A strain of Bacillus dysenteriae and its application in plant disease control

Abstract

The invention discloses a Bacillus vallismortis and its application in plant disease prevention and control, and belongs to the field of microbial technology. The Bacillus vallismortis of the present invention is Bacillus vallismortis (Bacillus vallismortis) YN‑1‑Q7, and the deposit number is CGMCCNo.29750. The bacterium has a high inhibitory effect on pathogens such as peanut white rot, peanut root rot, peanut damping-off, tobacco root rot, and tobacco layer sickle root rot, and the inhibition rate can reach more than 50%. The bacterium is prepared into a preparation, and tobacco and peanut are irrigated, which can effectively prevent and control plant diseases caused by pathogens such as peanut white rot, peanut root rot, peanut damping-off, tobacco root rot, and tobacco layer sickle root rot, and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Bacillus valerianus strain and application thereof in plant disease prevention and treatment. Background Art

[0002] Peanut is an important oil crop and cash crop, playing a pivotal role in ensuring national oil security and increasing farmers' income. In recent years, due to climate warming, the promotion of high-yield peanut varieties and changes in farming systems, especially the promotion of dense planting, the field microclimate and soil microecology have changed significantly, and the occurrence of peanut rhizomes and stem diseases has become increasingly serious. Peanut white rot fungus (Sclerotium rolfsii), peanut root rot fungus (Fusarium oxysporum), peanut sclerotinia (Rhizocfonia solani) and other diseases are becoming more and more serious. Rhizomes and stem diseases have become the most important diseases affecting peanut production due to the wide variety of pathogens and significant regional differences, and there is no effective prevention and control method in production.

[0003] Tobacco is one of the important economic crops in my country. As a global tobacco rhizome disease, tobacco Fusarium root rot is becoming more and more serious in my country. In some areas, it has become the main tobacco rhizome disease, seriously affecting the quality and yield of tobacco and restricting the sustainable development of the tobacco industry. Tobacco pests and diseases are one of the main factors restricting the safe production of the tobacco industry. The annual yield loss directly caused by tobacco pests and diseases accounts for 10% to 15% of the total output. Investigations and studies have found that there are more than 100 types of tobacco diseases worldwide, and 68 infectious diseases have been found in tobacco fields in my country. According to the damaged parts, they are mainly divided into leaf diseases and rhizome diseases, and Fusarium root rot is one of the main tobacco rhizome diseases.

[0004] At present, the prevention and control of peanut and tobacco pests and diseases mainly rely on plant-resistant varieties and chemical control, but the problem of pest resistance is becoming increasingly serious, and chemical pesticide residues cause serious harm, such as residual toxicity and environmental pollution, which pose a great threat to the human body and the ecology, and are not conducive to the sustainable development of the peanut and tobacco planting industries. Therefore, it is urgent to find an economical, safe and effective prevention and control measure. Biological control measures are a type of measures that use beneficial microorganisms to kill or reduce the number of pathogens to control the occurrence and development of plant diseases. These beneficial organisms are also called antagonistic microorganisms or biocontrol bacteria. Screening, separating and purifying more biocontrol bacteria can lay more research foundations and alternative materials for the biological control of peanut and tobacco pests and diseases. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a Bacillus dysenteriae and its application in plant disease prevention and control.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A strain of Bacillus vallismortis, wherein the Bacillus vallismortis is Bacillus vallismortis YN-1-Q7, and its deposit number is CGMCC No.29750.

[0008] The invention discloses an application of Bacillus dysenteriae with the deposit number of CGMCC No. 29750 in the prevention and treatment of plant diseases.

[0009] In a specific embodiment, the plant is peanut or tobacco.

[0010] In a specific embodiment, the plant disease is at least one of peanut Sclerotium rolfsii, peanut root rot, peanut damping-off, tobacco root rot, and tobacco Fusarium solani.

[0011] A method for preventing and controlling plant diseases, comprising: using a preparation containing Bacillus dysenteriae with a deposit number of CGMCC No. 29750 to perform root irrigation treatment on plants, wherein the concentration of Bacillus dysenteriae in the preparation is ≥ 1×10 7 cfu / mL.

[0012] In a specific embodiment, the preparation method of the preparation containing Bacillus dysvalleyi with a deposit number of CGMCC No. 29750 is as follows: the Bacillus dysvalleyi with a deposit number of CGMCC No. 29750 is activated and inoculated into LB liquid culture medium, and cultured at 28° C. and 180 r / min with shaking until the bacterial concentration is ≥ 1×10 10 cfu / mL, diluted 1000 times.

[0013] The invention discloses an application of Bacillus valerianus with the deposit number of CGMCC No. 29750 in the preparation of a preparation for preventing and controlling plant diseases.

[0014] A preparation for preventing and treating plant diseases, wherein the active ingredient comprises Bacillus valerianus with a preservation number of CGMCC No.29750.

[0015] In a specific embodiment, the concentration of Bacillus dysenteriae in the preparation is ≥ 1×10 7 cfu / mL.

[0016] In a specific embodiment, the preparation method of the preparation is as follows: Bacillus dysvalleyi with a preservation number of CGMCC No. 29750 is activated and inoculated into LB liquid culture medium, and cultured at 28°C and 180r / min shaking until the bacterial concentration is ≥1×10 10 cfu / mL, diluted 1000 times.

[0017] Advantages of the technical solution of the present invention

[0018] The present invention separates and obtains a dead valley bacillus, which has a high inhibitory effect on pathogens such as peanut white rot, peanut root rot, peanut damping-off, tobacco root rot, tobacco layered Fusarium root rot and the like, and the inhibition rate can reach more than 50%. The dead valley bacillus is prepared into a preparation, and watered on tobacco and peanuts, which can effectively prevent and control plant diseases caused by pathogens such as peanut white rot, peanut root rot, peanut damping-off, tobacco root rot, tobacco layered Fusarium root rot and the like, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Colony morphology of strain YN-1-Q7;

[0020] Figure 2 Phylogenetic tree of strain YN-1-Q7;

[0021] Figure 3 The inhibitory effect of strain YN-1-Q7 on peanut white rot fungus (Sclerotium rolfsii);

[0022] Figure 4 The inhibitory effect of strain YN-1-Q7 on peanut root rot pathogen (Fusarium oxysporum);

[0023] Figure 5 The inhibitory effect of strain YN-1-Q7 on Rhizoctonia solani;

[0024] Figure 6 The inhibitory effect of strain YN-1-Q7 on tobacco root rot pathogen (Fusarium oxysporum);

[0025] Figure 7 The inhibitory effect of strain YN-1-Q7 on tobacco root rot pathogen (Fusarium proliferatum). DETAILED DESCRIPTION

[0026] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0027] The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0028] Unless otherwise specified, the test materials, reagents, and drugs used in the following examples can be purchased through general channels.

[0029] Example 1

[0030] Isolation, purification and identification of strains

[0031] Rhizosphere soil of tobacco from Tengchong, Yunnan was collected, 5 g of rhizosphere soil was weighed and placed in a 45 mL triangular bottle with sterile water, mixed, and diluted 10 -1 ~10 -6 For soil suspension, use a micropipette to draw 50 μL of soil suspension of different dilution multiples onto the LB plate, apply it evenly with a sterile applicator, seal the plate and invert it in a 28°C constant temperature incubator for 48 hours. Pick a batch of single colonies on the plate, draw a line on the LB solid medium plate, invert it and incubate it in a 28°C constant temperature incubator for 1 to 2 days. Strain YN-1-Q7 was isolated and obtained.

[0032] Morphological identification: The colonies of strain YN-1-Q7 are irregular, light yellow, and wrinkled ( Figure 1 ), microscopic observation showed that the bacteria were Gram-positive and rod-shaped.

[0033] Physiological and biochemical identification: According to Bergey's Manual of Systematic Bacteriology, the physiological and biochemical characteristics of strain YN-1-Q7 were determined.

[0034] The results are shown in Table 1.

[0035] Table 1 Physiological and biochemical characteristics of strain YN-1-Q7

[0036]

[0037] Note: “+” indicates a positive result; “-” indicates a negative result.

[0038] As shown in Table 1, strain YN-1-Q7 is Gram-positive and has positive catalase activity, and can degrade starch. It is a fermentative strain and can utilize sugars such as glucose and sucrose.

[0039] Molecular Biology Identification:

[0040] The strain YN-1-Q7 colony was picked and the genomic DNA was extracted according to the instructions of the TIANGEN TIANamp BACTERia DNA Kit; then, PCR amplification was performed using the extracted genomic DNA as a template according to the instructions of the 16s rDNA Bacterial Identification PCR Kit of TaKaRa.

[0041] The forward primer is: 27F: 5'-AGAGTTTGATCATGGCTCAG-3' (SEQ ID NO: 2);

[0042] The reverse primer is: 1492R: 5'-AAGTCGTAACAAGGTAAGCG-3' (SEQ ID NO: 3).

[0043] The PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min; annealing at 53°C for 1 min; extension at 72°C for 90 s; and post-extension at 72°C for 5 min, for a total of 30 cycles.

[0044] The PCR products were separated by 1% agarose gel electrophoresis and observed under 3UVTM Transilluminator (UVP, USA) after EB staining. The electrophoresis detection showed that the size of the 16s rDNA fragment of the YN-1-Q7 strain was about 1500bp, which was consistent with the expected design of the kit.

[0045] The target DNA fragment was recovered using a recovery kit Silica Bead DNA Gel Extraction Kit and sequenced by Qingdao Qingke Biotechnology Co., Ltd. The sequencing result is shown in SEQ ID NO: 1, and the 16srDNA fragment of the YN-1-Q7 strain consists of 1435 nt base pairs.

[0046] SEQ ID NO:1(5'→3')

[0047]

[0048] The determined sequence was compared with the sequence in GenBank (NCBI, website http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) by BLAST program, and then the 16s rDNA sequence of species and genus similar to the sequence of strain YN-1-Q7 was obtained from GenBank. The comparison results showed that the 16s rDNA sequence of strain YN-1-Q7 was highly homologous to the 16s rDNA sequence of Bacillus vallismortis of the genus Bacillus in the GenBank gene library, with a homology rate of 100%. The results of constructing the phylogenetic tree of strain YN-1-Q7 showed ( Figure 2 ), strain YN-1-Q7 and Bacillus vallismortis (Accession No: MF177859) of the genus Bacillus constitute a separate branch, with the closest evolutionary distance, reflecting the closest relationship between them. The homology between the two was 100% obtained by DANMAN software analysis. Combining the results of morphological identification, physiological and biochemical characteristics identification and 16S rDNA sequence analysis, strain YN-1-Q7 was determined to be Bacillus vallismortis.

[0049] Bacillus vallismortis YN-1-Q7 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.29750, the deposit date is January 23, 2024, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0050] Example 2

[0051] Inhibitory effect of strain YN-1-Q7 on peanut or tobacco pathogens

[0052] A single colony of strain YN-1-Q7 was selected and inoculated into LB medium at 28°C and 200 r / min for 12 h. 100 μL of the culture solution was transferred into 20 mL of LB medium and incubated in a shaker at 28°C and 180 r / min until the bacterial suspension concentration reached 10 6 cfu / mL. Inoculate a peanut or tobacco pathogen cake with a diameter of 0.5 cm on the PDA plate culture medium in a sterile operating table, add 6 μL of YN-1-Q7 culture solution 2.5 cm away from the cake on the left and right sides, and culture it upside down in a 28°C incubator for 10-15 days. Results are shown in Figure 3-7 .

[0053] Among them, the peanut pathogens are peanut white rot fungus (Sclerotium rolfsii), peanut root rot fungus (Fusarium oxysporum), and peanut damping-off fungus (Rhizoctonia solani); the tobacco pathogens are tobacco root rot fungus (Fusarium oxysporum) and tobacco layer sickle root rot fungus (Fusarium proliferatum), and the above pathogens are all preserved in the laboratory of Shandong Peanut Research Institute.

[0054] The antibacterial rate was calculated according to the following formula:

[0055] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100

[0056] turn out:

[0057] The strain YN-1-Q7 was tested against tobacco root rot pathogen (Fusarium oxysporum) for three times, and the inhibitory effect was obvious, with the highest inhibition rate being 60.20%, the lowest being 54.23%, and the average inhibition rate being 57.20%.

[0058] The strain YN-1-Q7 was tested against Sclerotium rolfsii for 3 times, with the highest inhibition rate being 50.90%, the lowest being 50.82%, and the average inhibition rate being 50.85%.

[0059] In three confrontation tests of strain YN-1-Q7 against tobacco Fusarium proliferatum, the highest inhibition rate was 62.80%, the lowest was 59.32%, and the average inhibition rate was 61.33%.

[0060] In three confrontation tests of strain YN-1-Q7 against peanut root rot pathogen (Fusarium oxysporum), the highest inhibition rate was 50.30%, the lowest was 50.20%, and the average inhibition rate was 50.23%.

[0061] In three confrontation tests of strain YN-1-Q7 against Rhizoctonia solani, the highest inhibition rate was 59.61%, the lowest was 53.24%, and the average inhibition rate was 55.96%.

[0062] This shows that strain YN-1-Q7 has obvious antagonistic effect on peanut and tobacco pathogens, with inhibition rates exceeding 50%, and can be used as a potential biocontrol strain.

[0063] Example 3

[0064] Preparation of YN-1-Q7 fermentation broth: After the strain YN-1-Q7 was activated, it was inoculated into LB liquid culture medium and placed in a shaker at 28°C with a shaking speed of 180 r / min for 3 days to obtain YN-1-Q7 fermentation broth with a bacterial concentration of 1×10 10 cfu / mL.

[0065] Propagation of pathogens: Put oat grains into a triangular flask, soak in distilled water for 6 hours, pour out the water, and sterilize at 121℃ for 20 minutes; inoculate tobacco root rot fungus (Fusarium oxysporum), peanut white rot fungus (Sclerotium rolfsii), tobacco layer sickle root rot fungus (Fusarium proliferatum), peanut root rot fungus (Fusarium oxysporum), and peanut damping-off fungus (Rhizoctonia solani) into the sterilized oat grains, respectively, and culture at 28℃ for 7 days. Shake the inoculation bottle twice a day to ensure that all oat grains are infected with pathogens to obtain contaminated oat grains.

[0066] Pathogen inoculation:

[0067] Peanuts (variety Huayu No. 20) were planted in pots in an artificial climate chamber, with a mass ratio of soil: vermiculite: nutrient soil = 2:1:1. The culture temperature was 28°C during the day and 25°C at night, with 12h light / 12h dark. Peanuts were planted individually, with 3 seeds per pot. Peanuts were inoculated with pathogens 7 days after emergence; 10 wheat kernels with each pathogen were inoculated around the peanuts by spreading the topsoil, then covered with a thin layer of soil and watered.

[0068] Tobacco (Nicotiana benthamiana) was transplanted after emergence, with 3 plants in each pot. Pathogens were inoculated 7 days after tobacco transplanting. 10 wheat grains with each pathogen were inoculated around the tobacco by spreading the topsoil, and then covered with a thin layer of soil and watered.

[0069] YN-1-Q7 inoculation: Apply YN-1-Q7 fermentation liquid twice, at the time of peanut sowing and 7 days after seedling emergence; apply YN-1-Q7 fermentation liquid twice, at the time of tobacco sowing and 7 days after transplanting. Before application, the YN-1-Q7 fermentation liquid prepared above was diluted 1000 times, and the YN-1-Q7 fermentation liquid diluted 1000 times (1×10 7 cfu / mL) for root irrigation, 10 pots per treatment, 3 plants per pot, repeated 3 times. Set up two controls: 50% carbendazim 800 times solution and clean water. Observe the incidence of tobacco root rot, peanut white rot, tobacco sickle root rot, peanut root rot, and peanut damping-off 15 days after pathogen inoculation. Water peanuts and tobacco once every 2-3 days.

[0070] Grading standards for peanut white rot: Level 0: The plant has no symptoms; Level 1: Lesions only occur at the base of the stem; Level 2: Constriction symptoms occur at the base of the stem, and the infected parts showing systemic symptoms (wilt, death, wilt, etc.) account for less than one-third of the whole plant; Level 3: The infected parts showing systemic symptoms account for less than two-thirds of the whole plant; Level 4: The infected parts showing systemic symptoms account for more than two-thirds of the whole plant.

[0071] Peanut root rot classification standards:

[0072] Level 0: Plants are asymptomatic;

[0073] Level 1: Plant growth is basically normal or slightly dwarfed, a few roots are necrotic and black, and the middle and lower leaves are chlorotic;

[0074] Level 2: The diseased plant is 1 / 4 to 1 / 3 shorter than the healthy plant, or half of the roots are necrotic and black;

[0075] Level 3: The diseased plants are 1 / 3 to 1 / 2 shorter than healthy plants, most of the roots are necrotic and black, and more than 1 / 2 of the leaves are wilted;

[0076] Level 4: The diseased plant is more than 1 / 2 shorter than the healthy plant, all leaves of the plant are wilting, all roots are necrotic and black, and the secondary roots near the surface are obviously damaged. The diseased plant is basically dead.

[0077] Grading standards for peanut damping-off disease: Grade 0: the plant is asymptomatic; Grade 1: lesions only occur at the base of the stem; Grade 2: constriction symptoms occur at the base of the stem, and the infected parts showing systemic symptoms (wilt, death, wilt, etc.) account for less than one-third of the whole plant; Grade 3: The infected parts showing systemic symptoms account for less than two-thirds of the whole plant; Grade 4: The infected parts showing systemic symptoms account for more than two-thirds of the whole plant.

[0078] Grading standards for tobacco root rot:

[0079] Level 0: Plants are asymptomatic;

[0080] Level 1: Plant growth is basically normal or slightly dwarfed, a few roots are necrotic and black, and the middle and lower leaves are chlorotic;

[0081] Level 2: The diseased plant is 1 / 4 to 1 / 3 shorter than the healthy plant, or half of the roots are necrotic and black;

[0082] Level 3: The diseased plants are 1 / 3 to 1 / 2 shorter than healthy plants, most of the roots are necrotic and black, and more than 1 / 2 of the leaves are wilted;

[0083] Level 4: The diseased plant is more than 1 / 2 shorter than the healthy plant, all leaves of the plant are wilting, all roots are necrotic and black, and the secondary roots near the surface are obviously damaged. The diseased plant is basically dead.

[0084] Disease rate = number of diseased plants / total number of plants × 100%

[0085] Disease index = ∑ (representative value of disease level × number of diseased plants at each level) × 100 / (total number of plants surveyed × representative value of the highest disease level)

[0086] Control effect = [(control disease index - treatment disease index) / control disease index] × 100%

[0087] Pot test results: The fermentation liquid of strain YN-1-Q7 was used to treat peanut strains. The control effect of YN-1-Q7 on indoor potted peanut white rot was 63.5%, as shown in Table 2; the control effect of YN-1-Q7 on indoor potted peanut root rot was 49.6%, as shown in Table 3; the control effect of YN-1-Q7 on indoor potted peanut damping-off was 49.3%, as shown in Table 4, which was equivalent to the control effect of carbendazim. This shows that the biocontrol bacteria YN-1-Q7 has obvious control effects on peanut white rot, root rot and damping-off.

[0088] The fermentation liquid of strain YN-1-Q7 was used to treat tobacco strains. The control effect of YN-1-Q7 on indoor potted tobacco root rot was 51.7%, as shown in Table 5; the control effect of YN-1-Q7 on indoor potted tobacco sickle root rot was 51.7%, as shown in Table 6; this shows that the biocontrol bacteria YN-1-Q7 has obvious control effects on tobacco root rot and tobacco sickle root rot.

[0089] Table 2 Control effect of strain YN-1-Q7 on peanut white rot in potted plants

[0090]

[0091] Table 3 The control effect of strain YN-1-Q7 on peanut root rot in potted plants

[0092]

[0093] Table 4 Control effect of potted strain YN-1-Q7 on peanut damping-off disease

[0094]

[0095] Table 5 The control effect of potted strain YN-1-Q7 on tobacco root rot

[0096]

[0097] Table 6 The control effect of potted strain YN-1-Q7 on tobacco sickle root rot

[0098]

[0099]

[0100] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. The application of Bacillus dysenteriae with a deposit number of CGMCC No. 29750 in plant disease prevention and control, characterized in that: The Bacillus vallismortis is Bacillus vallismortis YN-1-Q7, and the plant disease is caused by Sclerotium rolfsii.

2. A method for preventing and controlling plant diseases, characterized in that: The plants were treated with a preparation containing Bacillus dysenteriae with a preservation number of CGMCC No. 29750, wherein the concentration of Bacillus dysenteriae in the preparation was ≥ 1×10 7 cfu / mL; the plant disease is caused by Sclerotium rolfsii.

3. The method for preventing and controlling plant diseases according to claim 2, characterized in that: The preparation method of the preparation containing Bacillus dysvalleyi with a preservation number of CGMCC No. 29750 is as follows: the Bacillus dysvalleyi with a preservation number of CGMCC No. 29750 is activated and inoculated into LB liquid culture medium, and cultured at 28° C. and 180 r / min with shaking until the bacterial concentration is ≥ 1×10 10 cfu / mL, diluted 1000 times.

4. The use of Bacillus dysenteriae with a deposit number of CGMCC No. 29750 in the preparation of a preparation for preventing and controlling plant diseases, characterized in that: The plant disease is caused by peanut Sclerotium rolfsii.

5. A preparation for preventing and controlling plant diseases, characterized in that: The active ingredient comprises Bacillus dysenteriae with a preservation number of CGMCC No. 29750, and the plant disease is caused by Sclerotium rolfsii of peanut.

6. The preparation for preventing and controlling plant diseases according to claim 5, characterized in that: The concentration of Bacillus dysenteriae in the preparation is ≥1×10 7 cfu / mL.

7. The preparation for preventing and controlling plant diseases according to claim 6, characterized in that: The preparation method of the preparation is as follows: Bacillus dysvalleyi with a preservation number of CGMCC No. 29750 is activated and inoculated into LB liquid culture medium, and cultured at 28°C and 180r / min shaking until the bacterial concentration is ≥1×10 10 cfu / mL, diluted 1000 times.

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