CN113088467B - Serratia strain and application thereof - Google Patents

Abstract

The invention discloses a Serratia strain and application thereof, wherein the strain is classified and named Serratia marcescens and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of the Ministry of microbiology of China academy of sciences, Beijing, North Chen Xilu No. 1 of the area facing the sun, the preservation date is 2021.03.09, and the preservation number is CGMCC NO. 21883. The strain is extracted from infected potato tuber moth larva. The bacterial colony of the strain is round, has a smooth surface and can produce red pigment. The strain is used for preparing biological insecticides and preventing and controlling lepidoptera pests such as potato tuber moths and the like.

Description

Serratia strain and application thereof

Technical Field

The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a serratia strain and application thereof.

Background

The tuber moth of potato (Phthyimaea operculella), also called tobacco leaf miner, belongs to the Insecta Lepidoptera Memmaleae, is a worldwide important agricultural pest, mainly harms solanaceous crops such as potatoes, tobacco, tomatoes and the like, and seriously harms the potato production regions such as clouds, Sichuan, precious and the like in China by using the larva leaf miner and eating tender stems and potato blocks, thereby forming a serious threat to the potato production in China. At present, chemical agents are mainly used for preventing and controlling the tuber moths, the biological prevention and control application is few, and the research and the development of efficient biological prevention and control products are urgently needed for preventing and controlling the tuber moths. A pathogenic bacterium is separated from a tuber moth larva, is identified to be Serratia marcescens (Serratia marcescens), has good pathogenic effect on the tuber moth larva, can be developed into a new biocontrol product, and is applied to control of the tuber moth and other lepidoptera pests.

Disclosure of Invention

The invention provides a serratia strain and application thereof, aiming at solving the technical problem that lepidoptera pests are difficult to control in the prior art. The purpose of the invention is mainly realized by the following technical scheme:

the invention provides a Serratia strain, which is classified and named as Serratia marcescens and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of the No. 1 Siro of Beijing Korean Chen, the preservation date is 2021.03.09, and the preservation number is CGMCC NO. 21883.

Further, the strain was extracted from diseased potato tuber moth larvae in vivo.

Furthermore, the bacterial colony of the bacterial strain is round, the surface is smooth, and the bacterial colony can produce red pigment.

The invention also provides application of the serratia strain in preparing a biological insecticide and preventing and treating lepidoptera pests.

Furthermore, the death rate of the larvae is up to 93.33 percent after 84 hours when the larvae of the lepidoptera pest potato tuber moth feed the potato slices stained with the serratia strain.

Further, the potato tuber moth larvae were bred with sterilized water-treated potato chips as a control, and the lethal medium concentration of the serratia strain to the pests was 1X 10⁴cfu/mL。

Further, as a control, potato tuber moth larvae were bred with LB medium-treated potato chips, and the lethal middle concentration of the Serratia strain was 8X 10⁴cfu/mL。

Compared with the prior art, the invention has at least one of the following practical effects:

1. the pathogenic Serratia marcescens strain Serratia marcescens OPT01 is separated from the body of the potato tuber moth;

2. the serratia strain can enter bodies of potato tuber moth larvae through food to cause the larvae to die;

3. the serratia strain can be used for developing microbial inoculum and is used for preventing and treating tuber moths of potatoes and other lepidoptera larvae.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and drawings.

Drawings

The drawings are only for purposes of illustrating particular embodiments and are not to be construed as limiting the invention, wherein like reference numerals are used to designate like parts throughout.

FIG. 1 is a diagram showing the isolated and cultured bacterial morphology from the larvae of the potato tuber moth, wherein A is the isolated and cultured bacterial colony; b is the colony morphology of the purified serratia strains; c is a bacterial morphology chart of the serratia strain;

FIG. 2 shows the molecular identification of Serratia strains, wherein A is the PCR amplification product of electrophoresis detection; b is a sequence determination peak chart; c is an NCBI sequence alignment chart; d is phylogenetic genetic analysis;

FIG. 3 shows the survival of the larvae of the potato tuber moth eating the potato slices stained with the bacteria for 84 hours;

FIG. 4 is a morphogram of a Helicoverpa punctifera larva, wherein A, B and E are normal larvae; c, D and F are larvae infected with Serratia;

FIG. 5 is a schematic diagram showing the mortality of potato chips fed with the serratia marcescens liquid from the larvae of the potato tuber moth at different times;

FIG. 6 is a schematic diagram showing the mortality rate of 3-4 instar potato tuber moth larvae after eating potato slices stained with Serratia bacteria of different concentrations for 72 hours.

Detailed Description

The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form a part hereof, and which together with the embodiments of the invention serve to explain the principles of the invention and not to limit its scope.

Example 1

A Serratia strain, the classification of which is named as: serratia marcescens, which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms. Address: west road No. 1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101, preservation date 2021.03.09, preservation number is CGMCC NO. 21883.

Example 2

A method for separating and purifying a serratia strain specifically comprises the following steps:

(1) the method comprises the following steps of sequentially disinfecting surfaces of diseased tuber moth larvae by using 75% ethanol and 3-5% sodium hypochlorite, and washing the surfaces of the tuber moth larvae by using sterilized water, wherein the sterilized water is specifically sterile water, namely sterilized and disinfected purified water;

(2) taking the coelomic tissue of the larvae of the tuber moth of potato tuber moth as a material under the aseptic condition, fully homogenizing the material by using normal saline, then diluting the material by using a dilution method, coating the diluted material on an LB culture medium plate, and culturing the plate at the constant temperature of 30 ℃ for 24-48 hours, wherein the colony morphology of the bacteria in the LB culture medium is shown in figure 1a and comprises two types of bacterial colonies of red and white, wherein the red is shown by shading in the figure, and the white is shown by unfilled shading in the figure;

the LB culture medium comprises the following components: 5g of yeast extract, 10g of tryptone, 10g of NaCl and 15g/L of agar.

(3) A single colony of the red bacterium of FIG. 1a was picked, purified and subcultured, and the red colony was purified and cultured as shown in FIG. 1 b. As can be seen, the red colony is round, the colony surface is smooth, and the deep red pigment can be produced. The morphology of the bacteria was observed in FIG. 1 c.

Example 3

The molecular identification method of the serratia strains is as follows, as shown in fig. 2:

extracting the red bacterium DNA in the example 2 by using a bacterium genome DNA extraction kit; then, the 16S rRNA gene sequence was PCR-amplified using the primer set 16S rDNA-27-F (5'-AGAGTTTGATCCTGGCTCAG-3')/16SrDNA-GSXJ2-R (5'-GGTTACCTTGTTACGACTT-3').

PCR reaction bodyIs (50 μ L): 5 μ L of 10 XBuffer, 4 μ L MgCl₂0.5. mu.L Taq enzyme (5U/. mu.L), 1. mu.L dNTPs (10mM/each), 1.5. mu.L forward primer (10 pmol/. mu.L), 1.5. mu.L reverse primer (10 pmol/. mu.L), 1.5. mu.L DNA template, and 35. mu.L sterile water.

The PCR amplification conditions were: 5min at 94 ℃, 30s at 55 ℃, 1min at 72 ℃ and 35 cycles; 10min at 72 ℃.

After the amplified product is separated and identified by 1% agarose gel electrophoresis, the PCR product is directly subjected to bidirectional sequencing. The resulting sequences were aligned using BLASTn to the 16S rRNA database at NCBI.

The results show that the sequence of the rhodobacter xylinum in example 2 is highly consistent with Serratia marcocens strain NBRC102203 and Serratia marcocens subsp. marcocens ATCC13880 strain JCM1239, so the rhodobacter xylinum in example 2 is named Serratia marcocens strain OPT01, namely Serratia marcescens OPT 01.

The following is the gene sequence of Serratia marcescens strain OPT 0116S rRNA:

ATTAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGTAGCACAGGGGAGCTTGCTCCCTGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAATGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAGGTGGTGAACTTAATACGTTCATCAATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGAAACTGGCAAGCTAGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCGATTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTATGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAGATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAA

example 4

The biological activity of Serratia OPT01 is determined as follows:

the method comprises the steps of coating a freshly cultured Serratia strain OPT01 on an LB medium plate through a diluent, culturing for 24 hours at the temperature of 30 ℃, counting colonies by adopting a plate counting method, determining the concentration of a bacterial liquid for later use, and taking cfu as the number of colony forming units.

Soaking fresh potato slices at a concentration of 1 × 10⁸After 2-3 seconds of cfu/mL serratia strain OPT01 suspension, placing the suspension in a sterilization culture dish paved with sterilization filter paper, placing 10 head of 3-4-year-old potato tuber moth larvae on a potato chip stained with serratia strain OPT01, placing the culture dish in a constant temperature incubator, and culturing at 26-28 ℃, with a light cycle of 16L:8D and a relative humidity of 80-90%.

The control group was larvae reared with potato flakes dipped in sterile water and a solution of the dominant intestinal bacteria (Enterococcus mundtii) of the larvae of the potato tuber moth at the same concentration.

In order to improve the accuracy of the test data, repeated tests are carried out for a plurality of times, specifically, 2 times of tests which use sterile water as a control group are carried out, and 3 groups of the same tests are carried out each time, namely P1, P2 and P3; tests using Enterococcus mundtii as a control group 3 identical tests were performed, namely P1, P2, P3; the assay for biological activity of Serratia OPT01 was performed 2 times, each time for 3 groups of the same assay, i.e., P1, P2, P3.

The survival status of the larvae of the tuber moth in each dish was observed every 12 hours, and the number of deaths of the larvae of the tuber moth was counted, and the statistics are shown in table 1, and the number of deaths of the larvae of the tuber moth in each dish was 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, 84 hours, and the total number of deaths.

TABLE 1 statistics of the number of deaths of the larvae of the potato tuber moth fed on the differently treated potato slices

The mortality and corrected mortality of the larvae of the potato tuber moth in each dish was calculated and corrected using the following formula:

the results showed that, as shown in fig. 3, the tuber moth larvae feed on potato chips infected with Serratia bacteria with significantly higher mortality rate than the control group. As shown in FIG. 4, diseased Phthorimaea operculella Zeller larvae infected with Serratia strain OPT01 appeared red, soft rot-like. As shown in FIG. 5, 48 hours after eating potato slices stained with Serratia strain OPT01, the number of dead larvae was over half, and 84 hours after eating, the death rate was over 90%. Table 2 shows the mortality of the larvae of the potato tuber moth fed on the potato slices treated differently, and as can be seen, the mortality of the larvae of the potato tuber moth fed on the potato slices stained with sterile water was only 11.67% after 84 hours; taking the potato tuber moth larvae which are fed with the potato slices stained with the non-pathogenic bacteria (enterococcus mundtii), wherein the mortality rate is only 23.33% after 84 hours, and the corrected mortality rate is only 13.2% after 84 hours by taking sterile water treatment as a control; when the potato tuber moth larvae are fed with the serratia strain OPT01 potato chips, the mortality rate after 84 hours reaches 93.33 percent, and the corrected mortality rate after 84 hours reaches 92.45 percent.

TABLE 2 mortality of Phlebopus armoricanus larvae after 84 hours on differently treated potato slices

	Sterile water	Serratia bacteria	Enterococcus mundtii
				Mortality rate	11.67％	93.33％	23.33％
Correcting mortality		92.45％	13.20％

Example 5

The determination method of the lethal middle concentration of Serratia OPT01 is as follows:

potato pieces were added at various concentrations (1X 10) as in example 4⁴，1×10⁵，1×10⁶，1×10⁷，1×10⁸cfu/mL) of the serratia strain OPT01, feeding the larvae for 2-3 seconds, observing the survival state of the larvae of the tuber moth after 72 hours, and counting the death number of the larvae of the tuber moth. Potato chips, which were dipped in sterile water and LB medium, were raised against the larvae of the tuber moth as a control.

The tuber moth larvae are bred on potato chips respectively soaked in sterile water, LB culture solution and Serratia strain OPT01 solution, each group of experiments are repeated for 3 times, and the death rate and corrected death rate of the tuber moth larvae are respectively calculated.

Note that, the lethal medium concentration means: the lowest concentration value at which Serratia OPT01 killed half of the total number of Helicoverpa punctifera larvae.

As a result, as shown in FIG. 6a, the control was made by feeding larvae with sterilized water-treated potato chips, and the lethal concentration of Serratia strain was 1X 10⁴cfu/mL; as shown in FIG. 6b, the larvae were fed with LB-treated potato chips as a control, and the lethal middle concentration of Serratia strains was 8X 10⁴cfu/mL。

It should be noted that the Serratia OPT01 can not directly enter the insect body from the insect body wall to kill the insect, but can only act after food enters the intestinal tract.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

<110> university of Beijing teachers

<120> Serratia strain and application thereof

<130> 2021.3.29

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attagagttt gatcctggct cagattgaac gctggcggca ggcttaacac atgcaagtcg 60

agcggtagca caggggagct tgctccctgg gtgacgagcg gcggacgggt gagtaatgtc 120

tgggaaactg cctgatggag ggggataact actggaaacg gtagctaata ccgcataacg 180

tcgcaagacc aaagaggggg accttcgggc ctcttgccat cagatgtgcc cagatgggat 240

tagctagtag gtggggtaat ggctcaccta ggcgacgatc cctagctggt ctgagaggat 300

gaccagccac actggaactg agacacggtc cagactccta cgggaggcag cagtggggaa 360

tattgcacaa tgggcgcaag cctgatgcag ccatgccgcg tgtgtgaaga aggccttcgg 420

gttgtaaagc actttcagcg aggaggaagg tggtgaactt aatacgttca tcaattgacg 480

ttactcgcag aagaagcacc ggctaactcc gtgccagcag ccgcggtaat acggagggtg 540

caagcgttaa tcggaattac tgggcgtaaa gcgcacgcag gcggtttgtt aagtcagatg 600

tgaaatcccc gggctcaacc tgggaactgc atttgaaact ggcaagctag agtctcgtag 660

aggggggtag aattccaggt gtagcggtga aatgcgtaga gatctggagg aataccggtg 720

gcgaaggcgg ccccctggac gaagactgac gctcaggtgc gaaagcgtgg ggagcaaaca 780

ggattagata ccctggtagt ccacgctgta aacgatgtcg atttggaggt tgtgcccttg 840

aggcgtggct tccggagcta acgcgttaaa tcgaccgcct ggggagtacg gccgcaaggt 900

taaaactcaa atgaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 960

tgcaacgcga agaaccttac ctactcttga catccagaga actttccaga gatggattgg 1020

tgccttcggg aactctgaga caggtgctgc atggctgtcg tcagctcgtg ttgtgaaatg 1080

ttgggttaag tcccgcaacg agcgcaaccc ttatcctttg ttgccagcgg ttcggccggg 1140

aactcaaagg agactgccag tgataaactg gaggaaggtg gggatgacgt caagtcatca 1200

tggcccttac gagtagggct acacacgtgc tacaatggca tatacaaaga gaagcgacct 1260

cgcgagagca agcggacctc ataaagtatg tcgtagtccg gattggagtc tgcaactcga 1320

ctccatgaag tcggaatcgc tagtaatcgt agatcagaat gctacggtga atacgttccc 1380

gggccttgta cacaccgccc gtcacaccat gggagtgggt tgcaaaagaa gtaggtagct 1440

taaccttcgg gagggcgctt accactttgt gattcatgac tggggtgaag tcgtaacaag 1500

gtaaccgtaa 1510

Claims (2)

1. A Serratia strain is characterized in that the strain is classified and named as Serratia marcescens and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of the West Lu 1 of the sunward Chen in Beijing, the preservation date of the microbial research institute of the Chinese academy of sciences is 2021.03.09, and the preservation number is CGMCC NO. 21883;

the strain is used for preparing a biological insecticide and preventing and controlling lepidoptera pests.

2. The serratia strain of claim 1, wherein the colony of the strain is round and has a smooth surface and produces a red pigment.

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