CN106613276A - Artemisia apiacea seedling cultivating method and special trichoderma atroviride fertilizer thereof - Google Patents
Artemisia apiacea seedling cultivating method and special trichoderma atroviride fertilizer thereof Download PDFInfo
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- CN106613276A CN106613276A CN201710013950.1A CN201710013950A CN106613276A CN 106613276 A CN106613276 A CN 106613276A CN 201710013950 A CN201710013950 A CN 201710013950A CN 106613276 A CN106613276 A CN 106613276A
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- trichoderma atroviride
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- 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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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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Abstract
The invention provides an artemisia apiacea seedling cultivating method which comprises the following steps: holing rhizosphere soil of artemisia apiacea seedlings, irrigating trichoderma atroviride K fertilizer and then covering soil. The invention provides trichoderma atroviride K fertilizer applied to cultivation of artemisia apiacea seedlings, and fills a blank of the artemisia apiacea planting field. Meanwhile, the growth and development of the artemisia apiacea seedlings can be effectively promoted by adopting the trichoderma atroviride K fertilizer, and the trichoderma atroviride K fertilizer is greener and more environment-friendly compared with the fertilizer.
Description
Technical field
The present invention relates to a kind of bacterial manure, more specifically to a kind of sweet wormwood seedling culture method and its special Trichoderma atroviride
K bacterial manure.
Technical background
Trichoderma (Trichoderma spp.) category Fungi Imperfecti clump stalk embraces mesh, a Cong Gengbao sections fungi, be widely present in soil,
In the ecological environments such as rhizosphere, Ye Wei, seed and bulb.Trichoderma colony growth is rapid, in indefinite form flocculence or fine and close clump pencil.
In green more than the color on its surface.Mycelia has every branch.Chlamydospore with or without.Conidiophore is the short lateral branch of mycelia.Side
Symmetrical or alternate branch on branch, forms 2 grades and 3 grades of branches.Crotch angle is acute angle or is bordering on right angle, in branch ends formation bottle
Shape stigma.Conidium mostly is oval.Colourless or green, cluster is born in stigma top.
Trichoderma is a kind of mesophilic fungi, and growth thermophilic is 20-28 DEG C, can be grown under the conditions of 37 DEG C, but in 48 DEG C of bars
Can not grow under part, in 6 DEG C or 32 DEG C still well-growns.The growth of trichoderma requires higher levels of humidity, its nutrient growth it is relatively wet
Degree requires more than 92%, and the formation of spore needs the humidity of 93%-95%, thus vitality of the trichoderma in slowly drained soil is more dry
Property soil in it is strong.Illumination is strengthened with logarithmic scale and can promote conidial generation. studies have found that 380nm and 440nm ripples
Long photoinduction power is most strong, and the effect that trichoderma Jing daylight processes 3min or Jing ultraviolets process 10-30S induction product spores is more preferable.
The most suitable growth pH value of trichoderma is 5-5.5, and growth is likely on the culture medium that pH value is 1.5 or 9.0, but acid condition compares alkali
Germination rate under the conditions of property is higher.CO2Impact to trichoderma growth depends on CO2Concentration and culture medium pH value, in basic group
In matter, the CO of high concentration2Be conducive to the growth of Trichoderma.
Trichoderma strain can be by the use of gas chromatography as carbon source, preferably monosaccharide and disaccharide, polysaccharide, purine, pyrimidine
With amino acid etc..Ammonium is the nitrogen source that Trichoderma most easily utilizes, and other nitrogen sources such as amino acid, urea, nitrate, nitrite also can
Maintain its normal growth.Especially good with the growth of asparagus fern Men Suwei nitrogen sources, nitrogen content is low, can promote to embrace son formation, to high concentration nitre
The negatively influencing of hydrochlorate can be compensated due to the presence of magnesium sulfate.Growth of the inorganic salts to trichoderma is critically important.To Trichoderma viride
For, magnesium ion can promote it to grow, and copper ion can promote conidiospore pigment to be formed, and iron ion is also very heavy to the formation for embracing son
Will.
Find that Trichoderma lignorum (T.lignorum) is parasitized after various plants disease fungus from Weindling in 1932, wood
Mould shows huge application potential as a class biocontrol microorganisms.Many researchers observe algae, Fungi Imperfecti, sac fungus and
There is trichoderma parasitic animal and plant in basidiomycetes.The parasitic phytopathogen of Trichoderma energy is that antagonism object includes Rhizoctonia
((Rhimetonia), pyrenomycetes ((Selerolium), Sclerotinia (Sclerotinia), long compacted armful of category (Helminth), sickle
Knife Pseudomonas (Fusarium), hair disc embrace category (Colletotriehum), wheel branch and embrace category (Yerticillium), Venturia
(Venturia), inner seat shell category (Endothia), ((Pythium), Phytophthora (Phytophlhora) hears seat shell category to pythium
And Fusicladium ((Fusicladium) etc. (Diaporthe).Therefore, there is trichoderma disease resistance to act on；Additionally, trichoderma has very
Strong decomposition of cellulose ability, is conducive to Crop to utilize, with the effect for promoting plant growth；Trichoderma also degrading pesticide；
And to environment non-hazardous.In a word, trichoderma is one of good quality strain of research and development organic fungi-manure.
Organic fungi-manure is a kind of new-type fertilizer that will be prepared from one or more beneficial microbe implantation organic matter, many
The organic matter of effect organic fungi-manure is both the basis of fertility, is again beneficial microorganism culture matrix.This new-type fertilizer has rush concurrently
Growth, it is disease-resistant, improve soil property, without the multiple advantage such as agriculture is residual, at home and abroad widely applied at present, and achieve significantly effect
Really.
Studies in China person king is intelligent inside, Zhao Peijie etc. obtains more thorough to the microbial manure Effect study of trichoderma, according to Zhao Pei
Clean report, the microorganism multiple-effect organic bacteria with Trichoderma harzianum zymotic fluid as main component of Zhejiang University's research and development from 1991
Fertilizer, to crop yield effect is significant, and in 1997 by provincial-level appraisal, obtains fertilizer registration and national patent.The height registered
Effect microbial biomass nitrogen is formed by Trichoderma harzianum submerged fermentation liquid Jing reduced pressure concentrations are refined, and protein content is 45%.Production procedure
For Trichoderma harzianum bacterial classification → flat board culture → agitated submerged culture → submerged fermentation → measure → filtration → sediment (discarding) → clear
Night → reduced pressure concentration preparation → drying → crushing → sample.Its product finds and uses traditional urea, matting through field experiment
Fertilizer is compared, obvious to the effect of increasing production of Chinese cabbage, and using simple, is had broad application prospects on vegetables.
The prestige of biotechnology research institute of Zhejiang University development and popularization it is green up to (VIRIDA) bacterial manure based on Trichoderma viride, hair shell
Mixture is made supplemented by bacterium, bacillus subtilis, is mainly used in green vegetables production, dry crop, Grassland protection, it is also possible to make
Flower fertilizer.The biologic product is blended into trichoderma, cupreum and beneficial bacteria with domestic waste as substratess, and it both contained
Organic and inorganic constituents needed for plant growth, and promote the effect of growth with diseases prevention, and can be a kind of with improved soil
Multi-functional bio-bacterial manure.Prestige is green up at home and abroad still belonging to pioneering, and product also has a good application prospect.
Sharp river board microorganism fertilizer (Trichoderma viride) with Trichoderma viride as main component, Shi Li rivers industrial corporation has in Li Jiang
On the basis of machine fertilizer, a kind of trichoderma microbial manure for developing of introducing technology.The bacterial manure to cucumber, celery, rape etc. all
There is significant production-increasing function, and precocity can be promoted, improve crop quality, may also suppress the generation of pest and disease damage.
It can be seen that, the trichoderma microbial manure with Trichoderma viride as main component has multi-effect, and meets current agriculture
Production environmental protection is nuisanceless without agriculture is residual and requirement of sustainable development, with wide market prospects.
Sweet wormwood ArtemisiaannuaL., also known as artemisia annua, are the annual medicinal herb plants of composite family.With heat-clearing solution
Heat, except steaming, effect of preventing malaria.For summer-heat evil heating, fever due to yin deficiency, night fever abating at dawn, hectic fever due to yin labor heat, malaria fever and chills, jaundice with damp-heat pathogen.
Its extract qinghaosu has the antimalarial performance of high-efficiency low-toxicity to cerebral malaria and chloroquine malaria, and WHO has assert Artequick
Patent medicine is at present most safely and effectively antimalarial.Although at present the drug artemisinin can be artificial synthesized, cost is very high, fails to produce
Industry.It is main to extract from sweet wormwood plant.Sweet wormwood raw material applies in a large number chemical fertilizer essentially from artificial cultivation because long-term in production,
Cause soil acidification, environmental pollution.Urgently find environmental protection to be substituted without the residual fertilizer of agriculture.And we isolate one plant it is deep
Green trichoderma (, Trichoderma aureoviride) and K bacterial strains, there are significant growth-promoting functions to sweet wormwood growth of seedling.Therefore,
Make bacterial manure with the bacterial strain, can reduce or even avoid the administration of sweet wormwood growth of seedling period chemical fertilizer, so as to reach soil protection and
The purpose of environment.
The content of the invention
For produced problem in sweet wormwood growth of seedling, while this area is also without a kind of bacterial manure suitable for sweet wormwood nursery
Problem, the invention provides a kind of sweet wormwood seedling culture method and its special Trichoderma atroviride K bacterial manure.
Wherein, a kind of sweet wormwood seedling culture method, is that duck eye is beaten in sweet wormwood seedling Rhizosphere Soil, and Trichoderma atroviride K bacterial manure is applied in filling
Blinding afterwards.
The amount of application of the Trichoderma atroviride K bacterial manure is 3g bacterium ball/strain.
The step of a kind of Trichoderma atroviride K bacterial manure for being exclusively used in sweet wormwood seedling culture, preparation of the Trichoderma atroviride K bacterial manure
For：
1) Trichoderma atroviride K bacterial classifications under aseptic technique, are taken and is inoculated in the fluid nutrient medium for preparing；
2) fluid nutrient medium for meeting Trichoderma atroviride K is placed in shaking table, 130 revs/min of rotating speed, 25 DEG C of environment temperature, during culture
Between 6-8 days, cover with fluid nutrient medium after bacterium ball take out；
3) surplus liquid culture medium is filtered off, with clear water suspended bacteria ball, obtains Trichoderma atroviride K bacterial manure.
The preparation method of the Trichoderma atroviride K bacterial classifications is comprised the following steps：
1) in June then, disease-free spot stalwartness sweet wormwood side shoot is taken, is cut into segment, wash away soil dust, soaked with washing powder water
1 minute, flowing water was standby after rinsing；
2) on superclean bench, 75% ethanol purge is used, the sterilization of 1/1000 mercuric chloride, sterile water wash 4-5 time, with aseptic
Filter paper suck dry moisture, is cut into the little stem section of 0.5-1 centimeter lengths；
3) little stem section is placed on potato glucose plating medium, and 25 DEG C are cultivated 7 ± 1 days, are grown around picking stem section
Hypha,hyphae；
4) hypha,hyphae is continued on potato dextrose culture-medium culture, until obtaining the fungus colony for purifying till；
5) above-mentioned fungus colony is done into Molecular Identification, chooses Trichoderma atroviride K bacterial strains as the bacterial classification of Trichoderma atroviride K bacterial manure.
The preparation method of the potato dextrose culture-medium is：Weigh 200 grams of potato and cut fritter, liquor water filtration is added
20 grams of glucose, 7 grams of agar powder is settled to 1 liter, after 121 DEG C sterilize 30 minutes, pours plate on superclean bench, cools down
It is standby afterwards.
The fluid nutrient medium preparation method is：200 grams of potato is weighed, fritter is cut, is boiled 20 minutes, removed slag and take juice,
20 grams of glucose is added, is added water and is settled to 1 liter.1 liter of triangular flask of packing, 250-500 milliliters/bottle.121 DEG C of sterilizings are obtained for 30 minutes
Arrive.
The method have the benefit that：The invention provides a kind of Trichoderma atroviride K for being applied to sweet wormwood seedling culture
Bacterial manure, has filled up the blank of sweet wormwood field of planting.Simultaneously sweet wormwood seedling can be effectively facilitated using Trichoderma atroviride bacterium K bacterial manure
Grow, and than chemical fertilizer environmental protection.
Specific embodiment
The preparation of the Trichoderma atroviride bacterial manure of embodiment 1
1st, the preparation of Trichoderma atroviride K bacterial manure
(1) screening of Trichoderma atroviride strain K
In June, disease-free spot stalwartness sweet wormwood side shoot is taken, cut 3 centimeter length segments, wash away soil dust, with washing powder water 1 point is soaked
Clock, flowing water is rinsed 40 minutes, and on superclean bench, with 75% ethanol purge 1 minute, 1/1000 mercuric chloride was sterilized 5 minutes, aseptic
Water is cleaned 4-5 time, uses aseptic filter paper suck dry moisture, is cut into 0.5-1 centimeter length segments, is placed in potato glucose PDA culture medium,
25 DEG C are cultivated 7 days or so, and the fungi grown around picking stem section continues culture in new PDA culture medium, until being purified
Fungus colony till.Gained bacterium colony send Huada gene company to do Molecular Identification, and it is Trichoderma atroviride to identify one plant of endogenetic fungus,
Tube propagation base (composition is with potato dextrose culture-medium) is inoculated in, 25 DEG C of cultures are standby, name as Trichoderma atroviride K.
(2) preparation of Trichoderma atroviride K bacterial manure
On superclean bench, under aseptic technique, the fritter of test tube strains one of Trichoderma atroviride K is taken, be inoculated in preparation
Good triangular flask fluid nutrient medium, is subsequently placed in shaking table, and condition of culture is set to 25 DEG C, and 130 revs/min of rotating speed cultivates 6-8
My god.Take out after bacterium ball is covered with fluid nutrient medium.With gauze surplus liquid culture medium is filtered off (because the liquid is given birth to sweet wormwood seedling
Length has inhibitory action), with a small amount of clear water suspended bacteria ball, obtain Trichoderma atroviride K bacterial manure.
2nd, the preparation of Trichoderma atroviride L bacterial manure is compareed
Trichoderma atroviride is Hyperparasite, can infect other fungies, therefore we cultivate the weaker Morciiella Esculeuta Mycelia of activity, with
Attract the Trichoderma atroviride spore in air, so as to isolated Trichoderma atroviride strain.Its incubation step is as follows：
(1) screening of Trichoderma atroviride strain L
1. hickory chick barley inoculum culture
Wheat berry is soaked in clear water 24 hours, soaks mistake heart, drains away the water, and fills Cans, and 121 DEG C sterilize 40 minutes,
Hickory chick test tube strains are inoculated with superclean bench, sealing is cultivated 7 days or so in 25 DEG C.After wheat covers with Morciiella Esculeuta Mycelia
It is standby.
2. the culture of Trichoderma atroviride L bacterial classifications
Fertile soil fills Cans, and 121 DEG C sterilize 40 minutes, and hickory chick barley inoculum is disperseed after granulating, to be buried in the earth about 1-2
Centimetre, plastic foil sealing is cultivated about 7 days in 15 DEG C, and Morciiella Esculeuta Mycelia to be covered with and open plastic foil behind native face, in 20 DEG C of cultures, is treated
Native face is grown after dark green spore, takes spore, and potato dextrose culture-medium plate (potato grape is inoculated on superclean bench
Sugar culture-medium preparation method：200 grams of potato cuts fritter, and liquor water filtration adds 20 grams of glucose, 7 grams of agar powder to be settled to 1
Rise, after 121 DEG C sterilize 30 minutes, pour on superclean bench standby after plate cooling), 25 DEG C are cultivated 4-7 days, grow bacterium colony
Afterwards, colony edge mycelium inoculation is taken in potato dextrose culture-medium plate, so repeatedly after purification, the bacterial classification for obtaining send Hua Da base
Because company carries out Molecular Identification, after confirming as Trichoderma atroviride, Tube propagation base (composition is with potato dextrose culture-medium) is inoculated in,
25 DEG C of cultures are standby, name as Trichoderma atroviride L.
(2) preparation of Trichoderma atroviride L bacterial manure
On superclean bench, under aseptic technique, the fritter of Trichoderma atroviride L test tube strains one is taken, be inoculated in and prepare
Triangular flask fluid nutrient medium, be subsequently placed in shaking table, condition of culture is set to 25 DEG C, and 130 revs/min of rotating speed is cultivated 6-8 days.
Take out after bacterium ball is covered with fluid nutrient medium.With gauze surplus liquid culture medium is filtered off (because the liquid is to sweet wormwood growth of seedling
Have inhibitory action), with a small amount of clear water suspended bacteria ball, obtain Trichoderma atroviride L bacterial manure.
Application of the Trichoderma atroviride K bacterial manure of embodiment 2 in sweet wormwood seedling culture
Test is carried out in the 4-6 months then, and now temperature is beneficial to Trichoderma atroviride mycelial growth.Sweet wormwood seedling is divided into into 3 groups,
55 plants per group, respectively control group, Trichoderma atroviride K bacterial manure groups and Trichoderma atroviride L bacterial manure groups beat little in sweet wormwood seedling Rhizosphere Soil
Hole, fills apply the Trichoderma atroviride K bacterial manure that obtains in embodiment 1 and Trichoderma atroviride L bacterial manure (about 3 grams of bacterium ball/strains) blinding afterwards respectively.Separately
If one group only with clear water pouring, used as control.After 25 days, statistics sweet wormwood seedling items Growth and development index is concrete such as the institute of table 1
Show.Significance analysis are carried out to the data of table 1, as a result as shown in table 2.As seen from Table 2, in addition to root length, remaining all growth
Development index, Trichoderma atroviride L bacterial manure relative comparison groups are without actual technique effect；Apply the sweet wormwood seedling of Trichoderma atroviride K bacterial manure
It is significantly better than Trichoderma atroviride L bacterial manure groups and control group sweet wormwood seedling.
Table 1 applies impact of the Trichoderma atroviride bacterial manure to sweet wormwood growth of seedling
The Trichoderma atroviride of table 2 is to sweet wormwood growth promoting function fertilizer efficiency significance analysis result
| Numbering | The long cm of root | Plant height cm | The thick mm of root | The thick mm of stem | Underground fresh weight g | Ground fresh weight g | Underground dry weight g | Ground dry weight g | Total fresh weight g | Gross dry weight g |
| Control group | 26.3036a | 3.0491b | 1.1302b | 1.0889b | 0.2582b | 0.2556b | 0.0296b | 0.0425b | 0.5138b | 0.0722b |
| Trichoderma atroviride K bacterial manure groups | 26.0882ab | 3.7418a | 1.3198a | 1.2138a | 0.3445a | 0.3009a | 0.0437a | 0.0515a | 0.6454a | 0.0952a |
| Trichoderma atroviride L bacterial manure groups | 25.7245ab | 3.1358b | 1.1332b | 1.1105b | 0.2748b | 0.2548b | 0.0302b | 0.0456b | 0.5203b | 0.0885b |
Claims (6)
1. a kind of sweet wormwood seedling culture method, it is characterised in that：Duck eye is beaten in sweet wormwood seedling Rhizosphere Soil, Trichoderma atroviride K bacterium are applied in filling
Blinding after fertilizer.
2. sweet wormwood seedling culture method according to claim 1, it is characterised in that：The administration of the Trichoderma atroviride K bacterial manure
Measure as 3g bacterium ball/strain.
3. a kind of Trichoderma atroviride K bacterial manure for being exclusively used in sweet wormwood seedling culture, it is characterised in that：The system of the Trichoderma atroviride K bacterial manure
Standby step is：
1）Under aseptic technique, Trichoderma atroviride K bacterial classifications are taken and is inoculated in the fluid nutrient medium for preparing；
2）The fluid nutrient medium for meeting Trichoderma atroviride K is placed in shaking table, 130 revs/min of rotating speed, 25 DEG C of environment temperature, incubation time 6-8
My god, cover with fluid nutrient medium and taken out after bacterium ball；
3）Surplus liquid culture medium is filtered off, with clear water suspended bacteria ball, Trichoderma atroviride K bacterial manure is obtained.
4. the Trichoderma atroviride K bacterial manure for being exclusively used in sweet wormwood seedling culture according to claim 3, it is characterised in that：The depth
The preparation method of green trichoderma K bacterial classifications is comprised the following steps：
1) in June then, disease-free spot stalwartness sweet wormwood side shoot is taken, is cut into segment, wash away soil dust, with washing powder water 1 point is soaked
Clock, flowing water is standby after rinsing；
2）On superclean bench, 75% ethanol purge, 1/1000 mercuric chloride sterilization, sterile water wash 4-5 time is used to use aseptic filter paper
Suck dry moisture, is cut into the little stem section of 0.5-1 centimeter lengths；
3）Little stem section is placed on potato glucose plate culture medium, and 25 DEG C are cultivated 7 ± 1 days, the fungi grown around picking stem section
Mycelia；
4）Hypha,hyphae is continued on potato dextrose culture-medium culture, until obtaining the fungus colony for purifying till；
5）Above-mentioned fungus colony is done into Molecular Identification, Trichoderma atroviride K bacterial strains is chosen as the bacterial classification of Trichoderma atroviride K bacterial manure.
5. the Trichoderma atroviride K bacterial manure for being exclusively used in sweet wormwood seedling culture according to claim 4, it is characterised in that：The soil
The preparation method of beans dextrose culture-medium is：200 grams of potato is weighed, fritter is cut, liquor water filtration adds 20 grams of glucose, fine jade
7 grams of cosmetics, is settled to 1 liter, after 121 DEG C sterilize 30 minutes, pours on superclean bench standby after plate cooling.
6. the Trichoderma atroviride K bacterial manure for being exclusively used in sweet wormwood seedling culture according to claim 3, it is characterised in that：The liquid
Body culture medium cuts fritter to weigh 200 grams of potato, boils 20 minutes, removes slag and takes juice, adds 20 grams of glucose, and add water constant volume
To 1 liter；
1 liter of triangular flask of packing, 250-500 milliliters/bottle；
121 DEG C of sterilizings are obtained for 30 minutes.
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| CN107200611A (en) * | 2017-07-17 | 2017-09-26 | 重庆市中药研究院 | A kind of special Trichoderma atroviride bacterial manure of sweet wormwood seedling and application |
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| CN111269838A (en) * | 2020-04-30 | 2020-06-12 | 福建省南平市农业科学研究所 | Method for inducing and separating antagonistic bacteria in soil by barley grains infected with gibberellic disease |
| CN113215000A (en) * | 2021-04-01 | 2021-08-06 | 慕恩（广州）生物科技有限公司 | Chaetomium gracile, seed soaking agent and application |
| CN113215000B (en) * | 2021-04-01 | 2023-11-28 | 慕恩（广州）生物科技有限公司 | Chaetomium, seed soaking agent and application |
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