CN110468084B - New burkholderia cepacia and application thereof in preventing and treating litchi frost blight and litchi anthracnose - Google Patents
New burkholderia cepacia and application thereof in preventing and treating litchi frost blight and litchi anthracnose Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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
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Abstract
The invention belongs to the technical field of plant disease biocontrol, and particularly relates to a new burkholderia cepacia XJYC-2 and application thereof in preventing and treating litchi frost blight and litchi anthracnose. The new Burkholderia cepacia is named as XJYC-2, and is preserved in Guangdong province microorganism culture collection, GDMCC for short, and the address is as follows: the microbial research institute of Guangdong province, No. 59 building, No. 5 building, of Zhongluo No. 100 prefecture, Guangzhou city, has the deposition number of GDMCC No. 60699 and the preservation time of 2019, 6 months and 17 days. The invention also provides a biological agent based on the novel burkholderia cepacia. The new Burkholderia cepacia and the biological preparation thereof have obvious inhibition effects on phytophthora litchi and colletotrichum litchi, and have good biocontrol effect and wide application prospect.
Description
Technical Field
The invention belongs to the technical field of plant disease biocontrol, and particularly relates to a new burkholderia cepacia XJYC-2 and application thereof in preventing and treating litchi frost blight and litchi anthracnose.
Background
Litchi (lichchi chinensis Sonn.) is an important tropical and subtropical fruit planted in southern areas of China, has good color, aroma and taste, rich nutrition, medicinal value after being eaten in a proper amount, shares the prime name of 'Chinese precious fruit', and is one of the most competitive export fruits in China. However, because the mature period of litchi is in midsummer, during picking, transporting and storing, great economic loss is easily caused by browning and rotting, and postharvest diseases are the main causes of rotting. The frost blight and anthracnose of litchi are the main diseases causing fresh litchi fruit rot in the post-harvest link.
However, the control of litchi peronophythora and litchi anthracnose in production is mainly chemical control. Not only remains potential risks for eating safety of litchi fruits, but also promotes pathogenic microorganisms to generate drug resistance, so that many researches are dedicated to developing safe and efficient biological control methods. Antagonistic microorganisms are screened from the litchi rhizosphere soil, the stability of the micro-ecological environment of the plant rhizosphere soil can be effectively maintained, and the method has important practical significance.
Research has shown that the novel burkholderia cepacia has an effect on the control of plant diseases caused by Rhizoctonia solani (Rhizoctonia solani), Fusarium oxysporum (Fusarium oxysporum), Pythium spp (Pythium spp.), Botrytis cinerea (Botrytis cinerea) and the like, as well as the control of postharvest diseases of citrus. The new Burkholderia cepacia not only has excellent control effect and broad spectrum and other excellent performances as biocontrol bacteria, but also can promote plant growth and decompose toxic substances. In order to fully utilize the bacteria, the mechanism of antibiotic antagonism generated by the bacteria can be researched by further experiments, the using mode is improved, and the possibility of putting the bacteria into commercial use is increased.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art in preventing and treating litchi frost blight and litchi anthracnose, the invention develops application of new Burkholderia cepacia, and the invention mainly aims to provide a new Burkholderia cepacia XJYC-2 which is obtained by separating rhizosphere microorganisms of litchi trees as screening objects and has better inhibiting effect on litchi frost blight and litchi anthracnose, is named as new Burkholderia cepacia XJYC-2, provides new resources and directions for biological prevention and chemical prevention and treatment instead of chemical prevention and treatment, and can be developed and utilized as biological pesticide in the future.
The invention also aims to provide a biological agent prepared on the basis of the novel burkholderia cepacia.
The invention also aims to provide the application of the new burkholderia cepacia in preventing and treating litchi frost blight and litchi anthracnose.
The purpose of the invention is realized by the following scheme:
a new Burkholderia cepacia is named as XJYC-2, which is deposited in Guangdong province microorganism culture collection center, GDMCC for short, and the address is as follows: the microbial research institute of Guangdong province, No. 59 building, No. 5 building, of Zhongluo No. 100 prefecture, Guangzhou city, has the deposition number of GDMCC No. 60699 and the preservation time of 2019, 6 months and 17 days.
The new burkholderia cepacia is cultured on an LB culture medium at 30 ℃ for 24 hours, and a single colony is circular and milky, has a smooth surface and is neat in edge.
A biological agent is prepared based on the above new Burkholderia cepacia.
The biological agent is prepared by liquid fermentation of the new burkholderia cepacia, and preferably comprises the following steps: inoculating the burkholderia cepacia to an LB liquid culture medium for culture to obtain the biological reagent.
The pH value of the LB liquid culture medium is 7.0.
The culture refers to the culture for 24 hours under the conditions of 28-30 ℃ and shaking rate of a shaking table of 180-220 rpm.
The invention also provides application of the biological agent in preventing and treating litchi frost blight and litchi anthracnose.
The invention also provides application of the new burkholderia cepacia in preventing and treating litchi frost blight and litchi anthracnose.
The new Burkholderia cepacia XJYC-2 and the biological preparation thereof have obvious inhibition effects on two fungi, namely peronophythora litchi and peronophythora litchi, have good biological control effects on peronophythora litchi and peronophythora litchi, are environment-friendly and nontoxic in biological sources, have small influence on the ecological environment, and show that the strain has wide application prospects in the aspects of developing biological pesticides to control fungal diseases, particularly peronophythora litchi and peronophythora litchi.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the new burkholderia cepacia obtained by the invention is derived from healthy litchi fruit tree rhizosphere soil, and can be symbiotic with litchi for a long time, so that the possibility of damaging the stability of the micro-ecological environment on the surface of a plant is avoided;
2. the new Burkholderia cepacia and the biological preparation thereof have strong inhibition effects on peronophythora litchi and colletotrichum litchi, and the biological preparation is environment-friendly and nontoxic in source and has small influence on the ecological environment;
3. the new Burkholderia cepacia obtained by the invention has low requirement on culture conditions and has good development and application prospects.
Drawings
FIG. 1 is a single colony of Burkholderia cepacia XJYC-2 on LB medium.
FIG. 2 is a graph showing the effect of Burkholderia cepacia XJYC-2 on the inhibition of Phytophthora litchi. Wherein A is the normal growth colony morphology of peronophythora litchi, and B is the inhibition map of new Burkholderia cepacia XJYC-2 on peronophythora litchi.
FIG. 3 is a diagram showing the effect of Burkholderia cepacia XJYC-2 on the inhibition of Colletotrichum litchi. Wherein A is the normal growth colony morphology of the litchi anthracnose pathogen, and B is the inhibition pattern of the new Burkholderia cepacia XJYC-2 on the litchi anthracnose pathogen.
FIG. 4 is a diagram showing the effect of a biological agent of Burkholderia cepacia XJYC-2 on the inhibition of Phytophthora litchi. Wherein A is the normal growth colony morphology of peronophythora litchi, and B is the inhibition map of new biological preparation of Burkholderia cepacia XJYC-2 on peronophythora litchi.
FIG. 5 is a diagram showing the effect of a biological agent of Burkholderia cepacia XJYC-2 on the inhibition of Colletotrichum litchi. Wherein A is the normal growth colony morphology of the litchi anthracnose pathogen, and B is the inhibition pattern of the biological agent of the new Burkholderia cepacia XJYC-2 on the litchi anthracnose pathogen.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto. The reagents mentioned in the examples are commercially available.
Example 1: isolation, purification and preservation of Burkholderia cepacia
(1) Preparing an LB culture medium: weighing 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding 1000mL of water, uniformly stirring, adding 15g of agar, fully heating to dissolve, subpackaging into 250mL triangular bottles, sterilizing at 121 ℃ for 20min by moist heat, and storing for later use.
(2) Separating and purifying burkholderia cepacia: a soil sample for separating antagonistic bacteria is collected from healthy fruit tree rhizosphere soil in a litchi orchard of southern China university of agriculture. Weighing 10g of soil sample, adding the soil sample into a triangular flask containing 90mL of sterile normal saline (0.9%) under aseptic condition, placing on a shaking table at 28 ℃ and 180rpm for 30min, and preparing to obtain the product with the dilution multiple of 10-1The soil dilution of (1) is allowed to stand at room temperature for 10min to separate solid impurities such as sand, stone, soil, etc. Take 1mL of 10-1Adding the soil diluent into a test tube of 9mL of normal saline, fully and uniformly mixing, and preparing the mixture to obtain the soil diluent with the dilution multiple of 10-2The soil dilution of (1). By parity of reasoning, gradually diluting the soil liquid to 10-5. 100 μ L of each dilution (dilution times: 10, respectively)-3、10-4And 10-5) And (3) coating the solution on an LB solid plate prepared in the step (1), setting each dilution multiple to be 3 times, and culturing the solution at the temperature of 30 ℃ for 3 days to separate various bacteria in the soil. Selecting bacteria with obvious characteristic difference of size, shape, color, texture, surface structure, etc., and purifying by plate-scribing method to obtain single colony for use.
The purified strains were subjected to 16S rDNA sequence analysis, wherein one of the bacterial sequences (shown below) had 99.86% homology with Burkholderia cenocepacia MC0-3 having accession number CP000960.1 in GenBank, and they were confirmed to be classified and named as Burkholderia cepacia (Burkholderia cenocepacia) and as XYJC-2.
The sequence fragments are as follows:
AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCCTTACACATGCAAGTCGAACGGCAGCACGGGTGCTTGCACCTGGTGGCGAGTGGCGAACGGGTGAGTAATACATCGGAACATGTCCTGTAGTGGGGGATAGCCCGGCGAAAGCCGGATTAATACCGCATACGATCCACGGATGAAAGCGGGGGACCTTCGGGCCTCGCGCTATAGGGTTGGCCGATGGCTGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAATCCTTGACTCTAATACAGTCGGGGGATGACGGTACCGGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGCTAAGACCGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTGGTGACTGGCAGGCTAGAGTATGGCAGAGGGGGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGCCAATACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTTGGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGGCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGGTCGGAATCCTGCTGAGAGGCGGGAGTGCTCGAAAGAGAACCGGCGCACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTTAGTTGCTACGCAAGAGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGAACAGAGGGTTGCCAACCCGCGAGGGGGAGCTAATCCCAGAAAACCGATCGTAGTCCGGATTGCACTCTGCAACTCGAGTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCG
the new Burkholderia cepacia XJYC-2 is cultured on LB culture medium at 30 ℃ for 24h, and the single colony is circular milky white, has smooth surface and neat edge (see figure 1).
(3) Preservation of New Burkholderia cepacia XJYC-2: the strain is preserved in Guangdong province microbial culture collection center (GDMCC) of Guangdong province microbial research institute of No. 59 building, No. 5 building, Guangdong province, of Mieli Zhou No. 100 college, Guangzhou city, 6.17.2019, and the preservation number is GDMCC No. 60699.
Example 2: determination of the Activity of Burkholderia cepacia by confrontation culture
(1) LB medium was prepared as in example 1.
(2) Preparing a PDA culture medium: cutting 200g of potato (peeled) into blocks, adding 1000mL of water, boiling for 20min, filtering with four layers of gauze, adding 10g of glucose and 15g of agar, stirring to fully dissolve, adding distilled water to reach a constant volume of 1000mL, subpackaging into 250mL triangular bottles, performing damp-heat sterilization at 121 ℃ for 20min, and storing for later use.
(3) Preparation of carrot culture medium: weighing 200g of carrot, fully juicing by using a juicer, filtering by using 16 layers of gauze, supplementing water to 1000mL of filtrate, adding 15g of agar, fully heating to dissolve, subpackaging in conical bottles, sealing, performing moist heat sterilization at 121 ℃ for 20min, and preserving for later use.
(4) Preparation of a single colony of Burkholderia cepacia: and (3) streaking and inoculating the bacillus onto an LB culture medium plate for activation culture for 24h, and then picking out a single colony for streaking preservation.
(5) Culturing Peronophythora litchi and colletotrichum litchi: respectively inoculating peronophythora litchi to a carrot plate and inoculating peronophythora litchi to a PDA plate, culturing for 7d, and punching bacterial cakes at the edges of bacterial colonies with a puncher with the diameter of 7 mm.
(6) And (3) activity determination:
firstly, the inhibition effect of the new burkholderia cepacia on the growth of peronophythora litchi is determined: and (3) streaking the new burkholderia cepacia prepared in the step (4) to the edge of a carrot culture medium flat plate, inoculating the peronophythora litchi cake prepared in the step (5) at a position 3cm away from the streaking area, taking the new burkholderia cepacia which is not inoculated as a control, repeating the steps, placing the mixture at a constant temperature of 25 ℃ for 5 days, and observing the hypha forms of the peronophythora litchi with the edge of an antibacterial zone and the control, wherein the result is shown in figure 2. As can be seen from the figure, the new Burkholderia cepacia XJYC-2 has obvious inhibition effect on the growth of phytophthora litchi and causes hypha malformation.
② the inhibition of the new onion burkholderia to the growth of litchi anthrax is determined: and (3) streaking the new burkholderia cepacia prepared in the step (4) to the edge of a PDA culture medium plate, inoculating the litchi colletotrichum cake prepared in the step (5) at a position 3cm away from the streaking area, taking the new burkholderia cepacia which is not inoculated as a control, repeating the steps, culturing at a constant temperature of 25 ℃ for 5 days, and observing hypha forms of litchi colletotrichum at the edge of a bacteriostatic zone and the control, wherein the result is shown in figure 3. As can be seen from the figure, the new Burkholderia cepacia XJYC-2 has obvious inhibition effect on the growth of litchi anthrax, and causes hypha malformation.
Example 3: oxford cup method for measuring activity of new burkholderia cepacia biological agent
(1) LB medium was prepared as in example 1; the LB liquid culture medium is prepared by dissolving the weighed medicine completely, subpackaging into conical flasks (each 100mL of culture solution), plugging and binding, sterilizing at 121 ℃ for 20min, cooling and storing for later use except that agar is not added.
(2) PDA medium and carrot medium were prepared as in example 2.
(3) Preparation of a new Burkholderia cepacia biological agent: and (2) inoculating the single colony of the new burkholderia cepacia prepared in the step (3) in the LB culture solution conical flask prepared in the step (1), and culturing at 30 ℃ and at the shaking table speed of 200rpm for 24 hours to obtain the biological preparation of the new burkholderia cepacia.
(4) The cultivation of Peronophythora litchi and Anthrax litchi was performed as in example 2.
(5) And (3) activity determination:
firstly, the inhibition effect of the new burkholderia cepacia biological agent on the growth of peronophythora litchi is determined: and (3) placing an Oxford cup at the edge of a carrot culture medium flat plate, sucking 200 mu L of the new Burkholderia cepacia biological agent prepared in the step (3) into the Oxford cup, inoculating the Phytophthora litchi cake prepared in the step (4) at a position 3cm away from the Oxford cup, taking the new Burkholderia cepacia as a control, repeating the steps for 3 times, placing the Nikholderia cepacia at a constant temperature of 25 ℃ for culturing for 5 days, and observing the hypha forms of the Phytophthora litchi at the edge of the bacteriostatic zone and the control, wherein the result is shown in figure 4. As can be seen from the figure, the biological preparation of the new Burkholderia cepacia XJYC-2 has obvious inhibition effect on the growth of Phytophthora litchi, and causes hypha malformation.
② the determination of the inhibition effect of the new onion burkholderia biological agent on the growth of litchi anthrax: and (3) placing an Oxford cup at the edge of a PDA culture medium flat plate, sucking 200 mu L of the new Burkholderia cepacia biological agent prepared in the step (3) into the Oxford cup, inoculating the litchi anthrax fungus cake prepared in the step (4) at a position 3cm away from the Oxford cup, taking the new Burkholderia cepacia not inoculated as a control, repeating the steps, placing the mixture at the constant temperature of 25 ℃ for 5 days, and observing the hypha forms of litchi anthrax at the edge of an antibacterial zone and the control, wherein the result is shown in figure 5. As can be seen from the figure, the biological preparation of the new Burkholderia cepacia XJYC-2 has obvious inhibition effect on the growth of the litchi anthrax, and causes hypha malformation.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> southern China university of agriculture
<120> a new burkholderia cepacia strain and application thereof in preventing and treating litchi frost blight and litchi anthracnose
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1385
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> 16S rDNA sequence of New Burkholderia cepacia XJYC-2
<400> 1
agagtttgat cctggctcag attgaacgct ggcggcatgc cttacacatg caagtcgaac 60
ggcagcacgg gtgcttgcac ctggtggcga gtggcgaacg ggtgagtaat acatcggaac 120
atgtcctgta gtgggggata gcccggcgaa agccggatta ataccgcata cgatccacgg 180
atgaaagcgg gggaccttcg ggcctcgcgc tatagggttg gccgatggct gattagctag 240
ttggtggggt aaaggcctac caaggcgacg atcagtagct ggtctgagag gacgaccagc 300
cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg gaattttgga 360
caatgggcga aagcctgatc cagcaatgcc gcgtgtgtga agaaggcctt cgggttgtaa 420
agcacttttg tccggaaaga aatccttgac tctaatacag tcgggggatg acggtaccgg 480
aagaataagc accggctaac tacgtgccag cagccgcggt aatacgtagg gtgcgagcgt 540
taatcggaat tactgggcgt aaagcgtgcg caggcggttt gctaagaccg atgtgaaatc 600
cccgggctca acctgggaac tgcattggtg actggcaggc tagagtatgg cagagggggg 660
tagaattcca cgtgtagcag tgaaatgcgt agagatgtgg aggaataccg atggcgaagg 720
cagccccctg ggccaatact gacgctcatg cacgaaagcg tggggagcaa acaggattag 780
ataccctggt agtccacgcc ctaaacgatg tcaactagtt gttggggatt catttcctta 840
gtaacgtagc taacgcgtga agttggccgc ctggggagta cggtcgcaag attaaaactc 900
aaaggaattg acggggaccc gcacaagcgg tggatgatgt ggattaattc gatgcaacgc 960
gaaaaacctt acctaccctt gacatggtcg gaatcctgct gagaggcggg agtgctcgaa 1020
agagaaccgg cgcacaggtg ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt 1080
taagtcccgc aacgagcgca acccttgtcc ttagttgcta cgcaagagca ctctaaggag 1140
actgccggtg acaaaccgga ggaaggtggg gatgacgtca agtcctcatg gcccttatgg 1200
gtagggcttc acacgtcata caatggtcgg aacagagggt tgccaacccg cgagggggag 1260
ctaatcccag aaaaccgatc gtagtccgga ttgcactctg caactcgagt gcatgaagct 1320
ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca 1380
caccg 1385
Claims (7)
1. A new burkholderia cepacia strain is characterized in that the new burkholderia cepacia strain is named as new burkholderia cepacia (B) (Burkholderia cenocepacia) XJYC-2, deposited in the Guangdong province culture Collection of microorganisms, GDMCC for short, address: the microbial research institute of Guangdong province, No. 59 building, No. 5 building, of Zhongluo No. 100 prefecture, Guangzhou city, has the deposition number of GDMCC No. 60699 and the preservation time of 2019, 6 months and 17 days.
2. A biological agent, which is produced by using the novel Burkholderia cepacia according to claim 1.
3. The biological agent according to claim 2, characterized by being prepared by a process comprising the steps of: inoculating the new burkholderia cepacia to an LB liquid culture medium for culture to obtain the biological reagent.
4. The biological agent according to claim 3, characterized in that: the pH value of the LB liquid culture medium is 7.0.
5. The biological agent according to claim 3, characterized in that: the culture refers to the culture for 24 hours under the conditions of 28-30 ℃ and shaking rate of a shaking table of 180-220 rpm.
6. The use of the biological agent of any one of claims 2-5 for the control of litchi frost blight and litchi anthracnose.
7. The use of the new burkholderia cepacia of claim 1 for the control of litchi frost blight and litchi anthracnose.
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| CN110468084A (en) | 2019-11-19 |
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