CN110423718A - Utilize the method and application of fermentation of bacillus liquid production trichoderma harzianum chlamydospore - Google Patents
Utilize the method and application of fermentation of bacillus liquid production trichoderma harzianum chlamydospore Download PDFInfo
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- CN110423718A CN110423718A CN201910744567.2A CN201910744567A CN110423718A CN 110423718 A CN110423718 A CN 110423718A CN 201910744567 A CN201910744567 A CN 201910744567A CN 110423718 A CN110423718 A CN 110423718A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N3/00—Spore forming or isolating processes
Abstract
The present invention relates to a kind of methods and application using fermentation of bacillus liquid production trichoderma harzianum chlamydospore；It the described method comprises the following steps: S1 trichoderma actication of culture；The preparation of S2 Trichoderma spore suspension；The preparation of S3 fermentation of bacillus liquid；S4 produces trichoderma harzianum chlamydospore.Fermentation of bacillus liquid 5-20mL/L is mainly utilized in the present invention, has used modified MS medium 4-5g/L, sucrose 10-20g/L, beef extract 2.5-5g/L, peptone 1-5g/L.Culture medium of the invention comprehensive, chlamydospore abundance full of nutrition, the advantage that spore germination rate is high, acquisition tissue-cultured seedling is healthy and strong, rooting efficiency is good, transplanting survival rate is high have good market prospects.
Description
Technical field
This application involves field of biotechnology, especially agricultural biological technical field.Specifically, this application involves utilizations
The method and application of fermentation of bacillus liquid production trichoderma harzianum chlamydospore.
Background technique
History of the Trichoderma for controlling plant diseases is more than 70 years existing, and biocontrol effect has gained public acceptance, and is the whole world
Widely used biocontrol microorganisms.Trichoderma glues spore mushroom, is in Deuteromycotina, Hyphomycetes, hyphomycetales, Moniliaceae
One kind is prevalent in the fungi in soil, is the important group of edaphon, can parasitize plant residue and animal excreta
Just it on, also can be often separated to from plant rhizosphere, blade and seed, bulb surface.Trichoderma can parasitize a variety of soil and pass
On phytopathogen.Since the eighties, with the fast development of molecular biology, the research of Trichoderma is mainly concentrated in
In terms of improving its biological preventive effect, such as by reinforcing chitinase, the expression of glucanase gene, Interspecific fusion means.
After Trichoderma PDA culture medium solid fermentation culture, bacterium colony starts as flocculence or fine and close clump pencil, and color is white
To canescence, no fixed shape.After conidium maturation, bacterium colony, to edge, graduates into different degrees of green Zi center,
Only a few is white powder.Conidiophore is born from the side shoot of mycelia, upright branch, and sprig often to life, do not expand by top, on
Raw spore ball estranged.Conidium is spherical, light or colourless.
Demand of most of Trichoderma to nutrition be not stringent, they can grow under various carbon sources and nitrogen source, while can
Convert and degrade some harmful or lasting harmful environmental contaminants；The derivative, organic of various monosaccharide, monosaccharide can directly be utilized
Acids；There are various polysaccharide of significantly degrading (cellulose, hemicellulose) and relevant polysaccharide (chitin)；Also it can convert and drop
Some pesticides are solved, such as: malathion, Dalapon, pentachloronitrobenzene.Trichoderma can utilize complicated and simple nitrogenous chemical combination
Object, casamino acids mixed liquor, asparatate, alanine, glutamic acid can utilize very well, have under the conditions of high nitrogen
Some enzymes, such as cellulase, lactase are generated conducive to Trichoderma.
Trichoderma is aerobic bacteria, and suitable oxygen is pressed with conducive to the growth of mycelia and spore generation；Optimal pH is 4.0-6.5；
Growth temperature is different due to kind, most of very rapid in 25 DEG C or so growths, and the pH of self-control environment, adapts to environment
Its growth conditions.The antagonism range of Trichoderma has broad spectrum activity, and correlative study is shown, Trichoderma at least belongs to 29 kinds to 18
Disease fungus has antagonism.Parasitic phytopathogen, that is, antagonism the object of Trichoderma energy includes Rhizoctonia
(Rhizotonia), pyrenomycetes (Sclerotium), Sclerotinia (Sclerotinia), long compacted armful of category (Helminth
Osporium), Fusarium (Fusarium), hair disc spore category (Colletotrichum), Verticillium (Verticillium),
Venturia (Venturia), inner seat shell category (Endothia), pythium (Pythium), Phytophthora (Phytophthora),
Seat shell category (Diaporthe) and Fusicladium (Fusicladium) etc..
Currently, many researchers have just carried out a large amount of exploration to the Sporulation condition of Trichoderma, to efficiently by such
Trichoderma is applied in production practices.From the point of view of the conidial Production conditions of Trichoderma, in many kinds of solids or Liquid Culture
Conidium, and the Coffee pulp of municipal refuse, corruption, the excrement of birds and the mixed coffee with cow dung can be generated in base
The inexpensive substances such as coffee pericarp, Banana Leaf, bagasse and wheat bran can be used as the solid medium of Trichoderma to produce conidium,
And spore output is all up 109Cfu/g or so.From this point of view, Trichoderma generate conidial working condition relative requirement compared with
It is low.
In recent years, the T.harzianum of research discovery both at home and abroad, the trichodermas such as T.viride strain is in liquid such as molasses-corn pulps
Submerged fermentation 15d or so is carried out in body culture medium can produce 107Chlamydospore；In wheat bran etc., sterile soil, soil extract
And 20d or so is cultivated in the solid mediums such as phytoclasts and also can produce chlamydospore up to 106.All in all, it reports at present,
The fermentation time of various trichoderma harzianum chlamydospores is longer (> 15d), and higher cost, and the yield of fermentation is lower, and after fermentation
The activity or ability to function of the part chlamydospore of generation may reduce or lose.
Bacillus (Bacillus) is a kind of mesophilous aerobic sporiferous bacillus, and physiological characteristic is abundant more
Sample, it is widely distributed, easily it is separately cultured.The bacterium is widespread in nature, nontoxic to people and animals, free from environmental pollution, energy
A variety of antibiotic and enzyme are generated, there is broad spectrum antibiotic activity and extremely strong anti-adversity ability.Bacillus not only can be in soil, plant
It is widely present in the external environments such as object rhizosphere, body surface, while endophytic bacterium still common in plant, is especially planting
The root of object, stem.The bacterium is own at present is shown on the crops such as rice, capsicum, cotton, wheat, cucumber, soybean, corn
Good disease-controlling effect.Bacillus feature most outstanding is that growth is fast, nutrition is simple, can generate the inverse bud of heat-resistant
Spore is conducive to the preparation of its fermentation liquid, not only with short production cycle, but also mass production processes are simple, and cost is relatively low, application side
Just, Storage period is long.In addition, bacillus generate have the active antibacterial material of biological and ecological methods to prevent plant disease, pests, and erosion, including rouge skin class, skin class, protide,
The multiple compounds such as phospholipid, polyenoid class, amino acids and nucleic acid, they, which have fungi, bacterium, virus etc., inhibits to make
With.
Summary of the invention
It is an object of the invention to for above-mentioned trichoderma harzianum chlamydospore existing in the prior art fermentation time compared with
Long (> 15d), and higher cost, the yield of fermentation is lower, and the activity of the part chlamydospore generated after fermenting or effect energy
The problems such as power may reduce or lose, provide it is a kind of using fermentation of bacillus liquid production trichoderma harzianum chlamydospore method and
Using.The trichoderma harzianum chlamydospore of this method production can be used in the prevention and treatment of controlling fungal diseases of crop, also can be used as microorganism
Fertilizer has the function of improving vegetation growth state.
The purpose of the present invention is achieved through the following technical solutions:
The present invention relates to a kind of methods for producing trichoderma harzianum chlamydospore, comprising the following steps:
S1, actication of culture: trichoderma strain is seeded in PDA solid medium, is inverted culture；By bacillus amyloliquefaciens
It is inoculated on LB solid medium, is inverted culture；
S2, the preparation of Trichoderma spore suspension: the Trichoderma conidium that S1 is cultivated is formulated as 3-4 × 107cfu/
The Trichoderma spore suspension of mL；
The preparation of S3, bacillus amyloliquefaciens fermentation liquid A1: the S1 bacillus picking single colonie cultivated is inoculated in LB
In fluid nutrient medium, fermentation liquid is centrifuged and is collected supernatant by shaking flask culture, and the supernatant after sterilization treatment is deposited as A1 fermentation liquid
- 20 DEG C are put in save for use；
S4, trichoderma harzianum chlamydospore production: A1 described in Trichoderma spore suspension and 5-20mL described in 1-5mL is taken to ferment respectively
Liquid, while being added in B1 culture medium, environment temperature is 25-32 DEG C, and shaking table speed control is in 150-200r/min, illumination cultivation
6-8d forms chlamydospore.
As one embodiment of the invention, the trichoderma strain in step S1 is Trichoderma asperellum (Trichoderma
asperellum)GDSF1009。
As one embodiment of the invention, the bacillus amyloliquefaciens in step S1 are bacillus amyloliquefaciens
(Bacillus amyloliquefaciens)ACCC11060。
As one embodiment of the invention, the inversion cultivation temperature of the Trichoderma in step S1 is 25-32 DEG C, is inverted
A length of 2-4d when culture.
As one embodiment of the invention, the inversion cultivation temperature of the bacillus amyloliquefaciens in step S1 is 30-
40 DEG C, it is inverted a length of 12-24h when culture.
As one embodiment of the invention, the PDA solid medium is obtained by being prepared via a method which: will
160-220g peeling potatoes are cut into small pieces, filtered through gauze after boiling, add 18-22g glucose, 15-25g agar powder, using going
Ionized water constant volume 1L, sterilize 30min at 121 DEG C.
As one embodiment of the invention, the LB solid medium is obtained by being prepared via a method which: will
Peptone 8-10g/L, yeast powder 3-5g/L, sodium chloride 5-10g/L control pH 6.5-7.5, use deionized water constant volume 1L, In
Sterilize 30min at 121 DEG C.
As one embodiment of the invention, the LB liquid medium includes peptone 5-10g/L, powdered beef 3-
5g/L, sodium chloride 5-10g/L control pH 6.5-7.5；It is 30-40 DEG C that shaking flask, which trains temperature,.
As one embodiment of the invention, in step S3, the method for the sterilization treatment are as follows: fermentation liquid is centrifuged,
It collects supernatant and passes through 0.22 μm sterile of PTFE miillpore filter, which deposits in -20 DEG C of preservations as A1 fermentation liquid
For use.
As one embodiment of the invention, in step S3, shaking flask culture 12-48h controls pH 6.5-8.5, culture
30-40 DEG C of temperature.
As one embodiment of the invention, in step S4, every 1 liter of B1 culture medium prescription includes consisting of: being changed
Good MS culture medium 4-5g/L, sucrose 10-20g/L, beef extract 2.5-5g/L, peptone 1-5g/L, and pH is demarcated by sodium hydroxide
To 6 ± 0.2.
As one embodiment of the invention, every 1 liter of modified MS medium includes following composition: MgSO4·7H2O
180-200mg/L, CaCl2·H2O 180-220mg/L, KNO3 300-700mg/L,(NH)4NO3500-600mg/L, KH2PO4
180-220mg/L, surplus are water.
The chlamydospore formed the invention further relates to a kind of method of aforementioned production trichoderma harzianum chlamydospore preparation to
Promote the purposes in the biological prevention and control agent of plant growth.
As one embodiment of the invention, the biological prevention and control agent is administered using soil pouring root mode.
As one embodiment of the invention, the biological prevention and control agent is used for controlling plant diseases.
Compared with prior art, the invention has the following beneficial effects:
1) after the improvement based on conventional medium, culture medium composition of the invention has modified the formula of MS, is added to beef
Cream and peptone have reached and had both shortened in the mycelia growth medium feed supplement fermentation of bacillus liquid of antibacterial peptide rich content
Trichoderma produces the period of chlamydospore, increases trichoderma harzianum chlamydospore yield；
2) antibacterial peptide of Trichoderma spore and remaining bacillus, the resistance for resisting disease to plant also increase；
3) in the present invention, the dosage of bacillus is controlled, the yield and quality of trichoderma harzianum chlamydospore can be made
Reach best, while retaining the activity of its most of antibacterial material；Therefore, research and utilization fermentation of bacillus liquid produces Trichoderma
Chlamydospore method is of great significance to the biological prevention and control agent developed based on trichoderma harzianum chlamydospore.
Specific embodiment
Technical solution of the present invention is described in detail below in conjunction with embodiment, the example is of the invention preferred
The case for producing chlamydospore is merely to illustrate the range that the present invention is not intended to limit the present invention, it will be understood by those skilled in the art that
Based on technical solution of the present invention, the other technologies scheme obtained under the premise of no creative both falls within protection of the invention
Range.Reagents or instruments used without specified manufacturer in embodiment is the conventional production that can be obtained by commercially available purchase
Product.It is shown in Table 1.
Embodiment 1
The present embodiment is related to a kind of method using fermentation of bacillus liquid production trichoderma harzianum chlamydospore, the method packet
Include following steps:
S1, actication of culture: Trichoderma asperellum (Trichoderma asperellum) GDSF1009 strain is seeded in and is put
It has set in the PDA culture dish of PDA solid medium, culture 3d is inverted in 28 DEG C of incubators；By bacillus amyloliquefaciens
(Trichoderma asperellum) GDSF1009 is inoculated on LB solid medium, and 1d is cultivated in 37 DEG C of inversions.
PDA solid medium: 200g peeling potatoes are cut into small pieces, and filtered through gauze after boiling adds 20g glucose, PDA
In additionally add 15g agar powder, using deionized water constant volume 1L, sterilize 30min at 121 DEG C；
LB solid medium: peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L control pH 7.5, use deionization
Water constant volume 1L, sterilize 30min at 121 DEG C.
S2, the preparation of Trichoderma spore suspension: Trichoderma is cultivated in PDA solid medium generates Trichoderma conidium,
It weighs 250mL deionized water to sterilize at 121 DEG C 30min, then rinses cultured PDA using the deionization after sterilizing and cultivate
Ware collects spore, detects spore under the microscope and is formulated as 3.5 × 107The spore suspension of cfu/mL.
The preparation of S3, bacillus amyloliquefaciens fermentation liquid: it cultivates the S1 to obtain bacillus amyloliquefaciens picking single colonie and connects
Kind in LB liquid medium, shaking flask culture 18h controls pH 7.0,37 DEG C of cultivation temperature, complete after culture by fermentation liquid with
6000r/min centrifugation collects supernatant and makes it through 0.22 μm sterile of PTFE miillpore filter, which ferments as A1
Liquid is deposited in -20 DEG C and is saved for use.
Above-mentioned LB liquid medium includes: peptone 8g/L, powdered beef 3g/L, sodium chloride 5g/L, controls pH 7.0.
S4, trichoderma harzianum chlamydospore production: the spore suspension and 10mL/L being separately added into B1 culture medium in 3mL S2
A1 fermentation liquid, environment temperature are 28 DEG C, and shaking table speed control forms chlamydospore, for giving birth in 180r/min, illumination cultivation 7d
Object prevents and treats preparation.
Above-mentioned B1 culture medium, every 1 liter of culture medium are grouped as by the group of following component and its concentration:
Modified MS medium 5g/L, sucrose 20g/L, beef extract 5g/L, peptone 3g/L, and pH by sodium hydroxide demarcate to
6±0.2。
Wherein, every 1 liter of modified MS medium includes following composition:
MgSO4·7H2O 200mg/L, CaCl2·H2O 180mg/L, KNO3600mg/L, (NH)4NO3550mg/L,
KH2PO4200mg/L, surplus are water.
Comparative example 1
This comparative example is related to a kind of method for producing trichoderma harzianum chlamydospore, and substantially with embodiment 1, the method includes such as
Lower step:
S1, actication of culture: Trichoderma asperellum (Trichoderma asperellum) GDSF1009 strain is seeded in and is put
It has set in the PDA culture dish of PDA solid medium, culture 3d is inverted in 28 DEG C of incubators.
PDA solid medium: 200g peeling potatoes are cut into small pieces, filtered through gauze after boiling, add 20g glucose PDA
In additionally add 15g agar powder, using deionized water constant volume 1L, sterilize 30min at 121 DEG C.
S2, the preparation of Trichoderma spore suspension: Trichoderma is cultivated in PDA solid medium generates Trichoderma conidium,
It weighs 250mL deionized water to sterilize at 121 DEG C 30min, then uses the cultured PDA culture dish of liquid-transfering gun repeated flushing,
Spore content is detected under the microscope, is formulated as 3.5 × 107The spore suspension of cfu/mL.
S3, trichoderma harzianum chlamydospore production: the spore suspension in 3mL S2, environment temperature 28 are added into B1 culture medium
DEG C, shaking table speed control forms chlamydospore in 180r/min, illumination cultivation 7d.
Above-mentioned B1 culture medium, every 1 liter of culture medium are grouped as by the group of following component and its concentration:
Modified MS medium 5g/L, sucrose 20g/L, beef extract 5g/L, and pH is demarcated by sodium hydroxide to 6 ± 0.2.
Wherein, every 1 liter of modified MS medium includes following composition:
MgSO4·7H2O 200mg/L, CaCl2·H2O 180mg/L, KNO3600mg/L, (NH)4NO3550mg/L,
KH2PO4200mg/L, surplus are water.
Comparative example 2
This comparative example is related to a kind of method for producing trichoderma harzianum chlamydospore, and substantially with embodiment 1, the method includes such as
Lower step:
S1, actication of culture: Trichoderma asperellum (Trichoderma asperellum) GDSF1009 strain is seeded in and is put
It has set in the PDA culture dish of PDA solid medium, culture 3d is inverted in 28 DEG C of incubators.
(Trichoderma asperellum) GDSF1009 is inoculated on LB solid medium, and 1d is cultivated in 37 DEG C of inversions.
PDA solid medium: 200g peeling potatoes are cut into small pieces, filtered through gauze after boiling, add 20g glucose PDA
In additionally add 15g agar powder, using deionized water constant volume 1L, sterilize 30mi at 121 DEG C.
S2, the preparation of Trichoderma spore suspension: Trichoderma is cultivated in PDA solid medium generates Trichoderma conidium,
It weighs 250mL deionized water to sterilize at 121 DEG C 30min, then uses the cultured PDA culture dish of liquid-transfering gun repeated flushing,
Spore content is detected under the microscope, is formulated as 3.5 × 107The spore suspension of cfu/mL.
S3, trichoderma harzianum chlamydospore production: the spore suspension in 3mL S2, environment temperature 28 are added into B1 culture medium
DEG C, shaking table speed control forms chlamydospore in 180r/min, illumination cultivation 7d.
Above-mentioned B1 culture medium, every 1 liter of culture medium are grouped as by the group of following component and its concentration:
Modified MS medium 5g/L, sucrose 20g/L, beef extract 5g/L, peptone 3g/L, and pH by sodium hydroxide demarcate to
6±0.2。
Wherein, every 1 liter of modified MS medium includes following composition:
MgSO4·7H2O 200mg/L, CaCl2·H2O 180mg/L, KNO3600mg/L, (NH)4NO3550mg/L,
KH2PO4200mg/L, surplus are water.
Comparative example 3
This comparative example is related to a kind of method using fermentation of bacillus liquid production trichoderma harzianum chlamydospore, basic with implementation
Example 1, the difference is that only:
What is selected in the B1 culture medium is MS culture medium, rather than modified MS medium；That is, every 1 liter of culture medium by with
The group of lower ingredient and its concentration is grouped as: MS culture medium 5g/L, sucrose 20g/L, beef extract 5g/L, peptone 3g/L, and pH by
Sodium hydroxide is demarcated to 6 ± 0.2.
Wherein, every 1 liter of MS culture medium includes following composition:
MgSO47H2O 370mg/L, CaCl2H2O 4400mg/L, KNO3 1900mg/L, (NH) 4NO3
1650mg/L, KH2PO4 170mg/L, surplus are water.
Embodiment 2
The present embodiment is related to a kind of method using fermentation of bacillus liquid production trichoderma harzianum chlamydospore, the method packet
Include following steps:
S1, actication of culture: Trichoderma asperellum (Trichoderma asperellum) GDSF1009 strain is seeded in and is put
It has set in the PDA culture dish of PDA solid medium, culture 3d is inverted in 28 DEG C of incubators；By bacillus amyloliquefaciens
(Trichoderma asperellum) GDSF1009 is inoculated on LB solid medium, and 18h is cultivated in 37 DEG C of inversions.
(Trichoderma asperellum) GDSF1009 is inoculated on LB solid medium, and 1d is cultivated in 37 DEG C of inversions.
PDA solid medium: 200g peeling potatoes are cut into small pieces, filtered through gauze after boiling, add 20g glucose PDA
In additionally add 15g agar powder, using deionized water constant volume 1L, sterilize 30min at 121 DEG C.
LB solid medium: peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L control pH 7.5, use deionization
Water constant volume 1L, sterilize 30min at 121 DEG C.
S2, the preparation of Trichoderma spore suspension: Trichoderma is cultivated in PDA solid medium generates Trichoderma conidium,
It weighs 250mL deionized water to sterilize at 121 DEG C 30min, then uses the cultured PDA culture dish of liquid-transfering gun repeated flushing,
Spore content is detected under the microscope, is formulated as 3.5 × 107The spore suspension of cfu/mL.
The preparation of S3, bacillus amyloliquefaciens fermentation liquid: it cultivates the S1 to obtain bacillus amyloliquefaciens picking single colonie and connects
Kind is in LB liquid medium, shaking flask culture 18 hours, controls pH 7.5,37 DEG C of cultivation temperature, completes fermentation liquid after cultivating
It is centrifuged with 6000r/min, collects supernatant and makes it through 0.22 μm sterile of PTFE miillpore filter, which sends out as A1
Zymotic fluid is deposited in -20 DEG C and is saved for use.
Above-mentioned LB liquid medium includes: peptone 8g/L, powdered beef 3g/L, sodium chloride 5g/L, controls pH 7.5.
S4, trichoderma harzianum chlamydospore production: the spore suspension being separately added into 3mL S2 into B1 culture medium contains and 5ml/L
A1 bacillus amyloliquefaciens fermentation liquid, environment temperature are 28 DEG C, and shaking table speed control is formed in 180r/min, illumination cultivation 7d
Chlamydospore is used for biological control agent.
Above-mentioned B1 culture medium, every 1 liter of culture medium are grouped as by the group of following component and its concentration:
Modified MS medium 4g/L, sucrose 10g/L, beef extract 2.5g/L, peptone 1g/L, and pH is demarcated by sodium hydroxide
To 6 ± 0.2.
Wherein, every 1 liter of modified MS medium includes following composition:
MgSO4·7H2O 180mg/L, CaCl2·H2O 180mg/L, KNO3300mg/L, (NH)4NO3500mg/L,
KH2PO4 170mg/L。
Embodiment 3
The present embodiment is related to a kind of method using fermentation of bacillus liquid production trichoderma harzianum chlamydospore, the method packet
Include following steps:
S1, actication of culture: Trichoderma asperellum (Trichoderma asperellum) GDSF1009 strain is seeded in and is put
It has set in the PDA culture dish of PDA solid medium, culture 3d is inverted in 28 DEG C of incubators；By bacillus amyloliquefaciens
(Trichoderma asperellum) GDSF1009 is inoculated on LB solid medium, and 1d is cultivated in 37 DEG C of inversions.
(Trichoderma asperellum) GDSF1009 is inoculated on LB solid medium, and 1d is cultivated in 37 DEG C of inversions.
PDA solid medium: 200g peeling potatoes are cut into small pieces, filtered through gauze after boiling, add 20g glucose PDA
In additionally add 15g agar powder, using deionized water constant volume 1L, sterilize 30min at 121 DEG C.
LB solid medium: peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L control pH 7.5, use deionization
Water constant volume 1L, sterilize 30min at 121 DEG C.
S2, the preparation of Trichoderma spore suspension: Trichoderma is cultivated in PDA solid medium generates Trichoderma conidium,
It weighs 250mL deionized water to sterilize at 121 DEG C 30min, then uses the cultured PDA culture dish of liquid-transfering gun repeated flushing,
Spore content is detected under the microscope, is formulated as 3.5 × 107The spore suspension of cfu/mL.
The preparation of S3, bacillus amyloliquefaciens fermentation liquid: it cultivates the S1 to obtain bacillus amyloliquefaciens picking single colonie and connects
Kind in LB liquid medium, shaking flask culture 18h controls pH 7.0,37 DEG C of cultivation temperature, complete after culture by fermentation liquid with
6000r/min centrifugation collects supernatant and makes it through 0.22 μm sterile of PTFE miillpore filter, which ferments as A1
Liquid is deposited in -20 DEG C and is saved for use.
Above-mentioned LB liquid medium includes: peptone 8g/L, powdered beef 3g/L, sodium chloride 5g/L, controls pH 7.5.
S4, trichoderma harzianum chlamydospore production: the spore suspension being separately added into 3mL S2 into B1 culture medium contains and 20mL/
L A1 bacillus amyloliquefaciens fermentation liquid, environment temperature are 28 DEG C, and shaking table speed control is in 180r/min, illumination cultivation 7d, shape
At chlamydospore, it to be used for biological control agent.
Above-mentioned B1 culture medium, every 1 liter of culture medium are grouped as by the group of following component and its concentration:
Modified MS medium 5g/L, sucrose 20g/L, beef extract 5g/L, peptone 5g/L, and pH by sodium hydroxide demarcate to
6±0.2。
Wherein, every 1 liter of modified MS medium includes following composition:
MgSO4·7H2O 200mg/L, CaCl2·H2O 220mg/L, KNO3700mg/L, (NH)4NO3600mg/L,
KH2PO4 200mg/L。
The corresponding trichoderma harzianum chlamydospore yield of above-described embodiment 1-3 and comparative example 1-3 is as shown in table 1:
Under 1 different formulations of table, trichoderma harzianum chlamydospore yield comparison (cfu/mL)
The microorganism formulation that embodiment 1-3 and comparative example 1-3 are obtained is calculated with the amount of chlamydospore, and amount of application is 4 ×
105A chlamydospore/square metre, any preparation will not added as control treatment (control group in table 2).Respectively with embodiment
The preparation of 1-3 and comparative example 1-3 carry out root irrigation to the cucumber in tri-leaf period, observe the disease incidence of powdery mildew of cucumber.As a result such as
Shown in table 2.It compared microorganism formulation embodiment 1-3 and comparative example 1-3, and the conventional non-treated prevention and treatment to powdery mildew of cucumber
Effect, wherein the relative control effect highest of embodiment 1.
Prevention and treatment of 2 microorganism formulation of table to powdery mildew of cucumber
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
Claims (10)
1. a kind of method for producing trichoderma harzianum chlamydospore, which comprises the following steps:
S1, actication of culture: trichoderma strain is seeded in PDA solid medium, is inverted culture；Bacillus amyloliquefaciens are inoculated with
In on LB solid medium, it is inverted culture；
S2, the preparation of Trichoderma spore suspension: the Trichoderma conidium that S1 is cultivated is formulated as 3-4 × 107The wood of cfu/mL
Mycotic spore suspension；
The preparation of S3, bacillus amyloliquefaciens fermentation liquid A1: the S1 bacillus picking single colonie cultivated is inoculated in LB liquid
In culture medium, fermentation liquid is centrifuged and is collected supernatant by shaking flask culture, and the supernatant after sterilization treatment is stored as A1 fermentation liquid
It is saved in -20 DEG C stand-by；
S4, trichoderma harzianum chlamydospore production: taking A1 fermentation liquid described in Trichoderma spore suspension and 5-20mL described in 1-5mL respectively,
It is added in B1 culture medium simultaneously, environment temperature is 25-32 DEG C, and shaking table speed control is in 150-200r/min, illumination cultivation 6-
8d forms chlamydospore.
2. the method for production trichoderma harzianum chlamydospore as described in claim 1, which is characterized in that the trichoderma strain in step S1
For Trichoderma asperellum (Trichoderma asperellum) GDSF1009.
3. the method for production trichoderma harzianum chlamydospore as described in claim 1, which is characterized in that the solution starch bud in step S1
Spore bacillus is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ACCC11060.
4. the method for production trichoderma harzianum chlamydospore as described in claim 1, which is characterized in that in step S3, the training of LB liquid
Supporting base includes peptone 5-10g/L, powdered beef 3-5g/L, sodium chloride 5-10g/L, controls pH6.5-7.5；Shaking flask trains temperature
30-40℃。
5. the method for production trichoderma harzianum chlamydospore as described in claim 1, which is characterized in that in step S3, the sterilizing
The method of processing are as follows: fermentation liquid is centrifuged, collects supernatant and by 0.22 μm sterile of PTFE miillpore filter, supernatant work
- 20 DEG C are deposited in for A1 fermentation liquid to save for use.
6. the method for production trichoderma harzianum chlamydospore as described in claim 1, which is characterized in that in step S4, every 1 liter of B1
Culture medium prescription includes consisting of: modified MS medium 4-5g/L, sucrose 10-20g/L, beef extract 2.5-5g/L, peptone
1-5g/L, and pH is demarcated by sodium hydroxide to 6 ± 0.2.
7. the method for production trichoderma harzianum chlamydospore as claimed in claim 9, which is characterized in that every 1 liter of improvement MS training
Supporting base includes following composition: MgSO4·7H2O 180-200mg/L, CaCl2·H2O 180-220mg/L, KNO3 300-700mg/
L,(NH)4NO3500-600mg/L, KH2PO4180-220mg/L, surplus are water.
8. a kind of chlamydospore that the method for production trichoderma harzianum chlamydospore as described in claim 1 is formed is in preparation to promote
Purposes into the biological prevention and control agent of plant growth.
9. purposes as claimed in claim 8, which is characterized in that the biological prevention and control agent is administered using soil pouring root mode.
10. purposes as claimed in claim 8, which is characterized in that the biological prevention and control agent is used for controlling plant diseases.
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