CN112063555A - Paenibacillus polymyxa BFP-1 strain, biological agent and application - Google Patents
Paenibacillus polymyxa BFP-1 strain, biological agent and application Download PDFInfo
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- CN112063555A CN112063555A CN202010946973.XA CN202010946973A CN112063555A CN 112063555 A CN112063555 A CN 112063555A CN 202010946973 A CN202010946973 A CN 202010946973A CN 112063555 A CN112063555 A CN 112063555A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/12—Bacillus polymyxa ; Paenibacillus polymyxa
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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
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- 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
Abstract
The invention discloses a paenibacillus polymyxa BFP-1 strain, a biological agent and application. The paenibacillus polymyxa BFP-1 strain is obtained by separation and screening, and is preserved in Guangdong province microbial strain collection center in 2020, 4 and 7 months, and the preservation number is GDMCC No. 60996. The paenibacillus polymyxa BFP-1 strain provided by the invention has a remarkable inhibiting effect on orchid leaf spot bacteria, the requirement on culture conditions is low, and the paenibacillus polymyxa BFP-1 strain also has a remarkable inhibiting effect on phytophthora, fusarium oxysporum or brown rot of white palm, and a biological agent obtained by culturing the strain in a liquid culture medium has a remarkable inhibiting effect on orchid leaf spot bacteria, and the strain is environment-friendly and nontoxic in source, has small influence on ecological environment, and has a good application prospect.
Description
Technical Field
The invention relates to the technical field of biological pesticides, and particularly relates to a paenibacillus polymyxa BFP-1 strain, a biological agent and application.
Background
Orchid belongs to high-grade potted flower, and the production of orchid plays an important role in the flower industry in China. In recent years, with the gradual expansion of the planting scale of orchids in China, diseases are increasingly serious. Among them, orchid leaf spot is a fungal disease caused by Fusarium sp, which has a serious impact on the production of orchids.
The leaf spot of orchid can be harmful to both Chinese orchid and hybrid orchid. The disease can damage the tender leaves and the grown leaves of the orchid, and dark brown small disease spots are formed on the leaves, which lead to withered leaves in severe cases. The leaf spot of orchid not only affects the growth of orchid plants, but also reduces the ornamental value of the plants, thereby reducing the economic benefit of orchid production. Investigation shows that the disease occurs in main orchid planting bases in Guangdong, and when the disease is serious, the disease incidence rate of individual flower fields reaches more than 50 percent, and the disease has great influence on production. Because the number of pathogenic bacteria of the disease is large, the disease spread is fast, and once the disease occurs in production, the prevention and control are difficult.
Paenibacillus polymyxa (Paenibacillus polymyxa) belongs to the genus Paenibacillus, is a gram-positive bacterium, and is a group of bacteria that produce spores. The strain is an environment-friendly strain, and is widely existed in nature in the forms of soil saprophytic bacteria, plant rhizobacteria, endophyte and the like. The paenibacillus polymyxa can produce various antibacterial substances such as antibacterial peptide, phenols, nucleosides, polymyxin and the like. The existing research shows that the Paenibacillus polymyxa has certain antagonistic action on plant pathogenic fungi and shows certain control effect on the control of plant fungal diseases of crops, vegetables, fruit trees and the like. In vivo inoculation of Liu Jing et al discovers that the separated Paenibacillus polymyxa biocontrol strain SHX301 can reduce the disease incidence rate of eggplant Verticillium wilt (Verticillium dahliae) by 30%, and the biocontrol strain can also promote the growth of eggplant plants (Liu Jing et al, pathogenic bacteria pathotype differentiation and biological control of eggplant Verticillium wilt, agricultural and life science version, university of Zhejiang university, 2019,45(4): 407) 417). However, no research on the control effect of biocontrol bacteria on orchid leaf spot is available at present. Therefore, aiming at the defects of the existing biological control technology of orchid leaf spot, a biological control bacterium capable of effectively controlling the disease needs to be found.
Disclosure of Invention
The invention provides a paenibacillus polymyxa BFP-1 strain, a biological agent and application in order to overcome the defect of difficult prevention and control of orchid leaf spot. The paenibacillus polymyxa BFP-1 strain provided by the invention has a remarkable inhibiting effect on orchid leaf spot bacteria, has low requirement on culture conditions, has a remarkable inhibiting effect on various pathogenic fungi of phytophthora, fusarium oxysporum or brown rot of white palm, and has a good application prospect.
The invention aims to provide a paenibacillus polymyxa BFP-1 strain.
The invention also aims to provide the application of the paenibacillus polymyxa in controlling the orchid leaf spot disease or preparing a biological agent for controlling the orchid leaf spot disease.
Another object of the present invention is to provide the use of Paenibacillus polymyxa for controlling plant diseases caused by Phytophthora, Fusarium oxysporum or Monilinia albiflora.
The invention also aims to provide a biological agent for controlling the leaf spot of orchid.
The invention also aims to provide application of the biological agent in preventing and treating orchid leaf spot.
The invention also aims to provide the application of the biological agent in preventing and treating plant diseases caused by phytophthora, fusarium oxysporum or brown rot of white palm.
In order to solve the technical problems, the invention adopts the technical scheme that:
the invention separates a Paenibacillus polymyxa (Paenibacillus polymyxa) BFP-1 strain from a pseudobulb of the national orchid, the strain is preserved in Guangdong province microorganism strain preservation center at 7 months 4 in 2020, with the preservation number of GDMCC No. 60996 and the preservation address of No. 59 building 5 of Michelia furiosa No. 100, Guangzhou city.
The morphological characteristics of the paenibacillus polymyxa BFP-1 strain are as follows: after the strain is cultured on a PDA culture medium for 48 hours, a single colony is circular and white, the surface is smooth, the strain is opaque and is raised, and the edge is neat.
16S rDNA sequence analysis of the paenibacillus polymyxa BFP-1 strain: the sequence of the strain is shown as SEQ ID NO:1, and the homology of the sequence and Paenibacillus polymyxa (accession numbers are CP006872 and GU369972) in GenBank reaches 99%.
The invention also protects the application of the paenibacillus polymyxa in controlling the orchid leaf spot disease or preparing a biological agent for controlling the orchid leaf spot disease.
The invention also protects the application of the paenibacillus polymyxa in preventing and treating plant diseases caused by phytophthora, fusarium oxysporum or brown rot of white palm.
Preferably, the plant disease is jaboticaba blight, orchid blight or white palm brown rot.
Preferably, the paenibacillus polymyxa is the above paenibacillus polymyxa BFP-1 strain.
The invention also discloses a biological agent for preventing and treating orchid leaf spot, which is obtained by culturing the paenibacillus polymyxa BFP-1 strain in a liquid culture medium. The biological agent has obvious inhibiting effect on orchid phyllosphaeria, and has the advantages of environment-friendly and nontoxic source and small influence on ecological environment.
Preferably, the liquid medium is LB broth comprising tryptone, yeast extract, NaCl and water.
Optionally, the LB culture solution comprises tryptone 10g, yeast extract 5g, NaCl 10g, and the balance of water, the pH is 7.2, and the total volume of the LB culture solution is 1000 mL. The LB medium may be adjusted to pH with 5mol/L NaOH.
Preferably, the conditions of the culture are: inoculating the paenibacillus polymyxa BFP-1 strain to LB culture solution, and carrying out shaking culture at the temperature of 36-38 ℃ at 180-200 r/min for 24-48 h.
More preferably, the conditions of the culture are: and inoculating the paenibacillus polymyxa BFP-1 strain to an LB culture solution, and performing shake culture at the temperature of 37 ℃ at 190r/min for 36 h.
The research of the invention proves that: the biological agent has obvious bacteriostatic action on orchid phyllosphaeria, and the average bacteriostatic zone is 5.1 mm; the culture solution of the strain has bacteriostatic effects on Phytophthora (Phytophthora sp.), Fusarium oxysporum (Fusarium oxysporum) and brown rot (Cylindrocladium spathiphyllum), the average bacteriostatic bands of the strain on the Phytophthora and the Fusarium oxysporum respectively reach 8.5mm and 7.0mm, and the strain has broad-spectrum bacteriostatic action.
Therefore, the application of the biological agent in preventing and treating orchid leaf spot and the application in preventing and treating plant diseases caused by phytophthora, fusarium oxysporum or brown rot pathogen also fall within the protection scope of the invention.
Preferably, the plant disease is a floral plant disease.
Compared with the prior art, the invention has the beneficial effects that:
the paenibacillus polymyxa provided by the invention has a remarkable inhibiting effect on orchid leaf spot bacteria, the requirement on culture conditions is low, and the strain also has an inhibiting effect on various pathogenic fungi (phytophthora, fusarium oxysporum and brown rot of white palm); in addition, the biological agent obtained by culturing the strain in a liquid culture medium has a remarkable inhibiting effect on orchid phyllosphaeria, is environment-friendly and nontoxic in source and has small influence on the ecological environment; therefore, the paenibacillus polymyxa has good application prospects in preventing and treating orchid leaf spot, preparing biological agents for preventing and treating orchid leaf spot and preventing and treating plant diseases caused by phytophthora, fusarium oxysporum or brown rot of white palm.
Drawings
FIG. 1 is a colony morphology diagram of Paenibacillus polymyxa BFP-1 strain culture.
FIG. 2 is a plate confrontation test chart of the inhibition effect of the paenibacillus polymyxa BFP-1 strain culture solution on orchid phyllosphaeria.
FIG. 3 is a plate confrontation test chart of the inhibition effect of the paenibacillus polymyxa BFP-1 strain culture solution on phytophthora.
FIG. 4 is a plate confrontation test chart of the inhibition effect of the paenibacillus polymyxa BFP-1 strain culture solution on Fusarium oxysporum.
FIG. 5 is a plate confrontation test chart of the inhibitory effect of Paenibacillus polymyxa BFP-1 strain culture solution on Monilinia albiflora.
Detailed Description
The present invention will be further described with reference to the following embodiments.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The culture medium and the formula thereof are as follows:
PDA culture medium: weighing 200g of potato, cleaning, peeling, cutting, adding 1000mL of water, boiling for half an hour, filtering with gauze, adding 18g of glucose and 18g of agar, fully dissolving, filtering with gauze while hot, sterilizing at 121 ℃ for 20 minutes, cooling, and storing for later use.
LB culture solution: 10g of tryptone, 5g of yeast extract and 10g of NaCl, adding deionized water to dissolve (about 900 mL), adding 5mol/L NaOH to adjust the pH value to 7.2, and continuously adding deionized water to 1000 mL.
The paenibacillus polymyxa BFP-1 strain is separated from the pseudo bulb of the cymbidium hybridum by a tissue separation method.
The colony morphology chart of the cultured paenibacillus polymyxa BFP-1 strain is shown in figure 1, and the morphological characteristics of the paenibacillus polymyxa BFP-1 strain can be seen as follows: after culturing for 48h on PDA culture medium, the single colony is round, white, smooth in surface, non-transparent, raised and neat in edge.
16S rDNA sequence analysis of the paenibacillus polymyxa BFP-1 strain: the sequence of the strain is shown as SEQ ID NO:1, and the sequence has 99 percent of homology with Paenibacillus polymyxa (accession numbers are CP006872 and GU369972) in GenBank.
Example 1 bacteriostatic Activity of Paenibacillus polymyxa (Paenibacillus polymyxa) BFP-1 Strain culture solution on orchid Blastomyces folliculorum
1. Experimental methods
The activity of the culture solution (containing living bacteria and strain culture filtrate) of the strain was measured by the plate confrontation method.
The bacterial strain is inoculated in LB culture solution and cultured for 48h at 37 ℃ under the condition of 180r/min oscillation. Punching a hole in the center of the PDA flat plate by using a puncher with the inner diameter of 6mm, adding 60 mu L of bacterial liquid into the hole, and respectively inoculating orchid phyllotreta germ blocks on two sides 3cm away from the hole.
2. Results of the experiment
Through determination, a plate confrontation test chart of the inhibition effect of the Paenibacillus polymyxa BFP-1 strain culture solution on orchid leaf spot bacteria is shown in figure 2, and it can be seen that the Paenibacillus polymyxa BFP-1 strain culture solution has an obvious inhibition effect on orchid leaf spot bacteria, and generates an inhibition zone with an average bandwidth of 5.1mm, so that the growth of orchid leaf spot bacteria hypha close to one side of the antagonistic bacteria is stopped.
Example 2 antimicrobial Spectroscopy of the culture broth of the Paenibacillus polymyxa BFP-1 Strain
1. Experimental methods
The antibacterial spectrum of the Paenibacillus polymyxa BFP-1 strain is determined by a plate confrontation method by taking a plurality of flower fungal disease pathogenic bacteria as objects.
The bacterial strain is inoculated in LB culture solution and cultured for 48h at 37 ℃ under the condition of 180r/min oscillation. A punch with an inner diameter of 0.6mm was used to punch a hole in the PDA medium, 60. mu.L of bacterial liquid was added to the hole, and Phytophthora sp, Fusarium oxysporum (Fusarium oxysporum) and Brown palmetto (Cylindrocarpon) were inoculated 3cm from the hole. Taking LB culture solution as a control, repeating each treatment for 3 times, culturing in a constant temperature incubator at 28 ℃, and measuring the width of the bacteriostatic zone when the diameter of a control colony reaches about 5 cm.
2. Results of the experiment
The test chart of the plate confrontation of the paenibacillus polymyxa BFP-1 strain culture solution on the inhibition effect of phytophthora is shown in figure 3, the test chart of the plate confrontation of the paenibacillus polymyxa BFP-1 strain culture solution on the inhibition effect of fusarium oxysporum is shown in figure 4, the test chart of the plate confrontation of the paenibacillus polymyxa BFP-1 strain culture solution on the inhibition effect of brown rot pathogen of anthurium is shown in figure 5, and the results of figures 3-5 show that: the paenibacillus polymyxa BFP-1 strain has good inhibition effects on jalapenosis caused by phytophthora, orchid wilt caused by fusarium oxysporum and brown rot of white palm caused by brown rot of white palm, and the inhibition bandwidths are 8.5mm, 7.0mm and 1.8mm respectively.
Therefore, the paenibacillus polymyxa BFP-1 strain has obvious bacteriostatic action on phytophthora, fusarium oxysporum or brown rot of white palm, and the antibacterial spectrum of the strain 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. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
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gttggtgggg taaaggccta ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg 300
ccacactggg actgagacac ggcccagact cctacgggag gcagcagtag ggaatcttcc 360
gcaatgggcg aaagcctgac ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta 420
aagctctgtt gccagggaag aacgtcttgt agagtaactg ctacaagagt gacggtacct 480
gagaagaaag ccccggctaa ctacgtgcca gcagccgcgg taatacgtag ggggcaagcg 540
ttgtccggaa ttattgggcg taaagcgcgc gcaggcggct ctttaagtct ggtgtttaat 600
cccgaggctc aacttcgggt cgcactggaa actggggagc ttgagtgcag aagaggagag 660
tggaattcca cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg 720
cgactctctg ggctgtaact gacgctgagg cgcgaaagcg tggggagcaa acaggattag 780
ataccctggt agtccacgcc gtaaacgatg aatgctaggt gttaggggtt tcgataccct 840
tggtgccgaa gttaacacat taagcattcc gcctggggag tacggtcgca agactgaaac 900
tcaaaggaat tgacggggac ccgcacaagc agtggagtat gtggtttaat tcgaagcaac 960
gcgaagaacc ttaccaggtc ttgacatccc tctgaccggt ctagagatag acctttcctt 1020
cgggacagag gagacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1080
taagtcccgc aacgagcgca acccttatgc ttagttgcca gcaggtcaag ctgggcactc 1140
taagcagact gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc 1200
cttatgacct gggctacaca cgtactacaa tggccggtac aacgggaagc gaaggagcga 1260
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cgcaaggagc cagccgccga aggtggagag ttt 1473
Claims (10)
1. A Paenibacillus polymyxa (Paenibacillus polymyxa) BFP-1 strain is characterized in that the strain is preserved in Guangdong province microbial strain collection center at 4-7 th of 2020, and the preservation number is GDMCC No: 60996.
2. Application of paenibacillus polymyxa in preventing and treating orchid leaf spot disease or preparing biological agent for preventing and treating orchid leaf spot disease.
3. Application of Paenibacillus polymyxa in preventing and treating flower plant diseases caused by phytophthora, Fusarium oxysporum or brown rot of white palm is provided.
4. The use according to claim 3, wherein the plant disease is Jade blight, orchid blight or brown rot of white palm.
5. The use according to claim 2 or 3, wherein the Paenibacillus polymyxa is the Paenibacillus polymyxa BFP-1 strain of claim 1.
6. A biological agent for controlling orchid leaf spot, which is obtained by culturing the paenibacillus polymyxa BFP-1 strain of claim 1 in a liquid medium.
7. The biological preparation according to claim 6, wherein the liquid medium is LB broth comprising tryptone, yeast extract, NaCl and water.
8. The biological agent according to claim 6, wherein the culture conditions are: inoculating the paenibacillus polymyxa BFP-1 strain to LB culture solution, and carrying out shaking culture at the temperature of 36-38 ℃ at 180-200 r/min for 24-48 h.
9. Use of a biological agent according to any one of claims 6 to 8 for controlling orchid leaf spot.
10. Use of a biological agent according to any one of claims 6 to 8 for controlling plant diseases caused by phytophthora, fusarium oxysporum or brown rot of white palm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114806947A (en) * | 2022-05-06 | 2022-07-29 | 兰州交通大学 | Paenibacillus polymyxa YF capable of preventing and treating root rot of angelica sinensis and application thereof |
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| CN114806947A (en) * | 2022-05-06 | 2022-07-29 | 兰州交通大学 | Paenibacillus polymyxa YF capable of preventing and treating root rot of angelica sinensis and application thereof |
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