CN113151062B - Bacillus belgii LJBV19 and application thereof - Google Patents
Bacillus belgii LJBV19 and application thereof Download PDFInfo
- Publication number
- CN113151062B CN113151062B CN202110312353.5A CN202110312353A CN113151062B CN 113151062 B CN113151062 B CN 113151062B CN 202110312353 A CN202110312353 A CN 202110312353A CN 113151062 B CN113151062 B CN 113151062B
- Authority
- CN
- China
- Prior art keywords
- ljbv19
- bacillus
- growth
- strain
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- 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
Abstract
The invention discloses a Bacillus beilai LJBV19 separated from grape rhizosphere soil, which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.21804 and the preservation date of 2021, 2 months and 2 days; the invention also discloses a broad-spectrum disease-preventing growth-promoting microbial inoculum and application thereof in preventing and treating plant diseases caused by plant pathogenic bacteria. The bacillus beilesensis LJBV19 has broader disease prevention and growth promotion effects, and can effectively control plant diseases caused by pathogenic bacteria of rice, grape, solanaceae crops and forest trees, wherein the average inhibition rate of the bacillus beilesensis LJBV19 on rice blast germs is as high as 75.75%; can also promote the growth of plants, improve the crop yield, improve the fruit quality, improve the crop resistance, develop agricultural economy and promote agricultural development.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to bacillus beilesiensis LJBV19 and application thereof.
Background
In order to increase the income and yield of crops, chemical pesticides and chemical fertilizers are beginning to be used in agricultural production in large quantities. The chemical pesticide is frequently used in a large amount, so that the problems of environmental pollution and pesticide residue are easily caused, and meanwhile, pathogenic bacteria are easily caused to generate drug resistance. The long-term excessive use of chemical fertilizers causes the enrichment of soil microorganisms and the reduction of soil pH, so that crops cannot obtain the fertilizers, the quality and the yield of the crops are reduced, and the environmental pollution is caused directly or indirectly. Therefore, it is very important to develop a bio-fertilizer and a bio-pesticide which are environment-friendly, improve soil fertility, enhance plant resistance and have combined action of multiple control mechanisms.
Biological control is to control the population density of harmful organisms on the basis of ecology, has the advantages of no environmental pollution, no public nuisance and no drug resistance, avoids the harm of pesticide residue to human and animals, promotes the healthy and sustainable development of agriculture, and is incomparable to non-biological methods for controlling plant diseases and insect pests such as pesticides. The biological fertilizer has the advantages of high product safety, low cost, high economic benefit and the like, and can effectively improve the quality and the yield of crops and promote the growth of the crops.
Among them, plant rhizobacteria are ideal biocontrol agents and biofertilizers because they can colonize plant roots, inhibit plant pathogens and promote plant growth. Plant rhizobacteria are a general term for useful microorganisms that promote plant growth or antagonize pathogenic bacteria. Plant rhizosphere bacteria generate endogenous auxin through tryptophan metabolism, assimilate and utilize nitrate and regulate ACC deaminase activity to promote plant growth; on the other hand, plant rhizobacteria increase plant resistance by activating plant pathogenesis-related gene expression, producing substances that increase plant resistance such as siderophiles, and producing auxin-activated biosynthetic signaling pathways.
With the abuse of chemical pesticides and chemical fertilizers, soil pollution of different areas occurs to different degrees. Therefore, plant growth-promoting rhizobacteria are receiving increasing attention as novel environmentally friendly fertilizers. The bacillus, the bacillus polymyxa, the streptomycete, the pseudomonas, the trichoderma, the mycorrhizal fungi and the like are successfully applied to commercial production at present.
Disclosure of Invention
Aiming at the problems, the invention discloses Bacillus belgii, which has wide antibacterial spectrum, can effectively inhibit plant diseases, has the inhibiting effect on rice pathogenic bacteria, grape pathogenic bacteria, solanaceae crop pathogenic bacteria and forest pathogenic bacteria, and has the average inhibiting rate of rice blast as high as 75.75 percent.
The invention discloses a Bacillus belgii, which is named as Bacillus belgii LJBV19 and is preserved in the China general microbiological culture Collection center, wherein the preservation address is No. 3 of Beijing Kogyang area Beichen Lu West Lu No. 1, the preservation number is CGMCC No.21804, and the preservation date is 2021, 2 and 2 days.
The Bacillus belgii LJBV19 is obtained by separating from rhizosphere soil of grapes, and strains which have disease prevention and growth promotion functions are screened from 151 strains.
The invention also discloses a broad-spectrum disease-preventing growth-promoting microbial inoculum which comprises the Bacillus beiLeisi LJBV 19.
The invention also discloses application of the Bacillus belgii LJBV19 or the broad-spectrum disease-preventing growth-promoting microbial inoculum in preventing and treating plant diseases caused by plant pathogenic bacteria.
Preferably, the plant pathogenic bacteria include one or more of rice pathogenic bacteria, grape pathogenic bacteria, solanaceous crop pathogenic bacteria, and forest pathogenic bacteria.
Preferably, the plant pathogenic bacteria include one or more of rice blast (Magnaporthe oryzae), grape anthracnose (Colletotrichum viniferum), Fusarium solani (Fusarium solani), Verticillium solani (Verticillium dahliae), Helicoverruca umbiliciformis (Exserohilum rosetatu), Phytophthora capsici (Phytophtora capsici), Fusarium graminearum (Fusarium graminearum), Botrytis cinerea (Botrytis cinerea), grape white rot (Coniella diplodiella), potato blight (Fusarium oxysporum), sunflower blight (Fusarium equisetii), and Rhizoctonia solani (Rhizoctonia solani).
The invention also discloses application of the bacillus beilesensis LJBV19 or the broad-spectrum disease-preventing growth-promoting microbial inoculum in promoting plant growth.
Preferably, the original liquid of the fermentation liquid of the Bacillus belgii LJBV19 or the original liquid of the fermentation liquid of the Bacillus belgii LJBV19 is diluted by different concentrations, and then the plant is subjected to root irrigation.
Preferably, the content of viable bacteria in the original fermentation liquid of the Bacillus beleisis LJBV19 is 1.0 multiplied by 10 after being diluted 7 cfu/mL～1.0×10 9 cfu/mL, more preferably 1.0X 10 8 cfu/mL。
Preferably, the plant comprises a plant of the solanaceae family, further preferably comprises tomato and/or tobacco.
Compared with the prior art, the invention has the beneficial effects that:
(1) the bacillus beilesensis LJBV19 is separated from the rhizosphere soil of grapes, and has the functions of disease prevention and growth promotion;
(2) the bacillus beilesensis LJBV19 has a broad-spectrum plant disease prevention and control function, and can effectively control plant diseases caused by rice pathogenic bacteria, grape pathogenic bacteria, solanaceae crop pathogenic bacteria and forest pathogenic bacteria, wherein the average inhibition rate of the bacillus beilesensis LJBV19 on rice blast (M.oryzae) is as high as 75.75%;
(3) the Bacillus beleisi LJBV19 can effectively inhibit the growth of hyphae of pathogenic bacteria such as grape anthracnose, potato fusarium wilt, grape white rot, grape gray mold and the like;
(4) The bacillus beilesiensis LJBV19 can also promote the growth of plants, improve the yield of crops, improve the quality of fruits, improve the resistance of crops, develop agricultural economy and promote agricultural development.
Drawings
FIG. 1 shows the inhibition counter-plot of strain LJBV19 against grape pathogens, wherein: a is botrytis cinerea (c.viniferum), b is botrytis cinerea (b.cinerea), and c is botrytis cinerea (c.dipnodiella); the strain LJBV19 is on the left side of the plate;
FIG. 2 shows the morphological feature of the strain LJBV19 on LB solid medium;
FIG. 3 shows a morphological feature map of a single-cell colony of strain LJBV19 on LB solid medium;
FIG. 4 shows a map of spore-stained strain LJBV19, in which the cells were red and the spores were green;
FIG. 5 shows a gram-stained map of strain LJBV19, wherein strain LJBV19 is gram-stained purple;
fig. 6 shows a phylogenetic tree between strain LJBV19 and other closely related bacillus strains, wherein No. boottrap Replication is 1000;
FIG. 7 shows a panel of inhibition of the tested phytopathogens by Bacillus belgii LJBV19, in which: (A) (B), (C), (D), (E), (F), (G), (H), (I), (J), (K) and (L) respectively represent treatment groups, and (a), (B), (C), (D), (E), (F), (G), (H), (I), (J), (K) and (L) respectively represent control groups; (A) and (a): rice blast (m.oryzae); (B) and (b): colletotrichum viticola (c.viniferum); (C) and (c): fusarium solani (f.solani); (D) and (d): verticillium dahliae (v.dahliae); (E) and (e): helminthosporium umbilicalis (e.rostratu); (F) and (f): phytophthora capsici (p.capsica); (G) and (g): fusarium graminearum (f.graminearum); (H) and (h): botrytis cinerea (b.cinerea); (I) (ii), (i): botrytis cinerea (c. diplodiella); (J) and (j): potato blight (f.oxysporum); (K) and (k): sunflower fusarium wilt (f.equiseti); (L), (L): rhizoctonia solani (r.solani);
FIG. 8 is a photograph showing the hyphal morphology of a test phytopathogen observed at a 40-fold objective, wherein: (A) the (A), (B), (C) and (D) represent treatment groups, and the (a), (B), (C) and (D) represent control groups; (A) (a) is the hyphal form of colletotrichum viticola (C.viniferum); (B) and (b) is the hypha form of Botrytis cinerea (B.cinerea); (C) and (c) is the hypha form of the fusarium oxysporum (F.oxysporum); (D) and (d) is the hypha form of white rot of grape (C.dipnodiella);
FIG. 9 is a graph showing the growth promoting effect of Bacillus beleisi LJBV19 on tomatoes, wherein CK is a schematic diagram of whole tomato plants in the blank group and LJBV19 is a schematic diagram of whole tomato plants in the experimental group;
FIG. 10 is a graph showing the growth promoting effect of Bacillus beleisis LJBV19 on tobacco, wherein A is a schematic diagram of the whole plant of tobacco: a1 is a schematic diagram of the whole blank group of tobacco, a2 is a schematic diagram of the whole experimental group of tobacco; b is a schematic diagram of tobacco leaves: b1 is a schematic diagram of blank tobacco leaves arranged from the top 1 st leaf to the 7 th leaf in sequence from left to right, and b2 is a schematic diagram of experimental tobacco leaves arranged from the top 1 st leaf to the 7 th leaf in sequence from left to right.
Detailed Description
The invention is explained in more detail below with reference to the figures and examples. The features and advantages of the present invention will become more apparent from the exemplary descriptions.
(1) Preparation of test Medium
1) PDA culture medium: 200g of potato, 20g of glucose, 15g of agar powder and 1000mL of distilled water, and sterilizing for 20min by high-pressure steam at 121 ℃.
2) LB liquid medium: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, adjusting the pH value to 7.0, and performing high-pressure steam sterilization at 121 ℃ for 20 min. The LB solid medium was supplemented with 15g agar powder per liter of distilled water.
3) Citrate medium: NaCl 5.0g, MgSO 4 ·7H 2 O 0.2g，NH 4 H 2 PO 4 1.0g，K 2 HPO 4 1.0g, 2.0g of sodium citrate, 0.04% phenol red I0 mL, 15g of agar and 1000mL of distilled water.
4) Starch agar medium: 2.0g of beef extract, 17.0g of dried egg white, 15.0g of agar, 2.0g of soluble starch and 1000mL of distilled water.
5) Gelatin culture medium: 5.0g of peptone, 120.0g of gelatin, 3.0g of beef extract and l000mL of distilled water.
(2) Culture of test plant pathogenic bacterial strains
Pyricularia oryzae (M.oryzae), colletotrichum botryoides (C.viniferum), botrytis cinerea (C.diplodiella), Fusarium solani (F.solani), Helicoveromyces rostratus (E.rostratu), Phytophthora capsici (P.capsica), Fusarium graminearum (F.graminearum) and Rhizoctonia solani (R.solani) were cultured in an incubator at 28 + -1 ℃ in the dark, and Pseudoperonospora solani (V.dahliae), Botrytis cinerea (B.cinerea), Fusarium solani (F.oxysporum) and Helianthus annuus (F.equiseti) were cultured in an incubator at 22 + -1 ℃ in the dark. PDA medium was used for all the above strains. The test strains are inoculated in a PDA culture medium plate, activated and cultured in an incubator at corresponding temperature in the dark and then used for inoculation experiments.
Example 1: isolation and selection of strains
(1) Isolation of the Strain
Soil was taken from the rhizosphere of grapes, and sampling required was within 20cm of the surface down, since the root system of grapes was mainly distributed in this block. Soaking soil in normal saline (m: v ═ 1:10), shaking, standing for 30min, collecting supernatant after soil precipitation, and bathing at 80 deg.C for 20 min. Diluting the supernatant after warm bath by 1, 10, 100 and 1000 times, spreading on LB solid culture medium, culturing at 37 deg.C for 1 day, selecting milky white single bacterial spot with biological membrane, and streaking for storage. Amplifying the 16SrRNA gene by using a 27F/1492R primer, sequencing, comparing and reserving a single colony belonging to the bacillus. The single colony is inoculated in LB liquid culture medium and is subjected to shaking culture on a shaking table at 37 ℃ and 180rpm for 24 hours to obtain bacterial suspension. Mixing the bacterial suspension with 50% sterile glycerol at equal ratio, and storing at-80 deg.C.
(2) Screening of Strain LJBV19
Screening of the strain LJBV19 adopts a confrontation culture method. Inoculating bacterial dishes of grape anthracnose pathogen (C.viniferum), grape botrytis cinerea and grape white rot pathogen (C.dipnodiella) in the center of a PDA culture medium plate respectively, and then inoculating the primarily screened bacillus strains around the bacterial dishes for opposite culture. Grape anthracnose bacterium (C.viniferum) and grape white rot bacterium (C.dipnodiella) were cultured at 28 ℃ in the dark, and grape gray mold bacterium (B.cinerea) was cultured at 22 ℃ in the dark, and the bacteriostatic condition was observed, and the bacteriostatic band width was measured, and each treatment was repeated 3 times. And selecting a strain with obvious bacteriostatic effect according to the bacteriostatic rate, and naming the strain as LJBV 19.
As shown in fig. 1, the strain LJBV19 has different degrees of inhibition effects on the 3 grape pathogenic bacteria, wherein the strain LJBV19 has the strongest inhibition activity on grape anthracnose pathogen (c.viniferum), and the inhibition rate is 48.25%; the bacteriostasis rate of the compound bactericide on botrytis cinerea (B.cinerea) and botrytis cinerea (C.dipnodiella) is also more than 20 percent.
Example 2: identification of Strain LJBV19
(1) Physiological and biochemical characteristics of strain LJBV19
Observation of colony morphology: selecting appropriate amount of lawn for 24h culture, inoculating in LB liquid culture medium, and shake culturing at 37 deg.C and 180rpm on shaking table for 48 h. 3 μ L of the solution was dropped onto a plate, and the colony shape, size, edge, surface, ridge shape, transparency, color, and the like were recorded.
Gram staining: uniformly coating a proper amount of lawn cultured for 24h on a clean glass slide with a drop of distilled water in the center, air-drying, fixing the smear above the flame for several times, and then carrying out crystal violet staining for 1 min; washing with running water, and adding iodine solution dropwise for 1 min; washing with running water, and decolorizing with 95% alcohol for 30 s; washing with running water, and re-staining with safranin for 2-3 min; after being washed by running water, the mixture is dried in the air and examined under the microscope. Gram-positive bacteria are purple and gram-negative bacteria are red.
Spore staining: selecting appropriate amount of lawn cultured for 24 hr, uniformly coating on clean glass slide with a drop of distilled water, air drying, fixing the smear several times above the flame, adding malachite green water solution, clamping the glass slide with wooden clamp, heating for 6-8s above the flame, washing with running water, dyeing with safranin solution for 1min, washing with running water, air drying, and examining under microscope. The thalli is red, and spores are green.
Requiring salinity, selecting a proper amount of lawn cultured for 24h, respectively inoculating the lawn into LB liquid culture medium containing 2%, 5%, 7%, 10% and 15% NaCl, and performing shake culture on a shaker at 37 ℃ and 180rpm for 72 h. Measuring OD of the upper culture solution at 600nm with ultraviolet spectrophotometer 600 The growth of the LJBV19 strain in different NaCl-containing culture media is judged according to the absorbance value.
Citrate utilization: inoculating the strain cultured for 48h on citrate medium plate, culturing at 37 deg.C for 5-7d, and determining that the culture medium is positive when the color of the culture medium turns to dark blue or pink, otherwise, determining that the culture medium is negative.
Contacting with enzyme: and (3) directly dripping 10% hydrogen peroxide on a strain inclined plane cultured for 48h, wherein if a large amount of bubbles are generated on the surface of the culture medium, the culture medium is positive, and if no bubbles are generated, the culture medium is negative.
V-P determination: taking 1ml of bacterial suspension which is cultured for 48 hours at 37 ℃ and 180rpm in a shaking way, putting the bacterial suspension into a test tube, mixing the bacterial suspension with 0.6ml of alpha-naphthol, adding 0.2ml of potassium hydroxide solution, mixing the mixture evenly, standing the mixture for 2 hours and observing the result. A positive result is pink, otherwise negative.
M-R determination: taking 1ml of bacterial suspension cultured for 48h at 37 ℃ and 180rpm in a shaking way, putting the bacterial suspension into a test tube, adding a little methyl red reagent, standing for 2h, and observing the result. The solution is positive when it turns red, otherwise it is negative.
Starch hydrolysis: inoculating the strain points cultured for 24h on a plate of a starch agar culture medium, culturing at 37 ℃ for 3d, dropping iodine on the plate after obvious colonies are formed, and if a transparent ring is generated around the colonies, indicating that the starch hydrolysis is positive, otherwise, indicating that the starch hydrolysis is negative.
Nitrate reduction: inoculating the strain cultured for 24h to a medium containing 0.1% KNO 3 After culturing the strain for 3 days at 37 ℃ and 180rpm with shaking, adding a solution A (8 g of sulfanilic acid dissolved in 1000mL of 5mol/L acetic acid) and a solution B (2.5 g of alpha-naphthol dissolved in 1000mL of 5mol/L acetic acid) into the LB liquid culture medium, wherein if the bacterial suspension is red or brown, the nitrate reduction is positive, and if the bacterial suspension is negative.
Acid and gas production: respectively preparing LB liquid culture medium containing 5% glucose, D (+) xylose, sucrose, D (-) mannitol, fructose and galactose, adjusting pH to 7.0, sterilizing, adding a little bromothymol blue, shaking and culturing at 37 deg.C and 180rpm for 48h, and observing the bacterial suspension of the measured bacteria. If the bacterial suspension turns yellow, it indicates that acid production is positive, and if it is still green, it is negative. If the Du's tubule floats up, the gas production is positive, otherwise, the gas production is negative.
Decomposition of tyrosine: and selecting a strain point cultured for 48 hours, inoculating the strain point on an LB solid medium plate containing 0.4% of tyrosine, culturing for 5 days at 37 ℃, and observing whether tyrosine particles around a colony become transparent, wherein the strain point is positive if the tyrosine particles around the colony become transparent, and the strain point is negative if the tyrosine particles around the colony become transparent.
Indole production: the test strain cultured for 48 hours was inoculated into a test tube containing LB liquid medium (pH 7.0), and after culturing at 37 ℃ for 48 hours with shaking at 180rpm, solution A (5.0 g of p-dimethylaminobenzaldehyde, 75.0mL of pentanol, and 25.0mL of concentrated hydrochloric acid) was added dropwise, and a red ring on the upper part of the medium was positive, otherwise negative.
H 2 S generation: and (3) puncturing and inoculating the to-be-detected strain cultured for 48h into a lead acetate culture medium, and culturing for 48h at 37 ℃. The black lead sulfide precipitate is generated to be positive, otherwise, the black lead sulfide precipitate is negative.
Anaerobic growth: and (3) puncturing and inoculating the to-be-detected bacterial strain cultured for 24 hours to the bottom of a test tube filled with an LB solid medium (pH 7.0) with the height of 4-5 cm, culturing at 37 ℃ for 3d, and observing the result, wherein if a bacterial colony grows along the surface of the medium, the bacterial colony is positive if the bacterial colony grows along the surface of the medium, and if the bacterial colony grows along a puncturing line, the bacterial colony is negative if the bacterial colony grows along the puncturing line, the bacterial colony is anaerobic.
Liquefaction of gelatin: and (3) puncturing and inoculating the strain to be tested which is cultured for 48h into a gelatin culture medium, loading 10mL of the strain into each test tube, culturing at 37 ℃, observing the liquefaction degree in 5 days, 10 days and 20 days respectively, namely, putting the test tube into a refrigerator at 4 ℃ to solidify gelatin, taking out the test tube during observation, and putting the test tube at room temperature to check the liquefaction condition of the gelatin due to decomposition, wherein if the gelatin is dissolved positively.
The results of the physiological and biochemical characteristics of the strain LJBV19 are shown in Table 1 and FIGS. 2-5.
As shown in FIGS. 2 to 4, the bacterial cells of the strain LJBV19 are rod-shaped and are endogenous to the spores. The strain LJBV19 grows on an LB solid culture medium, a single-cell colony is circular and milky, the surface is dry and has wrinkles, no gloss and unsmooth, and the edge of the colony is irregular and jagged; the strain LJBV19 is cultured in a clear LB liquid culture medium by shaking, a film layer is not formed on the surface, and the bacterial suspension is turbid.
As can be seen from Table 1 and FIG. 5, the strain LJBV19 is a gram-positive, aerobic bacterium; can tolerate 10% sodium chloride; the catalase and V-P reaction are positive, and the M-R reaction is negative; capable of reducing nitrate, hydrolyzing starch, gelatin and casein, but not tyrosine; can generate H 2 S，And the sugar alcohol can be fermented to produce acid but not indole.
TABLE 1 physiological and biochemical Properties of Strain LJBV19
Note: in the table, "+" indicates that the result is positive; "-" indicates negative results.
According to the morphological characteristics and physiological and biochemical characteristics, and by combining Bergey's bacteria identification manual and common bacteria system identification manual, the strain LJBV19 is very close to the Bacillus velezensis.
(3)16S rDNA sequence analysis
Determination of the 16S rRNA gene sequence was performed by Jinzhi Biotechnology, Inc., Suzhou. The determined sequences were subjected to Blast homology sequence search in GenBank (http:// www.ncbi.nlm.nih.gov /). The 16S rRNA sequences determined in the application and the 16S rRNA sequences of the species searched from GenBank are subjected to sequence analysis by using Clustal software, meanwhile, DNA and genetic distances among the 16S rRNA sequences of each strain are calculated by using MEGA-X software analysis, and a 16S rRNA phylogenetic tree is constructed by using a maximum likelihood method.
The 16S rRNA universal primer of bacteria and the genome DNA of a strain LJBV19 are used as templates for PCR amplification, a fragment with the length of about 1500bp is obtained as the amplification result, and the amplification product is subjected to gel cutting recovery and sequence determination. The sequencing result showed that the 16S r RNA of strain LJBV19 had a full sequence of 1342 bases, excluding the primer binding region.
The 16S rRNA sequence of the strain LJBV19 and the gene sequence downloaded from GenBank are compared by Clustal software, and MEGA 7.0 is used to construct the evolutionary tree. As can be seen from fig. 6, from the constructed phylogenetic tree, the strain LJBV19 and 2 b. Bacillus belgii (b. velezensis) strains (GenBank accession numbers KY694464 and EF433407, respectively) alone form a branch, while other species belonging to the same genus Bacillus (Bacillus) are gathered in other groups. It can be seen that the genetic relationship between strain LJBV19 and Bacillus belgii is closest.
According to the morphological culture characteristics, physiological and biochemical characteristics and the sequence analysis result of 16S rDNA, the strain LJBV19 is identified as Bacillus belgii (B.velezensis), therefore, the strain LJBV19 is named as Bacillus belgii velezensis LJBV19, namely Bacillus belgii LJBV19, and the strain is sent to the China general microbiological culture Collection center with the preservation number of CGMCC No.21804 and the preservation date of 2021 year, 2 months and 2 days.
(4) Whole genome alignment
Whole genome sequencing was performed by Shanghai Senno Biotech, Inc. The type (strain) Genome Server is used for whole Genome comparison, the similarity of the Bacillus belgii LJBV19 and the Bacillus belgii (B.velezensis) NRRL B-41580 whole Genome is the highest, the digital DNA-DNA hybridization (dDDH) is 92.1 percent, and the difference of the G + C content is 0.15 percent. The results are shown in table 2, and as a result of the whole gene alignment, no strain identical to bacillus beiLeisi LJBV19 was found, indicating that bacillus beiLeisi LJBV19 is a new strain in bacillus beiLeisi.
TABLE 2 Pair-wise comparison of Bacillus belgii LJBV19 genomes
Note: in table 2, the dDDH values of the b.belgii LJBV19 genome and the model strain genome are compared, and d0, d4, and d6 are three different genome alignment formulas, respectively, where:
d 0: the length of all HSPs (high scoring fragment base pairs) divided by the total genomic length;
d 4: the sum of all homologies in HSPs divided by the total length of HSPs;
d 6: the sum of all homologies in HSPs is divided by the total genome length.
Example 3: biocontrol effect of bacillus beilesiensis LJBV19 on phytopathogen
(1) Plate confrontation experiments with bacillus belgii LJBV 19: after the isolated Bacillus belgii LJBV19 was activated on LB solid medium for 24h, the plate center of PDA medium was streaked with activated Bacillus belgii LJBV19, and two sides of the plate distant from Bacillus belgii LJBV192.2cm were inoculated with phytopathogens, and the plate was not inoculated with Bacillus belgii LJBV19 as a control. Culturing at 28 deg.C in dark condition, repeating the treatment for 3 times, and calculating antibacterial rate when the control colony grows full.
DT: the diameter (cm) of the inhibition zone of the treatment group;
PC: blank diameter (cm) between two bacteria dishes of a control group;
DC: the distance (cm) between the two bacterial dishes.
Through a method of confronting culture on a flat plate, the Bacillus belgii LJBV19 grows on a PDA culture medium flat plate and releases antibiotic substances to kill or inhibit the growth of plant pathogenic bacteria, so that an inhibition zone is formed in a confronting area, and the radius of the inhibition zone is measured to evaluate the antagonistic capacity. The statistical bacteriostasis spectrum and bacteriostasis circle of the test strains on the 12 plant pathogenic bacteria are shown in figure 7 and table 3.
TABLE 3 inhibitory Effect of Bacillus belgii LJBV19 Strain on the phytopathogens tested
Note: data are presented as mean ± standard deviation (n ═ 3) and compared for significance of difference using LSD (least significant difference method), where: in different phytopathogens, the letters are the same, indicating that there is no significant difference between the two; letters different, indicating a significant difference (P ═ 0.05).
The results show that the bacillus beijerinckii LJBV19 has different degrees of inhibitory action on the pathogenic bacteria of the test plants, wherein the bacillus beijerinckii LJBV19 has the strongest inhibitory activity on the rice blast fungus (M.oryzae), and the average inhibitory rate is as high as 75.75%; the average inhibition rate of the anthracnose pathogen (C.viniferum) reaches 48.25 percent; the average inhibition rate of fusarium solani (F.solani), verticillium solani (V.dahliae), helminthosporium cucumerinum (E.rostratu), phytophthora capsici (P.capsica) and fusarium graminearum (F.graminearum) is more than 30%; the average inhibition rate of the bacillus subtilis on botrytis cinerea (B.cinerea), botrytis cinerea (C.diplodiella), potato blight (F.oxysporum), sunflower blight (Fusarium equiseti) and Rhizoctonia solani (Rhizoctonia solani) is more than 17%. The bacterium can prevent and treat plant diseases caused by various plant pathogenic bacteria.
(2) Hyphal morphology of phytopathogen under stress of bacillus beleisis LJBV 19: activated Bacillus beilis LJBV19 is inoculated in the center of a PDA culture medium plate in a streak way, phytopathogens are respectively inoculated on two sides of the position which is away from the Bacillus beilis LJBV192.2cm, and the influence of the Bacillus beilis LJBV19 on the hypha morphology of the phytopathogens is observed and compared under a microscope when the phytopathogens of a control group grow over a culture dish. The experiment was repeated 3 times. The results are shown in FIG. 8.
As can be seen from fig. 8, after bacillus belgii LJBV19 and plant pathogenic bacteria were oppositely cultured in a PDA medium plate for 5-7 days, microscopic observation results showed that hyphae of botrytis anthracnose (c.viniferum), botrytis cinerea (b.cinerea), potato fusarium wilt (f.oxysporum) and botrytis cinerea (c.diplediiella) were severely thickened and deformed, cell walls were thickened, and some hyphae were distorted and broken to cause intracellular protoplast leakage.
Example 4: growth promoting effect of Bacillus beleisi LJBV19 on plants
(1) Influence of Bacillus belgii LJBV19 fermentation liquor treatment on tomato seedling growth
The test plants were tomatoes, and the tomatoes used were Micro Tom tomatoes. Surface Disinfection of tomato seeds with 10% (v/v) sodium hypochlorite 15 min, then repeatedly washing in sterile distilled water for 4-5 times. Tomato seeds were cultivated in a greenhouse with a 1:1 ratio of vermiculite to soil at 25 ℃ day-night ratio of 16h/8 h. When the tomato grows to two true leaves, seedlings in the same germination state are selected to be placed in flowerpots (7cm multiplied by 7cm) formed by mixing vermiculite and soil in a ratio of 1:1, and 1 tomato seedling is planted in each flowerpot. After 3 days of transplanting and field planting of the tomato seedlings, the concentration of 15mL of root irrigation every 3 days of the experimental group is 10 8 cfu/mL Bacillus belgii LJBV19 fermentation broth, adding equal amount of LB liquid culture medium into blank group, and irrigating root for 5 times. After the tomato grows for 5 weeks, measuring the plant height, stem thickness, root length, dry weight and fresh weight of the tomato, and measuring the chlorophyll content of the leaf by adopting a Japanese Konika Mentada SPAD-502Plus hand-held chlorophyll meter. Each treatment was repeated 3 times, 8 seedlings per repetition. The relevant data for each treatment was statistically analyzed using SPSS 26.0 software.
The growth promoting effect of the tomatoes growing in the pots for 5 weeks was evaluated, and the plant height, root length, stem thickness, fresh weight, dry weight, chlorophyll content and other indicators of the tomatoes were measured, and the results are shown in fig. 9 and table 4.
As can be seen from fig. 9, the tomato plants of the experimental group were significantly higher than the tomato plants of the blank group; as can be seen from table 4, the plant height, root length, stem thickness, fresh weight above ground, fresh weight below ground, dry weight above ground, dry weight below ground and chlorophyll content of the tomatoes treated with bacillus belgii LJBV19 were increased by 48.09%, 19.42%, 20.02%, 62.94%, 71.32%, 65.36%, 69.67%, 75.22%, 71.03% and 7.80%, respectively, compared to the blank. Except for the root length, the plant height, stem thickness, overground fresh weight, underground fresh weight, overground dry weight, underground dry weight and leaf green content of the bacillus beilesiensis LJBV19 treated by the bacillus beilesiensis all have significant differences compared with the blank group. Therefore, the fermentation liquid of the bacillus beilesensis LJBV19 can promote the growth of the tomatoes, namely the bacillus beilesensis LJBV19 has the effect of promoting the growth of the tomatoes.
TABLE 4 evaluation of growth promoting effect of Bacillus beleisis LJBV19 on tomato
Note: data are mean ± sd of biomass replicates of 24 tomato plants. Indicates that the difference level is significant at the p less than or equal to 0.05 by the Duncan new double-pole difference method test.
(2) Influence of Bacillus belgii LJBV19 fermentation liquor treatment on growth of tobacco seedlings
The method specifically comprises the following steps of affecting the growth of tomato seedlings by treating the fermentation liquor of the same strain, wherein the differences are as follows: the test plants were tobacco, and the tobacco used was Nicotiana benthamiana.
The growth promoting effect of the tobacco grown in the pot for 5 weeks was evaluated, and the plant height, root length, stem thickness, dry weight, maximum leaf length, maximum leaf width, chlorophyll content and other indicators of the tobacco were measured, and the results are shown in fig. 10 and table 5.
As can be seen from FIG. 10, the overground tobacco of the experimental group is significantly more flourishing than the tobacco of the blank group, and the lamina is also larger; as can be seen from table 5, root length, stem thickness, above-ground dry weight, underground dry weight, maximum leaf length, maximum leaf width, and chlorophyll content of the burysia liiiensis LJBV 19-treated tobacco were increased by 30.38%, 22.04%, 35.51%, 66.33%, 24.40%, 24.08%, and 16.37%, respectively, compared to the blank group. The root length, stem thickness, fresh weight, dry weight, maximum leaf length, maximum leaf width and chlorophyll content of the tobacco treated by the Bacillus belgii LJBV19 are all significantly different from those of a blank group; there was no difference in plant height, and it can be presumed that there was no large difference in plant height, since the nicotiana benthamiana grew slowly and was mainly used for leaf growth. Therefore, the Bacillus beilesiensis LJBV19 fermentation liquid can promote the growth of tobacco, namely the Bacillus beilesiensis LJBV19 has the effect of promoting the growth of tobacco.
TABLE 5 evaluation of growth promoting effects of Bacillus beleisis LJBV19 on tobacco
Note: data are mean ± sd of 12 copies of nicotiana benthamiana biomass. Indicates that the difference level is significant at the p less than or equal to 0.05 by the Duncan new double-pole difference method test.
The present invention has been described above in connection with preferred embodiments, but these embodiments are merely exemplary and merely illustrative. On the basis of the above, the invention can be subjected to various substitutions and modifications, and the substitutions and the modifications are all within the protection scope of the invention.
Claims (6)
1. A bacillus beleisi bacterium, characterized in that: the Bacillus beilai is named as Bacillus beilai (Bacillus velezensis) LJBV19, the strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.21804 and the preservation date of 2021, 2 months and 2 days.
2. A broad-spectrum disease-preventing growth-promoting microbial inoculum is characterized in that: comprising the Bacillus belgii LJBV19 of claim 1.
3. Use of the bacillus belgii LJBV19 of claim 1 or the broad-spectrum disease-preventing growth-promoting inoculant of claim 2 for controlling plant diseases caused by phytopathogens; the plant pathogenic bacteria are one or more of rice blast (M.oryzae), grape anthracnose (C.viniferum), fusarium solani (F.solani), potato verticillium wilt (V.dahliae), peristropha umbiliciformis (E.rostratu), phytophthora capsici (P.capsica), fusarium graminearum (F.graminearum), botrytis cinerea (B.cinerea), botrytis cinerea (C.dipnodiella), potato fusarium wilt (F.oxysporum), sunflower fusarium wilt (F.equeiti) and rhizoctonia solani (R.solani).
4. Use of the bacillus belgii LJBV19 of claim 1 or the broad-spectrum disease-preventing growth-promoting inoculant of claim 2 for promoting the growth of plants, said plants being tomatoes and/or tobaccos.
5. Use according to claim 4, characterized in that: the live bacteria content of the original liquid of the fermentation liquid of the Bacillus beiLeisi LJBV19 or the original liquid of the fermentation liquid of the Bacillus beiLeisi LJBV19 is 1.0 multiplied by 10 after being diluted 7 cfu/mL～1.0×10 9 And (5) cfu/mL, performing root irrigation treatment on the plant.
6. Use according to claim 5, characterized in that: the viable bacteria content is 1.0 × 10 8 cfu/mL。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110312353.5A CN113151062B (en) | 2021-03-24 | 2021-03-24 | Bacillus belgii LJBV19 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110312353.5A CN113151062B (en) | 2021-03-24 | 2021-03-24 | Bacillus belgii LJBV19 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113151062A CN113151062A (en) | 2021-07-23 |
| CN113151062B true CN113151062B (en) | 2022-07-29 |
Family
ID=76888357
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110312353.5A Active CN113151062B (en) | 2021-03-24 | 2021-03-24 | Bacillus belgii LJBV19 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113151062B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215010B (en) * | 2020-01-21 | 2022-08-30 | 中国农业科学院蔬菜花卉研究所 | Bacillus belgii ZF128 and application thereof in preventing and treating potato wilt |
| CN113980846B (en) * | 2021-10-27 | 2023-04-21 | 河南省农业科学院烟草研究所 | High-efficiency stress-resistant bacillus subtilis for antagonizing fusarium oxysporum |
| CN113832083B (en) * | 2021-11-17 | 2023-03-17 | 千禾味业食品股份有限公司 | Bacillus beleisi and application thereof in vinegar brewing |
| CN114276957B (en) * | 2021-12-20 | 2023-09-05 | 中农新科(苏州)有机循环研究院有限公司 | Biocontrol strain with effect of preventing and controlling various plant pathogenic fungi and application thereof |
| CN114317371A (en) * | 2022-01-12 | 2022-04-12 | 中国科学院东北地理与农业生态研究所 | Biocontrol growth-promoting bacterium for rice blast and application thereof |
| CN114437972A (en) * | 2022-01-26 | 2022-05-06 | 云南农业大学 | Application of Bacillus beleisi YC89 or metabolite thereof in promoting plant growth |
| CN116121123B (en) * | 2022-12-09 | 2024-04-09 | 青岛农业大学 | Bacillus bailii and application thereof in grape gray mold and grape fresh-keeping |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101803678B1 (en) * | 2017-03-21 | 2017-12-01 | 충북대학교 산학협력단 | Primer sets for detecting Dyella thiooxydans ATSB10 and uses thereof |
| CN109022315A (en) * | 2018-08-13 | 2018-12-18 | 东北农业大学 | The biological and ecological methods to prevent plant disease, pests, and erosion bacillus of one plant of broad-spectrum disease resistance and its application |
| CN109136157A (en) * | 2018-10-19 | 2019-01-04 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | One plant of Bei Laisi bacillus for preventing and treating rice blast and its application |
| CN109266586A (en) * | 2018-10-18 | 2019-01-25 | 淮海工学院 | Bei Laisi bacillus BMF 03 and application thereof and fermentation process |
| CN110184221A (en) * | 2019-06-05 | 2019-08-30 | 中国农业科学院蔬菜花卉研究所 | One plant of Bei Laisi bacillus and its application in prevention and treatment tomato phytophthora root rot |
| CN111254093A (en) * | 2020-01-20 | 2020-06-09 | 上海交通大学 | Bacillus belgii 229-15 and application thereof |
| CN111471624A (en) * | 2020-04-25 | 2020-07-31 | 浙江师范大学 | Bacillus belgii CSQXDZ26 strain and application thereof |
-
2021
- 2021-03-24 CN CN202110312353.5A patent/CN113151062B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101803678B1 (en) * | 2017-03-21 | 2017-12-01 | 충북대학교 산학협력단 | Primer sets for detecting Dyella thiooxydans ATSB10 and uses thereof |
| CN109022315A (en) * | 2018-08-13 | 2018-12-18 | 东北农业大学 | The biological and ecological methods to prevent plant disease, pests, and erosion bacillus of one plant of broad-spectrum disease resistance and its application |
| CN109266586A (en) * | 2018-10-18 | 2019-01-25 | 淮海工学院 | Bei Laisi bacillus BMF 03 and application thereof and fermentation process |
| CN109136157A (en) * | 2018-10-19 | 2019-01-04 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | One plant of Bei Laisi bacillus for preventing and treating rice blast and its application |
| CN110184221A (en) * | 2019-06-05 | 2019-08-30 | 中国农业科学院蔬菜花卉研究所 | One plant of Bei Laisi bacillus and its application in prevention and treatment tomato phytophthora root rot |
| CN111254093A (en) * | 2020-01-20 | 2020-06-09 | 上海交通大学 | Bacillus belgii 229-15 and application thereof |
| CN111471624A (en) * | 2020-04-25 | 2020-07-31 | 浙江师范大学 | Bacillus belgii CSQXDZ26 strain and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 贝莱斯芽孢杆菌E69预防稻瘟病等多种真菌病害的潜力;沙月霞等;《中国农业科学》;20190601(第11期);第76-85页，参见全文 * |
| 贝莱斯芽孢杆菌S6拮抗物质分离纯化及抑菌机理;杨胜清等;《农药》;20170910(第09期);第24-27、39页，参见全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113151062A (en) | 2021-07-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113151062B (en) | Bacillus belgii LJBV19 and application thereof | |
| CN107136122B (en) | Biocontrol microbial inoculum for preventing and treating potato late blight | |
| CN108048354B (en) | Bacillus subtilis and application thereof | |
| CN113717901B (en) | Bacillus bailii and application thereof in preventing and controlling various vegetable diseases | |
| CN108641989B (en) | Bacillus methylotrophicus and application thereof | |
| CN111944716B (en) | Special compound microbial agent for tobacco seedling culture and preparation method and application thereof | |
| CN111808783B (en) | Enterobacter cloacae and production method and application of viable bacteria preparation thereof | |
| KR20130056585A (en) | Plant growth promotion by using bacterial strains isolated from roots of miscanthus sacchariflorus | |
| CN115261283A (en) | Bacillus cereus and application thereof in prevention and control of dry farming potato diseases | |
| CN109749953B (en) | Bacillus cereus, microbial inoculum and preparation method and application thereof | |
| CN114437994A (en) | Biological control bacterium Bacillus siamensis HT1 and application thereof in preparation of biological control bacterium agent | |
| CN108913621B (en) | Bacillus methylotrophicus YH-18 capable of effectively preventing and treating oriental cherry root cancer and application thereof | |
| CN112831443B (en) | Bacillus subtilis, microbial inoculum and application thereof in preventing and treating plant diseases | |
| CN108102972B (en) | Preparation method and application of composite biocontrol microbial inoculum for preventing and treating peanut diseases | |
| CN113755389A (en) | Bacillus belgii and application thereof | |
| CN112625957A (en) | Bacillus subtilis LJBS06 and application thereof | |
| CN112342173A (en) | Bacillus belgii and application thereof | |
| CN108913625B (en) | Salt-tolerant streptomycete, microbial inoculum thereof and application of microbial inoculum thereof in promoting plant growth | |
| CN108841752B (en) | Bacillus megaterium BM22 and application of spore liquid preparation thereof in preventing and treating cyclamen persicum radices | |
| CN113832071B (en) | Brevibacillus halotolerans strain and application thereof in preparation of biocontrol microbial inoculum | |
| CN114456973B (en) | Streptomyces rochei in tobacco and application thereof in prevention and control of tobacco diseases | |
| CN114085788B (en) | Pseudomonas amazonensis strain and application thereof | |
| CN116240126A (en) | Multifunctional bacillus belgium SB10 and application thereof | |
| CN115851479A (en) | Bacterium with antagonistic effect on botrytis cinerea and application thereof | |
| CN115058358A (en) | Salt-tolerant bacillus and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |