Bio-control inoculant for preventing and treating tobacco vein banding mosaic virus and application of bio-control inoculant

Abstract

The invention discloses a biocontrol microbial inoculum for preventing and treating tobacco vein banding mosaic virus and application thereof, and relates to the technical field of tobacco plant disease prevention and treatment, and the biocontrol microbial inoculum is prepared from bacillus siamensis fermentation liquor or / and penicillium reevesii fermentation liquor. Preferably, the composition is prepared by mixing bacillus siamensis fermentation liquor, penicillium reevesii fermentation liquor, calcium lignosulphonate and methylcellulose, has better prevention and treatment effects on tobacco vein banding mosaic virus than pesticides adopting chemical reagents such as moroxydine hydrochloride, and has the characteristics of low toxicity and high efficiency; the strain can be applied to preparation of a biocontrol inoculant for preventing and treating the tobacco vein banding mosaic virus and a product for preventing and treating the tobacco vein banding mosaic virus.

Description

Technical Field

[0001] The invention relates to the technical field of tobacco plant disease prevention and control, and in particular to a biocontrol agent for preventing and controlling tobacco vein mosaic virus and application thereof. Background Art

[0002] The pathogen of tobacco vein banding mosaic virus disease is tobacco vein banding mosaic virus (TVBMV), which belongs to the genus Potyvirus. The disease mostly occurs in the middle and late stages of tobacco fields. After the onset of the disease, the diseased leaves first show clear veins, then vein bands, and sometimes necrotic scars. The typical symptoms are narrow, dark green ribbon-like mosaic symptoms along the veins (especially the fine veins). The dark green bands on the leaves in the later stage are often net-like. Tobacco leaves infected with tobacco vein banding mosaic virus show narrow, dark green ribbon-like mosaic symptoms along the veins. The leaves of diseased plants in the field show vein bands and necrotic scars, which can be divided into 4 types according to the size and shape of the scars. The type, distribution and severity of necrotic scars on leaves are affected by environmental factors and the development period of tobacco plants in the field. Vein mosaic virus usually overwinters on potato tubers, year-round Solanaceae crops (tomatoes, peppers, etc.) and perennial weeds in the field. These are the main sources of initial infection of tobacco vein mosaic virus disease, and the infected tobacco plants in the field are the source of re-infection in the field. The virus is mainly transmitted by winged aphids, and cotton aphids and peach aphids have been reported. The virus can also be transmitted through contact and friction during agricultural activities.

[0003] At present, most of the registered pesticides in my country are for the prevention and treatment of mosaic disease caused by tobacco mosaic virus, such as guanidine hydrochloride. However, the prevention and treatment effect on tobacco vein mosaic virus is poor, which is far from solving the actual production problems.

[0004] Therefore, there is an urgent need to develop low-toxic and high-efficiency pesticides for this disease in production to improve the above problems. Summary of the invention

[0005] The object of the present invention is to provide a biocontrol agent for preventing and controlling tobacco vein mosaic virus and its application, so as to solve the problem that the pesticide in the prior art has poor control effect on tobacco vein mosaic virus.

[0006] The present invention is achieved through the following technical solutions: A biocontrol agent for preventing and treating tobacco vein mosaic virus is prepared by using siam bacillus fermentation liquid and / or penicillium rettgeri fermentation liquid.

[0007] Furthermore, the number of viable bacteria in the fermentation broth of Bacillus siamese is not less than 1×10 8 cfu / mL, the number of viable bacteria in the fermentation broth of Penicillium rettii is not less than 5×10 7 cfu / mL.

[0008] Furthermore, the siam bacillus fermentation broth is obtained by the following method steps: a1. The Siamese Bacillus was inoculated into LB solid medium and activated at 28°C to obtain the activated strain after 24h; b1. The strain obtained in step a1 was inoculated into LB liquid medium and cultured under shaking conditions of 30 ° C and 120 r / min to obtain a seed solution; c1. The seed solution obtained in step b1 was inoculated into LB liquid medium at an inoculum volume of 5 mL / 100 mL and cultured under shaking conditions of 30°C and 120 r / min to obtain a fermentation broth of Bacillus siamese.

[0009] Furthermore, the fermentation broth of Penicillium reiterii is obtained by the following method steps: a2. The Penicillium rei was inoculated into LB solid medium and activated at 32 ° C. After 36 hours, the activated strain was obtained; b2. The strain obtained in step a2 was inoculated into LB liquid medium and cultured under shaking conditions of 37 ° C and 200 r / min to obtain a seed solution; c2. The seed solution obtained in step b2 was inoculated into LB liquid culture medium at an inoculum volume of 5 mL / 100 mL and cultured under shaking conditions of 37°C and 200 r / min to obtain a fermentation broth of Penicillium rettii.

[0010] Furthermore, when the siam bacillus fermentation liquid and the penicillium rettgeri fermentation liquid are used for compounding, the siam bacillus fermentation liquid and the penicillium rettgeri fermentation liquid are uniformly mixed in a mass ratio of 1:1.

[0011] Furthermore, it also includes calcium lignin sulfonate and methyl cellulose, and the siam Bacillus fermentation broth, Penicillium rettii fermentation broth, calcium lignin sulfonate and methyl cellulose are compounded in a mass ratio of 3-4:3-4:3-5:2-4.

[0012] Furthermore, the siam Bacillus fermentation broth, the Penicillium rettgeri fermentation broth, calcium lignin sulfonate and methyl cellulose are compounded in a mass ratio of 3.5:3.5:4:3.

[0013] Furthermore, the Siamese Bacillus fermentation broth is deposited as CGMCC No.31958 and is classified as Siamese Bacillus Siamese Bacillus , obtained by activating Bacillus siamese with a strain number of snpv-z6; the fermentation broth of Penicillium rettii is deposited with a number of CGMCC No.41503 and is classified as Penicillium rettii Penicillium kidnapped , obtained by activating Penicillium reiterii with strain number SNPV-L10.

[0014] The use of the biocontrol agent for preventing and controlling tobacco vein mosaic virus as described in any of the above items in the preparation of the biocontrol agent for preventing and controlling tobacco vein mosaic virus and the product for preventing and controlling tobacco vein mosaic virus.

[0015] Furthermore, the application is performed at the early stage of tobacco vein mosaic virus onset and / or before the disease occurs, with each spraying amount being 80-120 mL / mu.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention proposes a biocontrol agent for preventing and controlling tobacco vein mosaic virus, which has a significant inhibitory effect on tobacco vein mosaic virus. According to experiments, the biocontrol agent has a passivation effect of 70.53% on tobacco vein mosaic virus, which is better than registered chemical pesticides such as morpholino hydrochloride. Preferably, when a compound reagent of Bacillus siamese fermentation broth, Penicillium rettii fermentation broth, calcium lignin sulfonate and methylcellulose is used, the passivation effect of the biocontrol agent on tobacco vein mosaic virus can be as high as 80.56%.

[0017] 2. In the present invention, a biocontrol agent for preventing and controlling tobacco vein mosaic virus is a biological agent, which can also achieve an effect of preventing and controlling tobacco vein mosaic virus that is equivalent to or even better than that of chemical agents. It has the potential to be a new type of biological pesticide for preventing and controlling tobacco vein mosaic virus disease in tobacco cultivation. It can replace or partially replace chemical agents, reduce the impact of chemical agents on the soil and other ecological environments, and is worthy of further research. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0019] In the following examples, the fermentation broth of Bacillus siamensis was deposited under the number CGMCC No. 31958 and was classified as Bacillus siamensis Siamese Bacillus , obtained by activation of Bacillus siamese with strain number SNPV-Z6.

[0020] The steps are: a1. The Siamese Bacillus was inoculated into LB solid medium and activated at 28°C to obtain the activated strain after 24h; b1. The strain obtained in step a1 was inoculated into LB liquid medium and cultured under shaking conditions of 30 ° C and 120 r / min to obtain a seed solution; c1. The seed solution obtained in step b1 was inoculated into LB liquid medium at an inoculum volume of 5 mL / 100 mL and cultured under shaking conditions of 30 ° C and 120 r / min to obtain a Siamese Bacillus fermentation broth with a viable count of not less than 1 × 10 8 cfu / mL.

[0021] The fermentation broth of Penicillium reiterii is deposited with the number CGMCC No.41503 and is classified as Penicillium reiterii I stole Penicillium , obtained by activating Penicillium reiterii with strain number SNPV-L10.

[0022] The steps are: a2. The Penicillium rei was inoculated into LB solid medium and activated at 32 ° C. After 36 hours, the activated strain was obtained; b2. The strain obtained in step a2 was inoculated into LB liquid medium and cultured under shaking conditions of 37 ° C and 200 r / min to obtain a seed solution; c2. The seed solution obtained in step b2 was inoculated into LB liquid medium at an inoculum volume of 5 mL / 100 mL and cultured at 37 ° C and 200 r / min to obtain a fermentation broth of Penicillium rettii, wherein the number of viable cells in the fermentation broth of Penicillium rettii was not less than 5 × 10 7 cfu / mL.

[0023] Example 1 Test agents: calcium lignin sulfonate, methylcellulose, Bacillus siamese fermentation broth, Penicillium rettii fermentation broth, guanidine hydrochloride as positive control, and sterile water as negative control.

[0024] Experimental method: Ordinary tobacco K326 plants with the same growth were selected and divided into 11 groups, with 9 plants in each group. They were sprayed with the biological agents or chemical reagents in Table 2 respectively. The control group 4 was sprayed with clean water. After 12 hours of spraying, the 4th leaf of each plant was selected and evenly sprayed with silicon carbide powder. Then, 100μg of tobacco vein mosaic virus was inoculated by gently wiping the leaves, and the leaves were rinsed with water 15 minutes after inoculation to remove all residues.

[0025] Then, the tobacco plants were uniformly cultured under the same conditions, and the symptoms were observed 7 days after virus inoculation. The experiment was repeated 3 times. The disease index of tobacco vein mosaic virus in each group was determined, and the inhibition rate was calculated. The results of the investigation are shown in Table 2. Table 1 is the disease level classification and corresponding characterization.

[0026] Table 1 Disease index = Σ (number of diseased plants × disease level) / (total number of plants × highest disease level representative value) Inhibition rate = (disease index of control group - disease index of treatment group) / disease index of control group × 100% Table 2 As shown in Table 2, the use of Bacillus siamense fermentation broth or Penicillium rettgeri fermentation broth alone, or the use of the biocontrol agent of Bacillus siamense fermentation broth and Penicillium rettgeri fermentation broth, or the use of Bacillus siamense fermentation broth and / or Penicillium rettgeri fermentation broth combined with calcium lignin sulfonate and methylcellulose all have a significant passivation effect on tobacco vein mosaic virus.

[0027] Among them, the biocontrol agent using the fermentation broth of Bacillus siamese or Penicillium rettii and their composite calcium lignin sulfonate and methylcellulose alone has a slightly better passivation effect on tobacco vein mosaic virus than the chemical reagent-guanidine hydrochloride. The biocontrol agent using the fermentation broth of Bacillus siamese and Penicillium rettii and their composite calcium lignin sulfonate and methylcellulose has a significantly better passivation effect on tobacco vein mosaic virus than the biocontrol agent using the guanidine hydrochloride reagent, with an inhibition rate of up to 80.56%.

[0028] After multiple rounds of tests, it was found that the biocontrol agent is best when it is a composite reagent of Bacillus siamense fermentation broth, Penicillium rettneri fermentation broth, calcium lignin sulfonate and methylcellulose, and calcium lignin sulfonate and methylcellulose are friendly to the environment and tobacco plants. The experiment found that the mass ratio of Bacillus siamense fermentation broth, Penicillium rettneri fermentation broth, calcium lignin sulfonate and methylcellulose should be controlled at 3~4:3~4:3~5:2~4, preferably 3.5:3.5:4:3.

[0029] When Bacillus siamensis fermentation broth and Penicillium ryanthellae fermentation broth are used alone as biocontrol agents, or when the two agents are used to prepare other biocontrol agents for controlling tobacco vein mosaic virus or products for controlling tobacco vein mosaic virus, it is better to control the mass ratio of the two bacterial broths, Bacillus siamensis fermentation broth and Penicillium ryanthellae fermentation broth, to be 1:1.

[0030] Example 2 In order to further investigate the control effect of the biocontrol agent proposed in the present invention on grass vein mosaic virus, this example further conducted a field test to investigate the effect of the biocontrol agent. The test site was the Tianzhushan Tobacco Experimental Field of Shenyang Agricultural University.

[0031] Test agents: calcium lignin sulfonate, methylcellulose, Bacillus siamese fermentation broth, Penicillium rettii fermentation broth, guanidine hydrochloride as positive control, and sterile water as negative control.

[0032] The area of ​​each row area in the test field was 30 square meters, and each plot had 90 plants. At the early stage of the onset of tobacco vein mosaic virus, the disease index of each row area was investigated, and then the biocontrol agent or the above-mentioned chemical agent was applied according to the formula in Table 3. In this embodiment, the composite biocontrol agent was tested using the formula of Group 6 in Example 1; and the control group 8 was applied with clean water. Five repetitions were performed. After 7 days, the disease index of each row area was investigated again, and the relative control effect was calculated. The results are shown in Table 3.

[0033] In this embodiment, the evaluation criteria for disease level classification are the same as Table 1 in Example 1.

[0034] The calculation formula of the disease index is: Disease index = Σ (number of diseased plants × disease level value) / (total number of plants × highest disease level representative value).

[0035] The calculation formula for relative protection effect is: Relative control effect = {1-(disease index of the treatment group after application × disease index of the control group before application) / (disease index of the treatment group before application × disease index of the control group after application)} × 100%.

[0036] Table 3 As shown in Table 3, the biocontrol agent of using Bacillus siamense fermentation broth or Penicillium rettii fermentation broth alone, or using Bacillus siamense fermentation broth and Penicillium rettii fermentation broth combined with calcium lignin sulfonate and methylcellulose, has good control effect on tobacco vein mosaic virus in the field. Among them, the control effect of using Bacillus siamense fermentation broth or Penicillium rettii fermentation broth alone is slightly better than the effect of using chemical reagent-morpholino hydrochloride. The control effect of using the composite reagent of Bacillus siamense fermentation broth, Penicillium rettii fermentation broth, calcium lignin sulfonate and methylcellulose is significantly better than the control effect of morpholino hydrochloride on tobacco vein mosaic virus, which can be as high as 74.44%. The test results are consistent with the experimental results of Example 1.

[0037] In this example, the protective effect of the composite reagent of Bacillus siamense fermentation broth, Penicillium rettii fermentation broth, calcium lignin sulfonate and methylcellulose on tobacco vein mosaic virus is slightly lower than that of the experimental group in Example 1, mainly because it is more affected by the environment during the test field experiment.

[0038] In practical applications, it is suitable to apply it at the early stage of tobacco vein mosaic virus and / or before the disease occurs, and the spraying amount each time should be 80~120mL / mu.

[0039] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A biocontrol agent for preventing and treating tobacco vein mosaic virus, characterized in that: The invention is prepared by using the fermentation liquid of Bacillus siamese and / or the fermentation liquid of Penicillium rettii.

2. A biocontrol agent for preventing and controlling tobacco vein mosaic virus according to claim 1, characterized in that: The number of viable bacteria in the fermentation liquid of Bacillus siamese is not less than 1×10 8 cfu / mL, the number of viable bacteria in the fermentation broth of Penicillium rettii is not less than 5×10 7 cfu / mL.

3. The biocontrol agent for preventing and controlling tobacco vein mosaic virus according to claim 1, characterized in that: The Siamese Bacillus fermentation broth is obtained by the following method steps: a1. The Siamese Bacillus was inoculated into LB solid medium and activated at 28°C to obtain the activated strain after 24h; b1. The strain obtained in step a1 was inoculated into LB liquid medium and cultured under shaking conditions of 30 ° C and 120 r / min to obtain a seed solution; c1. The seed solution obtained in step b1 was inoculated into LB liquid medium at an inoculum volume of 5 mL / 100 mL and cultured under shaking conditions of 30°C and 120 r / min to obtain a fermentation broth of Bacillus siamese.

4. The biocontrol agent for preventing and controlling tobacco vein mosaic virus according to claim 1, characterized in that: The Penicillium reiterii fermentation broth is obtained by the following method steps: a2. The Penicillium rei was inoculated into LB solid medium and activated at 32 ° C. After 36 hours, the activated strain was obtained; b2. The strain obtained in step a2 was inoculated into LB liquid medium and cultured under shaking conditions of 37 ° C and 200 r / min to obtain a seed solution; c2. The seed solution obtained in step b2 was inoculated into LB liquid culture medium at an inoculum volume of 5 mL / 100 mL and cultured under shaking conditions of 37°C and 200 r / min to obtain a fermentation broth of Penicillium rettii.

5. The biocontrol agent for preventing and controlling tobacco vein mosaic virus according to claim 1, characterized in that: When the siam bacillus fermentation liquid and the penicillium rettgeri fermentation liquid are used for compounding, the siam bacillus fermentation liquid and the penicillium rettgeri fermentation liquid are uniformly mixed in a mass ratio of 1:1 to obtain the compound.

6. The biocontrol agent for preventing and controlling tobacco vein mosaic virus according to claim 5, characterized in that: It also includes calcium lignin sulfonate and methyl cellulose, and the siam bacillus fermentation liquid, penicillium rettgeri fermentation liquid, calcium lignin sulfonate and methyl cellulose are compounded in a mass ratio of 3-4:3-4:3-5:2-4.

7. The biocontrol agent for preventing and controlling tobacco vein mosaic virus according to claim 6, characterized in that: The siam bacillus fermentation liquid, the Penicillium rettgeri fermentation liquid, the calcium lignin sulfonate and the methyl cellulose are compounded in a mass ratio of 3.5:3.5:4:

3.

8. The biocontrol agent for preventing and controlling tobacco vein mosaic virus according to claim 1, characterized in that: The Siamese Bacillus fermentation liquid is deposited with CGMCC No.31958 and is classified as Siamese Bacillus Bacillus siamensis , obtained by activating Bacillus siamese with a strain number of snpv-z6; the fermentation broth of Penicillium rettii is deposited with a number of CGMCC No.41503 and is classified as Penicillium rettii Penicillium raperi , obtained by activating Penicillium reiterii with strain number SNPV-L10.

9. Use of the biocontrol agent for preventing and controlling tobacco vein mosaic virus according to any one of claims 1 to 8 in the preparation of a biocontrol agent for preventing and controlling tobacco vein mosaic virus and a product for preventing and controlling tobacco vein mosaic virus.

10. The use according to claim 9, characterized in that: Apply it at the early stage of tobacco vein mosaic virus disease and / or before the disease occurs, with a spraying amount of 80~120mL / mu each time.

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