CN112980724A - Peanut endogenous burkholderia cepacia and application thereof - Google Patents
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Abstract
The invention discloses peanut endogenous burkholderia cepacia and application thereof, and relates to the technical field of microorganisms. The Burkholderia cepacia (Burkholderia cepacia) PRI08 is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2020, 9 and 29 days, and the preservation number is CGMCC No.: 20842. the invention successfully separates and screens a Burkholderia cepacia (Burkholderia cepacia) PRI08 strain from healthy peanut plants, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.: 20842, respectively; the Burkholderia cepacia PRI08 and the microbial agent prepared from the Burkholderia cepacia PRI08 bacterial suspension can be used for preventing and treating peanut rot, and are remarkable in preventing and treating effect, green, environment-friendly and environment-friendly.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to peanut endogenous burkholderia cepacia and application thereof.
Background
Peanuts are important oil plants and economic crops in the world, and also important crops for earning foreign exchange in export of China, and play an important role in national economy. Peanut rot, also known as rotten fruit disease, is a worldwide disease that mainly damages the pods of peanuts, causing the pods to rot.
At present, the prevention and control of peanut rot mainly comprises the prevention and control measures of chemical agent sterilization, crop rotation cultivation and the like. Because the pathogenic host range of the disease is wide, the disease is difficult to prevent and treat to a certain extent, the soil-borne characteristic further aggravates the difficulty of medicament prevention and treatment, and the problems of natural ecological environment pollution and peanut pesticide residue caused by long-term use of a large amount of pesticides have attracted great attention of people, so that a new way for safely preventing and treating peanut rot is urgently needed.
Biological control plays an increasingly important role in the control of plant diseases due to the advantages of no pollution, no residue, no ecological toxicity, good safety and the like. The biocontrol bacterium is adopted to prevent and control the peanut rot, is environment-friendly, and can avoid a series of problems caused by chemical prevention and control. In the prior art, no high-efficiency biocontrol bacterium for preventing and controlling peanut rot exists.
How to solve the technical problems is a technical problem to be solved in the technical field of microorganisms at present.
Disclosure of Invention
The Burkholderia cepacia (Burkholderia cepacia) PRI08 provided by the invention is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2020, 9 and 29 days, and the preservation number is CGMCC No.: 20842, deposit address: xilu No.1 Hospital No.3, Beijing, Chaoyang, North.
The Burkholderia cepacia PRI08 provided by the invention is used for preventing and treating peanut rot.
The Burkholderia cepacia PRI08 provided by the invention is used for preventing and treating peanut rot caused by infection of peanuts by pythium polycephalum.
The invention also provides a microbial agent, the effective active ingredient of which is the bacterial suspension of Burkholderia cepacia PRI08 as described above.
The invention also provides a microbial agent, wherein the concentration of the Burkholderia cepacia PRI08 in the Burkholderia cepacia PRI08 bacterial suspension is 107～108CFU/ml。
On the basis of the scheme, the Burkholderia cepacia PRI08 bacterial suspension is prepared by suspending Burkholderia cepacia PRI08 in a bacterial culture medium; preferably, the bacterial culture medium is an LB culture medium, and the formula components of the bacterial culture medium comprise: 5g of yeast extract, 10g of peptone, 5g of sodium chloride, 15-20 g of agar and 1000ml of water, wherein the pH value is 7.4-7.6.
The microbial agent provided by the invention is used for preventing and treating peanut rot.
The microbial agent provided by the invention is used for preventing and treating peanut rot caused by infecting peanuts with pythium polycephalum.
The invention has the beneficial effects that: the invention successfully separates and screens the roots of healthy peanut plants in a test base to obtain a Burkholderia cepacia (Burkholderia cepacia) PRI08 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.: 20842, respectively; the Burkholderia cepacia PRI08 and the microbial agent prepared from the Burkholderia cepacia PRI08 bacterial suspension can be used for preventing and treating peanut rot, are obvious in prevention and treatment effect, green and environment-friendly and are environment-friendly; meanwhile, the Burkholderia cepacia PRI08 has no obvious pathogenicity on peanuts, is suitable for being used as a biocontrol bacterium for preventing and controlling peanut rot, and has high use safety.
Drawings
FIG. 1 shows the results of in vitro antagonism of 5 active strains against Pythium herpotans;
wherein, the picture A is a plate confrontation picture; FIG. B is a histogram of the hyphal growth inhibition rate;
FIG. 2 shows the growth promoting effect of Burkholderia cepacia PRI08 on peanut at room temperature;
wherein the content of the first and second substances,
FIG. A is a graph showing the comparison of the angle of treated peanut seedlings with the control group;
FIG. B is a graph showing the results of another angle comparison of treated peanut seedlings for the treatment group and the control group;
FIG. C is a graph of peanut root results from post-anthesis harvest for treatment and control fractions, respectively;
FIG. D is a graph showing the comparison of the main stem length, side branch length and main root length of peanuts after treatment of the treated group and the control group respectively;
FIG. 3 shows the control results of Pythium arachidicola by Burkholderia cepacia PRI08 at room temperature;
wherein in panel B, the abscissa represents four different treated peanut shell samples;
the abscissa letter A is the peanuts irrigated with clear water, and the ungerminated species is pythium;
the abscissa letter AP is the peanut irrigated with clear water and inoculated with pythium nodosum;
the abscissa letter AB is used for irrigating peanuts with Burkholderia cepacia PRI08, and pythium is not inoculated;
the abscissa letter ABP is the pouring of peanuts with Burkholderia cepacia PRI08 and the inoculation of Pythium.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the 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.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Isolation and purification of the Strain
Healthy peanut plants were collected from the experimental field of the institute for peanut, Shandong province, Laixi, Shandong province, and root, stem and leaf tissues were cut, washed with tap water, and then washed with sterile water for 3 times. Soaking in 75% ethanol for 30s for sterilization, soaking in 2% sodium hypochlorite solution for 2.5min, taking out, and soaking and washing with sterile water for 5 times. Placing the mixture into a sterilized mortar, sequentially adding 1mL of sterile physiological saline and 1g of sterilized quartz sand, fully grinding at normal temperature, taking homogenate, and respectively carrying out 10-103The gradient dilution was performed by taking 100. mu.l of 10-4And 10-5The diluted solution is coated in nutrient agar culture medium, each dilution is repeated for 3 times, and after being cultured in a constant temperature incubator at 28 ℃ for 48 hours, single bacterial colonies with different culture characteristics are picked and used as test strains after being cultured and purified.
Example 2
Screening of bacterial strains with bacteriostatic activity
In order to obtain the endophytic strain with the highest antagonistic activity, all candidate isolated strains are tested for the antagonistic effect on the growth of the pythium aggregatum hyphae by adopting a disc diffusion method.
Through experiments, 5 active bacteria are screened out, namely a strain PRI08, a strain PRI20, a strain PRI16, a strain PRI19 and a strain PRI17, and all the 5 active bacteria show different hypha growth inhibition effects (shown in figure 1), wherein each experiment is repeated for 3 times, and CK is a blank control group.
As shown by different hypha growth inhibition effects of 5 active bacteria, the inhibition rate (PIRG) of the PRI08 strain on radial growth 2 days after inoculation is the highest (54% + -2%) (P is less than 0.05) in a hypha growth inhibition test; since the PRI08 strain has higher antagonistic activity against the growth of hyphae of Pythium hernianum than other strains, it is considered as an effective antagonistic biocontrol bacterium and was selected for further study.
Example 3
Identification of PRI08 Strain
And (3) morphological identification: the PRI08 strain is cultured on nutrient agar medium at 28 deg.c to form circular, yellow, smooth, raised and complete colony. The bacteria are gram-negative in staining and strictly aerobic in staining, and are positive in both catalase and oxidase. Meanwhile, peanut seedlings inoculated with the cell suspension of the strain do not show obvious disease symptoms.
And (3) molecular identification: a single colony on LB plate was picked, dissolved in 10. mu.L of sterile water, and the 16S sequence was amplified using bacterial suspension as a template with bacterial 16S universal primers. The upstream sequence is shown as SEQ ID No.2, and the downstream sequence is shown as follows: a fragment of the bacterium was amplified as shown in SEQ ID No. 3.
The PCR system is 50 μ L system, 2 μ L bacterial suspension, 1.5 μ L upstream primer, 1.5 μ L downstream primer, (primer concentration is 10 μmol/L), 5 μ L10 XPCR buffer, 1 μ L dNTPs, 2 μ L Taq DNA polymerase, ddH2O 37 μ L. PCR reaction conditions of 95 ℃ for 3 min; 30s at 95 ℃, 30s at 55 ℃, 80s at 72 ℃ and 32 cycles; 5min at 72 ℃. And detecting the PCR reaction product by 1% agarose gel electrophoresis, and sending to sequencing analysis after detection. The sequencing result of the amplified fragment is shown as SEQ ID No. 1.
The PRI08 strain belongs to Burkholderia cepacia through BLAST alignment, and the homology is 99%. Its 16S rRNA GenBank accession number is MW 045429.
Example 4
Test for determining peanut growth promoting effect of Burkholderia cepacia PRI08 under greenhouse condition
The experimental conditions are as follows: sieving sandy loam with 2mm sieve, mixing, sterilizing with high pressure steam at 121 deg.C for 2 hr, and loading into 20cm diameter flowerpot. Selecting a disease susceptible variety of peanut pythium rot, namely, a medium flower No. 12 (the medium flower No. 12 is abbreviated as zh12 in the invention), selecting healthy and plump peanut seeds, soaking the peanut seeds in 75% alcohol for 0.5-1 min, soaking the peanut seeds in 10% sodium hypochlorite for 15min, sterilizing and washing the peanut seeds for 3 times, sowing 3 seeds in each flowerpot, putting the plants in a climatic chamber for cultivation, and alternating light and dark for 14/10 h.
Grouping experiments: treatment group (designated zh12+ burkholderia cepacia) and control group (designated zh 12);
wherein the treatment group is irrigated with bacterial suspension of Burkholderia cepacia PRI08 with OD value of 0.5 for 3 times every 10 days for 1 time; the control group was irrigated with clear water.
When the peanuts grow to the next stage, 10 oat grains full of pythium aggregatum hyphae are inoculated in the rhizosphere soil of the peanuts, after the peanuts are ripe, the main stem length, the side branch length and the tillering number of the peanuts are investigated, and the peanut growth promoting result of Burkholderia cepacia PRI08 is shown in figure 2.
In fig. 2, panel D, the right bar graph of the two comparative bar graphs is the treatment group result, and the left bar graph is the control group result.
Example 5
Test for measuring control of peanut rot by Burkholderia cepacia PRI08 under greenhouse condition
The experimental method is as follows: sieving sandy loam with 2mm sieve, mixing, sterilizing with high pressure steam at 121 deg.C for 2 hr, and loading into 20cm diameter flowerpot. Selecting a disease susceptible variety (zh12) of rotten fruit diseases of peanut pythium, selecting healthy and plump peanut seeds, soaking the peanut seeds in 75% alcohol for 0.5-1 min, soaking the peanut seeds in 10% sodium hypochlorite for 15min, sterilizing and washing the peanut seeds for 3 times, sowing 3 seeds in each flowerpot, culturing the plants in a phytotron, and alternating light and dark for 14/10 h.
Grouping experiments: the treatment groups (3 groups, respectively named as zh12+ colony, zh12+ primary and zh12+ primary colony, wherein the zh12+ colony is the peanuts irrigated with clear water and inoculated with pythium cepacia, the zh12+ primary group is the peanuts irrigated with Burkholderia cepacia PRI08 and inoculated with pythium cepacia, and the zh12+ colony is the peanuts irrigated with Burkholderia cepacia PRI08 and inoculated with pythium cepacia) and the control group (named as zh 12);
wherein the treatment group is irrigated with bacterial suspension of Burkholderia cepacia PRI08 with OD value of 0.5 for 3 times every 10 days for 1 time; the control group was irrigated with clear water and no pythium was colonized.
When the growth reaches the next stage of peanuts, 10 oat grains full of pythium aggregatum hyphae are inoculated on the rhizosphere soil of the peanuts, after the peanuts are ripe, physiological indexes of the peanuts, such as main stem length, side branch length, tiller number and the like, and the attack situation of pod rot are investigated, and the control results of the Burkholderia cepacia PRI08 on the pythium arachnid are shown in A and B in figure 3.
Example 6
Test for determining control of peanut rot by Burkholderia cepacia PRI08 in field test
The test is carried out in 2019 in a peanut rot disease identification resource garden of peanut research institute Lai West test base in Shandong province, and the resource garden is a natural disease garden of peanut rot disease due to continuous cropping for many years; the identification materials are arranged randomly, the fruit rot of the plot is 10m long, the plot of four rows is 0.6m, and the plot seedling is about 200.
And (3) performing field investigation 120 days after peanut sowing, randomly selecting 20 plants which are not at the edge of the cell and have consistent growth vigor from each cell as investigation objects, investigating the attack condition of pod rot of peanuts, recording the disease grade and the fresh weight of the pods of each peanut, and calculating the disease index of each treatment.
The growth promoting effect is shown in Table 1, and the antagonistic effect is shown in Table 2.
Wherein zh12-1, zh12-2 and zh12-3 are control groups, and bacterial suspension of Burkholderia cepacia PRI08 is not irrigated; zh12+ PRI08-1, zh12+ PRI08-2, zh12+ PRI08-3 as treatment groups, and pouring bacterial suspension of Burkholderia cepacia PRI08 according to experimental requirements.
Disease grading standard
Referring to Wheeler et al, studies have classified the grading criteria for peanut rot into:
level 1, no rotten fruit, namely the rotten fruit rate is 0;
grade 3, the rotten kernel rate is less than 2.5 percent, and the rotten fruit rate is less than 10 percent when the rotten kernel rate is more than 0;
grade 7, rotten kernel rate is less than 2.5%, and rotten fruit rate is less than 50% when the percentage is more than 25%;
grade 9, rotten kernel rate > 2.5%, or rotten fruit rate > 50%.
TABLE 1 growth promoting action
TABLE 2 antagonistic Effect
The experimental results of example 2, example 4, example 5 and example 6 are substantially consistent, and show that burkholderia cepacia has a strong inhibition effect on the growth of peanut rot fungi and can be used for controlling the peanut rot fungi, which indicates that the direct antagonism is probably the main mechanism for biologically controlling the peanut rot fungi by the burkholderia cepacia.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> institute for peanut research in Shandong province
<120> peanut burkholderia endophytic and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 687
<212> RNA
<213> 16S rRNA of Burkholderia PRI08 (16S rRNA of Burkholderia cepacia PRI08)
<400> 1
ggggagacac cgcagcgaac ggcagcacgg ggcgcaccgg ggcgagggcg aacggggaga 60
aacacggaac agccgagggg ggaagcccgg cgaaagccgg aaaaccgcaa cgacacggag 120
aaagcggggg acccgggccc gcgcaagggg gccgaggcga agcagggggg gaaaggccac 180
caaggcgacg acagagcggc gagaggacga ccagccacac gggacgagac acggcccaga 240
cccacgggag gcagcagggg gaaggacaag ggcgaaagcc gaccagcaag ccgcggggaa 300
gaaggcccgg ggaaagcacg ccggaaagaa accgaccaaa cagcggggga gacggaccgg 360
aagaaaagca ccggcaacac ggccagcagc cgcggaaacg aggggcaagc gaacggaaac 420
gggcgaaagc ggcgcaggcg ggcaagaccg aggaaacccc gggccaaccg ggaacgcagg 480
gacggcaggc agagaggcag agggggggag aacccgggag caggaaagcg agagagggga 540
ggaaaccgag gcgaaggcag cccccgggcc aaacgacgcc agcacgaaag cgggggagca 600
aacaggaaga accccggagc cacgcccaaa cgagccaaca ggggggggac accagaacga 660
gcaacggcgg aaggacggcc gggggag 687
<210> 2
<211> 14
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agaggacmgg ccag 14
<210> 3
<211> 8
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
rggaccgc 8
Claims (8)
1. Burkholderia cepacia (Burkholderia cepacia) PRI08 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.: 20842.
2. use of the burkholderia cepacia PRI08 of claim 1, wherein the burkholderia cepacia PRI08 is for peanut kernel rot control.
3. The use of burkholderia cepacia PRI08 according to claim 2, wherein the burkholderia cepacia PRI08 is used for the control of peanut rot caused by infection of peanuts with pythium nodosum.
4. A microbial agent, characterized in that the effective active ingredient is the bacterial suspension of Burkholderia cepacia PRI08 according to claim 1.
5. The microbial inoculant according to claim 4, wherein the concentration of Burkholderia cepacia PRI08 in the bacterial suspension of Burkholderia cepacia PRI08 is 107～108CFU/ml。
6. The microbial inoculant according to claim 4 or 5, wherein the bacterial suspension of Burkholderia cepacia PRI08 is prepared by suspending Burkholderia cepacia PRI08 in a bacterial culture medium.
7. The use of the microbial inoculant according to claim 4 or 5, wherein the microbial inoculant is used for peanut rot control.
8. Use of a microbial inoculant according to claim 4 or 5 for the control of peanut rot caused by infection of peanuts by pythium colonization.
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2021
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| US6077505A (en) * | 1997-06-11 | 2000-06-20 | Wisconsin Alumni Research Foundation | Biological seed treatment to improve emergence, vigor, uniformity and yield of sweet corn |
| CN105695364A (en) * | 2016-03-28 | 2016-06-22 | 福建农林大学 | New burkholderia cenocepacia and application thereof |
| CN109182198A (en) * | 2018-09-29 | 2019-01-11 | 信阳师范学院 | Burkholderia cepacia and its application |
| CN109913393A (en) * | 2019-04-03 | 2019-06-21 | 贵州大学 | One plant of Burkholderia P10 and its application |
| CN110468084A (en) * | 2019-09-20 | 2019-11-19 | 华南农业大学 | One plant of new Burkholderia cepacia and its application in prevention and treatment lichee frost epidemic disease and lichee anthracnose |
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