CN109355351B - Bacillus subtilis for preventing and treating tobacco target spot disease and microbial inoculum thereof - Google Patents
Bacillus subtilis for preventing and treating tobacco target spot disease and microbial inoculum thereof Download PDFInfo
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- CN109355351B CN109355351B CN201811296722.0A CN201811296722A CN109355351B CN 109355351 B CN109355351 B CN 109355351B CN 201811296722 A CN201811296722 A CN 201811296722A CN 109355351 B CN109355351 B CN 109355351B
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Abstract
The invention discloses bacillus subtilis for preventing and treating target spot of tobacco and a microbial inoculum thereof, and relates to the technical field of microorganisms. The bacillus subtilis Kb-01 is a biocontrol bacterium, and the preservation number is CGMCC No. 12589. The invention also provides a bacillus subtilis microbial inoculum prepared from the bacillus subtilis Kb-01 and a preparation method thereof. The bacillus subtilis Kb-01 and the microbial inoculum prepared from the bacillus subtilis Kb-01 have obvious inhibition effect on the tobacco target spot, the prevention and treatment effect can reach 79.86%, and the bacillus subtilis Kb-01 has good biological pesticide development prospect.
Description
Technical Field
The invention relates to the technical field of microorganisms. More particularly relates to bacillus subtilis and a microbial inoculum thereof for preventing and treating tobacco target spot.
Background
The tobacco target spot (tobaco target spot) is a newly reported outbreak epidemic disease of tobacco leaf parts at home and abroad in recent years, has obvious influence on the yield and quality of tobacco, and is extremely harmful. The current prevention and treatment method of the disease mainly uses chemical pesticides, but the long-term use of the chemical pesticides can easily cause the drug resistance of germs and cause more rampant harm; meanwhile, the problems of pesticide residue increase, environmental pollution and the like of the tobacco leaves can be caused. Therefore, the development, development and application of the environment-compatible biological pesticide are beneficial measures for relieving and changing the 'three R' (Resistance, Resistance and Source) problem caused by chemical pesticides, and are important means for promoting the sustainable and healthy development of the tobacco industry.
Disclosure of Invention
The first purpose of the invention is to provide bacillus subtilis which is pollution-free and has obvious prevention effect on tobacco target spot.
The second purpose of the invention is to provide a microbial inoculum capable of preventing and treating the target spot of tobacco.
The third purpose of the invention is to provide a preparation method of the microbial inoculum for preventing and treating the tobacco target leaf spot.
In order to achieve the first purpose, the invention provides a Bacillus subtilis Kb-01 for preventing and treating tobacco target spot, and the preservation number is CGMCC No. 12589.
In order to achieve the second object, the invention provides a microbial inoculum, which is prepared from the Bacillus subtilis Kb-01.
Preferably, the microbial inoculum is used at a concentration of 1.8 × 108cfu/mL。
In order to achieve the third object, the present invention provides a method for preparing the microbial inoculum, which comprises the following steps:
activating strains: inoculating Bacillus subtilis with the preservation number of CGMCC No.12589 on a slant culture medium for culture;
seed culture: picking single colony from a slant culture medium, inoculating the single colony into a seed culture medium, and culturing to obtain a seed solution;
fermentation culture: inoculating the seed solution into a fermentation culture medium for culture according to 6% of inoculation amount, and obtaining the bacillus subtilis microbial inoculum.
Preferably, the culture conditions are all 28 ℃ for 24 h.
Preferably, the slant culture medium comprises the following components in parts by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 20g of agar, and adding water to a constant volume of 1000mL, wherein the pH value is 7.0-7.5.
Preferably, the seed culture medium and the fermentation culture medium are both composed of the following components in parts by weight: 10g of peptone, 5g of yeast powder and 10g of sodium chloride, and adding water to a constant volume of 1000mL, wherein the pH value is 7.0-7.5.
The invention also provides application of the Bacillus subtilis Kb-01 or the microbial inoculum in preparation of a medicament for preventing and treating tobacco target spots.
The invention has the following beneficial effects:
experiments prove that the prevention and treatment effect of the bacillus subtilis Kb-01 and the microbial inoculum thereof on the tobacco target spot disease is as high as 76.18 percent, is obviously higher than that of the current domestic biological pesticide validamycin for preventing and treating the tobacco target spot disease, and is equivalent to that of the chemical pesticide dimethachlon, so that the popularization and application of the Kb-01 are expected to replace the chemical pesticide to prevent and treat the tobacco target spot disease, the environment can be protected, the pesticide residue is reduced, the safety of tobacco leaves is obviously improved, and the bacillus subtilis Kb-01 has a good biological pesticide development prospect.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is an electron micrograph of Bacillus subtilis Kb-01 according to example 1 of the present invention;
FIG. 2 is a photograph showing the indoor inhibition of Bacillus subtilis Kb-01 against tobacco target spot in example 3 of the present invention.
Detailed Description
The tobacco target spot (tobaco target spot) is a newly reported outbreak epidemic disease of tobacco leaf parts at home and abroad in recent years, has obvious influence on the yield and quality of tobacco, and is extremely harmful. The bacillus subtilis Kb-01 and the microbial inoculum prepared from the bacillus subtilis Kb-01 have obvious inhibition effect on tobacco target spot, and have good biological pesticide development prospect.
On one hand, the invention provides a Bacillus subtilis Kb-01 with a preservation number of CGMCC No.12589, and the preservation unit is as follows: china general microbiological culture Collection center (CGMCC for short), address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, with a preservation date of: 2016, 6 months and 2 days.
In another aspect, the invention also provides a Bacillus subtilis microbial inoculum, wherein the microbial inoculum is prepared from the Bacillus subtilis Kb-01.
The using concentration of the microbial inoculum for inhibiting the target leaf spot of the tobacco is preferably 1.8 multiplied by 108cfu/mL, the effect of inhibiting tobacco target spot disease was most pronounced at this use concentration. Other concentrations of the microbial inoculum can also inhibit the tobacco target spot to some extent.
The invention also provides a preparation method of the microbial inoculum, which comprises the following steps:
activating strains: inoculating Bacillus subtilis with the preservation number of CGMCC No.12589 on a slant culture medium for culture;
seed culture: picking single colony from a slant culture medium, inoculating the single colony into a seed culture medium, and culturing to obtain a seed solution;
fermentation culture: inoculating the seed solution into a fermentation culture medium for culture according to 6% of inoculation amount, and obtaining the bacillus subtilis microbial inoculum.
Preferably, the culture is carried out at 28 ℃ for 24 hours under all conditions, and the growth state of the cells is optimal under all conditions. Those skilled in the art can select other culture conditions in the actual operation process, and can successfully culture the bacteria.
Further, the slant culture medium for strain activation in the invention comprises the following components in parts by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 20g of agar, and adding water to a constant volume of 1000mL, wherein the pH value is 7.0-7.5. The seed culture medium for seed culture and the fermentation culture medium for fermentation culture in the invention comprise the following components in parts by weight: 10g of peptone, 5g of yeast powder and 10g of sodium chloride, and adding water to a constant volume of 1000mL, wherein the pH value is 7.0-7.5. It will be understood by those skilled in the art that the composition and ratio of the slant medium, the seed medium and the fermentation medium may be adjusted and selected according to the actual situation, and are not limited to the embodiments listed herein.
The invention also provides application of the Bacillus subtilis Kb-01 or the Bacillus subtilis microbial inoculum in preparation of a medicament for preventing and treating tobacco target spots.
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical suppliers, unless otherwise specified. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1
First, the acquisition and identification of Bacillus subtilis Kb-01
The bacillus subtilis Kb-01 is cultured in a planar culture medium for 4 days in 2016 (2 g) of rhizosphere soil samples of tobacco plants of a tobacco field in the national home of the range of Ningan county, the Danjiang province, the peony, the city, the Heilongjiang province, diluted tobacco plants are coated in the planar culture medium, single colonies are selected after the tobacco plants are cultured for 4 days, the single colonies are streaked in the planar culture medium, and the single colonies are subjected to inclined culture and preservation after purification.
As shown in attached figure 1, the bacterium is a gram-positive bacterium, the colony is round or irregular, the surface is rough, opaque and dirty white, and dirty white wrinkled molds are formed on the culture surface of an LB liquid culture medium. The size of a single cell is 0.7-0.7 um multiplied by 2-3.5 um, the periphytic flagellum can move, the spore is 0.6-0.9 um multiplied by 1.0-1.5 um, the ellipse is in a column shape, the bacteria can hardly grow under the condition that the pH value is lower than 5.5 and is more than 8.0, and the optimum pH value is 6.5. The optimal culture medium components are as follows: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 20g of agar, wherein the optimal growth temperature is 28 ℃.
The 16S rDNA sequence of the bacillus subtilis Kb-01 is shown as SEQ ID No: 1 is shown.
II, preservation of Bacillus subtilis Kb-01
According to the identification result, the strain Kb-01 is a strain of Bacillus subtilis, which is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of CGMCC No. 3 of Beijing university Hokko No.1 of sunward area, China academy of sciences microorganism research institute, postal code 100101) in 2016, 6.2.2016, and the preservation number is CGMCC No. 12589.
Example 2
Culture of Bacillus subtilis Kb-01
(1) Slant culture: incubation time 48h, incubation temperature 28 ℃, media components (1000 mL): 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 20g of agar, wherein the pH value is 7, and the high-temperature steam sterilization is carried out at 121 ℃ for 30 min.
(2) Seed culture: and (3) picking the activated single colony from the inclined plane in an ultra-clean workbench, inoculating the single colony into a seed culture medium, shaking the bacteria for 24h at 28 ℃, and rotating at the speed of 200rpm to obtain a seed solution. Seed medium composition (1000 mL): 10g of peptone, 5g of yeast powder and 10g of sodium chloride, wherein the pH value is 7.0, and the high-temperature steam sterilization is carried out at 121 ℃ for 30 min.
(3) Fermentation: inoculating the obtained seed liquid into a fermentation culture medium according to the inoculation amount of 5%, carrying out shake culture at 28 ℃ for 48h, and rotating at 150rpm to obtain bacillus subtilis fermentation liquid, namely the microbial inoculum. The fermentation medium has the same formula as seed culture medium.
Example 3
Indoor bacteriostasis test of bacillus subtilis Kb-01 on tobacco target spot bacteria
Preparing a plane culture medium: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 20g of agar, adding water to a constant volume of 1000mL, adjusting the pH value to 7.0-7.5, performing high-temperature steam sterilization at 121 ℃ for 30min, and pouring into a prepared sterilized culture dish for later use.
The treatment and the method are as follows:
a: four tobacco target spot pathogen fungus blocks are picked and connected around the culture dish, and Kb-01 bacterial strains are picked and connected at one point in the center;
CKA: four tobacco target spot pathogen fungus blocks are picked and placed on the periphery of the culture dish, and a little blank exists in the center;
b: four Kb-01 strains are picked and placed around the culture dish, and a bacterial block of the tobacco target leaf spot pathogen is picked and connected at one point in the center;
CKB: one spot in the center is picked and connected with the tobacco target spot pathogen block, and the periphery of the culture dish is blank.
Three replicates of the experimental setup were used.
The investigation method comprises the following steps: and (3) placing the culture dish in an incubator at 28 ℃ for 72h, observing the bacteriostatic effect after 7d, and measuring the width of the bacteriostatic band, wherein the figure is 2.
And (3) test results:
as shown in FIG. 2, in the case of not inoculating Kb-01 bacteria (see FIGS. 2b and 2d), four-peripheral inoculated and one-central inoculated plaque germs all grew over the petri dish, while after inoculating Kb-01 strain at one central point (see FIG. 2a), the expansion of the four-peripheral plaque germs is limited, the zone of inhibition is 6cm on average, and after inoculating Kb-01 strain at four peripheral points (see FIG. 2c), the expansion of the central plaque germs is also limited, and the zone of inhibition is 6cm on average. It can be seen that the Kb-01 strain can better inhibit the growth of the tobacco target leaf spot pathogen, and the next field control effect test can be carried out.
Example 4
Field control effect test of bacillus subtilis Kb-01(CGMCC No.12589) on tobacco target spot disease
Preparing inoculated target spot bacterial liquid: taking 10g of culture medium (purified and stored by Ministry of tobacco science research of peony and river) in which the pathogenic bacteria of the target spot have been cultured, cutting the culture medium, putting the cut culture medium into 500mLNA sterilized water, and preparing the culture medium for later use 30min before inoculation.
Preparing inoculated biocontrol bacterium bacillus subtilis Kb-01 fermentation liquor: selecting a little Bacillus subtilis Kb-01, placing into a triangular flask containing 500mL seed culture medium, placing the triangular flask into a constant temperature shaking incubator, shaking and culturing at 28 deg.C for 24h, and preparing into 3 × 10 concentration 30min before inoculation8cfu/mL of bacterial suspension is ready for use. And (3) experimental design: three treatments, one broth, one blank control, four replicates, 12 cells, 100 tobacco plants per cell were set up.
And (3) treatment A: bacillus subtilis Kb-01 fermentation broth (concentration about 16.0X 108cfu/mL)
Treating the chemical pesticide dimetachlone bactericide B, diluting by 500 times and spraying
Treating the biological pesticide validamycin C, diluting the biological pesticide validamycin C by 800 times and spraying
Treatment D-spraying of the culture solution
Treatment E-clear Water spray
The test method comprises the following steps: a, B, C, D, E five treatments are carried out by adopting a spraying method at intervals of 24h, and after the treatments are continuously carried out twice, the target spot bacterial liquid is inoculated by adopting the spraying method at intervals of 24 h.
The investigation method comprises the following steps: the lesion rate of each cell was investigated 7 days after inoculation.
The test results are shown in Table 1
TABLE 1 Bacillus subtilis Kb-01 preventive and therapeutic effects on plaque disease (test site: Linkou lotus village)
Investigation results show that the control effect of the bacillus subtilis Kb-01 on the tobacco target spot is as high as 79.86%; the prevention and treatment effect of the currently common biological pesticide polyoxin on tobacco target spots is only 31.57 percent, and is extremely obviously lower than the prevention and treatment effect of Kb-01; the control effect of the chemical pesticide mancozeb bactericide on the tobacco target spot is 79.22 percent, which is equivalent to Kb-01. The test result shows that the control effect of the bacillus subtilis Kb-01 on the tobacco target spot is obviously higher than that of the current domestic biological pesticide polyoxin for controlling the tobacco target spot and is equal to that of the chemical pesticide mancozeb bactericide, so that the popularization and application of the Kb-01 are expected to replace the chemical pesticide to control the tobacco target spot, the environment is protected, the pesticide residue is reduced, the safety of tobacco leaves is improved, and the application prospect is wide.
It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention.
Sequence listing
<110> peony river tobacco scientific research institute of Heilongjiang province of China tobacco general company
<120> bacillus subtilis for preventing and treating target leaf spot of tobacco and microbial inoculum thereof
<130> JLC18I0166E
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1402
<212> DNA
<213> Bacillus subtilis
<400> 1
cagtcgagcg gacagatggg agcttgctcc ctgatgttag cggcggacgg gtgagtaaca 60
cgtgggtaac ctgcctgtaa gactgggata actccgggaa accggggcta ataccggatg 120
gttgtctgaa ccgcatggtt cagacataaa aggtggcttc ggctaccact tacagatgga 180
cccgcggcgc attagctagt tggtgaggta acggctcacc aaggcgacga tgcgtagccg 240
acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc tacgggaggc 300
agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg cgtgagtgat 360
gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa caagtgccgt tcaaataggg 420
cggcaccttg acggtaccta accagaaagc cacggctaac tacgtgccag cagccgcggt 480
aatacgtagg tggcaagcgt tgtccggaat tattgggcgt aaagggctcg caggcggttt 540
cttaagtctg atgtgaaagc ccccggctca accggggagg gtcattggaa actggggaac 600
ttgagtgcag aagaggagag tggaattcca cgtgtagcgg tgaaatgcgt agagatgtgg 660
aggaacacca gtggcgaagg cgactctctg gtctgtaact gacgctgagg agcgaaagcg 720
tggggagcga acaggattag ataccctggt agtccacgcc gtaaacgatg agtgctaagt 780
gttagggggt ttccgcccct tagtgctgca gctaacgcat taagcactcc gcctggggag 840
tacggtcgca agactgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 900
gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ctgacaatcc 960
tagagatagg acgtcccctt cgggggcaga gtgacaggtg gtgcatggtt gtcgtcagct 1020
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca 1080
gcattcagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga 1140
cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gacagaacaa 1200
agggcagcga aaccgcgagg ttaagccaat cccacaaatc tgttctcagt tcggatcgca 1260
gtctgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag catgccgcgg 1320
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc 1380
gaagtcggtg aggtaacctt ta 1402
Claims (8)
1. A Bacillus subtilis Kb-01 for preventing and treating tobacco target spot disease has a preservation number of CGMCC No. 12589.
2. An agent prepared from the Bacillus subtilis Kb-01 according to claim 1.
3. The microbial preparation according to claim 2, wherein the microbial preparation is used at a concentration of 1.8X 108cfu/mL。
4. A method for producing the microbial agent according to claim 2, comprising the steps of:
activating strains: inoculating Bacillus subtilis with the preservation number of CGMCC No.12589 on a slant culture medium for culture;
seed culture: picking single colony from a slant culture medium, inoculating the single colony into a seed culture medium, and culturing to obtain a seed solution;
fermentation culture: inoculating the seed solution into a fermentation culture medium for culture according to 6% of inoculation amount, and obtaining the bacillus subtilis microbial inoculum.
5. The method according to claim 4, wherein the culturing is carried out at 28 ℃ for 24 hours.
6. The preparation method according to claim 4, wherein the slant culture medium comprises the following components in parts by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 20g of agar, and adding water to a constant volume of 1000mL, wherein the pH value is 7.0-7.5.
7. The preparation method according to claim 4, wherein the seed culture medium and the fermentation culture medium are prepared from the following components in parts by weight: 10g of peptone, 5g of yeast powder and 10g of sodium chloride, and adding water to a constant volume of 1000mL, wherein the pH value is 7.0-7.5.
8. Use of Bacillus subtilis Kb-01 according to claim 1 or a microbial inoculum according to claim 2 in the preparation of a medicament for preventing and treating tobacco target spots.
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| CN102367429A (en) * | 2011-10-24 | 2012-03-07 | 中国烟草总公司黑龙江省公司牡丹江烟草科学研究所 | Bacillus subtilis for controlling tobacco anthracnose |
| CN107151641A (en) * | 2017-07-04 | 2017-09-12 | 沈阳农业大学 | One plant suppression Rhizoctonia solani Kuhn Brevibacillus laterosporus and its application |
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| CN102367429A (en) * | 2011-10-24 | 2012-03-07 | 中国烟草总公司黑龙江省公司牡丹江烟草科学研究所 | Bacillus subtilis for controlling tobacco anthracnose |
| CN107151641A (en) * | 2017-07-04 | 2017-09-12 | 沈阳农业大学 | One plant suppression Rhizoctonia solani Kuhn Brevibacillus laterosporus and its application |
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