CN114097829B - Fungal metabolite dry powder for promoting plant growth, fungal metabolite active substance, preparation method and application thereof - Google Patents
Fungal metabolite dry powder for promoting plant growth, fungal metabolite active substance, preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of fertilizer preparation, and particularly relates to fungal metabolite dry powder for promoting plant growth, a fungal metabolite active substance, a preparation method and application thereof. The invention provides fungus metabolite dry powder for promoting plant growth, which can remarkably promote plant tillering, increase plant height, stem circumference and overground part fresh weight of crops and further achieve the effect of promoting plant growth by compounding the metabolite dry powder of penicillium citrinum, penicillium islandicum and penicillium erythraeum or the metabolite dry powder of the penicillium citrinum, penicillium islandicum, penicillium oxalicum and paecilomyces fumosoroseus. Meanwhile, the active ingredients of the fungus metabolite dry powder after being extracted by methanol, water, petroleum ether, ethyl acetate and n-butyl alcohol can further improve the growth promoting effect of the fungus metabolite, increase the crop yield and improve the economic benefit.
Description
Technical Field
The invention belongs to the technical field of fertilizer preparation, and particularly relates to fungal metabolite dry powder for promoting plant growth, a fungal metabolite active substance, a preparation method and application thereof.
Background
Tillering is an important component of the plant configuration of gramineous plants and is a key factor determining the density of the overground parts of the plants and the biological yield. The process of tillering generally involves two developmental stages. Firstly, axillary meristems in the axillary of the leaf or in the coronal primordium develop into initially dormant axillary buds; subsequently, axillary buds are activated to grow, producing new tillers. Tillering is closely related to the content of hormone in plants, cytokinin promotes tillering of gramineous plants, and auxin and strigolactone inhibit tillering. In addition, tillering has important agronomic significance for plant density, biological yield and adaptation to environmental stress.
The fertilizer is one of the important conditions for ensuring the agricultural harvest. However, in order to pursue high yield of crops, chemical fertilizers are used in large quantities, causing pollution to soil, water and atmosphere. When the chemical fertilizer is applied to soil, plants cannot absorb and utilize the chemical fertilizer completely, so that the chemical fertilizer remains in the soil to cause soil hardening; further, long-term use of chemical fertilizers reduces soil fertility and deteriorates soil physical properties. Chemical fertilizers which cannot be absorbed by plants are lost into a water area, so that the water area is eutrophicated, algae are propagated in a large quantity, aquatic animals are hindered from growing, and the aquatic animals are easily changed into foul water and dead water and develop to marsh; as most rural areas in China mainly take drinking underground water sources, if nitrogen in chemical fertilizers flows into underground water, water source pollution is caused, and the nitrate content in the water exceeds 45ppm, so that the human health is harmed; if the nitrite compound is formed by the action of microorganisms, there is a risk of carcinogenesis; meanwhile, the nitrogen oxide in the atmosphere is increased due to the pollution of the atmosphere.
The microbial fertilizer is a novel fertilizer, and improves the physical, chemical and biological properties of soil through the action of microbes such as bacteria, fungi and the like, so as to improve the soil environment. And various metabolites can be generated in the action of microorganisms, and the growth of the plants can be directly or indirectly promoted by the effects of promoting the division of plant cells, enhancing the capacity of absorbing water and nutrients, increasing the content of endogenous hormones in the plants and the like. However, the metabolites produced by different fungi are different, and the effect of combining different metabolites is not clear, and even the fertilizer efficiency of the microbial fertilizer is reduced due to the antagonism among different metabolites. Therefore, in order to solve the above technical problems and to better promote plant growth, it is necessary in the art to prepare a microbial fertilizer effective in promoting plant growth.
Disclosure of Invention
Based on the technical problems, the invention aims to provide fungal metabolite dry powder for promoting plant growth, a fungal metabolite active substance, a preparation method and application thereof. The fungus metabolite dry powder provided by the invention can obviously promote plant tillering and growth and increase crop yield.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a fungal metabolite dry powder for promoting plant growth, which comprises Penicillium citrinum metabolite dry powder, Penicillium islandicum metabolite dry powder and Penicillium rubrum metabolite dry powder
Or comprises a dried powder of a metabolite of Penicillium oxalicum, a dried powder of a metabolite of Penicillium citrinum, a dried powder of a metabolite of Penicillium islandicum and a dried powder of a metabolite of Paecilomyces fumosoroseus.
Preferably, the fungus metabolite dry powder comprises the following components in parts by mass: 10-30 parts of penicillium citrinum metabolite dry powder, 20-50 parts of penicillium islandicum metabolite dry powder and 10-40 parts of penicillium erythraeum product dry powder.
Preferably, the fungus metabolite dry powder comprises the following components in parts by mass: 20-50 parts of penicillium oxalicum metabolite dry powder, 10-40 parts of penicillium citrinum metabolite dry powder, 10-40 parts of penicillium islandicum metabolite dry powder and 10-40 parts of paecilomyces fumosoroseus metabolite dry powder.
The invention also provides a preparation method of the fungal metabolite dry powder for promoting plant growth, which comprises the following steps: and (3) mixing the Penicillium citrinum metabolite dry powder, the Penicillium islandicum metabolite dry powder and the Penicillium erythraeum metabolite dry powder, or mixing the Penicillium oxalicum metabolite dry powder, the Penicillium citrinum metabolite dry powder, the Penicillium islandicum metabolite dry powder and the Penicillium fumosoroseum metabolite dry powder to obtain the fungus metabolite dry powder.
Preferably, the preparation method of the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder, the penicillium erythraeum metabolite dry powder, the penicillium oxalicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder comprises the following steps: respectively inoculating activated bacteria of the penicillium citrinum, the penicillium islandicum, the penicillium erythraeum, the penicillium oxalicum and the paecilomyces fumosoroseus into a liquid culture medium, and performing oscillation culture, inactivation, drying and grinding to obtain dried powder of a penicillium citrinum metabolite, dried powder of a penicillium islandicum metabolite, dried powder of a penicillium oxalicum metabolite and dried powder of a paecilomyces fumosoroseus metabolite;
the oscillation rate of the oscillation culture is 120 r/min; the culture time is 3-7 days.
Preferably, when the penicillium citrinum metabolite dry powder is prepared, the liquid culture medium is a czochralski culture medium, the pH of the czochralski culture medium is 7.0-7.2, and the shaking culture temperature is 28 ℃;
when the penicillium islandicum metabolite dry powder is prepared, the liquid culture medium is a malt extract culture medium, and the shaking culture temperature is 25 ℃;
when the Penicillium erythraeum metabolite dry powder, the Penicillium oxalicum metabolite dry powder and the Paecilomyces fumosoroseus metabolite dry powder are prepared, the liquid culture medium is a PDA culture medium, and the shaking culture temperature is 25 ℃.
Preferably, the inactivation temperature is 140 ℃, and the inactivation time is 3 h.
The invention also provides an active ingredient of the fungal metabolite, which comprises the petroleum ether extract, the ethyl acetate extract, the n-butanol extract or the water-phase extract of the fungal metabolite dry powder in the technical scheme.
The invention also provides application of the active ingredients of the fungal metabolites prepared by the technical scheme in promoting plant growth.
Preferably, the promoting plant growth includes promoting tillering of a gramineous plant.
Preferably, the plant comprises one or more of wheat, rice, ryegrass, white clover, and tobacco.
Has the advantages that:
the invention provides a plant growth promoting fungus metabolite dry powder which comprises a Penicillium citrinum metabolite dry powder, a Penicillium islandicum metabolite dry powder and a Penicillium rubrum metabolite dry powder, or a Penicillium oxalicum metabolite dry powder, a Penicillium citrinum metabolite dry powder, a Penicillium islandicum metabolite dry powder and a Penicillium fumosoroseum metabolite dry powder.
According to the invention, the tillering of plants can be remarkably promoted by compounding the penicillium citrinum, the penicillium islandicum and the penicillium erythraeum or the metabolite dry powder of the penicillium citrinum, the penicillium islandicum, the penicillium oxalicum and the paecilomyces fumosoroseus, the plant height, the stem circumference and the fresh weight of the overground part of the plants are increased, and the effect of promoting the growth of the plants is further achieved.
Meanwhile, the active ingredients of the fungus metabolite dry powder after being extracted by methanol, water, petroleum ether, ethyl acetate and n-butyl alcohol can further improve the growth promoting effect of the fungus metabolite, increase the crop yield and improve the economic benefit.
And secondly, compared with chemical fertilizers and organic fertilizers, the microbial metabolite dry powder prepared by the invention is more green, safe and environment-friendly.
Drawings
In order to more clearly illustrate the technical solution in the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below.
FIG. 1 the procedure for extracting the active ingredients of fungal metabolites in example 4.
Detailed Description
The invention provides a fungal metabolite dry powder for promoting plant growth, which comprises a Penicillium citrinum metabolite dry powder, a Penicillium islandicum metabolite dry powder and a Penicillium rubrum metabolite dry powder or comprises a Penicillium oxalicum metabolite dry powder, a Penicillium citrinum metabolite dry powder, a Penicillium islandicum metabolite dry powder and a Penicillium fumosoroseum metabolite dry powder.
In the present invention, the fungal metabolite dry powder preferably includes penicillium citrinum metabolite dry powder, penicillium islandicum metabolite dry powder, and penicillium erythraeum metabolite dry powder; or preferably comprises Penicillium oxalicum metabolite dry powder, Penicillium citrinum metabolite dry powder, Penicillium islandicum metabolite dry powder and Paecilomyces fumosoroseus metabolite dry powder.
In the invention, when the fungus metabolite dry powder comprises penicillium citrinum metabolite dry powder, penicillium islandicum metabolite dry powder and penicillium erythraeum metabolite dry powder, the microbial metabolic microbial inoculum preferably comprises 10-30 parts by mass of the penicillium citrinum metabolite dry powder, more preferably comprises 13-25 parts by mass, and further preferably comprises 15 parts by mass.
The microbial metabolic microbial inoculum preferably comprises 20-50 parts of penicillium citrinum metabolite dry powder, more preferably 25-45 parts, even more preferably 30-42 parts, and most preferably 30 parts by mass of the penicillium citrinum metabolite dry powder.
The microbial metabolic microbial inoculum preferably comprises 10-40 parts of the penicillium citrinum product dry powder, more preferably 13-36 parts, even more preferably 20-30 parts, and most preferably 30 parts by mass of the penicillium citrinum metabolite dry powder.
In the invention, when the fungus metabolite dry powder comprises penicillium oxalicum metabolite dry powder, penicillium citrinum metabolite dry powder, penicillium islandicum metabolite dry powder and paecilomyces fumosoroseus metabolite dry powder, the microbial metabolic microbial inoculum preferably comprises 20-50 parts of penicillium oxalicum metabolite dry powder, more preferably 27-45 parts of penicillium oxalicum metabolite dry powder, even more preferably 33-40 parts of fungal metabolite dry powder, and most preferably 36 parts of fungal metabolite dry powder by mass.
The microbial metabolic microbial inoculum preferably comprises 10-40 parts of the penicillium citrinum metabolite dry powder, more preferably comprises 13-36 parts of the penicillium citrinum metabolite dry powder, even more preferably 17-28 parts of the penicillium citrinum metabolite dry powder, and most preferably 18 parts of the penicillium oxalicum metabolite dry powder.
The microbial metabolic microbial inoculum preferably comprises 10-40 parts of penicillium islandicum metabolite dry powder, more preferably 18-37 parts of penicillium oxalicum metabolite dry powder, and even more preferably 36 parts of penicillium oxalicum metabolite dry powder by mass.
The microbial metabolism microbial inoculum preferably comprises 10-40 parts of paecilomyces fumosoroseus metabolite dry powder, more preferably 13-36 parts, further preferably 17-30 parts, and most preferably 18 parts by mass of the paecilomyces oxalicum metabolite dry powder.
In the invention, the penicillium citrinum, the penicillium islandicum, the penicillium erythraeum, the penicillium oxalicum and the paecilomyces fumosoroseus are purchased from the China industrial microorganism culture collection management center, the preservation number of the penicillium citrinum in the China industrial microorganism culture collection management center is CICC 4008, the preservation number of the penicillium islandicum is CICC 40637, the preservation number of the penicillium erythraeum is CICC 4035, the preservation number of the penicillium oxalicum is CICC 2612, and the preservation number of the paecilomyces fumosoroseus is CICC 14005.
The invention also provides a preparation method of the fungal metabolite dry powder for promoting plant growth, which comprises the following steps: and (3) mixing the Penicillium citrinum metabolite dry powder, the Penicillium islandicum metabolite dry powder and the Penicillium erythraeum metabolite dry powder, or mixing the Penicillium oxalicum metabolite dry powder, the Penicillium citrinum metabolite dry powder, the Penicillium islandicum metabolite dry powder and the Penicillium fumosoroseum metabolite dry powder to obtain the fungus metabolite dry powder.
The method comprises the steps of preferably inoculating activated penicillium citrinum, penicillium islandicum, penicillium erythraeum, penicillium oxalicum and paecilomyces fumosoroseus into a liquid culture medium, carrying out oscillation culture, and correspondingly obtaining a penicillium citrinum metabolic bacterial liquid, a penicillium islandicum metabolic bacterial liquid, a penicillium erythraeum metabolic bacterial liquid, a penicillium oxalicum metabolic bacterial liquid and a paecilomyces fumosoroseus metabolic bacterial liquid. In the present invention, the amount of the activated Penicillium citrinum, Penicillium islandicum, Penicillium oxalicum and Paecilomyces fumosoroseus inoculated is preferably 3% by volume of the liquid medium.
In the invention, the speed of the shaking culture is preferably 120-160 r/min, more preferably 120-150 r/min, more preferably 120-135 r/min, and most preferably 120 r/min. In the present invention, the period of time for the shaking culture is preferably 3 to 7 days, and more preferably 5 days. In an embodiment of the invention, the shaking culture is performed in a shaker.
In the present invention, the liquid medium is preferably sterilized at high temperature before the penicillium citrinum, penicillium islandicum, penicillium erythraeum, penicillium oxalicum, and paecilomyces fumosoroseus are inoculated thereto. In the invention, the sterilization temperature is preferably 110-130 ℃, and more preferably 121 ℃; the time for sterilization is preferably 20-30 min, and more preferably 20 min. The sterilization is preferably carried out in an autoclave, the source and the type of the autoclave are not particularly limited, and the conventional autoclave in the field can be adopted.
In the present invention, the culture medium of penicillium citrinum is preferably a czochralski culture medium; the formulation of the Chaudou culture medium preferably comprises 30.0g of sucrose and NaNO in 1.0L of distilled water33.0g，MgSO4·7H2O 0.5g，KCl 0.5g，FeSO4·4H2O0.01 g and K2HPO41.0 g. In the invention, the pH of the Czochralski medium is preferably 7.0-7.2, and more preferably 7.0. In the invention, the temperature of the shaking culture is preferably 25-28 ℃, more preferably 27-28 ℃, and even more preferably 28 ℃.
In the present invention, the culture medium of penicillium islandicum is preferably a malt extract culture medium; the formulation of the malt extract medium preferably includes 20.0g of malt extract per 1.0L of tap water. The present invention does not specifically limit the pH value of the malt extract culture medium, and natural pH is used. In the present invention, the temperature for the shaking culture of penicillium islandicum is preferably 25 to 28 ℃, more preferably 25 to 26 ℃, and still more preferably 25 ℃.
In the present invention, the culture medium of penicillium erythraeum, penicillium oxalicum and paecilomyces fumosoroseus is preferably PDA culture medium; the PDA culture medium is preferably prepared by adding 200g of potato and 20g of glucose into 1L of deionized water. The pH value of the PDA culture medium is not specially limited, and natural pH is adopted. In the present invention, the shaking culture temperature of the penicillium rubrum, penicillium oxalicum, and paecilomyces fumosoroseus is preferably 25 to 28 ℃, more preferably 25 to 26.5 ℃, and still more preferably 25 ℃.
In the present invention, the activation method of the penicillium citrinum, penicillium islandicum, penicillium erythraeum, penicillium oxalicum, and paecilomyces fumosoroseus preferably comprises taking colonies of each of the above penicillium citrinum, penicillium islandicum, penicillium erythraeum, penicillium oxalicum, and paecilomyces fumosoroseus, performing shake culture in sterile water, counting, and obtaining sterile water-activated spore suspension bacteria solutions of the penicillium citrinum, penicillium islandicum, penicillium erythraeum, penicillium oxalicum, and paecilomyces fumosoroseus, respectively. The invention inoculates the sterile water activated spore suspension bacterial liquid of the penicillium citrinum, the penicillium islandicum, the penicillium erythraeum, the penicillium oxalicum and the paecilomyces fumosoroseus into a liquid culture medium, and carries out shake culture until the concentration of the spore suspension in the culture medium reaches 108cfu/mL, and the culture medium is full of cocci to respectively obtain the activated penicillium citrinum, penicillium islandicum, penicillium erythraeum, penicillium oxalicum and paecilomyces fumosoroseus.
In the present invention, the shake culture time of the penicillium citrinum, penicillium islandicum, penicillium erythraeum, penicillium oxalicum, and paecilomyces fumosoroseus in sterile water is preferably 30 min; the spore masses of the penicillium species were sufficiently dispersed during the shaking time. In the present invention, the counting preferably comprises sucking the spore suspension after shaking culture with a pipette and placing the spore suspension on a blood counting plate for counting. In the present invention, the concentration of the sterilized water-activated spore suspension is preferably 108cfu/mL。
In the present invention, the inoculum size of the sterilized water-activated spore suspension of penicillium citrinum, penicillium islandicum, penicillium erythraeum, penicillium oxalicum, and paecilomyces fumosoroseus is preferably 3% of the volume of the liquid medium.
In the present invention, the oscillation culture temperature, the rotation speed and the liquid culture medium during the activation process of the penicillium citrinum, penicillium islandicum, penicillium erythraeum, penicillium oxalicum and paecilomyces fumosoroseus are the same as the oscillation culture temperature, the rotation speed and the liquid culture medium for preparing the penicillium citrinum metabolic bacterial liquid, penicillium islandicum metabolic bacterial liquid, penicillium erythraeum metabolic bacterial liquid, penicillium oxalicum metabolic bacterial liquid and paecilomyces fumosoroseus metabolic bacterial liquid, and are not described herein again.
The bacterial liquids are inactivated respectively to obtain inactivated citrinin metabolic bacterial liquid, penicillium islandicum metabolic bacterial liquid, penicillium erythraea metabolic bacterial liquid, penicillium oxalicum metabolic bacterial liquid and paecilomyces roseus metabolic bacterial liquid. In the invention, the inactivation temperature is preferably 130-140 ℃, and more preferably 140 ℃; the inactivation time is preferably 2-4 h, more preferably 2.5-3.5 h, and even more preferably 3 h. The invention can make the above-mentioned fungi lose activity by high temperature, prevent the metabolic action of live bacteria from influencing the content and variety of metabolic product, and at the same time avoid the influence of live bacteria on plant and environment in the course of applying microbial metabolic product microbial inoculum.
After the inactivated penicillium citrinum metabolic bacteria liquid, penicillium islandicum metabolic bacteria liquid, penicillium oxalicum metabolic bacteria liquid and paecilomyces fumosoroseus metabolic bacteria liquid are obtained, the inactivated bacteria liquid is preferably dried and ground to obtain penicillium citrinum metabolic product dry powder, penicillium islandicum metabolic product dry powder, penicillium erythraeum metabolic product dry powder, penicillium oxalicum metabolic product dry powder and paecilomyces fumosoroseus metabolic product dry powder. In the invention, the drying temperature is preferably 55-65 ℃, more preferably 55-57 ℃, and more preferably 55 ℃; the drying time is preferably 48 to 72 hours, more preferably 48 to 56 hours, and even more preferably 48 hours. In the present invention, the drying mode is preferably drying; the present invention is preferably dried in an oven. The source and the model of the oven are not specially limited, and the oven can be a conventional oven in the field. The present invention preferably further comprises sieving after said grinding. In the present invention, the sieve mesh for sieving is preferably 100 to 500 mesh, more preferably 180 to 400 mesh, and still more preferably 200 mesh. The particle sizes of the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder, the penicillium erythraeum metabolite dry powder, the penicillium oxalicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder obtained by the invention are preferably less than or equal to 200 meshes.
The invention also provides an active component of the fungal metabolite, which comprises any one of petroleum ether extract, ethyl acetate extract, n-butanol extract or water-phase extract of the fungal metabolite dry powder in the technical scheme.
In the present invention, the preparation method of the petroleum ether extract preferably comprises extracting the fungus metabolite dry powder obtained by mixing the penicillium oxalicum metabolite dry powder, the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder with methanol, water and petroleum ether in sequence to obtain the petroleum ether extract.
According to the invention, the fungus metabolite dry powder is preferably mixed and extracted with methanol to obtain a methanol extract. In the present invention, the methanol is preferably anhydrous methanol; the mass volume ratio of the fungus metabolite dry powder to the methanol is preferably 10-12 kg: 50L, more preferably 10 kg: 50L. In the invention, the extraction time is preferably 24-36 h, and more preferably 24 h; the temperature of the extraction is preferably normal temperature. The invention preferably further comprises decompression concentration after the methanol extraction, wherein the decompression concentration pressure is preferably-0.07 Mpa. The methanol extract is obtained after extraction for 3 times.
After the methanol extract is obtained, the methanol extract is preferably mixed with water for extraction to obtain an aqueous phase extract A. In the present invention, the water is preferably distilled water; the mass-to-volume ratio of the methanol extract to the water is preferably 5.30 kg: 20L. In the present invention, the extraction temperature is preferably 25 ℃; the extraction time is preferably 12-24 h, and more preferably 12 h. The invention preferably extracts for 3 times, and the aqueous phase extract A is obtained after extraction.
After the aqueous phase extraction liquid A is obtained, the invention preferably uses petroleum ether to extract the aqueous phase extraction liquid A to obtain a petroleum ether extract and an aqueous phase extraction liquid B. The volume ratio of the aqueous phase extraction liquid A to the petroleum ether is not particularly limited, as long as the petroleum ether extract and the aqueous phase extraction liquid can be obtained by extraction. The present invention preferably further comprises chromatography and concentration under reduced pressure after the petroleum ether extraction. In the present invention, the time for the chromatography is preferably 4 hours. The chromatographic column is not particularly limited in the invention, and a chromatographic column conventional in the art can be adopted. In the present invention, the apparatus used for the concentration under reduced pressure is preferably a rotary evaporator; the temperature of the reduced pressure concentration is preferably 35 ℃; the rotation speed of the reduced pressure concentration is preferably 20-22 rpm, and more preferably 22 rpm; the pressure for the concentration under reduced pressure is preferably-0.07 MPa. According to the invention, petroleum ether is preferably adopted for extraction for 3 times, and the petroleum ether extract and the aqueous phase extraction liquid B are obtained after extraction.
In the present invention, the preparation method of the ethyl acetate extract preferably comprises subjecting the aqueous phase extract B obtained by preparing the petroleum ether extract to ethyl acetate extraction to obtain the ethyl acetate extract.
In the invention, the aqueous phase extraction liquid B is preferably extracted by ethyl acetate to obtain an ethyl acetate extract and an aqueous phase extraction liquid C. The volume ratio of the ethyl acetate to the aqueous phase extract B is not particularly limited in the present invention, as long as the ethyl acetate extract and the aqueous phase extract C can be obtained by extraction.
The invention preferably further comprises chromatography and concentration under reduced pressure after the ethyl acetate extraction. In the present invention, the time for the chromatography is preferably 12 hours. The chromatographic column is not particularly limited in the invention, and a chromatographic column conventional in the art can be adopted. In the present invention, the method of vacuum concentration is the same as the above-mentioned method of vacuum concentration of petroleum ether phase except that the temperature of vacuum concentration is preferably 40 ℃, and thus, the details thereof are not repeated. In the invention, ethyl acetate is preferably adopted for extraction for 3 times, and the ethyl acetate extract and the aqueous phase extraction liquid C are obtained after extraction.
In the present invention, the preparation method of the n-butanol extract preferably comprises extracting the aqueous phase extract C obtained by preparing the ethyl acetate extract with n-butanol to obtain the n-butanol extract.
In the invention, the aqueous phase extraction liquid C is preferably extracted by n-butanol to obtain n-butanol extract and aqueous phase extraction liquid D. In the present invention, the volume ratio of the n-butanol to the aqueous phase extract C is not particularly limited as long as the n-butanol extract and the aqueous phase extract D can be obtained by extraction.
The invention preferably also comprises chromatography and reduced pressure concentration after the n-butanol extraction. In the present invention, the time for the chromatography is preferably 12 hours; the chromatography temperature is preferably 65 ℃. The chromatographic column is not particularly limited in the invention, and a chromatographic column conventional in the art can be adopted. In the present invention, the method of concentration under reduced pressure is the same as that of the above-mentioned petroleum ether phase, and thus, the details thereof are not repeated. The invention preferably adopts n-butyl alcohol for extraction for 3 times, and the n-butyl alcohol extract and the aqueous phase extract C are obtained after extraction.
In the present invention, the method for preparing the aqueous phase extract preferably comprises concentrating the water tank extract D obtained by preparing the above-mentioned n-butanol extract under reduced pressure to obtain the aqueous phase extract.
In the present invention, the method of concentration under reduced pressure is the same as that of the above-mentioned petroleum ether phase, and thus, the details thereof are not repeated.
The invention also provides application of the active ingredients of the fungal metabolites prepared by the technical scheme in promoting plant growth. In the present invention, the promoting of plant growth preferably includes promoting tillering of gramineous plants.
In the present invention, the plant preferably includes one or more of wheat, rice, ryegrass, white clover and tobacco.
In order to further illustrate the present invention, the following embodiments are described in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparing a culture medium:
c, a Chao's medium: sucrose 30.0g, NaNO3 3.0g，MgSO4·7H2O 0.5g，KCl 0.5g，FeSO4·4H2O 0.01g，K2HPO41.0g, 1.0L of distilled water and 7.0-7.2 of pH;
malt extract medium: malt extract 20.0g, tap water 1.0L, natural pH.
PDA culture medium: 200g of potatoes and 20g of glucose, and the volume is set to 1000mL by deionized water.
Sterilizing the above culture medium in autoclave at 121 deg.C for 20 min.
Activating Penicillium rubrum, Penicillium islandicum, Penicillium oxalicum and Paecilomyces fumosoroseus at 25 deg.C for 5 days, and activating Penicillium citrinum at 28 deg.C for 3 days.
After the culture medium is cooled, respectively scraping bacterial colonies of the penicillium citrinum, the penicillium islandicum, the penicillium erythraeum, the penicillium oxalicum and the paecilomyces fumosoroseus into a 250mL triangular flask containing 100mL of sterile water, and oscillating for 30min under the condition of 120r/min to fully disperse spore groups, wherein the culture temperature of the penicillium citrinum is 28 ℃, and the culture temperature of the penicillium citrinum, the penicillium islandicum, the penicillium oxalicum and the paecilomyces fumosoroseus is 25 ℃. Sucking spore suspension with pipette, placing in blood count plate, counting to obtain final concentration of 108cfu/mL of spore suspensions of Penicillium citrinum, Penicillium islandicum, Penicillium erythraeum, Penicillium oxalicum and Paecilomyces fumosoroseus. Respectively inoculating spore suspension of Penicillium citrinum, Penicillium islandicum, Penicillium rubrum, Penicillium oxalicum and Paecilomyces fumosoroseus into a 250mL Erlenmeyer flask containing 100mL of liquid culture medium (the same as the following culture medium) at an inoculation amount of 3%, and performing shake culture under the same conditions until the spore suspension concentration in the liquid culture medium reaches 108cfu/mL。
Inoculating activated penicillium citrinum into a triangular flask containing 100mL of a Kirschner medium, inoculating penicillium islandicum into a triangular flask containing 100mL of a malt extract medium, inoculating penicillium rubrum, penicillium oxalicum and paecilomyces fumosoroseus into a triangular flask containing 100mL of a PDA medium, wherein the inoculation amounts are respectively 3% of the culture media, putting the three culture media into a 120r/min shaking table, and culturing for 3 days, wherein the culture temperature of the penicillium citrinum is 28 ℃, and the culture temperatures of the penicillium rubrum, the penicillium islandicum, the penicillium oxalicum and the paecilomyces fumosoroseus are 25 ℃.
And (3) inactivating the cultured penicillium citrinum liquid, penicillium rubrum liquid, penicillium islandicum liquid, penicillium oxalicum liquid and paecilomyces fumosoroseus liquid at a high temperature of 140 ℃ for 3 hours respectively, putting the inactivated penicillium citrinum liquid, penicillium rubrum liquid, penicillium islandicum liquid, drying the inactivated penicillium oxalicum liquid and paecilomyces fumosoroseus liquid in a 55 ℃ drying oven for 48 hours, grinding the inactivated penicillium citrinum liquid and the paecilomyces fumosoroseus liquid, and sieving the inactivated penicillium citrinum liquid, the paecilomyces rubrum metabolite dry powder, the penicillium islandicum metabolite dry powder, the penicillium oxalicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder by a 200-mesh sieve respectively.
Example 2
Taking 15g of the penicillium citrinum metabolite dry powder, 30g of the penicillium islandicum metabolite dry powder and 30g of the penicillium erythraeum metabolite dry powder prepared in example 1, mixing (the mass ratio of the penicillium citrinum metabolite dry powder to the penicillium islandicum metabolite dry powder to the penicillium erythraeum metabolite dry powder is 1: 2: 2) to prepare the fungus metabolite dry powder.
Example 3
Taking 36g of the penicillium oxalicum metabolite dry powder, 18g of the penicillium citrinum metabolite dry powder, 36g of the penicillium islandicum metabolite dry powder and 18g of the paecilomyces fumosoroseus metabolite dry powder prepared in example 1, mixing (the mass ratio of the penicillium oxalicum metabolite dry powder, the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder is 2: 1: 2: 1), and preparing the fungus metabolite dry powder.
Example 4
The fungal metabolite dry powder obtained in example 3 was extracted with 50L of anhydrous methanol at room temperature for 24h, stirred well, filtered to remove residue and extracted 3 times, and concentrated under reduced pressure of-0.07 MPa to obtain methanol extract.
Adding 20L distilled water solution into 5.30kg methanol extract, leaching at 25 deg.C for more than 12 hr, removing residue, and filtering for 3 times to obtain water phase extract A.
And fully and uniformly mixing the obtained aqueous phase extract A with petroleum ether, and standing in a chromatographic column at normal temperature for 4 hours. After the solution is layered, the aqueous phase and the petroleum ether phase are separated. And (3) filling the separated petroleum ether phase into a rotary steaming bottle, putting the bottle into a 35 ℃ water bath, starting a thermal reflux device and a rotary steaming instrument, and setting the rotating speed at 22rpm and the pressure at-0.07 Mpa. The petroleum ether phase was concentrated under reduced pressure on a rotary evaporator and the solvent was recovered. And adding petroleum ether into the recovered solvent again, fully and uniformly mixing the recovered solvent with the separated water phase, and concentrating the petroleum ether phase under reduced pressure under the same conditions. The extraction was repeated 3 times. Extracting the first water phase extract liquor for three times by using a petroleum ether solvent to obtain a petroleum ether extract and a water phase extract liquor B.
And fully and uniformly mixing the obtained aqueous phase extract B with ethyl acetate, and standing in a chromatographic column for 12 hours. After the solution was separated into layers, the ethyl acetate layer, the emulsion layer and the water layer were separated. The emulsion layer is discarded to obtain an ethyl acetate phase and a water phase. The temperature of the water bath kettle is 40 ℃, and the method of concentration under reduced pressure is the same as the method of petroleum ether extraction. Extracting the aqueous phase extract B with ethyl acetate solvent for three times to obtain ethyl acetate extract and aqueous phase extract C.
Mixing the obtained water phase extractive solution C with n-butanol, standing in chromatographic column for 8 hr at 65 deg.C in water bath, and concentrating under reduced pressure. And extracting the water-phase extract C with n-butanol for three times to obtain n-butanol extract and water-phase extract D.
Concentrating the water phase extractive solution D under reduced pressure, and extracting with petroleum ether to obtain water phase extract.
Comparative example 1
Taking 10g of the penicillium oxalicum metabolite dry powder, 50g of the penicillium citrinum metabolite dry powder, 50g of the penicillium islandicum metabolite dry powder and 50g of the paecilomyces fumosoroseus metabolite dry powder prepared in example 1, mixing (the mass ratio of the penicillium oxalicum metabolite dry powder, the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder is 1: 5: 5: 5) to prepare the fungus metabolite dry powder.
Comparative example 2
Taking 10g of the penicillium oxalicum metabolite dry powder, 50g of the penicillium citrinum metabolite dry powder, 60g of the penicillium islandicum metabolite dry powder and 50g of the paecilomyces fumosoroseus metabolite dry powder prepared in example 1, mixing (the mass ratio of the penicillium oxalicum metabolite dry powder, the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder is 1: 5: 6: 5), and preparing the fungus metabolite dry powder.
Comparative example 3
60g of the penicillium oxalicum metabolite dry powder, 50g of the penicillium citrinum metabolite dry powder, 50g of the penicillium islandicum metabolite dry powder and 50g of the paecilomyces fumosoroseus metabolite dry powder prepared in example 1 were mixed (the mass ratio of the penicillium oxalicum metabolite dry powder, the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder was 6: 5: 5: 5) to prepare a fungus metabolite dry powder.
Comparative example 4
Taking 60g of the penicillium oxalicum metabolite dry powder, 50g of the penicillium citrinum metabolite dry powder, 60g of the penicillium islandicum metabolite dry powder and 50g of the paecilomyces fumosoroseus metabolite dry powder prepared in example 1, mixing (the mass ratio of the penicillium oxalicum metabolite dry powder, the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder is 6: 5: 6: 5), and preparing the fungus metabolite dry powder.
Comparative example 5
Taking 70g of the penicillium oxalicum metabolite dry powder, 60g of the penicillium citrinum metabolite dry powder, 60g of the penicillium islandicum metabolite dry powder and 50g of the paecilomyces fumosoroseus metabolite dry powder prepared in example 1, mixing (the mass ratio of the penicillium oxalicum metabolite dry powder, the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder is 7: 6: 6: 5), and preparing the fungus metabolite dry powder.
Application example 1
Screening example 2 optimum concentration of fungal metabolite Dry powder in wheat production
Respectively adding 2.00 g/pot, 4.00 g/pot, 6.00 g/pot and 8.00 g/pot of fungus metabolite dry powder into 1.5L of conventional nutrient soil matrix to serve as a fungus metabolite dry powder treatment group, and simultaneously taking the fungus metabolite dry powder which is not added as a control group;
firstly, carrying out germination accelerating treatment on wheat seeds, then respectively and uniformly sowing the wheat seeds in plastic pots, 15 seeds in each pot, and placing the pots in a greenhouse for culture; thinning the wheat seedlings when the wheat seedlings grow to have 2 leaves, and leaving 10 wheat seedlings which have the same growth vigor, are disease-free and are not damaged as test materials. Three replicates of each treatment, 10 shoots per replicate. Sampling in the wheat green turning period, measuring various relevant indexes, detecting the influence of fungus metabolite inactivation dry powder treatment on the growth of wheat seedlings, and screening the optimal concentration for promoting the growth of the wheat seedlings, wherein the results are shown in table 1. The specific measurement indexes and methods are as follows:
plant height: the length of the plant from the base of the stem to the tip of the stem was measured with a ruler.
Fresh weight of aerial parts: the fresh weight of the aerial parts of the plants is measured by balance.
Tillering number: the tillering number was determined by counting.
Tillering time: and observing the tillering starting time of the wheat of the control group and the fungus metabolite inactivation dry powder treatment group.
TABLE 1 measurement results of various indexes of wheat treated with fungal metabolite dry soil mixture in example 2
As can be seen from Table 1, compared with the control group, the fungus metabolite dry powder in the embodiment 2 of the invention can promote the tillering of wheat, shorten the tillering time, obviously increase the plant height and the fresh weight of the overground part of wheat, and especially has obvious promotion effect on the growth of wheat under the concentration of 4.00 g/pot.
Application example 2
Screening example 2 optimum mode of administration of fungal metabolite Dry powder in wheat production
Soil mixing application: the fungal metabolite dry powders of example 2 at 2.00 g/pot, 4.00 g/pot, 6.00 g/pot and 8.00 g/pot, respectively, were applied by mixing into 1.5L of a conventional nutrient soil matrix as a fungal metabolite dry powder treatment group, while the fungal metabolite dry powder was not added as a control group. Firstly, carrying out germination accelerating treatment on wheat seeds, then respectively and uniformly sowing the wheat seeds in plastic pots, 15 seeds in each pot, and placing the pots in a greenhouse for culture. Thinning the wheat seedlings when the wheat seedlings grow into 2 leaves, and leaving 10 wheat seedlings which are consistent in growth vigor, free of diseases and damage as test materials. Three replicates per treatment, 10 seedlings per replicate;
irrigating roots and applying: firstly, uniformly sowing wheat seeds in a seedling raising tray, wherein the used matrix is a conventional nutrient soil matrix, and culturing in a greenhouse. Then selecting wheat seedlings which have consistent growth vigor, no diseases and no damage and have 2 leaves, transplanting the wheat seedlings into plastic pots, wherein 10 wheat seedlings are planted in each plastic pot. The wheat seedlings were treated by root irrigation with solutions of 2.00 g/pot, 4.00 g/pot, 6.00 g/pot and 8.00 g/pot of the fungal metabolite dry powder of example 2 mixed with 15mL of water, respectively, and the same volume of clear water was used as a control. Each treatment is repeated for three times, each time 10 seedlings are repeated, the root irrigation is carried out for 1 time every 7 days, and the root irrigation amount is 15mL for each plastic pot.
The growth indexes of the wheat seedlings in the soil-mixing application mode and the root-irrigation application mode are measured, the effects of the two application modes on the growth of the wheat seedlings are detected, the optimal application mode for promoting the growth of the wheat is determined, and the results are shown in table 2.
Table 2 example 2 measurement results of various indexes of wheat treated by dry powder root irrigation of fungal metabolite
The result shows that the fungus metabolite provided by the embodiment 2 of the invention is used for root irrigation treatment of wheat, the growth of wheat can be promoted, indexes such as plant height, tiller number and the like of wheat are obviously increased, but microorganisms such as fungus and the like are easy to breed on the surface of the substrate, and simultaneously, the odor is large, insects such as flies are easy to attract, and adverse effects are caused to the growth environment of wheat.
By combining application example 1 and application example 2, the optimal application concentration of wheat treated by the fungus metabolite dry powder is 4.00 g/pot; the best mode of application for treating wheat with the fungus metabolite dry powder is to mix the fungus metabolite dry powder into a substrate for application.
Application example 3
Screening example 2 optimum concentrations of fungal metabolite dry powder in rice production: the specific method is the same as the application example 1, the sampling time of the rice at different positions is 30 days after the rice seedlings emerge, and the results are shown in table 3.
TABLE 3 measurement results of various indexes of rice treated with inactivated soil mixed with fungal metabolites in example 2
As shown in Table 3, the fungus metabolite dry powder in example 2 of the present invention can promote rice tillering, shorten tillering time, and significantly increase plant height and fresh weight of overground part of rice, compared to the control group. In particular, the application amount of the fungal metabolite dry powder of 4.00 g/pot was the best for the growth promotion effect of rice.
Application example 4
Optimal application mode of screening fungus metabolite inactivated dry powder in rice production
The specific method is the same as application example 2, the sampling time of rice is 30 days after rice seedling emergence, and the results are shown in table 4.
TABLE 4 measurement results of various indexes of rice treated by fungus metabolite dry powder root irrigation in example 2
As can be seen from table 4, when the fungus metabolite dry powder provided in example 2 of the present invention is used for root irrigation treatment of rice, growth of rice can be promoted, indexes such as plant height and tiller number of rice can be significantly increased, but microorganisms such as fungi are easily bred on the surface of the substrate, and simultaneously, the odor is large, insects such as flies are easily attracted, and adverse effects are caused to the growth environment of rice.
According to the combination of application examples 3 and 4, the optimal application concentration of the fungus metabolite dry powder for treating rice is 4.00 g/pot; the best mode of application of the fungus metabolite dry powder for treating the rice is to mix the fungus metabolite inactivated dry powder into a matrix for application.
Application example 5
Screening example 2 optimum concentrations of fungal metabolite dry powder in ryegrass production were:
adding the fungus metabolite dry powders of example 2 of 2.00 g/pot, 4.00 g/pot, 6.00 g/pot and 8.00 g/pot into 1.5L of conventional humus soil respectively to serve as fungus metabolite dry powder treatment groups, and taking the fungus metabolite dry powder which is not added as a control group; uniformly sowing seeds of ryegrass in nutrient bags of 13X 12cm, 8 seeds per pot, and placing the seedlings in a greenhouse for culturing; and thinning the seedlings of the ryegrass when 2 leaves grow out, and leaving 5 seedlings of the ryegrass which have consistent growth vigor, no diseases and no damage as test materials. Three replicates of each treatment, 10 shoots per replicate. Sampling 60 days after the ryegrass seeds germinate, determining related indexes, detecting the influence of fungal metabolite dry powder treatment on the growth of the ryegrass, and screening the optimal concentration for promoting the growth of the ryegrass, wherein the results are shown in table 5.
TABLE 5 measurement of various indices of fungal metabolite in example 2 of rye grass treated with dry soil
As can be seen from Table 3, the fungus metabolite dry powder provided by the invention in example 2 has a good effect of promoting the growth of ryegrass, and particularly when the concentration is 2.00 g/pot, the fungus metabolite dry powder can remarkably promote the early tillering of ryegrass plants, and the tillering number, the plant height and the fresh weight of overground parts are remarkably increased.
Application example 6
Screening example 2 for optimum application of fungal metabolite Dry powder in rye grass production
Soil mixing application: the fungal metabolite dry powders of example 2 at 2.00 g/pot, 4.00 g/pot, 6.00 g/pot and 8.00 g/pot, respectively, were applied by stirring in 1.5L of humus soil as a fungal metabolite dry powder treatment group, while no fungal metabolite dry powder was added as a control group. Seeds of ryegrass were sown evenly in nutrient bags of 13X 12cm, 8 seeds per pot, and placed in a greenhouse for cultivation. Thinning the seedlings of ryegrass when 2 leaves grow out, and leaving 5 seedlings of ryegrass which have consistent growth vigor, no diseases and no damage as test materials. Three replicates per treatment, 10 seedlings per replicate;
irrigating roots and applying: the matrix is humus soil. Firstly, seeds of ryegrass are uniformly sowed in a seedling tray and placed in a greenhouse for cultivation. Then, selecting ryegrass seedlings which have consistent growth vigor, no diseases and no damage and 2 leaves, transplanting the ryegrass seedlings into nutrition bags of 10 multiplied by 10cm, wherein 5 nutrition bags are used for transplanting each seedling. The ryegrass is subjected to root irrigation treatment by using solutions obtained by mixing 2.00 g/basin, 4.00 g/basin, 6.00 g/basin and 8.00 g/basin of fungal metabolite dry powder with 15mL of water, and the same volume of clear water is used as a control. Three replicates of each treatment were irrigated 1 time every 7d, with 15mL per nutrition bag.
The seedling growth indexes of ryegrass under the modes of soil mixing application and root irrigation application are measured, the effects of the two modes of application on the growth of ryegrass are detected, and the optimal mode of application for promoting the growth of ryegrass is determined, and the results are shown in table 6.
TABLE 6 example 2 measurement of various indexes of rye grass treated by dry root irrigation of fungal metabolite
As shown in Table 6, the treatment of ryegrass by root irrigation with the fungal metabolite dry powder provided by the invention can promote the growth of ryegrass and remarkably increase indexes such as plant height and tiller number of ryegrass, but microorganisms such as fungi are easily bred on the surface of a substrate, and simultaneously, the fungal metabolite dry powder has a large smell, is easy to attract insects such as flies and causes a growing environment for ryegrass
As can be seen from application examples 5 and 6, the optimal application concentration of the ryegrass treated by the fungal metabolite dry powder provided by the invention is 2.00 g/pot; the best mode of application of the fungal metabolite dry powder treatment ryegrass is to mix the fungal metabolite inactivated dry powder into the matrix for application.
Application example 7
The seeds of tobacco were uniformly sprinkled in the matrix and placed in a greenhouse for cultivation. When 5 true leaves grow out from the tobacco, tobacco seedlings which grow uniformly and have no disease and damage are selected and transplanted into plastic pots (1 gallon, soil containing amount: 1.5L) respectively containing 1.50g, 3.00g, 4.50g and 6.00g of fungal metabolites, and clear water is mixed with soil to serve as a control. Each treatment was 3 replicates, 10 tobacco seedlings per replicate. Watering at a proper time without fertilizing, and measuring the growth related indexes of the tobacco plants 30 days after the tobacco seedlings are transplanted, wherein the results are shown in table 7. The specific measurement indexes and methods are as follows:
plant height: the height of the tobacco seedlings was measured using a tape measure.
Diameter of the stem: the diameter of the stem at the base of the stem was measured using a vernier caliper.
Fresh weight of aerial parts: the fresh weight of the aerial parts of the plants is weighed by using balance.
TABLE 7 detection results of various indexes of tobacco treated with fungal metabolites in example 3
As shown in Table 7, 3.00 g/pot of the fungal metabolite dry powder was able to promote the growth of tobacco seedlings, significantly increasing the plant height, stem diameter and fresh weight of aerial parts of tobacco.
Application example 8
Uniformly sowing seeds of ryegrass in nutrient bags of 13X 12cm, 8 seeds per pot, and placing the seedlings in a greenhouse for culturing; and (3) thinning the seedlings of the ryegrass when 2 leaves grow out, leaving 5 seedlings of the ryegrass with consistent growth vigor, no diseases and no damage as test materials, treating the ryegrass by using the petroleum ether extract, the n-butanol extract and the ethyl acetate extract in example 4 with certain concentration (the specific concentration is shown in table 8) and the aqueous phase extract obtained in the proportion 6 in a root irrigation manner, and irrigating the roots with clean water with the same volume as a control. Three replicates per treatment, 10 seedlings per replicate; the four extracts are respectively processed by ryegrass for 60 days and then sampled, related indexes are measured, and the influence of the fungal metabolite extract processing on the growth of the ryegrass is detected, and the results are shown in table 9. The measurement indexes and the specific measurement method are as follows:
plant height: the length of the plant from the base of the stem to the tip of the stem was measured with a ruler.
Fresh weight of aerial parts: the fresh weight of the aerial parts of the plants is weighed by using balance.
Table 8 application concentrations of fungal metabolite dry powder active ingredients in ryegrass, white clover, and tobacco in example 4.
Table 9 shows the results of measurements of the indices of ryegrass treated with four active ingredients of fungal metabolites in example 4
As can be seen from Table 9, compared with the control group, the application of the petroleum ether extract, the n-butanol extract and the water phase extract provided by the invention can promote the growth of ryegrass, especially the 20 mug/mL fungal metabolite ethyl acetate extract has the largest growth promotion effect on ryegrass, and can significantly increase the plant height and the fresh weight of the overground part of ryegrass.
Application example 9
Uniformly sowing seeds of white clover in nutrient bags of 13 x 12cm, 8 seeds per pot, and placing the pots in a greenhouse for culturing; after 1 true leaf of the white clover seedling grows out, thinning is carried out, 5 white clover seedlings which are consistent in growth vigor, free of diseases and damage are left as test materials, then the white clover is treated by the petroleum ether extract, the n-butyl alcohol extract and the ethyl acetate extract in the embodiment 4 with certain concentration (the specific concentration is shown in the table 8) and the water phase extract obtained in the proportion 6 in a root irrigation mode, and the root irrigation is carried out by clear water with the same volume as a control. Three replicates per treatment, 10 seedlings per replicate; after the four extracts are respectively treated for 60 days, the samples are taken, related indexes are measured, and the influence of the treatment of the fungal metabolite extract on the growth of the white clover is detected, wherein the results are shown in table 10. The measurement indexes and the specific measurement method are as follows:
plant height: the length of the plant from the base of the stem to the tip of the stem was measured with a ruler.
Fresh weight of aerial parts: the fresh weight of the aerial parts of the plants is weighed by using balance.
Number of blades: the leaf number of white clover is determined by a counting method.
Table 10 shows the results of measurement of each index of the four active ingredients of fungal metabolites treated white clover in example 4
As can be seen from table 10, the petroleum ether extract, n-butanol extract and water phase extract of the fungal metabolite provided by the present invention all promoted the growth of white clover, and in particular, the ethyl acetate extract of 20 μ g/mL had the best growth promoting effect on white clover, and significantly increased the plant height, leaf number and fresh weight of the aerial parts of white clover, compared to the control group.
Application example 10
The seeds of tobacco were uniformly sprinkled in the matrix and placed in a greenhouse for cultivation. When 5 true leaves grow out of the tobacco, selecting tobacco seedlings with consistent growth vigor and no diseases and no damage to the tobacco seedlings, transplanting the tobacco seedlings into a nutrition bag of 13 multiplied by 12cm, and then irrigating the tobacco seedlings with the petroleum ether extract, the n-butanol extract and the ethyl acetate extract in the embodiment 4 and the water phase extract obtained in the proportion 6 with certain concentrations (the specific concentrations are shown in a table 8), wherein the root irrigation amount is 5 mL/plant. Root drenching with the same volume of clear water was used as a control. Each treatment was 3 replicates, each replicate 10 tobacco seedlings. The root irrigation is carried out once a week, watering is carried out at proper time without fertilizing, the length is 25d from the first treatment, after 4 times of treatment, the tobacco plants are measured for relevant growth indexes, the influence of the treatment of the fungal metabolite extract on the growth of the tobacco is detected, and the results are shown in table 11. The measurement indexes and the specific measurement method are as follows:
plant height: the length of the plant from the base of the stem to the tip of the stem was measured with a ruler.
Number of blades: the number of leaves of the seedling was determined by counting.
Diameter of stem: the diameter of the stem at the base of the stem was measured using a vernier caliper.
Fresh weight of aerial parts: the fresh weight of the aerial parts of the plants is weighed by using balance.
Table 11 shows the measurement results of each index of four kinds of active-treated tobacco of fungal metabolites in example 4 and comparative example 6
As can be seen from table 11, the application of the petroleum ether extract, n-butanol extract, and water phase extract of the fungal metabolite provided by the present invention can promote the growth of tobacco, and in particular, the ethyl acetate extract of 10 μ g/mL has the best growth promoting effect on tobacco, and can significantly increase the plant height, stem diameter, and fresh weight of the aerial parts of tobacco.
Application example 11
The seeds of tobacco were uniformly sprinkled in the matrix and placed in a greenhouse for cultivation. When 5 main leaves grow out from the tobacco, selecting tobacco seedlings which grow uniformly and have no diseases and no damage, transplanting the tobacco seedlings into plastic pots (1 gallon, soil containing amount: 1.5L) respectively provided with fungal metabolite dry powder, and mixing soil with clear water as a control. Each treatment was 3 replicates, each replicate 10 tobacco seedlings. Watering at a proper time without fertilizing, and measuring the plant height, stem circumference and fresh weight of the overground part of the tobacco plant after the tobacco seedling is transplanted for 30 days.
Plant height: the length of the plant from the base of the stem to the tip of the stem was measured with a ruler.
Diameter of the stem: the diameter of the stem at the base of the stem was measured using a vernier caliper.
Fresh weight of aerial parts: weighing fresh weight of aerial part of plant by balance
The fungus metabolite dry powder in comparative examples 1-5 is applied to tobacco, and compared with a control group, the fungus metabolite dry powder has no significant difference, even inhibits the growth of tobacco, and the results are shown in table 12.
Table 12 shows the results of measuring the indexes of four kinds of actively treated tobacco of fungal metabolites in comparative examples 1 to 5
The embodiment shows that the fungus metabolite dry powder provided by the invention can obviously promote tillering of plants and increase the plant height, stem circumference and fresh weight of overground parts of crops. Meanwhile, the active ingredients of the fungus metabolite dry powder after being extracted by methanol, water, petroleum ether, ethyl acetate and n-butyl alcohol can further improve the growth promoting effect of the fungus metabolite and increase the crop yield.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (3)
1. A dry fungal metabolite powder for promoting plant growth, wherein the dry fungal metabolite powder comprises Penicillium citrinum (B) citrinumPenicillium citrinum) Metabolite dry powder, Penicillium islandicum (Penicillium islandicum) Metabolite dry powder and Penicillium rubrum: (Penicillium rubrum) Metabolite dry powder
Or comprises penicillium oxalicum (B)Penicillium oxalicum) Metabolite dry powder, Penicillium citrinum (B)Penicillium citrinum) Metabolite dry powder, Penicillium islandicum (Penicillium islandicum) Metabolite dry powder and Paecilomyces fumosoroseus (A)Paecilomyces fumosoroseus) A metabolite dry powder;
the fungus metabolite dry powder comprises the following components in parts by weight: 10-30 parts of penicillium citrinum metabolite dry powder, 20-50 parts of penicillium islandicum metabolite dry powder and 10-40 parts of penicillium erythraeum product dry powder;
or the fungus metabolite dry powder comprises the following components in parts by weight: 36 parts of penicillium oxalicum metabolite dry powder, 18 parts of penicillium citrinum metabolite dry powder, 36 parts of penicillium islandicum metabolite dry powder and 18 parts of paecilomyces fumosoroseus metabolite dry powder;
the collection number of the penicillium citrinum in the China industrial microorganism strain collection management center is CICC 4008, the collection number of the penicillium islandicum is CICC 40637, the collection number of the penicillium rubrum is CICC 4035, the collection number of the penicillium oxalicum is CICC 2612, and the collection number of the paecilomyces fumosoroseus is CICC 14005;
the preparation method of the fungal metabolite dry powder for promoting plant growth comprises the following steps: mixing the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder and the penicillium erythraeum metabolite dry powder, or mixing the penicillium oxalicum metabolite dry powder, the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder to obtain the fungus metabolite dry powder;
the preparation method of the penicillium citrinum metabolite dry powder, the penicillium islandicum metabolite dry powder, the penicillium erythraeum metabolite dry powder, the penicillium oxalicum metabolite dry powder and the paecilomyces fumosoroseus metabolite dry powder comprises the following steps: respectively inoculating activated bacteria of the penicillium citrinum, the penicillium islandicum, the penicillium erythraeum, the penicillium oxalicum and the paecilomyces fumosoroseus into a liquid culture medium, and performing shaking culture, inactivation, drying and grinding to obtain dried powder of the penicillium citrinum metabolite, the penicillium islandicum metabolite, the penicillium erythrosoroseum metabolite, the penicillium oxalicum metabolite and the paecilomyces fumosoroseus metabolite;
the oscillation rate of the oscillation culture is 120 r/min; the culture time is 3-7 d;
when the penicillium citrinum metabolite dry powder is prepared, the liquid culture medium is a Chaudou culture medium, the pH of the Chaudou culture medium is 7.0-7.2, and the shaking culture temperature is 25-28 ℃;
when the penicillium islandicum metabolite dry powder is prepared, the liquid culture medium is a malt extract culture medium, and the shaking culture temperature is 25-28 ℃;
when preparing the dried penicillium erythraeum metabolite powder, the dried penicillium oxalicum metabolite powder and the dried paecilomyces fumosoroseus metabolite powder, the liquid culture medium is a PDA culture medium, and the shaking culture temperature is 25-28 ℃;
the inactivation temperature is 130-140 ℃, and the inactivation time is 2-4 h.
2. An active ingredient of a fungal metabolite comprising a petroleum ether extract, an ethyl acetate extract, an n-butanol extract or an aqueous phase extract of the fungal metabolite dry powder of claim 1.
3. Use of the fungal metabolite dry powder of claim 1 or the fungal metabolite active ingredient of claim 2 for promoting plant growth;
the plant comprises one or more of wheat, rice, ryegrass, white clover and tobacco.
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