CN118956636A - Bacillus Velez LMY3-5 and its application - Google Patents
Bacillus Velez LMY3-5 and its application Download PDFInfo
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- CN118956636A CN118956636A CN202410393494.8A CN202410393494A CN118956636A CN 118956636 A CN118956636 A CN 118956636A CN 202410393494 A CN202410393494 A CN 202410393494A CN 118956636 A CN118956636 A CN 118956636A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01P3/00—Fungicides
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- A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
- A23B7/00—Preservation of fruit or vegetables; Chemical ripening of fruit or vegetables
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- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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Abstract
本发明公开了一株贝莱斯芽孢杆菌LMY3‑5，保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC N0:29700，本发明还提供了所述贝莱斯芽孢杆菌菌株的发酵液及其发酵产生的菌株次生代谢物。本发明的贝莱斯芽孢杆菌菌株及其菌株发酵产生的次级代谢物具有广谱抑菌活性，对猕猴桃软腐病菌、美澳型核果褐腐病菌、十字花科黑斑病菌、茶炭疽病菌具有良好的拮抗作用，抑菌率达到了52.18％以上，尤其对猕猴桃果实上病斑抑制率最高可达到73.59％，是猕猴桃软腐病防治的潜在生物制剂，具有良好的开发前景和应用价值。
The invention discloses a strain of Bacillus Velezii LMY3-5, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, and the deposit number is CGMCC NO: 29700. The invention also provides a fermentation liquid of the Bacillus Velezii strain and a secondary metabolite of the strain produced by fermentation. The Bacillus Velezii strain of the invention and the secondary metabolite produced by fermentation of the strain have a broad-spectrum antibacterial activity, and have a good antagonistic effect on kiwifruit soft rot fungi, American and Australian type drupe brown rot fungi, crucifer black spot fungi, and tea anthracnose fungi, and the antibacterial rate reaches more than 52.18%, especially the highest inhibition rate of lesions on kiwifruit can reach 73.59%, which is a potential biological agent for the prevention and treatment of kiwifruit soft rot, and has good development prospects and application value.
Description
Technical Field
The invention belongs to the technical field of biological control, and particularly relates to bacillus bailii LMY3-5 and application thereof.
Background
Kiwi fruits are also called kiwi fruits because of unique flavor of the fruits, are rich in vitamin C, dietary fibers and various minerals, and have the effects of clearing intestines and invigorating stomach and the like, and are internationally praised as 'fruit king'. At present, more than 30 countries exist in the world for producing kiwi fruits, wherein the cultivation area and the yield are the largest in China, along with the gradual expansion of the planting area of the kiwi fruits, the diseases of the kiwi fruits are increased increasingly, and especially the soft rot of the kiwi fruits after being picked can cause the average incidence rate of the fruits reaching 40-50% in the period of storage and sales to rot in a large quantity. The disease is not effectively prevented and treated in all areas of the world so far, the quality and the yield of the kiwi fruits are seriously influenced, and great economic loss is caused. The disease can be infected by fungi such as B.dothidea, D.eres, A.alternata, phomopsis phragmitis and the like singly or in combination, and the disease invades in young fruit stage and presents symptoms in storage stage, wherein B.dothidea is the main pathogenic bacteria. Currently, the use of chemical bactericides is still the main strategy for controlling post-harvest soft rot of kiwi fruits. The widespread and long-term use of bactericides leads to the emergence of resistant pathogenic bacteria and environmental pollution. Therefore, it is of great importance to find a safe, broad-spectrum and more effective agent for preventing and treating soft rot of kiwi fruits.
In recent years, biological control has been widely studied and applied in fruit and vegetable disease control due to the outstanding advantages of easy decomposition, no residue, difficult generation of drug resistance and the like. The most successful and widespread application among biocontrol bacteria is bacillus. Previous studies have shown that bacillus is capable of producing a variety of secondary metabolites, such as bacteriocins, cell wall degrading enzymes, lipopeptides antibiotics, polypeptides, and the like. Lipopeptides can damage fungal cell walls and membranes, cause leakage of cell contents, and cause cell death. Bacillus also synthesizes plant growth promoting metabolites such as indole-3-acetic acid, auxin, cytokinin and gibberellin. Yan Li et al isolated from healthy apples to strain 9001 (Bacillus amyloliquefaciens), which has an inhibitory effect on apple rot pathogen (B.dothidea). Strain JX-5 (Brevibacillus laterosporus) inhibits the growth of Botrytis by secreting an antibacterial active ingredient. Therefore, green, efficient and safe disease prevention and control measures are sought to replace the traditional chemical bactericides to reduce the risks of pesticide residues, environmental pollution and the like, so that the method has great research prospect and application potential.
Patent publication No. CN116970521A discloses Bacillus bailii GUMHT p and application thereof, bacillus bailii (B.velezensis) GUMHTp, and preservation number is CCTCCM N0:2023748. the invention also discloses application of bacillus beijerinus GUMHTp to prevention and treatment of soft rot and ulcer of kiwi fruits and application of bacillus beijerinus to production of lipopeptid antibacterial substances. The bacillus beijerinus GUMHTp of the invention has good antibacterial effect on kiwi soft rot and canker.
Patent publication No. CN115927082A discloses Bacillus belicus YJK1 and application thereof, wherein the Bacillus belicus YJK1 can remarkably inhibit the growth of tea anthracis (Colletotrichum Camelliae) and fruit anthracis (Colletotrichum fructicola) and reduce the germination rate of tea anthracis and fruit anthracis conidia. The strain fermentation liquor has good stability, ultraviolet radiation resistance, acid and alkali resistance and heat resistance, has obvious inhibition effect on leaf spot of anthracnose, and has 90.8% control effect on tea anthracnose.
However, none of the bacillus bailii has broad spectrum, which greatly limits the application range and application value of bacillus bailii.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a bacillus belicus strain, a strain secondary metabolite and application thereof, wherein the strain and the strain secondary metabolite produced by fermentation have broad-spectrum antibacterial activity and have good antagonism on various germs.
The method is realized by the following technical scheme:
The first object of the invention is to provide bacillus belicus named bacillus belicus (Bacillus velezensis) LMY3-5 which is preserved in China general microbiological culture collection center with the preservation number of CGMCC N0:29700.
The second object of the present invention is to provide a fermentation broth of Bacillus bailii LMY3-5 as described above.
It is a third object of the present invention to provide a secondary metabolite produced by fermentation of Bacillus bailii LMY3-5 as described above.
The fourth object of the invention is to provide the application of the bacillus beijerinus LMY3-5 and/or the fermentation broth in antagonizing plant pathogenic bacteria, wherein the plant pathogenic bacteria are kiwi fruit soft rot bacteria (B.dothida), kiwi fruit soft rot bacteria (D.eres), american and Australian brown rot bacteria (M.fructicola), cross black spot bacteria (A.alternata) and tea anthracnose bacteria (C.gloeosporides).
Specifically, the bacillus beleiensis LMY3-5 and/or the application of the fermentation broth in preparation of preparation for inhibiting plant pathogenic bacteria, wherein the plant pathogenic bacteria are kiwi fruit soft rot (B.dothida), kiwi fruit soft rot (D.eres), american and Australian brown rot (M.fructicola), cross black spot (A.alternata) and tea anthracnose (C.gloeosporioides).
The fifth object of the present invention is to provide an application of the bacillus beijerinus LMY3-5 and/or the fermentation broth in preparing a preparation for controlling diseases caused by plant pathogenic bacteria, wherein the plant pathogenic bacteria are kiwi fruit soft rot (b. Dothida), kiwi fruit soft rot (d. Eres), american and australia stone fruit brown rot (m. Fructicola), cruciferous black spot (a. Alternata), tea anthracnose (c. Gloosporides).
The sixth object of the invention is to provide the application of bacillus beijerinus LMY3-5 and/or fermentation broth in delaying the rot of kiwi fruits.
The seventh object of the invention is to provide an application of bacillus beijerinus LMY3-5 and/or fermentation broth in preparing a preparation for preventing and treating kiwi fruit rot.
The eighth object of the invention is to provide the application of the secondary metabolite produced by the fermentation of bacillus beijerinus LMY3-5 in delaying the rot of kiwi fruits.
The ninth object of the invention is to provide an application of the secondary metabolite produced by the fermentation of bacillus beijerinus LMY3-5 in preparing a preparation for delaying the decay of kiwi fruits.
The kiwi fruit rot is caused by kiwi fruit soft rot (b. Dothidea) infestation.
A tenth object of the present invention is to provide a biocontrol agent comprising bacillus belicus LMY3-5 as described above, and/or said fermentation broth, and/or said fermentation-produced secondary metabolite.
The beneficial effects are that:
The bacillus belicus LMY3-5 or fermentation liquor or secondary metabolite provided by the invention has broad-spectrum antibacterial activity, has good antagonism on kiwi fruit soft rot germs, american and Australian brown rot germs, cross black spot germs and tea anthracnose germs, and has an antibacterial rate of more than 52.18%, especially has a maximum inhibition rate of 73.59% on the kiwi fruit.
Drawings
Fig. 1: colony morphology observation chart in example 3;
fig. 2: the hypha growth inhibition effect of Bacillus bailii LMY3-5 fermentation broth in example 5 on kiwi fruit soft rot pathogens is shown;
fig. 3: in the example 6, the spore germination inhibition effect of bacillus beijerinus LMY3-5 fermentation liquor on kiwi fruit soft rot pathogenic bacteria is shown; wherein A is CK, B is 4.00%, C is 8.00%, D is 16.00%;
Fig. 4: in the embodiment 7, the control effect of bacillus bailii LMY3-5 fermentation liquor on kiwi fruits is shown; wherein A and B are the inhibitory effects on the spreading of B.dothidea lesions, and C and D are the inhibitory effects on the spreading of D.eres lesions;
Fig. 5: SEM image of B.dothidea hyphae morphology of Bacillus bailii LMY3-5 fermentation broth of example 8; wherein A is a control group, and B is a fermentation liquor treatment group;
Fig. 6: TEM image of B.dothidea cell ultrastructure of Bacillus bailii LMY3-5 fermentation broth in example 9; wherein A and B are control groups, and C and D are fermentation broth treatment groups;
Fig. 7: FIG. 10 shows the effect of Bacillus bailii LMY3-5 fermentation broth on B.dothidea cell membrane integrity;
fig. 8: FIG. 11 shows the fermentation broth of Bacillus bailii LMY3-5 against B.dothidea nucleic acid and protein release profile; wherein A is nucleic acid and B is protein;
fig. 9: effect of bacillus bailii LMY3-5 fermentation broth on b.dothidea glucanase and chitinase activity in example 12; wherein A is beta-1, 3 glucanase and B is chitinase;
fig. 10: antimicrobial activity profile of Bacillus bailii LMY3-5 against various pathogens in example 14.
Detailed Description
The following detailed description of the invention is provided in further detail, but the invention is not limited to these embodiments, any modifications or substitutions in the basic spirit of the present examples, which still fall within the scope of the invention as claimed.
Example 1
A biocontrol bacterial strain, named Bacillus bailii (Bacillus velezensis) LMY3-5 (hereinafter also referred to as LMY 3-5), is deposited at China general microbiological culture Collection center with the accession number CGMCC N0:29700, with the date of deposit being 2024, 01 and 24, and with the address of deposit at the institute of microbiology, national academy of sciences 3, proc. North Star, beijing, kogyo.
Example 2
The method for obtaining the bacillus bailii LMY3-5 comprises the following steps:
The first step is separation:
Washing kiwi fruits by adopting a flat dilution method, soaking the kiwi fruits in 70% ethanol for 30s, washing the kiwi fruits with sterile water for 3 times, sterilizing the kiwi fruits with 2% sodium hypochlorite for 3min, and washing the kiwi fruits with sterile water for 4-5 times. Grinding whole kiwi fruits in a sterile mortar, taking supernatant, diluting the supernatant with sterile water according to a proportion of 10 -2、10-3, coating the diluted supernatant on an LB solid culture medium, inversely culturing the mixture in a dark way for 24 hours in a 37 ℃ incubator, picking different single colonies, repeatedly purifying the single colonies on the LB culture medium by a streaking method, and preserving glycerol.
Second step of preliminary screening
The kiwi fruit soft rot (B.dothida) is used as a target bacterium, and the activity screening of the separated and purified bacteria is carried out by adopting a plate counter method. Inoculating kiwi fruit soft rot fungi cake (diameter of 0.5 cm) in the center of a PDA dish, taking the center of a flat plate as the center, inoculating a loop of bacteria at four equidistant positions 2.5cm away from the center of the flat plate, culturing for 7d at 28 ℃, setting a control, recording the antibacterial half-way of biocontrol fungi, and taking the strain with the radius of the antibacterial circle larger than 1.0cm as a candidate strain of a re-screening.
Through the primary screening, 14 strains of bacteria with primary screening activity are obtained, wherein the numbers are 5, 17, 18, 23, 29, 32, 33, 36, 37, 39, 40, 45, 61 and 62 respectively. As shown in table 1.
TABLE 1 radius of zone of inhibition for primary screening strains
Third step of re-screening
14 Strains with the radius of the inhibition zone larger than 1.0cm in the primary screen are respectively streaked on an LB culture medium to form single colonies, 1 loop of single colonies are picked up in 50mL of LB liquid culture medium, and then the single colonies are placed in a shaking incubator at 28 ℃ for 2d of culture. Inoculating a B.dothidea bacterial cake of activated 7d to the center of a PDA culture medium, respectively inoculating 5 mu L of the same bacterial strain liquid at 2 equidistant positions which are 2.5cm away from the center of the PDA culture medium, inoculating the bacterial cake only to the center of the PDA culture medium as a contrast, culturing in a culture box at 28 ℃ until a contrast bacterial colony grows on a flat plate, measuring the diameter of the pathogenic bacterial colony, calculating the bacteriostasis rate, and selecting bacillus beijerinus LMY3-5 with the highest bacteriostasis rate; the calculation formula of the bacteriostasis rate is as follows: antibacterial ratio (%) = [ (control group colony diameter-treatment group colony diameter)/treatment group colony diameter ] ×100;
The 14 strains of biocontrol bacteria screened at the beginning have the bacteriostasis rate of 56.35-70.63%, wherein the strain with the number of 3-5 (namely the strain with the number of LMY 3-5) has the best activity, and the mycelium growth inhibition rate reaches 70.63 percent, as shown in the table 2.
TABLE 2 inhibition of double screened strains
| Strain numbering | Inhibition ratio (%) |
| 3-5 | 70.63±0.69a |
| 3-17 | 65.48±1.19abc |
| 3-18 | 62.30±2.75cde |
| 3-23 | 68.65±0.69ab |
| 3-29 | 58.73±0.69ef |
| 3-32 | 61.90±1.19cde |
| 3-33 | 60.71±1.19cdef |
| 3-36 | 61.51±2.48cdef |
| 3-37 | 56.35±1.82f |
| 3-39 | 64.29±2.06bcd |
| 3-40 | 60.32±1.37cdef |
| 3-45 | 59.92±1.37def |
| 3-61 | 60.32±2.94cdef |
| 3-62 | 60.12±1.57def |
Example 3
Identification of bacillus bailii LMY3-5, including morphological and molecular biological identification, is specifically as follows:
Step 1 cell staining for morphology
The strain LMY3-5 is streaked on LB plates for activation, and cultured overnight in an incubator at 37 ℃. And observing the characteristics of the shape, the color, the size and the like, and photographing and recording. The strain was stained by gram staining and observed under a microscope.
The results showed that strain LMY3-5 forms milky colonies after 24h of culture on LB solid medium, and the surface was rough, wrinkled, irregular, and opaque (FIG. 1A). In gram staining, the staining result was purple, indicating that strain LMY3-5 was a gram positive bacterium (FIG. 1B).
Step 2 molecular biological identification
Referring to Biomiga company Bacteria DNA Kits, DNA of the biocontrol strain was extracted by the procedure of the extraction kit. The 16S rRNA and gyrA sequences of endophytes were amplified using the obtained DNA as a template, using the 16S rRNA gene primer (upstream primer: 5'-AGAGTTTGATCCTGGCTCAG-3', downstream primer: 5'-GGTTACCTTGTTACGACTT-3') and the gyrA gene primer (upstream primer: 5' -CAGTCAGGAAATGCGTACGTCCT, downstream primer: 5'-CAAGGTAATGCTCCAGGCATTGCT-3'), respectively. The PCR product was sent to the engineering (Shanghai) Co.Ltd for sequencing. Sequence comparison is carried out on GenBank in NCBI according to the sequencing result, and a sequence with higher homology is downloaded from a GenBank database as a reference sequence. Phylogenetic trees were constructed using MrBaye, raxML on CIPRES SCIENCEGATEWAY v.3.3 website. The obtained tree file is viewed with FigTree v.1.4.0.
The results show that: the 16S rRNA and gyrA sequences of LMY3-5 strain were submitted to NCBI, accession numbers PP231028 and PP239377, respectively. Blast comparison is carried out on the sequenced 16S rRNA and gyrA genes, corresponding sequences of 19 bacillus strains with higher homology are analyzed and selected as reference sequences, multi-gene system development analysis is carried out, and the strains LMY3-5 and Bacillus velezensis are gathered together, wherein the support rate is 100/1. The strain LMY3-5 was identified as Bacillus velezensis, combining morphological and molecular systematic results.
EXAMPLE 4 preparation of Bacillus bailii LMY3-5 fermentation broth
LMY3-5 strain preserved on glycerin is streaked on LB solid medium to activate strain, 1-loop fungus is selected from single colony obtained by activation and inoculated in 50mL of LB liquid medium, and shaking culture is carried out for 24h at 30 ℃ and 200r/min to obtain seed liquid. Inoculating 5mL of seed solution into 100mL of LB liquid medium, shaking and culturing at 30 ℃ and 200r/min for 3d, centrifuging at 12000r/min for 15min to obtain supernatant, and sterilizing the supernatant at 121 ℃ for 30min to obtain fermentation broth.
The formula of the LB solid medium is as follows: yeast powder 5g, peptone 10g, nacl 5g, agar 17g, distilled water 1000mL. Sterilizing at 121deg.C for 30 min.
The formula of the LB liquid medium comprises the following components: yeast powder 5g, peptone 10g, naCL 5g, distilled water 1000mL. Sterilizing at 121deg.C for 30 min.
EXAMPLE 5 inhibition of Bacillus bailii LMY3-5 fermentation broth on the growth of actinidia soft rot pathogen hyphae
Adding 1%, 2%, 4%, 8%, 16% (volume ratio) of LMY3-5 fermentation broth into PDA culture medium cooled to about 50deg.C, mixing, and making into flat plate with blank PDA as control. Inoculating pathogenic bacteria cake at the center of the plate, and culturing at 28deg.C. 3 replicates. Measuring the diameter of the colony after hypha in the blank PDA grows on a flat plate, and calculating the inhibition rate; inhibition ratio (%) = [ (control group colony diameter-treatment group colony diameter)/control group colony diameter ] ×100;
Results: the strain LMY3-5 fermentation liquor has remarkable inhibition effect on the growth of B.dothidea and D.eres hyphae (figure 2), and the antibacterial activity of the strain LMY3-5 fermentation liquor is gradually enhanced along with the increase of the concentration of the fermentation liquor in a culture medium. When the addition of the fermentation broth reached 16%, the inhibition rates for pathogenic bacteria B.dothida and D.eres reached 83.51% and 80.68%, respectively (Table 3)
TABLE 3 inhibitory Effect of bacterial Strain LMY3-5 fermentation broths on B.dothidea and D.eres hypha growth
EXAMPLE 6 inhibition of Bacillus bailii LMY3-5 fermentation broth against the germination of actinidia soft rot pathogen spores
The conidium formed on the B.dothidea plate was scraped off and diluted with sterile water to a 1X 10 6mL-1 spore suspension. 1mL of each of the fermentation broth of example 4 and the spore suspension of B.dothidea were mixed, and the mixture was incubated at 28℃for 12 hours with a sterile LB medium as a control, and the germination of spores of the two groups of treated pathogenic bacteria was observed under an optical microscope, and each treatment was repeated 3 times, and the spore germination rate and the spore germination inhibition rate were counted and compared.
Spore germination rate (%) =number of spores germinated/total number of spores×100
Spore germination inhibition (%) = (control germination rate-treatment germination rate)/control germination rate×100.
Results: when the addition amount of the fermentation liquor is 16.00% (volume ratio), the inhibition effect of the antagonistic bacteria LMY3-5 fermentation liquor is highest (figure 3), the spore germination rate is only 2.47% at the lowest, and the spore germination inhibition rate is 97.32% (table 4). The second was 8.00% and 4.00% by volume of the broth treated groups, respectively, with spore germination rates of 25.11% and 42.72% respectively, and spore germination inhibition rates of 72.76% and 53.65% respectively (Table 4). The result shows that the LMY3-5 fermentation liquor can effectively inhibit the spore germination of B.dothidea, and the inhibition effect of the strain LMY3-5 on the spore germination is stronger along with the increase of the concentration of the fermentation liquor in the PDB culture medium.
TABLE 4 inhibition of B.dothidea spore germination by LMY3-5 fermentation broth
Example 7 Bacillus bailii LMY3-5 fermentation broth for controlling Kiwi fruit
Picking fresh and healthy kiwi fruits with consistent sizes, sterilizing the surfaces, puncturing 3 wounds on the surfaces of each kiwi fruit by using a sterile toothpick, and soaking the kiwi fruits in 1%, 2%, 4%, 8% and 16% of fermentation broth of example 4 for 60min. Taking out, and naturally airing in a sterile culture dish. Inoculating pathogenic bacteria cake on wound of fructus Actinidiae chinensis, soaking in sterile water, and inoculating pathogenic bacteria as control. 10 kiwi fruits are treated each, and the kiwi fruits are placed in a 28 ℃ and 90% relative humidity incubator for cultivation, and the diameter of the disease spots is measured after 7 days. Fruit spot inhibition (%) = (control spot diameter-treatment spot diameter)/control spot diameter x 100.
Results: the kiwi fruits of the control group without fermentation liquid are serious in disease occurrence, the diameter of the disease spots is large, and the fruits are rotten. The kiwi fruits treated by the fermentation liquid have light disease occurrence and small disease spot diameter. The higher the concentration of the fermentation broth, the smaller the lesion diameter, and the better the inhibition effect (FIG. 4; table 5). The inhibition rates of 16% fermentation broth treated kiwi fruit inoculated with b.dothidea and d.eres were 73.59%,73.39%, respectively (fig. 4; table 5). The results show that the strain LMY3-5 can effectively inhibit the infection of B.dothidea and D.eres, slow down the spreading speed of the disease spots and has obvious control effect on the soft rot of the kiwi fruits.
TABLE 5 inhibition of B.dothidea and D.eres fruit lesions by LMY3-5 fermentation broth
EXAMPLE 8 B.beijerinus LMY3-5 fermentation broth for B.dothidea hypha morphology observation
The B.dothidea hyphae morphology was observed using a Scanning Electron Microscope (SEM) (SU-8010, hitachi, tokyo, japan). The mycelia of the pathogenic bacteria treated with the fermentation broth of example 4 were collected for 12 hours and completely immersed in glutaraldehyde fixative overnight at 4℃and rinsed 3 times with phosphate buffer (0.1M, pH to 7.2) 15min each after removal of the fixative. Gradient dehydration was then performed with 30%, 50%, 70%, 80%, 90% and 100% ethanol for 20min each. And immersing the sample into isoamyl acetate for replacement for 2 times for 30min each time, and placing the sample under a scanning electron microscope for observation and image acquisition after vacuum freeze drying treatment.
The result is shown in figure 5, the surface of the CK mycelium is full and complete, no shrinkage cavity exists, and the thickness is uniform; after fermentation liquid treatment, the surface of mycelium starts to have abnormal structure, collapse and collapse mycelium fracture. Therefore, the fermentation liquor damages the cell wall and membrane structure of hyphae, and influences the normal physiological metabolism of the hyphae, so that the hyphae are shrunken and gradually apoptosis.
EXAMPLE 9 ultra-structural observations of B.dothidea cells with Bacillus bailii LMY3-5 fermentation broths
The microstructure of b.dothidea cells was observed using a Transmission Electron Microscope (TEM) (EM 1200EX, JEOL). Samples were prepared as in example 8, and after 30%, 50%, 70%, 80%, 90% and 100% ethanol series gradient dehydration treatment, the samples were treated with pure acetone solution for 20min; embedding with Epon812 fixative, ultrathin slicing, double staining with uranium acetate and lead citrate, and observing and collecting images with a transmission electron microscope.
As shown in FIG. 6, the mycelium cells not treated with the fermentation broth of example 4 remained healthy, the cell walls and cell membranes were normal in shape, and the organelles were well defined and evenly distributed in the cytoplasm. In contrast, hyphal cells after 12h of fermentation broth treatment were not complete in structure, blurred in cell wall profile, contracted cell membranes, loose in internal structure, severely dissolved organelles, and large-area cavities were present. The fermentation broth of example 4 was shown to disrupt b.dothidea cell structure, damage cell membranes, promote increased cell membrane permeability, leakage of contents, and decrease in cytoplasmic density, resulting in limited growth and development of b.dothidea hyphae, and even death.
EXAMPLE 10 determination of B.dothidea PI staining from Bacillus bailii LMY3-5 fermentation broth
The mycelia of the pathogenic bacteria (B.dothidea) treated with the fermentation broth of example 4 for 12 hours were collected, 500. Mu.L of 1mg/mL PI dye solution was added, a light-resistant water bath was performed at 30℃for 30min, and the excess dye solution was washed with PBS. Images were observed and collected using a confocal fluorescence microscope (NE 910-FL, optical company, inc. Of zhejiang Ningbo Yongxin).
The result shows that Propidium Iodide (PI) can permeate the cell membranes of dead cells and cells in the middle and late stages of apoptosis to dye the cell nuclei, and can intuitively reflect the damage degree of hypha cell membranes. As shown in FIG. 7, after 12h of fermentation broth treatment, the B.dothidea hyphae showed red fluorescence under a fluorescence microscope, and the red fluorescence intensity increased with increasing concentration of fermentation broth. The results indicate that the fermentation broth has a significant disruption of b.dothidea cell membrane integrity (fig. 7).
EXAMPLE 11 Bacillus bailii LMY3-5 assay for B.dothida cell content release
The B.dothidea was placed in 150mL PDB and shake-cultured for 3d, and the culture broth was removed by filtration in an ultra clean bench, and the wet mycelia were collected. Equal amounts of mycelia (1 g) were weighed and resuspended in PDB medium containing 1%, 2%, 4%, 8%, 16% (volume ratio) of the fermentation broth of example 4 for 0.5h, 1h, 3h, 6h, 12h, 24h, and centrifuged at 6000rpm for 15min, and the supernatant was subjected to multifunctional enzyme-labeling apparatus (superMax 3100, shanghai flash Biotechnology Co., ltd.) to determine absorbance at 260nm and 280 nm. The relative leakage of nucleic acid and protein was calculated 3 times per treatment.
Nucleic acids and proteins are important cell components, and are indicators for detecting cell membrane permeability. As shown in FIG. 8, the extracellular protein and nucleic acid content of pathogenic bacteria increased with increasing treatment time and increasing concentration of fermentation broth, while the CK group did not change much. After 6h of fermentation broth treatment, the B.dothidea hypha intracellular nucleic acids and proteins begin to leak in large quantities. The fermentation liquid is proved to destroy the cell membrane, increase the permeability of the cell membrane, and lead to the increase of dosage effect of nucleic acid, protein and leakage.
EXAMPLE 12 determination of B.dothidea cell wall enzyme by Bacillus bailii LMY3-5 fermentation broth
Mycelia were prepared as in example 11, and after equal amounts of mycelia (1 g) were weighed and resuspended in PDB medium containing 1%, 2%, 4%, 8%, 16% (by volume) of the fermentation broth of example 4, each treatment was repeated 3 times for 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours. Referring to the beta-1, 3-glucanase and chitinase activity detection kit, the crude body fluid was extracted and its enzyme activity was detected using a multifunctional microplate reader (superMax, 3100, shanghai flash spectroscopy biotechnology Co., ltd.).
The results show that the overall β -1,3 glucanase and chitinase activity of b.dothidea showed a tendency to increase and then decrease. Chitinase activity reached a maximum after 12h of treatment and beta-1, 3 glucanase activity reached a higher level at 24h of treatment. And at each time point of determination, the activity of B.dothidea beta-1, 3 glucanase and chitinase is obviously higher than that of the control under the treatment of fermentation liquor. It was demonstrated that fermentation broth caused an increase in cell wall key enzyme activity, leading to progressive degradation of the cell wall (fig. 9).
EXAMPLE 13 identification of Bacillus bailii LMY3-5 GC/LC-MS fermentation broth composition
The components of the fermentation broth of example 4 were identified by GC/LC-MS. GC-MS the identity of the LMY3-5 broth was determined by Agilent 7890B Gas Chromatography (GC) combined with Agilent 5977B Mass Spectrometry (MS) (Agilent Technologies, USA) using a DB-5MS capillary column (30 m.times.0.25 mm, film thickness 0.25 μm). LC-MS was analyzed by LC-MS (API 2000 TM, AB Sciex, redwood City, calif., USA) eluting with a water/acetonitrile mobile phase containing 0.05% TFA. Mass spectrometry uses a triple quadrupole spectrometer equipped with an electrospray ionization (ESI) source.
GC-MS results showed that 181 differential metabolites were identified in total. It is reported in the literature that 12 of the metabolic components show antibacterial activity, see table 6.
LC-MS results showed that 1508 compounds were detected in the positive spectrum, 122 above Score 80. 1393 compounds were detected by negative spectra, 82 above Score 80. It is reported in the literature that 17 of the metabolic components show antibacterial activity, as shown in Table 7.
TABLE 6 GC-MS analysis of bacteriostatic Activity in LMY3-5 fermentation broth
TABLE 7 LC-MS analysis of bacteriostatic Activity in LMY3-5 fermentation broth
EXAMPLE 14 antibacterial Activity of Bacillus bailii LMY3-5 against various pathogenic bacteria
Selecting 1-loop biocontrol strain LMY3-5 single colony in 50mL of LB liquid medium, and then placing the culture medium in a shaking incubator at 28 ℃ for 2d at 200r/min to obtain LMY3-5 bacterial liquid. The method comprises the steps of taking pathogenic bacteria of kiwi fruit soft rot (B.dothidea), kiwi fruit soft rot (D.eres), american and Australian brown rot (M.fructicola), cross black spot (A.alternata) and tea anthracnose (C.gloeosporioides) as target pathogenic bacteria, inoculating 7d of activated pathogenic bacteria cakes to the center of a PDA culture medium (the diameter of the cakes is 0.5 cm), inoculating 5 mu L of LMY3-5 bacterial liquid at 2 equidistant positions which are 2.5cm away from the center of the PDA culture medium, taking the cakes as a control, culturing in a 28 ℃ incubator until the control colony grows into a flat plate, measuring the diameter of the pathogenic bacteria colony, and calculating the antibacterial rate.
The calculation formula of the bacteriostasis rate is as follows: antibacterial ratio (%) = [ (control group colony diameter-treatment group colony diameter)/treatment group colony diameter ] ×100;
The growth inhibition of Bacillus berryis LMY3-5 on mycelia of kiwi fruit soft rot (B.dothidea), kiwi fruit soft rot (D.eres), brown rot of American Australian stone fruit (M.fructicola), black spot of Cruciferae (A.alternata), and colletotrichum tea anthracnose (C.gloeosporioides) was observed and the results are shown in Table 8 and FIG. 10.
Table 8 growth inhibition ratio of Bacillus bailii LMY3-5 to mycelium of various pathogenic bacteria
Claims (7)
1. The bacillus beleiensis is characterized in that the bacillus beleiensis is named as bacillus beleiensis (Bacillus velezensis) LMY3-5 and is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC N0:29700.
2. The fermentation broth of bacillus belgium according to claim 1.
3. The secondary metabolite produced by the fermentation of bacillus belgium of claim 1.
4. The use of bacillus belgium according to claim 1 or of a fermentation broth according to claim 2 for the preparation of a formulation for inhibiting phytopathogenic bacteria, wherein the phytopathogenic bacteria are kiwi fruit soft rot (Botryosphaeria dothidea), kiwi fruit soft rot (Diaporthe eres), brown rot of the american australia type (Moniliniafructicola), black spot of the cruciferae (ALTERNARIA ALTERNATA), tea anthracnose (Colletotrichum gloeosporioides).
5. The use of bacillus belgium according to claim 1 or of a fermentation broth according to claim 2 for the preparation of a formulation for controlling diseases caused by phytopathogens, wherein the phytopathogens are kiwi fruit soft rot (Botryosphaeria dothidea), kiwi fruit soft rot (Diaporthe eres), mayonnaise brown rot (Monilinia fructicola), alternaria cross (ALTERNARIAALTERNATA) and tea anthracnose (Colletotrichum gloeosporioides).
6. Use of bacillus beljavensis according to claim 1 or a fermentation broth according to claim 2 or a secondary metabolite according to claim 3 for the preparation of a control formulation for the fruit rot of kiwi fruits.
7. A biocontrol agent comprising bacillus belicus according to claim 1, and/or the fermentation broth according to claim 2, and/or the fermentation-produced secondary metabolite according to claim 3.
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