Microbial fertilizer for promoting growth of switchgrass and preparation method thereof

Abstract

The invention belongs to the field of microbial fertilizers, and particularly relates to a microbial fertilizer for promoting growth of switchgrass seedlings and a preparation method thereof. The microbial fertilizer is characterized in that the effective components of the microbial fertilizer are Klebsiella micheliae, Bacillus flexus, Microbacterium naniii, and Leifsonia xyli subsp.Cynodonis, the preservation numbers of the Klebsiella micheliae, the Bacillus flexus, the Microbacterium naniii, and the Leifsonia xyli subsp.Cynodonis are respectively DSM 25444, DSM 1316, DSM 23203, and DSM 46306, and the preservation numbers of the Klebsiella micheliae, the Bacillus flexus, the Microbacterium naniii, and the Leifsonia xyli subsp.Cynodonis are respectively DSM 25444, DSM 1316, DSM 23203, and DSM 46306. Under the combined action of Klebsiella micrantha, bacillus flexus, microbacterium nucianus and Rafissi, the microbial fertilizer disclosed by the invention can promote nutrient absorption of switchgrass seedlings, improve the growth of overground parts and also improve the drought resistance of the switchgrass seedlings. The method has great potential for improving the agricultural production efficiency and realizing the sustainable development of agriculture.

Description

Technical Field

[0001] The invention belongs to the field of microbial fertilizers, and particularly relates to a microbial fertilizer for promoting the growth of switchgrass seedlings and a preparation method thereof. Background Art

[0002] Plant Growth Promoting Rhizobacteria (PGPR) refers to the general term for bacteria and fungi that exist in the rhizosphere soil or inside the roots and can synthesize metabolites to promote plant growth and development, improve plant absorption of soil mineral elements, and increase the ability of plants to resist abiotic stress. Plant growth and stress resistance are closely related to the bacterial community in and around its roots.

[0003] Switchgrass (Panicum virgatum L.) is a perennial plant of the Poaceae family. It can be used as a biomass energy raw material, forage and soil and water conservation plant. It has the characteristics of strong drought resistance, high biomass and good soil and water conservation function. It is a good ecological restoration and high-quality energy forage material. Under drought stress, the scientific and rational use of PGPR to improve the drought tolerance of plants and alleviate the stress suffered by plants has great potential for improving agricultural production efficiency and achieving sustainable agricultural development. Summary of the invention

[0004] The purpose of the present invention is to provide a microbial fertilizer for promoting the growth of switchgrass seedlings and a preparation method thereof, in order to address the problem that the existing microbial preparations or biological fertilizers have a narrow range of adaptability, screen soil beneficial microorganisms suitable for the germination and growth environment of switchgrass seedlings, promote nutrient absorption of switchgrass seedlings, improve the growth of the aboveground part, and overcome the shortcomings of the prior art.

[0005] First, the present invention provides a microbial composition, comprising Klebsiella michiganensis, Priestia flexa, Microbacterium yannicii, and Leifsonia xyli subsp. Cynodontis. The deposit numbers of Klebsiella michiganensis, Priestia flexa, Microbacterium yannicii, and Leifsonia xyli subsp. Cynodontis are DSM 25444, DSM 1316, DSM 23203, and DSM 46306, respectively. Secondly, the present invention provides an application of the microbial composition in the preparation of microbial fertilizer.

[0006] Furthermore, the microbial fertilizer is used to promote the growth of switchgrass seedlings.

[0007] Furthermore, the microbial fertilizer is used to improve the drought resistance of switchgrass seedlings.

[0008] Furthermore, in the microbial fertilizer, the ratio of live bacteria of Klebsiella michiganensis, Bacillus flexus, Microbacterium janini and Revsonella levison is 1-4:1-3:1-3:1-3.

[0009] Furthermore, the microbial fertilizer is in the form of a liquid for irrigation.

[0010] Again, the present invention provides a microbial fertilizer for promoting the growth of switchgrass seedlings, wherein the microbial fertilizer contains the microbial composition as an effective ingredient.

[0011] Furthermore, in the microbial fertilizer, the ratio of live bacteria of Klebsiella michiganensis, Bacillus flexus, Microbacterium janini and Revsonella levison is 1-4:1-3:1-3:1-3.

[0012] Furthermore, the microbial fertilizer is in the form of a liquid for irrigation.

[0013] Again, the present invention provides a method for preparing the microbial fertilizer for promoting the growth of switchgrass seedlings, which is characterized by comprising the following steps:

[0014] (1) Cultivation: Klebsiella michiganensis, Bacillus flexus, Bacillus yanini, and Revsonella rapa were inoculated into LB medium, respectively, and cultured at 28-37°C for 24-36 hours;

[0015] (2) Mixing: Mix the cultured bacterial suspension.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention prepares microbial fertilizer by using Klebsiella michiganensis, Bacillus flexus, Bacillus janinis and Revsonella. Under the joint action of Klebsiella michiganensis, Bacillus flexus, Bacillus janinis and Revsonella, the microbial fertilizer can promote the nutrient absorption of switchgrass seedlings, improve the growth of the aboveground part, and improve the drought resistance of the switchgrass seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The phenotypes of switchgrass inoculated and uninoculated with growth-promoting bacteria under drought and watered conditions.

[0019] Figure 2 The biomass of switchgrass inoculated and not inoculated with growth-promoting bacteria under watered and drought conditions. Among them, A, B, C, and D are the fresh weight of the aboveground part, the fresh weight of the underground part, the fresh weight of the aboveground part, and the dry weight of the underground part under watered conditions, respectively; E, F, G, and H are the fresh weight of the aboveground part, the fresh weight of the underground part, the fresh weight of the aboveground part, and the dry weight of the underground part under drought conditions, respectively.

[0020] Figure 3These are the morphological indicators of the aboveground parts of switchgrass inoculated and not inoculated with growth-promoting bacteria under watered and drought conditions. Among them, A and C are the plant height and leaf width under watered conditions, respectively; B and D are the plant height and leaf width under drought conditions, respectively.

[0021] Figure 4 These are the morphological indicators of the underground parts of switchgrass inoculated and not inoculated with growth-promoting bacteria under watered and drought conditions. Among them, A, B, C, D, and E are the root length, surface area, average diameter, number of root tips, and root volume under watered conditions, respectively; and F, G, H, I, and J are the root length, surface area, average diameter, number of root tips, and root volume under drought conditions, respectively.

[0022] Figure 5 These are the photosynthetic characteristics of switchgrass inoculated with and without growth-promoting bacteria under watered and drought conditions, where A, C, E, and G are the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate under watered conditions, respectively; B, D, F, and H are the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate under drought conditions, respectively.

[0023] Figure 6 Figure 3 is the relative chlorophyll content (SPAD) and leaf in vitro water loss rate of switchgrass inoculated and uninoculated with growth-promoting bacteria under watered and drought conditions, where A is the relative chlorophyll content under watered conditions, B is the relative chlorophyll content under drought conditions, C is the leaf in vitro water loss rate under watered conditions, and D is the leaf in vitro water loss rate under drought conditions.

[0024] Figure 7 Phenotypes of switchgrass mixed with and without growth-promoting bacteria under drought and watered conditions.

[0025] Figure 8 It is the biomass of switchgrass mixed with inoculation and without inoculation of growth-promoting bacteria under watered and drought conditions, among which, A, B, C, D are the fresh weight of the aboveground part, the fresh weight of the underground part, the fresh weight of the aboveground part, and the dry weight of the underground part under watered conditions, respectively; E, F, G, H are the fresh weight of the aboveground part, the fresh weight of the underground part, the fresh weight of the aboveground part, and the dry weight of the underground part under drought conditions, respectively.

[0026] Fig. 9 These are the morphological indicators of the aboveground parts of switchgrass mixed with and without growth-promoting bacteria under watered and drought conditions. A and C are the plant height and leaf width under watered conditions, respectively; B and D are the plant height and leaf width under drought conditions, respectively.

[0027] Fig.10These are the morphological indicators of the underground parts of switchgrass mixed with and without growth-promoting bacteria under watered and drought conditions. Among them, A, B, C, D, and E are the root length, surface area, average diameter, number of root tips, and root volume under watered conditions, respectively; and F, G, H, I, and J are the root length, surface area, average diameter, number of root tips, and root volume under drought conditions, respectively.

[0028] Fig.11 These are the photosynthetic characteristics of switchgrass mixed with and without growth-promoting bacteria under watered and drought conditions, where A, C, E, and G are the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate under watered conditions, respectively; B, D, F, and H are the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate under drought conditions, respectively.

[0029] Fig.12 The relative chlorophyll content (SPAD) and leaf in vitro water loss rate of switchgrass mixed inoculated with and without growth-promoting bacteria under watered and drought conditions, where A is the relative chlorophyll content under watered conditions, B is the relative chlorophyll content under drought conditions, C is the leaf in vitro water loss rate under watered conditions, and D is the leaf in vitro water loss rate under drought conditions. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0031] Klebsiella michiganensis, Priestia flexa, Microbacterium yannicii and Leifsoniaxyli subsp. Cynodontis in the following examples were purchased from German Collection of Microorganisms and Cell Cultures (DSMZ), and the accession numbers of Klebsiella michiganensis, Priestia flexa, Microbacterium yannicii and Leifsoniaxyli subsp. Cynodontis are DSM 25444, DSM 1316, DSM 23203 and DSM 46306, respectively.

[0032] Example 1: Preparation of microbial fertilizer

[0033] The preparation of microbial fertilizer refers to the following steps:

[0034] (1) Cultivation: Under sterile conditions, Klebsiella michiganensis, Bacillus flexus, Microbacterium janini, and Revsonella rapa were inoculated into LB medium and cultured at 28°C for 24 h;

[0035] (2) Mixing: The cultured bacterial suspension is mixed in a certain proportion to prepare a liquid dosage form for irrigation.

[0036] In this example, microbial fertilizers with several viable bacteria contents (CFU / mL) shown in Table 1 were prepared.

[0037] Table 1 Microbial fertilizer ratio

[0038]

[0039]

[0040] Example 2: Study on the effect of microbial fertilizer on switchgrass seedlings

[0041] In this example, the microbial fertilizer prepared in Example 1 was used to study the growth-promoting effect of switchgrass seedlings.

[0042] 1. Test materials

[0043] Alamo variety switchgrass seed.

[0044] 2. Methods

[0045] Alamo seeds were sterilized with 100% sodium hypochlorite solution plus 0.1% Tween, placed in a shaking table, and the shaking table culture conditions were set to run for 2.5h at 200rpm / min. After being taken out from the shaking table, the seeds were washed 5 times with sterile water. Sterilized again with 100% sodium hypochlorite solution, shaken at 200rpm for 20min, and washed 5 times with sterile water. The cleaned seeds were dotted in a culture pot filled with sterilized vermiculite, 12 seeds per pot, sterilized water was added to completely soak the vermiculite, and the culture pot was placed in a closed transparent plastic square box, with six culture pots placed in each transparent square box. After most of the seeds germinated and the seedlings emerged, transplanting was carried out, and eight switchgrass seedlings were transplanted in each culture pot to ensure that the growth and quantity of the seedlings in each culture pot were the same. The switchgrass seedlings were placed in an artificial climate chamber for cultivation. After transplanting, the growth of the seedlings was observed, and Hoagland nutrient solution was applied regularly, 5mL / plant each time. When the seedlings grew to 4 cm and the growth was basically the same, 5 mL of bacterial suspension was inoculated at the roots every three days for a total of two inoculations. After completing the two inoculations, the seedlings in the treatment group were subjected to drought treatment, and the seedlings in the control group (CK) were watered every three days. The indicators were measured three weeks after the seedlings germinated.

[0046] 3. Results

[0047] (1) Single inoculation treatment

[0048] like Figure 1 and Figure 2 As shown in the data, the aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight of the inoculated group under watering and drought were higher than those of the uninoculated group, and the aboveground biomass inoculated with Priestia flexa had more significant phenotypic indicators under watering conditions than that of switchgrass seedlings inoculated with other growth-promoting bacteria.

[0049] like Figure 3 As shown in the figure, the leaf width and plant height of the inoculated group were higher than those of the uninoculated group under watering and drought conditions, and the leaf width and plant height of the inoculated Priestiaflexa had more significant phenotypic indicators under watering conditions than those of the switchgrass seedlings inoculated with other growth-promoting bacteria. The leaf width and plant height of the inoculated Leifsonia xyli subsp.Cynodontis had more significant phenotypic indicators under drought conditions than those of the switchgrass seedlings inoculated with other growth-promoting bacteria.

[0050] like Figure 4 As shown in the results, the root length, surface area, average diameter, number of root tips and root volume of the inoculated group under water and drought were higher than those of the uninoculated group, and the switchgrass seedlings inoculated with Leifsonia xyli subsp.Cynodontis had more significant phenotypic indicators under water conditions. The root length, surface area, number of root tips and root volume of the switchgrass seedlings inoculated with Priestia flexa had more significant phenotypic indicators under drought conditions.

[0051] like Figure 5 As shown in the figure, the net photosynthetic rate of the inoculated switchgrass was higher than that of the uninoculated group under both watering and drought conditions, while the intercellular CO2 concentration was lower than that of the uninoculated group. Under watering conditions, the stomatal conductance and transpiration rate of the uninoculated switchgrass were significantly higher than those of the inoculated group.

[0052] like Figure 6 As shown, the SPAD of the inoculated group under watering and drought was higher than that of the uninoculated group, while the in vitro water loss rate of leaves was lower than that of the uninoculated group.

[0053] (2) Mixed inoculation treatment

[0054] like Figure 7 and 8 As shown, the aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight of the mixed inoculated group under watering and drought were higher than those of the uninoculated group, and the aboveground biomass of the switchgrass seedlings inoculated with the mixed bacterial agent Z2 had more significant phenotypic indicators under watering and drought conditions compared with the switchgrass seedlings inoculated with other mixed growth-promoting bacteria.

[0055] like Fig. 9As shown in the figure, the leaf width and plant height of the mixed inoculated group under watering and drought were higher than those of the uninoculated group, and the plant height of the mixed inoculated Z2 bacteria had more significant phenotypic indicators under watering and drought conditions compared with the switchgrass seedlings inoculated with other mixed growth-promoting bacteria. The leaf width of the inoculated Z3 bacteria had more significant phenotypic indicators under watering conditions compared with the switchgrass seedlings inoculated with other mixed growth-promoting bacteria. The leaf width of the inoculated Z1 bacteria had more significant phenotypic indicators under drought conditions compared with the switchgrass seedlings inoculated with other mixed growth-promoting bacteria.

[0056] like Fig.10 As shown in the figure, the root length, surface area, average diameter, number of root tips and root volume of the mixed inoculated group under watering and drought were higher than those of the uninoculated group, and the seedlings of switchgrass inoculated with Z3 inoculant had more significant phenotypic indicators under watering conditions except for the average diameter. Under drought, the root length and surface area of ​​switchgrass seedlings inoculated with Z1 and Z4 inoculants had more significant phenotypic indicators, and the average diameter and volume of the roots of switchgrass seedlings inoculated with Z2 and Z3 inoculants had more significant phenotypic indicators.

[0057] like Fig.11 As shown, the net photosynthetic rate of the mixed inoculated group was higher than that of the uninoculated group under both watering and drought conditions; the stomatal conductance of the uninoculated switchgrass under watering conditions was significantly higher than that of the mixed inoculated group, and there was no significant difference under drought conditions; the intercellular CO2 concentration of the mixed inoculated group was lower than that of the uninoculated group under watering and drought conditions; the transpiration rate of the uninoculated switchgrass under watering conditions was significantly higher than that of the mixed inoculated group, and there was no significant difference under drought conditions.

[0058] like Fig.12 As shown, the SPAD of the mixed inoculated group under watering and drought was higher than that of the uninoculated group, while the in vitro water loss rate of leaves was lower than that of the uninoculated group.

[0059] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0060] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0061] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A microbial composition, characterized in that: The invention comprises Klebsiella michiganensis, Priestia flexa, Microbacterium yannicii and Leifsonia xyli subsp. Cynodontis. The deposit numbers of Klebsiella michiganensis, Priestia flexa, Microbacterium yannicii and Leifsonia xyli subsp. Cynodontis are DSM 25444, DSM 1316, DSM 23203 and DSM 46306 respectively.

2. Use of the microbial composition according to claim 1 in the preparation of microbial fertilizer.

3. The use according to claim 2, characterized in that: The microbial fertilizer is used for promoting the growth of switchgrass seedlings.

4. The use according to claim 2, characterized in that: The microbial fertilizer is used for improving the drought resistance of switchgrass seedlings.

5. The use according to claim 2, characterized in that: In the microbial fertilizer, the ratio of live bacteria of Klebsiella michiganensis, Bacillus flexus, Microbacterium janini and Revsonella levison is 1-4:1-3:1-3:1-3.

6. The use according to claim 5, characterized in that: The microbial fertilizer is in the form of a liquid for irrigation.

7. A microbial fertilizer for promoting the growth of switchgrass seedlings, characterized in that: The microbial fertilizer comprises the microbial composition according to claim 1 as an effective ingredient.

8. The microbial fertilizer for promoting the growth of switchgrass seedlings according to claim 7, characterized in that: In the microbial fertilizer, the ratio of live bacteria of Klebsiella michiganensis, Bacillus flexus, Microbacterium janini and Revsonella levison is 1-4:1-3:1-3:1-3.

9. The microbial fertilizer for promoting the growth of switchgrass seedlings according to claim 8, characterized in that: The microbial fertilizer is in the form of a liquid for irrigation.

10. The method for preparing the microbial fertilizer for promoting the growth of switchgrass seedlings according to claim 9, characterized in that: The following steps are involved: (1) Cultivation: Klebsiella michiganensis, Bacillus flexus, Bacillus yanini, and Revsonella rapa were inoculated into LB medium, respectively, and cultured at 28-37°C for 24-36 hours; (2) Mixing: Mix the cultured bacterial suspension.

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