CN116042431B - Identification and application of bacillus bailii - Google Patents
Identification and application of bacillus bailii Download PDFInfo
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Classifications
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
Abstract
The invention discloses identification and application of bacillus. The bacillus is bacillus belicus (Bacillus velezensis) Rf21 strain which is preserved in China general microbiological culture collection center (CGMCC) for culture Collection of microorganisms (CGMCC) for 3-9 days in 2022, and the preservation number is CGMCC No.24487. The strain can inhibit xanthomonas strawberry (Xanthomonas fragariae), and can be used for preventing and treating bacterial angular leaf spot and 'cavitation' of strawberry.
Description
Technical Field
The invention belongs to the technical field of biology, and mainly relates to bacillus beijerinckii, an identification method thereof and application thereof in preventing and controlling strawberry diseases.
Background
The strawberry has unique and delicious flavor, wide cultivation and American name of berry queen, has good edible taste and high nutritive value, and is rich in multiple vitamins, mineral elements and antioxidant substances. At present, the strawberry production and planting area in China reaches 17.33 ten thousand hm 2 The total yield is about 500 ten thousand t, the total yield value reaches 600 hundred million yuan, and the method becomes the largest strawberry production and processing export country in the world. At present, strawberry in China is mainly cultivated by facilities, and in recent years, new hole diseases (also called empty heart disease, head breakage disease, marrow empty disease and the like) are novel destructive bacterial diseases. Recent studies have shown that the pathogen is xanthomonas strawberry (Xanthomonas fragariae). In recent years, the strawberry cavity disease has become an important disease of the China strawberry industry, and the continuous healthy development of the strawberry industry is severely restricted. As a novel disease, the strawberry cavity disease has no effective prevention and treatment method at present. The pathogenic bacteria of the disease are not known at first in production, so that a large amount of medicine is blindly taken, the natural environment is seriously destroyed, and the human health is threatened. Therefore, there is a need to find a sustainable, environmentally friendly means of biological control against strawberry void disease.
Disclosure of Invention
The invention aims to solve the technical problem of biological control of strawberry 'cavity disease' caused by xanthomonas strawberry (Xanthomonas fragariae).
The invention unexpectedly discovers and isolates a strain of bacillus beliensis (Bacillus velezensis) from healthy strawberry plant rhizosphere soil, which is named bacillus beliensis (Bacillus velezensis) strain Rf21. The bacillus belicus can effectively inhibit xanthomonas strawberry and prevent and treat the strawberry cavity disease.
The invention relates to a bacillus belicus (Bacillus velezensis) strain, which is named bacillus belicus Rf21 and is preserved in China general microbiological culture Collection center (CGMCC) No.24487.
The present invention also relates to a bacteriostatic agent comprising, as an active ingredient, the bacillus beliensis strain or a culture supernatant obtained after fermentation of the bacillus beliensis strain.
The invention also relates to the use of the bacillus belicus strain or a bacteriostat containing the bacillus belicus strain as an active ingredient in the prevention and treatment of strawberry bacterial angular leaf spot and strawberry cavity diseases caused by xanthomonas strawberry (Xanthomonas fragariae).
The invention also relates to the use of the bacillus belicus strain or a bacteriostat comprising the bacillus belicus strain as an active ingredient for preparing a biological agent for inhibiting xanthomonas strawberry (Xanthomonas fragariae).
The bacillus beijerinus Rf21 obtained by separation and purification has the following morphological characteristics (as shown in figure 1): the bacterial colony is milky white, semitransparent, smooth and regular in edge, thallus secretion can be dispersed in a cloud form, and after 24 hours of culture, the bacterial colony is provided with fold-shaped bulges.
The bacteriostat containing the fermentation liquor of the bacillus beljalis strain Rf21 has good stability in different temperatures, light treatment, ultraviolet treatment and pH, namely, the bacteriostat can remarkably inhibit the growth of xanthomonas strawberry on an agar plate after being treated under different conditions; in a potting test, the bacterial solution of the Rf21 strain is sprayed to effectively inhibit the incidence of bacterial angular leaf spot of strawberry and reduce the disease index.
Compared with the prior art, the invention has the advantages and advantages that:
1. bacillus belicus Rf21 and fermentation liquor thereof have remarkable inhibition effect on xanthomonas strawberry, which is a biocontrol strain provided for the first time for preventing and controlling the disease of strawberry 'cavity disease', and has good application prospect in strawberry production;
2. the bacteriostat containing the fermentation broth of bacillus belicus Rf21 has excellent bacteriostasis effect and stability, namely can play the bacteriostasis function in various environments such as temperature, acid and alkali, illumination and the like, is resistant to processing, and is suitable for popularization and use.
Drawings
FIG. 1 shows colony morphology of Bacillus belicus Rf21 grown on LB solid medium for 24 h.
FIG. 2 shows the BLAST alignment of the 16s rRNA sequence of Bacillus bailii Rf21.
FIG. 3 is a phylogenetic tree of the glpF, ilvD, tpiA, purH, rpoB, rpoD, pycA, ptA gene sequences of Bacillus beleiensis Rf21.
FIG. 4 is a chart showing the zone of inhibition of B.bailii Rf21 against Xanthomonas strawberry. Wherein the left part of the A diagram is the bacteriostasis circle of Rf21, and the right part is the bacteriostasis circle of Bacillus cereus; panel B is LB control.
FIG. 5 is a chart showing the circles of inhibition of Xanthomonas strawberry by the fermentation broth of Bacillus belicus Rf21. Wherein A is the bacteriostasis zone of the fermentation broth, B is a control, the upper is the bacteriostasis zone of the LB liquid medium, and the lower is the bacteriostasis zone of the bacillus subtilis (Bacillus subtilis) fermentation broth.
FIG. 6 shows the size of the inhibition zone of Bacillus bailii Rf21 after treatment in different environments. Wherein A is light treatment, B is ultraviolet treatment, C is temperature treatment, D is pH value treatment, E is treatment of each metal ion, wherein the last column in E is fermentation liquor without metal ions.
FIG. 7 shows the effect of Bacillus belicus Rf21 on inhibiting strawberry void disease and angular leaf spot in pot experiments. A, B, C is the symptom after 60d of the mixed treatment of the strain Rf21 and the xanthomonas strawberry liquid, D, E, F is the symptom after 60d of the treatment of the control group (the mixed treatment of the LB liquid culture medium and the xanthomonas strawberry liquid).
Detailed Description
The following is a specific embodiment of the present invention, and a detailed description of the technical solution of the present invention is provided. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified.
First, the culture media used in the examples are:
LB liquid medium: 10.0g of peptone, 5.0g of yeast powder and 5.0g of sodium chloride were dissolved in 1L of distilled water, and sterilized at 121℃for 25 minutes.
LB solid medium: 10.0g of peptone, 5.0g of yeast powder, 5.0g of sodium chloride and 15.0g of agar powder are dissolved in 1L of distilled water, and sterilized at 121 ℃ for 25min.
NB solid medium: 5.0g of peptone, 1.0g of yeast powder, 10.0g of sucrose and 3.0g of beef extract are dissolved in 1L of distilled water, the pH value is regulated to 7.0, 15.0g of agar powder is added, and sterilization is carried out for 25min at 121 ℃.
NB liquid medium: 5.0g of peptone, 1.0g of yeast powder, 10.0g of sucrose and 3.0g of beef extract are dissolved in 1L of distilled water, the pH value is regulated to 7.0, and the mixture is sterilized at 121 ℃ for 25min.
Example 1: the strain Rf21 was rapidly identified based on 16s rRNA and accurately identified based on glpF, ilvD, tpiA, purH, rpoB, rpoD, pycA, ptA gene.
After streaking the strain Rf21 on LB solid medium, picking single colony into a triangular flask filled with LB liquid medium, and culturing at 37 ℃ for 24 hours at 200r/min to obtain Rf21 bacterial liquid. Using Rf21 bacterial liquid as template, 16srRNA primer 27F:5'-AGAGTTTGATCCTGGCTCAG-3';1492R:5'-TACGGCTACCTTGTTACGACTT-3' PCR amplification was performed. The PCR reaction conditions were: 3min at 95 ℃;95℃15s,58℃15s,72℃1min,35 cycles; extension at 72℃for 5min, stopping at 4 ℃. The PCR product was sent to Beijing engine biotechnology Co. The sequencing results were BLAST aligned with the bacterial 16s rRNA sequences already present in NCBI, and the results are shown in FIG. 2. From the comparison of the colony morphology with 16s rRNA in FIG. 1, it was found that strain Rf21 belongs to the genus Bacillus.
In the classification and identification of bacteria, molecular identification by using 16s rRNA is widely used and the technology is mature, but has some limitations for some bacterial species with relatively close relatedness, so that the classification of genus by using 16s rRNA is relatively accurate and is not suitable for classification and identification of species. The bacillus genes are very similar, and it is important to accurately identify bacterial species by using a multi-site sequence typing (MLST) method. Therefore, the invention constructs a phylogenetic tree of Rf21 and other 17 closely related strains by using glpF, ilvD, tpiA, purH, rpoB, rpoD, pycA, ptA eight housekeeping genes to accurately identify the species.
PCR amplification was performed using the Rf21 bacterial liquid as a template, and the following primers were used, respectively:
glpF primer
glpF-F：5’-WTGACAGCATTTTGGGG-3’；
glpF-R：5’-GTAAAATACRCCGCCGA-3’；
ilvD primer
ilvD-F：5’-ATGAGATATTCGCTGCC-3’；
ilvD-R：5’-CTTCGTTAATGCGTTCTAAAGAG-3’；
tpiA primer
tpiA-F：5’-TCAGCTTCGTTGAAGAAGTGAAA-3’；
tpiA-R：5’-GGACTCTGCCATATATTCTTTA-3’；
purH primer
purH-F：5’-TTTGAGAAAAAACAATCGCT-3’；
purH-R：5’-TCGGCTCCCTTTTCGTCGG-3’；
rpoB primers
rpoB-F：5’-TTGACAGGTCAACTAGTTCAGT-3’；
rpoB-R：5’-CTATTCTTTCGTTACTGCGTCAC-3’‘
rpoD primer
rpoD-F：5’-GCCGAAGAAGAATTTGACCTTAA-3’；
rpoD-R：5’-CGTTTRCTTCTGCTHGGATGTCT-3’；
pycA primers
pycA-F：5’-AAATCAGARGCGAAAGC-3’；
pycA-R“5’-CCTGAGCGGTAAGCCAT-3’；
ptA primer
ptA-F：5’-ATACATATGAAGGSATGGAAGA-3’；
ptA-R：5’-TAGCCGATGTTTCCTGCT-3’。
The PCR system is shown in Table 1. The PCR reaction conditions were: 3min at 95 ℃;95℃15s,55℃15s,72℃1min,35 cycles; extension at 72℃for 5min, stopping at 4 ℃. The PCR product was purified and ligated into pMD19-T vector and sent to Beijing department of Biotechnology, inc. for sequencing. The above gene sequences of the other 17 kindred strains were derived from NCBI. After the MLST typing is established on the gene sequences of the strains, the MEGA7.0 software is utilized to construct a phylogenetic tree by using a K2 model, and the result is shown in figure 3. The comprehensive comparison shows that the strain Rf21 is in the same branch with Bacillus belicus (Bacillus velezensis) and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), has highest similarity with Bacillus belicus CSUSB3 (Bacillus velezensis strain CSUSB 3), can be determined to be Bacillus belicus (Bacillus velezensis), is formally named as Bacillus belicus Rf21 (Bacillus velezensis strain Rf), and is preserved in China general microbiological center (CGMCC) at 3 months 9 of 2022, with the preservation number of CGMCC No.24487.
TABLE 1 PCR System for amplifying Rf21 Gene of Strain
Example 2: antagonism of strain Rf21 with Xanthomonas strawberry was detected.
The experiment was repeated three times and the average value of the diameters of the inhibition zones was taken. The specific operation steps are as follows:
1. the xanthomonas strawberry is streaked on NB medium, single colony is selected to be placed in a triangular flask filled with 50ml of NB liquid medium, and the xanthomonas strawberry is cultivated at 25 ℃ and 200r/min until the bacterial liquid OD600 is approximately equal to 1.0. Mu.l to 50ml of melted NB solid medium (temperature about 50 ℃) were pipetted and mixed well, immediately followed by pouring into a plate to solidify.
2. The strain Rf21 is streaked on LB medium, single colony is picked up to a triangular flask filled with 50ml of LB liquid medium, and the strain Rf21 is cultured at 37 ℃ and 200r/min until the bacterial liquid OD600 is approximately equal to 0.8.
3. A sterile perforating ring with the aperture of 5mm is used for perforating on the mixed NB flat plate in the step 1, a sterile filter paper sheet is placed in the hole, and 5 mu l of the Rf21 bacterial liquid in the step 2 is injected into the filter paper sheet, so that the injected liquid LB culture medium is used as a control. The prepared counter plate is placed at 28 ℃ for 4d of culture, and then the size of the bacteriostasis zone is observed.
4. Results: the strain Rf21 has obvious inhibition effect on the xanthomonas strawberry (see table 2 and fig. 4), and the average inhibition zone size can reach 4.10 cm after 4 days of counter culture. While other bacillus (bacillus cereus La) has no obvious inhibition zone.
TABLE 2 diameter of inhibition zone of strain Rf21
Example 3: the inhibition of xanthomonas strawberry by the fermentation broth of strain Rf21 was detected on agar plates.
The inhibition effect detection of the fermentation broth of the strain Rf21 on the xanthomonas strawberry is realized by the size of a bacteriostasis circle of the xanthomonas strawberry on an agar plate. The experiment was repeated three times and the average was taken, and the specific implementation steps of each experiment were as follows:
1. preparation of strain Rf21 fermentation broth: after streaking the strain Rf21 on LB solid medium, picking single colony into a 2ml centrifuge tube filled with 1ml LB liquid medium, and culturing at 37 ℃ for 200r/min overnight to obtain a culture solution; taking 100 μl of the culture solution into a triangular flask filled with LB liquid medium, and culturing at 37deg.C and 200r/min for 60h for fermentation. Centrifuging the fermented bacterial liquid at 8000r/min for 25min, collecting supernatant, filtering with 0.45 μm filter head, and filtering with sterile 0.22 μm filter head to obtain sterile fermentation liquid.
2. The xanthomonas strawberry is streaked on NB medium, single colony is selected to be placed in a triangular flask filled with 50ml of NB liquid medium, and the xanthomonas strawberry is cultivated at 25 ℃ and 200r/min until the bacterial liquid OD600 is approximately equal to 1.0. 1ml to 50ml of melted NB solid medium (temperature about 50 ℃) was aspirated, mixed well, and immediately poured into a plate to allow solidification.
3. A sterile perforating ring with the aperture of 5mm is used for perforating at the center of a mixed NB flat plate, sterile absorbent cotton balls are placed in the holes, 300 μl of bacterial strain Rf21 sterile fermentation liquid is injected into the cotton balls, and the injected liquid LB culture medium is used as a control. The prepared counter plate is placed at 28 ℃ for 4d of culture, and then the size of the bacteriostasis zone is observed.
4. Results: the fermentation broth of the strain Rf21 has obvious inhibition effect on xanthomonas strawberry (see table 3 and figure 5), and the average inhibition zone size can reach 4.30 cm after 4 days of counter culture.
TABLE 3 diameter of zone of inhibition of fermentation broth of strain Rf21
Example 4: and (3) detecting the stability of the strain Rf21 fermentation broth.
The stability detection of the bacteriostasis effect of the strain Rf21 fermentation liquor is realized by the size of a bacteriostasis zone on an agar plate after the strain Rf21 fermentation liquor is treated in different environments. All experiments used a controlled variable method, i.e. when the stability of one environmental condition was checked, the other environmental conditions were consistent. The experiment was repeated three times and the average was taken, and the specific implementation steps of each experiment were as follows:
1. preparation of strain Rf21 fermentation broth: same as in example 3.
2. Detection of illumination stability: after the fermentation broths of the strain Rf21 are respectively placed under 10000Lx illumination for 2h, 4h, 6h, 8h and 10h, the size of a bacteriostasis zone is observed by the method of the steps 2-3 in the embodiment 3, and the bacteriostasis effect is verified. The fermentation broth without light treatment was used as a control.
3. Detection of ultraviolet stability: and (3) respectively exposing the fermentation broth of the strain Rf21 to ultraviolet rays for 15min, 30min, 45min, 60min and 75min, and observing the size of a bacteriostasis zone by using the method of the steps 2-3 in the embodiment 3 to verify the bacteriostasis effect. The fermentation broth without UV irradiation was used as a control.
4. Detection of temperature stability: the fermentation broths of the strain Rf21 were treated at-20 ℃,4 ℃, 15 ℃, 28 ℃, 40 ℃, 60 ℃, 80 ℃ and 100 ℃ for 1 hour, and then the size of the inhibition zone was observed by the method of steps 2 to 3 in example 3, and the inhibition effect was verified.
5. And (3) detection of acid-base stability: the pH value of the strain Rf21 fermentation broth is respectively adjusted to 2, 4, 6, 8, 10 and 12, and the size of a bacteriostasis zone is observed by the method of the steps 2-3 in the embodiment 3, so that the bacteriostasis effect is verified. Accordingly, each pH setting corresponds to a hydrochloric acid or KOH solution as a control.
6. Results: the fermentation broth of the strain Rf21 has good stability at different temperatures, light treatments, ultraviolet treatments and pH, and can remarkably inhibit the growth of xanthomonas strawberry on an agar plate even after different conditions are treated, as shown in figure 6.
Example 5: potted test of resistance of strain Rf21 to strawberry bacterial angular leaf spot
1. After streaking the strain Rf21 on LB solid medium, picking single colony into a triangular flask filled with 50ml of LB liquid medium, and shake culturing at 37 ℃ for 60h at 200r/min to obtain Rf21 bacterial liquid.
2. Culturing Xanthomonas strawberry on NB medium by streaking, picking single colony into triangular flask containing 50ml NB liquid medium, shake culturing at 25deg.C and 200r/min for 3d, centrifuging the bacterial solution at 5000r/min for 10min to collect thallus, removing supernatant, re-suspending with sterile distilled water, and diluting to a concentration of about 1×10 8 cfu/ml。
3. Healthy strawberries with four leaves and one heart and consistent growth vigor are selected, the variety is 'dazomet', the strawberries are planted in a sterile matrix, and 10 strains of Rf21 bacterial liquid are treated and controlled.
4. The treatment group is mixed with Rf21 bacterial liquid and xanthomonas strawberry heavy suspension and sprayed on the whole strawberry seedlings, and the control group is mixed with LB liquid culture medium and xanthomonas strawberry heavy suspension and sprayed on the whole strawberry seedlings. Once every 10d of spraying, the disease conditions of the statistical treatment group and the control group after 60d were continuously observed (see table 4 and fig. 7). The method for calculating the disease index of the strawberry bacterial angular leaf spot comprises the following steps: disease index = Σ (number of onset at each stage x representative value at each stage)/(total leaf count in investigation x highest representative value) ×100%. Control effect of the spraying strain Rf21 bacterial liquid= (control group disease index-treatment group disease index)/control group disease index x 100%. The disease grading criteria are shown in Table 5. As a result, after 30d of the inoculation treatment, the leaf angle spots of the plant leaf sprayed with the Rf21 bacterial liquid are stopped from spreading, the diseased leaf is gradually dried up, and new leaf continuously grows. The angular spots of the control group of leaves spread continuously, and the leaves appeared in the form of water spots and brown. The Rf21 strain can well control the development of strawberry bacterial angular leaf spot.
TABLE 4 incidence of bacterial angular leaf spot of strawberry after spraying Rf21 liquid
TABLE 5 criteria for grading the disease of bacterial angular leaf spot of strawberry
Claims (4)
1. A bacillus beleiensis (Bacillus velezensis) strain, named bacillus beleiensis Rf21, is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24487.
2. A bacteriostatic agent comprising the bacillus belicus strain according to claim 1 or a culture supernatant obtained after fermentation of the bacillus belicus strain as an active ingredient.
3. Use of a bacillus belgium strain according to claim 1 or a bacteriostatic agent according to claim 2 for controlling bacterial angular leaf spot and cavity disease of strawberries caused by xanthomonas strawberry (Xanthomonas fragariae).
4. Use of a bacillus bezeri strain according to claim 1 or a bacteriostatic agent according to claim 2 for the preparation of a biological agent for inhibiting xanthomonas strawberry (Xanthomonas fragariae).
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