CN114058521B

CN114058521B - Trichoderma atroviride C61

Info

Publication number: CN114058521B
Application number: CN202210046270.0A
Authority: CN
Inventors: 李红梅; 魏艳丽; 杨凯; 李纪顺; 扈进冬
Original assignee: Ecology Institute Of Shandong Academy Of Sciences (the Sino-Japanese Friendship Biotechnology Research Center Shandong Academy Of Sciences)
Current assignee: Ecology Institute Of Shandong Academy Of Sciences (the Sino-Japanese Friendship Biotechnology Research Center Shandong Academy Of Sciences); Rongcheng Hushan agricultural planting professional cooperative
Priority date: 2022-01-17
Filing date: 2022-01-17
Publication date: 2022-03-22
Anticipated expiration: 2042-01-17
Also published as: CN114058521A

Abstract

The invention relates to trichoderma atroviride C61, and belongs to the technical field of microorganisms. The Trichoderma atroviride (a)Trichoderma atroviride) C61 is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.21035, the preservation date is 2020, 12 months and 08 days, the preservation organization address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North. The invention also provides application of trichoderma atroviride C61 in degradation of phenolic acid and derivatives thereof. The trichoderma atroviride C61 of the invention degrades 9 mixed phenolic acids with the concentration of 100mg/L respectively as follows: ferulic acid, benzoic acid, salicylic acid and cinnamic acid are completely degraded in 2 days; p-hydroxybenzoic acid, syringic acid and p-coumaric acid are completely degraded in 3 days; vanillic acid and vanillin are completely degraded in 6 days. Meanwhile, the self-toxicity of the phenolic acid and the derivatives thereof on plants can be effectively relieved, and the continuous cropping obstacle soil can be repaired.

Description

Trichoderma atroviride C61

Technical Field

The invention relates to the technical field of microorganisms, and in particular relates to trichoderma atroviride C61.

Background

Autotoxic substances are one of main factors causing continuous cropping obstacles, phenolic acid and derivatives thereof are high in activity, and are released into the soil environment in the modes of plant volatilization, leaching, root secretion, litter and residue decomposition and the like, and are accumulated in the soil, so that the physicochemical properties of the soil are remarkably changed. The microbial community structure and diversity increase the proportion of soil-borne pathogenic microorganisms and increase the plant morbidity. Meanwhile, the phenolic acid and the derivatives thereof also influence the membrane system, photosynthesis and in-vivo enzyme activity of the plant, so that the growth of the plant is inhibited. The accumulation characteristics of phenolic acid and derivatives thereof in continuous cropping soil are proved in various plants such as rice, soybean, peanut, cucumber, traditional Chinese medicinal materials, forest trees and the like, one plant can simultaneously generate a plurality of phenolic acid autotoxic substances, and the difference of phenolic acid and derivatives thereof generated by different plants is large.

The method utilizes the microorganisms to decompose phenolic acid and derivatives thereof in the soil, thereby reducing the content of the phenolic acid and the derivatives thereof in the soil, relieving the self-toxicity of the phenolic acid and the derivatives thereof on crops, and being an effective way for solving the continuous cropping obstacles. And the microbial degradation is cheap and efficient, and the method is green and environment-friendly and has wide application prospect. Therefore, the screening of a bacterial strain capable of efficiently degrading various phenolic acids and the application of the bacterial strain in the remediation of the continuous cropping obstacle soil have important practical significance.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides trichoderma atroviride C61. The trichoderma atroviride C61 can efficiently degrade p-hydroxybenzoic acid, vanillic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid, benzoic acid, salicylic acid and cinnamic acid, and effectively relieve the self-toxicity of phenolic acid and derivatives thereof to plants.

Trichoderma atroviride (A) and (B)Trichoderma atroviride) C61, wherein the preservation number of the general microbiological center of China Committee for culture Collection of microorganisms is CGMCC number 21035, the preservation date is 2020, 12 months and 08 days, and the address of the preservation institution is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.

A method for preparing spore suspension containing trichoderma atroviride C61 comprises the steps of inoculating trichoderma atroviride C61 strain on a PDA (personal digital assistant) flat plate, placing the PDA flat plate in an incubator at 25 ℃ for growing for 4-5 days, scraping spores of one flat plate, adding the spores into sterile water, and then passing through 4 layers of sterile lens paper to obtain filtrate, namely the spore suspension.

A screening method of trichoderma atroviride C61 comprises the following specific steps: take 1X 10⁵Inoculating spore into phenolic acid liquid culture medium (PDB culture medium), and culturing in shaker at 25 deg.C and 150rpm for 5 days with the culture medium without inoculating bacteria as control; after the culture is finished, taking 1mL of culture medium, centrifuging for 2min at 10000g, taking 200mL of supernatant, adding 10mL of 0.1% bromocresol green for color development, and screening a strain with high degradation rate according to the shade of the solution color, namely trichoderma atroviride C61.

Preferably, the preparation method of the phenolic acid liquid medium (PDB medium) is as follows: boiling 200g of potato in distilled water for 30min, filtering with gauze, cooling to a constant volume of 1L to obtain potato water; 100mL of potato water, 900mL of distilled water, 100mg of each of p-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, benzoic acid, vanillin, salicylic acid and cinnamic acid are uniformly mixed to prepare a phenolic acid liquid culture medium, the liquid culture medium is subpackaged into test tubes, and autoclaving is performed to obtain the phenolic acid liquid culture medium.

The application of trichoderma aureoviride C61 in degrading phenolic acid and derivatives thereof by using trichoderma aureoviride C61 is disclosed.

Preferably, the phenolic acid and the derivatives thereof comprise at least one of p-hydroxybenzoic acid, vanillic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid, benzoic acid, salicylic acid or cinnamic acid.

Application of spore suspension containing Trichoderma atroviride C61 or preparation containing the spore suspension as effective component in relieving autotoxicity of phenolic acid and its derivatives on plants or repairing continuous cropping obstacle soil caused by phenolic acid and its derivatives is provided.

Preferably, the soil is used for planting tomatoes or American ginseng.

Preferably, the application also comprises dressing or soaking seeds by using spore suspension of trichoderma atroviride C61 or preparation with the spore suspension as the effective component during sowing.

Preferably, the application further comprises a root dipping treatment when the perennial plants are transplanted in a growing period using a preparation containing trichoderma atroviride C61 as an active ingredient.

The invention has the beneficial effects that:

the trichoderma atroviride C61 can efficiently degrade p-hydroxybenzoic acid, vanillic acid, syringic acid, vanillin, p-coumaric acid, ferulic acid, benzoic acid, salicylic acid and cinnamic acid, and effectively relieve the self-toxicity of phenolic acid and derivatives thereof to plants. The bacterial strain degrades 9 mixed phenolic acids with the concentration of 100mg/L in a PDB culture medium as follows: ferulic acid, benzoic acid, salicylic acid and cinnamic acid are completely degraded in 2 days; p-hydroxybenzoic acid, syringic acid and p-coumaric acid are completely degraded in 3 days; vanillic acid and vanillin are completely degraded in 6 days. The bacterial strain degrades 9 mixed phenolic acids with the concentration of 100mg/g dry soil in the soil as follows: ferulic acid, benzoic acid, salicylic acid and cinnamic acid are completely degraded within 5 days, the degradation rates of p-hydroxybenzoic acid, syringic acid and p-coumaric acid within 5 days respectively reach 81.28%, 80.45% and 82.14%, the degradation rates of p-hydroxybenzoic acid, syringic acid and p-coumaric acid within 10 days are completely degraded within 10 days, the degradation rates of vanillin and vanillic acid within 10 days are 83.82%, 78.78% and the degradation rates of p-coumaric acid within 15 days are completely degraded.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 is a graph showing the degradation tendency of Trichoderma atroviride C61 in PDB medium for 9 mixed phenolic acids in example 2 of the present invention.

FIG. 2 is a diagram showing the germination of lettuce in example 2 of the present invention using clear water as a medium.

FIG. 3 is a diagram showing the germination of lettuce in example 2 of the present invention using a degradation liquid as a medium.

FIG. 4 is a graph showing the germination of lettuce in example 2 of the present invention using a PDB medium (containing 9 phenolic acids) without inoculation as the medium.

FIG. 5 is a diagram showing the hypocotyl growth of a lettuce seedling cultured using clear water as a medium in example 2 of the present invention.

FIG. 6 is a diagram showing the hypocotyl growth of a lettuce seedling cultured using a degradation liquid as a medium in example 2 of the present invention.

FIG. 7 is a graph showing the degradation tendency of Trichoderma atroviride C61 in soil of 9 mixed phenolic acids in example 3 of the present invention.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The invention provides a trichoderma atroviride strainTrichoderma atroviride) C61, deposited at the china community collection and management committee for microorganisms, at 08 d 12/2020, address: no. 3 Xilu No. 1 Beijing, Chaoyang, North Chen, with a collection number of CGMCC number 21035. The specific screening method of trichoderma atroviride C61 is as follows:

preparing PDB culture medium (prepared by boiling potato 200g in distilled water for 30min, filtering with gauze, cooling to constant volume of 1L to obtain potato water, mixing potato water 100mL, distilled water 900mL, p-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, benzoic acid, vanillin, salicylic acid and cinnamic acid 100mg, mixing well to obtain phenolic acid liquid culture medium, and subpackaging the liquid culture medium with phenolic acid liquid culture mediumAutoclaving in test tube), scraping activated spore of strain to be detected, adding sterile water, shaking, passing through 4 layers of sterile lens paper to obtain filtrate as spore suspension, counting by using hemocytometer, and collecting 1 × 10⁵Inoculating the spores into PDB culture medium, setting culture medium without inoculating bacteria as control, and culturing in shaker at 25 deg.C and 150rpm for 5 days; after the culture is finished, 1mL of culture medium is taken, 10000g of culture medium is centrifuged for 2min, then 200mL of supernatant is taken, 10mL of 0.1% bromocresol green is added for color development, and strains with high degradation rate are screened according to the shade of the solution color, wherein the strain C61 is darkest in color development and is dark blue.

Example 2

1. Degradation of mixed phenolic acids in PDB medium by Trichoderma atroviride C61

1.1 preparation of standard solution and preparation of standard curve: accurately preparing 9 phenolic acid mixed standard substances (p-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, benzoic acid, vanillin, salicylic acid and cinnamic acid) by using 50% methanol as a solvent, wherein the concentrations of the p-hydroxybenzoic acid, the vanillic acid, the syringic acid, the p-coumaric acid, the ferulic acid, the benzoic acid, the vanillin and the salicylic acid are respectively 200, 100, 50, 25, 12.5, 6.25 and 3.125mg/L, the concentrations of the cinnamic acid are respectively 50, 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125mg/L, and the linear regression equation of the peak area X and the peak area Y of each phenolic acid is obtained by measuring the cinnamic acid with a high performance liquid chromatograph after passing through a filter membrane of 0.22 mu m;

1.2 determination of phenolic acid and its derivative content in culture medium: inoculating the stored trichoderma atroviride C61 strain to a PDA (PDA dextrose agar) plate, placing the plate in an incubator at 25 ℃ for 5 days, scraping spores of one plate, adding the plate into sterile water, and passing through 4 layers of sterile lens paper to obtain filtrate, namely spore suspension. Inoculating the spore suspension into PDB culture medium (containing 9 mixed phenolic acids with concentration of 100mg/L respectively) and non-inoculated PDB culture medium (containing 9 mixed phenolic acids with concentration of 100mg/L respectively) as control, performing shake culture at 25 deg.C and 150rpm for 7 days, repeating inoculation and control for three times, and sampling once every 24 h. Centrifuging the culture medium, mixing the supernatant with methanol at a ratio of 1:1, filtering with 0.22 μm filter membrane, and detecting phenolic acid content with high performance liquid chromatograph.

Chromatographic conditions are as follows: using Symmetry C18 chromatographic column (250 mm × 4.6mm, 5 μm), and mobile phase of 0.5% acetic acid water solution (A) and methanol (B); flow rate 0.7 mL.min^-1The column temperature is 35 ℃, the sample injection volume is 10 mu L, and the detection wavelength is 280 nm; gradient elution procedure: 0-10min, 95% -80% A; 10-13min, 80% -65% A; 13-33min, 65% -50% A; 33-48min, 50% -5% A; 48-50min, 95% A;

1.3 Effect of degradation liquid on lettuce Germination and seedling growth

The fermentation liquid obtained by culturing the inoculated strain in example 1.2 for 7 days was used as the degradation liquid of C61, and it was examined whether the degradation liquid had an effect on the germination and seedling growth of a phenolic acid sensitive plant, lettuce. Taking 10mL of degradation liquid and a control without inoculation, centrifuging, collecting supernatant, adding ethyl acetate with the same volume for extraction three times, combining extract liquid, rotating to evaporate to dryness, adding 10mL of water for dissolving, and using for a germination test. The experiment was set up with three treatments: degradation liquid; no inoculation control (i.e., phenolic acid control); and (4) comparing water, repeating every three times of treatment, adding 6mL of each treatment solution into a 9cm culture dish (3 layers of sterile filter paper are placed in the culture dish), adding 30 lettuce grains, placing the lettuce grains in a light incubator with the temperature of 25 ℃/20 ℃ and the light/dark of 16h/8h, counting the germination rate for 3 days, and counting the seedling height and fresh weight for 7 days. Analysis was performed using the Duncan's method in SPSS 21.0 statistical software, and the results were mean ± standard deviation of 3 replicates.

1.4 results

Calculating the residual amount of each phenolic acid in the culture medium according to a linear regression equation of each phenolic acid standard solution, and according to a formula: degradation rate (%) = (control concentration-treatment concentration)/control concentration × 100% the degradation rate of each phenolic acid was calculated.

Analysis shows that the strain C61 in example 2 has obvious degradation effect on 9 phenolic acids, the degradation rates of the phenolic acids are different, and ferulic acid, benzoic acid, salicylic acid and cinnamic acid are completely degraded in 2 days; p-hydroxybenzoic acid, syringic acid and p-coumaric acid are completely degraded in 3 days; vanillic acid, vanillin were completely degraded in 6 days (see figure 1). The degradation rate of vanillic acid in the next day is negative because one of metabolic intermediates of ferulic acid is vanillic acid, which is produced in the process of rapid degradation of ferulic acid, and the originally added vanillic acid accumulates in the culture medium, but is rapidly degraded by the strain.

After the PDB culture medium containing 9 mixed phenolic acids is inoculated with C61 and cultured for 7 days, the germination rate, plant height and fresh weight of degradation liquid to lettuce are shown in table 1, compared with a water control, the degradation liquid has no significant difference to the germination rate of lettuce, the fresh weight of seedlings and the water control, and has obvious promotion effect on seedling bud growth. As can be seen from the figures 2, 3, 4, 5 and 6, the degradation liquid has no influence on the germination rate and has obvious growth promotion effect on the hypocotyl of the vegetable seedling.

TABLE 1 Trichoderma atroviride C61 degradation liquid for lettuce germination and seedling

Note: different lower case letters after the same column of data indicate 0.05 level differences.

2. Trichoderma atroviride (Trichoderma atroviride) Degradation of phenolic acid and derivatives thereof in soil by C61

2.1 artificial phenolic acid soil treatment: the soil is naturally dried after being screened by a 10-mesh sieve, sterilized for three times at 121 ℃ for 1 hour each time, and is put into a tissue culture bottle according to 100 g/bottle, and a mixed solution of 9 phenolic acids (p-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, benzoic acid, vanillin, salicylic acid and cinnamic acid) is added to ensure that the concentration of each phenolic acid is 100mg/g of dry soil. Growing strain C61 on PDA plate for 5 days, scraping spores, adding sterile water, passing through 4 layers of sterile lens paper to obtain filtrate, counting the spore suspension with blood counting plate, inoculating into soil to make the spore concentration in soil be 10⁶CFU/g dry soil, phenolic acid soil without inoculation as control, soil humidity of 35%, sealing with ventilated membrane, placing in 25 deg.C incubator for dark culture, sampling once every 5 days, and detecting the residual amount of each phenolic acid in soil.

2.2 extraction of phenolic acid in soil: and naturally drying the soil sample, and grinding. Taking 5g, adding 8mL of 1mol/L NaOH solution, extracting for 12h at 25 ℃ by shaking at 200rpm, centrifuging, collecting supernate, adjusting the pH of the solution to 2.5, adding equal volume of ethyl acetate for extraction for three times, combining extracts, carrying out rotary evaporation, and dissolving residues with 80% methanol to constant volume.

2.3 determination of phenolic acid content in soil: the extract is measured by a high performance liquid chromatograph after passing through a 0.22 mu m filter membrane, the measuring method is the same as that of liquid degradation, and the degradation rate is calculated according to the formula: degradation rate (%) = (control concentration-treatment concentration)/control concentration × 100%

2.4 results

Trichoderma atroviride C61 was able to completely degrade phenolic acid in soil within 15 days (FIG. 7). Within 5 days, ferulic acid, benzoic acid, salicylic acid and cinnamic acid are completely degraded, the degradation rates of p-hydroxybenzoic acid, syringic acid and p-coumaric acid are 81.28%, 80.45% and 82.14% respectively, the three phenolic acids are also completely degraded by 10 days, the degradation rates of vanillic acid and vanillin reach 78.72% and 83.32% respectively by 10 days, and all the phenolic acids are completely degraded by 15 days.

Example 3

Trichoderma atroviride (Trichoderma atroviride) C61 relieving autotoxicity of phenolic acid and its derivatives on plants

Pot experiment: the test soil is taken from a test field of ecological research institute of academy of sciences of Shandong province, screened to remove impurities, tested for moisture content, loaded into a flowerpot (19 cm multiplied by 16 cm) according to 1000g of dry soil, and added with 100mL of 9 phenolic acid mixed solution of p-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, benzoic acid, vanillin, salicylic acid and cinnamic acid, so that the concentration of each phenolic acid in the soil is 30mg/g of dry soil; after the strain C61 grows for 5 days on a PDA plate, scraping spores, adding sterile water, passing through 4 layers of sterile lens paper, obtaining filtrate which is spore suspension, counting the spore suspension by a blood counting chamber, and adjusting the concentration of the spore suspension to 10⁸CFU/mL, placing tomato seeds for seed soaking for 1h, and setting the following treatment in the test: a first: blank control (no phenolic acid, clear water + tomato); II: treatment of group 1 (phenolic acid + tomatoes); no. three: treatment of group 2 (phenolic acid + tomato + trichoderma atroviride C61); each group of five repeats, each repeat comprises 4 plants, the plants are placed in a phytotron (25 ℃/18 ℃ and the illumination is 14 h) for conventional management, and after 4 weeks of growth, the plant height and the root length of the plants are measuredChlorophyll content, malondialdehyde content, above-ground dry weight, underground dry weight and the like.

As a result: after 4 weeks of growth, the growth indexes of each treatment are shown in table 2, after the exogenous phenolic acid is added into the soil, the growth of tomato plants is seriously influenced, the plants are short and small, particularly the growth of roots is seriously inhibited, and photosynthesis and cell membranes are also seriously influenced; after the trichoderma atroviride C61 is added, the stress of phenolic acid on the growth, photosynthesis and cell membranes of tomato plants can be remarkably relieved, and the growth of the plants is facilitated.

TABLE 2 Trichoderma atroviride C61 Effect on alleviating the autotoxicity of tomato by phenolic acids

Example 4

Trichoderma atroviride (Trichoderma atroviride) C61 restoration of soil with American ginseng continuous cropping obstacle

Field test: the experimental plot selects a demonstration base of American ginseng in the Huhai town of Weihai city in the main production area of American ginseng, and the following treatment is set: 1. a blank control zone; 2. a chemical pesticide treatment zone; 3. trichoderma atroviride C61 treatment area. Each zone has 1 ridge (2.5 m is multiplied by 40 m), three are repeated and randomly arranged, and the management is performed regularly at the same level.

When the seeds are planted at the bottom of 10 months, seed dressing treatment is carried out on the ginseng seeds, and a preparation used for seed dressing comprises the following components: the trichoderma atroviride C61 is fermented to prepare a conidium parent drug, 0.5 percent of glucose and 90 to 95 percent of medical stone are added according to the weight percentage of the parent drug to prepare the trichoderma seed dressing agent, and the effective spore content is 2 hundred million/g. The dosage of the preparation is 500 g/mu. The rate of emergence was counted in the second and third 5 months, and the roots were dipped in the next year after transplanting the one year ginseng, the used preparation was the same as the seed dressing preparation, the amount of spores was 2 hundred million/g, and the amount was 500 g/mu. Investigating rust and root rot diseases of roots when the ginseng is harvested for three years, calculating the prevention and treatment effect, and determining the yield and the saponin content of the American ginseng.

As a result: the emergence rate, the control effect and the yield increase rate of the American ginseng are calculated according to the following formulas, the results are shown in table 3, after the trichoderma atroviride C61 is applied into soil, the problem of continuous cropping obstacle of the American ginseng can be obviously improved, and the emergence rate is obviously different from the contrast; the prevention and treatment effect on main root diseases reaches 75.55%, the yield is increased to 205.24%, the content of ginsenoside (Rg1+ Re + Rb1) reaches 5.48%, the soil-borne diseases of the roots can be effectively prevented and treated, the yield is increased remarkably, the quality is improved, and the using amount of chemical pesticides is reduced greatly.

Emergence rate (%) = number of emergence/number of seeds sowed × 100%

Control effect (%) = (control disease rate-treated disease rate)/control disease rate × 100%

Yield increase (%) = (fresh weight of processed ginseng-fresh weight of control ginseng)/fresh weight of control ginseng × 100%

TABLE 3 repair of soil from American ginseng by Trichoderma atroviride C61 preparation

Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. Trichoderma atroviride C61, wherein Trichoderma atroviride (Trichoderma atroviride C61)Trichoderma atroviride) Is preserved in China general microbiological culture Collection center with the preservation number of CGMCC number 21035 and the preservation date of 2020Day 12 month 08, depository address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.