CN117866809A - Tobacco endophytic bacillus belicus and application thereof - Google Patents
Tobacco endophytic bacillus belicus and application thereof Download PDFInfo
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to the fields of microorganism and biological control, in particular to bacillus bailii with broad antibacterial spectrumBacillus velezensis) And applications thereof. The strain is endophytic antagonistic bacteria isolated from tobacco of continuous cropping for many years, and is safeTibetan number GDMCC No.64125. The strain has a wide antibacterial spectrum, has remarkable inhibiting effect on pathogenic bacteria such as tobacco bacterial wilt, tobacco black stem disease, tobacco brown spot and the like, can produce siderophores and indole-3-acetic acid (IAA), has the capability of dissolving organic phosphorus, has a 51.29% effect on preventing and treating the tobacco bacterial wilt, and has good disease resistance and growth promotion potential.
Description
Technical Field
The invention belongs to the fields of microorganism and biological control, and particularly relates to bacillus subtilis (Bacillus velezensis) capable of antagonizing bacterial wilt and application thereof.
Background
Tobacco is one of the economic crops with the largest planting area in China, tobacco is one of the dominant and characteristic industries in Hunan province, and the annual planting area is about 9 ten thousand hm 2 . However, as tobacco continues to crop year by year, tobacco soil-borne diseases in the Hunan province occur seriously year by year, wherein bacterial wilt and black stem disease are two most serious destructive soil-borne diseases on the tobacco in the province, pathogenic bacteria of the bacterial wilt and the black stem disease can survive in soil in a disease area for a long time and are difficult to cure radically, and the whole growth period of the tobacco can be endangered, so that serious economic loss is caused. Chemical pesticides and paddy-upland rotation are adopted as effective soil-borne disease control methods at present, but the method has the advantages of high cost, difficult operation, serious damage to soil micro-ecology and environmental pollution. Biological control of soil-borne diseases using antagonistic microbial agents is considered as an environmentally friendly control measure, and has become a hot spot of research in recent years. However, it is very difficult to obtain a good antagonistic micro-organism.
The research reports on antagonistic bacteria screening and biological control of tobacco soil-borne diseases are more, mainly focus on screening antagonistic bacteria, antagonistic streptomyces and the like in rhizosphere soil, and have little effect on screening endophytes. Compared with soil microorganisms, endophytes can enter plants through root systems besides being planted in rhizosphere soil, have a more stable living environment than microorganisms exposed to external environment, are easier to play the roles of disease resistance and growth promotion, and show a broad prospect of the plant endophytes as environment-friendly biopesticides.
Disclosure of Invention
The inventor tries to collect plants which remain healthy in the period of disease peak from a high disease tobacco field continuously cultivated for many years, separates an endophytic antagonistic bacterium HL-11 from the root system of the plants, identifies bacillus belicus (Bacillus velezensis), has obvious inhibition effect on Ralstonia tobacco (Ralstonia solanacearum), has obvious antibacterial activity on Ralstonia tobacco, carries out primary identification on the endophytic antagonistic bacterium, and determines growth promoting potential factors of the endophytic antagonistic bacterium. Finally, the present invention has been completed.
The present invention provides bacillus beleimeris (Bacillus velezensis) having accession No. GDMCC NO:64125.
the bacillus belicus is numbered HL-11, belongs to endophytic antagonistic bacteria, and is preserved in the microorganism strain collection center of Guangdong province (address: no. 100 building of laboratory building 5, GDMCC, for short) at 12 months 8 of 2023, wherein the preservation number is GDMCC NO:64125, classified under Bacillus velezensis.
The invention further provides application of the bacillus beijerinus in biological control. In particular, the biocontrol is for tobacco. More preferably, the biological control object is tobacco bacterial wilt.
The present invention also provides a biocontrol agent characterized by comprising the bacillus belicus as an active ingredient. Wherein preferably, the bacillus beijerinus strain after culture is used as an active ingredient.
The invention also provides a plant growth promoter, which contains the bacillus beijerinckii as an active ingredient. Specifically, it is added as a fertilizer additive to fertilizers.
Compared with the reported tobacco antagonistic bacteria, the endophytic antagonistic bacteria HL-11 separated by the invention can produce siderophores and indole-3-acetic acid (IAA) and has stronger capability of dissolving organic phosphorus, shows good disease resistance and growth promotion potential, and has the potential of being developed into a tobacco soil-borne disease biocontrol microbial agent or a disease resistance and growth promotion microbial fertilizer.
Drawings
FIG. 1 is a colony morphology of HL-11 strain.
FIG. 2 shows the spore morphology under a colony microscope of HL-11 strain.
FIG. 3 shows the antibacterial activity of antagonistic bacteria HL-11 against ralstonia solanacearum (Ralstonia solanacearum).
FIG. 4 shows the antibacterial activity of the supernatant of the fermentation broth of antagonistic bacteria HL-11 against ralstonia solanacearum (Ralstonia solanacearum).
FIG. 5 is a phylogenetic diagram of the antagonistic bacterium HL-11.
The control effect of the endophyte HL-11 microbial inoculum on tobacco bacterial wilt is shown in fig. 6.
Detailed Description
The invention separates 1 strain of endogenous antagonistic bacteria HL-11 from tobacco continuous cropping for many years, and the specific separation process is as follows.
1 materials and methods
1.1 test materials
In 2017, healthy tobacco plants are collected from high-incidence fields of the bacterial wilt of the Litsea river in Ningxiang province of Hunan, the tobacco variety is cloud tobacco 87 planted in a large area in Hunan, the test field is continuous 3a continuous cropping tobacco, and the bacterial wilt is serious.
The test plant pathogen Ralstonia solanacearum (Ralstonia solanacearum) GM1000 was provided by the teachings of Hunan agricultural university Chen Wufu.
Endophytic bacteria are separated by using LB culture medium and 1/10LB culture medium, conventional bacterial culture is performed by using LB culture medium, pathogenic fungi culture medium is performed by using PDA culture medium, ralstonia solanacearum is performed by using TTC culture medium (Jiang Huanhuan, cheng Kai, yang Xingming, etc.. Screening of antagonistic bacteria against tobacco bacterial wilt and biological control effect [ J ]. Soil theory, 2010, 47 (6): 1225-1231.), and liquid fermentation culture medium is performed by using Landy culture medium (Buddha, lv Fengxia, liu Zhaoxin, etc.. Bacillus subtilisfmbJ separation and identification of lipopeptides antibacterial substance [ J ]. Biological engineering report, 2006, 22 (4): 644-649.). Except PDA culture medium, the pH of the rest culture medium is 7.0-7.2.
LB medium: 10g of tryptone, 10g of sodium chloride, 5g of yeast extract, 20g of agar and 1L of distilled water. Landy medium:
PDA medium: 200g of potato, 20g of glucose and 15g of agar powder, and adding water to 1000mL.
1.2 test methods
1.2.1 isolation and purification of endophytes
Isolation of endophytes was finely tuned with reference to the prior art (Yuan Shanshan. Isolation of rice endophytes OsiSh-10 and study of rice blast resistance and plant growth promotion [ D ]. Changsha: university of Hunan, 2015). Cutting main roots and lateral roots of tobacco, cleaning sediment with clear water, putting the tobacco into an ultra-clean workbench for blow-drying, then putting the tobacco into disodium hydrogen phosphate for ultrasonic treatment for 1min to further remove surface impurities, taking out root systems, sequentially carrying out surface disinfection by absolute ethyl alcohol, 4% sodium hypochlorite and sodium thiosulfate, washing out residual disinfectants by a large amount of sterile water after disinfection, coating LB (liquid-solid) plates with cleaning liquid in the last step for detecting whether disinfection is thorough or not, and blow-drying thoroughly disinfected root systems in the ultra-clean workbench. Then put into a sterilized mortar to be ground into slurry, 1, 10 and 50 mu L of juice are taken to be coated on LB and 1/10LB plates, the slurry is cultured for 3 to 7 days in a 30 ℃ incubator, and single colony is selected to be streaked on the LB plates, thus obtaining pure cultures.
1.2.2 screening of endophyte antagonistic bacteria
The antagonism test of bacterial wilt adopts a double-layer flat plate method, bacterial wilt is added into a TTC culture medium, the flat plate is poured, the screened bacteria are inoculated on the flat plate, the culture is carried out for 2 days, and the size of a bacteriostasis zone is observed and measured.
1.2.3 determination of antibacterial Activity of endophyte fermentation supernatant
Taking a loop of HL-11 glycerol bacterial liquid from a refrigerator at the temperature of minus 80 ℃, streaking the glycerol bacterial liquid on an LB plate, and culturing the glycerol bacterial liquid in an incubator at the temperature of 30 ℃ for 24 hours; 1 single colony is selected and inoculated into 1.5mL NB culture medium, and cultured overnight at 30 ℃ and 900 rpm; 1mL of overnight bacteria are transferred into 50mL of NB medium, and cultured for 48 hours at 30 ℃ and 180 rpm; transferring the fermentation liquor into a 50ml centrifuge tube, and centrifuging at 8000rpm for 5-10 min; filtering the fermentation supernatant with a 0.22 μm filter membrane; 200 mL of LB solid medium is added with a certain amount (about 5-10 mu l) of pathogenic bacteria liquid (bacterial wilt), the plates are poured, and 15mL of medium is poured into each plate; after the plate is solidified, a plurality of holes are punched by a puncher, agar blocks in the holes are taken out, 100 mu l of fermentation filtrate is added, the plate is placed in a 30 ℃ incubator for culture for 24 hours, and the size (mm) of a bacteriostasis zone is measured.
1.2.4 preliminary identification of endogenous antagonistic bacteria
The genome of the endophyte is extracted by using a UNIQ-10 column type bacterial genome DNA extraction kit, and the method is referred to the kit instruction. After the endogenous antagonistic bacteria genome is extracted, sampling is carried out, agarose gel electrophoresis is carried out to detect the extraction quality of the genome, and a micro spectrophotometer Nano Drop One is used for measuring the DNA concentration. Then, the bacterial universal 16S rDNA 27F/1492R primer is used to PCR amplify the antagonistic bacterial 16S rDNA by using the endogenous antagonistic bacterial genome DNA as a template, and the amplified product is sent to sequencing. PCR amplification system, system: PCR Mix 25. Mu.L, primer 27F (10. Mu. Mol/L) 1. Mu.L, primer 1492R (10. Mu. Mol/L) 1. Mu.L, template DNA about 10pmoL, and weight distilled water to 50. Mu.L. Amplification conditions: 98 ℃ for 5min;94℃35s,55℃35s,72℃1min,35 cycles; and at 72℃for 10min. A DNA gel recovery kit is adopted, target DNA fragments are recovered through agarose gel electrophoresis, and sequencing is carried out by Hunan qinghao biotechnology Co-Ltd; the sequencing results were compared for homology in GenBank using BLAST software, and after multiple sequence alignment using ClustalX, multiple sequence homology analysis was performed using software Mega 6 to construct a phylogenetic tree.
1.2.5 preparation of Engineer antagonistic bacteria HL-11 containing microbial inoculum and potting test for controlling tobacco bacterial wilt
Activating living bacteria of endophytic antagonistic bacteria HL-11, inoculating the activated bacteria into an LB liquid culture medium, and culturing at 28 ℃ and 150rpm until logarithmic growth phase to obtain seed liquid; inoculating the seed solution into LB liquid medium at a volume of 2%, and heating at 28deg.CShake culturing at 150rpm for 40-48 hr to obtain fermentation broth; pouring out the fermentation liquor, counting the final concentration of the fermentation liquor by a plate counting mode to obtain the effective viable count, centrifuging the fermentation liquor, discarding the supernatant, and diluting the fermentation liquor with water to obtain 10 according to the evaluation result 8 cfu/mL of the solution was used for tobacco potting experiments. The potting test set up two groups, blank (CK) and treatment (HL-11), each treated 15 plants, potting soil used vegetable field soil from the field, 2Kg of soil per pot. Tobacco seedlings of the pre-transplanting treatment group (HL-11) are planted after being subjected to root dipping treatment by using HL-11 bacterial liquid for 30 seconds, and then tobacco plants are irrigated with the prepared bacterial agent once every 10 days (the bacterial liquid concentration is 10) 8 cfu/ml), the dosage of the microbial inoculum is 10 ml/plant, and the microbial inoculum is irrigated for 2 times; the blank group uses clear water to replace microbial inoculum to carry out root soaking treatment and irrigation of tobacco plants, other water and fertilizer management conditions of each treatment group are completely the same, fertilization is carried out according to the fertilization dosage of peasant habit, and medicines for preventing and treating diseases are not used uniformly. After 15 days of planting, all seedlings were artificially inoculated with bacterial wilt pathogen. After 14 days, the growth and disease conditions of tobacco are observed, and the disease rate and disease index are counted.
1.2.6 detection of plant growth promoting Activity of endogenous antagonistic bacterium HL-11
In order to predict whether the endophytic antagonistic bacteria HL-11 have the potential of promoting plant growth in addition to disease-resistant plants, the methods of rhizosphere literature (Li Yin, yu Li, li Huixin, etc.) are used for screening and identifying peanut rhizosphere growth-promoting bacteria, and characteristic study [ J ] ecological and rural environment school, 2012, 28 (4): 416-421; [16] Meng Yuan ] screening and growth-promoting effect study [ D ] SiAN: north Western university, 2011) of hydrogen oxidizing bacteria of siderophores and ACC deaminase, and the capabilities of HL-11 siderophores, indole-3-acetic acid (IAA) production, organic phosphorus dissolution, inorganic phosphorus dissolution, potassium dissolution, nitrogen fixation and the like are tested.
2 results and analysis
2.1 isolation and screening of antagonistic bacteria
65 endophytes are separated from healthy tobacco plant roots in severe-onset fields, 17 strains with antibacterial activity on tobacco bacterial wilt are screened, 1 strain with the number of HL-11 has strong antagonism on bacterial wilt (Ralstonia solanacearum) GM1000, the diameter of a bacteriostasis ring reaches 12.5mm under the condition of plate-facing culture (figure 3), and the antibacterial diameter of fermentation supernatant on bacterial wilt reaches 24.6mm (figure 4).
HL-11 was on LB plate medium, the colony was pale yellow, the center of the colony had obvious folds (FIG. 1), and the thallus was rod-shaped under the microscope to form spores (FIG. 2).
2.2 endogenous antagonistic bacteria 16S rDNA sequence and homology analysis result
In order to further identify the species of the strain, genomic DNA of the HL-11 strain mentioned by the kit is adopted, the genome of the PEB-23 strain is taken as a target, a primer 27F/1492R is successfully amplified to a band of about 1.5kb, the 16S rRNA gene segment of the endophytic antagonistic bacterium HL-11 after sequencing is 1 427bp, a phylogenetic tree (figure 5) is constructed by the sequence and the homologous sequence, and the closest relationship between the HL-11 and the Bacillus belicus (Bacillus velezensis) CBMB205 (accession number: NZ CP 011973.1) is shown, and the homology reaches 99.86%.
The nucleotide sequence of the 16S rRNA gene of HL-11 is as follows:
ggctccataaaggttacctcaccgacttcgggtgttacaaactctcgtggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgc
ggcatgctgatccgcgattactagcgattccagcttcacgcagtcgagttgcagactgcgatccgaactgagaacagatttgtgggattggctt
aacctcgcggtttcgctgccctttgttctgtccattgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccaccttc
ctccggtttgtcaccggcagtcaccttagagtgcccaactgaatgctggcaactaagatcaagggttgcgctcgttgcgggacttaacccaac
atctcacgacacgagctgacgacaaccatgcaccacctgtcactctgcccccgaaggggacgtcctatctctaggattgtcagaggatgtca
agacctggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggcccccgtcaattcctttgagtttcagtcttgcga
ccgtactccccaggcggagtgcttaatgcgtaagctgcagcactaaggggcggaaaccccctaacacttagcactcatcgtttacggcgtgg
actaccagggtatctaatcctgttcgctccccacgctttcgctcctcagcgtcagttacagaccagagagtcgccttcgccactggtgttcctcc
acatctctacgcatttcaccgctacacgtggaattccactctcctcttctgcactcaagttccccagtttccaatgaccctccccggttgagccgg
gggctttcacatcagacttaagaaaccgcctgcgagccctttacgcccaataattccggacaacgcttgccacctacgtattaccgcggctgct
ggcacgtagttagccgtggctttctggttaggtaccgtcaaggtgccgccctatttgaacggcacttgttcttccctaacaacagagctttacgat
ccgaaaaccttcatcactcacgcggcgttgctccgtcagactttcgtccattgcggaagattccctactgctgcctcccgtaggagtctgggcc
gtgtctcagtcccagtgtggccgatcaccctctcaggtcggctacgcatcgtcgccttggtgagccgttacctcaccaactagctaatgcgccg
cgggtccatctgtaagtggtagccgaagccaccttttatgtctgaaccatgcggttcagacaaccatccggtattagccccggtttcccggagtt
atcccagtcttacaggcaggttacccacgtgttactcacccgtccgccgctaacatcagggagcaagctcccatctgtccgctcgac。
2.4 control effect of endophyte HL-11 on tobacco bacterial wilt
A potting experiment is carried out, and a graph for determining the prevention and control effect of the HL-11 strain on the bacterial wilt of tobacco is shown in fig. 6. According to the statistical results of Table 1, the control effect of the HL-11 microbial inoculum on the tobacco bacterial wilt can reach 51.29%, which shows that the independently used HL-11 microbial inoculum has better control effect on the tobacco bacterial wilt.
Table 1 potted plant control effect of endophyte HL-11 microbial inoculum on tobacco bacterial wilt
| Group of | Incidence (%) | Index of disease condition | Control effect (%) |
| HL-11 | 40.38±12.35 | 47.50±8.55 | 51.29±33.92 |
| CK | 94.30±6.72 | 96.53±1.37 | \ |
2.5 determination of the potential of the endophytic antagonistic bacterium HL-11 to promote plant growth
The determination of the capacities of HL-11 siderophore production, indole-3-acetic acid (IAA) production, organic phosphorus dissolution, inorganic phosphorus dissolution, potassium dissolution, nitrogen fixation and the like is carried out. As shown in Table 2, the endophytic antagonistic bacteria HL-11 also have strong capability of producing siderophores, IAA and dissolving organic phosphorus, show good disease resistance and growth promotion potential, and have potential of being developed into a tobacco soil-borne disease biocontrol microbial agent or a disease resistance and growth promotion microbial fertilizer.
The bacteria siderophore is detected by using CAS plate, firstly preparing solution I,60.5mg of Chrome Azure (CAS) is dissolved in 50ml of ultrapure water, and 10ml of Fe 3+ Mixing the solutions, and adding the mixed solution into hexadecyl trimethyl ammonium bromide (HDTMA) solution; re-compounding solution II,0.3g KH 2 PO 4 ，0.5g NaCl、1.0g NH 4 Cl,15g of agar powder, 30.24g of sodium guazinedicarboxylate (pins) and adjusting the pH to 6.8; and then other solutions are prepared: a 20% sucrose solution; 10% acid hydrolyzed casein; 1mmol/L CaCl 2 ，10mmol/L MgSO 4 . And (3) sterilizing independently, and sterilizing all culture media at 121 ℃ for 30min for later use. The above solution was melted by heating before the CAS plate was placed, cooled to 50℃and poured into the plate after mixing at a certain ratio.
Orange-yellow halos were produced on the plates, indicating that they had siderophore production capability. And IAA can be produced in the case of tryptophan addition. In addition, larger transparent hydrolysis circles can be produced on the medium with organic phosphorus as the sole phosphorus source, but smaller hydrolysis circles can be produced on the medium with inorganic phosphorus as the sole phosphorus source. Can grow well on the culture medium of potassium feldspar and Apis Bei Modan, which indicates that the culture medium can have potassium dissolving and nitrogen fixing activities.
TABLE 2 determination of growth-promoting index of Engineer antagonistic bacteria HL-11
| Growth promoting index | Iron production carrier | IAA production | Dissolving organic phosphorus | Dissolving inorganic phosphorus | Potassium decomposing process | Nitrogen fixation |
| Horizontal level | ++ | ++ | +++ | + | + | + |
Note that: "+++" indicates that the activity of the product is very high, "++" indicates that the activity is strong, "+" indicates that the activity is low and "-" indicates that there is no activity.
Claims (10)
1. Bacillus bailii @ and its preparationBacillus velezensis) Its deposit number GDMCC NO:64125.
2. the use of bacillus belgium according to claim 1 for biological control.
3. The use according to claim 2, wherein the biocontrol is for tobacco.
4. The use according to claim 2, wherein the biological control object is tobacco bacterial wilt, tobacco black stem disease and/or tobacco brown spot.
5. Use of bacillus belgium according to claim 1 for promoting plant growth.
6. The use according to claim 2, wherein the plant is tobacco.
7. A biocontrol formulation comprising bacillus beljalis according to claim 1 as active ingredient.
8. The biocontrol formulation of claim 7, wherein the cultured bacillus subtilis form is used as an active ingredient.
9. A plant growth promoter comprising the Bacillus bailii according to claim 1 as an active ingredient.
10. The plant biological promoter according to claim 9, which is added as a fertilizer additive to a fertilizer.
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