CN115927066B - Bacillus belicus having antagonism to fusarium oxysporum and application thereof - Google Patents
Bacillus belicus having antagonism to fusarium oxysporum and application thereof Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Bacillus belicus having antagonism to fusarium oxysporum and application thereof belong to the technical field of agricultural biological control. In order to develop safe and efficient biocontrol microorganisms for biocontrol, the classification of the biocontrol bacillus is named as bacillus beliensis (Bacillus velveteen), and the biocontrol bacillus is preserved in the China general microbiological culture Collection center (China general microbiological culture Collection center) for 8 months and 5 days in 2022, the preservation address is North Chen West Lu No.1 in the Korean region of Beijing city, and the preservation number is CGMCC No.25497, and the biocontrol bacillus has the characteristics of wide antibacterial range, strong antagonism and obvious growth promoting effect, can effectively inhibit the growth of crop pathogenic bacteria such as fusarium oxysporum caused fusarium wilt, botrytis cinerea, cucumber fusarium wilt and the like, promote the germination of tomato seeds, promote the growth of radicle and promote the growth of seedling plants and root system; can secrete protease, amylase, cellulase and ferrite.
Description
Technical Field
The invention belongs to the technical field of microorganisms; in particular to bacillus belicus with antagonism to fusarium oxysporum and application thereof.
Background
Fusarium oxysporum (F.oxysporum) is a soil-borne pathogenic fungus distributed worldwide, has a wide host range, and can cause the occurrence of plant wilt of more than 100 kinds of melons, solanaceae, bananas, cottons, leguminosas, flowers and the like. Fusarium oxysporum (Imerfection fungi), fusarium (Moniliales), tuberculaceae (Tubercularia), fusarium (Fusarium) colonize the root vascular bundles of plants, resulting in blockage of the ducts, withering of aerial parts, brown necrotic spots on root surfaces, late root rot and even death of the whole plant, severely affecting the growth, development, yield and quality of the plants. At present, aiming at the prevention and treatment of fusarium oxysporum caused fusarium wilt, chemical reagents are mainly used, and researches show that the effect of part of chemical pesticides is gradually weakened, and even pathogenic bacteria generate drug resistance to the chemical pesticides used in a mixed mode. And with the use of chemical pesticides in large quantities, chemical agents remain in soil to cause unbalance of natural environment of the soil and influence the physical health of people, so that the biological control technology is important. However, at present, the biological control technology for tomato diseases still has some problems, and it is still necessary to develop a safer, more efficient and stable control effect of the biological control microorganism for controlling fusarium oxysporum.
Disclosure of Invention
In order to develop a biological control microorganism with higher and more efficient control effect on fusarium oxysporum, the invention provides bacillus belicus with antagonism on fusarium oxysporum and application thereof.
The invention is realized by the following technical scheme:
bacillus belicus having antagonism to fusarium oxysporum, wherein the biological control bacillus is classified and named as Bacillus belicus (Bacillus velezensis), and is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms at 8 months of 2022, wherein the preservation address is No. 3 of North Silu No.1 in the Korean yang area of Beijing city, and the preservation number is CGMCC No.25497.
Further, the nucleotide sequence of the 16S rDNA of bacillus beijerinus is shown as SEQ ID No:1, and the model number of the bacillus beijerinus is Y-4.
Further, bacillus belicus for preventing and controlling fusarium oxysporum is cultured on protease detection, cellulase and siderophore detection plates for 3-7 d to secrete protease, cellulase and siderophore, and auxin IAA is produced to form a biological film.
The application of the bacillus belicus with antagonism to the fusarium oxysporum is realized by virtue of the bacillus belicus with antagonism to the fusarium oxysporum, and the application of the bacillus belicus with antagonism to the agricultural biological control is realized.
Furthermore, the agricultural biological control comprises that the bacillus belicus is used for controlling fusarium oxysporum-induced fusarium wilt, tomato gray mold, cucumber anthracnose, cucumber fusarium wilt, fusarium verticillium, sunflower sclerotium bacteria or equisetum-induced crop diseases, and the specific method of application is that the bacillus belicus Y-4 bacterial suspension is used for spraying on the plant surface.
Further, the preparation method of the bacillus belicus Y-4 bacterial suspension comprises the following steps: collect the Bei LaiInoculating single colony of Bacillus to sterile LB culture solution, and culturing at 28deg.C and 200r/min for 2d to obtain bacterial solution with concentration of 1×10 8 cfu/mL of the bacterial suspension.
The application of the bacillus beleiensis with antagonism to the fusarium oxysporum is realized by virtue of the bacillus beleiensis with antagonism to the fusarium oxysporum, and the application of the bacillus beleiensis with antagonism to the fusarium oxysporum in the aspect of promoting the growth of crop seeds and seedlings.
Further, the specific method of application is to soak the crop seeds with the Bacillus bailii suspension diluted 500 times.
Further, the preparation method of the bacillus belicus Y-4 bacterial suspension comprises the following steps: collecting single colony of Bacillus bailii, inoculating into sterile LB culture solution, and culturing at 28deg.C and 200r/min for 2d to obtain bacterial solution with concentration of 1×10 8 cfu/mL of the bacterial suspension.
The invention relates to bacillus belicus with antagonism to fusarium oxysporum, which can secrete detected substances after being inoculated on protease, cellulase and ferriphilic detection plates for 3-7 d by using the bacillus belicus in the color development condition on an extracellular enzyme detection plate.
The beneficial effects of the invention are as follows:
1. the test starts with the flat plate opposition of fusarium oxysporum serving as pathogenic bacteria, a biocontrol bacterium Y-4 is screened out, the growth of fusarium oxysporum can be effectively inhibited, and the strain is identified as bacillus beijerinus (Bacillus velezensis) through morphological observation and 16S rDNA sequence analysis.
2. The bacteria Y-4 with antagonism to fusarium oxysporum provided by the invention has wide antibacterial spectrum and strong antagonism, can effectively inhibit the growth of fusarium oxysporum-induced fusarium wilt, tomato gray mold, cucumber fusarium wilt, cucumber anthracnose, fusarium equisetum, sunflower sclerotium germ and round-branched fusarium, can be widely applied to the field of agricultural biological control, and can be used for controlling crop diseases caused by fungi such as fusarium wilt, gray mold, anthracnose and sclerotium disease.
3. The bacillus belicus Y-4 provided by the invention can produce auxin IAA; can secrete protease, cellulase and ferrite; capable of forming a biofilm; HCN is not produced.
4. Can generate volatile substances to inhibit the growth of pathogenic bacteria.
5. The strain Y-4 has good promotion effect on tomato seeds and seedlings, and obviously increases morphological indexes, root length, surface area, node number, root tip number and seedling leaf related photosynthetic parameters of tomato seedlings relative to CK groups.
Drawings
FIG. 1 is a photograph showing colony morphology of Bacillus belicus Y-4 provided by the present invention;
FIG. 2 is a photomicrograph of Bacillus belicus Y-4 provided by the present invention;
FIG. 3 shows the 16S rDNAPCR amplification product of Bacillus bailii Y-4 provided by the invention;
FIG. 4 is a phylogenetic tree of Bacillus belicus Y-4 provided by the invention;
FIG. 5 is a photograph showing the bacteriostatic effect of Bacillus belicus Y-4 on 6 plant pathogenic bacteria;
FIG. 6 is a comparison photograph of the antibacterial effect of Bacillus belicus Y-4 provided by the invention on Fusarium oxysporum;
FIG. 7 is a photograph showing the result of detecting volatile matters in Bacillus bailii Y-4 provided by the invention;
FIG. 8 is a comparison photograph of IAA detection results of Bacillus bailii Y-4 provided by the present invention;
FIG. 9 is a photograph showing the result of the protease and cellulase detection of Bacillus belicus Y-4 provided by the invention;
FIG. 10 is a photograph showing the result of detecting the presence of ferrite of Bacillus belicus Y-4 according to the present invention;
FIG. 11 is a photograph showing the result of the biofilm assay of Bacillus belicus Y-4 provided by the present invention;
FIG. 12 is a photograph showing the result of HCN detection of Bacillus belicus Y-4 according to the present invention;
FIG. 13 is a graph showing the comparison of the effect of Bacillus belicus Y-4 in promoting germination of tomato seeds;
FIG. 14 is a graph showing the effect of Bacillus belicus Y-4 in promoting growth of tomato radicle for 3 days;
FIG. 15 is a graph showing the effect of Bacillus belicus Y-4 in promoting growth of tomato radicle for 6 days;
FIG. 16 is a graph showing the comparison of Bacillus belicus Y-4 obtained by the present invention after acting on tomato seedling plants
FIG. 17 is a photograph showing the result of the influence of Bacillus belicus Y-4 provided by the invention on root system index;
FIG. 18 is a graph showing the comparison of maximum light quantum efficiency Fv/Fm of Bacillus belicus Y-4 against tomato seedlings;
FIG. 19 is a graph showing the comparison of non-photochemical quenching of Bacillus belicus Y-4 provided by the present invention against tomato seedlings;
FIG. 20 is a graph showing the actual efficiency of the photochemical quenching qP of tomato seedlings by Bacillus belicus Y-4 provided by the invention.
Detailed Description
The following description of the present invention is provided with reference to the accompanying drawings, but is not limited to the following description, and any modifications or equivalent substitutions of the present invention should be included in the scope of the present invention without departing from the spirit and scope of the present invention.
Example 1
Bacillus belicus having antagonism to fusarium oxysporum, wherein the biological control bacillus is classified and named as Bacillus belicus (Bacillus velezensis), and is preserved in China general microbiological culture Collection center (China Committee) for 8 months and 5 days in 2022, wherein the preservation address is No. 3 of North Silu No.1 in the Korean region of Beijing city, and the preservation number is CGMCC No.25497.
Further, the nucleotide sequence of the 16S rDNA of bacillus beijerinus is shown as SEQ ID No:1, and the model number of the bacillus beijerinus is Y-4.
The present example provides screening and identification results of biocontrol bacteria.
Collecting a soil sample: the strain was isolated from material in a park in the state of san francisco. Selecting the positions around the root with strong plant growth and no disease, removing surface soil, and collecting soil samples at depths of 5-10 cm.
Isolation of strains: the method comprises the steps of separating and purifying rhizosphere soil of plants by adopting a flat plate dilution coating method to obtain a plurality of strains of bacteria, taking fusarium oxysporum as an indicator bacterium, adopting a flat plate counter method to obtain a strain of bacteria with antagonism to fusarium oxysporum, numbering the strain as Y-4, and carrying out morphological characteristics, physiological and biochemical characteristics and molecular biological identification on the strain.
(1) Morphological features
Inoculating the strain on LB solid medium, culturing at 28deg.C for 48 hr, and making colony in milky white, pasty, wrinkle-free, smooth in edge, slightly wrinkled in center, and opaque, wherein the colony morphology is shown in figure 1; the strain Y-4 is observed by a microscope oil microscope, and after gram staining, the thallus is purple and rod-shaped, is gram positive, and the microscopic morphology is shown in figure 2.
(2) 16SrDNA sequencing and phylogenetic analysis:
the Y-4 genome DNA is used as a template, and amplified into a general primer of 16S rDNA, so that a PCR product of about 1400bp is obtained. The amplified fragments were consistent with the length of conventional 16S rDNA sequences using column PCR products. As shown in FIG. 3, the PCR amplified product was recovered, and the recovered product was purified and sent to Beijing Liuhua macrogene technologies Co., ltd for sequencing.
The 16S rDNA sequence of strain Y-4 was submitted to the NCBI GenBank database and Blast aligned and homology aligned with the reported sequences. The MEGA6.06 software adopts a Neighbor-Joining method to construct a phylogenetic tree of strains Y-4 and 13 strains shown in FIG. 4, and the similarity is repeatedly calculated for 1000 times. "T" means a model strain. The homology with strain Y-4 was Bacillus velezensis strain Y W (MT 573877).
(3) Physiological and biochemical characteristics
The physiological and biochemical characteristics of strain Y-4 were identified from gram stain, contact enzyme, starch hydrolysis, aerobic or anaerobic tests, O/F glucose oxidative fermentation tests.
The results show that the strain Y-4 is gram positive bacterium, belongs to aerobic bacterium, contact enzyme, starch hydrolysis, and is positive and the O/F glucose oxidative fermentation test is alcogenicity bacterium.
Example 2
In this example, a bacteriostasis spectrum test was performed on Bacillus belicus having antagonism against Fusarium oxysporum as described in example 1 and various plant pathogenic bacteria.
Testing for phytopathogens includes: botrytis cinerea (Botrytis cinerea), cucumber fusarium wilt, cucumber anthracnose, fusarium equiseti, sunflower sclerotium germ and fusarium verticillatum; all were kept in the university of northeast agricultural biotechnology laboratory.
The biocontrol bacterium Y-4 is cultured for 48 hours at the constant temperature of 28 ℃ and at the speed of 200r/min, and bacterial suspension is obtained. Taking out 6 pathogenic bacteria cultured on a slant, placing the manufactured 5mm pathogenic bacteria cake in a sterilized and dried PDA solid culture center to the right, placing sterile oxford at the center to the left, sucking 80-100ul of bacterial suspension by a sterile gun head, injecting into oxford cups, sealing a number culture dish plate, culturing in a constant temperature oven at 28 ℃ for 7d, and measuring the antibacterial band broadband. The results are shown in table 1 and fig. 5:
as can be seen from the data in Table 1, the plant pathogenic fungi in pair 6 of Y-4 can generate a zone of inhibition, and the distance of inhibition is between 0.60cm and 0.90 cm. The biocontrol bacterium Y-4 has stronger antagonism to the growth of the pathogenic fungi, which fully indicates that the biocontrol bacterium Y-4 has wider bacteriostasis spectrum and stronger bacteriostasis when being used as biocontrol bacteria.
TABLE 1 bacterial strain confrontation results
Example 3
This example examined the effect of Fusarium oxysporum control of Bacillus belicus Y-4 having antagonism against Fusarium oxysporum as described in example 1.
(1) Plate counter effect
Culturing Bacillus bailii Y-4 at 28deg.C for 48h at 200r/min to obtain bacterial suspension. Taking out fusarium oxysporum pathogenic bacteria cultured on a slant, inoculating mycelia on a PDA culture medium, after the mycelia grow to be full of the flat plate, placing a manufactured 5mm pathogenic bacteria cake on a right side of a sterilized and dried PDA solid culture center, placing sterile oxford at a left side of the center, sucking 80-100ul of bacterial suspension by using a sterile gun head, injecting into oxford cups, sealing the flat plate of a culture dish, culturing for 7d in a constant temperature oven at 28 ℃, and measuring the width of a bacteriostatic zone. As shown in table 2, fig. 6.
TABLE 2 plate counter results
(2) Volatile flat panel detection effect
Pouring the prepared LB and PDA solid culture media into a double-cell culture dish, airing, picking Y-4 by an inoculating loop, inoculating the Y-4 into the LB culture medium, inoculating the fusarium oxysporum block into the PDA culture medium, and inoculating the fusarium oxysporum block only in contrast. Culturing at 28 ℃ at constant temperature, and observing the growth condition of the inoculated biocontrol bacteria plate when the control grows to half of the cell culture plate.
As a result, as shown in FIG. 7, growth of Fusarium oxysporum was inhibited in the petri dish inoculated with Y-4, indicating that Y-4 can produce volatile substances to inhibit growth of pathogenic bacteria.
Example 4
This example shows IAA detection of Bacillus belicus having antagonism against Fusarium oxysporum as described in example 1.
200mL of LB liquid medium is prepared, 1% of L-tryptophan is added, and then biocontrol bacteria Y-4 are inoculated, and the culture is carried out at a constant temperature of 28 ℃ and at 200rpm/min for 48 hours. The Y-4 bacterial liquid is taken out of a centrifuge tube and centrifuged at 12000rpm for 10min. 2mL of the supernatant was taken and 4mL of Salkowaski (0.5 mol/LFECl) 3 15 mL, concentrated sulfuric acid 300mL, distilled water 500 mL) reagent, followed by the addition of two drops of orthophosphoric acid. The mixed solution is put in a water bath at 28 ℃ for 2 hours, whether the solution changes color is observed, and if the color changes, the biological control bacteria are indicatedCan secrete IAA. The test was repeated 3 times. The results are shown in FIG. 8.
Example 5
This example describes extracellular enzyme secretion assays and ferrite secretion assays on Bacillus belicus having antagonism against Fusarium oxysporum as described in example 1.
Protease: preparing a protease detection plate, placing a sterile filter paper sheet with the diameter of 6mm at the center of the protease detection plate, dripping 2ul of biocontrol bacterial suspension cultured for 48 hours on the filter paper sheet, placing the detection plate in a constant temperature incubator at 28 ℃ for culturing for 3 days, and if transparent halos appear around the filter paper sheet, indicating that the strain Y-4 can produce protease, and repeating the test three times.
Cellulase: preparing a cellulase detection plate, placing a sterile filter paper sheet with the diameter of 6mm at the center of the cellulase detection plate, dripping 2ul of biocontrol bacterial suspension cultured for 48 hours on the filter paper sheet, placing the detection plate in a constant temperature incubator at 28 ℃ for culturing for 3 days, and if a transparent halo appears around the filter paper sheet, indicating that the strain Y-4 can produce protease, and repeating the test three times.
Basophilic iron core: the biocontrol bacteria adopt a ferrite solid culture medium, a sterile filter paper sheet with the size of 6mm is placed in the middle of the air-dried culture medium, and 2 mu L of biocontrol bacteria liquid is dripped; the test was repeated three times, at 28℃and daily for the presence of orange transparent halos.
As a result, the Y-4 strain was able to secrete protease, cellulase and siderophore as shown in FIGS. 9 and 10.
Example 6
This example demonstrates the biofilm formation ability and HCN detection of bacillus belicus having antagonism against fusarium oxysporum as described in example 1.
Biological film: bacillus belicus Y-4 strain was inoculated into LB liquid medium, and cultured at 28℃and 200rpm/min for 1d. 4mL of the bacterial liquid is placed at the bottom of a sterile test tube and is subjected to stationary culture for 2d at 28 ℃. The bacterial liquid is discarded, sterile water is used for cleaning the inner wall of the test tube for many times, then 5mL of 1% crystal violet dye liquid is added, standing is carried out for 15min at 28 ℃, crystal violet is discarded, the sterile water is used for cleaning for three times, and the bacterial strain can generate a biological film when the blue violet is generated on the test tube wall. The results are shown in FIG. 11.
After addition of crystal violet dye and rinsing with sterile water, blue-violet streaks formed on the tube wall, indicating that the Bacillus bailii Y-4 strain can produce a biofilm. The biocontrol bacteria exist in a form of biological film, and can effectively play roles of promoting growth, resisting diseases and the like of the biocontrol bacteria.
HCN detection: taking 10mL of bacterial liquid in a test tube, dropwise adding one drop of concentrated sulfuric acid, fully and uniformly mixing, and taking HCN test paper to immerse in the solution; waiting for 15min, waiting for overnight when the concentration is low, changing the test paper from light blue to dark blue according to the HCN concentration, and proving that HCN is not generated when the test paper is not discolored. The results indicate that Bacillus belicus Y-4 complements HCN production. The results are shown in FIG. 12.
Example 7
This example examined the growth promoting effect of Bacillus belicus Y-4 having antagonism against Fusarium oxysporum on tomato seeds and seedlings as described in example 1.
(1) Effects on seed germination Rate and radicle Length
The experiment is used for determining the effect of Y-4 on promoting the growth of tomato seeds and seedlings, wz-37 (Bacillus velezensis) is used as a control group and is stored by a laboratory of a garden institute of northeast agricultural university, and bacillus besseyi (Bacillus velezensis) wz-37 is stored in a China general microbiological culture collection center (CGMCC) with the storage number of CGMCC No.15766. The specific measurement method comprises the following steps: soaking tomato seeds in warm water at 55deg.C for 20min; sterilizing with 75% alcohol for 30s, and washing with sterile water for 3 times; 3% sodium hypochlorite for 3min, and washing with sterile water for 3 times.
Culturing Y-4 and wz-37 at 28deg.C and 200rpm/min for 2d, and regulating bacterial liquid concentration to 1×10 8 cfu/mL. Each group was then diluted 500-fold. Biocontrol Y-4 and wz-37 and sterile water are used for controlling 10mL of liquid in each group, 20 seeds are soaked respectively, the seeds are washed by sterile water after 3 hours, and then the seeds are put into a seed bag. The germination rates of the seeds and the radicle growth amounts of the seeds of 3d and 6d are recorded for 48h, 72h, 96h, 120h and 144h respectively. The test was repeated 3 times. The results are shown in fig. 13 and 14.
Fig. 13 shows: the germination rate of seeds under different treatments is different, compared with wz-37 and CK, the germination rate of seeds of the strain Y-4 in 48h, 72h, 96h, 120h and 144h is higher than that of the control, wherein the germination rate of seeds in 144h is up to Y-4, the germination rate is 100% and is 30% higher than that of the control, and 8% higher than that of the wz-37 group, and no obvious difference exists.
Fig. 14 and 15 show: the lengths of the radicle of the seeds treated by the Y-4 after 3d and 6d are 5.48cm and 7.48cm which are obviously higher than those of the control group, which shows that the Y-4 has promotion effect on the germination rate and radicle elongation of the seeds after 500 times dilution of the fermentation liquor.
(2) Influence on seedling morphology index
The Y-4 bacterial suspension and the sterilized tomato seeds are respectively soaked in sterile water for 3 hours and then sown in a nutrition pot. The two treatments are respectively injected into the soil for 10mLY-4 bacterial liquid and equal amount of sterile water during seedling transplanting, and are cultured for 30 days. The plant height, stem thickness, dry weight and fresh weight of the plants were recorded. The results are shown in Table 3 and FIG. 16.
TABLE 3 morphological criteria
| Treatment group | Height of plant (cm) | Stem thickness (mm) | Fresh weight (g) | Dry weight (g) |
| Y-4 | 30.00±0.29a | 0.49±0.01a | 5.09±0.69a | 1.36±0.05a |
| wz-37 | 28.23±0.98a | 0.41±0.03b | 3.64±1.13ab | 0.75±0.14b |
| CK | 23.67±1.62b | 0.39±0.01b | 3.52±0.92b | 0.48±0.11c |
The results are shown in Table 3 and FIG. 16, and the highest data of plant height, stem thickness, fresh weight and dry weight in the three groups of treatments are 30.00, 0.49, 5.09 and 1.36 respectively. Y-4 was significantly different from the Control (CK), Y-4 was significantly different from wz-37 on a dry weight basis, and the remainder was not significant. The treatment of the biocontrol bacterial suspension has good growth promoting effect on tomato seedlings.
(3) Influence of root system index and morphology of tomato seedlings
The Y-4 bacterial suspension and the sterilized tomato seeds are respectively soaked in sterile water for 3 hours and then sown in a nutrition pot. And (4) respectively injecting 10mLY-4 bacterial liquid and equal amount of sterile water into soil during seedling transplanting, and measuring root system index of the seedlings by using an image analysis system. The results are shown in Table 4 and FIG. 17.
TABLE 4 root system index
As shown in Table 4 and FIG. 17, the root length, root surface area, node number and root tip number Y-4 of tomato seedlings were significantly different from the control group (CK) and not significantly different from wz-37.
(4) Chlorophyll fluorescence parameter of tomato seedlings
The Y-4 bacterial suspension and the sterilized tomato seeds are respectively soaked in sterile water for 3 hours and then sown in a nutrition pot. The two treatments are respectively injected into the soil for 10mLY-4 bacterial liquid and equal amount of sterile water during seedling transplanting, and are cultured for 30 days. Chlorophyll fluorescence parameters of each treatment group were determined using a fluorocam chlorophyll imaging system. Chlorophyll fluorescence parameters are shown in fig. 17, 18, 19.
FIG. 18 shows the degree of influence of each treatment on the maximum photosynthetic efficiency (Fv/Fm) of tomato seedlings by the effect, and shows that the maximum photosynthetic efficiency values of the control group, strain Y-4 and strain wz-37 are 0.71, 0.78 and 0.74, respectively. The treated group of the strain Y-4 is obviously higher than the control group and the strain wz-37, which shows that the strain Y-4 can improve the photosynthetic efficiency of tomato seedlings.
FIG. 19 shows the degree of effect of each treatment on the non-photochemical quenching (NPQ) of tomato seedlings, showing that the non-photochemical quenching values of the control group, the strain Y-4 and the strain wz-37 are 0.41, 0.5 and 0.44, respectively, and that the non-photochemical quenching index of the strain Y-4 is highest and higher than that of the control group and the strain wz-37, indicating that the strain Y-4 has an accelerating effect on the non-photochemical quenching index of tomato seedlings.
FIG. 20 shows the extent of effect of treatment on photochemical quenching (qP) of tomato seedlings, showing that the photochemical quenching values of control, strain Y-4, strain wz-37 are 0.62, 0.79, 0.69, respectively, strain Y-4 treated group is significantly higher than control and strain wz-37 groups, demonstrating that strain Y-4 can enhance photoelectron transfer activity.
Example 8
Bacillus belicus having antagonism to fusarium oxysporum, wherein the biological control bacillus is classified and named as Bacillus belicus (Bacillus velezensis), and is preserved in China general microbiological culture Collection center (China Committee) for 8 months and 5 days in 2022, wherein the preservation address is No. 3 of North Silu No.1 in the Korean region of Beijing city, and the preservation number is CGMCC No.25497.
Further, the nucleotide sequence of the 16S rDNA of bacillus beijerinus is shown as SEQ ID No:1, and the model number of the bacillus beijerinus is Y-4.
Further, bacillus belicus for preventing and controlling fusarium oxysporum is cultured on protease detection, cellulase and siderophore detection plates for 3-7 d to secrete protease, cellulase and siderophore, and auxin IAA is produced to form a biological film.
Example 9
The application of bacillus belgium with antagonism to fusarium oxysporum is realized by virtue of bacillus belgium with antagonism to fusarium oxysporum in the embodiment 8, and is characterized by being applied to agricultural biological control.
Furthermore, the agricultural biological control comprises that the bacillus belicus is used for controlling fusarium oxysporum-induced fusarium wilt, tomato gray mold, cucumber anthracnose, cucumber fusarium wilt, fusarium verticillium, sunflower sclerotium bacteria or equisetum-induced crop diseases, and the specific method of application is that the bacillus belicus Y-4 bacterial suspension is used for spraying on the plant surface.
Further, the preparation method of the bacillus belicus Y-4 bacterial suspension comprises the following steps: collecting single colony of Bacillus bailii, inoculating into sterile LB culture solution, and culturing at 28deg.C and 200r/min for 2d to obtain bacterial solution with concentration of 1×10 8 cfu/mL of the bacterial suspension.
Example 10
The application of the bacillus belgium with antagonism to the fusarium oxysporum is realized by the bacillus belgium with antagonism to the fusarium oxysporum according to the embodiment 8, and is characterized in that the application of the bacillus belgium with antagonism to the fusarium oxysporum in the aspects of crop seed and seedling growth promotion.
Further, the specific method of application is to soak the crop seeds with the Bacillus bailii suspension diluted 500 times.
Further, the preparation method of the bacillus belicus Y-4 bacterial suspension comprises the following steps: collecting single colony of Bacillus bailii, inoculating into sterile LB culture solution, and culturing at 28deg.C and 200r/min for 2d to obtain bacterial solution with concentration of 1×10 8 cfu/mL of the bacterial suspension.
Claims (6)
1. Fusarium oxysporumFusarium oxysporum) Shellfish having antagonismBacillus riceBacillus velezensis) The bacillus beleiensis is characterized in that bacillus beleiensis is preserved under the preservation number of CGMCC No.25497 and is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the 8 th month of 2022, and the preservation address is the North Chen Xili No.1 and 3 of the Korean region of Beijing city.
2. The use of a bacillus belicus having antagonism against fusarium oxysporum according to claim 1, characterized in that the use of the bacillus belicus for controlling fusarium oxysporum-induced fusarium wilt, tomato gray mold, cucumber anthracnose, cucumber fusarium wilt, fusarium verticillium, sunflower sclerotium germ or fusarium equisetum is in agricultural biological control, in particular in the application of the bacillus belicus suspension to plant surfaces.
3. The use of bacillus belgium having antagonism against fusarium oxysporum according to claim 2, wherein the preparation method of the bacillus belgium bacterial suspension is as follows: collecting single colony of Bacillus bailii, inoculating into sterile LB culture solution, and culturing at 28deg.C and 200r/min for 2d to obtain bacterial solution with concentration of 1×10 8 cfu/mL of the bacterial suspension.
4. The use of a bacillus belgium having antagonism against fusarium oxysporum according to claim 1, wherein the use of bacillus belgium having antagonism against fusarium oxysporum is in the promotion of crop seed and seedling growth.
5. The use of bacillus beljalis having antagonism against fusarium oxysporum according to claim 4, characterized in that the specific method of use is to soak crop seeds with a 500-fold dilution of said bacillus beljalis suspension.
6. According to claimThe application of bacillus belgium with antagonism to fusarium oxysporum, which is described in 5, is characterized in that the preparation method of bacillus belgium bacterial suspension is as follows: collecting single colony of Bacillus bailii, inoculating into sterile LB culture solution, and culturing at 28deg.C and 200r/min for 2d to obtain bacterial solution with concentration of 1×10 8 cfu/mL of the bacterial suspension.
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