A Pseudomonas chlororaphis, a composite bacterial agent and its application in the restoration of degraded grasslands

Abstract

The present invention provides a strain of Pseudomonas chlororaphis, a composite bacterial agent and its application in the restoration of degraded grasslands, relating to the technical field of microorganisms. The Pseudomonas chlororaphis provided by the present invention is Pseudomonas chlororaphis PS08, and the preservation number is CGMCC No. 33616. This strain has the ability to decompose potassium and dissolve phosphorus, and also has the ability to produce siderophores, auxin and ACC deaminase, which can improve the seed germination rate, promote plant growth, increase plant biomass and promote plant root development, and thus be used for the restoration of degraded grasslands. The present invention uses the composite bacterial agent prepared from this strain to repair degraded grasslands. By constructing a multi-dimensional microhabitat of soil structure, nutrients and microorganisms, the density and vitality of underground propagules are enhanced, thereby improving the soil fertility. On this basis, Medicago falcata and wild native grass seeds are reseeded to achieve the synergistic restoration of vegetation and soil, increase the aboveground biomass and forage quality, and improve the soil fertility of grasslands.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Pseudomonas chlororaphis, a composite bacterial agent and their applications in the restoration of degraded grasslands. Background Art

[0002] The grassland ecosystem has ecological functions such as water conservation, soil and water conservation, windbreak and sand fixation, maintenance of biodiversity, carbon sequestration, etc., and provides production functions of various grass and livestock products. It is an important guarantee for realizing the transformation of modern agriculture and animal husbandry.

[0003] Due to the differences in the ecological niches of forage grasses, with different utilization times and spaces of light, heat, water, soil, and nutrients, there are technical problems that it is difficult for the sown forage grass seeds to emerge and establish. Therefore, it is necessary to select appropriate sowing methods and management measures, etc., to ensure the success of sowing and the establishment of the sown community.

[0004] Plant rhizosphere microorganisms play important roles in the effective utilization of nutrient elements, promotion of plant growth, formation of symbiotic relationships, and ecological restoration. However, the effects of rhizosphere microorganisms screened in nature on forage grasses and land vary. Therefore, how to screen and utilize rhizosphere microorganisms better for the application in the restoration of degraded grasslands is a technical problem to be solved urgently. Summary of the Invention

[0005] The purpose of the present invention is to provide a Pseudomonas chlororaphis, a composite bacterial agent and their applications in the restoration of degraded grasslands. This strain has the ability to decompose potassium and dissolve phosphorus, produce siderophores, plant growth hormones and ACC deaminase, and also has the effect of promoting plant growth. Based on the functions of this strain, the present invention also provides the applications of this strain.

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

[0007] The present invention also provides a bacterial agent containing the Pseudomonas chlororaphis PS08 or prepared from the Pseudomonas chlororaphis PS08.

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

[0009] The present invention also provides a biological fertilizer containing the Pseudomonas chlororaphis PS08 or prepared from the Pseudomonas chlororaphis PS08.

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

[0011] The present invention also provides the application of the Pseudomonas chlororaphis, the bacterial agent, the plant growth promoter, the biological fertilizer, or the seed dressing agent in at least one of the following

[0012] S1. Producing siderophores;

[0013] S2. Producing auxin;

[0014] S3. Producing ACC deaminase;

[0015] S4. Increasing soil phosphorus content;

[0016] S5. Promoting plant growth;

[0017] S6. Increasing plant biomass;

[0018] S7. Promoting plant root development;

[0019] S8. Increasing seed germination rate.

[0020] The present invention also provides a compound bacterial agent, which includes Bacillus brevis PS06 and the Pseudomonas chlororaphis PS08.

[0021] The present invention also provides the application of the Pseudomonas chlororaphis, the bacterial agent, the plant growth promoter, the biological fertilizer, the seed dressing agent, or the compound bacterial agent in restoring degraded grasslands.

[0022] The present invention also provides a method for restoring degraded grasslands, which includes the following steps:

[0023] (1) On the 30th to 45th day after the forage grass turns green, cut off the underground horizontal rhizomes of the rhizomatous gramineous forage grass in the grassland to be restored for root cutting;

[0024] (2) Spray the compound bacterial agent 2 to 3 times 15 to 60 days after the original forage grass turns green;

[0025] (3) From late May to late June, reseed grass seeds in the areas with insufficient forage grass coverage. The reseeded grass seeds are Medicago falcata seeds and wild native grass seeds; the reseeded grass seeds need to be dressed with the compound bacterial agent before reseeding;

[0026] (4) After the reseeded grass seeds grow out, spray and irrigate 2 to 3 times.

[0027] Preferably, in step (2), the spraying amount of the compound bacterial agent each time is 5 to 10 kg / hm2; in step (3), when dressing seeds, the reseeded grass seeds and the compound bacterial agent are dressed at a mass ratio of 15 to 25:1; the mass ratio of the Medicago falcata and the wild native grass seeds is 1:(1 to 4); the seeding rate of the reseeded grass seeds is 22.5 to 33 kg / hm2.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Biodeposit Information:

[0033] The Pseudomonas chlororaphis PS08 described in the present invention has a strain name of PS08, is classified as Pseudomonas chlororaphis, and is deposited in the General Microbiology Center of the China Microbiological Culture Collection (CGMCC). The deposit time is: February 24, 2025, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33616. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a diagram showing the growth status of Pseudomonas chlororaphis PS08 in Example 1 on the Montana organophosphate medium.

[0035] Figure 2 This is a diagram showing the effects of phosphate solubilization, siderophore production and potassium solubilization of Pseudomonas chlororaphis PS08 in Example 1.

[0036] Figure 3 This is the growth-promoting effect of the composite bacterial agent in Example 2 on alfalfa seedlings.

[0037] Figure 4 It is a process diagram of no-till reseeding during the restoration of degraded grassland in Example 3.

[0038] Figure 5 It is a comparison diagram of the effects of using the compound bacterium agent of Example 3 of the present invention compared with only treating with the Bacillus breviflora PS06 bacterial solution in Comparative Example 3 in Test Example 2. Detailed implementation manners

[0039] The present invention provides a strain of Pseudomonas chlororaphis, and the Pseudomonas chlororaphis is Pseudomonas chlororaphis PS08, with the preservation number of CGMCC No. 33616.

[0040] In the present invention, the Pseudomonas chlororaphis PS08 is isolated from the rhizosphere soil of Leymus chinensis. The colony morphology of the Pseudomonas chlororaphis PS08 is rod-shaped, oval, slightly yellowish, opaque, rough on the surface, aerobic, and the edge is irregular. In the present invention, the length of the 16S rDNA sequence of the Pseudomonas chlororaphis PS08 is 1432 bp, and the specific 16S rDNA sequence is shown as SEQ ID NO: 1.

[0041] In the present invention, the Pseudomonas chlororaphis PS08 has the functions of potassium solubilization and phosphorus solubilization, and also has the functions of producing siderophores, plant growth hormones and ACC deaminase, and also has the function of promoting plant growth, which can increase the soil phosphorus content, increase the seed germination rate, promote plant growth, increase the plant biomass, and promote the development of plant roots.

[0042] The present invention also provides a bacterium agent containing the Pseudomonas chlororaphis PS08 or prepared from the Pseudomonas chlororaphis PS08. In the present invention, the culture medium for preparing the Pseudomonas chlororaphis PS08 includes at least one of LB medium, NB medium, and Meng Jinna organic phosphorus medium. As an implementable manner, the fermentation conditions for preparing the bacterium agent are: 30 °C, 150 - 200 rpm. It is only necessary to ensure that the effective viable count of the Pseudomonas chlororaphis PS08 is greater than 1×108 cfu / mL.

[0043] The present invention also provides a plant growth promoter containing the Pseudomonas chlororaphis PS08 or prepared from the Pseudomonas chlororaphis PS08. In the present invention, the plant growth promoter can promote plant growth, increase the plant biomass, and promote the development of plant roots; the plant is preferably forage grass.

[0044] The present invention also provides a biological fertilizer containing the Pseudomonas chlororaphis PS08 or prepared from the Pseudomonas chlororaphis PS08. The Pseudomonas chlororaphis PS08 of the present invention has the characteristics of potassium solubilization and phosphorus solubilization, and can promote the decomposition and transformation of nutrients (nitrogen, phosphorus, potassium and iron) in the soil, which is beneficial to promoting the absorption of nutrients by plants.

[0045] In the present invention, the above-mentioned plant growth promoter or the biological fertilizer may further contain a microbial preparation or an adjuvant permitted in the field of biological fertilizers.

[0046] The present invention also provides a seed dressing agent containing the Pseudomonas chlororaphis PS08 or prepared from the Pseudomonas chlororaphis PS08. In the present invention, treating seeds with the Pseudomonas chlororaphis PS08 can significantly reduce the average germination time and the number of hard seeds, and significantly increase the seed germination rate and germination potential. The seeds are preferably forage seeds, and more preferably Medicago falcata seeds.

[0047] The present invention also provides the application 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,

[0048] S1. Producing siderophores;

[0049] S2. Producing plant auxin;

[0050] S3. Producing ACC deaminase;

[0051] S4. Increasing soil phosphorus content;

[0052] S5. Promoting plant growth;

[0053] S6. Increasing plant biomass;

[0054] S7. Promoting plant root development;

[0055] S8. Increasing seed germination rate.

[0056] In the present invention, the production of plant auxin is preferably the production of indole-3-acetic acid. The plant is preferably forage, and more preferably Medicago falcata.

[0057] The present invention also provides a compound microbial agent, which comprises Bacillus breviglumis PS06 and Pseudomonas chlororaphis PS08. In the present invention, as an implementable embodiment, the compound microbial agent is prepared by mixing Bacillus breviglumis PS06 and Pseudomonas chlororaphis PS08. Preferably, the volume ratio of Bacillus breviglumis PS06 to Pseudomonas chlororaphis PS08 is (1-3):(1-3). The compound microbial agent is further preferably a compound microbial agent formed by mixing the bacterial liquids of Bacillus breviglumis PS06 and Pseudomonas chlororaphis PS08 at a volume ratio of 1:1; more preferably, the effective viable count of Bacillus breviglumis PS06 is 1×108-4×108 cfu / mL, and the effective viable count of Pseudomonas chlororaphis PS08 is 1×108-4×108 cfu / mL. In the present invention, the use of the compound microbial agent has better effects than single Bacillus breviglumis PS06 or Pseudomonas chlororaphis PS08, reduces the average germination time and the number of hard seeds, 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 seedling; improves the aboveground biomass and forage quality of pasture and improves the soil fertility of grassland. The seeds or seedlings are preferably forage seeds or seedlings, and further preferably Medicago falcata.

[0058] The present invention also provides the application of Pseudomonas chlororaphis or the microbial agent or the plant growth promoter or the biological fertilizer or the seed dressing agent or the compound microbial agent in the restoration of degraded grassland.

[0059] The present invention also provides a method for restoring degraded grassland, comprising the following steps:

[0060] (1) On the 30th to 45th day after the forage turns green, cut off the underground horizontal rhizomes of rhizomatous gramineous forage in the grassland to be restored for root cutting;

[0061] (2) Spray the compound microbial agent 2-3 times on the 15th to 60th day after the original forage turns green;

[0062] (3) From late May to late June, reseed grass seeds in the areas with insufficient forage coverage. The reseeded grass seeds are Medicago falcata seeds and wild native grass seeds; the reseeded grass seeds need to be dressed with the compound microbial agent before reseeding;

[0063] (4) After the reseeded grass seeds grow out, irrigate by spraying 2-3 times.

[0064] In step (1) of the above-mentioned restoration method of the present invention, cutting the underground horizontal rhizomes of rhizomatous gramineous forage grasses in the grassland to be restored is to promote the vegetative propagation of the original forage grasses and achieve the colonization and renewal of the original forage grass population; the preferred root cutting depth is 10 - 15 cm, more preferably 12 cm, and the interval width is preferably 15 - 25 cm, more preferably 20 cm. After root cutting, apply 4000 - 5000 kg / hm² of organic fertilizer, preferably 4500 kg / hm² of organic fertilizer; the organic fertilizer is preferably pulverized sheep manure; the particle size of the pulverized sheep manure is preferably between 3 - 5 mm.

[0065] In step (2) of the above-mentioned restoration method of the present invention, the first spraying of the compound microbial agent is carried out on the 15th - 20th day after the original forage grasses turn green, and the next spraying is carried out at an interval of 20 days. The preferred spraying amount of the compound microbial agent each time is 5 - 10 kg / hm², more preferably 7.5 kg / hm².

[0066] In step (3) of the above-mentioned restoration method of the present invention, the mass ratio of Medicago falcata and the seeds of wild native grasses is preferably 1:(1 - 4), more preferably 1:1; the wild native grass seeds are preferably seeds collected from the natural grassland plant community of Baiyinxile Ranch in Xilingol League, Inner Mongolia, mainly including one or more of Leymus chinensis, Stipa krylovii, Stipa grandis, Agropyron cristatum, Achnatherum sibiricum, Bromus inermis, Elymus dahuricus, and no specific limitation is made on the proportion of each gramineous forage grass variety of Leymus chinensis, Stipa krylovii, Stipa grandis, Agropyron cristatum, Achnatherum sibiricum, Bromus inermis, Elymus dahuricus, as long as the purpose of restoring the degraded grassland is achieved. Before overseeding the grass seeds, mix the overseeding grass seeds with the compound microbial agent and dry them for later use. When mixing the seeds, the overseeding grass seeds and the compound microbial agent are mixed at a mass ratio of (15 - 25):1, more preferably 20:1. When overseeding, use a no-till overseeder to overseed the grass seeds. The preferred seeding row spacing is 20 - 40 cm, more preferably 30 cm; the seeding rate is 22.5 - 33 kg / hm², more preferably 30 kg / hm²; the seeding depth is 2 - 3 cm, and harrow the land 1 - 2 times after seeding.

[0067] In step (4) of the above-mentioned restoration method of the present invention, irrigate once when the overseeded grass seeds grow to 3 - 5 cm, and then irrigate the next time at an interval of 15 days - 20 days; the preferred number of irrigation times is 2 times; when irrigating, the irrigation amount is preferably 150 - 250 m³ / hm², more preferably 200 m³ / hm².

[0068] After the above-mentioned restoration method of the present invention is completed, carry out enclosure management. Enclose the overseeded grassland with a fence and do not carry out any further treatment, and carry out enclosure management for 2 years.

[0069] The restoration method provided by the present invention, compared with the plots without reseeding, can increase the aboveground biomass by 171%, the crude protein content of forage by 210%, the relative feeding value by 166%, the relative forage value by 130%, and the total digestible nutrients by 135%. At the same time, compared with the plots without reseeding, in the grassland after reseeding, the organic carbon increases by 41%, the total nitrogen and total phosphorus increase by 59.3% and 24.6% respectively, and the carbon, nitrogen, and phosphorus of the microbial biomass increase by about 58%, 242.1%, and 52.1% respectively. Therefore, the restoration method provided by the present invention can be used to increase the aboveground biomass and the quality of forage, and improve the soil fertility of the grassland to be restored. In addition, the root exudates of the native grass species reseeded will also affect the soil microbial flora, forming symbionts with beneficial microorganisms in the soil, which is beneficial to the colonization of the reseeded forage and the growth of other dominant forage grasses in the grassland. The restoration method provided by the present invention can achieve the effects of increasing the aboveground biomass and the quality of forage, and improving the soil fertility of the grassland to be restored only in two years, can significantly improve the productivity and soil fertility of degraded grasslands, can realize the coordinated restoration of forage and soil, and provides a basis for the rapid restoration of degraded grasslands.

[0070] The restoration method provided by the present invention can significantly improve the productivity and soil fertility of degraded grasslands, can realize the coordinated restoration of aboveground vegetation biomass and underground soil fertility, is applicable to improving the aboveground biomass and the quality of forage of Medicago falcata 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 as the moderately degraded grassland in Baiyinxile Ranch, Xilingol League, Inner Mongolia. According to "Classification and Evaluation of Grassland Resources" (GB / T 21010-2017), the degraded grassland is divided into three levels: light, medium, and heavy. The moderately degraded grassland needs to meet at least 3 core indicators (such as coverage, biomass, proportion of dominant species, etc.) reaching the degradation threshold of 30% - 50%. FAO (Food and Agriculture Organization of the United Nations): defines moderate degradation as "the ecosystem requires 10 - 20 years of intervention for restoration".

[0071] The full name of the ACC deaminase described in the present invention is 1-aminocyclopropane-1-carboxylic acid (1-Aminocyclopropane-1-carboxylic acid, abbreviated as ACC) deaminase.

[0072] The Bacillus PS06 described in the present invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC 1.17355T, and is publicly disclosed in the thesis [Shi Jiajia. Identification of a new strain of Bacillus PS06 isolated from the rhizosphere soil of Stipa breviflora and its growth promotion effect [D]. Hohhot: Inner Mongolia University, 2021.].

[0073] The rhizosphere soil of Leymus chinensis described in the present invention is taken from the moderately degraded grassland in Baiyinxile Ranch, Xilingol League, Inner Mongolia.

[0074] In the following examples, the experimental methods are conventional methods unless otherwise specified;

[0075] The reagents and materials can be obtained from commercial sources unless otherwise specified.

[0076] Example 1 Isolation and Identification of Pseudomonas chlororaphis PS08

[0077] (1) Isolation and Purification of Pseudomonas chlororaphis PS08

[0078] Take 1 g of rhizosphere soil of Leymus chinensis, dilute it with 10 mL of sterile water to suspensions with dilution factors of 10-1, 10-2, 10-3, and 10-4, and spread them on Meng Jinna organic phosphorus medium, and incubate them upside down in a constant temperature incubator at 30 °C for 48 h. Select the single colonies that are clearly distinguishable and grow evenly on the medium, transfer them to Meng Jinna organic phosphorus medium for 3 subcultures, then pick the single colonies and purify them on LB purification medium to obtain pure cultured bacteria, denoted as PS08.

[0079] (2) Physiological and Biochemical Characteristics of Pseudomonas chlororaphis PS08

[0080] Conduct physiological and biochemical experimental studies on the purified Pseudomonas PS08 strain. The individual colonies of this strain are rod-shaped, oval, slightly yellowish, opaque, rough on the surface, aerobic, and irregular at the edges after primary separation or subculture and observed under a microscope. The bacteria are Gram-negative when observed under an optical microscope.

[0081] (3) Molecular Identification of Pseudomonas chlororaphis PS08 Strain

[0082]

[0083] The SEQ ID NO: 1 sequence was subjected to sequence analysis and homology comparison in GenBank. Through alignment, the similarity between strain PS08 and Pseudomonas chlororaphis was 99.51%. At the same time, by constructing a phylogenetic tree and analyzing it, and through morphological identification of the strain, strain PS08 was determined to be Pseudomonas chlororaphis.

[0084] (4)Determination of the growth-promoting ability of Pseudomonas chlororaphis PS08

[0085] Detect the IAA content, ACC deaminase, siderophore production ability, potassium-solubilizing ability, and phosphorus-solubilizing ability of the purified Pseudomonas PS08 strain.

[0086] Determination of phosphorus-solubilizing ability

[0087] The purified Pseudomonas PS08 strain was inoculated in the center of Meng Jina organic phosphorus medium and PKO inorganic phosphorus medium, and cultured upside down at 28°C for 0 - 5 days. If a transparent circle appears around the colony, it is determined to have phosphorus-solubilizing ability. Measure the diameter of the phosphorus-solubilizing transparent circle (D) and the diameter of the colony (d), and calculate the ratio of the diameter of the phosphorus-solubilizing transparent circle to the diameter of the colony to judge the strength of the strain's phosphorus-solubilizing ability.

[0088] Determination of potassium-solubilizing ability

[0089] The purified Pseudomonas PS08 strain was inoculated in the center of silicate medium and potassium-releasing bacteria medium, and cultured upside down at 28°C for 0 - 5 days. If a transparent circle and a yellow transparent circle appear around the colony, it is considered to have potassium-solubilizing ability. According to the ratio of the diameter of the transparent circle to the diameter of the colony, judge the strength of the strain's potassium-solubilizing ability.

[0090] Determination of siderophore production ability

[0091] The purified Pseudomonas PS08 strain was inoculated in the center of the CAS solid detection medium, and cultured upside down at 28°C for 2 - 4 days. Observe whether an orange-yellow transparent circle appears around the bacterial lawn. If an orange-yellow transparent circle appears around the colony, it is considered to have siderophore production ability. Prepare MKB liquid medium, inoculate the purified Pseudomonas PS08 strain into the sterilized and cooled MKB liquid medium at an inoculation amount of 1%, culture at 28°C for 2 days, then centrifuge at 10000 rpm for 10 min, pipette 100 μL of the supernatant and add 100 μL of CAS detection solution, and measure the absorbance value A at 630 nm with an enzyme-linked immunosorbent assay (ELISA) after reacting in the dark for 60 min. Another 100 μL of CAS detection solution and 100 μL of the supernatant of the MKB liquid medium without inoculated bacteria were thoroughly mixed and reacted in the dark for 60 min, and the absorbance value (Ar) was measured in the same way. The siderophore expression level = (Ar - A) / Ar × 100%.

[0092] Determination of IAA production ability

[0093] Weigh 10 mg of indole-3-acetic acid (IAA), dissolve it in a small amount of ethanol first, and then make up the volume to 100 mL with deionized water to obtain a standard stock solution of indole-3-acetic acid (IAA) with a concentration of 100 mg / L. Prepare a series of standard solutions of indole-3-acetic acid (IAA) with standard concentrations of 0, 5, 10, 20, 40, 60, 80, 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 (mix 1 mL of 0.5 M FeCl3 and 49 mL of 35% HClO4), perform dark treatment for 30 min, detect the absorbance value with an enzyme-linked immunosorbent assay (ELISA) reader at 530 nm, use the standard concentration of indole-3-acetic acid (IAA) as the abscissa and the absorbance value as the ordinate to draw the standard curve of indole-3-acetic acid (IAA). After inoculating the purified Pseudomonas sp. PS08 strain into the Kings liquid medium containing 0.2 g / L tryptophan at a ratio of 1% and culturing it with shaking at 28 °C for 3 d, combine the standard curve of indole-3-acetic acid (IAA) concentration and use the Salkowski colorimetric method to determine the indole-3-acetic acid (IAA) content produced by the strain.

[0094] Determination of the ability to produce ACC deaminase

[0095] Prepare SM liquid medium, add filter-sterilized 1-aminocyclopropane-1-carboxylic acid ACC to the SM liquid medium to make its concentration 0.5 g / L to obtain SMA liquid medium. Add (NH4)2SO4 to the SM medium to make its concentration 0.5 g / L to obtain SMN liquid medium. Inoculate the purified Pseudomonas sp. PS08 strain into the SMN liquid medium at an inoculation amount of 1% and culture it with shaking for 20 h, centrifuge at 4 °C, use a pipette to aspirate the supernatant and collect the bacterial cells. Wash the centrifuged cells twice with SM culture solution, suspend the bacterial cells in the SM culture solution, and inoculate them into the SMA liquid medium at an inoculation amount of 5%, after culturing at 28 °C for 2 d, use the non-inoculated SM culture solution as a zero control and measure the absorbance value A at a wavelength of 600 nm using a spectrophotometer.

[0096] The growth state of Pseudomonas chlororaphis PS08 on the Meng Jina organic phosphorus medium is as Figure 1 shown, and its phosphorus solubilization, siderophore production and potassium release effects are as Figure 2 shown, and the specific detection results are shown in Table 1.

[0097]

[0098] From Table 1 and Figure 2It can be seen that Pseudomonas chlororaphis strain PS08 has the ability to produce IAA (indoleacetic acid), ACC deaminase and siderophores, and also has the characteristics of potassium solubilization and phosphorus solubilization, which can promote the decomposition and transformation of nutrients (nitrogen, phosphorus, potassium and iron) in the soil and is beneficial to promoting the absorption of nutrients by plants.

[0099] Example 2 Preparation of Compound Bacterial Agent

[0100] (1)Antagonism Detection between Pseudomonas chlororaphis PS08 and Bacillus breviflora PS06

[0101] The activated Pseudomonas chlororaphis strain PS08 and Bacillus breviflora PS06 (CGMCC 1.17355T) screened in Example 1 were respectively streaked on LB solid medium plates and cultured at a constant temperature of 30 °C for 1-2 d, and the growth conditions of the two strains at the cross-streaking area were observed.

[0102] If the growth of the two strains at the intersection point is relatively weak or does not grow, it indicates that there is an antagonistic effect between the two strains; if the bacteria at the intersection point grow well, it indicates that there is no antagonistic effect between the two strains and they can be used for mixed culture.

[0103] According to the antagonism detection results: there is no sterile area formed between Pseudomonas chlororaphis strain PS08 and Bacillus breviflora PS06, and they can grow in contact with each other, indicating that there is no inhibitory effect between them.

[0104] (2)Preparation of Compound Bacterial Agent for Restoring Degraded Grassland

[0105] The activated Pseudomonas chlororaphis strain PS08 and Bacillus breviflora PS06 (CGMCC 1.17355T) screened in Example 1 were respectively inoculated into LB liquid medium and cultured at a constant temperature of 30 °C to ensure that the bacterial liquid concentration of Bacillus breviflora PS06 and the effective viable count of Pseudomonas chlororaphis PS08 were both above 1×108 cfu / mL.

[0106] The above-mentioned bacterial liquid of Bacillus breviflora PS06 and bacterial liquid of Pseudomonas chlororaphis PS08 were mixed in equal volume (volume ratio 1:1) to obtain a compound bacterial agent.

[0107] Comparative Example 1

[0108] Referring to the preparation steps of the compound bacterial agent for restoring degraded grassland in (2) of Example 2, only a single Bacillus breviflora PS06 (CGMCC 1.17355T) was selected to prepare the bacterial liquid, and the effective viable count of Bacillus breviflora PS06 was ensured to be above 1×108 cfu / mL.

[0109] Comparative Example 2

[0110] Referring to the preparation steps of the compound microbial agent for restoring degraded grassland in (2) of Example 2, only the bacterial liquid prepared from the Pseudomonas chlororaphis PS08 screened in Example 1 alone was selected, ensuring that the effective viable count of Pseudomonas chlororaphis PS08 was above 1×108 cfu / mL.

[0111] Test Example 1

[0112] Determination of seed germination ability

[0113] Select 100 Medicago falcata grass seeds with uniform size and plump grains. After soaking in sterile water for 5 min, soak and disinfect them with 1% sodium hypochlorite for 30 s, then wash them with sterile water 3 times again, and add 5 mL of the cultured bacterial suspension and soak for 6 h. Place the treated seeds in a petri dish lined with 2 layers of filter paper (moistened with sterile water) for germination experiments, using sterile water treatment as a control, and set 4 parallels in each group.

[0114] The Medicago falcata seeds were cultured under the conditions of 20°C, 8 h of light, 16 h of darkness, and humidity of 70%-80%. The number of germinated seeds was counted starting from the first day until the tenth day.

[0115] The above-mentioned bacterial suspensions were different bacterial suspensions prepared in Example 2 and Comparative Examples 1-2.

[0116] Statistically analyze the effects of the different bacterial suspensions of Example 2 and Comparative Examples 1-2 on the germination and seedling growth of Medicago falcata seeds. The results are shown in Tables 2 and 3.

[0117] Table 2 Effects of different microbial agents on the germination of Medicago falcata seeds

[0118]

[0119] Table 3 Effects of different microbial agents on Medicago falcata seedlings

[0120]

[0121] Note: For the data in the same column of the table, those marked with the same lowercase letter indicate no significant difference at the P<0.05 level, and those marked with different lowercase letters indicate a significant difference at the P<0.05 level.

[0122] The test results show that treating Medicago falcata seeds with the compound microbial agent prepared in Example 2 can significantly (P<0.05) reduce the average germination time and the number of hard seeds, and significantly (P<0.05) increase the seed germination rate and germination potential, indicating that this compound microbial agent can better promote the germination of alfalfa seeds (Table 2).

[0123] In addition, the compound microbial agent also has a significant growth-promoting effect, which can significantly (P<0.05) increase the plant height, root length, stem weight and root weight of alfalfa seedlings (Table 3), being superior to Comparative Example 1 using only the single Bacillus PS06 bacterial solution, Comparative Example 2 using only the single Pseudomonas chlororaphis PS08 bacterial solution, and the sterile water control. Using only the single Pseudomonas chlororaphis PS08 bacterial solution or Bacillus PS06 bacterial solution can significantly (P<0.05) increase the germination rate, germination potential, plant height, root length, stem weight and root weight of Medicago falcata seeds compared with the sterile water control, and at the same time significantly (P<0.05) reduce the number of hard seeds.

[0124] In summary, the compound microbial agent prepared in Example 2 can promote the germination of alfalfa seeds and also the growth of alfalfa seedlings ( Figure 3 ), and can promote the growth of alfalfa seedlings and roots respectively.

[0125] Example 3 A method for restoring degraded grassland

[0126] Plot selection: Select a moderately degraded grassland in Baiyinxile Ranch, Xilingol League, Inner Mongolia. The vegetation coverage is 45%, the biomass is 380 kg / hm2, and the proportion of dominant species is 23%.

[0127] Root-breaking to promote tillering and colonization: On the 30th to 45th day after the forage grass turns green, use a grassland soil-breaking root-cutting machine to cut the underground horizontal rhizomes of rhizomatous gramineous forage grasses on the grassland to be restored for rhizome-breaking treatment, promoting the vegetative propagation of the original forage grasses and realizing the colonization and renewal of the original forage grass population; the root-breaking depth is 12 cm and the width is 20 cm.

[0128] Fertilization: After the root-breaking is completed, apply 4500 kg / hm2 of crushed sheep manure. The particle size of the crushed sheep manure is between 3 and 5 mm; spray the compound microbial agent prepared in Example 2 once every 15 to 60 days after the original forage grass turns green, and conduct the next spraying after an interval of 20 days, with a total of 2 sprayings, and the spraying amount each time is 7.5 kg / hm2.

[0129] Pretreatment of overseeding grass seeds: Before overseeding, mix the Medicago falcata + wild native grass seeds evenly at a ratio of 1:1 to obtain the mixed seeds. The mixed seeds are evenly mixed with the compound microbial agent prepared in Example 2 at a mass ratio (mixed seeds: compound microbial agent) of 20:1, and then dried for later use. The wild native grass seeds are the seeds collected from the natural grassland plant community in Baiyinxile Ranch, Xilingol League, Inner Mongolia, mainly including Leymus chinensis, Stipa krylovii, Stipa grandis, Agropyron cristatum, Achnatherum sibiricum, Bromus inermis, Elymus dahuricus.

[0130] No-till overseeding: From late May to late June, use a no-till overseeding machine to overseed the seeds obtained after the above pretreatment in the areas with insufficient forage grass coverage. The seeding row spacing is 30 cm, the seeding rate is 30 kg / hm2, the seeding depth is 2 to 3 cm, and rake the land 1 to 2 times after seeding. The process of no-till overseeding is shown inFigure 4 。

[0131] Irrigation: When the reseeded grass seeds grow to 3 - 5 cm, sprinkle irrigation is carried out once, and the next irrigation is carried out at intervals of 15 to 20 days. A total of 2 sprinkler irrigations are carried out, and the irrigation amount is 200 m3 / hm2.

[0132] Enclosure management: During the enclosure management period, the grassland to be restored after reseeding is fenced off and no further treatment is carried out. The enclosure management lasts for 2 years.

[0133] Example 4:

[0134] The difference from Example 3 is that Medicago falcata and wild native grass seeds are mixed in a ratio of 1:2.

[0135] Example 5:

[0136] The difference from Example 3 is that Medicago falcata and wild native grass seeds are mixed in a ratio of 1:3.

[0137] Example 6:

[0138] The difference from Example 3 is that Medicago falcata and wild native grass seeds are mixed in a ratio of 1:4.

[0139] Control Example 3

[0140] The difference from Example 3 is that the compound bacterial agent is replaced with the Bacillus breviflora PS06 bacterial liquid prepared only in Control Example 1.

[0141] Control Example 4

[0142] The difference from Example 3 is that the compound bacterial agent is replaced with the bacterial liquid of Pseudomonas chlororaphis PS08 prepared only in Control Example 2.

[0143] Control Example 5

[0144] No treatment is carried out, and the degraded grassland is fenced off for 2 years of enclosure management.

[0145] Control Example 6

[0146] Only the root cutting and fertilization treatments in Example 3 are carried out, and the enclosure management lasts for 2 years.

[0147] Test Example 2

[0148] The aboveground biomass and forage quality of the degraded Leymus chinensis grassland were detected, and the results are shown in the following table:

[0149] Table 4 Effects of aboveground - underground collaborative restoration on aboveground biomass and forage quality of degraded Leymus chinensis grassland

[0150]

[0151] Note: Different lowercase letters in the same column indicate significant differences between treatments (P<0.05).

[0152] As can be seen from Table 4, compared with the control group, each example group can significantly (P<0.05) increase the aboveground biomass and quality of forage grass, indicating that the restoration method provided by the present invention can improve grassland productivity. According to the results of the example group and the control group, it was found that after treating Medicago falcata and wild native grass seeds with the compound microbial agent (Examples 3-6) of the present invention and spraying the compound microbial agent for degraded grassland restoration, the aboveground biomass, crude protein, relative feeding value, relative forage value and total digestible nutrients of the forage grass in the experimental grassland were significantly improved compared with those without using the compound microbial agent (Control Examples 3-6). Compared with the non-seeded plot of Control Example 5, the restoration method of Example 3 of the present invention can increase the aboveground biomass by 171%, the crude protein content of the forage grass by 210%, the relative feeding value by 166%, the relative forage value by 130%, and the total digestible nutrients by 135% after seeding (Table 4). At the same time, mixing Medicago falcata + wild native grass seeds in a ratio of 1:1 and combining with the compound microbial agent for degraded grassland restoration in Example 3 significantly increased the relative forage value.

[0153] From Figure 5 It can be seen that the effect of degraded grassland restoration by spraying the compound microbial agent in Example 3 is better than that of degraded grassland restoration by only spraying the bacterial liquid of Bacillus brevis PS06, significantly improving the aboveground biomass and forage quality.

[0154] Table 5 Effects of aboveground-underground collaborative restoration on soil properties in degraded Leymus chinensis grassland

[0155]

[0156] Note: Different lowercase letters in the same column indicate significant differences between treatments (P<0.05).

[0157] As can be seen from Table 5, compared with the non-seeded plot of Control Example 5, after seeding, the organic carbon in the grassland restored by the method of Example 3 of the present invention 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. Compared with the control group, each example group can significantly (P<0.05) increase the soil nutrient content, indicating that the restoration method provided by the present invention can improve the 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 grassland, realize the collaborative restoration of vegetation and soil, and provide a basis for the rapid restoration of degraded grassland.

[0158] In summary, the restoration method provided by the present invention can be used to increase the aboveground biomass and forage quality and improve the soil fertility of the grassland to be restored.

[0159] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composite microbial agent, characterized in that, The composite bacterial agent is prepared by mixing Bacillus breviflora PS06 and Pseudomonas chlororaphis PS08, and the preservation number of Pseudomonas chlororaphis is CGMCC No. 33616.

2. Use of the composite bacterial agent according to claim 1 in restoring degraded grasslands.

3. A method for restoring degraded grassland, characterized in that, It includes the following steps: (1) 30 to 45 days after the forage grass turns green, sever the underground horizontal rhizomes of the rhizomatous gramineous forage grass in the grassland to be restored for root cutting; (2) Spray the composite bacterial agent described in claim 1 two to three times 15 to 60 days after the original forage grass turns green; (3) From late May to late June, reseed grass seeds in areas with insufficient forage grass coverage. The reseeded grass seeds are Medicago falcata seeds and wild native grass seeds; the reseeded grass seeds need to be dressed with the composite bacterial agent described in claim 1 before reseeding; (4) After the reseeded grass seeds grow out, spray and irrigate two to three times.

4. The method according to claim 3, wherein In step (2), the spraying amount of the compound microbial agent each time is 5 - 10 kg / hm 2 ; in step (3), when dressing seeds, the overseeding grass seeds and the compound microbial agent are dressed in a mass ratio of 15 - 25:1; the mass ratio of the Medicago falcata and the wild native grass seeds is 1:(1 - 4); the seeding rate of the overseeding grass seeds is 22.5 - 33 kg / hm 2 .

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