Biological prevention and control medicament for preventing both tobacco root rot and tobacco nematode disease

Abstract

The invention discloses a biological prevention and control medicament for preventing both tobacco root rot and tobacco nematode disease, and belongs to the field of tobacco disease prevention and control methods. The medicament is prepared from the following components in parts by weight: 4 to 6 parts of chloromethylsilatrane, 3 to 5 parts of bacillus marinus bacterial liquid and 2 to 5 parts of streptomyces microflavus bacterial liquid. The bacillus marinus is bacillus marinus, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) NO.25891; the streptomyces microflavus is streptomyces microflavus 168, the streptomyces microflavus is preserved in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, and the preservation number of the streptomyces microflavus is CGMCC No.15355. The streptomyces microflavus has the advantages that the streptomyces microflavus is a streptomyces microflavus strain 168; by means of the method, the tobacco root knot nematode disease and the tobacco cyst nematode disease can be prevented and treated, larvae are killed, the prevention and treatment effect is 75% or above, the tobacco can be induced to generate resistance, and meanwhile the treated tobacco can generate obvious disease resistance to nematodes; the composition can prevent and treat tobacco root rot, has a remarkable effect, does not destroy the soil environment, and has a good application prospect in prevention and treatment of tobacco diseases.

Description

Technical Field

[0001] The invention relates to the field of tobacco disease prevention and control methods, and more specifically to a biological control agent capable of preventing tobacco root rot and tobacco nematode disease. Background Art

[0002] Tobacco is a plant of the genus Nicotiana in the Solanaceae family, an annual or limited perennial herb. Tobacco can be used as a raw material for the tobacco industry; tobacco can also be used as a pesticide insecticide. The nicotine contained in tobacco has a wide insecticide spectrum, is fast-acting, and is easily degraded naturally. It is safe for most crops, has no or very little residue, and can kill more than 200 pests such as Lepidoptera, Diptera, Coleoptera, and Hemiptera; it can also be used as a medicine, as an anesthetic, sweating, sedative, and emetic. Tobacco cyst nematode disease was first reported by Lownsherry in Connecticut, USA in 1954. At present, tobacco cyst nematode disease only occurs sporadically in some tobacco-growing areas in Shandong and Henan, and the damage is relatively mild. Tobacco root knot nematode disease occurs in major tobacco-growing areas in the world, with serious damage in tropical, subtropical, and warm temperate zones. Tobacco root knot nematodes are relatively common in Yunnan Province, my country, and are more serious in tobacco-growing areas in Guangxi, Guizhou, Sichuan, Chongqing, Hubei, Henan, Shaanxi, Shandong and other provinces (autonomous regions, municipalities directly under the central government). Root-knot nematodes can cause complex infections with root rot, aggravating the damage.

[0003] The main nematodes that harm tobacco worldwide are root-knot nematodes ( Meloidogyne spp.), cyst nematodes ( Heterodera spp.) and root-rot nematodes ( Paratylenchus spp.) are the three major categories. At present, the root-knot nematodes are the most common in my country, with the largest number of cases and the largest losses. In addition to the direct damage caused by nematodes to tobacco, the wounds caused by nematodes are conducive to the infection of pathogens and can also aggravate the severity of diseases such as tobacco black shank and tobacco bacterial wilt. Some nematodes are also vectors of certain viruses, so the potential harm is even greater and needs to be paid great attention.

[0004] Tobacco root rot is a disease caused by the genus Fusarium, which occurs in tobacco. The disease can cause lesions in the roots of plants, with some only affecting one side, causing the top to bend to one side, small leaves, and rot at the roots of tobacco. When the diseased stems and roots are cut open, browning of the wood is observed, which destroys the tobacco's vascular system, making it difficult for the aboveground part to absorb water and the underground part to absorb nutrients, and eventually leading to the death of the tobacco plant. Summary of the invention

[0005] The invention provides a biological control agent capable of preventing both tobacco root rot and tobacco nematode disease, aiming to solve the problem of tobacco damage caused by tobacco root rot and tobacco nematode disease.

[0006] A biological control agent for preventing tobacco root rot and tobacco nematode disease, comprising the following components: 4-6 parts of silanol, 3-5 parts of marine bacillus liquid, and 2-5 parts of tenuiflavus liquid.

[0007] Furthermore, the marine Bacillus is marine Bacillus, and the strain collection number is CGMCC NO.25891; the Streptomyces leucodermi is Streptomyces leucodermi Liaoning variant ( Streptomyces microflavusvar.liaoning sword )168, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCCNo.15355.

[0008] Furthermore, the number of live bacteria in the marine Bacillus broth was 6×10 7 -6×10 9 cfu / mL. ; The number of viable Streptomyces flavonoids is 10 7 -10 9 cfu / mL.

[0009] In a second aspect, a method for preparing a biological control agent for preventing both tobacco root rot and tobacco nematode disease comprises: Step 1, bacterial culture: Bacillus marineus and Streptomyces tenuifolius were inoculated into LB solid medium: liquid medium plus 1.5% to 2% agar, transferred several times until a purified single colony appeared, and then the single colony was transferred into 250mL LB liquid medium, cultured at 200rpm and 37℃ for 48h, and then 20mL of bacterial solution was taken out and prepared with sterile saline to OD 600 The bacterial suspension is about 7, and the bacterial suspension is continued to be pipetted into LB liquid culture medium, and cultured at 200 rpm and 37°C for 36-48 hours to obtain bacterial liquid; Step 2: Evenly mix the silica ring, marine Bacillus solution and Streptomyces tenuifolius solution in a ratio of 1:1:1 to obtain a biocontrol agent.

[0010] Furthermore, the LB liquid culture medium includes: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 1000 mL of deionized water; The LB solid culture medium includes: liquid culture medium plus 1.5% to 2% agar.

[0011] The third aspect is the use of a biological control agent for preventing both tobacco root rot and tobacco nematode disease, which is used to prevent both tobacco root rot and tobacco nematode disease.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a biological control agent for preventing tobacco root rot and tobacco nematode disease, which can be used for root treatment, has a strong control effect on Fusarium, and also has a strong toxic effect on root-knot nematodes and cyst nematodes and larvae, and has a significant inhibitory effect on the hatching of egg capsules, thus solving the problem of tobacco nematode control. The biological control agent prepared by the biological control agent has good application prospects in preventing and controlling tobacco root diseases. DETAILED DESCRIPTION

[0013] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0014] The invention discloses a biological control agent for preventing tobacco root rot and tobacco nematode disease. The agent contains the following components by weight: 4-6 parts of silanol, 3-5 parts of marine bacillus bacterial liquid and 2-5 parts of tenuiflavous streptomyces bacterial liquid.

[0015] Example 1 This embodiment provides a method for preparing a biocontrol agent.

[0016] 1. Test strains The marine bacillus is marine bacillus Liaoning variant ( Bacillus marinus var. Liaoning ), isolated and purified from the plant phyllosphere, and deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 12, 2022, with the culture collection number CGMCC NO. 25891; The Streptomyces leucodermi is a Streptomyces leucodermi Liaoning variant ( Streptomyces microflavusvar.liaoni English )168, isolated and purified from rhizosphere soil, and deposited in the General Microbiology Center of China Microbiological Culture Collection on February 12, 2018, with the deposit number CGMCC No.15355.

[0017] 2. Bacterial culture LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 1000 mL deionized water; LB solid medium: liquid medium plus 1.5%~2% agar.

[0018] Bacillus marineus and Streptomyces tenuiflavos were inoculated on LB solid plates, transferred several times until a purified single colony appeared, and then transferred the single colony to 250 mL of LB liquid medium, cultured at 200 rpm and 37 °C for 48 h, and then 20 mL of the bacterial solution was taken out and prepared with sterile saline to OD 600 The bacterial suspension is about 7, and the bacterial suspension is continued to be pipetted into LB liquid culture medium, and cultured at 200 rpm and 37°C for 36-48 hours to obtain bacterial solution.

[0019] 3. Preparation of biocontrol agents The silica ring, marine Bacillus bacteria liquid, and fine yellow Streptomyces bacteria liquid are uniformly mixed in a ratio of 1:1:1. Specifically, 5 parts of silica ring, 5 parts of marine Bacillus bacteria liquid, and 5 parts of fine yellow Streptomyces bacteria liquid are uniformly mixed. The ratio is only one embodiment of the present invention. Specifically, the silica ring, marine Bacillus bacteria liquid, and fine yellow Streptomyces bacteria liquid can also be uniformly mixed in a ratio of 4~6:3~5:2~5.

[0020] Application Example 1 This application example provides a test for the activity of killing tobacco root-knot nematodes.

[0021] 1. Preparation of test preparations Prepare the biocontrol agent and avermectin according to the method provided in Example 1 2. Preparation of insect suspension Common root-knot nematodes that infect tobacco include the southern root-knot nematode ( Meloidogyne incognita )、Peanut root-knot nematode( M. arenaria ), javanic root-knot nematode ( M. javanica ) and northern root-knot nematode ( M. hapla ). This application example uses southern root-knot nematodes ( M.incognita ) oocysts, put them into a sterile culture dish, disinfect them with 0.5% sodium hypochlorite for 3 minutes, rinse them with sterile water, take half of the oocysts for later use, and add the other half into a petri dish with sterile water. After constant temperature culture at 25°C, add sterile water to the insect suspension and dilute it to a concentration of 200 oocysts / 500 μL for later use.

[0022] 3. Test method for killing root-knot nematodes 500 μL of the agent prepared in Example 1 and avermectin were pipetted into a Petri dish using a pipette, and 500 μL of the prepared worm suspension was added. Microscopic examination was performed after 24 h and 48 h to check the nematode mortality rate, and sterile water was used as a negative control.

[0023] 30 μL of the agent prepared in Example 1 and avermectin were separately pipetted and dropped into a 96-well plate, one oocyst was placed in each well, and the hatching rate was observed under a microscope after 24 h and 48 h.

[0024] 4. Experimental results The results of the determination of the lethality of the second-instar larvae of southern root-knot nematodes and the inhibition rate of the hatching of egg capsules of the biocontrol agent are shown in the table. It can be seen that the lethality of the second-instar larvae of southern root-knot nematodes by the biocontrol agent reached 67.39% at 24 hours, and the lethality is still increasing with the passage of time. The inhibition rate of the hatching of the egg capsules of southern root-knot nematodes is also as high as more than 75%; compared with avermectin, the control effect of the biocontrol agent on tobacco root-knot nematodes is very excellent.

[0025] Application Example 2 This application example provides a test for the activity of controlling Fusarium.

[0026] 1. Preparation of test preparations Preparation of culture medium: weigh 200 g of potatoes, wash, peel and cut into small pieces, steam for 30 min to obtain boiled potatoes; filter the boiled potatoes with gauze and heat them, add 20 g of agar during the heating process, stir and mix, after the agar is dissolved, add 20 g of glucose, stir evenly, add distilled water to 1000 mL, sterilize at 121° C. for 20 min, and obtain a sterilized PDA culture medium; cool the sterilized PDA culture medium to 45° C. to obtain a PDA culture medium; 25 mL of the biological agent described in Example 1, Bacillus subtilis (commercially available; positive control) and water (negative control) were added to the PDA medium and mixed evenly to obtain a mixture. 10 mL of the mixture was taken to prepare a solid plate; a control group was set up, and the control group was PDA medium.

[0027] 3) Take the Fusarium plate (deposited in the Tobacco Research Institute of the College of Plant Protection, Shenyang Agricultural University), punch out the bacterial cakes with a hole puncher, and put them on the above solid plate and the culture medium of the control group respectively. After culturing at 28°C for 60 hours, use a vernier caliper to measure the colony diameters of Fusarium oxysporum and Fusarium solani, and calculate the antibacterial results. The results are shown in Table 1; Inhibition rate = (colony diameter of control group - colony diameter of treatment group) / colony diameter of control group × 100%.

[0028] Table 1 The inhibition results of biological agents on Fusarium As can be seen from Table 1, both the biological agent and Bacillus subtilis have significant inhibitory effects on Fusarium, but the antibacterial effect of the biological agent is much higher than that of Bacillus subtilis. Therefore, the biological agent of the present invention can effectively inhibit tobacco Fusarium root rot.

[0029] Application Example 3 This application example provides a test for inducing systemic resistance in tobacco.

[0030] The root crack test was used to determine the induced systemic resistance of the biocontrol agent to root-knot nematodes and cyst nematodes. In the test, the challenge root system and the response root system were isolated from each other but the culture environment was completely the same. Since the response root system was only inoculated with the nematode suspension and had no direct contact with the agent, the control effect of the agent on nematodes was completely dependent on its induced resistance to the plant. This determination method is reliable and has been widely used in the study of systemic induced resistance of biocontrol microorganisms to soil-borne diseases.

[0031] 1. Preparation of test preparations Prepare the biocontrol agent according to the method provided in Example 1, and prepare avermectin 2. Preparation of Nematodes for Experimental Use Prepare a root-knot nematode suspension according to the method provided in Application Example 1; Cyst nematodes ( Heteroderaglycines ): Cyst nematode race 3 was obtained from the experimental base of the Northern Nematode Research Institute of Shenyang Agricultural University. Fresh, mature, and uniform cysts were picked under a stereoscope. They were disinfected with 0.5% sodium hypochlorite solution for 5 minutes, rinsed with sterile water, and cultured in a culture dish in a 25 ℃ constant temperature box. After incubation in a shallow dish for 15 days, the second-instar larvae were obtained, and the concentration of the suspension was adjusted to 200 larvae / mL.

[0032] 3. Test methods Wash the tobacco seedlings and divide the roots into two equal parts. Take three plastic pots of the same size and place them in a "pin" shape. The bottom plastic pots are placed in two 16cm diameter trays. Open two 1cm×2cm lines at the bottom of the top plastic pot, and place the seedlings in the top plastic pot so that the separated roots can reach the soil of the two lower plastic pots through the two cracks. The roots growing in the two bottom plastic pots are the challenge root system and the response root system. The three pots are filled with soil matrix to fix the plants. The system is placed in a greenhouse (25℃, 14h light). 10mL of biocontrol agent is applied below 2cm of the topsoil in the challenge root system, and avermectin and sterile water are applied to the control group. 1-2 weeks after the application, 5mL of root-knot nematode and cyst nematode suspensions are inoculated into the response root system, with avermectin and sterile water as the control. There are 3 treatments and 3 repetitions in total. After 25d, the number of invasive nematodes and cysts in the soil of the response root pots were investigated.

[0033] The roots were cleaned with running water, the fresh weight of the roots was weighed, and then the nematodes in the roots were stained with sodium hypochlorite-acid fuchsin staining method. The number of nematodes in the roots was observed and recorded under a stereomicroscope and converted into the number of nematodes per gram of root fresh weight.

[0034] For strains with significantly reduced nematode invasion after treatment, the reduction rate of nematode invasion in the root system was calculated. 5.3 Calculation of control effect Invasion reduction rate (%) = [(the number of invasive nematodes in the roots of the control response - the number of invasive nematodes in the roots of the response after strain treatment) / the number of invasive nematodes in the roots of the control response] × 100.

[0035] 6 Test results The test showed that the biocontrol agent can induce strong resistance to nematodes in tobacco. After applying the agent to the root system, the invasion of root-knot nematodes and cyst nematodes in the root system was significantly reduced, and the number of cysts was significantly reduced. As shown in the table, in the root system, the invasion of root-knot nematodes was reduced by 60.21%, the invasion of cyst nematodes was reduced by 60.10%, and the number of cysts in the soil was reduced by 73.79%. It can be seen that its induced resistance is much better than that of avermectin.

[0036] Application Example 4 This application example provides a field test on the effectiveness of controlling tobacco cyst nematode disease.

[0037] 1. Preparation of test preparations Prepare the biocontrol agent according to the method provided in Example 1, and prepare avermectin 2. Preparation of Nematodes for Experimental Use The cyst nematode suspension was prepared according to the method provided in Application Example 2.

[0038] 3. Test methods Tobacco was inoculated with naturally diseased soil in the field. Five replicates were conducted in Xinmin County, Liaoning Province. The field cyst count was 50 cysts / 100 mL soil. The field plot design consisted of 3 treatments, 4 replicates, and a total of 12 plots, arranged in random blocks, with 4 ridges per plot, 10 meters long, and a protective row set at the edge of the plot. Using avermectin and clean water as controls, 20 seedlings were randomly selected from each plot 7 days after application, and the topsoil (0-5 cm) was removed before the plants were dug out with their roots to keep the root system intact. The root cyst count of a single plant was investigated, and the cyst inhibition rate was calculated.

[0039] 4. Test results For tobacco treated with biocontrol agents, the root cyst attachment was investigated during the cyst-forming stage of tobacco cyst nematodes. The treated tobacco seedlings had a significant inhibitory effect on the cysts of cyst nematodes. As can be seen from the table, the inhibition rates reached 57.67% and 47.92%, which were much higher than those of the avermectin treatment group and the CK group.

[0040] Application Example 5 This application example provides a field test on the effectiveness of preventing and controlling tobacco root rot.

[0041] The experimental site was selected in Qingyishan Town, Kuandian County, Dandong City, Liaoning Province. 2The pesticide was sprayed on the day and 16 days after transplanting into the field, with a spraying rate of 0.2 g / m 2 ; The biological agents sprayed were: the biological agent described in Example 1 and Bacillus subtilis as positive controls; clean water as negative controls; and the disease index and control effect were calculated.

[0042] Disease classification standards: Level 0: The whole plant is disease-free; Level 1: The lower leaves turn green and yellow, and the tips of the leaves occasionally wilt; Level 3: The middle and lower leaves turn yellow and wilt, and the tips and edges of 1 / 3 to 1 / 2 of the leaves turn dry and withered; Level 5: The middle and lower leaves turn yellow and wilt, and the tips and edges of more than 1 / 2 of the leaves turn dry and withered. Level 7: The lower leaves are dead, and the tips and edges of the middle and upper leaves are withered downwards; Level 9: All leaves of the plant die.

[0043] Table 2 Control effect on Fusarium root rot It can be seen from the table that the control effect of the biological agent on tobacco Fusarium root rot is as high as 90.57%, and the control effect of the positive control on tobacco Fusarium root rot is 83.48%. Therefore, the biological agent of the present invention can effectively control tobacco Fusarium root rot.

[0044] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0045] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A biological control agent for preventing both tobacco root rot and tobacco nematode disease, characterized in that: The agent contains the following components by weight: 4-6 parts of silanol, 3-5 parts of marine bacillus bacterial liquid, and 2-5 parts of tenuiflavous streptomyces bacterial liquid.

2. The biological control agent for preventing both tobacco root rot and tobacco nematode disease according to claim 1, characterized in that: The marine Bacillus in the marine Bacillus liquid has a strain collection number of CGMCC NO. 25891; the Streptomyces pyogenes is a strain of Streptomyces pyogenes Liaoning variant ( Streptomyces microflavusvar.liaoningensis )168, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.15355.

3. The biological control agent for preventing both tobacco root rot and tobacco nematode disease according to claim 1, characterized in that: The number of live bacteria in the marine Bacillus solution is 6×10 7 -6×10 9 cfu / mL; the number of viable Streptomyces flavonoids is 10 7 -10 9 cfu / mL.

4. A method for preparing the biological control agent for preventing both tobacco root rot and tobacco nematode disease as claimed in claim 1, characterized in that: The preparation method comprises: Step 1, bacterial culture: Bacillus marineus and Streptomyces tenuifolius were inoculated into LB solid medium: liquid medium plus 1.5% to 2% agar, transferred several times until a purified single colony appeared, and then the single colony was transferred into 250mL LB liquid medium, cultured at 200rpm and 37℃ for 48h, and then 20mL of bacterial solution was taken out and prepared with sterile saline to OD 600 The bacterial suspension is about 7, and the bacterial suspension is continued to be pipetted into LB liquid culture medium, and cultured at 200 rpm and 37°C for 36-48 hours to obtain bacterial liquid; Step 2: Evenly mix the silica ring, marine Bacillus solution and Streptomyces tenuifolius solution in a ratio of 1:1:1 to obtain a biocontrol agent.

5. The biological control agent for preventing both tobacco root rot and tobacco nematode disease according to claim 4, characterized in that: in, The LB liquid culture medium includes: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 1000 mL of deionized water; The LB solid culture medium includes: liquid culture medium plus 1.5% to 2% agar.

6. Use of the biological control agent for preventing tobacco root rot and tobacco nematode disease as claimed in claim 1, characterized in that: Used to prevent tobacco root rot and tobacco nematode disease.

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