2 Organic fertilizer; the organic fertilizer is preferably crushed sheep manure; the particle size of the crushed sheep manure is preferably between 3 and 5 mm.

[0039] In step (2) of the above restoration method of the present invention, the composite bacterial agent is sprayed for the first time 15 to 20 days after the original pasture turns green, and the next spraying is performed after an interval of 20 days. The spraying amount of the composite bacterial agent each time is preferably 5 to 10 kg / hm 2 , more preferably 7.5kg / hm 2 .

[0040] In step (3) of the above-mentioned restoration method of the present invention, the mass ratio of the yellow lucerne and the wild native grass seeds is preferably 1: (1-4), and more preferably 1: 1; the wild native grass seeds are preferably seeds collected from the natural grassland plant community of Baiyinxile Pasture in Xilin Gol League, Inner Mongolia, mainly including one or more of Leymus chinensis, Stipa krylovii, Stipa grandis, Ice grass, Feather grass, Bromus inermis, and Elymus dactylis. The ratio of each grass species of the Poaceae family, Leymus chinensis, Stipa krylovii, Stipa grandis, Ice grass, Feather grass, Bromus inermis, and Elymus dactylis is not specifically limited, as long as the purpose of repairing the degraded grassland is achieved. Before reseeding the grass seeds, the reseeding grass seeds are mixed with the composite bacterial agent and dried for later use. When mixing the seeds, the reseeding grass seeds and the composite bacterial agent are mixed at a mass ratio of (15-25): 1, and more preferably 20: 1. When reseeding, use a no-tillage reseeder to sow grass seeds. The sowing row spacing is preferably 20-40 cm, and more preferably 30 cm. The sowing rate is 22.5-33 kg / hm2. 2 , more preferably 30kg / hm 2 ; The sowing depth is 2~3cm, and the ground should be harrowed 1~2 times after sowing.

[0041] In step (4) of the above-mentioned restoration method of the present invention, spray irrigation is performed once when the reseeded grass seeds grow to 3-5 cm, and then spray irrigation is performed again after an interval of 15-20 days; the number of spray irrigations is preferably 2 times; during spray irrigation, the irrigation volume is preferably 150-250m 3 / hm 2 , more preferably 200m 3 / hm 2 .

[0042] After the restoration method of the present invention is completed, the grassland after reseeding is fenced off and no further treatment is performed for 2 years.

[0043] The repair method provided by the present invention can increase the aboveground biomass by 171% after reseeding, increase the crude protein content of forage by 210%, increase the relative feeding value by 166%, increase the relative forage value by 130%, and increase the total digestible nutrients by 135% compared to the sample plot without reseeding. At the same time, compared to the sample plot without reseeding, the organic carbon in the grassland after reseeding increased by 41%, the total nitrogen and total phosphorus increased by 59.3% and 24.6% respectively, and the carbon, nitrogen and phosphorus of the microbial biomass increased by about 58%, 242.1% and 52.1% respectively. Therefore, the repair method provided by the present invention can be used to improve the aboveground biomass and forage quality, and improve the soil fertility of the grassland to be repaired. In addition, the root secretions of the native grass species reseeded will also affect the soil microbial flora, forming a symbiotic body with the beneficial microorganisms in the soil, which is conducive to the colonization of reseeded forage and the growth of other dominant forages in the grassland. The restoration method provided by the present invention can achieve the effect of increasing aboveground biomass and forage quality and improving soil fertility of the grassland to be restored in just two years. It can significantly improve the productivity and soil fertility of degraded grassland, realize the coordinated restoration of forage and soil, and provide a basis for the rapid restoration of degraded grassland.

[0044] The restoration method provided by the present invention can significantly improve the productivity and soil fertility of degraded grasslands, and can achieve the coordinated restoration of aboveground vegetation biomass and underground soil fertility. It is suitable for improving the aboveground biomass and forage quality of yellow lucerne and wild native grass species, and provides a basis for the rapid restoration of degraded grasslands. In the present invention, the degraded grassland is selected from the moderately degraded grassland of Baiyinxile Pasture in Xilin Gol League, Inner Mongolia. According to the "Grassland Resources Classification and Evaluation" (GB / T 21010-2017), degraded grasslands are divided into three levels: light, medium and heavy. The moderately degraded grassland must meet at least 3 core indicators (such as coverage, biomass, dominant species ratio, etc.) to reach a degradation threshold of 30%-50%. FAO (Food and Agriculture Organization of the United Nations): Moderate degradation is defined as "10 to 20 years of intervention required for ecosystem restoration."

[0045] The full name of the ACC deaminase described in the present invention is 1-aminocyclopropane-1-carboxylic acid (ACC for short) deaminase.

[0046] The Bacillus PS06 described in the present invention is deposited in the China General Microbiological Culture Collection Center (CGMCC) with a deposit number of CGMCC 1.17355. T , and published in the paper [Shi Jiajia. Identification of a new strain of Bacillus PS06 isolated from the rhizosphere soil of Stipa breviflora and its growth-promoting effect [D]. Hohhot: Inner Mongolia University, 2021.].

[0047] The rhizosphere soil of Leymus chinensis described in the present invention is taken from the moderately degraded grassland of Baiyinxile Pasture in Xilin Gol League, Inner Mongolia.

[0048] The experimental methods described in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials mentioned above can be obtained from commercial sources.

[0049] Example 1 Pseudomonas chlororaphis ( Pseudomonas chlororaphis )Isolation and identification of PS08 (1) Isolation and purification of Pseudomonas chlororaphis PS08 Take 1g of rhizosphere soil from Leymus chinensis and dilute it to 10 with 10mL of sterile water. -1 , 10 -2 , 10 -3 , 10 -4 The diluted suspensions were spread on the Montana organophosphate medium and placed in an incubator at 30°C for 48 hours. The single colonies that were clearly identifiable and grew evenly on the medium were selected and transferred to the Montana organophosphate medium for three passages. After that, the single colonies were picked and purified on the LB purification medium to obtain pure cultured bacteria, which were recorded as PS08.

[0050] (2) Physiological and biochemical characteristics of Pseudomonas chlororaphis PS08 The purified Pseudomonas PS08 strain was subjected to physiological and biochemical experimental research. The colony morphology of the individual strain under the microscope after primary division or subculture was rod-shaped, oval, slightly yellow, opaque, rough surface, aerobic, and irregular edges. Gram staining of the bacteria was negative under an optical microscope.

[0051] (3) Molecular identification of Pseudomonas chlororaphis PS08 strain

[0052] The sequence of SEQ ID NO: 1 was analyzed and homologously compared in GenBank. Through comparison, the similarity between PS08 strain and Pseudomonas chlororaphis was 99.51%. At the same time, a phylogenetic tree was prepared and analyzed. After morphological identification of the strain, it was determined that PS08 strain was Pseudomonas chlororaphis.

[0053] (4) Determination of the growth-promoting ability of Pseudomonas chlororaphis PS08 The purified Pseudomonas PS08 strain was tested for its IAA content, ACC deaminase, iron carrier capacity, and potassium and phosphorus solubilizing abilities.

[0054] Phosphate solubilization capacity determination The purified Pseudomonas PS08 strain was inoculated in the center of the Montkina organic phosphorus medium and the PKO inorganic phosphorus medium, and inverted and cultured at 28°C for 0-5 days. If a transparent circle is formed around the colony, it is judged to have the ability to solubilize phosphate. The diameter of the transparent zone (D) and the colony diameter (d) of the solubilized phosphate were measured, and the ratio of the transparent zone diameter to the colony diameter was calculated to determine the strength of the strain's ability to solubilize phosphate.

[0055] Potassium-dissolving capacity assay The purified Pseudomonas PS08 strain was inoculated in the center of the silicate medium and the potassium-releasing bacterial medium, and incubated at 28°C for 0-5 days. If transparent circles and yellow transparent circles were formed around the colonies, it was considered to have the ability to release potassium. The potassium-releasing ability of the strain was judged based on the ratio of the transparent circle diameter to the colony diameter.

[0056] Siderophore production capacity assay The purified Pseudomonas PS08 strain was inoculated in the center of the CAS solid detection medium, inverted and cultured at 28℃ for 2-4 days, and the orange-yellow transparent ring was observed around the bacterial lawn. If an orange-yellow transparent ring was produced around the colony, it was considered to have the ability to produce iron carriers. MKB liquid culture medium was prepared, and the purified Pseudomonas PS08 strain was inoculated into the sterilized and cooled MKB liquid culture medium at a 1% inoculation amount. After culturing at 28℃ for 2 days, ultracentrifuged at 10000rpm for 10 minutes, 100μL of supernatant was aspirated and added to 100μL of CAS detection solution, and the absorbance value A at 630nm was measured by an ELISA instrument after 60 minutes of reaction in the dark. Another 100μL CAS detection solution was taken and mixed with 100μL of the supernatant of the MKB liquid culture medium without inoculation, and the absorbance value (Ar) was measured by the same method after 60 minutes of reaction in the dark. The iron carrier expression amount = (Ar-A) / Ar×100%.

[0057] IAA production capacity determination Weigh 10 mg of indoleacetic acid (IAA), dissolve it with a small amount of ethanol, and then dilute it to 100 mL with deionized water to obtain a standard stock solution of indoleacetic acid (IAA) with a concentration of 100 mg / L. Prepare a series of standard solutions of indoleacetic acid (IAA) with standard concentrations of 0, 5, 10, 20, 40, 60, 80, and 100 mg / L, and store them in the dark. Take 1 mL of each concentration standard solution, add an equal volume of Salkowski reagent colorimetric solution (1 mL 0.5 M FeCl3 and 49 mL 35% HClO4 mixed), keep it away from light for 30 minutes, and use an enzyme reader to detect the absorbance at 530 nm. Use the standard concentration of indoleacetic acid (IAA) as the horizontal axis and the absorbance value as the vertical axis to draw the standard curve of indoleacetic acid (IAA). The purified Pseudomonas PS08 strain was inoculated at 1% into Kings liquid medium containing 0.2 g / L tryptophan and cultured at 28°C for 3 days. The indoleacetic acid (IAA) content of the strain was determined by the Salkowski colorimetric method based on the indoleacetic acid (IAA) concentration standard curve.

[0058] ACC deaminase production capacity assay Prepare SM liquid medium, add 1-aminocyclopropane-1-carboxylic acid ACC that has been filtered and sterilized to SM liquid medium to a concentration of 0.5 g / L to obtain SMA liquid medium. Add (NH4)2SO4 to SM medium to a concentration of 0.5 g / L to obtain SMN liquid medium. Inoculate the purified Pseudomonas PS08 strain into SMN liquid medium at a 1% inoculation rate and shake culture for 20 hours, centrifuge at 4°C, remove the supernatant with a pipette and collect the cells. Wash and centrifuge twice with SM culture medium, suspend the cells in SM culture medium, and inoculate them into SMA liquid medium at a 5% inoculation rate. After culturing at 28°C for 2 days, use the uninoculated SM culture medium as a zero control, and use a spectrophotometer to measure the absorbance value A at a wavelength of 600 nm.

[0059] The growth status of Pseudomonas chlororaphis PS08 on the Montkina organophosphate medium Figure 1 As shown in Figure 2, its effects on dissolving phosphorus, producing iron carriers and dissolving potassium are as follows: Figure 2 The specific test results are shown in Table 1.

[0060] Table 1 Results of the growth-promoting ability test of Pseudomonas chlororaphis PS08

[0061] From Table 1 and Figure 2It can be seen that the Pseudomonas chlororaphis PS08 strain has the ability to produce IAA (indoleacetic acid), ACC deaminase and iron carrier, and also has the characteristics of potassium and phosphorus solubilization, which can promote the decomposition and transformation of nutrients (nitrogen, phosphorus, potassium and iron) in the soil, which is beneficial to promote the absorption of nutrients by plants.

[0062] Example 2 Preparation of composite bacterial agent (1) Antagonism detection between Pseudomonas chlororaphis PS08 and Bacillus breviscapus PS06 The activated Pseudomonas chlororaphis PS08 strain and Stipa brevis PS06 (CGMCC 1.17355) obtained in Example 1 were T ) were streaked on LB solid culture medium plates and cultured at 30℃ for 1-2 days, and the growth of the two strains at the cross-streaked areas were observed.

[0063] If the two strains grow relatively weakly or not at the intersection, it means that there is antagonism between the two strains; if the bacteria at the intersection grow well, it means that there is no antagonism between the two strains and they can be used for mixed culture.

[0064] The results of antagonism test showed that no sterile zone was formed between the two strains of Pseudomonas chlororaphis PS08 and Bacillus brevisflower PS06, and they could grow in contact with each other, indicating that there was no inhibitory effect between them.

[0065] (2) Preparation of composite microbial agents for repairing degraded grasslands The activated Pseudomonas chlororaphis PS08 strain and Stipa brevis PS06 (CGMCC 1.17355) obtained in Example 1 were T ) were inoculated in LB liquid medium and cultured at 30°C to ensure that the bacterial concentration of Bacillus brevis PS06 and the effective viable count of Pseudomonas chlororaphis PS08 were both 1×10 8 cfu / mL or above.

[0066] The bacterial solution of the above-mentioned Bacillus brevisflower PS06 and the bacterial solution of Pseudomonas chlororaphis PS08 were mixed in equal volumes (volume ratio of 1:1) to obtain a composite bacterial agent.

[0067] Comparative Example 1 Referring to the preparation steps of (2) the composite bacterial agent for repairing degraded grassland in Example 2, only a single strain of Stipa breviflora PS06 (CGMCC 1.17355 T ) to prepare bacterial solution, ensuring that the effective viable count of Bacillus brevis PS06 is 1×10 8 cfu / mL or above.

[0068] Comparative Example 2 Referring to the preparation steps of (2) the composite bacterial agent for repairing degraded grassland in Example 2, only the single Pseudomonas chlororaphis PS08 screened in Example 1 was selected to prepare the bacterial solution, and the effective viable bacterial count of Pseudomonas chlororaphis PS08 was ensured to be 1×10 8 cfu / mL or above.

[0069] Test Example 1 Seed germination ability test Select 100 yellow alfalfa seeds of uniform size and full grains, soak them in sterile water for 5 minutes, then soak them in 1% sodium hypochlorite for 30 seconds, wash them again with sterile water for 3 times, add 5mL of cultured bacterial suspension and soak them for 6 hours. Place the treated seeds in a culture dish covered with 2 layers of filter paper (soaked in sterile water) for germination experiment, with sterile water treatment as the control, and set up 4 parallels in each group.

[0070] The yellow lucerne seeds were cultured at 20°C with 8 hours of light, 16 hours of darkness, and 70%-80% humidity. The number of germination seeds was counted from the first day until the tenth day.

[0071] The bacterial suspensions mentioned above are different bacterial suspensions prepared in Example 2 and Comparative Examples 1-2.

[0072] The effects of different bacterial suspensions of Example 2 and Comparative Examples 1-2 on the germination of alfalfa seeds and the growth of seedlings were statistically analyzed. The results are shown in Tables 2 and 3.

[0073] Table 2 Effects of different microbial agents on germination of alfalfa seeds

[0074] Table 3 Effects of different microbial agents on alfalfa seedlings

[0075] Note: The same lowercase letters after the data in the same column in the table represent P There was no significant difference at the <0.05 level. P The difference was significant at the <0.05 level.

[0076] The test results show that the composite bacterial agent prepared in Example 2 can significantly ( P <0.05) reduced the average germination time and number of hard seeds, significantly ( P <0.05) increased the seed germination rate and germination potential, indicating that the composite bacterial agent can effectively promote the germination of alfalfa seeds (Table 2).

[0077] In addition, the composite bacterial agent also has a significant growth-promoting effect, which can significantly ( P<0.05) increased the plant height, root length, stem weight and root weight of alfalfa seedlings (Table 3), which was better than the control example 1 using only a single Bacillus PS06 bacterial solution, the control example 2 using only a single Pseudomonas chlororaphis PS08 bacterial solution and the sterile water control. The use of only a single Pseudomonas chlororaphis PS08 bacterial solution or Bacillus PS06 bacterial solution was significantly higher than the sterile water control ( P <0.05) increased the germination rate, germination potential, plant height, root length, stem weight and root weight of yellow alfalfa seeds, and significantly ( P <0.05) to reduce the number of hard seeds.

[0078] In summary, the composite bacterial agent prepared in Example 2 can promote the germination of alfalfa seeds and the growth of alfalfa seedlings ( Figure 3 ), which can promote the growth of alfalfa seedlings and roots respectively.

[0079] Example 3 A method for restoring degraded grassland Sample site selection: The moderately degraded grassland of Baiyinxile Pasture in Xilin Gol League, Inner Mongolia, with a vegetation coverage of 45% and a biomass of 380kg / hm2 was selected. 2 , the proportion of dominant species is 23%.

[0080] Root cutting to promote tillering and colonization: On the 30th to 45th day after the grass turns green, use a grassland soil-breaking and root-cutting machine to cut off the underground horizontal rhizomes of the rhizomatous grass on the grassland to be repaired to promote the nutritional reproduction of the original grass and realize the colonization and renewal of the original grass population; the root cutting depth is 12cm and the width is 20cm.

[0081] Fertilization: 4500kg / hm2 after root removal 2 The crushed sheep manure has a particle size of 3-5 mm; the composite bacterial agent prepared in Example 2 is sprayed once 15-60 days after the original pasture turns green, and the next spraying is carried out at an interval of 20 days, for a total of 2 sprayings, with a spraying amount of 7.5 kg / hm2 each time 2 .

[0082] Pretreatment of reseeding grass seeds: Before reseeding, yellow alfalfa + wild native grass seeds were mixed evenly at a ratio of 1:1 to obtain mixed seeds, and the mixed seeds were mixed evenly with the composite bacterial agent prepared in Example 2 at a mass ratio (mixed seeds: composite bacterial agent) of 20:1, and dried for later use. Wild native grass seeds are seeds collected from the natural grassland plant community of Baiyinxile Pasture in Xilin Gol League, Inner Mongolia, mainly including Leymus chinensis, Stipa krylovii, Stipa grandis, Agropyron glabra, Feather grass, Bromus inermis, and Elymus dactylis.

[0083] No-tillage reseeding: From late May to late June, the areas with insufficient grass coverage were reseeded with the seeds obtained after the above pretreatment using a no-tillage reseeder. The row spacing was 30 cm and the sowing rate was 30 kg / hm2.2 The sowing depth is 2~3cm, harrow the land 1~2 times after sowing, and the no-tillage sowing process is shown in Figure 4 .

[0084] Irrigation: sprinkle once when the grass seeds grow to 3-5 cm, and sprinkle again after 15-20 days, a total of 2 sprinkles, irrigation volume 200 m 3 / hm 2 .

[0085] Closure and protection: During the closure and protection period, the grassland to be restored after reseeding will be enclosed with fences and no further treatment will be carried out. The closure and protection will last for 2 years.

[0086] Embodiment 4: The difference from Example 3 is that yellow clover and wild native grass seeds are mixed in a ratio of 1:2.

[0087] Embodiment 5: The difference from Example 3 is that yellow clover and wild native grass seeds are mixed in a ratio of 1:3.

[0088] Embodiment 6: The difference from Example 3 is that yellow clover and wild native grass seeds are mixed in a ratio of 1:4.

[0089] Comparative Example 3 The difference from Example 3 is that the composite bacterial agent is replaced with the bacterial solution of Bacillus brevisflower PS06 prepared in Comparative Example 1 alone.

[0090] Comparative Example 4 The difference from Example 3 is that the composite bacterial agent is replaced with the bacterial solution of Pseudomonas chlororaphis PS08 prepared in Comparative Example 2 alone.

[0091] Comparative Example 5 Without any treatment, the degraded grassland will be fenced off and managed for 2 years.

[0092] Comparative Example 6 Only the root cutting and fertilization treatments in Example 3 were performed, and the plants were sealed and managed for 2 years.

[0093] Test Example 2 The aboveground biomass and forage quality of degraded Leymus chinensis grassland were tested, and the results are as follows: Table 4 Effects of aboveground-underground synergistic restoration of degraded Leymus chinensis grassland on aboveground biomass and forage quality

[0094] Note: Different lowercase letters in the same column indicate significant differences among treatments ( P <0.05).

[0095] As shown in Table 4, each embodiment group can significantly ( P <0.05) increased the aboveground biomass and quality of pasture, indicating that the restoration method provided by the present invention can improve grassland productivity. According to the results of the embodiment group and the comparative example group, it was found that after the composite microbial agent of the present invention (Examples 3 to 6) was used to treat yellow alfalfa and wild native grass seeds and the composite microbial agent was sprayed to repair the degraded grassland, the aboveground biomass, crude protein, relative feeding value, relative forage value and total digestible nutrients of the experimental grassland forage were significantly improved compared with those not treated with the composite microbial agent (Comparative Examples 3 to 6). Compared with the sample plot without reseeding in Comparative Example 5, the aboveground biomass after reseeding can be increased by 171%, the crude protein content of forage can be increased by 210%, the relative feeding value can be increased by 166%, the relative forage value can be increased by 130%, and the total digestible nutrients can be increased by 135% (Table 4). At the same time, in Example 3, yellow alfalfa + wild native grass seeds were mixed in a ratio of 1:1 and combined with the composite microbial agent to repair the degraded grassland, which significantly increased the relative forage value.

[0096] Depend on Figure 5 It can be seen that the degraded grassland restoration by spraying the composite bacterial agent in Example 3 is more effective than the degraded grassland restoration by spraying only the bacterial solution of Stipa breviflora PS06, and significantly improves the aboveground biomass and forage quality.

[0097] Table 5 Effects of aboveground-underground synergistic restoration of degraded Leymus chinensis grassland on soil properties

[0098] Note: Different lowercase letters in the same column indicate significant differences among treatments ( P <0.05).

[0099] As shown in Table 5, the restoration method of Example 3 of the present invention was used, compared with the sample plot of Comparative Example 5 without reseeding, in the grassland after reseeding, the organic carbon increased by 41%, the total nitrogen and total phosphorus increased by 59.3% and 24.6% respectively, and the carbon, nitrogen and phosphorus of the microbial biomass increased by about 58%, 242.1% and 52.1% respectively. P <0.05) increased soil nutrient content, indicating that the restoration method provided by the present invention can improve grassland soil fertility. The above results show that the restoration method provided by the present invention can significantly improve the productivity and soil fertility of degraded grasslands, achieve coordinated restoration of vegetation and soil, and provide a basis for the rapid restoration of degraded grasslands.

[0100] In summary, the restoration method provided by the present invention can be used to increase aboveground biomass and forage quality, and improve soil fertility of grassland to be restored.

[0101] 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 strain of Pseudomonas chlororaphis, characterized in that: The Pseudomonas chlororaphis is Pseudomonas chlororaphis PS08, and its deposit number is CGMCC No. 33616.

2. A bacterial agent, characterized in that Contains the Pseudomonas chlororaphis PS08 according to claim 1 or is prepared from the Pseudomonas chlororaphis PS08 according to claim 1.

3. A plant growth promoter, characterized in that: Contains the Pseudomonas chlororaphis PS08 according to claim 1 or is prepared from the Pseudomonas chlororaphis PS08 according to claim 1.

4. A biological fertilizer, characterized in that: Contains the Pseudomonas chlororaphis PS08 according to claim 1 or is prepared from the Pseudomonas chlororaphis PS08 according to claim 1.

5. A seed dressing agent, characterized in that Contains the Pseudomonas chlororaphis PS08 according to claim 1 or is prepared from the Pseudomonas chlororaphis PS08 according to claim 1.

6. Use of the Pseudomonas chlororaphis according to claim 1, the bacterial agent according to claim 2, the plant growth promoter according to claim 3, the biological fertilizer according to claim 4, or the seed dressing agent according to claim 5 in at least one of the following, characterized in that: S1. Siderophore production; S2. Produces auxin; S3. Produces ACC deaminase; S4. Increase soil phosphorus content; S5. Promote plant growth; S6. Increase plant biomass; S7. Promote plant root development; S8. Improve seed germination rate.

7. A composite bacterial agent, characterized in that: The composite bacterial agent comprises Bacillus brevisflower PS06 and Pseudomonas chlororaphis PS08 described in claim 1.

8. Use of the Pseudomonas chlororaphis according to claim 1, the bacterial agent according to claim 2, the plant growth promoter according to claim 3, the biological fertilizer according to claim 4, the seed dressing according to claim 5, or the composite bacterial agent according to claim 7 in repairing degraded grassland.

9. A method for restoring degraded grassland, characterized in that: The steps include: (1) 30 to 45 days after the grass turns green, cut the rhizomes of the rhizomatous grass in the grassland to be repaired and remove the roots; (2) Spraying the composite bacterial agent of claim 7 2 to 3 times 15 to 60 days after the original pasture turns green; (3) From late May to late June, grass seeds are re-sown in areas with insufficient grass coverage, wherein the re-sown grass seeds are yellow clover seeds and wild native grass seeds; before the re-sown grass seeds are re-sown, the composite microbial agent described in claim 7 needs to be used for seed dressing; (4) After the reseeded grass seeds have grown, sprinkler irrigation 2 to 3 times.

10. The method according to claim 9, characterized in that: In step (2), the composite bacterial agent is sprayed at a rate of 5-10 kg / hm2 each time. 2 ; In step (3), when mixing seeds, the grass seeds and the composite bacterial agent are mixed at a mass ratio of 15-25:1; the mass ratio of the yellow alfalfa and the wild native grass seeds is 1:(1-4); the sowing rate of the grass seeds is 22.5-33 kg / hm 2 .

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