CN111139186B

CN111139186B - Trichoderma viride with disease prevention and growth promotion functions and application thereof

Info

Publication number: CN111139186B
Application number: CN201911389054.0A
Authority: CN
Inventors: 刘红彦; 千慧敏; 赵辉; 刘新涛; 倪云霞; 文艺; 李小杰; 李淑君; 赵新贝; 何碧珀
Original assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Henan Academy of Agricultural Sciences
Priority date: 2019-12-28
Filing date: 2019-12-28
Publication date: 2022-07-15
Anticipated expiration: 2039-12-28
Also published as: CN111139186A

Abstract

The invention relates to trichoderma virens capable of simultaneously preventing and treating tobacco black rot and root rot and promoting tobacco growth, and belongs to the technical field of biological prevention and treatment. The Trichoderma viride NKSF2019001F (Trichoderma virens NKSF2019001F) is preserved in China center for type culture Collection with the preservation number: CCTCC NO: m2019767; also provides a screening method of the trichoderma virens and biocontrol bacteria liquid. The strain can simultaneously prevent and treat the black rot and the root rot of tobacco roots, has the prevention effect on two tobacco diseases of more than 65 percent, can effectively reduce the morbidity of the root and stem diseases of the tobacco, and has the prevention effect equivalent to that of the conventional common chemical pesticide; meanwhile, the strain also has the function of promoting the growth of tobacco seedlings. The trichoderma virens of the invention can not generate drug resistance after being used, has no problems of pesticide residue, environmental pollution and the like, is safe and environment-friendly, and meets the requirements of ecological agriculture.

Description

Trichoderma viride with disease prevention and growth promotion functions and application thereof

Technical Field

The invention relates to trichoderma virens capable of simultaneously preventing and treating black rot and root rot of tobacco roots and promoting tobacco growth, and belongs to the technical field of biological prevention and treatment.

Background

The diseases and insect pests of the tobacco are one of the main factors restricting the high quality and stable yield of the tobacco, and particularly the diseases and the types of the roots and the stems of the tobacco are complex and seriously damaged. For a long time, tobacco root diseases cause tobacco yield reduction, and destructive loss is caused when serious diseases occur. In recent years, the tobacco root black rot and the tobacco root rot are mixed in the tobacco area in Henan, which causes great loss to the plant and tobacco farmers.

Tobacco root black rot is a common soil-borne fungal disease, the causative agent of which is rhizoctonia solani (Thielaviopsis basicola). The tobacco can generate black root rot from a seedling stage to a plant forming stage, the root of the tobacco is mainly infected, and the root of the plant is rotten during disease occurrence and presents specific black; damping-off is easy to occur when the seedlings are infected, the infected tobacco seedlings in the field grow slowly, tobacco plants are dwarfed, and the yield and the quality of tobacco are greatly influenced.

The root rot of tobacco can harm the root and stem of tobacco, and the pathogenic bacteria mainly include Fusarium oxysporum (Fusarium oxysporum), Fusarium solani (f.solani), etc., and have pathogenicity and parasitism to the root of tobacco. The disease can cause the root of the plant to be diseased, and only one side of the root is diseased, so that the top is bent to one side; after the disease occurs, the root of the tobacco is rotten, the xylem is browned, the absorption of moisture and nutrition of the tobacco is blocked, and finally, the tobacco plant is dead. In the middle and late field, there are no obvious stem symptoms, yellow leaf, obvious root, black main root and side root, and mildew layer from white to pink in wet state.

In recent years, the damage of soil-borne diseases such as tobacco black rot and tobacco root rot continues to increase, and the disease has become a main root and stem disease in partial tobacco areas. For a long time, the prevention and treatment of soil-borne diseases are mainly chemical prevention and treatment, and the use of a large amount of chemical pesticides can cause serious pollution of the chemical pesticides, thereby greatly influencing the food safety and the body health of people. With the concern of food safety and environmental safety in all countries in the world, the popularization and application of biological control of plant diseases and insect pests are vigorously advocated in all countries, so that biological pesticides obtain better development opportunity.

The currently reported biocontrol strains related to the tobacco root black rot mainly comprise bacillus strains, actinomycetes strains, trichoderma strains and other strains, the research on the biocontrol strains related to the tobacco root black rot is less, and meanwhile, the biocontrol strains for preventing and treating the tobacco root black rot and the root black rot are fewer, so that the screening of the trichoderma strains capable of effectively preventing and treating the two diseases and having a growth promoting effect on tobacco has important theoretical and practical significance.

Disclosure of Invention

The trichoderma virens strain can be used for simultaneously preventing and treating the black rot and the root rot of tobacco roots, can effectively reduce the morbidity of the root and stem diseases of tobacco, and has the function of promoting the growth of tobacco seedlings.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a Trichoderma virens strain with disease prevention and growth promotion functions is Trichoderma virens NKSF2019001F (Trichoderma virens NKSF2019001F), which is preserved in China center for type culture collection, with the preservation address as follows: wuhan university collection center, accession number: CCTCC NO: m2019767, deposit date: 20190929.

collecting soil in sesame field in Pingyu county of Youman shop, Henan province, adding 5g of soil sample into a triangular flask containing 45mL of sterile water, shaking for 30min at 160r/min on a shaking table, standing for 30s, taking supernatant as stock solution, and diluting by gradient of 10 times to 10 times^-4And coating 100 mu L of the diluent on a plate of a Bengal culture medium, culturing until bacterial colonies grow out, separating and purifying, screening strains with inhibiting effects on moniliforme rhizoctonia and fusarium oxysporum by using a plate antagonism test, and identifying to obtain Trichoderma viride NKSF 2019001F.

The preparation method of the biocontrol bacterial liquid of the trichoderma virens comprises the steps of taking trichoderma virens NKSF2019001F cultured for 5 days, and adding 0.05% (v/v) Tween 80 to obtain 2.0 multiplied by 10⁷cfu/mL conidium solution is inoculated into the culture solution by 3 percent of inoculum size, and the culture conditions are as follows: the fermentation temperature is 28 ℃, the fermentation time is 9d, the rotating speed is 210r/min, the initial pH is 8, and the concentration of chlamydospore liquid in the biocontrol bacterial liquid obtained after fermentation is 7.6 multiplied by 10⁷cfu/mL。

The formula of the culture solution is as follows: 25g of corn flour, 15g of glucose, 15g of corn protein powder, 1.3g of NaCl and MgSO₄0.4g、KH₂PO₄ 0.8g、CaCO₃2g/L and 1L of distilled water.

The application of the trichoderma virens in simultaneously preventing and treating the tobacco root black rot and the root rot is provided.

The application of the trichoderma virens in promoting the growth of tobacco.

The application of the trichoderma virens in preparing a biocontrol microbial inoculum for simultaneously preventing and treating tobacco root black rot and root rot and/or promoting tobacco.

The invention has the beneficial effects that:

according to the invention, biocontrol bacteria NKSF2019001F is screened from soil of sesame fields of Shimeji shop, Henan province for the first time, the strain is identified to be trichoderma viride, and antagonism, disease prevention and pot culture growth promotion experiments prove that the strain can prevent and control the black rot and the root rot of tobacco simultaneously, the prevention effect on two tobacco diseases reaches more than 65%, the morbidity of tobacco root and stem diseases can be effectively reduced, and the effect is equivalent to that of the conventional common chemical pesticide; meanwhile, the strain also has the function of promoting the growth of tobacco seedlings.

Disease prevention tests of the biocontrol bacterium disclosed by the invention show that the control effect of the biocontrol bacterium NKSF2019001F on tobacco root black rot is 74.36%, and the effect is superior to that of medicament control; the control effect of NKSF2019001F on tobacco root rot is 65.38%, and the effect is equivalent to that of medicament control.

The pot growth promotion test of the invention shows that the plant height, the maximum leaf area, the effective leaf number, the fresh weight, the dry weight, the root length, the fresh weight and the dry weight of the root of the tobacco are respectively increased compared with the control after the treatment of the spore liquid of the strain NKSF 2019001F; except for root length and fresh weight of roots, other characters have significant difference (P <0.05) compared with a control, which indicates that NKSF2019001F can promote tobacco growth to a certain extent.

The trichoderma virens of the invention can not generate drug resistance after being used, can not have the problems of pesticide residue, environmental pollution and other chemical pesticide residues, is healthy to human and livestock, environment-friendly, safe and environment-friendly, and meets the requirements of ecological agriculture.

Drawings

FIG. 1 shows the bacteriostatic effect of NKSF2019001F on Rhizobolus nivale and Fusarium oxysporum, in which A is a counter plate method and B is a drug-containing plate method;

FIG. 2 is a graph showing the effect of the strain NKSF2019001F on the plate of 12 pathogenic fungi,

wherein: 1-12 represent the bacteriostatic effect of NKSF2019001F and the 1-12 pathogenic bacteria plate confronting method in Table 3, and 1CK-12CK represents the growth condition of the 1-12 pathogenic bacteria plate confronting method in Table 3;

FIG. 3 is a graph showing the bacteriostatic effect of the NKSF2019001F strain on a drug-containing plate of 12 pathogenic fungi,

wherein: 1-12 represent the bacteriostatic effect of NKSF2019001F and the drug-containing plate method of 1-12 pathogenic bacteria in Table 3, respectively, and 1CK-12CK represents the growth of the control in the drug-containing plate method of 1-12 pathogenic bacteria in Table 3, respectively;

FIG. 4 growth pattern of NKSF2019001F on PDA plates;

FIG. 5NKSF2019001F under microscope;

FIG. 6 is a phylogenetic NKSF2019001F tree constructed in orthotopic fashion based on ITS sequences;

FIG. 7 shows a phylogenetic NKSF2019001F tree constructed in the ortho-position based on the Tef 1-alpha sequence;

FIG. 8 sporulation of NKSF2019001F on 3 media;

in the figure: different lower case letters on the bar graph indicate 5% significant difference levels; different capital letters indicate a 1% significant level of difference.

FIG. 9 Effect of different fermentation conditions on the chlamydospore productivity of NKSF 2019001F.

Detailed Description

The following examples further illustrate the embodiments of the present invention in detail.

Experimental materials:

bengal red medium: peptone 5g, glucose 10g, KH₂PO₄ 1g，MgSO₄·7H₂0.5g of O, 100mL of 1/3000(w/v) Bengal solution, 0.1g of chloramphenicol, 15-20 g of agar, 1000mL of distilled water and pH 7.2 +/-0.2.

PDA culture medium: 200g of potatoes (cutting the potatoes into small blocks with the side length of about 1cm, putting the small blocks into 800ml of distilled water, boiling for 15min, filtering by double-layer gauze, and then taking filtrate), 20g of glucose and 20g of agar, uniformly mixing, fixing the volume of the distilled water to 1000ml, and sterilizing at 121 ℃ for 30 min.

Culturing the gliotoxin culture solution: 25g of glucose, 2g of ammonium tartrate, 0.01g of ferrous sulfate, 1g of magnesium sulfate heptahydrate, adding distilled water to reach a constant volume of 1000mL, and sterilizing at 121 ℃ for 30min for later use. Pathogens that may be used in the present invention are shown in table 1.

Table 1 examples of possible pathogenic bacteria for use in the test

Other raw materials and reagents in the invention can be purchased from the market.

Example 1 screening of biocontrol bacteria (plate antagonism test)

Collecting soil in sesame field of Pingyu county of Chengma shop, Henan province, grinding, sieving, collecting soil sample 5g, adding into a triangular flask containing 45mL sterile water, shaking by 160r/min for 30min, standing for 30s, collecting supernatant as stock solution, and diluting by 10 times gradient to 10 times^-4g/mL. Taking out 100 μ L of the diluent, coating on a Bengal red culture medium plate, culturing at 28 deg.C, separating and purifying after bacterial colony grows out, selecting the edge of the bacterial colony, purifying on a new PDA plate to obtain purified strain, storing in 25% glycerol, and storing at-70 deg.C for use.

The method uses moniliforme and fusarium oxysporum as target bacteria, and adopts a plate confronting method and a drug-containing plate method to screen purified mould.

Plate confrontation method inoculates 0.6cm target bacteria cake at the center of a PDA plate with a diameter of 9cm, inoculates separated antagonistic strain at the edge of the plate with a distance of 2cm from the target bacteria, and takes singly inoculated target bacteria as control, each treatment is repeated for 3 times. Culturing in an incubator at 28 ℃ for 4d, measuring the radius of pathogenic bacteria colonies of the control and the treated pathogenic bacteria, and calculating the relative bacteriostasis rate.

Relative inhibition (%) (control colony diameter-treated colony diameter) × 100/(control colony diameter-cake diameter)

Preparing sterile fermentation filtrate by a drug-containing flat plate method: collecting Trichoderma strain NKSF2019001F cultured for 5d, and preparing 5 × 10 with 0.05% Tween 80⁶cfu/mL conidia solution was inoculated to the gliotoxin culture medium at an inoculum size of 1%, and fermented for 9 days at 28 ℃ and 160r/min on a shaker. Centrifuging the fermentation liquid, collecting supernatant, filtering with 0.22 μm microporous membrane for three times to obtain sterile fermentation filtrate, and storing at 4 deg.C.

PDA plates containing 20% sterile filtrate were prepared, and 1 cake of pathogenic bacteria (Leuconostoc rhizogenes and Fusarium oxysporum) with a diameter of 0.6cm was inoculated into the center of each dish for 3 times, respectively, using the medium containing no filtrate as a control. After the control pathogenic bacteria basically grow over the culture dish, the diameters of the colonies of the control pathogenic bacteria and the treated pathogenic bacteria are measured, and the relative bacteriostasis rate is calculated.

Finally, 1 strain of trichoderma NKSF2019001F with an inhibitory effect on both moniliforme and fusarium oxysporum is obtained by screening (see table 2 and figure 1).

Table 2 results of NKSF2019001F bacteriostasis on Leuconostoc rhizogenes and Fusarium oxysporum

Note: data are mean ± sem, the same below.

Example 2 measurement of bacteriostatic spectra of biocontrol bacteria

The bacterial inhibition spectra of NKSF2019001F were measured by plate counter-current and drug-containing plate methods, respectively.

The procedure is as in example 1. The results are shown in Table 3 and FIGS. 2 and 3.

As can be seen from table 3, fig. 2 and fig. 3, in the plate confrontation test, the strain NKSF2019001F has good bacteriostatic activity on 12 pathogenic bacteria to be tested, the average bacteriostatic rate on the 12 pathogenic bacteria reaches 61.67% -98.99%, wherein the bacteriostatic rate on the mulberry basidiomycetes in the plate confrontation is the highest (98.99%), and then the rhizoctonia cerealis (98.40%) in turn; in the drug-containing plate test, the bacteriostatic rates of the drug-containing plate test on other 7 pathogenic bacteria except for coccobacillus sphaericus, phacellaria phaseoloides, rhizoctonia solani, rhizoctonia cerealis and gluconobacter viticola are all more than 50%, wherein the bacteriostatic rate on thelephora funiculorum is the highest (76.47%). The isolated bacterium NKSF2019001F has better broad-spectrum bacteriostatic activity.

TABLE 3 bacteriostatic spectrum of biocontrol bacterium NKSF2019001F

Example 3 identification of biocontrol bacteria

Morphological observation results show that NKSF2019001F colonies are circular on a PDA plate, hyphae at the initial growth stage is white, aerial hyphae exist, after the mycelia are cultured for 2 days at 25 ℃, amorphous cotton flocculent white aerial hyphae are generated, conidia are generated, obvious conidium piles appear at the later stage, and the conidium piles are converged into wide grayish green concentric ring veins (figure 4). Observing under a microscope, wherein hyphae are colorless and fine, and have separation and multiple branches; conidiophores are grown from the lateral branches of hyphae, and generally have 2-3 recurrent branches, phialides in ampoule shape, are single or in opposite generation, and are arranged in a recurrent branch shape (fig. 5A). Most conidia were oval or subspherical with smooth walls (fig. 5B).

The genome of biocontrol trichoderma strain NKSF2019001F is used as a template, ITS fragments are amplified by universal primers ITS1 and ITS4, and Tef 1-alpha gene sequences are amplified by EF1T and EF 2T. The PCR product was detected as a single band by 1.0% agarose gel electrophoresis and was sequenced by Biotechnology engineering (Shanghai) Co., Ltd. Uploading the sequencing result to a NCBI database, wherein the accession numbers of ITS and Tef 1-alpha sequences are respectively MN493044 and MN447667, selecting similar sequences through Blast comparison on the NCBI, and constructing a phylogenetic tree, wherein the result shows that the strain NKSF2019001F is Trichoderma viride, see FIG. 6 and FIG. 7 (characters in parentheses in the figure represent the accession numbers of the strain NCBI sequence).

Example 4 Condition optimization of biocontrol bacteria

Preparation of trichoderma virens spore suspension: culturing 7d conidia of Trichoderma viride on PDA plate, eluting with 10mL sterile water, counting with blood counting plate, adjusting concentration to 2.0 × 10⁷cfu/mL。

On the basis of a fermentation industrial product culture medium formula, the following 3 culture media are selected as candidate culture media:

m1: corn flour 10g, bean pulp 4.7g, CaCO₃ 5g、(NH₄)₂SO₄ 1.5g、KH₂PO₄1.7g, 1L of distilled water;

m2: 5g of glucose, 5g of corn flour, 4.7g of corn protein powder and CaCO₃ 5g、(NH₄)₂SO₄ 1.5g、KH₂PO₄1.7g, 1L of distilled water;

m3: corn flour 25g, glucose 15g, corn protein powder 15g, NaCl 1.3g, MgSO₄ 0.4g、KH₂PO₄ 0.8 g、CaCO₃2g, 1L of distilled water;

basic fermentation conditions are as follows: the temperature is 28 ℃, the rotating speed is 180r/min, the initial pH of the fermentation is 7.0, the inoculation amount is 3%, the liquid loading amount is 200 mL/500mL, and the fermentation time is 7 d. And (3) detecting the final quantity of conidia and chlamydospores by adopting a blood counting plate, and determining the most suitable fermentation medium of the strain.

Determination of optimal fermentation temperature: the optimum culture medium is used as a basic culture medium, and the chlamydospore number of the antagonistic trichoderma is monitored at the culture temperature of 22 ℃, 25 ℃, 28 ℃ and 31 ℃.

Determination of optimal fermentation time: and monitoring the chlamydospore number of antagonistic trichoderma fermentations at 3d, 5d, 7d and 9d by taking the optimal culture medium as a basic culture medium at the optimal fermentation temperature.

Determination of the optimum fermentation speed: the optimal culture medium is used as a basic culture medium, the chlamydospore quantity fermented by antagonistic trichoderma at different rotating speeds of 150r/min, 180r/min, 210r/min, 230r/min and 250r/min is monitored under the optimal fermentation time and temperature, and the optimal fermentation rotating speed is determined.

Determination of optimum fermentation initial pH: the optimum culture medium is used as a basic culture medium, the quantity of conidia and chlamydospores fermented by antagonistic trichoderma under different pH values (pH values are respectively 5, 6, 7, 8 and 9) is monitored under the optimum fermentation time, temperature and rotating speed, and the optimum fermentation pH value is determined.

The fermentation optimization results (FIG. 8, FIG. 9) show that the M3 medium was used as the basic fermentation medium for NKSF2019001F, at 28 deg.C, 9d, 210r/min, and initial pH of 8.

Example 5 disease prevention test of biocontrol bacteria

The fermentation medium is M3, and the formula is as follows: corn flour 25g, glucose 15g, corn protein powder 15g, NaCl 1.3g, MgSO₄ 0.4g、KH₂PO₄ 0.8g、CaCO₃2g, 1L of distilled water;

preparing trichoderma virens biocontrol bacterial liquid: collecting Trichoderma viride NKSF2019001F cultured for 5d, adding 0.05% (v/v) Tween 80 to obtain 2.0 × 10⁷Inoculating each/mL conidium solution into a fermentation culture solution by 3% of inoculum size, wherein the culture conditions are as follows: the fermentation temperature is 28 ℃, the time is 9d, the rotating speed is 210r/min, the initial pH is 8, and the biological control bacteria liquid obtained after fermentationThe chlamydospore concentration of (b) was 7.6X 107 cfu/mL. When in use, the biocontrol bacterium liquid and the tobacco rhizosphere soil can be directly mixed uniformly.

5-6 true leaf tobacco seedlings with consistent growth vigor are taken for testing, and 6 treatments are set for the test:

t1: inoculating antagonistic bacteria and rhizomucor; t2: inoculating 70% thiophanate methyl wettable powder and moniliforme;

t3: inoculating sterile water and Rhizopus niveus; t4: inoculating antagonistic bacteria and fusarium oxysporum;

t5: inoculating 70% thiophanate methyl wettable powder and fusarium oxysporum; t6: inoculating sterile water and Fusarium oxysporum.

The antagonist bacteria inoculation method comprises the following steps: taking 10mL of trichoderma viride biocontrol bacterial liquid to inoculate the tobacco seedling root periphery, and uniformly mixing the tobacco seedling root periphery with soil;

the application method of the 70% thiophanate methyl wettable powder comprises the following steps: diluting the powder with 800 times of water, and irrigating 5mL of diluent to the root circumference;

the sterile water inoculation method comprises the following steps: directly irrigating root and inoculating to the periphery of root. 3d, firstly, carrying out root injury treatment on the tobacco seedlings by using a scalpel, cutting off tobacco roots (the depth is 3cm, the width is 2cm) at a position which is about 0.8cm away from the tobacco roots, and then inoculating pathogenic bacteria;

the rhizomucor inoculation method comprises the following steps: inoculating 5mL of conidium solution to the root periphery of the tobacco, wherein the concentration of the conidium solution is 5 multiplied by 10⁶cfu/mL；

The inoculation method of the fusarium oxysporum comprises the following steps: inoculating 5mL of conidium solution to the root periphery of the tobacco, inoculating 1mL of spore solution after carrying out 2mm root injury treatment on the stem base, wherein the concentration of the spore solution is 10⁷cfu/mL。

Each of the above treatments was performed in 4 replicates, each replicate of 4 tobacco shoots.

And (4) observing the pathogenic bacteria every day after inoculation, and investigating the disease occurrence condition at 21d according to a tobacco pest grading and investigation method GB/T23222-2008, and calculating the disease incidence, disease index and relative control effect.

The result shows that the control effect of NKSF2019001F on the tobacco root black rot is 74.36%, and the effect is superior to the medicament control; the control effect of NKSF2019001F on tobacco root rot is 65.38%, and the effect is equivalent to that of a medicament control (Table 4 and Table 5).

TABLE 4 potted plant disease prevention test results for treating root black rot differently

TABLE 5 potted plant disease prevention test results for different treatment of root rot

Example 6 potted plant growth promotion test

Transplanting 5-6 true leaf tobacco seedlings with consistent growth into a flowerpot filled with sterilized soil and a pindstrup matrix (pindstrup) in a volume ratio of 3:1, after 3d of seedling regrowth, inoculating NKSF2019001F inoculation liquid (prepared by inoculating trichoderma NKSF2019001F cultured for 5d and preparing 1.5 multiplied by 10 by using 0.05 percent of tween 80) prepared by a laboratory at rhizosphere⁸cfu/mL conidia), 10mL per strain, with an equal amount of sterile water as a control. Each treatment had 3 tobacco seedlings, which were replicated 3 times. After 30 days, carefully digging out the whole seedling, washing away soil at the root, measuring according to a tobacco agronomic character survey measuring method (industry standard YC/T142-2010), and recording the number of tobacco plant leaves, the maximum leaf length, the maximum leaf width, the plant height, the fresh weight, the dry weight, the root length, the fresh weight and the dry weight of the root.

The pot growth promotion test result shows that the plant height, the maximum leaf area, the effective leaf number, the fresh weight, the dry weight, the root length, the fresh weight and the dry weight of the root of the tobacco are respectively increased compared with the control after the treatment of the spore liquid of the strain NKSF 2019001F; except for root length and fresh weight of roots, other characters have significant difference (P <0.05) compared with the control, which shows that NKSF2019001F can promote tobacco growth to a certain extent (Table 6).

TABLE 6 growth promoting effect of biocontrol strain NKSF2019001F on tobacco

Note: the data in the table are mean ± sem, with different lower case letters representing less than 5%, a significant level of difference.

The foregoing is merely a preferred embodiment of this application and various modifications and changes will occur to those skilled in the art. All changes, equivalents and improvements which come within the spirit and scope of the application are desired to be protected by the following claims.

Claims

1. A trichoderma virens strain with disease prevention and growth promotion functions is characterized in that the trichoderma virens is trichoderma virens NKSF2019001F (A)Trichoderma virensNKSF2019001F), which has been deposited in the chinese culture collection, with the deposit address: wuhan university Collection, accession number: CCTCC NO: m2019767, deposit date: 20190929.

2. the method for preparing biocontrol bacterial liquid of Trichoderma viride of claim 1, wherein Trichoderma viride NKSF2019001F cultured for 5 days is added with Tween 80 of 0.05% (v/v) to obtain 2.0X 10⁷cfu/mL conidium solution is inoculated into the culture solution by 3 percent of inoculum size, and the culture conditions are as follows: the fermentation temperature is 28 ℃, the fermentation time is 9d, the rotating speed is 210r/min, the initial pH is 8, and the concentration of chlamydospore liquid in the biocontrol bacterial liquid obtained after fermentation is 7.6 multiplied by 10⁷ cfu/mL。

3. The method for preparing the biocontrol bacterial liquid of trichoderma virens according to claim 2, wherein the formula of the culture solution is as follows: corn flour 25g, glucose 15g, corn protein powder 15g, NaCl 1.3g, MgSO₄ 0.4 g、KH₂PO₄ 0.8 g、CaCO₃2g/L and 1L of distilled water.

4. Use of trichoderma virens according to claim 1 for the simultaneous control of black rot and root rot in tobacco.

5. Use of trichoderma virens according to claim 1 for promoting tobacco growth.

6. Use of trichoderma virens according to claim 1 in the preparation of a biocontrol agent for simultaneously controlling tobacco black rot, root rot and/or promoting tobacco.