CN106922349B - Method for promoting accumulation of chlorogenic acid in honeysuckle flower buds by using fungi - Google Patents
Method for promoting accumulation of chlorogenic acid in honeysuckle flower buds by using fungi Download PDFInfo
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- CN106922349B CN106922349B CN201710116176.7A CN201710116176A CN106922349B CN 106922349 B CN106922349 B CN 106922349B CN 201710116176 A CN201710116176 A CN 201710116176A CN 106922349 B CN106922349 B CN 106922349B
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- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 title claims abstract description 24
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G9/00—Cultivation in receptacles, forcing-frames or greenhouses; Edging for beds, lawn or the like
- A01G9/02—Receptacles, e.g. flower-pots or boxes; Glasses for cultivating flowers
- A01G9/029—Receptacles for seedlings
- A01G9/0299—Handling or transporting of soil blocks or seedlings
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Soil Sciences (AREA)
- Botany (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a method for promoting the accumulation of chlorogenic acid in honeysuckle buds by using fungi, which adopts a cutting seedling method to cultivate honeysuckle, and performs fungi inoculation on honeysuckle branches in the cutting process to improve the content of chlorogenic acid in the honeysuckle buds; the fungus is Phellinus Ribes and Trichoderma. According to the method, the honeysuckle plants are treated by the fungi, so that the growth of the fungi is accompanied in the growth process of the honeysuckle, the fungi grow on the stem base part of the honeysuckle, the growth of the fungi can promote the accumulation of secondary metabolites in honeysuckle buds, and the quality of the honeysuckle is improved. The method has the advantages of wide raw material source, convenient operation and easy implementation, and has important significance for improving the quality of the honeysuckle in production.
Description
Technical Field
The invention relates to a method for promoting accumulation of secondary metabolites in honeysuckle buds, in particular to a method for promoting accumulation of chlorogenic acid in the honeysuckle buds by using fungi.
Background
Along with the rise of the trend of returning to nature, the traditional natural botanical drug is more and more favored by people. Honeysuckle is derived from a dry flower bud or a flower with a primary bloom of Lonicera japonica Thunb of Caprifoliaceae, is cold in nature and sweet in taste, has the effects of clearing heat, removing toxicity, cooling and dispelling wind heat, is a common traditional Chinese medicine, is widely applied to Chinese patent medicines of 1/3 according to statistics, such as Shuanghuanglian, Yinqiao detoxification pills, Yinhuang oral liquid and the like, particularly plays a great role in prevention and treatment of severe epidemic diseases such as SARS and avian influenza and is known as 'penicillin in traditional Chinese medicine'. At present, the demand of the domestic and foreign markets for honeysuckle is increasing day by day, and the requirements on the quality of medicinal materials are also increasing. The secondary metabolites in the bodies of medicinal plants are generally the basis of important substances for the clinical efficacy of traditional Chinese medicinal materials, and the chlorogenic acid content in the bodies of honeysuckle specified in the 2015 edition of Chinese pharmacopoeia is one of important indexes for measuring the quality of honeysuckle medicinal materials, so that the promotion of the accumulation of chlorogenic acid in the bodies of honeysuckle by adopting effective measures is more and more important for improving the quality of the honeysuckle medicinal materials.
The Phellinus scriptus Phylloporia ribacter (Schumach.: Fr.) Ryvarden is a fungus from Lonicera japonica plants, which is produced mainly in the Shandong Malus hupehensis and parasitizes the old dry or bare roots of the Lonicera japonica plants, and the fruiting body of the fungus is called the "Yinhua moth". The fungus is a medicinal fungus, has a long history in local, is loaded in the traditional Chinese medicinal material standard of Shandong province in 2002, contains active ingredients such as triterpenes, sterols, polysaccharides and styryl pyrones, and has the effects of clearing away heat and toxic materials, relieving swelling and sore throat, reducing blood sugar, resisting tumors and the like. At present, few researches are carried out on the phyllobacterium ribirum which is mainly prepared into food or medicinal raw materials, and the purposes in other aspects are not reported.
Disclosure of Invention
The invention aims to provide a method for promoting the accumulation of chlorogenic acid in honeysuckle buds by using fungi.
The lamellar bacteria Phylloporia ribirus (Schumach.: Fr.) Ryvarden is an original wild fungus growing on a honeysuckle plant, and researches show that the lamellar bacteria Phylloporia ribirus growing on the honeysuckle plant in the original wild state has no significant influence on the accumulation of secondary metabolites in honeysuckle buds, which is probably related to the relative substance-ecological balance of the long-term coexistence of the lamellar bacteria Phylloporia ribirrorhii and the honeysuckle flower buds. Through research, the two fungi are inoculated together to promote the accumulation of secondary metabolites in honeysuckle buds, so that the content of the secondary metabolites in the honeysuckle buds is greatly improved.
The specific technical scheme of the invention is as follows:
a method for promoting the accumulation of chlorogenic acid in honeysuckle buds by using fungi comprises the following steps: cultivating honeysuckle by adopting a cutting seedling method, and performing fungus inoculation on honeysuckle branches in the cutting process so as to improve the content of chlorogenic acid in honeysuckle buds; the fungus is Phellinus Ribes and Trichoderma.
According to the method, the accumulation of the secondary metabolite chlorogenic acid in the honeysuckle buds is improved in a mode of carrying out fungus inoculation on the honeysuckle branches in the cuttage process, so that the content of the chlorogenic acid is improved. The cutting seedling raising method comprises the following steps:
(1) irrigating the substrate and the cutting bed with a carbendazim aqueous solution with the mass concentration of 1/3000 in the first 2 days of cutting until the substrate and the cutting bed are completely wet;
(2) then exposing the substrate and the slotting bed in the sun for 2 days, and placing the substrate in the slotting bed;
(3) irrigating the substrate and the cutting bed with fungus bacterial liquid 2 hours before cutting until the substrate and the cutting bed are completely irrigated, wherein the fungus bacterial liquid is a mixed liquid of a black currant leaf-shaped layer bacterium liquid and a trichoderma bacterial liquid;
(4) sterilizing and disinfecting a honeysuckle cutting branch, soaking the honeysuckle cutting branch in fungus liquid for 10-12h, taking out the honeysuckle cutting branch, dipping rooting powder, then cutting, and watering thoroughly after cutting;
(5) after cuttage, performing cuttage seedling raising management on the honeysuckle cuttage branches until the cuttage seedlings grow to meet the requirements of transplanting or outplanting, then performing transplantation and seedling stage management (namely field management) on the cuttage seedlings meeting the requirements, and picking buds 15-20 days after the plants bud.
In the method, a honeysuckle plant is inoculated by adopting a fungus bacterial liquid, wherein the fungus bacterial liquid is a mixed liquid of a black currant phylloplasmic bacteria bacterial liquid and an trichoderma bacterial liquid. Wherein, the liquid can be obtained by culturing the seed body of Pheyloporium ribirum (Schumach.: Fr.) growing on a wild honeysuckle plant, or by culturing the seed body of Pheyloporium ribirum (Schumach.: Fr.) with the collection number of CGMCC NO 1195; the Trichoderma liquid can be obtained by culturing Trichoderma viride, which can be Trichoderma viride (Trichoderma viride) or Trichoderma harzianum (Trichoderma harzianum). Trichoderma viride or Trichoderma harzianum can be obtained from China general microbiological culture collection center or China type culture collection center, such as Trichoderma harzianum with the collection number of CGMCC NO 3.5488 or CGMCC NO 3.5492, Trichoderma harzianum with the collection number of CCTCC No. M2010119, Trichoderma viride (Trichoderma viride) JUWE1002 with the collection number of CGMCC NO 7035, or can be purchased from the market.
Further, the preparation method of the bacterial liquid of the phyllobacterium ribrum used by the invention comprises the following steps:
A. phenylloporia ribirus ribwort (Schumach.: Fr.) Ryvarden is inoculated on a slant culture medium and cultured at 29 ℃ for 5-7 days; the slant culture medium comprises the following components in percentage by weight: potato 20%, agar 1.5%, glucose 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, and vitamin 1 × 10%-3Percent, the balance of water;
B. inoculating the cultured mycelium of Ribes nigrum leaf to liquid culture medium, culturing at 29 deg.C and 150rpm for 5-6 days to obtain Ribes nigrum leaf mycelium liquid, and inoculating 5-8 mycelium masses per bottle per 1000ml liquid culture medium; the liquid culture medium comprises the following components in percentage by weight: 30% of potato, 2% of glucose, 1.4% of agar, 10% of wort (10Bx), 1.5% of sucrose, 0.2% of peptone, 4% of bran, 0.05% of monopotassium phosphate, 0.025% of magnesium sulfate and the balance of water.
Further, the preparation method of the trichoderma liquid used by the invention comprises the following steps:
(1) inoculating Trichoderma into slant culture medium, and culturing at constant temperature of 25-30 deg.C for 2-3 days; the slant culture medium comprises the following components in percentage by weight: 20% of potato, 2% of glucose, 1.6% of agar, 0.2% of peptone, 0.2% of monopotassium phosphate and 0.1% of magnesium sulfate heptahydrate;
(2) inoculating the strain obtained by slant culture into liquid culture medium according to the inoculum size of 5-10wt%, and culturing on a shaker at 25-30 deg.C and 200r/min for 6-7 days to obtain Trichoderma bacteria liquid; the liquid culture medium comprises the following components in percentage by weight: 20% of potato, 1% of sucrose, 1% of glucose, 0.18% of anhydrous sodium acetate, 0.54% of magnesium sulfate heptahydrate, 0.06% of peptone, 0.02% of manganese chloride, 0.005% of defoaming agent and the balance of water.
In the fungus bacterial liquid, the volume ratio of the black currant phylloplast bacterial liquid to the trichoderma fungus bacterial liquid is 2-8:1, and preferably 5: 1. The fungus liquid of the phyllobacterium ribirum and the fungus liquid of the trichoderma asperellum are obtained according to the method, and the fungus liquids are mixed according to a specified volume ratio to obtain the fungus liquid.
In the cuttage method, the substrate used in the step (1) is obtained by mixing river sand, vermiculite and humus according to the mass ratio of 2: 1.
In the cutting method, the preparation method of the honeysuckle cutting branches comprises the following steps: collecting cutting branches at about 9:00 am on cloudy days, shearing 4 mm-thick shoots or hard shoots which are strong and have no diseases and insect pests and grow for two years into cuttings with the length of about 30cm, cutting 6-8 knots on each cutting, cutting 1/2 leaves from each leaf at each knot, flattening the lower end cut of each cutting shoot into an oblique opening, binding 50 cuttings into 1 bundle, spraying moisture on the cuttings by a sprayer, putting the cuttings into a foam heat insulation box after the cuttings are completely wetted, covering a layer of wet towel on the cuttings, and waiting for cutting.
In the cuttage method, the honeysuckle cuttage branches in the step (4) are soaked in 0.06wt% potassium permanganate aqueous solution for 10-12h, and sterilization and disinfection are carried out. And (3) soaking the honeysuckle cutting branches in fungus liquid after sterilization and disinfection so as to inoculate fungi to the cutting branches as much as possible, and dipping rooting powder after soaking so as to perform cutting. The rooting powder is a treating agent for promoting the rooting of the branches, is commonly used in the field, can be prepared by self, and can also be purchased from the market.
In the cuttage method, during cuttage, the bevel end of a cuttage branch is vertically inserted into a seedling bed, the lower end of the cuttage branch is inserted into a substrate of 8-12cm, and the cuttage density is 0.5 multiplied by 0.25 cm. And (5) tightly treading and compacting after cuttage, and completely watering for 1 time. And then carrying out conventional management such as watering, fertilizing and the like in time, generally watering 1 time every 5 days, rooting and sprouting to start growing in 7-15 days, and transplanting and carrying out conventional field management after survival.
In the method, after the cuttage branches meet the transplanting conditions, sandy loam with loose soil, rich and good drainage and a place with convenient irrigation and a water source are selected for transplanting. After land selection, deeply ploughing over 30cm, breaking soil blocks, leveling and harrowing, and applying enough base fertilizer. Then making high ridges or high ridges with the width of 1.3m for planting. The planting land can utilize sporadic land blocks such as barren slopes, land edges, ditch sides, houses and the like, and is generally transplanted before germination in early spring or in dormant period in autumn and winter. When transplanting, the plants are planted in a well-arranged mannerDigging holes on the ground at row spacing of 150cm and plant spacing of 120cm, the width and depth of each hole are 30-40cm, generally per hm2Planting 3750-4200 holes. Applying 5kg of mixed soil fertilizer into each hole, uniformly mixing the mixed soil fertilizer with the bottom soil, planting 1 strong seedling in each hole, filling fine soil, compacting, treading, thoroughly watering for root fixing, watering once every 30 days, performing conventional field management, and picking up 15-20 days after bud emergence. Picking in morning every day, not sandwiching impurities of branches and leaves, and lightly picking, and drying the picked fresh flowers at 40-50 deg.C in time.
The method uses the fungus to treat the honeysuckle plants, so that the growth of the fungus is accompanied in the growth process of the honeysuckle, the fungus grows at the stem base part of the honeysuckle, the growth of the fungus can promote the accumulation of a secondary metabolite-chlorogenic acid in honeysuckle buds, and the quality of the honeysuckle is improved. The method has the advantages of wide raw material source, convenient operation and easy implementation, and has important significance for improving the quality of the honeysuckle in production.
Detailed Description
The present invention is further illustrated by the following specific examples, which are intended to be purely exemplary of the invention and are not intended to be limiting thereof.
Example 1
The honeysuckle is cultivated by adopting the following method, and flower buds are adopted:
1. preparation of honeysuckle cutting branches
In the last ten days of 4 months, collecting cutting branches at about 9:00 am on cloudy days, selecting 4mm thick robust tender branches or hard branches which grow robustly, have no diseases, insects, shrink or damage to skins, shearing the robust tender branches or hard branches into cuttings with the length of about 30cm, wherein each cutting has 6-8 knots, 1/2 leaves are respectively sheared from the leaves at each knot, and the cuts at the lower ends of the cuttings are leveled into oblique mouths. Binding every 50 cut branches into 1 bundle, spraying moisture to the branches by using a sprayer, putting the branches into a foam heat insulation box after the branches are completely wetted, covering a layer of wet towel on the box for moisture preservation, and covering the box with a cover for waiting for cuttage.
2. Preparation of fungus liquid
2.1 preparation of liquid fungus of leaf layer of Ribes nigrum
A. Preparation of the culture Medium
Preparation of slant culture medium: purchasing potato on the market, peeling, cutting into 1cm square pieces, adding potato pieces into water, sterilizing at 121 deg.C, boiling for 30min (paying attention to control of fire and adding water), and filtering with gauze to obtain filtrate. Adding agar, glucose, potassium dihydrogen phosphate, magnesium sulfate, and vitamins into the filtrate, stirring with glass rod, heating to dissolve, adding water, adjusting pH to 6, placing into test tube, sterilizing, cooling, and making into slant culture medium; the formula of the slant culture medium is as follows (wt%): potato 20%, agar 1.5%, glucose 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, and vitamin 1 × 10%-3Percent, balance water.
Preparation of liquid medium: purchasing potato on the market, peeling, cutting into 1cm square pieces, adding potato pieces into water, sterilizing at 121 deg.C, boiling for 30min (paying attention to control of fire and adding water), and filtering with gauze to obtain filtrate. Adding glucose, agar, wort (10Bx), sucrose, peptone, bran, potassium dihydrogen phosphate and magnesium sulfate into the filtrate, supplementing water, adjusting pH to 6, and sterilizing to obtain liquid culture medium; the formula of the liquid culture medium is (wt%): 30% of potato, 2% of glucose, 1.4% of agar, 10% of malt extract (10Bx), 1.5% of sucrose, 0.2% of peptone, 4% of bran, 0.05% of monopotassium phosphate, 0.025% of magnesium sulfate and the balance of water.
B. Preparation of bacterial liquid of phyllobacterium ribis
Inoculation: the inoculation work is carried out on a clean bench. When Phenylloporia ribirus (Schumach.: Fr.) fruiting body of Phenylloporia ribirus growing on wild Lonicera japonica is used as strain, the fruiting body is sterilized with ultraviolet lamp for 40min before inoculation, part of tissue growing at the edge of fruiting body is inoculated into a test tube containing slant culture medium under sterile condition, and cultured at 29 deg.C for 5-7 days. When Phenylloporia ribirus ribwort (Schumach.: Fr.) with the collection number of CGMCC NO 1195 is used as a strain, the strain is directly inoculated into a test tube containing a slant culture medium and cultured for 5-7 days at 29 ℃.
Liquid culture: inoculating the solid cultured Ribes nigrum lamina mycelium in the slant test tube, inoculating into liquid culture medium, removing the slant culture medium containing agar at the bottom, wherein the diameter of the mycelium is about 1-2mm, keeping the size of the mycelium consistent as much as possible, inoculating 8 mycelium into each 1000ml of liquid culture medium, and culturing in a triangular flask at 29 deg.C and 150rpm for 6 days to obtain Ribes nigrum lamina mycelium liquid.
2.2 preparation of Trichoderma bacteria liquid
A. Preparation of the culture Medium
Preparation of slant culture medium: purchasing potato on the market, peeling, cutting into 1cm square pieces, adding potato pieces into water, sterilizing at 121 deg.C, boiling for 30min (paying attention to control of fire and adding water), and filtering with gauze to obtain filtrate. Adding glucose, agar, peptone, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate into the filtrate, stirring with glass rod, heating to dissolve, adding water, adjusting pH to 6, placing into test tube, sterilizing, cooling, and making into slant culture medium; the formula of the slant culture medium is as follows (wt%): 20% of potato, 2% of glucose, 1.6% of agar, 0.2% of peptone, 0.2% of monopotassium phosphate and 0.1% of magnesium sulfate heptahydrate.
Preparation of liquid medium: purchasing potato on the market, peeling, cutting into 1cm square pieces, adding potato pieces into water, sterilizing at 121 deg.C, boiling for 30min (paying attention to control of fire and adding water), and filtering with gauze to obtain filtrate. Adding sucrose, glucose, anhydrous sodium acetate, magnesium sulfate heptahydrate, peptone, manganese chloride and defoaming agent into the filtrate, supplementing water, adjusting pH to 6, and sterilizing to obtain liquid culture medium. The formula of the liquid culture medium is (wt%): 20% of potato, 1% of sucrose, 1% of glucose, 0.18% of anhydrous sodium acetate, 0.54% of magnesium sulfate heptahydrate, 0.06% of peptone, 0.02% of manganese chloride, 0.005% of defoaming agent and the balance of water.
B. Preparation of trichoderma liquid
Inoculation: the inoculation work is carried out on a clean bench. Trichoderma harziamum (Trichoderma harziamum) with the preservation number of CCTCC No. M2010119 is taken as a strain, the strain is directly inoculated into a test tube containing a slant culture medium, and the culture is carried out for 2-3 days at the temperature of 30 ℃.
Liquid culture: inoculating the strain obtained by slant culture into a liquid culture medium according to the inoculation amount of 8 wt%, and culturing for 7 days on a shaking table at 30 ℃ and 200r/min to obtain the trichoderma liquid.
2.3 preparation of fungal solutions
And (3) uniformly mixing the obtained bacterial liquid of the phyllobacterium ribirum and the obtained bacterial liquid of the trichoderma asperellum according to the volume ratio of 5:1 to obtain the fungal liquid.
3. Preparation of honeysuckle cutting seedling raising bed
The honeysuckle cutting and transplanting bed is a plastic container with water leakage holes at the bottom and the length of 2m, the width of 1m and the height of 0.25m, and culture mediums filled in the plastic container are river sand, vermiculite and humus which are mixed according to the mass ratio of 2: 1. And irrigating the substrate and the cutting bed with an aqueous solution of carbendazim with the mass concentration of 1/3000 in the first 2 days of cutting until the substrate and the cutting bed are completely wetted. The substrate and the bed were then exposed to the sun for 2 days, and the substrate was placed in the bed. Irrigating with fungus liquid 2 hours before cuttage until the substrate and the cutting bed are completely irrigated.
4. Cuttage of honeysuckle branches
Putting the honeysuckle stem cutting branches into 0.06wt% potassium permanganate aqueous solution, soaking for 10-12h, and sterilizing. Then soaking the mixture in fungus liquid (the volume ratio of the liquid of the fungus of the phyllopodium curriculum to the liquid of the fungus of the trichoderma is 5:1) for 12 hours, taking out the mixture, dipping the mixture in rooting powder, quickly dipping the mixture in the rooting powder for 20 seconds, and then carrying out cuttage. When in cuttage, the oblique mouth end of a cuttage branch is vertically inserted into a seedling bed, the lower end of the cuttage branch is inserted into a substrate by 8-12cm, and the cuttage density is generally 0.5 multiplied by 0.25 cm. And (5) tightly treading and compacting after cuttage, and completely watering for 1 time. And then carrying out conventional management such as watering, fertilizing and the like in time, generally watering 1 time every 5 days, rooting and sprouting to start growing in 7-15 days, and transplanting after survival.
5. Transplanting process
After the cuttage branches meet the transplanting conditions, sandy loam with loose soil, rich fertilizer and good drainage and a place with convenient irrigation and a water source are selected for transplanting. After land selection, deeply ploughing over 30cm, breaking soil blocks, leveling and harrowing, and applying enough base fertilizer. Then making high ridges or high ridges with the width of 1.3m for planting. The planting field can be used forTransplanting is generally carried out before germination in early spring or in dormancy stage in autumn and winter by barren slopes, ground edges, ditch sides and sporadic plots before and after houses. During transplanting, digging holes with the width and depth of 30-40cm respectively at the row spacing of 150cm and the plant spacing of 120cm on the prepared planting land, wherein the width and depth are generally per hm2Planting 3750-4200 holes. Applying 5kg of mixed soil fertilizer into each hole, uniformly mixing the mixed soil fertilizer with the bottom soil, planting 1 strong seedling in each hole, filling fine soil, compacting, treading, thoroughly watering for root fixing, watering once every 30 days, and performing conventional field management. The plants can be picked up 15-20 days after the plants grow to bud. Picking in morning every day, not sandwiching impurities of branches and leaves, and lightly picking, and drying the picked fresh flowers at 40-50 deg.C in time.
Example 2
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the Trichoderma strain is Trichoderma viride (Trichoderma viride) JUWE1002 with the preservation number of CGMCC NO. 7035.
Example 3
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the Trichoderma species used was Trichoderma harzianum YS-JZ0386, deposited at the American type culture Collection.
Example 4
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the volume ratio of the bacterial liquid of the phyllobacterium ribirum to the bacterial liquid of the trichoderma asperellum is 2: 1.
Example 5
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the volume ratio of the bacterial liquid of the phyllobacterium ribirum to the bacterial liquid of the trichoderma asperellum is 8: 1.
Comparative example 1
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the fungus bacterial liquid is single bacterial liquid of black currant leaf-shaped layer bacteria.
Comparative example 2
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the fungus bacterial liquid is single trichoderma bacterial liquid.
Comparative example 3
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the fungus bacterial liquid is a mixed liquid of a black currant leaf-shaped lamina bacterial liquid and a bacillus megaterium bacterial liquid in a volume ratio of 5: 1.
The preparation method of the bacillus megaterium liquid comprises the following steps:
inoculating Bacillus megaterium into liquid culture medium according to the inoculation amount of 1 wt%, adjusting the temperature to 35-38 deg.C, controlling the aseptic air quantity at 90-100m3/h, and fermenting for 2 days. The formula of the liquid culture medium is as follows: 1.2% of corn flour, 0.3% of yeast powder, 0.4% of bean cake powder, 0.05% of sodium chloride, 0.06% of magnesium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate and the balance of water, sterilizing at 121 ℃, and keeping the pH value at 7.0-7.2.
Comparative example 4
Honeysuckle plants were cultivated according to the cutting seedling method of example 1, with flower buds collected, except that: the substrate, the cutting bed and the honeysuckle cutting branches are not treated by fungus liquid.
In order to verify the influence of the cultivation method on the content of the secondary metabolite of the flower bud, a cultivation experiment is carried out on the Shandong Malus hupehensis. The experimental method comprises the following steps: selecting proper transplanting land, dividing the transplanting land into a plurality of cells, wherein each cell has an area of 66.7m2Honeysuckle was cultivated in a random block arrangement in each cell according to one of examples 1-5 and comparative examples 1-4, each of which was repeated 4 times. Besides different treatments of fungus liquid, other seedling management is kept consistent, such as fertilization, pesticide application, watering and weeding.
In the experimental process, the growth condition of the honeysuckle plant is recorded, chlorogenic acid in the flower buds is detected after the flower buds are collected, and the influence of various methods on the content of the chlorogenic acid is evaluated.
The chlorogenic acid content detection method comprises the following steps:
1. preparation of control solutions: precisely weighing appropriate amount of chlorogenic acid reference substance, placing in brown measuring flask, adding 50% methanol to obtain solution containing 40 μ g per 1ml (preservation at below 10 deg.C).
Preparation of sample solution: collecting bud sample, oven drying at 40-50 deg.C, grinding, collecting fine powder (sieved with No. 4 sieve) of about 0.5g, precisely weighing, placing in conical flask with plug, precisely adding 50ml of 70% ethanol, ultrasonic treating for 1 hr, cooling, weighing, supplementing lost weight with 70% ethanol, shaking, and filtering. The filtrate (10 ml) was measured with precision and filtered into a liquid phase vial with an organic membrane (pore size 0.2 μm) for use.
2. And (3) content detection: the detection was carried out by liquid chromatography using C18(4.60 mm. times.250 mm, 1.7 μm) as column, acetonitrile-0.4 wt% phosphoric acid solution as mobile phase (volume ratio 13: 87), detection wavelength of 327nm, sample size of 20uL and column temperature of 40 ℃. The number of theoretical plates is not less than 1000 calculated according to chlorogenic acid peak.
The specific detection method comprises the following steps: preparing the reference solution into different concentrations, and detecting by liquid chromatography to obtain a standard curve with the concentration and peak area as horizontal and vertical coordinates. And performing liquid chromatography detection on each sample solution by adopting the same method, and calculating the content of chlorogenic acid in each sample according to the peak area.
The experimental process shows that when the methods of examples 1-5 and comparative examples 2 and 3 are used for honeysuckle cultivation, the growth speed of honeysuckle plants is higher than that of comparative examples 1 and 4, the buds of the plants are earlier than that of comparative examples 1 and 4, and the average height of the plants is about 1.5 cm. And the disease and pest incidence rate of the honeysuckle plants of the examples 1-5 and the comparative examples 2 and 3 is low. The results of chlorogenic acid detection of the collected flower buds are shown in table 1 below, and it can be seen from the table that the chlorogenic acid content in the flower buds obtained by the methods of examples 1 to 5 is significantly higher than that in the methods of various proportions, but the effect of using only the bacterial liquid of the phyllobacterium triquetrum or the trichoderma liquid, or using other bacteria in combination with the bacterial liquid of the phyllobacterium triquetrum is not significant, and the fungal liquid of the invention has a good promotion effect on the accumulation of the secondary metabolites of the honeysuckle flower buds.
TABLE 1 content of chlorogenic acid in honeysuckle buds obtained by the example and comparative example cultivation method
| Chlorogenic acid content (%) | |
| Example 1 | 2.879 |
| Example 2 | 2.827 |
| Example 3 | 2.873 |
| Example 4 | 2.687 |
| Example 5 | 2.658 |
| Comparative example 1 | 1.605 |
| Comparative example 2 | 1.587 |
| Comparative example 3 | 1.656 |
| Comparative example 4 | 1.554 |
Claims (6)
1. A method for promoting the accumulation of chlorogenic acid in honeysuckle buds by using fungi adopts a cutting seedling method to cultivate honeysuckle, and is characterized in that: performing fungus inoculation on the honeysuckle branches in the cutting process to improve the content of chlorogenic acid in honeysuckle buds; the fungus is Phellinus Ribes and Trichoderma; the method specifically comprises the following steps:
(1) irrigating the substrate and the cutting bed with aqueous solution of carbendazim with the mass concentration of 1/3000 2 days before cutting until the substrate and the cutting bed are completely wetted;
(2) then exposing the substrate and the slotting bed in the sun for 2 days, and placing the substrate in the slotting bed;
(3) irrigating the substrate and the cutting bed with fungus bacterial liquid 2 hours before cutting until the substrate and the cutting bed are completely irrigated, wherein the fungus bacterial liquid is a mixed liquid of a black currant leaf-shaped layer bacterium liquid and a trichoderma bacterial liquid;
(4) sterilizing and disinfecting a honeysuckle cutting branch, soaking the honeysuckle cutting branch in fungus liquid for 10-12h, taking out the honeysuckle cutting branch, dipping rooting powder, then cutting, and watering thoroughly after cutting;
(5) after cuttage, performing cuttage seedling raising management on the honeysuckle cuttage branches until the cuttage seedlings grow to meet the requirements of transplanting or outplanting, then performing transplantation and seedling stage management on the cuttage seedlings meeting the requirements, and picking flower buds 15-20 days after the plants bud;
the fungus bacterial liquid is a mixed liquid of a blackcurrant phyllodes bacterial liquid and an trichoderma bacterial liquid, and the volume ratio of the blackcurrant phyllodes bacterial liquid to the trichoderma bacterial liquid is 2-8: 1;
the preparation method of the bacterial liquid of the phyllostachys ribis comprises the following steps:
A. inoculating Phellinus Linteus to slant culture medium, and culturing at 29 deg.C for 5-7 days; the slant culture medium comprises the following components in percentage by weight: potato 20%, agar 1.5%, glucose 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, and vitamin 1 × 10%-3Percent, the balance of water;
B. inoculating the cultured mycelium of Ribes nigrum leaf to liquid culture medium, culturing at 29 deg.C and 150rpm for 5-6 days to obtain Ribes nigrum leaf mycelium liquid, and inoculating 7-8 mycelium masses per 1000ml liquid culture medium; the liquid culture medium comprises the following components in percentage by weight: 30% of potato, 2% of glucose, 1.4% of agar, 10% of 10Bx malt extract, 1.5% of sucrose, 0.2% of peptone, 4% of bran, 0.05% of monopotassium phosphate, 0.025% of magnesium sulfate and the balance of water;
the preparation method of the trichoderma liquid comprises the following steps:
(1) inoculating Trichoderma into slant culture medium, and culturing at constant temperature of 25-30 deg.C for 2-3 days; the slant culture medium comprises the following components in percentage by weight: 20% of potato, 2% of glucose, 1.6% of agar, 0.2% of peptone, 0.2% of monopotassium phosphate and 0.1% of magnesium sulfate heptahydrate;
(2) inoculating the strain obtained by slant culture into liquid culture medium according to the inoculum size of 5-10wt%, and culturing on a shaker at 25-30 deg.C and 200r/min for 6-7 days to obtain Trichoderma bacteria liquid; the liquid culture medium comprises the following components in percentage by weight: 20% of potato, 1% of sucrose, 1% of glucose, 0.18% of anhydrous sodium acetate, 0.54% of magnesium sulfate heptahydrate, 0.06% of peptone, 0.02% of manganese chloride, 0.005% of defoaming agent and the balance of water.
2. The method of claim 1, further comprising: the bacterial liquid of the black currant phyllodes takes the fruiting body of the black currant phyllodes growing on a honeysuckle plant in a wild state as a strain, or takes the black currant phyllodes with the preservation number of CGMCC NO 1195 as the strain; the trichoderma liquid takes trichoderma as a strain, and the trichoderma is trichoderma viride or trichoderma harzianum.
3. The method of claim 1, further comprising: in the fungus bacterial liquid, the volume ratio of the black currant leaf-shaped layer bacterial liquid to the trichoderma fungus liquid is 5: 1.
4. The method of claim 1, further comprising: in the step (1), the matrix is obtained by mixing river sand, vermiculite and humus according to the mass ratio of 2: 1; in the step (4), the honeysuckle cutting branches are soaked in 0.06wt% potassium permanganate aqueous solution for 10-12h, and then are sterilized and disinfected.
5. The method of claim 1, further comprising: the preparation method of the honeysuckle cutting branch comprises the following steps: collecting cutting branches at about 9:00 am on cloudy days, shearing 4 mm-thick shoots or hard shoots which are strong and have no diseases and insect pests and grow for two years, shearing the shoots into cuttings with the length of about 30cm, cutting 6-8 knots on each cutting, respectively cutting 1/2 leaves from the leaves at each knot, flattening the cuts at the lower ends of the cutting sticks into inclined openings, binding 50 sticks into 1 bundle, spraying moisture on the branches by using a sprayer, putting the branches into a foam heat insulation box after the branches are completely wetted, covering a layer of wet towel on the branches for moisturizing, and covering a cover for waiting for cutting.
6. The method of claim 1 or 5, wherein: when in cuttage, the oblique mouth end of a cuttage branch is vertically inserted into a seedling bed, the lower end of the cuttage branch is inserted into a matrix of 8-12cm, and the cuttage density is 0.5cm multiplied by 0.25 cm.
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| CN103229716A (en) * | 2013-02-20 | 2013-08-07 | 张龙霏 | Method for seed selection of excellent honeysuckle flower Lonicera japonica Thunb II variety |
| CN106336329A (en) * | 2016-08-29 | 2017-01-18 | 佛山市艳晖生物科技有限公司 | Microbial bacterial fertilizer special for honeysuckles and preparation method thereof |
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| CN106336329A (en) * | 2016-08-29 | 2017-01-18 | 佛山市艳晖生物科技有限公司 | Microbial bacterial fertilizer special for honeysuckles and preparation method thereof |
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| 《丛枝菌根真菌对西南岩溶地区干旱及干湿交替下金银花根系生长的影响》;刘锦春 马晔 陶建平 高凯敏 梁千慧;《北京林业大学学报》;20151031;第37卷(第10期);第110-115页 * |
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