CN116200293A - Strain combination and application thereof - Google Patents
Strain combination and application thereof Download PDFInfo
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- CN116200293A CN116200293A CN202211546807.6A CN202211546807A CN116200293A CN 116200293 A CN116200293 A CN 116200293A CN 202211546807 A CN202211546807 A CN 202211546807A CN 116200293 A CN116200293 A CN 116200293A
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Abstract
The invention discloses a strain combination and application thereof, and relates to the microbial technology. The strain combination comprises bacillus subtilis and luxuriant bacteria; the bacillus subtilis is named as bacillus subtilis (Bacillus subtilis), and has a strain number T40 and a preservation number of CCTCC NO: m20221490; the luxuriant bacteria is named luxuriant bacteria (lecleria sp.), strain number T208, and the preservation number is CCTCC NO: m20221489. The biocontrol strains T40 and T208 provided by the invention have broad-spectrum inhibition effect on various rice pathogenic fungi, have good antibacterial effect and high control effect, can reduce the incidence rate of rice damping-off by about 47% compared with control treatment, and overcome the defect that chemical bactericides are easy to generate resistance.
Description
Technical Field
The invention relates to a microbial technology, in particular to a strain combination and application thereof.
Background
The rice is used as a main grain crop in China, and the safety, stability and high yield of the rice are ensured, so that the rice has great significance for the grain safety in China. With the development of the rice planting technology in China, the drought seedling raising technology plays an important role in cultivating strong rice seedlings, constructing high-yield group structures and exerting rice production potential. However, due to the influence of factors such as low temperature in seedling stage, the occurrence of soil-borne fungus diseases such as rice damping-off and the like seriously affects the safe production of rice.
In recent years, due to the influence of unreasonable use of chemical pesticides and the like, pathogenic bacteria gradually exhibit drug resistance to old pesticides, which is particularly prominent in the prevention and treatment of rice damping-off. The biocontrol bacteria and the like are utilized to prevent and control the rice damping off, so that not only can the occurrence of drug resistance be avoided, but also the growth of rice can be promoted, and the resistance of a rice system can be induced. However, although various biocontrol bacteria have been made into commercial agents at present, the use of biocontrol strains for germ control is explored, but the single biocontrol strain has the realistic problems of unstable control effect and weak persistence. The single strain has the defects of insignificant field application effect, low bacteriostasis broad spectrum, incapability of exerting the maximum advantage and the like, and in practical application, the bacteria agents are matched with each other to exert the maximum effect.
Patent application publication No. CN110713948A discloses a complete set of bacterial strain for preventing and treating verticillium wilt of tomatoes and application thereof. The complete set of strains disclosed by the invention comprises the endophyte NEAU-A5 and the endophyte NEAU-H7. The complete set of strains can effectively prevent and treat verticillium wilt of tomatoes, and the combined inner rhizogenes NEAU-A5 and NEAU-H7 of the complete set of strains have obvious antibacterial activity on verticillium wilt of tomatoes, the antibacterial rate reaches 67.65%, and the prevention and treatment effect on verticillium wilt of tomatoes can reach 62.16%. The invention lays a foundation for developing and developing the biocontrol combined preparation for effectively controlling the verticillium wilt of tomatoes.
The patent application with publication number of CN112574906A discloses a bacterial strain for preventing and treating common diseases of continuous cropping of tomatoes in greenhouse and a composite microbial agent thereof. The invention prepares the composite microbial agent by compounding YJY20-02 strain and YJY 19-01. After the compound microbial agent is applied, the compound microbial agent is easy to fix, is not easy to generate drug resistance, has obvious antibacterial activity on tomato continuous cropping germs, and has higher effect than single use of the strain. The method not only solves the practical problems of unstable control and weak persistence of a single biocontrol strain, but also has good control effect on common bacteria of continuous cropping of tomatoes, can quickly restore and stabilize the flora balance in soil, is a biocontrol strain with high efficiency for controlling common diseases of continuous cropping of tomatoes, and lays a foundation for developing and developing biocontrol combined preparations for effectively controlling the continuous cropping of tomatoes.
However, the use of bacillus subtilis and luxuriant bacteria for preventing and treating rice damping-off and inhibiting the growth of other rice pathogenic fungi has not been reported yet.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to design an application technical scheme for preventing and controlling rice damping-off by combining biocontrol bacteria.
The strain combination provided by the invention can be applied in a combined way, so that the ecological environment of soil can be regulated, and the occurrence of soil facsimile fungal diseases can be prevented and controlled. The method promotes the mutual coordination and the combined action of all the strains to prevent and treat plant diseases, effectively enhances the prevention and treatment capability and improves the ecological structure of soil to a greater extent.
The specific technical scheme of the invention is as follows:
the invention provides a strain combination, which comprises bacillus subtilis and luxuriant bacteria; the bacillus subtilis is named as bacillus subtilis (Bacillus subtiLis), and has a strain number T40 and a preservation number of CCTCC NO: m20221490; the luxuriant bacteria is named luxuriant bacteria (lecleria sp.), strain number T208, and the preservation number is CCTCC NO: m20221489, each of which was deposited with the China center for type culture Collection (China, 9, 26).
The invention also provides application of the strain combination in inhibiting growth of rice damping-off pathogenic fungi.
Preferably, the pathogenic fungus of the rice seedling blight is fusarium oxysporum or rice leaf spot.
The invention also provides a fungal inhibitor for the rice damping-off virus, and the active ingredients comprise the strain combination.
The invention also provides a preparation method of the rice damping-off pathogenic fungi inhibitor, which comprises the steps of inoculating the strain combination into a fermentation medium for culture, and removing the fermentation liquor of thalli to obtain the rice damping-off pathogenic fungi inhibitor.
Preferably, the incubation temperature is 25℃and the incubation time is 7 days.
The invention also provides a method for inhibiting the growth of pathogenic fungi of rice, wherein the strain combination or the fungal inhibitor of the damping-off disease of rice is applied to the soil where plants grow.
Preferably, the plant is in seedling stage.
The biocontrol strains T40 and T208 provided by the invention have broad-spectrum inhibition effect on various rice pathogenic fungi, have good antibacterial effect and high control effect, can reduce the incidence rate of rice damping-off by about 47% compared with control treatment, and overcome the defect that chemical bactericides are easy to generate resistance.
Drawings
FIG. 1 is a diagram showing the results of the screening of biocontrol bacteria T40 and T208, BLAST comparison analysis and the opposing culture of Fusarium oxysporum in the present invention.
FIG. 2 is a graph showing the effect of the combination of biocontrol strains T40 and T208 and two biocontrol strains on the opposite culture of Fusarium oxysporum and Hupencilia oryzae in the invention; wherein A is a graph of inhibition effect on fusarium oxysporum when biocontrol strains T40 and T208 are combined with two biocontrol strains; b is a graph of the inhibiting effect of biocontrol strains T40 and T208 on rice flax germs when the biocontrol strains are combined; c is a graph of the growth inhibition rate of fusarium oxysporum when biocontrol strains T40 and T208 are combined with two biocontrol strains; d is a graph of the growth inhibition rate of the biocontrol strains T40 and T208 on the rice flax germs when the biocontrol strains are combined with the two biocontrol strains.
FIG. 3 is a graph showing the disease condition and biocontrol effect of the seedling blight of rice after the biocontrol strains T40 and T208 are singly and jointly applied to a seedling tray of the rice; wherein A is the growth condition of rice seedlings under the condition of Contrast (CK); b is the growth condition of rice seedlings after T40 is applied to the rice seedling tray; c is the growth condition of rice seedlings after T208 is applied to the rice seedling tray; d is the growth condition of rice seedlings when two biocontrol strains T40 and T208 are combined; E-H are the emergence rate, disease index, biomass and seedling height of rice seedlings after the biocontrol strains T40 and T208 are singly and jointly applied to the rice seedling tray.
Detailed Description
Example 1: screening of biocontrol Strain
(1) Sample collection: samples used in the research are taken from a Zhejiang Fuyang paddy rice base, and are sampled by a sampling random sampling method. Taking 5 rice plants in different plots, and simultaneously retaining soil of rice root systems. And (5) placing the sample into a sterilized sealing bag, and carrying the sample back to a laboratory for biocontrol bacteria screening.
(2) Preparing a bacterial suspension: collecting 0.1g of collected rice plant root system soil, rice root system, rice stem, rice leaf, rice seed, etc., placing into sterilized 1.5mL centrifuge tube, adding 1mL sterilized ddH 2 And (3) putting the O and the small steel balls into a grinding instrument for oscillating and crushing. Standing on a centrifuge tube rack for 10-20min. The supernatant was placed in a fresh centrifuge tube and diluted 1:10 (V/V), 1:100 (V/V) and 1:1000 (V/V) to prepare bacterial suspensions of different concentrations.
(3) Isolation of bacteria: 200 mu L of each bacterial suspension with different diluted concentrations is evenly coated on NA (beef extract 5g, peptone 10g, sucrose 10g, agar powder 15g and deionized water 1L) solid culture medium, the culture dish is placed in a culture box at 25 ℃ for dark inversion culture after being dried, single bacterial colony is selected to a new NA culture dish for purification after 24 hours, and all the separated bacterial strains are numbered according to different parts.
(4) Screening of biocontrol strains: screening by adopting a counter culture method. The obtained monoclonal bacteria were cultured with LB liquid without antibody, and they were placed in an incubator at 37℃for shaking culture. 2 mu L of bacteria cultured overnight are dripped on one side of a potato dextrose culture medium (PDA potato 200g, dextrose 20g, agar powder 20 g), fusarium oxysporum (separated from the disease part of the rice damping-off disease plant of the Zhejiang Fuyang paddy) is placed at the symmetrical position on the other side, and the control is clear water. Culturing in dark at 25deg.C for 7 days, and measuring the longest growth diameter of Fusarium oxysporum as the indicator of antagonism of the strain to be detected. Through the experimental screening, 9 strains with obvious inhibition effect on fusarium oxysporum are obtained, the strain number T40 with the best inhibition effect is obtained, then other 8 strains of bacteria are co-cultured with the strain T40, and the strain number T208 capable of obviously improving the concentration of the mixed bacterial liquid is screened. 50% glycerol with strain T40 and T208 1:1 (V/V) were stored in a-80℃refrigerator.
Example 2: identification of biocontrol Strain
DNA extraction: genomic DNA of T40 and T208 was extracted using the Tiangen bacteria genome extraction kit (OSR-M502, high Ind. Co., beijing) technology Co., ltd.).
2mL of the overnight cultured bacterial liquid was collected, centrifuged at 10000rpm for 1min, and the supernatant was discarded. 200. Mu.L of buffer GA was added to the bacterial pellet, and the pellet was shaken until it was thoroughly suspended. Add 20. Mu.L of LProteinase K solution to the tube and mix upside down. Then adding 220 mu L of buffer B, shaking and mixing, standing at 70 ℃ for 10min, adding 220 mu L of absolute ethyl alcohol, shaking and mixing, and rapidly centrifuging to remove water drops on the inner wall of the tube cover. The solution in the tube was transferred to an adsorption column, centrifuged at 12000rpm for 30s, the waste liquid was discarded, and the adsorption column was put back into the collection tube. 500. Mu.L of buffer GD (with the proper ratio of absolute ethanol added in advance) was added to the column, centrifuged at 12000rpm for 30s, the waste liquid was discarded, and the column was returned to the collection tube. 600. Mu.L of the rinse PW (with the proper ratio of absolute ethanol added in advance) was added to the column, centrifuged at 12000rpm for 30s, the waste liquid was discarded, and the column was returned to the collection tube. The rinsing was repeated once. The waste liquid was discarded, and the adsorption column was put back into the collection tube and centrifuged at 12000rpm for 2min. The adsorption column was allowed to stand at room temperature for 2min, then transferred to a clean centrifuge tube, and 100. Mu.L of deionized water was added thereto, and allowed to stand at room temperature for 2min. The solution was collected into a centrifuge tube by centrifugation at 12000rpm for 2min, and the solution was T40 and T208 genomic DNA.
The 16S rRNA sequences were amplified using the primers T40 and T208 genomic DNA as templates, the primer sequences were as follows:
63F：CAGGCCTAACACATGCAAGTC；
13897R：GGGCGGTGATGTACAAGGC。
PCR reaction System (50. Mu.L): comprises 25. Mu.L of 2 XPCR mix, 2. Mu.L of 100 ng/. Mu.L of DNA template, 10. Mu.M primer 63F/13897R each 2.5. Mu.L, the balance ddH 2 O。
The reaction conditions are as follows: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s, amplification for 35 cycles, and extension at 72℃for 5min.
After the reaction was completed, a band of about 1300bp in size was detected by 1% agarose gel electrophoresis. And the remaining amplified product was subjected to sequencing analysis (entrusted to the well-known biotechnology company, hangzhou). A1306 bp DNA fragment is obtained by sequencing, and the nucleotide sequences of the DNA fragment are shown as SEQ ID NO.1 and SEQ ID NO. 2.
As shown in FIG. 1, the sequences were analyzed by BLAST alignment on the NCB site, and they were found to have the highest homology with Bacillus subtilis T40 (Bacillus subtilis) and Leclericia T208 (Leclericia sp.). Meanwhile, the T40 and T208 strains are preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of NO: m20221490 and CCTCC NO: m20221489, the preservation time is: the classification was Bacillus subtilis T and Leclercia sp.T208, month 9, day 26, 2022.
Example 3: plate confrontation of T40 and T208 strains with other rice pathogenic fungi and field inoculation experiments
The overnight cultured T40 and T208 strains were pipetted into 2. Mu.L of PDA solid medium at the distance edge 2em and ddH was pipetted onto the other PDA solid medium at the same location 2 O served as a control. Activated rice leaf spot bacteria (separated from rice leaf spot disease onset plants of Zhejiang Fuyang rice) are picked up by a puncher with the inner diameter of 6mm, and inoculated at symmetrical positions of strains T40 and T208 respectively. After 7 days of incubation at 25℃the colony growth was observed and the maximum growth diameter was measured.
Hypha growth inhibition ratio (%) = (maximum diameter of control colony growth-maximum diameter of treated colony growth)/maximum diameter of control colony growth x 100%,
each experiment was repeated 3 times, 3 parallel experiments were performed. The experimental results are shown in FIG. 2.
A greenhouse seedling test was arranged by applying the combination of T40 and T208 strains screened in example 1 to the soil where seedling blight of rice has occurred seriously. Investigation of rice seedling growth and development and disease conditions 14 days after inoculation; the experimental results are shown in FIG. 3.
As shown in the figure 2 and the figure 3, the combination of the T40 and the T208 shows better antibacterial growth and disease inhibition efficiency compared with the single use of two biocontrol bacteria, wherein the incidence rate of the rice damping-off can be reduced by about 47 percent compared with the control treatment, and therefore, the combined use of the T40 and the T208 is an effective rice damping-off prevention and treatment measure.
Claims (8)
1. A combination of strains, wherein the combination of strains comprises bacillus subtilis and luxuriant bacteria; the bacillus subtilis is named as bacillus subtilis (Bacillus Subtilis), and has a strain number T40 and a preservation number of CCTCC NO: m20221490; the luxuriant bacteria is named luxuriant bacteria (lecleria sp.), strain number T208, and the preservation number is CCTCC NO: m20221489.
2. Use of a combination of strains according to claim 1 for inhibiting the growth of pathogenic fungi of rice damping off.
3. The use according to claim 2, wherein the pathogenic fungus of rice damping off is fusarium oxysporum or fusarium graminearum.
4. A fungal inhibitor of the origin of rice damping-off, characterized in that the active ingredient comprises a combination of strains according to claim 1.
5. The method for preparing the fungal inhibitor for rice damping-off according to claim 4, wherein the bacterial strain of claim 1 is inoculated into a fermentation medium for cultivation, and the fermentation liquid from which the bacterial cells are removed is the fungal inhibitor for rice damping-off.
6. The method according to claim 5, wherein the culturing temperature is 25℃and the culturing time is 7 days.
7. A method of inhibiting the growth of pathogenic fungi in rice, characterized in that the combination of strains according to claim 1 or the fungal inhibitor of the plant damping-off disease according to claim 4 is applied to the soil in which the plants are growing.
8. The method of inhibiting the growth of a pathogenic fungus in rice of claim 8, wherein the plant is in seedling stage.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118147020A (en) * | 2024-05-11 | 2024-06-07 | 众乐中药功能成分工程技术研究中心(潍坊)有限公司 | Non-decarboxylated Leucol bacteria and application thereof in inhibition of zearalenone |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118147020A (en) * | 2024-05-11 | 2024-06-07 | 众乐中药功能成分工程技术研究中心(潍坊)有限公司 | Non-decarboxylated Leucol bacteria and application thereof in inhibition of zearalenone |
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