CN112410252B - Plutella xylostella malt aromatic Carnobacter PxCG2 strain and application thereof - Google Patents

Abstract

The invention discloses a plutella xylostella maltulinum aromaticum Carnobacterium maltulinum PxCG2 strain and application thereof, wherein the strain is preserved in Guangdong province microbial strain collection center (GDMCC) at 8-11 th month in 2020, and the strain number is GDMCC No: 61133. the research of the invention shows that the Carnobacterium maltuliformis PxCG2 strain has the functions of improving the insecticidal activity of Bt toxin and synergizing Cry1Ac protoxin to quickly kill plutella xylostella, and the Carnobacterium maltuliformis PxCG2 strain can be used as a novel biological control bacterium, is used for controlling cruciferous vegetable pests, and has good biological control potential and application prospect.

Description

Plutella xylostella malt aromatic Carnobacterium PxCG2 strain and application thereof

Technical Field

The invention relates to the technical field of agricultural microorganisms, in particular to a plutella xylostella maltulinosa Carnobacterium maltaromaticum PxCG2 strain and application thereof.

Background

Plutella xylostella (L) is one of the most serious insect pests harming cruciferous crops, and usually causes serious economic loss of many important vegetables such as cabbage, broccoli and cauliflower (Talekar and Shelton, 1993). Diamondback moth developed severe resistance to all insecticides against lepidopteran pests and bacillus thuringiensis (Bt), which became increasingly difficult to control (Talekar and Shelton, 1993; Baxter et al, 2005). Therefore, there is a need to find a method that can solve or alleviate the resistance of plutella xylostella to Bt, thereby improving the insecticidal activity of Bt proteins.

The various tissues and organs of the insects are distributed with insect symbiotic bacteria of different types and numbers, and the symbiotic bacteria are interdependent with the insect symbiotic bacteria in a long-term evolution process. Scientists have now explored the symbiotic flora of many insects, revealing the importance of the symbiotic flora to the host insects. The intestinal tract of the insect participates in the processes of feeding, digestion, excretion and the like of the insect, has extremely rich nutrient substances and unique physiological and biochemical environment, and is also a part on which numerous microorganisms live; the intestinal tract is mainly divided into the foregut, the midgut and the hindgut. Among them, the middle intestine is the most important site for food digestion and nutrient absorption. The species and the quantity of the intestinal microorganisms of the insects are extremely large, and the intestinal bacteria account for more than 90 percent of the total intestinal microorganisms (Yang Yun Qiu et al, 2018). Many scientific researches show that intestinal microorganisms of insects and host insects live and evolve together to participate in regulating a plurality of physiological functions and play an important role in the growth and development of the host insects, some scientists even show that the intestinal microorganisms are an organ of an insect digestive system, so that the significance of the intestinal microorganisms to the host can be seen, the midgut is taken as the most main part of the intestinal tract, and the structure and the function of the symbiotic microorganism population in the midgut gradually become research hotspots in recent years. For example, patent CN110982729A discloses a complex microbial inoculum for degrading poplar cellulose, a byproduct of wood processing, wherein the active ingredients of the microbial inoculum include Carnobacterium (Carnobacterium maltomonas strain TMW 2.158); patent CN101155910 discloses a method of using Carnobacterium maltulomaticum or related fermentates or bacteriocin compositions for the treatment of food, such as may be used for the treatment of fresh or processed meat against bacterial contamination; the above patents all relate to the research on the related functions of Carnobacterium maltulomaticum bacteria, but there are few reports on the effects thereof on the control of lepidopteran pests and on the insecticidal activity of Bt toxins.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings in the prior art and provide a Carnobacterium maltaromaticum PxCG2 strain.

The second purpose of the invention is to provide the application of the strain of the plutella xylostella maltulinosa Carnobacterium maltulinosum PxCG 2.

The above purpose of the invention is realized by the following technical scheme:

the strain of plutella xylostella maltobacter maltovaryticus PxCG2 is deposited in Guangdong province microbial strain collection center (GDMCC) at 8-11 th month in 2020, and the strain deposit number is GDMCC No: 61133.

specifically, the nucleotide sequence of the 16s rDNA of the strain is shown in SEQ ID NO. 1.

The strain is obtained by separating the midgut content of 3-instar larvae of the plutella xylostella, and a plutella xylostella feeding test is carried out on the plutella xylostella by using the Carnobacterium maltomonas mixis PxCG2 strain, so that the result shows that compared with a control, the death rate of the plutella xylostella is remarkably increased by feeding ciprofloxacin, levofloxacin and metronidazole antibiotics and removing the intestinal flora of the plutella xylostella after feeding Cry1Ac protoxin and the PxCG2 bacterial liquid. After the feed is added for 60 hours, the death rate of the diamondback moth is 90 percent, and the death rate of the diamondback moth is 98.33 percent after 72 hours; under the treatment of independently feeding Cry1Ac protoxin, the mortality rate is obviously reduced compared with that of simultaneously feeding Cry1Ac protoxin and PxCG2, the mortality rate of 60h plutella xylostella is 58.33% and the mortality rate of 72h is 71.67% when Cry1Ac protoxin is fed; the result shows that the Carnobacterium maltuliformis PxCG2 strain separated and identified by the invention has the function of quickly killing plutella xylostella by synergizing Bt prototoxins such as Cry1Ac and the like.

Therefore, the invention provides the application of the strain of the plutella xylostella maltobacter xylinum PxCG2 in improving the insecticidal activity of Bt protein.

Application of Carnobacterium maltophilium PxCG2 strain in preventing and treating cruciferous vegetable pests.

The invention also provides a method for controlling the pests of cruciferous vegetables, which is to feed the pests by the Carnobacterium maltophilium PxCG2 strain of the plutella xylostella and the Bt protein together.

The invention also provides application of the diamondback moth and aromatic bacillus carnobacter PxCG2 strain in preparing a medicament for controlling cruciferous vegetable pests.

The invention also provides a medicament for controlling brassicaceous vegetable pests, which comprises a strain PxCG2 of the plutella xylostella, namely, Carnobacterium maltulinosus, and the lethality of the strain to the pests can be improved by utilizing the synergistic effect of the strain PxCG2 on the Bt protein prototoxin.

Preferably, the Bt protein is Cry1Ac protoxin.

Preferably, the cruciferous vegetable pest is a diamondback moth.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a plutella xylostella maltulinosa Carnobacterium maltaromaticum PxCG2 strain and application thereof, wherein the strain is preserved in Guangdong province microbial culture collection center (GDMCC) at 8-11 th of 2020, and the strain number is GDMCC No: 61133. the research of the invention shows that the Carnobacterium maltuliformis PxCG2 strain has the effects of improving the insecticidal activity of Bt toxin and synergizing quick death of Cry1Ac protoxin to diamond back moth, and the Carnobacterium maltuliformis PxCG2 strain can be used as a novel biological control bacterium, is used for controlling cruciferous vegetable pests, and has good biological control potential and application prospect.

Drawings

FIG. 1 is a photograph of a plate with PxCG2 isolated microorganisms.

FIG. 2 is phylogenetic analysis of P.plutella intestinal isolate PxCG 216S rRNA. Note: labeled on each branch: GenBank sequence number + strain name.

FIG. 3 is the survival rate analysis of diamondback moth larvae treated differently. Note: Axenic-Cry1Ac was expressed as a sterile population and had no Cry1Ac protoxin added; axenic + Cry1Ac is indicated as a sterile population feeding Cry1Ac protoxin treatment; axenic + Cry1Ac + PxCG2 shows aseptic population treated with both Cry1Ac protoxin and PxCG2 bacterial solution.

Detailed Description

The invention is further described with reference to the drawings and specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

EXAMPLE 1 isolation culture of Strain PxCG2

(1) Preparation of a selective separation culture medium:

LB medium (10g peptone, 5g yeast extract, 10g NaCl, 15g agar, dissolved in 1L sterile ddH2O water, pH adjusted to 7.0);

nutrition agar medium NA (3g beef extract, 10g peptone, 5g NaCl, 15g agar, dissolved in 1L sterile ddH2O, pH 7.0).

(2) Dissecting and plating: dissecting 3-year larvae of diamondback moth under aseptic condition, collecting midgut content, centrifuging, collecting supernatant and precipitate, diluting by 5 concentration gradients, respectively coating NA flat plates after diluting by 10-4-10-5 times, culturing in 30 deg.C constant temperature incubator, and observing once every 24 hr;

(3) culture with continuous purification and photographing: after single colonies grow out in the selective culture medium, the single colonies are firstly selected according to the color, size and shape of the colonies, each single colony is continuously streaked and purified for more than 5 times on an LB (Langmuir-Blodgett) plate, then the streaked plate for separating the strains is photographed (figure 1) and is transferred to an LB liquid culture medium, and the streaked plate is preserved in 15 percent glycerol water solution when the strains are shaken to an exponential growth phase and is frozen and preserved in a refrigerator at minus 80 ℃ for later use.

Example 2 identification of Strain PxCG2 and phylogenetic analysis

1. Conventional biological assays

(1) Morphological characteristics of bacterial colony

The PxCG2 bacteria are round white colonies in morphology, have smooth and glossy surfaces, and are single elongated bacilli arranged in pairs or chains. Heterotypic fermentation, producing mainly L (+) -lactic acid from glucose. The optimal growth temperature is 30 ℃, the growth is aerobic, and the growth is slow under the anaerobic condition.

(2) Physiological and biochemical characterization of Bacillus cereus

The physiological and biochemical determination of bacteria is carried out by referring to a method of 'handbook for identifying common bacteria system', and a bacteria trace biochemical reaction tube is adopted. The results of the tests on available carbon sources are shown in Table 1, and galactose, sorbitol, rhamnose, arabinose, xylose, melibiose, D-ribose, sucrose and uranidin can be utilized, but lactose, inulin, raffinose, melezitose and starch can not be utilized.

Biochemical characteristics of table 1 PxG2

2. Molecular biological identification

(1) The stored genomic DNA of the monoclonal strain was extracted using a bacterial genomic DNA extraction Kit (TIANAmp Bacteria DNA Kit) from Tiangen organisms, and 16S rDNA of the Bacteria was amplified using the extracted DNA as a template and 16S rDNA universal primers 27F (5 '-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as upstream and downstream primers, and the PCR reaction system was as shown in Table 2. After gently mixing, briefly centrifuged, and placed on a PCR instrument according to: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 15s, and 35 cycles; 72 ℃ for 5 min; 4 ℃ end program reaction, detecting the PCR product by 1% agarose gel, cutting the gel, recovering and purifying, and sending to the department of biotechnology limited company (Guangzhou) for sequencing.

TABLE 2 bacterial 16S rDNA PCR amplification System (50. mu.L)

(2) Phylogenetic analysis of strain PxCG2

Based on the results of sequencing primers 27F and 1492R. The sequencing quality was analyzed and the sequence was spliced using SeqMan (DNAStar) and then blast aligned to the rRNA/ITS database in NCBI and uploaded to the GenBank database. Selecting a kindred sequence of a downloaded and sorted PxCG2 strain, then performing multi-sequence alignment analysis by using ClustalW software, then constructing a phylogenetic tree by using Mega7.0 software through a Neighbor-Joining method (Neighbor-Joining), adjusting a bootstrap value and checking the reliability of the phylogenetic tree. As shown in FIG. 2, the result indicates that PxCG2 has the highest similarity to Carnobacterium maltulationstrain DSM 20342. It was named as Plutella xylostella aromatic Carnobacterium (Carnobacterium maltophilium) PxCG2 strain, and classified into the kingdom of Bacteria (Bacteria), the phylum Firmicutes (Firmicutes), the class of Bacteria (Lactobacillales), the order of Lactobacillales (Bacillus), the family of Carnobacterium (Carnobacterium), the genus of Carnobacterium (Carnobacterium), and the strain of aromatic Carnobacterium maltophilium (Carnobacterium maltophilium). The strain is preserved in Guangdong province microorganism culture collection center (GDMCC) in 8-11 th month in 2020, and the strain preservation number is GDMCC No: 61133, classified and named as Carnobacterium maltulationcum PxCG2, and the preservation address is Mianfural Zhonglu 100, Guangzhou, Guangdong province.

Example 3 synergistic Effect of PxCG2 Strain on Bt

1. Elimination of enteric bacteria of diamondback moth

Preparing an antibiotic solution: 1mg/mL ciprofloxacin; 1mg/mL levofloxacin; 2mg/mL metronidazole;

a method for removing intestinal bacteria of diamondback moth and adding antibiotics. The specific method comprises the following steps: collecting and sterilizing diamondback moth egg cards: collecting the egg cards laid by the plutella xylostella in the egg laying prime period, firstly placing the egg cards in 5 per mill of sodium hypochlorite disinfectant for disinfection for 10 minutes, then placing the egg cards in clear water for soaking for two times, each time for 5 minutes, finally, placing the egg cards in an insect breeding box after being dried by absorbent paper, and adding feed for feeding after 24 hours;

and (3) antibiotic feeding treatment: weighing 4g of artificial feed for the plutella xylostella in an aseptic culture dish, respectively adding more than 333 mu L of prepared ciprofloxacin, levofloxacin and metronidazole antibiotic solution, uniformly stirring, placing in an insect breeding box, replacing the feed once a day, continuing to three-instar larvae from the initial incubation larva, and feeding normal feed to a control group;

and (3) detecting the intestinal bacteria clearing effect: randomly collecting 5 head worms from the treatment group and the control group, dissecting and collecting midgut contents, and sterilizing ddH₂Dilution gradient 10³～10⁴Taking 100 mu L of a plated LB solid medium (without resistance), and detecting whether bacteria grow or not after 48 hours; and grinding and extracting DNA of about 20 head worm midgut, detecting by using a 16S universal amplification primer PCR (polymerase chain reaction), and detecting whether an amplification band exists or not.

2. Test group treatment

Selecting three-instar plutella xylostella with consistent healthy development duration. Sensitivity determination of different treatments to Cry1Ac protoxin (20ug/mL) Using sub-lethal doses (LC)₂₅) Cry1Ac protoxin. Storing Cry1Ac protoxin with ddH₂O diluted to 20ug/mL and then 1mL toxin dilution per 4g feed was mixed. Before feeding 3-instar diamondback moth larvae, starving the diamondback moth for 4h, and counting the number of live insects every 12h from 24h after feeding until 132 h. The survival rate statistical analysis is shown in FIG. 3. Data processing and charting differential significance analysis was performed using Graphpad 7.0 software with Student's-T test. Therefore, when ciprofloxacin, levofloxacin and metronidazole antibiotics are used for feeding and removing the intestinal flora of the plutella xylostella, the mortality rate of the plutella xylostella is obviously increased by feeding Cry1Ac protoxin and PxCG2 bacterial liquid. After the feed is added for 60 hours, the death rate of the diamondback moth is 90 percent, and the death rate of the diamondback moth is 98.33 percent after 72 hours; under the treatment of independently feeding Cry1Ac protoxin, the mortality rate is obviously reduced compared with that of simultaneously feeding Cry1Ac protoxin and PxCG2, the mortality rate of 60h plutella xylostella is 58.33% and the mortality rate of 72h is 71.67% when Cry1Ac protoxin is fed; the PxCG2 strain separated and identified by the invention has the effect of synergizing Cry1Ac protoxin to quickly kill the plutella xylostella, and the separated plutella xylostella maltulinum aromatic Carnobacter (Carnobacterium maltulinum) PxCG2 strain can be used as a novel biological control bacterium for controlling cruciferous vegetable pests, and has good biological control potential and application prospect.

Sequence listing

<110> southern China university of agriculture

<120> plutella xylostella malt aromatic Carnobacterium PxCG2 strain and application thereof

<141> 2020-11-16

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1428

<212> DNA

<213> Plutella xylostella malt Aromatobacter aromatica (Carnobacterium maltaromaticum PxCG2)

<400> 1

ctatactgca gtcgaacgca cgaagttgaa gagcttgctc tttgaccaag tgagtggcgg 60

acgggtgagt aacacgtggg taacctgccc attagagggg gataacattc ggaaacggat 120

gctaataccg catagtttca ggaatcgcat gattcttgaa ggaaaggtgg cttcggctac 180

cactaatgga tggacccgcg gcgtattagc tagttggtga ggtaatggct caccaaggca 240

atgatacgta gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga 300

ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac 360

gccgcgtgag tgaagaaggt tttcggatcg taaaactctg ttgttaaaga agaacaagga 420

tgagagtaac tgctcatccc ctgacggtat ttaaccagaa agccacggct aactacgtgc 480

cagcagccgc ggtaatacgt aggtggcaag cgttgtccgg atttattggg cgtaaagcga 540

gcgcaggcgg ttctttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg 600

gaaactggag aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg 660

cgtagatatg tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg 720

aggctcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg 780

atgagtgcta agtgttggag ggtttccgcc cttcagtgct gcagctaacg cattaagcac 840

tccgcctggg gagtacggcc gcaaggctga aactcaaagg aattgacggg gacccgcaca 900

agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat 960

cctttgacca ctctagagat agagctttcc cttcggggac aaagtgacag gtggtgcatg 1020

gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta 1080

ttactagttg ccagcattta gttgggcact ctagtgagac tgccggtgac aaaccggagg 1140

aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca 1200

atggatggta caacgagtcg caaggtcgcg aggccaagct aatctcttaa agccattctc 1260

agttcggatt gtaggctgca actcgcctac atgaagccgg aatcgctagt aatcgcggat 1320

cagaacgccg cggtgaatac gttcccgggt cttgtacaca ccgcccgtca caccacgaga 1380

gtttgtaaca cccgaagccg gtgaggtaac cttttaggag ccagccgt 1428

Claims (7)

1. Diamondback moth malt aromatic carnobacter (A.B.)Carnobacterium maltaromaticum) PxCG2 strain, wherein the strain is deposited at Guangdong province microbial culture Collection (GDMCC) at 8/11/2020, and the strain deposit number is GDMCC No: 61133.

2. the use of the strain of plutella xylostella maltogenic Carnobacterium aromaticum PxCG2 as claimed in claim 1 for controlling plutella xylostella.

3. A method for controlling diamondback moth, characterized in that the diamondback moth is fed with the strain PxCG2 of the diamondback moth malt aromatic bacillus of claim 1 together with Bt protein.

4. Use of the strain Plutella xylostella Malachio & Acidobacterium PxCG2 in the preparation of a medicament for the control of Plutella xylostella.

5. An agent for controlling diamond back moth, comprising the diamond back moth malus aromatic carnobacterium PxCG2 strain of claim 1.

6. The agent of claim 5, further comprising a Bt protein.

7. The method of claim 3 or the agent of claim 6, wherein the Bt protein is Cry1Ac protoxin.

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