Microbial fertilizer for promoting growth of switchgrass and preparation method thereof

Abstract

The present application belongs to the field of microbial fertilizer, and particularly relates to a microbial fertilizer for promoting growth of switchgrass seedlings and a preparation method thereof. Effective components of the microbial fertilizer are Klebsiella michiganensis, Priestia flexa, Microbacterium yannicii and Leifsonia xyli subsp.Cynodontis, and the preservation numbers of the Klebsiella michiganensis, the Priestia flexa, the Microbacterium yannicii and the Leifsonia xyli subsp.Cynodontis are DSM 25444, DSM 1316, DSM 23203 and DSM 46306 respectively. Under the combined action of the Klebsiella michiganensis, the Priestia flexa, the Microbacterium yannicii and the Leifsonia xyli subsp.Cynodontis, the microbial fertilizer can promote nutrient absorption of the switchgrass seedlings, improve growth of the aboveground part, and improve drought resistance of the switchgrass seedlings. The present application has great potential for improving agricultural production efficiency and realizing sustainable development of agriculture.

Description

TECHNICAL FIELD

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

[0002] Plant Growth Promoting Rhizobacteria (PGPR) refers to bacteria, fungi and the like existing in rhizosphere soil or inside roots, capable of synthesizing 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 bacterial communities in and around roots.

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

[0004] The application aims to provide a microbial fertilizer for promoting growth of switchgrass seedlings and a preparation method thereof, screen soil beneficial microorganisms suitable for the germination and growth environment of switchgrass seedlings, promote the nutrient absorption of switchgrass seedlings, improve the growth of the aboveground part, and overcome the shortcomings of the prior art.

[0005] Firstly, the application provides a microbial composition, which comprises Klebsiella michiganensis, Priestia flexa, Microbacterium yannicii, Leifsonia xyli subsp. Cynodontis, Klebsiella michiganensis, Priestia flexa, Microbacterium yannicii and Leifsonia xyli subsp. Cynodontis, and the preservation numbers of the Klebsiella michiganensis, the Priestia flexa, the Microbacterium yannicii and the Leifsonia xyli subsp. Cynodontis are DSM 25444, DSM 1316, DSM 23203 and DSM 46306, respectively.

[0006] Further, the microbial fertilizer is used for promoting growth of switchgrass seedlings.

[0007] Further, the microbial fertilizer is used for improving 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 levesundii 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 levesundii 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, Microbacterium janini, and Revsonella rapa were inoculated into LB medium and cultured at 28-37°C for 24-36 h;

[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 a microbial fertilizer by using Klebsiella michiganensis, Bacillus flexus, Microbacterium janini and Revsonella. Under the combined action of Klebsiella michiganensis, Bacillus flexus, Microbacterium janini and Revsonella, the fertilizer can promote nutrient absorption of switchgrass seedlings, improve the growth of the aboveground part, and enhance the drought resistance of the switchgrass seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The phenotypes of switchgrass with and without growth-promoting bacteria under drought and watered conditions were analyzed.

[0019] Figure 2 is 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, fresh weight of the underground part, fresh weight of the aboveground part, and dry weight of the underground part under watered conditions, respectively; E, F, G, and H are the fresh weight of the aboveground part, fresh weight of the underground part, fresh weight of the aboveground part, and 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 uninoculated with growth-promoting bacteria under watered and drought conditions. 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; 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 and uninoculated with growth-promoting bacteria under watered and drought conditions. 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 are the relative chlorophyll content (SPAD) and leaf detachment 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 detachment water loss rate under watered conditions, and D is the leaf detachment water loss rate under drought conditions.

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

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

[0026] Figure 9 These are the morphological indicators of the aboveground parts of switchgrass with and without mixed inoculation of 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] Figure 10These are the morphological indicators of the underground parts of switchgrass with and without mixed inoculation of growth-promoting bacteria under watered and drought conditions. 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; 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] Figure 11 These are the photosynthetic characteristics of switchgrass with and without mixed inoculation of growth-promoting bacteria under watered and drought conditions. 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] Figure 12 The relative chlorophyll content (SPAD) and leaf detachment water loss rate of switchgrass with and without mixed inoculation of 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 detachment water loss rate under watered conditions, and D is the leaf detachment water loss rate under drought conditions. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they 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 the German Collection of Microorganisms and Cell Cultures (DSMZ). 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) Culture: Under sterile conditions, Klebsiella michiganensis, Bacillus flexus, Microbacterium yannini, and Rafsonella 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 seeds.

[0044] 2. Methods

[0045] Alamo seeds were sterilized with 100% sodium hypochlorite solution plus 0.1% Tween and placed in a shaking table with the shaking table culture condition set to 200 rpm / min for 2.5 h. After taking out from the shaking table, the seeds were washed 5 times with sterile water. Sterilized again with 100% sodium hypochlorite solution, the shaking table was shaken at 200 rpm for 20 min, and then washed 5 times with sterile water. The cleaned seeds were placed in a culture basin filled with sterilized vermiculite, with 12 seeds per basin, sterilized water was added to completely soak the vermiculite, and the culture basin was placed in a closed transparent plastic square box, with six culture basins 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 basin to ensure that the growth and quantity of the seedlings in each culture basin were the same. The switchgrass seedlings were placed in an artificial climate chamber and cultivated. After transplanting, the seedling growth was observed and Hoagland nutrient solution was regularly applied, 5 mL / strain at a time. Once the seedlings reached 4 cm and showed consistent growth, 5 mL of the bacterial suspension was inoculated into their roots twice every three days. After these two inoculations, the treated seedlings were subjected to drought stress, while the control (CK) seedlings were watered every three days. The parameters were measured three weeks after seedling germination.

[0046] 3. Results

[0047] (1) Single inoculation treatment

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

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

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

[0051] As shown in Figure 5 , the net photosynthetic rate of the inoculated groups was higher than that of the non-inoculated group, while the intercellular CO2 concentration was lower than that of the non-inoculated group. Under watering conditions, the stomatal conductance and transpiration rate of the non-inoculated group were significantly higher than those of the inoculated groups.

[0052] As shown in Figure 6 , the SPAD of the inoculated groups was higher than that of the non-inoculated group, while the leaf in vitro water loss rate was lower than that of the non-inoculated group.

[0053] (2) Mixed inoculation treatment

[0054] As shown in Figure 7 and 8 , the aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight of the mixed inoculated groups were higher than those of the non-inoculated group, and the aboveground biomass of the group inoculated with mixed inoculant Z2 had more significant phenotypic indicators than the groups inoculated with other mixed growth-promoting bacteria under watering and drought conditions.

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

[0056] like Figure 10 As shown, the root length, surface area, average diameter, number of root tips, and root volume of the mixed inoculated group under both watered and drought conditions were greater than those of the uninoculated group. Furthermore, the Z3 inoculated switchgrass seedlings had more significant phenotypic indicators under watered conditions, except for the average diameter. Under drought conditions, the root length and surface area of ​​switchgrass seedlings inoculated with the Z1 and Z4 inoculations showed more significant phenotypic indicators, while the average diameter and volume of the roots of switchgrass seedlings inoculated with the Z2 and Z3 inoculations showed more significant phenotypic indicators.

[0057] like Figure 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 was significantly higher than that of the mixed inoculated group under watering conditions, but 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 both watering and drought conditions; the transpiration rate of the uninoculated switchgrass was significantly higher than that of the mixed inoculated group under watering conditions, but there was no significant difference under drought conditions.

[0058] like Figure 12 As shown in the data, the SPAD of the mixed inoculated group was higher than that of the uninoculated group under both watering and drought conditions, 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 preferred embodiments.

[0060] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional 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 may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A microbial composition, characterized in that: The bacteria include 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 levesundii 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 contains the microbial composition according to claim 1 as an active 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 levesundii 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, Microbacterium janini, and Revsonella rapa were inoculated into LB medium and cultured at 28-37°C for 24-36 h; (2) Mixing: Mix the cultured bacterial suspension.

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