CN110423718B

CN110423718B - Method and application of producing Trichoderma chlamydospore by using Bacillus fermented liquid

Info

Publication number: CN110423718B
Application number: CN201910744567.2A
Authority: CN
Inventors: 孙佳楠; 陈捷; 贺安乐
Original assignee: Shanghai Jiao Tong University
Current assignee: Shanghai Jiao Tong University
Priority date: 2019-08-13
Filing date: 2019-08-13
Publication date: 2021-10-15
Anticipated expiration: 2039-08-13
Also published as: CN110423718A

Abstract

本发明涉及一种利用芽孢杆菌发酵液生产木霉菌厚垣孢子的方法及应用；所述方法包括以下步骤：S1木霉菌种活化；S2木霉菌孢子悬液制备；S3芽孢杆菌发酵液的制备；S4生产木霉菌厚垣孢子。本发明主要利用了芽孢杆菌发酵液5‑20mL/L，使用了改良MS培养基4‑5g/L，蔗糖10‑20g/L，牛肉膏2.5‑5g/L，蛋白胨1‑5g/L。本发明的培养基营养丰富全面、厚垣孢子产量丰富，孢子萌发率高、获得的组培苗健壮、生根效果好、移栽成活率高的优点，具有良好的市场前景。The invention relates to a method and application for producing Trichoderma chlamydospore by using Bacillus fermented liquid; the method comprises the following steps: S1 activation of Trichoderma species; S2 preparation of Trichoderma spore suspension; S3 preparation of Bacillus fermented liquid; S4 produces Trichoderma chlamydospores. The invention mainly utilizes 5-20 mL/L of Bacillus fermented liquid, 4-5 g/L of improved MS medium, 10-20 g/L of sucrose, 2.5-5 g/L of beef extract, and 1-5 g/L of peptone. The medium of the invention has the advantages of rich and comprehensive nutrition, rich chlamydospore yield, high spore germination rate, robust tissue culture seedlings obtained, good rooting effect and high transplanting survival rate, and has good market prospects.

Description

Method for producing trichoderma chlamydospore by using bacillus fermentation liquor and application

Technical Field

The present application relates to the field of biotechnology, in particular the field of agricultural biotechnology. In particular to a method for producing trichoderma chlamydospore by utilizing bacillus fermentation liquor and application thereof.

Background

The history of trichoderma used for preventing and controlling plant diseases has been over 70 years, the biocontrol effect of the trichoderma is well known, and the trichoderma is a biocontrol bacterium which is widely applied all over the world. Trichoderma belongs to Deuteromycotina, Hyphomycetes, Moniliaceae, myxosporium, is a fungus commonly present in soil, is an important community of soil microorganisms, can parasitize on plant residues and animal wastes, and can be isolated from the surface of plant rhizosphere, leaves, seeds and bulbs. Trichoderma can be parasitic on a variety of soil-borne plant pathogens. With the rapid development of molecular biology, the research on trichoderma has mainly focused on improving the biological control effect of trichoderma in the eighties, such as by enhancing the expression of chitinase and glucanase genes, interspecies fusion and other means.

After the trichoderma PDA culture medium is subjected to solid fermentation culture, colonies begin to be flocculent or compact cluster-shaped, and the color is white to offwhite without fixed shapes. When the conidia are mature, the bacterial colony gradually changes into green with different degrees from the center to the edge, and the rare bacterial colony is white powder. Conidiophores grow from lateral branches of hyphae, branch vertically, small branches are usually opposite, the top ends are not expanded, and conidiophores grow upwards. Conidiophores are spherical, light-colored or colorless.

Most trichoderma species are not critical in terms of nutrition, can grow on various carbon and nitrogen sources, and can convert and degrade some harmful or persistent harmful environmental pollutants; various monosaccharides, derivatives of monosaccharides, and organic acids can be directly used; the polysaccharide degrading agent has the advantages that various polysaccharides (cellulose and hemicellulose) and related polysaccharides (chitin) are remarkably degraded; can also be used for transforming and degrading pesticides, such as malathion, dalapon, quintozene, etc. The trichoderma can utilize complex and simple nitrogen-containing compounds, hydrolyzed casein amino acid mixed liquor, aspartic acid, alanine and glutamic acid can be well utilized, and the trichoderma is favorable for producing certain enzymes such as cellulase, lactase and the like under the high-nitrogen condition.

Trichoderma is an aerobic bacterium, and proper oxygen pressure is favorable for hypha growth and spore production; the optimal pH is 4.0-6.5; the growth temperature is different from species to species, most of the plants grow very quickly at about 25 ℃, and the pH value of the environment is self-regulated, so that the environment adapts to the growth conditions. The antagonism range of trichoderma has broad spectrum, and related research shows that trichoderma has antagonism to at least 18 pathogenic fungi of 29 genera. Antagonistic targets, which are phytopathogens parasitized by trichoderma include rhizoctonia (rhizobium), Sclerotinia sclerotiorum (sclerotiotium), Sclerotinia (sclerotirotinia), helminthium (helminthium), Fusarium (Fusarium), Colletotrichum (Colletotrichum), Verticillium (Verticillium), Venturia (Venturia), incrustation (Endothia), Pythium (Pythium), Phytophthora (Phytophthora), incrustum (Diaporthe), and nigrospora (Fusarium), and the like.

Currently, many researchers have been working on the spore production conditions of TrichodermaA great deal of research is carried out to apply this type of trichoderma efficiently in production practice. From the production condition of the conidia of the trichoderma, the conidia can be produced in various solid or liquid culture mediums, and urban garbage, putrefactive coffee pericarp, poultry excrement, coffee pericarp mixed with cow dung, banana leaf, bagasse, wheat bran and other cheap substances can be used as the solid culture medium of the trichoderma to produce the conidia, and the spore yield can reach 10⁹About cfu/g. Therefore, the production conditions for producing conidia by trichoderma are relatively low.

Recently, researches at home and abroad find that Trichoderma species such as T.harzianum, T.viride and the like can generate 10 in about 15 days of submerged fermentation in a liquid culture medium such as molasses-corn steep liquor and the like⁷Chlamydospores of (a); the chlamydospores can be produced by culturing in solid culture medium such as wheat bran, sterilized soil, soil leachate and plant debris for about 20 days to obtain chlamydospores of 10%⁶. In summary, the fermentation time of chlamydospores of various Trichoderma fungi reported at present is longer (>15d) And at higher cost, with lower yields of fermentation, and with partial chlamydospores produced after fermentation that may have reduced or lost activity or potency.

The Bacillus (Bacillus) is a mesophilic aerobic Bacillus-producing rod-shaped bacterium, has rich and various physiological characteristics and wide distribution, and is easy to separate and culture. The bacteria exist widely in nature, are non-toxic and harmless to human and livestock, do not pollute the environment, can generate various antibiotics and enzymes, and have broad-spectrum antibacterial activity and extremely strong anti-adversity capability. The bacillus can not only exist widely in external environments such as soil, plant rhizosphere, body surface and the like, but also is a common plant endophytic bacterium in a plant body, particularly in the root and stem of the plant. At present, the bacterium shows good disease control effect on crops such as rice, pepper, cotton, wheat, cucumber, soybean, corn and the like. The bacillus has the most prominent characteristics of quick growth, simple nutrition, capability of generating heat-resistant and stress-resistant spores, contribution to the preparation of fermentation liquor, short production period, simple batch production process, lower cost, convenient application and long storage period. In addition, antibacterial substances having biocontrol activity produced by bacillus include various compounds such as lipids, skins, proteins, phospholipids, polyenes, amino acids, nucleic acids, and the like, and they have inhibitory effects on fungi, bacteria, viruses, and the like.

Disclosure of Invention

The invention aims to provide a method for producing trichoderma chlamydospore by using bacillus fermentation liquor and application thereof, aiming at the problems that the trichoderma chlamydospore in the prior art has longer fermentation time (>15d), higher cost and lower fermentation yield, and the activity or action capacity of part of chlamydospore generated after fermentation is possibly reduced or lost. The trichoderma chlamydospore produced by the method can be used for preventing and treating fungal diseases of crops, can also be used as a microbial fertilizer, and has the effect of improving the growth condition of plants.

The purpose of the invention is realized by the following technical scheme:

the invention relates to a method for producing trichoderma chlamydospore, which comprises the following steps:

s1, strain activation: inoculating trichoderma strains in a PDA solid culture medium, and performing inverted culture; inoculating bacillus amyloliquefaciens on an LB solid culture medium, and performing inverted culture;

s2, preparation of trichoderma spore suspension: preparing 3-4 × 10 Trichoderma conidia obtained by culturing S1⁷cfu/mL trichoderma spore suspension;

s3, preparation of a bacillus amyloliquefaciens fermentation liquid A1: selecting a single colony of bacillus cultured in S1, inoculating the single colony in an LB liquid culture medium, performing shake flask culture, centrifuging a fermentation liquid, collecting a supernatant, and storing the sterilized supernatant serving as an A1 fermentation liquid at-20 ℃ for later use;

s4, Trichoderma chlamydospore production: respectively taking 1-5mL of the trichoderma spore suspension and 5-20mL of the A1 fermentation liquid, simultaneously adding the trichoderma spore suspension and the A1 fermentation liquid into a B1 culture medium, controlling the environmental temperature to be 25-32 ℃, controlling the shaking table speed to be 150-.

As one embodiment of the present invention, the Trichoderma species in step S1 is Trichoderma asperellum (Trichoderma asperellum) GDSF 1009.

As an embodiment of the present invention, the Bacillus amyloliquefaciens in the step S1 is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ACCC 11060.

As an embodiment of the present invention, the inverted cultivation temperature of Trichoderma in step S1 is 25-32 deg.C, and the inverted cultivation time is 2-4 d.

As an embodiment of the present invention, the temperature of the inverted culture of Bacillus amyloliquefaciens in the step S1 is 30-40 ℃, and the time of the inverted culture is 12-24 h.

As an embodiment of the invention, the PDA solid culture medium is prepared by the following method: peeling 160-220g of potatoes, cutting the potatoes into small pieces, cooking, filtering by using gauze, adding 18-22g of glucose and 15-25g of agar powder, fixing the volume to 1L by using deionized water, and sterilizing for 30min at 121 ℃.

As an embodiment of the present invention, the LB solid medium is prepared by the following method: controlling pH to 6.5-7.5 with peptone 8-10g/L, yeast powder 3-5g/L, sodium chloride 5-10g/L, diluting to constant volume of 1L with deionized water, and sterilizing at 121 deg.C for 30 min.

As an embodiment of the invention, the LB liquid medium comprises peptone 5-10g/L, beef powder 3-5g/L, sodium chloride 5-10g/L, pH 6.5-7.5; the temperature of the shake flask culture is 30-40 ℃.

In one embodiment of the present invention, in step S3, the method of the sterilization treatment includes: the broth was centrifuged and the supernatant was collected and passed through a sterile 0.22 μm PTFE microporous membrane and stored at-20 ℃ as A1 broth for further use.

As an embodiment of the invention, in step S3, the culture is performed for 12-48h in a shake flask, the pH is controlled to 6.5-8.5, and the culture temperature is controlled to 30-40 ℃.

As an embodiment of the present invention, in step S4, the formulation of B1 medium per 1 liter included the following composition: modified MS culture medium 4-5g/L, sucrose 10-20g/L, beef extract 2.5-5g/L, peptone 1-5g/L, and pH is calibrated to 6 +/-0.2 by sodium hydroxide.

As an embodiment of the present invention, the amount of the modification is 1 liter per literThe good MS culture medium comprises the following components: MgSO (MgSO)₄·7H₂O 180-200mg/L，CaCl₂·H₂O 180-220mg/L，KNO₃ 300-700mg/L,(NH)₄NO₃500-600mg/L，KH₂PO₄180-220mg/L, and the balance being water.

The invention also relates to the use of chlamydospores obtained by the method for producing trichoderma chlamydospores in the preparation of biocontrol agents intended to promote plant growth.

As one embodiment of the invention, the biocontrol agent is applied by soil drenching.

As one embodiment of the present invention, the biocontrol agent is used for controlling plant diseases.

Compared with the prior art, the invention has the following beneficial effects:

1) after the improvement of the conventional culture medium, the culture medium composition modifies the formula of MS, adds beef extract and peptone, and supplements bacillus fermentation liquor with rich antibacterial peptide in the middle growth period of hypha, so that the period of producing chlamydospore by trichoderma is shortened, and the chlamydospore yield of trichoderma is increased;

2) the antibacterial peptides of trichoderma spores and residual bacillus also have improved resistance to plant diseases;

3) in the invention, the output and quality of trichoderma chlamydospore can reach the best by controlling the dosage of bacillus, and the activity of most antibacterial substances is kept; therefore, the research on the method for producing trichoderma chlamydospore by using bacillus fermentation liquor has important significance for developing a biocontrol agent taking the trichoderma chlamydospore as a main body.

Detailed Description

The technical solution of the present invention will be described in detail with reference to the following examples, which are the case of the preferred chlamydospores of the present invention, and are intended to illustrate the invention only and not to limit the scope of the invention, and it will be apparent to those skilled in the art that other technical solutions, which can be obtained without inventive step, fall within the scope of the present invention. The reagents and instruments used in the examples are not indicated by the manufacturer, and are all conventional products available commercially. See table 1.

Example 1

The embodiment relates to a method for producing trichoderma chlamydospore by using bacillus fermentation liquor, which comprises the following steps:

s1, strain activation: inoculating Trichoderma asperellum GDSF1009 strain in PDA culture dish with PDA solid culture medium, and culturing in 28 deg.C incubator for 3 d; bacillus amyloliquefaciens (Trichoderma asperellum) GDSF1009 was inoculated on LB solid medium and cultured in 37 ℃ inversion for 1 day.

PDA solid medium: peeling 200g of potato, cutting into small pieces, steaming, filtering with gauze, adding 20g of glucose, adding 15g of agar powder into PDA, diluting with deionized water to a constant volume of 1L, and sterilizing at 121 deg.C for 30 min;

LB solid medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride, controlling the pH to be 7.5, using deionized water to fix the volume to 1L, and sterilizing at 121 ℃ for 30 min.

S2, preparation of trichoderma spore suspension: culturing Trichoderma in PDA solid culture medium to obtain Trichoderma conidium, sterilizing with 250mL deionized water at 121 deg.C for 30min, washing the cultured PDA culture dish with sterilized deionized water, collecting spores, detecting spores under microscope, and making into 3.5 × 10⁷cfu/mL spore suspension.

S3, preparing a bacillus amyloliquefaciens fermentation liquid: and (3) selecting a single colony of the bacillus amyloliquefaciens obtained by the culture of S1, inoculating the single colony in an LB liquid culture medium, carrying out shake culture for 18h, controlling the pH to be 7.0 and the culture temperature to be 37 ℃, centrifuging the fermentation liquor at 6000r/min after the culture is finished, collecting the supernatant, passing the supernatant through a sterile PTFE (polytetrafluoroethylene) microporous filter membrane with the diameter of 0.22 mu m, and storing the supernatant serving as the A1 fermentation liquor at the temperature of minus 20 ℃ for later use.

The LB liquid medium comprises: peptone 8g/L, beef powder 3g/L, sodium chloride 5g/L, and pH controlled at 7.0.

S4, Trichoderma chlamydospore production: adding 3mL of spore suspension in S2 and 10mL/L A1 of fermentation liquid into B1 culture medium respectively, culturing at 28 deg.C and 180r/min with light for 7d to form chlamydospore, and using in biological control agent.

The B1 culture medium comprises the following components in percentage by weight per 1 liter:

modified MS culture medium 5g/L, sucrose 20g/L, beef extract 5g/L, peptone 3g/L, and pH is calibrated to 6 +/-0.2 by sodium hydroxide.

Wherein, each 1 liter of the improved MS culture medium comprises the following components:

MgSO₄·7H₂O 200mg/L，CaCl₂·H₂O 180mg/L，KNO₃ 600mg/L，(NH)₄NO₃ 550mg/L，KH₂PO₄200mg/L, and the balance of water.

Comparative example 1

This comparative example relates to a process for the production of trichoderma chlamydospores, substantially as in example 1, comprising the following steps:

s1, strain activation: the strain Trichoderma asperellum GDSF1009 is inoculated in a PDA culture dish placed with PDA solid culture medium, and is inversely cultured in an incubator at 28 ℃ for 3 d.

PDA solid medium: peeling 200g of potato, cutting into small pieces, steaming, filtering with gauze, adding 20g of glucose PDA, adding 15g of agar powder, diluting with deionized water to a constant volume of 1L, and sterilizing at 121 deg.C for 30 min.

S2, preparation of trichoderma spore suspension: culturing Trichoderma in PDA solid culture medium to obtain Trichoderma conidium, sterilizing with 250mL deionized water at 121 deg.C for 30min, repeatedly washing the cultured PDA culture dish with liquid transfer gun, and detecting spore content under microscope to obtain 3.5 × 10⁷cfu/mL spore suspension.

S3, Trichoderma chlamydospore production: adding 3mL of spore suspension in S2 into B1 culture medium, culturing at 28 deg.C and 180r/min with light for 7d to form chlamydospore.

5g/L of modified MS culture medium, 20g/L of cane sugar and 5g/L of beef extract, and the pH is calibrated to 6 +/-0.2 by sodium hydroxide.

Comparative example 2

(Trichoderma asperellum) GDSF1009 was inoculated on LB solid medium and cultured in an inverted medium at 37 ℃ for 1 day.

PDA solid medium: peeling 200g of potato, cutting into small pieces, steaming, filtering with gauze, adding 20g of glucose PDA, adding 15g of agar powder, diluting to a constant volume of 1L with deionized water, and sterilizing at 121 deg.C for 30 mi.

Comparative example 3

The comparative example relates to a method for producing trichoderma chlamydospore by using bacillus fermentation liquor, which is basically the same as the example 1, and only differs from the following steps:

MS culture medium is selected from the B1 culture medium instead of modified MS culture medium; that is, the medium is composed of the following components and their concentrations per 1 liter: MS culture medium 5g/L, sucrose 20g/L, beef extract 5g/L, peptone 3g/L, and pH is calibrated to 6 +/-0.2 by sodium hydroxide.

Wherein each 1 liter of MS culture medium comprises the following components:

MgSO4 & 7H2O 370mg/L, CaCl2 & H2O 4400mg/L, KNO 31900 mg/L, (NH)4NO 31650 mg/L, KH2PO 4170 mg/L and the balance of water.

Example 2

s1, strain activation: inoculating Trichoderma asperellum GDSF1009 strain in PDA culture dish with PDA solid culture medium, and culturing in 28 deg.C incubator for 3 d; bacillus amyloliquefaciens (Trichoderma asperellum) GDSF1009 was inoculated on LB solid medium and cultured in an inverted state at 37 ℃ for 18 h.

S3, preparing a bacillus amyloliquefaciens fermentation liquid: and (3) inoculating a single colony of the bacillus amyloliquefaciens obtained by the culture of S1 into an LB liquid culture medium, carrying out shake culture for 18 hours, controlling the pH to be 7.5 and the culture temperature to be 37 ℃, centrifuging the fermentation liquor at 6000r/min after the culture is finished, collecting the supernatant, passing the supernatant through a sterile PTFE (polytetrafluoroethylene) microporous filter membrane with the diameter of 0.22 mu m, and storing the supernatant serving as the A1 fermentation liquor at the temperature of-20 ℃ for later use.

The LB liquid medium comprises: peptone 8g/L, beef powder 3g/L, sodium chloride 5g/L, and pH controlled at 7.5.

S4, Trichoderma chlamydospore production: adding 3mL of spore suspension containing S2 and 5mL/L A1 Bacillus amyloliquefaciens fermentation liquid into B1 culture medium, respectively, culturing at 28 deg.C and 180r/min with light for 7d to obtain chlamydospore for biological control agent.

modified MS culture medium 4g/L, sucrose 10g/L, beef extract 2.5g/L, peptone 1g/L, and pH is calibrated to 6 +/-0.2 by sodium hydroxide.

MgSO₄·7H₂O 180mg/L，CaCl₂·H₂O 180mg/L，KNO₃ 300mg/L，(NH)₄NO₃ 500mg/L，KH₂PO₄ 170mg/L。

example 3

S4, Trichoderma chlamydospore production: adding 3mL of spore suspension containing S2 and 20mL/L A1 Bacillus amyloliquefaciens fermentation liquid into B1 culture medium, respectively, culturing at 28 deg.C and 180r/min with light for 7d to obtain chlamydospore for biological control agent.

modified MS culture medium 5g/L, sucrose 20g/L, beef extract 5g/L, peptone 5g/L, and pH is calibrated to 6 +/-0.2 by sodium hydroxide.

MgSO₄·7H₂O 200mg/L，CaCl₂·H₂O 220mg/L，KNO₃ 700mg/L，(NH)₄NO₃ 600mg/L，KH₂PO₄ 200mg/L。

the Trichoderma chlamydospore productions of the above examples 1-3 and comparative examples 1-3 are shown in Table 1:

TABLE 1 comparison of Trichoderma chlamydospore production (cfu/mL) for different formulations

The microbial preparations obtained in examples 1-3 and comparative examples 1-3 were applied at a rate of 4X 10, calculated as chlamydospores⁵Chlamydospores per square meter, no formulation was added as a control treatment (control in table 2). The formulations of examples 1 to 3 and comparative examples 1 to 3 were used to treat cucumbers in the trefoil stage by root irrigation, and the incidence of powdery mildew of cucumbers was observed. The results are shown in Table 2. Compared with microbial preparations of examples 1-3 and comparative examples 1-3, and the control effect of conventional no-treatment on cucumber powdery mildew, the relative control effect of example 1 is the highest.

TABLE 2 prevention and treatment of cucumber powdery mildew by microbial preparation

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims

1. a method for producing Trichoderma thick chlamydospore, is characterized in that, comprises the following steps:

S1, bacterial classification activation: Trichoderma species is inoculated in PDA solid medium, inverted culture; Bacillus amyloliquefaciens is inoculated on LB solid medium, inverted culture;

S2. Preparation of Trichoderma spore suspension: The Trichoderma spores cultured in S1 were prepared into a Trichoderma spore suspension of 3.5×10 ⁷ cfu/mL;

S3. Preparation of Bacillus amyloliquefaciens fermentation broth A1: pick a single colony of the bacillus cultivated in S1 and inoculate it in LB liquid medium, culture in a shaker flask, centrifuge the fermentation broth and collect the supernatant, and the supernatant after sterilization treatment The liquid was stored at -20°C as fermentation broth A1 for later use;

S4, Trichoderma thick chlamydospore production: respectively take 3 mL of the Trichoderma spore suspension and 10 mL of the fermentation broth A1, and add them to the B1 medium at the same time, the ambient temperature is 28 ° C, and the shaker speed is controlled at 180 r/min , cultured in light for 7 days, chlamydospores were formed; each 1 liter of B1 medium formulation included the following components: modified MS medium 5 g/L, sucrose 20 g/L, beef extract 5 g/L, peptone 3 g/L, And the pH was calibrated to 6±0.2 by sodium hydroxide; each 1 liter of modified MS medium included the following composition: MgSO ₄ ·7H ₂ O 200 mg/L, CaCl ₂ ·H ₂ O 180 mg/L, KNO ₃ 600 mg/L L, NH ₄ NO ₃ 550 mg/L, KH ₂ PO ₄ 220 mg/L, and the balance is water;

In step S3, the LB liquid culture medium contains 8 g/L of peptone, 3 g/L of beef powder, 5 g/L of sodium chloride, and the pH is controlled to 7.0; the temperature of the shake flask culture is 37°C;

The Trichoderma species in step S1 is Trichoderma asperellum GDSF1009;

The Bacillus amyloliquefaciens in step S1 is Bacillus amyloliquefaciens ACCC11060.

2. the method for producing Trichoderma chlamydospore as claimed in claim 1, is characterized in that, in step S3, the method for described sterilization treatment is: the fermentation broth is centrifuged, supernatant is collected and passed through aseptic 0.22 μm The supernatant was stored at -20 °C as fermentation broth A1 for later use.

3. the purposes of the chlamydospore formed by the method for producing Trichoderma chlamydospore as claimed in claim 1 in the preparation of the biocontrol preparation for preventing and treating cucumber powdery mildew.