CN111363705B - Bacillus polymyxa strain and application thereof - Google Patents
Bacillus polymyxa strain and application thereof Download PDFInfo
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- CN111363705B CN111363705B CN202010276049.5A CN202010276049A CN111363705B CN 111363705 B CN111363705 B CN 111363705B CN 202010276049 A CN202010276049 A CN 202010276049A CN 111363705 B CN111363705 B CN 111363705B
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- 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
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Abstract
The invention discloses a bacillus polymyxa, which is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2019116, the bacillus polymyxa is named LTPP03, and the nucleotide sequence of the 16SrRNA of the bacillus polymyxa is shown as SEQ ID No. 1. LTPP03 is used as a new bacillus strain, and a biological agent prepared by taking the strain as a core has more remarkable control effect on citrus canker.
Description
Technical Field
The invention relates to the technical field of strain screening, in particular to a bacillus polymyxa strain and a screening method thereof.
Background
The bacillus polymyxa is a kind of rhizosphere growth promoting bacteria with wide host range, and has prevention and treatment effects on plant diseases caused by fungi, bacteria, nematodes and the like; it can produce a plurality of antibiotics, polymyxin, various hydrolytic enzymes and other antagonistic substances, and the antagonistic substances are one of the important mechanisms of the biological control function of the bacillus polymyxa. Meanwhile, the bacillus polymyxa can induce plants to produce salicylic acid, so that Induced Systemic Resistance (ISR) is triggered; the bacillus polymyxa can also synthesize auxin, cytokinin and exopolysaccharide, and has activities of dissolving phosphorus and fixing nitrogen.
Because the bacillus polymyxa can effectively prevent plant diseases caused by bacteria, fungi and nematodes, the bacillus polymyxa is usually screened out and used for preparing biocontrol microbial agents, but most of the bacillus polymyxa disclosed in the prior art have poor control effect on plant diseases and insect pests.
Therefore, how to provide a bacillus polymyxa capable of significantly improving the pest control effect is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of this, the invention provides a biological agent prepared from LTPP03 by using bacillus polymyxa (Paenibacillus polymyxa), which has a more significant effect of preventing and treating microorganisms such as bacteria.
In order to achieve the purpose, the invention adopts the following technical scheme:
the bacillus polymyxa is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2019116, the taxonomic name of the bacillus polymyxa is LTPP03, and the nucleotide sequence of the 16SrRNA of the bacillus polymyxa is shown as SEQ ID No. 1.
The technical effect achieved by the technical scheme is as follows: compared with bacillus of other species, the bacillus polymyxa LTPP03 has stronger disease resistance; it can effectively promote plant growth and enhance nitrogen fixation capacity of soil.
The application of the bacillus polymyxa LTPP03 in preventing and treating citrus diseases.
In conclusion, the invention achieves the technical effects that:
compared with other bacillus strains, the LTPP03 has better capabilities of resisting diseases, activating soil and promoting plant growth.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment discloses bacillus polymyxa, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2019116, which is taxonomically named as Bacillus polymyxa LTPP03, and the nucleotide sequence of 16SrRNA of Bacillus polymyxa LTPP03 is shown as SEQ ID No. 1.
EXAMPLE 2 screening of Bacillus polymyxa LTPP03
A method for screening bacillus polymyxa comprises the following steps:
1) collecting samples: collecting soil (four pond forest land in Xingning district of Nanning city), and placing the soil in a container;
2) diluting and heating in water bath: placing 10g of soil into a 250ml beaker, adding 90ml of sterile water into the beaker, and oscillating to obtain the soil with the concentration of 10-1The soil dilution A of (1); heating the soil diluent A in a water bath kettle at 80 ℃ for 10 min;
3) gradient dilution:
(1) 3 tubes containing 9mL of sterile water were taken and labeled: 10-2、10-3And 10-4；
(2) Shaking the soil diluent A, standing for 30s, sucking 1mL of soil diluent by a pipette, and adding the soil diluent marked with 10-2In the test tube, gently and repeatedly blowing and sucking the soil in the test tube for a plurality of times to ensure that the soil is fully and uniformly mixed to obtain a soil diluent B;
(3) similarly, 1mL of the soil diluent B is sucked and added with the serial number of 10-3Blowing and sucking the mixture evenly in the test tube to obtain a soil diluent C; obtaining soil diluent D by the same method.
4) Coating a flat plate:
respectively sucking A, B, C, D100 ul of soil diluent from low concentration, correspondingly coating the soil diluent on plates which are marked with A, B, C, D and are filled with culture medium, coating 2 plates on each diluent, uniformly coating the dilution by using a coater, and then placing the plates in an environment at 37 ℃ for constant-temperature culture for 20 hours; stopping culturing until bacterial colony grows out of the plate;
5) and (3) streak inoculation: selecting a flat plate with a growing bacterial colony for plate scribing, firstly, carrying out parallel scribing on 3 strips on one side of a plate culture medium, then, rotating a culture dish by an angle of 40 degrees, burning off residues on an inoculating loop, cooling, then, carrying out parallel scribing for the 2 nd time through a 1 st scribing part, carrying out the 3 rd time parallel scribing and the 4 th time parallel scribing through a 2 nd time parallel scribing part by the same method, placing the flat plate after scribing in an incubator for continuous culture until the bacterial colony grows out at the scribing part, and stopping culture;
6) gram staining microscopy:
(1) tabletting: selecting the bacterial colony obtained in the step 5), drying and fixing the bacterial colony on a glass slide to obtain a smear;
(2) primary dyeing: dripping ammonium oxalate crystal violet dye solution on the smear obtained in the step (1), dyeing for 0.5min, pouring out the dye solution, and washing with running water until the purple color fades;
(3) mordant dyeing: washing residual water on the smear obtained in the step (2) by using iodine solution, then covering the coated surface by using the iodine solution with the same concentration for 0.5min, and washing by using running water;
(4) and (3) decoloring: removing residual water of the smear obtained in the step (3), dripping 95% alcohol on the smear, decoloring for 15s, and immediately washing with running water;
(5) counterdyeing: dripping a safranine staining solution on the smear obtained in the step (4), staining for 3min, washing with water, and then blotting with absorbent paper;
(6) microscopic examination: under oil-lens observation, the nucleus was stained blue and the cytoplasm was stained pink.
7) Collecting the strain to obtain the bacillus polymyxa LTPP 03.
Example 3 identification of Bacillus polymyxa LTPP03
The strain of bacillus polymyxa LTPP03 was identified by the chinese type culture collection at 03.15.2019. The identification comprises morphological observation, physiological and biochemical characteristic test and 16SrRNA gene sequence determination and analysis of the strain LTPP03 of the bacillus polymyxa, and the classification status of the strain is determined according to the identification result. The strain is identified as follows: bacillus polymyxa LTPP03(Paenibacillus polymyxa LTPP 03). The detection results of the physiological and biochemical characteristics are shown in tables 1-2;
TABLE 1 physiological and biochemical characteristics of strain LTPP 03-enzyme activity, carbon source assimilation
+: positive reaction; -: negative reaction; w.weak positive reaction
TABLE 2 physiological and biochemical characteristics of Strain LTPP 03-production of acid Using carbon sources
+: positive reaction; -: negative reaction;
example 4 plate antagonism assay
The antagonistic bacterium against banana fusarium oxysporum provided by bacillus polymyxa LTPP03, university of guangxi (isolated from soil samples collected from jinling towns in western villages): performing a plate bacteriostasis test on 4 strains of bacillus subtilis K1, bacillus polymyxa XK2 and bacillus subtilis purchased from Deqiang organisms, respectively activating 4 strains to be detected and citrus canker bacteria on an NA culture medium, performing amplification culture by using an NA liquid culture medium, measuring the OD value of the bacteria liquid by using an ultraviolet spectrophotometer, and respectively diluting the citrus canker bacteria liquid and the bacteria liquid to be detected to the concentration of about 108cfu/mL, adding 100uL of citrus canker pathogenic bacteria liquid into every 100mL of NA culture medium to prepare a bacterium-containing plate, sucking 5uL of bacterium liquid to be detected by using a trace adjustable liquid-transferring gun, adding the bacterium liquid to the bacterium-containing plate, adding 5 points to each bacterium-containing plate, placing the blank control into a bacterium-containing plate without the bacterium liquid, placing the plate in a constant temperature box at 28 ℃ for inverted culture for 2-5 days, observing and measuring the diameter of a bacterium-inhibiting ring by using a scale. The larger the inhibition zone is, the better the inhibition effect is, and the antagonism to the bacillus ulcer is stronger.
In an antagonistic test on citrus canker pathogenic bacteria, bacillus polymyxa LTPP03, bacillus subtilis strain K1, bacillus polymyxa XK2 and bacillus subtilis purchased from Delong organisms have the average diameters of inhibition zones of 21.00mm, 12mm, 10mm and 14.2mm, so that the bacillus polymyxa LTPP03 has an obvious antagonistic effect on citrus canker.
Example 5 Ex vivo control Effect of Bacillus polymyxa LTPP03 on Citrus canker
The activated antagonistic bacteria (4 strains described in example 4) and Citrus canker germ were respectively subjected to amplification culture with NA liquid medium, and the antagonistic bacteria liquid and Citrus canker germ liquid cultured for 24 hours were respectively adjusted to 1 × 108cfu/mL, 3 treatments are set for each antagonistic bacterium, 3 times of repetition are carried out, 3 leaves are repeated, each leaf has 6 acupuncture points, and after treatment, the treated leaves are moisturized by absorbent paper and placed at the bottom of a culture dish to be cultured in a constant temperature box at 28 ℃.
Treatment 1: inoculating antagonistic bacteria (spraying the leaf surface), inoculating pathogenic bacteria by needling after 2 days, and observing and recording the disease condition after 7 days;
and (3) treatment 2: inoculating pathogenic bacteria, spraying antagonistic bacteria after 2 days, and observing and recording the disease condition after 7 days;
and (3) treatment: directly inoculating pathogenic bacteria after spraying antagonistic bacteria, and observing and recording the disease condition after 10 days;
clear water control: in each treatment, clear water spray is used to replace the antagonistic bacteria to be tested as a control.
The disease occurrence of 4 treatments was recorded and the control effect was calculated.
TABLE 3 in vitro prevention and treatment effects of Bacillus polymyxa on Citrus canker
The test result shows that the 4 strains of antagonistic bacteria have certain prevention and treatment on the canker of the citrus in vitro leaves
The effect (table 3) is that in treatment 1, the control effects of the 4 tested antagonistic bacteria are all over 60%, wherein the control effects are better that LTPP03 and Bacillus subtilis (Deqiang organism) are 90.74% and 85.19%, respectively; in the treatment 2, the control effect of LTPP03 is the best, namely 55.56 percent, and the control effects of the rest 3 treatments are poor, namely 15 to 35 percent; in the treatment 3, XK2 has no control effect on citrus canker, while the control effect of LTPP03 is 33.33%, the control effect of K1 and Bacillus subtilis (Deqiang organisms) is only 16.67%, and the incidence rate of the control treatment is 100%. The control effects of different strains are different by the same treatment, and the control effect of LTPP03 is the best and the control effect of strain XK2 is the worst by comprehensive comparison.
In the present specification, the embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Guangxi Letu Biotechnology Ltd
<120> bacillus polymyxa strain and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1444
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggcggggtgc tatacatgca agtcgagcgg ggttagttag aagcttgctt ctaactaacc 60
tagcggcgga cgggtgagta acacgtaggc aacctgccca caagacaggg ataactaccg 120
gaaacggtag ctaatacccg atacatcctt ttcctgcatg ggagaaggag gaaaggcgga 180
gcaatctgtc acttgtggat gggcctgcgg cgcattagct agttggtggg gtaaaggcct 240
accaaggcga cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatgggc gaaagcctga 360
cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt tgccagggaa 420
gaacgctttg tagagtaact gctcttgaag tgacggtacc tgagaagaaa gccccggcta 480
actacgtgcc agcagccgcg gtaatacgta gggggcaagc gttgtccgga attattgggc 540
gtaaagcgcg cgcaggcggc tctttaagtc tggtgtttaa tcccgaggct caacttcggg 600
tcgcactgga aactggggag cttgagtgca gaagaggaga gtggaattcc acgtgtagcg 660
gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct gggctgtaac 720
tgacgctgag gcgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc 780
cgtaaacgat gaatgctagg tgttaggggt ttcgataccc ttggtgccga agttaacaca 840
ttaagcattc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacgggga 900
cccgcacaag cagtggagta tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960
cttgacatcc ctttgaccgg tctagagata gatctttcct tcgggacaga ggagacaggt 1020
ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080
aacccttatg cttagttgcc agcaggtcaa gctgggcact ctaagcagac tgccggtgac 1140
aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac 1200
acgtactaca atggccggta caacgggaag cgaaggagcg atctggagcc aatcctagaa 1260
aagccggtct cagttcggat tgtaggctgc aactcgccta catgaagtcg gaattgctag 1320
taatcgcgga tcagcatgcc gcggtgaata cgttcccggg tcttgtacac accgcccgtc 1380
acaccacgag agtttacaac acccgaagtc ggtgaggtaa ccgcaaggag ccagccgccg 1440
aagt 1444
Claims (2)
1. The bacillus polymyxa is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2019116, which is named as Bacillus polymyxa LTPP03, and the 16srRNA sequence is shown as SEQ ID No. 1.
2. The bacillus polymyxa according to claim 1, wherein the bacillus polymyxa LTPP03 is used for controlling citrus diseases.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101018854B (en) * | 2004-08-09 | 2011-05-04 | 科研制药株式会社 | Novel strains belonging to the genus paenibacillus and method of controlling plant disease by using these strains or culture thereof |
| WO2017137353A1 (en) * | 2016-02-09 | 2017-08-17 | Basf Se | Mixtures and compositions comprising paenibacillus strains or metabolites thereof and other biopesticides |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101018854B (en) * | 2004-08-09 | 2011-05-04 | 科研制药株式会社 | Novel strains belonging to the genus paenibacillus and method of controlling plant disease by using these strains or culture thereof |
| WO2017137353A1 (en) * | 2016-02-09 | 2017-08-17 | Basf Se | Mixtures and compositions comprising paenibacillus strains or metabolites thereof and other biopesticides |
Non-Patent Citations (2)
| Title |
|---|
| Biocontrol potential of Paenibacillus polymyxa against Verticillium dahliae infecting cotton plants;Fan Zhang等人;《Biological Control》;20181231;70-77 * |
| 一株抗真菌内生多粘芽孢杆菌的分离鉴定及对水稻恶苗病菌的抑制作用;张淑梅等人;《中国生物工程杂志》;20100228;84-88 * |
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