CN112120043A - A kind of preparation method of compound biocontrol agent for preventing and treating tobacco black shank - Google Patents

Abstract

The invention relates to the technical field of tobacco agricultural microbial fermentation, in particular to a preparation method of a composite biocontrol microbial inoculum for preventing and treating tobacco black shank, wherein the strains of the composite biocontrol microbial inoculum comprise paenibacillus polymyxa-LB-9 and trichoderma viride-YCS-Tr 2; culturing a paenibacillus polymyxa-LB-9 strain by using an LB culture medium, and culturing a trichoderma brevifolium-YCS-Tr 2 strain by using a potato glucose agar culture medium, thereby respectively preparing a paenibacillus polymyxa-LB-9 fermentation broth and a trichoderma brevifolium-YCS-Tr 2 fermentation broth; mixing the prepared paenibacillus polymyxa-LB-9 fermentation liquor and the prepared trichoderma brevicompactum-YCS-Tr 2 fermentation liquor according to the volume ratio of 1:1 to 1:2, and concentrating the prepared mixed liquor by 10 times to prepare biocontrol bacteria fermentation liquor; preparing an assistant formula, wherein the raw materials of the assistant formula comprise naphthalenesulfonate, magnesium aluminum silicate, xanthan gum, glycerol and kasons, and mixing the prepared biocontrol bacterium fermentation liquor with the assistant formula to prepare the composite biocontrol bacterium agent.

Description

A kind of preparation method of compound biocontrol agent for preventing and treating tobacco black shank

technical field

The invention relates to the technical field of tobacco agricultural microorganism fermentation, in particular to a preparation method of a composite biocontrol agent for preventing and treating tobacco black shank.

Background technique

Tobacco black shank (tobacco black shank) is one of the most serious diseases in tobacco production in the world, especially in temperate, subtropical and tropical regions. Tobacco black shank caused by Phytophthora infestans is the main disease of tobacco in my country. One of the rhizome diseases, which first occurred in Huanghuai Tobacco Area, and currently occurs in various major tobacco-producing areas. The average annual economic loss caused by Tobacco Black Shank in my country is more than 100 million yuan; It will lead to the increase of pesticide residues in tobacco leaves, the increase of pathogen resistance, the reduction of drug efficacy, the destruction of farmland microecological balance and environmental pollution, thus posing a serious threat to the quality of tobacco leaves and the safety of human life. Moreover, biological pesticides are an important guarantee and fundamental way out for replacing chemical pesticides to solve the problem of environmental pollution and realize pollution-free and green agricultural production; At present, many Bacillus sp., Streptomyces sp., Pseudomonas sp., Trichoderma sp. with excellent biocontrol function have been isolated, and some of them have been made into commercial preparations. However, biopesticides have a low market share in my country's pesticide market, especially the research and development and industrialization of microbial pesticides are still very weak. In terms of biological control of tobacco diseases, there is no special biocontrol preparation.

At present, there are many studies on the screening and control effect of biocontrol bacteria on tobacco blackleg, but there are few studies on the optimization of the fermentation conditions of efficient biocontrol bacteria and the formulation of biocontrol bacteria and fungi. The invention takes the dominant physiological race No. 1 of Tobacco black shank in Henan Province as the target, screened out high-efficiency biocontrol Bacillus LB-9 and Trichoderma YCS-Tr2, and clarified the optimal fermentation medium, fermentation temperature and fermentation time. , the initial fermentation PH value, the establishment of the fermentation process. The best combination and compound ratio of biocontrol strains were screened out, and a new high-efficiency compound biocontrol preparation for the control of tobacco black shank was developed. The growth-promoting effect of compound biocontrol agents on tobacco seedlings and the control effect on tobacco black shank were studied through seedbed matrix mixing bacteria and indoor pot experiment system, which is of great significance to the biological control of tobacco black shank.

Therefore, to provide a method for preparing a composite biocontrol agent for preventing and treating tobacco blackleg, which is convenient for prevention and treatment, has good bacteriostatic effect, avoids chemical pesticide pollution, and can promote the growth of floating tobacco seedlings, which has broad market prospects.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides a preparation method of a composite biocontrol agent for preventing and treating tobacco blackleg, which is convenient for prevention and treatment, has good bacteriostatic effect, avoids chemical pesticide pollution, and can promote the growth of floating tobacco seedlings. For overcoming the deficiencies in the prior art.

The technical scheme adopted in the present invention is as follows: a preparation method of a composite biocontrol agent for preventing and treating tobacco black shank, wherein the strains of the compound biocontrol agent include Paenibacillus polymyxa-LB-9 and nearly greenish green Trichoderma-YCS-Tr2; its preparation method specifically includes the following steps: 1) using LB medium to cultivate Paenibacillus polymyxa-LB-9 strain, and using potato dextrose agar medium to cultivate Trichoderma eutropha-YCS-Tr2 Bacterial strains, thereby preparing Paenibacillus polymyxa-LB-9 fermentation broth and Trichoderma near chloroticus-YCS-Tr2 fermentation broth respectively; 2) Step 1) to prepare Paenibacillus polymyxa-LB-9 fermentation broth and The fermented liquid of Trichoderma chinensis-YCS-Tr2 is mixed according to the volume ratio of 1:1 to 1:2, and the obtained mixed liquid is concentrated by 10 times, so as to obtain a biocontrol fermented liquid; 3) Preparation of auxiliary formulations, the raw materials of auxiliary formulations include naphthalene sulfonate, magnesium aluminum silicate, xanthan gum, glycerol and carson, and step 2) preparing biocontrol bacteria fermentation broth and auxiliary formulations are mixed, Thereby the compound biocontrol agent is prepared.

The LB medium includes 10-15g of glucose, 10-15g of soybean meal and 0.2-0.3g/L of MgSO ₄ , the fermentation initial pH of the LB medium is 7.0, and the LB medium is inoculated with Paenibacillus polymyxa-LB-9 The inoculation amount of the strain is 1-2%, the culture temperature of Paenibacillus polymyxa-LB-9 strain is 25-30 ℃, and the shaking culture is carried out at 170-190r/min speed for 48-50h.

The potato dextrose agar medium comprises corn flour 25-30g, glucose 15-20g, corn gluten meal 15-20g, NaCl 1.3-1.5g, MgSO ₄ 0.4-0.5g/L, K ₂ HPO ₄ 0.8-1g And CaCO ₃ 2-2.2g/L, the initial pH of fermentation of potato dextrose agar medium is 6.0, and the inoculum amount of potato dextrose agar medium inoculated with Trichoderma near chlorotic-YCS-Tr2 is 6-8%, and nearly gradually green The culture temperature of Trichoderma-YCS-Tr2 was 25-30°C, the bottling volume of potato dextrose agar medium was 150mL/500mL, and the fermentation time was 7-8d.

The said Paenibacillus polymyxa-LB-9 fermentation broth and the Trichoderma near chloroticum-YCS-Tr2 fermentation broth are respectively poured into the liquid stirring equipment for stirring, and the mixed liquid after stirring and mixing passes through the feeder Connect to a concentrated evaporator for concentration treatment.

The proportion of each raw material of the composite antibacterial agent is 92-93% of biocontrol bacteria fermentation broth, 5-5.5% of naphthalene sulfonate, 1-1.3% of magnesium aluminum silicate, 0.2-0.3% of yellow Original Gum, 3-3.5% Glycerin, and 0.1-0.15% Casone.

The beneficial effects of the present invention are as follows: firstly, the strains of the composite biocontrol agent include Paenibacillus polymyxa-LB-9 and Trichoderma near chlorophyll-YCS-Tr2; the preparation method specifically includes the following steps: 1) using LB to cultivate The Paenibacillus polymyxa-LB-9 strain was cultured on the base, and the Trichoderma parachlorophyte-YCS-Tr2 strain was cultured using potato dextrose agar medium to prepare Paenibacillus polymyxa-LB-9 fermentation broth and A. Trichoderma aeruginosa-YCS-Tr2 fermentation broth; 2) step 1) to prepare Paenibacillus polymyxa-LB-9 fermentation broth and Trichoderma subtilis-YCS-Tr2 fermentation broth in a volume ratio of 1:1 to 1 The ratio of: 2 is mixed, and the mixed solution obtained is concentrated 10 times, so as to prepare the biocontrol bacteria fermentation liquid; 3) preparation of auxiliary agent formula, the raw materials of auxiliary agent formula include naphthalene sulfonate, magnesium silicate Aluminum, xanthan gum, glycerin and carson, and step 2) preparing the biocontrol bacteria fermentation broth and mixing the auxiliary formulations, so as to facilitate the preparation of composite biocontrol agents and avoid the pollution of chemical pesticides; secondly, the LB-9 fermentation broth and YCS-Tr2 fermentation broth were mixed at a ratio of 1:1 and 1:2 and diluted to 100 times, respectively, and 200 μL of the dilution was spread on the agar medium, and then the pathogenic bacteria cake (diameter 5mm) ) was placed in the middle of the medium, and the diameter of the pathogenic bacteria was investigated after 5 days, and the inhibition rate of the biocontrol bacteria mixture on the pathogenic bacteria was calculated [bacteriostatic rate (%) = (radius of control colony − radius of confrontation culture Phytophthora colonies)/control colony Radius × 100%], the strain combination and compounding ratio with the best inhibitory effect on Phytophthora nicotiana were screened out. The bacteriostatic rate of Phytophthora has reached more than 90%, that is, the present invention has a good bacteriostatic effect; thirdly, through the pot experiment in the artificial climate room, the control effect of the compound biocontrol agent on tobacco black shank can reach 70%. % or more, that is, the present invention has a good preventive effect, so that the present invention has good social and economic benefits, and is a product that is easy to popularize and use.

Detailed ways

A preparation method of a composite biocontrol fungicide for preventing and treating tobacco black shank, wherein the bacterial species of the composite biocontrol fungicide include Paenibacillus polymyxa-LB-9 and Trichoderma subtilis-YCS-Tr2; The preparation method specifically includes the following steps: 1) using LB medium to cultivate Paenibacillus polymyxa-LB-9 strain, and using potato dextrose agar medium to cultivate Trichoderma euphorbia-YCS-Tr2 strain, thereby preparing polymyxa respectively Paenibacillus polymyxa-LB-9 fermentation broth and Trichoderma parachlorophyll-YCS-Tr2 fermentation broth; 2) Step 1) was used to prepare Paenibacillus polymyxa-LB-9 fermentation broth and Trichoderma parachlorophyll-YCS- Tr2 fermentation broth is mixed according to the volume ratio of 1:1 to 1:2, and the prepared mixed liquor is concentrated 10 times to prepare biocontrol bacteria fermentation broth; 3) preparation of auxiliary agent formula, auxiliary agent The raw materials of the formula include naphthalene sulfonate, magnesium aluminum silicate, xanthan gum, glycerin and cassone, and the step 2) preparing the biocontrol bacterial fermentation broth and the auxiliary formulation are mixed to prepare a composite biocontrol bacterial agent .

The LB medium includes 10-15g of glucose, 10-15g of soybean meal and 0.2-0.3g/L of MgSO ₄ , the fermentation initial pH of the LB medium is 7.0, and the LB medium is inoculated with Paenibacillus polymyxa-LB-9 The inoculation amount of the strain is 1-2%, the culture temperature of the Paenibacillus polymyxa-LB-9 strain is 25-30°C, and the shaking culture is 48-50h at a rotational speed of 170-190r/min; the potato dextrose agar medium Including corn flour 25-30g, glucose 15-20g, corn gluten meal 15-20g, NaCl 1.3-1.5g, MgSO ₄ 0.4-0.5g/L, K ₂ HPO ₄ 0.8-1g and CaCO ₃ 2-2.2g/ L, the initial pH of fermentation of potato dextrose agar medium was 6.0, the inoculum amount of potato dextrose agar medium inoculated with Trichoderma albivara-YCS-Tr2 was 6-8%, and the cultivation of Trichoderma alba viridis-YCS-Tr2 The temperature is 25-30° C., the bottling volume of the potato dextrose agar medium is 150mL/500mL, and the fermentation time is 7-8d; The Trichoderma viridans-YCS-Tr2 fermentation liquid is poured into the liquid stirring equipment for stirring, and the mixed liquid after stirring and mixing is connected to the concentration evaporator through the feeder for concentration treatment; the proportion of each raw material of the composite antibacterial agent is: 92-93% biocontrol bacteria fermentation broth, 5-5.5% naphthalene sulfonate, 1-1.3% magnesium aluminum silicate, 0.2-0.3% xanthan gum, 3-3.5% glycerol and 0.1-0.15% % Casson.

Embodiment 1: use glucose 10g, soybean meal 10g and MgSO ₄ 0.2g/L to make LB medium, the corresponding inoculum of 1% Paenibacillus polymyxa-LB-9 strain is cultivated, and use corn flour 25g, glucose 15g , corn gluten meal 15g, NaCl 1.3g, MgSO ₄ 0.4g/L, K ₂ HPO ₄ 0.8g and CaCO ₃ 2g/L to make potato dextrose agar medium, correspondingly inoculated with 8% Trichoderma near chloroticus-YCS- The Tr2 strain was cultured to prepare Paenibacillus polymyxa-LB-9 fermentation broth and Trichoderma subchlorophylla-YCS-Tr2 fermentation broth respectively; LB-9 fermentation broth and YCS-Tr2 fermentation broth were in a ratio of 1:1 After mixing, it was diluted to 100 times, and 200 μL of the dilutions were spread on the agar medium, and then the pathogenic bacteria cake (diameter 5 mm) was placed in the middle of the medium. The inhibition rate [bacteriostatic rate (%) = (radius of control colony − radius of confrontation culture Phytophthora colony)/radius of control colony × 100%], and the strain combination with the best inhibitory effect on Phytophthora nicotianae was screened out. The results showed that the antibacterial rate reached 97.72%.

Embodiment 2: use glucose 13g, soybean meal 15g and MgSO ₄ 0.3g/L to make LB medium, the corresponding inoculum of 2% Paenibacillus polymyxa-LB-9 strain is cultivated, and use corn flour 30g, glucose 20g , corn gluten meal 20g, NaCl 1.5g, MgSO ₄ 0.5g/L, K ₂ HPO ₄ 1g and CaCO ₃ 2.2g/L to make potato dextrose agar medium, correspondingly inoculated with 8% Trichoderma near chloroticus-YCS- The Tr2 strain was cultured to prepare Paenibacillus polymyxa-LB-9 fermentation broth and Trichoderma subchlorophylla-YCS-Tr2 fermentation broth respectively; LB-9 fermentation broth and YCS-Tr2 fermentation broth were in a ratio of 1:1 After mixing, it was diluted to 100 times, and 200 μL of the dilutions were spread on the agar medium, and then the pathogenic bacteria cake (diameter 5 mm) was placed in the middle of the medium. The inhibition rate [bacteriostatic rate (%) = (radius of control colony − radius of confrontation culture Phytophthora colony)/radius of control colony × 100%], and the strain combination with the best inhibitory effect on Phytophthora nicotianae was screened out. The results showed that the antibacterial rate reached 97.23%.

Embodiment 3: use glucose 10g, soybean meal 10g and MgSO ₄ 0.2g/L to make LB medium, the corresponding inoculum of 1% Paenibacillus polymyxa-LB-9 strain is cultivated, and use corn flour 25g, glucose 15g , corn gluten meal 15g, NaCl 1.3g, MgSO ₄ 0.4g/L, K ₂ HPO ₄ 0.8g and CaCO ₃ 2g/L to make potato dextrose agar medium, correspondingly inoculated with 8% Trichoderma near chloroticus-YCS- The Tr2 strain was cultured to prepare Paenibacillus polymyxa-LB-9 fermentation broth and Trichoderma subchlorous-YCS-Tr2 fermentation broth respectively; the LB-9 fermentation broth and YCS-Tr2 fermentation broth were in a ratio of 1:2 After mixing, it was diluted to 100 times, and 200 μL of the dilutions were spread on the agar medium, and then the pathogenic bacteria cake (diameter 5 mm) was placed in the middle of the medium. The inhibition rate [bacteriostatic rate (%) = (radius of control colony − radius of confrontation culture Phytophthora colony)/radius of control colony × 100%], and the strain combination with the best inhibitory effect on Phytophthora nicotianae was screened out. The results showed that the antibacterial rate reached 94.95%.

Embodiment 4: use glucose 15g, soybean meal 13g and MgSO ₄ 0.3g/L to make LB medium, the corresponding inoculum of 2% Paenibacillus polymyxa-LB-9 strain is cultivated, and use corn flour 30g, glucose 20g , corn gluten meal 20g, NaCl 1.5g, MgSO ₄ 0.5g/L, K ₂ HPO ₄ 0.9g and CaCO ₃ 2.2g/L to make potato dextrose agar medium, correspondingly inoculated with 8% Trichoderma near chloroticus-YCS -Tr2 strain was cultivated to prepare Paenibacillus polymyxa-LB-9 fermentation broth and Trichoderma eutropha-YCS-Tr2 fermentation broth respectively; LB-9 fermentation broth and YCS-Tr2 fermentation broth were prepared according to 1:2 After mixing, dilute to 100-fold solution, take 200 μL of the dilutions and spread them on the agar medium, then place the pathogenic bacteria cake (5 mm in diameter) in the middle of the medium, investigate the diameter of the pathogenic bacteria after 5 days, and calculate the amount of the biocontrol bacteria mixture. The inhibition rate of pathogenic bacteria [bacteriostatic rate (%)=(radius of control colony−radius of confrontation culture Phytophthora nicotianae colony)/radius of control colony×100%], and the strain combination with the best inhibitory effect on Phytophthora nicotianae was screened out And the compound ratio, the results show that the antibacterial rate reached 95.33%.

In order to study whether the compound biocontrol agent can promote the growth of floating tobacco seedlings, the raw materials of the compound biocontrol agent are 92.5% biocontrol bacteria fermentation broth, 5% naphthalene sulfonate, 1% silicon Magnesium-aluminum acid, 0.2% xanthan gum, 3% glycerol and 0.1% cassone were mixed, and the method of floating seedlings was used in the greenhouse of the Tobacco Institute of Henan Academy of Agricultural Sciences. The floating tray for the test was a 200-hole seedling tray (length, width and height: 66cm×33cm×5.5cm), and the seedling substrate was specially used for tobacco floating seedlings. A total of 4 treatment schemes were set up, and the experiments were repeated 3 times for each treatment scheme. For a 200-hole seedling tray. Treatment scheme 1 [T1] uses 30ml of compound bio-proofing water agent and 250 mL of clean water, and mixes it with 750g of substrate before placing it on a plate; treatment scheme 2 [T2] uses 60 ml of compound bio-proofing water agent and 250 mL of clear water, and after mixing Mix well with 750g of matrix and put on a plate; for treatment plan 3 [T3], use 90ml of compound bio-proofing water agent to add 250mL of clean water, after mixing, mix well with 750g of matrix and put on a plate; for treatment plan 4 [T4 (CK)], use 250mL of clean water Mix well with 750g base and plate.

50 days after sowing, 20 tobacco seedlings with uniform growth were selected from the floating trays of each treatment plan, and their agronomic traits were determined with reference to the survey method of tobacco agronomic traits (YC/T142-2010), and the significance was performed using DPS7.05 software. Test, using Duncan's new multiple range method to compare significant differences, Table 1 is the growth rate and growth table of tobacco seedlings, and Table 2 is the agronomic traits of tobacco seedlings. As can be seen from Table 1, the emergence rate of T1-T2 treatment is slightly higher than that of the control treatment, the seedling stage is 2 days earlier than the control treatment, the growth is strong, and the seedling color is green; the emergence rate of T3 treatment is slightly lower than that of the control treatment, and the seedling stage 3 days later than the control, and the seedling color was yellow-green. This shows that the compound biocontrol agent of suitable concentration can promote the germination of tobacco seeds and the growth of tobacco seedlings; as can be seen from Table 2, the agronomic traits indicators (plant height, stem circumference, root fresh weight, The fresh weight of shoots) were higher than those of the control treatment, and the plant height, fresh weight of roots and fresh weight of shoots in T1 treatment were significantly different from those in T4 (CK) treatment. This shows that the compound biocontrol agent can significantly promote the growth of tobacco seedling roots and tobacco plants, and has a good growth-promoting effect.

In order to test the indoor potted control effect of compound biocontrol agents on tobacco black shank, a potted test was carried out in an artificial climate room. The tobacco variety was selected from China Tobacco 100. First, Phytophthora infestans was inoculated on the oat medium, and the temperature was 25-30 ℃. Cultivated in the dark environment for 7-14 days, when the mycelium grows vigorously, the edge of the colony is basically close to the periphery of the petri dish (φ9cm), and the asexual sporangia are formed in large numbers but have not fallen off, scrape the Phytophthora infestans from the plate. Put it into a sterilized 10 mL centrifuge tube, add 5 mL of sterile water, shake it on a shaker for 2 minutes, and then filter it with a 300-mesh sieve to remove the mycelium to obtain the supernatant, the sporangia suspension, and adjust the concentration by microscopy. It is 104 spores/mL, which is used for inoculation test; then the tobacco seedlings at the 4-5 leaf stage are transplanted into flower pots (diameter is 100mm × height 90mm), and the cultivation environment of tobacco seedlings is room temperature 25-28 ℃ , Humidity 50-60%, light for 12h a day and darkness for 12h, pour 10mL of compound biocontrol agent on each plant after 5-7d; then, inoculate the seedlings with Phytophthora nicotianae after 24h, using the root-wounding method, each strain Phytophthora spore suspension 10mL.

The incidence rate and incidence grade were investigated 20 days after inoculation. The grading standard was implemented according to the tobacco industry standard of the People's Republic of China (YC/T39-1996). , disease index [disease index = ∑ (number of diseased plants at all levels × representative value of disease levels at all levels) / (total number of plants under investigation × representative value of the highest level) × 100] and prevention effect [control effect (%) = (disease index in control area) - Disease index in the prevention and control area) / disease index in the war zone × 100], at the same time, statistics are made according to the chemical treatment data and water treatment data of the investigation, and the statistical data is made into statistical table 3; The control effect of bacterial agent treatment on tobacco black shank can reach more than 70%, although it is slightly lower than that of chemical agent treatment, but the biocontrol agent is of great significance to farmland microecological balance, environmental protection and the realization of pollution-free green agricultural production. The biological control of tobacco blackleg has important guiding value.

To sum up, the present invention provides a method for preparing a composite biocontrol agent for preventing and controlling tobacco blackleg, which has good control effect, good bacteriostatic effect, avoids chemical pesticide pollution, and can promote the growth of floating tobacco seedlings. Inventions have broad market prospects.

Claims (5)

1. a preparation method of a composite biocontrol fungicide for preventing and treating tobacco black shank, it is characterized in that: the bacterial classification of described composite biocontrol fungus comprises Paenibacillus polymyxa-LB-9 and Trichoderma near chloroticus -YCS-Tr2; Its preparation method specifically comprises the following steps: 1) using LB medium to cultivate Paenibacillus polymyxa-LB-9 strain, and using potato dextrose agar medium to cultivate Trichoderma euphorbia-YCS-Tr2 strain, so as to separately prepare polymyxa-LB-9 strains. Paenibacillus myxobacterium-LB-9 fermentation broth and Trichoderma subtilis-YCS-Tr2 fermentation broth; 2) mixing step 1) the preparation of Paenibacillus polymyxa-LB-9 fermentation broth and Trichoderma subtilis-YCS-Tr2 fermentation broth according to the volume ratio of 1:1 to 1:2, and mixing; The prepared mixed solution is concentrated 10 times to prepare biocontrol bacteria fermentation broth; 3) Preparation of auxiliary agent formula, the raw material of auxiliary agent formula includes naphthalene sulfonate, magnesium aluminum silicate, xanthan gum, glycerol and carson, and step 2) is prepared for biocontrol bacteria fermentation liquid and auxiliary agent formula to carry out Mixing to prepare composite biocontrol agent. 2. the preparation method of the composite biocontrol agent for preventing and treating tobacco black shank according to claim 1, is characterized in that: described LB medium comprises glucose 10-15g, soybean meal 10-15g and MgSO ₄ 0.2-0.3 g/L, the fermentation initial pH of LB medium is 7.0, the inoculum amount of LB medium inoculated with Paenibacillus polymyxa-LB-9 strain is 1-2%, and the culture of Paenibacillus polymyxa-LB-9 strain The temperature was 25-30°C, and the culture was shaken at 170-190r/min for 48-50h. 3. the preparation method of the composite biocontrol agent of preventing and treating tobacco black shank according to claim 1, is characterized in that: described potato dextrose agar medium comprises corn meal 25-30g, glucose 15-20g, corn gluten Powder 15-20g, NaCl 1.3-1.5g, MgSO ₄ 0.4-0.5g/L, K ₂ HPO ₄ 0.8-1g and CaCO ₃ 2-2.2g/L, the initial pH of fermentation of potato dextrose agar medium is 6.0 , the inoculum amount of potato dextrose agar medium inoculated with Trichoderma epichlorous-YCS-Tr2 is 6-8%, the culture temperature of Trichoderma epichloropsis-YCS-Tr2 is 25-30 ℃, and the inoculation of potato dextrose agar medium is 6-8%. The bottle volume is 150mL/500mL, and the fermentation time is 7-8d. 4. the preparation method of the composite biocontrol agent for preventing and treating tobacco black shank according to claim 1, it is characterized in that: the described Paenibacillus polymyxa-LB-9 fermentation broth and the fermented liquid that will be produced respectively are The Trichoderma aeruginosa-YCS-Tr2 fermentation liquid is poured into the liquid stirring equipment for stirring, and the mixed liquid after stirring and mixing is connected to the concentration evaporator through the receiver for concentration treatment. 5. the preparation method of the composite biocontrol agent for preventing and treating tobacco black shank according to claim 1, it is characterized in that: the ratio of each raw material of described compound antimicrobial agent is the biocontrol bacteria fermentation of 92-93% liquid, 5-5.5% naphthalene sulfonate, 1-1.3% magnesium aluminum silicate, 0.2-0.3% xanthan gum, 3-3.5% glycerin and 0.1-0.15% carson.