A method for inoculating a tobacco plant with a biocontrol agent

Abstract

The present application relates to the technical field of tobacco biocontrol bacteria inoculation, and particularly relates to a tobacco biocontrol bacteria inoculation method.The present application provides a tobacco biocontrol bacteria inoculation method, which comprises the following steps: (1) when tobacco seedlings grow to five leaves with a heart, the tobacco seedlings are pruned, biocontrol bacteria are inoculated to the pruned wounds of the tobacco seedlings, the inoculation is repeated three times, and a promoting agent is sprayed after each inoculation; (2) the tobacco seedlings are transplanted 7-9 days after the last pruning of the tobacco seedlings, the roots of the tobacco seedlings are dipped in the biocontrol bacteria for root dipping when the tobacco seedlings are transplanted, then the tobacco seedlings are transplanted to a field, and a promoting agent is used for root irrigation.The tobacco seedling inoculation method provided by the present application has high inoculation colonization amount of bacteria, good disease prevention and control effect of biocontrol bacteria after inoculation, can effectively prevent and control tobacco bacterial wilt, black shank disease and tobacco mosaic virus and various tobacco diseases, and has practical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tobacco biocontrol bacteria inoculation, and particularly relates to a tobacco biocontrol bacteria inoculation method. BACKGROUND

[0002] At present, tobacco bacterial wilt is mainly prevented and treated by cultivating disease-resistant varieties, improving tillage system and adopting chemical control in production. However, the disease-resistant varieties have a long cultivation period and are easily affected by the environment, and the prevention and treatment effect is not ideal. Improving tillage system and adopting crop rotation are effective measures against the disease, but it is difficult to achieve in actual production. Chemical pesticides can reduce the harm of the disease to a certain extent, but the prevention and treatment effect is not satisfactory, and long-term use of chemical pesticides will also cause environmental pollution, pesticide residues and continuous increase of disease resistance of the bacteria, etc. Therefore, it is urgent to find an effective and safe prevention and treatment measure.

[0003] Biological control has the advantages of no toxicity, no residue and rich resources, and has become an effective way to solve soil-borne diseases. Among them, using antagonistic microorganisms to prevent and control plant diseases has become an important direction in biological control. So far, the screened biocontrol bacteria mainly include Pseudomonas, Bacillus, mycorrhizal fungi and a few actinomycetes. However, in general, the research basis for using antagonistic strains to prevent and control tobacco bacterial wilt is relatively weak, especially the efficient strains are obviously scarce. Moreover, there are the following problems in the prevention and treatment process of biocontrol bacteria; the inoculation process of biocontrol bacteria lacks standardized operation process, resulting in low inoculation success rate; the colonization and survival rate of biocontrol bacteria in the plant body after inoculation is low, the disease prevention effect is poor, which leads to that the biocontrol bacteria cannot play the expected role.

[0004] Therefore, how to provide a tobacco biocontrol bacteria inoculation method to solve the technical problems of low inoculation success rate of tobacco biocontrol bacteria and poor disease prevention effect of biocontrol bacteria after inoculation in the prior art is a problem that needs to be solved by those skilled in the art. SUMMARY

[0005] The purpose of the present application is to provide a tobacco biocontrol bacteria inoculation method.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical scheme:

[0007] The present application provides a tobacco biocontrol bacteria inoculation method, which comprises the following steps:

[0008] (1) when the tobacco seedlings grow to five leaves and one heart, the tobacco seedlings are pruned, the biocontrol bacteria are inoculated into the tobacco seedling pruning wounds, and the process is repeated three times, and a promoting agent is sprayed after each inoculation;

[0009] (2) the tobacco seedlings are transplanted 7-9 days after the last pruning of the tobacco seedlings, the roots of the tobacco seedlings are dipped in the biocontrol bacteria for root dipping when transplanting, then the tobacco seedlings are transplanted to the field, and a promoting agent is used for root irrigation.

[0010] The promoting agent comprises the following components in mass fraction: amino oligosaccharide 1-2 parts, nicotinamide 1-2 parts, auxin 2-4 parts, magnesium sulfate 2-4 parts, potassium dihydrogen phosphate 4-6 parts, ferrous sulfate 2-4 parts, carrageenan 2-4 parts, scutellaria extract 6-10 parts, cnidium extract 6-10 parts and water 60-70 parts.

[0011] Preferably, the interval time of each inoculation in step (1) is 5-7 days.

[0012] Preferably, the promoting agent is sprayed 12-24 hours after each inoculation in step (1).

[0013] Preferably, the biocontrol agent in step (1) is prepared by mixing bacillus amyloliquefaciens liquid, bacillus pumilus liquid and alkaligenes faecalis liquid.

[0014] Preferably, the concentration of the bacillus amyloliquefaciens liquid, bacillus pumilus liquid and alkaligenes faecalis liquid is 2-8x10 8 cfu / mL.

[0015] Preferably, the volume ratio of the bacillus amyloliquefaciens liquid, bacillus pumilus liquid and alkaligenes faecalis liquid is 4-8:7-11:10-16.

[0016] Preferably, the bacillus amyloliquefaciens is bacillus amyloliquefaciens YN48, the bacillus pumilus is bacillus pumilus B011 and the alkaligenes faecalis is alkaligenes faecalis L1.

[0017] Preferably, the preparation method of the promoting agent is mixing amino oligosaccharide, nicotinamide, auxin, magnesium sulfate, potassium dihydrogen phosphate, ferrous sulfate, carrageenan, scutellaria extract, cnidium extract and water.

[0018] Preferably, the use amount of the promoting agent in step (2) is 15-25 ml / plant when root irrigation is performed.

[0019] Preferably, the root dipping time in step (2) is 5-7 seconds.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] Among the accelerators used in the present application, nicotinamide contains rich vitamin B, not only has the effect of promoting wound healing, but also can increase the immunity of tobacco plants and improve their disease resistance. The use of amino oligosaccharide can promote the reproduction of biocontrol bacteria, and it also contains rich nutrient elements that can be absorbed and utilized by tobacco seedlings. Auxin can promote cell division and root differentiation. The purpose of adding magnesium sulfate, potassium dihydrogen phosphate and ferrous sulfate is to provide trace elements, and carrageenan plays a thickening role. Scutellaria extract and Cnidium extract have bactericidal effect and can prevent the invasion of external bacteria. The prepared accelerator of the present application has a synergistic effect between each component, which plays the roles of promoting the reproduction of biocontrol bacteria, promoting the wound healing of tobacco seedling plants, promoting growth and inhibiting bacteria.

[0022] The biocontrol bacterial strains used in the present application, Bacillus amyloliquefaciens YN48 can prevent and control tobacco black shank disease; Brevibacillus brevis B011 can prevent and control tobacco bacterial wilt; Alcaligenes faecalis L1 can prevent and control tobacco common mosaic virus disease.

[0023] The inoculation method provided by the present application has high colonization amount of bacterial species of tobacco seedlings after inoculation, and the biocontrol bacteria have good disease prevention effect after inoculation, can effectively prevent and control tobacco bacterial wilt, black shank disease and tobacco mosaic virus and other tobacco diseases, and has practical application value. DETAILED DESCRIPTION

[0024] The present application provides a tobacco biocontrol bacterial inoculation method, comprising the following steps:

[0025] (1) When the tobacco seedlings grow to five leaves and one heart, the tobacco seedlings are pruned, the biocontrol bacteria are inoculated into the tobacco seedling pruning wounds, and the process is repeated three times, and the accelerator is sprayed after each inoculation;

[0026] (2) After the last tobacco seedling pruning 7-9 days, the tobacco seedlings are transplanted, the roots of the tobacco seedlings are dipped in the biocontrol bacteria for root dipping when transplanting, then the tobacco seedlings are transplanted to the field, and the accelerator is used for root irrigation;

[0027] The accelerator comprises components in the following mass fractions: 1-2 parts of aminooctanose, 1-2 parts of nicotinamide, 2-4 parts of auxin, 2-4 parts of magnesium sulfate, 4-6 parts of potassium dihydrogen phosphate, 2-4 parts of ferrous sulfate, 2-4 parts of carrageenan, 6-10 parts of scutellaria extract, 6-10 parts of cnidium extract, and 60-70 parts of water; preferably 2 parts of aminooctanose, 2 parts of nicotinamide, 3 parts of auxin, 3 parts of magnesium sulfate, 5 parts of potassium dihydrogen phosphate, 3 parts of ferrous sulfate, 3 parts of carrageenan, 7-9 parts of scutellaria extract, 7-9 parts of cnidium extract, and 60-70 parts of water; further preferably 2 parts of aminooctanose, 2 parts of nicotinamide, 3 parts of auxin, 3 parts of magnesium sulfate, 5 parts of potassium dihydrogen phosphate, 3 parts of ferrous sulfate, 3 parts of carrageenan, 8 parts of scutellaria extract, 8 parts of cnidium extract, and 64-66 parts of water; more preferably 2 parts of aminooctanose, 2 parts of nicotinamide, 3 parts of auxin, 3 parts of magnesium sulfate, 5 parts of potassium dihydrogen phosphate, 3 parts of ferrous sulfate, 3 parts of carrageenan, 8 parts of scutellaria extract, 8 parts of cnidium extract, and 65 parts of water.

[0028] In the present application, step (2) is transplanting tobacco seedlings 8 days after the last time of cutting leaves of tobacco seedlings.

[0029] In the present application, the interval time of each inoculation in step (1) is 5-7 days; preferably 6 days.

[0030] In the present application, the accelerator is sprayed 12-24 hours after each inoculation in step (1); preferably 14-20 hours; further preferably 16-18 hours; more preferably 17 hours.

[0031] In the present application, the biocontrol agent in step (1) is mixed by Bacillus amyloliquefaciens liquid, Bacillus pumilus liquid and Alcaligenes faecalis liquid.

[0032] In the present application, the concentration of the Bacillus amyloliquefaciens liquid, Bacillus pumilus liquid and Alcaligenes faecalis liquid is 2-8 x 10 8 cfu / mL; preferably 3-7 x 10 8 cfu / mL; further preferably 4-6 x 10 8 cfu / mL; more preferably 5 x 10 8 cfu / mL.

[0033] In the present application, the volume ratio of the Bacillus amyloliquefaciens, Bacillus pumilus and Alcaligenes faecalis is 4-8:7-11:10-16; preferably 5-7:8-10:11-15; further preferably 6:9:12-14; more preferably 6:9:13.

[0034] In the present application, the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens YN48; the Bacillus pumilus is Bacillus pumilus B011; and the Alcaligenes faecalis is Alcaligenes faecalis L1.

[0035] In the present application, the preparation method of the promoter is mixing aminoo- ligosaccharide, nicotinamide, auxin, magnesium sulfate, potassium dihydrogen phosphate, ferrous sulfate, carrageenan, Scutellaria extract, Cnidium extract and water.

[0036] In the present application, the amount of the promoter used in the step (2) is 15-25 ml / plant; preferably 17-23 ml / plant; further preferably 19-21 ml / plant; more preferably 20 ml / plant.

[0037] In the present application, the time for the step (2) is 5-7 s; preferably 6 s.

[0038] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0039] The preparation method of Scutellaria extract and Cnidium extract is mixing Scutellaria and Cnidium with 8 times of water by mass, decocting for 2 h, and filtering the obtained filtrate to obtain Scutellaria extract and Cnidium extract.

[0040] Example 1

[0041] A tobacco biocontrol agent inoculation method, the steps are as follows:

[0042] (1) mixing aminoo- ligosaccharide 1 part, nicotinamide 1 part, auxin 2 parts, magnesium sulfate 2 parts, potassium dihydrogen phosphate 4 parts, ferrous sulfate 2 parts, carrageenan 2 parts, Scutellaria extract 6 parts, Cnidium extract 6 parts and water 60 parts to obtain a promoter;

[0043] (2) when the tobacco seedlings grow to five leaves and one heart, the tobacco seedlings are pruned, the biocontrol agent is inoculated to the tobacco seedling pruning wound in a smearing manner, repeated three times, the interval time of each inoculation is 5 d, and the promoter is sprayed 12 h after each inoculation until the leaf surface just drips water;

[0044] (3) transplanting the tobacco seedlings 7 d after the last pruning of the tobacco seedlings, dipping the roots of the tobacco seedlings in the biocontrol agent for 5 s, then transplanting the tobacco seedlings to the field on the same day, and using the promoter to irrigate the roots after 3 d; the amount used is 15 ml / plant;

[0045] The biocontrol agent is Bacillus amyloliquefaciens YN48, Brevibacillus brevis B011 and Alcaligenes faecalis L1, and the concentration of the bacterial liquid is 2×10 8 cfu / mL; the volume ratio is 4:7:10.

[0046] Example 2

[0047] A tobacco biocontrol agent inoculation method, the steps are as follows:

[0048] (1) mixing 2 parts of amino-oligosaccharide, 2 parts of nicotinamide, 4 parts of auxin, 4 parts of magnesium sulfate, 6 parts of potassium dihydrogen phosphate, 4 parts of ferrous sulfate, 4 parts of carrageenan, 10 parts of Scutellaria extract, 10 parts of Cnidium extract, and 70 parts of water to obtain a promoting agent;

[0049] (2) when the tobacco seedlings grow to five leaves with one heart, the tobacco seedlings are pruned, and the biocontrol bacteria are inoculated to the pruning wounds of the tobacco seedlings in a smearing manner, repeated three times, the interval time of each inoculation is 7 days, and the promoting agent is sprayed 24 hours after each inoculation until the leaf surface just drips water;

[0050] (3) the tobacco seedlings are transplanted 9 days after the last pruning, the roots of the tobacco seedlings are dipped in the biocontrol bacteria for 7 seconds, then the tobacco seedlings are transplanted to the field on the same day, and the promoting agent is used to irrigate the roots 5 days later; the dosage is 25 ml / plant;

[0051] The biocontrol bacteria are Bacillus amyloliquefaciens YN48, B. brevis B011, and Alcaligenes faecalis L1, and the concentration of the bacterial liquid is 8×10 8 cfu / mL; the volume ratio is 8:11:16.

[0052] Example 3

[0053] A method for inoculating tobacco biocontrol bacteria, the steps are as follows:

[0054] (1) mixing 2 parts of amino-oligosaccharide, 2 parts of nicotinamide, 4 parts of auxin, 4 parts of magnesium sulfate, 6 parts of potassium dihydrogen phosphate, 4 parts of ferrous sulfate, 4 parts of carrageenan, 10 parts of Scutellaria extract, 10 parts of Cnidium extract, and 70 parts of water to obtain a promoting agent;

[0055] (2) when the tobacco seedlings grow to five leaves with one heart, the tobacco seedlings are pruned, and the biocontrol bacteria are inoculated to the pruning wounds of the tobacco seedlings in a smearing manner, repeated three times, the interval time of each inoculation is 7 days, and the promoting agent is sprayed 24 hours after each inoculation until the leaf surface just drips water;

[0056] (3) the tobacco seedlings are transplanted 9 days after the last pruning, the roots of the tobacco seedlings are dipped in the biocontrol bacteria for 7 seconds, then the tobacco seedlings are transplanted to the field on the same day, and the promoting agent is used to irrigate the roots 5 days later; the dosage is 25 ml / plant;

[0057] The biocontrol bacteria are Bacillus amyloliquefaciens YN48, B. brevis B011, and Alcaligenes faecalis L1, and the concentration of the bacterial liquid is 8×10 8 cfu / mL; the volume ratio is 8:11:16.

[0058] Experimental Example 1

[0059] Single factor experiment

[0060] Example 3 as the experimental group;

[0061] Control group 1: other methods are the same as example 3, the only difference is that no accelerator is used.

[0062] Control group 2: other methods are the same as example 3, the only difference is that the amino oligosaccharide and nicotinamide in the accelerator are replaced by an equal amount of distilled water.

[0063] Control group 3: other methods are the same as example 3, the only difference is that the scutellaria extract and cnidium extract in the accelerator are replaced by an equal amount of distilled water.

[0064] Control group 4: other methods are the same as example 3, the only difference is that no accelerator is used to irrigate the roots after transplanting tobacco seedlings.

[0065] Prior art group 1: 72% streptomycin sulfate is used to prevent tobacco bacterial wilt.

[0066] Prior art group 2: 58% metalaxyl is used to prevent tobacco black shank.

[0067] Prior art group 3: 20% moroxydine ethyl copper is used to prevent tobacco mosaic virus.

[0068] The experimental group, control group 1 to control group 4 respectively according to the corresponding method to deal with tobacco seedlings, the prior art group does not carry on the inoculation of biocontrol bacteria.

[0069] In Hunan Yongzhou region, choose a piece of tobacco planting field to plant tobacco after each test group processing, tobacco planting field is divided into 24 regions on average, each test group randomly plant three regions.

[0070] After 15 days of tobacco seedling transplanting, the endophytic bacteria of seedlings are isolated by using the conventional microbial isolation method, and the colonization amount of three strains in the experimental group, control group 1 to control group 4 in tobacco root, stem and leaf is calculated. The test statistical data is taken as average value, and the results are shown in table 1.

[0071] In Hunan Yongzhou region, choose a piece of tobacco planting field to plant tobacco after each test group processing, tobacco planting field is divided into 24 regions on average, each test group randomly plant three regions in each planting field.

[0072] The prior art group is used at the early stage of tobacco disease, and the prevention effect of each test group on tobacco bacterial wilt, black shank and tobacco mosaic virus is counted 90 days after tobacco seedling transplanting. The blank control is to replace the reagent used in the prior art group with an equal amount of distilled water. The test statistical data is taken as average value, and the results are shown in table 2 to table 4.

[0073] Incidence = number of diseased plants / number of investigated plants x 100%

[0074] Control efficiency (%) = incidence of blank control - incidence of agent treatment

[0075] Table 1: Different bacterial species colonization amount (unit x 10 4 cfu / g)

[0076]

[0077] Table 2: Control efficiency on tobacco bacterial wilt (unit %)

[0078] Group Bacterial wilt Experimental group 78.66 Control group 1 63.52 Control group 2 67.55 Control group 3 72.58 Control group 4 76.25 Prior art group 1 67.85

[0079] Table 3: Control efficiency on tobacco black shank (unit %)

[0080]

[0081]

[0082] Table 4: Control efficiency on tobacco mosaic virus (unit %)

[0083] Group Tobacco mosaic virus Experimental group 90.62 Control group 1 65.45 Control group 2 72.16 Control group 3 83.45 Control group 4 86.33 Prior art group 3 71.23

[0084] From the content described in Table 1 to Table 4, it can be seen that, by the method of the present application, three kinds of biocontrol bacteria can be inoculated at the same time, and the inoculation effect is good. The use of the promoter also plays its due role in promoting the reproduction of biocontrol bacteria, preventing the invasion of external pathogens, and helping the wound healing of tobacco seedlings. The use effect of the inoculation method provided by the present application is obviously better than that of the prior art drug. From the data of control groups 1 and 2, it can also be seen that, if the promoter provided by the present application is not used or some components are discarded, it will significantly affect the inoculation effect of biocontrol bacteria, and further affect the effect of biocontrol bacteria. Moreover, if the use method of the promoter is not appropriate, it will also significantly affect the use effect of biocontrol bacteria.

[0085] From the data of control group 3, it can be seen that the Scutellaria extract and Cnidium extract play their due auxiliary role, and if they are not added, it will also affect the use effect of biocontrol bacteria.

[0086] The above is only the preferred embodiment of the present application, and it should be pointed out that, for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method of inoculating a tobacco plant with a plant growth promoting bacteria, the method comprising, The method comprises the following steps: (1) when the tobacco seedlings grow to five leaves with one heart, the tobacco seedlings are pruned, the biocontrol bacteria are inoculated to the pruning wounds of the tobacco seedlings, and the process is repeated three times, and a promoting agent is sprayed after each inoculation; (2) the tobacco seedlings are transplanted 7-9 days after the last pruning of the tobacco seedlings, the roots of the tobacco seedlings are dipped in the biocontrol bacteria for root dipping when the tobacco seedlings are transplanted, then the tobacco seedlings are transplanted to the field, and the promoting agent is used for root irrigation; The promoting agent comprises the following components in mass fractions: 1-2 parts of amino oligosaccharide, 1-2 parts of nicotinamide, 2-4 parts of auxin, 2-4 parts of magnesium sulfate, 4-6 parts of potassium dihydrogen phosphate, 2-4 parts of ferrous sulfate, 2-4 parts of carrageenan, 6-10 parts of scutellaria extract, 6-10 parts of cnidium extract, and 60-70 parts of water; The biocontrol bacteria in step (1) are prepared by mixing bacillus amyloliquefaciens liquid, bacillus pumilus liquid and alkaligenes faecalis liquid; The concentration of the bacterial solution of Bacillus amyloliquefaciens, B. pumilus and Alcaligenes faecalis is 2-8x10 8 cfu / mL. The volume ratio of the bacillus amyloliquefaciens, the bacillus pumilus and the alkaligenes faecalis is 4-8:7-11:10-16; The bacillus amyloliquefaciens is bacillus amyloliquefaciens YN48, the bacillus pumilus is bacillus pumilus B011, and the alkaligenes faecalis is alkaligenes faecalis L1.

2. The inoculation method for tobacco biocontrol bacteria according to claim 1, characterized in that, The interval time of each inoculation in step (1) is 5-7 days.

3. The method of claim 1 or 2, wherein the method comprises the steps of: a) inoculating the tobacco plant with the biocontrol bacteria; and b) applying the at least one compound to the tobacco plant. The promoting agent is sprayed 12-24 hours after each inoculation in step (1).

4. The method of claim 1, wherein the biocontrol bacteria is Bacillus amyloliquefaciens. The preparation method of the promoting agent is that the amino oligosaccharide, the nicotinamide, the auxin, the magnesium sulfate, the potassium dihydrogen phosphate, the ferrous sulfate, the carrageenan, the scutellaria extract, the cnidium extract and the water are mixed.

5. The method of claim 1, wherein the biocontrol bacteria is Bacillus amyloliquefaciens. The use amount of the promoting agent for root irrigation in step (2) is 15-25 ml / plant.

6. The method of claim 1, wherein the biocontrol bacteria is Bacillus amyloliquefaciens. The root dipping time in step (2) is 5-7 seconds.

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