CN114287448A - Compound medicament for improving heat resistance of Lanzhou lily and preparation method thereof - Google Patents
Compound medicament for improving heat resistance of Lanzhou lily and preparation method thereof Download PDFInfo
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- 241000377871 Trichoderma longibrachiatum SMF2 Species 0.000 claims abstract description 21
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- 108010061784 Peptaibols Proteins 0.000 claims description 4
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Abstract
The invention provides a compound medicament for improving heat resistance of Lanzhou lily and a preparation method thereof, and the compound medicament comprises the following steps: (1) preparing a trichoderma longibrachiatum SMF2 spore suspension; (2) solid culture of trichoderma longibrachiatum SMF 2; (3) preparing TKs; (4) TKs, CaCl2And SA, preparing a compound medicament according to a preset concentration. The medicament prepared by the invention is prepared from Trichoderma secondary metabolite TKs, exogenous SA and CaCl2And (4) compounding. After the compound medicament is used for irrigating the Lanzhou lily, the heat resistance of the Lanzhou lily is obviously enhanced, and the survival rate after heat shock is improved by more than 50 percent compared with a control. The compound medicament has obvious and timely effect, simple and convenient method operation and safety to the environment.
Description
Technical Field
The invention belongs to the technical field of plant cultivation, and particularly relates to a compound medicament for improving heat resistance of Lanzhou lily and a preparation method thereof.
Background
Lilium davidii var. unicolor is a variety of Lilium davidii of liliaceae, has large and white bulbs, fine texture, sweet taste, rich nutrition and beautiful flowers, and has high edible, medicinal and ornamental values. Lilium davidii var unicolor is weak in heat resistance, and high temperature is one of the main abiotic stress factors limiting the production thereof. In recent years, the yield and quality of bulbs are seriously affected by the high-temperature weather in summer of the main producing area of Lanzhou lily. In addition, the introduction and cultivation of the Lanzhou lily are also severely restricted in hot summer in most areas of China. Therefore, the improvement of the heat resistance of the Lanzhou lily is of great significance for the production of the Lanzhou lily.
The breeding of heat-resistant varieties by cross breeding is a main way to improve the heat resistance of lily, but the breeding process is complicated and slow. The application of exogenous protective agents can rapidly enhance the heat resistance of plants, but is not reported in Lanzhou Baihe.
Salicylic Acid (SA) is a small molecular phenolic substance widely existing in plants, participates in various physiological processes of plants, and particularly plays an important role in disease resistance and heat resistance of the plants. Exogenous salicylic acid can improve the heat resistance of chrysanthemum, begonia senilis, ornamental pepper, lily bulb and other plants. Calcium is a second messenger of plant essential nutrients and plant cells, and is also involved in plant response to heat stress. The external application of calcium can improve the heat resistance of plants such as tillandsia, oriental lily, radish, pinellia ternate, vine rose and the like. TKs (Trichokinins) are peptaibols generated by Trichoderma longibrachiatum SMF2(Trichoderma longibrachiatum SMF2), have various biological activities of resisting bacteria and viruses, inducing plant resistance, promoting plant growth and the like, can effectively control plant diseases, but have no function report of the TKs in the aspect of plant abiotic stress.
At present, no report of an exogenous protective agent for improving the heat resistance of the Lanzhou lily is found. In order to solve the problems, a medicament capable of obviously improving the heat resistance of the Lanzhou lily is needed to be invented, and the medicament can be applied in a high-temperature period in summer and can effectively improve the heat resistance of the Lanzhou lily in time.
Disclosure of Invention
Aiming at the problem that the high temperature in summer affects the yield and the quality of the Lanzhou lily bulb, the invention provides a compound medicament consisting of microbial secondary metabolites TKs, exogenous salicylic acid and calcium, and the compound medicament can jointly enhance the heat resistance of the Lanzhou lily. The compound preparation is timely and effective, convenient to use, time-saving and labor-saving, safe to environment and free of pesticide residue on agricultural products.
A compound medicament for improving heat resistance of Lanzhou lily comprises: TK with concentration of 1-4 mg/Ls，1～2g/L CaCl2，100～400mg/LSA。
Preferably, the concentration is 2mg/L TKs, 1g/L CaCl2100 mg/LSA. The preparation method comprises the following steps:
(1) preparation of trichoderma longibrachiatum SMF2 spore suspension: trichoderma longibrachiatum SMF2(Trichoderma longibrachiatum SMF2) was cultured on PDA plates for 5 d. Scraping an appropriate amount of spores from PDA plates and adding to sterile ddH containing 0.1% (v/v) Tween-802In O, the mixture is blown and beaten by a pipette and is mixed evenly to prepare the mixture with the spore content of 1 multiplied by 108Spore suspension of one/ml.
(2) Solid culture of trichoderma longibrachiatum SMF 2: 1ml of spore suspension of Trichoderma longibrachiatum SMF2 (1X 10) was added to a 500ml Erlenmeyer flask containing 10g of solid fermentation medium8Pieces/ml) of the strain, placing the strain in a mold incubator at 28 ℃ and culturing for 5 days.
The solid fermentation culture medium: bran 9g, rice hull 1g, liquid A (1.2% KH)2PO4,1.2％(NH4)2SO4,0.5％MgSO4·7H2O)4ml, B liquid (0.5% CaCl)2)4 ml. Sterilizing at 121 deg.C for 20 min.
(3) Preparation of Trichokinins (TKs): adding 100ml of absolute ethyl alcohol into the cultured solid culture of the trichoderma longibrachiatum SMF2, fully shaking, standing for 4h, filtering by using four layers of gauze, centrifuging the filtrate at 10000rpm for 10min, performing rotary evaporation and concentration on the supernatant to obtain 1/4-1/3 of the original volume, centrifuging the supernatant at 10000rpm for 10min, and taking the supernatant to obtain the crude TKs extract. A small amount of TKs mother liquor was collected, filtered through a 0.22 μm syringe filter, and subjected to HPLC to detect the production of peptaibols. Chromatographically pure methanol and ultrapure water (0.1% TFA) as mobile phases, methanol: and (3) adding water at a flow rate of 1ml/min to 84:16, selecting an Agilent C18 reverse phase chromatographic column, and detecting the wavelength of 203 nm.
(4) Preparing a compound medicament: TKs, CaCl2And SA, preparing a compound medicament according to a preset concentration.
Preferably: the first medicament: 5mg/L TKs; a second medicament: 2.5g/L CaCl2(ii) a Medicament III: 0.5g/L SA. The medicament is prepared into a compound medicament according to the volume ratio of 2:2: 1.
The medicament prepared by the invention is prepared from Trichoderma secondary metabolite TKs, exogenous SA and CaCl2And (4) compounding. After the compound medicament is used for irrigating the Lanzhou lily, the heat resistance of the Lanzhou lily is obviously enhanced, and the survival rate after heat shock is improved by more than 50 percent compared with a control. The compound medicament has obvious and timely effect, simple and convenient method operation and safety to the environment.
Detailed Description
The specific technical scheme of the invention is described by combining the embodiment.
The preparation method of the compound medicament for improving the heat resistance of the Lanzhou lily comprises the following steps:
(1) preparation of trichoderma longibrachiatum SMF2 spore suspension:
trichoderma longibrachiatum SMF2(Trichoderma longibrachiatum SMF2) was cultured on PDA plates for 5 d. Scraping an appropriate amount of spores from PDA plates and adding to sterile ddH containing 0.1% (v/v) Tween-802In O, the mixture is blown and beaten by a pipette and is mixed evenly to prepare the mixture with the spore content of 1 multiplied by 108Spore suspension of one/ml.
(2) Solid fermentation medium:
bran 9g, rice hull 1g, liquid A (1.2% KH)2PO4,1.2％(NH4)2SO4,0.5％MgSO4·7H2O)4ml, B liquid (0.5% CaCl)2)4 ml. Sterilizing at 121 deg.C for 20 min.
(3) Solid culture of trichoderma longibrachiatum SMF 2:
1ml of spore suspension of Trichoderma longibrachiatum SMF2 (1X 10) was added to a 500ml Erlenmeyer flask containing 10g of solid fermentation medium8Pieces/ml) of the strain, placing the strain in a mold incubator at 28 ℃ and culturing for 5 days.
(4) Preparation of Trichokinins (TKs):
adding 100ml of absolute ethyl alcohol into the cultured solid culture of the trichoderma longibrachiatum SMF2, fully shaking, standing for 4h, filtering by using four layers of gauze, centrifuging the filtrate at 10000rpm for 10min, performing rotary evaporation and concentration on the supernatant to obtain 1/4-1/3 of the original volume, centrifuging the supernatant at 10000rpm for 10min, and taking the supernatant to obtain the crude TKs extract. A small amount of TKs mother liquor was collected, filtered through a 0.22 μm syringe filter, and subjected to HPLC to detect the production of peptaibols. Chromatographically pure methanol and ultrapure water (0.1% TFA) as mobile phases, methanol: and (3) adding water at a flow rate of 1ml/min to 84:16, selecting an Agilent C18 reverse phase chromatographic column, and detecting the wavelength of 203 nm.
(5) Preparing a compound medicament:
the first medicament: 5mg/L TKs; a second medicament: 2.5g/L CaCl2(ii) a Medicament III: 0.5g/L SA. The above medicines are prepared into compound medicines (the final concentration is 2mg/L TKs and 1g/L CaCl) according to the volume ratio of 2:2:12，100mg/L SA)。
(6) Influence of the compound medicament on heat resistance of Lanzhou lily:
preparing plant materials of lilium davidii var unicolor:
selecting healthy and consistent lily bulbs, planting the bulbs in a nutrition pot, and using peat and river sand as a culture medium (V: 1). The seed balls and the matrix are disinfected by 800 times of mancozeb liquid before use. The culture conditions of the potted Lanzhou lily in the greenhouse are 25 ℃ in the daytime, 16 hours in the illumination time and 20 ℃ at night. After a period of growth, healthy, uniform size plantlets were selected as test material.
Medicament and heat stress treatment:
respectively using TKs (0.5, 1, 2, 4, 8mg/L) and CaCl at different concentrations2(0.5, 1, 2g/L) and SA (25, 100, 400mg/L) were applied to the Lanzhou lily root-drench with 30ml of distilled water as a control. After treatment, the culture medium is cultured for 12 hours at the temperature of 22 ℃ and then placed in an artificial incubator for stress treatment for 72 hours at the temperature of 40 ℃. After high-temperature stress treatment, the plants are restored and cultured in a greenhouse for 30d, and the survival rate is counted. 12 strains per treatment, 3 replicates. After the optimal concentration of a single medicament is determined, the combination of comparative medicaments (2mg/L TKs +1g/L CaCl)22mg/L TKs +100mg/L SA and 2mg/L TKs +1g/L CaCl2+100mg/L SA), the same procedure was followed.
TABLE 1 Effect of Single Agents on Lily, Lanzhou, Heat resistance
TABLE 2 Effect of the compounded Agents on Lily Lanzhou Heat resistance
③ the test result:
table 1 shows that 1-4 mg/LTKs can remarkably improve the survival rate of the Lanzhou lily after heat stress, and the optimal concentration is 2 mg/L; CaCl2The proper concentration range of the compound is 1-2 g/L, and the optimal concentration is 1 g/L; the proper concentration range of SA is 100-400 mg/L, and the optimal concentration is 100 mg/L; the survival rate of the 2mg/L TKs treatment is obviously higher than 1g/L CaCl2And 100mg/L SA.
Table 2 shows that 2mg/L TKs +1g/L CaCl2The survival rate of SA treatment of +100mg/L is the highest, and is improved by more than 50 percent compared with a control. The results show that the compound medicament (containing 2mg/L TKs and 1g/L CaCl)2And 100mg/L SA) can significantly improve the heat resistance of lilium davidii.
Claims (3)
1. The compound medicament for improving the heat resistance of the Lanzhou lily is characterized by comprising the following components: the concentration of the TKs is 1-4 mg/L, and the CaCl is 1-2 g/L2，100～400mg/LSA。
2. The preparation method of the compound medicament for improving the heat resistance of the lilium davidii var davidii as claimed in claim 1, wherein the method comprises the following steps:
(1) preparation of trichoderma longibrachiatum SMF2 spore suspension: culturing Trichoderma longibrachiatum SMF2 on a PDA plate for 5 d; scraping an appropriate amount of spores from PDA plates and adding to sterile ddH containing v/v 0.1% Tween-802In O, the mixture is blown and beaten by a pipette and is mixed evenly to prepare the mixture with the spore content of 1 multiplied by 108Spore suspension of one/ml;
(2) solid culture of trichoderma longibrachiatum SMF 2: adding 1ml of spore suspension of trichoderma longibrachiatum SMF2 into a 500ml triangular flask filled with 10g of solid fermentation medium, placing the flask in a 28 ℃ mould incubator, and culturing for 5 days;
(3) preparation of TKs: adding 100ml of absolute ethyl alcohol into a solid culture of the cultured trichoderma longibrachiatum SMF2, fully shaking, standing for 4 hours, filtering by using four layers of gauze, centrifuging a filtrate at 10000rpm for 10 minutes, performing rotary evaporation and concentration on a supernatant to obtain 1/4-1/3 of the original volume, centrifuging at 10000rpm for 10 minutes, and taking the supernatant to obtain a crude TKs extracting solution; taking a small amount of TKs mother liquor, filtering by using a 0.22-micron syringe filter, and detecting the generation condition of peptaibols by HPLC; chromatographically pure methanol and ultrapure water 0.1% TFA as mobile phases, methanol: water is 84:16, the flow rate is 1ml/min, an Agilent C18 reversed phase chromatographic column is selected, and the detection wavelength is 203 nm;
(4) preparing a compound medicament: TKs, CaCl2And SA, preparing a compound medicament according to a preset concentration.
3. The preparation method of the compound medicament for improving the heat resistance of the lilium davidii as claimed in claim 2, wherein the solid fermentation medium is prepared according to the following proportion: 9g of bran, 1g of rice hull, 4ml of A liquid and 4ml of B liquid; sterilizing at 121 deg.C for 20 min; the solution A comprises 1.2 percent of KH2PO4,1.2％(NH4)2SO4,0.5％MgSO4·7H2O; the solution B comprises 0.5 percent of CaCl2。
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