CN107937326B - Paenibacillus polymyxa for preventing and treating root rot of American ginseng and application thereof - Google Patents
Paenibacillus polymyxa for preventing and treating root rot of American ginseng and application thereof Download PDFInfo
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Abstract
The invention relates to a paenibacillus polymyxa for antagonizing fusarium root rot of American ginseng and application thereof. Belongs to the technical field of biological control of plant diseases and the field of development and utilization of microbial resources, and the preservation number of the paenibacillus polymyxa is as follows: CGMCC No. 15112. The application is the application of the paenibacillus polymyxa in antagonizing fusarium and preventing and treating American ginseng root rot, watermelon fusarium wilt, wheat scab and solanaceae fusarium wilt. Compared with the existing pesticide and biological agent, the paenibacillus polymyxa DN67 has high-efficiency antagonistic action on fusarium.
Description
Technical Field
The invention belongs to the technical field of biological control of plant diseases and the field of development and utilization of microbial resources, and particularly relates to paenibacillus polymyxa and application thereof.
Background
American ginseng (scientific name: Panax quinquefolius) is a perennial plant of Panax of Araliaceae, namely American ginseng, which is native to Dakuibek in Canada and Wisconsin in America, Beijing Huarou in China, Shandongdong and Changbai mountain. The American ginseng has the double effects of nourishing yin and tonifying qi, calming the nerves and promoting intelligence, clearing heat and promoting fluid, reducing pathogenic fire and relieving summer heat, and has wide application and great market demand. However, the continuous cropping obstacle of American ginseng is very serious, and when American ginseng is continuously planted in the same land, the diseases and insect pests are serious, and the yield is reduced or even the yield is no longer high. Researches find that the root rot disease which threatens the American ginseng the most is mainly caused by Fusarium sp, and the Fusarium sp is also the pathogenic bacteria of crops such as bananas, watermelons, cucumbers and the like.
With the development of green agriculture concept, the limit of crops on pesticide residue is stricter in China, the use types and the range of pesticides are greatly limited in the American ginseng planting management process, pentachlorinated nitrobenzene pesticides most effective on fusarium are forbidden, special medicines are lacked when root rot occurs, and great economic loss is brought to growers. Many biological control preparations including microbial agents are researched, and various types of reported antagonistic bacteria are reported aiming at diseases of other crops caused by fusarium, for example, bacillus subtilis for antagonizing special fusarium oxysporum of radix pseudostellariae reported by Fujian agriculture and forestry university has been issued by patent of invention, but radix pseudostellariae is dry root tuber of Pseudostellaria heterophylla (Miq.) Pax of caryophyllaceae, which has a far relationship with American ginseng of Araliaceae, and the separated strains in the research are different in source and strain compared with the strains.
Disclosure of Invention
The invention screens a paenibacillus polymyxa strain for antagonizing American ginseng root rot fusarium from sea bottom mud near Bohai sea of a smoke station. The fermentation liquor of the paenibacillus polymyxa can efficiently inhibit fusarium root rot of American ginseng, and has wide application prospect in the field.
The Paenibacillus polymyxa for antagonizing American ginseng root rot fusarium provided by the invention is Paenibacillus polymyxa DN67, and has been registered and preserved in the general microorganism center of the China general microbiological culture Collection Committee on 21.12.2017, wherein the preservation number is as follows: CGMCC No.15112, preservation address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The fermentation culture condition of the paenibacillus polymyxa DN67 is 28-37 ℃, and the time is 18-24 h.
The formula of the fermentation medium of the strain is as follows: 1-2% of glucose, 0.5-1.0% of yeast extract powder, 0.5-1.0% of dipotassium hydrogen phosphate, 0.2-0.5% of sodium chloride, 0.5-1.0% of ammonium sulfate, 0.03-0.05% of magnesium sulfate and adjustment of pH to 6.5-7.5.
The invention also provides application of the paenibacillus polymyxa DN67 in medicines for preventing and treating American ginseng root rot fusarium.
The invention also provides a biocontrol microbial inoculum containing the paenibacillus polymyxa DN 67.
Further, the concentration of the paenibacillus polymyxa DN67 in the biocontrol microbial inoculum is 1 multiplied by 108～1×1010CFU/mL or 1X 108～1×1010CFU/g。
The invention also provides a preparation method of the biocontrol microbial inoculum, which comprises the following steps:
(1) inoculating the paenibacillus polymyxa into a liquid culture medium for culture until the OD600 is 0.3-0.8 after the culture solution is diluted by 20 times, and obtaining a seed solution;
(2) inoculating the seed liquid into fermentation culture medium, and culturing until the thallus concentration in the fermentation liquid reaches 1 × 109～1×1010CFU/mL to obtain the biocontrol microbial inoculum.
Further, the volume ratio of the inoculation amount of the seed liquid is 1-10%.
Further, the components and final concentration of the liquid medium were 1.6% glucose, 0.25% sodium chloride, 0.05% magnesium sulfate, 1% yeast extract powder, 0.7% ammonium chloride, 0.1% dipotassium hydrogen phosphate, and pH 7.
Further, the formula of the fermentation medium is as follows: 1-2% of glucose, 0.5-1.0% of yeast extract powder, 0.5-1.0% of dipotassium hydrogen phosphate, 0.2-0.5% of sodium chloride, 0.5-1.0% of ammonium sulfate, 0.03-0.05% of magnesium sulfate and adjustment of pH to 6.5-7.5.
Further, the temperature of the amplification culture is 28-37 ℃.
Compared with the prior art, the invention has the beneficial effects that:
before or during planting of American ginseng, uniformly spraying the paenibacillus polymyxa suspension on leaf surfaces of American ginseng or dripping the American ginseng in soil, or mixing the paenibacillus polymyxa with other bacteria or organic fertilizer and applying the mixture to an agricultural field, wherein the concentration of the paenibacillus polymyxa suspension is 1 multiplied by 108～1×1010CFU/mL. Can effectively inhibit the breeding of fusarium of the American ginseng root rot.
Drawings
FIG. 1 is a photograph of a colony of Paenibacillus polymyxa (Paenibacillus polymyxa) DN67 in LB medium.
FIG. 2 shows the bacteriostatic effect of Paenibacillus polymyxa (Paenibacillus polymyxa) DN67 on Fusarium root rot of American ginseng.
FIG. 3 is a phylogenetic tree of Paenibacillus polymyxa (Paenibacillus polymyxa) DN67 based on the 16S rRNA sequence.
Preservation information: paenibacillus polymyxa (Paenibacillus polymyxa) DN67, which is registered and preserved in the China general microbiological culture Collection center on 21.12.2017, and the preservation numbers are: CGMCC No.15112, preservation address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
Detailed Description
Example 1 selection of Paenibacillus polymyxa (Paenibacillus polymyxa) DN67
Collecting sample from offshore seabed surface sediment of cigarette stage city of Shandong province in 2016 (7 months and 2 days), separating and purifying microorganism by dilution coating method, weighing 10g of mud sample, adding into a triangular flask containing 90mL of sterile water, oscillating at 180r/min for 30min, and diluting the obtained suspension to 10-1、10-2、10-3、10-4、10-5And (3) respectively coating the strains on LB plate culture media with different dilutions, repeating each dilution for 2 times, performing inverted culture in an incubator at 30 ℃ for 24 hours, selecting single colonies in the culture media, performing plate streaking and purification for 3 times to obtain cultured pure strains, respectively inoculating the pure strains to PDA culture media coated with American ginseng root rot fungi fusarium solani, culturing for 2-3 days at 28 ℃, and selecting strains with obvious inhibition zones for purification and preservation.
The strain is inoculated into an LB plate culture medium and cultured for 2-3 days at 30 ℃, a puncher is used for transferring the strain into a PDA culture medium, meanwhile, American ginseng root rot fungus fusarium solani with the same size is placed at the other end of the same plate, plate confrontation culture is carried out, and the bacteriostasis effect is shown in figure 2. Strains with good antagonistic effect are screened and identified, and are determined to be Paenibacillus polymyxa (Paenibacillus polymyxa) DN67 through morphological, physiological and biochemical analysis (see table 1) and 16S rRNA molecular biological identification, and a phylogenetic tree based on a 16S rRNA sequence is shown in figure 3.
The paenibacillus polymyxa DN67 forms milky colonies on an LB culture medium, and the colonies are semitransparent, smooth, slightly convex in the middle, uneven in the edge and sticky when picked up (see figure 1).
TABLE 1 physiological and biochemical characteristics of Strain DN67
16S rRNA sequence of paenibacillus polymyxa DN 67:
gggctttgggcggaggccgctaatacatgcagtcgagcggggttaggttagaagcttgcttctaacctaacctagcggcggacgggtgagtaacacgtaggcaacctgcccacaagacagggataactaccggaaacggtagctaatacccgatacatccttttcctgcatgggagaaggaggaaaggcggagcaatctgtcacttgtggatgggcctgcggcgcattagctagttggtggggtaaaggcctaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccgcaatgggcgaaagcctgacggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgccagggaagaacgcttgatagagtaactgctcttgaagtgacggtacctgagaagaaagccccggctaactacgtgccagcagccgcggtaatacgtagggggcaagcgttgtccggaattattgggcgtaaagcgcgcgcaggcggctctttaagtctggtgtttaatcccgaggctcaacttcgggtcgcactggaaactggggagcttgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctgggctgtaactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatgaatgctaggtgttaggggtttcgatacccttggtgccgaagttaacacattaagcattccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacggggacccgcacaagcagtggagtatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatccctctgaccggtctagagatagatctttccttcgggacagaggagacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttatgcttagttgccagcaggtcaagctgggcactctaagcagactgccggtgacaaaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacacacgtactacaatggccggtacaacgggaagcgaaggagcgatgtggagccaatcctagaaaagccggtctcagttcggattgtaggctgcaactcgcctacatgaagtcggaattgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggtcttgtacacaccgcccgtcacaccacgagagtttacaacacccgaagtcggtgaggtaaccgcaaggagccagccgctcgtatagatgggatacgcagggc。
example 2 determination of the bacteriostatic Effect of Paenibacillus polymyxa (Paenibacillus polymyxa) DN67
The embodiment provides a biocontrol microbial inoculum, which is fermentation liquor of paenibacillus polymyxa DN67, and the preparation method comprises the following steps:
(1) inoculating the paenibacillus polymyxa DN67 into a liquid culture medium for culture until OD600 is 0.3-0.8 after the culture solution is diluted by 20 times, and obtaining a seed solution;
the components and final concentration of the liquid culture medium are 1.6 percent of glucose, 0.25 percent of sodium chloride, 0.05 percent of magnesium sulfate, 1 percent of yeast extract powder, 0.7 percent of ammonium chloride, 0.1 percent of dipotassium phosphate and pH 7.
(2) And (3) 1-10% of the seed liquid by volume ratio. The amount of the strain is inoculated into a fermentation medium for amplification culture at the temperature of 30 ℃ until the concentration of the strain in the fermentation liquor reaches 1 x 109～1×1010CFU/mL to obtain the biocontrol microbial inoculum.
The formula of the fermentation medium is as follows: 1-2% of glucose, 0.5-1.0% of yeast extract powder, 0.5-1.0% of dipotassium hydrogen phosphate, 0.2-0.5% of sodium chloride, 0.5-1.0% of ammonium sulfate, 0.03-0.05% of magnesium sulfate and adjustment of pH to 6.5-7.5.
The biocontrol microbial inoculum is centrifuged to take the supernatant, the supernatant is respectively diluted by 1 time, 10 times and 100 times, 100 microliters of the supernatant is respectively used for antagonistic experiments of American ginseng root rot fungus fusarium solani, the result is shown in figure 2, the fermented supernatant diluted by 10 times still has obvious bacteriostatic effect, and the diameter of a bacteriostatic zone is 21 mm.
Example 3 inhibition of spore germination of Fusarium solani by Paenibacillus polymyxa (Paenibacillus polymyxa) DN67
Respectively adding 10% sucrose solution and fermentation supernatant of Paenibacillus polymyxa DN67 to obtain final product4each/mL spore suspension of fusarium solani is taken from each treatment group, 10 mu L spore suspension is placed on a glass slide, the glass slide is placed in a heat-preservation and moisture-preservation incubator at 28 ℃ for static culture for 10h, the spore germination condition of each treatment group is observed, and the germination rate is calculated. Microscopic observation shows that fusarium solani spores in the fermented supernatant of paenibacillus polymyxa DN67 can not form germ tubes, the spores have the phenomena of abnormal expansion, breakage and the like, the germination rate is 0, and the fusarium solani in 2% sucrose solutionThe germination rate of the bacterial spores is 98%.
Example 4 field test for controlling root rot of American Ginseng
Selecting a field of three-year-old American ginseng, dividing the field into three fields with the same size, taking one field as a CK blank control, and irrigating roots with fusarium solani spore liquid without applying any microbial inoculum; the second plot is treatment group 1, the roots are irrigated by fermentation liquor of Paenibacillus polymyxa DN67, and then the roots are irrigated by fusarium solani spore liquid; and the third plot is a treatment group 2, roots are irrigated by fusarium solani spore liquid, then roots are irrigated by fermentation liquid of Paenibacillus polymyxa DN67, and the incidence rate of American ginseng root rot is compared, so that the incidence rate of American ginseng root rot in plots which are applied with the fermentation liquid of Paenibacillus polymyxa DN67 is obviously lower than that in a control group which is not applied with the bacterial liquid.
Table 2 incidence rates of different treatment groups
Sequence listing
<110> university of Ludong
<120> Paenibacillus polymyxa for preventing and treating American ginseng root rot and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1474
<212> DNA
<213> Paenibacillus polymyxa (Paenibacillus polymyxa)
<400> 1
gggctttggg cggaggccgc taatacatgc agtcgagcgg ggttaggtta gaagcttgct 60
tctaacctaa cctagcggcg gacgggtgag taacacgtag gcaacctgcc cacaagacag 120
ggataactac cggaaacggt agctaatacc cgatacatcc ttttcctgca tgggagaagg 180
aggaaaggcg gagcaatctg tcacttgtgg atgggcctgc ggcgcattag ctagttggtg 240
gggtaaaggc ctaccaaggc gacgatgcgt agccgacctg agagggtgat cggccacact 300
gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccgcaatgg 360
gcgaaagcct gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc gtaaagctct 420
gttgccaggg aagaacgctt gatagagtaa ctgctcttga agtgacggta cctgagaaga 480
aagccccggc taactacgtg ccagcagccg cggtaatacg tagggggcaa gcgttgtccg 540
gaattattgg gcgtaaagcg cgcgcaggcg gctctttaag tctggtgttt aatcccgagg 600
ctcaacttcg ggtcgcactg gaaactgggg agcttgagtg cagaagagga gagtggaatt 660
ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct 720
ctgggctgta actgacgctg aggcgcgaaa gcgtggggag caaacaggat tagataccct 780
ggtagtccac gccgtaaacg atgaatgcta ggtgttaggg gtttcgatac ccttggtgcc 840
gaagttaaca cattaagcat tccgcctggg gagtacggtc gcaagactga aactcaaagg 900
aattgacggg gacccgcaca agcagtggag tatgtggttt aattcgaagc aacgcgaaga 960
accttaccag gtcttgacat ccctctgacc ggtctagaga tagatctttc cttcgggaca 1020
gaggagacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080
cgcaacgagc gcaaccctta tgcttagttg ccagcaggtc aagctgggca ctctaagcag 1140
actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg ccccttatga 1200
cctgggctac acacgtacta caatggccgg tacaacggga agcgaaggag cgatgtggag 1260
ccaatcctag aaaagccggt ctcagttcgg attgtaggct gcaactcgcc tacatgaagt 1320
cggaattgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggtcttgtac 1380
acaccgcccg tcacaccacg agagtttaca acacccgaag tcggtgaggt aaccgcaagg 1440
agccagccgc tcgtatagat gggatacgca gggc 1474
Claims (8)
1. A strain of Paenibacillus polymyxa for antagonizing American ginseng root rot fusarium is characterized in that the Paenibacillus polymyxa (Paenibacillus polymyxa) DN67 is registered and preserved in the general microbiological center of China Committee for culture Collection of microorganisms at 21.12.2017, and the preservation number is as follows: CGMCC number 15112, storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
2. The application of the paenibacillus polymyxa DN67 in the medicine for preventing and treating the fusarium root rot of American ginseng.
3. A biocontrol bacterial agent containing the paenibacillus polymyxa DN67 as claimed in claim 1.
4. The biocontrol microbial inoculum according to claim 3, wherein the concentration of paenibacillus polymyxa DN67 in the biocontrol microbial inoculum is 1 x 108～1×1010CFU/mL or 1X 108～1×1010CFU/g。
5. The method for preparing the biocontrol microbial inoculum of claim 4 is characterized by comprising the following steps:
(1) inoculating the paenibacillus polymyxa DN67 into a liquid culture medium for culture until OD600 is 0.3-0.8 after the culture solution is diluted by 20 times, and obtaining a seed solution;
(2) inoculating the seed liquid into fermentation culture medium, and culturing until the thallus concentration in the fermentation liquid reaches 1 × 109～1×1010CFU/mL to obtain the biocontrol microbial inoculum.
6. The method of claim 5, wherein the seed solution is inoculated in an amount of 1-10% by volume.
7. The method according to claim 5, wherein the fermentation medium has a composition and final concentration of: 1-2% of glucose, 0.5-1.0% of yeast extract powder, 0.5-1.0% of dipotassium hydrogen phosphate, 0.2-0.5% of sodium chloride, 0.5-1.0% of ammonium sulfate, 0.03-0.05% of magnesium sulfate and adjustment of pH to 6.5-7.5.
8. The method according to claim 5, wherein the temperature of the scale-up culture is 28 to 37 ℃.
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| CN111733120B (en) * | 2020-08-25 | 2020-11-24 | 鲁东大学 | Bacillus aryabhattai and application thereof in prevention and treatment of American ginseng diseases |
| CN112795507A (en) * | 2021-01-07 | 2021-05-14 | 河南省农业科学院植物保护研究所 | Paenibacillus polymyxa capable of preventing and treating root rot of salvia miltiorrhiza and application thereof |
| CN112812995B (en) * | 2021-01-07 | 2022-07-26 | 河南省农业科学院植物保护研究所 | Paenibacillus polymyxa capable of preventing and treating root rot of salvia miltiorrhiza and application thereof |
| CN114717155B (en) * | 2022-05-06 | 2023-05-02 | 兰州交通大学 | Paenibacillus polymyxa for preventing and treating root rot of codonopsis pilosula and application thereof |
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