CN113265360B - Brevibacillus brevis and application thereof - Google Patents
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Abstract
The invention discloses a brevibacillus brevisBrevibacillus brevisCGMCCNo.21880, the strain is finally identified as Brevibacillus brevis by morphological feature observation, physiological and biochemical reaction and 16S rDNA sequence comparisonBrevibacillus brevis(ii) a The strain and the fermentation filtrate thereof have antagonistic activity on the alternaria mali; the strain has simple culture conditions, the preparation process of the fermentation filtrate is simple, the application is convenient, and the strain has the potential of developing a novel and efficient apple-related disease biocontrol microbial inoculum from the fermentation filtrate.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to Brevibacillus brevis, and application of a strain and fermentation liquor thereof in preventing and treating plant diseases.
Background
Cercospora leaf spot of apple is a major disease in apple production, and was first discovered in north america in 1903, and then it was also reported in japan, brazil, korea, italy, and the like. The earliest source of record for this disease in our country was the 1913 record of Miyake in Hubei. The disease is caused by infection of apple Pantoea persilica (Marssonina coronaria Davis), and is generally and seriously harmful in the major apple producing areas in China at present. When the disease is epidemic, a large number of apple leaves can fall off in advance, so that the nutrition accumulation of trees is insufficient, and the growth and development of fruit trees and the yield and quality of apples are seriously affected.
At present, the prevention and treatment measures of the cercospora brown spot of the apple mainly comprise spraying of chemical pesticides, strengthening of orchard cultivation management and the like, and the problems of pathogenic bacteria drug resistance, pesticide residue harm, ecological balance and the like caused by long-term use of the chemical pesticides are more and more serious. Therefore, the development of the high-efficiency biocontrol agent for replacing chemical pesticides meets the requirement of modern agricultural production and has important significance for promoting the green, high-quality and healthy development of the apple industry.
Bacillus is a gram-positive rod-shaped bacterium capable of producing spores, and is developed into a bactericide for biological control of plant diseases due to the advantages of wide distribution, safety to human, livestock and environment, high stress resistance, wide antibacterial spectrum, relatively low production cost and the like. At present, the application of bacillus to control various plant diseases at home and abroad is reported more, wherein large-area commercial strains such as GB03, QST713, Bs-916 and the like are not lacked, but the application of bacillus to the practice of controlling the cercospora leaf spot of the apple is less. Therefore, it is necessary to isolate and develop a bacillus strain for controlling cercospora leaf spot of apple.
Disclosure of Invention
The invention aims to provide bacillus and application thereof, and the bacillus and a fermentation liquid thereof have a remarkable inhibition effect on apple brown spot germs.
A Bacillus brevis with antagonistic activity against apple brown spot pathogen is separated from healthy apple tree rhizosphere soil in township, Shandong province, Haiyang, Shandong province, tobacco Taiwan, etc. The preservation unit: china general microbiological culture Collection center (CGMCC), preservation date: 2021, 3/9, accession number: CGMCC No.21880, preservation address: western road No. 1, north chen west road, north kyo, chaoyang, institute of microbiology, china academy of sciences, zip code 100101. The strain is classified and named as Brevibacillus brevis.
The Brevibacillus brevis provided by the invention has the advantages that the culture conditions are simple, the Brevibacillus brevis grows well on most culture media, bright white and nearly circular colonies are formed on a beef extract peptone agar plate, the colonies are opaque, the centers of the colonies are raised, the edges of the colonies are regular, and the surfaces of the colonies are smooth and glossy. The thallus is short and rod-shaped, produces oval spores and is gram-positive. The strain is an aerobic strain, the periphery of a bacterial colony in a nitrate reduction test is red, a gelatin test tube culture medium can be liquefied, the glucose fermentation reaction has no color change, a starch hydrolysis reaction does not generate a hydrolysis ring, the strain cannot grow on a flat plate added with 5% of sodium chloride, and the strain can slowly grow at the temperature of 4 ℃.
The genome DNA of the strain is taken as a template, the bacterial 16S rDNA sequence universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') are taken as primers, a fragment of about 1.5kb is amplified by PCR, the sequencing result shows that the length of the amplified sequence is 1444bp, and the sequencing result shows the nucleotide sequence in a sequence table. The sequence is compared with 16S rDNA gene nucleotide sequence of representative strain recorded in GenBank database for homology, the homology with Brevibacillus brevis AY887081.2 reaches 99%, and MEGA5 is used for constructing phylogenetic tree. Combining the results of morphological observation, physiological and biochemical determination and molecular identification, the strain is named Brevibacillus brevis.
The fermentation filtrate based on the strain is realized by the following steps: activating the strain, inoculating the activated strain into a 250mL triangular flask containing 100mL fermentation medium according to the volume ratio of 2%, performing shake culture at 30 ℃ and 200rpm for 72h to obtain fermentation liquor, centrifuging the fermentation liquor for 10min at 5000r/min, taking supernatant, and filtering and sterilizing by using a 0.22-micron microporous filter to obtain fermentation filtrate. The formula of the fermentation medium is as follows: 10g of peptone, 3g of beef extract, 5 NaCL5g and distilled water, wherein the volume is fixed to 1L, the pH value is adjusted to 7.0-7.5, and the sterilization is carried out for 20min at 121 ℃.
The antagonistic activity of Brevibacillus brevis and the fermentation filtrate thereof on apple brown spot pathogen is realized by adopting a plate antagonistic method and a growth rate method respectively. The strain and the fermentation filtrate thereof have good antagonistic activity on the apple brown spot pathogen.
The determination of the prevention effect of the fermentation filtrate of the brevibacillus brevis on the apple brown spot germs on the leaves in vitro is realized by adopting a plate leaf method. The brevibacillus brevis fermentation filtrate can obviously reduce the disease index of the apple brown spot germ on the leaves in vitro, and has good prevention effect.
The Brevibacillus brevis provided by the invention has strong antagonistic activity on apple brown spot pathogen, the fermentation filtrate has good in-vitro leaf control effect on apple brown spot pathogen, the preparation process is simple, the application is convenient, and the Brevibacillus brevis has the potential of developing a novel and efficient apple-related disease biocontrol microbial inoculum.
Drawings
FIG. 1: the colony morphology of the Brevibacillus brevis in example 1 when cultured on a beef extract peptone agar medium for 2 d;
FIG. 2: the phylogenetic tree of Brevibacillus brevis and related strains constructed according to 16S rDNA sequence in example 1;
FIG. 3: the antagonistic effect of Brevibacillus brevis on apple brown spot pathogen by plate antagonism in example 2 is shown (A is control, B is antagonistic plate).
FIG. 4: the growth inhibition of Brevibacillus brevis fermentation filtrate on Malus pumilus growth using the growth rate method in example 3 was plotted (A is a control, and B is a plate containing Brevibacillus brevis fermentation filtrate).
The Brevibacillus brevis is deposited in China general microbiological culture Collection center on 3-9 months in 2021, which is abbreviated as follows: CGMCC, preservation number: CGMCC No. 21880.
Detailed Description
The invention is further described below in conjunction with the drawings and the detailed description so that those skilled in the art can understand the invention.
The media used in the following examples are as follows:
the potato carrot culture medium (PCDA) comprises the following components in g/L: 100g of potato, 100g of carrot, 20g of glucose, 15g of agar powder and distilled water, wherein the volume is fixed to 1L, and the sterilization is carried out for 20min at 121 ℃. The method is used for culturing and storing alternaria mali and analyzing the antagonistic activity of Brevibacillus brevis on the alternaria mali.
The beef extract peptone agar medium (NA) comprises the following components in g/L: 10g of peptone, 3g of beef extract, 5g of sodium chloride, 15g of agar powder and distilled water, wherein the volume is fixed to 1L, and the sterilization is carried out for 20min at 121 ℃. Is used for culturing and preserving the slant and the plate of Brevibacillus brevis.
The beef extract peptone liquid medium (NB) comprises the following components in g/L: 10g of peptone, 3g of beef extract, 5g of sodium chloride and distilled water are added to a constant volume of 1L, the pH value is adjusted to 7.0-7.5, and sterilization is carried out at 121 ℃ for 20 min. Is used for the fermentation culture of Brevibacillus brevis.
Example 1
The invention relates to the separation and identification of Brevibacillus brevis
A. Separation and preservation of Brevibacillus brevis
Step 1) specimen collection: collecting healthy apple tree rhizosphere soil of township in Shandong province tobacco terrace, Haiyang city, and filling the healthy apple tree rhizosphere soil in sterile bags for later use.
Step 2) separation and purification of Brevibacillus brevis: and (3) drying the collected soil sample in the air and then grinding the soil sample. Weighing 10g of soil sample, placing the soil sample into a conical flask, adding 90mL of sterile water, and shaking the table for 30min to obtain soil sample suspension. Sucking 1mL of suspension, adding into a small test tube containing 9mL of sterile water, and mixing to obtain a suspension with a concentration of 10-2g/mL of soil sample diluent, and sequentially obtaining the soil sample diluent with the concentration of 10 by adopting the same method-5g/mL、10-6g/mL of a soil sample diluent. Suction 10-5g/mL、10- 6Coating 100 μ L of each dilution solution on NA culture medium with coating bar, air drying, repeating for 3 times, culturing at 30 deg.C for 2d, picking out single colony with inoculating needle, inoculating strain on NA culture medium by scribing method, culturing at 30 deg.C for 2d, picking out single colony, and storing.
Step 3), strain preservation: and (3) storing the purified strain in a refrigerator at 4 ℃, wherein the storage culture medium is an NA culture medium.
B. Identification of Brevibacillus brevis
The Brevibacillus brevis is identified according to the colony morphology characteristics, physiological and biochemical characteristics and a phylogenetic tree of a 16S rDNA gene sequence.
Step 1) morphological characteristics
The strain forms bright white and nearly circular colonies on an NA flat plate, the colonies are opaque, the centers of the colonies are raised, the edges of the colonies are regular, the surfaces of the colonies are smooth and glossy (shown in figure 1), and the bacteria are short-rod-shaped, produce oval spores and are gram-positive.
Physiological and biochemical characteristics of the strain in step 2)
TABLE 1 physiological and biochemical characteristics of Brevibacillus brevis
| Detecting items | Results | Detecting items | Results |
| Oxygen demand | + | Starch hydrolysis | - |
| Nitrate reduction | + | 2％NaCl | + |
| Liquefaction of gelatin | + | 5％NaCl | - |
| Fermentation of glucose | - | Growth at 4 deg.C | + |
Step 3) molecular identification of the strains
The genome DNA of the strain is taken as a template, the bacterial 16S rDNA sequence universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') are taken as primers, a fragment of about 1.5kb is amplified by PCR, the sequencing result shows that the length of the amplified sequence is 1444bp, and the sequencing result shows the nucleotide sequence in the sequence table. Homology comparison of the sequence and 16S rDNA gene nucleotide sequence of representative strain recorded in GenBank database shows that the homology of the sequence and Brevibacillus brevis (Brevibacillus brevis AY887081.2) reaches 99%, and MEGA5 is used to construct phylogenetic tree (figure 2). Combining the results of morphological observation, physiological and biochemical determination and molecular identification, the strain is named Brevibacillus brevis.
Example 2
Analysis of antibacterial Activity of Brevibacillus brevis of the present invention
And (3) carrying out antibacterial activity analysis on the Brevibacillus brevis by adopting a plate opposite growth method: the method comprises the steps of taking apple brown spot pathogen as an indicator bacterium, punching an activated indicator bacterium cake by using a 6mm puncher, placing the indicator bacterium cake in the center of a flat plate, inoculating Brevibacillus brevis by adopting a point inoculation method, enabling the two bacteria to be separated by about 2cm, inoculating the indicator bacterium cake only in a control group on one diameter, repeating for 3 times, carrying out standing culture in the dark at 28 ℃, observing the growth condition of pathogenic bacteria in a test group after 30 days, and measuring a bacteriostasis zone (figure 3 and table 2).
TABLE 2 antibacterial Activity analysis of Brevibacillus brevis against apple brown spot pathogen
| Indicator bacterium | Radius of control group (mm) | Width of antibacterial belt (mm) | Inhibition ratio (%) |
| Brown spot of apple | 10±1.22 | 5.78±0.97 | 82.68±4.21 |
Example 3
Analysis of antibacterial Activity of fermentation filtrate of Brevibacillus brevis of the present invention
Activating Brevibacillus brevis, inoculating the activated Brevibacillus brevis into a 250mL triangular flask containing 100mL fermentation medium according to the volume ratio of 2%, carrying out shake culture at 30 ℃ and 200rpm for 72h to obtain fermentation liquor, centrifuging the fermentation liquor for 10min at 5000r/min, taking supernatant, filtering and sterilizing by using a 0.22 mu m microporous filter to obtain fermentation filtrate. Fermenting filtrate according to the proportion of 1: mixing 50 volume ratio with PCDA culture medium sterilized at about 50 deg.C, pouring into a flat plate, taking Malus pumila as indicator bacteria, punching activated indicator bacteria cake with 6mm puncher, placing in the center of the flat plate, analyzing antibacterial activity of the bacteria fermentation filtrate by growth rate method, taking the flat plate without fermentation filtrate as control, repeating for 3 times, and calculating antibacterial rate: the bacteriostatic ratio (%) (control pathogen colony diameter-treated pathogen colony diameter)/control pathogen colony diameter × 100% (fig. 4, table 3).
TABLE 3 antibacterial Activity of Brevibacillus brevis fermentation filtrate on 2 indicator bacteria
| Indicator bacterium | Control group diameter (mm) | Treatment group diameter (mm) | Bacteriostatic ratio (%) |
| Brown spot of apple | 19.59±0.73 | 6.46±0.24 | 67.04±0.32 |
Example 4
The prevention effect of the fermentation filtrate of Brevibacillus brevis on in vitro leaves of apple brown spot pathogen is determined
Determining the biocontrol effect of the fermentation filtrate of the Brevibacillus brevis on the apple brown spot pathogen by adopting a plate leaf method: the gauze is soaked and laid on the bottom of the culture dish, and the leaf stalks are wrapped by the soaked absorbent cotton, and then the healthy and fresh apple leaves with consistent leaf age and size are placed on the gauze. The leaves were sprayed with the fermentation filtrate every 24h for 3 times, with sterile water spray as control. Scraping leaves with a scalpel 24h after the fermentation filtrate spray treatment is finished, and preparing the concentration to be 106And (3) dripping 60 mu L of spore suspension on each leaf of the pathogen spore suspension, uniformly smearing the spore suspension, and performing moisture culture, wherein each leaf is treated by 20 leaves and repeated for 3 times. After 10 days, investigation and statistics of prevention effect are carried out according to disease condition grading standards. The disease grading standard, disease index and prevention and treatment effect calculation formula is as follows:
the disease grading standard is as follows:
level 0: no disease spot on leaf surface
Level 1: the scab occupies less than 10% of the leaf area;
and 3, level: the scab occupies 11 to 25 percent of the area of the leaf;
and 5, stage: the scab occupies 26 to 40 percent of the area of the leaf;
and 7, stage: the scab accounts for 41-65% of the leaf area;
and 9, stage: the scab occupies more than 66% of the area of the leaf;
disease index (%) ═ Σ (present-grade representative value × present-grade leaf number)/(highest-grade representative value × total leaf number investigated) × 100%
The preventing and treating effect (%) is (contrast disease index-treatment disease index)/contrast disease index 100%
TABLE 4 determination of the prevention of the fermentation filtrate of Brevibacillus brevis
The results show that: the fermentation filtrate of Brevibacillus brevis can obviously reduce the morbidity degree of apple brown spot pathogen on the in vitro leaves of apples, the disease index of a treatment group is obviously reduced, and the prevention effect of the fermentation filtrate of the strain on the in vitro leaves of the apple brown spot pathogen is 64.25 percent respectively.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> forest resource monitoring and protecting service center of cigarette platform city (forest technology popularization center of cigarette platform city, provincial natural protection area management center of coastal protection forest of cigarette platform)
<120> Brevibacillus brevis and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1444
<212> DNA
<213> Brevibacillus brevis (Bacillus brevis)
<400> 1
cggcatggcg gcgtgcctat acatgcaagt cgagcgaggc gtcttcggac cctagcggcg 60
gacgggtgag taacacgtag gcaacctgcc tctcagactg ggataacata gggaaactta 120
tgctaatacc ggataggttt ttggatcgca tgatccgaaa agaaaagatg gcttcggcta 180
tcactgggag atgggcctgc ggcgcattag ctagttggtg gggtaacggc ctaccaaggc 240
gacgatgcgt agccgacctg agagggtgac cggccacact gggactgaga cacggcccag 300
actcctacgg gaggcagcag tagggaattt tccacaatgg acgaaagtct gatggagcaa 360
cgccgcgtga acgatgaagg tcttcggatt gtaaagttct gttgttaggg acgaataagt 420
accgttcgaa tagggcggta ccttgacggt acctgacgag aaagccacgg ctaactacgt 480
gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc 540
gcgcgcaggc ggctatgtaa gtctggtgtt aaagcccgga gctcaactcc ggttcgcatc 600
ggaaactgtg tagcttgagt gcagaagagg aaagcggtat tccacgtgta gcggtgaaat 660
gcgtagagat gtggaggaac accagtggcg aaggcggctt tctggtctgt aactgacgct 720
gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac 780
gatgagtgct aggtgttggg ggtttcaata ccctcagtgc cgcagctaac gcaataaaca 840
ctccgcctgg ggagtactct cgcaagagtg aaactcacag gaattgacgg gggcccgcac 900
aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca 960
tcccgctgac cgctctggag acagagcttc ccttcggggc agcggtgaca ggtggtgcat 1020
ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt 1080
atctttagtt gccagcattc agttgggcac tctagagaga ctgccgtcga caagacggag 1140
gaaggcgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac 1200
aatggttggt acaacgggat gctacctcgc gagaggacgc caatctctta aaaccaatct 1260
cagttcggat tgtaggctgc aactcgccta catgaagtcg gaatcgctag taatcgcgga 1320
tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacggg 1380
agtttgcaac acccgaagtc ggtgaggtaa ccgcaaggag ccagccgccg aaggtggtag 1440
ttgt 1444
Claims (3)
1. Brevibacillus brevis (A)Brevibacillus brevis) The strain is preserved in China general microbiological culture Collection center at 3 months and 9 days in 2021, with the preservation number: CGMCC No.21880, named as: brevibacillus brevis: (Brevibacillus brevis）。
2. The Bacillus brevis of claim 1, (b), (c) and (d)Brevibacillus brevis) The application of the thallus and the fermentation filtrate thereof in inhibiting the apple brown spot pathogen.
3. The use according to claim 2, wherein the method for preparing the fermentation filtrate of the bacterial cells comprises: activating the strain, inoculating the activated strain into a 250mL triangular flask containing 100mL fermentation medium according to the volume ratio of 2%, performing shake culture at 30 ℃ and 200rpm for 72h to obtain fermentation liquor, centrifuging the fermentation liquor for 10min at 5000r/min, taking supernatant, and filtering and sterilizing by using a 0.22-micron microporous filter to obtain fermentation filtrate.
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| CN101423812A (en) * | 2008-12-17 | 2009-05-06 | 河南省农业科学院 | Bacillus amyloliquefaciens and microbiological preparation and preparation method thereof |
| CN101993836A (en) * | 2010-06-13 | 2011-03-30 | 河南省农业科学院 | Bacillus subtilis strain YB-81, fungicide and preparation method and application thereof |
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| CN112175869A (en) * | 2020-09-29 | 2021-01-05 | 烟台市林业科学研究所 | Pseudomonas and application thereof |
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