CN114480188B - Marine source strain and application thereof in insect control - Google Patents
Marine source strain and application thereof in insect control Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention belongs to the technical field of screening and application of marine source biological control bacteria, and particularly relates to a marine source strain and application thereof in insect control. The lysobacter enzymogenes provided by the invention is a lysobacter enzymogenes (Lysobacter enzymogenes) SYZX2010 strain, and the preservation number is CGMCC No.24065. The lysobacter enzymogenes SYZX2010 strain provided by the invention is separated from deep sea environmental sediment, and has the capability of growing under the condition of high salt; the lysobacter enzymogenes SYZX2010 strain can inhibit the oviposition of nematodes, has a good effect of killing the nematodes, has strong viability in a high-salt environment, and can prevent and treat nematode diseases in environments such as saline-alkali soil. In addition, the soil environment is not harmed while the nematode control effect is achieved in the application.
Description
Technical Field
The invention belongs to the technical field of screening and application of marine source biological control bacteria, and particularly relates to a marine source strain and application thereof in insect control.
Background
At present, root-knot nematodes are the most serious agricultural hazard.
Root knot nematodes are a type of plant parasitic nematodes and are harmful to a wide range of plants, including more than thirty vegetables such as melons, solanaceae, beans, cabbages, radishes and the like, and fruits such as dragon fruits, grapes, citrus and the like. Nematode infected crops also increase the probability of being infected with other fungal and bacterial diseases.
Root knot nematodes mainly damage the roots of plants, destroy the normal differentiation of root tissues, cause abnormal growth of the root tissues, and form root nodules with uneven sizes; therefore, the moisture and the nutrients cannot be transported from the root, so that photosynthesis is reduced, leaves are withered and yellow, the yield is reduced, and even the particles are not harvested.
The root-knot nematodes are difficult to control, the root-knot nematodes are various in variety, flexible in proliferation mode and high in proliferation speed, good crop cultivation environments provide habitats for the nematodes, and the root-knot nematodes are infected from the root, so that the root-knot nematodes are difficult to find in the early stage of crop infection, and the control period is missed.
There are currently some physical methods for controlling nematodes, such as high temperature, low temperature or flooding, which can cause the nematode to die quickly but do not work with eggs. Eggs which are not hatched by nematodes and eggs in oocysts have strong ability to adapt to severe environments, can survive in soil in a dormant state and survive for 3 years, so that the effect of radical treatment is difficult to achieve. The strong pesticide can play a role in killing nematodes, but the side effects on soil and plants cannot be reversed.
Disclosure of Invention
The invention aims to provide a marine-source strain and application thereof in insect control, namely a marine-source lysobacter enzymogenes SYZX2010 strain. The strain has the capability of efficiently killing nematodes and the capability of surviving in a high-salt environment, so that the strain can be used for treating nematode diseases in saline-alkali soil and other environments and solves the problem that current nematodes are difficult to control.
The lysobacter enzymogenes provided by the invention is a lysobacter enzymogenes (Lysobacter enzymogenes) SYZX2010 strain which is preserved in the national institute of microbiological culture collection center of China academy of sciences microbiological culture collection center of China, north Chen Silu No. 1, no. 3, and the preservation number is CGMCC No.24065 in the year 2021, 12 and 09.
The lysobacter enzymogenes SYZX2010 strain provided by the invention is obtained by screening in a high-salt environment, and can be used for preventing and controlling nematodes;
in another aspect, the invention provides a method of controlling nematodes by using the isolated lysobacter enzymogenes synx 2010 strain;
the method for preventing and controlling the nematodes is to use fermentation liquor or fermentation extract of a lysobacter enzymogenes SYZX2010 strain to prevent and control the nematodes.
In another aspect, the invention also provides a preparation for preventing and controlling nematodes, wherein the preparation comprises viable bacteria of a lysobacter enzymogenes SYZX2010 strain;
furthermore, the product also comprises fermentation liquor and/or fermentation extract of the lysobacter enzymogenes SYZX2010 strain.
Compared with the reported lysobacter enzymogenes SYZX2010 strain, the lysobacter enzymogenes SYZX2010 strain provided by the invention has the following advantages:
1) The lysobacter enzymogenes SYZX2010 strain provided by the invention is separated from marine sediments and has the capability of growing under the high-salt condition;
2) The lysobacter enzymogenes SYZX2010 strain provided by the invention can inhibit the oviposition of nematodes, has a good effect of killing the nematodes, has strong viability in a high-salt environment, and can prevent and treat nematode diseases in saline-alkali soil and other environments. And the soil environment is not harmed when the nematodes are prevented and treated.
Drawings
Fig. 1: colony morphology map of lysobacter enzymogenes SYZX 2010;
fig. 2: growth profile of lysobacter enzymogenes SYZX 2010;
fig. 3: growth patterns of the lysobacter enzymogenes SYZX2010 and the lysobacter terrestris SYZX2023 under different salt concentrations;
fig. 4: after 3 days of culture, the caenorhabditis elegans is observed to obtain a photo, wherein a photo A is an experimental group and a photo B is a control group;
fig. 5: after 4 days of culture, the caenorhabditis elegans is observed to obtain a photo, wherein a photo A is an experimental group and a photo B is a control group;
fig. 6: photographs were observed after 4 days of caenorhabditis elegans culture, wherein plot a is the experimental group, plot B is the control group with DMSO addition, and plot C is the untreated control group;
fig. 7: photograph of the root of the crop in the experimental field is observed, wherein the photograph A is an experimental group, and the photograph B is a control group.
Detailed Description
The lysobacter enzymogenes belongs to the class gamma-anamorphic bacteria and the xanthomonas, is an environmental gram-negative bacterium and is visible everywhere in the natural environment. Lysobacter enzymogenes is known for its lytic effect on fungi, oomycetes, unicellular algae, gram-negative bacteria, gram-positive bacteria and other microorganisms.
The homology between the screened lysobacter enzymogenes SYZX2010 strain and the reported lysobacter enzymogenes YC36 is 98.55%, and the colony is light yellow; thus, it was designated as lysobacter enzymogenes (Lysobacter enzymogenes).
The embodiment of the invention describes that a 40% TSB (trypticase soytone) culture medium containing 2% NaCl is used for culturing a zymogenic lysobacter enzymogenes SYZX2010 strain, and the components and the proportions of the culture medium formula are as follows: tryptone 6g, soytone 2g, sodium chloride 20g, water 1L. However, other media for culturing nematodes may also be selected.
The nematodes used in the embodiment of the invention are caenorhabditis elegans, the culture medium is NGM (nematode growth medium), and the components and proportions of the culture medium are as follows: 2.5g peptone, 20g agar, 3g NaCl, 975mL distilled water, sterilizing with steam at 120deg.C for 30min, cooling in a water bath at 55deg.C, sequentially adding 5mg/mL cholesterol (dissolved in ethanol, not sterilized) 1mL, and sterilizing with 1M MgSO under high pressure 4 ，1M CaCl 2 1mL of 1M potassium phosphate buffer (pH 6.0) respectively, and shaking uniformly to obtain a complete culture medium;
the culture temperature of caenorhabditis elegans is 20 ℃; the caenorhabditis elegans is the OP50 strain of Escherichia coli, and the nematodes are cultured after coating OP50 on an NGM culture medium.
According to the invention, the zymobacter enzymogenes SYZX2010 fermentation liquid or a fermentation product thereof is added into the growth environment of the nematodes, so that the growth and spawning of the nematodes can be effectively inhibited, and the nematodes lose reproductive capacity and die gradually.
The present invention will be described in detail with reference to the following examples and the accompanying drawings.
Example 1: isolation and identification of lysobacter enzymogenes SYZX2010 strain
Media for screening of lysobacter enzymogenes were formulated at 40% TSA with NaCl concentration adjusted up to 2%.
A 1g sample of marine sediment was weighed, dissolved in distilled water, coated onto a prepared TSA plate and grown for one day at 28 ℃. Single colonies on the plates were then picked and grown for one day at 28℃on new 2% 40% TSA plates to give single colonies designated SYZX2010 strain.
Selecting SYZX2010 single colony, dissolving with 200 mu L of 1 xTE, blowing and mixing uniformly, boiling for 10min, immediately putting into a cold storage at the temperature of minus 20 ℃ to obtain the bacterial DNA.
Using DNA as template, the primer is 16SrDNA full-length universal primer 8F and 1492R, and making 16S rDNA amplification: premix Taq 15. Mu.L, double distilled water 13.4. Mu.L, 8F 0.3. Mu.L, 142R 0.3. Mu.L, template 1. Mu.L; the PCR procedure was as follows: pre-denaturation at 94℃for 5min, denaturation at 55℃for 1min, annealing at 72℃for 1min, extension for 1.5min, extension at 72℃for 10min,30 cycles. Sending the PCR product to a biological engineering Co., ltd for sequencing; the sequencing results (SEQ ID NO: 1) were subjected to BLAST alignment.
The comparison result shows that the homology between the SYZX2010 strain and the lysobacter enzymogenes YC36 is 98.55 percent, and the bacterium is identified as the lysobacter enzymogenes. Seed-retaining and enzyme-producing lysobacter SYZX2010 strain, and then storing in a refrigerator at-20 ℃.
The strain of the screened lysobacter enzymogenes SYZX2010 strain is light yellow (figure 1), the optimal culture temperature is 28 ℃, the applicable culture medium is TSB culture medium, and the strain enters a logarithmic phase (figure 2) after 8 hours of growth, and has the characteristic of being capable of survival in a high-salt environment.
Example 2: growth effect of lysobacter enzymogenes SYZX2010 strain under high salt concentration
Culturing the lysobacter enzymogenes SYZX2010 strain and the lysobacter terrestris SYZX2023 strain in 40% TSB containing NaCl with a concentration gradient of 0-2%, and growing at 28 ℃ for 24 hours; the absorbance at 600nm was then measured with an ultraviolet spectrophotometer, and the data in Table 1 are absorbance at 600nm for detecting the concentration of bacteria. It can be seen that when the salt concentration was increased to 2%, the growth of the terrestrial lysobacter enzymogenes synx 2023 strain was more significantly inhibited than that of the lysobacter enzymogenes synx 2010 strain (fig. 3).
Table 1: absorbance table of lysobacter enzymogenes SYZX2010 at different NaCl concentration gradients
| Strain | 0％NaCl | 0.5％NaCl | 1％NaCl | 1.5％NaCl | 2％NaCl |
| SYZX2010 | 0.4595 | 0.475 | 0.3665 | 0.189 | 0.13 |
| SYZX2023 | 0.434 | 0.4015 | 0.355 | 0.1155 | 0.0755 |
Example 3: experiment for inhibiting nematode oviposition by using lysobacter enzymogenes SYZX2010 strain
1) Culturing zymogenic lysobacter SYZX2010 strain fermentation liquor
The lysobacter enzymogenes SYZX2010 strain is transferred from a refrigerator at the temperature of minus 20 ℃ to a 40% TSA flat plate with the NaCl concentration of 2% for activation, and is transferred to a 40% TSB liquid culture medium with the NaCl concentration of 2% for growth for 24 hours, and is grown for 3 days at the temperature of 28 ℃ to obtain a fermentation broth.
2) Culturing caenorhabditis elegans
Preparing an NGM plate, coating escherichia coli OP50 on the plate, culturing for one day at 37 ℃, transferring 3-5 caenorhabditis elegans to the plate by using a picker, and culturing for 6 days at 20 ℃;
3) Experiment for inhibiting oviposition of nematodes
Ultraviolet irradiating the NGM culture medium with OP50 for 30min, coating 200 μl of fermentation broth of the strain SYZX2010 of the lysobacter enzymogenes on a plate, and air drying; the plates of the control group were not coated with the zymogen syn x2010 broth, and three replicates were set up for each group.
Nematodes on NGM medium were washed off with M9 solution, blotted 50 μl onto plates of experimental and control groups, incubated at 16 ℃, and observed daily for caenorhabditis elegans growth with microscopy.
Three days after the culture, caenorhabditis elegans on the plates of the experimental group were not able to lay eggs normally, whereas caenorhabditis elegans on the plates of the control group had already begun to lay eggs. Four days after incubation, caenorhabditis elegans on the plates of the experimental group still failed to lay eggs and lost their ability to move and die gradually, while eggs on the plates of the control group had grown into larvae (figures 4 and 5).
Example 4: fermentation product HSAF of lysobacter enzymogenes SYZX2010 inhibits nematode growth
1) Preparation of fermentation product of lysobacter enzymogenes SYZX2010
The lysobacter enzymogenes SYZX2010 strain is transferred from a refrigerator at the temperature of minus 20 ℃ to a 40% TSA flat plate with the NaCl concentration of 2% for activation, is transferred to a 40% TSB liquid culture medium with the NaCl concentration of 2% after 24 hours of growth, and is grown for 3 days at the temperature of 28 ℃ to obtain fermentation liquor.
Centrifuging the fermentation liquor, taking supernatant, and adsorbing with macroporous resin overnight; sequentially eluting macroporous resin with 30%, 50%, 60% and 100% methanol, collecting 100% methanol eluate, rotary evaporating, concentrating, dissolving DMSO, and purifying with high performance liquid chromatography to obtain fermentation product HSAF.
2) Culturing caenorhabditis elegans
Preparing an NGM plate, coating the escherichia coli OP50 on the plate, culturing for one day at 37 ℃, transferring 3-5 caenorhabditis elegans to the plate by using a picker, and culturing for 6 days at 20 ℃.
3) Experiment for inhibiting nematode growth
Ultraviolet irradiating NGM culture medium with OP50 for 30min, coating 200 μl of HSAF with final concentration of 12 μg/mL on the plate, and air drying; the control groups were each coated with 200 μl DMSO and without any addition of solution, and each group was set in triplicate.
Four days after incubation, the growth rate of caenorhabditis elegans on the plates of the experimental group was significantly inhibited, and DMSO had no effect on caenorhabditis elegans growth (figure 6).
Example 5 field test of lysobacter enzymogenes SYZX2010 strain
Transferring a lysobacter enzymogenes SYZX2010 strain from a refrigerator at the temperature of minus 20 ℃ to a 40% TSA flat plate with the NaCl concentration of 2% for activation, transferring the strain into a 40% TSB liquid culture medium with the 2L NaCl concentration of 2% after 24 hours of growth, and culturing the strain at the temperature of 28 ℃ for 2 days; diluting the bacterial liquid by 5 times with water for later use.
The test field is divided into two parts, one part is set as an experimental group, the bacterial liquid is sprayed, and the other part is set as a control group, and the treatment is not carried out. After spraying the bacterial liquid for 4-5 days, starting to plant crops; after a period of time of crop growth, the growth condition of root-knot nematodes at the roots of plants is observed, and in the experimental group sprayed with the bacterial liquid, the root-knot nematodes at the roots of plants are fewer, and the root-knot nematodes in the control group are more (figure 7).
Experimental results show that the screened lysobacter enzymogenes SYZX2010 strain has an efficient nematode killing effect.
Sequence listing
<110> university of ocean in China
<120> a marine source strain and application thereof in insect control
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<170> SIPOSequenceListing 1.0
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gcgagtggcg gacgggtgag gaatacgtcg gaatctgcct atttgtgggg gataacgtag 120
ggaaacttac gctaataccg catacgacct acgggtgaaa gtgggggacc gcaaggcctc 180
acgcagatag atgagccgac gtcggattag ctagttggcg gggtaaaggc ccaccaaggc 240
gacgatccgt agctggtctg agaggatgat cagccacact ggaactgaga cacggtccag 300
actcctacgg gaggcagcag tggggaatat tggacaatgg gcgcaagcct gatccagcca 360
tgccgcgtgt gtgaagaagg ccttcgggtt gtaaagcact tttgtccgga aagaaaagct 420
tagggttaat aaccttgagt catgacggta ccggaagaat aagcaccggc taacttcgtg 480
ccagcagccg cggtaatacg aagggtgcaa gcgttactcg gaattactgg gcgtaaagcg 540
tgcgtaggtg gtttgttaag tctgatgtga aagccctggg ctcaacctgg gaatggcatt 600
ggaaactggc ttactagagt gcggtagagg gtagcggaat tcccggtgta gcagtgaaat 660
gcgtagatat cgggaggaac atctgtggcg aaggcggcta cctggaccag cactgacact 720
gaggcacgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccctaaac 780
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catggctgtc gtcagctcgt gtcgtgagat gtgggttaag tccgcaacga gccgcaaacc 1080
cttgtccttt aggttgccta gca 1103
Claims (7)
1. A marine source strain is characterized in that the strain is lysobacter enzymogenesLysobacter enzymogenes) The preservation number is CGMCC No.24065.
2. Use of the lysobacter enzymogenes according to claim 1 for controlling root-knot nematodes.
3. Use of the lysobacter enzymogenes according to claim 1 for the preparation of a product for controlling root-knot nematodes.
4. A method for controlling root-knot nematodes, which is characterized in that the method comprises using the lysobacter enzymogenes according to claim 1 for controlling root-knot nematodes.
5. A method for controlling root-knot nematodes, which is characterized in that the fermentation broth of the lysobacter enzymogenes of claim 1 is used for controlling root-knot nematodes.
6. A product for controlling root-knot nematodes, comprising the lysobacter enzymogenes according to claim 1.
7. The article of manufacture of claim 6, wherein the article of manufacture comprises a fermentation broth of the lysobacter enzymogenes of claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101177671A (en) * | 2007-12-05 | 2008-05-14 | 南京农业大学 | Biocontrol bacteria strain preventing and curing plant disease |
| CN102943061A (en) * | 2012-11-28 | 2013-02-27 | 南京农业大学 | Construction and application of unmarked lysobacter enzymogenes engineering strain capable of preventing plant bacteriosis |
| CN113337424A (en) * | 2021-05-28 | 2021-09-03 | 山东链云网络科技有限公司 | Live bacterium agent for preventing and treating agricultural diseases and application thereof |
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| EP3011833A1 (en) * | 2014-10-23 | 2016-04-27 | Futureco Bioscience, S.A. | Bacteria with nematicidal activity and the ability to promote plant growth |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101177671A (en) * | 2007-12-05 | 2008-05-14 | 南京农业大学 | Biocontrol bacteria strain preventing and curing plant disease |
| CN102943061A (en) * | 2012-11-28 | 2013-02-27 | 南京农业大学 | Construction and application of unmarked lysobacter enzymogenes engineering strain capable of preventing plant bacteriosis |
| CN113337424A (en) * | 2021-05-28 | 2021-09-03 | 山东链云网络科技有限公司 | Live bacterium agent for preventing and treating agricultural diseases and application thereof |
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