CN110172422B - Bacillus proteolicus and application thereof in preventing and controlling root-knot nematodes - Google Patents
Bacillus proteolicus and application thereof in preventing and controlling root-knot nematodes Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The disclosure relates to a bacillus proteolyticus and application thereof in preventing and controlling root-knot nematodes. The strain is Bacillus proteolyticus (Bacillus proteoliticus) Bp-196, which is preserved in the common microorganism center of China general microbiological culture Collection center in 2018, 9 and 21 months, and the strain preservation number is CGMCC NO. 16525. The strain is a bacterium separated from rhizosphere soil of cucumbers suffering from root-knot nematode diseases, has a good prevention and control effect on the root-knot nematodes, is beneficial to early prevention and control of the harm of the root-knot nematodes, and is beneficial to realizing safe and pollution-free production of vegetables.
Description
Technical Field
The disclosure relates to the field of microorganism application, in particular to a bacillus proteoliposphilus and application thereof in preventing and controlling root-knot nematodes.
Background
Plant parasitic nematodes exist in a wide variety of countries and regions of the world. The root-knot nematode is one of the most important plant parasitic nematodes, and with the rapid increase of the planting area of facility vegetables and the continuous improvement of vegetable multiple cropping indexes in China, the generation area of the root-knot nematode is gradually enlarged, so that the yield of the vegetables can be reduced by 20-30 percent, even 60-70 percent, and the development of the facility vegetable industry is severely restricted. Hosts of root-knot nematodes (m.incognita) are more than 3000 plants, and are able to infect the roots of almost all cultivated plants.
At present, chemical nematocides are mainly used for preventing and controlling root-knot nematodes in production, but the chemical nematocides generally have the problems of high toxicity, environmental pollution, easy generation of drug resistance and the like, so that the use amount of chemical agents is gradually reduced in practice. The biocontrol strain has the characteristics of no environmental pollution, no drug resistance and safety to people and livestock, so the screening of the biocontrol strain for efficiently preventing and treating the root-knot nematode becomes a hot spot of the current domestic and foreign research.
Disclosure of Invention
In order to overcome the problems in the prior art, the disclosure provides a bacillus poproteolyticus Bp-196 strain, which is a bacterium separated from rhizosphere soil of cucumbers suffering from root-knot nematode diseases, has a good prevention and control effect on the root-knot nematodes, is beneficial to early prevention and control of the harm of the root-knot nematodes, and is beneficial to realizing safe and pollution-free production of vegetables.
The strain provided by the disclosure is Bacillus proteolyticus (Bacillus proteoliticus) Bp-196, which is obtained by separating and purifying cucumber rhizosphere soil with root knot nematode disease in cucumber field blocks planted in corridor city of North river, and is preserved in the general microbiological center of China Committee for culture Collection of microorganisms in 2018, 9 and 21 days, and the address is as follows: the number of the strain preservation is CGMCC NO.16525 at Xilu No.1 of Beijing, Chaoyang, North Chen, China academy of sciences and microbiology. It has the following biological properties: after 3 days of culture on an LB culture medium plate at 30 ℃, the bacterial colony of the strain is large, the surface is rough, flat and irregular, the color of the inner circle is darker, and all the bacterial strains are gram positive. The observation under a scanning electron microscope shows that the cells of all bacteria are rod-shaped and have abundant flagella, and oval spores are formed in swollen sporangia.
In one example, the present disclosure provides that the bacillus proteolicus Bp-196 strain is a gram-positive strain.
In one example, the present disclosure provides a method for fermenting a bacillus proteolicus Bp-196 strain, wherein the method comprises: the Bacillus proteolicus Bp-196 strain was inoculated into a fermentation medium and cultured at 30 ℃ for 3 days at a rotation speed of 220 rpm.
In one example, the fermentation medium comprises: 5g/L yeast powder, 10g/L peptone and 10g/L sodium chloride, and the volume is fixed to 1L by using distilled water.
In one example, the bacillus proteolicus Bp-196 strain provided by the present disclosure is used in the manufacture of a medicament for controlling root knot nematode disease.
The bacillus proteolicus Bp-196 strain provided by the present disclosure is a bacterium isolated from cucumber rhizosphere soil in which root knot nematode disease occurs. On one hand, the strain is safe and harmless to crops, and is beneficial to realizing safe and pollution-free production of vegetables; on the other hand, the strain has good control effect on the root-knot nematode. Experiments show that the lethality of the strain fermentation liquor to the second-instar larvae of the root-knot nematodes reaches 100%, in a prevention and treatment test, the prevention and treatment effect on the root-knot nematodes reaches 86.5%, and the prevention and treatment effect is efficient and stable.
Drawings
FIG. 1 is a colony map of the Bacillus proteolicus Bp-196 strain in some embodiments of the present disclosure;
FIG. 2 is a bacterial morphology of the Bacillus proteolicus Bp-196 strain shown in FIG. 1.
Detailed Description
The principles and spirit of the present disclosure will be described with reference to a number of exemplary embodiments. It is understood that these embodiments are given solely for the purpose of enabling those skilled in the art to better understand and to practice the present disclosure, and are not intended to limit the scope of the present disclosure in any way.
Example 1: separation, purification and identification of Bacillus proteolicus (Bacillus proteoliticus) Bp-196 strain
1. Isolation and purification of Bacillus proteolicus (Bacillus proteoliticus) Bp-196 Strain
The Bacillus proteolyticus (Bacillus proteolyticus) Bp-196 strain disclosed by the invention is obtained by separating a soil dilution method in cucumber rhizosphere soil which generates root knot nematode diseases in cucumber fields planted in corridor cities of Hebei province, wherein the separation method comprises the following steps:
soil samples are uniformly collected from cucumber rhizosphere in cucumber planting fields of corridor city in Hebei province, six parts are calculated, 10g of each part is taken, and 6 parts of collected soil are uniformly mixed. And adding the mixed 60g of soil sample into 1000mL of distilled water, stirring uniformly, standing for 1min, taking the supernatant, putting the supernatant into a water bath kettle at 80 ℃ for heating in a water bath for 30min, and fully stirring the supernatant in the water bath process to ensure that the supernatant is heated uniformly. After heating in the water bath, the supernatant was cooled to room temperature, shaken up, and 1mL of the supernatant was taken and diluted 100-fold with sterile water. And then 0.25ml of diluted clear liquid is taken and coated on an LB flat plate, the clear liquid is uniformly coated, the clear liquid is placed in a constant-temperature incubator at 30 ℃ for culture for 2-4 d, and the growth condition of the bacterial colony is observed regularly.
The LB plate was produced by the following method: putting 5g/L yeast powder, 10g/L peptone, 10g/L sodium chloride and 0.13-0.15g/L agar powder into a culture dish, and fixing the volume to 1L by using distilled water to obtain LB plate base solution; then, the LB plate base solution was autoclaved at 121 ℃ for 20 minutes and plated in a petri dish, thereby obtaining an LB plate.
After the tissue block grows out, performing single spore separation, and selecting a single colony to be purified on an LB culture medium.
2. Identification of Bacillus proteolicus (Bacillus proteoliticus) Bp-196 Strain
(1) Microbiological characteristics:
as shown in FIG. 1-2, the obtained strain was cultured on LB medium plate at 30 ℃ for 3 days, and the colony of the strain was large, rough, flat, irregular, dark in inner circle color, and all strains were gram-positive. The observation under a scanning electron microscope shows that the cells of all bacteria are rod-shaped and have abundant flagella, and oval spores are formed in swollen sporangia. The strain is morphologically identified according to Bergey's Manual of identification of bacteria, and the strain is determined to be Bacillus proteolicus (Bacillus proteoliticus).
(2) Molecular biological characteristics:
a single colony was picked with a sterilized toothpick, and placed in an EP tube containing 300. mu.L of bacterial fine lysate SDS, lysed at 65 ℃ for 2 hours, and then bacterial DNA was extracted with a bacterial genomic DNA extraction kit, and 16s rRNA sequence amplification was performed with primers 27F and 1492R using DNA as a template. The PCR amplification reaction system is 50. mu.L, and contains 25. mu.L of ExTaq enzyme, 1. mu.L of forward primer, 1. mu.L of reverse primer, 3. mu.L of DNA template, and 20. mu.L of sterile water. The amplification conditions were: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 45 ℃ for 30s, extension at 72 ℃ for 2.5min, and 30 cycles; extension at 72 ℃ for 10 min. And separating and identifying the amplification product by 1% agarose gel electrophoresis, and directly performing bidirectional sequencing on the PCR product. The above-mentioned bacterial genomic DNA extraction kit is selected from Tiangen Biotech Ltd.
16S rRNA sequence amplification was performed using primers 1492R and 27F, and the PCR product was subjected to 1% agarose gel electrophoresis. The sequence of the 16s rRNA amplified by the strain is 1509bp by sequencing analysis. After BLAST analysis is carried out on the 16S rRNA sequence of the strain, the similarity of the strain and Bacillus proteolicus (Bacillus proteoliticus) reaches 100 percent. The strain is identified as Bacillus proteolicus (Bacillus proteoliticus). The sequence determination result of the 16s rDNA gene of the strain is AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACTGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGGTGAAGTCGTAACAAGGT (SEQ No. 1).
The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 9 and 21 months, and the address is as follows: the number of the strain preservation is CGMCC NO.16525 at Xilu No.1 of Beijing, Chaoyang, North Chen, China academy of sciences and microbiology.
Example 2: nematicidal effect of Bacillus proteolicus (Bacillus proteoliticus) Bp-196 strain
1. Preparation of fermentation broth of Bacillus proteolicus (Bacillus proteoliticus) Bp-196 Strain
The Bacillus proteolicus Bp-196 strain was inoculated into a fermentation medium and cultured at 30 ℃ for 3 days at a rotation speed of 220rpm to obtain a fermentation broth of the Bacillus proteolicus Bp-196 strain. Wherein, the fermentation medium comprises: 5g/L yeast powder, 10g/L peptone and 10g/L sodium chloride, and the volume is fixed to 1L by using distilled water.
2. Preparation of nematodes for testing
The root-knot nematodes were collected from the greenhouse of vegetable and flower institute of agricultural sciences, China. Taking out root systems of the peppers with the root-knot nematode diseases, slightly washing the roots with water, carefully taking off egg masses from the surfaces of the root systems, sterilizing the egg masses in 1% sodium hypochlorite for 3min, washing the eggs with sterile water for 3 times, putting the eggs in a culture dish containing a small amount of sterile water, culturing the eggs in a thermostat at 25 ℃, collecting hatched second-instar larvae of the root-knot nematode for 24-48 h, and suspending the larvae with the sterile water for experimental study.
3. Test method
And (3) respectively treating the second-instar larvae of the meloidogyne with fermentation liquor of the bacillus proteolicus Bp-196 strain and 10-time and 100-time diluent thereof, and determining the killing effect of the bacillus proteolicus Bp-196 fermentation liquor on the meloidogyne.
Taking 12 sterile 24-hole cell culture plates, dividing the 12 cell culture plates into four groups, repeating each group of experiments for 3 times, adding fermentation liquor, diluted 10-time fermentation liquor, diluted 100-time fermentation liquor and sterile water into holes of each group of cell culture plates respectively for 1mL, wherein the fermentation liquor group is added, the diluted 10-time fermentation liquor group is added, and the diluted 100-time fermentation liquor group is added as a test group, the sterile water group is added as a control group, then 100 mu L of nematode suspension (about 100 nematodes) is added into the four groups of cell culture plates respectively, the cells are cultured for 24 hours at room temperature, the death condition of the root-knot nematodes is observed, and the corrected mortality is calculated, namely the nematode killing effect is obtained.
Corrected mortality (%) - (average mortality of test group root knot nematodes-average mortality of control group root knot nematodes)/(average mortality of control group root knot nematodes) 100%
Table 1 shows the results of experiments on the two-stage meloidogyne larvae killing of fermentation broth of Bacillus proteus Bp-196 strains with different concentrations.
TABLE 1 test results of two-stage larvae killing of Meloidogyne incognita with fermentation broth of Bacillus proteolicus Bp-196 strain at different concentrations
| Mortality (%) | Fermentation liquor group | 10-fold dilution fermentation liquor group | 100 times diluted fermentation liquor group | Sterile water set |
| Repeat test 1 | 100 | 94.5 | 10.2 | 1.2 |
| Repeat test 2 | 100 | 90.7 | 9.8 | 1.1 |
| Repeat test 3 | 100 | 92.2 | 9.6 | 1.1 |
| Average mortality | 100 | 92.5 | 9.9 | 1.1 |
| Correcting mortality | 100 | 92.5 | 9.9 | — |
The experiments show that the effect of the bacillus proteolicus Bp-196 fermentation liquor with different dilution times on the nematodes is different. When the fermentation liquor is not diluted, the insecticidal effect on the root-knot nematodes is 100 percent; when the fermentation liquor is diluted by 10 times, the insecticidal effect on the root-knot nematodes still reaches 92.5 percent; when the fermentation liquor is diluted by 100 times, the insecticidal effect on the root-knot nematodes is very low, only 9.9 percent, and the difference with a control group is very small, which shows that the fermentation liquor of the bacillus proteolicus Bp-196 strain has good effect of preventing and treating the root-knot nematodes under high concentration.
Example 3: nematicidal effect of Bacillus proteolicus (Bacillus proteoliticus) Bp-196 strain
1. Preparation of fermentation broth of Bacillus proteolicus (Bacillus proteoliticus) Bp-196 Strain
The Bacillus proteolicus Bp-196 strain was inoculated into a fermentation medium and cultured at 30 ℃ for 3 days at a rotation speed of 220rpm to obtain a fermentation broth of the Bacillus proteolicus Bp-196 strain. Wherein, the fermentation medium comprises: 5g/L yeast powder, 10g/L peptone and 10g/L sodium chloride, and the volume is fixed to 1L by using distilled water.
2. Preparation of nematodes for testing
The root-knot nematodes were collected from the greenhouse of vegetable and flower institute of agricultural sciences, China. Taking out root systems of the peppers with the root-knot nematode diseases, slightly washing the roots with water, carefully taking off egg masses from the surfaces of the root systems, sterilizing the egg masses in 1% sodium hypochlorite for 3min, washing the eggs with sterile water for 3 times, putting the eggs in a culture dish containing a small amount of sterile water, culturing the eggs in a thermostat at 25 ℃, collecting hatched second-instar larvae of the root-knot nematode for 24-48 h, and suspending the larvae with the sterile water for experimental study.
3. Test method
180 cucumber seedlings are taken for testing, the 180 cucumber seedlings are divided into two groups, each group of tests is repeated for 3 times, fermentation liquor of bacillus poproteolyticus Bp-196 strain and sterile water are uniformly irrigated in soil of each group of cucumber seedlings respectively for 15mL, wherein the cucumber seedling group irrigated with the fermentation liquor of the bacillus poproteolyticus Bp-196 is taken as a test group, and the cucumber seedling group irrigated with the sterile water is taken as a control group. After 1 day, inoculating 500 juveniles of the second instar larvae of the root-knot nematodes to the cucumber seedlings of the test group and the control group, normally culturing at room temperature, and detecting the number of the root knots on the cucumber seedlings of the test group and the control group after 5 weeks, thereby calculating the effect of the fermentation liquid of the bacillus proteolicus Bp-196 strain on preventing the root-knot nematodes.
The prevention and treatment effect calculation formula is as follows:
control effect (%) - (1-test group average root knot number/control group average root knot number) × 100%
Table 2 shows the results of the experiments on the prevention of root-knot nematode by the fermentation broth of Bacillus proteolicus Bp-196.
TABLE 2 test results of the prevention of Meloidogyne by fermentation broth of Bacillus proteolicus Bp-196 strain
| Root knot number | Fermentation liquor group | Sterile water set |
| Repeat test 1 | 18.9 | 135.7 |
| Repeat test 2 | 17.1 | 124.5 |
| Repeat test 3 | 16.2 | 127.1 |
| Mean root number of knots | 17.4 | 129.1 |
According to the control effect calculation formula, the control effect of the fermentation liquor of the bacillus proteolicus Bp-196 strain on the root-knot nematode reaches 86.5 percent.
The experiment shows that the number of root knots on cucumber plants treated by the fermentation liquor of the bacillus proteolicus Bp-196 strain is obviously reduced. In the control group, the number of root knots in cucumber averaged 129.1 per plant, whereas the number of root knots in cucumber treated with the fermentation broth of Bacillus proteolicus Bp-196 strain averaged 17.4 per plant. The calculation results show that the control effect of the fermentation liquid of the bacillus proteolyticus Bp-196 strain on the root-knot nematodes reaches 86.5 percent, which indicates that the bacillus proteolyticus Bp-196 strain can effectively control the cucumber root-knot nematodes. Has important value in preventing and controlling root knot nematode disease in agricultural production.
The foregoing description of the implementations of the disclosure has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the disclosure. The embodiments were chosen and described in order to explain the principles of the disclosure and its practical application to enable one skilled in the art to utilize the disclosure in various embodiments and with various modifications as are suited to the particular use contemplated.
Sequence listing
<110> vegetable and flower institute of Chinese academy of agricultural sciences
<120> bacillus proteoliposophilus and application thereof in prevention and treatment of root-knot nematodes
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Claims (4)
1. Bacillus proteolicus (B.proteolicus)Bacillus proteolyticus) The Bp-196 strain has the preservation unit of China general microbiological culture Collection center (CGMCC) and the preservation number of CGMCC No. 16525.
2. The method for fermenting the bacillus proteolicus Bp-196 strain of claim 1, wherein the method comprises:
the Bacillus proteolicus Bp-196 strain was inoculated into a fermentation medium and cultured at 30 ℃ for 3 days at a rotation speed of 220 rpm.
3. The method for fermenting the bacillus proteolicus Bp-196 strain of claim 2, wherein the fermentation medium comprises: 5g/L yeast powder, 10g/L peptone and 10g/L sodium chloride, and the volume is fixed to 1L by using distilled water.
4. Use of the bacillus proteolicus Bp-196 strain of any one of claims 1 to 3 in the manufacture of a medicament for the control of root knot nematode disease.
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| KR20180091594A (en) * | 2017-02-07 | 2018-08-16 | 안동대학교 산학협력단 | Bacillus subtilis strain YGB70 and microbial agent for prevention of ginseng root rot pathogens comprising the same |
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| CN106754493B (en) * | 2016-12-09 | 2019-12-31 | 中国农业科学院蔬菜花卉研究所 | Bacillus subtilis strain and application thereof |
| GB201712908D0 (en) * | 2017-08-11 | 2017-09-27 | Univ Exeter | Biopesticides |
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| CN106479938A (en) * | 2016-12-09 | 2017-03-08 | 中国农业科学院蔬菜花卉研究所 | A kind of Brevibacillus brevis bacterial strain and its application |
| KR20180091594A (en) * | 2017-02-07 | 2018-08-16 | 안동대학교 산학협력단 | Bacillus subtilis strain YGB70 and microbial agent for prevention of ginseng root rot pathogens comprising the same |
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| Systemic nematicidal activity and biocontrol efficacy of bacillus firmus against the root-knot nematode Meloidogyne incognita.;Jing Xiong等;《World J Microbiol Biotechnol》;20150430;第31卷(第4期);第661-667页 * |
| 植物寄生线虫生防细菌的研究进展;黄金玲等;《广西农业生物科学》;20080915;第27卷(第03期);第288-293页 * |
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