Tobacco biocontrol bacteria, biocontrol bacterial agent and propagation method thereof

Abstract

The application discloses a tobacco biocontrol bacterium, a biocontrol bacterium agent and a propagation method thereof, the bacterium is a strain of Sphingobium sp. PE05 with a preservation number of CCTCC No: M20221104; the strain of Sphingobium sp. PE05 is used for preventing and treating black shank and / or antagonizing Pythium aphanidermatum. The bacterium is separated from healthy tobacco leaves of a cigar tobacco variety of Yunnan Province Puer City Jiangcheng County Yunxue No. 2 and identified as Sphingobium sp., the bacterium is not used for preventing and treating black shank and related bacteria, and the bacterium is not used for antagonizing Pythium aphanidermatum; experiment verification shows that the inhibition rate of the bacterium to Pythium aphanidermatum mycelium growth is as high as 73.5%, the bacterium propagation product has a prevention effect on tobacco black shank of more than 80%, and the bacterium propagation product has the characteristics of low use cost, no residue and safety to human and livestock.

Description

Technical Field

[0001] This application relates to the fields of microbial pesticides and microbial fertilizers, and in particular to a tobacco biocontrol bacterium, a biocontrol agent, and a method for propagating it. Background Technology

[0002] Phytophthora indicum ( Phytophthora ramorum Black shank disease, caused by infection, is one of the most serious diseases in tobacco cultivation (Ma Guosheng et al., 2001). The average incidence rate of tobacco black shank is 10%–15%, reaching up to 75% in high-incidence fields. Severely affected tobacco fields can even result in total crop failure. The annual affected area of ​​tobacco black shank in my country is approximately 76,372 hectares. 2 The economic losses amounted to 130 million yuan (Wang H et al., 2012).

[0003] The existing biocontrol bacteria used for the biological control of black shank include: Penicillium (CN201510153971.4), Bacillus subtilis (CN201710060669.3), Paecilomyces lilacinus (CN200710300087.4), and Trichoderma longifolia (CN202110207287.5). The existing types of biocontrol bacteria are limited and cannot meet the needs of black shank control in different regions.

[0004] Currently, chemical control remains the primary method for controlling tobacco black shank, but biological control is highly valued due to its unique safety and sustainability, forming the basis for developing new biocontrol resources and biological agents. The use of various biocontrol microorganisms to control tobacco black shank has been widely reported (Huang et al., 2015), but "low efficacy and high cost" are global bottlenecks limiting the widespread application of microbial agents in tobacco cultivation.

[0005] References

[0006] Huang Y, et al. 2015. Isolation and characterization of rhizospherebacteria active against Meloidogyne incognita, Phytophthora ramorum and theroot knot-black shank complex in tobacco. Pest Management Science, 71:415-422.

[0007] Wang H, et al. 2012. A rapid microbioassay for discovery ofantagonistic bacteria for Phytophthora ramorum var. nicotianae.Phytopathology, 102:267-271.

[0008] Ma Guosheng, et al. Research progress on tobacco black shank. Tobacco Science and Technology. 2001, 9:44-48. Summary of the Invention

[0009] This application provides a strain of *Sphingomonas comatus* PE05 that can be used as a biocontrol bacterium for tobacco black shank disease and the *Phytophthora indicum* that causes black shank disease.

[0010] This application provides a tobacco biocontrol bacterium, namely the *Sphingomonas comatus* PE05 strain (accession number: CCTCC No: M20221104); the *Sphingomonas comatus* PE05 strain is used to control black shank disease and / or antagonize *Phytophthora indicum*.

[0011] The inventors isolated the *Sphingomyelinatoria coli* strain PE05 from healthy tobacco leaves of the Yunxue No. 2 cigar tobacco variety in Jiangcheng County, Pu'er City, Yunnan Province. Indoor bioassays showed that this strain has a strong antagonistic effect on the mycelial growth of *Phytophthora indicum*, with the inhibition rate of PE05 strain on mycelial growth reaching as high as 73.5%. The inoculum prepared using PE05 strain and the fermentation broth obtained by propagating PE05 strain using the propagation method of this patent both have good preventive effects against tobacco black shank disease.

[0012] Sphingomyelin-1, coccidioidomycin ( Sphingobacterium faecale This is a newly published bacterial species in 2022. For details, please refer to Chen M, Li N, Zhang XF, et al. 2022. Sphingobacterium faecale sp.nov., a 1-aminocyclopropane- 1-carboxylate deaminase producing bacteriumisolated from camel faeces. Int. J. Syst. Evol. Microbiol. 2022;72:005215.

[0013] Preferably, the tobacco strain infected with Phytophthora indicum is a cigar tobacco strain.

[0014] Preferably, the tobacco strain infected with Phytophthora indicum is either Cigar Smoking Yunxue 2 or Flue-cured Tobacco Yunyan 87. Specifically, this strain has a better biocontrol effect against Cigar Smoking Yunxue 2.

[0015] Another aspect of the present application also provides a biocontrol agent, comprising at least one of a) to c) below:

[0016] a) the strain of Sphingobium sp. PE05 as described above;

[0017] b) the intracellular substance of the strain of Sphingobium sp. PE05 as described above; or

[0018] c) the fermentation culture of the strain of Sphingobium sp. PE05 as described above. Any of the above has the effect of preventing and controlling the black shank and / or antagonizing Phytophthora parasitica.

[0019] Preferably, the intracellular substance of the strain of Sphingobium sp. PE05 is at least one of the supernatant or the precipitate of ultrasonic lysis. The intracellular substance is obtained by conventional ultrasonic lysis of cells, and has the effect of preventing and controlling the black shank and / or antagonizing Phytophthora parasitica.

[0020] Preferably, the method for preparing the fermentation culture comprises the following steps:

[0021] After the slant culture, the seed liquid culture and the fermentation liquid culture, a fermentation liquid containing the bacterial cells and metabolites of the strain is obtained.

[0022] Specifically, the test tube slant seed culture: the strain of PE05 is inoculated into a conventional LB medium slant, and a slant seed is obtained after 2 days of culture at 35°C.

[0023] The strain liquid seed culture: the obtained slant seed is inoculated into a flask containing LB liquid medium, and a liquid seed is obtained after 3 days of culture at 35°C and 120 rpm.

[0024] The strain fermentation tank mass culture: the liquid seed is inoculated into LB culture liquid at a ratio of 3% to 5% (V / V), and the fermentation is carried out in a 500 to 1000 liter fermentation tank. The culture conditions are controlled as follows: temperature 32°C to 36°C, stirring speed 120 rpm to 160 rpm, and fermentation time 48h to 72h.

[0025] Preferably, the method for preparing the biocontrol agent comprises the following steps: mixing the fermentation culture with a binder to obtain the biocontrol agent.

[0026] Specifically, the binder used is soluble corn starch. The water content of the obtained biocontrol agent is less than 5%, and the effective viable cell number of the strain of PE05 in the agent is more than 10 billion CFU / gram.

[0027] Preferably, the method for using the biocontrol agent is as follows: the biocontrol agent is diluted with water at a ratio of 2kg / acre to 3kg / acre, and then applied to the roots of transplanted tobacco plants.

[0028] Preferably, the biocontrol agent is applied after the liquid expansion in the field to obtain the expansion liquid.

[0029] Preferably, the liquid expansion method in the field comprises the following steps: after preparing the liquid medium and adding the fungicide, sealing and standing, inoculating the S. scabriuscula PE05 strain, and expanding for 7-10 days, with regular stirring during the expansion, to obtain the expansion liquid with a viable bacterial count of 1x10 8

[0030] The fungicide is trichloroisocyanuric acid.

[0031] Preferably, the amount of fungicide added is 0.2%-1% of the volume of the liquid medium; the liquid medium is prepared in a plastic bucket or a reservoir as the container and expanded; and the amount of PE05 agent used is inoculated at a volume-to-mass ratio of 1500-1800:1 of the culture liquid to the mass of the PE05 agent.

[0032] After using the bioagent by the field expansion method, the control effect on black shank disease is significantly improved, which can reach more than 80%, and the control effect is higher than that of chemical pesticides.

[0033] The medium used can be the expansion medium for S. scabriuscula, and preferably, the liquid medium is diammonium hydrogen phosphate 1 g / L, corn flour 20 g / L, sodium chloride 10 g / L, potassium dihydrogen phosphate 0.6 g / L, manganese ions 0.25 g / L, yeast powder 5 g / L, and peptone 5 g / L, with a pH of 7.2.

[0034] Specifically, the expansion operation comprises the following steps: taking the reservoir and the plastic bucket as the fermentation containers respectively, mixing the medium raw materials according to the formula with tap water at a mass ratio of 1:500, and stirring until the medium components are dissolved.

[0035] The beneficial effects that can be produced by the present application include:

[0036] 1) The tobacco biocontrol agent provided by the present application is first isolated from healthy tobacco leaves of the Cigar Tobacco Yunnan Snow No. 2 variety in Jiangcheng County, Pu'er City, Yunnan Province. The strain is identified as S. scabriuscula, which has not been used for the prevention and antagonism of black shank disease and related strains. Experimental verification shows that the inhibition rate of the tobacco strain on the mycelial growth of P. nicotianae is as high as 73.5%, and the control effect of the bacterial product on tobacco black shank disease is more than 80%, with the characteristics of low use cost, no residue, and safety to humans and animals.

[0037] ​2) The method for propagating tobacco biocontrol bacteria provided in this application utilizes the inoculum prepared by strain PE05 as a seed culture. The culture medium is then placed in readily available field fermentation containers such as water tanks or plastic buckets, using trichloroisocyanuric acid as a sterilizing agent. This method kills over 99% of bacteria and fungi in the culture medium, and the residual trichloroisocyanuric acid does not adversely affect the normal growth of strain PE05. Furthermore, strain PE05 can propagate normally in this culture medium, achieving a biomass of 4.6 × 10⁻⁶ after propagation. 8 CFU / g can effectively increase the number of live bacteria in the biocontrol agent, enabling the bacteria to multiply rapidly and effectively increase the content of live bacteria per gram in the soil after application.

[0038] The *Sphingomyelinus* in this application ( Sphingobacterium faecale The PE05 strain was deposited at the China Center for Type Culture Collection on July 13, 2022; Address of depositary institution: Wuhan University, Bayi Road, Hongshan District, Wuhan, Hubei Province; Accession number: CCTCC No: M20221104. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Technical means not detailed in this application and not used to solve the technical problems of this application are all set according to common general knowledge in the field, and multiple common general knowledge setting methods can be implemented.

[0041] Example

[0042] Unless otherwise specified, all materials and equipment used in the following embodiments were obtained through commercial channels.

[0043] Example 1: Obtaining strain PE05

[0044] The inventors obtained the PE05 strain of *Sphingomyelinatoria coli* from healthy tobacco leaves of the Yunxue No. 2 cigar variety in Jiangcheng County, Pu'er City, Yunnan Province, using existing endophytic bacteria isolation methods.

[0045] The fermentation broth of strain PE05 was obtained by culturing in existing LB liquid medium at 37°C and 120 rpm for 48 h.

[0046] From the fresh culture of tobacco Phytophthora plate with puncher (diameter 5 mm) to take 1 mycelium block inoculation in PDA plate, 28℃ under culture 5 d after mycelium full plate, in this PDA plate distance inoculation block 2 cm position punch, in the punch respectively join 100 μl PE05 strain fermentation liquor, with equal amount of LB medium instead of bacteria liquid as control group.

[0047] After the above obtained control group and experimental group PDA plate is cultured at 28℃ for 5d, the diameter of tobacco Phytophthora colony around each hole is measured, and the inhibition rate of each hole is calculated according to the following formula:

[0048] Inhibition rate (%) = (tobacco Phytophthora colony diameter of control-tobacco Phytophthora colony diameter of treatment) / tobacco Phytophthora colony diameter of control x 100%.

[0049] The arithmetic mean of the inhibition rate of each hole of the experimental group added with PE05 strain fermentation liquor is calculated. The average inhibition rate obtained is 73.5%. The inhibition rate of each hole in the control group is 0. It shows that PE05 strain has significant inhibition and antagonistic effect on the growth of tobacco Phytophthora.

[0050] Example 2 Culture of PE05 strain and preparation of bacterial agent

[0051] Test tube slant seed culture: inoculate PE05 strain to the conventional LB medium slant, cultivate at 35℃ for 2d to obtain slant seed.

[0052] Strain liquid seed culture: inoculate the obtained slant seed to the triangular flask containing LB liquid medium, cultivate at 35℃, 120 rpm in the shaking bed for 3 days to obtain liquid seed.

[0053] Strain fermenter mass culture: inoculate the liquid seed into LB culture solution at a ratio of 3% (V / V), and use 1000L fermenter for fermentation, and control the culture conditions at temperature 32℃, stirring speed 120 rpm, and fermentation time 48h.

[0054] Preparation of bacterial agent of PE05 strain: mix the bacterial body and metabolites obtained by fermenter culture with appropriate amount of soluble corn starch, and then spray dry to obtain bacterial body powder. The water content of the bacterial agent is less than 5%, and the effective viable count of PE05 strain in the bacterial agent is more than 10 billion CFU / g.

[0055] Example 3 Field propagation of PE05 bacterial agent

[0056] Propagation medium formula: diammonium phosphate 1 g / L, corn flour 20 g / L, sodium chloride 10 g / L, potassium dihydrogen phosphate 0.6 g / L, manganese ion 0.25 g / L, yeast powder 5 g / L, peptone 5 g / L, pH 7.2.

[0057] Sterilization method of culture medium: take the pool and plastic bucket as the fermentation container respectively, mix the culture medium raw materials with tap water according to the mass ratio of 1:500 after being weighed according to the formula, and stir until each component of the culture medium is dissolved.

[0058] Add the sterilizing agent trichloroisocyanuric acid (the effective ingredient content of the used trichloroisocyanuric acid reagent is ≥50%) to the obtained liquid, and after the addition is completed, the final concentration of trichloroisocyanuric acid in the obtained culture medium solution is 0.2%, and after stirring for 10 min, cover the container with a plastic film and seal it, and stand still for more than 72 h under the environment.

[0059] PE05 agent propagation method: inoculate the PE05 agent prepared in the present application into the sterilized culture medium according to the weight ratio of 1500:1, stir uniformly at room temperature (20-30°C), and stand still indoors for 7 d, and stir once every 24 h during the period. The biomass of PE05 strain in the fermentation broth obtained by the propagation method can reach more than 1×10 8 individuals / gram.

[0060] Here, the biomass of PE05 strain in the propagation system = the biomass of bacteria in the culture solution obtained after inoculating PE05 agent for propagation - the biomass of bacteria in the culture solution obtained after inoculating PE05 agent for propagation.

[0061] Example 4: Preparation effect of PE05 agent

[0062] The bacteria were extracted from the slant seed of PE05 strain, inoculated on LB plate, and cultured at 35°C for 2 d to activate the strain; a single colony was picked from the plate and inoculated into a triangular flask containing LB liquid medium, and cultured at 35°C and 120 rpm for 3 d to obtain liquid seed; the liquid seed was inoculated into a 500 L fermenter containing 300 L LB medium at a proportion of 3% (V / V), and cultured at a temperature of 35°C, a stirring speed of 140 rpm, and a fermentation time of 72 h; the obtained bacteria and metabolites were mixed with an appropriate amount of soluble corn starch, and then spray-dried to obtain the agent, the water content of which was controlled to be less than 5%. 1 gram of the agent was diluted in 9 mL of sterile water, mixed uniformly, and then coated on the LB medium plate by the conventional dilution coating method, and cultured at 35°C for 2 d to count the number of colonies, and the biomass of PE05 strain per gram of agent was counted. The results showed that the effective viable count of PE05 strain in the prepared agent was 1.15 billion CFU / g.

[0063] Example 6: Sterilization experiment of trichloroisocyanuric acid on bacteria and fungi in culture medium

[0064] Take 1 kg of propagation medium dissolved in 500 kg of tap water, stir for 5 min to fully dissolve the medium, then add 1 kg of trichloroisocyanuric acid (effective ingredient content ≥ 50%), stir for 10 min, then cover with plastic film and seal the container. The resulting medium solution is the treatment group;

[0065] The medium solution without trichloroisocyanuric acid is used as the control.

[0066] After standing for 72 h, take the solutions of the treatment group and the control group, respectively. The solutions of the treatment group and the control group are respectively coated on LB plates for bacterial culture and PDA plates for fungal culture according to the conventional dilution coating method, with 10 repeats for each treatment.

[0067] The LB plates are cultured at 35°C for 2 d; the PDA plates are cultured at 28°C for 5 d. The number of bacterial and fungal colonies on each plate is counted, respectively. The sterilization rate of each repeat is calculated according to the following formula, and the arithmetic mean of the sterilization rate of the treatment group and the control group is calculated, respectively:

[0068] Sterilization rate (%) = number of colonies in the treatment group / number of colonies in the control group x 100%.

[0069] The results show that the bacterial and fungal sterilization rates of the culture solution in the treatment group are 98.4% and 99.6%, respectively. The sterilization rate in the control group is 0. This method can effectively kill bacteria and prevent other bacteria from contaminating the medium and hindering the propagation of PE05. This sterilization method is simple to operate and is especially suitable for killing bacteria and fungi in various containers in environments with high bacterial content.

[0070] Example 7: Field propagation biomass detection experiment of PE05 inoculant

[0071] In 500 L of the culture solution sterilized by the method in Example 6, inoculate 333.33 g of PE05 inoculant as the treatment group; use the sterilized culture solution obtained in Example 6 without PE05 inoculant as the control.

[0072] Both the treatment group and the control group are operated according to the following steps: after the culture solution is stirred evenly, it is left to stand in the room for 7 d, during which it is stirred every 24 h; on the 7th day, samples of the treatment group and the control group are taken, respectively, and are coated on LB plates for bacterial culture according to the conventional dilution coating method, which are cultured at 35°C for 2 d. The number of bacterial colonies on the plates of the treatment group and the control group is counted. The bacterial biomass in the treatment group and the control group is calculated, respectively.

[0073] The biomass of PE05 strain in the propagation system = bacterial biomass in the treatment group - bacterial biomass in the control group.

[0074] The results show that the biomass of PE05 strain in the propagation medium obtained by this propagation method is 4.6 x 108 CFU / g. It is illustrated that the propagation method can effectively realize the rapid propagation of PE05 inoculant in the complex environment of microbial system in the field, improve the activity of the strain, activate the strain, increase the number of active bacteria in the soil, and effectively play the prevention and control effect of the inoculant on the black shank disease in the soil.

[0075] Example 8: Greenhouse test of PE05 inoculant and PE05 inoculant propagation product on tobacco black shank disease

[0076] 1. Test agent

[0077] I: PE05 propagation product, the viable bacterial content is 460 million CFU / g, which is prepared according to the method in Example 7;

[0078] II: PE05 inoculant, the viable bacterial content is 1150 million CFU / g, which is prepared according to the method in Example 4;

[0079] III: 58% metalaxyl-m zinc wettable powder, the dosage is 500 g / acre, produced by Xi'an Dingsheng Biological Chemical Co., Ltd.

[0080] 2. Flue-cured tobacco variety

[0081] Yunyan 87.

[0082] 2. Test treatment setting

[0083] Treatment I: PE05 propagation product, root irrigation, 110 kg / acre, equivalent to 100 g / plant (1100 plants per acre), interval 10 days, root irrigation again, the total amount of 2 times is 220 kg / acre;

[0084] Treatment II: PE05 inoculant, 2 kg / acre, equivalent to 1.66 g / plant (1100 plants per acre), diluted with water and then root irrigation;

[0085] Treatment III: 58% metalaxyl-m zinc, 0.5 kg / acre, equivalent to 0.45 g / plant (1100 plants per acre), diluted with water according to the dilution multiple on the product instruction and then root irrigation.

[0086] Treatment IV: equal amount of water as the control group.

[0087] Each treatment is repeated 6 times, randomly arranged, inoculated with 5000 Phytophthora parasitica oospores per plant on the 3rd day after application according to the conventional method, and the fertilizer and water management is carried out according to the conventional method. The tobacco plants are pulled out at 60 d, and the disease classification, disease index calculation and control effect are carried out according to the method introduced in GB / T 23222-2008 "Tobacco Disease Grading and Investigation Method".

[0088] The test results are shown in Table 1.

[0089] Table 1: Effect of PE05 inoculant and its propagation on tobacco black shank and cost analysis

[0090]

[0091] Note: the same letter in the same column indicates no significant difference; otherwise, the difference is significant. The cost accounting is based on:

[0092] 58% metalaxyl-m wettable powder 80 yuan / kg, 0.5 kg / acre, a total of 40 yuan / acre;

[0093] PE05 inoculant 30 yuan / kg, 2 kg / acre, a total of 60 yuan / acre;

[0094] 250 kg of PE05 inoculant propagation cost: PE05 inoculant 0.35 kg, 10.5 yuan; propagation medium 1.6 kg, 10 yuan / kg, a total of 16 yuan; 1 kg of trichloroisocyanuric acid, 12 yuan / kg, a total of 12 yuan, container and labor cost about 3.6 yuan / acre, a total of about 42.1 yuan / acre.

[0095] As can be seen from Table 1, treatment I uses PE05 inoculant propagation product with 4.6 billion CFU / g of effective viable bacteria, 100 ml / plant, a total of 2 times, a total of 220 kg / acre, the control effect on tobacco black shank is as high as 85.6%, which is significantly higher than the control effect (82.4%) of 58% metalaxyl-m wettable powder as a chemical agent in treatment III.

[0096] Treatment II uses PE05 inoculant with 11.5 billion CFU / g of effective viable bacteria, and the control effect on tobacco black shank is only 66.4% at a dosage of 2 kg / acre.

[0097] From the cost analysis, the cost of treatment I, II and III is 42.1 yuan, 60 yuan and 40 yuan per mu, respectively. The cost of treatment I and II is higher than that of treatment III, but both of them are biological inoculants, and the ecological benefits and tobacco quality are higher than that of treatment III. Comparing the control effect and cost of treatment I and II, it shows that the inoculant can significantly improve the control effect of the inoculant after propagation according to the present application, and can reduce the use cost.

[0098] Example 9

[0099] The difference from Example 2 is that the strain fermentation tank mass culture: the liquid seed is inoculated into LB culture solution at a proportion of 5% (V / V), and the fermentation is carried out in a 500L fermentation tank, and the culture conditions are controlled at: temperature 36℃, stirring speed 160 rpm, and fermentation time 72h.

[0100] The difference from Example 2 is that the strain fermentation tank mass culture: the liquid seed is inoculated into the LB culture solution at a ratio of 5% (V / V), the fermentation is carried out using a 500L fermentation tank, and the culture conditions are controlled at: temperature 36℃, stirring speed 160 rpm, and fermentation time 72h.

[0101] Example 10

[0102] The difference from Example 3 is that the fungicide addition amount is 0.1% of the volume of the liquid culture medium; the liquid culture medium is prepared and expanded in a plastic bucket or a water reservoir as a container; and the use amount of the PE05 microbial agent is inoculated at a volume to mass ratio of 1500:1 of the culture solution to the mass of the PE05 microbial agent.

[0103] The propagation method of the PE05 microbial agent: after stirring evenly, it is left to stand in the room for 10d.

[0104] Example 11

[0105] The difference from Example 3 is that the fungicide addition amount is 0.2% of the volume of the liquid culture medium; the liquid culture medium is prepared and expanded in a plastic bucket or a water reservoir as a container; and the use amount of the PE05 microbial agent is inoculated at a volume to mass ratio of 1800:1 of the culture solution to the mass of the PE05 microbial agent.

[0106] Example 12

[0107] The difference from Example 4 is that the liquid seed is inoculated into a 500L fermentation tank containing 300L LB culture medium at a ratio of 3% (V / V), at a temperature of 32℃, a stirring speed of 120 rpm, and a fermentation time of 72h.

[0108] Example 13

[0109] The difference from Example 4 is that the liquid seed is inoculated into a 500L fermentation tank containing 300L LB culture medium at a ratio of 3% (V / V), at a temperature of 36℃, a stirring speed of 160 rpm, and a fermentation time of 72h.

[0110] Example 14

[0111] The difference from Example 8 is that the use method of the biocontrol microbial agent in the corresponding treatment II: the biocontrol microbial agent is diluted with water at a rate of 2kg / acre and applied by root irrigation.

[0112] Example 15

[0113] The difference from Example 8 is that the use method of the biocontrol microbial agent propagation in the corresponding treatment I: the propagation culture is applied by root irrigation at a rate of 50kg / acre, and after 10d, it is applied again twice.

[0114] Although the present application has been described in detail with reference to the foregoing embodiments, the technical solutions recorded in the foregoing embodiments can be modified, or some of the technical features can be replaced by equivalent features, by those skilled in the art, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A biocontrol agent for controlling black shank and / or antagonizing Phytophthora nicotianae, characterized in that, for a or b) : a) Sphingobium faecale PE05 strain; or b) a fermentation culture of Sphingobium faecale PE05 strain; The preservation number of the Sphingobium terrigena PE05 is CCTCC No: M20221104; the classification name is Sphingobacterium faecale .

2. The biocontrol agent of claim 1, characterized in that, The preparation method of the fermentation culture comprises the following steps: After the slant culture, the seed liquid culture and the fermentation liquid culture, a fermentation liquid containing the bacterial strain and metabolites is obtained.

3. The biocontrol agent of claim 1, wherein, The preparation method of the biocontrol agent comprises the following steps: granulating the fermentation culture and the binder to obtain the biocontrol agent.

4. The biocontrol agent of claim 1, wherein, The use method of the biocontrol agent: the biocontrol agent is diluted with water at 2 kg / acre~3 kg / acre and then applied to the root of the transplanted tobacco plant.

5. The biocontrol agent of claim 1, wherein, The biocontrol agent is used after the liquid expansion in the field to obtain the expanded liquid.

6. The biocontrol agent of claim 5, wherein, The field liquid propagation method comprises the following steps: preparing a liquid culture medium, adding a fungicide, sealing and standing, inoculating the saprophytic sphingobium PE05 strain, propagating for 7-10 days, regularly stirring during the propagation, and obtaining a propagation liquid with a viable bacterial count of 1×10 8 9 / g or more. The fungicide is trichloroisocyanuric acid.

7. The biocontrol agent of claim 6, wherein, The addition amount of the fungicide is 0.1%~0.2% of the volume of the liquid culture medium; the liquid culture medium is prepared in a plastic bucket or a reservoir as a container and is expanded; the use amount of the PE05 agent is 1500~1800:1 of the volume of the culture liquid to the mass of the PE05 agent.

8. The biocontrol agent of claim 6, wherein, The liquid culture medium is diammonium hydrogen phosphate 1 g / L, corn flour 20 g / L, sodium chloride 10 g / L, potassium dihydrogen phosphate 0.6 g / L, manganese ion 0.25 g / L, yeast powder 5 g / L, and peptone 5 g / L, and the pH value is 7.2.

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