CN114854618B - Bacillus bailii SF327 and application thereof - Google Patents
Bacillus bailii SF327 and application thereof Download PDFInfo
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- CN114854618B CN114854618B CN202210198705.3A CN202210198705A CN114854618B CN 114854618 B CN114854618 B CN 114854618B CN 202210198705 A CN202210198705 A CN 202210198705A CN 114854618 B CN114854618 B CN 114854618B
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Classifications
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- 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
-
- 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
- C12N1/02—Separating microorganisms from their culture media
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to bacillus beijerinus SF327 and application thereof. The bacillus belicus SF327 strain is preserved in China Center for Type Culture Collection (CCTCC) on the 2 nd month 18 of 2022, and the preservation number is CCTCC M2022130. The bacillus berliner has remarkable growth promoting effect on green vegetables, can produce indole-3-acetic acid auxin, has remarkable inhibiting effect on rice bacterial leaf blight bacteria and strip spot bacteria of xanthomonas oryzae, and also has broad-spectrum antibacterial activity on plant pathogenic fungi such as rice blast bacteria, cucumber fusarium wilt bacteria, phytophthora capsici, rubber gum spore anthracnose bacteria, rubber tree acutus bacteria and the like. The result of the invention shows that the strain has the application prospect of preventing diseases and promoting growth, provides good biocontrol resources for preventing and controlling bacterial leaf blight, leaf spot and rice blast of the rice and lays a foundation for developing novel microbial agents for anthracnose of rubber trees.
Description
Technical Field
The invention belongs to the field of application of microbial technology, and particularly relates to bacillus beijerinckii SF327 and application thereof.
Background
The Cruciferae (Cruciferae) has a total of 375 species, about 3200 species, and is widely distributed and worldwide. There are 95 genera, 425, 124 varieties distributed in our country, introduction of 7 genera, 20 more than one species (Li Ming, gu Jianguang, etc. identification of crucifer host range of brassica napus. 2011). The brassica and the radish in the cruciferous plants are main vegetables and oil crops in China, or are used as spicy seasonings, some are important medicinal plants (such as Isatis tinctoria), some are ornamental plants (such as Violet), and some belong to honey plants, and are important economic crops.
The brassica cluba is a pathogenic bacterium which is extremely serious in harm on cruciferous plants, and the plant cluba caused by the brassica cluba is called plant cancer. The root (Dekhuijzen H M.The occurrence of free and bound cytokinins in clubroots and Plasmodiophora brassicae.infected turnip tissue cultures.Physiologia plantarum,1980,49(2):169-176.), of a host crop is primarily infested by the brassica oleracea and the plant can be infested in each growth period, the earlier the infestation occurs, the greater the damage to the plant and the more serious the extent of the attack (Zhang Tinglin, dai Pingping. Occurrence and control measures of brassicaceous vegetable clubroot; shanghai vegetables, 2014 (4): 48-49.). The basal leaves of the diseased plants become wilted in a yellow and serious state, and the whole plants die, so that great economic loss is caused. In late stage root rot, dormant spores are released into soil, and generally the dormant spores can survive in the soil for more than 20 years, if the continuous cropping farmland is extremely threatening (Dixon G R.Plasmodiophora brassicae in its environment.Journal of Plant Growth Regulation,2009b,28(3):212-228.)., more than 3 years are needed for eradication through rotation, the feasibility is low, and therefore once the plant is in place, the root swelling disease is caused, and the influence is serious and long-term. The long-term use of common chemical prevention and treatment means not only can improve the drug resistance of pathogenic bacteria, but also can bring about negative effects such as soil degradation (Zhang Ribo. Survey of cruciferous clubroot in Chengdu city and screening of drug prevention and treatment, university of Sichuan agriculture, 2010.).
In recent years, more and more researches are carried out on plant growth-promoting bacteria (PGPR), and the application of the PGPR can replace chemical fertilizers to a certain extent, so that the agronomic soil environment is improved (Zhang Motong, li Chaoqun, and the like). For example, dunaliella salina can improve the tolerance of the cabbages to salt stress (Cui Gongli, xu Wenxin, etc.), dunaliella salina can influence the salt tolerance of the cabbages, shanxi university agricultural university report (natural science edition), 2021, 41 (5): 112-120.) can improve the agronomic characters of agricultural products (Huang Shuchao, hou Dong, etc.), three growth promoting bacteria and mixed microbial agents thereof can influence the growth and quality of the lettuce, zhejiang agricultural report, 2021, 33 (7): 1212-1221.) can further improve the root microecology of the crops (Wang Yanyu, liu Shuang, etc., 3 saline and alkaline tolerant growth promoting bacteria can influence the root microecology of the mung beans while relieving the injury of the saline and alkaline stress.
The comprehensive development and utilization of the growth-promoting bacteria are important in recent research, the development of the growth-promoting bacteria resources not only can play a role in real time, but also can reduce the use of pesticides, improve the ecological condition of soil, and is an important way for promoting the development of ecological agriculture and the recovery of ecological environment (Liu S L,Maimaitiaili B,et al.Response of soil microorganisms after converting a saline desert to arable land in central Asia.Applied Soil Ecology,2016,98:1-7).
Disclosure of Invention
The invention aims to provide bacillus beijerinckii SF327 and application thereof.
The bacillus bernatis has a growth promoting effect on the green vegetables, and has a remarkable antagonism on rice bacterial leaf blight bacteria, rice strip spot bacteria, rice blast bacteria, cucumber fusarium wilt bacteria, phytophthora capsici, colletotrichum gloeosporioides and colletotrichum gloeosporioides. The bacillus belicus of the invention can provide help for promoting growth of green vegetables, preventing and controlling rice diseases and preventing and controlling anthracnose diseases of rubber trees.
The aim of the invention can be achieved by the following technical scheme:
The first aspect of the invention is: there is provided a strain of bacillus beleiensis, which was isolated from the soil of the sea marshland, the sea harbour garden, the sea harbour, the sea bacillus beleiensis (Bacillus velezensis) SF327, and which was preserved in the China center for type culture collection (cctccc) at 18 of 2022, with a preservation number of cctccc M2022130.
The bacterial colony of the bacillus beijerinckii is observed to be milky white in color, uneven in edge, dry and rough in surface and opaque through plate culture of a solid culture medium; the result of gram staining showed positive results.
By physiological and biochemical tests, it is clear that the bacillus belicus is a gram positive bacterium, short rod-like, capable of producing spores, aerobic or facultative anaerobic bacteria.
The bacillus belicus is capable of secreting 3-hydroxy butanone to produce acetyl methyl methanol and gelatinase; cannot hydrolyze o-nitrobenzene-galactoside, arginine, lysine, ornithine, sodium citrate, sodium thiosulfate and the like; glucose, mannitol, rhamnose and the like cannot be oxidized; can produce acid by using 22 carbon sources such as glycerol, L-arabinose, ribose, D-xylose, glucose, fructose, mannose and the like.
The bacillus beleiensis has growth promoting effect on the green vegetables.
The growth promoting effect comprises that the overground fresh weight of the green vegetables can be obviously increased by using the bacillus beleiensis which can produce indole-3-acetic acid (1H-Indole-3-ACETIC ACID, IAA) auxin.
The bacillus belicus has antagonism to xanthomonas oryzae.
The xanthomonas oryzae comprises two pathogenic varieties of bacterial leaf blight bacteria (Xanthomonas oryzae pv. Oryzae, xoo) and rice stripe bacteria (Xanthomonas oryzae pv. Oryzicola, xoc).
The antagonism of bacillus beijerinckii SF327 on rice bacterial leaf blight and rice bacterial leaf spot is detected by a plate opposite culture method, and the antagonism of the bacillus beijerinckii SF327 on the 2 pathogenic bacteria is proved to be obvious.
The second aspect of the invention is: providing a method for separating bacillus belicus: and (3) adopting a gradient dilution plating method, adding sterile water into a soil sample, dissolving and vibrating for 20min, sequentially diluting the soil sample into soil bacterial suspensions with different gradients, respectively coating the soil bacterial suspensions on an NA (NA) plate inoculated with rice leaf spot bacteria, observing the formation of a bacteriostasis zone, streaking bacterial colonies capable of generating an obvious bacteriostasis zone on the NA plate to obtain single bacterial colonies, carrying out PCR (polymerase chain reaction) amplification by utilizing a bacterial 16S rRNA gene, constructing a phylogenetic tree, and determining the classification status of the bacterial strains.
The 16S rRNA gene of Bacillus bailii SF327 is shown in SEQ ID NO. 1.
The invention uses 16S rRNA gene to construct phylogenetic tree, carries out comparative analysis of genetic relationship, and combines physiological and biochemical characteristics of the strain to prove that the strain is bacillus bailii (Bacillus velezensis).
A third aspect of the invention is: the application of the bacillus belgium is provided, which comprises the following applications:
In one specific embodiment of the invention, the bacillus beijerinus SF327 has a remarkable antagonism on both rice bacterial blight bacteria and rice stripe bacteria.
In one embodiment of the invention, the bacterial leaf spot of rice comprises PXO99 A and the bacterial leaf spot of rice comprises RS105.
In one embodiment of the invention, the bacillus belicus SF327 has antagonism to a plurality of plant pathogenic fungi, and specifically comprises rice blast germ (Magnaporthe oryza), cucumber fusarium wilt germ (Fusarium oxysporum f.sp cucumerinum), phytophthora capsici (Phytophthora capsici), colletotrichum rubber (Colletotrichum gloeosporioides) and colletotrichum rubber (Colletotrichum acutatum).
In one specific embodiment of the invention, the bacillus beljavensis SF327 has a growth promoting effect on the green vegetables, and particularly comprises the step of obviously increasing the fresh weight of the green vegetables on the ground.
In one embodiment of the invention, the pro-growth effect on the broccoli comprises that the Bacillus belicus is capable of producing auxin of the indole-3-acetic acid (1H-Indole-3-ACETIC ACID, IAA) type.
In one embodiment of the invention, the bacillus beleiensis is used as a green vegetable growth promoting agent, and simultaneously, the bacillus beleiensis is used as a xanthomonas oryzae (Xanthomonas oryzae) antagonistic bacterium and the bacillus beleiensis is used as a plant pathogenic fungi antagonistic bacterium.
Compared with the prior art, the bacillus belgium SF327 belongs to plant growth promoting bacteria (PGPR), has obvious growth promoting effect on green vegetables, can produce indole-3-acetic acid auxin, has obvious inhibiting effect on rice bacterial blight bacteria and leaf spot bacteria of Xanthomonas oryzae, and also has broad-spectrum antibacterial activity on plant pathogenic fungi such as rice blast bacteria, cucumber fusarium wilt bacteria, phytophthora capsici, rubber gum spore anthracnose bacteria, rubber tree acuminate spore anthracnose bacteria and the like.
The result of the invention shows that the strain has broad-spectrum bacteriostasis and growth promotion capability, has application prospect of disease prevention and growth promotion, provides good biocontrol resources for preventing and treating bacterial leaf blight, leaf spot and rice blast of the white vegetables, and lays a foundation for developing novel microbial agents for anthracnose of rubber trees. Meanwhile, the biocontrol potential is required to be further developed, and a foundation is laid for developing a multifunctional biocontrol microbial inoculum.
Drawings
FIG. 1 is a photograph of Bacillus belicus SF327 observed under a microscope (1000X) and colony morphology.
FIG. 2. Alignment of Bacillus bailii SF327 16S rRNA gene sequences the Neighbor-Joining phylogenetic tree was constructed with Virgibacillus pantothenticus NCDO 1765 (X60627) as the outer branch.
FIG. 3.16 gel electrophoresis results of S rRNA gene. Wherein 1 represents 2k Marker;2 represents the product of the 16S rRNA gene.
FIG. 4 is a graph showing the effect of Bacillus belicus SF327 on the production of indole-3-acetic acid auxin.
FIG. 5 is a graph showing the effect of Bacillus belgium SF327 on growth promotion of green vegetables.
FIG. 6 shows the antagonistic effect of Bacillus belicus SF327 on bacterial leaf blight of rice PXO99 A.
FIG. 7 shows the antagonistic effect of Bacillus belicus SF327 on rice stripe bacteria RS 105.
FIG. 8 shows the antagonistic effect of Bacillus bailii SF327 on Pyricularia oryzae.
FIG. 9 shows the antagonistic effect of Bacillus belicus SF327 on cucumber fusarium wilt.
FIG. 10 shows the antagonistic effect of Bacillus bailii SF327 on phytophthora capsici.
FIG. 11 is a graph showing the antagonistic effect of Bacillus bailii SF327 on colletotrichum gloeosporioides.
FIG. 12 is a graph showing the antagonistic effect of Bacillus belicus SF327 against colletotrichum gloeosporioides.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention.
The strain medium used in the following examples is as follows:
NA solid medium (g/L): 3g of beef extract, 5g of polypeptone, 10g of sucrose, 1g of yeast powder and 15g of agar powder, adding water for dissolving and fixing the volume to 1000mL, adjusting the pH to 7.0-7.2, and sterilizing under high pressure (121 ℃ for 20 min).
NB liquid Medium (g/L): 3g of beef extract, 5g of polypeptone, 10g of sucrose and 1g of yeast powder, adding water for dissolving and fixing the volume to 1000mL, adjusting the pH to 7.0-7.2, and sterilizing under high pressure (121 ℃ for 20 min).
PDA solid medium (g/L): 200g of potato, 20g of glucose and 15g of agar, adding water for dissolution, finally fixing the volume to 1000mL, and sterilizing under high pressure (121 ℃ for 20 min) at the pH of 7.0-7.2.
Auxin assay YM Medium (g/L): m (g/L): 5g of mannitol, 0.25g of K 2HPO4 g, 0.05g of NaCl, 1.5g of yeast extract and 0.05g of tryptophan, adding water for dissolution, fixing the volume to 1000mL, adjusting the pH to 7.0, and sterilizing under high pressure (121 ℃ for 20 min).
Example 1 acquisition of Bacillus bailii SF327
1. Soil source
Hainan province sea-mouth city sea-meadow harbor garden orchard land soil collected on 11 months and 8 days of 2011
2. Screening of strains
(1) Soil sample collection
Collecting soil samples of about 10cm from the root surrounding surface layer of the fruit tree plant, and collecting 200g of soil samples at each point. 3 soil samples are collected in each block, and the time, place and kind of sampling are recorded. The collected soil sample is preserved at normal temperature for bacterial separation.
(2) Isolation of bacteria
Plate dilution method: 10g of soil sample is weighed into an conical flask, 90mL of sterile water is added, then the mixture is oscillated in a shaking table at 200rpm and 28 ℃ for 20min, and then the mixture is taken out and is kept stand at room temperature for 10min to prepare the soil fungus suspension stock solution. The stock solution is subjected to gradient dilution to obtain 10 0、10-1、10-2、10-3、10-4、10-5 of diluted solutions with 6 gradients respectively. 200. Mu.L of each bacterial suspension dilution was spread evenly on NA plates containing Brevibacterium oryzae RS105 (OD 600 = 2.0) and 3 replicates were performed per gradient. The plate was placed in a 28℃incubator, cultured for 48 hours, and observed.
(3) Bacterial purification
Single colonies with obvious inhibition zones are observed and selected, streak purification is carried out on an NA plate, the single colonies are selected after inversion culture in a 28 ℃ incubator for 12 hours, and the single colonies are numbered in sequence.
(4) Preservation of bacteria
The single colony of the strain is inoculated in NB liquid culture medium, and after culturing for 12 hours in a shaking table at 28 ℃ and 180rpm, 1mL of bacterial liquid and 1mL of 50% sterile glycerol are sucked, mixed by gentle shaking, and placed at-80 ℃ for long-term storage.
(5) Screening of growth-promoting bacteria
Auxin assay:
And screening and content determining the strain of the auxin IAA by adopting a spectrophotometry method. The prepared modified YM medium was sterilized by pouring into a test tube. Each tube was inoculated under sterile conditions with the strain to be tested. Culturing at 28deg.C under 135r/min for 96 hr. The supernatant was kept by centrifugation, mixed with the colorimetric solution, and incubated at room temperature for 30min, and pink color was developed, indicating IAA production. OD value is measured at 530nm wavelength, and IAA standard curve is substituted to calculate IAA content of endophyte.
FIG. 4 shows the effect of Bacillus belicus SF327 on indole-3-acetic acid (IAA) production.
Fresh weight measurement method of green vegetables:
picking the overground part of the green vegetables (removing the underground part of the green vegetables by scissors) and weighing immediately, and calculating the fresh weight of the overground part.
The effect of Bacillus belicus SF327 on growth promotion of green vegetables is shown in FIG. 5.
Finally screening bacillus beleiensis (Bacillus velezensis) SF327, wherein the bacillus beleiensis is preserved in China Center for Type Culture Collection (CCTCC) on the 2 nd month 18 of 2022, and the preservation number is CCTCC M2022130.
A photograph of Bacillus belicus SF327 observed by a microscope (1000X) and colony morphology are shown in FIG. 1.
EXAMPLE 2 16S rRNA Gene identification of Bacillus bailii SF327
Genomic DNA of strain SF327 was extracted by CTAB method using Shanghai Paenox BioCo., ltd.) and bacterial 16S rRNA gene primer: 27F 5 '-AGAGTTTGATCCTGGGCTCAG-3' and 1499R 5'-TACGGCTACCTTGTTACGACTT-3' are subjected to PCR amplification by using the extracted genomic DNA as a template to obtain a target fragment. The PCR reaction system is as follows: the system is arranged according to the actual situation.
TABLE 1 Ex-Taq polymerase chain reaction System
The basic conditions of the PCR reaction are as follows: pre-denaturation at 95℃for 3min, denaturation at 95℃for 30s, annealing at 65℃for 30s, extension at 72℃for 30s, pre-extension at 72℃for 10min, and preservation at 4℃for 35 cycles. After the reaction is finished, the PCR products are all checked by 1% agarose gel electrophoresis, the result is detected and recorded by a gel imager, the gel electrophoresis result of the 16S rRNA gene is shown in figure 3, and 1 in figure 3 represents Marker;2 represents the product of the 16S rRNA gene. The PCR stock was sent to platinum Biotechnology (Shanghai) Inc. for sequencing. Sequencing results were analyzed using DNA Star and BLAST alignment was performed on NCBI website to determine the species of the near-border bacteria.
The 16SrRNA gene of bacillus bailii SF327 is shown in SEQ ID NO. 1.
The alignment result of Bacillus bailii SF327 16S rRNA gene sequence is shown in FIG. 2, and a Neighbor-training phylogenetic tree constructed by using Virgibacillus pantothenticus NCDO 1765 (X60627) as an outer branch shows that: the 16SrRNA gene of SF327 has 99.86% similarity to Bacillus velezensis.
Example 3 physiological and Biochemical identification of Bacillus bailii SF327
The bacillus belgium SF327 of the invention has the physiological and biochemical characteristics that: capable of secreting 3-hydroxy butanone to produce acetyl methyl methanol and gelatinase; cannot hydrolyze o-nitrobenzene-galactoside, arginine, lysine, ornithine, sodium citrate, sodium thiosulfate and the like; glucose, mannitol, rhamnose and the like cannot be oxidized; can produce acid by using 22 carbon sources such as glycerol, L-arabinose, ribose, D-xylose, glucose, fructose, mannose and the like. See tables 2 and 3.
TABLE 2 physiological and biochemical characteristics of SF327 strain-enzyme activity and carbon source oxidation
+: A positive reaction; -: a negative reaction;
TABLE 3 physiological and biochemical characteristics of strain SF 327-acid production Using carbon
+: A positive reaction; -: a negative reaction; weak positive response W
Example 4 antagonistic Spectrum assay of Bacillus belicus SF327
1) Determination of antagonistic Activity of Bacillus bailii SF327 against Xanthomonas oryzae
Bacterial strain PXO99 A and rice stripe bacteria RS105 and Bacillus besseyi SF327 are respectively cultured in a single colony NB culture medium at 28 ℃ and 180rpm shaking table for 12 hours, and then the bacterial concentration is uniformly adjusted to OD 600 =2.0. 200 mu L of bacterial liquid of PXO99 A and RS105 are respectively absorbed and fully mixed with about 25mL of NA solid culture medium, the mixture is poured into a plate, then an oxford cup with the diameter of 6mm is placed in the center of a NA flat plate, 10 mu L of SF327 bacterial liquid is inscribed in each oxford cup, 3 repetitions are carried out on each pathogenic bacteria, the mixture is placed in an incubator at 28 ℃, after 48 hours of culture, the existence of a bacteriostasis zone is observed, the size of the bacteriostasis zone is recorded, and the arrangement and the photographing are carried out.
The graph of antagonism effect of bacillus belicus SF327 on rice bacterial leaf blight bacteria PXO99 A is shown in fig. 6, the graph of antagonism effect on rice stripe bacteria RS105 is shown in fig. 7, and the antibacterial effect is shown in table 4.
The result shows that the bacteriostasis rate of bacillus beijerinus SF327 on rice bacterial leaf blight bacteria PXO99 A is 75.92%, and the bacteriostasis rate of bacillus beijerinus on rice stripe bacteria RS105 is 73.93%. This indicates that SF327 has a significant antagonism against xanthomonas oryzae.
Table 4 bacteriostatic effects of Bacillus bailii SF327 on Xanthomonas oryzae
2) Antagonistic activity assay of Bacillus belicus SF327 against 5 plant pathogenic fungi
The filter paper is used for the opposite culture method. Culturing Pyricularia oryzae, pyricularia cucumber, phytophthora capsici, rubber gum spore anthracnose and rubber tree point spore anthracnose on PDA culture medium respectively, taking fungus blocks on the plate by using a hole puncher after hyphae grow on the whole plate, taking one fungus cake with the hyphae facing downwards, inoculating to the center of a new PDA plate, placing filter paper sheets at equal distance (35 mm) on the left and right sides of the fungus cake, taking bacillus bescens SF327 5 mu L drop filter paper sheets, and treating 3 repetitions each time, and simultaneously taking the plate without bacillus bescens SF327 as a control. Culturing was performed in an incubator at 28℃for 5 days, and bacteriostasis was observed and recorded.
TABLE 5 bacteriostatic effects of Bacillus bailii SF327 on 5 plant pathogenic fungi
The antagonism effect of bacillus berryis SF327 on rice blast is shown in figure 8, the antagonism effect of bacillus berryis on cucumber fusarium wilt is shown in figure 9, the antagonism effect of phytophthora capsici is shown in figure 10, the antagonism effect of colletotrichum gloeosporioides is shown in figure 11, and the antagonism effect of colletotrichum gloeosporioides is shown in figure 12.
The results show that the antibacterial rate of bacillus berryis SF327 against Pyricularia oryzae is 60.91%, the antibacterial rate of bacillus berryis against cucumber fusarium wilt is 64.68%, the antibacterial rate of bacillus berryis against phytophthora capsici is 58.57%, and the antibacterial rates of bacillus berryis against rubber gum and colletotrichum acutum are 59.72% and 67.42%, respectively. This indicates that SF327 has a better antagonism against all 5 pathogenic fungi.
Therefore, the bacillus beleiensis SF327 provided by the invention can generate auxin IAA, has a growth promoting effect on green vegetables, has a remarkable antagonism on rice bacterial leaf blight bacteria and rice leaf spot bacteria, has antagonism on various plant pathogenic fungi important in production, and provides new resources for biological control of rice bacterial diseases, fungal diseases and rubber tree anthracnose.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Sequence listing
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<120> Bacillus bailii SF327 and application thereof
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tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60
acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120
atggttgtct gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat 180
ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag 240
ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360
gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata 420
gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540
tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660
tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720
gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780
agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960
tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020
gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080
ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140
ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1200
caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260
gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380
cccgaagtcg gtgaggtaac cttttaggag ccagccgccg aag 1423
Claims (3)
1. The application of bacillus beleiensis as a green vegetable growth promoting microbial agent, a xanthomonas oryzae (Xanthomonas oryzae) antagonistic bacteria and a plant pathogenic fungi antagonistic bacteria is characterized in that the bacillus beleiensis is named as bacillus beleiensis (Bacillus velezensis) SF327, and the bacillus beleiensis is preserved in China center for type culture collection (China center for type culture collection) at the month 18 of 2022, and the preservation number is CCTCC M2022130;
The bacillus belicus can produce indole-3-acetic acid auxin, and the fresh weight of the overground parts of the green vegetables is increased;
The pathogenic fungi include Pyricularia oryzae (Magnaporthe oryza), pyricularia cucumber (Fusarium oxysporum f.sp cucumerinum), phytophthora capsici (Phytophthora capsici), alternaria rubber (Colletotrichum gloeosporioides) and Alternaria rubber (Colletotrichum acutatum).
2. The use according to claim 1, wherein said brassica napus is of the long sign No. two variety.
3. The use according to claim 1, wherein the xanthomonas oryzae comprises bacterial blight (Xanthomonas oryzae pv. oryzae) PXO99 A and rice stripe germ (Xanthomonas oryzae pv. oryzicola) RS105.
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