CN110684700B - Brevundimonas 2B and application thereof - Google Patents
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Abstract
The invention discloses Brevundimonas sp 2B and application thereof, relating to the technical field of edible fungi biology, wherein the Brevundimonas sp 2B is classified and named as Brevundimonas sp 2B and is preserved in China center for type culture collection with the preservation number of CCTCC No. M2019638; the preservation date is as follows: 8, 8 and 16 in 2019; and (4) storage address: when the bacteria disclosed by the invention are applied to the promotion of oyster mushroom growth in Wuhan university in China, the average growing speed of hyphae per day, the yield of a single bag and the thickness of a pileus are increased, the number of days for filling the bag with hyphae is shortened, the oyster mushroom hyphae growth can be effectively promoted, the production period of oyster mushrooms is shortened, the problems of drug residue and environmental pollution can be effectively avoided while the fungus growth is promoted, and the development and the utilization in the commercial field in the future are facilitated.
Description
Technical Field
The invention relates to the technical field of edible fungus biology, and particularly relates to brevundimonas sp.2B and application thereof.
Background
Pleurotus ostreatus (Pleurotus ostreatus) also called Pleurotus ostreatus, oyster mushroom, and Salvia nigricans is one of Pleurotaceae of Agaricales under Basidiomycotina, is a common edible gray mushroom, and is well popular with consumers. The oyster mushroom contains rich nutrient substances, each hundred grams of oyster mushroom contains 20 to 23 grams of protein, and the amino acid components and the types are complete. The oyster mushroom has delicious taste because it contains many kinds of amino acids, can stimulate human taste organs to produce delicious taste, is much better than the delicate taste of sodium glutamate contained in pure monosodium glutamate, and does not cause thirst. Research shows that the oyster mushroom also contains physiologically active substances such as oyster mushroom extract (protein sugar) and acidic polysaccharide, and has certain effects on health, longevity, hepatitis prevention and treatment and cancer prevention; the MUSHROOm ribonic acid (MUSHROOm-vrna) contained in Pleurotus ostreatus can strongly inhibit virus proliferation, so that when fungus food such as Pleurotus ostreatus is frequently eaten, viral infection diseases such as influenza and hepatitis can be reduced, and epidemic virus invasion can be resisted.
The oyster mushroom is the edible mushroom with the largest cultivation amount and the widest cultivation range in China. In order to increase the yield of oyster mushrooms, farmers generally take measures of additional manuring, covering soil or applying plant ash, using growth regulators, spraying pesticides, etc. to increase the yield by chemical agents. Although these methods can increase the yield, they also cause problems such as deterioration of soil shape, deterioration of product quality and environmental pollution, but if biological control techniques using growth-promoting bacteria capable of promoting fungal growth are used, problems such as drug residues and environmental pollution can be effectively avoided. It is therefore of great importance to find a microorganism which promotes an increase in the yield of Pleurotus ostreatus strains without causing human health hazards and environmental pollution.
Disclosure of Invention
Aiming at the defects of the prior art, the technical problem to be solved by the invention is to provide a bacterium brevundimonas 2B for promoting the growth of oyster mushroom, which is a suitable edible fungus growth-promoting bacterium; the invention aims to solve another technical problem of providing the application of brevundimonas sp.2B in promoting the growth of oyster mushrooms so as to solve the problems that the prior art can not effectively avoid medicine residue and environmental pollution while promoting the growth of fungi.
In order to solve the problems, the invention adopts the technical scheme that:
a bacterium for promoting growth of oyster mushroom is classified and named as Brevundimonas sp 2B, is preserved in China center for type culture collection with the preservation number of CCTCC No: m2019638; the preservation date is as follows: 8, 8 and 16 in 2019; and (4) storage address: wuhan university in Wuhan, China.
The brevundimonas sp 2B is applied to promoting the growth of oyster mushrooms.
The brevundimonas sp 2B and the oyster mushroom strain are co-inoculated in the culture material.
In the application, the weight ratio of the oyster mushroom inoculation amount to the culture material is 1: 80.
The brevundimonas brevundii 2B fermentation liquid is applied to promoting the growth of oyster mushrooms.
The application is that the concentration of Brevundimonas 2B in the fermentation liquor is 1.0-2.0 multiplied by 108cfu/mL。
The application of the brevundimonas sp 2B bacterial suspension in promoting the growth of oyster mushrooms.
Has the advantages that: compared with the prior art, the invention screens, separates and identifies a bacterial brevundimonas brevurica 2B for promoting the growth of oyster mushroom from the soil samples of fruiting bodies, mushroom bags and earthing cultivation edible mushrooms, the fermentation liquor of the bacteria and the oyster mushroom strains are inoculated in the culture material together, the number of days for filling the mushroom bags with the added mushroom groups, the daily average growth speed of the mushroom, the yield of single bags and the thickness of mushroom caps are obviously improved, the production cycle of the oyster mushroom can be effectively shortened, and the economic benefit of the oyster mushroom production is increased.
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FIG. 1 is a graph showing the results of co-culturing the selected bacteria with Pleurotus ostreatus strains on a plate for 7 days; the upper left panel shows a bacterial liquid group, and the lower left panel shows a control group; the upper part of the right figure is a bacterial liquid filtrate group, and the lower part of the right figure is a control group;
FIG. 2 is a result chart of the effect of Brevundimonas solution on Pleurotus ostreatus hypha length;
FIG. 3 is a graph showing the effect of Brevundimonas filtrate on Pleurotus ostreatus hypha length;
FIG. 4 is a graph of the result of a phylogenetic tree constructed from 16s rDNA sequences;
FIG. 5 is a graph showing the effect of Brevundimonas on the number of days for which the bags of mycelia were overgrown by Pleurotus ostreatus;
FIG. 6 is a graph showing the effect of Brevundimonas on the average growth rate of hyphae per day;
FIG. 7 is a graph showing the effect of Brevundimonas on Pleurotus ostreatus single bag yield;
FIG. 8 is a graph showing the results of the effect of Brevundimonas on pileus thickness.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to be limiting.
Example 1
1) Sample collection and preservation
Randomly collecting soil samples of different fruiting bodies, fungus bags and soil-covered cultivated edible fungi in a planting base of an edible fungus science and technology industrial park of Taian congratulations and towns in Shandong, adopting aseptic operation for all the soil samples, and respectively putting the collected soil samples into aseptic bags for preservation at-20 ℃.
2) Purification and preservation of bacteria
Accurately weighing 1g of soil sample to be tested, placing into sterilized test tube containing 9mL of sterile water, placing on shaking table, oscillating for 20min at 28 deg.C, rotating at 170r/min to disperse microbial cells, standing for 20-30s to obtain 10-1Diluting the solution; then pipette 10 with 1mL sterile pipette-1Diluting with 1mL of the diluted solution, transferring into a test tube containing 9mL of sterile water, sucking the bacteria solution by blowing, and mixing uniformly to obtain 10-2Diluting the solution; then replacing a sterile suction tube to suck 10-21mL of diluent is transferred into a test tube filled with 9mL of sterile water, and the solution is evenly blown and sucked to obtain 10-3Diluting the solution; by analogy, continuously diluting to obtain 10-4、10-5、10-6And a series of diluted bacteria liquid is obtained.
Melting the prepared NA culture medium, pouring the culture medium into a sterile plate while the culture medium is hot, numbering the culture medium after solidification, and then sucking 1mL of bacterial liquid by using a sterile pipette to inoculate the bacterial liquid on agar plates with different dilution numbers in a number-by-number mode (three times are set for each numbering). And (3) uniformly coating the bacterial liquid on a flat plate by using a sterile spatula, using a sterilization spatula for each dilution, and burning and sterilizing the spatula when the dilution is changed. The coated flat plate is placed on a table for 20-30min to allow the bacterial liquid to permeate into the culture medium,the plates were then inverted and incubated at 28 ℃. This example selects 10-4、10-5、10-6Three gradient smear plates.
And (3) obtaining 63 single colonies through a gradient dilution method, selecting the single colonies, carrying out streaking separation, carrying out purification culture until the single colonies are detected to be single microorganisms through microscopic smear staining, simultaneously carrying out corresponding numbering on the primarily screened bacteria, storing the bacteria to be cultured at a constant temperature of 28 ℃, and observing after 24 hours.
3) Screening for growth-promoting bacteria
PDA plate experiment to identify whether the bacteria living body has growth promoting function
Inoculating the strains which are separated and purified by primary screening and stored into a conical flask of NA liquid culture medium, culturing for 48h at 28 ℃ at 180r/min in a shaking table to obtain cultured saturated bacterial liquid, taking 0.2mL of the bacterial liquid respectively into enriched PDA plates (20 g of glucose, 200g of potato, 10g of peptone, 3g of yeast extract, 15-20g of agar, 1000mL of water and pH7.0) according to the requirement of aseptic operation by using a pipette, uniformly coating by using a coater, taking 1cm of oyster mushroom hyphae by using a puncher, and placing the oyster mushroom hyphae in the center of the plates coated with the bacteria. 0.2mL of sterile water was added to the control group, the control group was inoculated and cultured in an incubator at 28 ℃ and the growth of the mycelia was observed every day, and the results of 7 days of culture are shown in FIG. 1, in which the length of the mycelia in the added group 1 was 3.6cm, the length of the mycelia in the added group 2 was 3.4cm, the length of the mycelia in the added group 3 was 3.5cm, the average length of the mycelia in the added group was 3.5cm, and the length of the mycelia in the control group was 3.1 cm.
b. Growth promoting function of bacterial metabolites
Taking 20mL of cultured bacterial liquid, adding the bacterial liquid into a sterile conical flask through a bacterial filter membrane (0.22 mu m) to obtain bacterial suspension, taking 0.2mL of bacterial suspension by a liquid transfer gun, adding the bacterial suspension into a PDA (personal digital assistant) plate, taking oyster mushroom hyphae by a puncher, and placing the oyster mushroom hyphae in the center of the plate coated with the bacterial suspension. The test groups were subjected to 3-group parallel tests, and the control group was 1 group to which 0.2mL of sterile water was added, inoculated and cultured in an incubator at 28 ℃ and the growth of hyphae was observed every day, and the results at 7 days of culture are shown in FIG. 1.
After the hyphae grow well, comparing with the control group, the bacteria which grow more vigorously than the control group are taken as the bacteria with the growth promoting function, and the influence of the bacteria on the length of the hyphae of the oyster mushroom is shown in figures 2 and 3. The length of the hypha of the added group 1 is 4.0cm, the length of the hypha of the added group 2 is 3.9cm, the length of the hypha of the added group 3 is 3.6cm, the average hypha length is 3.8cm, and the hypha length of the control group is 2.8 cm.
4) Biological characteristics and physiological and biochemical results of oyster mushroom growth promoting bacteria
And (3) observing the biological characteristics of the growth-promoting bacteria of the oyster mushrooms by adopting methods such as morphological characteristics, gram staining, 3% KOH solubility test, spore staining and the like. The morphological characteristics are as follows: the thallus is short rod-shaped, the colony is oval, the surface is smooth, the thallus is opaque, the color is grey white, and no spore exists. The biochemical characteristics of the Pleurotus ostreatus growth promoting bacteria were measured by citrate using the methods of experiment, indole experiment, methyl red experiment, Vop (VP) experiment, etc., and the results are shown in Table 1.
TABLE 1 physio-biochemical characteristics of Pleurotus ostreatus growth-promoting bacteria
Item | Results | Item | Results |
v.P. reaction | - | Acid production from sucrose | - |
Oxidase reaction | + | Fructose acid production | - |
Indole formation | - | Citric acid salt | - |
Gram stain | - | Hydrolyzed starch | - |
Nitrate reduction | - | Hydrolyzed gelatin | - |
Methyl Red test | + | Hydrogen sulfide generation | - |
5) 16s rDNA identification of Pleurotus ostreatus growth-promoting bacteria
Extracting total DNA of bacteria, amplifying and purifying 16s rDNA gene by PCR technology. Phylogenetic trees were then sequenced and constructed (as shown in FIG. 4) for analysis. The 16S rDNA sequence is shown in SEQ ID NO.1, and BLAST comparison is carried out on the tested 16S rDNA gene sequence and sequences in a GenBank database. The result shows that the strain has a closer relationship with Brevundimonas sp, and is identified as Brevundimonas sp 2B by combining morphological characteristics and 16S rDNA gene sequence analysis.
Example 2
Application of growth-promoting bacteria of oyster mushroom
The Pleurotus Ostreatus strain is selected from Taian edible fungi research institute, and is cultured in sunlight greenhouse of edible fungi matrix. The temperature in the base is proper, the water content is moderate, and the method is suitable for normal growth of oyster mushroom strains.
In the examples, three replicate groups of bacteria and one control group were used.
And (3) filling the stirred culture materials into polyethylene bags of 23cm multiplied by 36cm by a machine bagging method, filling 2kg of culture materials into each bag, then adding a sleeve ring at one end of each bag, sealing the bag by using a plastic film, and sterilizing for 8 hours at the normal pressure and the temperature of 100 ℃. After sterilization, the control group is inoculated when the temperature of the material to be prepared is naturally reduced to the normal temperature.
TABLE 2 growth promoting effect of Brevundimonas sp.2B on Pleurotus ostreatus
Number of days/d for filling hypha bag | Hypha day average speed/(cm/d) | Yield per g of single bag | Pileus thickness/cm | |
Bacterium application group A | 21 | 0.548 | 1.224 | 1.70 |
Group B for application of bacteria | 18 | 0.573 | 1.240 | 1.73 |
CK | 23 | 0.524 | 1.106 | 1.67 |
The results show that: the number of days for adding the mycelium bags is full, the average growth speed of the mycelium days is high, the yield of a single bag is high, the thickness of the mushroom cap is increased obviously, and the production period of the oyster mushroom can be shortened effectively.
It should be noted that the present invention is not limited to the above embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
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<120> brevundimonas sp.2B and application thereof
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<213> Brevundimonas sp.2B
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cgggggggca gctaacacat gcaagtcgaa cggacccttc ggggttagtg gcggacgggt 60
gagtaacacg tgggaacgtg cctttaggtt cggaatagct cctggaaacg ggtggtaatg 120
ccgaatgtgc ccttcggggg aaagatttat cgcctttaga gcggcccgcg tctgattagc 180
tagttggtga ggtaacggct caccaaggcg acgatcagta gctggtctga gaggatgacc 240
agccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt ggggaatctt 300
gcgcaatggg cgaaagcctg acgcagccat gccgcgtgaa tgatgaaggt cttaggattg 360
taaaattctt tcaccgggga cgataatgac ggtacccgga gaagaagccc cggctaactt 420
cgtgccagca gccgcggtaa tacgaagggg gctagcgttg ctcggaatta ctgggcgtaa 480
agggcgcgta ggcggatcgt taagtcagag gtgaaatccc agggctcaac cctggaactg 540
cctttgatac tggcgatctt gagtatgaga gaggtatgtg gaactccgag tgtagaggtg 600
aaattcgtag atattcggaa gaacaccagt ggcgaaggcg acatactggc tcattactga 660
cgctgaggcg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 720
aaacgatgat tgctagttgt cgggctgcat gcagttcggt gacgcagcta acgcattaag 780
caatccgcct ggggagtacg gtcgcaagat taaaactcaa aggaattgac gggggcccgc 840
acaagcggtg gagcatgtgg tttaattcga agcaacgcgc agaaccttac caccttttga 900
catgcctgga ccgccacgga gacgtggctt tcccttcggg gactaggaca caggtgctgc 960
atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1020
tcgccattag ttgccatcat ttagttggga actctaatgg gactgccggt gctaagccgg 1080
aggaaggtgg ggatgacgtc aagtcctcat ggcccttaca gggtgggcta cacacgtgct 1140
acaatggcaa ctacagaggg ttaatcctta aaagttgtct cagttcggat tgtcctctgc 1200
aactcgaggg catgaagttg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata 1260
cgttcccggg ccttgtacac accgcccgtc acaccatggg agttggttct acccgaaggc 1320
ggtgcgctaa ccagcaatgg aggcagccga ccacgtagtt ccggcc 1366
Claims (7)
1. A bacterium for promoting growth of oyster mushroom is classified and named as Brevundimonas sp 2B, is preserved in China center for type culture collection with the preservation number of CCTCC No: m2019638; the preservation date is as follows: 8, 8 and 16 in 2019; and (4) storage address: wuhan university in Wuhan, China.
2. The use of brevundimonas sp.2B of claim 1 to promote the growth of oyster mushroom.
3. The use according to claim 2, characterized in that brevundimonas sp.2B is co-inoculated with an oyster mushroom strain in a culture material.
4. The application of claim 3, wherein the weight ratio of the oyster mushroom inoculation amount to the culture material is 1: 80.
5. The use of brevundimonas sp.2B fermentation broth of claim 1 to promote the growth of oyster mushroom.
6. The use according to claim 5, wherein the concentration of Brevundimonas 2B in the fermentation broth is 1.0 to 2.0X 108cfu/mL。
7. The use of a bacterial suspension of brevundimonas sp.2B of claim 1 to promote the growth of oyster mushroom.
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CN115927064B (en) * | 2022-08-31 | 2024-07-30 | 河南省农业科学院植物营养与资源环境研究所 | Pseudomonas taiwanensis PC18 and application thereof |
CN116836833A (en) * | 2023-03-23 | 2023-10-03 | 曲阜师范大学 | Application of pseudomonas Kangshi FHX3 in promoting pleurotus geesteranus growth |
CN116904337A (en) * | 2023-03-23 | 2023-10-20 | 曲阜师范大学 | Application of pseudomonas Mitigrina LMJ4 in promoting growth of mushrooms |
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