CN115725457A - Bacillus amyloliquefaciens for soil-borne disease control and soil remediation and application thereof - Google Patents
Bacillus amyloliquefaciens for soil-borne disease control and soil remediation and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of probiotic screening and application, and particularly provides a novel bacillus amyloliquefaciens strain（Bacillus amyloliquefaciens) And provides the application of the compound in plant disease control and soil remediation. The bacillus amyloliquefaciens is selected from rhizosphere soil of tomatoes in a greenhouse for planting vegetables in drinking maltowns in shou-guan city of Shandong province, and the preservation number is CCTCC NO: m20211421 has remarkable control effect on soil-borne diseases of crops such as cucumbers, tomatoes and the like, and can effectively improve soil acidification, degrade pesticide residues in soil, improve crop yield and ensure crop food safety.
Description
Technical Field
The invention relates to the technical field of functional microorganism screening, in particular to bacillus amyloliquefaciens for preventing and treating soil-borne diseases and repairing soil and application thereof.
Background
Under the current situation, the problem of agricultural non-point source pollution is taken as an agricultural negative product, which is increasingly prominent for the sustainable development of agriculture and is highly valued by the whole society. A great deal of research shows that excessive use of chemical fertilizers is one of the main causes of the agricultural non-point source pollution problem at the present stage. The use intensity of the chemical fertilizer in the current Chinese agricultural production obviously exceeds that of internationally recognized chemical fertilizer application safety on line, the use amount of the chemical fertilizer is strictly controlled and reduced, and the work of preventing and controlling the non-point source pollution of the chemical fertilizer is imperative.
The agricultural microbial agent is a live bacterial preparation prepared by processing target microorganisms (effective bacteria) after industrial production and propagation. It has the functions of improving soil, restoring soil fertility, maintaining rhizosphere microflora balance, degrading toxic and harmful matter, etc; the microbial fertilizer is applied to agricultural production, and can increase the supply of plant nutrients or promote the growth of plants and improve the quality of agricultural products and the agricultural ecological environment through the life activities of microorganisms contained in the microbial fertilizer. Compared with the traditional fertilizer, the microbial fertilizer has the advantages of protecting ecology and utilizing agricultural waste resources, maintaining soil health, improving the utilization rate of the fertilizer and the quality of agricultural products and the like.
The agricultural microbial agent comprises bioremediation bacteria, mycorrhizal fungi, growth promoting bacteria, a fertilizer decomposition agent, a photosynthetic bacteria agent, azotobacter, rhizobium, a silicate microbial agent and the like, and the granular, powdery and liquid microbial agents are types of the preparation. Microbial bactericides are classified into bacterial bactericides, fungal bactericides and the like.
The most widely studied and applied fungal fungicides are the trichoderma, and secondly the gliocladium species. In addition, some nematophagous fungi can be used for preventing and controlling soybean cyst nematode and root-knot nematode diseases, such as paecilomyces lilacinus is used for preventing and controlling banana nematodosis and potato nematodosis, and the yield is improved.
The bacteria used for biocontrol are mainly bacillus, and mainly include bacillus subtilis (b.subtilis), bacillus amyloliquefaciens (b.amyloliquefaciens), bacillus polymyxa (b.polymyxa), bacillus cereus (b.cereus), bacillus licheniformis (b.licheniformis), bacillus megaterium (b.megaterium), bacillus pumilus (b.pumilis), and the like. Because the bacillus has the capability of inhibiting plant diseases, is a non-pathogenic bacterium widely existing in nature, has no harm to people and livestock, and does not pollute the environment, the bacillus is concerned. For example, the chenchenchenchenfang provides a bacillus megaterium strain HB, which has strong stress resistance, can tolerate high-salt and high-temperature environments, has strong colonization capacity, can be quickly propagated into dominant floras when applied to soil, decomposes organic phosphorus in the soil, converts ineffective phosphorus into effective phosphorus, and effectively improves the absorption efficiency of the phosphorus in the soil. The yield of the polypeptide agricultural microbial agent prepared by the bacillus megatherium is 7.2-12.5% in greenhouse strawberries, the Vc content is improved by 6.2-7.9%, the tomato yield is increased by 7.3-11.2%, and the Vc content is improved by 6.9-9.1%. Yan Longxiang and the like provide a bacillus subtilis LVLE14 with disease inhibiting activity, and the bacillus subtilis and fermentation liquor and fertilizer prepared by the same have effective biocontrol effect and excellent control effect on powdery mildew and gray mold of cucumbers.
The development of biopesticides and agricultural microbial agents with biocontrol property has become an internationalization trend, the biopesticides in China are far from developed countries in terms of variety, dosage form, quality and quantity, a plurality of strains only aim at a certain type of pathogenic bacteria, the bacteriostatic spectrum is very narrow, the variety is single, and the ever-increasing agricultural microbial agent market cannot be met, so that the technical barrier is broken through by screening multifunctional and efficient agricultural microbial strains.
Disclosure of Invention
The invention provides a novel Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and application thereof in plant disease control and soil remediation. The bacillus amyloliquefaciens is selected from rhizosphere soil of tomatoes in a greenhouse for planting vegetables in drinking maltown in shou-guan city, shandong province, has obvious control effect on soil-borne diseases of crops such as cucumbers and tomatoes, and can effectively improve soil acidification, degrade pesticide residues in the soil, improve crop yield and ensure crop food safety.
The invention provides a Bacillus amyloliquefaciens, which is named as Bacillus amyloliquefaciens 14-21 (Bacillus amyloliquefaciens 14-21) and is preserved in China center for type culture collection, china university of Wuhan, in 2021, 11 months and 15 days, and the preservation number is CCTCC NO: M20211421.
The invention provides an application of the bacillus amyloliquefaciens in soil-borne disease control.
The soil-borne disease comprises any one of root knot nematode disease, gray mold, epidemic disease, powdery mildew, root rot, blight, sclerotinia sclerotiorum, gummy stem blight, seedling damping-off disease, rhizoctonia rot and brown streak disease.
The invention provides an application of the bacillus amyloliquefaciens in soil remediation.
The invention also provides a microbial preparation which comprises the bacillus amyloliquefaciens 14-21.
The microbial preparation further comprises any one or more of pseudomonas, agrobacterium, azotobacter, rhizobium, aspergillus and rhizopus.
The viable bacteria amount of Bacillus amyloliquefaciens 14-2 in the microbial preparation is at least 10 8 CFU/g。
The invention also provides application of the microbial preparation in preventing and treating soil-borne diseases.
The invention also provides application of the microbial preparation in soil remediation.
The screened bacillus amyloliquefaciens 14-21 has strong inhibition effect on fusarium oxysporum, and the width of an inhibition zone exceeds 24mm; meanwhile, the strain has obvious contact killing effect on nematodes.
The bacillus amyloliquefaciens 14-21 can effectively prevent and control the root knot nematode diseases, the prevention and control effects on the root knot nematode diseases of cucumbers, melons and loofah are respectively as high as 71.1%, 68.7% and 64.5%, meanwhile, the growth vigor and the yield of crops can be effectively improved, and unexpected technical effects are obtained.
The bacillus amyloliquefaciens 14-21 has remarkable control effect on tomato root rot, the control efficiency reaches up to 83.6 percent, and unexpected technical effect is achieved.
The bacillus amyloliquefaciens 14-21 can effectively degrade pesticide residues in soil while preventing and treating cucumber nematode diseases and tomato root rot, the degradation rates of pesticides acetamiprid, imidacloprid and cyhalothrin respectively reach 92.8%, 75.8% and 85.1%, and the degradation rates of fungicides triazolone and chlorothalonil respectively reach 95.4% and 95.1%, so that unexpected technical effects are achieved.
The bacillus amyloliquefaciens 14-21 also has a better improvement effect on acidified soil.
The bacillus amyloliquefaciens 14-21 can be independently used as a biocontrol microbial inoculum, a biological fertilizer, a soil conditioner and the like to be widely applied to the field of agricultural production, can be combined with any one or more of other bacillus, pseudomonas, agrobacterium tumefaciens, azotobacter, rhizobium, aspergillus and rhizopus to be used for preventing and treating common soil-borne crop diseases, improving the soil environment and improving the crop quality, and has wide application prospect.
Drawings
FIG. 1 is a peak protein spectrum of 14-21 strain.
FIG. 2 is the gene fingerprint spectrum of 14-21 strain.
FIG. 3 is a picture of the growth vigor and root system of a cucumber pot.
Detailed Description
The invention is further illustrated by the following specific examples. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.
The equipment and reagents used in the present invention may be selected from any commercially available ones.
EXAMPLE 1 isolation and screening of the strains
1. Sample source:
greenhouse tomato rhizosphere soil is planted in drinking Mazhen vegetables in shou city of Shandong province.
2. Strain separation:
by gradient dilution coating, 10g of the soil sample is weighed into a 250mL Erlenmeyer flask containing sterile glass beads and 90mL sterile water and shaken well. Standing, and performing gradient dilution on an ultraclean workbench to obtain 10 -4 、10 -5 、10 -6 Three dilutions, 100 μ L each, were spread on nutrient agar plates and repeated 3 times. Culturing in 37 deg.C incubator for 24 hr, observing growth condition, classifying according to colony size, shape and color, selecting single colony, purifying and culturing to obtain 8 strains of bacteria with numbers of PS1, PS2, PS3, 8230, PS8, and preserving 8 strains of bacteria in liquid glycerol.
3. Strain screening:
screening dominant biocontrol strains through a plate confronting antibacterial test, respectively inoculating the 8 separated and purified strains into a nutrient broth culture medium, and culturing at 37 ℃ and 220r/min for 14h to prepare a test bacterial liquid for later use.
The pathogenic bacterium fusarium oxysporum (provided by the plant protection institute of the agricultural academy of Shandong province) is cultured on a PDA culture medium for 5 days for later use.
Inoculating fusarium oxysporum cakes in the center of a nutrient agar culture medium, respectively inoculating the test bacteria liquid at the positions 2.5cm away from the center of a culture dish on the two sides of the cakes, culturing for 72 hours in an incubator at 30 ℃, and taking out to measure the width of a bacteria inhibiting zone so as to judge the bacteria inhibiting effect. Specific results are shown in table 1.
TABLE 1 inhibitory Effect of different strains on Fusarium oxysporum
| Strain numbering | The width of the bacteriostatic strip is mm |
| PS1 | 16.0±1.0 |
| PS2 | 20.0±0.5 |
| PS3 | 18.0±1.5 |
| PS4 | 21.0±0.5 |
| PS5 | 23.0±1.0 |
| PS6 | 20.0±1.0 |
| PS7 | 24.0±1.0 |
| PS8 | 21.0±0.5 |
As can be seen from the data in Table 1, the strains selected from the soil according to the invention have better PS2, PS6, PS7 and PS8 inhibition effects on Fusarium oxysporum. Wherein, the PS7 strain has the best bacteriostatic effect, and the width of the bacteriostatic band exceeds 24mm. 4. Strain screening:
screening nematode-prevention dominant strains by using a laboratory nematode model: and (3) coating 8 separated and purified strains on LB culture medium plates, inoculating 3 plates to each strain, inoculating L4-stage nematode larvae subjected to synchronous culture to the 8 culture medium plates, inoculating about 15 nematodes to each plate, taking E.coli OP50 as a blank control, culturing all the plates at 16 ℃, observing at the same time every day, and counting the number of dead nematodes until day 12.
Nematode mortality (%) = number of dead nematodes/number of test nematodes × 100%.
TABLE 2 Effect of different strains on the mortality of nematodes
| Strain numbering | Mortality of nematodes |
| PS1 | 45.60％ |
| PS2 | 41.91％ |
| PS3 | 14.17％ |
| PS4 | 29.30％ |
| PS5 | 47.78％ |
| PS6 | 62.86％ |
| PS7 | 65.28％ |
| PS8 | 14.85％ |
| Blank control | 14.10％ |
As can be seen from the results in Table 2, the PS7 strain fermentation broth among the 8 strains screened by the present invention has the best effect on contact killing of nematodes, and the lethality of the nematodes at the end of the culture reaches 65.28%.
The plate confronting bacteriostasis test of the 8 separated strains on fusarium oxysporum pathogenic bacteria and the lethal test on nematodes in a nematode model show that the strain PS7 has the best control effect on soil-borne diseases and nematode diseases, so that the strain is screened to be a strain for dominantly biologically controlling the soil-borne diseases and the nematode diseases, and the applicant names the strain as 14-21 and further identifies the strain.
EXAMPLE 2 identification of strains 14-21
1. And (3) colony morphology identification:
the bacterial colony of the strain on a nutrient agar culture medium is round beige, the diameter of the bacterial colony is 3-5mm, the edge of the bacterial colony is smooth, the bacterial colony is wet and glossy, the middle of the bacterial colony is raised, the thallus is in a shape of a short straight rod, gram-positive bacteria can produce spores. The spore is oval, and the part is round, and the sporangium is not expanded. The cells exist individually, in pairs or in short chains.
2.16 molecular characterization of S rRNA:
and extracting the genome of the strain 14-21 by using the kit. Then, 16S rRNA was amplified using the genome as a template and specific primers 27F and 1492R.
27F：5’-AGAGTTTGATCATGGCTCAG-3’；
1492R：5’-TAGGGTTACCTTACGACTT-3’。
The PCR system comprises: mu.l 27F, 0.7. Mu.l 1492R, 4. Mu.l template DNA, 17.5. Mu.l SuperMiX and 12.1. Mu.l water. The PCR reaction conditions were set as follows: (1) 5min at 94 ℃; (2) pre-denaturation at 94 ℃ for 30s; (3) 30s at 55 ℃; (4) 72 ℃ for 1min; executing the loop of the steps (2) to (4) 35; (5) 72 ℃ for 10min. And (3) carrying out 1% agarose gel electrophoresis detection on the PCR product obtained by amplification, wherein the result shows that the size of the PCR product is about 1500bp and meets the requirement.
The PCR amplification product is sent to a sequencing company for sequencing, and the obtained sequence is subjected to BLAST comparison in an NCBI database, so that the similarity of the sequence and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is highest. Therefore, the 14-21 strain was preliminarily determined to be Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
MALDI-TOF-MS protein mass spectrum identification:
coating a small amount of 14-21 single colonies on a target plate in a thin film mode; adding 1 mu L of lysate in the mass spectrum sample pretreatment kit, and naturally airing at room temperature; adding 1 mu L of matrix solution in the mass spectrum sample pretreatment kit to cover the sample, and naturally airing at room temperature; and putting the sample target into a mass spectrometer for identification. The identification result shows that the 14-21 strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and the protein spectrum peak diagram of the strain is shown in figure 1.
RiboPrinter full-automatic microbial gene fingerprint identification:
the strains 14-21 were identified on-machine according to the full-automatic microbial gene fingerprint identification system operating instructions to obtain their rRNA gene fingerprint maps, as shown in FIG. 2. The similarity between the strain 14-21 and the Bacillus amyloliquefaciens is up to more than 96 percent by comparing with a known standard strain library fingerprint map, so that the strain is identified as the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
In conclusion, the applicant identifies the strains 14-21 by using three molecular biological methods of 16S rRNA identification, MALDI-TOF-MS protein mass spectrum and RiboPrinter full-automatic microbial gene fingerprint identification, and the identification results are consistent. And then, by combining the colony morphological characteristics of the strain 14-21, the applicant determines that the strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and is named as Bacillus amyloliquefaciens 14-21 (Bacillus amyloliquefaciens 14-21).
The applicant has deposited the above Bacillus amyloliquefaciens 14-21 (Bacillus amyloliquefaciens 14-21) in the China center for type culture Collection, CCTCC NO: M20211421, at 11/15/2021.
Example 3 evaluation of nematode-proofing efficacy of Bacillus amyloliquefaciens 14-21 on potted cucumber
1. Preparation of a microbial inoculum sample:
and (3) performing liquid fermentation on the bacillus amyloliquefaciens 14-21 in a 5-ton fermentation tank, stopping fermentation when the microscopic spore rate reaches more than 90%, centrifuging, and performing spray drying to obtain a powder product with the bacterial load of 10 hundred million/g.
2. Collecting and processing disease-free soil:
collecting soil which is used for natural planting in the field and has no root knot nematode, and sieving to ensure uniform fineness; and then the soil sample is sterilized to avoid the influence of pathogenic bacteria or existing nematodes. Meanwhile, the sterilization soil is mixed into a certain amount of sand, and the volume ratio of the sand to the sterilization soil is 1:2 preparing the spare soil.
3. Preparation of worm egg suspension:
taking plant roots with root-knot nematode attack, adding water by using a wall breaking machine to crush the plant roots to obtain a crude suspension, counting the number of egg masses of 1ml of the crude suspension under a microscope, repeating for more than 5 times, calculating the egg content of the crude suspension for subsequent inoculation, wherein the inoculation amount is 2000-4000 egg masses per pot.
4. Seedling culture:
the experimental crop is a root-knot nematode susceptible variety of cucumber, a seedling culture substrate is filled into a seedling culture basin with 50 holes, seeds of a root-knot nematode susceptible host are sown into the seedling culture substrate, 1 seed is planted in each seedling culture hole, and the seedling can be transplanted when the seedling grows to 3-5 true leaves.
5. Inoculation:
1/2 to 2/3 of sterile spare soil is added at the bottom of an inoculation flowerpot, then a quantitative egg suspension liquid is added to ensure that the inoculation amount is more than 2000 eggs, then a layer of spare soil is paved, then a microbial inoculum (the viable count is 10 hundred million/g) is added according to 5 per mill of the weight of the soil, the microbial inoculum is mixed with the soil and spread for a layer, then a layer of spare soil is paved, and finally spare seedlings are transplanted. And (4) watering a proper amount of planting water for the potted plant after the inoculation and the transplantation are finished.
6. Culturing:
culturing the treated potted plant in a greenhouse at 20-25 deg.C, watering once for about 3 days, and properly adjusting the soil humidity according to the soil humidity, wherein the soil humidity is not more than 50%. After culturing for 35-45d, the pot is buckled, and the root is taken out, and the statistical effect is investigated.
7. Root knot index and prevention effect investigation and calculation:
TABLE 3 root knot grading Standard
| |
Root knot is not found in the |
| Level | |
| 1 | By carefully identifying a few root knots |
| Stage 2 | The main root has no root knot, and the fibrous root has a few root knots which can be clearly distinguished |
| Grade 3 | The main root has no root knot, and a slightly larger root knot can be found on the fibrous root |
| Grade 4 | The main root has no root knot, and the fibrous root is mainly provided with larger |
| Grade | |
| 5 | 50% of the roots are infected, and the main roots are infected in a very small amount |
| Grade 6 | Root knots found on the main root |
| Stage 7 | The majority of the main root is infected by the root knot |
| Stage 8 | All major roots are infected/few are not infected |
| Grade 9 | All roots can be severely infested/plants usually die |
| |
All roots were severely infested and rootless |
The root knot index and relative control were calculated as follows:
disease index = [ Σ (disease grade value × number of diseased leaves at that grade)/(investigation total number of leaves × highest disease grade value) ] × 100.
The calculation formula of the prevention and treatment efficiency is = [1- (disease index of treatment group/disease index of control group) ] × 100%.
The experimental results are as follows: the growth vigor and root system conditions of the cucumber potted plant test plant are shown in figure 3, the root knot index and the control effect are calculated according to statistical experimental data, the bacillus amyloliquefaciens 14-21 treatment group obviously reduces the occurrence of root knot nematode diseases of potted cucumber, the control effect on the root knot nematode diseases is up to 87.7 percent, and meanwhile, the growth promotion effect on the root system and the overground part growth vigor of the seedling stage of the cucumber can be well realized.
Example 4 evaluation of nematode-proofing Effect of Bacillus amyloliquefaciens 14-21 on cucumber planted in field
1. The experimental site:
zhonghua county cucumber greenhouse of Qingdao, riexi, institute.
2. Cucumber planting experiment:
the area of the experimental plot is 10m multiplied by 10m, each experimental plot has 10 ridges of cucumbers, about 500 +/-10 plants, and 15 experimental plots are totally arranged, and each treatment group is subjected to 3 times of repetition through random block groups.
(1) Blank control group: normal field management, no microbial inoculum is used, and clear water is applied;
(2) 14-21 microbial inoculum treatment group: the 14-21 microbial inoculum (the amount of viable bacteria is 10 hundred million/g) is applied with water after the cucumber seedlings are transplanted according to the dosage of 3-10 kg/mu, and is applied once after 7 days. Wherein:
treatment group 1: the dosage of 14-21 microbial inoculum is 3 kg/mu;
treatment group 2: the dosage of 14-21 microbial inoculum is 5 kg/mu;
treatment group 3: the dosage of the 14-21 microbial inoculum is 8 kg/mu;
treatment group 4: the dosage of the 14-21 microbial inoculum is 10 kg/mu.
The cucumber is investigated every 1 month after being transplanted and planted, and the investigation is carried out for three times. During each investigation, 10 cucumbers are dug in each cell, the height of each cucumber is measured, the weight of each cucumber is weighed, the number of root knot stages is counted and recorded, and the average weight of each cucumber, the average height of each cucumber and the root knot prevention effect are calculated; in the picking period, sampling is carried out in different batches to count the weight of a single fruit, and the yield of a cell is calculated, and the result is shown in a table 4.
TABLE 4 prevention and treatment effects of Bacillus amyloliquefaciens 14-21 on cucumber root-knot nematode disease
| Experiment grouping | Plant height (cm) | Yield (kg) | Increase of yield | Root knot index | Relative prevention and cure effect |
| Blank control group | 154.65 | 93.8 | - | 67.5 | - |
| |
199.32 | 98.5 | 5.0％ | 38.6 | 42.8％ |
| Treatment group 2 | 212.56 | 108.2 | 15.4％ | 35.5 | 47.4％ |
| Treatment group 3 | 243.1 | 110.6 | 17.9％ | 30.8 | 54.4％ |
| Treatment group 4 | 255.2 | 115.8 | 23.5％ | 19.5 | 71.1％ |
From the experimental results in table 4, it can be known that the growth vigor of cucumber plants can be remarkably improved and the plant height and plant weight can be increased by applying the bacillus amyloliquefaciens 14-21 microbial inoculum in the seedling stage of the cucumber. Compared with a control group, the cucumber yield of the treatment group is improved by 5.0-23.5%, and the yield increase effect is obvious; meanwhile, the occurrence of root-knot nematode diseases can be effectively reduced, the disease index is reduced, the highest prevention effect on cucumber root-knot nematodes reaches 71.1%, and the prevention and treatment effect is good.
In addition, nematode control experiment results carried out on other cucurbitaceae crops such as melons, loofah and the like show that the control effects of the bacillus amyloliquefaciens 14-21 on root-knot nematodes of melons and loofah are also very remarkable, and the control efficiencies reach 68.7% and 64.5% respectively.
The bacillus amyloliquefaciens 14-21 can effectively improve the growth vigor and the yield of crops and simultaneously has obvious control effect on root-knot nematode diseases of cucurbitaceae crops such as cucumbers, melons, luffa and the like.
Example 5 evaluation of control Effect of Bacillus amyloliquefaciens 14-21 on tomato root rot
1. Test site:
shou guang city for drinking from horse town.
2. And (3) experimental design:
the test was carried out with 4 treatments in total, each treatment area being 100m 2 3 replicates for a total of 12 test cells. The blank control is conventional fertilization without using a microbial inoculum.
Treatment group 1: 0.5kg of 14-21 microbial inoculum (the amount of live bacteria is 10 hundred million/g) is applied to the tomato rhizosphere along with water drop irrigation;
treatment group 2: 1.0kg of 14-21 microbial inoculum (the amount of live bacteria is 10 hundred million/g) is applied to the tomato rhizosphere along with water drop irrigation;
treatment group 3: 1.5kg of 14-21 microbial inoculum (the amount of live bacteria is 10 hundred million/g) is applied to the tomato rhizosphere along with water drop irrigation.
In the tomato harvesting period, the number of the plants with the root rot of each group is respectively counted, the disease incidence rate and the control efficiency of bacillus amyloliquefaciens 14-21 on the root rot of the tomatoes are calculated, and specific results are shown in table 5.
Incidence (%) = number of diseased plants/total number of plants × 100%.
Control efficiency (%) = (blank control group morbidity-treatment group morbidity)/blank control group morbidity × 100%.
TABLE 5 prevention and treatment effects of Bacillus amyloliquefaciens 14-21 on tomato root rot
| Experimental groups | Incidence of disease | Efficiency of prevention and cure |
| Blank control group | 25.6％ | - |
| |
12.2％ | 52.3％ |
| Treatment group 2 | 6.4％ | 75.0％ |
| Treatment group 3 | 4.2％ | 83.6％ |
As can be seen from the data in Table 5, the tomato root rot disease incidence rate of each treatment group applied with the Bacillus amyloliquefaciens 14-21 microbial inoculum is only 4.2% -12.2%, which is far lower than that of the control group. Therefore, the bacillus amyloliquefaciens 14-21 provided by the invention has remarkable control effect on the tomato root rot, the control efficiency reaches 83.6 percent at most, and unexpected technical effect is achieved.
In addition to root knot nematode disease and root rot, the bacillus amyloliquefaciens 14-21 can also effectively prevent and treat the degradation effect experiment of the bacillus amyloliquefaciens 14-21 on soil pesticide residues in the embodiment 6
1. Test site:
shouguanluo town.
2. And (3) experimental design:
the test was carried out with 6 treatments in total, each treatment area being 50m 2 3 repetitions for a total of 18 cells. Aiming at 7 common herbicides, bactericides and insecticides in the market, 5 chemical pesticides of acetamiprid, imidacloprid, cyfluthrin, triazolone and chlorothalonil which have higher pesticide residue and great harm to human bodies are screened for field test. Pouring pesticide and flushing 14-21 bacteria (the amount of live bacteria is 10 hundred million/g) of bacillus amyloliquefaciens on the day of cucumber transplanting, flushing the bacteria again on the 10 th day, wherein the using amount of the bacteria is 10 kg/mu (calculated by 10 hundred million/g), respectively taking crop rhizosphere soil samples on the 15 th day, the 30 th day and the 60 th day after transplanting, detecting chlorothalonil and cyfluthrin by using a gas chromatography-mass spectrometer, detecting the contents of triazolone, acetamiprid and imidacloprid by using a liquid chromatography-mass spectrometer, and calculating the degradation rate. The control group was flushed with clear water. The specific results are shown in Table 6.
3. Test results and analysis:
TABLE 6 degradation rates of Bacillus amyloliquefaciens 14-21 on different pesticide residues
| Time | Acetamiprid | Imidacloprid | Cyhalothrin | Triazolones | Chlorothalonil |
| 15d | 64.3％ | 45.2％ | 28.3％ | 50.2％ | 44.9％ |
| 30d | 78.5％ | 68.6％ | 42.6％ | 79.2％ | 86.4％ |
| 60d | 92.8％ | 75.8％ | 85.1％ | 95.4％ | 95.1％ |
As can be seen from the experimental data in Table 6, the Bacillus amyloliquefaciens 14-21 microbial inoculum can obviously reduce the pesticide residue in the soil; after the pesticide composition is applied for 60 days, the degradation rates of acetamiprid, imidacloprid and cyhalothrin insecticides in the soil reach 92.8%, 75.8% and 85.1% respectively, and the degradation rates of triadimefon and chlorothalonil fungicides reach 95.4% and 95.1% respectively, so that unexpected technical effects are achieved.
In conclusion, the bacillus amyloliquefaciens 14-21 has a strong degradation effect on common insecticides and bactericides in crop planting, can effectively reduce pesticide residues in soil, guarantees the soil safety, obviously reduces crop pesticide residues and guarantees the crop food safety.
Example 7 experiment of the repairing and improving effects of Bacillus amyloliquefaciens 14-21 on soil acidification
1. Test site:
the peace degree is ren Mei Zhen.
2. And (3) experimental design:
the test was carried out with 4 treatments in total, each treatment area being 100m 2 3 replicates for a total of 12 test cells. The blank control is conventional fertilization without using a microbial inoculum. The planted crops are two crops of tomatoes, three years of tests are continuously carried out, soil samples are taken at the same period every year, and the pH values of the soils of different test groups are detected.
Treatment group 1: 0.5kg of 14-21 microbial agents (the amount of live bacteria is 10 hundred million/g) are applied to the rhizosphere of the tomato along with water drop irrigation;
treatment group 2: 1.0kg of 14-21 microbial inoculum (the amount of live bacteria is 10 hundred million/g) is applied to the tomato rhizosphere along with water drop irrigation;
treatment group 3: 1.5kg of 14-21 microbial inoculum (the amount of live bacteria is 10 hundred million/g) is applied to the tomato rhizosphere along with water drop irrigation.
3. The restoration effect of the acidified soil is as follows:
TABLE 7 improving effect of Bacillus amyloliquefaciens 14-21 on soil acidification
| Sample numbering | Blank control | Treatment group | 1 | Treatment group 2 | Treatment group 3 |
| |
5.86 | 5.95 | 6.02 | 6.09 | |
| Year 2 | 5.80 | 6.01 | 6.10 | 6.18 | |
| Year 3 | 5.76 | 6.09 | 6.16 | 6.22 |
As can be seen from the results in Table 7, the continuous application of Bacillus amyloliquefaciens 14-21 for 3 years has a good effect of improving the soil acidification, and particularly the acidification remediation effect of the experimental group 3 is most obvious.
In conclusion, the bacillus amyloliquefaciens 14-21 can be used for preventing and treating root-knot nematode diseases easy to occur to cucurbitaceous crops such as cucumbers, melons, towel gourds and the like and common root rot diseases of tomatoes, and can also obviously promote the growth of the crops and improve the quality and the yield of the crops. The strain can effectively degrade chemical bactericides and pesticides which are remained in soil, has an obvious repairing effect on the acidification condition of tomato planting soil, and can be applied to common facility vegetable planting.
The bacillus amyloliquefaciens 14-21 provided by the invention can be independently used as a biocontrol microbial inoculum, a biological fertilizer, a soil conditioner and the like to be widely applied to the field of agricultural production, can be combined with any one or more of other bacillus, pseudomonas, agrobacterium tumefaciens, azotobacter, rhizobium, aspergillus and rhizopus to be used for preventing and treating common soil-borne crop diseases, improving the soil environment, improving the crop quality, ensuring the food safety and having wide application prospect.
Claims (9)
1. The bacillus amyloliquefaciens is characterized in that the preservation number of the bacillus amyloliquefaciens is CCTCC NO: m20211421.
2. Use of the bacillus amyloliquefaciens of claim 1 for controlling soil-borne diseases.
3. The use of claim 2, wherein the soil-borne disease is any one of root knot nematode disease, gray mold, epidemic disease, powdery mildew, root rot, blight, sclerotinia, gummy stem blight, seedling damping off, damping off or brown streak disease.
4. Use of the bacillus amyloliquefaciens of claim 1 in soil remediation.
5. A microbial preparation comprising the Bacillus amyloliquefaciens of claim 1.
6. The microbial preparation of claim 5, further comprising any one or more of pseudomonas, agrobacterium, azotobacter, rhizobium, aspergillus, and rhizopus in combination.
7. A microbial preparation according to claim 5 or 6 wherein the viable count of Bacillus amyloliquefaciens in the microbial preparation is at least 10 8 CFU/g。
8. Use of a microbial preparation according to any one of claims 5 to 7 for the control of soil-borne diseases.
9. Use of a microbial preparation according to any one of claims 5 to 7 for soil remediation.
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