CN114190404B - Biological control method for tobacco target spot and application - Google Patents

Biological control method for tobacco target spot and application Download PDF

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CN114190404B
CN114190404B CN202111612846.7A CN202111612846A CN114190404B CN 114190404 B CN114190404 B CN 114190404B CN 202111612846 A CN202111612846 A CN 202111612846A CN 114190404 B CN114190404 B CN 114190404B
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张崇
李斌
谢云波
江连强
闫芳芳
徐传涛
杨洋
何佶弦
吴元华
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Shenyang Agricultural University
China National Tobacco Corp Sichuan Branch
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract

The invention relates to the field of tobacco disease control methods, and particularly discloses a biological control method and application of tobacco target spot disease, wherein the tobacco target spot disease is controlled by spraying a biological control microbial inoculum containing water Rhizomyidae bacteria on tobacco leaves in a vigorous growing period, and the preservation number of the water Rhizomyidae bacteria is CCTCC NO: M2015245. The method can prevent and treat the target spot disease of the tobacco, and the prevention effect can reach 68.8-86.2%.

Description

Biological control method for tobacco target spot and application
Technical Field
The invention relates to the field of tobacco disease control methods, in particular to a biological control method for tobacco target spot and application thereof.
Background
Tobacco belongs to the family Solanaceae (Solanaceae) genus Nicotiana (Nicotiana) annual herbaceous plant. The tobacco is an important economic crop, the planting area is wide, tobacco planting brings benefits to a large number of tobacco growers, and the development of local economy is promoted.
The tobacco target spot damages tobacco leaves, is a small round water-stain-shaped spot at first, then quickly expands, has irregular diseased spots, has the diameter of 2-10cm, has light brown tissue in the diseased spots, and often has concentric ring veins, and necrotic parts of the diseased spots are fragile and fall off to form through holes which are shaped like holes left on a target after bullet shooting, so the target spot is called as the target spot. The tobacco target spot disease originally discovered in Liaoning province in 2005 in China is separated and identified as Rhizoctonia solani. Tobacco target spot disease mainly damages tobacco leaves in the mature period, causes perforation and necrosis of the leaves, reduces the quality and grade of tobacco leaves, and seriously loses the baking value of the tobacco leaves in the field, even produces no crop and receives no crop, thus causing serious loss to tobacco growers.
At present, most of the prevention and treatment of the tobacco target spot disease are carried out by spraying chemical agents, and the prevention and treatment mainly comprises azoxystrobin, azoxystrobin and the like, so that the prevention and treatment effect on the tobacco target spot disease is certain; however, long-term use of chemical agents for controlling diseases is easy to cause pathogenic bacteria to generate drug resistance, and meanwhile, long-term use of chemical agents also causes harm to the environment, so that the search for new biological control methods is urgent.
Disclosure of Invention
In order to solve the technical problems, the invention provides a biological control method and application of tobacco target spot disease, wherein the tobacco target spot disease can be controlled by spraying biocontrol microbial inoculum diluent on tobacco leaves, and the control efficiency is as high as 68.8-86.2%.
The first purpose of the invention is to provide a biological control method for tobacco target spot disease, which comprises the following steps: spraying biocontrol microbial inoculum diluent on tobacco leaves;
the active ingredient of the biocontrol microbial inoculum is rhinestone bacteria;
the preservation number of the water Rhizomenon ehrlatiae is CCTCC NO of M2015245;
the reinhardtia in the dilution of the biocontrol microbial inoculumThe effective viable count of the Erecta bacteria is 6 x 10 6 -6ⅹ10 8 cfu/mL。
Further, the spraying period of the biocontrol microbial inoculum diluent is from nine-leaf period to vigorous period of tobacco.
Further, the biocontrol microbial inoculum diluent is obtained by diluting the biocontrol microbial inoculum by 0-100 times with sterile water.
Further, the effective viable count of the riemerella reinhardtii in the biocontrol microbial inoculum is 6 x 10 8 cfu/mL。
Further, the preparation process of the biocontrol microbial inoculum comprises the following steps:
s1, preparing bacterial suspension: performing streak activation culture on the rhinestone bacteria on an LB solid culture medium flat plate, selecting a single colony after purification, transferring the single colony to an LB liquid culture medium for culture for 24 hours, centrifuging to obtain thalli, adding the thalli into sterile normal saline, and uniformly mixing to obtain OD 600 A bacterial suspension equal to 0.7;
s2, preparing a biocontrol microbial inoculum: and adding the bacterial suspension into an LB liquid culture medium according to the volume ratio of 3mL to 100mL, and culturing at 32 ℃ at 140r/min for 48h to obtain the biocontrol microbial inoculum.
Further, in S1, the culture conditions of the rhymenia reinhardtii are 32 ℃ and 140r/min.
Further, in S1, the centrifugation conditions were 4 ℃ and 4000r/min, and the centrifugation was carried out for 15min.
The second purpose of the invention is to provide the application of the biocontrol microbial inoculum in the prevention and treatment of tobacco target spot disease.
Further, the tobacco target spot is caused by rhizoctonia solani.
The third purpose of the invention is to provide the application of the rhinestone bacilli in the prevention and treatment of the tobacco target spot.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the tobacco target leaf disease is effectively prevented and controlled by spraying the anti-bacterial agent diluent on the tobacco leaves, the prevention effect can reach 68.8% -86.2%, and a technical thought is provided for disease prevention and control in the tobacco planting process;
2. the biocontrol microbial inoculum prepared by the invention can inhibit the growth of rhizoctonia solani, the antibacterial effect can reach 92.1 percent, and a basis is provided for preventing and treating the target spot of tobacco;
3. the active component of the biocontrol microbial inoculum prepared by the invention is the water Rhizomenon ehrlani (CCTCC NO: M2015245), the strain can not be used for preventing and treating the tobacco target spot disease at present, but the invention finds the new application of the strain in preventing and treating the tobacco target spot disease.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a colony morphology of Himerella reinhardtii in example 1 of the present invention.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
The embodiment provides a biological control method and application of tobacco target spot.
1. Strain preparation
1. Test strains
(1) Tobacco target leaf spot pathogen (Rhizoctonia solani AG-3) was provided by plant virus research laboratory of Shenyang university plant protection institute;
(2) The water Rhizomyir reinhardtii is purchased from China center for type culture collection, and the preservation number is CCTCC NO: M2015245.
LB liquid medium: 10g of Tryptone (Tryptone), 5g of Yeast extract (Yeast extract), 10g of sodium chloride (NaCl) and 1000mL of deionized water. LB solid medium: agar (Agar) is added into the liquid culture medium by 1.5 to 2 percent.
PDA culture medium: 200g of potato, 20g of glucose, 15-20 g of agar and 1000mL of distilled water.
2. Preparation of biocontrol microbial inoculum
(1) Activation and identification of rhinestone
Inoculating the purchased bacterial strain on an LB solid culture medium to perform streak activation culture under the culture conditions of 32 ℃, 140r/min and 24 hours; the activated single colony morphology was white colony, dry-shrunken surface, irregular edge (as shown in FIG. 1).
Transferring for 3-4 times, selecting single bacterial colony, culturing in LB liquid culture medium at 32 deg.C and 140r/min for 24h to obtain bacterial liquid, then sucking 2.5mL bacterial liquid, centrifuging at 4 deg.C and 4000r/min for 15min, discarding supernatant, collecting thallus, adding 100 μ L TE Buffer into thallus, mixing, adding 10 μ L lysozyme, treating in a constant temperature water bath at 37 deg.C for 12h, and extracting DNA according to the above-mentioned steps.
After the DNA extraction is successful, the strain DNA is used as a template, and the nucleotide sequence of the conserved segment of the strain DNA is subjected to PCR amplification by using a universal primer of a 16S rRNA gene sequence. The bacterial PCR reaction system is a 25 mu L system, the upstream primer is 0.5 mu L, the downstream primer is 0.5 mu L, the template is 1 mu L, ddH 2 O10.5. Mu.L, PCRmix 12.5. Mu.L, and the obtained PCR product was sent to a sequencing company for sequencing.
The general primer sequence is as follows:
27F：5'-AGAGTTTGATCCTGGCTCAG-3'
1492R：5'-GGTTACCTTGTTACGACTT-3'
according to the sequencing result, BLAST comparison analysis is carried out in GenBank in NCBI, the target strain is determined to be the rhinestone bacteria, the similarity of the target strain and the rhinestone bacteria in GenBank reaches 99%, and the target strain is the required target strain without pollution in the purchasing activation process.
(2) Preparation of biocontrol microbial inoculum
Performing streak activation culture on the rhinestone on an LB solid culture medium under the culture conditions of 32 ℃, 140r/min and 24 hours; transferring for 3-4 times, selecting single colony, culturing in LB liquid culture medium at 32 deg.C and 140r/min for 24 hr to obtain bacterial liquid, centrifuging at 4 deg.C and 4000r/min for 15min, discarding supernatant, collecting thallus, adding sterile physiological saline into thallus, and making into OD 600 A bacterial suspension equal to 0.7;
adding the obtained bacterial suspension into a fresh LB liquid culture medium according to the volume ratio of 3 8 cfu/mL。
3. Inhibitory effect of biocontrol microbial inoculum on rhizoctonia solani AG-3
(1) Detecting by using a plate confronting culture method.
Inoculating rhizoctonia solani AG-3 on a PDA solid culture medium, culturing for 48h, punching a bacterial cake from the edge of a bacterial colony by using a puncher with the diameter of 5mm, inoculating the bacterial cake at the central position of a fresh blank PDA solid culture medium flat plate, respectively inoculating 2 mu L of biocontrol microbial inoculum at two sides 2.0cm away from the bacterial cake, using sterile water as a control, culturing at a constant temperature of 28 ℃ and observing the bacteriostatic condition, repeating each treatment for three times, measuring the growth radius of the bacterial colony when the rhizoctonia solani AG-3 mycelia in the control group PDA solid culture medium flat plate fully grow on the whole plate, and calculating the bacteriostatic rate by using the following formula.
Relative inhibition = (control colony radius-treated colony radius)/control colony radius x 100%
(2) Diluting the biocontrol microbial inoculum by 10 times, 30 times, 50 times, 60 times, 80 times and 100 times by using sterile water, and respectively detecting the inhibition effect of a biocontrol microbial inoculum stock solution, a biocontrol microbial inoculum 10 times solution, a biocontrol microbial inoculum 30 times solution, a biocontrol microbial inoculum 50 times solution, a biocontrol microbial inoculum 60 times solution, a biocontrol microbial inoculum 80 times solution and a biocontrol microbial inoculum 100 times solution on rhizoctonia solani AG-3 by using a plate confrontation culture method;
each group was run in triplicate and the average was calculated.
4. Research on prevention and treatment effect of prevention and treatment method on tobacco target spot
(1) The obtained raw liquid of the anti-biological agent, 10 times of the liquid of the anti-biological agent, 30 times of the liquid of the anti-biological agent, 50 times of the liquid of the anti-biological agent, 60 times of the liquid of the anti-biological agent, 80 times of the liquid of the anti-biological agent and 100 times of the liquid of the anti-biological agent are used for spraying tobacco to detect the control effect on the target spot disease of the tobacco;
(2) Selecting and potting tobacco NC89, and when the tobacco is grown to a 9-leaf stage, respectively spraying a raw solution of a biological control agent, a 60-time solution of the biological control agent, an 80-time solution of the biological control agent and a 100-time solution of the biological control agent onto tobacco leaves, properly wetting the leaves until the leaves drip, spraying an equal amount of sterile water onto a control group, repeating each treatment for 3 times, inoculating rhizoctonia solani AG-3 fungus cakes (the diameter is 5 mm) on the tobacco leaves of an experimental group after 24 hours, inoculating 4 th, 5 th and 6 th true leaves on each plant, inoculating 3 fungus cakes (the three fungus cakes are uniformly distributed on the leaves) on each leaf, picking off the fungus cakes after 48 hours, culturing for 10 days, measuring the diameters of the scabs of the leaves, calculating the area of the scabs, and calculating the control effect by using the following formula;
control efficacy = (control group plaque area-experimental group plaque area)/control group plaque area x 100%
In the formula, the experimental group sprays the scab area of the tobacco leaves of a raw solution of the anti-biological agent, a solution of 10 times of the anti-biological agent, a solution of 30 times of the anti-biological agent, a solution of 50 times of the anti-biological agent, a solution of 60 times of the anti-biological agent, a solution of 80 times of the anti-biological agent or a solution of 100 times of the anti-biological agent;
2. results of the experiment
1. Research on bacteriostatic effect of biocontrol microbial inoculum on rhizoctonia solani AG-3
TABLE 1 bacteriostatic effect of biocontrol microbial inoculum prepared by the invention on rhizoctonia solani AG-3
Figure BDA0003435952410000061
As can be seen from Table 1, the biocontrol microbial inoculum prepared by the invention can inhibit Rhizoctonia solani, the relative inhibition rate reaches 92.1%, and the dilution of the biocontrol microbial inoculum with each multiple can also effectively inhibit Rhizoctonia solani, and the inhibition efficiency is 74.3% -92.1%. The biocontrol microbial inoculum prepared by the invention can be used for preparing a reagent for inhibiting rhizoctonia solani and can be applied to preventing and treating the tobacco target spot disease caused by the rhizoctonia solani.
2. Research on control effect of control method on tobacco target spot
TABLE 2 prevention and control of tobacco target leaf spot by the method of the present invention
Grouping Diameter of lesion (mm) Area of lesion (mm) 2 ) Control effect (%)
Control group 8.6 58.1 ―
Biocontrol microbial inoculum stock solution 3.2 8.0 86.2
10 times of solution of biocontrol microbial inoculum 3.8 11.3 80.6
30 times of liquid of biological control agent 4.0 12.6 78.3
50 times liquid of biocontrol microbial inoculum 4.2 13.8 76.2
60 times liquid of biocontrol microbial inoculum 4.3 14.5 75
80 times liquid of biological control agent 4.6 16.6 71.4
100 times liquid of biological control agent 4.8 18.1 68.8
As can be seen from Table 2, the biocontrol microbial inoculum prepared by the method and the 10-100 times of diluent are sprayed on tobacco leaves, so that the tobacco target spot can be effectively prevented and treated, the prevention effect on the tobacco target spot is 68.8-86.2%, and the biocontrol microbial inoculum has great significance for preventing and treating diseases in the tobacco planting process.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such 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 such modifications and variations.

Claims (10)

1. A biological control method for tobacco target spot is characterized by comprising the following steps: spraying biocontrol microbial inoculum diluent on tobacco leaves;
the active ingredient of the biocontrol microbial inoculum is rhinestone bacteria;
the preservation number of the water Rhizomenon ehrlatiae is CCTCC NO of M2015245;
the effective viable count of the rhinestone bacteria in the biocontrol microbial inoculum diluent is 6 x 10 6 -6ⅹ10 8 cfu/mL。
2. The method for biologically controlling target spot disease of tobacco according to claim 1, wherein the dilution of the biocontrol microbial inoculum is sprayed during the period from nine leaves to vigorous.
3. The method for biologically controlling tobacco target spot disease according to claim 2, wherein the biocontrol microbial inoculum dilution is obtained by diluting the biocontrol microbial inoculum with sterile water by 0 to 100 times.
4. The method for biocontrol of tobacco target spot disease according to claim 3, wherein the effective viable cell number of the Salmonella reinhardtii in said biocontrol bacterial agent is 6 x 10 8 cfu/mL。
5. The method for biologically controlling tobacco target spot disease according to claim 1, wherein the biocontrol microbial inoculum is prepared by the following steps:
s1, preparing a bacterial suspension: performing streak activation culture on the rhinestone bacteria on an LB solid culture medium flat plate, selecting a single colony after purification, transferring the single colony to an LB liquid culture medium for culture for 24 hours, centrifuging to obtain thalli, adding the thalli into sterile normal saline, and uniformly mixing to obtain OD 600 A bacterial suspension equal to 0.7;
s2, preparing a biocontrol microbial inoculum: and adding the bacterial suspension into an LB liquid culture medium according to the volume ratio of 3mL to 100mL, and culturing at 32 ℃ at 140r/min for 48h to obtain the biocontrol microbial inoculum.
6. The method according to claim 5, wherein the culture conditions of the Eremophilus rhinelloides in S1 are 32 ℃ and 140r/min.
7. The method for biological control of tobacco target spot according to claim 5, wherein in S1, the centrifugation conditions are 4 ℃,4000r/min, and 15min.
8. The use of the biocontrol microbial inoculum of claim 1 in the prevention and treatment of tobacco target spot disease.
9. The use of the biocontrol microbial inoculum of claim 8 in the control of tobacco target spot disease, wherein said tobacco target spot disease is caused by rhizoctonia solani.
10. Use of the rhymenia reinhardtii of claim 1 for the control of tobacco target spot disease.
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