CN115725457B - 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 screening and application of probiotics, in particular to a novel bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and application thereof in plant disease control and soil remediation. The bacillus amyloliquefaciens sieve is selected from tomato rhizosphere soil in a vegetable planting greenhouse in the Shandong Shouguang city, 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, 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 soil-borne disease control and soil remediation and application thereof.
Background
Under the current situation, the problem of agricultural non-point source pollution is taken as a negative product of agriculture, the growing significance of the pollution is on the growing variety of sustainable development of agriculture, and the pollution is highly valued by the whole society. A great deal of research shows that excessive use of chemical fertilizers is one of the main reasons for the problem of agricultural non-point source pollution at the present stage.
The agricultural microbial agent is a viable bacteria preparation prepared by processing target microorganisms (effective bacteria) after industrial production and propagation. It has the functions of directly or indirectly improving soil, recovering soil fertility, maintaining balance of rhizosphere microorganism systems, degrading toxic and harmful substances and the like; is applied to agricultural production, and can increase the supply of plant nutrients or promote plant growth, improve the quality of agricultural products and the agroecological environment through the vital activities of microorganisms contained in the microbial biomass. Compared with the traditional fertilizer, the microbial fertilizer has the advantages of protecting ecology, utilizing agricultural waste resources, maintaining soil health, improving fertilizer utilization rate, improving agricultural product quality and the like.
Agricultural microbial agents are of a large variety and include bioremediation bacteria, mycorrhizal bacteria, growth promoting bacteria, fertilizer decomposing agents, photosynthetic bacteria agents, azotobacter, rhizobium, silicate microbial agents and the like, and are of the type of preparations in granular, powdery and liquid forms. Microbial bactericides are classified into bacterial bactericides, fungal bactericides and the like.
The most widely studied and used fungal bactericides are trichoderma, and then the mucor-like. In addition, some nematophagous fungi can be used for preventing and controlling diseases of soybean cyst nematodes and root-knot nematodes, such as paecilomyces lilacinus for preventing and controlling radopholus similis and potato gold nematode, and improving the yield of the nematophagous fungi.
Bacteria used for biocontrol mainly include Bacillus subtilis, bacillus amyloliquefaciens, bacillus polymyxa, bacillus cereus, bacillus licheniformis, bacillus megaterium, bacillus pumilus, and Bacillus pumilus. Bacillus has the capability of inhibiting plant diseases, is a non-pathogenic bacterium widely existing in nature, is harmless to people and livestock and does not pollute the environment, so that the bacillus is paid attention to. For example Xing Fangfang and the like provide a bacillus megaterium strain HB which is strong in stress resistance, can resist a high-salt and high-temperature environment, has strong colonization capacity, can be quickly propagated into dominant bacterial groups when being applied into soil, can decompose organic phosphorus in the soil, can convert ineffective phosphorus into effective phosphorus, and can effectively improve the absorption efficiency of phosphorus in the soil. The yield of the polypeptide agricultural microbial agent prepared by using the bacillus megatherium is 7.2-12.5%, 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%. Longxiang and the like provide bacillus subtilis LVLE with disease-inhibiting activity, and the bacillus subtilis and fermentation liquor and fertilizer prepared by using the bacillus subtilis have effective biocontrol effect and particularly good control effect on powdery mildew of cucumber and gray mold of cucumber.
The development of biopesticides and agricultural microbial agents with biocontrol property has become an internationalized trend, the biopesticides in China are far from developed countries in variety, dosage form, quality and quantity, and many strains are only aimed at a certain type of pathogenic bacteria, the bacteriostasis spectrum is narrow, the variety is single, and the growing market of agricultural microbial agents 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 provides application thereof in plant disease control and soil remediation. The bacillus amyloliquefaciens sieve is selected from the rhizosphere soil of the tomato in the vegetable planting greenhouse of the urban area of the Shuguang in Shandong province, has remarkable control effect on soil-borne diseases of crops such as cucumbers and tomatoes, can effectively improve soil acidification, degrade pesticide residues in the soil, improve crop yield and ensure crop food safety.
In one aspect, the invention provides a bacillus amyloliquefaciens, named bacillus amyloliquefaciens 14-21 (Bacillus amyloliquefaciens-21), which is preserved in China center for type culture collection (CCTCC NO: M20211421) of university of Wuhan, china at 11-15 days of 2021.
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, gray mold, epidemic disease, powdery mildew, root rot, fusarium wilt, sclerotinia sclerotiorum, gummy stem blight, seedling stage damping-off, damping-off and brown streak.
The invention provides an application of the bacillus amyloliquefaciens in soil remediation.
The invention also provides a microbial preparation comprising the bacillus amyloliquefaciens 14-21.
The microbial preparation further comprises any one or a combination of a plurality of pseudomonas, agrobacterium, azotobacter, rhizobium, aspergillus and rhizopus.
The viable bacteria amount of the 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 bacillus amyloliquefaciens 14-21 screened by the invention has strong inhibition effect on fusarium oxysporum, and the width of a bacteriostatic zone exceeds 24mm; meanwhile, the strain has remarkable contact killing effect on nematodes.
The bacillus amyloliquefaciens 14-21 can effectively prevent and treat root knot nematode diseases, has the prevention and treatment effects on cucumber, melon and towel gourd root knot nematode diseases as high as 71.1%, 68.7% and 64.5%, can effectively improve the growth vigor and yield of crops, and achieves unexpected technical effects.
The bacillus amyloliquefaciens 14-21 has remarkable control effect on tomato root rot, the highest control efficiency reaches 83.6%, and unexpected technical effects are achieved.
The bacillus amyloliquefaciens 14-21 can effectively degrade pesticide residues in soil while preventing and controlling cucumber nematode diseases and tomato root rot, and has degradation rates of 92.8%, 75.8% and 85.1% for acetamiprid, imidacloprid and cyhalothrin, and 95.4% and 95.1% for triadimefon and chlorothalonil, respectively, thus unexpected technical effects are achieved.
The bacillus amyloliquefaciens 14-21 also has a good effect of improving acidified soil.
The bacillus amyloliquefaciens 14-21 can be independently used as a biological antibacterial agent, 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, azotobacter, rhizobium, aspergillus and rhizopus, is used for preventing and controlling common soil-borne crop diseases, improves the soil environment, improves the crop quality and has wide application prospect.
Drawings
FIG. 1 is a protein mass spectrum peak diagram of 14-21 strain.
FIG. 2 is a graph of gene fingerprint patterns of strains 14-21.
FIG. 3 is a picture of the growth vigor and root system of a potted cucumber.
Detailed Description
The invention is further illustrated below in connection with specific examples. With respect to the specific methods or materials used in the embodiments, those skilled in the art may perform conventional alternatives based on the technical idea of the present invention and are not limited to the specific descriptions of the embodiments of the present invention.
The equipment and reagents selected for use in the present invention may be selected from any of those commercially available.
EXAMPLE 1 isolation and screening of strains
1. Sample source:
greenhouse tomato rhizosphere soil for planting vegetables in the urban area of Shouguang in Shandong province.
2. Isolation of strains:
10g of soil sample was weighed by gradient dilution coating method and put into a 250mL conical flask containing sterile glass beads and 90mL of sterile water, and shaken well. After standing, carrying out gradient dilution on an ultra-clean workbench to obtain 10 -4、10-5、10-6 three gradient dilutions, respectively taking 100 mu L of dilutions to be coated on a nutrient agar plate, and repeating the steps for 3 times. Culturing in a 37 ℃ incubator for 24 hours, observing growth conditions, classifying according to the size, shape, color and other characteristics of bacterial colonies, selecting single bacterial colonies, purifying and culturing to obtain 8 bacterial strains, numbering the 8 bacterial strains as PS1, PS2, PS3, … … and PS8, and preserving the 8 bacterial strains with liquid glycerol.
3. Strain screening:
the dominant biocontrol strain is screened through a plate counter bacteriostasis test, and the separated and purified 8 strains are respectively inoculated into a nutrient broth culture medium for culture for 14 hours at 37 ℃ and 220r/min to prepare a test bacterial liquid for later use.
The pathogenic bacteria Fusarium oxysporum (provided by plant protection institute of agricultural sciences, shandong province) were cultured on PDA medium for 5 days for use.
Inoculating Fusarium oxysporum bacterial cake in the center of nutrient agar medium, inoculating the above test bacterial liquid at the position 2.5cm away from the center of the culture dish on both sides of bacterial cake, culturing in a 30 deg.C incubator for 72 hr, taking out and measuring the width of antibacterial zone, and judging antibacterial effect. The specific results are shown in Table 1.
TABLE 1 inhibition of Fusarium oxysporum by different strains
| Strain numbering | Antibacterial zone width 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 screened from soil according to the invention have better inhibition effect on Fusarium oxysporum by PS2, PS6, PS7 and PS 8. Wherein, the antibacterial effect of the PS7 strain is best, and the antibacterial bandwidth exceeds 24mm. 4. Strain screening:
Screening dominant strains for preventing wireworms by using a laboratory nematode model: and (3) coating 8 strains separated and purified on LB (liquid) culture medium plates, inoculating 3 plates to each strain, inoculating L4-stage nematode larvae subjected to synchronous culture on the 8 culture medium plates, inoculating about 15 nematodes on 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 death number of the nematodes until 12 days.
Nematode mortality (%) = number of dead nematodes/number of test nematodes x 100%.
TABLE 2 lethality effects of different strains on nematodes
As shown in Table 2, the PS7 strain fermentation broth of 8 strains selected by the method has the best effect of killing the insects, and the mortality rate of the insects reaches 65.28% at the end of cultivation.
The comprehensive separated 8 strains have the best effects on soil-borne diseases and nematode disease prevention in the plate-stand bacteriostasis test of fusarium oxysporum and the killing test of nematodes in a nematode model, so that the strains are screened to be the strains for dominant biocontrol of soil-borne diseases and nematode diseases, and the applicant names the strains as 14-21 and further identifies the strains.
Example 2 identification of strains 14-21
1. Colony morphology identification:
the bacterial colony of the strain is round-beige on a nutrient agar culture medium, has the diameter of 3-5mm, smooth and moist edge, luster and middle bulge, and the bacterial colony is short straight rod-shaped and gram positive bacteria, so that spores can be generated. The spore is elliptical, the part of the spore is circular, and the sporangium is not inflated. Cells are present singly, in pairs or in short chains.
2.16 Identification of S rRNA molecules:
The genome of strain 14-21 was extracted using the kit. The genome was then used as a template to amplify its 16S rRNA using specific primers 27F and 1492R.
27F：5’-AGAGTTTGATCATGGCTCAG-3’；
1492R：5’-TAGGGTTACCTTACGACTT-3’。
The PCR system comprises: 0.7. Mu.l 27F, 0.7. Mu.l 14992R, 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) 94 ℃ for 5min; (2) pre-denaturation at 94℃for 30s; (3) 55 ℃ for 30s; (4) 72 ℃ for 1min; performing the loop of steps (2) to (4) 35; (5) at 72℃for 10min. The amplified PCR product is subjected to 1% agarose gel electrophoresis detection, and the result shows that the size of the PCR product is about 1500bp, thereby meeting the requirements.
The PCR amplified product was sent to sequencing company for sequencing, and the obtained sequence was BLAST aligned in NCBI database, which showed the highest similarity to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Thus, strain 14-21 was initially identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
MALDI-TOF-MS protein Mass Spectrometry identification:
A small amount of 14-21 single colonies are coated on a target plate in a film form; 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 (5) placing 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 mass spectrum peak diagram is shown in figure 1.
RiboPrinter full-automatic microbial gene fingerprint identification:
The bacterial strain 14-21 is subjected to on-machine identification according to the operation instruction of the full-automatic microorganism gene fingerprint identification system, and rRNA gene fingerprint images of the bacterial strain are obtained, as shown in figure 2. By comparison with the fingerprint patterns of known standard strain libraries, the similarity between the strains 14-21 and the bacillus amyloliquefaciens is up to more than 96%, so that the strain is identified as the bacillus amyloliquefaciens (Bacillus amyloliquefaciens).
In summary, the applicant uses three molecular biology methods of 16S rRNA identification, MALDI-TOF-MS protein mass spectrum and RiboPrinter full-automatic microorganism gene fingerprint identification to identify the strain 14-21, and the identification results are consistent. In combination with the morphological characteristics of the colonies of the strain 14-21, the applicant determined that the strain was Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), designated Bacillus amyloliquefaciens 14-21 (Bacillus amyloliquefaciens 14-21).
The applicant has preserved the above bacillus amyloliquefaciens 14-21 (Bacillus amyloliquefaciens 14-21) in China center for type culture collection, with the preservation number of CCTCC NO: M20211421, of university of Wuhan, china on the 11 th month 15 of 2021.
EXAMPLE 3 evaluation of the protective effect of Bacillus amyloliquefaciens 14-21 against line insects on potted cucumber
1. Preparation of a microbial inoculum sample:
And (3) fermenting bacillus amyloliquefaciens 14-21 in a 5-ton fermentation tank, stopping fermentation when the microscopic examination spore rate reaches more than 90%, centrifuging, and then spray-drying to obtain a powder product with the bacterial load of 10 hundred million/g.
2. Collecting and treating disease-free soil:
Collecting soil which is used for natural planting in the field and is free of root knot nematodes, and sieving to ensure uniform fineness; and then sterilizing the soil sample to avoid the influence of pathogenic bacteria or existing nematodes. Meanwhile, the sterilizing soil is mixed with certain sand, and the volume and the dosage ratio of the sand to the sterilizing soil are 1:2, preparing the soil for standby.
3. Preparation of insect egg suspension:
Taking plant roots with root knot nematode disease, adding water into a wall breaking machine, crushing to obtain a crude suspension, counting the number of 1ml of crude suspension egg masses under a microscope, repeating for more than 5 times, calculating the egg content of the crude suspension, and using the crude suspension for subsequent inoculation, wherein the inoculation amount is 2000-4000 egg masses per basin.
4. Seedling raising:
the experimental crop is the root-knot nematode susceptible variety of cucumber, the seedling substrate is filled into a 50-hole seedling pot, seeds of the root-knot nematode susceptible host are sown into the seedling substrate, 1 seed is planted in each seedling hole, and the seedling can be transplanted when 3-5 true leaves grow.
5. Inoculating:
1/2 to 2/3 of sterile standby soil is firstly added at the bottom of an inoculation flowerpot, then a quantitative insect egg suspension is added to ensure that the inoculation amount is more than 2000 insect eggs, then a layer of standby soil is additionally paved, then a layer of microbial inoculum (the viable bacteria amount is 10 hundred million/g) is added according to 5 per mill of the soil weight, the microbial inoculum is mixed with the soil and is broadcasted, then a layer of standby soil is additionally paved, and finally standby seedlings are transplanted. And (5) pouring a proper amount of field planting water into the pot after the inoculation and transplanting are finished.
6. Culturing:
culturing the treated potted plant in a greenhouse at 20-25 deg.c, watering for 3d for one time, and regulating the soil humidity to 50% or less. After culturing for 35-45d, buckling the basin and taking the root investigation statistical effect.
7. Root knot index and prevention effect investigation and calculation:
TABLE 3 root knot grading Standard
| Level 0 | Root knot is not found at root |
| Level 1 | By carefully identifying, a small number of root knots can be found |
| Level 2 | The main root has no root knot, and the fibrous root has a small number of root knots and can be clearly distinguished |
| 3 Grade | The main root has no root knot, and the fibrous root can find a slightly larger root knot |
| Grade 4 | The main root has no root knot, and the fibrous root is mainly provided with a larger root knot |
| Grade 5 | 50% Of the roots are infected, the main roots are infected in very small amounts |
| Grade 6 | Finding root knot on principal root |
| Level 7 | The main root is mostly infected by root knots |
| Level 8 | All principal roots are infected/few are not infected |
| Grade 9 | All roots can be severely infested/the normal plant dies |
| Grade 10 | All roots are severely infected, no roots |
Root knot index and relative control are calculated as follows:
disease index = [ Σ (disease grade value x number of leaves of the grade)/(total leaf number of investigation x highest disease grade value) ]x100.
Calculation formula of control efficiency= [1- (treatment group disease index/control group disease index) ]x100%.
Experimental results: the growth vigor and root conditions of the potted cucumber test plants are shown in fig. 3, root index and control effect are calculated according to statistical experimental data, the bacillus amyloliquefaciens 14-21 treatment group obviously reduces the occurrence of the root knot nematode disease of the potted cucumber, the control effect on the root knot nematode disease is as high as 87.7%, and meanwhile, the growth promoting effect on the root system and overground part growth vigor of the cucumber in the seedling stage can be good.
EXAMPLE 4 evaluation of the effects of Bacillus amyloliquefaciens 14-21 on the protection against Linear insects of field-grown cucumber
1. Experiment site:
the Qingdao Laiyi city institute of Leisha is placed in Guxu village cucumber greenhouse.
2. Cucumber planting experiment:
The area of each experimental cell is 10m multiplied by 10m, 10 rows of cucumbers are arranged in each experimental region, about 500+/-10 strains are arranged in each experimental region, 15 experimental cells are arranged in total, and each treatment group is repeated for 3 times through a random group.
(1) Blank control group: normal field management, no microbial inoculum is used, and fresh water is poured;
(2) 14-21 microbial inoculum treatment groups: 14-21 microbial inoculum (the viable bacteria amount is 10 hundred million/g) is applied with water after transplanting cucumber seedlings according to the dosage of 3-10 kg/mu, and is used once again after 7 days. Wherein:
Treatment group 1: the dosage of the 14-21 microbial inoculum is 3 kg/mu;
treatment group 2: the dosage of the 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 transplanting and planting, and the total investigation is three times. When each investigation is carried out, 10 cucumber plants are dug in each district, the plant heights are measured, the plant weights are weighed, the root knot progression is counted and recorded, and the average plant weight, the average plant height and the root knot prevention effect of the single plant are calculated; sampling was performed in different batches during the picking period to count single fruit weights, and cell yields were calculated, and the results are shown in table 4.
TABLE 4 control effect of Bacillus amyloliquefaciens 14-21 on cucumber root knot nematode diseases
| Experimental grouping | Height of plant (cm) | Yield (kg) | Yield enhancement | Root knot index | Relative prevention effect |
| Blank control group | 154.65 | 93.8 | - | 67.5 | - |
| Treatment group 1 | 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 is known that the growth vigor of cucumber plants can be remarkably improved and the plant height and the plant weight can be increased by applying the bacillus amyloliquefaciens 14-21 microbial inoculum in the cucumber seedling stage. Compared with the control group, the cucumber yield of the treatment group is improved by 5.0-23.5%, and the yield increasing effect is obvious; meanwhile, the occurrence of root-knot nematode diseases can be effectively reduced, the disease index is lightened, the highest control effect on cucumber root-knot nematodes is 71.1%, and the control effect is good.
In addition, the nematode control experiment results carried out on other cucurbitaceae crops such as muskmelon, towel gourd and the like show that the control effect of the bacillus amyloliquefaciens 14-21 on the melons and the towel gourd root knot nematodes is very obvious, and the control efficiency reaches 68.7% and 64.5% respectively.
The comprehensive effect of the bacillus amyloliquefaciens 14-21 provided by the invention can effectively improve the growth vigor and yield of crops, and has obvious control effect on root-knot nematode diseases which are easy to occur in cucumbers, melons, luffa and other cucurbitaceae crops.
EXAMPLE 5 evaluation of control Effect of Bacillus amyloliquefaciens 14-21 on tomato root rot
1. Test site:
the goddess tea is a kind of tea for the goddess of the god of longevity.
2. And (3) test design:
The test was run with a total of 4 treatments, each 100m 2 per treatment area, 3 replicates, and a total of 12 test cells. The blank control is conventional fertilization without microbial inoculum.
Treatment group 1: 0.5kg of 14-21 microbial inoculum (the viable bacteria amount is 10 hundred million/g) is applied to the rhizosphere of tomatoes by drip irrigation with water;
treatment group 2: 1.0kg of 14-21 microbial inoculum (the viable bacteria amount is 10 hundred million/g) is applied to the rhizosphere of tomatoes by drip irrigation with water;
treatment group 3: 1.5kg of 14-21 microbial inoculum (viable bacteria amount 10 hundred million/g) was applied to the rhizosphere of tomatoes by drip irrigation with water.
In the tomato harvesting period, the number of plants with root rot in each group is counted, the morbidity and the control efficiency of bacillus amyloliquefaciens 14-21 on the tomato root rot are calculated, and the specific results are shown in table 5.
Incidence (%) =number of diseased plants/total number of plants×100%.
Control efficiency (%) = (incidence of placebo-treated group)/incidence of placebo x 100%.
TABLE 5 control Effect of Bacillus amyloliquefaciens 14-21 on tomato root rot
| Experimental grouping | Incidence of disease | Efficiency of control |
| Blank control group | 25.6％ | - |
| Treatment group 1 | 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 incidence of tomato root rot was only 4.2% -12.2% for each treatment group to which Bacillus amyloliquefaciens 14-21 bacteria were applied, much lower than for the control group. Therefore, the bacillus amyloliquefaciens 14-21 provided by the invention has obvious control effect on tomato root rot, the highest control efficiency reaches 83.6%, and unexpected technical effects are achieved.
In addition to root knot nematode and root rot, the bacillus amyloliquefaciens 14-21 of the invention can also effectively prevent and treat the degradation effect experiment of the bacillus amyloliquefaciens 14-21 of the embodiment 6 on soil drug residues
1. Test site:
the birthday present in the town.
2. And (3) test design:
The test was run with a total of 6 treatments, each treatment area of 50m 2, 3 replicates, and a total of 18 cells. Aiming at 7 common herbicides, bactericides and pesticides in the market, 5 kinds of acetamiprid, imidacloprid, cyhalothrin, triazolone and chlorothalonil chemical pesticides with higher drug residues and larger harm to human bodies are screened for field tests. And (3) on the day of transplanting the cucumber, filling and flushing Shi Jie bacillus amyloliquefaciens 14-21 microbial inoculum (the viable bacteria amount is 10 hundred million/g), on the 10 th day, flushing the microbial inoculum again, wherein the microbial inoculum amount is 10 kg/mu (based on 10 hundred million/g microbial inoculum amount), on the 15 th day, 30 days and 60 days after transplanting, respectively taking a crop rhizosphere soil sample, detecting chlorothalonil and cyhalothrin by using a gas chromatography-mass spectrometer, detecting the triazolone, acetamiprid and imidacloprid content by using a liquid chromatography-mass spectrometer, and calculating the degradation rate. The control group was Chong Shi Qingshui. The specific results are shown in Table 6.
3. Test results and analysis:
TABLE 6 degradation Rate of Bacillus amyloliquefaciens 14-21 for different pesticide residues
| Time of | 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％ |
From the experimental data in Table 6, the bacillus amyloliquefaciens 14-21 microbial inoculum can obviously reduce pesticide residues in soil; after 60 days of application, the degradation rates of three pesticides of acetamiprid, imidacloprid and cyhalothrin in the soil respectively reach 92.8%, 75.8% and 85.1%, and the degradation rates of two bactericides of triazolone and chlorothalonil respectively reach 95.4% and 95.1%, so that unexpected technical effects are achieved.
In the comprehensive view, the bacillus amyloliquefaciens 14-21 has a strong degradation effect on pesticides and bactericides commonly used in crop planting, can effectively reduce pesticide residues in soil, ensure soil safety, obviously reduce pesticide residues of crops and ensure crop food safety.
EXAMPLE 7 repair improvement Effect of Bacillus amyloliquefaciens 14-21 on soil acidification
1. Test site:
Flat kernel is a megatown.
2. And (3) test design:
The test was run with a total of 4 treatments, each 100m 2 per treatment area, 3 replicates, and a total of 12 test cells. The blank control is conventional fertilization without microbial inoculum. The planted crops are two-crop tomatoes, three-year test is continuously carried out, and soil samples are taken in the same period each year to detect the pH value of soil in different experimental groups.
Treatment group 1: 0.5kg of 14-21 microbial inoculum (the viable bacteria amount is 10 hundred million/g) is applied to the rhizosphere of tomatoes by drip irrigation with water;
treatment group 2: 1.0kg of 14-21 microbial inoculum (the viable bacteria amount is 10 hundred million/g) is applied to the rhizosphere of tomatoes by drip irrigation with water;
treatment group 3: 1.5kg of 14-21 microbial inoculum (viable bacteria amount 10 hundred million/g) was applied to the rhizosphere of tomatoes by drip irrigation with water.
3. Acidizing soil restoration effect:
TABLE 7 Effect of Bacillus amyloliquefaciens 14-21 on improvement of soil acidification
| Sample numbering | Blank control group | Treatment group 1 | Treatment group 2 | Treatment group 3 |
| Year 1 | 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 3 years of application of Bacillus amyloliquefaciens 14-21 had a better effect of improving soil acidification, and in particular, the acidification repair effect of experimental group 3 was most obvious.
In conclusion, the bacillus amyloliquefaciens 14-21 can be used for preventing and controlling root knot nematode diseases and common root rot diseases of tomatoes, which are easy to occur in cucumbers, melons, luffa and other cucurbits, and can also remarkably promote the growth of crops and improve the quality and yield of the crops. The strain can also effectively degrade residual chemical bactericides and pesticides in soil, has obvious restoration 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 biological control microbial inoculum, a biological fertilizer, a soil conditioner and the like, can be widely applied to the field of agricultural production, can be combined with any one or more of other bacillus, pseudomonas, agrobacterium, azotobacter, rhizobium, aspergillus and rhizopus, is used for preventing and controlling common soil-borne crop diseases, improves the soil environment, improves the crop quality, ensures the food safety, and has wide application prospect.
Claims (7)
1. Bacillus amyloliquefaciens (Bacillusamyloliquefaciens), which is characterized in that the preservation number of the Bacillus amyloliquefaciens is CCTCC NO: m20211421.
2. The use of bacillus amyloliquefaciens according to claim 1 for controlling cucumber root knot nematode disease or tomato root rot.
3. The use of bacillus amyloliquefaciens according to claim 1 in soil acidification remediation.
4. A microbial preparation comprising the bacillus amyloliquefaciens of claim 1.
5. The microbial preparation of claim 4, wherein the viable count of bacillus amyloliquefaciens in the microbial preparation is at least 10 8 CFU/g.
6. Use of the microbial preparation according to claim 4 or 5 for controlling cucumber root knot nematode or tomato root rot.
7. Use of a microbial preparation according to claim 4 or 5 in soil acidification remediation.
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